Agent Of Bacterial Fruit Blotch Research Articles

Effect of Acetic Acid on Biofilm Formation in Paracidovorax citrulli, Causal Agent of Bacterial Fruit Blotch.

The unique tissue structure of pathogenic bacteria biofilm plays an important role in its pathogenicity and bactericide resistance. Inhibition or destruction of biofilm formation of pathogenic bacteria is of great significance for the control of plant bacterial diseases. In this study, Paracidovorax citrulli was inoculated into KB medium containing acetic acid, and after shaking at 28°C and 55 r/min for 48 h, it was found that the content of extracellular polysaccharide, extracellular protein and extracellular DNA (eDNA) decreased with the increase of acetic acid concentration, which resulted in the decrease of biofilm formation, it is not even possible to form biofilms on plastic slides. When the final concentration of acetic acid in the culture medium was greater than or equal to 0.5 mg/mL, there was no biofilm on the plastic slides. Therefore, the use of acetic acid as an inhibitor of P. citrulli has a good potential for control of bacterial fruit blotch.

Molecular tracking of Acidovorax citrulli: Unveiling pathogen dynamics and blotch disease outbreaks through specific markers

Acidovorax citrulli (Ac), a gram-negative bacterium, is the causal agent of bacterial fruit blotch (BFB), which poses a significant threat to cucurbit crop production worldwide. Understanding the genetic diversity of Ac is crucial for identifying sources of resistance and implementing effective disease management strategies. In this study, we conducted the first genetic characterization of Ac strains collected in Turkey using Inter-Simple Sequence Repeat (ISSR) markers. These markers were selected based on repetitive domains mapped on the complete reference genome sequence of Acidovorax citrulli strain NWB SC196. The identity of the Turkish strains was confirmed through molecular (PCR) and serological (Immunofluorescence test and ELISA) methods, while the selected ISSRs, which exhibited similarity to flanked regions in the pathogen's whole genome sequence, were employed to assess the genetic diversity among Ac strains. We compared the profiles of Turkish strains with those of a collection of Ac strains from various countries, including the US, to explore a possible common origin. Specifically, we considered the dissemination of these strains through rootstocks used for grafted seedling production (Cucurbita maxima ? Cucurbita moschata). The results demonstrated a shared genetic profile, suggesting a potential link between Ac strains collected in Turkey and foreign strains. The Mauve analysis, utilizing whole genome sequences of various Ac strains available in the NCBI database, displayed similar clustering patterns to those obtained using our selected molecular markers, confirming the discriminatory efficiency of our method. Based on the high discriminatory power of the selected markers, our proposed method offers a rapid and straightforward approach for genetic analysis of intraspecific variation and monitoring Ac gene flow across countries. The characterized strains and markers presented in this study serve as valuable resources and reference materials for further genetic investigations and tracking contamination sources associated with Ac.

PilA Gene Contributes to Virulence, Motility, Biofilm Formation, and Interspecific Competition of Bacteria in Acidovorax citrulli.

Acidovorax citrulli, the causative agent of bacterial fruit blotch, can be divided into two main groups based on factors such as pathogenicity and host species preference. PilA is an important structural and functional component of type IV pili (T4P). Previous studies have found significant differences in pilA DNA sequences between group I and group II strains of A. citrulli. In this study, we characterized pilA in the group I strain pslb65 and the group II strain Aac5. pilA mutants, complementation strains, and cross-complementation strains were generated, and their biological phenotypes were analyzed to identify functional differences between pilA in the two groups. pilA deletion mutants (pslb65-ΔpilA and Aac5-ΔpilA) showed significantly reduced pathogenicity compared with the wild-type (WT) strains; pslb65-ΔpilA also completely lost twitching motility, whereas Aac5-ΔpilA only partially lost motility. In King's B medium, there were no significant differences in biofilm formation between pslb65-ΔpilA and WT pslb65, but Aac5-ΔpilA showed significantly reduced biofilm formation compared to WT Aac5. In M9 minimal medium, both mutants showed significantly lower biofilm formation compared to the corresponding WT strains, although biofilm formation was recovered in the complementation strains. The biofilm formation capacity was somewhat recovered in the cross-complementation strains but remained significantly lower than in the WT strains. The interspecies competitive abilities of pslb65-ΔpilA and Aac5-ΔpilA were significantly lower than in the WT strains; Aac5-ΔpilA was more strongly competitive than pslb65-ΔpilA, and the complementation strains recovered competitiveness to WT levels. Furthermore, the cross-complementation strains showed stronger competitive abilities than the corresponding WT strains. The relative expression levels of genes related to T4P and the type VI secretion system were then assessed in the pilA mutants via quantitative PCR. The results showed significant differences in the relative expression levels of multiple genes in pslb65-ΔpilA and Aac5-ΔpilA compared to the corresponding WT stains. This indicated the presence of specific differences in pilA function between the two A. citrulli groups, but the regulatory mechanisms involved require further study.

