MATERIALS AND METHODS

Area of study: The experiment was conducted at the Laboratory and the planthouse, the Department of Crop and Soils Sciences of the Kwame Nkrumah University of Science and Technology (KNUST), Kumasi-Ghana in August-September, 2017. The University is located between Latitude 6.6745°North and 1.5716°West, the second largest city of Ghana, West Africa.

Sources and maintenance of microbial antagonists: In this study, microbial antagonists viz: Bacillus amyloliquefaciens, Aspergillus sp. and Penicillium sp. were used. The B. amyloliquefaciens was previously isolated from yam (Dioscorea sp.) rhizosphere soils, kept on nutrient agar and refrigerated at 4°C till needed whilst the fungal antagonists (Aspergillus sp. and Penicillium sp.) were obtained as anti-Phytophthora laboratory contaminants of P. palmivora and maintained on Green Cocoa Mucilage Agar (GCMA) and kept at 26±2°C with frequent sub-culturing till needed.

Protection of cocoa seeds from damping-off disease with the Bacillus amyloliquefaciens (plastic tube experiment): Plastic centrifuge tubes (50 cc) measuring about 30 mm in diameter and 115 mm deep were filled with five grams of oven-sterilized topsoil and infested with a mixture of mycelia and sporangia of P. palmivora (10 mL sterile distilled water) suspension prepared from a 14 days-old culture. The tubes were incubated on a planthouse bench. After two weeks, the infested soils were separately drenched with (i) 10 mL NB suspension of the B. amyloliquefaciens prepared from a 24 hrs culture of the bacterium and diluted to a concentration of 10⁸ CFU mL^–1 and (ii) Fungicide (seed star) containing 20% Metalaxyl, 20% Imidacloprid and 4% Anthraquinone (Nova Agro Ltd, Hong Kong), prepared according to the manufacturer’s instruction. Two cocoa seeds were then sown into each tube and tinned to one seedling after germination. Four replicate tubes per treatment were established. Sterilized infested soils with P. palmivora drenched with NB and infested soils with only P. palmivora served as controls. The setups were kept in a planthouse for six weeks and disease assessments were made on seed decay and the resulting seedlings. For the recovery of the bacterium from rhizosphere soil, representative tubes of each treatment were assayed for B. amyloliquefaciens by sprinkling particles of soil on NA in Petri plates and were observed after 24 hrs. Roots of plants were also biopsied by plating pieces on NA plates and the resulting cultures observed for the diagnostic characteristics of B. amyloliquefaciens.

Evaluation of microbial antagonists for the prevention of damping-off/seedling blight and plant-growth promotion of cocoa seedlings (Pot experiments): Steamed sterilized topsoil (20 kg) was placed on a black polyethylene sheet on a laboratory floor and infested manually by mixing with two liters of Green Cocoa Mucilage Broth (GCMB) suspension of P. palmivora. The suspension was prepared by homogenizing a two-week-old culture of the fungus with a laboratory blender at low speed for 10 min. The infested soil was covered with another polyethylene sheet and left to incubate on a laboratory floor at room temperature of 28±2°C. An uninfested control soil was also established. Particles of the infested soil were bioassayed after two weeks, by sprinkling on GCMA to ensure that the P. palmivora had been established in the soil. Uninfested control soil was similarly assayed to ensure that it did not contain any Phytophthora inoculum. Ten kilograms of the P. palmivora-infested soil was distributed into 450 cc (90 mm diameter and 70 mm deep) plastic pots (250 g of soil/pot) and each of five pots drenched with a NB suspensions of the bacterium B. amyloliquefaciens (100 mL/pot). Sets of five pots were similarly, but separately drenched with Potato Dextrose Broth (PDB) suspensions of an Aspergillus sp. and a Penicillium sp. (10⁸ spores mL^–1). Treated soils were incubated for 14 days in a planthouse. Infested soil drenched with the fungicide Seed star and undrenched control soils were also established as positive and negative controls respectively. Soils with only NB, GCMB, PDB and sterilized water were similarly setup as other controls.

