1. Introduction
Cherry leaf spot (CLS), caused by the fungus
Blumeriella jaapii (Rehm) Arx, is a significant disease problem on sour/tart cherry (
Prunus cerasus L.). Ascospores of
B. jaapii are produced in apothecia on overwintered leaves on the orchard floor. The ascospores are typically released near petal fall and infect leaves via the stomata. In years with moderately warm and wet weather during bloom, initial infections can occur on bract leaves adjacent to flowers; this situation commonly results in CLS epidemics that can have a major negative impact on fruit ripening. On untreated tart cherry trees of the highly-susceptible cultivar ‘Montmorency’ (~95% of the tart cherry industry in Michigan, USA), the incidence of CLS can increase from ~5% to ~100% within one month [
1]. As leaves accumulate CLS lesions, they become chlorotic and abscise; CLS-mediated defoliation typically follows initial leaf infection by about two to four weeks [
1]. With CLS disease in Michigan, in situations where defoliation levels of >50% occur prior to September, affected trees are at significant risk of winter injury and possible tree death [
1].
Blumeriella jaapii is a prolific sporulator on ‘Montmorency’, and masses of conidia, visible to the naked eye, are formed on the abaxial leaf surface in acervuli. The conidia are readily spread by rain and wind causing secondary infections also via the stomata, making management of CLS extremely difficult under favorable weather conditions. Because of the high susceptibility of ‘Montmorency’, CLS management is entirely dependent upon the use of fungicides, with six to eight full cover applications used in a typical season. Broad-spectrum fungicides including chlorothalonil, captan, and coppers are effective in CLS control; however, growers have historically favored single-site fungicides that are either systemic or translaminar because these fungicides can control other diseases including powdery mildew (Podosphaera clandestina (Wallr.:Fr.) Lev.) and brown rot (Monilinia fructicola (G.Winter) Honey), and can be used with longer interval times between applications.
The evolution of fungicide resistance in populations of
B. jaapii in Michigan has been an ongoing problem over the past 15–20 years. Resistance to the sterol demethylation inhibitor fungicide class emerged in the mid-2000s [
2], and resulted from overexpression of the target
CYP51 gene due to insertion of an outwardly-directed promoter sequence carried by a transposable element [
3]. Resistance to the succinate dehydrogenase inhibitor (SDHI) boscalid, a component of the premix fungicide Pristine (BASF Corporation; Research Triangle Park, NC) was first detected in 2010–2011 [
4]. Frequent use of Pristine at this time in tart cherry orchards in Michigan quickly led to practical resistance in most commercial orchards in the state. These boscalid-resistant
B. jaapii strains possessed the H260R mutation in the
SdhB target gene (B-H260R) [
4]; mutation of this conserved histidine residue to either arginine or tyrosine is very common among other boscalid-resistant fungi [
5,
6,
7].
The SDH complex is encoded by four genes (
SdhA,
SdhB,
SdhC, and
SdhD) and comprises a critical component of aerobic respiration in fungal mitochondria [
8]. Structural analyses of these proteins have shown that a complex of the
SdhB,
SdhC, and
SdhD proteins generates a ubiquinone-binding pocket [
6]. This pocket is targeted by most fungicides of the SDHI class, and mutations that alter the structure of the pocket can result in SDHI fungicide resistance [
5]. However, because of the variety of chemical structures of SDHI fungicides discovered to date, mutations such as B-H277Y and B-H277R that changed the structure of the ubiquinone-binding pocket and prevented boscalid from targeting the SDH complex did not necessarily result in cross resistance to other SDHI fungicides such as fluopyram [
9]. Similarly, we found that the boscalid-resistant
B. jaapii strains isolated in Michigan were still susceptible to other SDHI fungicides, including fluopyram and fluxapyroxad [
4], and so Pristine was quickly replaced in CLS disease control programs by the fungicides Luna Sensation (Bayer CropScience; St. Louis, MO, USA), which is a premix of fluopyram and the quinone outside inhibitor (QoI) trifloxystrobin, and Merivon (BASF), which is a premix of fluxapyroxad and the QoI pyraclostrobin. Both of these fungicides were registered for CLS disease control in 2012.
Reports of resistance to fluopyram and fluxapyroxad in fungi such as
Alternaria alternata, Botrytis cinerea, and
Sclerotinia homoeocarpa have been appearing in recent years [
10,
11,
12,
13,
14,
15,
16]. Several
Sdh gene mutations have been identified and shown to be correlated with fluopyram and/or fluxapyroxad resistance including C-G91R and C-G150R with fluxapyroxad resistance in
S. homoeocarpa [
15], B-P225F and B-N230I with fluopyram and fluxapyroxad resistance in
B. cinerea [
13,
14], B-I229V in
SdhB from a single fluopyram-resistant field isolate of
Stagonosporopsis citrulli [
17], and B-I280V in
SdhB from fluopyram-resistant
Corynespora cassiicola [
18]. The location for both of the isoleucine to valine mutants discovered in
SdhB is only two amino acids away from the conserved His residue, where mutation had been associated with boscalid resistance.
