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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]

Botrytis cinerea isolates with multi-fungicide resistance have frequently been isolated from small fruit fields such as strawberries and blackberries. Individual B. cinerea isolates have been found resistant to up to seven chemical classes of fungicides. Fitness costs and less competitiveness have been observed in multi-fungicide resistant isolates, but this has not been examined under field conditions. In the spring of 2016, flowers of field-grown blackberries were either not inoculated or inoculated with B. cinerea isolates sensitive (0CCR), resistant to five or six chemical classes excluding phenylpyrroles (5CCR), or resistant to six or seven chemical classes including phenylpyrroles (6CCR/MDR1h). The experimental field was left unsprayed for the duration of this study and isolates of B. cinerea were collected from flowers and/or fruit in each of the three experimental years. Isolates collected in summer of 2016 revealed resistance phenotypes in each plot closely matching those of the respective inoculum, with 95% 0CCR, 55% 5CCR, and 91% 6CCR/MDR1h isolates recovered from 0CCR, 5CCR, and 6CCR/MDR1h inoculation plots, respectively. In the 2017 and 2018 isolate collections, 6CCR/MDR1h resistance phenotypes were found in plots inoculated and non-inoculated with this phenotype, indicating their persistence and movement between plots. Resistance phenotypes different from the inoculum were also recovered each year, indicating that the inoculum was successfully competing with a native Botrytis population. Despite the competition, 6CCR/MDR1h isolates were recovered in high frequency from all inoculated plots in 2018. G3pdh and mrr1 sequences of 6CCR/MDR1h isolates collected in 2018 were identical to the sequences of the inoculum, indicating that these isolates likely descended from the inoculum. This study demonstrates that isolates carrying multi-fungicide resistant phenotypes, specifically 6CCR/MDR1h, are competitive in the absence of fungicide selection pressure. Unlabelled Image • Multi-fungicide resistant Botrytis cinerea inocula competed with native population. • 6CCR/MDR1h phenotype was recovered frequently in third year amidst dry weather. • Recovered isolates were genotypically and phenotypically identical to the inoculum. [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]

Anthracnose fruit rot is one of the main diseases affecting strawberries especially in warm and humid climates. In commercial settings, producers are relying on fungicides to control the disease. One of the most potent chemical classes of fungicides for anthracnose control are the quinone outside inhibitors (QoIs), but resistance in field isolates due to a point mutation in the target gene cytb has recently been reported in multiple states of the USA. During a routine screening, two Colletotrichum nymphaeae isolates (OH_S1 and OH_S2) obtained from strawberry plants of a commercial field in Marietta-Ohio were found to be resistant to QoI fungicide azoxystrobin. The EC50 values determined in poison agar tests were greater than 100 μg/L. Both isolates were not controlled on detached strawberry fruit treated with label rates of Abound fungicide (a.i. azoxystrobin) and revealed disease severity of 86% and 96% on detached fruit. A baseline isolate (Cn_ebel2) of the same species revealed an EC50 value of 1.2 μg/L and no disease developed on Abound-treated fruit. In contrast to other reports, sequencing of the cytb gene from all three isolates used in this study revealed no amino acid changes in cytb, indicating the existence of a different and still unknown resistance mechanism. [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]

Succinate dehydrogenase inhibitor (SDHI) fungicides are important tools to control preharvest and postharvest brown rot of stone fruits. In this study we determined the mycelial growth rate of Monilinia fructicola isolates on various media and picked the three that allowed the fastest growth to assess the sensitivity of mycelium to SDHI fungicides boscalid, fluopyram, and penthiopyrad. Minimal medium (MM) supported mycelial growth the best and yielded lowest EC50 values for three SDHI fungicides. EC50 values corresponded with disease incidence data obtained from detached fruit assays. Penthiopyrad had significantly greater intrinsic activity in vitro compared to fluopyram at the α = 0.05 level and compared to boscalid at the α = 0.1 level. However, detached fruit assays revealed that this 'advantage' did not carry over in vivo. In conclusion, MM appears to be the best medium currently available to assess the sensitivity of M. fructicola mycelium in vitro. [ABSTRACT FROM AUTHOR]

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 µg/ml thiophanatemethyl 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 µg/ml azoxystrobin; only the mutant but not the parental isolate was able to adapt to 5 µg/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 Qol 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. [ABSTRACT FROM AUTHOR]

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.

