Techniques in mycorrhizal studies springer netherlands

TECHNIQUES
IN
MYCORRHIZAL
STUDIES
Techniques
in
Mycorrhizal
Studies
Edited by
K.G. Mukerji
Department
of
Botany, University
of
Delhi,
Delhi, India
C.Manoharachary
Department
of
Botany, Osmania University,
Hyderabad, India
and
B.P.
Chamola
Department
of
Botany, University
of
Delhi,
Delhi, India
SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

A C.I.P. Catalogue record for this book
is
available from the Library
of
Congress.
ISBN 978-90-481-5985-7 ISBN 978-94-017-3209-3 (eBook)
DOI 10.1007/978-94-017-3209-3
Printed on acid-free
paper
AII
Rights Reserved
© 2002 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers
in
2002
Softcover reprint
of
the hardcover 1 st edition 2002
No part
of
this work may be reproduced, stored in a retrieval system,
or
transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming, recording
or
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of
being entered

and executed on a computer system, for exclusive use by the purchaser
of
the work.
Preface
The
importance of mycorrhiza
for
the
improvement
of
plant growth
is
increasingly being realised
in
Agriculture
and
Forestry and several
mycorrhizal
fungi
have
been
commercially
recognised
for
the
purpose.
The
aim
of
this

book
is
to
describe
the
various
techniques used
to
study
the
mycorrhizal
biology.
Problems
with
preparing
such
a
book
are
many.
Mainly
mailing
of
manuscripts
to
and
from
authors
resulted
in

irregular
peer review
and
final
editing.
Every
effort
was
made
not
to
change
the
original
manuscript
to
ensure
accuracy.
Our
sole
aims
is
to
communicate
to
the
greatest
extent
possible
a

current
world
need
in
mycorrhizal
research.
Plant
productivity
is
regulated
by
microbial
associations
established
in
the
plant root
systems.
The
interactions of microorganisms
and
plant
roots
are
especially
important
in
providing
nutritional
requirements

of
the
plant
and
the
associated
microorganisms.
Plant
growth
and
development
are
controlled
largely
by
the
soil
environment
in
the
root
region
-
rhizosphere.
This
is
a
very
complex
environment

in
which
the
effects
of
the
plant
on
soil
microorganisms
and
the
effects
of
microorganisms
on
the plant
are
interacting,
interdependent
and
highly
complex.
Plant
root
exudates
and
breakdown products
feed
the

microbes
and
the microbe
in
tum often
benefit
the
plant.
Mycorrhizal
fungi
are
important
tools
for
increasing
growth,
development
and
yield
of economically important
plants,
they play important
role
of
biofertilizer which
can
help
establish plants
in
nutrient deficient soils,

particularly
phosphorus
deficient
soils,
arid,
semi-arid
and
waste
lands.
Mycorrhizal
plants
grow
well
under
stress
conditions
and
are
resistant
to
pathogens (bioloical control). Mycorrhizal technology, therefore,
has
assumed
greater
relevance
in
forest
production of
exotics
and

artificial
regeneration
of
rare plants.
Its
potential
in
agriculture, social
and
agroforestry
programmes
aimed
at
increasing
productivity of
food,
fuel,
fodder
and
fibre
for
exanding
popUlation
is
attracting
serious
attention
in
vi
Techniques

in
Mycorrhizal
Studies
recent
years.
Mycorrhizaresearch
thus
contribute
an
important
component
in
the
field
of
biotechnology,
the
practical
application
of
which
can
lead
to
successful afforestation, additional timber production with the modest
increase
in
survival
and
growth

rate
of
trees.
Since
Mycorrhizal
biology
has
become
important
in
Agriculture,
Forstry
and
Horticulture, a
good
book
on
techniuqes
in
mycorrhizal
studies
was
needed. There
is
already a book
on
molecular aspects
of
mycorrhizal
research, there

was
great
demand
for
a book
on
fundamental and basic
aspects
of
mycorrhiza, without knowledge
of
which
no
research
on
mycorrhiza can be initiated.
It
is
with this
aim
that this book has been
compiled covering
all
major
aspects
of
mycorrhizal biology. An entire
gneration
of
mycorrhizologists

have
been
trained
primarily
in
molecular
biology,
and
more
specifically
in
cloning
and
sequencing.
Although
such
tools
will
remain
fascination
of
investigators
for
some
time
to
come,
there
is
a critical need

to
carry
out
multidisciplinary investigations. It
was
set
against this back ground that
we
conceived the idea
for
this book.
We
wanted
to
bring
together
the
diversity
oftoolsltechniques
that
are
necessary
for
contemporary study
of
mycorrhizal biology into a single volume.
Although
assimilating
the
theoretical

background
and
wealth
of
literature
in
all
of
the
necessary
disciplines
would
have
been
an
impossible
job,
we
considered
that
this
was
ofless
importance
than
providing,
simple,
well-
explained laboratory
protocols

of
the
relevant
procedures.
After
all,
it
is
at
the
front
line
of
mycorrhizal
research
that
we
want
this
book
to
be
used
by students and learners
at
all
stages
of
their growth
from

graduate
to
post-doctoral
level,
nursery
workers
to
field
reserachers.
The
book contains twenty
four
Chapters
and starts with
an
overview
of
Mycorrhizal Studies
and
its
scope.
The
Chapters
1
to
5
deal
about basic
knowledge
of

soil
and
root
microflora,
their
ecology,
techniques used
in
their
isolation
and
identification.
Emphasis
has
been
given
on
mycorrhizal
fungi
in
relation
to
soil
factors
and
root
exudation.
Methods
used
to

collect
and
analyse root exudate
has
also
been
given.
Chapters 6
to
8 deal with
ectomycorrhizal
fungi,
their isolation,
mass
culture
and
identification.
Chapters
9
to
17
deal
with
vesicular-arbuscu1armycorrhiza
with
particular
emphasis
on
techniques
to

isolate,
mass-culture
and
identifying
V
AM
fungi.
Chapter
17
is
slightly
drifted
form
the
main
theme
of
the
book
but
significant
Vll
as
it
pertains
to
degradation
of(V
A)
mycorrhizal

root
leading
to
soil
fertility.
Chapters
18
to
21
give
current
status
of
the
other
types
of
mycorrhiza
i.e.
ericord, orchidoid
and
monotropoid.
No
account
has
been given
on
arbutoid mycorrhiza
as
we

considered
them
less
significant. Last three
chapters
i.e.
22
to
24
deal
with
two
very important biotechnological
applications
of
mycorrhiza i.e. their role
in
in vitro establishment
of
micropropagated
plants
from
laboratory
to
field
conditions
and
mycorrhiza
as
'biocontrol

agents'.
All
chapters
are
new
but
some
(20,
22, 23,
24)
have been especially
written
for
our book
and
probably
no
other publication
has
earlier dealt
with these topics.
Other biotechnological applications have not been
included
in
this
book
since
these
are
already

given
in
earlier
pUblications
by
us
and
others.
Each
chapter
has
been
written
by
experienced scientists
in
the
field.
Our
hope
in
assembling
this
variety
of
experimental
approaches
together,
in
a

