The plant material, including Cymbopogon citratus and Blumea balsamifera leaves, was collected in Bokito, a small village located approximately 132 km from Bafia. The extracts were prepared in the chemistry laboratory of the University of Yaounde I (Ngoa Ekele). Diseased cocoa pods were obtained from a plot without recent fungicide application in Nkoemvone, one of Cameroon’s high-incidence cocoa pod rot disease basins [4] [9] [25] [26] , located in the southern region of Cameroon. Laboratory work including the isolation and identification of fungal pathogens, Koch’s postulates, and evaluation of extracts in vitro and in vivo, were carried out at the Innovative Plant Health Laboratory of the FASA Annex in Bafia.

Isolates of Phytophthora megakarya were obtained from cocoa pods exhibiting well-developed brown rot symptoms. The pods were collected from one of the plots at Nkoemvone, Southern Cameroon, belonging to the Institute of Agricultural Research for Development (IRAD). Prior to collection, it was ensured that the plot had not been recently treated with any fungicide. To isolate the fungus, the surfaces of the pods were initially cleaned with tap water, disinfected using 95% ethanol, and then cut into fragments from the marginal zones of the lesions (between healthy and diseased tissue) using a flamed scalpel [27] . The fragments were dried on blotting paper and placed on V8 agar medium [26] [27] . Five pieces of cubic fragments were placed on each V8 plate and then incubated at room temperature in darkness. After 4 - 5 days, a single mycelial fragment with distinct morphology was transferred to a fresh V8 medium. This process was repeated until a pure culture of P. megakarya was obtained [16] [26] [27] . To confirm the pathogenicity of the isolate, the Koch postulate test was conducted.

The Koch postulate test involved artificially infecting healthy cocoa pods disinfected with a 1% sodium hypochlorite solution and 95% ethanol. The cortex of the pods was then divided into four pieces and disinfected once again. Using an 11 mm diameter punch, holes were created on each disinfected cortex. A mycelial explant from the pure culture of P. megakarya was placed in each hole, covered with sterile cotton wool soaked in distilled water, and incubated in darkness at room temperature (24 ± 2˚C) for seven days. The development of disease symptoms on the cocoa pod cortex confirmed that P. megakarya was the causal agent of brown rot [16] [26] [27] .

Fresh leaves of Cymbopogon citratus and Blumea balsamifera were collected from the Bokito locality and transported to the Innovative Plant Health Laboratory of the FASA Annex in Bafia. The leaves were shade-dried for two weeks and then crushed using a mortar and pestle. A total of 500 grams of powdered C. citratus and B. balsamifera were mixed with 1 Lof 95% ethanol (solvent) and macerated for 48 hours [28] . The mixture was filtered using Whatman No. 1 filter paper, and the ethanol was removed from the extract using a rotary evaporator (Buchi-R-200) at 60˚C, resulting in the crude extract [26] [28] . The extracts of C. citratus and B. balsamifera were stored at 4˚C for future use.

Antifungal tests were conducted in Petri dishes using V8 medium amended with separate concentrations of crude extracts of C. citratus and B. balsamifera: 25 µl/ml, 50 µl/ml, 100 µl/ml, 250 µl/ml, and 500 µl/ml. Pure colonies of P. megakarya (with a diameter of approximately 8 mm) were placed in the center of the amended V8 medium. The positive control consisted of V8 medium supplemented with the fungicide Ridomil (5000 µg/ml, as recommended by the manufacturer), while the negative control contained V8 medium without any additions. Each treatment was replicated four times. All the V8 plates were incubated in darkness at room temperature (24 ± 2˚C) for seven days [26] . The colony diameter was measured daily using two perpendicular lines drawn on the V8 plates. The percentage inhibition (PI) of mycelial growth was calculated using the formula: PI = [(A - B)/A] × 100, where PI represents percentage inhibition, A is the colony diameter in the negative control, and B is the colony diameter in the treatment.

The most effective concentration of the crude extract tested was prepared from individual plants of C. citratus and B. balsamifera, following the method described above. The extract was then added to V8 medium alone or combined at different ratios (1:1, 1:3, and 3:1). The positive control and negative control consisted of V8 medium supplemented with Ridomil (fungicide) and V8 medium without any additions, respectively. The V8 plates were incubated at room temperature (24 ± 2˚C) in darkness, and the colony diameter was measured daily for seven days [26] . Additionally, mycelial explants from V8 plates where no colony growth occurred at the end of the assay were transferred to a freshly prepared V8 medium without extract or fungicide and incubated for an additional 7 days at room temperature. The treatment that allowed growth resumption was considered fungistatic, while the treatment that prevented mycelial growth resumption was assumed to be fungicidal [29] .

