Human Security and Sustainability: Sharing Reverence for ...
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A Project Report
On
”Study the effects of various PGPRs and Organic Manures on Growth and Yield attributes of two different varieties ( CIM-Suvas & CIM-
Shishir) of Ocimum basilicum.”
Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science
In
ENVIRONMANTAL SCIENCES
Of
Integral University
Submitted By
Km. Anushree Srivastava
Under the guidance of
Dr.Rajesh Kumar Verma
Division of agronomy and soil sciences
CSIR-Central Institute of Medicinal and
Aromatic Plants, Lucknow 2020.
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ACKNOWLEDGEMENT
Firstly, I bow my head with utmost reverence before the
Almighty whose eternal blessing has enabled me to
accomplish this noble effort.
I am privileged to work under the supervision of Dr.Rajesh
Kumar Verma, Principal Scientist, Division of Agronomy
and Soil Sciences, CSIR-CIMAP, Lucknow. It was my
opportunity to be guided by a person of caliber, whose
blessings brings best in every one of my endeavors. His
keen interest, constructive discussions, clear
understanding and great support provided me an excellent
atmosphere for completing this study. Working under him
has been a great experience and I shall forever treasure
this association.
I pay my sincere regards to Dr. Saudan Singh, chief
scientist (division of agronomy and soil science, CSIR-
CIMAP) for his encouragement, inspiration, parental
affection, keen interest and advice throughout my work.
I am also thankful to Dr. Abdul Samad, Acting Director and
Dr. Laiq Ur Rahman, senior principal scientist of CIMAP,
who provided me an opportunity to carry out my three
months project work in this prestigious institution.
I express my heartfelt thanks to Dr. M.A.Khalid (Professor
and Head of Department), Dr. Saema, Dr. Rahila
(Professors ) of Environmental Science, Integral
University.
I owe my unpayable debt and special thanks to all my
senior lab mates, Miss. Jahnvi Pandey (senior research
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fellow),Mr. Vishnu , Miss Riya and Mr. Shivam Dangi who
helped me at several stages during the work. The
encouragement rendered by them has helped me to
overcome several hurdles to complete this dissertation.
I would also like to thank Miss. Neelu, Mr. Arvind, Mr.
Dhananjay , Mr.Mazeed, Mr. Nikhil, Mr.Shubham and my
friend Miss.Nidhi for their kind support and it was
pleasure working with them in the same lab.
I am thankful to my respected family for their constant
words of encouragement, deep affection and heartful
blessings that enabled me to this stage of career . friends
are always a moral support which is extremely important
when one is feeling low . I take great pleasure in thanking
my friends Shreeda, Nidhi, Monazza and Divya for giving
me moral support , sharing the burden of my work and
making things smooth .
My sincerest thanks to Mr. Suresh and Mr. Sandeep for
their invaluable and generous help in the laboratory. I feel
proud to be a part of CSIR-CIMAP, where I learnt a lot and
spent some unforgettable moments of my life.
ANUSHREE SRIVASTAVA
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TABLE OF CONTENTS
1. Introduction 05-10
2. Review of literature 10-17
3. Materials and Methods 18-31
4. Results and Discussions 32-43
5. Conclusions 44-46
6. References 47
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1.INTRODUCTION
1.1 Tulsi (Ocimum spp.) :
Tulsi is an aromatic shrub in the basil family Lamiaceae
(tribe ocimeae) that is thought to have originated in north
central India and now grows native throughout the eastern
world tropics. Within Ayurveda, tulsi is known as “The
Incomparable One,” “Mother Medicine of Nature” and
“The Queen of Herbs,” and is revered as an “elixir of life”
that is without equal for both its medicinal and spiritual
properties.
Tulsi tastes hot and bitter and is said to penetrate the
deep tissues, dry tissue secretions and normalize kapha
and vata. Daily consumption of tulsi is said to prevent
disease, promote general health, wellbeing and longevity
and assist in dealing with the stresses of daily life,In
addition to these health-promoting properties, tulsi is
recommended as a treatment for a range of conditions
including anxiety, cough, asthma, diarrhea, fever,
dysentery, arthritis, eye diseases, otalgia, indigestion,
hiccups, vomiting, gastric, cardiac and genitourinary
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disorders, back pain, skin diseases, ringworm, insect, snake
and scorpion bites and malaria.
1.2 Types of Tulsi and its Varieties:
1. Ocimum bacilicum- CIM-Snigdha , CIM-Saumya,CIM-
Surabhi and CIM-Sharda
2. Ocimum santum- CIM-Ayu , CIM-Angna
3. Ocimum africanum- CIM-Jyoti
4. Ocimum kilimunduscharicum- CIM-Okay11
5. Hybrid- CIM-Shishir, CIM-Suvas
CIM-Shishir CIM-Suvas
1.3 Soil and climate:
The plant is sufficiently hardy and it can be grown on any
type of soil except the ones with highly saline, alkaline or
water logged conditions. However, sandy loam soil with
good organic matter is considered ideal. The crop has a
wide adaptability and can be grown successfully in tropical
and sub-tropical climates.
