Volatile Metabolite Profiling for the Discrimination of Onion Bulbs Infected by Erwinia carotovora ssp. carotovora, Fusarium oxysporum and Botrytis allii

The volatile metabolites of the headspace gas of onion bulbs inoculated with three different pathogens, Erwinia carotovora ssp. carotovora, Fusarium oxysporum and Botrytis allii, were profiled using gas chromatography/mass spectrometry. Differences in the number and amount of volatile metabolites were observed. Two hundred and fifty three volatile metabolites were detected in bulbs inoculated with three pathogens or sterile distilled water. On day three, 202 volatile metabolites were observed, compared to 166 on day six. Of the 253 compounds, however, only 59 occurred relatively consistently over replications, of which 25 compounds were specific to one or more pathogens, including 10 that were unique to a pathogen. Metabolites such as 1-Oxa-4,6-diazacyclooctane-5-thione and 4-mercapto-3-(methylthio)--(thio-lactone)-crotonic acid were exclusive to onions inoculated with F. oxysporum. Acetone, acetic acid-hydrazide, propylcarbamate, 1-bromo-1-propene, thiirane, 1-(methylthio)-E-1-propene and 1-ethenyl-4-ethyl-benzene were specific to B. allii. 3-bromo-furan was specific to E. carotovora ssp. carotovora. Sterile water-inoculated bulbs produced 3,3-dioxy-1,2-propanediol-tetranitrate. Highest amount of sulfurs was found in pathogen-inoculated, while highest amounts of terpenes, aromatics and aliphatics were found in sterile distilled water-inoculated bulbs. The possible use of these differences in the volatile metabolites for detecting and discriminating diseases of onion in storage is discussed.     

Flagella-mediated motility is required for biofilm formation by Erwinia carotovora subsp. carotovora

To elucidate the role of flagella in biofilm formation by Erwinia carotovora subsp. carotovora EC1, we used a nonflagellate, nonmotile mutant (ΔfliC) and a flagellate, nonmotile mutant (ΔmotA). A biofilm-inducing medium, which contains the yeast peptone (YP) medium plus the salts of M-63 minimal medium, supported biofilm formation to a greater extent than either the YP or Luria Bertani (LB) medium alone. We demonstrated that both the ΔfliC and ΔmotA mutants greatly reduced their ability to form a biofilm on the surface of the wells of polyvinyl chloride (PVC) microtiter plates. The inability of both mutants to form biofilm on the PVC surface was further confirmed with phase-contrast microscopy. Both aflagellate (ΔfliC) and flagellate (ΔmotA) nonmotile mutants were equally defective in attachment to the PVC surface. The treatment of bacteria with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which inhibits the motility of this organism, reduced greatly the biofilm formation. Based on these results, flagella-mediated motility may play an important role in biofilm formation of E. carotovora subsp. carotovora EC1.     

Re-infection by Erwinia carotovora (Jones) Bergey et al. in Potato Stocks derived from Stem Cuttings1

Propagation of potato stocks by the stem cutting method has produced crops almost entirely free from infection with the soft rot coliform bacteria, Erwinia carotovora (Jones) Bergey et al var. atroseptica (Hellmers et Dowson) Dye and E. carotovora var. carotovora Dye. However, low levels of re-infection have occurred and evidence indicates that a source of the organisms was infected insects. Air-borne spread of bacterial aerosols generated by raindrop impaction on infected stems might also result in healthy stocks becoming re-infected.     

Erwinia carotovora Infection Enhances the Expression of Two Novel Abiotic Stress-inducible Genes in Potato

In this study, cDNAs of two Erwinia carotovora-induced potato genes, designated Solanum tuberosum–Erwinia-induced-1 and 2 (Stei1 and Stei2) were isolated by differential display technique. Stei1 and Stei2 were detected in low copy number in the potato genome and found to encode putative proteins with no significant homology to any genes with known function. Treatment of the leaves with salicylic acid, methyl jasmonate and ethylene elevated neither Stei1 nor Stei2 mRNA levels. However, Stei1 and Stei2 expression were induced not only by E. carotovora but also by infiltration of water in leaves, albeit to a lesser extent. In addition, Stei2 was up-regulated by NaCl, wounding, dehydration and abscisic acid. Thus Stei1 and Stei2 define novel genes belonging to the family of those pathogenesis-related genes whose expression can be induced both by biotic and abiotic stresses.     

