“Selection of Effective AZOTOBACTER Isolates for Tomato … · 2018-12-06 · “Selection of Effective Azotobacter Isolates for Tomato (Lycopersicon esculentum Mill.)’’Thesis

“Selection of Effective AZOTOBACTER Isolates for

Tomato (LYCOPERSICON ESCULENTUM MILL.)’’

M.SC. (AG.) THESIS

BY

SURENDRA SINGH

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY

COLLEGE OF AGRICULTURE

INDIRA GANDHI KRISHI VISHWAVIDYALAYA

RAIPUR (C.G.)

2011

“Selection of Effective Azotobacter Isolates for Tomato

(Lycopersicon esculentum Mill.)’’

Thesis

Submitted to the

Indira Gandhi Krishi Vishwavidyalaya, Raipur (C.G.)

by

SURENDRA SINGH

IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR THE

DEGREE OF

MASTER OF SCIENCE

in

AGRICULTURE (AGRICULTURAL MICROBIOLOGY)

Roll No. 12517 ID No. 112105082

JUNE, 2011

Certificate - I

This is to certify that the thesis entitled “SELECTION OF EFFECTIVE

Azotobacter ISOLATES FOR TOMATO (Lycopersicon esculentum Mill.)’’,

submitted in partial fulfilment of the requirements for the degree of “MASTER

OF SCIENCE IN AGRICULTURE (Agricultural Microbiology)” of the

Indira Gandhi Krishi Vishwavidyalaya, Raipur, is a record of the bonafide

research work carried out by Mr. SURENDRA SINGH under my guidance and

supervision. The subject of the thesis has been approved by the Student's

Advisory Committee and the Director of Instructions.

No part of the thesis has been submitted for any other degree or

diploma (certificate, award etc.) or has been published / published part

has been fully acknowledged. All the assistance and help received during

the course of the investigations have been duly acknowledged by him.

Date: Chairman

Advisory Committee

Thesis approved by the student's advisory committee

Chairman Dr. Tapas Chowdhury ____________________

Member Dr. S.B. Gupta ____________________

Member Dr. C.P. Khare ____________________

Member Dr. (Smt.) G. Chandrakar ____________________

Member Dr. Rajendra Lakpale ____________________

CERTIFICATE - II

This is to certify that the thesis entitled “SELECTION OF EFFECTIVE

Azotobacter ISOLATES FOR TOMATO (Lycopersicon esculentum Mill.)’’

submitted by Mr. SURENDRA SINGH to the Indira Gandhi Krishi

Vishwavidyalaya, Raipur (C.G.) in partial fulfillment of the requirements for the

degree of ―M.Sc. (Ag)‖, in the Department of Agricultural Microbiology has

been approved by the external examiner and student‘s advisory committee after

oral examination.

Date: External Examiner

Major Advisor

____________________

Head of the Department/ Section

____________________

Dean Faculty

____________________

Director of Instruction

____________________

ACKNOWLEDGEMENT

“Education plays fundamental role in personal and social development and

teacher plays a fundamental role in imparting education. Teachers have crucial role in

preparing young people not only to face the future with confidence but also to build up it

with purpose and responsibility. There is no substitute for teacher pupil relationship”. I start

in the name of God-who has bestowed upon me all the physical and mental attributes that I

possess and skills to cut through and heal a fellow human.

With a sense of high resolve and reverence I express my sincere and deep sense of

gratitude to adorable Dr. Tapas chowdhury, Scientist (Agricultural Microbiology), College of

Agriculture, Raipur (C.G.) who is chairman of my advisory committee. I have no word to

express my heartfelt thanks to him for his blessings, invaluable inspiring guidance, unfailing

encouragement, suggestions, research insight, unique supervision, constructive criticism,

scholarly advice, sympathetic attitude and keen interest, throughout the investigation and

preparation of this manuscript.

I have immense pleasure in expressing my whole hearted sense of gratitude and

appreciation for Dr. S.B. Gupta, Head of Department (Agricultural Microbiology), member

of my Advisory Committee for his blessings, inspiring suggestions, enthusiastic interest and

encouragement which provided me solace during the tenure of investigation and preparation

of this manuscript.

I would be ever grateful to the other members of my Advisory Committee, Dr.

C.P. Khare, Scientist (Plant Pathology), and Dr.(Smt.) G. Chandrakar, Senior Scientist

(Agril. Stat. Maths. and Computer Science), Dr. Rajendra Lakpale, Senior Scientist

(Agronomy), for providing proper guidance and encouragement throughout the research work.

Without their kind cooperation, it would not have been easy for me to complete this

manuscript.

I am deeply obliged to Mrs. Deepti Mayee Das, Scientist (Agricultural

Microbiology) and Mr. Porte, Scientist (Soil Science), for providing me inspiring suggestions

and encouragement during the tenure of investigation.

I am highly obliged to Hon`ble Vice chancellor Dr. M.P. Pandey, Dr. O.P.

Kashyap, Dean, College of Agriculture, Raipur, Dr. S.K. Patil, Director Research Services,

Dr. R.B.S. Sengar, Director Extension Services and Dr. U.K. Mishra, Director of

Instructions, IGKV, Raipur for providing necessary facilitates to conduct the investigation.

I wish to express my grateful thanks to Mr. Hari, Mr. Gangu, Mr. Devanand,

Mr. Hiralal, Mrs. Padama and all staff members of Department of Agricultural

Microbiology, College of Agriculture, I.G.K.V., Raipur for their co-operation.

I express my thanks to my friends Onasish, Ashish, Nirala, Mishra, Anuj, Ram,

Samel, Narayan, Amit, ,Sandeep, Kavach, Sanjay, Anil, Deepak, Yogash, Bala, Tarun,

Shyam, Jitu sir, Vijay, Mohan, Rakesh, Shukla, Ganesh, Khare, Sanjeet, ,Rivanchal, Sujata,

Yuvraj ,Tushar sir and Tiwari sir for their care, love and support they have given to me

during my study period.

I am also thankful to my juniors Suvesh, Rakesh Patel, Smriti, Moorat, Sarju,

Sanjay & Subham.

Words can hardly express the heartfelt gratitude to my beloved Father Mr.Babulal

Singh, Mummy Mrs.Sudha Singh, Brother dear Rinku, Anurag, Sentu, and Rajesh whose

selfless love, filial affection, obstinate sacrifices and blessing made my path easier.

There is no substitute for the love and affection bestowed on me by my

Grandfather Mr.Thakur Prasad Singh. & Uncle Mr.Bhola Singh.

I would like to convey my cordial thanks to all those who helped me directly or

indirectly to fulfill my dream.

How can I express my thanks to “God” because there is no word to express it.

So, my lord, please realize and accept my feelings.

College of Agriculture, Raipur (C.G.)

Date: _______Surendra Singh

CONTENTS

CHAPTER

NO.

PARTICULERS PAGE NO.

I. INTRODUCTION

II. REVIEW OF LITERATURE

III. MATERIALS AND METHODS

IV. RESULTS AND DISCUSSION

V. SUMMARY, CONCLUSIONS AND

SUGGESTIONS FOR FUTURE WORK

VI. ABSTRACT

VII. BIBLIOGRAPHY

VIII. APPENDICES

LIST OF TABLES

TABLE NO. PARTICULARS PAGE

4.1 Nitrogen fixation capacity of local Azotobacter isolates and

standard check in the N free Jensen‘s liquid medium

4.2 Influence of various Azotobacter isolates and different levels of

nitrogen on plant height of tomato


nitrogen on fruit yield of tomato


nitrogen on dry matter yield of tomato


nitrogen on N-accumulation by tomato fruit


nitrogen on N-accumulation by tomato shoot at harvest


nit nitrogen on total N-uptake (fruit + shoot) by tomato


nitrogen on Dehydrogenase activity in soil at 30 DAT

4.9 Effect of local isolates & standard check of Azotobacter on

Fusarium oxysporium

LIST OF FIGURES

FIGURE

NO.

PARTICULARS PAGE

4.1 Nitrogen fixation capacity of local Azotobacter isolates and

standard check in the N free Jensen‘s liquid medium


nitrogen on plant height of tomato




nitrogen on dry matter yield of tomato


nitrogen on N-accumulation by tomato fruit


nitrogen on N-accumulation by tomato shoot at harvest


nitrogen on total N-uptake (fruit + shoot ) by tomato


nitrogen on Dehydrogenase activity in soil at 30 DAT

4.9 Effect of local isolates & standard check of Azotobacter on

Fusarium oxysporium.

LIST OF PLATES

PLATE

NO. PARTICULARS AFTER PAGE

1 (a) Growth performance of some Azotobacter isolates

during initial screening

1 (b) Estimation of N-fixing capacity of Azotobacter

isolates by Microkjeldhal method

2 General view of tomato crop during second stage

screening of Azotobacter isolates

3 (a) Growth performance of tomato crop during second

stage screening of Azotobacter isolates (30DAT)

3 (b) Growth performance of tomato crop during second

stage screening of Azotobacter isolates (60DAT)

4 (a) Influence of promising local Azotobacter isolate

AZOT-B-33 over Control CI, CII, and CIII on

tomato (70DAT)

4 (b) Growth performance of tomato crop inoculated with

promising local Azotobacter isolates AZOT-B-33

(70DAT).

5 Antifungal activity of promising Azotobacter isolates

and standard check in dual culture against Fusarium

oxysporium

LIST OF ABBREVIATIONS

ABBREVIATIONS FULL FORM

% percent

@ at the rate

CD critical difference

cm centimeter

DAT days after transplanting

et al. and co-workers/ and others

Fig. figure

g gram

ha hectare

hr hours

i.e. that is

kg kilogram

l litre

mg milligram

mm millimeter

ml millilitre

NPK Nitrogen, phosphorus and potassium

0C degree Celsius

pH potentiality of hydrogen

SEm+ standard error of mean

Viz. for example

100:60:80 100 Kg N: 60 Kg P2O5:80 Kg K2O / ha.

CHAPTER-I

INTRODUCTION

Soil the natural habitat for all microorganisms; harbour both bandits and

benefactors of the plant kingdom. Beneficial microorganisms are those that can

stimulate plant growth by fixing atmospheric nitrogen, solubilizing unavailable

phosphates, decomposing organic wastes and enhance nutrient recycling by producing

bioactive substances such as vitamins, hormones, enzymes etc. (Brown, 1975).

Abundance and versatility of such organisms are very high in rhizosphere, which is the

volume of soil influenced by plant roots. Using these beneficial microorganisms,

various microbial inoculants have been prepared for use in crop production to reduce

the cost on chemical fertilizers and to minimize environmental pollution. Since

microorganisms are useful in eliminating the problems associated with use of chemical

fertilizers and pesticides, they are now widely applied in organic farming.

Tomato (Lycopersicon esculentum Mill.) is one of the most important

vegetable crop. It belongs to family solanaceae and is believed to be a native of western

South America. This crop is also known as an industrial crop because of its outstanding

processing qualities. Tomato is rich source of minerals, vitamins and organic acid and

fruit provides 3-4% total sugar, 4-7% total solids, 15-30 mg/100g ascorbic acid, 7.5-10

mg/100 ml titrable acidity and 20-50 mg/100g fruit weight of lycopene. Also in 100g of

edible part of fruit composed of 93.1g moisture, protein 1.9g, fat 0.1g, minerals 0.6g,

fiber 0.7g, carbohydrates 3.6g, sodium 45.8mg, potassium 114mg, copper 0.19mg,

sulphur 24mg, chlorine 38mg, vitamin A 320 I.U, thiamine 0.07mg, riboflavin 0.1mg,

nicotinic acid 0.4mg, vitamin C 31mg, calcium 20mg, magnesium 15mg, oxalic acid

2mg, phosphorus 36mg, and iron 1.8mg. Several epidemiological studies indicated

beneficial effects of tomato consumption in the prevention of some major chronic

disease, such as cancer and cardiovascular disease (Giovannucci, 1999).

It is cultivated in an area of 52.55 million hectares world over producing

130.53 million tonnes of tomato with an average yield of 27.98 tonnes/ha (Anon,

2009). In India, it is mainly grown in Bihar, Karnataka, Uttar Pradesh, Orissa, Andhra

Pradesh, Maharashtra, Madhya Pradesh, Assam and Chhattisgarh, accounting for a total

production of 11149 thousand MT from an area of 599 thousand hectares with an

average productivity of 18.6 MT per hectare. In Chhattisgarh, tomato is being

cultivated as commercial crop in Raipur, Durg, Sarguja, Bilaspur, Jashpur, Raigarh and

Bastar districts occupying an area 39.2 thousand hectares with production and

productivity of 420.4 thousand MT and 10.7 MT per hectare, respectively (Anon,

2009).

During last one hundred years, large numbers of aerobic and anaerobic

bacteria have been identified as free-living nitrogen fixers. Their N fixing potential

ranging from 2mg to 25mg per gram of carbon source utilized. Amongst these potential

N-fixer Azotobacter is one that fixes nitrogen in non-legumes. Azotobactor is a

heterotrophic free living nitrogen fixing bacteria present in alkaline and neutral soils.

Azotobactor chrococcum is the most commonly occurring species in arable soils of

India. Apart from its ability to fix atmospheric nitrogen in soils, it can also synthesize

growth promoting substances viz., auxins, and gibberellins and also to some extent the

vitamins. Many strains of Azotobactor also exhibit fungicidal properties against certain

species of fungus. Response of Azotobactor has been seen in rice, maize, cotton,

sugarcane, pearl millet, vegetable and some plantation crops. Its population is very low

in uncultivated lands. Presence of organic matter in the soil promotes its multiplication

and nitrogen fixing capacity. Field experiments carried out on Azotobacter indicated

that this is suitable when inoculated with seed or seedling of crop plants like onion,

brinjal, tomato and cabbage under different ago-climatic conditions. Azotobacter

inoculation curtails the requirement of nitrogenous fertilizers by 10 to 20% under

normal field conditions.

Area of Chhattisgarh state with is bigger than many states of atmospheric N2

fixing and P mobilizing microbial inoculation, as has a demand for identified by

analysis of soil samples of various district of this state. The low population density of

above heterotrophs are mainly due to high air temperature up to 480C, soil surface

temperature beyond 600C and low humidity up to 3-4% for prolonged period of

summer season resulting to loss of organic matter and population of beneficial

microbes (Anonymous, 1996). Azotobacter spp. are also sensitive to acidic pH, high

salts and temperature above 350C, so its population is very poor in soils of

Chhattisgarh. The soil of Chhattisgarh are low to medium in available nitrogen thus N

is one of the most limiting plant nutrients. In the light of ever increasing prices coupled

with increasing demand of chemical fertilizers and depleting soil fertility necessitates

developing effective bioinoculant of Azotobacter for tomato crop. In this view of above

it may worthwhile to develop the specific location effective Azotobacter isolates for

tomato.

So an attempt was made to develop a suitable Azotobacter inoculant for

tomato growers of Chhattisgarh with the following objectives.

