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Studies on extracellular enzyme production in Beauveria bassiana isolates.

Introduction

The entomopathogenic fungi Beauveria bassiana is widely attributed for its potential as a promising candidate for bio pest management [1]. Unlike other microbial pesticides, fungi need not be consumed by their hosts to cause an infection, but instead, the germinating fungal spores have capacity of penetrating directly through the insect's cuticle via enzymatic hydrolysis [2]. The entomopathogenic fungi produce a range of cuticle degrading enzymes corresponding to the different polymers of the insect cuticle [3]. Laboratory experiments indicate a well documented isolation, identification and purification of extra-cellular cuticle hydrolyzing enzymes from entomopathogenic fungi and their induction in cultures containing cuticle as substrates has been studied [4]. Pedrini et-al., [5] have thoroughly reviewed the biochemistry of insect epicuticle degradation by enzymes secreted by entomopathogenic fungi. In most studies, a positive correlation has been reported between extracellular enzyme secretion and pathogenecity [6].

In-vitro production of extracellular enzymes by entomopathogens has been the subject of many studies [7]. St. Leger et al. [8] performed his studies on two isolates each of B. bassiana and M. anisopliae. The results of these enzymes are difficult to compare because of differences in isolates and culturing methods [9, 10]. The entomopathogenic fungi are widespread and ubiquitous in occurrence. In nature, the different isolates exhibit varied response in terms of virulence potential, and enzyme secretion is one of the key determinants in this variation. The purpose of this study has been to identify those naturally occurring entomopathogenic fungi which are high secretors and thus possess high potential for enzymatic pathogenesis. In-vitro laboratory studies were conducted to investigate the enzyme secretion by seventeen B. bassiana isolates for the enzymes chitinase, protease, caseinase, lipase and amylase. An enzyme assay was also conducted for chitinase and protease activity for all the seventeen isolates. Morphological characteristics were also taken into account to ascertain their pathogenic potential.

Materials and Methods

Fungal cultures

The different isolates were either procured from ARSEF (USDA-ARS Plant Protection Unit) or isolated locally in India. The seventeen B. bassiana isolates were designated as UB1-UB16 and AB1. The isolates with their accession no., their geographical origin and the host insect from which they were isolated are detailed in table 1. The isolates were routinely sub cultured on SDA (Sabouraud dextrose Agar) slants at 28[degrees]C in incubators and maintained at 4[degrees]C.

Studies on growth characteristics and colony morphology

For germination assay agar slide technique was used. Petri plates were lined with blotting paper discs and 2 glass slides were placed in each of the plates and autoclaved. One ml medium (SDA) was evenly spread on each of the glass slides using micropipette. Conidial suspension was prepared from seven day old cultures with concentrations maintained at [10.sup.6] conidia/ ml. Around 100[micro]l of [10.sup.6] spores of fungal isolates were spread on the media coated slides. The slides were placed back in the Petri plates and the blotting paper discs were moistened with sterilized water. The Petri plates were kept for incubation at 28[degrees]C. The slides were observed under compound microscope (40X) for germination, every 2 hours, starting from 4th hour and for each hour different slides were used. A conidia was considered germinated when a distinct germ tube projected from it, and was at least twice the diameter of the conidia [11]. Approximately 300 conidia were scored per replicate for each of the treatments.

For assay of colony growth and sporulation seven day old cultures on SDA slants were used for preparing spore suspension in 0.02% Tween 80 solution at 1x[10.sup.6] spores/ ml. About 200[micro]l of [10.sup.6] spores were spread plated on SDA medium and were incubated for 3 days at 28[degrees]C. At the end of 3 days, 5mm agar disc with mycelia was retrieved with the help of a cork borer and placed in middle of fresh test substrates 9cm plates and incubated at 28[degrees]C and five replicates were maintained for each isolate. Radial growth was measured every 2nd day till 8th day. Radial growth rate ([mmd.sup.-1]) was calculated from the linear portions of the curves plotted from these values. At the end of 8th day, 5mm agar discs were randomly taken with the help of a cork borer. The discs were placed in 10ml of 0.02% (v/v) Tween 80 solution and vortexed to suspend the spores. Spore yield was determined using a Haemocytometer. The plates (8 day old) prior to spore count were studied for colony morphology characteristics like the growth was fluffy/powdery and sector formation.

