ISOLATION, IDENTIFICATION AND SCREENING OF ENDOPHYTIC NITROGEN FIXING BACTERIA FROM SUGARCANE AND SELECTION OF EFFICIENT STRAINS FOR THEIR MASS PRODUCTION AS LIQUID STATE BIOINOCULANT WITH FORMULATIONS BY FERMENTATION BASED BIOTECHNOLOGY.

Project Report

Submitted to the

Jai Dhaneshwari Education SocietyCollege OF Agriculture Biotechnology

Raipur-492006 (C.G) INDIA.

By

Laxman A. Savalkar

Roll No. 4537 ID No. UG/BIO/03/18A-37

JUNE 2007

CERTIFICATE

This is to certify that Project entitled “ Isolation, Identification and Screening

of Endophytic nitrogen fixing bacteria from sugarcane and selection of

efficient strains for their mass production as liquid state bioinoculant with

formulations by Fermentation based biotechnology.” Submitted by Mr.

Laxman A. Savalkar to the Jai Dhaneshwari Education Society, Collage of

Agricultural Biotechnology, Raipur ( C.G.) has been approved by the students

advisory committee after on oral examination in collaboration with the external

examiner.

Major Advisor: Mrs. Chaitali Niratker

Members of advisory committee

1. Mrs. Archana Prasad

2. Mrs. Kiran Kumari

“Dedicated to

My beloved

ParentsWho gave me a

solid foundation in life.”

Acknowledgement

I take this golden opportunity to express heartfelt and deepest sense of gratitude to those who have helped me to complete this thesis. My debts to many individuals can warmly be acknowledged but never fully recompensed. Any project report is the culmination of any course of study for undergraduate degree. As such it forms the crown piece; the crown piece of my B.Sc. (Ag) Biotechnology degree would take its shape due to the assiduous efforts of my major advisor, Mrs. R. R. More, Scientific officer, Plant pathology and Agril. Microbiology,& Mr. D. B. Phonde Scientist, soil Science Department, Vasantdada Sugar Institute(VSI), Manjari(Bk), Pune, Completion of my thesis is the result of his cooperative labour and intellect of honorable guidance.

Most humbly and respectfully I wish to express my profound sense of gratitude to Dr.A.S. Patil, Directorate Of Research, V.S.I. Pune.

I wish to express my profound sense of gratitude to Mrs. Chaitali Niratker, for her guidance, patience and encouragement towards my work and in my studies.

I am indebted to all my teachers for having shared their wisdom, especially Mr. Chavan, Mrs. Archana Prasad, Miss. Aditi Sharma, Mr. Suhas kadam, Mr. Niraj kumar, Mr.Rupesh Deshmuk, Mr. Krishna, Mr. Amit Deokar and other staff members of the College of Ag-Biotechnology for their kind help and co-operation during my study period. Working under a single roof, it was a good company of Mr.V.C.Vasekar. Scientist, P.R.surve. Senier boiler, S.D. Ghule assistant of plant pathology & Agril. Microbiology laboratory, who have always helped me and made me enthusiastic to come up with good results in one way or the other.

It will be a sin if I forget love affection and cooperation of my beloved one Miss. Sayali Pungaonkar who care, support, guided whenever me needed. I take this opportunity to offer my emotional thanks in works to Mrs. Reshu and Mr. Alok Shrivastava, Miss Ashu, Mr. Alok Verma, Mr. Sanjay Dvivedi, Mr. & Mrs. Mishra, Shanu & Golu for their encouraging words and their cooperation throughout my work.

It is indeed a great pleasure to acknowledge the love, affection, cooperation and inspiration rendered by my batch mates and friends Vishvajeet, Pravin, Anurag, Amol, Harish, Ram, Sarjerao, Vikrant, Vivek, Vinod, Ankur, Ajay, Tripti, Mohan, Pavan2, Ashish, for their continued affection and unending encouragement during the course of this research work.

Diction is not enough to express my gratitude to my beloved parents Shri. Ashok Savalkar and Smt. Sagarbai A. Savalkar and brother Lakhan Savalkar. Whose selfless love, constant encouragement, obstinate sacrifices, sincere prayers, expectations and blessings has always been the most vital source of inspiration and motivation in my life. I am highly indebted to my beloved Parents whose affection has been the source of inspiration and encouragement throughout my career.

I would like to thank all those who helped me directly or indirectly to fulfill this huge task.

Indeed the words at my command are not adequate, either in form of spirit, to express the depth of my humility and humbleness before Almighty God without whose endless benevolence and blessings this tedious task could not have been accomplished.

Place:

Date: Laxman Savalkar

Content

ChapterNo

Topics Page No.

01 Introduction 02 Review of literature 03 Material and Method 04 Result & Discussion 05 Summery & Conclusion

AbstractBibliography

List of Figures/Graphs

Figure No.

Particulars Page No.

4.1 Isolated Strain Of Agrobacterium

Diazptrophicus4.2 Isolated Strain Of Azosperrillum

4.3 Isolated Strain Of Azoarcus

4.4 Pure Strain of Endophytes Given by

Institute45 Dilution scheme for reducing sugar by

DNSA method.4.6 Dilution scheme for sucrose by phenol

sulphuric acid method.4.7 Dilution scheme for protein by Folin-

Lowery method.

List of tables

Table

No.

Particulars Page No.

3.1 Dilution scheme for reducing sugar by

DNSA method.3.2 Dilution scheme for sucrose by phenol

sulphuric acid method.

3.3 Dilution scheme for protein by Folin-

Lowery method.4.3 Biochemical characteristics of

endophytes

4.4 Utilization of different carbon source by

endophytes.4.5 Screening of Endophytes for N2 fixation

in vitro.4.6 Temperature range for growth of

Endophytic bacteria.

4.7 pH range for growth of Endophytic

bacteria.

4.8 Response of Endophytes to various

sucrose concentrations.4.9 Dilution scheme for reducing sugar by

DNSA method.4.10 Dilution scheme for sucrose by phenol

sulphuric acid method.4.11 Dilution scheme for protein by Folin-

Lowery method.4.12 Chemical analysis

4.13 Microbial analysis

ABREVATIONS

Agr. - Agro bacterium Diazotrophicus

Azr. - Azoarcus

Act - Acetobacter Diazotrophicus.

i.e. – That is.

ha-1. - Per hectare

BNF - Biological Nitrogen Fixation.

D/W - Distilled Water

BTB - Bromothymol Blue Indicator

Fig. - Figure

Introduction

Chapter 1

Introduction

Endophytic bacterial Nitrogen fixing liquid Bioinoculant is a

unique agro-based product in liquid state, formulated with

growth boosters and cell protectants and it is a consortium of

group of efficient Endophytic Nitrogen fixing bacteria in live

form. Endophytic bacterial Nitrogen fixing Bioinoculant is

special product with newly developed A4H medium with high

cell count, zero contamination, longer shelf life, greater

protection against environment stresses, increased field

efficiency with respect to spreading and penetration and

convenience of handling are main features of the this product.

(Baldani et al., 1978 ).

Endophytic bacteria are those bacteria that fix nitrogen

internally in plant tissues; mostly they are present in the

apoplast i.e. intercellular spaces and xylem vessels. Hence they

are called as endophytic bacteria. Endophytic bacteria such as

Acetobacter, Azoarcus, Herbaspirillum, Azosperrillum, and

Agrobacterium are present in all parts of sugarcane plant

including leaf, stem, roots and juice. These endophytic bacteria

actively participate in biological nitrogen fixation and fix more

nitrogen as compare to ectophytic bacteria. (Dobereiner, J.

1998).

“Biofertilizers” are products consisting of selected, efficient

and beneficial live or latent (resting stage- spores)

microorganisms, which help to improve plant growth and

productivity mainly through supply of plant nutrients.

Biofertilizers are also known as microbial inoculants or bio-

inoculants. Biofertilizers have been introduced in Indian

agriculture since last three decades in view of their cost

effectiveness, contribution to crop productivity, soil

sustainability, and eco-friendly characteristics. Biofertilizers

form an integral part of integrated plant nutrient supply system

(IPNS or INM) and organic farming which constitutes the present

as well as the future mandate of Indian agriculture. (Bellone,et

al., 1989)

Nitrogen is the most essential nutrient required in fairly

large amount for increased productivity of sugarcane and other

cereal crops. It is universal fact that atmosphere is highly rich in

nitrogen (78% N) but without an aid of microorganisms not a

single molecule of atmospheric nitrogen can be utilized by the

plant can utilize the plant. Biological nitrogen fixation (BNF)

is a process either carried out symbiotically or non-symbiotically

by ectophytic and endophytic bacteria which converts

atmospheric nitrogen into “ammonia” and further converted

into “nitrate” readily available form of nitrogen through agency

of nitrifying bacteria or taken up by the plants for their growth

and development. Unless atmospheric nitrogen is fixed it is not

available to plant. So, Biological Nitrogen Fixation through

agency of microbes plays important role in agriculture from

economic point of view. Use of BNF bacteria along with organic

matter and reducing dose of inorganic fertilizer is best source

for maintaining soil fertility as well as achieving the potential

crop yield. . (Bellone,et al., 1989).

Introduction of Endophytic nitrogen fixing

bacterial Bioinoculant in Indian agriculture for monocotyledons

will completely change the concept of symbiotic nitrogen

fixation restricted to dicotylydons like legumes with root nodule

formation. These five major groups of nitrogen-fixing bacteria

and their interaction with the host plants are compared and

many scientists have reported the potential of their use in

agriculture. Hence mass production of these endophytic

nitrogen-fixing bacteria as liquid bioinoculants will be a mile

stone in field of Agriculture with respect to biological nitrogen

fixation and will be a road map for organic farming for all crops.

