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Resistance to fludioxonil in Botrytis cinerea and B. fragariae was previously found to be linked to either overexpression of the drug efflux pump atrB activated by mutations in transcription factor mrr1 or to mutations in the osmoregulation gene os1 . In the present study, isolates of B. cinerea , Botrytis group S, or B. fragariae collected from strawberry fields in the United States were resistant to fludioxonil with half-maximal effective concentration values ranging from 0.04 to 0.43 µg/ml for B. cinerea , 0.03 to 1.03 µg/ml for Botrytis group S, and 0.28 to 3.48 µg/ml for B. fragariae . Analyses of mrr1 sequences revealed various mutations linked to fludioxonil resistance in B. cinerea and Botrytis group S isolates. However, no mutations in mrr1 correlated with atrB overexpression-mediated resistance in B. fragariae isolates. Neither nucleotide variations in the 1,370-bp upstream region of atrB nor increased atrB copy numbers could explain the atrB overexpression in these B. fragariae isolates. Mutations in os1 conferred resistance to iprodione in B. cinerea and Botrytis group S isolates; none correlated with resistance to fludioxonil in B. fragariae . In contrast to European isolates, U.S. B. fragariae isolates contained a 3-bp insertion in the coding region of os1. These isolates were more sensitive to osmotic stress but it is unclear whether the insertion is responsible for this phenotype. Our findings suggest that atrB overexpression-associated fludioxonil resistance is an across-species mechanism of resistance to fludioxonil that can be induced by mutations in mrr1 and other, still-unknown mechanisms.

Background: The increasing use of demethylation inhibitor (DMI) fungicides for the control of peach brown rot has resulted in resistance in Monilinia fructicola. Resistance in the southeastern USA is caused by overexpression of the MfCYP51 gene due to the presence of a 65-bp inserted element 'Mona' located in the upstream regulatory region of MfCYP51. A rapid diagnostic assay would be useful to detect the presence and monitor further spread of this resistance mechanism., Results: A loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of 'Mona'-based DMI resistance. The assay was optimized for specificity and sensitivity, and was shown to detect the presence of 10 fg of purified target DNA per reaction within 85 min. Only DNA isolated from DMI-resistant isolates containing 'Mona' resulted in a fluorescent signal after LAMP assay amplification. DNA from sensitive isolates from China and the USA and six other common fungal species of peach did not yield a signal. The method also positively identified 'Mona' from crude DNA extracts (using Lyse and Go reagents heated to 100 °C for 10 min) obtained from the mycelium and conidia of symptomatic fruit., Conclusion: Considering its specificity, stability and repeatability, the LAMP assay could be a valuable tool for rapid on-site diagnosis of M. fructicola isolates resistant to DMI fungicides in the southeastern USA. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Anthracnose disease, caused by Colletotrichum truncatum, affects marketable yield during preharvest production and postharvest storage of fruits and vegetables worldwide. Demethylation inhibitor (DMI) fungicides are among the very few chemical classes of single-site mode of action fungicides that are effective in controlling anthracnose disease. However, some species are inherently resistant to DMIs and more information is needed to understand this phenomenon. Isolates of C. truncatum were collected from the United States and China from peach, soybean, citrus, and begonia and sensitivity to six DMIs (difenoconazole, propiconazole, metconazole, tebuconazole, flutriafol, and fenbuconazole) was determined. Compared with DMI sensitive isolates of C. fructicola, C. siamense, and C. fioriniae (EC 50 value ranging from 0.03 to 16.2 µg/ml to six DMIs), C. truncatum and C. nymphaeae were resistant to flutriafol and fenbuconazole (with EC 50 values of more 50 µg/ml). Moreover, C. truncatum was resistant to tebuconazole and metconazole (with resistance factors of 27.4 and 96.0) and displayed reduced sensitivity to difenoconazole and propiconazole (with resistance factors of 5.1 and 5.2). Analysis of the Colletotrichum spp. genome revealed two potential DMI targets, CYP51A and CYP51B, that putatively encode P450 sterol 14α-demethylases. Both genes were identified and sequenced from C. truncatum and other species and no correlation between CYP51 gene expression levels and fungicide sensitivity was found. Four amino acid variations L208Y, H238R, S302A, and I366L in CYP51A, and three variations H373 N, M376L, and S511T in CYP51B correlated with the DMI resistance phenotype. CYP51A structure model analysis suggested the four alterations may reduce azole affinity. Likewise, CYP51B structure analysis suggested the H373 N and M376L variants may change the conformation of the DMI binding pocket, thereby causing differential sensitivity to DMI fungicides in C. truncatum.

