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

Document Type : Research Article

Authors

• S. Sameie-Shirkadeh ¹
• Morteza Golmohammadi ²
• S.A. Alahinia ¹
• S. Bashiri ³

¹ Guilan University

² Citrus and Subtropical Fruit Research Center

³ Kurdistan University

10.22067/jpp.v30i3.30987

Abstract

Introduction: 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.
Materials and Methods: 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 King'B 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.
Results and Discussion: 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.
Conclusion: 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.

Keywords

• Fingerprinting
• Genetic diversity
• Pss
• rep-PCR

20.1001.1.20084749.1395.30.3.1.5

---

References

1- Abbasi V., Rahimian H., Tajick-Ghanbari M.A., and Rezaian V. 2011. The assessment of genetic diversity of strains of Pseudomonas syringae pv.syringae causing bacterial canker in stone fruit in some northern province of IRAN. Iranian Journal of Plant Patholology, 47: 133-135. (in Persian with English abstract)

2- Aldaghi M., Rahimian H., and Mohamadi M. 2010. Comparison of phenotypic, serological and molecular characteristics of Pseudomonas syringae pv. syringae strains, the causal agent of bacterial canker of stone fruits and blight of cereals. Iranian Journal of Plant Pathology, 45(4): 91-93. (in Persian with English abstract)

3- Beigi F., Rahimian H., Goltapeh-mohammadi E., Shams-Bakhsh M., Barzegar A., Busquets A., Garcia- Valdes E., and Lalvkat G. 2012. Phenotypic and Pathogenecity Characteristics of the Agents Causing Citrus Blast Disease in the Northern Provinces of Iran. Iranian Journal of Plant Protection, 43(2):211-222. (in Persian)

4- Bradbury J.F. 1986. Pseudomonas syringae pv. syringae. p.175-177. In: Guide to Plant Pathogenic Bacteria. CAB International Mycological Institute, Kew, England.

5- Cheng G.Y., Legard D.E., Hanter J.E., and Barr T.J. 1989. Modified bean pod assay to detect strains of Pseudomonas syringae pv. syringae that cause bacterial brown spot snap bean. Plant Disease, 73: 419-423.

6- Cirvilleri G., Bonaccorsi A., Scuderi G., and Scortichni M. 2005. Potential biological control activity and genetic diversity of Pseudomonas syringae pv. syringae strains. Phytopathology, 153: 633-750.

7- Golmohammadi M. 2004. Study the distribution and identification of blast disease of citrus in the West of Mazandaran and Gilan East. Citrus Research Institute of the country. The final report of the research project.

8- Gross D.C., Cody Y.S., Proebsting E.L., Radamaker J.W., and Spots R.A. 1984. Ecotypes and pathogenicity of Ice- nucleation active Pseudomonas syringae isolated from deciduous fruit tree orchards. Phytopathology, 74: 241-284.

9- Gross D.C. 1991. Molecular and genetic analysis of toxin production by pathovars of Pseudomonas syringae. Annual Review of Phytopathology, 29:247-278.

10- Hassanzadeh N. 1995. Incidence and progress of different diseases incited by pathovars of Pseudomonas syringae. Journal of Agricultural Sciences Islamic Azad University, 1:5-14.

11- Hildebrand D.C., Schroth M.N., and Huisman O.C. 1982.The DNA homology matrix and non-random variation concepts as the basis for the taxonomic treatment of plant pathogenic and other bacteria. Annual Review of Phytopathology, 20: 235-256.

12- Hirano S.S., and Upper C.D. 2000. Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae pathogen, ice nucleus and epiphyte. Microbology and Molecular Biology Reviews, 624–653.

13- Jones J.B., Chase A.R., and Harris G.K. 1993. Evaluation of the Biolog GN Microplate system for identification of some plant-pathogenic bacteria. Plant Disease, 77:553–558.

14- Klement Z., Farkas G.L., and Loverkovich L. 1974. Hypersensitive reaction induced by phytopathogenic bacteria in tobacco leaf. Phytopathology, 64: 474-477.

15- Kovacs N. 1956. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature, London, 178-703.

16- Lilley A.K., Bailey M.J., and Fry J.C. 1996. Diversity of mercury resistance plasmid obtained by exogenous isolation from the bacteria of sugar beet in three successive seasons. FEMS Microbiology Ecology, 20: 211-227.

