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Fungicides are commonly applied to prevent diseases in eastern North American cherry orchards at the same time that honey bees (Apis mellifera L. (Hymenoptera: Apidae)) are rented for pollination services. Fungicide exposure in honey bees can cause negative health effects. To measure fungicide exposure, we sampled commercial honey bee colonies during orchard bloom at two commercial tart cherry orchards and one holding yard in northern Michigan over two seasons. Nurse bees, foragers, larvae, pollen, bee bread, and wax were screened for captan, chlorothalonil, and thiophanate-methyl. We also looked at the composition of pollens collected by foragers during spring bloom. We found differences in fungicide residue levels between nurse bees and foragers, with higher captan levels in nurse bees. We also found that residue levels of chlorothalonil in workers were significantly increased during tart cherry bloom, and that nurse bees from hives adjacent to orchards had significantly higher chlorothalonil residues than nurse bees from hives kept in a holding yard. Our results suggest that fungicide exposure of individual honey bees depends greatly on hive location in relation to mass-flowering crops, and worker role (life stage) at the time of collection. In some pollen samples, captan and chlorothalonil were detected at levels known to cause negative health effects for honey bees. This study increases our understanding of exposure risk for bees under current bloom time orchard management in this region. Further research is needed to balance crop disease management requirements with necessary pollination services and long-term pollinator health.

Erwinia amylovora, the causative agent of fire blight, uses flagella-based motilities to translocate to host plant natural openings; however, little is known about how this bacterium migrates systemically in the apoplast. Here, we reveal a novel surface motility mechanism, defined as sliding, in E. amylovora. Deletion of flagella assembly genes did not affect this movement, whereas deletion of biosynthesis genes for the exopolysaccharides (EPSs) amylovoran and levan resulted in non-sliding phenotypes. Since EPS production generates osmotic pressure that potentially powers sliding, we validated this mechanism by demonstrating that water potential positively contributes to sliding. In addition, no sliding was observed when the water potential of the surface was lower than -0.5 MPa. Sliding is a passive motility mechanism. We further show that the force of gravity plays a critical role in directing E. amylovora sliding on unconfined surfaces but has a negligible effect when cells are sliding in confined microcapillaries, in which EPS-dependent osmotic pressure acts as the main force. Although amylovoran and levan are both required for sliding, we demonstrate that they exhibit different roles in bacterial communities. In summary, our study provides fundamental knowledge for a better understanding of mechanisms that drive bacterial sliding motility.

Populations of the fire blight pathogen Erwinia amylovora Ea110 on apple flower stigmas were tracked over the course of apple bloom in field studies conducted between 2016 and 2019. In 18 of 23 experiments, flower stigmas inoculated on the first day of opening were found to harbor large (106 to 107 cells per flower) populations of E. amylovora when assessed 3 to 5 days postinoculation. However, populations inoculated on stigmas of flowers that were already open for 3 days did not reach 106 cells per flower, and populations inoculated on stigmas of flowers that were already open for 5 days never exceeded 104 cells per flower. During this study, ≥10-fold increases in E. amylovora stigma populations in a 24-h time period (termed population surges) were observed on 34.8, 20.0, and 4.0% of possible days on 1-, 3-, and 5-day-open flowers, respectively. Population surges occurred on days with average temperatures as high as 24.5 and as low as 6.1°C. Experiments incorporating more frequent sampling during days and overnight revealed that many population surges occurred between 10:00 p.m. and 2:00 a.m. A Pearson’s correlation analysis of weather parameters occurring during surge events indicated that population surges were significantly associated with situations in which overnight temperatures increased or remained constant, in which wind speed decreased, and in which relative humidity increased. This study refines our knowledge of E. amylovora population dynamics and further indicates that E. amylovora is able to infect flowers during exposure to colder field temperatures than previously reported.

The enterobacterial pathogen Erwinia amylovora uses multiple virulence-associated traits to cause fire blight, a devastating disease of apple and pear trees. Many virulence-associated phenotypes have been studied that are critical for virulence and pathogenicity. Despite the in vitro testing that has revealed how these systems are transcriptionally regulated, information on when and where in infected tissues these genes are being expressed is lacking. Here, we used a high-throughput sequencing approach to characterize the transcriptome of E. amylovora during disease progression on apple flowers under field infection conditions. We report that type III secretion system genes and flagellar genes are strongly co-expressed. Likewise, genes involved in biosynthesis of the exopolysaccharide amylovoran and sorbitol utilization had similar expression patterns. We further identified a group of 16 genes whose expression is increased and maintained at high levels throughout disease progression across time and tissues. We chose five of these genes for mutational analysis and observed that deletion mutants lacking these genes all display reduced symptom development on apple shoots. Furthermore, these induced genes were over-represented for genes involved in sulphur metabolism and cycling, suggesting the possibility of an important role for maintenance of oxidative homeostasis during apple flower infection.

