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Characterization of eld isolates of Trichoderma antagonistic against Rhizoctonia solani

ronique EDEL-HERMANNc, Muhammad ANEESa, Arne TRONSMOb, Ve cile HERAUDc, Christian STEINBERGc,* Linda Gordon HJELJORDb, Ce
a

Kohat University of Science and Technology, Kohat, Pakistan Department of Chemistry, Biotechnology and Food Science, PO Box 5003, NO-1432 Aas, Norway c INRA, Universite de Bourgogne, UMR 1229 Microbiologie du Sol et de lEnvironnement, CMSE, 17 rue Sully, BP 86510, 21065 Dijon, France
b

article info
Article history: Received 21 August 2009 Received in revised form 27 May 2010 Accepted 28 May 2010 Available online 9 June 2010 Corresponding Editor: Nicholas P. Money Keywords: Biocontrol Coiling Fungal interactions Trichoderma gamsii Trichoderma velutinum Volatile inhibitors Water-soluble inhibitors

abstract
The aim of the present study was to characterize sixteen isolates of Trichoderma originating from a eld of sugar beet where disease patches caused by Rhizoctonia solani were observed. Use of both molecular and morphological characteristics gave consistent identication of the isolates. Production of water-soluble and volatile inhibitors, mycoparasitism and induced systemic resistance in plant host were investigated using in vitro and in vivo tests in both sterilized and natural soils. This functional approach revealed the intra-specic diversity as well as biocontrol potential of the different isolates. Different antagonistic mechanisms were evident for different strains. The most antagonistic strain, T30 was identied as Trichoderma gamsii. This is the rst report of an efcient antagonistic strain of T. gamsii being able to reduce the disease in different conditions. The ability to produce water-soluble inhibitors or coil around the hyphae of the pathogen in vitro was not related to the disease reduction in vivo. Additionally, the strains collected from the high disease areas in the eld were better antagonists. The antagonistic activity was not characteristic of a species but that of a population. 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction
Trichoderma spp. are cosmopolitan and abundant fungi in soil in a wide range of ecosystems and climatic zones. They are characterized by rapid growth, capability of utilizing diverse substrates and resistance to noxious chemicals (Klein & Eveleigh 1998). Their economic importance includes their roles as primary decomposers, producers of antibiotics and enzymes as well as biocontrol agents against a wide range of plant pathogens

(Rossman 1996; Hjeljord & Tronsmo 1998; Kubicek & Penttila 1998; Sivasithamparam & Gisalberti 1998). Trichoderma spp. may inhibit plant pathogenic fungi either by inducing resistance and plant defence reactions or by direct confrontation through mycoparasitism and antibiosis as well as competition (Papavizas 1985; Howell 1998, 2003; Verma et al. 2007). A wide range of defence mechanisms against invasion is triggered in plants following contact with pathogenic and non-pathogenic microorganisms. This kind of resistance

* Corresponding author. INRA, Universite de Bourgogne, UMR 1229 Microbiologie du Sol et de lEnvironnement, CMSE, 17 rue Sully, BP 86510, 21065 Dijon, France. Tel.: 33 (0) 380 69 30 50; fax: 33 (0) 380 69 32 24. E-mail address: [email protected] 1878-6146/$ e see front matter 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.05.007

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may include the localized acquired resistance (LAR), systemic acquired resistance (SAR) or the induced systemic resistance (ISR) (van Loon et al. 1998; Vallad & Goodman 2004). In LAR, only the tissues exposed to the primary infection become more resistant while in the other two cases, a signal propagates the enhanced defensive capacity throughout the plant. SAR is commonly triggered by local infection and is correlated with activation of pathogenesis-related (PR) genes that generally requires the involvement of the signal molecule salicylic acid (SA). In contrast, ISR is generally triggered by non-pathogenic rhizobacteria followed by the expression of the genes involved in the jasmonate/ethylene signalling pathways, which does not involve the accumulation of PR proteins. The types of resistance induced by Trichoderma spp. are mainly considered as LAR or ISR (Harman et al. 2004; Shoresh et al. 2005). To assess ISR induced by Trichoderma spp., the biocontrol fungus is inoculated at different times or sites than the pathogenic attack. In the direct interactions between Trichoderma spp. and the plant pathogenic fungi, mycoparasitism is one of the mechanisms observed whereas the antagonist coils around the hyphae of the pathogen, develops hook like structures known as appressoria coupled with production of lytic enzymes and then penetrates the pathogen hyphae (Chet 1987; Kubicek et al. 2001). Coiling of the plant pathogenic fungal hyphae by Trichoderma spp. is one parameter used to characterize the mycoparasitism (Rocha-Ramirez et al. 2002; Howell 2003). Trichoderma spp. have also been reported to produce a plethora of secondary metabolites showing anti-microbial activity (Vinale et al. 2008). The chemical composition of these compounds depends on the strains and they may be classied as volatile, water-soluble or water-insoluble compounds (Ghisalberti & Sivasithamparam 1991). The competition for space, infestation sites and nutrients has also been shown to be possible mechanisms involved in the biocontrol activities of Trichoderma spp. (Dennis & Webster 1971a, b; Chet 1987; Tronsmo & Hjeljord 1998). The knowledge of mechanisms of interaction of Trichoderma spp. with plant pathogenic fungi and the plant host is of importance to enhance the practical application of these benecial microorganisms. Trichoderma spp. are among the microorganisms most frequently used as antagonists against Rhizoctonia solani (Hyakumachi 1996; Hjeljord & Tronsmo 1998). R. solani is a soil-borne plant pathogenic fungus known worldwide for causing root diseases in diverse cultures (Ogoshi 1996). Rhizoctonia-diseased areas have been shown previously to become more suppressive towards coming invasion by the same pathogen (Hyakumachi 1996; Guillemaut 2003; Anees et al. 2009, 2010a) and hence, may present a potential reservoir of benecial microorganisms. Comparing the structure of fungal communities in healthy and diseased areas in elds affected by R. solani revealed that Trichoderma spp. accumulated in the latter and were proposed to be responsible for the increased suppressiveness (Mghalu et al. 2007; Anees et al. 2010b). Similarly sclerotia of Rhizoctonia on potatoes have been shown to be a source of benecial antagonistic microorganisms including Trichoderma spp. (Grosch et al. 2006). These ndings suggest that putative active antagonistic populations of Trichoderma are more likely to be found within diseased areas than in healthy areas. Therefore, it would be worthwhile to check for naturally occurring Trichoderma in soils where R. solani is active to determine their putative intrinsic abilities to control this pathogenic fungus.

