Thèse Nadine Ali

Communaut
es de n
ematodes phytoparasites associ
es `
a
lolivier : r
eponse aux for
cages anthropiques et
environnementaux
Nadine Ali
To cite this version:

Nadine Ali. Communautes de nematodes phytoparasites associes a` lolivier : reponse aux
forcages anthropiques et environnementaux. Phytopathologie et phytopharmacie. Ecole
nationale superieure agronomique de montpellier - AGRO M, 2015. Francais. <NNT :
2015NSAM0015>. <tel-01398778>
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Dlivr par
Centre international dtudes suprieures en sciences
agronomiques de Montpellier (SUPAGRO)
Prpare au sein de lcole doctorale
Systmes Intgrs en Biologie, Agronomie, Gosciences,
Hydrosciences, Environnement (SIBAGHE)
et de lunit de recherche
Centre de biologie pour la gestion des populations (CBGP)
Spcialit :
cologie, Evolution, Ressources Gntique, Palobiologie (EERGP)
Direction de thse : Thierry MATEILLE, DR IRD
Co-encadrement : Elodie CHAPUIS, CR IRD
Prsente par Nadine ALI
Communauts de nmatodes phytoparasites

associes lolivier : rponses aux forages
anthropiques et environnementaux
Soutenue le 08 octobre 2015 devant le jury compos de :
Mr Philippe CASTAGNONE-SERENO
Directeur de recherches
INRA, Sophia Antipolis
Rapporteur
Mr Serge KREITER
Professeur
Montpellier SupAgro
Examinateur,
Prsident du jury
Mr Thierry MATEILLE
Directeur de recherches
IRD, Montpellier
Directeur de thse
Mr Manuel PLANTAGENEST
Professeur
Agrocampus-Ouest, Rennes
Rapporteur
Mr Thierry TATONI
Professeur
Universit Aix-Marseille
Examinateur
Remerciements
S'engager dans un projet aussi long et complexe quune thse ne peut tre une dmarche solitaire. Cette gageure,
apporte un lment scientifique contribuant la connaissance et au dveloppement du savoir, ncessite de
la part du "thsard" une volont inflexible mais aussi la confiance de la communaut des chercheurs dans ses
capacits et ses comptences.
Tout dabord, je remercie les financeurs de ma thse : luniversit de Tichrine Lattaqui en Syrie ainsi que le projet
PESTOLIVE (ARIMNET).
Je tiens remercier chaleureusement les Professeurs Serge Kreiter (Montpellier SUPAGRO), Thierry Tatoni (UAM,
Marseille), Manuel Plantagenet (AgroCampus Ouest, Rennes) et le Docteur Philippe Castagnone-Sereno
(INRA, Sophia-Antipolis) pour le temps et lintrt quils ont consacr lvaluation de ce travail ainsi que
pour leurs propositions pour lamlioration de ce travail. A Philippe et Manuel encore une fois, merci
davoir accept den tre les rapporteurs.
Mes sincres remerciements vont Mon directeur de thse Thierry Mateille et mon encadrante Elodie Chapuis...
Merci Thierry pour tout : merci dtre plus quun directeur de thse, dtre lami quand il fallait, merci pour lattention
et le grand soutien que vous mavez apport au quotidien, merci pour tous les conseils, tout le temps et les
efforts que vous avez dpenss pour moi et d'avoir su me remonter le moral, mencourager jusqu la fin. Je
pense aussi votre pouse pour lui dire merci de tout cur
A toi Elodie, je reconnais tout ce que tu as fait pour moi, tout ce que jai appris avec toi. Je texprime ma profonde
gratitude davoir t mes cts tout au long de ce travail, pour ton encadrement scientifique et rdactionnel
merci pour tout..
Johannes Tavoillot, la main magique qui ma accompagne tout au long des manips, merci pour ton soutien
personnel et professionnel. Merci pour ton amiti, pour ton appui technique qui a rendu l'achvement de mes
travaux possible. Sans ton aide, je ne sais pas comment ma thse aurait pu voir le jour
Un grand merci aux membres de mon comit de thse : Marie-Laure Navas (Montpellier, SUPAGRO), Guillaume
Besnard (CNRS-UPS, Toulouse), Eric Grenier (INRA, Rennes), Benoit Facon (INRA, Montpellier) et Michel
Nicole (IRD, Montpellier). Merci galement aux membres extrieurs qui ont particip lun ou lautre des
comits : Stphane Bllafiore (IRD, Montpellier) et Raphal Leblois (INRA, Montpellier)
Guillaume Besnard et Stphane Bllafiore avec qui jai eu de prcieuses collaborations. Merci beaucoup Guillaume
pour tout ce que tu as fait dans ma thse, pour toute aide, conseils et soutien sur tout ce qui concerne la
gntique et tes connaissances sur la gntique de lolivier. Merci Stphane pour ton sens du partage de
lexprience et pour ta bonne humeur.
Tous mes remerciements vont aussi Bouchaib Khadari (INRA, Montpellier) pour tout effort dans le cadre de ma
thse et pour tout ce qui concerne les analyses de la diversit gntique de lolivier..
A Dr. Pablo Castillo (Institute for Sustainable Agriculture, CSIC, Espagne), j'adresse mes sincres remerciements pour
lamabilit et votre collaboration. Merci davoir consacr du temps rpondre mes questions et me
donner des conseils. Je tiens galement remercier Dr. Juan Emilio Palomares-Rius et Carolina Cantalapiedra
pour la collaboration que nous avons eue, couronne par la description dune nouvelle espce de nmatodes.
J'adresse aussi ma reconnaissance tous les collaborateurs du projet PESTOLIVE au Maroc qui nous ont beaucoup
aid pendant lchantillonnage et la suite du travail. Je pense en particulier au Pr. Abdelhamid El Mousadik et
Mohamed At Hamza (UIZ-FSA, Agadir), au Pr. Mohamed Ater (UAE-FST, Ttouan), Pr. Cherkaoui EL
Modafar (UCA-FSTG, Marrakech), aux Dr. Abdelmajid Moukhli et Dr. Acha El Oualkadi (INRA, Marrakech),
Dr. Ahmed El Bakkali (INRA, Mekns) et Dr. Zahra Ferji (IAV, Agadir). Je tiens galement remercier Dr.
Ewa Dmowska et Grayna Winiszewska (MIZ-PAS, Lomianki, Pologne) pour leur collaboration et leur
investissement dans ce travail
Un merci tout particulier Odile Fossati (IRD, Montpellier) pour ton amabilit et de mavoir encadre pendant une
grande partie de mes analyses de donnes.
Je noublierai jamais sa prsence bien aime au laboratoire, lui qui ma initie lidentification des nmatodes et
lensemble des techniques en Nmatologie, celui qui a toujours eu le sens de lamiti, Bernard Martiny (IRD,
Montpellier). Merci toi et ton pouse.
Je suis particulirement reconnaissante du bon accueil que jai reu au Centre de la Biologie pour la Gestion des
Populations (UMR CBGP) au sein duquel jai effectu mon stage de Master 2 puis ma thse. Durant ces annes
au CBGP, je me sentais toujours que je suis au sein de ma famille avec lamiti et le soutien quon ma apport.
Je tiens remercier chaleureusement la direction : Flavie Vanlerberghe, Jean-Yves Rasplus et Jean-Pierre
Rossi (INRA, Montpellier), Jean-Marc Duplantier et Laurent Granjon (IRD, Montpellier) qui nont jamais
manqu defforts pour maider rsoudre tous mes problmes administratifs.. Flavie et Laurent, de tout
cur, merci beaucoup pour tout..
Je tiens galement remercier toute lquipe de gestion Samira Ouabbi-Saleh, Corine Seguy, Julien Moulin (INRA,
Montpellier), Julie Martin, Sylvie Hart, Charlotte Moreau, et Sigrid Neveu (IRD, Montpellier). Un grand merci
particulirement adress Vincent Danneville (INRA, Montpellier) qui ma beaucoup soutenue et qui a,
depuis son arrive dans lunit, ma aide dbloquer de nombreuses situations difficiles..
A Astrid Cruaud, Simon Fellous, Julien Foucaud, Emmanuelle Jousselin, Carole Kerdelhu, Gal Kergoat, Sylvain Piry,
Jean-Pierre Rossi (INRA, Montpellier), Carine Brouat (IRD, Montpellier), Jean-Franois Martin et MarieStphane Tixier (Montpellier, SUPAGRO), Guillaume Fried (ANSES, Montpellier), qui ont tous
chaleureusement rpondu mes demandes de discussions et qui mont clarifi certains points. Merci vous
Un grand merci tous les chercheurs et les chercheuses, tous les techniciens et les techniciennes avec qui jai pass de
bons moments au cours de ces dernires annes. Merci Alain M., Anne L., Anne X., Anne-Laure, Andrea,
Antoine, Armelle, Arnaud, Berhnard, Bruno, Bruno M., Caroline, Christine S., Denis, Emma, Eric P., Fabrice,
Franck, Hlne, Gauthier, Guillaume, Gwen, Jean-Franois, Jean L., Jean-Marie, Jean-Marc T., Laure S., Laure B.,
Laurent, Mathieu, Max, Maria, Maria N., Marie-Pierre, Martial, Nathalie CH., Nathalie G., Nathalie Vieira,
Nicolas, Philippe Auger, Philippe G., Philippe A., Raphaelle, Rjane, Renaud, Sabine N., Stphane, Yannick
Sussmuth. Un Merci bien particulier Alex pour lamiti et pour tout encouragement et conseil
A celles qui mont toujours fait sentir que jai ma mre mes cts, qui mont beaucoup aide pour rsoudre mes
problmes, Florence Baussant (INRA, Montpellier) et Manuelle Rival (IRD, Montpellier), merci beaucoup..
Merci aux collgues avec qui jai cohabit dans le bureau B1.14, dont chacun ma apport une aide et un savoir-faire.
Merci Allan, Arnaud, Aurlien, Ashraf, Claudius, Elsa, Florian, Homa, Marion, Nicolas, Olivier, Valentin,
Jadresse un grand merci galement Guillaume Laugier pour toute laide que tu mas apporte.
A Ghais, Allan, et Sabine, je vous adresse toutes ma reconnaissance et mon amiti, Merci dtre toujours mes
cts, de me soutenir, de mencourager surtout la dernire ligne droite.
Un grand merci galement Karmadine, Denise, Victor, Muckherjy dtre mes cts durant les dernires
priodes les plus difficiles, merci pour les moments de dtente que jai eus avec vous.
Un grand merci aux thsards avec qui jai partag la souffrance de faire une thse , je pense surtout Julie, Joa,
Martin, Christophe, Antoine,..
Un grand merci Dr. Philippe Castagnone-Sereno, Dr. Nicole Viaene, Dr. Fateh Toumi et Nancy De Sutter (IAFR,
Merelbeke, Belgique), pour la fourniture des matriels biologiques dont javais besoin pour certains de mes
exprimentations
Tous mes remerciements Martine Barraud et Elisabeth Bozsonyik (charge des tudes doctorales Montpellier,
SupAgro) qui mont beaucoup aid et mont conduit pendant mes dmarches dinscriptions chaque anne et
toi Elisabeth pour toute aide pendant toutes les procdures la dernire ligne droite..
A Dr. Manuel Mota (NemaLab-ICAAM, Portugal), Dr. Saad Hafez (University of Idaho, Etats-Unis) qui mont
beaucoup soutenu et encourag. A Dr. Lieven Waeyenberge (IAFR, Merelbeke, Belgique) et Pr. Dr. Gerrit
Karssen (NOOP, Pays-Bas) pour leurs conseils
Encore lui, Serge Kreiter qui ma beaucoup aide depuis mon arrive Montpellier pour le master 2 en sant des
plantes. Je pense aussi adresser un grand merci tous les professeurs et les enseignants que jai eus
pendant ce master
A Michel Nicole (IRD, Montpellier), Henri-Pierre Aberlenc et Grard Delvard (CIRAD, Montpellier) pour lamiti et
les discussions riches et diverses avec vous
Je souhaite galement adresser tous mes remerciements Dr. Caroline-Djian Caporalino et Dr. Pierre Abad (INRA,
Sophia-Antipolis) qui mont beaucoup aide pendant ma premire anne en France. Jai galement une
grande pense tous les collgues du Centre INRA de Sophia-Antipolis avec qui jai vcu des grands
moments. Je pense Ariane, Laurent, Jol, Nathalie, Karine, Samira, Daniel, Gilbert, Paulo, Mohamed. Je vous
exprime tous mes remerciements.. Je pense aussi Nicole Bareste, merci pour tout..
Je pense aussi Ali pour, de tout cur pour tout, merci beaucoup
A toi Serge, merci davoir partag mes moments de doute et de dcouragement en me disant souvent "qu'une chose
commence mrite d'tre acheve"
Jai galement une pense chaleureuse Simon Benateau et Marie, merci pour tout
A Jean-Jacques et sa famille, Linda en particulier, jadresse tous mes remerciements..
A Ahlam, Myriam, Marie-Ccile, Elise, merci pour tout..
A toi, Olivier, jai une grande reconnaissance pour tout le soutien et laide que tu mas apport
Merci ceux, trop nombreux pour tre cits, qui ont crois mon chemin durant cette priode intense, en me prtant
cette attention rconfortante pour me pousser avancer, mes compatriotes syriens de mavoir aide dans les
moments difficiles ainsi que dans les moments de joie. Grce eux, je me suis cr une deuxime famille
ltranger. Parmi vous, de tout cur, merci tous ce qui mont soutenue le jour de ma soutenance, par la
prsence ou par la pense..
A toi, Mudar, tous mes reconnaissances pour tout conseil et encouragement...
A ma grande famille, mes ami(e)s en Syrie, mes professeurs la facult dagronomie luniversit de Tichrine
Lattaqui, qui mont beaucoup soutenue toujours fiers de moi, parmi vous, Dr. S. El Moughrabi, qui a suivi
lavancement de mes tudes en France, et tous mes professeurs et collgues au Dpartement de Protection
des Plantes, Facult dagronomie, Lattaqui..
Enfin, pour leur amour et le courage de subir la sparation de ces longues annes malgr les vnements qui frappent
mon pays, malgr la peur du malheur, j'prouve aujourd'hui la vnration la plus profonde pour mes parents,
mes frres, ma sur, mes belles-surs et les petits adors Koki, Zozo, Aloushi, Shahd, Hiba..
Sommaire
Figures. vi
Tableaux.. ix
Chapitre I : Introduction gnrale
Contexte de ltude 2
1. La biodiversit : notion retenir.. 4
2. La communaut, lieu dinteractions multiples 9
3. Le sol : trsor de biodiversit 11
4. Les nmatodes comme organismes modles 13
4.1. Gnralits 13
4.2. Les nmatodes phytoparasites : un enjeu pour l'agriculture. 15
4.3. Diversit des communauts de nmatodes phytoparasites... 19
4.4. Diversit interspcifique : le cas des nmatodes galles du genre Meloidogyne. 21
4.4.1. Cycle biologique standard des nmatodes du genre Meloidogyne.. 22
4.4.2. Importance agro-conomique 23
4.4.3. Diversit biologique et gntique des Meloidogyne.. 24
5. Lolivier : modle cologique dtude de la structuration des communauts de nmatodes
phytoparasites 27
5.1. Quelques lments de botanique.. 27
5.2. Distribution... 27
5.3. Elments culturels. 28
5.4. Histoire volutive.. 29
6. Objectifs de la thse et questions de recherche... 32
7. Rfrences bibliographiques. 34
Chapitre II : Les nmatodes phytoparasites associs lolivier au niveau mondial
41
Rsum 42
Abstract... 43
1. The olive tree: origins, dissemination and cultivation 43
2. Plant-parasitic nematodes associated with olive trees 44
2.1. General considerations.. 44
2.2. Diversity of plant-parasitic nematodes associated with olive trees.. 45
2.3. Distribution of plant-parasitic nematodes. 45
2.4. Impacts of plant-parasitic nematodes on olive trees. 51
3. The dominant plant-parasitic nematodes associated with olive trees... 54
3.1. Meloidogyne spp. (root-knot nematodes).54
3.2. Pratylenchus spp. (lesion nematodes)... 55
3.3. Helicotylenchus spp. (spiral nematodes)... 55
3.4. Xiphinema spp. (dagger nematodes). 56
3.5. Tylenchulus spp. (citrus nematodes). 56
3.6. Rotylenchulus spp. (reniform nematodes) 56
3.7. Heterodera spp. (cyst nematodes) ... 56
3.8. Other plant-parasitic nematodes57
4. Management of plant-parasitic nematodes on olive... 57
4.1. Exclusion of PPN infection... 57
4.2. Plant resistance.. 57
4.3. Soil solarisation. 57
4.4. Biological control.. 58
5. Prospects: better knowledge on PPN biodiversity for better management.. 58
Acknoledgements... 58
References.. 58
Chapitre III: Mthodologie gnrale (a)

Etude de la diversit des nmatodes phytoparasites associs lolivier au Maroc
63
64
1. Sites dtude 65
2. Echantillonnages et prlvements 66
3. Traitements des chantillons. 67
3.1. Extraction des nmatodes..
67
3.2. Caractrisation morphologique des nmatodes au rang de la famille, du genre et de lespce 68
3.3. Caractrisation gntique des oliviers...69
3.4. Analyses physico-chimiques du sol. 69
3.5. Caractristiques climatiques. 70
4. Typologie du dispositif dchantillonnage au Maroc.. 71
4.1. Cartographie des variables dans les sites chantillonns au Maroc.. 71
4.1.1. Systmes olicoles. 72
4.1.2. Diversit chloroplastique gntique de lolivier72
4.1.3. Types de sols chantillonns. 73
4.1.4. Variables climatiques 73
4.2. Structure des donnes environnementales 73
5. Analyse de la diversit des nmatodes phytoparasites... 80
5.1. Diversit taxonomique.. 80
5.2. Dominance des taxons dans les chantillons 81
5.3. Diversit fonctionnelle et trophique. 81
5.4. Patrons des communauts. 82
6. Analyses statistiques.. 82
6.1. Jeux de donnes 82
6.2. Analyses 82
7. Rfrences... 83
Chapitre IV: Diversit taxonomique des nematodes en communauts
85
Rsum 86
Abstract... 87
1. Introduction 88
2. Materials and Methods.. 91
2.1. Site description.. 91
2.2. Genetic characterisation of the olive trees 92
2.3. Soil sampling 93
2.4. Nematode extraction, identification and quantification 94
2.5. Climate and soil characteristics 94
2.6. Definition of biodiversity and environmental variables for analyses.. 95
2.7. Data analyses 95
3. Results. 96
3.1. Taxonomical diversity of plant-parasitic nematodes 96
3.2. Biodiversity structure between putative refuge areas of wild olive.. 97
3.2. PPN biodiversity in communities according to anthropogenic changes and environmental conditions.. 98
4. Discussion99
4.1. Diversified PPN communities associated with olive trees in Morocco 100
4.2. Role of human activities in the structure of PPN diversity in communities. 100
4.3. Persistence of PPN diversity in communities associated with wild olive areas... 102
5. Concluding remarks.. 103
6. References... 104
Chapitre V: Diversit fonctionnelle
125
Rsum 126
Abstract... 127
1. Introduction 128
ii

2.3. Nematode extraction, identification and quantification... 130
2.4. Nematode ecological indices 130
3. Results. 133
3.1. Functional diversity of the plant-parasitic nematode communities.. 133
3.2. Trophic diversity of plant-parasitic nematode communities.134
4. Discussion137
5. Concluding remarks.. 140
Chapitre VI: Patrons des communauts de nematodes phytoparasites
143
Rsum 144
Abstract.. 145
1. Introduction 146
2.3. Nematode extraction, identification and quantification 151
2.4. Genetic characterisation of the olive trees 151
2.5. Climate and soil characteristics 152
2.6. Definition of environmental variables 152
3. Results. 154
3.1. Dominance of the plant-parasitic nematodes 154
3.2. Plant and environmental typology of the sampling device.. 155
3.3.Correspondences between plant-parasitic nematodes community patterns and environmental variables 158
3.3.1. Patterns related to olive tree variables... 158
3.3.2. Patterns related to climatic modalities... 158
3.3.3. Patterns related to soil data 159
3.3.4. Patterns related to all environmental variables. 159
3.4. Species diversity of a widespread genus: the case of Helicotylenchus spp.. 166
4. Discussion168
Chapitre VII. Mthodologie gnrale (b)

Etude de la variabilit interet intra spcifique du genre Meloidogyne spp.
175
176
1. Caractrisation spcifique des nmatodes du genre Meloidogyne. 176

1.1. Traitement des chantillons.. 176
1.2. Caractrisation biochimique. 178
1.3. Caractrisation molculaire.. 178
2. Etude de la diversit interet intraspcifique des nmatodes du genre Meloidogyne. 179
2.1. Diversit molculaire 179
2.1.1. Marqueurs microsatellites. 179
2.1.2. Analyses des squences ITS2 180
2.1.3. Analyses des squences COI 180
2.1.4. Analyses des squences 63R 181
2.2. Diversit morphologique.. 181
3. Rfrences... 181
Chapitre VIII: Description dune nouvelle espce de Meloidogyne : M. spartelensis n.sp.
184
Rsum.... 185
iii
Abstract... 186
1. Introduction........ 187
2. Materials and Methods...... 187
2.1. Soil sampling, nematode extraction, and Meloidogyne spp. detection and rearing.. 187
2.2. Nematode morphological identification
188
2.3. Isozyme phenotype analysis..
188
2.4. Nematode molecular identification... 188
2.5. Phylogenetic analyses... 189
3. Results. 189
3.1. Meloidogyne spartelensis n. sp. 189
3.2. Associated host and locality.. 194
3.3. Diagnosis and relationships.. 195
3.4. Isozyme analysis and molecular characterization. 195
4. Discussion198
Acknoledgements... 201
References... 202
Chapitre IX: Diversit des Meloidogyne associs lolivier au Maroc
204
Rsum............ 205
Abstract........... 206
1. Introduction.... 207
2. Materials and Methods...... 209
2.1. Site description. 209
2.3. Soil and climate characteristics. 211
2.4. Nematode extraction and root-knot nematode cultures 211
2.5. Identification of Meloidogyne species.. 212
2.5.1. Isozyme phenotype analysis.. 212
2.5.2. Molecular identification 212
3. Results......... 214
3.1. Species characterization 214
3.2. Distribution of the Meloidogyne populations215
3.3. Correspondences between the abundance of Meloidogyne species and environmental factors... 216
4. Discussion....216
Chapitre X: Variabilit gntique chez Meloidogyne spp. associ lolivier
235
Rsum............ 236
A. Morphological intraspecific diversity of Meloidogyne javanica juveniles parasitizing olive trees in
Morocco
237
Abstract.. 237
1. Introduction... 238
2. Materials and Methods..... 241
2.1. Sites sampling and Meloidogyne javanica acquisition. 241
2.2. Establishment of nematode subcultures 242
2.3. Preparation of dead material. 243
2.4. Morphometric study. 243
2.5. Statistical analyses 243
3. Results......... 245
4. Discussion... 250
5. References.. 253
iv
B. Etude de la diversit molculaire des populations de Meloidogyne spp.

associes lolivier au Maroc
257
1. Introduction.... 257
2. Matriels et Mthodes.... 260
3. Rsultats...... 261
3.1. Analyses des squences ITS2... 261
3.2. Analyses des squences COI262
3.3. Analyses des squences 63R. 262
4. Discussion... 267
5. Rfrences... 269
Chapitre XI: Synthse et discussion gnrale
273
1. Rappel de la problmatique et des objectifs.... 274

2. Principaux rsultats obtenus durant cette thse..... 276
2.1. Diversit globale des nmatodes phytoparasites.. 276
2.2. Impacts des forages anthropiques et environnementaux..... 276
3. Analyse critique...... 279
3.1. Limites exprimentales. 279
3.2. Biais mthodologiques. 280
4. Perspectives court terme.... 283
4.1. Diversit nmatologique...... 283
4.2. Diversit gntique des Meloidogyne... 284
5. Perspectives long terme.. 286
5.1. De la diversit gntique la diversit fonctionnelle... 286
5.2. Histoire volutive de lolivier mditerranen et de ses parasites associs... 287
5.3. Approche cologique de la gestion des nmatodes phytoparasites.. 287
6. Conclusion.. 289
7. Rfrences.. 290
Annexes
1. Valorisation scientifique 293
2. Annexes techniques 295
Annexe 1: Extraction des nmatodes du sol par Elutriation (Elutriateur de Seinhorst). 295
Annexe 2: Prparation de nmatodes pour les tudes morphologiques... 298
Annexe 3: Prparation des nmatodes pour les tudes molculaires... 302
Annexe 4: Extraction des nmatodes des racines en chambre brouillard 304
Annexe 5 : Caractrisation de nmatodes du genre Meloidogyne par lectrophorse
isoenzymatique (PAGE).. 305
Annexe 6: Procdure dextraction ADN Kit QIAgen DNeasy Blood & Tissue. 310
Annexe 7: Procdure damplification ADN mthode SCARS 312
Annexe 8: Procdure damplification ADN gne 28S portion D2D3 314
Annexe 9: Amorces et conditions damplification utilises pour les analyses microsatellites....................... 315
Annexe 10 : Procdure damplification ADN ribosomique gne Internal Transcriber Spacer 2 (ITS2).316
Annexe 11: Procdure damplification ADN mitochondrial gne Cytochrome Oxydase 1 317
Annexe 12 : Procdure damplification ADN mitochondrial gne 63R.. 318
Figures
Figure 1: Les diffrents niveaux d'organisation de la biodiversit.. 4
Figure 2: Effets des changements globaux et rponses potentielles de lorganisme... 8
Figure 3: Classification des organismes du sol selon leur taille.. 12
Figure 4: Nmatode phytoparasite du genre Pratylenchus spp15
Figure 5: Phylum des Nematoda.. 16
Figure 6: Correspondance entre le patron statistique des communauts de nmatodes et
la production de canne sucre. 20
Figure 7: Galles dues Meloidogyne javanica sur racine de melon 21
Figure 8: Cycle biologique de Meloidogyne spp. (cas dune parthnogense mitotique. 23
Figure 9: Modes de reproduction des espces de Meloidogyne spp 25
Figure 10: Distribution mondiale de lolivier.. 28
Figure 11: Tablette cuniforme portant mention l'huile d'olive 29
Figure 12: Distribution de la diversit chloroplastique des trois lignes E1, E2, E3
de lolivier mditerranen 31
Figure 13 : Objectifs de la thse...
34
Chapitre II : Les nmatodes phytoparasites associs lolivier (revue bibliographique)
Figure 1: Simplified diagram of the Olea genus (Oleaceae) and biogeography of taxa. 44
Figure 2: World distribution of Olea europaea L. producing areas 45
Figure 3: Species diversity in each plant-parasitic nematode genus 52
Figure 4: Frequency of the plant-parasitic nematode genera detected (% of countries). 53
Figure 5: World and Mediterranean distribution of the most frequent plant-parasitic nematode genera
associated with olive tree (i.e. present in more than 25% of countries).. 53
Figure 6: Genus richness of plant-parasitic nematode recorded in each Mediterranean country
and associated publications.. 54
Figure 7: World and Mediterranean distribution of root-knot nematode species (Meloidogyne spp.)
associated with olive tree. 54
Chapitre III : Mthodologie gnrale (a)
Figure 1: Organigramme gnral des procdures suivies au cours de la thse 64
Figure 2 : Sites dchantillonnage dolivier avec indication des rgions et des modalits chantillonnes 66
Figure 3: Procdures dchantillonnage suivies dans des vergers dolivier cultiv et dans des sites
dolivier fral ou sauvage 67
Figure 4: Dispositif dlutriation du sol pour lextraction des nmatodes.. 68
Figure 5: Triangle des textures de sol qui dcrit le type du sol selon les proportions dargiles,
de limons et de sables.. 70
Figure 6: Distribution des sites dchantillonnage et des modalits des diffrents systmes olicoles
rencontrs et les lignes chloroplastiques au Maroc 75
Figure 7 : Distribution des sites dchantillonnage des oliviers sauvages et fraux et les haplotypes
chloroplastiques dtects..
76
Figure 8: Types de sols dans les sites chantillonns au Maroc.. 77
Figure 9: Distribution des isothermes maximaux moyens annuels, des isothermes minimaux moyens
annuels et des isohytes annuels.. 78
Figure 10: Plan factoriel Hill and Smith des principales variables environnementales caractristiques
des sites dchantillonnage...
79
Chapitre IV: Diversit taxonomique des nmatodes en communauts
Figure 1: Sites sampled in Morocco and Southern Spain. Olive growing modality is given for each site..111
Figure 2: A between classes analysis for the putative refugia. 112
Figure 3: Principal Component Analysis PCA results using the nematode species identified data,
for each group defined by the between classes analyse... 113
Figure 4: Multivariate analyses on the plant-parasitic nematode diversity indices and
environmental factors... 114
vi
Figure 5: PPN diversity indices according to environmental variables studied.. 115

Figure S1: cpDNA haplotypes in olive trees sampled. 124
Figure 1: Abundance of plant-parasitic nematodes in cp-3 and cp-2 functional groups according to
the olive growing modality.. 137
Chapitre VI: Patrons des communauts de nmatodes phytoparasites
Figure 1: Localization of sampling sites in Morocco with indications on the olive growing modalities
surveyed in each site 150
Figure 2: Dominance and structure of plant-parasitic nematode genera in communities
in the olive areas surveyed in Morocco... 156
Figure 3: Environmental patterns in the olive areas surveyed in Morocco. 157
Figure 4: Correspondences between plant-parasitic nematode and olive tree data in the areas
surveyed in Morocco 161
Figure 5: Correspondences between plant-parasitic nematode and climate data in the olive areas
Figure 6: Correspondences between plant-parasitic nematode and soil data in the olive areas
Figure 7: Co-inertia loading plot for plant-parasitic nematode communities according to
the most discriminant environmental data in the olive areas surveyed in Morocco 164
Figure 8: Diversity of Helicotylenchus species in the olive areas surveyed in Morocco 167
Chapitre VII. Mthodologie gnrale (b)
Figure 1: Procdure de traitement et de production du matriel biologique pour la caractrisation
biochimique et molculaire ainsi que pour ltude de la diversit des nmatodes du genre Meloidogyne.. 177
Figure 1: Line drawings of Meloidogyne spartelensis n. sp. from Morocco... 190
Figure 2: Light micrographs of Meloidogyne spartelensis n. sp. from Morocco... 191
Figure 3: Scanning electron microscope photographs of Meloidogyne spartelensis n. sp. from Morocco. 193
Figure 4: Esterase (a) and malate dehydrogenase (b) phenotype electrophoresis patterns for
Meloidogyne spartelensis n. sp 197
Figure 5: Phylogenetic relationships within Meloidogyne species using D2 and D3 expansion
segments of 28S rDNA sequences alignments 198
Figure 6: Phylogenetic relationships within Meloidogyne species using ITS rDNA gene
sequence alignment.. 199
Figure 7: Phylogenetic relationships within Meloidogyne species using partial 18S gene
sequence alignment.. 200
Figure 8: Phylogenetic relationships within Meloidogyne species using partial coxII-16S rRNA gene
sequences alignment 201
Figure 1: Electrophoretic esterase patterns of Meloidogyne populations collected from olive soils
in Morocco... 232
Figure 2: Spatial distribution and abundance of the Meloidogyne species identified in Morocco
with indication of olive growing modalities on which the Meloidogyne populations were detected.. 233
Figure 3: Co-inertia analyses between the abundance of the Meloidogyne species and
environmental factors.. 234
A. Morphological intraspecific diversity of Meloidogyne javanica parasitizing olive trees in Morocco
Figure 1: Location of the Meloidogyne javanica populations recovered on olive trees in Morocco.. 242
Figure 2: Experimental procedures from subcultures of Meloidogyne javanica populations to
measurements of morphological traits. 244
Figure 3: Morphological traits measured on Meloidogyne javanica second-stage juveniles.. 245
Figure 4: Box plot of the body length, of the stylet length, of the maximum body width and of the body
width at median bulb among the Meloidogyne javanica... 247
Figure 5: Similarities between the Meloidogyne javanica populations according to the morphological
vii
measurements of juveniles.. 249
B. Etude de la diversit molculaire des populations de Meloidogyne spp. associes lolivier au Maroc
Figure 1 : Arbre phylogntique du gne ITS2 ralis avec lensemble des populations despces
de Meloidogyne identifies sur olivier au Maroc. 263
Figure 2 : Arbre phylogntique du gne COI ralis avec lensemble des populations despces
de Meloidogyne dtectes sur olivier au Maroc, des populations despces de rfrence et
doutgroups GenBank.. 264
Figure 3 : Matrice des squences du gne COI gnres partir des populations despces
de Meloidogyne dtectes sur olivier au Maroc... 265
Figure 4 : Distribution des lignes (A) et haplotypes (B) 63R des populations de M. arenaria et de
M. javanica dtectes sur olivier au Maroc. 266
viii
Tableaux
Tableau 1 : Exemples de dure du cycle de vie, de fcondit, de modes de reproduction et de caryotypes
chez quelques espces de nmatodes phytoparasites... 17
Tableau 2 : caractres biologiques distinguant les nmatodes colonisateurs des nmatodes persistants 18
Chapitre II: Les nmatodes phytoparasites associs lolivier (revue bibliographique)
Table 1: Plant-parasitic nematode taxa associated with olive tree in the word... 46
Chapitre III: Mthodologie gnrale (a)
Tableau 1 : Tests et mthodes statistiques utilises pour les analyses des donnes 71
Tableau 2: Nombre dchantillons par modalit pour chacune des principales variables
Environnementales caractristiques des sites dchantillonnage au Maroc.83
Chapitre IV: Diversit taxonomique des nmatodes en communauts
Table 1: Agricultural and environmental variables and their corresponding codes 109
Table S1. Location of the olive sampling sites surveyed in Morocco and southern Spain. 117
Table S2. Data matrix of the 17 plastid DNA olive haplotypes revealed in this study... 118
Table S3. Plastid DNA lineages and haplotypes detected within spontaneous and cultivated olive trees
collected in Morocco and in some wild olive populations from Southern Spain 119
Table S4. Plant-parasitic nematode taxa associated with olive identified in the study... 120
Table1: Location of the olive sampling sites surveyed in Morocco 132
Table 2: Plant-parasitic nematode genera detected in the olive growing areas sampled in Morocco,
with indication of the functional (cp-value) and trophic groups to which they belong... 135
Table 3: Mean plant-parasitic index (PPI) and relative mean abundance (%) of each cp-i value and
of each trophic group... 136
Table 4: Richness of plant-parasitic nematode genera within each functional (cp-value) and
trophic group according to olive growing modalities.. 136
Chapitre VI: Patrons des communauts de nmatodes phytoparasites
Table 1: Environmental variables and their corresponding codes... 153
Table 2: Nematodes genera and their corresponding codes. 154
Table 3: P-value of GLM analyses for each nematode genera according to the most significant
environmental variables... 165
Table 1: Morphometrics of adult males and second-stage juveniles (J2) of Meloidogyne spartelensis n. sp. 194
Table 2: Morphological and morphometric differences among Meloidogyne species closely
related to Meloidogyne spartelensis n. sp. and parasite species to olive. 196
Table 1: Environmental variables analysed in the co-inertia analysis, and corresponding codes... 229
Table 2: Location of the olive sampling sites surveyed in Morocco and infested by Meloidogyne spp. 230
Table 3: Biochemical and molecular diagnostics of the Meloidogyne species associated with olive trees
in Morocco and their abundance (number of juveniles/dm3 of soil. 231
A. Morphological intraspecific diversity of Meloidogyne javanica parasitizing olive trees in Morocco
Table 1: Mean and standard deviation (SD) for each morphological trait in each population 248
Table 2: Mean and standard deviation (SD) for each morphological trait in each hierarchical group 250
ix
Chapitre I
Introduction gnrale
Aperu gnral sur :
la biodiversit, les enjeux qui lui sont associs, ses organisations,
le sol, trsor de diversit biologique,
les nmatodes, invertbrs principaux du sol,
Les nmatodes phytoparasites et lolivier : modles biologiques de ltude.
Page 1
Contexte de ltude
Mon projet de thse porte sur les nmatodes phytoparasites (NPP), vers ronds filiformes
microscopiques vivant dans le sol et/ou lintrieur des tissus vgtaux. Ces parasites
sattaquent, le plus souvent, aux organes souterrains des plantes (racines, tubercules). Ils
peuvent causer aux cultures des dgts agronomiques et conomiques trs importants par la
rduction de la qualit et la quantit de la production agricole. La lutte contre ces parasites
savre difficile en raison de leur vie souterraine ou de leur prsence lintrieur des tissus
vgtaux. La lutte chimique reste jusqu prsent la mthode la plus efficace et la plus facile
mettre en uvre. Nanmoins, son cot reste lev avec un fort impact cologique et sanitaire
sur lenvironnement et la sant humaine, do linterdiction croissante des nmaticides.
Les autres mthodes de lutte disponibles (gntique [rsistance varitale], biologique,
physique, etc.) sont peu efficaces et souvent spcifiques quelques espces emblmatiques
conomiquement importantes qui induisent des dgts significatifs. Ces mthodes ne tiennent
pas compte de la complexit des communauts de NPP (au sens de mlange despces). Le fait
de cibler ces quelques espces emblmatiques et de les liminer perturbe la structure des
communauts et dautres espces dans la communaut prennent le relai en se dveloppant des
niveaux potentiellement nuisibles. Les interventions humaines dans les milieux (dforestation,
intensification des cultures, changement dusage du sol, pesticides, etc.) sont des causes
majeures de perturbation des communauts dorganismes et daggravation des dgts induits
par les ravageurs des cultures.
Les recherches menes par lquipe de recherche IRD dans laquelle jai ralis ma thse
portent sur des tudes comparatives de communauts de NPP dans divers cosystmes, en
valuant les changements de structure et les forages impliqus (analyse descriptive), et en
analysant aussi la part de lhistoire volutive des NPP dans les mcanismes dassemblage des
communauts (analyse mcaniste). Donc, par la comprhension des mcanismes dinteraction
des NPP entre eux et avec leur milieu de vie, lquipe entend dvelopper un nouveau concept
de gestion des NPP par approche conservatoire de leur diversit.
Dans ce cadre, je me suis intresse ltude des communauts de NPP associes la
culture de lolivier dans le Bassin Mditerranen et au Maroc en particulier. Ltude sest
articule sur deux axes. Le premier a consist dcrire la diversit des NPP rencontrs, leur
abondance, leur richesse spcifique et les patrons
des communauts, ainsi qu valuer
limpact de la domestication, de lanthropisation des milieux, des pratiques culturales, et des
Page 2
conditions environnementales sur la diversit des nmatodes en communauts. Le deuxime

axe sest concentr sur les nmatodes galles des racines (Meloidogyne spp.), considrs parmi
les nmatodes les plus nuisibles en agriculture au niveau mondial. Ltude a consist analyser
la diversit spcifique des Meloidogyne associs lolivier et les facteurs qui peuvent
influencer leur distribution. Elle a galement port sur une analyse de la diversit
morphologique et molculaire des populations de M. javanica en particulier.
Ce travail sest intgr dans le programme Communauts de nmatodes phytoparasites :
rponses aux forages volutifs et environnementaux dvelopp par lIRD dans lUMR
CBGP (Centre de la Biologie pour la Gestion des Populations) Montferrier sur Lez. La thse
a t finance par lUniversit de Tichrine Lattaqui en Syrie (bourse de doctorat) et par le
projet PESTOLIVE : Contribution of olive history for the management of soil-borne
parasites in the Mediterranean basin (financement EU-ARIMNet KBBE219262,
www1.montpellier.inra.fr/CBGP/pestolive).
Page 3
1. La biodiversit : notion retenir

Thomas E. Lovejoy un spcialiste de lAmazonie semble tre le premier avoir utilis, en
1980, le terme de diversit biologique , devenu plus tard biodiversit (Le Guyader,
2008). La diversit biologique est dfinie par la variabilit des organismes vivants de toute
origine. Ceci inclut la variabilit gntique lintrieur des espces et de leurs populations, la
variabilit des espces et de leurs formes de vie, la diversit des complexes despces associes
et de leurs interactions, et celle des processus cologiques quils influencent et dont ils sont les
acteurs, dite diversit cosystmique (Le Guyader, 2008 ; Gaston & Spicer, 2013). Autrement
dit, cette variation peut sexprimer trois niveaux de la biodiversit : diversit gntique,
diversit taxonomique et diversit cosystmique (Solbrig, 1991 ; Watson et al., 1995). Elle
couvre donc un champ trs large : des espces sauvages aux espces domestiques, des
bactries aux grands mammifres, mais galement les habitats et les interactions entre ces
diffrentes composantes, des gnes aux cosystmes, et mme les services rendus par les
cosystmes (Commissariat Gnral au Dveloppement Durable, 2010). Par consquent, la
biodiversit sapprhende diffrents niveaux dorganisation biologique : lindividu, la
population, la communaut, lcosystme et la biosphre (Figure 1).
Figure 1: Les diffrents niveaux d'organisation de la biodiversit

(http://max2.ese.upsud.fr/epc/conservation/EE/pdf/Ecologie_Communautes_15112009.pdf).
Page 4
Elle se dcrit laide de plusieurs indicateurs: le nombre despces (richesse), l'abondance, la

composition trophique, la distribution spatiale, les interactions entre gnotypes, entre
populations d'une mme espce, entre espces, entre types fonctionnels et entre traits dont la
variabilit pourrait sexprimer entre et l'intrieur des espces, et les units paysagres dans un
systme donn (Diaz et al., 2006). La structure des peuplements et la distribution de la
biodiversit sont souvent le rsultat de linfluence dun ensemble de facteurs biotiques et
abiotiques (Encadr 1) et de processus cologiques agissant diffrentes chelles spatiales et
temporelles (Levin et al. 2001 ; Neubert et al. 2006).
Encadr 1 : Dfinition des facteurs biotiques et abiotiques influenant la structure des
peuplements
Facteurs biotiques : ce sont les facteurs induits par les interactions entre les individus (e.g.
comptitions, prdation, parasitisme).
Facteurs abiotiques : sont plutt lis aux interactions de lindividu avec son milieu de vie
(e.g. composants physico-chimiques du sol, temprature, pluviomtrie).
La diversification des organismes pourrait tre galement influence positivement ou
ngativement par des facteurs (Butlin et al., 2011) tels que :
La mutation est la modification de l'information gntique dans le gnome, et peut tre
un mcanisme lorigine des diffrentes versions possibles dun gne. La mutation comme
source de la biodiversit est la cl de la biodiversit gntique et phnotypique des individus
dune population ou dune espce. La drive gntique comme la slection naturelle peuvent
conduire lapparition de nouvelles espces. Deux populations dune mme espce
initialement interfcondes peuvent tre isoles et voluer diffremment. La drive gntique et
la slection naturelle sont deux mcanismes d'volution qui peuvent modifier la diversit
alllique au sein d'une population ce qui peut conduire au cours du temps, la diffrenciation
des populations et la gense de nouvelles espces (Encadr 2).
Encadr 2 : Dfinition dune espce
Une espce est une population ou un ensemble de populations dont les individus peuvent
effectivement ou potentiellement se reproduire entre eux et engendrer une descendance
viable et fconde, dans des conditions naturelles. Ainsi, l'espce est la plus grande unit de
population au sein de laquelle le flux gntique est possible et les individus d'une mme
espce sont donc gntiquement isols dautres ensembles quivalents du point de vue
reproductif.
Page 5
Le mode de reproduction est galement un des facteurs de structuration de la

biodiversit, en fonction de l'importance relative des stratgies de reproduction sexue ou
asexue (croissance clonale) dans le maintien des populations (Lbel et al., 2009). Comme
exemple, un nombre croissant de donnes indiquent que les clonales populations peuvent
maintenir des quantits considrables de la diversit gntique (Eckert & Barrett, 1993).
Le rle cl de la biodiversit dans la vie terrestre et son rapport aux humains a t lun
des sujets principaux de la Confrence de Rio-de-Janeiro (Brsil) en 1992, qui a abouti la
Convention sur la Diversit Biologique. Cette convention a soulign un devoir de conservation
de la diversit biologique et dutilisation durable de ses composantes (Blondel, 2005). La
biodiversit au sens large affecte les proprits des cosystmes, donc les services que lhomme
tire des cosystmes. (MEA, 2005 ; Diaz et al., 2006). Le concept de biodiversit, avec tous les
enjeux et dfis quil vhicule sur les plans scientifique, sociologique, conomique et politique,
est directement li la crise de lenvironnement. La crise de l'environnement est devenue en
quelques dcennies un sujet majeur d'inquitude. Elle rsulte de la conjonction entre l'explosion
dmographique, le gaspillage des ressources naturelles et les dsordres cologiques induits en
particulier par les pollutions. Cette crise sest peu peu cristallise dans le monde scientifique
et politique ainsi quauprs du grand public au point de devenir aujourdhui un problme de
socit majeur (Blondel, 2005).
Cest pourquoi, si nous voulons prserver les services actuels et potentiels que les
cosystmes fournissent aux humains, nous devrions nous concentrer sur la prservation ou le
rtablissement de leur intgrit biotique en termes de composition, d'abondance relative,
d'organisation fonctionnelle, et de richesse des espces tout en prenant en compte nanmoins
ladaptation de la biodiversit inhrente aux changements globaux naturels (e.g. climat) ou aux
accidents gologiques et climatiques (e.g. tsunami, cyclones). Cependant, avec le succs
cologique et conomique de lespce humaine, nous vivons actuellement la sixime crise
dextinction (Encadr 3). Les cinq crises prcdentes furent la consquence de catastrophes
gologiques (ruptions volcaniques, etc.) ou astronomiques (chutes de mtores), gnralement
suivies et amplifies par des changements climatiques (e.g. glaciation) et donc cologiques. La
crise actuelle sen distingue puisquelle serait due aux interventions humaines dans les
cosystmes, menaant les fondements dun dveloppement durable des socits humaines
(Barbault, 2005).
Page 6
Encadr 3 : Dfinition dune crise dextinction

La Terre a connu cinq grandes crises dextinction despces. De nombreux scientifiques
considrent que nous vivons aujourdhui la sixime grande crise dextinction et quelle est
due laction de lespce humaine sur son environnement. Une crise est estime partir dun
taux dextinction puisque chaque espce a une dure de vie limite (5 10 millions
dannes). A partir de lesprance de vie des espces et de leur nombre, il est possible de
calculer un taux dextinction global qui correspond la proportion despces qui disparat
pendant un intervalle de temps donn. Il est principalement li, dans un contexte naturel ,
au nombre dindividus : plus le nombre dindividus au sein dune espce est faible, plus les
risques de disparition de cette dernire sont importants car elle a peu de chance de sadapter
aux changements environnementaux (Teyssdre, 2004).
Nous dsignons donc les changements globaux comme des consquences des
changements climatiques et des interventions humaines dans les cosystmes telles que
lurbanisation, la dforestation, la pollution, les invasions biologiques et les pratiques
culturales. Ces changements ont un impact sur la biodiversit et sur lorganisation des
cosystmes. Cependant, limpact des activits humaines sur la biodiversit devient
prpondrant; elles ont accentu l'extinction des espces dun taux 1000 fois suprieur celui
que la plante a pu connatre auparavant, et menacent d'extinction aujourdhui 10 30% des
mammifres, des oiseaux et des amphibiens (MEA, 2005). Ces changements globaux peuvent
causer de fortes pressions de slection sur des traits biologiques importants en terme de valeur
slective, comme par exemple le dcalage des priodes de ponte chez les oiseaux cause du
rchauffement climatique (Garant et al., 2008).
Les organismes soumis linfluence des changements globaux peuvent ragir par
diffrents types de rponses : migration et installation dans de nouveaux environnements o les
conditions de vie sont plus favorables leur survie et leur reproduction (Parmesan, 2006 ;
Lenoir & Svenning, 2013), adaptation aux nouvelles conditions par plasticit phnotypique
(Encadr 4 ; Meril & Hendry, 2014), ou par slection naturelle (Parmesan, 2006 ; Hoffmann &
Sgr, 2011). Par un exemple chez les plantes, la figure 2 illustre les diffrentes provenances de
changement global et les rponses potentielles de lorganisme pour y faire face (Matesanza &
Valladares, 2014). L'importance relative de ces diffrentes rponses pour chaque espce
dpendra de nombreux facteurs tels que l'intensit et la direction du changement de
l'environnement, des traits dhistoire de vie de lespce, sa variabilit gntique ainsi que les
Page 7
interactions entre les espces co-existantes (Jump et al., 2009 ; Matesanz et al., 2010 ; Nicotra
et al., 2010 ; Matesanza & Valladares, 2014). Les consquences peuvent se traduire donc par
diffrents phnomnes comme la modification des aires de distribution des espces,
lapparition et ladaptation despces envahissantes dans des milieux nouveaux, etc.
Figure 2 : Effets des changements globaux et rponses potentielles de lorganisme pour y

faire face (daprs Matesanza & Valladares, 2014).
Encadr 4 : Quelques dfinitions

La plasticit phnotypique est la capacit d'un organisme exprimer diffrents phnotypes
partir dun gnotype donn selon des conditions biotiques et/ou abiotiques
environnementales (Agrawal, 2001), ou comme consquence des ractions physiologiques
dues des facteurs environnementaux (Wharton, 2011). Comme exemple, les espces du
nmatode Caenorhabditis dmontrent une variation de la taille du corps des adultes selon la
prise alimentaire par les juvniles.
Lvolution rapide : elle pourrait tre dfinie comme un changement gntique se
produisant assez rapidement (dans une chelle danne quelques dcennies) pour avoir un
impact mesurable sur les changements cologiques simultans (Hairston et al., 2005).
Page 8
En agriculture, les changements globaux induits par les interventions humaines sont lis
aux forages imposs par la demande mondiale en matire de production agricole (e.g.
lorientation actuelle vers des systmes de culture intensive). Ils causent d'normes pertes de
biodiversit, y compris la perturbation de processus cosystmiques (tels que la formation du
sol, la rgulation du climat, la production de loxygne), la destruction des habitats, l'extinction
despces, la dstructuration des communauts despces et l'rosion de la diversit gntique
au sein des espces (par exemple, la fixation des allles induite par la drive gntique dans une
population pourrait entraner au sein de celle-ci une baisse de la diversit gntique qui est
pourtant essentielle ladaptabilit des espces aux changements de milieu, de conditions de
vie). Ces perturbations ont ainsi un impact sur la structure et la productivit des cosystmes,
pouvant ainsi mener une modification de la composition des faunes et flores, de la
physiologie, de la dmographie et de la gntique des espces vivantes mais aussi avoir de
lourdes consquences sur la sant et la qualit de vie des communauts.
2. La communaut, lieu dinteractions multiples

Les organismes vivants se rpartissent en une multitude despces distinctes runies dans une
communaut ou biocnose . Chaque individu de chacune de ces espces est soumis
linfluence des facteurs abiotiques et dautres biotiques. Cet assemblage dindividus des
populations despces diffrentes coexistant et potentiellement en interaction constitue une
communaut cologique . Au sein de cette communaut, deux types d'interactions peuvent
tre observs selon le niveau considr : entre individus de diffrentes espces (interactions
interspcifiques) ou entre individus de la mme espce (interactions intra-spcifiques). Ces
interactions peuvent affecter labondance, la diversit ainsi que la distribution des espces dans
la communaut, ce qui en dtermine la structure de la communaut en patrons. Ces patrons sont
la consquence de processus qui sexpriment dans un contexte dhtrognit des habitats
(Shea et al., 2004) ainsi que des pratiques agronomiques (Drijber et al., 2000 ; Kennedy et al.,
2004). Les espces coexistent donc en quilibre interactif dont la prsence ou l'absence et
mme labondance relative de ces espces peut tre dduite de rgles d'assemblage . Cet
quilibre est important pour la stabilit de la communaut et pour sa rsistance aux
perturbations (Pontin, 1982). Afin de comprendre ces rgles, la gntique des communauts
savre tre un outil qui cherche expliquer la variation gntique prsente entre populations
de mme et/ou de diffrentes espces dune communaut et qui en impacte la structure. Elle
analyse galement lorigine de cette variabilit : des processus slectifs (drive gntique,
Page 9
mutation, etc.), des facteurs biotiques et/ou abiotiques ainsi que le rle des changements
globaux dans cette variation gntique. Ces rgles dassemblages tiennent aussi compte des
niches cologiques ou des rles fonctionnels de chaque espce de la communaut (MacArthur
1970 ; Weiher & Keddy, 1999). La plupart des espces sont distribues de faon ingale sur un
territoire donn, peu importe lchelle spatiale considre (Bell, 2001 ; Bell et al., 2006).
Comprendre cette distribution ainsi que la structure de la communaut en espces, ont capt
lattention des cologistes afin den arriver une explication. Plusieurs thories ont t
proposes dont les plus frquentes: (i) la thorie de la diffrentiation des niches qui considre
que chaque espce occupe une niche pluridimensionnelle, qui lui est propre, dfinie en
corrlant le plus de dtails dabondance, de croissance et dinteractions interspcifiques
possibles avec les caractristiques de lenvironnement (Boulanger, 2007). Cela dit que chaque
espce occupe seulement les sites pour laquelle elle est bien adapte et o elle est capable
dexclure ses comptiteurs. Deux processus biologiques fondamentaux sont proposes : 1)
lvolution de la spcialisation cologique par la slection naturelle, et 2) le dveloppement
dune structure de communaut par la slection des espces les mieux adaptes dans un
environnement htrogne (Bell et al. 2006). (ii) La thorie neutre, qui est loppos de la
thorie des niches, suggre que les espces sont identiques et exercent et subissent des
comptitions gales dans leur habitat. Toutes les espces peuvent donc se dvelopper sur un
site donn, faisant en sorte que la composition de la communaut est dtermine largement par
la dispersion stochastique des individus (Boulanger, 2007). Cette thorie neutre mise avant par
Hubbell (2001) avec notamment la parution de son ouvrage The unified neutral theory of
biodiversity and biogeography eut leffet dune bombe en cologie des populations. En
effet, elle va expliquer la diversit et l'abondance relative d'espces dans les communauts
cologiques en se basant principalement sur le concept dquivalence appele redondance
fonctionnelle . Ce concept stipule que des espces similaires appartenant au mme niveau
trophique sont identiques dmographiquement, cest--dire, par rapport leur taux de
naissance, de mortalit, de dispersion et de spciation (Boulanger, 2007). Cette thorie a t
applique sur plusieurs types de communauts, notamment sur des organismes sessiles (plantes
herbaces, arbres, bivalves) (McGill et al. 2006).
En conclusion, comprendre les mcanismes de coexistence de diffrentes espces dans
une communaut, valuer les interactions existant entre les individus dune part et entre les
individus et leur environnement dautre part, comprendre comment ces interactions peuvent
affecter labondance, la distribution et la diversit des diffrentes espces au sein de la
communaut sont parmi les questions fondamentales de lcologie des communauts .
Page 10
Ltude de la biologie des populations et celle de lcologie des communauts ont longtemps
constitu deux traditions de recherche disjointes ce qui a limit la comprhension des
mcanismes de coexistence des espces (Holoak et al., 2005). Nanmoins, un rapprochement
est actuellement en cours et lun des objectifs de lcologie des communauts est de
comprendre comment les processus neutres ou slectifs (e.g. drive gntique, migration,
slection naturelle, comptition, adaptation une niche cologique) influencent lindividu et les
populations, et dterminent les patrons des communauts pour enfin conclure sur lvolution de
la communaut en question.
3. Le sol : trsor de biodiversit

Le sol reprsente un des rservoirs de biodiversit les plus importants. Milieu complexe, il est
le sige dinteractions multi-trophiques impliques dans son fonctionnement et dans la
production vgtale. Il apparat de plus en plus vident aux instances politiques nationales et
internationales de prendre en compte la biodiversit du sol dans leurs programmes de
dveloppement durable. Ainsi, diffrentes organisations et projets ont soulign la place de la
biodiversit des sols comme une composante majeure de l'intgrit et du devenir des
cosystmes naturels et agricoles. Plusieurs projets ont port attention la biodiversit des sols,
tels que le programme GESSOL Fonctions environnementales et GEStion du patrimoine
SOL cofinanc par l'Agence Nationale de la Recherche Agronomique (INRA) et le ministre
de l'Agriculture (CASDAR), le projet SOFIA Agrosystmes et biodiversit fonctionnelle des
Sols anim par l'Agence Nationale de la Recherche Agronomqiue, le projet BiodivERsA
2015 Biodiversit continentale et amlioration des plantes, Biodiversit marine et halieutique,
Ressources en eau et sols , cofinanc par la Commission europenne. Diversit et fonctions
lies au sol ont conduit rcemment au concept de qualit des sols dfinie par sa capacit
fonctionner dans le cadre dun cosystme donn pour entretenir la production biologique,
maintenir la qualit environnementale et favoriser la sant des plantes et des animaux (Doran,
2002). La qualit des sols englobe trois composantes de base : les proprits physiques,
chimiques et biologiques. Les proprits biologiques sont directement dtermines par
ldaphon du sol (i.e. lensemble des organismes y vivant) qui a t dfini, selon le gradient de
la chane trophique, en tenant compte de la taille de ses organismes. Lestimation du nombre
des espces (dcrites et non dcrites) dans le sol est value actuellement : 30000 pour les
bactries, 1500000 pour les champignons, 60000 pour les algues, 10000 pour les protozoaires,
500000 pour les nmatodes (Hawksworth & Mound, 1991) et 3000 pour les vers de terre (Lee,
Page 11
1994 ; Figure 3).

Plusieurs fonctions essentielles du sol (comme la dgradation de la matire organique), et
certaines de ses proprits physico-chimiques, dpendent de laction de ces tres vivants (e.g.
les fourmis, les termites, les vers de terre et les nmatodes). Ce qui fournit des services
cosystmiques impliqus dans la formation du sol, la nutrition vgtale, animale ou
humaine, la rgulation biologique du cycle hydrique, lvolution de la matire organique, les
flux dnergie, etc. (Chaussod et al., 1996). La diversit de ces organismes et leurs interactions
ont un impact direct positif sur la fertilit des sols, sur leur capacit de rsilience lorsquils ont
t perturbs, et sur leur tat sanitaire (Chaussod, 2002). Malgr cette prise de conscience,
l'tude de la biodiversit des sols est encore trop nglige. De plus, la rpartition des
organismes du sol, les liens fonctionnels quils entretiennent entre eux et les effets des usages et
des pratiques sur cette ressource biologique demeurent encore peu documentes (Cluzeau et al.,
2009).
Figure 3 : Classification des organismes du sol selon leur taille (d'aprs Swift et al.,
1979).
Dans le domaine agronomique, la prise en compte de ltat sanitaire des sols a conduit au
concept de sant des sols , un tat sanitaire satisfaisant tant en gnral li des cycles de
nutriments, une forte stabilit (la capacit de rsistance ou de rsilience) ainsi qu une
variabilit leve de ses composants biologiques (Doran & Zeiss, 2000 ; Gros, 2002).
Page 12
Ltat de la faune du sol permet donc de mettre en vidence des conditions

environnementales particulires (pollutions, changements microclimatiques). Ainsi, ces
organismes jouent un rle de bio-indication de qualit des sols, puisque chaque organisme a sa
niche cologique (exigences physico-chimiques, biologiques et environnementales), et donc
chaque organisme dfinit sa niche (Gobat et al., 2003). Le choix pertinent dun bio-indicateur
pour qualifier un sol dpend donc dune bonne connaissance de la systmatique de ces
organismes, de leur physiologie, de leur cologie et de leurs interactions. La production
vgtale rsulte des interactions hypoges (sol) / piges (milieu arien) (concept aboveground-below-ground interactions , Bardgett & Wardle, 2010) auxquelles participent
activement les plantes (racines vs tiges et feuilles). On sait aujourdhui, quindpendamment
des mcanismes physiologiques dadsorption hydrique et minrale par la plante, les organismes
du sol influencent la croissance des plantes la fois quantitativement (quantit totale de la
production vgtale) et qualitativement (architecture racinaire, proportion de la biomasse
alloue aux graines, composition chimique, etc.). Au sein de ces interactions hypoges/piges,
des organismes appartenant un mme groupe taxonomique peuvent jouer des rles
antagonistes lun de lautre comme cest le cas des nmatodes telluriques.
4. Les nmatodes comme organismes modles
4.1. Gnralits
Les nmatodes (Nmathelminthes) ou "vers ronds", forment un groupe zoologique
dorganismes vermiformes. Ils sont dpourvus de membres et de squelette et ont la forme dun
fuseau. Ils prsentent une symtrie bilatrale. Le corps est non segment et recouvert dune
paisse cuticule. La majorit des nmatodes telluriques mesurent entre 0,6 et 0,8 mm de long et
entre 0,025 0.50 mm de large (Sumenkova, 1988),
certains comme les Longidoridae
atteignant 4 mm (Coyne et al., 2010). Ils sont ubiquistes (toutes latitudes et tous climats) en
ayant colonis tous les types de milieux : les eaux continentales et ocaniques (sales et
douces) et les sols. Certains dentre eux se sont adapts la vie parasitaire chez lhomme, les
animaux et les vgtaux.
Organismes aquatiques arobies, ils se dplacent dans le sol dans leau porale en ondulant
grce leurs muscles longitudinaux. Les nmatodes ont une capacit de colonisation trs
leve, et peuvent tre chantillonns toutes les saisons. En outre, certaines espces de
nmatodes peuvent rsister des conditions extrmes puisquil est possible de les congeler ou
de les dshydrater des fins exprimentales (Bongers, 1990). Les nmatodes telluriques sont
microscopiques, donc difficiles voire impossibles observer lil nu, et par consquent
Page 13
largement inconnus du grand public. Ils sont majoritairement prsents dans les couches
superficielles du sol (horizon A), mais peuvent galement se trouver dans les zones de
prospection racinaire profonde (cas des espces ligneuses). Ils constituent lun des groupes
danimaux les plus nombreux et les plus rpandus dans le sol, et les mieux caractriss dun
point de vue taxonomique (Lee, 2002). Cest le plus grand phylum animal, avec environ 27 000
espces dcrites ce jour. Cependant, une grande portion (300000 500000 espces) reste
encore dcrire (Hugot et al., 2001). Dans le sol, les nmatodes sont les animaux les plus
abondants aprs les protozoaires. Ils comptent de 100 1000 individus/g de terre, soit de 1 30
millions /m2 (biomasse = 1 30 g/m2) (Floyd et al. 2002). On peut compter plus de 100000
individus appartenant une centaine despces dans un cm3 de sol (Gobat et al., 2003). On les
diffrencie en fonction de leur mode trophique : ils peuvent tre bactriophages, mycophages,
saprophages, prdateurs dautres organismes du sol, zooparasites et phytoparasites (Yeates et
al., 1993). Dun point de vue cologique, les nmatodes du sol, et en particulier les omnivores
et les bactrivores, sont impliqus dans diffrentes fonctions du sol telles que le cycle du
carbone et de lazote, le cycle des lments minraux et la dcomposition de la matire
organique (Ingham et al., 1985 ; Ferris et al., 2004 ; Briar et al., 2007).
Les communauts de nmatodes du sol ont fait lobjet de plusieurs tudes dans le but
destimer limpact de certaines perturbations sur lactivit biologique des sols par lutilisation
dindices de biodiversit (Encadr 5). Les conclusions fournies par Neher et al. (1995)
montrent que les meilleurs indicateurs de ltat sanitaire des sols sont bass sur la structure des
communauts de nmatodes. De mme lutilisation des nmatodes phytoparasites et prdateurs
servent dindicateurs de linfluence de lactivit humaine sur le sol (Wasilewiska, 1997) et de
ses caractristiques physico-chimiques (Pankhurst et al., 2002). En raison de leur impact direct
sur la production vgtale, la recherche agronomique met laccent sur les nmatodes
phytoparasites.
Encadr 5 : Indices de biodiversit
Indices de biodiversit : ce sont des mesures qui prennent en compte le nombre dindividus
et celui despces dans un cosystme. Elles indiquent la distribution de ces individus entre
espces.
Diffrents indices sont disponibles pour mesurer la diversit de la communaut : la diversit
des espces (e.g. le nombre total dindividus, la richesse en genres ou espces, la rgularit
de la distribution des espces dans une communaut), leur dominance (dfinie par la
frquence et labondance des espces dans la communaut), la composition trophique et
fonctionnelle ainsi que la structure des communauts en patrons.
Page 14
4.2. Les nmatodes phytoparasites : un enjeu pour l'agriculture

Les nmatodes phytoparasites (NPP) (Figure 4A) se trouvent gnralement dans le sol ou
lintrieur des cellules vgtales racinaires et parfois foliaires, des tiges ou dans les semences.
Ils se distinguent des autres groupes de nmatodes du sol par la prsence dune pice buccale
spcialise : le stylet (Figure 4B) qui leur sert perforer les cellules vgtales, y injecter des
enzymes lytiques et en extraire la sve vgtale, dune manire trs semblable aux aphids
(pucerons) (Coyne et al., 2010). Leurs glandes salivaires excrtent des substances qui peuvent
produire des modifications dans les tissus. Ces secrtions semblent jouer un rle crucial dans
l'infection et la formation de cellules de la plante hte en site nourricier comme cest le cas
pour les nmatodes galles Meloidogyne et les nmatodes kystes Heterodera (Hussey et al.,
1994 ; Williamson & Gleason, 2003). Les NPP pondent leurs ufs dans le sol ou dans les tissus
racinaires.
Figure 4 : Nmatode phytoparasite du genre Pratylenchus spp.

A : forme gnrale ; B : stylet (Photos IRD, UMR CBGP).
Plusieurs critres peuvent expliquer la trs grande diversit des espces de NPP (environ
5000 espces dcrites ce jour) :
- ils ont trois origines volutives distinctes (Blaxter et al. 1998) (Figure 5).
- ils prsentent tous les modes de reproduction (Encadr 6) : amphimixie, parthnogense
mitotique et miotique, hermaphrodisme (Perry & Moens, 2006). Plusieurs modes de
reproduction peuvent tre rencontrs mme au sein dun mme genre (e.g. chez les
Page 15
nmatodes lsion Pratylenchus) (Tableau 1).

- ils montent galement une diversit importante dans leur caryotype (Tableau 1).
- ils se distinguent en deux modes trophiques principaux : spcialiss (strictement infods
aux plantes) et gnralistes (se nourrissent la fois sur les plantes, sur des champignons
et des bactries). Ils se distinguent galement en cinq modes parasitaires par rapport
leur position sur ou dans les racines des plantes : ectoparasites, semi-endoparasites
(migrateurs/sdentaires) et endoparasites (migrateurs/sdentaires) (Siddiqi, 2000).
- leur cycle de vie est simple mais varie selon lespce. Ils prsentent galement une grande
variabilit dans leur fcondit (Tableau 1).
Figure 5 : Phylum de Nematoda (daprs Blaxter et al., 1998), (Les clades dans lesquels
les nmatodes phytoparasites se trouvent sont entours).
Page 16
Encadr 6 : Modes de reproduction chez les nmatodes phytoparasites (daprs Weischer & Brown,
2000)
Reproduction amphimictique: mode de reproduction sexue qui implique la fusion d'un gamte mle
et dun gamte femelle haplode.
Reproduction parthnogntique : mode de reproduction asexue, qui ne ncessite pas lintervention
des spermatozodes du mle (pas de fcondation). Dans la parthnogense miotique, la descendance
nest pas gntiquement identique la mre (recombinaison aprs miose) ; dans la parthnognse
mitotique, tous les descendants sont gntiquement identiques leur mre (reproduction clonale).
Hermaphrodisme : cas particulier de la reproduction bisexuelle. Un nmatode hermaphrodite est autofertile, il produit des spermatozodes et des ovules en une seule gonade. Les spermatozodes sont
dabord produits et stocks pour fertiliser les gamtes qui sont produits par la suite.
Tableau 1 : Exemples de dure du cycle de vie, de fcondit, de modes de reproduction

et de caryotypes chez quelques espces de nmatodes phytoparasites.
Espce
Cycle de
vie
Fcondit
(nombre dufs)
Mode de reproduction
Nb de
chromosomes
Ditylenchus dipsaci
70 jours
500
amphimixie
>50
Heterodera schachtii
30 jours
400
amphimixie
18
Meloidogyne hapla
30 jours
500
amphimixie/parthnogense
45
Meloidogyne javanica
30 jours
500-1000
parthnogense (mitotique)
43-48
Paratrichodorus minor
16 jours
57-86
Xiphinema diversicaudatum
1.2 an
200
* Pratylenchus brachyurus
5-10
jours
Pratylenchus coffeae
27 jours
Pratylenchus neglectus
28 jours
* Pratylenchus penetrans
30-86
jours
Pratylenchus scribneri
29 jours
hermaphodisme
amphimixie
10
parthnogense mitotique
30-40
30-32
amphimixie
parthnogense mitotique
20
amphimixie
10
parthnogense miotique
12
* : la dure de cycle de vie de ces espces de Pratylenchus est variable selon la temprature.
Page 17
Ils prsentent galement des stratgies adaptatives varies (dfinies par la cp-value). Ces
stratgies distinguent les nmatodes en colonisateurs (stratgie r dfinie par la c value) ou
persistants (stratgie K dfinie par la p value). La cp-value est un indice de diversit
fonctionnelle attribu aux familles de nmatodes du sol (Bongers, 1990). Tous les nmatodes
telluriques sont rpartis en cinq classes de cp-value selon leur sensibilit aux perturbations des
milieux. La cp-value varie de 1 (espces opportunistes, colonisatrices, peu sensibles au stress,
cycles de vie courts (quelques jours quelques semaines), produisent de nombreux petits ufs)
5 (espces sensibles aux perturbations, persistantes, cycles de vie longs de plusieurs mois,
produisent peu de gros ufs). Les nmatodes phytoparasites noccupent que les classes cp-2
cp-5 (Tableau 2). Quelques exemples cits dans le tableau 1 pourraient clarifier les caractres
biologiques chez les colonisateurs et les persistants. Comme exemples, Meloidogyne est un
nmatode colonisateur, alors que Xiphinema est un nmatode persistant
Tableau 2 : Caractres biologiques distinguant les nmatodes colonisateurs des nmatodes
persistants (Bongers, 1990).
Caractre
Colonisateurs (r)
Persistants (k)
Cycle de vie
court
long
Capacit de colonisation
leve
faible
Tolrance aux perturbations
tolrants
sensibles
Dominance dans les chantillons
dominants
non dominants
ufs
beaucoup de petits ufs
peu de gros ufs
Taux de reproduction
fort
faible
Les NPP parasitent tous les types de cultures (cralires, marachres, florales et
fruitires, etc.) (Dijan-Caporalino et al., 2009). Leurs activits parasitaires se traduisent par
diffrents types de symptmes sur la plante tels que le fltrissement, le nanisme, le
jaunissement, les carences nutritives, etc. (symptmes totalement atypiques). Sur les racines, ils
causent gnralement des ncroses racinaires, et, dans certains cas spcifiques, la formation de
galles sur les racines (e.g. pour le genre Meloidogyne). Les blessures racinaires causes par les
NPP permettent galement la pntration de champignons phytopathognes (Powell, 1971).
Certains nmatodes peuvent aussi transmettre des maladies virales (Taylor & Brown, 1997).
Ainsi, le parasitisme par les NPP dprcie les fonctions racinaires vitales de la plante
(absorption hydrique et minrale), rduisant par l-mme la croissance et la production
Page 18
vgtale. Les dgts occasionns par ces parasites sont trs importants au niveau mondial,
puisquils sont estims 20 30 % de la production agricole (Cayrol et al., 1994), reprsentant
des pertes annuelles de rcolte values plusieurs millions de tonnes (Agrios, 2005). Les
pertes causes par les nmatodes phytoparasites sont gnralement plus prononces dans les
rgions tropicales que les rgions tempres. Cela tient une diversit des pathognes plus
importante, des conditions environnementales plus favorables leur dveloppement, leur
reproduction et leur dispersion, et une moindre utilisation de moyens efficaces pour les
combattre (De Waele & Elsen, 2007).
Face ces impacts agronomiques et conomiques, le contrle de ces parasites est
impratif. Mais les producteurs ne souponnent pas damble les NPP comme responsables des
dgts observs parce que les NPP ninduisent gnralement pas de symptmes spcifiques. En
outre, la matrise de ces parasites est difficile du fait de leur confinement dans le sol ou
lintrieur des tissus vgtaux, et de leur grande variabilit physiologique qui leur permet de
dvelopper des adaptations, voire des rsistances, de nombreuses mthodes de lutte (e.g.
contournement de la rsistance comme cest le cas du gne de rsistance Me3 vis--vis de M.
arenaria et M. incognita (Castagnone-Sereno et al., 2001) ou adaptation aux nmaticides
(Viglierchio, 1990). Diffrentes stratgies ont t mises en uvre afin de les contrler, telles
que des pratiques culturales (varits rsistantes, rotations de cultures), des mthodes physiques
(solarisation des sols ou dsinfection la vapeur), biologiques ( laide dantagonistes
microbiens), et chimiques qui demeurent les plus efficaces et les plus faciles utiliser.
Nanmoins, la majorit des nmaticides de synthse sont interdits progressivement
cause de leurs effets nfastes sur lenvironnement (pollution des sols et des nappes phratiques,
rsidus dans les denres alimentaires), facteurs risque pour les sants animale et humaine, eu
gard des rsultats souvent insuffisants (volution de la lgislation europenne sur les
pesticides, e.g. Plan Ecophyto 2018 qui vise rduire lutilisation des produits phytosanitaires
50% pour 2018.).
4.3. Diversit des communauts de nmatodes phytoparasites
Dans la majorit des cas, la gestion des NPP est apprhende vis--vis despces
emblmatiques sur chacune des cultures : nmatodes galles (Meloidogyne spp.) sur cultures
marachres, nmatodes kystes (Heterodera spp. et Globodera spp.) sur crales, pomme de
terre et soja, nmatodes lsion (Pratylenchus spp.) sur ananas, cafier, bananier, mas,
cultures fruitires, etc. (Jones et al., 2013). Et empiriquement, afin dimpacter le moins possible
le biotope tellurique, la recherche-dveloppement a labor des mthodes de contrle
Page 19
spcifiques de ces taxons (e.g. rsistance varitale, antagonistes microbiens). Or les NPP sont
partout prsents en communauts (au sens mlanges despces) associant des espces aux
histoires volutives (Blaxter et al., 1998) et aux caractristiques biologiques (cycle de
dveloppement, taux de reproduction, gamme de plantes htes, etc.) trs varies. Par
consquent, ces mthodes de lutte spcifiques mnent le plus souvent la cration de vides
cologiques (par diminution des effectifs des espces cibles) ou exercent des pressions
dadaptation, qui conduisent conscutivement une rorganisation de la structure des
communauts et, par consquent, de nouveaux phnomnes invasifs ; autrement dit, moins
defficacit et non durabilit cologique de la gestion des NPP).
En outre, il a t dmontr que la production vgtale est plus lie la structure des
communauts de NPP que strictement aux effectifs des espces qui la composent. Par exemple,
une tude sur limpact de la structure dune communaut de NPP sur la production de canne
sucre (Lavelle et al., 2004) a montr que pour une mme diversit despces de NPP mais pour
des proportions diffrentes, lexpression de la pathognicit nest pas la mme (Figure 6).
Inversement, la perturbation des cosystmes induit des modifications de la structure des
communauts de NPP, qui se traduit trs souvent par une rosion de la richesse (trait de
rponse). Ainsi, la richesse spcifique est-elle leve (S > 20) dans les cosystmes peu
anthropiss comme les dunes littorales Ammophila arenaria (Mateille et al., 2011) ou les
forts (Baujard et al., 1979), sans dgt apparent sur la plante, alors quelle est plus faible (S <
10) dans les agrosystmes trs contraints comme le marachage sous abri (Djian-Caporalino et
al., 2015) o sexpriment des dgts importants
Figure 6 : Correspondance entre le patron statistique des communauts de nmatodes et la

production de canne sucre (Lavelle et al., 2004).
Page 20
Or, les mthodes de gestion actuelles (rsistances et agents de lutte biologique) sont
spcifiques despces cibles. On peut donc considrer que, si des communauts de NPP sont
peu ou pas pathognes, une perte de diversit due une mthode de contrle peut constituer un
risque pertinent valuer. Inversement, valuer le gain dune gestion approprie de leur
biodiversit sur la production vgtale devient un enjeu tout fait adquat (Mateille et al.,
2008).
4.4. Diversit interspcifique : le cas des nmatodes galles du genre Meloidogyne
Les Meloidogyne, appels communment nmatodes galles des racines , nmatodes des
racines noueuses (ou root-knot nematodes RKN), reprsentent un genre de nmatodes
phytoparasites sdentaires obligatoires. Ces nmatodes tirent leur appellation commune du fait
quils entranent sur les racines des plantes parasites la formation de renflements
caractristiques ou galles, trs facilement reconnaissables (Figure 7). Le genre Meloidogyne
spp. appartient la classe de Secernentea, lordre de Tylenchida, la sous-ordre
dHoplolaimina, la superfamille dHoplolaimoidea, la famille de Meloidogynidae et la
sous-famille de Meloidogyninae (Siddiqi, 2000). Ce genre contient une diversit spcifique
importante avec plus de 97 espces dcrites ce jour (Hunt & Handoo, 2009).
Figure 7 : Galles dues Meloidogyne javanica sur racine de melon (Photo IRD).
Page 21
4.4.1. Cycle biologique standard des nmatodes du genre Meloidogyne

Le cycle de vie des RKN comporte trois niveaux de dveloppement (uf, quatre stades
juvniles (J1, J2, J3, J4) et adulte (femelles et trs rarement des mles cause de leur mode de
reproduction parthnogntique). Leur cycle se droule en deux phases (Figure 8) :
- une phase libre mobile qui ne concerne que le stade juvnile J2 qui se dplace dans le
sol la recherche des racines de dune plante hte.
- une phase sdentaire de maturation des J2 en femelles qui se droule lintrieur des
racines aprs la pntration des J2.
Le cycle commence partir des ufs runis en une masse dans une gangue
mucilagineuse lintrieur de laquelle on peut trouver des ufs diffrents stades de
dveloppement, du stade unicellulaire au juvnile J1 et ensuite au J2 prt clore. Le
dveloppement embryonnaire dure de sept neuf jours 28C au cours duquel intervient une
premire mue. Une fois luf clos, le J2 se dplace dans le sol la recherche de sa plante hte,
il pique une racine et y pntre. Il traverse lpiderme, puis le cortex pour arriver au cylindre
central o il se fixe et tablit un site nourricier induit par les scrtions salivaires du nmatode.
Ce site est constitu de 5 6 cellules gantes polynucles. En 3 8 semaines selon la
temprature, le J2 subit trois autres mues en J3, J4 pour atteindre ensuite le stade adulte en une
femelle piriforme (ce qui conduit la dformation des racines en galles) qui pond ses ufs (300
3000) dans la gangue mucilagineuse (Djian-Caporalino, 2009) ou en mle qui reste filiforme
et qui quitte les racines pour le sol.
Page 22
Figure 8 : Cycle biologique de Meloidogyne spp. (cas dune parthnogense mitotique). a :

apex racinaire infest de juvniles ; b : galles racinaires ; c : coupe histologique dune galle
montrant une femelle mature et des cellules gantes (*) (daprs Castagnone-Sereno et al, 2013).
4.4.2. Importance agro-conomique

Lattention particulire porte aux Meloidogyne comme modle biologique pour de nombreuses
tudes est due leur impact agro-conomique majeur et leur rpartition gographique
mondiale sur une large gamme dhtes. On les rencontre dans toute la zone intertropicale et
dans les rgions tempres chaudes (e.g. le bassin mditerranen) comme par exemple M.
incognita, M. javanica, M. arenaria. Certaines espces (e.g. M. hapla, M. chitwoodi et M.
fallax) remontent mme dans des latitudes Nord faibles et on les trouve frquemment dans les
serres des pays temprs froids (De Guiran & Netscher, 1970). Ces nmatodes sont de
redoutables bio-agresseurs parasitant plus de 5500 espces de plantes (Blok et al., 2008). Ils
s'attaquent aussi bien aux grandes cultures (crales, pommes de terre, betterave, etc.), qu'aux
cultures marachres, florales et fruitires (Djian-Caporalino et al., 2009). Les galles quils
provoquent sur les racines peuvent envahir tout le systme racinaire, induisant un fltrissement
et un retard de la croissance. En cas dattaque prononce, ces dgts peuvent conduire la mort
de la plante. Au niveau mondial, on estime les pertes dues Meloidogyne environ 100
Page 23
milliards de dollars par an (Bird & Kaloshian, 2003). En Europe, ils sont responsables de 10%
de la rduction de la production cralire et de 20 30% de diminutions de rcoltes dans les
vergers d'agrumes mditerranens (Feldmesser, 1971). En outre, par les lsions quils
provoquent, ces nmatodes entranent des infestations secondaires dautres pathognes
fongiques ou bactriens (e.g. Phytophthora, Rhizoctonia, Pythium, Fusarium, Pseudomonas,
Agrobacterium). Les problmes phytoparasites causs par les Meloidogyne se rvlent de plus
en plus proccupants et peuvent devenir dramatiques dans les annes venir (Djian-Caporalino
et al., 2009). En plus, des espces de Meloidogyne telles que M. chitwoodi et M. fallax sont
dclares espces de quarantaine en Europe par lOrganisation Europenne de Protection des
Plantes (OEPP) (liste A2) (www.eppo.int/QUARANTINE/listA2.htm). Une attention
particulire est porte quatre espces principales : M. arenaria, M. incognita, M. javanica et
M. hapla qui sont les plus disperses au niveau mondial et qui ont la plus large gamme dhtes.
4.4.3. Diversit biologique et gntique des Meloidogyne
Les espces de Meloidogyne prsentent une diversit importante plusieurs gards. Ces
espces peuvent par exemple tre divises en deux groupes selon leurs exigences thermiques :
cryophiles (e.g. M. hapla, M. chitwoodi, M. naasi) et thermophiles (e.g. M. arenaria, M.
javanica, M. exigua) au seuil de 10C (Lyons et al., 1975). Les Meloidogyne dmontrent une
variabilit en nombre de gnrations produites au cours de lanne. Les espces tropicales (i.e.
M. arenaria, M. javanica et M. incognita) peuvent atteindre plusieurs gnrations par an alors
que dautres espces comme M. naasi ne ralisent quune seule gnration par an (Rivoal et al.,
1993). Ces nmatodes montrent galement plusieurs dgrs de spcialisation vis--vis des
plantes htes. Ainsi, en gnral, les plantes cultives favorisent plus leur dveloppement que les
adventices (Hillocks et al., 1995).
Mais leur diversit touche essentiellement leur mode de reproduction. Ils peuvent se
reproduire par amphimixie (e.g. M. megatyla et M. microtyla) (Chitwood & Perry, 2009) ou par
parthnogense (miotique ou mitotique, cf. Encadr 6) comme M. javanica, M. arenaria, M.
incognita. Dautres espces (comme M. hapla) sont mme capables de se reproduire soit par
amphimixie soit par parthnogense miotique, en fonction de la prsence ou de l'absence des
males (Figure 9 ; Castagnone-Sereno et al., 2013). Les Meloidogyne prsentent galement des
caryotypes variables : la plupart des espces ont un nombre de chromosomes somatiques qui
varie de 30 50, ils sont surtout considrs comme des organismes diplodes ou triplodes.
Certaines formes ttraplodes ont t rarement rapportes chez M. hapla (Triantaphyllou,
1984). Cette variation chromosomique a t galement observe entre des populations
Page 24
appartenant la mme espce ; ainsi, au sein de lespce M. arenaria, la plupart des

populations sont diplodes (avec 30 39 chromosomes) ou hypotriploides (40 48
chromosomes). Dautres populations triplodes (50 56 chromosomes) sont frquemment
observes (Janati et al., 1982 ; Triantaphyllou, 1985).
Figure 9: Modes de reproduction des espces de Meloidogyne spp.

