Ron Gitaitis

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.

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. [ ] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The soil-borne pathogens, particularly Fusarium oxysporum f. sp. niveum (FON) and southern root-knot nematode (RKN, Meloidogyne incognita) are the major threats to watermelon production in the southeastern United States. The role of soil micronutrients on induced resistance (IR) to plant diseases is well-documented in soil-based media. However, soil-based media do not allow us to determine the contribution of individual micronutrients in the induction of IR. In this manuscript, we utilized hydroponics-medium to assess the effect of controlled application of micronutrients, including iron (Fe), manganese (Mn), and zinc (Zn) on the expression of important IR genes (PR1, PR5, and NPR1 from salicylic acid (SA) pathway, and VSP, PDF, and LOX genes from jasmonic acid (JA) pathway) in watermelon seedlings upon inoculation with either FON or RKN or both. A subset of micronutrient-treated plants was inoculated (on the eighth day of micronutrient application) with FON and RKN (single or mixed inoculation). The expression of the IR genes in treated and control samples was evaluated using qRT-PCR. Although, significant phenotypic differences were not observed with respect to the severity of wilt symptoms or RKN galling with any of the micronutrient treatments within the 30-day experimental period, differences in the induction of IR genes were considerably noticeable. However, the level of gene expression varied with sampling period, type and concentration of micronutrients applied, and pathogen inoculation. In the absence of pathogens, micronutrient applications on the seventh day, in general, downregulated the expression of the majority of the IR genes. However, pathogen inoculation preferentially either up- or down-regulated the expression levels of the IR genes at three days post-inoculation depending on the type and concentration of micronutrients. The results demonstrated here indicate that micronutrients in watermelon may potentially make watermelon plants susceptible to infection by FON and RKN. However, upon infection the IR genes are significantly up-regulated that they may potentially aid the prevention of further infection via SA- and JA-pathways. This is the first demonstration of the impact of micronutrients affecting IR in watermelon against FON and RKN infection.

The soil-borne pathogens, particularly Fusarium oxysporum f. sp. niveum (FON) and southern root-knot nematode (RKN, Meloidogyne incognita) are the major threat to watermelon production in the south-eastern United States. The role of soil micronutrients on induced resistance (IR) to plant diseases is well-documented in soil-based mediums. However, soil-based mediums limit the determination of the role(s) of individual micronutrients in IR. In this manuscript, we utilized hydroponics to assess the effect of controlled application of micronutrient, including iron (Fe), manganese (Mn), and zinc (Zn) on the expression of some IR genes (PR1, PR5, and NPR1 from salicylic acid (SA) pathway, and VSP, PDF, and LOX genes from jasmonic acid (JA) pathway) in watermelon seedlings upon inoculation with either FON or RKN or both. Plants were treated with higher (3X) or lower (0.5X) concentrations of micronutrients in Steiner solution (X= standard dose of micronutrient) for 7 days in a hydroponics system under greenhouse conditions. A subset of micronutrient-treated plants was inoculated (on the 8th day of micronutrient application) with FON and RKN (single and mixed). The expression of the IR genes in treated and control samples were evaluated using qRT-PCR. Although, significant phenotypic differences were not observed with respect to the severity of wilt symptoms or RKN galling with any of the micro-nutrient treatments within the 30 day-experimental-period, differences in the induction of IR genes were observed. However, the level of gene expression varied with sampling period, type and concentration of micro-nutrients ap-plied, and pathogen-inoculation. In the absence of pathogens, no significant changes were observed in the expression level of IR genes on 7th day of micronutrient treatment. However, pathogen inoculation affected the expression levels of the IR genes at 3-day post-inoculation. In FON inoculated plants, PDF was upregulated in high Fe treatment, whereas in RKN inoculated plants, low Mn treatment resulted in up-regulation of VSP. In the case of mixed inoculation with FON and RKN, the plants with low Zn treatment resulted in the upregulation of PR1. These observations suggest that the type and concentration of micronutrients in watermelon may potentially induce systemic resistance against FON and RKN through SA and JA pathways.

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 2632^T) 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.

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.

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 2632^T and P. stewartii subsp. stewartii LMG 2715^T 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 2632^T 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.

The phytopathogenic bacterium Xanthomonas euvesicatoria causes bacterial leaf spot (BLS) of pepper and has a worldwide distribution. BLS is difficult to control and an integrated management strategy that incorporates crop rotation, use of clean seed and clean plants, weed control, resistant varieties, applications of bactericides, biocontrol agents, and systemic acquired resistance (SAR) inducers is generally recommended. However, even with that arsenal of weapons, BLS can still be responsible for severe losses under favorable environmental conditions. Thus, additional tools need to be added to an overall integrated management strategy to combat BLS. In this article, we developed several models from 2012 to 2014 that were based on how macronutrients, micronutrients, and micronutrient ratios affect BLS severity. Factors used to select a model for validation included highly significant P values, high adjusted R² values, low variance inflation factor values (2 value of 0.92. A significant relationship between observed AUDPC versus predicted values was also observed in Tift County in 2016 (P < 0.001; adjusted R² = 0.98). Relative gene expression of both NPR1 and PR1 genes was significantly (P < 0.01) higher in pepper grown in predicted low-risk sites compared with pepper from high-risk sites in Colquitt, Worth, and Tift Counties. Although BLS severity will fluctuate depending on environmental conditions, the data indicate that the level of risk at a particular location may be influenced by how macronutrient and micronutrient concentrations affect plant disease resistance genes in the SAR pathway.

Although seed production has been moved to semiarid regions to escape seedborne pathogens, seedborne bacterial diseases continue to be problematic and cause significant economic losses worldwide. Infested seeds are responsible for the re-emergence of diseases of the past, movement of pathogens across international borders, or the introduction of diseases into new areas. Considerable attention has been paid to improving the sensitivity and selectivity of seed health assays by using techniques such as flow cytometry and the polymerase chain reaction. There has also been progress in understanding infection thresholds and how they influence seed sample size determination and ultimately the reliability of seed health testing. Disease development and dissemination of pathogens from contaminated seedlots can be predicted using formulas that take into account inoculum density and environmental pressures. In general, seeds infested with bacterial pathogens are distributed within a Poisson distribution. In a subset of contaminated seeds, bacteria are distributed in non-Gaussian distributions, e.g., a lognormal distribution.

S are listed in alphabetical order by the last name of the senior author.