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1.
Four Gram-negative, rod-shaped pectinolytic bacterial strains designated as 2M, 9M, DPMP599 and DPMP600 were subjected to polyphasic analyses that revealed their distinctiveness from the other Pectobacterium species. Strains 2M and 9M were isolated from Calla lily bulbs cultivated in Central Poland. DPMP599 and DPMP600 strains were isolated from Calla lily leaves from plants grown in Serbia. Phylogenetic analyses based on nine housekeeping genes (gapA, gyrA, icdA, pgi, proA, recA, recN, rpoA, and rpoS), as well as phylogeny based on the 381 most conserved universal proteins confirmed that Pectobacterium zantedeschiae strains were distantly related to the other Pectobacterium, and indicated Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium parmentieri and Pectobacterium wasabiae as the closest relatives. Moreover, the analysis revealed that Pectobacterium zantedeschiae strains are not akin to Pectobacterium aroidearum strains, which were likewise isolated from Calla lily.The genome sequencing of the strains 2M, 9M and DPMP600 and their comparison with whole genome sequences of other Pectobacterium type strains confirmed their distinctiveness and separate species status within the genus based on parameters of in silico DNA–DNA hybridization and average nucleotide identity (ANI) values. The MALDI-TOF MS proteomic profile supported the proposition of delineation of the P. zantedeschiae and additionally confirmed the individuality of the studied strains. Based on of all of these data, it is proposed that the strains 2M, 9M, DPMP599, and DPMP600 isolated from Calla lily, previously assigned as P. atrosepticum should be reclassified as Pectobacterium zantedeschiae sp. nov. with the strain 9MT (PCM2893 = DSM105717 = IFB9009) as the type strain.  相似文献   

2.
Pectobacterium carotovorum subsp. carotovorum, a member of the Enterobacteriaceae family, is an important plant-pathogenic bacterium causing significant economic losses worldwide. P. carotovorum subsp. carotovorum bacteriophage My1 was isolated from a soil sample. Its genome was completely sequenced and analyzed for the development of an effective biological control agent. Sequence and morphological analyses revealed that phage My1 is a T5-like bacteriophage and belongs to the family Siphoviridae. To date, there is no report of a Pectobacterium-targeting siphovirus genome sequence. Here, we announce the complete genome sequence of phage My1 and report the results of our analysis.  相似文献   

3.
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5.
Fifty-three plant-associated microorganisms were investigated for their ability to convert sucrose to its isomers. These microorganisms included one Dickeya zeae isolate and 7 Enterobacter, 3 Pantoea, and 43 Pectobacterium species. Eleven out of the 53 strains (21%) showed the ability to transform sucrose to isomaltulose and trehalulose. Among those, Pectobacterium carotovorum KKH 3-1 showed the highest bioconversion yield (97.4%) from sucrose to its isomers. In this strain, the addition of up to 14% sucrose in the medium enhanced sucrose isomerase (SIase) production. The SIase activity at 14% sucrose (47.6 U/mg dcw) was about 3.6-fold higher than that of the negative control (13.3 U/mg dcw at 0% sucrose). The gene encoding SIase, which is comprised a 1776 bp open reading frame (ORF) encoding 591 amino acids, was cloned from P. carotovorum KKH 3-1 and expressed in Escherichia coli. The recombinant SIase (PCSI) was shown to have optimum activity at pH 6.0 and 40 °C. The reaction temperature significantly affected the ratio of sucrose isomers produced by PCSI. The amount of trehalulose increased from 47.5% to 79.1% as temperature was lowered from 50 °C to 30 °C, implying that SIase activity can be controlled by reaction temperature.  相似文献   

