Agrobacterium Diazotrophicus As A Liquid Biofertilizer.

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 Society College OF Agriculture Biotechnology Raipur-492006 (C.G) INDIA.

By Laxman A. Savalkar Roll No. 4537 37

ID No. UG/BIO/03/18A-

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:

Members of advisory committee 1. Mrs. Archana Prasad

2. Mrs. Kiran Kumari

Mrs. Chaitali Niratker

“Dedicated

to

My beloved

Parents

Who 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 AgBiotechnology 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 Chap ter No 01 02 03 04 05

Topics

Page No.

Introduction Review of literature Material and Method Result & Discussion Summery & Conclusion Abstract Bibliography

List of Figures/Graphs Figure No.

Particulars

4.1

Isolated Strain Of Agrobacterium

Page No.

4.2

Diazptrophicus Isolated Strain Of Azosperrillum

4.3

Isolated Strain Of Azoarcus

4.4

Pure Strain of Endophytes Given by

45

Institute Dilution scheme for reducing sugar by

4.6

DNSA method. Dilution scheme for sucrose by phenol

4.7

sulphuric acid method. Dilution scheme for protein by FolinLowery method.

List of tables Table

Particulars

No. 3.1

Dilution scheme for reducing sugar by

3.2

DNSA method. Dilution scheme for sucrose by phenol sulphuric acid method.

Page No.

3.3

Dilution scheme for protein by Folin-

4.3

Lowery method. Biochemical characteristics of endophytes

4.4

Utilization of different carbon source by

4.5

endophytes. Screening of Endophytes for N2 fixation

4.6

in vitro. Temperature range for growth of Endophytic bacteria.

4.7

pH range for growth of Endophytic bacteria.

4.8

Response of Endophytes to various

4.9

sucrose concentrations. Dilution scheme for reducing sugar by

4.10

DNSA method. Dilution scheme for sucrose by phenol

4.11

sulphuric acid method. Dilution scheme for protein by Folin-

4.12

Lowery method. 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 bioinoculants. 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.

Review of Literature

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

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 nonrenewable 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

Azospirillum

J.

Dobereiner. are

capable

1980. of

Members nitrogen

of

the

fixation

genus 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,

Azospirillum

seropedicae

was

renamed

seropedicae

(Baldani

al.,

1986a).

et

as

thus

Herbaspirillum

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 N 2-

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

N

15

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 showed that

N study by Lima et al., (1987)

15

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 nonlegume 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-JuniorFB-dos;

Reis-VM;

Urquiaga-S;

Dobereiner-Brazil

(2000).

Comparison of benefit to sugarcane plant growth and incorporation

following

inoculation

of

sterile

Acetobacter diazotrophicus wild type and nif

plants

N2

15

with

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

N study by Lima et al.,

15

(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).

Materials and Methods

Chapter 3 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.

10 ml sample from bottle first and transferred it to next

3.

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,

reactions were studied in the laboratory.

1)

Shape

motility,

staining

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.

Slide was carefully turns so that the drop remains

c)

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

immersion lens.

3.2.2.3 Biochemical characteristics 1.

Hydrolysis of starch

observed

under

oil

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

2.

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 0.2 0.4 0.6 0.8 1 -

D.W. in Final ml conc. in µg 0.8 20 0.6 40 0.4 60 0.2 80 100 1 -

DNSA ml 1 1 1 1 1 1

Boil For 10-15 Min And Cool

D.W. ml 8 8 8 8 8 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.

Sucros e ml 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 -

D/ W ml 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 1

5% Pheno l 1 1 1 1 1 1 1 1 1 1

Conc. H2SO4 ml 5 5 5 5 5 5 5 5 5 5

Mix Thoroughly And Let It Cool & Measure the O.D.

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 BS A ml

D/W Final Alkalin ml conc e . µg solutio n ml

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 -

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 1

10 20 30 40 50 60 70 80 90 -

5 5 5 5 5 5 5 5 5 5

Mix Thoroughl y And Incubate At RT For 10 min.

Folin coicaltea n reagent ml 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5.

Mix Thoroughl y And Incubate At RT for 30 min.& Measure the optical density at 750 nm.

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 CaCl2 x 2 H20

- o.o2gm - 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 Distil water

- 15gm - 1000ml

3. Agrobacterium Diazotrophicus medium (for 1 liter) (pH-5.56.0) For (Agrobacterium Diazotrophicus) Sucrose

- 100gm

Nacl

- 0.2 gm

MgS04

4.

