Fumonisin production by and biological control of Fusarium species associated with cowpea seed

Abstract

Cowpea, Vigna unguiculata (L.) Walp. is an important edible legume crop gaining recognition in the world generally and in Africa particular. The seed is a very good source of vegetable protein for millions of people living in sub-Saharan Africa. Cowpea seed are susceptible to global post-harvest losses caused by insect damage, microbial deterioration and other factors especially when stored at high ambient temperatures and relative humidities. The detection of Fusarium species and fumonisins associated with cowpea seed was primarily investigated in this study. Secondly, the antifungal activity of extracts from three Rubiaceae plant species was evaluated against five Fusarium species. Column chromatography was used in an attempt to isolate potential compounds from the plant extracts. Cowpea seed samples were obtained from a local market of a rural community in Mpumalanga Province, South Africa and were analysed for seed mycoflora and various fungal genera including, Aspergillus spp., Fusarium spp. (Fusarium oxysporum. F. solani, F. subglutinans, F. verticillioides and F. proliferatum), Penicillium spp., Chaetomium spp., Chrysonilia spp., Cladosporium spp., Monascus spp., Phoma spp., Mucor spp., Alternaria alternata, Epicoccum spp. and Lasiodiplodia theobromae were isolated from the seed samples. Some Fusarium species including F. oxysporum, F. verticillioides, F. subglutinans, F. solani and F. proliferatum were cultured on maize patty media and analysed for fumonisin production. Fumonisin detection was conducted using the VICAM Affinity method. Only Fusarium subglutinans and F. oxysporum produced fumonisin B analogues ranging between 1.1 - 4.3 ìg/g and 3.47 - 31.66 μg/g, respectively. Neither Fusarium verticillioides, F. solani nor F. proliferatum revealed any production of fumonisin B. Fumonisin B analogues were found to be present in all the cowpea seed samples with levels ranging between 6.2 - 59 μg/g. Purified fumonisin B1 was used to investigate the phytotoxic effects of the toxin in vitro and in vivo on cowpea seedling growth and emergence and also on the ultrastructure of the seed tissue. Surface-disinfected seeds were imbibed in sterile distilled water amended with FB1 at various concentrations (10, 25, 50 and 100 μg/ml) and dry seeds and seeds imbibed in sterile distilled water served as the controls. Percentage germination was determined in vitro and in vivo according to the International Seed Testing Association (ISTA) rules. Root and shoot length was measured after 8 days. Compared to the controls all the concentrations significantly decreased seed germination and the highest concentrations of FB1 (50 and 100 μg/ml) inhibited root and shoot elongation. Transmission electron microscopy (TEM) was used to study sections of the cotyledon and embryonic tissue of cowpea seed. Samples were treated with different concentrations of FB1. Compared to the controls, the degree of damage to the vacuoles, cell walls and lipid bodies of cowpea seed tissue was directly proportional to the concentrations. The efficacy of crude extracts from Morinda citrifolia, Gardenia brighamii, and Psychotria capensis was investigated against various Fusarium species by two methods. The microtitre plate doubling dilution method and bioautography method were used to test methanol, acetone, and dichloromethane extracts of the leaves for their antifungal activity. Psychotria capensis acetone extracts showed the best inhibition against F. proliferatum and F. verticillioides with an MIC of 1.5 mg/ml and an MFC of 1.5 mg/ml. The dichloromethane extracts exhibited no activity against the Fusarium species tested. Column chromatographic purification of methanolic extracts of the leaves of P. capensis led to the isolation and identification of two compounds namely β-sitosterol and a carotenoid derivative.