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Elimination of Cassava Mosaic Disease Virus by Thermotherapy and Meristem

Culture in Côte D'ivoire Abbreviations MS-Murashige and Skoog DNA-
Deoxyribonucleic Acid PCR-Polymerase Cha...

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 International Journal of Agriculture and Biological Sciences- ISSN (2522-6584) Jan & Feb 2022
February 28, 2022
Elimination of Cassava Mosaic Disease Virus by Thermotherapy and Meristem
Culture in Côte D'ivoire
Author’s Details :
DJAHA Konan Engueran1*, DOUBI Bi Tra Serges1, ABO Kouabenan2, KONE Mongomaké3
1
Centre National de Recherche Agronomique, Port-Bouet, Côte d’Ivoire,
2
Institut National Polytechnique Félix Houphouët-Boigny, Laboratoire de Phytopathologie et de Biologie
Végétale, Yamoussoukro, Côte d'Ivoire,
3
UFR des Sciences de la Nature, Université Nangui Abrogoua, Abidjan, Côte d’Ivoire
*Corresponding Author : Djaha Konan Engueran1*, 07 BP 13 Abidjan 07, Port-Bouet (Côte d’Ivoire), Phone :
(00225) 0777958928. E-mail: [email protected]

Abbreviations
MS-Murashige and Skoog
DNA-Deoxyribonucleic Acid
PCR-Polymerase Chain Reaction
TAS-ELISA- Triple Antibody Sandwich Enzyme-Linked Immuno Sorbent Assay
ACMV-African Cassava Mosaic Virus
BAP-6-Benzyle Amino-Purine
NAA-Naphthalene Acetic Acid
CTAB-Cetyltrimethylammonium Bromide
MS-Murashige and Skoog
2.4-D-Dichlorophenoxyacetic acid

Received Date: 09-Dec-2021 Accepted Date: 16-Jan-2022 Published Date: 28-Feb-2022

__________________________________________________________________________________________

Abstract
Grown in Côte d'Ivoire by smallholders, cassava takes great part of food security and poverty decreasing.
Cassava is most important crops in climate change context. Cultivation is done with cuttings derived from
local varieties. Unfortunately, these cuttings are susceptible to cassava mosaic disease which is transfer
from one crop to another one. Infected cassava plants produce low yields and tubers are bad quality. The
aim of this paper is to evaluate performance of thermotherapy engineering associated with meristem culture
for cassava cuttings sanitation. Ten cultivars of cassava were used. Fifteen cuttings of each cultivar were
collected and subjected to a temperature varying from 32 to 39°C under a photoperiod of 16/8h and
evaluated after three weeks. Thermotherapy associated with meristem culture was used to treat variety Yacé.
The results obtained showed that thermotherapy allowed a cleaning of cassava cuttings at percentages
ranging from 6 to 33%. In addition, thermotherapy and meristem culture of cultivar Yacé allowed to obtain
a percentage of sanitation of 92 %.
Keywords : Cassava - Côte d’Ivoire - Meristem culture - Sanitation - Thermotherapy
__________________________________________________________________________________________

INTRODUCTION
Once used as a subsistence crop during times of hunger, cassava has become a cash crop today (Thresh,
2006). This change is the result of high demand for derived products from cassava tubers such as attiéké
(fermented cassava semolina), for local consumption and sub saharan export (N’zué B, 2007). Cassava plant
adapts well to the effects of climate change and can grow on different soil types. Indeed, cassava is resistant
to periods of prolonged drought and produces average yields on marginal soils (FAO, 2013). Despite the
agronomic and economic benefits of cassava for food security and poverty reduction, the cassava during its
development faces to major constraints such as viral diseases. In Côte d'Ivoire, cassava mosaic disease is
caused by two major viruses, African Cassava Mosaic Virus (ACMV) and East African Cassava Mosaic

