Jo Ann E. Asselin

Pantoea allii , one of four Pantoea species known to cause onion centre rot, is infrequently isolated from onion compared to its closely related onion-pathogenic species in the same genus. To better understand the genomic diversity and genetic determinants of pathogenicity in this species, we analysed a collection of 38 P . allii strains isolated from 2 primary ecological niches, plants and rainwater, across North and South American and African continents using comparative genomics and phylogenetic approaches. Core-genome phylogeny, average nucleotide identity and gene presence–absence analyses revealed three genetically distinct lineages. All strains harboured conserved biosynthetic gene clusters (BGCs) for quorum sensing, carotenoid production, siderophores and thiopeptides. In contrast, two phosphonate BGCs, key determinants of onion pathogenicity, exhibited lineage-specific distributions. Onion-associated strains from lineages 1 and 2 carried the Halophos BGC responsible for onion tissue necrosis and also encoded the alt gene cluster that confers tolerance to thiosulfinates. Lineage 3 strains, isolated from both onion and rainwater, either lacked a phosphonate BGC entirely or possessed the HiVir phosphonate BGC. In addition, lineage 3 strains lacked the alt cluster altogether. The localization of these virulence genes in the genome varied, with Halophos integrated in the chromosome, HiVir encoded on the conserved Large Pantoea Plasmid, and alt located on small, variable plasmids (plasmid B). The type IV secretion system (T4SS) and type VI secretion system (T6SS) showed variable genomic architectures, with plasmid-borne T4SSs and two chromosomal T6SS loci differing in conservation and gene content. Additionally, conserved Pantailocin phage islands were detected in most genomes. Overall, this study reveals that while core metabolic and competitive traits are conserved across P. allii , virulence-associated loci display lineage-specific distribution, reflecting ecological differentiation and evolutionary plasticity within the species.

Pantoea allii , one of four Pantoea species known to cause onion center rot, is infrequently isolated from onion compared to its closely related onion-pathogenic sister taxa. To better understand the genomic diversity and genetic determinants of pathogenicity in this species, we analyzed a collection of 38 P. allii strains isolated from two primary ecological niches, plants and water, across three continents using comparative genomics and phylogenetic approaches. Core-genome phylogeny, average nucleotide identity (ANI), and gene presence–absence analyses revealed three genetically distinct lineages. All strains harbored conserved biosynthetic gene clusters (BGCs) for quorum sensing, carotenoid production, siderophores, and thiopeptides. In contrast, two phosphonate BGCs, key determinants of onion pathogenicity, exhibited lineage-specific distributions. Onion-associated strains from Lineages 1 and 2 carried the Halophos BGC associated with onion tissue necrosis, and onion isolates encoded the alt gene cluster conferring thiosulfinate tolerance. Lineage 3 strains, isolated from both onion and rainwater, either lacked a phosphonate BGC loci or carried the HiVir phosphonate BGC. In addition, Lineage 3 strains lacked the alt cluster altogether. The localization of these virulence genes in the genome varied, with Halophos integrated in the chromosome, HiVir encoded on the conserved Large Pantoea Plasmid, and alt located on a small, variable plasmids (plasmid B). The Type IV and Type VI secretion systems showed variable genomic architectures, with plasmid-borne T4SSs and two chromosomal T6SS loci differing in conservation and gene content. Additionally, conserved Pantailocin phage islands were detected in most genomes. Overall, this study reveals that while core metabolic and competitive traits are conserved across P. allii , virulence-associated loci display lineage-specific partitioning, reflecting ecological differentiation and evolutionary plasticity within the species. Impact Statement: This study presents a comprehensive comparative genomic analysis of available Pantoea allii genomes, a known onion pathogen. By analyzing 38 strains isolated from plant and water sources across three continents, we uncovered lineage-specific distributions of key virulence genes, alongside conserved genetic traits associated with competition and environmental resilience. These findings clarify the genetic basis of P. allii pathogenesis and highlight its potential as a biocontrol agent, offering broader insights into how ecologically significant loci contribute to the dual roles of plant-associated bacteria as both pathogens and beneficial microbes. Data Summary: PENDING

Bacteria isolated from onion bulbs suffering from bacterial decay in the United States and Norway were previously shown to belong to the genus Rahnella based on partial housekeeping gene sequences and/or fatty acid analysis. However, many strains could not be assigned to any existing Rahnella species. Additionally, strains isolated from creek water and oak as well as a strain with bioremediation properties were assigned to Rahnella based on partial housekeeping gene sequences. The taxonomic status of these 21 strains was investigated using multilocus sequence analysis, whole genome analyses, phenotypic assays and fatty acid analysis. Phylogenetic and phylogenomic analyses separated the strains into five clusters, one of which corresponded to Rahnella aceris . The remaining four clusters could be differentiated both genotypically and phenotypically from each other and existing Rahnella species. Based on these results, we propose the description of four novel species: Rahnella perminowiae sp. nov. (type strain SL6^T=LMG 32257^T=DSM 112609^T), Rahnella bonaserana sp. nov. (H11b^T=LMG 32256^T=DSM 112610^T), Rahnella rivi sp. nov. (FC061912-K^T=LMG 32259^T=DSM 112611^T) and Rahnella ecdela sp. nov. (FRB 231^T=LMG 32255^T=DSM 112612^T).

