Biochemical characteristics that presented variable response among the bacterial isolates. Molecular characterization is also showed (see below).
1. Introduction
The fall armyworm,
The
During a screening programme of
2. Biochemical characterization of B. thuringiensis isolates and assessment of toxicity
Crystalliferous spore-forming bacteria were isolated from both
Bacteria -characterized by conventional microbiological methods- possessed typical cellular and colonial morphologies, as well as physiological, biochemical and nutritional features that resembled
TRC11* | TMAN2* | THM8* | NN1** | TRC10* | RT** | TSA2* | TRC12* | N28** | MAN8** | MAN1** | THM30* | LSM** | LQ** | ||
Central spore | + | + | + | + | + | + | + | - | + | + | + | - | + | + | + |
Sub terminal spore | - | - | - | - | - | - | - | + | - | - | - | + | - | - | - |
Growth at pH 9 | + | + | - | + | + | + | + | + | + | + | + | - | + | + | + |
b e Growth in 0.2 % chitin | - | + | + | + | - | + | + | + | - | + | - | - | + | + | + |
b c e CMC hydrolysis (0.5 %) | + | + | + | + | + | - | - | + | + | + | - | + | + | - | - |
b e Chitin hydrolysis (0.2 %) | - | - | + | - | - | + | + | - | - | - | - | - | + | + | + |
b Gelatin hydrolysis (12 %) | + | - | - | - | - | - | + | - | + | - | - | + | - | - | - |
b Starch hydrolysis (2 %) | - | - | + | + | + | + | + | - | + | - | + | + | + | + | + |
Gas production in glucose | - | + | - | + | - | + | - | + | - | - | - | - | + | + | + |
d clindamycin | + | - | + | + | + | + | + | + | + | + | - | + | + | + | + |
d gentamicin | + | + | - | - | + | - | - | + | + | - | - | - | + | - | - |
d rifampicin | + | + | + | + | - | + | - | + | - | + | + | + | + | + | + |
- | + | - | + | - | + | - | - | - | - | + | + | + | + | ND | |
- | + | + | - | + | + | - | + | + | - | - | + | + | + | ND | |
- | + | + | |||||||||||||
+ | + | + | |||||||||||||
+ | + | + | |||||||||||||
- | + | + | |||||||||||||
+ | + | + |
From a biochemical point of view, the 14 strains were catalase-positive, reduced nitrate and produced acetyl methyl carbinol in Voges-Proskauer broth; growth was observed at pH 7 on LB agar supplemented with 2, 3 and 5% NaCl and on LB agar at 30, 37 and 45 ºC. The strains also hydrolyzed casein and were motile on soft LB agar. Negative results for all strains were obtained in several tests: no growth was observed on LB agar at pH 4 or at 50 ºC and none of the strains hydrolyzed carboxymethyl cellulose (CMC) and urea. Antibiotic sensitivity tests revealed a resistance profile to penicillin, oxacillin, trimethoprim and a sensitive profile to erythromycin, vancomycin, levofloxacin, minocycline, chloramphenicol and teicoplanin. Phenotypic features that presented variability among the strains are showed in Table 1. The positive or negative result of each biochemical assay was entered in a 1-0 matrix. These data were subsequently analyzed through correspondence multivariate analysis, using Multivariate Statistical Package (MSVP) software (version 3.13). A cluster diagram based on these variable biochemical properties (that represented 54% of data variability) revealed that the strains formed two main groups
Although most of the native isolates presented similar biochemical and phenotypical characteristics compared with reference strain
RT | 100 ± 0 a | 9.2 (10.4 –16.0) | 1.98 ± 98 b |
LSM | 90.0 ± 7.3 a | 37.7 (27.8 – 46.2) | 1.80 ± 93 b |
LQ | 73.0 ± 5.7 c | 79.6 (68.2 – 90.7) | 1.14 ± 25 a |
86.0 ± 15.1 b | 58.7 (50.4 – 66.0) | 946 ± 14 a | |
control | 1.0 ± 3.1 d |
3. Numerical analysis of insecticidal crystal proteins of B. thuringiensis
In order to differentiate native crystalliferous isolates and to evaluate the relationship between the toxicity assays against
4. Molecular characterization of B. thuringiensis strains and crystal morphology
Although the presence of parasporal crystals is a diagnostic characteristic of
