Genome-wide association and dissociation studies in Pantoea ananatis reveal potential virulence factors affecting Allium porrum and Allium fistulosum × Allium cepa hybrid.

Pantoea ananatis is a member of a Pantoea species complex that causes center rot of bulb onions (A. cepa) and also infects other Allium crops like leeks (Allium porrum), chives (Allium schoenoprasum), bunching onion or Welsh onion (Allium fistulosum), and garlic (Allium sativum). This pathogen relies on a chromosomal phosphonate biosynthetic gene cluster (HiVir) and a plasmid-borne thiosulfinate tolerance cluster (alt) for onion pathogenicity and virulence, respectively. However, pathogenicity and virulence factors associated with other Allium species remain unknown. We used phenotype-dependent genome-wide association (GWAS) and phenotype-independent gene-pair coincidence (GPC) analyses on a panel of diverse 92 P. ananatis strains, which were inoculated on A. porrum and A. fistulosum × A. cepa under greenhouse conditions. Phenotypic assays showed that, in general, these strains were more aggressive on A. fistulosum × A. cepa as opposed to A. porrum. Of the 92 strains, only six showed highly aggressive foliar lesions on A. porrum compared to A. fistulosum × A. cepa. Conversely, nine strains showed highly aggressive foliar lesions on A. fistulosum × A. cepa compared to A. porrum. These results indicate that there are underlying genetic components in P. ananatis that may drive pathogenicity in these two Allium spp. Based on GWAS for foliar pathogenicity, 835 genes were associated with P. ananatis' pathogenicity on A. fistulosum × A. cepa whereas 243 genes were associated with bacterial pathogenicity on A. porrum. The Hivir as well as the alt gene clusters were identified among these genes. Besides the 'HiVir' and the alt gene clusters that are known to contribute to pathogenicity and virulence from previous studies, genes annotated with functions related to stress responses, a potential toxin-antitoxin system, flagellar-motility, quorum sensing, and a previously described phosphonoglycan biosynthesis (pgb) cluster were identified. The GPC analysis resulted in the identification of 165 individual genes sorted into 39 significant gene-pair association components and 255 genes sorted into 50 significant gene-pair dissociation components. Within the coincident gene clusters, several genes that occurred on the GWAS outputs were associated with each other but dissociated with genes that did not appear in their respective GWAS output. To focus on candidate genes that could explain the difference in virulence between hosts, a comparative genomics analysis was performed on five P. ananatis strains that were differentially pathogenic on A. porrum or A. fistulosum × A. cepa. Here, we found a putative type III secretion system, and several other genes that occurred on both GWAS outputs of both Allium hosts. Further, we also demonstrated utilizing mutational analysis that the pepM gene in the HiVir cluster is important than the pepM gene in the pgb cluster for P. ananatis pathogenicity in A. fistulosum × A. cepa and A. porrum. Overall, our results support that P. ananatis may utilize a common set of genes or gene clusters to induce symptoms on A. fistulosum × A. cepa foliar tissue as well as A. cepa but implicates additional genes for infection on A. porrum.

Pan-Genome-Wide Analysis of Pantoea ananatis Identified Genes Linked to Pathogenicity in Onion.

Pantoea ananatis, a gram negative and facultative anaerobic bacterium 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 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 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-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 diverse P. ananatis strains. 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 of P. ananatis. A total of 1182 HGT events including the HiVir/PASVIL and alt cluster 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 in P. ananatis.

Pan-Genome of Novel Pantoea stewartii subsp. indologenes Reveals Genes Involved in Onion Pathogenicity and Evidence of Lateral Gene Transfer.

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 2715T, P. ananatis LMG 2665T, P. agglomerans LMG L15, and P. allii LMG 24248T. 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.

Neofusicoccum caryigenum, a new species causing leaf dieback disease of pecan (Carya illinoinensis).

