The fruiting body of Ganoderma sp. located at the base of the oil palm stem was collected using a sterile knife, then transported to the Plant Biology Laboratory of the University of Douala.

These fruiting body samples were cleaned with the tap water using a brush, the external part was removed, and the internal structures were cut into cubes. Theses fragments surface were sreilised with 70% alcohol for 30 seconds, then rinsed twice with sterile distilled water. The samples pieces were inoculated into petri dishes (9 cm diameter) containing 15 ml of PDA medium [5] . Incubation was carried out in darkness at 27˚C. Data were recorded every daily over a 4-weeks period. Fungal samples were subjected to successive subcultures until pure mycelum was obtained.

Soil samples were collected from 4 different blocks (A3/06; B4/74; D4/74; G7/75) of socapalm Dibombari (Littoral-Cameroon) Plantation where the disease incidence is high. Within each block, 4 asymptomactic plants located near disease foci were selected. An 800 g soil samples was collected within 1 meter of the root system of each plant in the 4 cardinal directions. Before each sampling, the soil surface was cleaned of leaves and other organic debris. The soil samples were collected from the 30 - 40 cm soil layer [6] , carrefully mixed, and sent to the laboratory.

Roots sampling was conducted in 4 differents blocks (Rao. 2018) of socapalm dibombari with high disease incidence. For each block, one asymptomatic oil palm located in the disease foci was selected. Roots were collected aseptically at a depth of 0.2 - 20 cm [7] . The roots were stored with rhizospheric soil in bags at 4˚C until use to prevent druying out and preserve the integrity of microoganisms associeted with the root.

The collected soil samples were crushed and sieved, the microorganisms were isolated according to the method of [6] , which consisted of taking 10 g of crushed sample and transferring it to 90 ml of sterile distilled water, then shaking for 30 minutes, which constituted a 10⁻¹ dilutions. From that solution, other solutions were prepared by transferring 1 ml of solution into 9 ml of sterile distilled water up to 10⁻⁸ [8] . Only dilutions 10⁻⁴, 10⁻⁵, 10⁻⁶ and 10⁻⁷ were used for inoculation of the solid medium [9] .

Inoculation consisted of taking 0.1 ml of each dilution using a pipette and depositing it into a petri dish containaing the culture medium; three replicates were performed for each dilution.

Roots samples were washed with tap water, cut into 2 cm pieces, disinfected with 70% ethanol for 30 seconds, and rinsed three times with distilled water to remove any residual disinfectant. The pieces xere then cultured on PDA medium in petri dishes and incubated in the dark for 7 days for fungal growth [6] and 2 days for bacterial growth.

Bacteria that grew in the petri dishes were subcultured and purified several times by streaking on PCA medium. The method used was the quadrant technique [10] .

Colonies showing fine filaments (hyphae) in the incubated petri dishes were identified as fungi and were isolated and subcultured using a sterilized platinum loop on the PDA medium. Incubation was performed at 28˚C for 4 to 6 days. This process was repeated until pure strains were obtained [11] .

All isolated microorganisms underwent a direct confrontation test to assess their competiviness against the pathogen responsible for the disease [12] . This test identifies the most competitive microorganism showing potential for use in biological control.

1) Fungi

This technique consists of placing a Ganoderma sp. mycelium disk and an antagonistic disk to be tested in a diamtrically opposite position in the same 90 mm diameter petri dish containaing PDA medium. The two 6 mm mycelium disks of the same age (4 days old) were placed about 30 mm apart from each other relative to the center of petri disk [12] . The control consisted of placing a 6mm diameter of Ganoderma sp. (4 days old) alone at the center of the petri dish containaing PDA culture medium.

Incubation was performed at 27˚C for 7 days with daily observations. After 3 days of incubation, the percentage of inhibition of diameter growth (PIDG) of the pathogen was assessed using the method of [13] . Three repititions were made per microorganisms.

PIDG (%) = (1 − C_n/C_o) × 100

C_o = control fungi diameter

C_n = fungal diameter confronted by the antagonistic microorganism

2) Bacteria

The direct confrontation assay was conducted in Petri dishes containing PDA medium. A 6 mm diameter mycelial disk of Ganoderma sp. (4 days old) was placed at the center of the petri dish surrounded by two streaks of bacterial (2 days old) culture spaced 2 cm apart. Three replicates were performed for each confrontation. The dishes were incubated at 27˚C in dackness for 7 days and observed daily. The control consisted of monoculture of Ganoderma sp. Inoculated at the center of the petri dish. The results were evaluated by measuring the distance of mycelial growth of Ganoderma sp. towards the bacterial antagonist, and the percentage of inhibition was calculated using the equation reported by [14] .

