The document discusses a study on the effect of different carbon sources on the production of hydrolytic enzymes in Trichoderma sp strains T13 and T14 and their ability to act as mycoparasites. Key findings include: - Chitin was found to induce maximum production of chitinase, protease and beta-glucanase enzymes. - T. reesei was able to produce hydrolytic enzymes like chitinase, protease and beta-glucanase when grown in soil in the presence of various fungal pathogens like Fusarium moniliforme, Fusarium solani, Rhizoctonia solani, Sclerotium rolfsii

1. Effect of different carbon sources on production
of
hydrolytic enzymes in Trichoderma sp strain T13
and T14 with reference to mycoparasitism.
Prof. MANJUNATH K.
DEPARMENT OF MICROBIOLOGY
AND BIOTECHNOLOGY, BANGALORE.
By

2. INTRODUCTION
In 1932 Weindling discovered that Trichoderma lignorum
parasitized a number of soil borne fungi in culture and thus
suggested controlling certain pathogenic fungi by
augmenting soil with an abundance of this mycoparasite.
Biocontrol is a process where, a living antagonistic
micro-organism is used to control a harmful pathogenic
micro-organism causing plant diseases in a specific
ecological niche without any ecological imbalance.

3. MECHANISMS INVOLVED IN BIOCONTROL
•The mechanisms involved in biological control can be of three
types, parasitism or lysis, antibiosis and competition.
ANTIBIOSIS
•Is the inhibition or destruction of one microorganism by a
metabolic product of another. Various Trichoderma strains
possess the ability to excrete toxic or inhibitory metabolites
•Weindling and Emerson 1936 isolated a toxic substance from
the culture filtrate of Trichoderma, which has inhibitory effect
on Rhizoctonia solani and other soil fungi.

4. * COMPETITION
It is a mechanism, where two or more organisms compete for
the limited amount of substrate space and oxygen available.

5. MYCOPARASITISM
Is defined as a direct attack of a parasite on a fungal
thallus. Mycoparasitism is a complex process, which
includes
(i) Chemotropic growth of the mycoparasite.
(ii) Recognition of pathogen by the mycoparasite.
(iii) Excretion of extra-cellular enzymes.
(iv) Lysis of the host.
Sivan and Chet (1989) studied the degradation of fungal
cell walls by lytic enzymes of Trichoderma harzianum.

6. Different strains of Trichoderma spp. exhibiting
mycoparasitism against F. solani

7. Trichoderma exhibiting mycoparasitism against F. oxysporum

8. Mycelium of Rhizoctonia solani (control)

9. Mycelial swellings of Rhizoctoni solani caused T.reesei

10. Rupturing of the mycelium of Rhizoctonia solani caused by
T. harzianum

11. Mycelium of Sclerotium rolfsii (control)

12. Cytolysis of Sclerotium rolfsii caused by T.harzianum

13. Lysis of Sclerotia of Sclerotium rolfsii caused by T.reesie

14. *Physically it is a fabric of interwoven microfibrils
embedded in an amorphorous matrix.
*Chitin, cellulosic and non-cellulosic polysaccharides are
microfibrillar components or skeletal components of the
wall
*while proteins and other short chained polysaccharides
like galactan, mannan, constitute the matrix
HYDROLYTIC ENZYME OF TRICHODERMA SPP.
INVOLVED IN MYCOPARASITISM
*Rigid walls enclose fungal cells. The cell wall protects
cells from the hazards of the environment

15. Chitin is an unbranched homopolymer of 1, 4-β-linked N-
acety-D-glucosamine (GlcNAc) a structural polymer in
most fungi and insects, including those that are
agricultural pests
Chitinases are chitin degrading enzymes and they play a
vital role in biological control.
T.harzianum attacks pathogens by excreting lytic
enzymes, including glucanases, chitinases, proteases and
lipases, which enable it to degrade host cell walls and thus
reduce disease incidence
CHITINASE.

16. Chitinolytic enzymes are divided into three principal categories.
(i) 1, 4-β-N-acetyl glucosaminidase, which split the chitin
polymer into GlcNAc monomers in an exo-type fashion.
(ii) Endochitinases, which cleave randomly at internal sites
over the entire length of the chitin microfibril.
Exochitinases, which catalyse the progressive release of
diacetylchitobiose in a stepwise fashion such that no
monosaccharides or oligosaccharides are formed.