Isolation and Identification Antagonistic Bacterium Paenibacillus tianmuensis YM002 against Acidovorax citrulli.

In this study, we aimed to screen antagonistic microorganisms against Acidovorax citrulli, the causal agent of bacterial fruit blotch, which is known to induce sever diseases in cucurbit crops. From 240 bacterial strains isolated, only one unknown bacterial isolate, named YM002, showed significant antagonistic activity against A. citrulli KACC17909. Further experiments revealed that YM002 shows antagonistic activity against all tested A. citrulli strains, including KACC17000, KACC17001 and KACC17005, to different degrees. The phylogenetic analysis of 16S rRNA sequences identified YM002 as Paenibacillus tianmuensis. Importantly, pretreatment of cucumber (Cucumis sativus) leaves with YM002 enhanced disease resistance as observed by significantly reduced necrotic symptom development and bacterial growth. YM002-induced resistance accompanied by enhanced expression of defense-related genes, such as PAL1, PR1-1a and CTR1. Importantly, culture filtrate of YM002 significantly suppressed biofilm formation and swimming motility of A. citrulli, which is indispensable for its full virulence. In addition to its antagonistic activity, YM002 showed a various plant growth promotion (PGP)-related traits, such as production of ammonia production, amylase production, ACC deaminase production, inodole-3-acetic acid production, extracellular protease production, siderophore production, and zinc solubilization activities. Indeed, treatment of cucumber roots with YM002 significantly enhanced plant growth parameters, such as fresh and dry weight of leaves or roots. This study suggests the potential of YM002 as an effective PGPR with biological control activity against Acidovorax citrulli in cucumber plants.

DETECTION OF Acidovorax citrulli IN WATERMELON SEEDLINGS IN HOPELCHÉN, CAMPECHE, MEXICO

In Mexico, there are no reports of the presence of Acidovorax citrulli, the causal agent of bacterial fruit blotch and seedling blight in cucurbits. The introduction of A. citrulli is a latent risk due to the importation of seeds and seedlings. Fruits and plants with typical symptoms of this disease have been observed in watermelon (Citrullus lanatus) production fields of small growers and commercial nurseries in the municipality of Hopelchén, Campeche. The objective was to detect A. citrulli by PCR in watermelon seedlings in a production area in Hopelchén, with the hypothesis that the bacterium is present in this municipality. In December 2018, 51 samples of nine watermelon varieties were collected from two seedling production nurseries. Each sample contained ten symptomatic seedlings ranging in age from 12 to 41 days. End-point PCR analysis with primers WFB1 and WFB2 indicated the presence of A. citrulli in 68.6 % of the samples; immuno-PCR results confirmed the presence of the bacterium in 80.9 % of these samples. Consensus 16S rRNA gene fragment sequences from eight samples aligned with 98 % similarity on average to A. citrulli sequences deposited in the GenBank-NCBI database. The dark, sunken, elongated lesions on cotyledonal leaves, as well as the black, spreading lesions from the margin to the base of true leaves observed on watermelon seedlings, were caused by A. citrulli. This study constitutes a new report of A. citrulli in Campeche, which should be considered to delimit and prevent the spread of the bacterium to other cucurbit-producing areas in Mexico.

