BREEDING OF COCOA TREES (THEOBROMA CACAO L.) RESISTANTTO PHYTOPHTHORA MEGAKARYA , AGENT OF BLACK POD DISEASE IN COTE D'IVOIRE

Backgrownd : Black pod disease is the cause of significant production losses of cocoa trees. This work aims to select tolerant and resistant genotypes to Phytoththoramegakarya within the main collection of cocoa trees of the National Center for Agronomic Research. Methods : The artificial inoculation


ISSN: 2320-5407
Int. J. Adv. Res. 9(09), 793-803 794 increasingly facing many production constraints. These include the low level of use of improved plant material (Koua et al., 2018), the aging of the orchard (Assiri et al., 2009;2016;Koua et al., 2018) and the high pressure parasitic. The latter is caused by pests such as mirids [SahlbergellasingularisHagl. (Mirideae)] (Kouamé et al., 2014) and diseases such as black pod rot, the main agent of which in Côte d'Ivoire is Phytophthorapalmivora but the most damaging agent in the field is Phytophthoramegakarya. In Côte d'Ivoire, black pods disease contributes around 10 to 25% of production losses. However, it can cause up to 60% losses in some regions when agro-ecological conditions are favorable for the development of the pathogen (Coulibaly, 2014).
The disease begins on the pods with the appearance of a brownish-colored spot that spreads quickly and can gradually cover the entire surface of the pod. In humid weather, the spots become covered with a whitish mycelial felting. Examination of the mycelial felting covering the diseased pod reveals the presence of numerous conidia which are the asexual reproduction organs of the fungus (Coulibaly, 2014). Conidia release ciliated zoospores which, dispersed by water, wind or insects, can contaminate new fruits. The germination of zoospores requires the presence of water so that contamination often begins at the apex of the fruit which, due to its shape, retains water in a hanging droplet in which the zoospores can move, germinate and infect the fruit (Braudeau, 1969). Infection begins with zoospores (germs) entering the stomata or through the epidermis. Germination of zoospores can also occur in water retained on fruit at the stalk attachment or between two adjacent pods.
One of the most using ways to control black pod disease is the use of phytosanitary products. However, this method is expensive and dangerous for the environment. To remedy this state of affairs, research is increasingly turning to genetic control to select cocoa trees resistant to brown rot (Nyassé et al., 1995;Tahi et al., 2000). The evaluation of the resistance of cocoa trees to pod rot by the leaf disc test represents an efficient, rapid and reliable technic tested by several authors (Nyassé et al., 1995;Tahi et al., 2000) for selecting cocoa trees resistant to this disease. Indeed, a positive and significant correlation has been demonstrated between the classification of genotypes by evaluation via test on cocoa leaves in the laboratory and the rate of brown rot of pods of these same genotypes observed in the field (Tahi et al., 2003). Therefore, the artificial inoculation test makes it possible to quickly assess the level of sensitivity of cocoa trees to black pod, which makes it possible to shorten the selection cycles of cocoa trees resistant to Phytophthorasp (Charbierski, 2000). Furthermore, the use of leaf discs instead of whole leaves is justified by the results obtained by Tahi et al. (2000). Indeed, these authors showed a non-significant interaction between clones and organ size. These results thus indicate that the behavior of the plant material on leaf discs does not vary significantly compared to their behavior on whole leaves. Thus, leaf discs, less bulky and easily allowing the use of a high number of clones per tank were used for the evaluation.
In the context of this study, the resistance of 52 clones to P.megakarya, an agent of brown pod rot was measured by artificial inoculations with a calibrated suspension of zoospores on leaf discs (Nyassé et al., 1995) following the protocol proposed by Tahi et al. (2000).

Plant material
The plant material is composed of 52 potentially high-producing clones from the CNRA collection including three clones of variable sensitivity to brown pod rot, used as reference controls. It isis NA 32, susceptible to disease; PA 150, moderately resistant to disease and SCA 6, resistant to disease (Tahi, 2003). The list of plant material is presented in Table 1.

Fungal material
The fungal material used for this study is a strain of P. megakarya, isolated from a pod naturally infected with brown rot. The strain was subcultured, maintained and stored on an artificial medium based on pea agar (Figure 1). The strain was cloned by mono-zoospore subculturing and its aggressiveness was re-tested on healthy pods.

