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LuxR-type transcriptional regulators are essential for many physiological processes in bacteria, including pathogenesis. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes great losses in melon and watermelon worldwide. However, the LuxR-type transcriptional factors in A. citrulli have not been well studied, except for the previously reported LuxR-type regulatory protein, AcrR, involved in regulating virulence and motility. Here, we characterized a second LuxR-type regulator, AclR, in the group II strain Aac-5 of A. citrulli by mutagenesis, virulence and motility assays, and transcriptomic analysis. Deletion of aclR resulted in impaired twitching and swimming motility and flagellar formation and diminished virulence but increased biofilm formation. Transcriptomic analysis revealed that 1,379 genes were differentially expressed in the aclR mutant strain, including 29 genes involved in flagellar assembly and 3 involved in pili formation, suggesting a regulatory role for AclR in multiple important biological functions of A. citrulli. Together, our results not only indicate that AclR plays a global role in transcriptional regulation in A. citrulli influencing motility, biofilm formation, and virulence but also provide perspective regarding the regulatory network of biological functions in A. citrulli. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

This study was conducted to check the sterilization efficacy of microwave plasma (MWP) against the watermelon seeds infected with Acidovorax citrulli 11-251. Watermelon seeds were artificially vacuum inoculated to produce A. citrulli 11-251 infected seeds. Aac ImmunoStrip and scanning electron microscope (SEM) results suggests that, seeds (coat and endosperm) were infected under the concentration of 1×107/30 min. MWP sterlization process was carried out at 50 W (3 min, 5 min, and 10 min), 80 W (3 min, 5 min, and 10 min), and 100 W (3 min, 5 min, and 10 min). According to the results, MWP sterilized the artificially inoculated seed coats by 95.96% at 80 W/10 min and seed endosperms by 100% at 100 W/10 min respectively. Although, seeds were sterlized by MWP, germination rate of seeds were low as compared to non treated (negative control) seeds. Moreover, cell membrane of A. citrulli 11-251 was damaged while observed in SEM after sterilized with MWP. Further studies regarding the appropriate sterilization condition by MWP against A. citrulli infected seeds for germination will be conducted in our next study.

Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB) in watermelon, a disease that poses a serious threat to watermelon production. Because of the lack of resistant cultivars against BFB, virulence factors or mechanisms need to be elucidated to control the disease. Glycerol-3-phosphate dehydrogenase is the enzyme involved in glycerol production from glucose during glycolysis. In this study, we report the functions of a putative glycerol-3-phosphate dehydrogenase in Ac (GlpdAc) using comparative proteomic analysis and phenotypic observation. A glpdAc knockout mutant, AcΔglpdAc(EV), lost virulence against watermelon in two pathogenicity tests. The putative 3D structure and amino acid sequence of GlpdAc showed high similarity with glycerol-3-phosphate dehydrogenases from other bacteria. Comparative proteomic analysis revealed that many proteins related to various metabolic pathways, including carbohydrate metabolism, were affected by GlpdAc. Although AcΔglpdAc(EV) could not use glucose as a sole carbon source, it showed growth in the presence of glycerol, indicating that GlpdAc is involved in glycolysis. AcΔglpdAc(EV) also displayed higher cell-to-cell aggregation than the wild-type bacteria, and tolerance to osmotic stress and ciprofloxacin was reduced and enhanced in the mutant, respectively. These results indicate that GlpdAc is involved in glycerol metabolism and other mechanisms, including virulence, demonstrating that the protein has pleiotropic effects. Our study expands the understanding of the functions of proteins associated with virulence in Ac.

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

Acidovorax citrulli is a seed‐transmitted gram‐negative bacterium that can cause substantial economic yield loss in watermelon and melon production worldwide. Four small‐molecule libraries containing 4,952 compounds were selected for high‐throughput screening against A. citrulli wild‐type strain Xu3‐14 by evaluation of growth inhibition. One hundred and twenty‐seven molecules (2.5% hit rate) were identified as bactericidal or bacteriostatic against A. citrulli at 100 μM. Secondary screens indicated that 27 candidate compounds were more effective against A. citrulli Group II strains than Group I strains (classified using repetitive element PCR). Several compounds were inhibitory to other pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis and Xanthomonas campestris pv. campestris, but did not affect the growth of plant beneficial bacteria Pseudomonas fluorescens and Bacillus subtilis. More than half of the compounds did not inhibit germination of Arabidopsis or watermelon seeds. The effect of small molecules on A. citrulli seed‐to‐seedling transmission was evaluated by applying each compound to inoculated watermelon seeds and assessing seedling infection. Nine compounds were chosen for further investigation based on their reduction of percentage seedling infection and compiling scores on their specificity, sensitivity, and phytotoxicity obtained in the secondary screens. The five best compounds were selected (thiamphenicol, nadifloxacin, pipemidic acid, ciclopirox, and zinc pyrithione) for greenhouse tests and were found to effectively reduce the seed‐to‐seedling transmission of A. citrulli in both artificially and naturally infested seeds. These top five compounds provide a basis for future development of an A. citrulli‐specific bactericide.

Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide. The pathogen is seed-transmitted, so seed detection to prevent distribution of contaminated seed is crucial in disease management. In this study, we adapted a quantitative real-time PCR (qPCR) assay to droplet digital PCR (ddPCR) format for A. citrulli detection by optimizing reaction conditions. The performance of ddPCR in detecting A. citrulli pure culture, DNA, infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR, qPCR, and dilution plating method. The lowest concentrations detected (LCD) by ddPCR reached up to 2 fg DNA, and 102 CFU mL−1 bacterial cells, which were ten times more sensitive than those of the qPCR. When testing artificially infested watermelon and melon seed, 0.1% infestation level was detectable using ddPCR and dilution plating method. The 26 positive samples were identified in 201 commercial seed samples through ddPCR, which was the highest positive number among all the methods. High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.

