Biocontrol

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

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.

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.

* COMPETITION

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

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.

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

Trichoderma exhibiting mycoparasitism against F. oxysporum

Mycelium of Rhizoctonia solani (control)

Mycelial swellings of Rhizoctoni solani caused T.reesei

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

Mycelium of Sclerotium rolfsii (control)

Cytolysis of Sclerotium rolfsii caused by T.harzianum

Lysis of Sclerotia of Sclerotium rolfsii caused by T.reesie

*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

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.

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.

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

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.

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).

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.

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

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.

Sl.No.

SUBSTRATE Total Protein Contentmg-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

0.1750.0001(±0.00003)

0.6460.0041(±0.0013)

9.130.28(±0.095)

20.580.16(±0.053)

2. Chitin

0.0075 0.0001(±0.00003)

8.180.1252(±0.0417)

158.331.52(±0.509)

860.91.93(±0.64)

3. Chitin + Sucrose +Soil

0.10.07(±0.0033)

6.8350.0567(±0.0789)

26.970.25(±0.083)

64.560.137(±0.045)

4. Chitin + Yeast extract + soil.

0.06240.00015(±0.00003)

2.6630.015(±0.005)

3.200.16(±0.053)

63.820.53(±0.17)

5. Chitin + soil.

0.00750.0001(±0.00003)

4.060.087(±0.029)

146.52.5(±0.83)

455.62.51(±0.84)

6. Gelatin

0.1350.0001(±0.00003)

0.3000.0075(±0.0025)

11.030.075(±0.025)

Nil Activity

7. Gelatin + Glucose

0.3210.001(±0.0003)

0.1180.006(±0.002)

4.150.0037(±0.0012)

29.610.37(±0.123)

8. Chitin + Sucrose

0.1870.0003(±0.0001)

2.770.045(±0.015)

20.740.191(±0.063)

13.170.09(±0.033)

9. Yeast- extract

0.1550.00005(±0.00016)

0.5360.007(±0.0023)

11.610.12(±0.04)

55.150.125(±0.041)

10. Gelatin + Sucrose.

0.16690.0003(±0.0001)

0.3000.003(±0.001)

7.240.09(±0.03)

123.30.73(±0.24)

EFFECT OF EXTRACELLULAR HYDROLYTIC ENZYME PRODUCTION BY T.REESEI IN SYNTHETIC MEDIA AMENDED WITH DIFFERENT CARBON SOURCES

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

+C

hit

Ch

it

Ch

it+S

uc+

So

il

Ch

it + Y

E+

soil

Ch

it+ so

il

Gel

Gel +

Glu

Ch

it+ S

uc

YE

Gel +

Su

c

EN

ZY

ME

UN

ITS

β-Glucanase

Protease

Chitinase

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.

Sl.No.


SD (±SE)


CHITINASE *SD (±SE)

PROTEASE**SD (±SE)


1. CONTROLT-14

0.0670.0001(±0.00003)

NIL ACTIVITY 2.380.03(±0.001)

7.930.055(±0.078)

2. Fm +T-14 0.05 0.001(±0.00033)

0.1240.0036(±0.0012)

5.650.128(±0.042)

11.390.075(±0.025)

3. Fs + T-14 0.060.002(±0.00067)

0.1370.0025(±0.00083)

9.630.03(±0.01)

15.190.065(±0.021)

4. Rs + T-14 0.06250.001(±0.00033)

0.1650.0017(±0.0005)

5.750.035(±0.011)

32.810.091(±0.03)

5. Sr + T-14 0.08250.001(±0.00033)

0.1510.003(±0.001)

5.1530.06(±0.020)

91.180.072(±0.024)

6. Pe + T-14 0.080.001(±0.00033)

0.23360.0025(±0.0008)

4.0250.066(±0.022)

65.130.155(±0.051)

EXTRACELLULAR HYDROLYTIC ENZYME PRODUCTION BY T.REESEI

AGAINST VARIOUS PATHOGENS IN LOAMY SOIL

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

ENZY

ME

UNIT

S

Chitinase

Protease

β-Glucanase

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.

Sl.No.

SUBSTRATE

Total Protein Contentmg-1 ml-1

SD (±SE)

Specific Activity (μ moles min-1 mg-1)

CHITINASE*SD (±SE)

PROTEASE**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)

42.340.36 (±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.

