Identifying and studying the genetic diversity of pathogenic Pseudomonas strains isolated from pepper, onion, and potato in east and west Azerbaijan provinces

Document Type : Research paper-Persian

Authors

1 Ph.D. Student of Plant Pathology, Department of Plant Protection, Tabriz branch, Islamic Azad University, Tabriz, Iran

2 Associate Professor, Department of Plant Protection, Tabriz branch, Islamic Azad University, Tabriz, Iran

3 Associate Professor, Department of Plant Protection, Mahabad branch, Islamic Azad University, Mahabad, Iran

Abstract

Background and Objectives
The bacterial rot of pepper, onion, and potato is one of the most crucial diseases in the East and West Azerbaijan provinces of Iran. Therefore, accurate identification of the causes of these diseases and the study of their genetic diversity can be beneficial in preventing their occurrence. In the present study, in addition to studying and identifying their morphological and molecular characteristics, the genetic diversity of bacterial species from selected hosts in the provinces of East and West Azerbaijan is studied to obtain comprehensive information.
Material and Methods
Infected potatoes, onions, and peppers with soft rot symptoms were collected from different areas in the East and West Azarbaijan provinces of Iran in the timeframe between 2019 and 2020. Infected samples were cultured on the NA medium. Moreover, purified bacterial colonies were streaked on the Kings’B medium. All isolates that produced fluorescent pigments were identified using biochemical, nutritional, and physiological tests, including LOPAT tests (levan production from sucrose (L), the presence of oxidase (O), the ability to cause rot on potato tubers (P), the presence of arginine dihydrolase (A), and the ability to induce hypersensitivity reaction (HR) on tobacco leaves (T)), nitrate reduction, and the utilization of various carbon sources. All isolates that produced fluorescent pigments were identified as Pseudomonas using phenotypic properties. To assess the genetic diversity within the strains, BOX and ERIC-PCR were analyzed. The UPGMA method was employed to evaluate the similarity matrix and clustering between the strains. For genetic identification, 16S rRNA, gyrB, and ropD genes of strains were amplified and sequenced.
 
Results
A total of 59 isolates were obtained from infected onion, pepper, and potato plants. The bacterial isolates revealed cream colonies with a low convex height, jagged edge, and rugged surface on the nutrient agar medium. All strains produced fluorescent pigments on the King’s B medium and demonstrated Gram-negative and positive reactions to the catalase. In pathogenicity tests, all strains were pathogenic on healthy plants. Moreover, clustering ERIC and BOX-PCR results with UPGMA and Jaccard’s similarity coefficients showed that the strains of potato, pepper, and onion were clustered into five and four main groups, respectively. The gyrB, rpoD, and 16S rRNA nucleotide sequences showed high similarity between the strains and Pseudomonas cichorii.
Discussion
The results showed that Iranian P. marginalis strains are very diverse and are related to the isolation site and the host plant. This high diversity can be due to the long history of cultivating these plants in the provinces of Iran and the high genetic diversity in the bacterial population. Based on previous studies published on Iranian P. marginalis, this is the first report of isolation and identification of P. marginalis, the agent that causes soft rot in pepper, onion, and potato in East and West Azarbaijan provinces.