Acidovorax citrulli causes bacterial fruit blotch in Cucurbitaceae, including watermelon. Although A. citrulli is a seed-borne pathogen, it can cause diverse symptoms in other plant organs like leaves, stems and fruits. To determine the infection routes of A. citrulli, we examined the virulence of six isolates (Ac0, Ac1, Ac2, Ac4, Ac8, and Ac11) on watermelon using several inoculation methods. Among six isolates, DNA polymorphism reveals that three isolates Ac0, Ac1, and Ac4 belong to Clonal Complex (CC) group II and the others do CC group I. Ac0, Ac4, and Ac8 isolates efficiently infected seeds during germination in soil, and Ac0 and Ac4 also infected the roots of watermelon seedlings wounded prior to inoculation. Infection through leaves was successful only by three isolates belonging to CC group II, and two of these also infected the mature watermelon fruits. Ac2 did not cause the disease in all assays. Interestingly, three putative type III effectors (Aave_2166, Aave_2708, and Aave_3062) with intact forms were only found in CC group II. Overall, our results indicate that A. citrulli can infect watermelons through diverse routes, and the CC grouping of A. citrulli was only correlated with virulence in leaf infection assays.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit fruit and seed production worldwide. In recent years, the BFB has spread to many areas of China, mainly via the inadvertent distribution of contaminated commercial seeds. To assess the prevalence of seedborne A. citrulli in commercial watermelon and other cucurbitaceous seedlots in China, a 9-year survey was conducted between 2010 and 2018. A total of 4,839 seedlots of watermelon and other cucurbitaceous species were collected from 13 major seed production areas of China and tested by a semiselective media-based colony PCR technique for A. citrulli. Overall, A. citrulli was detected in 18.00% (871/4,839) of all cucurbitaceous seedlots. The bacterium was detected in 21.59% (38/176), 19.19% (33/172), 23.44% (214/913), 40.76% (247/606), 13.28% (85/640), 15.40% (95/617), 13.25% (73/551), 8.03% (48/598), and 6.71% (38/566) of all commercial seedlots tested from the 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, and 2018 growing seasons, respectively. Additionally, the prevalence of A. citrulli in cucurbit seedlots was determined for different seed production areas. The prevalence of A. citrulli in cucurbitaceous seedlots produced in Xinjiang, Gansu, Ningxia, Inner Mongolia, and 9 other provinces was 18.76% (582/3103), 26.34% (103/391), 21.47% (82/382), 11.11% (14/126), and 10.75% (90/837), respectively. This is the first survey for A. citrulli in commercial cucurbit seeds in China, and the relatively high prevalence suggests that commercial seeds represent a substantial source of primary inoculum that can threaten cucurbit seed and fruit production in China.

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., AcrR, a LuxR‐type regulator, acts as a global regulator affecting multiple important biological functions, especially swimming motility and flagellum assembly of Acidovorax citrulli.

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.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, is an economically devastating disease in melon. Molecular characterization of causal strains and identification of sources of resistance are necessary to control this important disease. Many strains of BFB were identified by partial sequencing and documented in GenBank. Recently, there was an outbreak of BFB strain NIHHS15-280 in melon fields (Goksung, Jeonnam, Korea) that severely hampered melon production in Korea. For this reason, we characterized NIHHS15-280 by partially sequencing the 16S rRNA gene using PCR-based markers. The partial sequence of the 16S rRNA gene of NIHHS15-280 was much more similar to A. citrulli strains associated with melon and watermelon hosts than other cucurbit hosts. A PCR-based assay identified the strain (NIHHS15-280) as belonging to A. citrulli group I, which predominantly infects non-watermelon cucurbits (virulent to melon). Thirty-five melon genotypes were screened against NIHHS15-280 in a detached-leaf inoculation assay. The lack of visible disease symptoms 11 days after inoculation in lines PI353814 and PI140471 indicated these to be highly resistant lines, whereas moderate resistance was observed in lines PI420145 and SCNU1154 and cultivars ME6 and ME5. Also, the most resistant lines were corroborated by a fruit inoculation assay. The characterized A. citrulli strain NIHHS15-280 (group I) and identified resistant lines will serve as a valuable resource for further genetic studies and improvement of BFB resistance in melon.

Acidovorax citrulli, the gram-negative bacteria that causes bacterial fruit blotch (BFB), has been responsible for huge worldwide economic losses in watermelon and melon production since 1980. No commercial cultivar resistant to BFB has been reported. Of the two reported genotypes of A. citrulli, genotype I is the main causal agent of BFB in melon and genotype II causes disease in watermelon. After the isolation of the first bacteriophage against A. citrulli (ACP17), efforts have been made to isolate bacteriophages with wider host ranges by collecting samples from watermelon, pumpkin, and cucumber. The newly isolated phage ACPWH, belonging to the Siphoviridae family, has a head size of 60 ± 5 nm and tail size of 180 ± 5 nm, and can infect 39 out of 42 A. citrulli strains. ACPWH has genome size of 42,499 and GC content of 64.44%. Coating watermelon seeds with bacteriophage ACPWH before soil inoculation with A. citrulli resulted in 96% germination and survival, compared to 13% germination of uncoated control seeds. These results suggest that phage ACPWH may be an effective and low-cost biocontrol agent against BFB.

Summary 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.

Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax citrulli, is a major threat to commercial watermelon and melon production worldwide. At present, there are at least two genetically distinct sub-populations (group I and II) of A. citrulli that differ in host preference among cucurbit species and copper sensitivity. In this study, we analyzed the pilA gene sequences of 103 A. citrulli strains from China and other countries. Based on these data, we classified all tested A. citrulli strains into three types. The pilA-based type 1 strains in this study coincided with the previously established group I strains; while the type 2 strains coincided with group II strains. Ten strains that did not cluster with group I or II strains were classified into a new type, designated type 3. Based on differences in pilA sequences, we designed a multiplex PCR assay to distinguish the three A. citrulli pilus types. This multiplex PCR assay has proven to be viable for strain typing of 139 A. citrulli strains and for the detection of this pathogen in artificially inoculated seeds and leaves and naturally infected leaves and fruits. This assay proved to be rapid, accurate, reliable and applicable for early distinction of A. citrulli types associated with BFB epidemics. It may also inform the judicious and environmentally sound use of bactericides, especially copper-based compounds.

