Synergistic Inhibition — Strobel and Porter 



127 



Table 9. Synergistic inhibition of mycelial growth oi' Pesfalotia sp. by sodium salicylate (SA) and a ready-to-use com- 

 mercial formulation of a neeni oil extract (NMRTU). NMRTU dilutions were achieved by adding 0-100 |jl1 NMRTU 

 to wells containing sufficient liquid glucose-salts medium (GS) to yield 0.5 ml total volumes. SA concentrations were 

 achieved by adding 10 or 20 |jl1 of a 100 mM stock to yield final concentrations of 2.0 mM or 4.0 mM. The extent of 

 mycelial growth (mm) from inoculum plugs was measured after a 48 hour incubation period. Data are mean ± standard 

 error of four (SA 0.0 mM) or two (SA 2.0 mM) repUcate wells per treatment. No PESP growth occurred in the 

 presence of 4.0 mM SA (n = 2 per combination with NMRTU), and these data are omitted from the body of the 

 table. Other abbreviations: OPC, observed percent of control; ADD and SYN denote additive and synergistic interac- 

 tions, respectively. 





NMRTU 0 



NMRTU l/l()()x 



NMRTU l/3()x 



NMRTU 1/20 X 



NMRTU 1/1 OX 



NMRTU 1/5 X 



SA 0.0 mM 



6.0 ± 0.2 



4.0 ± 0.0 



3.5 ± 0.1 



2.8 ± 0.1 



1.9 ± 0.1 



0.9 ± 0.1 



OPC 



100 



67 



58 



47 



32 



15 



SA 2.0 mM 



4.5 ± 0.4 



3.0 ± 0.0 



2.0 ± 0.0 



1.5 ± 0.4 



1.0 ± 0.4 



0.0 ± 0.0 



OPC 



75 



50 



33 



25 



17 



0 







ADD 



SYN 



SYN 



SYN 



SYN 



to those commonly used for the experimental 

 induction of disease resistance in plants (2.0- 

 10.0 mM) are potentially directly antifungal as 

 well. Sensitivity of plant-pathogenic fungi to 

 SA is consistent with prior published reports 

 of the inhibition of saprophytic fungi by SA 

 (Cruess and Irish 1931). However, concentra- 

 tions of SA equivalent to those reported to oc- 

 cur in infected plant tissues (10.0-100.0 |jlM 

 endogenous SA) did not inhibit the growth of 

 BC in vitro. Thus, it seems unUkely that en- 

 dogenous levels of SA in infected plant tissues 

 (10-100 |jlM) would alone be capable of in- 

 hibiting growth of pathogens in plants, as pro- 

 posed by Ruffer et al. (1995). This does not 

 rule out the possibility that SA may act in con- 

 cert with other substances produced by plants 

 to hmit pathogen activity (see below). 



We observed multiple instances of in vitro 

 synergism of SA with diverse other antifungal 

 materials (PQ, Cu, BCF, and NMRTU). The 

 mechanisms underlying these synergistic in- 

 teractions remain to be determined. BC pro- 

 duces a wide variety of enzymes that degrade 

 reactive oxygen species (Steel and Nair 1993; 

 Choi et al. 1997; Gil-ad et al. 2000) that might 

 be inhibited by SA as are some related plant 

 enzymes (Chen et al. 1993; Durner and Kles- 

 sig 1995; Slaymaker et al. 2002). Another plau- 

 sible mechanism for the observed effects of 

 SA is the suppression by SA of mitochondrial 

 generation of ATP (Norman et al. 2004), 

 which supplies energy necessary for many as- 

 pects of cellular Ufe, including the production, 

 operation, and maintenance of various detox- 

 ification mechanisms. In the case of Cu, SA 



may also increase entry of the toxicant into 

 fungal cells via chelation. However, this would 

 not likely account for the observed antagonis- 

 tic interactions of lower concentrations of SA 

 with Cu seen with CG but not with PESP. In 

 the case of NMRTU, matters seem more com- 

 plex, and may involve multiple interactions of 

 NMRTU active or inert ingredients, contami- 

 nating bacteria, and SA. It is also plausible that 

 acidification of the growth medium by anti- 

 fungal materials or by fungi in response to 

 same (not examined in the present studies) 

 may result in increased uptake of salicylate, 

 which is more lipid-soluble and more fungi- 

 toxic in the protonated form (Cruess and Irish 

 1931). Whether SA may usefully synergize the 

 antifungal activities of commercial synthetic 

 fungicides also remains to be determined. 



We speculate that SA may be of practical 

 utility as a multi-functional component (induc- 

 er of local disease resistance, direct antifungal 

 agent, modifier of the composition and size of 

 microfloral populations on plant surfaces, and 

 synergist of other antifungal materials) of new 

 plant disease-control formulations that com- 

 bine ingredients of synthetic chemical and/or 

 biological origin. Such combinations may delay 

 or prevent development of pathogen resis- 

 tance to conventional fungicides, and may also 

 enable reduced application rates for these fun- 

 gicides (Ye et al. 1995). For example, fixed 

 copper fungicides such as Bordeaux mixture 

 are often applied at rates that deliver approx- 

 imately 1-2 kg of elemental copper per acre 

 per application, sometimes resulting in the ac- 

 cumulation of copper to phytotoxic levels in 



