Thiosulfinate Tolerance Gene Clusters Are Common Features of Burkholderia Onion Pathogens.

Burkholderia gladioli pv. alliicola, B. cepacia, and B. orbicola are common bacterial pathogens of onion. Onions produce organosulfur thiosulfinate defensive compounds after cellular decompartmentalization. Using whole-genome sequencing and in silico analysis, we identified putative thiosulfinate tolerance gene (TTG) clusters in multiple onion-associated Burkholderia species similar to those characterized in other Allium-associated bacterial endophytes and pathogens. Sequence analysis revealed the presence of three Burkholderia TTG cluster types, with both Type A and Type B being broadly distributed in B. gladioli, B. cepacia, and B. orbicola in both the chromosome and plasmids. Based on isolate natural variation and generation of isogenic strains, we determined the in vitro and in vivo contribution of TTG clusters in B. gladioli, B. cepacia, and B. orbicola. The Burkholderia TTG clusters contributed to enhanced allicin tolerance and improved growth in filtered onion extracts by all three species. TTG clusters also made clear contributions to B. gladioli foliar necrosis symptoms and bacterial populations. Surprisingly, the TTG cluster did not contribute to bacterial populations in onion bulb scales by these three species. Based on our findings, we hypothesize onion-associated Burkholderia may evade or inhibit the production of thiosulfinates in onion bulb tissues. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Linkage Mapping and Genome-Wide Association Study Identified Two Peanut Late Leaf Spot Resistance Loci, PLLSR-1 and PLLSR-2, Using Nested Association Mapping.

Identification of candidate genes and molecular markers for late leaf spot (LLS) disease resistance in peanut (Arachis hypogaea) has been a focus of molecular breeding for the U.S. industry-funded peanut genome project. Efforts have been hindered by limited mapping resolution due to low levels of genetic recombination and marker density available in traditional biparental mapping populations. To address this, a multi-parental nested association mapping population has been genotyped with the peanut 58K single-nucleotide polymorphism (SNP) array and phenotyped for LLS severity in the field for 3 years. Joint linkage-based quantitative trait locus (QTL) mapping identified nine QTLs for LLS resistance with significant phenotypic variance explained up to 47.7%. A genome-wide association study identified 13 SNPs consistently associated with LLS resistance. Two genomic regions harboring the consistent QTLs and SNPs were identified from 1,336 to 1,520 kb (184 kb) on chromosome B02 and from 1,026.9 to 1,793.2 kb (767 kb) on chromosome B03, designated as peanut LLS resistance loci, PLLSR-1 and PLLSR-2, respectively. PLLSR-1 contains 10 nucleotide-binding site leucine-rich repeat disease resistance genes. A nucleotide-binding site leucine-rich repeat disease resistance gene, Arahy.VKVT6A, was also identified on homoeologous chromosome A02. PLLSR-2 contains five significant SNPs associated with five different genes encoding callose synthase, pollen defective in guidance protein, pentatricopeptide repeat, acyl-activating enzyme, and C2 GRAM domains-containing protein. This study highlights the power of multi-parent populations such as nested association mapping for genetic mapping and marker-trait association studies in peanuts. Validation of these two LLS resistance loci will be needed for marker-assisted breeding.

Aggressive Alternaria brassicicola with Reduced Fungicide Sensitivity Can Be Associated with Naturally Infested Broccoli Seeds.