Fire blight, caused by Erwinia amylovora, is a devastating disease of apple. Blossom Protect, a product consisting of Aureobasidium pullulans as the active ingredient, is one of the most effective biological controls of fire blight. It has been postulated that the mode of action of A. pullulans is to compete against and to antagonize epiphytic growth of E. amylovora on flowers, but recent studies have found that flowers treated with Blossom Protect harbored similar to only slightly reduced E. amylovora populations compared to non-treated flowers. In this study, we tested the hypothesis that A. pullulans-mediated biocontrol of fire blight is the result of induced host resistance. We found that PR genes in the systemic acquired resistance pathway, but not genes in the induced systemic resistance pathway, were induced in hypanthial tissue of apple flowers after Blossom Protect treatment. Additionally, the induction of PR gene expression was coupled with an increase of plant-derived salicylic acid in this tissue. After inoculation with E. amylovora, PR gene expression was suppressed in non-treated flowers, but in flowers pre-treated with Blossom Protect, the heightened PR expression offset the immune repression caused by E. amylovora, and prevented infection. Temporal and spatial analysis of PR-gene induction showed that induction of PR genes occurred 2 days after Blossom Protect treatment, and required direct flower-yeast contact. Finally, we observed deterioration of the epidermal layer of the hypanthium in some of the Blossom Protect-treated flowers, suggesting that PR-gene induction in flowers may be a result of pathogenesis by A. pullulans.

Erwinia amylovora is a plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. This bacterium colonizes host vascular tissues via the production of exopolysaccharides (EPSs) including amylovoran. It is well-established that the nearly ubiquitous plasmid pEA29 of E. amylovora is an essential virulence factor, but the underlying mechanism remains uncharacterized. Here, we demonstrated that pEA29 was required for E. amylovora to produce amylovoran and to form a biofilm, and this regulation was dependent on the thiamine biosynthesis operon thiOSGF. We then conducted carbohydrate and genetic analyses demonstrating that the thiamine-mediated effect on amylovoran production was indirect, as cells lacking thiOSGF produced an EPS that did not contain glucuronic acid, one of the key components of amylovoran, whereas the transcriptional activity and RNA levels of the amylovoran biosynthesis genes were not altered. Alternatively, addition of exogenous thiamine restored amylovoran production in the pEA29-cured strain of E. amylovora and positively impacted amylovoran production in a dose-dependent manner. Individual deletion of several chromosomal thiamine biosynthesis genes also affected amylovoran production, implying that a complete thiamine biosynthesis pathway is required for the thiamine-mediated effect on amylovoran production in E. amylovora. Finally, we determined that an imbalanced tricarboxylic acid cycle negatively affected amylovoran production, which was restored by addition of exogenous thiamine or overexpression of the thiOSGF operon. In summary, our report revealed a novel signaling pathway that impacts E. amylovora virulence in which thiamine biosynthesis enhances bacterial respiration that provides energetic requirements for the biosynthesis of EPS amylovoran. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Fire blight, caused by the bacterial phytopathogen Erwinia amylovora, is an economically important and mechanistically complex disease that affects apple and pear production in most geographic production hubs worldwide. We compile, assess, and present a genetic outlook on the progression of an E. amylovora infection in the host. We discuss the key aspects of type III secretion–mediated infection and systemic movement, biofilm formation in xylem, and pathogen dispersal via ooze droplets, a concentrated suspension of bacteria and exopolysaccharide components. We present an overall outlook on the genetic elements contributing to E. amylovora pathogenesis, including an exploration of the impact of floral microbiomes on E. amylovora colonization, and summarize the current knowledge of host responses to an incursion and how this response stimulates further infection and systemic spread. We hope to facilitate the identification of new, unexplored areas of research in this pathosystem that can help identify evolutionarily susceptible genetic targets to ultimately aid in the design of sustainable strategies for fire blight disease mitigation.