(More et al., 2007)

The proposed investigation was carried out with following

objectives:

1) Isolation, Identification and screening of efficient strains

of Endophytes liquid Bioinoculant production and for Biological

Nitrogen Fixation.

2) Formulation of liquid endophytic Bioinoculant with cell

protects ants.

3) Efficiency test for Liquid Bioinoculant.

4) Growth and Chemical analysis.

Chapter 2

REVIEW OF LITERATURE:

Biological Nitrogen Fixation (BNF) is a vital component of

agricultural sustainability. ‘Sustainability’ is defined as ‘

successful management of resources for agriculture to satisfy

Review of Literature

human needs while maintaining or enhancing the quality of the

environment and conserving resources’ (TAC, CGIAR, 1988).

Economists measure sustainability as the ratio of output to

input taking into account stock depletion. Stocks in agriculture

include soil, water, non-renewable energy resources and

environmental quality.

Modern agriculture is based on maximum output in the

short term with inadequate concern for input efficiency or stock

maintenance (Odum, 1989). Nitrogen fertilizer ranks first

among the external inputs to maximize output in agriculture.

Input efficiency of Nitrogen fertilizer is one of the lowest among

the plant nutrients and in turn contributes sustainability to

environment pollution. The continued and unabated use of N

fertilizer would further accelerate depletion of stocks of non-

renewable energy resources used in fertilizer production. The

removal of large quantities of crop produce from the land

additionally depletes soil of its native N reserves. On the other

hand, BNF, a microbiological process in the biosphere, converts

atmospheric dinitrogen into a plant usable form through the

microbial enzyme nitrogenase.

This chapter gives the comprehensive review of literature

of the project work & review is summarized under following

headings.

2.1 N2 fixing Endophytic bacteria.

2.1.1 Acetobacter Diazotrophicus

2.1.2 Agrobacterium Diazotrophicus

2.1.3 Azoarcus

2.1.4 Azospirrillum

2.1.5 Herbaspirrillum

2.2 Importance of N2 fixing endophytic bacteria.

2.3 Endophytes as a Biofertilizer.

2.1 N2 fixing Endophytic bacteria.

Nitrogen input through BNF can help maintain soil

N reserves as well as substitute for N fertilizer to attain large

crop yields. (Peoples and Croswell, 1992). Biological Nitrogen

Fixation (BNF) can therefore be a major source of N in

agriculture when symbiotic N fixing systems are used. The

amount of N input reported to be as high as 360 kg N ha-1. On

the other hand, a contribution for non-symbiotic (associative

and free-living) N2-fixation in upland agriculture is generally not

Substantial, although N2-fixation to the order of 160kg N ha-1

has been reported for sugarcane (Ladha et al., 1992). Brazilian

cultivars of sugarcane rarely respond to N fertilizer applications

during the plant crop. Among 135 NPK fertilizer trials all over

the country, only 19% showed significant increase in cane yield

due to N application. This indicates that some endophytic

bacteria may contribute for Biological Nitrogen fixation. Initially

reported by Azeredo et al., (1986). Tremendous progress in

BNF has been made during the last more than 30 years and yet

we are still hoping for breakthrough during the years to come

and as such abundant literature is already available especially

on the BNF except BNF in sugarcane associations and therefore

the review of literature is especially is confined to the

rhizospheric associative diazotrophs in sugarcane. Johanna

Döbereiner initiated research on BNF with grasses in Brazil

when she joined the research team at the National Center of

Education and Agricultural Research of the Ministry of

Agriculture, in the fifties. The first studies were Dedicated to the

memory of Dr. Johanna Döbereiner by two of her disciples who

learned through working with her that research could be done

with simplicity, perseverance, honesty, ethics and sagacity.

2.1.1 Acetobacter:

Acetobacter is Gram negative, Micro aerophilic

bacteria motile with 1-3 lateral flagella present in high number

in roots and stems showing optimum growth with 19% sugar

and pH around 5.5 precisely this condition prevailing in

sugarcane. First isolation of Acetobacter diazotrophicus strains

from roots and stems of sugarcane and classified them under

the genera, viz., Gluconobacter and Acetobacter (De Ley et al.,

1984) or to the genus Frateuria (Swings et al., 1984) on

observing that this organism has capacity to grow at low pH

values and their ability to form acetic acid from ethanol by De

Ley and Swings (1984) from Brazil. A new N2 fixing bacterium

Acetobacter diazotrophicus found in high numbers in roots and

stem of Sugarcane samples from all over the Brazil and also in

Australia and Mexico,It was reported by Cavalcante and

Dobereiner, 1988 and Gills et al. Dobereiner et al., (1988) and

Paula et al., (1989) observed that these bacteria were not

however found in soil between rows of sugarcane plants or

roots of 12 different weed species, which grew in sugarcane

fields. It was also not found in grain of sugar sorghum but was

isolated from few samples of washed roots and aerial parts of

Pennisetum purpureum CV Cameroon and from sweet potatoes.

Gillis et al., (1988, 1989) reported that this nitrogen fixing

bacterium that seems to be specific for sugarcane (Dobereiner

et al., 1988) has been identified through DNA-rRNA

hybridizations and DNA-DNA homology values, as a new species

of Acetobacter diazotrophicus. Reis et al., (1988) also observed

that A. diazotrophicus occur only in plants propagated

vegetatively but not in plants grown from seeds. Dobereiner et

al., (1988) reported the endophytic occurrences of Acetobacter

diazotrophicus in sugarcane, sweet potatoes and Cameroon

grass; all plants that are propagated vegetatively and that

contain high sugar concentrations which was later confirmed by

Li and Mac Rae (1992). Boddey et al., (1991) from the above

observations concluded that this Acetobacter diazotrophicus

must be considered as an endophytic in nature, which

propagated within stem cuttings. Mahesh Kumar-KS; Krishnaraj

Dharwad, India (1999) carried out mineral phosphate

solubilizing activity of Acetobacter diazotrophicus a bacterium

associated with sugarcane, Li and Mac Rae (1992) reported the

presence of A. diazotrophicus in soil samples collected between

cane rows. This was later confirmed by Reis et al., (1993). Paula

et al., (1992); Reis and Dobereiner (1991) reported the

presence of A. diazotrophicus in stems, tubers and roots of

sweet potato collected from various regions in Brazil.Dobereiner

et al., (1993) further reviewed the work on endophytic

diazotroph in sugarcane, cereals and tuber plants.

2.1.2 Agrobacterium Diazotrophicus:

Agrobacterium diazotrophicus are rod in shape &

motile by 1-6 peritrichous flagella. They are gram negative,

microaerophillic, showing optimum growth with 10-20% sugar,

pH around 5.5-6.0, temperature 250C & highly obtained from

internode region of a sugarcane plant. They cause proliferation

in many plants. Study on influence of nitrogen fertilization on

the population of diazotrophic bacteria A. diazotrophicus in

sugar cane (Saccharum spp.) by Reis-Junior-FB-dos; Reis-VM;

Urquiaga-S; Dobereiner-Brazil (2000). Stephen et al., (1991)

studied the physiology and dinitrogen fixation of Acetobacter

diazotrophicus. Reis et al.,(1994) have therefore reexamined

several alternatives and gave the most successful methods and

some results on the specific occurrence of this diazotroph in

sugar rich plants (sugarcane, sweet sorghum, sweet potato,

beet root, etc.), which are being propagated vegetatively.

2.1.3 Azoarcus:

Azoarcus is a gram negative, aerobic in nature,

highly present in juices of stem & leaves of sugarcane plants.

Showing optimum growth at pH 6.6-7.0 & temperature 250C.

They were firstly isolated from salt tolerant plant. They are

belonging to proteobacter beta super family. & They are

aerobic in nature. Azoarcus sp.and their interactions with grass

roots, by B. Reinhold- Hurek and T. Hurek. (1980)

2.1.4 Azosperrillum:

Azospirillum is gram negative, microaerophillic

bacteria, motile with flagella & highly present in roots & leaves

of sugarcane plants. Showing optimum growth with

temperature 300C, pH around 6.6-7.0. They were firstly isolated

from rhizosphere of C4 plants & widely studied as rhizosphere

bacteria. They are belonging to proteobacter alpha super

family. & They are aerobic in nature. Host-plant specificity in

the infection of cereals with Azospirillum spp by Baldani, V.

L. D., and J. Dobereiner. 1980. Members of the genus

Azospirillum are capable of nitrogen fixation under

microaerophillic conditions in association with the roots of

several grasses Dobereiner et al., (1991). Azospirillum appears

to form several different types of cyst-like cell: pleomorphic

cyst-like forms associated with cultured sugarcanes-callus

tissue and with root colonization. (Bashan et al., 1991; Berg et

al., 1979.;1980; Whallon et al ., 1985.)

2.1.5 Herbaspirillum:

Herbaspirillum has been fund in maize, sorghum,

sugarcane & other graminous plants. They are usually vibroid,

occasionally helical in shape, gram negative & motile by 1-3

flagella at one or both poles& they showing optimum growth on

temperature 300C, optimum pH 7.0 & highly present in roots of

sugarcane plant. They are belonging to proteobacter beta

super family & they are aerobic in nature. Herbaspirillum

seropedicae the first nitrogen-fixing bacterium with endophytic

characteristics was isolated in 1984 from the rhizosphere,

washed roots and surface sterilized roots of maize, sorghum

and rice plant and named as Azospirillum seropedicae (Baldani

et al., 1984). Although this group of bacteria showed several

morphological and physiological characteristics similar to the

genus Azospirillum, DNA: DNA homology studies showed that

they formed a new genus named Herbaspirillum, thus

Azospirillum seropedicae was renamed as Herbaspirillum

seropedicae (Baldani et al., 1986a). Characterization of

Herbaspirillum seropedicae gen. nov. Sp. nov. a root-associated

nitrogen-fixing bacterium. (Baldani et al., 1986a).