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-1

4

Fs +

T -

14

Rs

+ T

- 14

Sr +

T -

14

Pe +

T -

14

1% C

hit+

T-14

1% G

el+T

-14

1% S

oy+T

-14

ENZY

ME

UN

ITS

β-Glucanase

Protease

Chitinase

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.

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)


1. Fs + T - 13 0.020.001(±0.00033)

0.6230.008(±0.0027)

4.050.07(±0.023)

45.530.29(±0.96)

2. Rs + T - 13 0.01246 0.00005(±0.00002)

0.6630.015(±0.005)

19.200.16(±0.053)

66.870.30(±0.010)

3. Sr+ T - 13 0.0050.0001(±0.000033)

0.2490.036(±0.012)

8.00.2(±0.067)

140.470.58(±0.193)

4. D+ T- 13 0.0100.001(±0.00033)

0.6040.021(±0.007)

32.00.4(±0.13)

858.8 0.76(±0.025)

5. C+ T -13 0.0050.0001(±0.000033)

0.430.01 (±0.0033)

3.971.2(±0.4)

22.750.06(±0.02)

6. Fs + T – 14 0.017460.0005(±0.000019)

1.0730.0057(±0.0019)

10.280.115(±0.038)

193.160.326(±0.108)

7. Rs + T – 14 0.0150.0002(±0.000067)

1.260.01(±0.0033)

9.38 0.08(±0.026)

129.190.25(±0.08)

8. Sr + T- 14 0.02750.0001(±0.000033)

0.6090.0036(±0.0012)

10.670.066(±0.022)

339.30.63(±0.21)

9. D+ T- 14 0.0150.001(±0.00033)

0.9720.014(±0.0046)

5.080.075(±0.025)

310.90.13(±0.045)

10. C+ T -14 0.0050.0001(±0.000033)

0.420.01(±0.0033)

40.02(±0.008)

30.350.086(±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

FIG:15. THE PRODUCTION OF EXTRACELLULAR HYDROLYTIC ENZYMES BY T.REESEI AND T.HARZIANUM IN SYNTHETIC MEDIA INCORPORATED WITH HOMOGENIZED AND KILLED CELL WALLS OF VARIOUS

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

EN

ZYM

E U

NIT

S

Chitinase

Protease

β-Glucanase

Sl.No.

SUBSTRATE Total Protein Content.mg-1 ml-1

SD(±SE)


CHITINASE*SD (±SE)

PROTEASE**SD (±SE)

1. CHITIN + GLUCOSE +T-4

0.0225 0.0001 (±0.00003)

0.4790.028 (±0.0094)

35.530.44 (±0.148)

2. CHITIN+GLUCOSE +T-7

0.0150 0.001 (±0.00033)

2.7430.051 (±0.017)

53.30.7 (±0.23)


0.0190.001 (±0.00033)

1.0390.05 (±0.0168)

10.00.5 (±0.16)


0.0250.001 (±0.00033)

2.080.04 (±0.013)

20.00.4 (±0.133)


ND ND ND


ND ND ND

7. CHITIN+T-4 0.0250.001 (±0.00033)

3.330.04 (±0.0133)

64.0 0.4 (±0.133)

8. CHITIN+T-7 0.0300.001 (± 0.00033)

3.510.111 (±0.0371)

98.31.7 (±0.57)

9. CHITIN+T-10 0.02750.0001 (±0.000033)

2.260.026 (±0.0088)

18.170.36 (±0.12)

10. CHITIN+T-11 0.02250.0001 (±0.00003)

5.100.070 (±0.023)

124.260.46 (±0.153)

11. CHITIN+T-13 0.0050.0001 (±0.000033)

43.810.315 (±0.105)

220.02.0 (±0.67)

12. CHITIN + T-14 0.00750.0001 (±0.00003)

22.210.14 (±0.046)

158.51.50 (±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.

FIG:17. SCREENING FOR EXTRACELLULAR CHITINASE AND PROTEASE PRODUCTION BY DIFFERENT STRAINS OF TRICHODERMA SP. IN SYNTHETIC MEDIA

0

50

100

150

200

250

300

CH

IT+G

LU

+T

-4

CH

IT+G

LU

+T

-7

CH

IT+G

LU

+T

-10

CH

IT+G

LU

+T

-11

CH

IT+T

-4

CH

IT+T

-7

CH

IT+T

-10

CH

IT+T

-11

CH

IT+T

-13

CH

IT+T

-14

EN

ZY

ME

UN

ITS

Protease

Chitinase

Sl.No.


