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.     

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.     

Identification and ecology of bacterial communities associated with necroses of three cactus species.

To compare the bacterial communities residing in necrotic tissues of columnar cacti of the Sonoran Desert, isolates from 39 organ pipe, 19 saguaro, and 16 senita cacti were obtained. The isolates were clustered into 28 conspecific groups on the basis of their fatty acid profiles. The distributions of the individual bacterial isolates varied among cactus species. Seven of the 28 species groups were unique to a particular cactus species, whereas 8 species groups were found in all three cacti. The effective number of bacterial species for each cactus species was positively correlated with both the chemical complexity and glucose concentration of the plant tissues. The effective number of bacterial species and bacterial distribution patterns were compared with those known for communities of cactophilic yeasts. The observed bacterial distribution patterns are most likely due to differences in the chemical compositions of the three cactus species.     

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.     

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.     

A rapid method for identifying and quantifying soft rot erwinias directly from plant material based on their temperature tolerances and sensitivity to erythromycin

A method is described for identifying and quantifying three soft rot erwinias directly from plant tissue and from other sources that is particularly useful in epidemiological studies. Colonies of these bacteria form characteristic deep cavities on selective-diagnostic crystal violet pectate (CVP) medium. Bacteria from individual presumptive erwinia colonies on CVP plates spot inoculated on plates of CVP medium with or without erythromycin (35 μg/ml) added and incubated at 27, 33°5 and 37°C can be identified according to the pattern of cavity formation. Erwinia carotovora pv. atroseptica forms the characteristic cavities only at 27°C and E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C on CVP with or without erythromycin. Erwinia chrysanthemi forms cavities at all temperatures and can also be identified by failure to grow at 27°C on CVP with erythromycin. Similarly, erwinias in mixed populations can be quantified by dilution plating on CVP with or without erythromycin and incubating at the different temperatures. Using this method, ca 80% of 183 erwinia strains in a culture collection were correctly identified, the precision increasing to over 95% when recently isolated erwinia strains were examined.     

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.     

Properties of Pseudomonads Causing Spoilage of Vegetables Stored at Low Temperature

Twenty-four strains of pectolytic, fluorescent pseudomonads were isolated from soft rots of celery stored at 0.4-1°C and five strains were isolated from soft rots in cabbage stored at 1°C. When inoculated into the vegetable from which they were isolated these bacteria caused soft rot of wounded, but not of unwounded tissue. According to their biochemical reactions, the organisms were divided into three groups; Group 1 (15 strains) were identified with Pseudomonas fluorescens Biotype II (Doudoroff & Palleroni 1974) ( Ps. marginalis ); Group 2 (12 strains) and Group 3 (two strains) would be included in the 'Miscellaneous strains'of Ps. fluorescens described by the above authors. One strain biochemically representative of Group 1 showed a maximum growth rate at 27°C (doubling time, 0.88 h) and a doubling time at 0.2°C of 14.9 h. A strain representative of Group 2 showed a maximum growth rate at 29°C (doubling time 0.96 h) and a doubling time at 0.2°C of 16.6 h. Neither strain grew at 36°C. The temperature characteristics (calculated for the range 0.2-20.8°C) were 83 011 and 79 534 J/mol, respectively. The mean doubling time for the remaining Group 1 strains at 0.2°C was 17.6 h and for remaining Group 2 strains was 17.1 h.     

DNA characterization of Lyme disease spirochetes.

Lyme disease spirochetes (LDS) have phenotypic characteristics of both treponemes and borreliae. To ascertain whether one or more species of LDS exist, as well as their taxonomic status, we determined the DNA base (G + C) content for three strains of LDS, the DNA relatedness of ten strains isolated in the United States or Europe, and the DNA relatedness of LDS to other spirochetes. The G + C content of the three LDS strains was 28.1-29.0 mol%, most similar to those of Borellia hermsii (30.6 mol %) and Treponema hyodysenteriae (25.6 mol %) among the other spirochetes tested. DNA hybridization studies of nine LDS strains to a reference strain isolated from human blood revealed divergence (unpaired bases) within related nucleotide sequences of only 0.0-1.0 percent, indicating the strains were one species. Similarly, relatedness values of seven strains to the reference strain were high: 58-98 percent (mean, 71 percent) in 50 degrees C reactions and 50-93 percent (mean, 69 percent) in 65 degrees C reactions. Labeled DNA from B. hermsii was 30-40 percent related to three Lyme disease spirochete strains in 50 degrees C reactions and 8-10 percent related in 65 degrees C reactions. In contrast, DNA from the reference LDS strain showed relatedness of only 1 percent to DNAs of two leptospires and only 16 percent to DNA from T. hyodysenteriae. We conclude that LDS are a single species, genetically unlike treponemes or leptospires, which belong in the genus Borrelia.     

