“selection of effective azotobacter isolates for tomato … · 2018-12-06 · “selection of...
TRANSCRIPT
“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
4.3 Influence of various Azotobacter isolates and different levels of
nitrogen on fruit yield of tomato
4.4 Influence of various Azotobacter isolates and different levels of
nitrogen on dry matter yield of tomato
4.5 Influence of various Azotobacter isolates and different levels of
nitrogen on N-accumulation by tomato fruit
4.6 Influence of various Azotobacter isolates and different levels of
nitrogen on N-accumulation by tomato shoot at harvest
4.7 Influence of various Azotobacter isolates and different levels of
nit nitrogen on total N-uptake (fruit + shoot) by tomato
4.8 Influence of various Azotobacter isolates and different levels of
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
4.2 Influence of various Azotobacter isolates and different levels of
nitrogen on plant height of tomato
4.3 Influence of various Azotobacter isolates and different levels of
nitrogen on fruit yield of tomato
4.4 Influence of various Azotobacter isolates and different levels of
nitrogen on dry matter yield of tomato
4.5 Influence of various Azotobacter isolates and different levels of
nitrogen on N-accumulation by tomato fruit
4.6 Influence of various Azotobacter isolates and different levels of
nitrogen on N-accumulation by tomato shoot at harvest
4.7 Influence of various Azotobacter isolates and different levels of
nitrogen on total N-uptake (fruit + shoot ) by tomato
4.8 Influence of various Azotobacter isolates and different levels of
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
T2 Azotobacter isolate No. : AZOT-B-32 +
100:60:80
T3 Azotobacter isolate No. : AZOT-B-18 +
100:60:80
T4 Azotobacter isolate No. : AZOT-B-39 +
100:60:80
T5 Azotobacter isolate No. : AZOT-B 123 + 100:60:80
T6 Azotobacter isolate No. : AZOT-B-33 +
100:60:80
T7 Azotobacter isolate No. : AZOT-B 109 + 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.
BIBLOGRAPHY
Abbass, Z. and Okon, Y. 1993. Plant growth promotion by Azotobacter paspali in the
rhizosphere. Soil Biology and Biochemistry. 25(8):1075-1083.
Aeon, R. and Barea. J.M. 1975. Synthesis of auxins, gibberellins and cytokinins by
Azotobacter vinelendil and Azotobacter beijerinckii related to effects product on
tomato plants. Plant Soil.43:96-103.
Agrawal, Nirmala. and Singh, H.P. 2000. Selection of Azotobacter strains for growth
and survival under stress condition. Biotecnology of microbes and sustainable
utilization .75-80.
Amer, A.H., Shimi, I.Z.E. and Zayad, G.A. 2003. Response of tomato plants grown in
newly reclaimed sandy soils to bio and mineral fertilization. Annals of
Agricultural Science. 41(2):925-938.
Anonymous 1996. Status report on absence of rhizobia in Chhattisgarh soils. Submitted
to the Director General I.C.A.R., New Delhi.
Anonymous. 2009. National horticulture board. Ministry of Agriculture Government of
India, Gurgaon.
Azcon, R. and Barea, J.M. 1975. Synthesis of auxins, gibberellins and cytokinins by
Azotobacter vinelandii and Azotobacter Beijerinckii related to effects produced
on tomato plants. Plant and Soil. 43: 609-619.
Badawy, F.H., Dsouky, M.M.E., Sadiek, H.S. and Baker, A.A.A. 2003. Response of
tomato to inoculation with single and multi-strain inoculants of different
bacterial species. Assiut Journal of Agricultural Sciences. 34(5): 275-286.
Badoni, A. 2006. Effect of inorganic fertilizers and organic manure on seed
germination and seedling growth of Solanum melongena L. under
environmental condition. M. Sc. Thesis, Department of Seed Science and
Technology, H. N. B. Garhwal University, Srinagar.
Bagyaraj, D.J. and Menge, J.A. 1978. Interaction between a VA mycorrhiza and
Azotobacter and their effects on rhizosphere microflora and plant growth. New
Phytologist. 80: 567-573.
Balasubramanian, A. 1992. Quality of biofertilizer. National conference on biofertilizer
and organic farming. SPIC science foundation, Madras.
Barbara j. taller and Tit-yee wong .1989.Cytokinins in Azotobacter vinelandii culture
media, Applied and environmental microbiology.55(1):266-267.
Barea, J. M. and Azcon, R. 1975. Possible synergistic interactions between soils and
nitrogen fixing bacteria. In: Mosse, B. and Tinker, P. B., eds. Endomycorrhizas.