Chemicals used for plant health may have negative impacts on humans, animals and environment, thus antagonistic microorganisms have become popular for the control of plant diseases. Acidovorax citrulli (Ac)-induced melon bacterial fruit blotch (BFB) disease poses serious threats worldwide to the production of cucurbits, especially melon (Cucumis melo) and watermelon (Citrullus lanatus). In this study, effects of 14 different antagonistic bacteria isolated from the phyllosphere of melon and watermelon were tested against BFB through seed treatments under growth chamber conditions and through foliage sprays under greenhouse conditions. When seeds were treated with antagonistic bacteria, seven of them reduced disease incidence by over 50% and disease severity by about 70%. Then, these seven antagonists were sprayed on melon leaves twice (1 and 15 days after transplanting) under greenhouse conditions and disease development was monitored weekly for five weeks. As compared to the control, Pseudomonas oryzihabitans Antg-12, isolated from watermelon leaf, reduced both disease severity (55.85%) and the area under the disease curve (AUDPC) (66.85%), and increased yield by about 41%. In the present study it was shown that certain bacterial biocontrol agents can substantially reduce disease and, when added into integrated management of BLB, may well provide very useful solutions to disease control. To further prove this, studies using the antagonists under open field conditions are planned.

Bacterial fruit blotch (BFB) is a serious disease of melon and watermelon caused by the Gram-negative bacterium Acidovorax citrulli. The strains of the pathogen can be divided into two major genetic groups, I and II. While group I strains are strongly associated with melon, group II strains are more aggressive on watermelon. Like many pathogenic bacteria, A. citrulli secretes a variety of protein effectors to the host cell via the type III secretion system. In the present study, we characterized AopW1, an A. citrulli type III-secreted effector that shares similarity with the actin cytoskeleton-disrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. aopW1 is present in group I and II strains, encoding products of 485 amino acids. Although highly conserved in most of the sequence, AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, including 14 amino acid differences between groups. Here we show that group I AopW1 is more toxic to yeast and plant cells than group II AopW1, having a stronger actin filament disruption activity, and increased ability to reduce plant callose deposition. We demonstrate the role of some of these 14 amino acid positions in determining the phenotypic differences between the two versions of the effector. Moreover, cellular analyses revealed that in addition to the interaction with actin filaments, AopW1 is localized to the endoplasmic reticulum, chloroplasts, and early and recycling plant endosomes, with differences observed between the two AopW1 versions. Finally, we show that overexpression of the endosome-associated protein EHD1 that increases cellular recycling, attenuates the toxic effects exerted by AopW1 and increases defence responses. This study provides insights into the HopW1 family of bacterial effectors and their interactions with the plant cell and provides first evidence on the involvement of EHD1 in response to biotic stress.

Bacterial fruit blotch (BFB) is a bacterial disease that devastates Cucurbitaceae crops worldwide, causing significant economic losses. Currently, there is no means to treat or control the disease. This study focused on exploring the antibacterial properties of endophytic fungi against Acidovorax citrulli (Aac), the causative agent of BFB. Based on disc diffusion, time kill and MIC microdilution broth assays, four endophytes showed promise in controlling Aac. Nonetheless, only one strain, Neocosmospora sp. MFLUCC 17-0253, reduced the severity of disease on watermelon and melon seedlings up to 80%. Structure analysis revealed production of several compounds by the fungus. Three of these secondary metabolites, including mixture of 2-methoxy-6-methyl-7-acetonyl-8-hydroxy-1,4-maphthalenedione and 5,8-dihydroxy-7-acetonyl-1,4-naphthalenedione, anhydrojavanicin, and fusarnaphthoquinones B exhibited antagonistic activity against Aac. The chemical profile data in planta experiment analyzed by LC-Q/TOF-MS suggested successful colonization of endophytic fungi in their host plant and different metabolic profiles between treated and untreated seedling. Biofilm assay also demonstrated that secondary metabolites of Neocosmospora sp. MFLUCC 17-0253 significantly inhibited biofilm development of Aac. To the best of our knowledge, secondary metabolites that provide significant growth inhibition of Aac are reported for the first time. Thus, Neocosmospora sp. MFLUCC 17-0253 possesses high potential as a biocontrol agent for BFB disease.

Bacterial fruit blotch, which is caused by Acidovorax citrulli, is one of the most common diseases of melon crops. To date, there is no disease-resistant commercial cultivar, and management strategies have not been efficient. Therefore, the objectives of this study were to identify melon accessions with genetic resistance to bacterial fruit blotch at different phenological stages of plant growth and to multiple strains of the pathogen. The disease severity was estimated using descriptive scales and evaluated by inoculating A. citrulli into seeds (55 accessions), seedlings, and plants before flowering (38 accessions). After assessing the stability of seven accessions to three strains of the bacterium, two accessions were selected to evaluate their resistance at the flowering and fruiting stages and to perform a transmission assay and optical and scanning electron microscopic analyses. Most accessions showed varied responses (resistance vs. susceptibility) at the different phenological stages of melon growth. However, accessions I-136 and A-43 showed high levels of resistance in most assays and in the stability assay. The fruits of these accessions showed no symptoms of bacterial fruit blotch, and seed transmission of A. citrulli showed values of only 0.6% (I-136) and 2.5% (A-43). Microscopy showed few structural and temporal changes in the colonization of A. citrulli in the leaf mesophyll tissues of these accessions, which can be associated with resistance. The results suggest that accessions A-43 and I-136 can act as donors of bacterial fruit blotch resistance genes, which can be introduced into commercial cultivars through genetic improvement programs.

Bacillus subtilis, a plant-beneficial bacterial species exhibiting good biocontrol capabilities, has been widely used in agricultural production. The endophytic strain 9407 can efficiently control bacterial fruit blotch (BFB) caused by the gram-negative bacterium Acidovorax citrulli. However, the mechanism underlying its biocontrol ability remains poorly understood. Given the genomic diversity of B. subtilis, strain 9407 was sequenced and assembled in this study to determine the genome information associated with its biocontrol traits. A combination of core genome phylogenetic analysis and average nucleotide identity (ANI) analysis demonstrated that the 9407 strain belonged to B. subtilis. Various functional genes related to biocontrol traits, i.e., biofilm formation, motility, pathogen inhibition, plant growth promotion, and induction of systemic resistance, were identified in B. subtilis 9407. Four secondary metabolite biosynthesis gene clusters with antibacterial ability were also found in the B. subtilis 9407 genome, including newly identified subtilosin A, bacilysin, and bacillaene, and the previously reported surfactin. Mutants lacking sboA or bacG, which are defective in synthesizing subtilosin A or bacilysin, showed decreased inhibitory activity against A. citrulli MH21, and the triple mutant with deleted sboA, bacG, and srfAB almost completely lost its inhibitory activity. The biofilm formation and swarming motility of the sboA and bacG mutants also decreased, in turn leading to decreased colonization on melon roots and leaves. Under greenhouse conditions, the biocontrol efficacy of the sboA and bacG mutants against BFB on melon leaves decreased by 21.4 and 32.3%, respectively. Here, we report a new biocontrol pathway of B. subtilis 9407 against BFB, in which subtilosin A and bacilysin contributed to the biocontrol efficacy by improving antibacterial activity and colonization ability of the strain. The comprehensive genomic analysis of B. subtilis 9407 improves our understanding of the biocontrol mechanisms of B. subtilis, providing support for further research of its biocontrol mechanisms and field applications.