Cocoa seeds were extracted from pods (Hybrid: Amelonado×Amazon) obtained from the Seed Production Unit of Cocobod, Jamasi in the Ashanti region. Pods, collected from the same tree were used. The seeds were bulked and sown in the pots (one seed per pot) and kept in a planthouse. They were equally watered with 10 mL water when needed. On germination, the seedlings were grown for an additional six weeks and the following data were taken on each plant (i) the girth of stem-taken with Vernier calipers two centimeters from the soil line, (ii) the plant heights were taken with a 30 cm meter rule from the soil line to the tip of growing stem, (iii) the number of leaves per plant and (iv) the dry matter. Dry matter analysis was done according to the method of Awuah¹⁴ by carefully removing plants from soil, washing soil off the roots, drying the entire plants in an oven at 80°C to constant weight and dry matter obtained by weighing the plants.

Concurrently, cocoa seeds were dipped in suspensions of the microbial antagonists before sowing in P. palmivora- infested soils contained in 450 cc plastic pots (five replicate pots per treatment). Seeds dipped in a fungicide (Seed star) suspension, NB, GCMB, PDB and sterilized distilled water were also setup as control. Plants were grown as in the drenching experiment and similar plant growth data taken after 6 weeks.

Recovery of microbial antagonists: For the recovery of microbial antagonists (B. amyloliquefaciens, Aspergillus sp. and Penicillium sp.) and the pathogen (P. palmivora), soils were assayed before sowing and 6 weeks later at the end of the experiment to confirm the presence or the absence of the antagonists in the soil. Assays were done by introducing a sterile wire loop into the soil and streaking it on Nutrient Agar (NA) plates (for the bacterium), Chloramphenicol Potato Dextrose Agar (CPDA) plates (for the Aspergillus sp. and the Penicillium sp.) and green cocoa mucilage agar (GCMA) plates for P. palmivora. The experiments were repeated in the same soils and pots after six weeks (which constituted Pot experiment II).

Determination of antimicrobial metabolite production in soil by the rhizobacterium, B. amyloliquefaciens, ESI: In an initial experiment, plastic pots (150 cc; 65 mm diameter and 60 mm deep) each containing 125 g sieved, oven sterilized topsoil were either drenched with 50 mL, 24 hrs old ESI Nutrient Broth (NB) culture or ESI in sterilized distilled water suspension. Potted soils were maintained by watering with an equal amount (10 mL) of water when needed to keep them moist. There were four sets of treatments viz. (i) Potted soil amended with 0.7 g of urea (40% N) and drenched with ESI NB culture, (ii) Soil amended with 0.7 g of urea (40% N) and drenched with ESI in Sterilized Distilled Water (SDW) suspension, (iii) Soil drenched with ESI NB culture and (iv) Soil drenched with ESI SDW suspension. Controls consisted of the above treatments but without ESI were maintained. Also maintained as controls were ESI NB culture and NB alone without passing through the soil. Four replicates pots were maintained per treatment. The experiment was repeated once but in the repeat experiment, controls consisted of only ESI NB culture and NB alone without passing through soil were maintained. After three weeks, soil in each pot was mixed thoroughly, 10 g added to eight milliliters of distilled water into 25 cc vials and left to stay for 1 hr. The decanted supernatant was autoclaved and allowed to stand for 24 hrs for the fine soil particle to settle. Five milliliters of the resulting clarified supernatant was placed in 25 cc vials and autoclaved again to obtain the final ESI filtrates from the soils. Each vial was seeded with a 20 μL of seven-day-old A. niger conidial suspension (1×10⁶ conidia mL^–1) and incubated on a laboratory bench at room temperature (28±2°C) for 28 days. Growths of mycelial mats from the resulting cultures, if any were carefully harvested by using loop onto pieces of A4 paper, oven-dried at 20°C for 3 hrs and weighed. The experiment was repeated simultaneously with non-sterilized soil.

Statistical analysis: The study was conducted using a Complete Randomized Design (CRD) with three replicates where necessary. The analysis of variance (ANOVA) was performed on the data using GenStat statistical package¹⁵. When there was significance, mean separation was accomplished using the Least Significant Differences (LSD) test at 5% probability. For the basic statistical analysis of data, MS Excel 2010 was used.

