1. Introduction
Cocoa production plays a critical role in Cameroon’s economy, particularly for smallholder farmers, contributing approximately 1.2% to the GDP and 8.2% to the agricultural GDP. It generates a significant direct and indirect added value of over 200 billion CFA francs [1]. Cameroon ranks among the top cocoa producers in Africa, with an annual production exceeding 290,000 metric tons (ICCO2020). However, this vital sector faces a severe threat from black pod disease, caused by the aggressive fungus Phytophthora megakarya. This disease can devastate cocoa pods, leading to yield losses of over 80% under favorable conditions [2] [3]. In Cameroon, it is considered one of the primary phytosanitary problems affecting cocoa production [4]. Current control methods, such as chemical fungicides, resistant cultivars, and sanitation practices, have not been able to fully manage the disease [5]. Although chemical fungicides remain the primary approach, their use can potentially lead to the emergence of fungicide-resistant strains of P. megakarya [6] [7], raising concerns about their long-term effectiveness. Moreover, the environmental impact, human health risks, and increasing costs associated with chemical fungicides necessitate the exploration of alternative and sustainable solutions [8]. This urgency is further amplified by the specific threat posed by P. megakarya in Cameroon. Unlike the more widespread but less destructive P. palmivora [9], P. megakarya causes significant yield losses [4] [9] [10]. This highlights the need for innovative and eco-friendly management strategies [11]. Previous research has demonstrated the effectiveness of plant extracts in controlling fungal diseases, including studies showing the inhibitory activity of extracts from Parthenium hysterophorus, Nerium oleander, Oscimum basilicum, and Thevetia peruviana against various Phytophthora species, including P. megakarya and P. capsici [12]-[17].
This study aims to investigate the potential of ethanolic extracts from two aromatic plants, Cymbopogon citratus L. (lemongrass) and Blumea balsamifera L. (Sambong), known for their antimicrobial properties [18]. The objective is to evaluate the efficacy of these extracts in inhibiting the growth of the fungal pathogen in a controlled environment and explore their potential as a sustainable management strategy for black pod disease in Cameroon. Lemongrass extracts contain various bioactive compounds with antioxidant, antimicrobial, and anti-inflammatory properties [19]. Sambong extracts also possess a range of biological activities, including antimicrobial, antioxidant, and anti-inflammatory effects, suggesting their potential for plant-based disease control [20]-[24]. By exploring plant-based alternatives, this study aims to contribute to the development of ecological solutions to combat black pod disease, promote sustainable cocoa production practices, and improve the livelihoods of cocoa farmers in Cameroon.
2. Materials and Methods
2.1. Experimental Site and Source of Materials
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.
2.2. Isolation and Purification of Phytophthora megakarya
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].
2.3. Sources of Plant Materials and Preparation of Extracts
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.
2.4. Effect of Plant Extracts on Phytophthora megakarya Growth Inhibition in Vitro
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.
2.5. Evaluation of the Antifungal Potential of Combined Extracts in Vitro
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].
2.6. Evaluation of the Antifungal Potential of Combined Extracts in Vivo
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).
2.7. Effect of Storage Time on the Antifungal Potency of the Best Combined Extract in Vivo
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.
2.8. Statistical Analysis
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].
3. Results
3.1. Concentration Response of Cymbopogon citratus and Blumea balsamifera Extracts in Inhibiting the Growth of Phytophthora megakarya
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.
Table 1. Analysis of variance (ANOVA) of the effect of concentration on the growth of four P. megakarya isolates.
Effect |
Df |
SS |
MS |
F |
P |
Treatments |
3 |
15463.20 |
5154.40 |
3303.40 |
<0.0001 |
Isolates |
3 |
675.50 |
225.20 |
144.31 |
<0.0001 |
Concentration |
4 |
12644.80 |
3161.20 |
2025.98 |
<0.0001 |
Treatments × Isolates |
9 |
292.80 |
32.50 |
20.84 |
<0.0001 |
Treatments × Concentration |
4 |
30.30 |
7.60 |
4.86 |
0.001 |
Isolates × Concentration |
12 |
235.50 |
19.60 |
12.58 |
<0.0001 |
Treatments × Isolates × Concentration |
12 |
173.60 |
14.50 |
9.27 |
<0.0001 |
Residuals |
144 |
224.70 |
1.60 |
- |
- |
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.
