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Trichoderma march 14th

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Pradnyarani Nidagundi

This document discusses the use of biocontrol agents, specifically Trichoderma species, for managing plant pathogens and diseases. Some key points: - Pathogens threaten global crop production and excessive fungicide use pollutes the environment and leads to resistance, so alternative biological control methods are needed. - Trichoderma is an effective biocontrol agent that controls pathogens through mycoparasitism, antibiosis, competition, and other mechanisms without environmental pollution. - Mass production of Trichoderma uses liquid fermentation or solid substrates like wheat bran to grow the fungus, which is then mixed with carriers like talc or vermiculite before application to seeds, soil, or plants.

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W E L C O M E
In agriculture, world wide, pathogens are threat to crop production (Sarah and Paul, 2005)  The extensive use of fungicides in various parts of the world for years has increased the pollution level in soil and water, and adverse effect on food quality and human health  Apart from this, the chemicals tend to become less efficient due to the development of resistance among the pathogen a over time Hence, it is necessary to look for alternative disease management practices, which include the use of eco-friendly biological control agents .
KITTUR RANI CHANNAMMA COLLEGE OF HORTICULTURE, ARABHAVI - 591 218 Presented by: PRADNYARANI P. N USH11PGM135 DEPT. OF HORTICULTURE PLANT PATHOLOGY 3
What are BIO-CONTROL AGENTS ? Control of plant pathogens and diseases caused by them through antagonistic microorganisms or botanicals is termed biological control agents  According to Baker and Cook’s (1974) defn:- “Biological control is the reduction of inoculum or disease producing activity of a pathogen accomplished by or through one or more organisms other than man.”  Antagonistic microorganisms like species of Trichoderma, Penicillium, Bacillus, Pseudomonas etc.
Trichoderma is….  Very effective biological agent  Free living  Ubiquitous  Highly proliferating  Non- pollutive  Easily accessible  Non phytotoxic  Systemic ephemeral  Readily biodegradable  Cost effective  Synergistic effect  Longer shelf life  Greater compatibility
History of Trichoderma 1671 – First found in Germany 1794 – Identified by Persoon almost 218 years ago 1927 – Gilman and Abbott recognized four species based on colour, shape of conidia and colony appearance >75 years ago the potential use of Trichoderma by Weindling (1932) and first to demonstrate the parasitic activity in wilt of Pigeon pea Best known mycoparasite against many soil borne plant pathogens
Trichoderma  Free living fungus common in soil and root ecosystem  Highly interactive in root, soil and foliar environment  Suppresses the pathogen by different mechanism of biocontrol Trichoderma harzianum
Taxonomical position of Trichoderma Kulkarni and Sagar (2007) mentioned the Trichoderma as asexual stage and Hypocrea as sexual stage Position Asexual stage (conidia) Sexual stage (ascospore) Kingdom Fungi Fungi Phylum Ascomycota Ascomycota Sub-division Deuteromycotina Ascomycotina Class Hyphomycetes Pyrenomycetes Order Monilliales Sphariales Family Monilliaceae Hypocreaceae Genus Trichoderma Hypocrea
General Characters of Trichoderma spp.  Cultures are fast growing at 25-30 C  Conidia forming within on week in compact or loose tufts in shades of green or yellow or less frequently white  Yellow pigment may be secreted into the agar, specially on PDA  A characteristic sweet or ‘coconut’ odour is produced by some species
Fig. 1 (A) Trichoderma on solid media (B) microscopic view (C) Trichoderma in liquid medium
Conidiophores Conidia Hyphae Morphological structure of Trichoderma
Fig 2: Photograph shows colonies of Trichoderma strains on PDA plate (dorsal view) and conidiophore with conidia. 1a & 1b. T. virens (IMI-392430), 2a & 2b. T. pseudokoningii (IMI-392431), 3a & 3b. T. harzianum (IMI-392432), 4a &4b. T. harzianum (IMI- 392433) and 5a & 5b.T. harzianum (IMI-392434).
Conidiophores characteristics of Trichoderma spp.  Highly branched, difficult to define or measure  Loosely or compactly tufted  Main branches of the conidiophores produce lateral side branches  The branches may rebranch, with the secondary branches and longest secondary branches being closest to the main axis
Fig3 : View of T. harzianum through a Stereo microscope (1mm to 10μm) Samuels et al., 2006, USA
Conidia  Typically appear dry but in some species they may be held in drops of clear green or yellow liquid (e.g. T. virens, T. flavofuscum) Round to oval in shape
Hypocrea teliomorph of Trichoderma spp. 1mm Mature perithecia 20 μm Viewed through stereo microscope Samuels et al., 2006, USA
Where do they come from? They can be easily isolated from soil, root, decaying wood and other forms of plant organic matter
Singh et al., 2007.
enzyme production Antibiotic production Mycoparas itism SAR Competition Growth promotion Effective antagonist Rapid substrate colonization Potential bio control activities exhibited by Trichoderma Kamala and Indira, 2012, Manipur
Competition  For space and nutrients under specific condition do not get substrate  Suppress growth of pathogen population e.g: Soil treatment with Trichoderma harzianum spore suppressed infestation of Fusarium oxysporum f. sp. vasinfectum and F. oxysporum f. sp. melonis (Perveen and Bokhari, 2012) Mechanisms of action
Mycoparasitism  Antagonist fungi parasitize other pathogenic fungi  Hyphae of Trichoderma either grow along the host hyphae or coil around it E.g. : T. harzianum and T. hamatum were mycoparasite of both Scelerotium rolfsii and R. solani
Interaction –  Coiling of hyphae around the pathogen,  Vacuolization,  Penetration by haustoria and  lysis (Omero et al., 1999).  Recognize and attach to the pathogenic fungus and excrete extra-cellular lytic enzymes like β-1,3-glucanase, chitinase, proteases and lipase (Schlick et al., 1994).
Trichoderma coils around, penetrates, and kills other fungi that are pathogenic (i.e. cause disease) to crops. It can digest their cell walls A clear view with an electron microscope Trichoderma spp.(T) fungal strands coil (C) around the Rhizoctonia (R) Initial stages of degradation (D) as a result of Trichoderma generated enzymes. T: Trichoderma R: Rhizoctonia
Antibiosis  It is the condition in which one or more metabolites excreted by an organism have harmful effect on one or more other organisms  In such antagonistic relationship spp. A produces a chemical substance that is harmful to Spp. B without a Spp. A deriving any direct benefit e.g: Trichoderma secreted - Trichodermin, viridine, Trichothecin, Sesqiterpine etc.
