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.     

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.     

Characterization of Monoclonal Antibodies Specific for Erwinia carotovora subsp. atroseptica and Comparison of Serological Methods for Its Sensitive Detection on Potato Tubers

Seven monoclonal antibodies (MAbs) to Erwinia carotovora subsp. atroseptica have been produced. One, called 4G4, reacted with high specificity for serogroup I of E. carotovora subsp. atroseptica, the most common serogroup on potato tubers in different serological assays. Eighty-six strains belonging to different E. carotovora subsp. atroseptica serogroups were assayed. Some strains of serogroup XXII also reacted positively. No cross-reactions were observed against other species of plant pathogenic bacteria or 162 saprophytic bacteria from potato tubers. Only one strain of E. chrysanthemi from potato cross-reacted. A comparison of several serological techniques to detect E. carotovora subsp. atroseptica on potato tubers was performed with MAb 4G4 or polyclonal antibodies. The organism was extracted directly from potato peels of artificially inoculated tubers by soaking or selective enrichment under anaerobiosis in a medium with polypectate. MAb 4G4 was able to detect specifically 240 E. carotovora subsp. atroseptica cells per ml by indirect immunofluorescence and immunofluorescence colony staining and after soaking by ELISA-DAS (double-antibody sandwich enzyme-linked immunosorbent assay) after enrichment. The same amount of cells was detected by using immunolectrotransfer with polyclonal antibodies, and E. carotovora subsp. atroseptica and subsp. carotovora were distinguished by the latter technique. ELISA-DAS using MAb 4G4 with an enrichment step also efficiently detected E. carotovora subsp. atroseptica in naturally infected tubers and plants.     

Erwinia soft rot resistance of potato cultivars expressing antimicrobial peptide tachyplesin I

Tachyplesin I is a 2.3 kDa antimicrobial peptide isolated from Southeast Asian horseshoe crabs. Bacterial suspensions containing 1×10⁶ colony-forming units/ml of six isolates of pectolytic Erwinia spp., the causal pathogens of potato soft rot and blackleg, were killed in vitro by 1.4 to 11.1 g/ml of tachyplesin I. In an attempt to enhance resistance to Erwinia spp., each of the potato cultivars Bintje, Karnico and Kondor were transformed with two gene constructs encoding different precursor tachyplesin I proteins under the control of a cauliflower mosaic virus 35S promotor. Northern and western blot analysis showed that the tachyplesin I gene was expressed in transgenic plants. Small tubers of 17 transgenic clones were screened twice for soft rot resistance to Erwinia carotovora ssp. atroseptica. Under aerobic or anaerobic conditions, transgenic clones showed slightly less rot than control tubers.Abbreviations AP acidic carboxyl terminal polypeptide - Eca Erwinia carotovora ssp. atroseptica - Ecc E. carotovora ssp. carotovora - Ech E. chrysanthemi - IF intercellular fluid - SP signal peptide - TPNI (tpnI) tachyplesin I     

Role of Antibiosis in Competition of Erwinia Strains in Potato Infection Courts

Erwinia carotovora subsp. betavasculorum strains produced a bactericidal antibiotic in vitro that inhibited a wide spectrum of gram-negative and gram-positive bacteria. The optimum temperature for production was 24°C, and the addition of glycerol to culture media enhanced antibiotic production. Antibiotic production by these strains in the infection court of potato was the principal determinant enabling it to gain ascendancy over competing antibiotic-sensitive Erwinia carotovora subsp. carotovora strains. There was a complete correlation between antibiotic production by E. carotovora subsp. betavasculorum in vitro and inhibition of competing E. carotovora subsp. carotovora strains in planta. Inhibition of the latter by the former was apparent after 10 h of incubation in potato tuber wounds. Population densities of sensitive E. carotovora subsp. carotovora strains in mixed potato tuber infections with E. carotovora subsp. betavasculorum were approximately 10⁶-fold lower after 48 h of incubation than in corresponding single sensitive strain infections. E. carotovora subsp. carotovora were not inhibited in tuber infections that were incubated anaerobically. This correlated with the absence of antibiotic production during anaerobic incubation in vitro. Antibiotic-resistant strains of E. carotovora subsp. carotovora were not inhibited in planta or in vitro by E. carotovora subsp. betavasculorum. Moreover, isogenic antibiotic-negative (Ant⁻) mutant E. carotovora subsp. betavasculorum strains were not inhibitory to sensitive E. carotovora subsp. carotovora strains in tuber infections.     

