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1.
We constructed a promoter-trap plasmid, pEGFP-V1, to isolate various promoters for foreign gene expression in the leaf-colonizing bacterium Erwinia ananas NR-1. A library was constructed in pEGFP-V1 by introducing genomic DNA fragments upstream of the promoterless EGFP gene to transform E. ananas cells. The library, which consisted of 3500 E. ananas transformants was screened for GFP expression. We found nine strong GFP-expressing clones from the library. Furthermore, we characterized the clones by restriction analysis, sequencing, primer extension analysis, and then quantification of promoter activity. Selected promoters, specifically two (PCF9 and PCF53), gave strong gene expression in E. ananas. Our results indicate that pEGFP-V1 is a useful tool for screening DNA fragments with strong promoter activity in E. ananas.  相似文献   

2.
We recently reported that two diverse types (types 1 and 2) were identified among strains of Erwinia carotovora from mulberry trees. Type 1 strains were similar to E. carotovora subsp. carotovora (Ecc), whereas type 2 strains were distinct from Ecc and other E. carotovora strains. In this study, seven more mulberry strains of type 2 and reference strains were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and randomly amplified of polymorphic DNA (RAPD). On the basis of SDS-PAGE profiles of whole-cell proteins, type 2 strains had high similarity with one another. In addition, they had an unique peptide band with a molecular mass of approximately 28kDa. RAPD analysis showed that they were also effectively differentiated by a strong, specific RAPD fragment for type 2 strains. Based on these two approaches, we have confirmed that the present type 2 strains from mulberry can be discriminated clearly from other soft rot Erwinia species.  相似文献   

3.
To elucidate the role of flagella in biofilm formation by Erwinia carotovora subsp. carotovora EC1, we used a nonflagellate, nonmotile mutant (ΔfliC) and a flagellate, nonmotile mutant (ΔmotA). A biofilm-inducing medium, which contains the yeast peptone (YP) medium plus the salts of M-63 minimal medium, supported biofilm formation to a greater extent than either the YP or Luria Bertani (LB) medium alone. We demonstrated that both the ΔfliC and ΔmotA mutants greatly reduced their ability to form a biofilm on the surface of the wells of polyvinyl chloride (PVC) microtiter plates. The inability of both mutants to form biofilm on the PVC surface was further confirmed with phase-contrast microscopy. Both aflagellate (ΔfliC) and flagellate (ΔmotA) nonmotile mutants were equally defective in attachment to the PVC surface. The treatment of bacteria with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which inhibits the motility of this organism, reduced greatly the biofilm formation. Based on these results, flagella-mediated motility may play an important role in biofilm formation of E. carotovora subsp. carotovora EC1.  相似文献   

4.
A total of 88 strains of Erwinia carotovora subsp. carotovora (Ecc) isolated from various host plants in several geographic regions were screened for production of antibacterial substances using the same strains as indicators. Of the 88 strains, 72 produced antibacterial substances. One of these 72 strains, a Brazilian strain Ecc 32, produced an antibacterial substance active against all tested Ecc strains on TSA medium. The antibacterial spectrum of the compound from Ecc 32 strain was limited to closely related strains of soft-rot Erwinia species. Such a narrow spectrum of activity is typical of bacteriocins. The compound produced by Ecc 32 strain, however, was resistant to some enzymes and detergents. Moreover, the compound was heat-stable and active over a wide pH range. The physical characteristics of the compound were not in agreement with those of bacteriocin or carotovoricin.  相似文献   

5.
We report the construction of a clone library of the Erwinia carotovora subsp. atroseptica genome and the isolation of a gene for endo-pectate lyase. The library, inserted into the PstI site of plasmid pBR322, contains approximately 1700 clones. Five of these produce pectolytic enzymes as detected by a plate screening assay. Using a cloned endo-pectate lyase gene from the related bacterium, E. carotovora subsp. carotovora, as a probe, we found that one pectolytic E. carotovora subsp. atroseptica clone had strong homology to the probe. We characterized that clone by restriction endonuclease mapping and studied its pectolytic protein product. The purified enzyme is an endo-pectate lyase with a cofactor preference for Co2+. The molecular weight of the protein is 31 000 and it has an isoelectric focusing point of 9·2, corresponding to an endo-pectate lyase produced by E. carotovora subsp. atroseptica, but not to the protein product of the E. carotovora subsp. carotovora probe DNA, which has a pI of 9·5. Restriction endonuclease site polymorphisms in the two cloned endo-pectate lyase genes suggest substantial sequence divergence between these two loci.  相似文献   

