**2.5 Data analyses**

306 Biodiversity Loss in a Changing Planet

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

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

factor 1-alpha (*tef1*) obtained on sequences IST 1 and 2 of the DNAr.

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

*Phytophthora* in the spots was confirmed by microscopic observations.

were scored according to the scale of Blaha (Nyassé *et al*., 1995).

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

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 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 Student Newman and Keuls test at 5%.
