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Objective: To describe a standard approach to provide a support structure for pharmacy resident research that emphasizes self-identification of a residency research project. Methods: A subcommittee of the residency advisory committee was formed at our institution. The committee was initially comprised of 2 clinical pharmacy specialists, 1 drug information pharmacist, and 2 pharmacy administrators. The committee developed research guidelines that are distributed to residents prior to the residency start that detail the research process, important deadlines, and available resources. Instructions for institutional review board (IRB) training and deadlines for various assignments and presentations throughout the residency year are clearly defined. Residents conceive their own research project and emphasis is placed on completing assignments early in the residency year. Results: In the 4 years this research process has been in place, 15 of 16 (94%) residents successfully identified their own research question. All 15 residents submitted a complete research protocol to the IRB by the August deadline. Four residents have presented the results of their research at multi-disciplinary national professional meetings and 1 has published a manuscript. Feedback from outgoing residents has been positive overall and their perceptions of their research projects and the process are positive. Conclusion: Pharmacy residents selecting their own research projects for their residency year is a feasible alternative to assigning or providing lists of research projects from which to select a project.

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., Competing Interests: The author(s) declare no conflict of interest.

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., Competing Interests: The author(s) declare no conflict of interest.

The Global Plant Health Assessment (GPHA) is a collective, volunteer-based effort to assemble expert opinions on plant health and disease impacts on ecosystem services based on published scientific evidence. The GPHA considers a range of forest, agricultural, and urban systems worldwide. These are referred to as (Ecoregion × Plant System), i.e., selected case examples involving keystone plants in given parts of the world. The GPHA focuses on infectious plant diseases and plant pathogens, but encompasses the abiotic (e.g., temperature, drought, and floods) and other biotic (e.g., animal pests and humans) factors associated with plant health. Among the 33 (Ecoregion × Plant System) considered, 18 are assessed as in fair or poor health, and 20 as in declining health. Much of the observed state of plant health and its trends are driven by a combination of forces, including climate change, species invasions, and human management. Healthy plants ensure (i) provisioning (food, fiber, and material), (ii) regulation (climate, atmosphere, water, and soils), and (iii) cultural (recreation, inspiration, and spiritual) ecosystem services. All these roles that plants play are threatened by plant diseases. Nearly none of these three ecosystem services are assessed as improving. Results indicate that the poor state of plant health in sub-Saharan Africa gravely contributes to food insecurity and environmental degradation. Results further call for the need to improve crop health to ensure food security in the most populated parts of the world, such as in South Asia, where the poorest of the poor, the landless farmers, are at the greatest risk. The overview of results generated from this work identifies directions for future research to be championed by a new generation of scientists and revived public extension services. Breakthroughs from science are needed to (i) gather more data on plant health and its consequences, (ii) identify collective actions to manage plant systems, (iii) exploit the phytobiome diversity in breeding programs, (iv) breed for plant genotypes with resilience to biotic and abiotic stresses, and (v) design and implement plant systems involving the diversity required to ensure their adaptation to current and growing challenges, including climate change and pathogen invasions., Competing Interests: The author(s) declare no conflict of interest.

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/m 2 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/m 2 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., Competing Interests: The author(s) declare no conflict of interest.

The Threshold of Toxicological Concern (TTC) is an approach for assessing the safety of chemicals with low levels of exposure for which limited toxicology data are available. The original TTC criteria were derived for oral exposures from a distributional analysis of a dataset of 613 chemicals that identified 5 th percentile no observed effect level (NOEL) values grouped within three tiers of compounds having specific structural functional groups and/or toxic potencies known as Cramer I, II and III classifications. Subsequent assessments of the TTC approach have established current thresholds to be scientifically robust. While the TTC has gained acknowledgment and acceptance by many regulatory agencies and organizations, use of the TTC approach in evaluating drinking water chemicals has been limited. To apply the TTC concept to drinking water chemicals, an exposure-based approach that incorporates the current weight of evidence for the target chemical is presented. Such an approach provides a comparative point of departure to the 5 th percentile TTC NOEL using existing data, while conserving the allocation of toxicological resources for quantitative risk assessment to chemicals with greater exposure or toxicity. This approach will be considered for incorporation into NSF/ANSI/CAN 600, a health effects standard used in the safety evaluation of chemicals present in drinking water from drinking water contact additives and materials certified to NSF/ANSI/CAN 60 and 61, respectively.