Further Characterization of Host Preference of Acidovorax citrulli Based on Growth Competition between Group I and Group II Strains

Acidovorax citrulli, the causal agent of bacterial fruit blotch, can be divided into two groups. Group I is mainly isolated from melon, and group II is mainly isolated from watermelon. In this study, group I and II strains were used to assess competition in vivo and in vitro by evaluating inhibition activity assays and by measuring population growth dynamics. Our results indicated that there is no mutual inhibition of growth between the two groups of strains in King’s B medium. The bacterial populations of A. citrulli strains were counted within 7 days after inoculation in melon and watermelon cotyledons and plotted against time to generate population growth curves. Area under the population growth curves was calculated. When the two groups of strains were inoculated separately into melon and watermelon cotyledons, the results of analysis of variance show that there was no significant difference. In this study, for the first time in an experimental setting, we inoculated two A. citrulli groups into melon and watermelon cotyledons at the same time and determined the population dynamics of each strain. The results showed that there was no significant difference between two group strains in melon cotyledons. However, in watermelon cotyledons, the area under population growth curves of group II strain were significantly higher than those of group I strain. Three-way analysis of variance results showed that there was interaction between host and grouping on the influence of strain population level (p < 0.0001). These data provide additional information on the host preference of different groups of A. citrulli.

Host-specific activation of a pathogen effector Aave_4606 from Acidovorax citrulli, the causal agent for bacterial fruit blotch

RipAY, an effector protein from the plant bacterial pathogen Ralstonia solanacearum, exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the host cellular glutathione (GSH) when stimulated by host eukaryotic-type thioredoxins (Trxs). Aave_4606 from Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbit plants, shows significant homology to RipAY. Based on its homology, it was predicted that the GGCT activity of Aave_4606 is also stimulated by host Trxs. The GGCT activity of a recombinant Aave_4606 protein was investigated in the presence of various Trxs, such as yeast (ScTrx1), Arabidopsis thaliana (AtTrx-h1, AtTrx-h2, AtTrx-h3, and AtTrx-h5), or watermelon (Cla022460/ClTrx). Unlike RipAY, the GGCT activity of Aave_4606 is stimulated only by AtTrx-h1, AtTrx-h3, AtTrx-h5 and ClTrx from a watermelon, the primary host of A. citrulli, but not by ScTrx1, AtTrx-h2. Interestingly, GGCT activity of Aave_4606 is more efficiently stimulated by AtTrx-h1 and ClTrx than AtTrx-h5. These results suggested that Aave_4606 recognizes host-specific Trxs, which specifically activates the GGCT activity of Aave_4606 to decrease the host cellular GSH. These findings provide new insights into that effector is one of the host-range determinants for pathogenic bacteria via its host-dependent activation.

Polyphasic Characterization of Acidovorax citrulli Strains Originating from Serbia

Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB), is an economically important pathogen of watermelon and related plant species worldwide. In the period 2014–2018, several outbreaks of BFB were observed in major watermelon production regions in Serbia. A total of 43 strains, isolated from symptomatic watermelon tissue, were analyzed by biochemical, pathogenic and molecular tests. Based on the phenotypic characteristics, PCR assay, and 16S rRNA gene sequence analysis, all strains were identified as A. citrulli. A multilocus sequence analysis of the four housekeeping (adk, gyrB, pilT and gltA) and three virulence genes (Aave_1548, avrRx01 and luxR) revealed that studied A. citrulli strains represent the homogeneous population and they clustered together with group II reference strain AAC00-1. Strain affiliation to group II was confirmed by PCR based on the putative type III secretion effector gene and by duplex PCR test. High homogeneity of studied strains was also confirmed by BOX-PCR. Differences were observed for two strains in their pathogenicity as well as susceptibility to copper compounds. Moreover, six major watermelon varieties grown in Serbia showed high sensitivity to the pathogen, while cross inoculation assay revealed that the strains were able to infect other species within the Cucurbitaceae family.

Identification of a Cupin Protein Gene Responsible for Pathogenicity, Phage Susceptibility and LPS Synthesis of Acidovorax citrulli.

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King’s B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

Napthoquinones from Neocosmospora sp.-Antibiotic Activity against Acidovorax citrulli, the Causative Agent of Bacterial Fruit Blotch in Watermelon and Melon.

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.

