Spore germination assays. Explorato

Spore germination assays. Exploratory tests were performed with isolates Cg-917 and Cg-935 in order to determine spore germination and standardize incubation conditions (Cerón et al., 2005). Fresh colonies of the two isolates were produced on Marthur´s medium incubated at 28°C in darkness for 10 d. Spore suspensions (1·105 spores/ mL) were prepared on either a) sterile water amended with Tween®-80 (0.05% v/v) or b) a 10% aqueous sucrose solution amended with Tween-80 (0.05% v/v); and incubated at 28°C and 100% relative humidity on glass slides. Germination counts were carried out every 2 h and for up to 24 h, germination was higher on water (10-15%) as compared to the sucrose solution (3%). Based on these results, all germination assays used 1.5 mL of spore suspensions of 1·105spores/mL in water + Tween®-80 (0.05% v/v), using Eppendorf tubes incubated at 27°C for 18 h.

A completely randomized design with three repetitions was used to compare the inhibitory activity of crude filtrates from liquid cultures of 14 microbial isolates (six fungi, four bacteria, two actinomycetes, and two yeasts) on spore germination of the isolate Cg-917. For this, spore suspensions were mixed at a 1:1 ratio with the culture filtrates in 2 mL Eppendorf tubes, and incubated as described above. Following incubation, 50 µ:L were taken from each tube and poured into a Neubauer chamber to determine germination.

A randomized block design displayed in a factorial arrangement with three repetitions was used to compare six plant extracts in four concentrations (Tab.2) by their inhibitory activity on spore germination of the isolate Cg-935 of the mango pathogen. Spore suspensions prepared as described were mixed at a 1:1 ratio with each one of the plant extract treatments, incubated and evaluated as described above.