A Conjugative Plasmid Carrying the efe Gene for the Ethylene-Forming Enzyme Isolated from Pseudomonas syringae pv. glycinea

ABSTRACT Previous work suggested that the efe gene encoding the ethylene-forming enzyme was present in the plasmids of three pathovars of Pseudomonas syringae including glycinea, phaseolicola (kudzu strains), and cannabina. However, no direct evidence to support this assumption had been presented. In the current study, we isolated the conjugative plasmid harboring the efe gene (ethylene plasmid) designated pETH2 from P. syringae pv. glycinea MAFF301683. pETH2 was detected by Southern blot hybridization using the efe probe, marked with the transposon mini-Tn5-Km1, and transferred into P. syringae Ni27(n), which does not produce ethylene. The transconjugant Ni27(n) (pETH2) produced ethylene at a level similar to pv. glycinea MAFF301683. In addition, the plasmid designated pCOR2, which encodes coronatine biosynthesis genes, was detected in the same strain. Although the molecular size of the plasmid pCOR2 was not easily distinguishable from pETH2, pCOR2 transferred independently into Ni27(n) and the transconjugants produced coronatine. These findings suggested that the efe gene has been horizontally dispersed among pathovars of P. syringae by plasmid-mediated conjugation in nature.     

Influence of rootstock on the susceptibility of sweet cherry scions to bacterial canker, caused by Pseudomonas syringae pvs morsprunorum and syringae

In a field trial to determine whether the rootstock influenced the susceptibility of cherry cultivars to bacterial canker three cultivars (Napoleon, Roundel and JI 14039), each grafted on two rootstocks (F 12/1 and Colt), were subjected to natural infectionand to inoculation with three bacterial canker pathogens (Pseudomonas syringae pv. morsprunorum races 1 and 2 and P. syringae pv. syringae). Inoculations were made through leaf scars and through wounds. The high susceptibility of Napoleon and high resistance of JI 14039 were confirmed. Napoleon was more susceptible to inoculation through branches when on F12/1 than when on Colt but the reverse was true for leaf scar inoculations. JI14039 was more susceptible to race 1 inoculated through leaf scars when grown on F12/1 than when on Colt. No rootstock/scion interaction was detected with Roundel.
The complexity of the relationships between the pseudomonad pathogens and their cherry hosts is briefly discussed.     

Rapid and Specific Detection of Virulent Pseudomonas avellanae Strains by PCR Amplification

Specific oligonucleotides, based on hrpW (hypersensitive response and pathogenicity) gene sequences encoding harpin protein in phytopathogenic bacteria, were designed to detect and identify virulent strains of Pseudomonas avellanae by polymerase chain reaction (PCR). A population of virulent P. avellanae strains, isolated in central Italy (Viterbo region), was assessed with hrpW-derived primers, producing a specific band of about 350 base pairs in length. This target was successfully amplified from purified genomic DNA, from bacterial culture and from hazelnut bark tissue. No amplification was obtained when the PCR assay was performed on other plant-pathogenic species from the following genera Agrobacterium, Erwinia, Brenneria, Pseudomonas, Ralstonia, Xanthomonas or from hazelnut-associated bacteria, indicating the specificity of these primers. Moreover DNA from strain ISPaVe-MCB-596, isolated from north Italy (Piedmont region) and belonging to the less aggressive population of P. avellanae, did not amplify in PCR. The PCR assay with the primers described here provides a rapid, specific and sensitive diagnostic method for virulent P. avellanae strains and a useful tool to evaluate the progress of sanitation of the area.     

The Role of Ethylene Production in Virulence of Pseudomonas syringae pvs. glycinea and phaseolicola

ABSTRACT The importance of ethylene production for virulence of Pseudomonas syringae pvs. glycinea and phaseolicola was assayed by comparing bacterial multiplication and symptom development in bean and soybean plants inoculated with ethylene-negative (efe) mutants and wild-type strains. The efe mutants of Pseudomonas syringae pv. glycinea were significantly reduced in their ability to grow in planta. However, the degree of reduction was strain-dependent. Population sizes of efe mutant 16/83-E1 that did not produce the phototoxin coronatine were 10- and 15-fold lower than those of the wild-type strain on soybean and on bean, and 16/83-E1 produced very weak symptoms compared with the wild-type strain. The coronatine-producing efe mutant 7a/90-E1 reached fourfold and twofold lower population sizes compared with the wild-type strain on soybean and bean, respectively, and caused disease symptoms typical of the wild-type strain. Experiments with ethylene-insensitive soybeans confirmed these results. The virulence of the wild-type strains was reduced to the same extent in ethylene-insensitive soybean plants as the virulence of the efe mutants in ethylene-susceptible soybeans. In contrast, the virulence of Pseudomonas syringae pv. phaseolicola was not affected by disruption of the efe gene.     