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., Competing Interests: The author(s) declare no conflict of interest.

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., Competing Interests: The author(s) declare no conflict of interest.

Adult hearts are characterized by inefficient regeneration after injury, thus, the features that support or prevent cardiomyocyte (CM) proliferation are important to clarify. Diploid CMs are a candidate cell type that may have unique proliferative and regenerative competence, but no molecular markers are yet known that selectively identify all or subpopulations of diploid CMs. Here, using the conduction system expression marker Cntn2-GFP and the conduction system lineage marker Etv1Cre ERT2 , we demonstrate that Purkinje CMs that comprise the adult ventricular conduction system are disproportionately diploid (33%, vs. 4% of bulk ventricular CMs). These, however, represent only a small proportion (3%) of the total diploid CM population. Using EdU incorporation during the first postnatal week, we demonstrate that bulk diploid CMs found in the later heart enter and complete the cell cycle during the neonatal period. In contrast, a significant fraction of conduction CMs persist as diploid cells from fetal life and avoid neonatal cell cycle activity. Despite their high degree of diploidy, the Purkinje lineage had no enhanced competence to support regeneration after adult heart infarction.

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.

Microplastics (MPs) contamination in marine environments is of increasing concern, as plastic particles are globally ubiquitous across ecosystems. A large variety of aquatic taxa ingest MPs, but the extent to which animals accumulate and transfer MPs through food webs is largely unknown. In this study, we quantified MP uptake in bivalves, crabs, echinoderms, and fish feeding at different trophic levels at three sites on southern Vancouver Island. We paired stable-isotope food web analysis with MP concentrations in digestive tracts across all trophic levels and in fish livers. We then used Bayesian generalized linear mixed models to explore whether bioaccumulation and biomagnification were occurring. Our results showed that MPs (100-5000 μm along their longest dimension) are not biomagnifying in marine coastal food webs, with no correlation between the digestive tract or fish liver MP concentrations and trophic position of the various species. Ecological traits did, however, affect microplastic accumulation in digestive tracts, with suspension feeder and smaller-bodied planktivorous fish ingesting more MPs by body weight. Trophic transfer occurred between prey and predator for rockfish, but higher concentrations in full stomachs compared with empty ones suggested rapid excretion of ingested MPs. Collectively, our findings suggested the movement of MP through marine food webs is facilitated by species-specific mechanisms, with contamination susceptibility a function of species biology, not trophic position. Furthermore, the statistical methods we employ, including machine learning for classifying unknown particles and a probabilistic way to account for background contamination, are universally applicable to the study of microplastics. Our findings advance understanding of how MPs enter and move through aquatic food webs, suggesting that lower-trophic-level animals are more at risk of ingesting >100-μm MPs, relative to higher-trophic-level animals. Our work also highlights the need to advance the study of <100-μm MPs, which are still poorly understood and may need to be considered separately in ecological risk assessments., (© 2022 The Authors. Ecological Applications published by Wiley Periodicals LLC on behalf of The Ecological Society of America.)