6.
Agave tequilana is the raw material for the production of the alcoholic beverage tequila. A bacterial disease has affected the A. tequilana crop in recent years. Previous reports based on colony and cell morphology, Gram stain and potato rot indicated that Erwinia sp. is the main pathogen. We isolated a several bacterial isolates capable of producing soft-rot symptoms in greenhouse pathogenicity assays. An extensive characterisation involving pathogenicity tests, fatty acid profile, metabolic and physiological properties, ribosomal DNA sequence and intergenic transcribed spacer amplification (ITS-PCR) and restriction banding pattern (ITS-RFLP) was made of each isolate. Three different species: Erwinia cacticida, Pantoea agglomerans and Pseudomonas sp. were identified. Fatty acid and metabolic profiles gave low similarity values of identification but 16S rDNA sequence, ITS-PCR and ITS-RFLP confirmed the identification of E. cacticida. In the phylogenetic tree, E. cacticida from blue agave was grouped neither with E. cacticida type strains nor with Erwinia carotovora. This is the first report that associates E. cacticida with A. tequilana soft-rot symptoms.  相似文献   

7.
In 1991 and 1992, 12 potato cultivars were screened at two locations for resistance to blackleg, after vacuum infiltration of the seed withErwinia carotovora subsp.atroseptica orE. chrysanthemi. Cultivar differences for resistance toE.c. subsp.Atroseptica andE. chrysanthemi were found which were consistent over locations and years. Seed tubers of the same cultivars were also screened for resistance to bothErwinia spp. by using a tuber slice inoculation method. Correlation coefficients for comparisons between resistance to blackleg in the field and tuber tissue resistance under aerobic or anaerobic conditions were not significant. This could partly be explained by drastic changes in relative tuber tissue resistance of the cultivars within a 5 weeks period after planting in the field. Presprouting of seed tubers in diffuse daylight had a less pronounced effect on relative tuber tissue resistance than planting in the field. Monitoring the process of mother tuber decay during the growing season of 1993 after vacuum infiltration withE.c. subsp.atroseptica andE. chrysanthemi revealed that cultivars differed in the extent to which these bacteria enhanced the process of mother tuber decay. These differences partly explained the cultivar differences for resistance to blackleg in the field.Abbreviations Eca Erwinia carotovora subsp.atroseptica - Ech Erwinia chrysanthemi - NOP Noordoostpolder - Wag Wageningen  相似文献   

8.
Erwinia soft-rot is an important disease of many ornamental potted crops and is one of the most limiting factors in greenhouse calla lily (Zantedeschia spp.) production. Experiments were conducted to test the effect of phosphorous added to soil-less mixes or to nutrient solutions used for irrigation on soft-rot caused by Erwinia carotovora subsp. carotovora (Ecc). Soft-rot incidence increased to 51% when soil-less mix was amended with superphosphate in comparison to regular soil-less mix (no superphosphate added) (31%). In contrast, addition of phosphorous in the nutrient solution met the phosphorous needs of the plant without enhancing soft-rot. Plant height, fresh mass, and number of flowers per plant were greater in calla lilies irrigated with nutrient solution containing phosphorous than no phosphorous treatments. Similar results were obtained in tests conducted in a commercial greenhouse with larger sample size. No statistical differences were found between tubers sprayed with water (control) or with a 0.5 mM solution of KH2PO4 in laboratory experiments to determine the effect of phosphorous on tuber root development. In other experiments, tubers were sprayed with either water, a bacterial cell suspension 1 × 102 cfu ml–1, a solution of 0.5 mM KH2PO4, or a suspension of bacteria in KH2PO4. The results from these tests showed a significant increase of soft-rot development in tubers treated with the suspension of Ecc prepared in a solution of KH2PO4 relative to other treatments. Further laboratory tests indicated that enzymatic activity (polygalacturonase and pectate lyase) of Ecc increased when grown in the presence of phosphorous. These experiments suggest that increased soft-rot in the presence of phosphorous is due to increased virulence of Ecc.  相似文献   

9.
A mulberry epiphytic Enterobacter cloacae MUL1 harbors plasmid pMUL1 encoding five drug-resistance genes. This plasmid was examined upon its conjugal transfer into epiphytic Erwinia herbicola on the phylloplane of mulberry and 12 species of weeds. The plasmid was transferred into Er. herbicola at a frequency of 10–5–10–3/recipient in mulberry and Lolium multiflorum LAM. 1–8 days after wound inoculation with 106–108/ml suspensions. In Chenopodium album L. and C. album L. var. centrorubrum, however, it was transferred only after wound inoculation with a 108/ml suspension, but not with 107/ml or 106/ml suspensions, owing to the weak epiphytic fitness of Ent. cloacae on these weeds. Transconjugants were also obtained for seven other species of weeds in the case of inoculation with a 108/ml suspension. In contrast, when bacterial suspensions were sprayed on mulberry leaves with or without fresh wounds, transconjugants were obtained only in wounded leaves, which were considered suitable for bacterial conjugation. These findings suggest that epiphytic bacteria, including Ent. cloacae and Er. herbicola, may be carriers of drug-resistance genes distributed among plant pathogenic bacteria in nature.  相似文献   