(%) Inhibition = (1 − D_n/D_o) × 100

D_o = control fungi diameter

D_n = fungal diameter affected by the bacteria

1) Morphological description

The macroscopic appearance of fungi was evaluated from cultures on PDA medium, and the following characteristics were examined:

2) Microscopic description

The method consisted of cutting a small piece of scoth tape and applying it with its adhesive side to the colony using tweeters, then placing it on a clean slide. Observations were made at 40× magnification, and the following critteria observe:

1) Macroscopic observations of colonies

The initial step in bacterial strain identification is the macroscopic examination of islated colonies. The main characteristic evaluated are: shape, color, elevation, contour, size, surface, and opacity [15] .

2) Microscopic description

a) Biochemical characterization

i) Catalase test

It involves the search for catalase, an important enzyme for the elimination of hydrogen peroxide according to the following rection:

In bacteriology, the catalase test is used to identify and chacterize bacteria, particlarity Gram-poisitive bacteria. The test consisted of transferring a bacterial colony from a solid agar plate to a sterile slide, followed by the addition of a drop of hydrogen peroxide (H₂O₂). The presence of catalase is demonstrated by the immediate evolution of gas bubbles [16] .

ii) Oxydase test

The oxydase test is a diagnostic tool used to detect the presence of oxydase enzyme in bacteria [17] . This test is crucial for identifying Gram-negative bacteria, as it helps to differentiate oxidase-positive bacteria, such as Pseudomonas species, and oxidase-negative bacteria [18] .

The oxydase test involves adding a reagent typically tetramethyl-P-Phenylenediamine, to a bacterial colony [18] . If the bacteria possess the oxydase enzyme, the reagent will be oxydized and change color. The oxydase test results are interpreted as follow:

Positive result: bacterium is oxydase positive, indicating the presence of the oxydase enzyme, and the reagent changes color.

Negative result: the bacterium is oxidase-negative indicating the absence of oxydase enzyme, and the reagent does not change the color.

Genomic DNA was extracted from the samples received using the Quick-DNA^TM Fungal/Bacterial kit (Zymo Research, Catalogue No. D6005). The ITS target region was amplified using OneTaq^® Quick-Load^® 2X Master Mix (NEB, Catalogue No. M0486) with the primers presented in Table 1 for fungi and Table 2 for bacteria. The PCR products were run on a gel and cleaned up enzymatically using the EXOSAP method. The extracted fragments were sequenced in the forward and reverse direction (Nimagen, BrilliantDye^TM Terminator Cycle Sequencing Kit V3.1, BRD3-100/1000) and purified (Zymo Research, ZR-96 DNA Sequencing Clean-up Kit^TM, Catalogue No. D4050). The purified fragments were analyzed on the ABI 3500xl Genetic Analyzer (Applied Biosystems, ThermoFisher Scientific) for each reaction for every sample, as listed in Section 1. DNASTAR was used Genomic DNA was extracted from the samples received using the Quick-DNA^TM Fungal/Bacterial kit (Zymo Research, Catalogue No. D6005). The ITS target region was amplified using OneTaq^® Quick-to analyse the ab1 files generated by the ABI 3500XL Genetic Analyzer and results were obtained by a BLAST search (NCBI).

Data analysis was performed using SAS (Statistical Analysis System) software for statistical analyses. The result of qualitative and quantitative variables were expressed as percentages and illustrated using bar/ graphs generated with microsoft Excel. For the isolated microorganisms, analysis of variance (ANOVA) was conducted. To compare average values of groups student’s t-test, Newman and Keuls tests were performed at a significance level of 5%.

The fruiting body of Ganoderma sp. located at the base of the oil palm tree stem ( Figure 1 ) has a semi-circular shape, with a shiny color ranging from orange to reddish-brown coloration, and its fresh is colored and woody ( Figure 2 ).