single
volume
is
that
any
researcher
studying
mycorrhiza
can
use
this
book
as
their
first
reference
for
starting
a
new
avenue
of
investigation.
With
each
procedure
we
have given theoretical information about the topic.
Collectively,
we

believe
there
is
a
powerful
combination
of
experimental
tools
here
that
will
be
broadly
applicable.
Weare greateful
to
all
the
authors
for
their contribution
to
this
book
and
for
accepting suggestions
to
produce

the
final
balance ofthe
book.
The
articles
are
original
and
some
aspects
have
been
included
for
the
first
time
in
any
book.
Since
these
chapters
have
been
written
by
independent
authors,

there
is
the
possibility
of a
slight
overlap
or
repitition
of
certain
statements,
but
this
is
difficult
to
avoid
in
assignment
like
this.
It
is
our
hope
that
this
book
will

be
useful
to
all
students
and
researchers
in
microbial
biotechnology,
microbial
ecology,
soil
microbiology, applied
mycology,
agriculture
and
forestry.
17th
November,
2001
K.G. Mukerji
C.Manoharachary
B.P. Chamola
Table of Contents
Preface v
Introduction:
Mycorrhizal
Studies
C.

Manoharachary and
K.
G.
Mukerji



1
1.
Soil
microbes
K.
G.
Mukerji 7
2.
Soil
microflora
:
isolation,
enumeration
and
identification
Rani
Gupta
and
H.
Mohapatra

15
3.
Soil
factors
in
relation
to
distribution
and
occurrence
of
vesicular
arbuscular
mycorrhiza
Rupam
Kapoor,
B.
Giri
and
K.
G.
Mukerji


51
4.
Rhizosphere

biology
K.
G.
Mukerji






87
5.
Root
exudate
biology
Rajni
Gupta
and
K.
G.
Mukerji



103
6.
Isolation
of
ectomycorrhizaJ.
fungi:

methods
and
techniques
S.
Kumar and
T.
Satyanarayana



133
7.
Production
ofincoulum
of
ectomycorrhizaJ.
fungi
Sanjeev Kumar and
T.
Satyanarayana



143
8.
Identification
of
ectomycorrhizas
K.
Natarajan and

V.
Mohan




167
9.
Tehniques
for
the
isolation
of V
AMI
AM
fungi
in
soil
Rajni
Gupta
and
K.G.
Mukerji
201
10.
Methods
in
study
of
viability

of V
AM
fungal
spores
M Bansal and
K.
G.
Mukerji






217
11.
Root-Clearing
techniques
and
quantification
of
arbuscular
.
myconhizal
fimgi
C.
Manoharachary and IK. Kunwar .


231

x
12.
Arbuscular
mycorrhizal
fungi
-
identification,
taxonomic
criteria,
classification,
controversies
and
tenninology
C.
Manoharachary,
l.K.
Kunwar and
K.
G.
Mukerji


249
13.
Tebniques
of
AM
fungus
inoculum
production

Geeta
Singh
and
K.
V.B.R.
Tilak

273
14.
Multiplication
of
arbuscularmycorrhizal
fungi
on
roots
P.
Chellappan,
S.A.
Anitha Christry and
A.
Mahadevan

285
15.
Biotechnological
approaches
for
mass
production
of

arbuscular
mycorrhizal
fungi
:
current
scenario
and
future
strategies
Abdul-Khaliq, ML.
Gupta
and
M.
A
lam


299
16.
V
AM
technology
in
establishment
of
plants
under
salinity
stress
conditions

B.
Giri,
Rupam
Kapoor
and
K.
G.
Mukerji
.
313
17.
Method
in
study
of
degradation
of
mycorrhizal
roots
M Bansal and
K.G.
Mukerji
329
18.
Ericoid
mycorrhiza
-
isolation
and
identification

Sumeet and
K.
G.
Mukerji

345
19.
Ericoid
mycorrhizae
-
current
status
Geeta
Singh
and
K.
G.
Mukerji



365
20.
Orchidoid
mycorrhiza
and
techniques
to
investigate
S.P.

Vij,
T.N.
Lakhanpal and Ashish
Gupta

385
21.
Some
aspects
of
monotropoid
mycorrhizas
C.
Manoharachary,
l.K.
Kunwar and
K.
G.
Mukerji
435
22.
Role
of
mycorrhizae
in
in
vitro
micropropagation
of
plant

P.S.
Srivastava,
Nisha
Bharti,
Deepshikha
Pande
and
Sheela
Srivastava




443
23.
Evaluating
performance
of
plants
infected
with
vescicular
arbuscular
mycorrhizal
fungi
for
alleviating
abiotic
stresses
P.

Sharmila
and
P.
Pardha
Saradhi


469
24.
Mycorrhizae
as
biocontrol
agents
Lisette
J.
C.
Xavier and
Susan
M Boyetchko




493
Index 537
List
ofContributers
551
Introduction: Mycorrhizal Studies
C. MANOHARACHARY

and
K.G. MUKERJI
There
is
a
vast
microbial
flora,
inheriting
the
earth
and
they
are
found
in
all
types
of
soils
which
are
virgin
or
cultivated,
sands,
deserts,
thennal
soil,
snow

covered
soils
and
others.
The
dominating
groups
of
microorganisms
are
bacteria,
actinomycetes,
fungi,
nematodes
and
protozoans.
But
most
ofthese organisms
share
a
common
character
in
being
heterotrophic
in
their
nutrition
and

there
by
depend
on
other
organic,
dead
or
living
organism
and
inorganic
source
of
nutrition
for
their
sUlVival
and
multiplication.
Most
of
the
fungi,
bacteria
and
actinomycete
are
microscopic
and

show vast
variation
quantitatively
and
qualitatively
in
different
sites
of
collection
and
at
different
depths,
considerable
variation
occurs
even
between
soil
samples
taken
a
few
inches
apart.
Biotrophic,
saprobic
and
symbiotic microbes

are
found
in
soil.
The
microbial
population
of
soil
is,
therefore,
dependent
on
composite
micro-ecological
environments.
Mycorrhizae
are
the
structures
fonned
by
the
association
of
root
in
plant
with
fungi.

