microbial resource

8/10/2019 Microbial Resource

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Microbial resource


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Soil health, also referred to as soil

quality, is defined as the continuedcapacity of soil to function as a vitalliving ecosystem that sustains plants,animals, and humans.


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Anthropogenic factors

Unsustainable Agricultural Practice

Inappropriate Technology and Advice,


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CRDT-


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PLANT GROWTH

PROMOTINGRHIZOBACTERIA


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PGPR

Soil is a complex living food web, where a variety of microorganisms such asbacteria, actinomycetes, fungi, protozoa, and algae reside and are involved inkey environmental processes such as degradation of organic matter andbiogeochemical cycling of nutrients through which they participate inmaintaining health and productivity of soil.

Rhizosphere is defined by Hiltner (1904) as volume of soil surrounding theroot, influenced chemically, physically, and biologically by the presence ofliving plant roots.

Rhizosphere is highly favorable habitat for the proliferation ofmicroorganisms and exerts a potential impact on plant health and soil fertility(Sorensen 1997). Root exudates are complex mixtures of carbon containingcompounds (Carvalhais et al. 2011), which serve as primary source ofnutrients, and support the dynamic growth and activities of variousmicroorganisms within the vicinity of the roots.


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PGPR

An important group of bacterial communities that exert beneficial

effects on plant growth upon root colonization were first defined by

Joseph Kloepper and Milton Schroth and termed as plant growth-

promoting rhizobacteria (PGPR) (Kloepper and Scroth 1978).

These free-living and root-colonizing bacteria, when applied to seeds or

roots, enhance the growth of the plant, reduce the damage from

phytopathogens, and impart resistance against abiotic stress.

Basically, PGPR are defined by three intrinsic characteristics

(1) they must be able to colonize the root,

(2) they must survive and multiply in microhabitats associated with theroot surface, in competition with other microbiota, at least for the time

needed to express their plant promotion/protection activities,

(3) they must promote plant growth.


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PGPR

Strains of the genera such as Aeromonas,Agrobacterium, Alcaligens, Azoarcus, Azospirillum,Azotobacter, Arthrobacter, Bacillus, Cellulomonas,

Clostridium, Enterobacter, Erwinia, Flavobacterium,Gluconacetobacter, Klebsiella, Microbacterium,Micromonospora, Panibacillus, Pseudomonas, Rhizobia,

Serratia, Streptomyces, and Xanthomonas have beenidentified as PGPR, while the search foradditional strains is still continued


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Different tomato growing districts of

Karnataka surveyed in the present study

Mysore

Mandya

Hassan

Chikmagalur

Ramnagara

Banagalore urban

Bangalore ruralChikballapur

Kolar

186 rhizospheric soil samples

were collected


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Plant growth promoting rhizobacterial cultures

a. Bacillus subtilis,

b. Bacillussp.,

c. Azotobactersp.,

d. Arthrobactersp.,

e. Serratia marcescens,

f. Pseudomonas fluorescens,

g. Enterobacter sp.,

h. Unknown rhizobacteria,i. Rhizobacterial cultures on LB agar slants.

0

100

200

300

400

500

600

700

800

a b c d e

Root colonizing property of isolated

rhizobacteria and their effect on plant growtha: Total number of rhizosphere soil samples collected,

b: Total number of rhizobacteria isolated, c: Root

colonizing rhizobacteria, d: Total number of

rhizobacteria which promote or did not affect the plant

growth, e: Plant growth inhibiting rhizobacteria.

Totally 752 rhizobacterial isolates were isolated

659 isolates were found colonizing tomato roots

568 isolates were found not inhibiting or promoting the plant growth

Remaining 91 isolates were inhibited the normal tomato growth


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Serratia marcescens

Pseudomonas aeruginosa

Cell wall fatty acid mehtyl ester

(FAME) analysis of selectedrhizobacteria 16s

ribosomalRN

A

geneamplification

and

sequencewa

sdone

forselected

rhiz

obacterialisolt

es

Biochemical characterization


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Rhizobacteria

1.99

7.313

5.452

2.925

2.26

8.51

1.728

4.12

3.19

23

20.07

5.313.19

3.85

5.31

1.72

Bacil lus spp.