(daprs Castagnone-Sereno et al., 2013).
Considrant limportance agronomique et conomique de ces parasites, connaitre cette

diversit semble dimportance pour comprendre leur grande diversit spcifique, leur
adaptation cologique, leur rpartition gographique mondiale, leur capacit de parasiter une
large gamme de plantes htes. Cest aussi la raison pour laquelle la dtermination des espces
de Meloidogyne a toujours pos des problmes aux spcialistes en raison de leur variabilit
physiologique (Karssen, 2002), de leur large gamme dhtes pour une grande partie dentre
elles, de lexistence despces cryptiques morphologiquement indistinguables, de lexistence
dun dimorphisme sexuel chez certaines espces, de cas de diplodie, etc. (Janati et al., 1982 ;
Page 25
Blok & Powers, 2009). La caractrisation dune espce fait gnralement appel une analyse
multicritres intgrant des approches morphologiques, biologiques, biochimiques et
molculaires :
diversit morphologique : les mles tant rares la plupart du temps et les juvniles
ayant trs peu de caractres diffrencis entre les espces, la morphomtrie est essentiellement
applique aux femelles. Mais dans le cas de Meloidogyne, les femelles sont piriformes ; on fait
donc appel lempreinte prinale. Ces structures cuticulaires montrent des stries entourant la
vulve et lanus et formant des sortes dempreintes digitales. Cependant, ces plaques prinales
sont trs variables lintrieur dune espce et se ressemblent souvent beaucoup dune espce
lautre, si bien quil est parfois impossible de se prononcer sur une dtermination si lon ne
dispose pas au minimum dune dizaine de plaques prinales (De Guiran & Netscher, 1970).
diversit biologique : le test North Carolina Differential Host Test est
communment utilis dans l'identification des races sauvages ou virulentes (contournement des
rsistances naturelles ou acquises par amlioration varitale) appartenant aux quatre espces
principales, savoir M. incognita, M. javanica, M. arenaria et M. hapla (Taylor & Sasser,
1978 ; Hartman & Sasser, 1985). Ce test consiste confronter les populations de chaque espce
une gamme de plante htes de rfrence (Taylor & Sasser, 1978).
diversit biochimique : elle est base sur la sparation lectophortique des protines
de femelles et la rvlation disoenzymes (Estrase EST, Malate-dshydrognase MDH,
Glutamate-oxaloacetate-transaminase
GOT,
Superoxyde-dismutase
SOD)
aux
poids
molculaires spcifiques (Esbenshade & Triantaphyllou, 1985 ; 1990). La migration

lectrophortique des bandes enzymatiques est compare celle des bandes dune population
tmoin de M. javanica et value par un rapport de migration (Rm%). Cette mthode est
largement utilise en routine. Mais elle nest pas fiable en cas de mlange despces dans un
chantillon. Elle ne peut tre utilise que sur femelle pour des raisons de seuil de concentration
protique (Esbenshade & Triantaphyllou, 1990).
diversit molculaire : les mthodes molculaires deviennent indispensables pour
identifier les nmatodes surtout dans le cas despces cryptiques (Gamel et al., 2014). Ces
mthodes sont fiables, rapides, prcises et relativement simples appliquer. Elles sont bases
sur la dtection de polymorphismes d'ADN diffrents entre les espces laide de la technique
PCR, telles que la RFLP (Restriction Fragment Length Polymorphism), la RAPD (Random
Amplified Polymorphic DNA), (Adam et al., 2007; Blok & Powers, 2009 ; Holterman et al.,
Page 26
2012). Des marqueurs molculaires de type SCARS (Sequence Characterized Amplified

Region) sont largement utiliss pour le diagnostic des espces. Cette dtermination est base
sur amplification dune rgion dADN dune espce de Meloidogyne avec des amorces issues
de fragments RAPD (ADN polymorphique amplifi au hasard) spcifiques certaines espces.
Des amorces SCARS sont disponibles pour dterminer M. arenaria, M. javanica, M. incognita
(Zijlstra et al., 2000), M. hapla, M. chitwoodi et M. fallax (Zijlstra, 2000). La rgion ITS,
localise entre le gne 18S et 28S, offre une variation gntique suffisante entre genres, entre
espces et mme au niveau intra-spcifique (Ziljstra et al., 1995). Cette rgion a t largement
utilise pour construire des arbres phylogntiques, estimer la structure
gntique des
populations, valuer les processus volutifs des populations et pour la taxonomie (Powers et al.,
1997).
5. Lolivier : modle cologique dtude de la structuration des communauts de

nmatodes phytoparasites
5.1. Quelques lments de botanique
Lolivier Olea europaea L. appartient la famille des Oleaceae. Il est partag en six sous
espces : europaea, laperrinei, cuspidata, guanchica, maroccana, cerasiformis (Mdail et al.,
2001; Green, 2002). Dans le Bassin Mditerranen, lolivier appartient la sous-espce O. e.
subsp. europaea, qui se distingue en deux varits botaniques : O. e. subsp. europaea var.
europaea qui regroupe les formes cultives et O. e. subsp. europaea var. sylvestris qui regroupe
les formes sauvages et spontanes (des formes non cultives qui poussent partir des graines
transportes par des oiseaux par exemple), habituellement nomms olastres (Green, 2002).
Cest une espce arborescente feuillage persistant, grande longvit et dont la variabilit
gntique reflte une grande gamme de caractres morphologiques et physiologiques (vigueur,
taille des fruits, rendement, teneur en huile, etc.) et de traits dadaptation aux stress
environnementaux (contraintes climatiques et daphiques qui caractrisent son aire dorigine
mditerranenne) (Fernndez, 2014). Cest pourquoi, il est considr comme un des meilleurs
indicateurs biologiques du climat mditerranen (Blondel et al., 2010).
5.2. Distribution
Cet arbre est la source principale dhuile comestible depuis des milliers dannes, ce qui
explique son importance conomique et sa distribution quasi-mondiale. Cette espce arrive au
Page 27
24me rang des 35 espces botaniques les plus rpandues (Ellstrand, 2003). Cest une culture
largement distribue en Europe du Nord, en Afrique du Nord, au Moyen-Orient et sur le
continent amricain ; elle pousse extensivement en Australie subtropicale et en Afrique
Centrale (Figure 10). La surface cultive en oliviers est estime plus de 10 Mha dans le
monde, dont environ 97% sont localiss dans le Bassin Mditerranen qui produit 98% de
lhuile dolive mondiale (FAOSTAT, 2012).
Figure 10 : Distribution mondiale de lolivier (IOC, 2000).
5.3. Elments culturels

Dans le monde mditerranen, lolivier est considr comme l'espce la plus emblmatique en
raison de son importance cologique, conomique et culturelle (Zohary et al., 2012 ; Kaniewski
et al., 2012). Lolivier demeure le tmoin de l'mergence des premires civilisations
mditerranennes (Kaniewski et al., 2012) puisquil fait partie des cultures les plus anciennes
dans cette rgion (Zohary & Spiegal-Roy, 1975). Historiquement, cet arbre accompagne les
mythes fondateurs des cultures mditerranennes, comme symbole de force, de longvit et de
paix. Dans lhistoire de lhumanit, lolivier est synonyme de soleil et de convivialit
symbolisant lesprit des populations prsentes depuis des millnaires autour de cette tendue
maritime. De nombreuses reprsentations mythologiques mettent en scne lolivier avec Isis en
Egypte, Athna (desse de la sagesse) en Grce et Minerve chez les Romains. En Egypte,
lolivier est reprsent dans les dcorations des stles funraires et des pyramides (Estin &
Laporte, 2004). Il est cit avec dautres plantes fruitires (vigne et figuier) dans les critures
saintes confirmant sa grande valeur dans lhistoire des civilisations et la vie des hommes
(Moldenke & Moldenke, 1952 ; Janick, 2007).
Page 28
5.4. Histoire volutive

Cest un lment fondamental dans les paysages mditerranens, exploit depuis au moins
9000 ans (Figure 11).
Figure 11 : Tablette cuniforme (ARM 7238) dcouverte sur le site archologique de

Mari (Est de la Syrie), portant mention l'huile d'olive I ZI.IR.DU (Bottro, 1956).
Cependant, nos connaissances sur son origine gographique et sa domestication sont
incompltes et ont souvent fait lobjet de controverses (Kaniewski et al., 2012 ). Les travaux
archologiques ont montr la prsence de restes fossiliss de feuilles dolivier datant des res
palolithique et nolithique vers 37000 avt. JC dans lIle de Santorin en Grce (Liphschitz et
al., 1991; Therios, 2009). Il apparat tout de mme que les origines de cette culture et de sa
domestication sont bien antrieures lAntiquit, avec une mergence 5000 ans avant notre re
(Terral et al., 2012). Dautres avis ont dclar que la culture de l'olivier est originaire de
Palestine pendant la priode chalcolithique (Zohary & Spiegel-Roy, 1975; Davies & Fall,
2001). Cependant, lanalyse de la variabilit gntique de lolivier nont pas clairement soutenu
cette hypothse (Besnard et al., 2001). La domestication de l'olivier semble donc tre perdue
Page 29
dans la brume des temps (Besnard et al., 2007). Il est gnralement accept par des preuves
archologiques, historiques et molculaires que la premire domestication de lolivier a
commenc dans le Levant : Jordanie, Liban, Ouest de la Syrie, Sud-Est de la Turquie, valles
du Tigre et de lEuphrate, Irak et Ouest de l'Iran (Kaniewski et al., 2012; Zohary et al., 2012;
Besnard et al ., 2013). Cependant, dautres rfrences proposent que la domestication de
lolivier a t un processus long et continu qui sest dvelopp dans de nombreuses rgions de
manire indpendante et non pas partir dun unique foyer proche-oriental (Terral et al., 2012).
La domestication de lolivier a t caractrise par la multiplication vgtative des arbres les
plus valoriss (qui ont par exemple de plus gros fruits) et l'tablissement de vergers (Zohary &
Spiegel-Roy, 1975). L'olivier cultiv tire son origine de l'olivier sauvage, ce qui est attest la
citation du pollen et des macrofossiles dans de nombreux documents datent au Chalcolithique
(Zohary et Hopf, 2000; Carrion et al., 2010). Ces processus continus de slection des cultivars
ont probablement t raliss partir de graines ou de la culture de formes spontanes ou
dolastres. Ceci est encore frquemment pratiqu dans certains endroits, par exemple au nord
du Maroc (Haouane & Khadari, 2011).
Des travaux rcents utilisant des marqueurs molculaires (e.g. ADNcp, marqueurs
chloroplastiques) suggrent que la diversification des ressources gntiques englobe des formes
issues de louest du Bassin Mditerranen (Breton, 2006), ce qui est aussi le cas de la vigne
(Arroyo-Garca et al., 2006) et du figuier (Khadari et Kjellberg, 2009). Les analyses
molculaires indiquent que la domestication de lolivier a t un processus long et continu et
que les cultivars proviennent de populations sources multiples (Kaniewski et al., 2012). Cela a
t confirm plus tard par lanalyse des gnomes d'origine maternelle (chloroplastiques et
mitochondriaux) qui a montr une diversit gntique rpartie en 48 profils distincts (ou
haplotypes) et trois lignes gntiques E1, E2, E3. Cette diversit semble tre trs structure
entre lEst et lOuest de la Mditerrane (E1 vs E2/E3), indiquant que ces lignes sont bien
issues de ces deux rgions (Figure. 12 ; Besnard et al., 2013). Une quatrime ligne M a t
aussi dcouverte dans lolivier sauvage O. e. ssp. maroccana, endmique la rgion
macaronsienne du Maroc (Besnard et al., 2006). Cette diversit gntique chloroplastique est
plus leve chez lolivier sauvage que chez lolivier cultiv (Besnard et al., 2013). De ces
foyers, les varits dolivier ont t largement dissmines par les activits humaines autour du
Bassin Mditerranen. Les analyses gntiques ont galement dmontr que la plupart des
varits modernes (85%) possdent le fond gntique maternel E1 qui est le plus distribu sur
l'ensemble de la rgion mditerranenne (Besnard et al., 2002). Aujourdhui, plus de 2000
cultivars, diffrencis par leur port, leur phnologie ainsi que par la morphologie des feuilles,
Page 30
des fruits et des noyaux, sont connus (Moutier et al., 2004). La slection des varits que l'on
trouve aujourd'hui serait le rsultat d'un isolement ancestral (dernire glaciation, Encadr 7) de
trois populations d'oliviers : Afrique du Sud, Asie et Bassin Mditerranen. Considrant
lolastre O. e. europaea var. sylvestris comme un bio-indicateur climatique mditerranen, la
domestication de lolivier a permis une adaptation de larbre des altitudes et des latitudes plus
leves (Carrion et al., 2010).
Encadr 7 : Dfinition de la dernire priode glaciaire
La dernire priode glaciaire est une priode de refroidissement global ou glaciation qui
caractrise la fin du Plistocne. Ces priodes de glaciation sont souvent dues la variation
des paramtres orbitaux de la terre : excentricit et obliquit de lcliptique, et prcession
des quinoxes. Quatre priodes glaciaires se sont succdes durant les 600.000 dernires
annes. Spares par des priodes interglaciaires, elles portent les noms de Gnz, Mindel,
Riss et Wrm. Cette dernire sest acheve voici 11.600 ans.
Figure 12 : Distribution de la diversit chloroplastique des trois lignes E1, E2, E3 de lolivier
mditerranen (daprs Besnard et al., 2013).
Page 31
Paralllement ces deux formes sauvages et cultives vraies , une troisime forme est
reprsente par lolivier fral qui dsigne des oliviers ensauvags ou fral dans des vergers
abandonns ou issus de graines dolivier sauvage ou cultiv propages par les oiseaux. L'olivier
fral peut tre caractris par lune ou lautre des trois lignes E1, E2, E3, et peut tre trouv
dans des agrosystmes comme dans des environnements naturels.
Cette diversit botanique et gntique de lolivier reflte une diversit de ses parasites
dont les nmatodes phytoparasites. Une forte diversit des NPP avec 153 espces et 56 genres a
t rvise sur lolivier (Ali et al., 2014). La domestication de lolivier mditerranen depuis
lAntiquit sest faite pour partie partir de boutures enracines. En consquence, la
propagation de lolivier a t accompagne par un transport de sol et donc de parasites
telluriques. Donc, nous faisons lhypothse que les communauts locales de NPP sont la
rsultante historique des communauts natives avant la propagation de lolivier et des
communauts introduites par boutures enracines, soumises aux forages volutifs et
environnementaux.
6. Objectifs de la thse et questions de recherche

Lanalyse des mcanismes dassemblage des communauts de NPP et de coexistence des
espces saborde par une approche comparative des communauts entre divers milieux. Le projet
de la thse a pour ambition dapporter des connaissances sur les leviers biotiques et abiotiques
qui dterminent la structuration des communauts de nmatodes phytoparasites.
Deux approches exploratoires sont distingues :
lune qui consiste analyser la part des filtres anthropiques et environnementaux sur la
structuration des communauts de NPP. Ltude portera sur la diversit taxonomique en
NPP (indices de diversit), la composition fonctionnelle et trophique ainsi que des patrons
statistiques des communauts de NPP.
une autre qui consiste analyser la part des filtres volutifs en explorant les interactions
rciproques entre la diversit morphologique et molculaire de diffrentes populations dun
genre de nmatodes (Meloidogyne spp.) et la structure des communauts de NPP.
Ces objectifs de la thse sont illustrs dans la figure 13.

Page 32
Cette tude a port sur le modle cologique de lolivier mditerranen au Maroc. Plusieurs
arguments justifiaient ce choix :
-
Le Maroc est un des premiers pays producteurs avec plus de 1,5 million de tonnes
d'olives produites sur plus de 780000 Ha (El Mouhtadi et al., 2014). En outre, le Plan
Maroc Vert a programm le doublement des surfaces cultives en trs haute densit dici
2020 (www.agriculture.gov.ma/pages/acces-fillieres/filiere-oleicole). Il va sen dire que
ces agrosystmes seront rapidement perturbs par une rsurgence de plusieurs bioagresseurs, dont les PPN, dont la diversit et la distribution spatiale est totalement
inconnue au Maroc.
Le Maroc offre de vastes zones de culture de lolivier, (i) soumises aux climats
ocanique, mditerranen ou montagnard, (ii) en plaine ou sur les piedmonts atlasiens,
(iii) dans divers types de sols, (iv) mene de manire traditionnelle (faible densit de
culture et culture pluviale le plus souvent) ou de manire industrielle (trs haute densit et
culture irrigue). Les quatre lignes gntiques E1, E2, E3 et M y sont prsentes.
Afin datteindre ces objectifs, nous tenterons de rpondre aux questions suivantes :
1.
Quelle est la diversit des communauts de NPP (famille, genre, espces) sur lolivier au
Maroc ?
2.
En quoi les modalits de culture de lolivier, sa diversit botanique et gntique, les

paramtres physico-chimiques du sol et le climat contribuent-elles la diversit et la
structure de ces communauts ?
3.
Au sein de ces communauts de NPP, quelle est la diversit spcifique du genre

Meloidogyne ?
4.
Comment les espces de Meloidogyne sont-elles distribues gographiquement et

comment peut-on expliquer cette distribution ?
5.
Quelle variabilit gntique morphologique et molculaire caractrise ce taxon et quel

niveau : intra et inter populations ? Y-a-t-il congruence de ces deux marqueurs de
diversit ?
Suite aux rponses ces questions, nous proposerons, en perspectives, danalyser la

correspondance potentielle entre la diversit gntique des Meloidogyne et la structure des
communauts qui les hbergent. Nous proposons galement danalyser la congruence entre la
diversit taxonomique des communauts de NPP et/ou la diversit gntique de Meloidogyne
avec celle de lolivier.
Page 33
Figure 13 : Objectifs de la thse (traits pleins = tudes ralises ; traits pointills = perspectives).
Note : Nous informons le lecteur que tous les chapitres de rsultats ont t rdigs sous
forme de manuscrits prts tre soumis pour publication. Certains manuscrits sont
prsents aux normes recommandes par certaines revues (Plos ONE, OIKOS, etc.)
7. Rfrences
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Bardgett, R.D., Wardle, D.A., 2010. Abovegroundbelowground linkages: Biotic interactions,
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Baujard P., Compas B., Scotto La Massese C., 1979. Introduction ltude cologique de la
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Page 40
Chapitre II
Les nmatodes
phytoparasites associs
lolivier au niveau mondial
Revue bibliographique
Page 41
Rsum
Ce chapitre traite dune analyse bibliographique des nmatodes phytoparasites (NPP) associs
la culture de lolivier au niveau mondial avec un focus sur la rgion Mditerranenne, la
rgion principale de cette culture. Cette revue bibliographique a fait lobjet dune publication
en premier auteur publie dans la revue Comptes Rendus Biologies en 2014. Elle
complte dautres analyses bibliographiques plus anciennes ayant recens les espces de NPP
associes lolivier comme plante hte. Outre cette mise jour, cette revue avait comme
objectifs danalyser leur diversit taxonomique, leur composition trophique ainsi que leur
dominance, de rviser les mthodes disponibles de lutte pour combattre contre ces parasites,
et enfin de discuter de nouvelles stratgies de contrle base sur la gestion de la structure des
communauts de NPP.
Cette publication se devise en quatre parties : (i) une premire traitant de lolivier comme
plante hte des nmatodes, en sappuyant sur son origine, sa rpartition mondiale, son
importance conomique et culturelle dans la rgion mditerranenne en particulier, ainsi que
sa diversit botanique et gntique qui pourrait impacter la diversit de ses parasites dont les
NPP ; (ii) Une deuxime focalisant sur les nmatodes parasitaires des plantes, avec quelques
indications sur leur morphologie, les pertes conomiques quils induisent sur les plantes
attaques, les NPP mentionns dans la littrature associs lolivier regroups dans une liste
de 153 espces et de 56 genres. Ces NPP sont diffremment distribus dans les pays
mditerranens dans lesquels des prospections nmatologiques sur lolivier ont t ralises.
Limpact des NPP sur lolivier ainsi que la raction de la plante hte vis--vis de ces attaques
ont t dcrites ; (iii) un focus port sur les quelques genres de NPP considrs comme
dominants sur lolivier mditerranen : Criconemoides, Helicotylenchus, Longidorus,
Meloidogyne, Pratylenchus, Rotylenchulus, Rotylenchus, Tylenchorhynchus, Tylenchulus,
Tylenchus, et Xiphinema. Certains de ces nmatodes tels que les nmatodes galles
Meloidogyne sont considrs comme des parasites importants, surtout en conditions
dirrigation de lolivier et en ppinires ; (iv) La dernire partie a dtaill les mthodes de
lutte (exclusion des NPP, rsistance varitale, solarisation, contrle biologique, etc.). Une
nouvelle vision sur la lutte contre les NPP a t galement aborde. Cette vision est base sur
la gestion de la diversit des NPP prsents partout en communauts (au sens de mlange
despces) via lexploitation des interactions entre les espces ainsi que linfluence des
paramtres environnementaux sur cette diversit.
Page 42
!
!!
C. R. Biologies 337 (2014) 423442
Contents lists available at ScienceDirect
Comptes Rendus Biologies

www.sciencedirect.com
Review/Revue
Plant-parasitic nematodes associated with olive tree

(Olea europaea L.) with a focus on the Mediterranean Basin:
A review
Nadine Ali a,b,*, Elodie Chapuis b,c, Johannes Tavoillot b, Thierry Mateille b
a
Tichreen University, Faculty of Agriculture, Plant Protection Department, PO Box 230, Latakia, Syrian Arab Republic
IRD, UMR CBGP (Center for Biology and Management of Populations) (INRA/IRD/CIRAD/Montpellier SupAgro), campus de Baillarguet,
755, avenue du Campus-Agropolis, CS30016, 34988 Montferrier-sur-Lez cedex, France
c
IRD, UMR RPB, 911, avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 4 March 2014
Accepted after revision 16 May 2014
Available online 26 July 2014
The olive tree (Olea europaea ssp. europaea.) is one of the most ancient cultivated trees. It is an
emblematic species owing to its ecological, economic and cultural importance, especially in
the Mediterranean Basin. Plant-parasitic nematodes are major damaging pests on olive trees,
mainly in nurseries. They significantly contribute to economic losses in the top-ten oliveproducing countries in the world. However, the damages they induce in orchards and
nurseries are specifically documented only in a few countries. This review aims to update
knowledge about the olive-nematode pathosystem by: (1) updating the list of plant-parasitic
nematodes associated with olive trees; (2) analysing their diversity (taxonomic level, trophic
groups, dominance of taxa), which allowed us (i) to assess the richness observed in each
country, and (ii) to exhibit and describe the most important taxa able to induce damages on
olive trees such as: Meloidogyne, Pratylenchus, Helicotylenchus, Xiphinema, Tylenchulus,
Rotylenchulus, Heterodera (distribution especially in the Mediterranean Basin, pathogenicity
and reactions of olive trees); (3) describing some management strategies focusing on
alternative control methods; (4) suggesting new approaches for controlling plant-parasitic
nematodes based on the management of the diversity of their communities, which are
structured by several environmental factors such as olive diversity (due to domestication of
wild olive in the past, and to breeding now), cropping systems (from traditional to highdensity orchards), irrigation, and terroirs.
2014 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.
Keywords:
Communities
Control methods
Diversity
Geographical distribution
Mediterranean Basin
Olive
Pathogenicity
Plant-parasitic nematodes
1. The olive tree: origins, dissemination and cultivation

Six Olea europaea subspecies with distinct geographic
areas are distinguished [1,2] (Fig. 1):

subsp. europaea present in the Mediterranean Basin, with

two botanical varieties: O. europaea subsp. europaea
var. europaea for the cultivated forms, and O. e. subsp.

* Corresponding author.
E-mail address: [email protected] (N. Ali).
e. var. sylvestris for wild and spontaneous trees (usually

named oleasters) [2];
subsp. laperrinei in the Saharan mountains;
subsp. cuspidata from southern Africa to southern Egypt
and from Arabia to China;
subsp. guanchica in the Canary Islands;
subsp. maroccana in southern Morocco;
subsp. cerasiformis in Madeira.
The Mediterranean olive (Olea europaea L. subsp.

europaea; Oleaceae) is one of the first domesticated tree
http://dx.doi.org/10.1016/j.crvi.2014.05.006
1631-0691/ 2014 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.
Page 43
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N. Ali et al. / C. R. Biologies 337 (2014) 423442
424
Family
Oleaceae
Genus
Olea
Subgenus
Olea
Section
Olea
Ligustrodes
Species
Olea europaea
Olea capensis
O. woodiana
O. lancea
Subspecies
O. europaea
O. laperrinei
O. cerasiformis
O. guanchica
O. maroccana
O. cuspidata
Est Africa & Madagascar
Mediterranean basin
Sahara
Madeira
Canary Islands
Morocco
Asia, Arabia, South & East Africa
Fig. 1. Simplified diagram of the Olea genus (Oleaceae) and biogeography

of taxa [11].
species [3,4]. Its origins and domestication history are still

highly debated, but archaeological and molecular data
sustain that the first cultivars originated from the Levant,
very likely 6000 years ago, in a region presently located at
the border between southwestern Turkey and northwestern
Syria [5,6]. Genetic studies indicated also that olive
domestication is a long and on-going process and cultivars
have originated from multiple source populations, as for
grape and figs [79]. It is currently established that western
wild olives secondarily contributed to the cultivated gene
pool, while most of present cultivars remain mainly related
to eastern wild olives [10].
The genetic diversity of oleaster is somewhat greater
than that of the cultivated olive [6,10,11]. This diversity is
highly structured between the East and the West of the
Mediterranean Basin [6,12,13]. The analysis of plastid DNA
polymorphisms allowed the identification of 48 distinct
profiles delineating three diverging lineages. Lineage E1 is
currently distributed over the whole Basin, whereas
lineages E2 and E3 exclusively occur in western and
central Mediterranean areas [6,14].
The olive tree has long been considered as the most
typical Mediterranean tree. It has been widely disseminated through conquests and exchanges that accompanied
the expansion of the Mediterranean civilizations (Phoenicians, Greeks, Romans, and Arabs) [15]. Populations of wild
olives are restricted to a few isolated areas of native
Mediterranean forests, where pollen/stones may be
spread by natural factors (wind and birds) [13]. Most
other wild-looking forms of olive may include feral forms
(either cultivated olive abandoned and becoming wild or
olive from stones of cultivated olive spread by birds). The
olive dissemination was accompanied by a secondary
diversification that has led to a high genetic variability
that reflects a wide range of morphological (tree vigour,
fruit shape, yield, oil content, etc.) and physiological
(adaptation traits to environmental stresses, particularly
those related to climate and soil constraints) characteristics that can explain its large distribution in the
Mediterranean Basin [16,17]. This diversity can also result
from a series of events related to human practices made to
!!
meet microclimate and terroir requirements and to satisfy

consumer taste [12].
More than 2000 Mediterranean varieties have been
recognised based on fruit, pit, and leaf shapes and colours,
tree architecture and phenology (i.e. flowering time)
[1821]. Olive cultivars are propagated vegetatively by
cutting or grafting. Most of the modern varieties display a
maternal lineage (E1) that is distributed all around the
whole Mediterranean area [6,14,22]. However, the genetic
diversity of cultivated populations exhibits a complex
patchy pattern [23,24].
For cultural (traditions, landscapes) and economic (oil
and olives) reasons, this tree is regarded as one of the most
cultivated plant in the world, ranking the 24th among 35
species [25]. It is now distributed in the five continents. It
grows extensively in Mediterranean climate regions in
Australia, South Africa, North and South America and
covers about 11 Mha, with 98% located in the Mediterranean Basin [26,27]. Eighty percent of the cultivated olive
surface is located in northern Mediterranean countries
(Spain, Italy, Greece, Turkey), 17% in the Middle East
(Jordan, Syria, Iraq, Iran) and in North Africa (Morocco,
Algeria, Tunisia, Egypt), and only 2% is located in North
(USA) and South (Mexico, Argentina, Peru) America [28,29]
(Fig. 2).
Olive diversity may also reflect a significant diversity of
associated pests, some of them being specific. Major olive
diseases are due to insects (e.g., the fruit fly Bactrocera oleae,
the black scale Saissetia oleae, the moth Prays oleae, the borer
Palpita unionalis), and to air-borne (e.g., the peacock spot
Spilocaea oleaginea, the knot Pseudomonas syringae) or soilborne (e.g., verticillium wilt Verticillium dahliae and plantparasitic nematodes) parasites and pathogens.
2. Plant-parasitic nematodes associated with olive trees

2.1. General considerations
Nematodes are roundworms of the phylum Nematoda, Metazoa having unsegmented, cylindrical bodies
with a non-ciliated tough outer cuticle. The group
includes free-living forms and disease-causing parasites.
Plant-parasitic nematodes (PPN) are soil-borne microscopic worms that mainly feed on plant-root cells using a
spear-like structure, called a stylet. They introduce it in
cells to inject digestion secretions and suck cell contents
[30].
Thereby, these parasites cause significant economic
damages to many types of crops as cereals, vegetables,
tubers, fruit, and floral crops [31]. Annual losses caused by
PPN are estimated from 8.8 to 14.6% of the world crop
production (100157 billion USD/year) [32,33]. In Europe,
economic losses are estimated to 10% of the grain
production and lead to reduce by 20 to 30% Mediterranean
citrus production [34].
Olive trees are hosts of these parasites. But very few
information is available about PPN communities associated with olive [27]. The first record was from the USA,
where root-knot nematodes Meloidogyne spp. were
detected [35]. Since then, some researches on olive PPN
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!N. Ali et al. / C. R. Biologies 337 (2014) 423442
425
Fig. 2. World distribution of Olea europaea L. producing areas [26].
were developed in several parts of the world, mainly in

Italy [3638], Chile [39], Jordan [40], Libya [41], Spain
[27,42], Portugal [43], Egypt [4446], Iran [47,48], Turkey
[49,50], Argentina [51], and Syria [52].
Most of these studies have focused on PPN description
without studying their potential effects on the crop [53].
However, studies focused on taxa that represent a real
threat to olive tree, particularly in the Mediterranean
Basin, such as root-knot nematodes Meloidogyne [5457],
lesion nematodes Pratylenchus [58], spiral nematodes
Helicotylenchus [59,60], reniform nematodes Rotylenchulus
[61,62], cyst nematodes Heterodera [63,64], and dagger
nematodes Xiphinema [60].
Other studies focused on assessing the impact of
these parasites in nurseries, especially in Spain [27] and
Iran [48].
2.2. Diversity of plant-parasitic nematodes associated with
olive trees
Previous reviews updated knowledge about nematode
diversity associated with olive tree [53,6568].
Moreover, the analysis of all literature sources let to
record 153 species (some as still unidentified) belonging to
56 genera related to the PPN orders Aphelenchida (4
genera and 3 species), Dorylaimida (5 genera and 36
species) and Tylenchida (48 genera and 114 species) (Table
1). Most of the species have been detected on other crops
[28]. They belong to three trophic groups: plant feeders,
fungal feeders (this group usually feeds on fungi but may
alternatively feeds on plants, e.g. Aphelenchida nematodes) and root-hair feeders. Considering their adaptive
strategies defined by colonizer/persister values (= cpvalues) ranking from 1 (colonizers/r strategy) to 5
(persisters/K strategy) [69], most of these species show
high reproduction rates and rapid adaptation capacities to
environmental changes (cp-3).
While many species such as Helicotylenchus digonicus,
Meloidogyne javanica, M. incognita, Pratylenchus vulnus are
able to parasite a wide range of cultivated and wild plants,
others, such as H. oleae, H. neopaxilli, M. baetica,

M. lusitanica, Rotylenchulus macrosoma, are specific to
olive tree [28].
Considering the species richness encountered in each
genus, the genera Xiphinema, Tylenchorhynchus, Pratylenchus, Helicotylenchus, Longidorus, Meloidogyne and
Paratylenchus are the most diversified with 16, 13, 12,
10, 9, and 8 species respectively, while 41.4% of the genera
are monospecific (Fig. 3).
2.3. Distribution of plant-parasitic nematodes
Plant-parasitic nematodes were encountered in most of
the olive-producing countries. But 81% of the genera were
scattered in less than 25% of the countries (Fig. 4). Twentyone genera were detected in only one country. The
dominant genera (Criconemoides, Helicotylenchus, Longidorus, Meloidogyne, Pratylenchus, Rotylenchulus, Rotylenchus, Tylenchorhynchus, Tylenchulus, Tylenchus, and
Xiphinema) are each present in at least six countries
(Fig. 5), especially around the Mediterranean Basin. Rootknot nematodes Meloidogyne spp. are distributed in 19
countries. That could explain the economic importance of
this group that can represent a real threat for olive.
Modelling the number of genera recorded in each
country (Fig. 6A), the highest diversities were recorded in
Spain (38 genera), in Jordan (28), in Italy (24), in Greece
(20), and in Turkey (16). Ten to twelve genera were
detected in Chile, Egypt, Iran, Portugal, and Syria. Only one
to five genera were recorded in the other countries.
However, these data can in no way reflect the true diversity
of the PPN associated with olive trees in each country.
Indeed, modelling the research effort by the number of
publications as an indicator (Fig. 6B), the diversity found in
each country is close to this indicator. Moreover, considering the Mediterranean Basin only, PPN diversity is
totally unknown in France, in Morocco, in Adriatic
countries, and in most of the Mediterranean islands.
The most common nematode genera detected in
Jordan were [40]: Filenchus, Helicotylenchus, Merlinius,
Page 45
!
426
!!
Table 1
Plant-parasitic nematode taxa associated with olive tree.
Order
Family
Species
Country
Reference
Aphelenchida
Aphelenchidae
Aphelenchus avenae Bastian 1865
Greece
Irana
Jordan
Spain
Iran
Italy
Spain
Egypt
Italy
Jordan
Spain
Syria
Spain
Egypt
Portugal
Greece
Greece
Egypt
Spain
Jordan
Portugal
Egypt
Italy
Spaina
Syria
Italy
Italy
Italy
USA
Portugal
Egypt
Greece
Italy
Jordan
Italy
Jordan
Italy
Portugal
Italy
Spain
Italy
Jordan
Portugal
Spain
Spaina
Spain
Spain
Spain
Chile
Egypt
Iran
Israel
Italy
Jordan
Spain
Italy
Spaina
Spaina
Jordan
Spain
Spainb
Spain
Spaina
Portugal
Spain
Portugal
Spainb
Italy
Italy
[103]
[48]
[40]
[42]
[47]
[36]
[42]
[46]
[36]
[40]
[42]
[52]
[42]
[108]
[79]
[167]
[168]
[46]
[42]
[94]
[79]
[60]
[36]
[27]
[52]
[110]
[112]
[113]
[114]
[115]
[60]
[169]
[121]
[40]
[116]
[94]
[78]
[79]
[117]
[17]
[68]
[94]
[79]
[17]
[27]
[118]
[42]
[118]
[120]
[46]
[47]
[170]
[36]
[171]
[172]
[142]
[27]
[27]
[40]
[42]
[140]
[140]
[27]
[143]
[140]
[143]
[140]
[141]
[142]
Aphelenchus sp. Bastian 1865
Aphelenchoididae
Dorylaimida
Longidoridae
Paraphelenchus pseudoparietinus Micoletzky 1921

Aphelenchoides sp. Fischer 1984
Aprutides guidettii Scognamiglio 1974

Longidorus africanus Merny 1966
L. belloi Andres and Arias 1988
L. closelongatus Stoyanov 1964
L. cretensis Tzortzakakis et al., 2001
L. elongatus De Man 1876
L. macrosoma Hooper 1961
L. siddiqii Aboul-Eid 1970
L. vinearum Bravo and Roca 1995
Longidorus sp. Micoletzky 1922
Paralongidorus sp. Siddiqi et al., 1963

Xiphinema aequum [112]
X. barense Lamberti et al., 1986
X. californicum Lamberti and Bleve-Zacheo 1979
X. diversicaudatum Micoletzky, 1927
X. elongatum Schuurmans Stekhoven and Teunissen, 1938
X. index Thorne and Allen 1950
X. ingens Luc and Dalmasso 1964

X.
X.
X.
X.
X.
italiae Meyl 1953

lusitanicum Sturhan, 1983
macroacanthum Lamberti et al., 1990
nuragicum Lamberti et al., 1992
pachtaicum Tulaganov 1938
X. sahelense Dalmasso, 1969

X. turcicum Luc and Dalmasso, 1964
X. vuittenezi Luc et al., 1964
Xiphinema sp. Cobb, 1913
Trichodoridae
Paratrichodorus alleni Siddiqi, 1974

P. minor Colbran 1956
P. teres Hooper 1962
Paratrichodorus sp. Siddiqi 1974
Trichodorus aequalis Allen 1957
T. andalusicus Decraemer et al., 2012
T. iliplaensis Decraemer et al., 2012
T. giennensis Decraemer et al., 1993
T. lusitanicus Siddiqi, 1974
T. parasilvestris Decraemer et al., 2012
T. primitivus De Man 1880
T. silvestris Decraemer et al., 2012
T. taylori Mancine 1980
T. variopapillatus Hooper, 1972
Page 46
!
427
!!
Table 1 (Continued )
Order
Tylenchida
Family
Anguinidae
Species
Country
Reference
Trichodorus sp. Cobb 1913
Chile
Italy
Spain
Jordan
Spain
Spain
Italy
Jordan
Spain
Spain
Portugal
Greece
Spain
Spain
Jordan
Spain
Spain
Jordan
Spaina
Chile
Egypt
Greece
Italy
Jordan
Zimbabwe
Turkey
Spain
Brazil
Italy
Spain
Spaina
Jordan
Portugal
Zimbabwe
Spain
Jordan
Italy
Portugal
Spain
Italy
Italy
Italy
Italy
Turkey
Chile
Spaina
Spaina
Italy
Spain
Italy
Spain
Spain
Cyprus
Greece
Jordan
Spain
Spaina
Turkey
Brazil
Cyprus
Egypt
Italy
Jordan
Spaina
Spain
Zimbabwe
Italy
Jordan
Italy
Italy
[120]
[36]
[42]
[40]
[173]
[172]
[36]
[171]
[42]
[42]
[68]
[169]
[42]
[42]
[40]
[27]
[42]
[94]
[27]
[120]
[60]
[103]
[36]
[171]
[174]
[49]
[68]
[175]
[176]
[68]
[27]
[177]
[142]
[174]
[42]
[40]
[148]
[146]
[68]
[110]
[178]
[110]
[36]
[49]
[120]
[27]
[27]
[63]
[64]
[36]
[42]
[147]
[179]
[103]
[180]
[17]
[27]
[49]
[181]
[179]
[108]
[111]
[180]
[27]
[182]
[109]
[59]
[40]
[70]
[70]
Ditylenchus anchiliposomus Tarjan 1958

D. dipsaci Kuhn 1857
D. virtudesae Tobar-Jimenez 1964
Ditylenchus sp. Filipjev 1936
Criconematidae
Criconema annuliferum De Man 1921

C. princeps Andrassy 1962
Criconema sp. Hofmanner and Menzel 1914
Criconemella informis Micoletzky 1922
C. sphaerocephala Taylor 1936
C. xenoplax Raski 1952
Criconemella sp. De Grisse and Loof 1965
Criconemoides informis Micoletzky 1922
Criconemoides sp. Taylor 1936
Crossonema multisquamatum Kirjanova 1948

Hemicriconemoides gaddi Loos 1949
Mesocriconema curvatum Raski 1952
M. onoense Luc 1959
M. sicula Loof and De Grisse 1989
M. sphaerocephalum Taylor 1936
M. xenoplax Raski 1952
Neolobocriconema olearum Hashim 1984
Nothocriconema princeps De Grisse and Loof 1965
Ogma civellae Steiner 1949
O. rhomobosquamatum Mehta and Raski 1971
O. seymouri Wu 1965
Dolichodoridae
Hemicycliophoridae
Heteroderidae
Hoplolaimidae
Ogma sp. Mehta and Raski 1971

Dolichodorus heterocephalus Cobb 1911
Dolichodorus sp. Cobb 1915
Trophurus sp. Loof 1956
Hemicycliophora sturhani Loof 1984
Hemicycliophora sp. De Man 1921
Heterodera avenae Filipjev 1934
H. mediterranea Vovlas et al., 1981
Heterodera sp. Filipjev and Schuurmans Stekhoven 1941
Heteroderinae Filipjev and Schuurmans Stekhoven 1941
Aorolaimus perscitus Doucet 1980
Helicotylenchus digonicus Perry 1959
H. dihystera Cobb 1893
H.
H.
H.
H.
erythrinae Zimmermann 1904

minzi Sher 1966
neopaxili Inserra et al., 1979
oleae Inserra et al., 1979
Page 47
!
428
!!
Order
Family
Species
Country
Reference
H. pseudorobustus Steiner 1914
Greece
Irana
Italy
Jordan
Spaina
Spain
Israel
Jordan
Portugal
Spain
Turkey
Jordan
Portugal
Spaina
Spain
Turkey
Algeria
Chile
Egypt
Greece
Iran
Italy
Jordan
Syria
Portugal
Turkey
Egypt
Syria
Turkey
Turkey
Jordan
Turkey
Portugal
Cyprus
Greece
Italy
Spaina
Spain
China
Chile
China
Spaina
Spainb
Chile
Israel
Portugal
Argentina
Brazil
Chile
China
Egypt
India
Israel
Italy
Jordan
Lebanon
Lybia
Portugal
Spaina
[103]
[48]
[68]
[94]
[27]
[27]
[109]
[94]
[146]
[173]
[49]
[40]
[146]
[27]
[173]
[49]
[104]
[120]
[46]
[103]
[47]
[36]
[171]
[52]
[146]
[49]
[60]
[52]
[49]
[49]
[40]
[49]
[142]
[179]
[103]
[36]
[27]
[42]
[89]
[39]
[89]
[27]
[90]
[39]
[88]
[96]
[68]
[181]
[39]
[89]
[45]
[183]
[88]
[184]
[185]
[186]
[41]
[187]
[27]
H. tunisiensis Siddiqi 1963
H. vulgaris Yuen 1964
Helicotylenchus sp. Steiner 1945
Hoplolaimus aorolaimoides Siddiqi 1972

H. galeatus Cobb 1913
Hoplolaimus sp. Daday 1905
Plesiorotylenchus striaticeps Vovlas et al., 1993
Rotylenchus buxophilus Golden 1956
R. cypriensis Antoniou 1980
R. robustus De Man 1876
Rotylenchus sp. Filipjev 1936
Meloidogynidae
Hoplolaiminae Filipjev 1934

Meloidogyne acrita Chitwood 1949
M. arenaria Neal 1889
M. baetica Castillo et al. 2003

M. hapla Chitwood 1949
M. incognita Kofoid and White 1919
Page 48
!
429
!!
Order
Family
Species
Country
Reference
M. javanica Treub 1885
Argentina
Chile
China
Egypt
Greece
Irana
Israel
Italy
Jordan
Lybia
Pakistan
Portugal
Spaina
Portugal
Spaina
Chile
Cyprus
Iran
Israel
Italy
Jordan
Portugal
Syria
USA
Italy
Portugal
Spain
Spain
Italy
Spain
Spaina
Turkey
Spain
Spain
Spain
Spain
Spain
Italy
Italy
Jordan
Syria
Turkey
Spaina
Iran
Jordan
Australia
Jordan
Italy
Spaina
Turkey
USA
Greece
Italy
Iran
Jordan
Spain
Spaina
Australia
Italy
Jordan
Spain
Turkey
Irana
Jordan
Spaina
Algeria
Australia
Italy
Spaina
USA
Turkey
[51]
[39]
[89]
[60]
[103]
[48]
[87]
[184]
[94]
[41]
[68]
[68]
[27]
[43]
[27]
[120]
[179]
[47]
[87]
[59]
[171]
[188]
[52]
[35]
[36]
[146]
[68]
[147]
[37]
[42]
[27]
[49]
[42]
[17]
[173]
[173]
[173]
[95]
[36]
[171]
[52]
[49]
[27]
[190]
[171]
[101]
[40]
[190]
[27]
[49]
[102]
[103]
[95]
[189]
[40]
[173]
[27]
[191]
[95]
[40]
[42]
[68]
[48]
[94]
[27]
[104]
[191]
[58]
[27]
[102]
[49]
M. lusitanica Abrantes and Santos 1991

Meloidogyne sp. Goeldi 1887
Paratylenchidae
Gracilacus peratica Raski 1962
G. teres Raski 1976

Gracilacus sp. Raski 1962
Paratylenchus arculatus Luc and de Guiran 1962
P. baldacii Raski1975
P. ciccaronei Raski 1975
P. microdorus Andrassy 1959
P. nanus Cobb 1923
P. sheri Raski 1973
P. vandenbrandei De Grisse 1962
Paratylenchus sp. Filipjev 1936
Pratylenchidae
Pratylenchoides erzurumensis Yuksel 1977

P. ritteri Sher 1970
Pratylenchoides sp. Winslow 1958
Pratylenchus coffeae Zimmerman 1898
P.
P.
P.
P.
P.
crenatus Loof 1960

fallax Seinhorst 1968
mediterraneus Corbett 1983
musicola Cobb 1919
neglectus Rensch 1924
P. penetrans Cobb 1917
P. thornei Sher and Allen 1953
P. vulnus Allen and Johnson 1951
P. zeae Graham 1951
Page 49
!
!!
430
Order
Family
Species
Country
Reference
Pratylenchus sp. Thorne 1949
Algeria
Chile
Egypt
Greece
Iran
Italy
Jordan
Spain
Syria
Greece
Spain
Spaina
Turkey
Irana
Jordan
Spain
Greece
Iran
Greece
Italy
Israel
Jordan
Spainb
Turkey
Egypt
Greece
Algeria
Syria
Portugal
Turkey
Jordan
Spaina
Spain
Turkey
Cyprus
Greece
Irana
Jordan
Spain
Jordan
Spain
Jordan
Turkey
Spain
Turkey
Greece
Italy
Spaina
Jordan
Spaina
Cyprus
Turkey
Turkey
Greece
Spain
Spaina
Jordan
Spaina
Spaina
Turkey
Greece
Jordan
Turkey
[104]
[120]
[46]
[103]
[47]
[36]
[171]
[42]
[52]
[103]
[42]
[27]
[49]
[48]
[40]
[42]
[169]
[47]
[133]
[131]
[134]
[40]
[62]
[49]
[192]
[103]
[104]
[52]
[193]
[49]
[94]
[27]
[42]
[49]
[179]
[103]
[48]
[94]
[42]
[40]
[173]
[40]
[49]
[42]
[49]
[103]
[36]
[27]
[94]
[27]
[68]
[49]
[49]
[103]
[42]
[27]
[94]
[27]
[27]
[49]
[103]
[194]
[49]
Radopholus sp. Thorne 1949

Zygotylenchus guevarai Tobar-Jimenez 1963
Psilenchidae
Psilenchus hilarulus De Man 1921

P. iranicus Kheiri 1970
Psilenchus sp. De Man 1921
Rotylenchulidae
Rotylenchulus macrodoratus Dasgupta et al., 1968

R. macrosoma Dasgupta et al., 1968
R. reniformis Linford and Oliveira 1940

Rotylenchulus sp. Linford and Oliveira 1940
Telotylenchidae
Amplimerlinius amplus Siddiqi 1976

A. dubius Steiner 1914
A. macrurus Goodey 1932
A. paraglobigerus Castillo et al., 1990
Amplimerlinius sp. Siddiqi 1976
Bitylenchus goffarti Sturhan 1966
Merlinius brevidens Allen 1955
M. microdorus Geraert 1966

M. nothus Allen 1955
Quinisulcius acutus Allen 1955
Paratrophurus loofi Arias 1970
Scutylenchus lenorus Brown 1956
Telotylenchus sp. Siddiqi 1960
Trophurus sp.
Tylenchorhynchus aduncus De Guiran 1967
T. clarus Allen 1955
T. claytoni Steiner 1937
T. cylindricus Cobb 1913
T. dubius Buetschli 1873
T.
T.
T.
T.
T.
T.
T.
goffarti Sturhan 1966

huesingi Paetzold 1958
mamillatus Tobar-Jimenez 1966
penniseti Gupta and Uma 1980
striatus Allen 1955
tenuis Micoletzky 1922
tritici Golden et al., 1987
Page 50
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431
!!
Order
Family
Tylenchidae
Species
Country
Reference
Tylenchorhynchus sp. Cobb 1913
Algeria
Chile
Egypt
Greece
Iran
Italy
Jordan
Spaina
Syria
Spain
Spain
Jordan
Irana
Iran
Jordan
Spain
Spain
Greece
Spain
Spain
Jordan
Spain
Greece
Spain
Jordan
Spain
Irana
Spain
Spain
Spain
Chile
Greece
Iran
Italy
Jordan
Spain
Jordan
Australia
Chile
Egypt
Greece
Italy
USA
Chile
Italy
Syria
[104]
[120]
[45]
[103]
[47]
[36]
[171]
[27]
[52]
[42]
[42]
[40]
[48]
[47]
[40]
[42]
[42]
[103]
[42]
[42]
[40]
[42]
[103]
[17]
[40]
[17]
[48]
[42]
[42]
[17]
[120]
[103]
[47]
[36]
[94]
[17]
[40]
[101]
[68]
[60]
[169]
[38]
[195]
[120]
[110]
[52]
Aglenchus agricola De Man 1884

Basiria duplexa Hagemeyer and Allen 1952
Basiria sp. Siddiqi 1959
Boleodorus thylactus Thorne 1941
Boleodorus sp. Thorne 1941
Coslenchus cancellatus Cobb 1925

C. costatus De Man 1921
C. lateralis Andrassy 1982
Coslenchus sp. Siddiqi 1978
Discotylenchus discretus Siddiqi 1980
Filenchus discretus Siddiqi 1979
F. filiformis Buetschli 1873
F. sandneri Wasilewska 1965
Filenchus sp. Andrassy 1954
Irantylenchus sp. Kheiri 1972
Neopsilenchus magnidens Thorne 1949
Tylenchus arcuatus Siddiqi 1963
T. davainei Bastian 1865
Tylenchus sp. Bastian 1865
Tylenchulidae
Trophotylenchulus saltensis Hashim 1983

Tylenchulus semipenetrans Cobb 1913
Tylenchulus sp. Cobb 1913
a
b
In nurseries.
On wild olive.
Pratylenchus, and Xiphinema. A more recent study conducted in Iran revealed other frequent genera, such as
Aphelenchus,
Boleodorus,
Meloidogyne,
Psilenchus,
Tylenchus, and Tylenchorhynchus [47].
The spatial distribution of PPN species in different fields
sampled in Spain is homogeneous [42]. However, this
author noticed seasonal fluctuations according to local
climate. High-level PPN populations were observed in
autumn, low levels in summer and intermediate levels in
winter and spring. He hypothesized that microclimatic soil
variations could modify root tissue plasticity, which
consequently could change the aptitude of nematodes to
penetrate roots [70].
Nematode occurrence usually differs in nurseries:
the most common species detected in southern Spain
are M. incognita, M. javanica, M. arenaria, Mesocriconema
xenoplax, Pratylenchus penetrans, and P. vulnus [27]. In
contrast, the most common nematodes detected in nurseries in northern Iran are [48]: Aphelenchus avenae,
M. javanica, Irantylenchus sp. (a new genus reported for
the first time as associated with olive), Pratylenchus thornei,
Helicotylenchus pseudorobustus, Boleodorus thylactus,
Psilenchus hilarulus, and Merlinius brevidens. But in nurseries,
the occurrence of PPN is directly related to the origin of the
cutting substrates that are often not sterilized, and to the
susceptibility of olive cultivars (e.g., P. vulnus is more
frequent on cv. Manzanilla than on other cultivars [27]).
2.4. Impacts of plant-parasitic nematodes on olive trees
Hostparasite interactions between PPN and olive trees
have been scarcely studied. However, controlled experiments showed that root-knot nematodes Meloidogyne (e.g.,
M. arenaria, M. incognita, and M. javanica) significantly
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432
!!
Fig. 3. Species diversity in each plant-parasitic nematode genus.
reduce tree growth [5557,71] and may be responsible for

5 to 10% crop losses [33], while these damages are often
difficult to assign to PPN [28]. Indeed, the symptoms due to
PPN parasitism are not clear, because
the hardiness of this plant is able to hide them;
population levels are usually low in the rhizosphere [68].
In any case, this is commonly observed in traditional
olive cropping systems.
Three species of fruit crop infecting nematodes that
could damage olive (Longidorus diadecturus, Xiphinema
americanum, X. californicum) are absent in Europe [68].
These species have been declared as quarantine pests in
Europe (EPPO/OEPP). Heterodera mediterranea and X. italiae
are also quarantine pests in Chile.
The nematode parasitism impacts directly the plant
growth and yield. Basically, root functions are disturbed
by histological alterations of root tissues. Physiological
disorders limit water and mineral absorption, leading to
major deficits [66]. These phenomena are less prominent
on wild olive trees that seems more tolerant to PPN
infection [28].
The impact of nematodes is much more significant in

nurseries where irrigation conditions favour the development of roots and therefore PPN multiplication [27,28,68].
Humidification of the substrates moderates the temperature, two factors that support the development of nematodes [72]. Moreover, roots of olive seedlings are more
susceptible to PPN than older plants [27]. Thus, infestation
levels with Meloidogyne arenaria, M. incognita, M. javanica,
Mesocriconema xenoplax, Pratylenchus penetrans and
P. vulnus are significantly higher in nurseries than in
orchards [27,48]. But although the same genera are known
for their harmfulness on other fruit trees such as apple and
pear, the damaging threshold of PPN is poorly evaluated on
olive [55,58,73].
Indirect root damages are also related to nematode
infection, whose penetration opens pathways to other soilborne pathogens (bacteria, fungi) [67,74]. The best
example is the association of PPN, such as M. incognita
and P. vulnus, with the fungal pathogen Verticillium dahliae
(Verticillium wilt). The presence of nematodes enhances
the symptoms induced by the fungus [7577]. Experiments have demonstrated a positive correlation between
the number of root galls induced by M. incognita and the
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! N. Ali et al. / C. R. Biologies 337 (2014) 423442
433
Fig. 4. Frequency of the plant-parasitic nematode genera detected (% of countries).
Fig. 5. World and Mediterranean distribution of the most frequent plant-parasitic nematode genera associated with olive tree (i.e. present in more than 25%
of countries).
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434
Genus richenss in each country
40
A
30
20
10
Number of publications
0
20
15
10
!!
M. javanica/Fusarium sp. complex would also be responsible for the Seca disease that induces drying of young
olive trees in nurseries [51].
Virus transmission by some PPN species is also a major
risk to the olive tree. Thus, the olive tree is a natural host to
Nepoviruses such as the Arabis Mosaic Nepovirus
(ArMV), the Strawberry Latent Ringspot Sadwavirus
(SLRSV) and the Cherry Leaf Roll Virus (CLRV) transmitted by Xiphinema diversicaudatum and X. vuittenezi
[78,79]. These viruses do not produce symptoms on olive
[80], except SLRSV that is responsible for leaf and fruit
distortion on the variety Ascolana tenera [81].
Biochemical reactions in olive tree roots are diverse,
depending on the nematode species that induce specific
physiological mechanisms, as for lesion nematodes as
P. vulnus [58,82], for root-knot nematodes as M. javanica
[55,56], or for dagger nematodes as Xiphinema index [83].
The secondary metabolism would be involved in plant
reactions, which would confer resistance (i.e. lower
infestation and reproduction) on some varieties [84].
China
India
Lebanon
Libya
Pakistan
Australia
Argentina
Portugal
Egypt
Syria
Chile
Cyprus
Algeria
Israel
USA
Brazil
Zimbabwe
Spain
Jordan
Italy
Greece
Turkey
Iran
Countries
Fig. 6. Genus richness of plant-parasitic nematode recorded in each

Mediterranean country (A) and associated publications (B).
extent of root rots and vascular discoloration, and the

M. incognita/V. dahliae combination reduced rhyzogenesis
[76]. When the three parasites M. incognita/P. vulnus/
V. dahliae were associated, the reproduction of each of two
nematode species was affected by the presence of the other
species and/or by the fungus.
Similarly, damages induced by Heterodera mediterranea
could be intensified by the presence of V. dahliae [64]. The
3. The dominant plant-parasitic nematodes associated

with olive trees
3.1. Meloidogyne spp. (root-knot nematodes)
Root-knot nematodes (RKN) are sedentary endoparasites. Ninety-seven species have been described [85].
They have developed reproduction and parasitism strategies that allow them to infect thousands of plant species.
Most RKN species induce galls that can invade the whole
root system. They are widespread all over the world
[66,74,86].
These nematodes have been firstly reported on olive
trees in the United States [35]. Thereafter, several Meloidogyne species were detected: M. javanica [87], M. incognita,
and M. hapla [88], M. arenaria [89], M. lusitanica [43], and
Fig. 7. World and Mediterranean distribution of root-knot nematode species (Meloidogyne spp.) associated with olive tree.
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M. baetica [90] recently. These nematodes occur on wild and

cultivated olive and also in nurseries (except for M. baetica
[27] (Table 1). Among these species, M. baetica was
detected on wild olive in southern Spain, and could infect
olive orchards. Experiments have shown that this species
does not reproduce on tomato (on which it induces small
galls without any egg mass) or on legumes. That would
confirm its specificity to olive, which led to call it
Mediterranean olive root-knot nematode [90]. This
species was also detected on Pistacia lentiscus and
Aristolochia baetica [91].
RKN are very well adapted to a wide range of
temperatures, which explains their worldwide distribution
(Table 1 and Fig. 7). However, M. incognita and M. javanica
are the most widespread species in the world [53,92]
especially in the Mediterranean Basin [93].
But RKN are at least the less frequent species in olive
orchards: they were detected in less than 4% of the samples
in Jordan, mainly in irrigated areas and nurseries [40,94], in
2% of the samples in southern Italy (the main production
area of the country) and in less than 1% in the other
producing areas of the country [95]. That supports other
observations made elsewhere in the world [54,55,60,92].
In spite of their scarce distribution, RKN are considered
as major pests on olive [53]. Thus, M. javanica is the main
cause of Seca syndrome in new olive orchards in
Argentina [51]. This species is responsible for olive decay
in nurseries [40]. RKN reduce plant growth [27,43,55,56].
Pathogenicity tests showed that M. arenaria, M. incognita
and M. javanica grow as well on rootstocks [56,57] and on
plants cv. Picual and cv. Arbequina, which are widely
distributed in the Mediterranean Basin [71]. Controlled
experiments showed that six months after inoculation
with 5000 or 10,000 M. javanica juveniles per plant, plant
growth was significantly reduced: 28% [60] and 4552%,
according to the cultivar [55], respectively. The stem
diameter is the most sensitive parameter to infestation
[57,71]. Olive trees infected by Meloidogyne spp. show
yellowing of the highest branches, and then defoliation
[68]. These symptoms are observed everywhere, as well as
in China [89], Portugal [96], and Chile [39].
Histological observations of roots infected with
M. incognita [97], with M. baetica [90,91], or with
M. javanica and M. lusitanica [92] indicate different sizes
and different localisations of the galls they induce.
M. javanica galls are usually located on the root tip, but
some galls are also distributed along roots. Clusters of
several galls were also observed and no secondary roots
seen near them [92]. M. lusitanica galls are usually
distributed as individual galls along roots. M. baetica
induces small galls, frequently located along the taproot
and very rarely on secondary roots, without the development of egg masses [90].
3.2. Pratylenchus spp. (lesion nematodes)
Lesion nematodes (LN) are migratory endoparasites.
They are worldwide distributed, and are able to infect a
wide range of plants. They are involved with other soilborne pathogens (e.g., fungi) in parasitic complexes. LN are
the most damaging nematodes with RKN [98100].
435
Several Pratylenchus species have been detected on

olive (Table 1 and Fig. 5): P. coffeae [101], P. crenatus [95],
P. mediterraneus and P. musicola and P. vulnus [102],
P. neglectus [103], P. penetrans [95], P. thornei [104] and
P. zeae [49]. Some of them (P. neglectus, P. penetrans,
P. thornei, P. vulnus and P. fallax) have been also observed in
nurseries [27,48].
Among these species, P. vulnus and P. penetrans are
considered the most frequent and pathogenic on olive
[58,71].
Infection with LN induces large necrotic areas on roots,
which may cover all the root system. This may rot the
cortical parenchyma [66,102] and reduce both number and
size of infected roots [105]. Other symptoms such as leaf
chlorosis, internode shortening and defoliation can be
observed [106].
Controlled inoculations revealed that most of the LN
species do not reproduce on olive, leading one to minimize
the impact of these nematodes on this crop [71]. Field
observations pointed that LN reproduce primarily in weeds
without injuring olive trees [107]. Nevertheless, P. vulnus is
the most pathogenic species [58]. It is responsible for tree
decline [53,106]. Significant damages attributed to this
species have been recorded in several olive orchards in
Italy [38,95,106], in the USA [102], and in Algeria [104].
Inoculations with P. vulnus performed on several olive
cultivars (Arbequina, Ascolana, Manzanilla, Picual) confirmed its strong pathogenic potential [58,71]. Competitions between RKN and LN have been recorded on olive.
Reproduction of P. vulnus populations was reduced with
root-knot nematode co-infection [76].
3.3. Helicotylenchus spp. (spiral nematodes)
Spiral nematodes (SN) are migratory ectoparasites.
They are also widely distributed in the world.
Several Helicotylenchus species were detected in the olive
rhizosphere such as H. digonicus [103], H. erythrinae [59],
H. dihystera [108], H. minzi [40], H. neopaxili and H. oleae [70],
H. pseudorobustus [103], H. tunisiensis [109], and H. vulgaris
[40]. Some species, such as H. digonicus, H. pseudorobustus
and H. vulgaris were also detected in nurseries [27,48]. These
species have been observed in different olive-producing
areas in the world (Table 1 and Fig. 5).
H. digonicus is dominant in many olive orchards [17,42].
For example, it occurs in more than 45% of the orchards in
Jordan [40]. Nevertheless, H. oleae is widely distributed in
Italy [70], where H. digonicus was not detected [42]. Other
studies also demonstrate the dominance of other spiral
nematodes, as Rotylenchus spp. in Italy [37,110,111].
Pathogenicity processes caused by SN are still unclear
[53]. However, some few studies indicate that H. dihystera,
H. erythrinae or H. oleae induces brown lesions on roots and
chlorosis of leaf tips, leading to root-mass and plant-growth
reduction [59,60,66,70]. Growth delay was observed on
plants infected by H. digonicus [93]. A 78% plant-growth
reduction was measured on plants inoculated with 1000
H. dihystera six months before [60]. Some species (e.g.,
H. digonicus and H. oleae) have a semi-endoparasitic
behaviour (i.e. introduction of the anterior body part into
roots) [40,70]. H. oleae penetrates up to the root epidermis
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436
and to the cortical parenchyma. It induces cell wall necroses,

feed from place to place, leaving cavities in the cortex.
3.4. Xiphinema spp. (dagger nematodes)
Dagger nematodes (DN) are ectoparasites, frequently
found in the olive rhizosphere [66]. Different species have
been recorded (Table 1 and Fig. 5): Xiphinema aequum
[112], X. barense [113], X. californicum (X. americanum)
[114], X. diversicaudatum [115], X. elongatum [60], X. index
[40], X. ingens [116], X. italiae [78], X. lusitanicum [79],
X. macroacanthum [117], X. nuragicum [17] and X.
pachtaicum [94], X. sahelense and X. vuittenezi [118], and
X. turcicum [42]. Despite the low population densities,
X. pachtaicum is the most common DN species on olive
[40,94].
X. diversicaudatum and X. pachtaicum show different
distribution areas [115]. X. diversicaudatum could be
considered as an Atlantic species, distributed in both
cultivated and uncultivated habitats, but is more abundant
in uncultivated ones with weak environmental stresses.
X. pachtaicum is rather a Mediterranean species, associated
also with other fruit trees (apple and peach). This would
explain why X. diversicaudatum has been reported on olive
in central and northern Spain [115], but not in the South
[119].
These nematodes are considered as major pathogens on
olive trees in Chile [120]. In the USA, they are responsible
for 5 to 10% of the damages, which corresponds to an
estimated $ 38.9 million loss [33]. X. pachtaicum could be a
real nuisance in olive orchards [42]. DNs affect plant
growth initiated by root necrosis [60,121].
A 65% plant-growth reduction was observed on plants
inoculated with 500 X. elongatum six months before [60].
However, significant development of X. californicum populations did not affect plant growth [114]. In fact, DNs are
usually difficult to rear in controlled conditions because of
their susceptibility to moisture and temperature variations
[122]. That could explain failures in pathogenicity experiments [53].
3.5. Tylenchulus spp. (citrus nematodes)
Usually known as citrus parasites, T. semipenetrans was
reported on olive in California, Chile and Italy [123] (Table
1 and Fig. 5). Studying several life history traits associated
with infection and reproduction on specific host plants, six
biotypes were identified: four in California [124], one in
Florida [125], and one in Italy [126]. This diversity was
revised and reduced to four biotypes [127]:
the Poncirus biotype that reproduces on Poncirus
trifoliate and grapevine but not on olive (= California
biotype C3);
the Citrus biotype that reproduces on Citrus spp.,
grapevine and olive (= California biotype C1)both
previous C2 and C4 California biotypes were included
in this biotype group, since they cannot reproduce on
P. trifoliate; but they were detected on olives in Italy;
the Mediterranean biotype is very close to the Citrus
biotype for its host range except olivethis biotype has
!!
been found in all olive-producing Mediterranean countries, in South Africa and possibly in India;
the Grass biotype was reported in Florida only, where it
reproduces only on Andropogon rhizomatus, but this
biotype corresponds to the new species T. graminis that
only parasites monocots [128].
The limits of these biotypes are rather unclear. C1 and C2
reproduce on grapevine and olive, whereas C3 affects
grapevine but not olive, and C4 reproduces only on olive
[124]. Furthermore, C1 does not reproduce on P. trifoliate and
olive [126], while other studies showed that it is the only
biotype able to reproduce on olive [127]. Moreover,
T. semipenetrans was found in olive roots in northern Italy
where citrus is not cultivated [127]. C1 has not been reported
in Spain [129] either in orchards or in nurseries [27].
Histological disturbances induced by these nematodes
in olive roots are similar to those observed on citrus [127].
However, life history traits vary from one variety to
another. C1 is more aggressive and reproduces faster on
cvs. Manzanilla and Ascolana than on other cultivars, and
much more than on Citrus sinensis, delaying plant growth
[130]. Similarly, C1 populations significantly reduced cv.
Frangivento root growth, even no reproduction was
observed one year after infection [126].
3.6. Rotylenchulus spp. (reniform nematodes)
Reniform nematodes (RN) are semi-endoparasites that
infect herbaceous and woody plants, especially in tropical
and subtropical regions. Three RN species were detected
on olive (Table 1 and Fig. 5): R. macrodoratus [131],
R. macrosoma [62,132] and R. reniformis [103,133]. These
species were registered in Algeria [104], Greece [103],
Israel [134], and Italy [95,131,135], but their pathogenicity
on olive remains poorly documented [53].
Non-mature females penetrate the roots and remain in
the epidermis on feeding sites [61,136,137]. Mature female
produce a gelatinous matrix, and lays eggs within it [62].
Distribution of R. macrodoratus is restricted to Mediterranean countries, especially to France, Greece, Italy, and
Malta. It is the most common RN species in Italy, where it
has been detected in about 20% of the samples in the South
and 22% somewhere else [38,95].
R. macrosoma is more abundant on wild olive in southern
Spain [62], but it has not been detected in nurseries in the
same region [27]. It is able to reproduce on common olive
cultivars (Picual, Arbequina). Potential damages may occur
in irrigated sandy soils for 0.51 nematode/cm3 population
levels, but infected plants show no symptoms [62].
Feeder cell disturbances are different according to RN
species: R. macrodoratus and R. reniformis induce extended
mononucleated cells [131], whereas R. macrosoma induces
syncytia [62]. High infection with R. reniformis leads to
weak tree growth [103].
3.7. Heterodera spp. (cyst nematodes)
Cyst nematodes (CN) are sedentary endoparasites.
Females lay eggs inside them which remain as a
persistence structure, and/or in a yellow gelatinous matrix.
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437
When they die, their cuticle hardens, forming a cyst that

may persist in the soil for several years in the absence of host
plant [66], and hatch when soil conditions are favourable.
CN are widespread and economically important nematodes.
Very few species infect woody plants [138]. They were not
recorded as olive parasites until H. mediterranea was
described on cultivated olive (cv Manzanilla) in Spain
[64]. More recently, H. avenae was detected in olive
nurseries on grasses associated with substrates [27].
H. mediterranea differs from other CN species by its
semi-endoparasitic behaviour which would be explained
by the woody structure of olive roots [64]. Identical
observations have been reported on pistachio tree [63].
Infected olive trees do not exhibit any symptoms on
leaves, even in heavily infested soils. However, necrosis
and root distortion were observed near root penetration
sites [64], suggesting that H. mediterranea could contribute
to plant decline in young orchards and nurseries
[64,66,139], where high densities occur [66].
infestations by RKN and LN nematodes in olive orchards

originate from unsanitized plant material produced in
uncertified nurseries [149]. Chemical disinfection of
substrates is a suitable approach for production of
nematode-free nursery stock [68]. Although laws have
been established by the European Union for healthy olive
seedlings certification [150], their application is unfortunately still limited [68]. Cultural practices such as
fertilization, weed control with herbicides, could lead to
increased populations of NPP [17]. On the other hand,
intensive production systems (high-yield varieties, highdensity plantations, permanent grassing, irrigation, fertilization, etc.) induce environments suitable for the
development of soil-borne diseases caused directly or
indirectly (e.g., verticillium wilt) by PPN [68]. These
cropping systems could lead to PPN communities dominated by the most damaging species to olive tree.
3.8. Other plant-parasitic nematodes
The use of resistant cultivars is an effective alternative

to chemical control of PPN [151], but their productivity in
olive fruits and oil can be a limiting factor to use them [53].
Some cultivars are resistant or tolerant to some species of
Meloidogyne, Pratylenchus, and Tylenchulus.
Olive varieties can be distinguished as susceptible
(nematode reproduction and plant disturbance), tolerant
(nematode reproduction without plant disturbance) and
resistant (no nematode reproduction) to RKN. Cvs. Manzanillo and Ascolano are very susceptible to M. javanica [54].
But they are resistant to M. arenaria and M. hapla [55]. In
nurseries, Arbequina, Cornicabra, Hojiblanca, Manzanilla
and Picual are the most susceptible cultivars to M. arenaria,
M. incognita, and M. javanica [27].
Olive cultivars show also various reactions towards
P. vulnus. This species does not reproduce on cv. Verdalion
[82] while cvs. Ascolana and Manzanilla are very
susceptible [55]. However, cv. Ascolana is more tolerant
than cv. Manzanilla [53]. The hostparasite interaction
would also depend on the diversity of both P. vulnus
geographical strains and host plants on which they were
detected [152].
Several olive cultivars are susceptible to X. index except
cvs. FS17 and DA12I that exhibited resistance [121].
Comparing CN reproduction traits, such as soil (juveniles and cysts) and root (juveniles) infestation levels and
reproduction rates on different olive cultivars, cv. Arbequina appeared as the most susceptible cultivar to
H. mediterranea [68].
These resistant varieties can be also used as stocks for
susceptible cultivars that have yield interests.
In that way, resistance screenings were developed, and
in vitro tests have proved to be valid alternatives to
traditional resistance screenings trials [151].
Trichodoridae nematodes are also frequently detected

on olive trees. They are polyphagous root ectoparasites and
are distributed worldwide. They cause damage to a wide
range of crops and natural vegetation by direct feeding on
roots, causing stunted roots. Some species (13%) are known
as Tobraviruses vectors, resulting in important yield losses
[140]. Three species of Paratrichodorus and 10 species of
Trichodorus were recorded on olive trees, especially in Italy
[141,142], Portugal [143], and Spain [27,42,140] (Table 1).
Nematodes belonging to the Telotylenchidae family are
not major crop parasites, although they are mostly
associated with several crops. The genus Tylenchorhynchus
seems to be the most diverse, with 13 species recorded in
different olive-producing areas, mainly in the Mediterranean Basin (Table 1 and Fig. 5).
Gracilacus peratica (family Paratylenchidae) is an ectoparasite detected on olives in Italy [36,95,144,145], Spain
[42], and Portugal [146] (Table 1). Juveniles, young females
and males were detected only in soil, while mature females
were observed in roots. Cell walls become thick and lignified
[145]. Pathogenicity processes on olive are not known [53].
G. teres was also detected on olive in Spain [147].
Ogma spp. nematodes are sedentary ectoparasites.
O. rhombosquamatum has been reported in different
orchards in Spain [42], Italy [38,148], Jordan [40], and
Portugal [146] (Table 1). Nematode infection induces cortical
cell wall thickening and cortex and epidermis necrosis.
4. Management of plant-parasitic nematodes on olive
4.1. Exclusion of PPN infection
Given that PPN are parasites living in the soil and/or
in roots, olive tree protection relies first on the use of
healthy plant material transplanted in a soil free of these
parasites. That is why the first step in avoiding PPN on olive
starts in nurseries where the best conditions for PPN
multiplication are met, and from where they could be
widely disseminated in orchards [53]. In that way, most
4.2. Plant resistance
4.3. Soil solarisation

This method was successfully used to control soil-borne
parasites: fungi [153,154], bacteria [155], and PPN [156]. It
can be an efficient method especially in the Mediterranean
Basin, where climatic conditions are optimal with high
Page 57
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438
summer temperatures. In nurseries, soil solarisation can

therefore be used for controlling RKN in substrates [157],
affecting 95% of M. incognita eggs up to a 40-cm depth after
a 10-day application. However, the gelatinous egg mass
matrix protects them against several environmental
stresses. Then, Meloidogyne spp. is sometimes heattolerant and difficult to control by soil solarisation
[158]. This method may also be useful to control other
PPN, e.g., P. vulnus and P. penetrans, and other soil-borne
pathogens as Verticillium dahliae [157].
4.4. Biological control
4.4.1. Organic residues
The addition of forest residues provides organic matter
that could contribute to PPN suppression, by increasing
soil microflora [159]. Composted dry cork, especially in
nurseries, may be effective for M. incognita suppression,
due to the toxicity of released products (e.g., ammonia,
phenolic compounds) [160].
4.4.2. Biofumigation
Some plants (e.g., Brassica oleracea, B. nigera, B. juncea,
Raphanus sativum, Crambe abyssinica, Sorghum spp.) are
able to release volatile biocides after ploughing in soil that
could have toxic effects on PPN. Such soil biofumigations
are ecological alternatives to chemical fumigation against
PPN and other soil-borne pathogens [161]. Trials to assess
the effect of Sorghum sudanense on the suppression of
M. incognita showed a significant reduction in final
nematode populations 30 to 60 days after treatment
[68]. Brassicaceae species showed suppressive effects on
M. incognita parasitizing tomato plants by reducing root
galls and nematode reproduction [162]. The association of
these plants with olive trees could be a way to reduce PPN
populations.
4.4.3. Microbial control
Soil inoculation with mycorrhizal fungi can be a useful
practice to reduce RKN populations. An early inoculation of
olive stocks cvs. Arbequina and Picual with Glomus
intraradices, G. mosseae and G. viscosum enhanced plantgrowth and controlled M. incognita race 1 and M. javanica
populations by inhibiting nematode reproduction and
reducing root galling [163]. Other microbial antagonists to
PPN (such as nematophagous fungi and Pasteuria penetrans)
can be also used for nematode suppression.
5. Prospects: better knowledge on PPN biodiversity for
better management
This review clearly shows that the diversity of the PPN
communities strongly differ across countries. Considering
olive protection, PPN control strategies appear to be
developed in each Mediterranean producing country
according to its own ecological and agronomical constraints, and the PPN diversity encountered.
As seen above, most of the researches emphasized
studies targeting some PPN species. Because resistance and
biocontrol are specific to emblematic species and because
PPN are everywhere found in communities (i.e. species
!!
mixtures), these control strategies may induce changes in

nematode communities, but do not necessarily decrease
their overall pathogenicity. Consequently, such biological
strategies may be unsustainable from an ecological point
of view [164], and they must focus on community
rearrangements instead of reducing the most pathogenic
species.
Considering olive tree history and diversity of associated PPN around the Mediterranean Basin, local PPN
communities could be originating from historical mixtures
set up with native (before olive introduction) and invasive
(with rootstocks from oleasters) communities. Olive
domestication in the past, and breeding and cropping
now could explain the present distribution of PPN.
Consequently, management strategies must take into
account the spatial distribution of the PPN diversity all
around the Mediterranean Basin (regional scale) and in
each olive-producing country (local scale). A special focus
on PPN communities associated with wild olive would give
information about trade-offs between PPN communities
and olive trees in non-disturbed situations. Moreover,
investigation about co-adaptation between PPN and olive
tree (wild and varieties) would provide new insights into
assembling mechanisms between PPN and olive trees. In
this way, a large survey with high sampling efforts is
initiated [165] in order to evaluate as precisely as possible
the diversity of PPN communities parasitizing wild and
cultivated olive, and to target the historical (evolution) and
environmental (cropping systems, olive varieties, soil
factors) filters involved in community patterns. As a
confirmation, interesting effects of olive genotypes on PPN
community patterns were observed [17]. Such observations could lead to the development of new management
strategies in a diversity-conservation approach.
Acknowledgements
This work was part of the PESTOLIVE project: Contribution of olive history for the management of soil-borne
parasites in the Mediterranean Basin (KBBE 219262),
funded by the ARIMNet action (www.arimnet.net) [166]
supported by EU and non-EU Mediterranean countries. The
authors thank Dr. G. Besnard (CNRS, Universite PaulSabatier, Toulouse, France) for his critical reading of this
manuscript.
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Page 62
Chapitre III
Mthodologie gnrale (a)

Ce chapitre dcrit la mthodologie gnrale suivie dans le cadre de ltude
de la diversit des nmatodes phytoparasites en communauts associes
lolivier. Cette mthodologie est suivie dune description et dune
prsentation
graphique
du
dispositif
dchantillonnage
et
de
la
caractrisation pdologique et climatique des sites chantillonns.

