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Apple orchards with minimal or reduced fungicide inputs in the Mid-Atlantic region of the United States have experienced outbreaks of severe premature defoliation with symptoms that matched those of apple blotch disease (ABD) caused by Diplocarpon coronariae. Fungal isolates obtained from symptomatic apple leaves and fruit produced uniform slow-growing, dark-gray colonies on peptone potato dextrose agar and had conidia. Internal transcribed spacer DNA sequences matched with D. coronariae and Koch’s postulates were fulfilled when typical ABD symptoms occurred when reinoculated onto apple leaves and fruit. Spore dispersal in nonfungicide-treated orchards detected with quantitative PCR was low in early spring and dropped to undetectable levels in late May and early June before rising exponentially to highs in July and August, which coincided with symptom development. Only low spore numbers were detected in fungicide-treated orchards and nearby forests. In preliminary fungicide tests, fluxapyroxad, thiophanate methyl, and difenoconazole effectively inhibited mycelial growth of isolates in vitro. When apple cultivars Fuji and Honeycrisp were inoculated with D. coronariae, Honeycrisp showed delayed onset of symptoms and lower disease severity, and the transcription profile of seven host defense-related genes showed that PR-2, PR-8, LYK4, and CERK1 were highly induced in Honeycrisp at 2 and 5 days postinoculation. This is the first report of ABD in the Mid-Atlantic United States, which includes studies of seasonal D. coronariae spore dispersal patterns, preliminary fungicide efficacy, and host defense-related gene expression to assist development of best ABD management practices.

Bitter rot, caused by Colletotrichum species, is one of the most devastating summer rot diseases affecting apple production in the Eastern United States. Given the differences in virulence and fungicide sensitivity levels between organisms belonging to the acutatum species complex (CASC) and the gloeosporioides species complex (CGSC), monitoring their diversity, geographic distribution, and frequency % are essential for successful bitter rot management. In a 662-isolate collection from apple orchards in Virginia, isolates from CGSC were dominant (65.5%) in comparison to the CASC (34.5%). In a sub-sample of 82 representative isolates, using morphological and multi-locus phylogenetic analyses, we identified C. fructicola (26.2%), C. chrysophilum (15.6%), C. siamense (0.8%) and C. theobromicola (0.8%) from CGSC and C. fioriniae (22.1%) and C. nymphaeae (1.6%) from CASC. The dominant species were C. fructicola, followed by C. chrysophilum and C. fioriniae. C. siamense followed by C. theobromicola developed the largest and deepest rot lesions on ‘Honeycrisp’ fruit in our virulence tests. Detached fruit of 9 apple cultivars and one wild accession (Malus sylvestris) were harvested early and late season and tested in controlled conditions for their susceptibility to C. fioriniae and C. chrysophilum. All cultivars were susceptible to both representative bitter rot species, with ‘Honeycrisp’ fruit being the most susceptible and Malus sylvestris, accession PI 369855, being the most resistant. We demonstrate that the frequency and prevalence of species in Colletotrichum complexes are highly variable in the Mid-Atlantic and provide regional-specific data on apple cultivar susceptibility. Our findings are necessary for the successful management of bitter rot as an emerging and persistent problem in apple production both pre- and postharvest.

Apple growers in the Mid-Atlantic region of the U.S.A. have reported increased losses to bitter rot of apple. We tested the hypothesis that this increase is because the Colletotrichum population has developed resistance to commonly used single-mode-of-action (single-MoA) fungicides. We screened 220 Colletotrichum isolates obtained from 38 apple orchards in the Mid-Atlantic region for resistance to 11 fungicides in Fungicide Resistance Action Committee (FRAC) groups 1, 7, 9, 11, 12, and 29. Eleven (5%) of these isolates were resistant to FRAC group 1 with confirmed β-tubulin E198A mutations, and two (25 values (concentrations that reduce growth by 25%) are better predictors of fungicide efficacy in normal field conditions than EC50 values. We present these results as a guideline for choosing single-MoA fungicides for bitter rot control in the Mid-Atlantic U.S.A.