17. β -GLUCANASE
*β-Glucans are homopolymers of D-glucose linked in a β-
configuration.
*The primary role of cell wall β-glucans in fungi is as structural
polymers, maintaining rigidity and conferring protection.
*Exo-β-glucanases hydrolyse the β-glucan chain by sequentially
cleaving glucose residues from the non-reducing end.
Consequently, the sole hydrolysis product is a monomer, usually
glucose.
*Endo-β-glucanases cleave β-linkages at random sites along the
polysaccharide chain, releasing smaller oligosaccharides

18. A number of Trichoderma isolates secrete 1,3 β-glucanases.
Along with chitinases and proteases they are involved in the
cell lysis of several phytopathogenic fungi during the
mycoparasitic process.
Since chitin and β-glucan are embedded in a matrix of
amorphous material, successful cell-wall degradation may
depend on the activity of more than one enzyme.

19. PROTEASES
Besides chitin and glucan, the skeleton of filamentous
fungal cell walls contains lipids and proteins (Hunsley and
Burnett, 1970).
Fungal proteases may therefore play a significant role in
the cell-wall lysis that occurs during pathogen–host
interactions (Haran et al., 1996).

20. THE OBJECTIVES OF THE PRESENT RESEARCH
*Isolation of soil-borne plant pathogens from diseased forest
nursery seedlings
*Screening of different soil samples for the isolation of
antagonistic fungi
*Study the antagonistic effect of selected strains of
Trichoderma sp. against the various soil-borne fungal
pathogens isolated from diseased nursery seedlings in vitro
and in vivo conditions.
* To determine the presence of different hydrolytic enzymes
involved in biocontrol of various pathogens.

21. ISOLATION OF PATHOGENS FROM VARIOUS FOREST NURSERY SEEDLINGS
Sl. No. PATHOGENS HOST DISEASE SYMPTOMS
1. (a) Fusarium solani Tectona grandis (teak) Root rot
(b) Fusarium solani Tamarindus indica (Tamarind) Collar rot
(c) Fusarium solani Michelia champaca (Champaca) Damping-off
(d) Fusarium solani Calamus rotang (Calamus) Damping-off
(e) Fusarium solani Phyllanthus embilica (Goose berry) Collar rot
(f) Fusarium solani Areca catecheu (Arecanut) Collar rot
2. (a) Fusarium moniliforme Michelia champaca (Champaca) Damping-off
(b) Fusarium moniliforme Grevillea robusta (Silver oak) Wilting
3. Fusarium oxysporium Persea Americana (Avocado) Root rot
4 (a) Rhizoctonia solani Eucalyptus grandis (Eucalyptus) Damping-off
(b) Rhizoctonia solani Swietenia macropylla Collar rot
5. Sclerotium rolfsii Eucalyptus grandis (Eucalyptus) Collar rot
6. Pestalotia sp. Tectona grandis (Teak) Leaf spot
7. Drechslera sp. Areca catecheu (Arecanut) Damping-off

22. Sl.No. ISOLATION SITE HOST FUNGAL ANTAGONISTS
TRICHODERMA SPECIES
STRAIN No.
1. Forest nursery, Kerala Forest Research
Institute, Peechi, Kerala.
Allenquium salvolum 1
2. Western Ghats region [Dakshina Karnataka ] Areca catecheu
(Arecanut)
4
3. Forest nurseries,
Plantations and
Dhanvanthrivana, Bangalore.
Syzygium jambolanum
(Jaman)
2
Musa (Banana) 3
Persea americana
(Avocado)
5
Psidium guajava (Guava) 6
Phyllanthus emblica
(Goose berry)
7
4. Mysore, Mandya and
Maddur (Nuseries and
Cultivated soil).
Zea may (Maize) 8and 9
Saccharum officinarum
(Sugarcane)
10 and 11
Mangifera indica
(Mango)
12 and 14
Tamarindus indica
(Tamarind)
13
Euphorbia geniculata
(Euphorbia)
15
LIST OF FUNGAL ANTAGONISTS ISOLATED FROM RHIZOSPHERE AND
RHIZOPLANE REGION OF VARIOUS NURSERY SEEDLINGS AND
CULTIVATED SOIL.