Generally, B. thuringiensis insecticidal protein toxin genes (cry) reside on large self-transmissible plasmids, and individual B. thuringiensis strains can harbor a diverse range of plasmids that can vary in number from 1 to 17 and in size from 2 to 80 MDa [29,30], although it has also been suggested that they are present in the chromosome [31]. In this context, to study the plasmid profiles of Bt strains is an important parameter to determine their identity, since the number and size of these is associated with a particular Bt strain. Comparison between strains belonging to the same serotype showed a great difference in variability [30]. Some serotypes (e.g., israelensis) showed the same basic pattern among all its strains, while other serotypes (e.g., morrisoni) showed a great diversity of patterns. These results indicate that plasmid patterns are valuable tools to discriminate strains below the serotype level [30]. The profile of extrachromosomal elements in Bt is influenced by a number of stressful growth conditions, which determine its stability and heritability (e. i. high temperatures determine the plasmid loss), therefore it is neccesary to take some care. In this study, cultures were routinely grown at 30 °C to avoid this phenomenon. Detection and isolation of plasmid DNA was conducted following the method of Kado and Liu [32]. DNA plasmid samples were electrophoresed on 0.8 % (wt vol-1) agarose gel. Our results showed that selected Bt strains present a complex plasmid profile (Figure 6).
In this experiment, the plasmid DNA was not linearized and therefore the same plasmid can produce as many as three different bands in the agarose gel. This made it difficult to determine the precise number of plasmids present in each complex plasmid profile. For this reason, we will refer to the number and size of plasmidic bands rather to plasmids themselves. An intense band above the chromosomal band (C) was observed in
Identification of cry genes by means of PCR has been used to predict insecticidal activity of the strains [17,18] and to determine the distribution of cry genes within a collection of B. thuringiensis strains [20, 33]. In this context, our crystalliferous strains were characterized in terms of presence of cry1 and cry2 genes by amplification with general primers. The most toxic Bt strains RT, LSM and LQ were characterized through additional PCR with specific
primers to identify the presence of
In addition, amplified fragments corresponding to
As mentioned before,
16S: 27F 1492R | 5´-GGTTACCTTGTTACGACTT-3´ | [34] |
ITS: ISR-1494 ISR-35 | [35] | |
Gral- | 5´-TGAGTCGCTTCGCATATTTGACT-3´ | [36] |
Gral-cry2 | [37] | |
Spe- | [38] | |
Spe- | ||
Spe- | ||
Spe- | ||
Spe- | [17] | |
Spe- | ||
Spe- |
From a methodological point of view, washing of crystal suspensions with absolute ethanol/distilled water (Figure 9B) was more appropriate for microscopic observation than washing with distilled water (Figure 9A).
As mentioned above, identification of
5. Assessment of enzyme activities in B. thuringiensis
Phenotypic characterization of selected strains allows identification of properties that are relevant at the moment of selecting bacteria for their use in environmental and agricultural microbiology. Synthesis of lytic enzymes by
In
Protein profiles are a useful tool to discriminate among strains, as they provide information about the proximity between species, subspecies and biovars [18, 52]. Considering this, characterization of microorganisms by means of their extracellular isoenzymes showing high polymorphism, as is the case of esterase, is particularly appealing. To determine extracellular esterase profiles,
6. Conclusion
Lepidoptera causes some of the most devastating insect pests in important crops in America. Since economy of these regions depends largely of agriculture, their
Fourteen
The discovery of a highly toxic isolates reveals the usefulness of screening studies for novel
References
- 1.
Konecka E. Kaznowski A. Ziemnicka J. Ziemnicki K. 2007 Molecular and phenotypic characterization of isolated during epizootics in Cydia pomonella L. J. Invertebr. Pathol.94 56 63 - 2.
