**2.2.2 Soil microorganisms**

The microorganisms were obtained by direct isolation from the soil according to the method described by Davet and Rouxel (1997) and from fragments of pod husk buried in the soil samples. The soil samples were beforehand dried, ground and calibrated by sieving. The fragments of husk were ground in a porcelain mortar to separate each living propagule, because of the gelatinous consistency of the decomposing pod husk.

In both cases, 10 g of ground soil were transferred in 90 ml of sterile distilled water contained in an Erlenmeyer. The mixture is then put in agitation for 30 minutes to obtain a good separation of the particles. To obtain a variable concentration of propagules and facilitate the enumeration of the colonies, a series of dilution was performed from the initial solution whose concentration was 10-1 (Rapilly, 1968). To obtain a solution of 10-2, 1 ml of the initial solution was mixed in 9 ml of sterile distilled water. Thus, a series of dilution from 10- 2 to 10-9 was performed in hemolytic tubes. For each dilution, 100µl was pipetted and spread onto the surface of the culture media in Petri dishes. For each dilution and for each medium, 4 Petri dishes were inoculated. The incubation was also made in the dark in a steam room at 26 °C for 2 days for the bacteria and 7 days for the fungi.

### **2.3 Conservation and identification of the microorganisms**

The microorganisms isolated were first purified by two or three successive monospore transplantings on specific culture media. Once purified, each isolate was designated by a code number. For strains of bacteria, the code numbers were preceded by the letter B, followed by an order number. The nomenclature of the fungi isolates begins with one or several first letters of the name of the genus, followed by an order number. The conservation of the microorganisms was then made in a freezer at a temperature of - 80°C for the bacteria, and - 10°C for the fungi. In both cases, agar disks taken near the edge of the purified culture were transferred to 1.5 ml sterile Eppendorf microtubes containing glycerol at 50 %. The identification of the isolated microorgamisms was based on the macroscopic, microscopic, biochemical and molecular characters. The molecular characterization was made by the

Isolation and Identification of Indigenous Microorganisms of Cocoa Farms in

samples were taken on the treated trees every 15 days.

Student Newman and Keuls test at 5%.

**3.1 Microorganisms isolated** 

**2.5 Data analyses** 

**3. Results** 

plantation and the pod cortex

Occurence (%)

Côte d'Ivoire and Assessment of Their Antagonistic Effects Vis-À-Vis *Phytophthora palmivora…* 307

group were chosen randomly in the field, numbered and marked with the same color. However, care was taken so that each test tree was surrounded by 8 border trees. The *Trichoderma* based biological fungicides were applied using a knapsack sprayer at the concentration of 107 conidia ml-1. The entire cocoa tree was treated. Six applications at 21 days interval were made. Over the duration of the trial, weekly count were made for healthy mature pods, rotting pods, wilting pods and pods damaged by squirrels. During the trial, the survival of *Trichoderma* on treated pods and flower cushions was evaluated. Thus, pod

The SAS program (Statistical Analysis System, SAS Institute, Cary, NC) was used for all the statistical analyses. For the isolation of the microorganisms and the leaf disk test, the analyses of variances (ANOVA) were performed on the mean number of microorganisms colonies counted on the culture media and the mean rating score of leaf susceptibility to *Phytophthora* in the presence of bacteria and fungi. The normality of residuals and the homogeneity of the variances were verified. The mean comparisons were realized with the

The exploration of the biodiversity of microorganisms obtained from soils of cocoa field and endophytes of pods revealed 2 categories of microorganisms: fungi and bacteria. On the pods, the fungi represented 66.31% of the positive isolation, against 33.04 for the bacteria. In the soil, the two categories of microorganisms were present in inverted proportion: 55.8% for the bacteria and 29.8% for the fungi. In the group of the fungi, the yeast that represented 11.6% of the population on the pods, represented only 3.7% in the soil. Among the bacteria, the Actinomycetes which represented only 0.64% on the pods, reached 10.5% in the soil (Fig.1).

