Insights into the cumulative effect of Colletotrichum gloeosporioides and Fusarium acutatum causing anthracnose-twister disease complex of onion.

Colletotrichum is an important plant pathogenic fungi that causes anthracnose/-twister disease in onion. This disease was prevalent in the monsoon season from August to November months and the symptoms were observed in most of the fields. This study aimed to investigate the pathogenicity and cumulative effect, if any of Colletotrichum gloeosporioides and Fusarium acutatum. The pot experiment was laid out to identify the cause responsible for inciting anthracnose-twister disease, whether the Colletotrichum or Fusarium or both, or the interaction of pathogens and GA3. The results of the pathogenicity test confirmed that C. gloeosporioides and F. acutatum are both pathogenic. C. gloeosporioides caused twisting symptoms independently, while F.acutatum independently caused only neck elongation. The independent application of GA3 did not produce any symptoms, however, increased the plant height. The combined treatment of C. gloeosporioides and F. acutatum caused twisting, which enhanced upon interaction with GA3 application giving synergistic effect. The acervuli were found in lesions infected with C. gloeosporioides after 8 days of inoculation on the neck and leaf blades. Symptoms were not observed in untreated control plants. Koch's postulates were confirmed by reisolating the same pathogens from the infected plants.

Description and management of Aspergillus section Nigri causing post-harvest bulbs rot of onion.

When onions are improperly stored, a post-harvest disease known as black mold of onion bulbs can result in considerable economic losses. Aspergillus section Nigri, one of many species, has been implicated in the development of black mold. In the present study, rot onion bulbs were collected from markets in Qena, Egypt. Thirteen Aspergillus section Nigri isolates were obtained and identified by morphological and molecular characterization. The ochratoxins potential of isolated A. section Nigri was tested, and three isolates were producers at the range of 1.5-15 ppm. For the presence of pks gene, no amplification product was detected. Using the fungal growth inhibition test, the isolates of A. niger were inhibited by eco-friendly materials Cement and Zeolite. Cement exhibited maximum percentage growth inhibition against the tested isolates at 74.7-86.7%. The pathogenicity activity of the A. niger isolates was tested by inoculation of healthy onion bulbs, other onion bulbs covered with Cement and Zeolite before inoculation by A. niger was used. The two treatments significantly reduced bulbs rot disease of onion than untreated bulbs. Seven and nine isolates showed 0% rot on covered bulbs by Cement and Zeolite, respectively as compared with inoculated onions, which exhibited rot ranging from 55 to 80%. Using eco-friendly materials with efficiency against post-harvest bulbs rot of onion was evaluated in this study.

Enabling Population Biology Studies of Stemphylium vesicarium from Onion with Microsatellites.

Stemphylium leaf blight (SLB), caused by the fungus Stemphylium vesicarium, is dominant within the foliar disease complex affecting onion production in New York (NY). The disease causes premature defoliation and significant reductions in bulb weight and quality. Foliar diseases of onion are usually managed by an intensive fungicide program, but SLB management is complicated by resistance to multiple single-site modes of action. The design of integrated disease management strategies is limited by incomplete knowledge surrounding the dominant sources of S. vesicarium inoculum. To facilitate genomic-based studies of S. vesicarium populations, nine microsatellite markers were developed. The markers were multiplexed into two PCR assays containing four and five fluorescently labeled microsatellite markers. Initial testing of the S. vesicarium isolates found the markers were highly polymorphic and reproducible with an average of 8.2 alleles per locus. The markers were used to characterize 54 S. vesicarium isolates from major NY onion production regions in 2016 (n = 27) and 2018 (n = 27). Fifty-two multilocus genotypes (MLGs) were identified between these populations. Genotypic and allelic diversities were high in both the 2016 and 2018 populations. A greater degree of genetic variation was observed within populations than between years. No distinct pattern of MLGs according to population was identified and some MLGs were closely related between 2016 and 2018. The lack of evidence for linkage among loci also was strongly suggestive of clonal populations with only minor differences between the two populations. These microsatellite markers will be a foundational resource for the testing of hypotheses surrounding the population biology of S. vesicarium and therefore informing disease management.

