Cultivated bacteria associated with tomato seeds

Relevance. Bacterial diseases that reduce tomato yield in both open and protected ground occupy a special place due to their harmfulness, intensity of transmission in agrocenosis, and difficulty of treatment. The widespread distribution of bacterial phytopathogens on tomatoes worldwide is due to their ability to remain viable for a long time on the surface and inside seeds. The aim of this study is to clarify the composition of cultivated bacteria associated with seeds of different tomato varieties and to identify pathogenic species.

Material and Methodology. The work was conducted in the Bacteriology Department of the All- Russian Plant Quarantine Center (Moscow Region, town of Bykovo). To study the composition of cultivated bacteria, seeds from 24 varieties and hybrids of tomatoes were used. Seed samples were inoculated onto YDC nutrient medium in duplicate using the Drigalski method. Petri dishes were incubated at 27°C. DNA extraction from pure bacterial cultures was performed using the boiling method. Isolates were identified using Sanger sequencing. For pathogenicity testing, a bacterial suspension of isolates Pseudomonas sp. and Curtobacterium sp. was prepared from a two-day pure culture in sterile distilled water at a concentration of 10⁶ CFU/ml. Tomato seedlings were grown from seeds of three hybrids. Artificial infection of the plants was conducted after the appearance of 2-3 true leaves by injecting into the stem between the cotyledons and the first true leaf in triplicate.

Results and Discussion. During the phytosanitary examination of the obtained tomato seed material, bacterial isolates belonging to 10 genera were identified: Sphingomonas, Micrococcus, Phyllobacterium, Ralstonia, Frigoribacterium, Arthrobacter, Devosia, Agrococcus, Pseudomonas, and Curtobacterium. Using artificial infection of tomato seedlings, it was demonstrated that representatives of the genus Pseudomonas and Curtobacterium were pathogenic to tomato plants. It was found that plants of the Bellioso F1 hybrid are most susceptible to infection with Pseudomonas bacteria, with more numerous and large necrosis on the leaves compared with the Callanzo F₁ hybrid and especially with the Senserno F₁ hybrid. The bacteria of the genus Curtobacterium were less aggressive, and the necroses they caused on the leaves were smaller than those caused by Pseudomonas bacteria. Significant growth inhibition of the tested varieties was noted. Upon inoculation with Pseudomonas sp., the plants lagged behind the control by 40-50%, while infection with Curtobacterium sp. resulted in a growth delay of 44-54%, depending on the hybrid. Thus, qualitative and timely diagnosis of phytopathogenic agents of bacterial diseases, along with the culling or disinfection of seeds, is an effective way to reduce yield losses and increase the profitability of tomato production.

1. Bulgarelli D., Rott M., Schlaeppi K., Ver Loren van Themaat E., Ahmadinejad N., Assenza F., Schulze-Lefert P. Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature. 2012;488(7409):91-95. https://doi.org/10.1038/nature11336

2. Mendes R., Garbeva P., Raaijmakers J. M. The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS microbiology reviews. 2013;37(5):634-663. https://doi.org/10.1111/1574-6976.12028

3. Lundberg D.S., Lebeis S.L., Paredes S.H., Yourstone S., Gehring J., Malfatti S., Dangl J.L. Defining the core Arabidopsis thaliana root microbiome. Nature. 2012;488(7409):86-90. https://doi.org/10.1038/nature11237

4. Van Der Heijden M.G., Bardgett R.D., Van Straalen N.M. The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecology letters. 2008;11(3):296-310. https://doi.org/10.1111/j.1461-0248.2007.01139.x

5. Nelson E.B. The seed microbiome: origins, interactions, and impacts. Plant and Soil. 2018;422:7-34. https://doi.org/10.1007/s11104-017-3289-7

6. Muller D.B., Vogel C., Bai Y., Vorholt J.A. The plant microbiota: systems-level insights and perspectives. Annual review of genetics. 2016;50(1):211-234. https://doi.org/10.1146/annurev-genet-120215-034952

7. Lugtenberg B., Kamilova F. Plant-growth-promoting rhizobacteria. Annual review of microbiology. 2009; 63(1):541-556. https://doi.org/10.1146/annurev.micro.62.081307.162918

8. Haas D., Defago G. Biological control of soil-borne pathogens by fluorescent pseudomonads. Nature reviews microbiology. 2005;3(4):307-319. https://doi.org/10.1038/nrmicro1129