6.
《Process Biochemistry》2010,45(2):223-229
Bacterial l-asparaginase has been widely used as therapeutic agent in the treatment of various lymphoblastic leukemia diseases. Studies on localization and production of novel glutaminase-free l-asparaginase were performed using Pectobacterium carotovorum MTCC 1428. The localization of l-asparaginase was carried out using cell fractionation techniques. The activity of l-asparaginase was found to be 85 and 77% in the cytoplasm of P. carotovorum MTCC 1428 grown on medium containing l-asparagine and combination of l-asparagine and glucose respectively. Among the tested carbon sources, l-asparagine or the combination of l-asparagine and glucose was found to be the most suitable carbon sources to maximize the production of l-asparaginase. The maximum production of l-asparaginase was observed to be 14.56 U/ml (26.92 U/mg of protein) at 4 and 2 g/l of l-asparagine and glucose respectively. Yeast extract, l-asparagine and peptone have shown significant effect on the production of l-asparaginase. P. carotovorum MTCC 1428 has assimilated l-asparagine as an essential carbon source for maximizing the production of l-asparaginase.  相似文献   

7.
The assessment of bacterial diversity and bioprospection of the high-altitude lake Suraj Tal microorganisms for potent antimicrobial activities revealed the presence of two Gram-stain-variable, endospore-forming, rod-shaped, aerobic bacteria, namely IHBB 9852T and IHBB 9951. Phylogenetic analysis based on 16S rRNA gene sequence showed the affiliation of strains IHBB 9852T and IHBB 9951 within the genus Paenibacillus, exhibiting the highest sequence similarity to Paenibacillus lactis DSM 15596T (97.8% and 97.7%) and less than 95.9% similarity to other species of the genus Paenibacillus. DNA-DNA relatedness among strains IHBB 9852T and IHBB 9951 was 90.2%, and with P. lactis DSM 15596T, was 52.7% and 52.4%, respectively. The novel strains contain anteiso-C15:0, iso-C15:0, C16:0 and iso-C16:0 as major fatty acids, and phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol were predominant polar lipids. The DNA G+C content for IHBB 9852T and IHBB 9951 was 52.1 and 52.2 mol%. Based on the results of phenotypic and genomic characterisations, we concluded that strains IHBB 9852T and IHBB 9951 belong to a novel Paenibacillus species, for which the name Paenibacillus ihbetae sp. nov. is proposed. The type strain is IHBB 9852T (=MTCC 12459T = MCC 2795T = JCM 31131T = KACC 19072T; DPD TaxonNumber TA00046) and IHBB 9951 (=MTCC 12458 = MCC 2794 = JCM 31132 = KACC 19073) is a reference strain.  相似文献   

8.
Four novel Gram-stain-positive, non spore forming and fructose-6-phosphate phosphoketolase-positive strains were isolated from the faeces of a cotton top tamarin (Saguinus oedipus) and an emperor tamarin (Saguinus imperator). Phylogenetic analyses based on 16S rRNA revealed that bifidobacterial strains TRE 1T exhibit close phylogenetic relatedness to Bifidobacterium catulorum DSM 103154 (96.0%) and Bifidobacterium tissieri DSM 100201 (96.0%); TRE DT and TRE HT were closely related to Bifidobacterium longum subsp. longum ATCC 15708T with similarity values of 97.4% and 97.5%, respectively; TRI 7T was closely related to Bifidobacterium tissieri DSM 100201 (96.0%). The Average Nucleotide Identity (ANI) and in silico DDH (isDDH) analysis with closest neighbour supported an independent phylogenetic position of all strains with values ranged from 74 to 85% for ANI and from 24 to 28% for isDDH. DNA base composition of the four strains was in the range of 58.3–63.5 mol% G + C. Based on the phylogenetic, genotypic and phenotypic data, the strains TRE 1T, TRE DT, TRE HT and TRI 7T clearly represent four novel taxa within the genus Bifidobacterium for which the names Bifidobacterium primatium sp. nov. (type strain TRE 1T = DSM 100687T = JCM 30945T), Bifidobacterium scaligerum sp. nov. (type strain TRE DT = DSM 103140T = JCM 31792T), Bifidobacterium felsineum sp. nov. (type strain TRE HT = DSM 103139T = JCM 31789T) and Bifidobacterium simiarum sp. nov. (type strain TRI 7T = DSM 103153T = JCM 31793) are proposed.  相似文献   