London: Academic Press. pp. 409-417.
Bashan, Y. and Levanony, H. 1995. Migration, colonization and absorption of
Azospirillum brasilense to wheat roots. In: Lectins, Biology, Biochemistry,
Clinical Biochemistry,. 6 :69-84.
Begum, H. 1998. Effect of nitrogen fixing rhyzobacteria and chemical fertilization on
yield of Lycopersicon esculentum Mill var. Santa Clara. South Indian
Horticulture. 46(1&2):88-90.
Beijerinck, M.W. 1901. Über ologonitrophile mikroben. Zentralbl. Bakteriol.
Parasitenkd. Infektionskr. II Abt.: 561-582.
Bergersen, F.J. 1970. The quantitative relationship between nitrogen fixation and
acetylene reduction assay. Aust. J. Biol. Sci., 23:1015-1025.
Bescking, J.H. 1961. Studies on nitrogen-fixing bacteria of the genus Beijerinckia. I.
Geographical and ecological distribution in soil. Plant Soil. 14: 49-81.
Bhadauria, K.K.S., Dwivedi, Y.C. and Kushwah, S.S. 2005. Effect of bio- inoculants
and its methods of inoculation in combination with different levels of nitrogen
on growth yield and economics of tomato. JNKVV Research Journal.
39(2):112-115.
Bhadoria ,S.K.S.,Dwivedi, Y.C. and Kushaeah,S.S. 2007.Flowring and fruiting
behaviour of tomato as affected by Azotobacter and nitrogen. Indian J.of
Hotriculture 64.(3): 366-368.
Bhriguvanshi, S.R. and Gangawar, B.R. 1984. Effect of temperature on the survival of
bacterial species. J. Indian Soc. Soil Sci., 32: 172-74.
Bilal, K.S. and Rakhshanda, Y.L. 1990. World Journal of Microbiology and
Biotechnology. 6(1): 112-114.
Boddey, R.M. and Dobereiner, J. 1982. Association of Azospirillum and other
diazotrophs in Tropical graminae. Non symbiotic nitrogen fixation. symposia
papers: Transactions of the 12th
International Congress of Soil Science, New
Delhi, pp. 190-226.
Bottini, R. Cassan, F. and Piccoli. P. 2004. Gibberellin production by bacteria and its
involvement in plant growth promotion and yield increase. Appl Microbial
Biotechnol. 65:497-503.
Bowen, G.D., Rovira, A.D., 1999. The rhizosphere and its management to improve
plant growth. Advances in Agronomy. 66: 1-102.
Brakel, J. Hilger, F. 1965. Etude qualitative quantitative synthese de substances de
nature auxinique par Azotobacter chroococcum in vitro. Bull. Inst. Agron. Stns.
Rech. Gembloux. 33: 469-487.
Bremner, J.M. 1965. Nitrogen availability indexes. In C.A. Black (ed.). Methods of soil
analysis part-2. Agronomy, Am. Soc. Of Agro. Madison, Mis. 9:1324-1345.
Brian, W. and Hemmingh, G. 1955. The effect of gibberellic acid on shoot growth of
pea seedlings. Physiologia PI. 8: 669.
Brockwell, J. 1977. Application of legume seed inoculants. In a treatise on Dinitrogen
fixation (Eds) R.W.F. Hardy an A.H. Gibson section Agronomy and Ecology.
John wiley and sons. Newyork.
Brown M.E., Burlingham S.K. 1968. Production of plant growth substances by
Azotobacter chroococcum. J. Gen. Microbiol., 53: 135-144.
Brown, M. 1975. Role of Azotobacter paspali in association with Paspalum notatum. J.
Appl. Bacteriol., 40: 341-348.
Brown, M.E. Burlingham, S.K. Jackson, R.M.,1964. Studies on Azotobacter species in
soil. Populations of Azotobacter in the rhizosphere and effects of artificial
inoculation. Plant and Soil 17.
Brownm, E. Jackson, R.M. and Burlinghams, K.T.L. 1968. Effects produced on tomato
plants, Lycopersicum esculentum, by seed and root treatment with gibberelljc
acid and indoleacetic acid. J. exp. Bot. (in the Press)
Burgmann, H, Pesaro, M., Widmer, F. and Zeyer, J. 2003. Strategy for optimizing
quality and quantity of DNA extracted from soil. Bacteriological Reviews. 36:
(2): 295-341.
Burlingham. S.K. 1964. Growth regulators produced by Azotobacter in culture media.
Ann. Rep. Rothamsted. Exp. Stat., p. 92.