Pseudom onas spp.

Serratia spp .

Azotobacter spp.

Azospir i l lum spp.

Arthor bacter spp.

Enterobacter spp .

Acetobacter sp p.Flavobacter spp .

A lcalygens spp.

Agr obacter ium spp.

Rhizobium spp.

Proteus spp.

Ralstonia spp .

Burkho lderia spp.

Unknwon

Percentage of occurrence of rhizobacterial genus in tomato rhizosphere across

tomato growing regions of Karnataka


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Bacillusspp.

Pse

udomonasspp.

Serratiaspp.

Azo

tobacterspp.

Azospirillums

pp.

Arthorb

acterspp.

Enteroba

cterspp.

Acetobacterspp.

Flavobacte

rspp.

Alcalygens

spp.

Agrobacteriums

pp.

Rhizobiums

pp.

Proteusspp.

Ralstoniaspp.

Burkholderiaspp.

Unknwon

Mys

Man

Has

Chkm

Bru

Bur

Ram

ChkbKol0

5

10

15

20

25

30

35

40

No.ofrhizobacteriaisolate

d

Rhizobacteria

Pla

ceofC

oll

ectio

n

Mys

Man

Has

ChkmBru

Bur

Ram

Chkb

Kol

Rhizobacterial diversity across tomato growing regions of Karnataka.

Mys: Mysore, Man: Mandya, Has: Hassan, Chi: Chikkamagalore, Bur: Bangalore urban, Bru:Bangalore rural, Ram: Ramnagara, Chk: Chikkabalapura, Kol: Kolar.


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Rhizobacteria Mys Man Has ChkM Bru Bur Ram ChkB Kol

No. of

isolates

recovered

Frequency

of dominant

RB

Bacillusspp.

100.0

100.0

91.30

53.84

78.57

70.0

93.30

100.0

96.87

173

23.76

Pseudomonasspp. 95.0 100.0 60.86 76.92 42.85 90.0 33.33 95.0 68.75 151 20.35

Serratiaspp. 48.0 41.17 13.04 23.07 - - - 25.0 9.37 40 4.71

Azotobacterspp. 28.0 11.76 21.73 15.38 - 20.0 6.66 5.0 6.25 24 3.38

Azospirillum spp. 24.0 5.88 21.73 23.07 7.14 10.0 - 15.0 25.0 29 3.89

Arthrobacterspp. 4.0 11.76 8.69 - 7.14 10.0 6.66 10.0 9.37 15 1.99

Enterobacterspp.

48.0

26.47

30.43

23.07

-

-

-

25.0

12.50

40

4.88

Acetobacterspp. 8.0 5.88 4.34 - 7.14 20.0 - 15.0 6.25 13 1.96

Flavobacterspp. 32.0 14.70 - - 7.14 20.0 - 15.0 15.62 24 3.08

Alcalygensspp. 20.0 14.70 13.04 15.38 - 10.0 20.0 25.0 21.82 31 4.13

Agrobacteriumspp. 8.0 14.70 8.69 7.69 - - - - 9.37 13 1.43

Rhizobiumspp. 48.0 44.11 43.47 23.07 14.28 20.0 - 30.0 43.75 64 7.87

Proteusspp.

