ASSAM AGRICULTURAL UNIVERSITYCredit Seminar of Post-Graduate Student M.Sc. (Agri.)

Name of the Student : Munmi Bora

Roll No. : 11-AMJ-83

Programme of study : M.Sc (Agri.)

Major Discipline : Plant Pathology

Supporting discipline : Agril. Statistics and Soil

Major Advisor : Dr. B.C. Das

Professor

Deptt. of Plant Pathology

AAU, Jorhat-13

A. Title of the seminar: How much we know about Trichoderma???

Introduction:

Focus on Trichoderma:

Among thousands of fungal genera, Trichoderma is one with the broadest impact on mankind. The application of Trichoderma species in contemporary biotechnologies is mainly the result of the ability of some species to hydrolyze cellulose. The production of hydrolytic enzymes (cellulases and hemicellulases) has been mainly studied and commercially exploited in T.reesei (teleomorph H.jecorina). Mutants of the isolate QM6a have been used for years for cellulases. In a clinical context, a pair of genetically related species, a strictly clonal T. longibrachiatum and H. orientalis has been shown to occur as an opportunistic pathogen of immune-compromised or immune-suppressed mammals, including humans (Kredics et al.,2003; Druzhinina et al.,2008).

Although some properties, like mycoparasitism, are characteristic of a wide variety of species in the genus, other properties, like opportunistic attacks on immune-compromised mammals or green

mold disease, seem to be restricted to certain taxa. Thus, the great diversity of applications combined with ecological adaptability of the genus Hypocrea/Trichoderma makes a deeper understanding of its ecology and diversity worthwhile.

What are Trichoderma???

Fungi of the genus Trichoderma are soil borne, green spored ascomycetes that can be found all over the world. Trichoderma spp. are characterized by rapid growth, mostly bright green conidia and a repetitively branched conidiophore structure. As opportunistic plant symbionts and effective mycoparasites, numerous species of this genus have the potential to become commercial biofungicides (Harman et al.,2004). The fungi of the genus Trichoderma are favored by the presence of high level of plant roots, in which they can colonize readily. Trichoderma is present nearly in all soils and other diverse habitats. Species of Trichoderma are among the most frequently detected mitosporic fungi (teleomorph Hypocrea, Ascomycota, Dykaria) in cultivation based surveys.

Definition of Trichoderma-

A form genus of imperfect fungi (Deuteromycetes) family Moniliaceae having non-septate conidia borne in heads on 2-branched or 3-branched conidiophores.

Scientific Classification-

Kingdom-Fungi

Division-Ascomycota

Sub- division-Pezizomycotina

Class-Sordariomycetes

Order-Hypocreales

Family-Hypocreaceae

Genus-Trichoderma

Species-100 phylogenetically defined species (Druzhinina et al.,2006).

Popularly known Trichoderma spp are-

Trichoderma aggressivum Trichoderma amazonicum Trichoderma asperellum Trichoderma atroviride Trichoderma aureoviride Trichoderma austrokoningii Trichoderma brevicompactum Trichoderma candidum

Trichoderma caribbaeum var. aequatoriale Trichoderma caribbaeum var. caribbaeum Trichoderma catoptron Trichoderma cremeum Trichoderma ceramicum Trichoderma cerinum Trichoderma chlorosporum Trichoderma chromospermum Trichoderma cinnamomeum Trichoderma citrinoviride Trichoderma crassum Trichoderma cremeum Trichoderma dingleyeae Trichoderma dorotheae Trichoderma effusum Trichoderma erinaceum Trichoderma estonicum Trichoderma fertile Trichoderma gelatinosus Trichoderma ghanense Trichoderma hamatum Trichoderma harzianum Trichoderma helicum Trichoderma intricatum Trichoderma konilangbra Trichoderma koningii Trichoderma koningiopsis Trichoderma longibrachiatum Trichoderma longipile Trichoderma minutisporum Trichoderma oblongisporum Trichoderma ovalisporum Trichoderma petersenii Trichoderma phyllostahydis Trichoderma piluliferum Trichoderma pleuroticola Trichoderma pleurotum Trichoderma polysporum Trichoderma pseudokoningii Trichoderma pubescens Trichoderma reesei Trichoderma rogersonii Trichoderma rossicum Trichoderma saturnisporum Trichoderma sinensis Trichoderma sinuosum Trichoderma sp. MA 3642 Trichoderma sp. PPRI 3559

