Thiosulfinate Tolerance Gene Clusters Are Common Features of Burkholderia Onion Pathogens.

Burkholderia gladioli pv. alliicola, B. cepacia, and B. orbicola are common bacterial pathogens of onion. Onions produce organosulfur thiosulfinate defensive compounds after cellular decompartmentalization. Using whole-genome sequencing and in silico analysis, we identified putative thiosulfinate tolerance gene (TTG) clusters in multiple onion-associated Burkholderia species similar to those characterized in other Allium-associated bacterial endophytes and pathogens. Sequence analysis revealed the presence of three Burkholderia TTG cluster types, with both Type A and Type B being broadly distributed in B. gladioli, B. cepacia, and B. orbicola in both the chromosome and plasmids. Based on isolate natural variation and generation of isogenic strains, we determined the in vitro and in vivo contribution of TTG clusters in B. gladioli, B. cepacia, and B. orbicola. The Burkholderia TTG clusters contributed to enhanced allicin tolerance and improved growth in filtered onion extracts by all three species. TTG clusters also made clear contributions to B. gladioli foliar necrosis symptoms and bacterial populations. Surprisingly, the TTG cluster did not contribute to bacterial populations in onion bulb scales by these three species. Based on our findings, we hypothesize onion-associated Burkholderia may evade or inhibit the production of thiosulfinates in onion bulb tissues. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Genetic Requirements for HiVir-Mediated Onion Necrosis by Pantoea ananatis, a Necrotrophic Plant Pathogen.

Pantoea ananatis is an unusual bacterial pathogen that lacks typical virulence determinants yet causes extensive necrosis in onion foliage and bulb tissues. The onion necrosis phenotype is dependent on the expression of the phosphonate toxin, pantaphos, which is synthesized by putative enzymes encoded by the HiVir (high virulence) gene cluster. The genetic contributions of individual hvr genes in HiVir-mediated onion necrosis remain largely unknown, except for the first gene, hvrA (phosphoenolpyruvate mutase, pepM), whose deletion resulted in the loss of onion pathogenicity. In this study, using gene-deletion mutation and complementation, we report that, of the ten remaining genes, hvrB to hvrF are also strictly required for the HiVir-mediated onion necrosis and in-planta bacterial growth, whereas hvrG to hvrJ partially contributed to these phenotypes. As the HiVir gene cluster is a common genetic feature shared among the onion-pathogenic P. ananatis strains that could serve as a useful diagnostic marker of onion pathogenicity, we sought to understand the genetic basis of HiVir-positive yet phenotypically deviant (non-pathogenic) strains. We identified and genetically characterized inactivating single nucleotide polymorphisms in the essential hvr genes of six phenotypically deviant P. ananatis strains. Finally, inoculation of cell-free spent medium of the isopropylthio-ß-galactoside (IPTG)-inducible promoter (Ptac)-driven HiVir strain caused P. ananatis-characteristic red onion scale necrosis as well as cell death symptoms in tobacco. Co-inoculation of the spent medium with essential hvr mutant strains restored in-planta populations of the strains to the wild-type level, suggesting that necrotic tissues are important for the proliferation of P. ananatis in onion. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Shoot Maturation Strengthens FLS2-Mediated Resistance to Pseudomonas syringae.

Temporospatial regulation of immunity components is essential for properly activating plant defense response. Flagellin-sensing 2 (FLS2) is a surface-localized receptor that recognizes bacterial flagellin. The immune function of FLS2 is compromised in early stages of shoot development. However, the underlying mechanism for the age-dependent FLS2 signaling is not clear. Here, we show that the reduced basal immunity of juvenile leaves against Pseudomonas syringae pv. tomato DC3000 is independent of FLS2. The flg22-induced marker gene expression and reactive oxygen species activation were comparable in juvenile and adult stages, but callose deposition was more evident in the adult stage than the juvenile stage. We further demonstrated that microRNA156, a master regulator of plant aging, does not influence the expression of FLS2 and FRK1 (Flg22-induced receptor-like kinase 1) but mildly suppresses callose deposition in juvenile leaves. Our experiments revealed an intrinsic mechanism that regulates the amplitude of FLS2-mediated resistance during aging. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Novel Biosynthetic Gene Cluster Across the Pantoea Species Complex Is Important for Pathogenicity in Onion.

