-By- AMOL VIJAY SHITOLE

1. 1

3. Seminar presentation
on
“Significant Achievements and
Present Status of Trichoderma spp.
in
Biocontrol of Plant Diseases”
3

4. Chairman Dr. R. M. Gade
Member Dr. R. W. Ingle
Member Dr. A. M. Charpe
Member Dr. R. N. Katkar
DEPARTMENT OF PLANT PATHOLOGY,
POST GRADUATE INSTITUTE ,
DR.P.D.K.V,AKOLA.
Advisory Committee
4

5. Contents
Introduction
Mechanism of Trichoderma
Type of mutation & mutagen
Reviews
Impact of mutation on morphological
characters
Impact of mutation on biochemical
changes
Impact of mutation on mycoparasitism
5

6. INTRODUCTION

7. Position Asexual stage
(Conidia)
Sexual
stage
(Ascospore)
Kingdom Fungi Fungi
Phylum Deuteromycota Ascomycota
Sub-Division Deuteromycotina Ascomycotina
Class Hyphomycetes Pyrenomycetes
Order Monilliales Sphariales
Family Monilliaceae Hypocreaceae
Genus Trichoderma Hypocrea
Table: Taxonomic position of Trichoderma spp.
TRICHODERMA
SPP.

8. The word Trichoderma is taken from
thrix = hair and derma = skin.
Trichoderma is free living, asexually
reproducing and filamentous fungi.
The genus Trichoderma was first
proposed as a genus more than two
hundred years ago by Persoon in 1794
at Germany.
8

9. Species
Characteristics
 Tolerant to cool and warm temperatures.
 Tolerant to low moisture
 Tolerant to many fungicides
 Prefers acidic soils (pH 3.5 to 4.5)
 Rhizosphere competent
Life cycle
Habitat
9

10. 10
USES OF TRICHODERMA
Biocontrol agent-
In recent years, genetic improvement of Trichoderma by induction of mutation
using mutagens has successfully been attempted for improvement of potential ability of
biocontrol agent. Mutation in Trichoderma results in beneficial and harmful effect on
morphological, physiological, biochemical and molecular properties.
Medical uses- Cyclosporine A (CsA) a calcineurin inhibitor
Industrial use-
T. reesei is used to produce cellulase and hemicellulase
T. longibratum is used to produce xylanase
T. harzianum is used to produce chitinase.
Plant growth promotion
As a source of transgenes

11. Mode of Action: Competition
 Mycoparasitism
-Chemotaxis to target
-Coiling
-Extracellular enzymes (cellulases and chitinases)
-Appresoria like structures and penetration
 Induced resistance
 Antibiosis
-Diffusible inhibitors (toxins, antibiotics)
-Volatile inhibitors (alcohols, ketones, sesquiterpenes)
11

12. 12

13. 13
Trichoderma activities (a) mycoparasitism, (b) competition, (c) mycelial growth
in rhizosphere and production of metabolite

14. Fig. 3 (A): Mycoparasitism by a
Trichoderma strain on the plant
pathogen (Pythium) on the surface of
pea seed. The Trichoderma strain was
stained with an orange fluorescent
dye while the Pythium was strained
green.
Fig. 3(B): Scanning electron micrograph
of the surface of a hyphae of the plant
pathogen Rhizoctonia solani after
mycoparasitic Trichoderma hyphae were
removed. Erosion of the cell wall due to
the activity of cell wall degrading
enzymes from the biocontrol fungus is
evident, as are holes where the
mycoparasitic Trichoderma hyphae
penetrated the R. solani.

15. Trichoderma spp. showing different mechanism of action on plant pathogens
T. harzianum coils around
Rhizoctonia solani.
15
Trichoderma spp. coiling around
Rhizoctonia solani hyphae

16. Figure 3: Colonization of root hairs of corn by the highly rhizosphere
competent of T. harzianum T22.
16

17. Figure 4: Antagonistic activity of T. viride on Bortytis cinerea.
17

18. Colony morphology of the wild type (WT) and a vel1 mutant (ve3) of T. virens.
Mukherjee P K , Kenerley C M Appl. Environ. Microbiol.
2010;76:2345-2352

19. Growth of wild-type (WT), mutant (ve3), and complemented (Comp) strains in shake cultures.
Mukherjee P K , Kenerley C M Appl. Environ. Microbiol.
2010;76:2345-2352

20. WHAT IS
MUTATIONAn abrupt appearance of a new characteristic in an
individual as the result of an accidental change in a
gene or chromosome.
20

