Botrytis cinerea on grapes causes bunch rot at both pre- and post-harvest stages, where losses can reach up to 100%. Chemical control primarily relies on the prophylactic use of site-specific fungicides. Repeated applications of these products raise the risk of fungicide resistance development in B. cinerea populations, in which can result in disease control failures. To determine the extent of resistance, B. cinerea isolates were collected from grape clusters in the northwest and southwest grape growing regions of Michigan in 2014 (n = 115) and 2018 (n = 125). These isolates were phenotyped using discriminatory doses of 8 fungicides to determine the levels of resistance. Fungicide resistance increased from 2014 to 2018 mostly affecting the active ingredients fenhexamid, fluopyram, and iprodione. B. cinerea isolates resistant to multiple fungicides were detected in 2014 and 2018, with a higher frequency of resistance in 2018. TaqMan real time PCR has been developed to detect B. cinerea fungicide resistance to fenhexamid and to differentiate the erg27 F412S/I/V alleles. The assay was tested for sensitivity, specificity, and reproducibility on purified DNA and infected grape tissue samples. Our data provide essential information to growers about the efficacy for B. cinerea control using the available botryticides. Further, the developed fenhexamid markers will be transferred to diagnostic clinics to assist growers in the management of bunch rots before resistance related control failures occur.
Phytophthora capsici is a destructive pathogen of cucurbits that causes root, crown, and fruit rot. Winter squash (Cucurbita spp.) production is limited by this pathogen in Michigan and other U.S. growing regions. Age-related resistance (ARR) to P. capsici occurs in C. moschata fruit but is negated by wounding. This study aimed to determine whether structural barriers to infection exist in the intact exocarp of maturing fruit exhibiting ARR. Five C. moschata cultivars were evaluated for resistance to P. capsici 10, 14, 16, 18, and 21 days postpollination (dpp). Scanning electron microscopy imaging of Chieftain butternut fruit exocarp of susceptible fruit at 7 dpp and resistant fruit at 14 and 21 dpp revealed significant increases in cuticle and epidermal thicknesses as fruit aged. P. capsici hyphae penetrated susceptible fruit at 7 dpp directly from the surface or through wounds before 6 h postinoculation (hpi) and completely degraded the fruit cell wall within 48 hpi. Resistant fruit remained unaffected at 14 and 21 dpp. The high correlation between the formation of a thickened cuticle and epidermis in maturing winter squash fruit and resistance to P. capsici indicates the presence of a structural barrier to P. capsici as the fruit matures.
As sequencing costs continue to decrease, new tools are being developed for assessing pathogen diversity and population structure. Traditional marker types, such as microsatellites, are often more cost effective than single-nucleotide polymorphism (SNP) panels when working with small numbers of individuals, but may not allow for fine scale evaluation of low or moderate structure in populations. Botrytis cinerea is a necrotrophic plant pathogen with high genetic variability that can infect more than 200 plant species worldwide. A panel of 52 amplicons were sequenced for 82 isolates collected from four Michigan vineyards representing 2 years of collection and varying fungicide resistance. A panel of nine microsatellite markers previously described was also tested across 74 isolates from the same population. A microsatellite and SNP marker analysis of B. cinerea populations was performed to assess the genetic diversity and population structure of Michigan vineyards, and the results from both marker types were compared. Both methods were able to detect population structure associated with resistance to the individual fungicides thiabendazole and boscalid, and multiple fungicide resistance (MFR). Microsatellites were also able to differentiate population structure associated with another fungicide, fluopyram, while SNPs were able to additionally differentiate structure based on year. For both methods, AMOVA results were similar, with microsatellite results explaining a smaller portion of the variation compared with the SNP results. The SNP-based markers presented here were able to successfully differentiate population structure similar to microsatellite results. These SNP markers represent new tools to discriminate B. cinerea isolates within closely related populations using multiple targeted sequences.
Succinate dehydrogenase inhibitors (SDHIs) are keystone synthetic fungicides used to manage Botrytis cinerea in several hosts. In this study, we investigated the cross-resistance between five new SDHIs, including pyraziflumid, isofetamid, benzovindiflupyr, fluxapyroxad, and pydiflumetofen with commonly used SDHIs, boscalid and fluopyram. Different SdhB mutations were detected in a B. cinerea collection from Michigan grapes, and their frequency and EC50 value were determined. Among 216 B. cinerea isolates, five different mutations were detected, including H272R/Y, P225F/H, and N230I at frequencies 82.6, 4.3, 11.5, 0.4, and 5.3 %, respectively. Five isolates of each genotype were used to screen the cross-resistance of the SDHIs. We have classified the resistant profile of our mutants into five patterns. We report that all tested mutants were sensitive to benzovindiflupyr, indicating that it can be used as an effective fungicide against all B. cinerea mutants. In addition, fluopyram, pydiflumetofen, and isofetamid can provide effective control according to which type of mutation is present in the field. We also developed and compared two molecular diagnostic tools, rhAMP and TaqMan assays, for rapid detection of SDHI resistance-associated mutants in B. cinerea. We report that the TaqMan assay was more successful than the rhAMP assay in detecting the B. cinerea mutant DNA at ≤ 10 pg, and in a single assay was capable of monitoring two amino acids positions. Our results provide essential information about new SDHIs and provide molecular tools for monitoring SDHI resistance mutations, which will assist in gray mold disease control.
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