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University of Pune

Farokh Rokhbakhsh-Zamin
& Department of Microbiology
Institute of Bioinformatics and Biotechnology
Agricultural Biotechnology
))Biofertilizers
:Soil health
Biological, Chemical and
Physical
features to long term,
sustainable
agricultural productivity with
minimal environmental impact
.)Arias, 2005(
:Soil Fertility
Are: soil organic matter )including
microbial Biomass(, Soil texture, soil
structure, soil depth, content of
nutrients, storage capacity
adsorption capacity(, soil reactions(
& absence of toxic elements
FERTILIZERS:
2. Soils may be naturally low in nutrients
3. Deficient due to nutrient removal by crops
4. When high yielding varieties are grown
(In order to obtain high yields, Fertilizers are needed).

FERTILIZERS

Chemical Fertilizers Biological Fertilizers


)Conventional Farming( )Organic Farming(
The threat of chemical Fertilizers:
1. Threaten Human Health.
2. Threaten Agricultural soils,
Food safety and Waterways.
• Soil quality
• Plant uptake
• Water quality
The threat of chemical pesticides:
*Health & Environmental Problems.
WHO-3million acute sever cases of poisoning
Although, during last 50 years, farmers
have dramatically increased crop yields
through the use of chemical fertilizers &
pesticides, and improved varieties, today,
the rising costs of chemical inputs and a
host environmental concerns have
caused farmers to consider alternative
agri-industrial managements (e.g.Organic
Farming) to reduce costs, protect human
health, and conserve the resource base.
(Kritcher, 1993)
Organic Farming by
Bioorganic fertilizers:

• If continuous exploitation of land for


cultivation has caused a progressive
decline in soil health, it can be restored and
maintained to a greater extend by the use of organic manures.

• Further improvement in this regard was observed by


incorporating microorganisms in organic manures to develop
Bioorganic Fertilizers (Chakradhar, 2004).

• So, Utilization of microbial inoculants specially PGPR for


sustainable agri-industrial applications has been
subjected of a number of recent reviews to manipulate
rhizosphere conditions by innovative techniques for a
better plant growth and plant health (Bloemberg, 2001).
Biofertilizers:
• Fertilizers:
Any of large number of natural & synthetic material
like manure, N, P, K compounds, spread on or
worked into soil to increase its fertility.
• Biofertilizers:
6. Living fertilizers compounds of Microbial
inoculants:
eg,Plant growth promoting substances like
Hormones and Auxins.
7. Group of microorganisms which are able to fix
atmospheric nitrogen or solubilize Phosphorus,
decompose organic material or oxidize sulfur in
soil.
• Importance of Biofertilizers:
• Eco friendly.
• In addition to N2 ,Provide certain PGP
substances like hormones , vitamins,… .
• Supplying N2 , continuously throughout the
entire period of crop growth in the field
under favorable conditions.
• Without toxic effects.
• When applied to soil improve the soil
structure.
• Low production cost.
Plant Growth
Rhizobacteria

Endophytic .1 Exophytic .2
Bacteria Bacteria
Bacterial Endophytes:
Why Are They There?
• Opportunists?
–Some have no apparent effect
on plant performance
• Mutualists?
–Evidence is accumulating to
support this possibility
Bacterial Endophytes: Another
Mutualistic Symbiosis
• Benefit to microorganism:
– Provides an environment buffered from external
stresses
– Steady source of nutrients and water
• Benefit to the plant host:
– Nitrogen fixation
– Biological control of plant pathogens and pests
– Enhanced uptake of nutrients and water
Bacterial Genera With
Endophytes:
Acetobacter Acidovorax Achromobacter

Acinetobacter Actinomyces Agrobacterium

Alcaligenes Arthrobacter Azoarcus

Azorhizobium Azospirillum Bacillus

Bordetella Chryseobacter Clavibacter


ium
Comamonas Corynebacterium Curtobacterium
Deleya Enterobacter Escherichia
Erwinia Flavobacterium Herbaspirillum
Kingella Klebsiella Lactobacillus
Leuconostoc Methylobacterium Micrococcus
Moraxella Pantoea Pasteurella
Photobacterium Phyllobacterium Providencia
Pseudomonas Psychrobacter Rahnella
Rhizobium Rhodococcus Serratia
Shewanella Sphingomonas Staphylococcus

