Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato

Twenty-eight strains of Pseudomonas syringae pv. actinidiae isolated in 1984, 1987 and 1988 from kiwifruit orchards in Japan were tested for their resistance to copper sulfate. All strains isolated in 1984 were copper sensitive with a minimum inhibitory concentration (MIC) of cupric sulfate of 0.75 mM. However, some strains isolated in 1987 and 1988 were resistant, with the MIC ranging from 2.25 to 3.0 mM. All copper-resistant strains contained at least one of two plasmids, pPaCul (about 70.5 kb) or pPaCu2 (about 280 kb), or both. In a copper-resistant strain Pa429, the location of the copper-resistance gene(s) was examined by insertional inactivation with Tn5. The MIC of copper sulfate in the copper-sensitive mutant obtained by Tn5 tagging decreased from 2.75 to 0.75 mM. The 14.5 kb BamHI fragment, designated pPaCuB14, containing the same locus mutagenized with Tn5 was cloned from pPaCu1. However, pPaCuB14 did not confer copper resistance in the transformant of copper-sensitive strain Pa21R, suggesting that this clone did not contain a full set of copper-resistance gene(s). Then a cosmid library of pPaCu1 was constructed and six cosmid clones hybridized with pPaCuB14 were selected. One of the six cosmids, designated pPaCuC1, conferred a near wild-type level of copper resistance in the transformant of the copper-sensitive strain. pPaCuC1 had a homologous region that hybridized with all of the PCR-amplifled fragments of copA, copB, copR, and copS genes of P. syringae pv. tomato. DNA sequence analysis of the homologous region revealed the existence of four open reading frames (ORF A, B, R and S) oriented in the same direction. The predicted amino acid sequences of ORF A, B, R and S had 80, 70, 97 and 95% identity with CopA, B, R and S of P. syringae pv. tomato, respectively. Received 5 July 2001/ Accepted in revised form 27 September 2001     

Bacterial Apical Necrosis of Mango in Southern Spain: A Disease Caused by Pseudomonas syringae pv. syringae

ABSTRACT A necrotic bacterial disease of mango trees (Mangifera indica) in Spain affecting buds, leaves, and stems is described for the first time. Necrosis of flower and vegetative buds on commercial trees during winter dormancy was the most destructive symptom of the disease. The apical necrosis is caused by Pseudomonas syringae, which was always isolated from mango trees with disease symptoms. Of 95 bacterial strains isolated from symptomatic tissues and characterized from 1992 to 1997, over 90% were identified as P. syringae pv. syringae. Additional strains were isolated from healthy mango trees, and they were identical to the isolates from diseased tissues. Pathogenicity tests on mango plants showed that P. syringae pv. syringae incited the apical necrosis, but that climatic conditions determined the onset of disease development. Populations of total bacteria and of P. syringae and the number of active ice nuclei were monitored over a 3-year period. The largest populations of P. syringae were associated with cool, wet periods that coincided with the highest disease severity, whereas P. syringae was only occasionally detected on healthy trees. The median effective dose was estimated from infectivity titration assays.     

Nontoxigenic Strains of Pseudomonas syringae pv. phaseolicola Are a Main Cause of Halo Blight of Beans in Spain and Escape Current Detection Methods

ABSTRACT From a collection of 152 pseudomonads isolated from diseased beans in Spain, 138 (91%) of the strains were identified as Pseudomonas syringae pv. phaseolicola and the rest as P. syringae pv. syringae. The P. syringae pv. phaseolicola strains produced typical water-soaked lesions on bean pods, although 95 of them did not produce phaseolotoxin in vitro. Ninety-four of these isolates did not produce the expected 0.5-kb product after polymerase chain reaction (PCR) amplification using primers specific for open reading frame (ORF) 6 of the phaseolotoxin (tox) gene cluster and did not contain DNA homologous to ORF 6 in Southern hybridization experiments. To our knowledge, this is the first report of the widespread occurrence in the field of strains of P. syringae pv. phaseolicola lacking the tox cluster, which contrasts sharply with the general belief that Tox(+) isolates are the only ones with epidemiological importance. Additionally, the tox(-) isolates were not specifically detected by a commercial polyclonal antisera in an enzyme-linked immunosorbent assay. Accordingly, it is possible that the certification of seed lots as free of the pathogen cannot be reliably done in Spain, or in any other country where tox(-) strains might occur frequently, using current PCR or serological protocols. The amplification of three avirulence genes by PCR allowed us to make predictions of the P. syringae pv. phaseolicola race structure, as confirmed by plant assays. Six races (races 1, 2, 5, 6, 7, and 9) were identified, with race 7 being the most prevalent (46.1%) followed by races 6 (21.3%) and 1 (9.0%). All the tox(-) isolates contained gene avrPphF, typical of races 1, 5, 7, and 9.     

