Effect of mixed inoculations of Plant Growth Promoting

Rhizobacteria of chilli on Growth and Induced Systemic

Resistance of Capsicum fruitescence

Amrutha V Audipudi, Nokku Pradeep kumar and Sudhir Allu

Department of Microbiology

Acharya Nagarjuna University,Guntur 522510,A.P India

Email:audipudi_amrita@yahoo.com

4th Asian Plant Growth-Promoting Rhizobacteria (PGPR) for Sustainable Agriculture (biofertilizers and biofungicides)

Conference (4th ASIAN PGPR) Hanoi, Vietnam, May 3-6, 2015,

Homage to my beloved Professor

Prof. V.S. Rama Das F.N.A

Development of sustainable agricultural systems

Eco-friendly,

Low input of non-renewable resources and

Less cost gaining

Strategy is

PGPR.

Objective

To study the effect of combined inoculations

of Plant Growth-Promoting Rhizobacteria

(PGPR) On

PGP and ISR of chilli.

Given the negative environmental impact of chemical fertilizers

and their increasing costs, the use of PGPB as natural fertilizers is

advantageous for the development of sustainable agriculture.

The use of plant growth promoting rhizobacteria (PGPR) in

sustainable agriculture has been increased in the Last decades in

several regions of the world.

Various bacterial genera are included in PGPR group, such as

Pseudomonas, Azospirillum, Bacillus, Enterobacter, Xanthomonas

and Serratia (Khalid et al. 2004).

The mechanisms of action of PGPR may initially linked

to inhibition of soil plant pathogens and there by stimulate

plant growth indirectly (Gupta et al. 2000).

It is often difficult to recognize the mechanisms and

relate directly to promotion of plant growth, since more than

one mechanism produced by bacteria (Araujo et al. 2005).

PGPR MECHANISMS

There are few studies about evaluation of phosphate solubilising bacteria

strains and their efficiency in plant growth promotion in sub-tropical soils.

The aims of this paper were

(1)To find novel phosphate solubilising bacteria strains

isolated from chilli fields

(2) to evaluate their plant growth promoting activities

and

(3) to assess their efficacy on plant growth promotion

and ISR.

Isolation of plant growth promoting rhizosphere bacteria: Screening of Phosphate solubilisation:(Gaur, 1990)..

Production of Indole acetic acid:. (Brick et al.1991).

Production of ammonia:(Cappuccino & Sharma1992).

HCN production:(Lorck,1948).

Characterization of siderophore: (Briskot,G. et al., 1986).

Quantitative estimation PGP traits: Inorganic phosphate Solubilization (Nautiyal and Jackson 2001).

IAA (Loper & Scoth 1986).

Ammonia production (Demutskaya and Kalinichenko 2010).

Molecular characterization of bacterial isolates: 16S rRNA gene was done by using universal bacterial primer 1492R (5´-TACGGYTACCTTGTTACGACTT-3´) and 27F (5´

AGAGTTTGATCMTGGCTC AG3´) sequenced by Macrogen Incorporation, Seuol Korea.

Seed treatment and nursery experiments: Seeds of chilli were treated with the 48-h-old culture (approximately 108 CFU/ml) of the selected isolate 30 min and were shade-

dried at 28 + 28C for 1 h. The treated seeds (100) were sown in pots containing coco peat in a greenhouse. Observations were recorded

on germination percentage in the beginning, root length, shoot length and wet weight of the seedlings ever two weeks interval of

sowing by removing 10 seedlings from each replication.

Induced systemic resistance: Estimation of defence enzymes. (Sudhir et al.2015).

Perooxidase, Poly phenol oxidase and Phenylalanine lyase

Results and Discussion

FUNCTIONAL DIVERSITY OFPHOSPHATE SOLUBILSERS OF CHIILLI RHIZOSPHERE

RHIZOSPHERE BACTERIA

Phosphate Solubilisers

ANTAGONISM AGAINST Colletotrichum

MULTIPLE PGP TRAITS

55 isolates

AVP1-AVP13 13 isolates

AVP7

AVP1-AVP13

IDENTIFICATION OF PHOSPHATE SOLUBILISERS

PSEUDOMONAS AVP1,2,3,4

AVP- 5,6,7,8,9,10

AVP22

AVP27

AVP23

BACILLUS

KLEBSIELLA

STENOTROPHOMONAS

ACHROMOBACTER

AVP3

AVP7

AVP27

SELECTED ISOLATES

Biochemical Tests AVP 3 AVP 7 AVP27 Indole -ve +ve +ve Methyl Red +ve +ve +ve Vogues proskeur +ve -ve +ve Citrate test +ve -ve +ve Catalase +ve +ve -ve Oxidase +ve -ve -ve H2S -ve -ve +ve Urease +ve +ve +ve Amylase -ve -ve -ve Lipase -ve -ve -ve Glucose +ve +ve +ve Lactose +ve +ve -ve Maltose +ve +ve -ve Sucrose +ve +ve +ve Tentative genus Pseudomonas Bacillus Stenotrophomonas