Other fungi Bacteria Yeast Actinomycetes

Fig. 1. Group of micro-organisms and their relative importance in the soil under cacao-

Microorganisms

Endophyte of pods Soil under cacao tree

method developed by Druzhinina *et al*. (2005) to identify the various species of fungi isolated. This method uses baits specific targeting genes encoding for translation elongation factor 1-alpha (*tef1*) obtained on sequences IST 1 and 2 of the DNAr.

#### **2.4 Evaluation of the antagonistic effect of the microorganisms**

The antagonistic effect of the isolated microorganisms against *P. palmivora* was evaluated using three tests. The first test conducted *in vitro*, is a test of direct confrontation between *P. palmivora* and the microorganism in a mixed culture. The second test was realized *in vivo* on leaf disks of cacao tree. The test consists in measuring the leaf susceptibility to *P. palmivora* in the presence of the microorganism according to the scoring scale of Blaha which varies from 0 to 5 (Nyassé *et al*., 1997). The third test was carried out in the field on cacao trees.

The confrontation between *Trichoderma* and *P*. *palmivora* was realized in Petri dishes containing agar culture media made of potato broth (PDA medium). A fragment of mycelium, 6 mm in diameter, was taken around the edge of the cultures of each fungus. These fragments were transplanted face to face in the same Petri dish, 2 cm from the center of the dishes (Benhanou and Chet, 1996). The controls were monocultures of each of the 2 fungi being confronted. In this case, the fragment of mycelium was placed in the center of the Petri dishes. Each treatment was conducted in 6 replications. The incubation was done in the dark in a cryptogrammic steam room. Twenty four hours after the start of the cultures, the mycelial growth of each fungus was measured daily until the Petri dish was full with the fungal development. Seven days after the mycelial strands of both fungi have met, the survival of the spores of *P. Palmivora* was evaluated by taking fragment of mycelium in the Petri dishes containing the mixed culture according to an axis which passes by the center and the transplanting sites of both fungi. Two consecutive samples were taken 0,5 cm apart. Thus, nine samples were taken in every Petri dish of mixed culture. The fragments of mycelium taken were placed in lesions made on healthy pods collected from trees of the same clone, making sure the pods were not attacked by *Phytophthora* sp. These inoculated pods were placed in crystallizers in which the humidity was maintained by a plug of sterile cotton wool soaked with sterile distilled water. The incubation was done at the ambient temperature of the laboratory. The survival of *Phytophthora* was evaluated by recording the number of brown spots on the surface of the inoculated pods after 15 days of daily observation. The presence of *Phytophthora* in the spots was confirmed by microscopic observations.

The effect of the microorganisms on *P. palmivora* was evaluated through the leaf disk test. This test was performed on disk of cocoa leaves 15 mm in diameter. The disks were beforehand dipped into the bacterial suspension or the suspension of *Trichoderma* for 1 min, and then arranged in containers on a plate of foam soaked with water. Each disk received 10 µl of a suspension of zoospores of *P. palmivora* calibrated to 3.105 zoospores / ml. The controls were not dipped in bacterial suspension or suspension of *Trichoderma* before receiving the suspension of zoospores of *P. palmivora.* The leaf disks were obtained from 3 cocoa clones, the reaction of which to *Phytophthora* sp. is known (Tahi et *al*., 2000). Thus the susceptible clone (IFC5), the moderately resistant clone (P7) and the resistant clone (SCA6) were tested. For each clone, 40 leaf disks were inoculated and placed in 4 containers, each representing a replicate. The incubation was done in the dark at 26°C for 7 days. The results were scored according to the scale of Blaha (Nyassé *et al*., 1995).

In the field, the random target method was used to assess the effect of the microorganisms on *P. palmivora*. This method consisted in following the development of the disease on groups of 100 Cacao trees with different treatments. The 100 trees corresponding to each group were chosen randomly in the field, numbered and marked with the same color. However, care was taken so that each test tree was surrounded by 8 border trees. The *Trichoderma* based biological fungicides were applied using a knapsack sprayer at the concentration of 107 conidia ml-1. The entire cocoa tree was treated. Six applications at 21 days interval were made. Over the duration of the trial, weekly count were made for healthy mature pods, rotting pods, wilting pods and pods damaged by squirrels. During the trial, the survival of *Trichoderma* on treated pods and flower cushions was evaluated. Thus, pod samples were taken on the treated trees every 15 days.