Aims Bacterial decays of onion bulbs have serious economic consequences for growers, but the aetiologies of these diseases are often unclear. We aimed to determine the role of Rahnella , which we commonly isolated from bulbs in the United States and Norway, in onion disease. Methods and Results Isolated bacteria were identified by sequencing of housekeeping genes and/or fatty acid methyl ester analysis. A subset of Rahnella spp. strains was also assessed by multilocus sequence analysis (MLSA); most onion strains belonged to two clades that appear closely related to R. aquatilis . All tested strains from both countries caused mild symptoms in onion bulbs but not leaves. Polymerase chain reaction primers were designed and tested against strains from known species of Rahnella. Amplicons were produced from strains of R. aquatilis , R. victoriana , R. variigena , R. inusitata and R. bruchi , and from one of the two strains of R. woolbedingensis . Conclusions Based on binational testing, strains of Rahnella are commonly associated with onions, and they are capable of causing mild symptoms in bulbs. Significance and Impact of the Study While Rahnella strains are commonly found within field‐grown onions and they are able to cause mild symptoms, the economic impact of Rahnella‐associated symptoms remains unclear.

Pantoea ananatis, a cause of center rot of onion, is problematic in the United States and elsewhere. The bacterium lacks disease determinants common to most other bacterial pathogens of plants. A genomic island containing the gene pepM was detected within many onion-pathogenic strains of P. ananatis of diverse origins. The pepM gene of P. ananatis putatively encodes a protein that converts phosphoenolpyruvate to phosphonopyruvate, the first step in the biosynthesis of phosphonates and related molecules. This gene appears to be essential for center rot disease. Deletion of pepM rendered the mutant strain unable to cause lesions in leaves of growing onions and water-soaking of inoculated yellow onion bulbs. Furthermore, growth of the deletion mutant in onion leaves was significantly diminished compared with wild-type bacteria, and the mutant failed to cause cell death in tobacco. Complementation of the mutated strain with pepM restored the phenotype to wild-type capability. The pepM gene is the first pathogenicity factor identified that affects bacterial fitness as well as symptom development in both leaves and bulbs in a pathogen causing center rot of onion.

Several members of the lactic acid bacteria group were isolated from diseased onion plants and bulbs. Based on growth characteristics and sequence analysis of 16S rRNA and rpoA genes, the strains were identified as Lactococcus lactis, Lactobacillus plantarum, and three species of Leuconostoc, i.e., citreum, mesenteroides, and pseudomesenteroides. Pathogenic potential to onion leaves and mature onion bulbs was assessed. L. plantarum and all three Leuconostoc species caused symptoms in both leaves and bulbs. L. lactis caused scale discoloration in bulbs but failed to cause lesions on leaves. Leuconostoc citreum caused bulb decay in 7 days at 18°C as well as 37°C. This is the first report of a group of gram-positive bacteria able to cause disease in onion leaves.

Bacterial decays of onion bulbs cause sporadic and sometimes serious losses to onion (Allium cepa). In New York, three groups of bacteria were identified as problematic: Burkholderia spp., Pantoea ananatis, and Enterobacter spp. To aid in efficient detection and diagnosis of these pathogens, pairs of specific polymerase chain reaction primers were designed and validated, based on a strategy that utilized various genome sequences now available in public databases. Primer pairs were tested against numerous strains of target bacteria, closely related bacteria, and other onion-pathogenic bacteria. Each primer pair yielded a single, apparently highly specific amplicon from aqueous suspensions of the target bacteria. Minimum sensitivities were approximately 10³ CFU per 25-μl reaction mixture for all three primer pairs.

AvrE family type III effector proteins share the ability to suppress host defenses, induce disease-associated cell death, and promote bacterial growth. However, despite widespread contributions to numerous bacterial diseases in agriculturally important plants, the mode of action of these effectors remains largely unknown. WtsE is an AvrE family member required for the ability of Pantoea stewartii ssp. stewartii (Pnss) to proliferate efficiently and cause wilt and leaf blight symptoms in maize (Zea mays) plants. Notably, when WtsE is delivered by a heterologous system into the leaf cells of susceptible maize seedlings, it alone produces water-soaked disease symptoms reminiscent of those produced by Pnss. Thus, WtsE is a pathogenicity and virulence factor in maize, and an Escherichia coli heterologous delivery system can be used to study the activity of WtsE in isolation from other factors produced by Pnss. Transcriptional profiling of maize revealed the effects of WtsE, including induction of genes involved in secondary metabolism and suppression of genes involved in photosynthesis. Targeted metabolite quantification revealed that WtsE perturbs maize metabolism, including the induction of coumaroyl tyramine. The ability of mutant WtsE derivatives to elicit transcriptional and metabolic changes in susceptible maize seedlings correlated with their ability to promote disease. Furthermore, chemical inhibitors that block metabolic flux into the phenylpropanoid pathways targeted by WtsE also disrupted the pathogenicity and virulence activity of WtsE. While numerous metabolites produced downstream of the shikimate pathway are known to promote plant defense, our results indicate that misregulated induction of phenylpropanoid metabolism also can be used to promote pathogen virulence.

Strains of Erwinia amylovora, the bacterium causing the disease fire blight of rosaceous plants, are separated into two groups based on host range: Spiraeoideae and Rubus strains. Spiraeoideae strains have wide host ranges, infecting plants in many rosaceous genera, including apple and pear. In the field, Rubus strains infect the genus Rubus exclusively, which includes raspberry and blackberry. Based on comparisons of limited sequence data from a Rubus and a Spiraeoideae strain, the gene eop1 was identified as unusually divergent, and it was selected as a possible host specificity factor. To test this, eop1 genes from a Rubus strain and a Spiraeoideae strain were cloned and mutated. Expression of the Rubus-strain eop1 reduced the virulence of E. amylovora in immature pear fruit and in apple shoots. Sequencing the orfA-eop1 regions of several strains of E. amylovora confirmed that forms of eop1 are conserved among strains with similar host ranges. This work provides evidence that eop1 from a Rubus-specific strain can function as a determinant of host specificity in E. amylovora.