The document discusses Trichoderma, a group of asexually reproducing fungi, highlighting its biological characteristics, methods of pathogen interaction, and its role in plant growth promotion. It elaborates on the mechanisms of mycoparasitism, antibiosis, and competition in plant-pathogen dynamics, as well as the various secondary metabolites produced that contribute to these actions. Additionally, the paper emphasizes the applications of Trichoderma in biocontrol and horticulture, alongside precautions for field application and future prospects for commercial products.

1. University of Horticultural science, Bagalkot
College of Horticulture, Bagalkot, 587104
Presentation on : Trichoderma a Potential Bio fungicidal to Plant Pathogens
Submitted to: Dr. Kiran Kumar K. C.
Asst. Prof. Plant Pathology
UHS, Bagalkot
Presented By: Nagesh
Ist Ph.D Plant Pathology
UHS,Bagalkot

2. • Trichoderma - asexually reproducing fungi - isolated soil fungi.
• Also colonize woody and herbaceous plant materials, in which the sexual TELEOMORPH
(genus Hypocrea) has most often been found.
• 1794-Erected by Persoon.
• 1865-sexual/Teleomorph state Hypocrea species was suggested Tulasne and Tulasne.
• 1932- Weindling -1st demonstrated parasitic activity against Rhizoctonia solani.
• All species earlier named T. viride-oligonucleotide barcode (TrichOKEY) and a customized
similarity search tool (TrichoBLAST).
• Now more than 200 species recorded (Only about 20 recorded from soil/ rhizosphere)
• Some species are endophytes.

3. •A few are opportunistic human pathogens.
•Common species are
1. Trichoderma reesei,
2. Trichoderma virens,
3. Trichoderma atroviride,
4. Trichoderma harzianum,
5. Trichoderma asperellum,
6. Trichoderma longibrachiatum,
7. Trichoderma citrinoviride

4. Trichoderma biology:
Trichoderma species are fast growing under the optimum range between 25-30 ºC.
(Latifian et al., 2007).
Trichoderma used a variety of compounds such as carbon and nitrogen sources as a growth medium for its sporulation.
(Gao et al., 2007; Seyis and Aksoz, 2005).
T. harzianum favors warm climate while T. viride and T. polysporum characteristic of cool temperature.
(Sarhy-Bagnon et al., 2000).
T. citrinoviride has been reported in South East Asia but not found in India (Zhang et al., 2005).
BLR-1 and BLR-2 are known to be photoreceptor-orthologs and light regulatory protein ENVOY that regulates
expression of cellulase gene established connection between nutrient signaling and light response in Trichoderma.
(Schmoll et al., 2009).
PDA medium was the best for biomass and spore production of Trichoderma species (Mustafa et al., 2009).
(PDA/ Waksman Agar/Agar Agar/ Corn Meal Agar/ Czepak’s Agar).
Mycelial growth was good at the temperature range of 30-37ºC while sporulation was favored by 30-45ºC. Moreover,
maximum growth and sporulation of T. viride was observed at 4.5-6.0 pH. (Singh et al., 2014).

5. Plant Protection, 02 (03) 2018. 109-135

6. Mechanisms
I. Trichoderma- pathogen interaction
1. Mycoparasitism
2. Antibiosis
3. Competition
II. Trichoderma- Plant interaction
1. Plant root colonization
2. Plant growth promotion
3. Induced resistance
4. Symbiosis and Endophytism

7. Mycoparasitism :
1. Chemotropic growth of Trichoderma.
2. Recognition of the host.
3. coiling and appresoria formation.
4. secretion of hydrolytic enzymes. (glucanases, chitinases and proteases)
5. penetrations of the hyphae and lysis of the host.
Coilling
Root
A
A

8. The cellwall degrading enzymes of Trichoderma
1.ß-1,3-glucanases and different chitinolytic enzymes
2.Endochitinase (42- kDa),
3. chitobiosidase (40-kDa) and
4.N-acetyl-b- D-glucosaminidase (73-kDa)
5.Celluases
Druzhinina et al., 2011

9. Antibiosis
Two different mechanisms of action.
1. The production of low molecular weight, non-polar, volatile compounds (i.e. 6PP) results in a high concentration of
antibiotics in the soil environment, that have a relatively long distance range of influence on the microbial
community.
2. Short distance effect may be due to the polar antibiotics and peptaibols acting in close proximity to the producing
hyphae.
(i)Volatile antibiotics, i.e. 6-pentyl-a-pyrone (6PP) and most of theisocyanide derivates.
(ii)Water-soluble compounds, i.e. heptelidic acid or koningic acid.
(iii)Peptaibols, which are linear oligopeptides of 12–22 amino acids rich in a-aminoisobutyric acid, N-acetylated at the N-
terminus and containing an amino alcohol (Pheol or Trpol) at the C-terminus.