Proteomic and Phenotypic Analyses of a Putative Glycerol-3-Phosphate Dehydrogenase Required for Virulence in Acidovorax citrulli

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.

ПРИМЕНЕНИЕ НОВЫХ МЕТОДОВ ВЫЯВЛЕНИЯ И ИДЕНТИФИКАЦИИ ACIDOVORAX CITRULLI И ОПТИМИЗАЦИЯ СХЕМЫ ЛАБОРАТОРНОЙ ДИАГНОСТИКИ

The phytopathogenic bacterium Acidovorax citrulli is included in the quarantine lists of many countries, including the unified list of quarantine objects of the Eurasian Economic Union. The control of the introduction and spread of the quarantine organism into the territory of Russia is carried out through laboratory diagnostics. Diagnostic methods should ensure quality and the high speed of product health testing. The study aimed to use new methods for the detection and identification of A. citrulli and to optimize the laboratory diagnostic scheme. In the course of the work, cucumber seeds with an infectious load were prepared and tested. Testing was carried out according to an optimized diagnostic scheme using methods not previously used to diagnose A. citrulli. At each stage of work with samples, the time spent was recorded. The researchers tested the prepared infected seeds at the first stage using the standard qPCR method and then the qPCR method PAS F/R, PAS P, which had not previously been used to diagnose A. citrulli. The new qPCR test PAS F/R, PAS P showed stable results at testing each sample in a row of each replication. The sample enrichment method was successfully applied, not previously used in diagnosing A. citrulli. The use of the method made it possible to separate samples containing live target bacteria from those containing only A. citrulli DNA within 2.5 days. After enrichment, the isolation and identification of the A. citrulli culture from the samples were successfully carried out. Based on the data obtained as a result of this study, the scheme for detecting and identifying the causative agent of bacterial fruit blotch of cucurbits was optimized. The proposed scheme will reduce diagnosing a dangerous quarantine organism from four weeks to one week.

Acidovorax citrulli Type III Effector AopP Suppresses Plant Immunity by Targeting the Watermelon Transcription Factor WRKY6.

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.

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

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

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.

ITRAQ-based proteomic analyses of the plant-pathogenic bacterium Acidovorax citrulli during entrance into and resuscitation from the viable but nonculturable state

Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) disease, infects cucurbit crops including watermelon and melon. This bacterium can enter the viable but nonculturable (VBNC) state following exposure to copper sulfate. Moreover, copper-induced VBNC A. citrulli cells can be resuscitated by EDTA. In this study, isobaric tag for relative and absolute quantification (iTRAQ) was used to compare protein profiles of VBNC cells, resuscitated cells at different stages and log-phase cells of the A. citrulli model strain AAC00-1. A total of 2672 proteins were identified, with 60 being differentially abundant in VBNC cells compared with log-phase cells, and 469 being differentially abundant in resuscitated cells compared with VBNC cells. Proteins involved in the arginine and proline metabolism pathway and degradation of aromatic compounds could be important for the VBNC cells. In the early resuscitation process, proteins associated with carbon metabolism, and degradation of naphthalene and aromatic compounds were significantly enriched, while proteins involved in oxidative phosphorylation, bacterial chemotaxis, ABC transporters and quorum sensing were significantly enriched at the late resuscitation stages. This is the first study reporting thorough protein profile analyses of VBNC and resuscitating cells of a plant-pathogenic bacterium. Biological significanceThe VBNC state is a dormant-like condition that was reported to occur in many bacterial species, upon facing a variety of environmental stresses. Acidovorax citrulli is a seed borne pathogenic bacterium that threatens cucurbit production worldwide. Moreover, A. citrulli can enter into the VBNC state after treatment of copper sulfate, thus increasing its survival and dissemination probabilities. This study enriches our understanding of the mechanisms of entrance into and resuscitation from the VBNC state of this important plant-pathogenic bacterium. This knowledge could be exploited in the future to develop novel approaches to interfere with these processes, thus contributing to a more efficient management of this pathogen. In a broader perspective, the knowledge emerging from this study has implications to the general understanding of the VBNC state in bacteria.

Prevalence of Acidovorax citrulli in Commercial Cucurbit Seedlots During 2010-2018 in China.