9. Berendsen R.L., Pieterse C.M., Bakker P.A. The rhizosphere microbiome and plant health. Trends in plant science. 2012;17(8):478-486. https://doi.org/10.1016/j.tplants.2012.04.001

10. Compant S., Clement C., Sessitsch A. Plant growth-promoting bacteria in the rhizo-and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biology and Biochemistry. 2010;42(5):669-678. https://doi.org/10.1016/j.soil-bio.2009.11.024

11. Glick B.R. Plant growth-promoting bacteria: mechanisms and applications. Scientifica. 2012;(1):963401. https://doi.org/10.6064/2012/963401

12. Vessey J.K. Plant growth promoting rhizobacteria as biofertilizers. Plant and soil. 2003;255:571-586. https://doi.org/10.1023/A:1026037216893

13. Yang J., Kloepper J.W., Ryu C.M. Rhizosphere bacteria help plants tolerate abiotic stress. Trends in plant science. 2009;14(1):1-4. https://doi.org/10.1016/j.tplants.2008.10.004

14. Beloshapkina O.O., Pisareva I.N. Determination of the analytical sensitivity and specificity of PCR methods for the diagnosis of bacterial spot of tomato. Izvestiya of Timiryazev Agricultural Academy. 2024;(3):78-94. https://doi.org/10.26897/0021-342X-2024-3-78-94 https://www.elibrary.ru/nefuxa (In Russ.)

15. Obolenksy R.R, Slovareva O.Yu., Dorofeeva L.V. Evaluation of the real-time PCR applicability for the identification of bacterial ear rot of wheat Rathayibacter tritici. Plant Health and Quarantine. 2025;1(22):26-39. (In Russ.) https://doi.org/10.69536/FKR.2025.85.45.003 https://www.elibrary.ru/wiuyiq

16. Pisareva I.N., Beloshapkina O.O. Modern diagnosis of bacterioses in seeds for tomato protection. News of FSVC. 2024;(2):7-13. (In Russ.) https://doi.org/10.18619/2658-4832-2024-2-7-13 https://www.elibrary.ru/ehksoe

17. Xhemali B., Giovanardi D., Biondi E., Stefani E. Tomato and pepper seeds as pathways for the dissemination of phytopathogenic bacteria: A constant challenge for the seed industry and the sustainability of crop production. Sustainability. 2024;16(5):1808. https://doi.org/10.3390/su16051808

18. Peritore-Galve F.C., Tancos M.A., Smart C.D. Bacterial canker of tomato: revisiting a global and economically damaging seedborne pathogen. Plant Disease. 2021;105(6):1581-1595. https://doi.org/10.1094/PDIS-08-20-1732-FE

19. Gebeyaw M. Review on: Impact of seed-borne pathogens on seed quality. American Journal of Plant Biology, 2020;(5):77-81. https://doi.org/10.11648/j.ajpb.20200504.11

20. Kim H., Nishiyama M., Kunito T., Senoo K., Kawahara K., Murakami K., Oyaizu H. High population of Sphingomonas species on plant surface. Journal of applied microbiology. 1998;85(4):731-736. https://doi.org/10.1111/j.1365-2672.1998.00586.x

21. Asaf S., Numan M., Khan A.L., Al-Harrasi A. Sphingomonas: from diversity and genomics to functional role in environmental remediation and plant growth. Critical Reviews in Biotechnology. 2020;40(2):138-152. https://doi.org/10.1080/07388551.2019.1709793

22. Zhang J.Y., Liu X.Y., Liu S.J. Agrococcus terreus sp. nov. and Micrococcus terreus sp. nov., isolated from forest soil. International journal of systematic and evolutionary microbiology. 2010; 60(8):1897-1903. https://doi.org/10.1099/ijs.0.013235-0

23. Mergaert J., Cnockaert M. C., Swings J. Phyllobacterium myrsinacearum (subjective synonym Phyllobacterium rubiacearum) emend. International Journal of Systematic and Evolutionary Microbiology. 2002;52(5):1821-1823. https://doi.org/10.1099/00207713-52-5-1821

24. Stelzmueller I., Biebl M., Wiesmayr S., Eller M., Hoeller E., Fille M., Bonatti H. Ralstonia pickettii – innocent bystander or a potential threat? Clinical Microbiology and Infection. 2006;12(2):99-101. https://doi.org/10.1111/j.1469-0691.2005.01309.x