23. Sl.
No.
SUBSTRATE Total Protein
Content
mg-1
ml-1
SD (±SE)
Specific Activity (μmoles min-1
mg-1
)
CHITINASE
*
SD (±SE)
PROTEASE**
SD (±SE)
β-GLUCANASE
***
SD (±SE)
1. Glucose +Chitin 0.175
0.0001
(±0.00003)
0.646
0.0041
(±0.0013)
9.13
0.28
(±0.095)
20.58
0.16
(±0.053)
2. Chitin 0.0075
0.0001
(±0.00003)
8.18
0.1252
(±0.0417)
158.33
1.52
(±0.509)
860.9
1.93
(±0.64)
3. Chitin + Sucrose +Soil 0.1
0.07
(±0.0033)
6.835
0.0567
(±0.0789)
26.97
0.25
(±0.083)
64.56
0.137
(±0.045)
4. Chitin + Yeast extract
+ soil.
0.0624
0.00015
(±0.00003)
2.663
0.015
(±0.005)
3.20
0.16
(±0.053)
63.82
0.53
(±0.17)
5. Chitin + soil. 0.0075
0.0001
(±0.00003)
4.06
0.087
(±0.029)
146.5
2.5
(±0.83)
455.6
2.51
(±0.84)
6. Gelatin 0.135
0.0001
(±0.00003)
0.300
0.0075
(±0.0025)
11.03
0.075
(±0.025)
Nil Activity
7. Gelatin + Glucose 0.321
0.001
(±0.0003)
0.118
0.006
(±0.002)
4.15
0.0037
(±0.0012)
29.61
0.37
(±0.123)
8. Chitin + Sucrose 0.187
0.0003
(±0.0001)
2.77
0.045
(±0.015)
20.74
0.191
(±0.063)
13.17
0.09
(±0.033)
9. Yeast- extract 0.155
0.00005
(±0.00016)
0.536
0.007
(±0.0023)
11.61
0.12
(±0.04)
55.15
0.125
(±0.041)
10. Gelatin + Sucrose. 0.1669
0.0003
(±0.0001)
0.300
0.003
(±0.001)
7.24
0.09
(±0.03)
123.3
0.73
(±0.24)
EFFECT OF EXTRACELLULAR HYDROLYTIC ENZYME PRODUCTION BY T.REESEI IN
SYNTHETIC MEDIA AMENDED WITH DIFFERENT CARBON SOURCES

24. FIG: 10. EFFECT OF EXTRACELLULAR HYDROLYTIC ENZYME PRODUCTION BY T.REESEI IN
SYNTHETIC MEDIA AMENDED WITH DIFFERENT CARBON SOURCE
0
200
400
600
800
1000
1200
Glu+Chit
Chit
Chit+Suc+Soil
Chit+YE
+soil
Chit+soil
Gel
Gel+Glu
Chit+Suc
YE
Gel+Suc
ENZYMEUNITS
β-Glucanase
Protease
Chitinase

25. RESULTS
*The maximum amount of total protein was produced in
synthetic media supplemented with gelatin +glucose with
0.321mg-1
of protein.
* The maximum amount of all the three hydrolytic enzymes i.e.,
chitinases, proteases, β -glucanase activity was produced in
synthetic media with 1% chitin
*The maximum chitinase activity of 8.18 μmoles min-1
mg-1
,
maximum protease of 158.33 μmoles min-1
mg-1
and maximum β
-glucanase of 860 μmoles min-1
mg-1
.

