Thesis titled "Development of Biopesticide for the control of Root Pathogenic Fungi in Chickpea using Plant Growth Promoting Rhizobacteria ". • Supervised by Prof. Dr. M. Inam-ul-Haq. • Isolation and Characterization of Rhizbacterial isolates from Rawalpindi District • Utilization of PGPR antagonistic potential in the form of biopesticide formulation against Fungal Root Infecting Pathogens. • The Developed formulations with best shelf life and Rhizobacterial viability were evaluated for their efficacy under open field conditions for disease control and plant growth enhancement.

2. Shazia Shahzaman
Ph.D.
Plant Pathology
05-arid-207
Development of Biopesticide for the Control
of Root Pathogenic Fungi in Chickpea using
Plant Growth Promoting Rhizobacteria

3. Prof. Dr. M. Inam-ul-Haq
Department of Plant Pathology
Prof. Dr. Tariq Mukhtar
Department of Plant Pathology
Prof. Dr. M. Naeem
Department of Entomology
Supervisor
Member
Member

4. INTRODUCTION TO CROP
 Chickpea (Cicer arietinum L.) belongs to Fabaceae
family and ranked third after dry beans and peas
(FAO, 2013)
 Significant in Human diet and animal feed.
 Chickpea cultivated area is 1068 thousand ha-1 with
production of 523 thousand tons during 2013-14 with
an average yield of 685 kg ha-1
Agri. Stat. of Pak., 2014

5. PAKISTAN RANKING IN CHICKPEA PRODUCTION
77%
9%
7%
4% 3%
India
Turkey
Pakistan
Islamic republic of Iran
Mexico
Pakistan ranked 3rd in world chickpea
production 757.1 (Kg/Ha).
WORLD PRODUCTION
Data Source
http://www.factfish.com/statistic country/Pakistan/chickpea,+ production+ quantity
The Punjab province alone contributed 900.1 thousand
ha which was 84% of the to the total chickpea area
grown in the country.

6. Area and Production of Chickpea Crop
Crop
2012-2013 2013-2014
% change in
ProductionArea
(000 ha)
Production
(000 tons)
Area
(000 ha)
Production
(000 tons)
Chickpea 1067 562 1068 523 -6.9
670
680
690
700
710
2012-13 2013-14
Yield (kg/ha)

7.  About 10-50% losses by Fungal Root Disease
have been reported on chickpea in the dry areas of
Pakistan during the past several years
Woltz and Jones, 2012
 Estimated yield losses in Chickpea due to
biotic and abiotic factors range from 15-80%
Kuku et al., 1996

8. Wet Root rot Fusarium Wilt
Verticillium Wilt
Important Fungal Diseases of Chickpea
Dry Root rot

9. Need of the Project
• 311 compounds have been registered as
fungicides Milne, 2010
• Fungicides results into acute and chronic toxicity.
Goldman, 2008
• Environmental pollution result in human
exposure through consumption of residues of
pesticides in food and, possibly, drinking water
Suprapta, 2012
• Millions of people suffer from pesticide
problems and 18,000 die every year. WHO/
Miller, 2012

10. • Various reports on the utilization of potential
microbes as bio-control.
Yang et al., 2008
• Several species of Rhizobacteria antagonize
the fungal pathogens.
Walsh et al., 2001
• Development of Bio-formulation using Plant
growth promoting rhizobacteria (PGPR).
Kumar et al., 2011

11. PGPR Mode of action

12. Hypothesis
Rhizobacteria naturally present in soils may interfere with the
extent of root colonization, Disease suppression and plant growth
promotion by Plant Growth Promoting Rhizobacteria (PGPR)

13. • Isolation of fungal root pathogens and rhizobacteria from chickpea
roots and rhizospheric soil.
• Selection of rhizobacterial isolates antagonistic to root pathogenic
fungi of chickpea and their characterization.
• Evaluation of PGPR based formulations against fungal root pathogens
under various conditions.
• Selection of suitable formulation as a biopesticide.

15. Plant growth promoting rhizobacteria (PGPR) were first defined as
the soil bacteria that colonize the roots of plants by following
inoculation on to seed and that enhance plant growth
Kloepper and Schroth
(1978)
At present, the use of biological approaches is becoming more popular
as an additive to chemical fertilizers for improving crop yield in an
integrated plant nutrient management system. In this regard, the use of
PGPR has found a potential role in developing sustainable systems in
crop production.
(Sturz et al. 2000;
Shoebitz et al.2009).
P. fluorescens strains isolated from rhizosphere of rice, wheat, pigeon
pea, groundnut and chili crops produced extra cellular siderophores
which were antagonistic to fungal pathogens like Fusarium
oxysporum, Alternaria sp and Colletotrichum capsicii.
(Suryakala et al.,2004)
Important genera of bacteria used in natural and man-created
bioremediation includes Bacillus, Pseudomonads, Methanobacteria,
Ralstonia and Deinococcus, etc.
Milton 2007
The production of siderophores with fungicidal properties of
Pseudomonas fluorescens and neem cake against Fusarium wilt
disease of chickpea. The potential them is worthy of further evaluation
as a biocontrol system for chickpea wilt.
Inam et al., 2010

16. Utilization of these organisms has no negative effect on
environment and also on other non-target organisms.
Various reports are available on the utilization of these
potential microbes as bio-control agents. Yang et al., 2008
Several soil borne non-pathogenic species of rhizobacteria
are reported to antagonize the disease causing fungal
pathogens and can be utilized as alternative to chemical
control measure Walsh et al., 2001
PGPR belonging to various genera, such as Azotobacter,
Azospirillum, Pseudomonas, Acetobacter, Burkholderia and
Bacillus and have been repeatedly reported by many
researchers. Bashan and Ulangatan, 2002
Many bio-control based formulations have been prepared
and commercialized in America and Europe. Many of the
pesticide companies aimed to develop bio-control gents
based bio-pesticide formulations as commercial product.
Suprapta, 2012
Increase in soil fertility, plant growth promotion, plant
pathogen suppression and development of eco-friendly
bio-formulation is the matter of major concern. Arora et al., 2010

