Molecular characterization of the chitinase genes of native Bacillus thuringiensis isolates and their antagonistic activity against three important phytopathogenic fungi

Abstract

Bacillus spp. can promote the growth of plants and reduce plant disease incidence or severity by triggering induced systemic resistance to plant pathogens. In addition, bacteria of this genus are chitinase enzyme producers. Chitinase inhibits the growth of fungi by breaking down the chitin-containing cell wall of plant pathogenic fungi. In this study, 270 native Bacillus spp., isolated from various habitats in Kayseri and Adana, Turkey, were screened by PCR for the chitinase gene and 66 of them were found to have this gene. Nine Bacillus thuringiensis (Bt) isolates showing high insecticidal activity against insect pests in our previous studies were selected from 66 isolates containing the chitinase gene. Chitinases with different molecular weights ranging from ~ 40 to 113 kDa were determined by SDS-PAGE. To determine the antagonistic effects against plant pathogenic fungi (Fusarium oxysporum f.sp. niveum, Verticillium dahliae and Aspergillus niger), a dual culture assay was used with nine native and two standard strains of Bt, and radial growth inhibition was calculated as a percentage. Of all the tested isolates, SY33.3 showed the strongest antagonistic activity and thus, can be used as an effective biological control agent against plant pathogenic fungi.

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Code availability

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Abbreviations

SY:

Semih Yilmaz

U:

Ugur Azizoglu

Bt :

Bacillus thuringiensis

chi :

chitinase

PCR:

Polymerase Chain Reaction

PIRG:

Percentage Inhibition of Radial Growth

LB:

Luria-Bertani

mM:

mili Molar

TAE:

Tris-Acetate-EDTA

L:

Liter

rpm:

revolutions per minute

SDS-PAGE:

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Tm:

melting temperature

kb:

kilobase

F:

Forward

R:

Reverse

References

  1. Akintokun PO, Okuwa AO, Oloyede AR, Adebajo SO, Akintokun AK (2020) Potentials of indigenous Bacillus thuringiensis isolates from the soil in controlling Fusarium wilt of cucumber cause by Fusarium oxysporum f.sp cucumerinum. Nig J Biotech 37(1):129–137. https://doi.org/10.4314/njb.v37i1.14

    Article  Google Scholar 

  2. Arora N, Ahmad T, Rajagopal R, Bhatnagar RK (2003) A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1. Biochem Biophys Res Commun 307:620–625. https://doi.org/10.1016/S0006-291X(03)01228-2

    CAS  Article  PubMed  Google Scholar 

  3. Azizoğlu U (2009) Effect of the isolates of Bacillus thuringiensis isolated from agricultural fields on Ephestia kuehniella and Plodia interpunctella larvae irradiated by ultraviolet radiation. Erciyes University, Graduate School of Natural and Applied Sciences, pp 50 M. Sc. Thesis, Kayseri, Turkey

  4. Azizoglu U (2019) Bacillus thuringiensis as a biofertilizer and biostimulator: A mini–review of the little–known plant growth–promoting properties of Bt. Curr Microbiol 76:1379–1385. https://doi.org/10.1007/s00284-019-01705-9

    CAS  Article  PubMed  Google Scholar 

  5. Azizoglu U, Karabörklü S (2021) Role of recombinant DNA technology to improve the efficacy of microbial insecticides. In: Khan MA, Ahmad W (eds) Microbes for sustainable insect pest management. Sustainability in Plant and Crop Protection, vol 17. Springer, Cham. https://doi.org/10.1007/978-3-030-67231-7_8

  6. Basha S, Ulaganathan K (2002) Antagonism of Bacillus species (strain BC121) towards Curvularia lunata. Curr Sci 82:1457–1463

    CAS  Google Scholar 

  7. Bradford MM (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3

    CAS  Article  Google Scholar 

  8. Bravo A, Sarabia S, Lopez L, Ontiveros H, Abarca C, Ortiz A, Ortiz M, Lina L, Villalobos FJ, Peña G, Nuñez-Valdez ME, Soberón M, Quintero R (1998) Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl Environ Microbiol 64:4965–4972. https://doi.org/10.1128/AEM.64.12.4965-4972.1998

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Casique-Arroyo G, Bideshi D, Salcedo-Herna´ndez R, Barboza-Corona JE (2007) Development of a recombinant strain of Bacillus thuringiensis subsp. kurstaki HD-73 that produces the endochitinase ChiA74. Antonie Van Leeuwenhoek 92:1–9. https://doi.org/10.1007/s10482-006-9127-1

