Biomolecular characterizing the sequence diversity of ACC deaminase of rhizobacterial strains associate with leguminous plants of arid, semi arid regions of Rajasthan

1. Characterizing the sequence diversity of
ACC deaminase of rhizobacterial strains
associate with leguminous plants of arid,
semi arid regions of Rajasthan
Presented by:-
Suman Kumar
Deptt:-Biotechnology
2013MSBT022

2.  Legumes are members of the plant family Fabaceae
 Legume plant are important as they constitute one of the
richest source of dietary source of proteins.
 Legume crops constitute one of the most important
commercial crops of arid, semi- arid regions of Rajasthan.

3. Rhizosphere- Soil layer bound to the plant root. It is a
zone of many beneficial plant - microbe interactions.
Rhizobacteria- Soil bacteria residing in the rhizosphere
are termed as rhizobacteria.
Some rhizobacteria are capable of enhancing plant growth
and they are called
Plant growth promoting rhizobacteria (PGPR)

4. Modified from: XiteBio Technology Inc.
Plant stress control

5. Salinity stress
pH stress
Heavy metals stress
Organic contamination stress
 Extreme temperature stress

6. ACC (1-Aminocyclopropane-1-carboxylase) deaminase

7. 10/13/2015
• Whether this mechanism also exist in Legume plants
growing in arid, semi- arid regions of Rajasthan?
• If so, what is the sequence diversity of ACC deaminase
present in PGPRs associated with legume plants of arid,
semi- arid regions of Rajasthan?

8.  Diversity of rhizobacterial strains isolated from
leguminous plants of arid, semi arid regions of Rajasthan.

9. Soil sample collection and processing
Determining the of physico-chemical characteristics
of soil samples
Isolation, purification and
preservation of culturable bacterial
isolates from legume rhizosphere
Optimization of soil
DNA procedure
Soil DNA PCR
RFLP analysis of acdS amplicons
Optimization of colony PCR for
acdS amplification
Sequencing of representative acdS amplicons

10. Sample
Area
Sample Name
(Legume
Plants)
No. of
collected
Sample
Temperature
during Sampling
Crop age at time
of sample
collection
Village- Untwaliya
District- Churu
State-Rajasthan
(Altitude: 75.02)
(Latitute: 28.29o
N)
(Longitude: 74.96o
E)
Tilhan 10 Env-38ºC
Soil-37ºC
90 Days
Gaur 10 Env-40ºC
Soil-38ºC
90 Days
Mung 10 Env-43ºC
Soil-41ºC
90 Days
Moth 5 Env-42ºC
Soil-42ºC
90 Days
Farmer Name:- Mula Ram &
Sharvan Kumar.

11. Sample
Name
(Legume
Plants)
Soil type
No. of
irrigation
times
Fertilizer
used
Pesticide
used
Harvesting Time
Tilhan Arid and
Semi-arid
2 Rain No No Late OCT-2014
Gaur Arid and
Semi-arid
3Rain Yes,
Only
Compost
No Mid NOV-2014
Mung Arid and
Semi-arid
1 Rain Yes,
Only
Compost
No Late OCT-2014
Moth Arid and
Semi-arid
1Rain Yes,
Only
Compost
No Late OCT-2014
Sample collection report Farmer Name:- Mula Ram &
Sharvan Kumar.

13. pH analysis of soil samples Organic carbon result of the soil samples

14. Physico-
chemical
characteristic
Samples Name
Guar
Rhizosphere
soil
Moong
Rhizosphere
soil
Moth
Rhizosphere
soil
Tilhan
Rhizosphere
soil
pH ~ 9 ~ 9 ~ 9.5 ~ 9
Oxidizable
Organic
carbon
Medium-Low
(0.300-0.500)
Medium-Low
(0.300-0.500)
High
(1.001-50)
Medium-Low
(0.300-0.500)
Phosphate 22-56kg/hac 22-56kg/hac 22-56kg/hac 22-56kg/hac
Potassium Medium
112-280kg/hg
High
280-390kg/hg
Low
112kg/hg
High
280-390kg/hg
Ammonical
Nitrogen
Medium
About-15kg/hg
Medium
About-15kg/hg
Medium
About-15kg/hg
Medium
About-15kg/hg
Nitrate
nitrogen
Very Low
About-4kg/hg
Very Low
About-4kg/hg
Very Low
About-4kg/hg
Very Low
About-4kg/hg

15. 1. Spreading of diluted samples
on Nutrient Agar medium
1. 2.
Purification of bacterial isolates
on Nutrient Agar medium
All Soil Samples were isolate and purified on Nutrient agar media.
Total 232 rhizobacterial isolate are purified
M.G.R.001,(44) M.G.S.001,(76) M.R.001,(40) M.S.001,(34) T.R.001,(36)
T.S.001,(40) G.R.(36) G.S.001,(70) M.S.002,(32) T.S.002,(56).