Trichoderma spirale Trichoderma stramineum Trichoderma strigosum Trichoderma stromaticum Trichoderma surrotundum Trichoderma taiwanense Trichoderma thailandicum Trichoderma thelephoricolum Trichoderma theobromicola Trichoderma tomentosum Trichoderma velutinum Trichoderma virens Trichoderma viride Trichoderma viridescens

Morphological Characters of Trichoderma spp.

1. Septate fungus which produces highly branched conidiophores with a conical or pyramidal outline (Rifai,1969)

2. Phialides are flask shaped structure found at the tip of the conidiophores.3. Phialospores (conidia) are produced at the end of the phialides where they accumulate to

form a conidial head (Gams & Bissett,1998)4. Asexual reproduction through intense sporulation or clonal growth from hyphae fragments

(Gams & Bissett,1998)5. Sexual (Teleomorph) stage known Hypocrea in the Ascomycetes order Hypocreales

(Samuels,1996)6. Forms floccose or tufted colonies of various colours (white, yellow, green) which in the past

were used to identify species (Rifai,1969)

Key to some of the species-

1. Sterile hyphal elongations absent; conidia globose, hyaline- T.piluliferum2. Conidia green, short ellipsoidal, surrounded by a wide irregular veil and conidia are smooth

walled or finely punctuated- T.saturnisporum3. Conidia hyaline,small 2.4-3.8x 1.8-2.2 µm- T.polysporum4. Conidia green, small to large 3.8-6.0x2.2-2.8 µm- T.hamatum5. Conidia roughened 3.6-4.8x 3.5-4.5µm- T.viride6. Conidia ellipsoidal or oblong often appearing angular 3.0-4.8x1.9-2.8µm- T.koningii7. Conidia obovoid, with truncate base 3.0-4.8x2.0-3.0 µm; reverse of colonies often

discoloured; colonies reaching 3 cm diameter in 5 days at 20˚c on OA – T.aureoviride8. Conidia globose, subglobose or short obovoid, with length:width ratio of less than 1.25,2.8-

3.2x2.5-2.8µm colony reverse uncoloured: colonies reaching >9 cm diameter in 5 days at 20˚c in OA.- T.harzianum

9. Conidia sub-globose to ovoid, 3.8-4.5x 2.5-3.0µm, colony ellipsoidal- T.reesei

10. Phialides usually only slightly attenuate at the base ; conidia large, partly dark green to 7 µm long, mostly ellipsoidal- T.longibrachiatum

11. Phialides usually distinctly attenuate at the base:conidia smaller, pale green 2.8-4.8µm long mostly oblong ellipsoidal- T.pseudokoningii.

History of Trichoderma:

Persoon(1794)- Introduced the generic name Trichoderma more than 200 years ago on the basis of material collected in Germany.

Tulasne and Tulasne(1865)- Determined that T.viride is the asexual stage of Hypocrea rufa. The fact that this relationship is still true today renders their finding a milestone in the taxonomy of conidial fungi.

Gilman and Abbott(1927)- Recognized four well-defined groups among the integrating Trichoderma isolates from soil and were identified as T.lignorum (=T.viride) because of its globose conidia or as T.koningii because of its oblong conidia.

Weindling(1932)- First to demonstrate the parasitic activity of members of this genus to pathogens such as R.solani. But the use of Trichoderma spp as biological control agents has been investigated for over 70 years.

Bisby(1939)- Reduced all species of Trichoderma to the single species T.viride, a system that was followed until 1969.

Rifai(1969)- Recognized 9 aggregate species, some of which were isolated from Hypocrea specimens.