Disc preparation for inoculation on different enzymatic substrates

Seven to ten day old cultures on SDA slants were used for preparing spore suspension in 0.02% Tween 80 solution at 1x [10.sup.6] spores/ ml. 200[micro]l of [10.sup.6] spores were plated on SDA medium and was incubated for 3 days at 28[degrees]C. At the end of 3 days, 5mm mycelial disc with agar was retrieved with help of cork borer and placed in middle of fresh test substrate plates (5 replicates/ isolate were maintained) and incubated at 28[degrees]C for 8 days. The control plates did not have the substrate on them; they just had minimal media solidified on 2% agar. Enzyme activities were calculated as an index of the total diameter of the colony + halo divided by the diameter of the colony [8]. Enzymatic index value >1.0 indicates enzyme activity.

Chitinase plate assay

Colloidal chitin was prepared by a modification of the method suggested by [12]. Ten Grams of practical-grade crab shell chitin (Sigma) were mixed with 100ml 12N hydrochloric acid with a continuous stirring for about 2hrs at 4[degrees]C. The suspension was mixed with 1lt water and filtered. This step was repeated 4-5 times and then the pH of the suspension was brought to neutralization by addition of 5N NaOH and again colloidal suspension was washed several times with ddH2O for desalting.

2% colloidal chitin was used to prepare chitin agar plates that were inoculated with 5mm agar disc with mycelia and incubated at 28[degrees]C for 8 days. At the end of incubation period, a thin layer of 0.002% Calcofluor white was spread on the culture plates and left for incubation for few more hours after which they were observed under UV transilluminator [13,14].. Presence of a zone of clearance indicated chitinase activity. The diameter of the fungal colony was measured both exclusively and inclusively of the surrounding halo and enzymatic index values were ascertained.

Protease plate assay

1% Gelatin extract in minimal media (0.003% NaCl, 0.03% MgS[O.sub.4] and 0.015% [K.sub.3]P[O.sub.4]) in conjunction with 2% agar (for solidifying), pH adjusted to 7.0 before autoclaving, was used for in vitro estimation of protease. The plates were inoculated with 5mm agar disc with mycelia and incubated at 28[degrees]C for 8 days. At the end of the incubation period, the culture plates were flooded with a solution of 15% Mercuric Chloride in 2N HCl. A distinct transparent zone of clearance could be seen around the colony while the rest of the plate appeared translucent white in color.

Caseinase plate assay

Milk Agar @ 24g/ 1000 ml was used as substrate (pH 7.2 at 25[degrees]C) for caseinase activity. The plates were inoculated with 5mm agar disc with mycelia and incubated at 28[degrees]C for 8 days. At the end of the incubation period, a clear transparent halo could be seen around the colony while the rest of the plate appeared opaque white in color.

Lipase plate assay

1% Tween 20 was used for in vitro lipase estimation. The other components of the media included 1% peptone, 500mg NaCl, 10mg Ca[Cl.sub.2] and 2% agar (for solidifying), pH adjusted to 6.0 before autoclaving. The plates were inoculated with 5mm agar disc with mycelia and incubated at 28[degrees]C for 8 days. At the end of the incubation period, formation of lipolytic enzymes by a colony was seen as either a visible precipitate due to the formation of crystals of the calcium salt of the lauric acid liberated by the enzyme, or as a clearing of such a precipitate around a colony due to complete degradation of the salt of the fatty acid.

Amylase plate assay

Starch Agar (@ 30g/ 1000 ml), containing starch as the sole carbon source was used as substrate with pH 7.4 at 25[degrees]C [15]. About 20ml media was poured in 9cm Petri plates. The plates were inoculated with 5mm agar disc with mycelia and incubated at 28[degrees]C for 8 days. Colony growth of the fungus implies that starch has been degraded and utilized. At the end of the incubation period, the culture plates were flooded with Lugol's Iodine solution (1g Iodine and 2g Potassium Iodide in 300ml distilled water) and a yellow colored halo could be seen around the colony in an otherwise blue medium indicating amylolytic activity.

