**2. Materials and methods**

#### **2.1 Samples and culture media**

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

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

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 brought to the laboratory for isolation of endophytes.

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*  (Burbage, 1982), and the NYD medium (nutrient yeast dextrose) (Guizzardi and Pratella, 1996), adapted to a larger spectrum of bacteria were used. For the isolation of fungi, the TME (*Trichoderma* medium E), specific to fungi belonging to the genera *Trichoderma* and *Gliocladium* (Papavizas and Lumsden, 1982) were used. The PDA (potato dextrose agar), adapted to a larger spectrum of fungi was used for the isolation of the other fungi.
