AKSF1 Isolated From the Rice-Virulent Strain Acidovorax avenae K1 Is a Novel Effector That Suppresses PAMP-Triggered Immunity in Rice.

Microbial pathogens deliver effectors into plant cells to suppress plant immune responses and modulate host metabolism in order to support infection processes. We sought to determine if the Acidovorax avenae rice-virulent K1 strain can suppress pathogen-associated molecular pattern-triggered immunity (PTI) induced by flagellin isolated from the rice-avirulent N1141 strain. The flagellin-triggered PTI, including H2O2 generation, callose deposition, and expression of several immune-related genes were strongly suppressed in K1 preinoculated cultured rice cells in a type III secretion system (T3SS)-dependent manner. By screening 4,562 transposon-tagged mutants based on their suppression ability, we found that 156 transposon-tagged K1 mutants lost the ability to suppress PTI induction. Mutant sequence analysis, comprehensive expression analysis using RNA sequencing, and the prediction of secretion through T3SS showed that a protein named A. avenae K1 suppression factor 1 (AKSF1) suppresses flagellin-triggered PTI in rice. Translocation of AKSF1 protein into rice cells is dependent on the T3SS during infection, an AKSF1-disruption mutant lost the ability to suppress PTI responses, and expression of AKSF1 in the AKSF1-disruption mutant complemented the suppression activity. When AKSF1-disruption mutants were inoculated into the host rice plant, reduction of the disease symptoms and suppression of bacterial growth were observed. Taken together, our results demonstrate that AKSF1 is a novel effector that can suppress the PTI in a host rice plant.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.

Identification and Functional Analysis of AopN, an Acidovorax Citrulli Effector that Induces Programmed Cell Death in Plants.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, seriously affects watermelon and other cucurbit crops, resulting in significant economic losses. However, the pathogenicity mechanism of A. citrulli is not well understood. Plant pathogenic bacteria often suppress the plant immune response by secreting effector proteins. Thus, identifying A. citrulli effector proteins and determining their functions may improve our understanding of the underlying pathogenetic mechanisms. In this study, a novel effector, AopN, which is localized on the cell membrane of Nicotiana benthamiana, was identified. The functional analysis revealed that AopN significantly inhibited the flg22-induced reactive oxygen species burst. AopN induced a programmed cell death (PCD) response. Unlike its homologous protein, the ability of AopN to induce PCD was dependent on two motifs of unknown functions (including DUP4129 and Cpta_toxin), but was not dependent on LXXLL domain. More importantly, the virulence of the aopN mutant of A. citrulli in N. benthamiana significantly decreased, indicating that it was a core effector. Further analysis revealed that AopN interacted with watermelon ClHIPP and ClLTP, which responds to A. citrulli strain Aac5 infection at the transcription level. Collectively, these findings indicate that AopN suppresses plant immunity and activates the effector-triggered immunity pathway.

Characterization, identification and expression profiling of genome-wide R-genes in melon and their putative roles in bacterial fruit blotch resistance.

BACKGROUND: Bacterial fruit blotch (BFB), a disease caused by Acidovorax citrulli, results in significant economic losses in melon. The causal QTLs and genes for resistance to this disease have yet to be identified. Resistance (R)-genes play vital roles in resistance to plant diseases. Since the complete genome sequence of melon is available and genome-wide identification of R-genes has been performed for this important crop, comprehensive expression profiling may lead to the identification of putative candidate genes that function in the response to BFB. RESULTS: We identified melon accessions that are resistant and susceptible to BFB through repeated bioassays and characterized all 70 R-genes in melon, including their gene structures, chromosomal locations, domain organizations, motif distributions, and syntenic relationships. Several disease resistance-related domains were identified, including NBS, TIR, LRR, CC, RLK, and DUF domains, and the genes were categorized based on the domains of their encoded proteins. In addition, we profiled the expression patterns of the genes in melon accessions with contrasting levels of BFB resistance at 12 h, 1 d, 3 d, and 6 d after inoculation with A. citrulli. Six R-genes exhibited consistent expression patterns (MELO3C023441, MELO3C016529, MELO3C022157, MELO3C022146, MELO3C025518, and MELO3C004303), with higher expression levels in the resistant vs. susceptible accession. CONCLUSION: We identified six putative candidate R-genes against BFB in melon. Upon functional validation, these genes could be targeted for manipulation via breeding and biotechnological approaches to improve BFB resistance in melon in the future.