With the goal to develop management tools to mitigate the severity of shoot blight on pear caused by Erwinia amylovora and prevent fire blight canker development from infected shoots on pear tree wood, we evaluated five preventive treatments in inoculated experiments over 2-years. We focused on foliar spray and trunk injection applications of extract of giant knotweed, Reynoutria sachalinensis (RSE), as a plant resistance activator candidate and on comparisons to antibiotics. Five foliar applications of RSE (2.24 L/ha) on 6-year-old trees of cultivar ‘Bartlett’ resulted in 100% control of shoot blight severity and canker incidence in both years of evaluation. The RSE trunk injection in fall of the first year did not control the disease allowing 75% of shoot severity and 38.5% of canker incidence. The opposite happened in the second year, when we injected RSE in spring, providing 85.7% control of shoot blight severity and 89.8% control of canker incidence. Antibiotics oxytetracycline (OTC) and streptomycin (STM) did not provide consistent control in the second year in comparison to the first year. In the first year, both antibiotics, OTC (trunk injection and foliar spray) and STM were effective, providing 94.8%, 100% and 86.3% control of shoot blight, respectively. They also provided 100, 91 and 84% control of canker incidence, respectively. However, in the second they were not effective allowing 69 – 96% of shoot blight severity and 70 – 92% of canker incidence. Regardless of the inconsistency in the effectiveness of antibiotics, which are not recommended for shoot blight control due to risks of promoting antibiotic resistance, five preventive spray applications of RSE could be used on pear trees to prevent shoot blight and canker development. Thus, the RSE spray program we developed could serve as a valuable control tool that could help prevent sudden outbreaks of shoot blight during summer and pear tree death from fire blight cankers with no risk for pathogen resistance.

To reduce the severity of shoot blight and prevent the resulting development of cankers on perennial apple wood, we evaluated eight fire blight postinfection spray programs of prohexadione-calcium (PCA) alone or with acibenzolar-S-methyl (ASM) over 2 years. On mature trees of cultivar Royal Court, a single application of the high PCA rate (247 mg/liter) at 2 to 3 days after inoculation resulted in 89.5 and 69.5% reduction of shoot blight severity after inoculation. Two applications of PCA 247 mg/liter 12 or 14 days apart, with the first one applied 2 to 3 days after inoculation, resulted in 78.8 and 74.5% reduction of shoot blight severity in both years. A 100% control of canker incidence on perennial wood from infected shoots in both years was achieved with a single application of PCA (247 mg/liter) applied at 2 or 3 days after the inoculation, and three applications of PCA (125 mg/liter) + ASM (25 mg/liter) 12 to 16 days apart reduced canker incidence by 83.5 and 69% in the 2 years. The other programs with lower PCA rates and frequencies of application reduced shoot blight severity 50.8 and 51.8% (PCA) and 62.6 to 72% and 59.3% (PCA + ASM) over 2 years, respectively. Reduction of canker incidence on wood by the other programs was 66.5% and 69 to 90.4% in the two years, respectively. As fire blight cankers lead to death of dwarf apple trees and serve as primary sources of inoculum, our effective PCA and PCA + ASM programs could serve as viable postinfection management options. These treatments can reduce or prevent canker development and thus significantly abate tree losses in high-density apple orchards after fire blight epidemics occur. Accepted version