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.

Isolation and Characterization of Bacteria Associated with Onion and First Report of Onion Diseases Caused by Five Bacterial Pathogens in Texas, U.S.A.

Bacterial diseases pose a severe challenge to growers and cause significant loss to the billion-dollar onion industry in the United States. Texas is the sixth largest onion producing state, yet the bacterial communities associated with short-day onion crops grown in Texas have not been studied. This study was conducted to identify, characterize, and understand the diversity of bacteria associated with onion production in Texas. In 2020, 190 foliar and 210 bulb samples were collected from onion crops in the Rio Grande Valley and Winter Garden regions of Texas. Sequencing of the 16s rRNA gene was used to identify each bacterial strains to a genus. The pathogenicity to onion of each bacterial strain was tested using three assays: a red onion scale assay, a yellow onion bulb assay, and a foliar assay. Whole genome sequencing was done to identify the onion-pathogenic strains to species. Collectively, isolates of 24 genera belonging to three phyla were detected, including 19 genera from foliar samples and nine genera from bulb samples. Isolates in the Phylum Proteobacteria, including 15 genera of Gram-negative bacteria, were the most abundant of the taxa, comprising 90.0% of the strains isolated. The diversity of foliar isolates was evenly distributed between Gram-positive and Gram-negative bacteria, while Gram-negative bacteria dominated the isolates from bulb samples. In total, 83.9% of the bacterial isolates were not pathogenic on onion, with only isolates of Pantoea, Pseudomonas, Burkholderia, Erwinia, Enterobacter, and Curtobacterium proving pathogenic. Strains of Burkholderia gladioli, Pseudomonas alliivorans, Pantoea agglomerans, P. ananatis, and P. allii are the first documented cases of these pathogens of onion in Texas. Identifying and characterizing the nature of onion microflora, including pathogens of onion, is vital to developing rapid disease detection techniques via pathogenomics and minimizing losses through the application of effective disease management measures.

Bi-national outbreak of Salmonella Newport infections linked to onions: the United States experience.

From 2016-2019, dry bulb onions were the suspected cause of three multistate outbreaks in the United States. We investigated a large multistate outbreak of Salmonella Newport infections that caused illnesses in both the United States and Canada in 2020. Epidemiologic, laboratory and traceback investigations were conducted to determine the source of the infections, and data were shared among U.S. and Canadian public health officials. We identified 1127 U.S. illnesses from 48 states with illness onset dates ranging from 19 June to 11 September 2020. Sixty-six per cent of ill people reported consuming red onions in the week before illness onset. Thirty-five illness sub-clusters were identified during the investigation and seventy-four per cent of sub-clusters served red onions to customers during the exposure period. Traceback for the source of onions in illness sub-clusters identified a common onion grower in Bakersfield, CA as the source of red onions, and onions were recalled at this time. Although other strains of Salmonella Newport were identified in environmental samples collected at the Bakersfield, CA grower, extensive environmental and product testing did not yield the outbreak strain. This was the third largest U.S. foodborne Salmonella outbreak in the last 30 years. It is the first U.S. multistate outbreak with a confirmed link to dry bulb onions, and it was nearly 10-fold larger than prior outbreaks with a suspected link to onions. This outbreak is notable for its size and scope, as well as the international data sharing that led to implication of red onions as the primary cause of the outbreak. Although an environmental assessment at the grower identified several factors that likely contributed to the outbreak, no main reason was identified. The expedient identification of the outbreak vehicle and response of multiple public health agencies allowed for recall and removal of product from the marketplace, and rapid messaging to both the public and industry on actions to protect consumers; these features contributed to a decrease in cases and expeditious conclusion of the outbreak.

Preliminary Studies on Detection of Fusarium Basal Rot Infection in Onions and Shallots Using Electronic Nose.

