Rhizobacteria as potential bio-inoculants and biocontrol agents to promote black pepper plant cultivation -

Rhizobacteria as potential bio-inoculants and biocontrol agents to promote black pepper plant cultivation

In this study, isolated seven indigenous rhizobacteria antagonistic to soil-borne Fusarium solani, the causal fungus of slow decline, the most serious debilitating disease of black pepper. Isolated bacteria Bacillus subtilis, Bacillus siamensis, Brevibacillus gelatini, Pseudomonas geniculata, Pseudomonas beteli, Burkholderia ubonensis and Burkholderia territorii and these bacteria were effective in production of antifungal siderophore with the amount of 53.4 % –73.5 % per 0.5 mL of cell-free supernatants and also produced chitinase with chitinolytic index was ranged from 1.19 to 1.76. The bacteria have shown phosphate solubilizing index within 1.61 to 2.01. The isolated antagonists were efficacious in stimulation of black pepper plant growth and root development through IAA (10.5 μg/mL–42.6 μg/mL) secretion. In conclusion, the isolated rhizobacteria are potent to be developed as biocontrol agents and also minimize the utilization of hazardous chemicals in black pepper disease management. It also developed as bio-fertilizers to improve black pepper plant growth due to their capabilities in plant growth-promotion.

Antagonistic effects of rhizobacteria in against F. solani strain FS010 were evaluated through dual culture plate assay. Rhizobacteria was spot inoculated at one side of agar plate with 3 cm in distance from mycelium plug. Control plate contained fungal mycelium plug without bacterial inoculation. The percentage of inhibition of radial growth (PIRG) was calculated through formula PIRG (%) = [(Rc – Ri) / Rc] x 100, where Rc denotes fungal mycelium growth at opposite side of bacterial colonies and Ri denotes fungal mycelium growth towards bacterial colonies.

Inhibition of fungal infection

Pathogenicity tests were carried out in two experimental designs to study the antagonistic effects of isolated bacteria on F. solani strain FS010. Fungus inoculation on black pepper cultivar Semengok Aman (PnSA) and cultivar Kuching (PnKch) roots was carried out. The three month-old black pepper cuttings were carefully uprooted from the soil medium. The roots and collar regions of the cuttings were washed carefully with adequate tap water to remove excess soils prior wounded by using sterile scalpels. In Trial 1, the washed pepper roots were dipped in overnight grown bacterial cultures (LB) for 10 min prior transplanted into polybags filled with sterile soil medium. The cuttings were subsequently treated with same bacterial cultures for three days before fungal infection was introduced on the 4^th day of the experiments. Bacteria inoculation was carried out by drenching soil surrounding collar regions of black pepper cuttings with overnight grown bacterial cultures. Meanwhile, in Trial 2, the cuttings were directly transplanted into new polybags without bacterial treatment. No activity was carried out on black pepper cuttings in Trial 2 for the first three days of the experiments. Fungal inoculation was introduced in both trials on the 4^th day of the experiments by drenching the soil surrounding collar regions of black pepper cuttings with F. solani conidial suspensions. The experiments were left for three months until black pepper cuttings have shown clear symptoms of slow decline disease such as foliar yellowing, damage of collar regions and loss of feeder roots. The percentage of fungal colonization on the infected black pepper roots was subsequently calculated using the formula, Colonization (%) = (Nf/Nt) x 100, where Nf denotes number of root segments with fungal colonization and Nt denotes the total number of root segments.

Antagonism

In the dual culture plate assay, the strongest antagonism was exhibited by Br. gelatini strain JD04 with PIRG value of 68.5 %. The bacteria suppressed the growth of F. solani through iron competition. However, by supplying FeCl₃ to the culture medium, there is no privilege for the bacteria to compete iron with F. solani through siderophore production as there was sufficient amount of iron in culture medium to support the growth of both organisms. The siderophore-mediated antagonism assay revealed that the percentage of PIRG without FeCl₃ supplement was varied from the lowest 61.6 % in P. beteli strain JS05 up to the highest 68.5 % in Br. gelatini strain JD04.

Inhibition of fungal infection

Three month-old black pepper cuttings of PnSA and PnKch were infected by F. solani strain FS010, the diagnosed causal fungus of slow decline incident reported in disease farm. Foliar yellowing was observed on cuttings in Trial 2 after 2 weeks of infection. As the experiment progressed, damage of collar regions and loss of feeder roots was also observed on infected cuttings in Trial 2. However, there is no disease symptoms were observed on cuttings in Trial 1 after the same trial periods. The results of fungal colonization analysis have shown that F. solani strain FS010 was present in root tissues of the infected cuttings in Trial 2. The fungus was detected in root segments of both black pepper cultivars with PnKch roots were more susceptible to fungal invasion than PnSA roots. The mean values of fungal colonization on PnSA and PnKch roots were 40.51 % and 48.31 %, respectively. However, no or very little amount of fungus was colonized on black pepper root tissues that have been treated with isolated PGPB in Trial 1. These results revealed that the invasion of F. solani strain FS010 in black pepper root tissues was restricted by the selected antagonistic rhizobacteria in Trial 1.

Discussion

Plant growth-promoting bacteria are of great importance to agriculture due to their abilities in promoting plant growth and suppress pathogens. The rhizobacteria and their secondary metabolites such as antibiotic, enzyme and hormone have constituted valuable biological stimulants in regulating plant stress and defence response. Bacteria were more effective in restriction of fungal growth when the content of ferric iron in agar medium was reduced.

In the presence of ACC deaminase producing bacteria, it is able to convert ACC and reduce ethylene levels in the black pepper cuttings during the sowing process and subsequently promote the growth of the cuttings. The released ammonia can be used as nitrogen source for the bacteria and also uptake by black pepper cuttings for root development. Moreover, the selected bacteria isolates are potent to be developed as bio-fertilizer as they are an effective deaminase producer. Application of these bacteria to nature environment could improve nitrogen availability in soil as these bacteria are able to hydrolyse amino compounds of the organic substances in soil to release ammonia through deamination.

This study has proposed biocontrol strategies as an alternative solution instead of synthetic fertilizers and agrochemicals in combating slow decline on black pepper. In total, seven indigenous PGPB isolates potential in restriction of pathogenic soil-borne F. solani strain FS010 have been identified in this study. These antagonists are potent to be developed as biocontrol agents due to their capabilities in the production of siderophore and chitinase. Moreover, these antagonists also have great potential to be developed as bio-fertilizers as they were an effective ammonia producers and phosphate solubilizers. These antagonists were also efficient in stimulating black pepper root growth and improving nutrient uptake status in black pepper plants through IAA synthesis ACC deaminase activity.

Citation:

Lau, E.T., Tani, A., Khew, C.Y., Chua, Y.Q. and San Hwang, S., 2020. Plant growth-promoting bacteria as potential bio-inoculants and biocontrol agents to promote black pepper plant cultivation. Microbiological research, 240, p.126549.