Cette mthodologie sera suivie ultrieurement par une mthodologie
spcifique ltude de la diversit des nmatodes galles Meloidogyne spp.
Page 63
Etude de la diversit des communauts des nmatodes phytoparasites associes

lolivier
Lorganigramme gnral de la mthodologie suivie dans le cadre de la thse est illustr dans
la figure suivante (Figure 1).
Figure 1: Organigramme gnral des procdures suivies au cours de la thse. A: diversit

des communauts de nmatodes phytoparasites; B: diversit du genre Meloidogyne.
Page 64
1. Sites dtude
Une prospection a t effectue sur olivier au Maroc en mars et avril 2012. Le choix du Maroc
est d des raisons cologiques puisque ce pays offre une grande varit de systmes de
culture olicoles (traditionnel en faible densit, irrigu et pluvial; haute densit irrigu, Mdail
et al., 2001; Green, 2002). Il hberge galement plusieurs sites refuge doliviers sauvages
(olastres Olea europaea subsp. sylvestris) et de la sous espce Olea europaea subsp.
maroccana endmique la rgion macaronsienne du sud-ouest marocain (Kaniewski et al.,
2012). Ces zones refuges dolivier sauvage au Maroc seront dcrites ultrieurement. La forme
frale est galement partout observe: proche de la forme sauvage, elle est issue doliviers
sauvages ou cultivs par dissmination de graines par les oiseaux ou est observe dans des
parcelles abandonnes doliviers cultivs.
Un total de 213 chantillons de sol ont t collects dans 94 sites rpartis sur un axe
Nord-Est / Sud-Ouest (environ 900km). Les sites chantillonns sont rpartis entre plusieurs
rgions gographiques (Figure 2) : dans la rgion du Rif au Nord du Maroc (33 sites) sur les
deux piedmonts Ouest (faade atlantique) et Est (faade mditerranenne) du Rif dans lesquels
la plupart des oliviers sont sauvages (13 sites) et fraux (12 sites), avec la prsence de quelques
vergers de culture traditionnelle (8 sites); dans la rgion du Jel (5 sites), situe l'Est du pays,
proche de la ville de Taza, les oliviers chantillonns sont cultivs de manire traditionnelle;
dans la rgion du Kandar (5 sites), situe dans le Nord du Moyen Atlas, au sud de la ville de
Fs, o les oliviers sont cultivs de manire traditionnelle; dans la rgion du Guerouane (13
sites de culture traditionnelle (5 sites) et haute densit (6 sites) et 2 sites fraux) et du Zaane (3
sites d'oliviers sauvages), situes au Sud de la ville de Mekns; dans la rgion du Tadla (5
sites), situe sur le piedmont nord du Moyen Atlas mridional, prs de la ville de Beni Mellal,
les arbres chantillonns taient sauvages (2 sites), fraux (1 site) ou cultivs traditionnellement
(2 sites); dans la rgion du Haouz (15 sites) situe sur le piedmont Nord du Haut Atlas, prs de
la ville de Marrakech, o les arbres chantillonns taient cultivs (culture traditionnelle et
haute densit) ou fraux; dans la rgion du Souss (15 sites) situe sur le piedmont Sud du Haut
Atlas mridional, prs de la ville d'Agadir. Les arbres chantillonns provenaient de sites
d'oliviers sauvages (9 sites y compris quelques chantillons dO. maroccana), dun site fral et
de parcelles d'oliviers cultivs traditionnellement (5 sites). Ces diffrentes rgions prsentent
une grande variabilit pdoclimatique, avec diffrents types de sols (e.g. podzols forestiers
associs des sols bruns, ferreux et humifre dans la rgion du Rif, sols argileux ou argilosableux ou calcaires dans les rgions du Haut et du Moyen Atlas), des rgimes de prcipitations
annuelles variant de 800 1000 mm dans le Nord moins de 200 mm dans le Sud, des
Page 65
!
tempratures moyennes variant de 14-21C dans le Souss, 12-24C dans le Rif, 10-28C dans
le Moyen et le Haut Atlas, et des altitudes variant de 200 m 1700 m (Ouazzani et al., 1996).
Figure 2 : Sites dchantillonnage dolivier avec indication des rgions et des modalits
chantillonnes.
2. Echantillonnages et prlvements
Considrant quune grande partie du cycle biologique de tous les nmatodes phytoparasites
est tellurique (Cadet et Thioulouse, 1998), les prlvements ont t uniquement raliss sur le
sol. Les prlvements du sol ont t raliss avec une petite pelle sous la frondaison de
lolivier, dans la rhizosphre suprieure ( 15-20 cm de profondeur), raison de 5
prlvements sous chaque arbre.
Lchantillonnage na pas t ralis de la mme faon sur olivier cultiv et sur olivier
sauvage ou fral :
-
sur olivier cultiv : considrant labondance des vergers et que les labours et dautres
activits humaines y sont frquents, ce qui conduit par consquent une homognisation des
communauts de NPP dans le verger, chaque verger a t considr comme une rptition
statistique. Les 5 prlvements de sol rcolts sous chaque arbre ont t rpts sur au moins
Page 66
quatre arbres par verger, tous les 10m environ le long dun transect (Coyne et al., 2010). Les
20 chantillons collects ont t rassembls en un chantillon de rfrence de 1-dm3 par
verger (Figure 3A). Selon la disponibilit des vergers, 2 5 vergers ont t choisis pour
chaque modalit tudie.
-
sur olivier fral ou sauvage, o les interventions humaines sont rares ou absentes, chaque
arbre a t considr comme une rptition statistique. Les 5 prlvements rcolts sous
chaque arbre ont t rassembls en un chantillon de rfrence de 1-dm3 par arbre (Figure
3B). Selon la disponibilit des arbres dans les sites fraux ou sauvages, jusqu' 25 arbres ont
t chantillonns sparment.
En parallle, 3 rameaux feuills ont t prlevs dans chacun des vergers visits ou sur les
arbres fraux et sauvages pour la caractrisation des lignes chloroplastiques des arbres.
Figure 3: Procdures dchantillonnage suivies dans des vergers dolivier cultiv (A) et dans
des sites dolivier fral ou sauvage (B).
3. Traitements des chantillons
3.1. Extraction des nmatodes
Tous les chantillons de sol ont t expdis temprature ambiante par transport express au
Laboratoire de Nmatologie de lUMR CBGP "Centre de Biologie pour la Gestion des
Page 67
Populations" Montferrier sur Lez (laboratoire agr Quarantaine Vgtale par la Prfecture
de lHrault n80622).
Les nmatodes ont t extraits par la technique dlutriation du sol dveloppe par
Seinhorst, (1962) (Figure 4, annexe 1). Elle est fonde sur le principe de la sdimentation des
particules dans une colonne deau. Cette technique est divise en trois tapes : une premire
tape permet, en appliquant un courant deau ascendant de faible dbit (60cm3/min) dans la
colonne, de sparer les particules grossires du sol des particules fines dont les nmatodes.
Les nmatodes sont rcuprs donc en suspension dans leau. Une deuxime tape
(concentration) consiste rduire le volume de la suspension en rcuprant les refus dune
batterie de quatre tamis (50m de mailles). Une troisime tape (purification) consiste
liminer les impurets de la suspension par passage actif des nmatodes travers un filtre de
cellulose. Cette mthode entrane uniquement lextraction des formes mobiles de nmatodes).
Cette mthode est applique une aliquote de 250 cm3 de chaque chantillon de sol.
Figure 4: Dispositif de Seinhorst (1962) dlutriation du sol pour lextraction des nmatodes.
3.2. Caractrisation morphologique des nmatodes au rang de la famille, du genre et de

lespce
Les nmatodes phytoparasites (NPP), caractriss par la prsence dun stylet au niveau des
pices buccales, ont t identifis et dnombrs aux rangs de la famille et du genre, dans une
Page 68
cellule de comptage de 5-cm3 (Merny & Luc, 1969) sous un stromicroscope (grossissement
60x), selon les cls dichotomiques de Mai & Mullin (1996) et les nomenclatures Systema
Naturae (2000) et Siddiqi (2000). Les nmatodes non phytoparasites ont t galement
numrs mais sans distinction taxonomique. Les effectifs de nmatodes ont t exprims en
nombre de nmatodes /dm3 de sol frais.
Ensuite, pour lidentification morphologique des NPP au rang de lespce, les suspensions de
nmatodes ont t fixes et conserves dans du glycrol (De Grisse, 1969 ; cf. protocole
annexe 2) et des spcimens ont t monts sur lames (Cobb, 1917). Les identifications
spcifiques ont t ralises par Dr. E. Dmowska (CBE-PAN, Lomianki, Pologne).
Lidentification spcifique de certains taxa comme les nmatodes galles Meloidogyne spp. a
t ralise laide de marqueurs biochimiques et molculaires (dtails au chapitre III-B).
3.3. Caractrisation gntique des oliviers
Elle a t ralise par caractrisation des lignes chloroplastiques. Les chloroplastes tant
hrits de la mre, la variation de leur ADN permet une identification des lignes maternelles
(Besnard et al., 2000). L'ADN a t extrait avec un procd CTAB (Cetyltrimethyl
ammonium bromide) partir des feuilles fraches dessches en gel de silice (Besnard et al.,
2000). Une mthode de profilage du gnome de plaste base sur des microsatellites, des indels
(insertion, dltion) et des polymorphismes nuclotidiques simples (SNP) ont t utiliss pour
caractriser chaque chantillon (Besnard et al., 2011). Les caractrisations chloroplastiques
des oliviers ont t ralises et valides par Dr. B. Khadari (UMR Amlioration Gntique et
Adaptation des Plantes, Montpellier) et Dr. G. Besnard (UMR Evolution et Diversit
Biologique, Toulouse). Ces donnes ont t utilises pour confirmer la distinction entre
olivier fral et sauvage.
3.4. Analyses physico-chimiques du sol
Des chantillons de 200 g de sols collects lchelle du verger (olivier cultivs) ou du site
(oliviers fraux et sauvages) ont t schs et envoys lINRA dArras afin danalyser les
proprits physico-chimiques de ces sols. Les analyses standardises selon Anderson &
Ingram (1993) ont port sur :
!
La granulomtrie : proportions en argiles (Cl = clay, 0.2-2m), en limons fins (FSi =

fine silts 2-20m) et grossiers (CSi = coarse silts, 20-50m), et en sables fins (FSa =
fine sands 50-200m) et grossiers (Csa = coarse sands, 200-2000m) selon la technique
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!
Textol (NF X31-107, 2003). Elle a permis de dfinir les types de sols selon le triangle
des textures de Jamagne (1967) (Figure 5).
!
La teneur en azote N (ISO 13878, 1998) et en carbone organique C (ISO 10694, 1995)
par chromatographie gazeuse.
La teneur en matire organique (OM) est ensuite
calcule selon OM = 1.724 x C.

!
La teneur en phosphore assimilable P2O5 par la mthode dOlsen (ISO 11263, 1995).
Les teneurs en lments solubles : calcium (Ca), magnsium (Mg), potassium (K),
sodium (Na), fer (Fe), manganse (Mn), cuivre (Cu) et zinc (Zn) par dissolution fluoronitro-perchlorique (ISO 14869-1, 2001).
! Lacidit (pHH2O) (ISO 10390, 2005).

! La capacit dchange cationique (CEC) mesure par la conductivit (mS/cm ; ISO
11265, 1995).
Figure 5: Triangle des textures de sol qui dcrit le type du sol selon les proportions dargiles,
de limons et de sables Jamagne (1967).
3.5. Caractristiques climatiques

Les isothermes annuels moyens maximaux (Tmax) et minimaux (Tmin) ainsi que les
isohytes (pluviomtrie) annuels (annual rainfall = AR) ont t valus partir des donnes
recueillies sur lanne 2012 dans les stations climatiques les plus proches des sites
chantillonns par Dr. M. Ater (Facult des Sciences, Universit Abdelmalek Essaadi,
Ttouan, Maroc).
Page 70
4. Typologie du dispositif dchantillonnage au Maroc

Comme
dj
mentionn
dans
la
mthodologie
gnrale,
diffrentes
variables
environnementales ont t prises en considration afin d'valuer leur impact sur la diversit
des communauts de NPP associes lolivier au Maroc, ainsi que sur la distribution des
espces de nmatodes galles du genre Meloidogyne spp. Ces variables se rpartissent en
trois groupes :
- Les variables lies la plante hte telles que :
!
les conditions de vie de lolivier : sauvage, fral, cultiv (traditionnel ou haute densit).
sa diversit gntique dfinie par les lignes chloroplastiques E1, E2, E3 et M.
- Les paramtres physico-chimiques du sol : texture, matire organique, acidit, minralogie

de la solution du sol, types de sols.
- Les variables climatiques : isohytes et isothermes maximaux et minimaux.
4.1. Cartographie des variables dans les sites chantillonns au Maroc
La distribution des chantillons par variable indique a priori une rpartition trs htrogne de
lchantillonnage pour chacune des principales variables mesures, except celle lie la
temprature minimale et la pluviomtrie (Tableau 1). En fait, cela est directement li aux deux
modes dchantillonnages distincts dans les sites cultivs et dans les sites sauvages ou fraux
(cf. 3.2.). Cette htrognit est galement lie la raret de certaines modalits (e.g.
ligne chloroplastique M pour O. e. maroccana).
Tableau 1: Nombre dchantillons par modalit pour chacune des principales variables
environnementales caractristiques des sites dchantillonnage au Maroc :
Modalit de culture: HD = olivier cultiv haute densit; TR = olivier cultiv traditionnel; FO
= olivier fral; WO = olivier sauvage
Lignes chloroplastiques: E1, E2, E3 = lignes O. e. ssp. e. var. europaea et O. e. ssp. e. var.
sylvestris ; M = O. e. ssp. maroccana
Tmax= isothermes maximaux moyens annuels rpartis en trois classes: Tmax1= (21 -
23)C; Tmax2= (> 23- 25)C; Tmax3= (> 25- 27)C
Tmin= isothermes minimaux moyens annuels rpartis en trois classes: Tmin1= (7.5 -
10)C; Tmin2= (> 10 - 12.5)C; Tmin3= (>12.5- 15)C
AR = pluviomtrie annuelle rpartie en trois classes: AR1= (200 - 400)mm; AR2= (>400 700)mm; AR3= (>700 - 900)mm
Page 71
4.1.1. Systmes olicoles

Les foyers doliviers sauvages chantillonns sont surtout localiss dans les rgions
montagneuses du Rif au Nord du Maroc et du Haut-Atlas mridional au Sud (Figure 6)
Dautres foyers ont t chantillonns dans le Plateau Central dans les rgions du Zaane et du
Tadla. Les sites doliviers cultivs en haute densit dominent dans les rgions du Haouz au
Sud et du Guerouane au Centre, alors que les sites doliviers traditionnels et fraux se
distribuent dans toutes les zones olicoles du Maroc. Donc, excepts lolivier cultiv
traditionnellement et lolivier fral, que lon peut trouver aussi bien dans des agrosystmes
quen milieu naturel, le dispositif dchantillonnage rvle une forte partition des systmes
naturels olivier sauvage et des systmes cultivs en haute densit.
4.1.2. Diversit chloroplastiques gntique de lolivier
La combinaison des polymorphismes chloroplastiques dtects a permis de dfinir 17
haplotypes appartenant aux quatre lignes chloroplastiques de lolivier mditerranen (selon
Besnard et al., 2007; 2013). Dans le dispositif dchantillonnage, elles se distribuent de la
manire suivante (Figure 7):
-
La ligne E1 (un seul haplotype) a t dtecte dans 97 arbres de tous les oliviers
cultivs, et de certaines populations dolivier fral. Elle est donc prsente dans toute la zone
olicole marocaine.
-
La ligne E2 (6 haplotypes) a t trouve dans 66 arbres de sites fraux et sauvages,
rpartis dans la rgion du Rif, du Guerouane, du Tadla et du Souss.

-
La ligne E3 (7 haplotypes) a t dtecte dans 45 arbres de sites sauvages et fraux,
dans la rgion du Rif, du Zaane et du Souss.

-
La ligne M spcifique O. e. maroccana (3 haplotypes) na t rpertorie que dans
5 chantillons seulement, tous situs dans le haut-Atlas mridional.

Ces donnes chloroplastiques ont permis de confirmer la qualification des modalits de
lolivier, en particulier de distinguer gntiquement les oliviers sauvages des oliviers fraux.
Elles ont galement permis de dfinir des zones biogographiques doliviers sauvages
chantillonns au Maroc. Les zones refuges sont situes dans le Rif (faades occidentale
ocanique et orientale mditerranenne), le Moyen Atlas et le Haut Atlas. Ces zones ont t
dfinies sur la base des connaissances des refuges glaciaires (Mdail & Diadema, 2009), et
sur les modles de diversit gntique de l'olivier au niveau du Bassin Mditerranen
(Besnard et al., 2013).
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!
4.1.3. Types de sols chantillonns

Les rgions du Rif et du Kandar, et plus rarement des sites situs dans les rgions du
Guerouane, du Haouz et du Souss, sont caractrises par des sols argileux (Figure 8). Les sols
limoneux se rencontrent plus frquemment dans les rgions du Haouz, du Souss et du Jel, et
dans quelques sites dans les rgions du Rif, du Guerouane et du Tadla. Enfin, les sites
caractriss par des sols sableux sont plus rares, et dissmins dans les rgions du Guerouane,
du Zaane, du Tadla et du Souss.
4.1.4. Variables climatiques (Figure 9)
Tempratures maximales (moyenne annuelle) : les rgions montagneuses du Rif et du Souss
sont sous linfluence de tempratures maximales basses (21 23C), alors que les
tempratures les plus leves (25 27C) ont t enregistres dans les plaines du Haouz et du
Tadla. Les tempratures maximales moyennes (23 25C) ont t enregistres dans le Plateau
Central du Maroc (rgions du Zaane, du Guerouane, et du Kandar), et galement dans le Rif
occidental.
Tempratures minimales (moyenne annuelle) : les tempratures minimales les plus basses (7.5
10C) ont t enregistres dans le Plateau Central du Maroc. Les rgions montagneuses du
Rif et du Souss, et quelques sites du Haouz, sont sous linfluence des tempratures minimales
les plus leves (12.5 15C). Les tempratures minimales intermdiaires (10 12C) ont t
enregistres dans les rgions du Haouz, du Tadla, et du Rif, et dans quelques sites localiss
dans les rgions du Souss, du Guerouane et du Jel.
Pluviomtrie (cumul annuel) : les isohytes sont globalement rpartis selon un gradient
latitudinaire du Nord (900mm) au Sud (200mm). Les isohytes levs sont renforcs par
laltitude (Rif et quelques sites dans le Tadla). Une forte aridit est galement enregistre dans
le Maroc oriental (rgions du Kandar et du Jel).
4.2. Structure des donnes environnementales
Une analyse Hill & Smith (HS) a t applique lensemble des jeux de donnes afin
den analyser la structure et slectionner les variables les plus structurantes.
Le plan factoriel indique que les deux premiers axes de lanalyse expliquent 64.4%
(valeurs propres) de la variance (Figure 10). Selon le premier axe HS1, les sols sableux (SA)
sopposent aux sols limoneux (SI) et argileux (CL). Les sites chantillonns sont donc trs
structurs entre des sols lgers (sableux) dune part et des sols lourds (argileux) dautre part.
Les tempratures maximales (Tmax) et minimales (Tmin) sont galement trs structurantes,
mais elles divergent sur le second axe HS2, indiquant une opposition des extrmes
Page 73
!
thermiques, et diffrencient les sites chantillonns dans des rgions continentales, des
rgions sous influence maritime, et des rgions montagneuses. L'axe HS2 est surtout expliqu
par les prcipitations annuelles (AR), ce qui sinterprte en grande partie par lamplitude des
isohytes qui caractrisent les sites (de 200mm au Sud 900mm au Nord) et par leur
distribution latitudinaire. Enfin, les systmes olicoles (SYS) et la diversit chloroplastique de
lolivier (Lineage) distinguent les donnes factorielles sur les deux axes.
Par consquent, on en dduit que toutes ces variables, sans exception, ont un poids
important dans la structure des jeux de donnes environnementales enregistres.
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!
Figure 6: Distribution des sites dchantillonnage et des modalits des diffrents systmes olicoles rencontrs (A) et les lignes
chloroplastiques (B) au Maroc.
Page 75
!
Figure 7 : Distribution des sites dchantillonnage des oliviers sauvages et fraux et les haplotypes chloroplastiques dtects (les oliviers cultivs
ne sont pas indiqus sur la figure parce quils ont quun seul haplotypes E1-1).
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!
Figure 8: Types de sols dans les sites chantillonns au Maroc.
Page 77
!
Figure 9: Distribution des isothermes maximaux moyens annuels Tmax (A), des isothermes minimaux moyens annuels Tmin (B) et des
isohytes annuels AR (C).
Page 78
!
Figure 10: Plan factoriel Hill and Smith des principales variables environnementales
caractristiques des sites dchantillonnage
(SYS = modalit de lolivier; Ligne = ligne chloroplastique de lolivier; AR =
isohytes annuels ; Tmax = isothermes maximaux moyens annuels ; Tmin = isothermes
minimaux moyens annuels ; SA = sol sableux; SI = sol limoneux; CL = sol argileux). Les
valeurs indiques sur les histogrammes correspondent la fraction de la valeur propre
conserve par les deux premiers axes de lanalyse Hill and Smith.
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!
5. Analyse de la diversit des nmatodes phytoparasites

La diversit des nmatodes phytoparasites en communauts a t dcrite avec plusieurs indices
de diversit :
5.1. Diversit taxonomique
Des indices de diversit taxonomique prennent en compte le nombre dindividus et ceux des
espces dans un cosystme. Ils indiquent la distribution de ces individus entre espces. Les
indices utiliss sont :
Le nombre total de nmatodes (N) : il sagit du nombre total dindividus dans une
communaut.
Les indices de richesse : il sagit du nombre total de genres (richesse gnrique G) et
despces (richesse spcifique S) dans une communaut. Toutefois, ces indicateurs
dpendent du nombre dchantillons (sampling effort) et ne prennent pas en compte
l'abondance relative des diffrents genres ou espces.
La diversit locale (indice de Shannon H) : il permet de quantifier lhtrognit de la
diversit. Il est calcul partir de la formule suivante :!
avec S = richesse spcifique ou G = richesse gnrique
ni = nombre d'individus pour l'espce i
N = nombre total dindividus dans la communaut
H varie de 0 ln(S).
Lquitabilit (E) ou indice de Simpson : cet indice value la rgularit de la distribution des
espces (evenness en anglais), lment important de la diversit. Une espce reprsente
abondamment ou par un seul individu napporte pas la mme contribution la communaut.
Pour un mme nombre despces (mme richesse spcifique entre deux communauts ou
deux cosystmes), la prsence despces trs dominantes entrane mathmatiquement la
raret de certaines autres; on comprend donc assez intuitivement que le maximum de
diversit est atteint lorsque les espces ont une rpartition rgulire. Cet indice est calcul
par la formule suivante : E= H/lnS.
Il varie entre 0 et 1. Plus lquitabilit est proche de 1, plus les espces sont qui-rparties,
cest--dire quelles prsentent un nombre dindividus quivalent et quaucune delles nest
dominante.
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!
Lintrt des deux derniers indices (Shannon et Simpson) est quils prennent en compte la
fois le nombre despces (richesse spcifique) et labondance relative des espces.
5.2. Dominance des taxons dans les chantillons

Cet indice est une reprsentation de la rgression entre labondance et la frquence de
chaque genre ou espce dans les chantillons (Fortuner & Merny, 1973).
Labondance (A) : nombre moyen dindividus dun genre (ou dune espce) dans tous les
chantillons dans lesquels il a t trouv.
La frquence (F) : proportion dchantillons dans lesquels le genre (ou lespce) a t
trouv.
La dominance indique la capacit dun organisme survivre et se reproduire dans un
milieu donn.
5.3. Diversit fonctionnelle

Labondance relative des classes fonctionnelles (Rcpi) : un indice de diversit fonctionnelle
(cp-value) est attribue aux familles de nmatodes du sol (Bongers, 1990). La cp-value
correspond des stratgies adaptatives varies, allant de comportements colonisateurs
(stratgie r) des comportements persistants (stratgie K). Elle varie de 1 (espces
opportunistes, colonisatrices, peu sensibles au stress, cycles de vie courts de quelques jours
quelques semaines, produisent de nombreux petits ufs) 5 (espces sensibles aux
perturbations, persistantes, cycles de vie longs de plusieurs mois, produisent peu de gros
ufs). Les nmatodes phytoparasites occupent les cp-value 2 5. Rcpi reprsente
labondance relative de chacune des cpi dans une communaut. Il est calcul par : Rcpi =
nicpi/N.
Le Plant Parasitic Index (PPI) : cet indice value la cpi moyenne dune communaut et
se calcule par :
La diversit trophique de la communaut : les nmatodes phytoparasites sont classs en
diffrents groupes trophiques selon Wasilewska (2006) : phytoparasites stricts (OPF pour
Obligate Plant Feeders en anglais) ; phytoparasites facultatifs (FPF= Facultative Plant
Feeders, nmatodes principalement phytoparasites et accessoirement fongivores) ;
Fongivores (F=Fungivorous, nmatodes principalement fongivores et accessoirement
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!
phytoparasites). La diversit trophique de la communaut est alors value par l'abondance

relative moyenne (%) des individus dans chaque groupe trophique (Neher et al., 2004).
5.4. Patrons des communauts

Nous avons construit des patrons statistiques des communauts laide de diffrentes
mthodes danalyses multivaries dans le but de rechercher des correspondances avec les
facteurs environnementaux du milieu.
6. Analyses statistiques
6.1. Jeux de donnes
Diffrents jeux de donnes ont t construits : nmatodes (effectifs des genres et des
espces, indices de diversit taxonomique, fonctionnelle et trophique) ; olivier (mode de
conduite ou modalit, irrigation, lignes chloroplastiques) ; sol (type de sols et paramtres
physico-chimiques), climat (temprature et pluviomtrie).
6.2. Analyses
Sur les jeux de donnes indiqus prcdemment, diffrentes mthodes danalyses
multivaries ont t utilises dans un premier temps pour dcrire la structure des jeux de
donnes. Ces analyses ont galement permis dtudier la correspondance entre des indices
de diversit ou des effectifs et les variables environnementales. Des analyses infrentielles
(ANOVA, Wilcoxon, MLG, etc.) ont t galement utilises. Les mthodes statistiques sont
dtailles dans chacun des chapitres du manuscrit.
Le tableau 2 montre les mthodes et tests statistiques utiliss. Le choix des tests statistiques
et des analyses multivaries dpend de lobjectif de lanalyse et du type de variable
explique (quantitatives, qualitatives), la distribution des variables explicatives (normales ou
pas), le nombre des variables explicatives intgres.
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!
Tableau 2 : Tests et mthodes statistiques utilises pour les analyses des donnes.
Modles
Mthodes
Comparaisons de moyennes
Test t, ANOVA, Wilcoxon, Kruskal Wallis
Modles Linaires Gnraliss

Corrlations multiples
Correspondances
ACP, AFC, AFCM, ACM, Hill and Smith

Co-Inertie
ANOVA
ACP
AFC
AFCM
ACM
Analyses de variance (sans s je crois)

Analyse en Composantes Principales
Analyse Factorielle (sans s) des Correspondances
Analyse Factorielle (sans s) des Correspondances Multiples
Analyse des Correspondances Multiples
7. Rfrences
Anderson J.M., Ingram J.S.I., 1993. Tropical Soil Biology and Fertility: A Handbook of Methods, 2nd
Edition. CABI. Wallingford, UK.
Besnard, G., Bervill, A., 2000. Multiple origins for Mediterranean olive (Olea europaea L. ssp.
europaea) based upon mitochondrial DNA polymorphisms. Comptes Rendus de l'Acadmie des
Sciences-Series III-Sciences de la Vie, 323: 173-181.
historical processes of isolation and reticulation in the olive tree complex (Olea europaea L.).
Besnard, G., Hernndez, P., Khadari, B., Dorado, G., Savolainen, V., 2011. Genomic profiling of plastid
DNA variation in the Mediterranean olive tree. BMC Plant Biology, 11: 80.
Besnard, G., Khadari, B., Navascues, M., Fernandez-Mazuecos, M., El Bakkali, A., et al., 2013. The
complex history of the olive tree: from Late Quaternary diversification of Mediterranean lineages
to primary domestication in the northern Levant. Proceedings of the Royal Society of London.
Series B, Containing papers of a Biological character. Royal Society, 280: 20122833.
Bongers, T., Bongers, M., 1998. Functional diversity of nematodes. Applied Soil Ecology, 10: 239-251.
Cadet, P., Thioulouse, J., 1998. Identification of soil factors that relate to plant parasitic nematode
communities on tomato and yam in the French West Indies. Applied Soil Ecology, 8: 35-49.
Cobb, N.A 1917. Notes on nemas. Intra vitam color reactions in nemas. Contributions to a science of
Nematology 5: 120-124.
Coyne, D.L., Nicol, J.M., Claudius-Cole, B., 2010. Les nmatodes des plantes: Un guide pratique des
techniques de terrain et de laboratoire. 82 pp.
De Grisse, A.T., 1969. Redescription ou modifications de quelques techniques utilises dans ltude des
nmatodes phytoparasites. Meded. Rijksfakulteit Landbouwwetenschappen Gent,, 34: 351-369
Fortuner, R., Merny, G., 1973. Les nmatodes parasites des racines associs au riz en Basse-Casamance
(Sngal) et en Gambie. Cahiers ORSTOM, Srie Biologie, 21: 3-20.
Green, P.S., 2002. A revision of Olea L. (Oleaceae). Kew Bulletin, 91-140.
ISO 10390, 2005. Soil quality - Determination of pH. AFNOR, Paris.
ISO 10694, 1995. Soil quality - Determination of organic and total carbon after dry combustion
(elementary analysis), AFNOR, Paris.
ISO 11263, 1995. Soil quality - Determination of phosphorus - Spectrometric determination of
phosphorus soluble in sodium hydrogen carbonate solution, AFNOR, Paris.
ISO 11265, 1995. Soil quality - Determination of the specific electrical conductivity, AFNOR, Paris.
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ISO 13878, 1998. Soil quality - Determination of total nitrogen content by dry combustion (elemental
analysis), AFNOR, Paris.
ISO 14869-1, 2001. Soil quality - Dissolution for the determination of total element content - Part 1:
Dissolution with hydrofluoric and perchloric acids, AFNOR, Paris.
Jamagne, M., 1967. Bases et techniques d'une cartographie des sols. In Annales agronomiques (Vol.
18). Paris: INRA. 142 pp.
Kaniewski, D., Van Campo, E., Boiy, T., Terral, J.F., Khadari, B., Besnard, G., 2012. Primary
domestication and early uses of the emblematic olive tree: palaeobotanical, historical and
molecular evidence from the Middle East. Biological Reviews, 87: 885-899.
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significance of Olea europaea L. ssp. maroccana (Greuter & Burdet) P. Vargas et al., a relictual
olive tree in south!west Morocco. Botanical Journal of the Linnean Society, 137: 249-266.
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!
Chapitre IV
Diversit taxonomique des

nmatodes phytoparasites en
communauts
Page 85
Rsum
Ltude prsente dans ce chapitre avait pour but de dcrire la diversit des communauts de
nmatodes phytoparasites (NPP) associes lolivier au Maroc o cette diversit tait
totalement inconnue. Elle avait galement comme objectifs de dcrire cette biodiversit dans
des zones dfinies comme des zones de refuge pour lolivier sauvage; et de dterminer
limpact potentiel des activits humaines (la domestication et lintensification de la culture de
lolivier) ainsi que des variables daphiques et climatiques dans la structuration de la diversit
en communauts. Les prospections ralises ont rvl une grande diversit taxonomique de
NPP appartenant 48 genres et 122 espces. Les rsultats ont galement indiqu la
dtection de 60 espces dcrites pour la premire fois en association avec lolivier au niveau
mondial. La biodiversit des communauts a t dcrite par des indices de biodiversit: le
nombre total des individus, la richesse spcifique, la diversit locale et lquitabilit. Un des
rsultats majeurs de ce travail est lopposition entre labondance en NPP et les autres indices
de biodiversit. Les analyses ralises dans les zones refuges de lolivier sauvage ont indiqu
que les communauts associes sont similaires en diversit, mais que la composition
spcifique des communauts est compltement distincte entre certains refuges surtout entre
les deux cts du Rif. La diversit des nmatodes en communauts est fortement affecte par
la domestication de lolivier (sauvage vs cultiv) avec des communauts riches en espces
dans les milieux non cultivs (sauvage et fral), et une qui-rpartition des espces au sein des
communauts, contrairement ce qui a t observ dans les milieux cultivs (plus faible
richesse spcifique et dominance de certains taxons). Cependant, lintensification de la culture
(faible vs haute densit) ne semble pas avoir deffet. Les analyses des variables daphiques et
climatiques ont rvl un impact significatif du type de sol et de son contenu en matire
organique, ainsi que des tempratures minimales. Ltude compare la diversit dtecte en
NPP celle observe lors dautres prospections et tudes ralises sur lolivier. Elle discute
ensuite la persistance des communauts de NPP dans les refuges d'olivier sauvage, et attire
l'attention sur l'impact des pratiques culturales et les interventions humaines dans les milieux
sur la multiplication, la composition et la rpartition des espces dans les communauts.
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How species diversity in biological communities is structured in response to

anthropogenic changes and environmental conditions? The plant-parasitic
nematode communities associated with olive trees as a case study
Abstract
The plant-parasitic nematodes (PPN) contribute to economic losses in cropping systems.
Their communities diversity and structure may vary in response to environmental and
anthropogenic forces. The olive tree (Olea europaea) is a good host model to understand the
impact of such potential forces on PPN diversity because it grows according to different
environmental modalities (as wild, feral, or cultivated). In Morocco and southern Spain, wild
olives persist in long-term refugia and grow in various soil and climate conditions.
A wide soil survey was conducted in several olive growing areas in Morocco and southern
Spain. Putative refugia areas of wild olive were defined. A high specific diversity of 122
species of PPN was encountered among which 60 were recorded for the first time on the olive
tree. The PPN diversity was described within the putative refugia areas and according to some
agronomic, soil and climate factors. The first result of this work is the systematic opposition
of the richness, the local diversity and the evenness of species in communities to the total
number of individuals they hosted. Rich PPN communities were observed in wild olive areas.
Although PPN communities associated to putative refugia areas share similar species
diversity, their composition of species was very distinct. The anthropogenic changes were
revealed as main factors impacting the PPN diversity because a low specific richness in the
favour of the multiplication of some nematode taxa was recorded in cultivated conditions.
This study provides the first analysis of PPN diversity within putative refugia areas of wild
olive. Isolation of these regions promoted divergence between their PPN communities. The
study proposes further research focusing on the composition of PPN communities and of
others communities of soil biota in order to determinate potential trade-off interactions that
could be used for biological control of these parasites.
Key-words: Anthropogenic changes, Biodiversity, Olive tree, Plant parasitic nematode

community, Refugia.
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1. Introduction
A biological community refers to an assemblage of populations from different organisms
found living together in a habitat. This biological assemblage within a community could be
describe by several traits such as the number of species (richness), their relative abundance
(evenness), the kind of present species (composition), the interactions among them as well as
their temporal and spatial variation [1]. This species diversity is important for the stability of
the community and consequently that of the ecosystems [2]. Functional consequences on
ecosystem processes were revealed related to species diversity because the number and kinds
of present species determine organismal traits influencing these processes. In addition to its
effects on current functioning of ecosystems, species diversity can play a crucial role in the
resilience and resistance of ecosystems to human disturbances and to environmental change
[1].
In the soil, various communities of living organisms such as plant roots, bacteria, fungi,
protozoa, invertebrates and vertebrates are hosted [3]. These soil biotas were also documented
as important factors for essential services to ecosystems and to human society [4]. However,
the activity and diversity of soil organisms are regulated by a hierarchy of abiotic and biotic
factors. Within its community, every organism sits in such a mixture of living (include all
forms of life with which it interacts) and nonliving (temperature, water, nutrients, topography,
salinity, pH, etc.) elements [4-6]. Considering the abiotic factors only, they may represent
new challenges for many organisms, which consequently would determine the above and
below ground diversity of plants, animals and microbes communities [7,8]. Any change in
these factors could be thus accompanied by several soil biotic variations (e.g. reproduction
rates, sex ratios, migration, occupation of micro-habitats, use of resources, etc.) [9]. For
instance, invertebrate communities could change in response to land-change uses [10]; to
plant species or to soil factors that were demonstrated impacting rhyzospheric microbial
communities [11]. Agricultural practices (e.g. crop intensification), revealed as important
sources of soil disturbances, could leading to impact soil biotas [12].
Within the soil, nematodes, microscopic round worms, are ubiquitous and numerous
members of this biosphere. They count about 100 to 1000 individuals / per gram of soil [13].
Soil nematodes are believed to have profound effects on soil processes such as decomposition
of organic matter and nutrient recycling [14-16]. They consequently have an important role in
fertility and productivity, and to measure functional status of the soil [17-19]. The nematodes
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have been developed several life strategies (r-colonizer to K-persister strategies, 20]. These
various strategies could explain their ability to survive as well as to rapidly response to
disturbances in their environment better than others soil organisms. In this context, soil
nematodes are usually affected by human disturbances and by environmental changes. As
example, any perturbation that affects plant species composition or physiology, soil texture,
chemistry, or climatic factors (soil moisture and temperature) may alter the specific diversity
of nematode and the composition of their functional groups [21,22]. Soil nematodes also
reflect several feeding behaviors that allow allocating them into different trophic groups
(bacterivores, fungivores, carnivores, and plant feeders [23]). Considering the plant-parasitic
nematodes (PPN), the diversity and structure of their communities, as for the others groups of
soil nematodes, are subject to the influence of biotic and abiotic factors. Because PPN are
usually obligate plant-parasites, and as they spend all (ectoparasites nematodes) or almost
(endo- and semi-endoparasites nematodes) their life cycle in the soil, their diversity and
structure in communities are obviously affected by soil parameters [24], or by cultural
practices and human activities [25]. The vegetal composition in a parcel was demonstrated as
a major determinant of community diversity [26] because nematode abundance, diversity and
trophic structure in nematode community can vary either in function of presence or absence of
a plant [27], or according to plant diversity [28] or to in response to land-change use [29].
Little is known about the effect of genetic variation of the host plant on nematode
communities [30], but, the infection by PPN initiates complex changes in plant gene
expression [31].
The PPN can devastate a wide range of crop plants, causing annual crop losses
estimated at multibillion dollars on a worldwide scale [32] to many types of crops as cereals,
vegetables, tubers, fruit and floral crops [33]. On the olive tree (Olea europaea L.), PPN are
able to reduce tree growth [34] and may be responsible for 5 to 10% yield losses [35]. Their
impact is strengthened in nurseries and in intensive cultivation systems (high-density
cultivation) where irrigation conditions favour the development of roots and therefore
nematode multiplication [36,37].
In the Mediterranean Basin, the olive tree is a good example of ecological, botanical
and genetic diversity. Different botanical entities of olive have been described in this region
[38,39]. Spontaneous trees are distinguished under three different forms: (i) Firstly,
autochthonous wild trees that are usually named oleasters [O. e. subsp. europaea var.
sylvestris (Mill.) Lehr.] are common in coastal and mountainous regions (frequently present in
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southern Spain, and the foothills of Moroccan mountains) [40]); (ii) secondly, an hexaploid
subspecies, the Moroccan olive [O. e subsp. maroccana (Greut. & Burd.) P. Vargas et al.], is
endemic to the High Atlas Mountains [38,41]; (iii) and lastly, feral forms are wild-looking
olives that correspond either to cultivated olive abandoned and becoming wild or to olive
issued from cultivated olive seeds spread by birds. Additionally, cultivated forms (O. e. subsp.
e. var. europaea) are also widespread. The cultivated olive is culturally and economically
very significant within the Mediterranean basin and remains integral to the economy of this
region [42]. Different olive cropping systems can be distinguished according to tree density
[43]: traditional orchards (ca. 80-400 trees/ha) vs high density orchards (that may reach a
density up to 1800 trees/ha). The olive genetic diversity has been investigated in order to
distinguish wild and cultivated forms. The polymorphism of the chloroplast genome revealed
four Mediterranean lineages [44]: E1 is distributed from the Aegean area to the Levant and
has been spread over the whole Mediterranean Basin, and most of the modern varieties and
feral forms harbour an haplotype of this lineage (namely E1.1); E2 and E3 that show a hotspot
of diversity on the Gibraltar Strait are currently distributed in the western and central
Mediterranean regions, and a few cultivars show haplotypes of these lineages; M is specific to
the Macaronesian area (including High Atlas; subsp. maroccana). Several Last Glacial
Maximum (LGM) refugia areas have been described in Morocco and Southern Spain [45].
Genetic patterns and species distribution modelling suggest major long-term refugia for the
wild olive in westernmost Mediterranean regions in particular on both sides of the Gibraltar
Strait and in the Atlas Mountains [40]. Within these areas, a clear influence of Pleistocene
climatic cycles on patterns of species richness has been shown [46].
According to the above, and based on its distribution in distinct geographic areas and its
persistence as wild forms in several putative refugia areas around the Mediterranean Basin, on
its distinction in different growing modalities and its growth according to wide range of
edaphic and climatic conditions, the olive tree reveals as a good model of host plant for PPN
to prove the hypothesis that the diversity and the structure of these nematodes in communities
could be changed in response to these different ecological factors. Based on the distribution of
wild olive in putative glacial refugia in Morocco and southern Spain, we hypothesize that
these isolated regions (presenting high barriers for organisms dispersion) could host PPN
communities different in their diversity as a potential consequence of limited exchanges
and/or specific evolutionary pressures in the distinct refugia. Considering the olive
domestication, it is generally performed from rooted cuttings that could be transplanted in
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nematodes-contaminated soil. The PPN could spread by soil transport or from un-sanitized
plant material (from uncertified nurseries as example). The local PPN populations in olive
cropping areas could be therefore originating from historical mixtures set up with native
(before olive introduction) and invasive (with root-stocks from oleasters) communities. In this
context, we hypothesize that PPN communities may have adapt to olive domestication
processes and to human activities. These anthropogenic forces could be evident in Morocco
where programmed extensions of the cultivated areas with high-density practices and where
ancestral or traditional cultivars have been often discarded in favor of few highly productive
varieties [47]. These new conditions of cultivation might have to face a resurgence of several
pests, including PPN. To give first answers to these hypotheses, this study was undertaken in
order to (i) describe the species diversity of PPN communities in Morocco in particular where
this diversity is completely unknown; (ii) disclose for the first time the diversity and the
structure of PPN communities associated to isolated glacial refugia areas in Morocco and in
Southern Spain; (ii) assess, by comparing PPN communities between wild and cultivated
olive and according to different environmental variables, how the diversity could be
structured in response to anthropogenic practices, to soil physico-chemical and to climate
characteristics.
2. Materials and Methods

Ethics Statement
All the soil samples were air exported with a letter of authorization to the nematode
quarantine area (French Government Agreement n80622) of the Research Unit Centre de
Biologie pour la Gestion des Populations (Montpellier, France), where all analyses were
performed.
2.1. Site description

Sampling of soil and leaves olive took place from March to April 2012. Wild olives locations
were as far as possible from present orchards. In contrast, feral olive locations were collected
at proximity of cultivated olive stands or near main roads. Genetic olive data (based on plastid
DNA) was used to validate our distinction between wild and feral olives (see below). The
survey was conducted in 96 sites in several geographic regions all along a northeastsouthwest 900-km long transect (Fig 1, S1 Table). The main regions sampled in Morocco
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included (i) the Souss region (15 sites), located in the south facing side of the High Atlas
Mountains, near Agadir city, where trees sampled were either wild (9 sites, 2 of them
included trees of O. e. maroccana in sympatry with O. e. europaea var. sylvestris), feral (1
site) and traditionally cultivated (5 sites), (ii) the Haouz region (15 sites) located in the north
facing side of the High Atlas Mountains, near Marrakech city, where trees sampled were
traditionally (10) and high-density (3) cultivated or feral (2), (iii) the Tadla region (5 sites)
located along the north facing side of the southern Middle Atlas Mountains, near Beni Mellal
city where trees sampled were from wild (3 sites), feral (1 site) and traditionally cultivated
olives (1 site), (iv) the Zaane (3 sites of wild olive), situated to the South of Meknes city, (v)
the Guerouane (5 sites of traditional cultivation, 6 of high-density and 2 sites of feral olive),
(vi) the Kandar region (5 sites) located in the northern Middle Atlas Mountains, South of Fes
city and the Jel plain situated to the East of Taza city in eastern Morocco (5 sites) , where
trees in these two regions are traditionally cultivated, and (vii) both Atlantic and
Mediterranean facing sides of the Rif mountains in the North (33 sites) where most of trees
are wild (13 sites) with some sites of feral (12 sites) and traditionally cultivated at low density
(8 sites). The sampling was extended to the Spanish side of the Gibraltar Strait where two
sites of wild olives were sampled (in Tarifa and Algeciras). This Spanish side was used here
as another putative LGM refugia for wild olives to be compared to those from Morocco. In
the prospected regions, olive trees are found to grow over a large range of pedo-climates
conditions. As example, the Rif Mountains region was mainly characterized by forest podzols
soils associated with brown, ferrous and humus-bearing soils and by a high annual rainfall
rate (on average 800 mm). The Middle, High Mountains were characterized by clayey or
sandy-clay limestone soils, often from alluvial origin with less annual rainfall rate (on average
200 mm). These regions also exhibited variations in minimal/maximal temperatures (e.g. on
average 14-21C in the Souss region, 12-24C in the Rif region, 10-26C in the Middle Atlas,
11-28C in High Atlas) and altitudes (from 200 to 1700 m high).
2.2. Genetic characterisation of the olive trees

Three olive branches were collected in correspondence with soil samples to determine the
chloroplast haplotype of each tree. Chloroplasts are inherited by the mother, and their DNA
variation allows an identification of maternal lineages [48]. DNA was extracted with a CTAB
method from fresh leaves desiccated in silica gel [48]. A plastid genome profiling method
based on microsatellites, indels and single nucleotide polymorphisms (SNPs) were used to
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characterize each sample [49]. The combination of polymorphisms at 50 loci allowed us to

define 17 haplotypes (S2 and S3 Tables) belonging to the four Mediterranean olive plastid
lineages: E1 (one haplotype, E1.1, was detected in all cultivated trees, in some feral and in a
few wild olive individuals), E2 (six haplotypes in feral and wild olives), E3 (seven
haplotypes, in feral and wild populations) and M (three haplotypes, specific to O. e.
maroccana). This distribution of chlorotypes in Moroccan olives is congruent with previous
studies [40,44]. Feral olives either show only E1.1 or a mix of haplotypes including E1.1 and
common E2 or E3 haplotypes (i.e. E2.1, E2-2, E2-4 and E3-3, E3-4; these two chlorotypes
have been previously detected in Moroccan cultivars, but with frequencies below to 5% [40]).
In contrast, haplotypes characteristic of Moroccan-Iberian oleasters (i.e. E2-5, E2-6, E2-14,
E3-4, E3-7, E3-8) and O. e. maroccana (M1-1, M1-2, M1-7) were detected in wild
populations (Fig S1). These data confirmed the determination of olive modalities especially
the distinction between wild and feral olives; they also allowed us to define genetic groups
of wild olive populations according to their geographic distribution within putative long-term
refugia areas (see below).
2.3. Soil sampling

Considering that PPN spend all or almost all their life in the soil [50], the sampling only
included soil. A total of 220 soil samples were collected from the 96 sites. They were done
with a small spade under the foliage of each olive tree from the upper rhizosphere (the 15-20cm deep layer inhabited by pleiotropic roots). On cultivated olive (traditional and highdensity cultivation), the tillage and other human activities are frequent, that could lead to
homogenize the PPN communities in an orchard. For this reason, each orchard was
considered as a repetition per modality. The sampling was carried out in each orchard along
transects under four trees located at a distance of 10 meters approximately. Five sub-samples
were collected from each tree. These 20 sub-samples recovered were combined to form one 1dm3 reference sample per orchard. Contrary to cultivated orchards, heterogeneous PPN
communities were expected in wild and feral olives because human interventions are scarce or
absent, and wild trees are all propagated by seeds and thus genetically different. Each tree was
thus taken as repetition. Five sub-samples were also collected from each tree and then
combined to form one 1-dm3 reference sample per tree.
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2.4. Nematode extraction, identification and quantification

From each soil sample, a 250-cm3 wet aliquot was used for nematode extraction using the
elutriation procedure [51]. PPN belonging to the Aphelenchida, Dorylaimida, Triplonchida
and Tylenchida orders were enumerated in 5-cm3 counting chambers [52] and identified to the
genus level based on dichotomous keys [53] and to the species level [54]. The population
levels were expressed per dm3 of fresh soil. Concerning specific identification, the nematode
suspensions were preserved in glycerol [55] and specimens were mounted onto slides [56] for
microscopic observation. For root-knot nematodes (Meloidogyne spp.), the morphological
identification is difficult because (i) males are rare (most of the species are parthenogenic),
(ii) females are shapeless (they are embedded into roots), and (iii) juveniles dont provide
sufficient characters to distinguish species. Therefore, when juveniles were detected in soil
extract, tomato plants (a good host for several Meloidogyne species such as M. arenaria, M.
incognita, M. hapla and M. javanica already know to be associated with olive trees) were
transplanted in soils to rear the populations. These Meloidogyne populations were then
identified to species level by biochemical [57] and molecular approaches. The Meloidogyne
molecular identification was performed using SCAR markers (Sequence Characterized
Amplified Regions) [58, 59], and the D2-D3 expansion segments of 28S rDNA as described
in [60].
2.5. Climate and soil characteristics

Mean annual maximal temperature, minimal temperature and mean annual rainfall data were
collected from climate stations close to the sampling sites.
From each soil sample, a 100-cm3 dry and sieved (2 mm) aliquot was used for physicochemical analyses. They were performed by the Soil Laboratory of the INRA (Institut
National de la Recherche Agronomique) in Arras, France. The soil parameters analysed were:
(i) the proportion of clay (0-2 m), silt (2-50 m) and sand (50-2000 m) using the Textol
technique [61]; (ii) the proportion of organic carbon [62] using gas chromatography, making
possible to calculate organic matter content (OM = 1.724 x C); and (iii) the pHH2O [63]. These
soil parameters were chosen because they have been demonstrated as among the most
important factors explaining the presence and the abundance in plant parasitic nematode
communities [50].
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2.6. Definition of biodiversity and environmental variables for analyses

The PPN diversity in community was assessed by several ecological indices of biodiversity:
(i) the total number of PPN (N) that means the total number of individuals in a community,
(ii) the species richness (S) that represents the total number of species in a community, (iii)
the Shannon-Wiener diversity index H (H = -pi ln pi, where pi is the proportion of
individuals in each species i) that quantifies the local diversity or the heterogeneity of
diversity [H ranges from 0 to ln(S)], and (iv) the evenness (E = H / ln S) that quantifies the
regularity of species distribution within the community [E varies between 0 and 1].
The persistence of PPN in communities was investigated by comparing their diversity
between refugia areas of wild olive. Five biogeographic zones were thus defined: the Spanish
side of the Gibraltar Strait (SP), the East (ER) and West (WR) sides of the Rif, the Middle
Atlas (MA) and the High Atlas (HA).
The responses of PPN communities were analysed by comparing the diversity according
to olive modalities, to cultivation practices (intensification, irrigation) and to some pedoclimatic factors. Different types of environmental variables were thus defined: (i) olive
variables including: olive growing modalities (distinguished as wild (including O. e.
maroccana and O. e. europaea var. sylvestris), feral, traditionally and high-density
cultivation), and type of water supply such as irrigated (that concerned high-density and some
traditional cultivation) or rainfed (that concerns wild, feral and some traditionally cultivated
olive); (ii) climate variables (mean of maximum and minimum annual isotherms and of
annual rainfall); (iii) soil characteristics including the soil type (based on the proportion of
clay, silt and sand [64]), the content of organic matter and the pH. All The variables used in
the analyses are given in [Table 1].
2.7. Data analyses

All analyses presented in this study were done using R version 3.0.1 [65].
The four biodiversity indices were calculated using the Vegan library [66]. They were
calculated and the means were then compared using Wilcoxon test with Bonferroni
corrections between the five biogeographic areas. In order to inspect if these isolated regions
could present different PPN community structures, a between classes analysis was carried out
on nematodes data (at the species level) detected on wild olive. This analysis allowed seeing
if we can separate these five putative olive refugia areas into groups according to the specific
nematode composition hosted in communities. PCA analyses were thereafter applied on each
Page 95
class (putative refugia) separately in order to determinate which predominant species compose
the PPN community and can play a role in the putative groups made in the between classes
analyses. The a between classes and the PCA analyses were performed using ade4 [67],
FactoMineR [68] and Factoextra libraries [69].
In order to study the influence of environmental variables on PPN diversity in
communities, the first step aimed to describe the correlation between the different variables of
each data set. For that, multivariate analyses were applied: a principal component analysis
(PCA) was performed on biodiversity data and a multiple correspondence analysis (MCA) on
environmental data including olive (growing modality, water supply), climatic (mean
maximal and minimal temperatures, and mean annual rainfall) and soil characteristics (soil
type, organic matter content and pH). These multivariate analyses were carried out using the
ade4 library [67]. The putative link between each biodiversity index and environmental data
were then investigated by General Linear Model (GLM). The choice of GLM was because
several environmental variables are available to be tested on each biodiversity index. Each
biodiversity index variable was firstly tested for its distribution type in order to identify the
error family and the link function for each GLM model. Logarithmic transformation was
applied on the total number of PPN (N) to improve the distribution of this variable. A GLM
model was constructed for each biodiversity index with the environmental data. We used a
model element selection process according to the criterion of Akaike (AIC) using the MASS
package [70]. The used model was selected according to the low value of AIC. An ANOVA
test was then applied in order to analyse the significance of model elements. The residual
deviance and the normality of residuals were also investigated in order to estimate the quality
of the selected model. Lastly, the mean values of diversity indices were compared between
the significant variables according to GLM results. Wilcoxon test with Bonferroni corrections
were used for all pair-wise multiple comparisons. Differences obtained at levels of P < 0.05
were considered significant.
3. Results
3.1. Taxonomical diversity of PPN
The PPN communities associated with olive trees identified in this study were highly
diversified. A total of 122 species have been identified. They were belonging to 48 genera and
to two families of Aphelenchida, to one family of Dorylaimida, to one family of Triplonchida
Page 96
and to 14 families of Tylenchida (Table S4). Among these 48 identified genera, Filenchus
spp., Helicotylenchus spp., Merlinius spp., Paratylenchus spp., Pratylenchus spp.,
Rotylenchus spp., Tylenchorhynchus spp. and Xiphinema spp. revealed as the most
widespread in olive soils. Considering species level, Helicotylenchus species were highly
present in olive samples with 11 different species. Among them, H. crassatus was clearly the
most dominant species (occurring in 58% of the samples). It was present in all regions except
in the Jel and Kandar regions. Helicotylenchus dihystera and H. varicaudatus also occurred in
43% and 32% of the samples, respectively. In contrast, H. exallus and H. minzi detected in the
Guerouane region, and H. pseudorobustus, detected in the Haouz region, were scarcer. In
addition, Merlinius brevidens (Telotylenchidae) and Filenchus filiformis (Tylenchidae) were
also frequently observed, occurring in 51% and 40% of samples, respectively. Some
nematodes species such as Amplimerlinius paraglobigerus, Filenchus vulgaris, Rotylenchus
agnetis, R. robustus, and Sauertylenchus maximus were only detected in Southern Spain.
3.2. Biodiversity structure between putative refugia areas of wild olive

The means of biodiversity indices of PPN communities associated to wild olive were
compared between the five putative long-term refugia (the Spanish side of Gibraltar Strait, the
Western and the Eastern sides of the Rif Mountains, the Middle and the High Atlas
Mountains). These comparisons indicated some significant differences in the total number of
PPN (N). More nematodes were counted in wild olive areas from Spain and Middle Atlas than
from the High Atlas and the Eastern side of the Rif. No significant difference in the total
number of nematodes was recorded between the both sides of the Rif. In contrast, the richness
(S), the local diversity (H) and the evenness (E) were not different between these areas.
However, a between classes analysis (Fig 2, ABC) clearly separated the five putative refugia
in four groups: the Spanish side (SP), the Eastern Rif (ER), the Western Rif (WR) and the last
group with the Middle Atlas (MA) and the High Atlas (HA). PCA analyses were then applied
in order to explore the PPN composition within each group. For these analyses, the High
Atlas and the Middle Atlas were treated as a group (MHA). According to these analyses, each
putative refugia group had its specific patterns with different PPN dominant species (Fig 3,
ABCD). The surprising pattern was the difference between the Eastern and the Western sides
of the Rif that are geographically close. To investigate this pattern, we conducted a
discriminant analysis between the both sides. The analysis indicated that these micro-regions
may share the same taxa but they do not present the same dominant species. Looking data
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between these two sides demonstrated, as example, Helicotylenchus crassatus was strongly
present in the Western side but almost absent in the Eastern side. Helicotylenchus vulgaris
and Xiphinema pachtaicum, were highly present in the Western side but very few in Eastern
side, while Xiphinema sp. was observed in the other side. Some species were exclusively in a
side but not in the other: for instance, Pratylenchus neglectus, Helicotylenchus varicaudatus,
Tylenchorhynchus crassicaudatus were only present in the Western side but not in the East,
while Nothotylenchus geraerti was only detected in the East.
3.3. PPN Biodiversity in communities according to anthropogenic changes and

environmental conditions
A PCA was performed to describe the correlation between the biodiversity variables while a
MCA carried on environmental factors was to determine the correlation among these
variables. The evenness index (E) was not included in these analyses because its calculation
depends on the value of H index. The PCA indicated that the fraction of variance
(eigenvalues) accounted by the two first PC axes was 57.9% and 33.8%, respectively. The
loading plot of the PPN diversity indices (Fig 4A) showed that contribution of the richness (S)
and the local diversity (H) was opposite to that of the total number of PPN (N) according to
the PC1 axis, while the contribution of the total number of PPN (N) was higher on the PC2
axis.
Considering the MCA, the fraction of variance accounted by the two first MC axes
(eigenvalues) was 24.6% and 15.9%, respectively. The loading plot of the environmental
factors (Fig 4B) showed that the olive growing modalities contributed to the MC1 axis
structure with the wild olive modality (WO) in the negative values of the axis and the
cultivation modalities [high-density (HD) and traditional (TR) cultivation] and, at a lesser
extent, the feral olive modality (FO) in the positive values. The contribution of soil (especially
soil type) and climatic (Tmin in particular) factors was more important on the MC2 axis. The
high-density cultivation and the water supply (WS) contributed to the two axes. According to
this analysis, the olive growing modalities (on the first axis), the soil type and the minimal
temperature Tmin (on the second axis) were the variables the most contributing to the structure
of the factorial plan.
The GLM analyses indicated the environmental variables that had significant impact the
biodiversity indices. According to ANOVA tests (data not shown), the total number of PPN
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(N), the specific richness (S) and the local diversity (index of Shannon H) were significantly
impacted by olive growing modalities. Considering soil characteristics, the organic matter
content (OM) that was not highly contributed to the MCA loading plot structuration, it
showed, according to GLM analyses, having a significant effect on each of the specific
richness and the local diversity. The soil type was demonstrated significantly affecting the
specific richness. A significant impact of the minimal temperature (Tmin) was recorded on the
total number of individuals (N) and species (S). The means of the biodiversity indices were
thus only compared according to these variables (olive growing modalities, soil type and its
content of organic matter and minimal temperature). Considering olive modalities (Fig 5A),
the mean comparisons indicated that the total number of PPN (N) was about two times higher
on cultivated olives (in high-density and traditional orchards) than on non-cultivated olives (in
wild and feral sites). In contrast, the PPN communities on non-cultivated olives were
significantly richer in species (S) and more diversified (H) compared to cultivated olives. The
regularity of the distribution of PPN species in community (Evenness E) was higher on feral
olive and significantly different from traditionally and high-density cultivated olives. In order
to investigate the role of organic matter (OM) content, three OM classes (low, moderate and
high) were constituted. The analyses indicated a significant impact of the OM content on the
PPN richness and diversity of communities (Fig 5B), with the lowest values of S and H
recorded with the lowest OM content (i.e. OM1; <15 g/kg). The number of species was
significantly affected by soil type where more species were enumerated in clayed soil than in
sandy soil (Fig 5C). Concerning the minimal temperature (Tmin), three classes were also
defined; the analyses demonstrated that its effect was on the total number of nematodes (N)
and on the specific richness (S) where more nematodes and less species were recorded under
low minimal temperature (Tmin = 7-12C), (Fig 5D).
4. Discussion
The present study was focused on the diversity of PPN communities associated to olive trees
in wild and cultivated areas. It offers for the first time a description of the PPN associated to
olive trees in Morocco, of the structure of the specific diversity of nematodes within longterm refugia areas and in response to agricultural, edaphic and climatic factors. In the
following sections, we firstly discuss this diversity in the light of others surveys and reviews
conducted on olive trees. In the second part, we address the persistence of PPN communities
Page 99
among putative refugia of wild olive. We lastly draw attention to the impact of cropping
practices on the multiplication, the composition and the distribution of species in
communities.
4.1. Diversified PPN communities associated to olive trees in Morocco

Contrary to Spain or to others Mediterraneans countries, the global PPN fauna and their
distribution was totally unknown in Morocco before our study, except a few reports on some
root-knot nematodes such as Meloidogyne morocciensis [71] and cereal cysts nematodes [72].
This study describe for the first time the diversity of PPN communities associated to olive
trees in Morocco and clearly highlighted a high taxonomical diversity of PPN communities
where 117 species belonging to 47 genera were recorded in Morocco only. In addition, the
study added new taxa (corresponding to seven genera and 60 species) that were recorded for
the first time in association with olive trees. The taxonomical diversity analysed in this study
is the most important if compared with others surveys on olive trees that documented 223
species worldwide [as revised by 37, 73-77]. This high diversity and the detection of new taxa
could be essentially explained by: (i) a large sampling effort (220 soil samples corresponding
to 352 trees sampled), conducted along a long transect (about 900 km) covering a wide range
of olive growing regions in Morocco, and (ii) a large proportion of samples collected in wild
and feral olive areas. These non-cultivated olive habitats could be considered as reservoirs of
high diversity where a part remains unknown [78]. As evidence, new species of root-knot
nematodes, Meloidogyne spartelensis, was detected on wild olive in Northern Morocco [60].
However, other species could not be detected because they may occur only under uncharaterizable life stages (e.g. juveniles), or their development may relate to another periods
of the year or to specific microhabitats [79]. As an example, no Rotylenchulus could be
identified at the species level, because all the individuals were in juvenile stage (S4 Table).
4.2. Role of human activities in the structure of PPN diversity in communities

The diversity of PPN communities associated to olive trees was described through several
biodiversity indices. These indices were used to evaluate how this diversity could change
according to domestication (wild vs cultivated olive), and agricultural practices (cropping
intensification, irrigation) in olive areas. The impact of environmental factors (soil and
climate) was also investigated. The first main conclusion of this study was the significant
Page 100
opposition between the abundance of PPN in communities and their species diversity (specific
richness, local diversity and evenness). In other words, a high PPN species diversity within a
community may prevent the multiplication of the species as a potential effect of trade-off
interactions [80]. According to olive growing conditions, the diversity was also differently
distinct according to olive domestication gradients where the PPN diversity (high values of S,
H and E indicating richest and most diversified PPN communities) were found in noncultivated olive (wild and feral olive) compared to cultivated olive (traditional and high
density). These last were distinguished by reduction in PPN diversity. That underlines an
important role of human activities on the diversity patterns of communities. To explain this
contribution of human intervention, the wild and feral olive areas correspond to ecosystems
that are generally lowly or not affected by humans practices, and thus the PPN communities
associated to these ecosystems are not frequently disturbed. That agrees with others
ecological observations demonstrating that low disturbed ecosystems mainly host more
diverse communities of soil organisms, as demonstrated for earthworms [81] and for PPN
[82]. This image of the high diversity was completely inverted in cultivated olive orchards
where the PPN communities were characterized by high abundances and by low diversity of
these nematodes. The cropping systems are usually characterized to be disturbed by human
activities via agricultural practices (e.g. crop intensification, irrigation, tillage). These
anthropogenic practices lead to species decline as already demonstrated for soil biota
communities in intensive cropping systems [83]. That underlines the contribution of human in
ecosystems degradation that consequently leads to biodiversity extinction [84-86]. The impact
of human interference has been observed on bees, birds, and plants species [87] as well as on
nematodes [88]. Considering the nematodes, according to Yeates and Bongers [89], the
decrease in their diversity with increasing of human activities can be attributed to physical
disturbance, to change in quantity and quality of organic matter being returned to the soil and
to increase in numbers of specific plant feeding nematodes that are favoured by the selected
crops. Consequently, the study reveals the olive modality as the main factor driving the PPN
diversity in communities. Soil and climate factors were also contributed to this structuration
but as secondary factors. However, others observations on plant-parasitic nematode
communities associated to olive trees [77] demonstrated that the nematode diversity was
impacted by olive conditions (olive cultivar, orchard and soil management practices); but, the
main impact was related to olive variety and to soil texture.
Page 101
4.3. Persistence of PPN diversity in communities associated with wild olive areas
As for the others Mediterranean plants, the olive trees are persisted in long term refugia in
several parts of the Western the Mediterranean Basin (including Southern Spain and Morocco
[40]). These refugia correspond to phylogeographical hotspots of diversity [45]. The
analyses of PPNs associated to wild olive in these refugia allowed us to underline rich and
diversified communities. The PPN communities characterized by a high species richness, high
local diversity and high evenness (regular distribution of the species) in wild olive, are
probably a consequence of relative ecosystem stability. That could suggest that the PPN
communities may survive for a long term with olive trees in these areas and could be
considered as native or historical communities of these regions.
Although diversity indices are useful in describing community characteristics, they do
not provide information on compositional features of biodiversity relating to the abundances
of shared taxa [90]. That was confirmed in the putative refugia areas, where the communities
were composed by a similar specific richness, but not by the same specific composition.
Others analyses (PCA) were thus required to describe the specific structure of communities.
The PCA within each refugia demonstrated that communities are strongly heterogeneous in
their specific composition and the identity of the dominant species. A good and surprising
example on this heterogeneity was observed between the both eastern and western sides of the
Rif Mountains that are geographically very close. This difference in species composition
could be explained by the presence of Rif Mountains that limited the dispersion of nematodes
between communities from a side to the other for a long time. Furthermore, the olive trees
from these two regions are highly differentiated according to maternally inherited
polymorphism. As example, the haplotypes E2-14 has only been detected on the Eastern side,
while haplotypes E3-4 and E3-5 are specific to the Western side [see 40, Fig S1]. This genetic
differentiation of olive between the two sides of the Rif could also impact the structure of
hosted communities. In this regard, the effect of olive genotype on soil or endophyte biota has
also been shown in recent studies which demonstrated that it can significantly influence the
nematode assemblages present in their soil rhizosphere [91], the communities of Arbuscular
Mycorhizal Fungi [92] as well as the bacterial assemblage colonize Mediterranean olive trees
[93].
Page 102
5. Concluding remarks
The present study concluded that the diversity of PPN communities is a result of the interplay
between complex historical processes and heterogeneous environmental related to olive
growing conditions. The domestication (wild vs cultivated olive) were revealed as important
factors able to drive the diversity of PPN in communities. These practices (from wild to
cultivated ecosystems or cropping intensification) could lead to community rearrangements in
favour to high pathogenic species defined as major agricultural pests [94]. In this vein, the
cultivated olive areas were characterized by a sharp reduction of specific diversity in PPN
communities as a consequence of the anthropogenic disturbance. This reduction was related
to the development of harmful taxa on cultivated olive such as root-knot (Meloidogynidae),
root-lesion (Pratylenchidae) and spiral (Hoplolaimidae) nematodes that are known to affect
olive production in the world [76]. The development of these taxa should be attracting more
attention to ensure the use of healthy olive seedlings (rootstocks) free of nematodes in order
to avoid the introduction of plant-parasitic nematodes to olive orchards. This study underlined
the biological diversity as relevant indicator to assess the soil health and to investigate the
impact of human intervention in the ecosystems. However, further studies targeted the
description of the free-living nematodes or the others soil organisms should be considered.
Olive tree, based on its longevity and its high genetic variability within each of oleaster and
cultivars, should contain a high diversity of microbes and of free-living nematodes, providing
antagonist interactions against the PPN. These interactions could be potentially used in their
control as viable and environmentally friendly alternative method [95].
The study was also considered the structure of PPN communities associated to wild
olive refugia areas in Morocco and in Southern Spain. It revealed a different specific
composition of PPN persisted with olive trees within the considered refugia areas. That
underlines the importance of refugia areas as a good tool to study the evolutionary history of
olive domestication around the Mediterranean Basin by comparing the phylogeography of the
olive (its genetic differentiation) and its parasites. In this way, including others putative
refugia form other parts of the Mediterranean Basin, could help to decipher the history of PPN
communities associated to the olive tree across this region. Based on the hypothesis that the
parasite shares a common history with its host [96], these phylogeography studies may clarify
the mechanisms that underlie the co-evolution of olive-PPN. The sedentary endoparasites are
usually characterized by an intimate interaction with their hosts [97]. Then nematode genera
Page 103
such as Meloidogyne and Rotylenchulus (having a sedentary position on the plant roots) could
be thus the best positioned for such studies.
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Antalya, Turkey, 21-23 October; 2009. pp. 45-50.
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Cadet P. Gestion cologique des nmatodes phytoparasites tropicaux. Cahiers Agricultures.
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Postma-Blaauw MB, de Goede RGM, Bloem J, Faber JH, Brussaard L. Soil biota community
structure and abundance under agricultural intensification and extensification. Ecology. 2010;
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Vitousek PM, Mooney HA, Lubchenco J, Melillo JM. Human domination of Earth's
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2003; 18: 326-330.
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monitoring human impacts on biodiversity. Ecol Indic. 2012; 17: 58-67.
Yamaura Y, Royle JA, Shimada N, Asanuma S, Sato T, et al. Biodiversity of man-made open
habitats in an underused country: a class of multispecies abundance models for count data.
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Pan F-J, Xu Y-L, McLaughlin NB, Xue AG, Yu Q, et al. Response of soil nematode community
structure and diversity to long-term land use in the black soil region in China. Ecol Res. 2012;
27: 701-714.
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113-135.
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Palomares-Rius JE, Castillo P, Montes-Borrego M, Mller H, Landa BB. Nematode community

populations in the rhizosphere of cultivated olive differs according to the plant genotype. Soil
Biol Biochem. 2012; 45: 168-171.
Montes-Borrego M, Metsis M, Landa BB. Arbuscular mycorhizal fungi associated with the
olive crop across the Andalusian landscape: factors driving community differentiation. PLoS
ONE. 2014; 9: e96397.
Mller H, Berg C, Landa BB, Auerbach A, Moissl-Eichinger C, et al. Plant genotype-specific
archaeal and bacterial endophytes but similar Bacillus antagonists colonize Mediterranean olive
trees. Front Microbiol. 2015; 6, 138.
Mateille T, Cadet P, Fargette M. Control and management of plant-parasitic nematode
communities in a soil conservation approach. In: Ciancio A, Mukerji KG, editors. Integrated
management and biocontrol of vegetable and grain crops nematodes. Dordrecht: Springer;
2008. pp. 79-97.
Aranda S, Montes-Borrego M, Jimnez-Daz RM, Landa BB. Microbial communities associated
with the root system of wild olives (Olea europaea L. subsp. europaea var. sylvestris) are good
reservoirs of bacteria with antagonistic potential against Verticillium dahliae. Plant and Soil.
2011; 343: 329-345.
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in plant biology. 2011; 14: 435-443.
Page 108
Table 1. Agricultural and environmental variables and their corresponding codes.
Variables
Olive variable
Growing modality
- wild olive
- feral olive
- traditional cultivation
- high-density cultivation
Water supply
- rainfed
- irrigated
Climate variables
- Maximal temperature (C)
- Minimal temperature (C)
- Annual rainfall (mm)
Soil factors
- Sandy soil
- Silty soil
- Clayed soil
- Organic matter content (g/kg)
- pH soil
Code
WO
FO
TR
HD
RF
WS
Tmax
Tmin
AR
SA
SI
CL
OM
pH
Page 109
Legends for figures
Figure 1: Sites sampled in Morocco and Southern Spain. Olive growing modality is given for
each site.
Figure 2: Analyses of PPN diversity between putative refugia areas of wild olive using a
between classes analysis (see text for details).
A: scores and classes for the five groups: SP holds for Spanish side of the Gibraltar Strait,
WR for Western Rif, ER for Eastern Rif, HA for High Atlas and MA for Middle Atlas. B:
only classes for the same analysis are represented for a more easy representation. C: the
permutation test (permuting data between groups) is a Monte-Carlo test on the percentage of
explained inertia. The statistic is the ratio of the inertia (sum of eigenvalues) of the
constrained analysis divided by the inertia of the unconstrained analysis. The test shows that
those classes are significantly different (the bar is the observed value while the histogram
represents the simulated values).
Figure 3: Principal Component Analysis PCA results within group defined by the between
classes analyse (i.e. 4 groups).
A. HMA for High Atlas together with Middle Atlas, B. ER for Eastern Rif, C. WR for
Western Rif and D. SP for Spanish side. The values of each cos2 on the first two axes for
each of the 122 species are represented by a colour going to light grey to black. For clarity,
we chose to represent only the name of the dominant species on the two first axes of the PCA
(i.e. cos2 0.75).
Figure 4: Multivariate analyses on the PPN diversity indices and environmental factors.
A: PCA loading plot of the diversity indices (N = total number of nematodes; S = species
richness; H = local diversity); B: MCA loading plot of the environmental variables (codes for
environmental factors are given in Table 1).
Figure 5: Mean of PPN biodiversity indices between some environmental variables.
A: between olive modalities WO = wild olive, FO = feral olive, TR = traditional olive
cultivation, HD = high density olive cultivation; B: between the three classes of organic
matter content: poor (OM1; 6-15 g/kg), moderate (OM; > 15-30 g/kg), and high (OM3; > 30
g/kg); C: between soil types (clay, silt, sand); ; D: between the three classes of minimal
temperature: Tmin1 [7 > Tmin 9], Tmin 2 [9 > Tmin 12], and Tmin 3 [12 > Tmin 15].
Page 110
Figure 1
Page 111
Figure 2
Page 112
Figure 3
Page 113
0.1
Figure 4
HD
CL
H
0
1.5
Tmin
2.0
A B
S
1.0
-0.1
Tmax TR
FO
WO
RF
0.0
-0.2
OM
-0.3
-0.5
ppH
SI
Dim 2 (15.9%)
AR
0.5
Dim 2 (33.8%)
WS
-1.0
SA
N
-0.3
-0.2
-0.1
Dim 1 (57.9%)
0.1
-1
Dim 1 (24.6%)
Page 114
Figure 5
Page 115
Supplementary information
SI Appendix includes:
Table S1. Location of the olive sampling sites surveyed in Morocco and southern Spain.
Table S2. Data matrix of the 17 plastid DNA olive haplotypes revealed in this study.
Table S3. Plastid DNA lineages and haplotypes detected within spontaneous and cultivated
olive trees collected in Morocco and in some wild olive populations from Southern Spain.
Table S4. Plant-parasitic nematode taxa associated with olive identified in the study.
Figure S1: Plastid DNA lineages and haplotypes detected within wild and feral olive in
Morocco and in some wild olive in Sothern Spain.
Page 116
Table S1. Location of the olive sampling sites surveyed in Morocco.

