A new semi-selective medium for Fusarium graminearum, F. proliferatum, F. subglutinans and F. verticillioides in maize seed

Zea mays L., known also as corn and maize, is the most important crop according to the amount of tonnes produced each year. 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. 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. Currently no method has been developed to test for Fusarium species in maize seed that has been validated and published by the International Seed Testing Association (ISTA). 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, 1/2 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 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% NaOCl) and for different time intervals (1 min, 3 min, 5 min and 10 min). The effect of black-blue light (365 nm) on sporulation of the fungi was also investigated. Surface disinfection of maize seeds with 1% NaOCl for 5 min provided consistent results. PDA, 1/2 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. The Fusarium species growing on these media formed underdeveloped morphological structures, thereby obscuring accurate identification. MGA 2.5 showed better hindering of the saprophytes in some instances. MGA 2.5 amended with sterile carnation leaf pieces was the most satisfactory medium in hindering the growth of the saprophytes while allowing adequate sporulation by the four Fusarium species to permit accurate identification. The media also resulted in higher F. verticillioides and lower saprophytic fungal isolation frequency when compared to the other media tested.

► We develop a new test for isolation of Fusarium species in maize seed.
► Eight different media compositions (new and existing) are compared using four Fusarium species.
► MGA 2.5 media with sterile carnation leaf pieces hinder growth of fungal saprophytes.
► MGA 2.5 and sterile carnation leaf media permit accurate identification of four Fusarium species.
► First test of Fusarium spp. on maize seeds was submitted to the International Seed Testing Association.