The physiological and molecular effects of fumonisin B1 on cowpea (Vigna unguiculata L. Walp)

Abstract

Cowpea (Vigna unguiculata) is an edible annual leguminous crop cultivated by many subsistence and rural communities in developing countries in subtropical areas of the world. Cowpea is a very versatile, well adapted and nutritious grain legume. However, cowpea seed is prone to fungal infestation under suboptimal storage conditions. Some of these fungi, including Aspergillus and Fusarium spp., produce their associated mycotoxins under these conditions. Fumonisins are produced primarily by Fusarium verticillioides and F. proliferatum and are known to be toxic to vertebrates and plants. Fumonisin B1 is phytotoxic to cowpea seeds and is speculated to inhibit ceramide synthase in the sphingolipid pathway in plants. This study was done to determine the phytotoxic effects of FB1 on cowpea seedlings and to provide insight on the mode of action of the toxin at a molecular level. Surface-disinfected seeds were imbibed for 10 h in sterile distilled water amended with FB1 to yield final concentrations of 2, 20 and 40 mg/L. Slow imbibed seeds (placed in moist paper towels) and seeds placed in sterile distilled water for 10 h served as the positive and negative controls, respectively. Additionally cowpea seeds were inoculated with the conidia of three different FB1-producing Fusarium verticillioides strains. Percentage emergence was determined after seven dpi whereas, seedling mass, length and seedling vigour index where determined after seven and 21 dpi. Total chlorophyll content was measured after 14 and 21 dpi. Stunted growth was observed in FB1 treated seedlings. Emergence was reduced by all three FB1 concentrations as well as in seeds treated with F. verticillioides strain MRC 8265. Seedlings imbibed in 40 mg/L FB1 had reduced seedling length. Seedling mass was reduced by all three FB1 concentrations as well as all three Fusarium strains. Total chlorophyll content was higher for seeds imbibed in all three FB1 concentrations when compared to both controls. This is in contrast to other studies which reported that FB1 causes chlorosis in plants. In order to evaluate the effect of FB1 on ceramide synthase gene expression seeds were imbibed in a 20 mg/L FB1 solution and the control seeds were imbibed in sterile distilled water. RNA was extracted from untreated and treated samples after 0, 3 and 12 dpi with the latter being divided into shoot and roots samples. cDNA was synthesised from the extracted RNA samples. Amplification of the ceramide synthase gene was done using primers that were designed to the conserved regions of Glycine max and Phaseolus vulgaris ceramide synthase gene homologues. Sequence analysis revealed that the designed primers did amplify the correct gene from cowpea seedlings. The expression levels of the ceramide synthase gene from cowpea were thus tested using semi-quantitative PCR amplification. No significant differences in ceramide synthase gene expression were observed between the control and toxin treated samples. Two FB1 unrelated differences were observed. A possible developmental difference was observed as ceramide synthase gene expression decreased over time in both the control and treated samples. It thus seems that FB1 did not influence ceramide synthase gene expression in cowpea.