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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]

Quantitative (partial) and qualitative (complete) resistance responses to quinone outside inhibitor (Qol) fungicides have been documented for the apple scab pathogen Venturia inaequalis. Resistance monitoring efforts have traditionally focused on the detection of qualitative resistance based on a single point mutation, G143A, within the cytochrome b (cyt b) gene. In order to better understand the role of heteroplasmy of the cyt b gene in the Qol resistance response for isolates and populations of V inaequalis, an allele-specific quantitative polymerase chain reaction was developed to quantify the relative abundance of the A143 (resistant) allele in 45 isolates of V. inaequalis with differing in vitro resistance responses to the Qol fungicide trifloxystrobin. Although a high relative abundance of the A143 allele (>62%) was associated with isolates with high resistance responses (50 to 100% relative growth on trifloxystrobin-amended medium), heteroplasmy of the cyt b gene was not the primary factor involved in isolates with moderate resistance responses (29 to 49% relative growth). The relative abundance of the A143 allele in isolates with moderate resistance to trifloxystrobin rarely exceeded 8%, suggesting that other resistance mechanisms are involved in moderate resistance and, therefore, that the Qol resistance response is polygenic. In research orchards where Qol fungicides failed to control apple scab (practical resistance), field trials were conducted to demonstrate the link between practical resistance and the abundance of the A143 allele. Relative abundance of the A143 allele in these orchard populations exceeded 20% in 2011 and 2012. Similarly, of the eight additional commercial orchards screened in 2011, the relative abundance of the A143 allele always exceeded 20% in those with Qol practical resistance. Although heteroplasmy of the cyt b gene did not entirely explain the response of isolates with moderate resistance to Qols, the relative abundance of A143 in orchard populations of V inaequalis helps to explain the point of emergence for practical resistance to trifloxystrobin across several orchard populations with differing production histories. [ABSTRACT FROM AUTHOR]

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), b-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. [ABSTRACT FROM AUTHOR]

Demethylation inhibitor (DMI) resistant populations of Monilinia fructicola, the causal agent of brown rot of stone fruit, and the presence of the genetic DMI resistance determinant 'Mona' have been reported throughout the eastern United States. In this study, we endeavored to conduct a comprehensive investigation of DMI sensitivity, the prevalence of 'Mona', and implications of DMI use for M. fructicola populations from New York and Pennsylvania. Of the 18 orchards surveyed, only 9 were primarily composed of isolates with either resistance or reduced sensitivity to fenbuconazole and propiconazole. The DMI resistance determinant 'Mona' was only found in 5 orchards, present in isolates with a range of sensitivity phenotypes, and not always present in resistant isolates. These results suggested that 'Mona' only contributes to a portion of the quantitative resistance response to DMI fungicides. On detached blossoms and fruit, protective applications of fenbuconazole (Indar 2F) against isolates with resistance or reduced sensitivity resulted in significantly (P < 0.05) lower brown rot incidence compared to applications of propiconazole (Orbit 3.6EC) and water controls. By comparison, therapeutic applications of fenbuconazole and propiconazole against isolates with resistance or reduced sensitivity provided little to no reduction in brown rot incidence on blossoms and fruit. [ABSTRACT FROM AUTHOR]

Biotechnology has several advantages over conventional breeding for the precise engineering of gene function and provides a powerful tool for the genetic improvement of agronomically important traits in crops. In particular, it has been exploited for the improvement of multiple traits through the simultaneous introduction or stacking of several genes driven by distinct tissue-specific promoters. Since transcriptional enhancer elements have been shown to override the specificity of nearby promoters in a position- and orientation-independent manner, the co-existence of multiple enhancers/promoters within a single transgenic construct could be problematic as it has the potential to cause the mis-expression of transgene product(s). In order to develop strategies with, which to prevent such interference, a clear understanding of the mechanisms underlying enhancer-mediated activation of target promoters, as well as the identification of DNA sequences that function to block these interactions in plants, will be necessary. To date, little is known concerning enhancer function in plants and only a very limited number of enhancer-blocking insulators that operate in plant species have been identified. In this review, we discuss the current knowledge surrounding enhancer-promoter interactions, as well as possible means of minimizing such interference during plant transformation experiments. [ABSTRACT FROM AUTHOR]