1.4 Manures and fertilizers:
The plant requires about 15t/ha of FYM which is to be
applied as basal dose at the time of land preparation.
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Regarding the inorganic fertilizers application of 120:60:60
kg/ha of NPK is recommended.
1.5 Irrigation:
Irrigation is provided twice a week till one month so that
the plants establish themselves well. Later, it is given at
weekly interval depending upon the rainfall and soil
moisture status.
1.6 An Overview of Plant Growth Promoting
Rhizobacteria (PGPR) for Sustainable Agriculture
Soil bacteria beneficial to plant growth usually referred to
as plant growth promoting rhizobacteria (PGPR), are
capable of promoting plant growth by colonizing the plant
root. The mechanisms of PGPR-mediated enhancement of
crop growth includes :
(i) a symbiotic and associative nitrogen fixation;
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(ii) solubilization and mineralization of other
nutrients;
(iii) production of hormones e.g. auxin i.e. indole
acetic acid (IAA), abscisic acid (ABA), gibberellic
acid and cytokinins;
(iv) production of ACC-deaminase to reduce the level
of ethylene in crop roots thus enhancing root
length and density;
(v) ability to produce antagonistic siderophores, ß-1-
3-glucanase, chitinases, antibiotics, fluorescent
pigment and cyanide against pathogens and
(vi) enhanced resistance to drought and oxidative
stresses by producing water soluble vitamins
niacin, thiamine, riboflavin, biotin and pantothenic
acid. Increased crop production through biocontrol
is an indirect mechanism of PGPR that results in
suppression of soil born deleterious
microorganisms PGPR can play an essential role in
helping plants to establish and grow in nutrient
deficient conditions. Their use in agriculture can
favour a reduction in agro-chemical use and
support ecofriendly crop production.
1.7AIM: On the application of five different stains of
rhizospheric bacteria, namely;
1. CRC-1(Pseudomonas monteilii),
2. CRC-2(Cedecea davisae),
3. CRC-3(Cronobacter dublinensis),
4. CRC-4(Advencca spp.),
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5. PSB-2(Unknown);
The aim of my project is to achieve better yield and growth
attributes of those plants which are treated with above
given PGPR Stains in comparison with controls and FYM
treated plants.
1.8 OBJECTIVE:
To achieve the specific aim we need to take following
steps into action:
• plantation of tulsi (Suvas and Shishir) varieties sapliings
in the sterlised soil in replication of three.
• measure the initial length of each sapling.
• test initial soil parameters i.e. Ph, EC, Nitrogen, Organic
Carbon, Phosphorous, Sulfur, DHA, Micro-nutrients.
• prepare culture media from Nutrient Broth and after 24
hrs inoculate with 5 different PGPRs labelled as CRC-1,
CRC-2, CRC-3, CRC-4 and PSB-2 and incubate it for 2-3
days; then prepare dose of PGPRs in 80% saline solution
and pour the doses in the pots several times , freshly
prepared each time, as treatment-2 replications were
dosed with CRC-1 , treatment-3 replications with CRC-2,
treatment-4 with CRC-3, treatment-5 with CRC-4 and
treatment-6 with PSB-2respectively.
• put ( 2.5 tonns per hect) vermin-compost in each of the
three replications of pots of treatment-2 to treatment-6; 5
tonns per hect in treatment-7 and 10 tonns per hect FYM
in treatment-8.
• finally note down the final length of all plants and also
take out its biomass just after harvesting after 3months.
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• test the final soil parameters i.e. . Ph, EC, Nitrogen,
Organic Carbon, Phosphorous, Sulfur, DHA, Micro-
nutrients.
• after harvesting perform distillation to measure the yield
through its oil extraction.
2. REVIEW OF LITERATURE
1.The potentiality of PGPR in agriculture is steadily
increased as it offers an attractive way to replace the use
of chemical fertilizers, pesticides and other supplements.
Growth promoting substances are likely to be produced in
large quantities by these rhizosphere microorganisms that
influence indirectly on the overall morphology of the
plants.
2.Bacterial strains PsF84 were isolated from tannery
sludge polluted soil, Jajmau, Kanpur, India. 16S rRNA gene
sequence and phylogenetic analysis confirmed the
taxonomic affiliation of PsF84 as Pseudomonas
monteilii . A greenhouse study was carried out with rose-
scented geranium (Pelargonium graveolens cv. Bourbon)
grown in soil treated with tannery sludge to evaluate the
effects of bacterial inoculation on the heavy metal uptake.