Objectives of the investigation are as follows:

1. Testing of native isolates of Azotobacter for their N- fixing ability.

2. Influence of different Azotobacter isolates on performance of tomato crop.

3. Dehydrogenase enzyme activity of Azotobacter isolates in soil.

CHAPTER-II

REVIEW OF LITERATURE

There are several beneficial rhizomicroorganisms in the rhizosphere, which

can improve soil quality, enhance crop production and protection, conserve natural

resources and ultimately create more sustainable agricultural production and safe

environment. Effective techniques have been developed to isolate and enumerate these

organisms from the rhizosphere of crop plants and test their efficiency for beneficial

effects on soil and plant as well. Enormous literature are available on the relationship of

annual crop plants with soil microorganisms on the nitrogen fixation and phosphate

solubilisation but at the same time, literature on tomato crop with relation to

biofertilizer is scanty. Literature pertaining to utilization of these beneficial organisms

as bioinoculants in crop production and their effects as single or mixed inoculants on

crop growth, production and nutrient uptake have been reviewed in this chapter :-

2.1. Azotobacter – A potential Nitrogen fixer

2.2. Characterization of Azotobacter

2.3. Importance of strain selection

2.4. Isolation techniques of Azotobacter

2.5. Method of inoculation and benefit

2.6. Azotobacter population and Soil Environment

2.7. Production of plant growth promoting substances

2.8. N fixation by Azotobacter

2.9. Yield enhancement by Azotobacter inoculation

2.10. Field Response of Azotobacter

2.1. Azotobacter – A potential Nitrogen fixer:

Nitrogen is a fundamentally important element in biologically mediated

production and nutrient cycling processes. N2 containing constituents of organic

molecules often confer bioactivity to these molecules. Major cellular, structural, and

functional constituents have essential and often highly specific requirements for N2.

Nitrogen fixation is the reduction of N2 (atmospheric nitrogen) to NH3 (ammonia). Free

living prokaryotes with the ability to fix atmospheric dinitrogen (diazotrophs) are

ubiquitous in soil but our knowledge of their ecological importance and their diversity

remains incomplete. In natural ecosystems, biological N2 fixation is most important

source of N. The capacity for nitrogen fixation is widespread among bacteria and

archaea. The estimated contribution of free-living N-fixing prokaryotes to the N input

of soil ranges from 0-60 kg/ha /year (Burgmann et al., 2003).

Azotobacter is used as a biofertilizer in the cultivation of most crops.

Azotobacter is an obligate aerobic diazotrophic soil-dwelling organism with a wide

variety of metabolic capabilities, which include the ability to fix atmospheric nitrogen

by converting it to ammonia. Azotobacter naturally, fixes atmospheric nitrogen in the

rhizosphere. There are different strains of Azotobacter each has varied chemical,

biological and other characters. However, some strains have higher nitrogen fixing

ability than others besides (Burgmann et al., 2003).

Azotobacter sp. is a gram-negative bacterium, which grows in aerobic

environments and fixes atmospheric nitrogen. Azotobacter plays a remarkable role,

being broadly dispersed in different environments, such as soil, water and sediments

(Chan et al., 1986). In addition it is a bacterium with a broad metabolic diversity, this

feature enables it to degrade numerous highly resistant substrates to increase plant yield

(Jackson et al., 1964; Rovira, 1965; Denarie and Blanchere, 1966) due to the increase

of fixed nitrogen content in soil (Gouri and Jagasnnatathan, 1995; Maltseva et al.,

1995).

Azotobactor is a heterotrophic free living nitrogen fixing bacteria present in

alkaline and neutral soils. Azotobactor chrococcum is the most commonly occurring

species in arable soils of India. Apart from its ability to fix atmospheric nitrogen in

soils, it can also synthesize growth promoting substances viz., auxins, gibberellins and

also to some extent the vitamins. Many strains of Azotobactor also exhibit fungicidal

properties against certain species of fungus. Response of Azotobactor has been seen in

rice, maize, cotton, sugarcane, pearl millet, vegetable and some plantation crops. Its

population is very low in uncultivated lands. Presence of organic matter in the soil

promotes its multiplication and nitrogen fixing capacity. Field experiments carried out

on Azotobacter indicated that this is suitable when inoculated with seed or seedling of

crop plants like onion, brinjal, tomato and cabbage under different ago-climatic

conditions. Azotobacter inoculation curtails the requirement of nitrogenous fertilizers

by 10 to 20% under normal field conditions (Tom et al., 2007).

Area of Chhattisgarh state is bigger than many states having actual need of

effective location specific atmospheric N2 fixing and P mobilizing microbial inoculants

has been identified by analysis of soil samples of various district of this state

(Anonymous, 1996). The low population density of above heterotrophs is mainly due to

high air temperature up to 480C, soil surface temperature beyond 60

0C and low

humidity up to 3-4% for prolonged period of summer season resulting to loss of organic

matter and population of beneficial microbes. Azotobacter spp., are also sensitive to

acidic pH, high salts and temperature above 350C so its population is very poor in soils

of Chhattisgarh. In the light of ever increasing prices coupled with increasing demand

of chemical fertilizers and depleting soil fertility necessitates to develop effective

bioinoculant of Azotobacter for vegetable crops in general & tomato in particular. In

this view of above it may worthwhile to develop the location specific effective

Azotobacter isolate.

2.2. Characterization of Azotobacter:

Azotobacter is a genus of usually motile, oval or spherical bacteria that form

thick –walled cysts , and may produce large quantities of capsular slime , elongated,

1.4-2.0 um diameter and rod shaped cell. These bacteria being single and also couple,

irregular colony, and sometime from a long chain. Azotobacter does not produce

endospore, but form cyst. This chemorganotrophy bacteria, is gram negative, show

motility using flagella, or non motile, aerobic, but can also grow under oxygen

pressure. Azotobacter can be fixed N (non symbiotic) at least 10mg N2 per gram of

carbohydrate (usually in the form of glucose) is consumed. In certain species these

bacteria use nitrate, ammonia, certain amino acids as nitrogen sources, and able to grow

in the PH. range 4.8-8.5 while the pH optimum for nitrogen fixation and growth is 7.0-

7.5 in soil and water. This species may be associated with the root of plant. (Holt et

al.,1994).

Menuke (1964)& Brown (1975) found that Azotobacter reproduce very well

on nitrogen-free nutrient mediums which marked the beginning of a new phase in

Azotobacter research. Many authors tried to find a practical application of this ability

but their results turned out widely different and the conclusion was that the positive

effects of this bacteria had on the plant were more due to their production of certain

growth substances than to their nitrogen-fixing activity.

In 1997 Kanungo et al. reported that Azotobacter species (Azotobacter

vinelandii and A. chroococcum) are free-living, aerobic heterotrophic diazotrophs that

depend on an adequate supply of reduced C compounds such as sugars for energy.

Their activity in rice culture can be increased by straw application presumably as a

result of microbial breakdown of cellulose into cellobiose and glucose.

Shehata and El-Khawas (2003) reported that isolated Azotobacter sp were

subjected to different physical parameters like sugar concentration, pH, and

temperature which influence the growth and morphological properties (Cappuccino

and Sherman. 1996). Hence the strains were grown in varying sugar (Sucrose)

concentrations like 0.5, 1.0, 2.0, 3.0, 4.0 % and the influence of sugar was recorded

with Burk‘s broth using spectrophotometer at 520 nm. Similarly the influence of pH

(5.0, 6.0, 7.0, 8.0 and 9.0) and temperature (20, 28, 37 and 45°C) were recorded.

2.3 Importance of strain selection:

Improvement of yield and yield attributes were noted by A. chrocococcum due

to its nutritional, stimulatory and therapeutic capabilities that include availability of

nitrogen and phosphorus to plants, initiation of seed emergence by producing growth

promoting substance (IAA,GIA,auxins,vitamins etc.) and its antagonistic approach to

plant pathogen , respectively. However, still we lag behind selecting anappropriate

efficient strains of A.chroococcum for a particular crop plant .The following steps

should be given weightage to select an efficieant strains for its actual performance:

a) it should be homologous (specific to crop),

b) It should be large sized, chromogenic (brown to black pigment),

c) It should have moderate capacity to fix nitrogen,

d) It should be able to initiate seed germination,

e) It should properly establish in the rhizosphere and on the root of crop plant,

f) It should be screened for crops under different agro-climatic conditions.

The first and most important step in biofertilizer production is the selection of

efficient strain. This involves careful experimentation in the laboratory and field, which

is possible only in well-equipped laboratories and field with trained experts. Presently,

Biofertilizers strains are selected by

(a) Extensive screening

(b) Mutagenesis and

(c) Genetic engineering methods.

However most of biofertilizer strains used in India have been obtained

through screening techniques while a few strains have been selected by mutation as

reported by Balasubramanian, 1992; Tilak, 1991; Siddharmiah and Bagyaraj, 1981.The

only strain of Azospirillum developed by Tamilnadu Agricultural University Scientists

through modern genetic engineering has been released for mass production. In 1992,

Balasubramanian concluded that the performance of selected strains have to be tested in

field and hence take nearly 3 to 5 years for releasing an efficient strain for field

application.

Even though we have national strains identified as efficient, there is still scope

for improving the efficiency or for identifying a location specific strain which proves to

be better one (Bergersen, 1970; and Halliday, 1984).One has to search continuously for

more efficient strains, which can be accomplished by isolating and screening numerous

wild type strains or by genetic manipulation and screening numerous mutants. Since the

former method is time consuming, we are trying the mutagenesis approach for strain

improvement. Initially the inoculants strains are selected for good performance under

field condition; such strains are subjected to mutagenesis using chemical mutagens and

transposoon elements (Palaniappan, 1992).

Tilak (1991), Palaniappan, (1992) and Subba Rao et al. (1993) concluded that

strain efficiency reflects the ability for survival and multiplication in the carrier and

soil, growth rate, tolerance to environmental stress, symbiotic properties such as

nitrogen fixation, growth stimulant production etc. and competition with native flora

existing in soil.

El-Dsouky et al. (2003) studied the strains of Azotobacter, Azospirillum and

Pseudomonas locally isolated from rhizosphere soils of different plants grown at

Aswan area, Egypt, whereas Bacillus polymyxa [Paenibacillus polymyxa] strain no.37,

a phosphate dissolving bacteria, was isolated from the rock phosphate of Sebaeia mine.

Azotobacter strains no.5 and 11 induced the most pronounced effects in all plant growth

parameters. The highest N% and total N content were found in plant shoots inoculated

with these two strains.

Considerable variations in all Azotobacter spp. strains were found for the

different inoculated isolates in fine-textured soil (clay soil). The basic reasons for this

are increasing surface area and nutrient contents (Stotzky 1972;& Haris, 1981. Clay

particles have a million fold more surface area per mass than silt. Clay is capable of

holding large amounts of water and nutrients such as P, K but may prevent the release

of water for Azotobacter spp.

2.4. Isolation techniques of Azotobacter:

Azotobcter chroococcum (family : Azotobacteraceae) had been isolated by

Beijerinck in 1901. At present seven species of this bacterium are known. Species of A.

chroococcum most frequently occurring in different soils. Due to its multiple

physiological attributes of broad- spectrum utility, the use of the Azotobacter is

recommended for various area.

The characteristics of Azotobacter which are to be taken into account during

isolation process : Pleomorphic, gram negative, often motile (polar or peritrichous

flagella), non- spore forming, relatively large rods or even yeast like appearance,

showing variation in shape and size, mesophilic (optimum growth temperature 30oC),

obligate aerobes ,use carbohydrate as an energy source, catalase positive ,macrocyst

forming, cultures grow best with free nitrogen or simple forms of combined nitrogen,

capable of fixing atmospheric nitrogen or simple forms of combined nitrogen ,capable

of fixing atmospheric nitrogen asymbiotically , widely distributed in soil (PH 6.0-7.5).

there is only one species i.e. Azotobacter paspalum , which specifically associated with

Paspalum notarum ,a grass ,which is classed as a case of associative symbiosis

(Dobereiner,1970).

Mahalakshmi and Reetha (2009) isolated 44 bacterial isolates from the

rhizosphere of tomato grown in Cuddalore and Nagapattinam districts of Tamil Nadu,

India. These bacterial isolates were grouped into Azospirillum (18 isolates) Azotobacter

(9) Pseudomonas (12) and Bacillus (5) based on their morphological and biochemical

characteristics. All the isolates were screened for their plant growth promoting

activities viz., IAA production, phosphate solubilization, siderophore production, HCN

production, ACC deaminase activity and antifungal activity. They found that not all the

isolates possessed all the PGP activities. The range of percentage of positive isolates of

Azospirillum, Azotobacter, Pseudomonas and Bacillums for each of PGP activities

varies greatly. Among the 44 isolates, three isolates of Azospirillum, 2 from

Azotobacter, one from Bacillus and four from Pseudomonas were selected and the IAA

production, siderophore production and antifungal activity against R. solani and

Fusarium oxysporum were determined quantitatively.

Mary et al.(1985) used different selective media for the isolation of

Azotobacter sp. from marine source. Azotobacter strains used for this study were

maintained and cultured in Burk medium. As the isolates are of marine origin, the

media were prepared by the 3.5% sodium chloride (NaCl). Media used for the isolation

of nitrogen fixing organism (Azotobacter) from marine sources were Jensen‘s agar

medium, Azotobacter agar medium, Burks Medium and marine agar medium. Gram

staining, motility determination and biochemical test like catalase and starch hydrolysis

test were carried out to confirm Azotobacter spp.

Lophnez & Tejran (2005) reported that bacteria with the ability to grow on

Nfb and with nitrogenase activity under aerobic or micro aerobic conditions were

isolated from sugarcane roots, collected from four different agricultural locations in

Granada (Spain). Isolates were Gram negative rods and were identified as Azotobacter

chroococcum and Azospirillum brasilense. It was concluded from the study that

Azotobacter isolates do not have a particular affinity for sugarcane rhizospheres and

that, on the contrary, Azospirillum isolates show specific association and perhaps

endophytic colonization of sugarcane.

Stotzky (1972) examine the role of environment on the inoculation of

Azotobacter strains isolated from different soil samples and their incubation for 8

weeks in optimal environmental conditions. He found there were considerable

variances in N fixation capacities between isolated Azotobacter strains and the soil

where it is located. It was also found that Azotobacter strains add more N in sandy clay

loam soils than that of loam and clay soils. The soil pore system which consists of

various amounts of water and air has to be characterized quantitatively in order to

describe the soil as a habitat for Azotobacter spp. population and their activity. Soil

pore size as determined by soil texture may be as important for the transport of gases

and nutrients.

Different N free media are used for the isolation, cultivation and maintenance

of Azotobacter with different carbon sources. Brown and Burlingham (1968) suggested

the use of starch medium as a selective medium for isolating Azotobacter. Other media

which could be used for the isolation of this organism are Waksman No. 77, Ashby‘s

mannitol phosphate agar medium, Jensen‘s medium and Burk‘s nitrogen free medium.

2.5. Method of inoculation and benefit:-

The use of Azotobacter biofartilizer was started late in 1970s in india. In

transplanted crops like tomato and brinjal, treatment of seedling with Azotobacter

during transplantation resulted in a significant increase in yield of respective vegetables

from 2 to29 percent (tomato) & 1-42 percent (brinjal) (Mehrotra and Lehri, 1971).

Inoculation with Azotobacter sp. complements the symbiotic relationship

between plant roots and AM (Abasicular micoriza) fungi due to its nitrogen fixation,

phytohormones production and phosphate solubilization properties (Kumar et al., 2001,

Narula et al. 1980). The beneficial effects of dual inoculation have been reported by

many workers (Mandhare et al., 1998, Sreeramula et al., 2000, Vassilev et al, 2001) for

certain plant species.

Bagyaraj and Menge (1978) studied the interaction between a mycorrhizal

fungus and Azotobacter and their effects on rhizosphere micro flora and plant growth.

