Исследование инсектицидного и фунгицидного потенциала бактерий эндофитов пшеницы, сои и рапса методами биоинформатического анализа

Эндофитные бактерии способны влиять на доступность различных соединений, урожайность и рост сельскохозяйственных растений, а также обеспечивать устойчивость к болезням и вредителям. Поэтому исследование эндофитов сельскохозяйственно значимых культурных растений является перспективной задачей в области биологической защиты растений. В данной работе рассмотрены изоляты штаммов бактерий, полученные из листьев и/или корней пшеницы, рапса и сои. Было произведено полногеномное секвенированиеизолятов. С помощью аналитического конвейера собраны и охарактеризованы геномы 15 штаммов бактерий эндофитов культурных растений, проанализирован их инсектицидный и фунгицидный потенциал. Анализ генного репертуара с помощью программы GenAPI показал высокую степень соответствия между генным репертуаром штамма BZR 585 относительно Alcaligenes phenolicus, BZR 762 и BZR 278 относительно Alcaligenes sp., BZR 588 и BZR 201P относительно Paenochrobactrum pullorum. Во всех штаммах, за исключением BZR 162, BZR 588 и BZR 201P, найдены гены, кодирующие белки, обладающие фунгицидной активностью, такие как итурины, фенгицины и сурфактины. Также во всех штаммах найдены гены, кодирующие белки с инсектицидной активностью, а именно: GroEL, Spp1Aa1, Spp1Aa2, Vpb1Ab1, Vpb4Ca1, HldE, фенгицин, микосубтилин и бацилломицин. Полученные геномные данные подтверждены экспериментальными испытаниями: ранее показана высокая инсектицидная активность штаммов BZR 1159, BZR 936, BZR 920 и др. против Galleria mellonella, Tenebrio molitor и Cydia pomonella, а также фунгицидные свойства против Fusarium, Alternaria, Trichothecium. Это демонстрирует практическую значимость выявленных генетических детерминант для создания новых агентов биоконтроля.

1. Abdul Salam S., Parthiban V.K., Paranidharan V., Johnson I., Karthi keyan M., Kavitha C. Leucobacter aridicollis strain SASBG215: a novel biocontrol agent against Colletotrichum orbiculare. Biol Forum Int J. 2022;14(2):905-911

2. Ali M.A., Ahmed T., Ibrahim E., Rizwan M., Chong K.P., Yong J.W.H. A review on mechanisms and prospects of endophytic bacteria in biocontrol of plant pathogenic fungi and their plant growth-promot ing activities. Heliyon. 2024;10(11):e31573. doi 10.1016/j.heliyon.2024.e31573

3. Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Ku likov A.S., Lesin V.M., … Sirotkin A.V., Vyahhi N., Tesler G., Alek seyev M.A., Pevzner P.A. SPAdes: a new genome assembly algo rithm and its applications to single-cell sequencing. J Comput Biol. 2012;19(5):455-477. doi 10.1089/cmb.2012.0021

4. Bateman A., Martin M.J., Orchard S., Magrane M., Agivetova R., Ah mad S., Alpi E., … Vinayaka C.R., Wang Q., Wang Y., Yeh L.S., Zhang J. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021;49(D1):D480-D489. doi 10.1093/nar/gkaa1100

5. Berry C., O’Neil S., Ben-Dov E., Jones A.F., Murphy L., Quail M.A., Holden M.T.G., Harris D., Zaritsky A., Parkhill J. Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis. Appl Environ Micro biol. 2002;68(10):5082-5095. doi 10.1128/AEM.68.10.5082-5095.2002

6. Blanco-Míguez A., Beghini F., Cumbo F., McIver L.J., Thompson K.N., Zolfo M., Manghi P., … Franzosa E.A., Pasolli E., Asnicar F., Hut tenhower C., Segata N. Extending and improving metagenomic tax onomic profiling with uncharacterized species using MetaPhlAn 4. Nat Biotechnol. 2023;41(11):1633-1644. doi 10.1038/s41587-02301688-w

7. Bolotin A., Gillis A., Sanchis V., Nielsen-LeRoux C., Mahillon J., Le reclus D., Sorokin A. Comparative genomics of extrachromosomal elements in Bacillus thuringiensis subsp. israelensis. Res Microbiol. 2017;168(4):331-344. doi 10.1016/j.resmic.2016.10.008

8. Chaumeil P.-A., Mussig A.J., Hugenholtz P., Parks D.H. GTDB-Tk v2: memory friendly classification with the genome taxonomy data base. Bioinformatics. 2022;38(23):5315-5316. doi 10.1093/bioinformatics/btac672

9. Chen S. Ultrafast one‐pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta. 2023;2(2):e107. doi 10.1002/imt2.107

10. Chen S., Zhou Y., Chen Y., Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34(17):i884-i890. doi 10.1093/bioinformatics/bty560