Currant cane dieback, caused by the fungus Botryosphaeria ribis (syn. Neofusicoccum ribis), devastated currant production in the northeastern United States approximately a century ago. Later epidemics of this disease were halted by bans on the planting of Ribes spp. to control white pine blister rust, but since regulations restricting the planting of currants were removed in the 1980s and 1990s, currant cane dieback has reemerged in the northeastern United States. In this study, we obtained fungal isolates from diseased canes at four production operations in New York and Connecticut. We confirmed the isolates as N. ribis on the basis of morphology and ribosomal DNA sequence analysis, and verified pathogenicity through inoculation of healthy currant bushes. Furthermore, field trials to study the effects of cultivar, pruning of symptomatic canes, and fungicide applications were conducted over 2 years. Differences in Ribes cultivar susceptibility to N. rihis were demonstrated, with a pink fruiting cultivar (Pink Champagne) having lower levels of dieback compared to two red cultivars (Rovada and Jonkheer van Tets) and one white cultivar (Blanka) evaluated. While there were no consistent reductions in cane dieback incidence over both years and for all cultivars when pruning or fungicide treatments (copper hydroxide and sulfur applications) were used alone, fungicide treatment combined with pruning resulted in significant (P < 0.05) reductions (>80%) in the incidence of dieback compared to bushes not treated with fungicide or pruned. [ABSTRACT FROM AUTHOR]

Management of brown rot of stone fruit relies upon the application of effective fungicides that may be compromised by the development of fungicide resistance. We evaluated fungicide resistance in the brown rot pathogen, Moniliniafructicola, using Alamar blue (AB) dye, or resazurin, a chromogenic substrate that can be used as an indicator of respiration, in a 96-well microtiter format. We compared the AB method to traditional mycelial growth assays for resistance screening using 10 isolates of M. fructicola that represented a range of sensitivities to fenbuconazole. Using traditional mycelial growth assays, isolate sensitivity ranged from 17.7 to 115.3% growth on medium amended with fenbuconazole at 0.03 μg/mI relative to that on nonamended medium. Concordant results between both assays were obtained (R² = 0.9943, P < 0.0001), but the AB method provided results within 24 h, as opposed to the 3- to 5-day period required for mycelial growth assays. We found that sensitive isolates reduced AB less than resistant isolates in the presence of fungicide. Spore density influenced the reduction of AB by M. fructicola; spectrophotometric discrimination of fungicide sensitivity was best achieved at a density of 105 spores/mI. [ABSTRACT FROM AUTHOR]

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

Fire blight, caused by Erwinia amylovora, is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause disease. Compounds that inhibit the function of the T3SS (T3SS inhibitors) have emerged as alternative strategies for bacterial plant disease management, as they block bacterial virulence without affecting growth, unlike traditional antibiotics. In this study, we investigated the mode of action of a T3SS inhibitor named TS 108, a plant phenolic acid derivative, in E. amylovora. We showed that adding TS 108 to an in vitro culture of E. amylovora repressed the expression of several T3SS regulon genes, including the master regulator gene hrpL. Further studies demonsttated that TS 108 negatively regulates CsrB, a global regulatory small RNA, at the posttranscriptional level, resulting in a repression of hrpS, which encodes a key activator of hrpL. Additionally, TS 108 has no impact on the expression of T3SS in Dickeya dadantii or Pseudomonas aeruginosa, suggesting that its inhibition of the E. amylovora T3SS is likely species specific. To better evaluate the performance of T3SS inhibitors in fire blight management, we conducted five independent field experiments in four states (Michigan, New York, Oregon, and Connecticut) from 2015 to 2022 and observed reductions in blossom blight incidence as high as 96.7% compared with unseated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight. [ABSTRACT FROM AUTHOR]

Nighttime applications of germicidal UV light (UV-C) have been used to suppress several fungal diseases of plants, but less is known of UV-C's potential to suppress bacterial plant pathogens. Fire blight of apple and pear, caused by the bacterium Erwinia amylovora, is difficult to suppress using cultural practices, antibiotics, and host resistance. We therefore investigated the potential of UV-C as an additional means to manage the disease. Laboratory assays confirmed that in vitro exposure of cultures E. amylovora to UV-C at doses ranging from 0 to 400 J/m² in the absence of visible light was more than 200% as effective as cultures exposed to visible light after the same UV-C treatments. In a 2-year orchard study, we demonstrated that with only two nighttime applications of UV-C at 200 J/m² made at bloom resulted in an incidence of blossom blight and shoot blight equivalent to the results viewed when antibiotic and biopesticide commercial standards were applied. In vitro dose-response studies indicated consistency in pathogen response to suppressive UV-C doses, including pathogen isolates that were resistant to streptomycin. Based on these results, UV-C may be useful in managing bacterial populations with antibiotic resistance. Concurrent measurements of host growth after UV-C applications indicated that the dose required to suppress E. amylovora had no significant (P > 0.05) effects on foliar growth, shoot extension, internode length, or fruit finish but substantially reduced epiphytic populations of E. amylovora on host tissues. [ABSTRACT FROM AUTHOR]

