**3.1 Microorganisms isolated**

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).

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

Isolation and Identification of Indigenous Microorganisms of Cocoa Farms in

**3.3 Effect of the microorganisms on** *Phytophthora palmivora* **3.3.1 Effect of** *Trichoderma* **on the mycelial growth of** *P. palmivora*

high and the growth of *P. palmivora* practically stops (Fig.2).

identified yet.

Diameter (mm)

Côte d'Ivoire and Assessment of Their Antagonistic Effects Vis-À-Vis *Phytophthora palmivora…* 309

One hundred and two (102) colonies of bacterial endophytes of pods were identified. These bacteria belong to two groups based on Gram-coloration response to chemicals: 56 Grampositive bacteria (45 bacilli and 11 cocci) and 46 gram-negative bacteria (9 bacilli and 37 cocci). The bacterial colonies B105 and B116 were identified as belonging to the genus

With regard to the soil, amongst 455 isolates collected and purified, 254 bacteria, 136 fungi, 48 actinomycetes and 17 yeasts were identified. In the group of the fungi, 44 isolates belonging to the genus *Trichoderma* were identified. These were *T. virens* (32 isolates), *T. harzianum* (4 isolates), *T. spirale* (6 isolates) and *T. asperellum* (2 isolates). Three isolates belonging to the genus Clonostachys were also identified. The other isolates have not been

The direct confrontation tests realized *in vitro*, between the isolates of *Trichoderma* sp. and of those of *Phytophthora palmivora* revealed an inhibitory effect of *Trichoderma* on *P. palmivora* in mixed culture. After 3 days of confrontation, the inhibition of the growth speed became very

**12345**

Fig. 2. Influence of *Trichoderma* on the mycelial growth of *Phytophthora palmivora.* Legend. PP : *Phytophthora palmivora*, Tricho: *Trichoderma*, P/Tricho : *Phytophthora/Trichoderma*

The capacity of *Trichoderma* to stop the mycelial growth of *P. palmivora* reveals a deep fungistatic effect. From the fourth day, we note a progressive disappearance of the mycelium of *P. palmivora*. This degradation of the mycelium of *P. palmivora* which is more accentuated at the fifth day, with all the isolates, reveals a mycoparasitic effect of

Time (day)

PP Tricho P/Tricho1 P/Tricho 2 P/Tricho 3 P/Tricho 4 P/Tricho 5 P/Tricho 6

*Bacillus*. Finally, 55 yeast strains and 2 isolates of actinomycetes were identified.

The results of the statistical analyses showed that the mean number of microorganisms colonies counted per gram of soil varied significantly (P<0.05) with the isolation method (with baits or no bait). This result revealed that the use of fragment of pods infected by *Phytophthora* as bait, significantly improved the isolation of the microorganisms on PDA, TME and PCAT media (Table 1).


Means within the same column followed by the same letter are not significantly different according to Newman & Keuls's test at 5 % probability

Table 1. Mean numer of colonies for unit (cfu/g) according to the isolation methods and the culture media

For the samples taken in Divo and Abengourou, the statistical analyses did not show any significant differences (P>0.05) between regions with regard to the mean number of microorganisms colonies for the different culture media except for the NYD. There was no clear relationship between the density of microorganisms isolated and the age of field except for the bacteria on the NYD medium in Abengourou (Table 2).


Means within the same column followed by the same letter are not significantly different according to Newman & Keuls's test at 5 % probability

Table 2. Mean number of colonies for unit (cfu/g) according to the locations and the age of the farms

#### **3.2 Identification of the microorganisms isolated**

Regarding the pods, 313 fungi isolates were purified. Among the purified isolates, 58 isolates belonging to 9 genera were identified. These were *Penicillium* sp (6), *Fusarium* sp (7), *Botrytis* sp. (9), *Pestalotia* sp. (24). The remaining isolates identified belong to the genera *Nigrospora* (2), *Physoderma* (1), *Polynema* (1), and *Botryodiplodia* (8). The other isolates (255) belong to diverse species or genera, but the relationship has not been established yet.

One hundred and two (102) colonies of bacterial endophytes of pods were identified. These bacteria belong to two groups based on Gram-coloration response to chemicals: 56 Grampositive bacteria (45 bacilli and 11 cocci) and 46 gram-negative bacteria (9 bacilli and 37 cocci). The bacterial colonies B105 and B116 were identified as belonging to the genus *Bacillus*. Finally, 55 yeast strains and 2 isolates of actinomycetes were identified.

With regard to the soil, amongst 455 isolates collected and purified, 254 bacteria, 136 fungi, 48 actinomycetes and 17 yeasts were identified. In the group of the fungi, 44 isolates belonging to the genus *Trichoderma* were identified. These were *T. virens* (32 isolates), *T. harzianum* (4 isolates), *T. spirale* (6 isolates) and *T. asperellum* (2 isolates). Three isolates belonging to the genus Clonostachys were also identified. The other isolates have not been identified yet.