Such
root
fungus
associations
were
found
to
be
nonnal
on
the
root
system.
The
studies
ofGennan
Botanist
Frank
(1)
had
initiated
worldwide
interest
on
myconbizae.
Myconhizae
fonn
symbiotic
association
with

many
plants
under
natural
conditions.
Fungi
receive
carbohydrates
from
host
plant
while
plants
receive
fastly
mobilized
phosphorus
and
other
nutrients
throughmyconhizal
roots.
Seven
types
of
mycorrhizal
associations
are
known
namely

-
ectomycorrhizae,
arbuscular
mycorrhizae,
ect-endo
mycorrhiza,
arbutoid
mycorrhiza,
monotropoid,
ericoid
and
orchidoid
mycorrhizae (3,4,5). Mycorrhizal plants suggest saving
of
50%
of
phosphorus
if
proper management
is
observed by deriving maximum
mycorrhizal
response.
Ectomycorrhizal
fungal
inoculation
is
a
common
practice

and
it
is
of
immerse
utility
in
forestry,
silvicuture
and
agroforestry
operations.
K.
G.
Mu'kerji
et
al."(
eels.),
Techniques
in
Mycorrhizal
Studies,
1"'{).
© 2002
Kluwer
Academic
Publishers.
2 Techniques
in
Mycorrhizal Studies

The
endomycorrhizal
associations
produce
minor
changes
in
the
root
morphology.
The
mantle
of the root
is
absent.
Orchids
have
endomycorrhiza
which
is
called
glomerate
endomycorrhiza
because
of
the
presence
of
glomerules.
Members

ofEricaceae
develop
a different
type
of
endo-mycorrhizal association. Ectendo-mycorrhiza are
characterized
by
the
presence
of a
small
mantle
and
mycelium
penetrating
the
root cells.
AM
fungi
are
found
associated
with
crop
plants, forest
plants,
horticultural
plants,
ornamental

plants
and
other
plants.
These
fungi
are
not
host
specific
but
differ
in
their
characteristic
association
with
host
rhizosphere.
AM
fungi
produce
azygospore,
chlamydospore.
Most
AM
fungi
sporulate outside
the
plant root

in
soil
but
for
Glomus
tenuis
and
Glomus
intraradices.
However
there
are
around
10
species
of
Glomus
which
can
be
multiplied
in
soil
pot
culture.
Endomycorrhizae
can
not
be
cultured

on
agar
media
or
in
axenic
culture
as
AM
fungi
are
obligate
symbionts.
Ectomycorrhizae
can
be
distinguished
from
endomycorrhizae
because
in
them
the
root
system
is
heterorhizic,
comprising
long
and

short
roots.
The
ectomycorrhizal
plants
are
characterized
by
the
presence
of
mantle
and
the
hartig
net.
Morpho-anatomical features, sporophore
morphology,
basidiospore
structure
and
other
characters
fonn
important
criteria
in
recognizing
ectomycorrhizal
fungi.

Various
techniques
have
been
used
for
establishing
the
mycorrhizal
association
of
trees
and
fungal
fruit
bodies.
Taxonomically
the
ectomyconhizal
fungi
belong
to
Basidiomycotina
and
few
are
also
members
of
AscomycotinalDeuteromycotina.

Around
2000
wooden
plant
species
are
colonized
by
5000
fungal
species/strains.
These
fungi
can
be
easily
identified
from
their
fruit
bodies.
The
pure
culture
isolations
of
ectomycorrhizal
fungi
have
been

tried
from
the
fruiting
bodies,
surface sterilized mycorrhizal roots
and
sclerotia. Pisolithus
and
Thelephora
have
proved
as
the
most
efficient
and
beneficial
bioinoculants
as
they
have
shown
dramatic
improvements
in
survival
and
growth
of

pine
and
other
seedlings.
Ectomycorrhizal
fonnations
are
confined
to
forest
and
ornamental
tree
species
belonging
to
Pinaceae,
Salicaceae,
Betulaceae,
some
members
of
Rosaceae,
Leguminosae,
Ericaceae,
Juglandaceae
and
Dipterocarpaceae.
Experimental
synthesis

of
ectomycorrhizae
for
field
inoculation
has
undergone
change
so
as
to
adopt
to
desired
soil
and
environmental
conditions.
With
the
discovery
of
molecular
tools
it
has
become
easy
to
C.

Manoharachary and
K.G.
Mukerji 3
introduce
new
traits
that
can
be
advantageous
to
host.
The
fungi
that
fonn
mycorrhizae with vascular plants belong
to
the zygomycotina,
ascomycotina,
basidiomycotina
and
to
the
fungi
imperfecti.
Around
5000
species
of

ascomycotina
and
basidiomycotina fonn ectomycorrhizaes.
Around
150
species
of
AM
fungi
belonging
to
zygomycotina
infect
90%
plants.
Arbuscular
mycorrhizal
fungi
are
the
naturally
occurring
fungal
component
of
soil
biota
in
most
terrestrial

ecosystems.
It
also
represents
a
obligate
symbiotic
group
distinct
from
the
rest
of
soil
microbial
biomass.
Endomycorrhizal
symbiosis
is
a
dynamic
process
and
interaction
that
affects
all
physiological
aspects
of

the
host.
These
fungi
are
unique
as
they
are
partly inside and partly outside the
root.
The
vesicles, arbuscules and
hyphae that
are
fonned inside the root
does
not encounter competition
and
antagonism
from
soil
micro
organisms
to
the
host
rhizosphere,
soil
conditions

and
host
genotype
are
some
of
the
factors
that
affect
biological
interactions
in
that
specialized
ecological
niche.
The
arbuscularmycorrhizal
fungi
are
ubiquitous
in
distribution
and
occur
abundantly.
Ninety percent plants
ranging
from

non
flowering
to
flowering
plants
have
the
dynamic
association
of
AM
fungi.
It
is
easy
to
identify
and
count
plants
which
are
not
mycorrhizal
than
the
plants
which
are
mycorrhizal.

Unlike
the
ectomycorrhizal
fungi,
Arbuscularmycorrhizal
fungi
belong
to
Zygomycotina
which
are
obligate
biotrophs
that
can
not
be
cultured.
The
arbuscular
mycorrhizal
fungi
are
the
most
complex
group
of
mycorrhizae
which

fonn
intradical
structures
(i)
vesicleslarbuscules
inter
and
intra
cellular
hyphae
inside
the
root
tissue.
(ii)
spores/sporocarp
and
branched
hyphae
outside
the
root
and
fonned
in
soil.
Arbuscules
are
the key sites
for

nutrient exchange and remain
active
upto
fifteen
days
depending
upon
host
response.
There
are
six
valid
genera namely -
Acaulospora,
Entrophospora,
Glomus,
Gigaspora,
Sclerocystis
and
Scutellospora.
Vesicles
are
not
reported
in
Gigaspora
and
Scutellospora,
but

extramatrical
vesicles
were
reported.
The
fossil
evidence
clearly
indicates
that
the
invasion ofland by
the ancestors
of
the present
day
vascular plants clearly seems
to
have
been
facilitated
by
the
origin
of
symbiotic
association
between
the
plants

and
mycorrhizal
fungi.
Fossil data demonstrates that ectomycorrhizal
association
was
established
from
the
later
Ordovician
and
Silurian
period.
There are about
150
AM
fungal
species reported colonizing 30,000
receptive
hosts.
4 Techniques
in
Mycorrhizal Studies
Mycorrhizal
fungi
are
abundant
in
soils which

are
deficient in
phosphorus
and
other
mineral
elements.
Mycorrhizal
fungi
play
significant
role
in
phosphorus
mobilization.
In
fact
mycorrhizal
effect
decreases
with
increased
supply
of
soluble
phosphate.
Therefore
non-mycorrhizal
plants
or non-inoculated plants

