Jannatul Farthouse
MS Student
Reg. No. 08-05-2068
Department of Biotechnology
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Background and Justification
Materials and Methods
Results and Discussion
Conclusions
Recommendations
2
Background and Justification
Rice (Oryza sativa L) is the most important
cereal crop in the world, feeding more than
50% of the world’s Population.
It is the staple food of about 160 million
people of Bangladesh accounting for about
75 percent of agricultural land use.
3
Background and Justification continued
However, increased rice production results
in higher production cost and environment
degradation because of excessive use of
chemical fertilizers and pesticides which is
not sustainable.
Sustainable production of rice will hence
mean increasing the rice yield without the
mass use of chemical fertilizers and
pesticides.
4
Background and Justification continued
 Biofunctional endophytic bacteria live within plant
tissues and stimulate growth as well as protect host
plant through various direct or indirect mechanisms.
 Considered as one of the potential alternatives of
the hazardous chemicals for low input sustainable
agriculture.
The aim of my research was to isolate and
characterize
biofunctional
endophytic
bacteria and apply them as plant growth
promoter for low cost sustainable rice
production in Bangladesh.
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 Isolate biofunctional endophytic bacteria from
indigenous rice seeds.
 Screen bacterial isolates for various traits of plant
growth promotion including IAA production, and
antagonisms to the phytopathogens.
 Evaluate performances of some selected strains on
growth promotion and yield of rice.
 Identify potential strains using 16S rRNA gene
sequencing and submit their sequence data to the
GENBANK.
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Materials & Methods
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COLLECTION OF RICE SEEDS
Seeds of 48 indigenous/local rice varieties were
collected from UDB. Seeds of one high yielding
variety of rice was collected from BINA.
Nunia
UDB-Unnoyan Dhara, Bangladesh
Lalzira
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Steps of Isolation of bacteria
Surface sterilization of seeds sample
Crush seeds with mortar & pestle
Dilute the extract with SDW up to 1 × 10-6
Spread extract onto PDA & NBA plates
Observe & isolate distinct bacterial colony
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Isolation of bacteria
Single colony on NBA
Sub culture on NBA
Pure culture on NBA
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Biochemical Characterization
• KOH Test
• Gliding Motility test
• Catalase Test
• Gram staining test
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 Screening for phosphate solubilizing bacteria on agar assay
•
•
A
Point inoculation on NBRIP media
Incubated at 28˚C for 7 days
B
Phosphate Solubilizing Index (PSI) = A/B
A= Diameter (colony + halo)
B = Diameter of the colony
 Quantification of phosphate solubilizing activity
• 10 ml NBRIP broth inoculated with the bacteria
• Incubated for 2 d at 28˙C on a shaker at 180 rpm
• Phospho-molybdate blue complex colorimetric method
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Determination of Indole 3 Acetic
Acid (IAA) Production
 Yeast Mannitol Broth (YMB) - Bacterial isolates were
inoculated in YMB at 29 ± 20C for 5 days
 2 ml of culture solution was centrifuged at 7,000 rpm for 6
minute
 Salkowsky’s reagent -One ml of the supernatant was mixed
with 2 ml of Salkowsky’s reagent to observe color change
 IAA concentration produced by different bacteria was
determined by plotting on a standard graph
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Test pathogens
Phytophthora capsici
Source: Professor W. Yuanchao,
Nanjing Agricultural University,
China.
Sclerotium rolfsii
Source: Plant pathology laboratory
of BSMRAU, Bangladesh.
