Blackleg potential of potato seed: determination of tuber contamination by Erwinia carotovora subsp. Atroseptica by immunofluorescence colony staining and stock and tuber sampling

Two methods to determine numbers of the blackleg pathogen, Erwinia carotovora subsp. atroseptica, in tuber peel extract were compared; (1) growth and cavity formation on crystal violet pectate (CVP) medium (Pérombelon, Lumb & Hyman, 1987) and (2) immunofluorescent colony (IFC) staining (Van Vuurde & Roozen, 1990) using an antiserum against the bacterium conjugated with fluorescein isothiocyanate. Detection, identification and quantification of the bacterium based on the differential effect of temperature on growth in the CVP method were severely restricted and in some cases could not be done at low peel extract dilutions containing > 106 saprophytic bacteria ml“1 and > 103 cells ml-1 of E. carotovora subsp. carotovora. In contrast, although recovery was c. 50% relative to growth of E.c. atroseptica alone on nutrient agar, numbers of the bacteria could be determined by the IFC method regardless of numbers of saprophytic bacteria and E.c. carotovora present. Moreover, the tedium of counting colonies on a UV microscope could be avoided by automation using an imaging system on photograph film negatives of the microscope fields. Readily accessible tubers from the top layer of one tonne boxes in commercial stores were c. 10 times less contaminated than those from the middle of the boxes. For the two methods of peel extract preparation examined, the estimated sample size needed with an allowable error of log1010 E.c. atroseptica cells ml“1 extract with 95% confidence, was c. five tubers per box and 14 boxes for extract prepared from individual tubers and c. three lots of 10 tubers per box and 10 boxes for extract from 10 pooled tubers. A blackleg potential index for seed stocks was proposed based on the summation of the weighted number of individually tested tubers in different classes of contamination level.     

A competitive PCR-based method for the detection and quantification of Erwinia carotovora subsp. atroseptica On potato tubers

A PCR-based method was developed for the simultaneous detection and quantification of the potato pathogen Erwinia carotovora subsp. atroseptica (Eca) on potato tubers. The method incorporates a competitor PCR template cloned into Escherichia coli in vector pGEM-T (E. coli 4R l/l). Predetermined numbers of E. coli 4R were added to potato peel extract, either pre-inoculated with Eca or from naturally contaminated tubers, and Eca numbers estimated by comparing the ratio of products generated from Eca target DNA and competitor template DNA following PCR. Estimates of Eca numbers were consistent with counts obtained on crystal violet pectate medium and immunofluorescence colony staining. Unlike these methods, however, the PCR-based method is not affected by the presence of other erwinias and saprophytes and is able to detect all serogroups of Eca. Based on this method, a key was produced relating product ratios, obtained following PCR from contaminated tuber stocks, to the likelihood of blackleg disease incidence. This is the first quantitative PCR-based detection method described for Eca and is the first for any bacterial plant pathogen to incorporate a DNA extraction control.     

Comparisons between Phytoalexin Concentrations and the Extent of Rotting of Potato Tubers Inoculated with Erwinia carotovora sub sp. atroseptica, E. carotovora sub sp. carotovora or E. chrysanthemi

Isolates of Erwinia carotovora sub sp. atroseptica, E. carotovora sub sp. carotovora and E. chrysanthemi were compared for their ability to rot potato tubers and to elicit the accumulation of phytoalexins. With three incubation temperatures (15, 22 and 30°C) tubers were more susceptible at higher temperatures and mean rot weights were inversely correlated with mean rishitin concentrations. However, within treatment variation between tubers in the extent of rotting was not correlated with rishitin concentration suggesting that other factor(s) also affect resistance. Phytoalexins, including 15-dihydrolubimin, 3-hydroxylubimin, phytuberin and solavetivone, were measured by high pressure liquid chromatography using a silica column.     

