Phylogenetic Analysis of Phytopathogenic Bacteria Affecting Walnuts (Juglans sp.) in Ukraine
DOI:
https://doi.org/10.15407/microbiolj88.01.003Keywords:
16S rRNA gene, phylogenetic analysis, pathogens of bacterial diseases of walnut, Pseudomonas syringae, Xanthomonas arboricola, Agrobacterium tumefaciensAbstract
Walnuts (Juglans sp.) are affected by a wide range of bacterial pathogens belonging to different taxa, and their accurate identification is necessary for understanding both the etiology of diseases and the development of effective control measures. Phylogenetic analysis based on the analysis of the 16S rRNA gene is widely used in bacterial taxonomy. However, taxonomic studies of pathogens of bacterial diseases of walnuts in Ukraine are poorly known. The aim of this study was to determine the taxonomic position of phytopathogenic bacteria isolated from walnuts (Juglans sp.) in Ukraine using comparative analysis of nucleotide sequences of the 16S rRNA gene. Methods. The 16S rRNA gene was amplified by universal primers pA and pH. Sequence assembly, editing, and alignment were performed using Multalin and Sequence Manipulation Suite software. The rate of the 16S rRNA gene nucleotide sequences similarity of isolated strains with homology nucleotide sequences of typical strains located in GenBank was established using the BLASTN program. Phylogenetic relatedness was established and graphically displayed using the MEGA program. Results. Phylogenetic analysis of 16S rRNA gene sequences showed that isolated Agrobacterium spp. strains have the highest percentage of nucleotide sequence similarity (99.4–99.9%) with Agrobacterium tumefaciens LGM 196. Nucleotide sequences of the 16S rRNA gene of isolated Xanthomonas spp. strains are 98.51–99.65% related to the identical sequence of Xanthomonas arboricola pv. juglandis NCPPB 411. Pseudomonas spp. isolates are phylogenetically related (98.31–99.51%) to Pseudomonas syringae pv. syringae UCM B-1027ᵀ and P. syringae pv. syringae ATCC 19310. Conclusions. The high level of similarity of the 16S rRNA gene sequences isolated from the type strains confirms their taxonomic affiliation to these taxa and emphasizes the importance of phylogenetic analysis in the taxonomy of walnut pathogenic bacteria. Comparative analysis of the 16S rRNA gene sequences in combination with phenotypic characteristics confirms that the bacteria isolated from walnut belong to the species Agrobacterium tumefaciens, Xanthomonas arboricola, and Pseudomonas syringae.
Downloads
References
Baltrus, D. A., McCann, H. C., & Guttman, D. S. (2016). Evolution, genomics and epidemiology of Pseudomonas syringae. Molecular Plant Pathology, 18(1), 152-168. Portico. https://doi.org/10.1111/mpp.12506
Bansal, K., Kumar, S., & Patil, P. B. (2020). Phylogenomic Insights into Diversity and Evolution of Nonpathogenic Xanthomonas Strains Associated with Citrus. MSphere, 5(2). https://doi.org/10.1128/mSphere.00087-20
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(9), e105547. https://doi.org/10.1371/journal.pone.0105547
Costechareyre, D., Rhouma, A., Lavire, C., Portier, P., Chapulliot, D., Bertolla, F., Boubaker, A., Dessaux, Y., & Nesme, X. (2010). Rapid and Efficient Identification of Agrobacterium Species by recA Allele Analysis. Microbial Ecology, 60(4), 862-872. https://doi.org/10.1007/s00248-010-9685-7
Dankevych, L. A., Zarudniak, M. I., Patyka, V. P., Dankevych, A. E., & Mychnik, F. V. (2024). Phenotypical Characteristics of Walnut's (Juglans sp.) Bacterial Diseases Agent. Mikrobiolohichnyi Zhurnal, 86(5), 20-31. [In Ukrainian]. https://doi.org/10.15407/microbiolj86.05.020
De Cleene, M., & De Ley, J. (1976). The host range of crown gall. The Botanical Review, 42(4), 389-466. https://doi.org/10.1007/BF02860827