9.
Three strains, H01100409BT, H01100413B, and H27100402HT, were isolated from several internal organs of diseased redbanded seabream (Pagrus auriga) reared in Andalusia (Southern Spain). All strains were studied by phenotypic, including chemotaxonomy, and genomic characteristics. Phylogenetic analysis based on concatenated sequences of six housekeeping genes (gyrB, ftsZ, topA, mreB, gapA, and 16S rRNA) supported the inclusion of the strains within the clade Phosphoreum of the genus Photobacterium, and two of the strains (H27100402HT and H01100409BT) formed a tight group separated from the closest species P. aquimaris. Genomic analyses, including average nucleotide identity (ANIb and ANIm) and DNA–DNA hybridization (DDH), clearly separated strains H27100402HT and H01100409BT from the other species within the clade Phosphoreum with values below the thresholds for species delineation. The chemotaxonomic features (including FAME analysis and MALDI-TOF-MS) of H27100402HT and H01100409BT strains confirmed their differentiation from the related taxa. The results demonstrated that strain H01100413B was classified as P. aquimaris and the strains H27100402HT and H01100409BT represented a new species each in the genus Photobacterium, for which we propose the names Photobacterium malacitanum sp. nov., type strain H27100402HT (=CECT 9190T = LMG 29992T), and Photobacterium andalusiense sp. nov., type strain H01100409BT (=CECT 9192T = LMG 29994T).  相似文献   

10.
Two non-pathogenic strains R89-1 and R90T isolated from poppy seed (Papaver somniferum L.) wastes were phenotypically and genotypically characterized. Multilocus sequence analysis (MLSA) was conducted with six genes (atpD, glnA, gyrB, recA, rpoB, 16S rRNA). The strains represented a new species which clustered with Agrobacterium rubi NBRC 13261T and Agrobacterium skierniewicense Ch11T type strains. MLSA was further accompanied by whole-genome phylogeny, in silico DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses for both strains. ANI and dDDH values were deep below the species delineation threshold. Phenotypic features of the novel strains unequivocally allowed their differentiation from all other Agrobacterium species. Unlike other agrobacteria, the strains were salt sensitive and were able to biotransform morphine alkaloids. The dominant cellular fatty acids are 18:1 w7c, 16:0 and 12:0 aldehyde/16:1 iso I/14:0 3OH summed in feature 2 and the major respiratory quinine is Q-10 (87%). The DNA G + C content is 56 mol%. Microbial community analysis indicated probable association with P. somniferum plant material. Altogether, these characteristics showed that strains R90T and R89-1 represent a new species of the genus Agrobacterium which we propose to name Agrobacterium bohemicum. The type strain of A. bohemicum is R90T (=CCM 8736T = DSM 104667T).  相似文献   

11.
To identify bacteria causing soft rot and blackleg in potato in Finland, pectinolytic enterobacteria were isolated from diseased potato stems and tubers. In addition to isolates identified as Pectobacterium atrosepticum and Dickeya sp., many of the isolated strains were identified as Pectobacterium carotovorum subsp. carotovorum. Phylogenetic analysis and biochemical tests indicated that one of the isolates from potato stems resembled Pectobacterium wasabiae. Furthermore, two blackleg‐causing P. carotovorum strains recently isolated in Europe clustered with P. wasabiae, suggesting that at least some of these isolates were originally misidentified. All the other Finnish P. carotovorum isolates resembled the subsp. carotovorum type strain in biochemical tests but could be clustered into two distinct groups in the phylogenetic analysis. One of the groups mainly contained strains isolated from diseased tubers, whereas the other mainly included isolates from potato stems. In contrast to the tuber isolates, the stem isolates lacked genes in Type III secretion genes, were not able to elicit a hypersensitive response in tobacco leaves and produced only small amounts of autoinducers in the stationary phase in vitro. P. wasabiae isolate was able to cause similar amount of blackleg‐like symptoms as P. atrosepticum in a field experiment with vacuum‐infiltrated tubers, whereas both P. atrosepticum and P. carotovorum isolates reduced emergence and delayed growth more than P. wasabiae. Our findings confirm the presence of P. wasabiae in Finland and show that the Finnish P. carotovorum subsp. carotovorum isolates can be divided into two groups with specific characteristics and possibly also different ecologies.  相似文献   