They found larger population of bacteria and actinomycetes in the rhizosphere of

tomato plants inoculated with mycorrhizal fungus Glomus fasciculatum and

Azotobacter chroococcum than uninoculated treatment. Inoculation of G. fasciculatum

increased the population of A. chroococcum in the rhizosphere and maintained the same

for a longer period.

Subba Rao et al.(1993) reported that besides Peat based inoculants other

forms of biofertilizer used are granular soil inoculants where marbles and calcite grains

are wetted by peat based cultures using adhesives. Such granular inoculants could be

broadcasted by aeroplane. Most of these improvements tend to be expensive and their

possible use in developing countries is hence limited.

Peppler and Perlman (1992) reported that there different types of cultures are

used for inoculation but commonly used are agar-based cultures. Agar based cultures

are the quickest way to inoculate plants in small experiments. Azotobater were applied

to seed because this was an easy, convenient way to establish the bacteria in the root

zone of developing seedling.

In USA the normal rate of application is 4.4gm of inoculum per kilogram of

seed regardless of seed size. According to Burton & Curley (1965), they need more

inoculum for effective growth. In USA and Australia, pelleted seeds are often used to

establish legumes and oilseeds in acid soils or to avoid to the hazards of pesticides or

fertilizers (Brock well, 1977 and Burton, 1979). The usual method of pelleting involves

addition of 40% gum arabic or 5% carboxyl methylcellulose to the inoculant slurry

before application to seeds. Besides lime other pelleting agents are as dolomite,

gypsum, bentonite, rock phosphate, talc, charcoal and basic slag have been used to

establish soybean in problem soils (Chonkar et al., 1971).

Bhadauria et al. (2005) conducted experiment on method of inoculation of

Azotobacter culture with different levels of nitrogen on growth, yield and economics of

tomato. They observed application of 75 kg N/ ha along with seedling inoculation with

Azotobacter culture recorded the highest plant height (52.43 cm), branch number

(13.50), leaf number (166.16), number of fruits per plant (23.84) and yield per hectare

(440.26 q). However, plant height, number of branches and leaves per plant were at par

with the application of 100 kg N/ha along with Azotobacter culture. The highest

benefit: cost ratio (2.08) was also observed upon the application of 75 kg N/ha along

with seedling inoculation with Azotobacter culture.

Poi and Kabi (1983) observed that seed inoculation of sorghum significantly

increased fresh weight and N content of pot grown plants. Pod yield consistently

increased by inoculation. The increase was from 18 to 34 % in Hyderabad.

2.6. Azotobacter population and soil environment:-

The Azotobater population in soil varied to a greater extent by different

environmental conditions like soil moisture, temperature, humidity, concentration of

oxygen and carbon di oxide in soil and air, organic matter, presence of different

elements and other chemicals in soil and due to other soil microbes which directly or

indirectly affect the Azotobacter growth and population. Rajasekaran (1998) reported

that the pH of the soil is known to affect the growth and activity of microorganisms.

The selected efficient micro-bacteria were capable of fixing nitrogen over a wide range

of pH from 5.0 to 7.8. There have been a large number of investigations on the effect of

environmental condition on the Azotobacter population and its ultimate effect on plant

growth.

Zafar, Malik and Niemann (1997) observed the effects of different levels of

combined nitrogen (NO 3 – & NH 4

+), pH (5.5–9.0) and salt (NaCl) on nitrogenase

activity of the isolates were determined at various time intervals. All isolates exhibited

nitrogenase activity even in the presence of 5 mmol/l NO 3 – or NH 4

+ in a semi-solid

medium after 24 h of growth. Maximum nitrogenase activity was observed at alkaline

pH and all isolates were able to tolerate up to 3% NaCl in the medium. Studies on N2-

fixing bacteria associated with the salt-tolerant grass, Leptochloa fusca.

Lal and Khanna (1996) found that in winter the activity of Azotobacter was

almost nil whereas on onset of spring and rise in temperature, activity became faster.

Our country comes under tropical zone where the temperature sometimes shoots up

very high. So the viability of Azotobacter is greatly reduced. A favorable temperature

for multiplication of most of species of Azotobater is up to 400C as reported by

Bhriguvanshi and Gangwar (1984).Similarly Day et al. (1978) and Kritovich et al.

(1981) reported that bacterial growth is optimum at 37 to 42 ºC after which there is a

sharp decline. The effect of carbon di oxide‘s concentration in atmosphere was studied

by Thomas et al. (1991). They reported that atmospheric CO2 partial pressures will

enhance nitrogen fixing ability of Azotobacter but that will depend on soil and nutrient

status. Schoroyemeyer et al. (1996) and Zanetti et al. (1998) also reported similar

types of results.

Bilal & Rakhshanda (1990) reported the characterization of Azotobacter and

related diazotrophs associated with roots of plants growing in saline soils and found

that the pH is most prominent factor affecting the population of Azotobacter in

rhizosphere.

Narula&Vasudeva(2006) showed that Azotobacter is characteristically

sensitive to high hydrogen ion concentrations. Their absence is associated directly with

pH. As a rule, environments more acid than pH 6.0 are free of the organism or contain

very few Azotobacter cells. Similarly, the bacteria generally, will neither grow nor fix

N2 in culture media having a pH below 6.0. Beijerinckia spp. do not possess the acid

sensitivity like Azotobacters and they develop and fix N2 from pH 3 to 9.

In soils, Azotobacter spp. populations are affected by soil physico-chemical

(eg. organic matter, pH, temperature, soil depth, soil moisture) and microbiological (eg.

microbial interactions) properties. As far as physico-chemical soil properties are

concerned, numerous studies have focused on the nutrients (i.e. P, K, Ca) and organic

matter content and their positive impact on Azotobacter spp. populations in soils

(Pramanix and Misra, 1955; Bescking, 1961; Jensen, 1965 and Burris, 1969). In

contrast, little information is available on the relationships among Azotobacter spp.

populations, their activities and microbiological properties of soils such as microbial

biomass C, basal soil respiration, and enzyme activities (dehydrogenase, catalase,

glucosidase, urease, phosphatase and sulphatase). Since soil biological properties are

indicators for soil quality, soil health and fertility, examining the relationships between

these parameters and Azotobacter spp. populations have vital role for agricultural

practices and management application.

2.7 Production of plant growth promoting substances

A diverse group of microbes have been found to synthesize phytohormones

including soil, epiphytic and tissue colonizing bacteria. In fact, it has been suggested

that up to 80 percent of bacteria isolated from the rhizosphere can produce IAA (Pattern

and Glick, 1994).

Margaret et al.(1968) showed that cultures of Azotobacter chroococcum strain

A6 were grown for 14 days in a nitrogen-deficient mineral medium, the supernatant

fluid and bacteria extracted and examined by paper partition chromatography with two

solvent systems which separate authentic gibberellin (GA 3) and indolyl-3-acetic acid

(IAA). Gibberellin-like substances were not detected on the chromatograms examined

under ultraviolet (u.v.) radiation, but were detected when chromatograms were cut into

ten equal strips representing a sequence of RF values and the eluates tested in dwarf

pea and lettuce hypocotyl bioassays. Certain eluates applied to the roots of tomato

seedlings also altered the lateral growth of stems, leaves and flowers. The Azotobacter

cultures contained three gibberellin-like substances, of which probably the dominant

was one with an RF value similar to that of GA 3; the other two were not identified.

The average concentration of gibberellin/ml culture was 0.03 pg. GA 3 equivalent. The

gibberellins in Azotobacter cultures probably cause therapeutic effects on plant

development and yield when seeds or roots are inoculated with Azotobacter. Plant

growth may also be affected by synthesis of further gibberellins in the root zone when

the Azotobacter inoculum colonizes developing roots. Of the three gibberellin-like

substances detected in the present work in cultures of Azotobacter chroococcum strain

6th, one with the same RF value as GA I or GA3 was probably the most important.

Although the amount was too small to detect by fluorescence on paper chromatograms,

bioassays readily detected it and suggested that the concentration in 14-days old

cultures ranged between 0.01 and 0-1 pg. GA 3 equivalent/ ml. This amount of

gibberellin-like substance was seemingly enough, when an inoculum of Azotobacter

was added to seeds or roots, to alter the lateral development of tomato plants, possibly

because it was taken up by the seedlings at a critical stage of development, when

vegetative and reproductive primordia were differentiating. However, not all the

gibberellin taken up by the seedlings may have come from the initial inoculum, for

gibberellins may have continued to be synthesized for a short period when the roots

were being colonized by the Azotobacter inoculum which moved from the seed to the

germinating root and multiplied (Jackson & Brown, 1966). Only 14-day Azotobacter

cultures grown in a nitrogen-deficient mineral medium have so far been studied; it has

yet to be determined whether the conditions of cultivation affect the production of

gibberellins by Azotobacter.

Brown, Jackson & Burlingham (1968) have found that after treating tomato

seeds or seedling roots with small amounts (0.5-0.01 pg.) of commercially produced

gibberellins GA 3, the plants responded in the same way as after treatments with 14-

day cultures of Azotobacter chroococcum strain A 6 , had no effect on plant

development, and adding 0.5 pg. IAA with GA 3 had no greater effect on growth than

GA 3 alone. These results indicated that the active substance in Azotobacter culture was

a gibberellin.

Barbara et al.( 1989) reported that the biological significance of cytokinin

production by Azotobacter spp. is not known. As discussed above, the growth-

promoting activity of this organism is commonly attributed to its production of plant

growth substances. There is considerable commercial interest in plant growth regulators

that increase yield, commonly microbial fermentation products which contain

cytokinins among their components. The amount of cytokinin in our cultures was low,

but cytokinin synthesis in the rhizosphere may be influenced by factors from the plant.

Many soil bacteria such as Azotobacter sp,.Azospirillum sp. and Pseudomonas

sp. can be promoted plant growth by production of phytohormon such as

auxin,cytokinin,gibberellins and abisic acacid(Bottini et al. 2004, Safak &Nilfer., 2006)

which can be beneficial to stimulate plant growth and increase plant production.

Brakel and Hilger (1965) showed that Azotobacteria produced indol-3-acetic

acid (IAA) when tryptophan was added to the medium. Vancura and Macura (1960),

Burlingham (1964), and Hennequin and Blachere (1966), on the other hand, found only

small amounts of IAA in old cultures of Azotobacteria to which no tryptophan was

added. Three gibberelin-like substances were detected by Brown and Burlingham

(1968) in an Azotobacter chroococcum strain. The amounts found in the 14-dayold

cultures ranged between 0.01 and 0.1 μg GA3 equivalent/ml.

A study by Govedarica et al. (1993) on the production of growth substances

by nine Azotobacter chroococcum strains isolated from a chernozem soil has showed

that these strains have the ability to produce auxins, gibberelins, and phenols and, in

association with the tomato plant, increase plant length, mass, and nitrogen content.

Abbass and Okon (1993) studied the effect of Azotobacter paspali on plant growth

promotion concluded that treatment of seedling hypocotyls and roots of rapeseed

(Brassica campestris), wheat (Triticum aestivum) and tomato (Lycopersicum

esculentum) with cultures of Azotobacter paspali changed plant growth and

development and significantly increased weight of shoot and roots. Morphological

changes of root tips were observed 5 days after inoculation. After 21 days the main

effect was on the root surface area. Plant growth promotion was dependent on the

inoculum size, indicating that for any given growth condition there is an optimal

number of A. paspali for a positive effect on the plant. Plant growth promotion effects

of A. paspali were similar in morphology to those obtained following Azospirillum

lipoferum or A. brasilense inoculation.

2.8. N fixation by Azotobacter:-

The Azotobacter is an aerobic, heterotrophic, asymboiotic free living nitrogen

fixing bacteria, isolated and described by Beijerinck (1901). In addition to fixing

nitrogen asymbiotically, it is also known to produce plant growth hormones and

fungistatic substances. This organism grows well in nitrogen free medium utilizing

molecular nitrogen for its cell protein synthesis. The dead cells on subsequent

mineralization contribute towards the nitrogen availability of plants.

Azotobacter spp. is free-living aerobic bacteria dominantly found in soils.

They are non symbiotic heterotrophic bacteria capable of fixing an average 20 kg

N/ha/per year. Besides, it also produces growth promoting substances and are shown to

be antagonistic to pathogens. Azotobacter spp. are found in the soil and rhizosphere of

many plants and their population ranges from negligible to 104 g-

1 of soil depending

upon the physico-chemical and microbiological (microbial interactions) properties.

Azotobacter chroococcum is the most prevalent species found but other species

described include A.agilis, A.vinelandii, A. beijerinckii, A.insignis, A.macrocytogenes

and A.paspali (FAO, 1982).

Burgmann et al. (2003) reported that Azotobacter is a heterotroph bacterium

of aerobic character having the capability of fixation of dinitrogen as nonsymbiont.

However; some strains have higher nitrogen fixing ability than others . Haris, (1981)

studied and also addressed the physical properties of differently textured soils in

undisturbed and remolded state and their effect on N fixation by different Azotobacter

spp. strains. Research results showed that the maximum N fixation by Azotobacter spp.

was in coarse-textured (sandy clay loam) soils. The probable reason is the water and air

rapidly penetrates coarse soils with granular subsoil, which tend to be loose when moist

and don‘t restrict water or air movement.

Kader et al. (2002) observed that in addition to nitrogen fixation, Azotobacter

also produces, thiamin, riboflavin, indole acetic acid and gibberellins. When

Azotobacter is applied to seeds, seed germination is improved to a considerable extent,

so also it controls plant diseases due to above substances produced by Azotobacter.

Mishustin (1966) had proposed that Azotobacter inoculants acted not by

stimulating N‘ ñxation, but by affecting plant growth through gibbeïellin or

cytotokinin-like substances. However, young seedlings can absorb such growth

regulators produced by A. puspali . It does not, however, exclude the possibility .that

old, mature roots can fix N2 or that a wide range of other substances of bacterial origin

might effect plant growth (Lynch J.M. White, 1977).

Kizilkaya(2009) made study with the objectives to count and culture

Azotobacter spp. in sampled soils, to determine the nitrogen (N) fixing capacity by

Azotobacter spp. in pure culture and different soils, and to explore the relationships

between N fixation capacity of Azotobacter spp. and microbiological properties of soils

in Northern Anatolia, Turkey. Statistically significant relationships were found between

the population of Azotobacter spp. in soils and microbial biomass C (Cmic ),

dehydrogenase (DHA), b-glucosidase (GA), alkaline phosphatase (APA) and

arylsulphatase (ASA) activities. However, relationships between the population of

Azotobacter spp. and basal soil respiration (BSR), urease (UA) and catalase (CA)

activities were insignificant. The N fixation capacities of native 3 day old Azotobacter

chroococcum strains added to Ashby Media varied from 3.50 to 29.35 μg N ml-1 on

average 10.24. In addition, N fixation capacities of Azotobacter spp. strains inoculated

with clayey soil, loam soil, and sandy clay loam soil during eight week incubation

period were 4.78-15.91 μg N g-1, 9.03- 13.47 μg N g-1 and 6.51-16.60 μg N g-1,

respectively. It was concluded that the most N fixation by Azotobacter spp. was in

sandy clay loam soils.

2.9. Yield enhancement by Azotobacter inoculation

The inoculation with Azotobacter alone significantly increased the root depth,

shoot height, fresh and dry weights of roots and shoots (P = 0.05) and root/shoot of

fresh and dry weights .The beneficial effect of Azotobacter on tomato plants might be

due to nitrogen fixation and secretion of a high quantity of plant growth regulators

(Azcon & Barea 1975, El-shourbagy et al., 1979& EL-shanshoury,1979).