Background: Metalaxyl is a phenylamide fungicide, introduced for oomycete management in the 1970s. RPA190 gene has been reported to be associated with metalaxyl resistance, although other genes may also be involved in the process. This study assessed the evolutionary association of amino acid (AA) mutations in RPA190 with metalaxyl resistance in Phytophthora infestans., Results: All isolates from Yunnan were resistant to metalaxyl with multiple AA mutations in RPA190. Two main evolutionary pathways were found in metalaxyl resistance: in one pathway, RPA190 from sensitive isolates (SAA) underwent a single type AA mutation at position 1476, changing from valine to glycine (V1476G); the second pathway involved multiple steps of point mutations. In a phylogenetic tree, nucleotide sequences of the gene clustered into six clades largely corresponding to AA isoform groups. Among the clusters, sequences carrying mutations V1476G and P980S formed two independent clades; other clades shared a common mutation of F382Y. All three mutations together with another two were shown to be under positive selection and the number of AA mutations in an isolate was positively correlated with EC 50 values., Conclusion: Multiple mutations such as V1476G, P980S, and F382Y in RPA190 contribute to metalaxyl resistance, and resistance to metalaxyl can emerge in at least two independent pathways in P. infestans. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

The genetic element 'Mona' has been shown previously to be associated with resistance to demethylation inhibitors (DMIs) in Monilinia fructicola. In this study, the promoter activity of the 'Mona' element was demonstrated genetically and the activity was narrowed down to a 20-bp active region through a series of deletions. 'Mona' knockout transformants (ΔMona) were generated from DMI-resistant isolate Bmpc7, and EC 50 values and expression of the MfCYP51 gene were found to be reduced in transformants compared with the parental isolate. When the 'Mona' element was inserted into the upstream region of the MfCYP51 gene of the DMI-sensitive isolate HG3, the EC 50 values and expression of the MfCYP51 gene increased in the transformants compared with the parental sensitive isolate. These results indicate that the 'Mona' element determines the DMI fungicide resistance through the up-regulation of the expression of the downstream MfCYP51 gene. No fitness penalty was observed in knockout and insertion transformants, i.e. transformants showed similar mycelial growth rate, sporulation and ability to cause lesions on fruit compared with their parental isolates, suggesting that the 'Mona' element does not affect basal life activities., (© 2016 BSPP and John Wiley & Sons Ltd.)

Previous research has shown that Botrytis cinerea isolates with resistance to multiple chemical classes of fungicides exist in eastern strawberry fields. In this study, the fungicide resistance profiles of 2,130 isolates from flowers of commercial strawberry fields located in multiple states was determined over four consecutive strawberry production seasons. Producers were asked to alternate single-site fungicides that were considered low risk in their specific location based on resistance monitoring results in their fields. This recommendation led to an increase of chemical class diversity used in the spray programs. Results indicated that simultaneous resistance in individual isolates to two, three, four, five, six, and seven classes of fungicides increased over time. The increase in chemical class resistances within isolates was likely due to a process we termed "selection by association", where fungicide resistance traits were often linked to the trait being selected rather than the selectable trait itself. Data analysis also indicated that the odds were highest for isolates resistant to one chemical class (1CCR) to be resistant to thiophanate-methyl; for 2CCR isolates to be resistant to thiophanate-methyl and pyraclostrobin; and for 3CCR isolates to be resistant to thiophanate-methyl, pyraclostrobin, and either cyprodinil or fenhexamid. We hypothesize that the more chemical classes are used in a spray program, the faster isolates will be selected with increasing numbers of chemical class resistances by virtue of selection by association if such isolates preexist in the population.