The isolates solubilized inorganic phosphorus and were
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capable of producing indole acetic acid (IAA) and
siderophore. The isolate PsF84 increased the dry biomass
of shoot by 44%, root by 48%, essential oil yield 43% and
chlorophyll by 31% respectively over uninoculated control.
3.Cd-resistant strains were isolated from the roots of Cd
accumulators, and their plant growth-promoting activities
were characterized. Isolates were able to produce indole-
3-acetic acid (IAA) (28–133 mg L−1) and solubilize
phosphate (65–148 mg L−1). In a pot experiment, the
inoculation of isolates Cedecea davisea LCR1 significantly
enhanced the growth of and uptake of Cd by the Cd
hyperaccumulator S. plumbizincicola. 454 pyrosequencing
revealed that the inoculation of the PGPR lead to a
decrease in microbial community diversity in the
rhizopshere during phytoextraction. Inoculation of
strains Cedecea davisae LCR1 could promote S.
plumbizincicola growth and enhance the remediation
efficiency.
4.Two year field studies indicated that seed treatment of
Ocimum basilicum var. CIM-Saumya with efficient
bioinoculants (Pseudomonas monteilii – strain CRC1,
Cronobacter dublinensis – strain CRC3 and Bacillus spp. –
strain
AZHGF1) can significantly improve the essential oil yield
(45–56%); maximum essential oil yield was observed in
plants inoculated with P. monteilii (56%) followed by C.
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dublinensis (49%) and Bacillus spp. (45%). The content of
essential oil was also significantly improved (15%) when
inoculated with P. monteilii compared to un-inoculated
control. The higher concentrations of linalool (40.40%) and
β-caryophyllene (14.15%) were observed in the plants
inoculated with P. monteilii. P. monteilii also produced
maximum biomass yield; an increase of about 55%
followed by C. dublinensis (42%) and Bacillus spp. (30%).
To the best of our knowledge this might be an exclusive
report suggesting the use of bioinoculants for higher yields
and disease management for organic growers of sweet
basil.
4.Increased crop production through biocontrol is an
indirect mechanism of PGPR that results in suppression of
soil born deleterious microorganisms. Biocontrol
mechanisms involved in pathogen suppression by PGPR
include substrate competition, antibiotic production, and
induced systemic resistance in the host. PGPR can play an
essential role in helping plants to establish and grow in
nutrient deficient conditions. Their use in agriculture can
favour a reduction in agro-chemical use and support
ecofriendly crop production. Trials with rhizosphere-
associated plant growth-promoting P-solubilizing and N2-
fixing microorganisms indicated yield increase in rice,
wheat, sugar cane, maize, sugar beet, legumes, canola,
vegetables and conifer species.
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5. A range of beneficial bacteria including strains
of Herbaspirillum, Azospirillum and Burkholderia are
closely associated with rhizosphere of rice crops. Common
bacteria found in the maize rhizosphere are Azospirillum
sp., Klebsiella sp., Enterobacter sp., Rahnella aquatilis,
Herbaspirillum seropedicae, Paenibacillus azotofixans,
and Bacillus circulans. Similarly, strains of Azotobacter,
Azorhizobium, Azospirillum, Herbaspirillum,
Bacillus and Klebsiella can supplement the use of urea-N in
wheat production either by BNF or growth promotion.
6. The commonly present PGPR in sugarcane plants
are Azospirillum brasilense, Azospirillum lipoferum,
Azospirillum amazonense, Acetobacter
diazotrophicus, Bacillus tropicalis, Bacillus borstelensis,
Herbaspirillum rubrisubalbicans and Herbaspirillum
seropedicae. Symbiotic N2-fixing bacteria collectively
known as Rhizobia are currently classified into six genera;
Rhizobium, Allorhizobium, Azorhizobium, Bradyrhizobium,
Mesorhizobium and Sinorhizobium and 91 species. Their
inoculation may increase nodulation and N2-fixation in
legumes. All these Rhizobiumn spp. Can minimize chemical
N fertilizers by BNF, but only if conditions for expression of
N2-fixing activity and subsequent transfer of N to plants
are favourable. In this Chapter, PGPR role has been
discussed in the process of crop growth promotion, the
mechanisms of action and their importance in crop
production on sustainable basis.
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7. TIURIN'S method for the determination of organic
carbon in soil is modified to give results practically
identical with those of the dry combustion method. The
standard deviation of a single determination is only 12%.
By using 50 mg of soil and 10 ml of 0.2 N dichromate
solution, soils with a carbon, content up to 12% can bo
analysed. The method is suitable for all soils except those
containing much chloride or reducing substances other
than organic carbon Carbonates do not interfere.