Okon (1985) and Subba Rao (1982) found that significant impact of

Azotobacter inoculation on biomass, yield and nitrogen economy of different crops

grown under field and pot conditions. Three pot experiments were conducted on tomato

(Lycopersicon esculentum) cv. Castle Rock during the growing seasons of 1998/99,

1999/2000, and 2000/2001 to test its response to inoculation with selected single or

multi-mixed strains compared with the uninoculated control. In the first season

(1998/99), with the exception of Pseudomonas fluorescens, all single inoculation

treatments with the selected strains Azotobacter chroococcum strains 5 and 11,

Azospirillum lipoferum and Bacillus polymyxa [Paenibacillus polymyxa] produced

significant or highly significant increases in shoot and root fresh and dry weights of

tomato plants. Inoculation with either of Azotobacter chroococcum strains 5 or 11

scored the highest shoot and root dry weights, number of branches and fruit yield per

plant. The mixed co-inoculation treatments with double of the selected strains showed

superior effects on plant growth and yield in two cases: first when Azotobacter

chroococcum strain 11 was mixed with B. polymyxa and second when Azotobacter

chroococcum strain 5 was mixed with Azospirillum lipoferum. In the second and third

seasons, 1999/2000 and 2000/01, the results of the single inoculation treatments

showed similar trends to those recorded in the first season. Fruit yield in both seasons

was significantly increased by single inoculation with Azotobacter chroococcum and B.

polymyxa as well as by co-inoculation with their mixture. The increases in fruit yield

per plant in the second season were 55, 63 and 39%, respectively, for the above

inoculation treatments compared with the uninoculated control. In the third season, the

corresponding values were 36, 28 and 36%, respectively. In the third season, co-

inoculation of Azotobacter chroococcum plus Azospirillum lipoferum increased fruit

yield by 55.5% compared with the uninoculated control. Generally, the results of the

three seasons showed that the mixed inoculation treatments, except those with

Pseudomonas fluorescens, were usually more promotive than the single inoculation

treatments. It is also indicated that co-inoculation of Pseudomonas fluorescens with

either Azotobacter chroococcum or Azospirillum lipoferum prevented their promotive

effect. (Badawy et al., 2003). Bowen and Rovira (1999) reviewed the biology of the

rhizosphere and its management to improve plant growth, summarising their interest in

this area from an agronomic point of view. Their review commenced with the increases

in growth when tomatoes were inoculated with Azotobacter (Brown et al., 1964).

Gajbhiye et al. (2003) studied the effect of Azotobacter & P.S.B.on the growth and

yield parameter of tomato. They concluded that Azotobacter was more effective than

phosphobacteria (P.S.B.) in the improvement of plant height, number of primary

branches per plant, number of fruits per plant, weight of fruits per plant, fruit size and

yield. Azotobacter in combination with the recommended fertilizer rate was superior in

the enhancement of the aforementioned parameters of tomato.

Azotobacter chroococcurn has long been used in the Soviet Union to inoculate

seeds or roots of crop plants, and increases in yields from this practice have been

reported (Mishustin & Naumova, 1962). Recent pot trials and field trials outside the

Soviet Union have also shown that frequently plant growth was altered and sometimes

yield increased. Jackson et al. (1964) found that inoculation with Azotobacter

accelerated the stem and leaf growth of tomato and shortened the time between bud

appearance and petal fall.

Inoculation with Azotobacter can increase cotton yield by 15–28%

(Iruthayaraj, 1981) as a result of BNF, production of antibacterial and antifungal

compounds, growth regulators and siderophores (Pandey and Kumar, 1989). Patil and

Patil (1984) observed that seed inoculation with A. chroococcum plus 50–100 kg urea-

N ha1 gave higher cotton dry matter yield, N-uptake and soil N-content than those

obtained with N alone (50–100 kg urea- N ha1) in greenhouse conditions using non-

sterilised soils.

Sharma and Thakur (2001) conducted experiment on Azotobacter and

nitrogen to find out the effect on growth and yield of tomato. They observed that

application of Azotobacter significantly increased plant height, number of branches and

fruits per plant, fruit yield per plot, yield per hectare, nitrogen uptake at the flowering

stage and root biomass. Among treatment combinations, the maximum yield per

hectare was obtained when Azotobacter was applied in combination with 100 kg N/ha.

Begum (1998) made study to find out the response of tomato crop to Azotobacter

inoculation. She found fruit yields were highest from seedlings raised from Azotobacter

treated seeds given 150 kg N/ha into different splits at planting, and 30, 45 and 60 days

later. Control plants (no seed treatment, no N fertilizer) yielded 13.39 t/ha and those

dipped in Azotobacter inoculum 20.35-25.97 t/ha, depending on N rate.

Amer et al. (2003) experimented with three biofertilizers and four levels of

mineral fertilization on the yield and quality of tomato. They found the combined

application of mineral fertilizers and biofertilizers significantly increased the vegetative

growth, total fruit yield and fruit quality. The application of Azotobacter, Azospirillum

and Bacillus megatarium in tomato production in newly reclaimed sandy soils can

reduce the required amount of mineral fertilizer without reducing the productivity or

quality of tomatoes, thus reducing the high cost of chemical fertilizers and pollution of

the agriculture environment.

2.10. Field Response of Azotobacter :

Azotobacter chroococcum has been used by farmers to meet partly the

nutritional requirements for the better production of vegetable crops besides other

cultivated crop. Initial trials conducted in USSR and India had indicated beneficial

effect of Azotobacter on various vegetable crops like potato , beet root, tomato and

cucumber etc. In soviet union, Sheloumova (1935) reported an increase in yield of beet

root, corn and potato from 16 to 18 percent and Dorosinsiky (1964) obtained higher

yield of potato ,cabbage and tomato by 12.4, 75.0 & 28.0 percent respectively due to

Azotobacterization. Several mutliocational trial were conducted during 1956 and 1957

at scientific research institute , brodil , Czechoslovakia on the effect of Azotobacter on

potato yield . Result indicated an increase in potato yield by seed inoculation from 5.0

to 17.0 percent in first yield and 8.0 to 34.0 percent during next year. Mishustin and

Shilnikova (1969) reported that tuber bacterization with Azotobacter increase the yield

from 14.0 to 42.0 q/ha (12.8-25.3%). Seedling application with A. chroococcum also

increased the yield of cabbage, cauliflower and tomato by 19.0, 40 and 28-33.8 percent

respectively over control. The early ripening of tomato fruit and improvement in the

yield of first picking of the cucumber were also observed . The plants inoculated with

the Azotobacter were found less affected by disease, ripened sooner and yielded better.

El-Shaushuny et al. (1989) reported that Azotobacterization of tomato- seedling

enhanced root infected by Glomus fasciculatum and stimulated plant growth that

resulted in to an increased N,P,Ca & Fe in shoot compred to control plants.

Azotobacter species (Azotobacter vinelandii and A. chroococcum) are free-

living, aerobic heterotrophic diazotrophs that depend on an adequate supply of reduced

C compounds such as sugars for energy. Their activity in rice culture can be increased

by straw application (Kanungo et al., 1997), presumably as a result of microbial

breakdown of cellulose into cellobiose and glucose. Yields of rice in field trials

increased significantly (at 5% probability level) up to 0.9 t ha-1 (20% increase) with

applications of Azotobacter (Yanni and El-Fattah, 1999). The estimated N

accumulation by rice plant increased up to 15 kg ha-1 due to Azotobacter inoculation

(Yanni and El-Fattah, 1999). As 15N was not used as tracer, it is not possible to say

how much of the accumulated N was a result of BNF. Brown and Burlingham (1968)

and Eklund (1970) have demonstrated in their papers that the presence of Azotobacter

chroococcum in the rhizosphere of tomato and cucumber is correlated with increased

germination and growth of seedlings.

A study by Govedarica et al. (1993) concluded that the production of growth

substances by nine Azotobacter chroococcum strains isolated from a chernozem soil

has showed that these strains have the ability to produce auxins, gibberelins, and

phenols and, in association with the tomato plant, increase plant length, mass, and

nitrogen content.

Jackson et al. (1964) found that accelerated growth of tomato stem with

inoculation of Azotobacter. Mishutin (1966) demonstrated that bacterial fertilizers

slightly improved yield of a wide range of crop plants, especially vegetable. The yield

increases have been reported up to 28.56, 18.25, 19.33 and 55 per cent in case of

tomato, potato, cabbage and cucumber respectively.

Singh and Singh (1992) from their studies carried out at Faizabad, concluded

that plant height and number of branches per plant increased significantly and

maximum values were obtained at 125 kg N ha-1 in tomato cv. Pusa Ruby. They also

reported that fruits per plant and marketable fruit yield (q ha-1) increased in linear

fashion with increasing nitrogen levels.

Mohandas (1987) and EL-Shanshoury et al. (1989) observed that Bio-

fertilizer application significantly increased the nitrogen uptake in tomato at growth

stage. This may be because of better nitrogen fixation as result of accelerated bacterial

activity and better root system which might have resulted in more nitrogen

accumulation in tomato shoots. From the results of the experiment it is clear that bio-

fertilizer shows better results as compare to that of the inorganic fertilizers. The main

advantage of bio-fertilizer is that it does not pollute the soil and also does not show any

negative effect to environment and human health.

Taiwo (2004) observed the performance of Azotobacter croococcum in

enhancing growth and fruit production of tomato (Lycopersicum esculentum Mill.) in 3

greenhouse experiments and a field study. The first experiment assessed the appropriate

method of inoculation while the 2nd

study determined the relationship between

increases in the volume of inoculum and the yield parameters. Experiment 3

investigated the required number of doses needed for optimum yield.. The field study

attempted to validate positive responses obtained in the greenhouse. Seedling

inoculation and urea application at 2 weeks after transplanting (WAT) led to increases

ranging from 50% to over 160% in all the parameters assessed when compared with the

un-inoculated and unfertilized control. Soil and seed inoculation did not significantly

(p=0.05) impact positively on the height, stem girth as well as the number of fruits of

the test crop when compared with the control. There was a positive correlation between

increase in inoculum rates and plant height and girth with the regression coefficient (r2)

ranging from 0.74 to 0.96. Application of 2-3 doses of 50ml of the inoculum to the

seedling enhanced plant height and stem girth especially from 6WAT. Application of 2

doses at 2weeks interval gave about 10% increase in the number of fruits over the 2

dose-application. In the field, no significant (p=0.05) differences were obtained in plant

growth and yield when either the Azotobacter inoculum or urea was used. Each of the

treatments however, increased the growth and fruit yield of tomato when compared

with the control. Nitrogen fertilization promoted growth and yield of tomato. The use

of Azotobacter croococcum inoculum was an effective biological management option

in tomato fertilization programme.

Mahato et al.(2009) evaluated the response of bio-fertilizer and inorganic

fertilizer on germination and growth of tomato plant. They revealed that Azotobactor as

bio-fertilizer reported better than inorganic fertilizer in relation to seed germination and

all plant growth parameters. In the present study application of bio-fertilizer resulted

increase of shoot length and more number of leaves per plant. Similar observations

were observed by Martinej et al. (1993) in case of tomato. Bio-fertilizer application

significantly increased the nitrogen uptake in tomato at growth stage. This may be

because of better nitrogen fixation as result of accelerated bacterial activity and better

root system which might have resulted in more nitrogen accumulation in tomato shoots.

EL-Shanshoury et al. (1989) while working with Azotobacter in tomato have also

obtained similar results. From the results of the experiment it is clear that bio-fertilizer

shows better results as compare to that of the inorganic fertilizers. The main advantage

of bio-fertilizer is that it does not pollute the soil and also does not show any negative

effect to environment and human health.

Field trails with Azotobacter in USSR and India

Crop % increase in yield Reference (s)

Wheat, Rye, Oat, Barley,

Maize, Cotton.

10-17 Mishustin and Shilnikova,

1969

Tomato, Potato, Cabbage

& Sugarbeet

10-28 Mishustin and Shilnikova,

1969

Pea 60 Sundra Rao et al.,1963

Cabbage 33.5 IARI , 1963-64

Rice 17.7 Manna et al.1962

Maize (Fodder) 59.0 Shende, 1972

The effect of Azotobacter chrococcum inoculation in field on the yield of crop

Crop Location of

field trials in

India

Without

Azotobacter

With

Azotobacter

C.D.at

5%

% Increase

due to

Azotobacter

Sorghum

(kg/ha)

Pali 1280 1400 122 9.3

Dharwar 2360 3260 1056 38.1

Maize

(kg/ha)

I.A.R.I. 780 1340 480 71.1

Dharwar 320 4370 990 36.5

Cotton Surat 1254 1339 241 6.7

Indore 366 401 104 9.5

Khandwa 556 708 165 20.6

Source- Shende, 1972

CHAPTER-III

MATERIAL AND METHODS

The present study entitled ―Selection of effective Azotobacter isolates for

Tomato (Lycopersicon esculentum Mill.)‘‘ was carried out during 2010-11 at the

Department of Agricultural Microbiology, College of Agriculture, Raipur (C.G.). A

brief description of the materials used and the techniques adopted during the course of

study are presented in this chapter.

(A). LOCATION AND CLIMATE

3.1.1 Location of Experimental site

The Experiments were conducted in Soil Microbiology Laboratory and Glass

House of Department of Agricultural Microbiology, College of Agriculture, Raipur in

order to select effective Azotobacter isolate(s) for tomato crop.

3.1.2 Geographical situation

Raipur is situated in central parts of Chhattisgarh and lies at latitude, longitude

of 21o16 N, 81

o36 E, respectively with an altitude of 298.56 meters above mean sea

level.

3.1.3 Climate

Raipur the place of investigation comes under dry-sub humid to semi arid

agro climatic region under rice zone of the state. Out of the mean annual rainfall of

1200-1300mm, about 85% is received during third week of June to mid September.

Soil surface temperature of this region crosses 60oC, air temperature touches to 48

oC

and humidity drops down to 3 to 4% during summer season (Anonymous, 1996 and

Gupta et al., 2000 and 2002).

Ist Stage Screening:

This study was aimed at identifying effective Azotobacter isolates for tomato

growers of Chhattisgarh region. Under this Ist stage screening N-fixing capacity of

different Azotobacter isolates was estimated under in vitro condition in Soil

Microbiology Laboratory, Department of Agricultural Microbiology, CoA, IGKV

Raipur. In this direction forty Azotobacter isolates were collected from the Microbial

Culture Bank of Department of Agricultural Microbiology, College of Agriculture, and

Raipur and tested for their efficiency of nitrogen fixation in Jensen‘s N-free liquid

medium.

3.2.1 Testing of N-fixing capacity of different Azotobacter isolates:

Background of Azotobacter isolates

Forty local Azotobacter isolates and standard Azotobacter IARI isolate (standard

check) were collected from Microbial Culture Bank of Department of Agricultural

Microbiology, CoA, Raipur and were taken for the study.

Preparation of Broth Culture

Jensen‘s (1954) broth medium containing Sucrose: 20 g, K2HPO4 : 1.0 g,

MgSO4 : 0.5 g, NaCl : 0.5 g, FeSO4 : 0.1 g, Na2MoO4 :0.005 g, CaCO3 2.0 g, Agar :

15g, and distilled water 1000 ml was prepared and pH was adjusted to 7.0±0.2. Fifty

ml of Jensen‘s broth medium was transferred into each 150ml capacity conical flask

and plugged carefully with cotton wool. The conical flasks were sterilized at

20lbs/inch2 pressure for 30 minutes. After cooling, each conical flask containing broth

medium was inoculated aseptically by a loopful of individual isolate of Azotobacter.