Habitat of Trichoderma-

I. SoilII. Rhizosphere

III. Plant debrisIV. Decaying wood.

Trichoderma species are cosmopolitan soil fungi (Waksman,1952). They colonize a wide range of soil niches from cool temperate to tropical climates. These include agricultural orchards, forest, pasture and desert soils (Domsch et al.1980;Hagn et al.,2003, Roiger et al.,1991). The saprophytic nature of Trichoderma means that the fungi are commonly present in the soil’s top horizons (F and H) where high densities of mycelium can be recovered specially in the humid litters of deciduous and coniferous forests(Danielson and Davey,1973, Widden & Abitbol,1980). The high versatility of this genus means that some Trichoderma species have also been recovered in extreme environments like mangrove swamps, salt marshes and estuarine sediments (Borut & Johnson,1962; Domsch et al.,1980; Lee & Baker ,1972) where adverse osmotic potential conditions pose a real challenge for fungal survival. Yet, such environments are widely colonized by T.viride Pers.ex Gray which is probably the most wide-spread species of Trichoderma (Domsch et al.,1980).

Hypocrea or Trichoderma:

Fig: shows a species of the ascomycetes genus Hypocrea with all stages of its reproductive cycle present. The sexual structures are embedded within the orange cushions while the asexual structures are present in the green areas.

Characteristic features of Trichoderma spp.:

Fig: (a) T. reesei (b) T.atroviride growing on plates, (c) T. reesei or H. jecorina growing in daylight and showing light responsive conidiation, (d) fruiting body formation of T. reesei upon crossing with a nature isolate of H.jecorina , (e, f) T.longibrachiatum germinating and growing on human cells, (g, i ) T. reesei (left) during confrontation with Pythium ultimum (right), (h)T. atroviride (left) during confrontation with R. solani (right) (Appl Microbiol Biotechnol .2010 87:787-799)

Apart from those characters Trichoderma is also-

1. Ubiquitous colonizers of cellulosic materials. 2. Found in the rhizosphere of plants where they can induce systemic resistance (Harman

2000). 3. The search for potent biomass degrading enzymes. 4. Isolation of this fungi from unexpected sources, such a cockroaches (Yoder et al.,2008).

Marine mussels and shellfish (Sallenave et al. 1999; Sallenave-Namont et al. ,2000)and termite guts(Sreerama and Veerabhadrappa 1993)

5. Trichoderma spp is characterized by rapid growth, mostly bright green conidia and a repeatitively branched conidiophore structure.

Life cycle of Trichoderma spp-

The organisms grows and ramifies as typical fungal hyphae, 5- 10µm in diameter. Asexual sporulation occurs as single-celled, usually green ,conidia (typically 3-5µm in diameter) that are released in large numbers. Intercalary resting chlamydospores are also formed, these also are single celled, although two or more chlamydospores may be fused together.

Environmental Parameters-

The composition ,biomass and biological activity of microbe communities in the soil depend on important physical and chemical factors (Garbeva et al.,2004; Killham, 1994; Lavelle&Spain, 2001). Environmental parameters such as soil temperature, moisture, atmosphere, pH, organic matter (OM), nutrient content and plant types are key factors that influence soil colonization by Trichodema spp.(Carreiro &Koska,1992; Domsch et al.,1980).

Soil temperature:

Species like T.harzianum are generally isolated from warm tropical soils whereas T.polysporum and T.viride are mostly found in cool temperate regions (Danielson & Davey, 1973; Klein & Eveleigh,1998). The range of temperatures at which T.spp can grow is fairly wide, it can be low as 0˚c for T.polysporum and as high as 40˚c for T.koningii (Domsch et al.,1980; Tronsmo & Dennis,1978). Temperature not only affect the growth of T.spp, it also affects their metabolic activity especially the production of volatile antibiotics (Tronsmo & Dennis,1978) and enzymes.

Soil moisture:

Soil moisture or water potential is also an important factor which influences establishment of Trichoderma populations as well as other microorganisms in the soil (Lavelle & Spain, 2001). Water potential is closely dependent on the soil temperature and becomes more negative as the temperature rises (Dix & Webster, 1995). Hyphal growth, spore production and germination, and biological control activity of T.spp are generally negatively affected by high negative soil water potentials (Clarkson et al.,2004, Eastburn & Butler.,1991). Most T.spp require a low soil water potential to achieve optimal

growth and populations are generally more abundant in moist soil especially in humid litter (Danielson & Davey,1973). Despite this, some spp like T.viride have been known to achieve growth at soil water potentials of -240 MPa (Domsch et al.,1980).

