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1.
Phenotypic and genetic characteristics of nine bacterial strains isolated from mulberry ( Morus spp.), which were originally described as Erwinia carotovora ssp. carotovora (Ecc), were investigated. Based on the results of biochemical tests, these bacterial strains were divided into two different types, type 1 and type 2. Two strains of type 1 were similar to Ecc, whereas seven strains of type 2 were distinct from Ecc. A polyphasic study that included serological assay, specific PCR assay for E. carotovora ssp. atroseptica (Eca), PCR-RFLP of a pectate lyase ( pel ) gene and RAPD-PCR was performed on the type 2 strains, and the data were compared with those of related E. carotovora subspecies. The results of serological and specific PCR assays for Eca showed that the type 2 strains were distinct from Eca. In RFLP analysis of the pel gene using Sau 3AI, the type 2 strains showed a unique RFLP pattern. On the basis of RAPD analysis, similarity of RAPD patterns within the type 2 strains was very high. A unique RAPD fragment was isolated from the type 2 strains and used as a probe for Southern hybridization. This probe hybridized only with PCR products from the type 2 strains. Based on phenotypic, serological and genetic characteristics, the type 2 strains isolated from mulberry may belong to a distinct E. carotovora subspecies other than Eca or Ecc. 相似文献
2.
Soft rot erwiniae are a group of notorious plant pathogens for which currently available detection methods are inadequate. Based on the polymerase chain reaction, specific and sensitive detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in potato tubers has been achieved. The composition of the PCR primers used in two specific detection systems is based on identification of the consensus of sequences of metalloprotease-coding genes present in soft rot erwiniae. Bacterial DNA was extracted from the potato tuber matrix by differential centrifugation in order to avoid interference of potato-derived compounds with the performance of the PCR assay. The PCR assay jjerformed with the E. carotovora subsp. atroseptica specific primer set was found to be capable of distinguishing E. carotovora subsp. atroseptica from all other Erwinia species and the closely related subspecies E. carotovora subsp. carotovora. With the E. chrysanthemi specific primer set, agarose gel electrophoresis is required for unequivocal differentiation between E. chrysanthemi and other erwiniae. Combined with the efficient extraction procedure, the assay allowed specific detection of less than 103 culturable erwiniae per tuber. The specificity and sensitivity of the assay were not reduced in the presence of a 100-fold excess of DNA from both related and unrelated bacteria. This PCR-based method for detection of erwiniae in potato tubers provides a relatively fast and sensitive alternative to routinely applied serological methods. 相似文献
3.
J. M. VAN DER WOLF P. KASTELEIN J. R. C. M. VAN BECKHOVEN M. VAN DEN BRINK P. M. DE VRIES 《EPPO Bulletin》1996,26(3-4):707-715
In this study, the reliability and efficiency of three procedures for verification of IFC-positive colonies of Erwinia carotovora subsp. atroseptica were compared: (1) PCR amplification, (2) reisolation on a non-selective medium (trypticase soy agar) followed by direct immunodiffusion (TSA-DID), developed for isolation of target and cross-reacting bacteria, and (3) reisolation on a selective medium (crystal-violet pectate) and characterization of selected isolates with Ouchterlony double diffusion (DLCVP-ODD), developed for isolation of pectinolytic Erwinia spp. The reliability of a PCR amplification procedure for characterization of E.c. atroseptica was evaluated. Specific amplification products could be produced from DNA of all 187 European strains of the bacterium, while no amplification products were obtained from DNA of four distinctive serological groups of bacteria cross-reacting with antibodies against E.c. atroseptica , nor from DNA of randomly selected saprophytic bacteria isolated from potato peel extracts. All 60 immunofluorescent-positive target colonies from a potato peel extract with added E.c. atroseptica tested were positive by PCR compared with 68 and 72% successful determinations by TSA-DID and DLCVP-ODD, respectively. PCR enabled verification of fluorescent colonies from IFC preparations of naturally infected seed lots with an efficiency of 93%, compared with 48 and 71% successful determinations by TSA-DID and DLCVP-ODD, respectively. It is concluded that PCR is useful for routine confirmation of the identity of fluorescent colonies in IFC. 相似文献
4.