The flasks were shaken on a rotatory mechanical shaker for a week at 28± 2oC .

Efficacy of Nitrogenase

To determine the efficacy of nitrogenase enzyme of each isolate, amount of

nitrogen fixed by Azotobacter isolates was estimated by Microkjeldhal method given

by Jackson (1967). Triplicate samples were used for each isolate and standard check

including control.

After seven days of incubation (28-300C) the culture broth was homogenized.

Five ml of homogenized culture broth was withdrawn and digested with five ml

concentrated H2SO4 and five gram of digestion catalyst (K2SO4 and CuSO4 in ratio of

10: 1) until the contents became clear. After cooling, five ml of aliquot was transferred

to Microkjeldhal distillation unit. An aliquot of 10ml of 40 percent NaOH was added

and steam distilled. Ammonia evolved was collected in two percent boric acid (10 ml)

with mixed indicator (83.3 mg bromocresol green + 16.6 mg methyl red indicator

dissolved in 10 ml of 95 percent alcohol) and back titrated against 0.005N H2SO4.

Using titre value and the formula of one ml of 0.005N H2SO4 = 0.00007g of N, the

nitrogen fixed in vitro was calculated and expressed in N fixed/ gm of sucrose supplied.

IInd Stage screening:

3:3:1 Glass house Experiment:

The IInd stage screening was conducted in Glass House of Department of

Agricultural Microbiology, College of Agriculture, Raipur in pots containing, 8.5 kg

experimental soil. During this experiment, seven top performing isolates were

compared with the same standard check and three uninoculated control contained

100:60:80, 115:60:80 and 120:60:80 kg N, P2O5 and K2O, respectively. The number of

treatments was eleven replicated thrice in completely randomized design. Tomato C.V.

Pusa Rubi was taken as a test variety. Other details are as follows:

3.3.2 Treatment:

The following treatments were set up for II stage screening in glass house condition.

TREATMENTS ISOLATE No. + FERTILIZER

DOSES (N: P: K)

T1 Azotobacter isolate No. : AZOT-B-35 +

100:60:80


100:60:80


100:60:80


100:60:80

T5 Azotobacter isolate No. : AZOT-B 123 + 100:60:80


100:60:80


T8 Standard Check Azotobacter : IARI ,S.C. +

100:60:80

T9 Uninoculated control : (C-I) + 120:60:80

T10 Uninoculated control : (C-II) + 115:60:80

T11 Uninoculated control : (C-III) + 100:60:80

Analysis of experimental soil:

The physical and chemical characteristics of the experimental soil are

mentioned in Appendix-III

3.3.3 Determination of soil pH:

Twenty gram of soil was taken in a clean 100 ml beaker and 50 ml of distill

water was added to it. The suspension was stirred intermittently for thirty minutes or

continuously for ten minutes. The pH was recorded using pH meter.

For standardization of the pH meter, the instrument was switched on 15 - 20

minutes prior to estimation to warm up. The temperature adjusted to room temperature

by control knob. Then electrode was dipped in standard buffer solution of pH 4.0 and

the buffer control knob was set. The electrode was removed and washed with a jet of

distilled water and then the electrode was dipped in pH 7.0 or 9.2 standard buffer

solutions and then the instrument was calibrated. When the calibration was found

satisfactory, electrode was taken out and rinsed with distilled water and then electrode

was inserted in to soil suspension and the pH was recorded.

3.3.4 Electrical conductivity:

Electrical conductivity was determined in soil water suspension (1:2.5) by

Conductivity Bridge as described by Jackson (1973).

For this determination twenty gram of soil was weighed and taken in a 100ml

beaker. 50 ml distilled water was added and suspension was stirred intermittently for 30

minutes and allowed the suspension to settle for about one hour then measured EC in

supernatant solution using EC Bridge.

3.3.5 Determination of Available N:

Available N was determined by alkaline KMnO4 method of Subbiah and Asija

(1965) with slight modification. Twenty gram of soil sample was taken in one litre

boiling flask and 200 ml distilled water, 100 ml of 0.32 percent KmnO4 and 100 ml of

2.5 percent NaOH were then added in sequence. The flask was connected to the

condenser immediately after adding NaOH and the content was boiled on heater to

collect about 150ml distillate in 10 ml boric acid solution containing mixed indicator

(Bremmer, 1965). Ammonium-N in distillate was determined by titrating against 0.005

N H2SO4 (Bremmer, 1965).

3.3.6 Determination of Available P:

Soil phosphorus was extracted by 0.5M NaHCO3 as described by Olsen et al.

(1954) and phosphorus in the extract was determined by stannous chloride method.

Exactly 2.5 gram of soil was taken in a conical flask of 250 ml. to it one gram of

activated charcoal (Darco-G-60) was added. Then 50 ml of 0.5 M sodium bicarbonate

was added followed by shaking on a mechanical shaker for 30 minutes. The mixture

was filtered through whatman No.1 filter paper and 5 ml of this filtrate was pipetted in

to a fifty ml volumetric flask. Five ml of chloromolybdic acid was added followed by

shaking it slowly. Then it was allowed to stand for 5 minutes and then diluted to 40 ml.

then 1 ml of stannous chloride working solution was added and followed by shaking it

immediately and finally the volume was made up to 50 ml. After 10 minutes the

intensity of blue colour of the solution was read at 660nm using red filter in

spectrophotometer. The concentration of P in solution was found out by referring to a

standard curve.

3.3.7 Determination of Available K:

Potassium was estimated by flame photometer (Hanway and Heidel, 1952). Ten

gram air dried soil was taken in to conical flask. To it 50 ml of 1.0 N neutral

Ammonium acetate solution was added. The flask was shaken for 10 minutes. The soil

suspension was filtered through whatman No.40 filter paper. The soil was then leached

with an additional 50 ml of ammonium acetate solution. The standards of K were fed to

the flame photometer and the readings were noted down using K-filter. The standard

curve of the flame photometer reading was drawn against concentrations. The

ammonium acetate extract of soil was fed and the flame photometer readings were

noted down. Finally the concentrations were found from the standard curve.

3.3.8 Microbial analysis:

Microbial analysis of soil was done by serial dilution plating method (Subba

Rao, 1988). Soon after sampling, the samples were kept in polythene bags to prevent

moisture loss and were properly tagged, sealed and stored in refrigerator for

quantitative estimation of Azotobacter. For counting, serial dilutions of soil samples

were done by taking 1 gm of soil in 9 ml of sterilized water in a dilution tube

(Tuladhar, 1983). Jensen‘s agar media was used for enumeration of Azotobacter. After

counting of colonies, Azotobacter population was calculated on the basis of per gm of

soil using following formula (Schmidt and Caldwell, 1967).

Number of bacteria per gm of oven dry soil:

No. of colony forming units X Dilution

= ---------------------------------------------------------------

Dry weight of 1 gm moist soil X aliquot taken

The operations of making serial dilutions, inoculation, setting of plates with

appropriate media was done in sterilized atmosphere of laminar flow.

3.3.9 Pot preparation:

The medium used for growing tomato crop was soil (Vertisol) which was well

air dried and processed to good physical condition ideal for tomato growth. This soil

was filled in cement pots of capacity 12 kg at the rate of 8.5 kg soil per pot.

3.3.10 Seed treatment:

Healthy seeds of tomato (var. Pusa Rubi) were taken for experimentation. Just

before sowing, healthy seeds of tomato were treated with Thiram @ 3 gm/kg of seed.

3.3.11 Nursury Preparation:

About two hundred healthy, uniform sized, fungicide treated seeds of tomato

were sown in three cemented pots containing 8.5 kg soil for the development of quality

seedlings of tomato. Timely and uniform irrigation were provided to all the pots. The

nursery of tomato were maintained upto 20 days after emergence of seedlings as per the

standard procedure for tomato seedling preparation

3.3.12 Inoculum preparation:

Each Azotobacter isolate was inoculated to 25 ml nutrient broth in 50 ml conical

flask and incubated at 28 ± 20C for 48 hours. This broth culture was then used for the

purpose of seedling inoculation.

3.3.13 Seedling inoculation:

For root inoculation of seedling, mature broth of Azotobacter was diluted with

sterilized aqueous 0.5% sugar solution in such a way so that each and every seedling

received 106 viable cells of Azotobacter. After dipping of 20 days old seedling in this

broth they were transplanted in pots as per treatment. The control pots received same

amount of nutrient broth without Azotobacter population.

3.3.14 Application of fertilizer:

Nitrogen (N), Phosphorus (P2O5), and Potassium (K2O) were applied at the rate

of hundred to hundred twenty kg N (as per treatment) , sixty kg P2O5 and eighty kg K2O

per hectare through urea [Co(NH2)2], monocalcium phosphate [Ca(H2PO4).2H2O] and

potassium sulphate [K2SO4], respectively. Full dose of Phosphorus and Potassium was

applied as basal at the time of transplanting of seedlings. The Nitrogen was applied in

thee splits, 1/3rd

dose during transplanting and rest in two splits at 15 and 45 days after

transplanting.

3.3.15 Transplanting:

24 hours before sowing pots were irrigated with water. 20 days old seedlings

were transplanted in pots. Transplanting was done on 28/10/2010. Two seedlings per

pot were maintained.

3.3.16 Care after sowing:

Time to time uniform irrigation to all pots was provided. Weeding was done

timely so as to let the plant grow without competence for space and nutrients.

3.3.17 Plant protection:

Monocrotophos at the rate of 0.2% was sprayed for the control of insect when

required.

3.3.18 Harvestng of crop:

Harvesting of crop was done after 90 days of transplanting i.e. on 27/01/11.

Observations recorded during II stage screening:

1. Heights of the shoots:

Height of the plants were recorded at different days interval viz. 30, 60, and 90

days after transplanting (DAT) and expressed in centimeters per plant.

2. Fruit Yield:

As the ―Pusa Rubi‖ variety has the indeterminate fruiting behaviour and fruits

appeared throughout its growth phase so number of fruits was carefully counted and

their weight was recorded.

3. Dry weight of the fruits and shoots:

The fruits were harvested after its ripening and the shoots were harvested at

maturity i.e. 90DAT. They were then dried at 650C to get a constant weight and their

weight expressed in grams per plant. Biomass accumulated by fruits and shoots of the

plants recorded separately.

4. Nitrogen accumulation study:

The oven dried shoot samples were ground in stainless steel grinder for

subsequent chemical analysis. The ground samples were stored in envelopes and re-

dried before analysis.

The nitrogen content in the plant samples was estimated by Micro-kjeldhal

method as described by the Jackson (1973) using Gerhardt auto digestion and

distillation system (Vapodest-30).

5. Enzymatic study:

Soil samples were collected from each pot at 30DAT for estimation of

Dehydrogenase activity. The Dehydrogenase activity indirectly shows the microbial

density in soil

The procedure to evaluate the dehydrogenase activity of soil described by

Lenhard (1956) in which 1gm air dried soil sample was taken in a 15 ml airtight screw

capped test tube. 0.2 ml of 3% TTC solution was added in each of the tubes to saturate

the soil. 0.5 ml of distilled water was also added in each tube. Gently tap the bottom of

the tube to driven out all trapped oxygen so that a water seal was formed above the soil.

No air bubbles were formed that was ensured. The tubes were incubated at 37˚C for 24

hrs. Then 10 ml of methanol was added. Shake it vigorously and allowed to stand for 6

hrs. Clear pink coloured supernatant was withdrawn and readings were taken with a

spectrophotometer. The amount of TPF formed was calculated from the standard curve

drawn in the range of 10 µg to 90 µg TPF/ml.

6. Anti fungal study:

Antifungal activity of each Azotobacter isolate was checked against Fusarium

oxysporium by spot plate method including standard check ( Kaur and Seema, 2002).

Azotobacter isolates were spreaded over the plate with modified Martin‘s medium

(Appendix-III). Fungal disc ( Fusarium oxysporium) of 7.00 mm dia was then placed in

the center of plate already inoculated with Azotobacter isolates. Fungal disc without

Azotobacter inoculation servred as control. Each plate was replicated four times. Plates

were then incubated at 28±20

C for 96 hours and observed for the radial growth of

tested pathogenic fungus.

3.9 Statistical Analysis:

All the pre and post harvest observations were recorded and tabulated in a

systemic manner. The final observations were statistically analyzed by completely

randomized design (Panse and sukhatme, 1978).

CHAPTER-IV

RESULT AND DISCUSSION

The investigation was conducted at the Department of Agricultural

Microbiology, College of Agriculture, Raipur, Chhattisgarh during the year 2010-11.

It comprises of (i) Preliminary screening of forty local Azotobacter isolates on the basis

of their nitrogen fixation capacity in liquid medium comparing with standard check of

Azotobacter under in vitro condition (ii) Pot experiment with natural soil for second

stage screening of superior local Azotobacter isolates for tomato crop (iii) Enzymatic

activity of different local Azotobacter isolates in soil and (iv) Evaluation of promising

Azotobacter isolates for their antifungal property for disease suppression. The results

obtained from these studies are depicted and discussed in this chapter.

Background of the study:-

The present investigation is an important part of study which was carried out in

the Department of Agricultural Microbiology College of Agriculture, IGKV, Raipur in

order to develop location specific effective Azotobacter biofertilizer for tomato crop

grown under climatic condition of Chhattisgarh. Initially soils of Chhattisgarh have

shown the actual need of crop beneficial bacterial inoculations. Then after under this

present investigation, forty Azotobacter isolates were collected from the microbial

culture bank of Department of Agricultural Microbiology, College of Agriculture,

Raipur to select out effective Azotobacter isolate for production of Azotobacter

biofertilizer for tomato growers of Raipur district of Chhattisgarh. Similarly Kakkar

(2008) conducted isolation-screening experiment and selected effective location

specific Azospirillum isolate for mustard on the basis of BNF parameters.

4.1: Preliminary screening of Azotobacter isolates :-

Nitrogen fixing efficiency of Azotobacter isolates :

The nitrogen fixing ability of local Azotobacter isolates and standard check

was tested for initial screening of the isolates. For this purpose Azotobacter isolates

were grown on N-free Jensen‘s liquid medium (Appendix-I) for seven days (Plate 1a)

and then tested for their N-fixing efficiency (Plate 1b). The results obtained from the

above study are presented in Table 4.1,Fig. 4.1 and Appendix- II.

The range of nitrogen quantity fixed in the N-free Jensen‘s liquid medium

varied from 2.35 to 13.45 mg N/gm of sucrose (0.0047 to 0.0269 % N) after seven days

of incubation. Three local Azotobacter isolates i.e. AZOT-B-33, 32 and 18 were found

at par with standard check (standard Azotobacter IARI isolate). Among all isolates,

isolate number 33 fixed maximum quantity of nitrogen in the medium i.e. 13.45 mg

N/gm sucrose (0.0269% N), followed by isolate No.32 which fixed 13.15 mg N/gm

sucrose (0.0263% N) after seven days of incubation. The standard check released

13.10 mg N/gm sucrose( 0.0262 % N)after seven days of incubation. Agrawal and

Singh (2002) also conducted similar type of experiment to select effective Azotobacter

strains by their nitrogen fixing capacity, growth and survival under stress environment.