The evaluation of the antifungal potential of the combined extracts was performed in vivo using healthy detached cocoa pods. The same treatments and proportions as the in vitro tests (section 2.6) were applied. Before conducting the tests, the cocoa pods were disinfected according to the details mentioned in section 2.3. Each pod was cut into four pieces, approximately 4 cm × 4 cm in size. Using a paintbrush, 500 µl of each treatment was applied to the cortex of the pod pieces and left to dry for a few minutes. Once the pods were dry, a cylindrical hole with a diameter of 8 mm and a depth of approximately 0.5 cm was created at the center of each pod using a flamed cookie cutter. An 8 mm diameter mycelial fragment of P. megakarya was inserted into each pod and covered with a moist cotton pad soaked in water to maintain humidity [16] [26] . The treated pods were incubated at room temperature in a completely randomized design. The progression of necrotic lesions was monitored daily for seven days. The necrotic surface area was calculated using the formula:

S = D × d × Π

where: S represents the necrosis surface area in cm², D is the major diameter of the lesion in cm, d is the minor diameter of the lesion in cm, and Π is the value of pi (approximately 3.14).

The combined plant extract of B. balsamifera and C. citratus, which demonstrated the highest antifungal potential (i.e., complete inhibition), was prepared and stored at room temperature. After 3, 6, and 9 days of storage, the extract was retrieved and used to treat detached cocoa pod cortex following the protocol described in section 2.7. Mycelial growth was measured daily for seven days to assess the impact of storage time on the extract’s antifungal activity.

Data analysis for the experiments was conducted using R software version 4.3.0. A multivariate analysis of variance (ANOVA) was performed to compare the colony growth and inhibition rate obtained for the isolates in cultures with different treatments. The main treatment effects considered were treatment, isolates, and concentration. The mean colony growth and inhibition were compared using Tukey’s test with a significance level of 5%. The minimal inhibitory concentration of 50% was determined with 95% confidence limits (CL) using probit analysis with the “ecotox” package [30] .

The analysis of variance (ANOVA) results for the day 7 data revealed significant variations in colony growth among different treatments, concentrations, isolates, and their interactions ( Table 1 ). Moreover, the analysis indicated that as the concentration decreased, there was an increase in colony growth.

Colony Growth and Inhibition Percentage

Table 2 presents the efficacy of Blumea balsamifera and Cymbopogon citratus extracts in inhibiting the growth of P. megakarya. Both B. balsamifera and C. citratus extracts significantly reduced colony growth at all concentrations (25, 50, 100, 250, and 500 µl/ml) for all four isolates (M2-C, M2-O, M3-N, and M3-O) compared to the negative control (no extract). These findings indicate that these plant extracts possess antifungal properties against P. megakarya. In general, there was a trend of increasing inhibition with higher concentrations of both extracts for all isolates, indicating that higher extract concentrations hindered fungal growth.

For three out of the four isolates (M2-C, M2-O, and M3-N), there were no significant differences in the effectiveness of B. balsamifera and C. citratus at the highest concentration (500 µg/ml), suggesting similar inhibition of growth for these isolates at this concentration. However, at the highest concentration, Tukey’s test revealed a significantly greater reduction in colony growth for isolate M3-O treated with C. citratus (80.65 ± 1.49%) compared to B. balsamifera (59.28 ± 3.82%). This indicates that isolate M3-O is more sensitive to C. citratus. The positive control (ridomil) completely inhibited growth for all isolates, except for M3-N, which had a colony growth of 2.50 ± 0.00 mm and an inhibition percentage of 94.76 ± 0.06%, demonstrating its resistance to the treatment.

Means followed by the same letters by isolate in the same column are not significantly different with the Tukey multiple tests at threshold of 5%.