Growth inhibition of R. solani by the T. virens produced antibiotic gliotoxin . A: Gliotoxin amended B: non amended Cont…
Trichoderma strains solubilize phosphates and micronutrients The application of Trichoderma strains in rhizosphere of plants increases the number of deep roots, there by increasing the plants ability to resist drought Plant growth promoter
Fig.: Enhanced root development from field grown bean plants as a consequence of root colonization by the rhizosphere competent strain T. harzianum (Amin et al., 2010) Cont…
Evaluation under in vitro techniques  Dual culture or paired culture Upadhyay and Rai (1987)
Filter paper disc method
Procedure for isolation of Trichoderma from soil Isolation from soil on selective medium incubate 7 days at 250 C Sub culturing on PDA plates Purification Inoculation of purified culture on PDA slants Preservation in deep freezer (-200 C)
Mass production of biocontrol agents Liquid fermentation method Mix 30 gm molasses and 6gm Brewer’s yeast in 1 litre of water. Distribute 60 ml in each conical flask. autoclave Inoculate 8mm mycelial discs of Trichoderma in medium Incubate for 10 days at room temperature Use for multiplication in the fermentor Prepare 50 lit of molasses + yeast medium and sterilize for 30 min in the fermentor Transfer aseptically 1 lit of Trichoderma
Incubate for 10 days using haemocytometer (108 /ml spore) 500 ml of fungal biomass + 1 kg of talc powder Air dry & and carboxy methyl cellulose (CMC) + sticker 5 gm / kg Store in polythene bag
• Substrates for mass multiplication: wheat bran, wheat straw, FYM, press mud, coir pith, ground nut shell, rice bran, etc • Carrier/ food base materials: Talc, vermiculite, molasses, gypsum, kaolin, peat, sodium alginate, Cacl2
Advantages  Enhances yield along with quality of produce  Boost germination rate  Increase in shoot & Root length  Solubilising various insoluble forms of Phosphates  Augment Nitrogen fixing  Promote healthy growth in early stages of crop  Increase Dry matter Production substantially
 Harmless to humans and livestock  Act against a wide range of pathogenic fungi  Perpetuate themselves by producing ample spores  Grow rapidly and quickly colonize the soil They can promote nutrient uptake and enhance plant growth Provide natural long term immunity to crops and soil.
Disadvantages • Harmful parasite of mushrooms • Looses its effectivity if not placed in its native condition. • It cannot be used as foliar spray • It do not grow in alkaline pH (above 8). • Zone specific & slow growth
Methods of application 1. Seed treatment: Mix 6 - 10 g of Trichoderma powder per Kg of seed before sowing. 2. Nursery treatment: Apply 10 - 25 g of Trichoderma powder per 100 m2 of nursery bed. Application of neem cake and FYM before treatment increases the efficacy. 3. Cutting and seedling root dip: Mix 10g of Trichoderma powder along with 100g of well rotten FYM per litre of water and dip the cuttings and seedlings for 10 minutes before planting.
4. Soil treatment: Apply 5 Kg of Trichoderma powder per ha after turning of sun hemp or dhaincha into the soil for green manuring Or Mix 1kg of Trichoderma formulation in 100kg of farmyard manure and cover it for 7 days with polythene. Sprinkle the heap with water intermittently. Turn the mixture in every 3-4 days interval and then broadcast in the field. 5. Plant Treatment: Drench the soil near stem region with 10g Trichoderma powder mixed in a litre of water 6. Wound application 7. Furrow application Kulkarni and Sagar, 2007
Precautions Don't use chemical fungicide after application of Trichoderma for 4-5 days. Don't use Trichoderma in dry soil. Moisture is a essential factor for its growth and survivability. Don't put the treated seeds in direct sun rays. Don't keep the treated FYM for longer duration.
Compatibility Compatible with Organic manure, biofertilizers like Rhizobium, Azospirillum, Mycorrhizae, Azotobacter, Bacillus Subtilis and Phosphobacteria, Gliocladium virens, Pseudomonas fluorescens Trichoderma can be applied to seeds treated with Metalaxyl or Captan, Carboxin, Carbendazium but not Mercurials. Kulkarni and Sagar, 2007
Case studies
Fig1: Incidence of F. oxysporum f.sp. cepae in onion sets raised from seeds treated with procholaz and Trichoderma harzianum in artificially pathogen-inoculated pot soil. C+: sets raised from non-treated seeds in pot soil inoculated with FOC16. C-: sets raised from non treated seeds in non inoculated pot soil. Bars topped by the same letter do not differ significantly according to the Tukey-Kramer test at P<0.05 Coskuntuna and Ozer., 2008, Turkey
Fig2: Incidence of F.oxysporum f.sp. cepae sets raised from seeds treated with prochloraz and Trichoderma harzianum in naturally pathogen infested field soil. Control sets raised from seeds in field soil infested with FOC. Bars topped by the same letter do not differ significantly according to the Tukey-Kramer test at P<0.05. Coskuntuna and Ozer., 2008, Turkey
Fig3: Antagonistic activity of Trichoderma against different pathogens. A. R. solani. B. F. oxysporum, C. P. ultimum and D. P. aphanidermatum Kamala and Indira., 2012, Manipur
Fig4: Variation of the ratio of late blight infected leaves with respect to the leaf position on the main stem. The data were taken at the 14th day after the foliar inoculation of P. infestans. The values calculated as ratio of infected to total leaves on the main stem. Zegeye et al., 2011, Ethiopia
Storage days Talc Vermicompost Mean Room temperature Refrigerator temperature Mean Room temperature Refrigerator temperature Mean 30 143.33 132.00 137.67 186.00 147.00 166.50 152.08 60 113.00 89.00 101.00 187.00 89.00 138.00 119.50 90 98.00 77.00 87.50 152.00 76.33 114.17 100.83 120 68.00 64.00 66.00 108.00 41.33 74.67 70.33 150 46.00 39.00 42.50 51.00 27.33 39.17 40.83 180 28.00 25.00 26.00 24.00 16.00 20.00 23.25 Mean 82.72 71.00 76.86 118.00 66.17 92.08 84.47 Table1: Effect of different carrier materials on shelf life ( 106 cfu g -1 ) of T. harzianum (Th-2) at Different temperatures Bheemaraya et al., 2011, Raichur Comparing of means C.D @ 1% Carrier(A) 2.005 Temperature(B) 2.005 Storage days(C) 3.472 AXB 2.835 AXC 4.911 BXC 4.911 AXBXC 6.945 Room Temperature=28 1oc Refrigerated temperature=4 1oc
Storage days Agro-wastes/by-products( 106 cfu g-1) Sand maize meal Rice husk Saw dust Groundn ut cake Castor cake Mean 30 39.00 65.67 3.70 32.33 38.00 35.74 60 38.67 64.00 3.67 31.00 35.67 34.60 90 38.00 62.33 3.53 30.33 34.67 33.77 120 5.63 53.00 2.97 25.67 24.00 22.25 150 0.64 1.27 2.23 9.67 14.67 5.69 180 0.53 0.70 1.63 7.67 7.83 3.67 Mean 20.41 41.16 2.96 22.78 25.81 22.62 Table2: Effect of different agro-wastes/by-products on shelf life of Trichoderma piluliferum (Tp) Bheemaraya et al., 2011, Raichur C.D.at 1% Agro wastes(A) 0.439 Storage days(B) 0.481