Burris, R.H. 1969. Progress in the biochemistry of nitrogen fixation. Proc. Royal Soc.
London B. Biological Sci., 172: 339-354.
Burton, J.C. 1979. New development in inoculating legumes and oilseeds. In recent
advances in biological Nitrogen fixation (Ed.) N.S. Subba Rao. Oxford and IBH
publishing Co., New Delhi. pp. 380-405.
Cappuccino, J.G and Sherman, N. 1996.Microbiology - A laboratory manual. 6th
edition, Pearson education, USA, pp.150-175.
Casanovas, E. M., Barassi, C. A., Sueldo, R. J. 2000. Azospirillum inoculation of maize
seed during imbibition. Cereal Research Communications. 28 (1-2):25-32.
Chan, Y.K. 1986. Utilization of simple phenolics for dinitrogen fixation by soil
diazotrophic bacterial. Plants and Soil. 90: 87-94.
Chonkar, P.K. Iswaran, V. and Jauhari, K.S. 1971. Seed pelleting in relation to
nodulation and nitrogen fixation in saline alkaline soil. pl. soil., 35: 449-451.
condition. . Biotecnology of microbes and sustainable utilization.scientific
Curley, R.L Burton, J.C.. (1965). Indian Journal of Agronomy. 57:379-381.
Day, J.M., Roughely, R.J., Eulesham, A.I.J., Dye, M. and White, S.P. 1978. Ann.
Applied Biol., 88: 476.
Denarie, J. and Blanchere, H. 1966. Inoculation de graines de vegataux cultives al‘aide
de souches bacteriennes. Annales de l’Institut Pasteur & Actualites. 3:57-74.
Dobereiner, J. 1970. Further research on Azotobacter paspali and its varity specific
occurrence in the rhizosphere of Paspalum notatum Flugge. Zentbl bakt
parasitkde abt.II.124:224-230.
Dobereiner, J. and Day, J.M. 1976. First international symposium on nitrogen fixation.
In recent advances in Biological nitrogen fixation. Eds. Newton, W.E. and
Nyman, C.J., Washington state university press, Washington, pp. 518-538.
Dorosionsky, LM. 1964. Production, application and effectiveness of bacterial
fertilizers in USSR. In proc. symp. Bacterial fertilizers ed. LM Dorosinsky,
Kolos, Moscow. 35-49.
Eklund, E. 1970. Secondary effects of some Pseudomonads in the rhizosphere of peat
grown cucumber plant. In: Pharis R.P., Reid D.M., eds, Hormonal Regulation
of Development. Vol 3, Springer-Verlag, N.Y., p. 613.
El-dsouky, M.M., Farida, B.H., Sadiek, H.S. and Abo-baker, A.A. 2003. Isolation,
characterization and selection of rhizobacterial strains from plant rhizospheres
for use in inoculation tests. Assiut Journal of Agricultural Sciences. 34(6):89-
107.
El-shanshoury A.R. 1979.Production of plant growth hormones by certain
microorganism –M.scThesis,Faculty of Scince ,Tnta university,Egypt.
El-shaushouny, A.R., Hassan, M.A. and Abdel Ghaffar, B.A. 1989. Synergistic effect
of vesicular Arbusculr mycorrhizea and A. chroococcum on the growth and the
nutrient contents of tomato plant. Phyton (Horn) 29:704-706.
El-shourbagy, M.N., El-sayed, M.A. and El-shanshoury, A.R. 1979. Inoculation of soil
with Azotobacter chroococcum. - Egypt. J. Bot. 22 (3): 205-214.
FAO (1982) Application of Nitrogen-Fixing Systems in Soil Improvement and
Management. Food and Agriculture Organization of the United Nations. FAO
Soils Bulletin 49, Rome.
Franklandb and Wareingp, F. 1960. Effect of gibberellic acid on hypocotyl growth of
lettuce seedlings. Nature, Lond. 185, 255.
Gajbhiye, R. P., Sharma, R.R. and Tewari, R.N. 2003. Effect of biofertilizer on the
growth and yield parameter on tomato. Indian Journal of Horticulture.
60(4):368-371.
Gibson, A.H. 1987. Measurement of Nitrogen fixation by indirect means. In: Methods
for evaluating for BNF (Ed. F.J. Ergersen). pp. 17-35. (IAEA; Vienna).
Giovannucci, E. (1999). Tomatoes, tomato-based products, lycopene and cancer.
Review of the epidemiological literature. J. Natl. Cancer Inst. 91: 317-331.
