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Discovery of Biofunctional Endophytic Bacteria from Rice

MS THESIS PRESENTATION. Discovery of Biofunctional Endophytic Bacteria from Rice. Jannatul Farthouse MS Student Reg. No . 08-05-2068 Department of Biotechnology. Outline Of the Presentation. Background and J ustification Materials and Methods Results and Discussion Conclusions

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Discovery of Biofunctional Endophytic Bacteria from Rice

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  1. MS THESIS PRESENTATION Discovery of BiofunctionalEndophytic Bacteria from Rice JannatulFarthouse MS Student Reg. No. 08-05-2068 Department of Biotechnology

  2. Outline Of the Presentation • Background and Justification • Materials and Methods • Results and Discussion • Conclusions • Recommendations

  3. 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.

  4. 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.

  5. Background and Justification continued • Biofunctionalendophytic 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 biofunctionalendophytic bacteria and apply them as plant growth promoter for low cost sustainable rice production in Bangladesh.

  6. Objectives • 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 tothe • GENBANK.

  7. Materials & Methods

  8. Seeds of 48 indigenous/local rice varieties were collected from UDB. Seeds of one high yielding variety of rice was collected from BINA. Collection of RICE Seeds Lalzira Nunia UDB-UnnoyanDhara, 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. Phosphate solubilizationactivity • 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/B • A= Diameter (colony + halo) • B = Diameter of the colony • Quantification of phosphate solubilizingactivity • 10 ml NBRIP broth inoculated with the bacteria • Incubated for 2 d at 28˙C on a shaker at 180 rpm • Phospho-molybdateblue complex colorimetric method

  13. 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’sreagent -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

  14. Bioassays

  15. Test pathogens Phytophthora capsici Source:Professor W. Yuanchao, Nanjing Agricultural University, China. Sclerotium rolfsii Source: Plant pathology laboratory of BSMRAU, Bangladesh.

  16. Mycelial growth inhibition in dual culture assay • × 100 % Inhibition of growth = X Y Where, X = Mycelial growth of pathogen in absence of antagonist Y = Mycelial growth of pathogen in presence of antagonist

  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

  18. Molecular Identification of bacteria Extraction of DNA and polymerase chain reaction (PCR) Purification of PCR product Sequencing of 16S rRNA genes and analysis of the sequence data

  19. 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 μllysate in 50 μl bacterial suspension Run in PCR thermocycler for polymerase chain reaction

  20. Primers used in this study to identify bacteria Thermal profile for PCR amplification of the target gene

  21. Results & Discussion

  22. 60 Endophytic bacteria were isolated from 15 rice varieties * BTL-Biotechnology; L-Variety Name; 1-no of bacteria isolated **BNA-BINA released variety

  23. Endophyticbacteria showed distinct color and colony morphology in NBA medium

  24. Distinct biochemical & morphological characters of the isolated endophytic bacteria from rice +++ = Highly positive, ++ = Moderately positive, + = Weakly Positive, - = Negative

  25. Diverse growth plant promoting activities of the isolated endophytes + = Active, - = Inactive

  26. Characteristics halo zones generated by bacterial isolates around the colonies in the NBRIP media BNA3 BTLK15 BTLL6 BTLN10 BTLN9

  27. 5 endophytic bacteria displayed varying levels of phosphate solubilization in the NBRIP medium

  28. Varying levels (15.42-40.98 mg/ml) of tricalcium phosphate were solubilizedby the endophyticbacteria in the NBRIP broth assay 40.98 33.35 19.76 16.95 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 Sclerotiumrolfsii BTLK15 BNA1 BTLL6 BNA3 Control Interestingly, inhibition of mycelial growth by the endophytic bacteria is linked to the characteristic morphological alterations in the hyphae of S. rolfsiiapproaching 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 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

  33. Endophyticbacteria 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. 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. Effect 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 cepaciastrain ST10, T9-Bacillus subtilis strain BRtL2 C

  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. Data presented here are the Mean ± SE. Mean values in each column with the same superscript(s) differ significantly by DMRT (p≤0.05).

  37. 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 cepaciastrain ST10, T9- Bacillus subtilisStrain BRtL2

  38. Application of some endophytic bacteria significantly increased grain yield of rice. Data presented here are the Mean ± SE. Mean values in each column with the same superscript(s) differ significantly by DMRT (p≤0.05).

  39. 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

  40. Gel electrophoretic photograph of the PCR products of the endophyticbacteria showing DNA bands Ladder BTLN9 BTLN10 BTLK15 BTLN6 BNA 1

  41. Conclusions • Total 60 endophyticbacterial isolated were isolated and characterized from 15 rice varieties. • Five bacterial isolates, BTLL6, BTLN9, BTLN10, BTLK15 and BNA3 solubilizedinsoluablephosphates in both agar and NBRIP broth assays. • Four antagonistic bacterial strains (BTKL6. BTLK15, BNA1 and BNA4) inhibited mycelial growth of P. capsicithroughcharacteristic morphological alterations in the approaching hyphae

  42. Conclusions (cont.) • Interestingly, 4 bacteria (BTLL6, BTLK15, BNA1 and BN3) remarkably inhibited growth of Sclerotiumrolfsiiand 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 amyloliquefaciensBDR2 enhanced germination along with accumulation of higher amount of dry matter production of rice seedlings in laboratory assay.

  43. Conclusions (cont.) • BTLN8, BTLN9, BTLN10, BTLK15, BTL46, BTLL6, BTLA46 and two reference strains Burkholderia cepaciastrain 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 rRNAgene 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

  44. Recommendations • Isolated bacteria have high potentials for enhancing growth and yield of rice and they may be used as biofertilizer and biofungicidesas 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. Acknowledgements • Dr. Md. Tofazzal Islam, Professor, Department of Biotechnology • Dr. Abdul MannanAkanda, Professor, Department of Plant Pathology • Dr.AshrafulHaque, Associate Professor, Department of Biotechnology • Dr. Mahbubur Rahman, Associate Professor, Department of Biotechnology • Dr. DipaliRani Gupta,Assistant Professor, Department of Biotechnology • Professor W. Yuanchao, Department of Plant Pathology, Nanjing Agricultural University, China • Kbd. WahedMallik, Farm Manager, BSMRAU

  46. Thank You

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