Exogenous Regulators Enhance the Yield and Stress Resistance of Chlamydospores of the Biocontrol Agent Trichoderma harzianum T4

Abstract

Trichoderma strains have been successfully used in plant disease control. However, the poor stress resistance of mycelia and conidia makes processing and storage difficult. Furthermore, they cannot produce chlamydospores in large quantities during fermentation, which limits the industrialization process of chlamydospore preparation. It is important to explore an efficient liquid fermentation strategy for ensuring chlamydospore production in Trichoderma harzianum. We found that the addition of mannitol, glycine betaine, and N-acetylglucosamine (N-A-G) during liquid fermentation effectively increases the yield of chlamydospores. Furthermore, we provided evidence that chlamydospores have stronger tolerance to high temperature, ultraviolet, and hypertonic stress after the addition of mannitol and trehalose. Lipids are an important component of microbial cells and impact the stress resistance of microorganisms. We studied the internal relationship between lipid metabolism and the stress resistance of chlamydospores by detecting changes in the lipid content and gene expression. Our results showed that mannitol and trehalose cause lipid accumulation in chlamydospores and increase the unsaturated fatty acid content. In conclusion, we verified that these exogenous regulators increase the production of chlamydospores and enhance their stress resistance by regulating lipid metabolism. In addition, we believe that lipid metabolism is an important part of the chlamydospore production process and impacts the stress resistance of chlamydospores. Our findings provide clues for studying the differentiation pathway of chlamydospores in filamentous fungi and a basis for the industrial production of chlamydospores.

Keywords: Trichoderma harzianum T4; chlamydospore; exogenous regulators; lipid metabolism; stress resistance; unsaturated fatty acids.

Figure 1

Effects of different exogenous regulators on the chlamydospore yield. N-A-G: N-acetylglucosamine. The data are from three independent repeated experiments. The different letters above the bars indicate a significant difference in the chlamydospore yield for each treatment, according to the least significant difference test (p < 0.05).

Figure 2

Stress resistance ability of chlamydospores produced by adding different exogenous regulators: (A) heat stress, (B) UV stress, and (C) hypertonic stress. The data are from three independent repeated experiments. The different letters above the bars indicate that the survival rates of chlamydospores produced by adding different exogenous regulators are significantly different when facing different environmental pressures, according to the least significant difference test (p < 0.05).

Figure 3

Transmission electron microscopy (TEM) images of chlamydospores: (A) control, (B) mannitol, (C) glycine betaine, (D) N-A-G, and (E) trehalose. The scale bar is 1 μm. (a) Fat droplet, (b) diffuse outer layer, (c) outer wall, and (d) inner wall.

Figure 4

Images of chlamydospores produced by adding different exogenous regulators, as observed under a confocal microscope: (A) control group, (B) mannitol, (C) glycine betaine, (D) N-A-G, and (E) trehalose. The scale of the image is 20 μm.

Figure 5

Effects of different exogenous regulators on the total lipid and triglyceride accumulation: (A) lipid content and (B) TG content. The data are from three independent repeated experiments. The different letters above the bars indicate a significant difference in the lipid content or TG content for each treatment, according to the least significant difference test (p < 0.05).

Figure 6

Differences in the expression of genes related to lipid metabolism caused by different exogenous regulators: (A) TGL2 gene and (B) OLE1 gene. The data are from three independent repeated experiments. The different letters above the bars indicate a significant difference in the expression level of the TGL2 gene and the OLE1 gene for each treatment, according to the least significant difference test (p < 0.05).