26. Sl.
No.
SUBSTRATE Total Protein
Content
mg-1
ml-1
SD (±SE)
Specific Activity (μmoles min-1
mg-1
)
CHITINASE
*
SD (±SE)
PROTEASE
**
SD (±SE)
β-GLUCANASE
***
SD (±SE)
1. CONTROL
T-14
0.067
0.0001
(±0.00003)
NIL ACTIVITY 2.38
0.03
(±0.001)
7.93
0.055
(±0.078)
2. Fm +T-14 0.05
0.001
(±0.00033)
0.124
0.0036
(±0.0012)
5.65
0.128
(±0.042)
11.39
0.075
(±0.025)
3. Fs + T-14 0.06
0.002
(±0.00067)
0.137
0.0025
(±0.00083)
9.63
0.03
(±0.01)
15.19
0.065
(±0.021)
4. Rs + T-14 0.0625
0.001
(±0.00033)
0.165
0.0017
(±0.0005)
5.75
0.035
(±0.011)
32.81
0.091
(±0.03)
5. Sr + T-14 0.0825
0.001
(±0.00033)
0.151
0.003
(±0.001)
5.153
0.06
(±0.020)
91.18
0.072
(±0.024)
6. Pe + T-14 0.08
0.001
(±0.00033)
0.2336
0.0025
(±0.0008)
4.025
0.066
(±0.022)
65.13
0.155
(±0.051)
EXTRACELLULAR HYDROLYTIC ENZYME PRODUCTION BY T.REESEI
AGAINST VARIOUS PATHOGENS IN LOAMY SOIL

27. FIG: 11. EXTRACELLULAR HYDROLYTIC ENZYME PRODUCTION BY
T.REESEI AGAINST VARIOUS PATHOGENS IN LOAMY SOIL
0
10
20
30
40
50
60
70
80
90
100
C.T-14
Fm+T-14
Fs+T-14
Rs+T-14
Sr+T-14
Pe+T-14
ENZYMEUNITS
Chitinase
Protease
β-Glucanase

28. RESULTS
*T.reesei produces good amount of all three hydrolytic
enzymes in soil conditions, against various pathogens,
particularly in the case of S.rolfsii, R.solani and Pestalotia
species.
*These results substantiate the fact that they are good
biocontrol agents in both in vitro and in vivo conditions,
where they suppress the disease incidence completely.

29. Sl.
No
.
SUBSTRAT
E
Total Protein
Content
mg-1
ml-1
SD (±SE)
Specific Activity (μ moles min-1
mg-1
)
CHITINASE
*
SD (±SE)
PROTEASE**
SD (±SE)
β-GLUCANASE
***
SD (±SE)
1. CONTROL
+ T-14
0.0825 0.001(±0.0033) Nil activity 30.54
0.24(±0.080)
68.03
0.25 (±0.085)
2. Fm + T-14 0.0725
0.0043(±0.0014)
0.309
0.0085(±0.0028)
51.25
0.295 (±0.098)
94.64
0.38 (±0.127)
3. Fs + T - 14 0.070
0.001(±0.00033)
0.357
0.003(±0.001)
54.56
0.285 (±0.095)
96.11
0.21 (±0.07)
4. Rs + T - 14 0.0697
0.0037 (±0.00012)
0.4
0.003(±0.001)
77.91
0.29(±0.0967)
135.06
0.55 (±0.183)
5. Sr + T - 14 0.0797
0.00046 (±0.00015)
0.286
0.003(±0.001)
54.25
0.25 (±0.84)
73.51
0.121 (±0.04)
6. Pe + T - 14 0.075
0.001 (±0.00033)
0.28
0.003 (±0.001)
54.79
0.135 (±0.045)
61.33
0.157 (±0.052)
7. 1% Chitin +
T- 14
0.525
0.001 (±0.00033)
1.5067
0.0035(±0.0011)
56.06
0.294 (±0.098)
42.34
0.36 (±0.12 )
8. 1% gelatin +
T - 14
0.1125
0.001 (±0.00033)
0.1867
0.0015(±0.0005)
31.64
0.18 (±0.06)
18.26
0.259 (±0.086)
9. 1%
Soyatone
0.125
0.001 (±0.00033)
Nil activity 56.96
0.16(±0.053)
Nil activity
EXTRACELLULAR HYDROLYTIC ENZYME PRODUCED BY T.REESEI IN POTATO DEXTROSE
BROTH INCORPORATED WITH HOMOGENIZED AND KILLED CELL WALLS OF VARIOUS
PATHOGENS OR SUPPLEMENTED WITH NUTRIENT SOURCE SUCH AS 1% GELATIN, 1%
SOYATONE OR 1% CHITIN.