21. Point mutation :-
Altering the genetic code
Nonsense mutation
Change the genetic code and destroy the
information it contains.
Spontaneous mutation
Changes in frame shift.
Induced mutation
Artificial intervention.
TYPES OF MUTATION
21

22. TYPES OF MUTAGEN
Physical and chemical agents capable of bringing about
mutations are called mutagens.
Chemical mutagens
Include nitrous acid
This substance converts adenine to hypoxanthine, a
molecule that will not pair with thymine and thus
interrupts the genetic code.
A base analog is a chemical mutagen that resembles
a nitrogenous base and is incorporated by
error.
A DNA molecule cannot function in proteinsynthesis.
Certain dyes and fungal toxins (for example aflatoxin)
are known to be mutagens. 22

23. Physical mutagens :-
 Include X rays, gamma rays and ultraviolet light.
 They are bringing about genetic manipulation and
their by improving biocontrolling efficacy
23

24. METHOD OF DEVELOPING MUTANT OF Trichoderma sp.
BY USING UV LIGHT
Preparation of conidial suspension (5 day-old Trichoderma culture)
Spread on Petri plates (contain PDA medium)
Placed under UV light source for
different periods, quartz lamp, &
distance (i.e. 10,20,30 etc.)
Incubated at 250
C in normal condition
Comparison of changes with its parent
24

25. Impact of Mutation
Morphological changes
 Biochemical changes
 Mycoparasitism
25

26. 26

27. Isolates Phenotypic characters cfu/ml
TV5-(WT) Highly sporulating colony with yellow
pigmentation and aerial mycelium.
4.5x106
TV5-1 Highly sporulating colony where spores
were densely formed in the margin.
5.24x106
TV5-2 Initially colony with whitish mycelium less
number of spores appeared but assumed
heavy sporulation after interval of 4 days.
Yellow pigment appeared .
6.54x106
TV5-3 Colony with heavy sporulation and sporeless
marked zonation was also visible. Absence of
yellow pigment was prominent.
3.9x106
Table 1:- Phenotypic characteristics of Trichoderma viride
mutants and wild
Selvakumar et al. (2000)IARI, New Delhi
27

28. Table 2:- Comparison of T. viride parent and mutant strains
for the colony morphology, growth and sporulation
Character Trichoderma viride
Tv 34 Tv34-M4 Tv34-M5
Aerial mycelium Suppressed Fluffy Fluffy
Colony colour Yellowish to
greenish
Light orange to
greenish
Light orange to
greenish
Substrate
pigmentation
Light yellow Dark orange Dark orange
Sporulation 4.77×108
cfu/ml 7.45×108
cfu/ml 7.88×108
cfu/ml
Colony growth 1.92 mm 1.95 mm 1.96 mm
Mycelium dry
Weight (mg/100ml
broth culture)
146 187 193
Hunjan et al.(2004)P.A.U.,Ludhiana 28

29. Trichoderma
isolates
NaCl
(mM)
Reduction in linear
growth(%)
Description
Growth Sporulation
1st
7th
1st
7th
1st
7th
Wild type 0.0 0.0 0.0 Flat Flat + +
51 49.3 49.3 Cottony Cottony + +
69 53.1 53.1 Cottony Cottony + +
Th20M532
0.0 0.0 0.0 Flat Flat ++++ ++++
51 0.0 0.0 Flat Flat ++++ ++++
69 0.0 0.0 Flat Flat ++ ++
Th50M63
0.0 0.0 0.0 Flat Flat ++++ ++++
51 0.0 0.0 Flat Flat ++++ ++++
69 0.0 0.0 Flat Flat ++++ ++++
Th50M113
0.0 0.0 0.0 Flat Flat +++ ++++
51 0.0 0.0 Flat Flat +++ ++++
69 0.0 0.0 Flat Flat +++ +++
Table 3 :- Characterization of γ -induced mutants from T. harzianum grown
in different PDA supplemented with NaCl .
Table 3 :- Characterization of γ -induced mutants from T. harzianum grown
in different PDA supplemented with NaCl .
29
T. harzianum Wild type (WT).2&3
200 and 500 Gy γ- ray induced mutants,respectively.
(+) little disperse, (++) moderate disperse (+++) good disperse, (++++) copact.
(1st
) First subcultur and(7 th
) Seventh subcultures.
Mohmaed and Haggag(2005)N.R.C.,Dokki,Egypt