Phenotypic characterization presents a potential method to taxonomically identify Trichoderma spp. by using a morphological key (Samuels GJ, Chaverri P, Farr DF, McCray EB, http://nt.arsgrin.gov/taxadescriptions/keys/TrichodermaIndex.cfm). However, because of the increasing number of species recorded within the genus, the few morphological characters of anamorph and teleomorph have reached their limit for identifying species. Thus, these morphological characteristics need to be combined with molecular data resulting from DNA sequencing (Samuels 2006). As a single gene is generally not considered sufcient, a multigene approach using at least two unlinked loci is advised. The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) is one of the most reliable targets to identify a strain at the species level (Kullnig-Gradinger et al. 2002). However, some closely related species share the sequences of their ITS regions, such as in Trichoderma sect. Trichoderma, (Samuels 2006). Being more variable, the gene tef1, which encodes the translation elongation factor 1-a, is more variable and can be used to reect differences within and among groups of closely related species. A combination of the ITS region and tef1 allows most identications at the species level (Samuels 2006). For identication of Trichoderma strains, TrichOKEY and TrichoBLAST (www.isth.info) are convenient tools available online that are based on the sequence comparisons of ITS or tef1 regions (Druzhinina et al. 2005; Kopchinskiy et al. 2005). The primary aim of the present study was to characterize Trichoderma spp. originating from a sugar beet eld containing patches infested with R. solani, using a set of complementary approaches to investigate if there have been a change in the antagonistic ability of the Trichoderma isolates from the diseased and healthy area. These eld isolates of Trichoderma were tested for their antagonistic abilities towards R. solani AG 2-2 both in vitro and in vivo. In vitro tests were performed to characterize their mycoparasitism and ability to produce water-soluble metabolites or volatile inhibitors. In vivo tests were performed to characterize the isolates for induction of resistance in the host or direct antagonism of the pathogen. The isolates were identied using morphological and molecular characters. Morphological characterization was based on microscopic measurements of mycelial fragments as well as growth rates of different isolates on different media at different temperatures, while molecular identication was based on sequence comparisons of the ITS and tef1 regions.

Materials and methods

Fungal isolates
The study was conducted with sixteen isolates of Trichoderma (T30 to T37 and T40 to T47) collected in 2006 from soil samples originating from a sugar beet eld at the INRA Experimental Unit of Epoisses, Cote dOr, France (5 050 E; 47 140 N) (Table 1). The eld was chosen because patches of root rotting caused by Rhizoctonia solani were observed (Anees et al. 2009). The isolation was performed using malt extract agar (MEA) as previously described by Anees et al. (2010b). The Trichoderma isolates were stored in the collection Microorganisms of Interest for Agriculture and Environment (MIAE, INRA Dijon, France). Additionally, two external isolates of Trichoderma

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Table 1 e Morphological and molecular identication of the strains of Trichoderma used in this study. Strain MIAE accession no.a
MIAE00029 MIAE00030 MIAE00031 MIAE00032 MIAE00033 MIAE00034 MIAE00035 MIAE00036 MIAE00037 MIAE00038 MIAE00039 MIAE00040 MIAE00041 MIAE00042 MIAE00043 MIAE00044 MIAE00454 MIAE00220

Morphological identicationb
T. gamsii T. gamsii NI NI T. velutinum T. gamsii T. velutinum T. velutinum T. gamsii T. velutinum T. velutinum T. gamsii T. velutinum T. harzianum T. velutinum T. velutinum T. hamatum T. atroviride

Genbank accession no. ITS sequence

HM176559 HM176560 HM176561 HM176562 HM176563 HM176564 HM176565 HM176566 HM176567 HM176568 HM176569 HM176570 HM176571 HM176572 HM176573 HM176574 HM176576 HM176575

tef1 sequence
HM176577 HM176578 HM176579 HM176580 HM176581 HM176582 HM176583 HM176584 HM176585 HM176586 HM176587 HM176588 HM176589 HM176590 HM176591 HM176592 HM176594 HM176593

Molecular identication
T. gamsii T. gamsii T. tomentosum T. tomentosum T. velutinum T. gamsii T. velutinum T. velutinum T. gamsii T. velutinum T. velutinum T. gamsii T. velutinum T. harzianum T. velutinum T. velutinum T. hamatum T. atroviride

Denitive identication
T. gamsii T. gamsii T. tomentosum T. tomentosum T. velutinum T. gamsii T. velutinum T. velutinum T. gamsii T. velutinum T. velutinum T. gamsii T. velutinum T. harzianum T. velutinum T. velutinum T. hamatum T. atroviride

T30 T31 T32 T33 T34 T35 T36 T37 T40 T41 T42 T43 T44 T45 T46 T47 T38 P1

a Collection MIAE. Microorganisms of interest for agriculture and environment (INRA, Dijon, France). b NI: not identied.

were also included in this study: one isolate (T38) originating from another suppressive soil in Norway (Pythium suppressive soils) and one isolate (P1, ATCC74058) known for its antagonistic activity against R. solani (Tronsmo 1989).

