Biochemical and Molecular Characterization of  Sclerotium rolfsii: a Mycoherbicidal Agent      against Parthenium hysterop...
The Weed : Parthenium hysterophorus Alien, Invasive and Obnoxious weed. Potential hazard to our biodiversity ,human bein...
Infestation of Parthenium in ecosystemsA                          B       Agriculture land         Agriculture landC      ...
Human and Animal health hazards of                         Parthenium                                                 • Ph...
The various strategies developed are:-(I)    CONVENTIONAL  a. Physical Method- Uprooting: Health & Labor Problems         ...
Sclerotium rolfsii: Mycoherbicidal agentDevastating soil-borne pathogenRequires warm and humid environmentNecrotrophic, ad...
OBJECTIVES Recovery of geographically different strain from different hosts Determination of morphological variation and...
Colony characteristics produced by S. rolfsii isolates
Growth (cm)/day                                                                                  0                        ...
Maximum: Par#07 ; Minimum: Crotolaria
Fig.6a : Production of Oxalic acid, Biomass & sclerotia after 7 days of                                       incubation b...
Fig.6b: Production of Oxalic acid, Biomass & sclerotia after 14days of                                         incubation ...
Cellulolytic activity produced by S. rolfsii isolates Cellulases are cellulose degradingenzymes Plays a secondary role i...
0                                                                                                     20                  ...
0                                                                       2                                                 ...
Cellulolytic zymogram of different isolates of S. rolfsiilane 4 : Hyp#03 produced maximumnumber of bandsSubstrate used : 0...
Cellulase electromorph of different isolates of S. rolfsii                      No. of bands  6                      Band...
Pectinolytic activity of different isolates of S. rolfsii Pectinases play a significant role inS. rolfsii pathogenicity ...
0                                                                 5                                                       ...
0                                                                                                        2                ...
Pectinase electromorphs produced by different isolates of S. rolfsii                                Bands 8              ...
Mycelial Compatibility among S. rolfsii isolates                                          Anastomosis between             ...
Amplification of ITS region of rDNA of S. rolfsii isolates           Amplified ITS region ~ 650 base pairs
ITS – RFLP Fingerprint of different isolates of S. rolfsii Taq I                                                Hind IIIBa...
Random Amplified polymorphic DNA-polymerase chain reaction fingerprint ofdifferent isolates of S. rolfsii obtained using p...
Dendrogram of isolates of S. rolfsii revealed by cluster with the un-weighted    pair group methods with arithmetic averag...
Dendrogram of isolates of S. rolfsii revealed by cluster with the un-weighted    pair group methods with arithmetic averag...
Pathogenic Potential of S. rolfsii Isolates                               Pathogenicity      3.5       3      2.5       2 ...
Fig. 9a: Competence volume of S. rolfsii after 20 days of inoculation at                                            variou...
Fig. 9b: Competence volume of S. rolfsii after 30 days of inoculation at                                            variou...
MASS PRODUCTION OF S. rolfsiiWheat grains       Bagasse & Wheat bran        Maize Cover
Mycelial coverage (%)                                                                                                    1...
No. of Sclerotia Produced/50gm                                                                                      2000  ...
Mean disease intensity                                                                                                    ...
Amount of inoculum: 0.1 gm of each substrate : Observation recorded after 72 h
Host specificity depicted by fungal isolate
Profuse growth and Tip colonization inhibited flower and seed formation
Final Words All the isolates obtained form different hosts and region revealed remarkablevariation in most of the paramet...
PUBLICATIONS1. Shukla, R. and Pandey, A.K. (2006). Maximization of production of oxalic acid from Sclerotium   rolfsii, a ...
ACKNOWLEDGEMENTSDr. A. K. Pandey , Professor, Department of Biologicalsciences, R D University, Jabalpur, INDIAMadhya Prad...
DrRekhaShukla_PhDWork
DrRekhaShukla_PhDWork
DrRekhaShukla_PhDWork
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DrRekhaShukla_PhDWork

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This presentation contains the Doctoral Work of Dr Rekha Shukla at RD University, Jabalpur, INDIA.
