Insects
Larvae of G. pyloalis were collected from infested mulberry plantation of Rasht, northern Iran (37° 19′ N, 49° 37′ E; -9 m). Rearing of insect was carried out in a growth chamber at 24 ± 2 °C, 70 ± 5% R.H., and 16:8 (L:D) h of photoperiod on fresh mulberry leaves (Yazdani et al. 2013). Adult moths were placed in transparent plastic containers (20 × 12 × 12 cm3) and provided with fresh mulberry leaves for egg-laying and a cotton wool soaked in 10% honey for adult feeding. The containers were cleaned daily and fresh leaves were provided for the larvae.
Beauveria bassiana fungal culture
AM-118 isolate of B. bassiana was cultured on Potato Dextrose Agar (PDA) and kept at 25 ± 1 °C for 21 days. Then, the conidia were washed off by a 0.01% aqueous solution of Tween 80 to prepare a stock sample of fungal conidia. The isolate was collected from a rice field at Amol (26 25′ N, 52 21′ E; 17 m) and registered in herbarium of mycology.
Bioassay
The serial concentrations of 103, 104, 105, 106, and 107 conidia/ml of AM-118 were prepared in sterile distilled water containing Tween80 (0.02%); then, the early 4th instar larvae of G. pyloalis were randomly selected and separately dipped in each concentration for 5 s. The control larvae were dipped in aqueous solution of Tween 80 (0.02%) alone. After treatment, the larvae were placed on filter paper (Whatman No. 1), provided with fresh leaves, and kept at the rearing conditions. The bioassay was done in 3 replicates, containing 10 larvae/ replication. Mortality was recorded after 7 days and LC50 was determined using POLO-plus software. For estimation of LT50, the mortality was recorded until death of all larvae at the highest conidia concentration.
Liquid culture for enzyme production
A liquid medium, including KH2PO4, 0.02%; CaCl2, 0.01%; MgSO4, 0.01%; Na2HPO4, 0.02%; ZnCl2, 0.01%; and yeast extract, 0.01%, was used for enzymatic production of AM-118. Culture flasks were inoculated with a concentration of 108 conidia/ml and 5% (weight) of G. pyloalis larval cuticle and incubated for 8 days at 25 ± 1 °C on a rotatory shaker (70 rev/min) (Zibaee and Bandani 2009). In control flasks, starch and potato extract were added instead of larval cuticle.
Sample preparations for enzymatic assays
Eight days post-incubation, the mixture was harvested by centrifugation at 10,000×g and 4 °C for 30 min and washed in ice-cold Tris-HCl (25 mM, pH 8). Weighed mycelia were ground to a fine powder and suspended in distilled water. Then, the samples were homogenized and centrifuged at 20,000×g and 4 °C for 30 min to gain the supernatant for the enzymatic assays (Ramzi and Zibaee 2014).
Assay of proteases
Activities of subtilisin-like (Pr1) and trypsin-like (Pr2) as the 2 main fungal proteases were evaluated by 30 μl of succinyl-(alanine) 2-prolinephenylalanine-p-nitroanilide for Pr1 and benzoylphenylalanine-valine-arginine-p-nitroanilide for Pr2 in 100 μl of Tris–HCl buffer (20 mM, pH 8). Afterward, 20 μl of the enzyme solution was added to the mixture and incubated at 25 °C for 10 min. Then, 100 μl of trichloroacetic acid (TCA, 30%) was added and the absorbance was recorded at 405 nm (Zibaee and Bandani 2009).
Endo-chitinase assay
A reaction mixture, containing 50 μl of 0.5% colloidal chitin as substrate, 20 μl of enzyme sample, and 100 μl of Tris–HCl buffer (20 mM, pH 7), was prepared to assay endo-chitinase activity. The tubes containing reaction mixture were incubated in a water bath (30 °C) for 60 min. Then, 100 μl of DNS (dinitrosalicylic acid) was added and the incubation was prolonged for 10 min at boiling water before reading the absorbance at 545 nm (Miller 1959).
Exo-chitinase assay
Assay of exo-chitinase activity was carried out, using 200 μl of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNPg) solution (1 mg pNPg per ml of distilled water) as substrate, 500 μl of Tris-HCl (25 mM, pH 7), and 25 μl of culture filtrate. The mixture was incubated for 20 h at 30 °C, centrifuged at 20,000×g at 4 °C, and then, supernatant was added to 200 μl of sodium tetraborate-NaOH buffer (125 mM, pH 10) prior to reading the absorbance at 400 nm. The extinction coefficient of 18.5 mM−1 cm−1 was calculated.
Protein assay
The method of Lowry et al. (1951) was used to measure the amount of protein in the enzymatic preparations. Twenty microliters of the enzyme sample was added into 100 μl of reagent (Ziest Chem. Co., Tehran-Iran) and incubated for 30 min before reading the absorbance at 545 nm.
Hemolymph collection and hemocyte counts
Fourth instar larvae were injected in the latest segment of thorax by 1 μl of a soluble Tween 80 containing LC50 concentration of B. bassiana. Then, larval hemolymph was collected at intervals of 1, 3, 6, 12, and 24 h after injection. Control larvae remained intact, while other larvae were injected by Tween 80 solution. Samples of hemolymph were bled into ice cold anticoagulant buffer in a ratio of 1–3 (0.01 M ethylenediamine tetraacetic acid, 0.1 M glucose, 0.062 M NaCl, 0.026 M citric acid, pH 4.6) (Azambuja et al. 1991). Number of total hemocyte, granulocyte, plasmatocyte, and nodules were counted, using a Neubauer hemocytometer (Chemkind Co. China). For each treatment, 10 larvae were used and the experiment had 3 replicates.
Assay of phenoloxidase activity
The activity of phenoloxidase (PO) was assayed according to the procedure described by Wilson et al. (2002). Briefly, 10 μl of hemolymph was added into an Eppendorf tube (1.5 ml), centrifuged at 2000×g and 4 °C for 15 min. Supernatant was removed and 100 μl of ice-cold phosphate-buffered saline (20 mM, pH 7) was added to the pellets. To determine PO activity, samples were poured into each well of a plate containing 20 μl of 10-mM 3,4-dihydroxyphenylalanine (L-dopa). After 5 min of incubation at room temperature, the absorbance was recorded at 492 nm.
Statistical analysis
Probit analysis was performed to determine LC50 and LT50 values at the corresponding 95% confidence interval (CI) values by using POLO-Plus software. Biochemical data were compared by one-way analysis of variance (ANOVA), followed by t test and Tukey’s test, where applicable. Differences between control and treatments were statistically analyzed at a probability less than 5% and marked by different letters.
Also, the extinction coefficient of 18.5 mM−1 cm−1 was considered for activity calculation based on the following formula:
$$ \mathrm{Volume}\ \mathrm{activity}\ \left(\mathrm{U}/\mathrm{ml}\right)=\left[\Delta \mathrm{OD}\ \left(\mathrm{OD}\ \mathrm{test}-\mathrm{OD}\ \mathrm{blank}\right)\times {V}_{\mathrm{t}}\times \mathrm{df}\right]/\left(18.5\times t\times 1.0\times {V}_{\mathrm{s}}\right) $$
where Vt = total volume; Vs = sample volume; 18.5 = millimolar extinction coefficient of p-nitrophenol under the assay condition; 1.0 = light path length (cm); t = reaction time; and df = dilution factor (Miller 1959).