    CAS  Article  PubMed  Google Scholar 

  10. Chanpen W, Patcharapon S, Amarat B (1999) Purification and characterisation of chitinase from Bacillus circulans No.4.1. Curr Microbiol 39:134–140. https://doi.org/10.1007/s002849900434

    Article  Google Scholar 

  11. Cho MJ, Kim YK, Ka JO (2004) Molecular differentiation of Bacillus spp. antanistic against phytopathogenic fungi causing damping-off disease. J Microbiol Biotechn 14:599–606

    CAS  Google Scholar 

  12. Choi TG, Maung CEH, Lee DR, Henry AB, Lee YS, Kim KY (2020) Role of bacterial antagonists of fungal pathogens, Bacillus thuringiensis KYC and Bacillus velezensis CE 100 in control of root-knot nematode, Meloidogyne incognita and subsequent growth promotion of tomato. Biocontrol Sci Technol 30:685–700. https://doi.org/10.1080/09583157.2020.1765980

    Article  Google Scholar 

  13. Çolak A, Biçici M (2011) Determination differantiating of Fusarium oxysporum formae spseciales and determination incidence, severity and prevalence of fusarium wilt and crown-root rot in protected tomato growing areas of East Mediterranean Region of Turkey. Plant Prot Bull 51:331–345

    Google Scholar 

  14. Daulagala PWHKP (2017) Induction and expression of chitinases from four sub species of Bacillus thuringiensis. J Adv Microbiol 3:1–8. https://doi.org/10.9734/JAMB/2017/34084

    Article  Google Scholar 

  15. Dellavalle PD, Cabrera A, Alem D, Larrañaga P, Ferreira F, Rizza MD (2011) Antifungal activity of medicinal plant extracts against phytopathogenic fungus Alternaria spp. Chilean J Agric Res 71(2):231–239. https://doi.org/10.4067/S0718-58392011000200008

    Article  Google Scholar 

  16. Dervis S, Yetisir H, Yıldırım H, Tok FM, Kurt S, Karaca F (2009) Genetic and pathogenic characterization of Verticillium dahliae isolates from eggplant in Turkey. Phytoparasitica 37:467–476. https://doi.org/10.1007/s12600-009-0061-4

    Article  Google Scholar 

  17. Djenane Z, Nateche F, Amziane M, Gomis-Cebolla J, El-Aichar F, Khorf H, Ferré J (2017) Assessment of the antimicrobial activity and the entomocidal potential of Bacillus thuringiensis isolates from Algeria. Toxins 9:139. https://doi.org/10.3390/toxins9040139

    CAS  Article  PubMed Central  Google Scholar 

  18. Driss F, Kallassy-Awad M, Zouari N, Jaoua S (2005) Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki. J Appl Microbiol 99:945–953. https://doi.org/10.1111/j.1365-2672.2005.02639.x

    CAS  Article  PubMed  Google Scholar 

  19. Folders J, Tommassen L, Loon C, Bıtter W (2000) Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa. J Bacteriol 182:1257–1263. https://doi.org/10.1128/jb.182.5.1257-1263.2000

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Frankowski J, Lorıto M, Scala F, Schmid R, Berg G, Bahl H (2001) Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48. Arch Microbiol 176:421–426. https://doi.org/10.1007/s002030100347

    CAS  Article  PubMed  Google Scholar 

  21. Jabnoun-Khiareddine H, Daami-Remadi M, Barbara DJ, El Mahjoub M (2010) Morphological variability within and among Verticillium species collected in Tunisia. Tunis J Plant Prot 5:19–38

    Google Scholar 

  22. Ji SH, Paul NC, Deng JX, Kim YS, Yun BS, Yu SH (2013) Biocontrol activity of Bacillus amyloliquefaciens CNU114001 against fungal plant diseases. Mycobiology 41(4):234–242. https://doi.org/10.5941/MYCO.2013.41.4.234

    Article  PubMed  PubMed Central  Google Scholar 

  23. Jinantana J, Sariah M (1995) Antagonistic effect of Malaysian isolates of Trichoderma harzianum and Gliocladium virens on Sclerotium rolfsii. Pertanika J Trop Agric Sci 20:35–41