Soil pH:

Fungi from the genus Trichoderma seem to be positively affected by acidic substrates as most spp have an optimal pH range of 3.5-5.6 (Domsch et al.,1980) for both growth and spore germination (Danielson &Davey,1973) and can even grow at a pH of 2.1 (Waksman,1952). Soil pH is greatly dependant on the soil CO2 atmospheric content (Papavizas,1985). CO2 combines with water to produce a weak acid called carbonic acid. Some T.spp will grow better on slightly basic substrate at high atmosphere CO2 concentration (Danielson &Davey,1973). The concentrations of hydrogen ions also affects the solubilisation of salts in the soil solution and hence the availability of ions (Dix & Webster., 1995) and nutrients to the T.spp.

Taxonomy and genetics of Trichoderma:

Wild strains are highly adaptable, heterokaryotic (nuclei of dissimilar genotype) within a single organism and hence highly variable.

Strains used for bio-control in commercial agriculture are, or should be, homokaryotic (nuclei are all genetically similar or identical).

Trichoderma- a Micro fungi doing Macro role:

Trichoderma – Cosmopolitan saprophyte. Promote plant health Promote soil health Induce polygenic resistance Bioremediation

Trichoderma - A promising Bio-control agent

Biological control:

The use of specific microorganisms that interfere with plant pathogens and pests in a nature-friendly, ecological approach to overcome the problems caused by standard chemical methods of plant protection (Harman et al.,2004)

Mode of mechanism of Trichoderma as a biocontrol agent:

1. MYCOPARASITISM 2. ANTIBIOSIS 3. COMPETITION FOR NUTRIENTS OR SPACE

4. TOLERANCE TO STRESS THROUGH ENHANCED ROOT AND PLANT DEVELOPMENT 5. SOLUBILIZATION AND SEQUESTRATION OF INORGANIC NUTRIENTS 6. INDUCED RESISTANCE 7. INACTIVATION OF THE PATHOGEN’S ENZYME

1. Mycoparasitism:

Mycoparasitism is the process of one fungus parasitising another fungus of a different species. The degree of parasitism is strongly affected by the nutrients present in the substrate .

It can be divided into four sequential steps:-

The 1st step is the chemotrophic growth where the secretion of a chemical stimulus by the target fungus attracts an antagonist fungus.

The 2nd step is the specific recognition ,where the antagonist fungus identifies the cell surface of the pathogen.

The 3rd step involves 2 distinct process -(i) Coiling- when antagonist surrounds its host.(ii) It involves intimate hyphal interactions and contact where the

antagonist hyphae simply grows along the host surface. The 4th step is the secretion of specific lytic enzymes (chitinase, proteinases, ß-glucanase) which

degrades the host cell wall .

FIG: Mycoparasitism of Rhizoctonia solani by Trichoderma virens: A, parent strain coiling around host hyphae, and B, mycoparasitic-deficient mutant with no coiling or penetration of host hyphae.

Fig: Mycoparasitism by a Trichoderma strain on the plant pathogen (Pythium) on the surface of pea seed (Hubbard et al.,1983 phytopathology 73; 655-659 )

Fig: Mycelia of T.harzianum (Tri) and S.sclerotiorum (Smyc) opposite each other. Hyphae of S.sclerotiorum trying to breach the barrier of T.harzianum hyphae by forming “rhizomorph-like” (RM) structures

Fig: Scanning electron micrograph showing T. harzianum hyphae attached to S. sclerotiorum hypha. (a) “Hook-like” (HL) and “appressoria-like” (AP) structures on S. sclerotiorum hypha and (b) attachment, coiling, and penetration of T. harzianum hyphae into S. sclerotiorum hypha .

