Salicylate Activity. 1. Protection of Plants from Paraquat Injury F. PAUL SILVERMAN,* PETER D. PETRACEK,CHRISTINA M. FLEDDERMAN, ZHIGUO JU, ² DANIEL F. HEIMAN, AND PREM WARRIOR Valent BioSciences Corporation, 6131 Oakwood Road, Long Grove, Illinois 60047 Paraquat (1,1′-dimethyl-4,4′-bipyridinium; methylviologen) is a widely used, nonselective contact herbicide that rapidly stimulates free radical generation. It has been found that the addition of sodium salicylate (sodium 2-hydroxybenzoate; NaSA) to paraquat spray solutions significantly decreased herbicidal activity. This protection was observed in tobacco (Nicotiana tabacum) regardless of whether NaSA was foliar-applied along with or prior to paraquat application or NaSA was soil-applied prior to paraquat application. Because salicylic acid (SA) is an inducer of systemic acquired resistance (SAR) to plant disease, paraquat protection by three SAR inducers (acibenzolar-S-methyl, harpin, and probenazole) and selected salicylate derivatives was assessed. Twenty-two of 24 compounds tested decreased herbicidal activity when foliar-applied with paraquat. Protection from paraquat was greatest with 5-chlorosalicylate, and no protection was observed with benzoic acid. NaSA decreased paraquat activity on npr1-2, an Arabidopsis mutant that is compromised in NaSA-induced SAR, and on ein2-1, an ethylene-insensitive Arabidopsis mutant. Thus, salicylate protection from paraquat is independent of disease resistance and ethylene perception. This suggests the existence of an NaSA-mediated pathway capable of protecting plants from reactive oxygen stress. KEYWORDS: Photosystem I inhibitor; PSI; alternative respiration; salicylhydroxamic acid; SHAM; aminoethoxyvinylglycine; AVG; benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; BTH; 3-(2- propenyloxy)-1,2-benzisothiazole-1,1-dioxide INTRODUCTION Salicylic acid (SA) is a simple plant phenolic. The role of SA as an endogenous signal was first shown by the induction of thermogensis in Arum lilies (1). SA is an important signal molecule in the defense response of many plants and is integral in the establishment of resistance to pathogen attack known as systemic acquired resistance (SAR) (2). SA is also involved in plant stress tolerance. Exogenous applications of SA protect mustard plants from heat stress (3) and maize from chilling stress (4). SA application also induces antioxidant defenses, including superoxide dismutase (5). Moreover, SA levels have been shown to increase in response to the free radical generators ozone and UV illumination (6). Paraquat is a free radical-generating herbicide that inhibits photosysnthesis by accepting electrons from photosystem I, which in turn generates reactive oxygen species (ROS) in light (7). The ROS generated, which include superoxide anion, hydrogen peroxide, and the hydroxyl radical, cause lipid peroxidation and membrane damage (8). In the present study, we examined the relationship between salicylates and paraquat damage. Strobel and Kuc (9) found that SA pretreatment protects tobacco plants from paraquat injury. Ananieva et al. (10) and Kim et al. (11) also determined that pretreatment of plants with SA provided protection from subsequent paraquat treatment. In these papers, resistance to paraquat was significant, but all assumed a time element necessary for the induction of ROS-associated enzymes to quench paraquat activity. It is well-known that synergies and antagonisms of crop protection agents may significantly affect the performance of pesticides in the field. In our studies, we have determined that salicylate protection from paraquat injury does not require any pretreatment interval and that resistance to paraquat is independent of SAR and ethylene perception. MATERIALS AND METHODS Chemicals. Paraquat (1,1′-dimethyl-4,4′-bipyridinium; methylviolo- gen), sodium salicylate (sodium 2-hydroxybenzoate; NaSA), and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. 3-Fluorosalicylate, 6-methylsalicylate, and 3,5-di- fluorosalicylate were produced by synthesis as described elsewhere (12). Aminoethoxyvinylglycine (AVG) was obtained from Valent Bio- Sciences Corp. (Libertyville, IL). Actigard 50WG with the active ingredient acibenzolar-S-methyl [benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester] was obtained from Syngenta Crop Protection (Greensboro, NC). Messenger, with the active ingredient harpin, was obtained from Eden Bioscience (Bothell, WA). Oryzemate, with the active ingredient probenazole [3-(2-propenyloxy)-1,2-benzisothiazole- 1,1-dioxide], was obtained from Meiji Seika Kaisha Ltd. (Tokyo, Japan). The crop oil concentrate (COC) used in all foliar sprays comprised * Author to whom correspondence should be addressee (e-mail paul.silverman@valent.com). ² Present address: Shandong University of Technology, Zibo, Shandong, People’s Republic of China. 9764 J. Agric. Food Chem. 2005, 53, 9764-9768 10.1021/jf0513819 CCC: $30.25 © 2005 American Chemical Society Published on Web 11/15/2005