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Biological and Molecular Analyses of the Acibenzolar-S-MethylInduced Systemic Acquired Resistance in Flue-Cured Tobacco Against Tomato spotted wilt virus
B. Mandal, S. Mandal, A. S. Csinos, N. Martinez, A. K. Culbreath, and H. R. Pappu
First, second, third, fourth, and fifth authors: Department of Plant Pathology, University of Georgia, Coastal Plain Experiment Station,Tifton 31793; and sixth author: Department of Plant Pathology, P.O. Box 646430, Washington State University, Pullman 99164-6430. Current address of first and second authors: Plant Virology Unit, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India. Current address of fourth author: Department of Plant Pathology, University of Kentucky, Lexington. Acceptedfor publication 31 October 2007.

ABSTRACT Mandal, B., Mandal, S., Csinos, A. S., Martinez, N., Culbreath, A. K., and Pappu, H. R. 2008. Biological and molecular analyses of the acibenzolarS-methyl-induced systemic acquired resistance in flue-cured tobacco against Tomato spotted wilt virus. Phytopathology 98:196-204. Tomato spotted wilt virus (TSWV) is an economically important virus of flue-curedtobacco. Activation of systemic acquired resistance (SAR) by acibenzolar-S-methyl (ASM) in flue-cured tobacco was studied under greenhouse conditions by challenge inoculation with a severe isolate of TSWV. ASM restricted virus replication and movement, and as a result reduced systemic infection. Activation of resistance was observed within 2 days after treatment with ASM and a high level ofresistance was observed at 5 days onward. Expression of the pathogenesis-related (PR) protein gene, PR-3, and different classes of PR proteins such as PR-1, PR-3, and PR-5 were detected at 2 days post-ASM treatment which inversely correlated with the reduction in the number of local lesions caused by TSWV. Tobacco plants treated with increased quantities of ASM (0.25, 0.5, 1.0, 2.0, and 4.0 ga.i./7,000 plants) showed increased levels of SAR as indicated by the reduction of both local and systemic infections by TSWV. The highest level of resistance was at 4 g a.i., but this rate of ASM also caused phytotoxicity resulting in temporary foliar spotting and stunting of plants. An inverse correlation between the TSWV reduction and phytotoxicity was observed with the increase of ASM concentration.ASM at the rate of 1 to 2 g a.i./7,000 plants activated a high level of resistance and minimized the phytotoxicity. Use of gibberellic acid in combination with ASM reduced the stunting caused by ASM. Present findings together with previous field experiments demonstrate that ASM is a potential option for management of TSWV in flue-cured tobacco.

Systemic acquired resistance (SAR) is abroad-spectrum defense system present in plants, which was realized as early as 1901 (5,11,64), and was first demonstrated against a virus by Ross (66) in tobacco against Tobacco mosaic virus (TMV). Subsequent studies showed the SAR response to be long lasting and effective against diverse pathogens such as bacteria, fungi, nematodes, and parasitic plants, and was considered a potential tool for cropprotection (18,24,31,32,41,56,75). Activation of SAR is associated with a series of signal transduction events and the expression of the SAR gene family (24,69,76,77). The SAR genes encode various pathogenesis-related (PR) proteins, which play an active role in the resistance process against fungal pathogens (2,39,40). Several synthetic chemicals such as β-aminobutaric acid, isonicotinic acid, benzol[1,2,3] thiadiazol-7-carbothioic acid-Smethyl ester (BTH), acibenzolar-S-methyl (ASM, a derivative of BTH), salicylic acid (SA), and phosphates have been shown to be activators of SAR response in a variety of crop–pathogen systems (7,13,18,20,23,31,33,42,49,56,67,68,72,73,75). Of all these chemicals, only ASM was commercialized as an activator for disease resistance in plants. ASM was discovered in...