Advertisement

3 Biotech

, 9:117 | Cite as

Molecular characterization of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Thailand

  • Kesorn Boonmee
  • Sutticha Na-Ranong Thammasittirong
  • Anon ThammasittirongEmail author
Original Article
  • 61 Downloads

Abstract

A total of 511 local isolates of Bacillus thuringiensis from different geographical regions of Thailand were analyzed for the presence of the cry1A, cry1B, cry2A, cry9, and vip3A genes encoding for lepidopteran-specific toxins. PCR results revealed that 94.32% (482/511) of B. thuringiensis isolates harbored at least one of the detected genes, of which the cry1A, cry1B, cry2A, cry9, and vip3A genes were detected at frequencies of 90.61%, 89.63%, 76.32%, 40.70%, and 48.18%, respectively. Nineteen gene-combination profiles were discovered among 482 B. thuringiensis isolates, of which the most frequently detected profile contained the cry1A, cry1B, cry2A, and vip3A genes. Sixty-one isolates (12.66%), which harbored all of the detected insecticidal toxin genes, were further detected for the exochitinase (chi36) gene and chitinase activity. The results revealed that all 61 isolates contained the chi36 gene and exhibited chitinase activity. Insect bioassays showed that five isolates were highly toxic (more than 80% mortality) against second instar larvae of Spodoptera litura, of which the highest insect mortality (93%) was obtained from the B. thuringiensis isolates 225-15 and 417-1. Scanning electron microscopy revealed that the crystal morphologies of the five effective isolates were bipyramidal and cuboidal shapes. SDS-PAGE analysis of the spore–crystal mixture showed major bands of approximately 65 and 130 kDa. These five effective strains are alternative candidates for use as a microbial insecticide for the control of the S. litura pest.

Keywords

Bacillus thuringiensis Bacterial toxin Entomopathogenic bacteria Microbial insecticide 

Notes

Acknowledgements

This work was financially supported by the Kasetsart University Research and Development Institute (KURDI) and partially supported by the Research Promotion and Technology Transfer Center (RPTTC), and the Department of Microbiology (Grant year 2018) Faculty of Liberal Arts and Science, Kasetsart University, Nakhon Pathom, Thailand.

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

13205_2019_1646_MOESM1_ESM.docx (1.9 mb)
Fig. S1. Chitinase activity assay on colloidal chitin agar a): S. marcescens, b): B. thuringiensis serovar kurstaki, c): B. thuringiensis 314-2, d): B. thuringiensis 225-15, e): B. thuringiensis 349-4, f): B. thuringiensis 417-1, g): B. thuringiensis 831-2, h): B. thuringiensis 834-1. Table S1. Chitinase activity assay of B. thuringiensis isolates (DOCX 1977 KB)