30. FIG:12. EXTRACELLULAR HYDROLYTIC ENZYME PRODUCED BY T.REESEI IN POTATO DEXTROSE
BROTH INCORPORATED WITH HOMOGENISED CELL WALLS OF VARIOUS PATHOGENS OR
1%CHITIN 1% GELATIN OR 1% SOYATONE
0
50
100
150
200
250
C.T-14
Fm+T-14
Fs+T-14
Rs+T-14
Sr+T-14
Pe+T-14
1%Chit+T-
14
1%Gel+T-
14
1%Soy+T-14
ENZYMEUNITS
β-Glucanase
Protease
Chitinase

31. RESULTS
*The results indicate that though Potato Dextrose Broth
(PDB) is very rich in nutrient source, the presence of cell
walls of pathogens and chitin induces the production of
chitinases whereas the control PDB + T.reesei did not
produce any chitinase.
*The maximum amount of chitinase was produced in PDB +
1% chitin. T.reesei also produces all the three hydrolytic
enzymes against the killed cell walls of various pathogens in
PDB.
*This suggests that the very presence of the pathogen cell
wall or chitin acts as an inducer for chitinase production,
whereas the control containing only T.reesei did not produce
any chitinase activity.

32. Sl.
No.
SUBSTRATE Total Protein
Content
mg-1
ml-1
SD (±SE)
Specific Activity (μ moles min-1
mg-1
)
CHITINASE
*
SD (±SE)
PROTEASE**
SD (±SE)
β-GLUCANASE
***
SD (±SE)
1. Fs + T - 13 0.02
0.001
(±0.00033)
0.623
0.008
(±0.0027)
4.05
0.07
(±0.023)
45.53
0.29
(±0.96)
2. Rs + T - 13 0.01246
0.00005
(±0.00002)
0.663
0.015
(±0.005)
19.20
0.16
(±0.053)
66.87
0.30
(±0.010)
3. Sr+ T - 13 0.005
0.0001
(±0.000033)
0.249
0.036
(±0.012)
8.0
0.2
(±0.067)
140.47
0.58
(±0.193)
4. D+ T- 13 0.010
0.001
(±0.00033)
0.604
0.021
(±0.007)
32.0
0.4
(±0.13)
858.8
0.76
(±0.025)
5. C+ T -13 0.005
0.0001
(±0.000033)
0.43
0.01
(±0.0033)
3.97
1.2
(±0.4)
22.75
0.06
(±0.02)
6. Fs + T – 14 0.01746
0.0005
(±0.000019)
1.073
0.0057
(±0.0019)
10.28
0.115
(±0.038)
193.16
0.326
(±0.108)
7. Rs + T – 14 0.015
0.0002
(±0.000067)
1.26
0.01
(±0.0033)
9.38
0.08
(±0.026)
129.19
0.25
(±0.08)
8. Sr + T- 14 0.0275
0.0001
(±0.000033)
0.609
0.0036
(±0.0012)
10.67
0.066
(±0.022)
339.3
0.63
(±0.21)
9. D+ T- 14 0.015
0.001
(±0.00033)
0.972
0.014
(±0.0046)
5.08
0.075
(±0.025)
310.9
0.13
(±0.045)
10. C+ T -14 0.005
0.0001
(±0.000033)
0.42
0.01
(±0.0033)
4
0.02
(±0.008)
30.35
0.086
(±0.028)
EXTRACELLULAR HYDROLYTIC ENZYMES PRODUCED BY T.REESEI AND T.HARZIANUM
IN SYNTHETIC MEDIA INCORPORATED WITH HOMOGENIZED AND KILLED CELL
WALLS OF VARIOUS PATHOGENS

33. FIG:15. THEPRODUCTIONOFEXTRACELLULAR HYDROLYTIC ENZYMES BYT.REESEI AND T.HARZIANUM IN
SYNTHETIC MEDIA INCORPORATED WITHHOMOGENIZED AND KILLED CELL WALLS OFVARIOUS
PATHOGENS
0
100
200
300
400
500
600
700
800
900
1000
Fs+T-13
Rs+T-13
Sr+T-13
D+T-13
C+T-13
Fs+T-14
Rs+T-14
Sr+T-14
D+T-14
C+T-14
ENZYMEUNITS
Chitinase
Protease
β-Glucanase

