Study on the methods of identification of bacillus thuringiensis in biological pesticide siab bt

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS TITLE: STUDY ON THE METHODS OF IDENTIFICATION OF BACILLUS THURINGIENSIS IN BIOLOGICAL PESTICIDE “SIAMB BT” HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS STUDY ON THE METHODS OF IDENTIFICATION OF BACILLUS THURINGIENSIS IN BIOLOGICAL PESTICIDE “SIAMB BT” Student : Nguyen Thi Quynh Anh Class : K62CNSHE Student’s Code : 620654 Supervisor : Ninh Thi Thao, Ph.D HANOI – 2022 COMMITMENT I assure that the entire study procedure was carried out by myself under the scientific supervision of Ph.D Ninh Thi Thao I assure that all research contents, conclusions, and information in my graduation thesis are entirely honest and unpublished Hanoi, May 2022 Sincerely, Nguyen Thi Quynh Anh i ACKNOWLEDGEMENTS During the months of doing my graduation thesis at the Department of Plant Biotechnology - Vietnam National University of Agriculture, I had the opportunity to consolidate and practice my knowledge and study skills to be able to complete the thesis well mine In addition to my own efforts, I have received a lot of enthusiastic help from the teachers as well as people around First of all, I would like to express deeply my gratitude to my supervisor, Ph.D Ninh Thi Thao has supported me complete my thesis with her patience and knowledge Moreover, her guidance helped me in the period of writing this thesis Secondly, I would like to thank all my teachers in the Faculty of Biotechnology, especially the Department of Plant Biotechnology They provided useful scientific knowledge as well as technique for me This is one of the most important things to help me to successful my thesis Then, I would like to thank my lab companions for their significant assistance I will cherish the time we spent working together Last but not least, with all due respect and infinite gratitude, I would like to thank my family and friends who have always been there for me, taking care of me, encouraging me, creating conditions and motivating me to help me during the writing of this thesis My graduation thesis would not have been finished if it had not been for their support Hanoi, May 2022 Sincerely, Nguyen Thi Quynh Anh ii INDEX COMMITMENT i ACKNOWLEDGEMENTS ii INDEX iii LIST OF ABBREVIATIONS v LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT viii PART I INTRODUCTION 1.1 Preface 1.2 Objectives and requirements 1.2.1 Objectives 1.2.2 Requirements PART II LITERATURE OVERVIEW 2.1 Introduction of Bacillus thuringiensis species 2.1.1 Taxonomy and discovery 2.1.2 Morphological characteristics 2.1.3 Toxin and toxicity mechanisms of Bt 2.1.4 Bioinsecticide activity of Bacillus thuringiensis crystal proteins 12 2.2 Application of biological products of Bacillus thuringiensis 14 2.3 Current methods for quantification and determination of bacteria 17 2.4 Introduction of biological product “SIAMB BT” 19 PART III MATERIALS AND METHODS 20 3.1 Materials 20 3.2 Time and place of the study 20 3.3 Research contents and methods 21 3.3.1 Isolation of Bacillus thuringiensis from the sample 21 3.3.2 Identify Bt based on morphological characteristics 22 3.3.3 Identify Bt based on biochemical characteristics 23 3.3.4 Determination of efficient methods for DNA extraction from Bt 24 3.3.5 Identify Bt based on molecular analysis 25 3.4 Media and chemicals 27 iii PART IV RESULTS AND DISCUSSION 29 4.1 Isolation of Bacillus thuringiensis from “SIAMB BT” 29 4.1.1 Effect of initial sampling ratio on the isolation of Bt 29 4.1.2 Effect of sample dilution concentration on the isolation of Bt 30 4.1.3 Effect of temperature of sample incubation on the isolation of Bt 32 4.1.4 Effect of sample incubation time on the isolation of Bt 34 4.2 Identification Bt isolates based on the morphological characteristics 36 4.2.1 Colonial morphology 36 4.2.2 Cell morphology 37 4.2.3 Spore and crystal morphology 39 4.3 Identification Bt isolates based on the biochemical characteristics 41 4.3.1 Lecithinase test 41 4.3.2 Catalase test 42 4.3.3 Fermentation test 43 4.4 Identification suitable method for DNA extraction from Bt 45 4.5 Identification Bt at molecular level 48 PART V CONCLUSIONS AND SUGGESTIONS 51 5.1 Conclusions 51 5.2 Suggestions 51 REFERENCES 52 APPENDIX 57 iv LIST OF ABBREVIATIONS ABBREVIATION Bt CFU CTAB DNA LB EXPLANATION Bacillus thuringiensis Colony forming unit Cetyl trimethylammonium bromide Deoxyribonucleic Acid Luria Bertani broth MYP Mannitol Egg Yolk Polymyxin PCR Polymerase chain reaction subsp subspecies var variety βME Beta-Mercaptoethanol v LIST OF TABLES Table 3.1 PCR primers used in the study 26 Table 4.1 Effect of initial sampling ratio on bacterial growth after 18h of culture 29 Table 4.2 Effect of sample dilution on bacterial growth after 18h of culture 30 Table 4.3 Effect of temperature of sample incubation on bacterial growth after 24h of culture 32 Table 4.4 Effect of sample incubation time on bacterial growth after 24h of culture 34 Table 4.5 Morphology characterizations of Bt strains 40 Table 4.6 Results of biochemical testing 45 Table 4.7 Purity and yield of genomic DNA extracted from Bt 46 vi LIST OF FIGURES Figure 2.1 Colonial morphology of Bacillus thuringiensis Figure 2.2 Electron micrograph illustrating Bacillus thuringiensis var kurtsaki Figure 2.3 Scanning electron microscopy images of the different types of crystal morphologies and the spores (S) produced by Bt strains of the collection Figure 2.4 Spore morphology and crystals of some strains of Bacillus