Aims: Hydrogen sulfide (H2S) is a potent signalling molecule that activates diverse cardioprotective pathways by post-translational modification (persulfidation) of cysteine residues in upstream protein targets. Heart failure patients with reduced ejection fraction (HFrEF) exhibit low levels of H2S. Sulfide:quinone oxidoreductase (SQOR) catalyses the first irreversible step in the metabolism of H2S and plays a key role in regulating H2S-mediated signalling. Here, the aim of this study was to discover a first-in-class inhibitor of human SQOR and evaluate its cardioprotective effect in an animal model of HFrEF., Methods and Results: We identified a potent inhibitor of human SQOR (STI1, IC50 = 29 nM) by high-throughput screening of a small-molecule library, followed by focused medicinal chemistry optimization and structure-based design. STI1 is a competitive inhibitor that binds with high selectivity to the coenzyme Q-binding pocket in SQOR. STI1 exhibited very low cytotoxicity and attenuated the hypertrophic response of neonatal rat ventricular cardiomyocytes and H9c2 cells induced by neurohormonal stressors. A mouse HFrEF model was produced by transverse aortic constriction (TAC). Treatment of TAC mice with STI1 mitigated the development of cardiomegaly, pulmonary congestion, dilatation of the left ventricle, and cardiac fibrosis and decreased the pressure gradient across the aortic constriction. Moreover, STI1 dramatically improved survival, preserved cardiac function, and prevented the progression to HFrEF by impeding the transition from compensated to decompensated left ventricle hypertrophy., Conclusion: We demonstrate that the coenzyme Q-binding pocket in human SQOR is a druggable target and establish proof of concept for the potential of SQOR inhibitors to provide a novel therapeutic approach for the treatment of HFrEF., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

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.

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.

Apple scab is one of the most economically important diseases of apple in temperate production regions. In the absence of durable host resistance in commercially preferred cultivars, considerable applications of fungicides are needed to manage this disease. With the sequential development of resistance to nearly all classes of single-site fungicides in the apple scab pathogen Venturia inaequalis , synthetic multisite fungicides, such as mancozeb and captan, often comprise the core of chemical management programs for apple scab. Although these fungicides have demonstrable benefits for both disease and fungicide resistance management, the sustainability movement within agriculture aims to reduce reliance on such fungicides because of their broader environmental impacts. In this study, we establish a framework to enhance the feasibility of chemical management programs that do not rely on use of synthetic multisite protectant fungicides to manage apple scab. Specifically, we wish to evaluate chemical programs that integrate the biopesticide Bacillus subtilis QST 713 (Serenade Opti) in rotation with benzovindiflupyr (Aprovia), a single-site fungicide belonging to the class of succinate dehydrogenase inhibitors (SDHI), to circumvent the need for applications of synthetic multisite fungicides. During implementation of these programs, disease incidence data were taken at biweekly intervals. Regardless of the seasonal challenges presented in the 2 years of this study, when Bacillus subtilis QST 713 was used in place of captan and mancozeb mixtures, we did not observe any significant differences ( P > 0.05) in development of apple scab symptoms between any of the management programs for the vertical axis or super spindle orchards in either year. This potential for substituting synthetic multisite fungicides with biopesticides is best realized when the programs are used with a decision support system in a super spindle planting system, where trees have reduced canopy densities. This 2-year study shows the potential to achieve adequate disease control using the integration of SDHI fungicides and biological controls without the use of synthetic multisite fungicides.

Habitat heterogeneity shapes biological communities, a well-known process in terrestrial ecosystems but substantially unresolved within coral reef ecosystems. We investigated the extent to which coral richness predicts intra-family fish richness, while simultaneously integrating a striking aspect of reef ecosystems-coral hue. To do so, we quantified the coral richness, coral hue diversity, and species richness within 25 fish families in 74 global ecoregions. We then expanded this to an analysis of all reef fishes (4465 species). Considering coral bleaching as a natural experiment, we subsequently examined hue's contribution to fish communities. Coral species and hue diversity significantly predict each family's fish richness, with the highest correlations (> 80%) occurring in damselfish, butterflyfish, emperors and rabbitfish, lower (60-80%) in substrate-bound and mid-water taxa such as blennies, seahorses, and parrotfish, and lowest (40-60%) in sharks, morays, grunts and triggerfish. The observed trends persisted globally. Coral bleaching's homogenization of reef colouration revealed hue's contribution to maintaining fish richness, abundance, and recruit survivorship. We propose that each additional coral species and associated hue provide added ecological opportunities (e.g. camouflage, background contrast for intraspecific display), facilitating the evolution and co-existence of diverse fish assemblages., (© 2021. The Author(s).)

