Study on the methods of identification of bacillus thuringiensis in biological pesticide (khóa luận tốt nghiệp)

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS TITLE: STUDY ON THE METHODS OF IDENTIFICATION OF BACILLUS THURINGIENSIS IN BIOLOGICAL PESTICIDE HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS TITLE: STUDY ON THE METHODS OF IDENTIFICATION OF BACILLUS THURINGIENSIS IN BIOLOGICAL PESTICIDE Student : Pham Van Tuan Class : K62CNSHE Student’s Code : 620553 Supervisor : Dr Ninh Thi Thao HANOI – 2022 COMMITMENT I assure that the entire study procedure was carried out by myself under the scientific supervision of Dr Ninh Thi Thao I assure that all research contents, conclusions, and information in my graduation thesis are entirely honest and unpublished Hanoi, May 15th 2022 Sincerely, Pham Van Tuan i ACKNOWLEDGEMENTS During the months of doing my graduation thesis at the Department of Plant Biotechnology - Faculty of 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 I am highly grateful to supervisor Dr Ninh Thi Thao, for giving me much advice and spared no effort in helping me this work Many people have written many words in praise of her scientific attitude, untiring work but, I think I have no words for this renowned scientist as feelings are deep but unfortunately words are too shallow, that cannot fathom my feelings of respect for her I believe that whatever I learnt from his personality will guide me in every field of life I would like to express my sincere thanks to all the teachers who have enthusiastically taught in the biotechnology department of the Vietnam National University of Agriculture I am also grateful for the care and support of my family and friends That is a great source of encouragement for me to pursue and complete this Graduation essay I am thankful to all those who are Hanoi, May 15th 2022 Sincerely, Pham Van Tuan 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 History of Bacillus thuringiensis Discovery and Their Classification 2.1.2 Morphological characteristics 2.1.3 Biological toxin and toxicity mechanisms 2.2 Application of BACILLUS THURINGIENSIS biological products 15 2.2.1 In the world 15 2.2.2 In Vietnam 17 2.3 Methods currently used for identification of bacteria 20 2.4 Introduction of biological product “Delfin®WG” 22 PART III MATERIALS AND METHODS 25 3.1 Materials 25 3.2 Time and place of the study 25 3.3 Research contents and methods 25 iii 3.3.1 Isolation of Bacillus thuringiensis from the sample 25 3.3.2 Identify Bt based on morphological and biochemical characteristics 26 3.3.3 Determining effective techniques for DNA extraction from Bt 27 3.4 Media and chemicals 30 3.5 Data analysis 31 PART IV RESULTS AND DISCUSSION 32 4.1 Isolation of Bacillus thuringiensis from “Delfin®WG” 32 4.2 Identification Bt isolates based on the morphological characteristics 32 4.2.1 Colonical morphology 33 4.2.2 Cell morphology 34 4.2.3 Spore and crystal morphology 35 4.3 Identification Bt isolates based on the biochemical characteristics 37 4.3.1 Lecithinase test 37 4.3.3 Fermentation test 39 4.4 Identification Bt at molecular level 43 4.4.1 Quantification of Bt bacterial CFU in “Delfin®WG” 45 PART V CONCLUSION AND SUGGESTION 47 5.1 Conclusion 47 5.2 Suggestion 47 REFERENCES 49 iv LIST OF ABBREVIATIONS ABBREVIATION EXPLANATION Bt Bacillus thuringiensis CFU Colony forming unit CTAB Cetyl trimethylammonium bromide DNA Deoxyribonucleic Acid LB 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 29 Table 4.1 Morphology characterizations of Bt strains 36 Table 4.2 Results of biochemical testing 40 Table 4.3 Purity and yield of genomic DNA extracted from Bt 41 vi LIST OF FIGURES Figure 2.1 Colonies of Bacillus thuringiensis on Nutrient agar (NA) medium (Majdoub N, 2016) Figure 2.2 Colony morphology of Bt on NA medium Figure 2.3 Morphology of insecticidal crystals produced by the IS5056 isolate of B thuringiensis subsp thuringiensis Figure 2.4 Mode of action of Bacillus thuringiensis cry toxins 13 Figure 2.5 Insecticidal activity of Cry and Cyt δ-endotoxins against the orders Diptera, Coleoptera, Lepidoptera, Hemiptera, and Hymenoptera 14 Figure 2.6 Product packaging images Delfin®WG 22 Figure 3.1 Biological control product “Delfin®WG” 25 Figure 4.1 Colony image of microbial strains in Delfin WG preparation 32 Figure 4.2 Colony morphology of DT isolate on T3 medium 33 Figure 4.3 Gram staining of the isolates at 1000X magnification Vegetative form of Bacillus (rod shaped and thin), grampositive (violet) 34 Figure 4.4 Coomassie staining of the isolates at 100X magnification 35 Figure 4.5 Lecithinase reaction results 37 Figure 4.6 Catalase reaction results 38 Figure 4.7 Fermentation reaction results 39 Figure 4.8 Total DNA extraction using three different extraction techniques on n agarose gel electrophoresis 42 Figure 4.9 Amplification of PCR results of GroEL, GyrB, XRE and Cry2 from Bt