The use of the green fluorescent protein as a biomarker for sapstain fungi

To understand wood colonization by sapstain fungi and their potential biocontrol agents, it is necessary to differentiate these organisms directly on their natural substrates. In the present study the feasibility of transforming with the green fluorescent protein (GFP), the sapstain fungus Ophiostoma piceae and a potential biocontrol agent Cartapip®, an Ophiostoma piliferum albino strain was assessed. Transformants of the two fungal species were screened by polymerase chain reaction and Southern blot analyses. The GFP was expressed in spores, synnemata and mycelia of the transformants grown in artificial media or wood. The growth, pigmentation and wood colonization of the transformants were similar to that of the non-transformants, suggesting that the presence of the gfp gene had no negative effect on the biology of the transformants. Using fluorescence and confocal microscopy, the GFP-expressing fungi were easily differentiated from the wild-type strains and other fungal species in wood, even 4 months after inoculation. The results show that the use of the GFP system is feasible to monitor Ophiostoma fungi in wood.

[1]  K. Dewar,et al.  Analysis of a high frequency transformation system for Ophiostoma ulmi, the causal agent of Dutch elm disease , 2004, Molecular and General Genetics MGG.

[2]  M. Gold,et al.  The Green Fluorescent Protein Gene Functions as a Reporter of Gene Expression in Phanerochaete chrysosporium , 2001, Applied and Environmental Microbiology.

[3]  C. Breuil,et al.  Phylogenetic relationship of Ophiostoma piliferum to other sapstain fungi based on the nuclear rRNA gene. , 2001, FEMS microbiology letters.

[4]  C. S. Hoffman.  Preparation of Yeast DNA , 1997, Current protocols in molecular biology.

[5]  C. Breuil,et al.  Fungi that cause sapstain in Canadian softwoods , 1999 .

[6]  B. Dumas,et al.  Use of Green Fluorescent Protein To Detect Expression of an Endopolygalacturonase Gene of Colletotrichum lindemuthianum during Bean Infection , 1999, Applied and Environmental Microbiology.

[7]  C. Breuil,et al.  Rapid Detection of Ophiostoma piceae andO. quercus in Stained Wood by PCR , 1999, Applied and Environmental Microbiology.

[8]  C. Breuil,et al.  Transformation of sapstaining fungi with hygromycin B resistance plasmids pAN7-1 and pCB1004. , 1999 .

[9]  J. Trevors,et al.  Green fluorescent protein as a marker to monitor survival of phenanthrene-mineralizing Pseudomonas sp. UG14Gr in creosote-contaminated soil , 1998 .

[10]  R. Maor,et al.  Use of green fluorescent protein (GFP) for studying development and fungal-plant interaction in Cochliobolus heterostrophus , 1998 .

[11]  R. Blanchette,et al.  Biological Control of Blue Stain Fungi on Populus tremuloides Using Selected Ophiostoma Isolates , 1998 .

[12]  L. Leff,et al.  Use of green fluorescent protein to monitor survival of genetically engineered bacteria in aquatic environments , 1996, Applied and environmental microbiology.

[13]  J. Dooley,et al.  Thickness variation in coextruded layers : effect of polymer viscoleasticity on layer unformity , 1996 .

[14]  R. Kahmann,et al.  Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus , 1996 .

[15]  D W Galbraith,et al.  Green-fluorescent protein as a new vital marker in plant cells. , 1995, The Plant journal : for cell and molecular biology.

[16]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[17]  Robert A. Blanchette,et al.  Biological control of pitch in pulp and paper production by Ophiostoma piliferum , 1992 .