Comparative characterization of chitinolytic activity in five STREPTOMYCES strains and the metabolomic response of S. BAARNENSIS assi-23tb to phytopathogens IN PLANTA

Abstract

The wide species diversity of chitinolytic actinomycetes producing secondary metabolites and hydrolytic enzymes offers opportunities for effective soil biocontrol aimed at protecting plants from phytopathogens and preserving soil biodiversity. This study examined the chitinolytic potential of five soil actinomycete strains from the genus Streptomyces(S. flavovirens TT, S. sindenensis TK, S. baarnensis assi-23TB, S. anulatus TG, and S. xiamenensis TB) through integrated analytical and molecular biological approaches.The study demonstrated that the fundamental mechanism of Streptomycesstrain adaptation to chitin utilization is the targeted induction of functional chitinase genes.We identified distinct genome activation strategies and observed that high genetic activity leads to a robust physiological response, including active biomass accumulation and CO₂ emission. These findings confirm the high competitiveness of the investigated strains under conditions of limited access to readily available carbon and nitrogen sources. The study elucidated the interaction of the target strain Streptomyces baarnensis assi-23TB (VKPM Ac-2228) with two specific phytopathogens in both in vitro and in planta systems, using wheat seedlings (Triticum aestivum L. cv. Arkhat) and microclonal potato plants (Solanum tuberosum L. cv. Romano) as host models. A comparative analysis was performed to correlate the metabolic capabilities of S. baarnensis assi-23TB, as theoretically predicted by bioinformatic tools (antiSMASH, PATRIC), with its actual phenotypic metabolite production. The strain exhibited significant chitinolytic activity. Our analysis revealed dibutyl phthalate (DBP) to be a constitutive secondary metabolite responsible for the baseline antimicrobial activity.Notably, several predicted biosynthetic gene clusters (10-epi-HSAF, keivymicin, alkylresorcinol, desferrioxamine B, naringenin, and zeatin) remained in a "silent" state under the experimental conditions. We found that the S. baarnensis exometabolome strictly depends on the presence of the host plant and specific pathogens (Fusarium graminearum and Phytophthora infestans). The association with wheat exclusively triggers the synthesis of the phytostimulant putrescine and energy metabolism regulators, such as lactic and succinic acids. This symbiotic system also drives the production of amino acids, sugars, and myo-inositol.The study experimentally confirms the multifunctionality of the Streptomyces baarnensis assi-23TB strain, which integrates direct enzymatic lysis of pathogens with the biochemical stimulation of plant growth and adaptive responses

References

1. Зенова Г.М.Почвенные актиномицеты. М., 1992. 76 с. 2. Зенова Г.М., Звягинцев Д.Г. Разнообразие актиномицетов в наземных экосистемах. М., 2002. 132 с. 3. Калакуцкий Л.В., Зенова Г.М. Экология актиномицетов // Успехи микробиологии. 1984. № 19. С. 203–221. 4. Карамова Н.С., Зеленихин П.В., Мирошник Н.Б.и др. Апоптозиндуцирующее действие рибонуклеазы Bacillus pumilus и экстрактов лекарственных растений Египта на клетки аденокарциномы легких человека // Гены и Клетки. 2015. Т. 10, № 3. C. 62–67. https://doi.org/10.23868/gc120486 5. Кожевин П.А. Микробные популяции в природе. М., 1989. 175 с. 6. Манучарова Н.А., Власенко А.Н., Белова Э.В.и др.Методические аспекты определения использования хитина почвенными микроорганизмами // Известия РАН. Сер. Биол. 2008. № 5. C. 635–640. 7. Нетрусов А.И., Егоров М.