Alternaria brassicicola is a part of the Alternaria complex that causes leaf blight and head rot (ABHR) in brassica crops. Infested broccoli seeds can play an important role in introducing A. brassicicola in transplant houses and production fields. However, characterization of natural seed infestation and seed-to-seedling transmission of A. brassicicola in broccoli is yet to be demonstrated. In this research, we characterized Alternaria spp. isolates from commercial broccoli seedlots for their species identity, pathogenicity, and aggressiveness on broccoli and their sensitivity to a quinone-outside inhibitor (QoI) fungicide (azoxystrobin). Two hundred commercial seedlots from two broccoli cultivars, Cultivar 1 (EC; n = 100 seedlots) and Cultivar 2 (ED; n = 100 seedlots) were, evaluated for the presence of A. brassicicola under in vitro conditions using a seedling grow-out assay. Alternaria spp. was detected in 31 and 28% of the commercial seedlots of Cultivar 1 and Cultivar 2, respectively. The seed-to-seedling transmission (%) varied considerably within each positive-infested seedlot, which ranged from 1.3 to 17.3%. Subsequent molecular identification of single-spore cultures (n = 138) was made by sequencing four housekeeping genes: actin, the major allergen (Alta1), plasma membrane ATPase, and glyceraldehyde-3-phosphate dehydrogenase (GPD), and the sequences were concatenated and compared for the phylogenetic distance with diverse Alternaria species. Ninety-six percent (n = 133) of the isolates formed a cluster with a known A. brassicicola based on a multigene phylogeny, which were later confirmed as A. brassicicola using a species-specific PCR assay. One hundred percent of the A. brassicicola seed isolates (n = 133) were either highly or moderately aggressive on broccoli (cultivar Emerald Crown) based on a detached leaf assay. Sensitivity of representative A. brassicicola isolates (n = 58) to azoxystrobin was evaluated using a spore germination assay, and the EC50 values (effective fungicide concentration [ppm] at which germination of conidia of isolates were reduced by 50% compared to control) for each isolate was determined. A. brassicicola isolates from naturally infested commercial broccoli seeds were sensitive to azoxystrobin with considerably low EC50 values in the range of <0.0001 to 0.33 ppm; however, there were a few isolates (14%) that showed 100-fold reduced sensitivity from the most sensitive isolate (EC50 = 0.0001 ppm). Our results confirm that commercial broccoli seedlots can be naturally contaminated with pathogenic and aggressive A. brassicicola. We also provide evidence for the potential presence of A. brassicicola isolates with reduced azoxystrobin-sensitivity in naturally infested commercial broccoli seedlots, which has never been reported before. Together, these findings may have implications in considerations for seed-health testing, seed treatments, and greenhouse scouting to limit introduction of infested seedlots in commercial broccoli fields.

Characterization of Seed-to-Seedling Transmission of Alternaria brassicicola in Broccoli.

Alternaria brassicicola is part of a complex of Alternaria species that causes leaf blight and head rot in brassica crops such as broccoli, kale, cabbage, cauliflower, and collards. Seed can serve as a potential source of inoculum for the transmission of A. brassicicola in broccoli as demonstrated earlier; however, seed-to-seedling transmission of pathogen was never characterized empirically. So, the objectives of this study were to (i) re-evaluate the effect of artificial seed infestation on seed germination and seed-to-seedling transmission of A. brassicicola in broccoli; (ii) determine the effect of A. brassicicola-seed inoculum levels on seed-to-seedling transmission; (iii) evaluate if variations in A. brassicicola aggressiveness affect A. brassicicola seed-to-seedling transmission; and (iv) evaluate seed treatments that can reduce seed-to-seedling transmission of A. brassicicola in broccoli. Artificially infested seedlots were generated by inoculating broccoli seeds with a spore suspension of 1 × 105 conidia/ml of A. brassicicola using the vacuum infiltration method. Inoculated (n = 10 seedlots; 300 seeds/seedlot) or control seedlots in three replicates were planted on two layers of sterile blotter paper saturated with sterile water in transparent plastic boxes and incubated at 20°C and >90% relative humidity (RH) under continuous fluorescent light. Percent seed germination and percent seed-to-seedling transmission were recorded every other day for 21 days. Percent seed germination was significantly affected with artificial pathogen inoculation. One hundred percent of the seedlots transmitted the pathogen to broccoli seedlings, and seed-to-seedling percentages of the seedlots varied considerably. A strong linear and significant relationship between A. brassicicola inoculum level and seed-to-seedling transmission (%) within each seedlot was observed. Interestingly, variations in aggressiveness of A. brassicicola isolates did not affect seed-to-seedling transmission, as 100% of the seedlots were able to transmit the pathogen. Seed treatment with Miravis (a.i. pydiflumetofen 18.3%) significantly increased seed germination and reduced seed-to-seedling transmission percentages in A. brassicicola-inoculated seedlots. These results indicate that artificial seed inoculation with A. brassicicola can result in consistent seed-to-seedling transmission with significant impact on seed germination. Seed inoculum density of ≥104 conidia/ml is necessary for reliable transmission of A. brassicicola. Further seed-to-seedling transmission is not dependent on aggressiveness of A. brassicicola isolates and seed treatment with Miravis can significantly reduce pathogen transmission in broccoli seedings. Overall, this study provides detailed characterization of seed-to-seedling transmission of A. brassicicola in broccoli that can be further used to determine inoculum threshold, which has potential applications in seed-health testing and sample size determination. Furthermore, we also provide options for effective seed treatments that can significantly reduce A. brassicicola seed-to-seedling transmission and may potentially aid in managing seedborne fungal infection.