34. Sl.
No.
SUBSTRATE Total Protein
Content
.mg-1
ml-1
SD(±SE)
Specific Activity
(μmoles min-1
mg-1
)
CHITINASE*
SD (±SE)
PROTEASE**
SD (±SE)
1. CHITIN +
GLUCOSE +T-4
0.0225
0.0001 (±0.00003)
0.479
0.028 (±0.0094)
35.53
0.44 (±0.148)
2. CHITIN+
GLUCOSE +T-7
0.0150
0.001 (±0.00033)
2.743
0.051 (±0.017)
53.3
0.7 (±0.23)
3. CHITIN+
GLUCOSE +T-10
0.019
0.001 (±0.00033)
1.039
0.05 (±0.0168)
10.0
0.5 (±0.16)
4. CHITIN+
GLUCOSE +T-11
0.025
0.001 (±0.00033)
2.08
0.04 (±0.013)
20.0
0.4 (±0.133)
5. CHITIN+
GLUCOSE +T-13
ND ND ND
6. CHITIN+
GLUCOSE +T-14
ND ND ND
7. CHITIN+T-4 0.025
0.001 (±0.00033)
3.33
0.04 (±0.0133)
64.0
0.4 (±0.133)
8. CHITIN+T-7 0.030
0.001 (± 0.00033)
3.51
0.111 (±0.0371)
98.3
1.7 (±0.57)
9. CHITIN+T-10 0.0275
0.0001 (±0.000033)
2.26
0.026 (±0.0088)
18.17
0.36 (±0.12)
10. CHITIN+T-11 0.0225
0.0001 (±0.00003)
5.10
0.070 (±0.023)
124.26
0.46 (±0.153)
11. CHITIN+T-13 0.005
0.0001 (±0.000033)
43.81
0.315 (±0.105)
220.0
2.0 (±0.67)
12. CHITIN + T-14 0.0075
0.0001 (±0.00003)
22.21
0.14 (±0.046)
158.5
1.50 (±0.50)
SCREENING OF EXTRACELLULAR CHITINASE AND PROTEASE PRODUCTION
BY DIFFERENT STRAINS OF TRICHODERMA SP. IN SYNTHETIC MEDIA
SUPPLEMENTED WITH 0.5%CHITIN AND 0.5%GLUCOSE OR 1% CHITIN.

35. FIG:17. SCREENING FOR EXTRACELLULAR CHITINASE AND PROTEASE PRODUCTION BY DIFFERENT
STRAINS OFTRICHODERMA SP. IN SYNTHETIC MEDIA
0
50
100
150
200
250
300
CHIT+GLU+T-4
CHIT+GLU+T-7
CHIT+GLU+T-10
CHIT+GLU+T-11
CHIT+T-4
CHIT+T-7
CHIT+T-10
CHIT+T-11
CHIT+T-13
CHIT+T-14
ENZYMEUNITS
Protease
Chitinase

36. Sl.
No.
SUBSTRATE Total
Protein
Content
mg-1
ml-1
SD (±SE)
Specific Activity (μmoles min-1
mg-1
)
CHITINASE
*
SD (±SE)
PROTEASE
**
SD (±SE)
β-GLUCANASE
***
SD (±SE)
1. EPM + T-13 0.0125
0.0001
(±0.00003)
2.15
0.009
(±0.003)
25
0.2
(±0.067)
NIL ACTIVITY
2. EPM + T-14 0.0225
0.001
(±0.00033)
2.167
0.004
(±0.0015)
56.64
0.67
(±0.22)
60.79
0.165
(±0.055)
HYDROLYTIC ENZYME PRODUCTION BY T.REESEI AND
T.HARZIANUM IN ENZYME PRODUCTION MEDIA [EPM].

