Conidial Suspension and Viability

Beauveria bassiana isolates MHK and GHA were grown separately in Petri dishes (150 × 15 mm) containing PDA media and incubated at room temperature for 14 d. For each isolate of B. bassiana, conidia were scraped from 14 petri dishes containing PDA media colonized by fungal mycelia using sterile water and T-spreaders. Conidial suspensions were filtered through four layers of sterile cheesecloth and transferred to sterile conical tubes. Conidial concentrations were determined using a hemocytometer and adjusted to 2.5 × 10⁷ conidia/ml, 1.25 × 10⁸ conidia/ml, and 2.5 × 10⁸ conidia/ml, hereafter referred to as low, medium, and high concentrations. Fresh conidial suspensions were prepared for each bioassay and used immediately after suspension in water. Before each bioassay, the viability of conidia was determined by placing 100 µl of conidial suspensions, adjusted at 10⁵ conidia/ml, on PDA media. To do so, we observed by optical microscopy (400×) a total of 300 conidia 18 hr after inoculation. Only conidia with germ tubes twice their diameter were considered viable (Francisco et al. 2006). In all treatments, viability of conidia was above >92% at the time of exposure.