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Virus (EACMV) (Toualy et al, 2014). These are geminiviruses of the family Geminiviridae (Ogwok et al,
2015). Cassava mosaic disease is a systemic disease and is propagated by whitefly (Bemisia tabaci) by a
semi-persistent method (Jeremiah, 2015). After cassava plant infection, the using of infected cuttings as
planting materials becomes the second mode of disease spread (Munga et al, 2002). The use of cuttings
prone to infection promotes transmission of mosaic disease from one cassava field to another and from one
crop cycle to another over long distances (Legg JP et al, 2013). This results in a decrease in production
ranging from 20 to 90 % (IITA, 2000). This pandemic therefore becomes a real threat to food security in
Africa if it is not contained (Segnou et al, 2002). Various means of struggle exist. Those are : use of resistant
varieties, or virus-free cuttings, as well as sanitation of the plot limit the occurrence of diseases in newly
established plantations (Hillocks et al, 2003). Also, the systematic elimination of cassava shoots showing
symptoms of juvenile stage virosis greatly reduces the spread of the disease (Legg et al, 2011). However,
producers are reluctant to adopt improved varieties for reasons of late maturity and inadequate organoleptic
properties (Mwamachi et al, 2005). To address this, plant biotechnology techniques such as thermotherapy
and meristem culture are used as alternative ways to clean up local varieties of cassava (Zapata et al, 1995).
Meristematic areas of infected plants are generally virus-free because they have not differentiated cells or
contain minimal viral load (Wasswa et al, 2010). These same authors report that heat hinders the replication
of viruses. In Côte d'Ivoire, these techniques are not vulgarized or are little used for the remediation of local
plant species. The objective of this study is to contribute to improving the quality of cassava planting
material by using thermotherapy and meristem culture methods.

MATERIALS AND METHODS

Plant samples
The plant material consisted of cuttings and leaves collected on ten local cultivars of cassava namely
Nsakperne, Antenne, Six-month and Gnonrongouin (South-East), Zogloblé, Afery and Demoiselle (Center),
Diarassouba (North), Glagban (West) and Yacé (South). These cassava cultivars were grown in
experimental field at the University Nangui Abrogoua. The leaves of excised cuttings exhibited symptoms
of cassava mosaic disease.

Experimental design
The incubator is a cubic shape box of 1 m of side. The upper and lower faces are covered with plywood and
the other four (4) faces are covered with transparent plastic. Six incandescent bayonet bulbs (60-watt) were
installed indoors to serve as a source of heat and light (Figure 1). The electrical cable connecting the six
bulbs simultaneously was connected to the mains voltage via a timer. Inside the incubator, 2-liter jars
contain autoclaved soil at 121 ° C, at a pressure of 1 bar for 30 min.

Serological status of cuttings

The serological status of the different cultivars was verified with the young leaves of cultivars showing the
symptoms of the cassava mosaic disease. The TAS-ELISA test has been used (Clark et al, 1977). The
strains of ACMV (African Cassava Mosaic Virus) and EACMV (East African Cassava Mosaic Virus) were
detected using antibodies AS 0421-0421/2 and AS-0421-0421/4, respectively.

Cuttings thermotherapy
Cuttings were excised on plants with positive response to TAS-ELISA tests against the ACMV or the
EACMV. For each cultivar, five cuttings were used, and three replicates were performed. The cuttings were
planted in 2-liter pots in five rows in the incubator. The experiment was carried out with a photoperiod of
16/8 hours, a temperatures of 40 °C and 36 °C and a relative humidity of 65 %. Watering was done regularly
to keep the substrate moistened. After four weeks of incubation, the number of cuttings inducing shoot buds
was counted, then young leaves were excised to perform the TAS-ELISA.

Thermotherapy associated with meristem culture

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The cultivar Yacé was used in this experiment. It is the most cultivated cultivar by producers in Côte
d'Ivoire. Its tuber is used to prepare a dish called "Attiéké" very appreciated by most of the Ivorian
population. However, this cultivar is highly susceptible to cassava mosaic disease. In addition, compared to
other cultivars, the rate of recovery of healthy plants by the thermotherapy technique remains low in Yacé
cultivar. In order to improve this low rate, the apical meristems were isolated on the cuttings subjected to the
treatment of thermotherapy. A pre-disinfection was performed by washing segments with tap water, then
disinfection was carried out under a laminar flow hood, by soaking in 70 % ethanol for 3 min, followed by
immersion in a calcium hypochlorite solution (3 %) containing a drop of Tween 20 for a duration of 3 min.
The segments were subsequently rinsed four times in sterile distilled water. Then, the apical meristematic
domes were excised under a binocular microscope with scalpel blades n°11 and placed on culture media
(Figure 2).