应用PCR方法快速检测黄瓜细菌性角斑病菌

黄瓜细菌性角斑病是黄瓜上的一种重要细菌病害,其病原为丁香假单胞菌黄瓜致病变种(Pseudomonas syringae pv.lachrymans),目前未见到该病害特异性PCR检测方法的报道。通过分析丁香假单胞菌(P.syringae)不同致病变种glyceraldehyde-3-phosphate dehydrogenase 1(gap1)基因序列设计得到一对Psl特异性PCR引物。利用该引物对丁香假单胞菌不同致病变种、假单胞菌属其他种及其他属的共46株菌株进行了PCR扩增,结果表明,所有不同来源的12株黄瓜细菌性角斑病菌均得到179bp的目标片段,而所有其他参试菌株均无扩增条带,PCR检测的灵敏度为7.5×103cfu/mL。利用该方法可从接种后发病的黄瓜叶片总DNA中检测到特异条带,而健康叶片无条带。该引物的PCR检测方法可直接用于植株总DNA的检测,无需进行病原菌的分离培养,快速简便,适用于进出境检验检疫及种苗健康检测等。     

应用微滴数字PCR同时检测瓜类种子携带果斑病菌和角斑病菌

细菌性果斑病和角斑病是葫芦科作物两大重要细菌病害,病原菌分别为西瓜嗜酸菌Acidovorax citrulli和丁香假单胞菌黄瓜致病变种Pseudomonas syringae pv.lachrymans。两种菌均可通过种子、种苗带菌进行远距离传播。种子检测是预防和控制这两种病害发生的首要环节。本研究应用微滴数字PCR技术(droplet digital PCR,ddPCR)建立了同时检测种子携带西瓜嗜酸菌和丁香假单胞菌的方法。结果显示:两种细菌菌悬液和DNA样品等浓度混合时,ddPCR能同时检测到两种靶标菌的最低混合菌悬液浓度和最低DNA浓度分别为10³ cfu/mL和10^-3 ng/μL,其检测灵敏度是平行测试的real-time PCR方法的10倍;对于非等浓度混合的菌悬液和DNA样品,两种靶标菌菌悬液按浓度比1∶1000(10³∶10⁶ cfu/mL)混合或其DNA浓度比为1∶10000(2.28×10^-3 ng/μL∶22.8 ng/μL)条件下,ddPCR可检测到低浓度的靶标菌,检测灵敏度同样是real-time PCR的10倍。此外,在人工接菌种子测试中,西瓜、甜瓜单粒种子平均带菌量10⁵~10⁶ cfu/粒时,ddPCR方法可检测到带菌率0.2%(n=500)的西瓜、甜瓜种子样品。将分别携带两种菌的种子按比例1∶10混合时ddPCR方法可以准确检出浓度相对低的靶标菌;而使用相同检测引物的real-time PCR检测方法则只能检出西瓜嗜酸菌和丁香假单胞菌带菌率分别为0.2%和2%(n=500)的甜瓜种子混合样品中的西瓜嗜酸菌,未能稳定检出丁香假单胞菌。综上所述,本研究基于ddPCR技术建立了可同时检测两种重要葫芦科种传细菌的方法,检测结果稳定可靠,丰富了当前种传病原细菌的检测技术体系。     

黄瓜细菌性角斑病菌交叉引物恒温扩增检测技术

黄瓜细菌性角斑病是我国黄瓜生产上的重要病害之一,其病原菌为Pseudomonas syringae pv.lachrymans。根据该病原菌甘油醛-3-磷酸脱氢基因保守序列设计引物和探针,建立了交叉引物恒温扩增和核酸试纸条检测技术。菌体DNA检测灵敏度可达0.55 ng,纯菌直接检测灵敏度基本可达到单个细菌。所测试的5株黄瓜细菌性角斑病菌和染病黄瓜叶片均为阳性,其他13株对照菌株均为阴性。该方法灵敏度高,且操作简单,对设备要求低等,适合基层实验室应用。     

Bacterial leaf spot and blight of crucifer plants (Brassicaceae) caused by Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis

Bacterial leaf spot and blight diseases caused by Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pcal) are becoming a significant concern for producers of crucifer crops worldwide. Since Psm was first described in 1911, many have reported on its diverse phenotypic, genetic and pathogenic characteristics. Japanese isolates of Psm are also heterogeneous and differ in their host preferences. Pcal was first described in 2002 and has quickly spread globally. Recent work demonstrated that some isolates that had been identified as Psm are actually Pcal. Pcal was also shown to be split into two groups, A and B, based on bacteriological properties, genetic traits and pathogenicity. Group A of Pcal consists mostly of isolates from Japanese radish and radish, isolated before 1990s, that are more aggressive on radish leaves but less aggressive on other Brassica plants compared with group B. Group B of Pcal consists of recent isolates from various crucifer plants including the pathotype of Pcal. In this review, we suggest that group A of Pcal may have existed since the 1950s and survived as a relatively minor pathogen on radish or Japanese radish, whereas group B emerged in the late 1990s, causing global epidemics because of its stronger virulence on various Brassica crops. We also suggest that emergence of a new group of a pathogenic bacterium may cause a re-emergence or new epidemics of a disease that previously was of minor importance.     

Bacterial inflorescence rot of grapevine caused by Pseudomonas syringae pv. syringae

Molecular sequencing (rpoB) and standard pathological and microbiological methods identified Pseudomonas syringae pv. syringae (Pss) as the causal agent of bacterial inflorescence rot of grapevines (Vitis vinifera) in three vineyards in Tumbarumba, NSW, Australia in 2006 and 2007. Pss strains from shrivelled berries and necrotic inflorescences of diseased grapevines were used to inoculate leaves and inflorescences of potted cv. Semillon grapevines. Pss caused disease symptoms similar to those experienced in the field, including angular leaf lesions, longitudinal lesions in shoot tissues and rotting of inflorescences from before flowering until shortly after fruit set. High humidity promoted symptom severity. The necrotic bunch stem and leaf lesions were susceptible to the development of Botrytis cinerea infections. Cryo‐scanning electron microscopy (cryoSEM) indicated that Pss entered leaves and inflorescence tissues via distorted, open, raised stomata surrounded by folds of tissue that appeared as ‘star‐shaped’ callose‐rich complexes when viewed by UV light microscopy. In necrotic tissues, cryoSEM revealed Pss within petiole parenchyma cells and air‐filled rachis xylem vessels. This is the first report of inflorescence and hence fruit loss caused by Pss in grapevines. The disease is described as ‘bacterial inflorescence rot’ and regarded as one that expands the previously reported pathology of grapevines caused by P. syringae. This study also indicated that infection by Pss might promote destructive B. cinerea infections when the fungus is already present but latent, although further experimentation is needed to prove such an interaction.     

Nucleotide Sequence and Organization of Copper Resistance Genes from Pseudomonas syringae pv. actinidiae

Copper-containing bactericides have been used to control bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae. However, the efficacy of copper has been reduced by the occurrence of copper-resistant strains. Analysis of the DNA sequence of a cluster region containing the copper-resistance genes from P. syringae pv. actinidiae suggested the presence of three possible different systems for copper resistance: copper-trapping, copper-efflux and copper-transport systems. Transposon insertional inactivation analysis indicated that the copper-trapping system was essential for copper resistance.     

Specific Oligonucleotide Primers for the Rapid Identification and Detection of the Agent of Tomato Pith Necrosis, Pseudomonas corrugata, by PCR Amplification: Evidence for two Distinct Genomic Groups

Unique DNA bands from strains representative of two groups of Pseudomonas corrugata, as shown by amplification of their genomic DNA by polymerase chain reaction using short random sequence oligonucleotide primers (RAPD-PCR), were isolated, cloned and sequenced. Two pairs of specific primer sequences, based on the ends of the cloned unique DNA bands from strains IPVCT10.3 and IPVCT8.1, were used in multiplex PCR with a range of P. corrugata strains. All strains produced one of the two specific bands, 1100bp (from the IPVCT10.3-based primers) and 600bp (from the IPVCT8.1-based primers), representing groups designated I and II, respectively. The primers were also tested on a wider range of Pseudomonas species, including the closely-related fluorescent Pseudomonas genomospecies FP1, FP2 and FP3: none of these bacteria produced any bands following amplification by PCR with these primers. The primer sets detected P. corrugata in tomato pith necrosis-infected plants providing a useful tool for rapid identification and epidemiological studies.     