Evolved resistance to fungicides is a major problem limiting our ability to control agricultural, medical and veterinary pathogens and is frequently associated with substitutions in the amino acid sequence of the target protein. The convention for describing amino acid substitutions is to cite the wild-type amino acid, the codon number and the new amino acid, using the one-letter amino acid code. It has frequently been observed that orthologous amino acid mutations have been selected in different species by fungicides from the same mode of action class, but the amino acids have different numbers. These differences in numbering arise from the different lengths of the proteins in each species. The purpose of the present paper is to propose a system for unifying the labelling of amino acids in fungicide target proteins. To do this we have produced alignments between fungicide target proteins of relevant species fitted to a well-studied 'archetype' species. Orthologous amino acids in all species are then assigned numerical 'labels' based on the position of the amino acid in the archetype protein. © 2016 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2016 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Background: The dicarboximide fungicide dimethachlone has been widely used in China for more than 12 years to control the Sclerotinia stem rot caused by Sclerotinia sclerotiorum disease. First signs of resistance in the field are reported at low frequency. In this study, four resistant isolate/mutants were used to explore still unknown mechanisms leading to dimethachlone resistance., Results: The resistant isolate/mutants had significantly higher EC50 values compared with the sensitive control isolates. Cross-resistance was confirmed between dimethachlone and procymidone, iprodione and fludioxonil. The resistant isolate/mutants revealed a decreased mycelial growth rate, were less pathogenic on leaves of oilseed rape, were more sensitive to osmotic pressure and oxidative stress and released more electrolytes compared with the sensitive isolates. Only in one lab mutant did we find a point mutation (V238A) in the SsOs1 gene of the high-osmolarity glycerol (HOG) signalling pathway. The expression of this gene was lost in the field resistant isolate HN456-1-JBJ and decreased in mycelium that was subjected to either high osmotic pressure or dimethachlone; however, another key gene in the HOG pathway, SsHog1, could be induced in the resistant isolate and mutants with NaCl treatment., Conclusion: This study demonstrates that resistance to dicarboximide fungicide dimethachlone in S. sclerotiorum is emerging in China. Several fitness parameters, including mycelial growth rate, sclerotia formed in vitro, aggressiveness on leaves and osmotic and H2 O2 sensitivity, indicate that the resistant strains may not effectively compete with sensitive isolates in the field in the absence of selection pressure. Lost expression or the V238A point mutation in the SsOs1 gene may confer resistance to dicarboximide fungicide dimethachlone in S. sclerotiorum, but this study illustrates that other, yet unknown mechanisms also exist., (© 2015 Society of Chemical Industry.)

Gray mold, caused by the fungal pathogen Botrytis cinerea, is one of the most destructive diseases of small fruit crops and control is largely dependent on the application of fungicides. As part of a region-wide resistance-monitoring program that investigated 1,890 B. cinerea isolates from 189 fields in 10 states of the United States, we identified seven isolates (0.4%) from five locations in four different states with unprecedented resistance to all seven Fungicide Resistance Action Committee (FRAC) codes with single-site modes of action including FRAC 1, 2, 7, 9, 11, 12, and 17 registered in the United States for gray mold control. Resistance to thiophanate-methyl, iprodione, boscalid, pyraclostrobin, and fenhexamid was based on target gene mutations that conferred E198A and F200Y in β-tubulin, I365N/S in Bos1, H272R/Y in SdhB, G143A in Cytb, and T63I and F412S in Erg27. Isolates were grouped into MDR1 and MDR1h phenotypes based on sensitivity to fludioxonil and variations in transcription factor mrr1. MDR1h isolates had a previously described 3-bp deletion at position 497 in mrr1. Expression of ABC transporter atrB was increased in MDR1 isolates but highest in MDR1h isolates. None of the isolates with seven single resistances (SR) had identical nucleotide variations in target genes, indicating that they emerged independently. Multifungicide resistance phenotypes did not exhibit significant fitness penalties for the parameters used in this study, but MDR1h isolates produced more sclerotia at low temperatures and exhibited increased sensitivity to salt stress. In this study we show that current resistance management strategies have not been able to prevent the geographically independent development of resistance to all seven site-specific fungicides currently registered for gray mold control in the United States and document the presence of MDR1h in North America.