Enzyme assays for chitinase and protease Chitinase assay

Chitinase assay was done by the method of [16]. 500[micro]l of culture supernatant was incubated with 300[micro]l of 10 %(w/v) colloidal chitin and 200[micro]l of 0.2M acetate buffer (pH 4) at 37[degrees]C for 2hrs.The reaction product N-acetyl glucosamine was determined by using para-dimethyl-Amino benzaldehyde reagent [17]. Absorbance at 585nm was taken against water as blank. Sampling was done every two days till tenth day of culture incubation. One unit of chitinase activity was defined by the amount of enzyme that produced 1[micro]M of N- acetyl glucosamine per minute under the above conditions.

Protease assay

Proteolytic activity was assayed by a modified method of [18]. 100ul enzyme samples were incubated with 400[micro]l of 0.5% (w/v) gelatin in 50mM Tris- HCl buffer, pH 10.0, at 50[degrees]C for 20 minutes. The enzyme reaction was terminated by addition of 500[micro]l 10% (w/v) trichloroacetic acid and kept at room temperature for 10 minutes. The reaction mixture was centrifuged at 10,000g for 10 minutes at 4[degrees]C and the absorbance measured against water as blank at 280nm. One unit of proteases was defined as the amount of enzyme releasing the equivalent of 1 [micro]M of tyrosine per minute under the defined assay conditions.

Statistical analysis

Statistical analysis of all the data for fungal growth, sporulation and germination were subjected to one-way analysis of variance (ANOVA) and the means were separated by Student-Newman-Keuls multiple range test of comparisons of means at P= 0.05.

Results

Studies on growth characteristics and colony morphology on SDA medium

At 8th hr of incubation, isolate AB1 showed up to 62% germination and this was followed by isolate UB1 (61%), isolates UB2 and UB5 which had >35% of its conidia germinated. Isolate UB7, UB12- UB15 did not show any initiation of germination at 8th hr of incubation. Isolate UB1- UB6 and AB1 had all of their conidia germinated by 16th hr of incubation. Isolate UB2, UB6, UB9, UB15 and UB16 showed specific growth rate in the range of 1.5-1.6. Isolates UB1 and UB6 were the best sporulating isolates when compared to the rest of the isolates. Colony morphology was also studied and the isolates UB2, UB5 and UB11 showed sector formation when grown on SDA plates. Isolate UB1, UB2 and UB6 formed powdery colony whereas isolates UB7, UB9- 14 and UB16 formed fluffy colony. Isolates UB3- 5, UB8 and AB1 showed both fluffy and powdery traits.

In-vitro enzyme activity

It was observed that the in-vitro chitinase activity was exhibited in the range 1.05-1.35, the highest producer being isolate UB1 (1.34) followed by isolate AB1 (1.33). Isolates UB6, UB7, UB8, UB10 and UB11 exhibited similar activity with enzymatic index value of 1.1. The highest protease production was seen in UB1 with enzymatic index value of 2.11 followed by isolate UB6 (1.94). No protease activity was observed in isolates UB7, UB8, UB9, UB10 and UB11. Isolates UB3 and AB1 showed similar activity with an index of 1.56 (Table 3). The isolates exhibited caseinase index values in the range 1.50-3.11 (Table 3), the highest producer being UB12 with enzymatic index value of 3.11, followed by UB1 and UB6 with enzymatic index value of 2.63 and 2.35 respectively. Similar to isolate UB3, the isolate UB7 had an enzymatic index value of 1.71, but contrary to the former, the latter did not show any initiation of mycelial growth from the disc, it just produced a zone of clearance, suggestive of enzymatic activity. The isolates exhibited lipase activity with index values ranging from 2.06-3.89 (Table 3), the most potent secretor being isolate UB1. Six of the isolates, viz., UB1, UB3, UB6, UB7, UB12 and AB1 showed presence of two distinct zones of clearance around the fungal colony. Of the two zones, the first zone, immediately after the fungal colony was a rainbow-colored film with rosette like structures embedded in scattered fashion throughout the depth of the media. These structures when observed under 10X and 40X magnification of compound microscope, showed profuse leafy growth, which when teased with needle (taking care not to shear the filaments), showed the organism growing in a very organized manner. The second zone, which was present beyond the first zone, was a distinct halo with no special structures present. Both the haloes were considered as a whole for calculating the enzymatic index value. Isolate UB16 with enzymatic index value of 2.36 was the highest amylase producer (Table 3). Isolate UB1 did not show any amylase activity at all. Rest of the B. bassiana isolates showed amylase index value in the range of 1.14-1.2.