18. Activity 1
Survey, Sampling and Surveillance
Objective # 1

19. An extensive survey of Rawalpindi, Chakwal and
Attock District was done in 2012-13

20. Rhizospheric soil and root sample collection
Random fields in each visited Districts
Random samples from each visited field
Hierarchical sampling strategy was followed for sampling
(McDonald and Martinez, 1990)

21. Disease Severity rating scale (1-9)
• 1= highly resistant (0-10% plants wilted)
• 3= resistant (11-20% plants mortality)
• 5= moderately resistance (21-30% mortality)
• 7= susceptible (31-50% mortality)
• 9= highly susceptible (more than 50% mortality)
Iqbal et al. (2005)

22. Province District Areas Visited
No. of
Fields
Disease
Prevalence
%
Disease
Incidence
%
Disease
Severity
(1-9 rating
scale)
Punjab
Attock
FatehJang 20 83 74.5 7
Hasanabdal 11 80 82 5
Pindigheb 14 86 76 5
Chakwal Chakwal 19 95 89 9
Rawalpindi
Doltala 13 85 86 5
Tarnol 6 85 75.5 5
Taxila 10 60 47 3
Kahuta 9 32 20 3
Locations Surveyed and Surveillance
Table:1 Disease prevalence, incidence and severity of Fungal Root Pathogens in surveyed areas

23. Fig. 1 Disease prevalence, incidence and severity of Fungal Root Pathogen in different
areas.
0
1
2
3
4
5
6
7
8
9
10
0
10
20
30
40
50
60
70
80
90
100
Disease Prevelance % Disease Incidence % Disease Severity % (1-9 rating scale)

24. Activity 2
Isolation and Purification of Pathogens and Rhizobacteria

25. Isolation of Fungal Root Pathogen
• Infected root tissue at the advancing edge of a wilted area and
rhizosphere soil were used.
• Tissues were washed and cut into small pieces (2 mm), surface
disinfested in 1 % NaClO for 2 min, rinsed in distilled water,
and damp-dried on absorbent paper towels before being plated
on the PDA media.
(Jose et al , 2012)

26. Fig.2 Characterization of fungal root pathogen on the basis of colony shape and color A. Rhizoctonia
spp. B. Pythium spp. C. Fusarium spp. E. Macrophomina spp.
a b c
d e f

27. Fig. 3: Microscopic and visual observation of Fungal Root Pathogens
A- Colony growth of Fungal Root Pathogens
i.e., Fusarium spp., Rhizoctonia spp., Macrophomina spp.

28. Fig.4 Observation under microscope at 40 X (a) Rhizoctonia spp. (b) Fusarium spp. (c)
Fusarium spp. (d) Macrophomina spp. (e) Pythium spp.
a
b
c d e
a
d e

29. Morphological Identification of Fungal Root Pathogens
Fungal Isolates Colony Color Spore Shape Macroconidia
(µm)
Colony
Diameter(mm)
Media Used
Fusarium spp. Brick red to
violet
Blunt 21.49-29.23 3.2 Potato
Dextrose Agar
Rhizoctonia
spp.
Brownish to
blackish
Papillate 12.3-15.0 5 Potato
Dextrose Agar
Pythium spp. Slightly yellow Tapering 10.5-12.0 2.9 Corn Meal
Agar
Macrophomina
spp.
Creamy to pink Hooked 18.3-20.5 3.5 Potato
Dextrose Agar
(Arotupin, 2004)

30. Fungal Root Pathogen Isolated
Tehsil Fusarium
spp.
Rhizoctonia
spp
Pythium
spp.
Macrophomina
spp.
Total
FatehJang 9 3 5 4 21
Hasanabdal 9 3 3 3 18
Pindigheb 5 4 3 2 14
Chakwal 10 7 4 5 26
Doltala 6 5 3 2 16
Tarnol 5 4 3 2 14
Taxila 5 1 1 1 8
Kahuta 6 2 1 2 11
Total 55 29 23 21 128
Table.2 Total 128 fungal root pathogens isolated from rhizospheric soil and roots and most
of them from Chakwal i.e. 24

31. Isolation of Rhizobacteria
• Soil samples 1 g of each sample was suspended in 9 ml sterile
water and shaken vigorously for 2 min.
• The soil suspension was serially diluted (from 10-1 to 10-9)
• 0.1 ml of all dilutions were plated on selective media
supplemented with a commercial antifungal to inhibit fungi
growth.
• Petri dishes were incubated at 28±2°C
(Gerhardt, 1994)

32. Morphological Characterization of Rhizobacteria
Bacterial
Isolates
Colony
shape
Colony
Margin
Colony
Elevation
Colony
Color
Gram
staining
Media used
RB1 irregular flat flat white -ve Azotobacter
Agar
(Mannitol)
RB2 irregular round flat Creamy
white
+ve Nutrient Agar
RB3 irregular oval raised Yellowish
white
-ve King B
RB4 slightly
dome
shaped
round raised yellow -ve King B
RB5 Circular pointed raised yellow -ve Tryptic soy
agar

33. Fig.5 Various Rhizobacterial colonies Isolated on Nutrient agar media

34. Rhizobacteria Isolated from Rhizosphere of
Chickpea
Areas RB1 RB2 RB3 RB4 RB5 Total
FatehJang 8 6 8 9 6 37
Hasanabdal 7 5 6 6 4 28
Pindigheb 5 6 4 7 3 25
Chakwal 9 11 8 10 7 45
Doltala 5 4 5 6 3 23
Tarnol 6 5 7 8 5 31
Taxila 4 3 7 2 4 20
Kahuta 3 5 4 5 4 21
Total 47 45 49 53 36 230
Table.2 Total 230 bacterial isolates were isolated from rhizospheric soil.

35. Activity 3
Pathogenicity Test

36. • Inoculum of isolated fungal root pathogens was mixed with
1kg autoclaved soil in pots.
• Five seeds of chickpea cultivar were sown in each pot and
grown for 40 days at 25±2°C.
• Control plants were grown in a comparable mixture of
uninfected and autoclaved soil.

