Applications of Germicidal Ultraviolet Light as a Tool for Fire Blight Management (Erwinia amylovora) in Apple Plantings.

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.

Multisite field evaluation of bacteriophages for fire blight management: incorporation of UVR protectants, and impact on the apple flower microbiome.

Fire blight, a disease of pome fruits caused by the bacterium Erwinia amylovora, has become increasingly difficult to manage after the emergence of streptomycin-resistant strains. Alternative antibiotics and copper are available; however, these chemicals have use restrictions in some countries and also can carry risks of phytotoxicity. Therefore, there is growing interest in biological-based management options, with bacteriophage (phages) showing promise, as these naturally occurring pathogens of bacteria are easy to isolate and grow. However, there are several technical challenges regarding the implementation of phage biocontrol in the field as the viral molecules suffer from ultraviolet radiation (UVR) degradation and can die off rapidly in the absence of the host bacterium. In this work we assessed the efficacy of Erwinia phages and a commercial phage product for blossom blight control in the field across multiple locations in the eastern United States. In these tests, disease control ranged from 0.0 to 82.7%, and addition of a UVR protectant only resulted in significantly increased disease control in 2 of 12 tests. We also analyzed microbial community population changes in response to phage application. Changes in bacterial community diversity metrics over time were not detected, however relative abundances of target taxa were temporarily reduced after phage applications, indicating that these phage applications did not have deleterious effects on the flower microbiome. We have demonstrated that biological control of fire blight with phages is achievable, but a better understanding of phage:pathogen dynamics is required to optimize disease control efficacy.

Erwinia amylovora Type III Secretion System Inhibitors Reduce Fire Blight Infection Under Field Conditions.

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 TS108, a plant phenolic acid derivative, in E. amylovora. We showed that adding TS108 to an in vitro culture of E. amylovora repressed the expression of several T3SS regulon genes, including the master regulator gene hrpL. Further studies demonstrated that TS108 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, TS108 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 untreated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight.

Refining Management of Apple Powdery Mildew in New York State with Weather-Based Fungicide Application Timing Programs.

In the absence of durable host resistance among commercial cultivars, chemical management continues to be an essential component of disease control in apple production. Apple powdery mildew, caused by the ascomycete Podosphaera leucotricha, is largely managed with regular fungicide applications from the host phenological stages of tight cluster to terminal bud growth set, with applications typically being made in a prophylactic manner irrespective of existing disease pressure. Here we evaluated two management programs that aligned fungicide applications to specific weather thresholds conducive to powdery mildew development using a rotation of single-site fungicides and sulfur. In three separate orchards among four cultivars, we compared powdery mildew disease progression over the growing season for each of the weather factor-based programs and a typical calendar-based application program. In each year of the trial, we found that management programs with weather-based fungicide applications provided levels of disease control similar to the calendar program but required 50 to 83.3% fewer mildew-specific fungicide applications throughout the growing season. Our results provide a framework with which to evaluate future weather-based management programs for apple powdery mildew management. This knowledge could be implemented in the creation of a powdery mildew disease management decision support system to better inform and aid fungicide application programs for continued sustainable apple production in the northeast United States.

Would You Like Wood or Plastic? Bin Material, Sanitation Treatments, and Bin Inoculum Levels Impact Blue Mold Decay of Stored Apple Fruit.

Blue mold, caused primarily by Penicillium expansum, is a significant postharvest disease of apples. It not only causes economic losses but also produces mycotoxins that contaminate processed fruit products, which contributes to food waste and loss. Previous research has shown that packing and storage bins harbor Penicillium spores and that steam and hot water efficiently reduce spore inoculum levels. However, studies using wooden and plastic bins regarding their ability to harbor spores, the effect of chemical sanitation treatments on spore levels, and the impact of rinsate from treated bins on apple fruit decay have not been investigated for the Mid-Atlantic area (Okull et al. 2006; Rosenberger 2009). We evaluated different sanitation treatments (chemical and physical) to reduce P. expansum inoculum levels on wooden and plastic bins. We determined that wooden bins bound P. expansum spores four orders of magnitude higher than plastic. When both bin types were treated with steam (wooden) or sterile hot water (plastic), Thyme Guard, or Academy, all treatments resulted in significantly (P < 0.05) lower spore levels compared to untreated controls. Although, plastic bins retained lower numbers of spores after inoculation with contaminated spore rinsate and required much higher concentrations of P. expansum spores in rinsate to retain spores at levels that would lead to decay on apple fruit. Overall, we demonstrated that plastic bins retain fewer spores than wooden bins and that both can be sanitized by various physical or chemical treatments. We envision that our findings will be applicable in the future as the techniques implemented in this study were used to investigate industry-relevant questions. Our goal is that the research techniques and findings become feasible with advancements in technology and/or accompany other shifts in existing processes in commercial pome fruit packing and storage facilities.