Onion center rot is caused by at least four species of genus Pantoea (P. ananatis, P. agglomerans, P. allii, and P. stewartii subsp. indologenes). Critical onion pathogenicity determinants for P. ananatis were recently described, but whether those determinants are common among other onion-pathogenic Pantoea species remains unknown. In this work, we report onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. We identified two distinct secondary metabolite biosynthetic gene clusters present separately in different strains of onion-pathogenic P. stewartii subsp. indologenes. One cluster is similar to the previously described HiVir phosphonate biosynthetic cluster identified in P. ananatis and another is a novel putative phosphonate biosynthetic gene cluster, which we named Halophos. The Halophos gene cluster was also identified in P. allii strains. Both clusters are predicted to be phosphonate biosynthetic clusters based on the presence of a characteristic phosphoenolpyruvate phosphomutase (pepM) gene. The deletion of the pepM gene from either HiVir or Halophos clusters in P. stewartii subsp. indologenes caused loss of necrosis on onion leaves and red onion scales and resulted in significantly lower bacterial populations compared with the corresponding wild-type and complemented strains. Seven (halB to halH) of 11 genes (halA to halK) in the Halophos gene cluster are required for onion necrosis phenotypes. The onion nonpathogenic strain PNA15-2 (P. stewartii subsp. indologenes) gained the capacity to cause foliar necrosis on onion via exogenous expression of a minimal seven-gene Halophos cluster (genes halB to halH). Furthermore, cell-free culture filtrates of PNA14-12 expressing the intact Halophos gene cluster caused necrosis on onion leaves consistent with the presence of a secreted toxin. Based on the similarity of proteins to those with experimentally determined functions, we are able to predict most of the steps in Halophos biosynthesis. Together, these observations indicate that production of the toxin phosphonate seems sufficient to account for virulence of a variety of different Pantoea strains, although strains differ in possessing a single but distinct phosphonate biosynthetic cluster. Overall, this is the first report of onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pantailocins: phage-derived bacteriocins from Pantoea ananatis and Pantoea stewartii subsp. indologenes.

IMPORTANCE: Phage-derived bacteriocins (tailocins) are ribosomally synthesized structures produced by bacteria in order to provide advantages against competing strains under natural conditions. Tailocins are highly specific in their target range and have proven to be effective for the prevention and/or treatment of bacterial diseases under clinical and agricultural settings. We describe the discovery and characterization of a new tailocin locus encoded within genomes of Pantoea ananatis and Pantoea stewartii subsp. indologenes, which may enable the development of tailocins as preventative treatments against phytopathogenic infection by these species.

Pathogenic Bacteria Target Plant Plasmodesmata to Colonize and Invade Surrounding Tissues.

A hallmark of multicellular organisms is their ability to maintain physiological homeostasis by communicating among cells, tissues, and organs. In plants, intercellular communication is largely dependent on plasmodesmata (PD), which are membrane-lined channels connecting adjacent plant cells. Upon immune stimulation, plants close PD as part of their immune responses. Here, we show that the bacterial pathogen Pseudomonas syringae deploys an effector protein, HopO1-1, that modulates PD function. HopO1-1 is required for P. syringae to spread locally to neighboring tissues during infection. Expression of HopO1-1 in Arabidopsis (Arabidopsis thaliana) increases the distance of PD-dependent molecular flux between neighboring plant cells. Being a putative ribosyltransferase, the catalytic activity of HopO1-1 is required for regulation of PD. HopO1-1 physically interacts with and destabilizes the plant PD-located protein PDLP7 and possibly PDLP5. Both PDLPs are involved in bacterial immunity. Our findings reveal that a pathogenic bacterium utilizes an effector to manipulate PD-mediated host intercellular communication for maximizing the spread of bacterial infection.

Discovery of the Hrp Type III Secretion System in Phytopathogenic Bacteria: How Investigation of Hypersensitive Cell Death in Plants Led to a Novel Protein Injector System and a World of Inter-Organismal Molecular Interactions Within Plant Cells.