show
greater response towards fertilizer
application.
The
important
aspect
in
mycorrhizal
biotechnology
seems
to
be
the
utilization
of
unprocessed
phosphate
that
contain
sparingly
soluble
phosphorus.
Mycorrhizal
roots
provide
grater
exploration
of"P"
absorbing
area

resulting in increased
flow
of"P" into the plant.
The
unprocessed
phosphorus
gets
solubilized
by
phosphorus
solubilizing
fungi
and
other
microbes,
thus
making
soluble
phosphorus
available
to
the
plant.
Efficient
mobilization
of
phosphorus
is
possible
through

mycorrhizal
root
over
non-
mycorrhizal
plants.
Harley
(2)
suggested that production
of
phosphotases by
ectomycorrhizal
fungi
is
important
in
the
solubilization
of
inorganic
phytates,
which constitute a large fraction
of
total phosphate
in
humic soils.
Phosphotases
are
many
times

more
active
in
mycorrhizal
plants
than
those
on
non-mycorrhizal plants. Experiments using
32p,
it
was
found that
mycorrhizal
plants
accumulate
and
stores
"P".
This
accumulate
remains
consistent unless
there
is
a
dramatic
change
in
the

plant "P" status.
The
phosphorus
gets
accumulated
as
polyphosphate granules
and
later it
is
released
into
host
tissue
through
arbuscules
or
by
other
means.
Arbuscular mycorrhizal
fungal
taxonomy seems
to
be more
complicated.
The
motpho-taxonomic
criteria,
wall

layers,
ornamentation,
subtending hyphae and other character help
to
segregate genera and
species.
Presence
or
absence
of
the
sporacatp
is
also
an
important
criteria
for
identifying
AM
fungi.
It
has
been
suggested by
Mosse,
an
eminent
mycorrhizologist
that

arbuscule,
vesicles
and
hyphal
formation
have
to
be
established
in
the
concerned host root tissue
on
inoculation
so
that the
AM
fungal
species
entity
can
be
ascertained.
A complete and accurate description
and
identification
of
each
AM
fungus is necessary besides fixing its taxonomic status,

Motphotaxonomic criteria
no
doubt
will
help
in segregating
the
genera
and
species
of
AM
fungi
but
much
more
authenticity
and
scientific
accuracy
will
be
established
following
molecular
techniques.
Molecular
techniques
are
a major

impetus
in
mycorrhizal
research.
Molecular
and
biochemical
parameters
such
as
cell
wall
composition,
protein
immunogenicity,
isozyme
C.
Manoharachary and
K.
G.
Mukerji 5
electrophoretic
patterns,
fatty
and
methyl
ester
profiles,
direct
sequencing

of
I8S
RNA
gene
and
also
use
ofRAPD
approach
to
detect
polymorphism
and
others
have
been
proved
more
useful.
With
the
development
ofPCR,
it
is
now
possible
to
analyse
and

characterize
a
species
at
the
DNA
level.
PCR
-
RFLP
can
be
used
for
quantitative
comparison
oflevels
of
genetic
variation
and
interpretation
of
genetic
similarities or differences
among
isolates
of
AM
fungi.

Immunological
studies
have
also
added
new
strength
for
the
taxonomy
of
AM
fungi
particularly
in
physiological
and
ultrastructural
features during infection process or the ability
of
foreign
AM
fungi
to
compete
with
local
endophytes.
In
addition

to
the
pAbs,
mAbs
are
being
developed
or
adapted
from
heterologous
sources.
Although
the
advantages
of
pAbs
include
their
easy
production with considerably
less
apparative
expenditure
as
well
as
a higher possible titer
and
higher

affmity
for
most
antigens
a
number
of
problems
are
encountered
with
pAbs.
These
can
be
overcome
by
the
use
of
hybrid
om
a
technology.
The
benefits
derived
from
this
technology

include
the
virtually
unlimited
supply
of
specified
Abs,
fewer
problems
with
unspecific
binding
and
a
system
that
with
in
limits,
allows
a
selective
screening
for
those
mAbs
that
have
the

desired
specificities
and
affinities
towards
a
given
antigen.
AM
fungi can influence plant community composition by
differentially
affecting
the
growth
of
different plant
species.
AM
fungal
symbiosis
is
highly
dynamic
interaction
that
affects
nearly
all
physiological
aspects

of
the
host.
Restoration
of
disturbed
land
has
prompted
the
urgent
need
for
understanding
mycorrhizas.
Soils
which
are
disturbed
and
turned
into wastelands I unproductive land were found
to
have reduced
mycorrhizas
and
that
many
plant
species

dependent
of
mycorrhizas
failed
to
establish
or
survive
in
these
regions.
Therefore
greening
of
such
areas
is
possible
only
with
AM
fungal
symbiontc;.
Desert, arid and semiarid soils suffer most and pose technical
problems
with
discrete
patches
of
vegetation.

Therefore
mycorrhizal
fungi
can
be
important
in
the primary production
and
nutrient
flow
in
these
specialised ecosystems. Mycorrhizas
are
of
immense utility
in
the
establishment
of
forest
seedlings,
commercial
crops,
economically
viable
plants,
medicinal
and

aromatic
plants,
horticultural
and
ornamental
plants.
P uptake which
is
an
essential process
gets
meticulously monitored by
AM
fungi.
This
process
is
of
great
help
in
increasing
N,
P
and
K
status
of
host
tissues.

Mycorrhizal
fungal
net
work
enhances
soil
binding
capacity
and
soil
fertility.
6 Techniques
in
Mycorrhizal Studies
The
benefits
that
are
derived
by
plants
colonized
by
mycorrhizae
are:-
1.
Improvement
in
absorbance
area

followed
by
increased water
and
nutrient
uptake.
2.
Efficient
and
greater accumulation of phosphorus
and
other
elements
and
their
speedy
mobilization
to
the
host
tissue.
3.
Degradation
of
complex
and
organic
materials.
4.
Mycorrhizal

plants
offer
protection
against
pathogens.
5.
Provide
host
plant
with
growth
honnones
like
auxins,
cytokinins,
gibberellins
and
growth
regulators.
6.
Improves
plant
defense
mechanism
and
offer
tolerance
to
abiotic
and

biotic
stress
conditions.
7.
Help
in
nutrient
cycling
and
forest
biomass
increase.
8.
Increases
plant
growth,
biomass,
productively
and
yield.
9.
Provide
host
plant
with
more
survival
abilities.
10.
Increases

soil
binding
capacity
and
soil
fertility,
mycorrhizal
fungi
are
non
pollutants.
11.
Waste
land
development
and
reclamation
of
non
productive
soils.
References
1.
Frank,
A.B.
1885.
Uber
die
aufWurzelsymbiose
beruhende

Emahrung
gewisser
Baume durch Unterisdischr Pilze. Berichte der Deutschen Botanischen
Gesellschaft,3:
128-145.
2
Harley,
1.L.
1969.
Biology
of
mycorrhiza.
2nd
edn.
Leonard
Hill,
London.
p.483.
3.
MukeIji,
K.G.
(ed.)
1996.
Concepts
in
Mycorrhizal
Research.
Kluwer
Academic,
Publishers, Dordrecht,

Boston,
London
p.
374.
4.
MukeIji,
K.G.,
Chamola,
B.P.
and
Singh,
1.
(eds.)
2000.
Mycorrhizal
Biology.
Kluwer
Academic
/
Plenum
Publishers,
New
York.
p.336.
5.
Smith,
S.E.
and
Read,
D.J.