11. Dai R., Zhang J., Liu F., Xu H., Qian J.-M., Cheskis S., Liu W., … de Jonge R., Pieterse C.M.J., Levy A., Schlaeppi K., Bai Y. Crop root bacterial and viral genomes reveal unexplored species and microbiome patterns. Cell. 2025;188(9):2521-2539.e22. doi 10.1016/j.cell.2025.02.013

12. Deng X., Wang X., Li G. Nematicidal effects of volatile organic compounds from microorganisms and plants on plant-parasitic nematodes. Microorganisms. 2022;10(6):1201. doi 10.3390/microorganisms10061201

13. Dhaouadi S., Rouissi W., Mougou-Hamdane A., Nasraoui B. Evaluation of biocontrol potential of Achromobacter xylosoxidans against Fusarium wilt of melon. Eur J Plant Pathol. 2019;154(2):179-188. doi 10.1007/s10658-018-01646-2

14. Falqueto S.A., Pitaluga B.F., de Sousa J.R., Targanski S.K., Cam pos M.G., de Oliveira Mendes T.A., da Silva G.F., Silva D.H.S., Soares M.A. Bacillus spp. metabolites are effective in eradicating Aedes aegypti (Diptera: Culicidae) larvae with low toxicity to non target species. J Invertebr Pathol. 2021;179:107525. doi 10.1016/j.jip.2020.107525

15. Gabrielaite M., Marvig R.L. GenAPI: a tool for gene absence-presence identification in fragmented bacterial genome sequences. BMC Bio informatics. 2020;21(1):320. doi 10.1186/s12859-020-03657-5

16. Gong A.-D., Wu N.-N., Kong X.-W., Zhang Y.-M., Hu M.-J., Gong S.-J., Dong F.-Y., Wang J.-H., Zhao Z.-Y., Liao Y.-C. Inhibitory effect of volatiles emitted from Alcaligenes faecalis N1-4 on Aspergillus flavus and aflatoxins in storage. Front Microbiol. 2019;10:1419. doi 10.3389/fmicb.2019.01419

17. Guindon S., Dufayard J.-F., Lefort V., Anisimova M., Hordijk W., Gascuel O. New algorithms and methods to estimate maximum likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59(3):307-321. doi 10.1093/sysbio/syq010

18. Guizelini D., Raittz R.T., Cruz L.M., Souza E.M., Steffens M.B.R., Pe drosa F.O. GFinisher: a new strategy to refine and finish bacterial genome assemblies. Sci Rep. 2016;6(1):34963. doi 10.1038/srep34963

19. Gurevich A., Saveliev V., Vyahhi N., Tesler G. QUAST: quality assess menttool for genome assemblies. Bioinformatics. 2013;29(8):1072 1075. doi 10.1093/bioinformatics/btt086

20. Guyomar C., Delage W., Legeai F., Mougel C., Simon J.-C., Lemai tre C. MinYS: mine your symbiont by targeted genome assembly in symbiotic communities. NAR Genom Bioinform. 2020;2(3): lqaa047. doi 10.1093/nargab/lqaa047

21. Gyrnets (Bondarchuk) E.Yu., Asaturova A.M. Screening of bacterial strains on the criterion of entomopathogenic activity against Gal leria mellonella L. and Tenebrio molitor L. Dostizheniya Nauki i Tekhniki APK = Achievements of Science and Technology in Agro Industrial Complex. 2022;36(3):53-60. doi 10.53859/02352451_ 2022_36_3_53 (in Russian)

22. Gyrnets E.Yu., Asaturova A.M. Study of the polyfunctional proper ties of promising bacterial agents in relation to phytophages and pathogens of fruit cenosis. Dostizheniya Nauki i Tekhniki APK = Achievements of Science and Technology in AgroIndustrial Com plex. 2023;37(5):39-45. doi 10.53859/02352451_2023_37_5_39 (in Russian)

23. Hamane S., El Yemlahi A., Hassani Zerrouk M., El Galiou O., La glaoui A., Bakkali M., Arakrak A. Plant growth promotion and biocontrol potentiality of endophytes isolated from root nodules of Sulla flexuosa L. plants. Int J Agron. 2023;2023(1):1-9. doi 10.1155/2023/2451806

24. Hodgkin J., Félix M.-A., Clark L.C., Stroud D., Gravato-Nobre M.J. Two Leucobacter strains exert complementary virulence on Cae norhabditis including death by worm-star formation. Curr Biol. 2013;23(21):2157-2161. doi 10.1016/j.cub.2013.08.060