Keywords

References

Abbasi, V., Rahimian, H., & Tajick-Ghnbari, M. (2013). Genetic variability of Iranian strains of Pseudomonas syringae pv. syringae causing bacterial canker disease of stone fruits. European Journal of Plant Pathology, 135, 225–235.
Achbani, E. H., Sadik, S., Kahkahi, R. E., Benbouazza A., & Mazouz H. (2014). First report on Pseudomonas marginalis bacterium causing soft rot of onion in Morocco. Atlas Journal of Biology, 3, 218–23.
Ahmadi, K., Ebadzadeh, H., Hatami,F., Abdshah, H., & Kazemian, A. (2019). Agricultural Statistics of Iran in 2018-2019: Crops (Volume 1). Ministry agriculture – Jahad Publication, Tehran. (In Farsi).
Akramipour, N. (2013). Investigation of soft rot-causing pectobacteria from different hosts based on molecular properties. [Master of Science Thesis]. Yasuj University. Iran. (In Farsi)
Amanifar, N. (2019). Pseudomonas marginalis as a potential pathogen of greenhouse grown plants and crops with sprinkler irrigation. Iranian Journal of Plant Pathology, 55, 87-104. (In Farsi with English Abstract).
Ausuble, F., Brent, F. M., Kingestone, R. E., Moor, D. D., Smith, J. A., Seideman, J. G., & Struhl, K. (1992). Current Protocol in Molecular Biology. Greene Publishing Associates, Wiley Interscience, New York. 4757 p.
Baghaee-Ravari S., Shams-Bakhsh M., Rahimian H., & Safaie N. (2010). Phenotypic and genotypic diversity of pectolytic erwinias isolated from ornamental hosts in some northern parts of Iran. Iranian Journal of Plant Pathology, 46, 61-64.
Berge, O., Monteil, C. L., Bartoli, C., Chandeysson, C., Guilbaud, C., Sands, D. C., & Morris, C. E. (2014). A user’s guide to a data base of the diversity of Pseudomonas syringae and its application to classifying strains in this phylogenetic complex. PLoS One, 9,e105547.
Bilung , L.M., Pui, C., F., Su’ut, L., & Apun, K. (2018). Evaluation of BOX-PCR and ERIC-PCR as Molecular Typing Tools for Pathogenic Leptospira. Disease Markers, 5, 21-30.
Cottyn, B., Heylen, K., Heyrman, J., Vanhouteghem, K., Pauwelyn, E., Bleyaert, P., Van Vaerenbergh, J., Höfte, M., De Vos, P., & Maes, M. (2009). Pseudomonas cichorii as the causal agent of midrib rot, an emerging disease of greenhouse-grown butter head lettuce in Flanders. Systematic and Applied Microbiology, 32, 211–225.
Dahaghin, L, & Shams-Bakhsh, M. (2014). Identification and genetic diversity of pectolytic phytopathogenic bacteria of mono and dicotyledonous ornamental plants in Iran. Journal of Plant Pathology, 96(2): 271-279.
Danesh, D., & Bahar, M. (1984). Occurrence of bacterial wilt of potato in Iran. In: Proceedings of the 9th triennial Conference of the European Association for Potato Research, Interlaken, CH, 407-408.
Dawson, S. L., Fry, J., & Dancer, B. N. (2012). A comparative evalution of five typing techniques for determining the diversity of fluorescent Pseudomonads. Journal of Microbiology Methods. 50, 9-22.
Emami, P., Mehrabi-Koushki, M., Hayati, J., & Aeini, M. (2019). Detection and identification of some Pseudomonas species causing soft Rot using TUF gene. Biological Journal of Microorganism, 32, 81-93.
Ferrante, P., Takikawa, Y., & Scortichini, M. (2015). Pseudomonas syringae pv. actinidiae strains isolated from past and current epidemics to Actinidia spp. reveal a diverse population structure of the pathogen. European Journal of Plant Pathology, 142, 677–689.
Guilbaud, C., Morris, C.E., Barakat, M., Ortet, P., & Berge, O. (2016). Isolation and identification of Pseudomonas syringae facilitated by a PCR targeting the whole P. syringae group. FEMS Microbiology Ecology, 92, 1-7.
Girard, L., Lood, C., Roknizadeh, H., Van Voort, N., Lavingae, R., & De Mot, R. (2020). Reliable identification of environmental Pseudomonas isolates using the rpoD Gene. Microorganisms, 8, 1166- 1175.
Ghobakhloo, A., Shahriari, D., & Rahimian, H. (2002). Occurrence of bacterial leaf spot and blight of cucurbits in Varamin and evaluation of the resistance of some cultivars and lines of cucumber to the disease. Iranian Journal of Plant Pathology, 38, 59–60. (In Farsi with English Abstract).
Ilicic, R., Balaz, J., Stojsin, V., & Josic, D. (2016). Characterization of Pseudomonas syringae pathovars from different sweet cherry cultivars by RAPD analysis. Genetika, 48, 285–295.
Jinhua, L., Zhaxiang, C., Guoquan, L., & Wang, D. (2007). First report of Pseudomonas marginalis pv. marginalis as a cause of soft rot of potato in China. Australasian Plant Disease Notes, 2, 71–73.
 Kerkhof, L. 1997. A ribosomal RNA operon from Pseudomonas stutzeri Zobell. Gene, 192(2): 241-243.