Assessment of Prickly Sida as a Potential Inoculum Source for Sida Golden Mosaic Virus in Commercial Snap Bean Farms in Georgia, United States.

Sida golden mosaic virus (SiGMV), an obligate pathogen that infects snap beans (Phaseolus vulgaris), is known to infect prickly sida (Sida spinosa L.), which is a common weed in agricultural farms in Georgia. Prickly sida has also been reported as a suitable host of sweetpotato whitefly (Bemisia tabaci), the vector of SiGMV. Despite being a host for both SiGMV and its vector, the role of prickly sida as a reservoir and inoculum source for SiGMV in snap bean farms has not been evaluated. This study was conducted to document the occurrence of SiGMV-infected prickly sida plants and to assess its potential role as a source of SiGMV inoculum in snap bean farms. A survey of 17 commercial snap bean farms conducted in spring 2021 confirmed the presence of SiGMV-infected prickly sida in southern Georgia. In fall 2021 and 2022, on-farm field trials were conducted in four commercial farms where SiGMV-infected prickly sida plants were documented earlier as a part of survey in spring 2021. The spatial distribution and temporal patterns of adult whiteflies and SiGMV on snap bean were compared between macroplots (13.7 × 30.5 m) "with prickly sida" or "without prickly sida" that were at least 232 m apart from each other. We did not observe any consistent differences in counts of adult whiteflies between macroplots with or without prickly sida in the four commercial farms. SiGMV infection was detected earlier and with higher incidences in snap bean macroplots "with prickly sida" compared with macroplots "without prickly sida." An apparent disease gradient was observed in two of the four farms assessed. Higher SiGMV incidences were observed on the edges of macroplots "with prickly sida." These findings indicate prickly sida as a potential natural reservoir and a source for SiGMV spread in snap bean farms in southern Georgia.

The Genetic Requirements for HiVir-Mediated Onion Necrosis by Pantoea ananatis, a Necrotrophic Plant Pathogen.

Pantoea ananatis is an unusual bacterial pathogen that lacks typical virulence determinants yet causes extensive necrosis in onion foliage and bulb tissues. The onion necrosis phenotype is dependent on the expression of the phosphonate toxin, pantaphos, which is synthesized by putative enzymes encoded by the HiVir (high virulence) gene cluster. The genetic contributions of individual hvr genes in HiVir-mediated onion necrosis remain largely unknown, except for the first gene, hvrA (phosphoenolpyruvate mutase, pepM), whose deletion resulted in the loss of onion pathogenicity. In this study, using gene-deletion mutation and complementation, we report that, of the ten remaining genes, hvrB to hvrF are also strictly required for the HiVir-mediated onion necrosis and in-planta bacterial growth, whereas hvrG to hvrJ partially contributed to these phenotypes. As the HiVir gene cluster is a common genetic feature shared among the onion-pathogenic P. ananatis strains that could serve as a useful diagnostic marker of onion pathogenicity, we sought to understand the genetic basis of HiVir-positive yet phenotypically deviant (non-pathogenic) strains. We identified and genetically characterized inactivating single nucleotide polymorphisms in the essential hvr genes of six phenotypically deviant P. ananatis strains. Finally, inoculation of cell-free spent medium of the isopropylthio-ß-galactoside (IPTG)-inducible promoter (Ptac)-driven HiVir strain caused P. ananatis-characteristic red onion scale necrosis as well as cell death symptoms in tobacco. Co-inoculation of the spent medium with essential hvr mutant strains restored in-planta populations of the strains to the wild-type level, suggesting that necrotic tissues are important for the proliferation of P. ananatis in onion. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Novel Biosynthetic Gene Cluster Across the Pantoea Species Complex Is Important for Pathogenicity in Onion.

