Skip to main content
SpringerLink
Log in

Molecular and biological characterization of native Bacillus thuringiensis strains for controlling tomato leafminer (Tuta absoluta Meyrick) (Lepidoptera: Gelechiidae) in Colombia

  • Original Paper
  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Twenty-eight soil samples were obtained from open fields and greenhouses used for tomato cultivation in various regions of Colombia. For functional characterization, 99 Bacillus thuringiensis (Bt) strains were isolated and characterized by abundance and morphology of microscopic crystals, SDS–PAGE of protein extracts and M-PCR analyses of genes of the cry1 family, as well as for their insecticidal activity against Tuta absoluta second instar larvae. Native Bt strains had amorphous (5%), bi-pyramidal (27%), square (8%), spherical (38%) and triangular (22%) crystal forms. Based on the presence of 1–4 different crystal forms, 18 different profiles were established. The SDS–PAGE analyses of protein extracts established ten different strain groups based on their protein band weight and potential biological activity. The M-PCR technique identified 35 native Bt strains based on the presence of the 6 genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D, whose frequency of occurrence was 76, 26, 21, 35, 32 and 8.8%, respectively. Thirteen different PCR profiles were found in native Bt strains. Several gene combinations tended to co-occur with elevated frequency, such as the pairs cry1Ac/cry1C, cry1Ab/cry1Ac and cry1Ab/cry1B, for which Pearson correlation coefficients were 0.69, 0.52 and 0.54, respectively. Native strains ZBUJTL39 and ZCUJTL11 had up to three times higher biological activity against T. absoluta second instar larvae than the reference strain Bt var. kurstaki HD1, with an LD50 of 2.4 μg/ml (P < 0.05) for native Bt strain ZCUJTL11. This study suggests a high biodiversity of native Bt strains from tomato growing regions in Colombia, which has important implications for designing biological control strategies for T. absoluta.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Akio I, Yasuyuki S, Sakae K, Yoshitomo K, Kyoko K, Kenjiro M, Eiichi M, Tetsuyuki A, Michi O (2004) A Bacillus thuringiensis crystal protein with selective cytocidal action to human cells. J Biol Chem 272:21282–21286

    Google Scholar 

  • Ammons D, Rampersad J, Khan A (2002) Usefulness of staining parasporal bodies when screening for Bacillus thuringiensis. J Invertebr Pathol 79(3):203–204

    Article  Google Scholar 

  • Arango J, Romero M, Orduz S (2002) Diversity of Bacillus thuringiensis strain from Colombia with insecticidal activity against Spodoptera frugiperda Lepidoptera: Noctuidae. J Appl Microbiol 92:466–474

    Article  CAS  Google Scholar 

  • Aronson A, Beckman W, Dunn P (1986) Bacillus thuringiensis and related insects pathogens. Microbiol Rev 50:1–24

    CAS  Google Scholar 

  • Bahamondes L, Mallea A (1969) Biología en Mendoza de Scrobipalpula absoluta (Meyrick) Povolny (Lepidoptera-Gelechiidae), especie nueva para la República Argentina. Rev Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo 15(1):96–104

    Google Scholar 

  • Ben-Dov E, Eivan M, Perleg N, Boussiba S, Zaritsky A (1996) Restriction map of the 125-kilobase plasmid of Bacillus thuringiensis subsp israelensis carrying the genes that encode delta-endotoxins active against mosquito larvae. Appl Environ Microbiol 62(9):3140–3145

    CAS  Google Scholar 

  • Bernhard K, Jarret P, Meadows M, Butt J, Ellis D, Roberts G, Pauli S, Rodgers P, Burges H (1997) Natural isolation of Bacillus thuringiensis: worldwide distribution, characterization and activity against insect pests. J Invertebr Pathol 70:59–68

    Article  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Bravo A, Sarabia S, López L, Ontiveros H, Abarca C, Ortiz A, Ortiz M, Lina L, Villalobos F, Peña G, Núñez-Valdez M, Soberón M, Quintero R (1998) Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl Environ Microbiol 64(12):4965–4972

    CAS  Google Scholar 

  • Cáceres S (1992) La polilla del tomate en Corrientes. Biología y control. Estación Experimental Agropecuaria Bella Vista, INTA, Argentina