37. FIG:13. EXTRACELLULAR HYDROLYTIC ENZYME PRODUCTION BY T.REESEI
(T-14)AND T.HARZIANUM(T-13) IN ENZYME PRODUCTION MEDIA
0
10
20
30
40
50
60
70
1 2 3
CHITINASE PROTEASE β-GLUCANASE
ENZYMEUNITS
EPM+T-13
EPM+T-14

38. Sl.
No.
SUBSTRAT
E
Total Protein
Content
.mg-1
ml-1
SD(±SE)
Specific Activity (u moles min-1
mg-1
)
CHITINASE
* SD (±SE)
PROTEASE**
SD (±SE)
β-GLUCANASE
*** SD (±SE)
1. Fs v/s
T - 13
0.04
0.001 (±0.00033)
0.624
0.005 (±0.0018)
25.16
0.208 (±0.069)
257.36
0.27 (±0.90)
2. Fm v/s T -
13
0.0525
0.0001 (±0.00003)
0.436
0.04 (±0.0013)
21.01
0.145 (±0.048)
82.5
0.14 (±0.048)
3. Rs v/s
T - 13
0.0575
0.001 (±0.0003)
1.29
0.01 (±0.0033)
31.27
0.134 (±0.045)
100.45
0.13 (±0.043)
4. Sr v/s
T- 13
0.055
0.001 (±0.00033)
1.543
0.0057 (±0.0019)
24.31
0.55 (±0.103)
95.38
0.14(±0.048)
5. Pe v/s
T -13
0.0475
0.001(±0.00033)
0.48
0.014 (±0.005)
24.04
0.04 (±0.013)
145.62
0.122 (±0.040)
6. Fs v/s
T – 14
0.0875
0.00005(±0 .000019)
0.571
0.002 (±0.00067)
11.69
0.036 (±0.012)
136.3
0.125 (±0.041)
7. Fm v/s T –14 0.152
0.001 (±0.00033)
0.749
0.0011 (±0.00038)
7.75
0.023 (±0.076)
40.87
0.036 (±0.012)
8. Rs v/s
T – 14
0.140
0.001 (±0.00033)
0.726
0.011(±0.0037)
7.98
0.02 (±0.0067)
55.37
0.27 (±0.9)
9. Sr v/s T- 14 0.125
0.0001 (±0.00003)
0.979
0.004 (±0.0013)
11.03
0.015 (±0.050)
55.57
0.23 (±0.076)
10. Pe v/s
T -14
0.160
0.001 (±0.00033)
0.767
0.0015(±0.0005)
8.52
0.02 (±0.0067)
50.63
0.06 (±0.02)
THE PRODUCTION OF EXTRACELLULAR HYDROLYTIC ENZYMES BY
T.REESEI AND T.HARZIANUM AGAINST VARIOUS PATHOGENS IN POTATO
DEXTROSE BROTH.

39. FIG:14. EXTRACELLULAR HYDROLYTIC ENZYMEPRODUCTION BY T.REESEI AND
T.HARZIANUM AGAINST VARIOUS PATHOGENS IN POTATO DEXTROSEBROTH
0
50
100
150
200
250
300
Fsv/sT-13
Fmv/sT-
13
Rsv/sT-13
Srv/sT-13
Pev/sT-13
Fsv/sT-14
Fmv/sT-
14
Rsv/sT-14
Srv/sT-14
Pev/sT-14
ENZYMEUNITS
Chitinase
Protease
β-Glucanase

40. RESULTS
*The results indicated that though Potato Dextrose Broth is a
rich medium, chitinases, protease and β-glucanases were
produced by T.harzianum and T. reesei against the various
pathogens in varying amounts.
*T.harzianum produces comparatively higher amounts of all
the three hydrolytic enzymes than T.reesei.
* In the present study mycoparasitism was observed when
Trichoderma spp.and the Test pathogen was grown in dual
culture on PDA plates as well as in PD Broth, where
T.harzianum produced hydrolytic enzymes against various
pathogens in potato dextrose broth culture.