Bacteriocin-like substance inhibits potato soft rot caused by Erwinia carotovora

Soft rot is a major problem encountered in potatoes during postharvest storage. The soft rot bacterium Erwinia carotovora was inhibited by a novel bacteriocin-like substance (BLS) produced by Bacillus licheniformis P40. The BLS caused a bactericidal effect on E. carotovora cells at 30 microg mL(-1). Transmission electron microscopy showed that BLS-treated cells presented wrinkled bacterial surfaces and shrinkage of the whole cell, indicating plasmolysis. Erwinia carotovora cells treated with BLS were analyzed by FTIR showing differences in the 1390 cm(-1) and 1250-1220 cm(-1) bands, corresponding to assignments of membrane lipids. BLS was effective in preventing E. carotovora spoilage on potato tubers, reducing the symptoms of soft rot at 240 microg mL(-1) and higher concentrations. Soft rot development was completely blocked at 3.7 mg mL(-1). This BLS showed potential to protect potato tubers during storage.     

Soft rot Erwinia bacteria in surface and underground waters in southern Scotland and in Colorado, United States

An anaerobic liquid enrichment method followed by plating on a selective medium revealed that the soft rot coliform bacterium Erwinia carotovora subsp. carotovora was generally present in water from drains, ditches, streams, rivers and lakes (including reservoirs) in southern Scotland and in Colorado, United States, in mountainous, upland and arable areas through the year. Many sites were remote from susceptible or diseased crops. Erwinia carotovora subsp. atroseptica was isolated much less frequently and no Erwinia bacteria were isolated from underground waters. Erwinia bacteria were also found in rain-water in Scotland, in winter snow from mountain passes in Colorado, and in sea water from the west and east coasts of Scotland and from the coasts of Oregon, California, Texas, Louisiana and Florida. The significance of the occurrence of these bacteria in water is discussed in relation to the control of blackleg and soft rot diseases of potato by production of Erwinia -free stocks.     

Soft rot Erwinia bacteria in the rhizosphere of weeds and crop plants in Colorado, United States and Scotland

The soft rot coliform bacteria Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica were isolated by an enrichment method from the rhizosphere of many weed species and crop plants, collected in commercial potato fields either currently in potatoes or in a different crop as part of the rotation. Erwinia carotovora was isolated from 24 plant species in Colorado and 47 species in Scotland. Weeds contaminated with E. carotovora were found in fields growing other crops in which potatoes had not been grown for 1–2 and sometimes much longer. Weeds collected from virgin land in Colorado were not contaminated with E. carotovora but in Scotland virgin soils containing weed roots yielded E. carotovora subsp. carotovora . In general, the numbers of contaminated weeds rose from nil or low levels in spring and early summer to considerably higher levels during mid-season, and fell to progressively lower levels later. Erwinia carotovora subsp. carotovora was the predominant organism recovered from the rhizosphere, but E. carotovora subsp. atroseptica was less common, especially in Scotland, and its incidence varied in different seasons depending on factors such as temperature and moisture conditions. The bacteria could apparently persist in the root zone for an extended period of time and may be a source of inoculum to contaminate soft rot erwinia-free seed potato stocks; the origin of the bacteria was uncertain.     

The ineffectuality of potato protease inhibitor on the extracellular protease from Erwinia carotovora subsp. carotovora

Growth conditions are described for optimum production of extracellular protease in batch cultures of the soft rot pathogen Erwinia carotovora subsp. carotovora . This protease was inhibited by approximately 96% by 1 mmol/1 EDTA and by 55–6% by 10 mmol/l cysteine thereby classifying it as a metalloprotease. It was not inhibited by a chymotrypsin inhibitor extracted from potato tubers. This evidence suggests that the potato chymotrypsin inhibitor is not associated with resistance of potatoes to E. carotovora subsp. carotovora .     

