a novel bacillus aryabhattai ms3 promotes growth...

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

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n

Cat

alas

e

Oxi

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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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