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.
The fitness and the dynamics of demethylation inhibitor fungicide (DMI) sensitivity in isolates of Monilinia fructicola sensitive (no growth at 0.3 mg/liter propiconazole) and resistant (>/=50% relative growth at 0.3 mg/liter propiconazole) to propiconazole were investigated. Overall, there was no considerable compromise in the fitness of resistant isolates compared to sensitive isolates of M. fructicola at the time of collection. Resistant and sensitive isolates differed in their sensitivity to propiconazole (P < 0.001) and incubation period (P = 0.044), but not in latent period, growth rate, spore production, and spore germination frequency (P > 0.05). Consecutive transferring on potato dextrose agar had an impact on conidia production, conidial germination, and growth rate (P < 0.0001). Consecutive transferring also had an impact on propiconazole sensitivity in resistant isolates. In the resistant isolates, sensitivity to propiconazole increased (R(2) = 0.960, P = 0.0034) within the first eight transfers. Similarly, sensitivity to propiconazole increased by 273% over the course of 34 months in cold storage in propiconazole-resistant isolates. Our results show that propiconazole resistance is unstable in vitro and that standard subculturing and cold storage procedures impact propiconazole sensitivity of resistant isolates. The instability of propiconazole resistance in M. fructicola may have important implications for disease management in that a reversion to propiconazole sensitivity could potentially occur in the absence of DMI fungicide pressure in the field.
The involvement of overexpression of the CYP51A1 gene in Venturia inaequalis was investigated for isolates exhibiting differential sensitivity to the triazole demethylation inhibitor (DMI) fungicides myclobutanil and difenoconazole. Relative expression (RE) of the CYP51A1 gene was significantly greater (P < 0.0001) for isolates with resistance to both fungicides (MRDR phenotype) or with resistance to difenoconazole only (MSDR phenotype) compared with isolates that were resistant only to myclobutanil (MRDS phenotype) or sensitive to both fungicides (MSDS phenotype). An average of 9- and 13-fold increases in CYP51A1 RE were observed in isolates resistant to difenoconazole compared with isolates with MRDS and MSDS phenotypes, respectively. Linear regression analysis between isolate relative growth on myclobutanil-amended medium and log10 RE revealed that little to no variability in sensitivity to myclobutanil could be explained by CYP51A1 overexpression (R(2) = 0.078). To investigate CYP51A1 upstream anomalies associated with CYP51A1 overexpression or resistance to difenoconazole, Illumina sequencing was conducted for three isolates with resistance to difenoconazole and one baseline isolate. A repeated element, "EL 3,1,2", with the properties of a transcriptional enhancer was identified two to four times upstream of CYP51A1 in difenoconazole-resistant isolates but was not found in isolates with the MRDS phenotype. These results suggest that different mechanisms may govern resistance to different DMI fungicides in the triazole group.
Sterol demethylation inhibitor (DMI) fungicide resistance in isolates of Monilinia fructicola from Georgia has been linked to overexpression of the MfCYP51 gene and a corresponding 65-bp genetic element ‘Mona’ inserted in the upstream region of MfCYP51. In this study, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to detect the Mona element. Fourteen DMI fungicide-resistant (DMI-R) and six DMI fungicide-sensitive (DMI-S) isolates from Georgia, six DMI-R and 11 DMI-S isolates from South Carolina, seven DMI-R and nine DMI-S isolates from New York, and two DMI-R and three DMI-S isolates from Ohio were used in this study. The isolates from the southeastern United States and Ohio were collected from peach, whereas isolates from New York were collected from cherry. A 376-bp fragment containing the Mona element was consistently amplified with primer pair INS65-F and INS65-R from DMI-R isolates, and either a 311-bp or 1,815-bp fragment was amplified from DMI-S isolates. The primer pair did not amplify DNA fragments of similar sizes from isolates of five other common fruit rot pathogens of peach, including Alternaria alternata, Colletotrichum acutatum, Gilbertella persicaria, Penicillium expansum, and Rhizopus stolonifer. Gel electrophoresis of the PCR amplicon can distinguish between DMI-R and DMI-S isolates based on the 65-bp size difference of the amplicon; however, the restriction digestion assay can verify questionable results, especially in the absence of a positive control. Only the 376-bp fragment containing the Mona element was digestable with endonuclease BsrBI, resulting in two restriction fragments of 236 and 140 bp in size. In this study, a protocol for Mona detection from aerial fungal structures was developed that can yield results within a few hours of sampling. This study confirms that the Mona element is strongly linked to the DMI-resistance phenotype and reveals that overexpression of the MfCYP51 gene is a common DMI fungicide resistance mechanism in M. fructicola, not only in Georgia but throughout the eastern United States.