In recent years, great attention has been dedicated to study the role of soil

microorganisms that play in the dynamics of nitrogen (N), particularly those able to fix

nitrogen from atmosphere (Dobriener and Day, 1976). These microorganisms are

bacteria that inhabit the rhizosphere (Barea and Azcon, 1975 and Bowen and Rovira,

1999). The mechanisms involved in the microbial N fixation are the production of

organic acids and the release of protons to the soil solution (Weniger and Veen, 2004).

The results of this study are in line with the studies done by Boddey and

Dobriener, 1982; Okon et al., 1983; Gibson et al., 1987; Palaniappan, 1992 and

Ranjitha, 2000.

4.2 : Second stage screening of promising isolates:-

Based on the results of N- fixing capacity of local Azotobacter isolates, seven best

local isolates were selected out of the forty isolates. In this connection these seven

Azotobacter isolates and the standard check were further tested for their BNF efficiency in

respect of plant height, biomass accumulation, fruit yield and nitrogen accumulation by

tomato plants (Plate 2). The isolates were inoculated to the tomato seedlings and were

supplemented with N dose of 100 kg N / ha. The performances of the isolates were

compared with N dose of 100, 115 and 120 kg N/ha without any inoculation, which were

taken as control. In the same experiment, standard check with same dose of nitrogen (100

kg N / ha) was used to compare with the influence of local Azotobacter isolates on tomato

plants.

4.2.1 : Plant height study :-

The data pertaining to plant height study is presented in Table 4.2. and Fig.

4.2 .Under this study the tomato plants were allowed to grow up to ninety days after

transplanting (DAT) under uniform conditions of green house. The plant height was

measured at 30, 60 & 90 DAT. It is apparent from the data that plant height of tomato

crop inoculated with different local isolates and standard check of Azotobacter did not

vary significantly from each other at 30 DAT of the tomato seedlings (Plate 3a). The

application of higher doses of nitrogen did not show any significant effect on plant

growth in this stage of crop.

At 60 DAT there was significant variation was observed in the plant height

inoculated with different local isolates and standard check, compared with controls

(Plate 3b). At 60 DAT, maximum plant height (53.32 cm) was observed in the plants

grown with 100:60:80::NPK and local isolate AZOT-B-33, followed by treatment

100:60:80 NPK + standard check (51.25 cm). Minimum plant height 40.25 cm was

recorded in the treatment CIII i.e. 100 kg N. As compared to CIII, significantly

highest plant height was observed in AZOT-B-33 with 100:60:80 NPK level, followed

by standard check at the same level of nitrogen. Other local isolates 18, 32, 39 and 123

also increased the plant height significantly over CIII (100:60:80), however the plant

height of all the Azotobacter isolates including standard check was found at par with

the CI (120:60:80NPK)

Similarly at 90 DAT plant height significantly increased from 63.68 cm

(CIII) to 79.67, 77.43,76.37 and 71.97 cm due to inoculation with isolate AZOT-B-

33,32, standard check and AZOT-B-39, respectively. Maximum plant height was

recorded with isolate number AZOT-B-33, which was found at par with treatment CI.

Minimum plant height was recorded in case of control III, that was 63.68 cm. All the

local Azotobacter isolates and standard check with 100:60:80 NPK level showed at par

plant growth with control I (120:60:80 NPK)

The inoculations with different Azotobacter isolates were highly effective in

increasing the height of plants. Mahato et al. (2009) observed that application of

Azotobacter increased the shoot length and more number of leaves per plant. This

observation was also in line with that of Martinej et al. (1993) and Umar et al. (2009)

who clearly mentioned that application of Azotobacter resulted increase of shoot length

and more number of leaves. Selvarathi et al. (1010) also mentioned that addition of 3%

Azotobacter in the substrate increased the shoot length of tomato plants by 77%. The

beneficial effect of Azotobacter on tomato plants might be due to nitrogen fixation and

secretion of a high quantity of plant growth regulators (Azcon & Barea, 1975; El-

Shorbagy et al., 1979 and El-Shanshoury, 1979).

4.2.2 : Fruit yield study :-

Fruit number :

The data on the influence of different local Azotobacter isolates and different

levels of nitrogen on fruit number of tomato is presented in Table 4.3.

The data related to above parameter revealed that inoculation of tomato

seedlings with local Azotobacter isolates and standard check with NPK level of

100:60:80 significantly increased the fruit number per plant over control C-III

(100:60:80). Maximum number of fruits per plant was recorded due to inoculation of

local isolate AZOT-B-33 (22.90) followed by control C-I (120:60:80 NPK) (20.86) and

standard check inoculated plants (20.83). The number of fruit increased significantly in

plants due to inoculation with local local isolate AZOT-B-33 over standard check at the

same level of nitrogen (100kg N) and also over higher nitrogen doses, i.e.115 (C-II) &

120 kg /ha (C-I).

Fruit weight :

The data on fruit weight of tomato presented in Table 4.3 and illustrated in

Fig. 4.3 ,which revealed that inoculation with all local Azotobacter isolates significantly

increased the fruit weight over control CIII (100:60:80). The highest fruit weight was

observed under treatment 100:60:80 NPK + AZOT-B-33 (552.02 gm / plant) followed

by treatment C–I (120:60:80NPK) (518.58 gm) and standard check (505.13 gm)

with100:60:80 NPK level .Similarly the fruit weight of tomato inoculated with AZOT-

B-33 and standard check was found at par with uninoculated treatment C-I . However,

the fruit weight of tomato in plants inoculated with local Azotobacter isolates AZOT-B-

18,32,39 and standard check was found at par with control C-II (115:60:80 NPK) when

they were fertilized with NPK level of 100:60:80.

It is apparent from the fruit yield study that local isolate AZOT-B-33 and

standard check were able to fix 20 kg nitrogen per hectare. However, the isolate No.33

was found significantly superior over standard check to increase fruit weight per plant.

Local isolates AZO-B-18, 32, & 39 were also found to fix 15 kg or more nitrogen per

hectare.

Higher fruit yield of tomato in Azotobacter inoculated plants is associated

with the efficient fixation of nitrogen as well as metabolic products of Azotobacter like

gibberellins , indole acetic acid and cytokinin might have helped in inducing early

flowering, fruit setting, fruit picking and also increased number of flowers and fruits

per cluster ( Bhadoria et al., 2007). This view was corroborated with the observations

of Jackson et al.(1964) and Aeon & Barea (1975) who mentioned that favorable

environment, as the roots provide through proper aeration for better bacterial activity

resulting in more nitrogen fixation and higher growth attributes with seedling

inoculation with Azotobacter as compared to soil inoculation with Azotobacter. Tilak

et al. (2005) through their detailed study on soil health supporting bacteria concluded

that yield improvement of crops is attributed more to the ability of Azotobacter to

produce plants growth promoting substance such as phytoharmonce IAA and

siderophore azotobactin ,rather than to diazotrophic activity.

4.2.3 : Biomass accumulation study :-

Fruit dry matter :

The results on the effect of Azotobacter inoculation with local isolates as well

as standard Azotobacter check on fruit dry matter yield are presented in Table 4.4 &

illustrated in Fig. 4.4.

The results clearly elucidate that inoculation of tomato seedlings with local

Azotobacter isolates and standard check with NPK level of 100:60:80 significantly

increased the dry matter accumulation by fruit over only application of fertilizer @

100:60:80 (C-III). Highest dry biomass of fruit was found with isolate No. AZOT-B-33

(40.85 gm/pot), followed by uninoculated control C-I (37.34 gm) with fertilizer dose of

120:60:80 kg NPK. The local Azotobacter isolate AZOT-B-33 significantly

accumulated higher fruit dry matter over inoculation of standard check. Simultaneously

the same isolate AZOT-B-33 and standard check was found at par with the

performance of highest nitrogen dose i.e. 120 kg/ha to increase the fruit dry matter

yield. However, local isolate 18 and 32 had shown at par performance with

uninoculated control treatment C-II .

Shoot dry matter :

The effect of inoculation with different local Azotobacter isolates vis-à-vis

different levels of nitrogen on shoot dry matter yield was recorded and tabulated in

Table 4.4 and depicted in Fig. 4.4.

Data showed that inoculation of tomato crop with local Azotobacter isolates &

standard check with NPK leval of 100:60:80 significantly increased the shoot biomass

over uninoculated control CIII (100:60:80). Highest biomass accumulation (75.10

gm/pot) was recorded in treatment T6 (Plate) which received local Azotobacter isolate

AZOT-B-33, followed by control CI (120:60:80) (70.50 gm/pot), standard Azotobacter

check (68.72gm) and treatment T2 with local isolate AZOT-B-32(68.00gm/pot). The

dry matter yield of shoot increased significantly in plants due to inoculation with local

isolate AZOT-B-33 over standard Azotobacter check in presence of same level of

nitrogen. However, the same isolate AZOT-B-33 and standard check was found at par

with the performance of highest nitrogen dose i.e. 120 kg/ha to increase the shoot dry

matter yield. The shoot dry matter accumulation in control pot C-II(115 kgN/ha) was

found insignificant to that of local isolate No. AZOT-B-18,32 and 39 with 100:60:80

NPK level.

This increase in plant biomass might be due to the impact of Azotobacter on

tomato plants. Plant growth promoting rhizobacteria use one or more of direct or

indirect mechanisms of action to improve plant growth and health. Biological N-

fixation, P- solubilisation, improvement of other plant nutrients uptake and

phytohormone production like indole-3-acetic acid are some examples of mechanisms

that directly influence plant growth (Glick et al., 1995). Similar findings were also

reported by Raheem et al., (1989) and Puertas and Gonzales (1999) who clearly

mentioned that the inoculation with Azotobacter alone significantly increased the root

depth, shoot height, fresh and dry weights of root and shoots of tomato. Biological

control of plant pathogens and deleterious microbes, through the production of

antibiotics, lytic enzymes, hydrogen cyanide and siderophores or through competition

for nutrients and space can improve significant plant health and promote growth as

evidenced by I

ncreases in seedling emergence, vigor and yield (Hilal et al., 1997).

4.2.4 : Fruit N-accumulation study :-

N content study :

The data related to the effect of Azotobacter inoculation with lowest dose of

nitrogen comparing with higher nitrogen doses on nitrogen content of tomato are

tabulated in Table 4.5 and illustrated in Fig. 4.5.

It is revealed from the data that inoculation of tomato seedlings with local

Azotobacter isolates and standard check with 100:60:80 kg of NPK significantly

increased the N–content in tomato fruit over control C-III (100:60:80 NPK). Maximum

nitrogen content in fruit was recorded 1.95 % in treatment No. T6 (AZOT-B-

33+100:60:80 NPK) followed by 1.91 % in treatment No.T9 (standard check

+100:60:80 kg NPK) .The Azotobacter isolate AZOT-B-33 significantly increased the

percent N content in fruit over standard check when the plants were fertilized with the

NPK level of 100:60:80. However, the N content due to isolates No.33 and

standard check were found at par with the N content of fruits of uninoculated plants

grown with 120:60:80 kg NPK (C-I). Percent N content in fruits of plants raised with

115:60:80 NPK level was found statistically at par with N content due to local

Azotobacter inoculation of isolates 18,32,39 and 123 with C-III fertilizer level.

N-uptake study :

The result of the influence of Azotobacter inoculation vis-à-vis different levels

of nitrogen on nitrogen uptake by fruits are tabulated in Table 4.5 and illustrated in Fig.

4.6.

It is apparent from the data that inoculation of tomato plants with Azotobacter

isolates including standard check significantly increased the accumulation of nitrogen

by the fruits except isolate No. AZOT-B-109. The isolate No 33 showed the best

performance which was able to uptake 797.26 mg nitrogen per pot in presence of

100:60:80 NPK level followed by the uninoculated fertilization (716.94 mg/pot)

containing 120:60:80 kg of NPK (C-I). Minimum N- uptake by fruits was recorded

under uninoculated control treatment with 100:60:80 NPK level (C-III). The

Azotobacter isolate AZOTO-B-33 significantly increased nitrogen uptake in tomato

fruits over standard check of Azotobacter with the same level of NPK i.e. 100:60:80.

However, the above local isolate (33) alone showed at par result with control-I which

fertilized with 120:60:80 kg of NPK. The amount of nitrogen which was uptaken by

fruits due to inoculation with three other local Azotobacter isolates AZOT-B-18,32 and

39 and fertilization with 100:60:80 kg NPK was found at par with nitrogen

accumulated under uninoculated fertilizer treatment C-II (115:60:80) . Inoculation of

standard check was found significantly superior over uninoculated control C-II. Similar

type of study was also made by Govedarica et al. (1993) on the production of growth

substances by nine Azotobacter chroococcum isolated from sugarbeat rhizosphere, has

showed that these isolates have the ability to produce auxins, gibberellins and phenols

and, in association with tomato plants, increased plant length, mass and nitrogen

content. Mahato et al . (2009) also reported that Azotobacter application significantly

increased the nitrogen uptake in tomato. They mentioned this may be because of better

nitrogen fixation as a result of accelerated activity and better root system which might

have resulted in more nitrogen accumulation in tomato shoots.

Similar type of reports have also been obtained from several investigations by

Barea and Azcon, 1975; Dobriener and Day, 1976; Boddey and Dobriener, 1982; Okon

et al., 1983; Gibson et al., 1987; Bowen and Rovira, 1999; Palaniappan 1992; Ranjitha,

2000 and Weniger and Veen, 2004.

4.2.5 : Shoot N – accumulation study :-

N- content study :

The local Azotobacter isolates, standard check and different levels of nitrogen

exhibited a differential influence to enhance shoot N content of tomato plants, which is

presented in Table 4.6 and depicted in Fig. 4.5.

It is apparent from the data that inoculation of tomato seedlings with local

Azotobacter isolates and standard check with 100:60:80 NPK significantly increased

the N-content in tomato shoot at the time of harvest over uninoculated control C-I

(100:60:80). Maximum percent N content in shoot was found 0.79 % which was

recorded due to treatment of local Azotobacter isolate AZOT-B-33 followed by

uninoculated control C-I (0.77%).Minimum value was recorded in C-III (100:60:80)

i.e. 0.52%.The Azotobacter isolate AZOT-B-33 significantly increased the percent N

content in shoot over standard check when the plants were fertilized with the NPK level

of 100:60:80 . However, the level of N due to isolate 33 and standard check was found

at par with the nitrogen content found in uninoculated plants raised under NPK level of

120:60:80 (C-I). Nitrogen content in shoot under another uninoculated control

treatment C-II (NPK::115:60:80) was found statistically insignificant over local

Azotobacter isolates AZOT-B-18 , 32, 39 and 123 with CIII fertilizer level.

N-uptake study :

The data on the effect of Azotobacter inoculation vis-à-vis different nitrogen

levels on shoot nitrogen uptake at harvest stage of the crop are tabulated in Table 4.6

and illustrated in Fig. 4.6.

Data showed that inoculation of tomato plants with Azotobacter isolates

including standard check significantly increased the accumulation of nitrogen by the

shoot at the time of harvest. Maximum accumulation of nitrogen in plant shoot was

attributed to the inoculation of local Azotobacter isolate AZOT-B-33 (595.19 mg/pot)

with 100:60:80 NPK level, followed by uninoculated fertilized pot (542.57 mg/pot)

containing 120:60:80 kg NPK. Minimum N-uptake was found under uninoculated

control treatment with 100:60:80 NPK level (C-III).