10. Secondary metabolites isolated from
Trichoderma spp.
1.azaphilone;
2: butenolide;
3: harzianolide;
4: dehydro harzianolide;
5: harzianopyridone;
6: 6-pentyl-a-pyrone;
7: 1-hydroxy-3-methyl-anthraquinone;
8: 1,8-dihydroxy-3-methyl-anthraquinone;
9: harziandione;
10: koninginin A; 1
1: heptelidic acid;
12: trichoviridin;
13: harzianic acid;
14: gliotoxin;
15: gliovirin;
16: viridin;
17: viridiol;
18: trichorzianines.
T. asperellum strain produces two asperelines (A and E)
and five trichotoxins. (T5D2, T5E, T5F, T5G and 1717A)
which can be associated with antibiosis.
(Brito et al., 2014).

11. 1. Competition for substrates is the most important factor for fungi as is competition for light in the case of
evolution of plants (Garrett, 1956).
2. Root exudates and rhizosphere are rich source of nutrients such as sugar, amino acids, iron, vitamins,
organic acids etc.
3. The proficient mobilization of immobile nutrients and their utilization makes it more efficient and
competitive.
4. Production of organic acids, such as gluconic, citric and fumaric acids, which decrease soil pH and allow
the solubilization of phosphates, micronutrients and mineral cations like iron, manganese and magnesium.
(Vinale et al., 2008).
“In the aerobic environment (with oxygen and neutral pH) iron exists mainly as Fe3+ and tends to form
insoluble ferric oxide, making it unavailable for root absorption and microbial growth. (Miethke, 2013).
Iron act as cofactor of numerous enzymes
Trichoderma secrete siderophore, an iron chelating compounds which bind with insoluble iron (Fe+3) and
converted to soluble form (Fe+2) .
Competition

12. Trichoderma–plant interaction
Plant root colonization
The hyphae penetrate the root cortex but the colonization by Trichoderma is stopped, probably by the
deposition of callose barriers by the surrounding plant tissues. It appears that this interaction evolves into
a symbiotic rather than a parasitic relationship between the fungus and the plant, whereby the fungus
occupies a nutritional niche and the plant is protected from disease.
Direct molecular cross-talk occurs between the fungus and the plant.
Elicitors from Trichoderma activate the expression of genes involved in the plant defence response system,
and promote the growth of the plant, root system and nutrient availability. This effect in turn augments
the zone for colonization and the nutrients available for the biocontrol fungus, subsequently increasing
the overall antagonism to plant pathogens.

13. Plant growth promotional activity :
Trichoderma Secondary Metabolites: Koninginins, 6pentyl-a-pyrone, trichocaranes A – D, harzianopyridone,
cyclonerodiol, harzianolide and harzianic acid are examples of isolated compounds that affect plant growth in a
concentration dependent manner.
(Vinale et al., 2014).
Effect on seed germination :
Seed biopriming and seed treatment with Trichoderma spp. trigger the release and/or production of enzymes and
phytohormones which are involved in seed germination.
T.harzianum produces a metabolite as gliotoxin that may mimic the plant growth hormone gibberellic acid which is
involved seed germination process.
Effect on plant morphology :
Many lines of evidence strongly support a role for auxin in the regulation of root system architecture. T. virens is able to
produce auxins as indole-3acetic acid (IAA), indole-3-acetaldehyde (IAAld), and indole-3-ethanol (IEt).
(Contreras-Cornejo et al., 2009)

15. Trichoderma spp. have three MAPK cascades comprising MAPKKK, MAPKK and MAPK and
MAPK signaling pathways may act in mycoparasitism and biocontrol.
Earlier evidences suggested the involvement of chit42, chit3, bgn13.1, Bgn2 Bgn3 and
prb1 are involved in chitinases, glucanases and proteases in biocontrol (Mondizar et. al.,
2011).
T. atroviride produce the volatile metabolite 6-pentyl-2H-pyran-2-one (6-PP) which plays
an important role in Trichoderma– fungal interactions. (Hasan et al., 2008, Vinale et al.,
2009).
Earlier evidences suggested the involvement of chit42, chit3, bgn13.1, Bgn2 Bgn3 and
prb1 are involved in chitinases, glucanases and proteases in biocontrol (Mondizar et. al.,
2011).