8.Reports about the relationship between soil water
retention and organic carbon content are contradictory.
Adding information on taxonomic order and on taxonomic
order and organic carbon content to the textural class
brought 10% and 20% improvement in water retention
estimation, respectively, as compared with estimation
from the textural class alone. Using total clay, sand and silt
along with organic carbon content and taxonomic order
resulted in 25% improvement in accuracy over using
textural classes. Similar but lower trends in accuracy were
found for water retention at −1500 kPa and the slope of
the water retention curve. At low organic carbon contents,
the sensitivity of the water retention to changes in organic
matter content was highest in sandy soils. Increase in
organic matter content led to increase of water retention
in sandy soils, and to a decrease in fine-textured soils. At
high organic carbon values, all soils showed an increase in
water retention. The largest increase was in sandy and
silty soils. Results are expressed as equations that can be
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used to evaluate effect of the carbon sequestration and
management practices on soil hydraulic properties.
9. The reliability of the Kjeldahl method for the determination
of nitrogen in soils has been investigated using a range of soils
containing from 0·03 to 2·7% nitrogen. The same result was
obtained when soil was analysed by a variety of Kjeldahl
procedures which included methods known to recover
various forms of nitrogen not determined by Kjeldahl
procedures commonly employed for soil analysis. From
this and other evidence presented it is concluded that very
little, if any, of the nitrogen in the soils examined was in
the form of highly refractory nitrogen compounds or of
compounds containing N—N or N—O linkages. Results by
the method of determining nitrogen in soils recommended
by the Association of Official Agricultural Chemists were
10–37% lower than those obtained by other methods
tested. Satisfactory results were obtained by this method
when the period of digestion recommended was
increased. Ammonium-N fixed by clay minerals is
determined by the Kjeldahl method. Selenium and
mercury are considerably more effective than copper for
catalysis of Kjeldahl digestion of soil. Conditions leading to
loss of nitrogen using selenium are defined, and difficulties
encountered using mercury are discussed. The most
important factor in Kjeldahl analysis is the temperature of
digestion with sulphuric acid, which is controlled largely by
the amount of potassium (or sodium) sulphate used for
digestion. The period of digestion required for Kjeldahl
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analysis of soil depends on the concentration of potassium
sulphate in the digest. When the concentration is low (e.g.
0·3 g./ml. sulphuric acid) it is necessary to digest for
several hours; when it is high (e.g. 1·0 g./ml. sulphuric
acid) short periods of digestion are adequate. Catalysts
greatly affect the rate of digestion when the salt
concentration is low, but have little effect when the salt
concentration is high. Nitrogen is lost during Kjeldahl
analysis when the temperature of digestion exceeds about
400° C. Determinations of the amounts of sulphuric acid
consumed by various mineral and organic soils during
Kjeldahl digestion showed that there is little risk of loss of
nitrogen under the conditions usually employed for
Kjeldahl digestion of soil. Acid consumption values for
various soil constituents are given, from which the
amounts of sulphuric acid likely to be consumed during
Kjeldahl digestion of different types of soil can be
calculated. It is concluded that the Kjeldahl method is
satisfactory for the determination of nitrogen in soils
provided a few simple precautions are observed. The
merits and defects of different Kjeldahl procedures are
discussed.
10. The dehydrogenase activity (DHA) of the microflora of a
gleyic luvisol artificially contaminated by 1, 10, and 100 μg
tributylin (TBT)/g dry wt soil was compared with its ATP
content, long term CO‐ 2 evolution, and esterase activity. DHA
was measured by reduction of triphenyltetrazolium chloride.
This 203 day laboratory experiment comprised a phase of air‐
drying, a remoistening, and the addition of a substrate (alfalfa
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and yeast extract). The half life of TBT ranged from 40 (10 and‐
100 μg/g) up to 70 days (1 μg/g).
The microflora was not affected by 1 and 10 μg TBT/g.
After an initial 60% stimulation, the respiration and
esterase activity in the soil contaminated by 100 μg TBT/g
recovered to the level of the control soil in 2 and 14 days,
respectively. About 50% depression of DHA and ATP
content was observed throughout the 203 days of the
experiment. DHA inhibition was correlated with
depression of ATP content (r = 0.82). Air drying,
remoistening, and substrate addition had little influence
on the depression of DHA and of the ATP content.
Unlike long term CO‐ 2 evolution, DHA did not reflect the
total effective activity of soil microflora; rather, it reflected
its total potential activity. As for biomass estimates using
substrate induced respiration, a clear distinction should be‐
made between short term DHA and other measurements‐
of DHA. Short term DHA, as a substrate induced maximum‐ ‐
initial activity, appears mainly to reflect the biomass of soil
microflora. The measurement of DHA appears to be a
suitable low cost and sensitive tool for assessing side‐
effects of chemicals.