The evaluation of crop health status and early disease detection are critical for implementing a fast response to a pathogen attack, managing crop infection, and minimizing the risk of disease spreading. Fusarium oxysporum f. sp. cepae, which causes fusarium basal rot disease, is considered one of the most harmful pathogens of onion and accounts for considerable crop losses annually. In this work, the capability of the PEN 3 electronic nose system to detect onion and shallot bulbs infected with F. oxysporum f. sp. cepae, to track the progression of fungal infection, and to discriminate between the varying proportions of infected onion bulbs was evaluated. To the best of our knowledge, this is a first report on successful application of an electronic nose to detect fungal infections in post-harvest onion and shallot bulbs. Sensor array responses combined with PCA provided a clear discrimination between non-infected and infected onion and shallot bulbs as well as differentiation between samples with varying proportions of infected bulbs. Classification models based on LDA, SVM, and k-NN algorithms successfully differentiate among various rates of infected bulbs in the samples with accuracy up to 96.9%. Therefore, the electronic nose was proved to be a potentially useful tool for rapid, non-destructive monitoring of the post-harvest crops.

Pseudomonas uvaldensis sp. nov., a bacterial pathogen causing onion bulb rot.

A Gram-stain-negative, aerobic and non-spore-forming bacterial strain, designated 20TX0172T, was isolated from a rotting onion bulb in Texas, USA. The results of phylogenetic analysis based on the 16S rRNA sequence indicated that the novel strain represented a member of the genus Pseudomonas and had the greatest sequence similarities with Pseudomonas kilonensis 520-20T (99.3 %), Pseudomonas corrugata CFBP 2431T (99.2 %), and Pseudomonas viciae 11K1T (99.2 %) but the 16S rRNA phylogenetic tree displayed a monophyletic clade with Pseudomonas mediterranea CFBP 5447T. In the phylogenetic trees based on sequences of four housekeeping genes (gap1, gltA, gyrB and rpoD), the novel strain formed a separate branch, indicating that the strain was distinct phylogenetically from known species of the genus Pseudomonas. The genome-sequence-derived average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the novel isolate and P. mediterranea DSM 16733T were 86.7 and 32.7 %, respectively. These values were below the accepted species cutoff threshold of 96 % ANI and 70 % dDDH, affirming that the strain represented a novel species. The genome size of the novel species was 5.98 Mbp with a DNA G+C content of 60.8 mol%. On the basis of phenotypic and genotypic characteristics, strain 20TX0172T represents a novel species of the genus Pseudomonas. The name Pseudomonas uvaldensis sp. nov. is proposed. The type strain is 20TX0172T (=NCIMB 15426T=CIP 112022T).

Water Application Method Influences Survival or Growth of Escherichia coli on Bulb Onions during Field Curing.

ABSTRACT: The impact of water application method on bacterial survival at or after the final irrigation was evaluated in bulb onions during commercially relevant field drying (curing). A three-strain rifampin-resistant cocktail of Escherichia coli was introduced to onions via a single overhead spray application in two separate trials (5.22 [trial 1] or 2.40 [trial 2] log CFU per onion) 2 to 3 days after the final irrigation. Onions were lifted from the soil 8 days after spray inoculation and, in some cases, foliage was removed (topping); onions remained in the field for an additional ca. 2 weeks (total ca. 3 weeks of curing). E. coli populations declined on the onions in the first 4 h after spray inoculation. E. coli was recovered from 38 (48%) or 28 (35%) of 80 whole-onion enrichments at the end of curing in trials 1 or 2, respectively. Topping did not significantly impact the percentage of E. coli-positive onions detected at the end of curing. From 8 h to 21 days, E. coli populations on positive onions ranged from 1 CFU per onion to 7 log CFU per onion in both trials, representing a potential risk of E. coli growth with overhead application of contaminated water at the end of onion production. In trial 2, additional rows of onions were inoculated via a 22-cm subsurface or surface drip irrigation line (1.94 log CFU/mL for 2.5 h). E. coli was detected in 0 (subsurface) and 4 (surface) of 50 whole-onion enrichments 3 h after the initiation of drip irrigation. Positive onions were detected at days 1 (4 of 50) and 7 (1 of 50) with subsurface drip inoculation, and at days 1 (7 of 50), 7 (2 of 50), and 14 (2 of 50) with surface drip inoculation. E. coli was not detected in whole-onion enrichments at the end of curing when inoculated by subsurface (0 of 50) or surface (0 of 50) drip irrigation. Application of contaminated water through drip irrigation, when coupled with field curing, results in low rates of contamination of bulb onions at the time of harvest.

