**3.2 Effect of different sources of sugar and nitrogen on the blastospores growth of entomopathogenic fungi**

*M. anisopliae* growth observed from the shake flask cultures supported with different media differed significantly after 20 h (*F* = 96.535; df = 14, 45; *P* < 0.001), 40 h (*F* = 77.536; df = 14, 45; *P* < 0.001) and 60 h (*F* = 67.381; df = 14, 45; *P* < 0.001). As the incubation time elapsed, the concentration of the blastospores increased. After 20 h complete growth media (G + SM + CSL + PE + YE), afforded the highest growth of the yeast like hyphal bodies of the *M. anisopliae*.

While, G + PE, G + YE and G + PE + YE showed the lowest optical density of the growth media. After 40 and 60 h of incubation, the medium SM + CSL produced higher concentration of the blastospores (1.995) and remained significantly at higher level than any medium. While G + PE + YE media, showed the lowest values at both the studied time intervals (40 h and 60 h). The complete medium also afforded the growth of the fungi, which ultimately showed higher OD values of the blastospores but significantly at lower level than SM + CSL medium after 40 h and 60 h (Fig. 2a).

Current Status of Entomopathogenic Fungi

g g

G+PE

g

i i

G+YE

G+CSL+PE

h

bc g

G+CSL

0.00 0.30 0.60 0.90 1.20 1.50 1.80 2.10

(Fig. 2c).

**O .D** 

**(610 nm**

 **)**

as Mycoinecticides and Their Inexpensive Development in Liquid Cultures 113

ab

a

ef c

g f

d e

f g

G+CSL+PE+YE

Fig. 2c. Blastospores concentration (OD) of *I. fumosorosea* grown on different media supplemented with different sources of nitrogen and sugar after 20 h, 40 h and 60 h incubation. Mean ± SE values with the same letter along the bars of different growth media are not significantly different (*P <* 0.05). For detail of treatments see table 1.

G+PE+YE

to cause stimulant effect on the growth of the blastospores (Fig. 2b).

i

g

h

SM+CSL

Blastospores growth was highly variable and significant differences among the growth of *B. bassiana* on all the growth media after 20 h (*F* = 172.619; df = 14, 45; *P* <0.001), 40 h (*F* = 103.584; df = 14, 45; *P* <0.001) and 60 h (*F* = 62.153; df = 14, 45; *P* <0.001) were observed. After 20 h of incubation, media supplemented with SM showed higher growth compared to all the media containing glucose as sugar source in all the combinations with CSL, PE and YE. On the whole, the media SM + CSL after 40 h and 60 h showed the highest growth of blastospores. CSL in combination with PE and YE in the presence of G as sugar source enhanced the growth of the fungi. Comparatively, the media supplemented with SM found

There was a significant difference in the blastospores concentration of *I. fumosorosea* after 20 h (*F* = 36.819; df = 14, 45; *P* <0.001), 40 h (*F* = 145.915; df = 14, 45; *P* <0.001) and 60 h (*F* = 110.554; df = 14, 45; *P* <0.001) grown on different media supplemented with different sources of nitrogen and sugar in all possible combinations. *I. fumosorosea* grown on SM + CSL exhibited the highest growth not only after 20 h of incubation but also after 60 h (Fig. 2c). While, SM + CSL also promoted the growth resulting higher blastospores but remained significantly lower than SM + CSL + PE, which showed the highest growth after 40 h of incubation. On the whole, media supplemented with SM as sugar source showed higher growth of the blastospores compared with the media containing glucose as sugar source

**Growth Media**

SM+PE

SM+YE

SM+CSL+PE

e

ef def

G+CSL+YE

de

e

h

f

ab

b

def bc cd def de ab <sup>a</sup>

SM+CSL+YE

SM+PE+YE

SM+CSL+PE+YE

G+SM

a

c c

b

cd

d

e

+CSL+PE+YE

d

Fig. 2a. Blastospores concentration (OD) of *M. anisopliae* grown on different media supplemented with different sources of nitrogen and sugar after 20 h, 40 h and 60 h incubation. Mean ± SE values with the same letter along the bars of different growth media are not significantly different (*P <* 0.05). For detail of treatments see table 1.

