Identification and origin of races of Pseudomonas syringae pv. phaseolicola from Africa and other bean growing areas

Isolates of Pseudomonas syringae pv. phaseolicola from Africa and other bean growing areas were categorized into nine races on the basis of their reactions to eight differential cultivars following artificial inoculation. Eight hundred and ninety-three isolates representing 303 disease occurrences were initially identified as P.s. pv. phaseolicola by their pathogenicity to bean, cultural and serological characteristics and phage sensitivity. These tests also served to distinguish P.s. pv. phaseolicola from the closely related pathovars P.s . pv. glycinea and P.s. pv. syringae . Detailed race determinations were carried out on 175 selected isolates of p.s. pv. phaseolicola representative of the different geographical regions and hosts in which the pathogen was found and nine races were identified. A number of races (1,2,5,6 and 7) were distributed worldwide with race 6 predominant. Other races were found mainly in Africa; races 3 and 4 in East/Central Africa and races 8 and 9 in Southern Africa. Most isolates were obtained from the major host, Phaseolus vulgaris . Alternative natural hosts included 10 legume species representative of seven different genera ( Cajanus cajan, Desmodium sp., Lablab purpureus, Macroptilium atropurpureum, Neonotonia wightii, Phaseolus acutifolius, P. coccineus, P. lunatus, Vigna angularis and V. radiata ). Of these, Desmodium sp. constitutes a new host record.     

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.     

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.     

Identification of pathovars and races of <Emphasis Type="Italic">Pseudomonas syringae,</Emphasis> the main causal agent of bacterial disease in pea in North-Central Spain,and the search for disease resistance

A total of 298 bacterial isolates were collected from pea cultivars, landraces and breeding lines in North-Central Spain over several years. On the basis of biochemical-physiological characteristics and molecular markers, 225 of the isolates were identified as Pseudomonas syringae, either pv. pisi (110 isolates) or pv. syringae (112), indicating that pv. syringae is as frequent as pv. pisi as causal agent of bacterial diseases in pea. Most strains (222) were pathogenic on pea. Further race analyses of P. syringae pv. pisi strains identified race 4 (59.1% of the isolates of this pathovar), race 2 (20.0%), race 6 (11.8%), race 5 (3.6%) and race 3 (0.9%). Five isolates (4.6%) showed a not-previously described response pattern on tester pea genotypes, which suggests that an additional race 8 could be present in P. syringae pv. pisi. All the isolates of P. syringae pv. syringae were highly pathogenic when inoculated in the tester pea genotypes, and no significant pathogenic differences were observed. Simultaneous infections with P. syringae pv. pisi and pv. syringae in the same fields were observed, suggesting the importance of resistance to both pathovars in future commercial cultivars. The search for resistance among pea genotypes suitable for production in this part of Spain or as breeding material identified the presence of resistance genes for all P. syringae pv. pisi races except for race 6. The pea cultivars Kelvendon Wonder, Cherokee, Isard, Iceberg, Messire and Attika were found suitable sources of resistance to P. syringae pv. syringae.     

Genetic,biochemical and pathogenic diversity of Pseudomonas syringae pv. pisi strains

Pseudomonas syringae pv. pisi is a seedborne pathogen distributed worldwide that causes pea bacterial blight. Previous characterization of this pathogen has been carried out with relatively small and/or geographically limited samples. Here, a collection of 91 strains are examined that include strains from recent outbreaks in Spain (53 strains) and from 14 other countries, and that represent all races and the new race 8, including the type race strains. This collection was characterized on the basis of 55 nutritional tests, genetic analysis (rep‐PCR, amplification of AN3 and AN7 specific markers, and multilocus sequence typing (MLST)) and pathogenicity on the differential pea cultivars to identify races. Principal component analysis and distance dendrograms confirm the existence of two genetic lineages within this pathovar, which are clearly discriminated by the AN3/AN7 markers, rep‐PCR and MLST. Strains from races 1 and 7 amplified the AN3 marker; those from races 2, 6 and 8 amplified AN7, while strains of races 3, 4 and 5 amplified either AN3 or AN7. Nevertheless, strains were not grouped by race type by any of the genetic or biochemical tests. Likewise, there was no significant association between metabolic and/or genetic profiling and the geographical origin of the strains. The Spanish collection diversity reflects the variability found in the worldwide collection, suggesting multiple introductions of the bacteria into Spain by contaminated seed lots.     

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.     

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.     

Genetic relationship between races of Pseudomonas syringae pv.pisi and cultivars of Pisum sativum

Isolates of Pseudomonas syringae pv. pisi from the UK and overseas were categorized into six races on the basis of their reactions to a range of differential pea (Pisum sativum) cultivars. Race 2 was predominant among the isolates examined and this probably reflects its relative international importance. A previously uncharacterized race (race 6) was virulent on all cultivars tested. Resistance to races 1-5 was widespread in commercial cultivars and breeding lines with more than 75% showing resistance to one or more races. A preliminary study of the inheritance of resistance indicated that for races 1, 2 and 3, resistance was controlled by different dominant genes. The genetic basis for the relationship between races of P. syringae pv. pisi and pea cultivars was explained in terms of a gene-for-gene relationship involving five matching gene pairs. With further clarification of the genetics of resistance this host-pathogen association will meet most of the requirements of a model system for the study of the genetic and molecular basis of pathogenicity and host specificity.     

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.     