Development of Molecular Marker Linked with Bacterial Fruit Blotch Resistance in Melon (Cucumis melo L.).

Bacterial fruit blotch (BFB) causes losses in melon marketable yield. However, until now, there has been no information about the genetic loci responsible for resistance to the disease or their pattern of inheritance. We determined the inheritance pattern of BFB resistance from a segregating population of 491 F2 individuals raised by crossing BFB-resistant (PI 353814) and susceptible (PI 614596) parental accessions. All F1 plants were resistant to Acidovorax citrulli strain KACC18782, and F2 plants segregated with a 3:1 ratio for resistant and susceptible phenotypes, respectively, in a seedling bioassay experiment, indicating that BFB resistance is controlled by a monogenic dominant gene. In an investigation of 57 putative disease-resistance related genes across the melon genome, only the MELO3C022157 gene (encoding TIR-NBS-LRR domain), showing polymorphism between resistant and susceptible parents, revealed as a good candidate for further investigation. Cloning, sequencing and quantitative RT-PCR expression of the polymorphic gene MELO3C022157 located on chromosome 9 revealed multiple insertion/deletions (InDels) and single nucleotide polymorphisms (SNPs), of which the SNP A2035T in the second exon of the gene caused loss of the LRR domain and truncated protein in the susceptible accession. The InDel marker MB157-2, based on the large (504 bp) insertion in the first intron of the susceptible accession, was able to distinguish resistant and susceptible accessions among 491 F2 and 22 landraces/inbred accessions with 98.17% and 100% detection accuracy, respectively. This novel PCR-based, co-dominant InDel marker represents a practical tool for marker-assisted breeding aimed at developing BFB-resistant melon accessions.

A LuxR-type regulator, AcrR, regulates flagellar assembly and contributes to virulence, motility, biofilm formation, and growth ability of Acidovorax citrulli.

LuxR-type regulators regulate many bacterial processes and play important roles in bacterial motility and virulence. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes great losses in melon and watermelon worldwide. We identified a LuxR-type, nonquorum sensing-related regulator, AcrR, in the group II strain Aac-5 of A. citrulli. We found that the acrR mutant lost twitching and swimming motilities, and flagellar formation. It also showed reduced virulence, but increased biofilm formation and growth ability. Transcriptomic analysis revealed that 394 genes were differentially expressed in the acrR mutant of A. citrulli, including 33 genes involved in flagellar assembly. Our results suggest that AcrR may act as a global regulator affecting multiple important biological functions of A. citrulli.

Ferric Uptake Regulator (FurA) is Required for Acidovorax citrulli Virulence on Watermelon.

Acidovorax citrulli is the causal agent of bacterial fruit blotch, a serious threat to commercial watermelon and melon crop production worldwide. Ferric uptake regulator (Fur) is a global transcription factor that affects a number of virulence-related functions in phytopathogenic bacteria; however, the role of furA has not been determined for A. citrulli. Hence, we constructed an furA deletion mutant and a corresponding complement in the background of A. citrulli strain xlj12 to investigate the role of the gene in siderophore production, concentration of intracellular Fe2+, bacterial sensitivity to hydrogen peroxide, biofilm formation, swimming motility, hypersensitive response induction, and virulence on melon seedlings. The A. citrulli furA deletion mutant displayed increased siderophore production, intracellular Fe2+ concentration, and increased sensitivity to hydrogen peroxide. In contrast, biofilm formation, swimming motility, and virulence on melon seedlings were significantly reduced in the furA mutant. As expected, complementation of the furA deletion mutant restored all phenotypes to wild-type levels. In accordance with the phenotypic results, the expression levels of bfrA and bfrB that encode bacterioferritin, sodB that encodes iron/manganese superoxide dismutase, fliS that encodes a flagellar protein, hrcN that encodes the type III secretion system (T3SS) ATPase, and hrcC that encodes the T3SS outer membrane ring protein were significantly downregulated in the A. citrulli furA deletion mutant. In addition, the expression of feo-related genes and feoA and feoB was significantly upregulated in the furA mutant. Overall, these results indicated that, in A. citrulli, FurA contributes to the regulation of the iron balance system, and affects a variety of virulence-related traits.