A 14-day-old purified mycelium of Ganoderma sp. exhibits a development of dense, white, cottony filaments with a thin texture. The aerial filaments are abundant and slightly orange at the edge ( Figure 3 ). This mycelium has a moderate average growth. After 6 days, the filament reaches the edge of the 9 cm diameter pedri dish.

Microscopic examination reveals that the hyphae of isolated Ganoderma sp. are branched, forming a dense and complex network with secondary and tertiary branches. The hyphae are septate and narrow, measuring 5 - 15 µm in width, and display clamp connections ( Figure 4 ).

Spoores are spherical

The search for microorganisms from the rhizospheric soil of the palm grove and oil palm roots resulted in the isolation of 2 types of microorganisms: 14 fungal isolates (CR₁, CR₂, CR₃, CR₄, CR₅, CR₆, CR₈, CS₁, CS₂CS₄, CR₁₀, CR₉, CS₁₁, CS₁₂) and 16 bacterial isolates (IBS₁, IBS₂, IBS₃, IBS₄, IBS₅, IBS₆, IBS₇, IBS₈, IBS₉, IBS₁₀, IBS₁₁, IBS₁₂, IBR1, IBR2, IBS13, IBR4). CR refers to root-associted fungi, CS to soil-associeted fungi, IBS to soil-associeted bacteria and IBR to root-associeted bacteria. Fungi dominate root isolates for 64% compared to 36% for bacteria. However, these proportions are reversed in soil where bacteria account 75% and fungi 25% ( Figure 5 ).

1) Direct confrontation

The various microorganisms isolated were screened for their antififungal activity. Two microorganisms gave a PIRG value more than 50%. The fungus (CS₁) and the bacterium (IBS₆) showed significant antifungal activity, inhibiting pathogen growth by 66% and 72%, respectively ( Figure 6 , Figure 7 ). On control plates, colony of Ganoderma sp. covered the 9 cm diameter of petri dish in 6 days.

In direct dual culture with fungus (CS₁), Ganoderma sp. exhibits growth similar to the negative control during the first 3 days. However, starting from the 3^rd day, its growth slows down, reaching a maximum diameter of 40 mm on the 4th, followed by a reduction to 30 mm ( Figure 8 ). The hyphae of the phyopathogen swelled up, and the spores of the antagonistic fungus (CS1) were observed on it, followed by its rupture ( Figure 9 ).

In direct confrontation with the IBS6 bacterium, Ganoderma sp. growth is slow from the early days compared to the control. It reaches its maximum on the 5th day with a radial growth diameter of 25 mm and then stabilizes. No physical contact was oberved between the bacterium IBS6 and the pathogen Ganoderma sp. ( Figure 10 ).

1) Macroscopic description

The macroscopic characterization of the fungus (CS1) was conducted on 7-day-old cultures. This fungus has aerial hyphae forming a dense network. The colonies are initially whitish and then turn green after sporulation, which starts on the 4th day with the first concentric circle. The texture is cottony, and the mycelium is composed of branched filaments. Fungus (CS1) shows fast growth in culture medium and the development of conidia with green-yellow color ( Figure 11 ).

2) Microscopic description

Under the microscope, the fungus (CS1) has phialides, specialized cells that produce bottle-shaped conidia. The conidia are green and spherical, characteristic of asexual spores borne on branched conidiophores ( Figure 12 ).

1) Macroscopic characteristics

After inoculation on PCA medium and incubation for 48 hours at 27˚C, the macroscopic characteristic of IBS6 Bacterium are summarized in table below ( Figure 13 ).

2) Microscopic characteristics

Gram staining performed on colonies of bacterium (IBS6) reveals that this bacterium is Gram-positive and appears as violet rods.

a) Biochemical test

The biochemical test revealed that bacterium IBS₆ is catalase-positive and oxydase -negative.

The BLAST results correspond to the similarity between the queried sequences and biologiacal sequence in the NCBI databse. Two significant matches were obtained: for code IBS6, a similatity of 99.79% was observed with Bacillus subtilis (GenBank accesssion MT571500.1) and CS1, a perfect match of 100% identity was found with Trichoderma vierens Genank (accessionMN102106.1).

