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Fungal diseases, including downy mildew (caused by Plasmopara viticola) and gray mold (caused by Botrytis cinerea), significantly impact the marketable yield of grapes produced worldwide. Cytochrome b of the mitochondrial respiratory chain of these two fungi is a key target for Quinone outside inhibitor (QoI)-based fungicide development. Since the mode of action (MOA) of QoI fungicides is restricted to a single site, the extensive usage of these fungicides has resulted in fungicide resistance. The use of fungicide combinations with multiple targets is an effective way to counter and slow down the development of fungicide resistance. Due to the high cost of in planta trials, in silico techniques can be used for the rapid screening of potential fungicides. In this study, a combination of in silico simulations that include Schrödinger Glide docking, molecular dynamics, and Molecular Mechanism-Generalized Born Surface Area calculation were used to screen the most potent QoI and non-QoI-based fungicide combinations to wild-type, G143A-mutated, F129L-mutated, and double-mutated versions that had both G143A and F129L mutations of fungal cytochrome b. In silico docking studies indicated that mandestrobin, famoxadone, captan, and thiram have a high affinity toward WT cytochrome b of Botrytis cinerea. Although the QoIs mandestrobin and famoxadone were effective for WT based on in vitro results, they were not broadly effective against G143A-mutated isolates. Famoxadone was only effective against one isolate with G143A-mutated cytochrome b. The non-QoI fungicides thiram and captan were effective against both WT and isolates with G143A-mutated cytochrome b. Follow-up in silico docking and molecular dynamics studies suggested that fungicide combinations consisting of famoxadone, mandestrobin, fenamidone, and thiram should be considered in field testing targeting Plasmopara viticola and Botrytis cinerea fungicide resistance. [ABSTRACT FROM AUTHOR]

BACKGROUND: Stem‐end rot caused by Lasiodiplodia theobromae is one of the most important diseases of papaya in northeastern Brazil. It can be controlled effectively by demethylation inhibitor (DMI) fungicides, but the occurrence of DMI resistance may decrease fungicide efficacy. RESULTS: Detached fruit studies revealed that isolates with EC50 values of 6.07 and 6.28 μg mL−1 were not controlled effectively, but reduced virulence and ability to grow at temperatures ranging from 12 to 32 °C suggesting fitness penalties were observed. Cross‐resistance was observed only between difenoconazole and propiconazole. The entire cytochrome P450 sterol 14α‐demethylase (LtCYP51) gene and its flanking regions were cloned. The gene was 1746 bp in length and contained three introns. The predicted protein contained 525 amino acids. Phylogenetic tree analysis showed that the LtCYP51 belongs to the CYP51B clade. No amino acid variation was found between sensitive and resistant isolates; however, the gene was constitutively more highly expressed in resistant isolates. CONCLUSION: Resistance to DMI fungicides in L. theobromae is based on LtCYP51 gene overexpression and fitness penalties may be present in difenoconazole‐resistant isolates. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

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 This article presents the development of a LAMP method to on‐site detect DMI fungicide resistance in Monilinia fructicola. [ABSTRACT FROM AUTHOR]

Abstract: 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 EC50 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 [ABSTRACT FROM AUTHOR]

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 EC50 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 EC50 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. [ABSTRACT FROM AUTHOR]

BACKGROUND Colletotrichum species cause anthracnose diseases on many plants and crops. A new generation of succinate dehydrogenase inhibitors (SDHIs) was developed recently. The inhibitory activity of the five SDHI fungicides against Colletotrichum species was determined in this study. RESULTS Isolates of C. gloeosporioides, C. acutatum, C. cereale and C. orbiculare were insensitive (naturally resistant) to boscalid, fluxapyroxad and fluopyram on YBA agar medium. In contrast, these isolates were relatively sensitive to penthiopyrad, except for C. orbiculare. Most interestingly, benzovindiflupyr showed highest inhibitory activity against all of these four species. Benzovindiflupyr was effective against C. gloeosporioides and C. acutatum on apple and peach fruit, as well as on cucumber plants inoculated with C. orbiculare. The sdhB, sdhC and sdhD genes encoding the subunits of fungicide-targeted succinate dehydrogenase were sequenced, but, despite high polymorphisms, no apparent resistance mutations were found in Colletotrichum species. CONCLUSIONS This is the first report on the activity of benzovindiflupyr against Colletotrichum species. The broad-spectrum efficacy of benzovindiflupyr within the Colletotrichum genus might be exploited when designing disease management strategies against various pathogens on a wide range of crops. Other mechanism(s) than fungicide target-site modification may be responsible for differential sensitivity of Colletotrichum species to SDHI fungicides. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 H2O2 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 [ABSTRACT FROM AUTHOR]