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X Y
% Inhibition of growth =
× 100
X
X
Y
Where,
X = Mycelial growth of pathogen in absence of antagonist
Y = Mycelial growth of pathogen in presence of antagonist
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In vivo tests of bacteria on promotion
and growth of plants
Application of bacteria on rice seeds
Seeds were coated with bacteria by overnight soaking into
bacterial suspension
Application of bacteria on rice roots
Roots of seedlings were coated with bacteria by overnight
soaking in bacterial suspension
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Molecular Identification of bacteria
1. Extraction of DNA and
polymerase chain reaction (PCR)
2. Purification of PCR product
3. Sequencing of 16S rRNA genes
and analysis of the sequence
data
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Polymerase Chain Reaction (PCR)
Harvest single bacterial colony from tryptic soy broth (TSB)
media
Re-suspend in 100 μl sterile distilled water
Vortex for 10 sec
Use one μl lysate in 50 μl bacterial suspension
Run in PCR thermocycler for polymerase chain reaction
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Primers used in this study to identify bacteria
Target gene
Primer
Primer sequence (5’-3’)
Length
16S
24F
AGA GTT TGA TCM TGG CT
17bp
520R
GCGGCTGCTGGCACGAAGTT
20bp
Thermal profile for PCR amplification of the target gene
Target gene
16S rRNA
Initial
30 cycles
Final
Denaturing
Denaturing
Annealing
Extension
extension
94℃, 5 min
94℃, 40s
55℃, 40s
72℃, 1min
72℃, 10 min
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60 Endophytic bacteria were isolated from 15 rice varieties
SL.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Name of rice variety
Lalzira
Nunia
Joyna
Chinikanai
Kartikbalam
Dursar
Kalozira
Gore Kajal
Sadamata
Malsira
Aijong
Bashmoti
Bashgari
Bhojan
BINA-14
No. of bacterial
isolates
6
3
1
1
5
5
6
8
4
3
2
2
7
3
6
Name of bacterial
isolates
BTLL1*- BTLL6
BTLN7- BTLN10
BTLJ11
BTLC12
BTLK13 - BTLK18
BTLD19 - BTLD23
BTLK24 - BTLK30
BTLG31- BTLG38
BTLS39 - BTLS42
BTLM43 - BTLM45
BTLA46& A47
BTLB48 & B49
BTLBg50 -1Bg51
BTL-Bj52, Bj53, Bj54
BNA**1- BNA6
Total 60
* BTL-Biotechnology; L-Variety Name; 1-no of bacteria isolated **BNA-BINA released variety
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Endophytic bacteria showed distinct color
and colony morphology in NBA medium
Distinct biochemical & morphological characters
of the isolated endophytic bacteria from rice
Bacterial
Strain
BTLL6
Colony Color
Morphological and Biochemical Character
Margin
KOH
Motility Test
Gram Staining
test
Catalase
Test
Creamy
white
Light Brown
Irregular
+
+++
Gram ‘-’ve
+
Irregular
+
-
Gram ‘-’ve
++
Round
Round
+
+
-
Gram ‘-’ve
Gram ‘-’ve
++
+
Irregular
-
+++
Gram ‘+’ve
+
BTLK17
Brown
Whitish
brown
Yellowish
brown
Off White
+
++
Gram ‘-’ve
+
BTLA46
Yellow
+
-
Gram ‘-’ve
+
BNA1
Off White
-
+++
Gram ‘+’ve
+
BNA3
Cream color
+
++
Gram ‘-’ve
+
BNA4
Yellow
Round,
Smooth
Round,
Smooth
Round,
Smooth
Round,
Smooth
Convex
-
++
Gram ‘+’ve
+
BTLN8
BTLN9
BTLN10
BTLK15
+++ = Highly positive, ++ = Moderately positive, + = Weakly Positive, - = Negative
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Diverse growth plant promoting activities of the isolated endophytes
Strain name PSB
IAA producing
bacteria
BTLL6
+
+
+
+
-
BTLN8
BTLN9
BTLN10
BTLK15
BTLK17
BTLA46
BNA1
BNA3
BNA4
+
+
+
+
+
-
+ = Active, - = Inactive
Antagonistic Activity
Sclerotium sp. Phytophthora capsici
+
+
+
+
-
+
+
+
+
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Characteristics halo zones generated by bacterial isolates
around the colonies in the NBRIP media
BTLL6
BNA3
BTLK15
BTLN9
BTLN10
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5 endophytic bacteria displayed varying levels of
phosphate solubilization in the NBRIP medium
Phosphate solubilizing Index(PSI)
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
BTLK15
BNA3
BTLN10
BTLN9
BTLL6
Name of bacterial isolates
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Varying levels (15.42-40.98 mg/ml) of tricalcium
phosphate were solubilized by the endophytic
bacteria in the NBRIP broth assay
Phosphate Solubilization in broth
assay(μg/ml)
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40.98
40
33.35
35
30
25
19.76
20
16.95
15.42
BTLN9
BTLL6
15
10
5
0
BTLK15
BNA3
BTLN10
Name of bacterial isolates
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Variations in color from pink to dark pink
indicates different level of Indole 3 Acetic Acid
(IAA) produced by the endophytic bacteria
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Endophytic bacteria suppressed varying
levels of hyphal growth of Sclerotium rolfsii
BTLK15
BTLL6
BNA3
BNA1
Control
Interestingly, inhibition of mycelial growth by the endophytic
bacteria is linked to the characteristic morphological alterations
in the hyphae of S. rolfsii approaching to the bacterial colony 30
Endophytic bacteria suppress varying level of
hyphal growth of P. capcisi
Micrograph showing mycelial alternation due to In vitro interactions between endophytic
bacteria and P.capsici
Rewrite text similarly to the previous slide?