Brown rot, caused by Monilinia fructicola (G Wint) Honey, is a serious disease of peach in all commercial peach production areas in the USA, including South Carolina where it has been primarily controlled by pre-harvest application of 14-α demethylation (DMI) fungicides for more than 15 years. Recently, the Qo fungicide azoxystrobin was registered for brown rot control and is currently being investigated for its potential as a DMI fungicide rotation partner because of its different mode of action. In an effort to investigate molecular mechanisms of DMI and Qo fungicide resistance in M fructicola, the ABC transporter gene MfABC1 and the alternative oxidase gene MfAOX1 were cloned to study their potential role in conferring fungicide resistance. The MfABC1 gene was 4380 bp in length and contained one intron of 71 bp. The gene revealed high amino acid homologies with atrB from Aspergillus nidulans (Eidam) Winter, an ABC transporter conferring resistance to many fungicides, including DMI fungicides. MfABC1 gene expression was induced after myclobutanil and propiconazole treatment in isolates with low sensitivity to the same fungicides, and in an isolate with high sensitivity to propiconazole. The results suggest that the MfABC1 gene may be a DMI fungicide resistance determinant in M fructicola. [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]

Colletotrichum spp. isolates contain two paralogous CYP51 genes that encode sterol 14-demethylase enzymes; however, their role in sensitivity to demethylation inhibitor (DMI) fungicides is yet to be determined. In this study, each of the two genes from Colletotrichum fioriniae and C. nymphaeae was able to rescue the function of CYP51 in the yeast Saccharomyces cerevisiae, demonstrating their independent function. Deletion of CYP51A led to increased sensitivity to propiconazole, diniconazole, prothioconazole, cyproconazole, epoxiconazole, flutriafol, prochloraz, and difenoconazole in C. fioriniae, and to the same fungicides and tebuconazole in C. nymphaeae, with the exception of prochloraz. Deletion of CYP51B in C. fioriniae and CYP51B in C. nymphaeae made mutants increasingly sensitive to five of nine DMI fungicides tested. The results suggest species-specific, differential binding of DMI fungicides onto the two CYP51 enzymes. Pairing DMIs with different effects on CYP51A and -B deletion mutants resulted in synergistic effects, as determined in mycelial growth inhibition experiments. Deletion mutants showed no fitness penalty in terms of mycelial growth, sporulation, and virulence. Our study elucidates the effect of CYP51A and CYP51B of Colletotrichum spp. on DMI sensitivity, suggesting that using a mixture of DMIs may improve the efficacy for anthracnose management. [ABSTRACT FROM AUTHOR]

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 (EC50 value ranging from 0.03 to 16.2 pg/ml to six DMIs), C. truncatum and C. nymphaeae were resistant to flutriafol and fenbuconazole (with EC50 values of more 50 pg/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 14a-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. CYP5IA 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Succinate dehydrogenase inhibitor (SDHI) fungicides have been used to control gray mold of strawberry for more than a decade, and selection for resistance in the causal agent Botrytis cinerea has become a threat to producers. In total, 2,570 B. cinerea isolates were collected from strawberry fields in the eastern United States across three seasons and their sensitivity to the SDHI materials boscalid, fluopyram, fluxapyroxad, and penthiopyrad was assessed. Assays were based on visual assessment of presence or absence of mycelial growth on media amended with discriminatory fungicide doses to distinguish sensitive from resistant isolates, respectively. Overall frequencies of isolates resistant to boscalid, fluopyram, fluxapyroxad, and penthiopyrad increased over the 3 years to 30.0, 1.0, 5.5, and 7.4%, respectively. Four resistance patterns, designated A, B, C, or D, were found. Pattern A isolates were resistant to boscalid with the allele H272R at locus sdhB', pattern B isolates were resistant to boscalid and penthiopyrad with the allele H272R or H272Y at locus sdhB\ pattern C isolates were resistant to boscalid, fluxapyroxad, and penthiopyrad with the allele H272Y at locus sdhB\ and pattern D isolates were resistant to boscalid, fluopyram, fluxapyroxad, and penthiopyrad with alleles P225F or N230I at locus sdhB. Isolates with alleles H272Y, N230I, or P225F were sensitive to a new SDHI, benzovindiflupyr, with mean effective dose that inhibits 50% of mycelial growth values of less than 0.5 p-g/ml for each genotype, suggesting that this fungicide may be useful for resistance management. Our data show an increase of B. cinerea isolates resistant to SDHI fungicides over three consecutive production seasons. Resistance management practices must be implemented for the sustained efficacy of SDHI fungicides against gray mold of strawberry. [ABSTRACT FROM AUTHOR]