RESULTS

Protection of cocoa seeds with the B. amyloliquefaciens (Plastic tube experiment): Result from this study shows that cocoa seeds were totally protected when P. palmivora-infested soil in plastic tubes was drenched with the B. amyloliquefaciens (ESI) broth suspension giving a percentage of infection of zero (0%) indicating a 100% germination of seeds comparable to the P. palmivora- uninfested control soil and the fungicide-treated soil which recorded 100% disease infection (a total seedling decay/pre-emergence damping-off) in Table 1 and shown in Fig. 1a-e.

Prevention of damping-off/seedling blight in cocoa and promotion of plant growth by microbial antagonists: Similar results were obtained when the experiment was conducted in plastic pots that included two other microbial antagonists (Aspergillus and Penicillium spp.) apart from the rhizobacterium B. amyloliquefaciens (ESI), revealed that the percentage of damping-off was zero (0%) indicating 100% germination of seeds for all the three antagonists in both the drenched P. palmivora-infested soils as well as seeds dipped in antagonists before planted into P. palmivora-infested soils (five seed germinated out of the five replicate pots)and these were comparable to the uninfested soil and the fungicide treated soil (+ controls) which also recorded 0% damping-off (100% seed germination) that did not receive the antagonists. The P. palmivora-infested negative control soils, however, resulted in damping-off of 80% indicating 20% seeds germination and 60% damping-off (40% germination) in soils when drenched or seeds dipped in microbial suspension before sowing respectively in Fig. 2a-f and Table 2.

In the repeat experiment which was conducted in the same soils and pots after six weeks, total damping-off (100% damping-off) indicating 0% seed germination was recorded only when seeds were planted in soil that previously was infested with P. palmivora whiles 0% damping-off (100% germination) recorded in soils treated with microbial antagonists respectively in Fig. 3a-f and Table 3.

Fig. 1(a-e):

Cocoa seedlings growing in plastic tubes containing; (a) Untreated soil, (b) P. palmivora-infested soil drenched with B. amyloliquefaciens, ESI, (c) P. palmivora-infested soil drenched with fungicide (seed star), (d) P. palmivora-infested soil drenched with nutrient broth (NB) and (e) P. palmivora infested soil not receiving any protective drench, demonstrating the protective ability of the bacterial antagonist, ESI. The last two tubes had ungerminated, damped-off seeds

Plant-growth promotion with microbial antagonists (Pot experiments I and II)
Dry matter analysis: Dry matter weights of cocoa seedlings in P. palmivora-infested soils receiving ESI, Aspergillus and Penicillium spp. drenched soils ranged from 0.88-4.62 g and 0.88-4.69 g in seed dipped in microbial suspensions.

Fig. 2(a-f):

Stages of development of cocoa seedlings in drenched soils infested with P. palmivora; (a) Uninfested soil (positive control), (b) Infested soil treated with B. amyloliquefaciens ESI (bacterial antagonist), (c) Infested soil treated with Aspergillus sp. (fungal antagonist), (d) Infested soil treated with Penicillium sp. (fungal antagonist), (e) Infested soil treated with fungicide (seed star) (positive control) and (f) Untreated infested soil (negative control)

Fig. 3(a-f):

Stages of development of cocoa seedlings when seeds dipped in soils infested with P. palmivora; (a) Uninfested soil (positive control), (b) Infested soil with seeds dipped in the B. amyloliquefaciens, ESI suspension (bacterial antagonist, (c) Infested soil with seeds dipped in Aspergillus sp. suspension (fungal antagonist), (d) Infested soil with seeds dipped in Penicillium sp. suspension (fungal antagonist), (e) Infested soil with seeds dipped in fungicide suspension (seed star) (positive control) and (f) Infested soil (negative control)

The least dry matter of 0.88 g was associated with the infested soil that did not receive the microbial antagonists and this was significantly lower (p<0.05) than all the dry matter in Table 4. There were also no significant differences (p<0.05) between the three microbial antagonists. Similarly, the lowest result associated with infested soil was recorded when seeds were dipped in suspensions of the microbial antagonists in experiment I (Table 4).

