126 



Journal of the Kentucky Academy of Science 66(2) 



Table 8. Effect of timing of addition of sodium salicylate (SA) and a commercial ready-to-use formulation of a neem 

 oil extract (NMRTU), relative to inoculation with Botrytis cinerea, on synergistic inhibition of mycelial growth. Data 

 presented are growth incidence (GI, presence or absence) in three replicate wells per treatment combination (n = 3); 

 and growth rate (GR) as mean ± standard error for growth relative to controls, with a rating of 0 indicating no growth; 

 1,<10%; 2, 10-50%; 3, 51-75%; 4, 75-90%; or, 5, 91-100% growth relative to controls. No pretreatment indicates 

 that NMRTU and SA were added to wells on the same day as EC inoculation, whereas 3-day pretreatment indicates 

 that NMRTU and SA were introduced 3 days before EC inoculation. Other abbreviations: OPC, observed percent of 

 control; ADD, ANT, and SYN denote additive, antagonistic, and synergistic interactions, respectively. 



NMRTU dilutions 



0 1/100 X 1/20 X 1/5X 



GI 



GI 



GI 



GI 



GR 



No Pretreatment 

 SA 0.0 mM 3/3 

 OPC 



SA 2.0 mM 3/3 

 OPC 



Three Day Pretreatment 



SA 0.0 mM 

 OPC 



SA 2.0 mM 

 OPC 



3/3 

 3/3 



5.0 ± 0.0 



100 

 4.0 ± 0.0 

 80 



5.0 ± 0.0 



100 

 5.0 ± 0.0 



100 



3/3 

 3/3 



3/3 

 1/3 



5.0 ± 0.0 



100 

 4.0 ± 0.0 

 80 



ADD 



4.0 ± 0.0 



80 



0.3 ± 0.1 

 0.1 

 SYN 



3/3 

 3/3 



3/3 

 1/3 



4.0 ± 0.0 



80 



4.0 ± 0.0 

 80 

 ANT 



3.0 ± 0.0 

 60 



0.3 ± 0.1 

 0.1 

 SYN 



3/3 

 3/3 



3/3 

 0/3 



2.3 ± 0.1 

 46 



2.0 ± 0.0 

 40 

 ANT 



3.0 ± 0.0 

 60 



0.0 ± 0.0 

 0 

 SYN 



combinations of NMRTU and SA was most 

 consistent, frequent, and extensive when in- 

 oculation with BC had occasionally been de- 

 layed by one or more days after amendment 

 of wells with NMRTU and SA, the effect of 

 varied duration (0, 1, 2, or 3 days) of such 

 delay was formally investigated. Data regard- 

 ing the effects of a 0 or 3 d delay are pre- 

 sented in Table 8. Plates that received 

 NMRTU, SA, and BC on the same day exhib- 

 ited mycelial growth in all wells of each treat- 

 ment combination, and interactions of SA and 

 NMRTU were found to be either additive or 

 slightly antagonistic. In contrast, when BC in- 

 oculation was delayed until 3 days after addi- 

 tion of SA and NMRTU, synergistic inhibition 

 of BC growth was observed in all SA-NMRTU 

 treatment combinations. Turbidity associated 

 with bacterial growth was much greater in 

 plates that received NMRTU (with or without 

 SA) 3 days prior to inoculation with BC than 

 in wells that received NMRTU and SA im- 

 mediately prior to BC inoculation. These dif- 

 ferences in turbidity were observed both at 

 the time of inoculation with BC and at 3 days 

 post-inoculation, when BC growth was as- 

 sessed. Interestingly, the addition of SA and 

 NMRTU 1 day before BC inoculation resulted 



in only slight development of turbidity and no 

 inhibition of BC, whereas turbidity and extent 

 of BC inhibition were of intermediate degree 

 when BC inoculation occurred 2 days after ad- 

 dition of SA and NMRTU to wells (data not 

 shown). The interactive effects of NMRTU 

 and SA on mycelial growth of PESP were also 

 examined. Again, little impact of NMRTU 

 (alone or in combinations with SA) was ob- 

 served when NMRTU and SA amendment oc- 

 curred on the same day as BC inoculation 

 (data not presented). In an experiment in 

 which NMRTU and SA addition preceded BC 

 inoculation by 2 days (Table 9), NMRTU 

 alone reduced mean mycelial growth of PESP 

 relative to controls in a dose-dependent man- 

 ner, and the addition of SA resulted in a mod- 

 est synergistic enhancement of fungal inhibi- 

 tion with most of the NMRTU doses em- 

 ployed. 



DISCUSSION 



Millimolar concentrations of SA were found 

 to inhibit the growth and development of sev- 

 en plant-pathogenic fungi in vitro, suggesting 

 that sensitivity of fungal plant pathogens to 

 mM SA concentrations may be a general phe- 

 nomenon. Thus, SA concentrations equivalent 