We assessed the effect of three antibiotics (streptomycin, oxytetracycline, and kasugamycin) on populations of the fire blight pathogen Erwinia amylovora on apple flower stigmas during three field seasons. Application timing relative to E. amylovora presence on flower stigmas had little impact on population dynamics and subsequent disease incidence. Although E. amylovora populations on water-treated flowers increased to 106–7 cfu flower−1 after 4 to 5 days during each experiment, the antibiotics streptomycin and kasugamycin caused statistically significant reductions in stigma populations by as many as 4 to 5 logs over a 4- to 5-day period during two of the three experiments. In contrast, the effect of oxytetracycline on E. amylovora populations on stigmas was more variable, with reductions in E. amylovora populations only observed during one of the three experiments. In agreement with the population data, the disease incidence was significantly higher for oxytetracycline-treated flowers compared with the other antibiotic treatments during 2 of 3 years. Statistical analyses of the effects of weather parameters on antibiotic activity revealed that solar radiation and temperature negatively impacted the activity of both kasugamycin and oxytetracycline. We further assessed the potential for photodegradation of formulated kasugamycin (Kasumin 2L) and found that Kasumin 2L was susceptible to degradation in vitro after exposure to a 16-h photoperiod of daily light integrals (DLIs) varying from 6 to 35 mol⋅m−2⋅d−1. We further determined that exposure to three consecutive 16-h photoperiods of DLIs of 23 or 35 mol⋅m−2⋅d−1 reduced the available concentration of Kasumin 2L (assessed using a bioassay) from 100 μg⋅ml−1 to 10 to 20 μg⋅ml−1. Our results correlate the superior blossom blight control efficacy of kasugamycin and streptomycin with significant population reductions in E. amylovora on apple flower stigmas but indicate that, similar to oxytetracycline, kasugamycin is vulnerable to photodegradation, which would suggest that further considerations are necessary when applying this antibiotic.

Succinate dehydrogenase inhibitors (SDHIs) are a class of broad-spectrum fungicides used for management of diseases caused by phytopathogenic fungi. In many cases, reduced sensitivity to SDHI fungicides has been correlated with point mutations in the SdhB and SdhC target genes that encode components of the succinate dehydrogenase complex. However, the genetic basis of SDHI fungicide resistance mechanisms has been functionally characterized in very few fungi. Sclerotinia sclerotiorum is a fast-growing and SDHI fungicide–sensitive phytopathogenic fungus that can be conveniently transformed. Given the high amino acid sequence similarity and putative structural similarity of SDHI protein target sites between S. sclerotiorum and other common phytopathogenic ascomycete fungi, we developed an in vitro heterologous expression system that used S. sclerotiorum as a reporter strain. With this system, we were able to demonstrate the function of mutant SdhB or SdhC alleles from several ascomycete fungi in conferring resistance to multiple SDHI fungicides. In total, we successfully validated the function of Sdh alleles that had been previously identified in field isolates of Botrytis cinerea, Blumeriella jaapii, and Clarireedia jacksonii (formerly S. homoeocarpa) in conferring resistance to boscalid, fluopyram, or fluxapyroxad and used site-directed mutagenesis to construct and phenotype a mutant allele that is not yet known to exist in Monilinia fructicola populations. We also examined the functions of these alleles in conferring cross-resistance to more recently introduced SDHIs including inpyrfluxam, pydiflumetofen, and pyraziflumid. The approach developed in this study can be widely applied to interrogate SDHI fungicide resistance mechanisms in other phytopathogenic ascomycetes.

Resistance to sterol demethylation inhibitor (DMI) fungicides in Monilinia fructicola, causal agent of brown rot of stone fruit, has been reported in the southeastern and eastern United States and in Brazil. DMI resistance of some M. fructicola isolates, in particular those recovered from the southeastern United States, is associated with a sequence element termed “Mona” that causes overexpression of the cytochrome demethylase target gene MfCYP51. In this study, we conducted statewide surveys of Michigan stone fruit orchards from 2009 to 2011 and in 2019, and we determined the sensitivity to propiconazole of a total of 813 isolates of M. fructicola. A total of 80.7% of Michigan isolates were characterized as resistant to propiconazole by relative growth assays, but the Mona insert was not uniformly detected and was present in some isolates that were not characterized as DMI resistant. Gene expression assays indicated that elevated expression of MfCYP51 was only weakly correlated with DMI resistance in M. fructicola isolates from Michigan, and there was no obvious correlation between the presence of the Mona element and elevated expression of MfCYP51. However, sequence analysis of MfCYP51 from 25 DMI-resistant isolates did not reveal any point mutations that could be correlated with resistance. Amplification and sequencing upstream of MfCYP51 resulted in detection of DNA insertions in a wide range of isolates typed by DMI phenotype and the presence of Mona or other unique sequences. The function of these unique sequences or their presence upstream of MfCYP51 cannot be correlated to a DMI-resistant genotype at this time. Our results indicate that DMI resistance was established in Michigan populations of M. fructicola by 2009 to 2011, and that relative resistance levels have continued to increase to the point that practical resistance is present in most orchards. In addition, the presence of the Mona insert is not a marker for identifying DMI-resistant isolates of M. fructicola in Michigan.