Geographic region
City
Olive modality
No of sites
Latitude N
(decimal)
Longitude W
(decimal)
Tiguert
Aourir
wild
wild
wild
feral
traditional cultivation
wild
feral
high-density cultivation
feral
wild
feral
wild
wild
feral
wild
wild
wild
feral
wild
feral
wild
feral
wild
feral
wild
wild
2
1
1
1
1
5
2
1
1
1
1
1
1
2
7
1
1
2
1
2
2
1
2
2
4
3
2
5
3
2
1
2
1
1
3
1
1
5
3
2
1
7
5
1
1
30.63
30.52
30.81
30.42
30.42
30.74
30.61
30.65
30.55
32.15
31.37
31.46
31.70
31.69
31.63
31.57
31.28
32.58
32.21
32.32
33.32
33.33
33.70
33.77
33.88
33.85
33.63
33.87
34.25
34.26
35.79
35.78
35.54
34.79
35.07
35.03
35.05
35.07
35.07
35.38
35.54
34.79
34.79
36.02
36.11
9.86
9.59
9.14
9.02
9.02
8.77
9.34
8.18
8.66
7.26
7.95
7.98
7.69
8.11
8.10
7.97
7.96
5.98
6.83
6.39
6.07
6.00
5.63
5.71
5.41
5.39
5.83
4.88
3.80
3.74
5.92
5.37
5.62
5.77
5.33
5.32
5.35
5.33
5.32
5.37
5.62
5.77
5.77
5.60
5.43
Ouled Taima
Souss
Taroudant
Ouled Berhil
Aoulouz
El Kelaa Des Sraghna
Haouz
Tamellalt
Sidi Bou Othmane
Marrakech
Tahnaout
Asni
Tadla
Zaane
Beni Mellal
El Ksiba
Oulmes
Oued Zem
El Hajeb
Guerouane
Kandar
Jel
Meknes
Khemisset
Sefrou
Taza
Msoun
Tanger
Fnideq
Tetouan
Asilah
Rif
Chefchaouen
Bni Harchen
Ouazzane
Southern Spain
Tarifa
Algecira
Page 117
13
13 11 10 10 22 13 12
13 12
10 10 35
12 13 10 11 10 10 16 12 22 21
E1-1
10 11 10 12 20 10 12 10 15 10 21 15 11
15
13 10 11 10 21 18 13
13 12
11 10 33
12 12 11 11 10 10 16 11 21 21
E2-1
10 11 10 12 20 10 11
16 11 21 16 11
13
13 11 10 11 20 11 13
13 12
11 10 32
14 12 12 10 10 10 15 11 21 21
E2-2
10 11 10 12 20 10 11
16 11 21 16 11
13
13 11 10 11 20 11 13
13 12
11 10 33
14 12 12 10 10 10 15 11 21 21
E2-3
10 11 10 12 20 10 11
16 11 21 16 11
13
13 11 10 11 20 11 13
13 12
11 10 32
14 12 12 10 10 10 15 11 21 21
E2-4
10 11 10 12 20 10 11
16 11 21 16 11
13
13 11 10 11 20 11 13
13 12
11 10 32
14 12 12 10 10 10 15 11 21 21
E2-5
10 11 10 12 20 10 11
16 10 21 16 11
13
13 11 10 11 20 11 13
13 12
11 10 32
14 12 12 10 10 10 15 11 21 21
E2-6
10 11 10 12 20 10 11
16 10 21 16 11
13
13 11
11 20 11 13
13 12
11 10 32
14 12 12 10 10 10 15 11 21 21
E2-14
10 11 10 12 20 10 11
16 11 22 16 10
13
13 11 10 11 20 11 13
14 12
11 10 32
14 12 12 10 10 10 15 12 21 20
E3-1
10 11 10 12 20 11 10 10 16 11 22 15 12
13
13 11 11 10 21 18 12
11 11
10 11 10 34
11 12 13
11 11 15 11 21 21
E3-2
10 11 10 12 20 11
10 16 11 22 15 12
13
13 11 11 10 21 18 12
11 11
10 11 10 34
11 12 13
11 11 15 11 21 21
E3-3
10 11 10 12 20 11 10 10 16 11 22 15 12
13
13 11 11 10 21 18 12
11 11
10 11 10 33
11 12 13
11 11 15 11 21 21
E3-4
10 11 10 12 20 11 10 10 16 12 22 15 12
13
13 11 12 10 21 18 12
11 11
10 11 10 33
11 12 13
11 11 15 11 21 21
E3-5
10 11 10 12 20 11 10 10 16 12 22 15 12
13
13 11 12 10 21 18 12
11 11
10 11 11 33
11 12 13
11 10 15 11 21 21
E3-7
10 11 10 12 20 11 10 10 16 11 22 15 12
13
13 11 12 10 21 18 12
11 11
10 11 10 33
11 12 13
11 11 15 11 21 21
E3-8
10 11 10 11 20 11
13
13 11 11 10 21 18 12
11 11
10 11 10 34
11 12 13
11 11 15 11 21 21
10 16 11 22 15 12
61E
10 11 10 12 20 10 11 10 16 10 20 15 13
10D-2
11C-2
10D-1
11C-1
58C
19A
50C
33D
M1-7
29A
53D
56D
44A
49D
28C
17B
25A
16 12 22 21
41B
59C
12 13 10 11 10
47D
22C
12 13 10 11 10 10 16 12 22 21
42D
39C
10 10 35
21A
54B-2
10 10 36
51C
54B-1
9A
57B-2
13 12
23D
57B-1
14 12
46D
52B
13 11 11 10 21 13 12
38B
24A
13 11 11 10 21 13 12
36A
48B
13
2A
13
15B
31A
10 11 10 12 20 10 11 10 17 10 21 15 13
1B
10 11 10 12 20 10 11 10 16 10 21 15 13
27C
16A
26D
6C
5A
ECORI
M1-2
45C
XapI
M1-1
30B
Haplotype
Table S2. Data matrix of the 17 plastid DNA olive haplotypes revealed in this study using 50 loci (Besnard et al. 2011). Stretch size of the repeated motif is
given for each microsatellite locus, while a binary code (0/1) has been defined for indels and single nucleotide.
Page 118
Table S3. Plastid DNA lineages and haplotypes (coded according to 40) detected within spontaneous and cultivated olive trees collected in Morocco and in
some wild olive populations from Southern Spain. The number of haplotype occurrences is given for each olive modality.
Plastid lineage
Olive modality
Wild (sylvestris)
Wild (maroccana)
Wild (feral)
Traditional cultivated
High density cultivated
E1
#1
2
45
10
40
#1
35
13
-
#2
1
-
#4
7
-
E2
#5
12
-
#6
3
-
#14
5
-
#1
3
-
#2
2
-
#3
17
4
-
E3
#4
4
-
#5
9
-
#7
7
-
#8
1
-
#1
2
-
M1
#2
1
-
#7
2
-
Page 119
Table S4. Plant-parasitic nematode taxa associated to olives in our study
Aphelenchoididae
Dorylaimida
Longidoridae
Triplonchida
Trichodoridae
Tylenchida
Anguinidae
Criconematidae
Dolichoridae
Heteroderidae
Hoplolaimidae
Longidorus sp. Micoletzky, 1922

Xiphinema pachtaicum Tulaganov, 1938
X. turcicum Luc & Dalmasso,1964
X. vuittenezi Luc et al., 1964
Xiphinema sp. Cobb, 1913
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Aoulouz
Ouled Berhil
Aourir
Tiguert
Asni
Tahnaout
Marrakech
Sidi Bou Othmane
Tamellalt
Oued Zem
Oulmes
Khemisset
El Hajeb
Souss
Taroudant
Haouz
Ouled Teima
El Ksiba
+
+
Tadla
Beni Mellal
Sefrou
Taza
Ouazzane
Chefchaouen
Bni Harchen
Asilah
+
+
+
+
Paratrichodorus sp. Siddiqi, 1974

Trichodorus sp. Cobb, 1913
Ditylenchus emus Khan et al., 1969
D. equalis Heyns, 1964
Ditylenchus sp. Filipjev, 1936
Nothotylenchus acutus Khan, 1965
N. adasi Syces, 1980
N. geraerti Kheiri, 1971
N. medians Thorne & Malek, 1968
Ogma rhombosquamatus Mehta & Raski, 1981
Criconema sp. Hofmnner & Menzel, 1914
Criconemella sp. De Grisse & Loof, 1965
Macroposthonia sp. De Man, 1880
Neodolichorhynchus microphasmis Loof, 1960
Heterodera riparia Subbotin et al., 1997
Heterodera sp. Schmidt, 1871
Helicotylenchus canadensis Waseem, 1961
H. crassatus Anderson, 1973
H. crenacauda Sher, 1966
H. digonicus Perry, 1959
H. dihystera Cobb, 1893
H. exallus Sher, 1966
H. minzi Sher, 1966
H. pseudorobustus Steiner, 1914
H. tunisiensis Siddiqi, 1964
H. varicaudatus Yuen, 1964
H. vulgaris Yuen, 1964
Helicotylenchus sp. Steiner, 1945
Rotylenchus agnetis Szczygie 1968
Rotylenchus buxophilus Golden, 1956
R. goodeyi Loof & Oostenbrink 1958
R. pumilus Perry, 1959
R. robustus de Man, 1876
Rotylenchus sp. Filipjev, 1936
Tetouan
Aphelenchus avenae Bastian, 1865

A. helicus Heyns, 1964
A. isomerus Anderson & Hooper, 1980
Aphelenchoides graminis Baranovskaya & Haque, 1968
A. saprophilus Franklin, 1957
Aprutides guidetti Scognamiglio, 1974
Geographic regions
Guerouane
Zaane
Kandar
Jel
Fnideq
Aphelenchida
Aphelenchidae
Tanger
Spanish side
Rif
Meknes
Species
Guercif
Orders / Families
+
+
+
+
+
+
+
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Page 120
Neotylenchidae
Paratylenchidae
Pratylenchidae
Psilenchidae
Rotylenchulidae
Telotylenchidae
Aoulouz
Ouled Berhil
Taroudant
Ouled Teima
Aourir
Tiguert
Asni
Souss
Tahnaout
Tamellalt
Haouz
El Ksiba
Tadla
Beni Mellal
Oued Zem
Oulmes
Khemisset
El Hajeb
Sefrou
Taza
Ouazzane
Bni Harchen
Chefchaouen
Asilah
Tetouan
Fnideq
Geographic regions
Guerouane
Zaane
Kandar
Marrakech
Meloidogyne arenaria Neal, 1889

M. hapla Chitwood, 1949
M. javanica Treub, 1885
M. spartelensis Ali et al., 2015
Meloidogyne n.sp. Goeldi, 1892
Meloidogyne sp. Goeldi, 1892
Neotylenchus geraerti Kheiri, 1971
Cacopaurus sp. Thorne, 1943
Gracilacus sp. Raski, 1962
Paratylenchus (P.) microdorus b Andrssy, 1959
P. (P.) nanus Cobb, 1923
P.(P.) sheri Raski, 1973
P. (G.) straeleni de Coninck, 1931
P.(P.) vandenbrandei De Grisse, 1962
P.(P.) veruculatus Wu,1962
Paratylenchus (Paratylenchus) sp. Micoletzky, 1922
Pratylenchoides hispaniensis Troccoli et al. 1997
P. laticauda Braun & Loof, 1967
Pratylenchoides sp. Winslow, 1958
Pratylenchus crenatus Loof, 1960
P. mediterraneus Corbett, 1983
P. neglectus Rensch, 1924
P. pinguicaudatus Corbett, 1969
P. thornei Sher & Allen, 1953
Pratylenchus sp. Filipjev, 1936
Zygotylenchus guevarai Tobar Jimnez, 1963
Psilenchus aestuarius Andrssy, 1962
P. hilarulus de Man,1921
Rotylenchulus sp. Linford & Oliveira, 1940
Amplimerlinius globigerus Siddiqi, 1979
A. intermedius Bravo, 1976
A. paraglobigerus Castillo et al. 1990
Bitylenchus aerolatus Tobar Jimnez, 1970
Merlinius brevidens Allen, 1955
M. microdorus Geraert, 1966
M. nothus Allen, 1955
Merlinius sp. Siddiqi, 1970
Nagelus obscurus Allen, 1955
Paratrophurus loofi Arias, 1970
Sauertylenchus maximus Allen, 1955
Scutylenchus lenorus Brown, 1956
S. mamillatus Tobar- Jimnez, 1966
S. tessellatus Goodey, 1952
Telotylenchus avaricus Kleynhans 1975
T. paaloofi Tikyani & Khera, 1970
T. ventralis Loof, 1963
Trophurus sculptus Loof, 1956
Tylenchorhynchus clarus Allen, 1955
T. crassicaudatus Williams, 1960
Tylenchorhynchus sp. Cobb, 1913
Jel
Sidi Bou Othmane
Meloidogynidae
Tanger
Spanish side
Rif
Meknes
Species
Guercif
Orders / Families
+
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Page 121
Tylenchidae
Aglenchus agricola de Man, 1884

Basiria flandriensis Gerraert, 1968
B. graminophila Siddiqi, 1959
B. tumida Colbran, 1960
Boleodorus clavicaudatus Thorne, 1941
B. thylactus Thorne, 1941
B. volutus Lima & Siddiqi, 1963
Coslenchus gracilis Andrssy, 1982
Discotylenchus sp. Siddiqi, 1980
Filenchus andrassyi Szczygie, 1969
F. baloghi Andrssy, 1958
F. filiformis Btschli, 1873
F. hamatus Thorne & Malek, 1968
F. misellus Andrssy, 1958
F. sandneri Wasilewska, 1965
Filenchus vulgaris Brzeski, 1963
Filenchus sp. Andrssy, 1954
Irantylenchus vicinus Szczygie, 1970
Malenchus acarayensis Andrssy, 1968
M. andrassyi Merny 1970
M. exiguus Massey, 1969
Malenchus sp. Andrssy, 1968
Miculenchus salvus Andrssy, 1959
Ottolenchus discrepans Andrssy, 1954
O. facultativus Szczygie, 1970
Tylenchus elegans De Man, 1876
Tylenchus sp. Bastian, 1865
Aoulouz
Ouled Berhil
Taroudant
Ouled Teima
Aourir
Tiguert
Asni
Souss
Tahnaout
Marrakech
Sidi Bou Othmane
Tamellalt
Haouz
El Ksiba
Tadla
Beni Mellal
Oued Zem
Oulmes
El Hajeb
Khemisset
Geographic regions
Guerouane
Zaane
Kandar
Sefrou
Taza
Ouazzane
Bni Harchen
Jel
Chefchaouen
Asilah
Tetouan
Fnideq
Tanger
Spanish side
Rif
Meknes
Species
Guercif
Orders / Families
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
On this table, we can note that:

- Tylenchidae and Telotylenchidae were dispersed in all regions sampled; they were the most diversified families including 11 genera in
each, respectively. However, each genus was often represented by one or two species only (e.g. Amplimerlinus, Bitylenchus, Tylenchus).
Most of these species were very rare as they were detected in one or two sites only (e.g. Aglenchus agricola, Coslenchus gracilis and
Paratrophurus loofi in Rif, B. aerolatus and Miculenchus salvus in Haouz).
- Hoplolaimidae family was represented by two genera only (Helicotylenchus and Rotylenchus), but the number of species identified in
each genus was high (11 and 4 species, respectively), and they were distributed in all the regions, except in eastern Morocco (Kandar and
Jel regions).
- Longidoridae and Trichodoridae nematodes, which are known to be plant virus vectors, were detected mostly in the Rif region.
- Root-lesion nematodes (e.g. Pratylenchus spp.) and Paratylenchidae (e.g. Paratylenchus spp.) were dispersed in all the sites surveyed.
Page 122
Concerning root-knot nematodes, four identified species: Meloidogyne arenaria and M. hapla were detected in the Rif region, M.
javanica were detected mostly in southern Morocco (in Souss and Haouz), and Guerouane and Tadla regions. M. spartelensis is a new
species of Meloidogyne identified in the Rif region, another new species seems to occur in the Souss region (identification is in progress).
Others families such as Trichodoridae, Criconematidae and Psilenchidae were detected in a few sites.
Page 123
Figure S1
Page 124
Chapitre V
Diversit fonctionnelle
Page 125
Rsum
Ce chapitre a pour objectif de dcrire la diversit fonctionnelle et trophique des nmatodes
phytoparasites prsents dans les communauts associes lolivier au Maroc. Diffrents
indices cologiques ont t utiliss afin de dcrire cette diversit. Ces indices ont galement
permis de dterminer limpact de la domestication de lolivier (de lolivier sauvage vers
lolivier cultiv), les pratiques culturales (intensification de la culture, irrigation), et du
gnotype de lolivier (lignes chloroplastiques) sur la composition fonctionnelle et trophique
des communauts. Les rsultats montrent que les nmatodes phytoparasites dtects sur
lolivier se distribuent en quatre groupes fonctionnels (cp-2, cp-3, cp-4, cp-5) et en trois
groupes trophiques (phytoparasites stricts ou facultatifs et fongivores). La composition,
labondance relative et la richesse gnrique de ces diffrents groupes se rvlent impactes
par la domestication de lolivier puisque les nmatodes trs colonisateurs (cp-2) et fongivores
sont plus abondants sur oliviers sauvages alors que les nmatodes moins colonisateurs (cp-3)
et stricts sont plus abondants dans les vergers en haute densit. Les nmatodes persistants (cp5) ont t surtout dtects dans les milieux sauvages et compltement absents dans les vergers
haute densit. Une diffrence significative a t galement observe selon lintensification
de la culture entre les vergers faible et haute densit. Ltude a galement rvl un impact
du gnotype de lolivier sur ces diffrents groupes fonctionnels et trophiques. Les analyses de
la richesse gnrique de ces diffrents groupes entre les modalits de lolivier ont indiqu une
rduction de la richesse sur lolivier haute densit compare lolivier sauvage. Cette
rduction de richesse saccompagne du dveloppement de certains nmatodes tels que
Tylenchorhynchus spp. et Meloidogyne spp. dont les populations atteignent des niveaux
levs dans les vergers haute densit. Des dgts importants lis au dveloppement des
Meloidogyne pourraient tre donc attendus dans ces conditions. Ces rsultats soulignent
limportance de prendre en compte la structure trophique et fonctionnelle des communauts
afin de concevoir des stratgies de contrle des NPP bases sur la gestion de la diversit et de
la structure de leurs communauts.
Page 126
Functional and trophic diversity responses of plant-parasitic nematodes

between agro and ecosystems of olive trees
Abstract
Functional and trophic diversity had been proposed as important features for assemblages in
biological communities. This diversity could be structured as a response to biotic (interactions
between living organisms) and abiotic (interactions with the environment) factors. It could be
also impacted by disturbances in living environment. Human interventions in the ecosystems
could lead to disturb the functional and trophic diversity of biological communities such as
plant-parasitic nematodes (PPN). In this way, the present study was undertaken to firstly
describe the functional and the trophic diversity of PPN communities identified in several
olive growing areas in Morocco and to secondly investigate the impact of domestication and
cropping practices. Different ecological indices were used to describe this diversity. These
indices were compared according to olive domestication (wild vs cultivated olive) and
agricultural practices (intensification low vs high density and irrigation) and between different
olive chloroplast lineages. The results indicated that PPN communities were diversified
according to their reproduction strategies (four groups of cp-value) and their feeding (three
trophic groups). The composition, the relative abundance and the genus richness of these
different groups were mainly impacted by olive domestication and intensification practices.
Cp-2 and fungal feeder nematodes were abundant on wild olive, while cp-3 and obligate plant
feeders were highly abundant on high density olive cultivation. The abundance of these
groups was also significantly different between low (traditional) and high density cultivation.
The cultivated olive conditions (especially in high-density orchards) were related to reduced
genus richness in the different groups and enhanced the development of some taxa such as
root-knot nematodes (Meloidogyne spp.) that leads to expect crop damages. Consequently,
this study suggested that the factors favouring the development of these nematodes
(agricultural practices such as crop intensification, irrigation and tillage) should be controlled.
It also underlined the necessity to take into account the functional and the trophic structure of
PPN communities before designing control strategies. Further studies focused on the
functional and trophic diversity of the total nematode fauna associated to olive trees
(including free living nematodes) should be envisaged to investigate their interactions with
PPN and the potential impact of some of them as PPN predators.
Kew-words: community, functional diversity, Morocco, olive, plant-parasitic nematodes.

Page 127
1. Introduction
Since the last decades, ecosystems are subjected to increased human activities that endanger
the ecosystemic services they provide. Taxonomic diversity, functional status and diversity of
ecological communities are also known to influence ecosystem processes (Mouillot et al.
2011). Consequently, both changes in environmental conditions (e.g. global warming) and
modifications of biological communities (e.g. biodiversity erosion) affect ecosystem
processes (Chapin et al. 1997; Hooper et al. 2005). Functional diversity has been proposed as
an important feature of biological assemblages (Mason et al. 2005) and may offer a great
insight into how ecosystems are changing (Hopper et al. 2005). This impact of functional
diversity on ecosystem processes has been hypothesized for communities of microbes
(Griffiths et al. 1997), plants (Petchey et al. 2004) and of animals (Bremner et al. 2003).
Several essential functions of soil and some of its physicochemical properties, depend
on the action of the living organisms they host, since these organisms are involved in several
ecological processes such as the carbon cycling, the decomposition of the organic matter,
energy flows, etc. (Gobat et al. 2010). Any disturbance in the soil could reflect changes in the
composition of its living organisms. Soil organisms are consequently proposed to predict
disturbances in soil functions. Based on their sensitivity to pollutants and environmental
disturbance (Bongers and Ferris, 1999), on their rapid response to new resources, the
nematode community structure offers tools to assess changes in soils conditions (Bongers and
Bongers, 1998). They were widely used as indicators of soil health and of the influence of
human activities on its biological and physicochemical characteristics (Neher, 2001;
Pankhurst et al. 2005; Wasilewiska, 2006).
Soil nematodes are usually allocated to trophic and functional groups. According to
their food preferences, they encompass a range of trophic groups that include predators and
omnivorous species, as well as bacteria, fungus and plant feeder taxa (Yeates, 2003).
Considering the plant, these nematodes could be distinguished as non-plant-parasitic (freeliving) or as plant-parasitic nematodes. They all exhibit various reproductive strategies
(functional diversity) defined by five cp-values ranging from colonizer (r strategy, cp-1) to
persistent (K strategy, cp-5) nematodes. This range is assigned to the nematode families
(Bongers, 1990). As nematode species are in monophyletic families, the species belonging to
the same family exhibit similar responses to environmental perturbation (Bongers and Ferris,
1999). Most of the NPN (free-living nematodes) are included in the cp-values 1 to 5, while
the PPN (plant-parasitic nematodes) are included in cp-values 2 to 5. The study of functional
Page 128
diversity allows linking the species present in a community to the types and rates of
ecological functions that are occurring by measuring the traits of species (Cadotte et al. 2011).
Plant-parasitic nematodes PPN are obligate parasites. They feed exclusively on the
cytoplasm of living plant cells (Williamson and Gleason, 2003). They can decay a wide range
of crops, causing annual losses estimated at multibillion dollars on a worldwide scale (Barker
and Koenning, 1998; Williamson and Gleason, 2003; Agrios, 2005). Functional and trophic
compositions of communities were demonstrated to be controlled by the biophysical,
chemical and hydrological conditions of the soil (Yeates and Bongers, 1999). This
composition could be also structured according to disturbances in the environment (Ferris et
al. 2001) such as those caused by agricultural and intensification practices (Postma-Blaauw et
al. 2010; Barths et al. 2011; Villenave et al. 2013). In order to investigate how functional and
trophic composition of PPN communities could be structured in response to environmental
disturbances, olive tree is a good model of host plant because of its evolution from relic
refuges, its domestication around the Mediterranean Basin from wild to cultivated olive, its
various cultivation practices (e.g. intensification and irrigation practices), in addition of its
genetic diversity (four chloroplast lineages were defined in olive around the Mediterranean
Basin; Besnard et al. 2007; 2013). These different features are all available in Morocco where
no studies were already conducted to describe this composition of PPN communities on olive.
The aim of the present study was thus to describe the trophic and the functional diversity of
PPN communities associated to olive trees in Morocco, and to analyse how this diversity is
structured in communities and how it could be led by olive domestication and in response to
agroecosystem management.
2. Materials and Methods

Sampling took place in Morocco in March and April 2012. A total of 213 soil samples were
collected from 94 sites. The survey was conducted in several geographic regions all along a
northeast-southwest transect about 900km long (Tab. 1) : the Souss region (15 sites) located
in the south facing side of the High Atlas Mountains, near Agadir city, where trees sampled
were wild (some of them being O. maroccana), feral and traditionally cultivated; in the Haouz
region (15 sites) located in the north facing side of the High Atlas Mountains, near Marrakech
city, where trees sampled were cultivated (traditional and high density) and feral; in the Tadla
region (5 sites) located along the north facing side of the southern Middle Atlas Mountains,
near Beni Mellal city where trees sampled were wild, feral or traditionally cultivated; in the
Page 129
Guerouane (13 sites of feral, traditional and high density cultivation) and the Zaane (3 sites
of wild olive) regions situated to the South of Meknes city; in the Jel plain situated to the East
of Taza city in eastern Morocco (5 sites) and in the Kandar region (5 sites) located in the
northern Middle Atlas Mountains, South of Fes city where olive trees are traditionally
cultivated (low density of olive trees) in these two regions; on both Atlantic and
Mediterranean facing sides of the Rif mountains in the North (33 sites) where most of the
olive trees are wild, feral and some traditional cultivation.
2.2. Soil sampling

The soil samples were collected from the upper rhizosphere (the 20-30-cm deep layer
inhabited by plagiotropic roots) with a small spade. The sampling was conducted under the
foliage of each olive tree; five sub-samples were collected. On cultivated olive (traditional
and high-density cultivation), the sampling was conducted in orchards under four trees
located at a distance of 10 meters along transects. The 20 sub-samples recovered were
combined to form one 1-dm3 reference sample per orchard. For each cultivation modality, one
to five orchards were considered as repetitions according to their availability in each site. In
wild (including O. maroccana) and in feral olive sites, the sampling was conducted under 25
trees considered separately as repetitions.

elutriation procedure (Seinhorst, 1962). Plant-parasitic nematodes (PPN) belonging to the
Aphelenchida, Dorylaimida, Triplonchida and Tylenchida orders were identified by genus
(May and Mullin, 1996) and enumerated in 5-cm3 counting chambers (Merny and Luc, 1969)
under a stereomicroscope. PPN population levels were expressed per dm3 of fresh soil. All the
analyses processed on live material were performed in the nematode quarantine area (French
Government Agreement n80622) of the Research Unit Centre de Biologie pour la Gestion
des Populations (Montpellier, France).
2.4. Nematode ecological indices

Functional diversity of plant-parasitic nematodes in communities: plant-parasitic nematode
(PPN) genera detected in communities were distributed in functional groups according to the
cp-value of the family to which they belong. Then, several indices were used to describe the
functional diversity of the community: (i) the plant-parasitic index (PPI = cpini/N) which
Page 130
quantifies the plant-feeding diversity of the communities, (ii) the relative mean abundance
(%) of each cp-value class in communities calculated as following: Rcpi = cpini/N, (Bongers,
1990); (iii) the genus richness included in each cp-value class.
Trophic diversity in communities: PPN genera were assigned to the trophic groups according
to Wasilewska (2006): obligate plant feeders (OPF), facultative plant feeders (FPF) that
alternatively feeds on fungi, and fungal feeders (FF) that alternatively feed on plants. Then the
trophic diversity was described by (i) the relative abundance of individuals within each
trophic group, and (ii) the genus richness included in each trophic group (Neher et al. 2004).
2.5. Data analyses

These different indices were essentially compared according to olive data because our
analyses on biodiversity and patterns of PPN in communities were proven that these variables
are the most important in driving the structure and the diversity of PPN in communities (Ali et
al. in prep, Chapter IV). They were thus compared according to (i) olive growing modalities
as wild WO, feral FO, traditional TR and high density cultivation HD; (ii) type of irrigation
such as irrigated I (that concerns some traditional and high density cultivation) or rainfed R
(that concerns wild and feral and some traditional olive); (iii) olive lineages described in four
lineages E1 (essentially detected in all cultivated and in some feral olives), E2, E3 (detected
in wild and in some feral olives) and M specific to O. maroccana (lineage specific to O. e.
subsp. maroccana) (Besnard et al. 2007; 2013).
The mean values of these diversity data were analyzed between these variables
according to Wilcox test (non-parametric) with Bonferroni corrections for all pair-wise
multiple comparisons using the R software. Differences obtained at levels of P < 0.05 were
considered significant.
Page 131
Table1: Location of the olive sampling sites surveyed in Morocco.

Geographic region
City
Olive modality
No of sites
Latitude N
(decimal)
Longitude W
(decimal)
Tiguert
Aourir
wild
wild
wild
feral
wild
feral
feral
wild
feral
wild
wild
feral
wild
wild
wild
feral
wild
wild
feral
wild
wild
feral
2
1
1
1
1
4
2
1
1
1
1
1
1
2
7
1
1
2
1
2
2
1
2
2
4
3
2
5
3
2
1
2
1
1
4
1
8
3
2
2
1
2
5
30.63
30.52
30.81
30.42
30.42
30.74
30.61
30.65
30.55
32.15
31.37
31.46
31.70
31.69
31.63
31.57
31.28
32.58
32.21
32.32
33.32
33.33
33.70
33.77
33.88
33.85
33.63
33.87
34.25
34.26
35.79
35.78
35.54
34.79
35.07
35.05
35.07
35.07
35.38
35.54
34.94
34.79
34.79
9.86
9.59
9.14
9.02
9.02
8.77
9.34
8.18
8.66
7.26
7.95
7.98
7.69
8.11
8.10
7.97
7.96
5.98
6.83
6.39
6.07
6.00
5.63
5.71
5.41
5.39
5.83
4.88
3.80
3.74
5.92
5.37
5.62
5.77
5.33
5.35
5.33
5.32
5.37
5.62
5.53
5.77
5.77
Ouled Taima
Souss
Taroudant
Ouled Berhil
Aoulouz
Haouz
Tamellalt
Sidi Bou Othmane
Marrakech
Tahnaout
Asni
Tadla
Zaane
Beni Mellal
El Ksiba
Oulmes
Oued Zem
El Hajeb
Guerouane
Kandar
Jel
Meknes
Khemisset
Sefrou
Taza
Msoun
Tanger
Fnideq
Tetouan
Asilah
Rif
Chefchaouen
Bni Harchen
Ouazzane
Page 132
3. Results
Forty-seven plant-parasitic nematode genera were detected in the olive growing areas sampled
in Morocco. Their cp-value and their trophic preference are given in Table 2.
3.1. Functional diversity of the plant-parasitic nematode communities

The PPN identified on olive in Morocco were distributed in all the cp-2, cp-3, cp-4 and cp-5
groups. Comparing the functional indices between olive data, the wild olive areas (WO) and
the high-density orchards (HD) exhibited nematode communities with significantly highest
plant-parasitic indices (PPI) than in feral (FO) and traditional low-density (TR) conditions
(Tab. 3). That means that WO and HD olive areas inhabited significantly more plant-feeding
nematode communities than other olive systems. Yet, both olive lineages and irrigation did
not have any effect on the PPI index. The most opportunists PPN (cp-2 and cp-3) dominated
in all the PPN communities (44% and 48% respectively). The overall abundance and
occurrence of the persistent nematodes (cp-4 and cp-5) was very low (4% for each cp class).
The olive modalities did not have any effect on the cp-4 class. Cp-2 and cp-3 nematodes were
more abundant in cultivated olive areas (traditional and high-density cultivation), while cp-5
nematodes occurred more in wild olive areas and were completely absent in HD orchards. The
olive diversity (chloroplastic lineages) did not have any effect on the relative abundance of
colonizer nematodes (cp-2 and cp-3), except the M lineage (O. maroccana) that unbalanced
the cp-2 / cp-3 ratio in favour of cp-3. The most persistent nematodes (cp-5) were more
abundant on E2 and E3 olive genotypes (feral and wild olive) than on the other lineages. The
rainfed vs irrigation modality never impacted the colonizer/persistent ratios, except the cp-5
class that was decreased by irrigation.
The cp-2 and cp-3 groups were the most rich with genera (Tab. 2). Comparing this
richness in each group between olive modalities (Tab. 4), the PPN communities detected in
non-cultivated situations (WO and FO) demonstrated more richness and diversity compared
to those detected in the cultivated situations (TR and HD). But, more precise analyses on the
composition of cp-3 nematodes and their abundance demonstrated that some genera such as
Tylenchorhynchus spp., Meloidogyne spp. and Helicotylenchus spp. reached higher levels on
high-density cultivation than on wild olive (Fig. 1A). In the same way, high populations of
cp-2 nematodes were recorded in traditional olive cultivation and in feral olive sites
respectively, mainly due to the abundance of taxa such as Filenchus spp. and Boleodorus spp.
respectively (Fig. 1B).
Page 133
3.2. Trophic diversity of plant-parasitic nematode communities

The OPF nematodes were the most dominant in communities (62%), while the FPF and the
FF nematodes were the less frequent (26% and 12%, respectively). FF nematodes were
significantly more numerous in wild olive areas and on trees belonging to lineages E2 and E3
(Tab. 3). FPF and OPF nematodes were more abundant in traditional and high-density
orchards respectively. The ratio between FPF and OPF nematodes was unbalanced in favour
of OPF in high-density orchards and on M lineage olive trees, and in favour of FPF in
traditional orchards, in feral areas, and on E lineage olive trees. The rainfed vs irrigation
modality did not have any effect on the trophic groups.
Considering the genus richness recorded in each trophic group, the FPF and OPF were
represented by the highest number of genera (Tab. 2). Comparing this richness in each group
between olive modalities (Tab. 4), the PPN communities detected in non-cultivated situations
(WO and FO) demonstrated their richness and diversity compared to those detected in the
cultivated situations (TR and HD).
Page 134
Table 2: Plant-parasitic nematode genera detected in the olive growing areas sampled in
Morocco; with indication of the functional (cp-value) and trophic groups to which they
belong. FF= fungal feeders; FPF= facultative plant feeders; OPF= obligate plant feeders.
Order
Family
Genus
Cp -value
Trophic group
Aphelenchidae
Aphelenchus
FF
Aphelenchoididae
Aphelenchoides
FF
Aprutides
FF
Longidorus
OPF
Xiphinema
OPF
Paratrichodorus
OPF
Trichodorus
OPF
Ditylenchus
FPF
Nothotylenchus
FF
Ogma
OPF
Criconema
OPF
Criconemella
OPF
Macroposthonia
OPF
Dolichoridae
Neodolichorhynchus
OPF
Heteroderidae
Heterodera
OPF
Hoplolaimidae
Helicotylenchus
OPF
Rotylenchus
OPF
Meloidogynidae
Meloidogyne
OPF
Neotylenchidae
Neotylenchus
FF
Paratylenchidae
Cacopaurus
OPF
Aphelenchida
Dorylaimida
Longidoridae
Triplonchida
Trichodoridar
Tylenchida
Anguinidae
Criconematidae
Gracilacus
OPF
Paratylenchus
OPF
Pratylenchoides
OPF
Pratylenchus
OPF
Zygotylenchus
OPF
Psilenchidae
Psilenchus
FPF
Rotylenchulidae
Rotylenchulus
OPF
Pratylenchidae
Telotylenchidae
Tylenchidae
Amplimerlinius
OPF
Bitylenchus
OPF
Merlinius
OPF
Nagelus
OPF
Paratrophurus
OPF
Scutylenchus
OPF
Telotylenchus
OPF
Trophurus
OPF
Tylenchorhynchus
OPF
Aglenchus
FPF
Basiria
FPF
Boleodorus
FF
Coslenchus
FPF
Discotylenchus
FPF
Filenchus
FPF
Irantylenchus
FPF
Malenchus
FPF
Miculenchus
FPF
Ottolenchus
FPF
Tylenchus
FPF
Page 135
Table 3: Mean plant-parasitic index (PPI) and relative mean abundance (%) of each cp-i value
and of each trophic group (codes for trophic groups are listed in Table 2). Different letters (a, b,
etc.) indicate significant differences between olive modalities according to Wilcoxon test.
Olive data
Olive growing modality
wild olive WO
feral olive FO
traditional cultivation TR
high density cultivation HD
Olive lineage
E1
E2
E3
M
Water supply
Rainfed
irrigation
PPI
Rcp-2
Rcp-3
Rcp-4
Rcp-5
2.65 a
2.57 ab
2.49 b
2.74 a
45.58 a
46.19 a
52.62 a
25.96 b
48.89 b
52.19 ab
46.68 b
73.71 a
0.08
0.03
0,04
0,33
5.44 a
1.59 b
0.66 b
0.00 b
2.57
2.60
2.62
2.78
44.94 a
50.30 a
44.19 a
24.61 b
53.84 b
44.66 c
52.71 bc
74.00 a
0.05
0.13
0.00
0.00
2.61
2.55
45.91
46.27
50.69
53.11
0.05
0.11
FF
FPF
OPF
8.69 a
3.62 b
3.69 b
0.12 b
32.63 b
39.35 ab
46.89 a
25.13 b
58.68 ab
57.03 ab
49.42 b
74.76 a
1.17 b
4.91 a
3.10 a
1.39 ab
1.67 b
8.43 a
8.61 a
3.92 ab
39.99 a
39.02 a
31.49 a
17.18 b
58.34 b
52.55 b
59.90 ab
78.90 a
3.35 a
0.50 b
5.93
2.84
36.28
41.71
57.78
55.45
Table 4: Richness of plant-parasitic nematode genera within each functional (cp-value) and
trophic group according to olive growing modalities (codes for olive growing modalities are
listed in Table 3).
Olive variables
WO
FO
TR
HD
cp-2
24
13
11
5
cp-3
23
14
11
11
cp-4
1
1
1
2
cp-5
3
2
2
0
FF
5
3
4
1
FPF
14
8
6
3
OPF
32
19
15
14
Page 136
Figure 1: Abundance of plant-parasitic nematodes in cp-3 (A) and cp-2 (B) functional groups
according to the olive growing modality.
4. Discussion
This study demonstrated the allocation of PPN in several functional and trophic groups that
could be structured according to olive growing conditions. This study highlighted the impact
of olive domestication (wild vs cultivation) and of intensification (traditional vs high density
cultivation) practices in the structuration of functional and trophic groups of PPN in
communities. This impact was clear on the Plant-Parasitic Index value (PPI), on the relative
abundance of each group as well as on their genus richness.
Page 137
A clear distinction in functional and trophic composition was revealed over the olive
domestication gradient especially between wild and high-density olive cultivation. Cp-2 and
FF nematodes appeared to be enhanced in wild olive conditions, while, cp-3 and OPF
nematodes were highly present on high-density olive. Nematodes allocated to cp-5 appeared
to be especially related to wild olive, and were absent on high-density olive conditions. Feral
and traditional olives, which present intermediate positions on this domestication gradient,
generally exhibited intermediate content of these nematodes. The genus richness within each
group was also impacted by this gradient. As an example, OPF or cp-3 nematode
communities detected in high abundance on high-density olive hosted less genera compared
to those detected on wild olive. This was consistent with others that demonstrated that
cultivation intensification usually does not reduces the number of nematode trophic groups,
but may change the composition of the trophic group (Ferris et al. 1996; Berkelmans et al.
2003; Van Eekeren et al. 2008). The agroecosystem intensification from low to high-density
practices also impacted the functional and trophic diversity in communities. The PPI value
was significantly different between traditional (low-density) and high-density olive
cultivation. Furthermore, the functional and trophic groups were opposed between these two
modalities because abundant cp-2 and FPF nematodes were recorded on traditional olive,
while, cp-3 and OPF were more abundant on high-density olive. The genus richness was
usually higher in traditional than in high-density olive orchards. Agricultural intensification
has been demonstrated affecting the functional composition at different taxonomic levels
(Postma-Blaauw et al. 2010). Considering the biological characteristics of the nematodes and
their diversity within communities, variability would be expected in the response of members
of different trophic levels to disturbance (Kimenju et al. 2010). Usually, cultivation conditions
induce favorable environments for PPN multiplication, especially irrigation that enhances the
development of plant roots (El-Borai and Duncan, 2005; Castillo et al. 2010). These
conditions also increase the PPN infestation in nurseries (Nico et al. 2002; Sanei and
Okhovvat, 2011). That could explain the high abundance of OPF or cp-3 nematodes (rstrategy colonizers), and consequently the high value of PPI recorded in these cropping
conditions. Moreover, the olive cultivation enhances anthropogenic practices (tillage,
weeding, fertilization, removal of farm products, irrigation, etc.), which are very likely to
select and multiply the most competitive and harmful PPN species, such as root-knot
nematodes (Meloidogyne spp.) on HD olive. That should attract attention on the harmfulness
and the economic importance of these nematodes for olive cultivation that has increased in
recent years because most chemical control products have been banned due to environmental
and health concerns while other inputs increased (Castillo et al. 2010).
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On the opposite to cp-3 (r-strategy colonizer) nematodes, cp-5 nematodes (K-strategy

persisters) composed of Dorylaimidae nematodes (Xiphinema spp. and Longidorus spp.) were
numerous in wild olive areas. As they are very sensitive to environmental distributions, these
nematodes were not recorded in high-density orchards highly affected by agricultural
practices that correspond to disturbing stresses. In this vein, others observations (Ettema and
Bongers, 1993; Bongers and Ferris, 1999; Van Eekeren et al. 2008) demonstrated that the
persistent nematodes are reduced by intensive managements, while greater cp-value
nematodes are generally associated with low stress and undisturbed environments (Bongers
and Ferris, 1999; Ferris et al. 2001).
The wild olive conditions also hosted more fungal feeder nematodes. That could be related to
high organic matter contents in wild olive areas where the plant litter accumulation and
degradation increases food for fungi (Ferris et al. 2004) and then support large populations of
fungivorous nematodes (Bohlen and Edwards, 1994). The distinction between fungivorous
nematodes (Aphelenchidae or Aphelenchoididae) and facultative plant feeders is not always
obvious, and that varies according to the references. The allocation of Tylenchidae as fungal
feeders or epidermal cell feeders is still one of the most important discussion points among
nematologists (Bongers and Bongers, 1998). Anyways, these trophic groups, which mainly
corresponds to colonizer nematodes (cp-2) that are well adapted to various habitat conditions
(Tsiafouli et al. 2006) such as cropped soils (Freckman and Ettema, 1993; Ferris et al. 2004),
grasslands (de Goede and Bongers, 1998), and logged forests (McSorley, 1993). The absence
of agricultural practices or the reduction of land-use was demonstrated leading to increase the
abundance of these trophic groups (Barths et al. 2011). As an example, a higher fungal
component (fungal-feeders and facultative plant-feeders) leading to a significant higher part
of the fungal decomposition pathway in forests than in crops (Villenave et al. 2013).
In addition, an effect of the olive diversity (chloroplastic lineages) on the functional and
trophic diversity was confirmed. This effect was clearer on the diversity of the nematodes in
communities than on their abundance, especially on the M lineage (but M repetitions were
very low) and E3 wild olive lineages that hosted more cp-3 and obligate plant-feeding
nematodes (OPF). This correspondence between the olive genotype and the nematode
diversity could be related to different olive tree susceptibilities to PPN infections, as it was
previously established with cultivars (Palomares-Rius et al. 2012). These results may confirm
plant-parasite co-adaptation relationships (Nuismer and Otto, 2005), especially with
nematodes (Zinoviev, 2014).
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Consequently, these changes in nematode community structure could be indicative of wide

ranging changes in physical, chemical and biological properties of the soil that lead to
influence the abundance, distribution and structure of nematode communities (Kandji et al.
2001).
5. Concluding remarks
The present study indicates that the PPN communities identified on olive trees in Morocco
were diversified according to their reproduction strategies and their feeding. Their occurrence
and abundance significantly differ according to domestication and intensification olive
practices. The cultivated olive conditions (especially in high-density orchards) were
characterized by the development of some taxa such as root-knot nematodes (Meloidogyne
spp.) that leads to expect significant losses in these conditions. Consequently, factors
favouring the development of these nematodes including agricultural practices such as crop
intensification, irrigation and tillage should be controlled. That underlines the necessity to
take the PPN structure in communities into account in order to design control strategies based
on the management of their diversity and on their competitive interactions. Further studies
focused on the functional and trophic diversity of the total nematode fauna associated to olive
trees (including the free living nematodes) should be envisaged to investigate their interaction
and their potential impact on PPN as biological factors for their control.
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Chapitre VI
Patrons des communauts de

nmatodes phytoparasites
Page 143
Rsum
Le but de cette tude tait de dcrire les patrons des communauts de nmatodes
phytoparasites (NPP) associes lolivier au Maroc, et de dfinir les variables agronomiques
et environnementales susceptibles de contribuer la structuration des communauts. Ltude
a dabord procd une analyse de la dominance des 47 genres identifis dans lensemble des
oliveraies chantillonns. Cette dominance a t dcrite par la frquence et labondance totale
de chacun de ces genres dans les chantillons. Des analyses multivaries (ACP) ont t
ensuite appliques afin de dcrire ces patrons et dterminer les corrlations existantes entre
eux. Ces patrons ont t confronts des variables agronomiques (la modalit de lolivier
chantillonn
et
sa
ligne
chloroplastique,
lirrigation)
et
dautres
variables
environnementales (paramtres physico-chimiques du sol et du climat) afin de dterminer

comment ces patrons sont structurs en rponse ces variables. Les analyses de co-inertie
ralises ont relev une contribution majeure des variables lies lolivier dans cette
structuration. Cette contribution a reflt une structure diffrente selon un gradient de
domestication de lolivier (entre olivier sauvage et olivier cultiv, en particulier, lolivier
cultiv haute densit), et selon un gradient dintensification de la culture (entre faible
(culture traditionnelle) et haute densit). Les communauts de NPP dtectes dans les milieux
sauvages sont surtout structures par des nmatodes comme Xiphinema, Nothotylenchus et
Heterodera, alors que les milieux cultivs ( haute densit, en particulier) sont surtout
domins par les genres Meloidogyne (nmatode dimportance agronomique et conomique),
et Tylenchorhynchus. Les variables abiotiques testes ont permis de distinguer les nmatodes
en trois groupes selon leur sensibilit au type de sol, au contenu en matire organique, au pH
et aux tempratures. Le genre Helicotylenchus, qui sest rvl le plus largement distribu sur
olivier, a t trait en dtail afin de dcrire la contribution de ces diffrents facteurs dans la
distribution des espces du genre et dans leur structuration en communauts. Les observations
faites par cette tude rvlent le rle important de la domestication (olivier sauvage vs cultiv)
et de l'intensification (olivier cultiv faible densit vs cultiv haute densit) de la culture
conduisant les communauts de NPP vers des communauts plus pathognes pour la culture.
Ltude conclue sur la ncessit d'tudier et de comprendre l'cologie des communauts de
NPP afin de comprendre les processus d'assemblage des nmatodes en communauts. Ces
connaissances peuvent fournir des informations utiles pour une gestion durable des NPP et
pour une prservation des agro et des cosystmes.
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Patterns of plant-parasitic nematode communities in olive growing areas in

Morocco: responses to anthropogenic and environmental factors
Abstract
Assemblages of different species in communities could be the consequence of historical,
biotic and abiotic factors. Considering the plant-parasitic nematodes (PPN), which endanger
the agricultural production, their assemblage in communities could be led by factors such as
the host plant and soil characteristics. The olive trees, which are good hosts for PPN, exhibit a
high botanical and genetic variability; they also grow according to a wide range of soil and
climate conditions. These conditions could be hypothesized impacting the structure of PPN
communities. In that way, the present study aimed to describe the structure of PPN
communities associated with olive trees in Morocco and to investigate the impact of olive
growing conditions on the community patterns. Thus, 213 soil samples were collected from
different olive growing areas in Morocco. Multivariate analyses were applied to investigate
correspondences between nematodes genera and olive environmental data. The study clearly
indicated a significant role of the environmental characteristics measured (olive growing
modality, soil type, organic matter content, pH, minimal and maximal temperature, in
particular) that led to distinguish three situations: (i) some nematode genera such as
Helicotylenchus spp., appeared to be not impacted by these conditions because they are
widely distributed in all the olive growing areas (wild as well as feral and cultivated olive);
but, at the species level, these environmental variables could strongly impact the distribution
of the species; (ii) other genera, such as Meloidogyne spp., were very rare in wild olive areas,
while they dominate in orchards, especially cultivated according to high-density practices;
(iii) Xiphinema and Heterodera appeared to be more related to wild olive conditions. These
observations confirmed that land-use changes according to olive domestication (wild vs
cultivated) and intensification (low- to high density cultivation) are able to lead to more
pathogenic PPN communities. It is thus necessary to study ecology of nematode communities
which will aim at understanding assemblage processes; the knowledge of how populations
interact in communities (life trait evolution, adaptation) may provide clues and keys for
population management and sustainable use/preservation of agro-eco-systems.
Key-words: community, human practices, Morocco, olive, patterns, plant-parasitic

nematodes.
Page 145
1. Introduction
Ecology of communities has been one of the most compelling and attractive themes in
modern biology, and at the same time one of the most elusive to define (Cohen, 2013). It is
generally accepted that living organisms are distributed into distinct species constituting a
"community" or a "biota". Each individual within its community is subjected to the influence
of biotic and abiotic factors induced by interactions with others organisms and with the
environment. The assemblage of individuals of different species in communities depends on
both historical (biogeographical) and ecological phenomena (Webb et al. 2002). The complex
processes involved in species coexistence and community assemblage have long-fascinated
ecologists and evolutionary biologists (Mayfield and Levine, 2010). Community composition
and structure are important factors affecting diversity patterns in communities (Zhao et al.
2003). It has been hypothesized that community organization was the result of three main
filters operating at different levels (Belyea and Lancaster, 1999; Lortie et al. 2004): (i)
dispersion, which determines the pool of potential colonists available at a particular time and
place in relation to stochastic biogeographical events and landscape structure; (ii) abiotic
filters, which express the impacts of environmental conditions (soil pH, humidity,
temperature, etc.), resource availability and disturbance (as hosts for parasites); (iii) biotic
filters, leading to positive and negative interactions among organisms in the community.
Soil communities have been described as the "poor mans tropical rainforest", due to
the relatively high level of biodiversity and to the large proportion of undescribed species they
contain, and to the limited information that is known on community structure and dynamics
(Giller, 1996). Physical, chemical and biotic factors are known to determine the presence, size
and survival of invertebrate populations within the soil, resulting in a patchy distribution
(Curry and Spergel, 1988). Several reports have shown, as an example, the relationship
between bacterial communities and various environmental factors, such as the vegetation, the
organic matter content, the temperature, the soil moisture, the nutrient availability, pH, etc.
(Fierer and Jackson, 2006; Wallenstein et al. 2007; Coolen et al. 2011). Currently, crop
intensification affects the composition and the functions of soil biota in agro-systems
(Postma-Blaauw et al. 2010). Soil pH is also considered as the most important factor
influencing microbial community structure (Lauber et al. 2009; Chu et al. 2010; Shen et al.
2013; Bartram et al. 2014).
Within soil invertebrates, nematodes are ubiquitous soil inhabitants (Pen-Mouratov et
al. 2003) and are among the most abundant and diversified biota. They feed on plants, on
Page 146
fungi and bacteria; others are carnivores or omnivores. Due to their life history, short response
time and high turnover rates in nematode diversity and community structures are closely
related to their habitats with rapid responses to environmental changes (Bongers and Bongers,
1998; Bongers and Ferris, 1999). But the most difficult problem with nematode communities
is to distinguish the factors (such as soil, host and competition), that influence community
composition and structure (Norton, 1989).
Among soil nematodes, plant-parasitic nematodes (PPN) are one of the major soil-borne
crop pests. They cause signicant economic losses on a wide range of crops (Potter and
Olthof, 1993; Koenning et al. 1999). Their physiology is very diverse according to the species
and their confinement in the soil makes them very difficult to control. Chemical products are
the main means for their control. However, the negative impact of these products on both
environment and human health leads to their restriction or interdiction. Consequently, yield
reductions caused by PPN might become higher (Noling and Becker, 1994) and safe and
efficient control options are urgently needed (Nico et al. 2002). The most current alternative
practices (e.g. nematode-resistant crop varieties, crop rotations, biological control) seem to be
insufficient because all these methods usually target some emblematic nematode taxa, and
they ignore the diversity of PPN in communities. Furthermore, these methods could induce
biotic gaps and then rearrangements in the composition and the structure of community,
which could increase aggressiveness of minor species. The structure of PPN community has
been demonstrated as a key factor to expect the pathogenicity of the community (Lavelle et al.
2004). Because PPN are obligate parasites, the host plant is the most important driving force
in nematode populations, but abiotic factors are also important in maintaining the steady state
(Norton, 1989). The structure of PPN communities and the population levels of the most
pathogenic species could be influenced by agricultural practices (Freckman and Ettema,
1993), the host plant (Makulec, 2004), and the land-use such as the successional vegetation
changes (Niles, 1991; Villenave and Cadet, 1999; Villenave et al. 2001).
Some plants, such as olive tree (Olea europaea), were domesticated from a very long
time (Zohari et al. 2000), especially around the Mediterranean Basin. The olive model offers
different anthropogenic situations, ranging from wild olive (oleasters) usually observed in
relic plant refuge areas around the Mediterranean Basin (e.g. Strait of Gibraltar; Rif
Mountains, Middle and High Atlas Mountains in the West; Anatolia in the East; Mdail and
Diadma, 2009); to feral forms are wild-looking olives that correspond either to cultivated
olive abandoned and becoming wild or to olive issued from cultivated or wild olive seeds
spread by birds; to low density (100 trees /ha) and low input olive cultivation, and finally to
Page 147
super-high density (up to 2,200 trees /ha) and high input cultivation (Therios, 2009).
Furthermore, a high genetic diversity corresponding to four chloroplast lineages (E1, E2, E3,
M) was detected within olive trees in the Mediterranean Basin (Besnard et al. 2013). Olive
trees do not suffer from PPN in wild or in traditional orchards. But nematode damages to
olive are particularly expected in nurseries and in high-density orchards, where optimum
input practices favour root proliferation and increased nematode population growth that
subsequently impacts adult trees (El-Borai and Duncan, 2005). Indeed, olive trees serve as
hosts to a large number of PPN (up to 156 species according to Ali et al. 2014). Among them,
root-knot nematodes (Meloidogyne spp.), root-lesion nematodes (Pratylenchus spp.), spiral
nematodes (Helicotylenchus spp.), and Criconemoides xenoplax are pathogenic and widely
distributed in the Mediterranean Basin, in California and in South America (Castillo et al.
2010). Other ectoparasites, including dagger, needle, stunt, and stubby-root nematode species,
are considered weak pathogens to olive. Estimation of olive losses in the USA due to
Meloidogyne spp. and T. semipenetrans ranged from 5 to 10% (Koenning et al. 1999).
Considering the olive domestication history and nematode co-adaptation processes, we
hypothesize that local PPN populations in olive cropping areas could be originating from
historical mixtures set up with native (before olive introduction) and invasive (with rootstocks from oleasters) communities, subsequently forced by climatic changes and cultivation
techniques, and new intensification. Then, the past and recent olive ecological history could
explain the present distribution of the PPN communities (Ali et al. 2014). Facing the wide
spreading of high-input olive production that enhances soil-borne parasite populations,
enlightenment about historical co-adaptation of PPN communities to olive-tree domestication,
breeding and cultivation would help the development of new sustainable management
strategies.
In order to provide first adequate answers about responses of PPN communities to
anthropogenic and environmental factors, the present study was addressed to go on with the
analysis of the PPN communities structure in the light of olive domestication and
intensification practices, its chloroplast genetic structure and others interacting factors
depending on the cultivation techniques, on the soil, and on climatic variables. These different
environmental conditions are available in olive areas in Morocco where the intention is
oriented in favor of few highly productive varieties (El Mouhtadi et al. 2014). These new
conditions of cultivation might have to face a resurgence of several pests, including PPN. In
this way, this study aimed at (i) characterizing the PPN community patterns, (ii) typing the
ecological pattern of the survey device, and (iii) analysing correspondences between data sets
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in order to verify and hierarchize hypotheses bearing on the transition between PPN trade-offs
in oleasters to diversity erosion in cultivated areas and on the susceptibility of PPN to the
olive environment.
2. Material and methods

Sampling took place in Morocco in March and April 2012. A total of 213 soil samples were
collected from 94 sites. The survey was conducted in several geographic regions all along a
northeast-southwest transect about 900km long (Fig. 1): on both Atlantic and Mediterranean
facing sides of the Rif mountains in the North (33 sites) where most of the olive trees are wild
or feral, and some are cultivated according to traditional olive cultivation; in the Jel plain
situated to the East of Taza city in eastern Morocco (5 sites) where olive trees are cultivated
according to traditional practices; in the Kandar region (5 sites) located in the northern Middle
Atlas Mountains, South of Fes city, where trees are also traditionally cultivated; in the
Guerouane (13 sites), where olive sampled were traditional or high density cultivation or
feral; the Zaane (3 sites of wild olive) regions situated to the South of Meknes city; in the
Tadla region (5 sites) located along the north facing side of the southern Middle Atlas
Mountains, near Beni Mellal city where trees sampled were wild, feral and traditionally
cultivated; in the Haouz region (15 sites) located in the north facing side of the High Atlas
Mountains, near Marrakech city, where trees sampled were cultivated (traditional and high
density) and feral; in the Souss region (15 sites) located in the south facing side of the High
Atlas Mountains, near Agadir city, where trees sampled were wild (some of them being O.
maroccana), feral and traditionally cultivated.
In addition to this variation in olive growing modalities in Morocco, olive trees grow
over a large range of regional pedoclimates, such as soil types (forest podzols associated with
brown soils, ferrous and humus in the Rif region, clay soils or sandy clay or limestone in the
High and the Middle Atlas regions), annual precipitation regimes (from 800-1000 mm in the
North to less than 200 mm in the South), and minimal and maximal temperatures (e.g. on
average 14-21C in the Souss region, 12-24C in the Rif region, 10-26C in the Middle, 1128C in High Atlas).
Page 149
Figure 1: Localization of sampling sites in Morocco with indications on the olive growing
modalities surveyed in each site.
Page 150
2.2. Soil sampling

Considering that all PPN spend all or almost their life in the soil (Cadet and Thioulouse,
1998), the sampling was only focused on soil. It was conducted under the foliage of each
olive tree. Five sub-samples were collected from the upper rhizosphere (the 20-30-cm deep
layer inhabited by plagiotropic roots) with a small spade. On cultivated olive (traditional lowdensity and high-density cultivation), the sampling was conducted in orchards under four
trees located at a distance of 10 meters along transects, the 20 sub-samples recovered were
combined to form one 1-dm3 reference sample per orchard. For each cultivation modality, one
to five orchards were considered as repetitions according to their availability in each site. For
wild (including O. maroccana) and feral olive, the sampling was conducted under 25 trees
considered separately as repetitions in each site because these modalities were less numerous
than orchards and their growing areas were more heterogeneously and spot distributed.

elutriation procedure (Seinhort, 1962). PPN belonging to the Aphelenchida, Dorylaimida,
Triplonchida and Tylenchida orders were enumerated in 5-cm3 counting chambers (Merny
and Luc, 1969) and identified to genus level based on dichotomous keys as described by Mai
and Mullin (1996). The population levels were expressed per dm3 of fresh soil. Concerning
specific identification, the nematode suspensions were preserved in glycerol (De Grisse,
1969) and specimens were mounted onto slides (Cobb, 1917) for microscopic observation.
2.4. Genetic characterisation of the olive trees

Three olive leaves were collected in correspondence with soil samples to determine the
chloroplast haplotype of each tree. Chloroplasts are inherited by the mother, and their DNA
variation allows an identification of maternal lineages (Besnard et al. 2000). DNA was
extracted with a CTAB (cetyltrimethylammonium bromide) method from fresh leaves
desiccated in silica gel (Besnard et al. 2000). A plastid genome profiling method based on
microsatellites, indels and single nucleotide polymorphisms (SNPs) were used to characterize
each sample (Besnard et al. 2011). Four haplotype lineages E1, E2, E3 and M were reported
(Besnard et al. 2007; 2013).
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2.5. Climate and soil characteristics

Mean annual maximal and minimal temperatures and mean annual rainfall data were collected
from climate stations close to the sampling sites.
From each soil sample, a 100-cm3 dry and sieved (2 mm) aliquot was used for physicochemical analyses. They were performed by the Soil Laboratory of the INRA (Institut
National de la Recherche Agronomique) in Arras, France. The soil parameters analysed were:
(i) the soil texture defined by the clay (Cl), the silt (Si) and the sand (Sa) contents that allow
to distinguish three soil types (clayey, silty and sandy soils) according to the texture diagram
defined by Jamagne (1967); (ii) the organic matter content (OM); (iii) the pHH2O of soil; (iv)
the content each of nitrogen (N), assimilated phosphorus (P2O5); calcium (Ca), copper (Cu),
iron (Fe), magnesium (Mg), manganese (Mn), sodium (Na), potassium (K), zinc (Zn); (v) the
cation exchange capacity (CEC).
2.6. Definition of environmental variables

In order to analyse the response of the community structure to plant genetics, anthropogenic
and environmental factors, we defined different types of variables (Tab. 1): (i) olive growing
modalities as wild (O. e. ssp. maroccana and O. e. ssp. e. var. sylvestris), feral, traditional and
high density cultivation; (ii) type of irrigation such as irrigated (that concerns traditional and
high density cultivation) or rainfed (that concerns wild and feral olive); (iii) olive lineages
described in four lineages E1, E2, E3 and M. (iv) climate (maximum and minimum
temperatures, annual rainfall); (v) soil physico-chemical characteristics.
2.7. Data analyses

The dominance of the nematode genera in the samples was firstly estimated by modelling the
abundance (A) and the frequency (F) of each genus in the whole sampling (Fortuner and
Merny, 1973). The scarcest genera (total abundance less than 1%) were then excluded from
the data set prior to running analyses. Afterwards, PPN community patterns were described
according to multivariate statistical analyses. The first step aimed at describing the correlation
between variables within each data set: Principal Component Analyses (PCA) were
performed on quantitative data such as nematodes and soil factors, and Factorial
Correspondence Analyses (FCA) were performed on qualitative data such as cultivation and
climate data. The second step aimed at establishing the correspondences between nematode
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patterns and environmental data (olive, climate and soil variables) using co-inertia analyses
COA (Dray et al. 2003). Considering olive and climate data, the variables were used as a
whole for FCA analyses, but classes were defined for co-inertia analyses in order to
investigate the impact of each class on nematode genera. The multivariate analyses were
performed using the ade-4 package (Dray et al. 2007). The third step aimed at examining the
influence of the most significant environmental variables (selected according to co-inertia
analyses) on PPN communities using GLM models using the MASS package (Ripley et al.
2015). Differences obtained at levels of P < 0.05 were considered significant. All these
analyses were performed using the R v3.0.1 software (R Core Team, 2013).
Table 1: Environmental variables and their corresponding codes.

Olive data
Olive growing
wild olive
feral olive
high density cultivation
Code
WO
FO
TR
HD
Olive lineages
Lineage E1
Lineage E2
Lineage E3
Lineage M
E1
E2
E3
M
Water supply
rainfed
irrigation
R
I
Climate data
maximum temperature (C)
20>T23
23>T25
> 25
Code
Tmax1
Tmax2
Tmax3
minimum temperature (C)

7>T9
9>T12
> 12
Tmin1
Tmin2
Tmin3
annual rainfall (mm)

200>AR400
400>AR700
AR> 700
AR1
AR2
AR3
Soil data
Code
soil type
sandy
silty
clayey
organic matter ()
pHH2o soil
nutrients
nitrogen
assimilated phosphorus
calcium
copper
iron
magnesium
sodium
potassium
zink
cation exchange capacity
Sa
Si
Cl
OM
pH
N
P2O5
Ca
Cu
Fe
Mn
Na
K
Zn
CEC
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Table 2: Nematodes genera and their corresponding codes.

