Ethyl Phloretate and Ethyl p-Coumarate: Two Phytotoxins from Valsa mali and Their Pathogenic Activities

Valsa mali, the causal agent of apple Valsa canker, produces several phytotoxic metabolites to promote infection. Bioassay and ¹H nuclear magnetic resonance (NMR)-guided isolation from the culture filtrate of V. mali strain 03-8 led to the identification of seven compounds including three unreported ones, ethyl phloretate (1), ethyl p-coumarate (2), and 1-p-hydroxybenzoyl glycerol (3). Compounds 1 and 2 produced significant phytotoxicity, with average lesion areas of 6.22 and 3.74 mm², along with 2.96 and 3.47 mm² at 1 mg/ml on mature and tissue-cultured apple leaves, respectively, whereas compound 3 did not cause any symptoms on host plants. The necrotic lesion area of compounds 1 and 2 on tobacco leaves was 52.65 and 48.28 mm², respectively, compared with the negative control (0.46 mm²) at 1 mg/ml. At the same concentration, compounds 1 and 2 showed no significant influence on the germination rate of lettuce seeds while significantly decreasing the root length of lettuce seedlings to 6.74 and 4.67 mm, respectively, compared with that treated with sterile distilled water (22.01 mm). The discovery indicated that compounds 1 and 2 could be considered as non-host-specific toxins. Furthermore, compounds 1 and 2 could cause cell shrinkage, organelle damage, plasmolysis, and eventually ruptured protoplasmic membranes with cell death for their phytotoxicity in the host plants under optical microscopy and transmission electron microscopy. The results shed light on the mechanism for toxins 1 and 2 in V. mali-infected plants at the macroscopic and cellular levels.