Apple growers in the Mid-Atlantic region of the United States have been reporting an increase in losses to bitter rot of apple and are requesting up-to-date management recommendations. Management is complicated by variations in apple cultivar susceptibility, temperature, rainfall, and biology of the Colletotrichum spp. that cause bitter rot. Over 500 apple fruit with bitter rot were obtained from 38 orchards across the Mid-Atlantic and the causal species were identified as Colletotrichum fioriniae and C. nymphaeae of the C. acutatum species complex and C. chrysophilum, C. noveboracense, C. siamense, C. fructicola, C. henanense, and C. gloeosporioides sensu stricto of the C. gloeosporioides species complex, the latter two being first reports. Species with faster in vitro growth rates at higher temperatures were more abundant in warmer regions of the Mid-Atlantic, while those with slower growth rates at higher temperatures were more abundant in cooler regions. Regional bloom dates are earlier and weather data show a gradual warming trend that likely influenced but was not necessarily the main cause of the recent increase in bitter rot in the region. A grower survey of apple cultivar susceptibility showed high variation, with the increase in acres planted to the highly susceptible cultivar Honeycrisp broadly corresponding to the increase in reports of bitter rot. These results form a basis for future studies on the biology and ecology of the Colletotrichum spp. responsible, and suggest that integrated bitter rot management must begin with selection of less-susceptible apple cultivars.

Apple growers in the Mid-Atlantic region of the U.S.A. have reported increased losses to bitter rot of apple. We tested the hypothesis that this increase is because the

To reduce the severity of shoot blight and prevent the resulting development of cankers on perennial apple wood, we evaluated eight fire blight postinfection spray programs of prohexadione-calcium (PCA) alone or with acibenzolar

Background Apple blotch (AB) caused by Diplocarpon coronariae (Dc) has been established in Europe since 2010. AB is a serious apple disease, mostly in low input orchards and in cider production areas in Northern Italy, Switzerland, Austria and Germany. However, the epidemiology and population genetic structure of this pathogen is unknown. Methods We developed twelve Dc-specific microsatellite markers and screened DNA of both pure fungal isolates and infected apple leaves. The marker data of 313 European samples of Dc were compared to Dc isolates from Asia (n = 7) and the USA (n = 3). Results We found 31 distinct multilocus genotypes (MLGs) in European samples, and seven additional MLGs in the Asian and USA samples. The European samples had the typical genetic signature of a recently introduced species including high clonality, a low number of private alleles and one dominant MLG across all the sampling sites. All European MLGs were genetically distant from those MLGs of Asian and USA origin. Based on the lack of linkage disequilibrium observed, there is evidence that Dc undergoes regular cycles of sexual recombination in the European population, although the sexual stage (apothecia) has not been observed in Europe. Conclusions The twelve newly developed SSR markers reported here provide a useful tool to characterize the population genetic diversity and structure of Dc in Europe. Our study supports the hypothesis that Dc is a recently introduced pathogen in Europe, but of currently unknown origin. Dc has a large effective population size during field epidemics, so we believe that the pathogen has substantial evolutionary potential. Application of the SSR markers to large-scale and diverse Dc samples will help to better understand the epidemiology of AB, which has become a global apple disease, and will help guide effective mitigation strategies based on disease management and resistance breeding., CABI Agriculture and Bioscience, 2, ISSN:2662-4044

The samurai wasp, Trissolcus japonicus (Ashmead), has been proposed as a biocontrol agent against brown marmorated stink bugs (BMSB), due to its ability to parasitize and kill BMSB eggs. However, the wasps’ small size makes it challenging for those untrained in morphological identification to determine the wasps’ species. To circumvent this problem, a molecular method was created to identify T. japonicus. The method uses species-specific primers, designed in this study, which target the variable region of the mitochondrial Cytochrome Oxidase 1 (CO1) locus. After confirming successful DNA extraction from samples, the PCR amplification using our primers produced 227-bp PCR products for all T. japonicus specimens and no amplification in other microhymenoptera candidates. Additionally, DNA from BMSB-parasitized eggs gave positive PCR amplification, while the control BMSB samples showed no amplification. This indicates that PCR with our primers specifically and sensitively differentiates T. japonicus specimens from other similar wasp species and discriminates between T. japonicus-parasitized and non-parasitized BMSB eggs. Finally, an in silico analysis of CO1 sequences demonstrated that our primers match the sequences of four different haplotypes of T. japonicus, indicating that our diagnostic method could potentially be applied to analyze T. japonicus populations throughout North America, Europe, and parts of Asia.

