Tracking native and applied populations of Cryptococcus flavescens in the environment

Cryptococcus flavescens OH182.9_3C (3C) is a beneficial yeast strain that expresses significant biological control efficacy against Fusarium head blight, a globally important disease of wheat. In this study, traditional and quantitative PCR (qPCR) assays targeting one copy of the hsp70 gene were developed and applied to monitor the population dynamics of beneficial 3C-like C. flavescens in wheat and barley fields and on harvested grains. The assay was found to be specific to 3C-like C. flavescens, though detection of a different genotype of C. flavescens can occur with 104-fold lower sensitivity. The utility of the assays to detect native C. flavescens populations and track an inoculant strain was demonstrated in different field experiments. Across ten wheat growing states, up to 25% of wheat heads harbored 3C-like C. flavescens. However, by harvest those populations were found on no > 8% of dry wheat heads. Additional comparisons indicated that native populations of 3C-like C. flavescens preferentially colonized wheat heads, colonizing wheat foliage and barley heads at significantly (P < 0.05) lower levels. When inoculated onto growing wheat heads, strain 3C colonized and persisted at the magnitude of 104.5 to 106.5 target gene copies per gram fresh weight. Furthermore, the applied population apparently dispersed from the inoculated plots to non-inoculated areas in a stochastic fashion during the growing season. Populations of 3C-like C. flavescens were detected at levels of 102.5 to 105.5 copies per g on postharvest residues in inoculated areas during the winter and spring following the growing season, and higher populations were detected on non-rotting wheat stalks than on other sampled components of the field. On harvested grain from inoculated plots, 3C-like C. flavescens populations declined postharvest. In contrast, uninoculated wheat harbored over 100-fold lower levels of 3C-like yeasts at all tested pre- and post-harvest time points. In conclusion, the qPCR-based assay developed reveled rare but widespread occurrence of native populations of 3C-like yeasts and clarified the environmental fate of 3C when applied as a biopesticide to wheat.