Nanosecond-pulsed plasma protects against bacterial fruit blotch and promotes melon seedling growth.

BACKGROUND: Bacterial fruit blotch (BFB), known as the 'cancer' of cucurbits, is a seed-borne disease of melons caused by Acidovorax citrulli. Traditional chemical treatments for BFB are ineffective and adversely affect the environment. Using dielectric barrier discharge (DBD) nanosecond-pulsed plasma technology, melon seeds were treated to promote germination and growth and to control BFB. RESULTS: Based on the evaluation parameters of seed germination, seedling growth, leaf yellowing and bacterial infection after seed plasma treatments, 9 min at 20 kV was selected as the optimal plasma discharge parameter. In this study, seedling growth was significantly improved after treating melon seeds carrying A. citrulli using this discharge parameter. The number of first true leaves measured on the eighth day was 2.3 times higher and the disease index was reduced by 60.5% compared to the control group. Attenuated total reflectance-Fourier transform infrared measurements show that plasma treatments penetrate the seed coat and denature polysaccharides and proteins in the seed kernel, affecting their growth and sterilization properties. CONCLUSION: Pre-sowing treatment of melon seeds carrying A. citrulli using nanosecond-pulsed plasma technology can effectively control seedling BFB disease and promote melon seedling growth by optimizing DBD parameters. © 2024 Society of Chemical Industry.

Comparison of Copper-Tolerance Genes between Different Groups of Acidovorax citrulli.

Acidovorax citrulli populations exhibit genetic and phenotypic variations, particularly in terms of copper tolerance. Group I strains of A. citrulli generally exhibit higher copper tolerance compared to group II strains. This study aims to identify genes involved in copper tolerance to better understand the differences in copper tolerance between group I and group II strains. Representative strains pslb65 (group I) and pslbtw14 (group II) were selected for comparison. Deletion mutants of putative copper-tolerance genes and their corresponding complementary strains were constructed. The copper tolerance of each strain was evaluated using the minimum inhibitory concentration method. The results showed that the copA, copZ, cueR, and cueO genes played major roles in copper tolerance in A. citrulli, while cusC-like, cusA-like, and cusB-like genes had minor effects. The different expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 under copper stress indicated that they had different mechanisms for coping with copper stress. Overall, this study provides insights into the mechanisms of copper tolerance in A. citrulli and highlights the importance of specific genes in copper tolerance.

A CRISPR/LbCas12a-based method for detection of bacterial fruit blotch pathogens in watermelon.

Acidovorax citrulli is the main pathogen causing bacterial fruit blotch, which seriously threatens the global watermelon industry. At present, rapid, sensitive, and low-cost detection methods are urgently needed. The established CRISPR/LbCas12a visual detection method can specifically detect A. citrulli and does not cross-react with other pathogenic bacteria such as Erwinia tracheiphila, Pseudomonas syringae, and Xanthomonas campestris. The sensitivity of this method for genomic DNA detection is as low as 0.7 copies/µL, which is higher than conventional PCR and real-time PCR. In addition, this method only takes 2.5 h from DNA extraction to quantitative detection and does not require complex operation and sample treatment. Additionally, the technique was applied to test real watermelon seed samples for A. citrulli, and the results were contrasted with those of real-time fluorescence quantitative PCR and conventional PCR. The high sensitivity and specificity have broad application prospects in the rapid detection of bacterial fruit blotch bacterial pathogens of watermelon.IMPORTANCEBacterial fruit blotch, Acidovorax citrulli, is an important seed-borne bacterial disease of watermelon, melon, and other cucurbits. The lack of rapid, sensitive, and reliable pathogen detection methods has hampered research on fruit spot disease prevention and control. Here, we demonstrate the CRISPR/Cas12a system to analyze aspects of the specificity and sensitivity of A. citrulli and to test actual watermelon seed samples. The results showed that the CRISPR/Cas12a-based free-amplification method for detecting bacterial fruit blotch pathogens of watermelons was specific for A. citrulli target genes and 100-fold more sensitive than conventional PCR with quantitative real-time PCR. This method provides a new technical tool for the detection of A. citrulli.