The article focuses on research which aimed at sequencing and cloning of the cytochrome b (Cyt b) gene from Monilinia fructicola (M. fructicola). It reveals that the 1166 bp intron downstream of the G143 codon and Cyt b gene area covering the location of G143 and the adjoining 1166 bp intron was polymerase chain reaction (PCR) amplified. It notes that the study was the first complete sequence of Cyt b gene for M. fructicola.

The article presents a study which investigates the potential benefits of a sulfur/propiconazole mixture for the control of propiconazole-resistant isolates. A disease was developed by propiconazole-resistant isolates on detached fruit after protective or curative applications of propiconazole or its mixture with sulfur. According to the result, additive to slightly synergistic effects were observed for the mixture in protective treatments of peaches inoculated with propiconazole-resistant isolates. The addition of elemental sulfur to demethylation inhibitor (DMI) fungicide is likely to be a relatively inexpensive means to improve brown rot control in peach production area where reduced sensitivity to DMI fungicides is suspected.

The costs of two reduced-fungicide programs (5 applications each) and two micronized wettable-sulfur programs (6 or 11 applications each) and their impact on scab control and fruit quality were evaluated on 'Contender' and 'Cresthaven' peach over two growing seasons. The reduced-fungicide programs consisted of applications of chlorothalonil and captan or chlorothalonil, azoxystrobin, and captan. All programs provided excellent scab control when disease pressure was low to moderate; however, six sulfur applications did not adequately control scab under high disease pressure. The product cost of the reduced-fungicide chemicals was higher, but comparable with six applications of micronized wettable sulfur or nine hypothetical applications of nonmicronized wettable sulfur per season. The reduced-fungicide programs were preferable to weekly applications of sulfur (11 applications) because they equally controlled scab at a reduced cost and have greater potential to protect against other summer diseases. The fungicide program effects on fruit quality were inconsistent. A more detailed study is needed to determine if a relationship between fungicide programs and fruit quality exists. [ABSTRACT FROM AUTHOR]

The rapid emergence of resistance in plant pathogens to the limited number of chemical classes of fungicides challenges sustainability and profitability of crop production worldwide. Understanding mechanisms underlying fungicide resistance facilitates monitoring of resistant populations at large-scale, and can guide and accelerate the development of novel fungicides. A majority of modern fungicides act to disrupt a biochemical function via binding a specific target protein in the pathway. While target-site based mechanisms such as alternation and overexpression of target genes have been commonly found to confer resistance across many fungal species, it is not uncommon to encounter resistant phenotypes without altered or overexpressed target sites. However, such non-target site mechanisms are relatively understudied, due in part to the complexity of the fungal genome network. This type of resistance can oftentimes be transient and noninheritable, further hindering research efforts. In this review, we focused on crop pathogens and summarized reported mechanisms of resistance that are otherwise related to target-sites, including increased activity of efflux pumps, metabolic circumvention, detoxification, standing genetic variations, regulation of stress response pathways, and single nucleotide polymorphisms (SNPs) or mutations. In addition, novel mechanisms of drug resistance recently characterized in human pathogens are reviewed in the context of nontarget-directed resistance. [ABSTRACT FROM AUTHOR]

Although Botrytis fragariae was only recently identified as a new Botrytis species that causes strawberry gray mold, its prevalence extends to many states of the eastern United States. Compared with B. cinerea, which is known to be the causal agent of gray mold on many crop plants including strawberry, B fragariae appears to have specifically adapted to strawberry and exhibits distinct fungicide sensitivity. This is the first presentation of a high-quality genome assembly of B fragariae with gene annotation based on sequence homology and deep transcriptome data. The genome sequence information from B. fragariae is expected to help reveal genomic features underlying its host specialization and evolution of distinct fungicide resistance and other novel pathogenicity mechanisms. [ABSTRACT FROM AUTHOR]

