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...

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Published in:FEMS microbiology letters 2000-10, Vol.191 (2), p.249-254
Main Authors: Inglis, P.W, Aragão, F.J.L, Frazão, H, Magalhães, B.P, Valadares-Inglis, M.C
Format: Article
Language:English
Subjects:
Anticarsia gemmatalis
Aspergillus nidulans
bar gene
Biological control
EFGP gene
egfp gene
Entomopathogenic fungus
Fusarium oxysporum
gfp
glufosinate ammonium
gpd gene
green fluorescent protein
Metarhizium anisopliae
phosphinothricin
plasmid pBARKS1
Rhammatocerus schistocercoides
Streptomyces hygroscopicus
Tenebrio molitor
Transformation
trpC gene
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Summary: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 CG423 were transformed using microparticle bombardment. Plasmids used were: (1) pBARKS1 carrying the bar gene of Streptomyces hygroscopicus fused to the Aspergillus nidulans trpC promoter, encoding resistance to glufosinate ammonium (or phosphinothricin) and modified by addition of the telomeric repeat (TTAGGG) 18 of Fusarium oxysporum and 2.pEGFP/gpd/tel carrying a red-shifted variant gene for Aequorea victoria green fluorescent protein (EGFP) which we have fused to the A. nidulans gpd promoter and trpC terminator. Highly fluorescent co-transformants were selected on solid minimal medium containing 100 μg ml −1 glufosinate ammonium using an inverted microscope with 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. Transformants retained pathogenicity in bioassays against Rhammatocerus schistocercoides and showed unaltered lack of pathogenicity against larvae of the non-target insect Anticarsia gemmatalis. One co-transformant from four tested, however, showed a significant, but non-dose-dependent, elevation in virulence against Tenebrio molitor.
ISSN:0378-1097
1574-6968
DOI:10.1016/S0378-1097(00)00398-0