This PowerPoint Presentation intends to explore the isolation of Rhizobium and the contribution of microbes to soil fertility on a large scale.

1. General account about the microbes used as biofertilizer –
Rhizobium – isolation, identification, mass multiplication,
carrier based inoculants, Actinorrhizal symbiosis.

3. A Presentation by
Dr. N. Sannigrahi, Associate Professor,
Department of Botany,
Nistarini College, Purulia,
D.B. Road, Purulia (W.B) India-723101

4.  Microbes are the tiny, invisible biological world observed under
magnifying devices-microscope, ultra microscope like SEM etc.
 They include single- and multi-celled organisms, both prokaryotes and
eukaryotes can be microorganisms. They can be bacteria, archaea, fungi, or
protists.
 We can only see up to 100 micrometers (µm) with our bare eyes, so
organisms smaller than this size are considered microorganisms (Fig. 1).
 Microorganisms are found in every ecosystem and can also be closely
associated with many multicellular organisms.
 Bacteria are single-celled prokaryotic microorganisms that can live freely
or in association with a host and are omnipresent within our environment.
When bacteria are said to be omnipresent, it means that they are present in
virtually every habitat on Earth, from the soil to the ocean to within our
bodies. Bacteria even inhabit seemingly inhospitable environments, such as
within the hot soil in and near volcanoes and in radioactive waste.

5.  Archaea are another type of single-celled prokaryotic organism and, until
relatively recently, were believed to be another kind of bacteria known as
archaebacteria. This is due to the many similarities that archaea and
bacteria share.
 Fungi are eukaryotic organisms that can be microscopic or visible. Perhaps
the fungi you are most familiar with are mushrooms since they are visible
to the naked eye (and, sometimes, edible)!
 Despite the physical appearance of mushrooms, fungi are not plants and
are, instead, members of a separate biological kingdom unique from
animals and plants. For our purposes, we are only interested in microscopic
fungi, known as micro fungi.
 Protists are mostly microscopic, single-celled organisms, including algae
species, amoebas, ciliates, slime molds, and more. Protists appear to be a
very diverse group of organisms, and protist taxonomy is mainly in a state
of flux due to continually changing classifications and discoveries.
 All the microbe4s play a number of beneficial; & harmful attributes and the
functions can be summarized as below:

6.  Bacteria play a vital role in our environment and within our bodies. The
presence of bacteria is essential for all other life on Earth.
 Bacteria are both decomposers and producers - they decompose dead
organic matter and waste into inorganic matter rich in nutrients, thus
allowing plants to grow. These plants then feed herbivores, which in turn
feed carnivores.
 Inside and on animals, including humans, bacteria can be found as part of
the micro biome, playing vital roles such as aiding digestion. In fact,
disruption of the micro biome is believed to play a role in developing
specific syndromes and diseases. While most bacteria are harmless or
beneficial to humans, some can cause illness, disease, and even death.
These are known as pathogenic bacteria.
 They can act as the decomposers and to be used as the fertilizers of
biological origin. Some of the bacteria have the potential to act as bio-
fertilizers.
 Some of the fungi play a very significant role and acts as the fertilizers
with the close association of the higher plants- the symbiotic assessment is
considered as mycorrhizae.

8.  Rhizobium is a genus of bacteria belongs to Rhizobiaceae family acclaimed for
its ability to institute mutualism with leguminous plants and ultimately fix
elemental dinitrogen. With the furtherance of research, Rhizobium has gained
expeditious traction because of which legume–Rhizobium technology has also
risen to popularity. Amid such advancement, hydroponics is being applied to
screen symbiotic interactions of Rhizobium in order to obtain more information
on their genetic and molecular mechanisms including functionality in the host
plant. The bacteria enter the roots of leguminous plants and create nodules,
within which molecular nitrogen is reduced to ammonia, which is then used by
the plant to synthesize vitamins, proteins, and other nitrogen-containing
substances. As a result, these root nodules serve as ammonium-ion storage sites
(Flores-Félix et al., 2013). Rhizobium increases growth in non-leguminous
crops by changing their root shape and growth physiology. Furthermore,
Rhizobium spraying boosts crop productivity by increasing plant height, seed
germination, nitrogen content, and leaf chlorophyll (Sara et al., 2013). Rice
seed inoculation with various Rhizobium strains at increasing levels of nitrogen
increases the content of straw by 4%–19% and the yield of rice grain by 8%–
22% (Sammauria et al., 2020). Rhizobia include bacteria such as Rhizobium,
Bradyrhizobium , Azorhizobium, Mesorhizobium, and Sinorhizobium etc.

10.  Rhizobium are symbiotic diazotrophs,
 A gram (-) soil bacteria endo-symbiotic association with legumes,
 Rod shaped non-spore forming bacteria,
 Invade legume root through root hairs,
 Form effective red colored LHb in the root nodules and fix atmospheric
nitrogen,
 5 important genera- Rhizobium-slow-growing rhizobia forms acid,
Bradyrhizobium-fast –growing alkali, Azorhizobioum infects both stem &
root, Sinorhiozobium ,Photorhizobium,
 Different Rhizobium grows on different hosts like R. leguminosorum on
pea, R. phaseoli of Bean, R. trifoli on Clover, R. meliloti on alfa-alfa, R.
japonicum on soyabean, R. lupine on lupine groups.
 Most of the cases, the basic nature of the infections and the developmental
stages are almost identical and the multiplication in large steps are almost
identical.