Two application methods of calcium (Ca), fertigation and spray, were investigated regarding their effects on Botrytis blight on petunia (Petunia x hybrida) flowers. Plants were grown for 6 weeks with three nutrient solutions consisting of 0, 100, or 200 mg⋅L-1 Ca and weekly calcium chloride (CaCl2) sprays of 0, 750, or 1500 mg⋅L-1 Ca for a total of nine treatment combinations. Flowers were harvested, inoculated with Botrytis spores, placed in humidity chambers, and evaluated for Botrytis blight severity. Disease severity decreased by 57% and 70% when flowers were treated with Ca spray applications of 750 and 1500 mg⋅L-1 Ca, respectively; however, no change in disease severity occurred across the Ca fertigation applications. Ca concentration in the flower petal tissue increased with the Ca spray applications: the flower petal Ca concentration increased from 0.26% to 0.65% of tissue dry mass (DM) as the Ca spray application rate increased from 0 to 1500 mg⋅L-1. However, no change was observed across the Ca fertigation treatments. Leaf tissue Ca concentration increased from 2.1% to 3.2% DMas the fertigation solution increased from 0 to 200 mg⋅L-1 Ca, whereas spray application had no significant effects of leaf tissue Ca concentration. The results demonstrate that spray application is a more effective technique than fertigation application to provide higher Ca tissue concentrations in flowers, and that the Ca concentration in flower petal tissue is an important consideration when evaluating tissue susceptibility to Botrytis blight. Because of the high rate of fungicide resistance to Botrytis cinerea found in commercial greenhouses, spray applications of CaCl2 are an important disease management tool for commercial growers. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Sensitivity of Botrytis cinerea to seven fungicide chemical classes was determined for 888 isolates collected in 2016 from 47 California strawberry fields. Isolates were collected early season (minimum fungicide exposure) and late season (maximum fungicide exposure) from the same planting block in each field. Resistance was determined using a mycelial growth assay, and variable frequencies of resistance were observed to each fungicide at both sampling times (early season %. late season %): boscalid (12, 35), cyprodinil (12, 46), fenhexamid (53, 91), fludioxonil (1,4), fluopyram (2. 7), iprodione (25,8), isofetamid (0, 1). penthiopyrad (8,25), pyraclosfrobin (77,98), and thiophanate-methyl (81, 96). Analysis of number of chemical class resistances (CCRs) revealed an increasing shift in CCR from the early to late season. Phenotypes of 40 isolates that were resistant or sensitive to different chemical classes were associated with presence or absence of mutations in target genes. Fungicide-resistance phenotypes determined in the mycelial growth assay closely matched (93.8%) the genotype observed. Previously described resistance-conferring mutations were found for each gene. A survey of fungicide use from 32 of the sampled fields revealed an average of 15 applications of gray mold-labeled fungicides per season at an average interval of 12 days. The most frequently applied fungicides (average number of applications during the 2016 season) were captan (7.3), pyraclostrobin (2.5), cyprodinil (2.3), fludioxonil (2.3), boscalid (1.8), and fenhexamid (1.4). Multifungicide resistance is widespread in California. Resistance management tactics that reduce selection pressure by limiting fungicide use, rotating among Fungicide Resistance Action Committee codes, and mixing/rotating site-specific fungicides with multisite fungicides need to be improved and implemented. [ABSTRACT FROM AUTHOR]