Bravo A. Gill S. Soberón M. 2007 Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control 49 423 435 - 3.
Brar S. Verma M. Tyagi R. Surampalli R. Bernabé S. Valéro J. 2007 Bacillus thuringiensis proteases: Production and role in growth, sporulation and synergism Process Biochem.42 773 790 - 4.
Calderón M. Alcocer González. J. Molina M. Tames Guerra. R. Rodríguez Padilla. 2007 Adjuvant effects of crystal proteins from a Mexican strain of Bacillus thuringiensis on the mouse humoral response 35 271 276 - 5.
Virla E. Alvarez A. Loto F. Pera L. Baigorí M. 2008 Fall Armyworm strains (Lepidoptera: Noctuidae) in Argentina, their associate host plants and response to different mortality factors in laboratory Fla. Entomol.91 63 69 - 6.
Moreno Fajardo. O. Serna Cardona. F. 2006 Biología de (Lepidoptera: Noctuidae: Noctuinae) en flores cultivadas del híbrido comercial de Alstroemeria spp. Rev. Fac. Nal. Agr. Medellín.2 3435 3448 - 7. Berón C, Salerno G (2006) Characterization of Bacillus thuringiensis isolates from Argentina that are potentially useful in insect pest control. BioControl 51: 779-794.
- 8.
Gomes Monnerat. R. Cardoso Batista. A. Telles De Medeiros. P. Soares Martins. E. Melatti V. M. Praca I. B. Dumas V. F. Morinaga C. Demo C. Menezes Gomes. A. C. Falcao R. Siqueira C. B. Silva-Werneck J. O. Berry C. 2007 Screening of Brazilian thuringiensis isolates active against Spodoptera frugiperda, Plutella xylostella and Anticarsia gemmatalis. Biol. Control41 291 295 - 9.
Gassmann A. Carrière Y. Tabashnik E. 2009 Fitness Costs of Insect Resistance to Annu. Rev. Entomol.54 147 63 - 10.
Tabashnik B. Gassmann A. Crowder D. Carriére Y. 2008 Insect resistance to Bt crops: evidence versus theory Nat. Biotechnol.26 199 202 - 11.
Tabashnik B. Carrière Y. 2009 Environmental Impact of Genetically Modified Crops. In: Ferry N, Gatehouse A, editors. Insect Resistance to Genetically Modified Crops. Cambridge: CAB International.74 101 pp. - 12.
Christou P. Capell T. Kohli A. Gatehouse J. Gatehouse A. 2006 Recent developments and future prospects in insect pest control in transgenic crops Trends Plant. Sci.11 302 308 - 13.
Sauka D. Benintende G. 2008 Bacillus thuringiensis: generalidades. Un acercamiento a su empleo en el biocontrol de insectos lepidópteros que son plagas agrícolas. Rev. Argent. Microbiol.40 124 140 - 14.
Alvarez A. Virla E. Pera L. Baigorí M. 2011 Biological characterization of two Bacillus thuringiensis strains toxic against Spodoptera frugiperda World J. Microbiol. Biotechnol.27 2343 2349 - 15.
Carozzi N. Kramer V. Warren G. Evola S. Koziel M. G. 1991 Prediction of insecticidal activity of Bacillus thuringiensis strains by polymerase chain reaction product profiles. Appl. Environ. Microbiol.57 3057 3061 - 16.
Padidam M. 1992 The insecticidal crystal protein Cry1A (c) from is highly toxic for Heliothis Armigera. J. Invertebr. Pathol.59 109 111 - 17.
Ben-Dov E. Zaritski A. Dahan E. Barak Z. Sinai R. Manasherob R. Khamraeb A. Troitskaya E. Dubitsky A. Berezina N. Margalith Y. 1997 Extended screening by PCR for seven -group genes from field-collected strains of Bacillus thuringiensis. Appl. Environ. Microbiol.63 4883 4890 - 18.