Acidovorax citrulli is a seedborne pathogen that causes bacterial fruit blotch (BFB), a global threat to watermelon production. Treating watermelon seeds to eliminate A. citrulli is a critical component of BFB management, and several strategies have been evaluated to mitigate the impact of the disease. In China, watermelon seed producers routinely incubate seeds in watermelon juice (fermentation) to reduce the risk of seed infection by A. citrulli and seedling transmission of BFB. However, there has been limited effort to evaluate the efficacy of fermentation in mitigating A. citrulli seed infection. The current study showed that fermented watermelon fruit juice could inhibit A. citrulli population growth and demonstrated that the low pH conditions, not the temperature dynamic, generated during fermentation might play a major role in A. citrulli growth inhibition and could induce the viable but nonculturable (VBNC) state in A. citrulli. We developed an effective method that was based on propidium monoazide PCR to detect viable A. citrulli cells under low pH conditions or in fermented watermelon fruit juice. We also provided evidence that VBNC A. citrulli cells induced by fermented watermelon fruit juice could not be resuscitated and did not retain their virulence on watermelon seedlings. However, VBNC A. citrulli cells could be resuscitated in Luria–Bertani medium. Based on these observations, we conclude that fermentation in watermelon fruit juice may not be an effective seed treatment for BFB because it may increase the seed infection by A. citrulli.

Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB), and BFB poses a threat to global watermelon production. Despite its economic importance, the molecular mechanisms underlying Ac pathogenicity and virulence are not well understood, particularly with regard to its type III secreted effectors. We identify a new effector, AopP, in Ac and confirm its secretion and translocation. AopP suppresses reactive oxygen species burst and salicylic acid (SA) content and significantly contributes to virulence. Interestingly, AopP interacts with a watermelon transcription factor, ClWRKY6, in vivo and in vitro. ClWRKY6 shows typical nuclear localization, and AopP and ClWRKY6 co-localize in the nucleus. Ac infection, SA, and the pathogen-associated molecular pattern flg22Ac promote ClWRKY6 production, suggesting that ClWRKY6 is involved in plant immunity and SA signaling. Furthermore, ClWRKY6 positively regulates PTI and SA production when expressed in Nicotiana benthamiana. Importantly, AopP reduces ClWRKY6 mRNA and ClWRKY6 protein levels, suggesting that AopP suppresses plant immunity by targeting ClWRKY6. In summary, we identify a novel effector associated with the virulence mechanism of Ac, which interacts with the transcription factor of the natural host, watermelon. The findings of this study provide insights into the mechanisms of watermelon immune responses and may facilitate molecular breeding for bacterial fruit blotch resistance.

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.

Bacterial fruit blotch caused by Acidovorax citrulli is known to be the major threat to cucurbit crop production worldwide. The pathogen can penetrate into seed coat and cause disease symptoms at any stage of plant growth, which results in fruit loss. Two main genotypes (genotype I and II) are reported in A. citrulli, in which genotype II is the main cause of Bacterial Fruit Blotch (BFB) in watermelon and group I is known to be a causal agent of BFB in melon. To date, there are no commercially available cultivars resistant to BFB, and available strategies are not able to completely manage the disease. In this study, we aim to isolate bacteriophages to control BFB. Samples collected from watermelon, melon, and pumpkin were used to isolate bacteriophages. All isolated bacteriophages were tested against 42 strains of A. citrulli, among which two phages with the ability to lyse a greater number of hosts were selected and characterized. Bacteriophage ACP17 from the Myoviridae family, with a head size of 100 ± 5 nm and tail of 150 ± 5 nm, infected 29 strains of A. citrulli mostly belonging to genotype group I, whereas the second isolated bacteriophage, ACPWH from Siphoviridae, with a head size of 60 ± 5 nm and tail of 180 ± 5 nm, infected 39 A. citrulli strains. Genome analysis of both bacteriophages using Next generation Sequencing (NGS) showed that ACP17 and ACPWH have double-stranded DNA with sizes of 156,972 kb and 424,299 kb, respectively. Watermelon seeds coated with ACPWH showed a germination rate of up to 90% in the presence of A. citrulli in contrast to untreated seed, which showed no germination or germinated juveniles with BFB symptoms in the presence of A. citrulli. The results of this study show that the use of bacteriophages of A. citrulli represents a potential biocontrol method for controlling BFB.

Acidovorax citrulli, a seedborne bacterium and quarantine pest, causes the devastating bacterial fruit blotch disease in cucurbit plants. Immunological assays such as ELISA are widely used in routine field inspections for this bacterium. However, to the best of our knowledge, none of the currently available monoclonal antibodies (MAbs) can detect all common A. citrulli strains. We therefore aimed to produce a panel of MAbs and to develop an ELISA-based method capable of detecting all A. citrulli strains. We used a high-throughput bead array technique to screen and characterize A. citrulli-specific MAbs produced from hybridoma clones. The hybridoma library was simultaneously screened against five A. citrulli strains (PSA, KK9, SQA, SQB and P) and the closely related bacterium, Delftia acidovorans. Three MAbs exhibiting different binding patterns to A. citrulli were used to develop an ELISA-based method called "double antibody pairs sandwich ELISA" (DAPS-ELISA). DAPS-ELISA employing mixtures of MAbs was able to specifically detect all 16 A. citrulli strains tested without cross-reactivity with other bacteria. By contrast, our previously developed MAb capture-sandwich ELISA (MC-sELISA) and a commercial test kit detected only 15 and 14 of 16 strains, respectively. The sensitivity of the DAPS-ELISA ranged from 5×105 to 1×106 CFU/mL, while those of the MC-sELISA and the commercial test kit ranged from 5×104 to 1×107 CFU/mL and 5×104 to 5×105 CFU/mL, respectively. DAPS-ELISA thus represents an alternative method enabling rapid, accurate, and inexpensive detection of all A. citrulli strains. The method can be applied to seed testing prior to planting as well as to routine field inspections.

