It is a scientific seminar on bioinoculants application in medicinal plants to enhance the growth, development and metabolite content. It gives the idea of mechanism and effect of bioinoculants in medicinal plants. Why bioinoculants are very important in current scenario especially in medicinal plants production.

1. MASTER’S SEMINAR I
Application of Bioinoculants in medicinal
plants to enhance the growth, development
and metabolite contents
Presented By – Krishna Kumari
(Forest Biology and Tree Improvement)
College of Forestry , Sirsi. UAS Dharwad (Karnataka)
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2. Seminar Outline
• Introduction to medicinal plants
• Need for cultivation of medicinal plants
• Bioinoculants and their importance
• Mechanisms of bioinoculants.
• Case studies
• Conclusion
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3. Medicinal plants
DEFINITION –
There is nothing in this universe, which
is non-medicinal, which cannot be
made use of for many purposes and by
many modes.” 600AD ( Ashtaanga
Hardya).
World Health Organisation (WHO)
has defined medicinal plants as plants
that contain properties or compounds
that can be use for therapeutic
purposes or those that synthesize
metabolites to produce useful drugs.
3

4. • 3,500 species of both higher and lower plant
groups are of medicinal values.
• More than 80 % of medicinal and aromatic
plants (MAP) are collected from forest land.
• Due to over exploitation these became rare
(Rheum emodi, Aconitum deinorrhizum),
threatened (Rauvolfia serpentina, Berberis
artistata), or endangered ones (Sassurea
lappa, Dioscorea deltoidea).
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5. Need for cultivation of medicinal plants
 To reduce the pressure on wild vegetation.
 Ayurveda, Siddha and Unani systems >90% formulations
which are plant based
 To provide the livelihood for villagers and tribal.
 Domestic demand of medicinal plants has been estimated
1,95,000 MT for the year of 2014-15
 Export demand of medicinal plants estimated about 1,
34,500 MT with export value of ₹ 3211 Cr. during 2014-15
 International market for medicinal plants is @ US $60
billion per year, with annual growth rate of seven per cent.
5
Source- National medicinal Plant Board

6. Area and Production of Medicinal plants in India
Year Area(in ‘000 ha) Production (in ‘000MT)
2004-05 131 159
2005-06 262 202
2006-07 324 178
2007-08 397 396
2008-09 430 430
2009-10 509 573
2010-11 510 605
2011-12 506 566
2012-13 557 918
2013-14 493 895
2014-15 659 1000
2015-16 634 1022
2016-17 664 1042
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Source-Ministry of Agriculture and Farmers Welfare, Govt. of India

7. Current Scenario of Karnataka
• As per the survey conducted under National Horticulture Mission,
Department of Horticulture, Government of Karnataka, the total area
under cultivation of medicinal plants is 2420 ha and annual production is
4101 MT.
• Important medicinal plants like Sandalwood, Ashwagandha, Tulsi,
Coleus, Amla, Safedmusli, Aloevera, Acorus calamus, Kalmeg, Senna,
Muccuna prurita , Solanum viarum, Rauvolfia serpentina etc. have been
taken up under the Central sponsored scheme of National Mission on
Medicinal Plants since 2002 to till date.
7
Source - National mission on medicinal plants

8. Introduction to Bioinoculants
Bioinoculant is a ready-to-use live
formulation of such beneficial
microorganisms which on application to
seed, root, soil, foliage mobilize the
availability of nutrients by their biological
activity.
8
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9. • Arbuscular mycorrhizal
(AM) fungi is present in
90% of the vascular
plants of the world.
• AM fungi helps in
nutrient uptake,
especially phosphorous
(P) and growth of
plants. (Mac Donald
and Lewis, 1978; Fitter,
1985).
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10. Illustration of root colonization structures in ectomycorrhizal
(blue) and arbuscular mycorrhizal (pink) interactions.
(Bonfante and Genre, 2010)
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11. Bioinoculants
P-
solubilisation
N- fixation
Micronutrient
nutrients
absorption
Development
of systemic
Acquired
Resistance
IAA,
Gibbrelin
Production
Siderophore
Production
Antimicrobial
compounds
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Bioinoculants action mechanism