    Google Scholar 

  24. Juárez-Hernández EO, Casados-Vázquez LE, Brieba LG, Torres-Larios A, Jimenez-Sandoval P, Barboza-Corona JE (2019) The crystal structure of the chitinase ChiA74 of Bacillus thuringiensis has a multidomain assembly. Sci Rep 9:2591. https://doi.org/10.1038/s41598-019-39464-z

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Karabörklü S, Azizoglu U, Azizoglu ZB (2018) Recombinant entomopathogenic agents: a review of biotechnological approaches to pest insect control. World J Microbiol Biotechnol 34:14. https://doi.org/10.1007/s11274-017-2397-0

    CAS  Article  Google Scholar 

  26. Karaca İ, Karcıoğlu A, Ceylan B (1971) Wilt disease of cotton in the Ege Region of Turkey. J Tur Phytopathy 1:4–11

    Google Scholar 

  27. Kim HS, Timmis KN, Golyshin PN (2007) Characterization of a chitinolytic enzyme from Serratia sp. KCK isolated from Kimchi Juice. Appl Microbiol Biotechnol 75:1275–1283. https://doi.org/10.1007/s00253-007-0947-3

    CAS  Article  PubMed  Google Scholar 

  28. Kuzu SB (2008) Isolation of Bacillus producing chitinase, partial purification and characterization of enzyme. Çukurova University Institute of Natural And Applied Sciences Department of Biotechnology, MSc Thesis, pp.82

  29. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Lee YS, Park IH, Yoo JS, Chung SY, Lee YC, Cho YS, Ahn SC, Kim CM, Choi YL (2006) Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101. Bioresour Technol 98:2734–2741. https://doi.org/10.1016/j.biortech.2006.09.048

    CAS  Article  PubMed  Google Scholar 

  31. Lenardon MD, Munro CA, Gow NA (2010) Chitin synthesis and fungal pathogenesis. Curr Opin Microbiol 13(4):416–423. https://doi.org/10.1016/j.mib.2010.05.002

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Lin Y, Xiong G (2004) Molecular cloning and sequence analysis of the chitinase gene from Bacillus thuringiensis serovar alesti. Biotechnol Lett 26:635–639. https://doi.org/10.1023/B:BILE.0000023021.50213.ed

    CAS  Article  PubMed  Google Scholar 

  33. Mahmoudi E, Naderi D (2017) Anti-fungal and bio-control properties of chitinolytic bacteria against safflower Fusarium root rot. J Crop Prot 6:225–234

    Google Scholar 

  34. Martínez-Zavala SA, Barboza-Pérez UE, Hernández-Guzmán G, Bideshi DK, Barboza-Corona JE (2020) Chitinases of Bacillus thuringiensis: Phylogeny, modular structure, and applied potentials. Front Microbiol 14:10:3032. https://doi.org/10.3389/fmicb.2019.03032

    Article  Google Scholar 

  35. Maung CEH, Choi TG, Nam HH, Kim KY (2017) Role of Bacillus amyloliquefaciens Y1 in the control of Fusarium wilt disease and growth promotion of tomato. Biocontrol Sci Technol 27(12):1400–1415. https://doi.org/10.1080/09583157.2017.1406064

    Article  Google Scholar 

  36. Nagpure A, Choudhary B, Gupta RK (2014) Chitinases: in agriculture and human healthcare. Crit Rev Biotechnol 34:215–232. https://doi.org/10.3109/07388551.2013.790874

    CAS  Article  PubMed  Google Scholar 

  37. Okay S, Ozcengiz G (2011) Molecular cloning, characterization, and homologous expression of an endochitinase gene from Bacillus thuringiensis serovar morrisoni. Turk J Biol 35:1–7. https://doi.org/10.3906/biy-0905-5

    CAS  Article  Google Scholar 

  38. Pegg GF, Brady BL (2002) Verticillium Wilts. Plant Sci 79:80. https://doi.org/10.1079/9780851995298.0000

    Article  Google Scholar 

  39. Qin J, Tong Z, Zhan Y, Buisson C, Song F, He K, Nielsen-LeRoux C, Guo S (2020) A Bacillus thuringiensis chitin-binding protein is involved in insect peritrophic matrix adhesion and takes part in the infection process. Toxins 12(4):252. https://doi.org/10.3390/toxins12040252

    CAS  Article  PubMed Central  Google Scholar 

  40. Reyaz AL, Balakrishnan N, Udayasuriyan V (2019) Genome sequencing of Bacillus thuringiensis isolate T414 toxic to pink bollworm (Pectinophora gossypiella Saunders) and its insecticidal genes. Microb Pathog, 103553. https://doi.org/10.1016/j.micpath.2019.103553