Enzyme assays

The B. bassiana isolates showed maximum chitinase activity on fourth day of culture incubation (Table 4). Isolates UB1 and UB2 exhibited maximum chitinase activity when compared with other isolates on fourth day of incubation followed by isolates UB1 and AB1 on sixth day. On day two the chitinase activity was high for isolates UB2 and AB1. The chitinase activity decreased with the increase in days of incubation and the isolates showed less chitinase activity on days eight and ten.

Studies on protease activity demonstrated highest activity on eighth day for all the isolates studied (Table 5). On day two UB1 showed highest enzyme production (0.56U/ml) followed by the isolate UB6 (0.22U/ml). Isolate UB1 showed maximum activity on day six and eight followed by isolate UB6 on eighth day.

Discussion

For successful pathogenesis, the entomopathogenic fungi ought to breach the outer integument of their hosts by non-specific and specific events between the conidiospores and the insect cuticle [19, 20]. Entomopathogenic isolates of Metarhizium anisopliae, Beauveria bassaiana and Aspergillus flavus produce multiple extracellular enzymes [21]. Most of the members amongst the 17 isolates that were screened in the present study, produced an appreciable spectrum of protein and polysaccharide-hydrolyzing enzymes, which could be useful in the degradation of the complex living and non-living organic substrates and implies greatest genetic and biochemical versatility. This versatility reflects their ability to exploit resources as and when it becomes available to them [7]. In-vivo, the sequence of enzyme secretion corresponds to the sequence of the polymers present in the cuticle [2]. Depolymerization of the insect cuticle-components is an interactive phenomenon [22, 23]. Insect-pathogenic fungi could have evolved via adaptations in extracellular hydrolytic enzymes so as to accommodate hydrolysis of proteinaceous insect cuticle [24]. In in-vitro plate screening studies, enzyme production is typically indicated by formation of zones of clearance around the growing colony or by the formation of colored product. The most significant inference from the current study is the surprising degree of variability of enzyme secretion by the B. bassiana isolates. Isolate variability has been observed in the production of cuticle-hydrolyzing enzymes in various entomopathogens isolates [25, 23]. Entomopathogenesis entails involvement of proteases, chitinases and lipases [26]. Current understanding of the initial events of entomo-pathogenesis by fungi reflects that the enzyme system of the entomopathogenic fungi is unique and is of great interest as potential criteria for mycoinsecticide improvement. Cuticle-degrading enzymes like proteases and chitinases have been well characterized and a number of genes have been cloned and sequenced [27, 28]. Genetic optimization of entomopathogenic fungi by addition and expression of insecticidal genes in M. anisopliae has been successfully used to engineer a fungal isolate over-expressing a toxic protease, resulting in a considerable shortening in the time of [29]. Fang et al., [30]. stated that cloning chitinase gene from B. bassiana led to an increase in its virulence potential. Several workers have reported a positive correlation between production of extracellular enzymes and virulence potential [6]. Specific enzymes or several of them in an enzyme cascade may serve as key virulence determinants. Since enzymes were differentially expressed in test media, it is also possible that the enzymes may be involved in host range determination [31]. Information generated through such data, may be used to rationally screen and select a potent isolate from a population and thereafter go for its genetic optimization which might be host-specific/ broad host range or rhizosphere competent or has upgraded ability in the speed of kill [32].