    Google Scholar 

  • Carpenter J, Gianessi L (2001) Agricultural biotechnology: updated benefit estimates. National Center for Food and Agricultural Policy, Washington, DC. www.ncfap.org

  • Cely L, Cantor F, Rodríguez D, Cure J (2006) Niveles de daños ocasionados por diferentes densidades de Tuta absoluta (Lepidóptera: Gelechiidae) en tomate bajo invernadero. Abstracts, Socolen Encuentro con la Entomología en el Eje Cafetero. XXXIII Congreso de Entomología, Manizales, Colombia, 26–28 July

  • Cerón J, Covarrubias L, Quintero R, Ortíz A, Ortíz M, Aranda E, Lina L, Bravo A (1994) PCR, analysis of the cryI insecticidal crystal family genes from Bacillus thuringiensis. Appl Environ Microbiol 60:353–356

    Google Scholar 

  • Cerón J, Ortíz A, Quintero R, Güereca L, Bravo A (1995) Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection. Appl Environ Microbiol 61:3826–3831

    Google Scholar 

  • Chack K, Chao D, Tseng M, Kao S, Tuan S, Feng T (1994) Determination and distribution of cry-type genes of Bacillus thuringiensis isolates from Taiwan. Appl Environ Microbiol 60(7):2415–2420

    Google Scholar 

  • Charles JF, De Barjac H (1982) Sporulation et cristallogenese de Bacillus thuringiensis var. Israelensis en microscopie Electronique. Ann Microbiol 133:425–442

    CAS  Google Scholar 

  • CORPOICA (Corporación Colombiana de Investigación Agropecuaria) (2008) Librería virtual agropecuaria. Jorge Jaramillo: Tomate Bajo invernadero: July 2008. Bogotá, Colombia. http://intranet.corpoica.org.co. Accessed 24 July 2009

  • Crickmore N, Zeigler DR, Feitelson J, Schnepf E, Van Rie J, Lereclus D, Baum J, Dean HD (2010) Bacillus thuringiensis toxin nomenclature. http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html. Accessed 12 June 2010

  • De Vis R, Fuentes L, Escobar H, Lee R (2001) Manejo Integrado de Plagas y Enfermedades En: Producción de Tomate bajo Invernadero.Capítulo 5. Universidad de Bogotá Jorge Tadeo Lozano, CIAA, Colciencias, Bogotá, pp 59–90

    Google Scholar 

  • Estay P (2000) Polilla del tomate Tuta absoluta (Meyrick). Informativo La Platina 9:1–4

    Google Scholar 

  • Estay P, Bruna A (2002) Insectos y ácaros asociados al tomate en Chile. In: Estay P, Bruna A (eds) Insectos, ácaros y enfermedades asociados al tomate en Chile. Centro Regional de Investigación INIA-La Platina, Santiago, pp 9–22

    Google Scholar 

  • Feiltelson JS, Payne J, Kim L (1992) Bacillus thuringiensis: insects and beyond. Biotechnology 10:271–275

    Article  Google Scholar 

  • Fernández S, Montagne A (1990) Biología del tomate, Scrobipalpula absoluta (Meyrick) (Lepidoptera: Gelechiidae). Bol Entomol Venez 5(12):89–99

    Google Scholar 

  • Ferrandis M, Juárez-Pérez V, Frutos R, Ferre J (1999) Distribution of cryI, cryII and cryV genes within Bacillus thuringiensis isolates from Spain. Syst Appl Microbiol 22:179–185

    CAS  Google Scholar 

  • Finney DJ (1971) Probit analysis, 3rd edn. Cambridge University Press, Cambridge

    Google Scholar 

  • Gao M, Li R, Dai S, Wu Y, Yi D (2008) Diversity of Bacillus thuringiensis strains from soil in China and their pesticidal activities. Biol Control 44:380–388

    Article  CAS  Google Scholar 

  • García, F (1993) Control biológico de Scrobipalpula absoluta (Meyrick) plaga del tomate. In: Palacios F, Arciniegas I, Astrudillo AM (eds) Control Biológico en Colombia, Historias, Avances y Proyecciones. Lito-Támara Ltda, Palmira, Valle del Cauca, Colombia, pp 92–94

  • Gonçalves-Gervásio R, Ciociola A, Santa-Cecília L, Maluf W (1999) Aspectos biológicos de Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae) em dois genótipos de tomateiro contrastantes quanto ao teor de 2-tridecanona nos folíolos. Ciênc Agrotec 23:247–251