Site-specific fungicides, including the phenylpyrrole fludioxonil, are frequently used for gray mold control but are at risk for the development of resistance. In this study, field isolates that were low-resistant (LR) and moderately resistant (MR) to fludioxonil from blackberry and strawberry fields of North Carolina, South Carolina, and Virginia were characterized. Genes involved in osmoregulation, including bcsak1, BcOS4, bos5, and BRRG-1, were cloned and sequenced to detect potential target gene alterations; however, none were found. A previously described mutation (R632I) in transcription factor Mrr1, which is known to increase the expression of ATP-binding cassette transporter AtrB, was found in MR but not in sensitive (S) or LR isolates. Expression of atrB in MR isolates was ≈200-fold increased compared with an S isolate; however, 30- to 100-fold overexpression was also detected in LR isolates. Both MR isolates exhibited increased sensitivity to salt stress in the form of mycelial growth inhibition at 4% NaCl, indicating a disruption of osmoregulatory processes in those strains. However, the glycerol content was indistinguishable between S, LR, and MR isolates with and without exposure to fludioxonil, suggesting that the glycerol synthesis pathway may not be a part of the resistance mechanism in LR or MR strains. An investigation into the origin of LR and MR isolates from blackberry revealed two insertions in the mrr1 gene consistent with those found in the Botrytis clade group S. The emergence of strains overexpressing atrB in European and now in North American strawberry fields underscores the importance of this resistance mechanism for development of resistance to fludioxonil in Botrytis cinerea.

Gray mold, caused by the fungal pathogen Botrytis cinerea, is one of the most destructive diseases of strawberry. Control of the disease in commercial fields is largely dependent on the application of fungicides, including the dicarboximide iprodione. Single-spore isolates were collected from strawberry fields in Florida, North Carolina, and South Carolina and subjected to an assay using conidial germination that distinguished sensitive (S) isolates from isolates with various levels of resistance to iprodione. Of the 245 isolates, 1 was highly resistant (HR), 5 were moderately resistant (MR), and 43 had low resistance (LR) to iprodione. LR and MR strains were found in the Florida population and in 9 of 11 locations from North Carolina and South Carolina, indicating that resistance was widespread but accounted for only a relatively small percentage of the B. cinerea population. Sequence analysis of the target gene bos1, which codes for a class III histidine kinase, revealed that the MR phenotype was associated with Q369P and N373S mutations and that the LR phenotype was associated with either a I365S or a I365N mutation. The I365S and I365N mutations were also present in five additionally included HR isolates from North Carolina and South Carolina blackberry fields and one HR isolate from a Virginia strawberry field but no mutation or mutation combinations in bos1 were uniquely associated with the HR phenotype. Expression analysis of bos1 in S and HR isolates did not reveal convincing evidence of the gene's involvement in HR resistance either. The six HR isolates had three different phenotypes with respect to their sensitivity to fludioxonil; two were S, two were LR, and two were MR. The fludioxonil LR and MR isolates were also resistant to tolnaftate, an indication of multidrug efflux pump activity. These data suggest that, in addition to point mutations in bos1, drug efflux pump activity and potentially a third mechanism of resistance may be contributing to the iprodione HR phenotype. Detached fruit studies showed that field rates of Rovral 4 Flowable (iprodione) did not control iprodione MR and HR isolates.