25. Horwich A.L., Fenton W.A., Chapman E., Farr G.W. Two families of chaperonin: physiology and mechanism. Annu Rev Cell Dev Biol. 2007;23(1):115-145. doi 10.1146/annurev.cellbio.23.090506.123555

26. Hussain H.S., Tabbasum S., Mahreen N., Yahya M., Ejaz K., Aslam Z., Imtiaz M., ul Islam E., Sajid Z.I., Yasmin S. Integrated applica tion of drought-tolerant phosphate solubilizing bacteria and native Р source improves P availability with sustainable wheat production. J Soil Sci Plant Nutr. 2025;25(2):4723-4746. doi 10.1007/s42729-025-02425-9

27. Jain C., Rodriguez-R L.M., Phillippy A.M., Konstantinidis K.T., Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun. 2018;9(1):5114. doi 10.1038/s41467-018-07641-9

28. Ju S., Lin J., Zheng J., Wang S., Zhou H., Sun M. Alcaligenes faeca lis ZD02, a novel nematicidal bacterium with an extracellular se rine protease virulence factor. Appl Environ Microbiol. 2016;82(7): 2112-2120. doi 10.1128/AEM.03444-15

29. Knežević M., Dervišević M., Jovković M., Jevđenović G., Maksimović J., Buntić A. Versatile role of Bacillus velezensis: bio control of Fusarium poae and wireworms and barley plant growth promotion. Biol Control. 2025;206:105789. doi 10.1016/j.biocontrol.2025.105789

30. Kolmogorov M., Raney B., Paten B., Pham S. Ragout – a reference assisted assembly tool for bacterial genomes. Bioinformatics. 2014; 30(12):i302-i309. doi 10.1093/bioinformatics/btu280

31. Krzyżanowska D.M., Maciąg T., Ossowicki A., Rajewska M., Kaczyński Z., Czerwicka M., Rąbalski Ł., Czaplewska P., Jafra S. Ochrobactrum quorumnocens sp. nov., a quorum quenching bac terium from the potato rhizosphere, and comparative genome ana lysis with related type strains. PLoS One. 2019;14(1):e0210874. doi 10.1371/journal.pone.0210874

32. Kumar C.G., Sujitha P., Mamidyala S.K., Usharani P., Das B., Red dy C.R. Ochrosin, a new biosurfactant produced by halophilic Ochrobactrum sp. strain BS-206 (MTCC 5720): purification, cha racterization and its biological evaluation. Process Biochem. 2014; 49(10):1708-1717. doi 10.1016/j.procbio.2014.07.004

33. Kupper M., Gupta S.K., Feldhaar H., Gross R. Versatile roles of the chaperonin GroEL in microorganism-insect interactions. FEMS Microbiol Lett. 2014;353(1):1-10. doi 10.1111/1574-6968.12390

34. Letunic I., Bork P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res. 2024;52(W1):W78-W82. doi 10.1093/nar/gkae268

35. Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv. 2013. doi 10.48550/arXiv.1303.3997

36. Liu Y., Yin C., Zhu M., Zhan Y., Lin M., Yan Y. Comparative genomic analysis of Bacillus velezensis BRI3 reveals genes potentially as sociated with efficient antagonism of Sclerotinia sclerotiorum (Lib.) de Bary. Genes (Basel). 2024;15(12):1588. doi 10.3390/genes 15121588

37. Mohamadpoor M., Amini J., Ashengroph M., Azizi A. Evalua tion of biocontrol potential of Achromobacter xylosoxidans strain CTA8689 against common bean root rot. Physiol Mol Plant Pathol. 2022;117:101769. doi 10.1016/j.pmpp.2021.101769

38. Panneerselvam S., Mishra R., Berry C., Crickmore N., Bonning B.C. BPPRC database: a web-based tool to access and analyse bacte rial pesticidal proteins. Database. 2022;2022:baac022. doi 10.1093/database/baac022

39. Parthasarathy S., Parapatla H., Nandavaram A., Palmer T., Siddavat tam D. Organophosphate hydrolase is a lipoprotein and interacts with Pi-specific transport system to facilitate growth of Brevun dimonas diminuta using OP insecticide as source of phosphate. J Biol Chem. 2016;291(14):7774-7785. doi 10.1074/jbc.M116.715110

40. Pellegrinetti T.A., Monteiro G.G.T.N., Lemos L.N., dos Santos R.A.C., Barros A.G., Mendes L.W. PGPg_finder: a comprehensive and user friendly pipeline for identifying plant growth-promoting genes in genomic and metagenomic data. Rhizosphere. 2024;30:100905. doi 10.1016/j.rhisph.2024.100905

41. Pérez Ortega C., Leininger C., Barry J., Poland B., Yalpani N., Al tier D., Nelson M.E., Lu A.L. Coordinated binding of a two-compo nent insecticidal protein from Alcaligenes faecalis to western corn rootworm midgut tissue. J Invertebr Pathol. 2021;183:107597. doi 10.1016/j.jip.2021.107597