10.
Vascular plants have various inducible resistance mechanisms as defense against pathogens. Mosses, small nonvascular plants (subkingdom Bryophyta), have been little studied in regard to their pathogens or modes of defense. Data here show that Erwinia carotovora, a bacterial plant pathogen that causes softrot in many dicotyledonous plants, can also cause soft rot symptoms in the moss Physcomitrella patens. Infection of moss by E. carotovora required pathogenicity factors similar to those required to infect vascular plants and, again as in vascular plants, salicylic acid (SA) induced moss to inhibit tissue maceration by Erwinia. These data reveal that SA-dependent defense pathways may have evolved before differentiation of vascular and nonvascular plants.  相似文献   

11.
Following failure in control of fire blight with streptomycin, the distribution of streptomycin-resistant strains ofErwinia amylovora in Israel was surveyed. During 1994–1997 109 pear, apple, loquat and quince orchards were monitored. Streptomycin-resistant strains ofE. amylovora were recovered from flowers and from infected branches collected at 18 locations in the Sharon, Galilee and Golan Heights regions. In the Sharon region all the isolated strains ofE. amylovora were streptomycin-resistant, whereas in the Galilee and Golan Heights, resistant as well as sensitiveE. amylovora strains were recovered at different locations. In the southern coastal plain no resistance could be detected. Streptomycin-resistant strains ofE. amylovora did not hybridize with the DNA probe SMP3, and resistance could not be transferred by mating to a sensitive strain, suggesting that streptomycin resistance in Israel is not plasmid-mediated. Fire blight symptoms were observed, for the first time, on pear blossoms during the autumn of 1994. A high population of 2x 106-6x 107 CFU/flower in the autumn of 1995 and of 1996 was correlated with the appearance of blossom blight symptoms.  相似文献   

12.
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13.
Erwinia ananas, the causal agent of bacterial palea browning of rice, was transformed with bioluminescence genes to clarify their behavior on rice plants. Transformant CTB009T2 was used to inoculate rice plants, and the subsequent bioluminescence of CTB009T2 was observed using a two-dimensional luminometer. Luminous spots frequently appeared on anthers after flowering and on dead tissues such as leaf tips, lower leaf sheaths, and leaf blades. In spikelets that developed the disease symptom on the palea, luminous spots appeared 48h after flowering on stigmas, basal parts of ovaries, and lodicules. These results indicate that postflowering anthers and dead tissues on rice plants are important sites for a rapid increase in the pathogen population, and that the multiplication of the pathogen on internal tissues of spikelets after flowering is associated with the appearance of browning.  相似文献   

14.
A yellow pigmented, rod-shaped, Gram negative, peritrichously flagellated, fermentative bacterium was isolated from galls on the stems of the ornamental plant,Gypsophila paniculata. The galls appeared to be similar to those described on the plant by Brown (1934). The isolate proved pathogenic when inoculated into healthy stems; it was identified as a strain ofErwinia herbicola (Lohnis) Dye. It is suggested that the organism should be namedE. herbicola f. sp.gypsophilae.Samenvatting Een geel gepigmenteerde, staafvormige, Gram-negatieve, fermentatieve bacterie met petritriche flagellen werd geïsoleerd uit gallen op de stengels van de sierplantGypsophila paniculata. De ziektesymptomen kwamen overeen met de door Brown in 1934 van deze plant beschreven gallen. Het isolaat bleek na inoculatie in gezonde stengels pathogeen te zijn en werd geïdentificeerd als een stam vanErwinia herbicola. Voorgesteld wordt dit organism aan te duiden alsE. herbicola f. sp.gypsophilae.  相似文献   

15.
The phylogenetic relationships among Erwinia amylovora biovar 4 (the pathogen of bacterial shoot blight of pear in Japan), other biovars of E. amylovora, and Erwinia pyrifoliae were investigated using the sequences of 16S rRNA, gyrB, and rpoD genes. The tested isolates formed two distinct monophyletic groups in the phylogenetic trees constructed based on the gyrB gene, rpoD gene, or a combination of the three genes: group 1 contained E. amylovora biovars 1, 2, and 3; group 2 contained E. amylovora bv. 4 and E. pyrifoliae. This phylogenetic analysis showed that E. amylovora bv. 4 was more closely related to E. pyrifoliae than to other biovars of E. amylovora. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB242876 to AB242925.  相似文献   

