Hpa1 secretion via type III secretion system in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

In many Gram-negative plant pathogenic bacteria the type III secretion system (TTSS), encoded by hrp genes, is essential for pathogenicity in the host and induction of a hypersensitive reaction (HR) in nonhost plants. The expression of hrp genes has been suggested to be repressed in complex media, whereas it is induced in planta and under certain in vitro conditions. We recently reported that XOM2 medium allows efficient hrp expression by Xanthomonas oryzae pv. oryzae. In this study, we investigated hrp-dependent secretion of proteins by the bacteria in vitro. Using modified XOM2, in which bovine serum albumin was added and the pH was lowered to 6.0, we detected at least 10 secreted proteins and identified one as Hpa1. This is the first evidence of protein secretion via TTSS in X. oryzae pv. oryzae.     

Notes on the Occurrence of Pathogenic Races of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>Found in Hiroshima Prefecture in 1999

The pathogenic race of 59 cultures of Xanthomonas oryzae pv. oryzae, a pathogen of bacterial leaf blight of rice, isolated from six locations in the inland mountainous area of Hiroshima Prefecture in 1999, were determined by a set of traditional differentials. Four races—I, II, V and VII—were found across the area; however, we noticed the composition of the races as well as the dominant race in each location different. All races were avirulent on differential cultivar Te-tep. Races V and VII were new to Hiroshima. The rice cultivars infected with bacterial leaf blight in Hiroshima are thought to be grouped into the Kinmaze group, which does not have any resistance genes. Apparently, a variety of races occurred unexpectedly on the cultivars contrary to stabilizing selection theory. Received 25 February 2000/ Accepted in revised form 13 July 2000     

Structural conservation of the <Emphasis Type="Italic">hrp</Emphasis> gene cluster in <Emphasis Type="Italic">Xanthomons oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique.     

A diagnostic medium for the semi-selective isolation and enumeration of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis>pv. <Emphasis Type="Italic">vignicola</Emphasis>

A semi-selective medium for isolation of Xanthomonas axonopodis pv. vignicola from cowpea (Vigna unguiculata) plant and soil samples was developed. Twelve carbon and five nitrogen sources were tested with four strains of X. axonopodispv.vignicola, and 25 antibiotics were screened against saprophytes. -cellobiose (10g) was selected as the optimal carbon source. Among the antibiotics, cefazoline inhibited growth of most of the saprophytes with little effect on strains of the pathogen. ,-methionine enhanced growth of X. axonopodispv.vignicola. Boric acid along with ammonium chloride suppressed growth of Pseudomonas fluorescens. The semi-selective medium designated as cefazoline-cellobiose-methionine (CCM) medium contained K₂HPO₄ 1.34g, KH₂PO₄ 0.4g, MgSO₄ 0.3g, H₃BO₃ 0.2g, NH₄Cl 1.0g, -cellobiose 10g, cycloheximide 0.2g, ,-methionine 1.0g, cefazoline 10mg and agar 14g per l of water (pH 7.2). Colonies of X. axonopodispv.vignicola on CCM medium were whitish, round, raised and 0.2–1.8mm in diameter 96h after incubation. CCM medium generally inhibited growth of Pantoea agglomerans, Bacillus subtilis and saprophytes isolated from cowpea leaves. Colonies of Pseudomonas fluorescens and a saprophytic bacterium, which were not completely suppressed by CCM, could be differentiated from X. axonopodispv.vignicola by their smaller size and different color. The CCM medium proved useful for isolation of X. axonopodispv.vignicola from cowpea plant and soil samples. This is the first report of a semi-selective medium developed for detection of X. axonopodispv.vignicola.     

