Transformation with green fluorescent protein of Trichoderma harzianum 1051, a strain with biocontrol activity against Crinipellis perniciosa, the agentof witches'-broom disease of cocoa.

Abstract

A plasmid vector for fungal expression of an enhanced, red-shifted variant of the Aequoria victoriae green fluorescent protein was constructed by fusion of the EGFP gene to the highly expressed Aspergillus nidulans gpd promoter and the A. nidulans trpC terminator. This construction was introduced by cotransformation, using benomyl selection, into Trichoderma harzianum strain 1051, a strain being evaluated for the biological control of witches'-broom disease of cocoa caused by Crinipellis perniciosa. Epifluorescence microscopy was used to monitor germination and attachment of stable transformant conidia on the surface of C. perniciosa hyphae.

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

“…The plasmids used for co-transformation were pEGFP/gpd/tel and pBARKS/tel. pEGFP/gpd/tel [13] contains a red-shifted Aequorea victoria green £uores-cent protein gene, EGFP (GFPmut1; [14]) fused to an Aspergillus nidulans gpd promoter and A. nidulans trpC terminator and polyadenylation site and also contains a telomeric repeat (TTAGGG) 18 from Fusarium oxysporum [15]. pBARKS/tel was constructed by fusing the F. oxysporum telomeric element to pBARKS1, obtained from the Fungal Genetics Stock Center, University of Kansas Medical Center, Kansas City, KS, USA [16], carrying the phosphinothricin acetyltransferase (bar) gene from Streptomyces hygroscopicus under control of the A. nidulans trpC promoter and terminator [17].…”

“…Secondary phenotypic markers such as GUS expression [11] permit veri¢cation of the applied fungus at the microscopic level. More recently, green £uo-rescent protein (GFP) technology has been developed which allows non-invasive monitoring of transformed cells and has been applied to biocontrol fungi [12,13]. The current work describes the co-transformation of M. anisopliae var.…”

Biolistic co-transformation ofMetarhizium anisopliaevar.acridumstrain CG423 with green fluorescent protein and resistance to glufosinate ammonium

“…The plasmids used for co‐transformation were pEGFP/gpd/tel and pBARKS/tel. pEGFP/gpd/tel [13] contains a red‐shifted Aequorea victoria green fluorescent protein gene, EGFP (GFPmut1; [14]) fused to an Aspergillus nidulans gpd promoter and A. nidulans trpC terminator and polyadenylation site and also contains a telomeric repeat (TTAGGG) 18 from Fusarium oxysporum [15]. pBARKS/tel was constructed by fusing the F. oxysporum telomeric element to pBARKS1, obtained from the Fungal Genetics Stock Center, University of Kansas Medical Center, Kansas City, KS, USA [16], carrying the phosphinothricin acetyltransferase ( bar ) gene from Streptomyces hygroscopicus under control of the A. nidulans trpC promoter and terminator [17].…”

“…Secondary phenotypic markers such as GUS expression [11] permit verification of the applied fungus at the microscopic level. More recently, green fluorescent protein (GFP) technology has been developed which allows non‐invasive monitoring of transformed cells and has been applied to biocontrol fungi [12,13]. The current work describes the co‐transformation of M. anisopliae var.…”

Survival in soil and detection of co-transformed Trichoderma harzianum by nested PCR

“…The co-transformants, TE 10 and TE 41 (Inglis et al, 1999), were grown on potato dextrose agar (Difco, Detroit, Michigan, USA) containing 1.5 µg mL -1 benomyl at 28 o C for about five days until conidiation. Five mililiters conidial suspension (5.0x10 7 conidia mL -1 ) of TE 10 and TE 41 were mixed with 50 g non-sterile soil (sandy loam from the Brazilian cerrado) in 100 mL plastic pots with lids to preserve humidity.…”