Fig. 2b. Blastospores concentration (OD) of *B. bassiana* grown on different media supplemented with different sources of nitrogen and sugar after 20 h, 40 h and 60 h incubation. Mean ± SE values with the same letter along the bars of different growth media are not significantly different (*P <* 0.05). For detail of treatments see table 1.

cd

**Growth Media**

a

a

fg

SM+CSL

**Growth Media**

SM+PE

c

h

G+CSL+PE+YE

Fig. 2b. Blastospores concentration (OD) of *B. bassiana* grown on different media

supplemented with different sources of nitrogen and sugar after 20 h, 40 h and 60 h incubation. Mean ± SE values with the same letter along the bars of different growth media are not significantly different (*P <* 0.05). For detail of treatments see table 1.

e

SM+PE

SM+CSL

a

de de

SM+YE

<sup>c</sup> <sup>d</sup> <sup>e</sup> <sup>d</sup>

SM+YE

SM+CSL+PE

SM+CSL+YE

SM+PE+YE

SM+CSL+PE+YE

G+SM

fg ef

<sup>d</sup> <sup>d</sup>

de

c

SM+CSL+PE

SM+CSL+YE

a

g

fg

<sup>h</sup> <sup>i</sup> gh

G+CSL+PE+YE

Fig. 2a. Blastospores concentration (OD) of *M. anisopliae* grown on different media

supplemented with different sources of nitrogen and sugar after 20 h, 40 h and 60 h incubation. Mean ± SE values with the same letter along the bars of different growth media are not significantly different (*P <* 0.05). For detail of treatments see table 1.

<sup>h</sup> <sup>i</sup>

G+PE+YE

h h

<sup>g</sup> <sup>i</sup> <sup>i</sup>

G+YE

G+CSL+PE

g

de cd

fg

g

G+CSL+YE

<sup>f</sup> <sup>f</sup> <sup>e</sup> <sup>e</sup> <sup>f</sup> <sup>e</sup>

G+CSL+YE

ef

b

g

G+PE+YE

c

f

G+CSL

d

G+CSL

0.00 0.30 0.60 0.90 1.20 1.50 1.80 2.10

**O** 

**.D (610 nm**

 **)**

d

d

e

G+PE

hh

G+YE

G+CSL+PE

f

0.00 0.30 0.60 0.90 1.20 1.50 1.80 2.10

**O.D (610 nm**

 **)**

d

cd

ef

G+PE

<sup>a</sup> ab <sup>d</sup> bc <sup>d</sup> <sup>e</sup> <sup>f</sup>

SM+PE+YE

SM+CSL+PE+YE

G+SM

<sup>b</sup> ab <sup>a</sup>

<sup>c</sup> bc <sup>b</sup>

b

b

c

a

+CSL+PE+YE

ef

d

cd <sup>c</sup>

ef

<sup>c</sup> <sup>b</sup>

+CSL+PE+YE

b

ef <sup>e</sup> cd cd <sup>e</sup>

c

Fig. 2c. Blastospores concentration (OD) of *I. fumosorosea* grown on different media supplemented with different sources of nitrogen and sugar after 20 h, 40 h and 60 h incubation. Mean ± SE values with the same letter along the bars of different growth media are not significantly different (*P <* 0.05). For detail of treatments see table 1.

Blastospores growth was highly variable and significant differences among the growth of *B. bassiana* on all the growth media after 20 h (*F* = 172.619; df = 14, 45; *P* <0.001), 40 h (*F* = 103.584; df = 14, 45; *P* <0.001) and 60 h (*F* = 62.153; df = 14, 45; *P* <0.001) were observed. After 20 h of incubation, media supplemented with SM showed higher growth compared to all the media containing glucose as sugar source in all the combinations with CSL, PE and YE. On the whole, the media SM + CSL after 40 h and 60 h showed the highest growth of blastospores. CSL in combination with PE and YE in the presence of G as sugar source enhanced the growth of the fungi. Comparatively, the media supplemented with SM found to cause stimulant effect on the growth of the blastospores (Fig. 2b).

There was a significant difference in the blastospores concentration of *I. fumosorosea* after 20 h (*F* = 36.819; df = 14, 45; *P* <0.001), 40 h (*F* = 145.915; df = 14, 45; *P* <0.001) and 60 h (*F* = 110.554; df = 14, 45; *P* <0.001) grown on different media supplemented with different sources of nitrogen and sugar in all possible combinations. *I. fumosorosea* grown on SM + CSL exhibited the highest growth not only after 20 h of incubation but also after 60 h (Fig. 2c). While, SM + CSL also promoted the growth resulting higher blastospores but remained significantly lower than SM + CSL + PE, which showed the highest growth after 40 h of incubation. On the whole, media supplemented with SM as sugar source showed higher growth of the blastospores compared with the media containing glucose as sugar source (Fig. 2c).

