Identification and sequence analysis of partial 16s rrna of endophytic bacteria isolated from potato root

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS SUBJECT: “IDENTIFICATION AND SEQUENCE ANALYSIS OF PARTIAL 16S rRNA OF ENDOPHYTIC BACTERIA ISOLATED FROM POTATO ROOT” Hanoi - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS SUBJECT: “IDENTIFICATION AND SEQUENCE ANALYSIS OF PARTIAL 16S rRNA OF ENDOPHYTIC BACTERIA ISOLATED FROM POTATO ROOT” Student: NGUYEN THI NGOC ANH Student’s Code: 620514 Class: K62CNSHE Department: PLANT BIOTECHNOLOGY Supervisor: Ph.D DINH TRUONG SON Hanoi – 2022 COMMITMENT I guarantee that the graduate thesis: “Identification and sequenceanalysis of partial 16S rRNA of endophytic bacteria isolated from potato root” is my own research wich is done with the instruction of Mr Dinh Truong Son, PhD The parts where I use references in the thesis have been mentioned in the references section The data and results presented in the thesis are completely honest If wrong, I will take responsibility and all the discipline that the faculty and the academy offer Hanoi, September 11, 2022 Student Nguyen Thi Ngoc Anh i ACKNOWLEDGEMENT In order to complete this graduation thesis, in addition to my own efforts, i would like to express my sincere thanks to: Thanks to the Vietnam National University of Agriculture for supporting me about facilities with modern library system, diverse types of documents that are convenient for my research I would like to express my deepest gratitude to my instructor, Mr Dinh Truong Son, whose sincerity and encouragement I will never forget This thesis would not have been possible without Mr Dinh Truong Son, whose guidance from the initial step in research enabled me to develop an understanding of the subject I am thankful for the extraordinary experiences he arranged for me It is an honor to learn from Mr Dinh Truong Son I would also like to thank some students at the Department of Plant Biotechnology, Faculty of Biotechnology, Vietnam National University of Agriculture for creating favorable conditions for me to complete this thesis Finally, i look forward to receiving comments and suggestions from the teachers to make my graduation thesis more complete Hanoi, September 11, 2022 Student Nguyen Thi Ngoc Anh ii INDEX COMMITMENT i ACKNOWLEDGEMENT ii INDEX iii LIST OF ABBREVIATIONS vi LIST OF TABLES vii LIST OF FIGURES viii PART I: INTRODUCTION 1.1 Rationale of the Study 1.2 Research Objectives 1.3 Research requirements PART II: LITERATURE REVIEW 2.1 General introduction of potato 2.1.1 Overview of potato 2.1.2 Morphological characteristics of potato 2.1.3 Growth stages of potato 2.1.4 Factors affecting the growth and development of potato 2.1.5 Value of potato 2.2 Endophytic bacteria 2.2.1 Research history 2.2.2 Definition of endophytic bacteria 2.2.3 Origin of endophytic bacteria 2.2.4 The role of plant endophytes 2.3 Methods for identifying microorganisms 2.3.1 ITS fragment sequencing iii 2.3.2 16s rRNA gene sequencing technique 2.3.3 18s rRNA gene sequencing technique 10 2.3.4 26s rRNA gene sequencing technique 11 2.4 Some studies using 16s rRNA 11 2.4.1 Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes 11 2.4.2 Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments 12 PART III MATERIALS AND METHODS 14 3.1 Materials 14 3.2 Study place and time 14 3.3 Equipment 14 3.4 Chemicals 14 3.5 Contents and methods 15 3.5.1 Contents 15 3.5.2 Methods 15 3.5.2.1 Quantification of DNA by using spectrophotometer 15 3.5.2.2 PCR 16 3.5.2.3 Gel Electrophoresis 17 3.5.2.5 Data processing 18 Part 4: RESULTS AND DISCUSSION 19 4.1 PCR results 19 4.2 Sequencing results 19 iv Part V: CONCLUSIONS AND RECOMMENDATIONS 35 5.1 Conclusions 36 5.2 Recommendations 36 REFERENCES 37 v LST OF ABBREVIATION CTAB Cetyltrimethylammonium bromide DNA Deoxyribonucleic acid RNA Ribonucleic acid EDTA Ethylendiamin Tetraacetic Acid et al et alii (Latin), and others ml Milliliter PCR Polymerase chain reaction PVPP Polyvinylpyrrolidone TAE Tris-acetate EDTA °C Degree Celsius % Percent spp Species vi LIST OF TABLES Table 1: List of chemicals used in the study Table 2: Components of each PCR reaction Table 3: PCR produre Table 4: Identification of microorganisms Table 5: Secondary structure of 12 species of microorganisms determined by Unafold (https://www.unafold.org/) and R2DT (https://rnacentral.org/r2dt) vii LIST OF FIGURES Figure 2.1 Picture of potato