Abstract: Analysis of the genome sequence of Fusarium graminearum revealed three paralogous cyp51 genes (designated cyp51A, -B, and -C) encoding 14-α demethylases in this fungus. Targeted gene disruption showed that the cyp51A, -B or -C disruption mutants were morphologically indistinguishable from the parent isolate on potato dextrose agar medium, which indicates that none of these genes is essential for mycelial growth. The sensitivity of cyp51A deletion mutants to seven sterol demethylation inhibitor (DMI) fungicides increased significantly compared to the parent strain, while sensitivity of cyp51C deletion mutants increased to some but not all DMIs. No change in DMI sensitivity was observed for cyp51B deletion mutants. The parental phenotypes of cyp51A and cyp51C deletion mutants were completely restored by genetic complementation with the wild-type cyp51A and cyp51C genes, respectively. The sensitivity of F. graminearum isolates increased significantly when subjected in vitro to a mixture of DMI fungicides triadimefon and tebuconazole as compared to the individual components. These results indicate that different DMI fungicides target different CYP51 proteins in F. graminearum and that a mixture of DMI fungicides can result in synergistic effects. Our findings have directly implications on chemical management strategies of plant diseases caused by Fusarium species. [ABSTRACT FROM AUTHOR]

Isolation of Monilinia spp. from stone and pome fruit surfaces is difficult due to the presence of several fast-growing fungal species such as Rhizopus, Alternaria, and Penicillium spp. Therefore, a new selective medium (acidified potato dextrose agar [pH 3.6] amended with fosetyl-aluminum [fosetyl-AL] at 500 µg/ml) (APDA-F500) was developed for the recovery of Monilinia propagules. The antifungal agents fosetyl-Al, dichloran, ammonium molybdate, and 2-deoxy-D-glucose (2-dD-glucose) were tested in potato dextrose agar (PDA) for their selective activity against Monilinia fructicola and seven common fungal contaminants of peach, including Alternaria alternata, Aspergillus niger, Colletotrichum acutatum, Gilbertella persicaria, Penicillium expansum, Phomopsis amygdali, and Rhizopus stolonifer. Dichloran, ammonium molybdate, an 2-dD-glucose inhibited spore germination and mycelial growth of all test fungi, including M. fructicola, at comparable levels. Fosetyl-Al added t PDA (PDA-F) at 500 or 1,000 µg/ml did not inhibit germination of any of the fungi but had a strong effect on mycelial growth of six of eight test fungi at 1,000 µg/ml, with the exceptions being R. stolonifer and M. fructicola. Germination and mycelial growth of M. fructicola were least affected on APDA-F500 compared with the other test fungi. On APDA-F500 at pH 3.2 and 3.6, germination of M. fructicola was not inhibited but mycelial growth was reduced by 54.2 and 24.2%, respectively. In all, 17 M. fructicola, 6 M. fructigena, and 6 M. laxa isolates collected fro different geographic locations and diverse hosts were evaluated for their germination and mycelial growth on APDA-F500 (at pH 3.6). Germination was not inhibited for any isolate and relative mycelial growth was 45.8 to 83.3%. Field-grown peach fruit from South Carolina and Hungary and plum fruit from Hungary were used to test the selectivity of APDA-F500 for the recovery of three Monilinia spp. compared with PDA-F500 and Monilinia selective medium (MSM) previously developed for Monilinia spp. detection. Percent recovery of M. fructicola from South Carolinian peach fruit was highest on APDA-F500 (0, 17, and 69% in June, July, and August, respectively) compared with PDA-F500 (0, 3.5, and 50%, respectively) and MSM (0, 0, and 6.8%, respectively). Moreover, APDA-F500 selectively recovered M. fructigena and M. laxa propagules from the surfaces of Hungarian peach and plum fruit. Our results indicate that APDA-F500 is a useful medium for selective isolation and enumeration of the three most common Monilinia spp. attacking stone fruits worldwide. [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]