Hansen B. Damgaard P. Eilenberg J. Pedersen J. C. 1998 Molecular and phenotypic characterization of Bacillus thuringiensis isolated from leaves and insects J. Invertebr. Pathol.71 106 114 - 19.
Alvarez A. Virla E. Pera L. Baigorí M. 2009a Characterization of native Bacillus thuringiensis strains and selection of an isolate active against Spodoptera frugiperda and Peridroma saucia Biotechnol. Lett.31 1899 1903 - 20.
Porcar M. Juarez-Perez V. 2003 PCR-based identification of Bacillus thuringiensis pesticidal crystal genes. FEMS Microbiol. Rev.26 419 432 - 21.
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 - 22.
Soberón M. Bravo A. 2001 y sus toxinas insecticidas. In: Microbios en línea. UNAM México. Available vía DIALOG - 23.
://www.biblioweb.dgsca.unam.mx/libros/microbios /Cap12 Accessed 1 March2011 - 24.
Alvarez A. Pera L. Loto F. Virla E. Baigori M. 2009b Insecticidal crystal proteins from native Bacillus thuringiensis: Numerical analysis and biological activity against Spodoptera frugiperda Biotechnol. Lett.31 77 82 - 25.
Sharif F. Alaeddinoğlu N. 1988 A rapid and simple method for staining of the crystal protein of . J. Ind. Microbiol.3 227 229 - 26.
Arango J. Romero M. Orduz S. 2002 Diversity of Bacillus thuringiensis strains from Colombia with insecticidal activity against Spodoptera frugiperda (Lepidoptera: Noctuidae) J. Appl. Microbiol.92 466 474 - 27.
Del Rincón-Castro M. Méndez-Lozano J. Ibarra J. 2006 Caracterización de cepas nativas de con actividad insecticida hacia el gusano cogollero del maíz Spodoptera frugiperda (Lepidoptera: Noctuidae). Folia Entomol. Mex.45 157 164 - 28.
Seligy V. Rancourt J. 1999 Antibiotic MIC/MBC analysis of Bacillus-based commercial insecticides: use of bioreduction and DNA-based assays J. Ind. Microbiol. Biotechnol.22 565 574 - 29.
Gough J. Kemp D. Akhurst R. Pearson R. Kongsuwan K. 2005 Identification and characterization of proteins from Bacillus thuringiensis with high toxic activity against the sheep blowfly, Lucilia cuprina. J. Invertebr. Pathol.90 39 46 - 30.
González J. Carlton B. 1980 Patterns of plasmid DNA in crystalliferous and acrystalliferous strains of Bacillus thuringiensis. 3 92 98 - 31.
Reyes-Ramírez IJ 2008 Plasmid patterns of Bacillus thuringiensis type strains Appl. Environ. Microbiol.74 125 129 - 32.
(Kronstad J. Schnepf H. Whiteley H. 1983 ) Diversity of locations for Bacillus thuringiensis crystal protein genes. J. Bacteriol.154 419 428 . - 33.
Kado C. Liu S. 1981 Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol.145 1365 73 - 34.
Chak K. Chao D. Tseng M. Kao S. Tuan S. Feng T. 1994 Determination and distribution of cry-type genes of Bacillus thuringiensis isolated from Taiwan Appl. Environ. Microbiol.60 2415 2420 - 35.
Weisburg W. Barns S. Pelletier D. Lane D. 1991 16S Ribosomal DNA amplification for phylogenetic study. J. Bacteriol.173 697 703 - 36.
Daffonchio D. Borin S. Frova G. et al. 1998 PCR fingerprinting of whole genomes: the spacers between the 16S and 23S rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and Bacillus licheniformis. Int. J Syst Bacteriol48 107 116 - 37.
Bravo A. Sarabia S. Lopez L. Ontiveros H. Abarca C. Ortiz A. Ortiz M. Lina L. Villalobos F. J. Peña G. ME Nuñez-Valdez Soberón. M. Quintero R. 1998 Characterization of cry genes in Mexican strain collection. Appl. Environ. Microbiol.64 4965 4972 - 38.