Virulence-Related Assays for Investigation of the Acidovorax citrulli-Cucurbitaceae Pathosystem.

Acidovorax citrulli is one of the most important pathogens of cucurbit crops, mainly melon and watermelon. Although A. citrulli is able to infect all aerial parts of the plant, fruits are highly sensitive to the bacterium. Therefore, the disease is known as bacterial fruit blotch (BFB). The unavailability of effective tools for managing BFB, including the lack of resistant varieties, exacerbates the threat this disease poses to the cucurbit industry. However, despite the economic importance of BFB, still little is known about basic aspects of A. citrulli-plant interactions. Here, we present diverse techniques that have recently been developed for investigation of basic aspects of BFB, including identification of virulence determinants of the pathogen.

Natural variation in a short region of the Acidovorax citrulli type III-secreted effector AopW1 is associated with differences in cytotoxicity and host adaptation.

Bacterial fruit blotch, caused by Acidovorax citrulli, is a serious disease of melon and watermelon. The strains of the pathogen belong to two major genetic groups: group I strains are strongly associated with melon, while group II strains are more aggressive on watermelon. A. citrulli secretes many protein effectors to the host cell via the type III secretion system. Here we characterized AopW1, an effector that shares similarity to the actin cytoskeleton-disrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, showing 14 amino acid differences between group I and II variants. We show that group I AopW1 is more toxic to yeast and Nicotiana benthamiana cells than group II AopW1, having stronger actin filament disruption activity, and increased ability to induce cell death and reduce callose deposition. We further demonstrated the importance of some amino acid positions within the HVR for AopW1 cytotoxicity. Cellular analyses revealed that AopW1 also localizes to the endoplasmic reticulum, chloroplasts, and plant endosomes. We also show that overexpression of the endosome-associated protein EHD1 attenuates AopW1-induced cell death and increases defense responses. Finally, we show that sequence variation in AopW1 plays a significant role in the adaptation of group I and II strains to their preferred hosts, melon and watermelon, respectively. This study provides new insights into the HopW1 family of bacterial effectors and provides first evidence on the involvement of EHD1 in response to biotic stress.

A putative glucose 6-phosphate isomerase has pleiotropic functions on virulence and other mechanisms in Acidovorax citrulli.

Acidovorax citrulli (Ac) is a causal agent of watermelon bacterial fruit blotch (BFB) disease. Because resistance cultivars/lines have not yet been developed, it is imperative to elucidate Ac's virulence factors and their mechanisms to develop resistant cultivars/lines in different crops, including watermelon. The glucose-6-phosphate isomerase (GPI) is a reversible enzyme in both glycolysis and gluconeogenesis pathways in living organisms. However, the functions of GPI are not characterized in Ac. In this study, we determined the roles of GpiAc (GPI in Ac) by proteomic and phenotypic analyses of the mutant lacking GPI. The mutant displayed significantly reduced virulence to watermelon in two different virulence assays. The mutant's growth patterns were comparable to the wild-type strain in rich medium and M9 with glucose but not with fructose. The comparative proteome analysis markedly identified proteins related to virulence, motility, and cell wall/membrane/envelope. In the mutant, biofilm formation and twitching halo production were reduced. We further demonstrated that the mutant was less tolerant to osmotic stress and lysozyme treatment than the wild-type strain. Interestingly, the tolerance to alkali conditions was remarkably enhanced in the mutant. These results reveal that GpiAc is involved not only in virulence and glycolysis/gluconeogenesis but also in biofilm formation, twitching motility, and tolerance to diverse external stresses suggesting the pleiotropic roles of GpiAc in Ac. Our study provides fundamental and valuable information on the functions of previously uncharacterized glucose 6-phosphate isomerase and its virulence mechanism in Ac.

Acidovorax citrulli type III effector AopU interferes with plant immune responses and interacts with a watermelon E3 ubiquitin ligase.