Morphological Biochemical and Physiological Characteristics of AVP3, AVP7&AVP27

Isolate name

Phosphate solubilization

(ppm)

IAA µg/ml

Ammonia µg/ml

Siderophore production

HCN production

AVP 3 1640 53 72 POSITIVE POSITIVE AVP 7 1383 65 79 POSITIVE POSITIVE AVP 27 1733 72 60 POSITIVE POSITIVE

Plant Growth PromotingTraits of AVP3,AVP7&AVP27 isolated from chilli rhizosphere.

-0.050

0.050.1

0.150.2

0.250.3

0.350.4

0.450.5

sucrose Maltose Lactose Dextrose

O.D

of I

sola

te

Carbon Source

AVP3

AVP7

AVP23

0

0.05

0.1

0.15

0.2

0.25

0.3

250C 350C 500C

OD

of th

e Is

olat

e

TEMPERATURE

AVP3

AVP7

AVP 27

0

0.5

1

1.5

2

2.5

3

3.5

1 2 3 4 5

GRo

wth

of t

he is

olat

e

PH

AVP3

AVP7

AVP27

0

0.05

0.1

0.15

0.2

0.25

0.30% 0.50% 0.70% 0.70% 0.90% 1%

O.D

of I

sola

te

NaCl (%)

AVP3 AVP7 AVP23

3 isolates are subjected to optimization of growth for maximum production with

Temperatures(25˚C,35 ˚C and 50˚C),

pH ranging from 1-5,

Percentages of NaCl(0.3%,0.5%,0.7%,0.9% and 1%) and

carbon sources (Lactose, Sucrose, maltose and dextrose).

All the three isolates showed max. Growth (<1.0) at 35⁰C

Growth of AVP7 & 27 much higher than AVP3 .

All the three bacterial isolates showed maximum growth at

pH5 though they are native to tropical soils of chilli field with pH

ranging from 6.8-7.8 indicating the these isolates can tolerate

acidic pH conditions

Three isolates showed diversity to their growth pattern at

different concentrations of NaCl.

AVP3 is tolerant to salinity up to 0.9% of NaCl.

AVP7 and AVP27 showed to a level below the optimum.

3isolates showed maximum growth at 1% NaCl

AVP7&27 showed tolerance to 1% NaCl.

Three isolates showed diversity in their growth in response to

carbon source.

AVP27 showed maximum growth at lactose.

AVP7 showed maximum Growth at dextrose.

AVP3 is dextrose sensitive

Root length measured in chilli seedlings inoculated with AVP3,AVP7 and AVP27 and

the measurements are taken in weekly interval and in triplicates (P is <0.04))

shoot length measured in chilli seedlings inoculated with AVP3,AVP7 and AVP27 and the

measurements are taken in weekly interval and in triplicates (P is <0.04))

Oxidative stress enzymes as markers of induced systemic resistance

a)Phenyl Alanine Ammonia lyase

b) Peroxidase and

c) Polyphenol Oxidase

AVP3Treated seedling

AVP7 treated seedling

AVP27 treated seedling

AVP3+AVP7

Control

0.28

0.3

0.5

0.98

0.22

0.67

0.65

0.72

1.3

0.25

PAL Activity µmol trancinnamic acid/min/gm tissue

4th week 8th week

Conclusions

Seed bacterization of chilli by AVP3, AVP7 and mixed inoculation

of AVP3+AVP7 resulted in varied growth response and induced

systemic resistance (ISR) under greenhouse condition.

All inoculations positively influenced chilli growth.

ISR response was negative with isolate AVP3 and positive with

isolate AVP7 and very high with mixed inoculation of AVP3+AVP7.

PGP traits of bacterial isolates were highly specific and

application of mixed inoculation of bacterial isolates with varied

specificity can influence growth and ISR more efficiently than

application of individual PGP isolate.

Thank you

6/3/2015 DrAAV 32