Glick, B.R., Karaturovic, D.M., and Newell, P.C. 1995. A novel procedure for rapid
isolation of plant growth promoting bacteria. Can. J. Microbiol. 41:533-536.
Gouri, P.S.V.M. and Jagasnnatathan, R. 1995. Biotechnology in organic farming.
Biotechnology Review. 5: 34-47.
Govedarica, M., Mili, V., Gvozdenovi, D.J. 1993. Efficiency of the association between
Azotobacter chroococcum and some tomato varieties. Soil plant. 42: 113-120.
Govindan, M. and Bagyaraj, D. J. 1995. Field response of wetland rice to Azospirillum
inoculation. J. Soil Biol. and Ecol., 15: 17-22.
Gupta, S. B., Chowdhury, Tapas., Takur, Kapil S. and Thakur M. P. 2005. Exploting
Rhizobium isolates from Lathyrus as Biocontrol Agent Against Legume Rot
pathogen Sclerotium rolfsii. Jounaral of mycology and plant pathology .35 (2)
224-227.
Gupta, S.B., Tedia, K., Singh, A., Anurag, Lakpale, N., Pal, A. and Chhonkar, P.K.
2002. Isolation and identification of stress tolerant bio-inoculants for rainfed
cropping system. Soil and Crops. 12(1):11-16.
Halliday, J. 1984. Principles of Strain selection. In Biological nitrogen fixation (Ed. M.
Alexander). Plenum Press, Newyork. pp. 155-173.
Haris, R.F. 1981. Effect of water potential on microbial growth and activity. In: Water
potential relations in soil microbiology (Eds.: J.R. Parr, W.R. Gardner and L.F.
Eliot). Soil Science Society of America, Madison, Wisconsin. pp. 23-95.
Hemawathi, M. 1997. Effect of organic manures and biofertilizer on growth and
productivity of chrysanthemum (Chrysanthemum morifolium Ramat) cv. Local
yellow. M. Sc. (Agri.) Thesis, Uni. Agric. Sci. Bangalore (India).
Hennequin, J.R. and Blachere, H. 1966. Recherches sur la synthese de phytohormones
et de composes phenoliques par Azotobacter et des bacteries de la rhizosphere.
Ann. Inst. Pasteur, 3: 89-102.
Hilal, A.S.,Elian, M.I.,Matwally,A.H. and E.L.,Deeb,A.A.1997. Peanut diease in
Egypt. Factor affecting healthy survival plants pod rots and yield. Annals Agril.
Sci. Mashtophar, 28(3):1557-1567.
Holt, J.G. Krieg, N.R., Sneath, P.H.E., Staley, J.T. and Williams, S.T. 1994. Bergeys
manual of Determinative bacteriology . 9th
Ed Philadelphia : lippincot Williams
and wilkins.
Iruthayaraj, M.R. 1981. Let Azotobacter supply nitrogen to cotton. Intensive
Agriculture 19 (23): 320-332.
Jackson M.L.Soil 1958.chemical analysis. Prentice Hall of india.Pvt.Ltd.,New Delhi.
Jackson, M. and Brown, E. 1966. Behaviour of Azotobacter chroococcum introduced
into the plant rhizosphere. Annls Inst. Pasteur, Paris III. Suppl. to no. 3: 103.
Jackson, M.L. 1973. Soil Chemical Analysis. Prentice Hall of India (Pvt.) Ltd. New
Delhi.
Jackson, R.M., Brown, M.E. and Burlingham, S.K. 1964. Similar effects on tomato
plants of Azotobacter inoculation and application of gibberellins. Nature land.
203: 851-852.
Jarak, M., Hajnal, T., Djuric, S., and Zurkic, J., 1977. Survival ofrhizobia, Azotobacter
and actinomycetes in soils of different pH values. Annals of scientific work.
Faculty of Agriculture, Novi Sad (Yu) 29(1): 41-50.
Jensen, H.L. 1954. Acta Agric Scand. 4(2): 224-336.
Jensen, H.L. 1965. Non-symbiotic nitrogen fixation. In: Soil nitrogen (Eds.: W.V.
Bartholomew and F.E. Clark). American Society of Agronomy, Monograph No
10. Madison, Wisconsin. pp. 440-485.
Kader, M.A., Mian, M.H. and Hoque, M.S. 2002. Effect or Azotobacter inoculant on
the yield and nitrogen uptake by wheat. Department of soil science, Bangladesh
Agricultural University, Mymensigh, Bangladesh. 2(4): 251-261.
Kalpunik, Y. 1996. Plant growth promoting rhizosphere bacteria, in: Plant Roots The
Hidden Half, Waisel, Y., Eshel, A. and Kafkafi, U., eds., Marcel Dekker, N.Y.,
pp.769-781.
