Fungicide Resistance Profiles of Botrytis cinerea Isolates From Michigan Vineyards and Development of a TaqMan Assay for Detection of Fenhexamid Resistance

Abstract

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.

Population Genetic Analyses of Botrytis cinerea Isolates From Michigan Vineyards Using a High-Throughput Marker System Approach

“…Throughout the study, isolates were maintained on 20% clarified V8 agar media (100 ml of V8 juice, 1 g of CaCO 3 , 10 g of agar, and 400 ml of distilled water) at room temperature. All isolates were previously evaluated for fungicide resistance against eight fungicides with seven different chemical classes (Alzohairy et al, 2020). For each isolate, a multiple fungicide resistance (MFR) value was determined based on the total number of fungicides an isolate demonstrated resistance toward.…”

“…For each isolate, a multiple fungicide resistance (MFR) value was determined based on the total number of fungicides an isolate demonstrated resistance toward. For DNA extraction and quantification, we followed Alzohairy et al (2020); in brief, mycelia were collected from 1-to 2-week-old cultures. Mycelia were lyophilized, then approximately 5 mg of tissue was ground using a tissuelyser (Qiagen, Valencia, CA).…”

“…Botrytis cinerea is a necrotrophic pathogenic fungus that infects hundreds of plant species (Alfonso et al, 2000;Ma and Michailides, 2005;Williamson et al, 2007;Fillinger, 2016) including economically important crops such as fruits, ornamentals, and vegetables (Elad et al, 2004;Leroux, 2007). B. cinerea causes gray mold, a major worldwide destructive disease that results in significant yield loss of grapevines in the field and postharvest (Campia et al, 2017;Rupp et al, 2017b;Saito et al, 2019;Alzohairy et al, 2020). B. cinerea infects all grapevine plant parts, though fruit rot, known as botrytis bunch rot (preharvest) and gray mold (postharvest), is the most common (Gabler et al, 2003;Elmer and Michailides, 2007;Saito et al, 2019).…”

“…However, B. cinerea is considered a difficult pathogen to control due to its rapid spread by wind (Holz et al, 2007) and the high genetic variability (Leroux et al, 2002) that allow the pathogen to develop resistance against applied synthetic fungicides (Rupp et al, 2017b). In grapes, fungicide resistance has been reported in different countries worldwide (Leroch et al, 2011;Angelini et al, 2014;Panebianco et al, 2015;Yin et al, 2015) and in the US including Michigan (Alzohairy et al, 2020) and California (Saito et al, 2019;Avenot et al, 2020;Delong et al, 2020). An increasing number of isolates with resistance to not only a single fungicide but also to multiple fungicides of different chemical classes have been reported (Leroch et al, 2013;Fernández-Ortuño et al, 2015;Saito et al, 2019;Alzohairy et al, 2020;Delong et al, 2020).…”

“…In grapes, fungicide resistance has been reported in different countries worldwide (Leroch et al, 2011;Angelini et al, 2014;Panebianco et al, 2015;Yin et al, 2015) and in the US including Michigan (Alzohairy et al, 2020) and California (Saito et al, 2019;Avenot et al, 2020;Delong et al, 2020). An increasing number of isolates with resistance to not only a single fungicide but also to multiple fungicides of different chemical classes have been reported (Leroch et al, 2013;Fernández-Ortuño et al, 2015;Saito et al, 2019;Alzohairy et al, 2020;Delong et al, 2020). Fungicide resistance frequencies have been shown to differ between years, crop hosts, and locations (Fernández-Ortuño et al, 2015;Delong et al, 2020;Kozhar et al, 2020).…”