Farokh Rokhbakhsh-Zamin Department of Microbiology &

Institute of Bioinformatics and Biotechnology

University of Pune

Agricultural Biotechnology (Biofertilizers)

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: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:

AcetobacterAcidovoraxAchromobacter

AcinetobacterActinomycesAgrobacterium

AlcaligenesArthrobacterAzoarcus

AzorhizobiumAzospirillumBacillus

BordetellaChryseobacterium

Clavibacter

ComamonasCorynebacteriumCurtobacterium

DeleyaEnterobacterEscherichia

ErwiniaFlavobacteriumHerbaspirillum

KingellaKlebsiellaLactobacillus

LeuconostocMethylobacteriumMicrococcus

MoraxellaPantoea Pasteurella

PhotobacteriumPhyllobacteriumProvidencia

PseudomonasPsychrobacterRahnella

RhizobiumRhodococcusSerratia

ShewanellaSphingomonasStaphylococcus

VibrioXanthomonasYersinia

* 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 Well known PGPRs:PGPRs:

• Arthrobacter Arthrobacter

• AcetobacterAcetobacter

• Azotobacter Azotobacter

• AzosperillumAzosperillum

• BacillusBacillus

• EnterobacteriaEnterobacteria

• KlebsiellaKlebsiella

• ProteusProteus

•PseudomonasPseudomonas

•RhizobiumRhizobium

Pseudomonas spp.Pseudomonas spp. & related & related genera:genera:

• Although a range of different bacterial genera and Although a range of different bacterial genera and species have been studied, the overwhelming species have been studied, the overwhelming

number of papers have involved the use of number of papers have involved the use of PseudomonasPseudomonas species. It’s so because species. It’s so because PseudomonasPseudomonas and related genera are characteristically:and related genera are characteristically:

• Fast growingFast growing

• Easy to cultureEasy to culture

• Manipulate genetically in the laboratoryManipulate genetically in the laboratory

• Able to utilize a range of organic compoundsAble to utilize a range of organic compounds

• Produce many different metabolites which Produce many different metabolites which some are Plant Growth Promoting Substances 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.

Direct Direct Plant Growth Plant Growth PromotionPromotion

1.Microbial Production of Plant Hormones: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:1.1.Auxins:

Examples of IAA Examples of IAA production: production:

• Some strains of Some strains of AcinetobacterAcinetobacter isolated and characterized isolated and characterized from rhizosphere of wheat were showed indole-3-acetic acid from rhizosphere of wheat were showed indole-3-acetic acid (IAA) production. Pot experiments showed significant (IAA) production. Pot experiments showed significant increase in plant growth inoculated with eight increase in plant growth inoculated with eight AcinetobacterAcinetobacter genospecies as compared to control plants. IAA production genospecies as compared to control plants. IAA production was found to be encoded by plasmid PUP1126 and this is the was found to be encoded by plasmid PUP1126 and this is the first report of plasmid-encoded IAA production in the genus first report of plasmid-encoded IAA production in the genus Acinetobacter.Acinetobacter.

• The rhizobacterium The rhizobacterium Pseudomonas putidaPseudomonas putida GR 12-2 is a strong GR 12-2 is a strong candidate for development as a soil inoculant to enhance crop candidate for development as a soil inoculant to enhance crop yields. Inoculation of canola, tomato and other agriculturally yields. Inoculation of canola, tomato and other agriculturally important plants with this strain results in substantial important plants with this strain results in substantial promotion of seedling root growth. Characteristics that may promotion of seedling root growth. Characteristics that may contribute to the ability of contribute to the ability of P.PutidaP.Putida to enhance plant growth to enhance plant growth include the capacity to synthesize siderophores and thereby include the capacity to synthesize siderophores and thereby provide iron for the plant, the capacity to lower growth provide iron for the plant, the capacity to lower growth inhibiting levels of ethylene in plant tissues by production of inhibiting levels of ethylene in plant tissues by production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase , and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase , and capacity to secrete IAAcapacity to secrete IAA . .

1.2.Gibberellic 1.2.Gibberellic acids:acids: Azospirillum Azospirillum spp. are considered to be spp. are considered to be

important plant growth promoting important plant growth promoting rhizobacteria that can improve the growth rhizobacteria that can improve the growth and yield of at least several plant species and yield of at least several plant species (Labandera-Gonza´lez, 1994).(Labandera-Gonza´lez, 1994).

Phytohormone production, including Phytohormone production, including gibberellins (Bottini et al., 1989), is one gibberellins (Bottini et al., 1989), is one mechanism that has been proposed mechanism that has been proposed ( Cassan et al., 2001).( Cassan et al., 2001).

