A comprehensive comparison of assays for detection and identification of Ralstonia solanacearum race 3 biovar 2.

AIMS: To determine the reliable combination of protocols for specific detection and identification of R. solanacearum race 3 biovar 2 (R3bv2) through a comprehensive comparison among currently available techniques. METHODS AND RESULTS: Sensitivity and specificity of the conventional isolation, bioassay, serological assays, conventional and real-time PCR and multiplex PCR were assessed for the detection of 25 strains of R. solanacearum biovars 1, 2 and 3 (Phylotypes I, II, III and IV) in spiked potato saps. Results indicated that all assays evaluated varied in complexity and sensitivity and should be applied strategically in indexing schemes to maximize efficiency of testing without compromising accuracy of the results. CONCLUSIONS: The TaqMan PCR assay, with an internal reaction control and confirmation by melting curve and electrophoretic analysis, achieved best sensitivity at 10(2) -10(3 ) CFU ml(-1) for all eighteen strains of R. solanacearum R3bv2. Selective enrichment on mSMSA medium plates enhanced the detection sensitivity up to 10-100 CFU ml(-1) for the conventional PCR-based assays. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time nine different assays were compared side by side for their sensitivity and specificity in detection and identification of R. solanacearum R3bv2. The data accumulated here will provide basis for regulatory applications for low level detection and rapid identification of latently infections caused by R. solanacearum R3bv2.

Taxonomic relatedness between Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. odoriferum and Pectobacterium carotovorum subsp. brasiliense subsp. nov.

AIMS: Pectobacterium carotovorum is a heterogeneous species consisting of two named subspecies, P. carotovorum subsp. carotovorum and P. carotovorum subsp. odoriferum. A third subspecies, P. carotovorum subsp. brasiliense, was previously proposed. The study aimed to confirm the subspecies status and validate the proposed name of P. carotovorum subsp. brasiliense using a novel and standard microbial taxonomy. METHODS AND RESULTS: DNA-DNA hybridization confirmed that P. carotovorum subsp. brasiliense is a different species from P. wasabiae, P. betavasculorum and P. atrosepticum, with 28, 35 and 55% similarity values, respectively, but is a member of the P. carotovorum species with 73-77% similarity values. Sequencing the entire 16S rRNA gene of two polymorphic copies from strains of each of the P. carotovorum subspecies demonstrated that the average 16S rRNA gene sequence diversity between P. carotovorum subsp. brasiliense and P. carotovorum subsp. carotovorum was lower than the maximum genetic distances between two sequence types obtained from the same strain. Multilocus sequence analysis based on eight housekeeping genes (mtlD, acnA, icdA, mdh, pgi, gabA, proA and rpoS) differentiated the subspecies and delineated two P. carotovorum subsp. brasiliense clades. CONCLUSION: Pectobacterium carotovorum subsp. brasiliense clade I was comprised of strains isolated from Brazil and Peru, while clade II included strains from Asia, North America and Europe. Strains in clade I but not clade II were phenotypically consistent with the original description of P. carotovorum subsp. brasiliense in that they produced reducing substances from sucrose and acid from α-methyl glucoside. The type strain for P. carotovorum subsp. brasiliense 212(T) (= LMG2137(T) = IBSBF1692(T) = CFBP6617(T) ) was previously designated. The GC mol content of the type strain is 51·7%. SIGNIFICANT AND IMPACT OF THE STUDY: the study introduces a full description for the strains belonging to the two different clades assigned to P. carotovorum subsp. brasiliense.

Pectobacterium spp. associated with bacterial stem rot syndrome of potato in Canada.

Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, and P. wasabiae were detected in potato stems with blackleg symptoms using species- and subspecies-specific polymerase chain reaction (PCR). The tests included a new assay for P. wasabiae based on the phytase gene sequence. Identification of isolates from diseased stems by biochemical or physiological characterization, PCR, and multi-locus sequence typing (MLST) largely confirmed the PCR detection of Pectobacterium spp. in stem samples. P. atrosepticum was most commonly present but was the sole Pectobacterium sp. detected in only 52% of the diseased stems. P. wasabiae was most frequently present in combination with P. atrosepticum and was the sole Pectobacterium sp. detected in 13% of diseased stems. Pathogenicity of P. wasabiae on potato and its capacity to cause blackleg disease were demonstrated by stem inoculation and its isolation as the sole Pectobacterium sp. from field-grown diseased plants produced from inoculated seed tubers. Incidence of P. carotovorum subsp. brasiliensis was low in diseased stems, and the ability of Canadian strains to cause blackleg in plants grown from inoculated tubers was not confirmed. Canadian isolates of P. carotovorum subsp. brasiliensis differed from Brazilian isolates in diagnostic biochemical tests but conformed to the subspecies in PCR specificity and typing by MLST.