Kanungo, P.K., Ramakrishnan, B. and Rao, V.R. 1997. Placement effect of organic
sources on nitrogenase activity and nitrogen-fixing bacteria in flooded rice soils.
Biology and Fertility of Soils. 25: 103-108.
Katre R.K., Adil M.L. and Gupta S.B. (1997). Department of Biotechnology, New
Delhi sponsored project report, IGKV, Raipur.
Kaur,M. and Seema.2002. Antagonistic activity of Psuedomonas srtains isolated from
the rhizosphere of medicinal plants. In Proc. Nati.Sem.Role of Antimicrobials
for sustanabale Horticulture. I.G.A.U. 20 Jan.2002.pp 6-12.
Khammas, K.M., Ageron, E., Grimont, P.A.D. and Kaiser, P. 1989. Azospirillum
irakense spp. nov. a nitrogen fixing bacterium associated with rice roots and
rhizospheresoil. Res. Micribiol., 140: 679-693.
Kizikaya, R. 2009. Nitrogen fixation capacity of Azotobacter spp. strains isolated from
soils in different ecosystems and relationship between them and the
microbiological properties of soils. J. Environ. Biol. 30(1), 73-82.
Klein, D.A., Loh, T.C. and Goulding, R.L. (1971). A rapid procedure to evaluate the
dehydrogenase activity of soils low in organic matter. Soil Biol. Biochem. 3:
385-387.
kretrovich, W.L., Kariakina, T.I., Wwinowa, M.K., Sidelnikova, L.I. and Kazakova,
O.W. 1981 . Pl Soil. 61:145.
Kumar, A. and Shrivastava, M. 1994. Survey of local Azospirillum isolates for their
efficiency in nitrogen fixation and biomass production of field crops.
Kumar, V., Behl, R.K. and Narula, N. 2001. Establishment of phosphate solubilizing
strains of Azotobacter chroococcum in the rhizosphere and their effect on wheat
cultivers under green house conditions. Microbiological research. 156: 87-94.
Lal, B. and Khanna, S. 1996. Long term field study shows increased biomass
production in tree plants inoculated with Azospirillum. Plant and soil. 184 (1):
111-116.
Lophnez, H.W. and Tejran, D.N. 2005. World journal of Microbiology and
Biotechnology. Springer Netherlands. 5:1999-2001.
Lynch, J.M. and White, N. 1977. Effects of some non-pathogenic microorganisms on
the growth of gnotobiotic barley plants. Plant and Soil. 47: 161-170.
Mahalakshmi, S. and Reetha, D. 2009. Assessment of plant growth promoting activities
of bacterial isolates from the rhizophere of tomato (lycopersicon esculentum L.).
Recent Research in Science and Technology. 1(1): 026-029.
Mahato, P. Badoni, A. and Chauhan J.S. 2009. Effect of Azotobacter and Nitrogen on
Seed Germination and Early Seedling Growth in Tomato. Researcher. 1(4): 62-
66.
Maltseva, N.N., Nadkernichnaya, E.V. and Kanivets, N.A. 1995. Associations of
nitrogen-fixing bacteria with winter rye, Proceeding of the 10th
International
Congress on Nitrogen Fixation. St Petersburg, Russia, No. 614.
Mandhare, V.K., Patil, P.L. and Gadekar, D.A. 1998. Phosphorus uptake of onion as
inflenced by Glomus fasciculatum, Azotobacter and phosphorus levels. Agric.
Sci. Digest. 18(4): 228-230.
Manna, G.B., Mahapatra, I.C. and Vachani, M.V. 1962. Rice News Letter 10: 83.
Margaret e. Brown and late susan k. Burlingham.1968. Production of Plant Growth
Substances by Azotobacter chroococcum. J. gem Microbial.53: 135-144.
MartineJ, R. Dibut, B. and Gonzalez, R. 1993. Stimulation of tomato development and
yield by inoculation of red ferrallitic with Azotobacter chroococcum.
Mary, L.G. and Rita, R. 1985. Colwell, Enumeration, isolation and characterization of
N2 fixing bacteria from sea water . Department of microbiology, University of
Maryland. 50(2).
Mehrotra, O.L. and Lehri, C.K. 1971. Effect of Azotobacter inoculation on yield. J ind
soc soil sci. 19: 243-248.
