Acidovorax Avenae Research Articles

Bacteria occurring in onion (Allium cepa L.) foliage in Puerto Rico.

Bacteria associated with foliar symptoms of onion (Allium cepa L.) were examined in the southern region of Puerto Rico from January through April 2004. Different symptoms were observed in onion foliage of cultivars 'Mercedes' and 'Excalibur' at Juana Díaz and Santa Isabel, Puerto Rico. Ellipsoidal sunken lesions with soft rot and disruption of tissue were the most common symptoms observed in onion foliage in field conditions. From a total of 39 bacterial strains isolated from diverse symptoms in onion foliage, 38% were isolated from soft rotting lesions. Ninety-two percent of the bacteria isolated from onion foliage was Gram negative. Pantoea spp. with 25%, was the most frequently isolated genus, followed by Pasteurella spp. and Serratia rubidae with 10% each. Fifty- six percent of the strains held plant pathogenic potential; these strains belong to the genera Acidovorax sp., Burkholderia sp., Clavibacter sp., Curtobacterium sp., Enterobacter sp., Pantoea spp., Pseudomonas spp., and Xanthomonas spp. Pathogenicity tests showed that seven out of eight tested bacterial strains evaluated under field conditions caused symptoms in onion foliage for both cultivars. Acidovorax avenae subsp. citrulli, Burkholderia glumae, Pantoea agglomerans, P. dispersa, Pseudomonas sp., Xanthomonas sp., and Xanthomonas-like sp. were pathogenic to leaf tissues. Clavibacter michiganensis was not pathogenic to leaf tissues. Other bacteria identified as associated with onion leaf tissue were Curtobacterium flaccumfaciens, Cytophaga sp., Enterobacter cloacae, Flavimonas oryzihabitans, Mannheimia haemolytica, Pantoea stewartii, Pasteurella anatis, P. bettyae, P. langaaensis, Photobacterium damselae, Pseudomonas syringae pv. aptata, Rhizobium radiobacter, Serratia rubidae, Sphingobacterium spiritivorum, Sphingomonas sanguinis, and an unknown strain. This paper is the first survey of bacteria associated with onion foliage in Puerto Rico. The role of non- phytopathogenic bacteria associated with the life cycle of onion under field conditions remains unknown.

Effect of phosphate-solubilizing bacteria on the gene expression and inhibition of bacterial fruit blotch in melon

Pathogenic bacteria Acidovorax avenae subsp. citrulli can cause bacterial fruit blotch (BFB) in melon (Cucumis melo L.). However, the process in which melon plant inoculated with biocontrol bacteria cope with A. citrulli invasion remains unclear. This study aimed to analyze the expression of genes involved in the BFB with a biocontrol strain through RNA sequencing. Results showed that the disease incidence of BFB was reduced by 57.1 % when melon was inoculated with antagonistic phosphate-solubilizing bacteria (PSB) Burkholderia sp. ‘N3’ strain. The plant height, dry weight, leaf area, and uptake of nutrients of melon seedlings increased after it was inoculated with two strains (‘N3’ and ‘M01’). The P content in TN (inoculated with ‘N3’) significantly increased by 17.48 % compared with the control (TR1). Fe3+ significantly increased in melon plant inoculated with ‘N3’ and ‘Pslb24’ and might play an important role in the suppression of BFB in melon. A total of 129 upregulated genes and 33 downregulated genes were involved in the resistance of melon to A. citrulli infection (TP2 versus TP1). Gene ontology analysis revealed that most differentially expressed genes were located in biological regulation, cell component organization, binding, and catalytic activity functions. Kyoto Encyclopedia of Genes and Genomes analysis illustrated that pathways related to cell cycle, ribosome biogenesis, plant hormone signal transduction, starch and sucrose, and signal transmission mechanisms were involved. These findings will be helpful to elaborate and uncover the expression of resistance genes involved in controlling BFB in melon inoculated with beneficial PSB.

Comprehensive genomic analysis of Bacillus subtilis 9407 reveals its biocontrol potential against bacterial fruit blotch

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.

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.