The extent and survival of contamination of potato stocks in Scotland by Erwinia carotovora var. carotovora and E. carotovora var.atroseptica*

The following results were obtained when fifty-seven bulk and crate-stored commercial seed potato stocks from the East of Scotland were examined in 1966-8 for contamination by pectolytic Erwinia spp. (1) Most tubers of all the cultivars and stocks examined, irrespective of whether they were obtained from blackleg-infected or blackleg-free crops, were contaminated with E. carotovora; (2) some 80% of the Erwinia isolates obtained were identified as var. atroseptica, the rest being var. carotovora; (3) the organisms survived in and on tubers for 6–7 months of bulk storage over the winter and up to planting time the following spring; (4) contrary to what is generally thought, the high incidence of contamination of all stocks, while suggesting that the seed itself is the major source of E. carotovora for the growing crop, emphasizes that other factors affect manifestation of blackleg in the field and soft rot in store.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

Occurrence of bacteriophage T4 receptor in Erwinia carotovora

Summary We have tested for the presence of the receptor for the Escherichia coli phage T4 in different isolates of the plant pathogenic enterobacteria Erwinia carotora subsp. carotovora and subsp. atroseptica. Several of the isolates appeared to contain a functional T4 receptor as shown by phage adsorption and phage-induced lysis of the bacteria. Two of the isolates could even sustain lytic growth of T4. In addition, we show that the transducing derivative of T4, T4GT7, can be employed to transfer plasmids from E. coli to E. carotovora thus opening up new possibilities for genetic analysis of Erwinia.     

Bacterial soft rot of tomato in plastic greenhouses in Crete

During recent years a new disease has been noticed on tomatoes grown in Polythene greenhouses in Crete. Early symptoms are yellowing of the lower leaves, and a yellow brown discoloration of the pith and stem xylem. As leaves wilt and die there is progressive yellowing towards the top of the plants. A progressive disintegration of the cortical tissues follows which results in a soft rot and a longitudinal splitting of the stem running mainly upwards. Soft rot of the fruits rarely appears. Severely infected plants may wilt and die, but other less affected plants often survive and yield normally. Very vigorous plants grown under humid conditions are more susceptible. Often more than 20% of the plants are infected. Isolations were made from stem (xylem, cortex and pith), from leaf xylem and from fruits of infected tomato plants collected throughout the island from 1979 to 1985. Bacteria of the genus Erwinia and Pseudomonas were consistently isolated. On the basis of physiological and biochemical characters of 49 representative pathogenic isolates, 22 were identified as Erwinia carotovora subsp. carotovora, 10 as Erwinia carotovora subsp. atroseptica, four as Pseudomonas viridiflava and 13 as Pseudomonas fluorescens biotype I. All disease symptoms were reproduced when artificial inoculations were made with the above isolates in the laboratory (20°C and 100% r.h.) on 3–4 week tomato plants and in a commercial greenhouse on 4–5 months tomato plants. Bacteria used for inoculations were reisolated. Results indicated that the disease symptoms as described may be caused by four different bacteria species.     

Response of Endophytic Bacterial Communities in Potato Plants to Infection with Erwinia carotovora subsp. atroseptica

The term endophyte refers to interior colonization of plants by microorganisms that do not have pathogenic effects on their hosts, and various endophytes have been found to play important roles in plant vitality. In this study, cultivation-independent terminal restriction fragment length polymorphism analysis of 16S ribosomal DNA directly amplified from plant tissue DNA was used in combination with molecular characterization of isolates to examine the influence of plant stress, achieved by infection with the blackleg pathogen Erwinia carotovora subsp. atroseptica, on the endophytic population in two different potato varieties. Community analysis clearly demonstrated increased bacterial diversity in infected plants compared to that in control plants. The results also indicated that the pathogen stress had a greater impact on the bacteria population than the plant genotype had. Partial sequencing of the 16S rRNA genes of isolated endophytes revealed a broad phylogenetic spectrum of bacteria, including members of the α, β, and γ subgroups of the Proteobacteria, high- and low-G+C-content gram-positive organisms, and microbes belonging to the Flexibacter-Cytophaga-Bacteroides group. Screening of the isolates for antagonistic activity against E. carotovora subsp. atroseptica revealed that 38% of the endophytes protected tissue culture plants from blackleg disease.     

