Phenotypic and Genotypic Evaluation of Pseudomonas syringae pv. syringae Strains, Causing Citrus Blast in the West of Mazandaran and the East of Guilan

P. syringae pv. syringae (P.s.s), the causal agent of blast of citrus trees, is one of the most important plant pathogens in the world. P.s.s is unique among most P. syringae pathovars according to its ability to cause disease in over 180 species of plants in several unrelated genera. Traditionally, Strains of P.s.s are identified on the basis of biochemical and nutritional tests and symptom expression in host plants. Genomic fingerprinting methods based on the polymerase chain reaction (PCR) have been applied for identification and classification of plant-associated bacteria to the subspecies level. The objectives of this study were the phenotypic and molecular evaluation of P.s. pv. syringae strains causing citrus blast in the West of Mazandaran and the East of Guilan, and study of genetic diversity of P.s.s isolates of citrus by using ERIC and REP-PCR markers.
During 2011 to 2012, citrus infected tissues were sampled from different orchards in the West of Mazandaran and the East of Guilan. Bacterial phenotypes were studied based on standard physiological and biochemical tests. Gram reaction was determined by potassium hydroxide solubility test (KOH test). Strains were grown on KingB medium (KB) and fluorescent pigment production was evaluated. Levan formation, oxidase reaction, potato soft rot, Arginine dihydrolase and induction of the hypersensitive reaction in tobacco leaves (LOPAT tests), were done as described by Schadd et al. The standard strains of P.s. pv. syringae form IVIA were used as reference strains in this study. Pathogenicity Test was done as described by Yessad et al. Citrus seedlings were maintained in a greenhouse at 20°C. In addition, a PCR-based method was used to confirm the genus and species of bacteria by using bacterial specific primer pair’s designed for a specific gene of syringomycin B. Genetic diversity among the strains, was studied by rep-PCR fingerprinting. Genomic fingerprinting was carried out according to the methods of rep-PCR with ERIC primers of Little et al. and with REP primers of Zhao et al. The amplified PCR products were analyzed by gel electrophoresis on a gel containing 1.5% agarose in 1 × TAE buffer. Similarity analyses were done with the NTSYSpc ver. 2.02 software (Exeter Software, New York, USA) as described by Rohlf. Similar coefficients were compared using SM coefficient analysis according to the number and position of bands. Dendrograms were produced according to the unweighted pair-group mean arithmetic method (UPGMA) using NTSYSpc software.
Bacterial strains were identified on the basis of Phenotypic and pathogenicity tests. Twenty- seven isolates of bacteria identified as P. syringae pv. syringae. Strains in which the gram reaction was negative were investigated based on physiological and biochemical characteristics. All P.s.s strains used in this study were negative for oxidase, potato rot, and Arginine dihydrolase but, positive for levan production and the hypersensitive response in tobacco. Strains were positive in gelatin, casein, aesculin, fluorescent pigment production, and tolerant to 5% NaCl, syringomycin production tests, but negative for urease, hydrolysis of starch. Growth at 370C, reduction of nitrate to nitrite and the hydrolysis of tween 80 were variable. These strains produced acid from glucose, xylose, sorbitol, galactose, sucrose and mannitol, but did not produce acid from maltose, Ramnose and the use of lactose were variable. All of the P.s.s strains were pathogenic on seedlings and produced progressive necrotic symptom. To confirm biochemical identification of the bacterial strains, the specific markers were selected for PCR. All 27 isolates of the P.s.s and the standard strains of P.s.s form IVIA produced the expected 198 bp. fragment of the gene syrB with specific markers. The isolates were determinated as P. s. pv. syringae based on phenotypical features and molecular identification. Traditionally, strains of P.s.s are recognized based on biochemical, nutritional, and physiological characteristics of citrus in different parts of Mazandaran. To assess genetic diversity among the strains, ERIC and REP-PCR analysis were used. Strains formed 6 and 5 clusters in the ERIC-PCR and REP-PCR, at 75% similarity level respectively and by the combination data set of both ERIC and REP-PCR, strains formed 6 clusters. In addition, strains formed 2 clusters at 62% similarity level. Cluster one contained the strains of citrus from Kelarabad, Chaboksar, Ketalem. The second Cluster contained other strains with the standard strains of P.s.s (IVIA). Diversity among P.s.s strains using rep-PCR fingerprinting was considerable. The results of this study demonstrated that P.s.s strains isolated from citrus trees are genotypically heterogeneous.
In Iran, P.s.s strains were isolated from citrus and characterized. They emphasized phenotypic and nutritional. Whereas genotypic features of this pathogen have not been studied yet. In our study, according to phenotypic and molecular methods, strains were identified as P. s. pv. syringae. Despite similar phenotypic characteristics, the results indicated the existence of genetic heterogeneity among Pss strains causing citrus blast in the West of Mazandaran and the East of Guilan. The rep-PCR method is low cost, rapid, and reliable to discriminate plant-pathogenic bacteria at the pathovar level. Similar results have been reported in other studies of the strains isolated from other plants. Disclosing the population diversity of each pathovar, in turn, has implications for the implementation of breeding programs, disease management strategies, and ecological and epidemiological studies.