In the early 1960s, Pseudomonas syringae and other host-specific phytopathogenic proteobacteria were discovered to elicit a rapid, resistance-associated death when infiltrated at high inoculum levels into nonhost tobacco leaves. This hypersensitive reaction (or response; HR) was a useful indicator of basic pathogenic ability. Research over the next 20 years failed to identify an elicitor of the HR but revealed that its elicitation required contact between metabolically active bacterial and plant cells. Beginning in the early 1980s, molecular genetic tools were applied to the HR puzzle, revealing the presence in P. syringae of clusters of hrp genes, so named because they are required for the HR and pathogenicity, and of avr genes, so named because their presence confers HR-associated avirulence in resistant cultivars of a host plant species. A series of breakthroughs over the next two decades revealed that (i) hrp gene clusters encode a type III secretion system (T3SS), which injects Avr (now "effector") proteins into plant cells, where their recognition triggers the HR; (ii) T3SSs, which are typically present in pathogenicity islands acquired by horizontal gene transfers, are found in many bacterial pathogens of plants and animals and inject many effector proteins, which are collectively essential for pathogenicity; and (iii) a primary function of phytopathogen effectors is to subvert non-HR defenses resulting from recognition of conserved microbial features presented outside of plant cells. In the 2000s, Hrp system research shifted to extracellular components enabling effector delivery across plant cell walls and plasma membranes, regulation, and tools for studying effectors. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Evaluating Options to Increase the Efficacy of Biocontrol Agents for the Management of Pantoea spp. Under Field Conditions.

Center rot of onion is caused by a complex of plant pathogenic Pantoea species, which can lead to significant yield losses in the field and during storage. Conventional growers use foliar protectants such as a mixture of copper bactericides and an ethylene-bis-dithiocarbamate (EBDC) fungicide to manage the disease; however, organic growers have limited management options besides copper-protectants. Biocontrol agents (BCAs) provide an alternative; however, their efficacy could be compromised due in part to their inability to colonize the foliage. We hypothesized that pretreatment with peroxide (OxiDate 2.0: a.i., hydrogen peroxide and peroxyacetic acid) enhances the colonizing ability of the subsequently applied BCAs, leading to effective center rot management. Field trials were conducted in 2020 and 2021 to assess the efficacy of peroxide, BCAs (Serenade ASO: Bacillus subtilis and BlightBan: Pseudomonas fluorescens), and an insecticide program (tank mix of spinosad and neem oil) to manage center rot. We observed no significant difference in foliar area under the disease progress curve (AUDPC) between the peroxide pretreated P. fluorescens plots and only P. fluorescens-treated plots in 2020 and 2021. Peroxide pretreatment before B. subtilis application significantly reduced the foliar AUDPC as compared with the stand-alone B. subtilis treatment in 2020; however, no such difference was observed in 2021. Similarly, peroxide pretreatment before either of the BCAs did not seem to reduce the incidence of bulb rot as compared with the stand-alone BCA treatment in any of the trials (2020 and 2021). Additionally, our foliar microbiome study showed comparatively higher P. fluorescens retention on peroxide pretreated onion foliage; however, at the end of the growing season, P. fluorescens was drastically reduced and was virtually nonexistent (<0.002% of the total reads). Overall, the pretreatment with peroxide had a limited effect in improving the foliar colonizing ability of BCAs and consequently a limited effect in managing center rot.

Validation of Species-Specific PCR Assays for the Detection of Pantoea ananatis, P. agglomerans, P. allii, and P. stewartii.

Species of Pantoea represent a group of plant pathogenic bacteria that infect a variety of agro-economically important plant species. Among these, a complex of P. ananatis, P. allii, P. agglomerans, and P. stewartii subsp. indologenes cause center rot in onion, resulting in significant economic losses. As species of Pantoea are phenotypically closely related, identification of Pantoea species relies on the sequencing and phylogenetic analysis of housekeeping genes. To aid in rapid identification of Pantoea species, efforts have been made in developing species-specific primers to be used in PCR assays. In the current study, two P. ananatis, one P. allii, one P. agglomerans, and three P. stewartii published primers as well as newly developed P. agglomerans PagR primers were evaluated for their specificity against 79 Pantoea strains, belonging to 15 different species. To ensure that selected primers were evaluated against accurately identified species, sequencing and phylogenetic analysis of housekeeping gene infB were conducted. Thereafter, PCR assays using selected species-specific primers were performed. The results showed that previously described P. ananatis-specific PANA_1008; P. allii-specific allii-leuS; P. stewartii-specific PANST_rpoB, 3614galE, and DC283galE primers; and one newly designed P. agglomerans-specific PagR primer pair were highly specific for their target Pantoea species. They accurately identified these strains into their species and, in some cases, their subspecies level. The findings of the current study will facilitate rapid and reliable identification of P. ananatis, P. agglomerans, P. allii, and P. stewartii.

AlgU, a Conserved Sigma Factor Regulating Abiotic Stress Tolerance and Promoting Virulence in Pseudomonas syringae.

