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Avs significant achievements and present status of trichoderma spp. in
This document summarizes a seminar presentation on the significant achievements and present status of Trichoderma spp. in biocontrol of plant diseases. The summary includes: 1) Trichoderma spp. are filamentous fungi commonly used as biocontrol agents due to their ability to control plant pathogens through competition, mycoparasitism, and induced resistance. 2) Mutation induction using physical and chemical mutagens has been used to improve the biocontrol potential of Trichoderma spp. by impacting their morphological, biochemical, and mycoparasitic properties. 3) Studies presented show mutants exhibiting increased growth, sporulation, antibiotic production, and tolerance to stress
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Avs significant achievements and present status of trichoderma spp. in
- 1.
- 3. Seminar presentation on “Significant Achievementsand Present Status of Trichoderma spp. in Biocontrol of Plant Diseases” 3
- 4. Chairman Dr. R.M. Gade Member Dr. R. W. Ingle Member Dr. A. M. Charpe Member Dr. R. N. Katkar DEPARTMENT OF PLANT PATHOLOGY, POST GRADUATE INSTITUTE , DR.P.D.K.V,AKOLA. Advisory Committee 4
- 5. Contents Introduction Mechanism of Trichoderma Typeof mutation & mutagen Reviews Impact of mutation on morphological characters Impact of mutation on biochemical changes Impact of mutation on mycoparasitism 5
- 6.
- 7. Position Asexual stage (Conidia) Sexual stage (Ascospore) KingdomFungi Fungi Phylum Deuteromycota Ascomycota Sub-Division Deuteromycotina Ascomycotina Class Hyphomycetes Pyrenomycetes Order Monilliales Sphariales Family Monilliaceae Hypocreaceae Genus Trichoderma Hypocrea Table: Taxonomic position of Trichoderma spp. TRICHODERMA SPP.
- 8. The word Trichodermais taken from thrix = hair and derma = skin. Trichoderma is free living, asexually reproducing and filamentous fungi. The genus Trichoderma was first proposed as a genus more than two hundred years ago by Persoon in 1794 at Germany. 8
- 9. Species Characteristics Tolerant tocool and warm temperatures. Tolerant to low moisture Tolerant to many fungicides Prefers acidic soils (pH 3.5 to 4.5) Rhizosphere competent Life cycle Habitat 9
- 10. 10 USES OF TRICHODERMA Biocontrolagent- In recent years, genetic improvement of Trichoderma by induction of mutation using mutagens has successfully been attempted for improvement of potential ability of biocontrol agent. Mutation in Trichoderma results in beneficial and harmful effect on morphological, physiological, biochemical and molecular properties. Medical uses- Cyclosporine A (CsA) a calcineurin inhibitor Industrial use- T. reesei is used to produce cellulase and hemicellulase T. longibratum is used to produce xylanase T. harzianum is used to produce chitinase. Plant growth promotion As a source of transgenes
- 11. Mode of Action:Competition Mycoparasitism -Chemotaxis to target -Coiling -Extracellular enzymes (cellulases and chitinases) -Appresoria like structures and penetration Induced resistance Antibiosis -Diffusible inhibitors (toxins, antibiotics) -Volatile inhibitors (alcohols, ketones, sesquiterpenes) 11
- 12.
- 13. 13 Trichoderma activities (a)mycoparasitism, (b) competition, (c) mycelial growth in rhizosphere and production of metabolite
- 14. Fig. 3 (A):Mycoparasitism by a Trichoderma strain on the plant pathogen (Pythium) on the surface of pea seed. The Trichoderma strain was stained with an orange fluorescent dye while the Pythium was strained green. Fig. 3(B): Scanning electron micrograph of the surface of a hyphae of the plant pathogen Rhizoctonia solani after mycoparasitic Trichoderma hyphae were removed. Erosion of the cell wall due to the activity of cell wall degrading enzymes from the biocontrol fungus is evident, as are holes where the mycoparasitic Trichoderma hyphae penetrated the R. solani.