Comparative genomics-guided loop-mediated isothermal amplification for characterization of Pseudomonas syringae pv. phaseolicola.

AIMS: To design and evaluate a loop-mediated isothermal amplification (LAMP) protocol by combining comparative genomics and bioinformatics for characterization of Pseudomonas syringae pv. phaseolicola (PSP), the causal agent of halo blight disease of bean (Phaseolus vulgaris L.). METHODS AND RESULTS: Genomic sequences of Pseudomonas syringae pathovars, P. fluorescens and P. aeruginosa were analysed using multiple sequence alignment. A pathovar-specific region encoding pathogenicity-related secondary metabolites in the PSP genome was targeted for developing a LAMP assay. The final assay targeted a polyketide synthase gene, and readily differentiated PSP strains from other Pseudomonas syringae pathovars and other Pseudomonas species, as well as other plant pathogenic bacteria, e.g. species of Pectobacterium, Erwinia and Pantoea. CONCLUSION: A LAMP assay has been developed for rapid and specific characterization and identification of PSP from other pathovars of P. syringae and other plant-associated bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper describes an approach combining a bioinformatic data mining strategy and comparative genomics with the LAMP technology for characterization and identification of a plant pathogenic bacterium. The LAMP assay could serve as a rapid protocol for microbial identification and detection with significant applications in agriculture and environmental sciences.

Association of 'Candidatus Liberibacter solanacearum' with Zebra Chip Disease of Potato Established by Graft and Psyllid Transmission, Electron Microscopy, and PCR.

A new disease of potatoes, tentatively named zebra chip (ZC) because of the intermittent dark and light symptom pattern in affected tubers which is enhanced by frying, was first found in Mexico in 1994 and in the southwestern United States in 2000. The disease can cause severe economic losses in all market classes of potatoes. The cause of ZC has been elusive, and only recently has been associated with 'Candidatus Liberibacter' sp. Field samples of potato plants were collected from several locations in the United States, Mexico, and Guatemala to determine transmission to potato and tomato by grafting of ZC-infected scions and psyllid feeding. The disease was successfully transmitted, through up to three generations, by sequential top- and side-grafting ZC-infection scions to several potato cultivars and to tomato. The disease was also successfully transmitted to potato and tomato plants in greenhouse experiments by potato psyllids collected from potato plants naturally affected with ZC. Transmission electron microscopic observation of ZC-affected tissues revealed the presence of bacteria-like organisms (BLOs) in the phloem of potato and tomato plants inoculated by grafting and psyllid feeding. The BLOs were morphologically similar in appearance to BLOs associated with other plant diseases. Polymerase chain reaction (PCR) amplified 16S rDNA sequences from samples representing different geographic areas, including the United States, Mexico, and Guatemala, were almost identical to the 16S rDNA of 'Ca. L. solanacearum' previously reported from solanaceous plants in New Zealand and the United States. Two subclades were identified that differed in two single base-pair substitutions. New specific primers along with an innovative rapid PCR were developed. This test allows the detection of the bacteria in less than 90 min. These data confirm the association of 'Ca. L. solanacearum' with potatoes affected by ZC in the United States, Mexico, and Guatemala.

Absence of Potato spindle tuber viroid within the Canadian Potato Industry.