Erwinia amylovora is a plant-pathogenic bacterium that causes fire blight disease in many economically important plants, including apples and pears. This bacterium produces three exopolysaccharides (EPSs), amylovoran, levan, and cellulose, and forms biofilms in host plant vascular tissues, which are crucial for pathogenesis. Here, we demonstrate that ProQ, a conserved bacterial RNA chaperone, was required for the virulence of E. amylovora in apple shoots and for biofilm formation in planta. In vitro experiments revealed that the deletion of proQ increased the production of amylovoran and cellulose. Prc is a putative periplasmic protease, and the prc gene is located adjacent to proQ. We found that Prc and the associated lipoprotein NlpI negatively affected amylovoran production, whereas Spr, a peptidoglycan hydrolase degraded by Prc, positively regulated amylovoran. Since the prc promoter is likely located within proQ, our data showed that proQ deletion significantly reduced the prc mRNA levels. We used a genome-wide transposon mutagenesis experiment to uncover the involvement of the bacterial second messenger c-di-GMP in ProQ-mediated cellulose production. The deletion of proQ resulted in elevated intracellular c-di-GMP levels and cellulose production, which were restored to wild-type levels by deleting genes encoding c-di-GMP biosynthesis enzymes. Moreover, ProQ positively affected the mRNA levels of genes encoding c-di-GMP-degrading phosphodiesterase enzymes via a mechanism independent of mRNA decay. In summary, our study revealed a detailed function of E. amylovora ProQ in coordinating cellulose biosynthesis and, for the first time, linked ProQ with c-di-GMP metabolism and also uncovered a role of Prc in the regulation of amylovoran production. IMPORTANCE Fire blight, caused by the bacterium Erwinia amylovora, is an important disease affecting many rosaceous plants, including apple and pear, that can lead to devastating economic losses worldwide. Similar to many xylem-invading pathogens, E. amylovora forms biofilms that rely on the production of exopolysaccharides (EPSs). In this paper, we identified the RNA-binding protein ProQ as an important virulence regulator. ProQ played a central role in controlling the production of EPSs and participated in the regulation of several conserved bacterial signal transduction pathways, including the second messenger c-di-GMP and the periplasmic protease Prc-mediated systems. Since ProQ has recently been recognized as a global posttranscriptional regulator in many bacteria, these findings provide new insights into multitiered regulatory mechanisms for the precise control of virulence factor production in bacterial pathogens.

Erwinia amylovora is the causative agent of the devastating disease fire blight of pome fruit trees. After infection of host plant leaves at apple shoot tips, E. amylovora cells form biofilms in xylem vessels, restrict water flow, and cause wilting symptoms. Although E. amylovora is well known to be able to cause systemic infection, how biofilm cells of E. amylovora transit from the sessile mode of growth in xylem to the planktonic mode of growth in cortical parenchyma remains unknown. Increasing evidence has suggested the important modulatory roles of Hfq‐dependent small RNAs (sRNAs) in the pathogenesis of E. amylovora. Here, we demonstrate that the sRNA RprA acts as a positive regulator of amylovoran exopolysaccharide production, the type III secretion system (T3SS), and flagellar‐dependent motility, and as a negative regulator of levansucrase activity and cellulose production. We also show that RprA affects the promoter activity of multiple virulence factor genes and regulates hrpS, a critical T3SS regulator, at the posttranscriptional level. We determined that rprA expression can be activated by the Rcs phosphorelay, and that expression is active during T3SS‐mediated host infection in an immature pear fruit infection model. We further showed that overexpression of rprA activated the in vitro dispersal of E. amylovora cells from biofilms. Thus, our investigation of the varied role of RprA in affecting E. amylovora virulence provides important insights into the functions of this sRNA in biofilm control and systemic infection., The small RNA RprA is a positive regulator of pathogenicity factors in Erwinia amylovora, and overexpression of rprA activates the in vitro dispersal of E. amylovora cells from biofilms.