The Azotobacter isolate AZOT-B-33 significantly increased the nitrogen

uptake in tomato shoots over standard check of Azotobacter with the same level of

NPK i.e. 100:60:80. However , the above promising isolate (33) and standard check

showed at par with that of control –I which received only 120:60:80 kg NPK .The

amount of nitrogen which was accumulated by other local Azotobacter isolates AZOT-

B-18,32,39 & 123 in presence of 100:60:80 kg NPK was found statistically equal to

that of nitrogen accumulation under uninoculated fertilizer treatment C-II (115:60:80

NPK). The increment of nitrogen in tomato shoots may be attributed to N-fixation or

glutamase synthetase activity. This observation is in close agreement with Azcon &

Barea (1975), Smith et al. (1985), Mohandas (1987), El-Shanshoury et al. (1989) ,

Raheem et al. (1989), Martinez et al. (1993) and Mahato (2009). They clearly

mentioned that Azotobacter inoculation either individually or in combination with

other crop beneficial microbe significantly increased nitrogen concentration in the

root, shoot and whole plant, hence showed better results as compare to that of

inorganic fertilizer.

Table 4.2.6 : Total N-uptake study:-

The Azotobacter isolates exhibited a differential influence on total N-uptake by

tomato plants, which is presented in Table 4.7 and depicted in Fig. 4.7.

The data clearly showed that inoculation of tomato seedlings with local

Azotobacter isolates and standard check significantly enhanced the total nitrogen uptake

by the crop. It is observed from the data that maximum amount of N was accumulated

by tomato crop (1392.44 mg/pot) due to inoculation of local Azotobacter isolate

AZOT-B-33 followed by uninoculated treatment C-I (1259.52 mg/pot) with 120:60:80

NPK level. Significant increase in N-uptake by tomato crop varied from 341.10

mg/plant (C-III) to 1392.44, 1114.22, 1009.59, 918.12, 825.65, 730.35, 619.71 and

515.06 mg/pot as a result of inoculation with AZOT -B-33, standard check, AZOT-B-

32, 18, 39, 123, 35 and 109, respectively.

The local Azotobacter isolate AZOT-B-33 alone was found significantly

superior over control treatment C-I (120:60:80 NPK) and standard check. However, the

control treatment C-I was also found significantly superior over standard check. Two

other local Azotobacter isolates AZOT-B-32 and 18 were shown at par performance

with control treatment C-II (115:60:80 NPK).

The study of N accumulation by tomato crop concluded with the finding that

nitrogen accumulation in the crop was increased by inoculation with local Azotobacter

isolates and standard check. The isolate AZOT-B-33 was found to be most effective

inoculant for enhancing fruit yield of tomato and the plant nitrogen accumulation. The

possible mechanisms which facilitated more nitrogen uptake by crops are N2 fixation;

delivering combined nitrogen to the plant and the production of phytohormone-like

substances that alter plant growth and morphology, and bacterial nitrate reduction,

which increases nitrogen accumulation in inoculated plants (Mrkovacki and Milic,

2001). This finding was in close agreement with Raheem et al. (1989) who reported

that presence of Azotobacter inreased N content in plants rather than phosphorus.

Table 4.3 : Study of dehydrogenase activity:

The analysis data related to biochemical property of soil due to inoculation of

local Azotobacter isolates and standard check of Azotobacter are presented in Table 4.8

and illustrated in Fig. 4.8.

It is apparent from the data that inoculation of tomato seedling roots with crop

beneficial bacterium Azotobacter significantly increased the activity of dehydrogenase

enzyme (DHA) in soil at 30 DAT over uninoculated control C-III. It is clear from the

data that highest value of DHA was found due to local Azotobacter isolate AZOT-B-33

(42.95 µg TPF/h/g soil), followed by standard check of Azotobacter (41.34 µg TPF ).

Lowest DHA was recorded in uninoculated control pot C-III. Significant increase in

DHA of soil was noticed which varied from 25.57 µg TPF (C-III) to 42.95, 41.34,

40.21, 37.46, 35.91, 35.36, 33,50 and 32.63 µg TPF /h/g soil due to inoculation of crop

with AZOT-B-33, standard check, AZOT-B-32, 18, 39, 123, 35 and 109, respectively.

The local Azotobacter isolate AZOT-B-33 was found significantly superior over all the

three uninoculated control C-I, II & III but at par with the standard check . The

dehydrogenase activity of local Azotobacter isolate AZOT-B-32 was found

significantly superior over uninoculated control C-II & C-III but found at par with C-I

(120:60:80 NPK).

Enzymes in the soil are biologically significant as they participate in various

transformations and influence the availability of plant nutrients. The dehydrogenase

enzyme systems apparently fulfill a significant role in the oxidation of soil organic

matter as they transfer hydrogen from substrates to acceptors. Many different specific

dehydrogenase systems are involved in the dehydrogenase activity in soils; these

systems are an integral part of the microorganisms. Therefore, the result of the assay of

dehydrogenase activity would show the average activity of the active population

(Skujins, 1976). Meenakshi (2008) also expressed the similar views and mentioned that

in soil microorganisms, active roots and dead cells are the principal sources of

enzymes. They are likely to be influenced by fertilizers and manures. Chendrayan et al.

(1980) were also of the opinion that the increase in dehydrogenase activity was mainly

due to the higher microbial population. The earlier studies revealed that the enzyme

activities are often used as indices of microbial growth rather than the microbial

number, which further may reflect the microbial respiration and the potential capacity

of soil to perform biological transformations of importance to soil fertility.

Table 4.4 :Interaction study with Fusarium:-

The data recorded on the interaction of different local Azotobacter isolates &

standard chech of Azotobacter with Fusarium oxysporium are presented in Table 4.9

and depicted in Fig. 4.9.

Out of seven local Azotobacter isolates studied, four have shown complete

inhibition of the growth of the pathogen (Fusarium oxysporium). The standard check

has also shown complete suppression of the fungus against its 90 mm growth in

control. The promising local isolates of Azotobacter AZOT-B-33 and 32 found most

effective for hundred percent inhibition of Fusarium oxysporium (Plate 5) . Two other

local isolates (AZOT-B-18 and 123) also exhibited hundred percent performance to

control Fusarium oxysporium. Other three local Azotobacter isolate AZOT-B-35, 39

and 109 although found significantly superior over control with respect to inhibition of

fungal growth but found inferior to that of isolate AZOT-B-32, 18, 123, 33 and

standard check.These observations were also in close agreement with Mahalakshmi and

Reetha (2009), who found six out of nine isolates of Azotobacter of tomato rhizosphere

positive towards IAA production, phosphate solubilization, siderophore production,

HCN production, ACC deaminase activity and antifungal activity. They also reported

that two above isolates were effective in inhibiting the growth of fungal pathogen

Fusarium oxysporium, causing wilt of tomato. These finding are in agreement with

those of Bhattacharya and mukherrjee (1988) who reported seed bacterization with

Rhizobiam to inhibit the growth of Sclerotium spp. The fungal inhibition by Rhizobium

isolates may be due to production of secondary metabolites with antimicrobial activities

under different environment (Kaur and seema,2002).Similarly Rhizobium and brady

Rhizobium stains were also found to significantly suppress the mycelial growth of

Fusarium and other soil born pathogenic fungi under in vitro condition (Beigh et al.

1997, Nautiyal , 1997and Omar and Abd-Alla, 1998).

Keeping in view of above mentioned findings, it can be concluded that that

local Azotobacter isolate AZOT-B-33 was the most effective isolate for tomato as its

inoculation showed best results (Plate 4b) . The performance of local Azotobacter

isolate AZOT-B-33 was also found significantly superior over standard check to

increase yield, dry matter accumulation and nitrogen uptake by tomato crop. However,

the performance of both AZOT-B-33 and standard check was found at par with CI

(120:60:80 NPK level) (Plate 4a), which means that these organisms were able to

supplement 20 kg nitrogen per hectare. Other local Azotobacter isolates AZOT-B-32

and 18 were also found efficient to save 15 kg of mineral nitrogen per hectare. Similar

views were also expressed by Siddarmiah and Bagyaraj (1981) and Kumar and

Shrivastav (1994). Katre et al., (1997) also expressed similar views and mentioned that

local strains are more effective for a particular agroclimatic region than the strains

imported from other places. Applying this principle, it was possible to develop

Azotobacter inoculant, which performed best with tomato. Further the results obtained

from these preliminary studies are to be confirmed.

CHAPTER-V

SUMMARY, CONCLUSION AND SUGGESTIONS FOR

FUTURE WORK

Investigation was carried out in order to develop location specific effective

Azotobacter biofertilizer for tomato crop grown under climatic conditions of

Chhattisgarh. To achieve this target forty local Azotobacter isolates were collected from

the Microbial Culture Bank of Department of Agricultural Microbiology, College of

Agriculture, Raipur to select out effective Azotobacter isolate(s) for production of

Azotobacter biofertilizer for tomato growers of Raipur district of Chhattisgarh. The

inoculation effect of native Azotobacter isolates on growth parameters of tomato like

plant height, yield, biomass accumulation and nitrogen uptake were also studied.

Simultaneously, the dehydrogenase activity of Azotobacter isolates in soil and their

antifungal property were also evaluated. The highlights of the findings are summarized

in the following points:

1. In the first stage screening the Azotobacter isolates were tested for their

nitrogen fixing efficiency in N-free Jensen‘s liquid medium comparing with a

standard check (standard Azotobacter IARI isolate). The quantity of N-fixed in

the above liquid medium varied from 2.35-13.45 mg N /gm of sucrose (0.0047

to 0.0269% N) after seven days of incubation. Three local Azotobacter isolates

i.e. AZOT-B-33, 32, and 18 were found at par with standard check. Among all

isolates taken under study, isolate number 33 fixed maximum amount of N in

the medium i.e. 13.45 mg N /gm sucrose (0.0269 %N). The standard check was

released 13.10 mg N /gm sucrose after seven days of incubation.

2. Based on the results (nitrogen fixing ability) obtained from Ist stage screening,

top 7 local Azotobacter isolates were selected for further study (second stage

screening) compared with the standard check to find out their impact on fruit

yield, biomass and nitrogen accumulation by tomato crop. Under different

treatments, seedlings of tomato were inoculated with local Azotobacter isolates /

standard check and fertilized with NPK @ 100:60:80 kg /ha. To evaluate the N-

fixing capacity of Azotobacter isolates, three controls were incorporated in the

experiment viz.C-I, C-II and C-III contained thee levels of nitrogen @100,115

and 120kg /ha, respectively. All the controls having uniform dose of phosphorus

and potassium i.e.60 and 80 kg/ha, respectively. The observation on plant

height study revealed that the highest increase in plant height of tomato plant

was due to inoculation of plants with local Azotobacter isolate AZOT-B-33.

However, all the local Azotobacter isolates and standard check with 100:60:80

NPK level showed at par plant growth with control I (120:60:80 NPK) at 90

DAT.

3. Inoculation with all local Azotobacter isolates significantly increased the

number and weight of fruits over control CIII .The highest number of fruits and

their weight was observed under treatment AZOT-B-33 followed by treatment

C–I (120:60:80NPK) and standard check. The fruit weight of tomato inoculated

with AZOT-B-33and standard check was found at par with uninoculated

treatment C-I . However, the number of fruits in plants inoculated with local

Azotobacter isolates AZOT-B-33 found significantly superior over

uninoculated treatment C-I. The fruit weight due to other local Azotobacter

isolates AZOT-B-18,32,39 and standard check was found at par with control C-

II (115:60:80 NPK) when they were fertilized with NPK level of 100:60:80.

4. Inoculation of tomato seedlings with local Azotobacter isolates and standard

significantly increased the dry matter accumulation by fruit and shoot over

control C-III ( 100:60:80 ). Highest dry biomass yield of fruit and shoot was

found with isolate No. AZOT-B-33, followed by uninoculated control C-I and

standard check. The local Azotobacter isolate AZOT-B-33 significantly

accumulated higher fruit and shoot dry matter over standard check, however,

the promising isolate (33) showed at par performance with uninoculated

treatment C-I.. The local isolates 18,32 and 39 had shown at par performance

with uninoculated control treatment C-II .

5. The result of total nitrogen accumulation (Fruit + Shoot) study revealed that

inoculation of tomato seedlings with local Azotobacter isolates and standard

check significantly enhanced the total nitrogen uptake by the crop. It is

observed from the data that maximum amount of N was accumulated by tomato

crop (1392.44 mg/pot) due to inoculation of local Azotobacter isolate AZOT-B-

33 followed by uninoculated treatment C-I (1259.52 mg/pot) with 120:60:80

NPK level. The local Azotobacter isolate AZOT-B-33 alone was found

significantly superior over control treatment C-I (120:60:80 NPK) and standard

check. However, the control-I was found significantly superior over standard

check. Two other local Azotobacter isolates AZOT-B-32 and 18 had shown at

par performance with control treatment C-II (115:60:80 NPK).

6. The study on dehydrogenase activity showed that inoculation of tomato

seedling roots with crop beneficial bacterium Azotobacter significantly

increased the activity of dehydrogenase enzyme (DHA) in soil at 30 DAT over

uninoculated control C-III. It is clear from the data that highest value of DHA

was found due to local Azotobacter isolate AZOT-B-33 (42.95 µg TPF/h/g soil),

followed by standard check (41.34 µg TPF ). Lowest DHA was recorded in

uninoculated control pot C-III (25.57 µg TPF). The local Azotobacter isolate

AZOT-B-33 was found significantly superior over all the three uninoculated

control C-I, II & III but at par with the standard check. The local Azotobacter

isolate AZOT-B-32 was found significantly superior over uninoculated control

C-II & C-III but found at par with C-I (120:60:80 NPK).

7. The interaction study with Fusarium oxysporium revealed that out of seven

local Azotobacter isolates studied, four have shown complete inhibition of the

growth of the pathogen (Fusarium oxysporium). The standard check has also

shown complete suppression of the fungus. The promising local isolates of

Azotobacter AZOT-B-33 and 32 found most effective for hundred percent

inhibition of Fusarium oxysporium .

Keeping in view of above mentioned findings, it can be concluded that that

local Azotobacter isolate AZOT-B-33 was the most effective isolate for tomato as its

inoculation showed best results. The performance of local Azotobacter isolate AZOT-

B-33 was also found significantly superior over standard check to increase yield, dry

matter accumulation and nitrogen uptake by tomato crop. However, the performance of

both AZOT-B-33 and standard check was found at par with CI (120:60:80 NPK level),

which means that these organisms were able to supplement 20 kg nitrogen per hectare.

Other local Azotobacter isolates AZOT-B-32 and 18 were also found efficient to save

15 kg of mineral nitrogen per hectare. The dehydrogenase activity of AZOT-B-33 in

soil and its antifusarial property was found effective and supportive to declare it as the

most effective local Azotobacter diazotroph for tomato. This isolate (AZOT-B-33 ) has

the tremendous potential to develop it as a location specific Azotobacter biofertilizer

for tomato growers of Raipur district of Chhattisgarh. The results of this preliminary

study were very encouraging and were needed to be confirmed under field conditions.

SUGGESTIONS FOR FUTURE WORK

Azotobacter isolates have been shown to contribute a good portion of the total

N demand of various crops by fixing the atmospheric nitrogen but much work yet

remains to be done to search the location specific, crop and season specific effective

Azotobacter isolate(s) especially for rainfed region like Chhattisgarh. Based on the

highly significant findings of the present investigation, future line of work is hereby

suggested as follows:

1. The promising Azotobacter isolates of tomato should be tested at least for

three years under farmer‘s field conditions of Raipur region of Chhattisgarh

state before recommending them for location specific mass production of

the culture.