Growth of Glucose-uptake-deficient Mutant of Xanthomonas oryzae pv. oryzae in Rice Leaves

We isolated Xanthomonas oryzae pv. oryzae mutants deficient in the phosphoenolpyruvate : carbohydrate phos-photransferase system, a major glucose transport system in bacteria, using the glucose analogue 3-deoxy-3-fluoro-d-glucose (3FG). Glucose uptake by the mutants was decreased to 15–35% of the parental strain, and growth greatly decreased in synthetic media containing glucose as a sole sugar source. Growth of the mutants in rice leaves was, however, similar to the wild type. These findings suggest that glucose is not necessarily a major carbohydrate source for X. o. pv. oryzae in rice leaves. Received 11 August 2000/ Accepted in revised form 15 December 2000     

Development of a semi-selective medium for isolation of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv<Emphasis Type="Italic">. musacearum</Emphasis> from banana plants

Banana Xanthomonas wilt, caused by Xanthomonas campestris pv. musacearum, is a new threat to banana cultivation in eastern Africa. The causal bacterium grows slowly in culture and is easily overgrown by contaminants. A selective culture medium for isolation of X. c. pv. musacearum will facilitate disease study. A medium that suppressed saprophytic growth and possessed diagnostic characters for the pathogen was developed. Various carbon sources were tested with two isolates of X. c. pv. musacearum, and sucrose was selected as main carbon source. The susceptibility of X. c. pv. musacearum and other bacterial strains was tested with 29 different antibiotics. Cephalexin and cycloheximide had no effect on X. c. pv. musacearum but cephalexin inhibited most of the saprophytes and cycloheximide inhibited the fungal contaminants. Based on these studies, we have developed a semi-selective medium YTSA-CC containing yeast extract (1%), tryptone (1%), sucrose (1%), agar (1.5%), cephalexin (50 mg l⁻¹) and cycloheximide (150 mg l⁻¹), pH 7.0. The pathogen X. c. pv. musacearum was easily identified as yellowish, mucoid and circular colonies on YTSA-CC medium. This simple semi-selective medium was effective for isolation of X. c. pv. musacearum from infected banana tissues and soil, and it should be a valuable tool in ecological and epidemiological studies.     

<Emphasis Type="Italic">Bchex</Emphasis> virulence gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis>: characterization and functional analysis

We previously identified Bchex as a highly expressed gene during filamentous growth in Botrytis cinerea. The gene encodes the principal protein of the Woronin body and has been shown to seal septal pores in response to cellular damage. In the present study, Southern blot analysis of genomic DNA indicated that the gene exists as a single copy in the B. cinerea genome. The gene was differentially expressed during various developmental stages: expression was high in germinating conidia and the mycelial stage and lower in resting conidia and the appressorial stage. For functional analyses, homologous recombination was used to obtain a ΔBchex knockout mutant. Growth of the mutant was strongly reduced growth in complete medium and in defined media with sucrose, fructose or pectin as the carbon source. After detached tomato leaves were inoculated with the Bchex mutant, lesion development was markedly reduced compared to the control, suggesting that Bchex participates in normal growth, germination and virulence of this fungus.     

Detection and identification of the crucifer pathogen, <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis>, by PCR amplification of the conserved Hrp/type III secretion system gene <Emphasis Type="Italic">hrcC</Emphasis>

The development of a rapid detection method for Xanthomonas campestris pv. campestris (Xcc) in crucifer seeds and plants is essential for high-throughput certification purposes. Here we describe a diagnostic protocol for the identification/detection of Xcc by PCR amplification of fragments from the pathogenicity-associated gene hrcC. Under stringent conditions of amplification, a PCR product of 519 bp from hrcC was obtained from a collection of 46 isolates of Xcc, with the exception of two isolates from radish. No amplicons were obtained from 39 pure cultures of the phytopathogenic bacteria Xanthomonas campestris pv. cerealicola, X. campestris pv. juglandis, X. campestris pv. pelargonii, X. campestris pv. vitians, X. arboricola pv. pruni, X. axonopodis pv. phaseoli, X. axonopodis pv. vesicatoria, X. vesicatoria, Pseudomonas syringae pv. phaseolicola, P. syringae pv. syringae, P. syringae pv. tomato, P. fluorescens, P. marginalis, Pectobacterium atrosepticum, P. carotovorum subsp. carotovorum. In addition, PCR reactions were negative for fifty unidentified environmental isolates purified from the surface of crucifers. The PCR fragment was obtained from four strains previously classified as X. campestris pv. aberrans, X. campestris pv. armorociae, X. campestris pv. barbarae and X. campestris pv. incanae using pathogenicity assays. Our PCR protocol specifically detected Xcc in inoculated leaves, seeds and naturally infected leaves of crucifers.     