Blumeriella jaapii is the causal agent of cherry leaf spot (CLS), the most important disease of tart cherry in the Midwestern United States. Infection of leaves by B. jaapii leads to premature defoliation, which places trees at heightened risk of winter injury and death. Current management of CLS relies primarily on the application of three important fungicide classes, quinone outside inhibitors, sterol demethylation inhibitors, and succinate dehydrogenase inhibitors. Here, we present the first high-quality genome of B. jaapii through a hybrid assembly of PacBio long reads and Illumina short reads. The assembled draft genome of B. jaapii is 47.4 Mb and consists of 95 contigs with a N50 value of 1.5 Mb. The genomic information of B. jaapii, representing the most complete sequenced genome of the family Dermateaceae (Ascomycota) to date, provides a valuable resource for identifying fungicide resistance mechanisms of this pathogen and expands our knowledge of the phytopathogenic fungi in this family.

Erwinia amylovora is a plant pathogenic bacterium that causes the disease fire blight in a wide range of Rosaceous plants including apples and pears. This pathogen utilizes the hypersensitive response and pathogenicity (hrp)-type III secretion system (T3SS), an essential pathogenicity factor of E. amylovora, to deliver proteins from bacteria to plant apoplasts or the cytoplasm to regulate host immune responses and physiology. Proteins secreted by the E. amylovora T3SS include five effectors: DspA/E, Eop1, Eop3, Eop4 (AvrRpt2Ea), and HopPtoCEa, two harpins: HrpN and HrpW, and other proteins such as Eop2, HrpJ, and HrpK. Homologs of these T3SS-secreted proteins have been characterized in plant pathogens like Pseudomonas syringae, Xanthomonas spp., Ralstonia spp., plant symbionts like Rhizobium spp. and animal pathogens such as Yersinia spp., implicating a central role of these effector proteins in manipulating bacterial interactions with diverse host species. Translocation of effectors is crucial for T3SS-mediated disease development in hosts. It is thought to be mediated by cognate chaperones, but the translocation of E. amylovora DspA/E was recently shown to be regulated by several different chaperones as well as a previously reported harpin, HrpN, in addition to its cognate chaperone DspB/F. This suggests that the dynamics of effector translocation are more complicated than previously expected in E. amylovora. In this review, we summarize the current knowledge of E. amylovora T3SS effectors, chaperones, and harpins together with their homologs from other plant-associated bacterial species. We also discuss the roles of T3SS-secreted proteins in pathogenicity and fitness via exploring their localization, activity, and direct or indirect targets in host plants. Finally, we provide future perspectives on studying effector biology and the T3SS in E. amylovora and how this fundamental knowledge provides potential applications in preventing fire blight.

Cyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates biofilm formation and pathogenicity. To study the global regulatory effect of individual components of the c-di-GMP metabolic system, we deleted all 12 diguanylate cyclase (dgc) and phosphodiesterase (pde)-encoding genes in E. amylovora Ea1189 (Ea1189Δ12). Ea1189Δ12 was impaired in surface attachment due to a transcriptional dysregulation of the type IV pilus and the flagellar filament. A transcriptomic analysis of surface-exposed WT Ea1189 and Ea1189Δ12 cells indicated that genes involved in metabolism, appendage generation and global transcriptional/post-transcriptional regulation were differentially regulated in Ea1189Δ12. Biofilm formation was regulated by all 5 Dgcs, whereas type III secretion and disease development were differentially regulated by specific Dgcs. A comparative transcriptomic analysis of Ea1189Δ8 (lacks all five enzymatically active dgc and 3 pde genes) against Ea1189Δ8 expressing specific dgcs, revealed the presence of a dual modality of spatial and global regulatory frameworks in the c-di-GMP signaling network.

Erwinia amylovora is an economically devastating plant pathogen that causes fire blight disease in members of the Rosaceae family, most notably in apple and pear. The exopolysaccharide amylovoran is a pathogenicity determinant in E. amylovora and a major component of the extracellular matrix of biofilms formed within the xylem vasculature of the host plant. The second messenger cyclic-di-GMP (c-di-GMP) has been reported to positively regulate the transcription of amsG (the first gene in the 12-gene amylovoran [ams] biosynthetic operon), thus impacting amylovoran production. However, the regulatory mechanism by which this interaction occurs is largely unknown. Here, we report that c-di-GMP can bind to specific residues in the EAL domain of the E. amylovora protein CsrD. CsrD and RNase E regulate the degradation of the sRNA CsrB in E. amylovora. When CsrD is bound to c-di-GMP, there is an enhancement in the level of RNase E-mediated degradation of CsrB, which then alters amsG transcription. Additionally, csrD was also found to positively contribute to virulence and biofilm formation. We thus present a pathway of conditional regulation of amylovoran production mediated by changing intracellular levels of c-di-GMP, which impacts disease progression.