Effect of medium culture medium composition on plants regeneration from meristems

For this study, the basic medium used was Murashige and Skoog with MS vitamins supplemented with
sucrose 3 % and myo-inositol 0.1 g/l (Murashige and Skoog, 1962). Different hormones were added
individually to the basic medium. Depending on the nature and concentration of the growth regulators, four
media have been formulated. The meristems were placed on the respective media. The composition of each
medium is shown in Table 1.

Effect of medium culture composition on shoots rooting

After three weeks of culture, the shoots were counted and then transferred on rooting media. MS basal
medium without growth regulator and MS basal medium supplemented with NAA (0.01 mg/l) and BAP
(0.05 mg/l) were tested for root induction.

Frequencies of apical meristem regeneration

The frequency of shoot induction was evaluated according to the following formula:
% of shoot induction =

Rooting frequencies of shoot buds

After four weeks of culture in rooting medium, the percentage of rooted seedlings was calculated according
to the following formula:
% of rooting =

Health status of seedlings regenerated from apical meristem

Indexing was performed from leaf DNA extracts obtained with 26 plants regenrated from from apical
meristems. Positive samples were diagnosed by PCR, with JSP primers whose sequences are shown in Table
2. After six weeks of culture, leaves were excised from in vitro plantlets regenrated from apical meristem.
The leaves were used for indexing by enzymatic amplification test of nucleic acid sequences (PCR).

Cetyltrimethylammonium Bromide DNA Extraction Protocol (CTAB)

The protocol used for the extraction of DNA samples is that of Doyle and Doyle modified and adapted to
our plant material (Doyle and Doyle,1987). 0.3 g fresh leaf of each sample was milled in 1500 μl of CTAB
buffer, vortexed and the mixture is incubated at 60 °C in a water bath for 60 minutes with gentle stirring.
After which, the ground samples of the different samples are centrifuged at 20000 rpm for 10 min. Then 700
μl of the supernatant are collected in eppendorf tube (2 ml) to which 700 μl of chloroform isobutyl alcohol
(CIA) are added. The mixture is vortexed for 5 min and then centrifuged for 10 min at 20000 rpm for 10
min. Then, 400 μl of the supernatant was removed and poured into an eppendorf tube and 400 μl of
isopropanol were added. The whole was vortexed for 5 min to precipitate DNA. The mixture obtained is
incubated at -80°C. for 30 min and then centrifuged at 20000 rpm for a period of 10 min. The pellet
containing the DNA is taken up in 500 μl of 70 % ethanol. The mixture is centrifuged and the ethanol is
discarded and the eppendorf tubes containing the pellets of the DNA of each sample are left open air
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February 28, 2022
overnight for dehydration. The DNA is diluted in 100 μl of distilled water. This fraction was used to
evaluate the amount of DNA from each fresh leaf sample. The different reagents and their concentrations,
for a DNA sample, are shown in Table 2. Five µl of DNA solution were diluted to 1/50th and 20 µl of
reagents were added.

PCR program
The PCR program is constituted a first denaturation phase at 94 °C for 4 min. After the second, which lasted
1 min, the hybridization process was started at a temperature of 55 °C for 1 min. This phase was followed by
the elongation phase, carried out for 1 min at 72 °C. This is followed by another phase of elongation, done
for 10 min at the same temperature as before. Those three phases (denaturation, hybridization and
elongation) were carried out in 35 cycles. The PCR products were electrophoresed on 1.2 % agarose gel for
2 hours. Then, the gel was visualized using a transilluminator and then photographed.