Rahnella perminowiae sp. nov., Rahnella bonaserana sp. nov., Rahnella rivi sp. nov. and Rahnella ecdela sp. nov., isolated from diverse environmental sources, and emended description of the genus Rahnella.

Bacteria isolated from onion bulbs suffering from bacterial decay in the United States and Norway were previously shown to belong to the genus Rahnella based on partial housekeeping gene sequences and/or fatty acid analysis. However, many strains could not be assigned to any existing Rahnella species. Additionally, strains isolated from creek water and oak as well as a strain with bioremediation properties were assigned to Rahnella based on partial housekeeping gene sequences. The taxonomic status of these 21 strains was investigated using multilocus sequence analysis, whole genome analyses, phenotypic assays and fatty acid analysis. Phylogenetic and phylogenomic analyses separated the strains into five clusters, one of which corresponded to Rahnella aceris. The remaining four clusters could be differentiated both genotypically and phenotypically from each other and existing Rahnella species. Based on these results, we propose the description of four novel species: Rahnella perminowiae sp. nov. (type strain SL6T=LMG 32257T=DSM 112609T), Rahnella bonaserana sp. nov. (H11bT=LMG 32256T=DSM 112610T), Rahnella rivi sp. nov. (FC061912-KT=LMG 32259T=DSM 112611T) and Rahnella ecdela sp. nov. (FRB 231T=LMG 32255T=DSM 112612T).

Characterization of Alternaria porri causing onion purple blotch and its antifungal compound magnolol identified from Caryodaphnopsis baviensis.

Alternaria porri (Ellis) Clf. causes purple blotch disease on Allium plants which results in the reduction of crop yields and quality. In this study, to efficiently find natural antifungal compounds against A. porri, we optimized the culture condition for the spore production of A. porri and the disease development condition for an in vivo antifungal assay. From tested plant materials, the methanol extracts derived from ten plant species belonging to the families Cupressaceae, Fabaceae, Dipterocarpaceae, Apocynaceae, Lauraceae, and Melastomataceae were selected as potent antifungal agents against A. porri. In particular, the methanol extract of Caryodaphnopsis baviensis (Lec.) A.-Shaw completely inhibited the growth of A. porri at a concentration of 111 µg/ml. Based on chromatographic and spectroscopic analyses, a neolignan compound magnolol was identified as the antifungal compound of the C. baviensis methanol extract. Magnolol showed a significant inhibitory activity against the spore germination and mycelial growth of A. porri with IC50 values of 4.5 and 5.4 µg/ml, respectively. Furthermore, when magnolol was sprayed onto onion plants at a concentration of 500 µg/ml, it showed more than an 80% disease control efficacy for the purple blotch diseases. In terms of the antifungal mechanism of magnolol, we explored the in vitro inhibitory activity on individual oxidative phosphorylation complexes I-V, and the results showed that magnolol acts as multiple inhibitors of complexes I-V. Taken together, our results provide new insight into the potential of magnolol as an active ingredient with antifungal inhibitory action to control purple blotch on onions.

Production of transgenic Allium cepa by nanoparticles to resist Aspergillus niger infection.