Morphological identication
The identication was performed using an interactive key for strain identication (Samuels GJ, Chaverri P, Farr DF, McCray EB, http://nt.ars-grin.gov/taxadescriptions/keys/Trichoderma Index.cfm) based on morphology, differences in growth rates on potato dextrose agar (PDA) and synthetic nutrient agar (SNA), which is dened low-sugar medium containing 1 g of KH2PO4, 1 g of KNO3, 0.5 g of MgSO4$7H2O, 0.5 g of KCl, 0.2 g of glucose, 0.2 g of sucrose, and 20 g of agar per 1 l in distilled water (Nirenberg 1976), and microscopic measurements of mycelial parts. All measurements of morphological characters (shape and size of conidia, size of conidiophores, presence or absence of sterile hairs) were taken from slide mounts prepared by the tape touch method (Harris 2000) in a drop of lactofuchsin. Plugs (5 mm) from the edge of an actively growing colony of the Trichoderma spp. were placed in the center of three replicate 9 cm PDA and SNA plates and incubated at 20, 25, 30, 35, and 40  C in dark (Chaverri et al. 2003). Growth rates were measured every 24 h for 4 d. This experiment was repeated once.

Molecular identication
Molecular identication of Trichoderma isolates was based on DNA sequencing of two unlinked loci, the ribosomal ITS region and the tef1 gene. Fungal DNA was extracted from cultures on MEA using a rapid mini-preparation procedure (Edel et al. 2001). The ITS region was amplied by polymerase chain reaction (PCR) using primers ITS1F (Gardes & Bruns 1993) and

ITS4 (White et al. 1990) in a nal volume of 50 ml by mixing 2 ml of DNA with 0.5 mM of each of the primers, 150 mM of dNTP, 6 U of Taq DNA polymerase (Q-Biogen, Evry, France) and PCR reaction buffer. Amplications were conducted in a mastercycler (Eppendorf, Hamburg, Germany) with an initial denaturation of 3 min at 94  C followed by 35 cycles of 1 min denaturation at 94  C, 1 min primer annealing at 50  C, 1 min extension at 72  C and a nal extension of 10 min at 72  C. Aliquots of 2 ml of PCR products were checked by electrophoresis in a 2 % agarose gel and stained with ethidium bromide for visualization under UV light. The PCR products were cloned with ` pGEM@-T Easy Vector System (Promega, Charbonnieres, France) and sequenced using primers SP6 and T7 by Cogenics (Meylan, France). The tef1 fragment was amplied by PCR using the primers ef1 (50 -ATG GGT AAG GA (A/G) GAC AAG AC) and ef2 (50 -GGA (G/A)GT ACC AGT (G/C)AT CAT GTT) (Geiser et al. 2004) under the same PCR conditions stated above with the exception of the following parameters that differed: the concentration of primers (0.4 mM) and dNTP (250 mM), and the temperature of denaturation (95  C) and annealing (57  C). The PCR products were checked by electrophoresis as above and directly sequenced using primers ef1 and ef2 by Cogenics (Meylan, France). For each PCR product, sequences from both strands were assembled using SeqMan 6.0 (DNASTAR Inc., 2004). Sequence identities were determined using both a specic database for Trichoderma and the Genbank general database. We successively used the different tools available online from the International Subcommission on Trichoderma and Hypocrea (ISTH, www.isth.info): TrichOKEY v. 2.0 based on an oligonucleotide barcode within the ITS1 and ITS2 sequences, TrichoMARK to analyse both ITS and tef1 sequences, and TrichoBLAST to detect sequence similarity in the ITS region and the largest 4th intron sequence of tef1 gene (Druzhinina et al. 2005;

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Kopchinskiy et al. 2005). In some cases, BLAST analyses were also performed from the National Center for Biotechnology Information (NCBI) available online. Moreover, as per requirement, the alignments of sequences were performed with the help of the programme ClustalX v 2.0 (Larkin et al. 2007) and sequences were manually edited by visual adjustments by the help of the computer program Seaview (Galtier et al. 1996).

isolates with R. solani (Tronsmo & Dennis 1978). In addition to this, interactions between hyphae of Trichoderma isolates and R. solani were recorded as inhibition zones before contact between colonies, or as inhibition of the pathogen after contact with the antagonist, or no visible inhibition after contact between colonies.

