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1.
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   

2.
Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting many rosaceous plants and especially pear tree and apple tree. A protein named harpin, secreted through the Hrp secretion pathway and able to elicit an hypersensitive reaction (HR) on tobacco has recently been isolated. Mutants inhrpN, the gene encoding harpin were described as non pathogenic on immature pear fruit and unable to elicit an HR on tobacco [Weiet al., 1992; Wei and Beer, 1993]. In this paper, the phenotype on plant ofhrpN mutants was carefully determined.hrpN mutants expressed a weak but significant virulence on host plants. Furthermore, when infiltrated into tobacco leaf mesophyll, thehrpN mutants elicited varied responses that fluctuated from null reaction to full necrosis of the infiltrated area. These results show that harpin is not absolutely required neither for pathogenicity on host plant nor for elicitation of an hypersensitive reaction on tobacco. Furthermore, in all the tests performed, mutant blocked in harpin secretion remained non pathogenic and unable to elicit an HR on tobacco. This suggests that factor(s), different from harpin, involved both in pathogenicity and HR eliciting ability is (are) secreted through the Hrp secretion pathway.Abbreviations HR hypersensitive reaction - NSI necrosis severity index - CFU colonie forming units  相似文献   

3.
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4.
Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified fragments of recA, gyrA and rpoS genes was applied for the characterization of Erwinia amylovora and Erwinia strains, which cause fire blight and Asian pear blight in orchards. Primers, constructed on the basis of the published recA, gyrA and rpoS gene sequences of Erwinia carotovora, allowed us to amplify DNA fragments for RFLP differentiation of E. amylovora and E. pyrifoliae and finally to distinguish strains within these species and relate them to pear pathogens from Japan. Three to seven restriction endonucleases were applied for RFLP analysis of each gene fragment. The electrophoretic patterns generated after PCR–RFLP for each of the tested genes, were characteristic and specific for each species and allowed their differentiation. The data show that PCR–RFLP analysis of the recA, gyrA and rpoS gene fragments may be considered as a useful tool for the identification and differentiation of E. amylovora and E. pyrifoliae. Almost identical restriction patterns of the analyzed gene fragments indicated a high relationship of E. pyrifoliae strains from Korea and pear pathogens from Japan and a divergence to E. amylovora. For quick and effective differentiation of E. amylovora strains from Erwinia strains from Asia without nucleotide sequencing we recommend the amplification of recA and rpoS gene fragments and digestion of each of them with restriction endonuclease Hin6I.  相似文献   

5.
A genomic library of Erwinia amylovora isolate T was constructed in the cosmid pLAFR3 and maintained in Escherichia coli. Clones were transferred individually by conjugation into the non-pathogenic isolate P66 of E. amylovora. Transconjugants were screened for restoration of pathogenicity to pear by stab inoculation into sections of immature pear fruits. Three clones complemented P66 restoring pathogenicity and ability to cause the hypersensitive reaction (HR) in Phaseolus vulgaris. Restriction mapping and hybridization experiments showed that the three clones had a common 3·7 kb fragment of E. amylovora DNA. Sub-cloning and insertion mutagenesis with Tn5-lac confirmed that a determinant of pathogenicity and ability to cause the HR (hrp gene) was located on a 2·1 kb HindIII/BamHI fragment within the common DNA. Hybridization experiments using the 2·1 kb HindIII/BamHI fragment as a probe demonstrated that the hrp gene was located in the chromosome of isolate T and that homologous sequences were present in the non-pathogenic isolates P66 and S. Clones which restored hrp function did not affect the growth of isolate P66 in minimal or nutrient-rich media. Transconjugants of Pseudomonas syringae pv. phaseolicola race 1 harbouring the hrp gene(s) cloned from E. amylovora did not cause the HR in susceptible cultivars of bean but symptoms developed more slowly than in the absence of the clones or with pLAFR3 alone.  相似文献   

6.
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   

7.
A virulent strain of Erwinia amylovora, the causal agent of fire blight of Maloideae, and two of its non-virulent hrp mutants (a secretory and a regulatory mutant) were inoculated into apple cell suspensions either alone or in mixed inoculations. In single inoculations, death of 4- to 5-day-old apple cells occurred only when the concentration of the virulent strain of E. amylovora reached a threshold inoculum concentration of 104CFUml–1, while high concentrations of the hrp mutants were unable to kill apple cells. When mixed inoculated with the virulent parental strain, both hrp mutants protected apple cells from death caused by the virulent strain. The protective effect was associated with a decrease in the population level of the virulent strain and it was dependent on the non-virulent to virulent inoculum concentration suggesting a competition between the non-virulent mutant and the virulent strain. However, no differential protective ability between the two types of mutants could be noticed, contrary to previous results obtained with apple seedlings or apple flowers in which the regulatory mutant was significantly more effective than the secretory mutant. In conclusion, inoculation of apple cell cultures with E. amylovora does not seem to be a model suitable for investigating mechanisms leading to protection.  相似文献   