Assessment of Fungicide Resistance via Molecular Assay in Populations of Podosphaera leucotricha, Causal Agent of Apple Powdery Mildew, in New York.

Podosphaera leucotricha, causal agent of apple powdery mildew, is a pathogen endemic worldwide where apples are produced. In the absence of durable host resistance, the disease is most effectively managed in conventional orchards with single-site fungicides. In New York State, increasingly erratic precipitation patterns and warmer temperatures due to climate change may create a regional environment more conducive to apple powdery mildew development and spread. In this scenario, outbreaks of apple powdery mildew may supplant the apple diseases of current management concern: apple scab and fire blight. Presently, there have been no reports from producers of fungicide control failures for apple powdery mildew, though increased disease incidence has been reported to and observed by the authors. As such, action was needed to assess the fungicide resistance status of populations of P. leucotricha to ensure key classes of single-site fungicides (FRAC 3, demethylation inhibitors, DMI; FRAC 11, quinone outside inhibitors, QoI; and FRAC 7, succinate dehydrogenase inhibitors, SDHI) remain effective. In a 2-year survey (2021 to 2022), we collected 160 samples of P. leucotricha from 43 orchards, representing conventional, organic, low-input, and unmanaged orchards from New York's primary production regions. Samples were screened for mutations in the target genes (CYP51, cytb, and sdhB) historically known to confer fungicide resistance in other fungal pathogens to the DMI, QoI, and SDHI fungicide classes, respectively. Across all samples, no nucleotide sequence mutations that translated into problematic amino acid substitutions were found in the target genes, suggesting that New York populations of P. leucotricha remain sensitive to the DMI, QoI, and SDHI fungicide classes, provided no other fungicide resistance mechanism is at play in the population.

Optimizing Use of DMI Fungicides for Management of Apple Powdery Mildew Caused by Podosphaera leucotricha in New York State.

Powdery mildew, caused by the ascomycete Podosphaera leucotricha, is an endemic disease found wherever apples are grown that reduces both tree vigor and fresh market yield. In the absence of durable host resistance, chemical management is the primary means of disease control. Demethylation inhibitor (DMI) fungicides are widely used to manage apple powdery mildew, but members within this fungicide class have been observed to differ in efficacy with respect to disease control. Moreover, debate exists as to the optimal timing of DMI fungicide applications for management of apple powdery mildew. In this regard, the goal of this study was to determine the best-use practices for DMI fungicides to manage apple powdery mildew in New York State. Multiyear trials were conducted to evaluate the potential differential efficacy performance of four common DMI fungicides, and additional trials were conducted to assess optimal application timing. In all years, we observed that treatments of flutriafol and myclobutanil consistently had the lowest incidences of powdery mildew compared with difenoconazole and fenbuconazole. In the 2018 and 2021 trials, the newly registered mefentrifluconazole was more comparable to the difenoconazole program with respect to powdery mildew disease incidence. We hypothesize that differences in DMI efficacy may result from each fungicide's water solubility and lipophilicity characteristics and thus their ability to move systemically in the host or more easily penetrate the surface of germinating conidia. Applications timed between petal fall and first cover resulted in the lowest incidence of powdery mildew on terminal leaves of apple shoots compared with applications timed before petal fall. These observations are contrary to previous studies conducted in regions with differing climates. We also found that the incidence of secondary powdery mildew observed 2 weeks after petal fall was influenced by applications of DMI fungicides during the previous season. For example, management programs consisting of applications of flutriafol or myclobutanil in the previous season tended to have lower incidence of apple powdery in the next spring, presumably because of reductions in overwintering inoculum. Despite reports of DMI resistance in other apple pathosystems, the DMI fungicide class is still relevant for the successful management of apple powdery mildew in New York State.

Assessing and Minimizing the Development and Spread of Fire Blight Following Mechanical Thinning and Pruning in Apple Orchards.