11. The chemical characterizations or soil tests are most
apt to differentiate micronutrient availability when
nutrient element chemistry is carefully considered as part
of the soil test development. Successful calibration of
chemical soil extractants results in their use as soil tests
that can be interpreted in relation to adequacy of the
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nutrient. Soil test calibration must involve crop response
data from field studies that consider factors of the normal
growing environment that influence micronutrient soil
fertility and plant nutrition.
3. MATERIALS & METHODS
3.1 Instruments used:
Figure 1: top left-Vertical Autoclave,
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top lright- Cuvette Spectrophotometer,
bottom left- Incubator,
bottom right- Centrifuge
Figure 2: top left : Flame photometer,
top right: Std.Electromic Balance,
bottom left: Mechanical Shaker,
bottom right: Normal.Electronic Balance.
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Figure 3: top left- electro conductivity meter(EC meter),
top right- Ph meter,
bottom left- inside view of centrifuge with centrifuge tubes containing incubated
nutrient media with PGPG,
bottom right- Inductively Coupled Plasma (ICP).
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Figure 4: Vertical Laminr flow cabinet.
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3.2 Experimental setup
T1 P1 P2 P3 S1 S2 S3 T2 P1 P2 P3 S1 S2 S3 T3 P1 P2 P3 S1 S2 S3
T4 P1 P2 P3 S1 S2 S3
T5 P1 P2 P3 S1 S2 S3
T6 P1 P2 P3 S1 S2 S3
T7 P1 P2 P3 S1 S2 S3
T8 P1 P2 P3 S1 S3 S3
TREATMENT: Treatment Combination with
three replications
• T1 Soil only
• T2 PGPR stain-1 + 2.5t-1 Vermi
• T3 PGPR stain-2 + 2.5t-1 Vermi
• T4 PGPR stain-3 + 2.5t-1 Vermi
• T5 PGPR stain-4 + 2.5t-1 Vermi
• T6 PGPR stain-5 + 2.5t-1 Vermi
• T7 Only 5 t-1 vermi
• T8 10 t-1 FYM
1. We will first collect soil from different sites in bags for
auto clave, so that the soil become sterlised.
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TREATMENT CIM-Suvas
R1 R2 R3
CIM-Shishir
R1 R2 R3
2. Then we have to take 48 small pots with codes
namely;T1R1,T2R2…….T8S2,T8S3filled with above
sterlised soil and are arranged according to objective
protocol.
3. Plantation of tulsi (CIM-Suvas, CIM-Shishir) saplings
freshly harvested from nursery into each pots
according to the protocol; measure the initial length of
each plantlets in the pots.
4. Next step is to make dose for the plants from Nutrient
Broth. It is a general purpose medium used for
cultivating a broad variety of fastidious and non-
fastidious microorganisms with non-extracting
nutritional requirements.
COMPOSITION gl-1
Gelatin peptone 5
Beef extract 1Yeast extract 2
NaCl 5TOTAL 13
(13gms of nutrient broth in 1000ml of distill water).
First of all we will take five conical flasks of 1000ml and
one conical flask of 500ml, rinse well with detergent and
then with distill water twice and put it in oven for
complete drying; then in 1000ml beakers we will make
500ml of nutrient broth by mixing 6.5gms of media in
500ml warm distill water and mix well and all five conical
flasks as CRC-1,CRC-2,CRC-3,CRC-4 and PSB-2; and in one
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500ml beaker we will make 250 ml broth by mixing
3.25gms in 250ml warm distill water and marked as
control.
Now put cotton plugs on the beaker and wrap it with
aluminum foil and put it in auto clave at 121degree Celsius
at 15 atm for 30 mins and then after sterilization put the
broth in culture room for 24hrs to check for
contamination.
After 24hrs put the broth put the broth in laminar for
further sterlization for 5 mins then inoculate it with
bacteria and then put it in incubator(30-36 degree celsius)
for 24-72 hrs.
After two days check for the microbial growth in the broth
and perform further steps. Centrifuge( at 5000rpm at
14degree celsius for 10 mins) the broth and pour palette in
80% saline solution and discard supernatant.
80% saline solution is made by 8gms of NaCl in 100ml
distill water.
Dose is ready to pour in the pots according to the
protocol.