Potato spindle tuber viroid (PSTVd) causes a serious disease of potato, affecting yield and tuber quality. To control the disease, the Canadian seed certification program maintains a zero tolerance for the disease and a requirement that all nuclear stock, the micropropagated plantlets from which each lot of seed potatoes is initiated, is tested using reverse polyacrylamide gel electrophoresis (rPAGE) to ensure freedom from PSTVd. Moreover, seed potato fields are visually inspected during two or more annual field inspections for the presence of PSTVd and viruses. Symptoms of PSTVd have not been observed during field inspections for at least the last 25 years. Prior to 1989, seed potato stocks in the provinces of Prince Edward Island and New Brunswick were tested using rPAGE and nucleic acid dot blot hybridization for the presence of the viroid, and no infections were found (1). Similar surveys for PSTVd in Canada's western provinces of British Columbia, Alberta, and Saskatchewan also failed to detect the viroid (2). During 2000-2004, the PSTVd survey was extended to the provinces of Manitoba, Ontario, Quebec, Nova Scotia, and Newfoundland in which 211, 188, 95, 6, and 10 samples, respectively, were collected. Each sample consisted of 400 randomly selected leaves from selected potato fields representing seed lots registered in one of the four Elite seed classes or in the Foundation and Certified classes, except for a small number of samples (11%) that were from commercial nonseed fields. Leaves were tested using the dot blot procedure in composites of 50 leaves as described (2). Approximately 10% of the samples were retested using rPAGE followed by northern blotting to confirm dot blot results. All dot blot and rPAGE/northern blot results were negative for PSTVd. The cumulative results of the PSTVd surveys in all 10 Canadian provinces and the absence of the disease in the field as determined by annual visual inspection meets the International Standards of Phytosanitary Measures for the Requirements for the Establishment of Pest Free Areas (3). Hence, Canada declares that PSTVd is absent within its potato industry. A similar declaration was made by the United States recently on the basis of similar field inspection and survey data (4). References: (1) D. Coates-Milne. FAO Plant Prot. Bull. 37:130, 1989. (2) S. H. De Boer et al. Can. J. Plant Pathol. 24:372, 2002. (3) FAO. ISPM Pub. No. 4, 1996. (4) M. Sun et al. Am. J. Potato Res. 81:227, 2004.

First Report of Burkholderia andropogonis Causing Leaf Spots of Bougainvillea sp. in Hong Kong and Clover in Canada.

Burkholderia andropogonis has a broad host range including 52 species of 15 families of unrelated monocot and dicot plants such as white clover, carnation, bougainvillea, and other ornamental plants (2). In October 2003, a severely diseased Bougainvillea sp. was found in Kowloon, Hong Kong. Diseased leaves had circular lesions with brown centers surrounded by dark, red-brown margins bordered by chlorotic halos. A bacterium consistently isolated from such lesions using peptone yeast extract agar plus glucose plates was compared with several B. andropogonis strains, including the type strain as well as a B. andropogonis-like strain previously isolated from white clover in Vancouver, BC, Canada in June 1995. Pathogenicity of the isolates was determined by infiltrating greenhouse-grown white clover and carnation leaves with bacterial suspensions of ≈106 CFU/ml. Inoculated leaves developed lesions typical of those caused by B. andropogonis. Koch's postulates were fulfilled by isolating bacteria from typical lesions on inoculated plants that were identical to inoculated strains in colony morphology and biochemical characteristics. Using transmission electron microscopy, the Canadian and Hong Kong isolates, as well as authentic strains of B. andropogonis, were shown to have a single, polar sheathed flagellum, a unique feature of this bacterium (4). The two new isolates were compared with authentic strains of B. andropogonis using the Biolog system (Biolog Inc., Hayward, CA), whole cell protein profiles, and polymerase chain reaction (PCR) with species-specific primers. The two new isolates and authentic B. andropogonis cultures, including the type strain, were all identified as B. andropogonis using the Biolog system. The similarity in protein patterns of the new strains to those of authentic B. andropogonis strains supported their preliminary identification on the basis of morphology, pathogenicity, and the Biolog identification system. PCR amplification using primer pair Pf/Pr (Pf: 5'-AAGTCGAACGGTAACAGGGA-3', and Pr: 5'-AAAGGATATTAGCCCTCGCC-3'), which specifically targets B. andropogonis 16S rDNA (1), produced the expected 410-bp amplicon with genomic DNA templates from the two isolates, further confirming their identity. No sequence variation was observed between the amplicon and data (X67037) from GenBank, which confirmed the earlier observation that strains of B. andropogonis were phylogenetically homogenous (1). To our knowledge, this is the first report of B. andropogonis infection on Bougainvillea sp. in Hong Kong. The disease has been previously reported on this host only from Brisbane, Australia. This is also the first report of the isolation of B. andropogonis from clover in Canada, although the disease occurs on clover in other regions such as Australia and Hawaii. B. andropogonis has been previously reported in Canada only on greenhouse carnations (Dianthus sp.) (3). Usually, conditions of high humidity and high temperature are optimal for infection by B. andropogonis. On the basis of historical weather data, Hong Kong has tropical and subtropical coastal weather similar to Brisbane, Australia, while Vancouver, although mild, is cooler but has periods of high humidity. References: (1) R. D Bagsic et al. Lett. Appl. Microbiol. 21:87. 1995. (2) E. J. Cother et al. Plant Pathol. 53:129, 2004. (3) D. W. Creelman. Can. Plant Dis. Surv. 44:146, 1964. (4) X. Li. Ph.D. diss. The University of Queensland, St. Lucia, Australia. 1993.

Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil.

AIMS: To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia. METHODS AND RESULTS: Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E. carotovora subspecies and E. chrysanthemi. Pathogenicity and maceration ability of the Brazilian strains were greater than those of E. carotovora subsp. atroseptica, the causal agent of potato blackleg in temperate zones. Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E. carotovora subsp. atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia. Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E. carotovora and from E. chrysanthemi. A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR. CONCLUSIONS: The bacterium that causes the blackleg disease of potato in Brazil differs from E. carotovora subsp. atroseptica, the blackleg pathogen in temperate zones. It also differs from other subspecies of E. carotovora and from E. chrysanthemi and warrants status as a new subspecies, which would be appropriately named E. carotovora subsp. brasiliensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions. The Brazilian strain is more virulent than E. carotovora subsp. atroseptica, the usual causal agent of potato blackleg.

Detection and Confirmation of Potato mop-top virus in Potatoes Produced in the United States and Canada.

Potato mop-top virus (PMTV) was detected in potatoes grown in the United States and Canada during surveillance testing by a reverse transcription-polymerase chain reaction (RT-PCR) targeting the coat protein gene in RNA3. Out of 3,221 lots of seed and ware potatoes that were tested, 4.3% were positive for PMTV. The reliability of the survey results was confirmed by reextraction of selected samples and additional RT-PCR tests using two primer sets targeting gene segments in RNA2 and RNA3. Amplicons generated from RNA2 and RNA3 were identified by analysis of fragment length polymorphisms after digestion with BamHI and HindIII, respectively. PMTV was further identified by enzyme-linked immunosorbent assay, bioassay on Nicotiana debneyi, and transmission electron microscopy. Sequencing of a portion of the coat protein gene revealed near 100% identity among isolates from the United States and Canada and >97% homology of the North American isolates with European isolates.

Molecular characterization of DNA encoding 16S-23S rRNA intergenic spacer regions and 16S rRNA of pectolytic Erwinia species.

Sequences of 16S rDNAs and the intergenic spacer (IGS) regions between the 16S and 23S rDNA of bacterial strains from genus Erwinia were determined. Comparison of 16S rDNA sequences from different species and subspecies clearly revealed intraspecies-subspecies homology and interspecies heterogeneity. Phylogenetic analyses of 16S rDNA sequence data revealed that Erwinia spp. formed a discrete monophyletic clade with moderate to high bootstrap values. PCR amplification of the 16S-23S rDNA regions using primers complementary to the 3' end of 16S and 5' end of 23S rRNA genes generated two DNA fragments. The small 16S-23S rDNA IGS regions of Erwinia spp. examined in this study varied considerably in size and nucleotide sequence. Multiple sequence alignment and phylogenetic analysis of small IGS sequence data showed a consistent relationship among the test strains that was roughly in agreement with the 16S rDNA data that reflected the accepted species and subspecies structure of the taxon. Sequence data derived from the large IGS resolved the strains into coherent groups; however, the sequence information would not allow any phylogenetic conclusion, because it failed to reflect the accepted species structure of the test strains.

Relative Incidence of Erwinia carotovora subsp. atroseptica in Stolon End and Peridermal Tissue of Potato Tubers in Canada.