    Google Scholar 

  • González R (1989) Insectos y ácaros de importancia cuarentenaria en Chile. Ograma, Santiago

    Google Scholar 

  • González JM, Brown BJ, Carlton BC (1982) Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of Bthuringiensis and B. cereus. Proc Natl Acad Sci 79:6951–6955

    Article  Google Scholar 

  • Guedes R, Picanto M, Matoili A, Rocha D (1994) Efeito de inseticidas e sistemas de conducao do tomateiro no controle de Scrobipalpuloides absoluta (meyrick) (Lepidoptera: Gelechiidae). An Sociedad Entomologia de Brasil 23(2):321–325

    Google Scholar 

  • Henderson CF, Tilton EW (1955) Test with acaricides against the brown wheat mite. J Econ Entomol 48:157–161

    CAS  Google Scholar 

  • Hernández J, Mariño L, Orozco M, Narváez J (2000) Microscopic, biochemical and molecular characterization of native isolates of Bacillus thuringiensis in Colombia. El Astrolabio 2(2):13–18

    Google Scholar 

  • Herrnstadt C, Soares GG, Wilcox ER, Edwards DL (1986) A new strain of Bacilllus thuringiensis with activity against coleopteran insects. Bio Technol 4:305–308

    CAS  Google Scholar 

  • Höfte H, Whiteley H (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53:242–255

    Google Scholar 

  • Hongyu Z, Ziniu Y, Wangxi D (2000) Composition and ecological distribution of cry proteins and their genotypes of Bacillus thuringiensis isolates from warehouses in China. J Invertebr Pathol 76:191–197

    Article  CAS  Google Scholar 

  • IAN (Instituto Agronómico Nacional), JICA (Agencia de Cooperación Internacional de Japón) (1994) Control integrado de la palomilla del tomate Scrobipalpula absoluta (Meyrick, 1917). JICA, Caacupé

  • Ibarra JE, Federici BA (1986) Isolation of a relatively nontoxic 65 kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis var. israeliensis. J Bacteriol 165:527–533

    CAS  Google Scholar 

  • Jarrett P, Stephenson M (1990) Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis. Appl Environ Microbiol 56:1608–1614

    CAS  Google Scholar 

  • Kim HS, Lee DW, Woo SD, Yu YM, Kang SK (1998) Biological, immunological, and genetic analysis of Bacillus thuringiensis isolated from granary in Korea. Curr Microbiol 37:52–57

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685

    Article  CAS  Google Scholar 

  • Larraín P (1986) Plagas del tomate. Investigación y Progreso Agropecuario. Buenos Aires Argentina 39:30–35

    Google Scholar 

  • Lecadet MM (1970) Bacillus thuringiensis toxins. The proteinaceous crystal. In: Montie TC, Kadis S, Ajl SJ (eds) Microbial toxins, vol 3. Academic Press, New York, pp 437–471

    Google Scholar 

  • Lecadet MM, Dedondert R (1971) Biogenesis of the crystalline inclusion of Bacillus thuringiensis during sporulation. Eur J Biochem 23:282–294

    Article  CAS  Google Scholar 

  • Lietti MM, Botto EN, Alzogaray AR (2005) Insecticide resistat populations of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). Neotrop Entomol 34:113–119

    Article  Google Scholar 

  • López E (1991) Polilla del tomate: Problema crítico para la rentabilidad del cultivo de verano. Empresa y Avance Agríc 1(5):6–7

    Google Scholar 

  • Madigan MT, Martinko JM, Parker J (2003) BROCK biología de los microorganismos, 10th edn. Prentice Hall, Southern Illinois University, Carbondale

    Google Scholar 

  • Maeda M, Mizuki E, Hara M, Tanaka R, Akao T, Yamashita S, Ohba M (2001) Short communication Isolation of Bacillus thuringiensis from intertidal brackish sediments in mangroves. Microbiol Res 156:195–198

    Article  CAS  Google Scholar 

  • Martínez C, Ibarra J, Caballero P (2005) Association analysis between serotype, cry gene content and toxicity to Helicoverpa armigera larvae among Bacillus thuringiensis isolates native to Spain. J Invertebr Pathol 90:91–97