Biochemical and physiological characterisation of Erwinia species causing tip-over disease of banana

Tip-over disease has become a serious threat to banana plantations in the past decade. The disease is reported to be caused by Erwinia carotovorasubsp. carotovora and Erwinia chrysanthemi. We compared nine Erwinia strains of diseased banana plants from different agroclimatic zones of Karnataka and Andhra Pradesh, Southern India by conventional means. On the basis of morphological, cultural, physiological and biochemical characteristics and pathogenicity tests, the seven isolates I₁ to I₆ and I₈ showed similarities to Erwinia carotovorasubsp. carotovora. Isolate I₉ from Andhra Pradesh expressed characteristics similar to that of Erwinia chrysanthemi and was identified as Erwinia chrysanthemi. The isolate I₇ which showed wider variation, neither confirmed to the characteristics of Erwinia carotovorasubsp. carotovora nor with that of Erwinia chrysanthemi, and possessed characteristics in between the two species. Further we studied the host range of the bacterium causing tip-over disease of banana.     

Derivation of Mutants of Erwinia carotovora subsp. betavasculorum Deficient in Export of Pectolytic Enzymes with Potential for Biological Control of Potato Soft Rot

Erwinia carotovora subsp. betavasculorum Ecb168 produces an antibiotic(s) that suppresses growth of the related bacterium Erwinia carotovora subsp. carotovora in culture and in wounds of potato tubers. Strain Ecb168 also produces and secretes pectolytic enzymes and causes a vascular necrosis and root rot of sugar beet. Genes (out) involved in secretion of pectolytic enzymes by Ecb168 were localized to two HindIII fragments (8.5 and 10.5 kb) of Ecb168 genomic DNA by hybridization to the cloned out region of E. carotovora subsp. carotovora and by complementation of Out^- mutants of E. carotovora subsp. carotovora. Out^- mutants of Ecb168, which did not secrete pectate lyase into the culture medium, were obtained when deletions internal to either HindIII fragment were introduced into the genome of Ecb168 through marker exchange mutagenesis. Out^- mutants of Ecb168 were complemented to the Out⁺ phenotype by introduction of the corresponding cloned HindIII fragment. Out^- mutants of Ecb168 were less virulent than the Out⁺ parental strain on potato tubers. Strain Ecb168 and Out^- derivatives inhibited the growth of E. carotovora subsp. carotovora in culture, indicating that the uncharacterized antibiotic(s) responsible for antagonism was exported through an out-independent mechanism. Strain Ecb168 and Out^- derivatives reduced the establishment of large populations of E. carotovora subsp. carotovora in wounds of potato tubers and suppressed tuber soft rot caused by E. carotovora subsp. carotovora.     

Members of the amylovora group of Erwinia are cellulolytic and possess genes homologous to the type II secretion pathway

A cellulase-producing clone was isolated from a genomic library of the Erwinia rhapontici (Millard) Burkholder strain NCPPB2989. The corresponding gene, named celA, encodes an endoglucanase (EC?3.2.1.4) with the extremely low pH optimum of 3.4 and a temperature optimum between 40 and 50?°C. A single ORF of 999?nt was found to be responsible for the Cel activity. The corresponding protein, named CelA, showed 67% identity to the endoglucanase Y of E. chrysanthemi and 51.5% identity to the endoglucanase of Cellulomonas uda, and thus belongs to the glycosyl hydrolase family?8. The celA gene, or its homologue, was found to be present in all E. rhapontici isolates analysed, in E. chrysanthemi, and in E. amylovora. The presence of plant cell wall-degrading enzymes in the amylovora group of Erwinia spp. had not previously been established. Furthermore, the DNA of both E. rhapontici and E. amylovora was found to exhibit homology to genes encoding the type?II (GSP) secretion pathway, which is known to be responsible for extracellular targeting of cellulases and pectinases in Erwinia spp. that cause soft rotting, such as E. carotovora and E. chrysanthemi. Secretion of the CelA protein by E. rhapontici could not be verified. However, the CelA protein itself was found to include the information necessary for heterologous secretion by E. chrysanthemi.     