- 15. Trichoderma spp. showingdifferent mechanism of action on plant pathogens T. harzianum coils around Rhizoctonia solani. 15 Trichoderma spp. coiling around Rhizoctonia solani hyphae
- 16. Figure 3: Colonizationof root hairs of corn by the highly rhizosphere competent of T. harzianum T22. 16
- 17. Figure 4: Antagonisticactivity of T. viride on Bortytis cinerea. 17
- 18. Colony morphology ofthe wild type (WT) and a vel1 mutant (ve3) of T. virens. Mukherjee P K , Kenerley C M Appl. Environ. Microbiol. 2010;76:2345-2352
- 19. Growth of wild-type(WT), mutant (ve3), and complemented (Comp) strains in shake cultures. Mukherjee P K , Kenerley C M Appl. Environ. Microbiol. 2010;76:2345-2352
- 20. WHAT IS MUTATIONAn abruptappearance of a new characteristic in an individual as the result of an accidental change in a gene or chromosome. 20
- 21. Point mutation :- Alteringthe genetic code Nonsense mutation Change the genetic code and destroy the information it contains. Spontaneous mutation Changes in frame shift. Induced mutation Artificial intervention. TYPES OF MUTATION 21
- 22. TYPES OF MUTAGEN Physicaland chemical agents capable of bringing about mutations are called mutagens. Chemical mutagens Include nitrous acid This substance converts adenine to hypoxanthine, a molecule that will not pair with thymine and thus interrupts the genetic code. A base analog is a chemical mutagen that resembles a nitrogenous base and is incorporated by error. A DNA molecule cannot function in proteinsynthesis. Certain dyes and fungal toxins (for example aflatoxin) are known to be mutagens. 22
- 23. Physical mutagens :- Include X rays, gamma rays and ultraviolet light. They are bringing about genetic manipulation and their by improving biocontrolling efficacy 23
- 24. METHOD OF DEVELOPINGMUTANT OF Trichoderma sp. BY USING UV LIGHT Preparation of conidial suspension (5 day-old Trichoderma culture) Spread on Petri plates (contain PDA medium) Placed under UV light source for different periods, quartz lamp, & distance (i.e. 10,20,30 etc.) Incubated at 250 C in normal condition Comparison of changes with its parent 24
- 25. Impact of Mutation Morphologicalchanges Biochemical changes Mycoparasitism 25
- 26.
- 27. Isolates Phenotypic characterscfu/ml TV5-(WT) Highly sporulating colony with yellow pigmentation and aerial mycelium. 4.5x106 TV5-1 Highly sporulating colony where spores were densely formed in the margin. 5.24x106 TV5-2 Initially colony with whitish mycelium less number of spores appeared but assumed heavy sporulation after interval of 4 days. Yellow pigment appeared . 6.54x106 TV5-3 Colony with heavy sporulation and sporeless marked zonation was also visible. Absence of yellow pigment was prominent. 3.9x106 Table 1:- Phenotypic characteristics of Trichoderma viride mutants and wild Selvakumar et al. (2000)IARI, New Delhi 27
- 28. Table 2:- Comparisonof T. viride parent and mutant strains for the colony morphology, growth and sporulation Character Trichoderma viride Tv 34 Tv34-M4 Tv34-M5 Aerial mycelium Suppressed Fluffy Fluffy Colony colour Yellowish to greenish Light orange to greenish Light orange to greenish Substrate pigmentation Light yellow Dark orange Dark orange Sporulation 4.77×108 cfu/ml 7.45×108 cfu/ml 7.88×108 cfu/ml Colony growth 1.92 mm 1.95 mm 1.96 mm Mycelium dry Weight (mg/100ml broth culture) 146 187 193 Hunjan et al.(2004)P.A.U.,Ludhiana 28