Plasmids in Pseudomonas syringae pv. pisi carry genes for pathogenicity

All strains tested which are pathogenic to peas and which react with antiserum to Pseudomonas syringae pv. pisi contain two to four plasmids; non-pathogens contain none. Two plasmids from a pathogenic strain were transferred individually to a non-pathogenic recipient strain of Pseudomonas syringae: both plasmids converted the recipient to a pathogen on peas and from hypersensitivity negative to hypersensitivity positive on tobacco. Neither plasmid encoded homoserine catabolism.     

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.     

Detection of New Ethylene-Producing Bacteria, Pseudomonas syringae pvs. cannabina and sesami, by PCR Amplification of Genes for the Ethylene-Forming Enzyme

ABSTRACT Strains of Pseudomonas syringae (78 strains and 43 pathovars) and other strains (79) of plant and insect origin were examined for the presence of the ethylene-forming enzyme gene (efe) by polymerase chain reaction (PCR) assay. The sequence of the efe gene of P. syringae pv. phaseolicola PK2 was used to design two primer sets for amplification of the gene. In addition to P.syringae pv. phaseolicola (the "kudzu strain") and P.syringae pv. glycinea, which were efficient ethylene producers, several strains of P.syringae pvs. sesami and cannabina generated PCR products of the predicted size. A DNA probe of the efe gene, isolated from strain PK2, hybridized to these PCR products, indicating homology to the P.syringae pv. phaseolicola efe gene. PCR restriction fragment length polymorphism analyses suggested that these four pathovars harbor a similar efe gene. Furthermore, the probe hybridized to an indigenous plasmid of P.syringae pv. cannabina, suggesting that the efe gene could be located on a plasmid in this pathovar, but did not hybridize to plas-mids of P.syringae pv. sesami strains. P.syringae pvs. sesami and cannabina strains produced ethylene in King's medium B at levels similar to those of P.syringae pvs. phaseolicola and glycinea. Thus, two new ethylene-producing bacteria were detected by the PCR assay.     

Patterns of interaction between isolates of three pathovars of Pseudomonas syringae and accessions of a range of host and nonhost legume species

Isolates of three pathovars of Pseudomonas syringae were tested against 10 legume species. Some isolates of all pathovars showed cultivar-specific interactions with at least one legume species outside the expected host range. Lablab purpureus and Phaseolus lunatus were found to be hosts to isolates of both P. syringae pv. glycinea and P. syringae pv. phaseolicola, while Lathyrus latifolius was host to isolates of P. syringae pv. pisi and P. syringae pv. glycinea . Lens culinaris showed patterns of interaction with isolates of all three pathovars. Gene models based on mathematical estimates of minimum gene numbers agreed with those previously published for the interactions of P. syringae pv. pisi with Pisum sativum and P. syringae pv. phaseolicola with Phaseolus vulgaris. Two different gene-for-gene models based on five resistance/avirulence gene pairs were proposed to explain observed interactions between Glycine max and P. syringae pv . glycinea . Pathogen isolates which contained no known avirulences defined on their respective host species were found to carry cryptic avirulences recognized by other plant species. Estimates of minimum gene numbers required to explain the interactions of a plant species with all pathogen isolates or to explain the interactions of the isolates of one pathovar with all plant accessions were consistently lower than the sum of the minimum gene numbers required to explain the interactions of each individual component.     

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.     

Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley

Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.     

构树上一种新的丁香假单胞菌致病变种的研究

 构树细菌性疫病是一种荧光假单胞菌引起的新病害。主要症状有叶片角斑,嫩梢肿大和幼枝溃疡。从江苏一带分离获得的12个构树菌株和3个桑树对比菌株进行交互接种试验,发现构树菌株与桑树菌株之间不能交互侵染其寄主。细菌学特征和LOPAT试验及其它37项生理生化和营养特性试验表明,两种菌的表型特征基本相似,仅在7种化合物的利用上存在差异。两种菌的血清学反应无相关性。细胞全蛋白SDS一聚丙烯酰胺凝胶电泳图谱也略有不同。试验结果证明,构树细菌性疫病细菌属于假单胞菌属(Pseudomonas),丁香假单胞菌(P.syringae)的一个新致病变种,定名为Pseudomonas syrzngae pv.broussonetiae pv.nov.     

