Identification of Pseudomonas cichorii (Swingle 1925) Stapp 1928 in hydroponic lettuce production

Relevance. Lettuce (Latin: Lactúca satíva) is a species of annual herbaceous plant in the genus Lettuce of the Asteraceae family. As a vegetable crop, it is cultivated everywhere in the world, and its hydroponic cultivation technology has received special development in recent years. One of the common pathogens of lettuce is Pseudomonas cichorii, causing bacterial diseases of several important cultivated plants. In this regard, the study of the occurrence of this pathogen is important.

Material and methodology. The study was conducted on the basis of the Department of Agrobiotechnology of the ATI of RUDN University. The samples were provided by a commercial manufacturer of lettuce grown on a flow-through hydroponic line under conditions of minimal microbial contamination. The study of phytopathogenic bacteria includes a number of stages: isolation of bacteria on semi-selective culture media and obtaining a pure culture of bacteria; setting a test for pathogenicity (virulence); studying the phenotypic properties of bacteria; determining the taxonomic position of the isolated strains by molecular methods. All studies were conducted in accordance with the standard methods of identification of phytopathogenic bacteria.

Results. As a result of the work, the distribution of the species Pseudomonas cichorii in the hydroponic culture of lettuce in the Russian Federation was confirmed. Although, according to the EPPO database, P. cichorii was first described in Russia in 1965 by microbiological methods, but isolated bacteria are not available in microbiological collections to confirm this conclusion with appropriate diagnostic methods. Twelve isolates of P. cichoriiwere studied by a biochemical and phytopathological tests, and four isolates (01, 04, 06, and 12) that showed the greatest aggressiveness on host plants and tobacco leaves were identified by DNA sequencing of the 16S rRNA gene fragment. The obtained DNA fragments showed a high similarity (99-100%) with the sequences of P. cichoriifrom the Genebank. Evaluation of the virulence of the isolated isolates on a number of other cultivated plants, and the uniformity of their biochemical characteristics showed that they represent a group of bacteria specialized in lettuce.

1. Koike, Steven T. Vegetable diseases: a color handbook. Burlington, MA: Academic Press, 2007. 448 p. ISBN 978-0-12-373675-7, 978-0-12-373675-8).

2. Bradbury J.F. Pseudomonas cichorii. [Descriptions of Fungi and Bacteria]. In: IMI Descriptions of Fungi and Bacteria. 1981. Wallingford, UK: CAB International. Sheet 695. https://doi.org/10.1079/DFB/20056400695

3. Anzai Kim H., Park J.Y., Wakabayashi H. et al. Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence". Int J Syst Evol Microbiol. 2000;5(4):1563–89. https://doi.org/10.1099/00207713-50-4-1563. PMID 10939664.

4. Trantas E.A., Sarris P.F., Mpalantinaki E.E., Pentari M.G., Ververidis F.N., Goumas D.E. A new genomovar of Pseudomonas cichorii, a causal agent of tomato pith necrosis. European Journal of Plant Pathology. 2013;137(3):477-493. https://doi.org/10.1007/s10658-013-0258-8

5. European and Mediterranean Plant Protection Organization (EPPO) Global database, 2020. [Available: https://gd.eppo.int.].

6. Schaad N.W., Jones J.B., Chun W. Laboratory guide for the identification of plant pathogenic bacteria. Laboratory guide for the identification of plant pathogenic bacteria, 2001. Ed. 3:xii + 373 pp.; 24 ref

7. Ryu E. On the Gram-differentiation of bacteria by the simplest method. J. Jpn. Soc. Vet. Sci. 1938;(17):31.

8. Clinical Microbiology Procedures Handbook, 3rd edition. 2010. ASM. Washington, D.C.

9. Lane D.L., Pace B., Olsen G.J., Stahl D.A., Sogin M.L., Pace N.R.. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. USA. 1985;(82):6955-6959.

10. Eden P.A., Schmidt T.M., Blakemore R.P., Pace N.R. Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNA-specific DNA. International Journal of Systematic Bacteriology. 1991;41(2):324–325. https://doi.org/10.1099/00207713-41-2-324

11. Hall T.A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In: Nucleic acids symposium series. 1999;41(41):95-98.

12. Kumar S., Stecher G., Li M., Knyaz C., and Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution. 2018;(35):1547-1549

13. Lelliott R.A., Stead D.E. Methods for the diagnosis of bacterial diseases of plants. Oxford, UK: Blackwell Scientific Publications, 1987. 216 pp.

14. Saitou N. and Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution. 1987;(4):406-425.

15. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1987;(39):783-791.

16. Tamura K., Nei M., and Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA) 2004;(101):11030-11035.