А., Захарчук Л.М.и др. Практикум по микробиологии. М., 2005. 608 с. 8. Поздняков Л.А., Степанов А.Л., Гасанов М.Э.и др. Влияние лигногумата на биологическую активность почвы о. Бали, Индонезия // Почвоведение. 2020. № 5. C. 601–609. https://doi.org/10.31857/S0032180X20050111 9. Полянская Л.М., Гейдебрехт В.В., Степанов А.Л.и др.Распределение численности и биомассы микроорганизмов по профилям зональных типов почв // Почвоведение. 1995. № 2. С. 322–328. 10. Терегулова Г.А., Синёва О.Н., Маркелова Н.Н. и др. Оценка хитинолитической и антибиотической активности актиномицетов Streptomyces avidinii INA 01467 и Micromonospora aurantiaca INA 01468 // Почвоведение. 2023. №5. C. 594–602. https://doi.org/10.31857/S0032180X22601207 11. Терегулова Г.А. Пат. RU2839282, МПК C12N 1/20 (2006.01). Штамм хитинолитического актиномицета Streptomyces baarnensis для защиты растений от фитопатогенных грибов и стимуляции роста растений. 2025. Бюл. № 13. 12. Уразбахтина Н.А., Вахитова Р.К. Биотехнология в растениеводстве. Учебно-методическое пособие к лабораторным работам и практическим занятиям. Направление подготовки 35.03.04 «Агрономия». Уфа, 2019. 56 с. 13. Яруллина Л.Г., Цветков В.О., Бурханова Г. Ф. и др. Влияние бактерий Bacillus subtilis и сигнальных молекул на состояние про-/антиоксидантной системы и экспрессию генов защитных белков у растений картофеля при инфицировании возбудителем фитофтороза и недостатке влаги // Прикладная биохимия и микробиология. 2021. Т. 57, № 6. С. 594–604. https://doi.org/10.31857/S0555109921060131 14. Ahsan T., Chen J., Zhao X. et al. Extraction and identification of bioactive compounds (eicosane and dibutyl phthalate) produced by Streptomyces strain KX852460 for the biological control of Rhizoctonia solani AG-3 strain KX852461 to control target spot disease in tobacco leaf // AMB Express. 2017. Vol. 7, № 1.Article54. https://doi.org/10.1186/s13568-017-0351-z 15. Besset-Manzoni Y., Joly P., Brutel A. et al. Does in vitro selection of biocontrol agents guarantee success in planta? A study case of wheat protection against Fusarium seedling blight by soil bacteria // PloS One. 2019. Vol. 14, № 12. e0225655. https://doi.org/10.1371/journal.pone.0225655 16. Bettiol W., de Medeiros F.H.V., Barros Chiaramonte J. et al. Advances in screening approaches for the development of microbial bioprotectants to control plant diseases // Microbial bioprotectants for plant disease management. 2021. P. 33–86. https://doi.org/10.19103/AS.2021.0093.02 17. Blin K., Shaw S., Vader L.et al. antiSMASH 8.0: extended gene cluster detection capabilities and analyses of chemistry, enzymology, and regulation // Nucleic Acids Research. 2025. Vol. 53. P. W32–W38. https://doi.org/10.1093/nar/gkaf334 18. Chen Y. Application of Hydrocarbon Degrading Microorganism Enumeration and Catabolic Genes Detection for Soil Assessment. Master’s Thesis, University of Helsinki. Helsinki, 2013. 68 p. 19. Dilip C.V., Mulaje S., Mohalkar R. A review on Actinomycetes and their biotechnological application // International Journal of Pharmaceutical Sciences and Research. 2013. Vol. 4, № 5. P. 1730–1742. https://doi.org/10.13040/IJPSR.0975-8232.4(5).1730-42 20. Dow L., Gallart M., Ramarajan M.et al. Streptomyces and their specialised metabolites for phytopathogen control – comparative in vitro and in planta metabolic approaches // Front. Plant Sci. 2023. Vol. 14. Article 1151912. https://doi.org/10.3389/fpls.2023.1151912 21. El-Naggar M.Y.M. Dibutyl phthalate and the antitumour agent F5A1, two metabolites produced by Streptomyces nasri submutant H35 // Biomedical Letters. 1997. P.125–131. 