Evaluation of physiological and serological profiles of Pseudomonas syringae pv. pisi for pea blight identification

Phenotypic variability of the pea blight bacterium, Pseudomonas syringae pv. pisi, was studied on a large collection of strains isolated in France, as well as those obtained from foreign collections. Some other pseudomonads encountered on peas, particularly P.s. pv. syringae, were included in the study to evaluate differential tests for identification purposes. All the isolates that induced watersoaking on the pea cultivar Kelvedon Wonder after inoculation were considered to be P.s. pv. pisi. The other pseudomonads gave either no reaction or a hypersensitive reaction. When they corresponded to P. syringae according to the LOPAT test, they were referred to as P.s. pv. syringae.
P.s. pv. pisi did not show a single uniform phenotype. The variation of the different tests was estimated (fluorescence+ 93%; esculin-86%; dl-lactate-85%; homoserine + 75%; INA + 97%). Three O-sero-groups contained P.s. pv. pisi strains: APT-PIS (88.5%), HEL2 (11.4%) and RIB (0.1%). When the main criteria were combined, eight profiles were encountered within P.s. pv. pisi. This diversity was not linked to race structure or geographical origin of the strains. Profile PI was the most frequent (72.8%), and it was specific to the pathovar pisi . The strains belonging to the other profiles could be confused with some P.s. pv. syringae strains because of the serological heterogeneity of that pathovar. For instance, the pv. pisi strains belonging to profiles P2 and P4 resembled some of the P.s. pv. syringae found on peas and required pathogenicity tests on pea for their identification. The confusing pea isolates represented 12.8% of the total 4740 strains studied.     

Genetic characterization by fluorescent AFLP of Pseudomonas savastanoi pv. savastanoi strains isolated from different host species

The genetic diversity of 71 Pseudomonas savastanoi pv. savastanoi strains isolated from different host species and from diverse geographical regions was determined by fluorescent amplified fragment length polymorphism (f-AFLP) analysis. The study was carried out using three different selective primer combinations. Strains of P. syringae pv. syringae , P. syringae pv. phaseolicola , P. syringae pv. glycinea , P. syringae pv. tagetis and P. amygdali were also included as outgroups. Based on cluster analysis of f-AFLP data, all P. savastanoi pv. savastanoi strains showed a high degree of similarity, grouping in a cluster and forming a taxon clearly separate from outgroup strains. AFLP analyses failed to support placing strains of P. savastanoi pv. savastanoi , P. syringae pv. phaseolicola and P. syringae pv. glycinea in the same species. Strains of P. savastanoi pv. savastanoi formed subclusters that correlated with the host species. Strains identified within these subclusters were related to the geographical region where the strains were isolated. Strains of P. savastanoi pv. savastanoi from olive were divided into two subclusters. Strains from oleander were differentiated from those from ash and were divided into two additional subclusters, distinct from olive strains. Three strains isolated from jasmine showed a high level of similarity among them but, at a lower Dice similarity coefficient, were linked to a subcluster including olive strains. Finally, two strains isolated from privet were similar to strains from olive and were included in the same subcluster.     

Copper resistance in Xanthomonas campestris pv. campestris affecting crucifers in Trinidad

Fifty-six native isolates collected in 12 farming districts of Trinidad and seven reference strains of Xanthomonas campestris pv. campestris were evaluated for resistance to copper in buffered (pH 7.0) and unbuffered (pH 5.6) nutrient agar media. All isolates and reference strains were pathogenic and elicited typical black rot symptoms on a susceptible variety of Brassica olearceae, ‘Copenhagen Market’. Thirty-four and thirty-three native isolates were highly resistant to copper (growth on?≥?200 ppm copper) in buffered and unbuffered media, respectively; however, all the reference strains were highly susceptible to copper. The mean minimum inhibition concentration for the 56 native isolates was 224.6 ppm copper indicating that high levels of copper resistance are present in X. campestris pv. campestris in Trinidad. The association between growth of the 56 isolates and seven reference stains on buffered and unbuffered media was strong (Pearson’s and Spearman’s r?=?0.93; P?<?0.01) suggesting that either medium can be used to evaluate resistance to copper in X. campestris pv. campestris. There was also a strong association between length of time of continuous applications of copper formulations to treat black rot disease and proportion of the native X. campestris pv. campestris with resistance to copper (Pearson’s r?=?0.96; Spearman’s r?=?0.93); however, there was no association between resistance to copper and aggressiveness at 10 days after inoculation.     