Botrytis fragariae was recently described causing gray mold of strawberry in Germany and the United States. The goal of the present study was to determine its prevalence, distribution, and sensitivity to fungicides in strawberry fields of five states. In total, 188 Botrytis isolates were obtained from flowers and fruit collected from the states of Maryland (n = 35), Virginia (n = 38), North Carolina (n = 46), South Carolina (n = 4 1 ), and Georgia (n = 28). Only 13 of these were fruit samples and came from South Carolina (n = 5) and Georgia (n = 8). B. fragariae made up 35.1 % of the entire collection, and composed close to half of the Botrytis population in North Carolina (43.4%), South Carolina (61.0%), and Georgia (42.9%). One isolate of B. mali was also found, and the rest of the isolates were B. cinerea (sensu lato). B. fragariae and B. cinerea were found coexisting in 11 fields, while other field samples consisted of only B. fragariae (n = 3) or only B. cinerea (n =10) isolates. B. fragariae isolates with resistance to one or more fungicides were found, and resistance profiles differed from those of B. cinerea, in that no resistance to cyprodinil (FRAC 8) or boscalid and other FRAC 7 botryticides was detected. We detected B. fragariae resistance to the active ingredients thiophanate-methyl, iprodione, fludioxonil, and fenhexamid. We also detected B. fragariae isolates with resistance to up to four chemical classes of fungicides, though most isolates were resistant to one or two chemical classes. In conclusion, isolates of the newly detected species B. fragariae were commonly found on strawberry flowers in the Mid-Atlantic United States, and have developed resistance to many of the most commonly used botryticides. Though the relevance of this species to pre- and postharvest fruit infections is unknown, fludioxonil applications may give this species a competitive advantage over B. cinerea. Controlling this fungus with FRAC 7 fungicides may be an effective way of limiting its spread in strawberry fields. [ABSTRACT FROM AUTHOR]

Botrytis cinerea, the causal agent of gray mold disease, is one of the most important plant-pathogenic fungi affecting strawberry. During the last decade, control of gray mold disease in the southeastern United States has largely been dependent on captan and the use of at-risk fungicides with single-site modes of action, including a combination of the quinone outside inhibitor (Qol) fungicide pyraclostrobin and succinate dehydrogenase inhibitor (SDHI) fungicide boscalid formulated as Pristine 38WG. Reports about loss of efficacy of Pristine in experimental fields in North Carolina prompted us to collect and examine 216 single-spore isolates from 10 conventional fields and 1 organic field in North Carolina and South Carolina in early summer 2011. Sensitivity to pyraclostrobin or boscalid was determined using a conidial germination assay with previously published discriminatory doses. Pyraclostrobin- and pyraclostrobin+boscalid-resistant isolates were found in all conventional fields (with some populations revealing no sensitive isolates) and in the organic field. Among the isolates collected, 66.7% were resistant to pyraclostrobin and 61.5% were resistant to both pyraclostrobin and boscalid. No isolates were identified that were resistant to boscalid but sensitive to pyraclostrobin, indicating that dual resistance may have derived from a Qol-resistant population. The molecular basis of Qol and SDHI fungicide resistance was determined in a subset of isolates. Polymerase chain reaction-restriction fragment length polymorphism analysis of the partial cytochrome b (CYTB) gene showed that pyraclostrobin-resistant isolates possessed the G143A mutation known to confer high levels of Qol fungicide resistance in fungi. Boscalid-resistant isolates revealed point mutations at codon 272 leading to the substitution of histidine to arginine (H272R) or tyrosine (H272Y), affecting the third Fe-S cluster region of die iron-sulfur protein (SdhB) target of SDHIs. The results of the study show that resistance to Qol fungicides and dual resistance to Qol and SDHI fungicides is common in B. cinerea from strawberry fields in the Carolinas. Resistant strains were more frequent in locations heavily sprayed with Qol and SDHI fungicides. However, resistance to both fungicides was also found in the unsprayed, organic field, indicating that some resistant strains may have been introduced from the nursery. [ABSTRACT FROM AUTHOR]