Simple Summary The brown marmorated stinkbug Halyomorpha halys, is an invasive pest that causes millions of dollars of crop damage each year in the US. A promising biocontrol agent for this pest is the samurai wasp, Trissolcus japonicus, which is a potential long term control method with few negative ecological impacts. However, the wasps’ small size of only 1 mm in length can make it difficult to identify morphologically. We developed a DNA-based approach to determine whether a wasp specimen is T. japonicus using conventional methods of DNA extraction, PCR, and gel electrophoresis. When tested against eight families of Hymenoptera, including three Trissolcus species, our method identified samurai wasp samples with 100% accuracy. Additionally, in silico analyses of various T. japonicus sequences provide evidence that the method will work throughout the US, Europe, and parts of Asia. We expect that this method will be a valuable tool for reliably identifying T. japonicus during distribution and recapture efforts to assess its survival, establishment, and dispersal for biocontrol purposes. Abstract The samurai wasp, Trissolcus japonicus (Ashmead), has been proposed as a biocontrol agent against brown marmorated stink bugs (BMSB), due to its ability to parasitize and kill BMSB eggs. However, the wasps’ small size makes it challenging for those untrained in morphological identification to determine the wasps’ species. To circumvent this problem, a molecular method was created to identify T. japonicus. The method uses species-specific primers, designed in this study, which target the variable region of the mitochondrial Cytochrome Oxidase 1 (CO1) locus. After confirming successful DNA extraction from samples, the PCR amplification using our primers produced 227-bp PCR products for all T. japonicus specimens and no amplification in other microhymenoptera candidates. Additionally, DNA from BMSB-parasitized eggs gave positive PCR amplification, while the control BMSB samples showed no amplification. This indicates that PCR with our primers specifically and sensitively differentiates T. japonicus specimens from other similar wasp species and discriminates between T. japonicus-parasitized and non-parasitized BMSB eggs. Finally, an in silico analysis of CO1 sequences demonstrated that our primers match the sequences of four different haplotypes of T. japonicus, indicating that our diagnostic method could potentially be applied to analyze T. japonicus populations throughout North America, Europe, and parts of Asia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Apple bitter rot caused by Colletotrichum species is a growing problem worldwide. Colletotrichum spp. are economically important but taxonomically un-resolved. Identification of Colletotrichum spp. is critical due to potential species-level differences in pathogenicity-related characteristics. A 400-isolate collection from New York apple orchards were morphologically assorted to two groups, C. acutatum species complex (CASC) and C. gloeosporioides species complex (CGSC). A sub-sample of 44 representative isolates, spanning the geographical distribution and apple varieties, were assigned to species based on multi-locus phylogenetic analyses of nrITS, GAPDH and TUB2 for CASC, and ITS, GAPDH, CAL, ACT, TUB2, APN2, ApMat and GS genes for CGSC. The dominant species was C. fioriniae, followed by C. chrysophilum and a novel species, C. noveboracense, described in this study. This study represents the first report of C. chrysophilum and C. noveboracense as pathogens of apple. We assessed the enzyme activity and fungicide sensitivity for isolates identified in New York. All isolates showed amylolytic, cellulolytic and lipolytic, but not proteolytic activity. C. chrysophilum showed the highest cellulase and the lowest lipase activity, while C. noveboracense had the highest amylase activity. Fungicide assays showed that C. fioriniae was sensitive to benzovindiflupyr and thiabendazole, while C. chrysophilum and C. noveboracense were sensitive to fludioxonil, pyraclostrobin and difenoconazole. All species were pathogenic on apple fruit with varying lesion sizes. Our findings of differing pathogenicity-related characteristics among the three species demonstrate the importance of accurate species identification for any downstream investigations of Colletotrichum spp. in major apple growing regions.