12. PGPR mechanism 12

13. Tyler et al., 2011
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Bioinoculant beneficial effect on plant

14. Why Bioinoculants ?.....
• Pesticide residual free medicinal plants
• Cost Effective
• Nutrient availability
• Improves the growth and Yield of the Crop
• Improves the disease resistance power of the crop
• Improves uptake of minerals and water
• Eco-Friendly
• Saves Earthworm and other Micro organisms in the soil
• Maintains soil health
???
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15. Case study 1
Response of Bioinoculant Consortia on Nutrient
Uptake, Forskolin content (Diterpene) and Tuber
yield of Coleus (Coleus forskohlii ).
 (Coleus forskohlii) - an endangered medicinal plant.
 Study site- UAS, GKVK Bangalore.
 Elevation – 930m MSL
 Variety used- K8 ( IIHR)
 RCBD with 3 replications
 Recommended dose- 40:60:50 kg N:P:K per ha
Nawaz et al., 2017
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16. Treatment details
• Azotobacter chroococcum N2 fixer
(12 x 107 cfu g-1) 5g per plant.
• Bacillus subtilis (9 x 108 cfu g-1)
5g per plant.
• Pseudomonas fluorescens (6 x 108
cfu g-1) 5g per plant.
• Arbuscular Mycorrhizal fungus
Glomus fasciculatum (12500 IPg-1)
10g per plant.
• Trichoderma harzianum (2 x 106
cfu g-1) 2g per plant.
16

17. MATHODOLOGY
 Microbial parameters-
• Microbial population initial stage
• Microbial population after harvest
 Extraction and estimation of Forskolin content in coleus forskohlii
• Preparation of standard- 25 mg was weighed and dissolved acetonitrile and
volume was made up to 25 ml.
• Sample preparation- samples from each of the treatments (3 g) was weighed and
extract was obtained with 50 ml of acetonitrile by warming on a water bath for
about 20 minutes. ( 0.45µ membrane)
• Estimation of Forskolin content using HPLC.
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Nawaz et al., 2017

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Nawaz et al., 2017

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Nawaz et al., 2017

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Nawaz et al., 2017

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Nawaz et al., 2017

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Nawaz et al., 2017

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Nawaz et al., 2017

24. Conclusion
• Study reveal that Bioinoculants application
along with reduced levels of nitrogen and
phosphorus application helped in maintaining
rhizosphere microorganisms population,
forskolin content and tuber yield.
• Saving of 25 % N and P can be achieved by
using the microbial consortia.
24

25. Case study 2
Bioinoculants and vermicompost influence
on yield, quality of Andrographis paniculata
(kalmegh) and soil properties.
Objectives
• Study includes the different bioinoculants
application and effect on the growth and
metabolite contents.
• Correlation between the soil properties and
growth parameters.
Khan et al., 2015
25

26. Methodology
• Experiment was conducted in the field of CSIR- Central
Institute of Medicinal and Aromatic Plants, Lucknow.
• Experiment was arranged in CRBD with 3 replications.
• Genotype - Andrographis paniculata cv. CIM- Megha
(high yielding variety).
• Cylindrical RCC pots were used for experiment.
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Khan et al., 2015

27. Treatment details
T1 Control (Untreated)
T2 Vermicompost (VC)
T3 VC + Azotobacter chrococcum
T4 VC + Bacillus megaterium
T5 VC + Pseudomonas monteilii
T6 VC + Glomus intaradices ( AMF)
T7 VC + All the four bioinoculants (Azotobacter
chrococcum, Bacillus megaterium ,
Pseudomonas monteilii, Glomus intaradices )
VC in all the treatments is applied @ 5t per ha
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28. Harvesting and HPLC analysis-
• Above ground biomass was harvested after
125 days at the initial flowering time.
• Andrographolide content is optimum in this
stage.
• Plant material was dried and extract was
prepared.
• Standard andrographolide was used in HPLC
analysis.
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Khan et al., 2015

29. Soil sampling and analysis
• Initial and post harvest soil samples were
collected from 0-15 cm in all the treatments.
• One composite sample was prepared
• Sample was divided into two parts one for
chemical analysis and one for biological
analysis.
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Khan et al., 2015

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T1:Control; T2: vermicompost (VC); T3: VC + Azotobacter chrococcum;
T4: VC+Bacillus megaterium ; T5: VC+ Pseudomonas monteilii; T6:
VC+Glomus intraradices; T7: VC+ All bio-inoculants (Azotobacter
chrococcum + Bacillus megaterium + Pseudomonas monteilii + Glomus
intraradices).

