Vegetative compatibility between and within Epichloe species

Epichloe species (Clavicipitaceae, Ascomy- cota) are biotrophic symbionts of cool-season grasses and grow slowly on defined media. Nine Epichloe biological species (mating populations) have been identified. Vegetative compatibility within or between Epichloe species has not yet been investigated. We selected chlorate-resistant strains that were nitrate non-utilizing (nit) mutants from ten isolates representing four biological species. Each mutant grew appressed on a defined medium with nitrate as the sole nitrogen source, in contrast to the abundant aerial mycelia produced by the prototrophic wild type isolates on the same medium. Each mutant was putatively identified as nit1 (nitrate reductase structural gene mutant), nit3 (mutant in the pathway-specific regulatory locus or nitrite reductase gene), or NitM (mutant affecting molybdenum cofactor production) by growth phenotypes on medium with nitrite, hypoxanthine, uric acid or ammonium as the sole nitrogen source. When appropriate nit mutants were paired, heterokaryon formation and complementation were indicated by a zone of prototrophic growth. Complementation occurred between complementary mutants of most isolates, even from different species; however, complementation did not occur in all replications. Interspecific heterokaryon formation was confirmed by recovery of parental phenotypes and cosegregation of beta-tubulin gene polymorphisms among single-conidiospore isolates from the complementation zone. There was no indication that mating type interactions affected heterokaryon formation, and no discrete vegetative compatibility groups were identified.