-

2.4
Soil Microbes
Cultural pracdces
o Rotation of crops with different species of leguminous and
graminaceous plants bring out different stimulatory effects
of microflora.
o Other practices like tillage, irrigation have several chemical
and physical effects on soil that indirectly affect soil micro
populations. Waterlogged fields like rice fields promote
only anerobic and microaerophilic microbes
o Fertilizer application increase the application of green
manure and green leaf manure promote actinomycetes and
fungi microflora as the organism obtain ready nutrients.
Autotropic bacteria grow well, however addition of nitrates
inhibit activity of free living nitrogen fixing organism like
Azatobacter.
o Other practices like burning of the top soil which results in
the partial sterilisation of the soil. This results in the loss of
microbial equilibrium for s short period of time.

2.2 Major-groups of soil Microflora

The six major groups of microbes of soil are as follows:

• Bacteria and Cyanobaceria

• Fungi
• Actinomycetes
• Algae
• Protozoans
• Viruses

2.2.1 Bacteria
Bacteria population are the most dominant for .
of_the biomass of the soil comprising of ID' to IO~~e~:t1mated to be half
s011. The number and types of bacte . . fl s per grams of the
and their microenvironment Th na afire m uenced by the soil types
·1 h . . . . ey are ound maxim . .
so, s, t_ an m virgm soils due to th um m cultivated
availability. The number deer e p'.esence of nutrient and aeration
horizons down to D horizon) e~ses ~•th the _depth of soil. (from A
. n spite of therr small size and s1mp
. le
 Soil and Agricultural Microbiology
2.5
~orphology, they are highly significant because of their involvement in
h1ogeochemical cycles. They also have wide environm~ntal toler~ce and
have well established biotic relationship with other nucroorgamsms and
also with higher plants. Toe inner region of soil a~gregates co~~in
mostly gram negative, while the outer regions contam gram pos1t1ve
organisms.
Winogrodosky classified soil bacteria into three types:-
1. Autochthonous (Indigenous) - Tuey always present in large
numbers. Their number is always constant in ecosystem. This is
classified on the basis of the supply of nutrients.
2.. Zymogenous (Foreign) - They are present in low in numbers.
Their number will increase only when a particular substrate is
present in an ecosystem.
3. Allochthonous (Total strangers) -They are total strangers in the
ecosystem. Their number is purely transitional. They are not
permanent residents.
Bacteria are also classified based on their nutritional requirements.
Both autotrophs and heterotrophs are present in the soil. Phototrophs
utilising sunlight as source of energy and chemoautotrophs using
inorganic chemicals like nitrates and ammonia (Nitrosomonas) utilising
sulphur (Thiobacillus) utilising ferrous iron and converting to ferric like
Ferrobacilli etc are present. Bacteria exist in all forms like cocci, bacilli
and as spiral forms. Bacilli are more common and spiral are rare. Under
unfavourable conditions, these bacteria forms endospores which are
resistant, thick walled and help them to tide over unfavourable
conditions. The spores germinate under favourable conditions and give
rise to new bacterial populations. Enumeration of the bacteria population
in soil can be done by the serial dilution technique. However, this does
not give a real picture because a single medium is not suitable for a
variety of soil species .eg Rhizobium, Azatobacter, Pseudomonas
Nitorsomonas,_ Nitrobacteria, Xanthomonas, Mycobacterium:
Coryneb~ctenum, M1crococc1 etc. are quite predominant in soil.
S~orul~tmg for~ of Bacilli and Clostridium account for the 25% 0 f
ffilcrob1al populat10n.
Factors affecting bacterial population
Nutrient
H concentration - AutOtr ophs use CO2 for photosynthesis
etrotrophs depend on organic matter. '
Soil Microbes 2.6