Adsorption of OP<Subscript>1</Subscript>-related bacteriophages requires the gene encoding a TonB-dependent receptor-like protein in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Xanthomonas oryzae pv. oryzae strain T7174R is lysed by bacteriophage OP_1h and OP_1h2. Three mutants tolerant to both OP_1h and OP_1h2 were isolated by transposon mutagenesis. The mutants had an insertion of the transposon in XOO1687, which is predicted to encode a TonB-dependent receptor gene. Plasmid pHMIroNB that contained XOO1687 of T7174R was constructed, and the mutant was transformed with the plasmid. The transformant recovered sensitivity to OP_1h and OP_1h2. Electron microscopic analysis demonstrated that OP_1h and OP_1h2 can adsorb to the wild type and the transformant, but they could not adsorb to the phage-tolerant mutant. These results suggest that the TonB-dependent receptor gene relates to adsorption and infection of T7174R by OP_1h2 and OP_1h. Y. Inoue and S. Tsuge have contributed equally to this work.     

Epigallocatechin gallate,a major tea catechin,induces biofilm formation of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">theae</Emphasis>

Plants constitutively produce a variety of secondary metabolites that have antimicrobial activities against phytopathogens; however, interactions between these performed antimicrobial compounds and phytopathogens were poorly understood. In this study, interactions between epigallocatechin gallate (EGCg), which was a major tea catechin that had antimicrobial activities against varieties of bacteria, and Pseudomonas syringae pv. theae (P.s. theae), the causal of bacterial shoot blight of tea, were investigated. EGCg had less antimicrobial activity against P.s. theae; however, subinhibitory concentrations of EGCg induced biofilm formation. Because biofilms are induced in the presence of sucrose in the culture medium but not by P.s. theae strains deficient in exopolysaccharide levan production, biofilm induction by EGCg and levan production are closely related. EGCg increased survival of P.s. theae under dry conditions on nonwounded leaf surfaces in the presence of sucrose. These data indicate the possibility that tea catechins affect the survival of P.s. theae on the phyllosphere. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Visualisation of <Emphasis Type="Italic">hrp</Emphasis> gene expression in <Emphasis Type="Italic">Xanthomonas euvesicatoria</Emphasis> in the tomato phyllosphere

The plasmid pUFZ75 conferred constitutive GFP expression on the bacterial pathogen Xanthomonas euvesicatoria (syn. X. campestris pv. vesicatoria). Colonisation of the tomato phyllosphere and invasion of tomato leaves by X. euvesicatoria was examined using both fluorescence and confocal laser scanning microscopy. Xanthomonas euvesicatoria established a limited population on the tomato leaf surface, primarily occupying the depressions between epidermal cells and around the stomata, prior to invasion of the leaf via the stomata and subsequent growth within the substomatal chamber and the leaf apoplast. Additionally, hrp-gfp fusions were used to report on the temporal and spatial expression of hrp genes during epiphytic colonisation and invasion. Xanthomonas euvesicatoria cells carrying hrpG- and hrpX-gfp reporter constructs were not fluorescent in vitro on non-hrp-inducing LB agar but did exhibit a low level of fluorescence on the leaf surface within 24 h of inoculation, particularly in the vicinity of stomata. Cells carrying hrpG- and hrpX-gfp fusions exhibited high levels of fluorescence 72 h after inoculation in the substomatal chamber and the leaf apoplast. Cells carrying the hrpF-gfp fusion were slightly fluorescent on LB agar and showed no further increase in fluorescence on the leaf surface by 24 h after inoculation, but did show a significant increase in fluorescence 72 h after inoculation in the substomatal chamber and apoplast. The apparent low-level induction of the regulators hrpG and hrpX on the tomato leaf surface may suggest that some of the genes of the X. euvesicatoria HrpG/HrpX regulon are up- or down-regulated prior to invasion of the stomata while still on the leaf surface.     