Bio-agents Percent inhibition of mycelial growth* T. viride (TV-3) T. harzianum (TH-2) T. piluliferum (TP) B. subtilis (E) 100.00 (89.99)** 0.00 (0.00) 0.00 (0.00) P. flourescens (I) (Pf-4) 100.00 (89.99) 0.00 (0.00) 0.00 (0.00) A. quisqualis (E) 10.67 (18.63) 0.00 (0.00) 10.00 (18.42) control 0.00 (0.000 0.00 (0.00) 0.00 (0.00) mean 52.66 (49.65) 0.00 (0.00) 2.50 (4.6) C.D.at 1% 1.10 NS 0.69 Table3: Effect of different bio-agents on compatibility of Trichoderma spp. Bheemaraya et al., 2011, Raichur
Treatment Percent inhibition of mycelial growth* Mean Concentration(%) 2.5 5.0 T1- NSKE 0.00 (0.00)** 0.37 (2.02) 0.19 (1.01) T2 -Nimbicidine 81.48 (64.51) 83.70 (66.19) 82.59 (65.35) T3- Prosophis leaf extract 48.15 (43.93) 57.04 (49.04) 52.59 (46.49) T4- Pongamia leaf extract 0.37 (2.02) 10.37 (18.78) 5.37 (10.40) T5- Eucalyptus leaf extract 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) T6- control 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) Mean 21.67 (18.41) 25.25 (22.67) 23.46 (20.54) Table4: Effect of different plant extracts on compatibility of Trichoderma harzianum (Th-2) Bheemaraya et al., 2011, Raichur S.EM C.D.at 1% Plant extracts (P) 0.60 2.35 Concentration (C) 0.34 1.36
Treatment Percent inhibition of mycelial growth* Mean Concentration(%) 0.1 0.2 T1- Mancozeb 0.37 (2.02)** 5.19 (13.14) 2.78 (7.58) T2 - Carbendazim 100.00 (89.99) 100.00 (89.99) 100.00 (89.99) T3- Captan 100.00 (89.99) 100.00 (89.99) 100.00 (89.99) T4- Propiconozole 100.00 (89.99) 100.00 (89.99) 100.00 (89.99) T5- Metalaxyl- m+mancozeb 1.85 (4.54) 11.48 (19.59) 6.67 (12.07) T6- Control 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) Mean 50.37 (46.09) 52.78 (50.45) 51.57 (48.27) Table5: Effect of different fungicides on compatibility of Trichoderma harzianum (Th-2) Bheemaraya et al., 2011, raichur S.EM C.D.at 1% Fungicide (F) 1.12 4.42 Concentration (C) 0.65 2.55
Treatment Percent inhibition of mycelial growth* Mean Concentration(%) 0.1 0.2 T1-Chloropyriphos 96.30 (83.50) 100.00 (89.99) 98.15 (86.74) T2 –Carbofuron 100.00 (89.99) 100.00 (89.99) 100.00 (89.99) T3- Indoxocarb 87.78 (69.69) 100.00 (89.99) 93.89 (79.84) T4- Imidachloprid 12.96 (21.09) 59.26 (50.33) 36.11 (35.71) T5- Control 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) Mean 59.41 (52.85) 71.85 (64.06) 65.63 (58.46) Table6: Effect of different insecticides on compatibility of Trichoderma harzianum (Th-2) Bheemaraya et al., 2011, Raichur S.EM C.D.at 1% Insecticides (I) 1.53 6.14 Concentration (C) 0.96 3.88 Mean of four replications, **Figures in parentheses are arcsine transformed values
Table7: Effect of soil treatment with formulated Trichoderma species on incidence of fusarium wilt disease of giza 3 bean cultivar under greenhouse and field conditions. Nashwa et al., 2008, Egypt Wilt rating under green house conditions TREATMENT Time of application TH 1** TV 1 TS 3 infected control Mean Two weeks before planting 3.7 4.1 5.2 8.0 5.3 At time of planting 2.5 3.1 4.0 7.0 4.2 Mean 3.1 3.6 4.6 7.5 L.S.D at 0.05 time of application (A ):0.5 bioagents (B) :0.43. Interaction (AXB) :0.61 Wilt rating * under field conditions Time of application TH 1 TV1 TS 3 Infected control Mean Two weeks before planting 3.3 4.2 5.0 6.0 4.7 At time of planting 4.0 4.2 5.0 7.0 5.1 Mean 3.6 4.1 5.0 6.5 L.S.D at 0.05 time of application a :0.2 bioagents b :0.25. Interaction (AXB) :0.36 •According to CIAT scale (van schoonhoven and pastor-corrales,1987) TH 1: T. harzianum, TV 1: T. viride, TS 3: T. virens
Treatement Plant height(cm) Plant dry Weight(g) Disease incidence C 14.52a 2.45a 0.00a Fs 6.52b 1.06b 1.80b Fs+ Tv 12.68c 2.42a 1.06c Fs+ Th 14.18d 2.58a 0.80d Table8: Effect of T. harzianum and T. viride on height.dry weight and disease incidence of tomato plants inoculated with F. Solani under pot conditions. Perveen and Bokhari., 2012, Saudi Arabia Each value is average of six replicates. Data Followed by different letters in the column are significantly different (p< 0.05 ) according to Duncan’ s multiple range test . C, uninoculated control Fs ,F solani : Tv: T . viride , Th: T. harzianum Disease incidence graded on 0 to 3 scale where ,0= 25 %severity , 1 =26 to 50%, 2 = 51 to 75% and 3= 76 to 100%
Fig5: Pathogen growth inhibition by Trichoderma after 6 day of inoculation in dual culture Hajieghrari et al., 2008, Iran
Fig6: Pathogen growth inhibition Trichoderma volatile compounds Hajieghrari et al., 2008, Iran
Table9: Effect of pH and temperature on the mycelial growth(mm) of Trichoderma isolates Hajieghrari et al., 2008, Iran Treatment T. hamatum T612 T. harzianum T447 T. virens T523 T. harzianum T969 Trichoderma sp. T T. hamatum T614 pH 8 33.26* 23.89 41.15 31.07 30.19 33.78 pH 7 29.3 24.3 33.96 39.45 30.45 30.85 pH 5 33.78 34.8 45.52 34.7 32.22 27.68 30⁰c 29.15 17.2 43.67 35.41 36.89 31.37 25⁰c 36.04 38.89 37.56 36.44 35.3 30.96 20⁰c 31.14 26.89 39.41 33.63 31.67 29.96 *Values are means of four replicates.
Fig7 :Antagonistic activity of Trichoderma species against F.oxysporum evaluated by dual culture interaction (A)F.oxysporum alone , (B) F oxysporum +T.harzianum (T 1s), (C) F.oxysporum + T.viride (TvPDs) (D) F.oxysporum + T.harzianum(TDPs) Perveen and Bokhari, 2012, Saudi Arabia
Fig8: Inhibitory effect of the culture filtrate of Trichoderma spp. Incubated at different temperature (5, 15, 25, 35, 40⁰C). Each value is an average of three replicates.T1s= Trichodema harzianum isolate T1s, TvPD = T. viride isolate TvPD, TDPs = T. harzianum isolate TDPs Perveen and Bokhari, 2012, Saudi Arabia
Fig9: Average liner growth rate (ALG) of Trichoderma species on various culture media. Each value is an average of three replicates. T1s= Trichoderma harzianum isolate T1s, TvPDs= T. viride isolate TvPDs, TDPs=T harzianum isolate TDPs, PDA=Potato dextrose agar, SDA =Sabouraud dextrose agar, WA = Water agar (2% agar), CDA = Czapek dox agar, PDAL= natural media agar (PDA + 1% date palm leaves) Perveen and Bokhari, 2012, Saudi Arabia
Table10 : Effect of strains of Trichoderma species on the per cent inhibition of radial colony growth of P. aphanidermatum Mishra, 2010, India Trichoderma species Percent inhibition T.harzianum -4532 60.3 0.3 e T harzianum-4572 69.8 0.3 g T.viride -801 54.1 0.5 c T.viride-1763 52.2 0.5 b T.viride-1433 72.0 0.3 h T.viride-793 62.1 0.3 f T.Viride-2109 50.4 0.4 a T.koningii-2385 56.4 0.2 d T.virens-2023 53.5 0.6 c T.virens-2194 59.6 0.6 e Values are average of three replicates SEM Values in the column followed by same letter are not significantly different (P<0.05).