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Figure 5: top left- empty 5inch pot of 5Kgs.,
top right- Soil sample collected in sample bags from different sites of CSIR-CIMAP ,
bottom left- Autoclaved soil or sterlised soil,
bottom right- approx. 4Kgs of soil were filled in each of the 48 pots and arranged
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Figure 6: top left- plantation of tulsi varieties (CIM-Suvas,CIM-Shishir) saplings from nursery,
top right- Nutrient Broth in 5 conical flasks of 1000ml containing 500ml of it and
1 conical flask of 500ml containing 250 ml of it for blank, and strains of
CRC-1,CRC-2, CRC-3, CRC-4, PSB-2,
bottom left- pouring approx. 15ml of PGPR dose in each of the 48 pots,
bottom right- initial size of saplings of tulsi in pots.
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3.3 Testing
1. Ph Analysis: take 8gms of soil in a 50ml beaker add
20ml of distill water and stirred well and left for half an
hour; till then ph meter is calibrated by immersing the
electrode in different buffer of Ph-4.0,7.0 and 9.0 then the
electrode is immersed in the soil solution and reading is
taken.
Figure 7: Ph meter
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2. Electrical Conductivity: 8gms of soil sample is taken in
a 50ml beaker ; 20ml distilled water is added ;mixed well
and left for 30mins, the conductivity bridge was
calliberated with standard and the conductivity of the
sample was determined with conductivity meter.
Figure 8: EC meter (conductivity
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3. Nitrogen testing: take 100ml KmNO4 only in round
bottom flask (for blank) and for soil sample add 10gms of
soil also; In a 150ml conical flask take 100ml 1N H2SO4 ,
add 4-5 drops of indicator;
Fit kjeldahl apparatus and add 2.5N NaOH in round bottom
flask to start the reaction.
Sample + H2SO4 catalyst (NH4)2SO4 + CO2
+ H2O
(NH4)2SO4 + 2NaOH 2NH3 (gas) + Na2SO4
+ 2H2O
wait till the solution in conical flask increase upto 25ml;
then titrate the conical flask content with standard 0.1N
NaOH solution; solution colour turns from pink into green
at endpoint; note the value from the burette and perform
calculations.
Figure 9: kjeldahl apparatus on left and colour of nitrogen after titration on right
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4.Phosphorous testing: weigh 5gms of soil ;add 50ml of
NaHCO3 and pinch of activated charcoal to olsen the soil
solution; shake for ½ hrs (on mechanical shaker) then filter
with 42 No. wattman filter paper; take 5ml aliquate in
50ml volumetric flask; add a little water and 1ml of 5N
H2SO4 to maintain Ph of alliquate upto 5.5, shake well add
a little water , add 5ml mixed reagent and leave for some
time to develop blue colour , finally make up volume with
water and read its wavelength at 660nm
Figure 10: showing blue colouration sample solution due to prescence of
phosphorous of which absorbance is detected using spectrophotometer
at 660 λ.
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5.Organic carbon testing: take 0.5gms of soil in a 500ml
conical fask, add 10ml of 1N K2SO4 and swirl the flask 2-3
times and allow it to stand for 30mins for the reaction to
complete, pour 200ml of distill water to the flask to dilute
the suspension , add 5ml of H3PO4 (Orthophosphoric
acid ) and 1ml of Diphenylamine indicator and back titrate
the solution with 0.5N ferrous ammonium sulphate
solution , till the colour flashes from violet to blue to bright
green.
Figure 11: Green colouration showing the prescence of organic carbon
in the sample after titration.
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6.Sulfur testing: take 10gms soil in 150ml conical flask,
add 10ml of 0.15% of CaCl2 solution and put on
mechanical shaker for 30mins shaking , filter the solution
be 42No. filter paper, take 10ml of filtrate in 25ml
volumetric flask and add 1g BaCl2 in each flaskthen add
1ml of 0.25% gum acacia (emulsifier,stabilizer) and make
up the volume with distilled water, take the reading at
340nm from spectrophotometer, plot the curve
7.DHA Test: weigh 1gram of soil sample and place it in the
respective vials , add 0.2ml of 3% tripheny tetrazorium
chloride (TTC) solution in each of the tube of the soil, add
0.5ml of 1% glucose solution to the vials , cap it and shake
a little horizontally, incubate the tube at 28±0.5 °C for 24
hrs, after incubation add 10ml of methanol . Shake
vigorously the vials allow to stand for 6hours (min 1-
3hour), withdraw clear pink coloured supernatant liquid by
using whatsmann NO- 1 filter paper , read its absorbance
at 485nm,plot the graph.