Background: The long-term preservation of interesting phenotypes in plant pathogenic fungi allows for follow-up studies in the future. Twelve storage approaches were investigated to determine their effects on instability of propiconazole resistance for three demethylation inhibitor (DMI) fungicide-resistant and two DMI-sensitive isolates of Monilinia fructicola. They included mycelium in PDA slants under mineral oil, in PDA plugs under 10% glycerol, on dried filter paper and conidia on silica gel, each stored for 36 weeks at 4, - 20, and - 80 °C., Results: None of the storage approaches prevented the rapid decline of EC(50) values for propiconazole in the three resistant isolates, and no significant differences were found among storage approaches (P = 0.787) or between storage approaches and consecutive transfers (P = 0.053). Most of the decline in resistance occurred during the first 4 weeks of storage. The DMI resistance-associated genetic element Mona, located in the immediate upstream region of the MfCYP51 gene, was still present in the three resistant isolates after 36 weeks of storage and weekly transfers. Furthermore, the Mona element and a portion of the MfCYP51 gene, which encodes the target enzyme for DMIs, did not reveal signs of DNA methylation. Resistance to propiconazole was partially regained in resistant isolates after two growth cycles on fresh peach fruit., Conclusions: Obtained data indicate that the decline of DMI resistance in M. fructicola cannot be prevented using commonly employed storage methods at various temperatures. The number of consecutive transfers and the storage duration prior to fungicide sensitivity tests in M. fructicola should be indicated in scientific papers., (Copyright © 2011 Society of Chemical Industry.)

Background: The cytochrome b (Cyt b) gene is a key genetic determinant for quinone outside inhibitor (QoI) fungicide resistance in plant pathogenic fungi. A mutation at amino acid position G143 can cause qualitative resistance unless it is part of the recognition site for a self-splicing intron. The objective of this study was to clone and sequence the Cyt b gene from Monilinia fructicola (Wint.) Honey, the causal agent of brown rot of stone fruits, and to assess the risk for the development of a mutation at position 143., Results: The Cyt b gene of M. fructicola was 11 927 bp in size and contained seven introns located at cDNA positions (5'-3') 204, 395, 430, 491, 507, 780 and 812 with sizes of 1592, 1318, 1166, 1252, 1065, 2131 and 2227 bp respectively. Sequence analysis revealed that the above-mentioned 1166 bp intron, a self-splicing group I intron, was located just downstream of the G143 codon. The Cyt b gene region covering the G143 location and the adjacent 1166 bp intron was PCR amplified and sequenced from Chinese and US isolates, indicating that the intron may be omnipresent in M. fructicola., Conclusion: This is the first complete Cyt b gene sequence published for M. fructicola or any other Monilinia species, forming the basis for molecular analysis of QoI fungicide resistance. Sequence analysis revealed that the G143A mutation responsible for high levels of QoI fungicide resistance in many plant pathogenic fungi may not develop in M. fructicola unless genotypes emerge that lack the 1166 bp intron., (Copyright © 2010 Society of Chemical Industry.)

The ability to develop fungicide resistance was assessed in Monilinia fructicola isolates with different fungicide sensitivity phenotypes by adapting mycelium and conidia to increasing concentrations of selective fungicides and UV mutagenesis. Results showed that adaptation to Quinone outside inhibitor (QoI) fungicide azoxystrobin and sterol demethylation inhibitor (DMI) fungicide propiconazole was more effective in conidial-transfer experiments compared to mycelial-transfer experiments. DMI-resistant (DMI-R) isolates adapted to significantly higher doses of azoxystrobin in both, mycelial- and conidial-transfer experiments compared to benzimidazole-resistant (BZI-R) and sensitive (S) isolates. Adaptation to propiconazole in conidial-transfer experiments was accelerated in BZI-R isolates when a stable, nonlethal dose of 50 microg/ml thiophanate-methyl was added to the selection medium. One of two azoxystrobin-resistant mutants from DMI-R isolates did not show any fitness penalties; the other isolate expired before further tests could be carried out. The viable mutant caused larger lesions on detached peach fruit sprayed with azoxystrobin compared to the parental isolate. The azoxystrobin sensitivity of the viable mutant returned to baseline levels after the mutant was transferred to unamended medium. However, azoxystrobin resistance recovered quicker in the mutant compared to the corresponding parental isolate after renewed subculturing on medium amended with 0.2 and 1 microg/ml azoxystrobin; only the mutant but not the parental isolate was able to adapt to 5 microg/ml azoxystrobin. In UV mutagenesis experiments, the DMI-R isolates produced significantly more mutants compared to S isolates. All of the UV-induced mutants showed stable fungicide resistance with little fitness penalty. This study indicates the potential for QoI fungicide resistance development in M. fructicola in the absence of a mutagen and provides evidence for increased mutability and predisposition to accelerated adaptation to azoxystrobin in M. fructicola isolates resistant to DMI fungicides.

