Transformation of the entomopathogenic fungi, paecilomyces fumosoroseus and paecilomyces lilacinus (deuteromycotina: hyphomycetes) to benomyl resistence

Abstract

The entomopathogenic fungi Paecilomyces fumosoroseus and P. lilacinus have been transformed to resistance to the fungicide benomyl by a polyethylene glycol (PEG)-mediated procedure using a mutant <FONT FACE="Symbol">b</font>-tubulin gene from Neurospora crassa carried on plasmid pBT6. Benomyl-resistant transformants of P. lilacinus were obtained that could tolerate greater than 30 µg/ml benomyl and P. fumosoroseus transformants were obtained that could tolerate 20 µg/ml benomyl. Following 5 serial passages of transformants on benomyl-containing media and 5 serial passages on non-selective media, 100% of P. lilacinus transformants were found to be mitotically stable by a conidial germination test. In contrast, only 4 out of 9 transformants of P. fumosoroseus were mitotically stable. Southern blot analysis of genomic DNA from both species suggested that the mechanism of transformation in all transformants was by gene replacement of the <FONT FACE="Symbol">b</font>-tubulin allele. Non-homologous vector sequences were not detectable in the genomes of transformants.<br>Os fungos entomopatogênicos Paecilomyces fumosoroseus e P. lilacinus foram transformados com o plasmídio pBT6, contendo o gene de <FONT FACE="Symbol">b</font>-tubulina modificado, isolado do fungo Neurospora crassa, o qual confere resistência ao fungicida benomil. Transformantes resistentes ao benomil, obtidos da P. lilacinus, apresentaram tolerância a concentrações superiores a 30 <FONT FACE="Symbol">m</font>g/ml, enquanto transformantes da P. fumosoroseus apresentaram tolerância a 20 <FONT FACE="Symbol">m</font>g/ml. Após 5 passagens seriadas dos transformantes em meio contendo benomil e 5 passagens seriadas em meio não seletivo, 100% dos transformantes da P. lilacinus apresentaram estabilidade mitótica, através do teste de germinação de conídios. Em contraste, somente 4 dos 9 transformantes obtidos para P. fumosoroseus foram mitoticamente estáveis. Análise de Southern blot dos DNA genômicos de ambas as espécies sugere que o mecanismo de transformação foi por substituição alélica do gene de <FONT FACE="Symbol">b</font>-tubulina, para todos os transformantes obtidos. As seqüências não homólogas do vetor não foram detectadas nos genomas dos transformantes

Transformation of the entomopathogenic fungus Paecilomyces fumosoroseus with Agrobacterium tumefaciens

“…Transformation of P. fumosoroseus using protoplast‐PEG method yielded up to 2·5 transformants per microgram of plasmid DNA per 10 6 protoplasts (Barreto et al. 1997), while Inglis et al. (1999) described a transformation efficiency of 2·0–15 transformants per microgram of plasmid DNA per 5 × 10 7 protoplasts.…”

“…In previous transformation methods described by P. fumosoroseus multiple integration sites of tandem arrays of the vector (Barreto et al. 1997) or homologous gene replacement (Inglis et al. 1999) were common recombination events in transformants.…”

“…Gene transfer systems have previously been reported for P. fumosoroseus using a conventional method employing polyethylene glycol (protoplast‐PEG method) (Barreto et al. 1997; Cantone and Vandenberg 1999; Inglis et al. 1999) and particle bombardment (Barreto et al.…”

Sustainable Horticultural Systems

“…Modification of these genes may improve the virulence of fungal strains and shorten the killing time, which may be achieved by transformation systems (Bernier et al 1989;Goettel et al 1990;St. Leger et al 1995;Inglis et al 1999;Sandhu et al 2001) and strain improvement (St. Leger et al 1996;. For example, the overexpression of the pr1 gene by insertion of multiple copies resulted in increased speed of kill of a host insect, despite poor sporulation ability .…”

Development of Transformation System of Verticillium lecanii (Lecanicillium spp.) (Deuteromycotina: Hyphomycetes) Based on Nitrate Reductase Gene of Aspergillus nidulans

“…Modern molecular biology techniques offer powerful tools for analyzing interactions between organisms. Use of pBT plasmid as transformation vector is useful in developing a heterologous transformation system for entomopathogenic fungi based on the complementation of the nitrate reductase gene that would allow the genetic manipulation of this species [25,26]. With increasing understanding of biological phenomena, efficiency of biocontrol agents could be improved by transferring useful genes between micro-organisms.…”