Coast redwood (Sequoia sempervirens) is among the most widely planted landscape trees in California (CA) but is in decline outside its natural range due to factors including prolonged drought and plant pathogens. We investigated associations of Botryosphaeriaceae fungi with declining coast redwood trees throughout CA. More than 100 samples were collected from 11 coastal and inland locations in CA. Fifty-nine Botryosphaeria-like fungal strains were isolated and 18 were selected for further study. Phylogenetic analysis of ITS and EF-1α sequence data confirmed the presence of Botryosphaeria dothidea, Neofusicoccum australe, N. luteum, N. mediterraneum, and N. parvum. Pathogenicity testing showed that although the Neofusicoccum species vary in virulence, all are more virulent that B. dothidea. N. australe caused the largest lesions, followed by N. luteum, N. parvum, and N. mediterraneum. Of the species recovered, only B. dothidea has been previously confirmed as a pathogen of coast redwood in CA. These results confirm that multiple Botryosphaeriaceae species are associated with branch decline and dieback on coast redwood in CA, which agrees with similar studies on woody agricultural crops. Accurate diagnosis of fungal pathogens of coast redwood is important for the development of disease management strategies and may help improve horticultural practices in maintenance of urban stands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fire blight is a devastating disease of apple and pear caused by the bacterium Erwinia amylovora. One of its main symptoms is canker formation on perennial tissues which may lead to the death of limbs and/or the entire tree. E. amylovora overwinters in cankers which play an important role in initiating fire blight epidemics. However, knowledge of pathogen biology in cankers is scarce, in part due to limitations of classical microbiology methods and the inability of most molecular techniques to distinguish live from dead cells. In this work, a viability digital PCR (v-dPCR) protocol using propidium monoazide (PMA) was developed, allowing for the first time the selective detection and absolute quantification of E. amylovora live cells in apple and pear cankers collected in two time periods. Some key factors affecting the v-dPCR performance were the maceration buffer composition, the target DNA amplicon length, the thermal cycle number and the use of sodium dodecyl sulfate or PMA enhancer for Gram-negative bacteria to improve the effect of PMA. In the future, this methodology could shed light on E. amylovora population dynamics in cankers and provide clues on the effect of management practices, host cultivar, host water/nutritional status, etc., on bacterial survival.

To optimize the number and timing of trunk injections for season-long control of apple scab (Venturia inaequalis), we evaluated 1 to 2 and 4 seasonal and cross-seasonal injections of potassium phosphites and synthetic fungicides and quantified residues in leaves and fruit. Phosphites accumulated in the canopy at the highest concentrations, aligned well in time with scab suppression, and gave better leaf scab control of 41.8 to 73.5% than propiconazole (16.9 to 51.5%) or cyprodinil + difenoconazole (5.4 to 17.4%). More injections of phosphites controlled leaf scab better than fewer (23.7% versus 48.2%), and more fungicide injections resulted in 21.9 to 51.1% better leaf scab control than fewer. Leaf scab control with phosphites was only 3.2 to 13.9% better with 4 cross-seasonal compared with 4 seasonal injections, while 1 to 2 seasonal compared with 1 to 2 cross-seasonal injections improved scab control only for 4.2 to 22.1%. On shoots, injected phosphites provided comparable or for 4.4 to 10.5% and 22.3 to 41.4% better scab control than spray standards. On fruit, injected phosphites slightly improved control compared with sprayed phosphites or the sprayed fungicide standard (33.4 to 40.8%). Two seasonal injections of phosphites controlled shoot scab 5.7% better than 9 spray applications. Five sprays of cyprodinil + difenoconazole controlled scab better than their injections. Fruit residues of phosphites reached 2.8 ppm and declined in all treatments except in 2 seasonal injections and phosphite sprays. Cyprodinil and difenoconazole fruit residues reached 0.02 and 0.07 ppm and declined sharply toward the end of the season. These were far below the United States, Codex, and EU MRL-s of 1, 0.8, and 0.5 ppm for difenoconazole, and 1.7, 2, and 1 ppm for cyprodinil, respectively.