1997.
Mycorrhizal
Symbiosis.
2nd
edn.
Academic
Press,
London,
New
York.
p.
605.
1
Soil Microbes
K.G. MUKERJI
237 DDAISFS Mukerji Apartments, East Mukerji
Nagar,
Delhi-ll0009, India
ABSTRACT:
Different
techniques
for
isolation
of
soil
microorganisms
have
been
described.
Since

fungi
form
the
bulk
of
soil
micro
biota,
techniques
to
isolate
these
are
emphasized.
Dilution
Plate
technique
is
probably
the
best
for
quantitative
determination of
microbes.
1.
Introduction
Natural
environments
are

extremely
diverse
and
the
majority
contain a
wide
range
of
microorgnaisms
which
reflect
the
nature
of
the
habitat
and
the
ability
of
individual
members
to compete successfully
and
coexist
within
the
given
ecosystem.

In
general
terms
the
greater
the
heterogenecity
of
the
environment,
the
more
diverse
and
complex
will
be
the
microflora
(21).
For
example
in
garden
soil
with
numerous
microenvironments,
the
microbial

flora
is
extremely
complex
whereas
in
thermophilic
or
hypersaline
environments
where
one
physical
or
chemical
characteristic
dominates
over
all
others,
only
a
few
specialised
species
can
grow under
such
extreme
ecological

conditions
(12).
Some
organisms
are
present
in
very
low
number
and
isolation
of
such
microbes
is
possible
using
some
form
of
enrichment
of
the
medium.
The
particular
microorganism
which
develops

in
enrichment
culture
clearly
depends
upon
the
chemical
composition
of
the
medium
used,
along
with
other
factors
like
temperature,
pH,
presence
of
selective inhibitors,
light,
gas phase
and
others (13). The microbes
include
bacteria,
algae,

fungi
(including
mycorrhizal
fungi),
actinomycetes,
protozoa
and
nemtodes
etc.
7
K.
G.
Mukerji
et
al.
(eds.),
Techniques
in
Mycorrhizal
Studies,
7-13.
©
2002
Kluwer Academic Publishers.
8
Techniques
in
Mycorrhizal
Studies
2.

Soil Fungi
As
heterotrophic
organisms
the
fungi
in
soil
are
saprophytes,
symbionts
or parasites.
Thus
they
play important
roles
in
terrestrial
and
aquatic
ecoystems
as
decomposers
of
organic
matter
(in
nutrient cycling),
as
pathogens,

or
as
symbionts
with
terrestrial
plants.
The
ecologically
important
characteristics of terrestrial
fungi
(11)
are
as
follows:
1)
The fungi present
in
soil are influenced by the amount
of
organic matter
entering the soil through different types
of
vegetaion growing on it or
near it, soil-moisture, soil-temperature, soil-pH etc. and other
environmental factors (22). Different groups
of
fungi have been
isolated (2,3).
2)

The quality and quantity
of
fungi
is
governed by the microhabitats like
decomposing organic matter and living roots.
3)
The fungi exists in various morphological forms and physiological states,
e.g. spores, resting structures and hyphae (6).
4)
Methods which are known for the isolation
of
fungi from soil are all
selective (8) and therefore are incomplete for assessing quantitative
nature
of
soil mycoflora.
There
are
enormous
reports
on
fresh
water
and
marine
fungi.
They
are
equally important

like
soil
fungi
as
decoposers
of organic matter,
as
pathogens
and
also
serve
as
food
sources
for
aquatic
invertebrates
(15,16,17,18,28).
3. Isolation of Soil Fungi
3.1.
Direct
Observation
Glass
slides
are
burried
in
soil
for
known

amount
of
time
and
after
that
period
these
slides
are
observed
under
light
microscope
with
or
without
staining
(29).
3.2.
Soil
Immersion
Technique
The
fungi
present
in
soil
as
actively

growing
hyphae
are
isolated by
Immersion
Technique.
This
was
originally
reported
by
Chesters
(7)
which
K.G.
Mukerj; 9
was
later
modified
by
various
warkers
(1,19,21,22,25,30,35).
The
basic
principle
in
all
these
methods

is
same
i.e.
the
isolating
medium
is
placed
in
soil
in
such
a
way
that
it
is
separated
from
the
soil
through
an
air
gap.
The
medium
is
left
in

the
soil
for
5 to 8
days
after
which
it
is
brought
to
the
laboratory
where
small
portions
are
plated
on
to a
selective
medium.
Fungi
appearing
in
plates
from
the
inoculum
are

isolated
and
purified
in
Agar
slants
for
further
identification.
In
Mueller
and
Durrell's
(21)
technique
autoclavable
centrifuge
tubes
are
used.
Holes
are
bored
at
equal
distance
on
walls
of
each

centrifuge
tube.
These
holes
are
wrapped
(closed)
with
cellotapelscotch
tape
so
as
to
temporarily
closing
the
holes.
Isolating
medium
is
poured
in
each
tube
leaving
enough
space
at
the
top.

The
tubes
are
cotton
plugged
and
auotclaved.
In
the
field
the
plastic
tape
is
pierced
through
the
holes
in
the
tube
using
pre-sterilised
needles.
The
sterilsed
tubes
containg
medium
are

inserted
in
soil
and
kept
there
for
5-8
days
after
which
they
are
taken
to
laboratory.
The
fungi
are
isolated
by
taking
out
the
agar
core
from
each
tube
and

cutting
the
core
into
small
pieces
where
the
hole
was
in
contact
with
the
medium.
Individual
pieces
are
plated
on
agar
medium.
Disadvantages
of
immersion
methods
are
several:
i)
when

these
tubes
/
plates
are
inserted
in
soil
it
induces
germination
of
fungal
spores
in
the
vicinity
/ or contact
with
the
tube.
Hence
fungi
isolated
may
have
originated
from
spores
and

not
initially
active
hyphae,
ii)
small
animals
in
soil
may
enter
the
isolating
medium
through
poreslholes
in
the
immersion
apparatus
bringing
fungal
propagules
with
them;
(iii)
there
is
possibility
of competition between

fungi
for
entry
into
immersion
tube
and
for
subsequent
survival
in
the
medium.
3.3
Warcup's
Soil
Plate
Method:
Small
volumes
of
soil
are
dispersed
in
known
volume
of nutrient
agar,
using

sterile
needles
with
flat
tips.
5-15mg
of
soil
sample
is
plated
in
a
sterile
petridish,
a
drop
of
sterile
water
is
added
and
soil
sample
thoroughly
broken
up
and
dispersed