    Article  Google Scholar 

  • McPherson S, Perlack F, Fuchas R, Marrone P, Lavrik P, Fischhoff D (1998) Characterization of the coleopteran-specjfic protein gene of Bacillus thuringiensis var. tenebrionis. Biol Technol 6:61–66

    Google Scholar 

  • Mezquiriz N (2001) Control de la polilla del tomate (Tuta absoluta). Bol Hort UNLP 28:4–8

    Google Scholar 

  • Nazarian A, Jahangiri R, Salehi G, Seifinejad A, Soheilivand S, Bagheri O, Keshavarzi M, Alamisaeid K (2009) Coleopteran-specific and putative novel cry genes in Iranian native Bacillus thuringiensis collection. J Invertebr Pathol 102:101–109

    Article  CAS  Google Scholar 

  • Niedmann LL, Meza-Basso L (2006) Evaluación de cepas nativas de Bacillus thuringiensis como alternativa de manejo integrado de la polilla del tomate Tuta absoluta Meyrick; Lepidopetera: Gelechiidae en Chile. Agric Téc 66(3):235–246

    Google Scholar 

  • Pitre L, Hernández J, Bernal J (2008) Toxicidad de δ-endotoxinas recombinantes de Bacillus thuringiensis sobre larvas de la polilla guatemalteca (Tecia solanivora) (Lepidóptera: Gelechiidae). Rev Colomb Biotecnol 10(2):85–96

    Google Scholar 

  • Porcar M, Caballero P (2000) Molecular and insecticidal characterization of a Bacillus thuringiensis strain isolated during a natural epizootic. J Appl Microbiol 89:309–316

    Article  CAS  Google Scholar 

  • Prado E (1991) Artrópodos y sus enemigos naturales asociados a plantas cultivadas en Chile. Serie Bol. Técnico No 169. Instituto de Investigaciones Agropecuarias, Santiago, 207 pp

  • Quezada-Moraga E, García-Tovar P, Valverde-García P, Santiago-Álvarez C (2004) Isolation, geographical diversity and insecticidal activity of Bacillus thuringiensis from soils in Spain. Microbiol Res 159:59–71

    Article  Google Scholar 

  • Ramos F, Carmona A, Béres M, Méndez N (2004) Evaluación de asilamientos de Bacillus thuringiensis tóxicos a Diatrea saccharalis (Lepidoptera: Pyralidae). Bioagro 16(3):183–188

    Google Scholar 

  • Reddy A, Battisti L, Thorne C (1987) Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies. J Bacteriol 169:5263–5270

    CAS  Google Scholar 

  • Salazar E, Araya J (1997) Detección de resistencia a insecticidas en la polilla del tomate. Simiente 67:8–22

    Google Scholar 

  • Salazar E, Araya J (2001) Respuesta de la polilla del tomate, Tuta absoluta (Meyrick), a insecticidas en Arica. Agric Téc 61(4):429–435

    Article  Google Scholar 

  • Sanchis V, Lereclus D, Menou G, Chafaux J, Lecadet MM (1988) Multiplicity of delta-endotoxin genes with different specificities in Bacillus thuringiensis subesp. aizawai 7.29. Mol Microbiol 2:393–404

    Article  CAS  Google Scholar 

  • Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Fean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806

    CAS  Google Scholar 

  • Seifinejad A, Salehi JG, Hosseinzadeh A, Abdmishani C (2008) Characterization of Lepidoptera-active cry and vip genes in Iranian Bacillus thuringiensis strain collection. Biol Control 44:216–226

    Article  CAS  Google Scholar 

  • Sequeira HA, Guedes RN, Picanco MC (2000) Insecticide resistance in populations of Tuta absoluta (Lepidoptera: Gelechiidae). Agric For Entomol 23:431–434

    Google Scholar 

  • Souza JC, Reis PR, Gomes JM, Nacif AP, Salgado LO (1983) Traça-do-tomateiro, histórico, reconhecimento, biologia, prejuízos e controle. EPAMIG Bol 2, Belo Horizonte

  • 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 98:121–126

    Article  CAS  Google Scholar 

  • Theoduluz C, Vega A, Salazar M, González E, Meza-Basso L (2003) Expression of a Bacillus thuringiensis δ-endotoxin cry1Ab gene in Bacillus subtilis and Bacillus licheniformis strains that naturally colonize the phylloplane of tomato plants (Lycopersicon esculentum, Mills). J Appl Microbiol 94:375–381