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

Evaluating Options to Increase the Efficacy of Biocontrol Agents for the Management of Pantoea spp. Under Field Conditions.

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.

Species Identification and Fungicide Sensitivity of Fungi Causing Alternaria Leaf Blight and Head Rot in Cole Crops in the Eastern United States.

Alternaria leaf blight and head rot is an important disease of broccoli and other cole crops. With no resistant host varieties, fungicides are utilized to manage this disease. However, anecdotal evidence suggests that, in southeastern U.S. broccoli-producing states, there is a loss of disease control through the use of quinone outside inhibitor (QoI) fungicides. To understand why there is a reduced sensitivity to QoI fungicides in these states, we isolated Alternaria spp. from symptomatic lesions on cole crops from Georgia and Virginia (two states with observations of loss of fungicide sensitivity) as well as New York (a state with no observations of loss of fungicide sensitivity). Using multilocus sequencing and phylogenetic analysis, we identified two species, Alternaria brassicicola and A. japonica. Whereas A. brassicicola was isolated in all states, A. japonica was only isolated in Georgia. Next, we wanted to determine the sensitivity of these isolates to azoxystrobin-an active ingredient in some QoI fungicides-by estimating the effective concentration at which only 50% of spores germinate (EC50). The EC50 of A. brassicicola ranged from 0.01 to 0.17 ppm, whereas that of A. japonica was 8.1 to 28.1 ppm. None of the known target-site mutations that confer resistance to QoI fungicides were identified during screening of either species. A. japonica was first reported on the east coast of the United States in 2020 in South Carolina. The substantially higher EC50 value suggests that its emergence in the southeastern United States may play at least a part in the observed loss of disease control. However, further in planta and field studies are needed to thoroughly test this hypothesis.

Whitefly-Mediated Transmission and Subsequent Acquisition of Highly Similar and Naturally Occurring Tomato Yellow Leaf Curl Virus Variants.

Begomoviruses are whitefly-transmitted viruses that infect many agricultural crops. Numerous reports exist on individual host plants harboring two or more begomoviruses. Mixed infection allows recombination events to occur among begomoviruses. However, very few studies have examined mixed infection of different isolates/variants/strains of a Begomovirus species in hosts. In this study, the frequency of mixed infection of tomato yellow leaf curl virus (TYLCV) variants in field-grown tomato was evaluated. At least 60% of symptomatic field samples were infected with more than one TYLCV variant. These variants differed by a few nucleotides and amino acids, resembling a quasispecies. Subsequently, in the greenhouse, single and mixed infection of two TYLCV variants (variant #2 and variant #4) that shared 99.5% nucleotide identity and differed by a few amino acids was examined. Plant-virus variant-whitefly interactions including transmission of one and/or two variants, variants' concentrations, competition between variants in inoculated tomato plants, and whitefly acquisition of one and/or two variants were assessed. Whiteflies transmitted both variants to tomato plants at similar frequencies; however, the accumulation of variant #4 was greater than that of variant #2 in tomato plants. Despite differences in variants' accumulation in inoculated tomato plants, whiteflies acquired variant #2 and variant #4 at similar frequencies. Also, whiteflies acquired greater amounts of TYLCV from singly infected plants than from mixed-infected plants. These results demonstrated that even highly similar TYLCV variants could differentially influence component (whitefly-variant-plant) interactions.

Validation of Species-Specific PCR Assays for the Detection of Pantoea ananatis, P. agglomerans, P. allii, and P. stewartii.