Assessment of antagonistic activity using in vitro tests

Trichoderma isolates were evaluated for their potential to antagonize in vitro the plant pathogenic fungus Rhizoctonia solani using three different tests. R. solani AG 2-2 strain G6 (MIAE collection, INRA, France) was used. For all the in vitro tests, discs (5 mm) from the edge of growing fungal colonies were used to inoculate MEA in sterile plastic round 9 cm Petri dishes. The plates were incubated in the dark at 20  C. In the rst test, each isolate of Trichoderma was grown in dual culture with R. solani AG 2-2 strain G6. The strain G6 and the Trichoderma isolate were inoculated 6 cm apart on the same plate and incubated at 20  C. Radii of colony of R. solani approaching and not approaching the colony of Trichoderma isolate were measured twice a day for 3e4 d. Experiments were performed with three independent replicates. Inhibition of growth rate of R. solani was assessed as percentage of difference of radius not approaching and radius approaching Trichoderma, over radius not approaching and compared by analysis of variance (ANOVA) and Fisher least signicant difference (LSD) tests using XLSTAT-Version 2007.5 (Addinsoft). Part of the results from this test was presented in a previous publication (Anees et al. 2010b). The second test was designed to measure the ability of Trichoderma isolates to produce water-soluble inhibitors against R. solani strain G6. Trichoderma inoculum discs were placed in the center of MEA plates covered with a 50 mm thick cellophane membrane D and incubated for 5 d at 20  C. The cellophane membranes along with the mycelia of Trichoderma isolates were then removed and R. solani was cultured in the same plates (Dennis & Webster 1971a). Growth of R. solani was recorded after 24, 48 and 72 h. For the control plates, R. solani was cultured instead of Trichoderma spp. on MEA covered with the cellophane membranes under the conditions described above. The third test was designed to measure the ability of Trichoderma isolates to produce volatile inhibitors. The MEA plates were inoculated centrally with agar disks of Trichoderma isolates and the lid of each dish was replaced by a bottom dish containing MEA newly inoculated with R. solani. The two dishes were taped together with adhesive tape (Dennis & Webster 1971b). The growth of R. solani was recorded after 24, 48 and 72 h. In the control, R. solani was cultured in the same way but without Trichoderma isolates. For both volatile and water-soluble inhibitors tests, the percentage of inhibition was measured by dividing the difference between the radial growth of control and antagonised cultures of R. solani by the radial growth of the control and multiplied by 100. The experiments were performed with three independent replicates and results were compared by ANOVA as above. Finally, direct inspection by light microscopy was carried out before and after contact of the hypha of the Trichoderma

Assessment of antagonistic activity using in vivo tests

Bioassays were performed in climatic chambers in order to test the antagonistic activity of the selected Trichoderma isolates against Rhizoctonia solani AG 2-2 strain G6 causing disease on a susceptible host plant. Conidial suspensions of Trichoderma were prepared from 18-day-old cultures on MEA incubated at 25  C and adjusted to 4 106 ml1 conidia using a haemocytometer (Thoma, Preciss, France). Millet seed inoculum of R. solani was produced by adding ve pieces of MEA culture to millet seeds previously autoclaved for 1 h on three consecutive days at 105  C. The inoculated millet seeds were incubated for two weeks at 25  C with periodic shaking. Carrot (Daucus carota cv. Yukon) was used as a host plant susceptible to R. solani (Anees et al. 2010b). Twenty seeds of carrot were sown in each square plastic pot (8 8 7 cm) containing 260 g of sand covered by 20 ml of calcined clay to hold the irrigation water. To characterize resistance induced in the plant host by Trichoderma isolates, 15 ml of the conidial suspensions of Trichoderma isolates were added directly to the sand without soil at the time of sowing. Twelve days after sowing, 50 ml of heatsterilized or natural soil were placed around the crown of the carrot seedlings. Two days later, this soil was inoculated with millet seed inoculum of R. solani using four seeds per pot (one at each corner of the pot). Controls were setup in the same manner without adding the infected millet seed. To characterize direct interaction of Trichoderma isolates with R. solani in vivo, carrot seeds were sown as described above in sand without Trichoderma. After 12 d, 15 ml of conidial suspensions were mixed with 50 ml of sterilized or natural soil and placed around the crown of the seedlings. Two days later, this soil was inoculated with millet seed inoculum of R. solani as above. In all experiments, the temperature was maintained at 25  C day and 20  C night (16 h day length). Experiments were performed with three independent replicates such that 60 plants were used per treatment. Number of damped off seedlings were counted on alternate days for two weeks and the area under disease progress curve (AUDPC) measured as cumulative mortality rate was calculated. The percentage of inhibition of the disease by the Trichoderma isolates was calculated by subtracting AUDPC values of the treatments from those of the respective controls, dividing that by AUDPC values of the controls and multiplying by 100. The results were analysed by ANOVA as above.

Results
Identication of Trichoderma isolates
The isolates were identied using both morphological and molecular characters. Eight out of the 18 isolates could be

Characterization of Trichoderma isolates

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successfully identied using the morphological key. These isolates corresponded to one of the following species: Trichoderma gamsii (ve isolates), T. harzianum (one isolate), T. hamatum (one isolate) and T. atroviride (one isolate) (Table 2). The key failed to identify T34, T36, T37, T41, T42, T44, T46 and T47. However, the characteristics of producing small ellipsoidal smooth conidia and the presence of exuous or undulate conidiophores extensions made it possible to identify these eight isolates as T. velutinum (Bissett et al. 2003), a species not included in the key. Finally, the identication of T32 and T33 using the morphological key remained ambiguous. The presence of sinuous or spiral sterile hair, small subglobose to oblong conidia and very slow growth at 35  C on PDA and SNA after 72 h in the dark were characteristics of the species T. tomentosum, whereas presence of long phialides and high growth at 25  C and 30  C on PDA were not. The morphological identication was complemented by a molecular identication based on ITS and tef1 sequences. The identication of the strains T34, T36, T37, T41, T42, T44, T46 and T47 as T. velutinum, T45 as T. harzianum, T38 as T. hamatum, and P1 as T. atroviride was conrmed using TrichOKEY and was further conrmed by similarity search of tef1 intron-4 by trichoBLAST. For strains T32 and T33 the best match using trichoBLAST was T. tomentosum with 99 % and 94 % of similarity for ITS and the largest 4th intron regions, respectively. We compared these percentages of similarity with the intra-specic variability found within the species T. tomentosum. Concerning the ITS region, 1 % of mismatches is commonly found among sequences of T. tomentosum (for example between accession numbers AY605737 and AF149869). Concerning tef1 intron-4, 6 % of mismatches are commonly found between sequences of this species (for example between accession numbers AY605818 and AY605759). Thus, the variability observed between sequences of the isolates T32 and T33, and the sequences present in ISTH database may be considered as intra-specic variability. Therefore, T32 and T33 may be identied as T. tomentosum. The strains T30, T31, T35, T40, and T43, which were identied as T. gamsii by morphological key, could not be identied by TrichOKEY or TrichoBLAST because the sequences for this species were not included in the ISTH database at the time of the investigation. The identication of these strains was conrmed as T. gamsii by downloading the sequences of the ITS regions of the known T. gamsii strains (DQ315459, DQ315448, DQ841730, DQ845432) from the Genbank and aligned them with the sequences of the isolates used in the present study that were identied as T. gamsii morphologically. The percentages of mismatches between the probable T. gamsii strains and the reference strains were 0e0.3 %. Similarly, alignment of the tef1 intron-4 of the same strains (T30, T31, T35, T40 and T43) with the reference sequences (DQ790653, DQ790649, DQ790655, EF488117, EF488132) was performed as above. The intra-specic variability found in the tef1 intron-4 was up to 3 % among the reference strains while it was less than 2 % between the isolates under query and the reference strains. All these ndings, from both morphological and molecular identication conrmed that T30, T31, T35, T40 and T43 belong to T. gamsii.