The role of the seed tuber in determining partial resistance to potato blackleg caused byErwinia spp.

In 1991 and 1992, 12 potato cultivars were screened at two locations for resistance to blackleg, after vacuum infiltration of the seed withErwinia carotovora subsp.atroseptica orE. chrysanthemi. Cultivar differences for resistance toE.c. subsp.Atroseptica andE. chrysanthemi were found which were consistent over locations and years. Seed tubers of the same cultivars were also screened for resistance to bothErwinia spp. by using a tuber slice inoculation method. Correlation coefficients for comparisons between resistance to blackleg in the field and tuber tissue resistance under aerobic or anaerobic conditions were not significant. This could partly be explained by drastic changes in relative tuber tissue resistance of the cultivars within a 5 weeks period after planting in the field. Presprouting of seed tubers in diffuse daylight had a less pronounced effect on relative tuber tissue resistance than planting in the field. Monitoring the process of mother tuber decay during the growing season of 1993 after vacuum infiltration withE.c. subsp.atroseptica andE. chrysanthemi revealed that cultivars differed in the extent to which these bacteria enhanced the process of mother tuber decay. These differences partly explained the cultivar differences for resistance to blackleg in the field.Abbreviations Eca Erwinia carotovora subsp.atroseptica - Ech Erwinia chrysanthemi - NOP Noordoostpolder - Wag Wageningen     

Homolog of FlhDC, a master regulator for flagellum synthesis: required for pathogenicity in Erwinia carotovora subsp. carotovora

 Erwinia carotovora subsp. carotovora (Ecc) is a causal agent of soft-rot diseases in a wide variety of plants. Here, we have isolated nonmotile mutants in Ecc by in vivo insertional mutagenesis using a transposon Tn5. The sequence disrupted by the Tn5 insertion in YMU1 and YMU5 mutants was highly homologous to that of flhC and flhD genes, respectively. They are involved in the initiation of the expression of flagellum-related genes in many gram-negative bacteria such as Escherichia coli and Salmonella. With electron microscopy, the flhC and the flhD homolog mutants were shown to be aflagellate. Furthermore, the virulence of these mutants was greatly reduced in Chinese cabbage and potato compared to that of the parental strain. These results suggest that flagellar formation is required for the pathogenicity of Ecc. Received: November 5, 2002 / Accepted: December 2, 2002 Acknowledgments This research was supported in part by Grant-in-Aid (12052210) and by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (13073).     

An oligonucleotide probe for the detection of Erwinia herbicola and Erwinia ananas

An oligonucleotide probe targeting the rRNA of Erwinia herbicola and Erwinia ananas was designed to detect their cells using fluorescence in situ hybridization. The Cy3-labeled probe hybridized strongly with these species but very weakly with nontarget species such as Erwinia mallotivora, Erwinia nigrifluens, Erwinia cypripedii, Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Escherichia coli. This technique visualized E. herbicola cells after inoculation of kumquat fruits. The probe is promising as a tool for studying population dynamics of E. herbicola and E. ananas.     