Microplastic particles (MPs) occur widely in aquatic ecosystems and are ingested by a wide range of organisms. While trophic transfer of MPs is known to occur, researchers do not yet fully understand the fate of MPs in food webs. We explored the factors influencing reported ingestion of MPs in marine and freshwater fishes by conducting a literature review of 123 studies published between January 2011 and June 2020. We used Bayesian generalized linear mixed models to determine whether MP ingestion by fishes varies by Food and Agricultural Organization fishing area, trophic level, body size, taxa, and study methodology. After accounting for methodology, strong regional differences were not present, although ingested MP concentrations were slightly different among some FAO areas. According to the reviewed studies, MP concentrations in fish digestive tracts did not increase with either trophic level or body size, suggesting that biomagnification of MPs did not occur, although larger fish were more likely to contain MPs. Researchers reported higher concentrations of MPs in clupeids compared with other commonly studied taxonomic families, which could be due to their planktivorous feeding strategy. Methodology played an influential role in predicting reported concentrations, highlighting the need to harmonize methods among studies., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

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.

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.

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., (Copyright © 2020 American Society for Microbiology.)

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., (© 2019 Institute of Zoology, Chinese Academy of Sciences.)

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.

Chronic disturbance can disrupt ecological interactions including the foundational symbiosis between reef-building corals and the dinoflagellate family Symbiodiniaceae. Symbiodiniaceae are photosynthetic endosymbionts necessary for coral survival, but many Symbiodiniaceae can also be found free-living in the environment. Since most coral species acquire new Symbiodiniaceae from the environment each generation, free-living Symbiodiniaceae represent important pools for coral symbiont acquisition. Yet, little is known about the diversity of, or impacts of disturbance on, free-living Symbiodiniaceae. To determine how chronic and pulse disturbances influence Symbiodiniaceae communities, we sampled three reef habitat compartments - sediment, water, and coral (Pocillopora grandis, Montipora aequituberculata, Porites lobata) - at sites exposed to different levels of chronic anthropogenic disturbance, before, during, and after a major storm. Almost no (4%) Symbiodiniaceae amplicon sequence variants (ASVs) were found in all three compartments, and over half were found uniquely in coral. Sites experiencing chronic disturbance were typically associated with higher symbiont beta diversity (i.e., variability and turnover) across reef habitat compartments. Pulse stress, from the storm, exhibited some influence on symbiont beta diversity but the effect was inconsistent. This suggests that in this ecosystem, the effects of chronic disturbance are more prominent than temporal variability during a pulse disturbance for shaping symbiont communities.

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.

Microplastics are ubiquitous across ecosystems, yet the exposure risk to humans is unresolved. Focusing on the American diet, we evaluated the number of microplastic particles in commonly consumed foods in relation to their recommended daily intake. The potential for microplastic inhalation and how the source of drinking water may affect microplastic consumption were also explored. Our analysis used 402 data points from 26 studies, which represents over 3600 processed samples. Evaluating approximately 15% of Americans' caloric intake, we estimate that annual microplastics consumption ranges from 39000 to 52000 particles depending on age and sex. These estimates increase to 74000 and 121000 when inhalation is considered. Additionally, individuals who meet their recommended water intake through only bottled sources may be ingesting an additional 90000 microplastics annually, compared to 4000 microplastics for those who consume only tap water. These estimates are subject to large amounts of variation; however, given methodological and data limitations, these values are likely underestimates.

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 EC 50 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 EC 50 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.

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 EC 50 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.

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% (EC 50 ). Furthermore, fungicide sensitivity of P. niveus isolates was examined using relative growth assays at the mean baseline EC 50 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.

Diversity estimates play a key role in ecological assessments. Species richness and abundance are commonly used to generate complex diversity indices that are dependent on the quality of these estimates. As such, there is a long-standing interest in the development of monitoring techniques, their ability to adequately assess species diversity, and the implications for generated indices. To determine the ability of substratum community assessment methods to capture species diversity, we evaluated four methods: photo quadrat, point intercept, random subsampling, and full quadrat assessments. Species density, abundance, richness, Shannon diversity, and Simpson diversity were then calculated for each method. We then conducted a method validation at a subset of locations to serve as an indication for how well each method captured the totality of the diversity present. Density, richness, Shannon diversity, and Simpson diversity estimates varied between methods, despite assessments occurring at the same locations, with photo quadrats detecting the lowest estimates and full quadrat assessments the highest. Abundance estimates were consistent among methods. Sample-based rarefaction and extrapolation curves indicated that differences between Hill numbers (richness, Shannon diversity, and Simpson diversity) were significant in the majority of cases, and coverage-based rarefaction and extrapolation curves confirmed that these dissimilarities were due to differences between the methods, not the sample completeness. Method validation highlighted the inability of the tested methods to capture the totality of the diversity present, while further supporting the notion of extrapolating abundances. Our results highlight the need for consistency across research methods, the advantages of utilizing multiple diversity indices, and potential concerns and considerations when comparing data from multiple sources.