Herbaspirillum, an endophytic diazotroph colonizing vascular

tissue in leaves of Sorghum bicolor (Dobereiner et al., 1997).

Herbaspirillum lusitanum sp. Nov., a novel nitrogen fixing

bacterium associated with root nodules of phaseolus vulgaris.

2.1 Importance of N2 fixing Endophytic bacteria:

First observation on selective stimulation of N2-

fixating bacteria in sugarcane in Brazil by Dobereiner and

Alvahydo (1959) (Dobereiner, 1961). First time reported the

propagation of this organism in stem cuttings developing

sugarcane plants but this organism could not be identified later

by Patriquin et al., (1980). Lima (1981) conducted the 15N

dilution and nitrogen balance experiment with sugarcane and

reported that this crop was able to obtain more than 60% of its

Nitrogen needs from BNF, which was later reexamined by

Urquiaga et al., 1981,1992). In 15N study by Lima et al., (1987)

showed that after plant analysis it was revealed that 50% of

the plant N in cultivars CB-47-89 had been derived from the

atmosphere. Gillis et al., (1989) reported that within the genus

Acetobacter, seven species are described, however, until now,

Acetobacter diazotrophicus was the only one able to fix the

atmospheric nitrogen. Reis et al., (1990) reported that A.

diazotrophicus growing in 10 % sucrose showed an optimum

dissolved oxygen concentration for acetylene reduction in

equilibrium with 0.2 k PaO2 in the atmosphere but continued to

fix N2 up to 4.0 k Pa, showing a much higher oxygen tolerance

than Azospirillum spp. Paula et al., (1990) and Reis et al.,

(1990) reported the preliminary results on the synergistic

effects of G. clarum with A. diazotrophicus on sorghum and

sugarcane seedlings. R.M Boddey, et al., Brazil (1991)

confirmed that certain sugar cane varieties are capable of

obtaining large contribution of nitrogen from plant associated

N2 fixation. It was estimated that up to 60 to 80 % of plant N,

equivalent to over 200 kg n ha-1 /year, could be derived from

this source, under good conditions of water and mineral

nutrient supply. They also reported that incomplete inhibition of

N2 fixation by NH4 + in these organisms, as well as the lack of

nitrate reductase in Acetobacter diazotrophicus are of

considerable ecological and agronomic importance because

they may permit the complementation of plant associated BNF

with N fertilization. Paula et al., (1991) studied the effects of A.

diazotrophicus on VAM colonization and on the numbers of

spores within roots were also observed in sweet sorghum and

was the first to report the infection of a seed plant by A.

diazotrophicus. Reis (1991) isolated A. diazotrophicus from

sugarcane xylem sap. Stephen et al. (1991) studied the

physiology and dinitrogen fixation of Acetobacter

diazotrophicus. Reis et al., (1994) have therefore reexamined

several alternatives and gave the most successful methods and

some results on the specific occurrence of this diazotroph in

sugar rich plants (sugarcane, sweet sorghum, sweet potato,

beet root, etc.), which are being propagated vegetatively.

Burris-RH Wisconsin, USA (1994) carried out comparative study

of the response of Azotobacter vinelandii and Acetobacter

diazotrophicus to changes in pH. He reported that curves were

established for the pH response of respiration on eleven

substrates by Azotobacter vinelandii and Acetobacter

diazotrophicus. With every substrate the optimal pH for A.

diazotrophicus was lower than for A. vinelandii. The optimal

hydrogen ion concentration for A. diazotrophicus was 5 fold to

365 fold greater than for A. vinelandii depending upon the

substrate. In general, A. diazotrophicus supports respiration

over a wider pH range than does A. vinelandii. In Germany 1999

study was carried out for Analysis of nitrogen fixation and

regulatory genes in the sugarcane endophyte Acetobacter

diazotrophicus by Lee-S; Sevilla-M; Meletzus-D; Texeira-K;

Baldani-I; Kennedy-C; Martinez-E (ed.); Hernandez-G .The mcpA

gene product is involved in responses to extracellular

chemotactic signals, which may be important in plant-microbe

interactions. Study on the respiratory system and diazotrophic

activity of Acetobacter diazotrophicus PAL5 carried out by

Flores et al., (1999) The characteristics of the respiratory

system of Acetobacter diazotrophicus PAL5 were investigated.

Increasing aeration (from 0.5 to 4.0 liters of air/min per liter)

had a strong positive effect on growth and on the diazotrophic

activity of cultures. Cells obtained from well aerated and

diazotrophically active cultures possessed a highly active,

membrane-bound electron transport system with

dehydrogenases for NADH, glucose, and acetaldehyde as the

main electron donors. Ethanol, succinate, and gluconate were

oxidized but to only a minor extent. Fuentes-Ramirez et al.,

(1999) reported that colonization of sugarcane by Acetobacter

diazotrophicus is inhibited by high N-fertilization.

2.2 Endophytes as a Biofertilizer:

Herbaspirillum seropedicae the first nitrogen-fixing bacterium

with endophytic characteristics was isolated in 1984 from the

Rhizhosphere, washed roots and surface sterilized roots of

maize, sorghum and rice plant and named as Azospirillum

seropedicae (Baldani et al., 1984). Biological nitrogen fixation

in non-leguminous field crops: recent advances. By Kennedy,

I.R., and Y.T. Tchan. 1992. Recent advances in BNF with non-

legume plants. By Baldani et al., Nitrogen fixation in endophytic

and associative symbiosis. By James, E. K. 1999 Field Crop Res.

65:197-209. Infection and colonization of sugarcane and other

graminaceous plants by endophytic diazotrophs. By James et

al., Study on influence of nitrogen fertilization on the population

of diazotrophic bacteria Herbaspirillum spp. and Acetobacter

diazotrophicus in sugar cane (Saccharum spp.) by Reis-Junior-

FB-dos; Reis-VM; Urquiaga-S; Dobereiner-Brazil (2000).

Comparison of benefit to sugarcane plant growth and 15N2

incorporation following inoculation of sterile plants with

Acetobacter diazotrophicus wild type and nif mutant strains.

By Sevilla et al., Bacterial endophytes: potential role in

developing sustainable systems of crop production. By Sturz, A.

V., B. R. Christie, and J. Nowak. 2000. In 15N study by Lima et al.,

(1987) showed that after plant analysis it was revealed that

50% of the plant N in cultivars CB-47-89 had been derived from

the atmosphere. Gillis et al., (1989) reported that within the

genus Acetobacter, seven species are described, however, until

now, Acetobacter diazotrophicus was the only one able to fix

the atmospheric nitrogen. Certain sugar cane varieties are

capable of obtaining large contribution of nitrogen from plant

associated N2 fixation. It was estimated that up to 60 to 80 % of

plant N, equivalent to over 200 kg n ha-1 /year, could be

derived from this source, under good conditions of water and

mineral nutrient supply. R.M Boddey, et al., Brazil (1991).

Chapter 3

MATERIALS AND METHODS

Materials and Methods

The present investigation “isolation, Identification,

Characterizations And Screening of endophytic nitrogen fixing

bacteria from Sugarcane and selection of efficient strains for

their mass production as liquid state Bioinoculant with

fermentation based biotechnology” has been conducted at

Vasant Dada Sugar Institute, Pune.

3.1 Materials:

3.1.1 Plant Material:

Stem from Sugarcane variety CO.86032,

3.1.2 Microorganism:

The two pure strains of an endophytes viz. Acetobacter

diazotrophicus and Herbaspirrillum were provided by Institute.

3.2 Methods:

3.2.1 Isolation and Selection of Endophytic bacteria:

The help of following microbial techniques isolates the

three species of endophytes. And the inoculum used in the juice

form of all the three explants (leaf, stem, and Root).

3.2.1.1 Serial dilution (up to 10-12 dilutes):

1. 12 sterile bottles were taken with 90 ml distilled water in

three sets.

2. 10 ml of each respective sample was added in first bottle of

each set.

3. 10 ml sample from bottle first and transferred it to next

bottle that was carried up to 10-12 dilutions.

3.2.1.2 Pour Plate Method:

Pour plating was done for the selection for the isolates in respective

media.

1. 1 ml sample from each dilution was poured respective

dilution’s petriplate.

2. 10-15 ml media poured in each plate. And medias were

Azorcus media, Azospirrillum, media, Agrobacterium Diazotrophicus

media.

3. The plates of isolates were incubated at 30± 2°C for 7 days.

3.2.2. Identification and characterization:

For identification of isolates of Endophytes following

morphological characteristics, microscopic studies and

biochemical properties were carried out.

Colony Characters: -

The morphological characters viz., size, shape, colour,

consistency, opacity, margin, elevation, motility, staining

reactions were studied in the laboratory.

1) Shape

The culture growth of 48 hours of all eight isolates along with

the type culture of Endophytes grown in semi-solid medium

were taken as smear and were stained with Dorner’s nigrosin

solution (S.A.B, 1957) and observed under oil immersion for

detecting shape of bacterial isolates.

2) Size

The cell size of Endophytes was measured by using ‘Filar’

micrometer. Smears stained with Zeihl’s fuschion solution

(S.A.B, 1957) and recorded the size of isolates.