Field isolates of Alternaria alternata from peach were previously characterized for their sensitivity to succinate dehydrogenase inhibitor (SDHI) fungicides and the underlying molecular basis of resistance was determined. In the present study, we report that isolates resistant to the SDHI fungicide boscalid, regardless of genotype, were also resistant to pyraclostrobin and thiophanate-methyl. Resistance to pyraclostrobin was due to the G143A mutation in cytochrome b and resistance to thiophanate-methyl was due to 167Y in β-tubulin. Representatives of the two most commonly isolated SDHI resistance genotypes, H277Y in sdh subunit B and H134R in sdh subunit C, as well as genotype D123E in sdh subunit D, were selected for fitness evaluations. Genotypes H277Y and H134R suffered no fitness penalties based on mycelial growth on potato dextrose agar, spore production in vitro, osmotic sensitivity, oxidative sensitivity, germination ability, or the ability to cause disease on peach fruit. Hypersensitivity to oxidative stress and weak sporulation was observed only in genotype D123E. No competitive advantage was detected for sensitive isolates over the course of five consecutive transfers on peach fruit when spores were mixed with genotypes H277Y or H134R. Results suggest that, in the absence of fungicide pressure, A. alternata isolates resistant to methyl benzimidazole carbamate, quinone outside inhibitor, and SDHI fungicides carrying the H277Y mutation in SDHB and the H134R mutation in SDHC may effectively compete with the boscalid-sensitive populations. [ABSTRACT FROM AUTHOR]

Repeated applications of fungicides with a single mode of action are believed to select for pre-existing resistant strains in a pathogen population, while the impact of sub-lethal doses of such fungicides on sensitive members of the population is unknown. In this study, in vitro evidence is presented that continuous exposure of Monilinia fructicola mycelium to some fungicides can induce genetic change in form of transposon transposition. Three fungicide-sensitive M. fructicola isolates were exposed in 12 weekly transfers of mycelia to a dose gradient of demethylation inhibitor fungicide (DMI) SYP-Z048 and quinone outside inhibitor fungicide (QoI) azoxystrobin in solo or mixture treatments. Evidence of mutagenesis was assessed by monitoring Mftc1 , a multicopy transposable element of M. fructicola , by PCR and Southern blot analysis. Movement of Mftc1 was observed following azoxystrobin and azoxystrobin plus SYP-Z048 treatments in two of the three isolates, but not in the non-fungicide-treated controls. Interestingly, the upstream promoter region of Mf CYP51 was a prime target for Mftc1 transposition in these isolates. Transposition of Mftc1 was verified by Southern blot in two of three isolates from another, similar experiment following prolonged, sublethal azoxystrobin exposure, although in these isolates movement of Mftc1 in the upstream Mf CYP51 promoter region was not observed. More research is warranted to determine whether fungicide-induced mutagenesis may also happen under field conditions. [ABSTRACT FROM AUTHOR]

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 (3-tubulin, I365N/S in Bosl, H272R/Y in SdhB, G143A in Cytb, and T63I and F412S in Erg27. Isolates were grouped into MDR1 and MDRlh phenotypes based on sensitivity to fludioxonil and variations in transcription factor mrrl. MDRlh isolates had a previously described 3-bp deletion at position 497 in mrrl. Expression of ABC transporter atrB was increased in MDR1 isolates but highest in MDRlh 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, bui MDRlh 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 MDRlh in North America. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 fludi-oxonil 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. [ABSTRACT FROM AUTHOR]