Excessive tree wounding is a common concern regarding the use of trunk injection technology for tree protection purposes in landscapes and urban greening. We investigated the rate of healing of injection ports (points) in apple trees by monitoring parameters such as port diameters, the size of bark cracking, and port depths. We compared drilled injection ports from 4.4 and 9.5 mm drill bits, with latter being sealed with plastic-silicone plug (Arborplug®) or not, and the lenticular port from a double-edged blade. Depending on port size and type, port closure ranged from one to more than two years. Bark cracking around injection ports was more pronounced longitudinally. On the sealed 9.5 mm port, bark cracking was largely similar to all drilled ports. The depth of port wounds decreased faster on the port from the 4.4 mm drill bit and on lenticular injection port versus the unsealed port from the 9.5 mm drill bit. Plastic-silicone plugs, which simulate removed bark, slowed the healing of 9.5 mm drill port with callus and increased the port depths over time due to callus formation over the top of the plug. From fastest-healing to slowest-healing, on average the injection ports were: lenticular port from blade (70.8%), the unsealed 9.5 mm drill port (44.4%), 4.4 mm drill port (43.9%), and 9.5 mm drill port sealed with plastic-silicone plug (20.4%).

Field studies, laboratory bioassays, and residue profile analysis were used to determine the seasonal effectiveness of trunk injected pesticides against key apple disease and insect pests. Insecticides formulated for trunk injection, imidacloprid (Ima-jet™), rynaxypyr (XCL-r8™), and emamectin benzoate (TREE-age™) were injected into semi-dwarf Empire apple trees Malus domestica (Borkhausen) and evaluated for a wide range of insect pests. The fungicide compounds, propiconazole (Alamo®), phosphites (Phospho-jet), and penthiopyrad (Fontelis™), were injected into semi-dwarf MacIntosh (RedMax) apple trees M.domestica (Borkhausen) for control of apple scab fungus, Venturia inaequalis (Cooke). After the original single injection, imidacloprid was highly effective in controlling piercing and sucking pests such as the potato leafhopper, Empoasca fabae (Harris), and aphids (Aphididae), and emamectin benzoate was highly effective in controlling the oblique banded leaf roller, Choristoneura rosaceana (Harris), and potato leafhopper, E. fabae (Harris), and rynaxypyr was highly effective in controlling Oriental fruit moth, Grapholita molesta (Busck), and leafrollers all for two growing seasons. The residue profiles for insecticides showed that vascular delivery was predominantly to foliage, with fruit residues far below the EPA maximum residue limits (MRLs), and low to no residues detected in apple flower parts. Phosphites provided significant levels of apple scab control over two seasons for the single injection after the foliage recovered from the phytotoxicity damage in the first season. Propiconazole and penthiopyrad showed limited effectiveness for the control of apple scab. The residue profiles for fungicides showed phosphites to be delivered primarily to foliage, but inconsistent foliar residue levels for the other two compounds suggests possible incompatibilities may be responsible for poor product performance. These incompatibilities may include molecular or chemical properties. For example, on the molecular level such as the molecular size too large to fit through vascular tissue and chemical properties such as the viscosity of the compound resulting in poor translocation or pH.