Botrytis blight on petunia flowers causes significant losses in the postharvest environment. Infection occurs during greenhouse production, and symptoms are expressed during transport. This phenomenon is termed petunia flower meltdown because of the rapid collapse of flower petal tissue as the plants are transported from the production greenhouse to the retail store. The objective of this study was to determine the effect of calcium (Ca) spray applications on botrytis blight severity in petunia flowers. For the first experiment, petunia 'Pretty Grand Red' plants were sprayed twice per week for 2 weeks with calcium chloride (CaCl2) at rates of 0, 400, 800, and 1200 mg⋅L-1 Ca. A fungicide (cyprodinil, 37.5%; fludioxonil, 25%) was used as an additional control treatment. Twenty-four hours after the last treatment, freshly opened flowers were harvested, placed into a humidity chamber with 99% relative humidity, and inoculated with a Botrytis cinerea spore suspension (1 x 104 conidia/mL). Disease progression was recorded every 12 hours for 72 hours. The results showed a 96% reduction in botrytis blight severity as Ca concentration increased from 0 to 1200 mg⋅L-1 Ca. The Ca treatments provided better disease control than the fungicide treatment because of the fungicide resistance of the isolate used in the study. A second experiment was performed to determine whether the beneficial response to CaCl2 application was influenced by chlorine (Cl) or the electrical conductivity (EC) of the spray solutions, and no significant responses were observed. These studies prove Ca is the sole source of the reduction in botrytis blight severity following treatment with CaCl2 sprays, and demonstrate the benefit of using Ca as a tool for the management of botrytis blight on petunia flowers. [ABSTRACT FROM AUTHOR]

Stem-end rot caused by Lasiodiplodia theobromae is one of the most devastating diseases of papaya in northeastern Brazil. It is most effectively controlled by applications of fungicides, including site-specific fungicides at risk for resistance development. This study investigated the molecular mechanisms of reduced sensitivity to the QoI fungicide azoxystrobin and resistance to the MBC fungicide thiophanate-methyl in L. theobromae from Brazilian orchards. The EC 50 values for azoxystrobin in sixty-four isolates ranged from 0.36 μg/ml to 364.24 μg/ml and the frequency distribution of EC 50 values formed a multimodal curve, indicating reduced sensitivity to azoxystrobin. In detached fruit assays reduced sensitive isolates were not controlled as effectively as sensitive isolates at lowest label rate. Partial fragments were obtained from target genes β- tubulin (751 bp) and Cytb (687 bp) of isolates resistant to thiophanate-methyl and reduced sensitivity to azoxystrobin. Sequence analysis of the β- tubulin fragment revealed a mutation corresponding to E 198 K in all thiophanate-methyl-resistant isolates, while reduced sensitivity to axoxystrobin was not attributable to Cytb gene alterations. The target gene-based mechanism conferring resistance to thiophanate-methyl will likely be stable even if selection pressure subsides. However, the mechanism conferring reduced sensitivity to azoxystrobin is not based on target gene modifications and thus may not be as stable as other genotypes with mutations in Cytb gene. Unlabelled Image • Reduced sensitivity to azoxystrobin in Lasiodiplodia theobromae from papaya was observed. • Reduced sensitivity to azoxystrobin was not attributable to Cytb gene alterations. • Resistance to thiophanate-methyl was associated with E 198 K mutation in β-tubulin gene. • No-cross-resistance was found between azoxystrobin and thiophanate-methyl. [ABSTRACT FROM AUTHOR]

Methyl benzimidazole carbamate (MBC) fungicides were once widely used for brown rot (Monilinia fructicola) control of peach (Prunus persica (L.) Batsch) in the southeastern US, but their use was substantially reduced and often eliminated due to widespread resistance. In this study, 233 M. fructicola isolates were collected from major peach production areas in Alabama, Georgia, and South Carolina, and sensitivity to thiophanate-methyl was examined. Isolates were also collected from one organic and two experimental peach orchards. A discriminatory dose of 1 μg/ml was used to distinguish sensitive (S) and moderately sensitive (S-LR) isolates from low resistant phenotypes, while 50 and 500 μg/ml thiophanate-methyl concentrations were used to determine high resistant (HR) phenotypes. Sequence analyses were performed to identify mutations in the β-tubulin target gene and detached fruit assays were performed to determine the efficacy of a commercial product against isolates representing each phenotype. Results indicated 55.7%, 63.5%, and 75.9% of isolates from Alabama, Georgia, and South Carolina, respectively, were S to thiophanate-methyl; 44.3%, 36.5%, and 21.4% were S-LR; no isolates were LR; and only 3 isolates (1.3%) from South Carolina were HR. No mutations in S or S-LR isolates were found, but HR isolates revealed the E198A mutation, an amino acid change of glutamic acid to alanine conferring high resistance. The high label rate of a commercial product containing thiophanate-methyl controlled brown rot caused by S and S-LR isolates in detached fruit studies but was ineffective against HR isolates. The combinations of thiophanate-methyl with azoxystrobin or isofetamid, when mixed together and applied in an experimental orchard 14 days preharvest, significantly reduced brown rot incidence on pre and postharvest commercially ripe fruit and efficacy was comparable to that of a grower standard fungicide. These results indicate that thiophanate-methyl may again be useful to peach growers in the southeastern US for brown rot and fungicide resistance management. [Display omitted] • Monilinia fructicola isolates from three US states were largely sensitive or moderately sensitive to thiophanate methyl • Isolates resistant to thiophanate methyl contained E198A mutation in beta tubulin • Label rates of thiophanate methyl controlled brown rot caused by sensitive and moderately sensitive isolates • Field trials revealed thiophanate methyl applied in mixtures during preharvest controls brown rot [ABSTRACT FROM AUTHOR]