In the repeated experiment (experiment II) conducted in the same soils six weeks after the initial pot experiment (experiment I), the dry matter ranged from 0.00-4.92 g (drenched) and 0.00-4.41 g (dipped). The least of 0.00 g indicating no dry matter was obtained from P. palmivora infested soil. Dry matter of 4.92 and 4.41 g were associated with the sterilized uninfested soil of drenched and dipped respectively and this was significantly similar generally to those obtained with the microbial antagonists (Table 4).

Number of leaves (Pot experiment I): Infested soils drenched with suspensions of the microbial antagonists produce seedling with a significantly higher mean number of leaves of 4-10 (p<0.05) compared to those obtained from the soil infested with P. palmivora of 1-3 leaves in Fig. 4a. Similar results were obtained when the seed dipping method was used in Fig. 4b.

Plant height: Seedlings heights 14-17 cm were highest in P. palmivora-infested soils drenched with suspensions of microbial antagonists. There were significantly different (p<0.05) from those associated with the infested soil without biocontrol treatment of 4-5 cm in Fig. 4c. In the experiment where seeds were dipped in suspensions of the microbial antagonists before being sown, the results obtained with the microbial antagonists were generally similar (14-18 cm) to the drenched experiment with regard to the plant height and differed in plant height significantly (p<0.05) with P. palmivora-infested soil (control) of 3-5 cm in Fig. 4d.

Plant girth: The widest plant girths of 0.25-0.55 cm were obtained in infested soils drenched with suspensions of the three microbial antagonists and these were significantly higher (p<0.05) than those obtained with the P. palmivora-infested soil (-control) of 0.00-0.22 cm. Generally, there were also no significant differences in seedling girth among the microbial treatments in Fig. 4e. These trends were similar to those obtained using seeds dipping method with 0.22-0.48 and 0.00-0.18 cm for microbial antagonists and P. palmivora-infested soils respectively in Fig. 4f.

With respect to the growth parameters studied (number of leaves, plant height and plant girth) in the pot experiment II shown in Fig. 5a-f, conducted in the same soils, 6 weeks after the experiment I, the following results were obtained.

Number of leaves (Pot experiment II): Infested soils drenched with suspensions of the microbial antagonists produce seedling with a significantly higher mean number of leaves of 2-5 (p<0.05) compared to those obtained from the soil infested with P. palmivora of zero (0) leaves indicating that none of the seeds germinated in Fig. 5a. Similar results of 2-7 leaves were obtained for the microbial antagonists and zero (0) seeds for soil infested with P. palmivora when the seed dipping method was used respectively in Fig. 5b.

Fig. 4(a-f):

Graph showing plant growth promoting ability of the microbial antagonists (Pot experiment I); (a-b) Mean number of leaves for the drenching method and dipping method, (c-d) Drenching method and dipping method: plant height and (e-f) Plant girth for the drenching method and dipping method. Vertical bars represent standard errors of meanss

Fig. 5(a-f):

Graph showing plant growth promoting ability of the microbial antagonists (Pot experiment II); (a-b) Mean number of leaves for the drenching method and dipping method, (c-d) Plant height for the drenching method and dipping method and (e-f) Plant girth for the drenching method and dipping method. Vertical bars represent standard errors of mean

Plant height: Seedlings heights 14-18 cm were highest in P. palmivora-infested soils drenched with suspensions of microbial antagonists. There were significantly different (p<0.05) from those associated with the infested soil without biocontrol treatment of 0.00 cm in Fig. 5c. In the experiment where seeds were dipped in suspensions of the microbial antagonists before being sown, the results obtained with the microbial antagonists were generally similar (10-18 cm) to the drenched experiment with regard to the plant height and differed in plant height significantly(p<0.05) with P. palmivora infested soil (control) of 0.00 cm in Fig. 5d.

Plant girth: The widest plant girths of 0.25-0.55 cm were obtained in infested soils drenched with suspensions of the three microbial antagonists and these were significantly higher (p<0.05) than those obtained with the P. palmivora-infested soil (-control) of 0.00 cm. Generally, there were also no significant differences in seedling girth among the microbial treatments Fig. 5e. These trends were similar to those obtained using seeds dipping method with 0.22-0.48 and 0.00 cm for microbial antagonists and P. palmivora-infested soils respectively in Fig. 5f. The value of zero (0)recorded in the P. palmivora-infested soils that did not receive any microbial antagonists, the seedlings completely died indicating total or complete damping-off.