Vibrio Xanthomonas Yersinia


PGPR affect plant growth

Directly Indirectly
* Production of Plant hormones*Antibiosis
* Phosphorous solubilization *Induced resistance
* Enhanced iron availability *Iron scavenging
* Nitrogen Fixation *Competition for nutrients/niche
* Etcetera * Parasitism & Predation
*Etcetera
Well known
PGPRs:
• Arthrobacter
• Acetobacter
• Azotobacter
• Azosperillum
• Bacillus
• Enterobacteria
• Klebsiella
• Proteus
• Pseudomonas
• Rhizobium
Pseudomonas spp. & related
genera:
• Although a range of different bacterial genera
and species have been studied, the
overwhelming
number of papers have involved the use of
Pseudomonas species. It’s so because
Pseudomonas and related genera are
characteristically:
• Fast growing
• Easy to culture
• Manipulate genetically in the laboratory
• Able to utilize a range of organic
compounds
Other
rh izobacteria
• Sin ce o th er rhizobacte ria a re a lso
found i n t he rh iz osphere o f
many c rop p la nts L ik e w heat and th ere
were l it tle d etail ed stu dies o n t hem
fr om p lant r hizosphere:
• It becomes in teresting t o f in d o ut th e
probable role of oth er s i n r hizosphere
of wheat & o ther c rop p la nts.
Di r ect
Pla nt Gr owt h
Pr omotio n
1.Microbial Production of Plant Hormones:
• Plants themselves synthesize Auxin, Gibberellins, Cytokinins,
Ethylene, and Abscisic acid, but under less than ideal climatic
and environmental conditions, Plants may not synthesize
sufficient endogenous concentrations to sustain optimal
growth and development.

• Scientists have shown recently That Plant Growth can be


improved when specific microbial strains are used to
• ‘ inoculate’ seeds or roots of agricultural crops due to
microbe’s production of Plant Growth Hormones (Regulators).

• Exogenous Supplementation of PGPHs to plant roots is


reletively new approach to maximize crop yield.
Signals from under ground:
Bacterial volatiles
promote plant growth
1.1.Auxins:
Examples of IAA
production:
• Some strains of Acinetobacter isolated and characterized
Some strains of Acinetobacter isolated and characterized
from rhizosphere of wheat were showed indole-3-acetic
acid (IAA) production. Pot experiments showed significant
increase in plant growth inoculated with eight
Acinetobacter genospecies as compared to control plants.
IAA production was found to be encoded by plasmid
PUP1126 and this is the first report of plasmid-encoded IAA
production in the genus Acinetobacter.