SD (±SE)


CHITINASE *SD (±SE)

PROTEASE**SD (±SE)


1. EPM + T-13 0.01250.0001(±0.00003)

2.150.009(±0.003)

250.2(±0.067)

NIL ACTIVITY

2. EPM + T-14 0.0225 0.001(±0.00033)

2.1670.004(±0.0015)

56.640.67(±0.22)

60.790.165(±0.055)

HYDROLYTIC ENZYME PRODUCTION BY T.REESEI AND T.HARZIANUM IN ENZYME PRODUCTION MEDIA [EPM].

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

EN

ZY

ME

UN

ITS

EPM+T-13

EPM+T-14

Sl.No.

SUBSTRATE

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

0.040.001 (±0.00033)

0.6240.005 (±0.0018)

25.160.208 (±0.069)

257.360.27 (±0.90)

2. Fm v/s T - 13

0.0525 0.0001 (±0.00003)

0.4360.04 (±0.0013)

21.010.145 (±0.048)

82.50.14 (±0.048)

3. Rs v/s T - 13

0.05750.001 (±0.0003)

1.290.01 (±0.0033)

31.270.134 (±0.045)

100.450.13 (±0.043)

4. Sr v/sT- 13

0.0550.001 (±0.00033)

1.5430.0057 (±0.0019)

24.310.55 (±0.103)

95.38 0.14(±0.048)

5. Pe v/s T -13

0.04750.001(±0.00033)

0.480.014 (±0.005)

24.040.04 (±0.013)

145.620.122 (±0.040)

6. Fs v/s T – 14

0.08750.00005(±0 .000019)

0.5710.002 (±0.00067)

11.690.036 (±0.012)

136.30.125 (±0.041)

7. Fm v/s T –14 0.1520.001 (±0.00033)

0.7490.0011 (±0.00038)

7.75 0.023 (±0.076)

40.870.036 (±0.012)

8. Rs v/s T – 14

0.1400.001 (±0.00033)

0.7260.011(±0.0037)

7.980.02 (±0.0067)

55.370.27 (±0.9)

9. Sr v/s T- 14 0.1250.0001 (±0.00003)

0.9790.004 (±0.0013)

11.030.015 (±0.050)

55.570.23 (±0.076)

10. Pe v/s T -14

0.1600.001 (±0.00033)

0.7670.0015(±0.0005)

8.520.02 (±0.0067)

50.630.06 (±0.02)

THE PRODUCTION OF EXTRACELLULAR HYDROLYTIC ENZYMES BY T.REESEI AND T.HARZIANUM AGAINST VARIOUS PATHOGENS IN POTATO

DEXTROSE BROTH.

FIG:14. EXTRACELLULAR HYDROLYTIC ENZYME PRODUCTION BY T.REESEI AND T.HARZIANUM AGAINST VARIOUS PATHOGENS IN POTATO DEXTROSE BROTH

0

50

100

150

200

250

300

Fs v/sT-13

Fm v/sT-13

Rs v/sT-13

Sr v/sT-13

Pe v/sT-13

Fs v/sT-14

Fm v/sT-14

Rs v/sT-14

Sr v/s T-14

Pe v/sT-14

EN

ZYM

E U

NIT

S Chitinase

Protease

β-Glucanase

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.

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.

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.

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




SAMPLING OF TRICHODERMA FORMULATED BIOCONTROL AGENTS

( T.reesei and T.harzianum) AFTER SIX MONTHS OF STORAGE. STORED AT 4oC

STORED AT ROOM TEMPERATURE (28oC±3).

SAMPLING OF TRICHODERMA FORMULATED BIOCONTROL AGENT ( T.reesei and T.harzianum) AFTER THREE MONTHS OF

STORAGE. STORED AT 37oC

Sl.No. TRICHODERMA FORMULATED BIOCONTROL AGENT

C.F.U. gm-1


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





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





SAMPLING OF TRICHODERMA FORMULATED BIOCONTROL AGENTS ( T.reesei and T.harzianum) AFTER ONE YEAR OF STORAGE.

STORED AT 4oC

STORED AT ROOM TEMPERATURE (28oC±3).

Results

*The optimum temperature for storage is 4oC, in which case the formulating agent has a shelf life of one year.

*At 28oC ±3, the formulating agent can be stored for a period of six months.

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.

Thanks

Dr. Bharathi RPost Doctoral Fellow

National Centre for Biological Sciences

Bangalore

Biocontrol

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