a novel bacillus aryabhattai ms3 promotes growth...
TRANSCRIPT
A Novel Bacillus aryabhattai MS3 promotes growth
in rice under salinity stress
Muhammad Manjurul Karim, PhD Department of Microbiology, University of Dhaka, Bangladesh 1
2012
2020
2050
2100
Increased Flooding Increased Storm Surges Increased Moisture Stress Greater Temperature Extremes Increased Salinity Intrusion due to Sea Level Rise (SLR)
Sea Level Rise (SLR)
10 cm
25 cm
>100 cm
Land below SLR - 2% of land (2,500 km2)
Inundation of 0.2 million metric tons of production
(<1 % of current total)
Land below SLR - 4% of land (6,300 km2)
Inundation of 0.5 million metric tons of production
(2 % of current total)
Land below SLR – 17.5 % of land (25,000 km2)
Devastating floods may cause crop failure
Salinity effect on agriculture
↪ Reduces the ability of plants to absorb water, ↪ Reduces the growth and production of plants, ↪ Reduce N2-fixation ability by symbiotic and asymbiotic microorganisms
Source: World Bank, 2000
PROJECTED CLIMATE CHANGE IMPACTS IN BANGLADESH
2
Salinity problem: Current scenario of Bangladesh
3
Pakhimara village of Kalapara,
Patuakhali
Noble quote
A problem without a solution is a poorly stated problem.
AE
Prologue 4
Development of salt-resistant crop varieties
Application of salt-tolerant Plant growth promoting rhizobacteria (PGPR) takes the edge over the former (transgenic plants) because of ethical and environmental considerations.
WAY OUT for a sustainable development?
5 Prologue
Bioinnovation and Bioeconomy
Why PGPR?
Ro
les
of
PG
PR
Direct effect
N2 fixation
Phosphate solubilization
Siderophores production
K production
Phytohormones
• Cytokinin
• Ethylene
• IAA
Indirect effect
Antimicrobials production
Hydrolytic enzyme
Induced system resistance
EPS
BACKGROUND
BACKGROUND
Sampling site
Plant Growth
Promoting abilities
A Venn diagram to isolate potential bacteria
Bacillus aryabhattaiMS3
POT EXPERIMENTS under saline and non-saline condition
Materials Source of collection
Seed (Oryza sativa BR-28)
Laboratory of Plant Biotechnology, Department of BMB, DU
Agriculture field soil Gazipur
Earthen pot Local market
Biofertilizer
Preparation of bio-fertilizer • Bio-fertilizer was formulated according to the Bureau of
Indian Standards (BIS) guidelines. – Charcoal powder : Calcium carbonate : Gum acacia: Microbial culture =
7: 1: 0.2: 1×109
10
Tim
elin
e o
f in
viv
o e
xpe
rim
en
t 25-Jan 6-Mar 15-Apr 25-May 4-Jul
seeds incubated at 56⁰C
seeds transferred to a petriplatecontaining whatman filter paper
germinated seeds transferred tohypotonic solution
cultivation on pot under non-salinecondition
application of salt
(8 days)
(45 days)
Seeds incubated at 56⁰C (2 days)
Seeds transferred to a petriplate containing whatman filter paper (2 days)
Germinated seeds transferred to hydroponic solution (8days)
Cultivation on pot under normal condition (45 days)
Application of salt (25 days)
A 82 days long experiment continued from 9-March to 30 May (2017)
Collecting plants for physiological and phenotypic observation
In vivo experiment (PGPR application as biofertilizer on rice plant)
Control plants
After 45 days of biofertilizer application
MS3 applied plants
E.coli DH5α applied plants
25
84
7
12
78.13%
87.50%
Survivability of controledplants
Survivability of MS3 appliedplants
Live plantsnumberDead plantsnumber
Survival plant
Control plants MS3 Applied plants
E.coli DH5α applied plants
Salt application (200mM)
After 25 days
2
39
23
45
8.00%
46.00%
Survivability of controled plants
Survivability of MS3 applied plants
Observing physiological and phenotypic state of plants
Bar diagram representing the comparative length (cm) of plant stem and leaves under both
normal condition and saline condition
Plant length in cm
Plant dry weight
Bar diagram representing the dry weight of plants under both normal condition and saline
condition
Reduction rate
31.82%
Reduction rate
22.72%
Reduction rate
27.58%
Reduction rate
14.73%
0
10
20
30
40
50
60
None E.coli MS3
len
gth
in c
m
ID name
stem normal condition
stem saline condition
leaf normal condition
leaf saline condition
0
5
10
15
20
25
None E.coli MS3
len
gth
in c
m
plant dry weight (gm) normalcondition
plant dry weight (gm) salinecondition
Bar diagram representing the IAA
concentration of plants (stem and leaf) normal
and saline condition.
