**4. Discussion**

This study revealed the existence of a high biodiversity within the microorganisms' populations in the cocoa ecosystem. Regarding the two areas investigated, the soil microorganisms and pod endophytes, the biodiversity varied quantitatively and qualitatively. Endophytes are microorganisms that colonize plant tissues without causing visible symptoms in normal conditions (Carroll, 1998). Since the beginning of the 2000s, these microorganisms are usually studied in the tropical plants for their use in biological control or for the production of substances having pharmacological properties (Azevedo, 2002; Peixoto-Neto, 2002). The isolation carried out on cocoa pods showed a predominance of fungi (66.31%), against 33.04% of bacteria. Similar results were obtained by Evans *et al*. (2003) and Rubini *et al*. (2005). Regarding the soil, the results also showed a high proliferation of both categories of microorganisms with a greater proportion of bacteria. The soils of cocoa field and the pods are therefore the preferred sites of indigenous microorganism, potential antagonists of *P palmivora*, occupying the same ecological niche. The results showed that the use of baits, made by fragments of cocoa pod, infected by *P. palmivora*, significantly improved the isolations of microorganisms in the soil. This improvement could be explained by an affinity between *P. palmivora* and the collected

higher than 60 % for the isolate T54 of *T. asperellum*. The analysis of the evolution of the epidemic curves in each treated plot reveals that the isolate T54 of *T. asperellum* and the isolate T7 of *T. virens* substantially reduced the losses due to the black pod disease. Furthermore, these isolates delayed the onset of the epidemic of the black pod disease

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Fig. 5. Effect of the applications of biofungicide containing various species of *Trichoderma* on

This study revealed the existence of a high biodiversity within the microorganisms' populations in the cocoa ecosystem. Regarding the two areas investigated, the soil microorganisms and pod endophytes, the biodiversity varied quantitatively and qualitatively. Endophytes are microorganisms that colonize plant tissues without causing visible symptoms in normal conditions (Carroll, 1998). Since the beginning of the 2000s, these microorganisms are usually studied in the tropical plants for their use in biological control or for the production of substances having pharmacological properties (Azevedo, 2002; Peixoto-Neto, 2002). The isolation carried out on cocoa pods showed a predominance of fungi (66.31%), against 33.04% of bacteria. Similar results were obtained by Evans *et al*. (2003) and Rubini *et al*. (2005). Regarding the soil, the results also showed a high proliferation of both categories of microorganisms with a greater proportion of bacteria. The soils of cocoa field and the pods are therefore the preferred sites of indigenous microorganism, potential antagonists of *P palmivora*, occupying the same ecological niche. The results showed that the use of baits, made by fragments of cocoa pod, infected by *P. palmivora*, significantly improved the isolations of microorganisms in the soil. This improvement could be explained by an affinity between *P. palmivora* and the collected

Weeks

control

*T.spirale* T4

*T.virens* T7

*T.harzianum* T40

*T.asperellum* T54

compared to the untreated control (Fig. 5).

the evolution of black pod disease in the field.

**4. Discussion** 

Rotten pods (%)

microorganisms. An analysis of the list of the collected microorganisms revealed the existence of fungi and bacteria identified by several authors as having antagonistic effect against pathogens responsible for plant diseases. This is the case of the genus *Trichoderma* in which several species have been tested for the control of cocoa diseases (Sanogo *et al.,* 2002; Krauss and Soberanis, 2001; Tondje *et al.,* 2007; Samuels, 1996). In the group of the bacteria, the antagonistic effect of the genus *Bacillus* was demonstrated by Shari Fuddin, 2000. The direct confrontation tests carried out *in vitro* between *P. palmivora* and the *Trichoderma* isolates revealed an antagonistic effect going from the inhibition of mycelia growth (fungistatic effect), to the degradation and the disappearance of the mycelium of *P. palmivora* (mycoparasitic and fungicidal effect). Similar results were obtained with *Trichoderma harzianum* on *Fusarium oxysporum* (Hibar *et al.*, 2005). In Central America, similar results were also obtained with *Trichoderma stromaticum* in the control of witch's brooms disease of cacao and with *Trichoderma virens* against the black pod disease (Krauss and Soberanis, 2002). This study thus allowed to isolate and to purify several potential antagonists of *Phytophthora* sp, susceptible to be used in the control the black pod disease.

The evaluation of the effect of the bacterial strains on *P. palmivora* using the leaf disk test revealed a reduction of the leaf susceptibility to *P. palmivora* for resistant clones (SCA 6) and moderately resistant clones (P 7). On the other hand, this effect is less perceptible with the sensitive clone (IFC5). Similar results were obtained by Maurhofer et *al*. (1994) on tobacco, Duijff et *al*. (1997) on tomato and Chen et *al*. (1998) on cucumber. These results could reveal an increase in the level of the intrinsic resistance of the plant by the bacterial strains. The highest effects were obtained with bacterial strains B 105 and B 116, which makes them potential candidates for the biological control against *P. palmivora*. These two bacteria belong to the genus *Bacillus* endowed with an ability to sporulate, suggesting a good ability of dissemination within the framework of a biological control program. Also, on leaf disks, the effect of *Trichoderma* revealed a reduction of the size and the frequency of the necrotic lesions due to *P. palmivora*. Similar results were obtained by Bowers *et al*. (2001b) on leaf disk with *Phytophthora megakarya*. This effect of reducing leaf susceptibility results from the germination of the spores of *Trichoderma,* on the underside of the leaves, which probably inhibits or hampers the germination of the zoospores of *Phytophthora*. This germination would stimulate the mechanisms of defense, and consequently would strengthen the resistance to the penetration and the dissemination of the parasite. Similar results were obtained by Bigirimana et *al*. (1997), Howell et *al*. (2000), Sid Ahmed et *al*., 2000 and Harman et *al*. (2004) on bean, cotton, hot pepper and corn inoculated by *T. virens* and *T. harzianum*, subjected to the attacks of *Rhizoctonia* sp., *Colletrotrichum* sp. and *Phytophthora* sp.

The effects of the isolates T4 of *T. spirale*, T7 of *T. virens* and T40 of *T. harzianum* in the field and the isolate T54 of *T. asperellum* on *P. palmivora*, showed a reduction of the incidence of the black pod disease due to *P. palmivora*. However, this effect is more striking on cacao trees treated with *T. asperellum*. Similar results were obtained by Tondje et *al*. (2007) in Cameroon after evaluating the potential effect of the isolates of *T. asperellum* on the incidence of the black pod disease due to *P. megakarya*. This reduction would be due to the mycoparasitic effect of *T. asperellum* against *Phytophthora* sp. Indeed, *T. asperellum* penetrates and destroys the propagules of *Phytophthora* on the flower cushions, thus reducing the quantity of inoculums of this parasite. Besides the mycoparasitic effect on the propagules (mycelium and sporocystes) of *P. palmivora*, the production of cellulase by *T. asperellum* in the presence of *Phytophthora* sp. would also be determining. The synergy of the modes of action in the

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