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Enhancement of shoot and root dry weight of rice
seedlings by the endophytic bacteria in Petri dish assay
Name of bacterial isolates
Shoot Dry weight(mg)
Root dry wt.(mg)
BTLN8
5.73 ± 0.92a
2.23 ± 0.12a
BTLN9
2.30 ± 0.08c
1.76 ± 0.07b
BTLN10
5.00 ± 0.26b
1.03 ± 0.05c
BDR2
1.73 ± 0.09cd
0.90 ± 0.08c
BTL46
1.83 ± 0.04cd
0.80 ± 0.10c
BTLL6
2.10 ± 0.02cd
0.73 ± 0.13c
BTLK15
1.70 ± 0.01e
0.20 ± 0.02d
Control
1.16 ± 0.07f
0.80 ± 0.01c
BDR2-Bacillus amyloliquefaciens
Data presented here are the Mean ± SE. Mean values in each column with the same
superscript(s) differ significantly by DMRT (p≤0.05). The data presented are from
representative experiments that were repeated at least twice
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Endophytic bacteria promoted root and shoot growth of rice seedling (at day 15)
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 Endophytic bacteria significantly increased shoot dry weight
of rice compared to control with few exceptions.
 BTLN8 gave highest shoot dry weight (26.03 g) in half dose of
fertilizers, which is equivalent to full dose of fertilizer in control.
Treatment
BTLL6
BTLN8
BTLN9
BTLN10
BTLK15
BTLK17
BTLA46
Burkholderia cepacia
strain ST10
Bacillus subtilis Strain
BRtL2
Control
Full dose
24.03 ± 0.76h
37.15 ± 0.63a
35.03 ± 0.80c
29.96 ± 0.42e
26.50 ± 0.21g
26.90 ± 0.22f
24.10 ± 0.21h
36.03 ± 0.26b
Shoot dry weight (g)
Half dose
Zero dose
18.50 ± 0.34f
8.10 ± 0.32e
23.30 ± 0.03b
11.71 ± 0.46a
26.03 ± 0.21a
10.03 ± 0.87c
19.03 ± 1.2d
11.03 ± 0.61b
15.03 ± 0.69h
10.96 ± 0.53b
21.00 ± 0.35c
10.20 ± 0.54c
17.03 ± 0.45g
9.30 ± 0 .76d
20.03 ± 0.97d
7.63 ± 0.287e
34.03 ± 0.78d
18.46 ± .63f
12.11 ± 0.83a
27.03 ± 0.23f
17.00 ± 1.9g
9.00 ± 0.80d
Data presented here are the Mean ± SE. Mean values in each column with the
same superscript(s) differ significantly by DMRT (p≤0.05).
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Effect ofCendophytic bacteria on shoot growth & tillering of rice plant (Full dose fertilizer treatment;
Half dose fertilizer treatment; & Zero dose fertilizer treatment respectively). Scale bars = 1cm. T8Burkholderia cepacia strain ST10, T9- Bacillus subtilis strain BRtL2
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 Root dry weight of rice also significantly increased by the
application of endophytic bacteria.
 BTLN9 gave highest root dry matter in both half and full doses of
fertilizers.
Treatment
Full dose
Root weight (g)
Half dose
Zero dose
BTLL6
BTLN8
19.76 ± 1.48d
24.93 ± 0.45b
14.33 ± 0.020e
17.10 ± 2.00b
7.50 ± 0.09c
10.63 ± 0.07a
BTLN9
BTLN10
BTLK15
BTLK17
BTLA46
Burkholderia cepacia
strain ST10
Bacillus subtilis Strain
BRtL2
Control
27.13 ± 1.91a
24.14 ± 2.67c
17.04 ± 0.50g
17.25 ± 0.34g
17.96 ± 0.08f
18.07 ± 0.47f
18.10 ± 0.02a
15.00 ± 0.86d
11.03 ± 3.02 f
15.03 ± 0.12d
16.03 ± 0.140c
17.03 ± 0.04b
8.30 ± 0.14b
7.00 ± 0.13d
6.03 ± 0.04f
8.38 ± 0.05b
8.03 ± 0.86b
7.21 ± 0.56cd
18.96 ± 0.02e
18.15 ± 0.20a
7.21 ± 2.24cd
18.22 ± 0.27f
14.83 ± 0.87d
7.00 ± 1.34d
Data presented here are the Mean ± SE. Mean values in each column with the same
superscript(s) differ significantly by DMRT (p≤0.05).
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Full
dose
Half
dose
Zero
dose
Effect of endophytic bacteria on root growth of rice plant (Full dose frtilizer t eatment; Half dose
fertilizer treatment & Zero dose fertilizer treatment respectively). Scale bars = 1 cm.
T8- Burkholderia cepacia strain ST10, T9- Bacillus subtilis Strain BRtL2
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Application of some endophytic bacteria
significantly increased grain yield of rice.