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., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.)

Hydrogen sulfide (H 2 S) is an endogenously synthesized signaling molecule that is enzymatically metabolized in mitochondria. The metabolism of H 2 S maintains optimal concentrations of the gasotransmitter and produces sulfane sulfur (S 0 )-containing metabolites that may be functionally important in signaling. Sulfide:quinone oxidoreductase (SQOR) catalyzes the initial two-electron oxidation of H 2 S to S 0 using coenzyme Q as the electron acceptor in a reaction that requires a third substrate to act as the acceptor of S 0 . We discovered that sulfite is a highly efficient acceptor and proposed that sulfite is the physiological acceptor in a reaction that produces thiosulfate, a known metabolic intermediate. This model has been challenged by others who assume that the intracellular concentration of sulfite is very low, a scenario postulated to favor reaction of SQOR with a considerably poorer acceptor, glutathione. In this study, we measured the intracellular concentration of sulfite and other metabolites in mammalian tissues. The values observed for sulfite in rat liver (9.2 μM) and heart (38 μM) are orders of magnitude higher than previously assumed. We discovered that the apparent kinetics of oxidation of H 2 S by SQOR with glutathione as the S 0 acceptor reflect contributions from other SQOR-catalyzed reactions, including a novel glutathione:CoQ reductase reaction. We used observed metabolite levels and steady-state kinetic parameters to simulate rates of oxidation of H 2 S by SQOR at physiological concentrations of different S 0 acceptors. The results show that the reaction with sulfite as the S 0 acceptor is a major pathway in liver and heart and provide insight into the potential dynamics of H 2 S metabolism.

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.

Antibiotic applications are essential for fire blight management in the eastern United States. Recently, streptomycin-resistant Erwinia amylovora strains were found in New York. There are growing concerns that streptomycin resistance may develop from postbloom streptomycin applications in local orchards. Our goal was to investigate the impacts of increasing streptomycin and kasugamycin applications on bacterial epiphyte community composition and antibiotic resistance in the phyllosphere of 'Idared' apple plantings in 2014 and 2015. Rinsate samples from leaves treated with 0, 3, 5, and 10 applications of streptomycin and kasugamycin were collected to isolate, enumerate, and identify epiphytic bacterial species. The majority of isolated epiphytic bacteria were identified as Pantoea agglomerans and fluorescent Pseudomonas spp., whereas E. amylovora was rarely found. Overall, postbloom streptomycin use did not result in an increased recovery of streptomycin-resistant E. amylovora. However, other streptomycin-resistant epiphytes (P. agglomerans and Pseudomonas spp.) did increase with increasing streptomycin applications. Increasing kasugamycin applications reduced the overall number and percentage of streptomycin-resistant epiphytes in the phyllosphere, which has important implications regarding the use of kasugamycin in orchards where streptomycin resistance is a concern.

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.

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% (EC 50 ). Mean EC 50 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. EC 50 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.