    Article  Google Scholar 

  • Travers RS, Martin PA, Reichelderfer CF (1987) Selective process for efficient isolation of soil Bacillus spp. J Appl Environ Microbiol 53:1263–1266

    CAS  Google Scholar 

  • Tuta absoluta Information network (2010) http://www.tutaabsoluta.com. Accessed 10 March 2010

  • Urbaneja A, Vercher R, Navarro V, García MF, Porcuna JL (2007) La polilla del tomate, Tuta absoluta. Phytoma (Spain) 194:16–23

    Google Scholar 

  • Uribe D (2004) Ecología y distribución de Bacillus thuringiensis. In: Bravo A, Cerón J (eds) Bacillus thuringiensis en el control biológico, chap 4, 1st edn. Editorial Buena Semilla, Bogotá, Colombia, pp 101–122

  • Uribe D, Martinez W, Cerón J (2003) Distribution and diversity of cry genes in native strains of Bacillus thuringiensis obtained from different ecosystems from Colombia. J Invertebr Pathol 82:119–127

    Article  CAS  Google Scholar 

  • Vásquez M, Parra C, Hubert E, Espinoza P, Theoduloz C, Meza-Basso L (1995) Specificity and insecticidal activity of Chilean strains of Bacillus thuringiensis. J Invertebr Pathol 66:143–148

    Article  Google Scholar 

  • Vélez R (1997) Plagas agrícolas de impacto económico en Colombia: Bionomía y manejo integrado. Editorial Universidad de Antioquia, Medellín, pp 379–385

    Google Scholar 

  • Wang JH, Boets A, Van Rie J, Ren GX (2003) Characterization of cry1, cry2 and cry9 genes in Bacillus thuringiensis isolates from China. J Invertebr Pathol 82:63–71

    Article  CAS  Google Scholar 

  • Widner WR, Whiteley HR (1989) Two highly related insecticidal cry proteins of Bacillus thuringiensis subsp. kurstaki possess different host ratio specificities. J Bacteriol 171:965–974

    CAS  Google Scholar 

  • Xu-dong S, Chang-long S, Zhang J, Huang D, Tan J, Song F (2007) Identification and distribution of Bacillus thuringiensis isolates from primeval forests in Yunnan and Hainan Provinces and Northeast Region of China. Agric Sci China 6(11):1343–1351

    Google Scholar 

  • Yamamoto T, McLaughen RG (1981) Isolation of a protein from the parasporal crystal of Bacillus thuringiensis var. kurstaki toxic to the mosquito larvae Aedes tacniorhynchus. Biochem Biophys Res Commun 103:414–421

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. Michael Ahrens for their valuable comments and critical review of the manuscript. Also grateful to Dr. Leonardo Mariño, Dr. Alejandro López and Sally Wilson for critical reading. We would also like to thank Dr. Jorge Evelio Angel for his collaboration in the microscopic analyses. This work was supported by the Research Office of the Jorge Tadeo Lozano University.

Author information

Authors and Affiliations

  1. Facultad de Ciencias, Carrera de Biología Ambiental, GENBIMOL “Genética, Biología Molecular y Bioinformática”, Laboratorio de Biología Molecular, Universidad Jorge Tadeo Lozano, Cra. 4 No 22-61, Bogotá, D.C., Colombia

    J. Hernández-Fernández, L. Ramírez & N. Ramírez

  2. “Control Biológico de Plagas” Centro de Biosistemas, Universidad Jorge Tadeo Lozano, Km 3, Autopista Norte, Chía, Cundinamarca, Colombia

    L. S. Fuentes & J. Jiménez

Authors
  1. J. Hernández-Fernández
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. L. Ramírez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. N. Ramírez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L. S. Fuentes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J. Jiménez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. Hernández-Fernández.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hernández-Fernández, J., Ramírez, L., Ramírez, N. et al. Molecular and biological characterization of native Bacillus thuringiensis strains for controlling tomato leafminer (Tuta absoluta Meyrick) (Lepidoptera: Gelechiidae) in Colombia. World J Microbiol Biotechnol 27, 579–590 (2011). https://doi.org/10.1007/s11274-010-0493-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11274-010-0493-5

Keywords

Search

Navigation