Salicylic acid prevents Trichoderma harzianum from entering the vascular system of roots

Abstract

Trichoderma is a soil-borne fungal genus that includes species with a significant impact on agriculture and industrial processes. Some Trichoderma strains exert beneficial effects in plants through root colonization, although little is known about how this interaction takes place. To better understand this process, the root colonization of wild-type Arabidopsis and the salicylic acid (SA)-impaired mutant sid2 by a green fluorescent protein (GFP)-marked Trichoderma harzianum strain was followed under confocal microscopy. Trichoderma harzianum GFP22 was able to penetrate the vascular tissue of the sid2 mutant because of the absence of callose deposition in the cell wall of root cells. In addition, a higher colonization of sid2 roots by GFP22 compared with that in Arabidopsis wild-type roots was detected by real-time polymerase chain reaction. These results, together with differences in the expression levels of plant defence genes in the roots of both interactions, support a key role for SA in Trichoderma early root colonization stages. We observed that, without the support of SA, plants were unable to prevent the arrival of the fungus in the vascular system and its spread into aerial parts, leading to later collapse.

Keywords: Arabidopsis thaliana; Trichoderma; callose deposition; plant defence; root colonization; salicylic acid.

Figure 1

Confocal microscopy analysis of Arabidopsis roots from hydroponic cultures at 72 h post‐inoculation of 17‐day‐old seedlings with Trichoderma harzianum  GFP22. (a) Wild‐type Col‐0. (b) Salicylic acid (SA)‐impaired sid2 mutant. co, cortex; ep, epidermis, vt, vascular tissue.

Figure 2

Callose localization in Col‐0 (a, cross‐section; c, longitudinal section; e, control without Trichoderma) and salicylic acid induction deficient 2 (sid2) mutant (b, cross‐section; d, longitudinal section; f, control without Trichoderma) Arabidopsis roots from hydroponic cultures at 72 h post‐inoculation of 17‐day‐old seedlings with Trichoderma harzianum  GFP22. co, cortex; ep, epidermis, p, parenchyma, vt, vascular tissue. Scale bar. 200 μm.

Figure 3

Quantification of Trichoderma harzianum  GFP22 in Arabidopsis roots from Col‐0 and salicylic acid induction deficient 2 (sid2) mutant plants by real‐time polymerase chain reaction (PCR). Trichoderma harzianum and Arabidopsis actin genes were used. Bars represent standard deviations of the means of three biological replicates. Asterisks denote significant differences at P ≤ 0.05 between Arabidopsis  Col‐0 and the sid2 mutant.

Figure 4

Effect of Trichoderma harzianum  GFP22 treatment on Arabidopsis  Col‐0 (c, d) and the salicylic acid induction deficient 2 (sid2) mutant (a, b) grown in hydroponic culture. Photographs were taken 10 days after inoculation of 2 × 10⁷ Trichoderma spores (b, d).

Figure 5

Effect of Trichoderma harzianum  GFP22 on Arabidopsis  Col‐0 (a, b) and salicylic acid induction deficient 2 (sid2) mutant (c, d) roots grown in hydroponic culture, treated (b, d) or not (a, c) with the fungus. Representative sections of roots of both Arabidopsis lines treated or not with GFP22 were photographed with a Leica stereomicroscope. Scale bars, 0.15 mm.

Figure 6

Quantitative real‐time polymerase chain reaction (PCR) of the lipoxygenase 1 (Lox1), pathogenesis‐related protein‐1 (PR‐1), isochorismate synthase 1 (ICS1) and callose synthase 5 (Cals5) genes in Arabidopsis  Col‐0 and salicylic acid induction deficient 2 (sid2) mutant plant roots colonized or not with Trichoderma harzianum  GFP22. Values correspond to relative measurements against Arabidopsis incubated without GFP22 (2^{–ΔΔ Ct} = 1). The Arabidopsis actin gene was used as an internal reference gene. Bars represent standard deviations of the means of three biological replicates. Asterisks denote significant differences at P ≤ 0.05 between Arabidopsis  Col‐0 and the sid2 mutant.