Nicotiana species as surrogate host for studying the pathogenicity of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is one of the most important bacterial diseases of cucurbits worldwide. However, the mechanisms associated with A. citrulli pathogenicity and genetics of host resistance have not been extensively investigated. We idenitfied Nicotiana benthamiana and Nicotiana tabacum as surrogate hosts for studying A. citrulli pathogenicity and non-host resistance triggered by type III secreted (T3S) effectors. Two A. citrulli strains, M6 and AAC00-1, that represent the two major groups amongst A. citrulli populations, induced disease symptoms on N. benthamiana, but triggered a hypersensitive response (HR) on N. tabacum plants. Transient expression of 19 T3S effectors from A. citrulli in N. benthamiana leaves revealed that three effectors, Aave_1548, Aave_2708, and Aave_2166, trigger water-soaking-like cell death in N. benthamiana. Aave_1548 knockout mutants of M6 and AAC00-1 displayed reduced virulence on N. benthamiana and melon (Cucumis melo L.). Transient expression of Aave_1548 and Aave_2166 effectors triggered a non-host HR in N. tabacum, which was dependent on the functionality of the immune signalling component, NtSGT1. Hence, employing Nicotiana species as surrogate hosts for studying A. citrulli pathogenicity may help characterize the function of A. citrulli T3S effectors and facilitate the development of new strategies for BFB management.

Role of type IV secretion system genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae strain RS-2.

Type IV secretion system (T4SS) is a specialized nanomachine that is utilized for the pathogenicity of gram-negative bacteria. However, the role of T4SS genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae (Ao) strain RS-2 is not clear, which contains T4SS gene cluster based on genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type strain RS-2 and nine T4SS mutants, which were constructed in this study. Results indicated that mutation of pilT, pilM, pilQ, or pilZ3 genes not only significantly reduced bacterial virulence, but also caused a reduction of 20.4-62.0% in biofilm formation and 37.7-47.7% reduction in motility, but had no effect on exopolysaccharide (EPS) production or extracellular enzymatic activities when compared to the wild type. The four T4SS genes had a differential effect on bacterial growth after 24 h post-incubation. The complemented strains of the four T4SS mutants restored similar virulence symptom as the wild type. In addition, no change was observed in bacterial virulence by mutation of the other five T4SS genes. Totally, these results demonstrated that T4SS played vital roles in bacterial virulence, motility and biofilm formation in plant pathogen Ao strain RS-2.

L-Lysine-α-Oxidase: Acidovorax citrulli Bacterium Inhibitor.

Studies of the effects of Trichoderma harzianum Rifai F-180 culture fluid concentrate containing L-lysine-α-oxidase antitumor enzyme produced by the fungus and the homogenous enzyme, on ultrahazardous bacterium Acidovorax citrulli demonstrated the antibacterial activity of the concentrate. Trichoderma harzianum Rifai F-180 producing L-lysine-α-oxidase was cultured in a technological device at G. K. Skryabin Institute of Biochemistry and. Physiology of Microorganisms, Russian Academy of Sciences. Activity of L-lysine-α-oxidase in the resulted culture fluid concentrate was 0.54 U/ml, activity of the homogenous enzyme was 50 U/mg.

IcmF and DotU are required for the virulence of Acidovorax oryzae strain RS-1.

Acidovorax oryzae (Ao) cause bacterial brown stripe in rice that result in great economic loss. However, the pathogenic mechanism of this rice bacterial pathogen still remains unclear. Interestingly, transcriptomic and proteomic analysis of in vivo infection indicate that the pathogenicity of Ao strain RS-1 may be associated with the type six secretion system (T6SS), which was identified by in silico comparative genomic analysis in our previous studies. This makes it necessary to further examine the role of each core component of T6SS in the pathogenicity of Ao strain RS-1 to rice plants. Results from this study highlight the mutual interaction between IcmF and DotU, which was determined by bacterial two-hybrid and bimolecular fluorescence complementation assays. Furthermore, a difference was observed in bacterial pathogenicity, biofilm formation, secreted proteins identified by LC-MS/MS analysis and the expression of T6SS other genes examined by quantitative real-time PCR between the wild-type and both single-gene knockout mutants, which were respectively constructed based on the insertional mutagenesis of Ao strain RS-1 in this study. Overall, our results clearly revealed the importance of T6SS icmF and dotU in pathogenicity of Ao strain RS-1 to rice plants.