31. Fig.1. Plant growth image of the experiment of Andrographis paniculata
T1:Control; T2: vermicompost (VC); T3: VC + Azotobacter chrococcum;
T4: VC+Bacillus megaterium ; T5: VC+ Pseudomonas monteilii; T6:
VC+Glomus intraradices; T7: VC+ All bio-inoculants (Azotobacter
chrococcum + Bacillus megaterium + Pseudomonas monteilii + Glomus
intraradices).
31

32. LS: Leaf Stem ratio ;FHY : Fresh Herb yield; ANY: Andrographolide yield; SR:
Soil respiration; DHA: Soil Dehydrogenase enzyme; ALP: Soil Alkaline
Phosphatase enzyme; ACP: Soil Acid Phosphatase enzyme; SOC: Soil Organic
Carbon content; AVP : Available phosphorus; AVK: Available potassium.
* Correlation is significant at 0.05level., ** Correlation is significant at 0.01level.
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33. 33
Khan et al., 2015

34. Khan et al., 2015
34

35. Khan et al., 2015
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36. Conclusion
• More biomass and secondary metabolites can be
produced by application of bioinoculants and
organic fertilizers.
• Organic carbon content in soil is very important
for the soil microflora which makes the soil
healthy.
• Bioinoculants makes the nutrients available to
plants like N, P & K.
• Organic farming become cost effective in
medicinal plants production.
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37. Case study-3
Influence of PGPR on growth, Essential oil
and nutrient uptake of Sweet basil
• Study was conducted in Islamic Azad
University, Firoozabad (Iran).
• 1327 m above mean sea level.
• Randomized complete block design (RCBD)
with four replications.
Ordookhani et al., 2011
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38. Treatments
Treatment 1 Control
Treatment 2 Pseudomonas
Treatment 3 Azotobacter
Treatment 4 Azosprillum
Treatment 5 Pseudomonas + Azatobacter
Treatment 6 Pseudomonas + Azosprillum
Treatment 7 Azatobacter + Azosprillum
Treatment 8 Pseudomonas + Azatobacter +
Azosprillum
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(1x108 CFU per gram) concentration in all inoculants

39. Effect of diff. treatments on shoot fresh weight (g/Plant) at full bloom time
Ordookhani et al., 2011 39

40. Graph showing the effect of different treatments
on shoot dry weight (g/Plant) at full bloom time
Shootdryweight(g/Plant)
Ordookhani et al., 2011
40

41. Ordookhani et al., 2011
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42. Effect of different treatments on root dry
weight (g/Plant)
Ordookhani et al., 2011
42

43. Ordookhani et al., 2011
43

44. Ordookhani et al., 2011
44

45. Ordookhani et al., 2011
45

46. Conclusion
• Maximum root fresh weight (3.96 g/plant), Root dry weight
(0.87 g/plant)N, P, K content (4.72%), (0.8%), (4.2%)
respectively were observed in Pseudomonas + Azotobactor
+ Azosprillum significantly different from all other
treatments.
• Maximum shoot fresh weight (61 g/plant), shoot dry
weight (8.47g/plant) were found in
Pseudomonas + Azotobactor + Azosprillum
treatment which is significantly different from all
except Azotobactor + Azosprillum.
• Therefore bioinoculant consortia can be used to get the
higher yield with enhanced essential oil content from
Ocimum basilicum.
46

47. Case study- 4
Bioinoculants as a tool to improve total
bacoside content in Bacopa monnieri
• Experiment was conducted in CSIR- Central
Institute of Medicinal and Aromatic plants ,
Lacknow.
• CIM- Jagriti variety .
• CRD experimental design with 4 replications.
Singh et al., 2014
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48. Treatments
Treatment Source of isolate Name of Isolate Bioinoculant
Treatment 1 - - Uninoculated
Treatment 2 Rhizospheere of
Coleus
CRC 1 Pseudomonas
monteilii
Treatment 3 Vermicompost CRC 2 Cedecea davisae
Treatment 4 Vermicompost CRC 3 Cronobactor
dubilinensis
Treatment 5 Vermicompost CRC 4 Avenella spp.
Treatment 6 Rhizospheere of
Coleus CRC 5
Pseudomonas
aeruginosa
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49. Isolation, characterisation and mass
multiplication of isolates
• Isolation was done from coleus plant and
vermicompost.
• All the isolates were characterized by 16S
rRNA gene sequence of strains.
• Isolates were mass multiplied on broth media
• Seedling deep treatment and 5 ml per pot
( 2x108 bacterial cells per ml.)
Singh et al., 2014
49

50. Bacoside content estimation
• Fourier Transform Near Infra Red method (FT-NIR)
Absorbance spectra 10000 per cm – 4000 per cm
• 2mg sample (powder) subdivided into 40 subsamples of
50 µg in wt. and each subsample was analysed for 8 times
in FT-NIR.
• Every 2mg sample of Bacopa monneiri 320 FT-NIR
spectra were taken.
Singh et al., 2014
50