BACKGROUND: Transgenic plants are becoming a more powerful tool in modern biotechnology. Genetic engineering was used in biotech-derived products to create genetically modified (GM) plants resistant to diseases. The onion (Allium cepa, L.) is a common, important perennial vegetable crop grown in Egypt for food and economic value. Onions are susceptible to a variety of fungal infections and diseases. Aspergillus niger is a common onion phytopathogen that causes diseases such as black mould (or black rot), which is a major issue, particularly when exporting onions. A. niger grows between the bulb's outer (dead, flaky) skin and the first fleshy scales, which become water-soaked. Thionin genes produce thionin proteins, which have antimicrobial properties against a variety of phytopathogens, including A. niger. Chitosan nanoparticles act as a carrier for the thionin gene, which allows A. cepa to resist infection by A. niger. METHODS AND RESULTS: Thionin gene (Thio-60) was transformed into A. cepa to be resistance to fungal infection. The gene was loaded on chitosan nanoparticles to be transformed into plants. Transgenic A. cepa had a 27% weight inhibition compared to non-transgenic one, which had a 69% inhibition. The expressed thionin protein has a 52% inhibitory effect on A. niger spore germination. All these findings supported thionin protein's antifungal activity as an antimicrobial peptide. Furthermore, the data presented here demonstrated the efficacy of chitosan nanoparticles in gene transformation. CONCLUSION: The present study describes the benefits of producing transgenic onion resistance to black rot diseases via expression of thionin proteins.

Phylogeny and Identification of Pantoea Species Associated with Bulb Rot and Bacterial Leaf Blight of Onion Crops in Uruguay.

Onion is among the most consumed vegetables in Uruguay, grown in the northwestern and southern regions of the country. The onion supply presents interannual variations associated with significant postharvest losses, mainly caused by bacterial rots. Besides bulb rotting, onion leaf lesions as well as infections on seed-stalks during seed production may be devastating for some varieties under conducive conditions. This research aimed to identify the causal agents of bulb rots and leaf blight of onion crops in Uruguay. Symptomatic bulbs, seeds-stalks, and leaves were collected from commercial fields from 2015 to 2020. Bacterial colonies were isolated and identified at genera level using physiological tests and 16S rRNA gene sequence analysis. A collection of 59 Pantoea spp. isolates was obtained (11 from bulbs and 48 from leaves and seeds-stalks). Multilocus sequence analysis using four housekeeping genes (rpoB, gyrB, leuS, and fusA) allowed the assignment of the isolates to five Pantoea species: P. ananatis, P. agglomerans, P. allii, P. eucalypti, and P. vagans. The last two species were not previously reported as onion pathogens elsewhere. The ability to cause disease symptoms was tested by leaf inoculation and red onion scale assays. P. ananatis isolates showed the highest aggressiveness in both assays. Specific isolates from P. allii (MAI 6022), P. eucalypti (MAI 6036), P. vagans (MAI 6050), and Pantoea sp. (MAI 6049) ranked second in aggressiveness on onion leaves, whereas only three isolates belonging to P. eucalypti (MAI 6036 and MAI 6058) and P. agglomerans (MAI 6045) exhibited the same scale-clearing phenotype as P. ananatis. Leaf inoculation assays were also performed on a set of eight onion cultivars and breeding lines. Overall, P. ananatis MAI 6032 showed the highest aggressiveness in all tested cultivars, followed by P. eucalypti MAI 6036. The presence of new reported bacterial species leads to complex disease management and highlights the need for further studies on virulence factors and the epidemiology of these pathogens.

Evaluation of qualitative traits in modified Iranian Red Rey onion and comparison genetic resistance with primary mass selection and Red Azar-shahr cv to Fusarium oxysporum using laboratory and molecular markers.

BACKGROUND: The bulb onion (Allium cepa L.) is grown on all continents except Antarctica, and is prized by essentially all of the world's cultures for its flavor and health-enhancing attributes. Onion breeders focus primarily on bulb characteristics such as color, shape, soluble-solids content, pungency and flavor, storage ability, and health-enhancing attributes, as well as plant characters such as resistances to diseases. The use of breeding approaches, offers great promise for population improvement and hybrid development addressing changes in consumer preference and production environments. The aim of this study is to evaluate the storage and qualitative feature of modified Red Rey Iranian Onion. METHOD: Firstly, the modified population was obtained by the selection of superior bulbs, cultivation, its self-pollination and consequently the identification of the best families and implement open pollination between them. In next level, the Red Rey Iranian modified with basic population and Red Azar-shahr cultivar (comparative) was crossed. RESULTS: Our results showed that the selection procedure has leading to improvement in variety of traits in population. Also, the modified Red Rey is significantly superior to the base mass in qualitative traits such as: bulb stiffness, bulb dry matter, TSS, total sugar and glucose; So that the percentage of dry bulb content increased from 10.4% in the basal mass to 11.1% in the modified Red Rey; while spouring and rotting, minerals, and dry matter, vitamin C and fructose-reducing sugar was not affected by genotype. In the second step, resistances to Fusarium wilt disease (laboratory and molecular markers) were evaluated. Based on the results of phenotypic evaluation, the modified Red Rey had the lowest rate and level of infection and the highest score. According to the results of genotypic evaluation, there is a very high genetic affinity between resistant and susceptible cultivars.