Streptomycin is the most effective and widely used chemical control in the eastern United States for blossom blight of apple caused by Erwinia amylovora; however, resistance to this antibiotic has been a concern in New York since 2002. From 2011 to 2014, statewide collections of E. amylovora were conducted resulting in the isolation of streptomycin-resistant (SmR) E. amylovora from several commercial orchards. Further genetic analysis of isolates was necessary to understand the origins and the diversity of these bacteria. Clustered regularly interspaced short palindromic repeat (CRISPR) spacer sequencing was employed to explore the diversity and possible origins of New York SmR E. amylovora isolates. The spacer array CR1, CR2, and CR3 regions of 27 SmR E. amylovora isolates and 76 streptomycin-sensitive (SmS) E. amylovora isolates were amplified and subsequently sequenced, revealing 19 distinct CRISPR spacer profiles for New York isolates. The majority of SmR E. amylovora isolates had the same CRISPR profile as SmR E. amylovora isolates discovered in 2002. This may infer that eradication efforts in 2002 failed and the bacterial populations continued to spread throughout the state. Several CRISPR profiles for SmR E. amylovora were identical to SmS E. amylovora collected from the same orchards, leading to the hypothesis that resistance may be developing within New York. Profiles not unique to New York were identical to many isolates from the Midwestern, eastern, and western United States, implying that streptomycin resistance may be due to the introduction of SmR E. amylovora from other regions of the United States. The increased understanding as to how SmR E. amylovora isolates are introduced, evolve, or have become established afforded by CRISPR profiling has been useful for disease management and restricting the movement of streptomycin resistance in New York.

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.

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 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.

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% (EC 50 ) 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.

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.

Quantitative (partial) and qualitative (complete) resistance responses to quinone outside inhibitor (QoI) 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 QoI 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 QoI 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 QoI 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 QoI practical resistance. Although heteroplasmy of the cyt b gene did not entirely explain the response of isolates with moderate resistance to QoIs, 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.

Quinone-outside-inhibitor (QoI) 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) QoI 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 QoI fungicides from none to several applications a year. In all, 67% of these orchard populations of V. inaequalis were sensitive to QoI fungicides, 28% exhibited QoI practical resistance, and 5% were not sensitive QoI fungicides but had not become practically resistant. Isolates with qualitative QoI resistance, conferred by the G143A cytochrome b gene mutation, were found in 13 of the 34 QoI-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 QoI fungicide trifloxystrobin. All isolates that initially possessed qualitative resistance maintained the resistant genotype (G143A) for six transfers over 6 months in both the absence and presence of trifloxystrobin. Given the observed QoI 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 QoI resistance in the sensitive orchard populations remaining in the region.

The genetic transformation of plants has become a necessary tool for fundamental plant biology research, as well as the generation of engineered plants exhibiting improved agronomic and industrial traits. However, this technology is significantly hindered by the fact that transgene expression is often highly variable amongst independent transgenic lines. Two of the major contributing factors to this type of inconsistency are inappropriate enhancer-promoter interactions and chromosomal position effects, which frequently result in mis-expression or silencing of the transgene, respectively. Since the precise, often tissue-specific, expression of the transgene(s) of interest is often a necessity for the successful generation of transgenic plants, these undesirable side effects have the potential to pose a major challenge for the genetic engineering of these organisms. In this review, we discuss strategies for improving foreign gene expression in plants via the inclusion of enhancer-blocking insulators, which function to impede enhancer-promoter communication, and barrier insulators, which block the spread of heterochromatin, in transgenic constructs. While a complete understanding of these elements remains elusive, recent studies regarding their use in genetically engineered plants indicate that they hold great promise for the improvement of transgene expression, and thus the future of plant biotechnology.

