evalution, biodivetsity, different micro and macro flora and fauna, bateria, algae, fungi, actinomycetyes, protozoa,

1. Soil 609:
Recent trends in Soil Microbial Biodiversity (2+1)
Micro 604:
Recent Developments in Soil Microbiology (2+0)
Dr. HIREN K. PATEL
ASSISTANT PROFESSOR
DEPARTMENT OF AGRICULTURAL MICROBIOLOGY
B. A. COLLEGE OF AGRICULTURE
AAU, ANAND –388 110

2. Theory
• Unit I
Microbial evolution and biodiversity.
Microbial communities in ecosystems.
New insights in below ground diverse of plant performance.
• Unit II
Qualitative ecology of microorganisms.
Biomass and activities.
• Unit III
Nitrogen fixing organisms, trends in diversity of N fixing organisms.
Molecular approaches in characterizing N fixing microorganisms.
• Unit IV
Serology and molecular characterization.
Ecological aspects of bio determination.
Soil waste management.
• Unit IV
Biodegradability.
Testing and monitoring of the bioremediation of xenobiotic pollutants.
Bacterial fertilizers.

3. Practical
• Determination of soil microbes using classical
techniques.
• Determination of soil microbial diversity using
molecular techniques.
• Estimation of soil microbial biomass carbon,
nitrogen and phosphorus.
• Estimation of key soil enzyme activities.
• Community level physiological profiling of
microbial diversity.

4. Soil Biota

16. Nematodes
• Eukaryotic vermiform, microscopic, worm like
organisms, which does not possess respiratory and
circulatory systems.
• They are soft bodied heterotrophic and dioecious in
nature. They exhibit a serpentine motion.
• Nematodes are small invertebrates with smooth,
unsegmented bodies that are typically 50 µm in
diameter and 1 mm in length.
• Most nematode species are highly specialized parasites
of vertebrates, including humans, insects, and other
invertebrates.
• Nematodes are different from other worms in that they
are mostly parasitic with non-segmented bodies. These
also generally reside in soil surfaces and water bodies.
• Importance: Pathogenic to plants, animals and human,
they destroy roots of plants.

17. Where they live?
• Nematodes found in soil reside in the top layer of the soil with
organic matter even if they do not feed on the dead and
decaying matter.
• These feed on living microorganisms that are present on the
soil surfaces.
• Nematodes in the soil can be either free living or parasitic.
Most of the nematodes present in the soil include
roundworms that move through the soil if they are free-living.
• Soil nematodes can be classified into four different groups;
bacterial feeders, fungal feeders, protozoan feeders, and
omnivores.
• In the case of agricultural soil, about a teaspoon of soil
supports about 100 nematodes. However, the number differs
depending on the microbial community and the organic
content of the soil.

18. Positive Impact of Nematodes
• Soil nematodes, especially those feeding on bacteria and
fungi, help maintain the microbial community of the soil and
also ensure that enough nitrogen is available in the soil for the
plants.
• Some free-living nematodes are capable of mineralization
where they convert organic compounds into their inorganic
forms, aiding in the biogeochemical cycles.
• Nematodes might even enhance soil fertility by decomposing
complex organic compounds into simpler forms.
• Some of these animals feed on pests by either parasitizing
them or by feeding on them. The movement of nematodes
through the soil increases the porosity of the soil, thus
maintaining a balanced soil ecosystem.

19. Negative Impact of Nematodes
• Predatory nematodes in soils harm the useful
microbial community of the soil, decreasing
the soil health.
• Plant-parasitic nematodes feed on seedlings
and plant roots that cause crop loss in
different agricultural soil.

21. • Protozoa are unicellular eukaryotic
microorganisms that lack cell walls.
• They are motile heterotrophs that obtain food
by ingesting bacteria, yeasts, algae, small
protozoa, and organic matter.
• Free-living soil protozoa fall into three
categories: flagellates, amoebae, and ciliates.
• These single-celled animals differ in shape,
size, and distribution with some protozoan
species found in land habitats like soil.

22. Where they reside?
• Amoeba, ciliates, and flagellates are the three groups of protozoans found in soil.
• Numerous heterotrophic flagellates and naked amoebae are available in
agricultural soils, grassland, forest soil, bottom sediment of freshwater, coastal
and marine waters.
• The presence of protozoa in the soil is influenced by the presence of living and
dead plant roots and the organic content of the soil.
• The inhabitation of soil by protozoa depends on the structure and texture of the
soil.
• Mastigophorans (flagellates) tend to dominate in drier soils, while ciliophorans
(ciliates) are abundant in moist soil.
• Similarly, cultivated soil and clay soil are predominated by flagellates and
amoebae while the soil of coarse texture consists of large flagellates, testaceans,
and ciliates.
• Some protozoans might remain in a symbiotic relationship with other
microorganisms like bacteria and fungi.

