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Onion bulb rot can be caused by multiple plant pathogens including bacterial pathogens. During our routine survey of commercial onion farms in 2014, 2020, and 2021, seven putative Rouxiella spp. strains were isolated from symptomatic onion samples in Georgia, United States. Upon fulfilling Koch’s postulates on onion, a genome analysis was conducted. Whole-genome indices (ANI and dDDH) showed that the strains belonged to Rouxiella badensis. Although the seven R. badensis strains were not pathogenic on onion foliage, the strains were able to cause bulb rot and could also produce necrotic lesions in a red onion scale assay. R. badensis populations increased significantly and to a level comparable to P. ananatis PNA 97-1R in a red onion scale infection assay. The core-genome analysis grouped all onion R. badensis strains from Georgia together, and distinct from R. badensis strains isolated from other sources and locations. Based on the genome analysis of strains (from the current study and available genomes in the repository), type I, III (Ssa-Esc and Inv-Mxi-Spa types), and V secretion systems are present in R. badensis genomes, while type II, IV, and VI secretion systems are absent. However, various secondary metabolite gene clusters were identified from R. badensis genomes, and a thiol/redox-associated enzyme gene cluster similar to the Pantoea alt cluster mediating thiosulfinate tolerance was also present in onion strains of R. badensis. This is the first report of R. badensis as a plant pathogen.

A two-year study was conducted in 2019 and 2020 to characterize the spatial distribution of whitefly-transmitted virus complex (WTVC) in experimental and commercial yellow squash (Cucurbita pepo) fields in southern Georgia, USA. Field trials planted in Tifton, Georgia, USA were comprised of 30 rows of squash (variety: Gentry) divided into 300 quadrats. Six commercial fields in Tift, Worth, and Colquitt Counties in Georgia, USA were surveyed. In each field, 10 rows of plants per field were partitioned into 70 quadrats so that their approximate location could be mapped and disease incidence tracked in space over time. Plants in each quadrat were visually assessed for TVC symptoms at seven-day intervals (experimental fields) or at fourteen-day intervals (commercial fields). The spatial distribution of WTVC in a one-dimensional space was determined through ordinary runs analysis and in two-dimensional space through spatial autocorrelation analysis and spatial analysis by distance indices (SADIE). Ordinary runs analysis down and between rows suggested that disease was more likely to be transmitted from infected plants within rows than across rows. WTVC incidence in space was positively correlated with adult whiteflies on squash leaves taken one or two weeks prior to the assessment of virus incidence coinciding with latent period of Geminiviruses. SADIE generated disease severity maps indicated a shift from sparse and randomly distributed disease early in the infection cycle to denser, more aggregated patterns later in time in both experimental and commercial fields. Contour plots of interpolated indices from SADIE analysis suggested an edge effect on the spatial distribution of WTVC in experimental fields as well as in two of the commercial fields. An understanding of the shift from random to aggregated distribution and the edge effect on WTVC incidence are considerations for refining current management strategies.

Pantoea ananatis is a member of a Pantoea spp. complex that causes center rot of onion, which significantly affects onion yield and quality. This pathogen does not have typical virulence factors like type II or type III secretion systems but appears to require a biosynthetic gene-cluster, HiVir/PASVIL (located chromosomally), for a phosphonate secondary metabolite, and the onion-virulence regions, OVR (localized on a megaplasmid), for onion pathogenicity and virulence, respectively. We conducted a deep pan-genome-wide association study (pan-GWAS) to predict additional genes associated with pathogenicity in P. ananatis using a panel of diverse strains (n = 81). We utilized a red-onion scale necrosis assay as an indicator of pathogenicity. Based on this assay, we differentiated pathogenic (n = 51)- vs. non-pathogenic (n = 30)-strains phenotypically. Pan-GWAS revealed a large core genome of 3,153 genes and a flexible accessory genome of ≤5,065 genes. Phylogenomic analysis using pan-GWAS and presence and absence variants (PAVs) distinguished red-scale necrosis inducing (pathogenic) strains from non-scale necrosis inducing (non-pathogenic) strains of P. ananatis. The pan-GWAS also predicted 42 genes, including 14 from the previously identified HiVir/PASVIL cluster associated with pathogenicity, and 28 novel genes that were not previously associated with pathogenicity in onion. Of the 28 novel genes identified, eight have annotated functions of site-specific tyrosine kinase, N-acetylmuramoyl-L-alanine amidase, TraR/DksA family transcriptional regulator, and HTH-type transcriptional regulator. The remaining 20 genes are currently hypothetical. This is the first report of using pan-GWAS on P. ananatis for the prediction of novel genes contributing to pathogenicity in onion, which will be utilized for further functional analyses. Pan-genomic differences (using PAVs) differentiated onion pathogenic from non-pathogenic strains of P. ananatis, which has been difficult to achieve using single or multiple gene-based phylogenetic analyses. The pan-genome analysis also allowed us to evaluate the presence and absence of HiVir/PASVIL genes and 11 megaplasmid-borne OVR-A genes regarded as the ‘alt’ cluster that aid in P. ananatis colonization in onion bulbs. We concluded that HiVir/PASVIL genes are associated with pathogenic P. ananatis strains and the alt gene cluster alone is not responsible for pathogenicity on onion. The pan-genome also provides clear evidence of constantly evolving accessory genes in P. ananatis that may contribute to host-range expansion and niche-adaptation.Author summaryPantoea ananatis is a major bacterial pathogen that causes center rot of onion and diseases of a number of other plant species. In order to understand the genome architecture and identify genes responsible for pathogenicity in onion, a pan-genome analysis was performed. We used 81 strains of P. ananatis collected over 20 years from different regions of the state of Georgia, USA. The pan-genome study identified a core genome with a conserved set of genes and an accessory genome that displayed variation among strains. We conducted pan-GWAS (pan-genome-wide association study) using presence and absence variants (PAVs) in the genomes and associated onion-pathogenic phenotypes based on a red-onion scale necrosis assay. The study resulted in identification of genes, including a cluster of chromosomal HiVir/PASVIL genes, that are significantly associated with the onion pathogenic phenotype. In addition, we identified 28 novel genes, a majority of which (n = 20) have hypothetical functions. We concluded and further substantiated earlier findings that a cluster of genes is responsible for pathogenicity on onion. The pan-genome analysis also allowed us to evaluate the presence and absence of HiVir/PASVIL genes and 11 megaplasmid-borne OVR-A genes regarded as the ‘alt’ cluster that aid in bacterial colonization of onion bulbs by P. ananatis strains. We concluded that HiVir/PASVIL genes are associated with onion-pathogenic strains, and the alt gene cluster alone is not responsible for pathogenicity on onion. This study also provides potential evidence of constantly evolving accessory genes in P. ananatis which may help in host range expansion and adaptation to diverse niches.