Fig10: Efficiency of P solubility and biocontrol activity of T .harzianum isolates against Xanthomonas sp. Padmavathi and Madhumathi, 2011, Banglore
Fig11: Effect of initial pH on chitinase and ß-1,3-glucanase production (using 0.5% chitin or laminarin as carbon source, respectively ) by T.harzianum Katatny et al., 2000, Egypt
Fig12: Effect of different carbon sources on chitinase and ß-1,3-glucanase production by T. harzianum and on inhibition of S.rolfsii (100 % ß-1,3-glucanase activity correspond to 14.7 nkat/mL and 100% chitinase activity correspond to 59.8 pKat/mL) Katatny et al., 2000, Egypt
Fig13: Release of total reducing sugars(R,S),glucose and N-acetyl glucosamine from S.rolfsii(dried and fresh mycelium),T.harzianum and chitin by the T.harzianum enzymes Katatny et al., 2000, Egypt
Table11: Evaluation of Trichoderma isolates against soil borne fungal pathogens using dual culture Amin et al., 2010, Jammu and Kashmir Treatment Radial growth (mm)of test pathogens R.solani S.rolfsii S.sclerotiorum Trichoderma virens(Ts-1) 46.55 (48.11) 36.26 (59.71) 56.19 (37.56) Trichoderma harzianum(Th-1) 35.43 (60.51) 34.67 (61.47) 39.08 (56.57) Trichoderma harzianum(Th-2) 43.32 (51.71) 35.33 (60.75) 55.69 (38.12) Trichoderma viride(Tv-1) 30.67 (65.71) 28.88 (67.91) 30.41 (66.21) Trichoderma viride(Tv-2) 25.65 (71.41) 32.00 (64.44) 34.28 (61.91) Trichoderma viride(Tv-3) 41.59 (53.64) 34.93 (61.18) 55.65 (38.16) Control 89.72 90.00 90.00 C.D.(P=0.05) 2.52 1.23 3.59 Figures in parenthesis are per cent inhibition values
Table12: Evaluation of Trichoderma isolates against production of sclerotia in soil fungal pathogens using dual culture Amin et al., 2010, Jammu and Kashmir Treatment Rhizoctonia solani Sclerotrum rolfsii Sclerotinia sclerotiorum Sclerotioal count Inhibition over control(%) Sclerotial count Inhibition over control(%) Sclerotial count Inhibition over control(%) (Ts-1) 35.59 66.63 38.66 67.60 19.09 39.70 (Th-1) 23.66 77.81 28.73 75.92 12.07 61.87 (Th-2) 31.73 70.25 34.29 71.26 18.12 42.76 (Tv-1) 17.33 83.75 23.64 80.18 9.45 70.15 (Tv-2) 19.47 81.75 26.07 78.15 11.12 64.87 (Tv-3) 27.25 74.45 33.78 71.69 15.57 50.82 Control 106.66 - 119.3 - 31.66 - C.D.(P=0.05) 1.89 2.07 0.98
Fig14: The level of hydrolytic enzymes activities from three different samples E2- extracts from Botrytis mycelia;E3-extracts from Trichoderma mycelia :E4:Extract from a mixture of pathogen and antagonistic strains Cornea et al., 2009, Romania
Table13: Evaluation of volatile metabolites produced by Trichoderma isolates against production of sclerotia in different pathogens Amin et al., 2010, Jammu and Kashmir Treatment R. solani S. rolfsii S. sclerotiorum Sclerotial count Inhibition over control (%) Sclerotial count Inhibition over control (%) Sclerotial count Inhibition over control(%) (Ts-1) 60.09 39.30 91.43 29.30 10.12 57.24 (Th-1) 42.67 56.89 75.66 41.49 6.11 74.18 (Th-2) 54.67 44.77 89.06 31.13 8.73 63.11 (Tv-1) 38.42 61.19 67.00 48.19 5.00 78.87 (Tv-2) 34.00 65.65 71.04 45.07 5.11 78.41 (Tv-3) 49.52 49.97 84.93 34.33 7.22 69.49 Control 99.00 - 129.33 - 23.67 - C. D (P=0.05) 3.13 4.76 0.91 Figures in parenthesis are percent inhibition values
Fig16:Compatibility test between T. viride and P. fluorescens. The picture was taken on the 9th day after dual inoculation. Zegeye et al., 2011, Ethiopia
Table14 : Effect of foliar application of T.viride and P.fluorescens on the progress of late blight disease of potato Zegeye et al., 2011, Ethiopia. Treatments Mean AUDPC T. viride 260.0 190.0 c P. fluorescens 765.1 218.6 b Mixed culture 999.0 274.5 a Mancozeb 85.9 77.8 cd Negative control(inoculated/untreated) 1045.1 227.2 a Positive control (non- inoculated/untreated) 0.00 0.00 d Means followed by the same letter are not significantly different. The AUDPC was calculated from five consecutive weekly assessment of percentage of leaf area with symptoms of late blight. The nine replicates were arranged in a CRB design and the midpoint rule was used to calculate AUDPC values.
Mechanism of action against Phytonematodes • Secretion of Lytic enzyme chitinase help parasitism of Meloidogyne and Globodera eggs • T. viride releases Dermadin helps in destruction of nematode cuticle • Trichoderma spp. have high rhizosphere competency and easily colonize the roots, reduce the feeding sites for nematodes Jonathan, 2010, New Delhi
Healthy egg of Heterodera glycines Egg parasitized By fungus
 There are several reputable companies that manufacture government registered products. Trade Name Bio agent Manufacture Eco fit T. viride Hoechst and Schering AgroEvo Ltd, Mumbai India Super visit T. harzianum Fytovita, Czech Republic Soil guard T. virens Certis Inc,Columbia,MD,USA Root pro T. harzianum Efal Agri, Netanyl,Israel Tusal T. Viride +T. harzianum Tusal Carrera Ester, Lleida Spain Agroderma, Bio-cure, Bioderma, Ecofit, Rakshak, Trichosan Trichoderma viride Biocure (B&F) T. Viride and P. flourescens
Formulations  Powder formulations  Encapsulation in organic polymer like sodium alginate  As spray from emulsifiable concentrates  Molasses enriched clay granules  Pellating biomass and bran with sodium alginate
The use of Trichoderma has gained importance in managing most of the plant pathogens. However, there is still considerable interest in finding more efficient mycoparasitic fungi especially within Trichoderma harzianum strains, which differ with respect to their biocontrol effectiveness. The technique for mass production and use of these bio agents have been commercialized for the purpose of producers and farmers.