A Note on the Limitations of Identifying Soft Rot Erwinias by Temperature Tolerances and Sensitivity to Erythromycin on a Pectate Medium

Forty-five erwinia strains from 14 different hosts have been tested lor growth on a crystal violet pectate medium with or without 70 μ/ml erythromycin at 27, 33.5 and 37°C. On the basis of positive data in the literature it was tried to identify the strains as Erwnia carotovora subsp. atroseptica, E. carotovora subsp. carotovora or E. chrysanthemi using this method. It appeared that relatively few strains reacted differentially, apparently due to strain variation (all strains were identified also by biochemical and physiological tests). The conclusion is that for a reliable identification of field isolates of soft rot crwinias other (biochemical, physiological and serological) methods have to be applied.     

Formation of the blue pigment indigoidine by phytopathogenic erwinia

Most cultures belonging to the “Erwinia chrysanthemi group” of soft-rot bacteria form traces of a blue, extracellular, water-insoluble pigment. This pigment is generally not found in cultures of the other members of the genus Erwinia. The blue substance has been isolated and purified from three members of the E. chrysanthemi group; it has been identified as indigoidine, 5,5′-diamino-4,4′-dihydroxy-3,3′-diazadiphenoquinone-(2,2′).     

Genetic Regulation of Pathogenicity and Virulence Factors in Bacteria Erwinia carotovora subsp. atroseptica: Identification of Gene kduD

A mutant that cannot utilize pectin substances of plant cell walls was obtained via insertion of mini-Tn5xylE transposon into the chromosome of phytopathogenic bacteria Erwinia carotovora subsp. atroseptica. the inability of mutant cells to utilize these substrates was caused by a failure to accomplish the catabolism of unsaturated digalacturonic acid (UDA). Study of enzymatic activities has established that mutant bacteria lost the ability to produce 2,5-diketo-3-deoxygluconate dehydrogenase, an enzyme of intracellular UDA utilization. Molecular cloning of the mutant gene was conducted, and its nucleotide sequence was determined. It was shown that the nucleotide sequence of this gene had an 82% homology with the sequence of Erwinia chrysanthemi EC3937 kduD gene encoding 2,5-diketo-3-deoxygluconate dehydrogenase. The intergene kduI–kduD region in bacteria Erwinia carotovora subsp. atroseptica is shorter in length by 98 nucleotides than the corresponding region of Erwinia chrysanthemi and does not contain promoter sequences. The kduD gene was located at 126.8 min of the Erwinia carotovora subsp. atroseptica genetic map.     

Biosynthetic features and properties of xylose isomerases from Arthrobacter nicotianae, Escherichia coli, and Erwinia carotovora subsp. atroseptica

The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovora subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria produced the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovora subsp. atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the D-xylose-containing nutrient medium of 0.1–1.5% D-glucose did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovora subsp. atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.     

Effects of fluorescent pseudomonads on the potato blackleg syndrome

In four field trials in 1983 and 1984 potato tubers were inoculated by dipping them at planting in a suspension of Erwinia carotovora subsp. atroseptica and then treated with a powder formulation of two strains of fluorescent pseudomonads (B 10 and I 13) isolated from potatoes. lnoculation with E. carotovora increased blackleg and reduced emergence, plant growth, tuber size and weight compared with uninoculated controls. These effects were partially reversed by treatment of tubers with fluorescent pseudomonads which also reduced contamination by E. carotovora and the soft-rot potential of progeny tubers. In some trials a mixture of both pseudomonad isolates delayed the breakdown of the mother tuber although individual treatments did not.