Erwinia amylovora causes fire blight, the most devastating bacterial disease of apples and pears in the United States and worldwide. The model strain E. amylovora Ea1189 has been extensively used to understand bacterial pathogenesis and molecular mechanisms of bacterial-plant interactions. In this work, we sequenced and assembled the de novo genome of Ea1189, using a combination of long Oxford Nanopore Technologies and short Illumina sequence reads. A complete gapless genome assembly of Ea1189 consists of a 3,797,741-bp circular chromosome and a 28,259-bp plasmid with 3,472 predicted genes, including 78 transfer RNAs, 22 ribosomal RNAs, and 20 noncoding RNAs. A comparison of the Ea1189 genome to previously sequenced E. amylovora complete genomes showed 99.94 to 99.97% sequence similarity with 314 to 946 single nucleotide polymorphisms. We believe that the availability of the complete genome sequence of strain Ea1189 will further support studies to understand evolution, diversity and structural variations of Erwinia strains, as well as the molecular basis of E. amylovora pathogenesis and its interactions with host plants, thus facilitating the development of effective management strategies for this important disease.

A sensitive and specific assay was developed to detect bacterial leaf and flower spot disease on zinnia caused by Xanthomonas campestris pv. zinniae (Xcz). A draft genome sequence was determined for Xcz strain E4, isolated from zinnia in Jilin province, China. Comparative genomics analyses were utilized to identify a sequence specific to Xcz in the unique gene 0834. This sequence region was amplified as 1044 bp DNA fragment using designed primers 0834F and 0834R. Using this primer set, specific PCR products were only amplified from DNA from Xcz strains and not from other pathovars of X. campestris, other species of Xanthomonas, or from other genera such as Pseudomonas and Bacillus. The limit of PCR detection in pure culture suspension was approximately 1.7 × 103 CFU/ml per reaction. Specific amplification of the fragments for Xcz was sensitive relatively to the technique used, detecting as low as 0.1 pg template DNA. The PCR technique was also applied to detect the Xcz pathogen in naturally infected leaves and seeds of zinnia.

Trunk injections reduce pesticide inputs and environmental contamination, and recent work has addressed apple production systems. Insecticides and fungicides have been demonstrated for apple tree injection, however combinations of the two have not yet been tested. Trunk injections of the systemic insecticide emamectin benzoate and systemic acquired resistance (SAR) fungicide phosphorous acid were performed on mature apple trees to combine management strategies for foliar insect pests and apple scab (Venturia inaequalis). Injections of emamectin benzoate followed by phosphorous acid into the same set of injection ports resulted in higher mortality of Choristoneura rosaceana larvae and lower incidence of apple scab compared to the control. Scab incidence on trees in which phosphorous acid was injected into the same set of ports before emamectin benzoate were not different from control trees early in the growing season. Injections of emamectin benzoate and phosphorous acid into the same holes in either order showed higher mortality and reduced larval feeding in C. rosaceana bioassays compared with products injected into separate holes. This study demonstrates that two pesticides can interact dynamically within the vascular system of a tree, which has important implications for expanding the utility of trunk injection for fruit tree management.

Bacterial stalk and leaf necrosis of onion, Allium cepa L., is a concern of Michigan producers. Plants with symptoms of bacterial stalk and leaf necrosis were collected in 2013 and 2014 from 17 fields in six Michigan counties. Bacterial isolates were identified using Biolog and confirmed by sequencing the 16s rDNA gene. From a total of 414 isolates, 10 bacterial species were identified. Pantoea agglomerans (42.5%), Pantoea ananatis (17.4%), and Enterobacter cowanii (7.5%) were most prevalent and were tested for pathogenicity on onion foliage and bulbs. More P. ananatis isolates (92%) were pathogenic on seedlings than P. agglomerans or E. cowanii isolates (approximately 50%). When 197 bacterial isolates were tested for sensitivity to copper hydroxide (200 µg/ml), 41% of P. agglomerans isolates were found to be tolerant to copper hydroxide, whereas 19 and 22% of P. ananatis and E. cowanii isolates were tolerant, respectively. Identifying the bacterial species associated with foliar disease symptoms on onions in Michigan, their pathogenicity and tolerance to copper is an important step in developing improved disease management strategies.