Acidovorax citrulli is a seed-borne bacterium that causes bacterial fruit blotch of watermelon and other cucurbit plants worldwide. It uses a type III secretion system to inject type III effectors (T3Es) into plant cells, which affect the host immune responses and facilitate pathogen colonization. However, the current understanding of the specific molecular mechanisms and targets of these effectors in A. citrulli is limited. In this study, we characterized a novel T3E called AopU in A. citrulli group II strain Aac5, which shares homology with XopU in Xanthomonas oryzae. The Agrobacterium-mediated gene transient expression system was used to study the effect of AopU on host immunity. The results showed that AopU localized on the cell membrane and nucleus of Nicotiana benthamiana, inhibited reactive oxygen species burst induced by flg22 and the expression of marker genes associated with pathogen-associated molecular pattern-triggered immunity, but activated salicylic acid and jasmonic acid signal pathways. Further investigations revealed that AopU interacts with E3 ubiquitin ligase ClE3R in watermelon, both in vitro and in vivo. Interestingly, the deletion of aopU did not affect the virulence of A. citrulli, suggesting that AopU may have functional redundancy with other effectors in terms of its role in virulence. Collectively, these findings provide new insights into the mechanism of plant immune responses regulated by A. citrulli T3Es.

A novel double-ribonuclease toxin-antitoxin system linked to the stress response and survival of Acidovorax citrulli.

IMPORTANCE: Bacterial fruit blotch (BFB), which is caused by the seed-borne bacterium Acidovorax citrulli, is a devastating disease affecting cucurbit crops throughout the world. Although seed fermentation and treatment with disinfectants can provide effective management of BFB, they cannot completely guarantee pathogen-free seedstock, which suggests that A. citrulli is a highly stress-resistant pathogen. Toxin-antitoxin (TA) systems are common among a diverse range of bacteria and have been reported to play a role in bacterial stress response. However, there is currently much debate about the relationship between TA systems and stress response in bacteria. The current study characterized a novel TA system (Aave_1720-Aave_1719) from A. citrulli that affects both biofilm formation and survival in response to sodium hypochlorite stress. The mechanism of neutralization differed from typical TA systems as two separate mechanisms were associated with the antitoxin, which exhibited characteristics of both type II and type V TA systems. The Aave_1720-Aave_1719 system described here also constitutes the first known report of a double-ribonuclease TA system in bacteria, which expands our understanding of the range of regulatory mechanisms utilized by bacterial TA systems, providing new insight into the survival of A. citrulli in response to stress.

Antibacterial activities of coumarin-3-carboxylic acid against Acidovorax citrulli.

Coumarin-3-carboxylic acid (3-CCA), previously screened from natural coumarins, was found to possess strong antibacterial activity against Acidovorax citrulli (Ac). In order to further evaluate the activity of this compound against plant bacterial pathogens and explore its potential value as a bactericidal lead compound, the activity of 3-CCA against 14 plant pathogenic bacteria in vitro and in vivo was tested. Results showed that 3-CCA exhibited strong in vitro activities against Ac, Ralstonia solanacearum, Xanthomonas axonopodis pv. manihotis, X. oryzae pv. oryzae, and Dickeya zeae with EC50 values ranging from 26.64 µg/mL to 40.73 µg/mL. Pot experiment results showed that 3-CCA had powerful protective and curative effects against Ac. In addition, the protective efficiency of 3-CCA was almost equivalent to that of thiodiazole copper at the same concentration. The results of SEM and TEM observation and conductivity tests showed that 3-CCA disrupted the integrity of the cell membrane and inhibited polar flagella growth. Furthermore, 3-CCA resulted in reductions in motility and extracellular exopolysaccharide (EPS) production of Ac while inhibiting the biofilm formation of Ac. These findings indicate that 3-CCA could be a promising natural lead compound against plant bacterial pathogens to explore novel antibacterial agents.

Effects of di-(2-ethylhexyl) phthalate on growth, metabolism, and virulence of the plant pathogenic bacterium Acidovorax citrulli.