Species of Pantoea represent a group of plant pathogenic bacteria that infect a variety of agro-economically important plant species. Among these, a complex of P. ananatis, P. allii, P. agglomerans, and P. stewartii subsp. indologenes cause center rot in onion, resulting in significant economic losses. As species of Pantoea are phenotypically closely related, identification of Pantoea species relies on the sequencing and phylogenetic analysis of housekeeping genes. To aid in rapid identification of Pantoea species, efforts have been made in developing species-specific primers to be used in PCR assays. In the current study, two P. ananatis, one P. allii, one P. agglomerans, and three P. stewartii published primers as well as newly developed P. agglomerans PagR primers were evaluated for their specificity against 79 Pantoea strains, belonging to 15 different species. To ensure that selected primers were evaluated against accurately identified species, sequencing and phylogenetic analysis of housekeeping gene infB were conducted. Thereafter, PCR assays using selected species-specific primers were performed. The results showed that previously described P. ananatis-specific PANA_1008; P. allii-specific allii-leuS; P. stewartii-specific PANST_rpoB, 3614galE, and DC283galE primers; and one newly designed P. agglomerans-specific PagR primer pair were highly specific for their target Pantoea species. They accurately identified these strains into their species and, in some cases, their subspecies level. The findings of the current study will facilitate rapid and reliable identification of P. ananatis, P. agglomerans, P. allii, and P. stewartii.

Pseudomonas capsici sp. nov., a plant-pathogenic bacterium isolated from pepper leaf in Georgia, USA.

Three phytopathogenic bacterial strains (Pc19-1T, Pc19-2 and Pc19-3) were isolated from seedlings displaying water-soaked, dark brown-to-black, necrotic lesions on pepper (Capsicum annuum) leaves in Georgia, USA. Upon isolation on King's medium B, light cream-coloured colonies were observed and a diffusible fluorescent pigment was visible under ultraviolet light. Analysis of their 16S rRNA gene sequences showed that they belonged to the genus Pseudomonas, with the highest similarity to Pseudomonas cichorii ATCC 10857T (99.7 %). The fatty acid analysis revealed that the majority of the fatty acids were summed feature 3 (C16  :  1 ω7c/C16  :  1 ω6c), C16  :  0 and summed feature 8 (C18  :  1 ω7c/C18  :  1 ω6c). Phylogenomic analyses based on whole genome sequences demonstrated that the pepper strains belonged to the Pseudomonas syringae complex with P. cichorii as their closest neighbour, and formed a separate monophyletic clade from other species. Between the pepper strains and P. cichorii, the average nucleotide identity values were 91.3 %. Furthermore, the digital DNA-DNA hybridization values of the pepper strains when compared to their closest relatives, including P. cichorii, were 45.2 % or less. In addition, biochemical and physiological features were examined in this study and the results indicate that the pepper strains represent a novel Pseudomonas species. Therefore, we propose a new species Pseudomonas capsici sp. nov., with Pc19-1T (=CFBP 8884T=LMG 32209T) as the type strain. The DNA G+C content of the strain Pc19-1T is 58.4 mol%.

Foliar Chemical Protection Against Pantoea ananatis in Onion Is Negated by Thrips Feeding.

Center rot of onion, caused by Pantoea ananatis, is an economically important disease in onion production in Georgia and elsewhere in the United States. Growers rely on frequent foliar applications of bactericides and, in some cases, plant defense inducers to manage this disease. However, regular prophylactic application of these chemicals is not cost-effective and may not be environmentally friendly. Thrips (Thrips tabaci and Frankliniella fusca) are vectors of P. ananatis, and their feeding may compromise the effectiveness of foliar applications against P. ananatis. In this study, foliar treatments with acibenzolar-S-methyl (Actigard 50WG), cupric hydroxide (Kocide 3000), and Actigard plus Kocide were evaluated for their effectiveness in the presence and absence of thrips infestation at two critical onion growth stages: bulb initiation and bulb swelling. Onion growth stage had no impact on the effectiveness of either Kocide or Actigard. In the absence of thrips, Kocide application resulted in reduced center rot incidence compared with Actigard, regardless of the growth stage. However, when thrips were present, the efficacy of both Kocide and Actigard was reduced, with bulb incidence not significantly different from the nontreated control. In independent greenhouse studies in the presence or absence of thrips, it was observed that use of protective chemicals (Kocide, Actigard, and their combinations) at different rates also affected pathogen progression into internal neck tissue and incidence of bulb rot. These results suggest that thrips infestation can reduce the efficacy of protective chemical treatments against P. ananatis. Thrips feeding on onion foliage and resulting feeding scars could facilitate P. ananatis entry and subsequently compromise the efficacy of protective chemical treatments. Therefore, an effective center rot management strategy should likely include thrips management in addition to bactericides at susceptible growth stages of onion.