Current Status of Entomopathogenic Fungi

e

de

bcd

0.0

G+CSL

G+PE

G+YE

+ YE (0.20 µmol NA ml-1 min-1) (Fig. 3c).

G+CSL+PE

G+CSL+YE

G+PE+YE

**NA ml-1 min-1) of spore bound Pr1 of entomopathogenic fungi** 

G+CSL+PE+YE

SM+CSL

Fig. 3. Extracellular protease Pr1 activity (µmol NA ml-1 min-1) of (a) *M. anisopliae* (b) *B. bassiana* and (c) *I. fumosorosea* grown on media supplemented with different sources of nitrogen and sugar. Mean ± SE values with the same letter along the bars of different growth

**3.3 Effect of different sources of sugar and nitrogen on the enzymatic activity (µmol** 

Significant differences in enzymatic activity of spore bound Pr1 were observed when different sources of sugar and nitrogen in all possible combinations were used to grow *M. anisopliae* (*F* = 28.945; df = 14, 30; *P* < 0.001), *B. bassiana* (*F* = 20.302; df = 14, 30; *P* < 0.001) and *I. fumosorosea* (*F* =21.376; df = 14, 30; *P* < 0.001). The spores of all the studied entomopathogenic fungi cultivated from rice inoculated with medium supplemented with SM + CSL showed the highest activity of Pr1 (Fig. 3a-c). *M. anisopliae* spores grown on complete media also showed higher enzymatic activity (Fig. 3a). The growth medium SM + PE + YE in case of *M. anisopliae,* while SM + PE + YE and G + PE + YE, in case of *B. bassiana* showed the lowest enzymatic activity (Fig. 3a-b). The addition of PE and YE solely in combination with glucose produced spores of *B. bassiana* with relatively higher enzymatic activity compared to SM (Fig. 3b). In case of *I. fumosorosea*, CSL supplemented media showed comparatively higher enzymatic activity except the medium supplemented with SM

media are not significantly different (*P <* 0.05). For detail of treatments see table 1.

SM+PE

**Growth media**

SM+YE

0.2

0.4

0.6

**Spore bound Pr1 activity**

0.8

1.0

1.2

as Mycoinecticides and Their Inexpensive Development in Liquid Cultures 115

**(c)** 

a

bcd

e

bcd bcd

f

SM+CSL+PE

SM+CSL+YE

SM+PE+YE

SM+CSL+PE+YE

+CSL+PE+YE

G+SM

bcd bc

ab bc

a

cd

**Growth media**

**Growth media**

**(a)** 

a

b

cd <sup>d</sup>

G+PE+YE

G+CSL+PE+YE

SM+CSL

**Growth media**

a

**(b)** 

fg

SM+CSL

SM+PE

**Growth media**

SM+YE

SM+CSL+PE

SM+CSL+YE

g

G+CSL+PE+YE

def

fg

bcd

SM+PE

SM+YE

SM+CSL+PE

c

<sup>b</sup> <sup>b</sup> <sup>b</sup>

0

**S p ore b ou n d P** 

G+CSL

G+PE

ab abc

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

**Spore bound Pr1 activit**

**y**

G+CSL

G+PE

G+YE

G+CSL+PE

G+CSL+YE

G+PE+YE

G+YE

G+CSL+PE

G+CSL+YE

0.2

0.4

0.6

0.8

**r1 activity**

1

1.2

a

b

e

SM+CSL+PE+YE

+CSL+PE+YE

ab

def

g

SM+CSL+PE+YE

G+SM+CSL+PE+YE

fg

SM+PE+YE

def ef

cde

G+SM

d d

SM+CSL+YE

SM+PE+YE

b

c

**(c)** 

**Growth media**

Fig. 3. Extracellular protease Pr1 activity (µmol NA ml-1 min-1) of (a) *M. anisopliae* (b) *B. bassiana* and (c) *I. fumosorosea* grown on media supplemented with different sources of nitrogen and sugar. Mean ± SE values with the same letter along the bars of different growth media are not significantly different (*P <* 0.05). For detail of treatments see table 1.