IAA concentration in plants Chlorophyll conc.
Bar diagram represent the chlorophyll concentration of
plants (stem and leaf) normal condition and saline
condition. Carbohydrate concentration
Bar diagram representing the carbohydrate concentration of plants (stem and leaf) under saline and
normal condition.
Stem normal condition
Stem saline condition
Leaf normal condition
Leaf saline condition
0
2
4
6
8
10
12
None E.coli MS3
IAA
co
nce
ntr
atio
n((
µg/
g)
0
0.5
1
1.5
2
2.5
3
3.5
None E.coli MS3
chlo
rop
hyl
co
nc.
(µg/
g)
0
5
10
15
20
25
30
None E.coli MS3
carb
oh
ydra
te c
on
c (µ
M/g
)
0
2
4
6
8
10
12
14
16
normal condition saline condition normal condition saline condition
stem leaf
Me
lan
de
ald
eh
yde
co
nc.
(µM
/g)
0
50
100
150
200
250
300
350
Pro
line
co
nc.
(µ
M/g
)
Proline
mainly produced by
plants under saline
condition to
overcome the
oxidative damage of
tissues.
MDA
High MDA content
indicates membrane
lipid peroxidation.
sustainability of plant
growth under high
salinity is associated
with reduced MDA
formation
None
E.coli DH5α
MS3
MOLECULAR MECHANISM OF PLANT’S SALT RESISTANCE BY PGPR
18
Plant Gene Expression Analysis Why
To observe if the PGPR can confer salt tolerance ability in rice plant
by modulating in cellular transcription level.
3 salt responsive genes were selected to be analyzed.
Gene name Function
NHX1 Sodium proton exchanger; reduces Na+ concentration in cytosol during salt stress.
GIG Negative regulation of cellular protein translation.
BZ8 Regulate different transcriptional pathway through osmotic signaling
Ref: C.S. Nautiyal et al. / Plant Physiology and Biochemistry 66 (2013)
Transcriptomic analysis
Collect tissue
Total mRNA
RT PCR
cDNA
DNA pol
dNTP + buffer
Specific amplicon
pool of target cDNAs
C= control C+S=control
+salt B=biofertilizer B+S=biofertiliz
er +salt
Semi quantitative
RT-PCR
Analysis of plant gene expression upon salt stress
Pla
nt g
en
es
Keys:
C: Control
BF: Bio-fertilized
S: salt added, 200 mM
Plant vacuole membrane protein
• Na+ is uptaken into vacuoles in exchange of H+
during high salt conc.
Plant cell
Na+ Na+ Na+ Na+ Na+ Na+ Na+
Na+ Na+
Na+ Na+
Na+ Na+
Na+ Na+ Na+
Na+ Na+
Na+ Na+
Na+ Na+
Na+ Na+ Na+
Na+ Na+ Na+ Na+
• Nitrogen fixation • Phosphate solubilization • Siderophore production
• Phytohormone production
PGPR & Plant NHX1
Summery
Application of MS3 could induce plant growth
even under salinity stress conditions, as a
result of plant-microbe interaction by
increasing availability of nutrients (Fe, P)
decreasing reduction of IAA and chlorophyll
content
enhancing proline accumulation
avoiding MDA formation
We propose stimulation by Bacillus arybhattai
MS3 as a mechanism of inducing salt
tolerance in rice by modulating differential
transcription in a set of salt-tolerant genes.
Funding bodies: Ministry of Science and Technology, and Ministry of Education Government of the People’s Republic of Bangladesh
Thank you
24
My collaborator:
1. Dr Sirajul Hoq
Dept of Soil, Water and Environment
University of Dhaka
2. Dr Abidur Rahman
Iwate University, Japan
My students:
1. Sumonto C Paul
2. Shahnaz Sultana
3. Samia Rahman
4. Bushra Zannat and
5. Naziza Rahaman
25
Total 45 saline isolates
Total 8 non saline isolates Total 53 isolates
Enrichment & Isolation
27 Results
(a) Enrichment
of Soil samples.