4.0

8.82

8.69

15.38

14.28

-

20.0

5.0

9.37

17

2.52

Ralstoniaspp. 16.0 5.88 13.04 15.38 - 10.0 - 20.0 18.75 22 2.92

Burkholderia spp. 40.0 20.58 26.08 38.46 - 10.0 6.66 20.0 21.87 41 5.42

Unknwon 40.0 29.41 21.73 53.84 42.85 30.0 3.33 25.0 12.50 55 7.63

No. of isolates recovered 145 160 89 50 31 32 33 88 124 752 -

Total CF (%) 580.0 476.42 386.86 384.55 221.39 320 189.94 440 387.41 - -

Soil sample collected 25 34 23 13 14 10 15 20 32 - -

Colonization frequency of rhizobacteria isolates from tomato rhizosphere across tomato

growing regions of Karnataka


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LocationTotal

isolates

Total species

richnessDiversity indices

Sipmson Shannon

Mys 145 16 0.901 2.448Man 160 16 0.870 2.327

Has 89 15 0.892 2.393

ChikM 50 13 0.898 2.371

Bru 31 09 0.813 1.800

Bur 32 12 0.871 2.145

Ram 33 08 0.779 1.689

ChkB 88 15 0.875 2.326

Kol 124 16 0.881 2.391

Species richness and diversityof bacteria associated with rhizosphere across selected

tomato growing regions of Karnataka

0

2

4

6

8

10

12

14

16

18

0 20 40 60 80 100 120 140 160

Number of Isolations

ExpectednumberofspeciesE

(s)

Mys

Man

Has

ChkM

Bru

Bur

Ram

ChkB

Kol

Rarefaction curves of rhizobacteria

(Number of isolations Vs. Expected number of species,

E(s)obtained from different selected tomato growingregions of Karnataka

NMDS and UPGMA cluster diagram of rhizobacterial

profile of different locations

Pl h i d di i hi b i l i


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Plant growth promoting and disease suppressing rhizobacterial traits

a. Indole acetic acidb. hydrogen cyanide,c. Phosphatesolubilization, d. Siderophore, e. Chitinase, d. ,1-3,Glucanase, B. a. Antagonistic activity, b. Partially

purified antibiotics, c, d, e, and f, Growth promotion

studies.

Root colonization bioassaya. Screening rhizobacteria fortheir root colonizing ability,b. Seedlings raised fromtreated seeds showed

colonization of rhizobacteriaaround the roots as a opaquezone and in control the zoneis absent, c. d. & f.Aggressive root colonizationby Pseudomonassp., Bacillussp.

and Serratiasp. respectively.


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Diversity of PGPR traits of

selected rhizobacterial

isolates from tomatoacross tomato growing

districts of Karnataka


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Root disease

Fusarium wilt caused by

Fusarium oxyspo rumf. sp. lycopersic i

Foliar disease

Early blight caused by

Alternar ia solani


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PGPR TRAITS


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Root colonization and competition for space and

nutrition between PGPR and phytopathogens at

rhizosphere.

Expression of rhizobacterial ipdC gene by lowerconcentration of IAA released from root and

biosynthesis of IAA by rhizobacteria using tryptophan

as precursor present in root exudates.

Competition for iron between Plant, PGPR and phytopathogens. PGPR secreted siderophore complexes with available iron in

rhizosphere. Siderophore-iorn complex can be taken up by PGPR and plant, but phytopathogens are unable to use this complex.


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Lytic enzymes such as chitinase and -1,3-glucanase produced by

PGPR lyses fungal or oomycetes cell wall by releasing monomersor oligomers which can be used by PGPR as C source. On the

other hand degraded cell wall component act as elicitors

molecules which induce host defence response.

Degradation of ACC which is a immediate precursor of

ethylene by ACC deaminase producing PGPR reduced

available ACC to synthesize ethylene in root. Degradation

product Ammonia is further utilized as N source by plant and

PGPR.


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Total number of rhizobacteria offering

protection against Fusarium wilt and early

blight disease of tomato under greenhouse

conditions

Plant growth promoting activity of

rhizobacteria.

Under laboratory conditions seedlings vigor

was analyzed by subjecting the treated seeds to

Standard blotter method (ISTA, 2005).Under greenhouse conditions freshweight of

the seedlings was analyzed for the 30 day old

seedlings.