In May 2017, dead buds at pink and tight cluster growth stages were observed on young apple trees of cultivar SnapDragon at two orchard sites in Marlboro, NY. Disease symptoms included necrosis of lateral and terminal flower buds that were wilted and drooping. Bark below the dead buds on severely affected trees showed black streaking with or without interspersing white streaks. On the cross-section of branch wood, just below the buds, light brown xylem and glossy water-soaked sections resembling frost damage were observed. After bark shaving, dark xylem tissue was visible along the branch length, in contrast to the light or pale color of the healthy tissue. The incidence of the disease on trees was 3 to 5%, and 0.5% of trees died. There were five near-frost events recorded on 4, 8, 9, 10, and 11 May 2017, leading us to suspect that the observed symptoms were blossom blast caused by the ice-nucleating strains of Pseudomonas syringae. Isolation from the affected tree samples was performed on King’s B (KB) and nutrient sucrose agar (NSA) media. After 3 days at 25°C, most of bacterial isolates formed round, shiny, gray-white colonies producing green fluorescent pigment on KB medium and typical levan-positive colonies on NSA. Eight isolates, inducing a hypersensitive reaction on tobacco plants, were further characterized by conventional bacteriological and molecular tests (Braun-Kiewnick and Sands 2001). The analyzed bacterial isolates were Gram-negative, aerobic, hydrolyzed gelatin and aesculin, but not pectate, and were negative for oxidase and arginine-dihydrolase activity. All strains were ice-nucleation active and utilized sucrose, sorbitol, and inositol as carbon sources, but not tyrosine and tartaric acid. Results of levan, cytochrome c oxidase, potato soft rot, arginine dihydrolase, and tobacco hypersensitive reaction (LOPAT) tests indicated that the isolates belong to P. syringae group Ia, and gelatin hydrolysis, aesculin hydrolysis, tyrosinase activity, and tartrate utilization (GATTa) tests designated them as pathovar (pv.) syringae (Kaluzna et al. 2012). Polymerase chain reaction (PCR) with specific primers B1/B2 (Sorensen et al. 1998) and SyD1/SyD2 (Bultreys and Gheysen 1999) showed that isolates possessed genes responsible for synthesis (syrB) and secretion (syrD) of syringomycin, respectively. Partial sequencing of the 16S rDNA gene of three isolates (GenBank MG788015 to MG788017) showed 99 to 100% identity with sequences of P. syringae pv. syringae previously deposited in the NCBI GenBank database. To complete Koch’s postulates, pathogenicity of the four isolates was confirmed by inoculation of 1-month-old potted apple plantlets grown from seeds collected from mature apple fruit of cultivar SnapDragon. One plant per isolate was inoculated multiple times as follows: using a needleless syringe, two leaves per plant were spot-infiltrated with bacterial suspension (10⁶ CFU/ml); on the same plants, stem was also inoculated by inserting a sterile toothpick previously dipped into a bacterial colony. Sterile distilled water was used as a negative control. Inoculated plantlets were sealed within plastic bags for 48 h and further incubated at 22°C. Inoculation test was repeated in two independent experiments. Necrosis of the leaf and stem tissue developed at the point of inoculation, and its progression was observed daily for 2 weeks. No symptoms developed on the control plants. Bacteria were reisolated from inoculated tissues and confirmed as P. syringae pv. syringae by PCR for syrB and syrD genes. This is the first report of P. syringae pv. syringae causing blossom blast symptoms on developing apple buds in commercial orchards in the United States. Even though it rarely occurs on apples, with most recent frosts significantly lowering apple yields in Michigan (2012) and New York (2016), blossom blast might become a more common problem in U.S. orchards. Previously, P. syringae pv. syringae was isolated from dormant apple buds without and with internal necrosis in the United States (Burr and Katz 1984).

Pesticide use in orchards creates drift-driven pesticide losses which contaminate the environment. Trunk injection of pesticides as a target-precise delivery system could greatly reduce pesticide losses. However, pesticide efficiency after trunk injection is associated with the underinvestigated spatial and temporal distribution of the pesticide within the tree crown. This study quantified the spatial and temporal distribution of trunk-injected imidacloprid within apple crowns after trunk injection using one, two, four or eight injection ports per tree.The spatial uniformity of imidacloprid distribution in apple crowns significantly increased with more injection ports. Four ports allowed uniform spatial distribution of imidacloprid in the crown. Uniform and non-uniform spatial distributions were established early and lasted throughout the experiment. The temporal distribution of imidacloprid was significantly non-uniform. Upper and lower crown positions did not significantly differ in compound concentration. Crown concentration patterns indicated that imidacloprid transport in the trunk occurred through radial diffusion and vertical uptake with a spiral pattern.By showing where and when a trunk-injected compound is distributed in the apple tree canopy, this study addresses a key knowledge gap in terms of explaining the efficiency of the compound in the crown. These findings allow the improvement of target-precise pesticide delivery for more sustainable tree-based agriculture.

