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.     

Serological and biochemical variation among potato strains of Erwinia carotovora subsp. atroseptica and their taxonomic relationship to other E. carotovora strains

The serological and biochemical characteristics of 32 Erwinia carotovora subsp. atroseptica strains from potato were compared with 48 other pectolytic Erwinia strains. Biochemical characteristics were examined by the API 20E and API 50CHE systems. Numerical analysis using the Euclidean distance coefficients and clustering by the unweighted average pair group method indicated that these E. carotovora subsp. atroseptica strains formed a distinct cluster (subphenon A₁) that could be differentiated from other E. carotovora strains. Three non-potato strains also belonged to this group; two of these were from tomato and the other from Chinese cabbage. Named E. carotovora subsp. atroseptica strains from other hosts clustered into other phenons. Sixty-three per cent of subphenon A₁ strains tested in this study typed into serogroup I. One potato strain in another phenon also typed into this serogroup. The subphenon A₁ strains that did not type into serogroup I typed into serogroups XVIII, XX, or XXII. Many of these strains, however, expressed several different O antigens which were also expressed by E. carotovora strains in other phenons.     

Presence and population dynamics of Erwinia carotovora in irrigation water in south central Colorado

The presence of Erwinia carotovora in surface and underground (well) water was studied using filter concentration and anaerobic enrichment techniques. The organism was found in water samples collected at sites in mountainous (over 80 km from potato-producing regions), transitional (upland) and arable regions every month in 1982 and 1983. Filter concentration and anaerobic enrichment of 3-10 1 of water yielded E. carotovora from 82.8% of the water samples collected from streams, canals and lakes. The organism was detected by direct enrichment of 50 ml water samples in 56.3% of surface water samples collected. Erwinia carotovora subsp. carotovora was the predominant subspecies isolated. Of 1029 strains, 999 (97.1%) were identified as E. carotovora subsp. carotovora and 30 (2.9%) as E. carotovora subsp. atroseptica. Erwinia carotovora subsp. atroseptica was found primarily in water samples collected in arable regions during spring months. Erwinia chrysanthemi was never isolated. Quantitative bacteriological methods were used in 1982 and 1983 to monitor populations of E. carotovora in two streams in south central Colorado. These ranged from undetectable levels to 8.5 cfu/ml of water in Rio Grande River and Saguache Creek. Maximum populations were usually reached by August or September in both streams in both years. Erwinia carotovora was isolated from well water samples collected in the San Luis Valley, but only 15.6 and 15.4% of the samples yielded the organism during 1982 and 1983, respectively. Erwinia carotovora subsp. atroseptica was found only once, and E. carotovora subsp. carotovora was the predominant subspecies detected. Filter concentration of 3.4-10.0 1 of water plus anaerobic enrichment of the samples was usually necessary to detect E. carotovora in well water.     

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.     

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora.

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

Rapid detection and identification of Erwinia carotovora subsp. atroseptica by a conjugated Staphylococcus aureus slide agglutination test

A. MCLEOD AND M.C.M. PEROMBELON. 1992. A conjugated Staphylococcus aureus slide agglutination test was used to detect and identify the potato blackleg pathogen, Erwinia carotovora subsp. atroseptica. Agglutination was obtained with > 10⁸ cfu/ml of the homologous strain with a polyclonal antiserum (171) against E.c. atroseptica serogroup I which is the predominant E.c. atroseptica serogroup on potatoes in Scotland. The titre of antiserum 171 against live cells of E.c. atroseptica groups I and XXII was 2000 whereas that of other serogroups was considerably less; only 1 and 4 out of 22 serogroups of E. carotovora subsp. carotovora reacted at 1:1500 and 1:1000 antiserum dilutions, respectively and one of the three less common other E.c. atroseptica serogroups reacted at 1:1000. When tested against 24 different bacterial species including E. chrysanthemi and saprophytic bacteria present in potato tuber rots, negative results were obtained with 1:1000 antiserum dilution. The titre against heat-treated (1 h, 70°C) cells of E.c. atroseptica serogroups I and XXII was1700–2000 whereas it was < 10 against other bacteria including E.c. carotovora. Detection of E.c. atroseptica serogroups I and XXII in diseased potato tissues was achieved directly by the slide agglutination test, but lower antiserum dilutions (1:700–1000) were needed. Still lower antiserum dilutions were needed with heat-treated test material for E.c. atroseptica identification.     

Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica.

Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed.     