8.
The morphology of apple and pear stigma was investigated with confocal laser scanning microscopy and scanning electron microscopy. The floral colonization process by Erwinia amylovora was studied with gfp-labelled bacteria and confocal laser scanning microscopy to allow the in vivo observation of the pathogen colonization on intact, viable plant tissues without any kind of staining of the specimens. The interaction on the stigma between Erwinia amylovora and Pantoea agglomerans, both labelled with genes encoding for fluorescent proteins (DsRed-GFP), was also investigated. A stylar groove, covered by papillae and dwelling from the stigma along the style, was visualized. In laboratory conditions, this groove was shown to be an important way for E. amylovora migration towards the nectarthodes. Due to its anatomical structure the groove can sustain bacterial multiplication and thus may play an important role on the interactions between the pathogen and the bacterial antagonist P. agglomerans.  相似文献   

9.
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10.
Erwinia piriflorinigrans is a newly described pathogen causing necrosis of pear blossoms. Complete sequencing of the 37‐kb plasmid pEPIR37 common to 27 E. piriflorinigrans strains revealed homology to sequences of the ubiquitous plasmids pEA29 of the fire blight pathogen E. amylovora, plasmid pEP36 of E. pyrifoliae, plasmid pEJ30 of Erwinia sp. from Japan, and genomic regions of the related Rosaceae epiphytic Erwinia species E. tasmaniensis and E. billingiae. A second 5·5‐kb cryptic plasmid pEPIR5, found in 12 E. piriflorinigrans strains, was also sequenced revealing mobilization and replication proteins with similarities to many small ColE1‐type plasmids in Erwinia spp. and other enterobacteria. Functional analyses of pEPIR37 introduced into a strain of E. amylovora cured of pEA29 plasmid, which has a reduced virulence, showed a role in increasing symptom development similar to that observed in E. amylovora carrying plasmid pEA29.  相似文献   

11.
An oligonucleotide probe targeting the rRNA of Erwinia herbicola and Erwinia ananas was designed to detect their cells using fluorescence in situ hybridization. The Cy3-labeled probe hybridized strongly with these species but very weakly with nontarget species such as Erwinia mallotivora, Erwinia nigrifluens, Erwinia cypripedii, Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Escherichia coli. This technique visualized E. herbicola cells after inoculation of kumquat fruits. The probe is promising as a tool for studying population dynamics of E. herbicola and E. ananas.  相似文献   

12.
The interaction between Erwinia amylovora (the causal agent of fire blight) and the physiological status of pear trees was examined under orchard conditions. The physiological status of the trees was defined qualitatively, using host phenology and vigour as measures, and quantitatively, using the sorbitol content in annual shoots as a measure. Qualitatively, tree response to fire blight was governed by phenological stage at the time of infection and vigour: low vigour trees inoculated in the autumn (just before entering dormancy) and high vigour trees inoculated in the spring (soon after bloom) were more susceptible than high vigour trees inoculated in the autumn and low vigour trees inoculated in the spring. Quantitatively, the rate of symptom progression in perennial branches (SPR) was significantly (P ≤ 0.001) correlated to the absolute value of the rate of sorbitol content change (|SCR|). The relationship between hrp genes expression of transformed E. amylovora (estimated according to hrpE and hrpJ expression) and |SCR| was determined on 1 year-old trees. Expression of hrp genes was significantly correlated with |SCR| (P = 0.004) and 63.5% of the variability in the hrp genes expression was attributed to |SCR| values. The expression of hrp genes increased gradually and asymptotically with increasing |SCR| values; further increase in |SCR| did not affect the expression.  相似文献   

