Biolistic co-transformation of Metarhizium anisopliae var. acridum strain CG423 with green fluorescent protein and resistance to glufosinate ammonium

Metarhizium anisopliae var. acridum (syn. M. flavoviride) is recognized as a highly specific and virulent mycopathogen of locusts and grasshoppers and is currently being developed as a biological control agent for this group of insects in Brazil. Intact conidia of M. anisopliae var. acridum strain C G423 were transformed using microparticle bombardment. Plasmids used were: (1) pBARKS1 carrying the bar gene of Streptomyces hygroscopicus fused to th e Aspergillus nidulans trpC promoter, encoding resistance to glufosinate am monium (or phosphinothricin) and modified by addition of the telomeric repe at (TTAGGG)(18) of Fusarium oxysporum and 2.pEGFP/gpd/tel carrying a red-sh ifted variant gene for Aequorea victoria green fluorescent protein (EGFP) w hich we have fused to the A. nidulans gpd promoter and trpC terminator. Hig hly fluorescent co-transformants were selected on solid minimal medium cont aining 100 mu g ml(-1) glufosinate ammonium using an inverted microscope wi th 450-490 nm excitation/510 nm emission filter set. Southern blot analysis of co-transformants revealed varying multiple chromosomal integrations of both bar and egfp genes at both telomeric and non-telomeric loci. Transform ants retained pathogenicity in bioassays against Rhammatocerus schistocerco ides and showed unaltered lack of pathogenicity against larvae of the non-t arget insect Anticarsia gemmatalis. One co-transformant from four tested, h owever, showed a significant, but non-dose-dependent, elevation in virulenc e against Tenebrio molitor. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.