Cloning and characterization of a pectate lyase gene from Erwinia carotovora EC153

A pel gene cloned from strain EC153 of Erwinia carotovora encoded a pectate lyase that macerated plant tissue with moderate efficiency. This gene, called pel153, was sequenced and found to possess considerable homology with a pectate lyase gene from Yersinia pseudotuberculosis. The Yersinia protein, however, was truncated at the carboxyl terminal end relative to the Erwinia gene product and had a lower isoelectric point. The Erwinia pel153 gene was overexpressed in cells of Escherichia coli, and a 56-kDa protein was observed on sodium dodecyl sulfate-polyacrylamide gels. This compares with a molecular weight of 61 kDa for the mature, secreted protein as determined from sequencing data. Southern blot analysis disclosed the presence of the pel153 gene in three different strains of E. carotovora, but mutation of the gene in strain EC153 did not affect its ability to soft-rot potato tubers.     

PCR and restriction fragment length polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato diseases.

Using a sequenced pectate lyase-encoding gene (pel gene), we developed a PCR test for Erwinia carotovora. A set of primers allowed the amplification of a 434-bp fragment in E. carotovora strains. Among the 89 E. carotovora strains tested, only the Erwinia carotovora subsp. betavasculorum strains were not detected. A restriction fragment length polymorphism (RFLP) study was undertaken on the amplified fragment with seven endonucleases. The Sau3AI digestion pattern specifically identified the Erwinia carotovora subsp. atroseptica strains, and the whole set of data identified the Erwinia carotovora subsp. wasabiae strains. However, Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. odorifera could not be separated. Phenetic and phylogenic analyses of RFLP results showed E. carotovora subsp. atroseptica as a homogeneous group while E. carotovora subsp. carotovora and E. carotovora subsp. odorifera strains exhibited a genetic diversity that may result from a nonmonophyletic origin. The use of RFLP on amplified fragments in epidemiology and for diagnosis is discussed.     

Rapid identification and differentiation of the soft rot erwinias by 16S-23S intergenic transcribed spacer-PCR and restriction fragment length polymorphism analyses

Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from Erwinia and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III). Group I comprised all Erwinia carotovora subsp. atroseptica and subsp. betavasculorum isolates. Group II comprised all E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and E. cacticida isolates, and group III comprised all E. chrysanthemi isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp. atroseptica and subsp. betavasculorum (group I) and E. chrysanthemi (group III) isolates, while digestion with RsaI identified E. carotovora subsp. wasabiae, subsp. carotovora, and subsp. odorifera/carotovora and E. cacticida isolates (group II). In the latter case, it was necessary to distinguish E. carotovora subsp. odorifera and subsp. carotovora using the alpha-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as E. carotovora subsp. atroseptica, E. chrysanthemi, E. carotovora subsp. carotovora, and non-soft rot species. Ten "atypical" E. carotovora subsp. atroseptica isolates were identified as E. carotovora subsp. atroseptica, subsp. carotovora, and subsp. betavasculorum and non-soft rot species, and two "atypical" E. carotovora subsp. carotovora isolates were identified as E. carotovora subsp. carotovora and subsp. atroseptica.     

Quantitative Studies on the Generation of Aerosols of Erwinia carotovora var. atroseptica by Simulated Raindrop Impaction on Blackleg-infected Potato Stems

Quantitative studies on the generation of bacterial aerosols by simulated raindrop impaction on potato stems infected with the blackleg organism Erwinia carotovora var. atrosep:ica have shown that, theoretically, airborne transmission might result in re-contamination of blackleg-free potato stocks.     

Affinity purification of a siderophore that exhibits an antagonistic effect against soft rot bacterium

Bacterial colonies were isolated from different Egyptian soil samples. From these isolates, one bacterial species was found to produce siderophore. Using classical and biochemical identification methods, the siderophore producing isolate was identified as Pseudomonas fluorescens. Based on the affinity of siderophores for metal ions, an affinity chromatography system was designed for the purification of the siderophore in one step. It was possible to isolate 25 mg siderophore per liter of culture media. The purified siderophore was found to exist in two forms of approximately 30 and 90 kD. They are believed to be polymers of several siderophore molecules. Both forms were found to be active against the pathogen Erwinia carotovora var. carotovora, the causal bacteria of soft rot disease on potato tubers. The advantage of this method over other purification methods is that it uses metal ion so it can be applied for the purification of the known types of siderophores. Moreover, the purification is based on affinity chromatography, so the siderophore purity state permits several biotechnological applications without further treatments.     