In the eastern United States, Monilinia laxa (Aderh. & Ruhl.) Honey has only been reported on tart cherry in New York (NY) (1). As a result of considerable rain in May of 2009 and 2011, an ornamental planting of Kwanzan cherries in Middletown, Rhode Island (RI), a planting of sweet cherry cvs. Ulster, Hedelfingen, Sam, and Lapins in Lanesboro, Massachusetts (MA), and plantings of apricot cvs. Harcot and Hargrande in Albion, Aurora, and Geneva, NY, and Harogem in Lanesboro, MA developed severe shoot blight (>15 to 100% of first-year shoots). Blighted shoots were wilted with the blight encompassing the distal end and often extending into second-year tissue with a distinct sunken margin. Leaves on symptomatic shoots had flushed, but were blighted and light brown. Blossom spurs were often blighted and gummosis was frequently observed at the base. In these same years, sweet cherry cv. Black Gold in Walworth, NY and plum cv. Stanley in Olcott, NY developed severe fruit rot (35 to 70% incidence). Plantings suffering from fruit rot had fruit lesions that began as pale brown, soft lesions with indiscriminant margins that covered 15 to 85% of the fruit surface area. Many blighted spurs, shoot tissues, and infected fruit were sporulating with tan-to-buff colored conidia produced in chains. From each planting with shoot blight, shoot tips were removed for pathogen isolation. Sections of symptomatic shoots (5 cm long) were surface sterilized in 0.6% NaOCl for 1 min and rinsed in sterile dH 2 0. From plantings displaying blighted spurs or fruit rot, isolation was attempted directly from sporulating tissue. Cross sections of sterilized shoot tissue (3 mm thick) or tufts of sporulation from fruit and spurs were placed on potato dextrose agar amended with 50 μg/ml of streptomycin sulfate. After incubation at 24°C for 5 days, colonies with lobed margins, commonly described for M. laxa (4), were obtained. Several colonies resembling M. fructicola were isolated from all locations, but the majority of isolates from spurs and shoots resembled M. laxa. Conidia from both colony morphotypes were lemon shaped, but as expected, those from putative M. laxa isolates were smaller (10.75 × 12.0 μm) compared with those from putative M. fructicola isolates (15.75 × 18.25 μm) (4). Confirmation of M. laxa was further achieved by PCR amplification of the β-tubulin gene using M. laxa-specific primers as previously described (3). Pathogenicity of M. laxa isolates was proven by inoculating fruit of the stone fruit crop from which they were isolated as previously described (2). Fruit inoculated with M. laxa developed brown, soft sporulating lesions identical to the original observations, while those inoculated with water remained healthy. M. laxa was reisolated from symptomatic shoots and spurs, but not from water-inoculated tissues. The presence of M. laxa has been reported on tart cherries in NY (1), but to our knowledge, this is the first instance of economically devastating shoot blight on apricot in NY and MA, ornamental cherry in RI, and sweet cherry in MA and fruit rot on sweet cherry and plum in NY caused by M. laxa. In wet seasons, stone fruit growers may need to revise their chemical management programs to better prepare for M. laxa epidemics on several stone fruit species. References: (1) K. D. Cox and S. M. Villani. Plant Dis. 94:783, 2010. (2) K. D. Cox and S. M. Villani. Plant Dis. 95:828, 2011. (3) Z. Ma et al. Pest Manag. Sci. 61:449, 2005. J.M. (4) G. C. M. van Leeuwen and H. A. van Kesteren. Can. J. Bot. 76:2042, 1998.