Role of MRGs in Biocontrol and strain improvement:

MRGs (Mycoparasitism - Related Genes) are all the genes encoding cell-wall degrading enzymes.

In 1997, Flores and coworkers generated transgenic T.atroviride lines carrying multiple copies of prb1.

The resulting strains produced upto 20 times more proteinase and all strains tested were more effective in the control of R. solani.

The role of the Trichoderma 42kDa endochitinases in mycoparasitism has been addressed by genetic manipulation of the corresponding gene (ech42 and ech1) T. atroviride and T. virens. (Baek et al.1999;Carsolio et al.1999;Woo et al.1999).

2. Antibiosis/Enzymes:

The involvement of volatile and nonvolatile antibiotics in the antagonism by Trichoderma has been proposed(Dennis and Webster .,1971a,b). 1st characterized secondary metabolites was the peptide antibiotic-Paracelsin (Bruckner and Graf 1983). Trichoderma also produces linear oligopeptides of 12-22 amino acids (peptaibols), which are rich in aminoisobutyric acid ,N-acetylated at the N-terminus and containing an amino alcohol at the C-terminus (Rebuffat et al.1989;1991). These oligopeptides are known to form voltage-gated ion channels in black lipid membranes and modify the membrane permeability of liposomes(EI Hadjii et al.,1989). These suggested a scenario where cell wall degrading enzymes weaken the cell wall and peptaibol antibiotics inhibit synthesis of cell wall components,

impairing the capacity of the hyphae to repair the effect of cell wall degrading enzymes(Lorito et al.1996).

Fig: Growth inhibition of R.solani by the T.virens–produced antibiotic gliotoxin: A, gliotoxin- amended medium, and B, nonamended medium.

Fig: The pre-contact events of the mycoparasitic interaction Trichoderma—host fungus. Phase 1: the mycoparasite produces high molecular weight compounds that reach the host. Phase 2: low molecular weight-degradation products that are released from the host cell walls reach the mycoparasite and activate the mycoparasitic gene expression cascade. (Harman et al., 2004)

(Elan Chet, Hebrew University of Jerusalem )

Fig: Trichoderma sp. penetrate the host cell wall of plant pathogenic fungi by secreting lytic enzymes like Chitinases ,Proteases Glucanases, lipases when grown on cell walls of R.solani.

Enzymes:

Enzymes such as chitinases and /or glucanases produced by the biocontrol agent are responsible for the suppression of the plant pathogen. These enzymes function by breaking down the polysaccharides, chitin, and β –glucans that are responsible for the rigidity of fungal cell walls, thereby destroying cell wall integrity.

The purification and characterization of three endochitinases secreted by T.harzianum was 1st

reported by( De la Cruz et al., 1992). The reported isozymes to be 37, 33 and 42 kDa. Two genes showing similarity to the one encoding the 33kDa endochitinase described by (De la Cruz et al.,1992) have been cloned from T.virens (Kim et al. ,2002) . Two types of N-acetyl-β-D-glucosaminidases belonging to family 20 of the glycosyl hydrolases have been identified in T.harzianum and T.virens. Chit36 is another antifungal chitinases recently isolated from T.harzianum

Multi hydrolytic enzymes are:

Cellulase Esterase Protease Chitinases β1,6 Glucanases β1,3 Glucanases Chitobiosidases N acetyl β Dglucosaminidase

Secondary Metabolites:

In order to survive and compete in their ecological niche, fungi apply not only enzymatic weapons but also have a potent arsenal for chemical warfare at their disposal (Vinale et al.2008). Thereby, not only the potential antibiotics (peptaibols) but also mycotoxins and more than 100 metabolites with antibiotic activity including polyketides, pyrones, terpenes, metabolites derived from amino acids, and polypeptides (Sivasithamparam and Ghisalberti,1998).