The adoption of mechanical thinning and pruning in commercial apple orchards has been limited largely by the risk of development and spread of fire blight. This devastating disease, caused by the bacterial pathogen Erwinia amylovora, may be transmitted by mechanical injury such as pruning, especially under warm, moist conditions conducive to bacterial growth, infection, and disease development. However, risk may be mitigated by avoiding highest-risk times and applying a bactericide, such as streptomycin, after mechanical thinning or pruning. In 'Gala' and 'Idared' orchards, we evaluated the risk of fire blight development and spread after mechanical thinning early in bloom (20% bloom), when seasonal temperatures are cooler and there are few open flowers available for infection. In both orchards, we also evaluated the spread and development of fire blight by mechanical pruning in July and in August, before and after terminal bud set, when shoot growth is slowed and the tree is less susceptible to infection. We also assessed the potential efficacy of a streptomycin or Bacillus subtilis biopesticide application after mechanical thinning and pruning to mitigate the spread of fire blight. In the 'Gala' orchard, disease never developed beyond the inoculated tree after thinning or pruning, which was unexpected for this highly susceptible cultivar. In the 'Idared' orchard, incidence of blossom or shoot blight from the point source, represented as relative area under the disease progress curve, was rarely different for trees that underwent mechanical thinning or mechanical pruning compared with untreated trees, and it was often reduced or eliminated when the antibiotic streptomycin or the B. subtilis biopesticide was applied within 24 h of mechanical thinning or pruning. For both thinning and pruning, incidence of fire blight dropped off quickly beyond the inoculated tree in the 'Idared' orchard and generally was not observed in trees beyond 10 to 15 m from the inoculated point source or predicted beyond 10 m by exponential and power law models fit to the disease progress curves. The results of this work demonstrate the low risk for fire blight development and spread by mechanical thinning and pruning when practiced under low-risk conditions-early in bloom for mechanical thinning and after terminal bud set (in August) for mechanical pruning-especially when paired with a subsequent bactericide application. This study demonstrates the safe use of mechanical thinning and pruning in commercial apple production, corroborated by anecdotal evidence from apple growers in western New York State.

Investigating the Distribution of Strains of Erwinia amylovora and Streptomycin Resistance in Apple Orchards in New York Using Clustered Regularly Interspaced Short Palindromic Repeat Profiles: A 6-Year Follow-Up.

Fire blight, caused by the bacterium Erwinia amylovora, is one of the most important diseases of apple. The antibiotic streptomycin is routinely used in the commercial apple industries of New York (NY) and New England to manage the disease. In 2002 and again, from 2011 to 2014, outbreaks of streptomycin resistance (SmR) were reported and investigated in NY. Motivated by new grower reports of control failures, we conducted a follow-up investigation of the distribution of SmR and E. amylovora strains for major apple production regions of NY over the last 6 years (2015 to 2020). Characterization of clustered regularly interspaced short palindromic repeat (CRISPR) profiles revealed that a few "cosmopolitan" strains were widely prevalent across regions, whereas many other "resident" strains were confined to one location. In addition, we uncovered novel CRISPR profile diversity in all investigated regions. SmR E. amylovora was detected only in a small area spanning two counties from 2017 to 2020 and was always associated with one CRISPR profile (41:23:38), which matched the profile of SmR E. amylovora, discovered in 2002. This suggests the original SmR E. amylovora was never fully eradicated and went undetected because of several seasons of low disease pressure in this region. Investigation of several representative isolates under controlled greenhouse conditions indicated significant differences in aggressiveness on 'Gala' apples. Potential implications of strain differences include the propensity of strains to become distributed across wide geographic regions and associated resistance management practices. Results from this work will directly influence sustainable fire blight management recommendations for commercial apple industries in NY state and other regions.

Global transcriptomic responses orchestrate difenoconazole resistance in Penicillium spp. causing blue mold of stored apple fruit.

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.

The Effects of Succinate Dehydrogenase Inhibitor Fungicide Dose and Mixture on Development of Resistance in Venturia inaequalis.

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.IMPORTANCE Of much debate is the effect of fungicide application dose on resistance development, as fungicide resistance is a critical barrier to effective disease management in agricultural systems. Our field study in apples investigated the effect of fungicide application dose and mixture on the selection of succinate dehydrogenase inhibitor resistance in Venturia inaequalis, a fungal pathogen that causes the economically important disease apple scab. Understanding how to best delay the development of resistance can result in increased efficacy, fewer applications, and sustainable fungicide use. Results from this study may have relevance to other perennial crops that require multiple fungicide applications and that are impacted by the development of resistance.

A Genome Resource for Several North American Venturia inaequalis Isolates with Multiple Fungicide Resistance Phenotypes.

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.

Management of Fire Blight Using Pre-bloom Application of Prohexadione-Calcium.