Fire blight, caused by the bacterial phytopathogen

Cyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates the transition to biofilm formation in the phytopathogen Erwinia amylovora. The c-di-GMP system in E. amylovora is comprised of 12 diguanylate cyclase/Edc (dimerize cyclic-di-GMP) and phosphodiesterase/Pde (hydrolyze cyclic-di-GMP) proteins that are characterized by the presence of GGDEF and/or EAL motifs in their domain architecture. In order to study the global regulatory effect (without the inclusion of systemic regulatory impedance) of the c-di-GMP system in E. amylovora, we eliminated all 12 edc and pde genes in E. amylovora Ea1189Δ12. Comparisons between the representative transcriptomic profiles of Ea1189Δ12 and the combinatorial edc gene knockout mutant (Ea1189Δ5) revealed marked overall distinctions in expression levels for targets in a wide range of regulatory categories, including metabolic pathways involved in the utilization of methionine, isoleucine, histidine, etc. as well as critical signal transduction pathways including the Rcs phosphorelay and PhoPQ system. A complete loss of the cyclic-di-GMP signaling components resulted in the inability of Ea1189Δ12 cells to attach to and form biofilms in vitro and within the xylem vasculature in apple shoots. Using a flow-based in vitro biofilm system, we found that initial surface sensing was primarily dependent on the flagellar filament (FliC), following which the type IV pilus (HofC) was required to anchor cells to the surface to initialize biofilm development. A transcriptomic analysis of WT E. amylovora Ea1189 and Ea1189Δ12 cells in various stages of biofilm development revealed that cyclic-di-GMP based regulation had widespread effects on purine and pyrimidine biosynthesis pathways, amylovoran biosynthesis genes and the EnvZ/OmpR signal transduction system. Additionally, complementing individual eliminated genes back into Ea1189Δ12, and the collective evaluation of several virulence factors, enabled the correlative clustering of the functional effect rendered by each Edc and Pde enzyme in the system.SignificanceCyclic-di-GMP dependent regulation, in the context of biofilm formation, has been studied in several bacterial systems. However, the comprehensiveness of the studies exploring the role of individual genetic components related to cyclic-di-GMP is affected by the often large number of diguanylate cyclase and phosphodiesterase enzymes present within individual bacterial systems. To explore the evolutionary dependencies related to cyclic-di-GMP in E. amylovora, we used a collective elimination approach, whereby all of the enzymes involved in cyclic-di-GMP metabolism were eliminated from the system. This approach enabled us to highlight the critical importance of cyclic-di-GMP in plant xylem colonization due to its effect on surface attachment. Additionally, we highlight the global transcriptomic effect of cyclic-di-GMP dependent signaling at various stages of biofilm development. Our approach is aimed at exploring the regulatory role of individual cyclic-di-GMP related enzymes in a background that is free from any redundancy-based feedback.

Succinate dehydrogenase inhibitors (SDHIs) are a class of broad-spectrum fungicides used for management of diseases caused by phytopathogenic fungi. In many cases, reduced sensitivity to SDHI fungicides has been correlated with point mutations in the

Erwinia amylovora is the causal agent of fire blight, an economically impactful disease that affects apple and pear production worldwide. E. amylovora pathogenesis is comprised of distinct type III secretion-dependent and biofilm-dependent stages. Alterations in the intracellular levels of cyclic-di-GMP (c-di-GMP) regulate the transition between the different stages of infection in E. amylovora. We previously reported that hyper-elevation of c-di-GMP levels in E. amylovora Ea1189, resulting from the deletion of all three c-di-GMP specific phosphodiesterase genes (Ea1189ΔpdeABC), resulted in an autoaggregation phenotype. The two major exopolysaccharides, amylovoran and cellulose, were also shown to partially contribute to autoaggregation. In this study, we aimed to identify the c-di-GMP dependent factor(s) that contributes to autoaggregation. We conducted a transposon mutant screen in Ea1189ΔpdeABC and selected for loss of autoaggregation. Our search identified a peptidoglycan hydrolase, specifically, a D, D-endopeptidase of the metallopeptidase class, EagA (Erwinia aggregation factor A), that was found to physiologically contribute to autoaggregation in a c-di-GMP dependent manner. The production of amylovoran was also positively affected by EagA levels. An eagA deletion mutant (Ea1189ΔeagA) was significantly reduced in virulence compared to the wild type E. amylovora Ea1189. eagA is part of the znuABC zinc uptake gene cluster and is located within an operon downstream of znuA. The znuAeagA/znuCB gene cluster was transcriptionally regulated by elevated levels of c-di-GMP as well as by the zinc-dependent transcriptional repressor Zur. We also observed that with an influx of Zn2+ in the environment, the transcription of the znuAeagA/znuBC gene cluster is regulated by both Zur and a yet to be characterized c-di-GMP dependent pathway.