Evaluation Of Tests to Determine Resistance of Zantedeschia spp. (Araceae) to Soft Rot caused by Erwinia carotovora subsp. carotovora

Bacterial soft rot caused by Erwinia carotovora subsp. carotovora is a major disease in Zantedeschia spp., particularly in cultivars from the section Aestivae. The disease can be partly controlled by cultural measures, but by combining cultural methods with resistant plant material a promising strategy for control of soft rot can be developed. No tests are available for resistance testing in breeding Zantedeschia spp. Therefore, three tests developed for use in potato breeding were adapted for use on eight cultivars of Zantedeschia spp. Variation was found in all three tests. Resistant control cultivar Zantedeschia aethiopica Crowborough scored most resistant in all three tests. Within the section Aestivae, degrees of susceptibility were identified that were in agreement with each other and with field observations, indicating reliability of two of the methods in which tubers were used. The correlation coefficient of these two tests was high. A new non-destructive test method was developed for use on seedlings which involved immersion of leaf disks in a bacterial suspension. The percentage of decayed leaf area was a measure of resistance and results were in general agreement with the other tests. These methods will be useful for breeding for soft rot resistance and performing genetic analyses.     

Molecular cloning of an endo-pectate lyase gene from Erwinia carotovora subsp. atroseptica

We report the construction of a clone library of the Erwinia carotovora subsp. atroseptica genome and the isolation of a gene for endo-pectate lyase. The library, inserted into the PstI site of plasmid pBR322, contains approximately 1700 clones. Five of these produce pectolytic enzymes as detected by a plate screening assay. Using a cloned endo-pectate lyase gene from the related bacterium, E. carotovora subsp. carotovora, as a probe, we found that one pectolytic E. carotovora subsp. atroseptica clone had strong homology to the probe. We characterized that clone by restriction endonuclease mapping and studied its pectolytic protein product. The purified enzyme is an endo-pectate lyase with a cofactor preference for Co²⁺. The molecular weight of the protein is 31 000 and it has an isoelectric focusing point of 9·2, corresponding to an endo-pectate lyase produced by E. carotovora subsp. atroseptica, but not to the protein product of the E. carotovora subsp. carotovora probe DNA, which has a pI of 9·5. Restriction endonuclease site polymorphisms in the two cloned endo-pectate lyase genes suggest substantial sequence divergence between these two loci.     

Overwintering of Erwinia carotovora subsp. carotovora in Diseased Tissues in Soil and Its Role as Inoculum for Soft Rot of Chinese Cabbage (Brassica campestris, Pekinensis Group)

To examine whether the pathogenic bacterium, Erwinia carotovora subsp. carotovora, causal agent of soft rot of Chinese cabbage (Brassica campestris L., pekinensis group), can overwinter in plant debris and soil and serve as inoculum the following year, we monitored field populations of rifampicin-resistant, phage-sensitive strains of the bacterium. Chinese cabbage (cv. Matsushima Kohai W1116) were planted in field soil in pots that were sunk into the field on Aug. 2, 1996 and eventually reduced to one plant per pot. Outer petioles of the plants were inoculated with mixture of 13 bacterial strains of E. carotovora subsp. carotovora on Sept.5, 1996. After the soft rot spread throughout the plant, the diseased plant was buried in the potted soil. New seeds were sown in the pots on April 30, 1997, and the disease was observed in June and July. The bacterial strains were re-isolated from the potted soil, diseased tissue and rhizosphere soil by the dilution plating method on modified Drigalski's medium containing 100 ppm rifampicin and by the enrichment technique. In addition to rifampicin resistance, phage sensitivities of some of the re-isolated strains were identical to those of the strains buried in the soil with the diseased plant in the previous year. From these results, some of the 13 strains overwintered in the soil and infested plant tissue and acted as primary inoculum the following year. The frequency of re-isolation varied among the strains, perhaps because of competition among the strains, differences in epidemiological behavior and stabilizing selection among the strains, and the presence of different ecotypes of the organism. Received 21 July 2000/ Accepted in revised form 19 September 2000     

Digalacturonates from pectin degradation induce tissue responses against potato soft rot