Table 2. Colony growth and inhibition percentage of the four P. megakarya isolates seven days post-inoculation.
Isolates |
Treatments |
Concentration (µl/ml) |
Colony growth (mm) |
Inhibition (%) |
M2-C |
B. balsamifera |
25 |
35.75 ± 0.82bc |
17.80 ± 0.98fg |
50 |
28.87 ± 0.55d |
33.50 ± 2.17e |
100 |
19.62 ± 0.37ef |
54.84 ± 0.98cd |
250 |
18.37 ± 0.31f |
57.71 ± 0.95c |
500 |
8.75 ± 0.62g |
79.88 ± 1.38b |
C. citratus |
25 |
38.06 ± 0.48b |
12.43 ± 1.29g |
50 |
33.12 ± 0.37c |
23.70 ± 2.39f |
100 |
21.62 ± 0.37e |
50.26 ± 0.49d |
250 |
18.12 ± 0.89f |
53.54 ± 2.15cd |
500 |
11.12 ± 0.23g |
74.40 ± 0.62b |
Negative control |
0 |
43.50 ± 0.93a |
0.00 ± 0.0h |
Positive control |
5000 |
0.00 ± 0.00h |
100.00 ± 0.00a |
M2-O |
Blumea balsamifera |
25 |
36.37 ± 0.62bc |
16.31 ± 1.57fg |
50 |
28.87 ± 0.55d |
33.50 ± 2.17e |
100 |
19.62 ± 0.37ef |
54.84 ± 0.98cd |
250 |
18.37 ± 0.31f |
57.71 ± 0.95c |
500 |
8.75 ± 0.62g |
79.88 ± 1.38b |
C. citratus |
25 |
39.00 ± 0.35b |
10.26 ± 1.27g |
50 |
34.12 ± 0.51c |
21.40 ± 2.48f |
100 |
21.87 ± 0.23e |
49.67 ± 0.68d |
250 |
18.12 ± 1.46f |
58.45 ± 2.64c |
500 |
11.00 ± 0.54g |
74.71 ± 1.06b |
Negative control |
0 |
43.50 ± 0.93a |
0.00 ± 0.00h |
Positive control |
5000 |
0.00 ± 0.00h |
100.00 ± 0.00a |
M3-N |
B. balsamifera |
25 |
36.81 ± 0.40b |
13.12 ± 0.25f |
50 |
34.25 ± 0.25c |
19.14 ± 0.90e |
100 |
22.50 ± 0.61e |
46.93 ± 0.80c |
250 |
23.50 ± 0.35e |
44.49 ± 1.43c |
500 |
14.56 ± 0.52f |
65.58 ± 1.59b |
C. citratus |
25 |
35.00 ± 0.35bc |
17.39 ± 0.60ef |
50 |
30.00 ± 0.35d |
29.16 ± 1.39d |
100 |
24.00 ± 0.61e |
43.38 ± 0.85c |
250 |
23.25 ± 0.62e |
45.15 ± 1.01c |
500 |
15.75 ± 0.43f |
62.83 ± 0.84b |
Negative control |
0 |
42.37 ± 0.51a |
0.00 ± 0.00g |
Positive control |
5000 |
0.00 ± 0.0g |
100.00 ± 0.00a |
M3-O |
B. balsamifera |
25 |
41.62 ± 0.51b |
12.82 ± 9.44g |
50 |
34.62 ± 0.68c |
27.48 ± 1.13f |
100 |
29.00 ± 1.17d |
39.28 ± 2.19e |
250 |
25.87 ± 0.77de |
45.75 ± 2.05de |
500 |
19.37 ± 1.63f |
59.28 ± 3.82c |
C. citratus |
25 |
40.18 ± 0.23b |
15.80 ± 0.86g |
50 |
33.12 ± 0.71c |
30.64 ± 0.81f |
100 |
26.75 ± 0.25de |
43.95 ± 0.81e |
250 |
23.00 ± 0.54ef |
51.82 ± 1.02cd |
500 |
9.25 ± 0.77g |
80.65 ± 1.49b |
Negative control |
0 |
47.75 ± 0.59a |
0.00 ± 0.00h |
Positive control |
5000 |
2.50 ± 0.00h |
94.76 ± 0.06a |
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.