Acidovorax citrulli is a seed-borne bacterial pathogen that causes bacterial fruit blotch in cucurbits and severely affects the production of cucumbers and watermelons globally. In this study, we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP) on the growth, metabolism, and virulence of A. citrulli. Bacterial population was not affected by DEHP exposure; moreover, significant changes were not observed in lipid peroxidation, membrane permeability, and nucleic acid leakage. However, palmitoleic acid content was increased in the cell membrane of DEHP-exposed A. citrulli. Further, DEHP exposure increased the activity of TCA cycle-related enzymes, including α-ketoglutarate dehydrogenase and succinyl-CoA synthetase, along with increase in the content of glutamate, succinate, fumarate, and malate in TCA cycle. Additionally, total 270 genes were differentially expressed by the treatment, of which 28 genes were upregulated and 242 genes, including those related to translation, flagellum-dependent cell motility, and flagellum assembly, were downregulated. Regarding virulence traits, swimming activity was decreased in DEHP-exposed A. citrulli; however, biofilm formation was not affected in in vitro assay. Moreover, relative expression of pathogenicity genes, including hrpX and hrpG, were decreased in DEHP-exposed A. citrulli compared to that of unexposed A. citrulli. Therefore, these results suggest that DEHP accumulation in soil could potentially influence the metabolism and virulence traits of A. citrulli.

Proteomic and Phenotypic Analyses of a Putative YggS Family Pyridoxal Phosphate-Dependent Enzyme in Acidovorax citrulli.

Acidovorax citrulli (Ac) is a phytopathogenic bacterium that causes bacterial fruit blotch (BFB) in cucurbit crops, including watermelon. However, there are no effective methods to control this disease. YggS family pyridoxal phosphate-dependent enzyme acts as a coenzyme in all transamination reactions, but its function in Ac is poorly understood. Therefore, this study uses proteomic and phenotypic analyses to characterize the functions. The Ac strain lacking the YggS family pyridoxal phosphate-dependent enzyme, AcΔyppAc(EV), virulence was wholly eradicated in geminated seed inoculation and leaf infiltration. AcΔyppAc(EV) propagation was inhibited when exposed to L-homoserine but not pyridoxine. Wild-type and mutant growth were comparable in the liquid media but not in the solid media in the minimal condition. The comparative proteomic analysis revealed that YppAc is primarily involved in cell motility and wall/membrane/envelop biogenesis. In addition, AcΔyppAc(EV) reduced biofilm formation and twitching halo production, indicating that YppAc is involved in various cellular mechanisms and possesses pleiotropic effects. Therefore, this identified protein is a potential target for developing an efficient anti-virulence reagent to control BFB.

Differential plant cell responses to Acidovorax citrulli T3SS and T6SS reveal an effective strategy for controlling plant-associated pathogens.

Acidovorax citrulli is a gram-negative plant pathogen that employs the type Ⅲ secretion system (T3SS) to infect cucurbit crops and cause bacterial fruit blotch. This bacterium also possesses an active type Ⅵ secretion system (T6SS) with strong antibacterial and antifungal activities. However, how plant cells respond to these two secretion systems and whether there is any cross talk between T3SS and T6SS during infection remain unknown. Here, we employ transcriptomic analysis to compare cellular responses to the T3SS and the T6SS during in planta infection and report distinctive effects on multiple pathways. The T3SS-mediated differentially expressed genes were enriched in the pathways of phenylpropanoid biosynthesis, plant-pathogen interaction, MAPK signaling pathway, and glutathione metabolism, while the T6SS uniquely affected genes were related to photosynthesis. The T6SS does not contribute to the in planta virulence of A. citrulli but is critical for the survival of the bacterium when mixed with watermelon phyllosphere bacteria. In addition, T3SS-mediated virulence is independent of the T6SS, and the inactivation of the T3SS does not affect the T6SS-mediated competition against a diverse set of bacterial pathogens that commonly contaminate edible plants or directly infect plants. A T6SS-active T3SS-null mutant (Acav) could inhibit the growth of Xanthomonas oryzae pv. oryzae significantly both in vitro and in vivo and also reduce symptoms of rice bacterial blight. In conclusion, our data demonstrate the T6SS in A. citrulli is nonpathogenic to the plant host and can be harnessed as a pathogen killer against plant-associated bacteria. IMPORTANCE Chemical pesticides are widely used to protect crops from various pathogens. Still, their extensive use has led to severe consequences, including drug resistance and environmental contamination. Here, we show that an engineered T6SS-active, but avirulent mutant of Acidovorax citrulli has strong inhibition capabilities against several pathogenic bacteria, demonstrating an effective strategy that is an alternative to chemical pesticides for sustainable agricultural practices.