13.
Black lesions on shoots of European pear trees observed in an orchard in Yamagata Prefecture in May 2007 were suspected to be caused by a bacterial pathogen. The surface of the colonies isolated on a high sucrose medium did not have the crater morphology that is characteristic of E. amylovora bvs. 1–3, and a specific DNA fragment was amplified from the isolates in the PCR using the EprpoD primer set. The partial sequences of the 16S rRNA gene placed the isolates in the genus Erwinia. The isolates differed serologically from E. amylovora biovars and E. pyrifoliae in an Ouchterlony double-diffusion test although their bacterial properties suggested that they are closely related to E. amylovora biovars and E. pyrifoliae. In a DNA–DNA hybridization test, the relatedness between the isolates and E. amylovora biovars or E. pyrifoliae did not exceed 70% level, indicating that they are independent species. Thus, the isolates belongs to the genus Erwnia but are not E. amylovra or E. pyrifoliae. After succulent pear shoots were injected with bacterial suspensions (109, 108, 107 and 106 cfu/ml) of the isolates, lesions formed with 109 and 108 cfu/ml, but the disease incidence with 108 cfu/ml was much lower than with E. amylovora and E. pyrifoliae. Virulence of the present isolates is thus thought to be very weak. On the basis of these results, we consider that this is a new shoot disease of European pear. In the 2007 season, all affected trees were pulled out after harvest. No symptoms have been observed in field surveys since the fruitlet season in 2007.  相似文献   

14.
Invasion of apple fruit by Erwinia amylovora from fruit-bearing twigs through the abscission layer at fruit maturation was examined. Erwinia amylovora (ca. 105 cfu) tagged with bioluminescence genes from Vibrio fischeri was deposited in artificial wounds on fruit-bearing twigs of apple trees grown in a containment greenhouse on September 22, 27, or October 5, 2004. On October 22, 176 apples were harvested and cut horizontally in half. The upper halves were stamped on plates of selective medium, and the lower halves were flooded with iodine solution to assess maturity. All fruit were symptomless and fully mature. The pathogen was recovered from 19 (10.8%) apples. The result showed that if at least ca. 105 cfu of E. amylovora are present in fruit-bearing twigs at the time of fruit maturation, the bacteria can pass through the abscission layer into the fruit, even though the mature fruit lack symptoms.  相似文献   

15.
Erwinia amylovora, the causal agent of fire blight, is managed by application of bactericides to protect fruit tree blossoms from infection. Monitoring the response ofE. amylovora strains to bactericides is crucial for adequate disease management. The coliform agar medium produced by Merck was recently reported as an effective tool for rapid diagnosis ofE. amylovora (RD-medium). The objective of the present study was to examine the possibility of using the RD-medium forin situ determination of the response ofE. amylovora strains to oxolinic acid and streptomycin. The phenotypic response of 48E. amylovora strains isolated in 2002 to both bactericides was determined with the RD-medium and, for comparison, by a routine laboratory test. The results of 45 samples (93.7%) were in agreement with the findings of the routine laboratory test. Aχ 2 test rejected the null hypothesis that the phenotypic characteristics as determined by the two respective methods differed significantly (P=0.389). Thein situ test was implemented on a national scale in 2003 and the results were in agreement with those obtained in laboratory tests, which suggests that this medium can be usedin situ for monitoring the appearance of resistance inE. amylovora populations. http://www.phytoparasitica.org posting Feb. 11, 2004.  相似文献   

16.
Bacterial wilt is one of the most destructive diseases affecting a wide range of crops in the Cucurbits family including muskmelon (Cucumis melo), cucumber (Cucumis sativus), and squash (Cucubita pepo). The disease is caused by Erwinia tracheiphila, a Gram-negative and xylem-inhabiting species of Erwinia, which pathogenic mechanism is poorly understood. Many Gram-negative phytobacteria induce hypersensitive response (HR) in non-host plants, an immunity reaction triggered by pathogen recognition. With some exemptions, Erwinia species—notably E. amylovora, the causative agent of fire blight of rosaceous crops, and the reclassified soft rot pathogens, Pectobacterium and Dickeya species (formerly E. carotovora and E. chrysanthemi)—have been known to elicit HR in tobacco. However, concerning its pathogenic mechanism, the elicitation of classic HR has not been reported for some less-studied Erwinia species including E. tracheiphila. We characterized the induction of HR by the bacterial wilt pathogen in tobacco (Nicotiana tabacum cultivar ‘Xanthi’) using visual and physiological methods. We surveyed 21 E. tracheiphila strains and found that all of them elicited programmed cell death. Three strains (HCa1-5, UnisCu1-1, and MISpSq) fluorescently labeled with GFP could be visualized in the infiltrated leaves. We aligned the sequences of their HR-inducing protein, harpin (HrpN), predicted the secondary structures, and located the position of putative HR elicitors. We discovered differences between Cucurbita and Cucumis strains and found a close association of E. tracheiphila HrpN with those of Pantoea sp., Erwinia piriflorinigrans, and Erwinia pyrifoliae. Pre-infiltration of tobacco leaves with a lower cell population prevented HR following a subsequent challenge at the same area with HR-inducing levels of inoculum. The selected strains induced leaf conductivity levels similar to the HR-inducing E. amylovora strain E9, and their populations in the leaves decreased days after infiltration. Our results indicate that E. tracheiphila induces a classic HR in tobacco just like other HR-inducing Erwinia species.  相似文献   

