Management of aspergillus flavus link infections ingroundnut using bacterial endophytes as biological control agents.

Abstract

Microorganisms deteriorate groundnut seeds pre- and post-harvest. One of these microorganisms includes a fungus known as Aspergillus flavus Link. This pathogen reduces seed germination rate, see total oil content, carbohydrates, and proteins. Aspergillus flavus is a mycotoxigenic fungus that produces aflatoxins as secondary metabolites. As a result, seeds infected or colonized by A. flavus are considered not biologically unsafe for consumption and processing. Current management strategies have only provided temporary relief and increased susceptibility to the pathogen. Therefore, the use of environmentally friendly strategies is important. This study aimed to evaluate the efficacy of potential endophytic bacterial strains isolated from seeds, roots, leaves and stems of beans (Phaseolus vulgaris L.), peas (Pisum sativum L.), and groundnuts (Arachis hypogaea L.) in controlling A. flavus during storage and greenhouse trials. During the in vitro studies, 106 endophytic bacterial isolates were isolated and screened against A. flavus using the disc diffusion method. The best 10 isolates were selected for secondary screening against A. flavus in the dual culture assay. Only 13 (12%) of the isolates inhibited the growth of A. flavus in vitro after nine days of incubation. The best isolates from the screening trials include Isolate ALA (55.2%), followed by Isolates KI (40.9%) and KG (40.2%) inhibitions, respectively. Isolate ALA was obtained from Phaseolus vulgaris L. (dry bean) leaves, while Isolates KI and KG were isolated from groundnut stems and leaves. From the secondary screening, the best five isolates were sent to Inqaba Biotech Industries (Pty) Ltd for molecular characterization and identification at the species level. Three of the isolates KI, KG, and BB that were gram-positive were identified as either Bacillus siamensis, Bacillus velezensis or Bacillus amyloliquefaciens. Isolate BB also had similarities to B. subtilis. Isolate GNLA and ALA were identified as Pantoea dispersa and Pseudomonas fluorescens, respectively. Three of these strains (Bacillus siamensis/ Bacillus velezensis/Bacillus subtilis/Bacillus amyloliquefaciens strains BB, KG, and KI) were then used in in vivo experiments to assess their efficacy in controlling A. flavus on two groundnut cultivars (Akwa and Sellie-Plus) during a storage trial. In a short-term trial (7-day period), culture filtrate of B. amyloliquefaciens/B. siamensis/ B. velezensis strain KG showed potential as a biological control agent against A. flavus. An average percentage infection of 38.2% on Cultivar Akwa and 33.0% and Cultivar Sellie-Plus were observed. In contrast, the rest of the treatments (bacterial cells and crude lipopeptide extract) provided the least control. In the medium-storage trials (21-day period), the best endophytic bacterial treatment was the acidic cultured filtrate of B. amyloliquefaciens B. siamensis/ B. velezensis strain KG, which provided constant control for 21 days. The interaction of these isolates with A. flavus was studied using Scanning Electron Microscopy (SEM). In the greenhouse trials, three treatments were evaluated. These were (i) B. amyloliquefaciens/B. siamensis/ B. velezensis strain KG was used as a seed treatment, (ii) P. fluorescens strain ALA as a

foliar spray treatment, and (iii) the combination of both seed treatment and foliar spray to control A. flavus during the greenhouse trial using the two groundnut cultivar Akwa and Sellie-Plus. Both cultivars performed somewhat differently for each treatment. The highest number of seeds and seed weight was recorded for the seed treatment for Sellie-Plus. In contrast, the combination of seed and foliar spray treatment recorded the highest number of seeds and seed weight for Cultivar Akwa. However, the combination of seed and foliar spray treatment showed potential as the best treatment against A. flavus with an average percentage disease incidence of 54.8% and 46.8% for Akwa and Sellie-Plus respectively. In conclusion, the endophytic bacterial strains demonstrated potential in controlling A. flavus infection in groundnut seeds. Adjusting the pH of cultured filtrate provided a constant and enhanced control against the pathogen. Furthermore, the combination of seed and foliar spray treatment using the two bacterial strains showed potential as a biological control agent against A. flavus.