3) Motility

The 48 hours old culture was taken for observing motility

by hanging drop method (SAB, 1957) under oil immersion

objective.

Procedure: - (Hanging drop technique)

a) A hanging drop method was done with the help of glass

slide with a concavity.

b) Minute quantity of Vaseline was applied to the four

corners of a cover slip and loopfull of culture was sticked

to the corners of a cover slip and inert the cavity slides

and centers the concavity over the drop of the cultures.

c) Slide was carefully turns so that the drop remains

suspended in cavity and the edges of the hanging drop

focused under low power.

3.2.2.2. Staining

The staining reaction, viz., Gram’s staining of the three

isolates of entophytes were carried out by the method as

described by society of American Bacteriologist. (1957).

Procedure:

1. Smear of sample were prepared, air-dried and heat

fixed.

2. Crystal violet used as primary stain for half minute. the

excess stain was removed with minimum quantity of

water

3. The slide was flooded with Gram’s iodine for half

minute. The iodine solution removed and decolorizer

added drop by drop on the slide for half minute then

rinsed in tap water to stop the decolourization

reaction.

4. Counter stain with saffranin was applied for one

minute, washed, dried and observed under oil

immersion lens.

3.2.2.3 Biochemical characteristics

1. Hydrolysis of starch

The type strain of Endophytes (Azospirillum,

Agrobacterium diazotrophicus, Azoarcus) was inoculated in

petriplates containing 0.2% soluble starch in respected solid

media. The incubation of petriplate was at 30°C temperatures. The

plates were flooded with weak logust iodine solution after 3 days

of incubation at 30°C. (S.A. B. 1957)

2. Catalase test

The 48 hours old culture were taken on slide and emulsified

with few drops of H2O2 10% (v/v). Effervesces due to liberation

of free O2 was considered as catalase positive. (SAB, 1957)

3. Liquefaction of gelatin

LGI agar medium, which was modified, by Smith (1945) and

Frazier (1926) was employed for detection of liquefaction of

gelatin. The medium was prepared by adding 0.4 % gelatin.

The plates were inoculated by Endophytes (Azospirillum,

Agrobacterium diazotrophicus, Azoarcus) and incubated at

30°C for 3 days. After 2 days, the plates were flooded with 10

ml, solution of HgCl2 in 100 ml, distilled water and 200 ml conc.

HCl. The observations were recorded for production of ‘haloes’

around the colonies and the intensity of liquefaction was

recorded arbitrarily. (SAB, 1957)

3.2.2 Screening of Isolates:

Screening of isolates was done on the basis of phosphate

Solublising ability and Nitrogen fixation capacity.

3.2.3.1 Phosphate solubilizing ability of Endophytes:

Pikovskayas agar medium with pH 5.5 was used and poured in

sterilized petriplates. After solidification of medium Endophytes

(Azospirillum, Agrobacterium diazotrophicus, Azoarcus) were

streaked on the medium. (A.C Gaur, 1990) Plates were

incubated at 30°C temperature for 5 days and then observed for

transparent zones of phosphate solublization surrounding the

colony of endophytes.

3.2.3.2 Nitrogen fixation capacity

Screening of Endophytes isolates for N2 fixation in vitro (by

KJELDAHL method)

Procedure

A colony was selected from the plate having pure culture of

endophytes and used for inoculating the broth for Nitrogen

fixation. For this purpose, 50 ml aliquots of broth were taken in

250 ml conical flasks for inoculation. After 5 days growth at

30°C at 110 rpm, the contents of flask were checked for purity

by streaking on fresh medium and concentrating over a water-

bath (50 to 60°C) to dryness. The dried culture was washed and

taken as a sample. The contents of the flask in inoculated

control series were processed in a similar manner.

3.2.4. Purification:

The pure isolates of endophytes are obtained by follows. –

3.2.4.1. Streak plate technique:

1. An appropriate colony was selected and streaked it on

another plate containing a respective medium. Streaking was done

in Zigzag manner.

2. The plates were incubated at 30+2°c.

3.2.5. Growth Analysis:

Counter, the help of colony calculated the viable count of an

isolates, by following formula & these can be used for further

analysis.

3.2.6. Formulation:

The obtained three isolates of endophytes are formulated

for their mass production as a Liquid Bioinoculant with the

help optimization of following parameters -

3.2.6.1 Optimumization of temperature range for growth of

Endophytes The growth of endophytes with respect to

different temperature range was studied in test tubes containing

semi-solid N-free medium. The test tubes were inoculated with

culture of endophytes and incubated at different temperatures

ranging from 20°C to 50°C. The effect of temperature on growth was

recorded after 10 days of incubation period.

3.2.6.2 Optimumization Hydrogen ion concentration (pH)

for growth of Endophyte isolates.

The response of Endophytes strains were studied at

different pH ranging from 3.5 to 6.5 by adjusting pH with the

help of 1N HCl or 1NaOH of respected broth N-free medium.

After sterilization of broth the pH of broth may change which

was again checked and readjusted aseptically. The isolates

were inoculated separately. The growth and change in pH was

observed after 5 days of incubation at 30°C.

3.2.6.3 Response of Endophytes to various sucrose

concentrations

The respected broth containing various concentration of

sucrose, as 5 % to 40% were inoculated with Endophytes

and recorded the growth after 5 days of incubation at 30°C.

3.2.6.4 Utilization of different carbon compounds

The carbon requirement for growth of Endophytes

(Azospirillum, Agrobacterium diazotrophicus, Azoarcus) was

estimated by growing the organisms on 10% of different carbon

source in the medium viz., sucrose, fructose, D-glucose,

maltose, mannitol, ethanol (1%) keeping other basic

composition of medium (basal medium) and opt. conditions

same for growth. The growth was recorded after 5 days

incubation at 30° C (Cavalcante and Dobereiner, 1988). The test

tube containing 10 % sucrose with basal medium serves as

control.

3.2.7 Media Designing:

Mass production was done with three isolates and two pure

strains of Endophytes viz. Acetobacter diazotrophicus and

Herbaspirrillum & their broth cultures are provided by institute

for further study.

On considering above parameters with their results and by

comparing the selective media’s of three isolates and two pure

strain appropriate medium was designed for the mass

production of all the endophytes as a Liquid Bioinoculant, and

named as A4H media. It is used for mass production by scale up

of Fermentation technology and finally mass production by

Fermentation based Biotechnology.

3.2.7. Mass Production:

After formulation and designing a media the mass

production of Endophytes were carried. Mother culture

prepared was further inoculated in newly designed media

A4H (250ml) & its scale up of fermentation up to 5 liter

was carried out further.

Mass production was carried out with 100 liters of A4H

media by fermentation-based biotechnology, using 5-10%

inoculums of scale up of fermentation.

3.2.8. Growth and Chemical analysis:

3.2.8.1.CHEMICAL ANALYSIS AFTER FORMULATIONS of

Broth:

I. Estimation of reducing sugar by DNSA method:

Reducing sugar estimation was carried out by DNSA reagent method

and the dilution system is given in table 3.1 as the dilutions were

completed optical density measured for calculating the

concentration of reducing sugar present in the sample. Glucose was

used as standard sample (2000µ g/ml).

Table 3.1: Dilution scheme for reducing sugar by DNSA

method

Glucose stock in ml

D.W. in ml

Final conc. in µg

DNSA ml Boil

For 10-15 Min AndCool

D.W. ml

0.2 0.8 20 1 80.4 0.6 40 1 80.6 0.4 60 1 80.8 0.2 80 1 8

81 - 100 1- 1 - 1 8

II) Estimation of Non Reducing Sugar by phenol sulphuric acid method:

Non Reducing sugar estimation was carried out by Phenol sulphuric

acid method and the dilution system is given in table 3.2 as the

dilutions were completed optical density measured for calculating

the concentration of non reducing sugar present in the sample and

sucrose was used as standard in concentration of (500µ g/ml).

Table 3.2: Dilution scheme for sucrose by phenol sulphuric acid method.

Sucrose ml

D/W ml

5% Phenol

Conc. H2SO4 ml

MixThoroughlyAndLetItCool & Measure the O.D.

0.1 0.9 1 50.2 0.8 1 50.3 0.7 1 50.4 0.6 1 50.5 0.5 1 50.6 0.4 1 50.7 0.3 1 50.8 0.2 1 50.9 0.1 1 5- 1 1 5

III) Estimation of protein by Folin Lowry method:

Protein estimation in the given sample was carried out by Folin

Lowry method and the dilution system is given in table 3.3 as the

dilutions were completed optical density measured for calculating

the concentration of protein present in the sample and standard was

Bovine Serum Albumin (100µ g/ml).

Table 3.4: Dilution scheme for Protein by Folin- Lowry method

BSA ml

D/W ml

Final conc. µg

Alkaline solution ml

MixThoroughlyAndIncubateAt RTFor10 min.

Folin coicaltean reagent ml

MixThoroughlyAndIncubateAt RT for30 min.& Measure the optical density at 750 nm.

0.1 0.9 10 5 0.50.2 0.8 20 5 0.50.3 0.7 30 5 0.50.4 0.6 40 5 0.50.5 0.5 50 5 0.50.6 0.4 60 5 0.50.7 0.3 70 5 0.50.8 0.2 80 5 0.50.9 0.1 90 5 0.5- 1 - 5 0.5.