Botrytis cinerea causes pre- and postharvest decay of many fruit and vegetable crops. A survey of German strawberry fields revealed Botrytis strains that differed from B. cinerea in diagnostic PCR markers and growth appearance. Phylogenetic analyses showed that these strains belong to an undescribed species in Botrytis clade 2, named Botrytis fragariae sp. nov. Isolates of B. fragariae were detected in strawberry fields throughout Germany, sometimes at frequencies similar to those of B. cinerea, and in the southeastern United States. B. fragariae was isolated from overwintering strawberry tissue but not from freshly infected fruit. B. fragariae invaded strawberry tissues with an efficiency similar to or lower than that of B. cinerea but showed poor colonization of inoculated nonhost plant tissues. These data and the exclusive occurrence of this fungus on strawberry plants indicate that B. fragariae is host specific and has a tissue preference different from that of B. cinerea. Various fungicide resistance patterns were observed in B. fragariae populations. Many B. fragariae strains showed resistance to one or several chemical classes of fungicides and an efflux-based multidrug resistance (MDR1) phenotype previously described in B. cinerea. Resistance-related mutations in B. fragariae were identical or similar to those of B. cinerea for carbendazim (E198A mutation in tubA), azoxystrobin (G143A in cytB), iprodione (G367A_V368F in bos1), and MDR1 (gain-of-function mutations in the transcription factor mrr1 gene and overexpression of the drug efflux transporter gene atrB). The widespread occurrence of B. fragariae indicates that this species is adapted to fungicidetreated strawberry fields and may be of local importance as a gray mold pathogen alongside B. cinerea. IMPORTANCE Gray mold is the most important fruit rot on strawberries worldwide and requires fungicide treatments for control. For a long time, it was believed to be caused only by Botrytis cinerea, a ubiquitous pathogen with a broad host range that quickly develops fungicide resistance. We report the discovery and description of a new species, named Botrytis fragariae, that is widely distributed in commercial strawberry fields in Germany and the southeastern United States. It was observed on overwintering tissue but not on freshly infected fruit and seems host specific on the basis of its occurrence and artificial infection tests. B. fragariae has also developed resistance to several fungicides that is caused by mutations similar to those known in B. cinerea, including an efflux-based multidrug resistance. Our data indicate that B. fragariae could be of practical importance as a strawberry pathogen in some regions where its abundance is similar to that of B. cinerea. [ABSTRACT FROM AUTHOR]

