Genetic diversity of Pseudomonas syringae pv. actinidiae,pathogen of kiwifruit bacterial canker

Bacterial canker disease of kiwifruit currently occurs in at least 15 countries, causing serious damage. The causative agent of the disease is Pseudomonas syringae pv. actinidiae (Psa), which is genetically diverse and is currently classified into five biovars, namely, biovars 1, 2, 3, 5 and 6. In Japan, four biovars except biovar 2 have been found so far. These biovars have been confirmed to have differences in the virulence and composition of pathogenicity-related genes, such as toxin biosynthesis and type III effector genes. Biovars 1 and 6 possess the tox island, a genomic island of approximately 38 kb, which contains phaseolotoxin biosynthesis genes (argK-tox cluster) and is confirmed to have been acquired from other bacteria through horizontal transfers. Also, on the megaplasmid possessed by biovar 6, there exist coronatine biosynthesis genes, and biovar 6 has the ability to produce two phytotoxins, phaseolotoxin and coronatine. In 2014, biovar 3, considered to be of foreign origin, was confirmed for the first time in Japan. Biovar 5, whose virulence is relatively weak, is distributed only in a limited area. In addition to the tox island and various plasmids, a large number of mobile genetic elements are confirmed to be present on the Psa genomes, which might have played a major role in helping Psa to acquire new features. In order to understand how Psa acquired the ability to infect kiwifruit systemically, it is important to make polyphasic comparisons with related pathovars, such as P. syringae pv. theae and pv. actinidifoliorum.     

Pseudomonas syringae pv. avellanae pathovar nov., the bacterium causing canker disease on Corylus avellana

The results of an extensive study of 170 morphological, physiological, biochemical, serological and pathological characters of 18 representative isolates from the filbert canker pathogen are presented. These indicate that the bacterium responsible for the disease of filberts ( Corylus avellana ) in Greece is properly characterized as a typical member of the species Pseudomonas syringae. It is placed in group Ia according to the LOPAT tests. The differences in some characters between Pseudomonas syringae pv. syringae and the filbert Pseudomonas , as well as its host specificity, justify its classification as a new pathovar of Pseudomonas syringae and the name of Pseudomonas syringae pv. avellanae pv.nov. is proposed for the bacterium causing the disease 'bacterial canker' of Corylus avellana.     

猕猴桃溃疡病菌的分子检测技术研究

 猕猴桃溃疡病是猕猴桃生产上的主要病害,为建立该病的快速诊断技术,本实验通过RAPD分析获得一条1 300 bp左右的致病菌的特异片段,对该片段进行克隆测序,在测序的基础上设计并合成一对特异引物F7/R7,优化特异引物扩增条件,并验证引物的特异性和灵敏性。利用该特异引物对包括猕猴桃溃疡病菌在内的14个菌株基因组DNA进行PCR扩增表明,只有猕猴桃溃疡病菌能扩增出1条约为950 bp的特异条带,其他菌株及对照均未扩增出特异条带。对采自果园的染病枝干组织和接种致病菌的枝干组织的检测表明,该特异引物能特异性地检测到猕猴桃溃疡病菌的存在,其在组织中的检测灵敏度为100 fg/μL。因此,利用设计合成的特异引物F7/R7,参考优化的体系和程序,结合简单的试剂盒法提取猕猴桃溃疡病菌或植物组织DNA,可以在短时间内完成对该病原菌的分子检测。     

Identification of Pseudomonas syringae pv. syringae causing bacterial leaf blight of Miscanthus sinensis

Journal of Plant Diseases and Protection - In the late summer of 2015, severe leaf blight occurred on Miscanthus sinensis grown on natural riverside lands of the Han River in Seoul, South Korea....     

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.     

Potential spread of kiwifruit bacterial canker (Pseudomonas syringae pv. actinidiae) in Europe

Pest risk analyses (PRAs) are conducted to determine whether an organism is a pest and whether and how it should be regulated. Estimation of the potential area of establishment and pest spread are key factors of this analysis. Tools for modelling and mapping of these key factors have to be quick and easily applicable for a wide variety of organisms with limited data for parameterization. For this purpose, a dispersal kernel model based on a 2Dt‐distribution had been developed in a European Union project (PRATIQUE). The aim of the present study was the evaluation of this spread model hitherto tested on insects, plants, fungi and nematodes in order to determine its applicability to bacterial pests. Therefore, the potential distribution and spread of kiwifruit bacterial canker Pseudomonas syringae pv. actinidiae in Europe was investigated based on climatic suitability and host plant availability. The results of the modelling were compared with the spread history of the pest in Europe. It is shown that this generic spread model can also be applied to a bacterial pest.     

