Materials and methods
Fungal isolates
The Metarhizium strains used in this comparison study were M. anisopliae AUMC 3262 isolated from the great wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae), Egypt; M. brunneum ARSEF 4556 isolated from Boophilus sp. (Acari: Ixodidae), the USA; and M. brunneum V275 isolated from Cydia Pomonella L. (Lepidoptera: Torticidae), Austria. Prior to their use in these studies, all 3 fungal strains were first passed through the insect host, G. mellonella, in order to restore virulence, before being re-isolated on Sabouraud dextrose yeast agar (SDAY), composed of 40 g glucose, 10 neopeptone, 2 yeast extract, and 15 agar in 1 l dist. H2O (Sigma-Aldrich, UK) incubated in total darkness at 25 ± 2 °C for 15 days. Subcultures were maintained on SDAY at 4 °C for subsequent studies.
Insect rearing
Galleria mellonella cultures were maintained in 2-l glass jars covered with a muslin cloth at 27 ± 2 °C and 8:16 (L:D) h photoperiod, provided with honey and wheat germ as nutrient. All individuals used in this study were last larval instars, with the size range of 15–25 mm.
Effect of successive subculture on quality control parameters
Fungal plugs (8 mm) were taken from the edge of the colony of a 15-day-old culture and used to inoculate Petri dishes of SDAY incubated at 25 ± 2 °C for 7 days. This culture represented the first subculture. Successive subcultures were prepared by inoculating fresh SDAY plates with an inoculum of the previous subculture, with each subculture grown on SDAY for 7 days at 25 ± 2 °C. Following incubation, subcultures were stored at 4 °C until the 9th subculture had been produced. After the production of the 9th subculture, 1st, 3rd, 5th, 7th, and 9th subcultures were grown on new SDAY for 7 days. This procedure allowed the simultaneous generation of these subcultures under similar growth conditions (5 replicates per subculture). These cultures were then used for subsequent studies.
Monitoring fungal growth and sector formation
After the simultaneous growth, conidia of 7-day-old cultures were scraped from the simultaneously cultivated plates, spread on to 90 mm SDAY Petri dish, and incubated in darkness for 3 days at 25 ± 2 °C. Individual plugs (8 mm diameter) of un-sporulated mycelium from the 3-day-old cultures were placed upside down in the center of SDAY medium in 90-mm Petri dishes and incubated in darkness for 15 days at 25 ± 2 °C. Three replicates were prepared from each replicate of the five replicates previously prepared (total no. 15). Morphological changes and sector formation were observed daily, and surface radial growth was recorded from the 3rd to 15th days and was calculated following the equation:
$$ \frac{\mathrm{Colony}\ \mathrm{diameter}\ \mathrm{at}\ \mathrm{the}\ \mathrm{end}\ \mathrm{of}\ \mathrm{incubation}\ \mathrm{period}-\mathrm{Fungal}\ \mathrm{disc}\ \mathrm{diameter}}{\mathrm{Total}\ \mathrm{incubation}\ \mathrm{days}} $$
Conidial yield and viability
From the previous prepared 15 plates, 5-mm agar plugs were taken randomly after 15-day incubation from each plate and placed in 1 ml of 0.03% (v/v), Tween 80 then vortexed to suspend the spores. Spore yield was determined, using an Improved Neubauer hemocytometer, Germany. Germination and viability were assessed according to Inglis et al. (2012). 15 SDAY plates were inoculated with 50 μl of the previous conidial suspension (4 × 107 conidia/ml), then incubated in darkness at 25 ± 2 °C, after 20–24 h. Plates were examined for germination at × 40 (100 conidia per microscopic field). A spore was considered to have germinated if it had formed a germ tube that was as long as the spore width.
Determination of spore bound Pr1
Conidia-bound Pr1 activity was determined according to Shah et al. (2005) as follows: 10 mg of conidia was harvested from 15-day-old culture and washed once with 1 ml of 0.03% (v/v) aq. Tween 80 and twice with 1 ml distilled water, then incubated in 1 ml of 0.1 M Tris-HCl (pH 7.95) containing 1 mM Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma-Aldrich UK) for 5 min at room temperature. After incubation, conidia were pelleted by centrifugation at 12,000 rpm for 5 min (Sanyo, Harrier 18/80 centrifuge). The supernatant (200 μl) was transferred to a 96-well flat-bottom plate, optical grade (Greiner Bio-One) and absorbance measured, using a BioTek Synergy H1 multi-mode reader at 405 nm. Buffered substrate was used as a control.
Determination of fungal virulence
Assessment of conidial virulence for each subculture was carried out against the last larval instar of G. mellonella. Batches of 10 G. mellonella larvae were immersed for 10 s in 10 ml of 0.03% (v/v) aqueous Tween 80 conidial suspension (4 × 107 conidia/ml) prepared from each subculture. Excess conidial suspension was removed by transferring the inoculated insects to filter paper before moving them to a 90-mm Petri dish lined with filter paper moistened with distilled water and sealed with paradigm tape. No food was provided. Control insects were immersed in 0.03% (v/v) aqueous Tween 80 only. Treatments were carried out in triplicate. All dishes were incubated at 25 ± 2 °C in darkness and monitored daily for 5 days.
Statistical analysis
LT50 was calculated using Bio-Stat V5 (analyst soft inc. V.5.9.33). Other results were expressed as the mean ± standard error (SE). Statistical significance was determined by the way of analysis of variance (one-way ANOVA, SPSS software version 16), followed by the least significant difference LSD test.