References

  1. Arora N, Ahmad T, Rajagopal R, Bhatnagar RK (2003) A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1. Biochem Biophys Res Commun 307:620–625CrossRefGoogle Scholar
  2. Azizoglu U, Yılmaz S, Ayvaz A, Karabörklü S (2015) Effects of Bacillus thuringiensis subsp. kurstaki HD1 spore-crystal mixture on the adults of egg parasitoid Trichogramma evanescens (Hymenoptera: Trichogrammatidae). Biotechnol Biotechnol Equip 29:653–658CrossRefGoogle Scholar
  3. Baranek J, Konecka E, Kaznowski A (2017) Interaction between toxin crystals and vegetative insecticidal proteins of Bacillus thuringiensis in lepidopteran larvae. Biocontrol 62:649–658CrossRefGoogle Scholar
  4. Chakroun M, Banyuls N, Bel Y, Escriche B, Ferre J (2016) Bacterial vegetative insecticidal proteins (Vip) from entomopathogenic bacteria. Microbiol Mol Biol Rev 80:329–350CrossRefGoogle Scholar
  5. Chen L, Jiang H, Cheng Q, Chen J, Wu G, Kumar A, Sun M, Liu Z (2015) Enhanced nematicidal potential of the chitinase pachi from Pseudomonas aeruginosa in association with Cry21Aa. Sci Rep 5:14395CrossRefGoogle Scholar
  6. Chen WB, Lu GQ, Cheng HM, Liu CX, Xiao YT, Xu C, Shen ZC, Soberón M, Bravo A, Wu KM (2017) Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm. Transgenic Res 26:763–774CrossRefGoogle Scholar
  7. de Escudero IR, Banyuls N, Bel Y, Maeztu M, Escriche B, Munoz D, Caballero P, Ferre J (2014) A screening of five Bacillus thuringiensis Vip3A proteins for their activity against lepidopteran pests. J Invertebr Pathol 117:51–55CrossRefGoogle Scholar
  8. Ding X, Luo Z, Xia L, Gao B, Sun Y, Zhang Y (2008) Improving the insecticidal activity by expression of a recombinant cry1Ac gene with chitinase-encoding gene in acrystalliferous Bacillus thuringiensis. Curr Microbiol 56:442–446CrossRefGoogle Scholar
  9. González-Ponce KS, Casados-Vázquez LE, Salcedo-Hernández R, Bideshi DK, del Rincón-Castro MC, Barboza-Corona JE (2017) Recombinant Bacillus thuringiensis subsp. kurstaki HD73 strain that synthesizes Cry1Ac and chimeric ChiA74∆sp chitinase inclusions. Arch Microbiol 199:627–633CrossRefGoogle Scholar
  10. Gouffon C, Van Vliet A, Van Rie J, Jansens S, Jurat-Fuentes JL (2011) Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A Toxin. Appl Environ Microbiol 77:3182–3188CrossRefGoogle Scholar
  11. Herrero S, Bel Y, Hernández-Martínez P, Ferré J (2016) Susceptibility, mechanisms of response and resistance to Bacillus thuringiensis toxins in Spodoptera spp. Curr Opin Insect Sci 15:89–96CrossRefGoogle Scholar
  12. Jain D, Sunda SD, Sanadhya S, Nath DJ, Khandelwal SK (2017) Molecular characterization and PCR-based screening of cry genes from Bacillus thuringiensis strains. 3 Biotech 7:4CrossRefGoogle Scholar
  13. Juarez-Hernandez EO, Casados-Vazquez LE, del Rincon-Castro MC, Salcedo-Hernandez R, Bideshi DK, Barboza-Corona JE (2015) Bacillus thuringiensis subsp. israelensis producing endochitinase ChiA74Deltasp inclusions and its improved activity against Aedes aegypti. J Appl Microbiol 119:1692–1699CrossRefGoogle Scholar
  14. Kelkenberg M, Odman-Naresh J, Muthukrishnan S, Merzendorfer H (2015) Chitin is a necessary component to maintain the barrier function of the peritrophic matrix in the insect midgut. Insect Biochem Mol Biol 56:21–28CrossRefGoogle Scholar
  15. Lemes ARN, Figueiredo CS, Sebastião I, Marques da Silva L, da Costa Alves R, de Siqueira HÁA, Lemos MVF, Fernandes OA, Desidério JA (2017) Cry1Ac and Vip3Aa proteins from Bacillus thuringiensis targeting Cry toxin resistance in Diatraea flavipennella and Elasmopalpus lignosellus. from sugarcane. Peer J 5:e2866CrossRefGoogle Scholar