(b) Saline
isolates in
respective plate.
(c) Non saline
isolates in
respective plate.
(d) Isolation of a
pure culture
Salt
to
lera
nce
ass
ay
0.63% 1.25% 2.50% 5% 7.50% 10% 15%
DM-1.1 3 3 3 3 2 1 0
DM-1.3 3 3 3 3 2 1 0
DM-1.4 3 3 3 3 2 1 0
DM-1.7 3 3 3 3 2 1 0
DM-2.2 3 3 3 3 2 1 1
DM-2.5 3 3 3 3 2 1 0
DM-2.7 3 3 3 3 2 1 1
DM-2.9 3 3 3 3 2 1 0
DM-2.11 3 3 3 3 2 1 0
DK-1.2 3 3 3 3 2 1 1
DK-1.3 3 3 3 3 2 1 0
DK-1.6 3 3 3 3 2 1 1
DK-1.11 3 3 3 3 2 1 0
DK-1.12 3 3 3 3 2 1 0
DK-2.1 3 3 3 3 2 1 0
DK-2.2 3 3 3 3 2 1 1
DK-2.3 3 3 3 3 2 1 1
DK-2.4 3 3 3 3 2 1 0
DK-2.5 3 3 3 3 2 1 0
DK-2.8 3 3 3 3 2 1 0
DK-2.9 3 3 3 3 2 1 0
BP-1.1 3 3 3 3 2 1 1
BP-1.3 3 3 3 3 2 1 0
BP-1.8 3 3 3 3 2 1 0
BP-1.10 3 3 3 3 2 1 0
BP-1.12 3 3 3 3 2 1 0
BP-1.14 3 3 3 3 2 1 0
BP-2.1 3 3 3 3 2 1 1
BP-2.4 3 3 3 3 2 1 1
BP-2.6 3 3 3 3 2 1 1
BP-2.8 3 3 3 3 2 1 0
BP-2.9 3 3 3 3 2 1 0
KP-1.1 3 3 3 3 2 1 1
KP-1.2 3 3 3 3 2 1 1
KP-1.4 3 3 3 3 2 1 1
KP-1.6 3 3 3 3 2 1 1
KP-1.7 3 3 3 3 2 1 0
KP-1.8 3 3 3 3 2 1 0
KP-1.10 3 3 3 3 2 1 1
KP-2.4 3 3 3 3 2 1 1
KP-2.5 3 3 3 3 2 1 0
KP-2.6 3 3 3 3 2 1 0
KP-2.7 3 3 3 3 2 1 0
KP-2.8 3 3 3 3 2 1 0
KP-2.9 3 3 3 3 2 1 1
Salt tolerance assay
Saline
Isolate IDType
NaCl Concentration (%)
CL-1.1 3 3 2 1 0 0 0
CL-1.2 3 3 2 1 0 0 0
CL-1.4 3 3 2 1 0 0 0
CL-1.5 3 3 2 1 0 0 0
CL-2.2 3 3 3 2 1 0 0
CL-2.3 3 3 3 2 1 0 0
CL-2.4 3 3 3 2 1 0 0
CL-2.5 3 3 3 2 1 0 0
3 2 1 0
+ + + + + + No growth
Keys of
growth
Non- saline
0.63% 1.25% 2.50% 5% 7.50% 10% 15%
DM-1.1 3 3 3 3 2 1 0
DM-1.3 3 3 3 3 2 1 0
DM-1.4 3 3 3 3 2 1 0
DM-1.7 3 3 3 3 2 1 0
DM-2.2 3 3 3 3 2 1 1
DM-2.5 3 3 3 3 2 1 0
DM-2.7 3 3 3 3 2 1 1
DM-2.9 3 3 3 3 2 1 0
DM-2.11 3 3 3 3 2 1 0
DK-1.2 3 3 3 3 2 1 1
DK-1.3 3 3 3 3 2 1 0
DK-1.6 3 3 3 3 2 1 1
DK-1.11 3 3 3 3 2 1 0
DK-1.12 3 3 3 3 2 1 0
DK-2.1 3 3 3 3 2 1 0
DK-2.2 3 3 3 3 2 1 1
DK-2.3 3 3 3 3 2 1 1
DK-2.4 3 3 3 3 2 1 0
DK-2.5 3 3 3 3 2 1 0
DK-2.8 3 3 3 3 2 1 0
DK-2.9 3 3 3 3 2 1 0
BP-1.1 3 3 3 3 2 1 1
BP-1.3 3 3 3 3 2 1 0
BP-1.8 3 3 3 3 2 1 0
BP-1.10 3 3 3 3 2 1 0
BP-1.12 3 3 3 3 2 1 0
BP-1.14 3 3 3 3 2 1 0
BP-2.1 3 3 3 3 2 1 1
BP-2.4 3 3 3 3 2 1 1
BP-2.6 3 3 3 3 2 1 1
BP-2.8 3 3 3 3 2 1 0
BP-2.9 3 3 3 3 2 1 0
KP-1.1 3 3 3 3 2 1 1