Results suggest that resistance to QoI fungicides based on the G143A mutation is not likely to occur in M. fructicola or M. laxa. Conversely, M. fructigena may be at higher risk for developing QoI resistance owing to the absence of a G143-associated intron.
Xylosandrus germanus (Blandford) has caused increasing damage in high-density New York apple orchards since 2013, resulting in tree decline and death. We documented their occurrence and timing in > 50 orchards using ethanol-baited traps from 2014 to 2016. First captures ranged from 48 to 83 degree days (base 10 °C) from 1 January. Captures were numerically higher at the orchard-woods interface than within the orchard interior, but differences were not significant in locations with lower populations. Control using insecticide trunk sprays was tested in potted, waterlogged apple trees placed in orchards and nurseries, and inside wooded areas adjacent to orchards. A verbenone repellent was used in combination with trunk sprays to improve control. Overall, insecticide sprays were inconsistent and marginal in preventing new infestations. Chlorpyrifos significantly reduced infestations versus lambda-cyhalothrin and untreated trees at one location in the 2015 orchard trials, and versus untreated trees at one location in the 2016 nursery trials, but otherwise performed no better than other treatments. The addition of verbenone to either the check or permethrin treatments resulted in significantly fewer attack sites containing brood at one orchard site in 2016. Chlorpyrifos, lambda-cyhalothrin, and permethrin significantly reduced the number of attack sites containing adults compared with untreated trees at one nursery trial location in 2016, but were otherwise ineffective in reducing numbers of trees in other locations and infestation categories. We found several fungal and bacterial species associated with X. germanus and its infestation of apples. These microbes likely play a minimal role in apple decline.
Resistance to streptomycin in Erwinia amylovora was first observed in the United States in the 1970s but was not found in New York until 2002, when streptomycin-resistant (SmR) E. amylovora was isolated from orchards in Wayne County. From 2011 to 2014, in total, 591 fire blight samples representing shoot blight, blossom blight, and rootstock blight were collected from 80 apple orchards in New York. From these samples, 1,280 isolates of E. amylovora were obtained and assessed for streptomycin resistance. In all, 34 SmR E. amylovora isolates were obtained from 19 individual commercial orchards. The majority of the resistant isolates were collected from orchards in Wayne County, and the remaining were from other counties in western New York. Of the 34 resistant isolates, 32 contained the streptomycin resistance gene pair strA/strB in the transposon Tn5393 on the nonconjugative plasmid pEA29. This determinant of streptomycin resistance has only been found in SmR E. amylovora isolates from Michigan and the SmR E. amylovora isolates discovered in Wayne County, NY in 2002. Currently, our data indicate that SmR E. amylovora is restricted to counties in western New York and is concentrated in the county with the original outbreak. Because the resistance is primarily present on the nonconjugative plasmid, it is possible that SmR has been present in Wayne County since the introduction in 2002, and has spread within and out of Wayne County to additional commercial growers over the past decade. However, research is still needed to provide in-depth understanding of the origin and spread of the newly discovered SmR E. amylovora to reduce the spread of streptomycin resistance into other apple-growing regions, and address the sustainability of streptomycin use for fire blight management in New York.
Demethylation inhibitors (DMIs) are a class of single-site fungicides with high levels of protective and curative efficacy against Venturia inaequalis, the causal agent of apple scab. To determine the prevalence of resistance to the DMI fungicide myclobutanil, 3,987 single-lesion conidial V. inaequalis isolates from 141 commercial, research, and baseline orchard populations were examined throughout New England, the mid-Atlantic, and the Midwest from 2004 to 2013. Of these orchard populations, 63% had practical resistance, 13% had reduced sensitivity, and 24% were sensitive to myclobutanil. A sensitivity baseline for the recently introduced DMI fungicide difenoconazole was established to make comparisons with myclobutanil sensitivity in orchard populations. The mean effective concentration of difenoconazole at which mycelial growth was inhibited by 50% (EC50) was determined to be 0.002 μg ml−1 for 44 baseline isolates of V. inaequalis. From 2010 to 2013, 1,012 isolates of V. inaequalis from 37 of the 141 orchard populations above were screened for sensitivity to difenoconazole. In all, 1 orchard population had reduced sensitivity to difenoconazole, while the remaining 36 orchard populations were sensitive to the fungicide. In field experiments, difenoconazole demonstrated high levels of apple scab control on mature apple fruit, despite the fact that the population of V. inaequalis had practical resistance to difenoconazole. Although our results indicate widespread resistance to myclobutanil but not difenoconazole, due to the propensity for cross-sensitivity among DMI fungicides, growers with myclobutanil resistance should be cautious when using difenoconazole for disease management.
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