23. Positive Impact
• Protozoans are crucial in terrestrial ecosystems where
they act as bacterial consumers, leading to mineralization
of organic soil nitrogen to form ammonium.
• Protozoans community in the soil can also be used to
assess and monitor the changes in the biotic and abiotic
component of soil, thus acting as bioindicators of the
soil.
• Protozoa have been found to increase plant biomass
independently of nutrient contents in plant tissue.
• Many protozoan species feed on bacteria and other
microorganisms which enhances the nutrient cycles and
the energy flow between the microorganisms, animals,
and plants.

24. Negative Impact
• As most bacterial communities in the soil
are the source of food for protozoan, the
presence of protozoa in soil affects
bacterial diversity.
• Some protozoa might be harmful to the
plant, which decreases crop health and
crop yield

27. Algae
• It is eucaryotic photosynthetic, unicellular or multicellular
aquatic organisms.
• Size ranged from 1 µm to many feet.
• Reproduce by asexually as well as sexually.
• Importance: Production of food in aquatic environments,
used as food supplement and in pharmaceutical preparations.
• Source of agar for preparation of microbiological media (as a
solidifying agent), some produce toxic substances.

28. Cyanobacteria
• Cyanobacteria are phototrophic bacteria that are
important in soils where light and water are available.
• Cyanobacteria are autotrophic eukaryotes that consist of
both free-living photosynthetic bacteria and
endosymbiotic organisms.
• Blue-green algae exist in the form of motile filaments of
cells that travel away to form new colonies.
• Blue-green algae are found in colonial or filamentous
form, and the filamentous forms show heterocystous or
nonheterocystous filament.
• The heterocysts are thick-walled, large cells responsible
for nitrogen fixation under anaerobic conditions.

29. Where they reside?
• Blue-green algae in the soil are present in a wide variety of moist
soils, primarily present around the plant root in the form of the
symbiotic association.
• These organisms might either occur freely in the soil or in the
form of symbiotic relationships with plants of lichenforming
fungi.
• Cyanobacteria species have certain structures like heterocysts
that are involved in nitrogen fixation and thus, are present in the
anaerobic area of soil.
• Some of the common cyanobacteria include Nostoc,
Prochlorothrix, Anabaena, Nodularia, etc.
• Cyanobacteria have been reported from a wide range of soils,
thriving both on and below the surface.
• Blue-green algae in soil survive at the mesophilic temperature
that is sensitive to acidity/low pH (optimum pH range 6.5-8.0)
and waterlogged soil conditions.

30. Positive role
• Cyanobacteria are among the first microbial communities to
colonize terrestrial ecosystems.
• These microorganisms play essential roles in soil by fixing nitrogen
and carbon by the synthesis of exopolysaccharides that increase soil
fertility and water retention capacity.
• The application of cyanobacteria as inoculants to induce biocrust
formation on the soil is a novel technology that restores barren
degraded areas and prevents desertification processes.
• They play important roles in increasing soil physical structure,
porosity, and retaining soil moisture due to their filamentous
structure.
• These organisms also produce mucilaginous substances, release
phytohormones, vitamins, amino acids, and secondary metabolites
in the soil.
• Many of the cyanobacterial species have the intrinsic ability to fix
atmospheric nitrogen with the help of a very specialized cell called
heterocyst.

31. Negative role
• In some cases, blue-green algae might form algal blooms,
releasing toxins into the soil that directly or indirectly affects
the vegetation.
• The loss of a large amount of cyanobacteria community in the
soil affects the bacteria communities as it causes oxygen
depletion.

32. • Fungi (Sing: Fungus; Plu: Fungi): Fungi are nucleated, eukaryotic,
spore bearing, achlorophyllous (without chlorophyll) microscopic or
macroscopic organisms.
• Heterotrophic in nature
• Approx 1000 to 15000 kg/ha biomass in top soil
• Can tolerate dry environment and variation in pH
• Generally reproduce by asexually and sexually mean or both.
• Most other fungi have highly branched filaments with strands 2 to
30 mm in diameter and several centimeters long.
• Most fungi are aerobic except for yeasts, which can survive in
anaerobic environments by fermenting sugars into alcohol.
• The somatic structure (body) of fungi are usually filamentous
branched multi-cellular (Molds) and without filament unicellular
(yeast) and typically surrounded by cell walls, it (cell wall) contain
chitin and cellulose.