PPN genus
Code
PPN genus
Code
PPN genus
Code
Aglenchus
Agl
Helicotylenchus
Hel
Paratrophorus
pTro
Amplimerlinius
Amp
Heterodera
Het
Paratylenchus
pTyl
Aphelenchoides
Apo
Irantylenchus
Ira
Pratylenchoides
Pro
Aphelenchus
Apu
Longidorus
Lon
Pratylenchus
Pra
Aprutides
Apr
Macroposthenia
Mac
Psilenchus
Psi
Basiria
Bas
Malenchus
Mal
Rotylenchulus
Rol
Bitylenchus
Bit
Meloidogyne
Mel
Rotylenchus
Rot
Boleodorus
Bol
Merlinius
Mer
Scutylenchus
Scu
Cacopaurus
Cac
Miculenchus
Mic
Telotylenchus
Tel
Coslenchus
Cos
Nagelus
Nag
Trichodorus
Tri
Criconema
Cra
Neodolichorhynchus
Ned
Trophurus
Tro
Criconemella
Cre
Neotylenchus
Net
Tylenchorhynchus
Tyc
Disctylenchus
Dis
Nothotylenchus
Not
Tylenchus
Tyl
Ditylenchus
Dit
Ogma
Ogm
Xiphinema
Xip
Filenchus
Fil
Ottolenchus
Ott
Zygotylenchus
Zyg
Gracilacus
Gra
Paratrichodorus
pTri
3. Results
3.1. Dominance of the plant-parasitic nematode and structure of communities

The PPN fauna detected in the whole sampling survey conducted on olive trees in Morocco
was highly diversified with 47 genera belonging to Aphelenchidae (secondary plantparasites), Dorylaimidae, Triplonchida and Tylenchidae families. Modelling their dominance
(Fig. 2A), 83% of the genera were recorded as less frequent (F < 30%) according to the model
and 35% as occasional (F < 5%). 62.5% of the nematode genera were classified as highly
abundant according to the abundance threshold defined by the model (200 nematodes/dm3 of
soil). Eight genera were recorded dominant (F 30% and A 10,000 nematodes/dm3 of soil):
Filenchus and Helicotylenchus (F > 80%), Rotylenchus, Merlinius, Paratylenchus,
Xiphinema, Pratylenchus and Tylenchorhynchus (40 < F < 70%). Six other highly abundant
genera were less frequent, including root-knot nematodes (Meloidogyne spp., F = 12.2%) and
cyst nematodes (Heterodera spp., F = 10%). No genera was found frequent and in low
abundance.
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As mentioned above, the scarcest genera (total abundance less than 1%) were excluded
from the data set prior to running statistical analyses. As shown by the PCA loading plot of
the nematode taxa (Fig. 2B), Hoplolaimidae nematodes (Helicotylenchus and Rotylenchus),
and Paratylenchus, Filenchus and Pratylenchus genera to a lesser extent, were correlated to
the PC1 axis (negative values). The PC2 axis indicated contrasted positions of
Tylenchorhynchus spp. (negative values) and of Boleodorus, Xiphinema, Nothotylenchus,
Merlinius, Rotylenchulus, Meloidogyne, Heterodera and Telotylenchus (positive values).
3.2. Plant and environmental typology of the sampling device

Olive data: The FCA analysis (Fig. 3A) indicated contrasted positions of the olive growing
modality and the chloroplast lineage on the FCA1 axis. The irrigation modality was lowly
correlated to the FC2 axis that contributed to 5.9% of the variance only.
Climate variables: Three climate factors (minimal and maximal temperatures and annual
rainfall) were included in the analyses. FCA analysis indicated that the mean maximal
temperature and the annual rainfall were in contrasted positions on the FC1 axis (Fig. 3B),
while the mean minimal temperature was related to the FC2 axis (negative values).
Soil characteristics: PCA analysis (Fig. 3C) indicated that the PC1 axis was mainly structured
by the organic matter content (OM) (negative values) and by the soil texture gradient (from
sandy soils (Sa) in positive values to silty soils (Si) in null values and then to clayey soils (Cl)
in negative values). Most of the nutrients were also related to this axis. The cation exchange
capacity (CEC) was associated to clayey soils (Cl) on the PC1 axis. The soil pH and the sandy
texture on one hand and iron (Fe) and manganese (Mn) contents on the other hand mainly
contributed to the PC2 axis.
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Figure 2: Dominance and structure of plant-parasitic nematode genera in communities in the

olive areas surveyed in Morocco A: Dominance diagram of the nematode genera. Codes for
genera are listed in Table 2. Dotted lines indicate delineation between low and high
abundances and frequencies as described in Fortuner and Merny (1973). B: Plant-parasitic
nematode community patterns (PCA loading plot for the nematode genera).
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Figure 3: Environmental patterns in the olive areas surveyed in Morocco. A: FCA loading
plot for the olive data; B: FCA loading plot for the climate data; C: PCA loading plot for the
soil data.
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3.3. Correspondences between plant-parasitic nematodes community patterns and

environmental variables
These correspondences were investigated using co-inertia analyses (COA) that faced
nematode data to each environmental data set.
3.3.1. Patterns related to olive variables
The loading plot of the COA analysis between nematode and olive data (Fig. 4A) indicated
that the anthropogenic gradient (WO FO TR HD) highly contributed to the COA1 axis.
The irrigation variables were associated with the olive growing modality: irrigation supply
(I) is involved in high-density cultivated orchards (HD) and in some traditional cultivated
orchards (TR) while non-cultivated trees (FO and WO) grow under rainfed conditions. The
COA2 axis distinguished the feral growing modality (FO) from the others and clearly
established a chloroplastic lineage gradient (E1 E2 E3 M). The score plot of olive data
on the COA1 axis (Fig. 4B) confirmed that E2 and E3 lineages are associated with noncultivated olive trees growing under rainfed conditions, and that E1 lineages are associated to
cultivated olive trees mostly irrigated. Regarding the projection of the nematode genera in the
loading plot (Fig. 4A) and the score plot of nematode data on the COA1 axis (Fig. 4C), the
analysis indicated that the genera Xiphinema, Heterodera, Nothotylenchus, Rotylenchulus,
and Merlinius were correlated to wild olive (WO) and then to E2 and E3 lineages. In contrast,
root-knot nematodes (Meloidogyne spp.) and Tylenchorhynchus spp. were enhanced by
cultivation practices (especially HD) and irrigation (I). The other nematode genera
(Boleodorus,
Filenchus,
Helicotylenchus,
Merlinius,
Paratylenchus,
Pratylenchus,
Rotylenchus, Telotylenchus) were more related to rainfed traditional cultivated (TR) and feral
(FO) olive mostly characterized by the E1 chloroplastic lineage.
3.3.2. Patterns related to climatic modalities
The loading plot of the COA analysis between nematode and climate data (Fig. 5A) indicated
that the minimal temperature gradient (Tmin1-Tmin2-Tmin3) contributed to the COA1 axis.
The maximal temperature contributed to the two COA axes, with the lowest temperatures
(Tmax1) in COA1 positive values and the highest temperatures (Tmax3) in the COA2
negative values. The rainfall was less involved in the COA analysis but more related to COA1
axis. The score plot of the climate data on the COA1 axis (Fig. 5B) confirmed that the rainiest
regions (AR3) were associated with narrow thermal amplitudes (Tmin3 and Tmax1) while
more arid regions were under the influence of wider thermal amplitudes (Tmin1 and Tmax3).
Regarding nematode genera (Fig. 5A) and their range on COA1 axis (Fig. 5C), Xiphinema,
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Nothotylenchus and Heterodera nematodes were strongly related to wet and mild temperature
conditions. Root-knot nematodes (Meloidogyne spp.) were especially associated to warm
regions (Tmax3). Telotylenchus, Filenchus and Rotylenchus nematodes were mostly detected
in the coldest regions (Tmin1). Helicotylenchus and Paratylenchus genera were mostly
detected in arid regions (AR1). The other nematode genera did not seem to be affected by
specific climatic conditions.
3.3.3. Patterns related to soil data
The loading plot of the COA analysis between nematode and soil data (Fig. 6A) indicated that
the soil texture gradient (clay (Cl) and silt (Si) in the positive values and sand (Sa) contents in
the negative values) highly contributed to the COA1 axis. The cation exchange capacity
(CEC) fitted with the clay content. The soil pH also strongly contributed to the COA1 axis
(negative values) as well as the organic matter (OM) and the iron (Fe) contents (positive
values). The score plot of the soil characteristics on the COA1 axis (Fig. 6B) confirmed the
positions of variables on COA1 axis and indicated less or no contribution of the nutrients that
took parts more to the COA2 axis. Regarding nematode genera and their correspondence on
COA1 axis (Fig. 6A and 6C), Nothotylenchus and Xiphinema were more related to iron, clay
and organic matter contents. Meloidogyne spp. and Telotylenchus spp. were more associated
to sandy soils and depend on pH soil, while Merlinius spp. and Pratylenchus spp. were mostly
detected in silty soils. Boleodorus spp., Helicotylenchus spp. and Rotylenchus spp. were also
subjected to organic matter. The other genera were more or less subjected to soil nutrients.
3.3.4. Patterns related to all environmental variables
Here we took into account only the environmental variables that contributed to the COA1 axis
in each of the previous COA analyses. Consequently, all the soil nutrient variables were
excluded. Moreover, the organic matter and pH variables were defined into three classes (low,
moderate and high).
The loading plot of the COA analysis disclosed three groups of correspondences (Fig.
7). The first one was related to irrigated (I) olive conditions involving traditional (TR) and
high-density (HD) practices, to low organic soils (OM1), and to high maximal temperatures
(Tmax3). These conditions appeared favourable for nematode genera such as Meloidogyne,
Tylenchorhynchus and Pratylenchus. The second group was related to feral olive (FO) mostly
growing in alkaline (pH2 and pH3) coarse textured soils (Sa and Si) with intermediate organic
matter content (OM2), and mostly in very cold regions (Tmin1) subjected to intermediate
rainfall (AR2). Hoplolaimidae (Helicotylenchus spp. and Rotylenchus spp.), Telotylenchidae
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(Telotylenchus spp. and Merlinius spp. to a lesser extent), Filenchus Paratylenchus spp, and
Rotylenchulus spp. were more related to these conditions. The third group was related to wild
olive (WO) growing under narrow thermal amplitudes (Tmin3 and Tmax1) and rainy
conditions (AR3) in neutral (pH1) and organic (OM3) clayey (Cl) soils. The abundance of
Xiphinema, Nothotylenchus, Heterodera and Boleodorus genera was enhanced under these
conditions.
The GLM analysis confirmed the impact of these different variables on nematode
genera (Tab. 3). It indicated a significant impact of olive growing modalities on Filenchus,
Helicotylenchus, Meloidogyne and Tylenchorhynchus. Considering soil characteristics, the
soil type had a significant impact on Heterodera, Pratylenchus and Rotylenchulus.
Boleodorus, Meloidogyne, Rotylenchus, and Tylenchorhynchus were impacted by organic
matter content, while pH only impacted Telotylenchus and Xiphinema. Based on climate
factors, the mean maximal temperature highly impacted Helicotylenchus, Meloidogyne,
Paratylenchus and Telotylenchus. More impact of minimal temperature was observed on
Nothotylenchus, Rotylenchus, Rotylenchulus, Telotylenchus, Filenchus and Xiphinema. The
annual rainfall had a significant effect on Boleodorus and Xiphinema. No influence of olive
lineage was detected on all the nematode genera.
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Figure 4: Correspondences between plant-parasitic nematodes and olive tree data in the areas
surveyed in Morocco. A: COA loading plot for the nematode genera and the olive modalities;
B: Score plot of the eigenvalues for olive modalities on the COA1 axis; C: Score plot of the
eigenvalues for nematodes on the COA1 axis.
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Figure 5: Correspondences between plant-parasitic nematodes and climate data in the olive
areas surveyed in Morocco. A: COA loading plot for the nematode genera and the climate
data; B: Score plot of the eigenvalues for climate data on the COA1 axis, C: Score plot of the
eigenvalues for nematodes on the COA1 axis.
Page 162
Figure 6: Correspondences between plant-parasitic nematodes and soil data in the olive areas
surveyed in Morocco. A: COA loading plot for the nematode genera and the soil data; B:
Score plot of the eigenvalues for soil data on the COA1 axis. C: Score plot of the eigenvalues
for nematodes on the COA1 axis.
Page 163
Figure 7: Co-inertia loading plot for plant-parasitic nematode communities according to the
most discriminant environmental data in the olive areas surveyed in Morocco.
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Table 3: P-value of GLM analyses for each nematode genera according to the most significant environmental variables
***, ** and * mean very high, high and low significant effect of environmental variables on plant-parasitic nematodes.
Nematode genera
Sys
Lin
Sol
OM
pH
Tmax
Tmin
AR
Boleodorus
0.2993
0.4547
0.2491
0.001177 **
0.232
0.7945
0.8163
0.04951 *
Filenchus
5.357e-05 ***
0.3
0.4778
0.3449
0.3509
0.08736
0.007285 **
0.852
Helicotylenchus
0.02194 *
0.2154
0.7824
0.2592
0.6647
0.001144 **
0.1979
0.4179
Heterodera
0.2119
0.4022
0.008316 **
0.06324 .
0.08131
0.2541
0.7339
0.08824
Meloidogyne
1.229e-05 ***
0.4034
0.1622
0.04462 *
0.076
0.009346 **
0.3395
0.08659
Merlinius
0.5924
0.2015
0.1118
0.6353
0.9975
0.7435
0.09676 .
0.7086
Nothotylenchus
0.4387
0.1129
0.1781
0.9346
0.127
0.8884
0.02926 *
0.05355
Paratylenchus
0.296
0.7327
0.6644
0.05769
0.4696
0.0147 *
0.8348
0.2638
Pratylenchus
0.1302
0.1419
0.03263 *
0.3417
0.2446
0.05053 .
0.1917
0.9941
Rotylenchulus
0.1091
0.09929 7.235e-05 *** 0.8921
0.9192
0.3449
0.02043 *
0.6678
Rotylenchus
0.4021
0.2425
0.2532
0.02658 *
0.4042
0.5179
0.0005973 ***
0.8266
Telotylenchus
0.843
0.5297
0.07006
0.1316
0.02396 *
0.0253 *
0.02907 *
0.05208
Tylenchorhynchus
1.374e-11 ***
0.2255
0.1662
0.04915 *
0.315
0.2532
0.4006
0.3801
Xiphinema
0.1644
0.257
0.1388
0.2514
0.003393 ** 0.1881
0.001905 **
0.0006293 ***
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3.4. Species diversity of a widespread genus: the case of Helicotylenchus spp.

The dominance and the PCA analysis of the PPN community pattern (Fig. 2) indicated that
Hoplolaimidae nematodes, especially Helicotylenchus spp., were spread all over the olive
areas sampled. That was consequently confirmed by the COA analyses that shown no
significant correspondence between the abundance of Helicotylenchus spp. and the olive
growing modality and diversity (Fig. 4), the climate (Fig. 5), and the soil characteristics (Fig.
6) or according to the most significant environmental variables (Fig. 7). But this genus
exhibited 11 different species that were heterogeneously distributed in the olive areas in
Morocco. So, the impact of the environmental variables previously described was also
investigated on the Helicotylenchus species. A PCA analysis was performed with the most
dominant species (H. minzi and H. tunisiensis were thus excluded because of their very low
occurrence). The PCA loading plot (Fig. 8A) indicated that H. digonicus on one hand and H.
crenadauta, H. dihystera and H. vulgaris on the hand were opposed on the PC1 axis. The PC2
axis was strongly structured by H. varicaudatus (positive values) and by H. crassatus
(negative values).
As shown by the COA analysis (Fig. 8B), H. digonicus and H. varicaudatus were related to
wild olive, to neutral (pH1) and organic (OM3) sandy soils (Sa), and to low maximal
temperatures (Tmax1), while H. crenadauta, H. vulgaris, H. dihystera and H. pseudorobustus
fitted with cultivated olive (TR and HD), with alkaline clayey soils (pH3), and with high
maximal temperatures (Tmax3) and low rainfall (AR1). The others species (H. canadiensis,
H. crassatus and H. exallus) were more associated to feral olive (FO), to slightly alkaline
(pH2) and low to moderate organic (OM1 and OM2) silty soils (Si), and to low temperatures
and to high rainfall.
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Figure 8: Diversity of Helicotylenchus species in the olive areas surveyed in Morocco. A:

PCA loading plot for the Helicotylenchus species; B: COA loading plot for the species and
the environmental data.
Page 167
4. Discussion
Previous studies conducted on PPN diversity in communities (Ali et al, in prep; Chapter, IV)
demonstrated that the biodiversity indices are not sufficient to describe the communities,
because they give any information about the taxonomical composition of the communities.
The main questions addressed by this study were thus to describe the structure of PPN
communities associated to olive trees in Morocco (as well wild, feral and cultivated olive) and
to prospect how domestication, cultivation practices and environmental variables may impact
their structuration. The results revealed the detection of 47 genera distributed into three
groups of nematodes according to their dominance: high frequent and abundant, less frequent
but abundant and less frequent and less abundant. No nematode was widespread with low
population level. This dominance pattern was confirmed by PCA analyses that demonstrated
that the nematode data set was mainly structured by the most frequent and abundant genera,
and to less frequent but abundant nematodes to a lesser extent. That means that the frequency
is the main variable that leads the distribution of the nematode genera. The communities
observed were mainly dominated by Filenchus and Helicotylenchus, and other nematodes
such
as
Rotylenchus,
Merlinius,
Paratylenchus,
Xiphinema,
Pratylenchus
and
Tylenchorhynchus. Some of them have been previously reported as widespread on olive trees
in the word (Ali et al. 2014). The analysis of the dominance also revealed high population
levels of some nematode genera such as root-knot nematodes (Meloidogyne spp.) and cyst
nematodes (Heterodera spp.) considered as very dangerous soil-borne plant pests (Jones et al.
2013).
Biological hierarchy in communities is certainly controlled by trade-offs interactions
between facilitation and competition that result in spatio-temporal variability (Ledo et al.
2014). Moreover, assemblages within communities could also result from similar ecological
requirements (Rger et al. 2009), niche partitioning or existence of mechanisms that prevent
or favor the development of a particular species in the proximity of another species (Callaway
and Walker, 1997). Co-evolution processes may also interact with specific ecological
requirements (Turcotte et al. 2012). Such positive or negative interactions can be
hypothesized between nematodes through the PCA analysis that demonstrated a constant
correlation between Helicotylenchus spp. and Rotylenchus spp. that belong to the same
Hoplolaimidae family and no correlation between Merlinius spp., Telotylenchus spp. and
Tylenchorhynchus spp. that belong to the same Telotylenchidae family.
Environmental characteristics were revealed as important factors able to impact the
structure of the PPN communities associated with olive trees. But the statistical analyses
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showed that these environmental variables did not affect all the nematode taxa. One table
analyses (e.g. PCA, FCA) are able to show how data sets are structured and which variables
are the most discriminant. Two table analyses (e.g. COA) exhibit the contribution of two data
sets to a common co-structure. Then, comparing how nematode variables contributed to the
PCA and then to the COA structures, it was evident that dominant taxa, such as
Helicotylenchus, Rotylenchus, and Filenchus were not impacted by any environment variable,
while populations of other nematodes, such as Meloidogyne spp., that less contributed to
nematode communities, were enhanced in specific conditions (e.g. olive cultivation,
irrigation). Furthermore, these conditions appeared to be not impacted genera of the same
family (e.g. Tylenchorhynchus vs Merlinius of the family Telotylenchidae) or species of the
same genus (e.g. Helicotylenchus species) in the same way. The study established a clear
distinction between Merlinius and Tylenchorhynchus to environmental conditions. These
conditions separated the Helicotylenchus species into different groups. That could conclude
that the evolution of such species or genera is related to specific ecological requirements that
lead to repulsion between taxa.
Then, the co-inertia and the GLM analyses allocated the nematode genera to three
groups according to the environmental variables they showed positive correspondences.
These analyses also defined a gradient of olive domestication from wild, to feral and
cultivated olive. The PPN communities appeared to be structured according to this gradient:
Xiphinema spp., Nothotylenchus spp. and Heterodera spp. was correlated to wild olive
conditions; nematodes such as Telotylenchus spp., Rotylenchulus spp., Merlinius spp. were
enhanced in intermediate environmental conditions such as on feral and on traditional olive,
while, Meloidogyne spp. and Tylenchorhynchus spp. was mainly enhanced in orchards,
especially in high-density under irrigated conditions. The analyses also established a gradient
according to olive intensification between low density (traditional) to high density olive
cultivation. The contribution of high density olive was more important and related especially
to nematodes such as Meloidogyne spp., while, more nematodes appeared to be more
favoured under traditional cultivation.
The climatic variables also played a role in the structuration of the communities.
Coinertia analysis demonstrated that minimal and the maximal temperature gradients were
conserved, while the annual rainfall gradient was not conserved (that could be explained to
interference with irrigation). According to this coinertia analysis, communities especially
dominated by nematodes such as Rotylenchus spp., Filenchus spp. and Telotylenchus spp.
were related to regions characterized by high thermal amplitudes (Tmax3 and Tmin1), while
Page 169
regions with low thermal amplitudes (Tmax1 and Tmin3) emphazised nematode taxa such as
Xiphinema spp., Nothotylenchus spp. and Heterodera spp. Meloidogyne spp. appeared to be
favoured by high temperatures such as in the Haouz region. All the other nematode genera
were enhanced in regions subjected to moderate temperatures.
Among soil characteristics, organic matter content was revealed as an important factor
in community structuration. Meloidogyne spp., as example, arise high levels in low organic
soils. In contrast, Xiphinema spp., it was detected correlated to wild olive where the organic
matter content is usually high.
According to the bibliography, it had been demonstrated in other agro- and ecosystems,
that the abundance and the structure of nematodes communities are influenced by vegetation,
soil properties, soil temperature, moisture, season and nutrient dynamics (Goralczyk, 1998;
Boag et al. 1998) as well as by cultural practices and human activities (Yeates et al. 1993;
1999). A strong nematodesoil relationship could be expected because all parasitizing
nematodes carried out all or a part of their life in the soil (Cadet and Thioulouse, 1998).
The present study concluded on the main impact of olive domestication (wild vs
cultivated) and cropping intensification (low vs high density) on the structuration of the
nematode communities. The irrigation and the olive chloroplastic lineages did not contribute
in PPN communities structuration, because their impact is merged to that of the olive
modality. As example, E1 lineage or irrigated olive (I) is especially detected in cultivated
olive and in some feral olive, while rainfed olive (R) and E2 and E3 lineages were mostly
related to wild olive. Soil characteristics (soil type, organic matter content and pH) and
climate factors are involved as secondary impacts on community structure. However, their
impact was also merged with the impact of olive modalities. The role of the host plant in
structuring the nematode communities was expected because nematodes are obligate parasites
(Yeates, 1999). This effect of host plant on PPN populations agrees with others studies
(Nyhan et al. 1972; Niblack and Bernard, 1987; Thomas and Norton, 1986). This impact
could be direct (related to the quantity and quality of the food resource provided to the
nematodes) or indirect (due to the changes in soil properties) (Kandji et al. 2001). The study
also raises an important involvement of land-use changes and anthropogenic practices (wild
vs cultivated) in PPN patterns structure in communities. This involvement had been
confirmed on soil-borne pest communities (Mateille et al. 2008). These land-use changes
were supposed to reflect underlying changes in physical, chemical and biological properties
of soils (Kimenju et al. 2010). Agriculture intensification is frequently associated with
increased disturbance of the soil through tillage, indiscriminate use of mineral fertilizers and
Page 170
pesticides, manipulation of organic residues and of monoculture practices (Yeates et al.

1999). As an example, high-risk PPN such as root-knot nematodes (Meloidogyne spp.) that
were rarely detected on wild olive, were found at high population levels (> 10000 individuals
/dm3 of soil) in cultivated olive conditions marked by intense cultural practices (e.g high
density cultivation, irrigation, tillage). Their prevalence on cultivated olive could be explained
because these conditions create favourable conditions for their development. The irrigation
practices, as example, favor root proliferation and increased nematode population growth that
subsequently impacts adult trees (Nico et al. 2002). This was consistent with others
observations in Southern Morocco (At-Hamza et al. 2015). It was also reported that crops
involving agrochemical and tillage inputs had increased the total nematode biomass that was
dominated by herbivores (Bouwman and Zwart, 1994). Based on these observations, we can
expect significant olive losses due to highly abundant Meloidogyne spp. populations in olive
orchards. Facing this situation, some available options could be envisaged to reverse this
trend. That could include crop diversification (based on multiple cropping and crop rotation),
conservation agriculture (based on minimum tillage and cover cropping) and organic farming
(Kimenju et al. 2010). These practices could be introduced especially in high-density
orchards.
This study confirmed that variation between taxa is likely to be due to interactions
between scale-specific environmental, biotic and stochastic factors and individual taxa
biology, and emphasises the need to take scale into account when interpreting data from
ecological studies (Benefer et al. 2010). Although specific mechanisms that determine
community pattern are not identified, some ecological information can, nonetheless, be
discerned (Law et al. 2009; Brown et al. 2011). Information on the relative importance of
biotic, environmental and spatial factors to individual species distributions would be useful in
terms of managing pests and biodiversity (Benefer et al. 2010), and may allow better
predictive models to be produced.
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Chapitre VII
Mthodologie gnrale (b)

Cette partie suit la mthodologie gnrale prsente
prcdamment, elle dcrit la mthodologie suivie afin
dtudier la diversit des nmatodes galles Meloidogyne
spp.
Page 175

La deuxime partie de la thse sest concentre sur ltude de la diversit des nmatodes
galles du genre Meloidogyne associes lolivier au Maroc. Le choix de ce genre se justifie
des points de vue :
- conomique et agronomique puisque ce sont des parasites qui menacent la production
agricole. Ils ont t classs dans les espces top-ten les plus dommageables (Jones et
al., 2013) et certaines espces sont des espces de quarantaine.
- biologique puisque ces organismes prsentent une grande variabilit intra et inter
spcifique (Castagnone-Sereno et al., 2013).
- technique avec la disponibilit de plusieurs marqueurs morphologiques, biologiques,
biochimiques et molculaires.
1. Caractrisation spcifique des nmatodes du genre Meloidogyne

La dtermination des espces de Meloidogyne a toujours t problmatique. Leur diversit
spcifiques (plus de 97 espces dcrites), leur large gamme dhtes, prsence de complexe
despces cryptiques, le dimorphisme sexuel, la diplodie, etc., sont autant de caractres qui
rendent difficile leur identification spcifique (Janati et al., 1982 ; Blok & Powers, 2009). Par
exemple, l'identification morphologique est difficile parce que (i) les mles sont souvent rares (la
plupart des espces sont parthnogntiques), (ii) les femelles sont dformes (pyriformes parce
que elles sont sdentaires dans les racines), et (iii) les juvniles ne fournissent pas suffisamment de
caractres discriminants. Cest pourquoi, dans cette tude dautres approches de diagnostic sont
utilises: (i) diagnostic biochimique bas sur les patrons de migration lectrophortique
disoenzymes ralis sur les femelles, et (ii) diagnostic molculaire bas sur la migration
lectrophortique de squences dADN spcifiques, ou sur le squenage de gnes, et ralis sur
les juvniles clos des ufs pondus par les femelles identifies en biochimie.
1.1. Traitement des chantillons
Lorsque des juvniles de Meloidogyne ont t dtects dans des chantillons du sol (cf. 3.2.
Mthodologie gnrale a), des plants de tomate sensibles aux nmatodes galles (Lycopersicon
esculentum ; cv. Roma) gs de 5 semaines ont t transplants dans les sols afin de multiplier les
populations. Le choix de la tomate se justifie par le fait que cette plante est trs sensible la
plupart des espces de Meloidogyne, dont les espces dj identifies comme parasites de lolivier
au niveau mondial telles que M. javanica, M. arenaria, M. incognita (Ali et al., 2014), et quelle
constitue donc un excellent modle de plante dlevage au laboratoire. Dautre part, la dissection
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des racines pour rcuprer le matriel biologique ncessaire est beaucoup plus facile que sur des
racines ligneuses. Cinquante soixante jours aprs transplantation, les plants ont t dpots et les
racines ont t laves dans leau, puis observes sous stromicroscope pour vrifier la prsence
de galles et de masses d'ufs (cf. cycle biologique, 4.4.1, Introduction gnrale a). Par la suite,
deux protocoles ont t mis en place:
En cas de prsence de galles sur les racines : 24 masses dufs par plante ont t prleves
manuellement et mises clore individuellement dans un closoir lobscurit dans un
incubateur 25C (Madulu & Trudgill, 1994). Les juvniles de deuxime stade (J2) clos ont
t conditionns dans une goute de 6 l deau distille (cf. annexe 3, Protocole de prparation
des nmatodes pour des tudes molculaires) et stocks -20C. Les femelles
correspondantes ont t prleves par dissection des racines, conditionnes individuellement
dans des tubes Eppendorf de 250l contant 5 l de tampon de conservation de Trugdill (20%
sucrose), puis broyes avec un piston, et enfin conserves -20C.
En cas dabsence de galles sur les racines : les racines ont t fragmentes et introduites
dans une chambre brouillard (cf. annexe 4, Protocole dextraction des nmatodes des racines
en chambre brouillard ; Seinhorst, 1950) afin dextraire des juvniles susceptibles dclore
des masses dufs incluses dans les racines. Cette technique consiste pulvriser un
brouillard deau sur des fragments de racines et puis rcuprer les nmatodes qui quittent les
racines pourrissantes. Ces diffrentes tapes de traitement des chantillons et de prparation
du matriel biologique sont illustres en figure 1.
Figure 1: Procdure de traitement et de production du matriel biologique pour la caractrisation

biochimique et molculaire ainsi que pour ltude de la diversit de genre Meloidogyne.
Page 177
1.2. Caractrisation biochimique

Ce diagnostic est bas sur la sparation lectophortique des protines, la rvlation disoenzymes
(estrases EST, malate-dshydrognases MDH, glutamate-oxaloacetate-transaminases GOT,
superoxyde-dismutases SUD). Dans notre tude, les femelles ont t testes pour leur systme
estrasique EST, le plus rpandu dans le genre Meloidogyne (90% environ des espces) (Berg &
Dalmasso, 1975). Les broyats de femelles (cf. 1.1.) ont t centrifugs (9500 rpm, 10 min), et
injects sur gel de polyacrylamide vertical 7% (Maurer, 1971) (cf. annexe 5, procdure la
diagnostic biochimique des Meloidogyne). La migration est ralise 2-8C afin de prserver
lactivit enzymatique des protines. La rvlation des produits enzymatiques a t obtenue par
coloration au 1-naphtylactate (Brewer & Singh, 1970), ensuite fixe lacide actique (10%).
Les gels ont ensuite t schs entre deux feuilles cellophane pendant 48 heures sous hotte
chimique. Les rapports de migration (Rm) des bandes EST sont alors calculs pour chaque femelle
(Esbenshade & Triantaphyllou, 1985) et compars ceux dune population tmoin de M. javanica
conserve en levage au laboratoire. Les rsultats obtenus ont t ensuite compars tous ceux
dcrits dans la littrature.
1.3. Caractrisation molculaire

LADN des juvniles a t extrait par la technique de Kit QIAgen DNeasy Blood & Tissue (cf.
annexe 6, extraction dADN). Le diagnostic molculaire a t fait laide de deux marqueurs :
SCARS, puis D2D3 (28S rDNA) lorsque le diagnostic SCARS sest rvl ngatif:
SCARs : des marqueurs molculaires de types SCARs (Sequence Characterized Amplified
Region) sont disponibles pour dterminer quelques espces de Meloidogyne. LADN obtenu a
t amplifi par PCR QIAGEN (Multiplex) (cf. annexe 7) en prsence damorces de type
SCARs spcifiques aux espces : M. arenaria, M. javanica, M. incognita, M. hapla (Zijlstra,
2000 ; Zijlstra et al., 2000). Les produits amplifis ont t spars sur un gel dagarose
1.5%. La dtermination de lespce sest faite selon la taille des amplicons obtenus comme
par exemple, 4320pb pour M. arenaria, 700pb pour M. javanica, 1200pb pour M. incognita et
610pb pour M. hapla.
D2-D3 (28S rDNA) : plusieurs travaux ont confirm que cette rgion est un bon marqueur de
discrimination des espces de Meloidogyne (De Ley et al., 1999 ; Castillo et al., 2003). LADN
de juvniles a t amplifi par les amorces dcrites dans lannexe 8. Les produits PCR
amplifis ont t squencs (Socit EUROFINS Genomics, Allemagne) avec les amorces
utilises pour lamplification. La quantification, la concentration des chantillons et la
Page 178
purification de la PCR ont t ralises par le squenceur. Les squences obtenues ont t
rcupres sous forme de chromatogrammes, puis corriges, alignes et analyses avec les
logiciels Geneious R7 (v7.1.2) et BioEdit (v7.2.5) (Hall, 1999). Les squences alignes ont t
blastes sur GenBank afin de dterminer lespce.
2. Etude de la diversit interet intraspcifique des nmatodes du genre Meloidogyne

2.1. Diversit molculaire
Plusieurs outils molculaires ont t tests afin dtudier la variabilit gntique des populations
de Meloidogyne associes lolivier au Maroc:
Marqueurs microsatellites.
Analyses des squences dADN nuclaire de la rgion ITS2 (Internal Transcribed Spacer).
Analyse des squences dADN mitochondrial du gne COI (Cytochrome c oxidase subunit 1).
Analyse des squences dADN mitochondrial de la rgion non codante 63R (63 rptitions en
tandem).
2.1.1. Marqueurs microsatellites

Les microsatellites sont des squences courtes constitues de rptitions de deux, trois ou quatre
nuclotides. Leur longueur est variable et ils sont rpartis indiffremment dans le gnome. Chez les
Meloidogyne, 4880 locus de microsatellites ont t slectionns au sein de M. incognita
(Castagnone-Sereno et al., 2010). Certains ont t dtects chez dautres espces telles que M.
maritima (Richaud, 2004), M. hapla, M. javanica, M. paranensis, M. arenaria et M. exigua (Mulet
et al., 2011).
Des tests ont t effectus au laboratoire avec les microsatellites tudis par Richaud (2004)
pour ltude de la biodiversit au sein de M. maritima. La prparation des mix PCR, les conditions
damplifications rquises pour chaque raction et la liste des couples damorces avec les conditions
damplification requises pour chaque raction sont donnes dans lannexe 9. Les produits PCR
amplifis ont t dposs sur un gel dagarose 1.5 et 3 %. Ces gels sont ensuite colors au BET
et puis visualiss sous UV et les fragments la taille attendue ont t reprs. Ces essais ont t
effectus en collaboration avec Dr. S. Bellafiore (IRD, UMR IPME, Montpellier).
Page 179
2.1.2. Analyses des squences ITS2

Les ITS2 sont les marqueurs des squences internes non codantes de lADN ribosomal qui
sparent les gnes 5.8S et 28S.
Plusieurs travaux ont montr lutilit de cette rgion dADNr pour la caractrisation des
espces et galement pour tudier la diversit gntique chez les Meloidogyne (Zijlstra et al.,
1995 ; Clapp et al., 2000). Le protocole danalyse a t extrait du travail de Clapp et al., (2000) et
dtaill en annexe 10. Des appliquons de 292 298 paires de bases (pb) ont t rvls sur gel
dagarose 1.5%. Les produits PCR amplifis ont t squencs et analyss comme dcrit
prcdemment (cf. 1.3.).
2.1.3. Analyses des squences COI

Le gnome mitochondrial est souvent utilis pour des tudes de diagnostic (marqueur spcifique
diagnostiquer des espces), et a galement certaines applications phylogntiques. En effet,
lADNmt est particulirement intressant grce son grand nombre de copies, son taux de
mutations plus lev que dans le gnome nuclaire, son contenu en gnes relativement constant,
un taux de recombinaison rduit, et un taux d'volution lev. Son hritabilit maternelle est un
atout comme dans le cas des nmatodes reproduction parthnogntique (e.g. la plupart des
espces de Meloidogyne) (Anderson et al., 1998 ; Hoolahan et al., 2011 ; Garca & SnchezPuerta, 2015). LADNmt est un outil puissant pour valuer les profils phylogntiques
intraspcifiques chez les nmatodes phytoparasites (Hugall et al., 1994). Plusieurs tudes ont mis
en vidence sa variation chez les nmatodes galles (Powers & Sandall, 1988 ; Harris et al., 1990;
Okimoto et al., 1991 ; Blok et al., 2002).
Les amorces utilises pour lamplification ont t adaptes du travail de Lazarova et al.,
(2006). Le mix PCR ainsi que les amorces utilises et les conditions requises pour lamplification
sont dcrites en annexe 11. Des appliquons de 470 pb ont t rvls sur un gel dagarose de
1.5%. Les produits PCR amplifis ont t squencs et analyss comme dcrit prcdemment (cf.
1.3.).
Page 180
2.1.4. Analyses des squences 63R

Cette rgion a galement t utilise pour analyser la diversit gntique au sein des espces M.
arenaria et M. javanica (Okimoto et al., 1991), M. enterolobii (Blok et al., 2002). Le mix PCR
ainsi que les amorces utilises et les conditions requises pour lamplification sont dcrites en
annexe 12. Les produits PCR amplifis ont t squencs et analyss comme dcrit prcdemment
(cf. 1.3.). Les haplotypes entre les diffrentes populations de Meloidogyne ont t reprs selon le
nombre de rptions de la portion 63R dans la squence, selon la taille et les diffrents
polymorphisms des squences. Des calculs de la diversit nuclotidique haplotypique ont
galement t effectus.
2.2. Diversit morphologique

Cette tude est base sur des mesures de diffrents traits morphologiques chez des juvniles de
deuxime stade larvaire (J2), le stade le mieux adapt aux mesures morphobiomtriques puisque
les mles sont rares et les femelles sont dformes (pyriformes). Les J2 ont t largement utiliss
pour diffrencier les espces de Meloidogyne (Cliff & Hirschmann, 1985 ; Jepson, 1987).
Afin danalyser cette diversit, des masses dufs ont t rcoltes au hasard partir des
populations de Meloidogyne identifes sur olivier. Ces masses dufs ont t mises clore
individuellement. Des juvniles (60 J2) ont t prlevs de chaque ponte et inoculs des plants
de tomates. 50 60 jours plus tard, trois masses dufs ont t rcoltes sur chaque plant de
tomate et mises clore. Trois juvniles par ponte ont t fixs et conservs dans le glycrol, et
ont fait lobjet des photos sur lesquelles des mesures morphomtriques ont t ralises.
Les procdures exprimentales suivies dans cette tude sont dcrites en dtail dans le
chapitre portant sur ltude de la diversit morphologique des populations de M. javanica (chapitre
IX-A).
3. Rfrences
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olive tree (Olea europaea L.) with a focus on the Mediterranean Basin: A review. Comptes
Rendus Biologies, 337: 423-442.
Anderson, T.J., Blouin, M.S., Beech, R.N., 1998. Population biology of parasitic nematodes:
applications of genetic markers. Advances in Parasitology, 41: 219-283.
Berg, J.B., Dalmasso, A., 1975. Caractristiques biochimiques de quelques populations de
Meloidogyne hapla et Melodogyne spp. Cahiers ORSTOM. Srie Biologie: Nmatologie,
10 : 263-271.
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Besnard, G., Jhling, F., Chapuis, ., Zedane, L., Lhuillier, ., Mateille, T., Bellafiore, S., 2014.
Fast assembly of the mitochondrial genome of a plant parasitic nematode (Meloidogyne
graminicola) using next generation sequencing. Comptes Rendus Biologies, 337: 295-301.
Blok, V.C., Powers, T.O., 2009. Biochemical and molecular identification. Root-knot nematodes,
98-118. In: Perry, R.N., Moens, M., Starr, J.L., (eds.). Root-knot nematodes. CABI.
Blok, V.C., Wishart, J., Fargette, M., Berthier, K., Phillips, M.S., 2002. Mitochondrial DNA
differences distinguishing Meloidogyne mayaguensis from the major species of tropical
root-knot nematodes. Nematology, 4: 773-781.
Brewer, G.J. and Singh, C.F., 1970. An introduction to isozyme techniques. Academic Press,
New York, 161pp.
Castagnone-Sereno, P., Danchin, E.G., Deleury, E., Guillemaud, T., Malausa, T., Abad, P., 2010.
Genome-wide survey and analysis of microsatellites in nematodes, with a focus on the
plant-parasitic species Meloidogyne incognita. BMC genomics, 11: 598.
Castagnone-Sereno, P., Danchin, E.G., Perfus-Barbeoch, L., Abad, P., 2013. Diversity and
evolution of root-knot nematodes, genus Meloidogyne: new insights from the genomic era.
Annual Review of Phytopathology, 51: 203-220.
Castillo, P., Vovlas, N., Subbotin, S., Troccoli, A., 2003. A new root-knot nematode,
Meloidogyne baetica n. sp. (Nematoda: Heteroderidae), parasitizing wild olive in Southern
Spain. Phytopathology, 93: 1093-1102.
Clapp, J.P., Van der Stoel, C.D., Van Der Putten, W.H., 2000. Rapid identification of cyst
(Heterodera spp., Globodera spp.) and root-knot (Meloidogyne spp.) nematodes on the
basis of ITS2 sequence variation detected by PCR-single-strand conformational
polymorphism (PCR-SSCP) in cultures and field samples. Molecular Ecology, 9: 12231232.
Cliff, G.M., Hirschmann, H., 1985. Evaluation of morphological variability in Meloidogyne
arenaria. Journal of Nematology, 17: 445-459.
De Ley, I.T., Karssen, G., De Ley, P., Vierstraete, A., Waeyenberge, L., Moens, M., Vanfleteren,
J. 1999. Phylogenetic analyses of internal transcribed spacer region sequences within
Meloidogyne. Journal of Nematology, 31: 530-531.
Esbenshade, P.R. and Triantaphyllou, A.C. 1985. Use of enzyme phenotypes for identification of
Garca, L.E., Snchez-Puerta, M.V., 2015. Comparative and evolutionary analyses of
Meloidogyne spp. based on mitochondrial genome sequences. PloS ONE, 10: e0121142.
Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis
program for Windows 95/98/NT. In Nucleic acids symposium series, 41: 95-98.
Harris, T.S., Sandall, L.J., Powers, T.O., 1990. Identification of single Meloidogyne juveniles by
polymerase chain reaction amplification of mitochondrial DNA. Journal of Nematology,
22: 518-524.
Hoolahan, A.H., Blok, V.C., Gibson, T., Dowton, M., 2011. Paternal leakage of mitochondrial
DNA in experimental crosses of populations of the potato cyst nematode Globodera
pallida. Genetica, 139: 1509-1519.
Hugall, A., Moritz, C., Stanton, J., Wolstenholme, D.R., 1994. Low, but strongly structured
mitochondrial DNA diversity in root knot nematodes (Meloidogyne). Genetics, 136: 903912.
Janati, A., Berge, J.B., Triantaphyllou, A.C., Dalmasso, A., 1982. Nouvelles donnes sur
l'utilisation des isoestrases pour l'identification des Meloidogyne. Revue de Nmatologie,
5: 147-154.
Jepson, S.B., 1987. Identification of root-knot nematodes (Meloidogyne species). (continued).
Identification of root-knot nematodes (Meloidogyne species).
Jones, J.T., Haegeman, A., Danchin, E.G., Gaur, H.S., Helder, J., Jones, et al., 2013. Top 10
plant-parasitic nematodes in molecular plant pathology. Molecular Plant Pathology, 14:
946-961.
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Lazarova, S.S., Malloch, G., Oliveira, C.M., Hbschen, J., Neilson, R., 2006. Ribosomal and
mitochondrial DNA analyses of Xiphinema americanum-group populations. Journal of
Madulu, J.D., Trudgill, D.L., 1994. Influence of temperature on the development and survival of
Meloidogyne javanica. Nematologica, 40: 230-243.
Maurer, H.R., 1971. Disc electrophoresis and related techniques of polyacrylamide gel
electrophoresis. Walter de Gruyter, Publishers, Berlin-New York, 222 pp.
Mulet, K., Fargette, M., Richaud, M., Genson, G., Castagnone-Sereno, P., 2011. Isolation of
microsatellites from an enriched genomic library of the plant-parasitic nematode
Meloidogyne incognita and their detection in other root-knot nematode species. European
Journal of Plant Pathology, 129: 501-505.
Okimoto, R., Chamberlin, H.M., Macfarlane, J.L., Wolstenholme, D.R., 1991. Repeated
sequence sets in mitochondrial DNA molecules of root knot nematodes (Meloidogyne):
nucleotide sequences, genome location and potential for host-race identification. Nucleic
Acids Research, 19: 1619-1626.
Powers, T.O., Sandall, L.J., 1988. Estimation of genetic divergence in Meloidogyne
mitochondrial DNA. Journal of Nematology, 20: 505-511.
Richaud, M., 2004. Mise au point de marqueurs molculaires: Etude de la biodiversit au sein
dune espce de nmatodes, Meloidogyne maritima, parasite de loyat en milieu dunaire.
Mmoire de stage de 3me anne dIUP BBB : Biodtection, Biocertification, Biodiversit.
Universit Montpellier II.
Seinhorst, I.J., 1950. De betekenis van de toestand van de grond voor het optreden van aantasting
door het stengelaaltje (Ditylenchus dipsaci (Khn) Filipjev). Tijdschrift Over
Plantenziekten, 56: 289-348.
Zijlstra, C., 2000. Identification of Meloidogyne chitwoodi, M. fallax and M. hapla based on
SCAR-PCR: a powerful way of enabling reliable identification of populations or
individuals that share common traits. European Journal of Plant Pathology. 106: 283-290.
Zijlstra, C., Donkers-Venne, D.T., Fargette, M., 2000. Identification of Meloidogyne incognita,
M. javanica and M. arenaria using sequence characterised amplified region (SCAR) based
PCR assays. Nematology, 2: 847-853.
Zijlstra, C., Lever, A.E.M., Uenk, B.J., Van Silfhout, C.H.Y., 1995. Differences between ITS
regions of isolates of root-knot nematodes Meloidogyne hapla and M. chitwoodi.
Page 183
Chapitre VIII
Description dune nouvelle

espce de Meloidogyne:
M. spartelensis n. sp.
Page 184
Rsum
Meloidogyne spartelensis n.sp. est une espce de nmatodes galles nouvellement dcrite lors
de cette thse. La description de cette nouvelle espce a fait lobjet dune publication en
premier auteur publie en European Journal of Plant Pathology en 2015. Cette espce a t
dtecte sur un olivier sauvage au Cap Spartel, prs de la ville de Tanger, au nord du Maroc.
La nomenclature donne cette espce est inspire du nom du lieu o elle a t prleve. La
description de lespce a t effectue sur la base des mesures morphomtriques chez des
juvniles de deuxime stade larvaire, des mles et des femelles, des marqueurs biochimiques
(profils enzymatiques destrase et de malate dhydrognase) et des analyses des squences
des gnes ribosomiques rADN (18S, ITS1-5.8S-ITS2, D2-D3 de 28S rADN) et
mitochondriaux mtADN (coxII-16S rARN et coxI). Ces diffrentes analyses (surtout les
caractres morphologiques et les analyses phylogntiques) ont montr que cette nouvelle
espce est diffrente de toutes les espces dj connues de Meloidogyne. Cette espce nest
pas morphologiquement lie aux espces connues comme parasites de lolivier M. arenaria,
M. artiellia, M. baetica, M. hapla, M. incognita, M. javanica, et M. lusitanica. La
multiplication de cette nouvelle espce sur tomate indique que sa gamme dhte est sans
doute plus large que lolivier seul, cependant elle ne se multiplie pas sur la varit dolivier
Picholine du Languedoc. Cest pourquoi, des expriences de pathognicit devront tre
effectues afin dexaminer limpact de cette espce sur lolivier. Les analyses ont galement
montr que cette espce est proche de lespce M. silvestris qui a t galement identifie sur
un hte ligneux (Ilex aquifolium L.) dans la pninsule ibrique. Cela pourrait indiquer que ce
groupe despces semble tre li des processus de diversification au sein du Bassin
Mditerranen avec une rpartition gographique restreinte. Cela pourrait tre, en partie, d
une longue persistance des espces de Meloidogyne dans des rgions isoles aprs la dernire
glaciation (e.g. spciation allopatrique), surtout que la rgion o cette espce a t dtecte, a
t dfinie comme une zone refuge de la biodiversit au Maroc.
Ltude
souligne
l'importance
de
combiner
plusieurs
marqueurs
morphologiques,
biochimiques et molculaires pour une identification correcte de Meloidogyne au niveau de

l'espce.
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Eur J Plant Pathol
DOI 10.1007/s10658-015-0662-3
!!
A new root-knot nematode Meloidogyne spartelensis n. sp.

(Nematoda: Meloidogynidae) in Northern Morocco
Nadine Ali & Johannes Tavoillot & Thierry Mateille & Elodie Chapuis &
Guillaume Besnard & Ahmed El Bakkali & Carolina Cantalapiedra-Navarrete &
Gracia Libanas & Pablo Castillo & Juan E. Palomares-Rius
Accepted: 15 April 2015

# Koninklijke Nederlandse Planteziektenkundige Vereniging 2015
Abstract Low density of an unknown root-knot

nematode was found on wild olive soils at Cape
Spartel near Tanger city in northern Morocco.
Morphometry, esterase and malate dehydrogenase
electrophoretic phenotypes, as well as ribosomal
DNA (rDNA) and mitochondrial DNA (mtDNA)
sequences demonstrated that this nematode species
differs clearly from other previously described rootknot nematodes. The species is herein described,
illustrated and named as Meloidogyne spartelensis
n. sp. This new root-knot nematode can be morphologically distinguished from other Meloidogyne spp.
by: (i) roundish perineal pattern, dorsal arch low,
with fine, sinuous cuticle striae, lateral field faintly
visible; (ii) female excretory pore posterior to stylet
knobs, EP/ST ratio 1.4-2.0; (iii) second-stage
juveniles with hemizonid located 1 to 2 annuli anterior to excretory pore and long, sub-digitate tail; and
(iv) males with lateral field composed of four
incisures, with areolated outer bands. Phylogenetic
trees based on 18S, ITS1-5.8S-ITS2, D2-D3 of 28S
rDNA, and partial coxII-16S rRNA and coxI gene of
mtDNA showed that M. spartelensis n. sp. belongs
to an undescribed root-knot nematode lineage that is
clearly separated from other species with resemblance in morphology, such as M. dunensis,
M. kralli, and M. sewelli.
N. Ali
Plant Protection Department, Faculty of Agriculture, Tishreen
University, PO Box 230, Latakia, Syrian Arab Republic
A. El Bakkali
INRA, UMR APCRPG, BP 578, Mekns, Morocco
N. Ali : J. Tavoillot : T. Mateille : E. Chapuis

IRD, UMR CBGP (INRA/IRD/CIRAD/Montpellier
SupAgro), 755 avenue du Campus Agropolis,
CS30016 34988 Montferrier-sur-Lez Cedex, France
E. Chapuis
IRD, UMR IPME (IRD/Universit Montpellier 2/CIRAD),
911 avenue Agropolis, BP 64501 34394 Montpellier Cedex 5,
France
G. Besnard
CNRS, UMR EDB (UPS, CNRS, ENFA), Universit
Toulouse III Paul Sabatier, Btiment 4R1, 118 Route de
Narbonne, 31062 Toulouse cedex 9, France
Keywords 18S . Bayesian inference . Cytochrome

oxidase subunit II . ITS . Maximum likelihood . mtDNA .
New species . Plant-parasitic nematodes . rDNA
C. Cantalapiedra-Navarrete : P. Castillo :
J. E. Palomares-Rius (*)
Instituto de Agricultura Sostenible (IAS), Consejo Superior de
Investigaciones Cientficas (CSIC), Apdo. 4084, Campus de
Excelencia Internacional Agroalimentario, ceiA3,
14080 Crdoba, Spain
e-mail: [email protected]
G. Libanas
Departamento de Biologa Animal, Vegetal y Ecologa,
Universidad de Jan, Campus Las Lagunillas s/n, Edificio
B3, 23071 Jan, Spain
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Eur J Plant Pathol
Introduction
Root-knot nematodes (RKN) of the genus Meloidogyne
are among natures most successful plant parasites, being distributed worldwide and encompassing more than
95 nominal species (Moens et al. 2010). These nematodes infect thousands of different herbaceous and
woody monocotyledonous and dicotyledonous plants
and cause severe losses to numerous agricultural crops
and forest plants worldwide (Moens et al. 2010). Rootknot nematodes (Meloidogyne spp.) and root-lesion
nematodes (Pratylenchus spp.) are the most damaging
plant-parasitic nematodes of cultivated olive (Olea
europaea L. subsp. europaea var. europaea), especially
in nurseries (Castillo et al. 2010; Ali et al. 2014).
Meloidogyne species known to damage olive trees are
well adapted to temperate and subtropical areas and
include Meloidogyne arenaria (Neal 1889) Chitwood
1949; Meloidogyne baetica Castillo et al. 2003a,
Meloidogyne hapla Chitwood 1949, Meloidogyne incognita (Kofoid & White 1919) Chitwood 1949,
M. javanica (Treub 1885) Chitwood 1949 and
Meloidogyne lusitanica Abrantes and Santos 1991. In
orchards, olive trees infected by Meloidogyne spp. show
yellowing in the highest branches and defoliation
(Castillo et al. 2010). These species occur on wild and
cultivated olives in southern Spain, and also in nurseries
(except for M. baetica) (Castillo et al. 2003b). However,
plant-parasitic nematodes infecting wild olive have been
scarcely studied in the Mediterranean Basin, except for
the recent description of the root-lesion nematode
Pratylenchus oleae Palomares-Rius et al. 2014 in
Spain and Tunisia, and the reniform nematode
Rotylenchulus macrosoma in Spain (Castillo et al.
2003a). Yet, wild olive populations have persisted in
western and eastern parts of the Mediterranean Basin
since the Late Tertiary (Besnard et al. 2013), and in
particular in southern Spain and Morocco where Last
Glacial Maximum refugia have been described for many
organisms (Mdail and Diadema 2009). Natural dispersal of native phytoparasitic nematodes could be limited
(due to physical barriers and low migration ability) and
communities of such regions are thus expected to be
unique with high level of endemism.
Species determination of Meloidogyne is complex,
difficult and time-consuming even for experts. The application of molecular methods to studies of RKN population structure and systematics have revealed that
some long-assumed single species are in fact cryptic
species that are morphologically indistinguishable but

may be phylogenetically distant to one another (Gamel
et al. 2014). Sequences of nuclear ribosomal (rDNA)
and mitochondrial DNA (mtDNA) have been used for
molecular characterisation and reconstruction of phylogenetic relationships within the genus Meloidogyne during the last decade (Powers and Harris 1993; Ziljstra
et al. 2000; Adam et al. 2007; Adams et al. 2009;
Castillo et al. 2009). The analysis of isozyme electrophoretic patterns, in particular esterase (Est) and malate
dehydrogenase (Mdh), as well as several molecular
approaches have proved to be a valuable tool for precise
identification of Meloidogyne species (Blok and Powers
2009). Consequently, current availability of molecular
techniques integrated with classical morphological approaches may help to provide tools for differentiating
Meloidogyne species and can significantly improve and
facilitate the routine identification of these nematodes.
Nematode surveys in wild olive soils in northern
Morocco revealed very low population levels of a
RKN. This RKN appeared morphologically closerelated to M. kralli Jepson 1983, M. dunensis
Palomares-Rius et al. 2007 and M. sewelli Mulvey and
Anderson 1980, which prompted a comparative study
among related species. Some reliable diagnostic approaches commonly used to identify and compare certain RKN species such as analyses of isozyme phenotypes and phylogenetic analyses of ribosomal and mitochondrial DNA sequences were included in the study.
This work describes a new Meloidogyne species
associated with wild olive in northern Morocco as well
as its phylogenetic relationship with other RKNs based
on Bayesian inference (BI) and maximum likelihood
(ML) analyses of sequences from the 18S, ITS1-58SITS2, D2-D3 of 28S rDNA, and the partial coxII-16S of
rRNA and coxI gene of mtDNA. The undescribed rootknot nematode is herein described as Meloidogyne
spartelensis n. sp., the species epithet referring to Cap
Spartel where it was detected.
Material and methods

Soil sampling, nematode extraction, and Meloidogyne
spp. detection and rearing
A nematode survey was conducted in March 2012 in
wild olive (Olea europaea subsp. europaea var.
sylvestris (Mill.) Lehr) soils in northern Morocco
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Eur J Plant Pathol
(Cape Spartel; 35.790583N; 5.924983W; altitude

16 m), near Tanger city. Soil sub-samples were collected
with a shovel from the upper 20 cm of bare soil under
each olive tree canopy and mixed to form one 500 dm3
reference sample. Nematodes were extracted from
250 cm3 of fresh soil using the elutriation procedure
(Seinhorst 1962), and Meloidogyne juveniles and males
were observed under a stereomicroscope (Leica
M165C, 60 magnification). Tomato (cv. Roma) and
olive (cv. Picholine du Languedoc) plantlets were
transplanted in the remaining native soil and reared
under controlled greenhouse conditions in order to test
the infection rate and plant host range of this RKN. No
nematode survived on olive cv. Picholine du
Languedoc. But several juveniles and males were
extracted from the tomato roots placed in a mist
chamber (Seinhorst 1950). The Meloidogyne population was again reared on tomato in a sandy-peat
substrate (2/3-1/3).
Nematode morphological identification
For diagnosis and identification, females were collected
from tomato galled roots, while males, eggs and secondstage juveniles (J2) of nematodes were extracted from
the rhizosphere by centrifugal-flotation (Coolen 1979)
and from feeder roots by blending in a 0.5 % NaOCl
solution for 4 min (Hussey and Barker 1973).
Specimens for light microscopy (LM) were killed with
gentle heat, fixed in a 4 % solution of formaldehyde+
propionic acid and processed to glycerin by Seinhorsts
rapid method (Seinhorst 1966). Specimens were examined using a Zeiss III compound microscope with
Nomarski differential interference contrast at powers
up to 1,000 magnification. Randomly selected specimens of each life-stage were measured. Measurements
and drawings were made at the camera lucida on glycerine infiltrated specimens. All measurements were
expressed in micrometers (m). All other abbreviations
used are as defined in Siddiqi (2000).
Fixed specimens were dehydrated in a gradient ethanol series, critical-point dried, sputter-coated with gold
and observed by scanning electron microscopy (SEM)
according to Abolafia et al. (2002).
Perineal patterns of mature females were prepared
according to standard procedures (Hartman and Sasser
1985). Briefly, root tissues were teased apart with forceps and half spear to remove adult females. The lip and
neck regions of the nematode were excised, and the
posterior end was cleared in a solution of 45 % lactic

acid to remove remaining body tissues. Then, the perineal pattern was trimmed and transferred to a drop of
glycerin. At least 50 perineal patterns were examined for
species identification.
Isozyme phenotype analysis
!!
To obtain sufficient individuals of M. spartelensis n. sp.

for electrophoretic analyses, the nematode population
under study and a reference M. javanica population
from olive trees sampled at Crdoba, Spain (Nico
et al. 2002), were increased on tomato (cv. Roma) in a
glasshouse at 253 C. For that, a single egg mass of
M. spartelensis n. sp. was placed beneath the roots of
individual tomato seedling in 12 cm pots filled with
sterile loamy soil. Sixty days after inoculation, tomato
plants were uprooted, their roots gently washed free of
soil and the root tissues teased apart using forceps and
transfer needles to remove adult females.
Five young egg laying females of Meloidogyne
spartelensis n. sp. and of M. javanica (reference population) were macerated in microtubes containing 5 l of
20 % (wt/vol) sucrose, 1 % (vol/vol) Triton X-100 and 0
01 % (wt/vol) bromophenol blue. Electrophoresis was
carried out in 78 cm separating (pH 84) and stacking
(pH 68) homogeneous gels, 7 and 4 % polyacrylamide,
respectively, 075-mm thick, with Tris-glycine buffer in
a Mini Protean II electrophoresis unit (BioRad). Gels
were stained with the substrate -naphthyl acetate for
Est and with Fast Blue RR (Sigma-Aldrich) for Mdh.
Band patterns and relative migration of the bands (Rm)
were compared to M. javanica (Esbenshade and
Triantaphyllou 1985).
Nematode molecular identification
For molecular analyses, one female nematode was temporary mounted in a drop of 1 M NaCl containing glass
beads and after taking measurements and photomicrographs of diagnostic characters the slides were dismantled and DNA extracted. Nematode DNA was extracted
from single females and PCR assays were conducted as
described by Castillo et al. (2003b). The D2-D3 expansion segments of 28S rDNA was amplified using the
D2A (5-ACAAGTACCGTGAGGGAAAGTTG-3)
and D3B (5-TCGGAAGGAACCAGCTACTA-3)
primers (De Ley et al. 1999). The ITS region was
amplified using forward primers 18S (5-TTGATTAC
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Eur J Plant Pathol
GTCCCTGCCCTTT-3) and 26S (5-TTTCACTCGC

CGTTACTAAGGGAATC-3) (Vrain et al. 1992). The
18S region was amplified with MelF (5-TACGGACT
GAGATAATGGT-3) and MelR (5-GGTTCAAGCC
ACTGCGA-3) as described in Tigano et al. (2005).
The region of the mitochondrial genome between the
cytochrome oxidase subunit II (coxII) and 16S rRNA
mitochondrial DNA (mtDNA) genes was amplified
using primers C2F3 (5-GGTCAATGTTCAGAAATT
TGTGG-3) and 1108 (5-TACCTTTGACCAATCA
CGCT-3) (Powers and Harris 1993). A 450-pb portion
of the cytochrome oxidase subunit I gene (coxI) of
mtDNA was amplified using primers COX1F1 (5T T G RT T T T T T G G T C AT C C T G A R G - 3 ) a n d
COX1R1 (5-WSYMACWACATAATAAGTATCAT
G-3). COX1F1 and COX1R1 were designed using
PRIMER3 software (http://frodo.wi.mit.edu/primer3/
input.htm) from nematode mtDNA sequences
(Lazarova et al. 2006).
PCR products were purified after amplification using
ExoSAP-IT (Affmetrix, USB products), quantified
using a Nanodrop spectrophotometer (Nanodrop
Technologies, Wilmington, DE, USA) and used for
direct sequencing in both directions using the primers
referred above. The resulting products were purified and
run on a DNA multicapillary sequencer (Model 3130XL
genetic analyser; Applied Biosystems, Foster City, CA,
USA), using the BigDye Terminator Sequencing Kit
v.3.1 (Applied Biosystems, Foster City, CA, USA), at
the Stab Vida sequencing facilities (Caparica, Portugal).
CoxI PCR products were purified and sequenced in both
directions using the primers referred above by Eurofins
MWG Company (Germany). The resulting products
were corrected and aligned using Geneious R7 v. 7.1.2
(www.geneious.com) and BioEdit (Hall 1999) softwares. The newly obtained sequences were submitted
to the GenBank database under accession numbers indicated on the phylogenetic trees.
Phylogenetic analyses
D2-D3 expansion segments of 28S rDNA, ITS rDNA,
partial 18S and coxII-16S rRNA sequences of different
Meloidogyne species from GenBank were used for phylogenetic reconstruction. Outgroup taxa for each dataset
were chosen according to previous published data
(Castillo et al. 2009). The newly obtained and published
sequences for each gene were aligned using MAFFT
(Katoh et al. 2002) with default parameters. Sequence
alignments were manually edited using BioEdit (Hall

1999). Phylogenetic analyses of the sequence data sets
were performed based on maximum likelihood (ML)
using PAUP * 4b10 (Swofford 2003) and Bayesian
inference (BI) using MrBayes 3.1.2 (Ronquist and
Huelsenbeck 2003). The best fitted model of DNA
evolution was obtained using jModelTest v. 2.1.1
(Darriba et al. 2012) with the Akaike Information
Criterion (AIC). The Akaike-supported model, the base
frequency, the proportion of invariable sites, and the
gamma distribution shape parameters and substitution
rates in the AIC were then used in phylogenetic analyses. BI analysis under a general time reversible of invariable sites and a gamma-shaped distribution (GTR+
I+G) model for D2-D3 expansion segment of 28S
rDNA, (GTR+G) model for ITS1, (GTR+I+G) model
for partial 18S and (GTR+I+G) model for coxII-16S
rRNA, were run with four chains for 2106, 2106, 3
106 and 1106 generations, respectively. The Markov
chains were sampled at intervals of 100 generations.
Two runs were performed for each analysis. After
discarding burn-in samples and evaluating convergence,
the remaining samples were retained for further analyses. The topologies were used to generate a 50 % majority rule consensus tree. Posterior probabilities (PP)
are given on appropriate clades. Trees were visualised
using TreeView (Page 1996). In ML analysis the estimation of the support for each node was obtained by
bootstrap analysis with 100 fast-step replicates.
!
!
Results
Meloidogyne spartelensis1 n. sp. (Figs. 1, 2 and 3,
Table 1)
Holotype (female in glycerin) Body length L=712 m;

maximum body width=409 m; a=1.5; stylet length=
14.5 m; dorsal pharyngeal gland opening (DGO)=
3.5 m; excretory pore from anterior end=21.5 m;
excretory pore distance from anterior end/length of stylet (EP/ST)=1.5; vulva slit length=22 m; distance
from vulva to anus=26.5 m.
1
The species epithet is derived from Cape Spartel, the locality

from which the new species was collected.
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!
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Fig. 1 Line drawings of Meloidogyne spartelensis n. sp. from Morocco. Second-stage juvenile (a-f). a Whole body. b Pharyngeal region. c
Lip region. d-f Tail regions. Female (g-i). g Whole females. h Lip region. i Perineal patterns. Male (j-k). j Lip region. k Tail region
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Fig. 2 Light micrographs of Meloidogyne spartelensis n. sp. from
Morocco. Male (a-g). a Pharyngeal region. b Lip region. c Detail
of stylet. d-f Tail region showing spicules and gubernaculum. g
Lateral field at mid-body. Second-stage juvenile (h-n). h Whole
Second-stage juvenile. i Pharyngeal region. j Lip region. k-n Tail
regions. Mature female (o-s). o Whole female. p Female neck
region. q-s perineal pattern. (Scale bars: a, d-f, k-n, p-s=20 m;
b, c, g, j=10 m; h=50 m; o=100 m)
Female paratypes (n=16) L=62189 (489772) m;

maximum body width=38645 (267455) m; a=1.6
0.1 (1.41.8); stylet length 15.10.3 (14.515.5) m;
excretory pore from anterior end 23.12.2 (21.030.0)
m; EP/ST ratio (excretory pore to head end/stylet
length) = 1.5 0.1 (1.42.0); vulva slit = 22.0 1.6
(18.025.0) m; vulva-anus distance=21.32.2 (16.5
35.0) m.
!
Female Body usually completely embedded in galled
tissue in tomato, pearly white body, varying in shape
from ovoid to saccate and with a variable neck diam.
and length (Figs. 1g to 2o). Lip region continuous with
body contour. Head cap variable in shape, with labial
disk and post labial annulus not elevated. In SEM view,
the labial disc appears round-squared, slightly raised on
the medial and lateral sectors, which are all fused together (Fig. 3a and b). Labial framework weakly sclerotized. Amphidial apertures elongated, located between
labial disc and lateral lips. Stylet fairly long, with an
almost straight, rarely curved, cone, cylindrical shaft
and knobs oval and sloping posteriad, sometimes with
concave anterior surfaces. Excretory pore usually at
level of anterior end of procorpus. Pharyngeal gland
with a large mononucleate dorsal lobe and two subventral gland lobes, usually difficult to see. Perineal
pattern mostly rounded to oval, with moderately high
dorsal arch that is mostly rounded and sometimes squarish and generally low, with fine, sinuous cuticle striae,
which become coarser in the vicinity of perivulval region; lateral field not clearly visible. However, in some
specimens the lateral fields were slightly marked. In a
few specimens, the striae form two wings or shoulders
ending near the lateral field, which, in this case, are
made more visible by fine and small zigzag striae.
Phasmids distinct, located just above the level of anus.
Vulva slit in the middle of the unstriated area, almost as
long as the vulva-anus distance; anus fold clearly visible, but not always present (Figs. 1i and 2q to s).
Punctations and striae absent in perineum. Commonly,
large egg sac occurs outside the root gall, containing up

to 350450 eggs.
Male Body vermiform, tapering anteriorly; tail rounded,

with twisted posterior body portion. Lip region slightly
set off from body and with a high head cap. Lip framework strong and sclerotised. Prominent slit-like
amphidial openings between labial disc and lateral lips
(Fig. 3e to f). In SEM view, the labial disc is slightly
narrower and raised above the merged subventral and
subdorsal medial lip sectors, with a centred oval
prestoma into which opens a slit-like dorso-ventrally
oriented stoma; lateral lips reduced to a very narrow
strip, largely fused, in the middle part, with the postlabial annulus. Lip region high and lacking annulation.
Stylet robust and straight, with cone and shaft broadening slightly in the distal part. Stylet knobs mostly rounded, laterally or obliquely directed, merging gradually
with the base of the shaft. Lateral field consisting of
four incisures with areolations along body but only few
actually cross central field. Procorpus distinctly
outlined, three times larger than metacorpus.
Metacorpus ovoid, with a strong valve apparatus.
Excretory duct curved. Excretory pore distinct and usually located four to six annuli posterior to hemizonid.
Normally only one testis extending anteriorly. Spicules
of variable length, arcuate and with two pores clearly
visible at tip. Gubernaculum distinct. Phasmids at level
of cloacal aperture and located in central lateral field,
showing slit-like openings. All morphometric measurements of adult males are given in Table 1.
Second-stage juveniles Body vermiform (Figs. 1a and
2h), tapering more towards posterior than anterior end.
Lip region narrower than body and slightly set off
(Figs. 1b and 2i). Head cap slightly elevated. Lip framework weakly developed (Figs. 1c and 2j). Labial disc
and medial lips fused. In labial disc, a stoma-like slit
located in an ovoid prestoma and surrounded by six
inner labial sensilla. In SEM view, the labial disc appears oval to rectangular in shape, raised above medial
lips, to which it merges in a dumbbell-shaped structure.
Lip region smooth and lacking annulation (Fig. 3j to k).
Amphidial apertures elongated and located between
labial disc and lateral lips (Fig. 3l). Body annulated from
anterior end to terminus. Lateral field consisting of four
incisures, with areolations along body but only few
extending across field. Stylet delicate, with cone
straight, narrow, sharply pointed, shaft almost
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Fig. 3 Scanning electron microscope photographs of
Meloidogyne spartelensis n. sp. from Morocco. Female (a-d). a
anterior end in lateral view showing excretory pore. b Lip region in
en face view. c-d Perineal pattern. Male (e-i). e Anterior end in
ventro-lateral view. f Lip region in en face view. g Lateral field at
mid-body. h-i Tail region. Second-stage juvenile (j-o). j-k Lip
region in lateral view. l Lip region in en face view. m Lateral field
at mid-body. n-o Tail region. Abbreviations: a= anus; am =
amphid; ep=excretory pore; oa=oral aperture; v=vulva. (Scale
bars: a, g, h, m=5 m; b, j, l=1 m; c-e, n, o=10 m; f, i, k=
2 m;)
cylindrical, and knobs small, rounded, separate

from each other, laterally directed. Pharynx with
a long, cylindrical Procorpus (3.0-4.0 times length
of metacorpus), round-oval metacorpus, short isthmus and rather long gland lobe, with three equally
sized nuclei and overlapping intestine ventrally.
Hemizonid located anterior to excretory pore, extending for ca. two body annuli. Excretory pore
Table 1 Morphometrics of adult

males and second-stage juveniles
(J2) of Meloidogyne spartelensis
n. sp. All measurements in m
and in the format: means.d.
(range)*
Abbreviations are defined in

Siddiqi (2000)
located posterior to nerve ring. Excretory duct

curved and discernible when it reaches intestine.
Rectum slightly dilated. Tail long, conoid, terminus usually pointed, with several constrictions in
the hyaline region (Figs. 2 and 3). Tail annulation
fine, regular in the proximal two third, becoming
slightly coarser and irregular in the distal part.
Hyaline tail terminus clearly defined and long,
phasmids small, difficult to observe. All morphometric measurements carried out on second-stage
juveniles are given in Table 1.
!
!
Associated host and locality
Meloidogyne spartelensis n. sp. was found in a loamyclay soil around the roots of wild olive (Olea europaea
subsp. europaea var. sylvestris) at Cape Spartel, near
Tanger city, northern Morocco (35.790583N;
5.924983W, altitude 16 m).
Males
Second stage juveniles
15
1497154 (11441633)
45125.1 (398489)
Stylet
18.60.7 (17.519.5)
14.00.5 (13.014.5)
Stylet conus
9.40.6 (8.510.5)
7.70.5 (7.08.5)
Knobs width
4.50.6 (4.05.5)
2.00.3 (1.52.5)
D.G.O.
3.30.6 (2.54.0)
2.10.6 (1.53.0)
O (%)
17.63.1 (13.921.6)
15.24.5 (10.720.7)
Pharynx (to cardia)
12725.1 (85165)
Pharynx. (to end of gland lobe)
19420.7 (167226)
14117.1 (109165)
Pharyngeal overlap
458.2 (3454)
Lip end to excretory pore
1459.7 (133159)
8412.7 (60109)
Max body diam.
333.2 (2837)
15.31.1 (14.0-17.5)
Testis length
75783.8 (672894)
T (%)
54.56.4 (44.062.3)
Tail length
13.21.8 (10.016.0)
78.87.1 (6993)
Anal body diam.
25.52.4 (23.028.5)
10.81.0 (9.513.5)
Tail hyaline portion (J2)
28.110.8 (19.546.0)
Spicules
27.51.8 (25.030.5)
Gubernaculum
6.90.3 (6.57.5)
42.52.1 (40.146.3)
28.93.1 (22.734.9)
3.70.9 (2.85.2)
7.31.0 (6.49.1)
3.30.4 (2.73.8)
107.820.9 (75.0141.1)
5.80.5 (5.06.4)
0.50.1 (0.40.6)
7.30.8 (6.38.8)
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Type material
Holotype female, female perineal patterns, J2 and
paratype males, mounted on glass slides deposited in
the authors nematode collection at the Institute for
Sustainable Agriculture, CSIC, Crdoba, Spain.
Additional males and J2 paratypes were distributed to
the United States Department of Agriculture Nematode
Collection, Beltsville, MD, WANECO collection,
Wageningen, The Netherlands (http://www.waneco.
eu/), and IRD collection, Montpellier, France. Specific
D2-D3, ITS, 18S-rDNA and coxII-16S rRNA and coxI
of mtDNA sequences are deposited in GenBank with
accession numbers KP896292-KP896293, KP896294,
KP896295, KP896297, and KP997290-KP997301
respectively.
Diagnosis and relationships
Meloidogyne spartelensis n. sp. is characterized by a
stylet (14.515.5) m long, a perineal pattern mostly
rounded to oval, with moderately high dorsal arch that is
mostly rounded and sometimes squarish and generally
low, excretory pore usually at level of anterior end of
procorpus (EP/ST ratio=1.42.0), second-stage juveniles with smooth lip region, tail long, conoid, terminus
pointed, with several constrictions in the hyaline region
(19.546.0) m long, males with stylet 17.519.5 m
long, and specific D2-D3, ITS, 18S-rDNA, partial coxII16S rRNA and coxI sequences.
The female perineal pattern morphology of
M. spartelensis n. sp. is roundish without marked lateral
lines, which places it in Jepsons Group 3 (Jepson 1987).
While considering the species morphology,
M. spartelensis n. sp. is related to the Bgraminis-group^
studied by Jepson (1987), it is morphometrically closer
to M. kralli (Jepson 1983). Females differ by EP/ST
(1.42.0 vs. 2.03.0 in M. kralli). Juveniles were smaller
b ratio (3.7 vs 6.5), longer stylet (13.014.5 vs 10.5
11.5 m), and shorter DGO (2.1 vs 4.1). Males were
longer (1497 vs 1076 m), higher a ratio (42.5 vs 31.7),
shorter DGO (3.3 vs 4.4) and longer distance from
anterior end to excretory pore (145 vs 127.4). Partial
18S from M. kralli (KJ636370) is also different to
M. spartelensis n. sp. in 3 % differences (24 nucleotides
in difference).
Meloidogyne spartelensis n. sp. is also similar to
M. dunensis, from which it differs mainly in morphology of female perineal patterns (lateral field clear visible
on M. dunensis). Juvenile stylet is longer (13.014.5 vs

11.012.5 m) and as its hyaline part of tail (19.546.0
vs 9.516.5 m). In males, c ratio (107.8 vs 280.6) and
spicules (25.030.5 vs 29.038.0 m) were smaller. The
new species is also morphologically close to M. sewelli,
from which it differs mainly in higher female EP/ST
ratio (1.42.0 vs 1.2), longer length of J2 stylet (13.0
14.5 vs. 11.012.0 m), and in shorter length of male
gubernaculum (6.57.5 vs 8.09.0 m) and shorter
DGO (2.54.0 vs 5.06.0). D2-D3, ITS and partial
18S sequences from M. dunensis (EF612713,
EF612711 and EF612712, respectively) are also different to M. spartelensis n. sp. in 7 % differences (51
nucleotides), 6 % differences (25 nucleotides), and
2 % differences (16 nucleotides), respectively.
Meloidogyne spartelensis n. sp. can also be compared
with several other Meloidogyne species commonly infecting woody plants, such as M. arenaria, M. baetica,
M. hispanica, M. incognita and M. javanica. Their main
comparative diagnostic characters, useful to a rapid and
easy identification, are summarized in Table 2.
Meloidogyne spartelensis n. sp. also differs markedly
from the other known European root-knot nematode
species (Karssen and van Hoenselaar 1998; Karssen
2002; Castillo et al. 2003b).
In addition, M. spartelensis sp. n. differs from related
species in Est and Mdh phenotypes, as well as sequences of the ITS1-58S-ITS2 region, the small subunit 18S rDNA, the D2-D3 fragment of the 28S gene of
rDNA, the coxII-16S rRNA, and coxI gene sequences
(see below).
Isozyme analysis and molecular characterization

The isozyme electrophoretic analysis of five-specimen
groups of young egg-laying females of M. spartelensis
n. sp. revealed one strong Est band (Rm=42.65) after
prolonged staining (Fig. 4a) and one N1b Mdh band
(Fig. 4b) that did not occur in the Est and Mdh phenotypes of M. javanica, which showed J3 and N1 phenotypes, respectively (Fig. 4a and b) or in other Est and
Mdh phenotypes previously identified for other
Meloidogyne spp. Malate dehydrogenase phenotype of
M. spartelensis n. sp. clearly differs from that of
M. kralli (with N1c bands) (Karssen 2002), showing a
lower unique band for M. spartelensis n. sp in comparison to M. javanica and those of M. dunensis (with a
weak VS1 and N1c bands, respectively, Table 2), with a
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Morphological and morphometric differences among Meloidogyne species closely related to Meloidogyne spartelensis n. sp. and parasite species to olivea, b
spartelensis n. sp.
arenaria
baetica
dunensis
incognita
javanica
kralli
lusitanica
sewelli
Female
15.1 (14.515.5)
15.5 (14.415.8)
17.5 (1719)
14 (1316)
14 (1316)
17.0 (14.018.0)
18.2 (17.619.0)
17 (1619)
14.5 (14.015.0)
Knobs shape
oval and sloping

posteriorly,
sometimes
with concave
anterior
surfaces
1.5 (1.42.0)
rounded and
backwardly
directed
ovoid, sloping
backwards
rounded and/or
sloping
backwards
rounded or
drawn out
laterally
rounded
knobs set off from

shaft, rounded
and transversely
ovoid
ovoid and
backwardly
directed
knobs small,
rounded
2.4
0.7 (0.50.8)
1.6 (1.21.8)
1.4
(2.03.0)
2.6 (1.63.8)
1.2
striae interrupted
laterally by
conspicuous
double
incisures edging
lateral ridges
Circular to ovoid with striking, trapezoid with rounded dorsal arch

and many
coarse striae and
fine striae, lateral
medium to high
field faintly visible.
discontinuous,
dorsal arch
closely spaced
striae
EP/ST ratio
Perineal
pattern
rounded, with
round to oval with
distinct inner striae
numerous fine
dorsal arch low,
forming two distinct
sinuous dorsal and
striae fairly widely
longitudinal bands,
smooth ventral striae,
separated and
extending
with lateral fields
lateral field usually
throughout
usually not visible
marked by some
the perineum to just
forked and
below the vulva
broken striae
VS1
A1,A2,A3
VS1
Est
Isozymec
phenotype Mdh N1b
rounded to oval with striae closely spaced,

numerous fine
very wavy to
dorsal and ventral
zig-zag, dorsal
arch high
striae, lateral
field clear visible
Stylet length
VS1
I1
J3
A1
N1
N1c
N1c
N1
N1
N1c
P3
(450490)
738 (5101.012)
403 (394 to 422)
446 (417483)
371 (337403)
417 (387459)
439 (408476)
450 (390515)
505 (460540)
Second-stage juvenile
Body length
451 (398489)
Stylet length
14.0 (13.014.5)
11.6 (11.112.6)
13.5 (1314)
11.5 (11.012.5)
10.5 (9.611.7)
10.4 (9.411.4)
10.8 (10.511.5)
14.2 (1316)
(11.012.0)
Hemizonid
position
Tail length
23 annuli to EP
23 annuli anterior
to EP
50 (4754)
anterior, adjacent
to EP
68 (5482)
just anterior
to EP
48.7 (4552)
immediately
anterior to EP
49(3656)
anterior to EP
68.1 (61.078.0)
one or two annuli

anterior to EP
44 (3950)
78.8 (6993)
anterior, not adjacent

to EP
55.8 (52.259.9)
Tail hyaline
portion
Male
28.1 (19.546.0)
14.8 (10.819.8)
12 (1013)
14.0 (9.516.5)
8.9 (6.313.5)
13.7 (9.818.0)
17.4 (14.521.0)
12 (1014)
12.0 (9.013.5)
20.3 (18.923.4)
18.8 (18.020.0)
24.5 (2127)
19.0 (18.020.0)
76 (7082)
Stylet length
18.6 (17.519.5)
21.6 (20.723.4)
17 (1619)
20 (1622)
24.8 (24.326.1)
Knobs shape
smoothly rounded
to stylet
rounded and sloping

backwards
relatively small,
rounded, set off
knobs set off from

low and wide, often
rounded with flat,
shaft, rounded
anteriorly indented,
concave or toothed
and somewhat
and set off from
anterior margins
transversely ovoid
the shaft
Spicules
rounded, laterally or
obliquely directed,
merging gradually
with the base of
the shaft
27.5 (25.030.5)
25.7 (3134)
27 (2436)
35.3 (2938.1)
35 (2940)
29.5 (22.935.3)
26.3 (22.528.0)
37.9 (32.044.5)
29 (28.030.0)
Gubernaculum
6.9 (6.57.5)
8.2 (7.29.4)
12 (1014)
8.5 (6.010.5)
11.2 (9.413.7)
8.0 (5.710.1)
10.2 (8.512.0)
(8.09.0)
elongate, pear shaped, rounded

slightly set off from
shaft
All measurements are in m unless otherwise stated

Measurements in Abrantes and Santos 1991; Castillo et al. 2003b; Jepson 1983, 1987; Palomares-Rius et al. 2007
Isozyme phenotypes used are defined by Esbenshade and Triantaphyllou (1985)
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Eur J Plant Pathol
1979 (KF542870), M. ichinohei Araki 1992

(EF029862), M. artiellia Franklin 1961 (AY150369)
and M. baetica (AY150367). Meloidogyne spartelensis
n. sp. is closely related to M. dunensis (EF612712) and
M. silvestris Castillo et al. 2009 (EU570214) and in a
higher clade level with M. hapla (KJ645433 and
KJ598136).
Phylogenetic analysis (BI and ML) of M. spartelensis
n. sp. based on ITS sequences of a multiple edited
alignment including 71 sequences and 681 total characters showed one clade clearly separated and supported
(Fig. 6). Similarly to D2-D3 expansion segments of 28S
rDNA phylogeny M. artiellia (KC545880, JX393299
and AF248478), M. baetica (AY150366), M. camelliae
(KF542872, JX912885 and KF542871), M. mali
(JX978229) and M. panyuensis Liao et al. 2005
(AY394719) were not included in this major and well
supported clade. Meloidogyne spartelensis n. sp. formed
a well-supported clade with M. dunensis (EF612711).
n. sp. based on partial 18S sequences of a multiple
edited alignment including 76 sequences and 1641 bp
showed several nested clades clearly separated and supported (Fig. 7). A major clade including 62 sequences
was well supported while M. mali (KJ636400,
KC875395, KF895400 and JX978225) occupied a basal
position to this clade. Meloidogyne artiellia (KC875391
and KC875392) and M. baetica (KP896296) formed a
well-supported clade nested to the clades explained
before. Finally, M. ichinohei (KJ636350, KC875386
and KJ636349), M. camelliae (JX912884) and
M. coffeicola Lordello & Zamith 1960 (HE667739)
formed a well-supported basal clade to the other species.
Meloidogyne spartelensis n. sp. was related to
M. microtyla Lordello & Zamith 1960 (AF442198),
and M. ardenensis (AY593894) in a moderately supported clade that also includes M. graminis (Sledge &
Golden 1964) Whitehead 1968 (JN241856) in a moderately supported clade.
n. sp. based on the partial coxII-16S rRNA of a multiple
edited alignment including 62 sequences with 1633 bp
in length showed two moderate supported clades
(Fig. 8). One clade contained the majority of the species
while the other was formed by M. camelliae (JX912887)
and M. mali (KC112913). In the main clade,
Meloidogyne spartelensis n. sp. is sister to two wellsupported subclades: the first including M. arenaria,
M. thailandica Handoo et al. 2005, M. morocciensis
Fig. 4 Esterase (a) and malate dehydrogenase (b) phenotype

electrophoresis patterns of protein homogenates from five young,
egg-laying females of Meloidogyne spartelensis n. sp. from Morocco (Ms), and five young, egg-laying females of M. javanica
reference population (Mj)
lower esterase band and higher Mdh band, respectively,

taking as reference M. javanica for M. spartelensis n. sp.
Amplification of the 18S, ITS1-5.8S-ITS2, D2-D3
region of 28S rDNA, and partial coxII-16S rRNA and
coxI mtDNA from M. spartelensis n. sp. yielded single
fragments of approximately 900, 700, 840, 500 and
400 bp respectively. The partial D2-D3, ITS1-5.8SITS2, 18S, and partial coxII-16S rRNA sequences of
M. spartelensis n. sp. were clearly different from that
present in the GenBank database. Substantial sequence
divergence for D2-D3 region of 28S (7 % against
M. dunensis, (EF612712)), ITS1-5.8S-ITS2 (11 %
against M. hispanica Hirschmann 1986 (JX885742)),
partial 18S (1 % against M. ardenensis Santos, 1968
(AY593894)), and partial coxII-16S rRNA (19 %
against M. hapla, AY757889) sequences for the new
species distinguish M. spartelensis n. sp. from other
studied root-knot nematodes and support its separate
specific status. CoxI gene sequences from
M. spartelensis n. sp. (KP997290- KP997301) showed
a similarity of 89 % with M. hapla (JX683718 and
AY268113) and 86 % with M. javanica (KP202352),
M. arenaria (KP202350) and M. incognita (KJ476151).
Intraspecific variability for COI gene was of 99.6 % (1
nucleotide in 322).
n. sp. based on D2-D3 expansion segments of 28S
rDNA of a multiple edited alignment including 56 sequences and 765 bp showed one clade clearly separated
and supported (Fig. 5). The phylogenetic analysis
showed well supported groups at major and terminal
clades in both analyses. The phylogenetic tree resolved a
major clade excluding M. mali Itoh et al. 1969
(KF880399 and KF880400), M. camelliae Golden
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!
!
Fig. 5 Phylogenetic relationships within Meloidogyne species.