17- Little E.L., Bostock R.M., and Kirkpatrick B.C. 1998. Genetic characterization of Pseudomonas syringae pv. syringae strain from stone fruits in California. Applied and Environmental Microbiology, 64: 3818-3823.

18- Louws F.J., Fulbright D.W., Stephens C.T., and De Bruiin F.J. 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Applied and Environmental Microbiology, 80: 2286-2292.

19- Louws F.J., Rademarker J.W., and Brujin F.J. 1999. The three Ds of PCR-Base genoimic analysis of phytobacterial diversity, detection and diagnosis. Annual Review of Phytopathology, 37: 81-125.

20- Mosivand M., Rahimian H., and Shams-Bakhsh M. 2009. Phenotypic and genotypic relateness among Pseudomonas syringae pv. syringae strains isolates from sugarcane, stone fruits and wheat. Iranian Journal of Plant Patholology, 45: 75-85. (in Persian with English abstract)

21- Najafi Pour G., and Taghavi S.M. 2011. Comparison of P. syringae pv. syringae from different hosts based on pathogenicity and BOX-PCR in Iran. Journal of Agricultural Science and Technology, 13: 431-442.

22- Palleroni N.J., Kunisawa R., Contopoulou R., and Doudoroff M. 1973. Nucleic acid homologies in the genus Pseudomonas. International Journal of Systematic Bacteriology, 23:333-339.

23- Rademarker J.W., Hoste B., Louws F.J., kersters k., Swings J., Vauterine L., Vauterine P., and De bruijn F.J. 2000. Comparison of AFLP and rep-PCR genomic fingerprinting with DNA-DNA homology studies: Xanthomonas as a model system. International Journal of Systematic and Evolutionary Microbiology, 50: 665-677.

24- Rohlf F.J. 2000. NTSYSpc, Numerical taxonomy and multivariate analysis system. Version 2.1 Exete software. Applied Biostatistics INC., NY, USA. 493-505.

25- Schadd N.W., Jones J.B., and Chun W. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3thed. American Phytopathology Society Press.

26- Shams-Bakhsh M., and Rahimian H. 1990. Identification agents of Citrus Blast in Mazandaran. p. 150. In Proceedings of the 9th Iranian Plant Protection Congress. 1990. Ferdowsi University of Mashhad, Mashhad, Iran.

27- Sorensen K.N., Kim K.H., and Takemoto J.Y. 1998. PCR Detection of cyclic lipodepsinonapeptide-producing Pseudomonas syringae pv. syringae and similarity of strains. Applied and Environmental Microbiology, 64: 226-230.

28- Versalovic J., Koeuth T., and Lupski J.R. 1991. Distribution of repetitive DNA sequences in Eubacteria and application fingerprinting bacterial genomes. Nucleic Acids Research, 19: 6823-6831.

29- Versalovic J., Scheider M., De bruijn F.J., and Lupski J.R. 1994. Genomic fingerprinting of bacteria using repetitive sequence based polymerase chain reaction. Methods in Molecular and Cellular Biology, 5: 25-40.

30- Weingart H., and Volksch B. 1997. Genetic fingerprinting of Pseudomonas syringae pathovars using ERIC, REP and IS50-PCR. Phytopathology, 87: 339-345.

31- Whiteside L.O., Garnesy S.M., and Timmer L.W. 1989. Campendium of citrus disease. 2thed. American Phytopathology Society Press.

32- Yessad S., Manceau C., and Luisetti J. 1992. A detached leaf assay to evaluate virulence and pathogenicity of strains of Pseudomonas syringae pv. syringae on pear. Plant Disease. 76(4):370-373.

33- Young J.M. 1991. Pathogenicity and identification of the lilac pathogen Pseudomonas syringae pv syringae Van Hall 1902. Annual Applied Biology, 118: 283-298.

34- Young J.M., Takikawa Y., Gardan L., and Stead D.E. 1992. Changing concepts in the taxonomy of plant pathogenic bacteria. Annual Review of Phytopathology, 30: 67-105.

35- Zhao Y., Damicone J.P., Demezas D.H., Rangaswamy V., and Bender C.L. 2000. Bacterial leaf spot diseases of leafly crucifers in Oklahama caused by Pseudomonas syringe pv. maculicola. Plant Disease, 84: 1015- 1020.