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Revista Colombiana de Entomología
Print version ISSN 0120-0488On-line version ISSN 2665-4385
Abstract
RODRIGUEZ C., MARTHA LILIANA and GONGORA B., CARMENZA E. Tansformation of Beauveria bassiana Bb9205 with pr1A, pr1J, and ste1 genes of Metartzizium anisopliae and evaluation of the pathogenicity on the coffee berry borer. Rev. Colomb. Entomol. [online]. 2005, vol.31, n.1, pp.51-58. ISSN 0120-0488.
Beauveria bassiana strain 9205 (Bb9205) was transformed with protease genes (pr1A and pr1J ) and one sterase gene (ste 1) isolated from fletartiizium anisopliae that are involved in insect pathogenicity. The goal of this research was lo increase the pathogenicity of the strain against the coffee berry borer. !iypothenemus hampei. To do this, genes were cloned into the plasmid pBarOF’El that confers resistance to the herbicide ammonium-gluphosinate. A monosporic une of strain Bb9205 was obtained with pathogenicity of’ 78% mortality against 11. hampei, using 1 x 106 spore/ml. Protoplasts were produced from this monosporic line. The methodology for the protoplast production was optimized obtaining 3 x 10 protoplasts/ml after enzymatic pretreatment for 4 hours under slow agitation at 37°C. ‘flEansformation was carried out with the plasmids pBarGPEl- pr1A, pBarOPEI-priJ and pBarGPF.1-ste 1 by PEO and electroporation. Two strains transformed with the pr1A gene and one strain transformed with the .ste 1 gen were obtained, that showed resistance to 25 ¡tg/ml of ammonium glufosinate. These strains showed increased proteolytic and sterolytic activity, respectively. Under laboratory conditions, pathogenicity tests of 13. bassiana transformants was compared to untransformed monosporic 13b9205. Constitutive over-expression of protease in the transgenic strain Bb9205-pr 1A improved its insecticidal activity. The transformed strain showed a 21,7% increase in the insect mórtality rate with 14,3% reduction in the time of mortality. The Bb9205-ste 1 transformant strain showed a 9,5% reduction of the time of mortality compared with the untransforrned strain. The results indicate that breeding of 13eauveria strains by genetic transformation can be achieved for purposes of better biological control.
Keywords : F’athogenicity; Froteases; Sterases; t3iological control; I-Iypot/ienemus /zampei.