Paecilomyces rot of apples is a postharvest disease caused by the thermotolerant fungus Paecilomyces niveus (Byssochlamys nivea). The etiology of disease and the activity of fungicides against P. niveus are not yet well understood. This study evaluated the ability of P. niveus to infect 'Gala' apples growing in a conventionally managed orchard. In addition, the sensitivity of P. niveus isolates to postharvest fungicides ditenoconazole, tiudioxonil, and pyrimethanil was characterized for isolates from both agricultural and non-agricultural environments. Apples were wounded and mock-inoculated or inoculated with P. niveus in early July. At the time of harvest, 8 weeks after wounding, the inoculated apples had significantly larger lesions than mock-inoculated apples (P < 0.005). The average diameter of lesions on wound- inoculated apples was 11.17 mm ± 6.82 SD, while the average diameter of mock-inoculated lesions was 3.34 mm ± 1.85 SD. Disease symptoms in the orchard were similar to postharvest symptoms of Paecilomyces rot. Symptoms included a brown, flattened, circular lesion with faint concentric rings. The necrosis of mesocarp was firm and roughly U-shaped. Baseline isolates of P. niveus, from nonagricultural environments, were used to determine the effective fungicide dose at which growth was inhibited by 50% (EC50). Furthermore. fungicide sensitivity of P. niveus isolates was examined using relative growth assays at the mean baseline EC50 values to compare baseline isolates with isolates obtained from commercial apple orchards where they were likely exposed to fungicides. Among the exposed isolates, reduced sensitivity to all fungicides was observed, but significant differences between baseline and exposed isolates were only observed with fludioxonil (P < 0.0001). This is the first report demonstrating that P. niveus can infect apples that are wound-inoculated in the orchard and that isolates from agricultural environments are less sensitive to common fungicides, especially fludioxonil. This finding may have implications for the control of this postharvest diseases. Whether natural infections of apples by P. niveus is initiated in the orchard or during postharvest has yet to be determined. [ABSTRACT FROM AUTHOR]

The succinate dehydrogenase inhibiting (SDHI) fungicides are a class of single-site fungicides that are increasingly important in the management of Venturia inaequalis. In this study, the baseline sensitivity of V. inaequalis to penthiopyrad, fluopyram, and benzovindiflupyr was investigated. In all, 35 to 70 isolates with no prior exposure to single-site fungicides were used to determine the effective concentration at which growth was inhibited by 50% (EC50). Mean EC50 values for the conidial germ tube growth stage for penthiopyrad, fluopyram, and benzovindiflupyr were 0.086, 0.176, and 0.0016 μg ml-1, respectively. Linear correlation analysis revealed a significant and positive correlation between fluopyram and penthiopyrad (P ⩽ 0.0001, r = 0.66) and fluopyram and benzovindiflupyr (P = 0.0014, r = 0.52). Baseline sensitivities of V. inaequalis during the mycelial growth stage were also determined for fluopyram and benzovindiflupyr. EC50 values were higher for fluopyram and benzovindiflupyr during this stage compared with the conidial germ tube growth stage, with means of 0.043 and 2.02 μg ml-1, respectively. In addition, the sdhB gene was characterized for three isolates of V. inaequalis collected from a research, baseline, and commercial orchard population. No common mutation sites associated with SDHI resistance in other phytopathogenic fungi were discovered in these isolates or isolates that were recovered following field applications of SDHI fungicides. The results of this study suggest that SDHI fungicides have a high level of activity during the conidial germ tube elongation stage in V. inaequalis and provide a basis for phenotypic and genotypic monitoring of shifts toward resistance of V. inaequalis populations to the SDHI fungicide class. [ABSTRACT FROM AUTHOR]