thuringiensis Figure 2.5 Mode of action of Bacillus thuringiensis in Lepidoptera 11 Figure 2.6 Summarized view showing the known host spectrum of Bt δ-endotoxins (Cry and Cyt) 13 Figure 3.1 Biological control product “SIAMB BT” 20 Figure 4.1 Colonial morphology of standard and putative Bt strains 37 Figure 4.2 Gram staining of the isolates at 1000X magnification 38 Figure 4.3 Coomassie staining of the isolates at 1000X magnification 39 Figure 4.4 Lecithinase reaction results 42 Figure 4.5 Catalase reaction results 43 Figure 4.6 Fermentation reaction results 44 Figure 4.7 Agarose gel electrophoresis (2%) of total DNA extraction by three extraction methods 47 Figure 4.8 Agarose gel electrophoresis (2%) of PCR amplification of GroEL, GyrB, XRE and Cry2 from Bt isolates 49 vii ABSTRACT Currently, Bacillus thuringiensis (Bt) is one of the most extensively used to manage insect pests in many biological agents, produces different types of toxin that have potent and specific insecticidal activity “SIAMB BT” probiotic is one of the products containing Bacillus thuringiensis (Bt) which are expected to be able to replace or supplement chemical drugs because of their advantages such as high specificity for target insects, safety, and especially environmental friendliness In this study, we have focused on developing different approaches including morphological, biochemical and molecular analysis to identify and quantify Bt in the biological pesticide “SIAMB BT” The putative Bt isolates from “SIAMB BT” exhibited the characteristics of cell, colony spore and protein crystal as well as showed lethicinase-, sucrose- and catalase- postive that were similar to standard Bt strain 4T1 In addition, we indicated that the lysozyme and CTAB-based is the suitable method for DNA extraction from Bt The presence of four specific genes of Bt including XRE (transcriptional regulator gene), GroEL (chaperonin protein gene), GyrB (topoisomerase enzyme gene), and Cry2 (crystal protein gene) in the isolate were checked by PCR using specific primers The result revealed that the putative isolates displayed the specific band corresponding to the target genes, confirming that they are Bt Therefore we concluded that morphological, biochemical and molecular analysis can be used in combination for identification of Bacillus thuringiensis in “SIAMB BT” in particular and in other Bt-contained products in general viii Table 4.6 Results of biochemical testing Isolates Standard strain Putative isolate “4T1” “BT” Lecithinase production + + Catalase presence + + Sucrose fermentation + + Reactions Note: (+): Positive reaction As can be seen from Table 4.6, the putative isolate“BT” displayed similar biochemical properties to the standard strain “4T1” such as the ability to produce lecithinase, the ability to ferment sucrose and presence catalase in the cells In summary, it can be seen that the putative “BT" strain isolated from the microbial product “SIAMB BT” showed distinctive features of Bt bacteria including colony, cell, spore and crystal traits as well as biochemical activities (Baumann et al., 1984; Claus et al., 1986; Slepecky et al., 1992; Carlson et al., 1993; Hansen et al., 1998) Therefore, it is possible to initially confirm that the putative isolate could be Bacillus thuringiensis 4.4 Identification suitable method for DNA extraction from Bt Total DNA extraction is the fundamental and initial approach used to determine the genetic diversity of accessions Nucleic acid extraction has grown simpler because of technical breakthroughs and discoveries There are several techniques for complete DNA extraction available today However, a distinct procedure is required with each individual object As a result, it is critical to select and develop the procedure to fit each item In general, it is more difficult to lyse Gram-positive microorganisms such as Bacillus Thuringiensis than Gram-negative microorganisms because of the thick, 45 rigid cell walls In order to evaluate the extraction yields of methods, we measured the quantity of DNA obtained from a known amount of source material Pure DNA should have a ratio of approximately 1.8, rapid and efficient methods for the extraction of DNA specifically from bacterial cells, DNA with A260/280 nm ratio between 1.8 and 2.0 is considered pure (Nicklas et al., 2003) In this study, we tested three methods to extract DNA from the standard strain “4T1” and putative isolates “BT” namely from “BT1” to “BT6” The results of OD260/280, OD260/230 and the concentration of total DNA from these methods were presented in Table 4.7 Table 4.7 Purity and yield of genomic DNA extracted from Bt Concentration (ng/µl) OD260/280 OD260/230 Sample M1 M2 M3 M1 M2 4T1 120 149 254 1.23 BT1 324 376 125 BT2 340 187 BT3 345 BT4 M3 M1 M2 M3 1.45 1.96 1.02 1.05 2.31 1.04 1.02 1.99 1.39 0.97 1.86 105 1.04 1.10 1.90 1.02 1.32 2.22 498 309 1.54 1.09 2.05 0.66 1.06 2.12 475 448 136 1.00 1.03 2.00 0.97 1.02 2.33 BT5 142 328 169 0.98 1.24 1.85 1.04 0.94 1.92 BT6 264 208 136 1.05 1.34 2.10 0.76 0.88 1.91 Notes: (M1) Rapid boiling method; (M2) CTAB method; (M3) Lysozyme and CTAB-based method; 4T1: standard strain; BT1-BT6: putative Bt isolates The results in table 4.7 indicated that the lysozyme and CTAB-based method resulted the genomic DNA with the OD260/280 values ranging from 1.85 to 2.05 and OD260/230 values ranging from 1.86 to 2.31, proving that genomic DNA met the purity requirements Meanwhile, based on the rapid boiling method and the 46 CTAB method, the DNA accessions have OD260/280 values ranging from 0.98 to 1.54 (