Neofusicoccum species are endophytes and pathogens of woody hosts and members of the Botryosphaeriaceae. Leaf dieback is a new disease resulting in death of compound leaves and extensive defoliation of pecan trees (Carya illinoinensis) throughout the southeastern United States. Currently, the disease is consistently most severe on trees that are not managed with fungicides for pecan scab. Preliminary observations of the fungus isolated from symptomatic leaves indicated that it was a member of the genus Neofusicoccum. Our objectives were to confirm that this is the causal organism of leaf dieback disease of pecan and to determine whether this disease is caused by a new or previously described species of Neofusicoccum. Morphological observations of pure cultures, conidiomata, conidiogenous cells, and conidia were consistent with members of the genus Neofusicoccum. Using Koch's postulates, we established that Neofusicoccum sp. isolated from symptomatic leaves caused the disease. We sequenced the internal transcribed spacer of the rDNA (ITS), elongation factor 1-α (EF1-α), the second largest subunit of RNA polymerase II (RPB2), and ß-tubulin (TUB2) of 11 isolates collected from Georgia and Texas. Phylogenetic and network analyses of these sequences combined with publicly available sequences of 40 members of the N. parvum-N. ribis species complex and the outgroup N. australe revealed that this fungus is a member of the species complex but is genetically distinct from previously described species. We determined that leaf dieback of pecan is caused by a novel species, named herein N. caryigenum.

Pathogenicity and a TaqMan Real-Time PCR for Specific Detection of Pantoea allii, a Bacterial Pathogen of Onions.

Bacterial diseases of onion are reported to cause significant economic losses. Pantoea allii Brady, one of the pathogens causing the center rot on onions, has not yet been reported in Canada. We report the pathogenicity of P. allii on commercially available Canadian green onions (scallions). All P. allii-inoculated plants, irrespective of the inoculum concentration, exhibited typical leaf chlorotic discoloration on green onion leaves, which can reduce their marketability. Reisolation of P. allii from infected scallion tissues and reidentification by sequencing and phylogenetic analyses of the leuS gene suggest that the pathogen can survive in infected tissues 21 days after inoculation. This is the first report of P. allii as a potential pathogen of green onions. This study also reports the development and validation of a TaqMan real-time PCR assay targeting the leuS gene for reliable detection of P. allii in pure cultures and in planta. A 642-bp leuS gene fragment was targeted because it showed high nucleotide diversity and positively correlated with genome-based average nucleotide identity with respect to percent similarity index and identity of Pantoea species. The assay specificity was validated using 61 bacterial and fungal strains. Under optimal conditions, the selected primers and FAM-labeled TaqMan probe were specific for the detection of nine reference P. allii strains by real-time PCR. The 52 strains of other Pantoea spp. (n = 25), non-Pantoea spp. (n = 20), and fungi/oomycetes (n = 7) tested negative (no detectable fluorescence). Onion tissues spiked with P. allii, naturally infested onion bulbs, greenhouse infected green onion leaf samples, as well as an interlaboratory blind test were used to validate the assay specificity. The sensitivities of a 1-pg DNA concentration and 30 CFU are comparable to previously reported real-time PCR assays of other bacterial pathogens. The TaqMan real-time PCR assay developed in this study will facilitate reliable detection of P. allii and could be a useful tool for screening onion imports or exports for the presence of this pathogen.

Epiphytic Survival of Pantoea ananatis on Richardia scabra L. in Georgia.

Pantoea ananatis, the causal organism of center rot of onion (Allium cepa L.), can survive on different weeds but, in a previous survey, it was most commonly found on Florida pusley (Richardia scabra L.). The epiphytic survival of P. ananatis on R. scabra under different temperature and moisture regimes was investigated. Weed seedlings were spray inoculated with rifampicin-resistant strain PNA 97-1rif at either 103 or 108 CFU/ml and incubated in a growth chamber at 15.5 or 21.1°C at 65% relative humidity for 96 h postinoculation (hpi), which represented the mean environmental conditions during mid-March to mid-May in Vidalia, GA when onion production and R. scabra presence overlap. For plants inoculated with P. ananatis at 103 CFU/ml, the bacterium survived for 96 hpi when incubated at 21.1°C, with mean populations of 1.7 × 102 CFU/g of leaf tissue. In contrast, no viable bacteria were detected after 72 hpi at 15.5°C. For plants inoculated with P. ananatis at 108 CFU/ml, the bacterium survived for 96 hpi at 21.1°C (3.8 × 105 CFU/g) whereas, during the sample time period, viable bacterial populations were not detected at 15.5°C. Survival of P. ananatis on R. scabra was also monitored during alternating 12 h wet and 12 h dry periods, or continuous wet or dry periods for 96 hpi at 15.5 or 21.1°C. Compared with initial or continuous dry periods, P. ananatis survived significantly better with a 12 h wet/12 h dry cycle or a continuous 96 hpi wet period at both 15.5 and 21.1°C. Unlike at 15.5°C, P. ananatis populations (7.4 × 102 CFU/g) survived for 96 hpi at 21.1°C under a cycle of 12 h dry and 12 h wet. These results demonstrate that P. ananatis can survive on R. scabra leaves under conditions of 21.1°C and prolonged leaf wetness and may potentially serve as a source of inoculum to onion.