Associated Antimicrobials in Plant Health are:

Polyketides Alkyl pyrones Isonitriles Diketo piperazines Peptaibols

3. Competition and Rhizosphere Competence:

A mechanism that has gained adherents in recent years is that of competition through rhizosphere competence. Rhizosphere competence is important because a biocontrol agent cannot compete for space and nutrients if it is unable to grow in the rhizosphere . T.spp ,either added to the soil or applied as seed treatments, grow readily along with the developing root system of the treated plant. T.koningii that are excellent root colonizers exhibit little or no biocontrol activity against Rhizoctonia solani on cotton seedlings( C.R. Howell,unpublished). Competition occurs between microorganisms when space or nutrients (i.e., carbon,nitrogen and iron) are limiting and its role in the biocontrol of plant pathogens has been studied for many years with special emphasis on bacterial biocontrol agents(Weller1988). Some Trichoderma biological agents produce highly efficient siderophores that chelate iron and stop the growth of other fungi.

Fig: Colonization of root hairs of corn by the highly rhizosphere competent strain of T. harzianum T22. Used with permission of the American Phytopathological Society (Harman. 2000. Plant Disease 84:377-393)

Fig: Enhanced root development from field-grown corn and soybean plants as a consequence of root colonization by the rhizosphere competent strain T. harzianum T22.

Root colonization enhances root growth and development, crop productivity, resistance to abiotic stresses and the uptake and use of nutrients ( Arora et al.,1992).

Activity of biocontrol agents could also reduce the concentration of substances in soil that are inhibitory to plant growth (Windham et al.,1986).

Crop productivity in fields can increase upto 300% after addition of T.hamatum or T.koningii.

4. Tolerance to stress through enhanced root and plant development:

The plant-growth-promoting capacity of T.harzianum to solubilize in vitro some insoluble or sparingly soluble minerals via three possible mechanisms-

Acidification of the medium. Production of chelating metabolites. Redox activity( SOD and Antioxidant cycling enzymes) was recently investigated.

T.harzianum can solubilize MnO2, metallic zinc and rock phosphate(mostly calcium phosphate) .

FIG: Trichoderma initiating new root development in orchid.

Fig: Plant growth promotion effects of Trichoderma spp. strains on: pepper (top), lettuce (lower left), and tomato (lower right) plants grown in the greenhouse (Harman, 2006).

Fig: Effect of drought and Trichoderma colonization on rice seedling growth. Seedlings were grown in plastic pots in a controlled environment under glass house condition. The seedlings were either colonized with Trichoderma harzianum isolates Th 56, Th 69, Th 75, Th 82 and Th 89 or not colonized (Control). After transplanting at 21 d, seedlings were watered every other day for 42 d of growth in the plastic pots. After 42 d of growth, watering was stopped for subsequent days from 3 to 9 d (drought treatment), while control seedlings continued to be watered every other day. (A) 1 day before drought stress (B) 3 days drought stress (C) 5 days drought stress (D) 7 days drought stress (E) 9 days drought stress.

5. Induced Resistance:

Induced resistance is a plant response to challenge by microorganisms or abiotic agents such that following the inducing challenge de novo resistance to pathogens is shown in normally susceptible plants(de Wit 1985). Induced resistance can be localized, when it is detected only in the area immediately adjacent to the inducing factor or systemic, where resistance occurs subsequently at sites throughout the plant. Both localized and systemically induced resistances are non-specific.

The potential of T. harzianum T-203 to trigger plant defense responses was investigated by inoculating roots of cucumber seedlings with Trichoderma in an aseptic hydroponic system(Yedidia et al.,1999). Trichoderma-treated plants were more developed than nontreated plants throughout the experiment. Electron microscopy of ultrathin sections from Trichoderma- treated roots revealed penetration of T.harzianum, mainly to the epidermis and outer cortex. Strengthening of the epidermal and cortical cell walls was observed, as well as deposition of newly formed barriers. These typical host –reactions were found beyond the sites of potential fungal penetration.These root fungus associations also stimulate plant defensive mechanisms.The addition of Trichoderma metabolites that may act-

As elicitors of plant resistance or the expression in transgenic plants of genes whose products act as elicitors

Also result in the synthesis of phytoalexins, PR proteins and other compounds and in an increase in resistance against several plant pathogens.