Dr Shukla is currently at University of Georgia in Athens, (GA)USA

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DrRekhaShukla_PhDWork

  1. 1. Biochemical and Molecular Characterization of Sclerotium rolfsii: a Mycoherbicidal Agent against Parthenium hysterophorus Rekha Shukla Department of Biological Sciences R D University, Jabalpur 482001 INDIA
  2. 2. The Weed : Parthenium hysterophorus Alien, Invasive and Obnoxious weed. Potential hazard to our biodiversity ,human beings and Agriculture Prolific seed producer - 25,000 seedsper plant Weed of international Importance Can germinate and set flower and seedin 4-6 weeks
  3. 3. Infestation of Parthenium in ecosystemsA B Agriculture land Agriculture landC DD Residential Area Roadsides
  4. 4. Human and Animal health hazards of Parthenium • Photodermatitis • Drivers committed A B suicide C •12 buffalos died at Itarsi DA, B & C. Contact Dermatitis D. Asthma
  5. 5. The various strategies developed are:-(I) CONVENTIONAL a. Physical Method- Uprooting: Health & Labor Problems Stumping : Resurgence b. Chemical Method- 2,4-D, Environmental Pollution Tragedy :Bhopal Dec 3/4MIC(II) NON-CONVENTIONAL a. Biological control- Use of organisms and metabolites Eco-friendly, Economically Feasible
  6. 6. Sclerotium rolfsii: Mycoherbicidal agentDevastating soil-borne pathogenRequires warm and humid environmentNecrotrophic, advance pathogenCauses severe collar rot diseasePotential MycoherbicideHost specific isolate
  7. 7. OBJECTIVES Recovery of geographically different strain from different hosts Determination of morphological variation and mycelia compatibilityamong isolates Biochemical characterization of isolates Determination of the genetic diversity among strains Determination of mycoherbicidal potential of fungal isolate by field trials
  8. 8. Colony characteristics produced by S. rolfsii isolates
  9. 9. Growth (cm)/day 0 1 2 3 4 5 6 7 8 Lan#01 Lan#02 Lan#03 Par#01 Par#02 Par#03 Par#04 Par#05 Par#06 Par#07 Par#08 Par#09 Par#10 Growth (cm/d) Hyp#01 Hyp#02 Hyp#03 Hyp#04 z 5 Isolates rolfsii Crotolaria Cassia#01 Lentil Chkpea#01 Chkpea#02 Chkpea#03Sclerotia Dia. : Max; Par#08, Min; PeaGrowth: Max; Brinj#02 , Min; Cassia#01 Brinj#01 Brinj#02 Wht#01 Wht#02 Diam. (mm) Wht#03 Chestnut Pea Soyabean Fig. 5a : Growth and sclerotia diameter of different isolates of S. Grass 0 1 2 3 0.5 1.5 2.5 Diam. (mm)
  10. 10. Maximum: Par#07 ; Minimum: Crotolaria
  11. 11. Fig.6a : Production of Oxalic acid, Biomass & sclerotia after 7 days of incubation by different isolates of S. rolfsii 60 3 50 2.5 40 2 30 1.5 20 1 10 0.5 0 0 Chkpea#01 Chkpea#02 Chkpea#03 Pea Par#01 Par#02 Par#03 Par#04 Par#05 Par#06 Par#07 Pat#08 Par#09 Par#10 Hyp#01 Hyp#02 Hyp#03 Hyp#04 Crotolaria Cassia#01 Chestnut Grass Lentil Lan#1 Lan#2 Lan#3 Wht#01 Wht#02 Wht#03 Brinj#01 Brinj#02 Soyabean Isolates Biomass (g) Sclerotia (No.) Oxalic acid (mg/ml)Oxalic Acid; Max : Crotolaria Min:wht#02 Sclerotia; Max :chestnut Min: chkpea#02
