**1. Introduction**

26 Will-be-set-by-IN-TECH

302 Biodiversity Loss in a Changing Planet

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evaluate the effects of conservation and management actions, *Biological Conservation*

The black pod disease due to *Phytophthora* spp is a destructive disease of cocoa. Worldwide, yield losses have been estimated to 30% (Lass, 1985). Côte d'Ivoire, the first cocoa producing country in the world, with 44% of the world market (ICCO, 2000) is also concerned by this disease. Several species of *Phytophthora* are involved in the disease. In Africa, two species, *P. palmivora* and *P. megakarya*, are the most damaging. The first species, which is the most common, causes damage in all the cocoa producing countries in the world, with yield losses between 20 to 30% ; the second, endemic to central and west Africa, is the most aggressive. This pathogen may cause the loss of the whole pod production in some countries (Flood, 2006). In Côte d'Ivoire, since the discovery of *P. megakarya* in the western region in the 90s, the black pod disease problem became more serious (Koné, 1999; Kouamé, 2006). Yield losses increased from an average of 10% to 35-40% (Kébé *et al*., 1996). Thus, the control of the disease became also a priority.

Although chemical control was developed by the research scientists, the dissemination of this method to the farmers was little successful. The low level of adoption of this technology by the farmers could be explained by the high cost of the fungicides as well as the difficulties related to the provision of water and the application of the fungicides. In addition, the requirements of the international market in terms of bean quality, environmental constrains, health issues for the consumers, and the different moratoriums in this area from the market partners (Anonyme, 2006), are numbers of constraints that do not facilitate the development of the chemical control method.

The strategy adopted in Côte d'Ivoire to control the black pod disease is based on integrated management, which is cost effective and environmentally friendly. This approach combines the use of agronomic practices, resistant cocoa varieties and natural antagonistic microorganisms of *Phytophthora*. Research works continue in order to improve agronomic practices and varietal resistance. The use of natural antagonistic microorganisms of

Isolation and Identification of Indigenous Microorganisms of Cocoa Farms in

disinfectant (Arnold, 1999; Evans, et *al*., 2003; Rubini et *al.*, 2005).

because of the gelatinous consistency of the decomposing pod husk.

26 °C for 2 days for the bacteria and 7 days for the fungi.

**2.3 Conservation and identification of the microorganisms** 

**2.2 Isolation of the microorganisms** 

**2.2.1 Pod endophytes** 

**2.2.2 Soil microorganisms** 

Côte d'Ivoire and Assessment of Their Antagonistic Effects Vis-À-Vis *Phytophthora palmivora…* 305

(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),

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

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.

The microorganisms were obtained by direct isolation from the soil according to the method described by Davet and Rouxel (1997) and from fragments of pod husk buried in the soil samples. The soil samples were beforehand dried, ground and calibrated by sieving. The fragments of husk were ground in a porcelain mortar to separate each living propagule,

In both cases, 10 g of ground soil were transferred in 90 ml of sterile distilled water contained in an Erlenmeyer. The mixture is then put in agitation for 30 minutes to obtain a good separation of the particles. To obtain a variable concentration of propagules and facilitate the enumeration of the colonies, a series of dilution was performed from the initial solution whose concentration was 10-1 (Rapilly, 1968). To obtain a solution of 10-2, 1 ml of the initial solution was mixed in 9 ml of sterile distilled water. Thus, a series of dilution from 10- 2 to 10-9 was performed in hemolytic tubes. For each dilution, 100µl was pipetted and spread onto the surface of the culture media in Petri dishes. For each dilution and for each medium, 4 Petri dishes were inoculated. The incubation was also made in the dark in a steam room at

The microorganisms isolated were first purified by two or three successive monospore transplantings on specific culture media. Once purified, each isolate was designated by a code number. For strains of bacteria, the code numbers were preceded by the letter B, followed by an order number. The nomenclature of the fungi isolates begins with one or several first letters of the name of the genus, followed by an order number. The conservation of the microorganisms was then made in a freezer at a temperature of - 80°C for the bacteria, and - 10°C for the fungi. In both cases, agar disks taken near the edge of the purified culture were transferred to 1.5 ml sterile Eppendorf microtubes containing glycerol at 50 %. The identification of the isolated microorgamisms was based on the macroscopic, microscopic, biochemical and molecular characters. The molecular characterization was made by the

adapted to a larger spectrum of fungi was used for the isolation of the other fungi.

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

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 trees.
