Frequency of fungicide-resistant Botrytis cinerea populations isolated from ‘Thompson Seedless’ table grapes in the Central Valley of Chile

Cien. Inv. Agr. 44(3):295-306. 2017 www.rcia.uc.cl plant pathology DOI: 10.7764/rcia.v44i3.1721 research note Frequency of fungicide-resistant Botrytis cinerea populations isolated from ‘Thompson Seedless’ table grapes in the Central Valley of Chile Marcela Esterio11, Charleen Copier1, Andrea Román2, María José Araneda1, Mauricio Rubilar1, Isabel Pérez1, and Jaime Auger1 1 Universidad de Chile, Facultad de Ciencias Agronómicas, Departamento de Sanidad Vegetal, Laboratorio de Fitopatología Frutal y Molecular. Santiago, Chile. Código Postal 8820808. 2 Centro de Desarrollo de Tecnología para la Reducción y Racionalización de Agroquímicos (CEDETERRA). ESPOCH. Panamericana Sur km 1/2, Riobamba, Ecuador. Código Postal EC060155. Abstract M. Esterio, C. Copier, A. Román, M.J. Araneda, M. Rubilar, I. Pérez, and J. Auger. 2017. Frequency of fungicide-resistant Botrytis cinerea populations isolated from ‘Thompson Seedless’ table grapes in the Central Valley of Chile. Cien. Inv. Agr. 44(3): 295-306. During the 2013 and 2014 growth seasons, 526 single spore isolates of Botrytis cinerea were collected from naturally infected ‘Thompson Seedless’ table grape flowers from fifteen orchards in three regions of the Central Valley of Chile. The isolates were tested for resistance to azoxystrobin, boscalid, fenhexamid, fludioxonil and pyrimethanil. Among the 526 isolates, 106 (20.15%) were sensitive to all fungicides tested; from north to south, the frequency of sensitive isolates in the regions of Valparaiso, Metropolitana and O’Higgins ranged from 48.15% to 21.1% and 5.88%, respectively. Four hundred and twenty isolates (79%) showed resistance to single or multiple fungicides, 134 (25.4%) were simultaneously resistant to azoxystrobin and pyrimethanil. No fludioxonil-resistant isolates were found, indicating that fludioxonil has great potential for gray mold control in table grapes in Chile. From sixty randomly selected B. cinerea isolates, only the azoxystrobin-resistant isolates carried the G143A point mutation; according to the cytochrome b (cyt b) gene structure, the third intron Bcbi-143/144 was only detected in the azoxystrobin-sensitive isolates. The H272R and H272Y point mutations in the succinate dehydrogenase subunit B (sdhB) gene were associated only with the boscalid-resistant isolates. The F412S and F412V point mutations were found in the sequenced erg27 gene of randomly selected fenhexamid-resistant isolates. These results contribute to the knowledge of B. cinerea fungicide resistance for table grape vine crops in Central Chile, particularly for the development of multiple-resistance and the associated resistance mechanisms of azoxystrobin, boscalid and fenhexamid-resistant isolate populations. Antiresistance strategies are discussed in a general manner. Keywords: Anilino-pyrimidines, gray mold, multiple-resistance, phenylpyrroles, quinone outside inhibitors, sterol biosynthesis inhibitors class III, succinate dehydrogenase inhibitors. Introduction Grape vines, one of the major Chilean fruit crops, are mostly cultivated in the Central Valley because of its favorable regional agroecological conditions. Received December 27, 2016. Accepted October 10, 2017. Corresponding author: The vast majority of table grapes grown in Chile are exported to the northern hemisphere. Vitis vinifera cv. ‘Thompson Seedless’ (‘Th. Seedless’) ranks as the second most important commercial grape variety, with a total crop value of US$ 234 million (ODEPA, 2016). However, since most table grapes are trained onto overhead arbors 296 CIENCIA E INVESTIGACIÓN AGRARIA and due to the frequent changes to cool and wet weather conditions in the last growing seasons, ‘Th. Seedless’ table grape growers and fruit export traders have been affected by important pre- and postharvest Botrytis gray mold outbreaks. In fact, grape production decreased from 170,194 tons in 2012 to 132,465 tons in 2014 (ODEPA, 2016). In Chile, B. cinerea has caused blossom blight during the bloom period and fruit rot during the pre- and postharvest periods (Latorre et al., 2002). The application of fungicide sprays from blooming until harvest is the essential measure taken to reduce Botrytis infection-related losses. However, B. cinerea can develop resistance to commonly used fungicides, an ability that is partly due to its relatively large genetic diversity and enormous capacity for asexual reproduction by means of conidia (Leroux et al., 2002). Resistance to recently introduced fungicides, such as anilino-pyrimidines (APs), phenylpyrroles (PPs) and sterol biosynthesis inhibitors class III (SBIs-III; hydroxyanilides), has also been reported (Leroux et al. 2002; Moyano et al., 2004; Weber, 2010). Moreover, field resistance to novel succinate dehydrogenase inhibitor (SDHI) fungicides was detected soon after their introduction (Bardas et al., 2010). Molecular characterization of B. cinerea isolates that were sensitive or resistant to single-site fungicides showed the involvement of major genes of the fungus and a strong association between resistant phenotypes and point mutations (single nucleotide polymorphisms, SNPs) (De Miccolis Angelini et al., 2012). These results have been observed for several groups of fungicides, including SDHIs (Yin et al., 2012), SBIs-III (Fillinger et al., 2008) and quinone outside inhibitors (QoIs) (De Miccolis Angelini et al., 2012; Ishii et al., 2009). During the last decade, pyrimethanil, azoxystrobin, boscalid, fenhexamid and fludioxonil fungicides have been used extensively to control gray mold disease in table grapes in Chile, and fungicide resistance of B. cinerea has been reported (Este- rio et al., 2007, 2012, 2015; Latorre et al., 2002; Piqueras et al., 2014). We hypothesized that the frequency of multiple-resistance in B. cinerea populations would increase; thus, it would be crucial to monitor such frequency in table grape orchards to evaluate the risk of infection for specific areas and growing conditions and elaborate appropriate anti-resistance strategies. Therefore, the objectives of the present study were as follows: i) to screen the sensitivity of B. cinerea populations isolated from ‘Th. Seedless’ table grapes to azoxystrobin, boscalid, fenhexamid, fludioxonil and pyrimethanil and assess their resistance frequencies to these fungicides and ii) to investigate the point mutations associated with QoI, SDHI and SBI-III resistance in azoxystrobin-, boscalid- and fenhexamid-resistant isolates. Materials and methods Collection of B. cinerea isolates. To detect fungicide resistance in B. cinerea, ‘Th. Seedless’ naturally infected flowers were sampled during the 2013 and 2014 growing seasons from fifteen orchards in three regions of the Central Valley of Chile (Valparaiso, Metropolitana and O´Higgins). Two hundred flowers (n=50/plate) per orchard were incubated for 3–5 days on water agar medium at 25 °C. Mycelia from the colonized flowers were transferred to potato dextrose agar medium and i (...truncated)