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.

Using full chloroplast genomes of 'red' and 'yellow' Bixa orellana (achiote) for kmer based identification and phylogenetic inference.

BACKGROUND: Full chloroplast genomes provide high resolution taxonomic discrimination between closely related plant species and are quickly replacing single and multi-locus barcoding regions as reference materials of choice for DNA based taxonomic annotation of plants. Bixa orellana, commonly known as "achiote" and "annatto" is a plant used for both human and animal foods and was thus identified for full chloroplast sequencing for the Center for Veterinary Medicine (CVM) Complete Chloroplast Animal Feed database. This work was conducted in collaboration with the Instituto de Medicina Tradicional (IMET) in Iquitos, Peru. There is a wide range of color variation in pods of Bixa orellana for which genetic loci that distinguish phenotypes have not yet been identified. Here we apply whole chloroplast genome sequencing of "red" and "yellow" individuals of Bixa orellana to provide high quality reference genomes to support kmer database development for use identifying this plant from complex mixtures using shotgun data. Additionally, we describe chloroplast gene content, synteny and phylogeny, and identify an indel and snp that may be associated with seed pod color. RESULTS: Fully assembled chloroplast genomes were produced for both red and yellow Bixa orellana accessions (158,918 and 158,823 bp respectively). Synteny and gene content was identical to the only other previously reported full chloroplast genome of Bixa orellana (NC_041550). We observed a 17 base pair deletion at position 58,399-58,415 in both accessions, relative to NC_041550 and a 6 bp deletion at position 75,531-75,526 and a snp at position 86,493 in red Bixa orellana. CONCLUSIONS: Our data provide high quality reference genomes of individuals of red and yellow Bixa orellana to support kmer based identity markers for use with shotgun sequencing approaches for rapid, precise identification of Bixa orellana from complex mixtures. Kmer based phylogeny of full chloroplast genomes supports monophylly of Bixaceae consistent with alignment based approaches. A potentially discriminatory indel and snp were identified that may be correlated with the red phenotype.

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.

Prevalence and Genetic Diversity of Grabloviruses in Free-Living Vitis spp.

The distribution and diversity of grapevine red blotch virus (GRBV) and wild Vitis virus 1 (WVV1) (genus Grablovirus; family Geminiviridae) were determined in free-living Vitis spp. in northern California and New York from 2013 to 2017. Grabloviruses were detected by polymerase chain reaction in 28% (57 of 203) of samples from California but in none of the 163 samples from New York. The incidence of GRBV in free-living vines was significantly higher in samples from California counties with high compared with low grape production (χ2 = 83.09; P < 0.001), and in samples near (<5 km) to compared with far (>5 km) from vineyards (χ2 = 57.58; P < 0.001). These results suggested a directional spread of GRBV inoculum predominantly from vineyards to free-living Vitis spp. WVV1 incidence was also significantly higher in areas with higher grape production acreage (χ2 = 16.02; P < 0.001). However, in contrast to GRBV, no differential distribution of WVV1 incidence was observed with regard to distance from vineyards (χ2 = 0.88; P = 0.3513). Two distinct phylogenetic clades were identified for both GRBV and WVV1 isolates from free-living Vitis spp., although the nucleotide sequence variability of the genomic diversity fragment was higher for WWV1 (94.3 to 99.8% sequence identity within clade 1 isolates and 90.1 to 100% within clade 2 isolates) than GRBV (98.3% between clade 1 isolates and 96.9 to 100% within clade 2 isolates). Additionally, evidence for intraspecific recombination events was found in WVV1 isolates and confirmed in GRBV isolates. The prevalence of grabloviruses in California free-living vines highlights the need for vigilance regarding potential grablovirus inoculum sources in order to protect new vineyard plantings and foundation stock vineyards in California.

'Honeycrisp' Bitter Pit Response to Rootstock and Region under Eastern New York Climatic Conditions.