Persistence of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">vignicola</Emphasis> in weeds and crop debris and identification of <Emphasis Type="Italic">Sphenostylis stenocarpa</Emphasis> as a potential new host

The survival of Xanthomonas axonopodis pv. vignicola, incitant of cowpea bacterial blight and pustule, in residues of infested cowpea leaves was studied in the field in the forest savanna transition zone of South Benin and under variable controlled conditions. The pathogen survived for up to 60 days when placed on the soil surface, and up to 45 days buried at depths of 10 and 20 cm. In the glasshouse, bacteria survived in residue mixed with soil for at least 2 months in dry soil and less than 2 months in moist soil. The pathogen survived at least 30 days in the field after spray-inoculation on the weed species Euphorbia heterophylla, Digitaria horizontalis and Synedrella nodiflora; 20 days on Panicum subalbidum; 10 days on Euphorbia hirta; and 5 days on Talinum triangulare. After leaf-infiltration under glasshouse conditions, the pathogen was detected after 90 days in D. horizontalis; 75 days in T. triangulare, P. subalbidum and S. nodiflora; 60 days in E. hirta, and 30 days in E. heterophylla. Among 12 legume species tested as alternative hosts of X. axonopodis pv. vignicola, only Sphenostylis stenocarpa (African yam bean) showed typical symptoms of cowpea bacterial blight in a glasshouse experiment following artificial inoculation. This is the first time this legume species has been identified as a potential host of X. axonopodis pv.vignicola. Crop residue and weeds are likely sources of primary inoculum when planting two consecutive cowpea crops per year and they probably play a role in dissemination of the pathogen during the cropping season. The alternate host may form a bridge for primary inoculum between cropping seasons.     

Biofilm formation and resistance to bactericides of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">theae</Emphasis>

Biofilm-grown cells of Pseudomonas syringae pv. theae (P.s.theae) wild-type strain K9301 on abiotic surface had remarkable resistance to kasugamycin in comparison to planktonically grown cells; however, the biofilm-grown cells of K9301 had the same sensitivity to copper sulfate. Because both the lesser biofilm-forming strain K9301S3 and enhanced biofilm-forming strain K9301-6 also had remarkable biofilm resistance to kasugamycin just as K9301 did and because epigallocatechin gallate, which enhanced biofilm formation of P.s.theae, had no effect on biofilm resistance to kasugamaycin, the degree of biofilm formation was not correlated with the antibiotic susceptibilities. In addition, K9301 and K9301S3 had less sensitivity to kasugamycin but had high sensitivity to copper sulfate on nonwounded leaf surfaces. These results indicate a possibility that the mechanism of P.s.theae biofilm resistance to bactericide functions on both abiotic and nonwounded leaf surfaces.     

Discrimination of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> isolates from sweet and wild cherry using rep-PCR

Bacterial canker is one of the most important diseases of cherry (Prunus avium). This disease can be caused by two pathovars of Pseudomonas syringae: pv. morsprunorum and pv. syringae. Repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) was investigated as a method to distinguish pathovars, races and isolates of P. syringae from sweet and wild cherry. After amplification of total genomic DNA from 87 isolates using the REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX primers, followed by agarose gel electrophoresis, groups of isolates showed specific patterns of PCR products. Pseudomonas syringae pv. syringae isolates were highly variable. The differences amongst the fingerprints of P. syringae pv. morsprunorum race 1 isolates were small. The patterns of P. syringae pv. morsprunorum race 2 isolates were also very uniform, with one exception, and distinct from the race 1 isolates. rep-PCR is a rapid and simple method to identify isolates of the two races of P. syringae pv. morsprunorum; this method can also assist in the identification of P. syringae pv. syringae isolates, although it cannot replace inoculation on susceptible hosts such as cherry and lilac.