Pantoea ananatis is a gram-negative bacterium and the primary causal agent of center rot of onions in Georgia. Previous genomic studies identified two virulence gene clusters, HiVir and alt, associated with center rot. The HiVir gene cluster is required to induce necrosis on onion tissues via synthesis of a predicted small molecule toxin. The alt gene cluster aids in tolerance to thiosulfinates generated during onion tissue damage. Whole genome sequencing of other Pantoea species suggest that these gene clusters are present outside of P. ananatis. To assess the distribution of these gene clusters, two PCR primer sets were designed to detect the presence of HiVir and alt. Two hundred fifty-two strains of Pantoea spp. were phenotyped using the red onion scale necrosis (RSN) assay and were assayed using PCR for the presence of these virulence genes. A diverse panel of strains from three distinct culture collections comprised of 24 Pantoea species, 41 isolation sources, and 23 countries, collected from 1946-2019, were tested. There is a significant association between the alt PCR assay and Pantoea strains recovered from symptomatic onion (PP. ananatis strains but not among Pantoea spp., congeners. This may indicate a divergent HiVir cluster or different pathogenicity and virulence mechanisms. Last, we describe natural alt positive [RSN+/HiVir+/alt+] P. ananatis strains, which cause extensive bulb necrosis in a neck-to-bulb infection assay compared to alt negative [RSN+/HiVir+/alt-] P. ananatis strains. A combination of assays that include PCR of virulence genes [HiVir and alt] and an RSN assay can potentially aid in identification of onion-bulb-rotting pathogenic P. ananatis strains.

Pantoea ananatis is a member of the family Enterobacteriaceae and an enigmatic plant pathogen with a broad host range. Although P. ananatis strains can be aggressive on onion causing foliar necrosis and onion center rot, previous genomic analysis has shown that P. ananatis lacks the primary virulence secretion systems associated with other plant pathogens. We assessed a collection of fifty P. ananatis strains collected from Georgia over three decades to determine genetic factors that correlated with onion pathogenic potential. Previous genetic analysis studies have compared strains isolated from different hosts with varying diseases potential and isolation sources. Strains varied greatly in their pathogenic potential and aggressiveness on different cultivated Allium species like onion, leek, shallot, and chive. Using multi-locus sequence analysis (MLSA) and repetitive extragenic palindrome repeat (rep)-PCR techniques, we did not observe any correlation between onion pathogenic potential and genetic diversity among strains. Whole genome sequencing and pan-genomic analysis of a sub-set of 10 strains aided in the identification of a novel series of genetic regions, likely plasmid borne, and correlating with onion pathogenicity observed on single contigs of the genetic assemblies. We named these loci Onion Virulence Regions (OVR) A-D. The OVR loci contain genes involved in redox regulation as well as pectate lyase and rhamnogalacturonase genes. Previous studies have not identified distinct genetic loci or plasmids correlating with onion foliar pathogenicity or pathogenicity on a single host pathosystem. The lack of focus on a single host system for this phytopathgenic disease necessitates the pan-genomic analysis performed in this study.