In the present work carried out, selection of an isolate showing good response was studied. Colony morphology showed that sector formation was not a desirable attribute as it either did not form any spores or produced sterile spores. Isolate UB1 and AB1 showed good levels of extracellular enzyme secretion in in-vitro plate studies. These isolates showed similar response in chitinase and protease assays. Morphological and growth studies showed that these isolates had similar germination and growth rates, but isolate UB1 formed powdery colony while colony of isolate AB1 exhibited both powdery and fluffy traits. Powdery colony is preferred over fluffy colony as it produces more spores and aids in dispersal of mycopesticide. These isolates when further analyzed for spore yield showed that isolate UB1 had better sporulation in comparison to isolate AB1. Thus, in absolute terms, isolate UB1 was observed to be outstanding source of enzyme secretion, and can be rationally advocated towards production of improved myco-pesticides.

Acknowledgements

We acknowledge the financial support provided by Department of Science and Technology (Project No.SR/FT/L-144) and Ministry of Human Resource and Development (Project No. F. 26-11/2004 TS V). We also thank Dr. Richard A. Humber (USDA-ARS) for providing the fungal cultures.

References

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Uzma Mustafa and Gurvinder Kaur*

Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati-39, Assam, India *Corresponding author E-mail: gurvinder@iitg.ernet.in
Table 1: Source of B. bassiana isolates.

Isolates Code No. / Host Insect Geographical
 Accession No. Location
 Arsef/ Local

 UB1 USDA-ARS (1788) Helicoverpa virescens Spain
 UB2 USDA-ARS (2041) Cnaphalocrocis medinalis Philippines
 UB3 USDA-ARS (5278) Bemicia tabaci USA
 UB4 USDA-ARS (2417) Emmalocera depressella India
 UB5 USDA-ARS (2597) Hyblaea puer India
 UB6 USDA-ARS (6646) Spodoptera litura India
 UB7 USDA-ARS(4027) Coccinella septumpunctata Denmark
 UB8 USDA-ARS(1166) Helicoverpa armigera Spain
 UB9 USDA-ARS(2033) Coccinella sp. USA
 UB10 USDA-ARS(2034) Coccinella sp. USA
 UB11 USDA-ARS(4018) Coccinella septempunctata Denmark
 UB12 USDA-ARS(1886) Chilo infuscatellus India
 UB13 USDA-ARS(2412) Xyloryctes jamaicensis India
 UB14 USDA-ARS(8250) Basilepta fulvicornis India
 UB15 USDA-ARS(6650) Spodoptera litura India
 UB16 USDA-ARS(2660) Adult Coleoptera India
 AB1 Local Unknown India

Table 2: Studies on germination, growth, sporulation and colony
morphology on SDA.

Isolates Germination Specific Sporulation
 growth (x[10.sup.7]
 8th hr 16th hr (mm/day)

 UB1 61.00 (a) 100 (a) 1.00 (e) 3.60 (b)
 UB2 38.60 (b) 100 (a) 1.52 (a) (b) (c) 3.00 (c)
 UB3 36.64 (c) 100 (a) 1.13 (d) (e) 2.25 (f)
 UB4 26.97 (e) 100 (a) 1.22 (d) 2.60 (d)
 UB5 40.00 (b) 100 (a) 1.40 (c) 3.10 (c)
 UB6 30.20 (d) 100 (a) 1.59 (a) (b) 4.05 (a)
 UB7 0 (g) 92.70 (b) 0.75 (f) 1.90 (f)
 UB8 1.40 (g) 54.20 (e) 1.49 (b) (c) 0.90 (i)
 UB9 2.20 (g) 67.80 (d) 1.57 (a) (b) (c) 1.55 (g)
 UB10 6.60 (f) 35.80 (i) 1.18 (d) (e) 2.15 (e) (f)
 UB11 7.00 (f) 28.20 (j) 1.40 (c) 1.30 (g) (h)
 UB12 0 (g) 49.00 (f) 1.09 (d) (e) 1.00 (h) (i)
 UB13 0 (g) 46.20 (g) 1.00 (e) .95 (e) (h) (i)
 UB14 0 (g) 44.00 (h) 1.02 (e) 1.25 (g) (h) (i)
 UB15 0 (g) 46.40 (g) 1.66 (a) 2.50 (d) (e)
 UB16 30.40 (d) 83.40 (c) 1.48 (b) (c) 3.05 (c)
 AB1 61.80 (a) 100 (a) 1.13 (d) (e) 2.20 (e) (f)

