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Sterol demethylation inhibitor (DMI) fungicides continue to be essential components for the control of brown rot of peach caused by Monilinia fructicola in the United States and worldwide. In the southeastern United States, resistance to DMIs had been associated with overexpression of the cytochrome P450 14α-demethylase gene Mf CYP51 as well as the genetic element Mona, a 65 bp in length nucleotide sequence located upstream of Mf CYP51 in resistant isolates. About 20 years after the first survey, we reevaluated sensitivity of M. fructicola from South Carolina and Georgia to propiconazole and also evaluated isolates from Alabama for the first time. A total of 238 M. fructicola isolates were collected from various commercial and two experimental orchards, and sensitivity to propiconazole was determined based on a discriminatory dose of 0.3 μg/ml. Results indicated 16.2, 89.2, and 72.4% of isolates from Alabama, Georgia, and South Carolina, respectively, were resistant to propiconazole. The detection of resistance in Alabama is the first report for the state. All resistant isolates contained Mona, but it was absent from most sensitive isolates. It was unclear if the resistance frequency had increased in South Carolina and Georgia. However, the resistance levels (as assessed by the isolate frequency in discriminatory dose-based relative growth categories) did not change notably, and no evidence of other resistance genotypes was found. Analysis of the upstream Mf CYP51 gene region in the resistant isolate CF010 revealed an insertion sequence described for the first time in this report. Our study suggests that current fungicide spray programs have been effective against increasing resistance levels in populations of M. fructicola and suppressing development of new resistant genotypes of the pathogen., Competing Interests: The author(s) declare no conflict of interest.

Cytospora plurivora D.P. Lawr., L.A. Holland & Trouillas has been associated with recent premature peach tree decline in South Carolina, but very little is known about the pathogen or chemical control options. Ninety-three C. plurivora isolates were collected in 2016 and 2017 from 1-year-old peach wood and symptomatic scaffold limbs, respectively, from orchards in six towns in South Carolina. Six unique genotypes were identified based on substantial ITS1-5.8S-ITS2 sequence variability and classified G1 to G6. Three of the genotypes (G2, G3, and G6) were isolated in high frequency in multiple locations of both years. In addition to the genotypic variation, multiple phenotypes were observed between and within genotype groups. Species identity was determined using additional gene loci: ACT, TUB, and EF, and isolates were found to belong to C. plurivora for all genotype groups. All tested genotypes were sensitive to thiophanate-methyl (FRAC 1) but exhibited slightly lower sensitivity to propiconazole and difenoconazole (both FRAC 3). Boscalid, fluopyram (both FRAC 7s), azoxystrobin, and pyraclostrobin (both FRAC 11s) were ineffective in vitro at inhibiting mycelial growth of C. plurivora genotypes. Field inoculation of peach and nectarine trees revealed that all genotypes developed twig cankers with differences in virulence. G1 was most virulent, and G6 was least virulent. This study provides a link between the C. plurivora genetic variability and virulence and provides fungicide sensitivity information that could be used to improve disease management practices., Competing Interests: The author(s) declare no conflict of interest.

Isoprothiolane (IPT) resistance has emerged in Magnaporthe oryzae, due to the long-term usage of IPT to control rice blast in China, yet the mechanisms of the resistance remain largely unknown. Through IPT adaptation on PDA medium, we obtained a variety of IPT-resistant mutants. Based on their EC50 values to IPT, the resistant mutants were mainly divided into three distinct categories, i.e., low resistance (LR, 6.5 ≤ EC50 < 13.0 μg/mL), moderate resistance 1 (MR-1, 13.0 ≤ EC50 < 25.0 μg/mL), and moderate resistance 2 (MR-2, 25.0 ≤ EC50 < 35.0 μg/mL). Molecular analysis of MoIRR (Magnaporthe oryzae isoprothiolane resistance related) gene demonstrated that it was associated only with the moderate resistance in MR-2 mutants, indicating that other mechanisms were associated with resistance in LR and MR-1 mutants. In this study, we mainly focused on the characterization of low resistance to IPT in M. oryzae. Mycelial growth and conidial germination were significantly reduced, indicating fitness penalties in LR mutants. Based on the differences of whole genome sequences between parental isolate and LR mutants, we identified a conserved MoVelB gene, encoding the velvet family transcription factor, and genetic transformation of wild type isolate verified that MoVelB gene was associated with the low resistance. Based on molecular analysis, we further demonstrated that the velvet family proteins VelB and VeA were indispensable for IPT toxicity and the deformation of the VelB-VeA-LaeA complex played a vital role for the low IPT-resistance in M. oryzae, most likely through the down-regulation of the secondary metabolism-related genes or CYP450 genes to reduce the toxicity of IPT., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Meng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 EC 50 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., (© 2019 Society of Chemical Industry.)

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 E198K 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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A survey of fungal pathogens causing twig blight on commercial peach trees was conducted in South Carolina in the fall of 2016. Shoots with cankers, pycnidia, and dieback were collected from six locations around the state. Isolates obtained from these samples were identified as Botryosphaeria obtusa, Phomopsis amygdali, Leucostoma persoonii, and Cytospora sp., based on colony morphology, conidia size and shape, and ribosomal DNA sequence analysis. L. persoonii was the most prevalent species and was isolated in five of the six locations, followed by P. amygdali and B. obtusa. The sensitivity of representative isolates of B. obtusa, P. amygdali, and L. persoonii to fungicides of different FRAC codes was evaluated. All species tested were sensitive to thiophanate-methyl (FRAC 1) and pyraclostrobin and azoxystrobin (both FRAC 11), whereas all species were resistant to boscalid and fluopyram (both FRAC 7). P. amygdali and B. obtusa were sensitive to difenoconazole and propiconazole (both FRAC 3), whereas L. persoonii was moderately resistant. L. persoonii was the most virulent species based on expansion of mycelium in the cambium layer of 2-year-old, detached twig pieces. Bacterial spot (BS)-sensitive cultivar 'O'Henry' was most susceptible to B. obtusa compared with BS-sensitive 'Summerprince', brown rot (BR)-resistant 'Contender', and BR-sensitive 'Coronet' but was least susceptible to L. persoonii. Coronet was most susceptible to L. persoonii. There were no significant differences between susceptibility of the cultivars to P. amygdali. This study established that L. persoonii is currently the most frequent twig blight pathogen in South Carolina, perhaps owing to its superior fitness. Some fungicides were effective in controlling all twig blight pathogens and may therefore be useful for chemical management strategies. Our study also provides the first evidence that the genetic basis of resistance to BS and BR in peach trees is not necessarily linked to tolerance to wood pathogens.

