Optimization of zoospore production and inoculum concentration of Aphanomyces euteiches for resistance screening of pea and lentil

Abstract

Aphanomyces euteiches is an important root pathogen of several legume and forage crops worldwide. Disease management is challenging due to resilient oospores that can persist in the soil for long periods of time. Several research groups are working towards incorporating disease resistance into crop cultivars involving large-scale disease resistance screening and, for some crops, the use of molecular markers associated with quantitative trait loci for resistance. The refinement of laboratory protocols for efficient pathogen isolation, reliable and simple zoospore production, and a repeatable indoor screening assay are important tools needed to further research in this area. The standardization of these protocols among research groups would provide an efficient platform for research collaboration. The effect of different media and sporulation induction methods on zoospore production was assessed. The most effective and repeatable method of A. euteiches zoospore production involved culturing and incubation for 4 days on autoclaved wheat leaf segments placed on corn meal-yeast extract-phosphate buffer agar. Colonized wheat leaves were transferred into 100 mL of distilled water in 250 mL flasks and incubated at 100 rpm for 18 h at 24ºC to induce zoospore production. Five zoospore concentrations ranging from 100 to 10 000 zoospores mL−1 were evaluated using susceptible and partially resistant pea and lentil genotypes to optimize zoospore concentration for germplasm screening. The best differentiation of levels of resistance among lentil and pea genotypes was achieved with 1000 zoospores mL−1.