12. Name of the
group of host
plant
Name of
Rhizobium Sp.
Name of the
host plant
Nitrogen
fixation.kg/ year
Pea Group R. leguminosorium Pea ( Pisum
sativum), Lentil (
Lens culinaris)
62-132
Soybean Group R. Japonica Soybean ( Glycine
max)
57-105
Lupine Group R. lupini Lupinus arcticus 70-90
Alfalfa group R. meliloti Melilotus indicus 100-150
Beans Group R. phaseoli Phaseolus munga 80-110
Clover group R. trifolii Trifolium repens 130
Cowpea Group R. species Cicer arietinum,
Phaseolus munga
57-105

14.  Healthy nodules were isolated from the six month old D. sissoo seedlings
grown under net house conditions.
 The nodules were washed in tap water to remove the adhering soil particles
on its surface.
 Nodules were dipped in 0.1 % mercuric chloride (HgCl2) solution for 30
second and then washed successively eight to ten times with sterilized
distilled water to remove the traces of HgCl2.
 Surface sterilized nodules were crushed in sterilized distilled water by glass
rod to obtain a milky suspension of bacteriods.
 The suspension was streaked on YEMA medium and incubated at 28±2°C
for 2-5 days.
 The growth on YEMA medium was counted and expressed as cfu/g.
Isolates obtained from nodules of Dalbergia sissoo were purified on
YEMA medium by streak plate method
 Authentication Congo red test All the purified rhizobial isolates were
streaked on CRYEMA medium and were observed for absorption of Congo
red dye (Vincent, 1970).

15.  Bromothymol blue test The YEMA medium containing bro-mothymol blue
was streaked with isolated strains and was observed either for yellow color
due to production of acids or blue color due to production of alkali (Norris,
1965).
 Hofer’s alkaline test This test is based on the fact that Rhizobium is unable
to grow at higher pH 11.0 on yeast extract mannitol broth (Hofer, 1935).
Ketolactose agar test: Ketolactose agar plates were streaked with isolated
microbes. After incubation for 4-6 days at 28±2°C, the plates were flooded
with Benedict’s solution. This test is based on the fact that Rhizobium is
unable to utilize the lactose (Bernaerts and Deley, 1963).
 Plant infection test The different isolates were tested for their ability to
nodulate Dalbergia sissoo plants grown in plastic pots. Seeds of Dalbergia
sissoo were inoculated with Rhizobium isolates by soaking seeds. Plants
were carefully uprooted after 75 days and observed for nodulation.

17.  The multiplication of the Rhizobium is very important for the large scale
inoculation of the microbes in the host tissue to improve the plant health by
making the avenues accessible to the standing crops . Only the inoculums
of this particular species is not enough, it also needs the addressing of the
processing along with the packaging for transportation from the laboratory
to the fields.
 The mass production of Rhizobium comprises the following steps for the
successful multiplication of the microbes in the large scale:
 Inoculums preparation,
 Processing of carrier material,
 Selection of ideal carrier material,
 Preparation of carrier material,
 Mixing the carrier & broth culture and packing,
 Preparation of the inoculants packing.

18.  The inoculums preparation needs the preparation before the process and it
needs the in depth knowledge about the microbial pure culture techniques
garnished with the following apparatuses-
 Autoclave for sterilization- for making the media & glass goods along with
desired materials contamination free
 Hot air oven- Growth of the microbes at desired temperature
 Laminar Air Flow chamber- Transfer of inoculums in aseptic environment
 Incubator- Incubation of the microbes
 Rotary and Shaker- Aeration of the growth of the microbes
 pH meter- To maintain the desired pH of the microbes
 Refrigerator- To preserve the inoculants at low temperature
 Fermentor- A large vessel for mass production of microbes in controlled
condition
 In addition to these, the media preparation along with the desired growth
materials and continuous electric supply is essential in order to maintain the
aseptic environment for the successful execution of the entire microbiological
process.

19.  INOCULUMS PREPARATION:
 The inoculums preparation needs the understanding of the following
features of the isolation of the Rhizobium from the root nodules.
 Materials Required: Roots of the Legume plants, sterile distilled water,
pipettes, test tubes, YEMA plates, 70% ethanol, 0.1% Mercuric chloride
solution, aseptic conditions by using the disinfectants and sterilizing
materials along with the basic ingredients of the microbiological attributes.
 1. The root nodules are collected, washed with the running tap water and
treated with 0.1% mercuric chloride or 3-5% Hydrogen peroxide,
 2.After repeated running with the tap water, the nodules are passed through
70% ethyl alcohol,
 YEMA plates are prepared with the sterilized environment,
 1 gm of nodular extract is prepared with 10 ml of distilled water and mixed
properly,
 Serial dilution is made up to 10-8

20.  Suspension of 0.1 ml from 10-8 is taken and poured over the plates
prepared previously and made it, spreads throughout the plate,
 In the suitable environmental conditions at 32℃ is incubated for further
analysis.
 PREPARATION OF STOCK CULTURE
 After incubation, after 4-5 days, the Rhizobium colony will appeared on
the plate and it is identified with congored dye . Most of the Rhizobium
colony arte white in color and took the stain of congored.
 The colony took the strain are Rhizobium,
 The pH of the stock culture to be kept at 6.8.
 For large scale production, production of starter culture is required; the
starter culture is prepared in suitable broth,
 In the starter culture, the conical flasks are kept at the suitable temperature
for the same in the large scale production.