37. • Typical wilt symptoms were observed in most of the pots in 41
days after sowing, development of symptoms were recorded.
• Re-isolation of fungal root pathogens was made by taking
infected plant tissues on selective media, Identification of
these pathogens indicating that the plant mortality was
associated with these fungal root pathogens.
Nene & Haware (1980)

38. Fatehjang
Chickpea Varieties Bital-98 Wanhaar Desi-sp Noor-91 Punjab-2000
FFS-1 +++ ++ +++ + +
FFS-2 + ++ ++ - +++
FFS-3 - + +++ ++ -
FFS-4 +++ + + + +
FFS-5 + ++ - ++ +++
FFS-6 ++ + + - -
FFS-7 +++ - + + ++
FFS-8 ++ + ++ +++ +
FFS-9 +++ + + + +
FRS-1 ++ ++ +++ - +++
FRS-2 + - + +++ +
FRS-3 +++ + +++ + -
FPS-1 ++ ++ +++ - -
FPS-2 +++ ++ +++ - -
FPS-3 ++ - + ++ +
FPS-4 + ++ - + -
FPS-5 + +++ - + +++
FMS-1 +++ ++ +++ - +
FMS-2 +++ + - +++ +
FMS-3 - - +++ + -
FMS-4 +++ + + + +
FFS(Fatehjang Fusarium Spp.), FRS(Fatehjang Ralstonia spp.), FPS(Fatehjang Pythium Spp.), FMS(Fatehjang
Macrophomina Spp.), +++(most virulent) ++(moderately virulent) +(less virulent) –(no virulence)

39. Hasanabdal
Chickpea Varieties Bital-98 Wanhaar Desi-sp Noor-91 Punjab-2000
HFS-1 ++ - +++ + +
HFS-2 + ++ - + +
HFS-3 +++ ++ +++ ++ +
HFS-4 + + + - ++
HFS-5 + + + - ++
HFS-6 + + + - ++
HFS-7 + + + - ++
HFS-8 + + + - ++
HFS-9 + + + - ++
HRS-1 + - ++ + ++
HRS-2 +++ ++ + + +
HRS-3 + - +++ - +
HPS-1 +++ + - - -
HPS-2 - ++ ++ + ++
HPS-3 - + + - ++
HMS-1 + + ++ ++ -
HMS-2 +++ + + + +
HMS-3 - + ++ ++ +
HFS(Hasanabdal Fusarium Spp.), HRS(Hasanabdal Ralstonia spp.), HPS(Hasanabdal Pythium Spp.), HMS(Hasanabdal
Macrophomina Spp.)

40. Pindigheb
Chickpea Varieties Bital-98 Wanhaar Desi-sp Noor-91 Punjab-2000
PFS-1 +++ + +++ - -
PFS-2 + + +++ + ++
PFS-3 - - +++ + -
PFS-4 +++ + + - +
PFS-4 +++ + + - +
PFS-5 +++ + + - +
PRS-1 + - ++ - +++
PRS-2 - ++ + + +
PRS-3 +++ - +++ - -
PRS-4 - + - + ++
PPS-1 +++ + +++ - -
PPS-2 + - + ++ +
PPS-3 - ++ +++ - -
PMS-1 - ++ +++ ++ +
PMS-2 + + - + -
PFS(Pindigheb Fusarium Spp.), PRS(Pindigheb Ralstonia spp.), PPS(Pindigheb Pythium Spp.), PMS(Pindigheb
Macrophomina Spp.)

41. Chakwal
Chickpea Varieties Bital-98 Wanhaar Desi-sp Noor-91 Punjab-2000
CFS-1 ++ + +++ +++ -
CFS-2 + + ++ + ++
CFS-3 + +++ ++ + -
CFS-4 +++ ++ + - +
CFS-5 ++ + +++ +++ -
CFS-6 + + ++ + ++
CFS-7 + +++ ++ + -
CFS-8 +++ ++ + - +
CFS-9 ++ + +++ +++ -
CRS-1 + - +++ - +++
CRS-2 - - + + +
CRS-3 +++ - +++ +++ -
CRS-4 - + - + ++
CRS-5 ++ + - ++ -
CRS-6 - ++ ++ + ++
CPS-1 +++ +++ - - -
CPS-2 ++ - +++ ++ +
CPS-3 - + ++ - -
CPS-4 +++ ++ +++ + ++
CMS-1 ++ + + +++ +
CMS-2 ++ + - + -
CMS-3 +++ + ++ - +
CFS(Chakwal Fusarium Spp.), CRS(Chakwal Ralstonia spp.), CPS(Chakwal Pythium Spp.), CMS(Chakwal Macrophomina Spp.)

42. Doltala
Chickpea Varieties Bital-98 Wanhaar Desi-sp Noor-91 Punjab-2000
DFS-1 +++ + +++ ++ -
DFS-2 ++ +++ ++ + ++
DFS-3 +++ ++ +++ - +
DFS-4 - + +++ +++ -
DFS-5 ++ - + ++ +
DFS-6 ++ + ++ - +
DRS-1 + - + ++ ++
DRS-2 +++ ++ +++ - +
DRS-3 + + - - -
DRS-4 +++ - +++ +++ +++
DRS-5 + ++ +++ + -
DPS-1 + + + - +
DPS-2 +++ ++ +++ ++ -
DPS-3 ++ +++ - + +++
DMS-1 + ++ +++ - ++
DMS-2 +++ + - +++ -
DFS(Doltala Fusarium Spp.), DRS(Doltala Ralstonia spp.), DPS(Doltala Pythium Spp.), DMS(Doltala
Macrophomina Spp.)

43. Tarnol
Chickpea Varieties Bital-98 Wanhaar Desi-sp Noor-91 Punjab-2000
TFS-1 ++ + +++ - -
FFS-2 + + ++ +++ ++
TFS-3 +++ - ++ + -
TFS-4 ++ + ++ - +
TFS-5 + ++ - ++ +
TRS-1 + - ++ - +++
TRS-2 ++ ++ + + +
TRS-3 + - + ++ -
TRS-4 - + - + ++
TPS-1 ++ + - - -
TPS-2 ++ - + ++ +
TPS-3 - ++ ++ - -
TMS-1 - ++ +++ ++ +
TMS-2 + + - + -
TFS(Tarnol Fusarium Spp.), TRS(Tarnol Ralstonia spp.), TPS(Tarnol Pythium Spp.), TMS(Tarnol
Macrophomina Spp.)