30. Table 4:- Growth rate and sporulation of T. harzianum
mutant
(Th 38 M-7) and parent strain (Th-38)
Trichoderma
strain
Growth on agar medium Growth in broth medium
Colony diameter (mm)
after incubation period (hr.)
Mycelium dry
weight/flask
(mg/100ml)
Sporulation
(conidia/ ml)
24 48 72 Mean
Mutant
(Th 38 M-7)
22.00 57.00 90.00 56.33 628 5.57×107
Parent (Th 38) 14.00 44.00 85.60 47.89 611 7.67×106
Mean 18.00 50.50 87.84 619.5
C.D (P=0.05)
Strain (A) 4.23 9.0
3.92×107
Hours (B) 28.58 -
Strain × Hours
( A× B ) 11.66
Manav and Singh (2006)P.A.U.,Ludhiana 30

31. Organism Phenotypic character
Th-W High sporulating colony with green aerial
mycelium
Th-M1 Initial colony with whitish mycelia later turn
greenish , profuse sporulation and shows faster
growth rate and yellow pigment appeared later
Th-M3 Less sporulation, pale green in colony, spores are
densely formed in the margin, and sporeless
marked zonation is visible.
Mech et al.(2006)A.A.U.,Jorhat
Table 5:- Phenotypic characters of carbendazim tolerant
T. harzianum mutants
31

32. Table 6 : Cultural characteristics of some selected mutant isolates of
Gliocladium virens on PDA and OMA medium after 10th
generation
Mutant isolates Cultural characteristics of phenotypic mutants on
PDA OMA
50 Gv1 I Dark green, submerged colony Dark green, colony submerged,
little raised mycelial growth at the
edge of the colony
50 Gv1 V Albino (white), granular and submerged
colony with scanty mycelial growth
Albino (white) submerged
mycelial growth, nodular
radiating mycelial growth
75 Gv1 VI Yellowish green, fluffy colony with profuse
mycelial growth
Greyish green, granular mycelial
growth colony submerged
150 Gv1 II Green fluffy colony with white mycelial
growth at the margin
Deep green, colony granular,
submerged mycelial growth
75 Gv3 I
Greyish white, raised colony Dark green, submerged colony
with little raised mycelial growth
at the centre
Jash et al.(2006)Mohanpur,Nadia
32

33. 33

34. Trichoderma
spp.
(Wild type/
mutant)
Total
metabolite
Antibiotics Total
phenolic
compounds
Trichodermin Gliotoxin Virindin
T.harzianum 0.46 0.03 0.016 0.015 0.010
TH5053 0.67 0.39 0.148 0.020 0.027
TH508 0.81 0.50 0.355 0.295 0.025
T. viride 0.38 0.07 ND 0.217 0.022
TV203 0.48 0.14 ND 0.232 0.026
TV208 0.56 0.18 0.067 0.095 0.045
T. koningii 0.47 0.05 0.012 0.009 0.021
TK508 0.70 0.71 0.046 0.034 0.026
TK509 0.75 0.16 0.136 0.380 0.046
Table 7 :- Total metabolites, antibiotics and phenolic compound in the culture filtrates of
three Trichoderma spp. wild types and their mutants (as μg/ ml dry weight)
Haggag and Mohamed (2002)Egypt
Antibiotics and total phenolic compounds were quantified by HPLC analysis of filtrates of liquid culturaes
ad normalized to the dry weight of mycelium in the collected samples. ND, not detected.
34

35. Figure 7:- Growth inhibition of Sclerotium cepivorum inciting white rot of
onion by three Trichoderma spp. and their mutants
35

37. Table 10 :- Effect of culture filtrate of T. viride isolates on the
growth of Botrytis cinerea
Isolate Colony diameter
(mm)
Inhibition
(%)
T-15 29.0 53.2
T-15.M-1 45.0 27.4
T-15.M-3 18.7 69.8
T-15.M-5 23.3 62.4
T-15.M-11 20.7 66.6
T-15.M-14 23.3 62.4
Control 62.0 -
CD (P ≤ 0.05) 4.5 -
Mukherjee et.al (1997)B.A.R.C.,Mumbai
37