21.  PRODUCTION OF CARRIER BASED INOCULUM
 After the production of semi-liquid inoculums from the fermentor, the
carrier materials are taken,
 The carrier materials may be pit, lignite, vermiculite, charcoal of the
farmyard manure,
 The carrier materials must be cheap, easily available, low toxic contents
along with high organic contents to address the economic feasibility,
 Water holding capacity of the carrier materials must be more than 50%,
 The carrier materials must be transformed into dust and the pH must be
maintained with calcium carbonate powder,
 The all sorts of material to be autoclaved properly for making the
substances free from the contamination.
 The carrier materials must have some adhesive property so as to hold with
the root surface where the inoculation to be exercised

22.  PACKING:
 During the packaging, the bacterial culture must be kept in a metallic tray
with the suitable carrier and with the help of mixture or using hand gloves,
the mixture must be mixed properly before storage,
 The suitable measure to be adopted for the proper sterilization to do the
procedure for the optimum benefit that to be expected in this consequences.
 The polythene bags should be low density grade with a thickness of 50-75
micron,
 The package should contain the following information- the name of the
manufacturer, name of the strain, the crop which to recommended along
with the method of inoculation, date of manufacturer, date, batch number,
price etc,
 Full address of the manufacturer along with storage,
 Instructions to the farmers for its uses and application

23.  STORGAE OF BIO-FERTILIZER PACKET
 The packets should be stored in a cool place far from the heat & sunlight,
 The packets should be stored in room temperature or in cold storage
conditions in lots in plastic crates or the gunny bags,
 The population of the inoculants must be checked at the 15 days interval
for its optimum utilization,
 There should be more than 10 to the power 9 inoculants at the time of
packaging and 10 to the power 7 inoculants at the time of disposal,

24.  The term "actinorhiza" refers both to the filamentous bacteria Frankia, an
actinomycete, and to the root location of nitrogen-fixing nodules.
Actinorhizal plants are classified into four subclasses, eight families, and
25 genera comprising more than 220 species. Although ontogenically
related to lateral roots, actinorhizal nodules are characterized by
differentially expressed genes, supporting the idea of the uniqueness of this
new organ. Two pathways for root infection have been described for
compatible Frankia interactions:
 root hair infection or intercellular penetration. Molecular phylogeny
groupings of host plants correlate with morphologic and anatomic features
of actinorhizal nodules. Four clades of actinorhizal plants have been
defined, whereas Frankia bacteria are classified into three major
phylogenetic groups. Although the phylogenies of the symbionts are not
fully congruent, a close relationship exists between plant and bacterial
groups.

25.  A model for actinorhizal specificity is proposed that includes different
levels or degrees of specificity of host-symbiont interactions, from fully
compatible to incompatible. Intermediate, compatible, but delayed or
limited interactions are also discussed. Actinorhizal plants undergo
feedback regulation of symbiosis involving at least two different and
consecutive signals that lead to a mechanism controlling root nodulation.
 These signals mediate the opening or closing of the window of
susceptibility for infection and inhibit infection and nodule development in
the growing root, independently of infection mechanism.

27.  Soil fertility & soil health are important for crop productivity.
 The natural restoration of soil fertility is the call of the time due to
eutrophication and soil quality deterioration,
 The different microbes in general and some bacteria, BGA and fungi in
particular play a very significant role in this regard,
 Free living bacteria Azotobacter & clostridium etc play important role to fix
atmospheric nitrogen,
 Different symbiotic produce root nodules and the actively fox nitrogen by
Rhizobiun leguminosarum produce nodule in the legume plants,
 By means of biochemical process, the atmospheric free nitrogen are fixed by
the symbiotic mechanisms,
 Bacteria like Frankia fix nitrogen in association with angiosperms like Alnus,
Casuarina etc
 The huge demand of biofertilizers in the context of organic farming is
increasing day to day and the production of bio-fertlizers can be done by the
process of industrial process,
 The isolation followed by large scale production, packaging and to transport to
the fields are very important in this context.

28.  References:
 1. Fundamental Botany- Sen & Giri
 2. A text of Fungi- Vasistha,
 3. A Textbook of Microbiology- R.P. Singh,
 4.Textbook of Microbiology- Dubey & Maheswari
 5. Soil Microbiology- N.S. Subba Rao
 6. Agricultural Microbiology- G. Rangaswami
 7. Google for images
 8. Different WebPages for information.
 Disclaimer: This PPT has been made to enrich free online study
resources without any pleasure of financial interest.