- 29. Trichoderma isolates NaCl (mM) Reduction in linear growth(%) Description GrowthSporulation 1st 7th 1st 7th 1st 7th Wild type 0.0 0.0 0.0 Flat Flat + + 51 49.3 49.3 Cottony Cottony + + 69 53.1 53.1 Cottony Cottony + + Th20M532 0.0 0.0 0.0 Flat Flat ++++ ++++ 51 0.0 0.0 Flat Flat ++++ ++++ 69 0.0 0.0 Flat Flat ++ ++ Th50M63 0.0 0.0 0.0 Flat Flat ++++ ++++ 51 0.0 0.0 Flat Flat ++++ ++++ 69 0.0 0.0 Flat Flat ++++ ++++ Th50M113 0.0 0.0 0.0 Flat Flat +++ ++++ 51 0.0 0.0 Flat Flat +++ ++++ 69 0.0 0.0 Flat Flat +++ +++ Table 3 :- Characterization of γ -induced mutants from T. harzianum grown in different PDA supplemented with NaCl . Table 3 :- Characterization of γ -induced mutants from T. harzianum grown in different PDA supplemented with NaCl . 29 T. harzianum Wild type (WT).2&3 200 and 500 Gy γ- ray induced mutants,respectively. (+) little disperse, (++) moderate disperse (+++) good disperse, (++++) copact. (1st ) First subcultur and(7 th ) Seventh subcultures. Mohmaed and Haggag(2005)N.R.C.,Dokki,Egypt
- 30. Table 4:- Growthrate and sporulation of T. harzianum mutant (Th 38 M-7) and parent strain (Th-38) Trichoderma strain Growth on agar medium Growth in broth medium Colony diameter (mm) after incubation period (hr.) Mycelium dry weight/flask (mg/100ml) Sporulation (conidia/ ml) 24 48 72 Mean Mutant (Th 38 M-7) 22.00 57.00 90.00 56.33 628 5.57×107 Parent (Th 38) 14.00 44.00 85.60 47.89 611 7.67×106 Mean 18.00 50.50 87.84 619.5 C.D (P=0.05) Strain (A) 4.23 9.0 3.92×107 Hours (B) 28.58 - Strain × Hours ( A× B ) 11.66 Manav and Singh (2006)P.A.U.,Ludhiana 30
- 31. Organism Phenotypic character Th-WHigh sporulating colony with green aerial mycelium Th-M1 Initial colony with whitish mycelia later turn greenish , profuse sporulation and shows faster growth rate and yellow pigment appeared later Th-M3 Less sporulation, pale green in colony, spores are densely formed in the margin, and sporeless marked zonation is visible. Mech et al.(2006)A.A.U.,Jorhat Table 5:- Phenotypic characters of carbendazim tolerant T. harzianum mutants 31
- 32. Table 6 :Cultural characteristics of some selected mutant isolates of Gliocladium virens on PDA and OMA medium after 10th generation Mutant isolates Cultural characteristics of phenotypic mutants on PDA OMA 50 Gv1 I Dark green, submerged colony Dark green, colony submerged, little raised mycelial growth at the edge of the colony 50 Gv1 V Albino (white), granular and submerged colony with scanty mycelial growth Albino (white) submerged mycelial growth, nodular radiating mycelial growth 75 Gv1 VI Yellowish green, fluffy colony with profuse mycelial growth Greyish green, granular mycelial growth colony submerged 150 Gv1 II Green fluffy colony with white mycelial growth at the margin Deep green, colony granular, submerged mycelial growth 75 Gv3 I Greyish white, raised colony Dark green, submerged colony with little raised mycelial growth at the centre Jash et al.(2006)Mohanpur,Nadia 32
- 33.