Page 32 of 46
4 . RESULT & DISCUSSION
4.1 pH of soil
CIM-SUVAS
(INITIAL PH) (FINAL PH)
CONTROL 7.93 8.23
TREATMENT1 8.15 8.43
TREATMENT2 7.73 8.76
TREATMENT3 7.76 8.83
TREATMENT4 8.87 8.96
TREATMENT5 7.76 8.89
TREATMENT6 8.67 8.54
TREATMENT7 7.62 7.89
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CIM-SHISHIR
(INITIAL PH) (FINAL PH)
CONTROL 7.98 8.13
TREATMENT1 8.24 8.49
TREATMENT2 7.83 8.36
TREATMENT3 7.70 8.59
TREATMENT4 8.10 8.93
TREATMENT5 7.89 8.76
TREATMENT6 8.15 8.57
TREATMENT7 7.97 7.56
control t-1 t-2 t-3 t-4 t-5 t-5 t-76.5
7
7.5
8
8.5
9
9.5
ph of soil initial v/s final of both va-rieties of tulsi
initial final initial2 final2
Blue bars denote- initial ph readings of CIM-Shishir
Orange bars denote- final ph readings of CIM-Shishir
Grey bars denote- initial ph readings of CIM-Suvas
Yellow bars denote- final ph readings of CIM-Suvas.
Discussion-
• above graph clearly shows that the final ph readings of both varieties of tulsi i.e, CIM-Suvas and CIM-Shishir are comparatively more basic that the initial soil, that clearly shows that due to saline doses given to the plants shows its effect and thus makes the soil more alkaline.
Page 34 of 46
4.2 EC of soil
CIM-SUVAS
(INITIAL EC) (FINAL EC)
ΜS ΜS
CONTROL 93.70 112.14
TREATMENT1 109.90 116.24
TREATMENT2 96.45 105.34
TREATMENT3 123.56 137.12
TREATMENT4 133.14 145.21
TREATMENT5 121.09 145.00
TREATMENT6 107.00 116.23
TREATMENT7 102.34 112.23
Page 35 of 46
CIM-SHISHIR
(INITIAL EC) (FINAL EC)
ΜS ΜS
CONTROL 96.57 108.56
TREATMENT1 105.6 124.77
TREATMENT2 96.73 102.36
TREATMENT3 94.07 98.76
TREATMENT4 97.89 103.24
TREATMENT5 123.66 134.43
TREATMENT6 113.45 125.40
TREATMENT7 109.60 112.27
control t-1 t-2 t-3 t-4 t-5 t-6 t-70
20
40
60
80
100
120
140
160
EC of soil initial v/s final of both the varieties of tulsi
initial 1 final 1 initial 2 final 2
Blue bars denote- initial 1 EC readings of CIM-Shishir
Orange bars denote- final 1 EC readings of CIM-Shishir
Page 36 of 46
Grey bars denote- initial 2 EC readings of CIM-Suvas
Page 37 of 46
SHISHIR
Ni
Kg/hr
Nf
Kg/hr
CONTROL 338 369
TREATMENT 1 308 345
TREATMENT 2 246 302
TREATMENT 3 277 312
TREATMENT 4 215 289
TREATMENT 5 184 203
TREATMENT 6 154 245
TREATMENT 7 215 309
Blue bars denote- initial (N1 initial) Nitrogen content of
soil in Kg/hr of CIM-Shishir,
Orange bars denote- final (N1 final) Nitrogen content of
soil in Kg/hr of CIM-Shishir ,
Grey bars denote- initial (N2 initial) Nitrogen content of
soil in Kg/hr of CIM-Suvas,
Yellow bars denote- - final (N2 final) Nitrogen content of
soil in Kg/hr of CIM-suvas.
Page 38 of 46
SUVAS
Ni
Kg/hr
Nf
Kg/hr
CONTROL 345 356
TREATMENT 1 313 378
TREATMENT 2 256 308
TREATMENT 3 245 333
TREATMENT 4 267 314
TREATMENT 5 179 189
TREATMENT 6 289 345
TREATMENT 7 267 316
4.4 Soil organic carbon (%) content :
Blue bars denote- initial (C1 initial) O.C content of soil in %
of CIM-Shishir,
Page 39 of 46
SHISHIR
Ci
%
Cf
%
CONTROL 0.09 0.13
TREATMENT 1 0.15 0.28
TREATMENT 2 0.15 0.32
TREATMENT 3 0.39 0.45
TREATMENT 4 0.42 0.48
TREATMENT 5 0.36 0.48
TREATMENT 6 0.18 0.30
TREATMENT 7 0.36 0.48
SUVAS
Ci
%
Cf
%
CONTROL 0.3 0.24
TREATMENT 1 0.12 0.28
TREATMENT 2 0.09 0.18
TREATMENT 3 0.24 0.33
TREATMENT 4 0.33 0.45
TREATMENT 5 0.24 0.34
TREATMENT 6 0.21 0.31
TREATMENT 7 0.33 0.43
Orange bars denote- final (C1 final) O.C content of soil in
4.5 Absorbance of soil phosphorous :
Page 40 of 46
SHISHIR
Pi
λ.