Coexistence of ecologically similar colonising species III. Drosophila aldrichi and D. buzzatii: larval performance on,and adult preference for,three Opuntia cactus species

Two Drosophila species, D. buzzatti and D. aldrichi, coexist on several species of Opuntia cacti in Australia, primarily on O. tomentosa and O. streptacantha in the northern part of the cactus distribution, and on O. stricta in the south. Thorax length of field-collected adults was less, and the variance in length greater, than that for flies reared on simulated rots in the laboratory, indicating that these species are affected by crowding in nature. A larval performance index, measured on simulated cactus rots at low, moderate and high densities in single-species cultures, and at moderate and high densities in mixed-species cultures, was used to compare the relative intensity of intra- and interspecific competition at the same total larval density per 5 g necrotic cactus. Larval performance of both fly species was greatest on O. streptacantha, intermediate on O. tomentosa, and least on O. stricta in both single-species and mixed-species cultures. On O. stricta, the performances of D. aldrichi and D. buzzatii were not different when in single-species cultures, but that of D. aldrichi decreased significantly in mixed-species cultures. On the other two cactus species, the performances of D. aldrichi and D. buzzattii were not different in mixed-species cultures. The order of preferences by adult females for the cacti differed from that for larval performance, with females of both species prefering O. stricta. Analysis of microbial numbers growing on the cacti showed little difference among cacti at the rot age used for testing adult preference, but later growth was greater on O. tomentosa and O. streptacantha, the cacti that best supported larvae. Differential larval performance on O. stricta may contribute to the rare presence of D. aldrichi in the southern part of the cactus distribution, while the superior quality of O. tomentosa and O. streptacantha (larger rot size and higher microbial concentration) may reduce competition and facilitate cocxistence of the fly species in the north.     

Erwinia aphidicola,a new species isolated from pea aphid,Acyrthosiphon pisum

Five strains of Gram-negative, oxidase-negative, facultatively anaerobic, fermentative, motile, rod-shaped bacterium with the general characteristics of the family Enterobacteriaceae were isolated from the gut of multiple specimens of the pea aphid. All the strains caused aphid mortality when ingested by insects via a synthetic diet. The results of biochemical tests showed that these strains are most related to Erwinia herbicola and Pantoea agglomerans. According to DNA-DNA hybridization, the five strains showed more than 96% relatedness to each other, indicating that these organisms are members of a single species. These strains were most closely related to Erwinia herbicola (22% DNA relatedness). Phenotypic differentiation of these strains from Erwinia herbicola, which was also detected from aphid gut, was based on negative reactions in tests of yellow pigment production, gelatin liquefaction, acid production from inulin, starch and dulcitol, and positive acid production from melibiose, inositol, cellobiose and glycerol. On the basis of these data, the name Erwinia aphidicola is proposed for the new organism. The type strain is strain X 001 (=IAM 14479).     

Extracellular polysaccharide of Erwinia chrysanthemi CU643.

Erwinia chrysanthemi are gram-negative bacterial phytopathogens causing soft rots in a number of plants. The structure of the extracellular polysaccharide (EPS) produced by E. chrysanthemi strain CU643, pathogenic to Philodendron, has been determined using a combination of chemical and physical techniques including methylation analysis, high- and low-pressure gel-filtration and anion-exchange chromatography, high-pH anion-exchange chromatography, partial acid hydrolysis, mass spectrometry, and 1- and 2-D NMR spectroscopy. In contrast to the structures of the EPS reported for other strains of E. chrysanthemi, the EPS from strain CU643 is a linear polysaccharide containing L-Rhap, D-Galp, and D-GlcAp in the ratio 4:1:1. Evidence is presented for the following hexasaccharide repeat unit: -->3)-beta-D-Galp-(1-->2)-alpha-L-Rhap-(1-->4)-beta-D-GlcAp- (1-->2)-alpha-L- Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->(1 ).     

Study of Erwinia carotovora phage resistance with the use of temperate bacteriophage ZF40

The causes of the unique phage resistance of the pectinolytic phytopathogenic strains of Erwinia carotovora were studied with the use of temperate bacteriophage ZF40. It was shown that, in these bacteria, the bacteriophage-cell interaction can be substantially blocked at the adsorption level. An adequate indicator for studying the temperate bacteriophages of erwinias was developed on the basis of mutants resistant to macromolecular bacteriocins. Various restriction-modification systems, which influence cell resistance to bacteriophages, were revealed for the first time in E. carotovora. The phage resistance was shown to be determined by the wide occurrence of homoimmune temperate viruses in pectinolytic erwinias.     

