Regulation of morphogenesis and biocontrol properties in Trichoderma virens by a VELVET protein, Vel1

Abstract

Mycoparasitic strains of Trichoderma are applied as commercial biofungicides for control of soilborne plant pathogens. Although the majority of commercial biofungicides are Trichoderma based, chemical pesticides, which are ecological and environmental hazards, still dominate the market. This is because biofungicides are not as effective or consistent as chemical fungicides. Efforts to improve these products have been limited by a lack of understanding of the genetic regulation of biocontrol activities. In this study, using gene knockout and complementation, we identified the VELVET protein Vel1 as a key regulator of biocontrol, as well as morphogenetic traits, in Trichoderma virens, a commercial biocontrol agent. Mutants with mutations in vel1 were defective in secondary metabolism (antibiosis), mycoparasitism, and biocontrol efficacy. In nutrient-rich media they also lacked two types of spores important for survival and development of formulation products: conidia (on agar) and chlamydospores (in liquid shake cultures). These findings provide an opportunity for genetic enhancement of biocontrol and industrial strains of Trichoderma, since Vel1 is very highly conserved across three Trichoderma species.

FIG. 1.

Colony morphology of the wild type (WT) and a vel1 mutant (ve3) of T. virens. (A) Growth on VMS agar after 5 days of incubation at 26°C. (B) Microscope images of the cultures shown in panel A, showing conidiophores in the WT and chlamydospores in the mutant.

FIG. 2.

Production of conidia and chlamydospores by WT, mutant, and complemented (Comp) strains on 1% water agar. The cultures were incubated in the presence of light for 2 or 3 days (2d and 3d, respectively). Note that the WT produced conidiophores in large aggregates (indicated by arrows), compared to the small dispersed elements produced by the complemented strain. The mutants did not produce conidia but produced chlamydospores as early as 2 days after inoculation.

FIG. 3.

Growth of wild-type (WT), mutant (ve3), and complemented (Comp) strains in shake cultures. (A) Growth after 8 days of incubation. Note the absence of pigmentation in the mycelia of the mutant. Cultures were transferred to plates for photography. (B) Culture filtrates after 8 days. Note the absence of the yellow pigment in the supernatant from cultures of the mutant. (C) Colony morphology after 4 days of incubation of a shake culture. Note the smooth edges of the developing WT colonies compared to the striated growth of the mutant and the complemented strain. (Insets) Enlargements of single colonies.

FIG. 4.

Hydrophobicity of the wild type, the vel1 mutant, and the complemented strain (Comp). (A) Fifteen microliters of water or 0.5% aqueous aniline blue was spotted on colonies, which were photographed after 30 min. Note the disappearance of the solutions for the mutant (ve3). (B) Expression of the tvh1 hydrophobin gene in wild type, mutant, and complemented strains as determined by RT-PCR (lanes hd, hydrophobin primers; lanes h3, histone primers). (C) Confirmation of the absence of the tvh1 transcript in the mutant by real-time PCR (fold changes in mRNA expression relative to h3).

FIG. 5.

Production of gliotoxin. (A) TLC plates showing production of gliotoxin (arrow) (visualized under UV light) after 3 days (3d) and 6 days (6d) of incubation in Weindling medium. (B) Expression of the gliP gene as determined by RT-PCR (top panel) and real-time PCR (bottom panel). WT, wild type; ve3, vel1 mutant; CO, complemented strain.

FIG. 6.

Antagonistic interactions between the WT, the vel1 mutant, and the complemented strain in confrontation and simulated mycoparasitism assays. (A) Results of confrontation assay with P. ultimum (Pu) 6 days after coinoculation at the edges of VMS plates. (B) Results of confrontation assay with R. solani (Rs) 10 days after coinoculation of the organisms 4 cm apart. (C) Induction of serine protease (tvsp1/prb1) expression in a simulated mycoparasitism assay (RT-PCR). VM, sucrose used as the carbon source; Rs, R. solani cell walls used as the carbon source; Pu, P. ultimum cell walls used as the carbon source; W, wild type; 3, vel1 mutant; C, complemented strain; sp, tvsp1; and h3, histone. (D) Confirmation of downregulation in the mutant by real-time PCR.

FIG. 7.

Biocontrol of P. ultimum and R. solani in cotton. (A) Percentage of healthy seedlings in P. ultimum- or R. solani-infested soil. (B) Extent of root rot in R. solani-infested soil. Pu, P. ultimum; Rs, R. solani; UC, uninoculated control; Cont, pathogen-inoculated control; WT, T. virens wild type; ve3, vel1 mutant 3; ve5, vel1 mutant 5; Comp, complemented strain. The values for bars labeled with the same letter are not significantly different (P ≤ 0.01).