Phenotypic and molecular characterization of fenhexamid resistance in Botrytis cinerea isolates collected from pistachio orchards and grape vineyards in California
Introduction
Botrytis cinerea is a necrotrophic pathogen of immense economic importance causing pre- and postharvest diseases in more than 200 plant species worldwide, including grape and pistachio (Williamson et al., 2007). In wine and table grapes, B. cinerea is responsible for bunch rot in the field and gray mold in storage (Bulit and Dubos, 1988; Williamson et al., 2007; Smilanick et al., 2010; Weber and Hahn, 2011). In pistachio, B. cinerea causes Botrytis blossom, shoot, and fruit blight (Bolkan et al., 1984; Michailides, 1991). This disease is more prevalent during spring associated with wet and cool conditions and can cause significant damages by killing current season shoots, thus affecting the fruiting wood productivity for the following season. Later in the season (May/early June), if conditions remain favorable for infection, the pathogen can infect immature fruit clusters and kill parts or entire well-developed clusters (Bolkan et al., 1984; Michailides, 1991).
All plant part and growth stages in the respective host are sensitive to B. cinerea infection (Jersch et al., 1989; Michailides, 1991, 2002; Elad et al., 2004). Characteristic symptoms consist of the presence of gray mycelium often followed by sporulation, which is likely to develop when alternative conditions of high humidity and dryness prevail. The main source of inoculums for host infection are conidia released from over-wintering mycelium and sclerotia in early spring (Elad et al., 2004; Elmer and Michailides, 2007).
Although cultural practices and genetic resistance have been used, controls of diseases caused by B. cinerea are largely dependent on the application of fungicides (Michailides, 2002; Michailides and Elmer, 2000; Leroux, 2004; Leroux et al., 2002a, Leroux et al., 2002b). In vineyards, fungicides are typically applied to control bunch rot, but they are also applied in table grape for the control of postharvest decay (Rosslenbroich and Stuebler, 2000; Smilanick et al., 2010). Regarding Botrytis blossom, shoot and fruit blight management in pistachio, most available cultivars are susceptible to the disease. Cultural practices, such as pruning and removing blighted shoots, can contribute to reduce the inoculum in the orchards, but one to two fungicide sprays during bloom are recommended as the best approach to control this disease in commercial pistachio orchards (Michailides, 2002; Michailides et al., 2005).
Several fungicides are currently available for managing Botrytis blossom, shoot and fruit blight in pistachios and bunch rot and gray mold in vineyards. These include single-site fungicides belonging to seven chemical classes with different modes of action: anilinopyrimidines, benzimidazoles, dicarboximides, quinone outside inhibitors (QoIs), phenylpyrroles, succinate dehydrogenase inhibitors (SDHIs), and hydroxyanilide (Myresiotis et al., 2007; Adaskaveg et al., 2012). The risk for resistance development is higher for the single-site fungicides, especially for a high-risk pathogen such as B. cinerea, for which resistance has been reported to almost all registered fungicides (Ziogas and Klamarakis, 2001; Baroffio et al., 2003; Myresiotis et al., 2007; Thomidis et al., 2009; Malandrakis et al., 2011).
The hydroxyanilide derivative fenhexamid was introduced in spray program applied for gray mold control few years ago (Rosslenbroich and Stuebler, 2000; Myresiotis et al., 2007). It inhibits the germ tube elongation and mycelial growth with a broad-spectrum activity that includes Botrytis spp. and related fungi such as Monilinia sp. and Sclerotinia sp. (Rosslenbroich et al., 1998; Rosslenbroich and Stuebler, 2000; Myresiotis et al., 2007). Fenhexamid belongs to the SBI (Sterol Biosynthesis Inhibitor) fungicides and is involved in the inhibition of the 3-keto reductase enzyme (target gene erg27), which catalyzes C-4 demethylation during ergosterol biosynthesis (Debieu et al., 2001). It is classified as low to medium risk for resistance development. Due to its unique mode of action and good efficacy, fenhexamid has been used internationally to manage gray mold and as a reliable alternative to older fungicides in fungicide resistance management (Rosslenbroich and Stuebler, 2000; Brent and Hollomon, 2007; Grabke et al., 2013; Suty et al., 1999). However, due to the specificity of its mode of action and excessive use, B. cinerea fenhexamid-resistant field isolates soon emerged in various crops in France, Japan, USA and Germany, thus threatening its efficacy against gray mold (Forster et al., 2007; Fillinger et al., 2008; Mercier et al., 2009; Saito et al., 2011, 2014; Weber, 2011; Moorman et al., 2012; Grabke et al., 2013). Several B. cinerea fenhexamid-resistant phenotypes, ranging from medium to high resistance, have been identified. Four fenhexamid-resistant phenotypes named HydR1, HydR2, HydR3-, and HydR3+ have been characterized in Botrytis field isolates (Fillinger et al., 2008; Grabke et al., 2013) Resistance mechanisms associated with these phenotypes include target site modification, detoxification or increased efflux (Leroux et al., 2002a, Leroux et al., 2002b; Fillinger et al., 2008; Grabke et al., 2013). HydR3 phenotypes were found to be associated with genetic modifications in the erg27 target gene resulting in amino acid alterations in the 3-ketoreductase (Fillinger et al., 2008; Grabke et al., 2013).