2. Azotobacter is well known for N-fixation and also for producing growth

promoting substances. But it is still not popular among farmers of

Chhattisgarh region because of unavailability of its location specific

effective culture. Therefore efforts should be made to increase availability

of the location, crop and season specific effective cultures of Azotobacter at

right time among the farmers.

3. Efforts should be made at farmer fields to observe effect of Azotobacter

inoculation on seed germination and incidence of different diseases.

4. In this age of increasing cost of chemical fertilizers, efforts should be made

to find combinations of compatible beneficial microbes to be used as dual or

poly inoculants in tomato.

5. Even though we have national and local strains identified as efficient, there

is still scope for identifying a new location specific better one. Hence, one

has to search continuously for more efficient strains by isolating more local

microbial germplasm and their systematic screening.

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“SELECTION OF EFFECTIVE AZOTOBACTER ISOLATES FOR

TOMATO (Lycopersicon esculentum Mill.)’’

BY

SURENDRA SINGH

ABSTRACT

The investigation comprising (i) Preliminary screening of forty local

Azotobacter isolates on the basis of their nitrogen fixation capacity in liquid medium

comparing with standard check of Azotobacter under in vitro condition (ii) Pot

experiment with natural soil for second stage screening of superior local Azotobacter

isolates for tomato crop (iii) Enzymatic activity of different local Azotobacter isolates

in soil and (iv) Evaluation of promising Azotobacter isolates for their antifungal

property for disease suppression, was conducted at the Department of Agricultural

Microbiology, College of Agriculture, Raipur, C.G. during the year 2010-11. It was

planned especially in order to develop location specific effective Azotobacter

biofertilizer for tomato growers of Chhattisgarh.

During preliminary screening (First stage screening), forty local Azotobacter

isolates were collected from the Microbial Culture Bank of Department of Agricultural

Microbiology, College of Agriculture, Raipur. Then after the Azotobacter isolates were

tested for their nitrogen fixing efficiency in N-free Jensen‘s liquid medium comparing

with a standard check of Azotobacter. The quantity of N-fixed in the above liquid

medium varied from 2.35-13.45 mg N /gm of sucrose after seven days of incubation.

Among all isolates taken under study, isolate number 33 fixed maximum amount of N

in the medium i.e. 13.45 mg N /gm sucrose (0.0269 %N). Three local Azotobacter

isolates i.e. AZOT-B-33, 32, and 18 were found at par with standard check . However,

the standard check was released 13.10 mg N /gm sucrose after seven days of

incubation.

Based on the results (nitrogen fixing ability) obtained from Ist stage screening,

top 7 local Azotobacter isolates were selected for further study (second stage

screening), compared with the same standard check to find out their impact on fruit

yield, biomass and nitrogen accumulation by tomato crop raised from the inoculated

seedlings with respective Azotobacter isolates on Vertisol. The screening was done

along with three uninoculated fertilizer treatments i.e Control I, II and III with N doses

of 100, 115 and 120 kg / ha, respectively for comparison in order to estimate saving of

chemical N fertilizer. The observations related to yield, biomass accumulation and

nitrogen uptake by tomato crop clearly revealed that inoculation of local isolate

AZOT-B-33 significantly increased the yield, dry matter accumulation and nitrogen

uptake by the crop over standard check. However, the performance of both AZOT-B-33

and standard check was found at par with CI (120:60:80 ::NPK). These results

indicated that AZOT-B-33 and standard check were able to supplement 20 kg nitrogen

per hectare. The dehydrogenase activity of AZOT-B-33 in soil and its antifusarial

property was found effective and supportive to declare it as the most effective local

Azotobacter diazotroph for tomato.

Keeping in view of findings of present investigation, it can be concluded that

local Azotobacter isolate AZOT-B-33 holds tremendous potential for the development

of specific Azotobacter biofertilizer for tomato growers of Chhattisgarh state.

College of Agriculture, Raipur Dr. Tapas Chowdhury

Date: Chairman

Advisory Committee

APPENDIX -I

Chemical composition of media

N-free liquid Jensen’s medium (Jensen’s , 1954)

Sucrose : 20.g

K2HPO4 : 1.0g

MgSo4.7H2O : 0.5g

NaCl : 0.5g

FeSO4 : 0.1g

Na2MoO4 : 0.005g

CaCO3 : 2.0g

Agar : 15.0g

Distilled water : 1000ml

APPENDIX -II

Modified Martin medium

Glucose : 10g

Peptone : 5g

KH2PO4 : 1g

MgSO4.7H2O : 0.50g

Agar : 15g

Distilled water : 1000ml

APPENDIX-III

Physico-chemical, chemical and biological characteristics of the soil

used for pot experiment

Soil Vertisol

pH 7.2

E.C (dSm/m) 0.14

Organic corbon (%) 0.58

Available N (Kg/ha.) 225.3

Available P (Kg/ha.) 6.3

Available K (Kg/ha.) 385

Azotobacter Population

(cfu /gm soil)

4.34 x 103

APPENDIX-IV

N-fixation capacity of local Azotobacter isolates in the N- free Jensen’s

liquad medium

Name of Azotobacter isolates % N N-fixed

(mg N/gm of sucrose)

AZOT-B-121 0.0053 2.63

AZOT-B -115 0.0047 2.35

AZOT-B -33 0.0269 13.45

AZOT-B -32 0.0260 13.00

AZOT-B -39 0.0218 10.90

AZOT-B -46 0.0200 10.00

AZOT-B- 48 0.0050 2.50

AZOT-B -156 0.0195 9.75

AZOT-B -144 0.0196 9.80

AZOT-B- 154 0.0050 2.50

AZOT-B -127 0.0052 2.60

AZOT-B -133 0.0048 2.40

AZOT-B -155 0.0049 2.45

AZOT-B -34 0.0195 9.75

AZOT-B -35 0.0207 10.35

AZOT-B -126 0.0197 9.85

AZOT-B 1-46 0.0203 10.15

AZOT-B -109 0.0087 4.34

AZOT-B -108 0.0050 2.50

AZOT-B -125 0.0047 2.35

Name of Azotobacter isolates % N N-fixed

(mg N/gm of sucrose)

AZOT-B -145 0.0048 2.40

AZOT-B -31 0.0047 2.35

AZOT-B -51 0.0198 9.90

AZOT-B -58 0.0047 2.35

AZOT-B -18 0.0257 12.85

AZOT-B -47 0.0048 2.40

AZOT-B -38 0.0050 2.50

AZOT-B -83 0.0051 2.55

AZOT-B -159 0.0047 2.35

AZOT-B -44 0.0047 2.35

AZOT-B -129 0.0047 2.35

AZOT-B- 91 0.0048 2.40

AZOT-B -160 0.0048 2.40

AZOT-B -149 0.0050 2.50

AZOT-B -65 0.0052 2.60

AZOT-B 45 0.0047 2.35

AZOT-B- 137 0.0047 2.35

AZOT-B -162 0.0047 2.35

AZOT-B- 123 0.0209 10.45

AZOT-B -122 0.0049 2.45

Standard check 0.0258 12.90

Table 4.1 : Nitrogen fixation capacity of local Azotobactor isolates and

standard check in the N free Jensen’s liquad medium.

Azotobacter Isolates % N N – fixed

(mg N /gm of sucrose)

Standard check 0.0262 13.10

AZOT-B-33 0.0269 13.45

AZOT-B-32 0.0263 13.15

AZOT-B-18 0.0261 13.05

AZOT-B-39 0.0218 10.90

AZOT-B-123 0.0209 10.45

AZOT-B-35 0.0207 10.35

AZOT-B-109 0.0203 10.15

AZOT-B-46 0.0200 10.00

AZOT-B-51 0.0198 9.90

AZOT-B-126 0.0197 9.85

AZOT-B-144 0.0196 9.800

AZOT-B-34 0.0195 9.75

AZOT-B-156 0.0195 9.75

AZOT-B-146 0.0087 4.34

AZOT-B-121 0.0053 2.62

Rest isolates 0.0047-0.0052 2.35-2.60

C.D. (0.05) 0.0008 0.40

Table 4.2: Influence of various Azotobacter isolates and different levels of

nitrogen on plant height of tomato.

Treatment

Number

Treatment Height of tomato shoot (cm)

At 30 DAT At 60 DAT At 90 DAT

T1 100:60:80 + AZOT-B-35 12.58 45.85 70.65

T2 100:60:80 + AZOT-B-32 13.25 49.25 77.43

T3 100:60:80 + AZOT-B-18 13.00 47.67 71.23

T4 100:60:80 + AZOT-B-39 13.08 48.25 71.97

T5 100:60:80 + AZOT-B-123 12.87 47.10 71.27

T6 100:60:80 + AZOT-B-33 13.10 53.32 79.67

T7 100:60:80 + AZOT-B-109 12.67 44.56 69.43

T8 100:60:80 + S.C. 13.25 51.25 76.37

T9 N:P:K::120:60:80 (C-I) 14.17 51.02 74.00

T10 N:P:K::115:60:80 (C-II) 13.65 48.42 72.43

T11 N:P:K::100:60:80 (C-III) 9.65 40.25 63.68

C.D. (0.05)

N.S. 6.66 8.15

Table 4.3 : Influence of various Azotobacter isolates and different levels of


Treatment

Number

Treatment No. of fruit per

plant

Fruit weight per plant

(gm)

T1 100:60:80 + AZOT-B-35

15.16 325.33

T2 100:60:80 + AZOT-B-32

19.30 460.78

T3 100:60:80 + AZOT-B-18

18.83 430.45

T4 100:60:80 + AZOT-B-39

19.00 428.07

T5 100:60:80 + AZOT-B-123

17.16 379.06

T6 100:60:80 + AZOT-B-33

22.90 552.02

T7 100:60:80 + AZOT-B-109

13.83 292.37

T8 100:60:80 + S.C.

20.83 505.13

T9 N:P:K::120:60:80 (C-I)

20.86 518.58

T10 N:P:K::115:60:80 (C-II)

19.55 454.54

T11 N:P:K::100:60:80 (C-III)

10.16 211.63

C.D. (0.05)

2.02 45.78


nitrogen on dry matter yield of tomato.

Treatment

Number

Treatment Fruit dry matter

(gm/pot)

Shoot dry matter

(gm/pot)

T1 100:60:80 + AZOT-B-35

15.94 59.69

T2 100:60:80 + AZOT-B-32

29.03 68.00

T3 100:60:80 + AZOT-B-18

24.97 67.49

T4 100:60:80 + AZOT-B-39

23.54 62.13

T5 100:60:80 + AZOT-B-123

20.09 60.83

T6 100:60:80 + AZOT-B-33

40.85 75.10

T7 100:60:80 + AZOT-B-109

13.74 56.89

T8 100:60:80 + S.C.

34.35 68.72

T9 N:P:K::120:60:80 (C-I)

37.34 70.50

T10 N:P:K::115:60:80 (C-II)

27.73 67.54

T11 N:P:K::100:60:80 (C-III)

9.74 45.25

C.D. (0.05)

3.78 6.34

Table 4.5 : Influence of Azotobacter isolates and different levels of

nitrogen on N- accumulation by tomato fruits

Treatment

Number

Treatment N content

(%)

N-uptake

(mg / pot)

T1 100:60:80 + AZOT-B-35 1.49 238.43

T2 100:60:80 + AZOT-B-32 1.77 512.91

T3 100:60:80 + AZOT-B-18 1.72 430.97

T4 100:60:80 + AZOT-B-39 1.66 391.71

T5 100:60:80 + AZOT-B-123 1.60 322.45

T6 100:60:80 + AZOT-B-33 1.95 797.26

T7 100:60:80 + AZOT-B-109 1.28 174.22

T8 N:P:K::100:60:80 + S.C. 1.78 610.88

T9 N:P:K::120:60:80 (C-I) 1.91 716.94

T10 N:P:K::115:60:80 (C-II) 1.69 466.83

T11 N:P:K::100:60:80 (C-III) 1.06 105.76

C.D. at (0.05) 0.16 96.93


nitrogen on N- accumulation by tomato shoot at harvest.

Treatment

Number

Treatment N content

(%)

N-uptake

(mg / pot)

T1 100:60:80 + AZOT-B-35 0.64 381.27

T2 100:60:80 + AZOT-B-32 0.73 496.68

T3 100:60:80 + AZOT-B-18 0.72 487.14

T4 100:60:80 + AZOT-B-39 0.70 433.94

T5 100:60:80 + AZOT-B-123 0.67 407.89

T6 100:60:80 + AZOT-B-33 0.79 595.19

T7 100:60:80 + AZOT-B-109 0.60 340.84

T8 N:P:K::100:60:80 + S.C 0.73 503.33

T9 N:P:K::120:60:80 (C-I) 0.77 542.57

T10 N:P:K::115:60:80 (C-II) 0.72 486.69

T11 N:P:K::100:60:80(C-III) 0.52 235.33

C.D. (0.05) 0.05 90.01


nitrogen on total N-uptake (fruit+ shoot) by tomato

Treatment

Number

Treatment Total N-uptake

(mg/pot)

T1 100:60:80 + AZOT-B-35

619.71

T2 100:60:80 + AZOT-B-32

1009.59

T3 100:60:80 + AZOT-B-18

918.12

T4 100:60:80 + AZOT-B-39

825.65

T5 100:60:80 + AZOT-B-123

730.35

T6 100:60:80 + AZOT-B-33

1392.44

T7 100:60:80 + AZOT-B-109

515.06

T8 N:P:K::100:60:80 + S.C.

1114.22

T9 N:P:K::120:60:80 (C-I)

1259.52

T10 N:P:K::115:60:80 (C-II)

953.52

T11 N:P:K::100:60:80 (C-III)

341.10

C.D. (0.05)

102.32


nitrogen on dehydrogenase activity in soil at 30DAT

Treatment

Number

Treatment Dehydrogenase activity

(µg TPF / h / g soil)

T1 100:60:80 + AZOT-B-35

33.50

T2 100:60:80 + AZOT-B-32

40.21

T3 100:60:80 + AZOT-B-18

37.46

T4 100:60:80 + AZOT-B-39

35.91

T5 100:60:80 + AZOT-B-123

35.36

T6 100:60:80 + AZOT-B-33

42.95

T7 100:60:80 + AZOT-B-109

32.63

T8 N:P:K::100:60:80 + S.C.

41.34

T9 N:P:K::120:60:80(C-I)

37.84

T10 N:P:K::115:60:80(C-II)

33.63

T11 N:P:K::100:60:80(C-III)

25.57

C.D. (0.05)

4.45

Table 4.9 : Effect of different local isolates & standard check of Azotobacter

on Fusarium oxysporium

Azotobacter isolates Fusarium oxysporium (mm.)

AZOT-B-35 18

AZOT-B-32 00

AZOT-B-18 00

AZOT-B-39 12

AZOT-B-123 00

AZOT-B-33 00

AZOT-B-109 15

STANDARD CHECK 00

Control 90

C.D.(0.05) 1.05

LEGEND

TREATMENTS ISOLATE No. + FERTILIZER

DOSES (N: P: K)


100:60:80


100:60:80


100:60:80


100:60:80



100:60:80


T8 Standard Check Azotobacter : IARI ,S.C. +

100:60:80

T9 Uninoculated control : (C-I) + 120:60:80

T10 Uninoculated control : (C-II) + 115:60:80

T11 Uninoculated control : (C-III) + 100:60:80

Documents

“Selection of Effective AZOTOBACTER Isolates for Tomato … · 2018-12-06 · “Selection of Effective Azotobacter Isolates for Tomato (Lycopersicon esculentum Mill.)’’Thesis