Cotransformation of Trichoderma harzianum with beta-glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils.
Trichoderma harzianum was cotransformed with genes encoding green fluorescent protein (GFP), beta-glucuronidase (GUS), and hygromycin B (hygB) resistance, using polyethylene glycol-mediated transformation. One cotransformant (ThzID1-M3) was mitotically stable for 6 months despite successive subculturing without selection pressure. ThzID1-M3 morphology was similar to that of the wild type; however, the mycelial growth rate on agar was reduced. ThzID1-M3 was formed into calcium alginate pellets and placed onto buried glass slides in a nonsterile soil, and its ability to grow, sporulate, and colonize sclerotia of Sclerotinia sclerotiorum was compared with that of the wild-type strain. Wild-type and transformant strains both colonized sclerotia at levels above those of indigenous Trichoderma spp. in untreated controls. There were no significant differences in colonization levels between wild-type and cotransformant strains; however, the presence of the GFP and GUS marker genes permitted differentiation of introduced Trichoderma from indigenous strains. GFP activity was a useful tool for nondestructive monitoring of the hyphal growth of the transformant in a natural soil. The green color of cotransformant hyphae was clearly visible with a UV epifluorescence microscope, while indigenous fungi in the same samples were barely visible. Green-fluorescing conidiophores and conidia were observed within the first 3 days of incubation in soil, and this was followed by the formation of terminal and intercalary chlamydospores and subsequent disintegration of older hyphal segments. Addition of 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-Gluc) substrate to recovered glass slides confirmed the activity of GUS as well as GFP in soil. Our results suggest that cotransformation with GFP and GUS can provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil.
Bae
Y S
YS
Department of Plant, Soil, and Entomological Sciences, University of Idaho, Moscow, Idaho 83844-2339, USA.
Knudsen
G R
GR
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001203:Ascomycota / Q000502:physiology; D015139:Blotting, Southern; D004352:Drug Resistance, Microbial / Q000235:genetics; D005658:Fungi / Q000254:growth & development / Q000378:metabolism; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006921:Hygromycin B / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D011523:Protoplasts; D012988:Soil Microbiology; D014170:Transformation, Genetic; D014242:Trichoderma / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development*
D000900:Anti-Bacterial Agents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; D005966:Glucuronidase