3.2.8.2 Microbial Analysis:

By the help of colony counter, the viable count of an isolates were calculated, & these can be compared with before data. i.e., Growth ∝ rate of Nitrogen Fixation

COMPOSITION OF MEDIA

1. Azosperillum medium (for 1 liter.) (pH- 6.6-7)

(For Azosperillum)

Malic acid - 5gm

K2HPO4 -4gm

FeSo4 x 7 H20 – 0.05gm

Na2 Mo04 x 2 H20 – 0.002gm

MnSo4 x H20 - 0.01gm

MgS04 x 7 H20 -0.10gm

Nacl - o.o2gm

CaCl2 x 2 H20 - 0.01gm

Distil water - 1000ml

Agar-Agar - 30gm

2. Azoarcus medium (for 1 liter)(pH- 6.6-7)

(For Azoarcus)

Malic acid - 2-5gm

KOH - 2-5gm

KH2PO4 - 1.5gm

MgS04 x 7 H20 -1gm

Nacl -1gm.

Sodium Molybdate – 2mg.

CaCl2 -1gm

MnSo4 x H20 - 10mg

Fe EDTA - 66mg

Biotin - 1 mg

NH4Cl - 2mg

Beef Extract - 3gm

Yeast extract - 1gm

Agar-Agar - 15gm

Distil water - 1000ml

3. Agrobacterium Diazotrophicus medium (for 1 liter) (pH-5.5-

6.0) For (Agrobacterium Diazotrophicus)

Sucrose - 100gm

Nacl - 0.2 gm

MgS04 - 0.02 gm

CaCo3 - 1gm

Na MoO4 - 0.005gm

Agar - 15gm

4. LGI Medium (for 1 liter) (pH-5.5-6.0)

for Acetobacter Diazotrophicus.

Cane Sugar – 100 gm

KH2PO4 - 0.6 gm

K2HpO4 - 0.2 gm

MgS04 - 0.02 gm

Sodium molybdate – 0.002gm

Ferric Chloride - 0.01gm

CaCl2 - O.O2 gm

BTB - 5ml

Yeast Extract - 0.5 gm

Agar agar - 30gm

D/W - 1000ml.

5. Herbasperrillum medium (for 1 liter) (pH-7)

For HerbasperrillumKH2PO4 - 0.400 gm

K2HpO4 - 0.100 gm

MgS04 X 7 H20 - 0. 200 gm

Nacl - 0.100gm

Cacl2 - 0.020 gm

Fecl2 X 6 H20 - 0.010 gm

Sodium Molybdate - 0.002 gm

Yeast Extract - 0.025gm

D/W - 950 ml.

Agar Agar - 15 gm

Autoclave at 1200c for 15 min after sterilization add filter

sterilized solution A

Solution A = Sodium Malate 5.0 gm

Water 50ml (pH 7.0)

Results&

Discussion

Chapter 4

RESULT AND DISCUSSION

Endophytic bacteria are those bacteria that fix nitrogen

internally in plant tissues; Endophytic bacterial Nitrogen fixing

liquid Bioinoculant is a unique agro-based product in liquid

state, formulated with growth boosters and cell protectants and

it is a consortium of group of efficient Endophytic Nitrogen

fixing bacteria in live form.

This chapter gives the result and discussion of project work

under following headings.

4.1.Result

4.1.1 Isolation:

The three different isolates of an endophytes were obtained

i.e., Azospirillum, Agrobacterium diazotrophicus, Azoarcus.with

the help of respective selective media. Shows in Fig 4.1, 4.2 and

4.3

4.1.2 Identification and Characterization: - It was carried

out by morphological studies - Colony Characteristics and

microscopic studies.

The isolates are identified with Bergyess Manual.

4.1.2.1 Colony Characteristics of Endophytes: -

Table 4.1: Colony Characteristics of different isolates of Endophytes (Azospirillum, Agrobacterium diazotrophicus, Azoarcus) grown respective solid media at 30° c for 120 hrs

4.1.2 Microscopic Observations: -

Microscopic observation showed that the Endophytic bacteria

are Gram negative, short rods, motile with 2–3 lateral flagella.

Table 4.2 – Staining and Motility test of different isolates

of Endophytes.

M.Org/ Medium with temp. & time

Size (mm)

Shape Colour Margin Elevation

Consistency

Opacity

Azosprillium 300C for 120 hrs

0.4 Circular Greenish with white

metallic shine

Entire Convex Smooth Opaque

Azoarcus 300C for 120 hrs

0.3 Circular Insipid(Creamish white)

Entire Flat Smooth Opaque

Agrobacter diazotrophicus 300C for 120 hrs

1.0-2.0

Circular Dull white Entire Flat Moist Opaque

Organism Gram’s staining Motility

Azosperrillum Gram Negative Rods Sluggishly motile

Ag.diazotropicus Gram Negative Rods Sluggishly motile

Azoarcus Gram Negative Rods Sluggishly motile

4.2. Biochemical Characteristics: - The colony of

Endophytes (Azospirillum, Ag.diazotrophicus, and Azoarcus)

that was selected for colony characteristics was further selected

for biochemical study.

4.2.1.1. Hydrolysis Of starch: -

From the observations recorded (Table 3) shows that the

bacteria did not hydrolyze the starch, which is in agreement

worth report presented by Gills et al., (1989) and Bhowmik

(1995) who showed negative response of Endophytes

(Azospirillum, Ag. diazotrophicus, Azoarcus) to hydrolysis of

starch.

4.2.1.2. Catalase test: -

The investigations show (Table 4.3) that Endophytes

(Azospirillum, Ag. diazotrophicus, Azoarcus) isolates were

catalase positive further it was confirmed by the reports

mentioned by Dobereiner (1988); Stephan et al., (1991); L.E.

Fuentes – Ramirez et al., (1997).

4.2.1.3. Liquefaction of gelatin: -

The observation recorded (Tab 4.3) shows that Endophytes

(Azospirillum, Ag. diazotrophicus, Azoarcus) is weakly gelatin

liquefier. It showed a zone of clearance. Bhowmik, 1995 and

Gillis et al., (1989) showed that Acetobacter diazotrophicus was

unable to liquefy the gelatin.

Table 4.3: Biochemical Characteristics

TestM.org.

Hydrolysis of starch

Catalase test

Gelatin Liquefaction

Azospirillum -Ve +Ve +Ve

Ag.diazotrophicus -Ve +Ve +Ve

Azoarcus -Ve +Ve

+Ve

+Ve = Positive -Ve = Negative

4.2.1.4 Utilization of different carbon sources: -

From the Table 4.4 it was observed that Endophytes

(Azospirillum, Ag. diazotrophicus, Azoarcus) is able to utilize

different carbon sources such as sucrose, glucose, maltose,

fructose, mannose and ethanol (1%) with varying degree of

utilization. The most usable source was glucose, fructose, and

maltose, which also showed gas production.

Table 4.4: Utilization of different carbon source

Sugars >

Endo. Bacteria

Glucose Sucrose Fructose

Mannitol Mannose

Azosperrillum (+) + + + (+)

Ag.diazotrophicus

+ + + + +

Azoarcus + + + + -

Note: - + Indicates acid (+) Indicates acid and gas production.

4.2.2.4 Phosphate solubilizing ability of Endophytes:

The results indicate that Endophytes have ability to solublize

phosphate on Pikovskayas agar medium

Growth Analysis:

Table. 4.5 - Microbial Analysis

Dilutio

n no.

TVC of mother

cell

TVC of CP

added culture

after 7 days.

TVC of CP

added culture

after 15 days.Endo-

phytes

>

Agr Azr Azsp. Agr Azr Azsp. Agr Azr Azsp.

10-1 >30

0

>30

0

>300 >30

0

>30

0

>300 >30

0

>30

0

>300

10-2 >30

0

>30

0

>300 >30

0

>30

0

>300 >30

0

>30

0

>300

10-3 >30

0

>30

0

>300 >30

0

>30

0

>300 >30

0

>30

0

>300

10-4 >30

0

>30

0

>300 >30

0

>30

0

>300 >30

0

>30

0

>300

10-5 >30

0

>30

0

>300 >30

0

>30

0

>300 >30

0

>30

0

>300

10-6 234 265 188 >30

0

>30

0

>300 >30

0

>30

0

>300

10-7 232 238 154 >30 >30 >300 >30 287 >300

0 0 010-8 189 176 100 >30

0

>30

0

>300 >30

0

254 >300

10-9 176 166 98 >30

0

>30

0

>300 >30

0

232 214

10-10 123 122 76 >30

0

>30

0

209 234 212 209

10-11 100 98 65 >30

0

>30

0

167 212 198 189

10-12 65 53 34 193 >30

0

123 178 167 187

4.2.2.1. Screening of Endophytes for N2 fixation in vitro:

Nitrogen fixation was carried out in 50 ml broth (10 % Sucrose)

containing single colony of Endophytes selected for

identification and screening. Nitrogen, mg per gram of sucrose

consumed.

Table. 4.6 – Screening of Endophytes for N2 fixation in vitro

Sr.No.

Name of Endophyte

N2 fixed in mg/gm of sucrose consumedDry weight basis50 ml medium broth

Liquid weight basis50 ml medium broth

01. Agr. Diazotrophicus 38.46 11.36

02 Azospirillum 20.46 4.44

03 Azoarcus 46.66 10

Screening was further carried out for its efficiency of

Nitrogen fixation by Micro- Kjeldahl method and it was observed

that the selected colony of most efficient endophytic bacteria

fixes 49.30 mg of Nitrogen per gm of sucrose consumed (As per

calculation) in a respected broth containing 10% sucrose

incubated at 30°C for 120 hours at laboratory scale.

