**2.2 Isolation of the microorganisms**

### **2.2.1 Pod endophytes**

304 Biodiversity Loss in a Changing Planet

*Phytophthora* in the control of black pod disease is a new area of investigation explored by research scientists in several cocoa producing countries. Thus, some species in the genera *Trichoderma* and *bacillus* have been described by several scientific teams as potential biological agent for the control of *Phytophthora* spp. on coca (Bong *et al*., 1996; Krauss *et al*., 2003; Mpika, 2002). On other crops, fungi and bacteria belonging to several genera including *Pseudomonas, Burkholderia*, *Streptomyces*, *Serratia*, *Penicillium*, *Geniculosporiun, Gliocladium, Aspergillus*, *Coniothyrium, Ampelomyces, Phytophthora, Botrytis, Colletotrichum, Pythium, Rhizoctonia, Fusarium, Gaeunannomyces* and *verticillium* have been described as antagonists of many fungi, pathogens of plants. Members of these genera are pathogens of plants such tomato, rice, cucumber, maize, cotton and beans (Hebber *et al*., 1998 ; Benhamoun *et al*., 2000 ; Singh *et al*., 1999 ; de Cal *et al*., 1999 ; Paulitz and Linderman, 1991 ; Gerlagh *et al*., 1999 ; Vidhyasekaran

During this study, the biodiversity was explored in the cocoa ecosystems. Microorganisms, potential antagonists of *Phytophthora* spp., were collected from pods and soils of cocoa farms. A collection of microorganisms was established. The antagonistic effect of these microorganisms on *Phytophthora* was assessed in the laboratory and in the field on the cacao

The microorganisms used in this study were isolated from the cocoa ecosystems, either from soils or pods. The soil samples were collected from cocoa farms in Abengourou in the the Eastern region of the country, and in Divo in the West-central region. In each of the locations, the soil samples were taken in 8 cocoa farms grouped in two categories according to the age of the trees. The first category was made up by 4 young plots with trees being 3 to 5 years old. These newly established plots still have open canopies with little soil litters. The second category was made up by 4 olds farms (25 to 30 years old). These farms, with very mature and fully bearing trees have closed canopies and abundant litters on the soil. In each plot, a bulk sample of 800 g of soil was made-up with 4 samples taken at the base of 4 cacao

Before taking each soil sample, the litter was totally removed. The samples were then taken in the superficial zones colonized by the fine root system because of fertilizer application. It is in this horizon of 30 to 40 cm deep that the samples were taken (Davet and Rouxel, 1997). Each bulk sample was carefully mixed and divided in two equal parts which were put in plastic containers. In one of them, baits for the antagonists made-up by fragments of pod plugs infected by *Phytophthora palmivora*, were buried in the soil sample (Tim *et al*., 2003). The other was left without any bait. In order to obtain a good colonization of the pod plugs,

The pods used for the isolation of the microorganisms were collected in the main cocoa producing regions of Côte d'Ivoire. Thus, 390 healthy pods were collected from the 13 cocoa producing regions. In each region, pods were collected in 10 farms, at a rate of 3 pods per farm. The pods were kept in plastic bags labeled with information on the samples and

The diversity of fungi and bacteria in the soil of cocoa farms and in the pods were evaluated on selective culture media. For the isolation of bacteria, two selective media, including the PCAT medium (P. cepacia Azeaic acid tryptamine), specific to *Pseudomonas* and *Burkhoderia* 

and Muthamilan, 1999 ; Bong and Stephen, 1999 ; Tondje *et al.*, 2006a,b).

trees bearing many healthy pods and selected randomly in the plot.

the soil samples were kept in the laboratory at 20°C for 30 days.

brought to the laboratory for isolation of endophytes.

trees.

**2. Materials and methods 2.1 Samples and culture media**  The surface of the pods was beforehand washed with tap water, and then underwent a series of disinfection in ethanol at 95 % for 30 seconds, in sodium hypochlorite at 10 % for 2 minutes, and again in ethanol at 75 % for 2 minutes, in order to eliminate the microorganisms present on the husk. The pods were rinsed three times in sterile distilled water to eliminate any trace of disinfectant (Arnold, 1999; Evans, et *al*., 2003; Rubini et *al.*, 2005).

The sampling zone is chosen and the superficial tissues were removed using a sterile scalpel. Ten cubic shape fragments of 5 to 7 mm were taken per pod in the husk. The samples taken were put in culture on the selective media contained in Petri dishes. The incubation was made in the dark in a steam room, at 26 °C for 2 days for the bacteria and 7 days for the fungi.