- 0.02 gm

CaCo3

- 1gm

Na MoO4

- 0.005gm

Agar

- 15gm

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 Herbasperrillum KH2PO4 - 0.400 gm K2HpO4 MgS04

X

- 0.100 gm 7 H20

-

Nacl Cacl2 Fecl2 X 6 H20

0. 200 gm - 0.100gm

-

0.020 gm - 0.010 gm

Sodium Molybdate - 0.002 gm Yeast Extract D/W Agar Agar

- 0.025gm - 950 ml. - 15 gm

(for 1 liter) (pH-7)

Autoclave at 1200c for 15 min after sterilization add filter sterilized solution A Solution A = Water

Sodium Malate

5.0 gm

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 M.Org/ Size Medium (mm with ) temp. & time Azosprilli 0.4 um 300C for 120 hrs

Shape

Colour

Circular

Azoarcus 300C for 120 hrs Agrobact er diazotrop hicus 300C for 120 hrs

0.3

Circular

1.02.0

Circular

Greenish with white metallic shine Insipid (Creamis h white) Dull white

Margin

Elevat ion

Consist ency

Opacit y

Entire

Convex

Smooth

Opaque

Entire

Flat

Smooth

Opaque

Entire

Flat

Moist

Opaque

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.

Organism

Gram’s staining

Motility

Azosperrillum

Gram Negative Rods

Ag.diazotropicus

Gram Negative Rods

Azoarcus

Gram Negative Rods

4.2. Biochemical Characteristics: -

Sluggishly motile Sluggishly motile Sluggishly motile

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 Test

Hydrolysis Catalase of starch test

M.org.

Gelatin Liquefactio n

Azospirillum Ag.diazotrophicu s

-Ve

+Ve

+Ve

-Ve

+Ve

+Ve

+Ve

+Ve

Azoarcus -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 Azosperrillum

Glucose

Sucrose

Fructos e

Mannitol

Mannose

(+)

+

+

+

(+)

Ag.diazotrophicu s

+

+

+

+

+

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

TVC of mother TVC

n no.

cell

of

CP TVC

of

added culture added

CP

culture

after 7 days.

after 15 days.

Agr

Azr

Azsp.

Agr

Azr

Azsp.

Agr

Azr

Azsp.

10-1

>30

>30

>300

>30

>30

>300

>30

>30

>300

10

0 >30

0 >30

>300

0 >30

0 >30

>300

0 >30

0 >30

>300

-3

10

0 >30

0 >30

>300

0 >30

0 >30

>300

0 >30

0 >30

>300

10-4

0 >30

0 >30

>300

0 >30

0 >30

>300

0 >30

0 >30

>300

-5

10

0 >30

0 >30

>300

0 >30

0 >30

>300

0 >30

0 >30

>300

0

0

10-6

234

265

188

0 >30

0 >30

>300

0 >30

0 >30

>300

0 >30

>300

0 >30

0

154

0 >30

Endophytes >

-2

10

-7

232

238

287

>300

10-8

189

176

100

0 >30

0 >30

>300

0 >30

254

>300

0 >30

>300

0 >30

232

214

10

176

166

98

0 >30

10-10

123

122

76

0 >30

0 >30

209

234

212

209

0 >30

167

212

198

189

0

0 >30

123

178

167

187

-9

10

-11

10-12

100

98

65

0 >30

65

53

34

193

0

0

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 N2 fixed in mg/gm of sucrose consumed Sr.No Name of Dry weight basis Liquid weight . Endophyte 50 ml medium basis broth 50 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-

20˚c

25˚c

++

+++

++ + ++ +

+++

Endo. Bacteria Azospirilum Ag.diazotrophicus Azoarcus

++ + ++ + ++ +

+++

- = No growth

+

++ = Good growth 4.3.1.2.

30˚c 35˚c 40˚c 45˚c 50˚c ++ + ++ + ++

+

-

-

++

+

+

+

++ + -

= Poor growth

+++ = Excellent Growth

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. Ag.diazotropi cus Azoarcus

4.5

5.0

5.5

6.0

6.5

7.0

7.5

++

++

++

++

++

++

+

+

+

++

++

++ +

+++

++

Azospirillum

+

++

- = No growth ++ = Good growth 4.3.1.2.