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

Monilinia fructicola is a destructive pathogen causing brown rot on stone fruits worldwide. Though it is best known as a fruit rot pathogen, M. fructicola also causes blossom blight and, subsequently, twig cankers in the spring. Orchard management strategies often overlook cankers as an inoculum source, though they are an inoculum source of both blossom and fruit infections. In this study, we analyzed the role of cankers as storage structures for diverse genotypes of M. fructicola, examining whether multiple genotypes can be transmitted from blossom to canker. Fungal spores from blossoms, and 2 months later from their corresponding cankers, were collected from a conventional and an unsprayed orchard in 2015 and 2016. Simple sequence repeat markers were used to genotype 10 to 20 single spores from each of four blossom/canker pairs per orchard. Individual blossoms and cankers were detected containing up to four and five genotypes, respectively. The average number of genotypes in blossoms and corresponding cankers were not significantly different (P = 0.690) across both years and farms, showing that a bottleneck for genetic diversity was not generated during the transition from blossom to canker. The average number of genotypes unique to blossom or canker was not significantly different (P = 0.569) and no significant effect of farm (P = 0.961) or year (P = 0.520) was observed, although blossoms had a numerically greater number of unique genotypes in both cases. In conclusion, a single blossom may be infected by one or more genotypes of M. fructicola, and this diversity is being preserved in the corresponding canker. This information implicates M. fructicola cankers as diversity storehouses, and may also apply to other Monilinia spp. and fungal diseases that initiate in reproductive tissue.

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.

Background: Monilinia fructicola is a diverse pathogen of pome and stone fruits that causes severe economic losses each year. However, little is known about inoculum flow within or between orchards and pathogen establishment in an orchard, because few methods exist for detecting diversity or tracking isolates over time. SSR loci are an effective option, but may be confounded by a high degree of mutability and potential sensitivity to abiotic stress., Results: Through transcriptome analysis, we identified novel markers mrr1, DHFR and MfCYP01 and validated stability of these markers under fungicide stress in natural infection sites. Nucleotide variation within mrr1, DHFR and MfCYP01 sequences differentiated isolates at all spatial scales: within the same infection site, between trees and between two farms. Sequenced regions were also effective for matching isolates collected from blossoms at the beginning of the season to progeny in cankers obtained at the end of the season., Conclusions: Collectively, results show that mrr1, DHFR and MfCYP01 are able to accurately differentiate M. fructicola isolates at the population level, can be used to track isolates over time, and are more stable than SSRs under external stresses. Either by themselves or combined with SSR markers, these gene-encoding regions are a much-needed tool for better understanding M. fructicola population dynamics. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

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

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.

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

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 MfCYP51 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 MfCYP51 promoter region was not observed. More research is warranted to determine whether fungicide-induced mutagenesis may also happen under field conditions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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

In this study, 145 peaches and nectarines displaying typical brown rot symptoms were collected from multiple provinces in China. A subsample of 26 single-spore isolates were characterized phylogenetically and morphologically to ascertain species. Phylogenetic analysis of internal transcribed spacer (ITS) regions 1 and 2, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), β-tubulin (TUB2) revealed the presence of three distinct Monilinia species. These species included Monilinia fructicola, Monilia mumecola, and a previously undescribed species designated Monilia yunnanensis sp. nov. While M. fructicola is a well-documented pathogen of Prunus persica in China, M. mumecola had primarily only been isolated from mume fruit in Japan. Koch's postulates for M. mumecola and M. yunnanensis were fulfilled confirming pathogenicity of the two species on peach. Phylogenetic analysis of ITS, G3PDH, and TUB2 sequences indicated that M. yunnanensis is most closely related to M. fructigena, a species widely prevalent in Europe. Interestingly, there were considerable differences in the exon/intron structure of the cytochrome b (Cyt b) gene between the two species. Morphological characteristics, including spore size, colony morphology, lesion growth rate, and sporulation, support the phylogenetic evidence suggesting the designation of M. yunnanensis as a new species. A new multiplex PCR method was developed to facilitate the detection of M. yunnanensis and differentiation of Monilinia spp. causing brown rot of peach in China.

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

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.

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

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

Brown rot, caused by Moniliniafructicola (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-alpha 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. The alternative oxidase gene MfAOX1 from M fructicola was cloned and gene expression was analyzed. The MfAOX1 gene was 1077 bp in length and contained two introns of 54 and 67 bp. The amino acid sequence was 63.8, 63.8 and 57.7% identical to alternative oxidases from Venturia inaequalis (Cooke) Winter, Aspergillus niger van Teighem and A nidulans, respectively. MfAOX1 expression in some but not all M fructicola isolates was induced in mycelia treated with azoxystrobin. Azoxystrobin at 2 microg ml(-1) significantly induced MfAOX1 expression in isolates with low MfAOX1 constitutive expression levels.