Elsevier

Crop Protection

Volume 133, July 2020, 105133
Crop Protection

Phenotypic and molecular characterization of fenhexamid resistance in Botrytis cinerea isolates collected from pistachio orchards and grape vineyards in California

https://doi.org/10.1016/j.cropro.2020.105133Get rights and content

Highlights

  • 90 % of the isolates from grape were sensitive whereas the remaining 10% displayed low resistance to fenhexamid.

  • B. cinerea isolates from pistachio displayed low to high levels of resistance to fenhexamid.

  • The resistant phenotypes carried various amino acid alterations in the 3-ketoreductase erg27 target protein.

  • Inoculations on grape berries showed that Elevate® 50 WDG did not control the moderately and highly resistant phenotypes.

  • Continual monitoring of fenhexamid resistance in B. cinerea in pistachio and grape fields is therefore advised.

Abstract

Botrytis cinerea causes Botrytis blossom, shoot and fruit blight in pistachio and gray mold in grape. The hydroxyanilide (Hyd) fenhexamid, one of the most frequently used fungicides for Botrytis control, inhibits the 3-ketoreductase (Erg27) of the ergosterol biosynthesis pathway. Due to its site-specific mode of action, this fungicide is at-risk for resistance development. In this study, 74 and 58 B. cinerea isolates, collected from Californian pistachio orchards and grape vineyards, respectively, were evaluated for their in vitro sensitivity toward fenhexamid. Of the 58 grape isolates, 90% were sensitive to fenhexamid (HydS, EC50 < 1 μg/ml) whereas the remaining 10%displayed low resistance to this fungicide (HydLR, 1 < EC50 < 5 μg/ml). Among the 74 pistachio isolates, 69, 14, 1, 5, and 11% displayed sensitivity (HydS, EC50 < 1 μg/ml), low (HydLR, 1 < EC50 < 5 μg/ml), weak (HydWR, 5 < EC50 < 10 μg/ml), moderate (HydMR, 10 < EC50 < 50 μg/ml), and high resistance (HydHR, EC50 > 50 μg/ml) phenotypes toward fenhexamid, respectively. The 3-ketoreductase erg27 gene was sequenced from all the 29 B. cinerea fenhexamid-resistant phenotypes (HydLR, WR, MR, HR) detected in this study. The resulting sequences were then compared to corresponding sequences obtained from 21 fenhexamid-sensitive isolates (HydS). Of the 6 HydLR phenotype from grape, 3 had a substitution of proline to serine at position 238 (P238S). No mutations were found in the remaining 3 HydLR nor in the 10 HydLR pistachio isolates. The only HydWR phenotype from pistachio presented a change at position 309 resulting the replacement of valine to Methionine (V309M). Among the HydMR phenotype from pistachio, two had F412S alteration while the other two possessed the L400F alteration at position 400. Highly resistant isolates (HydHR) carried a substitution of the phenylalanine at position 412, either serine (F412S) or isoleucine (F412I). Inoculations on detached grape berries showed that field rates of Elevate® 50 WDG (fenhexamid) controlled HydS and HydLR, but not the HydMR and HydHR phenotypes. The presence of B. cinerea fenhexamid-resistant phenotypes in pistachio and grape fields in California must be monitored continually and resistance management practices implemented as needed for sustained Botrytis blossom, shoot and fruit blight and gray mold controls with Elevate® 50 WDG.

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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