1

Seminar presentation on

“Significant Achievements and Present Status of Trichoderma spp.

in Biocontrol of Plant Diseases ”

3

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

ContentsIntroduction

Mechanism of TrichodermaType of mutation & mutagen

ReviewsImpact of mutation on morphological charactersImpact of mutation on biochemical changesImpact of mutation on mycoparasitism

5

INTRODUCTION

Position Asexual stage (Conidia)

Sexual stage (Ascospore)

Kingdom Fungi FungiPhylum Deuteromycota AscomycotaSub-Division Deuteromycotina AscomycotinaClass Hyphomycetes PyrenomycetesOrder Monilliales SpharialesFamily Monilliaceae HypocreaceaeGenus Trichoderma Hypocrea

Table: Taxonomic position of Trichoderma spp.

TRICHODERMA SPP.

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

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

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

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

13

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

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.

Trichoderma spp. showing different mechanism of action on plant pathogens

T. harzianum coils around Rhizoctonia solani.

15

Trichoderma spp. coiling around Rhizoctonia solani hyphae

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

16

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

17

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

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

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

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

TYPES OF MUTAGEN Physical and chemical agents capable of bringing aboutmutations are called mutagens.

Chemical mutagensInclude 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

Physical mutagens :- Include X rays, gamma rays and ultraviolet light.

They are bringing about genetic manipulation and

their by improving biocontrolling efficacy

23

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 250C in normal condition

Comparison of changes with its parent24

Impact of Mutation

Morphological changes

Biochemical changes

Mycoparasitism

25

26

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 Delhi27

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/mlColony growth 1.92 mm 1.95 mm 1.96 mmMycelium dry

Weight (mg/100ml broth culture)

146 187 193

Hunjan et al.(2004) P.A.U.,Ludhiana 28

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 .

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

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

Organism Phenotypic characterTh-W High sporulating colony with green aerial

myceliumTh-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

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 OMA50 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,Nadia32

33

Trichoderma spp.

(Wild type/ mutant)

Total metabolite

Antibiotics Total phenolic

compoundsTrichodermin 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 0.025T. 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

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

35

36

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 27.4T-15.M-3 18.7 69.8T-15.M-5 23.3 62.4

T-15.M-11 20.7 66.6T-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

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 < 0.05Result are givaven as mean+ standared error.

Haggag and Mohamed (2002) N.R.C.,Dokki, Egypt38

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 0.010 12 24 38 58 55.6 36.8 44.1 35.620 8 24 36 49 70.4 36.8 47.1 45.630 8 20 34 47 70.4 47.4 50.0 47.850 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 0.010 8 19 44 48 70.4 50.0 35.3 46.720 7 19 33 44 74.1 50.0 51.5 51.130 8 18 29 42 70.4 52.6 57.4 53.350 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 0.010 9 19 46 52 66.7 50.0 32.4 42.220 8 18 34 49 70.4 52.6 50.0 45.630 7 18 32 41 74.1 52.6 52.9 54.450 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-M5-37.14C.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)

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- 2.9Strainx 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

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

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±0.18Bipolaris oryzae

7.0 2.0 77.7±0.23

Rhzoctonia solani 4.3 4.7 47.7±0.17Alternaria 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

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

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 growthSensitive MP-12 Resistant MP-8

UVTK-1 42.50 47.50

UVTK-2 46.67 52.97

UVTK-3 36.67 40.42UVTK-4 36.67 47.50

UVTK-5 21.17 52.80

TKWD 33.96 51.05

Patil and Kamble (2011)Shivaji University,Kolhapur 44

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.

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).

LIMITATIONSTrichoderma 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.

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