  41. Reyaz AL, Balakrishnan N, Udayasuriyan V (2021) A novel Bacillus thuringiensis isolate toxic to cotton pink bollworm (Pectinophora gossypiella Saunders). Microb Pathog 104671. https://doi.org/10.1016/j.micpath.2020.104671

  42. Reyes-Ramírez A, Escudero-Abarca BI, Aguilar-Uscanga G, Hayward-Jones PM, Barbozacorona JE (2004) Antifungal activity of Bacillus thuringiensis chitinase and Its potential for the biocontrol of phytopathogenic fungi in soybean seeds. J Food Sci 69:131–134. https://doi.org/10.1111/j.1365-2621.2004.tb10721.x

    Article  Google Scholar 

  43. Rojas Avelizapa LI, Cruz-Camarillo R, Guerrero MI, Rodríguez-Vázquez R, Ibarra JE (1999) Selection and characterization of proteo-chitinolytic strain of Bacillus thuringiensis able to grow in shrimp waste media. World J Microb Biot 15:299–308. https://doi.org/10.1023/A:1008947029713

    Article  Google Scholar 

  44. Srinivasan R, Shanmugam V (2006) Postharvest management of black mould rots of onion. Indian Phytopathol 59:333–339

    Google Scholar 

  45. Srivastava RAK (1987) Purification and chemical characterization of thermostable amylase produced by Bacillus stearothermophilus. Enzyme Microb Tech 9:749–754. https://doi.org/10.1016/0141-0229(87)90036-6

    CAS  Article  Google Scholar 

  46. Strange RN, Scott PR (2005) Plant disease: A threat to global food security. Annu Rev Phytopathol 43:83–116. https://doi.org/10.1146/annurev.phyto.43.113004.133839

    CAS  Article  PubMed  Google Scholar 

  47. Sumner DR (1995) Black mold. Compendium of onion and garlic diseases, Ed: Schwartz HF, Mohan SK, APS Press, St. Paul, pp 26–27

    Google Scholar 

  48. Tombolini R, Van der Gaag DJ, Gerhardson B, Jansson JK (1999) Colonization pattern of the biocontrol strain Pseudomonas chlororaphis MA 342 on barley seeds visualized by using green fluorescent protein. Appl Environ Microb 65:3674–3680. https://doi.org/10.1128/AEM.65.8.3674-3680.1999

    CAS  Article  Google Scholar 

  49. Usharani TR, Gowda TKS (2011) Cloning of chitinase gene Bacillus thuringiensis. Indian J Biotechnol 10:264–269

    CAS  Google Scholar 

  50. Yılmaz S (2010) Molecular characterization of Bacillus thuringiensis strains isolated from different locations and their effectiveness on some pest insects. Erciyes University, Graduate School of Natural and Applied Sciences, pp. 172, PhD. Thesis, Kayseri, Turkey

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Acknowledgements

Special thanks to Dr. Sibel Derviş for supplying phytopathogenic fungi. The authors would like to express special thanks to Dr. Hatice Korkmaz Güvenmez for supplying chitin. This study is the master thesis entitled ‘Screening of chitinase genes from local Bacillus isolates and testing their antifungal activity’ of Zehra Busra Azizoglu and presented on Ecology 2017 International Symposium held on 11–13 May 2017 in Kayseri, Turkey.

Funding

This project was funded by Erciyes University Scientific Project Unit under the codes of FBD-08-540.

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ZBA performed bioassays and molecular studies, and contributed to the planning of the study. SY contributed to the material supply, molecular studies, manuscript preparation and data analysis. UA participated in experimental studies, manuscript preparation and molecular studies. SK carried out statistical analysis of the data and contributed to the figures. RT contributed to experimental studies. AA conceived of the study, contributed to the design and coordination and helped to draft the manuscript. All authors read and approved the manuscript.

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Correspondence to Abdurrahman Ayvaz.

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Azizoglu, Z.B., Yilmaz, S., Azizoglu, U. et al. Molecular characterization of the chitinase genes of native Bacillus thuringiensis isolates and their antagonistic activity against three important phytopathogenic fungi. Biologia (2021). https://doi.org/10.1007/s11756-021-00802-0

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Keywords

  • Bt
  • Biological control
  • Chitinase
  • Plant pathogenic fungi