Table 3. Minimal Inhibitory Concentration (MIC) at 50% of extracts from Blumea balsamifera and Cybompogon citratus plant species against Phytophtora megakarya.
Fungal Isolates |
Treatments |
LC50 (µl mL−1) (95% FL) |
Slopes |
Chi-square |
M2-C |
B. balsamifera |
117 (100 - 137) |
1.9 |
30.4 |
C. citratus |
160 (136 - 190) |
2.02 |
36.4 |
M2-O |
B. balsamifera |
119 (101 - 140) |
1.9 |
34.2 |
C. citratus |
156 (135 - 182) |
1.95 |
35.1 |
M3-N |
B. balsamifera |
227 (183 - 296) |
2.39 |
43.1 |
C. citratus |
233 (197 - 283) |
1 |
16.5 |
M3-O |
B. balsamifera |
269 (219 - 346) |
1.38 |
24.8 |
C. citratus |
146 (126 - 170) |
1.6 |
28.8 |
FT = Fluicidal limits; LC50 = lethal concentration inhibiting fungal growth at 50%.
3.2. Combined Effect of Blumea balsamifera and Cybompogon citrates against P. mgakarya in Vitro
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.
Table 4. Combined effect of B. balsamifera and C. citratus on the growth and inhibition of P. megakarya colonies in vitro.
Isolates |
Treatments |
Colony growth (mm) |
Inhibition (%) |
M2-C |
T- |
26.25 ± 1.36b |
0.00 ± 0.00e |
T+ |
5.00 ± 0.00ef |
80.78 ± 1.06ab |
TB100 |
8.75 ± 0.62de |
66.34 ± 2.99bc |
TB50C50 |
31.62 ± 0.59a |
−21.61 ± 7.47f |
TC100 |
11.87 ± 0.65d |
54.08 ± 4.68c |
TC25B75 |
1.50 ± 1.06f |
93.61 ± 4.72a |
TC75B25 |
18.37 ± 0.82c |
29.10 ± 6.30d |
M2-O |
T- |
40.37 ± 1.85a |
0.00 ± 0.00d |
T+ |
3.00 ± 0.00d |
92.51 ± 0.37a |
TB100 |
13.75 ± 2.74c |
66.33 ± 6.12b |
TB50C50 |
28.56 ± 1.36b |
28.49 ± 6.01c |
TC100 |
11.00 ± 0.54c |
72.59 ± 1.74b |
TC25B75 |
0.81 ± 0.81d |
98.04 ± 1.95a |
TC75B25 |
9.50 ± 0.35c |
76.39 ± 0.86b |
M3-N |
T- |
42.37 ± 0.51a |
0.00 ± 0.00e |
T+ |
0.00 ± 0.00e |
100.00 ± 0.00a |
TB100 |
13.81 ± 1.16c |
67.30 ± 3.06c |
TB50C50 |
25.62 ± 0.55b |
39.47 ± 1.80d |
TC100 |
15.75 ± 0.43c |
62.83 ± 0.84c |
TC25B75 |
0.00 ± 0.00e |
100.00 ± 0.00a |
TC75B25 |
9.75 ± 1.16d |
77.07 ± 2.48b |
M3-O |
T- |
47.75 ± 0.59a |
0.00 ± 0.00e |
T+ |
2.50 ± 0.00e |
94.76 ± 0.06a |
TB100 |
19.37 ± 1.63bc |
59.28 ± 3.82cd |
TB50C50 |
24.56 ± 1.12b |
48.58 ± 2.08d |
TC100 |
9.25 ± 0.77d |
80.65 ± 1.49b |
TC25B75 |
3.43 ± 1.98e |
92.72 ± 4.20a |
TC75B25 |
14.37 ± 1.43cd |
69.96 ± 2.75bc |
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).