12.
Ten Gram-strain-negative, facultatively anaerobic, moderately halophilic bacterial strains, designated AL184T, IB560, IB563, IC202, IC317, MA421, ML277, ML318, ML328A and ML331, were isolated from water ponds of five salterns located in Spain. The cells were motile, curved rods and oxidase and catalase positive. All of them grew optimally at 37 °C, at pH 7.2–7.4 and in the presence of 7.5% (w/v) NaCl. Based on phylogenetic analyses of the 16S rRNA, the isolates were most closely related to Salinivibrio sharmensis BAGT (99.6–98.2% 16S rRNA gene sequence similarity) and Salinivibrio costicola subsp. costicola ATCC 35508T (99.0–98.1%). According to the MLSA analyses based on four (gyrB, recA, rpoA and rpoD) and eight (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA) concatenated gene sequences, the most closely relatives were S. siamensis JCM 14472T (96.8–95.4% and 94.9–94.7%, respectively) and S. sharmensis DSM 18182T (94.0–92.6% and 92.9–92.7%, respectively). In silico DNA–DNA hybridization (GGDC) and average nucleotide identity (ANI) showed values of 23.3–44.8% and 80.2–91.8%, respectively with the related species demonstrating that the ten isolates constituted a single novel species of the genus Salinivibrio. Its pangenome and core genome consist of 6041 and 1230 genes, respectively. The phylogeny based on the concatenated orthologous core genes revealed that the ten strains form a coherent phylogroup well separated from the rest of the species of the genus Salinivibrio. The major cellular fatty acids of strain AL184T were C16:0 and C18:1. The DNA G + C content range was 51.9–52.5 mol% (Tm) and 50.2–50.9 mol% (genome). Based on the phylogenetic-phylogenomic, phenotypic and chemotaxonomic data, the ten isolates represent a novel species of the genus Salinivibrio, for which the name Salinivibrio kushneri sp. nov. is proposed. The type strain is AL184T (= CECT 9177T = LMG 29817T).  相似文献   

13.
Pectobacterium carotovorum and Pectobacterium atrosepticum are dreadful causal agents of potato soft rot. Actually, there are no efficient bactericides used to protect potato against Pectobacterium spp. Biological control using actinobacteria could be an interesting approach to manage this disease. Thus, two hundred actinobacteria isolated from Moroccan habitats were tested for their ability to inhibit in vitro 4 environmental Pectobacterium strains and the two reference strains (P. carotovorum CFBP 5890 and P. atrosepticum CFBP 5889). Eight percent of these isolates were active against at least one of the tested pathogens and only 2% exhibited an antimicrobial activity against all tested Pectobacterium strains. Four bioactive isolates having the greatest pathogen inhibitory capabilities and classified as belonging to the genus Streptomyces species through 16S rDNA analysis were subsequently tested for their ability to reduce in vivo soft rot symptoms on potato slices of Bintje, Yukon Gold, Russet and Norland cultivars caused by the two pathogens P. carotovorum and P. atrosepticum. This test was carried out by using biomass inoculums and culture filtrate of the isolates as treatment. Among these, strain Streptomyces sp. OE7, reduced by 65–94% symptom severity caused by the two pathogens on potato slices. Streptomyces OE7 showed a potential for controlling soft rot on potato slices and could be useful in an integrated control program against potato soft rot pathogens in the objective to reduce treatments with chemical compounds.  相似文献   

14.
Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre‐ and post‐harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non‐pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop‐mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non‐target genera of plant‐associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular‐based detection assays.  相似文献   