An enrichment enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody 4F6, which is specific for the lipopolysaccharide antigen of serogroup I strains of Erwinia carotovora subsp. atroseptica, was used to detect the blackleg pathogen in potato seed lots after having confirmed that this serogroup is still the predominant strain of the pathogen in Canada. E. carotovora subsp. atroseptica was detected on <6% of tubers in most seed lots, but the level of tuber contamination increased with the number of field generations. The incidence of E. carotovora subsp. atroseptica was generally greater in tissue taken from the stolon end of tubers than from peel samples, although there was a positive correlation between the bacterium's presence in the two sample types. The incidence of E. carotovora subsp. atroseptica in seed tuber lots was estimated from the results of tests on multiple composite samples, providing a cost effective approach for indexing seed lots for blackleg risk. The incidence of E. carotovora subsp. atroseptica was two and three times greater for both stolon end and peel samples, respectively, after harvest and storage of seed lots compared with tubers hand dug just before commercial harvesting, which suggests that the process whereby tubers become contaminated has both field and postharvest components.

Classification, nomenclature, and plant pathogenic bacteria - a clarification.

ABSTRACT In a recent Letter to the Editor of Phytopathology, proposals were made for endorsement and for rejection of selected names of plant pathogenic Pseudomonas spp. and Xanthomonas spp. We believe that support for, and rejection of, several names was based on misconceptions concerning the Approved Lists of Bacterial Names and entails misinterpretations of several Rules of the International Code of Nomenclature of Bacteria. This letter aims to clarify those misconceptions and misinterpretations.

Occurrence of Potato Wart Caused by Synchytrium endobioticum on Prince Edward Island, Canada.

During harvest (October 2000) of a 26-ha field of processing potatoes (cv. Russet Burbank) on Prince Edward Island (PEI), a small number of tubers with symptoms of potato wart were found in a 1-ha area of the field. Resting sporangia of Synchytrium endobioticum were present in diseased tissue. Potato wart is not endemic in Canada outside of Newfoundland, where the disease has occurred since 1909, and has been under official quarantine control since 1912 (1). In the United States, the disease was eradicated from Pennsylvania and West Virginia by 1974 and, more recently, was eradicated from Maryland, where its presence had been reconfirmed in 1987 (2). Anecdotal information pertaining to the PEI field suggests that the source of infection may have been infected tubers from Newfoundland that were grown in this portion of the field many years ago. Cv. Russet Burbank is resistant to pathotype 1 which occurs in the United Kingdom, but is susceptible to pathotype 2, which predominates in Newfoundland (1). A soil survey confirmed the presence of S. endobioticum resting sporangia in the 1-ha area in which the symptomatic tubers were found. Concentrations of sporangia ranged from <1 to 124 sporangium per g of air-dried soil. Resting sporangia of S. endobioticum were not found in soil samples from fields within a 0.8-km radius of the infested field, nearby garden plots, or fields in which the same equipment had been used since 1984. References: (1) M. C. Hampson. Can. J. Plant Pathol. 15:223, 1993. (2) M. L. Putnam and A. B. Sindermann. Am. Potato J. 71:743, 1994.

Proficiency Testing in a Laboratory Accreditation Program for the Bacterial Ring Rot Pathogen of Potato.

Variability of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence tests for the detection of Clavibacter michiganensis subsp. sepedonicus in potato tissue was analyzed to determine the magnitude of repeatability (within analyst variation) and reproducibility (among analyst variation) components. The analysis was based on data generated by analysts in eight laboratories testing proficiency panel samples distributed under a laboratory accreditation program. The standard deviation for repeatability of the ELISA test was small but increased at higher absorbance readings, while the standard deviation for reproducibility was larger and also increased at high absorbances. For immunofluorescence, the standard deviation for repeatability and reproducibility were similar to one another and increased with increasing bacterial concentration, as might be expected for count data and the inherent subjectivity of the test. The reproducibility standard deviation provided the basis for calculating "z-scores" by the Association of Official Analytical Chemists' procedure to evaluate proficiency of chemical analyses. More than 90 and 80% of the z-scores for samples tested in this study by ELISA and immunofluorescence, respectively, were in the acceptable range. The rescaled sums of z-scores for individual analysts were used as single combination scores to evaluate each analyst's results over all samples of a proficiency panel. This measure may be useful for tracking analyst performance on process control charts as part of a quality control system.

Fimbrial-Specific Monoclonal Antibody-Based ELISA for European Potato Strains of Erwinia chrysanthemi and Comparison to PCR.