  16. Lone SA, Yadav R, Malik A, Padaria JC (2016) Molecular and insecticidal characterization of Vip3A protein producing Bacillus thuringiensis strains toxic against Helicoverpa armigera (Lepidoptera: Noctuidae). Can J Microbiol 62:179–190CrossRefGoogle Scholar
  17. Lu Q, Cao G, Zhang L, Liang G, Gao X, Zhang Y, Guo Y (2013) The binding characterization of Cry insecticidal proteins to the brush border membrane vesicles of Helicoverpa armigera, Spodoptera exigua, Spodoptera litura and Agrotis ipsilon. J Integr Agric 12:1598–1605CrossRefGoogle Scholar
  18. McNeil BC, Dean DH (2011) Bacillus thuringiensis Cry2Ab is active on Anopheles mosquitoes: single D block exchanges reveal critical residues involved in activity. FEMS Microbiol Lett 325:16–21CrossRefGoogle Scholar
  19. Monnerat RG, Batista AC, de Medeiros PT, Martins ÉS, Melatti VM, Praça LB, Dumas VF, Morinaga C, Demo C, Gomes ACM, Falcão R, Siqueira CB, Silva-Werneck JO, Berry C (2007) Screening of Brazilian Bacillus thuringiensis isolates active against Spodoptera frugiperda. Plutella xylostella and Anticarsia gemmatalis. Biol Control 41:291–295CrossRefGoogle Scholar
  20. Naqvi RZ, Asif M, Saeed M, Asad S, Khatoon A, Amin I, Mukhtar Z, Bashir A, Mansoor S (2017) Development of a triple gene Cry1Ac-Cry2Ab-EPSPS construct and its expression in Nicotiana benthamiana for insect resistance and herbicide tolerance in plants. Front Plant Sci 8:55CrossRefGoogle Scholar
  21. Negatu B, Kromhout H, Mekonnen Y, Vermeulen R (2016) Use of chemical pesticides in Ethiopia: a cross-sectional comparative study on knowledge, attitude and practice of farmers and farm workers in three farming systems. Ann Occup Hyg 60:551–566CrossRefGoogle Scholar
  22. Palma L, Munoz D, Berry C, Murillo J, Caballero P (2014) Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins (Basel) 6:3296–3325CrossRefGoogle Scholar
  23. Panuwet P, Siriwong W, Prapamontol T, Ryan PB, Fiedler N, Robson MG, Barr DB (2012) Agricultural pesticide management in Thailand: situation and population health risk. Environ Sci Policy 17:72–81CrossRefGoogle Scholar
  24. Patel KD, Chudasama CJ, Ingle SS (2012) Molecular characterization of Bacillus thuringiensis isolated from diverse habitats of India. J Basic Microbiol 52:437–445CrossRefGoogle Scholar
  25. Rangeshwaran R, Gorky A, Viswakethu V, Karkera A, Sivakumar G, Mohan M (2014) Cry gene and plasmid profiling of Bacillus thuringiensis isolated from Indian soils. J Biol Control 28:185–191Google Scholar
  26. Reyaz AL, Gunapriya L, Indra Arulselvi P (2017) Molecular characterization of indigenous Bacillus thuringiensis strains isolated from Kashmir valley. 3 Biotech 7:143CrossRefGoogle Scholar
  27. Ribeiro BM, Martins ÉS, de Souza Aguiar RW, Corrêa RFT (2017) Expression of Bacillus thuringiensis toxins in insect cells. In: Bacillus thuringiensis. and Lysinibacillus sphaericus. Springer, Cham, pp 99–110CrossRefGoogle Scholar
  28. Salama HS, Abd El-Ghany NM, Saker MM (2015) Diversity of Bacillus thuringiensis isolates from Egyptian soils as shown by molecular characterization. J Genet Eng Biotechnol 13:101–109CrossRefGoogle Scholar
  29. Sampson MN, Gooday GW (1998) Involvement of chitinases of Bacillus thuringiensis during pathogenesis in insects. Microbiology 144:2189–2194CrossRefGoogle Scholar