Quantitative proteomic analysis of the sugarcane defense responses incited by Acidovorax avenae subsp. avenae causing red stripe

The causal agent of red stripe, Acidovorax avenae subsp. avenae (Aaa), can incite significant economic losses during sugarcane production. Proteomics is a useful tool for the comprehensive identification of defense response proteins in plants. In this study, isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics was used to identify proteins that were produced in sugarcane in defense to Aaa in two Saccharum spp. cultivars: ROC22 (resistant to red stripe) and MT11−610 (susceptible). Defense response was evaluated at 0 h (mock-inoculation) and 24 h after inoculation with the bacterial pathogen, and 10,502 high-confidence proteins and 1962 differentially expressed proteins (DEPs) were identified. These DEPs included 1027 proteins (671 upregulated, 354 downregulated) in ROC22 and 1130 proteins (566 upregulated, 564 downregulated) in MT11−610. Gene ontology analysis revealed that most DEPs were clustered in the metabolic, single-organism metabolic, and oxidation-reduction processes, and in catalytic activity. In a KEGG analysis, these proteins belonged to common pathways such as secondary metabolite biosynthesis and biosynthesis of phenylpropanoids. Thirty-six DEPs were identified, especially epoxide hydrolase 1, dynamin-related protein 1C-like, cytochrome oxidase subunit I, FK506-binding protein, acetoacetyl CoA thiolase isoform 2, anthocyanidin 3-o-glucosyltransferase, ubiquitin carboxyl-terminal hydrolase 4, protein disulfide isomerase-like protein, and jumonji-like transcription factor family protein. These nine proteins were characterized by enhanced expression with ≥1.5-fold increases in resistant ROC22 versus susceptible MT11−610, and are therefore biomarker candidates in the sugarcane response against Aaa. These data could be very useful in molecular breeding for sugarcane resistance to red stripe.

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.

Novel qPCR-based test system for Acidovorax citrulli based on PAS domain S-box protein gene

<h3>ЦЕЛЬ ИССЛЕДОВАНИЯ</h3> Диагностика возбудителя бактериальной пятнистости тыквенных культур <i>Acidovorax citrulli</i> нуждается в новых высокоспецифичных методах, основанных на ПЦР в режиме «реального времени» (ПЦР-РВ). Целью исследования являлась разработка нового теста ПЦР-РВ для идентификации <i>A. citrulli</i>. <h3>МАТЕРИАЛЫ И МЕТОДЫ</h3> Методом кластеризации белковых последовательностей 9 геномов <i>A. citrulli</i> и 9 геномов <i>A. avenae</i> c помощью программы cd-hit были идентифицированы 153 белковых кластера, присутствующих во всех 9 штаммах <i>A. citrulli</i> и отсутствующих во всех штаммах <i>A. avenae</i>. Полученные кластеры сравнивали с известными белковыми последовательностями (nr) с помощью программы blastp с целью установления их специфичности для <i>A. citrulli</i>. Кодирующие последовательности специфичных для <i>A. citrulli</i> белков использовали для подбора праймеров и зонда. Специфичность ПЦР-РВ проверяли путем тестирования ДНК 6 штаммов <i>A. citrulli</i> и 188 штаммов нецелевых бактерий. <h3>РЕЗУЛЬТАТЫ</h3> В результате поиска специфичных для <i>A. citrulli</i> белковых последовательностей нами был идентифицирован белок S-box PAS домена. Проведенные ПЦР-тесты показали 100% специфичность как при тестировании целевых штаммов (все реакции положительны), так и с нецелевыми штаммами (все реакции отрицательны). <h3>ЗАКЛЮЧЕНИЕ</h3> Таким образом, был разработан новый тест ПЦР-РВ для идентификации <i>A. citrulli</i>, основанный на последовательности, кодирующей белок S-box домена PAS.

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

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

Screening for bacterial fruit blotch resistance in watermelon fruit

AbstractBacterial fruit blotch (BFB) caused by Acidovorax citrulli is a serious threat to the watermelon industry. Currently, there are no commercial watermelon cultivars with resistance to BFB. In this 3‐yr study, we conducted a screening of 1,452 Citrullus spp. cultigens (PIs and cultivars) for resistance to BFB at the immature fruit stage. However, due to high levels of missing data, many cultigens require additional testing to confirm resistance. Fruit BFB symptoms presented initially as raised bumps, then progressed to blotching, cracking, and internal necrosis. Resistant cultigens rarely proceeded beyond raised bumps at the infection site. Fruit resistance is significantly affected by environmental conditions and has low heritability. Among a subset of cultigens selected for high resistance and susceptibility during the first 2 yr of screening and retested using increased replications, PI 494819 [C. lanatus (Thunb.) Matsum. &amp; Nakai., Zambia], PI 596659 (C. amarus Schrad., South Africa), PI 596670 (C. amarus, South Africa), PI 490384 [C. mucosospermus (Fursa) Fursa, Mali], and PI 596656 (C. amarus, South Africa) had average disease ratings of &lt;1; significantly more resistance than the commercial check, Charleston Gray; low plant‐to‐plant variability within each PI; and infrequent incidence of advanced symptoms. These PIs are potential sources of fruit resistance for breeding programs as well as for research focused on understanding the underlying mechanisms and genetics.

