Parameters affecting the production of fumonisin B1 by fusarium verticillioides in culture
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Fi1sarium verticillioides is a very important mycotoxin-produeing fungus associated with maize. Fverticillioides produces a group of mycotoxins known as fumonisins under suitable environmental conditions. A series of studies was designed to provide information regarding some of the factors associated with the production offumonisin B1 (FB1) in maize patties and MYRO liquid medium. Our investigation together with previous studies have detailed the important influence of several factors on the production of fumonisins by F verticillioides strains. To understand why these strains are able to produce these toxins, an investigation into the complex interaction that occurs between biotic and abiotic parameters and their impact on fumonisin production was necessary. The results reflect the interacting factors and the intraspecific differences between strains, which may also be present in field conditions. The parameters that were varied under a predetermined set of culture conditions, included initial moisture content of maize patty cultures, temperature, initial pH and the addition of the fumonisin precursors, L-alanine and L-methionineto the cultures. Investigations into the three-way interactions of initial maize patty moisture content (30 ml water to 30g of maize), L-methionine (0.3 %) and temperature (25°C), resulted in the highest yield ofFB1 (5777.26 μgig) produced by MRC 4316. In contrast, MRC 826 was negatively affected, producing lower levels ofFB1 (3492.24 μg/g), compared to MRC 4316 at an initial moisture content (20 ml water to 30 g maize), L-methionine (0.3 %) and 25 °C. An American strain of F verticillioides MRC 7424 (= NRRL 13616), produced the highest levels of FB, (116 μg/ml), while the South African isolates, MRC 4316 and MRC 826, produced lower FB1 levels (93 and 62 μg/ml, respectively) in MYRO liquid medium. In general, FB1 production in maize patty cultures far exceeded levels obtained in liquid shake cultures. It appears that not only the ability of a particular strain of F. verticillioides, but the interaction of a variety of physiological and nutritional factors and the culture medium, are important in the production of FB,. Thus, variation of a single factor such as temperature under field conditions due to seasonal change, may therefore have a major effect on fomonisin production. A chain reaction may occur when changes in moisture, pH, etc. take place, which may influence fumonisin production further. Lyophilisation of fungal cultures proves to be an excellent method to preserve a wide range of fungi over long periods of time. It is, however, necessary to determine the viability of conidia stored in lyophilised vials at 4 ° Con a regular basis. At present, plate count methods remain the most valid technique for the detection of the viability of lyophilised conidia. Membrane-permeant nucleic acid-binding dyes (FUN-I) are viability stains that are relatively new flourescent probes for assessing the viability of metabolically active yeast cells. The purpose of this study was to microscopically determine the viability oflyophilised conidia of Fusarium and A lternaria species, using the yeast, Saccharomyces cerevisiae, as a control. FUN-1 viability stain was compared to two other staining methods, i.e. ethidium bromide (EB) and methylene blue (MB) and the viability of the conidia was compared to colony-forming units (CFU) on solid media as a control. For the purpose of determining or screening for percentage viability in a specific inoculum, results indicate that EB can be used in the case of lyophilised conidia, and MB in the case of freshly harvested conidia. Although FUN-I are recommended as a good way to determine the cell viability of a fungus, it needs relatively complicated procedures and has a time limit in which the stain can be used. The result of this study emphasize that the use of dyes to determine viability of lyophilised conidia require a critical definition of protocols for a specific fungal species, and that a good correlation with CFU needs to be demonstrated. The findings of this study could find useful applications in various studies on living and dead conidial populations. The diverse toxicological effects of fumonisins m animals and plants raised the possibility that fumonisins may also inhibit the growth of filamentous fungi. This study investigated the antifungal activity of FB1 to some h1sariu111 and other fungal species. The sensitivity of these fungi was tested by an agar-diffusion method on PDA plates. FB1 inhibited the myceliaJ growth of five of the nine fungi tested. The FB1-producing Fusarium species isolated from maize, i.e. F verticil/ioides, F glohosum and F proliferatum were resistant to FB1 even though a small inhibition zone at the highest FB1 concentration of 40mM was noted in the case of F. proliferatum. However, amongst two non-producing Fusarium spp. also isolated from maize, one (F subglutinans) was resistant and one (F graminearum) was sensitive. The most sensitive fungi tested were non-producing species not isolated from maize, i.e. A lternaria alternata, Botrytis cinerea and Penicillium expansum. The minimum inhibitory concentration ofFB1 ranged between 0.25-0.SmM for A. alternata, 1-SmM for P. expansum and B. cinerea and 5-1 OmM for F. graminearum, while the other fungi tested showed no sensitivity to FB1. This is the first report on the antifungal activity ofFB1 to filamentous fungi. Another study investigated the effect of FB1 on the germination of freshly harvested conidia of Fusarium and some other fungal species. The FB1 -producing F'usarium species isolated from maize, i. e. F vertici llioides, F. globosum and F. prolifer alum showed a decrease in germ tube length with an increase in FB1 concentrations. This indicated that these fungi can tolerate their own toxic metabolite to a ce11ain extent. However, amongst the two non-fumonisin producing Fi1sarium spp. examined, i.e. F. subglutinans and F. graminearum, isolated from maize, F. subglutinans was induced to genninate faster in the presence ofFB1 but soon developed stunted germ tubes, while F graminearum developed shorter germ tubes compared to the control cultures. The most sensitive fungi tested were species not isolated from maize, i.e. A. alternata, B. cinerea and P. expansum, which did not germinate at higher FB1 concentrations at all. Statistical analyses showed that the inhibiting effect of FB1 was highly significant (P <0.001). The conidial germination bioassay was more sensitive in the detection of the antifungal activity ofFB1 than the petri dish bioassay. The minimum inhibitory concentrations of FB1 for visible mycelial growth were closely comparable to those obtained from conidial germination. Results of these studies provide considerable information on the parameters affecting the production of FB1 and will be of great benefit in further studies focussing on fumonisin prodnction.