Antagonistic activity measured in vitro

Trichoderma strains T30, T40, T43 and P1 showed the highest inhibition (>38 %) of Rhizoctonia solani growth in dual cultures (Table 3). On the other hand, T36, T37, T46 and T47 showed the lowest inhibition (<10 %). The remaining strains showed intermediate values of percentage of inhibition of R. solani growth. Additionally, antagonism and hyphal interactions were observed with the naked eye and under the microscope in the dual culture experiments (Table 3, Fig 1). The strains T30, T31, T40, T43, and P1 were able to inhibit R. solani before contact. These strains did not coil over R. solani hyphae. On the other hand, T32, T33, T34, T35, T38, T41, T42, T44 and T45 were able to inhibit R. solani after contact. Additionally, these strains were able to coil around R. solani hyphae, except T42 and T38, for which coiling was not evident. Finally, the strains T36, T37, T46 and T47 did not inhibit R. solani, although they coiled around R. solani hyphae. The second part of the in vitro characterization of mechanisms of antagonism consisted of testing the strains for their ability to produce water-soluble metabolites that may inhibit the growth of R. solani (Table 3). T30, T40 and T43 inhibited R. solani growth by more than 50 %, with the highest inhibition produced by T43 (75.6 %). T35, T38, T41, and T45 showed a moderate inhibition (25e40 %) while T34, T46 and P1 were the poorest producers of water-soluble inhibitors with an inhibition of R. solani growth of less than 10 %. The reduction of R. solani growth by volatile inhibitors was not very remarkable (Table 3). The highest inhibition was produced by T39 (32.1 %) followed by T43 (14.4 %) and T31 (10.3 %). All other strains exhibited poor production of volatile inhibitors with an inhibition of less than 10 %.

Antagonistic activity measured in vivo

The in vivo tests for characterization of mechanisms included assessment of the induced resistance in plant host by the selected Trichoderma isolates and direct interaction of the isolates with Rhizoctonia solani in both the natural and sterile soil (Fig 2). Selection of strains for the in vivo tests was based on the in vitro results in dual cultures as explained above, such that T30, T40, T43 were the best inhibitors of R. solani; T36 and T47 showed the least inhibition while T45 was intermediate, along with the reference strain P1 which also performed well in dual culture. In bioassays characterizing direct interaction of Trichoderma isolates with R. solani in natural soil, highest antagonistic activities were observed for T30 (67.9 %) and T47 (75.2 %) (Fig 2). The strains T43 and P1 were moderately antagonistic while T36, T40 and T45 were not antagonistic in this case. The disease intensity was even increased to 19.7 % when T40 was present together with the pathogen in the natural soil. On the other hand, in the bioassays to assess the direct interaction in sterilized soil, T30, T45, and T47 proved to be highly antagonistic towards disease produced by R. solani while T36, T40 and T43 were not antagonistic under these conditions (Table 3). In bioassays aimed at characterization of induced resistance in the plant host in natural soil, T30, T40 and T43

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Table 2 e Characterization and identication of the Trichoderma isolates using morphological characters. Strain Shape
T30 T31 T32 T33 T34 T35 T36 T37 T40 T41 T42 T43 T44 T45 T46 T47 T38 P1 Ellipsoidal to oblong Ellipsoidal to oblong Subglobose to ovoid Subglobose to ovoid Ellipsoidal Ellipsoidal to oblong Ellipsoidal Ellipsoidal Ellipsoidal Ellipsoidal Ellipsoidal Ellipsoidal Ellipsoidal Globose to subglobose Ellipsoidal Ellipsoidal Ellipsoidal Globose to subglobose

Conidia Length Width L/W ratio (L e mm) (W e mm)

4.2e4.8 4.2e4.8 3e4 2.8e3.4 3.5e4.1 4.2e4.8 2.8e3.4 3e4 4.2e4.8 3.5e4.1 2.8e3.4 4.2e4.8 3.5e4.1 <2.7 2.8e3.4 2.8e3.4 3.5e4.1 2.8e3.4 2.7e3.2 2.7e3.2 2e2.5 2.2e2.7 2.2e2.7 2.7e3.2 2.2e2.7 2.2e2.7 2.7e3.2 2.2e2.7 2.2e2.7 2.7e3.2 2.2e2.7 2.2e2.7 2.2e2.7 2.7e3.2 2.7e3.2 2.7e3.2 1.5e1.8 1.5e1.8 1.2e1.5 1.2e1.5 1.2e1.5 1.5e1.8 1.2e1.5 1.2e1.5 1.2e1.5 1.2e1.5 1.2e1.5 1.5e1.8 1.2e1.5 <1.2 1.2e1.5 1.2e1.5 1.5e1.8 <1.2