The pathogenicity of the bacteriumErwinia carotovorasubsp.atroseptica, which causes potato soft rot, is triggered by short oligogalacturonates released by enzymic degradation of plant cell wall pectin. In the first stage unsaturated digalacturonate (uDG), produced by the action of pectate lyases, is degraded by oligogalacturonide lyase (OGL) to keto-deoxyuronate (DKI). The OGL encoding gene fromE. carotovoraand the corresponding recombinant enzyme were characterized. Measuring the changes in plant cell viability and tissue maceration during soft rot pathogenesis in tissue slices of sprouting potato tubers, it was observed that exposure to uDG and DKI, produced by recombinant OGL, killed up to 30% of the plant cells over a period of 16h. This protected the tissue against maceration byE. carotovorasubsp.atroseptica. Endogenous OGL activity was detected in extracts from sprouting tubers where it may be involved in the conversion of uDG into cell toxic compounds. The results indicate that an additional function of small, diffusable digalacturonates is to induce plant cell death during the rotting process, thus contributing to defence reactions againstE. carotovorasubsp.atroseptica.     

The interaction between bactericidal chemicals and the wound healing process of potato tuber tissue

In-vitro tests of dichlorophen against Erwinia carotovora, vars. atroseptica and carotovora, have shown good antibacterial activity; in-vivo tests on potato tubers, however, showed poor control of bacterial soft rotting. Under aerobic or near anaerobic conditions, wounded potato tubers dipped in dichlorophen showed significant increases in soft rotting over water-treated tubers. Under near anaerobic conditions, neither dichlorophen-dipped nor water-dipped tubers showed evidence of wound healing. However, when incubated aerobically, a distinct wound barrier was evident in the water-dipped but not the dichlorophen-dipped tubers. To investigate the possibility that dichlorophen was inhibitory to the initiation of wound healing, an image analysing computer was used to measure the accumulation of fluorescent products in the walls of sectioned tuber tissues treated with dichlorophen. Dichlorophen treatment resulted in very low levels of fluorescence, indicating little phenolic accumulation in the walls. Other bactericides also decreased fluorescence to some extent.     

三种化学物质诱导观赏百合青霉病抗性的研究

采用水杨酸、纳米硅、氯化铵等3种化学物质对观赏百合青霉病的抑菌活性和诱抗效果进行了测定。结果表明,3种化学物质对观赏百合青霉病菌抑菌率较低,最高仅为5.6%。3种化学物质处理百合鳞茎后,表现出较高诱导抗病性。在100、50和25μg/ml浓度下,水杨酸的诱导抗病效果最好,分别为79.09%、69.28%和61.96%。浓度为100μg/ml的水杨酸、纳米硅和氯化铵处理观赏百合鳞茎后,再进行挑战接种,1d后鳞茎组织内过氧化物酶、苯丙氨酸解氨酶、β-1,3-葡聚糖酶活性明显高于对照。浓度为100μg/ml的3种化学物质处理百合种球在2℃条件下贮存4个月后,青霉病的发病率和病情指数均低于对照;水杨酸、纳米硅和氯化铵处理的诱导防治效果分别为60.89%、55.56%和35.31%;处理种球种植后能够正常萌发,出苗率分别为91.67%、90.83%、90.83%。     

Characterization and differentiation of <Emphasis Type="Italic">Erwinia carotovora</Emphasis> strains from mulberry trees

We recently reported that two diverse types (types 1 and 2) were identified among strains of Erwinia carotovora from mulberry trees. Type 1 strains were similar to E. carotovora subsp. carotovora (Ecc), whereas type 2 strains were distinct from Ecc and other E. carotovora strains. In this study, seven more mulberry strains of type 2 and reference strains were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and randomly amplified of polymorphic DNA (RAPD). On the basis of SDS-PAGE profiles of whole-cell proteins, type 2 strains had high similarity with one another. In addition, they had an unique peptide band with a molecular mass of approximately 28kDa. RAPD analysis showed that they were also effectively differentiated by a strong, specific RAPD fragment for type 2 strains. Based on these two approaches, we have confirmed that the present type 2 strains from mulberry can be discriminated clearly from other soft rot Erwinia species.     

