For this project, seven different maize hybrids were used to isolate seed-borne Fusarium species. These included F. proliferatum, F. verticillioides, F. andiyazi, F. fujikuroi, and F. thapsinum. Their identity was confirmed morphologically, and by PCR amplification followed by sequencing of TEF-1α and β-tubulin genes.
Additionally, Agrobacterium tumefaciens mediated transformations of F. proliferatum (Green Fluorescent Protein) and F. verticillioides (monomeric Red Fluorescent Protein) species were done. These transformations were used to study the interaction (e.g. competition) of these species for a maize host by using a TaqMan multiplex real-time qPCR protocol. Sensitive and specific primer and probe sets were designed to identify and quantify the F. proliferatum and F. verticillioides by targeting the GFP or mRFP insertion.
Moreover, the fluorescent F. proliferatum was used to conduct hyphal growth experiments under different temperature, soil matric potential and distance combinations. The ability of F. proliferatum to grow by hyphae from a source of inoculum and colonize available organic matter (bait) was investigated. The presence/absence of F. proliferatum in the baits was determined by using a fluorescent microscope looking for the green fluorescence expression.
Genotyping by sequencing (GBS), amplified fragment length polymorphism (AFLP), and sequence analysis were used for population genetic analyses of the isolated Fusarium species. The GBS data was also used to design specific primer sets to accurately detect specific strains of F. proliferatum (GBS-PCR). This information was used to conduct experiments in the field, to track the characterized strains of F. proliferatum and study their life cycles. The AFLPs will be used to further confirm the genetic relatedness (by fingerprinting) of the isolated field strains with the strains characterized with GBS.