Figure 1. Progression of lesion due to P. megakarya during incubation time after application of treatments in vivo.
Table 5. Percentage of inhibition and surface lesions caused by P. megakarya on the cortex of cocoa pod seven days after application of both Blumea balsamifera and Cybompogon citratus.
Isolates |
Treatments |
Inhibition (%) |
Surface lesions (mm2) |
M2-C |
T- |
0 ± 0f |
196.93 ± 23.96a |
T+ |
100 ± 0a |
0 ± 0c |
TB100 |
83.34 ± 3.17bc |
5.45 ± 1.50c |
TB50C50 |
47.82 ± 3.80e |
52.07 ± 4.29b |
TC100 |
75.74 ± 3.21cd |
11.28 ± 1.90bc |
TC25B75 |
97.65 ± 1.46ab |
0.24 ± 0.18c |
TC75B25 |
62.21 ± 5.95de |
27.34 ± 5.80bc |
M2-O |
T- |
0.00 ± 0.00d |
46.59 ± 3.64a |
T+ |
100 ± 0a |
0 ± 0d |
TB100 |
40.33 ± 3.27c |
16.39 ± 1.14b |
TB50C50 |
62.39 ± 3.51b |
6.62 ± 1.11cd |
TC100 |
51.29 ± 6.88bc |
11.1 ± 2.30bc |
TC25B75 |
92.24 ± 4.53a |
0.50 ± 0.30d |
TC75B25 |
41.03 ± 4.15c |
15.96 ± 1.28b |
M3-N |
T- |
0 ± 0d |
388.52 ± 45.59a |
T+ |
100 ± 00a |
0 ± 0b |
TB100 |
85.97 ± 0.90c |
5.54 ± 0.94b |
TB50C50 |
65.02 ± 2.30b |
41.89 ± 5.93b |
TC100 |
83.76 ± 0.52b |
9.91 ± 1.80b |
TC25B75 |
98.75 ± 1.25a |
0.30 ± 0.3b |
TC75B25 |
62.13 ± 1.91c |
54.42 ± 3.64b |
M3-O |
T- |
0 ± 0e |
207.73 ± 11.27a |
T+ |
100 ± 0a |
0 ± 0b |
TB100 |
89.22 ± 1.07b |
2.47 ± 0.45b |
TB50C50 |
74.25 ± 1.16d |
13.89 ± 1.48b |
TC100 |
82.53 ± 2.56c |
6.53 ± 1.70b |
TC25B75 |
96.60 ± 1.53a |
0.40 ± 0.21b |
TC75B25 |
72.64 ± 1.17d |
15.51± 1.07b |
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.
4. Effect of Storage on the Fungicidal Potential of the Best Combination of the Two Extracts
During storage (0 to 9 days), inhibition percentages ranged between 91.38 ± 2.25% and 97.65 ± 1.46%, 71.55 ± 24.06% and 100 ± 00%, 92.70 ± 2.65% and 98.75 ± 1.25%, and 92.63 ± 0.9% and 96.60 ± 1.53% as shown in Figure 2. Importantly, statistical analysis revealed no significant differences in inhibition between isolates (df = 3; F = 0.473; P = 0.70), storage time (df = 3; F = 1.21; P = 0.31), or the interaction between isolates and storage time (df = 9; F = 1.165; P = 0.338). Notably, inhibition percentages remained above 75% for the entire storage period, suggesting good stability of the inhibitory effect.
Figure 2. Inhibition of fungi growth using treatment TC25B75 with Blumea balsamifera and Cybompogon citrates at ratio of 3:1, stored for 9 days at ambient temperature.