Silver Nanoparticles Complexed with Bovine Submaxillary Mucin Possess Strong Antibacterial Activity and Protect against Seedling Infection.

A simple method for the synthesis of nanoparticles (NPs) of silver (Ag) in a matrix of bovine submaxillary mucin (BSM) was reported previously by some of the authors of this study. Based on mucin characteristics such as long-lasting stability, water solubility, and surfactant and adhesive characteristics, we hypothesized that these compounds, named BSM-Ag NPs, may possess favorable properties as potent antimicrobial agents. The goal of this study was to assess whether BSM-Ag NPs possess antibacterial activity, focusing on important plant-pathogenic bacterial strains representing both Gram-negative (Acidovorax and Xanthomonas) and Gram-positive (Clavibacter) genera. Growth inhibition and bactericidal assays, as well as electron microscopic observations, demonstrate that BSM-Ag NPs, at relatively low concentrations of silver, exert strong antimicrobial effects. Moreover, we show that treatment of melon seeds with BSM-Ag NPs effectively prevents seed-to-seedling transmission of Acidovorax citrulli, one of the most threatening pathogens of cucurbit production worldwide. Overall, our findings demonstrate strong antimicrobial activity of BSM-Ag NPs and their potential application for reducing the spread and establishment of devastating bacterial plant diseases in agriculture.IMPORTANCE Bacterial plant diseases challenge agricultural production, and the means available to manage them are limited. Importantly, many plant-pathogenic bacteria have the ability to colonize seeds, and seed-to-seedling transmission is a critical route by which bacterial plant diseases spread to new regions and countries. The significance of our study resides in the following aspects: (i) the simplicity of the method of BSM-Ag NP synthesis, (ii) the advantageous chemical properties of BSM-Ag NPs, (iii) the strong antibacterial activity of BSM-Ag NPs at relatively low concentrations of silver, and (iv) the fact that, in contrast to most studies on the effects of metal NPs on plant pathogens, the proof of concept for the novel compound is supported by in planta assays. Application of this technology is not limited to agriculture; BSM-Ag NPs potentially could be exploited as a potent antimicrobial agent in a wide range of industrial areas, including medicine, veterinary medicine, cosmetics, textiles, and household products.

Host modification of a bacterial quorum-sensing signal induces a phenotypic switch in bacterial symbionts.

Bacterial communities colonize epithelial surfaces of most animals. Several factors, including the innate immune system, mucus composition, and diet, have been identified as determinants of host-associated bacterial communities. Here we show that the early branching metazoan Hydra is able to modify bacterial quorum-sensing signals. We identified a eukaryotic mechanism that enables Hydra to specifically modify long-chain 3-oxo-homoserine lactones into their 3-hydroxy-HSL counterparts. Expression data revealed that Hydra's main bacterial colonizer, Curvibacter sp., responds differentially to N-(3-hydroxydodecanoyl)-l-homoserine lactone (3OHC12-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL). Investigating the impacts of the different N-acyl-HSLs on host colonization elucidated that 3OHC12-HSL allows and 3OC12-HSL represses host colonization of Curvibacter sp. These results show that an animal manipulates bacterial quorum-sensing signals and that this modification leads to a phenotypic switch in the bacterial colonizers. This mechanism may enable the host to manipulate the gene expression and thereby the behavior of its bacterial colonizers.

Role of the Genes of Type VI Secretion System in Virulence of Rice Bacterial Brown Stripe Pathogen Acidovorax avenae subsp. avenae Strain RS-2.