Menuke, L. and Georgiu, V. 1964. Azotobacter chroococcum kak producent
auksinopodobnih. V. sb. Bakterijalne udobrenija, 166-176.microorganisms.—
M. Sc. Thesis, Faculty of Science, Tanta Univesity, Egypt.
Mishustin, E.N. 1966. Action Azotobacter of .végétaux supérieurs. Ann. Inst. Pasteur,
111, 121-135.
Mishustin, E.N. and Shilnikova, V.K. 1969. Free living nitrogen fixing bacteria of the
genus Azotobacter . In : soil biology reviws of Research , UNESCO publication.
8(65): 72-124.
Mishustine. N. and Naumovaa, N. 1962. Bacterial fertilizers, their effectivenessa and
mechanism of action. Mikro bio logiya 31: 543.
Mohandas, S. 1987. Field response of tomato (Lycopersicon esculentum Mill. ‗Pusa
Ruby‘) to inoculation with a VA mycorrhizal fungus Glomus fasciculatum and
with Azotobacter vinelandii. Plant and soil. 98: 295-29
Moreno, J., De-la-rubia, T., Ramos-cormenzana, A. and Vela, G.R. 1990. Growth and
nitrogenase activity of Azotobacter vinelandii on soil phenolics acids. Journal
of Applied Bacteriology. 69: 850-855.
Mrkovacki,N.Millc,V. 2001.Use of Azotobacter chroococcum as potentially useful in
agricultural application.Ann als of Microbiology.51:145-158.
Narayan,Kamal.2010.Risponce of soil and foliar application of nutrients on growth and
yield attributs of tomato.M.sc.thesis,IGKV.Raipur,C.G.
Narula, N. and Vasudeva, M. 2006. Terrestrial Environment. Environmental
Microbiology. 23.
Narula, N., Lakshminarayana, K. P. and Tauro. 1980. Ammonia excretion by
Azotobacter chroococuum. Biotechnol Sci., 23: 467-470.
Okon, Y. 1985. Azospirillum as a potential inoculant for agriculture. Trends in
Biotechnology. 3: 223-228.
Okon, Y., Heytler, P.G. and hardy, R.W.F. 1983. Nitrogen fixation by Azospirillum
brasilense and its incorporation in to its host Setaria italica. Applied and
Environmental Microbiology, 49: 694-698.
Okon,Y. and Labandera-gonzalez, C. 1994. Agronomic applications of Azospirillum: an
evolution of 20 year worldwide field inoculation. Soil Biol. Biochem 26: 159-
1601.
Olsen, S.R., Cole, C.V., Watanabe, F.S. and Dean, L.A. 1954. Estimation of available P
in soils by extraction with sodium bicarbonate. Circ. U.S. Deptt. Agric. p 939
Palaniappan, S.P. 1992. Strain improvement and quality control of inoculants. National
conference on biofertilizer and organic farming SPIC Science foundation,
Madras.
Pandey, A. and Kumar, S. 1989. Potential of Azotobacters and Azospirilla as
biofertilizers for upland agriculture: a review. Journal of Scientific and
Industrial Research. 48: 134-144.
Panse, V.G.S. and Sukhatme, V. 1978. Statistical Methods for Agricultural Workers.
Indian Council of Agricultural Research. New Delhi. pp. 145-156.
Pati, B.R., Sengupta, S. and Chjandra, A.K., 1995. Impact of selected phyllospheric
diazotrophs on the growth of wheat seedlings and assay of the growth
substances produced by the diazotrophs. Microbiological Research. 150: 121-
127.
Patil, P.L., Patil, S.P., 1984. Uptake of nitrogen by cotton inoculated with Azotobacter.
Journal of Maharashtra Agricultural Universities 9: 171-172.
Pattern, C.L. and Glick, B.R. 1994. Bacterial biosynthesis of indole-3-acetic acid.
Canadian J. of Microbiol. 46: 207-212.
Peppler, H.J. and Perlman, D. 1992. In Microbial technology. Academic press,
Newyork. 1: 4-55.
Poi, S.C. and Kabi, M.C. 1983. Tropical grain Bulletin No. 25: 20-22.
Pramanix, B.N. and Misra, A.N. 1955. Effect of continuous manuring with artificial
fertilizers on Azotobacter and soil fertility. Indian J. Agric. Sci., 25: 1-
13.publishes (India) Jodhpur.74-81.
Puertas A., Gonzalas L.M. (1999). Aislamiento de cepas natives de Azotobacter
chroococum.en la provincial gramna y evaluation de suactividad estimuladora
en plantulas detomate.Cell. Mol. Life Sci., 20: 4-7.
Rahhem, ADB EL. Shanshoury, E.L. Hassan, M.A. & Ghaffer-Abdel, B.A. 1988.