  12. 12. Fig.6b: Production of Oxalic acid, Biomass & sclerotia after 14days of incubation by different isolates of S. rolfsii 140 7 120 6 100 5 80 4 60 3 40 2 20 1 0 0 Chkpea#01 Chkpea#02 Chkpea#03 Pea Par#01 Par#02 Par#03 Par#04 Par#05 Par#06 Par#07 Pat#08 Par#09 Par#10 Hyp#01 Hyp#02 Hyp#03 Hyp#04 Crotolaria Cassia#01 Chestnut Grass Lentil Lan#1 Lan#2 Lan#3 Wht#01 Wht#02 Wht#03 Brinj#01 Brinj#02 Soyabean Isolates Biomass (g) Sclerotia (No.) Oxalic acid (mg/ml)Oxalic Acid; Max : Lan#03 Min: hyp #02 Sclerotia; Max :chestnut Min: chkpea#01
  13. 13. Cellulolytic activity produced by S. rolfsii isolates Cellulases are cellulose degradingenzymes Plays a secondary role in pathogenicityof fungi Isolates produced different type ofactivity Lan#02 and Par#08 producedmaximum zone Media: 0.5% CMC + 2% Agar Temp:450C Incubation : 3 hrs
  14. 14. 0 20 40 60 80 100 120 140 160 180 Lan#1 Lan#2 Lan#3 Par#01 Par#02 Par#03 Par#04 Par#05 Par#06 Par#07 Pat#08 Media: Basal salt; Temp.280C; pH:5 Par#09 Par#10 Hyp#01 Hyp#02 7d7D Max : Lan#01 Min: Par#05 Hyp#03 rolfsii Hyp#04 Isolates Crotolaria 14d Lentil Chkpea#01 Chkpea#02 Chkpea#03 Brinj#0114D Max : Grass Brinj#02 Wht#01 Wht#02 Wht#03 Chestnut Fig.7b Cellulase production (g/ml) by different isolates of S. Pea Soyabean Grass Cassia#01
  15. 15. 0 2 4 6 8 10 12 14 16 Lan#1 Lan#2 Lan#3 Par#01 Par#02 Par#03 Par#04 Par#05 Par#06 Par#07 0 Pat#08 Par#09 Media: Basal salt; Temp.28 C; pH:5 Par#10 Hyp#01 Hyp#027d Hyp#03 Hyp#04 Isolates Crotolaria14d Lentil isolates of S. rolfsii Chkpea#01 Chkpea#02 Chkpea#03 Brinj#01 Brinj#02 Wht#01 Wht#02 Wht#03 Fig.7a Cellulase production (Zone diameter mm) by different Chestnut Pea Soyabean Grass Cassia#01
  16. 16. Cellulolytic zymogram of different isolates of S. rolfsiilane 4 : Hyp#03 produced maximumnumber of bandsSubstrate used : 0.1% carboxy-methyl celluloseIncubation period : 3 hrsTemp: 45 0 C
  17. 17. Cellulase electromorph of different isolates of S. rolfsii No. of bands  6 Banding pattern s 13
  18. 18. Pectinolytic activity of different isolates of S. rolfsii Pectinases play a significant role inS. rolfsii pathogenicity Degrades pectin present in plantcell wall Act in concert with oxalic acid andcause disease Three different types of zones wererecorded. Media: 1% Pectin + 2% Agar Temp:450C Incubation : 3 hrs
  19. 19. 0 5 10 15 20 25 Lan#1 Lan#2 Lan#3 Par#01 Par#02 Par#03 Par#04 Par#05 Par#06 Par#07 Pat#087D Max : Soybean Min: Lan#02 Par#09 Par#10 Hyp#01 Hyp#02 7d Hyp#03 Hyp#04 S. rolfsii Isolates Crotolaria 14d Lentil Chkpea#01 Chkpea#02 Chkpea#03 Brinj#0114D Max : Hyp#01 Min: Par#01 Brinj#02 Wht#01 Wht#02 Wht#03 Fig.8b: Pectinase production (µM/ml) by different isolates of Chestnut Pea Soyabean Grass Cassia#01
  20. 20. 0 2 4 6 8 10 12 14 16 18 Lan#1 Lan#2 Lan#3 Par#01 Par#02 Par#03 Par#04 Par#05 Par#06 Par#07 Pat#08 Par#09 Media: Basal salt; Temp.280C; pH:5 Par#107D Max : Par#08 Min: Chkpea#01 Hyp#01 Hyp#02 7d Hyp#03 Hyp#04 Isolates Crotolaria 14d Lentil isolates of S. rolfsii Chkpea#01 Chkpea#02 Chkpea#03 Brinj#01 Brinj#02 Wht#0114D Max : Par#03 Min: Hyp#04 Wht#02 Wht#03 Chestnut Fig.8a: Pectinase production (Zone diameter mm) by different Pea Soyabean Grass Cassia#01
  21. 21. Pectinase electromorphs produced by different isolates of S. rolfsii Bands 8 Banding patterns 17