Fig1: Cultures of cotton roots from plants grown in Macrophomina phaseolina–infested soil and incubated at 25°C: A, roots from nontreated seed showing M. phaseolina, and B, roots from T.virens–treated seed showing only the biocontrol agent. Incubation of B at 40°C shows only M. phaseolina growing from cotton roots.

Mode of application of Trichoderma:

Seed Treatment Soil Application Foliar Application

Benefits of Seed Treatment:

1. Improve plant stands.2. Induce long-term improvements in plant quality.3. Easy to apply and cost effective etc.

Causal Agent of Diseases:

Causal Agent Disease Crop

T. Aggressivum (formerly T. harzianum biotype4)

Green mold disease Button mushroom

T.viride Green mold rot Onion

T. spp Respiratory problem, lethal threat to HIV infected persons and other immuno-compromised person

Human beings

Uses of Trichoderma:

As Industrial workhorse In Food Industry As a source of gene for crop improvement As Medicine

I. As Industrial workhorse: Shortly after the discovery of T.viride QM6a by the US army during World War II (Reese

1976), the outstanding efficiency of its cellulases led to extensive research toward industrial applications of these enzymes. Later on, this species was renamed T.reesei in honor of Elwyn T.Reese (Simmons 1977) and became the most important cellulase producer worldwide. Until now, this species is the most important one of the genus for industrial purposes. T. reesei is a

producer of heterologous proteins started more than 20 years ago with the production of calf chymosin.(Harkki et al.1989;Uusitalo et al.1991)

II. In Food Industry: Trichoderma spp. have been extensively applied for the production of food additives

and related products. Trichoderma enzymes are applied to improve the brewing process (β-glucanases),as macerating enzymes in fruit juice production(pectinases,cellulases,hemicellulases), as feed additive in livestock farming (xylanases) and for pet food. The first product isolated from T.viride was a chemical with characteristics coconut –like aroma, a 6-pentyl-α-pyrone with antibiotic properties.

III. Trichoderma as a source of genes for crop improvement: The use of potent chitinases with proven antifungal activity is thus an attractive

alternative. Because Ech42 from T.atroviride fulfills these criteria, the corresponding gene was introduced into tobacco and potato(Lorito et al.1998). An endochitinase or an exochitinase both from Trichoderma into apple plants(Bolar et al.2001). Plants expressing both enzymes simultaneously were more tolerant to V.inequalis that plants expressing either enzyme alone.

IV. Medical use of Trichoderma: Cyclosporine A (CsA), a calcineurin inhibitor produced by the fungi Trichoderma

polysporum and Cylindrocarpon lucidum, is an immunosuppressant prescribed in organ transplants to prevent rejection.

Mass production techniques Followed in India:

PDA and PDB preparation Maintenance of mother culture Multiplication of mother culture Inoculation of mother culture

Liquid fermentation is followed for multiplication of T.spp

Preparation of MYM Sterilization Inoculation Incubation Blending Quality control Package and labeling

Procedure for mass production of T.spp :

Autoclave both starter and fermented medium for 1 h. Inoculate starter medium with efficient strain of Trichoderma and shake on rotary shaker

Inoculate fermented media with starter inoculums and supply regularly ait for days by shaking/bubbling.

Filter through cotton muslin using funnel. Air dry or freeze dry the mat for 3 days fine grind into powder this contain mycelium and

chlamydospores. Powder is diluted with different diluents and used as dusts, granules, pellets, wettable powders,

emulsifiable concentration. Produce is viable for 6 months, if stored at 5˚c and 3 months if stored at 20˚c . Maximum

biomass is obtained generally after 15 days of inoculation.

How Trichoderma benefitted in Agriculture:

Control of root and foliar pathogens. a) Induced resistanceb) Biological control of diseases by direct attack of plant pathogenic fungi.

Changes in the micro-floral composition on roots. Enhanced nutrient uptake. Enhanced solubilization of soil nutrients. Enhanced root development. Increased root hair formation.