33. Molds:
• A fungus characterized by a filamentous
body structure (mycelium or thread like
structure) multicellular, eukaryotic.
• It can grow (cultivated) in laboratory on
artificial media. Size 2 to 10 µm x several
mm.
• Reproduction by both asexual (somatic or
vegetative reproduction) and by sexually.

34. Yeasts:
• It is one kind of fungus that is eukaryotic, unicellular and
lacks typical mycelium filamentous structure; it can grow
(cultivated) on media in laboratory. Size ranges from 5 to
10µm or more, width 1 to 5µm.
• Reproduce asexually by budding.
• The yeast cells are larger than most bacteria. They are
commonly egg shaped but some are spherical and
elongated.
• The best known and one of the most important use of yeast
is the production of the ethyl alcohol from carbohydrate
materials. In baking industry and brewing industry for
production of bakery, wine and beer e.g. Saccharomyces
cerevisiae.

35. Where they found in soil?
• In many ecosystems, fungi constitute the largest biomass of all the soil
organisms, ranging from 500 to 5000 kg/ha.
• An individual fungus can include many fruiting bodies scattered across a
large area with extensive underground hyphae.
• Among the soil fungi, one can find oomycetes, hyphochytriomycetes,
trichomycetes, chytridiomycetes, zygomycetes, ascomycetes,
basidiomycetes, and imperfect fungi
• Fungi found in soil fall mostly within three groups; decomposers,
mutualists (mycorrhizal fungi), and pathogens. Some of the common
fungi found in soil include Amanita, Tricholoma, Torrendia, Descomyces,
Thelephora, Verticillium, Phytophthora, Rhizoctonia, and Pythium.
• Since fungi prefer a rather low soil pH and a wide C/N-ratio, they
dominate in raw humus.
• Mycorrhizal fungi are mostly found around plant roots, whereas other
groups of fungi are found distributed throughout the soil.
• Fungi, in general, tend to dominate over bacteria and actinomycetes in
acidic soils as they can tolerate a wider range of pH levels.

36. Positive Role
• Fungi play essential roles in the soil where they help in nutrient cycling,
water dynamics, and disease suppression, all of which maintain the health of
the soil and increases crop yield.
• Fungal hyphae bind soil particles together and stabilize soil aggregates. Fungi
also form beneficial mycorrhizal symbioses with almost all terrestrial plants.
• Mycorrhizal fungi enhance the uptake of mineral nutrients (e.g., phosphorus
and zinc) to the plant in exchange for carbon compounds fixed by plant
photosynthesis.
• Fungi are important decomposers of organic matter in the soil here they use
nitrogen in the soil to decompose woody carbon residues.
• The large size of fungi protects plants against pests, diseases, and drought.
• Fungi are also producers of antibiotic compounds such as penicillin and
cyclosporins.
• Lichens are symbiotic associations of a fungus and an alga or a
cyanobacterium.
• They are important soil colonizers that play critical roles in soil formation
and stabilization.

37. Negative Role
• The pathogenic group of fungi in soil results in
various plant diseases by penetrating the plant
tissue and creating a weakened, nutrient-
deficient plant.
• In addition to the direct effects on the plants,
fungi also affect the interactions between
plants which change the competitive balance
between two species.
• Mycorrhiza and fungal pathogens also affect
seedlings, resulting in harmful consequences
for plant population dynamics.

38. Bacteria: (Sing. Bacterium, Plu. Bacteria)
• Bacteria, are prokaryotic, unicellular microorganisms.
• Size: 0.5 -2 um x 5-10 um.
• It can be grown on artificial media in laboratory, reproduce asexually by
simple cell division i.e. Binary fission.
• They have rigid cell wall, cells are rod, spherical (cocci), bacilli (cylindrical
rods), spiral and vibrios shape and some motile with flagella.
• They can be anaerobic, aerobic and facultative or obligate anaerobic or
microaerophilic in nature.
• On the basis of chemical composition of cell wall the bacteria are divided
in (1) Gram Positive and (2) Gram Negative.
• Importance: Some cause disease, some perform important role in
natural cycling of elements which contributes to soil fertility, useful in
industry for manufacture of valuable compounds, some spoil food and
some make foods.