Figure 2.2 Detailed image of potato Figure 4.1 PCR results Figure 4.2.1 Phylogenetic tree Figure 4.3 Nucleotide alignment and diversity viii 8) Group I predicted by Unafold 2) 4) 27 5) 7) 28 11) 12) 8) 29 B) Group II predicted by R2DT 3) 6) 9) 10) 30 Group II predicted by Unafold 3) 6) 9) 10) 31 In group II, using R2DT tools to predict the second order structure gave almost similar results Using the Unafold tool shows that at points 160, 200 and 240 there is the difference we have shown on the picture by the orange arrow, Here we still see that the structures predicted by R2DT are completely similar between the isolates in the group Also the sequence 2, 4, 5, 7, 11, 12 but when using Unafold, you can see that the secondary structure has a huge difference (circled as above) C) Group III predicted by R2DT and Unafold tools R2DT 1) Unafold 1) From the above results, we can see that the predicted structure from the R2DT tool showed high similarity between isolates in the same group Nevertheless, still we can see the difference in the secondary structure of different isolates 32 While secondary structures predicted by R2DT tool showed high similarity between isolates, Unafold tool showed significantly more differences in the secondary structure of the isolates 4.2.3 Species conservatism After identifying the polymorphisms by predicting the secondary structure, we conducted a more detailed analysis by alignment the sequences together based on MEGA7 software with toggle at 50% level, gap open at -400 and gap extend at Sequence alignment is the process of comparing and detecting similarities between biological sequences What “similarities” are being detected will depend on the goals of the alignment process Sequence alignment appears to be extremely useful in several bioinformatics applications Sequence alignments are useful in bioinformatics for identifying sequence similarity, producing phylogenetic trees, and developing homology models of protein structures The results are shown in the following pictures 33 A) B) C) D) E) F) G) H) 34 Figure 4.3 Nucleotide alignment and diversity The results showed that from the first nucleotide position to the 60th nucleotide position, only Bacillus thuringiensis showed difference compared to the remaining 11 strains of microorganisms (Figure 4.3A) Sequence in the region from 61 to 120: besides the difference of isolate 1, there was a difference between Bacillus paralicheniformis and Bacillus subtilis (Figure 4.3B) The most conservative region of the genus bacillus was on the area showed in Figure 4.3D and 4.3G 35 Part V: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions Twelve endophytic bacteria isolated from potato root were identified by using sequence of partial 16S rRNA The twelve endophytic bacteria belonged to the genus Bacillus The phylogenetic tree of 12 isolates were constructed using MEGA7 software and showed branches The first branch contented isolates which were all identified as Bacillus subtilis The second branch had isolates which were all annotated as Bacillus paralicheniformis The last branch had only one isolate (isolate 1) which was annotated as Bacillus thuringiensis Although the some isolates were grouped in one branch, the secondary structure of partial 16S rRNA by Unafold tool was able to reveal the high diversity of the isolates In addition, the details sequence analysis of partial 16S was also analyzed 5.2 Recommendations Other genetic diversity analysis of twelve isolates such as molecular markers should be applied to evaluate the divergence of the genetic 36 REFERENCES Bahmani, Kambiz, Nader Hasanzadeh, Behrouz Harighi, and Alireza Marefat 2021 'Isolation and identification of endophytic bacteria from potato tissues and their effects as biological control agents against bacterial wilt', Physiological and Molecular Plant Pathology, 116: 101692 Borges, Regis, Emilio C Miguel, Janice MR Dias, Maura da Cunha, Ricardo E Bressan-Smith, Jurandi G de Oliveira, and Gonỗalo A de Souza Filho 2004 'Ultrastructural, physiological and biochemical analyses of chlorate toxicity on rice seedlings', Plant science, 166: 1057-62 Chanway, CP 1996 'I Endophytes: they're not just fungi! CP Chanway', Can J Bot, 74: 321-22 Garbeva, P., L S van Overbeek, J W L van Vuurde, and J D van Elsas 2001 'Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments', Microbial Ecology, 41: 369-83 Kandel, Shyam L, Pierre M Joubert, and