Minimal Inhibitory Concentration (MIC)

Minimal inhibitory concentration (MIC) obtained with ethanolic extracts from each of the plant species showed MIC values ranging from 117 to 269 µl mL⁻¹ and 146 to 233 µl mL⁻¹ for B. balsamifera and C. citratus, respectively. The V8 amended with B. balsamifera showed the lowest MIC (117.0 µl mL⁻¹) against isolate M2-C as shown in Table 3 . However, C. citratus had a narrower range of MIC able to inhibit all fungal isolates, suggesting a more consistent inhibitory effect against different fungal isolates.

FT = Fluicidal limits; LC50 = lethal concentration inhibiting fungal growth at 50%.

Results of the experiment on evaluation of the combination of the two plant extracts showed significant differences between isolates (df = 3; F = 7.5; P < 0.0001), treatments (df = 6; F = 615.31; P < 0.0001) as well as interaction isolate-treatment (df = 18; F = 19.15; P< 0.0001). The colony growth ranged from 1.50 ± 1.06 to 31.62 ± 0.59 mm, 0.81 ± 0.81 to 40.37 ± 1.85 mm, 00 ± 00 to 42.37 ± 0.51 mm, 2.50 ± 0.00 to 47.75 ± 0.59 mm for M2-C. M2-O, M3-N, and M3-O, respectively. For the isolate M2-C, the low value of lesion diameter was obtained on the V8 medium amended with B. balsamifera and C. citrates (TC25B75) at ratio of 3:1, which was not significantly different from the positive control (T+). A similar trend was observed for the three other isolates ( Table 4 ). This treatment (TC25B75) drastically inhibited the colony growth of M3-N (100%), with high inhibition percentages of 92.72 ± 4.20, 93.61 ± 4.72, and 98.04 ± 1.95% for M3-O, M2-C, and M2-O respectively. This indicates that the combination of extracts can be highly effective in inhibiting fungal growth for some isolates but less effective for others. Compared to single extract treatments (TB100 and TC100), the combination treatment (TC25B75) generally exhibited superior antifungal activity. This suggests a synergistic effect on inhibiting the growth of Phytophtora megakarya, where the combination is more effective than either extract alone. However, the 1:1 combination of both extracts (TB50C50) showed limited antifungal activity for some isolates, suggesting that a specific ratio (3:1) might be crucial for optimal effect. The fact that fungal growth resumed after sub-culturing the mycelial explant from TC25B75 treatment suggests a fungistatic effect rather than fungicidal.

Means followed by the same letter in the same column for each isolate are significantly different using the Tukey multiple tests at threshold of 5%. T-: no amended PDA. T+: PDA amended with Ridomil. TB100: PDA amended with B. balsamifera extract only. TB50C50: PDA amended with B. balsamifera extract and C. citratus at 50% each. TC100: PDA amended with C. citratus extract only 500 µl. TC25B75: PDA amended with 75% B. balsamifera and 25% C. citratus TC75B25: PDA amended with 25% B. balsamifera and 75% C. citratus.

Comparison of Antifungal Activity of Combined Extracts in vivo

The results obtained following the application of the treatments in vivo demonstrated a significant difference in lesion diameter between the isolates (df = 3; F = 58.92; P < 0.0001) and between the treatments (df = 6; F = 593.35; P < 0.0001) ( Table 5 ). Furthermore, a significant difference was observed in the treatment-isolate interaction (df = 18; F = 32.39; P < 0.0001). Lesion diameter of plant extracts was notably lower than that of the negative control (T-) for all isolates ( Figure 1 ). This confirmed an antifungal effect of the combined extracts and suggested that the effectiveness of the extracts varied depending on the fungal strain.

The percentage inhibition followed a similar pattern to that observed for lesion diameter, with a significant difference between isolates (df = 3; F = 7.45; P < 0.0001), and between treatments (P < 0.05). However, no significant difference was observed in the treatment-isolate interaction (df = 18; F = 0.91; P = 0.56). A significant difference was observed in the necrotic area between isolates (df = 3; F = 38.03; P < 0.0001), between treatments (df = 6; F = 219.17; P < 0.0001), and in the treatment-isolate interaction (df = 18; F = 26.31; P < 0.0001).

Means followed by the same letter in the same column for each isolate are significantly different using the Tukey multiple tests at threshold of 5%. T-: distilled water. T+: positif control with Ridomil. TB100: Blumea balsamifera extract only. TB50C50: B. balsamifera extract and C. citratus at 50% each. TC100: C. citratus extract only. TC25B75: 75% B. balsamifera and 25% C. citratus TC75B25: 25% B. balsamifera and 75% C. citratus.