Demethylation inhibitor (DMI) fungicides are an effective means to manage apple scab caused by Venturia inaequalis. Unfortunately, practical resistance to DMI fungicide chemistries is prevalent in populations in New York and the New England states. Management practices that delay the development of DMI resistance in V. inaequalis populations are highly desired by regional apple producers. Trials were conducted in a New York apple orchard during the 2011 and 2012 growing seasons to determine the impact of delayed-dormant (after bud break, but prior to green tissue) chemical treatments on the DMI sensitivity of a V. inaequalis population with stable resistance to DMI fungicides. Delayed-dormant treatment programs consisted of either an application of a copper fungicide, a manganese sanitation product, a DMI fungicide (myclobutanil), or no fungicide. Sensitivity to the DMI fungicide myclobutanil was evaluated for a minimum of 25 V. inaequalis single lesion conidial isolates from each of four replicated treatment blocks. In both years, mean percent relative growth on myclobutanil amended media for V. inaequalis isolates from the copper treatment program were significantly (P < 0.05) lower than isolates from blocks did not receive a delayed dormant fungicide treatment. The effect of the manganese treatment was inconsistent between years. V. inaequalis isolates collected from the myclobutanil treatment program were not significantly (P > 0.05) different in myclobutanil sensitivity from isolates collected from the blocks that did not receive a delayed dormant fungicide treatment. Overall, the results suggest that delayed dormant treatments of copper may favorably impact the myclobutanil sensitivity for a population of V. inaequalis with resistance to DMI fungicides, and should be considered as a standard management practice in apple production. [ABSTRACT FROM AUTHOR]

Background: Blue mold is a globally important and economically impactful postharvest disease of apples caused by multiple Penicillium spp. There are currently four postharvest fungicides registered for blue mold control, and some isolates have developed resistance manifesting in decay on fungicide-treated fruit during storage. To date, mechanisms of fungicide resistance have not been explored in this fungus using a transcriptomic approach. Results: We have conducted a comparative transcriptomic study by exposing naturally-occurring difenoconazole (DIF) resistant (G10) and sensitive (P11) blue mold isolates to technical grade difenoconazole, an azole fungicide in the commercial postharvest product Academy (Syngenta Crop Protection, LLC). Dynamic changes in gene expression patterns were observed encompassing candidates involved in active efflux and transcriptional regulators between the resistant and sensitive isolates. Unlike other systems, 3 isoforms of cytochrome P450 monoxygenase (CYP51A-C) were discovered and expressed in both sensitive and resistant strains upon difenoconazole treatment. Active efflux pumps were coordinately regulated in the resistant isolate and were shown to mediate the global resistance response as their inhibition reversed the difenoconazole-resistant phenotype in vitro. Conclusions: Our data support the observation that global transcriptional changes modulate difenoconazole resistance in Penicillium spp. While the dogma of CYP51 overexpression is supported in the resistant isolate, our studies shed light on additional new mechanisms of difenoconazole resistance on a global scale in Penicillium spp. These new findings broaden our fundamental understanding of azole fungicide resistance in fungi, which has identified multiple genetic targets, that can be used for the detection, management, and abatement of difenoconazole-resistant blue mold isolates during long-term storage of apples. [ABSTRACT FROM AUTHOR]

Understanding how fungicide application practices affect selection for fungicide resistance is imperative for continued sustainable agriculture. Here, we examined the effect of field applications of the succinate dehydrogenase inhibitor (SDHI) fluxapyroxad at different doses and mixtures on the SDHI sensitivity of Venturia inaequalis, the apple scab pathogen. Fungicide applications were part of selection programs involving different doses (high or low) and mixtures (with a second single-site fungicide or a multisite fungicide). These programs were tested in two apple orchards over 4 years to determine potential cumulative selection effects on resistance. Each year after program applications, apple scab lesions were collected, and relative growth assays were conducted to understand shifts in fluxapyroxad sensitivity. After 4 years, there was a trend toward a reduction in sensitivity to fluxapyroxad for most selection programs in comparison to that in the non-selective-pressure control. In most years, the selection program plots treated with low-dose fluxapyroxad applications resulted in a larger number of isolates with reduced sensitivity, supporting the use of higher doses for disease management. Few significant differences (P < 0.05) in fungicide sensitivity were observed between isolates collected from plots where fungicide mixtures were applied compared to that in untreated plots, supporting the use of multiple modes of action in field applications. In all, appropriate doses and mixtures may contribute to increased longevity of SDHI fungicides used on perennial crops like apples. [ABSTRACT FROM AUTHOR]