A Quantal Method for Determining Numbers of Erwinia carotovora var. carotovora and E. carotovora var. atroseptica in Soils and Plant Material

SUMMARY: A quantal method is described, based on the maximum likelihood method, which was found especially suitable for quantitatively estimating small populations of Erwinia carotovora in soils and plant material. Drops from serial dilutions of the test material are placed on replicated defined areas of a solid, semiselective medium with a pectate overlayer. The presence or absence of one or more of the characteristic, deep cavities formed by E. carotovora colonies in the pectate layer is the basis of the quantal count from which estimates of the population size can be derived.     

Enumeration of two competing Erwinia carotovora populations in potato tubers by a membrane filter-immunofluorescence procedure

Relative increases in cell populations of specific Erwinia carotovora strains injected into potato tubers either singly or in combination of two strains were determined by depositing tissue extracts on membrane filters and staining cells of individual strains by immunofluorescence. The population increase of an Erw. carotovora subsp. atroseptica (Eca) strain was, in general, not affected by the presence of an Erw. carotovora subsp. carotovora (Ecc) strain. However, the increase of an Ecc strain was inhibited by the presence of an Eca strain, especially at incubation temperatures of 5° and 15°C but not at 26°C. One Ecc strain consistently increased in population over another Ecc strain at a greater rate when they were inoculated together at the same loci in comparison to when they were inoculated separately at different loci.     

The development of a conductimetric assay for automated detection of metabolically active soft rot Erwinia spp. in potato tuber peel extracts

Four media were tested for their ability to detect the soft rot potato pathogens Erwinia chrysanthemi (Ech) and Erwinia carotovora ssp. atroseptica (Eca) in potato tubers by means of automated conductance measurements. The specificity of the conductimetric assays was determined by testing a set of different Erwinia spp. and potato-associated saprophytes, including the genera Pseudomonas, Bacillus, Enterobacter and Flavobacterium. All bacteria tested produced conductance responses in Special Peptone Yeast Extract, whereas in minimal medium with L-asparagine only Erwinia spp. and Pseudomonas spp. were able to generate large conductance responses. In minimal medium supplemented with glucose and trimethylamine- N -oxide only Enterobacteriaceae, Erwinia spp. included, generated conductance responses, while with pectate as sole carbon source only Erwinia spp. produced distinct conductance responses. The pectate medium proved to be particularly useful for specific automated conductimetric detection of Erwinia spp. in potato peel extracts. Within 48 h, the detection threshold of the conductimetric assay for Eca varied between 10² and 10³ cfu per ml peel extract at both incubation temperatures of 20° and 26°C. Ech was detected at concentrations of 10⁴–10⁵ or 10³–10⁴ cfu ml^-1 at 20° and 26°C, respectively. To eliminate 'false'-positive reactions in conductimetry caused by Erwinia carotovora ssp. carotovora , results of the conductance measurements have to be confirmed by other techniques, like serology or DNA assays.     

Application of amplified fragment length polymorphism fingerprinting for taxonomy and identification of the soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi

The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted. Cluster 1 contained Erwinia carotovora subsp. carotovora (subclusters 1a and 1b) and Erwinia carotovora subsp. odorifera (subcluster 1c) strains, while cluster 2 contained Erwinia carotovora subsp. atroseptica (subcluster 2a) and Erwinia carotovora subsp. betavasculorum (subcluster 2b) strains. Clusters 3 and 4 contained Erwinia carotovora subsp. wasabiae and E. chrysanthemi strains, respectively. While E. carotovora subsp. carotovora and E. chrysanthemi showed a high level of molecular diversity (23 to 38% mean similarity), E. carotovora subsp. odorifera, E. carotovora subsp. betavasculorum, E. carotovora subsp. atroseptica, and E. carotovora subsp. wasabiae showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges. The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics. AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.     