Tratments
Total grain yield per pot (g)
Full dose
Half dose
Zero dose
BTLL6
BTLN8
BTLN9
BTLN10
BTLK15
BTLK17
BTLA46
Burkholderia cepacia
strain ST10
31.35 ± 0.50e
30.28 ± 0.5f
33.43 ± 0.29b
28.50 ± 1.65g
27.21± 1.71h
32.40 ± 0.79c
31.80 ± 0.17d
28.42 ± 2.95g
18.89 ± 0.15i
28.51 ± 1.49c
27.60 ± 2.50d
27.3 ± 0.50d
25.31 ± 0.0f
26.23 ± 0.06e
30.92 ± 2.31a
24.37 ± 0.33g
7.5733 ± 0.97g
9.38 ± 2.3d
11.81 0.16a
10.31± 2.61c
7.783 ± 0.72f
11.80 ± 1.16a
11.24 ± 1.73b
7.92 ± 0.85f
Bacillus subtilis Strain
BRtL2
Control
34.34 ± 0.76a
30.07 ± 1.07b
9.18 ± 0.92d
28.85 ± 0.87f
19.25 ± 0.16h
8.280 ± 0.47e
Data presented here are the Mean ± SE. Mean values in each column with the same
superscript(s) differ significantly by DMRT (p≤0.05).
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Molecular Identity of reference Bacterial Isolates
16S rRNA gene sequence similarity between bacterial isolates and
the closest strains of valid described species deposited in Genbank
Isolate
Closest strains from Similarity Acc. no. of
SL no.
no.
GeneBank
%
closest hit
BDR2
Bacillus
amyloliquefaciens
100
N/A
2.
ST10
Burkholderia cepacia
strain 4APE
99.00
KF921289
3.
BRtL2
Bacillus subtilis
99.00
N/A
1.
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Gel electrophoretic photograph of the PCR products
of the endophytic bacteria showing DNA bands
Ladder
BTLN9
BTLN10
BTLN6
BTLK15
BNA 1
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Conclusions
Total 60 endophytic bacterial isolated were isolated
and characterized from 15 rice varieties.
Five bacterial isolates, BTLL6, BTLN9, BTLN10,
BTLK15 and BNA3 solubilized insoluable
phosphates in both agar and NBRIP broth assays.
Four antagonistic bacterial strains (BTKL6.
BTLK15, BNA1 and BNA4) inhibited mycelial
growth of P. capsici through characteristic
morphological alterations in the approaching
hyphae
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Conclusions (cont.)
 Interestingly, 4 bacteria (BTLL6, BTLK15, BNA1 and
BN3) remarkably inhibited growth of Sclerotium rolfsii
and also induced characteristic morphological alterations.
 Four endophytic bacteria (BTLN8, BTLN9, BTLN10 and
BTLK17) produced higher amounts of IAA.
 BTLN8, BTLN9, BTLN10, BTLK15, BTL46, BTLL6 and
a reference strain Bacillus amyloliquefaciens BDR2
enhanced germination along with accumulation of higher
amount of dry matter production of rice seedlings in
laboratory assay.
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Conclusions (cont.)
 BTLN8, BTLN9, BTLN10, BTLK15, BTL46, BTLL6,
BTLA46 and two reference strains Burkholderia cepacia
strain ST10 and Bacillus subtilis strain BRtL2 remarkably
increased root length, shoot length, No. of tillers, dry matter
production and grain yield of rice in pot culture.
 Extractions of genomic DNA and PCR of five potential
isolates BTLN9, BTLN10, BTLK15 and BNA1 were done
for molecular identification through 16S rRNA gene
sequencing.
 This is the first report on isolation and characterization of
multifunctional plant growth promoting endophytic
bacteria from some local/indegenous rice varieties of
Bangladesh
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Recommendations
 Isolated bacteria have high potentials for enhancing
growth and yield of rice and they may be used as
biofertilizer and biofungicides as the alternative
hazardous synthetic chemicals which are commonly
used in conventional agriculture.
 Further study is needed for elucidation of the mode of
actions of these novel bacteria.
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Acknowledgements
 Dr. Md. Tofazzal Islam, Professor, Department of Biotechnology
 Dr. Abdul Mannan Akanda, Professor, Department of Plant Pathology
 Dr.Ashraful Haque, Associate Professor, Department of Biotechnology
 Dr. Mahbubur Rahman, Associate Professor, Department of Biotechnology
 Dr. Dipali Rani Gupta,Assistant Professor, Department of Biotechnology
 Professor W. Yuanchao, Department of Plant Pathology, Nanjing Agricultural University,
China
 Kbd. Wahed Mallik, Farm Manager, BSMRAU
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