U

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Trichoderma march 14th

  • 2. In agriculture, world wide, pathogens are threat to crop production (Sarah and Paul, 2005)  The extensive use of fungicides in various parts of the world for years has increased the pollution level in soil and water, and adverse effect on food quality and human health  Apart from this, the chemicals tend to become less efficient due to the development of resistance among the pathogen a over time Hence, it is necessary to look for alternative disease management practices, which include the use of eco-friendly biological control agents .
  • 3. KITTUR RANI CHANNAMMA COLLEGE OF HORTICULTURE, ARABHAVI - 591 218 Presented by: PRADNYARANI P. N USH11PGM135 DEPT. OF HORTICULTURE PLANT PATHOLOGY 3
  • 4. What are BIO-CONTROL AGENTS ? Control of plant pathogens and diseases caused by them through antagonistic microorganisms or botanicals is termed biological control agents  According to Baker and Cook’s (1974) defn:- “Biological control is the reduction of inoculum or disease producing activity of a pathogen accomplished by or through one or more organisms other than man.”  Antagonistic microorganisms like species of Trichoderma, Penicillium, Bacillus, Pseudomonas etc.
  • 5. Trichoderma is….  Very effective biological agent  Free living  Ubiquitous  Highly proliferating  Non- pollutive  Easily accessible  Non phytotoxic  Systemic ephemeral  Readily biodegradable  Cost effective  Synergistic effect  Longer shelf life  Greater compatibility
  • 6. History of Trichoderma 1671 – First found in Germany 1794 – Identified by Persoon almost 218 years ago 1927 – Gilman and Abbott recognized four species based on colour, shape of conidia and colony appearance >75 years ago the potential use of Trichoderma by Weindling (1932) and first to demonstrate the parasitic activity in wilt of Pigeon pea Best known mycoparasite against many soil borne plant pathogens
  • 7. Trichoderma  Free living fungus common in soil and root ecosystem  Highly interactive in root, soil and foliar environment  Suppresses the pathogen by different mechanism of biocontrol Trichoderma harzianum
  • 8. Taxonomical position of Trichoderma Kulkarni and Sagar (2007) mentioned the Trichoderma as asexual stage and Hypocrea as sexual stage Position Asexual stage (conidia) Sexual stage (ascospore) Kingdom Fungi Fungi Phylum Ascomycota Ascomycota Sub-division Deuteromycotina Ascomycotina Class Hyphomycetes Pyrenomycetes Order Monilliales Sphariales Family Monilliaceae Hypocreaceae Genus Trichoderma Hypocrea
  • 9. General Characters of Trichoderma spp.  Cultures are fast growing at 25-30 C  Conidia forming within on week in compact or loose tufts in shades of green or yellow or less frequently white  Yellow pigment may be secreted into the agar, specially on PDA  A characteristic sweet or ‘coconut’ odour is produced by some species
  • 10. Fig. 1 (A) Trichoderma on solid media (B) microscopic view (C) Trichoderma in liquid medium
  • 12. Fig 2: Photograph shows colonies of Trichoderma strains on PDA plate (dorsal view) and conidiophore with conidia. 1a & 1b. T. virens (IMI-392430), 2a & 2b. T. pseudokoningii (IMI-392431), 3a & 3b. T. harzianum (IMI-392432), 4a &4b. T. harzianum (IMI- 392433) and 5a & 5b.T. harzianum (IMI-392434).
  • 13. Conidiophores characteristics of Trichoderma spp.  Highly branched, difficult to define or measure  Loosely or compactly tufted  Main branches of the conidiophores produce lateral side branches  The branches may rebranch, with the secondary branches and longest secondary branches being closest to the main axis
  • 14. Fig3 : View of T. harzianum through a Stereo microscope (1mm to 10μm) Samuels et al., 2006, USA
  • 15. Conidia  Typically appear dry but in some species they may be held in drops of clear green or yellow liquid (e.g. T. virens, T. flavofuscum) Round to oval in shape
  • 16. Hypocrea teliomorph of Trichoderma spp. 1mm Mature perithecia 20 μm Viewed through stereo microscope Samuels et al., 2006, USA
  • 17. Where do they come from? They can be easily isolated from soil, root, decaying wood and other forms of plant organic matter
  • 18. Singh et al., 2007.
  • 20. Competition  For space and nutrients under specific condition do not get substrate  Suppress growth of pathogen population e.g: Soil treatment with Trichoderma harzianum spore suppressed infestation of Fusarium oxysporum f. sp. vasinfectum and F. oxysporum f. sp. melonis (Perveen and Bokhari, 2012) Mechanisms of action
  • 21. Mycoparasitism  Antagonist fungi parasitize other pathogenic fungi  Hyphae of Trichoderma either grow along the host hyphae or coil around it E.g. : T. harzianum and T. hamatum were mycoparasite of both Scelerotium rolfsii and R. solani
  • 22. Interaction –  Coiling of hyphae around the pathogen,  Vacuolization,  Penetration by haustoria and  lysis (Omero et al., 1999).  Recognize and attach to the pathogenic fungus and excrete extra-cellular lytic enzymes like β-1,3-glucanase, chitinase, proteases and lipase (Schlick et al., 1994).
  • 23. Trichoderma coils around, penetrates, and kills other fungi that are pathogenic (i.e. cause disease) to crops. It can digest their cell walls A clear view with an electron microscope Trichoderma spp.(T) fungal strands coil (C) around the Rhizoctonia (R) Initial stages of degradation (D) as a result of Trichoderma generated enzymes. T: Trichoderma R: Rhizoctonia
  • 24. Antibiosis  It is the condition in which one or more metabolites excreted by an organism have harmful effect on one or more other organisms  In such antagonistic relationship spp. A produces a chemical substance that is harmful to Spp. B without a Spp. A deriving any direct benefit e.g: Trichoderma secreted - Trichodermin, viridine, Trichothecin, Sesqiterpine etc.
  • 25. Growth inhibition of R. solani by the T. virens produced antibiotic gliotoxin . A: Gliotoxin amended B: non amended Cont…
  • 26. Trichoderma strains solubilize phosphates and micronutrients The application of Trichoderma strains in rhizosphere of plants increases the number of deep roots, there by increasing the plants ability to resist drought Plant growth promoter
  • 27. Fig.: Enhanced root development from field grown bean plants as a consequence of root colonization by the rhizosphere competent strain T. harzianum (Amin et al., 2010) Cont…
  • 28. Evaluation under in vitro techniques  Dual culture or paired culture Upadhyay and Rai (1987)
  • 30. Procedure for isolation of Trichoderma from soil Isolation from soil on selective medium incubate 7 days at 250 C Sub culturing on PDA plates Purification Inoculation of purified culture on PDA slants Preservation in deep freezer (-200 C)
  • 31. Mass production of biocontrol agents Liquid fermentation method Mix 30 gm molasses and 6gm Brewer’s yeast in 1 litre of water. Distribute 60 ml in each conical flask. autoclave Inoculate 8mm mycelial discs of Trichoderma in medium Incubate for 10 days at room temperature Use for multiplication in the fermentor Prepare 50 lit of molasses + yeast medium and sterilize for 30 min in the fermentor Transfer aseptically 1 lit of Trichoderma
  • 32. Incubate for 10 days using haemocytometer (108 /ml spore) 500 ml of fungal biomass + 1 kg of talc powder Air dry & and carboxy methyl cellulose (CMC) + sticker 5 gm / kg Store in polythene bag
  • 33. • Substrates for mass multiplication: wheat bran, wheat straw, FYM, press mud, coir pith, ground nut shell, rice bran, etc • Carrier/ food base materials: Talc, vermiculite, molasses, gypsum, kaolin, peat, sodium alginate, Cacl2
  • 34. Advantages  Enhances yield along with quality of produce  Boost germination rate  Increase in shoot & Root length  Solubilising various insoluble forms of Phosphates  Augment Nitrogen fixing  Promote healthy growth in early stages of crop  Increase Dry matter Production substantially
  • 35.  Harmless to humans and livestock  Act against a wide range of pathogenic fungi  Perpetuate themselves by producing ample spores  Grow rapidly and quickly colonize the soil They can promote nutrient uptake and enhance plant growth Provide natural long term immunity to crops and soil.