SOME FACTORS AFFECTING THE GERMINATION OF SEED OF THE SAGUARO CACTUS (CARNEGIEA GIGANTEA)

Alcorn , Stanley M. (U. S. Dept. of Agric., Tucson, Ariz.), and Edwin B. Kurtz , Jr . Some factors affecting the germination of seed of the saguaro cactus (Carnegiea gigantea). Amer. Jour. Bot. 46(7): 526–529. 1959.—Germination of saguaro cactus seeds is stimulated by red light (approx. 6550 A) or daylight and far-red light (approx. 7350 A) counteracts this effect. About 0.1% germinate in continuous darkness. A single exposure to red light was most effective when the seeds were imbibed 24 hr., but maximum germination resulted from multiple exposures to red light during a 72-hr. imbibition period. The optimum temperature for germination was 25°C.; no germination occurred at 15°C. and only slight germination at 35°C. Imbibition of light-treated seeds in 0.05 to 0.2% KNO₃ increased germination. Germination of seeds in either light or dark was increased by imbibing the seeds in 500 to 1000 p.p.m. gibberellic acid.     

Erwinia persicinus, a new species isolated from plants

Five strains of a gram-negative, oxidase-negative, facultatively anaerobic, fermentative, motile, rod-shaped bacterium with the general characteristics of the family Enterobacteriaceae were isolated from tomatoes (three strains), a banana, and a cucumber. All of the strains produced a water-soluble pink pigment. As determined by DNA hybridization (hydroxyapatite method) these five strains were 85 to 100% related in 60 and 75 degrees C reactions, and related sequences exhibited 1% or less base sequence divergence, indicating that the organisms are members of a single species. These bacteria were most closely related to Erwinia rhapontici (68 to 72% at 60 degrees C, 42 to 44% at 75 degrees C, 10.5% divergence) and to hybridization group VIII in the Enterobacter agglomerans (Pantoea agglomerans, Erwinia herbicola) complex (64% at 60 degrees C, 32% at 75 degrees C, 14.5% divergence). Phenotypic differentiation from Erwinia rhapontici, which also produces a water-soluble pink pigment, is based on negative reactions by the new species in tests for methyl red, N-acetylglucosamine, DL-tartrate assimilation, and acid production from amygdalin, dulcitol, D-fucose, beta-gentiobiose, alpha-methyl-D-glucoside, glycerol, D-lyxose, melezitose, D-turanose, xylitol, and D-xylose and a positive reaction for acetoin (Voges-Proskauer test). On the basis of these data, the name Erwinia persicinus is proposed for the new organism. The type strain is strain HK 204 (= AJ 2716 = CDC 9108-82 = IAM 12843 = JCM 3704 = ATCC 35998).     

Characterization of Further Erwinia amylovora Strains and the Application of the API 20E System in Diagnosis

The former phenotypic study of Erwinia amylovora (VANTOMME et al. 1982) was extended with a collection of 54 Erwinia amylovora strains from a broad plant and geographic origin. From the 85 phenotypic features studied, 72 (85%) were present in at least 90% of the strains. Only 49 (58%) of the features were shared by all strains. Thirty-eight strains were also examined by the API 20E system. The API 20E code numbers for E. amylovora are unique and, combined with an immature, (green) pear test, may be used for an accurate identification of Erwinia amylovora.     

Acceptance and transfer of plasmid Rts1 by bacteria of the genus Erwinia]

The ability of 13 Erwinia strains to accept, to inherit and to transmit the Rts1 factor by conjugation was studied. 11 strains accepted the Rts1 factor from Escherichia coli K-12 CSH-2 with the frequency of about 10(-7)--10(-3). The Rts1 factor was genetically stable in the Erwinia cells and was not eliminated by acriflavine and under the temperature of 37 and 42 degrees C. All the R+ exconjugants were characterized with more high degree of the resistance of kanamycin than E. coli cells harbouring the same R factor. Erwinia strains harbouring the Rts1 plasmid transferred it by conjugation into homologic (Erwinia) and heterologic (E. coli) bacteria. The study of kinetics of the transfer of the Rts1 factor in different mating systems showed that the transfer of this plasmid from R+ Erwinia into R- Erwinia and R- E. coli--in the liquid medium. It is concluded that Erwinia can be the host and the donor of the Rts1 factor.     

Defective lysogeny in Erwinia carotovora

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128-192 nm in length and 13-21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55-59, 66-75, and 92-98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid-induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.     

The ecology of the yeast flora in necroticOpuntia cacti and of associatedDrosophila in Australia

A survey was made of the yeast communities isolated from necrotic tissue of 4 species of prickly-pear cacti (Opuntia stricta, O. tomentosa, O. monacantha, andO. streptacantha) which have colonized in Australia. Yeast communities were sampled from a number of localities and at different times. Cactus specific yeasts accounted for 80% of the total isolates, and the 3 most common species contributed 63% of the total. Comparisons of the species compositions of the yeast communities indicated that the differences among communities were greater betweenOpuntia species than between different localities within a single cactus species, and also that differences between years were greater than average differences between localities within years. Multivariate statistical tests of association between yeast community and physical features of rots indicated that temperature, pH, and age of rot all exerted some influence on the structure of the yeast community. Similar analyses involvingDrosophila species inhabiting these cactus rots suggested the existence of complex associations betweenDrosophila community, yeast community, and physical and chemical attributes of the cactus necroses.