Sterile hair

Culture on SNA (mm) Coconut odour     Length Width Width 20 C 25 C 30 C 35 C 20  C 25  C 30  C 35  C at middle at base
8.5e10.2 8.5e10.2 14 15 4e7.5 10 7e12 7e12 7e10 7e10 7e10 8.5e10.2 5e10 8.5e10 7e10 7e14 ND 8.5e10 2e4 2.4e3.2 2.4e3.2 2.4e3.2 >3.5 3 3 2.5e3 3e3.6 2.4e3.2 2.4e3.2 2.4e3.2 2.4e3.2 3.2e3.5 2.4e3.2 1.8e2.4 ND 2.4e3.0 <2 <2 2e2.6 2e2.6 2e2.6 2.5 2e2.6 2.5 1.2e1.7 2.5 2e2.6 2e2.6 <2 2e2.6 2e2.6 1.8 ND <2 40.3 47 41.3 42.7 39.7 33.3 40.67 42 45.33 38.66 39.67 44.67 40 44.67 36 37.67 41.7 45 54 57 51 51 49 45 53 53 53 47 49 53 48 59 46 47 53 55 52 54 51 51 54 54 55 56 50 50 54 52 54 68 49 52 35 40 2.3 6 2 1.3 9.7 5 10 11 3.3 11 14 4 15 40 15 7 2.3 0.7 36 23 39 37 41 31 40 41 27 35 35 23 41 30 36 31 27 30 38.3 40.3 47 47.7 50 40.7 49.7 49 36.7 46.3 46.7 36 44.3 45.3 47.7 42 35.7 35.7 45 43 46 47 57 42 56 56 38 54 55 38 59 51 53 51 29 32 3 7 0.8 1 9 6.5 9.2 11 8.3 7.7 9.3 3.2 11 27 11 5.3 1.7 nil Present Present Absent Absent Absent Present Absent Absent Present Absent Absent Present Absent Absent Absent Absent Absent Present

Phialides (mm)

Culture on PDA (mm)

Speciesa

References

Absent Absent Flexuous Flexuous Flexuous Absent Flexuous Flexuous Absent Flexuous Flexuous Absent Flexuous Absent Flexuous Flexuous Straight Absent

T. gamsii T. gamsii NI NI

Jaklitsch et al. 2006 Jaklitsch et al. 2006

T. velutinum Bissett et al. 2003 T. gamsii Jaklitsch et al. 2006 T. velutinum T. velutinum T. gamsii T. velutinum T. velutinum T. gamsii T. velutinum T. harzianum Bissett et al. 2003 Bissett et al. 2003 Jaklitsch et al. 2006 Bissett et al. 2003 Bissett et al. 2003 Jaklitsch et al. 2006 Bissett et al. 2003

T. velutinum Bissett et al. 2003 T. velutinum Bissett et al. 2003 T. hamatum T. atroviride

The measurements of cultures on PDA and SNA media represent the mean of three independent values measured after 72 h in the dark. All the isolates showed green pigmentation of spores. No growth at 40  C on both media and pustules formation on PDA or SNA. a All the species were identied using the morphological key (http://nt.ars-grin.gov/taxadescriptions/keys/TrichodermaIndex.cfm) with the exception of the species T. velutinum which was not included in the key; NI not identied.

M. Anees et al.

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Table 3 e In vitro antagonistic activity of Trichoderma strains against Rhizoctonia solani AG 2-2 strain G6 measured as dual cultures (percentage of inhibition of G6 growth when grown together with Trichoderma strains); water-soluble metabolites (percentage of inhibition of G6 growth when grown on metabolites of Trichoderma strains); volatile inhibitors (percentage of inhibition of G6 growth when grown in the presence of separate cultures of Trichoderma strains); interactions between hyphae of the strains and G6 on agar media (interactions were recorded as inhibition zones before contact between colonies or as inhibition of R. solani growth after contact with Trichoderma or as visible inhibition after contact between colonies); microscopic interactions of colonies of the strains and G6. Strain Percentage of inhibition of Rhizoctonia solani growth Dual culture test
T30 T31 T32 T33 T34 T35 T36 T37 T40 T41 T42 T43 T44 T45 T46 T47 T38 P1 38.17abc 33.93bc 22.92def 16fghi 11.17ghij 19.29efgh 2.35j 7.3ij 46.31a 35.16bc 27.74cde 38.28abc 30.99bcd 15.93fghi 8.95hij 6.7ij 21.56defg 40.32ab

Soluble inhibitors
60.69ab 20.23de 15.61de 16.38de 4.05e 27.36de 22.25de 21.00de 58.12abc 30.62cde 18.74de 75.62a 20.31de 39.37bcd 6.24e 11.87de 29.87cde 9.25de

Volatile inhibitors
8.84bcd 10.32bc 5.92de 10.35e 1.83bcde 2.23cde 2.57bcde 2.94bcde 6.24bcd 3.13cde 3.13cde 14.37b 7.49bcd 4.38cde 0.01bcde 9.38e 10.69bc 32.09a

Visual interaction visible with naked eye

Inhibition before contact Inhibition before contact Inhibition after contact Inhibition after contact Inhibition after contact Inhibition after contact No inhibition No inhibition Inhibition before contact Inhibition after contact Inhibition after contact Inhibition before contact Inhibition after contact Inhibition after contact No inhibition No inhibition Inhibition after contact Inhibition before contact

Microscopic interaction
No coiling No coiling Coiling Coiling Coiling Coiling Coiling Coiling No coiling Coiling No coiling No coiling Coiling Coiling Coiling Coiling No coiling No coiling

Values in the same column followed by different letters are signicantly different (Fishers LSD, p < 0.05).

performed better in terms of percentage of inhibition of the disease caused by R. solani in the controlled environmental conditions (Fig 2). T36 produced 20.2 % of inhibition while all the other strains exhibited inhibition of less than 10 %. In the bioassays for characterizing induced resistance in the

plant host in sterilized soil, trends were similar to those noticed in natural soil for the different strains, except for T45 (Fig 2). T45 reduced the disease by 24.2 % in sterilized soil but only by 5.5 % in natural soil. T30, T40 and T43 were able to reduce the disease by more than 30 %.