ntrC Contributes to Nitrogen Utilization, Stress Tolerance, and Virulence in Acidovorax citrulli.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, severely damages watermelon, melon, and other cucurbit crops worldwide. Nitrogen, one of the most important limiting elements in the environment, is necessary for the growth and reproduction of bacteria. As a nitrogen-regulating gene, ntrC plays an important role in maintaining bacterial nitrogen utilization and biological nitrogen fixation. However, the role of ntrC has not been determined for A. citrulli. In this study, we constructed a ntrC deletion mutant and a corresponding complementary strain in the background of the A. citrulli wild-type strain, Aac5. Through phenotype assays and qRT-PCR analysis, we investigated the role of ntrC in A. citrulli in nitrogen utilization, stress tolerance, and virulence against watermelon seedlings. Our results showed that the A. citrulli Aac5 ntrC deletion mutant lost the ability to utilize nitrate. The ntrC mutant strain also exhibited significantly decreased virulence, in vitro growth, in vivo colonization ability, swimming motility, and twitching motility. In contrast, it displayed significantly enhanced biofilm formation and tolerance to stress induced by oxygen, high salt, and copper ions. The qRT-PCR results showed that the nitrate utilization gene nasS; the Type III secretion system-related genes hrpE, hrpX, and hrcJ; and the pili-related gene pilA were significantly downregulated in the ntrC deletion mutant. The nitrate utilization gene nasT, and the flagellum-related genes flhD, flhC, fliA, and fliC were significantly upregulated in the ntrC deletion mutant. The expression levels of ntrC gene in the MMX-q and XVM2 media were significantly higher than in the KB medium. These results suggest that the ntrC gene plays a pivotal role in the nitrogen utilization, stress tolerance, and virulence of A. citrulli.

Protocorm-like-body extract of Phalaenopsis aphrodite combats watermelon fruit blotch disease.

Bacterial fruit blotch, caused by the seedborne gram-negative bacterium Acidovorax citrulli, is one of the most destructive bacterial diseases of cucurbits (gourds) worldwide. Despite its prevalence, effective and reliable means to control bacterial fruit blotch remain limited. Transcriptomic analyses of tissue culture-based regeneration processes have revealed that organogenesis-associated cellular reprogramming is often associated with upregulation of stress- and defense-responsive genes. Yet, there is limited evidence supporting the notion that the reprogrammed cellular metabolism of the regenerated tissued confers bona fide antimicrobial activity. Here, we explored the anti-bacterial activity of protocorm-like-bodies (PLBs) of Phalaenopsis aphrodite. Encouragingly, we found that the PLB extract was potent in slowing growth of A. citrulli, reducing the number of bacteria attached to watermelon seeds, and alleviating disease symptoms of watermelon seedlings caused by A. citrulli. Because the anti-bacterial activity can be fractionated chemically, we predict that reprogrammed cellular activity during the PLB regeneration process produces metabolites with antibacterial activity. In conclusion, our data demonstrated the antibacterial activity in developing PLBs and revealed the potential of using orchid PLBs to discover chemicals to control bacterial fruit blotch disease.

A putative 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase is involved in the virulence, carbohydrate metabolism, biofilm formation, twitching halo, and osmotic tolerance in Acidovorax citrulli.