25. Kong D., Guo X., Zhou S., Wang H., Wang Y., Zhu J. Ruan, Z. Frigoribacterium salinisoli sp. nov., isolated from saline soil, transfer of Frigoribacterium mesophilum to Parafrigoribacterium gen. nov. as Parafrigoribacterium mesophilum comb. nov. International Journal of Systematic and Evolutionary Microbiology. 2016;66(12):5252-5259. https://doi.org/10.1099/ijsem.0.001504

26. Busse H.J. Review of the taxonomy of the genus Arthrobacter, emendation of the genus Arthrobacter sensu lato, proposal to reclassify selected species of the genus Arthrobacter in the novel genera Glutamicibacter gen. nov., Paeniglutamicibacter gen. nov., Pseudoglutamicibacter gen. nov., Paenarthrobacter gen. nov. and Pseudarthrobacter gen. nov., and emended description of Arthrobacter roseus. International journal of systematic and evolutionary microbiology. 2016;66(1):9-37. https://doi.org/10.1099/ijsem.0.000702

27. Yoon J.H., Kang S.J., Park S., Oh T.K. Devosia insulae sp. nov., isolated from soil, and emended description of the genus Devosia. International Journal of Systematic and Evolutionary Microbiology. 2007;57(6):1310-1314. https://doi.org/10.1099/ijs.0.65028-0

28. White III R.A., Gavelis G., Soles S.A., Gosselin E., Slater G.F., Lim D.S., Suttle C.A. The complete genome and physiological analysis of the microbialite-dwelling Agrococcus pavilionensis sp. nov; reveals genetic promiscuity and predicted adaptations to environmental stress. Frontiers in microbiology. 2018;9:2180. https://doi.org/10.3389/fmicb.2018.02180

29. Lalucat J., Gomila M., Mulet M., Zaruma A., & Garcia-Valdes E. Past, present and future of the boundaries of the Pseudomonas genus: proposal of Stutzerimonas gen. nov. Systematic and applied microbiology. 2022; 45(1):126289. https://doi.org/10.1016/j.syapm.2021.126289

30. Tarakanov R.I., Ignat’eva I.M., Beloshapkina O.O., Chebanenko S.I., Karataeva O.G., Dzhalilov F.S. Detection of the soybean bacterial blight pathogen Pseudomonas savastanoi pv. glycinea in seeds by the PCR method. Izvestiya of Timiryazev Agricultural Academy, 2024;(1):41-52. (In Russ.) https://doi.org/10.26897/0021-342X-2024-1-41-52 https://www.elibrary.ru/bjiwxk

31. Licciardello G., Bertani I., Steindler L., Bella P., Venturi V., Catara V. Pseudomonas corrugata contains a conserved N-acyl homoserine lactone quorum sensing system; its role in tomato pathogenicity and tobacco hypersensitivity response. FEMS microbiology ecology. 2007;61(2):222-234. https://doi.org/10.1111/j.1574-6941.2007.00338.x

32. Osdaghi E., Taghouti G., Dutrieux C., Taghavi S. M., Fazliarab A., Briand M., Jacques M. A. Whole Genome Resources of 17 Curtobacterium flaccumfaciens Strains Including Pathotypes of C. flaccumfaciens pv. betae, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiae. Molecular Plant-Microbe Interactions. 2022;35(4):352-356. https://doi.org/10.1094/MPMI-11-21-0282-A

33. Tarakanov R.I., Lukianova A.A., Pilik R.I., Evseev P.V., Miroshnikov K.A., Dzhalilov F.S. U., Tesic S., Ignatov A.N. First report of Curtobacterium flaccumfaciens pv. flaccumfaciens causing bacterial tan spot of soybean in Russia. Plant disease. 2023;107(7):2211. https://doi.org/10.1094/PDIS-08-22-1778-PDN

34. Tokmakova A.D., Tarakanov R.I., Lukianova A.A., Evseev P.V., Dorofeeva L.V., Ignatov A.N., Dzhalilov F.S.U., Subbotin S.A., Miroshnikov K.A. Phytopathogenic Curtobacterium flaccumfaciens strains circulating on leguminous plants, alternative hosts and weeds in Russia. Plants. 2024;13(5):667. https://doi.org/10.3390/plants13050667