Detection and identification of Pseudomonas savastanoi pv. phaseolicola, the causal agent of the bacterial halo blight of snap bean

Document Type : Research Article

Author

Isfahan University of Technology

Abstract

In March-April of 2020-2022, round and water-soaked spots surrounded by a brown border were observed on the green bean pods delivered to the markets of Isfahan from the southern provinces of the country. A bacterium with a fluorescent appearance and a spindle-shaped structure with irregular edges was routinely isolated from the 81 specimens collected. The genetic fingerprinting employing rep-PCR revealed a resemblance between the PCR product profiles of the isolates, which is consistent with their pathogenic and biochemical characteristics. The PCR reaction of representative isolates produced the expected-size fragments of 16Sr DNA through genus-specific primer sets. Subsequent analysis of the sequences of the fragments confirmed the affiliation of the isolates with Pseudomonas spp. The expected-sized amplicons were amplified and sequenced in nested-PCR tests using phaseolotoxin gene based primer pairs. The results showed that the nucleotide sequences were 99% homologous to Pseudomonas savastanoi pv. phaseolicola (Psp). By conducting a phylogenetic analysis with sequences of housekeeping genes gyrB and rpoD, the isolates were clustered with reference strains of Psp, confirming their identity. Considering the isolates’ morphological, biochemical and molecular characteristics, Psp was identified as the cause of halo blight disease in beans imported to Isfahan from the southern regions. In laboratory conditions, the persistence of the pathogen on seed was evaluated for more than one year. The long-term viability of Psp on seeds may lead to the ongoing prevalence of bean halo blight disease in the country in the forthcoming years, due to the cool and moist conditions resulting from spring rains.