Biofilm formation by strains of Burkholderia cenocepacia lineages IIIA and IIIB and B. gladioli pv. alliicola associated with onion bacterial scale rot.

The Burkholderia genus has high ecological and nutritional versatility, having species capable of causing diseases in animals, humans, and plants. During chronic infections in humans, biofilm formation is a characteristic often associated with strains from different species of this genus. However, there is still no information on the formation of biofilms by plant pathogenic strains of B. cenocepacia (Bce) lineages IIIA and IIIB and B. gladioli pv. alliicola (Bga), which are associated with onion bacterial scale rot in the semi-arid region of northeast Brazil. In this study, we performed an in vitro characterization of biofilm formation ability in different culture media by the phytopathogenic strains of Bce and Bga and investigated its relationship with swarming motility. Our results indicated the existence of an intraspecific variation in biofilm formation capacity in vitro by these bacteria and the existence of a negative correlation between swarming motility and biofilm formation for strains of Bce lineage IIIB. In addition, histopathological analyses performed using optical microscopy and scanning electron microscopy revealed the formation of biofilm in vivo by Bce strains in onion tissues.

Chemical or Genetic Alteration of Proton Motive Force Results in Loss of Virulence of Burkholderia glumae, the Cause of Rice Bacterial Panicle Blight.

Rice is an important source of food for more than half of the world's population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. B. glumae synthesizes toxoflavin, an essential virulence factor that is required for symptoms of the disease. The products of the tox operons, ToxABCDE and ToxFGHI, are responsible for the synthesis and the proton motive force (PMF)-dependent secretion of toxoflavin, respectively. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Our previous work has demonstrated that absence of certain DedA family members results in pleiotropic effects, impacting multiple pathways that are energized by PMF. We have demonstrated that a member of the DedA family from Burkholderia thailandensis, named DbcA, is required for the extreme polymyxin resistance observed in this organism. B. glumae encodes a homolog of DbcA with 73% amino acid identity to Burkholderia thailandensis DbcA. Here, we created and characterized a B. glumae ΔdbcA strain. In addition to polymyxin sensitivity, the B. glumae ΔdbcA strain is compromised for virulence in several BPB infection models and secretes only low amounts of toxoflavin (∼15% of wild-type levels). Changes in membrane potential in the B. glumae ΔdbcA strain were reproduced in the wild-type strain by the addition of subinhibitory concentrations of sodium bicarbonate, previously demonstrated to cause disruption of PMF. Sodium bicarbonate inhibited B. glumae virulence in rice, suggesting a possible non-toxic chemical intervention for bacterial panicle blight. IMPORTANCE Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Here, we constructed a B. glumae mutant with a deletion in a DedA family member named dbcA and report a loss of virulence in models of BPB. Physiological analysis of the mutant shows that the proton motive force is disrupted, leading to reduction of secretion of the essential virulence factor toxoflavin. The mutant phenotypes are reproduced in the virulent wild-type strain without an effect on growth using sodium bicarbonate, a nontoxic buffer that has been reported to disrupt the PMF. The results presented here suggest that bicarbonate may be an effective antivirulence agent capable of controlling BPB without imposing an undue burden on the environment.

Regulatory effects of nitric oxide on reproduction and melanin biosynthesis in onion pathogenic fungus Stemphylium eturmiunum.