4.3. Formulations:

In the beginning mother culture was prepared from

selected efficient strains of Endophytes and further mass

production was carried out by scale up of fermentation in new

A4H medium broth. Cell protectants 1 and 2 were added at

initial stage of inoculation during mass production in replicates

with control.

It has been observed that after 7 days of growth sucrose,

reducing sugar, protein and acidity (pH) estimated shows

normal growth with pH around 4.5. Hence it was decide to add

Cell protectant 1 and Cell protectant 2 after growth of 120

hours incubation at 30°C. Initial analysis of Cell protectant 1 and

cell protectant 2 showed that cell protectant 1 is liquid oil base,

when added to culture it shows a moiety of oil in which cells

were embedded under microscopic field. Cell protectant 1 is a

inert oil base cell protectant with pH around 6.8 to 7.0. Hence it

helps to increase pH of culture, which are around 3.5 to 4.5

after growth.

Cell protectant 2 is a amorphous (solid) cell protectant

with pH around 6.5 to 7.0. Both cell protectants 1 and 2 were

sterilized by filtration technique and added to the culture as per

formulation table. It has been observed that cell protectant 2

adjusted pH 7 with 10 gm quantity whereas cell protectant 1

adjusted pH 6.23 but considerable quantity required 100 ml and

its cost is also high. So it has been decide to continue the

experiment with cell protectant 2. In addition when both were

used in combination it has been observed that they did not

show effective result with respect to pH adjusted. Hence, cell

protectant 1 and

Cell protectant 2 was analyzed for sucrose, reducing sugar and

protein content. It has been observed that cell protectant do not

have any of the above constituent. Hence, it was decide as cell

growth booster or cell protectant whereas cell protectant 1 as a

cell protectant only at the final stage of packing of culture.

4.3.1. Optimization:

4.3.1.1. Optimum temperature range for growth of

Endophytes.

From the observation Table-4.5 it can be recorded that the growth of

Endophytes ranges between 20˚C to 50˚C temperature.

Table 4.7: Temperature range for growth of Endophytic

bacteria

Temp-

Endo. Bacteria

20˚c 25˚c 30˚c 35˚c 40˚c 45˚c 50˚c

Azospirilum + + + + + + + +

+ + +

+ - -

Ag.diazotrophicus + + +

+ + + + + +

+ + +

+ + + + + +

Azoarcus + + +

+ + + + + +

+ + + + -

- = No growth + = Poor growth

++ = Good growth +++ = Excellent Growth

4.3.1.2. Optimum Hydrogen ion concentration (pH) for

growth of Endophytic bacteria

From the Table No.4.6- it was revealed that the optimum

pH required for growth was between 5.5 to 6.5 The minimum

growth was observed at4.5 but the microbial population was

low, whereas the maximum pH tolerated at 7.5 with low density

of microbial population.

Table 4.8 - Optimum hydrogen ion concentration (pH) for

growth of Endophytic bacteria

Range of pH.

4.5 5.0 5.5 6.0 6.5 7.0 7.5

Ag.diazotropicus

+ + + + + + + + + + + + +

Azoarcus + + + + + + + + +

+ + + + +

Azospirillum + + + + + + + +

+ + +

+ +

- = No growth + = Poor growth

++ = Good growth +++ = Excellent Growth

4.3.1.2. Response of Endophytic bacteria to various sucrose

concentrations

For recording the response of Endophytic bacteria to different

sucrose concentration from 5% to 40% concentration range

were taken. (Table 4.7) The observations showed that there was

a good growth at 20% to 30% sucrose concentration of

Endophytes, whereas at 40% sucrose concentration and at 35%

sucrose concentration growth of A.diazotrophicus was

hampered.

Table. 4.9 -Response of Endophytes to various sucrose

concentrations.

Sucrose conc. 10% 20% 30% 40%

Azospirilum + + + + + + + +

Ag.diazotrophicus (+) (+)(+)

(+)(+)(+)

+ + +

Azoarcus + + + + + + +

(+) = Acid gas production - = No growth + = Poor

growth

++ = Good growth +++ = Excellent Growth

4.4. Growth and Chemical analysis:

4.4.2 Chemical Analysis:

4.4.2.1. Reducing sugar and Non Reducing Sugar

Initial A4H broth contained more concentration of sucrose

i.e. 10% = 10 gm (10 7 mg) but after sterilization sucrose

content were found to be inverted and sucrose contents

103 ,whereas reducing sugar content was 10 4 further after

inoculation and incubation sucrose were found to be decrease

to 10 2 and reducing sugar to 10 4. It suggest that bacteria

utilize some of the reducing sugar as well as some of the

sucrose during their growth and reducing pH from 5.5 to

3.65.After 7 days of inoculation formulation of sucrose content

was increasing to 7.3 x 10 3 with increase in reducing sugar to

1.1 x 10 4 at this stage increase in both sucrose as well as

reducing sugar suggest that cell protectant 2 acts as a source

of sucrose which may be resulting in to increase in sucrose

there was no any change in reducing sugar. Hence cell

protectant 2 more acts as a cell growth protectant. Microbial

counts during this stage suggest the same trend. After growth

when cell protectant 2 was added pH was adjusted to 7 with

increase in sucrose content from 10 2 to 10 3. Results were

shown in graph 4.5 and 4.6.

Table 4.10: Dilution scheme for reducing sugar by DNSA method

Glucose stock in ml

D.W. in ml

Final conc. in µg

DNSA ml Boil

For 10-15 Min AndCool

D.W. ml

O.D. at 540 nm

0.2 0.8 20 1 8 0.01120.4 0.6 40 1 8 0.05260.6 0.4 60 1 8 0.07290.8 0.2 80 1 8

80.1072

1 - 100 1 0.1525- 1 - 1 8 0

Table 4.11: Dilution scheme for sucrose by phenol sulphuric acid method.

Sucrose ml

D/W ml

5% Phenol

Conc. H2SO4 ml

MixThoroughlyAndLetItCool

O.D. at 480 nm

0.1 0.9 1 5 0.00540.2 0.8 1 5 0.05280.3 0.7 1 5 0.13510.4 0.6 1 5 0.17000.5 0.5 1 5 0.33380.6 0.4 1 5 0.33430.7 0.3 1 5 0.42180.8 0.2 1 5 0.49010.9 0.1 1 5 0.6106- 1 1 5 0

4.4.2.2. Protein estimation:

Protein in the initial broth was 10 4 mg inform of yeast

extract prior to sterilization but after sterilization it was found to

be decreased to 10 2 . It indicates sterilization denaturates the

protein content similar to sucrose inversion. But a cell

protectant 2 additions doesn’t affect protein content. Protein

shows slightly increase after 7 days from 1.9x10 1 to 2.4 x 10 1.

Further 15 days analysis shows that there is slightly decrease in

protein content to 1.8 x 10 1 and after 21 days study shows that

there was again slight increase in protein content. This

fluctuation in protein content may be due to cell division and

cell destruction. (Metabolism). Results were shown in graph 4.7.

Table 4.12: Dilution scheme for Protein by Folin- Lowry method

BSA ml

D/W ml

Final conc. µg

Alkaline solutio

Folin coicaltean

O.D. at 750 nm

n mlMixThoroughlyAndIncubateAt RTFor10 min.

reagent ml

MixThoroughlyAndIncubateAt RT for30 min.

0.1 0.9 10 5 0.5 0.22360.2 0.8 20 5 0.5 0.36460.3 0.7 30 5 0.5 0.47150.4 0.6 40 5 0.5 0.57530.5 0.5 50 5 0.5 0.71350.6 0.4 60 5 0.5 0.77970.7 0.3 70 5 0.5 0.87330.8 0.2 80 5 0.5 0.96050.9 0.1 90 5 0.5 0.995- 1 - 5 0.5. 0

DNSA Graph

y = 0.0002x - 0.0219

0

0.05

0.1

0.15

0.2

100 300 500 700 900 1100

ug Glucose

Ab

s at

550

nm

Graph. 4.5. Standard Graph for Reducing Sugar Estimation:

Sucrose Est. Graph

y = 0.0072x - 0.0805

00.10.20.30.40.50.60.7

5 25 45 65 85 105

ug conc. in Sucrose

Abs

at 4

80 n

m

Graph 4.6 Standard Graph for Sucrose Estimation:

Protein Std Graph

0

0.20.4

0.60.8

11.2

0 20 40 60 80 100

ug Conc. in protein

Ab

s a

t 7

50 n

m

Graph 4.7 Standard Graph For Protein Estimation:

Table 4.13 Chemical Analysis:

Observations

Reducing Sugar content

Sucrose content

Protein content

Acidity (pH)

(µ g/100 ml)

(OD at 550n)

(µ g/100ml)

(OD at 550nm)

(µ g/100ml)

(OD at 550nm)

Initial A4H BrothAfter sterilization

2.5 x104

1.0345

9.6 x104

3.730 9.6 x 103

0.252 6.5

A4H culture + G. booster/cell protectant

8.8 x104

3.600 4.6 x104

3.008 1.1 x 104

0.290 7.00

After 7 days of adding cell protectants

4.3 x105

17.590

4.9 x104

3.067 2.0 x104

0.458 5.35

After 15 days of adding cell protectants

5.1 x105

20.75 3.6 x104

2.900 1.4 x104

0.367 4.84

4.4.3. Microbial Analysis:

Microbial analysis of culture prior to addition of cell protectant 2

showed Acetobacter count in range of 53 x 10 –12 at pH 3.65.

After addition of cell protectant 2 after 7 days microbial count

was found to be more than 300 for 10 –12 dilution with decrease

in pH to 4.84 from 7. Further 15 days count in range of 19.2 x

10 –12 with pH 4.65.

