Carbon and Nitrogen Sources Influence Parasitic Responsiveness in Trichoderma atroviride NI-1

Abstract

Parasitic species of Trichoderma use hydrolytic enzymes to destroy the host cell wall. Preferent carbon and nitrogen sources suppress the expression of genes related to parasitism. Here, different nutrients were evaluated in the parasitic isolated NI-1, which was identified as Trichoderma atroviride. The genes cbh1 and chb2 (cellobiohydrolases), bgl3.1 (endoglucanase), and pra1 and prb1 (proteinases) were poorly expressed during the interaction between NI-1 and Phytophthora capsici on PDA. However, gene expression improved on minimal medium with preferent and alternative carbon sources. Dextrin and glucose stimulated higher transcript levels than cellulose, sucrose, and glycerol. Also, ammonium stimulated a stronger parasitic responsiveness than the alternative nitrogen sources. During interaction against different phytopathogens, NI-1 detects their host differentially from a distance due to the cbh1 and cbh2 genes being only induced by P. capsici. The pra1 and ech42 genes were induced before contact with Botrytis cinerea and Rhizoctonia solani, while when confronted with P. capsici they were stimulated until contact and overgrowth. The prb1 and bgl3.1 genes were induced before contact against the three-host assayed. Overall, T. atroviride prefers to parasitize and has the capacity to distinguish between an oomycete and a fungus, but nutrient quality regulates its parasitic responsiveness.

Keywords: biocontrol; catabolic repression; genes related to mycoparasitism; host sensing; hydrolytic enzymes; parasitism.

Figure 1

In vitro antagonism against P. capsici D3. (A) Growth inhibition by diffusible and volatile metabolites. Bars represent the average of at least three treatments. Line upper bars indicate the standard deviation (SD). (B) Mycoparasitic behavior of NI-1 against P. capsici D3 (10×). Hyaline hyphae of NI-1 coiled around D3 hyphae. Coiling is indicated by the red square.

Figure 2

Phylogenetic analysis of NI-1. Phylogram based on the combined ITS and TEF-1α sequences. Support values higher than 70% are shown at the nodes. Values are associated with BMP, BML, and PP, respectively. The accession numbers of the reference sequences are shown in Table 1. NI-1 is shown in red. The scale bar represents nucleotide substitutions per base.

Figure 3

Influence of carbon source on the transcript levels of parasitism-related genes during confrontation with NI-1 and D3. Dual culture assays were performed on PDA (-●-) or Vogel’s minimal media supplemented with dextrin (-▼-), cellulose (-●-), glucose (-▲-), glycerol (-◆-), and sucrose (-▅-). RNA was extracted from mycelia after contact in a dual culture assay. The color line identifies carbon sources. Symbols represent average signals, and lanes are SD. n = 3.

Figure 4

Influence of nitrogen source on the transcript levels of parasitism-related genes during confrontation with NI-1 and D3. Dual culture assays were performed on PDA (-●-) or Vogel’s minimal media supplemented with ammonium nitrate (-▅-), sodium nitrate (-▲-), and L-arginine (-▼-). RNA was extracted from mycelia after contact in a dual culture assay. Symbols represent the average signal, and bars are SD. n = 3.

Figure 5

The GRM expression during the confrontation of NI-1 against three different phytopathogens. (A) Gene expression profile during mycoparasitic interaction in dual cultures: before contact (2, 1, 0.5 cm), physical contact (0 cm), and overgrowth (OG). Interactions of T. atroviride against P. capsici (T-Pc), B. cinerea (T-Bc), and R. solani (T-Rs) were analyzed using as a control Trichoderma–Trichoderma (T-T). (B) Relative gene expression. The signal area detected before contact (2, 1, 0.5 cm), physical contact (C), and overgrowth (OG) in (A) was quantified, and gene expression was graphed for T-Pc (blue bars), T-Bc (gray bars), and T-Rs (red bars) relative to the control T-T, which was adjusted to the unit (green). The transcript levels of gpd were used to normalize the template amounts for each signal. Two-way ANOVA and multiple comparisons were performed using Dunnett’s test. Asterisks above the bar represent significant differences with respect to control relative expression p < 0.05 (*), p < 0.001 (**), and p < 0.0001 (***). Bars represent average transcript levels, and the lines indicate SD. n = 3.