Experimental design
The test wasrealisedusing a complete random block with 4 sub-blocks. Three repetitions of the test were carried out in order to have a solid database for statistical analyzes. Fifty-two cocoa clones (at the rate of 10 leaf discs per clone) were evaluated per tank. Forty leaf discs per clone were thus inoculated for all four tanks in a series. Thus, 2080 leaf discs for all 52 clones were inoculated per repeat.

Preparation of inoculum of strains of Phytophthoramegakarya
The inoculum usingis a suspension of zoospores of P.megakarya. The zoospore suspension was prepared from a strain grown on pea agar medium. The culture was incubated in the dark for six days at 26 °C. She was subsequently subjected to a 12-hour photoperiod for at least two days to induce sporocyst formation. Germination of the sporocysts was induced by heat shock by placing the culture for 15 min at 4 °C and then flooding it with 40 mL of sterile distilled water at room temperature. The suspension thus obtained was quantified using an optical microscope (Malassez cell) and the concentration was brought back to 3.105 zoospores per milliliter.

Collect of leaf samples
Two healthy, semi-august leaves located on the lower strata of the trees were taken very early in the morning (around 6.30 am) from each of the 52 clones (including the 3 controls). They were labeled, bagged and stored in a cooler containing foam soaked in distilled water. The plant material was then quickly transported to the laboratory to start the inoculations according to the protocol of Tahi et al. (2000).

Leaf inoculation
Healthy sampled leaves were preconditioned overnight to make the leaf blade more receptive. This step consisted of placing the underside of the sheet in a tray against a foam soaked in distilled water. After preconditioning, 40 leafs discs 15 mm in diameter per clone were cut from the leaf blades using a cookie cutter. The leaf discs were placed in the trays and then inoculated on the underside of the blade, by depositing 10 μL of zoospores suspension calibrated at 3.10 5 zoospores / mL using a micropipette ( Figure 2). Subsequently, the trays containing the inoculated leaves were sealed with black plastic wrap and incubated at 26 °C in the dark for seven (7) days.

Data collect
Seven days after incubation, data were collected on each leaf disc according to the scale proposed by Blaha (1974) and updated by Nyassé et al. (1995). Data collection consisted of observing each inoculated leaf disc and assigning it a sensitivity score according to the scale of Nyassé et al. (1995) presented in Table 2.
Statistical analyzes of data P. megakarya leaf sensitivity scores were analyzed with SAS 9.4 software. The statistical analysis consisted of a comparative study between the clones in order to highlight any differences between them. For this purpose, analysis of variance (ANOVA) was previously used. Any significant ANOVA (P<0.05) is followed by the test for the smallest significant difference (ppds) in order to classify the different clones for the characteristic considered. Furthermore, a Dunnett test was used in order to define the grouping of the clones according to each of the three controls (resistant, moderately resistant and sensitive). Indeed, the post-hoc test (or multiple comparison test) was used to determine the significant differences between the mean sensitivity scores of the clones and those of each of the three controls. Table 3 shows the results of Dunett's test showing the significance of the difference between the sensitive control NA 32 and the 49 clones analyzed. Analysis of the table indicates that all the clones analyzed, with the exception of IFC 1035 (sensitivity score = 3) and CC 39 (sensitivity score = 3.06) presented sensitivity scores significantly lower than the sensitive control NA 32 (sensitivity score = 3.31). Table 4 shows the results of Dunett's test showing the significance of difference between the moderately resistant control PA 150 and the 47 clones analyzed. Indeed, the two clones identified as sensitive were removed from the database before performing the statistical analyzes. The results indicate that four clones (IFC 1041; IFC 1027; GU 346 / R; GU 322 / B) exhibited respective sensitivity scores of 1.54; 1.63; 1.47; 1.36; significantly lower than the moderately resistant control PA 150 (NS = 2.59). These clones could be qualified as resistant or very resistant to brown pod rot. The remaining 43 clones [2.59 (PA150) <NS <3.31 (SCA6)] showed higher sensitivity scores than the moderately resistant control (PA150) (NS = 2.59) and lower than the resistant control (SCA6). These genotypes could therefore be qualified as moderately resistant

