Detection of Fusarium spp. on maize kernels

Abstract

In terms of amount of tonnes produced each year, Zea mays also known as maize and corn is the most important crop. Fungi cause significant destruction of maize in the field as well as during storage rendering the grain unsuitable for human consumption by decreasing its nutritional value and by producing mycotoxins that are detrimental to both human and animal health. Fusarium species are widely distributed and are amongst the most frequently isolated fungal species by plant pathologists and due to the fact that the Fusarium species involved in maize ear rot vary in fungicide sensitivity, pathogenicity as well as in their capability to produce mycotoxins, accurate quantification and identification is of paramount significance. There is currently no method developed for Fusarium detection in maize seed that has been validated by ISTA (the International Seed Testing Association). Malachite green agar 2.5 ppm (MGA 2.5) is a potent selective medium for isolation and enumeration of Fusarium spp. In this study, eight different media compositions, potato dextrose agar (PDA), PDA + malachite green oxalate, corn meal agar, ½ PDA + malachite green oxalate, 1% malt agar, carnation leaf agar supplemented with potassium chloride (KCLA), malachite green agar (MGA 2.5) and MGA 2.5 + sterile carnation leaf pieces (MGA 2.5 +) were compared using four Fusarium species (F. graminearum, F. proliferatum, F. subglutinans and F. verticillioides) and five commonly encountered saprophytic fungi (Aspergillus niger, Penicillium crustosum, P. digitatum, Trichoderma harzianum and Rhizopus stolonifer). The maize kernels were surface disinfected using three concentrations of sodium hypochlorite (0.5%, 1% and 1.5%) and for different time intervals (1min, 3min, 5min and 10min). The effect of black-blue light (365nm) on sporulation of the fungi was also investigated. 200 maize sees from two seed lots were surface disinfested and plated out on PDA, KCLA, MGA 2.5 and MGA 2.5 and incubated at 25oC for 7d under 12h black-blue light/12h darkness. PDA, ½ PDA, 1% malt agar and KCLA allowed profuse growth of the Fusarium species as well as saprophytes. Media that contained malachite green oxalate was most inhibitory to the radial colony growth of the saprophytes and the Fusarium species. Fusarium species growing on these media formed under-developed morphological structures, thereby obscuring accurate identification. MGA 2.5 supplemented with sterile carnation leaves was the most satisfactory medium in hindering saprophytic growth while allowing adequate sporulation of the Fusarium species to permit correct identification. The media also had a higher Fusarium verticillioides and less saprophytic fungal isolation frequency when compared to the other media tested. The location of Fusarium verticillioides within maize seed was also investigated and found to be only associated within the upper pedicel part of the kernel. Copyright