The Type VI secretion system (T6SS) is a class of macromolecular machine that is required for the virulence of gram-negative bacteria. However, it is still not clear what the role of T6SS in the virulence of rice bacterial brown stripe pathogen Acidovorax avenae subsp. avenae (Aaa) is. The aim of the current study was to investigate the contribution of T6SS in Aaa strain RS2 virulence using insertional deletion mutation and complementation approaches. This strain produced weak virulence but contains a complete T6SS gene cluster based on a genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type (RS-2) and 25 T6SS mutants, which were constructed using homologous recombination methods. The mutation of 15 T6SS genes significantly reduced bacterial virulence and the secretion of Hcp protein. Additionally, the complemented 7 mutations ΔpppA, ΔclpB, Δhcp, ΔdotU, ΔicmF, ΔimpJ, and ΔimpM caused similar virulence characteristics as RS-2. Moreover, the mutant ΔpppA, ΔclpB, ΔicmF, ΔimpJ and ΔimpM genes caused by a 38.3~56.4% reduction in biofilm formation while the mutants ΔpppA, ΔclpB, ΔicmF and Δhcp resulted in a 37.5~44.6% reduction in motility. All together, these results demonstrate that T6SS play vital roles in the virulence of strain RS-2, which may be partially attributed to the reductions in Hcp secretion, biofilm formation and motility. However, differences in virulence between strain RS-1 and RS-2 suggest that other factors may also be involved in the virulence of Aaa.

Recombination of Virulence Genes in Divergent Acidovorax avenae Strains That Infect a Common Host.

Bacterial etiolation and decline (BED), caused by Acidovorax avenae, is an emerging disease of creeping bentgrass on golf courses in the United States. We performed the first comprehensive analysis of A. avenae on a nationwide collection of turfgrass- and maize-pathogenic A. avenae. Surprisingly, our results reveal that the turfgrass-pathogenic A. avenae in North America are not only highly divergent but also belong to two distinct phylogroups. Both phylogroups specifically infect turfgrass but are more closely related to maize pathogens than to each other. This suggests that, although the disease is only recently reported, it has likely been infecting turfgrass for a long time. To identify a genetic basis for the host specificity, we searched for genes closely related among turfgrass strains but distantly related to their homologs from maize strains. We found a cluster of 11 such genes generated by three ancient recombination events within the type III secretion system (T3SS) pathogenicity island. Ever since the recombination, the cluster has been conserved by strong purifying selection, hinting at its selective importance. Together our analyses suggest that BED is an ancient disease that may owe its host specificity to a highly conserved cluster of 11 T3SS genes.

Further Evidence of Cucurbit Host Specificity among Acidovorax citrulli Groups Based on a Detached Melon Fruit Pathogenicity Assay.

Bacterial fruit blotch, caused by the gram-negative bacterium Acidovorax citrulli, is a serious economic threat to cucurbit crop production worldwide. A. citrulli strains can be divided into two genetically distinct groups, with group I strains infecting a range of cucurbit species and group II strains being predominantly associated with watermelon. Group I and II A. citrulli strains differ in their arsenal of type III secreted (T3S) effector proteins and we hypothesize that these effectors are critical for cucurbit host preference. However, the pathogenicity or virulence assays used for A. citrulli, including infiltration of seedling cotyledons and mature fruit rind tissues with cell suspensions and spray inoculation of seedlings, lack the sensitivity to consistently distinguish strains of the two groups. Here, we describe an immature, detached melon fruit assay based on 'Joaquin Gold' melon (Syngenta, Rogers Brand) that clearly indicates differences in host specificity between group I and II A. citrulli strains. Using this assay, four group I strains (M6, AAC213-52, AAC213-55, and XJL12) induced typical water-soaked lesions in melon fruit rind tissue 7 to 10 days after pinprick inoculation. In contrast, four group II strains (AAC00-1, AAC213-44, AAC213-47, and AAC213-48) did not induce water-soaked lesions on detached melon fruit rinds during the same period. These data suggest that group I A. citrulli strains have a specific capacity to infect immature Joaquin Gold melon fruit, whereas group II strains do not. Interestingly, this differential pathogenicity phenotype was not observed on foliar seedling tissues of the same melon cultivar, suggesting that host preference of A. citrulli strains is specific to immature fruit tissues. Using the immature melon fruit inoculation assay, a T3S system mutant of the group I A. citrulli strain, M6 (M6ΔhrcV), failed to induce water soaking. This indicates that T3S effectors are involved in A. citrulli cucurbit host preference, and that this assay is suitable for future studies of unique T3S effectors that distinguish group I and II strains.

Further Characterization of Genetically Distinct Groups of Acidovorax citrulli Strains.