16.
The pathogenicity of the bacteriumErwinia carotovorasubsp.atroseptica, which causes potato soft rot, is triggered by short oligogalacturonates released by enzymic degradation of plant cell wall pectin. In the first stage unsaturated digalacturonate (uDG), produced by the action of pectate lyases, is degraded by oligogalacturonide lyase (OGL) to keto-deoxyuronate (DKI). The OGL encoding gene fromE. carotovoraand the corresponding recombinant enzyme were characterized. Measuring the changes in plant cell viability and tissue maceration during soft rot pathogenesis in tissue slices of sprouting potato tubers, it was observed that exposure to uDG and DKI, produced by recombinant OGL, killed up to 30% of the plant cells over a period of 16h. This protected the tissue against maceration byE. carotovorasubsp.atroseptica. Endogenous OGL activity was detected in extracts from sprouting tubers where it may be involved in the conversion of uDG into cell toxic compounds. The results indicate that an additional function of small, diffusable digalacturonates is to induce plant cell death during the rotting process, thus contributing to defence reactions againstE. carotovorasubsp.atroseptica.  相似文献   

17.
The gene chiA, encoding for the endochitinase ChiA, was cloned from Serratia marcescens strain B2, a tomato epiphytic bacterium, and introduced into the epiphytic bacterium Erwinia ananas NR-1, isolated from rice phylloplane. The gene chiA was introduced under the control of two types of promoter into a broad-host-range plasmid vector. The vector contained various fragments with promoter activity isolated from E. ananas chromosomal DNA. The constructed vectors were designated pchiA-V1pcf9 and pchiA-V1pcf53 for their respective promoters. E. ananas NR-1 transformed with either of these vectors produced and secreted ChiA. The antifungal activity of ChiA produced by transformed E. ananas NR-1 was demonstrated in vitro by the inhibition of Pyricularia oryzae germ tube elongation such as bursting of the hyphal tip. Transformed E. ananas NR-1 suppressed the incidence of rice blast caused by P. oryzae under greenhouse conditions; however, the magnitude of the suppressive effect depended on which promoter was used. Both transformants and the nontransformant E. ananas NR-1 survived on rice phylloplane. It is expected that the rice epiphytic bacterium E. ananas NR-1 carrying a chitinolytic enzyme gene is an efficient biological control agent against rice blast.  相似文献   

18.
The infection frequency of mature apple fruit by Erwinia amylovora and the survival of E. amylovora in the fruit stored at low temperature were investigated. The fruit stems (pedicels) of 460 mature apple fruit were inoculated with 105 or 104 cfu of bioluminescent E. amylovora, tagged with lux genes. Nine days after inoculation, 43% and 27% of the fruit inoculated with 105 and 104 cfu, respectively, were infected. All infected fruit looked healthy. After 6 months of storage at 5°C, almost all of the 142 infected fruit had viable E. amylovora. Of the fruit containing E. amylovora internally, 19.5% had latent infections and the rest had blight symptoms. E. amylovora was not uniformly distributed in the fruit flesh, and internal brown lesions were observed where E. amylovora was densely distributed. These findings showed that mature apple fruit may be infected with E. amylovora, especially as latent infections, and act as a source for long-range dissemination.  相似文献   

19.
Bacterial soft rot caused by Erwinia carotovora subsp. carotovora is a major disease in Zantedeschia spp., particularly in cultivars from the section Aestivae. The disease can be partly controlled by cultural measures, but by combining cultural methods with resistant plant material a promising strategy for control of soft rot can be developed. No tests are available for resistance testing in breeding Zantedeschia spp. Therefore, three tests developed for use in potato breeding were adapted for use on eight cultivars of Zantedeschia spp. Variation was found in all three tests. Resistant control cultivar Zantedeschia aethiopica Crowborough scored most resistant in all three tests. Within the section Aestivae, degrees of susceptibility were identified that were in agreement with each other and with field observations, indicating reliability of two of the methods in which tubers were used. The correlation coefficient of these two tests was high. A new non-destructive test method was developed for use on seedlings which involved immersion of leaf disks in a bacterial suspension. The percentage of decayed leaf area was a measure of resistance and results were in general agreement with the other tests. These methods will be useful for breeding for soft rot resistance and performing genetic analyses.  相似文献   

20.
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   

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