Herbivorous insects may benefit from avoiding the smell produced by phytopathogens infecting plant host tissue if the infected tissue reduces insect fitness. However, in many cases the same species of phytopathogen can also infect host plant tissues that do not directly affect herbivore fitness. Thus, insects may benefit from differentiating between pathogen odors emanating from food and nonfood tissues. This is based on the hypothesis that unnecessarily staying attentive to pathogen odor from nonfood tissue may incur opportunity costs associated with not responding to other important survival functions. In this study adults of Drosophila suzukii Matsumura, an invasive larval frugivore, showed reduced attraction to the odor of raspberry fruit, a food tissue, when infected with Botrytis cinerea Pers., a ubiquitous phytopathogen, in favor of odors of uninfected raspberry fruit. Moreover, D. suzukii oviposited fewer eggs on infected raspberry fruit relative to uninfected raspberry fruit. Larval survival and adult size after eclosion were significantly reduced when reared on B. cinerea‐infected raspberry relative to uninfected fruit. Interestingly, when the behavioral choice experiment was repeated using Botrytis‐infected vs. ‐uninfected strawberry leaves, a nonfood tissue, in combination with fresh raspberry fruit, odor from B. cinerea‐infected leaves did not reduce D. suzukii attraction to raspberries relative to raspberries with uninfected leaves. These behavioral results illustrate the important role context can play in odor‐mediated interactions between insects, plants and microbes. We discuss implications of our findings for developing a repellent that can be useful for the management of D. suzukii. [ABSTRACT FROM AUTHOR]

The grape berry moth (GBM), Paralobesia viteana (Clemens) (Lepidoptera: Tortricidae), is a specialist pest insect of cultivated grape, Vitis spp. (Vitaceae), in the eastern USA. A blend of volatile compounds has been isolated from plant material that attracts female GBM in the flight tunnel and field. However, the origin of the volatile cue is potentially complicated by the presence of microbes (bacteria and fungi) living on the surface of the plant. Microbial volatile organic compounds can affect insect behavior, and therefore must be considered to fully understand olfaction‐mediated behaviors. We report here the chemical and behavioral analysis of the volatile profiles produced from both the sanitized and control shoot treatments. The sanitization treatment removed 96.4% of the surface microbes up to 24 h, covering the duration of the behavioral assays and volatile collections. Overall, the surface microbes did not significantly contribute to the volatile profile of the grape shoots, as all of the peaks in the volatile profile of sanitized shoots were found in the profile of control shoots. In flight tunnel assays, female GBM displayed the same level of upwind oriented flight to sanitized shoots (flew upwind 57.4%, landed 30.9%) as they did to control shoots (flew upwind 57.8%, landed 31.0%), suggesting further that surface microbes did not contribute to the production of the previously identified blend of behaviorally active volatiles for GBM. [ABSTRACT FROM AUTHOR]

Penicillium spp. cause blue mold of stored pome fruit. These fungi reduce fruit quality and produce mycotoxins that are regulated for processed fruit products. Control of blue mold is achieved by fungicide application, and in 2015 Academy (active ingredients fludioxonil and difenoconazole) was released for use on pome fruit to manage postharvest blue mold. Baseline sensitivity for fludioxonil but not difenoconazole has been determined for P. expansum. To establish the distribution of sensitivity to difenoconazole before commercial use of Academy, 97 unexposed single-spore isolates from the United States and abroad were tested in vitro. Baseline EC50 values ranged from 0.038 to 0.827 µg/ml of difenoconazole with an average of 0.16 µg/ml. Complete inhibition of mycelial growth for all but three isolates occurred at 5 µg/ml of difenoconazole, whereas 10 µg/ml did not support growth for any of the isolates examined. Hence, 5 µg/ml of difenoconazole is recommended for phenotyping Penicillium spp. isolates with reduced sensitivity. Isolates with resistance to pyrimethanil and to both thiabendazole and pyrimethanil were observed among the isolates from the baseline collection. Academy applied at the labeled rate had both curative and protectant activities and controlled four representative Penicillium spp. from the baseline population. This information can be used to monitor future shifts in sensitivity to this new postharvest fungicide in Penicillium spp. populations. [ABSTRACT FROM AUTHOR]

The apple scab pathogen, Venturia inaequalis, is among the most economically important fungal pathogens that affects apples. Fungicide applications are an essential part of disease management. Implementation of cultural practices and genetic sources of resistance in the host are vital components of scab management. This is the first presentation of multiple, high quality, well-annotated genomes of four North American V. inaequalis isolates having both sensitive and multiple fungicide resistance phenotypes. We envision that these isolates will enable investigations into fungicide resistance mechanisms, exploring fungal virulence factors and delineating phylogenomic relationships among apple scab isolates from around the world. [ABSTRACT FROM AUTHOR]