Acidovorax citrulli (Ac) is a gram-negative bacterium that causes bacterial fruit blotch (BFB) disease in cucurbit crops including watermelon. However, despite the great economic losses caused by this disease worldwide, Ac-resistant watermelon cultivars have not been developed. Therefore, characterizing the virulence factors/mechanisms of Ac would enable the development of effective control strategies against BFB disease. The 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (BdpM) is known to participate in the glycolysis and gluconeogenesis pathways. However, the roles of the protein have not been characterized in Ac. To elucidate the functions of BdpmAc (Bdpm in Ac), comparative proteomic analysis and diverse phenotypic assays were conducted using a bdpmAc knockout mutant (bdpmAc:Tn) and a wild-type strain. The virulence of the mutant to watermelon was remarkably reduced in both germinated seed inoculation and leaf infiltration assays. Moreover, the mutant could not grow with fructose or pyruvate as a sole carbon source. However, the growth of the mutant was restored to levels similar to those of the wild-type strain in the presence of both fructose and pyruvate. Comparative proteomic analyses revealed that diverse proteins involved in motility and wall/membrane/envelop biogenesis were differentially abundant. Furthermore, the mutant exhibited decreased biofilm formation and twitching halo size. Interestingly, the mutant exhibited a higher tolerance against osmotic stress. Overall, our findings suggest that BdpmAc affects the virulence, glycolysis/gluconeogenesis, biofilm formation, twitching halo size, and osmotic tolerance of Ac, suggesting that this protein has pleiotropic properties. Collectively, our findings provide fundamental insights into the functions of a previously uncharacterized phosphoglycerate mutase in Ac.

Development of one-step multiplex RT-PCR assay for rapid simultaneous detection of five RNA viruses and Acidovorax citrulli in major cucurbitaceous crops in China.

Cucurbitaceous fruits and vegetables are important crops. Viral and bacterial diseases cause substantial economic losses to cucurbit crops globally. For rapid detection of these pathogens and improved disease control, a one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) system was created. This method allowed for the concurrent detection of Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), and Acidovorax citrulli. Five pairs of specific primers were created according to the conserved regions around the coat protein (CP) genes of each virus, and one pair was based on the A. citrulli internal transcribed spacer (ITS). To limit false negatives, one pair of primers, created based on the Transcriptional elongation factor 1-α (EF1-α) from the major cucurbitaceous crop species, was put into the mRT-PCR reaction system. Primer concentrations, annealing temperature, extension time, and amplification cycles were optimized. Anticipated fragments of 152 bp (TMV), 205 bp (ZYMV), 318 bp (WMV), 419 bp (CGMMV), 529 bp (CMV), 662 bp (A. citrulli), and 821 bp (EF1-α) were amplified by the multiplex RT-PCR system, and their origin was established via DNA sequencing. This method was successfully used to examine field-collected seed samples of cucurbitaceous crops from China. The results demonstrated that the one-step mRT-PCR technique is a quick, efficient, and sensitive assay for the concurrent detection of six pathogens of cucurbits. It provides a method for monitoring and preventing these diseases.

Acidovorax citrulli Effector AopV Suppresses Plant Immunity and Interacts with Aromatic Dehydratase ADT6 in Watermelon.

Bacterial fruit blotch (BFB) is a disease of cucurbit plants caused by Acidovorax citrulli. Although A. citrulli has great destructive potential, the molecular mechanisms of pathogenicity of A. citrulli are not clear, particularly with regard to its type III secreted effectors. In this study, we characterized the type III secreted effector protein, AopV, from A. citrulli strain Aac5. We show that AopV significantly inhibits reactive oxygen species and the expression of PTI marker genes, and helps the growth of Pseudomonas syringae D36E in Nicotiana benthamiana. In addition, we found that the aromatic dehydratase ADT6 from watermelon was a target of AopV. AopV interacts with ADT6 in vivo and in vitro. Subcellular localization indicated ADT6 and AopV were co-located at the cell membrane. Together, our results reveal that AopV suppresses plant immunity and targets ADT6 in the cell membrane. These findings provide an new characterization of the molecular interaction of A. citrulli effector protein AopV with host cells.

OxyR contributes to virulence of Acidovorax citrulli by regulating anti-oxidative stress and expression of flagellin FliC and type IV pili PilA.