Genetic and physiological evidence for the production of N-acyl homoserine lactones by Pseudomonas syringae pv. syringae and other fluorescent plant pathogenic Pseudomonas species

N-acyl homoserine lactones (AHLs) function as cell density (quorum) sensing signals and regulate diverse metabolic processes in several gram negative bacteria. We report that strains of Pseudomonas syringae pvs. syringae (Pss), tabaci and tomato as well as P. corrugata and P. savastanoi produce difussible AHLs that activate the lux operons of Vibrio fischeri or the tra::lacZ fusion of Agrobacterium tumefaciens. In Pss strain B3A, AHL production occurs in cell density dependent manner. Nucleotide sequence and genetic complementation data revealed the presence of ahlI_Pss, a luxI homolog within the Ahl⁺ DNA of Pss strain B3A. The DNA expresses in AHL-deficient strains of P. fluorescens and E. carotovora subsp. carotovora (Ecc), and restores extracellular enzyme production and pathogenicity in the Ecc strain. The derivatives of Pss strains B3A and 301D carrying chromosomal ahlI::lacZ do not produce AHL, but like their wild type parents, produce extracellular protease and the phytotoxin syringomycin as well as elicit the hypersensitive reaction in tobacco leaves. While these strains also produce a basal level of -galactosidase activity, the expression of ahlI::lacZ is substantially stimulated in the presence of multiple copies of the DNA or by the addition of cell-free spent cultures containing AHL. The activation of -galactosidase production occurs with spent cultures of some, but not all Pseudomonas strains which produce AHL as indicated by the Lux and tra::lacZ assays. Pss strains deficient in the global regulatory genes, gacA or lemA, produce very low levels of AHL. Since inactivation of ahlI_Pss eliminates AHL production and since Ahl⁺ Pseudomonas strains carry the homolog of ahlI_Pss, we conclude that ahlI_Pss specifies a key step in AHL biosynthesis and it has been conserved in many plant pathogenic pseudomonads.     

Detection of Pseudomonas syringae pv. aptata in Irrigation Water Retention Basins by Immunofluorescence Colony-staining

Bacterial blight of cantaloupe (Cucumis melo) caused by Pseudomonas syringae pv. aptata was first observed in south-western France and has since spread to all cantaloupe-growing areas of this country. Use of pesticides registered for this disease has proved ineffective and no commercial cultivars of cantaloupe are resistant to this blight. To develop control strategies for this disease, the principal sources of inoculum were investigated. Among the different sources of inoculum studied, we report the isolation of P. syringae pv. aptata from irrigation water retention basins in south-western France using the immunofluorescence colony-staining (IFC) method. In this study, the pathogen was detected at a low concentration (12 and 70cful^–1) in two different retention basins. These results suggest that P. syringae pv. aptata can survive in water used to irrigate cantaloupe crops and could be a source of inoculum for epidemics of bacterial blight. To develop control strategies for this bacterial disease, the importance of water retention basins as sources of inoculum for bacterial blight of cantaloupe needs to be evaluated relative to other potential sources such as seeds, plants from nurseries and plant debris in the soil.     

Selective detection of Pseudomonas syringae pv. tomato using dot blot hybridization and real-time PCR

Two rapid detection methods based on dot blot hybridization with a nonradioactive DNA probe and molecular beacon-PCR were developed for the specific detection of Pseudomonas syringae pv . tomato , the causal agent of bacterial speck of tomato. A 1378 bp DNA fragment (Acc. No. AM039892), obtained from the extension of a 255 bp fragment generated by a RAPD protocol, was used to find a suitable combination of primers specific for the tomato pathovar. A 138 bp fragment from the genome of P. syringae pv. tomato DC 3000 was used as DNA probe. In dot blots of DNA extracted from either pure cultures or artificially contaminated seeds washes, the probe recognized specifically the tomato pathovar. A molecular beacon was designed from the same region for the specific detection and quantification of P. syringae pv . tomato by real-time PCR. A highly significant correlation was observed between the amount of target DNA and the cycle threshold (Ct). Using a fast protocol for DNA extraction, from pure cultures and from washes of artificially contaminated seeds, the limit of detection was about 1 × 10² CFU. The diagnostic tools developed proved highly specific for P. syringae pv. tomato and simple to use. They can therefore be applied to large-scale testing of tomato seeds and seedlings for the assessment of their phytosanitary condition in nurseries.     