15.
Two bacterial strains isolated from root nodules of soybean were characterized phylogenetically as members of a distinct group in the genus Ensifer based on 16S rRNA gene comparisons. They were also verified as a separated group by the concatenated sequence analyses of recA, atpD and glnII (with similarities ≤93.9% to the type strains for defined species), and by the average nucleotide identities (ANI) between the whole genome sequence of the representative strain CCBAU 251167T and those of the closely related strains in Ensifer glycinis and Ensifer fredii (90.5% and 90.3%, respectively). Phylogeny of symbiotic genes (nodC and nifH) grouped these two strains together with some soybean-nodulating strains of E. fredii, E. glycinis and Ensifer sojae. Nodulation tests indicated that the representative strain CCBAU 251167T could form root nodules with capability of nitrogen fixing on its host plant and Glycine soja, Cajanus cajan, Vigna unguiculata, Phaseolus vulgaris and Astragalus membranaceus, and it formed ineffective nodules on Leucaena leucocephala. Strain CCBAU 251167T contained fatty acids 18:1 ω9c, 18:0 iso and 20:0, differing from other related strains. Utilization of l-threonine and d-serine as carbon source, growth at pH 6.0 and intolerance of 1% (w/v) NaCl distinguished strain CCBAU 251167T from other type strains of the related species. The genome size of CCBAU 251167T was 6.2 Mbp, comprising 7,581 predicted genes with DNA G+C content of 59.9 mol% and 970 unique genes. Therefore, a novel species, Ensifer shofinae sp. nov., is proposed, with CCBAU 251167T (=ACCC 19939T = LMG 29645T) as type strain.  相似文献   

16.
Two closely related, thermophilic bacteria, designated strains YIM 76954T and YIM 76947, were isolated from the Rehai Geothermal Field, Tengchong, Yunnan province, south-west China. Polyphasic approach and whole genome sequencing were used to determine the taxonomy status and genomic profiles of the novel strains. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the two isolates were closely related to Thermus scotoductus SE-1T (97.1% sequence similarity), and T. amyloliquefaciens YIM 77409T (96.6%). The strains could be differentiated from most recognized Thermus species by their whitish to slight reddish colony color, distinct DNA fingerprinting profiles and low ANI values. Cells stained Gram-negative, rod-shaped of diameter 0.2–0.5 μm and length 1.5–5.0 μm. Growth occurred at 50–75 °C, pH 6.0–9.0 and in the presence of up to 1.0% (w/v) NaCl concentration. Thiosulfate was found to enhance cell growth, besides improving the intensity of its colony color. Oxygen, nitrate, sulfur, and Fe(III) could be used as terminal electron acceptors for growth. MK-8 was the major respiratory menaquinone. Major fatty acids were iso-C17:0, iso-C15:0, anteiso-C17:0, and anteiso-C15:0. The genome size was 2.26 Mbp with 65.5% average GC content. A total of 2374 genes was predicted, comprising 2322 protein-coding and 52 RNA genes. On the basis of the polyphasic evidence presented, it is proposed that strain YIM 76954T represents a novel species of the genus Thermus, for which the name Thermus tenuipuniceus sp. nov. is proposed. The type strain is YIM 76954T (=JCM 30350T = KCTC 4677T).  相似文献   

