Pantoea ananatis, a gram negative and facultative anaerobic bacterium is a member of aPantoeaspp. 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 comprised of 14 genes), for a phosphonate secondary metabolite, and the ‘alt’ gene cluster (located in plasmid and comprised of 11 genes) that aids in bacterial colonization in onion bulbs by imparting tolerance to thiosulfinates. We conducted a deep pan-genome-wide association study (pan-GWAS) to predict additional genes associated with pathogenicity inP. ananatisusing 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-genome analysis revealed a large core genome of 3,153 genes and a flexible accessory genome. Pan-GWAS using the presence and absence variants (PAVs) 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, conjugal transfer, and HTH-type transcriptional regulator. The remaining 20 genes are currently hypothetical. Further, a core-genome SNPs-based phylogeny and horizontal gene transfer (HGT) studies were also conducted to assess the extent of lateral gene transfer among diverseP. ananatisstrains. Phylogenetic analysis based on PAVs and whole genome multi locus sequence typing (wgMLST) rather than core-genome SNPs distinguished red-scale necrosis inducing (pathogenic) strains from non-scale necrosis inducing (non-pathogenic) strains ofP. ananatis. A total of 1182 HGT events including the HiVir/PASVIL andaltcluster genes were identified. These events could be regarded as a major contributing factor to the diversification, niche-adaptation and potential acquisition of pathogenicity/virulence genes inP. ananatis.

The production and quality of Phaseolusvulgaris (snap bean) have been negatively impacted by leaf crumple disease caused by two whitefly-transmitted begomoviruses: cucurbit leaf crumple virus (CuLCrV) and sida golden mosaic Florida virus (SiGMFV), which often appear as a mixed infection in Georgia. Host resistance is the most economical management strategy against whitefly-transmitted viruses. Currently, information is not available with respect to resistance to these two viruses in commercial cultivars. In two field seasons (2018 and 2019), we screened Phaseolus spp. genotypes (n = 84 in 2018; n = 80 in 2019; most of the genotypes were common in both years with a few exceptions) for resistance against CuLCrV and/or SiGMFV. We also included two commonly grown Lima bean (Phaseolus lunatus) varieties in our field screening. Twenty Phaseolus spp. genotypes with high to moderate-levels of resistance (disease severity ranging from 5%–50%) to CuLCrV and/or SiGMFV were identified. Twenty-one Phaseolus spp. genotypes were found to be highly susceptible with a disease severity of ≥66%. Furthermore, based on the greenhouse evaluation with two genotypes-each (two susceptible and two resistant; identified in field screen) exposed to viruliferous whiteflies infected with CuLCrV and SiGMFV, we observed that the susceptible genotypes accumulated higher copy numbers of both viruses and displayed severe crumple severity compared to the resistant genotypes, indicating that resistance might potentially be against the virus complex rather than against the whiteflies. Adult whitefly counts differed significantly among Phaseolus genotypes in both years. The whole genome of these Phaseolus spp. [snap bean (n = 82); Lima bean (n = 2)] genotypes was sequenced and genetic variability among them was identified. Over 900 giga-base (Gb) of filtered data were generated and >88% of the resulting data were mapped to the reference genome, and SNP and Indel variants in Phaseolus spp. genotypes were obtained. A total of 645,729 SNPs and 68,713 Indels, including 30,169 insertions and 38,543 deletions, were identified, which were distributed in 11 chromosomes with chromosome 02 harboring the maximum number of variants. This phenotypic and genotypic information will be helpful in genome-wide association studies that will aid in identifying the genetic basis of resistance to these begomoviruses in Phaseolus spp.

Pantoea stewartii subsp. indologenes (Psi) is a causative agent of leafspot on foxtail millet and pearl millet; however, novel strains were recently identified that are pathogenic on onions. Our recent host range evaluation study identified two pathovars; P. stewartii subsp. indologenes pv. cepacicola pv. nov. and P. stewartii subsp. indologenes pv. setariae pv. nov. that are pathogenic on onions and millets or on millets only, respectively. In the current study, we developed a pan-genome using the whole genome sequencing of newly identified/classified Psi strains from both pathovars [pv. cepacicola (n = 4) and pv. setariae (n = 13)]. The full spectrum of the pan-genome contained 7030 genes. Among these, 3546 (present in genomes of all 17 strains) were the core genes that were a subset of 3682 soft-core genes (present in ≥16 strains). The accessory genome included 1308 shell genes and 2040 cloud genes (present in ≤2 strains). The pan-genome showed a clear linear progression with >6000 genes, suggesting that the pan-genome of Psi is open. Comparative phylogenetic analysis showed differences in phylogenetic clustering of Pantoea spp. using PAVs/wgMLST approach in comparison with core genome SNPs-based phylogeny. Further, we conducted a horizontal gene transfer (HGT) study using Psi strains from both pathovars along with strains from other Pantoea species, namely, P. stewartii subsp. stewartii LMG 2715^T, P. ananatis LMG 2665^T, P. agglomerans LMG L15, and P. allii LMG 24248^T. A total of 317 HGT events among four Pantoea species were identified with most gene transfer events occurring between Psi pv. cepacicola and Psi pv. setariae. Pan-GWAS analysis predicted a total of 154 genes, including seven gene-clusters, which were associated with the pathogenicity phenotype (necrosis on seedling) on onions. One of the gene-clusters contained 11 genes with known functions and was found to be chromosomally located.

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 summary: Pantoea 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.

The production and quality of Phaseolus vulgaris (snap bean) have been negatively impacted by leaf crumple disease caused by two whitefly-transmitted begomoviruses; cucurbit leaf crumple virus (CuLCrV) and sida golden mosaic Florida virus (SiGMFV), which often appear as a mixed infection in Georgia. Host resistance is the most economical management strategy against whitefly-transmitted viruses. Currently, information is not available with respect to resistance to these two viruses in commercial cultivars. In two field seasons (2018 and 2019), we screened Phaseolus sp. genotypes (n = 84 in 2018; n = 80 in 2019; most of the genotypes were common in both years with a few exceptions) for resistance against CuLCrV and/or SiGMFV. Twenty genotypes with high- to moderate-levels of resistance (disease severity ranging from 5-50%) to CuLCrV and/or SiGMFV were identified. Twenty-one genotypes were found to be highly susceptible with a disease severity of ≥66%. Adult whitefly counts differed significantly among snap bean genotypes for both years. The whole genome of these Phaseolus sp. (n=82) genotypes was sequenced and genetic variability among them was identified. Over 900 giga-base (Gb) of filtered data were generated and >88% of the resulting data were mapped to the reference genome, and SNP and Indel variants in Phaseolus genotypes were obtained. A total of 645,729 SNPs and 68,713 Indels including 30,169 insertions and 38,543 deletions were identified, which were distributed in 11 chromosomes with chromosome 02 harboring the maximum number of variants. These phenotypic and genotypic information will be helpful in genome-wide association studies that will aid in identifying genetic basis of resistance to these begomoviruses in Phaseolus sp.