• The rhizobacterium Pseudomonas putida GR 12-2 is a


strong candidate for development as a soil inoculant to
enhance crop yields. Inoculation of canola, tomato and
other agriculturally important plants with this strain
results in substantial promotion of seedling root growth.
Characteristics that may contribute to the ability of
P.Putida to enhance plant growth include the capacity to
synthesize siderophores and thereby provide iron for the
plant, the capacity to lower growth inhibiting levels of
1.2.Gibberellic
 acids:
Azospirillum spp. are considered to be
important plant growth promoting
rhizobacteria that can improve the
growth and yield of at least several
plant species (Labandera-Gonza´lez,
1994).
 Phytohormone production, including
gibberellins (Bottini et al., 1989), is
one mechanism that has been
proposed ( Cassan et al., 2001).
 Other Gibberellin producing bacteria
in rhizosphere are as follows:
 Acetobacter
diazotropicus
1.3.Ethylene:
The effects of C2H4 have been observed in
practically all aspects of plant growth and
development, including seed germination
(Ketring et al., 1972), seedling growth (Burg et
al., 1968), root growth (Chadwick et al., 1970),
growth of leaves (Primrose, 1979), and ripening,
aging (Biale, 1960).
Agronomically, microbial production of C2H4
could have an impact on crop production under
certain management conditions. Ethylene
concentrations as low as 10 ,ug liter-' can evoke
plant responses, and concentrations of 25 pug
liter-' result in decreased fruit and flower
development (Primrose et al., 1979).
1.4. Cytokinins:
 the presence of micro-organisms capable
of producing cytokinins, can be expected
to raise the amounts of cytokinins in both the soil
solution and in plants growing there. In turn, this
may have an impact on the growth of these plants.
In support of this there are numerous reports that
certain micro-organisms affect plant growth through
their ability to produce phytohormones )Arshad and
frankenberger, 1991, 1998; Steenhoudt and
Vanderleyden, 2000(.
2.Phosphate solublization:
The ability of microorganisms to solubilize and
mineralize p in soils is vital. Phosphate availability
in soil is greatly enhanced through microbial
production of metabolites leading to lowering of
PH and release of phosphate from organic and
inorganic complexes.The species of
Pseudomonas,Micrococcus, Bacillus, Aerobacter,
Xanthomonas, brevibacterium, Alcaligenes,
Rhizobium have been reported to be active in
phosphate solubilization (Srivastav, 2004).
Although these PGPRs occur in soil, usually their
numbers are not high enough to compete with
other bacteria commonly established in the
rhizosphere. So, for agronomic utility, inoculation
of plants by target with such microorganisms at
higher concentration than those normally found in
 for example in this subject, the solubilization of
phosphatic compounds, one of the important
mechanisms of plant growth promotion shown by
PGPR Acinetobacter, increases its potential in the
development of future bioinoculum for crop
plants. In this investigation the phosphate
solubilization by Acinetobacter spp. was also
compared with other rhizosphere isolates like
Moraxella sp., Pesudomonas sp., Serratia sp., and
Pseudomonas putida NCIM1313, Escherichia coli
NCIM2810.
 All the phosphate solubilizing Acinetobacter
strains had zone diameter of dissolution in the
range 1-5cm while as control P. putida had
average zone diameter in the range 1-3.5cm.
Solubilization of insoluble phosphates started
along with the growth of strains and maximum
solubilization was achieved at logarithmic to late
stationary phase. Some cultures showed
reprecipitation of solubilized phosphate after
3. N 2
Fix ation:
N2 Fixation

Asymbiotic Symbiotic
Azotobacter Rhizobium
Azosperillum Bradyrhizobium
Bacillus Cyanobacteria
Klebsiella Anabaena
Clostridium
P.vulgaris
In Di r ect
Pla nt Gr owt h
Pr omotion
Biocontrol PGPR
• According to the United States
Department of Agriculture, biological
control of plant disease is defined as "
the involvement of the use of
beneficial microorganisms, such as
specialized fungi and bacteria, to
attack and control plant pathogens and
the diseases they cause.
• These "specialized" fungi and bacteria
are microorganisms that normally
inhabit most soils.
• Direct Competition with the Target Organism.

In this case the biocontrol agent out competes the target


organisms for nutrients and space.

Example:
• Iron competition in Pseudomonads has been intensively studied and
the role of the pyoverdine siderophore has been intensively studied
and the role of the pyoverdine siderophore produced by many
pseudomonas species has been clearly demonstrated in control of
Pythium and fusarium species.
2. Antibiosis:
The biocontrol agent produces an chemical compound such
as an antibiotic or some type of toxin that kills or has some
sort of detrimental effect on the target organism.

Example:
phenyazine-1-carboxylic acid (PCA) from Pseudomonas
aureofaciens kuyver tx-1 has even been used as a direct field
treatment of the control of dollar spot on creeping bent grass
(Powell et al., 2000).
3. Induced Resistance of the Host Plant.

It has been know for decades that once a plant is infected


with a pathogen, that infection triggers some sort of reaction
in the infected host plant that helps keep it from being
infected with other pathogens. The infected plant becomes
more "resistant" to other infections.
Changing that have been observed in plant roots exhibiting
Induced systemic resistance (ISR) include:

1. strengthening of epidermal and cortical cell walls and


deposition of newly formed barriers beyond infection
sites including callose, lignin and phenolics.
2. increased levels of enzymes such as chitinase,
peroxidase, polyphenol oxidase, and phenylalanine
ammonia lyase.
3. enhanced phytoalexin production.
4. enhanced expression of stress- related genes.

However, not all of these biochemical changes found in


all bacterial-plant combinations.

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