Polyoxin D is a Fungicide Resistance Action Committee (FRAC) code 19 fungicide that was recently registered for gray mold control of strawberry in the United States. In this study, we determined the sensitivity to polyoxin D zinc salt (hereafter, polyoxin D) of Botrytis cinerea isolates from 41 commercial strawberry farms in South Carolina, North Carolina, Maryland, Virginia, and Ohio and investigated the fitness of sensitive (S) and reduced sensitive (RS) isolates. Relative mycelial growth ranged between 0 and over 100% on malt extract agar amended with a discriminatory dose of polyoxin D at 5 p,g/ml. Isolates that grew more than 70% at that dose were designated RS and were found in three of the five states. The 50% effective dose (EC50) values of three S and three RS isolates ranged from 0.59 to 2.27 and 4.6 to 5.8 pg/ml, respectively. The three RS isolates grew faster on detached tomato fruit treated with Ph-D WDG at recommended label dosage than S isolates (P < 0.008). In all, 25 randomly selected RS isolates exhibited reduced sporulation ability (P < 0.0001) and growth rate (P < 0.0001) but increased production of sclerotia (P < 0.0386) compared with 25 S isolates. Of 10 isolates tested per phenotype, the number of RS isolates producing sporulating lesions on apple, tomato, and strawberry was significantly lower compared with S isolates (P < 0.0001 for each fruit type). The results of this study indicate that resistance management is necessary for fungicides containing polyoxin D. To our knowledge, this is the first study demonstrating reduced sensitivity to FRAC 19 fungicides in B. cinerea isolates from the United States. [ABSTRACT FROM AUTHOR]

Anthracnose fruit rot was observed in some late-season peach cultivars in South Carolina in the 2012 and 2013 production seasons as well as increased anthracnose leaf spot of blueberry in a commercial operation of the same state in 2012. Single-spore isolates of Colletotrichum siamense were either sensitive or resistant to both thiophanate-methyl and azoxystrobin with the concentration of the fungicide at which fungal development is inhibited by 50% of a 100 p,g/ml. Resistant isolates revealed the E198A mutation in (3-tubulin and the G143A mutation in cytochrome b. Nucleotide sequence analysis of the complete CYTB gene from genomic DNA of C. siamense isolates revealed an intronless genotype (Crl) and a genotype revealing two introns (CsII) at amino acid positions 131 and 164. Resistance to thiophanate-methyl or azoxystrobin was not found in isolates of C.fructicola collected from peach fruit. The CYTB gene of isolates of this species was of the C/II genotype or revealed a unique C/IIa genotype. Phylogenetic analysis of C. siamense isolates from different locations and different crops showed that the resistant isolates were genetically closer to each other than to sensitive isolates, suggesting that field resistance to thiophanate-methyl and azoxystrobin fungicides is derived from a common ancestor. [ABSTRACT FROM AUTHOR]

The fungicide resistance profiles to seven chemical classes of fungicides were investigated in 198 Botrytis cinerea isolates from five blackberry fields and 214 B. cinerea isolates from 10 strawberry fields of North and South Carolina. Populations of B. cinerea tended to have a single dominant, location-specific resistance profile that consisted of resistance to multiple fungicides in fields sprayed weekly with sitespecific fungicides. The most prevalent profile in blackberry fields consisted of resistance to thiophanate-methyl, pyraclostrobin, and boscalid. The most prevalent resistance profile found in conventional strawberry fields consisted of resistance to thiophanate-methyl, pyraclostrobin, boscalid, and cyprodinil. A statistical model revealed that multifungicide resistance patterns did not evolve randomly in populations from both crops. Instead, strains resistant to thiophanate-methyl were more likely to acquire resistance to pyraclostrobin, the resulting dual-resistant population was more likely to acquire resistance to boscalid, the resulting triple-resistant population was more likely to acquire resistance to cyprodinil, and the resulting quadruple-resistant population was more likely to acquire resistance to fenhexamid (strawberry population only) compared with random chance. Resistance to iprodione and fludioxonil evolved from a pool of strains with different fungicide resistance profiles. Resistance to thiophanate-methyl, pyraclostrobin, boscalid, and fenhexamid in blackberry isolates was, without exception, based on target gene mutations, including E198A and E198V in ß-tubulin, G143A in cytochrome b, H272Y and H272R in SdhB, and F412I in Erg27, respectively. A new genotype associated with fenhexamid resistance was found in one strain (i.e., Y408H and deletion of P298). Fungicide-resistant strains were present but rare in an unsprayed blackberry field, where some unique phenotypes, including low and medium resistance to fludioxonil, had emerged in the absence of fungicide pressure. The isolates resistant to fludioxonil had effective dose that inhibited 50% of mycelial growth values of 0.16 pg/ml (low resistance) and 0.32 and 0.38 pg/ml (medium resistance) and were also resistant to the anilinopyrimidine fungicide cyprodinil, indicating that this and similar phenotypes will eventually be selected by continued applications of the fludioxonil + cyprodinil premixture Switch. This study shows that multifungicide-resistant phenotypes are common in conventionally maintained strawberry and blackberry fields and that resistance to multiple fungicides evolved from stepwise accumulation of single resistances. [ABSTRACT FROM AUTHOR]