5. Discussion
Many phytoextracts from medicinal plants [31] or not including Rosmarinus officinalis, Thevethia puriviana, Azachitiracha indica, Allium sativum, Zingiber officinale, Thymus algeriensis, Chromoleana odorata, and Ageratum conyzoides, are already known for their antifungal properties against Phytophtora spp., causal agent of black pod [12] [13] [27] [32]. However, most studies have focused on assessing the efficacy of a single extract, whereas combining extracts can improve the efficacy of plant extracts. In the context of agroecology, there is a need to discover more fungicidal plants and their combined effects.
The present study examined the antifungal potential of Blumea basalmifera and Cymbopogon citratus against Phytophthora megakarya. The two plant extracts were assessed separately and in combination. Colony growth and inhibition percentages are the most widely quantified biological traits used to understand the interaction between plant extract and pathogen activity [27] [32].
The results obtained in this study suggest that extracts from both Blumea balsamifera and Cymbopogon citratus have the potential to inhibit the growth of P. megakarya fungi. The effectiveness of B. basalmifera and C. citratus may be due to bioactive components in the plant materials, such as secondary metabolites, which are known as important sources of microbicides, pesticides, and many pharmaceutical drugs. Ragasa et al. [33] reported that B. basalmifera contains icthyothereol acetate and cryptomeridiol, which have antifungal activities. Cymbopogon citrates also can contain 2 to 12 components depending on the geographic origin of the plant [34] [35]. Sawadogo et al. [34] reported a high content of citral (99.99%) and geranial (55%), which also have antimicrobial properties. The ethanolic extract of C. citrates may contain higher concentrations of bioactive components such as caffeic acid (1.432 mg/g), synapic acid (6.743 mg/g), and benzoic acid (7.431 mg/g), although the quantity and quality vary between geographic locations within different or even the same country [35]. Additionally, C. citrus has been widely used worldwide against various microbial diseases due to its antibacterial, antifungal, and analgesic properties.
Our study revealed that the antifungal activity of both plant extracts increased with increasing extract concentration. Different extract concentrations are typically obtained through dilution. However, this process does not increase the quantity of active components; it simply concentrates them in a smaller volume. This explains the observed trend of higher effectiveness at higher concentrations. Ambang et al. [32] also reported a similar increase in antifungal potential with increasing concentration of crude Thevetia peruviana seed extract against P. megakarya. Our findings are further supported by the reports of Djeugap et al. [36] and Adeyemi et al. [37], who observed a concentration-dependent increase in antifungal activity of ethanolic extracts of Thymus algeriensis and Chromoleana odorata, respectively, against P. megakarya.
Blumea balsamifera extract exhibited higher antifungal activity against three isolates, with the lowest Minimum Inhibitory Concentration (MIC) compared to C. citratus, which was highly effective against only one isolate. The greater effectiveness of B. balsamifera compared to C. citratus could be attributed to the qualitative and quantitative differences in their active components. These are generally affected by environmental conditions, especially the geographical location where the plants are grown [38]. The susceptibility of P. megakarya isolates to B. balsamifera and C. citratus varied. One of the four fungal isolates studied was less susceptible to both extracts. This difference in response among the fungal isolates could be related to their genetic variability, virulence, conidial production, and fungal growth speed [16]. Our findings highlight that isolates within the same fungal species can exhibit varying susceptibility. This suggests that validating plant extracts as an effective management strategy for cocoa black pod disease may require screening against various pathotypes from different agroecological zones. Our results align with those of Ambang et al. [32] who reported that one of the four P. megakarya isolates from the Centre region of Cameroon displayed resistance to Thevetia peruviana seed extract in vitro. Similarly, Mboussi et al. [27] showed that the inhibitory potential of T. peruviana and Azadirachta indica depended on the specific P. megakarya strain studied.