Bacterial fruit blotch (BFB) is an economically important disease in melons and watermelons for which no effective control method is available. Application of phytobacterium-infecting phage has been evaluated as an alternative means of preventing bacterial diseases in plants. Coating of seeds with bacteriophages infecting Acidovorax citrulli, the causal agent of BFB, is effective for controlling the disease, as shown in our previous study. We evaluated the transport of bacteriophage ACPWH from soil to the leaves of melon plants, and we also evaluated its effect on BFB. Leaves of melon plants were spray-inoculated with A. citrulli, and bacteriophage ACPWH was added to soil after symptoms had developed. ACPWH was detected by PCR in foliar tissue 8 h after addition to soil. DAPI-stained ACPWH accumulated at the leaf tip after 24 h. Melon treated with ACPWH showed 27% disease severity, compared to 80% for the non-treated control, indicating that ACPWH can be used to control BFB.

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.

Phytopathogenic bacteria are responsible for causing several losses in agriculture economic world. Biofilm formation presents itself as an important structure related to bacterial virulence. The objective of this study was to investigate the biofilm formation by Ralstonia solanacearum and Acidovorax citrulli isolates. Quantification of biofilm formation was performed by the crystal violet method, using NYD as the standard medium for both bacteria, TZC as a specific medium for R. solanacearum and YDC for A. citrulli. The biofilm was observed in SEM and CLSM. Under the tested conditions, B5-5, CGH8, CGM10 and CGH26 R. solanacearum isolates and Aac1.43 and Aac1.73 A. citrulli isolate formed moderately or strongly biofilm on both media tested. However, the amount of biofilm produced by R. solanacearum was higher than those produced by A. citrulli. The SEM and CLSM revealed structurally distinct biofilms among isolates of R. solanacearum, which did not occur for A. citrulli isolates. We conclude that R. solanacearum is a strong biofilm producer, while A. citrulli not seem to be well suited to this condition by not adhere well to the surface. This species depicts potentials to become natural models to study plant biofilm infections due to the high number of host species.

Acidovorax citrulli is the causal agent of bacterial fruit blotch of cucurbits. We have shown that functional type IV pili (T4P) are required for full virulence of this bacterium. To identify A. citrulli genes required for T4P activity, we screened a library of about 10,000 transposon mutants of A. citrulli M6 for altered T4P-mediated twitching motility. This screen led to the identification of 50 mutants impaired in twitching ability due to transposon insertions into 20 different genes. Representative mutants with disruptions in these genes were further characterized. All mutants were compromised in their virulence in seed transmission and stem inoculation assays and had reduced biofilm formation ability relative to wild-type M6. When grown on nutrient agar, most mutants produced colonies with a translucent and fuzzy appearance, in contrast to the opaque and smooth appearance of wild-type colonies. The colony morphology of these mutants was identical to that of previously reported phenotypic variants of strain M6. The exceptions were M6 mutants disrupted in genes tonB, pilT, pilW, and pilX that exhibited typical wild-type colony morphology, although lacking twitching haloes surrounding the colony. Transmission electron microscopy revealed that most mutants lacked the ability to produce T4P. The exceptions were mutants with disruptions in tonB, pilT, pilW, and pilX genes that were shown to produce these appendages. These findings support the idea that colony phenotypic variation in A. citrulli is determined by the lack of ability to synthesize T4P but not by lack of T4P functionality.

Bacteriophages of Acidovorax citrulli, the causal agent of bacterial fruit blotch, were isolated from 39 watermelon, pumpkin, and cucumber leaf samples collected from various regions of Korea and tested against 18 A. citrulli strains. Among the six phages isolated, ACP17 forms the largest plaque, and exhibits the morphology of phages in the Myoviridae family with a head diameter of 100 ± 5 nm and tail length of 150 ± 5 nm. ACP17 has eclipse and latent periods of 25 ± 5 min and 50 ± 5 min, respectively, and a burst size of 120. The genome of ACP17 is 156,281 base pairs with a G + C content of 58.7%, 263 open reading frames, and 4 transfer RNA genes. Blast search and phylogenetic analysis of the major capsid protein showed that ACP17 has limited homology to two Stentrophomonas phages, suggesting that ACP17 is a new type of Myoviridae isolated from A. citrulli.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is the most destructive bacterial disease affecting melon production in northeastern Brazil. Silicon (Si), which is widely recognized for suppressing plant diseases, has been proposed as a possible alternative for the control of BFB. Field experiments were conducted to evaluate the effects of slag fertilization on bacterial blotch development, soil chemical properties, plant growth and nutrition, and fruit quality. Melon hybrids AF 4945 and Medellin were grown in slag-fertilized (5.00 t ha−1) and non-fertilized plots, being inoculated with Ac 25 days after emergence. The slag fertilization significantly reduced the area under the disease progress curve (10%), disease index (14%) and disease incidence in fruit (12%) at harvest, regardless of hybrid. Slag fertilization improved the soil fertility, plant growth and nutrition after 55 days of cultivation. Tissue analyses showed no differences in Si accumulation between the melon hybrids. However, AF 4945 accumulated more Ca and Zn than Medellin, which was associated with higher reduction in disease intensity. Also, slag treatment increased fruit flesh thickness (8%) and soluble solids (7%). Our study confirms that the supply of slag to the soil previously assessed under greenhouse studies can reduce the severity and incidence of melon BFB while improving fruit quality under field conditions.

The gram-negative bacterium Acidovorax citrulli causes bacterial fruit blotch (BFB) disease of cucurbits, which represents a serious threat to melon and watermelon production worldwide. To date, there are no efficient means to manage the disease, and reliable resistance sources for cucurbit germplasm are lacking. Mineral nutrition markedly affects plant diseases. Recently, we reported that disease severity on melon foliage and A. citrulli growth in the leaf tissue were significantly influenced by the form of nitrogen supply. In the present study, we investigated the influence of potassium nutrition on BFB severity and A. citrulli establishment in the foliage of melon plants. Fertilization with relatively low concentrations of potassium increased these variables compared with higher potassium concentrations. Since establishment of A. citrulli during the growing season is assumed to increase the incidence of fruit infection, the fact that mineral nutrition influences BFB incidence in the plant foliage is of particular importance.