17.
The plant tumorigenic strain NCPPB 1650T isolated from Rosa × hybrida, and four nonpathogenic strains isolated from tumors on grapevine (strain 384), raspberry (strain 839) and blueberry (strains B20.3 and B25.3) were characterized by using polyphasic taxonomic methods. Based on 16S rRNA gene phylogeny, strains were clustered within the genus Agrobacterium. Furthermore, multilocus sequence analysis (MLSA) based on the partial sequences of atpD, recA and rpoB housekeeping genes indicated that five strains studied form a novel Agrobacterium species. Their closest relatives were Agrobacterium sp. R89-1, Agrobacterium rubi and Agrobacterium skierniewicense. Authenticity of the novel species was confirmed by average nucleotide identity (ANI) and in silico DNA–DNA hybridization (DDH) comparisons between strains NCPPB 1650T and B20.3, and their closest relatives, since obtained values were considerably below the proposed thresholds for the species delineation. Whole-genome-based phylogeny further supported distinctiveness of the novel species, that forms together with A. rubi, A. skierniewicense and Agrobacterium sp. R89-1 a well-delineated sub-clade of Agrobacterium spp. named “rubi”. As for other species of the genus Agrobacterium, the major fatty acid of the strains studied was 18:1 w7c (73.42–78.12%). The five strains studied were phenotypically distinguishable from other species of the genus Agrobacterium. Overall, polyphasic characterization showed that the five strains studied represent a novel species of the genus Agrobacterium, for which the name Agrobacterium rosae sp. nov. is proposed. The type strain of A. rosae is NCPPB 1650T (=DSM 30203T = LMG 230T = CFBP 4470T = IAM 13558T = JCM 20915T).  相似文献   

18.
Strain USBA-019T, an anaerobic and thermophilic strain, was identified as a new member of the genus Thermoanaerobacterium. USBA-019T cells are gram-positive, strictly anaerobic, thermophilic, chemoorganotrophic, moderately acidophilic, non-motile, endospore-forming, slightly curved, and rod-shaped. Cells measure 0.4 × 3.0–7.0 μm. Optimal growth occurs at 50–55 °C (35–65 °C). Optimum pH is 5.0–5.5 (4.0–8.5). Thiosulfate, elemental sulfur and nitrate were utilized as electron acceptors. Fermentation of glucose, lactose, cellobiose, galactose, arabinose, xylose, starch and xylan primarily produced acetate and butyrate. Xylan, starch and cellobiose produced ethanol and starch, cellobiose, galactose, arabinose and mannose produced lactic acid. Phylogenetic analyses based on 16S rRNA gene sequence comparison and genomic relatedness indices show the close relation of USBA-019T to Thermoanaerobacterium thermostercoris and Thermoanaerobacterium aotearoense (similarity value: 99%). Hybridization of USBA-019T, Th. thermostercoris DSM22141T and Th. aotearoense DMS10170T found DNA–DNA relatedness of 33.2% and 18.2%, respectively. Based on phenotypic, chemotaxonomic and phylogenetic evidence, along with low identity at whole genome level, USBA-019T is a novel species of the genus Thermoanaerobacterium which we propose to name Thermoanaerobacterium butyriciformans sp. nov. The type strain is USBA-019T (=CMPUJ U-019T = DSM 101588T).  相似文献   

19.
Twelve Acetobacter pasteurianus-related strains with publicly available genomes in GenBank shared high 16S rRNA gene sequence similarity (>99.59%), but average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values and multilocus sequence- and genome-based relatedness analyses suggested that they were divided into four different phylogenetic lineages. Relatedness analyses based on multilocus sequences, 1,194 core genes and whole-cell MALDI-TOF profiles supported that strains LMG 1590T and LMG 1591 (previously classified as the type strains of A. pasteurianus subsp. ascendens and paradoxus, respectively) and strain SLV-7T do not belong to A. pasteurianus. Strain SLV-7T, isolated from Korean traditional vinegar, shared low ANI (<91.0%) and in silico DDH (44.2%) values with all other Acetobacter type strains analyzed in this study, indicating that strain SLV-7T represents a new Acetobacter species. The phenotypic and chemotaxonomic analyses confirmed these results and therefore a new species named Acetobacter oryzifermentans sp. nov. is proposed with SLV-7T (= KACC 19301T = JCM 31096T) as the type strain. Strains LMG 1590T and LMG 1591 shared high ANI (99.4%) and in silico DDH (96.0%) values between them, but shared low ANI (<92.3%) and in silico DDH (<49.0%) values with other type strains analyzed in this study, indicating that strains LMG 1590T and LMG 1591 should be reclassified into a new single species that should be named Acetobacter ascendens sp. nov., comb. nov., with LMD 51.1T (= LMG 1590T = NCCB 51001T) as its type strain.  相似文献   