Bayesian 50 % majority rule consensus trees as inferred from D2
and D3 expansion segments of 28S rDNA sequences alignments
under the GTR+I+G model. Posterior probabilities more than
65 % are given for appropriate clades; bootstrap values greater

than 50 % are given on appropriate clades in ML analysis. Newly
obtained sequences in this study are in bold font
Handoo et al. 2005, M. javanica, M. incognita,

M. floridensis Handoo et al. 2004, M. ethiopica
Whitehead 1968, M. paranaensis Carneiro et al. 1996,
M. arabicida Lpez & Salazar 1989, M. enterolobii
Yang & Eisenback 1983, M. cf. haplanaria Eisenback
et al. 2003 plus other species not identified, and the
second including M. hapla and M. partytila
Kleynhans, 1986 in the other clade.
M. hapla, M. incognita, M. javanica, and

M. lusitanica (Castillo et al. 2010; Ali et al. 2014)
(see Table 2). The main character in its juveniles
associated with the long tail and hyaline region
indicates this species would be associated with the
Bgraminis^ group (Jepson 1987). However, its associated host plant (wild olive) is different to the main
species in this group (mainly associated to monocotyledon plants). Other plants such as arundo canes,
lentisc, Phillyrea, Mimosaceae schrubs, maritime
pine and eucalyptus trees, etc. were also detected
in the same site.
It also differs phylogenetically from this Bgraminis^
group of species but is related to M. dunensis and
M. silvestris associated in the Iberian peninsula with
dicotyledonous plants and with woody plants respectively (Palomares-Rius et al. 2007; Castillo et al. 2009).
The reproduction on tomato of this new species also
Discussion
The primary objective of this study was to identify
and molecularly characterize a species of
Meloidogyne detected in wild olive soils in northern
Morocco. However, this species was not related
morphologically to the other species associated with
olive such as M. arenaria, M. artiellia, M. baetica,
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Eur J Plant Pathol
!
!
Fig. 6 Phylogenetic relationships within Meloidogyne species. Bayesian 50 % majority rule consensus trees as inferred from ITS rDNA gene sequence alignment under the
GTR+G model. Posterior probabilities more than 65 % are
given for appropriate clades (in bold letters); bootstrap

values greater than 50 % are given on appropriate clades
in ML analysis. Newly obtained sequence in this study are
in bold font
expands its host-range, as for the polyphagous root-knot

nematode species M. incognita, M. javanica and
M. arenaria that are able to infect olive and other hosts.
However, the other group of species that parasite
olive (M. baetica and M. lusitanica) have a very
restricted host-range and limited prevalence (ElBorai and Duncan 2005). Despite no
M. spartelensis n. sp. surviving on olive cv.
Picholine du Languedoc (may be due to cultivar
incompatibility or low initial inoculum density),
pathogenicity experiments should be carried out with
M. spartelensis n. sp. in order to verify the olive host
suitability.
Phylogenetic analysis of the used molecular
markers (D2-D3 region, ITS, partial 18S, partial
coxII-16S rRNA and coxI sequences) demonstrates

that M. spartelensis n. sp. is different to the other
species with molecular markers in GenBank. It is
clearly phylogenetically related to M. dunensis,
found in Spain (Cullera, Valencia Province) with
sea rocket (Cakile maritima Scop.) in coastal
dunes. Both species are able to infect tomato and
possess long tails with similar morphological traits.
Probably both species evolved from the same ancestor species. This species is also related to
M. silvestris with only a woody host known (Ilex
aquifolium L.) and also described in the Iberian
Peninsula. These species group seems to be related
to a diversification process in the Mediterranean
basin with restricted areas of distribution in natural
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!!

Bayesian 50 % majority rule consensus trees as inferred from
partial 18S gene sequence alignment under the GTR+I+G model.
Posterior probabilities more than 65 % are given for appropriate
clades (in bold letters); bootstrap values greater than 50 % are

given on appropriate clades in ML analysis. Newly obtained
sequences in this study are in bold font
environments. This could be partially due to long

persistence of Meloidogyne spp. in isolated regions
(i.e., allopatric speciation), in particular during last
glacial maximum. Indeed, the place where
M. spartelensis n. sp. was detected belongs to
the Rif-Mountain plant-refuge area (Mdail and
Diadema 2009) and to the Gibraltar strait biodiversity hot-spot (Rodriguez-Sanchez et al. 2008). In
this specific area, wild olive consists in an ancestral wild gene pool with unique haplotypes
(Besnard et al. 2013) supporting refuge conditions.
The ability of some Meloidogyne species to infect
olive tree seems to be acquired in several events
in their evolution and not from a unique event,
because species with restricted ability to infect
other hosts, as M. baetica are related phylogenetically nor with them and nor the other group of
highly polyphagous species (M. incognita,
M. javanica and M. arenaria). Meloidogyne

lusitanica did not have molecular markers in order
to extend this supposition. However, this hypothesis is not completely tested with the lack of host
test for M. baetica, M. lusitanica and
M. spartelensis n. sp. Phylogenetic trees with the
markers used showed the ancestry of certain species in the different markers studied, i.e., M. mali,
M. camell iae , M . i c h i n o h e i, M. art i ellia ,
M. panyuensis, M. coffeicola and M. baetica.
This result is similar to other phylogenetic analysis
with a more limited number of Meloidogyne spp.
(Adams et al. 2009).
Meloidogyne spartelensis n. sp. also differs from Est
and Mdh isozyme electrophoretic patterns to the other
species closely related morphologically and specific to
olive parasitism. Differences in the number of bands and
their gel position give us the possibility to differentiate
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Eur J Plant Pathol
!!

Bayesian 50 % majority rule consensus trees as inferred from
partial coxII-16S rRNA genes sequences alignment under the
GTR+I+G model. Posterior probabilities more than 65 % are
given for appropriate clades (in bold letters); bootstrap values

greater than 50 % are given on appropriate clades in ML analysis.
Newly obtained sequence in this study are in bold font
it. The analysis of isozyme electrophoretic patterns, in

particular Est and Mdh, as well as several molecular
approaches have been proved to be a valuable tool for
precise identification of Meloidogyne species (Flores
Romero and Navas 2005; Blok and Powers 2009).
This accurate species description with molecular
markers associated to the type population (D2-D3 region, ITS, partial 18S and partial coxII-16S rRNA; coxI
of mtDNA) will provide accurate markers for the unequivocal species identification and monitoring the possibility of expansion or identification in other untested
areas.
In summary, present study establishes the importance
of using polyphasic identification highlighting the time
consuming aspect and difficulty of a correct
identification at species level within the Meloidogyne

spp. and describes a new root-knot nematode associated
with wild olive in the Mediterranean Basin area.
Acknowledgments This research was supported by grant
KBBE219262 ArimNET_ERANET FP7 20122015 Project
PESTOLIVE Contribution of olive history for the management
of soilborne parasites in the Mediterranean basin from Agence
Nationale de la Recherche (ANR, France) and Instituto Nacional
de Investigacin y Tecnologa Agraria y Alimentaria (INIA,
Spain), grant Long-Term Mission Program from Institut de
Recherche pour le Dveloppement (IRD, France), PhD grant from
Tishreen University (Syrian Arab Republic), grant ANR-10LABX-41 from LABEX TULIP (France), grant AGR-136 from
Consejera de Economa, Innvovacin y Ciencia from Junta de
Andaluca, and Union Europea, Fondo Europeo de Desarrollo
regional, BUna manera de hacer Europa^. The authors thank J.
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Martn Barbarroja and G. Len Ropero from IAS-CSIC for the
excellent technical assistance.
!!
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31, 263401.
Yang, B., & Eisenback, J. D. (1983). Meloidogyne enterolobii n.
sp. (Meloidogynidae), a root-knot nematode parasitizing
pacara earpod tree in China. Journal of Nematology, 15,
381391.
Ziljstra, C., Donkers-Venne, D. T. H. M., & Fargette, M. (2000).
Identification of Meloidogyne incognita, M. javanica and
M. arenaria using sequence characterised amplified region
(SCAR) based PCR assays. Nematology, 2, 847853.
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Chapitre IX
Diversit des Meloidogyne

associs lolivier au Maroc
Page 204
Rsum
Le chapitre suivant dcrit la diversit spcifique des nmatodes galles du genre Meloidogyne
associs lolivier au Maroc. Ces nmatodes ont t dtects dans 12% des chantillons
collects. Aprs levage sur tomate, les populations ont t identifies au niveau de lespce
par des marqueurs biochimiques (Estrase) et des marqueurs molculaires (SCARs, D2D3).
Les rsultats obtenus ont indiqu la dtection de quatre espces : M. javanica, M. arenaria, M.
hapla, M. spartelensis (rcemment dcrite comme une nouvelle espce de Meloidogyne), et
Meloidogyne n.sp. galement cense tre une nouvelle espce daprs les premiers rsultats
biochimiques, molculaires et biologiques (i.e. femelles fixes lextrieur des racines et
absence de masses dufs). La distribution de ces espces est impacte par les modalits de
lolivier sur lesquelles ont t collectes, puisque M. javanica (espce dominante dans les
chantillons) a t dtecte en forte abondance dans les vergers (haute densit et traditionnels)
et sur quelques oliviers fraux, alors que les autres espces ont t dtectes uniquement sur
olivier sauvage. Limpact des modalits de culture (sauvage, fral, culture traditionnelle et
haute densit), lirrigation (irrigu ou pluvial), des variables daphiques (type de sol, contenu
en matire organique, pH), et des variables climatiques (temprature maximale et minimale et
pluviomtrie) sur la distribution des espces a t analyse. Lanalyse a t renforce par une
analyse de dispersion via le calcul de lindice de dispersion VMR (variation mean ratio). Cette
tude a conclu sur une distribution agrge des Meloidogyne. La large dispersion de M.
javanica dans toutes les rgions de culture de lolivier est suppose tre lie une
introduction de ce nmatode partir des ppinires et une dispersion, par la suite, par les
pratiques culturales (irrigation, intensification de la culture), conditions particulirement
favorables la multiplication des Meloidogyne. Ltude suppose galement que les autres
espces de Meloidogyne dtectes sur olivier sauvage ont persist dans leurs zones refuge en
labsence dactivits humaines.
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Trend to explain the distribution of root-knot nematodes Meloidogyne spp.

associated with olive trees in Morocco.
Abstract
Knowing which factors affect the distribution of organisms provides useful insights for the
management of biodiversity. For root-knot nematodes Meloidogyne, a critical threat in
agriculture, an understanding of the constraints that impact their diversity is a fundamental
prerequisite for their control. They were reported to be major pests on olive trees, mainly in
high-density orchards and in nurseries. This study was undertaken in order to characterise the
Meloidogyne species associated with olive trees in Morocco and to consider how
anthropogenic stresses and some edaphic and climatic factors may be involved in their spatial
distribution. A soil sampling was conducted in the main olive growing areas in Morocco,
including wild, feral and cultivated olive trees. RKN were detected in 12.2% of the samples.
The analysis of biochemical (esterase patterns) and molecular (SCARs, D2D3) markers
revealed the occurrence of M. javanica (as the predominant species), M. arenaria, M. hapla,
M. spartelensis (newly described) and Meloidogyne n.sp. that also seems to be a new species.
Some esterase variability was detected in M. javanica and M. hapla populations. The RKN
species were distributed everywhere on an aggregated basis. However, M. javanica was
recorded in high abundance throughout the cultivated areas in Morocco in high-density and
traditionally cultivated olive orchards and rarely on some feral olive, while the other species
were detected in low abundance on wild olive in mountain plant refuge areas in northern and
southern Morocco. Nevertheless, the large distribution of M. javanica in cultivated areas
could be primarily due to widespread introductions from nurseries, followed by multiple
founder processes favoured by local conditions such as irrigation and planting density. In
contrast, the detection of the other species hosted by wild olive only in plant refuge areas
suggest that these areas host ancestral nematode species that did not disperse in the absence of
human activities.
Keywords: distribution; diversity; Meloidogyne; Morocco; olive, species identification.
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1. Introduction
The causes and consequences of the spatial distribution of plants and animals are considered
to be a main focus of biogeography and ecology researches. Dispersal, one of the fundamental
processes in biogeography, can explain organism distribution, similarities between distant
biota, as well as the impacts of human activities as means of transport for organisms.
Dispersal also has considerable consequences on individual fitness, allowing species to
colonise new habitats, on population dynamics and on genetics (Hanski, 1999; Bowler and
Benton, 2005). On the other hand, dispersal can have contrasting effects on species
coexistence and it can decrease local diversity by reducing refugia for less competitive species
(Chesson, 2000; Kneitel and Miller, 2003). Their dispersion ranges from passive transport
(with the help of humans, animals, wind, water, etc.) to active movement (in the case of
mobile forms) (Begon et al., 1996). However, some biological (ability of an organism to
reproduce and to disperse), physical (oceans or mountains, etc.) and environmental (soil type,
moisture conditions) factors could be barriers to dispersal (Parendes and Jones, 2000).
Determining the factors involved in the dispersal of individuals can be essential to
understanding how populations are spatially structured and how species respond to
environmental changes (Bowler and Benton, 2005). Furthermore, a better understanding of
these factors can provide useful insights for biodiversity management and conservation
biology in the context of exotic species invasion, the spread of pathogens and ecosystem
disturbances induced by anthropogenic influences (Pearson and Dawson, 2003; Wallace,
2012).
Nematodes are considered to be representative of soil invertebrates because they are
highly abundant in soils and encompass a wide range of trophic groups that include predators
and omnivorous species, as well as bacteria, fungus and plant feeder taxa that are
consequently involved in significant soil functions (Yeates, 2003). Soil nematodes are aerobic
and aquatic organisms that move actively but slowly over short distances (Prot and Nestcher,
1979) in poral water through large porosity (>50 m) (Carroll and Viglierchio 1981; Prot and
VanGundy, 1981a, b; Janiec, 1996; de Rooij-van der Goes et al., 1998). Among all the soil
nematodes, more attention is attributed to plant-parasitic nematodes (PPN) because of their
key role as agricultural pests (Neher, 2010). Since they depend on plants for their feeding
(obligate parasites), their dispersal over short distances is mainly from plant-to-plant
infestation through root networks in the soil. The distribution of PPN is typically aggregative
(Farias et al., 2002), often fitting with a negative binomial model (Goodell and Ferris, 1980;
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Herve et al., 2005). Their development is influenced by local soil factors such as pH, organic
matter content, texture and moisture (Norton and Hoffmann, 1974; Norton, 1978; Robertson
and Freckman, 1995; Zuckerman, 2012), and by local climatic conditions (Coomans, 2002;
Neher, 2010). Consequently, their dispersal over long distances is necessarily passive, via
water (contaminated irrigation or flooding), wind (White, 1953; Orr and Newton, 1971) or
human activities (Alenda et al., 2014) such as the introduction of infected planting material
(e.g., bulbs, tubers, rooted cuttings) or the displacement of dirty farm machines.
In that way, most infestations by root-knot nematodes (RKN) Meloidogyne spp. in olive
orchards (Olea europaea ssp. europaea) are caused by unsanitized plant material produced in
uncertified nurseries (Lehman, 1994). These nematodes were reported for the first time on
olive trees in the USA (Burher et al., 1933). Many species were thereafter detected: M.
javanica (Trajan, 1953), M. incognita and M. hapla (Minz, 1961), M. arenaria (Yang and
Zhong, 1980) and M. lusitanica (Abrantes and Santos, 1991). More recently, M. baetica
(referred to as the Mediterranean olive root-knot nematode) and M. spartelensis were
detected on wild olive trees (O. e. ssp. europaea var. sylvestris) in southern Spain (Castillo et
al., 2003) and in northern Morocco (Ali et al., 2015), respectively. Other unidentified species
were also reported in several countries (Ali et al., 2014). RKN have been reported as major
pests on olive trees, mainly in nurseries where irrigation conditions are favourable to their
multiplication (Nico et al., 2002; Sanei and Okhovvat, 2011), and controlled experiments
have proven the impact of RKN on olive growth (Afshar et al., 2014). They were reported in
19 olive-producing countries worldwide (Ali et al., 2014). Because of their economic
importance (Prez et al., 2001; Koenning et al., 1999), they were declared to be among the
top-ten impacting nematodes on plants (Jones et al., 2013).
Management of RKN in agriculture is a difficult challenge due to: (i) their adaptation to
a wide range of temperatures that allow them to have a ubiquitous distribution; (ii) their high
species diversity (98 Meloidogyne species reported as of February 2013, Jones et al., 2013);
(iii) different reproduction strategies (mitotic and meiotic pathogenicity and amphimixis;
Castagnone-Sereno et al., 2013) that might explain their wide host range estimated at 5,500
plants (Blok et al., 2008). Thus, in order to control their dispersion and their development,
identification of Meloidogyne species is a fundamental prerequisite for understanding their
physiology, their mode of reproduction and their ecology (Adam et al., 2007). However,
specific identification has always been a challenge, especially because of the existence of
species complexes, sexual dimorphism in some cases, etc. (Janati et al., 1982; Blok and
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Powers, 2009). Moreover, in practice, specific identification is made difficult by genetic

(virulence acquisition, for example), climatic and anthropogenic factors that are increasing in
response to the current global demand for agricultural production (Blok and Powers, 2009).
Consequently, several methods have been developed for the specific diagnosis of
Meloidogyne. Species characterisation is based primarily on morphological descriptions of
females (especially perineal patterns), males and second stage juveniles (Eisenback and
Hirschmann, 1979; 1980; Eisenback et al., 1980; Robertson et al., 2009). However,
morphological identification is difficult, especially for distorted pyriform females because
they may be embedded in the plant roots. The use of isozyme analyses and molecular methods
has thus increased due to the fact that they save time and to their accurate performance (Adam
et al., 2007; Blok and Powers, 2009). Some of these biochemical and molecular approaches
were used here in order to describe the diversity of RKN associated with olive trees in
Morocco where their diversity is totally unknown, especially in a context of increased acreage
and crop intensification (El Mouhtadi et al., 2014) that provides a favourable environment for
Meloidogyne multiplication and dispersion. The effects of cultivation conditions and of some
edaphic and climatic factors were also analysed since it is expected that they drive RKN
diversity. Another main objective of this study was to analyse how anthropogenic forces may
be involved in the distribution of RKN species from the perspective of two main olive
production systems in Morocco: (i) patchy natural ecosystems such as those found in plant
refuge areas dating from the Last Glacial Maximum, located in northern and southern
Morocco (Mdail and Diadma, 2009), and (ii) agrosystems such as traditional and highdensity orchards in agricultural plains and plateaus. The olive sampling device was
established in order to explain RKN species dispersal mechanisms in disturbed and nondisturbed environments and invasion processes in cultivated areas.
2. Materials and methods

Sampling took place in Morocco in March and April 2012. The survey was conducted in the
main olive growing areas, including: (i) wild and spontaneous trees that are usually referred to
as oleasters (O. e. ssp. europaea var. sylvestris) and O. e. ssp. maroccana, specific to the
Macaronesian Ecological Area; (ii) wild-looking forms of olive trees including feral forms
that correspond either to cultivated olive trees that were abandoned and became wild or to
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olive trees that grew from seeds of wild or cultivated olive that spread by birds; and (iii)
cultivated forms (O. e. ssp. e. var. europaea) distinguished according to tree density in
orchards as traditional (100 trees /ha) or high-density orchards (up to 2,200 trees /ha) (Mdail
et al., 2001; Green, 2002; Therios, 2009). A total of 213 soil samples were collected from 94
sites along a 900-km-long northeast-southwest transect, extending from the Strait of Gibraltar
to the city of Agadir: (i) along the edges of the Atlantic Ocean and the Mediterranean Sea
facing the Rif Mountains in the north; (ii) in the Jel Plains located to the east of Taza city in
eastern Morocco; (iii) in the Kandar region located in the northern Middle Atlas Mountains,
south of Fes city; (iv) in the Guerouane and Zaane regions located to the south of Meknes
city; (v) in the Tadla region located along the north side of the southern Middle Atlas
Mountains, near Beni Mellal city; (vi) in the Haouz region located on the north side of the
High Atlas Mountains, near Marrakech city; (vii) in the Souss region located on the south side
of the High Atlas Mountains, near Agadir city. Consequently, in Morocco, olive trees grow
over a large range of regional pedoclimates, including soil types (clayey to sandy soil), annual
precipitation regimes (from 800-1000 mm in the north to less than 200 mm in the south),
temperatures (12-24C in the Rif region, 10-26C in the Middle Atlas, 11-28C in High Atlas,
14-21C in the Souss region) and altitudes (from elevations of 200 m to 1700 m) (Ouazzani et
al., 1996).
2.2. Soil sampling

Considering that all PPN spend almost of their life cycle in the soil (Cadet and Thioulouse
1998), the sampling was focused on soil. It was conducted under the foliage of each olive tree
in the upper rhizosphere (the 20-30-cm deep layer inhabited by plagiotropic roots) with a
small spade. On cultivated olive trees (traditional and high-density cultivation), the sampling
was conducted in orchards under four trees located at a distance of 10 meters along transects.
Five sub-samples were collected from each tree. The 20 sub-samples recovered were
combined to form one 1-dm3 reference sample per orchard, considering that tillage and human
activities are frequent and could lead to homogeneous distribution of nematodes. On wild and
feral olive trees, the five sub-samples collected from each tree were then combined to form
one 1-dm3 reference sample per tree, considering that heterogeneous distribution of
nematodes were expected because of scarce or absent human interventions. Depending on the
availability of the orchards at each site or of trees in wild and feral sites, up to five orchards
were considered as repetitions for each cultivation category, and up to 25 trees were
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considered separately as repetitions at each wild and feral site.
2.3. Soil and climate characteristics

For soil physico-chemical analyses, a 100-cm3 dry and sieved (2 mm) aliquot from each soil
sample was used. The analyses were performed by the INRA Soil Laboratory (French
National Institute of Agricultural Research) in Arras, France. The soil parameters analysed
included: the proportion of clay (0-2 m), silt (2-50 m) and sand (50-2000 m) using the
Textol technique (NF X31-107, 2003); the proportion of organic carbon (ISO-10694, 1995)
using gas chromatography, making it possible to calculate organic matter content (OM =
1.724 x C); and the pHH2O (ISO-10390, 2005). These parameters have been shown to be
among the most significant factors affecting the life behaviour of PPN species (Cadet and
Thioulouse, 1998).
Mean annual maximal and minimal temperatures and mean annual rainfall data were
collected from climate stations close to the sampling sites.
2.4. Nematode extraction and root-knot nematode cultures

A 250-cm3 aliquot was used for nematode extraction from each soil sample using the
elutriation procedure (Seinhorst, 1962). Free forms of RKN in the soil (i.e., males and
juveniles) were identified by genus and enumerated under a stereomicroscope in 5-cm3
aliquots sampled from 25-cm3 suspensions (Merny and Luc, 1969). PPN population levels
were expressed per dm3 of fresh soil. When RKN were detected in soil samples, susceptible
tomato plants (cv. Roma) were transplanted in native soils in a greenhouse (12 hours light at
23C, 12 hours dark at 20) to grow and maintain the populations.
Approximately 60 days after transplantation (corresponding to two RKN life cycles),
the tomato roots were checked for the presence of RKN galls and egg masses. When no egg
mass was observed, roots and soil were placed in a mist chamber in order to collect juveniles
that may have remained in them (Seinhorst, 1950).
All the analyses carried out on live material were performed in the nematode quarantine
area (French Government Agreement n80622) of the Research Unit Centre de Biologie pour
la Gestion des Populations, in Montpellier, France).
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2.5. Identification of Meloidogyne species

2.5.1. Isozyme phenotype analysis
Infested tomato roots were gently washed free of soil and the root tissues teased apart
using forceps and transfer needles to remove adult females. Up to 50 females with their egg
masses were collected from each sample. Females were individually crushed in 250-L
microtubes containing 5 L of Trugdill buffer (Trudgill and Carpenter, 1971) supplemented
with 20% sucrose (pH 8.0), and then stored at -20C. Females from a pure M. javanica
population (provided by ANSES, Le Rheu, France) and reared on tomato plants were
prepared in the same way and used as the reference population for isozyme analyses.
Esterase electrophoresis was carried out in 78-cm homogeneous separating (pH 84)
and stacking (pH 67) gels, 7% and 3.5% Bis-acrylamide, respectively, 0.5-mm thick, with
Tris-glycine buffer in a Mini Protean II electrophoresis unit (BioRad). Microtubes with
single crushed females were centrifuged (10 min; 9,500 rpm) and supplemented with 0.01%
bromophenol-blue. The supernatants were transferred to the electrophoresis unit and the
electrophoresis process was performed at 2-8C (Maurer, 1971). Each gel included two
reference M. javanica females. The gels were stained with the substrate a-naphthyl acetate
and Fast Blue for esterase (Est) phenotypes (Brewer and Singh, 1970) and incubated at 37C
for one hour. The gels were then washed and placed in 10% acetic acid for several hours in
order to fix the stain of the bands, and then sandwiched between two cellophane sheets and
left to dry for 48 hours (Carneiro and Almeida, 2001). Band patterns and relative migration of
the bands (Rm) were compared to the M. javanica reference population (Esbenshade and
Triantaphyllou, 1985). Est-phenotypes were labelled with a letter for the Meloidogyne species
and a number referring to the number of bands detected (Esbenshade and Triantaphyllou,
1985; 1990).
2.5.2. Molecular identification

The egg masses corresponding to females used for the isozyme analysis were individually
placed in distilled water for hatching in order to produce juveniles and to clone populations
onto tomato plants. Five to ten juveniles were collected from each of them in order to provide
a sufficient amount of DNA. The molecular diagnostics were initially conducted using SCAR
markers (Sequence Characterized Amplified Regions) that are easy to use and do not require
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DNA sequencing. D2D3 markers (expansion segments of 28SrDNA) were then used when
Est or SCAR markers were not effective.
Nematode DNA was extracted using QIAgen DNeasy Blood & Tissue kits and PCR assays
were conducted with several primers according to the type of sequence targeted. The SCAR
fragments were amplified using pairs of primers: OPA-12 Fare/Rare (specific to M. arenaria),
OPB-06 Finc/Rinc (for M. incognita), OPA-01 Fjav/Rjav (for M. javanica) and OPI-14 Fh/Rh
(for M. hapla) (Zijlstra, 2000; Zijlstra et al., 2000). Amplifications were performed in a 10L
final volume containing 2L of DNA extracts, 5L of PCR QIAGEN kits (Multiplex-PCR), 1
L of SCAR primers and 2 l of sterile water, using the following temperature process: 95C
for 15min, 40 cycles at 94C for 30s, 58C for 90s and 72C for 90s, with a final extension at
72C for 10min. Amplified products were then verified on 1.5% agarose gel. A sized band
marker (GeneRuler, Eurogentec, AmphliSize, 200-10000 pb) was included in each gel in
order to measure the size of the amplified products, and the specific bands of the Meloidogyne
species were investigated.
The
D2-D3
fragments
were
amplified
using
the
D2A
(5-
ACAAGTACCGTGAGGGAAAGTTG-3) and D3B (5-TCGGAAGGAACCAGCTACTA3) primers (De Ley et al., 1999; Castillo et al., 2003; Ali et al., 2015). Amplifications were
performed in a 25L final volume containing 2ng DNA, 2.5L of 10x buffer, 0.75L of
MgCl2 (50mM), 0.25L dNTPs (10M), 0.75L of D2A and D3B primers, 0.2L of Taq
DNA polymerase (Qiagen) and sterile water (sqo). The D2D3 PCR reactions were set up at
94C for 3min, followed by 40 cycles at 92C for 30s, 55C for 1min and 72C for 90s, and a
final extension at 72C for 10min. As for SCAR assays, amplified products were verified on
1.5% agarose gel with a size marker. D2D3 PCR products were purified and sequenced in
both directions using the primers referred to above by Eurofins MWG (Germany). The
resulting products (17 to 24 sequences from each population) were corrected, aligned and
manually edited using Geneious R7 v. 7.1.2 and BioEdit Sequence Alignment Editor
Software (Hall, 1999).
2.6. Data analyses

In order to explore the distribution of the Meloidogyne species in the agro- and ecosystems
surveyed, multivariate analyses were applied on the abundance of RKN in the soil samples
where they were detected and on corresponding environmental factors such as: (i) agronomic
variables such as olive growing modality (wild, feral, traditional and high-density cultivation)
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and soil characteristics (soil type, organic matter content and pH); (ii) climatic variables such
as the mean annual maximum and minimum temperatures and the annual rainfall (Tab. 1).
PCA and MCA analyses were applied on RKN species and on environmental variables in
order to explore the correlation between each set of variables. A coinertia analysis was then
carried out in order to investigate the contribution of these environmental variables on the
Meloidogyne distribution. These different analyses were performed using the ade-4 and
FactoMineR packages (Dray and Dufour, 2007; L et al., 2008) using R software. The
dispersion of the Meloidogyne species was evaluated using the dispersion index calculated as
the variance-to-mean ratio VMR = s2/ (with s and = variance and mean of the abundance,
respectively) (Taylor, 1961). VMR gives us a reliable way to measure aggregation (Hariharan
et al., 2013): VMR=0 for undispersed populations that are uniformly distributed; VMR=1
when populations are randomly distributed; VMR>1 for aggregated or clumped distributions;
and 0<VMR<1 for binomial distributions.
3. Results
Meloidogyne spp. juveniles were detected in 26 out of 213 soil samples (12.2% occurrence)
distributed throughout the main olive growing areas in Morocco (except the Kandar and Jel
regions, Tab. 2) and occurred in all of the olive growing modalities (oleaster, feral, traditional
and high-density cultivation), except on O. e. subsp. maroccana. Twenty RKN isolates
reproduced on tomato plants, with roots that exhibited galls and egg masses. One isolate (I26)
detected on wild olive trees in the Souss region exhibited females feeding outside the roots of
tomato plants without galls or egg masses. Juveniles of Meloidogyne detected in isolates 255,
293, 340, 402 and 403 did not reproduce on tomato plants.
3.1. Species characterisation

The J3 Est phenotype of M. javanica used as a reference (Esbenshade and Triantaphyllou
1985) consisted of three bands with specific electrophoretic migration (a Rm of 46, 54.5 and
58.9%). Among all the females analysed, eight different phenotypes were observed,
designating ten different Est bands (Fig. 1). The reference M. javanica phenotype J3 was
detected as a pure population in most of the isolates (14 isolates). One J2 (J2a) Est phenotype
(bands 1 and 3 with a Rm of 46 and 58.9%, respectively), was detected as a pure population in
isolate (304) and mixed with J3 (1/3-2/3) in isolate (260). Another J2 (J2b) Est phenotype
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(bands 1 and 2 with a Rm of 46 and 54.5%, respectively) was mixed with J3 (1/4-3/4) in
isolate (252). These two J2 Est phenotypes labelled J2a and J2b were reported for M. javanica
Est phenotypes by Cofcewicz et al. (2004) and Tomaszewski et al. (1994), respectively. An
A2 Est phenotype, specific to M. arenaria (bands 4 and 5 with a Rm of 53.75 and 56.25%,
respectively; Esbenshade and Triantaphyllou, 1985), occurred in one isolate (313). All these
Est patterns were confirmed as M. javanica and M. arenaria using the species-specific SCAR
sequences (amplified fragments at 670 bp and 420 bp, respectively). Two Est phenotypes
were detected in isolate (373). One Est phenotype with one band (n6 with a Rm of 49.66%)
was close to the M. hapla pattern described by Esbenshade and Triantaphyllou (1985).
Another Est phenotype with two bands (one major band (n 7) and one minor band (n 8) with
a Rm of 49.83 and 54.37%, respectively) was detected. It did not correspond to Meloidogyne
patterns described in the literature. The SCAR markers processed on these two strains did not
provide any species-specific fragment. However, D2D3 amplified and sequenced fragments
blasted with M. hapla in the GenBank. The Est phenotypes were then referred to as H1 and
H2, respectively. A S1 Est phenotype specific to M. spartelensis (band n 9 with a Rm of
42.65%), confirmed by specific D2D3 sequences (Ali et al., 2015) occurred as a pure
population in isolate (312). Finally, another new Est pattern with a single band (n 10 with a
Rm of 61.12%) was detected in isolate (404). It did not correspond to Meloidogyne patterns
described in the literature and was therefore labelled NSP, considering this population as a
new species. However, not enough material was available for molecular diagnostics. Analyses
and results of all these different markers are summarized in Table 3.
3.2. Distribution of the Meloidogyne populations

Based on biochemical and molecular diagnostics, the occurrence of the Meloidogyne
populations detected in the olive areas surveyed in Morocco were: M. javanica in 65% of the
samples, and M. arenaria, M. hapla, M. spartelensis and Meloidogyne n.sp. in 4% of the
samples for each species. The M. javanica populations were the most widespread and were
never detected mixed with others species (Fig. 2). Nine J3 populations were detected on
cultivated olive trees (traditional and high-density) in the main producing areas (Haouz, Tadla
and Guerouane regions) with more than 102 nematodes/dm3 of soil (Tab. 3), and in lower
abundance (less than 102 nematodes/dm3 of soil) in the Souss valley (one J3 population). Four
J3 populations also occurred with quite high population levels (102-103 nematodes/dm3 of
soil) on feral olive trees in the Haouz and Tadla regions. Two J2a populations were observed
Page 215
either associated with a J3 population in a traditional orchard (Haouz region) or only on feral
olive trees (Tadla region); the abundance of these populations was less than 102
nematodes/dm3 of soil. Only one low-level J2b population was found mixed with J3 in a
traditional orchard in the Souss valley. On the other hand, M. javanica populations were
significantly more abundant in high-density orchards (average of 1.428 juveniles/dm3 of soil)
than in low-density orchards (traditional cultivation, average of 342 juveniles/dm3 of soil).
The populations of M. arenaria, M. spartelensis and M. hapla (H1 and H2 phenotypes) were
detected at low population levels (less than 102 juveniles) on wild olive O. europaea spp.
sylvestris only, in the Rif Mountains in northern Morocco, while Meloidogyne n.sp. was
found with low population levels in the western High Atlas Mountains in southern Morocco,
as well as on wild olive trees.
In order to describe the distribution pattern of these Meloidogyne populations, the VMR
index was calculated for the entire sampling device and in each olive growing area. All the
VMR measured were greater than 1, leading to the conclusion of an aggregated distribution
and low dispersion of all the Meloidogyne species.
3.3. Correspondences between the abundance of Meloidogyne species and environmental

factors
Considering the PCA analysis applied on the Meloidogyne species abundance, the loading
plot (Fig. 3A) showed that populations of M. javanica (jav) strongly contributed to the
negative values of the PC1 axis while M. arenaria (are), M. spartelensis (spa) and M. hapla
(hap) species contributed to its positive values. Meloidogyne n.sp. (nsp) contributed more to
PC2. The loading plot of the MCA analysis applied on the environmental factors (Fig. 3B)
indicated a high contribution of the wild olive modality (WO) (positive values) to MC1
opposed to the high-density (HD) and the traditional cultivation (TR) modalities (negative
values). The contribution of the feral olive modality (FO) was higher on the MC2 axis. The
water supply was also related to MC1 with rainfed olives in its positives values opposed to
irrigated olive in its negative values. Considering soil variables, the loading plot indicated a
high contribution of the soil type on the two axes. Clayey soils (CL) were strongly correlated
to MC1 opposed to silty soils (SI), while sandy soils (SA) were correlated to MC2. The other
soil variables such as the organic matter content (OM) and the acidity (pH) as well as the
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climatic variables (Tmax and Tmin temperatures, and annual rainfall AR) did not greatly
contributed to the MCA analysis. Moreover, the analysis showed a correlation between wild
olive (WO), clayey soils (CL) and rainfed conditions (R). According to the coinertia analysis
(Fig. 3C, 3D), these environmental variables were related to the occurrence of M. arenaria
(are), M. spartelensis (spa), M. hapla (hap) and Meloidogyne n.sp. (nsp). The coinertia
analysis also indicated a strong correspondence between the occurrence of M. javanica (jav)
and the high-density (HD) and traditional (TR) cultivation, the irrigated olive (I), the sandy
soils (SA), and the mean annual maximal temperature (Tmax).
4. Discussion
This investigation provided the first report on the diversity of Meloidogyne species associated
with olive trees in Morocco. RKN were one of the less frequent nematodes detected, with an
occurrence of 12%. This is consistent with other observations suggesting that RKN are not
frequently detected on olive areas (Diab and El-Eraki, 1968; Lamberti and Lownsbery, 1968;
Lamberti and Baines, 1969; Inserra et al., 1976; Abrantes et al., 1992). However, they are
considered as major pests on olive trees because high abundances were observed when they
were detected (Hashim, 1982). They are responsible for damages in nurseries (Hashim, 1983)
and for plant growth reduction in orchards (Lamberti and Baines, 1969; Abrantes and Santos,
1991; Sasanelli et al., 2002; Nico et al., 2002; Afshar et al., 2014).
The Meloidogyne species detected in Morocco were characterised using biochemical
(enzyme phenotypes Est) and molecular (SCAR and D2D3) approaches that are faster and
easier to use compared to morphological identification (Adam et al., 2007). These methods
demonstrated the occurrence of five species: M. javanica that was dominant and widespread
followed by M. arenaria, M. hapla and M. spartelensis, which were less frequent and
abundant. Meloidogyne n.sp. was also very rare. It seems to be a new species but it would be
complex to characterise because of the lack of biological material. It was impossible to
subculture this species on tomato (cv. Roma) or on olive (cv. Picholine du Languedoc),
probably because of host-plant incompatibility or varietal resistance. This species exhibited
ectoparasitic females without any egg mass. Similarly, no reproduction on tomato was
observed with M. beatica that was also detected on wild olive trees (Castillo et al., 2003). For
biochemical diagnostics, the esterase phenotype (Est) was investigated because of its
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usefulness (Dalmasso and Berg, 1978; 1983; Janati et al., 1982), its high occurrence (in 90%
of the RKN species) and its species-specificity within the Meloidogyne genus (Berg and
Dalmasso, 1976; Dalmasso and Berg, 1978; Esbenshade and Triantaphyllou, 1985; Carneiro
et al., 2004; Flores-Romero and Navas, 2005). Ten Est bands corresponding to eight different
phenotypes were detected. Some phenotypes such as J3 (M. javanica), A2 (M. arenaria) and
H1 (M. hapla) have already been reported on others crops (Janati et al., 1982; Esbenshade and
Triantaphyllou, 1985, Pais and Abrantes, 1989, Ibrahim and Perry, 1993; Carneiro et al.,
2000; 2008; Cofcewicz et al., 2005; Qunherv et al., 2011) as well as on olive (Castillo et
al., 2010). However, this study demonstrated some Est pattern variability detected within M.
javanica populations. The atypical phenotype J2a (M. javanica) with no intermediate band 2
(i.e., with a Rm of 54.5%) was reported by Yu and Chen (1997) and then confirmed as a M.
javanica variant by measuring morphological traits (Castro, 2001). This pattern was
subsequently reported by Cofcewicz et al. (2004) on banana and by Molinari et al. (2005), but
they designated it improperly as J3a. The other atypical phenotype J2b (M. javanica) with no
fast band 3 (i.e., a Rm of 58.9%) was previously defined in Meloidogyne populations from
Egypt detected on peanut and designated as J2 (Tomaszewski et al., 1994). It is assumed that
the presence/absence of these two faster Est bands 2 and 3 is due to the low esterase activity
of these bands, or to the female physiological conditions (Carneiro et al., 1996; 2000).
However, the slower main band 1 (i.e., a Rm of 46%), which is usually used to characterise
M. javanica, was present in all the corresponding phenotypes. Some Est infra-specific
variability was also detected within M. hapla with two distinct patterns mixed in the same soil
sample. The phenotype H1 was already reported on olive (Castillo et al., 2010). However, H2,
which was confirmed as M. hapla by molecular approaches, was never referenced in the
literature. Moreover, the J2a and J2b phenotypes of M. javanica and H2 of M. hapla had
never previously been reported on olive trees (Castillo et al., 2010).
When enzymatic patterns are difficult to read (e.g. new phenotypes) or need
confirmation, molecular techniques are required. SCAR markers have demonstrated their
specificity to M. arenaria and M. javanica (Zijlistra et al., 2000) and subsequently provided
confirmation for these two species, but SCAR markers developed for M. hapla detection
(Zijlstra, 2000) did not run with H1 and H2 individuals. That could be related to the
reproduction strategy of this species that can alternate between sexual and asexual
reproduction, leading to some recombination in genomes, or because H1 and H2 could
correspond to new races not previously detected. However, D2D3 (a variable region within
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the gene 28S that seems to be a good marker to distinguish Meloidogyne species (Landa et al.,
2008)) confirmed the identification of M. hapla.
This study showed that several Meloidogyne species are associated with olive trees in
Morocco. Some of them (M. arenaria, M. javanica and M. hapla) have been reported on olive
trees in Latin America, in several Mediterranean countries and in Asia (Ali et al., 2014).
However, M. beatica, M. incognita and M. lusitanica, which were also reported on olive trees,
were not recorded in Morocco. Conversely, new species such as M. spartelensis and
Meloidogyne n.sp. were detected. Several hypotheses could be proposed to explain the
diversity as well as the distribution of these Meloidogyne species. Sasser (1977) indicated that
the distribution and the economic importance of RKN species in agriculture are related to
favourable biological and environmental factors such as cultivation techniques, soil and
climate factors. M. javanica is the most frequent species in olive orchards worldwide (Ali et
al., 2014). Its widespread distribution in cultivated olive growing areas in Morocco
(traditional and high-density) agrees with previous observations that pointed its dominance in
irrigated orchards and nurseries (Hashim, 1979; 1983) where high levels of damage can be
expected especially in high-density conditions where M. javanica populations were ten time
more abundant than in traditional conditions. Nico et al. (2002) indicated that M. javanica and
M. incognita very frequently infect the major olive cultivars in Spain. Sanei and Okhovvat
(2011) observed that more than 20% of olive planting stocks sampled in Iran were infected
with M. javanica alone, and 10% were co-infected with M. javanica and M. incognita.
Regardless of their Est phenotype diversity, high temperatures appeared to be a favourable
factor for the multiplication of M. javanica. In fact, temperature is a key factor that affects the
fecundity of the nematodes and their ability to survive extreme heat and cold temperatures
(Evans and Perry, 2009). High temperatures are favourable for hatching, mobility and
invasion of M. javanica (Bird and Wallace, 1965). In contrast, M. hapla, usually found in
temperate regions (Bird and Wallace, 1965) or in altitude (Whitehead, 1969), was detected in
the coldest regions in northern Morocco also characterised by high annual rainfalls. The
effects of pH and temperature were confirmed by Taylor et al. (1982) who demonstrated that
soil alkalinity and heat favour the development of M. javanica. Moreover, M. javanica
occurred more often in coarse textured soils than the other species. RKN can occur in a wide
range of soil types but the damages they induce on crops are more pronounced in sandy soils
or in sandy patches within fields (Wesemael et al., 2011). In fact, sandy soils are generally
favourable for RKN development (Van Gundy, 1985; Wallace, 1989; Barker and Weeks,
Page 219
1991; Dabir and Mateille, 2004; Greco and Di Vito, 2009). RKN are frequently supressed in
clayey soils (Greco and Di Vito, 2009), which could be a limiting factor for nematode
movement. That could also explain the low abundance of M. arenaria, M. spartelensis and M.
hapla detected in clayey soils sampled in northern Morocco. Moreover, M. hapla and
Meloidogyne n.sp. could support organic soils in both northern and southern Morocco where
wild olive trees provide a high amount of organic matter under canopy.
Despite these potential interactions with environmental factors, it is assumed that
specific ecology traits were involved in the distribution of Meloidogyne species. As evidence,
M. javanica was detected throughout the center of the country in cultivated areas, generally
disturbed by agricultural practices and human activities, while M. arenaria, M. hapla, M.
spartelensis and Meloidogyne n.sp. occurred on wild olive trees growing in remaining plant
refuges either in the extreme north of the Rif Mountains or in the extreme west of the High
Atlas Mountains. First, the specific susceptibility (nematode reproduction rate) of wild and
cultivated forms of olive trees to RKN species can be assumed. No information is available
about the susceptibility of wild olive trees to plant-parasitic nematodes, but it is known that,
in cultivation conditions, olive varieties do not exhibit the same susceptibility to Meloidogyne
infection (Lamberti and Baines, 1969; Sasanelli et al.1997; Nico et al., 2003) and then lead to
specific nematode communities (Palomares-Rius et al., 2012; Palomares-Ruis et al., 2015).
Moreover, dispersion vs non-dispersion phenomena may be involved. Soil nematodes can
actively move over very short distances (e.g. no more than 75 cm in nine days in the case of
M. javanica juveniles (Prot and Nestcher, 1979)), but passive dispersion remains the main
means of spreading, usually via flooding, mudslides and human activities, in particular, as
was shown with the pinewood nematode (Robinet et al., 2009). In this case, the high VMR
ratios measured either at large (country) or at smaller (olive producing areas) scales indicated
that the distribution of the M. javanica populations is predominantly aggregated between and
within orchards. This aggregated distribution may be partly strengthened by specific local
habitat conditions (Begon et al., 1996). Thus, the extensive distribution of M. javanica in all
of the cultivated olive growing areas in Morocco may be the result of the widespread
introduction from nurseries (as source-sink processes), followed by multiplication due to
favourable local conditions such as irrigation, especially in high-density orchards cultivated
with fast-growing and RKN-susceptible cultivars. That was clearly illustrated in southern
Spain and northern Iran where high population densities of Meloidogyne spp. were at
potentially damaging levels in 28.6% and 22.3% of olive nurseries surveyed, respectively
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(Nico et al., 2002; Sanei and Okhovvat, 2011). In contrast, the long persistence of
Meloidogyne spp. in isolated regions can be considered, in particular during the Last Glacial
Maximum. Indeed, the places where the new species M. spartelensis (Ali et al., 2015) and M.
hapla H2 (new Est pattern) were detected belong to the Rif Mountain plant refuge area
(Mdail and Diadema, 2009) included in the Strait of Gibraltar biodiversity hotspot
(Rodriguez-Sanchez et al., 2008). M. spartelensis was detected on the west side of the Rif,
and M. hapla on the east side. In addition, the place in the western High Atlas Mountains
where Meloidogyne n.sp. was detected belongs to the Souss/W. Anti-Atlas plant refuge area
(Mdail and Diadema, 2009) for endemic plants specific to the Macaronesian area (Mdail
and Quzel, 1999), especially the wild olive O. e. ssp. maroccana (Mdail et al., 2001). In
these specific areas, wild olive consists in an ancestral wild gene pool with unique haplotypes
(Besnard et al., 2013) that support refuge conditions. The past co-adaptation of plant-parasitic
nematodes with wild olive forms and allopatric speciation (Inglis, 1971) can then be
hypothesized and could explain the diversity of the RKN species encountered in these natural
ecosystems. Moreover, species isolation in mountain habitats with no-to-low human activities
could explain the aggregation of these RKN species. Aggregation could be exacerbated by
specific biological traits leading to species competition, as was demonstrated between M.
javanica and M. arenaria (Olowe, 2004; Singh et al., 2012).
Because plant-parasitic nematodes are not long-distance moving pests, the detection of new
RKN taxa in wild olive ecosystems in Morocco suggested that these areas could host ancestral
nematode species that did not disperse in the absence of human activities. On the other hand,
olive anthropogenic forces intensify the introduction and the development of ubiquist RKN
taxa in agrosystems (Trudgill and Blok, 2001; Castagnone-Sereno et al., 2013), certainly of
non-olive origin (introduction through contaminated nursery substrates). Considering the
apomictic and the amphimictic modes of reproduction in Meloidogyne species (Chitwood and
Perry, 2009), these results encourage us to go further by investigating their phylogeography in
relation with the distribution of their wild and cultivated hosts (Castagnone-Sereno et al.,
2013) of olive RKN in Morocco in order to examine if and how they diverged from wild to
cultivated olive trees. From an agronomic point of view, colonisation processes from
nurseries and adaptation and dissemination in orchards would benefit from further study in
order to better manage technical itineraries for olive crop production.
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Legends for figures

Figure 1: Electrophoretic esterase patterns of Meloidogyne populations collected from olive
soils in Morocco. A: species-specific phenotypes detected; B: polyacrylamide gels showing
esterase phenotypes obtained from single females (F1 to F5) (R: M. javanica reference
population; A2: M. arenaria; H1 and H2: M. hapla; J2a, J2b and J3: M. javanica; S1: M.
spartelensis n.sp.; nsp: unknown pattern; Rm: relative migration; 1-10: number of specific
bands numerated according to M. javanica reference population).
Figure 2: Spatial distribution and abundance of the Meloidogyne species identified in
Morocco with indication of olive growing modalities on which the Meloidogyne populations
were detected.
Figure 3: Correspondence analysis between the abundance of the Meloidogyne species and the
environmental factors. A: PCA loading plot of the abundance of the Meloidogyne species
(labelled according to Est patterns, see Table 2); B: MCA loading plot of the environmental
variables (codes for environmental variables are listed in Table 3); C: COI score plot of the
eigenvalues for Meloidogyne species on the CO1 axis; D: COI score plot of the eigenvalues
for environmental variables on the CO1 axis.
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Table 1: Environmental variables analysed in the co-inertia analysis, and corresponding

codes.
Variables
Olive growing modality
wild olive
feral olive
Code
WO
FO
TR
HD
Water supply
irrigated
rainfed
I
R
Climatic variables
mean annual maximum temperature
mean annual minimum temperature
annual rainfall
Tmax
Tmin
AR
Variables
Soil factors
Soil type
sandy soil
silty soil
clayey soil
Code
organic matter
pH
OM
pH
SA
SI
CL
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Table 2. Location of the olive sampling sites surveyed in Morocco and infested by Meloidogyne spp. (Codes for isolates correspond to their
registration codes in laboratory).
Code
Isolate
312
313
340
373
410
304
305
308
309
255
258
259
260
261
262
285
383
293
296
300
301
252
253
402
403
404
Geographic
region
Rif
Province
Tanger-Asilah
Cap Spartel
Guerouane
Tadla
Ouezzane
Tanger-Asilah
Mekns
Beni Mellal
Zoumi
Dehar Kharroub Soufla
Sebaa Ayoune
Beni Mellal
Houaz
Marrakech
Ouled Mbarek
Fekhara
Tazakourte
Rehamna
Souss
Taroudant
Agadir Ida-Outanane
Site
El Afak
Douar Moulay Abbas
Douar Lahna
Ouled Jelal
Jaidate
Labraija
Khenafif
Taguergoust
Tiguert
Olive
growing category
wild
wild
feral
wild
high density
feral
feral
traditional
traditional
traditional
high density
high density
traditional
traditional
traditional
high density
high density
feral
feral
feral
feral
traditional
traditional
wild
wild
wild
Latitude N
(decimal)
35.7906
35.7909
34.8089
35.8460
33.8762
32.3157
32.3156
32.3149
32.2075
31.6293
31.6865
31.6872
31.6482
31.5078
31.5722
31.7023
31.7023
32.1498
32.1491
32.1478
32.1478
30.4211
30.6539
30.6269
30.6279
30.6279
Longitude W
(decimal)
5.9249
5.9255
5.3599
5.5292
5.4078
6.3925
6.3928
6.3931
6.8283
8.1007
8.1093
8.1104
8.1047
7.9394
7.9715
7.6875
7.6875
7.2589
7.2588
7.2602
7.2607
9.0222
8.1802
9.8493
9.8449
9.8445
Altitude
(m)
10
13
539
28
609
516
515
522
440
411
397
397
418
603
542
470
478
412
417
413
414
300
500
20
20
19
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Table 3: Biochemical and molecular diagnostics of the Meloidogyne species associated with
olive trees in Morocco and their abundance (number of juveniles/dm3 of soil). Patterns: Est =
esterase isozyme phenotype; SCAR = Sequence Characterised Amplified Regions; D2D3 =
rDNA 28S fragment; ns = not specific ((Codes for isolates correspond to their registration
codes in laboratory).
Isolate
312
313
340
373
410
304
305
308
309
255
258
259
260
261
262
285
383
293
296
300
301
252
253
402
403
404
Est
spartelensis S1
arenaria A2
no multiplication on tomato
hapla H1
hapla H2
javanica J3
javanica J3
javanica J2a
javanica J3
javanica J3
javanica J3
javanica J3
javanica J3
javanica J2a
javanica J3
javanica J3
javanica J3
javanica J3
javanica J3
javanica J3
javanica J3
javanica J3
javanica J2b
javanica J3
new pattern
Patterns
SCARs
ns
arenaria
Validated species
D2D3
spartelensis
-
COI
spartelensis
-
ns
ns
javanica
javanica
javanica
javanica
javanica
hapla
hapla
-
hapla
hapla
-
javanica
javanica
javanica
javanica
javanica
javanica
javanica
javanica
javanica
javanica
javanica
javanica
javanica
javanica
ns
no material
M. spartelensis
M. arenaria
Meloidogyne sp.
M. hapla
M. hapla
M. javanica
M. javanica
M. javanica
M. javanica
M. javanica
Meloidogyne sp.
M. javanica
M. javanica
M. javanica
M. javanica
M. javanica
M. javanica
M. javanica
M. javanica
Meloidogyne sp.
M. javanica
M. javanica
M. javanica
M. javanica
M. javanica
M. javanica
Meloidogyne sp.
Meloidogyne sp.
Meloidogyne n.sp.
Page 231
Figure 1
Page 232
Figure 2
Page 233
Figure 3
Page 234
Chapitre X
Variabilit gntique chez

Meloidogyne spp. associ
lolivier
Page 235
Rsum
Ce chapitre dcrit ltude de la diversit gntique chez des espces de Meloidogyne
identifies sur lolivier au Maroc et chez M. javanica en particulier (qui sest rvle lespce
dominante).
La diversit gntique de M. javanica identifi sur olivier au Maroc a t analyse par des
marqueurs morphologiques et molculaires. Concernant la diversit morphologique, ltude a
pris en compte des traits morphologiques frquemment utiliss pour lidentification
spcifiques des Meloidogyne, mesurs chez les juvniles de second stade. Ces traits taient la
taille du corps et celle du stylet ainsi que la largeur du corps mesure au niveau du bulbe
mdian et la largeur maximale estime 25% du dbut de la tte. Les mesures ont t
effectues chez des juvniles issus de lignes iso-femelles ou familles, en conditions contrle
de laboratoire afin dviter toute source de perturbation environnementale susceptible de
gnrer de la variabilit morphologique entre les individus mesurs et, par consquent, entre
les populations.
Les rsultats ont indiqu que ces diffrents traits sont significativement variables entre des
familles de mme population et entre les 18 populations tudies. Les longueurs du corps et
du stylet se sont rvles comme les traits les plus structurants dans la variabilit
morphologique. Ils ont permis de rpartir les populations en quatre groupes : deux
populations avec un long stylet, six populations avec un long corps, huit populations avec un
corps et un stylet de longueur moyenne, et deux populations avec un corps et un stylet courts.
Cette variabilit morphologique a t discute la lumire de plusieurs caractristiques
biologiques des Meloidogyne telles que le mode de reproduction, la variabilit des profils
enzymatiques, la diversit cytogntique (nombre de chromosomes), toutes susceptibles
dimpacter la variabilit des individus de mme espce. La contribution des facteurs
environnementaux (modalit de culture, paramtres climatiques et physicochimiques du sol)
ont galement un effet sur cette variabilit. Ltude met lhypothse dune origine gntique
de cette variabilit. Cette hypothse fera lobjet dune tude complmentaire (deuxime partie
de ce chapitre) afin de vrifier la congruence entre ces deux composantes de la variabilit,
congruence qui pourrait renseigner sur les mcanismes (e.g. effet relatif de la slection ou de
la drive) impliqus dans la diffrenciation gntique exprime entre les diffrentes
populations.
Page 236
Morphological intra-specific variation of Meloidogyne javanica juveniles

parasitizing olive trees in Morocco
Abstract
Phenotypic variation among species and within populations of the same species is generally
produced as a consequence of genetic variation among them or as an adaptive response to
environmental changes. Several populations of Meloidogyne javanica have been identified on
olive trees in Morocco. The aim of this study was to describe the phenotypic variation
exhibited in several morphological traits within and between these populations. In order to
investigate if the morphological variability is controlled by genetic variation among
populations: (i) the study was undertaken under uniform laboratory conditions in order to
avoid environmental perturbations, and (ii) experimental procedures were derived from the
isofemale line or family method where each line is founded by a single female and
constitutes a genetic unit. Body and stylet lengths and body width were measured on several
second stage juveniles from each line and from several families from each population. The
results revealed that these morphological traits were highly variable between populations and
between families within some populations. They strongly distinguished four groups of
populations: two populations with a long stylet, six populations with a long body, eight
populations with middle body and stylet lengths, and two populations with small body and
stylet lengths. More precise analyses indicated that the body lengths, and the stylet length at a
lesser extent, were the most discriminant characters between populations. Several factors
proposed to contribute to this morphological variability (e.g. the enzymatic variation among
populations, their cytogenetic variability and the mode of reproduction) are discussed.
However, a genetic variation is expected to generate morphological variation among
populations. The contribution of olive growing modality, soil and climatic conditions as
potential factors able to impact this morphological variability should be analyzed. Further
studies targeting molecular variability among these populations are expected in order to match
it to morphological variability, and then to provide information on the genetic mechanisms
involved in phenotypic evolution.
Key-words: intra-species, Meloidogyne, Morocco, morphological traits, olive, phenotypic

variation
Page 237
1. Introduction
During their life, living organisms exhibit several variations at inter- and intraspecific levels
(Roff, 1992). Their life traits are subjected to several evolutionary forces such as natural
selection that makes organisms to optimize their fitness (e.g. survival and reproduction)
facing to ecological changes inflicted by the environment (Roff, 1992; Stearns, 1992; 2000).
The evolution of life history traits by natural selection depends on genetic or phenotypic
variation on which selection can acts to produce adaptations to the environment (Fabian and
Flatt, 2012). Consequently, phenotypic variation within and among species may be due partly
to true genetic differences and partly to environmental influences (Van Der Beek et al. 1998).
Partitioning the phenotypic variance due to genetics or environmental variation and to their
interactions allow estimates of the heritability of the phenotypes, for a given population in a
particular environment (Falconer, 1989). However, understanding the role of environmental
and genetic interactions that generate phenotypic variations is thus a fundamental challenge in
biology (Braendle and Teotonio, 2015).
Considering evolutionary processes, the environment contributes firstly to establish the
relationship between the phenotype of an individual and its fitness, i.e. the fitness function. It
also plays a role in determining the phenotype and the strategies that maximize the match of a
phenotype to the prevailing environment. Better matching of phenotypes to environments can
be achieved by phenotypic plasticity (Scheiner, 1993; Viney and Diaz, 2012). This
phenotypic plasticity refers to the phenotypic changes induced by the environment that occurs
within an organisms lifetime (Stearns, 1989), and it is likely to play an important role in
diversification processes (West-Eberhard, 1989). This phenotypic plasticity is common
among nematodes. Remarkable examples are available among Caenorhabditis species such as
C. elegans characterized by the formation of the alternative dauer stage in response to
environmental variation (Cassada and Russell, 1975). However, not all plasticity is adaptive
and elucidating potentially adaptive benefits of phenotypically plastic traits requires an
understanding of how plasticity differs among genotypes and how it is expressed in diverse
environments (McGaughran and Sommer, 2014). Phenotypic variation could be also
controlled by genetic factors. That is generally true for phenotypic traits that are not plastic or
that may be plastic, but have been studied in a single, controlled environment, such that any
phenotypic plasticity is absent (Viney and Diaz, 2012).
As ubiquitous and ecologically-relevant species, nematodes represent a model
exhibiting a high level of variation in their biology, their morphology, their life strategies and
Page 238
their adaptive ability to environment changes. These variations have been associated with
some aspects of nematode behavior (Coomans, 1962; Bird, 1967) that allow them to
successful exploitation of diverse habitats (in marine, in fresh-water in terrestrial habitats, and
even in extreme environments such as polar ice) (Croll, 1970; Tahseen, 2012). All nematodes
are round worms and share a similar body plan, but they vary in length from 100 m to more
than 6 m (Blumenthal and Davis, 2004). Current accepted classification of nematodes is based
on morphological and ecological traits, primarily in the context of free-living or parasitic
phenotypes (Dorris and Blaxter, 1999). It has been suggested that all nematode parasites of
animals are evolved from arthropod parasitic ancestors. Similarly, plant-parasitic nematodes
(PPN) seem to be evolved independently in three times in Nematoda: Dorylaimida (clade I),
Triplonchida (clade II), and Tylenchida and Aphelenchida together (clade IV) (Blaxter et al.
1998). Based on this classification, these different groups of plant-parasitic nematodes display
a high variation in their morphology according to their different feeding habitats or trophisms
(as ectoparasites, semi-endoparasites and endoparasites and as obligate or facultative
parasites). They also display a morphological variability in their body size and their stylet
form and size. The morphological variation within parasitic nematode species presumably
reflects underlying genetic variation, although this should not be assumed without evidence
since it is clear that environment can play an important role in generating morphological
variation in nematodes (Hobbs et al. 1990).
Intraspecific morphological variability within PPN was considered in few taxonomic
reports and reviews (e.g. Coomans, 1962; 1963; 1971; De Grisse and Loof, 1970; Rodrigo
and Mai, 1976). Furthermore, molecular studies were carried out to investigate the intra- and
inter-population genetic variability or the genetic dispersal between populations of some
species such as Heterodera schachtii (Plantard and Porte, 2004), Globodera pallida (Picard
and Plantard, 2006), Bursaphelenchus xylophilus (Cheng et al. 2008; Mallez et al. 2013) and
Xiphinema index and X. pachtaicum (Villate et al. 2010; Gutirrez-Gutirrez et al. 2011). A
particular attention was given to root-knot nematodes (Meloidogyne spp.) because of their
economic importance, their widespread distribution, and their pathogenicity on a wide range
of higher plants (Moens et al. 2009). Meloidogyne species can reproduce via different sexual
(amphimixis) or asexual (meiotic and mitotic parthenogenesis) modes of reproduction
(Castagnone-Sereno et al. 2013). The transition from sexual to asexual reproduction has
happened many times during their evolution (Castagnone-Sereno, 2006). Various molecular
mechanisms are able to generate a high level of genetic variability that could, otherwise,
explain the extreme parasitic success of root-knot nematodes in nature. This high level of
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genetic diversity occurs at both species and population levels (Castagnone-Sereno et al.
2013). As an example, the development of biochemical and molecular markers led to confirm
that a high level of clonal diversity exists among populations within Meloidogyne species, in
particular within the M. javanica apomictic species (Castagnone-Sereno, 2006). This
nematode genetic diversity could be expressed by several biological traits such as their
morphology, their ability to parasite plants, their adaptation to different environmental
conditions, and their wide distribution.
The intraspecific variability of Meloidogyne was first detected during host-range
studies, and led to the concept of host race, that distinguishes populations according to their
ability to reproduce or not on selected differential hosts (Sasser, 1979). In that way, Cliff and
Hirschmann (1985) have demonstrated a morphological variability between and within seven
populations of M. arenaria. A great morphological and genetic variability was also
investigated within the Vietnamese populations of M. graminicola (Bellafiore et al. 2015).
Variation in virulence to resistant plant genotypes was demonstrated for M. incognita
populations against Mi resistance gene (Castagnone et al. 1994). Then selection in plant
parasites for virulence on resistant hosts and the resulting effects on parasite fitness may be
considered as a driving force in host-parasite coevolution (Castagnone et al. 2000; 2007). The
current observed diversity probably results from a combination (i) of genetic variation
accumulating during hybridization and persisting through subsequent polyploidization, (ii) of
several origins from a common sexual ancestor, (iii) of accumulation of independent
mutations in different clonal lineages (Castagnone-Sereno, 2006).
To investigate the genetic variability under uniform laboratory conditions in avoiding
environmental perturbations, experimental procedures derived from the isofemale line
methods developed by Parsons (1980) have been used (e.g. Castagnone et al. 1994). In this
method, each isofemale line (i.e. family or clone) is founded by a single female. The analysis
of the variance within and between isofemale lines had been demonstrated as useful to
compare different traits (Rockwell et al. 1983) and to estimate the relative proportions of
genetic and environmental variances in populations, from different habitats (Parsons, 1980).
This method is used in the present study in order to explore the contribution of genetic
variability to induce morphological variability among different populations of the nematode
Meloidogyne javanica.
M. javanica is one of the major PPN species on the basis of its worldwide distribution
and its wide host range (Lamberti, 1979). It reproduces exclusively by mitotic
parthenogenesis with no reduction in the chromosome number (Triantaphyllou, 1981; 1985).
Page 240
The life cycle of this nematode species takes about four weeks to complete. Eggs are
deposited by the female in a gelatinous matrix usually placed outside the root tissue. The eggs
hatch second-stage juveniles (J2s) in the soil that can invade roots. The J2s penetrate the
cortex and migrate to the vascular cylinder where they induce giant cells for their feeding.
After three further molts, the nematodes develop into pyriform females usually able to
produce 500 to 1000 eggs (Castagnone et al. 1994). Successive generations are thus
theoretically genetically alike and high genetic diversity is not expected (Castagnone-Sereno,
2006). In this way, the present study was undertaken to investigate the variability exhibited in
some morphometric traits among several populations of M. javanica identified on olive trees
in Morocco, in order to describe how this variability is structured between the different
populations, and to test the hypothesis that intra-specific morphology variation is a
consequence of a genetic variability.
2. Materials and methods

2.1. Site samplings and Meloidogyne javanica acquisition
Soil samples were collected from several olive growing areas in Morocco, as well as in low
(100 trees /ha) and high (2,200 trees /ha) density orchards, and in feral areas (cultivated olive
trees abandoned and becoming wild, or olive trees from seeds spread by birds). Plant-parasitic
nematodes were recovered from soil samples by using the Seinhorst (1962) elutriation
procedure. When root-knot nematodes (RKN) were detected in the soil samples, susceptible
tomato plants (Lycopersicum esculentum cv. Roma) were transplanted in native soils under
controlled greenhouse conditions (12 hours light at 23C, 12 hours dark at 20) to grow and
maintain the RKN populations. Approximately 60 days after transplantation (corresponding to
two RKN life cycles), the tomato roots were checked for the presence of Meloidogyne galls
and egg masses. Nematode females and egg masses were recovered from galls for
Meloidogyne identification by esterase phenotype (Esbenshade and Triantaphyllou, 1985) and
SCARS (Zijlstra et al. 2000) analyses. Then, when M. javanica species were identified, the
egg masses corresponding to females were individually placed in distilled water for hatching
in order to produce J2s and to clone mother populations onto tomato plants under the same
greenhouse conditions as described above. Heighteen mother populations were recovered:
two populations (P-252 and P-253) from the Souss region near Taroudant city; eleven
populations (P-258 to P-301 and P-383) from the Haouz region, near Marrakesh city (the
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population P-260 was distinguished into two sub-populations a and b according to the two
esterase patterns they displayed); four populations (P-304 to P-309) from the Tadla region,
near Beni Mellal city, and one population (P-410) from the Guerouane region, near Meknes
city (Fig. 1).
Figure 1: Location of the Meloidogyne javanica populations recovered on olive trees in