The variability within a collection of 100 isolates of Erwinia collected from various potato cultivars and locations in Algeria was studied using physiological, biochemical and molecular tests. The comparison of their biochemical characteristics with those of the type isolates CFBP 1526 ( E. carotovora ssp. atroseptica ), CFBP 2046 ( E. carotovora ssp. carotovora ) and CFBP 2048 ( E. chrysanthemi ) indicated that all the isolates collected in Algeria belonged to the species E . carotovora . They included 40 typical E. carotovora ssp. carotovora and 14 E. carotovora ssp. atroseptica ; the remaining 46 isolates could not be classified as E. carotovora ssp. atroseptica or ssp. carotovora , even though they were true Erwinia. Amplification of total genomic DNA with the primers Y1 and Y2, specific for E. carotovora , yielded an amplified fragment of the expected size in 99 isolates. The primers Y45 and Y46 specifically amplified a 439-bp DNA fragment in all E. carotovora ssp. atroseptica isolates tested, but not in isolates of the other E. carotovora subspecies or in atypical isolates, as expected from the characteristics of these primers . The digestion patterns of the 99 amplified products with the restriction enzymes Alu I, Hae II, Hpa II and Sau3A I yielded 12 RFLP groups, three of which were undescribed. The 14 isolates of E. carotovora ssp. atroseptica shared a single restriction pattern (RFLP group 1), while the typical isolates of E. carotovora ssp. carotovora and the atypical isolates composed the remaining groups (3, 4, 8–10, 12, 14, 22 and 25–27), reflecting the heterogeneity among these isolates. 相似文献
5.
彩色马蹄莲欧文氏菌PCR检测 总被引：1，自引：0，他引：1
采用聚合酶链式反应(PCR)技术检测彩色马蹄莲欧文氏菌纯培养和彩色马蹄莲样品,并用分离培养技术加以验证。结果表明,PCR能特异地检测出所有10个彩色马蹄莲欧文氏菌菌株,并证实了昆明地区侵染彩色马蹄莲的细菌为胡萝卜软腐欧文氏菌(Erwiniacarotovora subsp.carotovora)。PCR与分离培养技术检测结果基本一致,但总体上PCR检测阳性率稍高于分离培养检测的发病率。接种马铃薯、大白菜24h后接种点处均出现明显软腐症状。该项技术具有更高的灵敏度,适用于彩色马蹄莲种苗的检测和病害流行学研究。 相似文献
6.
Small plots of potatoes were inoculated with a mixture of Erwinia carotovora (E. c.) subsp. carotovora and E. carotovora subsp, atroseptica strains resistant to rifampicin. Subsequently the population off, c. subsp, carotovora and E. c. subsp, atroseptica (rifampicin-resistant and wild types) present as epiphytes on the surface of potato leaves was assessed using three methods, qualitative, semi-quantitative and quantitative, during 1986 and 1987. The population was generally low (< 102 colony forming units (> 104 cfu/g leaves) but reached higher levels (> 104 cfu/g) on occasions later in the growing season, Rifampicin-resistant erwinias were reisolated only infrequently throughout this study. Different methods of haulm destruction (herbicide, pulverization, sulphuric acid treatment and natural senescence) greatly influenced the number of erwinias present in the resulting plant debris. Pulverization resulted in the highest population (106 -107 wild-type cfu/g) in both seasons. In 1987. the wettest of the two seasons of this study, herbicide treatment resulted in similarly high populations. The results suggest that the high numbers of erwinias found in the haulm debris were probably derived from the generally low populations of epiphytic bacteria previously present on healthy leaves, E. c. subsp, carotovora was the most frequent subspecies in the rotting plant debris; E. c. subsp, atroseptica was more commonly found on healthy leaves. The implications of the results are discussed in relation to the production of seed potatoes with a low risk of blackleg. 相似文献
7.
The relationship between number of viable cells of Erwinia carotovora subsp. atroseptica on inoculated potato seed tubers and blackleg development was investigated in 2 years for five cultivars grown in the contrasting climates of Scotland and Israel. Blackleg, and to a lesser extent non-emergence, increased with higher numbers of bacteria on the seed tubers at planting. This relationship was also found for several commercial seed stocks of one cultivar naturally contaminated with different numbers of E. carotovora subsp. atroseptica.The threshold number of bacteria necessary for the development of blackleg declined during the growing season and was also higher for the cultivar Pentland Crown in comparison with the others. In general, yield declined linearly with blackleg incidence and there was a 0.8% reduction in yield for every 1 % blackleg at 13 weeks after planting. Yield loss was positively related to the incidence of blackleg late in the season, whereas the relationship between yield loss and the incidence of non-emergence was poor. 相似文献
8.