42. Prjibelski A., Antipov D., Meleshko D., Lapidus A., Korobeynikov A. Using SPAdes de novo assembler. Curr Protoc Bioinform. 2020; 70(1):e102. doi 10.1002/cpbi.102

43. Rabbee M.F., Ali M.S., Choi J., Hwang B.S., Jeong S.C., Baek K. Bacillus velezensis: a valuable member of bioactive molecules within plant microbiomes. Molecules. 2019;24(6):1046. doi 10.3390/molecules24061046

44. Richter M., Rosselló-Móra R., Oliver Glöckner F., Peplies J. JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics. 2016;32(6): 929-931. doi 10.1093/bioinformatics/btv681

45. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinfor matics. 2014;30(14):2068-2069. doi 10.1093/bioinformatics/btu153

46. Shi H., Zeng H., Yang X., Zhao J., Chen M., Qiu D. An insecticidal protein from Xenorhabdus ehlersii triggers prophenoloxidase acti vation and hemocyte decrease in Galleria mellonella. Curr Micro biol. 2012;64(6):604-610. doi 10.1007/s00284-012-0114-7

47. Sun Y., Ran Y., Yang H., Mo M., Li G. Volatile metabolites from Bre vundimonas diminuta and nematicidal esters inhibit Meloidogyne javanica. Microorganisms. 2023;11(4):966. doi 10.3390/microorganisms11040966

48. Topalović O., Elhady A., Hallmann J., Richert-Pöggeler K.R., Heuer H. Bacteria isolated from the cuticle of plant-parasitic nematodes at tached to and antagonized the root-knot nematode Meloidogyne hapla. Sci Rep. 2019;9(1):11477. doi 10.1038/s41598-019-47942-7

49. Walker B.J., Abeel T., Shea T., Priest M., Abouelliel A., Sakthikumar S., Cuomo C.A., Zeng Q., Wortman J., Young S.K., Earl A.M. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. 2014;9(11):e112963. doi 10.1371/journal.pone.0112963

50. Wang S., Jin P., Zheng Y., Kangkang W., Chen J., Liu J., Li Y. Bacil lus velezensis B105-8, a potential and efficient biocontrol agent in control of maize stalk rot caused by Fusarium graminearum. Front Microbiol. 2024;15:1462992. doi 10.3389/fmicb.2024.1462992

51. Wang Z., Lu K., Liu X., Zhu Y., Liu C. Comparative functional ge nome analysis reveals the habitat adaptation and biocontrol char acteristics of plant growth-promoting bacteria in NCBI databases. Microbiol Spectr. 2023;11(3):e0500722. doi 10.1128/spectrum.05007-22

52. Wu Y.-W., Simmons B.A., Singer S.W. MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets. Bioinformatics. 2016;32(4):605607. doi 10.1093/bioinformatics/btv638

53. Yamaç M., Şahin E., Ceyhan E., Amoroso M.J., Cuozzo S.A., Pilatin S. The screening of bacterial insecticides against Musca domestica L. (diptera: Muscidae). Fresenius Environ Bull. 2010;19(5):862-870

54. Yang K., Dai X., Maitikadir Z., Zhang H., Hao H., Yan C. Comparative genome analysis of endophytic Bacillus amyloliquefaciens MR4: a potential biocontrol agent isolated from wild medicinal plant root tissue. J Appl Genet. 2024;65(4):907-923. doi 10.1007/s13353-02400905-9

55. Yang T., Xin Y., Liu T., Li Z., Liu X., Wu Y., Wang M., Xiang M. Bac terial volatile-mediated suppression of root-knot nematode (Meloi dogyne incognita). Plant Dis. 2022;106(5):1358-1365. doi 10.1094/ PDIS-06-21-1139-RE

56. Yeo Y.J., Park A.R., Vuong B.S., Kim J.-C. Biocontrol of Fusarium head blight in rice using Bacillus velezensis JCK-7158. Front Micro biol. 2024;15:1358689. doi 10.3389/fmicb.2024.1358689

57. Zhang F., Liu Q., Wang Y., Yin J., Meng X., Wang J., Zhao W., Liu H., Zhang L. Effects of surfactin stress on gene expression and patho logical changes in Spodoptera litura. Sci Rep. 2024;14(1):30357. doi 10.1038/s41598-024-81946-2

58. Zhang M., Kong Z., Fu H., Shu X., Xue Q., Lai H., Guo Q. Rhizosphere microbial ecological characteristics of strawberry root rot. Front Microbiol. 2023;14:1286740. doi 10.3389/fmicb.2023.1286740