Distribution and Survival of Pseudomonas sp. on Italian Ryegrass and Curly Dock in Georgia.

Yellow bud, caused by Pseudomonas sp., is an emerging bacterial disease of onion. A polymerase chain reaction assay based on the coronafacate ligase (cfl) and HrpZ genes was used to detect initial suspected bacteria on weeds. Growth on an agar medium, ability to cause a hypersensitive response in tobacco, pathogenicity on onion, and sequence analysis of 16S ribosomal RNA and cfl genes were used to confirm the identity of Pseudomonas sp. recovered from 10 asymptomatic weed species in the Vidalia onion-growing zone (VOZ) of Georgia. Among the weeds identified as epiphytic hosts for Pseudomonas sp., Italian ryegrass (Lolium multiflorum) and curly dock (Rumex crispus) were prominent because ≥73% of the samples from five sample sites were positive for the bacterium. These weeds are commonly found throughout Georgia and, thus, were selected to assess their role in yellow bud epidemiology. Samples of the two weed species were collected from sites along the perimeter of and within the VOZ (n = 5 sites) during late June, August, and September 2012 and 2013, which represented the time interval between onion growing seasons. Samples (n = 10/weed species/site) were collected and processed for bacterial detection as described above. In June (2012 and 2013), Pseudomonas sp. was detected from Italian ryegrass and curly dock in 100 and 40% of the sample sites, respectively. During the months of August and September (2012), the bacterium was recovered from Italian ryegrass in 60 and 10% of the sample sites, respectively; whereas, in August (2013), Pseudomonas sp. was recovered from 40% of the sample sites. However, the bacterium was not recovered from any of the sites in September (2013). In contrast, during August and September (2012), Pseudomonas sp. was recovered from curly dock in 20 and 80% of the sample sites, respectively. Similarly, in August and September (2013), the bacterium was detected from 40 and 100% of the sample sites, respectively. These data demonstrated that the Pseudomonas sp. responsible for yellow bud can survive as an epiphyte on Italian ryegrass and curly dock between onion crops. Furthermore, using artificially infested onion seed, we demonstrated that Pseudomonas sp. can be transmitted through contaminated seed.

Molecular characterization of boscalid- and penthiopyrad-resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram.

BACKGROUND: Didymella bryoniae has a history of developing resistance to single-site fungicides. A recent example is with the succinate-dehydrogenase-inhibiting fungicide (SDHI) boscalid. In laboratory assays, out of 103 isolates of this fungus, 82 and seven were found to be very highly resistant (B(VHR) ) and highly resistant (B(HR) ) to boscalid respectively. Cross-resistance studies with the new SDHI penthiopyrad showed that the B(VHR) isolates were only highly resistant to penthiopyrad (B(VHR) -P(HR) ), while the B(HR) isolates appeared sensitive to penthiopyrad (B(HR) -P(S) ). In this study, the molecular mechanism of resistance in these two phenotypes (B(VHR) -P(HR) and B(HR) -P(S) ) was elucidated, and their sensitivity to the new SDHI fluopyram was assessed. RESULTS: A 456 bp cDNA amplified fragment of the succinate dehydrogenase iron sulfur gene (DbSDHB) was initially cloned and sequenced from two sensitive (B(S) -P(S) ), two B(VHR) -P(HR) and one B(HR) -P(S) isolate of D. bryoniae. Comparative analysis of the DbSDHB protein revealed that a highly conserved histidine residue involved in the binding of SDHIs and present in wild-type isolates was replaced by tyrosine (H277Y) or arginine (H277R) in the B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Further examination of the role and extent of these alterations showed that the H/Y and H/R substitutions were present in the remaining B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Analysis of the sensitivity to fluopyram of representative isolates showed that both SDHB mutants were sensitive to this fungicide as the wild-type isolates. CONCLUSION: The genotype-specific cross-resistance relationships between the SDHIs boscalid and penthiopyrad and the lack of cross-resistance between these fungicides and fluopyram should be taken into account when selecting SDHIs for gummy stem blight management.