Bioassay of soil for the microbial antagonists and P. palmivora: The microbial antagonists ESI, Aspergillus sp. and Penicillium sp. were recovered from soils into which they were incorporated in Table 5 whilst P. palmivora was recovered only from soils infested with the pathogen and did not receive anti-microbial treatments in both drenched soils and seeds dipped in microbial suspensions. The pathogen could not be isolated from soils that had been treated with the microbial antagonists. The presence of either the antagonists or the pathogen is indicated as positive (+) and absence as negative (-) (Table 5).

Anti-microbial metabolite production in soil by the B. amyloliquefaciens, ESI: In an initial study, when 24 hrs nutrient broth culture of ESI amended with urea fertilizer (43% N) or without urea fertilizer was used to drench the soil for three weeks, production of the anti-microbial metabolite was enhanced in the soil, resulting in complete growth inhibition (indicated as negative) of the test fungus, Aspergillus niger, grown in the soil extract in Table 6. Similarly, when sterilized distilled water was used instead of the nutrient broth and amended with either Urea or without Urea, the resulting soil extract also completely inhibited the growth of the test fungus (indicated as negative), compared to the control which was without the bacterial antagonist, B. amyloliquefaciens ESI (indicated as positive) (Table 6). In other control, traditionally the nutrient broth was used to culture the B. amyloliquefaciens ESI and to produce the antibiotics which was used as a positive control and also resulted in complete inhibition of the test fungus (also indicated as positive control). Similar results were obtained when the experiment was repeated with each of the treatments with its own control which did not have the metabolite produced by the bacterial antagonist, B. amyloliquefaciens ESI (also indicated as negative) (Table 7).

DISCUSSION

Seed decay and seedlings damping-off of cocoa (Theobroma cacao L.) caused by Phytophthora species continue to be a silent killer of cocoa seedlings at the nursery. Prevention of this menace is crucial using microbial antagonists for sustainable control. The results obtained in this study show that broth suspensions of the three microbial antagonists used as drenched or as seed dip in P. palmivora-infested soil, prevented damping-off and protected seedlings growth. The soil is a primary source of inoculum for Phytophthora palmivora⁵, therefore, soil with a history of Phytophthora infestation will require treatment before being used to raise seedlings. This, however, is rarely done but the soil is not the only source of inocula. According to Erwin and Ribiero⁷ and Opoku and Wheeler⁸, if soil infested with Phytophthora sp. is used in raising seedling, the roots of the seedlings become infected and if such seedlings are planted in the field, they would spread the pathogen to pathogen-free fields.

The three microbial antagonists also offered protection against cocoa seed decay and seedling damping-off, comparable to the fungicide Seed star. The ability of the microbial antagonists to protect cocoa seeds from decay and seedling damping-off is a positive development since healthy cocoa seedlings could be established not only in the nursery but even in the field during the early stages of transplanting for seedlings establishment, using the antagonists. When these microbial antagonists are introduced during the early stage of cocoa establishment, seedlings protection from Phytophthora root rot will be ensured and plant growth promoted.

Dipping the cocoa seeds in the suspensions of microbial antagonists or drenching the soil with microbial suspensions gave similar results with respect to protection against damping-off and plant growth, even when the soils were replanted after six weeks. This shows how early these microbes are established in the soil and how persistent these microbes are in the soil and this might due to how the microbial antagonists might have used the soil nutrients and root exudates for their maintenance in the soil. This study, therefore, suggests that the use of the seed dipping method of applying or introducing the microbial antagonists to the soil would be the most appropriate since it is easier to implement than the soil drenching method. Ahmad and Baker¹⁶ have indicated that biocontrol agents can colonize the rhizosphere when added as seed treatment and may protect the underground parts of plants from pathogen attack.