51. Table 1- Homology search of bacterial isolates
Singh et al., 2014
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52. Singh et al., 2014
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53. Fig. 1 Fourier Transform Near Infra Red analysis of B.
monneiri samples treated with bioinoculants for total
bacoside content
Singh et al., 2014
53

54. bar in graph shows the Standard error of mean, different
letters above bar shows the significant difference at P= 0.05
Singh et al., 2014
54

55. Conclusion
• Present study concluded that micro organisms isolates showed the
significantly higher herb yield ( 76.89g per pot) compared to control
treatments.
• Bacoside content was highest in Pseudomonas monteilii ( 0.75%)
which is significantly superior in all the treatments and all other
treatments was at par except control.
• FT-NIR is an economic and very fast method for biological
chemical estimations.
• Bioinoculants play very important role in medicinal plants where
chemicals cant be used because of health and residual
considerations.
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56. Case study – 5
Biochemical analysis and growth enhancement studies
of important medicinal plant, Rauvolfia serpentina
inoculated with Arbuscular mycorrhizal fungi in
nursery
• Experiment was conducted in Institute of Forest
Genetics and Tree breeding, Coimbtore (Tamilnadu).
• Studies was done at nursery level to investigate the
efficacy of different AM fungi on growth parameters.
• Complete Randomised design
• Replications - 4
Mohan et al., 2015
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57. Treatments
Treatments Arbuscular Mycorrhizal
Fungi (15 spore per gram)
Treatment 1 Control (Uninoculated)
Treatment 2 Glomus fasciculatum
Treatment 3 Glomus mosseae
Treatment 4 Glomus macrocarpum
Treatment 5 Glomus intraradices
Treatment 6 Scutellospora calospora
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58. DAT- Days After Transplanting
• Same letters in same column do not differ significantly
at P< 0.05 according to DMRT.
Mohan et al., 2015
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59. Table 3 Effect of different AM Fungi on Stem girth, leaf area and
number of leaves of Rauvolfia serpentina seedlings in glass house
DAT – Days After Transplanting
Mean in same column with same letters do not differ significantly
according to DMRT at P < 0.05.
Mohan et al., 2015
59

60. Table 4 – Effect of different AM fungi on biomass of Rauvolfia
serpentina seedling ( 120 DAT) in Glass house
DAT- Days After Transplanting
Mean in the same column with same letters do not differ significantly according to
DMRT at P < 0.05.
Fresh and Dry Weight (g/ plant)
Mohan et al., 2015
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61. Table 5- Effects of different AM fungi on Chlorophyll content
and P – content of Rauvolfia Serpentina
Treatment Chloroph
yll
content
( mg/g
fresh wt.)
P – content ( mg /
plant)
Shoot
Root
Total
Control (Uninoculated) 1.11 0.20 0.20 de 0.40 e
Glomus fasciculatum 3.09 12.14 2.81 a 14.95 a
Glomus mosseae 2.76 4.41 2.42 b 6.83 c
Glomus macrocarpum 1.98 5.26 0.80 bc 6.06 d
Glomus intraradices 1.40 0.30 0.26 cd 0.56 e
Scutellospora calospora 2.77 7.14 2.30 b 9.44 b
Means in the same column followed by same letters do not differ significantly at P< 0.05
Mohan et al., 2015
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62. Conclusion
• This study found that out of all the applied AM
fungi, Glomus Fasciculatum in R. Serpentina
gives maximum height (35.97 cm), Maximum
Stem girth (0.50 mm), Maximum Leaf Area
(90.67cm2), Maximum Plant Biomass ( FW-
51.24 g/ plant , DW- (9.08 g/plant) at 120 DAT.
• As the AM fungi play a very important role to
enhance the growth it can be used in fields for
development and metabolite enhancements.
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63. Seminar Conclusion
• Bioinoculant application showed the reduced
demand for inorganic fertilizers, which is more
economic and ecologically suitable for
medicinal plant production.
• More biomass and secondary metabolites can
be produced by bioinoculants and organic
fertilizers.
• Bioinoculants makes the nutrients available to
plants like N, P & K and help in absorption.
63

64. 64
• Bioinoculants play very important role in
medicinal plants where chemicals cannot be
used because of health and residual
considerations.
• Bioinoculants conserve the micro fauna of soil
to make the soil healthy.
• Bioinoculants are effective in disease and pest
management to overcome the residual effect in
medicinal plants.

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