39. Where they occur?
• Bacterial biomass found in soil ranges from 300 to 3000 kg/ ha.
• There are approximately 1010
bacteria in a gram of soil, most of them (>99%) have not been
or cannot be cultured in the laboratory.
• Common bacterial genera isolated from soil include Bacillus, Arthrobacter, Pseudomonas,
Agrobacterium, Alcaligenes, Clostridium, Flavobacterium, Corynebacterium, Micrococcus,
Xanthomonas, and Mycobacterium.
• In contrast to simple morphology, bacteria have the greatest metabolic diversity. In terms of
carbon utilization, there are autotrophs and heterotrophs, and in terms of energy
consumption, there are chemoorganotrophs, chemolithotrophs, and phototrophs.
• Aerobic bacteria use oxygen as an electron acceptor; anaerobic bacteria use alternate
electron acceptors such as nitrate, ferric iron, sulfate, carbonate, and organic matter.
• Since soil is an oligotrophic (nutrient-poor) environment, most bacterial cells are believed to
be dormant. T
• he one area in the soil where metabolically active microorganisms are commonly found is the
rhizosphere, where nutrients are not limiting.
• In waterlogged or heavily compacted soils, the number of aerobic bacteria is reduced,
whereas the microaerophilic and finally the anaerobic bacteria will increase.
• Unlike the other soil microorganisms, most bacteria prefer nutrient-rich soils of neutral or
slightly alkaline pH and a close C/N-ratio.

40. Positive Impact
• Bacteria are an important part of the biotic component of soil as they are
responsible for numerous physiological activities occurring in the soil.
• Soil bacterial communities provide a multitude of ecosystem services that
directly, and indirectly, affect the overall functioning of the soil environment.
• Many bacteria in the soil produce polysaccharides or glycoproteins that form a
layer on the surface of the soil particle.
• These substances thus, act as cementing agents and improve the soil
structure.
• Bacteria are a part of different biogeochemical cycles like the nitrogen cycle
and carbon cycle where they are involved in the production of a large number
of nutrients for the soil and the plants.
• The process of natural succession is also enhanced by bacteria that improve
the quality of soil so new plant communities can survive.
• Besides, bacteria are important for the enzymatic degradation of the complex
organic and Soil substances to nutrients and the release of nutrients and trace
• Enzymes elements from the mineral soil fraction. Bacterial communities in soil
act as indicators for the condition of the soil condition.

41. Negative Impact
• Pathogenic bacteria in the soil might have harmful
effects on the crops, resulting in poor crop health,
poor yields leading to crop loss.
• Some bacteria are known to disturb the ecological
balance of the soil, which results in soil infertility and
decreased soil health.
• Besides, pathogenic bacteria found in soil can cause
different forms of plant diseases.
• The release of different by-products might change
the chemical properties of soil, like the pH, cation
exchange capacity, and nutrient content.

43. Actinomycetes occurring in soil
• Actinomycete population is largest in the surface layer of soils and gradually
decreases with the depth; individual actinomycete strains are present in all soil
layers.
• They are widely distributed in the soil with estimated values ranging from 10 to 10
per gram of soil.
• They grow as hyphae like fungi, resulting in the characteristically “earth” smell of
freshly turned healthy soil.
• Streptomyces is the most abundant species of actinomycetes in soil, followed by
other species like Micromonospora, Thermoactinomycetes, and Nocardia.
• Besides, some actinomycetes are rarely isolated from desert soil, which includes
Microbispora, Microtetraspora, Amycolaptosis, Actinomadura, and Saccharothrix.
• Almost 90% of actinomycetes genera have been isolated from the soil where they
often form much-branched hyphae when growing, which then break up into spores,
either by the tip of the hyphae producing one or two spores.
• Actinomycetes in the soil are mesophilic organisms that are sensitive to acidity/low
pH (optimum pH range 6.5-8.0) and waterlogged soil conditions.
• Like other groups of microorganisms, some actinomycetes might be pathogenic,
resulting in different diseases in plants.

44. Positive role
• Actinomycetes decompose the more resistant and indecomposable organic
substances and produce several dark black to brown pigments which
contribute to the dark color of the soil humus.
• These organisms are also responsible for the subsequent decomposition of
humus (resistant material) in soil.
• Actinomycetes are important in agricultural soils because they contribute to
the carbon cycle by fixation (photosynthesis) and decomposition.
• Actinomycetes form associations with some non-leguminous plants and fix N ,
which is then available to both the host and plants in the near vicinity.
• Actinomycetes are the leading group of soil microorganisms that play a major
role in the recycling of organic matters in the environment by the production
of hydrolytic enzymes.
• They possess direct or indirect mechanisms that favor plant growth while
improving the availability of nutrients and minerals, synthesized plant growth
regulators, and suppression of phytopathogens.
• Many properties related to actinomycetes have the ability to act as biocontrol
tools.

