Discovery of Biofunctional Endophytic Bacteria from Rice

1

Discovery of Biofunctional Endophytic Bacteria from Rice

Jannatul FarthouseMS Student

Reg. No. 08-05-2068Department of Biotechnology

MS THESIS PRESENTATION

2

• Background and Justification• Materials and Methods• Results and Discussion• Conclusions• Recommendations

OUTLINE OF THE PRESENTATION

3

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.

Background and Justification

4

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.

Background and Justification continued

5

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.

Background and Justification continued

6

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.

Objectives

7

Materials & Methods

8

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 Lalzira

UDB-Unnoyan Dhara, Bangladesh

9

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

10

Isolation of bacteria

Single colony on NBA Sub culture on NBA Pure culture on NBA

11

Biochemical Characterization • KOH Test

• Gliding Motility test

• Catalase Test

• Gram staining test

12

Screening for phosphate solubilizing bacteria on agar assay

• Point inoculation on NBRIP media• Incubated at 28˚C for 7 days

Phosphate Solubilizing Index (PSI) = A/BA= Diameter (colony + halo) B = Diameter of the colony

A

B

Phosphate solubilization activity

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

13

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

Determination of Indole 3 Acetic Acid (IAA) Production

IAA concentration produced by different bacteria was determined by plotting on a standard graph

14

Bioassays

15

Phytophthora capsiciSource: Professor W. Yuanchao, Nanjing Agricultural University, China.

Sclerotium rolfsiiSource: Plant pathology laboratory of BSMRAU, Bangladesh.

Test pathogens

16

% Inhibition of growth = X

YX

Where, X = Mycelial growth of pathogen in absence of antagonist Y = Mycelial growth of pathogen in presence of antagonist

× 100

Mycelial growth inhibition in dual culture assay

X

Y

17

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

Molecular Identification of bacteria

18

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

Polymerase Chain Reaction (PCR)

19

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

Primers used in this study to identify bacteria

20

Target gene Primer Primer sequence (5’-3’) Length

16S 24F

520R

AGA GTT TGA TCM TGG CT

GCGGCTGCTGGCACGAAGTT

17bp

20bp

Target gene Initial

Denaturing

30 cycles Final

extensionDenaturing Annealing Extension

16S rRNA 94 , 5 min℃ 94 , 40s℃ 55 , 40s℃ 72 , 1min℃ 72 , 10 ℃min

Thermal profile for PCR amplification of the target gene

21

Results & Discussion

22* BTL-Biotechnology; L-Variety Name; 1-no of bacteria isolated **BNA-BINA released variety

SL. No.

Name of rice variety No. of bacterial isolates

Name of bacterial isolates

1 Lalzira 6 BTLL1*- BTLL62 Nunia 3 BTLN7- BTLN103 Joyna 1 BTLJ114 Chinikanai 1 BTLC125 Kartikbalam 5 BTLK13 - BTLK186 Dursar 5 BTLD19 - BTLD237 Kalozira 6 BTLK24 - BTLK308 Gore Kajal 8 BTLG31- BTLG389 Sadamata 4 BTLS39 - BTLS4210 Malsira 3 BTLM43 - BTLM4511 Aijong 2 BTLA46& A4712 Bashmoti 2 BTLB48 & B4913 Bashgari 7 BTLBg50 -1Bg5114 Bhojan 3 BTL-Bj52, Bj53, Bj5415 BINA-14 6 BNA**1- BNA6

Total 60

60 Endophytic bacteria were isolated from 15 rice varieties

Endophytic bacteria showed distinct color and colony morphology in NBA medium

24

Bacterial Strain

Morphological and Biochemical CharacterColony Color Margin KOH

testMotility Test Gram Staining Catalase

Test

BTLL6 Creamy white

Irregular + +++ Gram ‘-’ve +

BTLN8 Light Brown Irregular + - Gram ‘-’ve ++

BTLN9 Brown Round + - Gram ‘-’ve ++BTLN10 Whitish

brownRound + - Gram ‘-’ve +

BTLK15 Yellowish brown

Irregular - +++ Gram ‘+’ve +

BTLK17 Off White Round, Smooth

+ ++ Gram ‘-’ve +

BTLA46 Yellow Round, Smooth

+ - Gram ‘-’ve +

BNA1 Off White Round, Smooth

- +++ Gram ‘+’ve +

BNA3 Cream color Round, Smooth

+ ++ Gram ‘-’ve +

BNA4 Yellow Convex - ++ Gram ‘+’ve +

Distinct biochemical & morphological characters of the isolated endophytic bacteria from rice

+++ = Highly positive, ++ = Moderately positive, + = Weakly Positive, - = Negative

25

Strain name PSB IAA producing bacteria

Antagonistic ActivitySclerotium sp. Phytophthora capsici

BTLL6 + - + +BTLN8 - + - -BTLN9 + + - -BTLN10 + + - -BTLK15 + - + +BTLK17 - + - -BTLA46 - - - -BNA1 - - + +BNA3 + - + -BNA4 - - - +

+ = Active, - = Inactive

Diverse growth plant promoting activities of the isolated endophytes

26

Characteristics halo zones generated by bacterial isolates around the colonies in the NBRIP media