Meristem culture conditions

The cultures were placed on shelves in the culture room according to a completely randomized device and
incubated under a photoperiodic cycle of 12/12 hours and a temperature of 24 ± 2 °C. The light is provided
by fluorescent lamps with an intensity of 3000 lux, at a relative humidity of 60 %.

Data analysis
The data collected during heat therapy of the ten cultivars, the number of rooted seedlings and the number of
seedlings sanitized after thermotherapy coupled with the meristem culture were calculated with Excel 2010
software. The percentages of budburst and rooting were transformed by the Arcsinus √(x) angular formula
and analyzed using the STATISTICA version 7.1 software. The analysis of variance has a factor (ANOVA
1) was used to compare the averages. When a significant difference is observed, the Newman-Keuls test is
used to separate the means at the threshold α = 5 %.

RESULTS
Thermotherapy of cuttings
In this experiment, cuttings of different cultivars with symptoms of cassava mosaic disease were heat
treated. After four weeks of incubation, the severity index of cassava mosaic disease was estimated and
compare to severity before heat therapy. The results obtained are shown in Table 3. The analysis of this table
shows that the average severity index of the disease was reduced following the treatment of thermotherapy.
It ranged from 0.73 to 2.56 as opposed to a variation of 1.27 to 3.4 before thermotherapy. In addition, the
highest disease severity index before (3.40) and after (2.50) thermotherapy was recorded in the cultivar
Yacé.
The recovery rate in healthy plants after indexing by the TAS-ELISA test was calculated. The results
obtained are shown in Table 4. The analysis of this table shows that the rate varied from 6.66 to 33.33 %.
The highest rates of healthy plants recovery were recorded with the following cultivars: Afery, Diarassouba,
Nsakperne, Antenne, Demoiselle, Six-mois and Zogloblé. On the other hand, cultivars with low rates of
recovery of healthy plants are: Gnonrongouin (13.33 %), Glagban (13.33) and Yacé (6.66 %). The
appearance of the leaves of two cultivars (Six-mois and Yacé) after thermotherapy is shown in Figure 3. The
leaves of six-mois after heat treatment are colored uniform (green) and well full-blown without deformation
(Figure 3a). In Yacé cuttings non-sanitized after heat treatment, the leaf blade has deformities and a mosaic
color shown by red arrows (Figure 3b).

Association of thermotherapy with meristem culture

Effect of culture medium composition on shoot buds induction
Four (04) organogenesis media supplemented with different concentrations of growth hormones were used.
Meristems excised from cuttings with leaves exhibiting cassava mosaic symptoms after thermotherapy
treatment were placed on culture media. Four weeks later, experimental results were recorded in Table 5.
Composition of the culture medium significantly influenced the rate of shoot buds induction from meristem

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February 28, 2022
explants with frequencies varying between 0 and 63.25 %. M2 and M3 media favored the emergence of
shoot buds. The highest percentage (63.25 %) was recorded on M2 medium whereas no shoot induction was
recorded on the control medium (Cm) and the M1 medium (1/2 MS + 0.5 mg/l BAP + 0.1 mg/l 2.4-D).

Effect of culture medium composition on the rooting of shoot buds

The leafy shoots were transferred on different rooting media. After four weeks of culture, the percentage of
rooting was estimated. The results were recorded in Table 6. All the shoot buds (100 %) have rooted on the
MS basal medium devoid of phytohormone. MS medium supplemented with BAP (0.05 mg / l) + NAA
(0.01 mg / l) expressed a rooting frequency of 85.60 %.

Health status of the regenerated in vitro plants by PCR

Of a total of 26 samples, 22 were remediated, representing a remediation rate of 92.31 % (table 7). The
Figure 4 illustrates two profiles of electrophoresis migration of PCR products. Figure 4a shows the
migration pattern of virus free samples of cassava mosaic disease, while Figure 4b shows the migration
profile of virus free samples and an un-sanitized sample (20A).