- 34. Trichoderma spp. (Wild type/ mutant) Total metabolite Antibiotics Total phenolic compounds TrichoderminGliotoxin Virindin T.harzianum 0.46 0.03 0.016 0.015 0.010 TH5053 0.67 0.39 0.148 0.020 0.027 TH508 0.81 0.50 0.355 0.295 0.025 T. viride 0.38 0.07 ND 0.217 0.022 TV203 0.48 0.14 ND 0.232 0.026 TV208 0.56 0.18 0.067 0.095 0.045 T. koningii 0.47 0.05 0.012 0.009 0.021 TK508 0.70 0.71 0.046 0.034 0.026 TK509 0.75 0.16 0.136 0.380 0.046 Table 7 :- Total metabolites, antibiotics and phenolic compound in the culture filtrates of three Trichoderma spp. wild types and their mutants (as μg/ ml dry weight) Haggag and Mohamed (2002)Egypt Antibiotics and total phenolic compounds were quantified by HPLC analysis of filtrates of liquid culturaes ad normalized to the dry weight of mycelium in the collected samples. ND, not detected. 34
- 35. Figure 7:- Growthinhibition of Sclerotium cepivorum inciting white rot of onion by three Trichoderma spp. and their mutants 35
- 37. Table 10 :-Effect of culture filtrate of T. viride isolates on the growth of Botrytis cinerea Isolate Colony diameter (mm) Inhibition (%) T-15 29.0 53.2 T-15.M-1 45.0 27.4 T-15.M-3 18.7 69.8 T-15.M-5 23.3 62.4 T-15.M-11 20.7 66.6 T-15.M-14 23.3 62.4 Control 62.0 - CD (P ≤ 0.05) 4.5 - Mukherjee et.al (1997)B.A.R.C.,Mumbai 37
- 38. Trichoderma spp. (wild type/mutant) Antagonisticeffects against S. cepivorum Pathogen growth reduction(%) Inhibitory effect of culture filtrate 10% 50% T.harzianum 38.0±0.5bc2 6.7±0.1e3 58.5±0.4c4 TH5053 96.6±02a 48.8±0.4d 100.0a TH508 100.0a 64.4±0.5b 100.0a T. viride 33.3±0.4c 4.6±0.1e 48.0±0.3d TV203 96.3±0.5a 55.5±0.5c 96.6±0.6b TV208 100.0a 58.5±0.4c 100.0a T. koningii 44.4±0.2b 5.5±0.2e 57.5±0.4c TK508 100.0a 66.6±0.5b 100.0a TK509 100.0a 57.6±0.4a 100.0a Table 11:- Effect of wild types of Trichoderma spp. and their mutants on the growth of Sclerotium cepivorum Inhibition of S.cepivorum growth was determined on PDA containing 10 and 50% Trichoderma culture filtrates. Mean of 5 replicates Values in the the same raw followed by different letters are not statistically significant different at p < 0.05 Result are givaven as mean+ standared error. Haggag and Mohamed (2002)N.R.C.,Dokki, Egypt 38
- 39. 39 T .viride strainCulture filtrate concentration (%) Growth of R. solani (mm) after hours Colony growth inhibition (%) after hours 24 48 72 96 24 48 72 96 Parent 0 27 38 68 90 0.0 0.0 0.0 0.0 10 12 24 38 58 55.6 36.8 44.1 35.6 20 8 24 36 49 70.4 36.8 47.1 45.6 30 8 20 34 47 70.4 47.4 50.0 47.8 50 7 19 32 43 74.1 50.0 52.9 52.2 Mutant-Tv34 M4 0 27 38 68 90 0.0 0.0 0.0 0.0 10 8 19 44 48 70.4 50.0 35.3 46.7 20 7 19 33 44 74.1 50.0 51.5 51.1 30 8 18 29 42 70.4 52.6 57.4 53.3 50 7 13 24 29 74.1 65.8 64.7 67.8 Mutant-Tv34 M5 0 27 38 68 90 0.0 0.0 0.0 0.0 10 9 19 46 52 66.7 50.0 32.4 42.2 20 8 18 34 49 70.4 52.6 50.0 45.6 30 7 18 32 41 74.1 52.6 52.9 54.4 50 7 11 23 29 74.1 71.1 66.2 67.8 Table 12 :- Effect of culture filtrate of mutant and parent strains of T. viride on growth of R. solani causing black scurf of potato Mean growth (mm) : parent-41.72,Tv34-M4- 36.61 , Tv 34-M5-M5-37.14 C.D. (P=0.05) Strain (A) 0.70 Concentration (B) 0.91 Time (C) 0.91 AXB- 1.57 BXC 1.34 AXC 1.57 AXBXC 3.52 P.A.U.,Ludhiana Hunjan et al.(2004)