Organi~ matter ~f the soil - dead remains of plants and animal

population also influences the soil microflora.
Soil Moisture - optimum 50 - 70%. Moisture is present in the
form of film in the soil. Good combination of moisture and
aeration favours heterotrophs. These conditions can be altered
with irrigation, tilling, crop rotation etc which enhances the
bacterial population in the soil.
Temperature - optimum 25°C - 38 °C, with exceptions to some
thermophiles present in the hot springs.
pH - 4-10. Species showing wide tolerance from pH 3 - 11 is
. Accetobacter Chrococcum etc. However acidic and alkaline
conditions restrict growth. Neutral pH favours growth.
Cultivation practices - various agricultural practices like
ploughing, tillage improves soil porosity which favours bacteria
population.
Seasonal changes - During summer, there is low bacterial
density. The rainy season enhances bacterial activity. The dead
seasonal plants at the end of autumn contribute to the nutrients
of the soil.

Methods of isolation of Bacteria

Bacteria are very easy to isolate. They are easily isolated by growing
them in a medium containing soil extract.
Soil enrichment technique - Another simple method is by Soil
enrichment technique. This technique is normally used for
cellulose decomposing bacteria.
Tissue culture method - Plant pathogens are isolated by tissue
culture method using nutrient agar by preparing a bacterial
suspension from the infected tissues and streaking on the agar
medium. After incubation the colonies are isolated and
subcultures on agar slants.
Serial dilution and plating technique. To enumerate soil bacteria
from the rhizosphere, samples are collected and suspended in
serial dilutions and then plated in suitable media so as to allow
the development of not more than I00-150 colonies per plate of
IO cm dia. When the soil sample is spread on the solid agar
surface then it is called as spread plate, and when it is mixed
with melted agar medium and then allowed to solidify then it is
called as pour plate technique. The colonies developed are then
 2.7 ·1 d Agricultural Microbiology
S01an
counted . of soil bacteria
cann On a colony counter. En~er~tt~n soil dilution
ot be done accurately due to the Iurutattons of . fi
and plating. The inherent faculty of roanY bact~n~ todv~~:
e~dospores facilitates the survival of many bactena in a
situations.
rdentification of these bacteria can be done by ~_a:ng,
techniques like Gram Staining Acid fast staining etlcikw IMV1c~anc
1
· ' · · It ts e ,
be further confirmed by various b1ocbenuca es
TSI, enzymatic production etc.

2.2.2 Fungi th t
Next to bacteria fungi dominate the soil. Adanatz_ (!886) was e fi_rS
to isolate fungi from soil. Waksman reported that sod_1s a ~tural habi~t
for mycoflora. They play a very important role ID sod as organic
decomposers of plant and associations and are also many plant
pathogens. Saprophytic fungi are commonly active aro~d. woody pl~t
residue. Fungal hyphae have advantages over bacteria ID some soil
environments. Under dry conditions, fungi can bridge gaps between
pockets of moisture and continue to survive and grow, even when soil
moisture is too low for most bacteria to be active. Fungi are able to use
nitrogen up from the soil, allowing them to decompose surface residue
which is often low in nitrogen. Along with bacteria, fungi are important
as decomposers in the soil food web. They convert hard organic material
and digest them into forms that other organisms can use. Fungal hyphae
physicaHy bind soil particles together, creating stable aggregates that
help ~cre~e water _infiltration and soil water holding capacity. This
helps m.soil aggregation and formation of humus from raw residues. Soil
fungi can be _grouped into three general functional groups based on how
they get their energy. They are: Decomposers, Mutualists and Plant

:t f
pathogens
0 {~sers - saprophyt~c f~ngi - convert dead organic material into
ga_ I~mass, carbon dioxide (CO2), and small molecul
orgaruc acids These fun • es, sue
h
as
cellulose and. 11·gnm,· • g1 gend,era11y use complex substrates, such as the
1n woo and are · •
. carbon ring structures ins essentia1 m decomposing the
. fungi" because they use th~me poll~tants. A few fungi are called "sugar
Like bacteria fWigi _same sunple su~strates as do many bacteria.
. , are Important for b·1· . .
nutnents in the soil In dd. . unmo I 12mg, or retaining
fun · · a Itlon, many of the dary '
. gi '.11"e organic acids, so they h . secon metabolites of .
ac1d nch organic matter th t . e1 Increase the accumulation of humio-
the soil for hundreds of a Is ~es1stant to degradation and may stay in
years. FIiamentous fiungi· P1ay an important role
Soil Microbes 2.8