DISCUSSION
Cassava mosaic virus was removed from cassava cuttings treated with thermotherapy with a rate varying
between 6 and 33 %. This difference in sanitation rates could be explained by a cultivar effect. Some
cultivars are more susceptible to cassava mosaic disease than others. This is the case of the cultivar yacé
whose sanitation rate is 6 %. Concerning viral particles neutralization in plant material could be explained
by the temperature variation of 36 °C in the dark and 39 °C in the light (Sastry et al, 2014). Their work
revealed that temperature conditions above 30 °C slow or block the replication of viruses. In addition, heat is
important to the slowing or stopping of the multiplication of viral particles (Astier et al, 2001). The heat
hinders the activity of the replicase and creates an instability of the viral particles. Moreover, in a condition
of increase temperature, there is a weakening of the links between the protein subunits of the genetic
material of the viruses. This induces cracks that cause their destruction by nucleases (Allam, 2000). The
whole process of the viral cycle including replication and distribution that would be affected by the rise in
temperature in this virus-plant interaction (Del Toro et al, 2015).
This methodology is based on the principle that the apical meristem does not contain or has very few viruses
(Morel, 1948). In our study, the thermotherapy preceding the meristem culture would extend the apical zone
devoid of viral particles around the meristem. Indeed, the alternation of the temperature (32 ° C and 39 ° C),
would limit the movements of the viral particles towards the apical tissues. This facilitates the excision of a
large meristem to generate plantlets. This would explain the high percentage of remediation (92.31%).
Elimination of ACMV at 95 % by applying thermotherapy associated with meristem culture on four cassava
accessions have been reported (Yandian, 2015). Sanitizing of potato infected with potato virus X (Solanum
tuberosum) by combining thermotherapy and meristem culture have been successfully done (Li et al, 1994).
Concerning sweet potato (Ipomoea batatas), similar results have been reported (Mervat, 2009). The results
of their work showed the effectiveness of thermotherapy coupled with the meristem culture for the
elimination of the marbling virus from sweet potato.

CONCLUSION
Cassava mosaic disease caused by the ACMV is a real threat to a sustainable cassava crop. The
thermotherapy technique resulted in a recovery rate of 6 to 33 %, with the cultivar Yacé having the lowest
rate of recovery of healthy plants is 6 %. The thermotherapy associated with meristem culture in this cultivar
was able to neutralize the viral particles of cassava mosaic with a recovery rate of healthy plants of 92 %.
The healthy in vitro plants will be acclimatized after a mass production from nodal segments and deliver to
producers for field establishment.

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FIGURES LIST

Figure 1. Experimental device adopted for the thermotherapy of diseased cuttings of cassava mosaic

a b

c d

e f

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 International Journal of Agriculture and Biological Sciences- ISSN (2522-6584) Jan & Feb 2022
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Figure 2. Different steps of thermotherapy associated to meristem culture of cultivar Yacé.
(a) emission of young buds. (b) twigs containing the meristematic buds. (c) meristem sampling. (d) meristems on
culture medium. (e) leafy shoot from meristem after three weeks of culture. (f) evolution of the leafy shoot into
seedling.

a b
Figure 3. Leaves of cassava plants after a treatment of thermotherapy. (a) Cultivar six-mois sanitized with healthy
leaves; (b) non-sanitized Yacé cultivar, the red arrows indicate cassava mosaic disease symptoms.

1500 Pb
1000 Pb
500 Pb b

1500 Pb
1000 Pb
500 Pb

Figure 4. Electrophoresis of PCR products of cassava leaf samples after thermotherapy associated with meristem
culture. (a) gel showing seven virus free samples. (b) gel contract seven samples with CVMA-positive Sample 20. M
= molecular weight marker. 1Kb= DNA Ladder size. A = ACMV. E = EACMV. TA +(ACMV positive control = 783
bp). TE + (positive indicator EACMV = 780 bp). TA- (ACMV negative light). TE- (Negative Witness EACMV).
TABLES LIST

Table 1. Composition of culture media

Media Basal media BAP ANA GA3 2,4-D
Control media (Cm) MS + Vit MS - - - -
Medium 1 (M1) 1/2 MS + Vit MS 0.5 mg/L - - 0.1m g/L
Medium 2 (M2) MS + Vit MS 0.1 mg/l 0.2 mg/l 0.04 mg/l -
Medium 3 (M3) MS + Vit MS 0.15 mg/l 0.18 mg/l 0.02 mg/l -
MS : Murashige and Skoog, Vit MS : MS Vitamins