To employ a microsphere immunoassay (MIA) to simultaneously detect multiple plant pathogens (potyviruses, Watermelon silver mottle virus, Melon yellow spot virus, and Acidovorax avenae subsp. citrulli) in actual plant samples, several factors need to be optimized and rigorously validated. Here, a simple extraction method using a single extraction buffer was successfully selected to detect the four pathogens in various cucurbit samples (cucumber, cantaloupe, melon, and watermelon). The extraction method and assay performance were validated with inoculated and field cucurbit samples. The MIA showed 98-99% relative accuracy, 97-100% relative specificity and 92-100% relative sensitivity when compared to commercial ELISA kits and reverse transcription PCR. In addition, the MIA was also able to accurately detect multiple-infected field samples. The results demonstrate that one common extraction method for all tested cucurbit samples could be applied to detect multiple pathogens; avoiding the need for multiple protocols to be employed. This multiplex method can therefore be instrumental for high-throughput screening of multiple plant pathogens with many advantages such as a shorter assay time (2.5h) with single assay format, a lower cost of detection ($5 vs $19.7 for 4 pathogens/sample) and less labor requirement. Its multiplex capacity can also be expanded to detect up to 50 different pathogens upon the availability of specific antibodies.

Bacterial fruit blotch (BFB), which is caused by Acidovorax citrulli, is a serious threat to watermelon growers around the world. The present study was conducted to screen effective rhizobacterial isolates against 35 different A. citrulli isolates and determine their efficacy on BFB and growth parameters of watermelon. Two rhizobacterial isolates viz. Paenibacillus polymyxa (SN-22), Sinomonas atrocyanea (NSB-27) showed high inhibitory activity in the preliminary screening and were further evaluated for their effect on BFB and growth parameters of three different watermelon varieties under greenhouse conditions. The greenhouse experiment result revealed that SN-22 and NSB-27 significantly reduced BFB and had significant stimulatory effect on total chlorophyll content, plant height, total fresh weight and total dry weight compared to uninoculated plants across the tested three watermelon varieties. Analysis of the 16S ribosomal RNA (rRNA) sequences revealed that strains SN-22 belong to P. polymyxa and NSB-27 to S. atrocyanea with the bootstrap value of 99% and 98%, respectively. The isolates SN-22 and NSB-27 were tested for antagonistic and PGP traits. The result showed that the tested isolates produced siderophore, hydrolytic enzymes (protease and cellulose), chitinase, starch hydrolytic enzymes and they showed phosphate as well as zinc solubilizing capacity. This is the first report of P. polymyxa (SN-22) and S. atrocyanea (NSB-27) as biocontrol-plant growth promoting rhizobacteria on watermelon.

Summary The cucurbit pathogenic bacterium Acidovorax citrulli requires a functional type III secretion system (T3SS) for pathogenicity. In this bacterium, as with Xanthomonas and Ralstonia spp., an AraC‐type transcriptional regulator, HrpX, regulates expression of genes encoding T3SS components and type III‐secreted effectors (T3Es). The annotation of a sequenced A. citrulli strain revealed 11 T3E genes. Assuming that this could be an underestimation, we aimed to uncover the T3E arsenal of the A. citrulli model strain, M6. Thorough sequence analysis revealed 51 M6 genes whose products are similar to known T3Es. Furthermore, we combined machine learning and transcriptomics to identify novel T3Es. The machine‐learning approach ranked all A. citrulli M6 genes according to their propensity to encode T3Es. RNA‐Seq revealed differential gene expression between wild‐type M6 and a mutant defective in HrpX: 159 and 28 genes showed significantly reduced and increased expression in the mutant relative to wild‐type M6, respectively. Data combined from these approaches led to the identification of seven novel T3E candidates that were further validated using a T3SS‐dependent translocation assay. These T3E genes encode hypothetical proteins that seem to be restricted to plant pathogenic Acidovorax species. Transient expression in Nicotiana benthamiana revealed that two of these T3Es localize to the cell nucleus and one interacts with the endoplasmic reticulum. This study places A. citrulli among the ‘richest’ bacterial pathogens in terms of T3E cargo. It also revealed novel T3Es that appear to be involved in the pathoadaptive evolution of plant pathogenic Acidovorax species.

Watermelon (Citrullus lanatus Thunb.) is considered as a popular and nutritious fruit crop worldwide. Watermelon blood flesh disease caused by Cucumber green mottle mosaic virus (CGMMV) and bacterial fruit blotch caused by Acidovorax citrulli, are two major quarantine diseases of watermelon and result in considerable losses to global watermelon production. In this study, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) method was developed for simultaneous detection of CGMMV and A. citrulli in both watermelon leaves and seeds. Two pairs of specific primers were designed based on the conserved sequences of the genomic RNA of CGMMV and the internal transcribed spacer of A. citrulli, respectively. Transcriptional elongation factor-1α from watermelon was added as an internal reference gene to prevent false negatives. No cross-reactivity was detected with other viral or bacterial pathogens infecting watermelon. Moreover, the multiplex RT-PCR showed high sensitivity and could simultaneously detect CGMMV and A. citrulli as little as 102 copies of plasmid DNA. This method was successfully applied to test field-collected watermelon leaves and stored seeds of cucurbitaceous crops. These results suggested that the developed multiplex RT-PCR technique is a rapid, efficient, and sensitive method for simultaneous detection of CGMMV and A. citrulli, providing technical support for monitoring, predicting, and preventing these two quarantine diseases. To our knowledge, this is the first report on simultaneous detection of a virus and a bacterium by multiplex RT-PCR in watermelon.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit crop production worldwide. Based on genetic and phenotypic properties, A. citrulli strains are divided into two major groups: group I strains have been generally isolated from melon and other non-watermelon cucurbits, while group II strains are closely associated with watermelon. In a previous study, we reported the genome of the group I model strain, M6. At that time, the M6 genome was sequenced by MiSeq Illumina technology, with reads assembled into 139 contigs. Here, we report the assembly of the M6 genome following sequencing with PacBio technology. This approach not only allowed full assembly of the M6 genome, but it also revealed the occurrence of a ∼53 kb plasmid. The M6 plasmid, named pACM6, was further confirmed by plasmid extraction, Southern-blot analysis of restricted fragments and obtention of M6-derivative cured strains. pACM6 occurs at low copy numbers (average of ∼4.1 ± 1.3 chromosome equivalents) in A. citrulli M6 and contains 63 open reading frames (ORFs), most of which (55.6%) encoding hypothetical proteins. The plasmid contains several genes encoding type IV secretion components, and typical plasmid-borne genes involved in plasmid maintenance, replication and transfer. The plasmid also carries an operon encoding homologs of a Fic-VbhA toxin-antitoxin (TA) module. Transcriptome data from A. citrulli M6 revealed that, under the tested conditions, the genes encoding the components of this TA system are among the highest expressed genes in pACM6. Whether this TA module plays a role in pACM6 maintenance is still to be determined. Leaf infiltration and seed transmission assays revealed that, under tested conditions, the loss of pACM6 did not affect the virulence of A. citrulli M6. We also show that pACM6 or similar plasmids are present in several group I strains, but absent in all tested group II strains of A. citrulli.