Use of Trichoderma in Agriculture:

Crop Diseases and Nematodes Biocontrol Agent

Pigeon pea,cowpea, groundnut, gingelly,mulberry,cotton

Fusarium oxysporum f.sp. udum ,Macrophomina phaseolina

T. viride (10g/Kg)

Cotton Rotylenchulus reniformis+ M.phaseolina complex

T. viride (ST + SA)

Red gram ,Black gram Green gram Heterodera cajani Consortia of Pseudomonas and T. viride (ST)

Blackgram,Greengram,sunflower, groundnut,cotton

Rhizoctonia bataticola T. viride (ST)

Chickpea R.solani T. viride (ST)

Mulberry Fusarium moniliformae T. viride

Sugarcane Colletotrichum falcatum Ceratocystis paradoxa

T. harzianum – Sett treatment (10g/Litre)

Tobacco Pythium aphanidermatum T. viride (4g/Kg)Tomato Pythium aphanidermatum

Fusarium oxysporum f.sp. lycopersici

T. viride (10g/Kg) Soil – 2.5 Kg/ha Flowering, fruit formation

Brinjal Pythium aphanidermatum Rhizoctonia bataticola

T. viride (10g/Kg) Biohardening

Chillies Pythium aphanidermatum Colletotrichum capsici

T. viride (10g/Kg) Biohardening

Betelvine Phytophthora parasitica T. viride (50g/vine in Sorghum based)

Coconut Ganoderma lucidum T. viride (100g/tree)Fenugreek Root rot – R.solani Seed

Treatment,Soil Application4g/Kg (106 cfu/g) 1.5Kg/ha with 250Kg NC

Black Pepper – Foot rot Phytophthora capsici – Nursery Pre Plant/pit Field Application

T. harzianum 0.2% drenching 1 Kg NC+ T.V 50g/Pit 50g/vine – Twice

Pulses and Oil seeds Root rot – Seed Treatment and Soil Application

4g/Kg ,2.5 Kg/ha in 250Kg FYM

Ginger Dry rot – M.phaseolina Rhizome rot - Pythium ,Soil Application

TH -50g/Plant (Thrice – Multiplied in Sorghum grains)

Cardamom Phytophthora meadii – Field Application

TH or TV 50g/vine – Twice (Sorghum grains)

Turmeric Rhizome rot ,Soil Application TH or TV 2.5 Kg/ha + 250 Kg FYM on 3, 5, and 7th month

Cumin and Fenugreek Root rot – Seed Treatment, Soil Application

TH or TV 10g/Kg ,2.5 Kg/ha in 250Kg FYM

For biohardening –Trichoderma: Bio-hardening – Trichoderma – Protected Cultivation Mixing in Nursery mix: 1Kg/100 Kg of DCP. Soil Drenching: 5g/Litre .

For Bio-Fertigation- 1Kg of Trichoderma/1000 sq.ft. Frequency : At monthly intervals. Delivery sysytem : Through Drips.

Soil Application – Trichoderma – 2.5 Kg/ha for Annual crops Coconut and Arecanut – 100g/tree Betelvine – 50g/vine Noni – 100g .

Average requirement of T. spp. in India:- Four lakh tons(TNAU) .

Future prospects:

Production of second generation biofuels from agricultural, waste products by the aid of cellulases and hemicellulases produced by T.reesei and fermentation of the resulting oligosaccharides by yeast provides an alternative strategy(Merino and Cherry 2007).

Sustainability is also the major driving force for investigation of biocontrol with Trichoderma. As opportunistic plant symbionts and effective mycoparasites, numerous species of this genus have the potential to become commercial biofungicides.

Besides these major applications of T. spp., also the fields of green and white biotechnology become increasingly important for environmentally safe production of enzymes and antibiotics.

The extensive studies on diverse physiological traits available and still progressing for Trichoderma make these fungi versatile model organisms for research on both industrial fermentations as well as natural phenomena.

ASSAM AGRICULTURAL UNIVERSITY- (DEPARTMENT OF PLANT PATHOLOGY)

No. of M.Sc and phD Thesis submitted on Trichoderma (Biological Control):- 37 1st thesis on Trichoderma in AAU- “Potentiality of T.harzianum as a biocontrol agent of S. rolfsii

Sacc. Causing Basal rot of betel vine,Piper betel L. under field condition” (Shyam D. 1990).

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