16. M.S 001sample result
MG.R 001 sample result
s. no SIZE SHAPE COLOR TEXTURE HIGHT MARGIN
1 5 MM & MEDIUM SIZE IRREGULAR CREMISH & OPAQUE MUCOID & STICKY RISED ROUGH MARGIN
2 3-4 MM & SMALL SIZE IRREGULAR YELLOWISH & OPAQUE MUCOID & STICKY RISED ROUGH MARGIN
3 4-5 MM & MEDIUM SIZE IRREGULAR YELLOWISH & OPAQUE MUCOID RISED ROUGH MARGIN
4 4-5 MM & MEDIUM SIZE NON-CIRCULAR YELLOWISH & OPAQUE MUCOID & STICKY FLAT SMOTH MARGIN
5 6-7MM & MEDIUM LARGE IRREGULAR CREMISH & OPAQUE MUCOID FLAT FIBROUS MRGIN
6 5 MM & MEDIUM SIZE NON-CIRCULAR DULL YELLOWISH & OPAQUE MUCOID & STICKY FLAT SMOTH MARGIN
7 4-5 MM & MEDIUM SIZE NON-CIRCULAR CREMISH & OPAQUE MUCOID & STICKY FLAT SMOTH MARGIN
8 5-7 MM & MEDIUM LARGE NON-CIRCULAR DULL YELLOWISH & OPAQUE MUCOID & STICKY FLAT SMOTH MARGIN
9 3-4 MM & SMALL SIZE NON-CIRCULAR DARK YELLOWISH & OPAQUE MUCOID & STICKY FLAT SMOTH MARGIN
10 4-5 MM & MEDIUM SIZE NON-CIRCULAR DULL YELLOWISH & OPAQUE MUCOID & STICKY RISED ROUGH MARGIN
11 6-7 MM & MEDIUM LARGE NON-CIRCULAR DARK YELLOWISH & OPAQUE MUCOID & STICKY RISED ROUGH MARGIN
12 1-2 MM & SMALL SIZE NON-CIRCULAR YELLOWISH & OPAQUE MUCOID & STICKY RISED ROUGH MARGIN
13 4-5 MM & MEDIUM SIZE NON-CIRCULAR YELLOWISH & OPAQUE MUCOID FLAT SMOTH MARGIN
14 4-5 MM & MEDIUM SIZE NON-CIRCULAR ORANGESIH & TRANSLUCENT MUCOID RISED SMOTH MARGIN
15 5MM & MEDIUM SIZE NON-CIRCULAR YELLOWISH & OPAQUE MUCOID RISED SMOTH MARGIN
s.no SIZE SHAPE COLOR TEXTURE HIGHT MARGIN
1 8-9 MM & LARGE SIZE RHIZOID CREMISH & TRANSLUCENT MUCOID FLAT FIBROUS
2 7-8 MM & LARGE SIZE CIRCULAR CREMISH & OPAQUE MUCOID FLAT SMOOTH MARGIN
3 1-1.5 MM & VERRY SMALL CIRCULAR CREMISH TRANSLUCENT MUCOID RISED SMOOTH MARGIN
4 5-6 MM&LARGE SIZE CIRCULAR CREMISH & OPAQUE MUCOID DRIED FLAT SMOOTH MARGIN
5 6-8MM & LARGE SIZE CIRCULAR DULL CREMISH & OPAQUE MUCOID STICKY FLAT SMOOTH MARGIN
6 6-7MM & LARGE SIZE NON-CIRCULAR DULL CREMISH & OPAQUE MUCOIDSTICKY FLAT ROUGH MARGIN
7 3-4 MM & MEDIUM SIZE IRREGULAR CREMISH & OPAQUE MUCOID STICKY FLAT FIBROUS MARGIN
8 3-4 MM & MEDIUM SIZE IRREGULAR CREMISH & OPAQUE MUCOID STICKY FLAT ROUGH MARGIN

17. 1 2 3 4
Lane 1- DNA ladder 1kb
Lane 2- blank
Lane 3- Moong soil samples
S.NO. Name of sample DNA µg/ml
1 Moong sample 0.13 µg/ml
2 Gaur sample 0.11 µg/ml
5 6
Lane 4- gaur soil sample
Agarose gel analysis of soil DNA
Nanodrop spectrophotometer data

18. Lane 1- DNA ladder 1kb
Lane 2- blank
Lane 3- Garden soil samples
S.NO. Name of sample DNA µg/ml
1 Garden soil sample 31µg/ml
2 Gaur soil sample 18.01µg/ml
3 Moong soil sample 15µg/ml
Lane 4-5- Gaur soil sample
Lane 6-7- Moong soil sample
Agrose gel analysis in soil DNA isolation
1 2 3 4 5 6 7

19. Lane 3-7: Soil DNA PCR products for acdS
Agrose gel analysis of PCR
products from PCR for acdS
1 2 3 4 5 6 7
Lane 1: DNA ladder
Lane 2: blank

20.  Based on the results obtained, we conclude that a
microbial collection of ~ 235 culturable bacterial isolates
associated with rhizosphere of legume crops of arid and
semi- arid region of Rajasthan have been isolated.
 These culturable isolates as well as rhizospheric soil DNA
were tested with PCR for presence of acdS.
 However, due to lack of optimized protocols, it could not
get accomplished.

21.  The isolates will be screened for their role in other
plant growth promoting activities.
 Diversity of ACC deaminase gene these isolates and
unculturable diversity would be characterized with
PCR and cloning base approaches.

23. 1 2 3 4 5 6 7