  30. Sauka DH, Benintende GB (2017) Diversity and distribution of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Argentina. Rev Argent Microbiol 49:273–281PubMedGoogle Scholar
  31. Seifinejad A, Jouzani GRS, Hosseinzadeh A, Abdmishani C (2008) Characterization of Lepidoptera-active cry and vip genes in Iranian Bacillus thuringiensis strain collection. Biol Control 44:216–226CrossRefGoogle Scholar
  32. Tawatsin A, Usavadee T, Padet S (2015) Pesticides used in Thailand and toxic effects to human health. Med Res Arch 3:1–10Google Scholar
  33. Thammasittirong A, Attathom T (2008) PCR-based method for the detection of cry genes in local isolates of Bacillus thuringiensis from Thailand. J Invertebr Pathol 98:121–126CrossRefGoogle Scholar
  34. Thammasittirong A, Prigyai K, Thammasittirong SNR (2017) Mosquitocidal potential of silver nanoparticles synthesized using local isolates of Bacillus thuringiensis subsp. israelensis and their synergistic effect with a commercial strain of B. thuringiensis subsp. israelensis. Acta Tropica 176:91–97CrossRefGoogle Scholar
  35. Vachon V, Laprade R, Schwartz JL (2012) Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: a critical review. J Invertebr Pathol 111:1–12CrossRefGoogle Scholar
  36. Wang J, Boets A, Van Rie J, Ren G (2003) Characterization of cry1, cry2, and cry9 genes in Bacillus thuringiensis isolates from China. J Invertebr Pathol 82:63–71CrossRefGoogle Scholar
  37. Wang K, Yan P, Cao L (2014) Chitinase from a novel strain of Serratia marcescens JPP1 for biocontrol of aflatoxin: molecular characterization and production optimization using response surface methodology. Biomed Res Int 2014:1–8Google Scholar
  38. Wasano N, Saitoh H, Maeda M, Ohgushi A, Mizuki E, Ohba M (2005) Cloning and characterization of a novel gene cry9Ec 1 encoding lepidopteran-specific parasporal inclusion protein from a Bacillus thuringiensis serovar galleriae strain. Can J Microbiol 51:988–995CrossRefGoogle Scholar
  39. Yılmaz S, Ayvaz A, Akbulut M, Azizoglu U, Karabörklü S (2012) A novel Bacillus thuringiensis strain and its pathogenicity against three important pest insects. J Stored Prod Res 51:33–40CrossRefGoogle Scholar
  40. Yu X, Zheng A, Zhu J, Wang S, Wang L, Deng Q, Li S, Liu H, Li P (2011) Characterization of vegetative insecticidal protein vip genes of Bacillus thuringiensis from Sichuan Basin in China. Curr Microbiol 62:752–757CrossRefGoogle Scholar
  41. Zhang Q, Chen LZ, Lu Q, Zhang Y, Liang GM (2015) Toxicity and binding analyses of Bacillus thuringiensis toxin Vip3A in Cry1Ac-resistant and -susceptible strains of Helicoverpa armigera (Hubner). J Integr Agr 14:347–354CrossRefGoogle Scholar
  42. Zorzetti J, Ricietto APS, Fazion FAP, Meneguim AM, Neves PMOJ, Vilas-Boas LA, Rodrigues RB, Vilas-Bôas GT (2017) Selection and characterization of Bacillus thuringiensis (Berliner) (Eubacteriales: Bacillaceae) strains for Ecdytolopha aurantiana (Lima) (Lepidoptera: Tortricidae) control. Neotrop Entomol 46:86–92CrossRefGoogle Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  • Kesorn Boonmee
    • 1
  • Sutticha Na-Ranong Thammasittirong
    • 1
    • 2
  • Anon Thammasittirong
    • 1
    • 2
    Email author
  1. 1.Department of Microbiology, Faculty of Liberal Arts and ScienceKasetsart UniversityNakhon PathomThailand
  2. 2.Microbial Biotechnology Unit, Faculty of Liberal Arts and ScienceKasetsart UniversityNakhon PathomThailand

Personalised recommendations