in the degra~ation of cellulose, hemicelluloses, starch, pectin, lignin and

o!her pr~temceous substances and form organic matter, ammonium and
· simple mtrogen compounds added to the soil.
Mutualists- the mycorrhizal fungi - colonize pJant roots. In exchange
for car~on fro~ the ~]ant, mycorrhizal fungi help solubilize phosphorus
and bnng soil nutrients (phosphorus, nitrogen, micronutrients, and
perhaps water) to the plant. One major group of mycorrhizae, the
ectomycorrhizae grow on the swface layers of the roots and are
commonly associated with trees. The second major group of mycorrhizae
are the endomycorrhizae that grow within the root cells and are
commonly associated with grasses, row crops, vegetables, and shrubs.
Arbuscular mycorrhizal (AM) fungi. Ericoid mycorrhizal fungi can by
either ecto or endomycorrhizal.
Pathogens or parasitic fungi, the third group of fungi, cause reduced
production or death when they colonize roots and other organisms. Root-
pathogenic fungi, such as Verticillium, Phythophtora, Pythium, · and
Rhizoctonia, cause major economic losses in agriculture each year. Many
fungi help control diseases. For example, nematode-trapping fungi that
parasitize disease-causing nematodes, and fungi that feed on insects may
be useful as biocontrol agents maintaining the microbiological balance
in the soil.
All major classes of fungi are found in soil. Members of
Deuteromycetes are more frequent than the other groups. Higher fungi
like mushrooms are more predominant. Members of Ascomycetes and
Basidiomycetes are also present.eg Muc~r, Rhizopus, . Penci~lium,
Pythium, Aspergi!lus sps, Fusarium, Alternarza are present m maximum
populations.
Factors affecting fungal population
Organic matter - It has a direct effect on the fungal population.
Acidic pH- favours fungal species.
Tuey are strict aerobes.
Temperature - varies from 25°C - 40°C, mostly mesophilic.
Soil Moisture - 15 to 30%, excessive moisture is harmful
Increased NH 4+ concentration and fertilizer content favours the
fungal growth.
 2.9 Soil and Agricultural Microbiology
Seasonal chang H tbs are not suitable for
fun . . e - ot summer mon 1 related to the
g1. Vaned temperature and moisture are close Y
fungal population.
Vegetatio_n _ Regular supply of plant de~ris enhance the nutrient
status which increase the activity of fungi.
Me th0 ds of isolation offungi
Enumeration and isolation of soil fungi from soil is usually done ?Y
soil dilution and plating techniques. Martin's Rose Bengal agar with
Streptomycin is the best medium of this purpose.
The buried slide procedure is a convenient method of ~bs~rvin~
fungi. The technique involves the burial of the glas~ ~bde m sotl
for varying periods following which the excess sod 1s removed
by a gentle stream ~f water and the preparation is heat fixed and
stained with rose Bengal and examine microscopically. .Spatial
arrangement of the hyphal strands can be viewed.
The soil plate method developed by J.H. Warcup involves 5 - 15
mgs of soil, taken at the bottom of a sterile plate and melted agar
is poured over the soil. This method is important in qualitative
and ecological studies. ·
The immersion tube techniques consists of burying perforated
glass or plastic cylinders filled with agar in soil. After the
desired period of incubation the agar surfaces are examined and
isolations are made from the hyphae. This method is
adv~ntageous as it helps to study the fungal population in their
b~b1tat,. and to_ ge_t the non-sporulating forms which are often
missed m the .diJut10n plating techniques.
Tissue culture technique - Host tissue is cut and f:
t T db · sur ace
s en_ ise y usmg 95% alcohol and transferred to a suitable agar
medium (SDA agar) ·