Fig 1 e Coiling of hyphae of Rhizoctonia solani by Trichoderma spp. The arrows indicate the hyphae of T. tomentosum strain T33 coiling around the hyphae of R. solani AG 2-2 strain G6.

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100 90 80 a 70

Direct interaction in non disinfested soil

% age reduc tion of dis eas e

60 50 40 30 20 c bc 10 0 -10 -20 -30 T30 T40 T43 T45 T36 T47 P1 Direct interaction in disinfested soil ab ab abc bc a a ab

Host induced resistance in non disinfested soil Host induced resistance in disinfested soil

bc d

bc

cd cd

bc cd d

bc

Fig 2 e In vivo antagonistic tests of Trichoderma strains against infection of carrot seedlings by Rhizoctonia solani AG 2-2 strain G6. Direct interactions were measured as disease reduction when the strains were present in the crown and root zone at the time of pathogen introduction. Induced resistance was measured as disease reduction when root zones were inoculated with Trichoderma strains two weeks before the pathogen was introduced in the stem zone soil. The data are presented as percentage of reduction of disease as compared to controls without Trichoderma. ANOVA and LSD tests were performed separately for each of the four series. Bars designated by different small letters are signicantly different (Fishers LSD, p < 0.05).

Discussion
Based on the limited number of isolates taken from a sugar beet eld in which infectious patches occurred, we found an unexpected diversity by using three different approaches. The classical way to identify Trichoderma isolates relies on morphological and molecular approaches. Adding the functional approach, including various mechanisms of antagonistic activity towards Rhizoctonia solani pathogenic towards the sugar beet reveals the intra-specic diversity among the antagonists and brings some new information about the interactions as well as the biocontrol potential that is harboured by the Trichoderma group. Thus, the functional approach is inevitable for isolating better antagonistic strains, in contrast to mere identication, which renders information about interspecic diversity, as recently done by Migheli et al. (2009). The few morphological characters with limited variation may lead to an overlap and misidentication of the strains (Kullnig et al. 2001) showing the necessity of DNA based characters to complete identication evident from the present study. Moreover, the morphological key used in this study is an online interactive key and represents a way to make the morphological identication more convenient. However, it could be even more efcient if the newly identied species were regularly integrated in the key. For instance, in our case, the key could not identify T. velutinum because it was not yet included. Concerning molecular techniques, the Genbank database is generally referred, representing the largest reservoir of the sequences; however, it may not be safely used for identication as it contains many erroneous entries for Trichoderma (Druzhinina & Kubicek 2005). This fact

bc

emphasizes the need of a specic database for Trichoderma containing only vouchered sequences, such as the ISTH database used in this study which has recently been used successfully for identication of Trichoderma strains (Zhang et al. 2005; Migheli et al. 2009). The interactive morphological key and a specic molecular database coupled with tools for identication of Trichoderma strains represent an ideal way to identify the Trichoderma spp., although, they need regular updates to include the rapidly increasing number of species of this genus. The best strains in this study regarding antagonism against R. solani AG 2-2 belonged to Trichoderma gamsii. T. gamsii is a species that was recently identied and is a close relative morphologically and phylogenetically of T. viride to which many antagonistic strains belong (Brown et al. 1999; Jaklitsch et al. 2006). However, to our knowledge, this is the rst report of an efcient antagonistic strain of T. gamsii towards R. solani AG 2-2. The reason may be because before the reassessment of T. viride and denition of T. gamsii (Jaklitsch et al. 2006), this species might have been misidentied as T. viride or some other closely related species. In the present study, the most efcient strain overall was T. gamsii T30, which was able to reduce the growth of the pathogen and inhibit the disease both in vitro and in vivo. The results indicate that this strain is a good producer of water-soluble metabolites that can inhibit the growth of R. solani strain G6 in Petri dishes at a distance before contact between the colonies; however, T30 was not observed to coil around the hyphae of the strain G6. Hence, the principal mechanism of antagonism for this strain is most likely antibiosis or the production of catabolic enzymes that have already been reported to be involved in the antagonistic reaction (Harman et al. 2004). That may be why the strain was able to reduce the disease in

