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Plant Pathology

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Browsing Plant Pathology by Subject "Aflatoxins."

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    Management of aspergillus flavus link infections in groundnut seeds using hot water treatment and biological control agents.
    (2021) Mazibuko, Phiwokuhle Zasemangweni Phelele.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
    Groundnut (Arachis hypogaea L.) is the 13th most important crop and classified as a cash flow crop. Groundnuts’ economic importance includes food and fodder purposes in the agriculture and food industries. The major constraint in the cultivation of groundnuts is Aspergillus flavus Link contaminating the kernels, subsequently resulting in aflatoxin contamination. Aflatoxins are highly toxic secondary metabolites produced by fungi of the genus Aspergillus. Under favourable conditions, A. flavus grows and develop in groundnuts at pre and postharvest. A. flavus causes yellow mould disease and can cause severe damage to the kernel. Cultural management practices are used to control yellow mould; however, they are often ineffective. Fungicides are considered the best available method for managing yellow mould, but they are inadequate in achieving A. flavus inhibition; therefore, alternative control strategies and integrated strategies are needed to properly manage the disease. This study was conducted to evaluate the efficacy of using potential yeast and Bacillus spp. with hot water treatment (HWT) in controlling A. flavus in groundnut seeds at optimum temperature x time combination without affecting seed germination rate and seed vigour. The efficacy of the treatments in minimizing aflatoxin concentration in groundnuts were also evaluated. In vitro screening trials were conducted to select the best yeast and Bacillus spp. antagonists from 169 yeast isolates and 60 Bacillus spp. isolated from leaves of different plant species. In vitro screening was carried out using the dual culture technique, and data was presented as average percentage inhibition. Both Bacillus and yeast isolates were grouped according to their in vitro performance, and the percentage inhibition data was subjected to Analysis of Variance (ANOVA) using Statistical Analysis System (SAS Version 9.4.). The best 10 yeast and best 10 Bacillus isolates were selected for secondary screening. The best two Bacillus spp. and best two yeast isolates were used as potential biological control agents in the in vivo experiments. The in vivo trial was repeated once. From the screening trials, the best performing isolates were; Isolate CC1y (yeast) with 72.6% inhibition, Isolate PF3y (yeast) with 70.8% inhibition, Isolates LM1b (Bacillus spp.) with 70.3% inhibition and Isolate PTP1b (Bacillus spp.) with 68.6%. inhibition. Only 1.7% of the 169 yeast isolates provided A. flavus mycelial inhibition greater than 70%. The best performing yeast antagonists were isolates from the spider plant, Chlorophytum comusum (Thunb.), Jacques and protea flower, Protea cynaroides (L.) L. Moreover, only 1.6% of the 60 Bacillus isolates provided a more significant mycelial inhibition with average inhibition of 70%. The best performing Bacillus species were isolated from citrus, Citrus x limon (L.), and pink purslane, Portulaca pilosa (L.). For in vitro hot water treatment (HWT) experiments, the best temperature x time combination provided the least percentage infection, with a significant reduction in disease intensity over time (AUPDC) and a non-significant reduction in seed germination rate. The best temperature x time combination was at 40oC for 60 seconds, followed by 40oC for 20 seconds. The 40oC for 60 seconds showed the least mycelial growth of A. flavus (in vivo), with the least disease progress over time and stimulating the best germination rate of treated seeds. The 40oC for 60 seconds was the best combination of all the 17 treatments with the least/worst treatment at 75oC for 60 seconds. The number of seed infections was recorded over two weeks. From the in vitro screening trials, the best two performing yeast spp. and best two Bacillus spp. were sent to Inqaba Biotechnical Industries (Pty) Ltd for molecular characterization and identification to species level. Isolates were identified as follows: Isolate CC1y as Suhomyces kilbournensis KU751783, Isolate PF3y as Rhodotorula mucilaginosa MK267619.1, KY076610.1, Isolate LM1b as Bacillus cereus JX218990.1, and Isolate PTP1b as Alcaligenes faecalis MG746621.1. The best performing combination treatment was HWT + Suhomyces kilbournensis in the inhibition of A. flavus in groundnut seeds. HWT + S. kilbournensis achieved 52% control after 12 weeks storage. Bacillus cereus alone provided the lowest aflatoxin concentration of 0.00840 mgkg-1 in treated seeds. Under storage conditions, the best treatment for disease reduction did not necessarily produced the least concentration of AFB1 and the lowest percentage of infection. In conclusion, the tested yeast, Bacillus spp., and hot water treatment were effective in reducing A. flavus infections in groundnut seeds. Moreover, the combination of these treatments provided enhanced disease control. For the first time, this study reports the application of hot water treatment combined with biological control agents on groundnuts to manage aflatoxin B1 and A. flavus infections in groundnut seeds.
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    Management of aspergillus flavus link infections ingroundnut using bacterial endophytes as biological control agents.
    (2023) Sibisi, Nokulunga Nompilo.
    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.