Identification of Two Novel Pathovars of Pantoea stewartii subsp. indologenes Affecting Allium sp. and Millets.

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 2632T) 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.

Genetically Distinct Acidovorax citrulli Strains Display Cucurbit Fruit Preference Under Field Conditions.

Strains of Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits, can be assigned to two groups, I and II. The natural association of group I and II strains with different cucurbit species suggests host preference; however, there are no direct data to support this hypothesis under field conditions. Hence, the objective of this study was to assess differences in the prevalence of group I and II A. citrulli strains on cucurbit species in the field. From 2017 to 2019, we used group I and II strains to initiate BFB outbreaks in field plots planted with four cucurbit species. At different times, we collected symptomatic tissues and assayed them for group I and II strains using a group-specific PCR assay. Binary distribution data analysis revealed that the odds of melon, pumpkin, and squash foliage infection by group I strains were 21.7, 11.5, and 22.1 times greater, respectively, than the odds of watermelon foliage infection by the group I strain (P < 0.0001). More strikingly, the odds of melon fruit infection by the group I strain were 97.5 times greater than watermelon fruit infection by the same strain (P < 0.0001). Unexpectedly, some of the group II isolates recovered from the 2017 and 2019 studies were different from the group II strains used as inocula. Overall, data from these experiments confirm that A. citrulli strains exhibit a preference for watermelon and melon, which is more pronounced in fruit tissues.

Low Frequency of Horizontal and Vertical Transmission of Cucurbit Leaf Crumple Virus in Whitefly Bemisia tabaci Gennadius.

Cucurbit leaf crumple virus (CuLCrV), a bipartite begomovirus, is transmitted by whiteflies in a persistent and circulative manner. Like other begomoviruses, CuLCrV transmission via feeding is well understood; however, whether and how CuLCrV is transmitted by horizontal and vertical modes in its vector, Bemisia tabaci, remains unexplored. We studied transovarial and mating transmission of CuLCrV, and comparatively analyzed virus accumulation in whiteflies through feeding and nonfeeding modes. Furthermore, we quantified CuLCrV DNA A accumulation at different time points to determine whether this virus propagates in whiteflies. CuLCrV DNA A was transmitted vertically and horizontally by B. tabaci, with low frequency in each case. Transovarial transmission of CuLCrV DNA A was only 3.93% in nymphs and 3.09% in adults. Similarly, only a single viruliferous male was able to transmit CuLCrV DNA A to its nonviruliferous female counterparts via mating. In contrast, viruliferous females were unable to transmit CuLCrV DNA A to nonviruliferous males. Additionally, the recipient adults that presumably acquired CuLCrV transovarially and via mating were not able to transmit the virus to squash plants. We further report that the CuLCrV DNA A viral copy numbers were significantly lower in nonfeeding modes of transmission than in feeding ones. The viral copy numbers significantly decreased at succeeding time points throughout adulthood, suggesting no CuLCrV propagation in B. tabaci. Altogether, the low frequency of nonfeeding transmission, reduced virus accumulation in whiteflies, and absence of plant infectivity through nonfeeding transmission suggest that transovarial and mating CuLCrV transmission might not substantially contribute to CuLCrV epidemics.

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.

Isolation and Characterization of Novel Pantoea stewartii subsp. indologenes Strains Exhibiting Center Rot in Onion.

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

First Report of the Yellow Nutsedge Cyst Nematode, Heterodera cyperi, in Georgia, U.S.A.

