Effect of Fungicide Applications on Monilinia fructicola Population Diversity and Transposon Movement

Abstract

In this study, we investigated whether fungicide-induced mutagenesis previously reported in Monilinia fructicola could accelerate genetic changes in field populations. Azoxystrobin and propiconazole were applied to nectarine trees at weekly intervals for approximately 3 months between bloom and harvest in both 2013 and 2014. Fungicides were applied at half-label rate to allow recovery of isolates and to increase chances of sublethal dose exposure. One block was left unsprayed as a control. In total, 608 single-spore isolates were obtained from blighted blossoms, cankers, and fruit to investigate phenotypic (fungicide resistance) and genotypic (simple-sequence repeat [SSR] loci and gene region) changes. In both years, populations from fungicide-treated and untreated fruit were not statistically different in haploid gene diversity (P = 0.775 for 2013 and P = 0.938 for 2014), allele number (P = 0.876 for 2013 and P = 0.406 for 2014), and effective allele number (P = 0.861 for 2013 and P = 0.814 for 2014). Isolates from blossoms and corresponding cankers of fungicide treatments revealed no changes in SSR analysis or evidence for induced Mftc1 transposon translocation. No indirect evidence for increased genetic diversity in the form of emergence of reduced sensitivity to azoxystrobin, propiconazole, iprodione, and cyprodinil was detected. High levels of population diversity in all treatments provided evidence for sexual recombination of this pathogen in the field, despite apparent absence of apothecia in the orchard. Our results indicate that fungicide-induced, genetic changes may not occur or not occur as readily in field populations as they do under continuous exposure to sublethal doses in vitro.

Novel gene‐sequence markers for isolate tracking within Monilinia fructicola lesions

“…These markers also enable additional field studies of fungicide or other stressor‐induced mutagenesis over time. A proof of concept study using these markers was recently published, where markers were used to validate isolate identity when sampling the same infection site before and after application of a fungicide capable of causing mutagenesis in vitro …”

“…Results of this study will be important because the new gene regions will give researchers another and, in some experimental conditions, more effective tool for tracking isolates over time, as well as providing a complement to SSR genotyping. More broadly, isolate tracking with these markers enables a better understanding of the spread of inoculum and relative importance of sexual and asexual reproduction to disease development and M. fructicola spread in stone fruit orchards, as shown in our recent publication …”

“…Comparison of the SSR profile of the progenitor isolate (never exposed to fungicide) to the fungicide-exposed isolate showed allelic changes at SSR loci after as few as ten generations. 21 It is unknown whether changes at gene regions would also be observed for these isolates.…”

“…Isolates were grown on a gradient of the fungicide azoxystrobin (Abound Flowable; Syngenta Crop Protection, Greensboro, NC) and serially transferred from the 50–100% inhibition zone once a week for a total of 12 weeks, where each transfer was considered a generation. Comparison of the SSR profile of the progenitor isolate (never exposed to fungicide) to the fungicide‐exposed isolate showed allelic changes at SSR loci after as few as ten generations . It is unknown whether changes at gene regions would also be observed for these isolates.…”

“…More broadly, isolate tracking with these markers enables a better understanding of the spread of inoculum and relative importance of sexual and asexual reproduction to disease development and M. fructicola spread in stone fruit orchards, as shown in our recent publication. 21…”