38. Trichoderma spp.
(wild type/mutant)
Antagonistic effects against S. cepivorum
Pathogen growth
reduction(%)
Inhibitory effect of culture filtrate
10% 50%
T.harzianum 38.0±0.5bc2
6.7±0.1e3
58.5±0.4c4
TH5053 96.6±02a 48.8±0.4d 100.0a
TH508 100.0a 64.4±0.5b 100.0a
T. viride 33.3±0.4c 4.6±0.1e 48.0±0.3d
TV203 96.3±0.5a 55.5±0.5c 96.6±0.6b
TV208 100.0a 58.5±0.4c 100.0a
T. koningii 44.4±0.2b 5.5±0.2e 57.5±0.4c
TK508 100.0a 66.6±0.5b 100.0a
TK509 100.0a 57.6±0.4a 100.0a
Table 11:- Effect of wild types of Trichoderma spp. and their mutants on the
growth of Sclerotium cepivorum
Inhibition of S.cepivorum growth was determined on PDA containing 10 and 50% Trichoderma culture filtrates.
Mean of 5 replicates
Values in the the same raw followed by different letters are not statistically significant different at p < 0.05
Result are givaven as mean+ standared error.
Haggag and Mohamed (2002)N.R.C.,Dokki, Egypt
38

39. 39
T .viride strain Culture filtrate
concentration
(%)
Growth of R. solani (mm) after hours Colony growth inhibition (%) after
hours
24 48 72 96 24 48 72 96
Parent 0 27 38 68 90 0.0 0.0 0.0 0.0
10 12 24 38 58 55.6 36.8 44.1 35.6
20 8 24 36 49 70.4 36.8 47.1 45.6
30 8 20 34 47 70.4 47.4 50.0 47.8
50 7 19 32 43 74.1 50.0 52.9 52.2
Mutant-Tv34 M4 0 27 38 68 90 0.0 0.0 0.0 0.0
10 8 19 44 48 70.4 50.0 35.3 46.7
20 7 19 33 44 74.1 50.0 51.5 51.1
30 8 18 29 42 70.4 52.6 57.4 53.3
50 7 13 24 29 74.1 65.8 64.7 67.8
Mutant-Tv34 M5 0 27 38 68 90 0.0 0.0 0.0 0.0
10 9 19 46 52 66.7 50.0 32.4 42.2
20 8 18 34 49 70.4 52.6 50.0 45.6
30 7 18 32 41 74.1 52.6 52.9 54.4
50 7 11 23 29 74.1 71.1 66.2 67.8
Table 12 :- Effect of culture filtrate of mutant and parent strains of T. viride on growth
of R. solani causing black scurf of potato
Mean growth (mm) : parent-41.72,Tv34-M4- 36.61 , Tv 34-M5-M5-37.14
C.D. (P=0.05) Strain (A) 0.70
Concentration (B) 0.91
Time (C) 0.91
AXB- 1.57
BXC 1.34
AXC 1.57
AXBXC 3.52
P.A.U.,Ludhiana Hunjan et al.(2004)

40. Dose
(% wet
formulation)
Disease incidence
(%)
Yield (kg/plot) % Yield increase over
control
Mutant Parent Mutant Parent Mutant Parent
Seed treatment
1 76.1 79.6 6.1 5.0 25.0 9.0
1.5 69.3 76.7 5.9 5.1 21.9 12.0
2.0 69.3 74.0 6.8 5.4 31.2 18.1
Control 94.6 94.6 4.6 4.6 0.0 0.0
Mean 70.2 76.8 6.2 5.2 26.0 13.0
Seed treatment+ Spot Inoculation #
1 70.8 74.8 5.8 5.0 18.1 5.4
1.5 67.6 71.4 5.9 5.7 18.9 16.8
2.0 59.1 68.6 6.8 6.0 29.4 21.1
Control 94.5 94.5 4.8 4.8 0.0 0.0
Mean 65.8 71.6 6.2 5.6 22.1 14.4
Table 13:- Effect of seed treatment of T. viride parent (Tv 34) and mutant
(Tv34- M5) strains on black scurf development on potato under
field conditions.
C.D.(p=0.05) Strain- 2.4
Dose- 2.9
Strainx Dose- NS
Hunjan et al.(2004)P.A.U., Ludhiana
# spot inoculation was done with 50 g of fym and wet formulation mixture per seed tuber.
40