Soil samples collected during a survey for plant-parasitic nematodes in Tift County GA in summer 2017 were submitted for routine diagnosis of nematodes to the Extension Nematology Lab at the Department of Plant Pathology, University of Georgia, Athens, Georgia. Cyst nematodes recovered by centrifugal flotation technique were discovered in the samples from two research sites in a field with a history of tobacco and vegetable production. Cyst nematodes from tobacco (10 cysts/100 cm 3 of soil) and vegetable (2 cysts/100 cm 3 of soil) sites had similar morphological features. Morphology and morphometric measurements of the cysts and J2 ( Fig. 1A-C ) were in agreement with those of Heterodera cyperi ( Golden et al., 1962 ; Romero and López-Llorca, 1996 ). Measurements of J2 ( n = 12) included the length (range = 443-494 µm, mean = 467.4 µm) and width (18.3-24.4 µm, 20.6 µm) of body, stylet (19.1-20.8 µm, 20.3 µm), tail (61.6.0-66.4 µm, 64.2 µm), body width at anus (11.9-14.1 µm, 12.8 µm), and hyaline tail terminus (22.7-29.2 µm, 26.3 µm). The lateral field of J2 had three lines. Cysts ( n = 10; Fig. 1C ) were lemon-shaped, light to dark brown in color with protruding neck and vulval cone. The cysts had ambifenestrated vulval cone and no bullae was present. Morphometrics included body length excluding neck (370.5-714.4 µm, 555.7 µm); body width (165.6-411.1 µm, 310.9 µm); neck length (36.5-66.3 µm, 49.8 µm); fenestra length (26.3-42.5 µm, 35.8 µm), and fenestra width (19.1-31.5 µm, 23.8 µm). DNA was extracted from single cysts ( n = 3) and internal transcribed spacer (ITS) of rRNA and partial cytochrome oxidase I ( COI ) genes were amplified with primers TW81/AB28 and Het-coxiF/Het-coxiR, respectively ( Subbotin et al., 2001 ; Subbotin, 2015 ) and sequenced. The resulting sequences were deposited into the GenBank database (Accession no. MG825344 and MG857126) and also subjected to BLAST searches in the database. ITS sequence of H. cyperi showed 100% similarity (100% coverage) with that of a H. cyperi population from Spain (AF274388). COI sequence of H. cyperi showed 89% similarity (98% coverage) with that of H. guangdongensis (MF425735), and 88% similarity (83% coverage) with that of H. elachista (KC618473). The pathogenicity of H. cyperi was examined under greenhouse conditions using tobacco cv. K340, tomato cv. Tribute, cucumber cv. Thunder, and yellow nutsedge ( Cyperus esculentus L.). 3-wk-old seedlings of the test plants were transferred into Deepot D25L cell containers (5-cm-diam. × 25.4-cm deep) filled with sterilized sand: sand: soil mixture (1:2) and then inoculated with 1,000 eggs and J2 of H. cyperi . The plants were grown for 90 d in a greenhouse before examination of roots and extraction of cysts from the soil. Results showed that the nematode failed to reproduce on tobacco, tomato, and cucumber whereas white females and mature cysts of H. cyperi were observed on yellow nutsedge roots ( Fig. 1E ). The results confirmed that yellow nutsedeg was a host for the nematode, and tobacco, tomato, or cucumber were non-hosts. In the United States, H. cyperi was reported from Florida, North Carolina, and Arkansas ( Subbotin et al., 2010 ) infecting Cyperus spp. Yellow nutsedge is considered a serious weed problem in many cropping systems including peanut, cotton, tobacco, and vegetable crops in the Southern United States. To our knowledge, this is the first report of H. cyperi infecting yellow nutsedge in Georgia. Figure 1Photomicrographs of Heterodera cyperi from yellow nutsedge in Georgia. Whole body (A), the anterior region (B), and the posterior region (C) of J2. Cysts (D) recovered from the soil and the vulval cone of cyst with the ambifenestrate fenestra (E). A mature cyst (F) on the surface of yellow nutsedge root infected with the nematode.