Detailed analysis of the fatty acid composition of six plant-pathogenic bacteria

Bacteria show distinct and characteristic fatty acid (FA) patterns which can be modified by environmental conditions. In this study, we cultivated six plant-pathogenic bacteria of agricultural concern and performed a detailed analysis of the fatty acid composition. The study covered four strains of the gram-negative Xanthomonas campestris pathovar (pv) campestris (Xcc), Xanthomonas perforans (Xp), Acidovorax citrulli (Ac) and Pseudomonas syringae pv. tomato (Pst), and two strains of the gram-positive Clavibacter michiganensis subsp. michiganensis (Cmm) and Streptomyces scabies (Ssc). After cultivation, freeze-dried bacteria samples were transesterified and analysed by gas chromatography with mass spectrometry in full scan and selected ion monitoring (SIM) modes. Altogether, 44 different FAs were detected in the six strains with individual contributions of 0.01–43.8% to the total FAs. The variety in the six strains ranged between 12 and 31 individual FAs. The FA composition of Xcc, Xp, Cmm and Ssc were dominated by iso- and anteiso-fatty acids (especially i15:0, a15:0, i16:0), which is typical for most bacteria. In contrast to this, Ac and Pst showed only saturated and monounsaturated FAs. Four of the six bacteria showed similar FA patterns as reported before in the literature. Differences were observed in the case of Cmm where many more FAs were detected in the present study. In addition, to the best of our knowledge, the FA pattern of Xp was presented for the first time.

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.

Slag-based silicon fertilizer improves the resistance to bacterial fruit blotch and fruit quality of melon grown under field conditions

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 Medellín 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 Medellín, 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.

Assessment of inoculation techniques for screening sugarcane resistance to red stripe disease caused by Acidovorax avenae subsp. avenae

The red stripe disease caused by Acidovorax avenae subsp. avenae in sugarcane, has become a quite relevant issue in Argentina because of its high incidence in the sugarcane growing area. The resistance of host plants is the most promising method for controlling the disease. In that sense, the Estación Experimental Agroindustrial Obispo Colombres (EEAOC) has a Sugarcane Breeding Program, which generates new varieties with higher productivity and good sanitary behavior. The lack of an effective screening technique to select resistant sugarcane genotypes limits the cultivar selection process. To develop a practical and affordable method for achieving the expression of the red stripe disease, three available inoculation techniques were evaluated under controlled conditions over two sugarcane varieties, with a previously adjustment of soil composition and nutrition and relative humidity. They consisted in (i) scrubbing the leaf surface with a cotton ball soaked in the suspension of A. avenae subsp. avenae; and spraying inoculum under two conditions: (ii) leaves pre-treated with a refined sand scarification and (iii) leaves with no scarification. Fifteen plants were inoculated per cultivar and treatment according to a randomized protocol with three replicates and the severity of the disease was evaluated on a scale of 1- 9 according to the International Society of Sugarcane Technologists. The spray inoculation using a bacterial suspension of A. avenae subsp. avenae without abrasives was also field tested. These results contribute to sugarcane breeding programs, providing a tool to assess the resistance to red stripe of their materials, overcoming the lack of bacterial pressure or favorable conditions for the disease.

First report of Acidovorax citrulli causing a leaf spot disease on tomato plants in Greece

First report of Acidovorax citrulli causing a leaf spot disease on tomato plants in Greece

Genome-wide analysis of mitogen-activated protein (MAP) kinase gene family expression in response to biotic and abiotic stresses in sugarcane.