44. Taxila
Chickpea Varieties Bital-98 Wanhaar Desi-sp Noor-91 Punjab-2000
TxFS-1 ++ + +++ - -
TxFS-2 + +++ - +++ ++
TxFS-3 +++ - ++ + -
TxFS-4 ++ + + - +
TxFS-5 +++ ++ - ++ ++
TxRS-1 + - ++ - +++
TxPS-1 ++ + - - -
TxMS-1 + ++ +++ ++ +
TxFS(Taxila Fusarium Spp.), TxRS(Taxila Ralstonia spp.), TxPS(Taxila Pythium Spp.), TxMS(Taxila
Macrophomina Spp.)

45. Kahuta
Chickpea Varieties Bital-98 Wanhaar Desi-sp Noor-91 Punjab-2000
KFS-1 +++ + +++ - -
KFS-2 + ++ + + ++
KFS-3 +++ - - +++ -
KFS-4 ++ + ++ - +
KFS-5 - +++ + + -
KFS-6 ++ + + ++ ++
KRS-1 + - - - +++
KRS-2 +++ - +++ ++ +
KPS-1 +++ + +++ - -
KMS-1 - +++ +++ - +
KMS-2 ++ + ++ + ++
KFS(Kahuta Fusarium Spp.), KRS(Kahuta Ralstonia spp.), KPS(Kahuta Pythium Spp.), KMS(Kahuta
Macrophomina Spp.)

46. Pathogenicity confirmation
128 Isolates grouped according to their morphology and most
virulent isolates were selected for further studies i.e. 20

47. Activity 4
Zone Inhibition Test against Fungi
a)Rhizobacteria
b)Fungicides
Objective # 2

48. (Dual Culture Technique)
• Antagonism was tested by culturing both rhizobacteria and
Fungal Root Pathogens on same culture media containing plate.
• Dual culturing of rhizobacteria and pathogenic fungi
Incubation at 26±2oC.
• Control contain only pathogen
• Data collection by measuring zone of inhibition in mm.
Haine et al. 2008

49. (Poisoned Food Technique)
• The systemic fungicides viz., Tilt, Contaff, Bavistin and Folicur
were evaluated against the test fungus at the concentration of 15
ppm.
• Each media toxicated with fungicide was poured in three Petri plates
• Non toxicated media was poured into Petri plates kept as a check.
• A 5 mm mycelia disc of 6 days old culture of the test pathogen was
cut with sterile cork borer and placed in center of each Petri plate.
(Sharvelle, 1995)

53. Rhizobacteria (RB1, RB2, RB3, RB4, RB5), Fusarium spp. (FS), Propiconazole (Tilt),
Hexaconazole (Contaff), Carbendazim (Bavistin) and Tebuconazole (Folicur)
0
10
20
30
40
50
60
70
80
90
100
Fusarium spp.(FS) Inhibition(mm)

54. Rhizobacteria (RB1, RB2, RB3, RB4, RB5), Ralstonia spp.(RS), Propiconazole (Tilt),
Hexaconazole(Contaff), Carbendazim(Bavistin) and Tebuconazole(Folicur)
0
10
20
30
40
50
60
70
80
90
100
Rhizoctonia spp. (RS)Inhibition(mm)

55. Rhizobacteria (RB1, RB2, RB3, RB4, RB5), Pythium spp.(PS), Propiconazole (Tilt), Hexaconazole
(Contaff), Carbendazim (Bavistin) and Tebuconazole (Folicur)
0
10
20
30
40
50
60
70
80
90
100
Pythium spp. (PS)Inhibition(mm)

56. Rhizobacteria (RB1, RB2, RB3, RB4, RB5), Macrophomina spp.(MS), Propiconazole (Tilt),
Hexaconazole(Contaff), Carbendazim(Bavistin) and Tebuconazole(Folicur)
0
10
20
30
40
50
60
70
80
90
100
Macrophomina spp. (MS)Inhibition(mm)

57. Fungal Root
Pathogen
Fusarium
spp.
Rhizoctonia
spp.
Pythium
spp.
Macrophomina
spp.
Total
Pathogen Best
Growth
9 1 2 1 13
Rhizobacteria
Name
RB1 RB2 RB3 RB4 RB5 Total
Rhizobacteria
Inhibition
4 3 4 8 5 24
Out of 20 Fungal Isolates 8 found virulent and out of 230 Rhizobacteria
Isolated 24 showed best Antagonism against fungi

58. Activity 5
In-vivo Screening of Rhizobacterial Isolates

59. • A field trial was conducted with two local varieties of
Chickpea Bital-98, Desi-sp, in already infested field at
FatehJang in a randomized block design with three
replications.
• Plot size use was 2×2 m2. Chickpea seeds were treated with
rhizobacterial isolates (seed bacterization) following Jayraj et
al. (1999).
• The number of rhizobacterial population was maintained
through serial dilution and plating in NA. Treatments consist
of:

60. T1: RB-1(108 cfu/ml) @ 50ml/kg of seed
T2: RB-2(108 cfu/ml) @ 50ml/kg of seed
T3: RB-3(108 cfu/ml) @ 50ml/kg of seed
T4: RB-4(108 cfu/ml) @ 50ml/kg of seed
T5: RB-5(108 cfu/ml) @ 50ml/kg of seed
T6: control (no treatment)

61. Statistical Analysis
Data obtained from in vivo test were pooled for statistical
analysis and subjected to one ways ANOVA for determining any
significant differences among the treatments.