After 21 days analysis it was found to be 154 x 10 –12 with pH

4.3-6. Microbial count and pH studies indicates that after

formulation with cell protectant 2 there was sudden increase in

microbial count but gradual decrease after 15 days to 21 days

with respect to pH there was sudden decrease in pH during first

7 days after formulation with cell protectant 2 and further

gradual decrease in pH was observed up to 21 days.

Comparative Analysis Studies of chemical and microbial

parameter shows that initial sucrose content of A4H broth was

reduced during sterilization due to inversion of sucrose resulting

in formation of reducing sugar. Endophytes utilize both during

their growth period of 5 days. Further formulations of

Endophytic culture with cell protectant 2 to pH 7 increase the

sucrose content, which was further utilize by bacteria with

increase in reducing sugar. During this period it has been

observed that both reducing sugar and sucrose was utilized

simultaneously with minute fluctuation in pH. Further it was

observed that cell metabolism is leading to decrease in protein

content in minute quantity.

Microbial count of initial broth (53 x 10-12) has been boosted to

more than 300 x 10-12 it indicates that bacteria are utilizing

sucrose provided by cell protectant 2 and also reducing sugar

during metabolism and showing steady decrease in their count

up to 21 days.

Further studies will be carried out for 6 months of period for

sucrose, reducing sugar, protein, pH and microbial count in

order to estimate this contents and shelf life of product. Prior to

packing of the product depending on the final pH adjustment

will be carried out with some weak bases and antitox after

addition of 10 ml of cell protectant 1 per liter cell protectants.

Table.4.14- Microbial Analysis

Dilution no. TVC of mother

cultureTVC of cell protectant added culture After 7 days

TVC of cell protectant added culture After 15 days

10-1 >300 >300 >30010-2 >300 >300 >30010-3 >300 >300 >30010-4 >300 >300 >30010-5 >300 >300 >30010-6 245 >300 >300

10-7 232 >300 >30010-8 192 >300 >30010-9 178 >300 >30010-10 135 >300 >30010-11 96 >300 >30010-12 53 >300 192

DISCUSSUION

From the observations recorded (Table 3) shows that the

bacteria did not hydrolyze the starch, which is in agreement

worth report presented by Gills et al., (1989) and Bhowmik

(1995) who showed negative response of Endophytes

(Azospirillum, Ag. diazotrophicus, Azoarcus) to hydrolysis of

starch. The investigations show (Table 4.3) that Endophytes

(Azospirillum, Ag. diazotrophicus, Azoarcus) isolates were

catalase positive further it was confirmed by the reports

mentioned by Dobereiner (1988); Stephan et al., (1991); L.E.

Fuentes – Ramirez et al., (1997). The observation recorded

(Table 4.3) shows that Endophytes (Azospirillum, Ag.

diazotrophicus, Azoarcus) is weakly gelatin liquefier. It showed

a zone of clearance. Bhowmik, 1995 and Gillis et al., (1989)

showed that Acetobacter diazotrophicus was unable to liquefy

the gelatin. Calvalcate and Dobereiner (1988) reported that

besides 30% of sucrose, which was proved to be best carbon

source for growth of A.diazotrophicus, they also observed good

response for glucose, fructose, ethanol (1%), mannitol, and

maltose. They also found bet growth at high sucrose or glucose

concentration (10%) and strong acid production led to a final

pH of 3.0 or below. Bhowmik (1995) reported that glucose and

sucrose are best carbon source for growth of A.diazotrophicus.

The optimum temperature was observed at 30˚C (Bhowmik,

1995). There was a report that optimum temperature for

growth of A.diazotrophicus about 30˚C (Cavcalcante and

Dobereiner, 1988 and Gillis et al., 1989).Cavalcant and

Dobereiner (1988) reported that the suitability for the growth

of A.diazotrophicus at pH 4.5. The faster growth was obtained

at more acid i.e. pH 3.9. Stephan et al., (1988) reported that

pH 3.0 or below were suitable for growth and N2 fixation.

Further Stephan et al., (1991) revealed from their studies that

pH range was from 2.5 to 7.5, optimum pH of 5.5. Gillis et al.

(1989) found the excellent growth at pH 5.5 but no growth

occurs at pH 7.0.Therefore, the present investigation for

response to pH was in conformity with the above-mentioned

reports. (Cavalcante and Dobereiner 1988, Gillis et al., 1989;

Stephan et al., 1991) The same can be confirmed from report

of Bhowmik, (1995) that the optimum range of pH was between

5.6 to 6.6.There was report that the best growth occurred at

high sucrose concentration (10%) and even up to 30%

(Cavalcante and Dobereiner, 1988; Boddy et al., 1991).

Bhowmik, (1995) also reported that Nitrogen dependent growth

occurred between 1% to 30% cane sugar concentration with an

optimum between 10 and 15%.

Chapter v

SUMMARY AND CONCLUSION:

Isolation, Identification & Screening of different endophytic N2

fixing bacteria from sugarcane. Selection of efficient strains of

these endophytic N2 fixing bacteria was carried out for mass

production of endophytic N2 fixing bacterial bioinoculant through

fermentation based biotechnologies, by designing a new

common media and formulation of the same with cell

protectants & cell growth boosters. This unique product of

consortium of efficient endophytic N2 fixing bacteria.

A quality product formulated with Cell Growth Booster & Cell

Protectant with neutral pH, higher shelf life, easy in handling

storage and application with benefit ratio & ideal cost has been

developed. Such type of liquid formulation with CGB & CP will

Summary, Conclusion

also increase utilization efficiency of Liquid Bioinoculant by

stem leaves & plantlets, over average standard set or seed

treatment, foliar application & deeping of plantlets.

This endophytic N2 fixing Bioinoculant for different crops having

sucrose, including sugarcane for increasing yield and quality of

crop.

Considering the importance of endophytic bacteria in sugarcane

and other crops, with respect to biological nitrogen fixation,

present studies of isolation, screening and selection of efficient

strain of endophytic bacterial isolates and their mass production

as liquid bioinocolants with fermentation based biotechnology

has been undertaken.

In nonlegumes such as sugarcane from gramenecios family

endophytic diazotrophs such as Acetobacter, Azoarcus,

Herbaspirillum, Agrobacterium diazotrophicus and

Azosperrillum are presents in all parts of plant including leaf,

stem, roots and juice. The recent discovery of the endophytic

diazotrophs bacteria such as Acetobacter diazotrophicus,

Herbaspirillum spp. and Azoarcus spp. colonizing the interior of

sugarcane, rice and Kallar grass (Leptochloa fusca [Diplachne

fusca]), respectively, and other species of grasses as well as

cereals, has led to a considerable interest in exploring these

novel associations. There is a general consensus that plant

genotype is a key factor to higher contributions of BNF together

with the selection of more efficient bacterial strains. Nitrogen-

fixing bacteria are important in modern agriculture - exploiting

these bacteria would decrease the present dependency on

nitrogen fertilizers, which would have positive results for the

ecosystem and the health of humans and other animals.

CONCLUSION:

Endophytic bacterial Nitrogen fixing Bioinoculant is special

product with newly developed A4H medium with high cell count, zero

contamination, longer shelf life, greater protection against

environment stresses, increased field efficiency with respect to

spreading and penetration and convenience of handling are main

features of the this product.

In sugarcane endophytic diazotrophic bacteria like

Azospirillum, Azorcus, Agrobacterium diazotrophicus are present in

all parts of plant including left, stem, roots and juice. These

endophytic diazotrophs actively participates in biological nitrogen

fixation and fixes more Nitrogen as compare to ectophytic bacteria.

These bacteria’s were isolated successfully and they were

screened and compared with Bergyess manual.

Biological nitrogen fixing system offers an economically attractive and

ecologically sounds means, of externally reducing external inputs and improving

internal resources. Hence Biological Nitrogen fixation has been an interesting area of

research over several decades.

“Isolation, Identification and Screening of Endophytic NITROGEN FIXING bacteria from sugarcane and selection of efficient strains for their mass production as liquid state Bioinoculant with Formulations by fermentation based biotechnology.”

By Laxman Savalkar

ABSTRACT

Endophytic bacterial Nitrogen fixing Bioinoculant is special product with newly developed A4H medium with high cell count, zero contamination, longer shelf life, greater protection against environment stresses, increased field efficiency with respect to spreading and penetration and convenience of handling are main features of the this product. The proposed investigation was carried out with following objectives: Isolation, Identification and screening of efficient strains of Endophytes liquid Bioinoculant production and for Biological Nitrogen Fixation., Formulation of liquid entophytic Bioinoculant with cell protects ants., Efficiency test for Liquid Bioinoculant.,Growth and Chemical analysis.

Entophytic bacteria were isolated from different parts of various sugarcane Varieties, they are screened, and used for mass production as an liquid biofertiliser. These bacteria fix nitrogen internally on utilizing starch (byproduct of sugarcane and many cereal crops), as well as some strains fixes atmospheric nitrogen also. Formulation of Endophytic liquid Bioinoculant with cell growth booster and cell protectant may result into development of quality product with neutral pH, higher shelf life, ease in handling, storage and application with benefit ratio ideal cost help in application and its utilization by plant. It will increase

utilization efficiency of liquid Bioinoculant by stem; leaves and plantlets average standard treatment, foliar application and dipping in plantlets.

Date: Mrs.Chaitali Niratker

(Major Advisor)

Bibliography

BIBLIOGRAPHY

Bellone, C. H., De Bellone, S. D.C. Padrase R, and Monson, M.

