Role of Benzoic Acid and Lettucenin A in the Defense Response of Lettuce against Soil-Borne Pathogens

Abstract

Soil-borne pathogens can severely limit plant productivity. Induced defense responses are plant strategies to counteract pathogen-related damage and yield loss. In this study, we hypothesized that benzoic acid and lettucenin A are involved as defense compounds against Rhizoctonia solani and Olpidium virulentus in lettuce. To address this hypothesis, we conducted growth chamber experiments using hydroponics, peat culture substrate and soil culture in pots and minirhizotrons. Benzoic acid was identified as root exudate released from lettuce plants upon pathogen infection, with pre-accumulation of benzoic acid esters in the root tissue. The amounts were sufficient to inhibit hyphal growth of R. solani in vitro (30%), to mitigate growth retardation (51%) and damage of fine roots (130%) in lettuce plants caused by R. solani, but were not able to overcome plant growth suppression induced by Olpidium infection. Additionally, lettucenin A was identified as major phytoalexin, with local accumulation in affected plant tissues upon infection with pathogens or chemical elicitation (CuSO₄) and detected in trace amounts in root exudates. The results suggest a two-stage defense mechanism with pathogen-induced benzoic acid exudation initially located in the rhizosphere followed by accumulation of lettucenin A locally restricted to affected root and leaf tissues.

Keywords: benzoic acid; defense reaction; lettuce; lettucenin; phytoalexin; plant health; root exudates.

Figure 1

UHPLC-MS separation of benzoic acid in root washings of lettuce at 2.63 min retention time (A) and mass spectrum with [M+-H+], base peak at m/z= 121.09 (B).

Figure 2

Mycelial growth of R. solani AG1-1B after 72 hours on PDA medium, spiked with 0.0 mg L⁻¹ (A), 0.05 mg L⁻¹ (B) and 0.5 mg L⁻¹ (C) of benzoic acid. The red outline indicates the diameter of the mycelial growth. Means ± SE of three replicates per treatment. Different lowercase letters indicate significant differences between treatments by one-way ANOVA, Tukey’s test (p ≤ 0.05).

Figure 3

(A) Control plant of lettuce, non-infected by R. solani and Olpidium sp. (B) Stem base with browning of lower leaves (C) and stunted top-soil fine roots of lettuce (cv. Tizian) infected by R. solani AG1-IB (D) with fungal hyphae showing rectangular branching (E). Root system (F) and fine root tips of lettuce seedlings infected by naturally occurring Olpidium sp. with typical dark colored lines in xylem. (G) Sporangia formation in the root tissue by Olpidium sp.

Figure 4

Detection of lettucenin A in leaf and root extracts of lettuce (cv. Tizian), using (A) thin-layer chromatography: yellow fluorescent lettucenin spots after elicitation of lettuce leaves with CuSO₄ (left) and AgNO₃ (right). (B) Identification of lettucenin A in leaf and root extracts of lettuce by comparison of retention times and spectral characteristics using RP-HPLC-UV/VIS and (C) by UHPLC orbitrap-MS.