We hypothesized that the exposure of boscalid-resistant strains of
B. jaapii to fungicides containing fluopyram or fluxapyroxad would rapidly select for resistance to these compounds. However, we did not know if resistance evolution would occur sequentially, i.e., if
B. jaapii strains containing the B-H260R mutation would evolve a second mutation conferring resistance to fluopyram and/or fluxapyroxad, or if resistance evolution would occur via selection of a new mutation in previously SDHI-sensitive strains. In this study, we tracked the evolution of resistance in field populations of
B. jaapii at our test orchard at the Northwest Michigan Horticultural Research Center (NWMHRC) as fluopyram and fluxapyroxad lost CLS efficacy. We simultaneously performed a largescale orchard survey in Michigan of
B. jaapii for resistance to fluopyram and fluxapyroxad, and assessed instances of practical resistance within individual orchard populations. Practical resistance of a target fungal pathogen occurs when reductions in the level of disease control are caused by the selection of fungicide-resistant isolates [
19]. We further sequenced and identified mutations in the
SdhB and
SdhC genes of
B. jaapii that were correlated with different resistance phenotypes to fluopyram and fluxapyroxad.
4. Discussion
Our results show that resistance to boscalid, fluopyram, and fluxapyroxad in
B. jaapii is widespread in commercial tart cherry orchards in the major growing regions of Michigan. Assessments of the phenotypic composition of resistant isolates from individual orchards has also shown that practical resistance to fluopyram and fluxapyroxad occurs in most of the commercial orchards surveyed. Although both fluopyram and fluxapyroxad are marketed in fungicide premixes with the QoI fungicides trifloxystrobin and pyraclostrobin, respectively, the efficacy of these fungicides is impacted by SDHI fungicide resistance (
Table 3), even though resistance to QoIs has not been documented.
Assessment of a previous collection (2010–2011) of
B. jaapii sampled from commercial tart cherry orchards in Michigan (similar locations to orchards sampled in this study) revealed that 30.4% of 1189 isolates were resistant to boscalid [
4]. During this earlier time period, the collected isolates were sensitive to fluopyram and fluxapyroxad, and CLS was controlled with the premix fungicides Luna Sensation and Merivon [
4]. Because of the resistance issue, the boscalid-containing premix fungicide Pristine (BASF) was replaced with Luna Sensation and Merivon in commercial tart cherry orchards in Michigan beginning in 2012. The B-H260R mutation was detected in all ten Bosc
R B. jaapii isolates examined in our previous study [
4]. Because of the deployment of additional SDHI fungicides, and because the Bosc
R B. jaapii isolates all presumably contained a mutation that was not associated with fluopyram or fluxapyroxad resistance, we wondered if
B. jaapii isolates from 2016–2017 that recently evolved resistance to fluopyram, fluxapyroxad, and also possibly to boscalid, would have evolved by acquiring additional mutations to B-H260R. To address this question, we sequenced the
SdhB,
SdhC, and
SdhD genes from 19
B. jaapii isolates in an attempt to infer the subsequent evolution of SDHI resistance in Michigan populations of
B. jaapii.
Two mutations, B-I262V and C-S84L, were detected among eight Bosc
S Fluo
R Flux
S isolates examined (
Table 5). Homologous B-I229V and B-I280V mutations have been previously detected in fluopyram-resistant field isolates of
S. citrulli and
C. cassiicola, respectively [
17,
18]. The C-S84L mutation is a new fluopyram-resistance mutation. We hypothesize that both the B-I262V and C-S84L mutations evolved from SDHI-sensitive
B. jaapii strains. In addition, both B-I262V and C-S84L mutations conferred fluopyram resistance following expression of the mutant alleles in a heterologous
Sclerotinia sclerotiorum system [
23].