Morocco (colors correspond to geographic regions: orange= Souss, green=Haouz;
blue=Tadla; yellow= Guerouane).
All the analyses carried out on live material were performed in the nematode quarantine
laboratory (French Government Agreement n80622) of the Research Unit Centre de
Biologie pour la Gestion des Populations, in Montpellier (France).
2.2. Establishment of nematode subcultures

Tomato seeds (Lycopersicon esculentum Mill. cv Roma) were sowed in 50ml bottom drilled
plastic Corning tubes (35 mm diameter) filled with sand (33.22%), peat (66.44%) and
Page 242
sodium polyacrylamide crystals (0.33%, for water holding) according to Castagnone-Sereno

et al. (1993). Ten egg masses were randomly sampled from each M. javanica mother Pi
population (Fig. 2A) and individually placed in distilled water for hatching. One month after
sowing, 50 to 60 J2s of each progeny or clone (i.e. isofemale line or family) labelled F1 to
F10 were inoculated on the tomato plants (Fig. 2B). Subcultures were maintained in a
greenhouse (12 hours light at 23C, 12 hours dark at 20).
2.3. Preparation of dead material

Six weeks after inoculation, three egg masses labelled Eg1, Eg2 and Eg3 (= three replicates
per family) were isolated from each tomato plant. They were individually placed in distilled
water for hatching (Fig. 2C). The hatched juveniles were labelled as Pi-Fj-Egk (referred to the
population, to the family and to the replicate from each family). Several one-day old J2s were
randomly picked out, put a few hours in the fridge to get a straight habitus, and then preserved
in glycerol (De Grisse, 1969). Then five to seven J2s from each hatching were mounted in a
15 l glycerol drop between slide and slip cover, with the three hatchings Eg1 to Eg3 on the
same slide (Fig. 2D).
2.4. Morphometric study

Three J2s per hatching were used for morphological measurements. All the observations were
carried out under a microscope (Leica DMRD) with a camera (Leica DMF 20). Photos and
trait-marks were recorded using the Leica Application Suite (LAS) 2.5.0 R1 software. Four
morphological traits were measured on each juvenile (Fig. 2E & Fig. 3), as usually used for
morphological characterization of Meloidogyne species (Jepson, 1987; Siddiqi, 2000): (i) the
body length (L); (ii) the stylet length (S); (iii) the body width at the median bulb (Wh); the
maximum body width (Wm). The observations were made with x10 or x20 (for body length)
and x100 (for the others traits) magnification. Then, the ratios ab = L/ Wm and ac = L/ S were
calculated in order to test the dependence of the traits between individuals and/or between
populations.
2.5. Statistical analyses

Examination of the raw data indicated that the distribution of morphological traits was not
normal. Differences among and within populations were analyzed for the four morphological
Page 243
traits by multiple comparisons using the of Tukeys HSD (Honestly Significant Difference)
test. Bonferroni corrections were applied for all pair-wise multiple comparisons. A nonparametric Spearman's rank correlation test was performed between the four traits.
Differences obtained at levels of P < 0.05 were considered significant. Principal component
analyses (PCA) were performed to describe the distribution of the morphological variability
between populations. The PCA analyses were done using the ade-4 package software (Dray
and Dufour, 2007). Hierarchical cluster analyses (HCA) were used to display the hierarchical
relationship among the populations based on the distance matrix. All the statistical analyses
were performed using the R software (R Development Core Team, 2013).
Figure 2: Experimental procedures from subcultures of Meloidogyne javanica populations to

measurements of morphological traits.
Page 244
Figure 3: Morphological traits measured on Meloidogyne javanica second-stage juveniles

(from Jepson, 1987).
3. Results
A total of 1,532 J2s belonging to the 18 M. javanica populations were measured in order to
investigate the morphological variation within and between populations.
The body length L of all the J2s measured ranged from 336.3 to 554.5 m (average 413.4
m). J2s from populations P-253 and P-259 were the smallest in size (average 388.82 m and
392.73 m, respectively) while J2s from P-300 were the tallest (average 435.83 m) (Fig. 4A;
Tab. 1). The length of the stylet S in all the J2s ranged from 10 m to 14.5 m (average 12.9
Page 245
m). The shortest stylets were observed in the population P-253 (average 12.23 m), and the
longest in P-410 (average 13.43 m) (Fig. 4B; Tab. 1). The maximal body width Wm of all
the J2s ranged from 9.6 m to 16.3 m (average 13.2 m), mean at 13.2 m. The J2s of from
the population P-260a having were the less minimal body thinnest in size width (on average
12.70 m) compared to the J2s from population P-301 that having were the largest maximum
body width thickest (on average 13.77 m) (Fig. 4C; Tab. 1). The body width at median bulb
Wh of all the J2s ranged from 9.7 m to 14.1 m (average 11.7 m). J2s from the population
P-253 were the thinnest in size (average 11.45), while J2s from the population P-301 were the
thickest (average 12.05 m) (Fig. 4D; Tab. 1). Considering the body length / maximal body
width (ab) and the body / stylet length (ac) ratios, they ranged from 25 to 45.58 and from
26.56 to 43.38 respectively in all the J2s measured. The smallest ab ratio was observed in
populations P-259 and P-305 (average 29.91) and highest in P-260a (average 33.89). The
smallest ac ratio was observed in P-285 (average 31.11) and the highest in P-252 (average
33.60) (Tab. 1). The Tukeys test indicated a high significant variability of the morphological
traits between populations (P-value < 0.05) (Tab. 1). The small standard deviation values
suggested similarity of the traits within each population. However, some variability was
observed among families from the same population (indicated by * in Table 1).
According to Spearman's rank test, no correlation was found between each pair of these four
morphological traits (data not shown).
Page 246
Figure 4: Box plot of the body length (A), of the stylet length (B), of the maximum body
width (C) and of the body width at median bulb (D) among the Meloidogyne javanica
populations sampled on olive trees in Morocco.
Page 247
Table 1: Mean and standard deviation (SD) for each morphological trait in each population (different letters a to h indicate significant
differences between populations according to the Tukeys test; bold data refer to minimum and maximum values for each trait; (***), (**) and
(*) indicate very high, high and low significant trait differences between families in a same population respectively).
N
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Population
P-252
P-253
P-258
P-259
P-260a
P-260b
P-261
P-262
P-285
P-296
P-300
P-301
P-304
P-305
P-308
P-309
P-383
P-410
L= body length
(m)
mean
SD
416.7 *
20.7 de
16.7 a
388.82
426.20
19.5 ef
392.73
14.8 ab
429.39
20.1 f
406.12 *** 21.1 cd
409.01
16.1 cd
405.21
14.6 cd
410.54
23.0 cd
411.82 **
16.1 cd
435.83 *** 46.3 f
411.56
16.5 cd
430.55
12.5 f
400.42
18.5 bc
409.07 *
17.3 cd
426.75
25.6 ef
416.53
28.6 de
419.76*
23.4 df
S= stylet length
(m)
mean
SD
12.42
0.5 ab
0.6 a
12.23
12.89 *** 0.6 defg
13.06
0.5 efhi
13.24
0.4 hj
12.91
0.4 defg
12.53
0.6 ac
12.83
0.6 cf
13.19
0.5 ghj
12.77 *** 0.4 ce
13.39
0.3 j
13.18
0.3 efh
13.35
0.3 ij
12.55 *** 0.5 bc
12.65 *
0.7 bcd
13.03
0.4 efh
13.11
0.3 fhj
0.4 hj
13.43*
Wm = maximum
body width (m)
mean
SD
13.22
0.6 beg
13.01 * 0.8 abd
13.28
0.5 bef
13.15
0.5 be
0.7 a
12.70
13.09 ** 0.7 ae
13.26 ** 0.6 beg
13.06
0.7 ae
12.99
0.9 abc
13.66
0.5 f
13.39
0.4 def
0.6 fg
13.77
13.36 * 0.8 cdef
13.41 * 0.6 ef
13.17
0.7 be
12.89 ** 0.5 ab
13.11 * 0.9 be
13.17 * 0.9 aef
Wh = body width
at median bulb (m)
mean
SD
11.84
0.3 ce
0.4 a
11.45
11.76 * 0.2 bce
11.97
0.2 ce
11.59 * 0.5 bcd
11.73 * 0.4 bce
11.84
0.2 ce
11.65
0.3 ac
11.57
0.5 bcd
11.88 * 0.3 de
11.83
0.3 ce
0.4 e
12.05
11.76 * 0.2 bce
11.80
0.4 bce
11.59
0.5 bcd
11.49
0.2 ab
11.60 * 0.4 bcd
11.71
0.3 ace
ab ratio
mean
31.55***
29.97**
32.14
29.91
33.89
31.12***
30.88
31.11
31.71
30.18***
32.59***
29.92
32.34
29.91
31.14
33.13***
31.91*
32.09
SD
1.7
1.8
1.8
1.4
2.1
2.5
1.5
1.9
2.1
1.4
3.6
3.6
2.8
1.9
2.1
2.1
3.1
2.5
ac ratio
dfg
ab
fh
ab
i
cdf
bd
bef
dfg
abc
gh
a
egh
ab
cdf
hi
dfg
dfh
mean
33.60*
31.84
33.144**
30.094
32.45
31.49***
32.70*
31.62**
31.11**
32.29***
32.56***
31.23
32.26
31.94
32.43
32.75**
31.77
31.28*
SD
2.1
1.6
2.1
1.4
1.7
1.7
1.9
1.3
1.2
1.7
3.5
3.7
1.1
1.7
2.3
1.7
2.1
1.2
Nb of
individuals
g
bcd
fg
a
def
bce
dg
bcd
ac
def
dg
ab
def
cd
def
dg
bcd
acd
Page 248
90
103
90
90
90
89
90
66
90
90
90
80
90
87
90
90
90
27
In order to analyse similarities between populations and to describe how the morphological
variability is structured among populations, PCA analyses were performed on the body and
stylet lengths, and the body width at the median bulb (the maximum body width was excluded
because the two body widths were correlated). The PCA score plot showed two main groups
of populations according to the PC1 and PC2 axes (P-252, P-258, P-260a, P-300, P-304, P309, P-383 and P-410 on one hand, and P-260b, P-261 to P-296, P-305 and P-308 on the other
hand), and three populations apart (P-253, P-259, P-301) (Fig. 5A). The HCA analysis
displayed two main groups of populations, each group being divided into two sub-groups
(Fig. 5B). In order to investigate which traits are involved in the hierarchy, the morphological
trait averages were compared between the groups. The Tukeys (Tab. 2) test indicated that the
groups were more distinguished by the body length than by the stylet length, while no
significant difference was observed between populations according to the body width at the
median bulb (Tab. 2).
Figure 5: Similarities between the Meloidogyne javanica populations according to the

morphological measurements of juveniles (body length and width at median bulb, and stylet
length). PCA score plot (A) and HCA dendogram (B) of the measurements according to the
populations (Gi = hierarchical groups). Codes for populations are listed in Table 1.
Page 249
Table 2: Mean and standard deviation (SD) for each morphological trait in each hierarchical
group (HCA analysis) (different letters a, b, c indicate significant differences between groups
according to the Tukeys test, P < 0.05).
Hierarchical
groups
G1
G2
G3
G4
P-value
Body length
(m)
mean
SD
418.05 a
18.62
424.85 a
13.80
391.67 b
5.064
408.06 c
10.97
< 2.2e-16***
Stylet length
(m)
mean
SD
13.25 a
0.29
13.05 a
0.45
12.68 b
0.43
12.82 b
0.41
1.885e-06****
Body width
(m)
mean
SD
11.65
0.25
11.72
0.17
11.71
0.29
11.76
0.22
0.2888
Nb of
individuals
117
540
193
682
4. Discussion
This study was undertaken in order to investigate the variability of some morphological traits
observed in juveniles from 18 populations of Meloidogyne javanica parasitizing olive trees in
Morocco, and to examine if this variability is related to the genetic variation exhibited among
populations. The study was conducted on J2s derived from several isofemale lines from each
population under controlled laboratory conditions. Measurements of body length and width,
and of stylet length revealed that they are highly variable between populations and between
families within some populations. These traits were highly structured among populations and
allowed to define four population groups.
The comparisons of the trait means between these groups indicated that the body and
the stylet lengths at a lesser extent, were the most discriminant characters between
populations. The body length has been demonstrated variable among different populations of
M. javanica and M. incognita (Netscher, 1973), and both body and stylet lengths have already
shown intraspecific variations between populations of M. graminicola (Soriano et al. 1999;
Pokharel et al. 2010; Bellafiore et al. 2015). These results indicated no correlation between
the traits mesured, as it was previously demonstrated on M. graminicola (Soriano et al. 1999;
Pokharel et al. 2010), because the trait ratios (L/ Wm and L/ S) were not conserved among the
different populations.
The morphological traits used in this study are frequently used for morphological
characterization of Meloidogyne species (Jepson 1987; Siddiqi, 2000). However, this study
confirmed the high morphological variability between populations belonging to the same
species that makes them inadequate for Meloidogyne diagnostic (Bellafiore et al. 2015). That
is especially the case with populations P-260a and P-260b that were detected in mixture in the
same soil sample and exhibited two esterase patterns (three J3 and two J2a bands,
Page 250
respectively) (Ali et al. in prep, Chapter IX). These two populations exhibited a high
morphological variability among the four measured traits. Furthermore, Castillo et al. (2010)
gave different morphological traits among Meloidogyne species parasitizing olive trees. The
body and stylet lengths of M. javanica species noticed in this reference were noticed as
smaller than the same lengths measured on the Moroccan populations. That confirms that the
morphological diagnostic of M. javanica species must be extended (i) to nematode females
and males (Hirschmann, 1985; Jepson, 1987) and (ii) to biochemical and molecular traits.
Several hypotheses could be tackled to explain the morphological variability detected in
this study. Phenotypic variability has been described as a response to a given genotype
(Scheiner, 1993; Wharton, 2011) or as an adaptive response to unpredictable environments
(Donaldson-Matasci et al. 2010). However, experimental sources of environmental variability
were controlled in this study (rearing on the same host-plant species and cultivar, under the
same climatic conditions; measurements on juveniles of the same age). Moreover, the
isofemale line method should guarantee clonal progeny without any recombination event
(Castagnone-Sereno et al. 1994). That was why this method had been used to analyze the
phenotypic variation in natural populations of Drosophila melanogaster (Hoffmann and
Parsons, 1988) and the genetic variability in nematode virulence against the tomato Mi
resistance gene (Castagnone-Sereno et al. 1994). Considering that in selection experiments
conducted under controlled environments, the genetic variation, the host-plant specificity and
the inheritance of nematode virulence have been demonstrated (Castagnone-Sereno, 2002),
these arguments could conclude that the phenotypic variability is due to the occurrence of
genetic variability among populations.
However, this genetic variation could be influenced by biological features among
populations or by environmental characters related to their geographic origin. Grant (1994)
indicated that variability exists between strains within a species: isozyme and/or DNA
sequence were compared to define populations that may exhibit biological differences (e.g.
host range) but are still thought to be members of a single species. As an example, the
potential role of isozyme variability in morphological variability could be displayed with
populations P-260a and P-260b whose morphological traits and esterase phenotypes (J3 and
J2a patterns) were significantly different, although they initially derived from the same soil
sample (Ali et al. in prep, Chapter IX). In addition to enzymatic variability, Meloidogyne
species exhibit variability with respect to their chromosomal material, as a consequence of
structural rearrangements such as deletions, duplications, translocations, as well as polyploidy
and aneuploidy. Most populations have somatic chromosome numbers ranging from 30 to 50
Page 251
(Triantaphyllou, 1985). The chromosome number of M. javanica (2n=24-48) could vary

among the isolates (Triantaphyllou, 1962). Thus, an in-depth knowledge of cytogenetic is a
prerequisite for studying the genetic variation within Meloidogyne species (Van Der Beek et
al. 1998). The different modes of reproduction are also important factors influencing genetic
variability. Even M. javanica reproduces by obligatory mitotic parthenogenesis (apomictic)
that reduces genetic diversity, parthenogenesis does not prevent genetic variability in
Meloidogyne (Castagnone-Sereno, 2006). Genetic diversity patterns are mostly related to
crosses within one ancestral genetic pool, followed by the establishment of parthenogenesis
(Fargette et al. 2010). Parthenogenesis gives advantages in terms of colonization (males and
females do not need to copulate to give a progeny) and of protection of the advantageous
genotypes (Castagnone, 2002)
In this study, some morphological variation was detected between families within some
populations. Usually, individuals are rarely identical within populations, because natural
selection can act to optimize the fitness of the individuals (Morozov et al. 2013).
Uncontrolled conditions influencing female fecundity and egg hatching could induce different
phenotypes between juveniles produced by different females. This intra-population variability
could be also a source for morphological variability between populations.
The populations of M. javanica analyzed in this study were collected from several olive
growing areas in Morocco that exhibit a wide range of pedo-climatic conditions. These
various environmental conditions could lead to heritable differences since environmentallydriven selection can be responsible for differences among populations (McGaughran et al.
2014). Several examples on plants or fishes were demonstrated as inferring regional scales of
local adaptation, driven by distinct environmental conditions among populations (Temunovic
et al. 2012; Dionne et al. 2008). However, the geographic distance between populations did
not seem to be involved in the morphological variability analyzed between populations. For
example, the populations P-300 and P-301 were significantly different in size whereas they
were detected close to each other in the same region (Houaz region). Considering olive
growing modalities, five M. javanica populations were detected on feral olive, and the others
were detected in low and high density orchards (eight and five populations respectively).
These different cultivation conditions (low vs high input practices), or anthropogenic changes
(cropping intensification, irrigation, tillage, etc.) could impact the genetic and specific
diversity. These anthropogenic stresses could hinder dispersal (Kremer et al. 2012), alter
patterns of selection on complex traits (Cook et al. 2012), and influence genetic variation
(Jump and Peuelas, 2006; Willi et al. 2007; Hughes et al. 2008).
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The morphological variation in and within M. javanica populations detected in this

study highlights that taking into account the genetic variation of these nematodes is important
in order to develop control strategies such as new plant resistance genes or natural enemies,
and to ensure their durability. The genetic diversity within species could also be significant to
understand the coexistence of species within communities (Booth and Grime, 2003; Vellend,
2006). It has long been admitted that the organization and the structure of ecological
communities are partly determined by the genetic diversity of the populations involved
(Antonovics, 2003). As demonstrated the reduction in species diversity contribute to a decline
in ecosystem properties such as productivity and resistance to disturbance, the genetic
diversity may play an analogous role to species diversity. That could enhance community
resistance to natural and disturbances (Hughes and Stachowicz, 2004; Reusch et al. 2005).
The morphological variation described in this study, probably related to genetic variability
between populations, is thought to reflect important evolutionary processes proposing
evaluative bases for ecological adaptations of all or some of these traits to answer to the
constraints imposed by the interactions with the others populations or species in the
communities or by environmental changes. Then, the contribution of environmental factors
(e.g. olive growing modalities and edaphic and climatic constraints) in driving this variability
should be analyzed. We can predict that the genetic diversity will be most relevant in highly
variable environments or those subject to rapid anthropogenic change. As almost of the
populations of M. javanica studied were detected in cultivated areas, suspecting the role of the
nematode introduction from nurseries, further studies on phenotypic diversity of M. javanica
populations in nurseries should be initiated in order to compare them with populations
established in orchards.
These results clearly invite to go further with analyzing the molecular variability and
the genetic structure among populations in order to match them and to to understand the
genetic mechanisms that could explain the phenotypic evolution.
5. References
Ali, N., Tavoillot, J., Chapuis, E., Mateille, T., (in prep.). Trend to explain the distribution of root-knot
nematodes Meloidogyne spp. associated with olive trees in Morocco.
Bellafiore, S., Jougla, C., Chapuis, ., Besnard, G., Suong, M., Vu, P.N., et al. 2015. Intraspecific
variability of the facultative meiotic parthenogenetic root-knot nematode (Meloidogyne
graminicola) from rice fields in Vietnam. Comptes Rendus Biologies, 338: 471-483
Blumenthal, T., Davis, R.E., 2004. Exploring nematode diversity. Nature genetics, 36: 1246-1247.
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Braendle, C., Teotonio, H., 2015. Workshop report: Caenorhabditis nematodes as model organisms to
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B. Etude de la diversit molculaire des populations de Meloidogyne spp.

associes lolivier au Maroc
1. Introduction
La variabilit biologique se dfinit par des caractres volutifs (phylogntiques) ou
cologiques (fonctionnels) (Colwell, 2009), diffrents niveaux : au sein des espces (au
niveau des gnes et des populations), entre les espces (variation intra et inter spcifique), au
sein des cosystmes (Gaston & Spicer, 2013). Dans la nature, les individus de la mme
espce sont rpartis en plusieurs populations. Chaque population a pu voluer et acqurir une
individualit gntique, caractrise par une frquence alllique plus ou moins diffrente de
celle observe chez une population voisine / sur, ce qui caractrise une variabilit au sein de
lespce. Cependant, cette variation intraspcifique nest pas seulement contrle par une
variabilit gntique mais elle peut tre en partie due des influences environnementales
(Van Der Beek et al. 1998). Etudier la diversit gntique au sein dune population ou dune
espce est une tape indispensable pour comprendre ses processus volutifs ainsi que son
adaptation aux forages biotiques et abiotiques imposs par son environnement. En ce qui
concerne les bio-agresseurs des plantes, dterminer leurs biotypes (ou souches) ainsi que leur
variabilit gntique a notamment des implications importantes dans la comprhension des
processus impliqus dans leur adaptation au systme de dfense prsent chez lhte, et dans le
dveloppement de nouvelles races rsistantes aux pesticides (Stukenbrock & McDonald,
2008) ou la capacit du parasite contourner la rsistance de son hte (Castagnone-Sereno &
Djian-Caporalino, 2011). Connatre la structure gntique dun parasite peut donc aider
prvoir l'avenir volutif potentiel de ses populations (McDonald & Linde, 2002), et
dvelopper les bases dune gestion en utilisant des cultivars rsistants (Plantard & Porte,
2004). En outre, ltude de la structure gntique des populations permettent de rsoudre des
questions sur l'origine et les processus de dispersion (Mulet et al. 2011).
Les nmatodes phytoparasite (NPP), sont des parasites obligatoires des plantes. Ils se
nourrissent exclusivement sur le cytoplasme des cellules vgtales vivantes (Williamson &
Gleason, 2003). Ils parasitent une large gamme de plantes cultives, causant des pertes de
rcoltes annuelles estimes en plusieurs milliards de dollars l'chelle mondiale (Barker &
Koenning, 1998; Williamson & Gleason, 2003; Nicol et al. 2011). Peu d'tudes ont t
menes sur leur variabilit gntique intra et inter-populations ou sur la dispersion gntique
entre populations (Plantard & Porte, 2004). Cependant certains groupes de NPP ont fait
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lobjet dtudes de gntique des populations tels que Heterodera schachtii (Plantard & Porte,
2004), Globodera pallida (Picard & Plantard, 2006), Bursaphelenchus xylophilus (Zhou et al.
2007; Mallez et al. 2013), Xiphinema index et X. pachtaicum (Villate et al. 2010; GutirrezGutirrez et al. 2011).
La diversit gntique du nmatode galles Meloidogyne spp. a galement fait l'objet
de plusieurs tudes (Castagnone-Sereno et al. 1994; Zijlstra et al. 1995; Van Der Beek et al.
1998; De Luca et al. 2002; Adam et al. 2005; Fargette et al. 2005; 2010; Mulet et al. 2011;
Onkendi & Moleleki, 2013; Humphreys-Pereira & Elling, 2013). L'attention particulire
apporte ce groupe de nmatodes est due leur importance conomique ainsi qu leur
grande diversit. Ces nmatodes sont trs rpandus l'chelle mondiale et ont dtects sur
plus de 5500 espces de plantes (Blok et al. 2008) causant des pertes de rcoltes importantes.
Ils peuvent srieusement affecter la fois la quantit et la qualit de plusieurs cultures
(Wesemael et al. 2011). Le genre Meloidogyne englobe aujourdhui plus de 95 espces
nominales (Karssen, 2002; Moens et al. 2009). Ces espces sont significativement diffrentes
pour leur gamme dhtes, leur comportement parasitaire ainsi que pour leur distribution
gographique (Adam et al. 2005). Parmi ces diffrentes espces, Meloidogyne arenaria, M.
incognita, M. javanica, M. hapla, M. enterolobii, M. chitwoodi sont les plus rpandues et
responsables de la grande majorit des dgts occasionns aux cultures (Agrios, 2005).
Le choix des nmatodes galles comme modle biologique pour de nombreuses
tudes se justifie par le fait qu'ils peuvent tre facilement levs en conditions contrles et
facilement manipules des fins exprimentales, bien quils soient microscopiques. Ces
nmatodes reprsentent, en outre, un excellent modle deucaryote pour l'tude de l'volution
et de l'adaptation au parasitisme aux plantes (Castagnone-Sereno et al. 2013). Ils rvlent
diffrents niveaux de variation en termes :
-
de modes de reproduction (amphimixie, parthnogense miotique and mitotique

facultative et obligatoire).
de nombres de chromosomes qui varient de 30 50 chromosomes. Ce nombre varie

mme entre populations de la mme espce. Les espces de Meloidogyne sont
diplodes ou triplodes, quelques formes ttraplodes ont t rarement observes
(Triantaphyllou, 1966; 1984). Ces diffrents modes de reproduction et degrs de la
polyplodie/aneuplodie peuvent se traduire par une diversification cytogntique
(Triantaphyllou, 1985). Par exemple, les espces clonales telles que M. arenaria, M.
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incognita et M. javanica refltent une variation physiologique et cytologique au sein

des espces (Castagnone-Sereno, 2006).
-
molculaire comme par exemple la diversit des protines (e.g. estrases) qui pourrait
tre le rsultat d'une volution complexe du genre impliquant d'autres vnements
majeurs, tels que la polyplodisation et lhybridation interspcifique (CastagnoeSereno et al. 2013).
La diversit des Meloidogyne se traduit par une variabilit morphologique (Eisenback
et al. 1984), biochimique (e.g. profils isoenzymes, Esbenshade & Triantaphyllou, 1985 ;
1987), et molculaire. Lanalyse de la variabilit molculaire est principalement base sur les
techniques de PCR est particulirement utile pour l'tude de la structure gntique des
populations (Williams et al. 1990) et de la variabilit interet intra-espces. Plusieurs
marqueurs ont dj t mis au point tels que AFLP ou RAPD qui ont t utiliss pour
examiner la diversit gntique inter et intra-spcifiques (Fargette et al. 2005 ; Adam et al.
2005). Ces deux marqueurs ont rvl des niveaux inattendus de diversit des populations
clonales (Castagnone-Sereno, 2006). Les microsatellites ont dmontr un polymorphisme
intra-populations au sein de M. artiellia (De Luca et al. 2002) et de M. incognita (CastagnoneSereno et al. 2010 ; Mulet et al. 2011). Plusieurs gnes et rgions gntiques ont t cibls
afin dtudier cette diversit gntique chez les Meloidogyne. LADN ribosomique (rADN) a
t largement utilis parce que son occurrence en multi-copies offre une base suffisamment
stable et variable permettant de discriminer les espces et dtudier leur diversit
intraspcifique (Blok & Powers, 2009). La rgion (ITS2 Internal Transcribed Spacer 2) est un
bon outil dtude de la diversit gntique au sein de M. hapla et de M. chitwoodi (Zijlstra et
al. 1995), ainsi quau sein de M. arenaria, M. incognita and M. javanica (Hugall et al. 1999).
L'ADNmt mitochondrial se rvle galement appropri pour valuer les profils
phylogntiques intraspcifiques chez les nmatodes phytoparasites (Hugall et al. 1994).
Parmi les gnes mitochondriaux, le CoxI (cytochrome oxidase subunit I) a montr son utilit
pour ltude de la phylognie des NPP et de la diversit gntique au sein de diffrentes
espces de nmatodes comme par exemple chez Xiphinema americanum (Lazarova et al.
2006). La rgion non codante 63R (63 rptitions) rpte en tandem sest galement rvle
variable au sein de M. incognita, de M. arenaria et de M. javanica, (Okimoto et al. 1991), de
M. enterolobii (Blok et al. 2002). Ces trois marqueurs (ITS2, CoxI, 63R) ont t utiliss dans
le cadre de cette tude afin dtudier la diversit gntique des diffrentes populations de
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Meloidogyne identifis sur olivier au Maroc et de dterminer comment cette diversit est
structure entre les diffrentes populations.
2. Matriels et Mthodes
Comme dj prsent (chapitre IX), plusieurs espces de Meloidogyne (M. javanica, M.
arenaria, M. hapla, M. spartelensis et Meloidogyne n. sp. (cense tre une nouvelle espce
dcrire) ont t identifies en association avec lolivier au Maroc. Leurs populations ont t
leves sur tomate (comment dj dcrit dans le chapitre VII). Afin de prparer les matriels
biologiques ncessaires ltude, quinze masses dufs par population ont t prleves et
mises individuellement clore. Les juvniles clos ont t conditionns en tubes dans une
goutte de 6L deau osmose et conservs -20C. LADN de ces juvniles a ensuite t
extrait par la technique du Kit QIAgen DNeasy Blood & Tissue. Sur les ADNs extraits, les
diffrents marqueurs (ITS2, coxI et 63R) ont t tests afin dtudier la diversit gntique de
diffrentes populations de Meloidogyne. Les portions de ces diffrents gnes ont t
amplifies avec les amorces et selon les conditions indiques dans les protocoles joints en
annexes 10, 11 et 12.
Les amplicons, amplifis la taille attendue pour chacun des gnes utiliss, ont t
vrifis sur un gel dagarose 1.5% de concentration, puis envoys squencer (Socit
EUROFINS Genomics, Allemagne) avec les amorces utilises pour lamplification. Le
squenage a t effectu en utilisant la technologie de squenage de cycle (cycle sequencing
technology ou dideoxy chain termination / cycle sequencing), modifie de la technique
traditionnelle de squenage Sanger. Le dosage de la concentration des chantillons et leur
purification ont t ralises par le prestataire. Les squences obtenues ont t rcupres sous
forme de chromatogrammes, puis corriges, alignes et analyses avec les logiciels Geneious
R7 v7.1.2 et BioEdit v7.2.5 (Biological Sequences Editor) (Hall, 1999). Pour chaque analyse,
nous avons introduit des populations de rfrence fournies par lANSES de Rennes (M.
arenaria, M. incogita, M. javanica et M. hapla). Ensuite pour chaque gne, nous avons
ralis lalignement des squences corriges-contiges par la mthode ClustalW method
(Thompson et al. 1994) avec le logiciel Bioedit.
Afin danalyser les relations phylogntiques entre les populations de Meloidogyne,
nous avons utilis la mthode du maximum de vraissemblance (Maximum Likelihood- ML)
laide du logiciel RAxML v8.0 (Randomized Axelerated Maximum Likelihood) (Stamatakis,
2014). Un modle GTR + a t appliqu (quatre catgories de taux discrets ont t utiliss
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pour tablir approximativement la rpartition des taux dhtrognit (Yang, 1994).

Chaque phylogramme a t bootstrapp 1000 fois (1000 rptitions) afin dvaluer la
robustesse de chaque nud ou embranchement indique par l'arbre optimal (Stamatakis,
2006). Selon les gnes tudis, des squences Genbank de M. incognita, M. hapla, M.
hispanica, M. enterlobii, M. mali et de Pratylenchus oleae ont t galement introduites pour
construire des outgroups . Ces analyses phylogntiques ont t effectues avec le logiciel
CIPRES Science Gateway v3.3. (www.phylo.org/portal2/home.action; Miller et al. 2010). Les
arbres phylogntiques pour ITS2 et coxI ont t gnrs laide du logiciel FigTree v1.4.0
(disponibles sur le lien suivant : http://tree.bio.ed.ac.uk/software/figtree; Rambaut, 2012).
Les squences gnres aprs amplification de la portion 63R ont t galement corriges,
alignes comme dcrit prcdemment. Ces squences ont t analyses afin de reprer les
haplotypes de ce gne (selon le nombre de copies de ce gne dans la squence, la taille de la
squence, les bases nuclotidiques qui diffrent entre les haplotypes). Des indices de diversit
ont t calculs: la diversit totale HT dfinie comme HT = 1 - pi2 avec pi = frquence de
l'haplotype i, la richesse A des haplotypes afin de dcrire la rpartition des haplotypes dans
chacune des populations tudies.
3. Rsultats
3.1. Analyse des squences dITS2
La population de Meloidogyne n. sp. a t retire des analyses, parce quune seule squence
tait disponible mais dune qualit mauvaise. Lamplification du gne ITS2 a produit un seul
fragment de 292 298 paires de bases (pb) environ aprs avoir supprim les amorces utilises
pour lamplification. Au total, 307 squences ont t gnres partir des extraits dADN
amplifis des diffrentes populations de Meloidogyne. Les analyses de la variation au sein des
squences de lITS2 (Figure 1) ont dmontr que toutes les populations de M. javanica (252J
410J) et celle de M. arenaria (313A) partagent une homologie totale 100% entre elles, et
ont form un seul clade avec les populations de rfrence de M. arenaria (Mare) et M.
javanica (Mjav). Ces deux espces ont form un clade basal et indpendant de M. spartelensis
et de M. hapla avec un taux lev de boostraps ( 100%). La population M. spartelensis
(312S) est plus homologue celle de Pratylenchus oleae. La population de M. hapla (373H) a
form un seul clade avec la population homologue tmoin (MHap) trs distinct des autres
clades (boostraps 100%).
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3.2. Analyse des squences de coxI

Au total, la portion coxI a t amplifie dans 458 extraits dADN. Lamplification de cette
portion a produit un seul fragment de 470 pb environ. Parmi les individus amplifis et
squencs, 335 squences ont t exploites dans les analyses phylogntiques. Les analyses
phylogntiques (Figure 2) ont rvl cinq clades diffrents (boostraps > 85%) : un compos
des populations de M. javanica, de M. arenaria, de M. incognita et de M. hispanica (Cox1-I),
un deuxime compos de M. enterolobbi (Cox1-II), un troisime de M. mali (Cox1-III), un
quatrime de M. spartelensis (Cox1-IV), et un dernier avec M. hapla (Cox1-V). De faon
similaire ce qui a t observ pour lITS2, les squences gnres partir des populations
marocaines de M. javanica et de M. arenaria, et des populations de rfrence de ces deux
espces ont form un clade dhomologie totale de 100%. Contrairement aux rsultats
obtenus par lITS2, les squences gnres partir de M. hapla ont montr une forte
variabilit. Cette espce est spare en trois clades diffrents (boostraps > 85%) : la
population marocaine (373MH) se rpartit dans deux des trois clades, lun compos des
squences 373MH12, 13, 51, 52, 56 et de deux squences GenBank (MHap1 et MHap3),
lautre compos des autres squences de la population (373MH), des squences de la
population M. hapla tmoin (464MHTa, b) et de celle de la population de Meloidogyne n. sp.
(404M). Le troisime clade est compos des squences de la population tmoin (704MHT) et
des autres squences GenBank (MHap2 et MHap4).
Les bases nuclotidiques diffrentes entres les cinq clades et entre les diffrentes espces et
populations sont prsentes sur la figure 3.
3.3. Analyse des squences de 63R

Les squences de 63R ont t gnres partir de 165 individus appartenant une population
de M. arenaria (11 individus) et de 17 populations de M. javanica (154 individus). Les
haplotypes ont t reprs selon le nombre de copies de 63R dans les squences, selon la taille
des squences et selon les polymorphismes diffrencis entre les individus. Lanalyse des
squences a rvl un seul haplotype au sein de M. arenaria et 19 haplotypes regroups en
cinq lignes au sein des populations de M. javanica.
La distribution des lignes et des haplotypes sur la carte du Maroc et au sein de chaque
population est illustre dans les figures 4A et 4B. La population M. arenaria (P-313), dtecte
lextrme Nord du Maroc sur un olivier sauvage ou olastre, nest caractrise que par une
seule ligne et un seul haplotype. Elle se distingue des lignes de M. javanica dtectes
Page 262
partout ailleurs sur des oliviers fraux et cultivs. Considrant les cinq lignes (Figure 4A)
dans M. javanica, Mj-I et Mj-II taient chacune prsente par sept haplotypes; la ligne Mj-III
est reprsente par trois haplotypes; alors que les lignes Mj-IV et Mj-V ne sont reprsentes
que par un seul haplotype. Les lignes Mj-I et Mj-II sont presque partout prsentes (dtectes
dans toutes les populations sauf dans P-285), soit de manire unique (e.g. populations P-252,
P-259, P-261, P-262, P-304, P-309, P-383) soit en mlange (e.g. P-296, P-301). Les trois
autres lignes ont t dtectes en un seul exemplaire soit de manire unique (e.g. P-285), soit
en mlange avec les lignes Mj-I et Mj-II (e.g. P-253 et P-305).
Certaines populations (P-253, P-301 et P-305) sont composes jusqu cinq haplotypes
diffrents, tandis que chez d'autres (P-259, P-261, P-304 et P-309), un seul haplotype a t
dtect (Figure 4B).
Figure 1 : Arbre phylogntique du gne ITS2 ralis avec lensemble des populations
despces de Meloidogyne identifies sur olivier au Maroc, des populations despces de
rfrence et dun outgroup Genbank (Pratylenchus oleae).
Page 263
Figure 2 : Arbre phylogntique du gne coxI ralis avec lensemble des populations
despces de Meloidogyne dtectes sur lolivier au Maroc, des populations despces de
rfrence et doutgroups GenBank.
Page 264
Figure 3 : Matrice des squences du gne coxI gnres partir des populations despces de Meloidogyne dtectes sur olivier au Maroc, des
populations despces de rfrence et doutgroups GenBank.
Page 265
Figure 4 : Distribution des lignes (A) et des haplotypes (B) 63R des populations de M.
arenaria et de M. javanica dtectes sur olivier au Maroc.
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4. Discussion
Le but principal de cette tude tait danalyser la variabilit gntique molculaire au sein et
entre les diffrentes populations despces de Meloidogyne identifies sur olivier au Maroc.
Afin de rpondre cet objectif, notre premire intention tait danalyser cette diversit par des
marqueurs microsatellites qui sont gnralement considrs parmi les outils les plus
appropris pour tudier la variation au niveau individuel (Selkoe & Toonen, 2006). Comment
dj mentionn dans le chapitre VII, des marqueurs de type microsatellites dj dtermins
chez dautres espces de Meloidogyne (e.g. Meloidogyne artiellia, De Luca et al. 2002 ; M.
incognita, Mulet et al. 2011) ont t utiliss. Cependant les tests prliminaires raliss sur des
populations tmoin (M. arenaria, M. incognita et M. javanica) disponibles en levage au
laboratoire ont montr la ncessit dun dveloppement de marqueurs avant de les tester sur
les populations marocaines, dautant plus que certains tests ont montr que les produits
amplifis aprs clonage et squenage ne correspondaient pas des squences microsatellites.
Cest pourquoi, nous avons dcid dabandonner cette approche et de nous orienter vers des
gnes tels quITS2 (ADN nuclaire) et coxI (ADN mitochondrial).
Le choix de lITS2 sest justifi parce que ce gne comporte des squences moins
conserves qui permettraient la discrimination despces troitement lies et de sous-espces
(Power et al. 1997 ; Clapp et al. 2000). En outre, la rgion ITS a rvl une grande variabilit
gntique au sein de plusieurs genres de NPP dont Meloidogyne (Zijlstra et al. 1995 ; 1997;
Hugall et al. 1999). Le coxI a galement t largement utilis pour des tudes gntiques en
raison de la teneur relativement constante des gnes, lhritage maternel, le taux de
recombinaison rduite, et le taux d'volution lev (Anderson et al. 1998; Hoolahan et al.
2011). La variation mitochondriale au sein des Meloidogyne a t dmontre par plusieurs
tudes (e.g. Powers & Sandall, 1988; Harris et al. 1990; Okimoto et al. 1991; Powers &
Harris, 1993; Blok et al. 2002).
Nos analyses des squences ITS2 ont montr lutilit de ce marqueur pour distinguer le
groupe de M. javanica / M. arenaria des groupes de M. hapla ou de M. spartelensis. Par
contre, ce marqueur na pas permis de discriminer M. arenaria de M. javanica, indiquant une
proximit taxonomique de ces deux espces. Les squences gnres partir de M. hapla ne
prsentent aucune variation entre elles comme cela a t dj signal par dautres tudes
(Zijlstra et al. 1995 ; Hugall et al. 1999). Labsence de variabilit intraspcifique de lITS2 au
sein de M. arenaria, de M. hapla et de M. javanica saccordent avec les observations de Xue
et al. (1993) qui ont montr une homognit des squences ITS1 et ITS2 aux niveaux interet intra- espces de Meloidogyne (Blok et al. 1997).
Page 267
Dune faon similaire, le gne coxI ne permet pas non plus de sparer les populations de
M. javanica et de M. arenaria. Ces rsultats confirment ceux de Kiewnick et al. (2014) qui
ont dmontr lutilit de ce marqueur pour distinguer des espces de Meloidogyne et tudier
leur variabilit gntique, sauf dans le cas de M. javanica et de M. arenaria. Cela
sexpliquerait par un hritage maternel commun de ces espces partir dune seule espce
ancestrale (Hugall et al. 1999). Ce marqueur spare galement le groupe M. javanica / M.
arenaria de M. hapla et de M. spartelensis. Cependant contrairement ce qui a t observ
pour lITS2, une variabilit molculaire a t dtecte entre les individus de M. hapla,
puisque les individus squencs partir des populations marocaines ont t spars en deux
groupes. Ces observations ont t confirmes par lajout dautres squences.
Ces analyses sur lITS2 et sur le coxI confirment lutilit de ce marqueur comme un
outil de diagnostic des espces, nanmoins partielle (pas de distinction de M. javanica et de
M. arenaria) mais pas comme outil danalyse de la variabilit intraspcifique. Do la
ncessit de dvelopper un autre marqueur permettant la fois le diagnostic des espces et
lanalyse de leur variabilit gntique. Le marqueur 63R, rgion non codante rpte dans le
gnome mitochondrial, a t dvelopp dans le but de rpondre cet enjeu. Lanalyse des
squences ont premirement permis la distinction de M. javanica de M. arenaria (elles se
distinguent par 4 bases nuclotidiques ainsi que par le nombre de rptitions de 63R dans la
squence) et ont rvl une grande variabilit intraspcifique de M. javanica (19 haplotypes
identifis). En outre, de la variabilit gntique a t dtecte au sein mme de certaines
populations. La validation de ces rsultats est en cours : un complment de squences semble
ncessaire afin de confirmer certains rsultats comme les lignes Mj-IV et Mj-V dtectes
dans trs peu dindividus. Dans tous les cas, les rsultats ne sont pas facilement interprtables
surtout que de lhtroplasmie a t frquemment rencontre sur plusieurs rptitions de 63R.
Dans le mme sens, diffrentes copies de 63R ont t dtectes pour un seul individu
(htroplasmie) ou dans plusieurs populations de la mme espce pour M. javanica, M.
arenaria et M. incognita (Okimoto et al. 1991 ; Blok et al. 2002). Ces lments pourraient
remettre en cause la variabilit dtecte et donc limiter lexploration de ce marqueur (Lunt et
al. 1998 ; Blok et al. 2002 ; Garca & Snchez-Puerta, 2015).
Afin de donner suite ce travail, nous envisageons :
-
de poursuivre les analyses molculaires au sein de M. hapla : La diversit gntique

observe au sein des squences de cox1 dans la population de M. hapla nous a conduit
tester la fiabilit de ce marqueur sur dautres populations de M. hapla dorigines
gographiques diffrentes : trois populations collectes sur olivier et sur cultures
Page 268
marachres en France; deux populations originaires du Canada et des Pays-Bas en

levage en France (source : Dr. P. Castagnone-Sereno, INRA Sophia Antipolis); une
population dorigine hollandaise en levage en Belgique (source : Dr. N. Viaene,
IAFR Merelbeke). Les premiers rsultats sont encourageants et dmontrent une forte
diversit gntique au sein de ces populations.
-
de poursuivre les analyses sur le 63R, de valider les lignes et les haplotypes obtenus,
dtudier la distribution gographique des lignes (phylogographie), dtablir un
rseau dhaplotypes, de calculer lindice de la diffrentiation gntique Fst entre les
diffrentes populations. Cette variabilit molculaire pourrait ensuite nous fournir des
bases dinterprtation de la variabilit morphologique analyse dans le chapitre
prcdent afin de comprendre si la variabilit existante au sein des populations de
Meloidogyne dtectes sur olivier au Maroc est due des processus volutifs ou une
adaptation aux changements environnementaux. Dans ce sens, il est envisageable de
comparer la phylogographie du groupe M. javanica entre vergers et ppinires pour
confirmer les voies de dispersion de cette espce sur olivier au Maroc.
danalyser la diversit molculaire dtecte au sein de M. javanica en rapport avec les

modalits de culture et la variabilit environnementale des rgions o lespce a t
dtecte.
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Chapitre XI
Synthse & Discussion

gnrale
Page 273
1. Rappel de la problmatique et des objectifs

La biodiversit est considre comme une des sources de services cosystmiques
dapprovisionnement et comme moteur de rsilience cologique. Cest ce qui explique les grands
programmes de protection, de restauration et de conservation de la biodiversit. En revanche, les
antagonismes biologiques (dont les interactions parasitaires) appartiennent aux services
cosystmiques de rgulation. Or, il apparat vident que le contrle de ces antagonismes poursuit
des objectifs dextinction biologique. On peut alors se poser la question du risque cologique pos
par une diminution de la diversit parasitaire.
Dans le cas des nmatodes phytoparasites (NPP), ubiquistes et adapts toutes les
conditions pdoclimatiques, il a t tabli que, plus que la prsence et le dveloppement dune
espce sur une culture, la structure de leurs communauts est responsable, pour une grande part,
de la production vgtale. Ainsi, le programme de recherche dans lequel sinsre ce travail de
thse a pour but de mieux comprendre les facteurs impliqus dans lassemblage des espces en
communauts, pour ensuite inspirer une nouvelle stratgie de gestion des NPP. Cette stratgie
appuie sur la conduite de la diversit des NPP et de la structure de leurs communauts vers des
structures peu ou pas pathognes, comme cela existe dans les cosystmes naturels. Sachant que la
structuration des communauts dorganismes est sous la contrainte de forces volutives et
environnementales, il nous appartient de dterminer le rle de ces diffrentes forces sur les NPP
en communauts. Mais lanalyse des communauts de NPP dans des systmes cultivs
exclusivement ne suffit pas parce que ces milieux trs perturbs ne permettent pas de comprendre
les mcanismes dassemblage et de co-existence des espces dans des situations de trade-off
(quilibre dadaptation des espces dans une niche cologique). Le recours aux systmes naturels,
et mieux encore aux changements dusage entre milieux naturels et milieux cultivs, est donc
indispensable. Les modles cologiques rpondant ces exigences ne sont cependant pas
nombreux : e.g. agrosystmes forestiers des Landes de Gascogne (changements dusage fort vs
cultures, Baujard et al., 1979) et arganeraie marocaine (tude en cours par lquipe). Parmi ces
modles cologiques, lolivier mditerranen est particulirement congruent parce quil concerne
des agro-cosystmes anciens avec deux zones refuges post-glaciaires (Maroc et Turquie), de
nombreux spots dolastres et nombreux cas de fralit. Il offre galement une trs grande
diversit varitale issue de la domestication. Cultiv traditionnellement depuis des millnaires, la
mcanisation de sa culture et la cration de varits cycle court permettent aujourdhui
dintensifier ses modes de production, mais lexposent un dveloppement croissant des
bioagresseurs dont les NPP. Lhistoire volutive de lolivier et lintensification cologique de
loliveraie mditerranenne sont des atouts tout fait opportuns pour ltude comparative des
Page 274
patrons de communauts (structure et pathognie). Loliveraie marocaine, sur laquelle a port

cette thse, offre toutes ces composantes.
Afin datteindre nos objectifs (et rpondre aux questions poses dans lintroduction
gnrale), il a fallu, dans un premier temps, analyser la diversit et la structure des communauts
de NPP associes lolivier, dterminer ensuite quelles sont et dans quels sens les variables
cologiques ont un effet potentiel sur lorganisation de la diversit des nmatodes en
communauts. Pour ce faire, des analyses descriptives et comparatives de la diversit des NPP et
de la structure de leurs communauts entre diffrents systmes cologiques et agronomiques
prsentant des caractristiques variables ont t proposes.
Ltude a tenu compte de plusieurs caractristiques agronomiques et environnementales
dont certaines sont lies larbre lui-mme et dautres son environnement. Les caractristiques
propres lolivier ont t analyses sur un gradient: (i) de domestication de lolivier sauvage ou
olastre lolivier cultiv, en passant par lolivier fral ; (ii) dintensification de la culture en
faisant la distinction entre les vergers traditionnels (faible densit) et les vergers haute densit ;
(iii) dirrigation en distinguant les oliviers irrigus des oliviers pluviaux ; (iv) de diversit
gntique de lolivier (dcrite par des lignes chloroplastiques E1, E2, E3, M). Les
caractristiques environnementales taient reprsentes par les paramtres physicochimiques du
sol et les conditions climatiques des sites dchantillonnage.
Ces diffrentes variables ont t confrontes aux variables nmatodes dont les analyses
ont t ralises plusieurs chelles : (i) de la communaut : en analysant la diversit via des
indices simples de diversit taxonomique, de diversit fonctionnelle, de dominance des taxons
dcrite par leur abondance et leur frquence dans les chantillons, de structure des patrons (au
niveau du genre). (ii) du genre en analysant la diversit spcifique des nmatodes galles du genre
Meloidogyne spp. et des nmatodes du genre Helicotylenchus spp. ; (iii) de lespce en analysant
la diversit intraspcifique de diffrentes populations de Meloidogyne javanica par des traits
morphologiques et molculaires.
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2. Principaux rsultats obtenus durant cette thse

2.1. Diversit globale des nmatodes phytoparasites
Une grande diversit taxonomique (117 espces appartenant 47 genres) a pu tre dtecte
sur olivier au Maroc (chapitre IV). Ces espces se distinguent selon leur prfrence
trophique (phytoparasites stricts ou gnralistes) et selon leurs stratgies adaptatives aux
perturbations des milieux (colonisateurs et persistants) (chapitre V). Cette tude a permis
damender la liste des NPP dj enregistrs sur lolivier au niveau mondial de 60 espces
appartenant 7 genres).
Les communauts de NPP sont principalement domines par les nmatodes des genres
Filenchus spp., Helicotylenchus spp., Merlinius spp., Paratylenchus spp., Pratylenchus
spp., Rotylenchus spp., Tylenchorhynchus spp. et Xiphinema spp. (chapitre VI).
Bien que peu frquentes (12% doccurrence), plusieurs espces du genre Meloidogyne
(nmatode galles) ont t rapportes en association avec lolivier au Maroc : M. arenaria,
M. javanica, M. hapla (chapitre IX). En outre, deux nouvelles espces ont t dtectes sur
olivier sauvage : lune (M. spartelensis, chapitre VIII) a t trouve dans le Rif (Cap
Spartel) et lautre (en cours de description) a t trouve dans le Haut-Atlas. Parmi ces
espces, M. javanica est largement distribue lchelle du pays, essentiellement dans les
zones cultives et sur quelques oliviers fraux, alors que les autres espces sont confines
dans des refuges dolivier sauvage. Nous posons lhypothse que lintroduction de M.
javanica dans les zones cultives se ferait par dispersion partir des ppinires (substrats
non contrls).
Les populations de M. javanica prsentent une diversit de profils enzymatiques (trois

profils estrasiques dtects), une diversit morphologique des juvniles de deuxime
stade larvaire (surtout les tailles du corps et du stylet), et une diversit des squences du
gne mitochondrial 63R (rpartie en 5 lignes et 19 haplotypes) (chapitre X).
2.2. Impacts des forages anthropiques et environnementaux

Que ce soit lchelle de la communaut, du genre, ou de lespce, la diversit des NPP est
impacte par la domestication de lolivier, lanthropisation des milieux, par les pratiques
culturales, et par les paramtres daphiques et climatiques pris en compte dans ltude. Ces
impacts touchent aussi bien la diversit, la structure et la composition trophique et
fonctionnelle des communauts. Cependant, on peut distinguer deux grands groupes de
forages lis :
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La domestication de lolivier (olastre vs olivier cultiv) :
les indices de biodiversit ont rvl des communauts riches en espces dans les
milieux non cultivs (sauvage et fral), avec une qui-rpartition des espces au sein
des communauts, contrairement ce qui a t observ dans des milieux cultivs (plus
faible richesse spcifique et dominance de certains taxons). En revanche, les effectifs
en nmatodes sont trs suprieurs en vergers. Il y a donc opposition entre abondance
des nmatodes et diversit spcifique (chapitre IV).
la composition trophique des communauts est galement affecte : les olastres

hbergent plus de nmatodes gnralistes et moins de nmatodes phytoparasites stricts
que les oliviers cultivs (chapitre V).
tous les groupes fonctionnels ont t dtects dans les milieux naturels. Les espces
persistantes (cp-5) ont t surtout dtectes sur les olastres, alors que les milieux
cultivs sont surtout domins par des espces colonisatrices (cp-3) (chapitre V).
les patrons des communauts sont modifis : des genres tels que Xiphinema spp.,
Nothotylenchus spp. et Heterodera spp. sont plus spcifiques des cosystmes naturels
(olastres) alors que dautres genres tels que Meloidogyne spp. et Tylenchorhynchus
spp. sont plus infods aux milieux cultivs, en particulier dans les vergers haute
densit (chapitre VI).
la distribution des espces est aussi concerne : bien que le genre Helicotylenchus spp.
soit ubiquiste sur olivier au Maroc, des espces sont plutt infodes aux olastres (H.
digonicus et H. varicaudatus) et dautres aux oliviers cultivs (H. crenadauta, H.
vulgaris, H. dihystera et H. pseudorobustus) (chapitre VI). Il en est de mme pour des
espces plus rares comme celles du genre Meloidogyne, puisque M. javanica est trs
rpandu exclusivement sur olivier cultiv et sur quelques oliviers fraux, alors que les
autres espces rares, dont la nouvelle espce dcrite (M. spartelensis), ont t
uniquement dtectes dans les zones refuges post-glaciaires dolivier sauvage
(chapitre IX).
Lintensification de la culture :
bien que les indices de biodiversit ne semblent pas tre touchs par le degr
dintensification des systmes de cultures : faibles (culture traditionnelle faible
densit) ou forts (culture en haies haute densit) apports dintrants (chapitre IV).
une distinction importante apparat dans la composition trophique et fonctionnelle des
communauts : dominance des espces parasites facultatives dans les conditions de
culture traditionnelle et dominance des espces phytoparasites strictes dans les vergers
haute densit (chapitre V).
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la structure des communauts est galement touche par lintensification de la culture :

les genres Meloidogyne spp. et Tylenchorhynchus spp. dominent dans les vergers
haute densit tandis que les vergers faible densit sont plus favorables au
dveloppement dautres genres tels que Helicotylenchus spp. et Filenchus spp.
(chapitre VI).
Pour ce qui concerne le genre ubiquiste Helicotylenchus spp., les communauts sont
domines par les espces H. crenadauta, H. vulgaris, et H. pseudorobustus dans les
vergers haute densit, alors que H. dihystera est prpondrant dans les vergers
traditionnels (chapitre VI).
les vergers haute densit sont plus propices au dveloppement des populations de M.
javanica (7136 individus/dm3 en moyenne) que les vergers traditionnels (2524
individus/dm3 en moyenne) (chapitre IX).
Les autres variables mesures ont des impacts secondaires :
lirrigation na pas deffet notoire dans la mesure o cette variable se confond avec la
modalit de culture de lolivier : les vergers irrigus correspondent majoritairement
aux vergers haute densit et moins frquemment aux vergers traditionnels, tandis que
la modalit pluviale correspond majoritairement aux olastres, aux oliviers fraux et
frquemment aux vergers traditionnels.
la diversit gntique de lolivier se confond galement avec les modalits de culture :

la ligne chloroplastique E1 se dtecte soit sur olivier cultiv, soit sur olivier fral, les
lignes E2 et E3 soit sur olivier fral, soit sur olivier sauvage, et la ligne M
correspond exclusivement O. maroccana (olivier sauvage).
les variables du sol (en particulier le type de sol, le contenu en matire organique et le
pH) ainsi que les variables climatiques sont des facteurs secondaires de structuration
de la diversit. Le type du sol et son contenu en matire organique, ainsi que les
tempratures minimales, impactent les indices de biodiversit, dont la richesse
spcifique en particulier. Ces paramtres se sont rvls importants dans la
structuration des patrons de communauts. Labondance en Meloidogyne spp., par
exemple, semble ngativement corrle au contenu du sol en matire organique.
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3. Analyse critique
3.1. Limites exprimentales
Ces rsultats ont fourni des rponses certaines des questions initiales poses par la recherche.
Cependant, ces analyses nont pas tenu compte de toutes les interactions capables dimpacter la
diversit des communauts de nmatodes.
Une part de cette diversit et de la structure des communauts pourrait tre galement
dirige par divers types de comptitions (trade-off) tablies entre les nmatodes phytoparasites
eux-mmes. Dans ce sens, les analyses de lACP ont indiqu des corrlations positives ou
ngatives entre quelques genres de NPP. Ainsi, les caractristiques fonctionnelles des nmatodes
composant une communaut sembleraient tre impliques dans la coexistence des espces,
souvent regroupes en fonction de leurs traits fonctionnels. Ces traits fonctionnels interviennent
dans une varit dinteractions entre les espces (e.g. la concurrence, la facilitation, le mutualisme,
et la prdation) (Hooper et al., 2005). Chez les nmatodes phytoparasites, plusieurs tudes
indiquent que des espces proches coexistent souvent. La coexistence de Pratylenchus penetrans
et P. crenatus dans des conditions agricoles est bien connue (Bongers & Bongers, 1998). Ces
auteurs confirment que lhtrognit du sol (temprature, humidit, etc.) joue un rle important
dans la coexistence des espces. Cependant, elle peut galement tre lie des traits biologiques :
Yeates (1986) suggre que la longueur du stylet pourrait tre un indicateur de la diffrenciation de
niche, puisque les nmatodes ayant un long stylet se nourrissent sur les couches cellulaires
profondes des racines contrairement aux nmatodes ayant un stylet court.
Dautres interactions biologiques pourraient intervenir entre les NPP et les autres groupes de
nmatodes du sol (qui ncessiterait une caractrisation prcise) ou entre les NPP et les autres
organismes du sol (Piskiewicz et al., 2007 ; 2008) ou leurs produits de synthse. La plupart de ces
interactions sont de lordre de la prdation (nmatodes carnivores du genre Mononchus par
exemple, Wang et al., 2015), du parasitisme (champignons nmatophages et bactries du genre
Pasteuria, Siddiqui & Mahmood, 1999 ; Chen et al., 2004), de linhibition due des mtabolites
dorigine microbienne (Chen et al., 2000) ou vgtale.
En outre, les interactions biotiques et abiotiques dans un sol sont particulirement
complexes. Par exemple, l'activit des champignons nmatophages peut tre influence par le pH
du sol, son humidit, sa temprature ainsi que les nutriments disponibles (Jatala, 1986). Ces
facteurs abiotiques interagissent donc avec de nombreux organismes telluriques, modifiant les
trajectoires des rseaux trophiques, et ainsi impactant la comptitivit des espces de NPP. Dans
notre cas, la matire organique sest rvle comme un facteur dimpact significatif sur la diversit
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des NPP et la structure des communauts. Support de lactivit microbienne, elle stimule donc le
dveloppement des populations de nmatodes non phytoparasites qui pourraient entrer en
concurrence avec les NPP (Nahar et al., 2006). La dgradation de la matire organique peut
galement saccompagner de la libration dacides organiques toxiques pour les nmatodes
(Rodrigues-Kabana et al., 1981 ; Rodrigues-Kabana, 1986). Les amendements organiques sont
dailleurs efficaces pour rduire les populations de NPP en dessous de niveaux dommageables
pour la culture (Bohlen & Edwards, 1994 ; Akhtar & Mahmood, 1996 ; Nahar et al., 2006). Ainsi,
le taux lev de matire organique dans les sites dolivier sauvage pourrait expliquer la richesse
leve et les niveaux des populations faibles de NPP rencontrs dans ces conditions. Cela pourrait
justifier limpact significatif de la matire organique sur la diversit et la structure des
communauts de nmatodes.
La plante hte est videmment un facteur cl de structuration de la diversit des NPP. Bien
que la frondaison des oliviers en vergers abritait rarement des adventices, et, quen milieu
sauvage, la collecte des chantillons de sol a t ralise prfrentiellement sur des zones de sol
nu, on ne peut exclure la prsence de racines issues de plantes voisines (adventices dans les alles
de vergers, autres arbres et adventices proximit des oliviers sauvages). Or, ces plantes
secondaires pourraient tre galement htes pour certaines espces de nmatodes identifies
dans cette tude, parce que plusieurs dentre elles (e.g. Helicotylenchus digonicus, Meloidogyne
javanica) sont polyphages, et une large gamme de plantes sauvages sont capables de maintenir des
inocula en oliculture (El-Borai & Duncan, 2005). Donc, leffet potentiel de ces plantes
secondaires dans la structuration de la diversit des nmatodes en communauts devrait tre pris
en considration et analys comme variable explicative.
Des essais ont t raliss (collaboration avec Dr. G. Besnard, CNRS Toulouse et Dr. S.
Bellafiore, IRD Montpellier) afin de dvelopper des marqueurs microsatellites sur plusieurs
espces de Meloidogyne dont M. javanica et M. graminicola. Cependant, malgr la clonalit de la
reproduction asexue, des mutations dans les descendants dune mme femelle sont possibles
(Castagnone-Sereno et al., 1994) remettant en cause lutilisation de ces marqueurs sur des
juvniles dont il est en outre difficile dextraire suffisamment dADN systmatiquement (compte
tenu de leur taille).
3.2. Biais mthodologiques
Des biais mthodologiques peuvent galement intervenir dans les rsultats obtenus :
-
Puisque les nmatodes se distribuent dans le sol de manire agrge (Boag & Topham, 1984),
lchantillonnage, obligatoirement fait laveugle, se rvle alors comme une premire source
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d'erreur exprimentale. Lextraction des nmatodes est une deuxime source derreur,
puisquil est bien vident qu'aucune technique ne permettra d'extraire la totalit des
nmatodes contenus dans un chantillon (Merny & Luc, 1969). Ces sources derreurs ne
peuvent tre totalement limines. Elles peuvent donc contribuer lhtrognit des
donnes, influenant par consquent les paramtres de diversit considrs par ltude.
-
Leffort dchantillonnage a t diffremment men entre les sites et les modalits

chantillonnes en raison de la disponibilit en arbres et/ou en vergers. Par exemple, les
rgions du Kandar et du Jel ntaient reprsentes que par 5 chantillons. La modalit haute
densit ntait reprsente que par 10 chantillons, alors quil aurait t souhaitable de
disposer dun jeu de donnes plus consquent pour assurer une comparaison plus robuste avec
les oliviers sauvages. Daprs Mateille et al. (2011), lestimation de la diversit en NPP est
optimale quand le nombre dchantillons atteint au moins 20 rptitions par site.
Pour des raisons financires (cot danalyses), un seul chantillon de sol par site a t analys
pour ses proprits physico-chimiques.
Concernant les donnes climatiques utilises, elles ont t rcupres des stations mto les
plus proches des sites dchantillonnage. Des variations climatiques entre sites ne sont pas
exclure, dautant plus lorsque les sites dchantillonnage dans une rgion taient loigns lun
de lautre. En outre, les variables climatiques considres par ltude correspondaient des
moyennes mensuelles (tempratures) ou des cumuls annuels (pluviomtrie), alors que
lchantillonnage a t ralis durant deux mois de lanne. Dautres variables explicatives
lies au microclimat du sol, telles que lhumidit et la temprature du sol, nont pas t
analyses. Or ces variables microclimatiques sont connues pour leur influence sur la structure
trophique des nmatodes du sol (Ekschmitt et al., 2001). Enfin, ce ne sont pas des donnes
ponctuelles qui impactent le dveloppement des NPP, mais plutt des valeurs cumules (e.g.
degrs/jour, Trudgill, 1995).
Du fait que beaucoup danalyses multivaries sont fortement influences par les espces rares,
(Bouxin, 2014), celles-ci ont t retires des jeux de donnes analyss. Or bien que les taxons
dominants semblent jouer un rle cl comme moteurs de la dynamique de la communaut et de
la fonction de l'cosystme qui les hberge (Grime 2001, Dangles & Malmqvist 2004), des
espces rares peuvent avoir des effets significatifs dans la communaut. Elles pourraient, par
exemple, participer la rsistance/stabilit de la communaut vis--vis de linvasion de
nouvelles espces (Power et al., 1996 ; Lyons et al., 2005).
Ltude sest focalise sur la diversit des nmatodes au sein de la communaut et na pas
trait la diversit inter-communauts ou inter-habitats. Pour ce faire, dautres indices devront
tre pris en considration tels que lindice de Jaccard ou inversement lindice de Whittaker
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(diversit ) qui mesurent respectivement la similarit ou la dissimilarit entre deux habitats ou

deux communauts.
-
Une attention particulire a port sur la diversit des communauts de nmatodes associes
lolivier sauvage dans des zones refuges (Rif, Atlas) afin de caractriser la persistance des
nmatodes dans ces rgions. Les analyses des indices de biodiversit ont indiqu que la
diversit est quasiment identique entre ces diffrentes rgions. Bien que ces indices de
diversit (comme la richesse, lindice de Shannon et lquitabilit) se rvlent utiles pour
dcrire la diversit des communauts, ils ne donnent aucune information sur leur composition
taxonomique (Griffiths et al., 2011). Cest pourquoi, dautres analyses ciblant la composition
spcifique de ces communauts se sont avres ncessaires. Ces analyses ont dmontr que
des communauts de mme richesse et de mme diversit peuvent hberger des taxons
diffrents. Or il savre que les communauts rencontres dans les zones sauvages sont
beaucoup plus htrognes dun point de vue taxonomique que celles observes en vergers. En
outre, les milieux cultivs se distinguent par une diversit spcifique rduite des communauts
avec cependant une densit leve des populations de nmatodes. Les analyses de labondance
ont indiqu que ces niveaux de population concernent essentiellement les genres Meloidogyne
spp. et Tylenchorhynchus spp.. Cependant, ltude na pas trait la diversit taxonomique en
zones cultives, afin de savoir si elle est plus homogne que dans les zones sauvages.
Lors de ltude de la diversit spcifique des Meloidogyne, les populations dtectes ont t
multiplies sur tomate pour des raisons de simplification technique. Cependant, la tomate ne
semble pas tre la meilleure plante hte pour toutes les espces de Meloidogyne. Par exemple,
M. beaticae, espce dcrite sur olivier en Espagne, ne se multiplie pas sur tomate (Castillo et
al., 2003). Il est noter que cinq isolats de Meloidogyne spp. ne se sont pas multiplis sur
tomate (absence de galles et de masses dufs). On peut donc envisager que des populations
despces nont pas t dtectes cause dun effet slectif potentiel de la tomate.
Concernant la variabilit morphologique des populations de M. javanica, quelques limites

ltude peuvent tre voques :
Ltude a t uniquement ralise sur des juvniles de deuxime stade larvaire qui
offrent gnralement trs peu de caractres servant au diagnostic et la diffrenciation
des espces. Mais, les mesures sur le stade adulte sont difficilement ralisables sur les
femelles (difformes) et les mles (trs rares). Les juvniles restent le stade le plus facile
rcuprer et en grande quantit et le plus facile monter et mesurer. Malgr cela, la
prparation et lobservation (prises de photos et mesures) de 1532 juvniles a demand
plus dun an dtude.
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Le nombre des caractres morphologiques mesurs afin danalyser la variabilit

morphologique de M. javanica reste relativement limite. En fait, 10 variables avaient
t envisages sur chaque juvnile. Mais nous nous sommes limits aux variables les
plus fiables, cest dire systmatiquement reprables, et non perturbes par des
problmes optiques (e.g. variation de champ).
Les traits biologiques, comme la fcondit des femelles ou le taux dclosion des ufs,
nont pas t pris en compte parce quil nous tait impossible de slectionner des
masses dufs de mme taille, ou dtre sr que la masse dufs considre contenait
tous les ufs pondus par la femelle (juvniles dj clos dans le substrat).
4. Perspectives court terme

Pour faire suite ce travail, les rsultats dont nous disposons nous permettent denvisager :
4.1. Diversit nmatologique
-
Danalyser la diversit et la structure des communauts la lumire de modles de coexistence

des espces. Dans ce sens, le Checkerboard score (c-score, Stone & Roberts, 1990) semble
tre un bon outil statistique pour rpondre cet objectif. Cette analyse permet de dterminer la
distribution des espces et de quantifier son caractre alatoire entre biotopes. Lanalyse
calcule la cooccurrence des espces deux--deux dans un habitat donn. Elle est base sur une
matrice de prsence-absence des espces. Elle renseigne si les patrons de cooccurrence
observs rsultent dune exclusion comptitive entre espces ou dune htrognit
environnementale des sites dtude. Ici, il apparat important de considrer les processus la
fois locaux et rgionaux qui peuvent affecter la coexistence des espces diffrentes chelles
(Huston, 1994). Par exemple, les interactions comptitives sont des processus locaux, qui
peuvent influencer le nombre d'espces qui peuvent coexister l'chelle locale (chantillon de
sol). La coexistence des espces ainsi que la diversit des nmatodes une chelle plus grande
(celle du Maroc par exemple) peut tre influence par des processus rgionaux tels que la
dispersion, l'immigration, et des perturbations de lcosystme (Ettema, 1998).
Diffrentes zones refuges de lolivier sauvage ont t dfinies autour du Bassin Mditerranen
(Besnard et al., 2013). Mais, outre linfluence de facteurs pdo-climatiques trs divers, des
barrires naturelles ont pu gnrer une fragmentation et une drive de la diversit des
organismes. Par exemple, la diversit haplotypique de lolivier sauvage est diffrente entre les
deux berges du Dtroit de Gibraltar (dfini par Rodriguez-Sanchez et al., (2008) comme un
hot-spot de diversit et divis en deux zones refuges distinctes nord et sud par Mdail et
Diadma (2009)) et entre les deux faades atlantiques et mditerranennes du Rif (Besnard et
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al., 2013). Une analyse des co-structures des communauts de nmatodes associes permettrait
de voir si ces phnomnes de barrires ont affect galement la diversit des NPP et leur
persistance dans ces refuges. Cette tude pourrait stendre aux autres refuges mditerranens
de lolivier.
-
De quantifier la corrlation entre les facteurs environnementaux et la distribution des espces

en utilisant le modle SDM (Species Distribution Modeling) qui permet de mesurer
l'importance des facteurs environnementaux, de prdire la rpartition des espces dans des
zones non chantillonnes, et d'examiner les consquences cologiques des changements
environnementaux sur la distribution des espces (Miller, 2010). Le modle permet galement
de modliser lespace o les variables environnementales sont favorables la persistance
dune espce (Pearson, 2010).
4.2. Diversit gntique des Meloidogyne

Notre tude a entam une analyse de la diversit des Meloidogyne associs lolivier au Maroc.
Les populations dtectes ont montr une diversit de leurs profils enzymatiques estrasiques
(surtout au sein des populations de M. javanica et de M. hapla). Les quelques traits
morphologiques mesurs chez les juvniles ont galement montr une variabilit. La diversit
molculaire a t tudie via diffrents marqueurs (microsatellites, ITS2, cox1, 63R). Des rsultats
intressants ont t obtenus en analysant les squences de cox1 et de 63R.
Concernant les analyses morphologiques, elles ont t surtout focalises sur M. javanica
cause de sa dominance sur lolivier au Maroc. Mais les mmes mesures morphologiques ont t
effectues sur les autres espces M. arenaria, M. hapla et M. spartelensis, au sein desquelles une
variabilit morphologique a t galement t trouve. Mais comme nous ne disposions que dune
seule population pour chaque espce et quelles nont jamais t trouves en mlange dans une
communaut, il a paru statistiquement difficile den tirer des conclusions sur leur variabilit interet intraspcifique au sein dune mme communaut. A la suite des analyses morphologiques
ralises sur les populations de M. javanica, nous proposons de qualifier la distribution de la
variation gntique quantitative au niveau populationnel via le calcul de QST = FIT / (FIT + 2FIS),
FIT dfinissant la variabilit des individus entre les populations et FIS dfinissant la variabilit des
individus au sein dune population (Spitze, 1993).
Contrairement aux autres marqueurs, le gne 63R a permis de distinguer la population de M.
arenaria des populations de M. javanica. Il a galement permis de rvler une diversit gntique
importante inter et intra-populations de M. javanica avec la dtection de 19 haplotypes
correspondant 5 lignes. Cependant, il serait ncessaire dacqurir plus de donnes afin de
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valider les haplotypes dtects car les lignes Mj-IV et Mj-V ont t rarement dceles dans trs
peu dindividus, et surtout parce que de lhtroplasmie mitochondriale est souvent rencontre.
Nous proposons ensuite dterminer la distribution de cette variabilit molculaire entre les
diffrentes populations afin dtudier la structuration neutre de la diffrentiation molculaire via
des calculs de FST = FIT / (FIT + FIS) (Wright, 1952).
En combinant les variabilits gntiques molculaire et morphologique, ltude serait suivie
par lanalyse de la congruence entre ces deux composantes de la variation au sein et entre
populations. Lapproche envisage consisterait comparer les structures neutre et quantitative de
la diversit (comparaison QST / FST). Cette approche pourrait informer sur l'effet relatif de la
slection et de la drive dans cette diffrenciation gntique (voir encadr 1).
Cette approche pourrait tre galement applique lanalyse de la variation gntique entre
habitats o lespce est prsente. Dans notre cas, nous pourrions dfinir des habitats pour M.
javanica selon les biotopes de lolivier : oliviers fraux vs cultivs faible densit vs cultivs haute
densit, ou rgions pdoclimatiques.
Encadr 1:
Trois scnarios pourraient tre proposs pour interprter cette comparaison (Chapuis et al.,
2007 ; Leinonen et al., 2013 ):
QST = FST : cela suppose que le trait de divergence entre les sous-populations serait atteint par
la drive gntique seule.
QST > FST : cela suggre la prsence d'une adaptation locale, parce que le FST reprsente la
divergence neutre qui dpend de lquilibre entre le flux et la drive gntique. Autrement dit,
une slection directionnelle est attendue qui agit en favorisant diffrents phnotypes (et donc
gnotypes) chez diffrentes populations.
QST < FST : cela suggre que la diffrence des traits entre populations est infrieur aux attentes
par la drive gntique seule, savoir une slection uniforme ou une slection de stabilisation
travers les populations, i.e. des phnotypes similaires dans diffrentes populations.
De plus, la fcondit et la taille des populations sont des facteurs qui pourraient influencer la
diversit gntique observe. Les facteurs abiotiques lis lenvironnement o les populations ont
t dtectes (modalit de culture, proprits physicochimiques du sol, conditions climatiques)
devraient tre galement pris en considration. La correspondance entre diversit gntique et
facteurs environnementaux pourrait informer sur ladaptation des espces lolivier et aux
Page 285
conditions lies cette culture, surtout lorsque les espces sont introduites dans un nouvel habitat
par les activits humaines (comme lintroduction de M. javanica partir des ppinires). Chaque
espce occupe gnralement une niche spcifique et unique dans son environnement, et des
spcialisations morphologiques ou physiologiques sont souvent dveloppes pour maximiser la
fitness de lespce au sein de sa niche. Ladaptation du parasite son hte et aux conditions
environnementales associes peut conduire amliorer sa propre volution, ce qui pourrait tre
aussi une source majeure de diversit gntique, permettant une rponse aux changements
environnementaux (Kidwell & Lisch, 2000). Cependant, dans le cas de Meloidogyne spp. et en
absence de donnes cytogntiques et dinformations sur les plantes htes pour un nombre
important despces de Meloidogyne spp., il nest pas vident de relier lhistoire volutive des
Meloidogyne avec celle de la plante hte (Castagnone-Sereno, 2006).
Une diversit molculaire intressante a t galement rvle au sein des squences de cox1
de la population marocaine de M. hapla, puisque les individus squencs ont t spars en deux
groupes. Cette diversit molculaire semble tre aussi corrle la diversit enzymatique observe
chez cette espce. Cette corrlation entre la variabilit enzymatique et mitochondriale a t
galement rapporte pour M. arenaria, M. javanica et pour M. incognita et pourrait correspondre
des systmes gntiques indpendants, refltant la prsence d'entits gntiques distinctes au sein
de l'espce (Hugall et al., 1994). Afin de valider lefficacit de ce marqueur comme outil dtude
de la diversit gntique de M. hapla, nous avons initi une tude en ciblant plusieurs populations
de M. hapla dorigines gographiques diffrentes.
5. Perspectives long terme