Background: Management of demethylation inhibitor (DMI) fungicide resistance in Monilinia fructicola (G. Winter) Honey is a priority in peach orchards of the southeastern United States, but DMI fungicides are still an important component of antiresistance strategies in view of the few effective alternatives. The goal of this study was to investigate potential benefits of a sulfur/propiconazole mixture for the control of propiconazole-resistant isolates., Results: The mixture provided the best control for propiconazole-resistant isolates, regardless of protective or curative application timings, or the presence or absence of fruit injury. Propiconazole-resistant isolates developed disease on detached fruit after protective or curative applications of propiconazole or its mixture with sulfur, but protective applications of the mixture significantly reduced (P = 0.05) disease symptoms compared with the individual compounds. Additive to slightly synergistic effects were observed for the mixture in protective treatments of peaches inoculated with propiconazole-resistant isolates., Conclusion: The results suggest that the addition of elemental sulfur to a DMI fungicide is likely to be a relatively inexpensive means to improve brown rot control in peach production areas where reduced sensitivity to DMI fungicides is suspected but has not led to noticeable control failure.

Resistance in Monilinia fructicola to demethylation inhibitor (DMI) fungicides is beginning to emerge in North America, but its molecular basis is unknown. Two potential genetic determinants of DMI fungicide resistance including the 14alpha-demethylase gene (MfCYP51) and the ATP-binding cassette transporter gene MfABC1, were investigated in six resistant (DMI-R) and six sensitive (DMI-S) field isolates. No point mutations leading to an amino acid change were found in the MfCYP51 gene. The constitutive expression of the MfCYP51 gene in DMI-R isolates was significantly higher compared to DMI-S isolates. Gene expression was not induced in mycelium of DMI-R or DMI-S isolates treated with 0.3 mug of propiconazole/ml. A slightly higher average MfCYP51 copy number value was detected in DMI-R isolates (1.35) compared to DMI-S isolates (1.13); however, this difference could not be verified in Southern hybridization experiments or explain the up to 11-fold-increased MfCYP51 mRNA levels in DMI-R isolates. Analysis of the upstream nucleotide sequence of the MfCYP51 gene revealed a unique 65-bp repetitive element at base pair position -117 from the translational start site in DMI-R isolates but not in DMI-S isolates. This repetitive element contained a putative promoter and was named Mona. The link between Mona and the DMI resistance phenotype became even more apparent after studying the genetic diversity between the isolates. In contrast to DMI-S isolates, DMI-R isolates contained an MfCYP51 gene of identical nucleotide sequence associated with Mona. Still, DMI-R isolates were not genetically identical as revealed by Microsatellite-PCR analysis. Also, real-time PCR analysis of genomic DNA indicated that the relative copy number of Mona among DMI-S and DMI-R isolates varied, suggesting its potential for mobility. Interestingly, constitutive expression of the MfABC1 gene in DMI-R isolates was slightly lower than that of DMI-S isolates, but expression of the MfABC1 gene in DMI-R isolates was induced in mycelium after propiconazole treatment. Therefore, the MfABC1 gene may play a minor role in DMI fungicide resistance in M. fructicola. Our results strongly suggest that overexpression of the MfCYP51 gene is an important mechanism in conferring DMI fungicide resistance in M. fructicola field isolates from Georgia and that this overexpression is correlated with Mona located upstream of the MfCYP51 gene.