Acidovorax citrulli is a gram-negative bacterium that infects a wide range of cucurbits causing bacterial fruit blotch (BFB) disease. Copper-based compounds are the most widely-used chemicals for managing BFB and other bacterial diseases in the field. Many bacteria can enter a viable but non-culturable (VBNC) state in response to stress, including exposure to copper, and recover the culturability when favorable conditions return. The present study demonstrates that A. citrulli strain AAC00-1 is able to enter into the VBNC state by treatment with different concentrations of copper sulfate. It took 3 h, 5 and 15 days for all viable cells to lose culturability upon exposure to copper sulfate concentrations of 50, 10, and 5 μM, respectively. The VBNC A. citrulli cells regained culturability when the Cu2+ ions were removed by chelation with EDTA or by transfer of cells to LB broth, a cell-free supernatant from a suspension of AAC00-1, oligotrophic media amended with casein hydrolysate or watermelon seedling juice. We also found that the VBNC cells induced by Cu2+ were unable to colonize or infect watermelon seedlings directly, but the resuscitated cells recovered full virulence equivalent to untreated bacterial cells in the log phase. To the best of our knowledge, this is the first report on the VBNC state in A. citrulli and the factors that facilitate resuscitation and restoration of pathogenicity.

In September 1998, a fruit rot was reported affecting pumpkin (Cucurbita pepo) in a commercial field in Terrell Co., Georgia. Symptoms on the surface of fruit occurred as round, necrotic spots or cracks a few millimeters in diameter. With age, the tissue surrounding these lesions became soft and wrinkled. A soft rot expanded into the flesh of the pumpkin, originating from the lesions observed on the surface. In time, infected pumpkins totally collapsed. V-shaped, necrotic lesions occurred at the margin of the leaf and extended inward toward the mid-rib. Samples were collected from the field and bacteria were isolated from fruit and leaf lesions onto King's medium B (1). The bacterium isolated was rod shaped, gram negative, nonflourescent, oxidase positive, Tween 80 positive, carboxymethyl cellulose positive, β-OH butyrate positive, and malonate negative. The bacterium reacted positively with polyclonal antibodies specific for the watermelon fruit blotch pathogen Acidivorax avenae subsp. citrulli and was identified as A. avenae subsp. citrulli by MIDI (Microbial Identification System, Newark, DE) according to statistical analysis of fatty acid data. Results from polymerase chain reaction (PCR) amplification of the bacterium isolated from pumpkin yielded 360-bp fragments that, when digested with the restriction enzyme HaeIII, had DNA banding patterns identical to those of stock A. avenae subsp. citrulli DNA. Koch's postulates were completed successfully with 2-week-old watermelon seedlings. This is the first report of A. avenae subsp. citrulli causing fruit rot of pumpkin in Georgia. Reference: (1) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954.

In March 1999, a foliar bacterial disease was observed in a commercial crop of cucumber (Cucumis sativus L.) cv. Jetset in Gumlu in northern Queensland, Australia. Initial symptoms consisted of angular, chlorotic, water-soaked lesions that later dried to necrotic areas of light brown, dead tissue. White bacterial ooze was commonly found on the undersides of young water-soaked lesions. Lesions were delimited by veins and distributed uniformly over leaf surfaces, and more than 20% of the crop was affected. No symptoms were observed on plant stems or fruits. Bacterial streaming from the edges of freshly cut young lesions was clearly visible in a droplet of water under ×100 magnification in the laboratory. Isolations were made from young lesions on King's medium B (1). A slow-growing, white, gram-negative, nonfluorescent bacterium was consistently isolated. Three isolates of the bacterium were identified, using the Biolog software program (Biolog, Hayward CA), and in each instance, the bacterium was confirmed as Acidovorax avenae subsp. citrulli, with a similarity of >0.80. Koch's postulates were completed with 8-day-old glasshouse-grown cucumber (cv. Jetset) seedlings. Seedlings were misted until runoff with a bacterial suspension of 3 × 108 CFU/ml and enclosed in plastic bags for ≈30 h at 22°C. Water-soaked lesions were observed on cucumber cotyledons 4 days after inoculation. This is the first report of A. avenae subsp. citrulli as a pathogen of cucumber. Reference: (1) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954.

An immunomagnetic separation and polymerase chain reaction (IMS-PCR)-based assay was developed for detecting Acidovorax avenae subsp. citrulli in watermelon seed. IMS yielded a 10-fold increase in recovery of A. avenae subsp. citrulli over direct spread-plating on King's Medium B; however, the presence of seed debris reduced IMS efficiency. Synthetic oligonucleotide primers were designed based on the 16S rRNA gene of a known A. avenae subsp. citrulli strain and tested for specific DNA amplification by PCR. The primers amplified DNA from all A. avenae subsp. citrulli strains tested but also yielded amplicons with several closely related bacteria. IMS-PCR resulted in a 100-fold increase in A. avenae subsp. citrulli detection sensitivity over direct PCR and was unaffected by PCR inhibitors in watermelon seed. The threshold of A. avenae subsp. citrulli detection for IMS-PCR was 10 CFU/ml in watermelon seed wash, and seedlots with 0.1% infestation were consistently detected. IMS-PCR represents an efficient and sensitive approach to detecting A. avenae subsp. citrulli in watermelon seedlots.