62. Table. 3 Effect of different Rhizobacteria on various attributes in Chickpea varieties cultivated
in wilt affected field after 60 days V1: Bital-98, V2: Desi-sp; variety; data are mean of three
replications
Treatments Plant Height (cm) # of nodules/plant Dry weight (g/plant)
V1 V2 V1 V2 V1 V2
T1 32.2 60.8 4.66 13.00 1.8 3.0
T2 26.8 80.0 10.33 13.66 1.0 3.2
T3 31.0 71.5 7.33 11.00 2.1 3.5
T4 30.2 48.4 14.00 16.00 2.3 2.8
T5 37.2 91.2 6.00 12.66 2.7 6.5
T6 26.7 24.1 1.60 2.00 0.5 1.4
CD (p=0.05) 7.21 3.31 1.86

63. Table.4 Effect of various Rhizobacteria on disease parameters of Fungal Root disease in Chickpea
average value of occurrence of disease for three months
Treatments Disease Incidence (%) Disease Severity (%) Percentage Disease
Control
T1 52.67 53.11 35.03
T2 61.67 60.91 30.30
T3 50.67 54.08 34.48
T4 48.00 51.00 41.59
T5 47.00 49.37 43.63
Control 97.00 96.25
CD (p=0.05) 11.13 10.04
14 isolates of rhizobacteria were selected on the basis of their antagonism against
Fungal Root Pathogens i.e. 6 of T4 and 8 from T5

64. Activity 6
Biochemical Characterization of Rhizobacteria

65. Biochemical Test Performed
• Gram’s reaction
• Carbohydrate fermentation
• Oxidase test
• H2S production
• Catalase activity
• Casein hydrolysis
• Indole production
• Urease test
• Acid and gas production
• Starch and gelatin hydrolysis
(Cappuccino and Sherman, 1992)

66. s.no Parameters RB-1 RB-2 RB-3 RB-4 RB-5
1 Gram Staining -ve -ve -ve -ve -ve
2 Aerobic growth +ve +ve +ve +ve +ve
3 Anaerobic growth -ve -ve -ve -ve -ve
4 Florescent pigment +ve +ve +ve +ve +ve
5 Oxidase +ve +ve +ve +ve +ve
6 Catalase +ve +ve +ve +ve +ve
7 Arginine hydrolysis +ve +ve +ve +ve +ve
8 Starch hydrolysis +ve -ve +ve +ve +ve
9 Levan production -ve -ve -ve -ve -ve
10 Gelatin hydrolysis +ve +ve +ve +ve +ve
11 Nitrate reduction +ve +ve +ve +ve +ve
12 Inositol -ve -ve -ve -ve -ve
13 Trehalose -ve -ve -ve -ve -ve

67. Fig.6 Biochemical test for Rhizobacterial isolates i.e, a. Catalase activity c. KOH test d.
Gram staining e. Starch hydrolysis f. Gelatin liquefaction g. Indole production
a b c d
e f g
d
h

68. Activity 7
Molecular Characterization

69. Primer Designing
DNA Extraction
PCR
Sequencing & analysis

70. PRIMER USED FOR RHIZOBACTERIA
Primers designed based on already published universal primers (e.g. 16S rDNA)
were used
.
Van der Meer et al., 2010

71. PRIMER USED FOR FUNGAL ROOT PATHOGNS
Primers designed based on already published universal primers (e.g. 16S rDNA)
were used
.
Aoki T, et al., 2003

72. DNA Extraction
• Extraction buffer that contains
detergent cetyl-tri-methyl ammonium
bromide (CTAB) and 2-β-
mercaptoethanol, EDTA and polyvinyl
pyrolidone (PVP).
• Quantification of DNA was done
with spectrophotometer determination.
Working concentration of DNA was
adjusted to 20 ng/ml and stored at 4ºC
Martins et al., 2005

73. PCR (RHIZOBACTERIA)
The PCR thermal profile consisted of an initial denaturation step
(94°C, 3 min), followed by 30 cycles at 94°C (30 sec), 55°C (1
min), 72°C (90 sec) and a final elongation step of 10 min at 72°C.
Kuske et al. ,1997

74. • Five µ l of the amplification products (amplicons) were
analysed on 1% (w/v) agarose gels cast and run in TAE buffer
(0.04 M Tris, 0.001 M EDTA, 0.02 M acetic acid), stained with
ethidium bromide and photographed under UV translluminator.
M RB-1RB-2RB-3 RB-4 RB-5RB-6RB-7 RB-8 RB-9 RB-10RB-11
1 2 3 4 5 6 7 8 9 101 1211 13
M

75. PCR (FUNGAL ROOT PATHOGEN)
Reactions involved 1 cycle at 95°C for 5 min, followed by 35 cycles with a
denaturation step at 95°C for 30 s, an annealing step at 55°C for 1 min, and an
extension step at 72°C for 1 min, followed by 1 cycle at 72°C for 6 mins
F1 F2 F3 F4 F5 F6
1 2 3 4 5 6M

77. SEQUENCING AND ANALYSIS
• Sequencing (MACROGEN)
• Basic Local Alignment Standard Tools (BLAST)
• Nucleospin Extract Kit (Macherey Nagel, Germany) and
sequenced at Genelab Casaccia (S. M. Di Galeria, Italy).
MEGA software used for phylogenetic analysis to check
similarities.(Zheng et al.,2000), using default parameter
values, to give the percentage homology with known
sequences in the NCBI database.

78. Fungal Root Pathogen Identified
Isolates Accession number
Fusarium oxysporum EU091063
Macrophomina Phaseolina EU091058
F. oxysporum GU126793
F. oxysporum EU091056
F. oxysporum JF740777
Rhizoctonia solani DQ8376901
Fusarium sp. KJ776745

81. PGPR Identified
Bacterial Isolates Accession number
Pseudomonas sp. HF562431
Pseudomonas vancouverensis KJ956607
Bacillus subtilis KJ767347
Bacillus sp. KM678261
Pseudomonas sp. KM253109
Pseudomonas sp. JQ012959
Pseudomonas fluorescens JF327445
Bacillus subtilis KF863845

89. Activity 8
Development and Testing of Bio-formulation
Objective # 3

90. • Organic manure (O.M), vermi compost (V) Rice bran (Rb),
wheat bran (W) and decomposed mustard oil cake (D) were
collected from the local markets.
• Carboxymethyl cellulose (1% aq) was used as adhesives.
• The pH was adjusted to 7
• The mixture was then spread in a sterilized non sticky
disposable plate under sterile conditions Mannitol was added
as osmoticant.

