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Mixtures of fungicides with different modes of action are commonly used as disease and resistance management tools, but little is known of mixtures of natural and synthetic products. In this study, mixtures of metabolites from the rhizobacterium Pseudomonas chlororaphis strain ASF009 formulated as Howler EVO with below-label rates (50 µg/ml) of conventional sterol demethylation inhibitor (DMI) fungicides were investigated for control of anthracnose of cherry ( Prunus avium ) caused by Colletotrichum siamense . Howler mixed with metconazole or propiconazole synergistically reduced disease severity through lesion growth. Real-time PCR showed that difenoconazole, flutriafol, metconazole, and propiconazole induced the expression of DMI target genes CsCYP51A and CsCYP51B in C. siamense . The addition of Howler completely suppressed the DMI fungicide-induced expression of both CYP51 genes. We hypothesize that the downregulation of DMI fungicide-induced expression of the DMI target genes may, at least in part, explain the synergism observed in detached fruit assays., Competing Interests: The author(s) declare no conflict of interest.

Biological control agent Bacillus subtilis formulated as Theia is registered for control of fungal and bacterial diseases of fruit crops. Combinations of Theia and strategic concentrations of two demethylation inhibitor (DMI) fungicides were investigated to explore potential synergisms. Bacteria were cultured in nutrient broth and combined with technical grades and two formulations of propiconazole (emulsifiable concentrate [EC] and wettable powder) and metconazole (EC and water-dispersible granule) at 0, 10, 50, 100, and 150 µg/ml of active ingredient. After cocultivation, the optical density (OD 600 ) and colony forming units (CFU/ml) were evaluated. In contrast to EC formulations, the wettable powder or water-dispersible granule formulations at 10 or 50 µg/ml of both DMIs did not affect vegetative cell growth. The mixture of Theia and each formulated DMI at 50 µg/ml of active ingredient resulted in a significant reduction of Monilinia fructicola lesion development on apple, Colletotrichum siamense lesion development on cherry, and Botrytis cinerea lesion development on cherry. The combination of Theia with EC formulations showed weaker disease reduction due to antagonism. Only Theia plus non-EC formulated propiconazole and metconazole significantly reduced brown rot disease incidence of apple compared with the respective solo treatments and anthracnose disease incidence of cherry compared with the untreated control. Our results indicated that at least some DMI fungicides possess bactericidal effects depending on the formulation and concentration. The combination of Theia with a lower-than-label-rate concentration (50 µg/ml) of the DMI fungicides propiconazole and metconazole showed potential for synergistic effects, especially when non-EC formulations were used., Competing Interests: The author(s) declare no conflict of interest.

The fungicide fludioxonil is one of the most effective single-site fungicides available for managing flower blight caused by Botrytis cinerea on fruit and ornamental crops. Although low and moderate levels of resistance to fludioxonil have been reported in the pathogen across the United States and Europe, high resistance has been reported only from greenhouses in China. In this study, two B. cinerea isolates with high resistance (half maximal effective concentration >100 µg/ml) to fludioxonil were detected on ornamental calibrachoa flowers grown in a greenhouse. These isolates exhibited stable resistance for >20 generations, produced symptoms on calibrachoa flowers sprayed with label rates of fludioxonil, and displayed in vitro fitness penalties with decreased mycelial growth ( P < 0.0001) and sporulation ( P < 0.0001) compared with sensitive isolates. Highly resistant isolates were identified as MDR1h, containing the ΔL/V497 deletion in mrr1 . However, resistance levels and in vitro fitness parameter characteristics were not consistent with this phenotype. One isolate contained the mutation L267V between HAMP domains 1 and 2 of the Bos-1 gene, and both isolates exhibited high osmotic sensitivity and reduced glycerol accumulation in the presence of fludioxonil, indicating that high resistance of these isolates may be associated with the high-osmolarity glycerol mitogen-activated protein kinase pathway.

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.

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.

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.

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.

Fungi in the genus Monilinia cause brown rot disease of stone and pome fruits. Here, we report the draft genome assemblies of four important phytopathogenic species: M. fructicola, M. fructigena, M. polystroma, and M. laxa. The draft genome assemblies were 39 Mb (M. fructigena), 42 Mb (M. laxa), 43 Mb (M. fructicola), and 45 Mb (M. polystroma) with as few as 550 contigs (M. laxa). These are the first draft genome resources publicly available for M. laxa, M. fructigena, and M. polystroma.

In this study, we investigated whether fungicide-induced mutagenesis previously reported in Monilinia fructicola could accelerate genetic changes in field populations. Azoxystrobin and propiconazole were applied to nectarine trees at weekly intervals for approximately 3 months between bloom and harvest in both 2013 and 2014. Fungicides were applied at half-label rate to allow recovery of isolates and to increase chances of sublethal dose exposure. One block was left unsprayed as a control. In total, 608 single-spore isolates were obtained from blighted blossoms, cankers, and fruit to investigate phenotypic (fungicide resistance) and genotypic (simple-sequence repeat [SSR] loci and gene region) changes. In both years, populations from fungicide-treated and untreated fruit were not statistically different in haploid gene diversity (P = 0.775 for 2013 and P = 0.938 for 2014), allele number (P = 0.876 for 2013 and P = 0.406 for 2014), and effective allele number (P = 0.861 for 2013 and P = 0.814 for 2014). Isolates from blossoms and corresponding cankers of fungicide treatments revealed no changes in SSR analysis or evidence for induced Mftc1 transposon translocation. No indirect evidence for increased genetic diversity in the form of emergence of reduced sensitivity to azoxystrobin, propiconazole, iprodione, and cyprodinil was detected. High levels of population diversity in all treatments provided evidence for sexual recombination of this pathogen in the field, despite apparent absence of apothecia in the orchard. Our results indicate that fungicide-induced, genetic changes may not occur or not occur as readily in field populations as they do under continuous exposure to sublethal doses in vitro.

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.

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.

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 microg/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, and 2-dD-glucose inhibited spore germination and mycelial growth of all test fungi, including M. fructicola, at comparable levels. Fosetyl-Al added to PDA (PDA-F) at 500 or 1,000 microg/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 microg/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 from 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.

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.

ABSTRACT Agrobacterium tumefaciens-mediated transformation with plasmids carrying the hygromycin B resistance gene hph frequently is being used for inserting genes into fungal spores and mycelial cells and for conducting insertional mutagenesis to identify genes connected to a particular phenotype. In this article, we report that stable hygromycin B resistance can develop spontaneously in germinating conidia from Monilinia fructicola and that the mutants exhibit altered phenotypes. One spontaneously developing hygromycin B-resistant colony developed per 2.5 x 10(5) germinating conidia. Mutants grew significantly slower on potato dextrose agar, were 2.4- to 3.1-fold more sensitive to demethylation inhibitor fungicides, lacked melanization, and did not produce spores. The mode of action of hygromycin B resistance in the mutants seemed to be different from the hph transgene-mediated hygromycin B resistance based on different phenotypic characters. The ability of M. fructicola and possibly other fungi to spontaneously develop hygromycin B resistance associated with an altered phenotype may interfere with the selection of true transformants if hygromycin B is used as selective agent. This is particularly confounding if the hph gene is used as selectable marker in insertional mutagenesis experiments conducted for the identification of genes involved in melanization, sporulation, or fungicide resistance.