KP-1.2 3 3 3 3 2 1 1
KP-1.4 3 3 3 3 2 1 1
KP-1.6 3 3 3 3 2 1 1
KP-1.7 3 3 3 3 2 1 0
KP-1.8 3 3 3 3 2 1 0
KP-1.10 3 3 3 3 2 1 1
KP-2.4 3 3 3 3 2 1 1
KP-2.5 3 3 3 3 2 1 0
KP-2.6 3 3 3 3 2 1 0
KP-2.7 3 3 3 3 2 1 0
KP-2.8 3 3 3 3 2 1 0
KP-2.9 3 3 3 3 2 1 1
Salt tolerance assay
Saline
Isolate IDType
NaCl Concentration (%)
28
Growth on Johnson’s agar
Iso
late
d f
rom
sali
ne
are
as
Isolated from non-saline areas
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 2 4 6 8 10
OD
AT
60
0 N
M
TIME (HOURS)
CL-1.2 Growth Curve
0% NaCl
5% NaCl
10% NaCl
20% NaCl
Salt tolerance assay
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 5 10
OD
AT
60
0 N
M
TIME (HOURS)
CL-2.5 Growth curve
0% NaCl
5% NaCl
10% NaCl
20% NaCl
While non-saline isolates CL 1.2 (a) and CL 2.5 (b) exhibited no growth at 10% salt, the saline isolates BP 1.10 (c) and DK 2.2 (d) adapted the tolerance after an extended lag phase in LB broth supplemented with 0, 5, 10 and 20% NaCl.
a b
0
0.2
0.4
0.6
0.8
0 2 4 6 8 10
OD
A
T
60
0 N
M
TIME (HOURS)
BP-1.10 Growth Curve
0% NaCl
5% NaCl
10% NaCl
20% NaCl 0
0.2
0.4
0.6
0.8
0 5 10
OD
AT
60
0 N
M
TIME (HOURS)
DK-2.2 Growth curve
0% NaCl
5% NaCl
10% NaCl
20% NaCl
c d
29
Selection of bacterial isolates
Characterization of their growth promoting abilities viz. atmospheric nitrogen fixation, phosphate solubilization and IAA production.
30 Results
Plant growth promoting traits Method
Nitrogen fixation Kjeldahl method
Indole 3-acetic acid (IAA) production Gordon and Weber (1966)
Phosphate solubilization Molybdenum blue method in NBRIP broth
PGP activities
Based on those abilities, isolates were classified in their capacities of
Low, Moderate and High
31
Range of IAA production
Low ( 0-
150) µg/ml
Moderate
(151-250)
µg/ml
High (251-
400) µg/ml
Dm-1.1,
Dm-1.3,
Dm-1.4
Dm-2.2,
Dm-2.5, Dm-
2.7, Dm-
2.11,
Dm-1.7, Dm-
2.9,
Bp-1.1, Bp-
1.3, Bp-2.1,
Bp-2.4, BP-
2.9,
Bp-1.8, Bp-
1.12, Bp-2.6,
Bp-2.8, Bp-
1.10
Bp-1.14, Bp-
1.10
Dk-1.6, Dk-
2.3, Dk-2.8,
Dk-2.9,
Dk-1.11, Dk-
1.2
Dk-1.3, Dk-
2.1, Dk-2.2,
Dk-2.4, Dk-
2.5
Kp-1.6, Kp-
1.7 , Kp-2.4
, Kp-2.6,
Kp-2.7, Kp-
2.8, Kp-2.9,
Kp-1.1, Kp-
1.4, Kp-
1.10,
Kp-1.8, Kp-
2.5, Kp-1.2,
Kp-1.7.