91. • Subsequently, antagonistic rhizobacterial cell suspension of
concentration of 108 cfu/ml was pipetted into the mixture (1:10
v/w) and thoroughly mixed with the help of sterilized spoon.
• Formulation was divided into 3 parts, packed separately in
polypropylene bags (8 x 6.5 cm), heat sealed and stored at
28ºC temperature.
• Another set of bio-formulations was prepared for each
substrate carrier-adhesive bio-agent mixture and stored at 5°C
for comparative study.
Bora and Deka (2007)

93.  The population dynamics of the bioagents was determined
at different days after storage (DAS) in the two storage
conditions.
 At 7, 30, 60, 90 and 120 DAS at 5◦C and 28◦C temperature,
Population dynamics was examined by mixing 1 g of
formulations aseptically with 10 ml sterile distilled water
for 20 min in a rotary shaker.
 After incubating the plates at 28 ± 1°C for 48 h, the cfu/g
formulations were counted out. The population of Bioagents
in powder formulations (cfu/g formulation) recorded were
transformed and used for analysis in this study.

94. Abbreviations used in Tables and Graphs
Pseudomonas sp. (Ps), Pseudomonas vancouverensis (Pv), Bacillus subtilis
(Bs-1), Bacillus sp. (Bs-2), Uncultured Bacterium (Ub), Uncultured
Prokaryotes (Up), Pseudomonas sp. (Ps-1), Pseudomonas sp. (Ps-2),
Pseudomonas fluorescens (Pf), Bacillus subtilis (Bs-3), Organic matter (OM),
Vermiculite (V), Rice bran (Rb), wheat bran (W) and decomposed mustard oil
cake (D)

95. Table:5 Growth and Multiplication of antagonistic rhizobacteria on 7 Days After
Storage at 5ºC and 28ºC by using various Carrier Materials
S/No Treatment 5◦C 28◦C S.no Treatment 5◦C 28◦C
1 PsV 12.33 45 26 UpRb 14 41
2 PvV 11.33 48 27 Ps-1Rb 17 40
3 Bs-1V 22.33 56 28 Ps-2Rb 11 47
4 Bs-2V 15 54.33 29 PfRb 15 40.6
5 UbV 14.66 50 30 Bs-3Rb 16 41.5
6 UpV 10 49 31 PsWb 14 40.7
7 Ps-1V 15 51 32 PvWb 18 41.8
8 Ps-2V 15.33 55.33 33 Bs-1Wb 12 41.6
9 PfV 23.66 59 34 Bs-2Wb 16 40.6
10 Bs-3V 28.66 61 35 UbWb 13 46.2
11 PsOM 15 44 36 UpWb 14 47.1
12 PvOM 12 48 37 Ps-1Wb 17 41.9
13 Bs-1OM 33 85 38 Ps-2Wb 19 42.6
14 Bs-2OM 29 78 39 PfWb 19.5 40.3
15 UbOM 14 46 40 Bs-3Wb 14.9 41.8
16 UpOM 13 48 41 PsD 16.8 43.1
17 Ps-1OM 19 55 42 PvD 14.8 42.2
18 Ps-2OM 17 59 43 Bs-1D 16.7 41.3
19 PfOM 39 81 44 Bs-2D 18.2 40.7
20 Bs-3OM 45 90 45 UbD 12.1 42.1
21 PsRb 12 44 46 UpD 11.5 43.6
22 PvRb 10 42 47 Ps-1D 12.8 41.8
23 Bs-1Rb 16 43 48 Ps-2D 13.6 46.2
24 Bs-2Rb 15 41 49 PfD 12.8 40.8

96. Effect of various carrier based formulations of selected antagonistic rhizobacterial strains on 7th day after
storage. The growth responses were analysed at the end of 7th day.
0
10
20
30
40
50
60
70
80
90
100
5◦C 28◦C

97. Table:6 Growth and Multiplication of antagonistic rhizobacteria on 30 Days After Storage at
5ºC and 28ºC by using various Carrier Materials
S.no Treatment 5◦C 28◦C S.no Treatment 5◦C 28◦C
1 PsV 46 215 26 UpRb 37 169
2 PvV 44 199 27 Ps-1Rb 34.6 165
3 Bs-1V 69 223 28 Ps-2Rb 36.2 168
4 Bs-2V 72 232 29 PfRb 30.9 164
5 UbV 45 178 30 Bs-3Rb 30.6 163
6 UpV 48 187 31 PsWb 34 160
7 Ps-1V 42 201 32 PvWb 31 168
8 Ps-2V 62 198 33 Bs-1Wb 36.5 164
9 PfV 97 210 34 Bs-2Wb 35.8 139
10 Bs-3V 69 216 35 UbWb 36.2 140
11 PsOM 59 193 36 UpWb 36.4 145
12 PvOM 61 145 37 Ps-1Wb 36.2 148
13 Bs-1OM 115 286 38 Ps-2Wb 35.2 146
14 Bs-2OM 118 289 39 PfWb 36.4 141
15 UbOM 50 139 40 Bs-3Wb 32.4 139
16 UpOM 55 135 41 PsD 33.3 142
17 Ps-1OM 63 189 42 PvD 32.8 149
18 Ps-2OM 65 190 43 Bs-1D 39.5 148
19 PfOM 119 291 44 Bs-2D 36.5 144
20 Bs-3OM 121 311 45 UbD 30.5 123
21 PsRb 39 169 46 UpD 30 124
22 PvRb 35 142 47 Ps-1D 35 145
23 Bs-1Rb 36.5 162 48 Ps-2D 36 146
24 Bs-2Rb 30 172 49 PfD 32.6 142
25 UbRb 39 161 50 Bs-3D 32 147

98. Effect of various carrier based formulations of selected antagonistic rhizobacterial strains on 30th day after storage. The growth
responses were analysed at the end of 30th day.
0
50
100
150
200
250
300
350
400
450
500
5◦C 28◦C

