Farokh Rokhbakhsh-Zamin

Department of Microbiology &

Institute of Bioinformatics and Biotechnology
University of Pune
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
(FAO, 2000).

FERTILIZERS:
1. Soils may be naturally low in nutrients
2. Deficient due to nutrient removal by crops
3. When high yielding varieties are grown
(In order to obtain high yields, Fertilizers are needed).

FERTILIZERS
Chemical Fertilizers
(Conventional Farming)
Biological Fertilizers
(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
20,000 unintentional Deaths each year in D.C.

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:
1. Living fertilizers compounds of Microbial
inoculants:
eg,Plant growth promoting substances like
Hormones and Auxins.
2. Group of microorganisms which are able to fix
atmospheric nitrogen or solubilize Phosphorus,
decompose organic material or oxidize sulfur in
soil.


Importance of Biofertilizers:
1.Eco friendly.
2.In addition to N2 ,Provide certain PGP
substances like hormones , vitamins, .
3.Supplying N2 , continuously throughout the
entire period of crop growth in the field
under favorable conditions.
4.Without toxic effects.
5.When applied to soil improve the soil
structure.
6. Low production cost.


Plant Growth
Rhizobacteria
1. Endophytic
Bacteria
2. Exophytic
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:

Achromobacter
Acidovorax Acetobacter
Agrobacterium Actinomyces Acinetobacter
Azoarcus Arthrobacter Alcaligenes
Bacillus Azospirillum Azorhizobium
Clavibacter Chryseobacter
ium
Bordetella
Curtobacterium Corynebacterium
Comamonas
Escherichia Enterobacter Deleya
Herbaspirillum
Flavobacterium
Erwinia
Lactobacillus Klebsiella Kingella
Micrococcus
Methylobacterium
Leuconostoc
Pasteurella Pantoea Moraxella
Providencia
Phyllobacterium
Photobacterium
Rahnella Psychrobacter Pseudomonas
Serratia Rhodococcus Rhizobium
Staphylococcus Sphingomonas
Shewanella
Yersinia Xanthomonas Vibrio
* Production of Plant hormones*Antibiosis
* Phosphorous solubilization *Induced resistance
* Enhanced iron availability *Iron scavenging
* Nitrogen Fixation *Competition for nutrients/niche
* Etcetera * Parasitism & Predation
*Etcetera
PGPR affect plant growth
Directly Indirectly
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. Its 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
Produce many different metabolites which
some are Plant Growth Promoting Substances
Other rhizobacteria
Since other rhizobacteria are also
found in the rhizosphere of
many crop plants Like wheat and there were
little detailed studies on them from plant
rhizosphere:
It becomes interesting to find out the
probable role of others in rhizosphere of
wheat & other crop plants.

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
microbes 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 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 ethylene in plant
tissues by production of 1-aminocyclopropane-1-carboxylic acid
(ACC) deaminase , and capacity to secrete IAA .
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-Gonzalez, 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:
1. Acetobacter diazotropicus
2. Azosperillum lipoferum
3. Herbosperillum seropedicae
4. Rhizobium phaseoli
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 soil is necessary to take advantage of
their beneficial properties for plant yield enhancement
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 prolonged incubation
(Chopade, 2003).
2
N2 Fixation
Asymbiotic Symbiotic
Azotobacter Rhizobium
Azosperillum Bradyrhizobium
Bacillus Cyanobacteria
Klebsiella Anabaena
Clostridium
P.vulgaris
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.
1. 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.