J. Ky. Acad. Sci. 66(2): 118-128. 2005. 



Salicylate Inhibits Growth of Plant-Pathogenic Fungi and 

 Synergistically Enhances the Activity of Other 

 Antifungal Materials In Vitro 



Norman E. Strobel and Lawrence A. Porter 



Division of Natural Sciences, Bluegrass Community and Technical College, 470 Cooper Drive, 

 Lexington, Kentucky 40506-0235 



ABSTRACT 



We conducted dose-response studies of the toxicity of salicylate (SA; a putative signal molecule for en- 

 hancement of plant disease resistance [EPDR]) toward plant-pathogenic fungi. SA concentrations >10.0 

 mM were required for complete inhibition of fungal growth from mycelial plugs. SA doses of 2.0-5.0 mM 

 typically reduced fungal growth by 50%, whereas doses of 0.5 mM or lower had little or no effect on fungal 

 growth. However, growth of several test fungi was completely inhibited by 2.0 mM SA combined with 

 concentrations of cupric chloride, antifungal bacterial culture fluids, or neem extract that were otherwise 

 only slightly to moderately inhibitory. We conclude that (1) endogenous SA concentrations (up to 10.0-100.0 

 [jlM) are unlikely to directly inhibit fungi in plants, (2) concentrations of exogenous SA applied for EPDR 

 (2.0-10.0 mM) are likely to be only moderately inhibitory to fungi, and (3) additions of other antifungal 

 materials with which SA synergizes may enhance the antifungal activity of SA applied to plant surfaces for 

 EPDR. The latter conclusion provides a rationale for further study of the synergistic interactions of mod- 

 erately active antifungal materials for practical plant disease control. 



INTRODUCTION 



Salicylate (SA) has been intensively studied 

 as a putative endogenous signal molecule for 

 the activation of plant defenses against path- 

 ogens. Total endogenous SA may increase to 

 perhaps 10.0-100.0 fxM (locally) in infected 

 plant tissues, whereas 2.0-10.0 mM exogenous 

 SA is commonly applied to plants as a fohar 

 spray for the experimental enhancement of 

 plant disease resistance. The large discrepancy 

 between the concentrations of endogenous or 

 exogenous SA required for activation of plant 

 defenses has been discussed (Norman et al. 

 2004; Shirasu et al. 1997). The lower concen- 

 trations of endogenous SA may act via syner- 

 gistic enhancement of the generation of re- 

 active-oxygen species (ROS) that occurs upon 

 exposure of plants to pathogens or pathogen- 

 derived molecules (the "oxidative burst") 

 (Shirasu et al. 1997), and/or may trigger the 

 expression of an alternate oxidase in plant mi- 

 tochondria that results in reduced susceptibil- 

 ity of plant tissues to damage by ROS during 

 pathogenesis (Xie and Chen 1999). The higher 

 concentrations of exogenous SA used to acti- 

 vate disease resistance have been demonstrat- 

 ed to inhibit plant catalase activity, and the re- 

 sultant accumulation of hydrogen peroxide in 

 plant tissues has been proposed to be a trigger 



for the activation of anti-pathogen defenses 

 (Chen et al. 1993). Ruffer et al. (1995) chal- 

 lenged this concept based on several lines of 

 evidence, including their finding that SA can 

 inhibit a variety of enzymes of plant, animal, 

 and fungal origin, and the fact that concentra- 

 tions of endogenous SA comparable to those 

 required to inhibit plant catalase occur sel- 

 dom, if ever, in plant tissues. Ruffer et al. 

 (1995) also proposed that rather than serving 

 as a specific signal for activation of plant de- 

 fenses, SA may simply function as a phyto- 

 alexin (an inducible low-molecular weight an- 

 timicrobial compound made by plants in re- 

 sponse to pathogen invasion or certain other 

 stressors). Salicylate also is well known as an 

 uncoupler of mitochondrial electron transport 

 and chemiosmosis (Norman et al. 2004), and 

 has been used as an antimicrobial food addi- 

 tive (Cruess and Irish 1931). Thus, we hy- 

 pothesized that SA might also be directly in- 

 hibitory to plant pathogens via one or more 

 such mechanisms, particularly at the millimo- 

 lar concentrations a pathogen might encounter 

 on a plant leaf that has been sprayed with ex- 

 ogenous SA to activate plant defenses. We 

 tested this hypothesis with in vitro studies of 

 the effects of SA on the growth of seven plant- 

 pathogenic fungi. Further, we experimentally 



118 