  22. 22. Mycelial Compatibility among S. rolfsii isolates Anastomosis between the isolatesTotal pairings: 495Total compatible reaction: 81Total MCG s: 14 amongst 32 isolates Barrage zone between the isolates
  23. 23. Amplification of ITS region of rDNA of S. rolfsii isolates Amplified ITS region ~ 650 base pairs
  24. 24. ITS – RFLP Fingerprint of different isolates of S. rolfsii Taq I Hind IIIBands obtained: Bands obtained: 150 &250200 & 400 bp Banding pattern: 4Banding pattern : 5 Hpa I
  25. 25. Random Amplified polymorphic DNA-polymerase chain reaction fingerprint ofdifferent isolates of S. rolfsii obtained using primers OPE-12 and OPE-17 OPE-12 Bands obtained: 2000 & 450 bp Banding pattern : 4 OPE-17 Bands obtained: 1000 & 750 bp Banding pattern :3
  26. 26. Dendrogram of isolates of S. rolfsii revealed by cluster with the un-weighted pair group methods with arithmetic averages using primer OPE-12 Major groups: 2 Subgroups: 3
  27. 27. Dendrogram of isolates of S. rolfsii revealed by cluster with the un-weighted pair group methods with arithmetic averages using primer OPE-17 Major groups: 2 Subgroups: 5 Similarity coefficient
  28. 28. Pathogenic Potential of S. rolfsii Isolates Pathogenicity 3.5 3 2.5 2 1.5 1 0.5 0 Brinj#01 Brinj#02 Hyp#01 Hyp#02 Hyp#03 Hyp#04 Wht#01 Wht#02 Wht#03 Par#01 Par#02 Par#03 Par#04 Par#05 Par#06 Par#07 Par#08 Par#09 Par#10 Grass Lentil Chestnut Pea Soybean SEM Lan#01 Lan#02 Lan#03 Chkpea#01 Chkpea#02 Chkpea#03 Crotolaria Cassia#01 PathogenicityScale used: 0 no symptoms; 1Initial wilting and yellowing; 2 marginalblight and necrosis at collar region; 3: complete collapse of host seedlingAmount of Inoculum: 0.1 g of infective propagule and observation recordedafter 48 h
  29. 29. Fig. 9a: Competence volume of S. rolfsii after 20 days of inoculation at various depth & root radii. 0.6 120 Competence Volume (cm3) 0.5 100Roolt Radii cm 0.4 80 0.3 60 0.2 40 0.1 20 0 0 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 Depth Radius of roots 20d Competence volume(cm3) 20d
  30. 30. Fig. 9b: Competence volume of S. rolfsii after 30 days of inoculation at various depth & root radii. 0.6 100 98 Competence Volume (cm3) 0.5 96 94Roolt Radii cm 0.4 92 90 0.3 88 0.2 86 84 0.1 82 80 0 78 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 Depth Radius of roots 30d Competence volume(cm3) 30d
  31. 31. MASS PRODUCTION OF S. rolfsiiWheat grains Bagasse & Wheat bran Maize Cover
  32. 32. Mycelial coverage (%) 10 12 14 16 18 0 2 4 6 8 Rice bran Wheat Bran Tea leaves WSWB Pulses bran(Rahar) Chana Dal BT WWT TBWW WBB TPWS Substrates Wheat grains Wheat straw Cotton Tomato pulp(dry)Maximum: wheat grains ; Minimum: Tomato pulp Host leaves Maize cob Fig : Mycelial coverage of S.rolfsii on different substrates Orange peel Pomegranate Cob cover LSD
  33. 33. No. of Sclerotia Produced/50gm 2000 4000 6000 8000 10000 12000 14000 0 Rice bran Wheat Bran Tea leaves WSWB Pulses bran(Rahar) Chana Dal BT WWT TBWW WBB TPWS Substrates Wheat grains Wheat straw Cotton Tomato pulp(dry)Maximum: chickpea waste ; Minimum: wheat Bran Host leaves Maize cob Orange peel Fig : Sclerotia production of S.rolfsii on different substrates Pomegranate Cob cover LSD