17.
吴卓瑾  梁特  石娟 《植物保护学报》2023,50(6):1518-1527
为探究梨火疫病菌解淀粉欧文氏菌Erwinia amylovora在全球的潜在地理分布,基于其全球分布数据和筛选得到的环境变量,利用MaxEnt模型对其在当前气候和未来气候条件下的潜在地理分布进行预测,并利用刀切法和皮尔逊相关性分析法筛选对梨火疫病菌分布有重要影响的环境变量。结果显示,对梨火疫病菌分布有重要影响的环境变量包括2月平均最高温度、1月平均降水量、7月平均最低温度、温度变化方差、昼夜温差月均值和7月平均降水量,表明春季和夏季的温度和降水对梨火疫病菌的分布有较大影响。在当前气候条件下,梨火疫病菌在全球的适生区分布较广,适生区总面积达到5.58×107 km2,且高适生区主要分布在北美洲沿海地区、地中海沿岸和亚洲中部及东部的部分地区;梨火疫病菌在我国的适生区总面积为7.36×106 km2,占全国陆地总面积的76.70%;在未来气候SSP126和SSP585情景下,梨火疫病菌在全球的适生区总面积分别为5.52×107 km2和5.24×107 km2。表明梨火疫病菌对我国大部分地区有潜在威胁,应加强监测与防控。  相似文献   

18.
Conditional expression of harpinPsscauses yeast cell death that shares features of cell death pathway with harpinPss-mediated plant hypersensitive response (HR).Pseudomonas syringae pv.syringae 61 hrp Z gene encodes harpinPss, a 34.7 kD extracellular protein that elicits a hypersensitive response (HR) in plants. Conditional expression of either full-length or truncated hrp Z sequences under the GAL1 promoter caused cell death in Saccharomyces cerevisiae Y187. Plating of pYEUT- hrp Z transformants on a medium containing galactose resulted in complete inhibition of colony formation, whereas their growth on a glucose-based medium was unaffected. Western blot analysis confirmed the expression of harpinPssin yeast cells transformed with pYEUT- hrp Z and grown in galactose-containing medium. A time-dependent decline in the percentage of trypan blue-excluding cells in cultures of pYEUT- hrp Z transformants was observed when cultured on galactose-containing medium. Similarly, the number of viable cells reduced to about 50% within 6 h. There were similarities in the harpinPss-mediated cell death in plants and yeast cell death (YCD). Galactose-induced cell death in pYEUT-hrp Z transformants of S. cerevisiae Y187 was suppressed by a protein kinase inhibitor K252a (10 μ M). The viability of pYEUT- hrp Z transformants was prolonged in the presence of 100 U ml−1catalase suggesting a role for the oxidative burst in YCD that was further supported by the flow cytometric patterns of propidium iodide uptake by yeast cells. Overall, it appears that yeast provides a useful model system to understand the molecular mechanism of harpinPss-mediated cell death.  相似文献   

19.
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   

20.
In many Gram-negative plant pathogenic bacteria the type III secretion system (TTSS), encoded by hrp genes, is essential for pathogenicity in the host and induction of a hypersensitive reaction (HR) in nonhost plants. The expression of hrp genes has been suggested to be repressed in complex media, whereas it is induced in planta and under certain in vitro conditions. We recently reported that XOM2 medium allows efficient hrp expression by Xanthomonas oryzae pv. oryzae. In this study, we investigated hrp-dependent secretion of proteins by the bacteria in vitro. Using modified XOM2, in which bovine serum albumin was added and the pH was lowered to 6.0, we detected at least 10 secreted proteins and identified one as Hpa1. This is the first evidence of protein secretion via TTSS in X. oryzae pv. oryzae.  相似文献   

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