To systematically analyze mitogen-activated protein (MAP) kinase gene families and their expression profiles in sugarcane (Saccharum spp. hybrids; Sh) under diverse biotic and abiotic stresses, we identified 15 ShMAPKs, 6 ShMAPKKs and 16 ShMAPKKKs genes in the sugarcane cultivar R570 genome. These were also confirmed in one S. spontaneum genome and two transcriptome datasets of sugarcane trigged by Acidovorax avenae subsp. avenae (Aaa) and Xanthomonas albilineans (Xa) infections. Phylogenetic analysis revealed that four subgroups were present in each ShMAPK and ShMAPKK family and three sub-families (RAF, MEKK and ZIK) presented in the ShMAPKKK family. Conserved protein motif and gene structure analyses supported the evolutionary relationships of the three families inferred from the phylogenetic analysis. All of the ShMAPK, ShMAPKK and ShMAPKKK genes identified in Saccharum spp. R570 were distributed on chromosomes 1-7 and 9-10. RNA-seq and qRT-PCR analyses indicated that ShMAPK07 and ShMAPKKK02 were defense-responsive genes in sugarcane challenged by both Aaa and Xa stimuli, while some genes were upregulated specifically by Aaa and Xa infection. Additionally, ShMAPK05 acted as a negative regulator under drought and salinity stress, but served as a positive regulator under salicylic acid (SA) treatment. ShMAPK07 plays a positive role under drought stress, but a negative role under SA treatment. ShMAPKKK01 was negatively modulated by both salinity stress and SA treatment, whereas ShMAPKKK06 was positively regulated by both of the two stress stimuli. Our results suggest that members of MAPK cascade gene families regulate adverse stress responses through multiple signal transduction pathways in sugarcane.

Putative Bifunctional Chorismate Mutase/Prephenate Dehydratase Contributes to the Virulence of Acidovorax citrulli.

Acidovorax citrulli (Ac) is a plant pathogenic bacterium that causes bacterial fruit blotch (BFB) in cucurbit crops. Despite its importance in the cucurbit industry, resistant cultivars/lines against BFB have not yet been identified. Therefore, there is a need to characterize the virulence factors/mechanisms in Ac to control the disease. Chorismate mutase, a key enzyme in the shikimate pathway, produces aromatic amino acids. Here, we report the functions of putative bifunctional chorismate mutase/prephenate dehydratase in Ac (CmpAc) determined by proteomic analysis and phenotypic assays. Ac strain lacking CmpAc, AcΔcmpAc(EV), were significantly less virulent on watermelon in the germinated-seed inoculation and leaf infiltration assays. Sequence analysis revealed that CmpAc possesses two distinct domains: chorismate mutase and prephenate dehydratase, indicating that CmpAc is a bifunctional protein. Auxotrophic assays demonstrated that CmpAc is required for the biosynthesis of phenylalanine, but not tyrosine. The comparative proteomic analysis revealed that CmpAc is mostly involved in cell wall/membrane/envelop biogenesis. Furthermore, AcΔcmpAc(EV) showed reduced twitching halo production and enhanced biofilm formation. In addition, AcΔcmpAc(EV) was less tolerant to osmotic stress but more tolerant to antibiotics (polymyxin B). Thus, our study provides new insights into the functions of a putative bifunctional protein related to virulence in Ac.

Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.

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.

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

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

Silicon slag increases melon growth and resistance to bacterial fruit blotch

Melon bacterial fruit blotch (BFB) is the major bacterial melon disease in Northeastern Brazil. We evaluated the effects of applying a silicon (Si) slag on BFB suppressiveness in two melons cultivars as well as in soil chemical attributes and plant growth and nutrition. Slag was incorporated into the soil at concentrations equivalent to 0.00, 0.12, 0.24, 0.47, 0.71, and 1.41 g kg-1 of silicon. Plants were inoculated with Acidovorax citrulli 20 days after emergence. Results showed that amending the soil with Si slag improved the resistance of two melon cultivars against bacterial fruit blotch. Such an effect is probably related not only to the Si uptake by plants but also to changes in soil characteristics and improvement in plant nutrition. Both hybrid cultivars (AF4945 and Medellín) increased biomass, nutrient and Si accumulation as a function of Si doses applied to soil. According to Si concentration and Si to Ca ratio in plant tissue, both cultivars are regarded as intermediary Si-accumulators. We also observed that an intermediate dose of Si (0.71 g kg-1) posed better results on controlling melon bacterial fruit blotch than the highest dose tested

First Report of Bacterial Fruit Blotch on Watermelon Caused by Acidovorax citrulli in the Republic of North Macedonia

First Report of Bacterial Fruit Blotch on Watermelon Caused by <i>Acidovorax citrulli</i> in the Republic of North Macedonia