Hariprasad, P.Venkateswaran, G. and Niranjana, S. R. 2013. Diversity of cultivablerhizobacteria across tomato growing regions of Karnataka. Biological Control (In Press)

Split-pot bioassay


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Split-pot bioassay

IRB isolates

Tomato stems

No. of

roots/stemMRL (cm)

Rhizobacterial population

on adventitious roots

(1x106 cfu/g fresh weight)

Control 32 2.30e 3.8 0.21b -

IRB1 30 2.88e 4.9 0.23a 2.8 0.17a

IRB2 34 1.15e 4.0 0.17ab 2.3 0.08a

IRB3 44 2.88e 3.9 0.17ab 1.6 0.144a

IRB4 48 1.73e 3.7 0.08b 2.9 0.23a

IRB5 70 2.88d 4.1 0.29ab 2.6 0.57a

IRB6 76 3.46d 4.0 0.23ab 2.1 0.17a

IRB7 97 4.04bc 3.5 0.14b 2.4 0.80a

IRB8 80 2.88cd 3.8 0.23b 2.4 0.11a

IRB9 116 8.08ab 4.2 0.17ab 2.5 0.92a

IRB10 109 4.61ab 4.0 0.25ab 1.9 0.86a

IRB11 121 5.77a 4.1 0.13ab 2.4 0.14a

IRB12 115 4.04ab

3.9 0.11ab

2.7 0.02a

Treatments

Plant height

(cm)

Fresh weight

(g/plant)

Total chlorophyll

(mg/g tissue)

Number of

fruits/plantMean fruit weight (g)

Control 130.5 2.88b 220.5 5.19b 16.7 0.57a 26 1.44b 31 1.38a

IRB1 133.0 4.61b 235.0 2.88ab 16.6 0.0a 27 1.73ab 38 1.67a

IRB11

140.5 2.88a

250.0 4.04a

16.7 0.11a

32 0.34a

39 0.98a

Phosphate solubilizing rhizobacteria


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Detection of gluconic acid produced by isolate PSIRB2 on TLC plate afterspraying bromophenol blue. St: Standard Gluconic acid

Phytase zymogramanalysis of isolate PSRB21

and PSRB31. M: Protein

marker

Phosphate solubilizing rhizobacteria


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Phosphate solubilizing and indole acetic

acid producing rhizobacteria (PSIRB)

3 3.5

Chitinase activity (Units/ml) CZ/CS ratioChitinase producing rhizobacteria


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e

aa

aa

b

bcde

bc

bcdbcde

bcdecde

de

fef

defdededecd

bcbc

aba a

a

0

0.5

1

1.5

2

2.5

CRB1

CRB2

CRB11

CRB14

CRB20

CRB27

CRB33

CRB34

CRB39

CRB40

CRB46

CRB51

CRB55

Rhizobacterial isolates

Chitinaseac

tiv

ity

(Un

its

/ml)

-0.1

0.3

0.7

1.1

1.5

1.9

2.3

2.7

3.1

CZ/CSra

tios

y ( )

Ratio of clearance zone (CZ)/ colony size (CS)

and chitinase activity of selected rhizobacterial

isolates from tomato rhizosphere

decdebcde

bcdebcde

abcd abcd

abcdabc

ab

de

a

e

f

0

20

40

60

80

Control

CRB1

CRB2

CRB11

CRB14

CRB20

CRB27

CRB33

CRB34

CRB39

CRB40

CRB46

CRB51

CRB55

Rhizobacteria isolates

Dise

aseincidence(%)