Other Gibberellin producing bacteria in Other Gibberellin producing bacteria in rhizosphere are as follows:rhizosphere are as follows:

1.1. Acetobacter diazotropicusAcetobacter diazotropicus

2.2. Azosperillum lipoferumAzosperillum lipoferum

3.3. Herbosperillum seropedicaeHerbosperillum seropedicae

4.4. Rhizobium phaseoliRhizobium 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:1.4. Cytokinins: the presence of micro-organisms capable the presence of micro-organisms capable

of producing cytokinins, can be expected of producing cytokinins, can be expected to raise the amounts of cytokinins in both the soil to raise the amounts of cytokinins in both the soil solution and in plants growing there. In turn, this solution and in plants growing there. In turn, this may have an impact on the growth of these plants. may have an impact on the growth of these plants. In support of this there are numerous reports that In support of this there are numerous reports that certain micro-organisms affect plant growth through certain micro-organisms affect plant growth through their ability to produce phytohormones (Arshad and their ability to produce phytohormones (Arshad and frankenberger, 1991, 1998; Steenhoudt and frankenberger, 1991, 1998; Steenhoudt and Vanderleyden, 2000). Vanderleyden, 2000).

2.Phosphate solublization:2.Phosphate solublization: The ability of microorganisms to solubilize and The ability of microorganisms to solubilize and

mineralize p in soils is vital. Phosphate availability in mineralize p in soils is vital. Phosphate availability in soil is greatly enhanced through microbial production soil is greatly enhanced through microbial production of metabolites leading to lowering of PH and release of metabolites leading to lowering of PH and release of phosphate from organic and inorganic of phosphate from organic and inorganic complexes.The species of complexes.The species of Pseudomonas,Micrococcus, Pseudomonas,Micrococcus, Bacillus, Aerobacter, Xanthomonas, brevibacterium, Bacillus, Aerobacter, Xanthomonas, brevibacterium, Alcaligenes, RhizobiumAlcaligenes, Rhizobium have been reported to be have been reported to be active in phosphate solubilization (Srivastav, 2004). active in phosphate solubilization (Srivastav, 2004).

Although these PGPRs occur in soil, usually their Although these PGPRs occur in soil, usually their numbers are not high enough to compete with other numbers are not high enough to compete with other bacteria commonly established in the rhizosphere. So, bacteria commonly established in the rhizosphere. So, for agronomic utility, inoculation of plants by target for agronomic utility, inoculation of plants by target with such microorganisms at higher concentration with such microorganisms at higher concentration than those normally found in soil is necessary to take than those normally found in soil is necessary to take advantage of their beneficial properties for plant yield advantage of their beneficial properties for plant yield enhancementenhancement

for example in this subject, the solubilization of for example in this subject, the solubilization of phosphatic compounds, one of the important phosphatic compounds, one of the important mechanisms of plant growth promotion shown by mechanisms of plant growth promotion shown by PGPR PGPR AcinetobacterAcinetobacter, increases its potential in the , increases its potential in the development of future bioinoculum for crop plants. development of future bioinoculum for crop plants. In this investigation the phosphate solubilization In this investigation the phosphate solubilization by by AcinetobacterAcinetobacter spp. was also compared with spp. was also compared with other rhizosphere isolates like other rhizosphere isolates like Moraxella Moraxella sp., sp., Pesudomonas Pesudomonas sp., sp., Serratia Serratia sp., and sp., and Pseudomonas Pseudomonas putidaputida NCIM1313, NCIM1313, Escherichia coli Escherichia coli NCIM2810. NCIM2810.

All the phosphate solubilizing All the phosphate solubilizing AcinetobacterAcinetobacter strains strains had zone diameter of dissolution in the range 1-had zone diameter of dissolution in the range 1-5cm while as control 5cm while as control P. putida P. putida had average zone had average zone diameter in the range 1-3.5cm. Solubilization of diameter in the range 1-3.5cm. Solubilization of insoluble phosphates started along with the insoluble phosphates started along with the growth of strains and maximum solubilization was growth of strains and maximum solubilization was achieved at logarithmic to late stationary phase. achieved at logarithmic to late stationary phase. Some cultures showed reprecipitation of Some cultures showed reprecipitation of solubilized phosphate after prolonged incubation solubilized phosphate after prolonged incubation (Chopade, 2003).(Chopade, 2003).

3. N3. N22 Fixation: Fixation:

N2 Fixation

Asymbiotic Symbiotic

Azotobacter RhizobiumAzotobacter Rhizobium

Azosperillum BradyrhizobiumAzosperillum Bradyrhizobium

Bacillus CyanobacteriaBacillus Cyanobacteria

Klebsiella AnabaenaKlebsiella Anabaena

ClostridiumClostridium

P.vulgarisP.vulgaris

Azotobacter RhizobiumAzotobacter Rhizobium

Azosperillum BradyrhizobiumAzosperillum Bradyrhizobium

Bacillus CyanobacteriaBacillus Cyanobacteria

Klebsiella AnabaenaKlebsiella Anabaena

ClostridiumClostridium

P.vulgarisP.vulgaris

In Direct In Direct Plant Growth PromotionPlant Growth Promotion

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.