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

Abstract

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.

FIG. 1

Southern blot analysis of genomic DNAs from T. harzianum wild-type ThzID1 and cotransformant ThzID1-M3. (A) Probed with HindIII-EcoRI fragments from pAN7-2 including the hygromycin B resistance gene. (B) Probed with NcoI-NcoI fragments from pNOM102 including the GUS gene. (C) Probed with HindIII-BamHI fragments from pTEFEGFP including the GFP gene. Lanes: 1, λ-HindIII; 2, 3, and 4: pAN7-2, wild type, and cotransformant, respectively, digested with HindIII-EcoRI; 5, 6, and 7, pNOM102, wild type, and cotransformant, respectively, digested with NcoI-NcoI; 8, 9, and 10, pTEFEGFP, wild type, and cotransformant, respectively, digested with HindIII-BamHI.

FIG. 2

Growth of T. harzianum wild-type ThzID1 and cotransformant ThzID1-M3 on PDA after 1, 2, and 3 days of incubation at 25°C in the dark. All values for days and isolates were significantly different (P < 0.05) according to an LSD analysis.

FIG. 3

Colony diameter of T. harzianum ThzID1-M3 originating from alginate pellets in soil at 3, 5, and 10 days. Colony diameters were measured at a magnification of ×250 or ×400 under an epifluorescence microscope. Vertical bars represent ±1 standard error of the mean.

FIG. 4

Percentage of sclerotia colonized by Trichoderma spp. in soil at 14 days. Five alginate pellets of T. harzianum ThzID1 or ThzID1-M3 were applied in a 25-cm² pot containing 1 kg of natural soil. The dashed portion of ThzID1-M3 treatment represents the proportion of sclerotia colonized by T. harzianum ThzID1-M3. Means followed by the same letter are not significantly different (P > 0.05) according to an LSD analysis.

FIG. 5

Photomicrographs of GFP (A to C) and GUS (D to F) activities of T. harzianum ThzID1-M3 in natural soil. (A) Conidia; (B) conidial germination; (C and D) hyphal growth; (E and F) chlamydospore formation. Magnifications, ×400 (A to D and F) and ×250 (E).