Influence of seed bacterization with selected

CRB isolates on Fusarium wilt incidence oftomato under greenhouse conditions

Chitinase producing rhizobacteria(CRB) for the management of

plant diseases in tomato

Degradation of chitin on Renwicks medium

by different CRB isolates from therhizospheric soil sample of tomato


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SDS-PAGE of purified chitinase

from Bacillus subtilis isolate

CRB20. Standard protein marker

(A); Coomassie-stained chitinase

(B) Activity-stained Chitinase

a

aa

bb

b

b

b

a

0

20

40

60

80

100

Control Chitinase Chitin CRB CRB +

Chitinase

CRB +

Chitin

Treatments

Diseaseincidence(%

)

0

0.04

0.08

0.12

0.16

IsolateCRB20population(Log

10cfu/gsoil)

Disease incidence (%)

Isolate CRB20 population (Log10 cfu/g soil)

Influence of Bacillus subtilis CRB20 application

alone or in combination with chitinase or chitin on

Fusarium wilt incidence and rhizospheric

population of Bacillus subtilis isolate CRB20

under greenhouse conditions

Hariprasad, P., Divakara, S. T. and Niranjana, S. R. 2011. Isolation and

characterization of chitinolytic rhizobacteria for the management of fusarium wiltin tomato. Crop Protection 30: 1606-1612


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Plant disease suppression by PGPR through

antibiotics production

Antimicrobial activity of Pseudomonas aeruginosa isolate 2apa and its antibiotics.

Inhibition of a. Fusarium oxysporum and b. Ralstonia solanacearum on dual culture

technique by isolate 2apa; crude antibiotic inhibiting the growth of c. Fusarium oxysporum

and d. Ralstonia solanacearum on disc diffusion method.

Broad spectrum antimicorbial activity of Pseudomonas aeruginosa 2apa


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Extraction, purification and evaluation of

antibiotics from

Pseudomonas aeruginosastrain 2apa

Polymerase chain reaction

(PCR) tests for screening

of gene involved in

antibiotics production.


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Its a state of systemically enhanced host resistance by rhizospheric microorganisms

which offers protection against broad spectrum phytopathogens

PGPR mediated induction of systemic resistance (ISR) in tomato


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Influence of seed bacterization with P. aeruginosastrain 2apa on PAL, POX, PPO and LOX

activity in tomato challenged with or without Alternaria solani.T1: Seedlings raised from controlseeds, T2: Seedlings raised from control seeds followed by challenged with pathogen, T3: seedlings raised from

bacterized seeds, T4: seedlings raised from bacterized seeds followed by challenged with pathogen. Vertical bars

indicates the SE of three replicates.

PGPR mediated induction of systemic resistance (ISR) in tomato

T1 T2 T3 T4 T1 T2 T3 T4


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Native PAGE analysis for POX isoforms

induced by P. aeruginosastrain 2apa in tomato

challenged with or withoutA. solani.

Native PAGE analysis for PPO isoforms induced

by P. aeruginosastrain 2apa in tomato challenged

with or withoutA. solani.

e

de d

e

c

b

de

d d

e

aa

0

20

40

60

80

100

120

140

Phenolconc

entration(g/gfreshweight)

T1 T2 T3 T4

Different treatments

0 5 10

Native PAGE analysis for LOX isoforms

induced by P. aeruginosastrain 2apa in tomato

challenge inoculated with or withoutA. solni.

Influence of seed bacterization with

Pseudomonas aeruginosa 2apa on total phenol

content in tomato challenged with or withoutA. solani.

T1 T2 T3 T4 T1 T2 T3 T4

T1 T2 T3 T4

0.25T1 T2 T3 T4

10 T1 T2 T3 T4


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0

0.05

0.1

0.15

0.2

0 12 24 36 48 60 72 84 96

Hours after pathogen inoculation

S

alicy

licac

id(g

/gfw)

T1 T2 T3 T4

0

2

4

6

8

0 12 24 36 48 60 72 84 96


nmol/gfreshweight

0

0.2

0.4

0.6

0.8

1

0 12 24 36 48 60 72 84 96


nmol/g/h

T1 T2 T3 T4

Systemic production of Salicylic acid

(SA) in tomato leaves expressing

rhizobacteria-mediated ISR.Endogenous levels of total SA in leaves

of tomato plants harvested at different

hours after pathogen inoculation (HPI)

in different treatments

Systemic production of Jasmonic acid

(JA) in tomato leaves expressing

rhizobacteria-mediated ISR.