Bacterial fruit blotch of cucurbits (BFB) is caused by the gram-negative bacterium Acidovorax citrulli, whose populations can be distinguished into two genetically distinct groups, I and II. Based on visual assessment of BFB severity on cucurbit seedlings and fruit after inoculation under greenhouse conditions, group I A. citrulli strains have been reported to be moderately to highly virulent on several cucurbit hosts, whereas group II strains have exhibited high virulence on watermelon but low virulence on other cucurbits. Additionally, group I strains are recovered from a range of cucurbit hosts, while group II strains are predominantly found on watermelon. The goal of this research was to develop tools to characterize and rapidly distinguish group I and II A. citrulli strains. We first sought to determine whether quantification of A. citrulli colonization of cucurbit seedling tissue reflects the differences between group I and II strains established by visual assessment of BFB symptom severity. Spray inoculation of melon seedlings with cell suspensions containing approximately 1 × 104 CFU/ml resulted in significantly higher (P = 0.01) population growth of M6 (group I; mean area under population growth curve [AUPGC] = 43.73) than that of AAC00-1 (group II; mean AUPGC = 39.33) by 10 days after inoculation. We also investigated the natural spread of bacterial cells and the resulting BFB incidence on watermelon and melon seedlings exposed to three group I and three group II A. citrulli strains under mist chamber conditions. After 5 days of exposure, the mean BFB incidence on melon seedlings exposed to representative group II A. citrulli strains was significantly lower (25 and 3.98% in experiments 1 and 2, respectively) than on melon seedlings exposed to representative group I strains (94.44 and 76.11% in experiments 1 and 2, respectively), and on watermelon seedlings exposed to representative group I and II strains (70 to 93.33%). Finally, we developed a polymerase chain reaction assay based on the putative type III secretion effector gene, Aave_2166, to rapidly distinguish group I and II A. citrulli strains. This assay will be important for future epidemiological studies on BFB.

Strains of the Group I Lineage of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch of Cucurbitaceous Crops, are Predominant in Brazil.

Bacterial fruit blotch (BFB), caused by the seedborne bacterium Acidovorax citrulli, is an economically important threat to cucurbitaceous crops worldwide. Since the first report of BFB in Brazil in 1990, outbreaks have occurred sporadically on watermelon and, more frequently, on melon, resulting in significant yield losses. At present, the genetic diversity and the population structure of A. citrulli strains in Brazil remain unclear. A collection of 74 A. citrulli strains isolated from naturally infected tissues of different cucurbit hosts in Brazil between 2000 and 2014 and 18 A. citrulli reference strains from other countries were compared by pulsed-field gel electrophoresis (PFGE), multilocus sequence analysis (MLSA) of housekeeping and virulence-associated genes, and pathogenicity tests on seedlings of different cucurbit species. The Brazilian population comprised predominantly group I strains (98%), regardless of the year of isolation, geographical region, or host. Whole-genome restriction digestion and PFGE analysis revealed that three unique and previously unreported A. citrulli haplotypes (assigned as haplotypes B22, B23, and B24) occurred in Brazil. The greatest diversity of A. citrulli (four haplotypes) was found among strains collected from the northeastern region of Brazil, which accounts for more than 90% of the country's melon production. MLSA clearly distinguished A. citrulli strains into two well-supported clades, in agreement with observations based on PFGE analysis. Five Brazilian A. citrulli strains, representing different group I haplotypes, were moderately aggressive on watermelon seedlings compared with four group II strains that were highly aggressive. In contrast, no significant differences in BFB severity were observed between group I and II A. citrulli strains on melon and squash seedlings. Finally, we observed a differential effect of temperature on in vitro growth of representative group I and II A. citrulli haplotypes. Specifically, of 18 group II strains tested, all grew at 40 and 41°C, whereas only 3 of 15 group I strains (haplotypes B8[P], B3[K], and B15) grew at 40°C. Three strains representing haplotype B8(P) were the only group I strains that grew at 41°C. These results contribute to a better understanding of the genetic diversity of A. citrulli associated with BFB outbreaks in Brazil, and reinforce the efficiency of MLSA and PFGE analysis for assessing population structure. This study also provides the first evidence to suggest that temperature might be a driver in the ecological adaptation of A. citrulli populations.

New insights into virulence mechanisms of rice pathogen Acidovorax avenae subsp. avenae strain RS-1 following exposure to ß-lactam antibiotics.

