Agrobacterium diazotrophicus as a Liquid biofertilizer.
-
Upload
laxman-savalkar -
Category
Documents
-
view
1.702 -
download
0
description
Transcript of 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 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
Abstract
Bibliography
List of Figures/Graphs
Figure No.
Particulars Page No.
4.1 Isolated Strain Of Agrobacterium
Diazptrophicus
4.2 Isolated Strain Of Azosperrillum
4.3 Isolated Strain Of Azoarcus
4.4 Pure Strain of Endophytes Given by
Institute
45 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 Particulars Page No.
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
Chapter 1
Introduction
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.
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 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).
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.
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 302C 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.
3.2.2.1 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 30C temperatures. The
plates were flooded with weak logust iodine solution after 3 days
of incubation at 30C. (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
30C 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)
3.2.3 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 30C 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
30C at 110 rpm, the contents of flask were checked for purity
by streaking on fresh medium and concentrating over a water-
bath (50 to 60C) 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+2c.
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 20C to 50C. 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 30C.
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 30C.
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 (2000g/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 (500g/ml).
Table 3.2: Dilution scheme for sucrose by phenol sulphuric acid method.
Sucrose ml
D/W ml
5% Phenol
Conc. H2SO4 ml
MixThoroughlyAndLet0.1 0.9 1 5
ItCool & Measure the O.D.
0.2 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 (100g/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)
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.
Results&
Discussion
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/ 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
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 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
4.2.2. 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 >30 >300 >30 >30 >300 >30 >30 >300
0 0 0 0 0 0
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
0
>30
0
>300 >30
0
287 >300
10-8 189 176 100 >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 30C 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 30C. 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 solution ml
MixThoroughlyAndIncubateAt RTFor10 min.
Folin coicaltean reagent ml
MixThoroughlyAndIncubateAt RT for30 min.
O.D. at 750 nm
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
Graph. 4.5. Standard Graph for Reducing Sugar Estimation:
Graph 4.6 Standard Graph for Sucrose Estimation:
Graph 4.7 Standard Graph For Protein Estimation:
Table 4.13 Chemical Analysis:
Observations
Reducing Sugar content
Sucrose content
Protein content
Acidity (pH)
(g/100 ml)
(OD at 550n)
(g/100ml)
(OD at 550nm)
(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 culture
TVC of cell protectant added culture After 7 days
TVC of cell protectant added culture After 15 days
10-1 >300 >300 >300
10-2 >300 >300 >300
10-3 >300 >300 >300
10-4 >300 >300 >300
10-5 >300 >300 >300
10-6 245 >300 >300
10-7 232 >300 >300
10-8 192 >300 >300
10-9 178 >300 >300
10-10 135 >300 >300
10-11 96 >300 >300
10-12 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%.
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 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].