Cumulative ethylene (ET) productionover a 96 h time period in leaves of

tomato plants expressing rhizobacteria-

mediated ISR.

Hariprasad, P.,S. Chandrashekar, S. Brijesh Singh, S. R. Niranjana. 2013. Characterization

of a novel Pseudomonas aeruginosastrain 2apa from tomato rhizosphere as potential plantgrowth promoting and bioprotecting agent.Journal of basic microbiology (In Press).


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Figure Induction of resistance against Pseudomonas syringae pv. maculicola ES4326 in


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Arabidopsis exposed to bacterial VOCs.

Induced systemic resistance against P. syringaepv. maculicola ES4326 elicited by VOCs of P. polymyxaE681 and a water control, using the microtitresystem. Disease severity (0 = no symptom, 10 = severe chlorosis) was recorded seven days after pathogen challenge. Different letters indicate significantdifferences between treatments, according to least significant difference at P= 0.05. The error bars indicate SEM.doi:10.1371/journal.pone.0048744.g002

Lee B, Farag MA, Park HB, Kloepper JW, et al. (2012) Induced Resistance by a Long-Chain Bacterial Volatile: Elicitation of Plant Systemic Defense by a

C13 Volatile Produced by Paenibacillus polymyxa. PLoS ONE 7(11): e48744. doi:10.1371/journal.pone.0048744http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048744

strain E681 produced more

than thirty low molecular-

weight VOCs, of which

tridecane was only

produced by E681 and not

found in GB03 or IN937a

volatile blends. Thesestrain-specific VOCs

induced PR1 and VSP2 ge

nes.
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048744http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048744


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Development of bioformulations and field

studies

Bioformulations, a. Fresh culture, b.

Talcum powder formulation, c.

Preparing Talcum powder formulation

under aseptic conditions.

0.000001

0.00001

0.0001

0.001

0.010 1

530

45

60

75

90

105

120

Days after storage

Logcfu/gformulation

Serratia m arcescens Pan-9/c

Pseudom onas aerugino sa 2apa

Bacillus sub til is PSIRB2

Survival of selected PGPRs in talcum powder

formulation at room temperature.

Tomato seedlings cv. Suruchi F1 hybrid raised from the seeds treated with


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Tomato seedlings cv. Suruchi F1 hybrid raised from the seeds treated with

bioformulations, b. transplantation of tomato seedlings at Pandavapura (Mandya) at

Lakshmipura (Mysore).

Field experiments.


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e d e pe e s.

a. Healthy tomato plants raised form bioformulations treatment at Mandya, b. A women

worker picking the tomato fruits from experimental field, c. farmer grading the fruits and

analyzing the yield, d. plants showing early blight symptoms, e. tomato plant severely

infected with early blight disease, f. Tomato plant showing the typical wilt symptom and g. a

local farmer showing discoloration of vascular region of wilted plant.

Creating awareness regarding biofertilizers and biopesticides in Farmers and Public


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g g g p

Isolation and characterization of rhizobacteria for their beneficial traits to improve plant health


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Field survey and collection of rhizospheric soil sample from healthy plants

Isolation of rhizobacteria on common medium

(only those bacteria should be selected whose population is above 10% of total population)

Root colonization bioassay

Identification of rhizobacteria (16s rRNA gene sequence)

Characterization of plant growth promoting rhizobacteria for desired traits

Green house and field studies

(Plant growth promotion and disease suppression)

Mass Multiplication of rhizobacteriaPreparation of bioformulation (Talc based, lignite based, Liquid etc)

Shelf life period analysis

Controlled Field studies, Multi-location trials, Commercialization


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Thank You

microbial resource

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