Range of Phosphate solubilization
Low (0-150)
µg /ml
Moderate
(151-300) µg
/ml
High (300-450)
µg /ml
Dm-1.1, Dm-
1.3, Dm-1.4
Dm-2.5,
Dm-2.7, Dm-
2.9
Dm-2.11, Dm-1.7
Bp-1.1, Bp-
1.3, Bp-2.4,
Bp-2.9.
Bp-1.8, Bp-
1.10, Bp-1.12,
Bp-2.1, Bp-
2.6,
Bp-1.14, Bp-2.8,
Dk-1.6, Dk-
2.3, Dk-2.8
Dk-2.9,
Dk-1.11, Dk-
1.12, Dk-1.2,
D Dk-2.2, Dk-
2.5
Dk-1.3, Dk-2.1,
Dk-2.4,
Kp-1.1, Kp-
2.8,
Kp-1.4, Kp-
1.6, Kp-1.8,
Kp-2.6, Kp-
2.7, Kp-2.9
Kp-1.10, Kp-
1.2, Kp-1.7,
Kp-2.4, Kp-2.5
Range of Nitrogen fixation
Low (0-
1.5)%
Moderate (1.6-
2.5)% High (2.5-4)%
Dm-2.5,
Dm-1.1, Dm-1.3
Dm-1.4, Dm-2.2,
Dm-2.5, Dm-2.7,
Dm-2.11
Dm-1.7, Dm-
2.9,
Bp-2.4,
Bp-1.1,Bp-1.12,
Bp-1.14, Bp-2.1,
Bp-2.6, Bp-2.8 ,
Bp-2.9
Bp-1.3, Bp-1.8,
Bp-1.10
NILL
Dk-1.2, Dk-2.3,
Dk-2.4, Dk-2.5,
Dk-2.8, Dk-1.3,
Dk-1.11, Dk-1.12,
Dk-2.2
Dk-2.1,Dk-2.2,
Dk-2.9,
NILL
Kp-1.1, Kp-1.8,
Kp-2.4, Kp-2.5,
Kp-2.7, Kp-2.9
Kp-1.2, Kp-
1.4, Kp-1.6,
Kp-1.7, Kp-
2.6, Kp-2.8.
Kp-1.10
Short listing of potential isolates
0
2
4
6
8
10
12
14
Kp-1.2 Kp-1.7 Kp-1.10 Dk-2.1 Dm-1.7 Dk-1.2
Co
nce
ntr
atio
n o
f fi
xed
at
mo
sph
eri
c n
itro
gen
(%)
Isolates of PGPR
Non saline
saline
Nitrogen fixing ability
33
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
kp-1.2 kp-1.7 kp-1.10 Dk-2.1 Bp-1.8 Dm-1.7
IAA
co
nc.
(µ
g/m
L)
PGPR isolates
IAA concentration(µg/mL) non saline
IAA concentration(µg/mL) saline
IAA production
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
kp-1.2 kp-1.7 kp-1.10 Dk-2.1 Bp-1.8 Dm-1.7
solu
bili
zed
ph
osp
hat
e co
nc.
(µ
g/µ
L)
Strains of PGPR
normal condition
saline condition
Phosphate Solubilization
0
10
20
30
40
50
60
70
80
90
Kp-1.2 Dk-2.1 Bp-1.8 E.coli
sid
ero
ph
ore
un
it (
%)
PGPR strains
Minimal media
Minimal media+ 200 mMsalt
Siderophore production
Seeds sample Germinating seeds After 4 days of germination
Germinating seeds after 12 days
0
2
4
6
8
10
12
None E.coli Kp-1.2 Kp-1.7
IAA
co
nce
ntr
atio
n((
µg/
g)
stem normal condition
stem saline condition
leaf normal condition
leaf saline condition
Bar diagram representing the IAA
concentration of plants (stem and leaf) normal
and saline condition.
IAA concentration in plants
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
None E.coli kp-1.2 kp-1.7
chlo
rop
hyl
co
nc.
(µg/
g)
stem normal condition
stem saline condition
leaf normal condition
leaf saline condition
Chlorophyll conc.
Bar diagram represent the chlorophyll concentration of
plants (stem and leaf) normal condition and saline
condition.