Synergistic Effect of Vasicular-Arbuscular-Mycorrhizas and Azotobacter
chroococcum on the Growth and the Nutrient Contents of omato Plants.Phyton
(Austria).29(2):203-212.
Rajak,R.C.2002. Selection of Azotobacter strains for growth and survival under stress
Rajasekaran, R. 1998. Factors affecting the microbial fixation of nitrogen. In: Recent
Advances in nitrogen fixation and Mobilizing Microorganisms. TNAU,
Coimbatore. pp 29-35.
Ranjitha, K. 2000. Beneficial rhizosphere microflora of black pepper (Piper nigrum L.)
and their role in growth of the plant. MSC Thesis submitted to U.A.S. Dharwad.
Rovira, A.D. 1965. Interactions between plants roots and soil microorganisms. Annual
Review of Microbiology. 19: 241-266.
Safak, K. and Nilfer, A. 2006.Some optimal cultural parameter for gibberellic acid
biosynthesis by pseudomonas sp.Turk j Biol 30:81-85.
Schmidt, E.L. and Caldwell, A.C. 1967. A practical manual of Soil Microbiology
Laboratory Methods. Food and Agric. Organization of the United Natons Soils
Bull. pp. 72-75.
Schromeyer, M., Hartwig, U.A., Hendry, G.R. and Sadowsky, M.J. 1996. Microbial
community changes in the rhizospheres relating to crop growth. Soil biology
and Biochemistry. 28 (2): 1717-1724.
Selvarathi,P.Ramasubramanian,V.& Jeyaprakash,R.2010.Bioremedial effect of
Azotobacter and phosphobacterium on the growth and biochemical
characteristics of paper mill effluent treated Lycoparsicum esculentum
Mill.J.Biosci.Res.1(1): 58-64.
Sharma, S. K. and Thakur, K. S. 2001. Effect of Azotobacter and nitrogen on plant
growth and fruit yield of tomato. Vegetable Science 28(2):146-148.
Shehata, M.M. and El-khawas, S.A. 2003. Effect of two biofertilizers on growth
parameters, yield characters, nitrogenous components, nucleic acids content,
minerals, oil content, protein profiles and DNA banding pattern of sunflower
(Helianthus annuus L. cv. vedock) yield. Pakistan J. Biol. Sci., 6 (14): 1257-
1268.
Sheloumova, A.M. 1935. The use of Azotobacter as abacterial manure for non-
leguminous plants. Bull state inst Agric sci No.1M.
Shendhe, S.T. 1972. Azotobacter.A seed inoculation for maize. Symposium on soil
productivity,Varanasi and colcutta. pp 191-192.
Siddaramaiah,V.K..and Bagyaraj,D.J.1981.Isolation,testing and selection of an
inoculant strain of Rhizobium for Horsegram, Macrotyloma uniflorum(LMA.)
verde. I. Isolation and screening fo rsymbiotic response .Mysore J. Agric. Sci.,
15: 40-43.
Siddharmiah, V.K. and Bagyaraj, D.J. 1981. Isolation, testing and selection of an
inoculant strain of Azospirillum by screening for associative response. Mysore J.
Agri. Sci., 15: 40-43.
Singh, A.B. and Singh, S.S. 1992. Effect of various levels of nitrogen and spacing on
groeth, yield and quality of tomato. Vegetable Science. 19: 1-6.
Smith S.E., ST. John B.j.,Smith F.A. & nicholoas ,D.J.D.1985.Activity of glutamine
synthetese and glutamate dehydrogenase in Trifolium subterraneum and Allium
cepa: Effect of mycorrhizal infection and phosphate nutrition. – New
phytologist 99: 211-228.
Sreeramula, K.R., Hanumanthappa, M., Gowda, A., Kalyana, K.N. and Jayasheela, N.
2000. Dual inoculation of Azotobacter chroococcum and Glomus fasciculatum
improves growth and yield of sunflower under field conditions and saves N and
P fertilizer application. Environ Ecol., 18(2): 380-384.
Stotzky, G. 1972. Activity, ecology, and population dynamics of microorganisms in
soil. CRC Crit. Rev. Microbiol., 2, 59-137.
Subbarao, N.S. 1982. Phosphate solubilization by soil microorganisms. pp: 225-303. In
N.S. Subba Rao (ed.) Adv. Agric. Microbiol. Oxford and IBH Publ. Co., New
Delhi.
Subbarao, N.S. 1982. The effect of Azotobacter chrococcum inoculation in field on the
yield of crop. Soil Microbiology. 4: 130-131.