Fenhexamid has been part of the overall bunch rot and gray mold management programs in grape in the last several years; it is recommended as a single product (Elevate® 50 WDG) in alternation with other single-site fungicides or mixed with protectant fungicides (Adaskaveg et al., 2012). Similarly, Elevate® 50 WDG has been a key component of Botrytis blossom and shoot blight spray program in California pistachio orchards: one application during bloom lessens Botrytis blossom and shoot blight (Adaskaveg et al., 2012). Despite the reported cases of resistance to the site-specific inhibitor fenhexamid, major control failures resulting from resistance to this fungicide have not been reported. Nevertheless, in order to contend with potential resistance problems and develop anti-resistance strategies against B. cinerea, it is a necessity to monitor variations in fenhexamid sensitivity in pistachio and grape fields, and assess the risk of fenhexamid-resistance development in these California crops.
Thus, the objectives of this study were to: (i) assess the occurrence and frequency of fenhexamid-resistance in B. cinerea isolates collected from pistachio orchards and grape vineyards in California; (ii) investigate if known mutations in the erg27 gene are associated with resistance in fenhexamid-resistant phenotypes; iii) assess whether or not resistance is associated with fitness penalties and evaluate the in vivo efficacy of Elevate® 50 WDG in controlling gray mold.
Section snippets
Isolates of B. cinerea
In all, 132 single-spore Botrytis cinerea isolates collected between 2003 and 2014 from Californian grape and pistachio were used in this study. The number and characteristics of isolates from each host are presented in Table 1. Isolates were stored in glycerol (30%) as spore suspensions at −75 °C, transferred, and maintained onto in agar media as previously described by Avenot et al. (2018).
Determination of sensitivity of B. cinerea isolates to fenhexamid
Sensitivity of B. cinerea to fenhexamid was assessed in mycelial growth assay. Technical grade
In vitro sensitivity of B. cinerea isolates to fenhexamid
Based on the obtained EC50 values for fenhexamid, the 132 B. cinerea isolates tested were grouped into five different phenotype classes. Among the two crops, 103 isolates (78%) were sensitive to fenhexamid (HydS), while different levels of resistance were observed for the remaining 29 isolates, especially for the pistachio isolates. Among the 58 grape isolates 52 (90%) were sensitive to fenhexamid, while the other six (10%) showed low resistance (HydLR) (Fig. 1). Among the 74 pistachio
Discussion
The hydroxyanilide (Hyd) fenhexamid is one of the most frequently used site-specific fungicides against gray mold of grape and Botrytis blossom, shoot, and fruit blight of pistachio in the last several years. Our study confirmed the excellent in vitro and in vivo efficacies of fenhexamid (Smilanick et al., 2010), as most B. cinerea isolates from the two hosts were still sensitive to this fungicide. However, it also revealed the existence of isolates with different levels of resistance to
CRediT authorship contribution statement
Hervé F. Avenot: Conceptualization, Methodology, Formal analysis, Writing - original draft. David P. Morgan: Methodology. Joel Quattrini: Methodology, Formal analysis. Themis J. Michailides: Supervision, Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank the California Pistachio Research Board (CP CPRB; Res Agreem201302565) for their financial support.
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