ijrar.orgijrar.org/papers/IJRAR_225031.docx · Web viewPlants, Lucknow 2020. ACKNOWLEDGEMENT Firstly, I bow my head with utmost reverence before the Almighty whose eternal blessing

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.

Page 1 of 46

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





• 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

Page 25 of 46

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

Page 28 of 46

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

Page 29 of 46

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.

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4 . RESULT & DISCUSSION

4.1 pH of soil

CIM-SUVAS

(INITIAL PH) (FINAL PH)

CONTROL 7.93 8.23

TREATMENT1 8.15 8.43







Page 33 of 46

CIM-SHISHIR

(INITIAL PH) (FINAL PH)

CONTROL 7.98 8.13








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


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







SUVAS

Ci

%

Cf

%

CONTROL 0.3 0.24








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







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

Page 45 of 46

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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