Isolates Colony morphology

 Sector formation F/P

 UB1 No P
 UB2 Yes P
 UB3 Yes F+P
 UB4 No F+P
 UB5 Yes F+P
 UB6 No P
 UB7 No F
 UB8 No F+P
 UB9 No F
 UB10 No F
 UB11 Yes F
 UB12 No F
 UB13 No F
 UB14 No F
 UB15 No F+P
 UB16 No F
 AB1 No F+P

Values followed by the same lower case alphabets in the same column are
statistically equivalent (P<0.05) according to the Newman-Keul's
multiple range test. F= fluffy colony, P= powdery colony

Table 3: Enzymatic Index Values of extra-cellular enzymes produced by
B. bassiana isolates.

Isolates Enzymes

 Chitinase Protease Caseinase Lipase

 UB1 1.34 (a) 2.12 (a) 2.63 (b) 3.89 (a) *
 UB2 1.18 (d) (e) 1.44 (e) 1.73 (g) 2.63 (d)
 UB3 1.15 (e) (f) 1.56 (d) 1.71 (h) 2.62 (d) *
 UB4 1.18 (d) (e) 1.46 (e) 1.57 (i) 2.78 (c)
 UB5 1.12 (f) (g) 1.76 (c) 2.04 (d) 2.73 (c)
 UB6 1.11 (f) 1.94 (b) 2.5 (c) 2.17 (g) *
 UB7 1.10 (g) 0 (f) 1.71 (h) # 2.91 (b) *
 UB8 1.10 (g) 0 (f) 0 (k) 0 (i)
 UB9 1.14 (f) (g) 0 (f) 1.98 (e) 2.19 (g)
 UB10 1.10 (g) 0 (f) 1.50 (j) 0i
 UB11 1.10 (g) 0 (f) 0 (k) 2.24 (g)
 UB12 1.22 (c) 0 (f) 3.11 (a) 2.38 (f) *
 UB13 1.28 (b) 0 (f) 0 (k) 0 (i)
 UB14 1.20 (c) (d) 0 (f) 0 (k) 0 (i)
 UB15 1.05 (h) 0 (f) 0 (k) 0 (i)
 UB16 1.23 (c) 0 (f) 0 (k) 2.06 (h)
 AB1 1.33 (a) 1.56 (d) 1.80 (f) 2.50 (e) *

Isolates Enzymes

 Amylase

 UB1 0 (k)
 UB2 1.72 (d)
 UB3 1.41 (f)
 UB4 1.20 (i)
 UB5 1.32 (g) (h)
 UB6 1.37 (f) (g)
 UB7 2.12 (b)
 UB8 1.37 (f) (g)
 UB9 1.53 (e)
 UB10 1.52 (e)
 UB11 1.57 (e)
 UB12 1.80 (c)
 UB13 0 (k)
 UB14 1.14 (j)
 UB15 1.39 (f)
 UB16 2.36 (a)
 AB1 1.27 (h)

Values followed by the same lower case alphabets in the same column
are statistically equivalent (P<0.05) according to the Newman-Keul's
multiple range test. Enzymatic Index Value = (total diameter of the
colony + halo) / diameter of the colony.

# No growth from disc observed but halo (due to enzyme activity) was
present.

* Two distinct zones of clearances could be seen.

Table 4: Chitinase activity in B. bassiana isolates.

