Synergistic Inhibition — Strobel and Porter 



121 



The presence or absence of growth was first 

 assessed visually, and absence of growth then 

 verified by microscopic observation. Radial 

 mycehal growth from agar plugs was assessed 

 visually with a millimeter rule, by viewing 

 plates from beneath by transmitted ceiling 

 light, or sublit on a microscope stage upon 

 which the rule had been placed. In some ex- 

 periments ("NMRTU time-course studies"), 

 addition of SA and inoculation with fungi were 

 delayed relative to introduction of NMRTU. 

 Mycehal growth of EC in the NMRTU time- 

 course study presented in this paper was eval- 

 uated relative to that of controls according to 

 the following rating system: 0 = no growth; 1 

 = >90% inhibition (weak growth); 2 = 75% 

 inhibition (weak-moderate growth); 3 = 50% 

 inhibition (moderate growth); 4 = 10-25% in- 

 hibition (moderate-strong growth); 5 = 90- 

 100% of maximal growth (strong growth). 

 Germination of CG and PESP spores was as- 

 sessed with a microscope at 100 X total mag- 

 nification. In experiments with CG spores, a 

 minimum of 120 spores per well were evalu- 

 ated. 



The nature of interactions between SA and 

 these other antifungal materials was deter- 

 mined according to the method of Kosman 

 and Cohen (1996). For example, in Table 3, 

 the observed means of various treatments in- 

 volving doses of single agents (such as 2.0 mM 

 SA or 4.0 or 8.0 mM PQ) are first converted 

 into percentages of the observed mean values 

 (OPCs) for controls (which received neither 

 SA nor PQ). Then a simple additive model of 

 interaction (AMI) is employed to calculate the 

 values (predicted percent of control values, 

 PPG) one would expect to see (in the absence 

 of either antagonistic or synergistic interac- 

 tions between test compounds). The additive 

 model assumes the independent impact of 

 each agent on a test organism (such as the 

 fungus, EC), and the impact of two agents to- 

 gether is calculated as the product of the im- 

 pact of the first times the impact of the sec- 

 ond. Thus, in Table 3, we find that 2.0 mM 

 sahcylate alone reduced EG growth to 80% of 

 the water controls, whereas 4.0 mM PQ re- 

 duced EG growth to 31% of the water con- 

 trols. The PPG predicted by the AMI for the 

 combination of 2.0 mM SA and 4.0 mM PQ 

 is determined by multiplying 0.80 times 0.31, 

 which yields a PPG of 0.25, or 25%. An ob- 



served percent of control (OPG) value larger 

 than 25% would indicate a lesser degree of 

 inhibition than predicted by a simple additive 

 model of interaction, and would constitute ev- 

 idence of an antagonistic (ANT) interaction 

 between the two test substances. In the pres- 

 ent example, the OPG for the SA-PQ combi- 

 nation mentioned above (12%) is lower than 

 that predicted by the AMI (25%), and the in- 

 teraction is thus judged to have been syner- 

 gistic (a greater than predicted or expected 

 degree of inhibition of EG by the SA-PQ com- 

 bination was observed). 



RESULTS 



Dose-Dependent Inhibition of the Growth of 

 Plant-Pathogenic Fungi by SA 



A summary of the sensitivities of EG and 

 other fungi to SA added to PDA or GS is pre- 

 sented in Table 1. SA concentrations compa- 

 rable to those that may occur in infected plant 

 tissues (10.0-100.0 |jlM endogenous SA, data 

 not shown) had no observable effect on EG 

 mycelial growth in our experiments. Rather, 

 SA concentrations required for complete in- 

 hibition (IGioo) of mycelial growth on PDA or 

 GS media were typically >10.0 mM, whereas 

 between 1.0 and 5.0 mM SA were required to 

 inhibit mycefial growth on PDA by 50% (IGg,,) 

 for most fungi, and the IGg,, of PYSP was 5.0- 

 10.0 mM. Germinating GG spores were rela- 

 tively more sensitive to SA than were spores 

 of MF or PESP. The consistent nature of the 

 dose-dependent inhibition of fungal growth in 

 SA-amended PDA is shown by Table 2, which 

 summarizes data from three consecutive ex- 

 periments with EG. 



Synergistic Inhibition of EG by SA and the 

 Pro-oxidant Herbicide, PQ 



SA and PQ were found to inhibit growth of 

 EG on PDA in a synergistic manner (Table 3, 

 Figure 1). Growth of EG on PDA amended 

 with combinations of 2.0 mM SA and 4.0 or 

 8.0 mM PQ was substantially less than that 

 predicted by an additive model of interaction, 

 and these interactions were judged to be syn- 

 ergistic in nature. (See Material and Methods 

 section for details of the evaluation of inter- 

 actions.) Synergistic inhibition of EG on PDA 

 was also observed for other combinations of 

 SA and PQ (5.0 mM SA with 4.0 or 8.0 mM 

 PQ; data not presented). In a well-plate test 