Biocontrol of damping-off of seedlings with bacterial and fungal antagonists, especially Bacillus spp. is known for B. amyloliquefaciens¹⁶, B. subtilis^17-21, Pseudomonas flourescens²¹, P. putida²¹, Burkholderia cepacia^21-22, Trichoderma viride^21-22, Gliocladium virens^21-22. Bacillus spp. are capable of colonizing the root environmment^23-27. This study found this to be true as the rhizobacterium, ESI was detected on root surfaces of cocoa seedlings and seed coats perhaps providing physical protection as reported by¹⁶. This result also suggests that the rhizobacterium ESI would not only offer physical protection to the seeds but also may use other mechanisms such as antibiosis to achieve this objective since it is established that it possesses the genes for the production of secondary metabolites confirmed as antibiotics iturin A, bacillomycin D and surfactin in another studies²⁸. Therefore, the protection of cocoa seeds from decay and seedlings from damping-off by the bacterium could be attributed to the three antibiotics as well as the physical protection offered by the biofilm of the bacterium.

In this study, an indirect approach was used to determine the potency of antifungal metabolites production in soil by the rhizobacterium, ESI. The method involved drenching and leaching out treated soil with water and using a known amount of the leachate to culture the test fungus Aspergillus niger. Inhibition of the mycelial mat weight of the A. niger then would indicate that antifungal metabolites have been produced in the soil environments. This approach and/or its modifications were also used to determine whether metabolites were produced on root surfaces. Even though the soils were found to contain inhibitory substances (possibly antibiotics), such substances could not be detected on root surfaces possibly because small quantities were produced on such surfaces which rendered the method used insensitive for detection. Production of antibiotics in situ by biocontrol agents indicates that the effective quantities are difficult to estimate since they are produced in small amount relative to other, less toxic organic compounds such as salicylic acids, indole acetic acids, jasmonic acids, lipopolysaccharide, etc. present on root surfaces²⁹.

Plant-growth promotion is an attribute of biocontrol agents and is due to the production of growth hormones or regulators such as indole acetic acid, gibberellins, cytokinins and auxins that directly enhance growth^30-31 and indirectly through the production of antibiotics^32-33. In this study, cocoa seedlings growth, in terms of dry matter, the number of leaves, seedlings height and seedling girth were promoted when the antagonistic micro-organisms were added to the soil. Meena and Marimothu³⁴ observed the boosting effect of Pseudomonas fluorescens formulation on plant height, leaf area index, root length, nodules per plant and dry matter on beans. However, this study attributed the boosting effect to the suppression of Phytophthora palmivora in the soil to antibiotics production. This study has therefore demonstrated that the three microbial antagonists can be formulated to prevent damping-off and promote seedling growth of cocoa in the nursery environment.

CONCLUSION

This study has appreciated that seed decay and seedlings damping-off of cocoa (Theobroma cacao L.) caused by Phytophthora species which continues to be a silent killer of cocoa seedlings at the nursery, can no longer be considered unimportant. Prevention of this menace is therefore crucial using microbial antagonists for sustainable control. The findings of this study have demonstrated that broth suspensions of the three microbial antagonists used as a drench or as seed dip in P. palmivora-infested soil, prevented seed decay, seedling damping-off and promoted seedlings growth. However, the seed dip method is recommended over the drenched method due to ease of application. It is also noteworthy that the microbial antagonists as soil inhabitants, persisted in the soil, thereby offering lasting protection to cocoa seedlings. The microbial antagonist’s viz. Bacillus amyloliquefaciens (ESI), Aspergillus and Penicillium spp. have demonstrated inhibitory capacity against P. palmivora in the soil and therefore potential biocontrol agents against seed decay and seedling dumping-off of cocoa seedlings.

SIGNIFICANCE STATEMENT

This study has highlighted the role of microbial antagonists that could be beneficial for the prevention of seed decay, seedling damping-off and their growth promotion ability. The outcome has therefore helped to uncover the critical areas of the need to pay particular attention to protecting the cocoa seedlings at the nursery stage from eminent collapse and possible transfer of black pod disease to the field that many researchers in the field have not paid particular attention to.

ACKNOWLEDGMENT

I sincerely acknowledged the role of the Laboratory Technicians of the Department of Crop and Soils Sciences of the Kwame Nkrumah University of Science and Technology (KNUST), Kumasi-Ghana for their tireless effort to see this study materialized.