Pseudomonas syringae can rapidly deploy specialized functions to deal with abiotic and biotic stresses. Host niches pose specific sets of environmental challenges driven, in part, by immune defenses. Bacteria use a "just-in-time" strategy of gene regulation, meaning that they only produce the functions necessary for survival as needed. Extracytoplasmic function (ECF) sigma factors transduce a specific set of environmental signals and change gene expression patterns by altering RNA polymerase promoter specificity, to adjust bacterial physiology, structure, or behavior, singly or in combination, to improve chances of survival. The broadly conserved ECF sigma factor AlgU affects virulence in both animal and plant pathogens. Pseudomonas syringae AlgU controls expression of more than 800 genes, some of which contribute to suppression of plant immunity and bacterial fitness in plants. This review discusses AlgU activation mechanisms, functions controlled by AlgU, and how these functions contribute to P. syringae survival in plants.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.

Pseudomonas capsici sp. nov., a plant-pathogenic bacterium isolated from pepper leaf in Georgia, USA.

Three phytopathogenic bacterial strains (Pc19-1T, Pc19-2 and Pc19-3) were isolated from seedlings displaying water-soaked, dark brown-to-black, necrotic lesions on pepper (Capsicum annuum) leaves in Georgia, USA. Upon isolation on King's medium B, light cream-coloured colonies were observed and a diffusible fluorescent pigment was visible under ultraviolet light. Analysis of their 16S rRNA gene sequences showed that they belonged to the genus Pseudomonas, with the highest similarity to Pseudomonas cichorii ATCC 10857T (99.7 %). The fatty acid analysis revealed that the majority of the fatty acids were summed feature 3 (C16  :  1 ω7c/C16  :  1 ω6c), C16  :  0 and summed feature 8 (C18  :  1 ω7c/C18  :  1 ω6c). Phylogenomic analyses based on whole genome sequences demonstrated that the pepper strains belonged to the Pseudomonas syringae complex with P. cichorii as their closest neighbour, and formed a separate monophyletic clade from other species. Between the pepper strains and P. cichorii, the average nucleotide identity values were 91.3 %. Furthermore, the digital DNA-DNA hybridization values of the pepper strains when compared to their closest relatives, including P. cichorii, were 45.2 % or less. In addition, biochemical and physiological features were examined in this study and the results indicate that the pepper strains represent a novel Pseudomonas species. Therefore, we propose a new species Pseudomonas capsici sp. nov., with Pc19-1T (=CFBP 8884T=LMG 32209T) as the type strain. The DNA G+C content of the strain Pc19-1T is 58.4 mol%.

Patterns of Seed-to-Seedling Transmission of Xanthomonas citri pv. malvacearum, the Causal Agent of Cotton Bacterial Blight.

Cotton bacterial blight (CBB), caused by Xanthomonas citri pv. malvacearum, was a major disease of cotton in the United States in the early part of the twentieth century. The reemergence of CBB revealed many gaps in our understanding of this important disease. In this study, we employed a wild-type (WT) field isolate of X. citri pv. malvacearum from Georgia (U.S.A.) to generate a nonpathogenic hrcV mutant lacking a functional type-III secretion system (T3SS-). We tagged the WT and T3SS- strains with an auto-bioluminescent Tn7 reporter and compared colonization patterns of CBB-susceptible and CBB-resistant cotton seedlings using macroscopic image analysis and bacterial load enumeration. WT and T3SS- X. citri pv. malvacearum strains colonized cotton cotyledons of CBB-resistant and CBB-susceptible cotton cultivars. However, X. citri pv. malvacearum populations were significantly higher in CBB-susceptible seedlings inoculated with the WT strain. Additionally, WT and T3SS- X. citri pv. malvacearum strains systemically colonized true leaves, although at different rates. Finally, we observed that seed-to-seedling transmission of X. citri pv. malvacearum may involve systemic spread through the vascular tissue of cotton plants. These findings yield novel insights into potential X. citri pv. malvacearum reservoirs for CBB outbreaks.

Identification of Two Novel Pathovars of Pantoea stewartii subsp. indologenes Affecting Allium sp. and Millets.