- 40. Dose (% wet formulation) Disease incidence (%) Yield(kg/plot) % Yield increase over control Mutant Parent Mutant Parent Mutant Parent Seed treatment 1 76.1 79.6 6.1 5.0 25.0 9.0 1.5 69.3 76.7 5.9 5.1 21.9 12.0 2.0 69.3 74.0 6.8 5.4 31.2 18.1 Control 94.6 94.6 4.6 4.6 0.0 0.0 Mean 70.2 76.8 6.2 5.2 26.0 13.0 Seed treatment+ Spot Inoculation # 1 70.8 74.8 5.8 5.0 18.1 5.4 1.5 67.6 71.4 5.9 5.7 18.9 16.8 2.0 59.1 68.6 6.8 6.0 29.4 21.1 Control 94.5 94.5 4.8 4.8 0.0 0.0 Mean 65.8 71.6 6.2 5.6 22.1 14.4 Table 13:- Effect of seed treatment of T. viride parent (Tv 34) and mutant (Tv34- M5) strains on black scurf development on potato under field conditions. C.D.(p=0.05) Strain- 2.4 Dose- 2.9 Strainx Dose- NS Hunjan et al.(2004)P.A.U., Ludhiana # spot inoculation was done with 50 g of fym and wet formulation mixture per seed tuber. 40
- 41. Sr. No Treatment Radial growth(mm) at different hours of incubation Percent inhibition at different hours of incubation 24 48 72 24 48 72 1. T. harzianum (wild) + S.sclerotium 8.59 26.14c 43.57b 49.99 (45.00)d 48.97 (44.41)c 48.49 (44.13)c 2. Th-M1 + S. sclerotium 7.87 27.00b 42.29c 54.17 (47.40)c 45.92 (42.66)d 49.98 (44.99)c 3. Th-M2 + S. sclerotium 3.94 6.14d 7.55e 77.21 (62.10)a 88.16 (69.93)a 91.53 (73.05)a 4. Th-M3 + S. sclerotium 6.70 19.14c 30.43d 61.00 (51.37)b 63.16 (52.13)b 64.01 (53.13)b 5. S. sclerotium alone 17.29 51.86a 89.87a - - - SEM(+) 0.51 0.49 0.58 - - - CD(P=0.05) 1.01 0.97 1.18 - - - Table 14:- Radial growth and per cent inhibition of S. sclerotium in presence of wild and mutant T. harzianum in in vitro Mech et al. (2006)AAU., Jorhat Figure in parenthesis are Arcsine transformed value while outside are original values 41
- 42. Table 15 :-Biocontrol assay with wild and mutant strain of T. harzianum against phytopathogens Trichoderma Strains Phyto pathogens Growth of antagonist (cm) Growth of pathogen (cm) Pathogen inhibition (%) Wild Fusarium oxysporum 6.7 2.3 74.4±0.18 Bipolaris oryzae 7.0 2.0 77.7±0.23 Rhzoctonia solani 4.3 4.7 47.7±0.17 Alternaria sp 7.2 1.8 80.0±0.47 UV mutant F. oxysporum 7.9 1.1 87.7±0.35 B. oryzae 7.8 1.2 86.6±0.32 R. solani 6.1 2.9 67.7±0.19 Alternaria sp 8.0 1.0 88.8±0.47 Balasubramanian et al. (2010)Tamilnadu, India Values are mean of triplicates with ± standerd error. 42
- 43. Fig 6:- Antagonisticcapability of the selected T. harzianum mutants against F. oxysporum and R. solani (MT3, MT5, MD5, MS1 and MS6 are mutant arise from the wild type T3, T5, D5, S1 and S6, respectively). Hassan and Kareem ( 2011)Tikrit Univ., Iraq 43
- 44. Table 16 :-in vitro growth inhibition of sensitive and resistant isolates of M. phaseolina to carbedazim by mutant T. koningii in dual culture method Antagonist % Reduction of mycelial growth Sensitive MP-12 Resistant MP-8 UVTK-1 42.50 47.50 UVTK-2 46.67 52.97 UVTK-3 36.67 40.42 UVTK-4 36.67 47.50 UVTK-5 21.17 52.80 TKWD 33.96 51.05 Patil and Kamble (2011)Shivaji University,Kolhapur 44