Pantoea allii sp. nov., isolated from onion plants and seed.

Eight yellow-pigmented, Gram-negative, rod-shaped, oxidase-negative, motile, facultatively anaerobic bacteria were isolated from onion seed in South Africa and from an onion plant exhibiting centre rot symptoms in the USA. The isolates were assigned to the genus Pantoea on the basis of phenotypic and biochemical tests. 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA), based on gyrB, rpoB, infB and atpD sequences, confirmed the allocation of the isolates to the genus Pantoea. MLSA further indicated that the isolates represented a novel species, which was phylogenetically most closely related to Pantoea ananatis and Pantoea stewartii. Amplified fragment length polymorphism analysis also placed the isolates into a cluster separate from P. ananatis and P. stewartii. Compared with type strains of species of the genus Pantoea that showed >97 % 16S rRNA gene sequence similarity with strain BD 390(T), the isolates exhibited 11-55 % whole-genome DNA-DNA relatedness, which confirmed the classification of the isolates in a novel species. The most useful phenotypic characteristics for the differentiation of the isolates from their closest phylogenetic neighbours are production of acid from amygdalin and utilization of adonitol and sorbitol. A novel species, Pantoea allii sp. nov., is proposed, with type strain BD 390(T) ( = LMG 24248(T)).

Development of an improved isolation approach and simple sequence repeat markers to characterize Phytophthora capsici populations in irrigation ponds in southern Georgia.

Phytophthora capsici, the causal agent of Phytophthora blight, is a major concern in vegetable production in Georgia and many other states in the United States. Contamination of irrigation water sources by P. capsici may be an important source of inoculum for the pathogen. A simple method was developed in this study to improve the efficiency of recovering P. capsici from fruits used as baits in irrigation ponds. In contrast to direct isolation on agar plates, infected fruit tissues were used to inoculate stems of pepper seedlings, and the infected pepper stems were used for isolation on agar plates. With isolation through inoculation of pepper stems, the frequency of recovering P. capsici from infected eggplant and pear fruits increased from 13.9% to 77.7% and 8.1% to 53.5%, respectively, compared with direct isolation on agar plates. P. capsici was isolated from seven out of nine irrigation ponds evaluated, with most of the ponds containing both A1 and A2 mating types and a 4:5 ratio of A1 to A2 when isolates from all ponds were calculated. All P. capsici isolates were pathogenic on squash plants, and only a small proportion (8.2%) of the isolates were resistant or intermediately sensitive to mefenoxam. Simple sequence repeats (SSRs) were identified through bioinformatics mining of 55,848 publicly available expressed sequence tags of P. capsici in dbEST GenBank. Thirty-one pairs of SSR primers were designed, and SSR analysis indicated that the 61 P. capsici isolates from irrigation ponds were genetically distinct. Cluster analysis separated the isolates into five genetic clusters with no more than two genetic groups in one pond, indicating relatively low P. capsici genetic diversity in each pond. The isolation method and SSR markers developed for P. capsici in this study could contribute to a more comprehensive understanding of the genetic diversity of this important pathogen.

Control of Soilborne Pathogenic Fungi in Fields of Sweet Onion.

Populations of soil fungi from fields planted to sweet onion were assayed on selective media. In pathogenicity tests, Rhizoctonia solani AG-4, Pythium irregulare, and Phoma terrestris were the fungi most virulent on onion seedlings. Plots were fumigated with methyl bromide (MBR), chloropicrin (CP), MBR + CP (67% + 33%), metam sodium, 1,3,-dichloropropene (1,3-D), or 1,3-D + 17% CP in four field experiments in 2 years. Sweet onion was transplanted or direct seeded in October or November and harvested in April or May. MBR + CP and CP were effective in reducing populations of Phoma terrestris, Pythium spp., Fusarium spp., and R. solani AG-4 in soil. Metam-sodium and 1,3-D + 17% CP were less efficacious, and MBR and 1,3-D were ineffective. There were no differences in the percentage of bulbs with decay at harvest or after curing among treatments. Increased yield of marketable bulbs was associated with control of soilborne pathogenic fungi. In fields continuously cropped to onion, decreased yield was primarily associated with control of pink-root induced by Phoma terrestris, and P. terrestris was identified in soil from 74% of the fields assayed.