Pantoea stewartii subsp. indologenes is a causative agent of leafspot of foxtail millet and pearl millet; however, novel strains were recently identified that are pathogenic on onion. We phenotypically and genotypically characterized 17 P. stewartii subsp. indologenes strains from onion and other sources (pearl millet, foxtail millet, guar pulse, verbena, and corn). Based on the host range evaluation, we propose two pathovars: P. stewartii subsp. indologenes pv. cepacicola pv. nov. and P. stewartii subsp. indologenes pv. setariae pv. nov. P. stewartii subsp. indologenes pv. cepacicola pv. nov. causes symptoms on Allium spp. (leek, onion, chive, and Japanese bunching onion) and on foxtail millet, pearl millet, and oat. However, P. stewartii subsp. indologenes pv. setariae pv. nov. can only infect the members of Poaceae family (foxtail millet, pearl millet, and oat). We also propose that the type strain of P. stewartii subsp. indologenes (LMG 2632T) should be designated as a pathotype strain of P. stewartii subsp. indologenes pv. setariae and recommend that the strain PNA 14-12 be designated as the pathotype strain of P. stewartii subsp. indologenes pv. cepacicola. The digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and multilocus sequence analysis study showed that the two pathovars are genotypically closely related. Our study also showed that P. stewartii subsp. indologenes pathovars and P. stewartii subsp. stewartii share high genotypic relatedness and cannot be differentiated by dDDH and ANI values. Although the newly proposed pathovars are not clearly distinguishable by their fatty acid and methyl esterase profiles and substrate use patterns, a fatty acid (unknown with retention time: 10.9525) and a few metabolites (3-methyl glucose, Na butyrate, and fusidic acid) can be potentially used to distinguish them. We also report the distribution of previously known pathogenicity (HiVir, hrcC) and virulence (alt) factors of Pantoea spp. in the new pathovars. The impact of these new pathovars in the center rot pathosystem of onion is yet to be determined.

Transcriptome landscape of a bacterial pathogen under plant immunity.

Plant pathogens can cause serious diseases that impact global agriculture. The plant innate immunity, when fully activated, can halt pathogen growth in plants. Despite extensive studies into the molecular and genetic bases of plant immunity against pathogens, the influence of plant immunity in global pathogen metabolism to restrict pathogen growth is poorly understood. Here, we developed RNA sequencing pipelines for analyzing bacterial transcriptomes in planta and determined high-resolution transcriptome patterns of the foliar bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana with a total of 27 combinations of plant immunity mutants and bacterial strains. Bacterial transcriptomes were analyzed at 6 h post infection to capture early effects of plant immunity on bacterial processes and to avoid secondary effects caused by different bacterial population densities in planta We identified specific "immune-responsive" bacterial genes and processes, including those that are activated in susceptible plants and suppressed by plant immune activation. Expression patterns of immune-responsive bacterial genes at the early time point were tightly linked to later bacterial growth levels in different host genotypes. Moreover, we found that a bacterial iron acquisition pathway is commonly suppressed by multiple plant immune-signaling pathways. Overexpression of a P. syringae sigma factor gene involved in iron regulation and other processes partially countered bacterial growth restriction during the plant immune response triggered by AvrRpt2. Collectively, this study defines the effects of plant immunity on the transcriptome of a bacterial pathogen and sheds light on the enigmatic mechanisms of bacterial growth inhibition during the plant immune response.

Widely Conserved Attenuation of Plant MAMP-Induced Calcium Influx by Bacteria Depends on Multiple Virulence Factors and May Involve Desensitization of Host Pattern Recognition Receptors.

Successful pathogens must efficiently defeat or delay host immune responses, including those triggered by release or exposure of microbe-associated molecular patterns (MAMPs). Knowledge of the molecular details leading to this phenomenon in genuine plant-pathogen interactions is still scarce. We took advantage of the well-established Arabidopsis thaliana-Pseudomonas syringae pv. tomato DC3000 pathosystem to explore the molecular prerequisites for the suppression of MAMP-triggered host defense by the bacterial invader. Using a transgenic Arabidopsis line expressing the calcium sensor apoaequorin, we discovered that strain DC3000 colonization results in a complete inhibition of MAMP-induced cytosolic calcium influx, a key event of immediate-early host immune signaling. A range of further plant-associated bacterial species is also able to prevent, either partially or fully, the MAMP-triggered cytosolic calcium pattern. Genetic analysis revealed that this suppressive effect partially relies on the bacterial type III secretion system (T3SS) but cannot be attributed to individual members of the currently known arsenal of strain DC3000 effector proteins. Although the phytotoxin coronatine and bacterial flagellin individually are dispensable for the effective inhibition of MAMP-induced calcium signatures, they contribute to the attenuation of calcium influx in the absence of the T3SS. Our findings suggest that the capacity to interfere with early plant immune responses is a widespread ability among plant-associated bacteria that, at least in strain DC3000, requires the combinatorial effect of multiple virulence determinants. This may also include the desensitization of host pattern recognition receptors by the prolonged exposure to MAMPs during bacterial pathogenesis.