Possibility of V. inaequalis control on apple variety Idared was evaluated by ecologically acceptable products such as inorganic fungicides and acidified clays. The obtained results were compared with common, chemical protection. Year 2009 was one of the unfavorable for V. inaequalis, and 2010 was extremely favorable for apple scab development. Accordingly, in 2009 intensity of infection on apple leaves and fruits in control variant was low (leaves 15,4-34,9%; fruits 0-23,8%). In 2010 infection intensity on leaves and fruits was high (leaves 81,2%; fruits 100%). During period 2009-2010, in these different environmental conditions, common chemical protection showed high efficacy (leaves 96,2-100%; fruits 97,5-100%). High efficacy level in control of V. inaequalis on leaves of 87,4-99,7% was achieved also in variants in which copper products were applied in low concentration of 0,05%. It should be noted that in the most critical period, in April and May of 2010, the treatments were carried out in 5-6 days interval. Copper products showed differing efficacy on fruits (48,5-90,2%). In 2009 which is considered as one of the unfavorable for V. inaequalis development, product Ulmasud B applied alone on leaves was less efficiacy (51,8-58,6%), than on fruits (87,8%). In the same year, efficacy of this product in combination with sulfur, was better (leaves 71,4-76,6%; fruits 86,6). In 2010, which was especially rainy and favourable for spreading the disease, efficiacy of mention combination of products didn't satisfy (leaves 44,8%; fruits 41,0%). In the system of organic production, during vegetation period, program of apple protection in control of V. inaequalis should be supplemented by use of low concentrations of copper products (0,05%) and by various alternative products of different action mechanisms, such as mineral clays, sulfur products and etc. These researches should be continued. Ispitivana je mogućnost suzbijanja V. inaequalis na jabuci (ajdared) ekološki prihvatljivim preparatima (neorganski fungicidi i kisela glina). Dobijeni rezultati su poređeni sa uobičajenom, hemijskom zaštitom. Godina 2009. je bila jedna od najnepovoljnijih za V. inaequalis, a 2010. jedna od najpovoljnijih za širenje ovog patogena na jabuci. Shodno tome, u 2009. godini (tri lokaliteta) intenzitet zaraze u kontroli na listovima i plodovima jabuke bio je nizak (listovi 15,4-34,9%; plodovi 0-23,8%). U 2010. godini intenzitet zaraze na listovima i plodovima je bio visok (listovi 81,2%; plodovi 100%). Pri navedenim različitim uslovima zaraze, efikasnost uobičajene, hemijske zaštite, u obe godine je bila na visokom nivou (listovi 96,2-100%; plodovi 97,5-100%). Visoka efikasnost u suzbijanju V. inaequalis na listovima (87,4-99,7%), postignuta je i u varijantama sa primenom bakarnih preparata u niskoj koncentraciji (0,05%), uz napomenu da su u 2010. godini tretmani u najkritičnijem periodu izvođeni u intervalu 5-6 dana. Efikasnost bakarnih preparata na plodovima je bila različita (48,5-90,2%). U 2009. godini, koja je bila jedna od nepovoljnijih za širenje crne pegavosti na jabuci, preparat Ulmasud B primenjen samostalno, ispoljio je znatno slabiju efikasnost na listovima (51,9-58,6%), u odnosu na plodove (87,8%). Iste godine, ovaj preparat u kombinaciji sa sumpornim preparatima, obezbedio je bolju efikasnost (listovi 71,4-76,6%; plodovi 86,6%). Suprotno tome, u 2010. godini, koja je bila izrazito povoljna za širenje ovog patogena, efikasnost navedene kombinacije preparata nije bila zadovoljavajuća (listovi 44,8%; plodovi 41,0%). U sistemu organske proizvodnje, program zaštite jabuke od V. inaequalis tokom vegetacije treba dopuniti korišćenjem preparata na bazi bakra (u niskoj koncentraciji) i raznih alternativnih preparata (na bazi mineralnih glina, sumpornih preparata i sl.). Ova ispitivanja bi trebalo nastaviti.