A murine hybridoma cell line, named 6A6, was developed to produce monoclonal antibodies for serological detection of European potato strains of Erwinia chrysanthemi. The monoclonal antibodies were of the immunoglobulin G2b type and were shown to react with a fimbrial antigen by immuno-gold electron microscopy, and with the fibrillin protein by Western blotting. In enzyme-linked immunosorbent assay (ELISA), the monoclonal antibody reacted with all but two strains of E. chrysanthemi isolated from potato. One non-reactive strain originated from Australia and therefore was likely a different biovar, and the other strain was of unknown origin. The monoclonal antibody also reacted with 20 out of 36 strains of E. chrysanthemi isolated from hosts other than potato. A triple-antibody ELISA test utilizing monoclonal antibody 6A6 successfully detected E. chrysanthemi in infected potato stems and tubers but sensitivity was limited to about 107 CFU/ml, compared to a sensitivity of 103 CFU/ml for a polymerase chain reaction test using published primers directed to the pectate lyase gene.

Clavibacter michiganensis subsp. Sepedonicus Elicits a Hypersensitive Response in Tobacco and Secretes Hypersensitive Response-Inducing Protein(s).

ABSTRACT Strains of Clavibacter michiganensis subsp. sepedonicus, causal agent of bacterial ring rot of potato, showed marked differences in virulence on host plants. When infiltrated into tobacco leaves, virulent strains caused a rapid localized necrotic response (within 24 to 48 h) characteristic of the hypersensitive response (HR), whereas nonpathogenic strains did not. Concentrated cell-free culture supernatants (CCS) from virulent strains caused a necrotic reaction on tobacco, whereas CCS from nonpathogenic strains did not. The necrosis-inducing activity was heat stable and protease sensitive. Inhibitors of eukaryotic metabolism suppressed the necrotic reaction of tobacco to CCS. No necrotic response was observed when host plants were infiltrated with either cells or CCS from virulent strains. HR-inducing protein(s) from a virulent strain separated from the majority of other proteins on DEAE cellulose at 250 to 300 mM NaCl. Ammonium sulfate-precipitated proteins from a virulent strain produced a necrotic reaction at a total protein concentration of 18 mug/ml, whereas those from a nonpathogenic strain did not, even at a concentration of 180 mug/ml. We conclude that virulent strains of C. michiganensis subsp. sepedonicus elicit a typical HR in tobacco and secrete proteinaceous elicitor(s) of the nonhost HR.

Improved microscopic identification of Clavibacter michiganensis subsp. sepedonicus cells by combining in situ hybridization with immunofluorescence.

An oligonucleotide probe was selected from the 16S rRNA gene of Clavibacter michiganensis subsp. sepedonicus for specific in situ hybridization. The rhodamine-labelled oligonucleotide probe was used in conjunction with an indirect immunofluorescence procedure based on a specific monoclonal antibody detected with a fluorescein-labelled conjugate. Simultaneous labelling of bacterial cells with the oligonucleotide and antibody probes allows accurate microscopic identification of single cells when isolation and other methods of confirming bacterial identity are not possible.

Comparison of 16S ribosomal RNA genes in Clavibacter michiganensis subspecies with other coryneform bacteria.

Nearly complete sequences (97-99%) of the 16S rRNA genes were determined for type strains of Clavibacter michiganensis subsp. michiganensis, Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. sepedonicus, and Clavibacter michiganensis subsp. nebraskensis. The four subspecies had less than 1% dissimilarity in their 16S rRNA genes. Comparative studies indicated that the C. michiganensis subsp. shared relatively high homology with the 16S rRNA gene of Clavibacter xyli. Further comparison with representatives of other Gram-positive coryneform and related bacteria with high G+C% values showed that this group of bacteria was subdivided into three clusters. One cluster consisted of the Clavibacter michiganensis subsp., Clavibacter xyli, Arthrobacter globiformis, Arthrobacter simplex, and Frankia sp.; another cluster consisted of members of the corynebacteria-mycobacteria-nocardia (CMN) group of Mycobacteriaceae including Tsukamurella paurometabolum; and Propionibacterium freudenreichii alone formed a unique cluster, which was remote from other coryneform bacteria analyzed. The three clusters may reflect a systematic rank higher than the genus level among these bacteria.

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.