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

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. The Weed : Parthenium hysterophorus
 Alien, Invasive and Obnoxious weed.
 Potential hazard to our biodiversity ,
human beings and Agriculture
 Prolific seed producer - 25,000 seeds
per plant
 Weed of international Importance
 Can germinate and set flower and seed
in 4-6 weeks

3. Infestation of Parthenium in ecosystems
A B
Agriculture land Agriculture land
C DD
Residential Area Roadsides

4. Human and Animal health hazards of
Parthenium
• Photodermatitis
• Drivers committed
A B suicide
C •12 buffalos died at
Itarsi
D
A, B & C. Contact Dermatitis D. Asthma

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. Sclerotium rolfsii: Mycoherbicidal agent
Devastating soil-borne pathogen
Requires warm and humid environment
Necrotrophic, advance pathogen
Causes severe collar rot disease
Potential Mycoherbicide
Host specific isolate

7. OBJECTIVES
 Recovery of geographically different strain from different hosts
 Determination of morphological variation and mycelia compatibility
among isolates
 Biochemical characterization of isolates
 Determination of the genetic diversity among strains
 Determination of mycoherbicidal potential of fungal isolate by field trials

8. Colony characteristics produced by S. rolfsii isolates

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#03
Sclerotia Dia. : Max; Par#08, Min; Pea
Growth: 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. Maximum: Par#07 ; Minimum: Crotolaria

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. 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. Cellulolytic activity produced by S. rolfsii isolates
 Cellulases are cellulose degrading
enzymes
 Plays a secondary role in pathogenicity
of fungi
 Isolates produced different type of
activity
 Lan#02 and Par#08 produced
maximum zone
Media: 0.5% CMC + 2% Agar
Temp:450C
Incubation : 3 hrs

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
7d
7D Max : Lan#01 Min: Par#05
Hyp#03
rolfsii
Hyp#04
Isolates
Crotolaria
14d
Lentil
Chkpea#01
Chkpea#02
Chkpea#03
Brinj#01
14D 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. 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#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#01
Wht#02
Wht#03
Fig.7a Cellulase production (Zone diameter mm) by different
Chestnut
Pea
Soyabean
Grass
Cassia#01

16. Cellulolytic zymogram of different isolates of S. rolfsii
lane 4 : Hyp#03 produced maximum
number of bands
Substrate used : 0.1% carboxy-methyl cellulose
Incubation period : 3 hrs
Temp: 45 0 C

17. Cellulase electromorph of different isolates of S. rolfsii
No. of bands  6
Banding pattern s 13

18. Pectinolytic activity of different isolates of S. rolfsii
 Pectinases play a significant role in
S. rolfsii pathogenicity
 Degrades pectin present in plant
cell wall
 Act in concert with oxalic acid and
cause disease
 Three different types of zones were
recorded.
Media: 1% Pectin + 2% Agar
Temp:450C
Incubation : 3 hrs

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#08
7D 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#01
14D 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. 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#10
7D 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#01
14D 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. Pectinase electromorphs produced by different isolates of S. rolfsii
Bands 8
Banding patterns 17

22. Mycelial Compatibility among S. rolfsii isolates
Anastomosis between
the isolates
Total pairings: 495
Total compatible reaction: 81
Total MCG s: 14 amongst 32 isolates
Barrage zone between
the isolates

23. Amplification of ITS region of rDNA of S. rolfsii isolates
Amplified ITS region ~ 650 base pairs

24. ITS – RFLP Fingerprint of different isolates of S. rolfsii
Taq I Hind III
Bands obtained: Bands obtained: 150 &250
200 & 400 bp Banding pattern: 4
Banding pattern : 5
Hpa I

25. Random Amplified polymorphic DNA-polymerase chain reaction fingerprint of
different 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. 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. 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. 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
Pathogenicity
Scale used: 0 no symptoms; 1Initial wilting and yellowing; 2 marginal
blight and necrosis at collar region; 3: complete collapse of host seedling
Amount of Inoculum: 0.1 g of infective propagule and observation recorded
after 48 h

29. Fig. 9a: Competence volume of S. rolfsii after 20 days of inoculation at
various depth & root radii.
0.6 120
Competence Volume (cm3)
0.5 100
Roolt 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. 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
94
Roolt 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. MASS PRODUCTION OF S. rolfsii
Wheat grains
Bagasse & Wheat bran Maize Cover

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. 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. 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. Amount of inoculum: 0.1 gm of each substrate : Observation recorded after 72 h

38. Host specificity depicted by fungal isolate

39. Profuse growth and Tip colonization inhibited flower and seed formation

40. Final Words
 All the isolates obtained form different hosts and region revealed remarkable
variation in most of the parameters studied.
 Isolates did not reflect substantial difference in their genetic constitution
 Par #02 isolate obtained from Parthenium induced maximum symptoms
leading to the collapse of the weed after 48 HPT.
 TBWW recorded to be best substrate for mass production and maximum
mycoherbicidal effect was recorded at pre emergence stage by wheat grains.
 A decrease was observed in isolate efficiency during field trails in
comparison to Lab and green house studies.
 Host specificity was observed with this isolate and it can be develop as a
mycoherbicidal agent like other fungal pathogens after few more studies.
 It is eco-friendly and economically feasible.

41. PUBLICATIONS
1. 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).

42. ACKNOWLEDGEMENTS
Dr. A. K. Pandey , Professor, Department of Biological
sciences, R D University, Jabalpur, INDIA
Madhya Pradesh Council of Science and
Technology, Bhopal, INDIA
Institute of Microbial Technology, Chandigarh, INDIA