The sensitivity to seven chemical classes of fungicides was investigated in 1,810 Botrytis cinerea isolates collected from strawberry blossoms and fruit in 181 strawberry fields from seven southern states in the United States across 2 years. Ten isolates were examined from each field. Fungicide sensitivity assays were carried out based on visual assessment of diametrical mycelial growth after 4 days of incubation on media amended with discriminatory doses of fungicides in microliter plates. Results of visual assessments were verified with selected isolates using a previously published germination assay and by inoculating representative isolates with resistant phenotypes on fungicide-sprayed fruit. The overall resistance frequencies of 750 isolates collected in 2012 for thiophanate-methyl, pyraclostrobin, boscalid, cyprodinil, fenhexamid, iprodione, and fludioxonil were 76, 42, 29, 27, 25, 3, and 1%, respectively. Frequencies of 1,060 isolates collected in 2013 were 85, 59, 5, 17, 26, 2, and 1%, respectively. Resistance to thiophanate- methyl and pyraclostrobin was found in virtually every location in both years, whereas resistance to iprodione and fludioxonil was rarely found. Resistant isolates were resistant to either one (23%), two (18%), three (19%), four (14%), five (3%), or six (0.1%) chemical classes of fungicides in 2012. In 2013, this distribution was 24, 29, 26, 8, 2, and 0.3%, respectively. Multifungicide-resistant isolates of B. cinerea were widespread in southern states and evidence suggests that the frequency of isolates with multifungicide resistance increased from 2012 to 2013. The data also show that fungicide resistance in B. cinerea was already present in blossoms, indicating that resistance management needs to be implemented early in the season. [ABSTRACT FROM AUTHOR]

Sterol demethylation inhibitor (DMI) fungicide resistance in isolates of Monilinia fructicola from Georgia has been linked to overexpression of the MfCYP51 gene and a corresponding 65-bp genetic element 'Mona' inserted in the upstream region of MfCYP51. In this study, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to detect the Mona element. Fourteen DMI fungicide-resistant (DMI-R) and six DMI fungicide-sensitive (DMI-S) isolates from Georgia, six DMI-R and 11 DMI-S isolates from South Carolina, seven DMI-R and nine DMI-S isolates from New York, and two DMI-R and three DMI-S isolates from Ohio were used in this study. The isolates from the southeastern United States and Ohio were collected from peach, whereas isolates from New York were collected from cherry. A 376-bp fragment containing the Mona element was consistently amplified with primer pair INS65-F and INS65-R from DMI-R isolates, and either a 311 -bp or 1,81 5-bp fragment was amplified from DMI-S isolates. The primer pair did not amplify DNA fragments of similar sizes from isolates of five other common fruit rot pathogens of peach, including Alternaria alternata, Colletotrichum acutatum, Gilbertella persicaria, Penicillium expansum, and Rhizopus stolonifer. Gel electrophoresis of the PCR amplicon can distinguish between DMI-R and DMI-S isolates based on the 65-bp size difference of the amplicon; however, the restriction digestion assay can verify questionable results, especially in the absence of a positive control. Only the 376-bp fragment containing the Mona element was digestable with endonuclease BsrBI, resulting in two restriction fragments of 236 and 140 bp in size. In this study, a protocol for Mona detection from aerial fungal structures was developed that can yield results within a few hours of sampling. This study confirms that the Mona element is strongly linked to the DMI-resistance phenotype and reveals that overexpression of the MfCYP51 gene is a common DMI fungicide resistance mechanism in M. fructicola, not only in Georgia but throughout the eastern United States. [ABSTRACT FROM AUTHOR]