99. Table:7 Growth and Multiplication of antagonistic rhizobacterial on 60 Days After Storage at
5ºC and 28ºC by using various Carrier Materials
S.no Treatment 5◦C 28◦C S.no Treatment 5◦C 28◦C
1 PsV 182 342 26 UpRb 148 341
2 PvV 165 319 27 Ps-1Rb 148.2 362
3 Bs-1V 205 365 28 Ps-2Rb 147.6 352
4 Bs-2V 200 360 29 PfRb 146.5 349
5 UbV 138 310 30 Bs-3Rb 147.3 344
6 UpV 128 314 31 PsWb 146.5 346
7 Ps-1V 219 311 32 PvWb 146.7 351
8 Ps-2V 210 341 33 Bs-1Wb 146.2 356
9 PfV 210 382 34 Bs-2Wb 147.2 354
10 Bs-3V 201 389 35 UbWb 141 358
11 PsOM 195 346 36 UpWb 141.9 361
12 PvOM 182 312 37 Ps-1Wb 142 360
13 Bs-1OM 323 504 38 Ps-2Wb 142.4 359
14 Bs-2OM 333 508 39 PfWb 143 371
15 UbOM 121 301 40 Bs-3Wb 143.5 369
16 UpOM 111 300 41 PsD 143.6 361
17 Ps-1OM 147 318 42 PvD 143.7 360
18 Ps-2OM 161 325 43 Bs-1D 149.5 365
19 PfOM 313 501 44 Bs-2D 142.5 341
20 Bs-3OM 348 523 45 UbD 143.6 360
21 PsRb 145 328 46 UpD 147.7 356
22 PvRb 142 322 47 Ps-1D 143.1 352
23 Bs-1Rb 140 348 48 Ps-2D 146.5 341
24 Bs-2Rb 146 345 49 PfD 147.5 357
25 UbRb 142.9 344 50 Bs-3D 146.2 355

100. Effect of various carrier based formulations of selected antagonistic rhizobacterial strains on 60th day after
storage. The growth responses were analysed at the end of 60th day.
0
100
200
300
400
500
600
700
800
900
1000
5◦C 28◦C

101. Table:8 Growth and Multiplication of antagonistic rhizobacteria on 90 Days After Storage at
5ºC and 28ºC by using various Carrier Materials
S.no Treatment 5◦C 28◦C S.no Treatment 5◦C 28◦C
1 PsV 18.5 35.6 26 UpRb 12.9 41.8
2 PvV 15 27.2 27 Ps-1Rb 14 41.9
3 Bs-1V 25.9 49.3 28 Ps-2Rb 13.8 42.3
4 Bs-2V 35 48.8 29 PfRb 15 41.5
5 UbV 16 25 30 Bs-3Rb 17.2 42.8
6 UpV 17 29 31 PsWb 13.8 43.6
7 Ps-1V 23 31 32 PvWb 16.4 43.8
8 Ps-2V 27 33 33 Bs-1Wb 15.4 44
9 PfV 38 50.1 34 Bs-2Wb 13.9 44.7
10 Bs-3V 36 52.3 35 UbWb 14.2 43.6
11 PsOM 21 26 36 UpWb 14.8 45.7
12 PvOM 28 28 37 Ps-1Wb 15.9 46.1
13 Bs-1OM 45 98 38 Ps-2Wb 12.8 45.9
14 Bs-2OM 52 86 39 PfWb 14 45.5
15 UbOM 19 21 40 Bs-3Wb 13.4 41
16 UpOM 17 23 41 PsD 14.3 41.3
17 Ps-1OM 25.9 31 42 PvD 12.2 42.8
18 Ps-2OM 28.5 32.5 43 Bs-1D 15.8 41.6
19 PfOM 40 81 44 Bs-2D 16 43.8
20 Bs-3OM 39.9 92 45 UbD 12.8 39.8
21 PsRb 18.3 41 46 UpD 13 38.4
22 PvRb 16.9 40.8 47 Ps-1D 14.5 41.3
23 Bs-1Rb 17.5 40.5 48 Ps-2D 14.4 44.2
24 Bs-2Rb 15.2 40.2 49 PfD 13.7 43.1
25 UbRb 14.3 41.2 50 Bs-3D 16 46

102. Effect of various carrier based formulations of selected antagonistic rhizobacterial strains on 90th day after storage.
The growth responses were analysed at the end of 90th day.
0
10
20
30
40
50
60
70
80
90
100
5◦C 28◦C

103. Table:9 Growth and Multiplication of antagonistic rhizobacteria on 120 Days After Storage
at 5ºC and 28ºC by using various Carrier Materials
S.no Treatment 5◦C 28◦C S.no Treatment 5◦C 28◦C
1 PsV 2 9.5 26 UpRb 4.2 13
2 PvV 2.9 10.6 27 Ps-1Rb 4.8 12.5
3 Bs-1V 5.6 15.2 28 Ps-2Rb 4.3 14.1
4 Bs-2V 5.9 16.1 29 PfRb 3.9 14.5
5 UbV 1.9 14 30 Bs-3Rb 4.7 15.9
6 UpV 2.2 15.8 31 PsWb 2.5 15.6
7 Ps-1V 3.4 18 32 PvWb 2.3 15.4
8 Ps-2V 3.8 12 33 Bs-1Wb 3.1 15.6
9 PfV 8 14 34 Bs-2Wb 3.8 14.6
10 Bs-3V 9 16 35 UbWb 5.1 15.1
11 PsOM 3.5 18 36 UpWb 4.2 14.3
12 PvOM 4 15 37 Ps-1Wb 4.8 13.9
13 Bs-1OM 17 25 38 Ps-2Wb 4.3 14.7
14 Bs-2OM 21 28 39 PfWb 3.9 14.8
15 UbOM 5.1 12 40 Bs-3Wb 2.7 15
16 UpOM 4.9 11 41 PsD 1.5 12.6
17 Ps-1OM 5.1 9 42 PvD 1.3 12.1
18 Ps-2OM 5.3 10 43 Bs-1D 2.1 11.7
19 PfOM 15 22 44 Bs-2D 2.8 12.1
20 Bs-3OM 19 24 45 UbD 4.2 10.5
21 PsRb 2.5 12.8 46 UpD 3.2 11.8
22 PvRb 2.3 11.9 47 Ps-1D 3.6 12.3
23 Bs-1Rb 3.1 12.5 48 Ps-2D 3.3 11.5
24 Bs-2Rb 3.8 13.1 49 PfD 2.9 10.9
25 UbRb 5.1 12.9 50 Bs-3D 3.7 10

104. Effect of various carrier based formulations of selected antagonistic rhizobacterial strains on 120th day after
storage. The growth responses were analysed at the end of 120th day.
0
10
20
30
40
50
60
70
80
90
100
5◦C 28◦C