Resistance to sterol demethylation inhibitor (DMI) fungicides in

Background Toxin-antitoxin (TA) systems, abundant in prokaryotes, are composed of a toxin gene and its cognate antitoxin. Several toxins are implied to affect the physiological state and stress tolerance of bacteria in a population. We previously identified a chromosomally encoded hok-sok type I TA system in Erwinia amylovora, the causative agent of fire blight disease on pome fruit trees. A high-level induction of the hok gene was lethal to E. amylovora cells through unknown mechanisms. The molecular targets or regulatory roles of Hok were unknown. Results Here, we examined the physiological and transcriptomic changes of Erwinia amylovora cells expressing hok at subtoxic levels that were confirmed to confer no cell death, and at toxic levels that resulted in killing of cells. In both conditions, hok caused membrane rupture and collapse of the proton motive force in a subpopulation of E. amylovora cells. We demonstrated that induction of hok resulted in upregulation of ATP biosynthesis genes, and caused leakage of ATP from cells only at toxic levels. We showed that overexpression of the phage shock protein gene pspA largely reversed the cell death phenotype caused by high levels of hok induction. We also showed that induction of hok at a subtoxic level rendered a greater proportion of stationary phase E. amylovora cells tolerant to the antibiotic streptomycin. Conclusions We characterized the molecular mechanism of toxicity by high-level of hok induction and demonstrated that low-level expression of hok primes the stress responses of E. amylovora against further membrane and antibiotic stressors.

Summary Bacterial canker is a major disease of Prunus species, such as cherry (Prunus avium). It is caused by Pseudomonas syringae pathovars, including P. syringae pv. syringae (Pss) and P. syringae pv. morsprunorum race 1 (Psm1) and race 2 (Psm2). Concerns over the environmental impact of, and the development of bacterial resistance to, traditional copper controls calls for new approaches to disease management. Bacteriophage‐based biocontrol could provide a sustainable and natural alternative approach to combat bacterial pathogens. Therefore, seventy phages were isolated from soil, leaf and bark of cherry trees in six locations in the south east of England. Subsequently, their host range was assessed against strains of Pss, Psm1 and Psm2. While these phages lysed different Pss, Psm and some other P. syringae pathovar isolates, they did not infect beneficial bacteria such as Pseudomonas fluorescens. A subset of thirteen phages were further characterized by genome sequencing, revealing five distinct clades in which the phages could be clustered. No known toxins or lysogeny‐associated genes could be identified. Using bioassays, selected phages could effectively reduce disease progression in vivo, both individually and in cocktails, reinforcing their potential as biocontrol agents in agriculture., This study demonstrates a comprehensive analysis combining multiple elements that are significant for application of a bacteriophage as a potential biological control of bacterial canker of cherry. Bacteriophages were isolated from wide range of samples in cherry orchards in the UK. The phages had specific host range, with some able to lyse three strains of Pss, Psm1 and Psm2, causative agents of canker in cherry with an observed impact in reducing bacterial counts. Stability of the phages were tested at a range of different temperatures over a six‐month period and revealed they were capable of surviving in different environmental temperatures typical of the annual weather variations in the UK, which is a significant factor in choosing a phage for biocontrol. The phages could effectively reduce bacterial disease progression and infectivity in different plant systems. Genome sequencing of the bacteriophages revealed their novelty and divided the phages into five distinct clades, belonging to three different families, with no known toxins or lysogeny associated genes which indicate their use as an environmentally friendly biocontrol agent in cherry industry.

Antibiotics have been used for the management of relatively few bacterial plant diseases and are largely restricted to high-value fruit crops because of the expense involved. Antibiotic resistance in plant-pathogenic bacteria has become a problem in pathosystems where these antibiotics have been used for many years. Where the genetic basis for resistance has been examined, antibiotic resistance in plant pathogens has most often evolved through the acquisition of a resistance determinant via horizontal gene transfer. For example, the strAB streptomycin-resistance genes occur in Erwinia amylovora, Pseudomonas syringae, and Xanthomonas campestris, and these genes have presumably been acquired from nonpathogenic epiphytic bacteria colocated on plant hosts under antibiotic selection. We currently lack knowledge of the effect of the microbiome of commensal organisms on the potential of plant pathogens to evolve antibiotic resistance. Such knowledge is critical to the development of robust resistance management strategies to ensure the safe and effective continued use of antibiotics in the management of critically important diseases.