Real‐time and qualitative PCR for detecting Pseudomonas syringae pv. actinidiae isolates causing recent outbreaks of kiwifruit bacterial canker

Since 2008, Pseudomonas syringae pv. actinidiae virulent strains (Psa‐V) have quickly spread across the main areas of kiwifruit (Actinidia deliciosa and A. chinensis) cultivation causing sudden and re‐emerging outbreaks of bacterial canker to both species. The disease caused by Psa‐V strains is considered worldwide as pandemic. Recently, P. syringae strains (ex Psa‐LV, now called PsD) phylogenetically related to Psa‐V have been isolated from kiwifruit, but cause only minor damage (i.e. leaf spot) to the host. The different biological significance of these bacterial populations affecting kiwifruit highlights the importance of having a diagnostic method able to detect Psa‐V, which is currently solely responsible for the severe damage to the kiwifruit industry. In order to improve the specific molecular detection of Psa‐V, a real‐time PCR assay has been developed based on EvaGreen chemistry, together with a novel qualitative PCR (PCR‐C). Both methods are based on specific primer sets for the hrpW gene of Psa. The real‐time PCR and PCR‐C were highly specific, detecting down to 50 and 200 fg, respectively, and were applied to a range of organs/tissues of kiwifruit with and without symptoms. These methods are important tools for both sanitary and certification programmes, and will help to avoid the spread of Psa‐V and to check possible inoculum sources. In addition to being used as routine tests, they will also enable the study of the biology of Psa‐V and the disease that it causes, whilst avoiding the detection of other populations of related P. syringae present in kiwifruit.     

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.     

The problem of bacterial canker of hazelnut in Greece caused by Pseudomonas syringae pv. avellanae1

The present situation of the disease in Greece and its main aspects, causal agent, symptomatology, epidemiology and control measures are described. The characteristics differentiating the pathogen from some other Pseudomonas species are also reported.     

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.     

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 .     

Role of phaseolotoxin production by Pseudomonas syringae pv. actinidiae in the formation of halo lesions of kiwifruit canker disease

Pseudomonas syringae pv. actinidiae, the causal bacterium of kiwifruit canker, induces the formation of chlorotic halo lesions on infected leaves and inhibits the growth of Escherichia coli. The inhibition ofE. coli growth was shown to be reversed by L -arginine or L -citrulline, but not by L -glutamine, suggesting that the pathogen produces a toxin similar to phaseolotoxin, which inhibits ornithine carbamoyltransferase. The toxin was purified from culture broth of P. syringae pv. actinidiae strain Kw11, and was shown by nuclear magnetic resonance to be identical to phaseolotoxin. Assays based on inhibition of E. coli growth and on amplification of a phaseolotoxin fatty acid desaturase gene (ptx) fragment revealed that, among the plant pathogenic bacteria examined, the production of phaseolotoxin is restricted to strains of P. syringae pv. phaseolicola and pv.actinidiae . A non-toxigenic mutant of strain Kw11 generated by disruption of the ptx gene induced the formation of necrotic lesions on kiwifruit leaves; however, the lesions were not surrounded by a chlorotic halo as were those induced by the parent strain. The growth rate of the non-toxigenic mutant in leaf tissue was similar to that of the parent strain. These results suggest that phaseolotoxin production contributes to the formation of chlorotic halo lesions in kiwifruit canker but is not required for multiplication of the pathogenic bacterium during infection.     

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.     

Pseudomonas syringae pv. syringae on pepper seedlings in Italy

Pseudomonas syringae pv. syringae causing leaf spot on pepper seedlings grown in a plant bed is reported in Italy for the first time. The pathogen was identified by means of biochemical, physiological and pathogenicity tests as well as by SDS-polyacrylamide gel electrophoresis of whole-cell proteins. The bacterial isolates showed positive for ice nucleation and biocide production.     

Genetic fingerprinting of Iranian Xanthomonas campestris pv. campestris strains inducing black rot disease of crucifers

Northern Iran has one of the largest and most diverse populations of cultivated crucifers in Iran. Symptoms of black rot disease were observed in 40 % of fields. To assess the genetic diversity of Xanthomonas campestris pv. campestris (Xcc) strains, associated with black rot disease, 40 strains were isolated from infected samples of crucifers such as cabbage, radish, cauliflower, turnip and kohlrabi, and were collected from different geographic regions of northern Iran including West and East Azarbayjan and Ardabil provinces. Bacterial strains were characterized by their morphological, biochemical and physiological features and pathogenicity tests. Four races were found in northern Iran (1, 4, 5 and 6) and the majority of the tested strains belonged to either race 4 (45 %) or race 6 (20 %). To examine the distribution of dispersed repetitive DNA, Enterobacterial Repetitive Intergenic Consensus (ERIC), BOX, Repetitive Extragenic Palindromic (REP) and random amplified polymorphic DNA (RAPD) sequences in the genome of Xcc using conserved primers. The different markers produced characteristic banding patterns and the similarity matrices from binary banding data was derived with the similarity for qualitative data program (SIMQUAL). On the basis of the fingerprint patterns generated by the combination data set of both rep-PCR and RAPD, the Xcc strains were differentiated into seven clusters (A–G) at 76 % similarity level. The geographical origin of the Iranian strains does not seem to be correlated with the RAPD and rep-PCR clusters. The clusters seem to be more related to the race of the strains. This is the first study on genetic diversity of Xcc strains inducing black rot disease of crucifers in Iran.