From the experiments in 2021, we selected fungal isolates according to morphology from each treatment to confirm their identity using genetic analysis (simple sequence repeats; SSR). Isolates were spread on CM plates covered with filter paper. After 4–5 days of incubation, the mycelia were scraped off the filters, transferred to 2 mL Eppendorf tubes and frozen at -70°C. The frozen mycelia were lyophilized, and cells were disrupted with glass beads (3 mm and 1 mm) in a FastPrep-24 homogenizer (MP Biomedicals, Eschwege, Germany; 25 s at 6 m s-1). We extracted the DNA (sbeadex plant kit and King Fisher Flex Purification system, Thermo Fisher Scientific, Waltham, Massachusetts) and standardized the samples to 5 ng DNA μL-1.
We used six SSR markers in two primer pair sets for each species to analyze fungal genotypes (Bb1F4, Bb2A3, Bb2F8, Bb4H9, Bb5F4, Bb8D6 for B. brongniartii; 47 (link); Ma2049, Ma2054, Ma2063, Ma2287, Ma327, Ma195 for M. brunneum; 48 (link), 49 (link)). Reference strains were included for both species (B. brongniartii, Bip2 and Bip4; Metarhizium spp., Ma714, Ma500 and Bip5). Multiplex PCRs and fragment size analyses were performed as described by Mayerhofer et al. (50 (link)) and Fernandez-Bravo et al. (51 (link)).
We used six SSR markers in two primer pair sets for each species to analyze fungal genotypes (Bb1F4, Bb2A3, Bb2F8, Bb4H9, Bb5F4, Bb8D6 for B. brongniartii; 47 (link); Ma2049, Ma2054, Ma2063, Ma2287, Ma327, Ma195 for M. brunneum; 48 (link), 49 (link)). Reference strains were included for both species (B. brongniartii, Bip2 and Bip4; Metarhizium spp., Ma714, Ma500 and Bip5). Multiplex PCRs and fragment size analyses were performed as described by Mayerhofer et al. (50 (link)) and Fernandez-Bravo et al. (51 (link)).
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