A. (1997).

Cell colonization and infection thread formation in

sugarcane roots by Acetobacter diazotrophicus. Soil.

Biol-Biochem, 29;965-967.

Baldani, V.L.D., Baldani, J.I., Olivers, F., Doberenier, J. 1992

Identification of Herbaspirillum seriopicae and the

closely related Pseudomonas rubrisubalbicans.

Symbiosis; 13:65-73.

Baldani J. L, Baldani, V.L.D., Doberner J. 1986.

Characterization of Herbaspirillum seropedicae gen nov

sp. Nov:a root associated nitrogen fixing bacterium. Int.

J. Syst. Bacteriol; 36:86-93

Boddey R. M. 1993.

‘Green’ energy from sugarcane. Chem. And Ind.; 17 May

1993. pp 355-358.

Bellone, C. H., De Bellone, S. D.C. Padrase R, and Monson, M. A.

(1997). Cell colonization and infection thread formation

in sugarcane roots by Acetobacter diazotrophicus. Soil.

Biol-Biochem, 29; 965-967.

Baldani, J. I., B. Pot, G. Kirchhof, E. Falsen, V. L. D. Baldani, F. J.

Olivares, B. Hoste, K. Kersters, A. Hartmann, M. Gillis, and J.

Döbereiner. 1996.

Emended description of

Herbaspirillum; inclusion of [Pseudomonas] rubrisubalbicans,

a mild plant pathogen, as Herbaspirillum comb. nov.; and

classification of a group of clinical isolates (EF group 1) as

Herbaspirillum species 3. Int. J. Syst. Bacteriol. 46:802-810.

Baldani, J. I., L. Caruso, V. L. D. Baldani, S. Goi, and J.

Dobereiner. 1997.

Recent advances in BNF with non-legume plants. Soil

Biol. Biochem. 29:911-922.

Baldani, J. I., V. L. D. Baldani, L. Seldin, and J. Dobereiner.

Characterization of Herbaspirillum seropedicae gen.

nov., sp. nov., a root-associated nitrogen-fixing

bacterium. Int. J. Syst. Bacteriol. 34:451-456.

Baldani, V. L. D., and J. Dobereiner. 1980.

Host-plant specificity in the infection of cereals with

Azospirillum spp. Soil Biol. Biochem. 12:433-439.

Bani, D.,barberio, c.,Bazzicalupo,M.,Favilli, F.,Gallori,E. &

Polsinelli,M. (1980)

Isolation and characterization of glutamate synthase

mutants of Azospirillum brasilense.J Gen Microbial

119,239-244.

Barraquio, W.L.,L.Revilla, and J.K.Ladha. 1997.

Isolation of endophytic bacteria from wetland rice. Plant

Soil 194:15-24

Boddy, R.M.1995.

Biological nitrogen fixation in sugarcane: a key to

energetically biofuel production. Crit. Rev. Plant Sci.

14:263-279.

Bashan, Y., Levanony, H. & Whitmoyer, E. (1991).

Root surface colonization of non cereal crop plants by

pleomorphic Azospirillum brasilense Cd. J gen Microbial

137, 187-196.

Cavalcate V. A., Dobereiner, J. 1998.

A new acid-tolerant nitrogen fixing bacterium associated

with sugarcane. Plant & Soil; 108:23-31.

Dobereiner J.; A. C. S. Abbound, V.M. Reis, F.L. Dedivages, F.B.

Dos Reis Junior and R. M. Boddey (1993).

Elimination of fertilizer for sugarcane in Brazilian

nitrogen fixing cane Genotypes. Key to a high-energy

balance for biofuel production. Inter American sugarcane

Seminar, Miami,14-17 sept.

Dawe, D. 2000.

The potential role of biological nitrogen fixation in

meeting future demand for rice and fertilizer, p. 1-9. In J.

K. Ladha, and P. M. Reddy (ed.), The quest for nitrogen

fixation in rice. International Rice Research Institute, Los

Banos, Philippines.

Dong, Z., M. E. McCully, and M. J. Canny. 1997.

Does Acetobacter diazotrophicus live and move in the

xylem of sugarcane stems? Anatomical and physiological

data. Ann. Bot. 80:147-158

Egener, T., T. Hurek, and B. Reinhold-Hurek. 1998.

Use of green fluorescent protein to detect expression of

nif genes of Azoarcus sp. BH72, a grass-associated

diazotroph, on rice roots. Mol. Plant-Microbe Interact.

11:71-75

Elbeltagy, A., K. Nishioka, H. Suzuki, T. Sato, Y. Sato, H.

Morisaki, H. Mitsui, and K. Minamisawa. 2000.

Isolation and characterization of endophytic bacteria

from wild and traditionally cultivated rice varieties. Soil.

Sci. Plant Nutr. 46:617-629.

Engelhand, M., T. Hurek, and B. Reinhold-Hurek. 2000.

Preferential occurrence of diazotrophic endophytes,

Azoarcus spp., in wild rice species and land races of

Oryza sativa in comparison with modern races. Environ.

Microbiol. 2:131-141

Fujie, T., Y. D. Huang, A. Higashitani, Y. Nishimura, S. Iyama, Y.

Hirota, Y. Yoneyama, and R. A. Dixon. 1987.

Effect of inoculation with Klebsiella oxytoca and

Enterobacter cloacae on dinitrogen fixation by rice-

bacteria associations. Plant Soil 103:221-226.

Hiraishi, A., K. Furuhata, A. Matsumoto, K. A. Koike, M.

Fukuyama, and K. Tabuchi. 1995.

Phenotypic and genetic diversity of chlorine-resistant

Methylobacterium strains isolated from various

environments.

Hurek, T., B. Reinhold-Hurek, M. Van Montagu, and E.

Kellenberg. 1994.

Root colonization and systemic spreading of Azoarcus

sp. strain BH72 in grasses. J. Bacteriol. 176:1913-1923.

James, E. K. 1999.

Nitrogen fixation in endophytic and associative

symbiosis. Field Crop Res. 65:197-209.

James, E. K., V. M. Reis, F. L. Olivares, J. I. Baldani, and J.

Dobrereiner. 1994.

Infection of sugarcane by the nitrogen-fixing bacterium

Acetobacter diazotrophicus. J. Exp. Bot. 45:757-766

James, E. K., and F. L. Olivares. 1998.

Infection and colonization of sugarcane and other

graminaceous plants by endophytic diazotrophs. Crit.

Rev. Plant Sci. 17:77-119.

James, E. K., F. J. Olivares, J. I. Baldani, and J. Dobereiner.

Herbaspirillum, an endophytic diazotroph colonizing

vascular tissue in leaves of Sorghum bicolor L. Moench.

J. Exp. Bot. 48:785-797.

Kirchhof, G., B. Eckert, M. Stoffels, J. I. Baldani, V. M. Reis, and

A. Hartmann. 2001.

Herbaspirillum frisingense sp. nov., a new nitrogen-fixing

bacterial species that occurs in C4-fibre plants. Int.

J. Syst. Evol. Microbiol. 51:57-68.

Mae, T., and K. Ohira. 1981.

The remobilization of nitrogen related to leaf growth and

senescence in rice plants (Oryza sativa L.). Plant Cell

Physiol. 22:1067-1074.

Malmqvist, A., T. Welander, E. Moore, A. Ternstrom, G. Molin,

and I. Stenstrom. 1994.

Ideonella dechloratans, gen. nov., sp. nov., a new

bacterium capable of growing anaerobically with

chlorate as an electron acceptor. Syst. Appl. Microbiol.

17:58-64.

More R., Phonde D., Patil A.,

Endophytes as liquid bioinoculant boon for cane growers,

(Agro wan Sakal, 14th June 2007)

Olivares, F. L., V. L. D. Baldani, V. M. Reis, J. I. Baldani, and J.

Dobereiner. 1996.

Occurrence of the endophytic diazotrophs

Herbaspirillum spp. in root, stems, and leaves,

predominantly of Gramineae. Biol. Fertil. Soils 21:197-

200.

Reinhold-Hurek, B., and T. Hurek. 1998.

Life in grasses: diazotrophic endophytes. Trends

Microbiol. 6:139-144.

Rennie, R. J. 1981.

A single medium for the isolation of acetylene-reducing

(dinitrogen-fixing) bacteria from soils. Can J. Microbiol.

27:8-14.

Sevilla, M.,R.H. Burris, N. Gunapala, and C. Kennedy. 2001.

Comparison of benefit to sugarcane plant growth and

15N2 incorporation following inoculation of sterile plants

with Acetobacter diazotrophicus wild-type and nif

mutant strains. Mol. Plant-Microbe Interact. 14:359-366.

Smibert, R. M., and N. R. Krieg. 1981.

General characterization, p. 409-443. In P. Gerhardt, R.

G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N.

R. Krieg, and G. B. Phillips (ed.), Manual of methods for

general bacteriology. American Society for Microbiology,

Washington, D.C.

Sturz, A. V., B. R. Christie, and J. Nowak. 2000.

Bacterial endophytes: potential role in developing

sustainable systems of crop production. Crit. Rev. Plant

Sci. 19:1-30.

Tou, C., and F. Zhou. 1989.

Non-nodular endorhizospheric nitrogen fixation in

wetland rice. Can. J. Microbiol. 35:403-408.

valverde , A,., Velazquez, E., Gutierrez, C., Cervantes, E.,

Ventosa. A., Igual,J. M. (2003).

Herbaspillum lusitanum sp. Nov. , a novel nitrogen fixing

bacterium associated with root nodules of phaseolus

vulgaris. Int J Syst Evol Microbial 53: 1979-

1983[Abstract] [full text].