Conidia from all colonies, which were incubated for 10 days, were photographed under a phase-contrast microscope (400×) at the same light exposure. To compare the levels of light-penetrating activity of conidia, which was observed under a phase-contrast microscope, a densitometric analysis was
used to generate a relative densitometric value (RDV) of conidia. In each observation of the photographed conidia, the densitometric values at four spots of background (DV0) and four spots of conidia (DV1), randomly taken, were converted to RDV as follows: RDV = DV1/DV0. The highest RDV was arbitrarily given to 1.00 to compare it with the other RDVs. All conidial suspensions (c. 5 × 106 conidia mL−1) were transferred to fresh Eppendorf tubes (500 μL per Selleckchem Alectinib tube) and held in a water bath at 45 °C for 30, 60, 90 and 120 min. For each strain treatment (non-paired and paired), controls (non-exposed Selleckchem MI-503 conidial suspensions) were kept at room temperature (c. 25 °C). A 10-μL sample
was taken from each tube and dropped on ¼SDAY medium for a germination test prior to and after the exposures. After incubation of all plates at 20 °C for 24 h, percent germination was determined by randomly counting the number of germinated and ungerminated conidia among 100 counts microscopically (400×). A conidium was considered germinated if a germ tube was longer than the length of a conidium (Avery et al., 2004). In addition, the length of hyphae possibly including germ tubes was measured (10 hyphae per plate) after 24 h incubation. Each treatment was replicated three times (three tubes per treatment) and the entire test was repeated twice using different cultures. The virulence of conidia from the isolated colonies against WFT
larvae was investigated using a leaf dipping method in laboratory conditions (Butt & Goettel, 2000). Conidia from the non-paired ERL1578 and ERL1576 colonies Tyrosine-protein kinase BLK served as positive controls. Conidial suspensions were adjusted to 1 × 106 conidia mL−1 using 0.08% siloxane solution as a wetting agent. A siloxane solution (0.08%) served as a negative control. WFT were continuously reared on green beans, Phaseolus vulgaris L. at 25 ± 1 °C and a 16:8 (L/D) photoperiod with 40–50% relative humidity in wooden chambers (45 × 30 × 30 cm) in an insectary at the Entomology Research Laboratory, University of Vermont. Fresh green bean leaves were aseptically cut into 35 mm diam. circles using a cork borer sterilized with 70% ethanol. Three leaf discs were dipped for 10 s in a conidial suspension (15 mL) in a 35-mm Petri dish and dried at room temperature (c. 25 °C) for 20 min. All discs were placed on moistened filter papers (50 μL sterile distilled water per 35 mm diameter paper) in the lids of 35-mm Petri dishes (one disc/lid). Using an aspirator, 15 thrips 2 days old were placed on each leaf disc in the lid of a Petri dish.