2.2.3 Actinomycetes (Ray Fungi)

They are the transitional diverse b .
present numerously present . ..~ group, etween bactena and fungi
b m sw 1ace soil They b . .
num er. They are responsibl f; th · are next to actena m .
Actinomycetes are unknow: ; e ea~h~ smell. Several stages of
bacteria and funoi They ·bl ey exhibit characteristics of both
h · I:)'• resem e bacteri · ·
P ~s10Jogy and are susceptible to m size, morphology, staining,
therr appearance and are hence callrri;ny vrruses. !hey resemble fungi in
e as ray-fung1. They exhibit extreme
Soil Microbes 2.10

variability. Their vegetati~e mycelium_ is often co]ored green orange,

blue green or black. Therr cell wall 1s made up of chitin. They are
significant in having high % of G + C content. They are extremely
variable with non-septate mycelium which may be branched. Some
actinomycetes produce asexual spores ca11ed conidia. However sexual
stages of life cycle is not known. The majority of them are aerobic except
the class Actinomyces which are anerobic and micro-aerophilic.
Nocardia is the most significant pathogen causing mycetoma,
(Actinomycetaceae), for examp]e, .Streptomyces, the most important non
pathogenic genera producing antibiotics. Other examples include
Micromonospora, Thermoactinomycetae etc.

Factors affecting Actinomycetes population are

Organic matter - They are hetotrophilic in nature. Unusual
organimants like paraffin, phenol, steroids can also act as
substitutes.
Temperature Thermophilic actinomycetes like
Thermoactinomyces, Thermomonospora, Streptomyces are more
common.
pH - Their number decrease at pH 5; optimum 6.2
Other factors like depth, moisture content, soil reaction, so!l
vegetation also influence the growth of actinimonycetes. Therr
number decreases as depth increases.
They are high salt tolerant and occur frequently in saline soils
and sea water.
They are mostly present in cultivated aerated fields than in non-
cultivated soils.
. n and drier conditions of the upper layers of the
Better
. t
aera 10 'bl fior their dommance
• • A honzon.
m · .
sotl are responst e

Significance I t athogen and produce scabies . disease.

They act as b~ an !used by (Sterptomyces scabies)
Ex: (potato sea 1es - .
kn wn for their high production of
0
Antibiotics - They are_ d ce antiobiotics.75% of the
.b. t' Many species pro u
anti 10 1cs. S tOmyces
antibiotics are produced from trep •

I
 . Jtural Microbiology
2.11
_ _ _ _ _ _ _ _ _ _ _ ____:~~~ .n d S o i l a ts ~
like chitin and
They are also known for degrading recalcitran ·
cellulose and are active at high pH levels. . th
. spons1b1e 1or e
.i:-,

. b.iaI eqm·1 1bnum

M1cro · . - Actmomy . cetes are
. re of antunicro
. . b.ia I
balance of microbes with the produ~ion tomyces liberate
metabolites.eg Many species of . trep
extracellular protease which lyse bactena.

Methods of isolation ofActinomycetes . d fungi can

. d for bactena an
. The same methods of isolation mentione grow very slow than
. . Actinomycetes . .
be used to isolate a~tmomy~e~es. . which are low m ~1trogen
bacteria and fungi m spec1ahsed m~dia . or conn' s medium are
• ht'
content Media like Kennkn1g s m ' edia albummk's medium also ·gives
•
·. t or Czepec .
used to isolate them. Yeast extrac . ycetes can be isolated by
. h nic actmom
satisfactory growth. Plant pat oge t mvces seab.res
using tyrosine casein agar Ex. S'.trep O ._,,