41. Sr.
No
Treatment Radial growth (mm) at different
hours of incubation
Percent inhibition at different hours
of incubation
24 48 72 24 48 72
1. T. harzianum (wild) +
S.sclerotium
8.59 26.14c
43.57b
49.99
(45.00)d
48.97
(44.41)c
48.49
(44.13)c
2. Th-M1 + S. sclerotium 7.87 27.00b
42.29c
54.17
(47.40)c
45.92
(42.66)d
49.98
(44.99)c
3. Th-M2 + S. sclerotium 3.94 6.14d
7.55e
77.21
(62.10)a
88.16
(69.93)a
91.53
(73.05)a
4. Th-M3 + S. sclerotium 6.70 19.14c
30.43d
61.00
(51.37)b
63.16
(52.13)b
64.01
(53.13)b
5. S. sclerotium alone 17.29 51.86a
89.87a
- - -
SEM(+) 0.51 0.49 0.58 - - -
CD(P=0.05) 1.01 0.97 1.18 - - -
Table 14:- Radial growth and per cent inhibition of S. sclerotium
in presence of wild and mutant T. harzianum in in vitro
Mech et al. (2006)AAU., Jorhat
Figure in parenthesis are Arcsine transformed value while outside are original values
41

42. Table 15 :- Biocontrol assay with wild and mutant strain of
T. harzianum against phytopathogens
Trichoderma Strains Phyto pathogens Growth of antagonist
(cm)
Growth of pathogen
(cm)
Pathogen
inhibition (%)
Wild
Fusarium oxysporum 6.7 2.3 74.4±0.18
Bipolaris oryzae 7.0 2.0 77.7±0.23
Rhzoctonia solani 4.3 4.7 47.7±0.17
Alternaria sp 7.2 1.8 80.0±0.47
UV mutant
F. oxysporum 7.9 1.1 87.7±0.35
B. oryzae 7.8 1.2 86.6±0.32
R. solani 6.1 2.9 67.7±0.19
Alternaria sp 8.0 1.0 88.8±0.47
Balasubramanian et al. (2010)Tamilnadu, India
Values are mean of triplicates with ± standerd error.
42

43. Fig 6:- Antagonistic capability of the selected T. harzianum mutants against
F. oxysporum and R. solani (MT3, MT5, MD5, MS1 and MS6 are
mutant arise from the wild type T3, T5, D5, S1 and S6, respectively).
Hassan and Kareem ( 2011)Tikrit Univ., Iraq 43

44. Table 16 :- in vitro growth inhibition of sensitive and resistant
isolates of M. phaseolina to carbedazim by mutant
T. koningii in dual culture method
Antagonist % Reduction of mycelial growth
Sensitive MP-12 Resistant MP-8
UVTK-1 42.50 47.50
UVTK-2 46.67 52.97
UVTK-3 36.67 40.42
UVTK-4 36.67 47.50
UVTK-5 21.17 52.80
TKWD 33.96 51.05
Patil and Kamble (2011)Shivaji University,Kolhapur
44

45.  Several genes have been cloned from
Trichoderma spp. that offer great promise as
transgenes to produce crops that are resistant to
plant diseases.
(Tubulin,Chitin,Protease,Xylanase,Monooxygenase.)
 No such genes are yet commercially available,
but a number are in development.
 These genes, which are contained in
Trichoderma spp. and many other beneficial
microbes, are the basis for much of "natural" organic
crop protection and production.
Trichoderma spp. as a source of transgenes.

46. Fig. 7 : Some biocontrol genes from T. harzianum have been inserted
into plants, where they provide resistance to several diseases. Tobacco
and potatoes, shown in this figure, were transformed to express the
fungal endochitinase gene, which resulted in high levels of resistance
to Alternaria alternata (tobacco) and Rhizoctonia solani (potato).

47. LIMITATIONS
Trichoderma spp. can only be used against specific disease. They are
less effective than the fungicides.
At present, only few Trichoderma spp. are available for use and are
available only in few places.
This method is only a preventive measure and not a curative measure.
Trichoderma spp. should be multiplied and supplied without
contamination and this requires skilled persons.
The shelf life of Trichoderma spp. is short. Antagonists, Trichoderma
viride is viable for four months.
The efficiency of Trichoderma spp. is mainly decided by environmental
conditions.
A Trichoderma spp. under certain circumstances may become a
pathogen.
Mutation in Trichoderma results in harmful effect on morphological,
physiological, biochemical and molecular properties.

48. Conclusion
 Induced mutation is one of the most commonly
used practices to modify the genetic composition of
antagonistic fungi.
 Mutation on Trichoderma sp. by UV and gamma
radiation brought about changes in morphological features
like colony diameter, sporulation, dry mycelial weight and
enzymes like ß-1,3-glucanase, ß-1,4-glucanase, cellulase
and antibiotics like trichodermine, gliotoxin and virindin.
Selection of beneficial mutant of biocontrol agent
becomes a better option for management of plant
pathogens.
Adoption of Trichoderma Spp. in biological control in
Disease Management is need of time hence extensive studies
will be required on interaction of diseases and management
practices 48