over
the
base
of
the
dish.
10mI
of
molten
10
Techniques
in
Mycorrhizal
Studies
(c
45°C)
agar
is
poured
over
the
soil
in
each
petridish
and
slowly
roated
to
disperse

the
soil
into
the
agar
medium.
After
setting,
the
plates
are
incubated
at
22-28°C.
Fungi
appearing
in
these
plates
are
isolated
and
pure
cultures
grown
are
used
for
identification.
This

is
simple
method
but
like
Dilution
Plate
Method
it
is
disadvantageous
as
this
method
is
selective
for
fungi
present
as
spores.
Warcup
(33,34)
devised
a
more
precise
technique
for
isolation

offungi
present
in
active
state
in
soil.
Small
amounts
of
soil
are
saturated
with
sterile
water
and
dispersed
with
a
fine
jet of
sterile
water.
The
coarse
soil
particles
are
sedimented

and
the
finer
particles decanted
off.
The
supematent
is
resuspended
and
procedure repeated
several
times.
The
coarser particles
are
spread
in
a
film
of water
on
plate.
The
plates
are
observed
under
dissectinglbinocular
microscope

and
hyphal
bits
are
picked
with
fine
sterile
forceps
or a
needle.
The
hyphae
are
carefully
drawn
through
semi-solid
agar
to
trap other
fungal
spores,
bacteria or
organic
debris.
The
cleared
hyphae
are

then
plated
on
nutrient agar
plates.
The
position
of
each
hyphal
bit
is
marked
at
the
back
ofthe
plate.
The
plates
are
incubated
at
22-28°C
for
identification.
This
is
the
most

ideal
method
for
isolation
of
actively
growing
fungal
hyphae
in
soil
(4).
3.4.
Dilution
Plate
Method
(14,31)
The
soil
samples
are
made
into
suspensions
in
a
dilution
series
(Fig.
1)

i.e.
1:
100;
1:
1000,
1:
10,000
(27).
The
suitable
dilutions
are
plated onl
in
an
appropriate nutrient
medium.
The
soil
suspensions
are
made
of
known
amount
(by
weight
and
volume)
and

type
of
soil
sample.
This
method
is
not
useful
for
study
of
fungi
which
occur
in
hyphal
state.
The
hyphal
propagules
can
be
investigated
using
Warcup's
(32,33,34)
soil
plate
method

as
described
earlier.
This
method,
however
gives
a near
total count of spores
in
a particular
soil
sample.
The
number
offungil
gram
dry
weight
of
soil
is
calculated
by
multiplying
count
with
the
dilution
(31).

To
get a
complete
picture
of
the
soil
mycoflora
Dilution Plate
Method
and
Warcup's
Soil
Plate
Method
should
be
used
together
(14).
Aquatic
fungi
can
be
isolated
using
Baiting
technique
(5,17),
Dilution plate

technique
(24),
Particle Plating
method
(26)
and
Concentration
-
centrifugation,
Filtration
technique
(9,10,20)'
Weigh
Ig soil sample
l.Og
10'%
I
1.0ml
t
~
10-
3
Pour in each
petri plate
and
gently
swirl
K.G. Mukerji
11
' ;;::";;;=-=~

1. Oml
10-' 1
1.0ml
~
E§3
10-
7
Fig.
J.
Preparation
of
Soil
Dilution.
Detailed
account
of
isolation
of
soil
microflora
is
described
elsewhere
therefore
it
is
not
repeated
here
(23).

References
:
1.
Anderson,
AL.
and
Huber,
D.M.
1965.
The
plate
profile
technique
for
isolating
soil
fungi
and
studying
their
activity
in
the
vicinity of
roots.
Phytopathology,
!'!!'!:
592-594.
2.
Behera,

N.
and
Mukerji,
K.G.
1985a.
Seasonal
variation
and
distribution of
microfungi
in
forest
soils
of
Delhi.
Folia
Geobotanica
et
Phytotaxonomica,
20:
291-311.
12
Techniques
in
Mycorrhizal Studies
3.
Behera,
N.
and
Mukerji,

K.
G.
1985b.
Ecology
of
micromycetes
in
forest
soils
of
Delh.
Acta
Mycologica,
21:
101-108.
4.
Bissett,
J.
and
Widden,
P.
1972.
An
automatic
multichamber
soil
washing
apparatus
for
removing fungal

spores
form
soil.
Canadian Journal of
Microbiology,
18:
1399-1409.
5.
Booth,
C.
1971.
Fungal
culture
media.
In,
"Methods
in
Microbiology"
(ed.
Booth,
C.)
4:
49-94.
Academic
Press
London.
6.
Burges,
N.A
1960.

Dynamic
equilibria
in
the
soil.
In,
"The
Ecology
of
Soil
Fungi"
(eds.
Parkinson,
D.
and
Waid,
J.S.).
Liverpool
University
Press,
Liverpool.pp.185-191.
7.
Chesters,
C.G.
C.
1940.
A
method
for
isolating

soil
fungi.
Transactions
of
the
British
Mycological
Society.
24:
352-355.
8.
Crossan,
D.F.
1967.
Selective
isolation
of
soil
microganism
by
means
of
Differential
Media.
In,
"Source
Book
of
Laboratory
Exercises in Plant

Pathology"
(ed.
Kelman,
A)
W.H.
Freeman
and
Company.
San
Francisco
and
Londonpp.
19-21.
9.
Fuller,
M.S.
and
Poynton,
RO.
1964.
A
new
technique
for
the
isolation of
aquatic
fungi.
Bioscience,
14:

45-46.
10.
Gams,
W;
Vander
Aa
HA,
Vander-
Platts-Niterink,
AJ.,
Samson,
RA
and
Stalpers,
J.S.
1975.
C.B.S.
course of
mycology,
Centralbureau voor
schimelcultures,
Baarn,
Netherlands.
11.
Harley,
J.L.
1971.
Fungi
in
ecosystem.

Journal
of
Ecology,
59:
653-668.
12.
Herbert,
RA.
1982.
Procedures
for
the
isolation,
cultivation
and
identification
ofbacteria.
In,.
"Experimental
Microbial
Ecology"
(eds.
Burns,
R
G.
and
Slatter,
J.H.).
Blackwell
Scientific

Publication.
Oxford,
London.
pp.
3-21.
13.
Jannasch,
H.
W.
1967.
Enrichment
of
aquatic
bacteria in
continious
culture.
Archives
flir
Microbiologie
59:
165-173.
14.
Johnson,
L.F.
and
Curl,
E.A
1972.
Methods
for

Research
on
the
Ecology
of
Soil-borne
Plant
Pathogens.
Burges
Publishing
Co.
Minnessota,
U.S.A
15.
Jones,
F.B.G.
1971.
Aquatic
fungi.
In
"Methods
in
Microbiology"
(ed.
Booth,
C.
).
Vol.
4,
Academic

Press,
London,
pp.
335-365.
16.
Jones,
F.B.
G.
1974.
Aquatic
Fungi,
fresh
water
and
Marine,
In.,
"Biology
of
Plant Litter
Decoposition"
(eds.
Dickinson,
C.H.
and
Pugh,
GJ.F.),
Vol.
2.
Academic
Press,

London,
pp.
337-383.
17.
Jones,
F.B.G.
1976.
Recent
advances
in
aquatic
mycology.
Elek
Science,
London.
18.
Kohlmeyer,
J.
and
Kohlmeyer,
E.
1979.
Marine
Mycology.
The
Higher
Fungi.
Academic
Press,
London.