This study aimed firstly to highlight the microbial biodiversity within the palm grove ecosystem. The results showed a significant qualitative and quantitative diversity in the two studied components, soil and root endophytes. The isolation of potential biological control agents of phytopathogens requires sampling from the rhizosphere of plants to be protected that are growing in soil infested with the target pathogens [19] . Furthermore, it is important to select roots of vigorous and healthy plants located in the area where the disease is present, in order to identify microorganisms that have developed effective defense strategies against phytopathogens.

Isolation from healthy oil palm roots revealed a predominance of fungi (64%) compared to bacteria (36%). These results are similar to those that obtained by [20] who reported a proportion of 66.31% of fungi against 33.04% of bacteria during their search for indigenous microorganisms of caccao antagonistic to Phytophthora palmivora.

In the soil, a high proliferation of the two cathegories was observed, with bacteria being the dominant group (75%). [21] reported that bacteria are numerically abundant organisms in the soil, especially in rhizosphere. It is the preferential site for indigenous microorganisms with potential antagonistic activity against Ganoderma sp. because they occupy the same ecologique niche [20] [21] .

Secondly this study aimed to identify indigenous microoganisms with antagponistic activity against Ganoderma sp. To achieve this study, in vitro screening was conducted on microbial isolates to select those showing promising efficacy agaisnt this pathogen.

Mycelium isolated from the carpophore of Ganoderma sp. exhibited morphological characteristics consistent with those reported for Ganoderma sp. by [22] and [23] .

The result of the in vitro dual culture test showed that the bacterium Bacillus subtilis (IBS₆) and the fungus (CS₁) were able to inhibit the radial growth of Ganoderma sp. with PIRG value of 72% and 66% respectively. The selection of the two microorganisms as biological agents depends largely on their effectiveness in vitro result against Ganoderma sp. Some studies have also revealed that Ganoderma sp. has been inhibited to some extent by some biocontol agents, namely Actinomycetes, arbuscular mycorrhizal fungi, Bacillus spp., Diaporthe spp, mycoparasite, Penicillium, Streptomycetes spp., and Trichoderma spp. [24] ; [12] tested 3 different species of Trichoderma namely T. asparellum, T. virens, T. harzianum reporting that all the Trichoderma spp. tested significantly inhibited the growth of Ganoderma sp. with PIRG of more than 70% for all the species tested. The inhibition of Ganoderma sp. observed in direct confrontations suggests that the significant growth of antagonistic agents allow them to develop more rapidly, colonize the culture medium, and mobilize nutriments to their advantage [25] . This effect could be most likely due to the production of antibiotic, secondary metabolite compounds, or lytic enzymes which could contribute to the direct antagonistic effect degrading Ganoderma sp. cell wall and mycoparasitic activities [12] [24] . Moreover, antagonism could be also be caused by production of volatile and non volatile antibiotic acting synergistically with enzymes to contreract the pathogen. The main lytic enzymes produced by the biological control agents are chitinase, glucanase, cellulase and protease. The production of these cell wall degrading enzymes could be responsible for the inhibition of Ganoderma sp. in vitro. During direct confrontation between the Trichoderma virens (CS₁) and Ganoderma sp. significant differences were observed in antagonistic activity. The radical growth diameter of Ganoderma sp. control and the confronted were similar until 3rd day, suggesting a delayed antagonistic activity. However after the 3rd day, a significant difference between Control and the treatment diameters was noticed. By the 4th day, Ganodema sp. growth (40 mm) stopped, likely due to space occupation or nutrient depletion. On the 5th day, the diameter of Ganoderma sp. reduced to 30 mm, which could correspond to the destruction of a portion of Ganoderma mycelium by the antagonistic agent ( Figure 10 ).

For the treatment by Bacillus subtilis (IBS₆) the Ganoderma sp. growth was slowed from the first day compared to the contol, indicating early antagonistic activity. [26] reported that antibiotic production by Bacillus subtilis is a process that begins early in the growth of the bacterium, but it is during the stationary phase or in aged cultures that the bacterium releases the largest amount of antibiotic into the culture medium, maximizing its antimicrobial potential. The early antagonistic activity of the bacterium IBS₆ could be attributed to its rapid growth, allowing it to effectively colonize the environment and exert its inhibitory effect [27] . The diameter reached its peak on the 5th day and remainded stable until 7th ( Figure 10 ). The lack of physical contact between IBS₆ and Ganoderma sp. suggest a potential inhibitory action at a distance, likely due to the production of antibiotics and other secondary metabolites.