2.2.4 Algae · 'bl

nd eukaryotes. They are visi e
Soil algae includes both prokaryotes ath surface of the soil, where
to the naked eye as a green
. scum on e is predominantly
d unlight . . ble.
availa
/o
ade~uate amount _of mfro1st~;O a~ 000 per gram of soil. Due to the
Therr number vanes om ' · h · II th are
resence of the photosynthetic pigments m t elf ce s, ey ..
~utotrophic in nature using carbon dioxid~ ~?m the ~tm?sphere and give
out oxygen. Multiplication is by cell d1v1s10n which mvolves several
kinds of cells like exospores and akinites. Single celled blue green_ algae
show binary and multiple fission to releas~ exospores . apically.
Filamentous forms randomly fragment by vegetative reproduct10n. Each
fragment is known as harmogonia which grows into a new filament.
Eukaryotic soil algae are unicellular, filamentous or colonial and less
developed than aquatic algae. They are dominant in acidic soils and
numerous in neutral and alkaline soils. ex members belonging to
Chlorophyceae, Bacillarophyceae, Xanthophyceae and Diatoms are
n_um_erous in soil. Prokaryotic Cyanophycean (Blue Green) algae are
s1gmficant a~ th~y are photosynthetic and fix the atmospheric N2. Their
phot~s~nthet1c pigments (chlorophyJJ and phycocyanin) are located in
spec1allsed cells called chromatophores. They also possess specialised
cells called
. heterocysts
. wh1c
· h are mvo
· 1ved m • mtrogen
. . They are
fixation.
predommant m neutral and alkal me · soi·1s the dominant
• blue green algae
are C'hroococcus' Anabena, MosIoc, Q~ct'llatona, . Tolypothrix etc.
Soil Microbes 2.12

Factors affecting Algal population are

Light and carbon-dioxide - Two major factors which regulat
the algal population ( I03/ g of soil). e
Sa~ated soil is preferred and they do not grow in water-logged
soils.
Temperature-very wide,12- 87 °C

pH - prefer neutral/alkaline soils and are less in acidic soils.

Ca - Important element favourable algal growth. Calcium rich

soils are rich in NQ·3 and POlwhich favour the algal growth.

Fertilizers - Mixtures of Kand Pis quite effective while NH/ is

less effective.
Presence of other microbes is related to algal growth. If they are
continuously present, they get denatured and fungi dominate.
Soil fertility - Dead and live algae act as soil fertilizers. They
also act as binding agents and prevent soil erosion.

Methods of isolation of algae

It is not easy to isolate pure cultures of algae since most of them are
covered by mucilaginous matrix that harbours many contaminants. The
medium (Pringsheim's, Chu's, Benneck's or Bristol medium) is taken in
a half pint milk bottles, filled up to a depth of 5 cm plugged with cotton
wool and sterilised. After cooling, 1 gm of soil is taken, moistened with
distilled water and then introduced into the sterilised bottles containing
the medium. They are incubated at 30-35°C and lighted from above by
25 Watt lamps. The light intensity is adjusted to one third of the normal
sunlight. In about fortnight after incubation, algal growth may be seen.
The algae are sub cultured by transferring a few of the cells to Beneck's
agar in petridishes. They may be repeatedly streaked to get single
isolated colonies. Fogg's and Allen medium or Amon's medium is
generally used to grow nitrogen fixing algae like Anabena. For some
algae, soil extract or yeast extract, casein hydrolysate may also be added.

2.2.5 Protozoal)s
TI1ey are characterised by unicellular forms present actively in more
proportions in the rhizosphere. Their population ranges from I0,000 tO

I
 l
2.13 .Soil and Agricultural Microbiology
100,000 per gm of soil. They are abundant in the upper layers of soil and
the application of organic matter increases their nwnber.
They are relatively simple in _struct~e and are characterised by a cyst
in their life cycle which helps 1t to tide over adverse situations. Their
protoplasm may be naked or encased in shell.

They are of two types

Plant like - belonging to phytomastigophora, has chlorophyll.
The cells are motile with flagella.eg., Spiromonasa
Animal like - - Zoomastigophora, lack chlorophyll. They are
non motile. They have only pseudopodia. They lead an active
life during . favourable conditions and during unfavourable
conditions forms cysts which is the resting stage eg Amoeba.

They are also classified on the basis of locomotion Pseudopodia

belonging to the family Sarcodina (Fake foot) ex: Amoeba ciliates
belonging to the class ciliates (cilia) ex Paramoecium, Vorticella and
flagellates belonging to class Mastigophora (whip like structure)
ex: Cercomonas, Spiromonas Euglena. They act as predators regulating
the growth of bacteria, feed on them and release excess of nitrogen into
the soil to be utilised by the plants. They also act as a food source for
other soil organisms and feed on plant pathogens. They are significant in
predating soil bacteria and thereby maintaining the biological
equilibrium in soil.