Colletotrichum species cause diseases on many plants and are among the 'top 10' fungal plant pathogens. Species of the C. gloeosporioides and C. acutatum complexes are particularly important because they infect temperate fruit crops, but their control relies largely on chemical fungicides. In this study, differences in intrinsic fungicide sensitivity were determined in vitro using isolates of the C. gloeosporioides sp. complex (C. fructicola , C. siamense , and C. tropicale) and the C. acutatum sp. complex (C. fioriniae and C. nymphaeae), which had never been exposed to fungicides. Mycelial growth of all isolates was sensitive to the QoI azoxystrobin, the SDHI benzovindiflupyr, and the new DMI fungicide mefentrifluconazole. The isolates of C. nymphaeae were highly sensitive to the phenylpyrrole fungicide fludioxonil. The isolates of C. gloeosporioides sp. complex were sensitive to the bis-guanidine fungicide iminoctadine-albesilate, whereas those of C. acutatum sp. complex were inherently insensitive. These results are valuable when sensitivity of field populations is monitored in resistance management. Although SDHI fungicides are largely not effective against diseases caused by Colletotrichum species, benzovindiflupyr controlled anthracnose disease of various crops such as kidney bean, garland chrysanthemum, and strawberry, caused by C. lindemuthianum , C. chrysanthemi , and C. siamense , respectively, demonstrating this fungicide to be unique among SDHIs and having a broad control spectrum against anthracnose. To help understanding the reason for differential activity of benzovindiflupyr and boscalid, sdhB gene sequences were analyzed but those of C. lindemuthianum , C. chrysanthemi , and C. scovillei revealed no known mutations reported to be responsible for SDHI resistance in other fungi, indicating that other mechanism(s) than target-site modification may be involved in differential sensitivity to benzovindiflupyr and boscalid, found in Colletotrichum species. [Display omitted] • Azoxystrobin sensitivity was high in baseline isolates of Colletotrichum gloeosporioides and C. acutatum sp. complexes • Mefentrifluconazole, with favorable toxicity profile, was effective against Colletotrichum isolates regardless of the species • Mefentriconazole and benzovindiflupyr will become an effective tool to manage anthracnose on multiple crops [ABSTRACT FROM AUTHOR]

In the European Union (EU), regulation of sterol demethylation inhibiting (DMI) fungicides is tightened due to their suspected endocrine disrupting properties. However, the new DMI fungicide mefentrifluconazole was reported to have high fungicidal activity with minimal adverse side effects. In addition, some evidence suggests inconsistent cross resistance between mefentrifluconazole and other azoles. In this study, mefentrifluconazole and other triazoles were examined for activity to select pathogens sensitive or resistant to DMIs using mycelial growth tests on fungicide-treated culture medium or spray trials using cucumber plants. Cross-resistance was confirmed for all of the fungal species tested but activity levels varied. The sensitivity of Monilinia fructicola from peach to mefentrifluconazole was higher compared to other DMIs. In contrast, the inhibitory activity of mefentrifluconazole was equal or slightly inferior compared to difenoconazole, tebuconazole, propiconazole in Colletotrichum spp., Alternaria alternaria sp. complex and Cercospora beticola isolated from peach and sugar beet, respectively. Similar tendencies (i.e. equal or slightly inferior activity and cross-resistance) were observed for cucumber powdery mildew (Podosphaera xanthii) resistant to triflumizole, myclobutanil, and difenoconazole. Despite cross-resistance to other DMIs, mefentrifluconazole is a promising fungicide for fungal disease control on peach and other crops, with a reportedly more favorable toxicity profile. Unlabelled Image • Mefentrifluconazole exhibited cross resistance to other DMI fungicides both in vitro and in planta • The intrinsic efficacy of mefentrifluconazole is comparable to other DMI fungicides • Favorable toxicity profile and strong efficacy make this new azole an attractive IPM tool [ABSTRACT FROM AUTHOR]