Characterization of transposon insertion out- mutants of Erwinia carotovora subsp. carotovora defective in enzyme export and of a DNA segment that complements out mutations in E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi.

Soft-rotting Erwinia spp. export degradative enzymes to the cell exterior (Out+), a process contributing to their ability to macerate plant tissues. Transposon (Tn5, Tn10, Tn10-lacZ) insertion Out- mutants were obtained in Erwinia carotovora subsp. carotovora 71 by using plasmid and bacteriophage lambda delivery systems. In these mutants, pectate lyases, polygalacturonase, and cellulase, which are normally excreted into the growth medium, accumulated in the periplasm. However, localization of the extracellular protease was not affected. The Out- mutants were impaired in their ability to macerate potato tuber tissue. Out+ clones were identified in a cosmid library of E. carotovora subsp. carotovora 71 by their ability to complement mutants. Localization of cyclic phosphodiesterase in the periplasm indicated that the Out+ plasmids did not cause lysis or a nonspecific protein release. The Out+ derivatives of the E. carotovora subsp. carotovora 71 mutants regained the ability to macerate potato tuber tissue. Our data indicate that a cluster of several genes is required for the Out+ phenotype. While one plasmid, pAKC260, restored the Out+ phenotype in each of the 31 mutants of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi, it failed to render Escherichia coli export proficient. Homologs of E. carotovora subsp. carotovora 71 out DNA were detected by Southern hybridizations in subspecies of E. carotovora under high-stringency conditions. In contrast, E. chrysanthemi sequences bearing homology to the E. carotovora subsp. carotovora 71 out DNA were detectable only under low-stringency hybridization. Thus, although the out genes are functional in these two soft-rotting bacterial groups, the genes appear to have diverged.     

Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica

Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)     

Verification of ELISA results by immunomagnetic isolation of antigens from extracts and analysis with SDS-PAGE and Western blotting, demonstrated for Erwinia spp. in potatoes

Isolation of antigens on immunomagnetic beads and subsequent analysis with SDS-PAGE and Western blotting (immunomagnetic isolation-Western blotting (IMI-WB)) was used to verify positive ELISA results for Erwinia chrysanthemi and Erw. carotovora subsp. atroseptica in potato peel extracts. Direct analysis of highly contaminated extracts by Western blotting without previous immuno-isolation resulted in background reactions, whereas immunomagnetic isolation resulted in distinct bands of specific antigens. Target cells as well as antigenic cell products were captured in IMI-WB. Band patterns on IMI-WB of cell-free culture filtrates and cell suspensions were highly similar, but the removal of cells lowered the detection level by 10- to 100-fold. Threshold levels of IMI-WB were generally comparable with those of ELISA.
No differences in threshold levels and band patterns were found between a direct format and an indirect format of immuno-isolation.
In IMI-WB, blotting patterns differed between Erw. chrysanthemi and Erw. carotovora subsp. atroseptica. The patterns were identical for 15 Erw. chrysanthemi strains, isolated from potato peel extracts in The Netherlands. However, one of 15 strains of Erw. carotovora subsp. atroseptica from potato peel extracts in The Netherlands gave an aberrant pattern. Target bacteria could be easily distinguished from those of cross-reacting strains on the basis of band patterns.
Potato peel extracts naturally contaminated with Erw. chrysanthemi gave IMI-WB patterns that were similar to pure cultures of the homologous strains.     

Haemagglutinins and fimbriae of soft rot Erwinias.

Strains of phytopathogenic soft rot Erwinia spp. were examined for haemagglutinin (HA) production. Mannose-sensitive HA was found only in five of 15 strains of E. carotovora subsp. carotovora. Mannose-resistant HA (MRHA) was found in 12 of 15 strains of E.c. carotovora, ten of 13 strains of E.c. subsp. atroseptica and the single strain of E.c. subsp. betavasculorum, as well as all seven strains of E. chrysanthemi. MRHA, detectable only in a microtitre tray HA assay was of either broad- or narrow-spectrum activity when examined against blood of seven different animal species and could be inhibited by the beta-galactoside asialofetuin. Fimbriae of ca 10 nm diameter were found on MRHA(+) bacteria E.c. carotovora and E.c. atroseptica.     

Presence of Erwinia carotovora in surface water in North America

Erwinia carotovora was frequently isolated from samples of surface water collected from 66 rivers, springs, creeks, streams, lakes, reservoirs and ponds in 16 states in the US but was not found in the single fresh water sample collected in Canada. The organism was also isolated from water collected from the Pacific Ocean and the Gulf of Mexico. In Colorado and Wyoming, E. carotovora was isolated from water samples nearly every month of the year when monthly samples were collected from several streams. Erwinia carotovora subsp. carotovora represented 98–8% of the strains recovered from the water samples; E. carotovora subsp. atroseptica made up the remainder of the strains; E. chrysanthemi was not found.     