0
5
10
15
20
25
30
None E.coli Kp-1.2 Kp-1.7
carb
oh
ydra
te c
on
c (µ
M/g
)
stem normal condition
stem saline condition
leaf normal condition
leaf saline condition
Carbohydrate concentration
Bar diagram representing the carbohydrate concentration of plants (stem and leaf) under saline and
normal condition.
DO THE SALT RESISTANCE OF BACTERIA PROVIDE FURTHER RESISTANCE TO OTHER AGENTS?
Drug resistance
Heavy metal resistance
39
Antibiotics used
40
• (AMP) - Ampicillin 10µg,
• (AMK) - Amoxicillin with clavulanic acid
• (ATM) - Aztreonam 30µg
• (AZM)- Azithromycin 15µg,
• (C) - Chloramphenicol 30µg
• (CAZ) - Ceftazidime 30µg
• (CIP) - Ciprofloxacin 5µg
• (CN) - Gentamycin 120µg
• (CTX) - Cefotaxime 30µg
• (E) - Erythromycin 15µg
• (F) - Nitrofurantoin 300µg
• (FEP) - Cefepime 30µg
• (IPM) - Imipenem 10µg
• (K) - Kanamycin 30µg
• (OT) - Oxytetracycline 30µg
• (OX) - Oxacilin 1µg
• (P) - Penicilin G 10µg
• (RD) - Rifampicin 5µg
List of drugs used
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
KP-1.2 KP-1.7 BP-1.10 BP-2.8 DK2.2 DM-1.7 CL-1.2 CL-1.5 CL-2.5
Dru
g S
ensi
tiv
ity
Non-saline isolates
Sensitive
Resistant
Saline isolates
Antibiogram profile of isolates
Results
84%
16%
Average antibiotic sensitivity of isolates from saline
zone
Average Resistant Average Sensitive
9%
91%
Average antibiotic sensitivity of isolates from non-saline
zone
Average Resistant Average Sensitive
Average antibiotic sensitivity
Results
43
0
0.1
0.2
0.3
0.4
0.5
0.6
0 5 10 15
0% 0.63%1.25% 2.50%5% 10%20%
OD
at
600 n
m
Time (hour)
0
0.1
0.2
0.3
0.4
0.5
0.6
0 5 10 15
0% 0.63% 1.25%
2.50% 5% 10%
20%
OD
at
600 n
m
0
0.1
0.2
0.3
0.4
0.5
0.6
0 10 20
0% 0.63%1.25% 2.50%5% 10%20%
O
D a
t 600 n
m
Time (hour)
0
0.1
0.2
0.3
0.4
0.5
0.6
0 10 20
0% 0.63% 1.25%
2.50% 5% 10%
20%
O
D a
t 600 n
m
Time
(hour)
E. coli DH5 α at
various conc of
nickel chloride
E. coli DH5 α at
various conc of
copper chloride
Isolate Dk-2.1 at
various conc of
nickel chloride
Isolate Dk-2.1 at
various conc of
copper chloride
EPS
Presence of exopolysaccharide in saline isolate (DK-1.6) (a), which is absent in non-saline isolate (CL-1.2) (b)
(a) (b)
Scanning Electron Microscopy
Results
MOLECULAR IDENTIFICATION OF PGPR
Phylogenetic tree for the isolates based on 16S rDNA sequencing
46
16S rDNA electrophoresis
Strain Identification
47
Isolate ID Sequence
length Close similarity to
% identity (with accession numbers)
Kp 1.2 1384 Bacillus aryabhattai 100% strain CI5 (KU681035.1)
Kp 1.7 1355 Ochrobactrum intermedium 100% strain C13 (KT800883.1)
Kp 1.10 1365 Bacillus aryabhattai 100%
strain CI5 (KU681035.1)
Dk 2.1 1407 Bacillus megaterium 100% strain L43 (KU179346.1)
Dm 1.7 1354 Bacillus subtilis 100% strain SAN15 (KX098457.1)
Application of suitable PGPR could induce plant growth even under salinity stress conditions, as a result of plant-microbe interaction.
Genotypically diverse groups of PGPR species are distributed in the agricultural lands of Bangladesh.
We propose stimulation by PGPR as a mechanism of inducing salt tolerance in rice by modulating differential transcription in a set of salt-tolerant genes.