Recent research has shown that pathogen virulence can be altered by exposure to antibiotics, even when the growth rate is unaffected. Investigating this phenomenon provides new insights into understanding the virulence mechanisms of bacterial pathogens. This study investigates the phenotypic and transcriptomic responses of the rice pathogenic bacterium Acidovorax avenae subsp. avenae (Aaa) strain RS-1 to ß-lactam antibiotics especially Ampicillin (Amp). Our results indicate that exposure to Amp does not influence bacterial growth and biofilm formation, but alters the virulence, colonization capacity, composition of extracellular polymeric substances and secretion of Type VI secretion system (T6SS) effector Hcp. This attenuation in virulence is linked to unique or differential expression of known virulence-associated genes based on genome-wide transcriptomic analysis. The reliability of expression data generated by RNA-Seq was verified with quantitative real-time PCR of 21 selected T6SS genes, where significant down-regulation in expression of hcp gene, corresponding to the reduction in secretion of Hcp, was observed under exposure to Amp. Hcp is highlighted as a potential target for Amp, with similar changes observed in virulence-associated phenotypes between exposure to Amp and mutation of hcp gene. In addition, Hcp secretion is reduced in knockout mutants of 4 differentially expressed T6SS genes.

Identification of Pathogenicity-Related Genes in Biofilm-Defective Acidovorax citrulli by Transposon Tn5 Mutagenesis.

Biofilm formation is important for virulence of a large number of plant pathogenic bacteria. Indeed, some virulence genes have been found to be involved in the formation of biofilm in bacterial fruit blotch pathogen Acidovorax citrulli. However, some virulent strains of A. citrulli were unable to format biofilm, indicating the complexity between biofilm formation and virulence. In this study, virulence-related genes were identified in the biofilm-defective strain A1 of A. citrulli by using Tn5 insertion, pathogenicity test, and high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). Results from this study indicated that 22 out of the obtained 301 mutants significantly decreased the virulence of strain A1 compared to the wild-type. Furthermore, sequence analysis indicated that the obtained 22 mutants were due to the insertion of Tn5 into eight genes, including Aave 4244 (cation diffusion facilitator family transporter), Aave 4286 (hypothetical protein), Aave 4189 (alpha/beta hydrolase fold), Aave 1911 (IMP dehydrogenase/GMP reductase domain), Aave 4383 (bacterial export proteins, family 1), Aave 4256 (Hsp70 protein), Aave 0003 (histidine kinase, DNA gyrase B, and HSP90-like ATPase), and Aave 2428 (pyridoxal-phosphate dependent enzyme). Furthermore, the growth of mutant Aave 2428 was unaffected and even increased by the change in incubation temperature, NaCl concentration and the pH of the LB broth, indicating that this gene may be directly involved in the bacterial virulence. Overall, the determination of the eight pathogenicity-related genes in strain A1 will be helpful to elucidate the pathogenesis of biofilm-defective A. citrulli.

Antibody array in a multiwell plate format for the sensitive and multiplexed detection of important plant pathogens.

The global seed market is considered to be an important industry with a total value of $10,543 million US dollars in 2012. Because plant pathogens such as bacteria and viruses cause a significant economic loss to both producers and exporters, the seed export industry urgently requires rapid, sensitive, and inexpensive testing for the pathogens to prevent disease spreading worldwide. This study developed an antibody array in a multiwell plate format to simultaneously detect four crucial plant pathogens, namely, a bacterial fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), Chilli veinal mottle virus (ChiVMV, potyvirus), Watermelon silver mottle virus (WSMoV, tospovirus serogroup IV), and Melon yellow spot virus (MYSV, tospovirus). The capture antibodies specific to the pathogens were immobilized on each well at preassigned positions by an automatic microarrayer. The antibodies on the arrays specifically captured the corresponding pathogens present in the sample extracts. The presence of pathogens bound on the capture antibodies was subsequently detected by a cocktail of fluorescently conjugated secondary antibodies. The limits of detection of the developed antibody array for the detection of Aac, ChiVMV, WSMoV, and MYSV were 5 × 10(5) CFU/mL, 30 ng/mL, 1000 ng/mL, and 160 ng/mL, respectively, which were very similar to those of the conventional ELISA method. The antibody array in a multiwell plate format accurately detected plant pathogens in single and multiple detections. Moreover, this format enables easy handling of the assay at a higher speed of operation.