Table 2. PCR reagents for the preparation of master mix

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Reagents Initial Volume Final

concentration concentration
Tampon green colorless 5X 5µl 1X
MgCl2 25 mM 1 µl 1 mM
dNTP 10 mM 0,5 µl 0,2 mM
Primer JSP001 10 µM 1 µl 0,4 µM
Primer JSP002 (ACMV) or JSP003 (EACMV) 10 µM 1 µl 0,4 µM
GoTaq ® Polymerase 5 u/µl 0,125 µl 0,625 u
Distilled water - 11,375 µl -
(JSP001) F 5’-ATGTCGAAGCGACCAGGAGAT-3’ (JSP001) F 5’-ATGTCGAAGCGACCAGGAGAT-3’
(JSP002) R 5’-TGTTTATTAATTGCCAATACT-3’ (JSP003) R 5’-CCTTTATTAATTTGTCACTGC-3’
Primer sequence against ACMV Protein Shell Primer sequence against EACMV Protein Shell

Table 3. Effect of heat therapy on the disease severity index in ten cultivars showing symptoms of cassava mosaic
disease
Cultivars Average severity index before Average severity index after
heat therapy thermotherapy
Gnonrongouin 2,27±0,11b 1,60±0,21b
Glagban 2,80±0,35ab 1,73±0,27b
Zogloble 2,73±0,31ab 1,40±0,16b
Yacé 3,40±0,21a 2,56±0,28a
Antenne 2,73±0,11ab 1,80±0,24b
Nsakperne 1,93±0,34b 1,47±0,16b
Demoiselle 2,87±0,16ab 1,73±0,11b
Afery 2,58±0,12a 1,60±0,21b
Diarassouba 2,58±0,12a 1,60±0,13b
Six mois 1,27±0,22c 0,73±0,11c
F 6,39 8,25
P ˂ 0,000 ˂ 0,000
The figures followed by the same letter are statistically identical to the threshold α = 5% (Newman-keuls test); Mean ± standard
error.

Table 4. Indexing and recovery rates of healthy plants after thermotherapy treatment
Cultivars Serological Number of cuttings by serological status after Recovery rate of healthy plants after
status thermotherapy thermotherapy (%)
+ 13
Gnonrongouin 13,33
- 02
+ 13
Glagban 13,33
- 02
+ 10
Zogloblé 33,33
- 05
+ 14
Yacé 06,66
- 01
+ 12
Antenne 20,00
- 03
+ 11
Nsakperne 20,00
- 04
+ 12
Demoiselle 20,00
- 03
+ 05
Afery 33,33
- 10
+ 10
Diarassouba 33,33
- 05
+ 10
Six-mois 33,33
- 05
+: no virus-free sample. - : virus-free sample,

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 International Journal of Agriculture and Biological Sciences- ISSN (2522-6584) Jan & Feb 2022
February 28, 2022
Table 5. Shoot buds induction from meristem explants of cultivar Yacé after a treatment of thermotherapy
Culture media Percentage of meristem explants inducing
shoot buds (%)
Control medium(Cm) 0.00±0.00 c
Medium 1 (M1) 0.00±0.00 c
Medium 2 (M2) 63.25±9.00 a
Medium 3 (M3) 11.75±7.43 b
P ˂0.000
Statistics
F 26.720
In the same column, the mean ± standard deviation followed by the same letter are statistically identical at α = 5%
threshold (Newman-keuls test).

Table 6. Effect of the composition of the culture medium on the rooting of seedlings
Basal media BAP (mg/l) ANA(mg/l) Percentage of rooting (%)
MS 0 0 100 a
MS 0.05 0.01 85.60 b

Table 7. Percentage of sanited after thermotherapy combined with Yacé cultivar meristem culture

Number of treated Number of sick Number of viruses-free Percentage of recovery of

plantlets seedlings plantlets healthy plantlets (%)
26 04 22 92,31

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