Since the relaxation of federal sanctions on the planting of Ribes crops because of the development of white pine blister rust (WPBR) immune Ribes cultivars (3), a small industry for the production of Ribes fruit for fresh and processing markets was established in New York and surrounding New England states. The majority of Ribes acreage in the region is planted to a WPBR immune black currant R. nigrum cv. Titania. From 2008 to June 2011, symptoms resembling those caused by WPBR pathogen Cronartium ribicola were observed at a large (>12 ha) R. nigrum cv. Titania planting in Preston, CT. In 2008, infection was restricted to a single field (100% incidence), but in 2009, all fields suffered from premature defoliation by late July. In 2010 and 2011, there was considerable incidence (>85%), but premature defoliation was kept in check by chemical management. Symptoms began as chlorotic lesions (0.5 to 4.3 mm in diameter) on both sides of the leaf. These chlorotic lesions had margins delineated by leaf veins and many developed necrotic (0.3 to 0.9 mm in diameter) centers on the upper surface of leaves within 2 to 3 weeks. The undersides of lesions developed blisters containing orange uredinia (0.1 to 0.33 mm in diameter) with smooth peridia that broke with the production of yellow-orange urediniospores (30 × 15 to 25 μm). Symptoms and signs were consistent with published descriptions of C. ribicola (1) and with WPBR infections on highly susceptible R. nigrum cv. Ben Alder planted at the New York State Agricultural Experiment Station in Geneva. Additional confirmation was provided by sequencing the two internal transcribed spacer (ITS) regions and the 5.8S gene (GenBank Accession No. JN587805; 98% identity with No. DQ533975) in the nuclear ribosomal repeat using primers ITS1-F and ITS4 as described previously (2,4). Furthermore, an attempt was made to confirm pathogenicity in the greenhouse by inoculating shoots of potted nursery stock of R. nigrum cv. Titania. Shoots were inoculated by rubbing leaves with either an uninfected currant leaf or a currant leaf from Preston, CT sporulating with urediniospores. Plants were subsequently misted with dH20 and covered with plastic bags for 24 h. Plants were watered biweekly and kept in a greenhouse with 39 to 65% relative humidity at 21 to 26°C. Shoots were monitored for symptom development on a weekly basis. After 3 weeks, 2 of the 10 plants inoculated with infected leaves developed chlorotic lesions and uredinia identical to those on leaves from Preston, CT, while all others remained healthy. Although not easily reproducible in a greenhouse, the breakdown of immunity in R. nigrum cv. Titania was observed for the last 4 years in Connecticut. Given that WPBR immunity was one of the conditions for legalized planting of Ribes, the breakdown of immunity has potentially deleterious implications particularly for nearby states like Massachusetts and New York, in which the Ribes industries are expanding. Moreover, Ribes growers may need to rely on chemical management programs to manage WPBR in the future. References: (1) G. B. Cummins. Illustrated Genera of Rust Fungi. Burgess Publishing Company, Minneapolis, MN, 1959. (2) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (3) S. McKay. Hortic. Technol. 10:562, 2000. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

Transcriptional enhancers possess the ability to override the tissue-specificity and efficiency of nearby promoters, which is of concern when generating transgenic constructs bearing multiple cassettes. One means of preventing these inappropriate interactions is through the use of enhancer-blocking insulators. The 2-kb transformation booster sequence (TBS) from Petunia hybrida has been shown previously to exhibit this function when inserted between an enhancer and promoter in transgenic Arabidopsis thaliana. In this study, we attempted to further characterize the ability of this fragment to impede enhancer-promoter interference through an analysis of transgenic Arabidopsis and Nicotiana tabacum lines bearing various permutations of the TBS element between the cauliflower mosaic virus (CaMV) 35S enhancer and an assortment of tissue-specific promoters fused to the β-glucuronidase (GUS) reporter gene. The full-length TBS fragment was found to function in both orientations, although to a significantly lesser degree in the reverse orientation, and was operational in both plant species tested. While multiple deletion fragments were found to exhibit activity, it appeared that several regions of the TBS were required for maximal enhancer-blocking function. Furthermore, we found that this element exhibited promoter-like activity, which has implications in terms of possible mechanisms behind its ability to impede enhancer-promoter communication in plants.

Interest in phloem-specific promoters for the engineering of transgenic plants has been increasing in recent years. In this study we isolated two similar, but distinct, alleles of the Citrus sinensis sucrose synthase-1 promoter (CsSUS1p) and inserted them upstream of the β-glucuronidase (GUS) gene to test their ability to drive expression in the phloem of transgenic Arabidopsis thaliana and Nicotiana tabacum. Although both promoter variants were capable of conferring localized GUS expression in the phloem, the CsSUS1p-2 allele also generated a significant level of expression in non-target tissues. Unexpectedly, GUS expression was also instigated in a minority of CsSUS1p::GUS lines in response to wounding in the leaves of transgenic Arabidopsis. Deletion analysis of the CsSUS1p suggested that a fragment comprising nucleotides -410 to -268 relative to the translational start site contained elements required for phloem-specific expression while nucleotides -268 to -103 contained elements necessary for wound-specific expression. Interestingly, the main difference between the two CsSUS1p alleles was the presence of a 94-bp insertion in allele 2. Fusion of this indel to a minimal promoter and GUS reporter gene indicated that it contained stamen and carpel-specific enhancer elements. This finding of highly specific and separable regulatory units within the CsSUS1p suggests that this promoter may have a potential application in the generation of constructs for the use in the development of transgenic plants resistant to a wide variety of target pests.

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.