Genetic regulation of pathogenicity and virulence factors in bacteria Erwinia carotovora subsp. atroseptica: identification of kduD gene

A mutant that cannot utilize pectin substances of plant cell walls was obtained via insertion of mini-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 kdul-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.     

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.     

Thermodependence of growth and enzymatic activities implicated in pathogenicity of two Erwinia carotovora subspecies (Pectobacterium spp.)

Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora can cause substantial damage to economically important plant crops and stored products. The occurrence of the disease and the scale of the damage are temperature dependent. Disease development consists first of active multiplication of the bacteria in the infection area and then production of numerous extracellular enzymes. We investigated the effects of various temperatures on these two steps. We assayed the specific growth rate and the pectate lyase and protease activities for eight strains belonging to E. carotovora subsp. atroseptica and E. carotovora subsp. carotovora in vitro. The temperature effect on growth rate and on pectate lyase activity is different for the two subspecies, but protease activity appears to be similarly thermoregulated. Our results are in agreement with ecological data implicating E. carotovora subsp. atroseptica in disease when the temperature is below 20 degrees C. The optimal temperature for pathogenicity appears to be different from the optimal growth temperature but seems to be a compromise between this temperature and temperatures at which lytic activities are maximal.     

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.     

Haemagglutinins and fimbriae of soft rot Erwinias

A. WALLACE AND M.C.M. PÉROMBELON. 1992. Strains of phytopathogenic soft rot Erwinia spp. were examined for haemagglutinin (HA) production. Mannose-sensitive HA was found only in five of 15 strains of E. carotovora subsp. carotovora. Mannose-resistant HA (MRHA) was found in 12 of 15 strains of E.c. carotovora, ten of 13 strains of E.c. subsp. atroseptica and the single strain of E.c. subsp. betavasculorum, as well as all seven strains of E. chrysanthemi. MRHA, detectable only in a microtitre tray HA assay was of either broad- or narrow-spectrum activity when examined against blood of seven different animal species and could be inhibited by the β-galactoside asialofetuin. Fimbriae of ca 10 nm diameter were found on MRHA⁺ bacteria of E.c. carotovora and E.c. atroseptica.     

Characterization of a monoclonal antibody against active pectate lyase from Erwinia carotovora

A monoclonal antibody (2E2) produced against pectate lyase from Erwinia carotovora ssp. carotovora reacted with a 41- and a 44-kilodaltion protein on Western blots of concentrated Erwinia culture supernatants resolved by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. It was unequivocally shown that monoclonal 2E2 reacted with an active form of pectate lyase by affinity purifying the antigen with the monoclonal. The affinity-purified antigen was enzymatically active and moved as a single protein band in a nonequilibrium isoelectric focusing gel. Monoclonal 2E2 reacted with the pectate lyases of a diverse range of E. carotovora ssp. carotovora, ssp. atroseptica, and ssp. betavasculorum strains, as well as with one of three strains of E. chrysanthemi. The electrophoretic mobility of the major protein (44 kilodaltons) that reacted with 2E2 was identical within a subspecies but differed among subspecies.     

Differentiation of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica isolated from potato by Western blot and subsequent indirect ELISA

Electrotransfer of protein bands from a polyacrylamide gel to a hydrophobic poly-vinylidene difluoride (PVDF) membrane (Western blot) and their serological determination by indirect ELISA (immunoblotting) were used to differentiate Erwinia carotovora subsp. carotovora (Ecc) from Erwinia carotovora subsp. atroseptica (Eca). Ninety strains: 69 Ecc, 19 Eca and two Erwinia chrysanthemi (Echr) were examined. Eight polyclonal antisera against whole cells, glutaraldehyde fixed cells, glycopro-teins, and somatic antigens were prepared. Antisera produced with glutaraldehyde fixed cells did not recognize any band of the protein pattern. The remaining antisera recognized a limited number of bands. Two protein bands allowed differentiation of the two subspecies by the antisera against glycoproteins. One band with an estimated molecular weight of 36000 Da was present in the 19 Eca strains tested and another band with an estimated molecular weight of 35 000 Da was present in the 69 Ecc strains, except for three cases. The strains of Echr showed a band with an estimated weight of 33 000 Da.