19.
Luttrell,
E.S.
1967.
A
strip
bait
for
studying
the
growth
of
fungi
in
soil
and
aerial
habitats.
Phytopathology,
57:
1266-1267.
20.
Miller,
C.E.
1967.
Isolation
and
pure
culture
of

aquatic
phycomycetes
by
membrane
filtration.
Mycologia,
59:
524-527.
21.
Mueller,
K.E.
and Durrell,
L.W.
1957.
Sampling
tubes
for
soil
fungi.
Phytopathology,
47:
243.
K.
G.
Mukerji
13
22.
Mukerji,
K.
G.

1966.
Ecological
studies
on
the
microorganic
population
of
usar
soils.
Mycopathologia
et
Mycologia
Applicata,
29:
339-349.
23.
Mukerji, K.G., Mandeep and Varma,
A.
1998. Mycorrhizosphere
microorganisms:
Screening
and Evaluation:
In,
"Mycorrhiza Mannual"
(ed.
Vcmna,
A)
Springer,
Berlin,

pp.
85-97.
24.
Park,
D.
1972,
Methods
of Detecting
fungi
in organic detritus in
water.
Transactions of
the
British
Mycological
Society,
58:
281-290.
25.
Parkinson,
D.
1957.
New
methods
for
qualitative and quantitative
study
of
fungi
in therhizosphere.

Pedologia
Gand,
7:
146-154.
26.
Parkinson,
D.
1982.
Procedures
for
the
isolation,
cultivation
and identification
offungi.
In,
"Experimental
Microbial
Ecology"
(eds. Burns, RJ. and
Slatter,
J.H.)
Blackwell
Scientific
Publications,
Oxford,
Lodon,
pp.
22-30.
27.

Piper,
C.S.
1944.
Soil
and
Plant
Analysis.
University
Adelaide
Press,
Adelaide,
Australia.
28.
Sparrow,
F.K.
1968.
Ecology
of fresh water
fungi.
In "The fungi"
(eds.
Ainsworth,
G.C.
and
Sussman,
A.S.)
Vol.
3.
Academic
Press,

London,
pp.
41-
93.
29.
Starkey,
RL.
1938.
Some
influences
of
the
development
of higher plants
upon
the
microorganisms
in
the
soil.
Iv.
Microcopic
examination
of the
rhizosphere.
Soil
Science,
45:
207-249.
30.

Thornton, RH.
1952.
The
sereened
immersion plate. A
method
for
isolating
soil
microrganisms.
Research,
London
5:
190-191.
31.
Waksman,
S.A.
1922.
A tentative outline of plate
method
of determining the
number
of
microorganisms
in
soil.
Soil
Science,
14:
27-28.

32.
Warcup,
J.H.
1950.
The
soil
plate
method
for
isolation of fungi
from
soil.
Nature,
London
116:
117.
33.
Warcup,
J.H.
1955.
Isolation of
fungi
from
hyphae
present in
soil.
Nature,
London
175:
953-954.

34.
Warcup,
J.
H.
1957.
Fungi
in
soil.
In,
"Soil
Microbiology".
(eds.
Burges,
N.A.
and
Raw,
F.)
Academic
Press,
London,
pp.
51-110.
35.
Wood,
F.A
and
Wilcoxson,
RD.
1960.
Another

screening
immersion
plate
for
isolating soil fungi. Plant
Disease
Reporter,
44:
594.
2
Soil Microflora : Isolation, Enumeration
and
Identification
RANI
GUPTA
and
H.
MOHAPATRA
Department
of
Microbiology, University
of
Delhi South Campus, Benito
Juarez Road, New Delhi - no
021.
ABSTRACT: The soil and rhizosphere microorganic population
is
very varied
consisting
of

different groups
of
microbes including actinomycetes, bacteria,
fungi and micro-algae. They are essential for the healthy growth and development
of
plants. Various techniques
of
isolation and identification
of
these microbes
have been described. Modern methods
of
microbial identification have also been
described.
1. Introduction
There
is
considerable
diversity
in
the
population
of
rhizosphere
microflora
and
their
effects
on
plant

growth.
Hiltner
was
the first
to
recognize
the
importance
of
microbial activity associated with root systems
in
plant
nutrition
and
used
the
term
rhizosphere
to
desbcribe
the
zone
of intense
activity
around
the
roots
of
the
leguminaceae

(Fabaceae).
The
rhizosphere
is
a
zone
of
predominantly
commensal
and
mutualisitc
interactions
between
the
plant
and
microbes.
Interactions
between
soil
microorganisms
and
plant
roots
satisfy
important
nutritional
requirements
for
both

plant
and
the
associated
microorganisms
(9,
14,26).
This
interaction
of
plant
roots
and
rhizosphere
microorganism
is
based
largely
on
interactive
modification
of
the
soil
environment
by
processes
such
as
release

of
organic
chemicals
of
the
soil
by
roots,
water
uptake
by
plants, microbial production
of
plant
growth
factors
and
microbially
mediated
availability
of
mineral
nutrients.
The
plant
roots
directly
affect
the
density

and
microbial
community
of
the
rhizosphere.
This
rhizosphere effect
can
be
measured
as
a ratio
of
the
15
K.
O.
Mukerji et al. (eds.), Techniques in Mycorrhizal Studies, 15-50.
©
2002
Kluwer Academic Publishers.
16
Techniques
in
Mycorrhizal Studies
number
of
microorganisms
in

the
rhizosphere
soil
(R)
to
the
number
of
corresponding
microorganisms
in
soil
away
from
the
root (S)-the
RlS
ratio
(3).
Generally
RlS
ratios
range
from
5-20
but
it
may
reach
upto