Delamuta, J. R. M., Scherer, A. J., Ribeiro, R. A., & Hungria, M. (2020). Genetic diversity of Agrobacterium species isolated from nodules of common bean and soybean in Brazil, Mexico, Ecuador and Mozambique, and description of the new species Agrobacterium fabacearum sp. nov. International journal of systematic and evolutionary microbiology, 70(7), 4233-4244. https://doi.org/10.1099/ijsem.0.004278
Dia, N. C., Morinière, L., Cottyn, B., Bernal, E., Jacobs, J. M., Koebnik, R., Osdaghi, E., Potnis, N., & Pothier, J. F. (2022). Xanthomonas hortorum - beyond gardens: Current taxonomy, genomics, and virulence repertoires. Molecular plant pathology, 23(5), 597-621. https://doi.org/10.1111/mpp.13185
Edwards, U., Rogall, T., Blöcker, H., Emde, M., & Böttger, E. C. (1989). Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic acids research, 17(19), 7843-7853. https://doi.org/10.1093/nar/17.19.7843
Farrand, S. K., van Berkum, P. B., & Oger, P. (2003). Agrobacterium is a definable genus of the family Rhizobiaceae. International Journal of Systematic and Evolutionary Microbiology, 53(5), 1681-1687. https://doi.org/10.1099/ijs.0.02445-0
Flores-Félix, J. D., Menéndez, E., Peix, A., García-Fraile, P., & Velázquez, E. (2020). History and current taxonomic status of genus Agrobacterium. Systematic and Applied Microbiology, 43(1), 126046. https://doi.org/10.1016/j.syapm.2019.126046
Gasic, K., Prokic, A., Ivanovic, M., Kuzmanovic, N., & Obradovic, A. (2012). Differentiation of Pseudomonas syringae pathovars originating from stone fruits. Pesticidi i Fitomedicina, 27(3), 219-229. https://doi.org/10.2298/PIF1203219G
Giovanardi, D., Bonneau, S., Gironde, S., Saux, M. F.-L., Manceau, C., & Stefani, E. (2015). Morphological and genotypic features of Xanthomonas arboricola pv. juglandis populations from walnut groves in Romagna region, Italy. European Journal of Plant Pathology, 145(1), 1-16. https://doi.org/10.1007/s10658-015-0809-2
Gomila, M., Busquets, A., Mulet, M., García-Valdés, E., & Lalucat, J. (2017). Clarification of Taxonomic Status within the Pseudomonas syringae Species Group Based on a Phylogenomic Analysis. Frontiers in Microbiology, 8. https://doi.org/10.3389/fmicb.2017.02422
Hördt, A., López, M. G., Meier-Kolthoff, J. P., Schleuning, M., Weinhold, L.-M., Tindall, B. J., Gronow, S., Kyrpides, N. C., Woyke, T., & Göker, M. (2020). Analysis of 1,000+ Type-Strain Genomes Substantially Improves Taxonomic Classification of Alphaproteobacteria. Frontiers in Microbiology, 11. https://doi.org/10.3389/fmicb.2020.00468
Ilicic, R., Bagi, F., Blagojevic, M., Gosic, J., Milovanovic, P., & Popovic, T. (2021). Etiology of bacterial diseases of young walnut trees in Serbia. Pesticidi i Fitomedicina, 36(3), 101-109. https://doi.org/10.2298/PIF2103101I
Keshtkar, A. R., Khodakaramian, G., & Rouhrazi, K. (2016). Isolation and characterization of Pseudomonas syringae pv. syringae which induce leaf spot on walnut. European Journal of Plant Pathology, 146(4), 837-846. https://doi.org/10.1007/s10658-016-0962-2
Kim, H. S., Cheon, W., Lee, Y., Kwon, H.-T., Seo, S.-T., Balaraju, K., & Jeon, Y. (2021). Identification and characterization of Xanthomonas arboricola pv. juglandis Causing Bacterial Blight of Walnuts in Korea. The Plant Pathology Journal, 37(2), 137-151. https://doi.org/10.5423/PPJ.OA.12.2020.0217
Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology, 64(Pt_2), 346-351. https://doi.org/10.1099/ijs.0.059774-0
Kuzmanović, N., Puławska, J., Prokić, A., Ivanović, M., Zlatković, N., Jones, J. B., & Obradović, A. (2015). Agrobacterium arsenijevicii sp. nov., isolated from crown gall tumors on raspberry and cherry plum. Systematic and Applied Microbiology, 38(6), 373-378. https://doi.org/10.1016/j.syapm.2015.06.001
Lapage, S. P., Sneath, P. H. A., Lessel, E. F., et al., editors. International Code of Nomenclature of Bacteria: Bacteriological Code, 1990 Revision. Washington (DC): ASM Press; 1992.