Biological control of bacterial speck of tomato under field conditions at several locations in north america

ABSTRACT Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, continues to be a problem for tomato growers worldwide. A collection of nonpathogenic bacteria from tomato leaves plus P. syringae strains TLP2 and Cit7, P. fluorescens strain A506, and P. syringae pv. tomato DC3000 hrp mutants were examined in a greenhouse bioassay for the ability to reduce foliar bacterial speck disease severity. While several of these strains significantly reduced disease severity, P. syringae Cit7 was the most effective, providing a mean level of disease reduction of 78% under greenhouse conditions. The P. syringae pv. tomato DC3000 hrpA, hrpH, and hrpS mutants also significantly reduced speck severity under greenhouse conditions. The strains with the greatest efficacy under greenhouse conditions were tested for the ability to reduce bacterial speck under field conditions at locations in Alabama, Florida, and Ontario, Canada. P. syringae Cit7 was the most effective strain, providing a mean level of disease reduction of 28% over 10 different field experiments. P. fluorescens A506, which is commercially available as Blight-Ban A506, provided a mean level of disease reduction of 18% over nine different field experiments. While neither P. syringae Cit7 nor P. fluorescens A506 can be integrated with copper bactericides due to their copper sensitivity, there exist some potential for integrating these biological control agents with "plant activators", including Actigard. Of the P. syringae pv. tomato DC3000 hrp mutants tested, only the hrpS mutant reduced speck severity significantly under field conditions.     

广东南瓜细菌性叶枯病及其病原鉴定

 在广东省雷州市发生一种南瓜(Cucurbita moschata)叶枯病,病株叶片边缘开始出现水渍状病斑,逐步发展成大病斑,后期病斑焦枯;在叶片上也可形成近圆形水渍状病斑,伴有黄色晕圈,后期病斑联合形成不规则大枯斑;叶柄和匍匐茎被侵染后呈水渍状腐烂。从病斑上分离到一种细菌,在KB培养基上,菌落为椭圆形,乳白色,半透明,边缘参差不齐,紫外灯照射下产生荧光反应。致病性测定结果表明,该病原细菌可侵染6个南瓜品种引起与田间症状相同的叶枯病。生理生化试验结果表明,该病原细菌与丁香假单胞丁香致病变种(Pseudomonas syringae pv. syringae)的特性一致。应用假单胞菌属特异引物Ps-for/Ps-rev和丁香假单胞丁香致病变种组群特异性引物Group III-F/Group III-R,可从该病原细菌中扩增出预期大小分别为1 018 bp和750 bp的目的片段。应用丁香致病变种syrB基因特异性引物B1/B2,可从该病原菌中扩增出预期大小为750 bp的丁香霉素基因片段。基于16S rDNA与gyrB基因序列系统进化分析均表明,南瓜叶枯病菌株与已报道的P. syringae pv. syringae菌株HS191(CP006256)亲缘关系最近,二者聚类在一起形成一个小分支。人工接种条件下,该病原细菌还可侵染西葫芦、丝瓜、茄子、番茄、菜豆、扁豆等植物。这些结果表明,引起广东省南瓜叶枯病的病原为丁香假单胞丁香致病变种(Pseudomonas syringae pv. syringae)。这是首次在中国发现丁香假单胞丁香致病变种引起南瓜叶枯病。     

Genetic diversity of Pseudomonas syringae pv. lachrymans strains isolated from cucumber leaves collected in Poland

Several strains of Pseudomonas syringae pathovar (pv.) lachrymans and related bacterial pathogens were isolated from cucumber ( Cucumis sativus ) leaves collected in central and southern Poland in 2001 and 2002. Twenty five original strains, together with five reference strains of P. syringae pv. lachrymans , pv. syringae and pv. tomato , were genetically characterized by PCR-RFLP (polymerase chain reaction − restriction fragment length polymorphism), ADSRRS (amplification of DNA fragments surrounding rare restriction sites), and PCR-MP (PCR − melting profiles) fingerprinting techniques. Genetic similarity analyses of the PCR-RFLP and ADSRRS fingerprints showed that strains of P. syringae pv. lachrymans form distinct clusters. The results also indicated that the ADSRRS and the PCR-MP fingerprinting techniques may serve as more efficient tools for evaluating genetic similarity among pathovars and strains of P. syringae than PCR-RFLP. The 25 strains showed diverse pathogenicity to cucumber seedlings and biochemical tests were varied. The syrB gene was identified in four cucumber strains, characterized as P. syringae pv. syringae .     

