Management of aspergillus flavus link infections in groundnut seeds using hot water treatment and biological control agents.

Abstract

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.