Sharon L Doty 2017 'Bacterial endophyte colonization and distribution within plants', Microorganisms, 5: 77 Kumar, Senthil P 2011 'Efficacy of segmental stabilization exercise for lumbar segmental instability in patients with mechanical low back pain: A randomized placebo controlled crossover study', North American journal of medical sciences, 3: 456 Kumari, Pooja, Sanjay Kumar, Anupam Patra, Baljinder Singh, Vimal Pandey, and Sahil Mehta 2021 'Plant-Microbe Symbiosis led synthesis of Bioactive Compounds.' in, Antioxidants in Plant-Microbe Interaction (Springer) Lastochkina, Oksana, Liudmila Pusenkova, Darya Garshina, Cemal Kasnak, Recep Palamutoglu, Irina Shpirnaya, Il’dar Mardanshin, and Igor Maksimov 2022 'Improving the biocontrol potential of endophytic bacteria Bacillus subtilis with salicylic acid against Phytophthora infestans-caused postharvest potato tuber late blight and impact on stored tubers quality', Horticulturae, 8: 117 Lodewyckx, Cindy, Jaco Vangronsveld, Fiona Porteous, Edward RB Moore, Safieh Taghavi, Max Mezgeay, and Daniel van der Lelie 2002 'Endophytic bacteria and their potential applications', Critical reviews in plant sciences, 21: 583-606 McInroy, John A, and Joseph W Kloepper 1995 'Survey of indigenous bacterial endophytes from cotton and sweet corn', Plant and soil, 173: 337-42 Quadt-Hallmann, ANDREA, JW Kloepper, and N Benhamou 1997 'Bacterial endophytes in cotton: mechanisms of entering the plant', Canadian journal of microbiology, 43: 577-82 37 Rathod, Mayuri C, and DA Dhale 2016 'Plant Growth Promoting Endophytic Bacteria from Zyzypus mauritiana', Int J Curr Microbiol App Sci, 5: 5970 Ryan, John P, James FR Gower, Stephanie A King, W Paul Bissett, Andrew M Fischer, Raphael M Kudela, Zbigniew Kolber, Fernanda Mazzillo, Erich V Rienecker, and Francisco P Chavez 2008 'A coastal ocean extreme bloom incubator', Geophysical Research Letters, 35 38 APPENDIX >Iso_1 TTAGCGGCGGACAAGGTGAACTAACTGCTGCGGTAACCTGTCCCATAAGACTGGTGATAA CTACCGGGAAAACCCGCTTCTAATACAGAGATAACATTTTGAACTGCATGGTTCGAAATT GAAAAGCTGCTTCGGCTGGCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAG GTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGG GACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCACTACGGAATCTTCCGCAATGGAC GAAAGTCTGACGGATCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGT TGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAA AGCCACGGCTAACTACATGCCAACAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGG AATTATTCC > Iso_2 TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG > Iso_3 TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATAAAAGGTGG CTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGC TCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGA CACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGG AAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGG CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTG G > Iso_4 TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG 39 > Iso_5 TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG > Iso_6 TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATAAAAGGTGG CTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGC TCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGA CACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGG AAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCATAAAGCCACGG CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTG > Iso_7 TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG > Iso_8 TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTATGTGGCAAGCGTTGTCCGGAATTATTGG > Iso_9 TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATAAAAGGTGG CTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGC TCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGA CACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGG AAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCGCGG CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTG G 40 > Iso_10 TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATAAAAGGTGG CTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGC TCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGA CACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGG AAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGG CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTG G > Iso_11 TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG > Iso_12 TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG 41