In the greenhouse, 1,344 Citrullus spp. and Praecitrulllus fistulosus accessions were screened for resistance to Acidovorax avenae subsp. citrulli. Seedlings were inoculated at the first true leaf stage by misting with a water suspension of a virulent strain of A. avenae subsp. citrulli originally isolated from commercial watermelon in Florida in 1989. Seedlings were considered resistant if less than 20% of the cotyledons were necrotic and there were no lesions on the true leaves 10 days after inoculation. Twelve accessions had individual seedlings that were resistant to A. avenae subsp. citrulli. Selfs of seven of these accessions were susceptible in greenhouse and field tests. Selfs were obtained from five accessions from Zimbabwe and Zambia that possessed a level of greenhouse and field resistance to A. avenae subsp. citrulli that could provide control of bacterial fruit blotch of watermelon. Based on field evaluations, plant introduction (PI) 482279 and PI 494817 were judged to contain plants with the best sources of resistance; however, PI 500303, PI 500331, and PI 482246 also had plants with high levels of resistance.

Acidovorax citrulli is a seed-borne pathogen causing bacterial fruit blotch of cucurbits including melon and watermelon. We investigated the roles of quorum sensing in the wild-type group II strain Aac-5 of A. citrulli by generating aacR and aacI knockout mutants and their complementation strains. We found that twitching motility and virulence were reduced, but biofilm formation and seed attachment were increased significantly in the two mutants as compared to the wild type strain. Deletion of aacR and aacI, however, had no effect on swimming motility and polar flagella formation of Aac-5. Furthermore, deletion of aacR resulted in reduced gene expression of hrpE, hrcN and pilT, while deletion of aacI affected only the expression of hrpE and pilT, not hrcN.

Watermelon (Citrullus lanatus) is an important crop of the Cucurbitaceae family in fruit production worldwide. During its production, bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Acidovorax avenae subsp. citrulli) is an important limiting factor on the volume and value of crops. This pathogen is known as a seed-borne pathogen, and the infested seeds can be a primary source of inoculum. Hence, a rapid and sensitive method for detecting A. citrulli on seeds would be an important tool in the management of BFB. In this study, we sought to develop a method to detect A. citrulli bacterial cells based on a TaqMan probe-based insulated isothermal PCR (TiiPCR) assay. Firstly, the specific primers and probe were designed based on a specific DNA fragment from the genome of A. citrulli. Then, PCR amplification was performed with the plasmid DNA to adjust the components of the PCR reagents, such as the concentrations of primers, magnesium chloride, and Taq DNA polymerase. Results revealed that 10 copies of plasmid DNA were detectable within the modified reagents by TiiPCR. Moreover, 10 bacterial cells in each reaction tube were detectable at a 100 % detection rate in this condition with a fluorescent signal intensification over 1.8. Based on these results, we concluded that a specific, rapid, and sensitive method based on TiiPCR had been successfully developed to detect bacterial cells of A. citrulli.

Plant-derived essential oil is an alternative to antibiotics, eliminating the concern of developing antibiotic-resistant bacterial strains. In this study, using the half-divided Petri plate assays, 32 volatile essential oils were screened for their antibacterial activity against Acidovorax citrulli (Acc). Sweet basil and peppermint oils were the most effective against Acc, with subsequent trials showing that peppermint oil to be the most active. Using gas chromatography–mass spectrometry, the major compositions of peppermint oil were analysed. Among the various compositions of peppermint oil, menthol, neomenthol, isopulegone and 1,8-cineole were significantly active against Acc and each component at 0.2% concentration inhibited all bacterial growth. This study demonstrated in vitro and in vivo antibacterial activities of peppermint oil and its active components against Acc. These results suggest the use of peppermint oil as a potential antibacterial agent to treat seed with Acc.

Bacterial fruit blotch (BFB) caused by Acidovorax avenae subsp. citrulli is defective disease towatermelon cultivated areas. To control of BFB, we investigated control efficiency to use commercial anti-bacterial pesticides. Growth inhibition zone on medium were formed as oxolinic acid WP and oxytetracyclineWP. Control efficacy of four anti-bacterial pesticides on seed and seedling stage were performed. As aresults, oxytetracyclin WP is shown over 90% control efficiency on seed and acibenzolar-S-methyl +mancozeb WP shown over 90% control efficiency on seedling stage Hot-water treatment method could bepossible to reduced infection rate on seed. The conditions of hot-water treatments are 50~55 o C on 20~30minutes. These results suggested that the methods were helpful watermelon seedling nursery to control of thebacterial fruit blotch by A. avenae subsp. citrulli.Key words Anti-bacterial pesticides, Bacterial fruit blotch, Control, Hot-water treatment 서론 수박 과일썩음병(bacterial fruit blotch)은 Acidovorax avenaesubsp. citrulli에 의해 발병하는 병으로, 1980년대 미국Indiana 남서부 지역의 수박 재배지역에서 대발생하여 경제적 피해를 일으켰다(Latin and Fane, 1990). 그 이후에 남아메리카, 호주, 아시아 등에서 멜론, 수박, 오이 등 박과작물에서 과일썩음병이 보고되었다(Cheng et al., 2000; Isakeitet al., 1997; Martin and O'Brien 1999; Shirakawa et al.,2000). 우리나라는 1991년 전북 고창에서 그 발생이 보고된이후로수박에서주요병해로 인식되고 있으며(Song et al.,1991), 전남 광주, 나주지역의 멜론재배 농가에서도 발생하였다(Seo et al., 2006). 전 세계적으로 과일썩음병에 대한연구는 박과작물에 발생하는 발생보고와 진단기술 개발이주로 수행되었다(Ha et al., 2009; Latin and Hopkins, 1995;Schaad and Freedrick, 2002; Walcott et al., 2000). 최근, 국내에서는 과일썩음병균 신속하고 정확한 검출을 위해 nestedPCR법과 real-time PCR법이 개발되었다(Cho et al., 2015;Kim et al., 2015). 수박 과일썩음병 방제를 위해 멀칭 종류별 발병 억제효과 검정, 나노 화학물을 이용한 항균활성 효과를 검정한 연구가 진행되었다(Lee et al., 2014; Kim etal., 2015). 최근, 국내에서는 과일썩음병균을 수집하고, 분자계통학적 유연관계를 분석하여 국내산 과일썩음병균에 유전적 변이가 있음을 보고하였다(Song et al., 2015). 수박 과일