  • 36. Disadvantages • Harmful parasite of mushrooms • Looses its effectivity if not placed in its native condition. • It cannot be used as foliar spray • It do not grow in alkaline pH (above 8). • Zone specific & slow growth
  • 37. Methods of application 1. Seed treatment: Mix 6 - 10 g of Trichoderma powder per Kg of seed before sowing. 2. Nursery treatment: Apply 10 - 25 g of Trichoderma powder per 100 m2 of nursery bed. Application of neem cake and FYM before treatment increases the efficacy. 3. Cutting and seedling root dip: Mix 10g of Trichoderma powder along with 100g of well rotten FYM per litre of water and dip the cuttings and seedlings for 10 minutes before planting.
  • 38. 4. Soil treatment: Apply 5 Kg of Trichoderma powder per ha after turning of sun hemp or dhaincha into the soil for green manuring Or Mix 1kg of Trichoderma formulation in 100kg of farmyard manure and cover it for 7 days with polythene. Sprinkle the heap with water intermittently. Turn the mixture in every 3-4 days interval and then broadcast in the field. 5. Plant Treatment: Drench the soil near stem region with 10g Trichoderma powder mixed in a litre of water 6. Wound application 7. Furrow application Kulkarni and Sagar, 2007
  • 39. Precautions Don't use chemical fungicide after application of Trichoderma for 4-5 days. Don't use Trichoderma in dry soil. Moisture is a essential factor for its growth and survivability. Don't put the treated seeds in direct sun rays. Don't keep the treated FYM for longer duration.
  • 40. Compatibility Compatible with Organic manure, biofertilizers like Rhizobium, Azospirillum, Mycorrhizae, Azotobacter, Bacillus Subtilis and Phosphobacteria, Gliocladium virens, Pseudomonas fluorescens Trichoderma can be applied to seeds treated with Metalaxyl or Captan, Carboxin, Carbendazium but not Mercurials. Kulkarni and Sagar, 2007
  • 42. Fig1: Incidence of F. oxysporum f.sp. cepae in onion sets raised from seeds treated with procholaz and Trichoderma harzianum in artificially pathogen-inoculated pot soil. C+: sets raised from non-treated seeds in pot soil inoculated with FOC16. C-: sets raised from non treated seeds in non inoculated pot soil. Bars topped by the same letter do not differ significantly according to the Tukey-Kramer test at P<0.05 Coskuntuna and Ozer., 2008, Turkey
  • 43. Fig2: Incidence of F.oxysporum f.sp. cepae sets raised from seeds treated with prochloraz and Trichoderma harzianum in naturally pathogen infested field soil. Control sets raised from seeds in field soil infested with FOC. Bars topped by the same letter do not differ significantly according to the Tukey-Kramer test at P<0.05. Coskuntuna and Ozer., 2008, Turkey
  • 44. Fig3: Antagonistic activity of Trichoderma against different pathogens. A. R. solani. B. F. oxysporum, C. P. ultimum and D. P. aphanidermatum Kamala and Indira., 2012, Manipur
  • 45. Fig4: Variation of the ratio of late blight infected leaves with respect to the leaf position on the main stem. The data were taken at the 14th day after the foliar inoculation of P. infestans. The values calculated as ratio of infected to total leaves on the main stem. Zegeye et al., 2011, Ethiopia
  • 46. Storage days Talc Vermicompost Mean Room temperature Refrigerator temperature Mean Room temperature Refrigerator temperature Mean 30 143.33 132.00 137.67 186.00 147.00 166.50 152.08 60 113.00 89.00 101.00 187.00 89.00 138.00 119.50 90 98.00 77.00 87.50 152.00 76.33 114.17 100.83 120 68.00 64.00 66.00 108.00 41.33 74.67 70.33 150 46.00 39.00 42.50 51.00 27.33 39.17 40.83 180 28.00 25.00 26.00 24.00 16.00 20.00 23.25 Mean 82.72 71.00 76.86 118.00 66.17 92.08 84.47 Table1: Effect of different carrier materials on shelf life ( 106 cfu g -1 ) of T. harzianum (Th-2) at Different temperatures Bheemaraya et al., 2011, Raichur Comparing of means C.D @ 1% Carrier(A) 2.005 Temperature(B) 2.005 Storage days(C) 3.472 AXB 2.835 AXC 4.911 BXC 4.911 AXBXC 6.945 Room Temperature=28 1oc Refrigerated temperature=4 1oc
  • 47. Storage days Agro-wastes/by-products( 106 cfu g-1) Sand maize meal Rice husk Saw dust Groundn ut cake Castor cake Mean 30 39.00 65.67 3.70 32.33 38.00 35.74 60 38.67 64.00 3.67 31.00 35.67 34.60 90 38.00 62.33 3.53 30.33 34.67 33.77 120 5.63 53.00 2.97 25.67 24.00 22.25 150 0.64 1.27 2.23 9.67 14.67 5.69 180 0.53 0.70 1.63 7.67 7.83 3.67 Mean 20.41 41.16 2.96 22.78 25.81 22.62 Table2: Effect of different agro-wastes/by-products on shelf life of Trichoderma piluliferum (Tp) Bheemaraya et al., 2011, Raichur C.D.at 1% Agro wastes(A) 0.439 Storage days(B) 0.481
  • 48. Bio-agents Percent inhibition of mycelial growth* T. viride (TV-3) T. harzianum (TH-2) T. piluliferum (TP) B. subtilis (E) 100.00 (89.99)** 0.00 (0.00) 0.00 (0.00) P. flourescens (I) (Pf-4) 100.00 (89.99) 0.00 (0.00) 0.00 (0.00) A. quisqualis (E) 10.67 (18.63) 0.00 (0.00) 10.00 (18.42) control 0.00 (0.000 0.00 (0.00) 0.00 (0.00) mean 52.66 (49.65) 0.00 (0.00) 2.50 (4.6) C.D.at 1% 1.10 NS 0.69 Table3: Effect of different bio-agents on compatibility of Trichoderma spp. Bheemaraya et al., 2011, Raichur
  • 49. Treatment Percent inhibition of mycelial growth* Mean Concentration(%) 2.5 5.0 T1- NSKE 0.00 (0.00)** 0.37 (2.02) 0.19 (1.01) T2 -Nimbicidine 81.48 (64.51) 83.70 (66.19) 82.59 (65.35) T3- Prosophis leaf extract 48.15 (43.93) 57.04 (49.04) 52.59 (46.49) T4- Pongamia leaf extract 0.37 (2.02) 10.37 (18.78) 5.37 (10.40) T5- Eucalyptus leaf extract 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) T6- control 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) Mean 21.67 (18.41) 25.25 (22.67) 23.46 (20.54) Table4: Effect of different plant extracts on compatibility of Trichoderma harzianum (Th-2) Bheemaraya et al., 2011, Raichur S.EM C.D.at 1% Plant extracts (P) 0.60 2.35 Concentration (C) 0.34 1.36