Lassalle, F., Planel, R., Penel, S., Chapulliot, D., Barbe, V., Dubost, A., Calteau, A., Vallenet, D., Mornico, D., Bigot, T., Guéguen, L., Vial, L., Muller, D., Daubin, V., & Nesme, X. (2017). Ancestral Genome Estimation Reveals the History of Ecological Diversification in Agrobacterium. Genome Biology and Evolution, 9(12), 3413-3431. https://doi.org/10.1093/gbe/evx255
Loper, J. E., Hassan, K. A., Mavrodi, D. V., Davis, E. W., Lim, C. K., Shaffer, B. T., Elbourne, L. D. H., Stockwell, V. O., Hartney, S. L., Breakwell, K., Henkels, M. D., Tetu, S. G., Rangel, L. I., Kidarsa, T. A., Wilson, N. L., van de Mortel, J. E., Song, C., Blumhagen, R., Radune, D., … Paulsen, I. T. (2012). Comparative Genomics of Plant-Associated Pseudomonas spp.: Insights into Diversity and Inheritance of Traits Involved in Multitrophic Interactions. PLoS Genetics, 8(7), e1002784. https://doi.org/10.1371/journal.pgen.1002784
LPSN - List of Prokaryotic names with Standing in Nomenclature https://lpsn.dsmz.de/
Martins, L., Fernandes, C., Blom, J., Dia, N. C., Pothier, J. F., & Tavares, F. (2020). Xanthomonas euroxanthea sp. nov., a new xanthomonad species including pathogenic and non-pathogenic strains of walnut. International Journal of Systematic and Evolutionary Microbiology, 70(12), 6024-6031. https://doi.org/10.1099/ijsem.0.004386
Miller, H. N., Miller, J. W., & Crange, G. L. (1975). Leaf, stem, crown, and root galls induced in chrysanthemum by Agrobacterium tumefaciens. Phytopathology, 65(7), 805-811. https://doi.org/10.1094/Phyto-65-805
Mousavi, S. A., Österman, J., Wahlberg, N., Nesme, X., Lavire, C., Vial, L., Paulin, L., de Lajudie, P., & Lindström, K. (2014). Phylogeny of the Rhizobium-Allorhizobium-Agrobacterium clade supports the delineation of Neorhizobium gen. nov. Systematic and Applied Microbiology, 37(3), 208-215. https://doi.org/10.1016/j.syapm.2013.12.007
Osdaghi, E. (2022). Xanthomonas arboricola pv. juglandis (walnut blight) [dataset]. In CABI Compendium. CABI Publishing. https://doi.org/10.1079/cabicompendium.56946
Parkinson, N., Aritua, V., Heeney, J., Cowie, C., Bew, J., & Stead, D. (2007). Phylogenetic analysis of Xanthomonas species by comparison of partial gyrase B gene sequences. International Journal of Systematic and Evolutionary Microbiology, 57(12), 2881-2887. https://doi.org/10.1099/ijs.0.65220-0
Pasche, J., Kalil, A., & Markell, S. (2024). Bacterial blight: Pseudomonas syringae pv. syringae (Lentil Disease Diagnostic Series, Publication PP1913). North Dakota State University Extension. https://www.ndsu.edu/agriculture/sites/default/files/2024-02/pp1913_0.pdf
Ramírez-Bahena, M. H., Vial, L., Lassalle, F., Diel, B., Chapulliot, D., Daubin, V., Nesme, X., & Muller, D. (2014). Single acquisition of protelomerase gave rise to speciation of a large and diverse clade within the Agrobacterium/Rhizobium supercluster characterized by the presence of a linear chromid. Molecular Phylogenetics and Evolution, 73, 202-207. https://doi.org/10.1016/j.ympev.2014.01.005
Rouhrazi, K., & Rahimian, H. (2014). Biochemical and genetic characterisation ofAgrobacterium tumefaciensthe causal agent of walnut crown gall disease in Iran. Archives of Phytopathology and Plant Protection, 47(20), 2493-2500. https://doi.org/10.1080/03235408.2014.880575
Shams, M., Vial, L., Chapulliot, D., Nesme, X., & Lavire, C. (2013). Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR. Systematic and applied microbiology, 36(5), 351-358. https://doi.org/10.1016/j.syapm.2013.03.002
Stackebrandt, E. (2006). Taxonomic parameters revisited: tarnished gold standards. Microbial Today, 33, 152.
Stackebrandt, E., & Goebel, B. M. (1994). Taxonomic Note: A Place for DNA-DNA Reassociation and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology. International Journal of Systematic and Evolutionary Microbiology, 44(4), 846-849. https://doi.org/10.1099/00207713-44-4-846
Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution, 38(7), 3022-3027. https://doi.org/10.1093/molbev/msab120
Temperini, C. V., Pardo, A. G., & Pose, G. N. (2017). First Report of Apical Necrosis in Walnut Cultivars from Northern Argentinean Patagonia. Journal of Plant Pathology and Microbiology, 08(07). https://doi.org/10.4172/2157-7471.1000414
Vauterin, L., Hoste, B., Kersters, K., & Swings, J. (1995). Reclassification of Xanthomonas. International Journal of Systematic Bacteriology, 45(3), 472-489. https://doi.org/10.1099/00207713-45-3-472
Wayne, L. G., Moore, W. E. C., Stackebrandt, E., Kandler, O., Colwell, R. R., Krichevsky, M. I., Truper, H. G., Murray, R. G. E., Grimont, P. A. D., Brenner, D. J., Starr, M. P., & Moore, L. H. (1987). Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics. International Journal of Systematic and Evolutionary Microbiology, 37(4), 463-464. https://doi.org/10.1099/00207713-37-4-463
Young, J. M., Kuykendall, L. D., Martínez-Romero, E., Kerr, A., & Sawada, H. (2003). Classification and nomenclature of Agrobacterium and Rhizobium - a reply to Farrand et al. (2003). International Journal of Systematic and Evolutionary Microbiology, 53(5), 1689-1695. https://doi.org/10.1099/ijs.0.02762-0
Downloads
Published
Issue
Section
License
Copyright (c) 2026 Mikrobiolohichnyi Zhurnal
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