Biological properties of some nitrogen-fixing associative enterobacteria

Enterobacteria isolates from potato tubers were able to fix nitrogen, to protect plants against phytopathogens and to produce phytohormones thus increasing the plant yield. These isolates were previously phenotypically identified as Erwinia carotovora; however, they differed from typical E. carotovora in a number of biological characteristics and were found to be nonphytopathogenic (avirulent) due to the lack of pectate lyase activity. A data matrix, containing 31 strains and 105 biological characteristics was used for computer cluster analysis. The avirulent strains formed a separate cluster more closely related to Klebsiella spp. strains (with a 0.67 level of similarity) than to typical phytopathogenic bacteria of the E. carotovora group (with a 0.48 level of similarity). A phylogenetic analysis based on restriction polymorphisms of an amplified ribosomal DNA spacer region revealed that the avirulent strains studied here were different from all Erwinia, Klebsiella and other enterobacteria species strains. The AP PCR/hybridization technique showed cross homology of amplified DNA of these avirulent strains and a lack of such homology with the DNA from strains of other species. Numerical taxonomy data, rDNA analysis and AP PCR/hybridization assays confirmed that these avirulent bacteria may be regarded as an independent group of enterobacteria.     

Soft Rot Erwinia Bacteria in the Atmospheric Bacterial Aerosol

Using a Casella High Volume Airborne Bacteria Sampler, the soft rot coliform bacteria Erwinia carotovora var. carotovora and E. carotovora var. atroseptica were caught from the open air during rainfall in mid to late summer, autumn and early winter, but not in late winter, spring or early summer. They were not found every time it rained, and never when the weather was dry. The bacteria were caught close to potato crops, but there were several occasions when they were found at sites where there were no such crops or at times of year after potato crops had been harvested. The sources of the organisms are uncertain, but those obtained close to potato crops may have originated there. The results are considered to provide further evidence that airborne spread and subsequent deposition of viable bacteria could cause contamination of Erwinia -free potato stocks, and suggest that rainfall is a major generator of the general atmospheric bacterial aerosol.     

魔芋软腐病菌分子鉴定与遗传多样性

通过对分离的魔芋软腐病菌株和其它参试菌株的致病性测定、选择性培养基培养性状观察和16S-23S rDNA转录间隔区PCR(ITS-PCR)分析,将测试的33株软腐病菌株主要分为3个组群。第1组群为胡萝卜软腐欧文氏杆菌胡萝卜软腐亚种(Erwinia carotovorasubsp.carotovora,E.c.c.);第2组群为菊欧文氏杆菌(Erwinia chrysanthemi,E.ch.);还有一组未能确定的菌株。利用细菌基因组重复序列通用引物BOX和J3进行Rep-PCR特异性扩增,引起软腐病的菌株E.c.c.和E.ch.(ITS-PCR鉴定)种内的Rep-PCR指纹存在明显的遗传分化,经聚类分析,在0.1水平上把E.c.c.13株区分为5个类群。     

Novel quorum-sensing-controlled genes in Erwinia carotovora subsp. carotovora: identification of a fungal elicitor homologue in a soft-rotting bacterium

Seven new genes controlled by the quorum-sensing signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) have been identified in Erwinia carotovora subsp. carotovora. Using TnphoA as a mutagen, we enriched for mutants defective in proteins that could play a role in the interaction between E. carotovora subsp. carotovora and its plant hosts, and identified NipEcc and its counterpart in E. carotovora subsp. atroseptica. These are members of a growing family of proteins related to Nep1 from Fusarium oxysporum which can induce necrotic responses in a variety of dicotyledonous plants. NipEcc produced necrosis in tobacco, NipEca affected potato stem rot, and both affected virulence in potato tubers. In E. carotovora subsp. carotovora, nip was shown to be subject to weak repression by the LuxR family regulator, EccR, and may be regulated by the negative global regulator RsmA.