BTLL6 BTLK15 BNA3

BTLN9 BTLN10

27

5 endophytic bacteria displayed varying levels of phosphate solubilization in the NBRIP

medium

BTLK15 BNA3 BTLN10 BTLN9 BTLL60

0.5

1

1.5

2

2.5

3

3.5

4


Phos

phat

e so

lubi

lizin

g In

dex(

PSI)

28

BTLK15 BNA3 BTLN10 BTLN9 BTLL60

5

10

15

20

25

30

35

40

45


Phos

phat

e So

lubi

lizat

ion

in b

roth

as

say(

μg/m

l)

Varying levels (15.42-40.98 mg/ml) of tricalcium phosphate were solubilized by the endophytic bacteria in the NBRIP broth assay

40.98

33.35

19.7616.95 15.42

29

Variations in color from pink to dark pink indicates different level of Indole 3 Acetic Acid (IAA) produced by the endophytic bacteria

30

Endophytic bacteria suppressed varying levels of hyphal growth of Sclerotium rolfsii

BTLL6 BTLK15 BNA1

BNA3 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

31

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?

32

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 amyloliquefaciensData 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

33Endophytic bacteria promoted root and shoot growth of rice seedling (at day 15)

34

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 Shoot dry weight (g)Full dose Half dose Zero dose

BTLL6 24.03 ± 0.76h 18.50 ± 0.34f 8.10 ± 0.32eBTLN8 37.15 ± 0.63a 23.30 ± 0.03b 11.71 ± 0.46aBTLN9 35.03 ± 0.80c 26.03 ± 0.21a 10.03 ± 0.87cBTLN10 29.96 ± 0.42e 19.03 ± 1.2d 11.03 ± 0.61bBTLK15 26.50 ± 0.21g 15.03 ± 0.69h 10.96 ± 0.53bBTLK17 26.90 ± 0.22f 21.00 ± 0.35c 10.20 ± 0.54cBTLA46 24.10 ± 0.21h 17.03 ± 0.45g 9.30 ± 0 .76dBurkholderia cepacia strain ST10

36.03 ± 0.26b 20.03 ± 0.97d 7.63 ± 0.287e

Bacillus subtilis Strain BRtL2

34.03 ± 0.78d 18.46 ± .63f 12.11 ± 0.83a

Control 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).

35

CEffect of endophytic bacteria on shoot growth & tillering of rice plant (Full dose fertilizer treatment; Half dose fertilizer treatment; & Zero dose fertilizer treatment respectively). Scale bars = 1cm. T8- Burkholderia cepacia strain ST10, T9- Bacillus subtilis strain BRtL2

36

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 Root weight (g)Full dose Half dose Zero dose

BTLL6 19.76 ± 1.48d 14.33 ± 0.020e 7.50 ± 0.09cBTLN8 24.93 ± 0.45b 17.10 ± 2.00b 10.63 ± 0.07a

BTLN9 27.13 ± 1.91a 18.10 ± 0.02a 8.30 ± 0.14bBTLN10 24.14 ± 2.67c 15.00 ± 0.86d 7.00 ± 0.13dBTLK15 17.04 ± 0.50g 11.03 ± 3.02 f 6.03 ± 0.04fBTLK17 17.25 ± 0.34g 15.03 ± 0.12d 8.38 ± 0.05bBTLA46 17.96 ± 0.08f 16.03 ± 0.140c 8.03 ± 0.86bBurkholderia cepacia strain ST10

18.07 ± 0.47f 17.03 ± 0.04b 7.21 ± 0.56cd


18.96 ± 0.02e 18.15 ± 0.20a 7.21 ± 2.24cd

Control 18.22 ± 0.27f 14.83 ± 0.87d 7.00 ± 1.34d


37

Full

dose

Half 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

Zero dose

38

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 31.35 ± 0.50e 18.89 ± 0.15i 7.5733 ± 0.97gBTLN8 30.28 ± 0.5f 28.51 ± 1.49c 9.38 ± 2.3dBTLN9 33.43 ± 0.29b 27.60 ± 2.50d 11.81 0.16aBTLN10 28.50 ± 1.65g 27.3 ± 0.50d 10.31± 2.61cBTLK15 27.21± 1.71h 25.31 ± 0.0f 7.783 ± 0.72fBTLK17 32.40 ± 0.79c 26.23 ± 0.06e 11.80 ± 1.16aBTLA46 31.80 ± 0.17d 30.92 ± 2.31a 11.24 ± 1.73bBurkholderia cepacia strain ST10

28.42 ± 2.95g 24.37 ± 0.33g 7.92 ± 0.85f


34.34 ± 0.76a 30.07 ± 1.07b 9.18 ± 0.92d

Control 28.85 ± 0.87f 19.25 ± 0.16h 8.280 ± 0.47e


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

SL no.Isolate

no.

Closest strains from

GeneBank

Similarity

%

Acc. no. of

closest hit

1. BDR2Bacillus

amyloliquefaciens 100 N/A

2. ST10Burkholderia cepacia strain 4APE

99.00 KF921289

3. BRtL2 Bacillus subtilis 99.00 N/A39

40

Gel electrophoretic photograph of the PCR products of the endophytic bacteria showing DNA bands

Ladder

BTLN9

BTLN10

BTLN6

BTLK15

BNA 1

41

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

42

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.

Conclusions (cont.)

43

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

Conclusions (cont.)

44

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.

45

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

Acknowledgements

Thank You

Documents

Discovery of Biofunctional Endophytic Bacteria from Rice