100
in
certain
cases.
Such
an
increase
represents
the
direct
influence
of
the
plant
root
exudates
on
soil
microorganisms,
which
favour
organisms
with
high
intrinsic
growth
rates
(4).
Just
as

the
plant
roots
have
a
direct
effect
on
the
surrounding
microbial
populations,
rhizosphere
microbial
community
also
has
a
marked
influence
on
the
growth
of
the
plants.
The
rhizobial
microbial
community

benefit
the
plant
in
a
variety
of
ways
including
recycling
and
solubilization
of
mineral
nutrients,
synthesis
of
vitamins,
amino
acids,
auxins,
cytokinins
and
gibberellins,
which
stimulate
plant
growth
and
antagonism

with
potenitial
plant
pathogens through competition
and
development
of
amensal
relationships
based
on
production
of
antibiotics
(2).
Among
the
various
types
of
plant-microbial
associations,
the
myconhizal
association
and
nitrogen
fixing
ability
are

by
far
the
most
important
ones
(see
pp.
88-101
this
book).
Some
fungi
enter
into
mutualistic
relationship
with
plant
roots
called
'mycorrhizae'
in
which
the
fimgi
actually
become
integrated
into

the
physical
structure
of
the
roots.
The
fungus
derives
nutrition
(photosynthates)
from
the
plant
and
in
return
contributes
to
its
nutrition
without
causing
disease.
The
mycorrhizal
associations
are
more
specific

and
organized
than
the
rhizosphere
association.
In
this
association
the
fungi
becomes
integrated
with
the
plant
roots
forming
distinct
morphological
units.
One
of
the
most
important
and
technologically
explored
mutualistic

relationships
of
the
plant
and
microorganisms
is
that
of
the
'nitrogen
fixing
bacteria'
and
plant
roots.
These
bacteria
infect
the
host
plant
and
form
'nodules'
where
the
conversion
of
atmospheric

nitrogen
to
ammonia
takes
place.
The
nodulating
bacteria
associated
with
the
leguminous
plants
were
edier
placed
into
a
single
genus,
Rhizobium
but
recently
two
new
genera
Azorhizobium
and
Bradyrhizobium
have

been
recognized.
Table
1
lists
the
characterstics
of
all
the
above
three
genera.
The
conversion
efficiency
of
atmospheric
nitrogen to
ammonia
depends
upon
the
nitrogenase
enzyme
system
Nitrogenase
being
oxygen
sensitive

is
restricted
to
habitats,
which
are
aerobic.
In
addition
to
nitrogen
reduction, the nitrogenase complex forms one
~
for every
N2
reduced
and
can
also
reduce
other
substrates
such
as
acetylene
to
ethylene.
Only
a
few

strains of
Rhizobium
and
Bradyrhizobium
possess hydrogenase
Rani
Gupta
and
H.
Mohapatra
17
TABLE
1
Characteristics
of
Rhizobium,
Bradyrhizobium
and
Azorhizobium
Characters
Rhizobium
BradJ::.rhizobium
Azorhizobium
Flagella
in
liquid
None None
One
lateral
medium

solid
Peritrichous
One
polar
or
subpolar
One
lateral
medium
Nitrogen
fixing
ability
None
None
All
strains
of strains
outside
host
plant
Growth
rate
in
culture
Fast
Slow
Fast
Range
of
host

specificity
Narrow
Broad
Only
one
species
identified
so
far
Specificity
of
hosts
Legumes
and
forage
Soybeans
None
cro~s
activity
and
can
utlize
hydrogen.
The
infection
of
Rhizobium
and
Bradyrhizobium
is

greatly host
specific.
The
association between the
leguminous
host
and
the
bacteria
is
based
on
specific
chemostatic
response
and
specific
binding
to
the
root
hair
prior
to
invasion
and
establishment
of
the
root

nodule
respectively.
Apart
from
the
leguminous
plants
nitrogen
fixation
also
takes
place
in
non-legumes
by
Rhizobium, cyanobacteria
and
actinomycetes.
To
cite
a
few
examples,
Rhizobium
can
fix
nitrogen
in
association
with

Trema,
a tree
found
in
tropical
and
sub-tropical
regions.
Similarly,
actinomycete
(Franlda)
symbiosis
is
more
frequent
in
various
woody
shrubs,
small
trees
as
Alnus,
Myrica,
Hippophae,
Comptonia,
Casuarina
and
Dryas.
These

trees
are
common
in
the sub-tropical
and
tropical
regions.
The
liverworts,
mosses,
pteridophytes,
gymnosperm
and
angiosperms
are
able
to
establish
mutualistic
relationships
with
nitrogen
fixing
cyanobacteria
Nostoc
or Anabaena.
The
gymnosperm
Cycas

develops
corolloid
roots
while
the
angiosperm
Gunera
has
stem
nodules.
Inspite
of
all
the
above
beneficial
effects
tapping
of
these
microbial
resources
still
pose
a
big
challenge.
Measurement
of
microbial

biomass
or
population
size
in
soil
is
difficult
and
the
techniques
commonly
used
have
limited
accuracy.
For
example,
direct
microscopic
observation
of
the
soil
samples
tends
to
over
estimate
the

number
of
viable
cell
propagules
because
non-viable
cells
are
included
in
counts.
Alternatively,
the
indirect
plate
count
technique
tend
to
under
estimate
the
number
of
soil
micro-organisms,
due
to factors
such

as
incompatibility
between
microbes
in
the
soil
sample
and
the
nutrient
medium
18
Techniques in Mycorrhizal Studies
used
for
incubation
or
incomplte
suspension
of
microbial
propagules
in
original
soil
dilution
(see
pp.
7-13

this
book).
Recent
molecular
techniques
based
on
the
analysis
of
ribosomal
RNA
(rRNA)
offers
a culture
independent
method
for
microbial
identification
and
should
result
in
a
more
objective
assessment
of
variability

and
population
size
in
natural
mocrobial
community.
This
chapter
deals
with
the
isolation,
purification
and
identification
of
the
naturally
occurring
microbes
and
envisages
the
importance
of
modem
molecular
taxonomy
in

the
flawless
positioning
of
these
microbes
in
the
taxonomical
hierarchy.
The
isolation,
purification
and
identification
of
bacteria
and
fungi
have
been
dealt
with
at
the
beginning
of
the
chapter
and

it
concludes
with
an
account
of
procedures
for
isolation,
and
identification
of
actinomycetes
as
a
separate
section.
2.
Isolation
The
preparation of a pure culture
involves
the
isolation
of
a
given
microorganism
from
a

mixed
natural
microbial
population.
Pure
cultures
may
simply
be
obtained
by
pour
or
spread
plate
method.
This
involves
the
separation
and
immobilization
of
individual
organism
on
solidified
nutrient
medium.
Prior

to
isolation,
the
soil
sample
should
be
homogenized
to
produce
a
well-dispersed
suspension
of
soil
or
sediment
that
can
be
diluted
readily
for
inoculation.
The
method
also
serves
to
release

microbial
cells
from
particle
surfaces,
but
the
extnet
to
which
it
does
so,
depends
on
the
physical
and
chemical
nature
ofthe
sample
being
processed.
Sample
preparation
Procedure
a Aseptically transfer
109
of

sample
material
to
a sterile blender
head.
Note:
Metal
"semimicro"
(25-250ml)
blender
heads
are
recommended
because
they
blend
100ml
samples
more
effectively
than
do
the
larger
head
that
come
with
most
blenders.

b.
Add
95ml
of
sterile
O.
1%
sodium
pyrophosphate
(NalP,.
10~O;pH7.0)to
the
sample
in
the
blender.
This
produces
a I:
10
suspension,
assuming
approximately
50%
pre
space
in
the
sample.
c.

Blend
the
sample
for
I
min
(two
30s
bursts
separated
by
a
30s
rest interval).
d.
Rapidly
transfer
the
blended
material
to
a
sterile
container
that
can
be
stoppered
tightly
and

shaken
(e.g.
a
flask
or
bottle).