20.
Pectobacterium species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of Pectobacterium species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two Pectobacterium clades were present in each field examined, although representatives of only three of five Pectobacterium clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in Pectobacterium strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in Pectobacterium strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers.The genus Pectobacterium (formerly Erwinia) contains both narrow- and broad-host-range bacterial plant pathogens that cause soft rot, stem rot, wilt, and blackleg in species belonging to over 35% of plant orders (20). Four Pectobacterium species have been described: Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium carotovorum, and Pectobacterium wasabiae (9). The recently described organism P. carotovorum subsp. brasiliensis is genetically distinct from previously described Pectobacterium taxa; approximately 82% of its genes are shared with P. atrosepticum, and 84% of its genes are shared with P. carotovorum subsp. carotovorum, while 13% of its genes are found in neither P. atrosepticum nor P. carotovorum subsp. carotovorum (7, 10, 20). To date, only P. carotovorum subsp. carotovorum and P. atrosepticum have been reported to occur in the same field (14, 21). P. carotovorum subsp. carotovorum is found worldwide, and P. atrosepticum is found in cool climates; while P. carotovorum subsp. brasiliensis has been found only in Brazil, Israel, and the United States, it is likely to have a wider distribution (20). Compared to the ecology and genetics of P. carotovorum subsp. carotovorum and P. atrosepticum, little is known about the ecology and genetics of P. betavasculorum, P. wasabiae, or P. carotovorum subsp. brasiliensis.Pectobacterium strains isolated from potato are diverse based on serology, genome structure, and fatty acid composition (5, 35). Previous epidemiological studies of pectolytic Enterobacteriaceae were complicated by the diversity of this group and the lack of tools capable of placing all isolates into clades. For example, Gross et al. (14) were unable to classify over 50% of Pectobacterium isolates obtained from potato, and Pitman et al. (23) were unable to type 13% of their isolates. Novel PCR-based methods potentially capable of classifying all Pectobacterium isolates have been described, but they were developed prior to the recognition of P. carotovorum subsp. brasiliensis (1, 34).The main virulence determinants of Pectobacterium are the pectolytic enzymes secreted through the type II secretion system. Although these enzymes are required for development of symptoms, many other virulence genes have been shown to contribute to Pectobacterium pathogenicity, including the type III secretion system (T3SS) genes, the cfa gene cluster, and the type IV secretion system genes (3, 15, 19). Recent genomic analysis showed that some of these gene clusters, such as the cfa and type IV secretion system cluster genes, as well as genes important for interactions with insects, are present in only some Pectobacterium species (10). Thus, Pectobacterium species appear to use different genetic tools to overcome plant host barriers and to interact with insect vectors.Many gram-negative pathogenic bacteria secrete virulence proteins, known as effectors, through the T3SS into host cells. Once inside host cells, the effectors manipulate host defenses and promote bacterial growth (13). Unlike many other gram-negative plant pathogens, Pectobacterium does not require the T3SS for pathogenicity. Rather, this secretion system makes a small, but measurable, contribution to the early stages of P. carotovorum growth in leaves of the model plant Arabidopsis thaliana (26) and contributes to the virulence of P. atrosepticum on potato (15). Recently, we isolated Pectobacterium strains that lack the T3SS from potatoes and also found P. wasabiae and P. carotovorum subsp. brasiliensis on potatoes in Wisconsin (35). The first goal of this study was to determine if P. wasabiae and P. carotovorum subsp. brasiliensis are common in agricultural fields or if soft rot disease is typically caused by P. carotovorum subsp. carotovorum and P. atrosepticum, which have been the focus of nearly all previous studies of potato soft rot, stem rot, and blackleg disease. Second, since we recently isolated a strain lacking the T3SS (35), we also aimed to determine if strains lacking the T3SS are common in infected potatoes and if these strains tend to be less virulent on potato stems and tubers than strains encoding a T3SS.  相似文献   

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