An oligonucleotide array for the identification and differentiation of bacteria pathogenic on potato
ABSTRACT Oligonucleotides, 16 to 24 bases long, were selected from the 3' end of the 16S gene and the 16S-23S intergenic spacer regions of bacteria pathogenic on potato, including Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, and the pectolytic erwinias, including Erwinia carotovora subsp. atroseptica and carotovora and E. chrysanthemi. Oligonucleotides were designed and formatted into an array by pin spotting on nylon membranes. Genomic DNA from bacterial cultures was amplified by polymerase chain reaction using conserved ribosomal primers and labeled simultaneously with digoxigenin-dUTP. Hybridization of amplicons to the array and subsequent serological detection of digoxigenin label revealed different hybridization patterns that were distinct for each species and subspecies tested. Hybridization of amplicons generally was restricted to appropriate homologous oligonucleotides and cross-hybridization with heterologous oligonucleotides was rare. Hybridization patterns were recorded as separate gray values for each hybridized spot and revealed a consistent pattern for multiple strains of each species or subspecies isolated from diverse geographical regions. In preliminary tests, bacteria could be correctly identified and detected by hybridizing to the array amplicons from mixed cultures and inoculated potato tissue. 相似文献
9.
M. C. M. PÉROMBELON Y. BERTHEAU M. CAMBRA D. FRECHON M. M. LOPEZ F. NIEPOLD P. PERSSON A. SLETTEN I. K. TOTH J. W. L. VAN VUURDE J. M. VAN DER WOLF 《EPPO Bulletin》1998,28(1-2):141-155
Contamination of seed potato tubers by Erwinia carotovora subsp. atroseptica is widespread with the bacteria usually sited superficially in lenticels and suberized wounds. As seed contamination level is related to blackleg incidence, seed health is best assessed by determining the number of cells of E. c. atroseptica per mL of tuber-peel extract. The relative specificity, sensitivity and ease of use of four recently developed microbiological, immunological and molecular methods to detect and/or quantify tuber contamination are discussed in relation to the testing of commercial seed stock. Sensitivities of all four methods are at or below the threshold level for blackleg development (< 103 cells mL-1 ), but there are differences regarding their specificity and ease of use. Three of them allow enumeration of most live cells of the bacterium, using specific monoclonal and polyclonal antibodies against the predominant serogroup I: (a) immunomagnetic separation of E. c. atroseptica before viable count on a selective-diagnostic growth medium, crystal violet pectate, (b) immunofluorescence staining and counting of colonies in pour-plate medium in tissue culture plates and (c) enrichment of the bacterium in peel-extract dilutions directly in microtitre plates prior to DAS-ELISA. In the fourth method, both live and dead cells are detected, but not quantified, by PCR amplification of target sequences using specific primers for E. c. atroseptica regardless of serogroup. 相似文献
10.
The susceptibility of tubers of different potato cultivars to soft rot by Erwinia carotovora subspp. uroseptica and carotovora was assessed in 3 years by two methods. In one method, whole tubers inoculated at wounds with either bacterium were incubated under anaerobic conditions for 5 or o days at 15°C. In the other method, wounds made in tuber slices were allowed to heal or not, before inoculation with different concentrations of each bacterium and were then incubated under aerobic conditions for 3 days at 15°C. Most cultivars gave consistent reactions in repeated experiments using the same method, but there was some seasonal variation. A few cultivars were consistently susceptible (Klondyke and Manna) or resistant (Drayton) in both methods but others gave completely contrasting results (Record). In both methods and with all cultivars more rotting was caused by subsp. atroseptica than by subsp. carotovora because of the temperature of Incubation. 相似文献
11.
B.A. Fraaije M. Appels S.H. De Boer J.W.L. Van Vuurde R.W. Van den Bulk 《European journal of plant pathology / European Foundation for Plant Pathology》1997,103(2):183-193
Automated conductance measurements in polypectate medium were used for the detection of pathogenic soft rot Erwinia spp. in potato peel extracts. The detection threshold for Erwinia carotovora subsp. atroseptica (Eca) in inoculated peel extracts was ca. 104 colony forming units (cfu) ml-1 when samples were considered positive on the basis of a response within 48 h at 20 °C. Detection of E. chrysanthemi (Ech) was less sensitive, only 105 cfu ml-1 peel extract were detected within 36 h at 25 °C. The linear correlation between detection times in conductimetry and inoculum levels of Eca and Ech in peel extracts was used for a quantitative estimation of Eca and Ech in naturally contaminated peel extracts. Samples giving a positive conductimetric response had to be confirmed with an enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) for the presence of Eca and Ech, because E. carotovora subsp. carotovora (Ecc) also generated a conductance response. Conductimetry was sensitive and efficient for detection of contamination levels of Eca higher than 104 cfu ml-1 peel extract. For Ech, conductimetric detection was less sensitive and inefficient due to low contamination levels of Ech and the presence of high numbers of Ecc in many samples after enrichment, which interfered with the test. Immunofluorescence cell staining (IF) combined with enrichment and immunofluorescence colony staining (IFC) were suited to detect and quantify low numbers of Eca and Ech at less than 104 cfu ml-1 in peel extracts. However, since false positive and negative reactions in serology were observed, the use of PCR after enrichment, or in combination with IFC to confirm positive results, was required for accurate detection. 相似文献
12.