When compared to their non-saline counterparts, the PGPR isolates from saline zones have distinction of higher salt tolerance,
possess drug and heavy-metal resistance,
Such a resistance could be attributed to their EPS & biofilm formation
Conclusion
Conclusions
Model interpreted • Based on the IPCC
AR4 projections, the worst future scenario (March 2050)
• Creating bio-bank composed of suitable AIMO could be a novel entrepreneurship taken forward for combating CC effects.
Concluding remarks
Concluding
Remarks
Blood hemolysis characteristics of bacterial isolates under study
Strain ID Hemolysin
Kp-1.2 γ hemolysin
Kp-1.7 α hemolysin
Kp-1.10 γ hemolysin
Dk-2.1 γ hemolysin
Bp-1.8 γ hemolysin
Dm-1.7 α hemolysin
Siderophore details info…
4 mm
E.Coli DH5α
7 mm 5 mm
CAS solution Kp-1.2 Kp-1.2 Salt
Dk-2.1 Dk-2.1 Salt
Bp-1.8 Bp-1.8 salt
E.coli
CAS liquid assay :
Strain ID Relative absorbance of CAS solution at 630 nm
(Ar)
Absorbance of culture supernatant
+ CAS solution at 630 nm
(As)
Siderophore unit (%)
Ar-As = ×100 Ar
Kp-1.2 0.2 0.045 77.5
Kp-1.2 (200mM salt) 0.16 20
Dk-2.1 0.078 61
Dk-2.1(200 mM salt) 0.18 10
Bp-1.2 0.13 50
Bp-1.2(200 mM salt) 0.2 0
E. coli 0.06 70
E.Coli(200 mM salt) 0.21 0
Siderophore unit produced by selected PGPR
Effect of Biofertilizer component on PGPR growth:
To observe this…
Selected PGPR (kp-1.2,Kp-1.7 with E.coli DH5α as control) were grown in 5 ml nutrient
broth with different combinatorial media composition
• Calcium carbonate =25%,
• Charcoal powder = 35%,
• Gum= 10%,
• Calcium carbonate + Charcoal powder = 13%+17%,
• Calcium carbonate + Gum = 13%+ 5%,
• Charcoal powder + Gum= 17%+ 5%
• CaCO3+ Cp+ Gum = 8%+ 12%+ 4%
After 24 hr of incubation period
Cfu/mL was measured through drop plate count method
Component E. coli DH5α ( cfu/ml)
KP-1.7 (cfu/ml) Kp-1.2 (cfu/ml)
CaCO3 (Ca) 3×103 3×103 3×103
Gum (G) 1.3×106 1.2×106 1.7×106
Charcoal powder (CP)
4×104 9×104 8×104
Ca + G 2.1×106 2.4×106 2.6×106
Ca + CP 2.5×104 2.1×104 2.5×104
G + CP 1.8×106 1.8×106 2.1×106
Ca + CP + G 6.1×106 7.2×106 7.5×106
Nutrient broth 5.3 ×108 5.5×108 6.1×108
Normal saline 3×103 3×103 3×103
The components , in any combination, do not support the
growth of PGPR significantly and so they are not growth
additives for the PGPR
CULTURE & MICROSCOPY Short-listed isolates
60
Iso
late
s ID
Co
lon
y m
orp
ho
logy
Gra
m s
tain
ing Biochemical properties
Co
lor
Ap
pea
ran
ce
MR
VP
Mo
tilit
y
Nit
rate
re
du
ctio
n
Ind
ole
p
rod
uct
ion
Sucr
ose
fe
rmen
tati
on
Man
nit
ol
pro
du
ctio
n
Cat
alas
e
Oxi
das
e
Kp-1.2 Off white
Gummy + + - + + + + + + +
Kp-1.7 Transparent
Highly
gummy _ + - _ + + + _ + +
Kp-1.10 Off white
Gummy + + - + + + + + + +
Dk-2.1 White Gummy + + - + + + + + + +
Dm-1.7 Transparent
Gummy + + - _ + + + + + +
Dk-1.2 White Gummy _ + - + + + + + - +
• PGPR treatment, with or without salt gave similar expression for all tested genes as compared to control.
• We propose stimulation by osmoprotectant utilizing microbial population as a mechanism of inducing salt tolerance in rice by modulating differential transcription in a set of at least 14 genes.
• RNA was isolated from rice leaves after 25th day of salt stress in pot grown.
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