A rapid method to identify and quantify soft rot erwinias on seed potato tubers1

Potato tubers are usually contaminated by more than one species or pathovar of soft rot erwinia and, because blackleg incidence is related to the contamination level of seed tubers, the disease potential of seed stocks may be assessed by determining seed-tuber contamination level. A method is described for identifying and quantifying directly from tubers the three soft rot erwinias commonly associated with potatoes. Replicate lots of 10–15 tubers are peeled by dry abrasion in a commercial potato peeler and the peel weight determined by weighing the tubers before and after peeling. Sap is expressed from the peel, an antioxidant (0.075% dithiothreitol) added, and the sap is dilution-plated on a diagnostic selective medium (crystal violet pectate [CVP]). After incubating for 24 h at 20°C, the plates are velvet-replicated onto fresh plates of CVP with or without 35 μg ml^-1 erythromycin and incubated for 48 h at 27°C and 24 h at 33.5 or 37°C. Soft-rot erwinias typically form deep cup-like cavities on CVP and they can be identified and enumerated according to the pattern of cavity formation. Cavities are formed by Erwinia carotovora pv. atroseptica only at 27°C, by E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C, whereas E. chrysanthemi forms cavities at all temperatures but fails to grow in the presence of erythromycin. Contamination levels can be expressed as the number of different erwinias per tuber or per g peel.     

Detection,quantification and classification of soft rot erwinias associated with potato tubers

An improved method for detection, quantification and classification of soft rot bacteria associated with potato seed tubers, plant material, soil and water has been developed. The method is based on the use of a modified version of the crystal violet pectate selective medium (CVP), enrichment cultures under anaerobic conditions using pectate as the sole carbon source for recovery improvement, and the quantitative estimation ofErwinia spp. by employing a new solid medium - most probable number (MPN) method. The use of this method enabled an improvement in the recovery and identification of specificErwinia spp. in mixed populations. This was done by incubating CVP plates — used for the MPN counting — at three different temperatures (15, 28 and 39°C). These combined techniques were used for estimating low level populations at less than one cell per gram or ml tested ofErwinia carotovora subsp.carotovora, E. carotovora subsp.atroseptica, andE. chrysanthemi.     

Methods for detection of the blackleg,ring rot and gangrene pathogens in potato nuclear stock mother tubers and plantlets1

Methods are described for the detection of latent infection by Erwinia carotovora ssp. atroseptica (blackleg and soft rot), Corynebacterium sepedonicum (ring rot) and Phoma exigua vat. foveata (gangrene) which are regarded as the most important tuber-borne pathogens of potato. The methods comprise selective media for E. carotovora ssp. atroseptica and P. exigua var. foveata, indirect fluorescence antibody staining for E. carotovora ssp. atroseptica and C. sepedonicum, and the eggplant test for C. sepedonicum and P. exigua var. foveata. These are used in Northern Ireland to detect pathogen contamination in nuclear stock mother tubers prior to micropropagation. Marked reductions in disease levels of blackleg have been observed in present pre-basic stocks raised by micropropagation compared with those previously propagated from stem cuttings. It was found that Pseudomonas stutzeri, a saprophytic contaminant bacterium, can pass through the micropropagation process in a latent form.     

Comparison of immunofluorescence colony-staining in media,selective isolation on pectate medium,ELISA and immunofluorescence cell staining for detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in cattle manure slurry