Keshtkar, A., Khodakaramian, G., and Rouhrazi, K. 2016. Isolation and characterization of Pseudomonas syringae pv. syringae which induce leaf spot on walnut. European Journal of Plant Pathology, 146: 837–846.
Marcelletti, S., & Scortichini, M. 2014. Identification of plant pathogenic Pseudomonas genomospecies of the Pseudomonas syringae complex through multiple comparative approaches. Bacteriology, 104, 1274-1283.
Marques, E., Borges, D. F., & Uesugi, C. (2016). Identification and pathogenicity of Pseudomonas cichorii associated with a bacterial blight of gerbera in the Federal District. 2016. Horticultura Braseleira. 7, 22-31.
Mulet M., Lalucat J., & García-Valdés, E. (2010). DNA sequence-based analysis of the Pseudomonas species. Environmental Microbiology, 12, 1513-1530.
Nordstedt, N. P., Chapin, L. J., Christopher, G., & Jones, M. L. 2020. Identification of Pseudomonas Spp. that increase ornamental crop quality during abiotic stress. Frontiers in Plant Science, 10, 1-12.
Nosratnezhad, F., & Rouhrazi, K. (2018). Characterization and genetic diversity of Pseudomonas syringae isolates from stone fruits in north-western Iran. Journal of Phytopathology, 166, 516-524.
Nouioui, I., Carro, L., Marina, G., Jan P., Tanja, W., Nikos, K., Rüdiger, P., & Markus, G. (2018). Genome-Based Taxonomic Classification of the Phylum Actinobacteria. Frontiers in mocrobilogy, 9, 1-19.
Palleroni, N. j. (2010). The Pseudomonas Story. Environmental Microbiology, 12, 1377–1383.
Popovic, T., Menkovic, J., Prokic, A., Zlatkovic, N., & Obradovic, A. (2021). Isolation and characterization of Pseudomonas syringae isolates affecting stone fruits and almond in Montenegro. Journal of Plant Diseases and Protection, 21, 1-15.
Prakash, O., Verma, V., Sharma, P., Kumar, M., Kanna, M. and Lal, R. (2007). Polyphasic approach of bacterial classification - An overview of recent advances. Indian Journal of Microbiology, 42, 98-108.
Rajwar, A., & Sahgal, M. (2016). Phylogenetic relationships of fluorescent pseudomonads deduced from the sequence analysis of 16S rRNA, Pseudomonas-specific and rpoD genes. 3 Biotech, 6, 80-90.
Rohlf, F. J. (1993). NTSYS-pc: Numerical taxonomy and multivariate analysis system. Version 2.0. New York, NY: Exeter Software.
Rouhrazi, K., & Rahimian, H. (2012). Characterization of Iranian grapevine isolates of Rhizobium (Agrobacterium) spp. Journal of Plant Pathology, 94, 555–560,
Ruan, H., Shi, N., Du, Y., Chen, F., Yang, X., Gan, L., & Dai, Y. (2018). First report of Pseudomonas cichorii causing tomato pith necrosis in Fujian Province, China. Plant disease, 103(1), 145.
Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406–425.
Schaad, N. W., Jones, J. B., & Chun, W. (2001). Laboratory Guide for the Identification of Plant Pathogenic Bacteria. American Phytopathology Society, St. Paul, MN.
Scortichini, M., Marchesi, U., Dettori, M. T., & Rossi, M. P. (2003). Genetic diversity, presence of the syrB gene, host preference and virulence of Pseudomonas syringae pv. syringae strains from woody and herbaceous host plants. Plant Pathology, 52, 277-286.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular and Biology Evolution, 30, 2725-2729.
Tareke, E. T., Karlsoon, P., Sune Eriksson, S., & Margareta, T. (2002). Analysis of acrylamide, a carcinogen formed in heated foodstuffs. Journal of Agricore and Food Chemistry, 50, 4998-5006.
Trantas, E. A., Sarris, P. F., Mpalantinaki, E. E., Pentari, M. G., Ververidis, F., & Goumas, D. E. (2013). A new genomovar of Pseudomonas cichorii, a causal agent of tomato pith necrosis. European Journal of Plant Pathology, 137, 477–493.
Vandamme, P., Pot, B., Gilis, M., de Vos, P., Kersters, K., & Swings, J. (1996). Polyphasic taxonomy, a consensus approach to bacterial systematic. Microbiological reviews, 60, 407-38.
Versalovic, J., Koeuth, T., & Lupski, J. R. (1991). Distribution or repetitive DNA– sequences in eubacteria and application of fingerprinting of bacterial genomes. Nucleic Acid Research, 19: 6823-6831.
Versalovic, J., Schneider, M., de Bruijn, F.J., & Lupski, J. R. (1994). Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods in Molecular Cell Biology, 5, 25–40.
Weisburg, W.G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173, 697-703.
Yamamoto, S., Kasai, H., Arnold, D. L., Jackson, R. W., Vivian, A., & Harayama, S. (2000). Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes. Microbiology, 146, 2385-2394.
 © 2022 by the authors. Licensee SCU, Ahvaz, Iran. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0 license) (http://creativecommons.org/licenses/by-nc/4.0/
Plant Protection (Scientific Journal of Agriculture)
Volume 45, Issue 2
July 2022
Pages 63-83

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