Figure 3

Bioassays of the mycelial growth inhibition activity of purified and renatured His ₆ -tagged mature SnOLP. A-B. Representative bioassay Petri dishes. Filter paper discs containing 10 μL of test proteins were placed on plates 3 days after inoculation with fungal mycelia (plugs). Petri dishes were incubated at 29°C during the entire bioassay. Inhibitory effects of purified and renatured His₆-tagged mature SnOLP upon the fungi are observed as areas lacking mycelial growth. A. Phytophthora nicotiana var. parasitica; 1 day after adding protein (a.a.p.); B. Macrophomina phaseolina, 5 days a.a.p. 1–3. Corresponds to the doses of 1 μg, 2 μg and 3 μg of purified and renatured His₆-tagged mature SnOLP, or to the concentrations of 0,1 μg/μL, 0,2 μg/μL and 0,3 μg/μL, respectively; 4. Corresponds to the dose of 10 μg of Bovine Serum Albumin (BSA), or to the concentration of 1 μg/μL; 5. Corresponds to the dose of 2000 U Nistatin, or to the concentration of 200 U/μL. C-F. Average area (mm²) of mycelial growth inhibition caused by renatured His₆-tagged mature SnOLP, as measured (software UTHSCSA Image Tool, Version 3.00 [57]) in three replicates, similar to the bioassay Petri dishes shown in A and B, for each fungus/oomycete and for each dose/concentration of SnOLP separately. Standard deviation bars are shown for each average column. The averages were statistically compared by using ANOVA and Tukey Test at the probability level of 1% (software Genes [58]). Different letters above the average columns (i.e. a, b and c), indicate that the average values were considered to be statistically different among each other, whereas statistically identical average values are indicated by the same letter. C. Phytophthora nicotiana var. parasitica; 1 day after adding protein (a.a.p.); D. Macrophomina phaseolina, 5 days a.a.p. ; E. Colletotrichum gloeosporioides, 12 days a.a.p.; F. Colletotrichum gossypii var. cephalosporioides, 5 days a.a.p.; G. Fusarium solani f. sp. glycines, 5 days a.a.p.