There are still unknown factors at play in the causation of bitter pit in 'Honeycrisp' as well as in other apple varieties. To investigate some of these factors, we conducted a survey of 34 'Honeycrisp' orchard blocks distributed across two disparate production regions in eastern New York State, representing a variety of rootstocks, over three growing seasons. Weather, soil, horticultural traits, fruit quality traits, pick timing, leaf and peel minerals were evaluated for their impact on bitter pit (BP) incidence; factors were further evaluated for their interaction with region and rootstock. 'Honeycrisp' trees on B.9 rootstock were smaller but with comparable terminal shoot growth when compared to those on M.26 and M.9 rootstocks. B.9 fruits, which had similar fruit size to M.26 and M.9 and had good fruit quality at harvest and after storage, were much less likely to express bitter pit symptoms compared to M.9 and M.26 rootstocks. Not all traits evaluated individually correlated significatively with bitter pit incidence after a period in storage. Depending on rootstock and region, the correlation could be significant in one situation, with no correlation at all in another. In this study, peel Mg/Ca ratio and peel Ca correlated with BP for all three rootstocks, with the strongest correlations associated with the M.9 clones. These same traits correlated with BP for both regions. Pick timing had a significant influence on BP incidence following storage, with later picks offering better bitter pit storage performance. While excessively large fruits, those in the 48 and 56 count size categories, were found to be highly susceptible to BP regardless of rootstock, B.9 BP fruit susceptibility for smaller sizes was found to be size neutral. A PLSR prediction model for each rootstock and each region showed that different variables correlated to BP depending on the situation. Thus, the results could suggest that in addition to the variables considered in this study, there are other less studied factors that can influence the expression of BP symptoms. We strongly suggest that rootstock BP performance be considered a critical parameter when planning a commercial 'Honeycrisp' orchard and be evaluated in rootstock breeding and development programs prior to wide commercial release.

Xylosandrus germanus (Coleoptera: Curculionidae: Scolytinae) Occurrence, Fungal Associations, and Management Trials in New York Apple Orchards.

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.

Solanum lycopersicum (tomato) hosts robust phyllosphere and rhizosphere bacterial communities when grown in soil amended with various organic and synthetic fertilizers.

Due to the intimate association between plants and their microbial symbionts, an examination of the influence of agricultural practices on phytobiome structure and diversity could foster a more comprehensive understanding of plant health and produce safety. Indeed, the impact of upstream crop producti006Fn practices cannot be overstated in their role in assuring an abundant and safe food supply. To assess whether fertilizer type impacted rhizosphere and phyllosphere bacterial communities associating with tomato plants, the bacterial microbiome of tomato cv. 'BHN602' grown in soils amended with fresh poultry litter, commercially available sterilized poultry litter pellets, vermicompost or synthetic fertilizer was described. Culture independent DNA was extracted from bulk and rhizosphere soils, and washes of tomato blossoms and ripe fruit. PCR amplicons of hypervariable regions of the 16S rRNA gene were sequenced and profiled using the QIIME pipeline. Bulk and rhizosphere soil, and blossom and fruit surfaces all supported distinct bacterial communities according to principal coordinate analysis and ANOSIM (R=0.87, p=0.001 in year 1; R=0.93, p=0.001 in year 2). Use of microbiologically diverse organic fertilizers generally did not influence bacterial diversity, community structure or relative abundance of specific taxa on any plant organ surface. However, statistically significant differences in sand and silt contents of soil (p<0.05) across the field and corresponding shifts in water activity were positively (R2=0.52, p=0.005) and negatively (R2=0.48, p=0.009) correlated with changes in bacterial community structure in the rhizosphere, respectively. Over two harvest seasons, this study demonstrated that the application of raw poultry manure, poultry litter pellets and vermicompost had little effect on the tomato microbiome in the rhizosphere and phyllosphere, when compared to synthetically fertilized plants. Plant anatomy, and other factors related to field location, possibly associated with edaphic and air characteristics, were more influential drivers of different tomato organ microbiomes than were diverse soil amendment applications.

Audiovisual distraction as a useful adjunct to epidural anesthesia and sedation for prolonged lower limb microvascular orthoplastic surgery.

Lower limb orthopedic operations are frequently performed under regional anesthesia, which allows avoidance of potential side effects and complications of general anesthesia and sedation. Often though, patients feel anxious about being awake during operations. To decrease intraoperative anxiety, we use multimedia equipment consisting of a tablet device, noise-canceling headphones, and a makeshift frame, where patients can listen to music, watch movies, or occupy themselves in numerous ways. These techniques have been extensively studies in minimally invasive, short, or minor procedures but not in prolonged orthoplastic operations. We report 2 cases where audiovisual distraction was successfully applied to 9.5-hour procedures, proved to be a very useful adjunct to epidural anesthesia + sedation, and made an important contribution to positive patients' outcomes and overall patients' experience with regional anesthesia for complex limb reconstructive surgery. In the era when not only patients' safety and clinical outcomes but also patients' positive experiences are of paramount importance, audiovisual distraction may provide a simple tool to help improve experience of appropriately informed patients undergoing suitable procedures under regional anesthesia. The anesthetic technique received a very positive appraisal by both patients and encouraged us to study further the impact of modern audiovisual technology on anxiolysis for major surgery under regional anesthesia. The duration of surgery per se is not a contraindication to the use of audiovisual distraction. The absolute proviso of successful application of this technique to major surgery is effective regional anesthesia and good teamwork between the clinicians and the patients.