Erwinia amylovora, the causal agent of fire blight, causes considerable economic losses in young apple plantings in New York on a yearly basis. Nurseries make efforts to only use clean budwood for propagation, which is essential, but E. amylovora may be present in trees that appear to have no apparent fire blight symptoms at the time of collection. We hypothesized that the use of infected budwood, especially by commercial nursery operations, could be the cause, in part, of fire blight outbreaks that often occur in young apple plantings in New York. Our goal was to investigate the presence of E. amylovora in asymptomatic budwood from nursery source plantings as it relates to trees with fire blight symptoms. From 2012 to 2015, apple budwood was collected from two commercial budwood source plantings of 'Gala' and 'Topaz' at increasing distances from visually symptomatic trees. From these collections, internal contents of apple buds were analyzed for the presence of E. amylovora. E. amylovora was detected in asymptomatic budwood in trees more than 20 m from trees with fire blight symptoms. In some seasons, there were significant (P ≤ 0.05) differences in the incidence of E. amylovora in asymptomatic budwood collected from symptomatic trees and those up to 20 m from them. In 2014 and 2015, the mean E. amylovora CFU per gram recovered from budwood in both the Gala and Topaz plantings were significantly lower in budwood collected 20 m from symptomatic trees. Further investigation of individual bud dissections revealed that E. amylovora was within the tissue beneath the bud scales containing the meristem. Results from the study highlight the shortcomings of current budwood collection practices and the need to better understand the factors that lead to the presence of E. amylovora in bud tissues to ensure the production of pathogen-free apple trees. [ABSTRACT FROM AUTHOR]

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 (MSDS 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To determine the impact of white pine blister rust (WPBR) following the recent breakdown of the Cr resistance dominant gene in cultivated Ribes spp., 255 plants of 19 Ribes cultivars and 445 neighboring eastern white pine (Pinus strobus) from 42 sites across New Hampshire were evaluated. Of the 19 Ribes cultivars evaluated, 15 were WPBR resistant, and 4 of these were labeled as black currant (Ribes nigrum) with the Cr gene (Cr Ribes cultivars). Incidence of WPBR ranged from 0 to 88% for WPBR-resistant Ribes cultivars. Mean WPBR severity was 14 and <6% of leaf area for Cr Ribes and partially resistant cultivars, respectively. The presence of Cronartium ribicola was confirmed on 17 of the 19 Ribes cultivars screened with polymerase chain reaction analysis and DNA sequencing. Reference accessions of Cr Ribes cultivars from the Canadian Clonal Genebank were successfully infected with C. ribicola inoculum collected in New Hampshire from Cr Ribes cultivars and P. strobus, confirming that the vCr race of C. ribicola that has overcome the Cr resistance dominant gene in cultivated Ribes spp. is present in New Hampshire. The probability of finding pine trees with WPBR was greater for trees neighboring infected Cr Ribes cultivars (0.18) than trees neighboring WPBR-free Ribes cultivars (0.02). Results from this study suggest that the breakdown of Cr-based resistance in Ribes spp. poses a threat to the white pine resource and to cultivated Ribes production. [ABSTRACT FROM AUTHOR]

Quinone-outside-inhibitor (Qol) fungicides are a safe and effective means of managing apple scab caused by Venturia inaequalis. To determine the prevalence of both quantitative (partial) and qualitative (complete) Qol resistance in V. inaequalis in the northeastern United States, we sampled single-lesion conidial isolates (n = 4,481 ) from 120 commercial and research orchards from 2004 to 2011 with a range of exposure to Qol fungicides from none to several applications a year. In all, 67% of these orchard populations of V. inaequalis were sensitive to Qol fungicides, 28% exhibited Qol practical resistance, and 5% were not sensitive Qol fungicides but had not become practically resistant. Isolates with qualitative Qol resistance, conferred by the G143A cytochrome b gene mutation, were found in 13 of the 34 Qol-resistant orchard populations. To evaluate the stability of the G143A mutation, 27 isolates were selected from different orchard populations to represent the scope of regional populations. These isolates were subcultured continuously in the presence or absence of the Qol fungicide trifloxystrobin. All isolates that initially possessed qualitative resistance maintained the resistant genotype (Gl43A) for six transfers over 6 months in both the absence and presence of trifloxystrobin. Given the observed Qol resistance in orchard populations of V. inaequalis and the stability of the G143A mutation in individual isolates, apple scab management paradigms must encompass strategies to limit selection of Qol resistance in the sensitive orchard populations remaining in the region. [ABSTRACT FROM AUTHOR]