A simplified slice method for assessing tuber susceptibility of potato cultivars to Erwinia carotovora subsp atroseptica 总被引：1，自引：1，他引：0
The susceptibility of 24 potato cultivars to soft rot caused by Erwinia carotovora subsp. atroseptica was assessed in a simplified form of a tuber slice test described previously. Freshly made wounds on tuber slices were inoculated with drops (0–02 ml) containing 108 , 107 or 106 cells/ml and incubated aerobically for 3 days at 15° or 20°C. At 20°C differences between cultivars were clear at all concentrations but at 15°C less so at the lowest concentration unless incubation time was extended. The rank order of cultivar susceptibility was similar to that found in previous seasons. 相似文献
13.
Ampaabeng Gyedu KYEREMEH Toshio KIKUMOTO Duen-yau CHUANG Yuichi GUNJI Yoshiyuki TAKAHARA Yoshio EHARA 《Journal of General Plant Pathology》2000,66(3):264-268
Two pathogenic strains of E. carotovora subsp. carotovora 2T-2 and TT-4 with high bacteriocin activity but low sensitivity to the bacteriocins of other strains were treated with ethyl
methane sulphonate (EMS). Two avirulent mutants A-f-39 and B-e-19 developed from 2T-2 and TT-4, respectively, by this treatment
had the same bacteriocin activity as their respective parents and inhibited the in vitro growth of pathogenic strains of this species. The disease control of these two mutant strains were compared in the field in
1995 and 1997 to the control by CGE234M403 (M403) (a commercialized biocontrol agent), a mixture of A-f-39 and M403, and an
agrochemical (basic dithianon-copper chloride). The protection obtained with A-f-39 was comparable to M403 and was better
than that with the chemical. The mixture of A-f-39 and M403 consistently gave the best results in all the field trials.
Received 17 September 1999/ Accepted in revised form 22 February 2000 相似文献
14.
Heat treatment of seed tubers for control of potato blackleg (Erwinia carotovora subsp. atroseptica) and other diseases 总被引：1，自引：1，他引：0
The thermal death points of Erwinia carotovora subsp. atroseptica and subsp. carotovora were determined in relation to duration of heat treatment, age of culture and culture medium. No isolates cultured in liquid media survived heating at 53°C for 5 min while those on solid media were killed by heating at 54°C for 10 min. After immersing naturally contaminated potato tubers for 10 min in water at 55°C, Erwinia could not be detected. The same treatment of naturally or artificially contaminated seed tubers gave complete absence of blackleg infection in the field and decreased the amounts of powdery scah(Spongospora subterranea) and black scurf (Rhizoctonia solani) on progeny tubers. 相似文献
15.
To examine whether the pathogenic bacterium, Erwinia carotovora subsp. carotovora, causal agent of soft rot of Chinese cabbage (Brassica campestris L., pekinensis group), can overwinter in plant debris and soil and serve as inoculum the following year, we monitored field
populations of rifampicin-resistant, phage-sensitive strains of the bacterium. Chinese cabbage (cv. Matsushima Kohai W1116)
were planted in field soil in pots that were sunk into the field on Aug. 2, 1996 and eventually reduced to one plant per pot.
Outer petioles of the plants were inoculated with mixture of 13 bacterial strains of E. carotovora subsp. carotovora on Sept.5, 1996. After the soft rot spread throughout the plant, the diseased plant was buried in the potted soil. New seeds
were sown in the pots on April 30, 1997, and the disease was observed in June and July. The bacterial strains were re-isolated
from the potted soil, diseased tissue and rhizosphere soil by the dilution plating method on modified Drigalski's medium containing
100 ppm rifampicin and by the enrichment technique. In addition to rifampicin resistance, phage sensitivities of some of the
re-isolated strains were identical to those of the strains buried in the soil with the diseased plant in the previous year.