5.1. De la diversit gntique la diversit fonctionnelle
La diversit gntique est une source fondamentale de biodiversit. Elle se rvle avoir des effets
significatifs sur des processus cologiques tels que la productivit primaire, la comptition
interspcifique, la structure de la communaut (Hughes et al., 2008). Il a longtemps t admis que
l'organisation et la structure des communauts biologiques sont en partie dtermines par la
diversit gntique des populations qui les composent (Antonovics, 2003). La contribution de la
diversit gntique est de plus en plus vidente dans plusieurs processus cologiques tels que la
coexistence des espces (Booth & Grime, 2003), et la rsistance/rsilience des communauts aux
perturbations environnementales (Hughes & Stachowicz, 2004 ; Reusch et al., 2005).
Page 286
Que ce soit au niveau des gnes, des espces ou des communauts, la biodiversit affecte
clairement le fonctionnement des cosystmes. Ces effets pourraient tre ngatifs, neutres ou
positifs (Wardle, 2002 ; Wardle et al., 2004). Notre comprhension des rles fondamentaux que la
biodiversit peut jouer dans les proprits des cosystmes peut aider dvelopper une gestion
plus efficace de l'environnement et amliorer notre capacit prvoir les changements
environnementaux (Hooper et al., 2005). Plusieurs tudes ont mis l'accent sur la faon dont le
nombre d'espces et de groupes fonctionnels au sein des communauts affecte la stabilit et le
fonctionnement des cosystmes (Elton 1958; May, 1974; Chapin et al., 1997 ; Chapin et al.,
2000 ; Hooper et al., 2005).
Afin de comprendre les mcanismes par lesquels la biodiversit peut affecter le
fonctionnement de lcosystme, nous avons intrt dterminer la relation entre la diversit
taxonomique et la diversit fonctionnelle des communauts. Cependant, la relation entre la
diversit fonctionnelle et la diversit spcifique nest pas toujours vidente. Par exemple, la
diversit des espces peut tre en augmentation ou en diminution suite un changement d'usage
des terres, alors que la diversit fonctionnelle peut rester identique si aucun nouveau type
fonctionnel nest ajout ou supprim de la communaut. Il est galement thoriquement possible
d'avoir une diminution de la diversit fonctionnelle avec une augmentation de la diversit des
espces et inversement (Mayfield et al., 2010).
5.2. Histoire volutive de lolivier mditerranen et de ses parasites associs

Lolivier est un des cultures les plus anciennes et dimportance conomique, agronomique et
culturelle dans la rgion mditerranenne. Comprendre lhistoire volutive de sa domestication
empirique autour du Bassin Mditerranen reste un enjeu pour la cration varitale. Des tudes de
co-phylognie olivier-nmatodes sont envisages en collaboration avec les partenaires du projet
PESTOLIVE qui regroupe plusieurs laboratoires de recherches rpartis dans plusieurs pays
mditerranens. Parmi les bioagreseurs de lolivier, les nmatodes phytoparasites sont un bon
modle pour apprhender les mcanismes de co-adaptation entre le parasite et sa plante-hte. Les
nmatodes endoparasites plus particulirement sont un bon modle biologique dans la mesure o
les espces endoparasites sdentaires maintiennent une relation plus intime avec leur hte
(Reinhold-Hurek & Hurek, 2011) que des endoparasites migrateurs ou des ectoparasites.
Rotylenchulus spp., nmatode semi-endoparasite sdentaire largement distribu sur olivier autour
de la Mditerrane (alors que loccurrence de Meloidogyne spp. est faible), pourrait tre un bon
candidat pour ces tudes, pour autant que des marqueurs molculaires, pour linstant
indisponibles, soient dvelopps.
Page 287
5.3. Approche cologique de la gestion des nmatodes phytoparasites

Nous avons vu que les conditions de culture de lolivier saccompagnent dune rduction de la
diversit taxonomique (essentiellement la richesse) et dune augmentation des effectifs de taxons
fort potentiel parasitaire (espces colonisatrices cycle court, trs fcondes et tolrantes aux
perturbations environnementales). Donc, on sattend bien videmment un dveloppement
croissant des populations et des dgts agronomiques (le dveloppement des nmatodes galles
en vergers haute-densit en est la preuve). En consquence, une conclusion majeure simpose : les
activits humaines (changement dusage du sol, intensification de culture, travail du sol, rduction
de la diversit varitale, etc.) conduisent des changements dramatiques dans la structure et la
composition des communauts biologiques, par la perte d'espces ou l'introduction d'espces
dominatrices comme M. javanica et conduisent finalement dstabiliser les niches cologiques.
Ltude souligne le rle majeur de la diversit et de la structure des nmatodes en
communauts. Les stratgies de gestion de ces parasites devraient donc sorienter vers la gestion
de la diversit des communauts afin de conserver ou rtablir (rsilience) des structures plus
diversifies et par consquent peu ou pas pathognes pour la culture. Certaines options pourraient
tre proposes pour rpondre cette stratgie dans les systmes haute-densit qui vont connatre
une forte expansion dans les prochaines annes : systmes de culture diversifis (diversification et
rotation des cultures dans le temps, association de cultures [e.g. Brassicaceae]), agriculture de
conservation (travail du sol minimal, cultures de couverture), agriculture biologique (Kimenju et
al., 2010). Des modifications des pratiques culturales peuvent tre galement envisages telles que
lajout de la matire organique (corrlation ngative avec des nmatodes, e.g. Meloidogyne),
enherbement des parcelles dont les adventices pourraient tre des htes alternatifs des NPP,
augmentant par consquent leur diversit. La diversit gntique de la plante hte pourrait tre
galement un facteur dvelopper en proposant des gnotypes rsistants aux espces de
nmatodes les plus agressives (Altieri, 1990 ; Zhu et al., 2000). La recherche de sources de
rsistance aux NPP chez lolivier sauvage pourrait tre prospecte.
Une attention particulire devrait aussi tre porte aux interactions entre espces de
nmatodes en comptition vis--vis de la ressource trophique. Le genre Helicotylenchus est trs
diversifi sur olivier (11 espces recenses au niveau mondial) et prsent partout autour du bassin
mditerranen. Notre tude a montr quil tait prsent dans toutes les zones olicoles au Maroc,
avec cependant une distribution spcifique htrogne. En revanche, ce taxon tend disparatre
dans les vergers fortement contraints par les activits humaines (haute-densit), alors quil ne
semble pas provoquer de dgts particuliers dans les zones naturelles (olastres et olivier fral).
Cela nous amne prendre en considration des tudes ralises sur la canne sucre qui ont
Page 288
dmontr le caractre modrateur de ce taxon au sein de communauts de NPP (Lavelle et al.,

2004; Spaull et al., 2007). Ce rle modrateur a t dfini comme tel : en considrant des
communauts de mme composition taxonomique, celles domines par H. dihystera sont moins
pathognes (forte production vgtale) que celles domines par Meloidogyne spp. En outre,
lintroduction exprimentale de H. dihystera dans des communauts pathognes les rend moins
pathognes (Villenave & Cadet, 1998). Appliqu lolivier, ce concept devrait nous inciter
dvelopper des approches de gestion des nmatodes par conservation de leur diversit plutt
que par contrle direct de leur dveloppement.
6. Conclusions
Cette tude confirme que les communauts de NPP sont au centre denjeux environnementaux,
puisque nous avons montr que leur diversit et la structure de leurs communauts se rvlent tre
des indicateurs pertinents pour valuer ltat sanitaire des agro et des cosystmes. Les nmatodes
rpondent aux forages imposs par leur environnement. Leurs traits ces forages se traduisent
par des modifications dans la diversit taxonomique des communauts, dans la structure des
patrons de diversit, dans leur composition trophique et fonctionnelle, par la dominance de taxons
fort potentiel pathogne.
Les tudes diligentes dans le cadre de ce travail de thse permettent de faire ressortir deux
conditions fondamentales qui caractrisent laspect positif de la biodiversit :
Des descripteurs tels que niveaux des populations , richesse spcifique , structure
des communauts , pourraient tre considrs comme des bioindicateurs de ltat de sant
des sols et des cosystmes (incluant la production vgtale).
Les contraintes agronomiques actuelles sont des facteurs de dsquilibre biologique qui
contribuent laccroissement des contraintes parasitaires. La conservation de la diversit
des espces de nmatodes et dinteractions stables entre elles (trade-offs) doit tre intgre
dans les stratgies de production vgtale comme facteur de dveloppement agronomique.
La diversit parasitaire doit tre considre comme une variable prioritaire prendre en compte
avant de dvelopper une stratgie de gestion des parasites. Ce raisonnement devrait conduire
llaboration de stratgies intgratives cohrentes, bases sur le concept de rsilience de la
diversit (mme sil sagit de parasites), mieux adaptes la gestion durable des communauts de
NPP et la prservation des milieux.
Page 289
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Bakkali A, Moukhli A, Khadari B, El Modafar C, Ater M, Ferji Z, Mateille T. 2013. Les
communauts de nmatodes phytoparasites associes la culture de lolivier au Maroc.
Journes des Doctorants SPE, 26-28 juin 2013, Montpellier, France.
Ali N, Chapuis E, Tavoillot J, Mateille T. 2013. Les communauts de nmatodes
phytoparasites associes la culture de lolivier au Maroc. 6me Edition du Printemps de
Baillerguet, 06-08 juin 2013, Montpellier, France.
Bakkali A, Moukhli A, Khadari B, El Modafar C, Ater M, Ferji Z, Mateille T. 2013. Plantparasitic nematodes associated with olive in Morocco. 1st International AMAS Conference,
Rabat, Morocco, 18-19 march 2013.
Ali N, Chapuis E, Tavoillot J, Martiny B, Plas S, Edel-Hermann V, Gautheron N, Hraud C,
Jimenez D, Granereau G, Barbier S, Rulliat, E, Steinberg C, Mateille T. 2012. Response of
plant-parasitic nematode communities to land-use changes. The case of a heath-forest-crop
succession. 31st ESN International Symposium, Adana, Turkey, 23-27 Sept. 2012.
Ali N, Chapuis E, Tavoillot J, Martiny B, Plas S, Jimenez D, Granereau G, Barbier S,
Mateille T. 2011. Rponse des communauts de nmatodes phytoparasites au changement
d'usage des sols. Cas d'une succession lande-fort-culture. 9me CIRA, Montpellier 26-27
octobre 2011.
Encadrements
- Simon BENATEAU, M2 Protection des Plantes et Environnement (PPE), Agro-Campus
Ouest, Rennes : Mars-septembre 2012.
-
Malyna SUONG : M2 Biologie des Plantes et des Micro-organismes, Biotechnologies,

Bioprocds, Spcialit : Biologie des Plantes, Parcours : Biologie Fonctionnelle des
Plantes, Universit Montpellier 2 : avril-juin 2013.
Mohamed AIT HAMZA : Doctorant, Facult des sciences, Universit Ibn Zohr, Agadir,
Maroc : stages effectus en 2012, 2014, 2015.
Caroline OJEIL, chercheur Institut de Recherche Agronomique Libanaise (LARI) : mars

2013
Joachim SICARD, BTS Bioanalyses et contrles, Lyce Jean Mermoz : juin- juillet 2015.
Organisation dvnements
- Journes des doctorants SPE, Montpellier 26-28 juin 2013.
-
Printemps de Baillarguet, Montpellier 04-05 juin 2015.
Page 294
Annexes techniques
Annexe 1 : Extraction des nmatodes du sol par Elutriation (Elutriateur de Seinhorst)
Rsum :
Le prsent document dcrit un mode dextraction des nmatodes vivant dans le sol.
Ce procd permet dextraire des nmatodes partir de sol, et de les rcuprer en suspension dans leau. Il est
adapt de la Norme ISO 23611-4: 2008 (section 6.3 Extraction)
I. Ractifs et Matriels ncessaires
A. Ractifs
- Echantillons de sol tudier, numrots et annots et rpertoris dans le cahier denregistrement
lutriation prsent au laboratoire.
- Oxalate de sodium.
B. Matriels :
- Elutriateur de Seinhorst compos (cf. schma joint cette annexe) :
dun Erlenmeyer de 2L (plastique ou verre) avec col taraud.
dune ttine en verre avec col taraud.
dune colonne en verre souffl.
dun bouchon silicone.
dune alimentation en eau rgule par un dbitmtre.
- Passoire domestique mtallique (maille 2mm ; 15cm).
- Entonnoir plastique 15cm.
- Seaux (10 15L).
- Minuteur.
- Boites de Ptri 10cm).
- Bchers 250mL.
- Verres pied 500mL.
- Tamis de passage actif (disque de toile moustiquaire d1mm de maille coll sur une rondelle de PVC int.
80mm, H15mm).
- Mouchoirs Kleenex blanc.
- Colonne de tamisage : 4 tamis inox 25cm maille 50M superposs.
- Tamis inox 25cm maille 100M.
- Pissette deau osmose.
- Balance 500g.
II. Protocole standard
Seinhorst, J.W. 1962. Modifications of the elutriation method for extracting nematodes from soil. Nematologica, 8 :
117-128.
A. Prparation de lchantillon de sol
A partir de lchantillon de sol prlev,
- Verser la totalit de lchantillon dans un plateau.
- Retirer les racines, les dbris vgtaux et les graviers.
- Homogniser manuellement et dlicatement lchantillon dans le plateau.
B. Mesure du taux dhumidit dun prlvement de sol
Afin de rapporter le nombre de nmatodes au poids de sol sec, il est indispensable de connatre le taux dhumidit
du sol. Cette mesure ne peut tre ralise sur le prlvement de sol lutri. Elle doit donc tre faite sur le reliquat de
sol du prlvement correspondant.
PSE = PFE x (1-H%) = PFE x PSP / PFP.
avec : PSE = poids sec de sol lutri.
PFE = poids frais de sol lutri.
PFP = poids frais de sol du reliquat de sol du prlvement.
PSP = poids sec de sol du reliquat de sol du prlvement.
Mesure du Poids frais du sol lutri (PFE).
- Mettre la balance sous tension (bouton On/Off).
- Poser un bcher plastique vide de 250 cc sur la balance et faire la tare.
- Remplir un bcher de 250 cc avec le sol frais lutrier.
- Peser le bcher plein sur la balance.
- Porter la mesure du poids frais mesur PFE dans la case Poids frais lutri de la feuille correspondante
du carnet souche des prlvements.
Mesure des Poids frais (PFP) et secs (PSP) des prlvements.
- Mettre la balance sous tension (bouton On/Off).
- Poser un sac plastique de prlvement vide sur la balance et faire la tare
Page 295
- Peser le prlvement de sol frais restant (= PFP).

- Porter la mesure du poids frais PFP dans le Cahier poids frais sec. Sols restants aprs lutriation .
- Ouvrir le sac au maximum et laisser scher le sol temprature ambiante.
- Aprs 2 semaines minimum, poser un sac plastique de prlvement vide sur la balance et faire la tare.
- Peser le prlvement de sol sec restant (= PSP).
- Porter la mesure du poids sec PSP dans le Cahier poids frais sec. Sols restants aprs lutriation .
Enregistrement
- Reporter ces donnes dans les fichiers de collecte.
C. Tamissage pralable et mise en suspension de lchantillon de sol
- Joindre le numro de lchantillon au bcher
- Passer travers un tamis grossier (passoire denviron 1 mm de maille), au-dessus dun entonnoir dans un
erlen de 2 L de llutriateur, avec un courant deau.
- Jeter le refus du tamis
- Remplir lerlen deau ras et fermer avec la ttine.
- Appliquer le numro de lchantillon sur lerlen.
Cas dun sol lourd
- Verser laliquote de 250 mL de terre sur un tamis grossier au-dessus dune bassine de 2 L environ.
- Faire passer la terre manuellement travers le tamis
- Verser le contenu de la bassine dans lerlen de 2 L
Cas dun sol trs argileux
- Verser laliquote de 250 mL de terre dans une bassine contenant 1 L deau environ.
- Ajouter une pince doxalate de sodium et laisser reposer quelques minutes
- Faire passer la suspension travers le tamis au-dessus dun entonnoir dans un erlen de 2 L, avec un
courant deau.
D. Prparation de llutriateur
- Boucher le bas de la colonne deau avec le bouchon silicone.
- Remplir la colonne deau jusquau trop-plein.
- Ouvrir le dbimtre au maximum et purger lair.
- Placer le seau en position A (cf schma joint cette annexe)
- Rgler le dbimtre 60 mL/min.
E. Extraction
- Agiter lerlen contenant la suspension de sol.
- Renverser lerlen sur le haut de la colonne.
- Appliquer le numro de lchantillon sur la paroi interne du seau.
- Laisser agir pendant 20 min.
Rem : Surveiller le dbit deau
Veiller viter toutes prises dair ventuelles au niveau du col de lerlen.
Veiller ce que lchantillon de sol tombe bien dans la colonne
- Retirer lerlen et verser son contenu dans le seau.
- Laisser le seau en position A et laisser agir de nouveau pendant 10 min.
- Arrter le courant deau.
- Placer le seau en position B (cf schma joint cette annexe).
- Vider le contenu de la colonne dans le seau.
- Laisser dcanter la suspension dans le seau environ 15 min.
Rem : Nettoyer imprativement la colonne leau osmose en fin de manipulation avant toute nouvelle utilisation
afin dviter toute contamination entre 2 chantillons).
F. Concentration de la suspension par tamisage
- Verser le contenu du seau travers la colonne de 4 tamis inox maille 50M.
- Rcuprer le refus des 4 tamis dans un verre pied (entre 50 et 100 mL environ), par rinage avec la
pissette deau osmose.
- Appliquer le numro de lchantillon sur la paroi interne du verre pied.
G. Purification par passage actif
- Tapisser un tamis de passage actif (PVC + toile moustiquaire) de 2 feuilles de papier Kleenex. Prvoir des
bords retombant lextrieur.
- Humidifier le papier Kleenex avec de leau distille.
Rem : Veiller ce que le papier Kleenex adhre bien la paroi du tamis.
- Verser trs dlicatement le contenu du verre pied sur le tamis, au dessus dun vier.
- Dposer le tamis dans une bote de Ptri.
Page 296
Rincer le fond du verre pied avec une pissette deau distille (quelques mL) dans le tamis.
Remplir la bote de Ptri deau distille jusqu affleurement de la surface du tamis, pour viter tout
desschement des nmatodes et pour permettre leur diffusion travers le tamis.
Appliquer le numro de lchantillon sur le couvercle de la bote de Ptri.
Laisser diffuser au moins 48h temprature ambiante.
III. Protocole spcifique aux nmatodes de grande taille (e.g. Longidoridae)

F.Concentration
- Verser le contenu du seau travers un tamis inox maille 100M.
- Rcuprer le refus du tamis dans un verre pied (entre 50 et 100 mL environ), par rinage avec la pissette
deau osmose.
- Appliquer le numro de lchantillon sur la paroi interne du verre pied.
G. Purification
- Tapisser un tamis de passage actif (PVC + toile moustiquaire) dune seule feuille de papier Kleenex.
Prvoir des bords retombant lextrieur.
Rem : Veiller ce que le papier Kleenex adhre bien la paroi du tamis
- Verser trs dlicatement le contenu du verre pied sur le tamis, au dessus dun vier.
- Rincer le fond du verre pied avec une pissette deau distille (quelques mL) dans le tamis.
- Remplir la bote de Ptri deau distille jusqu affleurement de la surface du tamis, pour viter tout
- Appliquer le numro de lchantillon sur le couvercle de la bote de Ptri.
- Laisser diffuser 24h temprature ambiante.
Page 297
Annexe 2: Prparation de nmatodes pour les tudes morphologiques

Rsum :
Le prsent document dcrit un procd de prparation de nmatodes permettant ltude morphologique.
I. Ractifs et matriels ncessaires
A. Ractifs :
- Suspensions de nmatodes extraits du sol ou des racines.
- Formaldhyde 30%.
- Eau osmose.
- Ethanol absolu.
- Gel de silice.
- Glycrine bidistille anhydre.
- Paraffine.
- Carbonate de sodium CO3Na2.
B. Matriels :
- Plaque chauffante pour histologie.
- Bain-marie.
- Pince en bois.
- Tubes hmolyse 10 mL et 20 mL (sans anticoagulant).
- Tubes en verre gradus 25, 50, 100 mL.
- Stromicroscope.
- Canne pche pour nmatode (poil de porc mont sur manche bois).
- Godets ( coloration) avec couvercles.
- Pompe vide.
- Pipettes.
- Dessicateurs.
- Etuve thermostate.
- Lampe alcool.
- Sorbonne.
- Emporte-pice 12mm.
- Lamelles rondes (18 mm) et rectangulaires (24x50mm).
- Lames de Cobb = lames microscopiques en aluminium (Cobb, N.A., 1917. Notes on nemas. Contr. Sci.
Nematol., 5 : 117-128).
- Carton blanc 1mm.
II. Protocole
A. Tri des nmatodes
- Placer un aliquote de suspension de nmatodes dans une cellule de comptage sous le stromicroscope.
- Pcher les nmatodes avec la canne pche.
- Regrouper les individus par famille ou par genre suivant les critres morphologiques observables en les
distribuant dans un tube hmolyse de 10 ml contenant 50 L deau osmose raison de 10 individus
adultes minimum.
- -Garder le reste de la suspension (dnomm culot ) dans un tube en verre gradu de 100 mL, laisser
dcanter et retirer le surnageant pour garder environ 5 mL.
Rem : Les tapes suivantes seront toutes ralises sous une sorbonne chimique avec aspiration car le formaldhyde
est trs toxique.
B. Fixation
Fixateur de De-Grisse (De Grisse, A.T., 1969. Redescription ou modifications de quelques techniques utilises
dans ltude des nmatodes phytoparasites. Meded. Fakulteit Landbouw. Gent, 34 : 351-369).
- 133 mL de Formaldhyde 30 %.
- 10 ml de glycrol Bidistill.
- ajuster pH 7 avec solution de CO3Na2 1%.
- qsp 1L deau osmose.
Fixation des nmatodes tris
- Faire chauffer 9 mL (environ) de fixateur de De-Grisse au bain marie dans un tube hmolyse de10mL.
Rem : Tenir le tube avec une pince en bois et ajouter un thermomtre.
Attendre que la temprature atteigne environ 45C.
Retirer le tube du bain marie et attendre que la temprature de 45C soit stable.
- Verser le fixateur dans le tube hmolyse contenant les nmatodes.
Rem : attendre le refroidissement complet avant de reboucher les tubes.
- Laisser dcanter 3 4 jours temprature ambiante.
Page 298
Rem : ce stade, les nmatodes peuvent tre conservs durant plusieurs mois.
Fixation des nmatodes en suspension (culot)
- Faire chauffer 25 mL (environ) de fixateur de De Grisse au bain marie dans un grand tube gradu de100
mL.
Rem : Tenir le tube avec une pince en bois et ajouter un thermomtre.
Attendre que la temprature atteigne environ 45C.
Retirer le tube du bain marie et attendre que la temprature de 45C soit stable.
- Verser le fixateur dans le tube gradu 100 mL contenant les nmatodes.
Rem : attendre le refroidissement complet avant de boucher les tubes avec du parafilm.
- Laisser dcanter 3 4 jours temprature ambiante.
- Retirer le surnageant, sans aspirer le culot de nmatodes.
- Pipeter tout le culot et le transfrer dans un tube hmolyse de 20 mL.
Rem : Rincer le cne de la micropipette plusieurs fois avec le fixateur pour ne pas perdre de nmatodes.
- Complter le tube avec du fixateur et boucher le tube.
Rem : ce stade les nmatodes peuvent tre conservs durant plusieurs mois. Les nmatodes pourront tre repris
sous stromicroscope plac sous sorbonne chimique aspiration pour viter de respirer les vapeurs de formol
reprsentant un danger pour la sant humaine).
C. Dshydratation
Solutions de dshydratation
Solution 1
Solution 2
200 mL Ethanol 95%
950 mL Ethanol 95%
10 mL Glycrol bidistill
50 mL Glycrol bidistill
qsp 1 L eau osmose
pour la fabrication de lthanol 95 % : cf Tableau 1 joint lannexe.
Procdure de dshydratation (nmatodes tris)
- Retirer le surnageant dans les tubes hmolyse, sans aspirer le culot de nmatodes.
- Pipeter le culot avec tous les nmatodes et le dposer dans un godet ( coloration).
Rem : Rincer le cne de la micropipette plusieurs fois avec la solution 1 pour ne pas perdre de nmatodes.
Verser rapidement 1.5 mL de solution S1 (= moiti du volume du godet).
- Placer le godet (sans couvercle) dans un dessicateur contenant de lthanol 95%.
- Placer le tout ltuve 35-40C pendant 12h minimum.
- -Retirer le godet du dessicateur et retirer 1mL de surnageant S1 (sans aspirer de nmatodes).
- Rajouter 1 mL de S2.
- Replacer le godet moiti couvert par son couvercle (pour permettre lvaporation lente de lthanol
95%) dans le dessicateur et replacer le tout ltuve 35-40C pendant 48h minimum.
- Placer le godet non couvert dans un dessicateur contenant du gel de silice, pendant 24h minimum.
Rem : avant toute utilisation pour montage sur lame, vrifier que la suspension est bien dshydrate (goutte
visqueuse).
D. Montage sur lame de Cobb
- Nettoyer les lamelles avec un papier absorbant.
Rem : Vrifier quil ne reste aucun dpt de graisse ou de papier.
- Chauffer lextrmit de lemporte-pice avec la lampe alcool.
- Tremper lemporte-pice dans la paraffine sur une profondeur de 5mm maximum et dposer un anneau
rgulier et fin de paraffine la surface dune lamelle rectangulaire.
- Tremper lextrmit dun embout de micro-pipette (cne jaune) dans le Glycrol bidistill anhydre et
dposer le Glycrol au centre de lanneau de paraffine.
Rem : aliquoter environ 50 mL de glycrol bidistill anhydre dans un flacon et le dboucher.
Passer ltuve et conserver le flacon dbouch dans un dessicateur afin dviter toute rhydratation.
Sortir le godet du dessicateur et pcher les individus un un (entre 3 et 6 individus par lame).
Dposer les individus paralllement les uns aux autres dans la goutte de glycrol afin de faciliter leur
observation.
Rem : vrifier chaque dpt (sous le stromicroscope) que les nmatodes sont bien prsents dans la goutte.
Dposer dlicatement une lamelle ronde sur le cercle de paraffine.
Placer lensemble sur la plaque chauffante (70C maximum).
Rem : ds que la paraffine fond, appuyer lgrement sur la lamelle ronde pour chasser les bulles dair.
Placer lensemble des 2 lamelles sur une lame de Cobb.
Dcouper et placer de chaque ct de la lame deux morceaux de carton blanc.
Sertir le tout.
Identifier la lame comme ci-dessous.
Page 299
- Genre
- Espce
- Nombre individu
- Sexe
- N de lame
- N Echantillon
- Plante hte
- Lieu
- Date
Archiver la lame dans une bote lames labri de la lumire et de la poussire.

Rem : toutes les donnes sont galement enregistres sur le Tableau de Rfrencement (cf Tableau 2 joint
lannexe).
Tableau 1 de dilution de lalcool par Gay-Lussac (volume deau (mL) ajouter 100 mL dalcool diluer)
Page 300
Tableau 2 de Rfrencement
Page 301
Annexe 3 : Prparation des nmatodes pour les tudes molculaires

Rsum :
Le prsent document dcrit deux procds de prparation de nmatodes permettant ltude molculaire.
I. Ractifs et matriels ncessaires
A. Ractifs :
- Eau osmose
- Ethanol Absolu
- NaCl
B. Matriels :
- Plaque chauffante et rcipient pour bain-marie
- Pince en bois
- Tubes hmolyse de 20 mL
- Tubes gradus
- Loupe binoculaire
- Canne pche pour nmatode
- Pipettes
- Plaque chauffante
- Tube Eppendorf de 1.5 mL
- Boite de conglation avec compartiment
- Conglateur 80C
- Etiquette pour tube
II. Protocole
A partir des suspensions de nmatodes obtenues aprs lutriation de sol (ou extraction de racines en chambre
brouillard.
Procder lidentification taxons prsents dans les chantillons suivant les critres morphologiques observables la
loupe binoculaire.
La conservation peut se faire sous diffrents modes (-80C, NaCL, Alcool) sur du matriel tri ou en culot.
i.
Conservation 80C:
1) Dposer une goutte (6 L) deau osmose au fond dun tube eppendorf de1.5 mL.
2) Pcher le nmatode (individus adultes et ou juvniles) et dposer dans la goutte, au fond du tube eppendorf.
Rem : Vrifier chaque dpt (sous la loupe binoculaire) que le nmatode soit bien prsent dans la goutte.
Suivant ltude molculaire raliser :
Faire des tubes contenant un seul individu et/ ou des tubes avec 5 ou 10 individus.
3) Identifier le tube (N Echantillon, genre, (espce si connue), nombre dindividus, date de mise en tube ) et la
boite de conglation.
Rem : Les tubes sont conservs dans des botes de conglation compartimentes et numrotes.
4) A chaque entre dun nouveau tube, le positionnement du tube dans la bote est archiv informatiquement, pour
permettre un reprage des tubes plus rapide (cf Annexe 1) et une meilleure gestion du matriel congel.
5) Pour chaque nouveau tube, linformation relevant du taxon et de son chantillon dorigine, de sa bote de
conglation, est classe dans une base de donnes (fichier Excel, ou base de donnes informatise).
Cf. Tableau de Rfrencement, Annexe 2
6) Conserver au conglateur 80C.
ATTENTION : Veiller ne jamais avoir de rupture de la chane du froid (panne dlectricit)
Rem : Aucun envoi nest possible avec du matriel stock de la sorte.
ii.
Conservation dans une solution saline:
Prparation de la solution NaCl 1M
- 58.54 g NaCl
QSP 1000 ml deau.
Rem: Peser la quantit requise de NaCl requise, dissoudre ce sel dans les du volume deau final et aprs
dissolution complte, ajuster avec leau jusquau volume final.
Rem : Le sel de cuisine convient en cas de ncessit.
1) Dposer 1ml de NaCl 1M dans un tube eppendorf de1.5 mL.
2) Pcher le nmatode (individus adultes et ou juvniles) et dposer dans le tube eppendorf (en plongeant la canne
pche dans la solution).
Page 302
Transfrer autant de nmatodes que souhait ou que le matriel biologique extrait le permet.
Rem : Si ncessaire et afin daider ajuster ses gestes, vrifier chaque dpt (sous la loupe binoculaire) que le
nmatode est bien prsent dans la solution.
3) Identifier le tube (N Echantillon, genre, (espce si connue), nombre dindividus, date de mise en tube ).
4) Pour chaque nouveau tube, linformation relevant du taxon et de son chantillon dorigine est classe dans une
base de donnes (tableau papier ou tableau de rfrencement, ou base de donnes informatise).
Cf. Tableau de Rfrencement Annexe 2
Conserver temprature ambiante ou bien entre 2 et 8C.
Conservation des chantillons dans ces conditions, valable jusqu 6 mois.
Rem : Les chantillons peuvent ainsi tre partitionns ultrieurement.
Eviter de conserver ces chantillons au-del de 2 mois.
Ils peuvent voyager par courrier et la rception au laboratoire de destination, les chantillons peuvent tre
partitionns puis congls (Cf II-A), ou bien directement congls (avec lobligation, dan ce cas, dutiliser tous
les nmatodes ou de perdre ceux qui ne sont pas directement utiliss aprs dconglation. Do lutilit de
partitionner lchantillon avant conglation. )
iii.
Conservation dans de lalcool 80%
Dans ce cas partager le culot :

- 2/3 rserv et conserv dans lthanol 80% (pour tude molculaire),
- 1/3 rserv la fixation au Formaldhyde (pour tude morphologique).
1) Prparer la solution de conservation :
Solution dEthanol 80 % (cf Tableau de dilution de lalcool par Gay-Lussac )
Exemple : A partir dEthanol absolu
100 mL Ethanol Absolu
20.54 mL Eau osmose
Volume final obtenu dthanol 80 % = 120,54 mL
- Faire chauffer 25 mL (environ) dEthanol 80% au bain marie dans un grand tube gradu de 100mL.
Rem : Tenir le tube avec une pince en bois et ne pas faire bouillir lthanol, arrter de chauffer ds lapparition de
petites bulles.
- Verser lthanol dans le tube gradu contenant les nmatodes (2/3 culot initial).
Rem : Le culot trait reprsente 2 ml au maximum.
- Attendre refroidissement complet avant de boucher les tubes avec du parafilm.
- Laisser dcanter 3 4 jours TA.
Rem : A cette tape les nmatodes peuvent tre conservs plusieurs mois.
Afin de gagner de la place et contrler lvaporation de lalcool, nous procdons une tape supplmentaire de
transfert dans des tubes hmolyse plus petits (de 20 mL) et moins encombrants.
- Pipeter lexcs dthanol, pour ne garder quenviron. 1 2 ml de culot et en prenant soin de ne pas aspirer
les nmatodes.
- Pipeter tout le culot et le transfrer dans un tube hmolyse de 20 mL.
Rem : Rincer le cne plusieurs fois avec de lthanol 80 % pour ne pas perdre de nmatodes.
- Couvrir nouveau le culot avec 19 mL (environ) dthanol 80 % et boucher les tubes.
Rem : A cette tape, les nmatodes peuvent tre conservs durant plusieurs mois.
Il est important de contrler les niveaux dalcool dans les tubes (vaporation frquente).
Conserver les tubes soit temprature ambiante soit 2-8C dans des rfrigrateurs quips de systme antidflagrant afin dviter tout risque dexplosion.
Page 303
Annexe 4 : Extraction des nmatodes des racines en Chambre brouillard

Rsum : Le prsent document dcrit un mode dextraction de nmatodes vivants, partir de fragments de racines.
I.
A.
Ractifs et matriels ncessaires

Ractifs
Echantillons de racine tudier, identifis (cf. Procdure NEM / PG QUAR) et rpertoris (cf. Procdure
NEM/ PS EN HISTO) dans le cahier denregistrement Asperseurs prsent au laboratoire.
B. Matriels :
- Chambre brouillard (Cf. Feuille de fonctionnement).
- Tamis dextraction (disque de toile moustiquaire d1mm de maille coll entre 2 tubes PVC int. 45mm
H50mm).
- Flacons plastique 100 mL munis dun orifice de trop plein.
- Verres pied 500 mL
- Boites de Ptri 10cm)
- Tamis de passage actif (disque de toile moustiquaire d1mm de maille coll sur une rondelle de PVC int.
80mm, H15mm)
- Mouchoirs Kleenex blanc
- Pissette deau osmose
II. Protocole
Seinhorst, J.W. 1950. De betekenis van de toestand van de grond voor het optreden van aantasting door het
stengelaatje (Ditylenchus dipsaci (Khn) Filipjev). Tijdschr. Plant Ziekt. 56: 291-349.
1. Prparation de lchantillon
- Laver les racines sous leau du robinet.
- Dcouper les racines en fragments (longueur maximale 1 cm).
- Dposer les racines sur le tamis dextraction.
2. Extraction des nmatodes
- Placer le tamis dans le flacon plastique de 100mL.
- Placer le tout dans la chambre brouillard.
- Retirer les flacons de la chambre aprs 1 semaine (R1) puis aprs 2 semaines (R2) dextraction.
- Laisser dcanter la suspension pendant 2 heures.
3. Purification
Rem : elle nest ncessaire que si la suspension est trop trouble.
- Tapisser un tamis de passage actif (PVC + toile moustiquaire) de 2 feuilles de papier Kleenex.
- Prvoir des bords retombant lextrieur.
Rem : Veiller ce que le papier Kleenex adhre bien la paroi du tamis
- Verser trs dlicatement le contenu du verre pied sur le tamis, au-dessus dun vier.
- Rincer le fond du verre pied avec une pissette deau distille (quelques mL) dans le tamis.
- Remplir la bote de Ptri deau distille jusqu affleurement de la surface du tamis, pour viter tout
- Appliquer le numro de lchantillon sur le couvercle de la bote de Ptri.
- Laisser diffuser au moins 48h temprature ambiante.
Page 304
Annexe 5 : Caractrisation de nmatodes du genre Meloidogyne par lectrophorse de leurs isoenzymes

(PAGE).
Rsum :
Le prsent document dcrit un mode de caractrisation de nmatodes du genre Meloidogyne, par ltude de leurs
profils isoenzymatiques. Le principe repose sur la prparation dchantillons (dissection et broyat) tudis en
lectrophorse (migration et rvlation).
Esbenshade, P., Triantaphyllou, A. 1985. Use of enzyme phenotypes for identification of Meloidogyne species.
Journal of Nematology, 17 : 6-20.
I. Ractifs et matriels ncessaires :
I- A. Ractifs
- Persulfate dAmmonium (PrSO4).
- Mlange dAcrylamide et de Bis-Acrylamide (ratio 37:1).
- Ttra Mthyl Ethylne Diamine (Temed).
- PhotoFlo.
- HCl.
- TRIS hydroxy mthyl amino mthane.
- Saccharose.
- Glycine.
- Bleu de Bromophnol.
- Acide Ascorbique.
- Cystine Hydrochlorure.
- NaH2PO4 et Na2HPO4.
- Naphtyl Actate.
- Fast Blue.
- Acide Actique.
- Acide Ktoglutarique.
- Acide L Aspartique.
- PVP (Poly Vinyl Pyrrolidone).
- EDTA (Ethylne Diamine Ttra Actique Acide).
- Carbonate de sodium.
- Acide L Malique.
- NAD ( Nicotinamide Adnine Dinuclotide).
- NBT (Nitro Blue Ttrazolium).
- PMS (Phnazine Mtho Sulfate).
- Ethanol.
- Glycrine.
- Riboflavine.
I- B. Matriels
Matriel de dissection / Seringue Hamilton (25L ou 50 L)/ Piston / Tubes micro hmatocrites (sans
anticoagulant)
Systme complet dlectrophorse sur gel vertical
- Cuve et gnrateur
- Systmes de fixation = support de plaques et support de cuve
- Plaques verre petit et grand modles
- Espaceurs de 0.5 mm dpaisseur et peignes
Ph-mtre calibr et control (cf Procdure NEM / MAT PS MAT pHm)
Agitateurs magntiques
Balances contrles
Etuve 37C.
Lampe halogne 60W
Systme complet de schage des gels (cadres, pinces, feuilles cellophane )
Centrifugeuse ventile avec adaptateurs pour tubes microhmatocrites
Matriel biologique
II. Matriel biologique et prparation des chantillons pour lectrophorse
Ltude porte sur le stade femelle de nmatodes du genre Meloidogyne.
- Dissquer la racine infeste sous loupe binoculaire et extraire la femelle.
Page 305
Placer la femelle dans un tube micro hmatocrite contenant du tampon Trugdill1.(tubes remplis laide
dune seringue Hamilton) contenant 20% de sucre.
- Broyer les femelles laide dun piston.
- Identifier les tubes
Rem : Lchantillon peut tre utilis directement ou bien conserv au conglateur -20C
Tampon Trugdill :
1/ Fabrication Solution mre :
- TRIS
1.314 g
- A.Ascorbique
0.018 g
- Cystine Hydrochlorure 0.014 g
- Eau distille
40 mL
Ajuster pH 8 avec solutions HCl / NaOH 1 mol/L
QSP 100 mL Eau distille
Aliquoter la solution mre et conserver < - 20C.
2/ Ajout sucrose pour obtenir une solution finale 20% de sucrose (poids/volume):
Rem : Aprs dconglation et ajout du sucrose, le tampon Trugdill doit tre conserv
2-8 C pendant 1 semaine maximum.
-
III- Protocole
III-1 Sparation par lectrophorse en conditions natives (Vertical PAGE)
III-1- A. Montage plaques sur support de cuve :
- Au pralable laver les plaques dans une solution de PhotoFlo et scher avec papier absorbant.
Rem : Le PhotoFlo est un agent mouillant, facilitant louverture des plaques lors du dmoulage du gel (vite
dchirure).
Attention de ne pas laisser de dpt de papier sur les plaques ou des marques de doigts.
Solution PhotoFlo :
- 750 L PhotoFlo.
- 100 mL Eau distille.
- Appliquer les deux plaques de verre l'une contre l'autre, avec les espaceurs.
- Monter les plaques sur le support de plaques.
Rem : Vrifier le niveau du plan de travail lors du coulage des gels afin dassurer lhorizontalit de la limite entre
le gel de sparation et de tassement.
III-1- B. Prparation des gels :
* Gel stop
2 Gels
Solution PrSO4 50 mg/mL
60 L
Bis/Acrylamide
1500 L
Temed
30 L
Rem : Ajouter imprativement les ractifs dans cet ordre.
Polymrisation rapide du gel.
* Gel de sparation
Solution A : - HCL 1 N
- TRIS
- Temed
20
mL
12 g
0.23 mL
Ajuster pH 8.4 ou 8.9 (suivant systmes enzymatiques tudis) avec solutions HCl / NaOH 1 mol/L. QSP 100
mL Eau distille.
Prparer pour 2 Gels :
- Eau distille
1.92 mL
- Solution PrSO4 1.4 mg /mL
4.8 mL
- Bis/Acrylamide
1.68 mL
- Solution A
1.2 mL
Rem : Ajouter imprativement les ractifs dans cet ordre.
Bien homogniser la solution finale sans faire de bulles.
1
Trudgill, D.L. & Carpenter, J.M. 1971. Disc electrophoresis of proteins from Heterodera species and pathotypes of Heterodera rostochiensis.
Ann. Appl. Biol., 69 : 35-41.
Page 306
Aprs avoir coul le gel, ajouter de leau sa surface pour viter les ridules de surface lors de la
polymrisation.
Temps de polymrisation : 1 H
*Gel de tassement
Solution de SPA :
- HCL 1 N
40 mL
- Eau distille 20 mL
- TRIS
5.98 g
- Temed
0.46 mL
Ajuster pH 6.7 HCl / NaOH 1 mol/L. QSP 100 mL Eau distille.
Prparer pour 2 Gels : - Solution Saccharose 400 mg/mL
2.65 mL
- Solution PrSO4
5.6 mg/mL
0.5 mL
- Bis/Acrylamide
0.35 mL
- Solution SPA
0.5 mL
Rem : Avant de couler le gel, ter leau la surface du gel de sparation avec du papier absorbant.
Couler le gel et placer les peignes (en vitant la formation de bulles sous et entre les dents).
Temps de polymrisation : 20 min
* Fabrication tampons complmentaires
Tampon de Cuve :
Solution mre (conserver 2-8C) diluer au 1/10 avant utilisation et changer rgulirement.
Solution Mre : - TRIS
6g
- Glycine 28.8 g
QSP 1L Eau distille.
Marqueur de front de Migration :
Dissoudre quelques g de Bleu de Bromophnol dans du tampon de cuve et conserver 2-8C afin de conserver
lactivit enzymatique des protines en cours dtude.
III 2 Migration
La migration doit avoir lieu 2-8C afin de prserver lactivit enzymatique des protines en cours dtude. La
diffrence de potentiel permet la migration des protines charges ngativement vers llectrode positive situe dans
la cuve infrieure.
Le pH du gel de sparation varie avec le systme enzymatique tudi.
- Centrifuger chantillons biologiques congels 9500 rpm (10 000 g) 10 min.
- Clipper le support de plaque avec les gels sur le support de cuve.
Rem : Vrifier le joint du support de cuve pour viter toute fuite lors du remplissage avec le tampon de cuve.
-
Placer le tout dans la cuve et remplir doucement de tampon de cuve (le tampon doit toujours couvrir les
puits afin de toujours assurer le contact avec llectrode suprieure) ainsi que le fond de la cuve (sans faire
de bulle sous le gel afin de toujours assurer le contact avec llectrode infrieure).
Collecter les chantillons avec seringue Hamilton (rincer entre chaque prlvement avec tampon de cuve)
et charger les gels.
Rem : Penser distinguer les 2 gels et reprer le sens de chargement.

-
Ajouter quelques gouttes de marqueur du front de migration (Bleu de bromophnol)

Connecter la cuve au gnrateur 40 V - 15 min puis 160 V 1h 30 min.
Rem : Arrter la migration lorsque le front de migration (ligne violette de bleu de bromophnol) atteint 5 cm dans le
gel de sparation. La distance parcourue dans le gel de tassement nest pas prise en compte.
III - 3 Coloration spcifique du systme enzymatique cibl
Rvlation des enzymes
- Vider le tampon de la cuve.
- Retirer les plaques de la cuve.
- Dcoller les 2 plaques.
- Dcouper toujours le mme (ex : langle infrieur gauche) des gels comme repre.
- Retirer le gel de sparation (enlever le gel de tassement et dcouper le gel de sparation au niveau de la
ligne du front de migration = ligne violette).
- Placer le gel dans la cuve contenant la solution de coloration .
- Placer ltuve.
Page 307
Rem : La composition de la solution de coloration, le temps et le mode dincubation varient suivant le systme
enzymatique rvl.
- Systme enzymatique ESTERASE (EST)1
* Condition dlectrophorse :
Gel de SEPARATION pH 8.4
* Condition de coloration des gels :
Tampon Phosphate :
1- Solution Stock A NaH2PO4 Solution 0.2 M soit m = 27.8 g, QSP 1L eau distille
2- Solution Stock B Na2HPO4 Solution 0.2 M *
* Attention : Suivant le flacon utilis, les produits sont disponibles sous diffrents taux dhydratation (TH) et sont
donc de poids molculaires (PM) diffrents.
Si lindication TH = 7 H2O et le PM de 268.1 alors la quantit peser = 53.65 g (formulations utilises au
laboratoire).
Si lindication TH = 2 H2O et le PM de 182.5 alors la quantit peser = 35.6 g.
Si lindication TH = 12 H2O et le PM de 358.5 alors la quantit peser = 71.7 g.
3- Tampon utilise (pH = 7,2) obtenu en mlangeant =
Solution Stock A 56 mL
Solution Stock B 144 mL
Solution coloration prparer pour 2 gels
- Tampon PHOSPHATE 50 mL
- Naphtyl Actate
40 mg (dissout dans 1 mL Actone).
- Fast Blue
30 mg
Rem : Prparation faire au dernier moment car Fast Blue photosensible.
- Dissoudre correctement le Fast Blue (pour viter tout sous dosage de ce ractif) dans la solution et filtrer
avant de mettre en contact avec le gel.
- Placer ltuve ( lobscurit) 37C 1 H.
- Rincer leau distille et conserver (temporairement avant schage) dans Acide Actique 10% (pour
fixer la coloration).
- Systme enzymatique Glutamate Oxaloacetate transaminase( GOT)2
Solution coloratio (pour 2 gels)
- Acide Ktoglutarique
0.037g
- Acide L Aspartique
0.133g
- PVP
0.5g
- EDTA
0.05g
- Na2HPO4
1.42 g
- Fast Blue
0.1 g
QSP 100 ml Eau distille.
- Placer ltuve 37C 1 H
- Rincer leau distille et conserver (temporairement) dans Acide Actique 10 % (pour fixer la coloration).
- Systme enzymatique Malate dshydrognase (MDH)1
Solution 1 : - Carbonate de Sodium 10.6 g
- Acide L Malique
1.34 g
Solution 2 : - TRIS 6.06 g
Ajuster pH 7.1 avec solutions HCl / NaOH 1 mol/L
Solution coloration finale (pour 4 gels)
1
Harris, H. & Hopkinson, D.A. 1976. Handbook of enzyme electrophoresis in human genetics. North-Holland Publ. Co., NewYork, 356pp.
Page 308
- Prparation n1
Solution 1 10 mL
Solution 2 15 mL
NAD
0.05 g
NBT
0.03 g
- Prparation n2
PMS 0.002g
Mlanger le contenu des 2 prparations au dernier moment.
Placer ltuve 37C 1 h.
Rincer leau distille et conserver (temporairement) dans Acide Actique 10 % (pour fixer la coloration).
- Systme enzymatique Superoxyde dismutase (SOD)1
Solution Tris stock TRIS 0.61 g
Ajuster pH 8.2 avec solutions HCl / NaOH 1 mol/L
QSP 100 mL eau distille
Solution coloration finale ( pour 4 gels)
- EDTA
0.0075 g
- Riboflavine 0.004 g
- NBT
0.01 g
- Solution TRIS 100 mL
Placer ltuve 37C 10 min (maximum) puis 10 min (minimum) la lumire vive (lampe).
Rincer leau distille et conserver (temporairement) dans Acide Actique 10 % (pour fixer la coloration).
A- Schage des gels ( 6 gels par plaques)
Solution de schage : - Ethanol 50 % 100 mL
- Glycrine
6g
- Faire tremper les 2 feuilles dans la solution (1 min).
- Dposer la 1ere feuille sur le fond du cadre sans faire de bulles.
- Faire tremper les gels dans solution ( 5min) puis les dposer sur la feuille sans faire de bulles.
- Recouvrir avec la seconde feuille.
- Fermer le systme avec le cadre vid et les 6 pinces.
- Laisser scher 48h
Rem : Acclration du schage sous hotte aspirante.
Page 309
Annexe 6: Procdure extraction ADN Kit QIAgen DNeasy Blood & Tissue
Rsum :
Le prsent document dcrit le mode dextraction dADN partir de nmatodes du genre Meloidogyne au stade J2.
I. Produits et matriels ncessaires
A. Produits
- Kit dextraction Qiagen DNeasy Blood & Tissue
- Protinase K.
- Tampon AL.
- Tampon AW1.
- Tampon AW2.
Rem: Les tampons AW1 et AW2 sont repris avant utilisation dans de lthanol 100% (cf. tiquettes flacons).
- PBS pH 7.4.
- Ethanol 100%.
- Eau pure chauffe 70C.
Fabrication du PBS:
- NaCl (137 mM) 8g.
- KCl (2.7mM) 0.2g.
- Na2HPO4 (10 mM) 1.44g.
- KH2PO4 (1.8 mM) 0.24g.
- Ajouter 800 ml deau distille.
- Mlanger (sur agitateur magntique) pendant 20 min.
- Faire le pH 7.4.
- QSP 1L deau.
B. Matriels
- Bain Marie.
- Plaque chauffante.
- Centrifugeuse (jusqu 13000rpm) et centrifugeuse de paillasse.
- Vortex.
- pHmtre talonn.
- Pipette P200, P1000 et cnes striles filtre.
- Tubes eppendorf 1.5 mL.
- Portoirs.
- Colonnes dextraction et tubes de collecte 2 mL Qiagen.
- Agitateur magntique et barreau aimant.
- Balance, spatules de pese et papier aluminium.
II. Protocole
- Placer 10 nmatodes J2 (minimum) au fond dun tube eppendorf de 1,5 ml dans une goutte deau distille
(environ 6l).
Rem : Pour le moment cette technique na pas t teste sur un seul nmatode au stade J2.
- Centrifuger (pulse) et vrifier que les nmatodes soient bien au fond du tube.
- Centrifuger 3 min 13000 rpm pour culoter les nmatodes.
- Ajouter 90 L de PBS.
- Ajouter 10 L de Protinase K.
- Ajouter 100 L de tampon AL.
- Vortexer pour mlanger.
Rem : Tenir le tube bien verticalement
- Centrifuger 5 min 13000 rpm pour sassurer que les nmatodes soient bien immergs dans les tampons,
puis homogniser de nouveau en tapotant sur le tube.
- Incuber 4 H 56C au bain marie
- Ajouter 100 L dthanol 100% et homogniser en vortexant
- Centrifuger 1 min- 8000 rpm.
Rem : Un prcipit blanc peut apparaitre lors de lajout de lalcool mais il ninterfre en rien sur la manipulation
dextraction.
- Dposer le contenu du tube (y compris le prcipit) sur une colonne/ tube de collecte.
Rem : Annoter le capuchon de la colonne au marqueur.
- Centrifuger 1 min 8000 rpm.
- Jeter lluat et le tube de collecte en sortie de colonne.
Rem : Tous les dchets doivent tre collects dans une poubelle spciale et non vids dans lvier.
Page 310
- Placer la colonne dans un nouveau tube de collecte de 2 mL.

- Ajouter 250 L de tampon AW1.
- Ajouter 250 L de tampon AW2.
- Centrifuger 3 min 13000 rpm pour scher la membrane.
Rem : Il est trs important dliminer toutes traces dalcool dans la colonne. Pour cela aspirer laide dune
pipette (P200) lalcool rsiduel prsent sur la collerette de la colonne.
- Placer la colonne sur un tube eppendorf de 1.5 mL, ajouter 50 L deau pure chauffe pralablement
70C.
- Laisser en contact pendant 3 min sur la paillasse TA.
- Centrifuger 1 min - 8000 rpm.
- Rcuprer lluat dans le tube eppendorf et le refaire passer dans la mme colonne.
Rem : Pour rcuprer tout lADN extrait.
- Centrifuger 1 min - 8000 rpm
- Aliquoter les 50 L dans de tubes eppendorf tiquets et les stocker au conglateur -20C.
Page 311
Annexe 7 : Procdure damplification ADN mthode SCARS

Rsum :
Le prsent document dcrit un mode damplification de lADN (technique des SCARS) partir de nmatodes du
genre Meloidogyne au stade J2.
A. Produits
- Tampon QIAGEN Multiplex (fournisseur Qiagen)
- Primers :
M. arenaria:
OPA-12 Fare 5'- TCG GCG ATA GAG GTA AAT GAC -3'
OPA-12 Rare 5'- TCG GCG ATA GAC ACT ACA ACT -3'
M. javanica
OPA-01 Fjav 5'- GGT GCG CGA TTG AAC TGA GC -3'
OPA-01 Rjav 5'- CAG GCC CTT CAG TGG AAC TAT AC -3'
M. incognita
OPB-06 Finc 5'- CTC TGC CCA ATG AGC TGT CC -3'
OPB-06 Rinc 5'- CTC TGC CCT CAC ATT AAG -3'
M. chitwoodi
OPI-09 Fc2 5'- GGC ATT GAC GTG CTC CGA GAG T -3'
OPI-09 Rc 5'- GGT CTG AGT GAG GAC AAG AGT A -3'
M. fallax
OPI-01 Ff2 5'- CCA TTT CTG CTA AAT GCC AAA CTA -3'
OPI-01 Rf 5'- GGA CAC AGT AAT TCA TGA GCT AG -3'
M. hapla
OPI-14 Fh 5'- TGA CGG CGG TGA GTG CGA -3'
OPI-14 Rh 5'- TGA CGG CGG TAC CTC ATA G -3'
- Eau pure (milliQ) et strile (passer au UV 10 min).
- ADN extrait et si possible dos (quantit estime sur Nanodrop).
B. Matriels
- Tubes eppendorf 1.5 mL
- Pipettes de prcision et cnes
- Centrifugeuse de paillasse
- Plaque PCR
- Bac avec de la glace
II. Protocole
Carolien ZIJLSTRA et al. Identification of Meloidogyne incognita, M. javanica and M. arenaria using sequence
characterised amplied region (SCAR) based PCR assays.Nematology, 2000, Vol. 2: 847-853.
Nous nous sommes bass sur larticle ci-dessus et nous avons ralis des mises au point et amliorations au niveau
des ractifs utiliss pour la ralisation du mix PCR (condition multiplexage) ainsi quau niveau des conditions de
run de la PCR.
- Evaluer la quantit dADN extraite sur NanoDrop ou estimer celle-ci.
Rem : 1 juvnile de Meloidogyne = 40 60 ng
Quantit maximale dADN amplifiable lors dune PCR 100 ng.
Si la concentration excde les 100 ng, diluer lADN avec de leau pure et strile avant de raliser lamplification.
- Raliser le mix PCR :
Ractifs :
Concentration initiale
Concentration finale
Volume ractif pour 1 test
-Tampon QUIAGEN
2X
1X
5L
(Multiplex)
- Amorces*
100M
0.2M
1L
- Eau
2 L
- ADN
2 L
Volume total ractionnel par puits = 10 L (8 L mix + 2 L ADN)
Rem : Pour obtenir la bonne molarit (2 M), diluer les amorces au 1/50 avant utilisation dans le mix PCR.
Page 312
Ex1 : Raction avec 1 couple damorces

2 L damorces1 Forward
2 L damorces1 Reverse
QSP 100 L deau pure et strile
Ex2 : Raction avec 3 couples damorces
Multiplexage possible avec les amorces suivantes :
Triplette 1 :
M. arenaria
OPA-12 Fare 5'- TCG GCG ATA GAG GTA AAT GAC -3'
OPA-12 Rare 5'- TCG GCG ATA GAC ACT ACA ACT -3'
M. javanica
OPA-01 Fjav 5'- GGT GCG CGA TTG AAC TGA GC -3'
OPA-01 Rjav 5'- CAG GCC CTT CAG TGG AAC TAT AC -3'
M. incognita
OPB-06 Finc 5'- CTC TGC CCA ATG AGC TGT CC -3'
OPB-06 Rinc 5'- CTC TGC CCT CAC ATT AAG -3'
Triplette 2 :
M. chitwoodi
OPI-09 Fc2 5'- GGC ATT GAC GTG CTC CGA GAG T -3'
OPI-09 Rc 5'- GGT CTG AGT GAG GAC AAG AGT A -3'
M. fallax
OPI-01 Ff2 5'- CCA TTT CTG CTA AAT GCC AAA CTA -3'
OPI-01 Rf 5'- GGA CAC AGT AAT TCA TGA GCT AG -3'
M. hapla
OPI-14 Fh 5'- TGA CGG CGG TGA GTG CGA -3'
OPI-14 Rh 5'- TGA CGG CGG TAC CTC ATA G -3'
- Distribuer dans chacun des puits de la plaque PCR, 2 L dADN et 8 L de mix
- Fermer la plaque avec film thermosoudable
- Centrifuger la plaque (pulse)
- Placer la plaque dans le thermocycleur et lancer le programme adquat.
Conditions PCR requises :
Dnaturation initiale 95C-15min
Nombre de cycle 40 cycles
Dnaturation 94C-30s
Hybridation 58C-1,30min
Elongation 72C-1,30min
Elongation finale 72C-10min
Page 313
Annexe 8 : Procdure damplification ADN Gne 28S portion D2D3

Rsum :
Le prsent document dcrit un mode damplification de lADN ribosomique du gne 28S (portion D2D3 700pb)
partir de nmatodes du genre Meloidogyne au stade J2.
A. Produits
- Tampon 10X DreamTaq green buffer (Thermoscientific)
- Primers (Eurogentec):
D2A_forward 5- ACA AGT ACC GTG AGG GAA AGT TG -3
D3B_reverse 5-TCG GAA GGA ACC AGC TAC TA-3
- MgCl2 (Qiagen) 25 mM.
- dNTP (Qiagen) 2.5 mM
- Dream Taq (Thermoscientific) 5U
- Eau pure (milliQ) et strile (passer au UV 10 min)
- ADN extrait et si possible dos (quantit estime sur Nanodrop)
B. Matriels
- Vortex plaque
- Plaque PCR et film thermosoudable ou autocollant
II. Protocole
A new root knot nematode, Meloidogyne baetica n. sp. (Nematoda: Heteroderiadae) paraziting wild olive in
southern spain. Castillo & al 2003. Phytopathology vol 93 : 1093- 1102.
des ractifs utiliss pour la ralisation du mix PCR ainsi quau niveau des conditions de run de la PCR.
Ractifs :
- Tampon 10X
- MgCl2 supplmentaire
- dNTP
-Amorces Forward
- Amorces Reverse
- Dream Taq
- Eau pure
- ADN
10X
25mM
2.5mM
100M
100M
5U
1X
1.5mM
0.2mM
0.3M
0.3M
1U
2.5 L
1.5 L
2 L
0.075 L
0.075 L
0.2 L
16.65 L
2 L

- Distribuer dans chacun des puits de la plaque PCR, 2 L dADN et 8 L de mix.
- Fermer la plaque avec film thermosoudable ou autocollant.
- Homogniser (pour bien mlanger les ractifs) et Centrifuger la plaque (pulse).
- Placer la plaque dans le thermocycleur et lancer le programme adquat.
Dnaturation initiale 94C 3 min
Nombre de cycle 40 cycles puis
Dnaturation 92C 30 s
Hybridation 55C 1 min
Elongation 72C - 1,30 min
Elongation finale 72C - 10min
Page 314
Annexe 9 : Les amorces et les conditions damplifications utilises pour les tests des microsatellites raliss au cours de ltude
Page 315
Annexe 10 : Procdure damplification ADN ribosomique Gne Internal Transcriber Spacer 2 (ITS2)
Rsum :
Le prsent document dcrit un mode damplification de lADN ribosomique du gne Internal Transcriber Spacer 2
(ITS2) partir de nmatodes du genre Meloidogyne au stade J2.
I.
A.
Produits et matriels ncessaires

Produits
- Tampon 10X DreamTaq green buffer (Thermoscientific)
- Primers (Eurogentec):
CysFwd1 5'- GAT CGA TGA AGA ACG CAG C -3'
CysNRvs1 5'- TCC TCC GCT AAA TGA TAT G -3'
- MgCl2 (Qiagen) 25 mM
- dNTP (Qiagen) 2.5 mM
- Dream Taq (Thermoscientific) 5U
- Solution Q (Qiagen) 5X
B.
Matriels
- Vortex plaque
II. Protocole
Clapp et al 2000. Rapid identification of cyst and root knot nematodes on the basis of ITS2 sequence variation
detected by PCR single strand conformational polymorphism in cultures and fields samples. Molecular Ecology, 9:
1223-1232.
des ractifs utiliss pour la ralisation du mix PCR ainsi quau niveau des conditions de run de la PCR.
Rem : 1 juvnile de Meloidogyne = 40 60 ng.
Ractifs :
- Tampon 10X
- MgCl2 supplmentaire
- dNTP
-Amorces Forward
- Amorces Reverse
- Dream Taq
- Solution Q
- Eau pure
- ADN
10X
25mM
2.5mM
100M
100M
5U
5X
1X
1.5mM
0.2mM
0.3M
0.3M
1U
0.25X
2 L
1.2 L
0.4 L
0.14 L
0.14 L
0.125 L
1 L
10.995 L
4 L

- Fermer la plaque avec film thermosoudable ou autocollant
- Homogniser (pour bien mlanger les ractifs) et Centrifuger la plaque (pulse)
- Placer la plaque dans le thermocycleur et lancer le programme adquat
Hybridation 50C 1 min
Elongation finale 72C - 10min
Page 316
Annexe 11 : Procdure damplification ADN mitochondrial Gne Cytochrome Oxydase 1

Rsum :
Le prsent document dcrit un mode damplification de lADN mitochondrial gne Cytochrome Oxydase 1 partir
de nmatodes du genre Meloidogyne au stade J2.
I.
A.

Produits
- Tampon QIAGEN Multiplex (fournisseur Qiagen).
- Primers :
COX1_Forward1 5'- TTG RTT TTT TGG TCA TCC TGA RG -3'
COX1_reverse1 5'- WSY MAC WAC ATA ATA AGT ATC ATG -3'

- ADN extrait et si possible dos (quantit estime sur Nanodrop).
B. Matriels
-
Tubes eppendorf 1.5 mL.

Pipettes de prcision et cnes.
Centrifugeuse de paillasse.
Vortex pour plaque.
Plaque PCR et film thermosoudable ou autocollant.
Bac avec de la glace.
II. Protocole
Blouin et al. 2002. Molecular prospecting for cryptic species of nematodes: Mitochondrial DNA versus internatl
transcribed spacer. International Journal for parasitology.
Lazarova, S. S., et al. 2006. Ribosomal and mitochondrial DNA analyses of Xiphinema americanum-group
populations. Journal of Nematology, 38, 404410.
Nous nous sommes bass sur les articles ci-dessus et nous avons ralis des mises au point et amliorations au
niveau des ractifs utiliss pour la ralisation du mix PCR (condition multiplexage) ainsi quau niveau des
conditions de run de la PCR.
Ractifs :
- Tampon QUIAGEN
2X
1X
12.5 L
(Multiplex)
- Solt Amorces 1/50
100M
2M
5 L
- Eau
7.5 L
- ADN
4 L
Ex : Prparation solution damorces Forward et Reverse au 1/50
Hybridation 51C - 1,30 min
Elongation 72C 30 s
Elongation finale 72C 10 min
Page 317
Annexe 12 : Procdure damplification ADN mitochondrial Gne 63R

Rsum :
Le prsent document dcrit un mode damplification de lADN mitochondrial gne 63R (appellation 63 R sur
gnome Meloidogyne javanica) partir de nmatodes du genre Meloidogyne au stade J2.
A. Produits
- Tampon QIAGEN Multiplex (fournisseur Qiagen)
- Primers :
63R_ forward1 5'- TTG AAA TTG CTT TAT TGT TAC TAA GAA G -3'
63R_ formard2 5'- GAA TTC CAT AAA TCA GAA AAT TGA GG -3'
63R_reverse 5'- ACC TTA GAA ATA TGA AWC CTA WAG AC -3'
B. Matriels
- Vortex pour plaque
II. Protocole
Besnard, G.et al 2014. Fast assembly of the mitochondrial genome of a plant parasitic nematode (meloidogyne
graminicola) unsing next genaration sequencing. C. R. Biologie Vol. 337, 295-301.
des ractifs utiliss pour la ralisation du mix PCR (condition multiplexage) ainsi quau niveau des conditions de
run de la PCR. Et nous avons redessin des amorces spcifiques Meloidogyne javanica.
Ractifs :
- Tampon QIAGEN
2X
1X
10 L
- Solt Amorces 1/50
100M
2M
2 L
- Eau
4 L
- ADN
4 L
Ex : Prparation solution damorces Forward et Reverse au 1/50
Hybridation 55C - 1,30 min
Elongation finale 72C 10 min
Page 318
Rsum
Les interventions humaines, de plus en plus frquentes et persistantes dans les cosystmes, dune part, et
lintensification des systmes de cultures dautre part saccompagnent pour partie de mthodes radicales pour combattre
les bio-agresseurs des cultures. Elles nous posent de multiples questions au sujet des risques cologiques lis aux
changements des milieux, dont les perturbations induites sur les communauts dorganismes vivants. Les nmatodes
phytoparasites (NPP), vers ronds microscopiques telluriques, qui occasionnent des pertes de production vgtale
importantes, sont partout prsents en communauts. Ces nmatodes rpondent rapidement aux forages extrieurs (e.g.
anthropiques et environnementaux) par des modifications de la structure de leurs communauts. Par ce travail de thse,
nous cherchons mieux comprendre leffet des facteurs impliqus dans lassemblage des espces de NPP en
communauts associes lolivier mditerranen et dterminer la rponse de ces communauts aux forages imposs
par la domestication de lolivier, par lintensification de sa culture et par diffrents facteurs environnementaux. Ltude
a t ralise au Maroc dans toutes les rgions olicoles (vergers traditionnels faible densit et vergers hautedensit), dans des zones refuge dolivier sauvage (olastre) et sur olivier fral. Les facteurs pdoclimatiques qui
caractrisent les sites dchantillonnage ont galement t pris en considration. Lanalyse de la nmatofaune a rvl
dune grande diversit spcifique, de nombreuses espces tant dcrites pour la premire fois sur olivier, et une nouvelle
espce (Meloidogyne spartelensis) ayant t dcouverte. La diversit, la composition taxonomique, trophique et
fonctionnelle, la dominance des taxons, les patrons de communauts sont fortement affects par les diffrents forages
pris en compte. Le gradient danthropisation croissante (sauvage vs fral vs cultiv traditionnel vs cultiv haute-densit)
est la variable qui impacte le plus la diversit par rduction de la richesse spcifique et laugmentation de labondance
en NPP. Ltude a galement port une attention particulire sur la diversit des nmatodes galles des racines du genre
Meloidogyne, un des principaux ravageurs de lolivier. Elle a indiqu la dispersion de M. javanica dans les vergers et
sur olivier fral, alors que dautres espces (M. arenaria, M. hapla et M. spartelensis) sont confines dans les zones
refuge des olastres. Afin danalyser la diversit gntique, des marqueurs morphologiques et molculaires ont dvoil
dune diversit importante entre et au sein des diffrentes populations de Meloidogyne. Les tudes diligentes dans le
cadre de cette thse confirment que la diversit et la structure des communauts de NPP pourraient tre des indicateurs
pertinents pour valuer la sant des sols dans les agro et cosystmes, en corrlant diversit et pathognicit des
communauts. Elles soulignent donc limportance de la diversit parasitaire comme variable prioritaire prendre en
compte pour inspirer des stratgies de gestion des parasites bases sur le concept de rsilience de la diversit (mme sil
sagit de parasites), pour une gestion durable des communauts de NPP et la prservation des milieux.
Mots-cls : anthropisation, biodiversit, communauts, diversit gntique, Meloidogyne, nmatodes phytoparasites,
olivier.
Abstract
Human activities increasingly frequent and persistent in ecosystems on one hand, and cropping system intensification
on the other hand partly with radical methods to control pests and diseases of crops, raise questions about the ecological
risks related to biotop changes, including disturbances of living organism communities. Plant-parasitic nematodes
(PPN), microscopic round soil-borne worms that cause significant losses of crop production, are everywhere present in
communities. They rapidly respond to external forces (e.g. anthropogenic and environmental) by changing the structure
of their communities. The aim of this thesis was to better understand the effect of the factors involved in the assemblage
of PPN species in communities associated with the Mediterranean olive tree and to determine the response of these
communities to forces imposed by olive domestication, by crop intensification and by various environmental factors.
The study was conducted in all olive-growing regions in Morocco (traditional low density and high density orchards), in
refugia areas for wild olive (oleaster) and on feral olive trees. The soil and climatic factors that characterize the
sampling sites were also considered. The analysis of the nematofauna revealed a wide species diversity, many species
being described for the first time on olive tree, and a new species (Meloidogyne spartelensis) have been discovered. The
diversity, the species / trophic / functional structure of the communities, the dominance of the taxa, and the community
patterns are strongly affected by the various forces considered. The increasing anthropogenic gradient (wild vs feral vs
traditional cultivation vs high-density cultivation) is the variable that mostly impacts the diversity by reducing the
species richness and increasing the abundance of PPN. A particular attention was attributed in this study to the diversity
of root-knot nematodes Meloidogyne, a major pest of the olive tree. It revealed that M. javanica is widespread in
orchards and olive feral, while other species (M. arenaria, M. hapla and M. spartelensis) are confined in oleaster refuge
areas. In order to analyse the genetic diversity, morphological and molecular markers have revealed a significant
diversity between and within different Meloidogyne populations. These thesis studies confirm that both the diversity
and the structure of the PPN communities could be relevant indicators to assess soil health in agro and ecosystems, by
correlating diversity and pathogenicity of communities. They therefore emphasize the importance of parasite diversity
as a main variable to consider for inspiring pest control strategies based on the concept of diversity resilience (even if it
concerns parasites) for sustainable management of PPN communities and for environment preservation.
Keywords: anthropisation, biodiversity, communities, genetic diversity, Meloidogyne, olive tree, plant-parasitic
nematodes.
Nadine Ali
UMR CBGP Centre de Biologie pour la Gestion des Populations, Campus International de Baillarguet, CS30016, 34988
Montferrier sur Lez, France.
Tishreen University, Faculty of Agriculture, Plant Protection Department, Latakia, Syrian Arab Republic.
[email protected]

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Communauts de nmatodes phytoparasites

Chapitre I : Introduction gnrale

Chapitre II : Les nmatodes phytoparasites associs lolivier au niveau mondial

Chapitre III: Mthodologie gnrale (a)

Chapitre IV: Diversit taxonomique des nematodes en communauts

Chapitre V: Diversit fonctionnelle

2. Materials and Methods.. 129

Chapitre VI: Patrons des communauts de nematodes phytoparasites

Chapitre VII. Mthodologie gnrale (b)

1. Caractrisation spcifique des nmatodes du genre Meloidogyne. 176

Chapitre VIII: Description dune nouvelle espce de Meloidogyne : M. spartelensis n.sp.

Chapitre IX: Diversit des Meloidogyne associs lolivier au Maroc

Chapitre X: Variabilit gntique chez Meloidogyne spp. associ lolivier

B. Etude de la diversit molculaire des populations de Meloidogyne spp.

Chapitre XI: Synthse et discussion gnrale

1. Rappel de la problmatique et des objectifs.... 274

Figure 5: PPN diversity indices according to environmental variables studied.. 115

measurements of juveniles.. 249

la biodiversit, les enjeux qui lui sont associs, ses organisations,

le sol, trsor de diversit biologique,

les nmatodes, invertbrs principaux du sol,

Les nmatodes phytoparasites et lolivier : modles biologiques de ltude.

des communauts, ainsi qu valuer

limpact de la domestication, de lanthropisation des milieux, des pratiques culturales, et des

conditions environnementales sur la diversit des nmatodes en communauts. Le deuxime

1. La biodiversit : notion retenir

Figure 1: Les diffrents niveaux d'organisation de la biodiversit

Elle se dcrit laide de plusieurs indicateurs: le nombre despces (richesse), l'abondance, la

Le mode de reproduction est galement un des facteurs de structuration de la

Encadr 3 : Dfinition dune crise dextinction

Figure 2 : Effets des changements globaux et rponses potentielles de lorganisme pour y

Encadr 4 : Quelques dfinitions

2. La communaut, lieu dinteractions multiples

3. Le sol : trsor de biodiversit

1994 ; Figure 3).

Ltat de la faune du sol permet donc de mettre en vidence des conditions

certains comme les Longidoridae

4.2. Les nmatodes phytoparasites : un enjeu pour l'agriculture

Figure 4 : Nmatode phytoparasite du genre Pratylenchus spp.

nmatodes lsion Pratylenchus) (Tableau 1).

Tableau 1 : Exemples de dure du cycle de vie, de fcondit, de modes de reproduction

Tolrance aux perturbations

Dominance dans les chantillons

beaucoup de petits ufs

peu de gros ufs

Figure 6 : Correspondance entre le patron statistique des communauts de nmatodes et la

4.4.1. Cycle biologique standard des nmatodes du genre Meloidogyne

Figure 8 : Cycle biologique de Meloidogyne spp. (cas dune parthnogense mitotique). a :

4.4.2. Importance agro-conomique

appartenant la mme espce ; ainsi, au sein de lespce M. arenaria, la plupart des

Figure 9: Modes de reproduction des espces de Meloidogyne spp.

Considrant limportance agronomique et conomique de ces parasites, connaitre cette

molculaires spcifiques (Esbenshade & Triantaphyllou, 1985 ; 1990). La migration

2012). Des marqueurs molculaires de type SCARS (Sequence Characterized Amplified

5. Lolivier : modle cologique dtude de la structuration des communauts de

subsp. europaea present in the Mediterranean Basin, with