Subbarao, N.S. 1993. Biofertilizers in Agriculture and Forestry. Oxford and IBH
publishing Co. Pvt. Ltd., New Delhi, p. 242.
Subbiah, B.V. and Asija, G.L. 1965. A rapid procedure for estimation of available
nitrogen in soils. Curr. Sci. 25:259-260.
Sundrarao, W.V.B., Mann, H.S., Pal N.B. and Mathur, R.S. 1963. Bacterial inoculation
experimentes with special reference to Azotobacter .ind j Agric sci 33:279-290.
Taiwo, L.B. 2004. Growth response of tomato (Lycopersicum esculentum Mill)
inoculated with Azotobacter croococcum in an alfisol. Moor Journals of
Agriculture Research. vol-1.
Taller, B.J. and Wong, T. 1989. Cytokinins in Azotobacter vinelandii culture media.
Applied and environmental microbiology. 55(1):266-267.
Thomas, G.V., Iyer, R.D. and Bopiah, B.M. 1991. Beneficial microbes in the nutrition
mustard. Journal of oilseed Research, 14: 45-51
Tilak, K.V.B.R. 1991. Bacterial fertilizers. ICAR Publications, New Delhi.
Tilak,K.V.B.R., Ranganayaki,N., Pal,K.K., De,R., Saxena,A.k., Shekhar ,C., Nautiyal,
Shilpi Mittai, Tripathi A.K.,&Johari.2005.Current scince, 89(1):136-150.
Tom,Miles.2007. Bio-Fertilizers. Information on the intentional use of BioChar
(charcoal from biomass) to improve soils.
Tuladhar, K.D.Y. 1983. Interaction of soil microorganisms with Rhizobium. Ph.D.
thesis, submitted to Post Graduate School, IARI, New Delhi.
Umar,lqbal.WaliKumar,Vinod.Kher,Ravi.&Jamwal,Mahital.2009.Effect of FYM,Urea
and Azotobacter on Growth,Yield and Quality of Strawbery Cv.
Chandler.Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 37(1),139-134.
Vancura, V., and Macura, J. 1960. Indole derivatives in Azotobacter cultures. Folia
Microbiol., 5: 293.
Vassilev, N., Vassileva, M., Azcon, R. and Medina, A. 2001. Interactions of an
arbuscular mycorrhizal fungus with free or co-encapsulated cells of Rhizobium
trifoli and Yarowia lipolytica inoculated into a soil-plant system. Biotechnol.
Lett., 23: 149-151.
Verma, O.P. and Pal, S. 1998. Isolation and characterization of Azotobacter
chroococcum,Soil-plant-microbe interaction in relation to integrated nutrient
management. pp.112-214.
Weniger ,C. Christiansen and Veen, J. A. van 2004. Biology and Fertility of Soils
Springer link Publications. 12(2):210-218.
Wu, F.J., Moreno, J. and Vela, G.R. 1987. Growth of Azotobacter vinelandii. Applied
and Environmental Microbriology. 58: 78-82.
Yanni, Y.G. and El-fattah, F.K.A. 1999. Towards integrated biofertilization
management with free living and associative dinitrogen fixers for enhancing
rice performance in the Nile delta. Symbiosis 27, 319–331.
Zafar, Y., Malik, K. A. and Niemann, E. G. 1997. World journal of Microbiology and
biotechnology. Springer Netherlands, 3:1987-1992
Zanetti, S., Hartwig, U.A. and Nosberger, J. 1998. Elevated atmospheric CO2 does not
affect symbiotic nitrogen as opposed to mineral nitrogen of Trifolium repens L.
Plant cell and Environment. 21(6): 623-630.
“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
nitrogen on fruit yield of tomato
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
Table 4.4 : Influence of various Azotobacter isolates and different levels of
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
Table 4.6 : Influence of Azotobacter isolates and different levels of
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
Table 4.7 : Influence of Azotobacter isolates and different levels of
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
Table 4.8 : Influence of various Azotobacter isolates and different levels of
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)
T1 Azotobacter isolate No. : AZOT-B-35 +
100:60:80
T2 Azotobacter isolate No. : AZOT-B-32 +
100:60:80
T3 Azotobacter isolate No. : AZOT-B-18 +
100:60:80
T4 Azotobacter isolate No. : AZOT-B-39 +
100:60:80
T5 Azotobacter isolate No. : AZOT-B 123 + 100:60:80
T6 Azotobacter isolate No. : AZOT-B-33 +
100:60:80
T7 Azotobacter isolate No. : AZOT-B 109 + 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