Piperonylic acid

Pseudomonas strains were cultured at 28°C on King's medium B [24] (link). Pseudomonas syringae pv. syringae 61-1530B strain (kind gift of A. Collmer, Cornell University, Ithaca, USA) was cultured on antibiotic-containing (kanamycin, 50 µg/ml) King's medium B. These bacteria are mutant in their hrcC gene, are compromised in their pathogenicity, and are unable to cause HR. Challenge inoculations in the HR-inhibition tests were carried out with Pseudomonas syringae pv. syringae 61 strain, an incompatible wild type strain normally causing HR in tobacco. Overnight cultures of both bacteria were suspended in distilled water and adjusted to 10⁸ CFU/ml before using for infiltration of leaves. PIP (piperonylic acid, Sigma) was dissolved in dimethyl sulfoxide (DMSO) to gain a 200 mM stock solution. Inoculations were carried out with a final concentration of 1 mM. PIP was used either alone, or in combination with Pseudomonas syringae pv. syringae hrcC⁻ (10⁸ CFU/ml) suspension.
Flagellin 22 (flg22) peptide (Genescript, USA) was dissolved in double-distilled H₂O to a stock solution of 1 mM. This was further diluted to inoculate at a concentration of 1 µM.

Rice plants were treated with 10 or 20 mM of DHA (L-dehydroascorbic acid, Sigma-Aldrich, Cat. No. 261,556) (Chavan et al. 2022 (link)) and/or 300 µM PA (piperonylic acid, Sigma-Aldrich, Cat. No. P49805) (Desmedt et al. 2021 (link)). The chosen 20 mM concentration of DHA was previously optimized for best efficacy against M. graminicola and lack of phytotoxicity (Chavan et al. 2022 (link)). In order to potentially reduce the dose of DHA, the half dose, i.e., 10 mM was used while combining it with PA. Since PA was dissolved in DMSO, an additional mock treatment with only DMSO was included. Each plant was treated with 6.25 ml solution or distilled water containing 0.02% (v/v) of Tween20 (Sigma-Aldrich, Cat. No. P1379) for efficient spread and uptake of chemicals. Plants were treated by spraying the above-ground parts using a hand automizer sprayer.