Light Regulates Secreted Metabolite Production and Antagonistic Activity in Trichoderma

Abstract

Secondary metabolism is one of the main mechanisms Trichoderma uses to explore and colonize new niches, and 6-pentyl-α-pyrone (6-PP) is an important secondary metabolite in this process. This work focused on standardizing a method to investigate the production of 6-PP. Ethanol and ethyl acetate were both effective solvents for quantifying 6-PP in solution and had limited solubility in potato-dextrose-broth media. The 6-PP extraction using ethyl acetate provided a rapid and efficient process to recover this metabolite. The 6-PP was readily produced during the development of Trichoderma atroviride growing in the dark, but light suppressed its production. The 6-PP was purified, and its spectrum by nuclear magnetic resonance and mass spectroscopy was identical to that of commercial 6-PP. Light also induced or suppressed other unidentified metabolites in several other species of Trichoderma. The antagonistic activity of T. atroviride was influenced by light, as suppression of plant pathogens was greater in the dark. The secreted metabolite production on potato-dextrose-agar was differentially regulated by light, indicating that Trichoderma produced several metabolites with antagonistic activity against plant pathogens. Light has an important influence on the secondary metabolism and antagonistic activity of Trichoderma, and this trait is of key relevance for selecting antagonistic Trichoderma strains for plant protection.

Keywords: antagonism; biocontrol; crop protection; fungal biotechnology; plant pathogen; secondary metabolism.

Figure 1

The absorbance of 6-pentyl-α-pyrone in different solvents. (A) Absorbance spectrum of 6-PP (100 mg/L) dissolved in n-hexane, ethanol, ethyl acetate, and PDB. Absorbance was scanned using a UV-1600PC VWR Spectrophotometer for 200 nm to 500 nm wavelengths. (B,C) Linear dynamic range. Absorbance was measured at 320 nm using a gradient of concentrations of 6-PP dissolved in ethanol (B) and ethyl acetate (C).

Figure 2

Effect of mixing time and media concentration on extraction of 6-pentyl-α-pyrone. (A) Mixing time for recovery of 6-PP (100 mg/L) from the PDB media. The 6-PP dissolved in PDB was mixed with the solvent ethyl acetate, vortexed for the time indicated, and the supernatant recovered by centrifuging. (B) Media:Solvent (M:S) ratio for recovery of 6-PP (20 mg/L). Volume relation was 1M:1S (X1), 2M:1S (X2), 3M:1S (X3), and 4M:1S (X4). Mixtures were vortexed for 30 s and centrifuged for 3 min at 3000 rpm. The 6-PP was quantified using the equation in Figure 1C.

Figure 3

Effect of incubation in the dark or light on T. atroviride IMI206040 growth and secretion of organic compounds. (A) Dry weight of mycelium after growth in PDB for 7 days at 27 °C. (B) Absorbance following ethyl acetate extraction from the T. atroviride IMI206040 cultures. (C,D) Compounds extracted from cultures of T. atroviride IMI206040 growing in light or dark for 7 days. The TLCs were exposed to short-wave UV (C, 254 nm) or long-wave UV (D, 350 nm) to detect the compounds produced in light (LC) or dark (DC).

Figure 4

A 6-pentyl-α-pyrone purified from T. atroviride IMI206040. (A) The Nuclear magnetic resonance of 6-PP purified. (B) Stacked NMR spectra of commercially available 6-PP and the 6-PP-like compound isolated from a submerged culture grown in the dark.

Figure 5

Effect of incubation in the light on growth of Trichoderma species/ strains and metabolite production. (A) Dry weight of mycelium of the Trichoderma strains after growing in PDB for 3 days at 27 °C under light or dark. (B,C), Metabolite pattern of different strains of Trichoderma growing for 3 days at 27 °C in light (L) and dark (D). (B) The TLCs exposed to short-wave UV (254 nm) (left) or long-wave UV (350 nm) (right) to detect the compounds produced in light (LC) or dark (DC). The strains used were T. atroviride IMI206040, Trichoderma sp. atroviride B LU132, LU584, and LU633, T. hamatum LU592, T. asperellum LU697, T. gamsii LU755, and T. viridescens LU1369. The bars have the (+/−) standard deviation of data generated from three replicates, and different letters over the bars indicate significant differences between dark and light.

Figure 6

Effect of growth in the dark and light on antagonistic activity of T. atroviride IMI206040 as assessed by plant pathogen growth. (A) Antagonistic activity on PDA plates. (B) Colony growth: colony diameter was measured using ImageJ, and the average diameter of the control was taken as 100 % growth. The vertical bars on each bar are (+/−) standard deviation of data generated from three replicates, and different letters over the bars indicate significant differences between dark and light.

Figure 7

Effect of light on antagonistic activity of several species of Trichoderma against four plant pathogens. Antagonistic activity was assayed by growing Trichoderma strains on PDA plates covered with a cellophane sheet for 48 h in the dark or light at 27 ° C. After removing the Trichoderma colony, the plant pathogens were inoculated, and as a control, fresh PDA was inoculated. Each treatment had three replicates (Table S1). The heat map matrix shows the average inhibition percentage of two treatments: dark and light.

Figure 8

Metabolites secreted by Trichoderma spp. grown on PDA in the dark or light. PDA plates holding Trichoderma growth for 60 h in the dark or light were treated to isolate the metabolites produced by Trichoderma strains and analyzed by TLC. The compounds from three cultures were detected using short-wave UV. As a control, metabolite extraction was performed using fresh PDA, and as a reference, 5 µg 6-PP was used.