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.

Complete Assembly of the Genome of an Acidovorax citrulli Strain Reveals a Naturally Occurring Plasmid in This Species.

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.

Induction and Resuscitation of the Viable but Non-culturable (VBNC) State in Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch of Cucurbitaceous Crops.

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.

First Report of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch, on Melon (Cucumis melo) in Guadeloupe (France)

HomePlant DiseaseVol. 103, No. 5First Report of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch, on Melon (Cucumis melo) in Guadeloupe (France) PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch, on Melon (Cucumis melo) in Guadeloupe (France)A. Cunty, C. Audusseau, S. Paillard, V. Olivier, C. François, C. Rivoal, and F. PoliakoffA. Cunty†Corresponding author: A. Cunty; E-mail Address: [email protected]http://orcid.org/0000-0001-8174-587XLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, C. AudusseauLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, S. PaillardLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, V. OlivierLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, C. FrançoisLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, C. RivoalLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, and F. PoliakoffLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this authorAffiliationsAuthors and Affiliations A. Cunty † C. Audusseau S. Paillard V. Olivier C. François C. Rivoal F. Poliakoff Laboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, France Published Online:15 Mar 2019https://doi.org/10.1094/PDIS-10-18-1825-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In March 2015, symptomatic melon (Cucumis melo) plants and fruits were received from the French West Indies Island, Guadeloupe, at the Anses Plant Health Laboratory (LSV). The leaves showed brown necrotic spots surrounding by a chlorotic halo. The fruits presented small and large water-soaked lesions on the rind that did extend into the flesh, and dark brown necrotic spots into the flesh were observed under some of the lesions. The yield loss was estimated at around 50%. Symptoms were first suspected to be associated with the presence of a pathovar of Pseudomonas syringae, but the isolated strain was identified as Acidovorax citrulli and the pathogenicity was confirmed. Because leaf samples were damaged during transport (development of rot), isolations were only performed from lesions on the fruits on modified King’s medium B (mKB) supplemented with cycloheximide (50 mg/liter). A gram-negative, motile, oxidative, nonfluorescent bacterium was isolated (LSV 46.36 and then registered in the French CIRM–Plant Associated Bacteria collection as CFBP 8568). It induced a hypersensitive reaction on tobacco, had a cytochrome c oxidase activity, did not display an arginine dihydrolase activity nor hydrolyze aesculin or gelatin, did not produce levan, used l-arabinose and glucose, but did not use cellobiose and rhamnose. When plated on mKB, the colonies grew at 41°C. The strain CFBP 8568 was compared with the A. citrulli type strain CFBP 4459. Whole cell DNA, extracted by boiling bacterial suspension (1 × 106 CFU/ml) for 10 min at 100°C, was amplified by the duplex polymerase chain reaction (PCR) developed by Bahar et al. (2008). In specific, two fragments of 476 and 279 bp were obtained for both strains, indicating that the strain CFBP 8568 was A. citrulli. The real-time PCR described in appendixes 4 and 6 of the EPPO protocol PM 7/127 (EPPO 2016), which targeted complementary genes, were also applied to the DNA extract and confirmed the previous results. Application of the PCR assay developed by Zivanovic and Walcott (2017) allowed classifying the strain CFBP 8568 in group I. To fulfill all Koch’s postulates, a pathogenicity test was performed. Three melon seedlings (Charentais type) were inoculated by infiltrating a first true leaf with a cell suspension (1 × 107 CFU/ml) of the strain CFBP 8568. The type strain CFBP 4459 was used as a positive control. After 13 days, brown necrotic spots surrounded by a chlorotic halo were observed at the inoculation points for both strains. From those necrotic spots, strains with all the characteristics of A. citrulli (as previously described) were isolated. No symptoms were observed on the stem, which may be because the test was stopped just after the appearance of necrotic leaf spots. To our knowledge, this is the first report of bacterial fruit blotch (BFB) in Guadeloupe. Prophylactic measures and a strong sanitary survey of this crop allowed eradication of the outbreak. Indeed, no symptoms have been reported in 2018. However, this first report of BFB showed that it could represent a threat in Caribbean area, were it was never reported before.