22. Jiang D., Hou J., Gao W.et al. Exogenous spermidine alleviates the adverse effects of aluminum toxicity on photosystem II through improved antioxidant system and endogenous polyamine contents // Ecotoxicology and Environmental Safety. 2021. Vol. 207. Article 111265. https://doi.org/10.1016/j.ecoenv.2020.111265 23. Hasegawa S., Meguro A., Shimizu M.et al. Endophytic actinomycetes and their interactions with host plants // Actinomycetologica. 2006. Vol. 20, № 2. P. 72–81. https://doi.org/10.3209/saj.20.72 24. Hobel C.F., Marteinsson V.T., Hreggvidsson G.O. et al.Investigationofthemicrobialecologyofintertidalhotspringsby usingdiversityanalysisof16SrRNAandchitinasegenes//AppliedandEnvironmentalMicrobiology.2005.Vol.71, № 5.P.2771–2776. https://doi.org/10.1128/AEM.71.5.2771-2776.2005 25. Köhl J., Kolnaar R., Ravensberg W.J. Mode of action of microbial biological control agents against plant diseases: relevance beyond efficacy // Front. Plant Sci. 2019. Vol. 10. Article 845.https://doi.org/10.3389/fpls.2019.00845 26. Landete J.M., Arena M.E., Pardo I.et al. The role of two families of bacterial enzymes in putrescine synthesis from agmatine via agmatine deiminase // International microbiology. 2010. Vol. 13,№4. P. 169–177. https://doi:10.2436/20.1501.01.123 27. Mahmud F., Lai N.S., How S.E. et al. Bioactivities and mode of actions of Dibutyl Phthalates and Nocardamine from Streptomyces sp. H11809 // Molecules. 2022. Vol. 27, №7. Article 2292. https://doi.org/10.3390/molecules27072292 28. Murashige T., Skoog F. A revised medium for rapid growth and Bio Assays with tobacco tissue cultures // Physiologia Plantarum. 1962. Vol. 15. P. 473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x 29. Olson R.D., Assaf R., Brettin T. et al.Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): a resource combining PATRIC, IRD and ViPR // Nucleic Acids Research. 2023. Vol. 51(D1). P. D678–D689. https://doi.org/10.1093/nar/gkac1003 30. Roy R.N., Laskar S., Sen S.K. Dibutyl phthalate, the bioactive compound produced by Streptomyces albidoflavus 321.2 // Microbiol Research.2006. Vol. 161, № 2. P. 121–126. https://doi.org/10.1016/j.micres.2005.06.007 31. Silva G.C., Kitano I.T., Ribeiro I.A.F.et al. The potential use of actinomycetes as microbial inoculants and biopesticides in agriculture // Frontiers in Soil Science. 2022. Vol. 2. Article 833181.https://doi.org/10.3389/fsoil.2022.833181 32. Tatusova T., DiCuccio M., Badretdin A. et al. NCBI prokaryotic genome annotation pipeline // Nucleic Acids Research. 2016. Vol. 44, № 14. P. 6614–6624. https://doi.org/10.1093/nar/gkw569 33. Terlouw B., Blin K., Navarro-Muñoz J. C. et al. MIBiG 3.0: a community-driven effort to annotate experimentally validated biosynthetic gene clusters //Nucleic Acids Research. 2022. Vol. 51(D1). P. D603-D610. https://edepot.wur.nl/584330 34. Tian C., Ni J., Chang F.et al. Bio-Source of di-n-butyl phthalate production by filamentous fungi // Scientific reports. 2016. Vol. 6. Article 19791. https://doi.org/10.1038/srep19791 35. Wohlleben W., Mast Y., Stegmann E. et al. Antibiotic drug discovery // Microbial Biotechnology. 2016. Vol. 9, №5. P. 541548. https://doi.org/10.1111/1751-7915.12388 36. Yamunarani D.K., Sundaram D.A., Pandiyan D.M. Streptomycetes as a potential biocontrol agent // Journal of entomology and zoology studies. 2019. Vol. 7. P. 637–644. https://api.semanticscholar.org/CorpusID:108966831 37. Zhang H., Hua Y., Chen J. et al. Organism-derived phthalate derivatives as bioactive natural products // Journal of Environmental Science and Health. 2018. Part C. Vol. 36,№ 3. P. 125–144. https://doi.org/10.1080/10590501.2018.1490512