- 45. Several geneshave been cloned from Trichoderma spp. that offer great promise as transgenes to produce crops that are resistant to plant diseases. (Tubulin,Chitin,Protease,Xylanase,Monooxygenase.) No such genes are yet commercially available, but a number are in development. These genes, which are contained in Trichoderma spp. and many other beneficial microbes, are the basis for much of "natural" organic crop protection and production. Trichoderma spp. as a source of transgenes.
- 46. Fig. 7 :Some biocontrol genes from T. harzianum have been inserted into plants, where they provide resistance to several diseases. Tobacco and potatoes, shown in this figure, were transformed to express the fungal endochitinase gene, which resulted in high levels of resistance to Alternaria alternata (tobacco) and Rhizoctonia solani (potato).
- 47. LIMITATIONS Trichoderma spp. canonly be used against specific disease. They are less effective than the fungicides. At present, only few Trichoderma spp. are available for use and are available only in few places. This method is only a preventive measure and not a curative measure. Trichoderma spp. should be multiplied and supplied without contamination and this requires skilled persons. The shelf life of Trichoderma spp. is short. Antagonists, Trichoderma viride is viable for four months. The efficiency of Trichoderma spp. is mainly decided by environmental conditions. A Trichoderma spp. under certain circumstances may become a pathogen. Mutation in Trichoderma results in harmful effect on morphological, physiological, biochemical and molecular properties.
- 48. Conclusion Induced mutationis one of the most commonly used practices to modify the genetic composition of antagonistic fungi. Mutation on Trichoderma sp. by UV and gamma radiation brought about changes in morphological features like colony diameter, sporulation, dry mycelial weight and enzymes like ß-1,3-glucanase, ß-1,4-glucanase, cellulase and antibiotics like trichodermine, gliotoxin and virindin. Selection of beneficial mutant of biocontrol agent becomes a better option for management of plant pathogens. Adoption of Trichoderma Spp. in biological control in Disease Management is need of time hence extensive studies will be required on interaction of diseases and management practices 48
Editor's Notes
- #19 Colony morphology of the wild type (WT) and a vel1 mutant (ve3) of T. virens. (A) Growth on VMS agar after 5 days of incubation at 26°C. (B) Microscope images of the cultures shown in panel A, showing conidiophores in the WT and chlamydospores in the mutant.
- #20 Growth of wild-type (WT), mutant (ve3), and complemented (Comp) strains in shake cultures. (A) Growth after 8 days of incubation. Note the absence of pigmentation in the mycelia of the mutant. Cultures were transferred to plates for photography. (B) Culture filtrates after 8 days. Note the absence of the yellow pigment in the supernatant from cultures of the mutant. (C) Colony morphology after 4 days of incubation of a shake culture. Note the smooth edges of the developing WT colonies compared to the striated growth of the mutant and the complemented strain. (Insets) Enlargements of single colonies.