Detecting the Hormonal Pathways in Oilseed Rape behind Induced Systemic Resistance by Trichoderma harzianum TH12 to Sclerotinia sclerotiorum

Abstract

Plants have the ability to resist pathogen attack after infection or treatment with biotic and abiotic elicitors. In oilseed rape plant Brassica napus AACC and in the artificially synthesized Raphanus alboglabra RRCC, the root-colonizing Trichoderma harzianum TH12 fungus triggers induced systemic resistance (ISR), and its culture filtrate (CF) triggers a systemic acquired resistance (SAR) response against infection by the Sclerotinia sclerotiorum. Salicylic acid (SA) and jasmonic acid/ethylene (JA/ET) are plant hormone signals that play important roles in the regulation of ISR and SAR. In this study, at six different time points (1, 2, 4, 6, 8 and 10 days post-infection [dpi]), six resistance genes were used as markers of signaling pathways: JA/ET signaling used AOC3, PDF1.2 and ERF2 genes, while PR-1, TGA5 and TGA6 genes were used as markers of SA signaling. The results of quantitative real-time polymerase chain reaction (qRT-PCR) showed that AOC3, PDF1.2 and ERF2 expression levels in infected leaves of AACC and RRCC increase at 1 and 2 dpi with S. sclerotiorum or inoculation with TH12. PR-1, TGA5 and TGA6 expression levels increased at 8 and 10 dpi in infected leaves. PR-1, TGA5 and TGA6 expression levels increased early in plants treated with CF in both of the healthy genotypes. Furthermore, induction of SA- and JA/ET-dependent defense decreased disease symptoms in infected leaves at different times. The results suggest that the RRCC genotype exhibits resistance to disease and that the ability of TH12 and its CF to induce systemic resistance in susceptible and resistant oilseed rape genotypes exists. In addition, the results indicate for the first time that in RRCC the SA signaling pathway is involved in resistance to necrotrophic pathogens.

Fig 1. After 30 days of growth, pots of AACC and RRCC plants were treated with 10-mL suspensions of T. harzianum TH12 (1.5 ×10⁷ CFU/mL) or with its CF by blending with the upper soil surface, and other pots containing each genotype were not treated (served as controls).

After 1 day, all leaves of the genotypes were infected with 1-cm² mycelial agar discs of S. sclerotiorum. Leaves were collected at 1, 2, 4, 6, 8 and 10 dpi to measure the size of necrotic lesions or pooled for RNA extraction. Five seedlings per pot in three replicates (pots) for each time point and for each treatment were used in this study.

Fig 2

Disease progression of S. sclerotiorum infection on leaves of two genotypes: (A) B. napus AACC and (B) R. alboglabra RRCC. The leaves of both genotypes (30 days of growth) were infected with 1-cm² mycelial agar discs of S. sclerotiorum. (C) Lesion size was measured 1, 2, 4, 6, 8 and 10 dpi.

Fig 3

Disease progression of S. sclerotiorum infection on leaves of two genotypes: (A) B. napus AACC and (B) R. alboglabra RRCC. The leaves of both genotypes (90 days of growth) were infected with 1-cm² mycelial agar discs of S. sclerotiorum. (C) Lesion size was measured 1, 2, 4, 6, 8 and 10 dpi.

Fig 4

Symptoms on stems at (A) 1, 2, 4, 6, 8, 10 and 30 dpi; (B) 10 dpi; (C) 30 dpi, inside of stems; and (D) 30 dpi in partially resistant checks of B. napus AACC and R. alboglabra RRCC.

Fig 5. Lesion size was measured at 1, 2, 4, 6, 8 and 10 dpi of 10 mL of TH12 (1.5 ×10⁷ CFU/mL) and its CF for disease progression of S. sclerotiorum infection on leaves (30 days of growth) of two genotypes: B. napus AACC and R. alboglabra RRCC.

Leaves of plants were inoculated with 1-cm² mycelial agar discs of S. sclerotiorum 1 day after treatment with TH12 or CF.

Fig 6. Relative gene expression in leaves (30 days of growth) of both AACC and RRCC genotypes infected with 1-cm² mycelial agar discs of S. sclerotiorum.

Five leaves from each pot of infected and non-infected plants were sampled 1, 2, 4, 6, 8 and 10 dpi. JA/ET-responsive genes (AOC3, PDF1.2 and ERF2) and SA-inducible genes (PR-1, TGA5 and TGA6) were analyzed with qRT-PCR and compared with GAPDH expression levels.

Fig 7. Relative gene expression in leaves (30 days of growth) of both AACC and RRCC genotypes treated or not treated with 10 mL of TH12 and its CF.

Five leaves from each pot of treated and un-treated plants were sampled at 1, 2, 4, 6, 8 and 10 dpi. JA/ET-responsive genes (AOC3, PDF1.2 and ERF2) and SA-inducible genes (PR-1, TGA5 and TGA6) were analyzed with qRT-PCR and compared with GAPDH expression levels.

Fig 8. Relative gene expression in leaves (30 days of growth) of both AACC and RRCC genotypes treated or not treated with 10 mL of TH12 and its CF separately by soil drenching; after 1 day the leaves were inoculated with 1-cm² mycelial agar discs of S. sclerotiorum.

Five leaves from each pot of treated and un-treated plants were sampled at 1, 2, 4, 6, 8 and 10 dpi. JA/ET-responsive genes (AOC3, PDF1.2 and ERF2) and SA-inducible genes (PR-1, TGA5 and TGA6) were analyzed with qRT-PCR and compared with GAPDH expression levels.