**2.2 Influence of liquid media composition on the** *in vitro* **growth of entomopathogenic fungi**

The three sources of nitrogen: peptone (Sigma), yeast extract (Sigma) and corn steep liquor (Shanghai Xiwang Starch Sugar Co., Ltd.), and two sources of sugar: glucose (Sigma) and sugar molasses, were used in different combinations as shown in Table 2. In all the treatments, the following salts were used at the concentration, CaCl2 . 2H2O (0.06%), KCl (0.28%), MgCl2 . 6H20 (0.16%), MgSO4 . 7H2O (0.2%), NaHCO3 (0.03%) and NaH2PO4 . H2O (0.1%). To avoid reactions among the salts, they were prepared in compatible pairs: CaCl2. 2H2O with KCl; MgCl2. 6H20 with NaH2PO4. H2O; MgSO4. 7H2O with NaHCO3. The other precautionary measures to avoid precipitation were adopted as described by Leite et al. (2005). Media preparations were finalized by adjusting the pH to 6.2, with filter-sterilized HCl (0.1%) and NaOH (10%).

Current Status of Entomopathogenic Fungi

extracellular protease Pr1.

nitroanilide (NA) released per minute.

**2.6 Statistical analyses** 

**entomopathogenic fungi** 

**3. Results** 

as Mycoinecticides and Their Inexpensive Development in Liquid Cultures 109

separately. Inoculated rice granules were mixed thoroughly from the outside of the bag. Inoculated rice bags were then incubated for 18 days at ambient conditions (24 ± 2 ºC, 75- 85% RH). The sporulating rice in bags from each growth medium was then allowed to dry for 10 d at 30 °C. The spores were separated from the rice by sieving through a 300 µm mesh. A collecting vessel, such as a bucket was fitted to the plastic sheeting at the bottom of the sieve to create a funnel into the collecting vessel. The sieve was shaken until all the loose spores had been removed from the rice and had collected in the vessel below. The spores were then further sieved using a 106 μm sieve to separate the larger rice dust particles from the spores. The spores as powder were kept at 4 ºC for subsequent analysis of the activity of

**2.5 Influence of artificial media on the activity of extracellular protease Pr1** 

The activity of Pr1 protease bound to 10 mg spores harvested from the rice of above mentioned media were assayed using a modified method of St. Leger et al. (1987). Briefly, 10 mg spores were washed once in 0.3% aq Tween 80 solution and twice in distilled water, were then incubated in 1 ml of 0.1M Tris HCl (pH 7.95), containing 1mM Succinyl-ala-alapro-phe-*p*-nitroanilide (Sigma) for 5 min at room temperature. The spores were pelleted by centrifugation for 5 min at 12000 g (Fastwin Bio-Tech Company Limited). A 200 µL of supernatant was transferred to quartz cuvette. The absorbance was measured at 405 nm by using a spectrophotometer (Shimadzu UV-1800). Buffered substrate was used as a reference. The amount of spore bound extracellular protease Pr1 is expressed as micromoles of

All experiments were repeated four times except the activity of spore bound Pr1, repeated three times. Data were analyzed by analysis of variance using the ANOVA procedure of SAS (SAS Institute, 2000) for a completely randomized design. When the effect was

**3.1 Effect of different sources of sugar and nitrogen on the dried fungal biomass of** 

Significant differences in dried biomass of *M. anisopliae* (*F* = 10.632; df = 14, 45; *P* < 0.001), *B. bassiana* (*F* = 9.286; df = 14, 45; *P* < 0.001) and *I. fumosorosea* (*F* = 9.596; df = 14, 45; *P* < 0.001) were observed when fungi were grown on media containing different sources of nitrogen and sugar in different combinations. The media supplemented with sugarcane molasses (SM) afforded comparatively higher growth of *M. anisopliae* and *I. fumosorosea* than glucose (Fig. 1a, c). The growth medium contained G + CSL + YE, showed the lowest growth (12.95 mg/ml) of *M. anisopliae*. The glucose in combination with CSL, PE and YE showed higher growth; while their combination among them did not afford higher fungal growth (Fig. 1a). The growth medium such as G + CSL + PE, G + CSL + YE and G + PE + YE, afforded the lowest growth of *I. fumosorosea* and non significant differences were observed among them (Fig. 1c). *B. bassiana* grown on media supplemented with SM in combination with CSL, PE

significant (*P* < 0.05), means were separated using Duncan's Multiple Range Test.


Table 2. Composition of the media of shake flask cultures in agitated liquid cultures of entomopathogenic fungi

The media were poured into 250 ml Erlenmeyer flasks and autoclaved. After cooling, all the flasks were inoculated with one milliliter of spore suspension (1 × 106 spores/ml) in 0.03 % Tween 80 (Sigma-Aldrich, St Louis, MO, US) from 24-day-old cultures of *B. bassiana*, *M. anisopliae* and *I. fumosorosea* grown on PDA. Four replicates were used for each growth medium. After inoculation, cultures (100 ml) were grown in 250 ml shake flasks at 120 rpm on a rotary shaker, 25 ± 0.5 ºC and 16 h fluorescent light photophase. After 120 h of growth, fungal biomass of each flask was evaluated separately. The fungal biomass was filtered through Whatman No. 1 filter paper. After filtration, the filtrates were dried for 24 h at 70 ºC and weighed.