Isolates Enzyme activity (U/ml) as on day

 2 4 6

 UB1 18.44 (b) (D) 45.53 (a) (A) 33.25 (a) (B)
 UB2 19.20 (a) (C) 46.49 (a) (A) 28.59 (c) (B)
 UB3 6.55 (f) (D) 22.93 (f) (A) 17.10 (f) (B)
 UB4 4.34 (J) (B) 6.74 (l) (A) 6.61 (j) (k) (A)
 UB5 5.25 (h) (i) (C) 8.08 (k) (A) 5.89 (k) (B)
 UB6 4.88 (i) (E) 15.42 (J) (A) 13.57 (h) (B)
 UB7 3.73 (k) (C) 6.96 (l) (A) 6.57 (j) (k) (A)
 UB8 5.98 (g) (C) 8.25 (k) (A) 7.40 (j) (B)
 UB9 6.70 (f) (D) 19.06 (g) (A) 12.65 (i) (B)
 UB10 5.42 (g) (h) (i) (E) 16.24 (i) (A) 13.51 (h) (B)
 UB11 5.79 (g) (h) (e) 16.75 (h) (i) (A) 12.50 (i) (B)
 UB12 10.35 (d) (D) 34.10 (d) (A) 20.59 (a) (B)
 UB13 15.33 (c) (C) 33.76 (d) (A) 22.66 (d) (B)
 UB14 5.21 (h) (i) (e) 17.24 (h) (A) 14.70 (g) (B)
 UB15 7.61 (a) (D) 19.15 (e) (A) 17.47 (f) (B)
 UB16 7.51 (a) (D) 23.68 (e) (A) 17.32 (f) (B)
 AB1 18.07 (b) (D) 42.81 (c) (A) 32.01 (b) (B)

Isolates Enzyme activity (U/ml) as on day

 8 10

 UB1 20.92 (a) (C) 10.26 (b) (E)
 UB2 17.87 (b) (D) 8.54 (c) (E)
 UB3 11.28 (e) (C) 5.30 (g) (E)
 UB4 4.15 (k) (B) 1.58 (k) (C)
 UB5 5.25 (j) (C) 2.02 (k) (D)
 UB6 9.45 (g) (C) 7.95 (d) (D)
 UB7 4.96 (j) (B) 1.85 (k) (D)
 UB8 7.04 (i) (B) 5.56 (g) (C)
 UB9 9.00 (h) (C) 4.07 (i) (E)
 UB10 10.57 (f) (C) 8.00 (d) (D)
 UB11 10.25 (f) (C) 794 (d) (D)
 UB12 11.33 (e) (C) 7.36 (e) (E)
 UB13 12.32 (d) (D) 6.62 (f) (E)
 UB14 13.98 (c) (C) 11.35 (a) (D)
 UB15 11.07 (e) (C) 4.66 (h) (E)
 UB16 12.54 (d) (C) 3.38 (j) (E)
 AB1 20.60 (a) (C) 10.97 (a) (E)

Values followed by the same lower/ upper case alphabets in the same
column/ row are statistically equivalent (P<0.05) according to the
Newman-Keul's multiple range test.

Table 5: Protease activity in B. bassiana isolates.

 Enzyme activity (U/ml) as on day

Isolates 2 4 6

 UB1 0.56 (a) (D) 0.58 (a) (C) 0.75 (a) (A)
 UB2 0.02 (e) (C) 0.11 (f) (A) (B) 0.12 (f) (A)
 UB3 0.16 b(d) (E) 0.17 (a) (D) 0.10 (e) (C)
 UB4 0.01 (f) (D) 0.06 (c) (B) 0.11 (g) (B)
 UB5 0.20 (c) (D) 0.24 (c) (B) 0.26 (c) (A)
 UB6 0.22 (b) (C) 0.29 (b) (A) 0.29 (b) (A)
 AB1 0.16 (d) (D) 0.19 (d) (C) 0.21 (d) (B)

 Enzyme activity (U/ml) as on day

Isolates 8 10

 UB1 0.75 (a) (A) 0.66 (a) (B)
 UB2 0.12 (f) (A) 0.11 (a) (B)
 UB3 0.23 (a) (A) 0.20 (d) (B)
 UB4 0.12 (f) (A) 0.11 (c) (C)
 UB5 0.27 (c) (A) 0.22 (c) (C)
 UB6 0.30 (b) (A) 0.25 (b) (B)
 AB1 0.24 (d) (D) 0.21 (c) (d) (B)

Values followed by the same lower/ upper case alphabets in the
same column/ row are statistically equivalent (P<0.05) according
to the Newman-Keul's multiple range test.
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Author:Mustafa, Uzma; Kaur, Gurvinder
Publication:International Journal of Biotechnology & Biochemistry
Date:Nov 1, 2010
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