Keywords

References

Aguilera S., Morales A. A., Murillo J. Flores J. L. H., Bravo J. and Zavala S. D. T. 2017.Temperature-mediated biosynthesis of the phytotoxin phaseolotoxin by Pseudomonas syringae pv. phaseolicola depends on the autoregulated expression of the phtABS genes. PLoS One 12: e0178441. doi:10.1371/journal.pone.0178441
Arnold D. L., Lovell H. C., Jackson R. W. and Mansfield J. W. 2011. Pseudomonas syringae pv. phaseolicola: from has been to supermodel. Mol. Plant Pathol.12: 617-627. doi:10.1111/j.1364-3703.2010.00697.x
Bardaji L., Prez-Martinez I., Rodriguez-Moreno L., Rodriguez-Palenzuela P., Sundin G. W. and Murillo J. 2011. Sequence and role in virulence of the three plasmid complement of the model tumor-inducing bacterium Pseudomonas savastanoi pv. savastanoi NCPPB 3335. PLoS One. 6: e25705.
Dell’Olmo, E.; Tiberini, A.; Sigillo, L. 2023. Leguminous Seedborne Pathogens: Seed Health and Sustainable Crop Management. Plants 12, 2040. https:// doi.org/10.3390/plants1210
Ferguson A. R. and Johnston J. S. 1980. Phaseolotoxin-chlorosis, ornithine accumulation and inhibition of ornithine carbamoyltransferase in different plants. Physiological Plant Pathology 16:269–75. doi:10.1016/0048-4059(80)90041-7
Frank J. A., Reich C.I., Sharma S., Weisbaum J. S., Wilson B. A. and Olsen G. J. 2008. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied and Environmental Microbiology74: 2461-2470.
Garibaldi J. A. 1967. Mediafor the enhancement of fluorescent pigment production by Pseudomonas species. Journal of Bacteriology 94: 1296-1299.
Gardan L., Bollet C., Abu Ghorrah M., Grimont F. and Grimont P. A. D. 1992. DNA relatedness among the pathovar strains of Pseudomonas syringae subsp. savastanoi Janse (1982) and proposal of Pseudomonas savastanoi sp. nov. International Journal of Systematic Bacteriology 42: 606–612. doi:10.1099/00207713-42-4-606.
Gomila M., Busquets A., Mulet M., García-Valdés E. and Lalucat J. 2017. Clarification of taxonomic status within the Pseudomonas syringae species group based on a phylogenomic analysis. Front. Microbiol. 8:2422. doi: 10.3389/fmicb.2017.02422  
Hirano S. S. and Upper C. D. 1983. Ecology and epidemiology of foliar bacterial plant pathogens. Annual Review of Phytopathology 21: 243-270.
Lelliott R. A., Billing E., and Hayward A. C. 1966. A determinative scheme for the fluorescent plant pathogenic pseudomonads. Journal of Applied Bacteriology 29:470-489.
Marques A.S.d.A., Corbière R., Gardan L., Toute C., Manceau C., Taylor J. D. and Samson, R. 2000. Multiphasic approach for the identification of the different classification levels of Pseudomonas savastanoi pv. phaseolicola. European Journal of Plant Pathology 106: 715–734. doi:10.1023/A:1026563831461.
Mitchell, R. E. 1978. Halo blight of beans: toxin production by several Pseudomonas phaseolicola isolates. Physiological Plant Pathology 13: 37-49.
Mulet M., Bennasar A., Lalucat J. and Garcia-Valdes E. (2009) An rpoD based PCR procedure for the identification of Pseudomonas species and for their detection in environmental samples. Mol.Cell Probes 23:140–147.
Murray M. G. and Thompson W. F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research. 8: 4321-4325.
Nobel T. J., Young A. J., Douglas C., Williams B. and Mundree S. 2019. Diagnosis and management of halo blight in Australian mung beans. Crop Pasture Science 70: 195–203.
Nobel T. J., Young A. J., Kelly L. A., Barrerro R. A., Douglas C. A., Long H., Williams B. and Mundree, S. 2020.Characterisation of the Pseudomonas savastanoi pv. phaseolicola population found in Eastern Australia associated with halo blight disease in Vigna radiata. Australasian Plant Pathology 49: 515-524. doi:10.1007/s13313-020-00722-8.
 Oguiza J. A., Rico A., Rivas L.A., Sutra L., Vivian A. and Murillo. J. 2004. Pseudomonas syringae pv. phaseolicola can be seprated into two genetic lineages distinguished by the possession of the phaseolotoxin biosynthetic cluster. Microbiology 150: 473-482.
Owen J. G. and Ackerley D. F. 2011. Characterization of pyoverdine and achromobactin in Pseudomonas syringae pv. phaseolicola 1448a. BMC Microbiology 11: 218.  doi:10.1186/1471-2180-11-218.
Prosen D., Hatziloukas E., Schaad N. W. and Panopoulos N. J. 1993. Specific detection of Pseudomonas syringae pv. phaseolicola DNA in bean seed by polymerase chain reaction-based amplification of a phaseolotoxin gene region. Phytopathology 83: 965-970.
Rico A., Lopez R., Asensio C., Aizpun M., Asensio S., Manzanera C. and Murill J. 2003. Nontoxigenic strains of P. syringae pv. phaseolicola are a main cause of halo blight of beans in Spain and escape current detection methods. Phytopathology 93: 1553-1559.
Sawada H. and Fujikawa T. 2019. Genetic diversity of Pseudomonas syringae pv. actinidiae, pathogen of kiwifruit bacterial canker. Plant Pathology 68:1235–1248.
Schwartz H., Steadman J., Hall R. and Forster R. 2005. Compendium of bean diseases. 2nd Ed. American Phytopathological Society Press. Minnesota, USA.
Spilker T., Coenye T., Vandamme P. and Li Puma J. J. 2004. PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. Journal of Clinical Microbiology 42: 2074- 2079.
Taylor J. D., Dudley C. L. and Presly L. 1979a. Studies of halo blight seed infection and disease transmission in dwarf beans. Annals of Applied Biology 93: 267–277. doi:10.1111/j.1744-7348.1979.tb06541.x
Taylor J. D., Phelps K., and Dudley C. L. 1979b. Epidemiology and strategy for the control of halo-blight of beans. Annals of Applied Biology 93: 167–172. doi:10.1111/j.1744-7348.1979.tb06527.x‏
Turner, J. G. 1986. Effect of phaseolotoxin on the synthesis of Arginine and protein. Plant Physiology 86: 760-765.
Widmer F., Seidler R. J., Gillevet P. M., Watrud L. S. and Di Giovanni G. D. 1998. A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (sensu stricto) in environmental samples. Applied and Environmental Microbiology 64: 2545-2553.
Versalovic J., Schneider G. M., De Bruijn F. 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.
Yamamoto S., Kasai H., Arnold D.L., Jackson R.W., Vivian A. and Harayama S. 2000. Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes. Microbiology 146:2385–2394.
Young J. M. 2010. Taxonomy of Pseudomonas syringae. Journal of Plant Pathology:92: S 1.5–S1.14.
Iranian Journal of Plant Pathology
Volume 59, Issue 3
October 2024
Pages 217-234

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