From these results, some of the 13 strains overwintered in the soil and infested plant tissue and acted as primary inoculum
the following year. The frequency of re-isolation varied among the strains, perhaps because of competition among the strains,
differences in epidemiological behavior and stabilizing selection among the strains, and the presence of different ecotypes
of the organism.
Received 21 July 2000/ Accepted in revised form 19 September 2000 相似文献
16.
ABSTRACT The lysozyme from Erwinia amylovora phage PhiEa1h was investigated for its ability to inhibit growth of bacteria and compared with the lysozyme from Escherichia coli phage T4. The assays to measure lysozyme activity included cell lysis and growth inhibition of bacteria. Bacterial strains with kanamycin resistance were not affected by lysates containing the PhiEa1h-enzyme. The titer of Micrococcus luteus but not of Erwinia amylovora was diminished by cell extracts containing T4 lysozyme. In contrast, PhiEa1h lysozyme preferentially inhibited E. amylovora, exceeding the T4 lysozyme activity at least one million-fold. Spherical cells were formed after application to E. amylovora similar to lyz-gene expression in Escherichia coli. Heating of cell extracts destroyed the murami-dase activity, but retained an antibacterial activity. Other plant-associated bacteria related to Erwinia amylovora also were inhibited for growth when cell extracts with PhiEa1h lysozyme were applied to soak pear slices and potato slices. Ooze formation and soft rot caused by E. amylovora or E. carotovora subsp. atroseptica, respectively, were strongly reduced and the PhiEa1h lysozyme was more efficient compared with extracts containing T4 lysozyme. 相似文献
17.
The resistance of eight potato cultivars to tuber soft rot caused by E. carotovora subsp. atroseptica was assessed in 2 years using three different test methods. Similar cultivar resistance rankings were obtained for any one method within a year and between years for two methods (single site, infectivity titration), but not for the third (vacuum infiltration). However, the ranking of cultivars differed for the three methods. Ranking was not affected by inoculating the cortex or the more susceptible medullary tissue, or by assessing rotting in terms of infection frequency or lesion size, but it was affected by oxygen concentration during incubation. Differences among cultivars were greater when inoculated tubers were incubated anaerobically than when incubated with 5% oxygen. There was no relationship between the relative susceptibilities of cultivars to tuber soft rot in storage in January/February and those of mother tubers after planting. 相似文献
18.
Malgorzata Waleron Krzysztof Waleron Joanna Kamasa Wlodzimierz Przewodowski Ewa Lojkowska 《European journal of plant pathology / European Foundation for Plant Pathology》2011,131(2):341-354
The utility of polymorphism analysis was determined for differentiation of the following subspecies of the Gram-positive plant
pathogenic bacterium, Clavibacter michiganensis: C. m. subsp. michiganensis, C. m. subsp. sepedonicus, C. m. subsp. insidiosus C. m. subsp. nebraskensis, and C. m. subsp. tessellarius. Specific primers designed for amplification of the housekeeping genes recA, rpoB, and rpoD generated 827-, 1037-, and 862-bp DNA fragments, respectively. PCR products obtained from 40 C. michiganensis strains were analysed using RFLP with four restriction endonucleases, and those PCR products with specific RFLP patterns
were sequenced. The genotypes discriminated after PCR–RFLP were specific for each subspecies and also allowed for differentiation
of C. m. subsp. michiganensis strains. Sequence analysis of the recA, rpoB, and rpoD gene fragments also distinguished C. michiganensis subspecies and was useful for phylogenetic analysis of all subspecies. For rapid, inexpensive, and effective differentiation
of the five subspecies in this research, we recommend the amplification of recA and/or rpoD gene fragments and digestion of the PCR products with the restriction endonuclease FnuDII. 相似文献
19.
菊欧文氏菌分子检测技术的研究 总被引：1，自引：0，他引：1
蝴蝶兰细菌性软腐病对蝴蝶兰的生长危害严重, Erwinia chrysanthemi(菊欧文氏菌)、Erwinia carotovora subsp. carotovora(胡萝卜软腐欧文氏菌胡萝卜软腐亚种)是引起蝴蝶兰软腐病的主要病原细菌, 其中E.chrysanthemi被列入我国三类检疫性有害生物。本文对菊欧文氏菌分子检测技术进行了研究, 设计出针对该病原细菌的特异性引物, 应用实时荧光PCR方法检测样品中存在的菊欧文氏菌, 检测灵敏度达到102cfu/mL。 相似文献