  • 50. Treatment Percent inhibition of mycelial growth* Mean Concentration(%) 0.1 0.2 T1- Mancozeb 0.37 (2.02)** 5.19 (13.14) 2.78 (7.58) T2 - Carbendazim 100.00 (89.99) 100.00 (89.99) 100.00 (89.99) T3- Captan 100.00 (89.99) 100.00 (89.99) 100.00 (89.99) T4- Propiconozole 100.00 (89.99) 100.00 (89.99) 100.00 (89.99) T5- Metalaxyl- m+mancozeb 1.85 (4.54) 11.48 (19.59) 6.67 (12.07) T6- Control 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) Mean 50.37 (46.09) 52.78 (50.45) 51.57 (48.27) Table5: Effect of different fungicides on compatibility of Trichoderma harzianum (Th-2) Bheemaraya et al., 2011, raichur S.EM C.D.at 1% Fungicide (F) 1.12 4.42 Concentration (C) 0.65 2.55
  • 51. Treatment Percent inhibition of mycelial growth* Mean Concentration(%) 0.1 0.2 T1-Chloropyriphos 96.30 (83.50) 100.00 (89.99) 98.15 (86.74) T2 –Carbofuron 100.00 (89.99) 100.00 (89.99) 100.00 (89.99) T3- Indoxocarb 87.78 (69.69) 100.00 (89.99) 93.89 (79.84) T4- Imidachloprid 12.96 (21.09) 59.26 (50.33) 36.11 (35.71) T5- Control 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) Mean 59.41 (52.85) 71.85 (64.06) 65.63 (58.46) Table6: Effect of different insecticides on compatibility of Trichoderma harzianum (Th-2) Bheemaraya et al., 2011, Raichur S.EM C.D.at 1% Insecticides (I) 1.53 6.14 Concentration (C) 0.96 3.88 Mean of four replications, **Figures in parentheses are arcsine transformed values
  • 52. Table7: Effect of soil treatment with formulated Trichoderma species on incidence of fusarium wilt disease of giza 3 bean cultivar under greenhouse and field conditions. Nashwa et al., 2008, Egypt Wilt rating under green house conditions TREATMENT Time of application TH 1** TV 1 TS 3 infected control Mean Two weeks before planting 3.7 4.1 5.2 8.0 5.3 At time of planting 2.5 3.1 4.0 7.0 4.2 Mean 3.1 3.6 4.6 7.5 L.S.D at 0.05 time of application (A ):0.5 bioagents (B) :0.43. Interaction (AXB) :0.61 Wilt rating * under field conditions Time of application TH 1 TV1 TS 3 Infected control Mean Two weeks before planting 3.3 4.2 5.0 6.0 4.7 At time of planting 4.0 4.2 5.0 7.0 5.1 Mean 3.6 4.1 5.0 6.5 L.S.D at 0.05 time of application a :0.2 bioagents b :0.25. Interaction (AXB) :0.36 •According to CIAT scale (van schoonhoven and pastor-corrales,1987) TH 1: T. harzianum, TV 1: T. viride, TS 3: T. virens
  • 53. Treatement Plant height(cm) Plant dry Weight(g) Disease incidence C 14.52a 2.45a 0.00a Fs 6.52b 1.06b 1.80b Fs+ Tv 12.68c 2.42a 1.06c Fs+ Th 14.18d 2.58a 0.80d Table8: Effect of T. harzianum and T. viride on height.dry weight and disease incidence of tomato plants inoculated with F. Solani under pot conditions. Perveen and Bokhari., 2012, Saudi Arabia Each value is average of six replicates. Data Followed by different letters in the column are significantly different (p< 0.05 ) according to Duncan’ s multiple range test . C, uninoculated control Fs ,F solani : Tv: T . viride , Th: T. harzianum Disease incidence graded on 0 to 3 scale where ,0= 25 %severity , 1 =26 to 50%, 2 = 51 to 75% and 3= 76 to 100%
  • 54. Fig5: Pathogen growth inhibition by Trichoderma after 6 day of inoculation in dual culture Hajieghrari et al., 2008, Iran
  • 55. Fig6: Pathogen growth inhibition Trichoderma volatile compounds Hajieghrari et al., 2008, Iran
  • 56. Table9: Effect of pH and temperature on the mycelial growth(mm) of Trichoderma isolates Hajieghrari et al., 2008, Iran Treatment T. hamatum T612 T. harzianum T447 T. virens T523 T. harzianum T969 Trichoderma sp. T T. hamatum T614 pH 8 33.26* 23.89 41.15 31.07 30.19 33.78 pH 7 29.3 24.3 33.96 39.45 30.45 30.85 pH 5 33.78 34.8 45.52 34.7 32.22 27.68 30⁰c 29.15 17.2 43.67 35.41 36.89 31.37 25⁰c 36.04 38.89 37.56 36.44 35.3 30.96 20⁰c 31.14 26.89 39.41 33.63 31.67 29.96 *Values are means of four replicates.
  • 57. Fig7 :Antagonistic activity of Trichoderma species against F.oxysporum evaluated by dual culture interaction (A)F.oxysporum alone , (B) F oxysporum +T.harzianum (T 1s), (C) F.oxysporum + T.viride (TvPDs) (D) F.oxysporum + T.harzianum(TDPs) Perveen and Bokhari, 2012, Saudi Arabia
  • 58. Fig8: Inhibitory effect of the culture filtrate of Trichoderma spp. Incubated at different temperature (5, 15, 25, 35, 40⁰C). Each value is an average of three replicates.T1s= Trichodema harzianum isolate T1s, TvPD = T. viride isolate TvPD, TDPs = T. harzianum isolate TDPs Perveen and Bokhari, 2012, Saudi Arabia
  • 59. Fig9: Average liner growth rate (ALG) of Trichoderma species on various culture media. Each value is an average of three replicates. T1s= Trichoderma harzianum isolate T1s, TvPDs= T. viride isolate TvPDs, TDPs=T harzianum isolate TDPs, PDA=Potato dextrose agar, SDA =Sabouraud dextrose agar, WA = Water agar (2% agar), CDA = Czapek dox agar, PDAL= natural media agar (PDA + 1% date palm leaves) Perveen and Bokhari, 2012, Saudi Arabia
  • 60. Table10 : Effect of strains of Trichoderma species on the per cent inhibition of radial colony growth of P. aphanidermatum Mishra, 2010, India Trichoderma species Percent inhibition T.harzianum -4532 60.3 0.3 e T harzianum-4572 69.8 0.3 g T.viride -801 54.1 0.5 c T.viride-1763 52.2 0.5 b T.viride-1433 72.0 0.3 h T.viride-793 62.1 0.3 f T.Viride-2109 50.4 0.4 a T.koningii-2385 56.4 0.2 d T.virens-2023 53.5 0.6 c T.virens-2194 59.6 0.6 e Values are average of three replicates SEM Values in the column followed by same letter are not significantly different (P<0.05).