45. Negative role
• Some products of Actinomycetes might affect the
microorganisms of the soil, resulting in decreased
diversity.
• Pathogenic species of actinomycetes result in plant
diseases that affect plant health and crop loss.

46. Protozoa
• Eukaryotic, unicellular with animal like
characteristics.
• Size ranged from 1 to 200 µm.
• Some cultivated in laboratory.
• Reproduce by asexually as well as by sexual
methods.
• Importance: Food for aquatic animals and some
cause disease in human e.g. Malaria –
Plasmodium vivax.

47. Phytoplasmas (Mycoplasmas)
• A member of a group of bacteria, those lacking cell
walls, the outer boundary of the cells being the
cytoplasmic membrane. As a result the cells have
plasticity and can assume many different shapes ranging
from sphere to branched filaments. Reproduce by
binary fission or budding. The plasticity allows many of
the cells to pass through bacteriological filters even
through the smallest cells are about 0.3 µm in diameter.
• Importance: These organisms are parasites of the
mucous membranes and joints. Mainly pathogens of
animals but some species are pathogenic for human.
The sp. Mycoplasma pheumoniae is causative agent of
primary typical pneumonia (walking pneumonia) in
human beings. They require cholesterol for growth. It
cause disease in plant sesamum phyllody.

48. Rickettsias
• Obligately parasitic bacteria of arthropods, many are
pathogenic for human and other animals.
• The bacteria small rod shaped or cocoid, which are often
pleomorphic.
• It contain nucleic acid RNA and DNA. Multiplication by binary
fission, inhibited by antibacterial drugs.
• They transmitted to human by an arthropod vector like lice,
fleas, ticks and mites.
• The organism multiplies within the cytoplasm and some times
within the nucleus of host cell.
• In laboratory, they are cultured on host animals, pigs or mice.
• Diseases : Typhus-fever (lice); Murin typhus fever (fleas).

49. Viruses
• Non-cellular infectious, an obligate
intracellular parasitic microorganisms, that is
smaller than bacteria. They are so small that
they can be visualized only by electron
microscope. Size of the virus particles are
ranged from 0.0015 to 0.2 µm. Viruses can be
cultivated only in living cells i.e. they are
obligate parasite and whose genomes are a
nucleic acid either DNA or RNA, which is
typically surrounded by protein coat. The
virus particles pass through the pores of
filters, which do not permit the passage of
most bacteria.

50. • Reproduction (Production): Virus particles out
side a host cell have no independent metabolic
activity and are incapable of reproduction like
other organisms.
• Multiplication takes place by replication in which
the viral protein and nucleic acid components
are reproduced within susceptible host cells.
• Importance: They can cause serious disease in
animals, plants and fungi (mycovirus), TMV-
Tobacco Mosaic Virus.

51. Bacteriophage
• “It is virus that infects bacteria and causes the lysis of
bacterial cells”.
• Bacterial viruses (phages) are widely distributed in
nature.
• Phages exist for most but not all bacteria.
• With the proper techniques these phages can be
isolated quite easily in the laboratory.
• Bacteriophages, like all viruses are composed of nucleic
acid surrounded by a protein coat.
• Bacterial viruses occur in different shapes, although
many have a tail through which they inoculate the host
cell with viral nucleic acid.

52. Where they occur?
• Viruses are the most abundant biological entities on our planet and
exceed the number of cellular organisms in marine and soil habitats.
• The concentration of viruses in soil has been estimated to be 10 virus
particles per gram dry weight.
• The majority of the soil viruses are tailed bacteriophages that prefer
wetland forest soil over drier agricultural soils.
• Some of the common viruses inhabiting soil include small spherical virus
particles similar in size to single-stranded (ss) RNA containing
bacteriophages of the Leviviridae family or to some plant viruses, and
larger spherical viruses similar to the double-stranded (ds) DNA containing
viruses of the Partitiviridae, Chrysoviridae, and Totiviridae families.
• Diverse viruses of the Myoviridae, Siphoviridae, and Podoviridae families
with genomes ranging in size from 45 to 270 kb are found in the Sahara
desert surface sands.
• Soils act as reservoirs of viruses, but these are probably not entirely static
reservoirs as at least some viruses seem to move readily between
environments.