Pattern-Triggered Immunity Alters the Transcriptional Regulation of Virulence-Associated Genes and Induces the Sulfur Starvation Response in Pseudomonas syringae pv. tomato DC3000.

Pattern-triggered immunity (PTI) can confer broad defense against diverse microbes and pathogens with disparate lifestyles through the detection of microbial extracellular signatures by surface-exposed pattern recognition receptors. However, unlike recognition of pathogen effectors by cytosolic resistance proteins, PTI is typically not associated with a host-cell programmed cell death response. Although host PTI signaling has been extensively studied, the mechanisms by which it restricts microbial colonization are poorly understood. We sought to gain insight into the mechanisms of PTI action by using bacterial transcriptomics analysis during exposure to PTI. Here, we describe a method for bacterial cell extraction from inoculated leaves that was used to analyze a time course of genome-wide transcriptional responses in the pathogen Pseudomonas syringae pv. tomato DC3000 during early naïve host infection and exposure to pre-induced PTI in Arabidopsis thaliana. Our analysis revealed early transcriptional regulation of important bacterial metabolic processes and host interaction pathways. We observed peak induction of P. syringae virulence genes at 3 h postinoculation and that exposure to PTI was associated with significant reductions in the expression of virulence genes. We also observed the induction of P. syringae sulfur starvation response genes such as sulfate and sulfonate importers only during exposure to PTI.

Validation of RT-qPCR Approaches to Monitor Pseudomonas syringae Gene Expression During Infection and Exposure to Pattern-Triggered Immunity.

Pseudomonas syringae pv. tomato DC3000 is an important model plant pathogen, with a fully annotated genome and multiple compatible plant hosts. Very few studies have examined the regulation of DC3000 gene expression in vivo. We developed a quantitative reverse transcription-polymerase chain reaction assay to monitor transcriptional changes in DC3000 inoculated into Arabidopsis thaliana leaves during disease and exposure to pattern-triggered immunity (PTI). In our approach, bacterial RNA concentrations in total tissue RNA are standardized using P. syringae-specific 16S ribosomal RNA primers. We validated multiple stable reference genes for normalization in calculating the relative expression of genes of interest. We used empirically derived rates of amplification efficiency to calculate relative expression of key marker genes for virulence-associated regulation. We demonstrated that exposure to PTI alters DC3000 expression of type III secretion system, coronatine synthesis genes, and flagellar marker genes.

Isolation and Characterization of Novel Pantoea stewartii subsp. indologenes Strains Exhibiting Center Rot in Onion.

Center rot of onion is an economically important disease caused by three Pantoea spp.: Pantoea ananatis, P. agglomerans, and P. allii. Symptoms caused by these three species are similar and include white streaking and necrosis of foliage; and, in some cases, the bacterium may enter the bulb, causing liquefaction and rot of bulb scales. Two bacterial strains were isolated from onion expressing symptoms indicative of center rot from two different outbreaks in Toombs County, GA in 2003 (PNA 03-3) and 2014 (PNA 14-12). These strains were initially identified as P. ananatis based on physiological and specific polymerase chain reaction (PCR) assays; however, further 16S ribosomal RNA (rRNA) and multilocus sequence analysis showed that the strains were more closely related to P. stewartii subsp. stewartii and P. stewartii subsp. indologenes. Further characterization using phylogenetic analysis, a P. stewartii subsp. indologenes-specific PCR assay, indole test, and pathogenicity on onion and pearl millet were conducted. Phylogenetic analyses (16S rRNA and atpD, gyB, infB, and rpoB genes) revealed that these strains formed a distinct cluster with the type strains of P. stewartii subsp. indologenes LMG 2632T and P. stewartii subsp. stewartii LMG 2715T separate from P. ananatis, P. agglomerans, and P. allii. Furthermore, onion strains were amplified with the P. stewartii subsp. indologenes-specific PCR assay. The pathogenicity assays with onion strains showed that they were pathogenic on onion and pearl millet, a known host of P. stewartii subsp. indologenes. However, the type strain of P. stewartii subsp. indologenes LMG 2632T was pathogenic only on pearl millet but not on onion. These results suggest that the onion strains PNA 03-3 and PNA 14-12 can potentially be novel P. stewartii subsp. indologenes strains capable of producing symptoms on onion. Hence, we recommend the inclusion of P. stewartii subsp. indologenes as the fourth member in the center rot complex of onion, along with P. ananatis, P. agglomerans, and P. allii.