The Relationship of Host Range, Physiology, and Genotype to Virulence on Cantaloupe in Pseudomonas syringae from Cantaloupe Blight Epidemics in France

ABSTRACT In 1993, a bacterial blight caused important losses of cantaloupe (Cucumis melo var. cantalupensis) in southwestern France and has now been reported in all cantaloupe-growing regions of France. The causal agent of this blight is Pseudomonas syringae, although on a worldwide basis this bacterium has not been a major pathogen of melon for over 50 years. To identify the pathovar of the cantaloupe pathogen, we employed biochemical tests, plasmid and chromosomal profiling, and host range studies for 23 strains from cantaloupe and 47 reference strains of 14 pathovars of P. syringae. Numerical analysis of 119 traits, serological typing, syringomycin production, and BOX-polymerase chain reaction profiles did not allow us to differentiate among pathovars related to P. syringae pv. syringae. Host range studies of cantaloupe and references strains on 18 plant species showed that virulence to sugar beet was a common feature of strains virulent on cantaloupe, but was not common to strains avirulent on cantaloupe. Virulence to other species of plants varied among strains, but the overall extent of the host range was proportional to aggressiveness to cantaloupe. We propose that the strains attacking cantaloupe in France be considered P. syringae pv. aptata and that adequate host range testing may reveal that this pathovar is the cause of cantaloupe blight reported in other parts of the world.     

海南芒果蒂腐病菌对吡唑醚菌酯的抗药性测定

从海南芒果主产区采集分离蒂腐病菌Botryodiplodia theobromae。首先采用区分剂量法检测供试菌株对吡唑醚菌酯的抗药性,然后再采用生长速率法测定吡唑醚菌酯对蒂腐病菌的EC_(50)。结果表明:95株芒果蒂腐病菌中有56株在含10mg/L吡唑醚菌酯的PDA培养基中菌落直径大于空白对照的40%,抗性频率为58.95%,来自不同产区的芒果蒂腐病菌对吡唑醚菌酯的抗性频率差异不大。吡唑醚菌酯对供试菌株的EC50在3.12~1525.43mg/L之间,呈连续性正态分布,而抗性最高的菌株的EC_(50)是最敏感菌株EC50的488.92倍。敏感菌株、低抗菌株、中抗菌株及高抗菌株的平均EC_(50)分别为:5.39mg/L、45.34mg/L、144.72mg/L和514.46mg/L。海南芒果蒂腐病菌对吡唑醚菌酯杀菌剂已经产生严重的抗药性。     

Biochemical and pathogenic differences between Kenyan and Brazilian isolates of pseudomonas syringae pv. garcae

Isolates of Pseudomonas syringae pv. garcae from Kenya and Brazil differed in pathogenic and biochemical characters. In inoculations on Coffea arabica var. SL28 from Kenya, only the Kenyan isolates were virulent. The Kenyan isolates were not bacteriocin producers while the Brazilian isolates were active producers comparable to P. s. syringae from lilac ( Syringa vulgaris ). Pigment production separated the two types of P. s. garcae isolates distinctly. The Kenyan isolates produced the UV fluorescent yellow-green siderophore while the Brazilian isolates produced a nonfluorescent brown diffusible pigment on King's B medium. API-20NE diagnostic kits were largely ineffective in distinguishing between biochemical reactions of P. s. garcae isolates from Kenya and Brazil or between these and P. s. syringae . Syringomycin activity on lemon and Geotrichum candidum distinguished P. s. syringae from P. s. garcae isolates. It is concluded that P. s. garcae (as represented by the seven cultures from the National Collection of Plant Pathogenic Bacteria, Harpenden, UK) exists in at least two strains, the Kenyan isolates comprising one strain while the Brazilian isolates comprise one or more distinct strains.