Results:-
The results of Dunett's test showing the significance of the difference between the resistant control SCA 6 and the four clones analyzed are presented in Table 5. Indeed, the 43 clones identified as being moderately resistant were removed from the database before to perform statistical analyzes. The table Indicates that the two clones (IFC 1041 and IFC 1027) presented respective sensitivity scores of 1.54 and 1.63, higher than that of the resistant control SCA 796 6 and lower than that of the moderately resistant control [1.73 (SCA6) <NS <2.59 (PA 150)]. These clones could be described as resistant to brown pod rot. Finally, the two remaining clones (GU 346 / R; GU 322 / B) presented respective sensitivity scores of 1.47 and 1.36, lower than those of the resistant control SCA 6 (NS = 1.73). These clones could be described as very resistant to brown rot. Table 6 presents the mean values of the susceptibility scores to P. megakarya of potentially high-producing clones. Table indicates that the leaf sensitivity scores of the clones to this pathogen are between 1.36 (GU 322 B) and 3.31 (NA 32) with an average of 2.38 ± 0.74 and a coefficient variation of 14.18. The results indicates a very highly significant difference (P<0.0001) between the 52 clones analyzed for P. megakarya infection scores. The results of this table show the structuring of the genotypes according to the 3 different groups of sensitivities.

Discussion:-
This study consisted of the evaluation of the resistance by leaf disc test to P. megakarya of 52 clones potentially high producers and resistant to brown pod rot in the field. This work was undertaken to better appreciate the tolerance to brown rot of the clones evaluated.
The results of this study showed a very highly significant difference (P<0.001) between the 50 clones analyzed for the scores of susceptibility to P. megakarya. Three levels of sensitivity make it possible to structure the analyzed clones. Indeed, the results made it possible to highlight three groups of sensitivity according to the reference controls. The first group is composed of two clones (IFC 1035 and CC 39) qualified as susceptible to brown rot. The second group is made up of 43 clones qualified as moderately resistant. The third group is composed of four clones qualified as resistant (IFC 1041 and IFC 1027) and very resistant (GU 346 / R; GU 322/B) to the pathogen. These results confirm the horizontal nature of the resistance to brown pod rot that would characterize cocoa trees, as indicated by Tahi (2003). In addition, the clones qualified as very resistant belong to the "Guiana" genetic group (GU 346 / R; GU 322 / B). These results are in agreement with those of Paulin et al. (2008) whose work focused on identifying new sources of resistance to P. megakarya in cocoa trees. These authors identified seven (07) and 29 new clones of the Guiana genetic group, respectively very resistant and resistant to P. megakarya. The results of these authors also showed that the clones of this genetic group would constitute sources of resistance to P. megakarya. Their study thus confirmed the good level of resistance of the clones of the "Guiana" group to P. megakarya and the important role that the clones of this genetic group could play in a breeding program. This program would aim to control the pathogen and select clones tolerant to brown pod rot (Dzahini-Obiatey& Fox, 2010). Moreover, based on the existence of a positive and significant correlation highlighted by the work of Tahi et al. (2000), clones GU 346 / R and GU 322 / B, which presented lower sensitivity scores than the resistant control SCA6, will be characterized by a high resistance toBlack pod disease.

Conclusion:-
This work consisted of an evaluation of resistance to P. megakarya of 52 cocoa clones notable for production and resistance in the field to brown pod rot.
The results revealed three groups of genetic diversity, within the population studied, according to their susceptibility to P. megakarya. The first group is composed of two clones (IFC 1035 and CC 39) sensitive to P. megakarya. The second group consists of 43 clones moderately resistant to brown rot. The third group is composed of four clones qualified as resistant (IFC 1041 and IFC 1027) and very resistant (GU 346 / R; GU 322 / B) to the pathogen. In addition, the group qualified as very resistant to brown rot was marked by a strong contribution from clones of the Guiana genetic group.
These results constitute an important source of information for the breeder and an indicator in the choice of clones to be proposed for variety release. In addition, these high-performance clones constitute broodstock to be integrated into a breeding program for plant material resistant to brown pod rot.    Marble spots (large, uniform spots) sensitive 5

List of tables
True spots (very large necrotic spots, sometimes exceeding the limits of the inoculation drop) Very sensitive