Improved identification of Pseudomonas savastanoi pv. phaseolicola at the molecular level

Polymerase chain reaction (PCR) technology was used to identify Pseudomonas savastanoi pv. phaseolicola at the DNA level. Oligonucleotide primers were designed on the basis of the DNA sequence of the phaseolotoxin gene cluster of P. s . phaseolicola . Identification of the pathogen was significantly improved and appearance of unspecific bands was greatly diminished by experimentally selecting the most suitable annealing temperature (Tm) value. We obtained a single, strong DNA band of 1.4 kb length, specific for the identification of P. s . phaseolicola , using a PCR programme based on a Tm value of 80 °C.     

Discrimination of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> isolates from sweet and wild cherry using rep-PCR

Bacterial canker is one of the most important diseases of cherry (Prunus avium). This disease can be caused by two pathovars of Pseudomonas syringae: pv. morsprunorum and pv. syringae. Repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) was investigated as a method to distinguish pathovars, races and isolates of P. syringae from sweet and wild cherry. After amplification of total genomic DNA from 87 isolates using the REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX primers, followed by agarose gel electrophoresis, groups of isolates showed specific patterns of PCR products. Pseudomonas syringae pv. syringae isolates were highly variable. The differences amongst the fingerprints of P. syringae pv. morsprunorum race 1 isolates were small. The patterns of P. syringae pv. morsprunorum race 2 isolates were also very uniform, with one exception, and distinct from the race 1 isolates. rep-PCR is a rapid and simple method to identify isolates of the two races of P. syringae pv. morsprunorum; this method can also assist in the identification of P. syringae pv. syringae isolates, although it cannot replace inoculation on susceptible hosts such as cherry and lilac.     

Comparison of Ethylene Production by Pseudomonas syringae and Ralstonia solanacearum

ABSTRACT Strains of Pseudomonas syringae pv. pisi and Ralstonia solanacearum produced ethylene at rates 20- and 200-fold lower, respectively, than strains of P. syringae pvs. cannabina, glycinea, phaseolicola, and sesami. In the current study, we investigated which ethylene biosynthetic pathways were used by P. syringae pv. pisi and R. solanacearum. Neither the activity of an ethylene-forming enzyme nor a corresponding efe gene homolog could be detected in R. solanacearum, suggesting synthesis of ethylene via 2-keto-4-methyl-thiobutyric acid. In contrast, 2-oxoglutarate-dependent ethylene formation was observed with P. syringae pv. pisi, and Southern blot hybridization revealed the presence of an efe homolog in this pathovar. The efe genes from P. syringae pvs. cannabina, glycinea, phaseolicola, pisi, and sesami were sequenced. Nucleotide sequence comparisons indicated that the efe gene in pv. pisi was not as highly conserved as it was in other P. syringae pathovars. The pv. pisi efe homolog showed numerous nucleotide substitutions and a deletion of 13 amino acids at the C-terminus of the predicted gene product. These sequence alterations might account for the lower rate of ethylene production by this pathovar. All ethylene-producing P. syringae pathovars were virulent on bush bean plants. The overlapping host range of these pathovars suggests that horizontal transfer of the efe gene may have occurred among bacteria inhabiting the same host.     

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。     

Genetics of specific resistance in pea (Pisum sativum) cultivars to seven races of Pseudomonas syringae pv. pisi

Seven races of Pseudomonas syringae pv. pisi were distinguished using eight differential cultivars of pea (Pisum sativum). Segregation among F₂ populations of crosses between differential cultivars sequentially inoculated with races of P.s. pv. pisi provided evidence for four and possibly six putative resistance(R)/avirulence(A) gene pairs. R1, R2 and R3 are dominant resistance alleles at single loci, R4 is a dominant allele at a single locus which exhibits variable expression possibly dependent on genetic background. There is evidence that R3 and R4 are at linked loci. Homology tests provided proof of the occurrence of the alleles R2, R3 and R4 in more than one cultivar. Two other alleles, R5 and R6, were postulated to explain the observed segregation ratios in certain crosses.
It can be inferred that P.s. pv. pisi races 2, 3 and 4 each carry a different single a virulence gene, race 6 carries no apparent avirulence genes, and race 7 carries at least A2, A3 and A4. Race 1 carries Al, A3, A4 and possibly A6; race 5 carries A2, A4 and possibly A5 and A6.     

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.     

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.     

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 .     

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.     

Copper Resistance in Pseudomonas syringae Strains Isolated from Mango Is Encoded Mainly by Plasmids

ABSTRACT Bacterial apical necrosis of mango, elicited by Pseudomonas syringae pv. syringae, limits fruit production in southern Spain and Portugal. Examination of a collection of P. syringae pv. syringae isolates for copper resistance showed that 59% were resistant to cupric sulfate. The survey of a mango orchard revealed an increase in frequencies of copper-resistant bacteria after repeated treatments with Bordeaux mixture. These data suggest that selection of copper-resistant strains could be a major reason for control failures following management with copper bactericides. Most copper-resistant isolates harbored plasmids, although the majority of them contained a 62-kb plasmid that also was present in copper-sensitive strains. The 62-kb plasmids were differentiated by restriction enzyme analysis and hybridization to copABCD DNA. The most frequently found copper-resistant plasmid type (62.1) was transferable by conjugation. Southern blot hybridizations showed that genetic determinants partially homologous to copABCD were present in all the copper-resistant strains examined, and usually were associated with plasmids; these determinants were not detected in copper-sensitive strains. The selective pressure exerted by copper bactericide sprays on the diversity of copper resistance determinants in bacterial populations of mango is discussed.