Comparison of ethylene-producing Pseudomonas syringae strains isolated from kudzu (Pueraria lobata) with Pseudomonas syringae pv. phaseolicola and Pseudomonas syringae pv. glycinea

The relationships among strains of Pseudomonas syringae pv. glycinea (Psg) and Pseudomonas syringae pv. phaseolicola (Psp) isolated from kudzu ( Pueraria lobata) and bean ( Phaseolus vulgaris) were investigated. All strains tested showed a close phenotypic similarity, with the exception of the utilization of inositol and mannitol as well as the production of toxins. On this basis the strains could be divided into three groups. Group 1 consists of all strains of pathovar glycinea, group 2 includes all Psp strains isolated from kudzu, and all Psp strains isolated from bean belong to group 3. This grouping was also reflected in the genetic fingerprints using the polymerase chain reaction (PCR) with primers that anneal to dispersed repetitive bacterial sequences (rep-PCR). The rep-PCR generated fingerprints were unique for each of the three groups. The strains of group 2, Psp strains isolated from kudzu, possess certain characteristics of group 1 (ethylene production) and group 2 (phaseolotoxin production). The Psp strains from kudzu can be clearly differentiated from Psp strains isolated from bean. They utilize mannitol, produce ethylene, and are strongly pathogenic to kudzu, bean, and soybean. The results obtained show that the Psp strains from kudzu should be separated from the pathovar phaseolicola and should represent their own pathovar.     

Factors affecting the severity of bacterial canker of pear caused by Pseudomonas syringae pv. syringae

Several factors affecting the severity of bacterial canker of pear were studied. In the orchard, infection of shoots by Pseudomonas syringae pv. syringae occurred only when the inoculum dose exceeded 10⁶ colony-forming units/shoot. However, under favourable conditions in a growth chamber, cankers formed on detached shoots inoculated with 5 cfu/shoot. A second-order polynomial relationship was established between log₁₀ transformed canker length and log₁₀ transformed inoculum dose. In orchard and growth chamber experiments, shoots were susceptible from the time of bud swell until after fruit harvest. The severity of Pseudomonas canker of detached shoots increased if they were frozen at – 10°C for 24 h before inoculation. Shoots were most susceptible when inoculated immediately after wounding, and no cankers developed in the orchard when 3-day-old wounds were inoculated. Additionally, no cankers resulted from inoculation of leaf scars at leaf drop. Actively growing, current-season shoots were more susceptible than shoots that had set a terminal bud. The practical implications of these results are discussed as a basis for control of bacterial canker of pear.     

菜豆晕疫病菌的检测技术研究

通过选择性培养基的筛选,利用菜豆萎蔫毒素基因序列扩增引物和菜豆萎蔫毒素基因缺失菌株独有的ORF6序列的扩增引物,采用双重PCR技术实现了菜豆晕疫病菌的快速检测。     

Occurrence of specific reactions induced by Pseudomonas syringae pv. syringae on bean pods, lilac and pear plants

A study on the pathogenicity of 81 strains of Pseudomonas syringae pv. syringae (PSS) isolated from 16 different hosts was conducted on lilac plants, bean pods and pear seedlings, using artificial inoculation.
Only 55 among the 81 strains induced a necrotic lesion when inoculated on lilac leaves. On bean pods, all but one of the bean isolates, and only eight strains among the 52 strains isolated from other hosts, induced typical green water-soaked lesions. On pear leaves, only pear isolates incited a typical progressive necrotic reaction, the isolates from other origins inducing no symptoms or a weak reaction limited to the inoculation point. This study indicates that in addition to the large variability observed in aggressiveness of PSS strains, host specificity occurred on bean and pear.     

Infection of horse chestnut (Aesculus hippocastanum) by Pseudomonas syringae pv. aesculi and its detection by quantitative real-time PCR

Pseudomonas syringae pv. aesculi is a pathogenic bacterium causing bleeding canker disease of horse chestnut ( Aesculus hippocastanum ). This is a serious disease which has been affecting horse chestnut in several European countries over the last five years; however, very little is known about the biology of the causal agent. One of the obstacles to studying this pathogen is the lengthy procedure associated with confirming its presence on the host. In this study, P. syringae pv. aesculi was isolated from lesions on different parts of horse chestnut and its pathogenicity confirmed on horse chestnut saplings using two inoculation techniques. Real-time PCR primers were developed based on gyrase B gene sequence data for the specific detection of P. syringae pv. aesculi . Primer specificity was tested on isolates of the target pathogen as well as on a broad range of related non-target bacteria and other bacterial spp. which inhabit horse chestnut. The real-time primers reliably amplified P. syringae pv. aesculi down to 1 pg of extracted DNA, with and without the presence of host DNA, and also amplified unextracted DNA in whole cells of the bacterium down to at least 160 colony forming units. Detection and quantification of the target pathogen in phloem and xylem of both naturally infected and inoculated horse chestnut tissues was also demonstrated. This quantitative real-time PCR assay provides the facility to study several important aspects of the biology of P. syringae pv. aesculi on horse chestnut including its potential for dissemination in different substrates.