  34. 34. Mean disease intensity 100 0 10 20 30 40 50 60 70 80 90 Rice bran Wheat Bran Tea leaves WSWB Pulses bran(Rahar) Chana Dal BT WWT TBWW WBB TPWS Wheat grains Substrates Wheat straw Cotton Fig : Effect of fungus on host seedlings Tomato pulp(dry)Maximum: wheat grains ; Minimum: BT Host leaves Maize cob Orange peel Pomegranate Cob cover LSD
  35. 35. Amount of inoculum: 0.1 gm of each substrate : Observation recorded after 72 h
  36. 36. Host specificity depicted by fungal isolate
  37. 37. Profuse growth and Tip colonization inhibited flower and seed formation
  38. 38. Final Words All the isolates obtained form different hosts and region revealed remarkablevariation in most of the parameters studied. Isolates did not reflect substantial difference in their genetic constitution Par #02 isolate obtained from Parthenium induced maximum symptomsleading to the collapse of the weed after 48 HPT. TBWW recorded to be best substrate for mass production and maximummycoherbicidal effect was recorded at pre emergence stage by wheat grains. A decrease was observed in isolate efficiency during field trails incomparison to Lab and green house studies. Host specificity was observed with this isolate and it can be develop as amycoherbicidal agent like other fungal pathogens after few more studies. It is eco-friendly and economically feasible.
  39. 39. PUBLICATIONS1. Shukla, R. and Pandey, A.K. (2006). Maximization of production of oxalic acid from Sclerotium rolfsii, a mycoherbicidal agent against Parthenium. Ann. Pl. Protect. Sc. 14(1): 202-205.2. Shukla, R., Prahalad, S. and Pandey, A.K. (2006) Herbicidal activity of cell free culture filtrate of Sclerotium rolfsii FGCC#105 against Hyptis suaveolens. Vislesana. 10 B (2): 55-61.3. Shukla, R. and Pandey, A.K. (2007). Diversity in isolates of Sclerotium rolfsii isolates from central India. J.Mycol.Pl Pathol. 37(3): 514-518.4. Shukla, Rekha and Pandey, A.K. (2008). Pathogenic diversity of Sclerotium rolfsii isolates, a potential biocontrol agent against Parthenium hysterophorus L. Afr. J. Env. Sc. Tech. 2(4): 124-126.5. Shukla Rekha and A.K.Pandey (2008). Formulation and evaluation of agrochemicals from Sclerotium rolfsii FGCC#02 against Parthenium hysterophorus. J. Plant Protect. Res. Vol. 48(4) : 487-494.6. Shukla Rekha and A.K. Pandey (2009). Evaluation of different substrate for mass production and field performance of collar rot fungi strain for the management of Parthenium Indian J. Weed Sci. 41(1&2): 67-69.7. Shukla Rekha and A.K. Pandey (2011). Variation in Cellulases produced by Sclerotium rolfsii isolates: a herbicidal agent against Parthenium hysterophorus. Journal of Plant Protection Research (Accepted).
  40. 40. ACKNOWLEDGEMENTSDr. A. K. Pandey , Professor, Department of Biologicalsciences, R D University, Jabalpur, INDIAMadhya Pradesh Council of Science andTechnology, Bhopal, INDIAInstitute of Microbial Technology, Chandigarh, INDIA

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