Ibarra J. del Rincón M. Ordúz S. Noriega D. Benintende G. Monnerat R. Regis L. Oliveira C. Lanz H. Rodriguez M. Sánchez J. Peña G. Bravo A. 2003 Diversity of strains from Latin America with insecticidal activity against different mosquito species Appl. Environ. Microbiol.69 5269 5274 - 39.
Cerón J. Covarrubias L. Quintero R. Ortiz M. Aranda E. Lina L. Bravo A. 1994 PCR Analysis of the cryI Insecticidal Crystal Family Genes from Bacillus thuringiensis. Appl. Environm. Microbiol.60 353 356 - 40.
Al-Momani F. Saadoun I. Obeidat M. 2002 Molecular characterization of local Bacillus thuringiensis strains recovered from Northern Jordan. J. Basic Microbiol.2 156 161 - 41.
Noguera P. Ibarra J. 2010 Detection of New cry Genes of Bacillus thuringiensis by Use of a Novel PCR Primer System Appl. Environ. Microbiol.76 6150 6155 - 42.
Benintende G. López-Meza J. Cozzi J. Ibarra J. E. 1999 Novel non-toxic isolates of Bacillus thuringiensis Lett.Appl. Microbiol.29 151 155 - 43.
Daffonchio D. Raddadi N. Merabishvili M. 2006 Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis. Appl. Environ. Microbiol.72 1295 1301 - 44.
Ramírez R. Ibarra J. 2005 Fingerprinting of Bacillus thuringiensis type strains and isolates by using Bacillus cereus group-specific repetitive extragenic palindromic sequence-based PCR analysis. Appl. Environ. Microbiol.71 1346 1355 - 45. Stefanova M, Leiva M, Larrinaga L, Coronado M (1999) Metabolic activity of Trichoderma spp. isolates for a control of soilborne phytopatogenic fungi. Rev. Fac. Agron. (LUZ) 16: 509-516.
- 46.
MJ Lengyel Pekár. S. Felföldi G. Patthy A. Gráf L. Fodor A. Venekei I. 2004 Comparison of Proteolytic Activities Produced by Entomopathogenic Photorhabdus Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes. Appl. Environ. Microbiol.70 7311 7320 - 47. Cokmus C, Elcin M (1995) Stability and controlled release properties of carboxymethylcellulose-encapsulated Bacillus thuringiensis var. israelensis. Pest. Sci. 45: 351-355.
- 48.
barra J. E. Del Rincón Castro. M. C. Galindo E. Patiño M. Serrano L. García R. Carrillo J. A. Pereyra-Alférez B. Alcázar-Pizaña A. Luna-Olvera H. Galán-Wong L. Pardo L. Muñoz-Garay C. Gómez I. Soberón M. Bravo A. 2006 Los microorganismos en el control biológico de insectos y fitopatógenos. Rev. Latinoam. de Microbiol.48 113 120 - 49.
González C. Martínez A. Vázques F. Baigorí M. Figueroa L. 1996 New method of screening and differentiation of exozymes from industrial strains. Biotechnol. Tech.10 519 522 - 50.
Secades P. Guijarro A. 1999 Purification and Characterization of an Extracellular Protease from the Fish Pathogen Yersinia ruckeri and Effect of Culture Conditions on Production Appl. Environ. Microbiol.65 3969 3975 - 51.
Heussen C. Dowdell E. 1980 Electrophoretic analysis of plasminogen activators in polyacrilamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem.102 196 202 - 52.
Ferrero M. Castro G. Abate M. Baigorí M. Siñeriz F. 1996 Thermostable alkaline proteases of MIR 29: Isolation, Production and Characterization. Appl. Microbiol. Biotechnol.45 327 332 - 53.
Berber I. 2004 Characterization of species by numerical analysis of their SDS-PAGE protein profiles. J. Cell Mol. Biol.3 33 37 - 54.
Norris J. 1964 The classification of Bacillus thuringiensis J. Appl. Bacteriol.27 39 447