The formation of propagules is the critical stage for transmission of the pathogenic fungus Stemphylium eturmiunum. However, how the development of these propagules is regulated remains to be fully understood. Here, we show that nitric oxide (NO) is necessary for reproduction in S. eturmiunum.Application of NO scavenger carboxy-CPTIO (cPTIO) or soluble guanylate cyclase (sGC) inhibitor NS-2028 abolishes propagules formation, which was increased by a supplement of sodium nitroprusside (SNP). SNP supplement also triggered increased biosynthesis of melanin, which can be inhibited upon the addition of arbutin or tricyclazole, the specific inhibitors for DOPA and DHN synthetic pathway, respectively. Intriguingly, enhanced melanin biosynthesis corelates with an increased propagules formation; The SNP-induced increment propagules formation can be also compromised upon the supplement of cPTIO or NS-2028. RT-PCR analysis showed that SNP promoted transcription of brlA, abA and wetA at 0.2 mmol/L, but inhibited at 2 mmol/L. In contrast, SNP increased transcription of mat1, and mat2, and the synthetic genes for DHN and DOPA melanins at 2 mmol/L. However, the increased transcription of these genes is down-regulated upon the supplement of cPTIO or NS-2028. Thus, NO regulates reproduction and melanin synthesis in S. eturmiunum possibly through the NO-sGC-GMP signaling pathway.

Interactive effects of plant growth-promoting rhizobacteria and a seaweed extract on the growth and physiology of Allium cepa L. (onion).

Detrimental effects caused by the overuse of synthetic agrochemicals have led to the development of natural biostimulants such as seaweed extracts and plant growth-promoting rhizobacteria (PGPR) being used as an alternative, environmentally-friendly technology to improve crop growth and increase agricultural yields. The present study aimed to investigate the interactions between PGPR and a commercial seaweed extract on the growth and biochemical composition of onion (Allium cepa). A pot trial was conducted under greenhouse conditions where onion plants were treated individually with the two PGPR, namely Bacillus licheniformis (BL) and Pseudomonas fluorescens (PF) and a seaweed extract Kelpak® (KEL) and combinations of KEL + BL and KEL + PF. Growth and yield parameters were measured after 12 weeks. KEL-treated plants showed the best growth response and overcame the inhibitory effects of BL treatment. KEL-treated plants also had the highest chlorophyll content. PGPR application improved the mineral nutrition of onion with these plants having the highest mineral content in the leaves and bulb. All biostimulant treatments increased the endogenous cytokinin and auxin content with the highest concentrations generally detected in the PF-treated plants. These results suggest that co-application of different biostimulant classes with different modes of action could further increase crop productivity with an improvement in both growth and nutrition content being achieved in onion with the co-application of a seaweed extract and PGPR.

Visualization of Three Sclerotiniaceae Species Pathogenic on Onion Reveals Distinct Biology and Infection Strategies.

Botrytis squamosa, Botrytis aclada, and Sclerotium cepivorum are three fungal species of the family Sclerotiniaceae that are pathogenic on onion. Despite their close relatedness, these fungi cause very distinct diseases, respectively called leaf blight, neck rot, and white rot, which pose serious threats to onion cultivation. The infection biology of neck rot and white rot in particular is poorly understood. In this study, we used GFP-expressing transformants of all three fungi to visualize the early phases of infection. B. squamosa entered onion leaves by growing either through stomata or into anticlinal walls of onion epidermal cells. B. aclada, known to cause post-harvest rot and spoilage of onion bulbs, did not penetrate the leaf surface but instead formed superficial colonies which produced new conidia. S. cepivorum entered onion roots via infection cushions and appressorium-like structures. In the non-host tomato, S. cepivorum also produced appressorium-like structures and infection cushions, but upon prolonged contact with the non-host the infection structures died. With this study, we have gained understanding in the infection biology and strategy of each of these onion pathogens. Moreover, by comparing the infection mechanisms we were able to increase insight into how these closely related fungi can cause such different diseases.