Our results showed three genotypes among eight Bosc
R Fluo
R Flux
R isolates examined, none of which contained the B-H260R mutation (
Table 5). Four of the isolates harbored a C-N86S mutation; this mutation has been detected in field populations of
Zymoseptoria tritici in Europe [
24], and the homologous C-N75S mutation in
SdhC has been detected in field populations of
Pyrenophora teres in Europe [
25]. However, the C-N86S (C-N75S) mutation has generally been associated with low to moderate levels of resistance to SDHIs [
24,
26]. Likewise, we were not able to confirm the function of the C-N86S mutation in conferring resistance to fluopyram and fluxapyroxad following expression in the heterologous
S. sclerotiorum system, but did find that this mutation confers resistance to two other SDHI compounds, pyraziflumid and inpyrfluxam [
23]. The four Bosc
R Fluo
R Flux
R and three other Bosc
S Fluo
R Flux
R B. jaapii isolates harboring the C-N86S mutation displayed high levels of resistance to fluopyram and fluxapyroxad, but these isolates may have an additional mutation that was undetected. Three Bosc
R Fluo
R Flux
R isolates harbored the N86S mutation in
SdhC with an additional B-I262V mutation, and one isolate had a combination of the B-I262V mutation along with a C-S84L mutation. While the B-I262V and C-S84L mutations were each individually correlated only with fluopyram resistance, it is possible that co-association of either of these mutations with the C-N86S mutation changes the fungicide-binding pocket resulting in additional fluxapyroxad resistance. We hypothesize that each of these Bosc
R Fluo
R Flux
R isolates evolved from an original genotype that was sensitive to all three SDHI fungicides.
Thus, we hypothesize that there were a few evolutionary routes in the
B. jaapii population ending in the fully-resistant Bosc
R Fluo
R Flux
R phenotype, and that all of these routes were initiated from a fully-sensitive Bosc
S Fluo
S Flux
S isolate (
Figure 2). The first route involved the selection of the C-N86S mutation, and possibly an additional mutation(s) that we have not detected (
Figure 2). The second route involved an intermediate Bosc
S Fluo
R Flux
S isolate harboring the B-I262V mutation in which second mutations, either C-S84L or C-N86S, were selected (
Figure 2). An alternative evolutionary pathway to the Bosc
R Fluo
R Flux
R phenotype with B-I262V + C-S84L genotype would be via an intermediate Bosc
S Fluo
R Flux
S isolate harboring the C-S84L mutation in which the B-I262V mutation was subsequently selected (
Figure 2). Older Bosc
R Fluo
S Flux
S strains in the population, prior to the use of Luna Sensation and Merivon in commercial orchards, could have given rise to Bosc
S Fluo
R Flux
R isolates through selection of the additional C-N86S mutation (
Figure 2). The B-H260R + C-N86-S genotype was detected in one isolate, and we hypothesize that addition of the second mutation altered the structure of the SDHI-binding pocket resulting in the altered resistance phenotype.
It is interesting that resistance to three separate SDHI fungicides has evolved in
B. jaapii populations in Michigan, even though each of these fungicides has been only applied in fungicide premixes, and the mixing partner QoI fungicide is effective in CLS management [
4]. However, in the case of Luna Sensation, the preferred fungicide rate utilized by growers of 365.4 mL hectare
−1 (5.0 fluid ounces per acre) contains only 91.4 g hectare
−1 of trifloxstrobin, which is used at a rate of 136.1 g hectare
−1 (3.8 fluid ounces per acre) when applied as the single fungicide Flint Extra (Bayer). This 49% reduction in amount of trifloxystrobin in the Luna Sensation premix likely resulted in a reduction in disease control by the trifloxystrobin component of the mixture, putting more selection pressure on the fluopyram component. The effect of dose rates, and the use of two single-site fungicides together in a mixture for fungicide resistance management have been studied in multiple pathosystems (ex. [
27,
28]), but optimal use strategies are clearly not universal. One idea emerging from the study of Ayer et al. [
28] is that “managing population size may be one of the best strategies for reducing resistance development”. These authors conducted a four year field study on fungicide management of apple scab, caused by
Venturia inaequalis. In this study, dose rate was important, as the authors noted a higher probability of reduced sensitivity or resistance emerging in populations exposed to lower doses of fungicides, including fluxapyroxad [
28]. However, fungicide resistance to fluxapyroxad never evolved when this fungicide was used as part of a premix with various other fungicides [
28]. While the apple scab and CLS diseases are similar in disease cycle [
29,
30], the successful management of apple scab early in the season can effectively eliminate this pathogen for the rest of the growing season [
31]. In contrast, the incidence of CLS will continue to increase after harvest and into the fall, as it is simply not economically feasible to try to manage this pathogen throughout the entire growing season. This difference in pathogen population size may be a factor in the rapid evolution of SDHI resistance in
B. jaapii in Michigan and the lack of SDHI resistance evolution in
V. inaequalis, even though resistance to the QoI component of the SDHI premix fungicides Luna Sensation and Merivon is widespread in
V. inaequalis [
32,
33].