When combining plant extracts, synergism is often the most desirable effect because it optimizes efficacy, can affect resistant pathogens, and broadens the spectrum of activity compared to a single extract [39]. Previous research on using combinations of plant extracts against cocoa pathogens has shown promising results. For example, African panaxia extract and combinations like Cymbopogon citratus and C. schoenanthus essential oil or C. citratus combined with Azadirachta indica extract all demonstrated improved antimicrobial potential [36] [40]. In our study, a synergistic effect was observed when B. balsamifera and C. citratus were applied in a 3:1 ratio in V8 media (resulting in 92.72 ± 4.20% to 100% growth inhibition) and on detached pod cortex artificially infected (achieving 92.24 ± 4.53% to 98.75 ± 1.25% inhibition) against P. megakarya. This suggests that in vitro assays could be reliable for screening plant extracts against pathogens. Synergism may have been induced by several mechanisms, including the inhibition of a common biochemical pathway and protective enzymes, increased concentration of antifungal compounds at the inoculation sites, and the use of cell wall active agents that enhance the uptake of other antimicrobials [39]. One of the extracts may have facilitated entry into the pathogen by serially blocking metabolic pathways or inactivating microbial enzyme [39].
Our study observed high stability in the combined extract during the 9-day storage period, with antifungal activity against P. megakarya remaining above 75%. The content of bioactive components in plant extracts can vary depending on the plant species and storage duration. Some plants exhibit higher phenolic and flavonoid content in fresh material compared to stored material, while the opposite is true for others. Additionally, some species maintain similar levels of bioactive components in both fresh and stored material [41]. The consistent antifungal activity observed in our study, before and after storage, could be attributed to the presence of stable bioactive components in both the fresh and stored extracts.
Generally, microbial proliferation within plant extracts can accelerate decomposition or fermentation processes [42]. The observed stability of our extract could be due to the potential presence of natural biopreservatives within the plant material itself [43]. Further studies are warranted to optimize extraction formulations for long-term preservation.
6. Conclusion
The current study revealed that extracts of both C. citratus and B. basalmifera, alone or in combination, exhibited antifungal potential against P. megakarya. However, the antifungal effect was optimized when the two extracts were combined synergistically. This synergistic effect was concentration-dependent and varied among fungal isolates. Fungal growth inhibition increased with increasing extract concentration. Notably, the combination of 25% C. citratus and 75% B. basalmifera exhibited the strongest synergistic effect. The observed consistency between in vitro and in vivo results suggests that in vitro assays can be a reliable tool for screening plant extracts with high potential for controlling various phytopathogens. Further research is warranted to explore the underlying mechanisms of this synergy between C. citrates and B. basalmifera. Additionally, investigating the potential for optimizing the extract ratio for different P. megakarya isolates could improve the overall efficacy of this approach.
Authors’ Contributions
Conceptualization: G. Membang, J.M. Tchotet Tchoumi; Methodology: G. Membang, J.M. Tchotet Tchoumi; Investigation and Data curation: G. Membang, J.M. Tchotet Tchoumi, E.Y. Mba Ela, S.L. Lontsi Dida, L. Tchuenkam Tsango, P.L. Ekango Mbonjo; Formal analysis: G. Membang, J.M. Tchotet Tchoumi, E.E.Y. Mba; Resources: F.O. Tabi, G. Ntsomboh Ntsefong, G. Membang, J.M. Tchotet Tchoumi, E.Y. Mba Ela, S.L. Lontsi Dida,; Supervision: F.O. Tabi, G. Ntsomboh Ntsefong; Writing original draft: J.M. Tchotet Tchoumi, G. Membang; Manuscript revision: J.M. Tchotet Tchoumi, G. Membang, F.O. Tabi, G. Ntsomboh Ntsefong. All authors read and approved the final manuscript.
Acknowledgements
The Authors thank the Phytopathology and Plant Protection laboratory of the University of Yaounde 1 and the Institute of Agricultural Research for Development (IRAD) (Nkolbisson) for the protocol in maceration and technical platforms provided for the identification of Phytophtora magakarya in this work.