RESUMO
Twelve Bacillus thuringiensis (Bt) strains, isolated from larvae and soil samples in Argentina, were molecularly and phenotypically characterized and their insecticidal activities against Spodoptera frugiperda and Peridroma saucia were determined. One isolate--Bt RT--produced more than 93% mortality on first instar larvae of both species, which was higher than that produced by the reference strain Bt 4D1. Bt RT carried a different cry gene profile than Bt 4D1. Scanning electron microscopy showed the presence of bipyramidal and cuboidal crystals. Phenotypic characterization revealed lytic enzymes that could contribute to Bt pathogenicity.
Assuntos
Bacillus thuringiensis/isolamento & purificação , Bacillus thuringiensis/patogenicidade , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Infecções por Bactérias Gram-Positivas/veterinária , Proteínas Hemolisinas/toxicidade , Lepidópteros/efeitos dos fármacos , Lepidópteros/microbiologia , Animais , Argentina , Bacillus thuringiensis/ultraestrutura , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Endotoxinas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/ultraestrutura , Larva/microbiologia , Microscopia Eletrônica de Varredura , Microbiologia do Solo , Spodoptera/efeitos dos fármacos , Spodoptera/microbiologia , Análise de SobrevidaRESUMO
Bacillus thuringiensis HD-73 was transformed with the endochitinase gene chiA74 under the control of a strong promoter (pcytA) and a 5' mRNA stabilizing (STAB-SD) sequence (HD-73-pEBchiA74). Expression levels were compared with those observed from the wild type strain (HD-73) and the recombinant HD-73 strain expressing chiA74 under the control of its native promoter (HD-73-pEHchiA74). The chitinolytic activity of HD-73-pEBchiA74 was markedly elevated, being ~58- and 362-fold higher than, respectively, HD-73-pEHchiA74 and parental HD-73, representing the highest levels of chitinase expression in recombinant B. thuringiensis reported to date. Parasporal crystals measured under transmission electron microscopy showed that HD-73 produced crystals of 1.235 (+/-0.214) and 1.356 (+/-0.247) mum in length when the bacterium was grown in respectively, NBS and NBS with glucose. Otherwise, HD-73-pEBchiA74 synthesized crystals of 1.250 (+/-0.222) and 1.139 (+/-0.202) mum in length when cultivated in NBS and NBS with glucose, respectively, values that showed a diminution of ~10 and 20% compared with crystals produced by HD-73-pEHchiA74 grown under the same conditions. Comparison of viable spore counts per ml showed that HD-73-pEBchiA74 produced fewest viable spores (1.5 x 10(9), 1.3 x 10(9)), compared to HD-73-pEHchiA74 (4.9 x 10(9), 5.3 x 10(9)) and HD-73 (6.8 x 10(9), 8.8 x 10(9)) when grown in NBS and NBS supplemented with glucose, respectively. No change in cellular protease activity was observed despite the overproduction of the chitinase.