41. CONCLUSIONS
* The enzyme studies prove that T.reesei and T.harzianum
produce good amount of all the three hydrolytic enzymes i.e.,
Chitinases, Proteases and β-Glucanases.
* The synergistic action of these enzymes is required for
successful mycoparasitic action against the various pathogens
and thus can be an effective biocontrol agent.
* Thus these two Trichoderma spp. Trichoderma strain no.
T-13 T.harzianum and strain no. T-14 T.reesei can be
considered for biological control of various pathogens causing
diseases in forest nursery seedlings.

42. THE PREPARATION AND STUDY OF DIFFERENT
TRICHODERMA FORMULATIONS.
*The development of an effective, stable and cost effective
formulation that is compatible with agricultural, agro-technical,
forestry technology and disease management is critical for the
successful commercialization of the antagonist
*The two species, Trichoderma harzianum Rifia and Trichoderma
reesei E.Simmons were selected for preparing various formulations
such as Talc, Wheat bran, Sawdust and a combination of these
formulating agents.

43. Sl.No TRICHODERMA FORMULATED BIOCONTROL
AGENTS
C.F.U. gm-1
1. Sawdust 14 x 107
2. Talc 10 x107
3. Wheat bran. 15 x 107
4. Talc + Sawdust 20 x 107
5. Talc + Wheat bran 18 x 107
6. Talc + Wheat bran + Sawdust 8 x 107
7. Press mud 10 x 107
Sl.No. TRICHODERMA FORMULATED BIOCONTROL AGENTS. C.F.U. gm-1
1. Sawdust 20 x 107
2. Talc 10 x 107
3. Wheat bran 4 x 107
4. Talc + Sawdust 16 x107
5. Talc + Wheat bran 12 x 107
6. Talc + Wheat bran + Sawdust 2 x 107
7. Press mud 14 x 107
SAMPLING OF TRICHODERMA FORMULATED BIOCONTROL AGENTS
( T.reesei and T.harzianum) AFTER SIX MONTHS OF STORAGE.
STORED AT 4o
C
STORED AT ROOM TEMPERATURE (28o
C±3).

44. SAMPLING OF TRICHODERMA FORMULATED BIOCONTROL
AGENT ( T.reesei and T.harzianum) AFTER THREE MONTHS OF
STORAGE. STORED AT 37o
C
Sl.No. TRICHODERMA FORMULATED
BIOCONTROL AGENT
C.F.U. gm-1
1. Talc + Wheat bran 2 x 104

45. Sl.No TRICHODERMA FORMULATED BIOCONTROL AGENTS C.F.U. gm-1
1. Sawdust 2 x 107
2. Talc 9 x 107
3. Wheat bran. 14 x 107
4. Talc + Sawdust 12 x 107
5. Talc + Wheat bran 11 x 107
6. Talc + Wheat bran + Sawdust 8 x 107
7. Press mud 2 x 107
Sl.No. TRICHODERMA FORMULATED BIOCONTROL AGENTS. C.F.U. gm-1
1. Sawdust 4 x 107
2. Talc 2 x 107
3. Wheat bran 2 x 107
4. Talc + Sawdust 4 x 107
5. Talc + Wheat bran 2 x 107
6. Talc + Wheat bran + Sawdust 4 x 107
7. Press mud 6 x 107
SAMPLING OF TRICHODERMA FORMULATED BIOCONTROL
AGENTS ( T.reesei and T.harzianum) AFTER ONE YEAR OF STORAGE.
STORED AT 4o
C
STORED AT ROOM TEMPERATURE (28o
C±3).

46. Results
*The optimum temperature for storage is 4o
C, in which
case the formulating agent has a shelf life of one year.
*At 28o
C ±3, the formulating agent can be stored for a
period of six months.

47. CONCLUSION
*The enzyme studies carried out reveal the production of all three
hydrolytic enzymes by both T.harzianum and T.reesei in varying
amounts in different media used.
*These hydrolytic enzymes play a very important role in biological
control of various pathogens and thus suppress the disease
incidence.
*The two Trichoderma spp. i.e. T.harzianum and T.reesei were
found to be potential bio-control agents in vitro and in vivo
conditions.
*They were chosen for preparation of formulation using various
substrates and carrier materials.

48. Thanks
Dr. Bharathi R
Post Doctoral Fellow
National Centre for Biological
Sciences
Bangalore