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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,
Development of sustainable agricultural systems
Eco-friendly,
Low input of non-renewable resources and
Less cost gaining
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).
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
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.
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