105. Somasegran and Hoben (1994)
Fig.6 Colony count after different days after storage to check efficacy of selected antagonistic rhizobacterial strains

106. Selected
antagonistic
rhizobacteria
Ps Pv Bs-1 Bs-2 Ub Up Ps-1 Ps-2 Pf Bs-3
Carrier Materials
Organic matter
(OM)
1 0 5 5 0 0 0 1 5 4
Vermiculite (V)
1 0 5 5 0 1 2 0 4 5
Rice bran (Rb) 0 0 0 0 0 0 1 0 0 1
wheat bran (Wb)
1 1 1 0 0 2 2 1 2 1
Decomposed
mustard oil cake
(D)
1 0 1 0 1 0 0 0 0 1
3 Bacillus subtilis isolates and 1 Pseudomonas fluorescens
shows best results on Organic matter and Vermiculite

107. Activity 9
Bio-formulation Testing in Greenhouse
Objective # 4

108. POT EXPERIMENT
• Surface sterilized chickpea seeds with disinfested in 1 % NaClO for
2 min, rinsed in distilled water, and damp-dried on absorbent paper
towels before sowing
• Seed bacterization 5g/kg of seeds
• Plants were inoculated with pathogen at 36 days after sowing
• Disease intensity was assessed 20 days after inoculation
• Control pots with only infested soil.
• All experiments were repeated with three replicates of each
treatment. Experiments were carried out in green house in a
complete randomized design (CRD).

109. Table.10 Effect of bio-inoculant formulations on growth responses of
chickpea plants under glasshouse conditions
Treatment Germination shoot length (cm) Root length Fresh weight (g) Dry weight (g)
Control 53.75c 16.75e 3.92e 0.47e 0.075e
Bs-1OM 61.25b 24.1a 5.47c 0.73bc 0.16c
Bs-2OM 73.25b 28.55b 9.25ab 1.36a 0.37b
BS-3OM 89.25a 32.75a 12a 1.61a 0.51a
PfOM 69ab 26.4b 7.425bc 0.92c 0.24b
Bs-1V 41d 19.1ef 4.1e 0.55f 0.1de
Bs-2V 33.8d 20.2d 4.3f 0.61e 0.08ef
Bs-3V 37.2e 19.6ef 4 0.59f 0.07f
PfV 42.1d 20.7d 4.5de 0.54d 0.11de

110. 4 antagonistic rhizobacteria which perform best under
greenhouse conditions were selected for field
experiment
0
10
20
30
40
50
60
70
80
90
100
Control Bs-1OM Bs-2OM BS-3OM PfOM Bs-1V Bs-2V Bs-3V PfV
Germination
shoot length (cm)
Root length
Fresh weight (g)
Dry weight (g)

111. Activity 10
Bio-formulation Testing in Field

112. • Seeds were surface-sterilized with 0.1% sodium hypochlorite .Later,
50 g of chickpea seeds were treated with 0.5 g of bio-inoculant
carrier formulations.
• Chickpea variety Bital-98 was sown with 4 replications, 4
treatments in 4 selected fields.
• The controls included only the pathogen inoculated treatment with
no bio-formulation applied (control)
• The experiment was laid out in a randomized complete block design
with factorial arrangement. The net plot size for the experiment was
1.6 x 5 m.
• Seed was drilled in 40-cm spaced rows and Plant-to-Plant distance
of 15 cm was maintained by thinning ten days after germination in
experiments.

113. Treatment
Disease
Prevalence %
Disease Incidence
%
Disease Severity
(1-9 rating scale)
Control
83 89.5 9
Bs-1OM 57 41 5
Bs-2OM 53 37 7
BS-3OM 23 20 3
PfOM 48.8 45.3 7
Field-1

114. Fig.7 Symptoms appear on plants and in field after application of bio-formulation

115. Treatment
Disease
Prevalence %
Disease Incidence
%
Disease Severity
(1-9 rating scale)
Control
89 79 9
Bs-1OM 51 52 7
Bs-2OM 49 49 5
BS-3OM 20 19 3
PfOM 53.8 48.3 7
Field-2

117. Treatment
Disease
Prevalence %
Disease Incidence
%
Disease Severity
(1-9 rating scale)
Control
86 75 9
Bs-1OM 56 61 7
Bs-2OM 55 54 5
BS-3OM 21 18 3
PfOM 54 49 7
Field-3

119. Treatment
Disease
Prevalence %
Disease Incidence
%
Disease Severity
(1-9 rating scale)
Control
89 79 9
Bs-1OM 51 52 7
Bs-2OM 49 49 5
BS-3OM 20 20 3
PfOM 53.8 48.3 7
Field-4

120. Growth Parameters Analyzed
Treatment Germination shoot length (cm) Root length Fresh weight (g) Dry weight (g)
Control 53.75c 16.75e 3.92e 0.47e 0.075e
Bs-1OM 61.25b 24.1a 5.47c 0.73bc 0.16c
Bs-2OM 73.25b 28.55b 9.25ab 1.36a 0.37b
BS-3OM 89.25a 32.75a 12a 1.61a 0.51a
PfOM 69ab 26.4b 7.425bc 0.92c 0.24b
The growth responses were analysed at the end of 100 days. The values represent the mean
of 20 replicates (±SD). Means followed by the same letter in a column are not significantly
different from each other according to Duncan’s multiple range test.

121. Conclusion
• Selection of different antagonistic rhizobacteria by using
various cheap carrier materials.
• Bacillus subtilis (Bs-3) used with Organic Matter (O.M)
showed best results in Lab, green house as well as in Field
experiments among all.
• So, we recommend selected bio-formulation for the control of
fungal root pathogens to get the best results with maximum
yield.
• As this bio control is very cheap and environment friendly, so
it will help the poor farming community

122. Expected Outcomes
They are generally less destructive to Beneficial's, cause less
environmental pollution than conventional pesticides.
Biopesticide may provide a satisfactory alternative to chemical
pesticides when used as part of an overall IPM plan.

123. Acknowledgment

124. Experimental Work

125. Faculty Members at CRA-PAV, Rome (Italy)

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