16. Induced-resistance
Interaction of Trichoderma with the plant, different classes of metabolites may act as elicitors
or resistance inducers.
These molecules include:
(i) proteins with enzymatic activity, such as xylanase
(ii) avirulence-like gene products able to induce defence reactions in plants
(iii) low-molecular-weight compounds released from fungal or plant cell walls by the activity
of Trichoderma enzymes.

18. 9 Secretion of effector-like proteins during
plant-Trichoderma interactions.
Trichoderma spp. constitutively secrete
proteins which are detected by plant

19. Biocontrol genes of Trichoderma :
The genome sequencing of Trichoderma species has provided exclusive data for phylogenetic and bioinformatic analyses
toward understanding the roles of these opportunists in agroecosystems.
At present the, genome sequences of seven species: Trichoderma harzianum, T. asperellum, T. reesei, T. virens,
T . atroviride, T. longibrachiatum and T. citrinoviride are available.
(Sharma et al., 2011).
Plant growth promotional activities and their environmental adaptation (drought, salt, heavy metal tolerance etc.).
Several laboratories have recently started or planned to use proteomic and genomic analysis to frame the changes that
occur in the Trichoderma.

20. Table.1 Biocontrol genes of Trichoderma spp

21. Symbiosis and Endophytism
Endophytism: a fungus, or a bacterium living within plant tissues for a part of its life without causing
apparent damage/injury.
The saprophytic fitness of Trichoderma species has enabled their establishment in soil and rhizosphere and
often within roots where hyphae grow between cortical cells
Although well known for their ability to colonize the rhizosphere with limited root penetration, some
Trichoderma species are known to reside in plants as typical endophytes, entering through trichomes by
producing appressoria-like structures.
T. Asperellum, T. virens, T. koningiopsis, T. stilbohypoxyli and T. stromaticum

24. Isolation of
Trichoderma

28. Precautions:
Some precautionary measures should be kept in mind regarding the application of Trichoderma inoculums in
the field condition.
1. Don’t settle treated Farm Yard Manure (FYM) for a longer time.
2. Don’t place Trichoderma treated seed in direct sunlight.
3. Don’t apply chemical pesticides or fungicides after application of Trichoderma for 5-6 days.
4. Moisture is an important factor for Trichoderma growth and reproduction so don’t try to use it in dry soil.

29. Singh. et al., 2018

30. Trichoderma based commercial products against various diseases

32. Future Prospects :
Omic studies on Trichoderma spp. are regarded as a successful case of translational research, where data are
quickly applied to:
(a) improved agent-selection methods that provide new active principles for commercial products;
(b) new types of formulations;
(c) optimized application protocols;
(d) safer use, etc.
More than 100 Trichoderma-based agriculture products are today on the market, in spite of the difficulties
encountered with the registration process

33. Enhancement of Lytic Enzymes Activity and AntagonisticTraits of
Trichoderma harzianum
Using γ-Radiation Induced Mutation
J. Agr.Sci.Tech. (2019)Vol. 21(4): 1035-1048
Ghasemi. et al., 2019

34. MATERIAL AND METHODS:
Isolation and Identification of T. harzianum
Determination of Growth Rates of Fungi
Antagonistic Activity of T. harzianum and Its Mutants against R. solani
I= (1-Cn/Co)×100.
Chitinase and Cellulase Enzyme Production
Extracellular Enzyme Production
Estimation of Extracellular Protein Production and Enzyme Activity
Electrophoresis and Molecular Size Determination. (SDS– PAGE)

35. Table 1. Percentage of growth inhibition of R. solani by T. harzianum mutants after 7 days' incubation in dual culture assay

36. Table 2. Extracellular protein production assay and the rate of mycelial growth of T. harzianum wild type
(T. h control) and its mutant isolates

37. Table 3. Specific Cellulase (Endo- and Exo-glucanase) and Chitinase enzyme assay of T. harzianum and its
mutant isolates in TFM supernatant after 48 h incubation at 180 rpm and 28 ˚C

38. SDS-PAGE Analysis and Molecular Size Determination
Figure 1. The profile of chitinase enzyme protein and optical density measurement of protein bands in the T. harzianum
wild type and its mutant isolates by Gel –Pro (Ver. 6).

39. Figure 2. The profile of cellulase enzyme protein and optical density measurement of protein bands in the T. harzianum
wild type and its mutant isolates by Gel –Pro (Ver.6).