Factors affecting Protozoan population are

Adequate supply of nutrients, organic matter and moisture.

Present on the surface of soil up to pH 6.

Agricultural pra~tices and rich manure favours growth. .

Aerobic - 02 is essential
Aquatic water supply is also essential.
Indifferent to pH variations (mostly)

Cool and damp conditions favour

water is low they protoz . ~owth .. When the supply of
environment becomes c o~re~am ma cyst condition until the
on uc1ve for their growth.
Soil Microbes 2.14

Methods of ;solation ofProtozoans'

Enumeration and isolation is generally done by glass ring method.
Aerobacterium is grown on a suitable medium. Bacterial cells are taken
from this culture and spread in small circles on the surface of the
sol~dified a~ar. (m_ade from 5 % Sodium Chloride and and 1 % agar)
Smtable soil dilutions are prepared on 0.5% NaCl solution and 0.5 ml
aliquots of each dilutions are transferred to bacterial circles.
Subsequently small glass rings are pressed on the agar around the
bacterial circles. When the protozoans grow, bacterial colonies disappear
indicating the presence of protozoans.

2.2.6 Viruses
Soil viruses are sub microscopic and obligate parasites in soil. Viruses
do not form a major soil population, but play a important role as _they
infect other microbes. They remain in soils for a periods ranging up to
several months. They do not multiply in soil but persists for more than a
year. They pass through bacterial filters. The tail of the viruses attack
host, gain entry into the cytoplasm of the host. Even though they are seen
only under the electron microscope the lysogenic effect of specific
viruses on their hosts can be seen in the form of plaques on agar plates.
The strain of viruses affecting other microbes are called as phages as
follows:-
Bacteria Bacteriophages

Fungi Mycophages
Actinomycetes Actinophages
Blue green algae - Cyan ophages

Nematodes Nematophages
Lysis sets in when the bacterium multiplies in the host and the phages are
released capable of infecting other cells. Importance of the phages in the
soil is still under study. They are recognised only by their symptoms on
the plant surface. Big Vein disease of lettuce are important viral
pathogens.
Factors affecting viral population are
They are generally present in rich organic soils with humus
Sandy soil is not favourable.

I
2.15
~ ~ - - -______JS~o~iland Agricultural Microbiology
-
--
The~ adversely affect the bacteria population affecting the N2
fixation brings down the soil fertility. ·
They are also significant as they uansfer genetic material into
new b~cteria (transduction). ·

Mel hods of isolation ofviruses

A sample of soil is incubated with the host bacteirum, allow it to
m~ltiply for increase in population for that specific viruS, 50 g of soil is
mixed with 150 ml of distilled water and then filtered through a
bacteriological filter. The filtrate is added to a broth culture of the host
bacterium. After addition the filtrate is spread over a nutrient agar plate
and incubated at 37°C .The plates are frequently .examined. Within 48 -
72 hrs the bacterial colonies are lysed by and patches of clear areas are
seen on the plate. The Iysed area contains a· large population of
bateriophages. This area of Iysis is called as plaque. By the dilution plate .
technique the number of bacteriophages per plate can be ·counted under
an electron microscope. By this method, bacteriophages specific for
Arthrobacter, Arthrobacter, Pseudomonas, Rhizobium Nocardia
Streptomyces have been demonstrated in a variety of soils. '
Review Questions
Two marks
1. Define a) Autochthonous b) Zymogenous c) Allocthonous
2. What are fungi?
3.What are ray fungi?
4. What are phages?
5• What is a plaque?
Essay Questions
I. Mention
. . the different types of m1crob1al
. . population in soil and th .
s1gmficance. elf
2. What are the various facts affecti . . .
3. What are the v . ng m1crob1al population in soil? .
ar10us methods of iso1atmg. m1crobiaJ
. population in soil?

J1
11
tr