Various isolation and serological techniques were compared for the detection ofErwinia carotovora subsp.atroseptica (Eca) andE. chrysanthemi (Ech) in cattle manure slurry containing c. 10⁸ colony forming units (cfu) per ml. The slurry samples could be preserved at –80°C for 8 months without reduction in the number of bacteria but not at –20°C. Samples stored at –80°C were inoculated with concentrations of the target bacterium ranging from 10² to 10⁸ per ml. Only immunofluorescence colony-staining (IFC) in combination with selective media was able to detect the target organism at a concentration of 100 cells per ml. No IFC-positive colonies were found in pour plates of the non-inoculated cattle slurry. The recovery of the target bacterium from slurry inoculated with 10² cfu of Ech per ml was 64% in PT medium (containing polygalacturonic acid) and 19% in crystal violet pectate medium (CVP). Recoveries of Eca were 32% and 82%, respectively. Ech and Eca could be detected at levels of 10³ cfu per ml of slurry by isolation on CVP. Crude filtration procedures were necessary for analysis of slurry samples with immunosorbent immunofluorescence (ISIF) cell staining. The detection level of ISIF for Ech was 10⁵ cells per ml of slurry. IF-positive cells were incidentally observed in the non-inoculated slurry. Detection of Ech and Eca with ELISA was only possible in slurry inoculated with 10⁸ cells of the target bacterium per ml.Samenvatting Verschillende isolatie- en serologische methoden werden vergeleken om de doelbacteriënErwinia carotovora subsp.atroseptica (Eca) enE. chrysanthemi (Ech) aan te tonen in runderdrijfmest met een natuurlijke bacterieflora van ca 10⁸ kolonievormende eenheden(cfu) per ml. De mestmonsters konden gedurende de onderzoekperiode tenminste 8 maanden bij –80°C worden bewaard zonder dat er een afname van het aantal levende mestbacteriën werd geconstateerd, terwijl bij bewaring bij –20°C wel een afname werd gevonden. De ontdooide mestmonsters werden geïnoculeerd met de doelbacterie in concentraties tussen 10² en 10⁸ per ml. De laagste concentratie van de doelbacterie, 10² cfu per ml, kon alleen worden aangetoond met de immunofluorescentie-kleuring van bacteriekolonies (IFC) in een selectief medium. Met deze techniek was het percentage herisolatie vanuit drijfmest geïnoculeerd met 10² Ech cfu per ml respectievelijk 64% in PT-medium (bevat polygalacturonzuur) en 19% in kristalviolet pectine medium (CVP). Voor Eca bedroegen deze percentages respectievelijk 82% en 32%. In de niet-geïnoculeerde mestmonsters werden geen IFC-positieve kolonies gevonden. Via isolatie op CVP konden 10³ of meer cfu van Eca en Ech worden aangetoond. Ruwe filtratie van de mestmonsters was nodig voor het aantonen van Eca- en Ech-cellen met immunoadsorptie immunofluorescentie microscopie. De detectiedrempel lag voor deze techniek op 10⁵ bacteriecellen per ml mestmonster. In niet-geïnoculeerde mest werden incidenteel IF-positieve bacteriën gevonden. Het aantonen van Ech en Eca met ELISA was slechts mogelijk in mest geïnoculeerd met 10⁸ of meer cellen van de doelbacterie per ml.     

Moss-Erwinia pathosystem reveals possible similarities in pathogenesis and pathogen defense in vascular and nonvascular plants

Vascular plants have various inducible resistance mechanisms as defense against pathogens. Mosses, small nonvascular plants (subkingdom Bryophyta), have been little studied in regard to their pathogens or modes of defense. Data here show that Erwinia carotovora, a bacterial plant pathogen that causes softrot in many dicotyledonous plants, can also cause soft rot symptoms in the moss Physcomitrella patens. Infection of moss by E. carotovora required pathogenicity factors similar to those required to infect vascular plants and, again as in vascular plants, salicylic acid (SA) induced moss to inhibit tissue maceration by Erwinia. These data reveal that SA-dependent defense pathways may have evolved before differentiation of vascular and nonvascular plants.     

吉林省人参细菌性软腐病病原

对分离自吉林省主要参场的软腐病参根和厌气诱发产生软腐症状的参根的菌株进行了致病性测定、电镜形态观察和生理生化测定等。结果表明,造成吉林省人参细菌性软腐病的病原种类为:胡萝卜软腐欧文氏菌胡萝卜软腐亚种(Erwinia carotovora subsp, carotovora Dye)、胡萝卜软腐欧文氏菌黑胫亚种(Erwinia carotovora subsp, atroseptica Dye)和石竹假单胞菌[Pseudomonas caryophylli(Burkholder) Starr & Burkholder]。