Fire blight, a bacterial disease of rosaceous plants caused by Erwinia amylovora, is one of the most important diseases affecting commercial apple production worldwide. Antibiotics, applied at bloom to protect against blossom infection, are the most effective means of management but raise concern due to the potential for antibiotic resistance in both the pathogen population and nontarget organisms. In addition, most fire blight outbreaks in New York State often emerge in late June to July as shoot blight, calling into question the role of blossom infections and the antibiotic applications made to manage them. Prohexadione-calcium (PhCa) is a gibberellic acid inhibitor used post-bloom to control shoot vigor and to manage shoot blight. However, the magnitude of shoot blight management is directly related to the suppression of shoot growth, which is undesirable, especially in young orchards during establishment years. PhCa is believed to control shoot blight by thickening cell walls in cortical parenchyma, preventing invasion of host tissues by E. amylovora. We hypothesize that PhCa applied pre-bloom could similarly prevent invasion of blossom pedicels following infection, leading to reduced disease incidence. We evaluated novel pre-bloom PhCa programs for their effects on disease management (blossom and shoot blight) as well as their impact on shoot growth for three years in a mature 'Gala' orchard in New York. In all three years of the study, all PhCa programs resulted in less than 27% incidence (71% control) of blossom blight and less than 13% incidence (77% control) of shoot blight with minimal effect on tree growth. Inclusion of a biopesticide during bloom further reduced the incidence of blossom blight in one year of three. Using light microscopy, we found that cell walls in the cortical parenchyma of fruitlet pedicels on trees receiving pre-bloom PhCa applications were significantly thicker than those of untreated trees 40 days after full bloom and inoculation. Overall, we found that pre-bloom applications of PhCa had utility in reducing blossom blight and shoot blight with minimal impacts on tree growth. These pre-bloom programs would fit with standard production practices and may contribute toward the development of fire blight management programs without the use of antibiotics.

Characterization of the VisdhC and VisdhD Genes in Venturia inaequalis, and Sensitivity to Fluxapyroxad, Pydiflumetofen, Inpyrfluxam, and Benzovindiflupyr.

Succinate dehydrogenase inhibitors (SDHI) are an important class of fungicides for management of apple scab, especially as resistance to other classes of fungicides has become prevalent in the northeastern United States. Considering their single-site mode of action, there is high risk of resistance development to SDHI fungicides. Such risk mandates the need for proper monitoring of shifts in population sensitivity. This study aims to provide a means for phenotypic and genotypic characterization of SDHI fungicide resistance for Venturia inaequalis, the causal agent of apple scab. To complement the published sequence of VisdhB, target genes VisdhC and VisdhD were identified using sequences of homologous genes in other fungal organisms and a draft genome of V. inaequalis. Using mycelial growth and conidial germination assays, baseline sensitivities and cross sensitivities of V. inaequalis were determined for several SDHI fungicides. Mean baseline EC50 values for conidial germination of benzovindiflupyr, fluxapyroxad, pydiflumetofen, and inpyrfluxam were found to be 0.0021, 0.0284, 0.014, and 0.0137 µg ml-1, respectively. Mean baseline EC50 values for mycelial growth of benzovindiflupyr, fluxapyroxad, pydiflumetofen, and inpyrfluxam were found to be 0.0575, 0.228, 0.062, and 0.0291 µg ml-1, respectively. A significant and positive correlation in sensitivity was found between benzovindiflupyr, fluxapyroxad, pydiflumetofen, and inpyrfluxam as well as penthiopyrad and fluopyram, with the highest correlation between benzovindiflupyr and penthiopyrad for mycelial inhibition of V. inaequalis (r = 0.950, P < 0.001). For inhibition of conidial germination, the highest correlation was observed between penthiopyrad and fluopyram (r = 0.775, P < 0.001). Furthermore, the sequences of the VisdhC and VisdhD genes were identified and characterized for baseline isolates of V. inaequalis. Residues of similar position to mutations found in other systems that confer resistance to SDHI fungicides were identified in baseline isolates, but no mutations were identified in baseline isolates or those previously exposed to SDHI fungicides. This study will serve as a reference for future monitoring of resistance to SDHI fungicides in V. inaequalis at both a phenotypic and genotypic level.

Paecilomyces niveus: Pathogenicity in the Orchard and Sensitivity to Three Fungicides.

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 difenoconazole, fludioxonil, and pyrimethanil was characterized for isolates from both agricultural and nonagricultural 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.

Baseline Sensitivity of Penicillium spp. to Difenoconazole.

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.

Fire Blight Symptomatic Shoots and the Presence of Erwinia amylovora in Asymptomatic Apple Budwood.

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.

Resistance to Increasing Chemical Classes of Fungicides by Virtue of "Selection by Association" in Botrytis cinerea.

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.

Overexpression of the CYP51A1 Gene and Repeated Elements are Associated with Differential Sensitivity to DMI Fungicides in Venturia inaequalis.

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.

Molecular Characterization of the sdhB Gene and Baseline Sensitivity to Penthiopyrad, Fluopyram, and Benzovindiflupyr in Venturia inaequalis.

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.