  • 61. Fig10: Efficiency of P solubility and biocontrol activity of T .harzianum isolates against Xanthomonas sp. Padmavathi and Madhumathi, 2011, Banglore
  • 62. Fig11: Effect of initial pH on chitinase and ß-1,3-glucanase production (using 0.5% chitin or laminarin as carbon source, respectively ) by T.harzianum Katatny et al., 2000, Egypt
  • 63. Fig12: Effect of different carbon sources on chitinase and ß-1,3-glucanase production by T. harzianum and on inhibition of S.rolfsii (100 % ß-1,3-glucanase activity correspond to 14.7 nkat/mL and 100% chitinase activity correspond to 59.8 pKat/mL) Katatny et al., 2000, Egypt
  • 64. Fig13: Release of total reducing sugars(R,S),glucose and N-acetyl glucosamine from S.rolfsii(dried and fresh mycelium),T.harzianum and chitin by the T.harzianum enzymes Katatny et al., 2000, Egypt
  • 65. Table11: Evaluation of Trichoderma isolates against soil borne fungal pathogens using dual culture Amin et al., 2010, Jammu and Kashmir Treatment Radial growth (mm)of test pathogens R.solani S.rolfsii S.sclerotiorum Trichoderma virens(Ts-1) 46.55 (48.11) 36.26 (59.71) 56.19 (37.56) Trichoderma harzianum(Th-1) 35.43 (60.51) 34.67 (61.47) 39.08 (56.57) Trichoderma harzianum(Th-2) 43.32 (51.71) 35.33 (60.75) 55.69 (38.12) Trichoderma viride(Tv-1) 30.67 (65.71) 28.88 (67.91) 30.41 (66.21) Trichoderma viride(Tv-2) 25.65 (71.41) 32.00 (64.44) 34.28 (61.91) Trichoderma viride(Tv-3) 41.59 (53.64) 34.93 (61.18) 55.65 (38.16) Control 89.72 90.00 90.00 C.D.(P=0.05) 2.52 1.23 3.59 Figures in parenthesis are per cent inhibition values
  • 66. Table12: Evaluation of Trichoderma isolates against production of sclerotia in soil fungal pathogens using dual culture Amin et al., 2010, Jammu and Kashmir Treatment Rhizoctonia solani Sclerotrum rolfsii Sclerotinia sclerotiorum Sclerotioal count Inhibition over control(%) Sclerotial count Inhibition over control(%) Sclerotial count Inhibition over control(%) (Ts-1) 35.59 66.63 38.66 67.60 19.09 39.70 (Th-1) 23.66 77.81 28.73 75.92 12.07 61.87 (Th-2) 31.73 70.25 34.29 71.26 18.12 42.76 (Tv-1) 17.33 83.75 23.64 80.18 9.45 70.15 (Tv-2) 19.47 81.75 26.07 78.15 11.12 64.87 (Tv-3) 27.25 74.45 33.78 71.69 15.57 50.82 Control 106.66 - 119.3 - 31.66 - C.D.(P=0.05) 1.89 2.07 0.98
  • 67. Fig14: The level of hydrolytic enzymes activities from three different samples E2- extracts from Botrytis mycelia;E3-extracts from Trichoderma mycelia :E4:Extract from a mixture of pathogen and antagonistic strains Cornea et al., 2009, Romania
  • 68. Table13: Evaluation of volatile metabolites produced by Trichoderma isolates against production of sclerotia in different pathogens Amin et al., 2010, Jammu and Kashmir Treatment R. solani S. rolfsii S. sclerotiorum Sclerotial count Inhibition over control (%) Sclerotial count Inhibition over control (%) Sclerotial count Inhibition over control(%) (Ts-1) 60.09 39.30 91.43 29.30 10.12 57.24 (Th-1) 42.67 56.89 75.66 41.49 6.11 74.18 (Th-2) 54.67 44.77 89.06 31.13 8.73 63.11 (Tv-1) 38.42 61.19 67.00 48.19 5.00 78.87 (Tv-2) 34.00 65.65 71.04 45.07 5.11 78.41 (Tv-3) 49.52 49.97 84.93 34.33 7.22 69.49 Control 99.00 - 129.33 - 23.67 - C. D (P=0.05) 3.13 4.76 0.91 Figures in parenthesis are percent inhibition values
  • 69. Fig16:Compatibility test between T. viride and P. fluorescens. The picture was taken on the 9th day after dual inoculation. Zegeye et al., 2011, Ethiopia
  • 70. Table14 : Effect of foliar application of T.viride and P.fluorescens on the progress of late blight disease of potato Zegeye et al., 2011, Ethiopia. Treatments Mean AUDPC T. viride 260.0 190.0 c P. fluorescens 765.1 218.6 b Mixed culture 999.0 274.5 a Mancozeb 85.9 77.8 cd Negative control(inoculated/untreated) 1045.1 227.2 a Positive control (non- inoculated/untreated) 0.00 0.00 d Means followed by the same letter are not significantly different. The AUDPC was calculated from five consecutive weekly assessment of percentage of leaf area with symptoms of late blight. The nine replicates were arranged in a CRB design and the midpoint rule was used to calculate AUDPC values.
  • 71. Mechanism of action against Phytonematodes • Secretion of Lytic enzyme chitinase help parasitism of Meloidogyne and Globodera eggs • T. viride releases Dermadin helps in destruction of nematode cuticle • Trichoderma spp. have high rhizosphere competency and easily colonize the roots, reduce the feeding sites for nematodes Jonathan, 2010, New Delhi
  • 72. Healthy egg of Heterodera glycines Egg parasitized By fungus
  • 73.  There are several reputable companies that manufacture government registered products. Trade Name Bio agent Manufacture Eco fit T. viride Hoechst and Schering AgroEvo Ltd, Mumbai India Super visit T. harzianum Fytovita, Czech Republic Soil guard T. virens Certis Inc,Columbia,MD,USA Root pro T. harzianum Efal Agri, Netanyl,Israel Tusal T. Viride +T. harzianum Tusal Carrera Ester, Lleida Spain Agroderma, Bio-cure, Bioderma, Ecofit, Rakshak, Trichosan Trichoderma viride Biocure (B&F) T. Viride and P. flourescens
  • 74. Formulations  Powder formulations  Encapsulation in organic polymer like sodium alginate  As spray from emulsifiable concentrates  Molasses enriched clay granules  Pellating biomass and bran with sodium alginate
  • 75. The use of Trichoderma has gained importance in managing most of the plant pathogens. However, there is still considerable interest in finding more efficient mycoparasitic fungi especially within Trichoderma harzianum strains, which differ with respect to their biocontrol effectiveness. The technique for mass production and use of these bio agents have been commercialized for the purpose of producers and farmers.
  • 76. U