53. Positive impact
• The main way in which viruses in soils act beneficially is by
transferring genes between microbial hosts by horizontal gene
transfer.
• The gene transfer enables the transfer of beneficial
characteristics between different communities.
• Another way that viruses in soils have potential benefits for
plants is by infecting organisms that are pathogenic for plants.
• Viruses of different microbes in the soil as pathogens have an
essential role in regulating the population structure of their
microbial hosts.
• Virus populations may also act as reservoirs of genes involved
in all the biochemical functioning of their microbial hosts, and
by recombination among themselves during co-infections,
could be a source of new gene variants.

54. Negative impact
• Among the virus community in soil, distinct
proportions are plant pathogens that reach the plant
via mechanical means, nematode vectors, or fungal
vectors.
• Viruses also affect other microbial communities of
bacteria, fungi, and protozoa that cause an
imbalance in the biotic component of the soil.
• Viruses might even affect the physical and chemical
properties of soil by affecting the biotic and abiotic
components of the soil.

55. Spiroplasmas:
• These organisms are helical and exhibit a
swimming motility.
• The spiroplasmas are pathogenic for
citrus and other plants. They can be
isolated from plant fluids and plant
surfaces and from arthropods that feed
on plants.

56. • There are two main types of bacterial viruses-
lytic (virulent) and temperate (nonvirulent or
lysogenic).
• When lytic phages infect cells, the cells respond
by producing large number of new viruses. This is
at the end of the incubation period; the host
(bacterial) cell bursts, releasing new phages to
infect other host cells. This is called as lytic cycle.
• In the temperate type of infection the results is
not so readily apparent. These organisms are
beneficial because they control the harmful
bacteria.

57. Viroids
• Viroids are group of virus but are smaller than virus.
• Viroids are nucleic acid entities of naked RNA only.
• They do not possess a protein coat like viruses. In spite
of their small size viroids replicate autonomously in cells
of susceptible host plants. They are resistant to heat,
U.V. radiation and ionizing radiation.
• Importance: Viroids cause several important diseases of
cultivated plants i.e. Potato Spindal tuber, Citrus
exocortis and Coconut cadang-cadang.

59. Important discoveries of Agril.
Microbiology
S. A. Waksman published the book “Principles of soil Microbiology"
and discovered the antibiotic "Streptomycin" produced
by Streptomyces griseus, a soil actinomycets (1944).
Van Niel- studied chemoautotrophic bacteria and bacterial
photosynthesis.
Bortels demonstrated the importance of molybdenum in accelerating
nitrogen fixation by nodulating legumes.
Kubo proved-the role and importance of “leghaemoglobin” (Red
pigment) present in root nodules of legumes in nitrogen fixation.

60. Ruinen (1956) coined the term "Phyllosphere" to denote the region of leaf
influenced by microorganisms.
Jensen (1942) developed the method of studying nodulation on agar
media in test tubes.
Barbara Mosse and J. W. Gerdemann (1944) reported occurrence of VAM
(vesicular-arbuscular Mycorrhiza) fungi (Glomus, Aculopora genera) in
the roots of agricultural crop plants which helps in the mobilization
of phosphate.
Alexander Fleming developed the antibiotic "Penicillin" from the
fungus Penicillium notatum (1929).
Hardy & Associates developed the technique of measurement of
nitrogenase activity by “acetylene-reduction test” coupled with gas
chromatography and thereby estimation of biological nitrogen
fixation.

61. Dobereiner and associates (1975, Brazil) studied nitrogen fixing
potential of Azospirillum in some tropical forage grasses like Digitaria,
Panicum and some cereals like maize, sorghum, wheat, rye etc. in their
roots.
He coined the term “Associative Symbiosis” to denote the
association between nitrogen fixing Azospirillum and cereal roots.
Recently this terminology has been changed and renamed as
“Diazotrophic Biocoenocis”.
Dommergues & associates had discovered / reported nodules on
stem of Sesbania rostrata which could fix nitrogen and can be used as
an excellent green manure crop in low land rice cultivation.
They also discovered N2 fixing stem nodules on Casuarina
sp. caused by Frankia, an actinomycete.
Brefeld Introduced the practice of isolating soil fungi by "Single
Cell" technique and cultivating / growing them on solid media. He used
gelatin (first solidifying agent) in culture media as solidifying agent.