++

+

++ +

++ +

+

+

= Poor growth

+++ = Excellent Growth

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. Azospirilum

10% +++

20% ++

30% ++

Ag.diazotrophicus

(+)

Azoarcus

++

(+) (+)(+) (+) (+) +++ +

40% + +++ +

(+) = Acid gas production

-

= No growth

+

= Poor

growth ++ = Good growth 4.4.

+++ = Excellent Growth

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 1.1 x 10

4

3

with increase in reducing sugar to

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 0.2 0.4 0.6 0.8 1 -

D.W. in ml 0.8 0.6 0.4 0.2 1

Final conc. in µg 20 40 60 80 100 -

DNSA ml 1 1 1 1 1 1

D.W . ml

Boil For 1015 8 Min And 8 Cool 8 8 8 8

O.D. at 540 nm 0.0112 0.0526 0.0729 0.1072 0.1525 0

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

Sucro se ml 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 -

D/ W ml 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 1

5% Pheno l 1 1 1 1 1 1 1 1 1 1

Conc. H2SO4 ml 5 5 5 5 5 5 5 5 5 5

Mix Thoroughly And Let It Cool

O.D. at 480 nm 0.0054 0.0528 0.1351 0.1700 0.3338 0.3343 0.4218 0.4901 0.6106 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 BS A ml

D/W Final Alkalin ml conc e . µg solutio

Folin coicaltea n

O.D. at 750 nm

n ml 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 -

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 1

10 20 30 40 50 60 70 80 90 -

5 5 5 5 5 5 5 5 5 5

reagent Mix ml Thoroug 0.5 hly 0.5 And 0.5 Incubate 0.5 At 0.5 RT 0.5 For 0.5 10 min. 0.5 0.5 0.5.

Mix Thorou ghly And Incubat e At RT for 30 min.

0.2236 0.3646 0.4715 0.5753 0.7135 0.7797 0.8733 0.9605 0.995 0

DNSA Graph

y = 0.0002x - 0.0219 Abs at 550 nm

0.2 0.15 0.1 0.05 0 100

300

500

700

900

1100

ug Glucose

Graph. 4.5. Estimation:

Standard

Graph

for

Reducing

Sugar

Sucrose Est. Graph

Abs at 480 nm

y = 0.0072x - 0.0805 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 5

25

45

65

85

105

ug conc. in Sucrose

Graph 4.6 Standard Graph for Sucrose Estimation:

Abs at 750 nm

Protein Std Graph 1.2 1 0.8 0.6 0.4 0.2 0 0

20

40

60

80

100

ug Conc. in protein

Graph 4.7 Standard Graph For Protein Estimation:

Table 4.13 Chemical Analysis: Observatio ns

Initial A4H Broth After sterilizatio n A4H culture + G. booster/cel l protectant After 7 days of adding cell protectant s After 15 days of adding cell protectant s

Reducing Sucrose Sugar content content

Protein content (OD at 550n m)

Acid ity (pH)

(µ g/1 00 ml)

(OD at 550n )

(µ g/ 100ml )

(OD at 550n m)

(µ g/1 00ml)

2.5 x104

1.034 5

9.6 x104

3.730

9.6 103

x 0.252

6.5

8.8 x104

3.600

4.6 x104

3.008

1.1 104

x 0.290

7.00

4.3 x105

17.59 0

4.9 x104

3.067

2.0 x104

0.458

5.35

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.

10-1 10-2 10-3 10-4 10-5 10-6

TVC of mother TVC of cell culture protectant added culture After 7 days >300 >300 >300 >300 >300 245

>300 >300 >300 >300 >300 >300

TVC of cell protectant added culture After 15 days >300 >300 >300 >300 >300 >300

10-7 10-8 10-9 10-10 10-11 10-12

232 192 178 135 96 53

>300 >300 >300 >300 >300 >300

>300 >300 >300 >300 >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%.

Summary, Conclusion 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

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

Herbaspirillum,

such

as

Agrobacterium

Acetobacter,

Azoarcus,

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. Nitrogenfixing 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 Herbaspirillum; inclusion of

description

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 nov.,

sp.

nov.,

a

seropedicae gen.

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,

Polsinelli,M. (1980)

c.,Bazzicalupo,M.,Favilli,

F.,Gallori,E.

&

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 ricebacteria 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 Herbaspirillum

of

the

spp.

in

endophytic root,

stems,

diazotrophs and

leaves,

predominantly of Gramineae. Biol. Fertil. Soils 21:197200. 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 N2 incorporation following inoculation of sterile plants

15

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

1983[Abstract] [full text].

Evol

Microbial

53:

1979-