In many bacteria, OxyR acts as a transcriptional regulator that facilitates infection via degrading hydrogen peroxide (H2O2) generated by the host defense response. Previous studies showed that OxyR also plays an important role in regulating biofilm formation, cell motility, pili relate-genes expression, and surface polysaccharide production. However, the role of OxyR has not been determined in Acidovorax citrulli strain xjl12. In the current study, the qRT-PCR and western blot assays revealed that the expression level of oxyR was significantly induced by H2O2. The oxyR deletion mutant of A. citrulli was significantly impaired bacterial tolerance to oxidative stress and reduced catalase (CAT) activity. In addition, oxyR mutant resulted in reduced swimming motility, twitching motility, biofilm formation, virulence, and bacterial growth in planta by significantly affecting flagellin and type IV pili-related gene (fliC and pilA) expression. The qRT-PCR assays and western blot revealed that OxyR positively regulated the expression of fliC and pilA. Furthermore, bacterial one-hybrid assay demonstrated that OxyR directly affected pilA and fliC promoter. Through bacterial two-hybrid assay, it was found that OxyR can directly interact with PilA and FliC. These results suggest that OxyR plays a major role in the regulating of a variety of virulence traits, and provide a foundation for future research on the global effects of OxyR in A. citrulli.

Hcp of the Type VI Secretion System (T6SS) in Acidovorax citrulli Group II Strain Aac5 Has a Dual Role as a Core Structural Protein and an Effector Protein in Colonization, Growth Ability, Competition, Biofilm Formation, and Ferric Iron Absorption.

A type VI secretion system (T6SS) gene cluster has been reported in Acidovorax citrulli. Research on the activation conditions, functions, and the interactions between key elements in A. citrulli T6SS is lacking. Hcp (Hemolysin co-regulated protein) is both a structural protein and a secretion protein of T6SS, which makes it a special element. The aims of this study were to determine the role of Hcp and its activated conditions to reveal the functions of T6SS. In virulence and colonization assays of hcp deletion mutant strain Δhcp, tssm (type VI secretion system membrane subunit) deletion mutant strain Δtssm and double mutant ΔhcpΔtssm, population growth was affected but not virulence after injection of cotyledons and seed-to-seedling transmission on watermelon. The population growth of Δhcp and Δtssm were lower than A. citrulli wild type strain Aac5 of A. citrulli group II at early stage but higher at a later stage. Deletion of hcp also affected growth ability in different culture media, and the decline stage of Δhcp was delayed in KB medium. Biofilm formation ability of Δhcp, Δtssm and ΔhcpΔtssm was lower than Aac5 with competition by prey bacteria but higher in KB and M9-Fe3+ medium. Deletion of hcp reduced the competition and survival ability of Aac5. Based on the results of Western blotting and qRT-PCR analyses, Hcp is activated by cell density, competition, ferric irons, and the host plant. The expression levels of genes related to bacterial secretion systems, protein export, and several other pathways, were significantly changed in the Δhcp mutant compared to Aac5 when T6SS was activated at high cell density. Based on transcriptome data, we found that a few candidate effectors need further identification. The phenotypes, activated conditions and transcriptome data all supported the conclusion that although there is only one T6SS gene cluster present in the A. citrulli group II strain Aac5, it related to multiple biological processes, including colonization, growth ability, competition and biofilm formation.

The GDSL-Lipolytic Enzyme Lip1 Is Required for Full Virulence of the Cucurbit Pathogenic Bacterium Acidovorax citrulli.

Bacterial fruit blotch caused by Acidovoraxcitrulli is a serious disease of cucurbit crops. Here we report characterization of a mutant strain of A. citrulli M6 defective in lip1, a gene encoding a lipolytic enzyme. The M6-lip1- mutant was detected in a mutant library screen aimed at identifying M6 mutants with altered levels of twitching motility. In this screen M6-lip1- was the only mutant that showed significantly larger twitching motility haloes around colonies than wild-type M6. Sequence analyses indicated that lip1 encodes a member of the GDSL family of secreted lipolytic enzymes. In line with this finding, lipolytic assays showed that the supernatants of M6-lip1- had lower lipolytic activity as compared with those of wild-type M6 and a lip1-complemented strain. The mutant was also affected in swimming motility and had compromised virulence on melon seedlings and on Nicotiana benthamiana leaves relative to wild-type and complemented strains. Lip1 contains a predicted N-terminal signal sequence for type II secretion. Evidence from our study confirms Lip1 is indeed secreted in a type II secretion-dependent manner, and this is required for full virulence of A. citrulli. To the best of our knowledge this is the first study reporting contribution of lipolytic activity to virulence of a plant-pathogenic Acidovorax species.