Pf
λ.
CONTROL 0.0823 0.2784
TREATMENT 1 0.1090 0.3286
TREATMENT 2 0.0639 0.3126
TREATMENT 3 0.1733 0.0151
TREATMENT 4 0.0655 0.2426
TREATMENT 5 0.0849 0.2814
TREATMENT 6 0.1230 0.1670
TREATMENT 7 0.2688 0.3470
Blue bars denote- initial (P1 initial) Phosphorous content
of soil absorbance of CIM-Shishir,
Orange bars denote- final (P1 final Phosphorous content of
soil absorbance of CIM-Shishir,
Grey bars denote- initial (P2 initial) Phosphorous content
of soil absorbance of CIM-Suvas,
Yellow bars denote- - final (P2 final) Phosphorous content
of soil absorbance of CIM-Suvas.
4.5 Absorbance of soil Sulfur :
Page 41 of 46
SUVAS
Pi
λ.
Pf
λ.
CONTROL 0.0786 0.2433
TREATMENT 1 0.2430 0.2230
TREATMENT 2 0.0327 0.1659
TREATMENT 3 0.1657 0.2286
TREATMENT 4 0.2489 0.2788
TREATMENT 5 0.3478 0.3276
TREATMENT 6 0.3230 0.5270
TREATMENT 7 0.2749 0.3370
Blue bars denote- initial (S1 initial) Sulfur content of soil
absorbance of CIM-Shishir,
Page 42 of 46
SHISHIR
Si
λ.
Sf
λ.
CONTROL 0.0752 0.2645
TREATMENT 1 0.1270 0.3147
TREATMENT 2 0.0584 0.3275
TREATMENT 3 0.1845 0.0237
TREATMENT 4 0.0467 0.2366
TREATMENT 5 0.0237 0.2816
TREATMENT 6 0.3267 0.1765
TREATMENT 7 0.2476 0.3743
SUVAS
Si
λ.
Sf
λ.
CONTROL 0.0676 0.2678
TREATMENT 1 0.2326 0.2775
TREATMENT 2 0.0754 0.1864
TREATMENT 3 0.1567 0.2567
TREATMENT 4 0.2985 0.2957
TREATMENT 5 0.3567 0.3276
TREATMENT 6 0.3675 0.5964
TREATMENT 7 0.2866 0.3965
Orange bars denote- final (S1 final Sulfur content of soil
absorbance of CIM-Shishir,
Grey bars denote- initial (S2 initial) Sulfur content of soil
absorbance of CIM-Suvas,
Yellow bars denote- - final (S2 final) Sulfur content of soil
absorbance of CIM-Suvas.
5.CONCLUSION
5.1 growth attribute:
Suvas
(gms)
shishir
(gms)
control 68 67
treatment 1 59 58
treatment 2 63 61
treatment 3 62 61
treatment 4 73 72
treatment 5 57 56
treatment 6 60 59
treatment 7 58 57
Page 43 of 46
Graph 1: it is found maximum biomass is of
treatment 4 i.e. Advencca Spp.
5.2 yield attribute:
Suvas
(ml)
shishir
(ml)
control 0.23 0.25
treatment 1 0.20 0.20
treatment 2 0.24 0.24
treatment 3 0.13 0.13
treatment 4 0.25 0.26
treatment 5 0.14 0.13
treatment 6 0.25 0.00
treatment 7 0.25 0.20
Page 44 of 46
Graph 2: it is found that yield from treatment 4 i.e.
Advencca Spp. Is slightly more than rest of the treatments.
5.REFERENCE
1. P. N. Bhattacharyya & D. K. Jha World Journal of Microbiology and Biotechnology volume 28, pages1327–1350(2012)
2. .S.Darni,A.K.Srivastava,A Samad, D.D.Patra-Chemosphere,2014-Elsevier.
3. . Wuxing Liu, Qingling Wang, Beibei Wang, Jinyu Hou, Yongming Luo, Caixian Tang & Ashley E. Franks Journal of Soils and Sediments volume 15, pages1191–1199(2015)
4. .R Singh, SK Soni, RK Patel, A Kalra- Industrial crops and production,2013-Elsivier
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5. Geoderma Volume 116, Issues 1–2, September 2003, Pages 61-76
6. The Journal of Agricultural ScienceVolume 55, Issue 1August 1960 , pp. 11-33
7. Micronutrient Soil Tests J. T. Sims G. V. Johnson Book Editor(s): J. J. Mortvedt First published:01 January 1991
8. Dehydrogenase activity of soil microflora: Significance in ecotoxicological tests
9. D. Rossel J. Tarradellas First published:February 1991.
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