Characterization of Trichoderma isolates

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the bioassays conducted to assess the direct interactions of the isolate with the strain G6 in the sterilized as well as natural soils. Additionally, the Trichoderma strain appeared to be able to induce resistance in the host against the infectious activity of G6, as application of the antagonist to the root zone resulted in reduced disease following subsequent exposure of the plant crown and roots to the pathogen. In the present set of bioassays to assess the disease resistance induced in the plant host by Trichoderma spp. we attempted to avoid direct interaction between the antagonist and the pathogen by inoculating them at different times and sites. Induced resistance is a phenomenon often suggested to be related to biocontrol by Trichoderma spp. and has been hypothesized that the mechanism may be production of molecules such as enzymes or antibiotics that are sensed by the plant which leads to ISR (Harman et al. 2004; Woo et al. 2006). In our case, the other two strains of T. gamsii, T40 and T43 also inhibited the growth of the strain G6 by producing water-soluble inhibitors in vitro and inhibited the disease in vivo principally through induced resistance, as less disease expression resulted from application of the antagonist to soil around the stem. Hence, our experiments indicate that strains able to produce water-soluble metabolites were also able to induce the disease resistance in the plant. T. velutinum strain T36 and T. harzianum strain T45, which produced from moderate to low extent of water-soluble inhibitors in vitro, also seemed to produce moderate to low disease suppression through induced resistance. Antibiosis in vitro was not related to the disease reduction in vivo by direct interactions. For instance, T40 and T43 did not consistently reduce the disease in the direct interaction bioassays. T. harzianum T45 was unable to reduce disease in natural soils. The same strain could coil around the pathogen hyphae in vitro and reduced the disease in vivo signicantly by direct interaction, but only in sterile soil. These results support the hypothesis that T45 is a poor competitor under natural conditions. The interaction between different isolates and the R. solani strain G6 was observed under the microscope. Coiling of hyphae was noticed in some instances. Two important conclusions may be drawn from the present results. First, not all Trichoderma species or even strains within a given species, coiled around strain G6. For instance, the only strain of T. gamsii that was observed to coil was T35. Similarly, coiling was evident for all strains of T. velutinum except T42. Secondly, coiling action of hyphae of Trichoderma was not related to their higher antagonistic abilities. T37 and T46 (T. velutinum) could not inhibit the growth of the strain G6 in vitro; nevertheless these strains were able to coil around the hyphae of the pathogen. T. velutinum strain T36 was able to coil around R. solani but did not reduce disease through direct interaction. However, T. velutinum strain T47, which was observed to coil around the hyphae of the strain G6 but was considered to be a poor antagonist in dual culture inhibited the disease by direct interaction with the strain G6 in vivo both in sterile or natural soils. These results are in accordance with previous reports where no correlation between coiling frequencies and cell wall degrading enzymes was found (Almeida et al. 2007). T. atroviride strain P1 is a known mycoparasite of different fungal plant pathogens (Hjeljord & Tronsmo 1998). It has been

reported that this strain produces diffusible hydrolytic enzymes and inhibits R. solani when the fungi are grown together (Kullnig et al. 2000). In the present study, P1 showed pre-contact inhibition of the strain G6 when the fungi were grown together in the confrontation assays and produced volatile inhibitors against the strain G6. In the absence of the pathogen, P1 did not produce strongly inhibitory water-soluble metabolites. Coiling of hyphae around strain G6 by P1 was not evident which is consistent with the previous observations where it has not been reported as a serious coiler of fungi. In the bioassays, P1 did not strongly reduce disease by direct antagonism in natural or sterile soils. These results indicate that T. atroviride P1 is a poor mycoparasite of the strain G6. From the present results, it is clear that antagonism is not a property of a species, as different strains of the same species can exhibit varying potentials of biocontrol. The strains which can express rapidly and efciently their genes involved in antagonistic activities in the presence of host are in fact better antagonists (Scherm et al. 2009). For instance, although the most efcient strain in our case belonged to T. gamsii (T30), we also isolated a strain of T. gamsii (T35) that was a poor antagonist of the pathogen used in this study. This fact is also evident from the literature where strains of different species have been used for biocontrol with varying mechanisms (Bentez et al. 2004). This highlights the necessity to use a func tional approach to characterize the mechanisms and to identify the good biocontrol strains from the eld. Recently Scherm et al. (2009) characterized a few strains based on subtraction hybridization approach and identied the genes which could be used as markers for rapid screening and preidentication of T. harzianum strains for their biocontrol potential. The main objective of the present study was to characterize randomly isolated strains of Trichoderma originating from a eld. Among the 16 strains isolated from the same eld, the most abundant species were T. velutinum and T. gamsii, representing 50 and 31 % of the strains, respectively. Until now, few data are available concerning the biodiversity of Trichoderma in soil. Nevertheless, previous studies have rather reported T. harzianum as the dominant species among soil isolates (Zhang et al. 2005; Migheli et al. 2009). In our study, the number of isolates is too small to draw a denite conclusion in terms of diversity. However, only one out of the 16 isolates belongs to the species T. harzianum. Migheli et al. (2009) found very little correlation between species distribution and soil abiotic parameters, with the exception of T. velutinum and soil pH. Indeed, they only found T. velutinum in soils with pH 5 or less. But this explanation does not t with our results since the soil pH was 7.1 (Anees et al. 2010b). Another factor that could affect the distribution of fungal species in soil is the cultivated plant. But until now, no data are available to conclude whether the crop and especially sugar beet inuence the diversity of Trichoderma species in soil. Our results indicate that antagonistic Trichoderma strains are stimulated by the presence of R. solani and may increase in numbers during disease outbreaks in the eld. In general the isolates picked from the disease foci (T30, T40 and T43) induced host resistance more than isolates from healthy areas (T36, T45 and T47), which supports the hypothesis that Trichoderma may be partly responsible for the increased disease

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suppression inside patches (Anees et al. 2010a, b). Additionally, the strains of T. gamsii found in the diseased areas were better antagonists than the strain of the same species found in the healthy area. Hence the increased suppressiveness may not be related to a particular species, i.e., the distribution of different species in the eld seems to be random regardless the disease condition. Our results therefore support the theory that soils from diseased sugar beet areas are potential sources of antagonists that may be further exploited in the future.

Acknowledgements
This work was made possible by funding from Higher Education Commission, Government of Pakistan and French Government supporting PhD Studentship in INRA Dijon, France. A part of work was carried out in the Department of chemistry, Biotechnology and Food Science, Aas, Norway.

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