strains on agarose gel electrophoresis 44 vii ABSTRACT Bacillus thuringiensis (Bt) is one of the most widely utilized biological agents to manage insect pests because it produces a variety of toxins with strong and targeted insecticidal activity One of the products containing Bacillus thuringiensis (Bt), "DELFIN® WG" probiotic, is anticipated to be able to supplement or replace chemical medications due to its benefits, including excellent specificity for target insects, safety, and therefore most especially environmental friendliness To identify and quantify Bt in the biological insecticide "DELFIN® WG," we analyzed the morphological, biochemical, and molecular characteristics of putative isolate and compared with reference strain The results showed that the putative Bt isolate from "DELFIN® WG" shared traits with reference Bt strain 4T1 in terms of lethicinase, sucrose, and catalase positivity as well as cell, colony spore, and protein crystal properties PCR was used to determine whether the putative isolates contained the four specific genes of Bt including XRE (transcriptional regulator), GroEL (chaperonin protein), GyrB (topoisomerase enzyme), and Cry2 (crystal protein) The findings demonstrated that the putative isolate had the distinct band corresponding to the target genes, proving that they are Bt As a result, we concluded that by morphological, biochemical, and molecular analyses can be utilized in combination to identify Bacillus thuringiensis in "DELFIN® WG" specifically, as well as in other Bt-contained products generally viii The following results show the positive reactions which reveal the presence of catalase due to the forming of air bubbles (O2) in both DT isolate and the reference strain 4T1 The catalase test mainly demonstrates that DT is Gram-positive and belongs to the species of Bacillus (catalase-positive) 4.3.3 Fermentation test A fermentation test is used to assess a microorganism's capacity to utilize a specific carbon source for growth The three categories of this carbon source are simple sugars, poly sugars, and alcohols The Phenol red dye does not change color if the bacteria not ferment it If fermentation takes place, acid is created, the pH drops, and the medium turns red as a result The findings of the fermentation test were displayed in figure 4.7 Figure 4.7 Fermentation reaction results Positive reaction (+): the medium changes color from red-orange into magenta-pink 39 Figure 4.7 Shows that the bacteria in the culture consumed sugar as food for development and produced acid, which lowered the pH of the medium As a result, two medium cultures that contained reference strain 4T1 and DT isolates changed color due to the phenol red indicator, going from red-orange to magenta-pink whereas The medium that only contain H2O (negative control) retained the red-orange colour Table 4.2 Showed the results of three biochemical tests on DT isolate in comparision with reference strain 4T1 Table 4.2 Results of biochemical testing Isolates Reference strain Putative isolate 4T1 DT Lecithinase production + + Catalase presence + + Sucrose fermentation + + Reactions Note: (+): Positive reaction As shown in Table 4.2, DT isolate exhibited biochemical traits that were similar to those of the reference strain 4T1, such as the capacity to make lecithinase, the capacity to ferment sucrose, and the presence of catalase in the cells In conclusion, it can be shown that DT isolated from the microbial product "DELFIN® WG" displayed recognizable Bt bacteria characteristics, such as 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) It is therefore possible to initially confirm that DT isolate might be Bacillus thuringiensis 40 4.3 IDENTIFYING THE EFFECTIVE 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 Gram-positive microbes, such as Bacillus thuringiensis, typically have thick, solid cell walls, making it more challenging to lyse them than Gramnegative microorganisms We assessed the amount of DNA extracted from a known quantity of source material in order to assess the extraction yields of different procedures Rapid and effective techniques for the extraction of DNA from bacterial cells particularly, DNA with an A260/280 nm ratio between 1.8 and 2.0 is deemed pure Pure DNA should have a ratio of roughly 1.8 (Nicklas et al., 2003) In this investigation, we evaluated three methods for obtaining DNA from the reference strain 4T1 and DT isolates Table 4.3 displays the outcomes of OD260/280, OD260/230, and the concentration of total DNA from these methods Table 4.3 Purity and yield of genomic DNA extracted from Bt Concentration (ng/µl) OD260/280 OD260/230 Sample M1 M2 M3 M1 M2 M3 M1 M2 M3 4T1 120 149 254 1.23 1.45 1.96 1.02 1.05 2.31 DT 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: reference strain; DT: putative Bt isolates According to the findings in table 4.3, lysozyme and CTAB-based method generates genomic DNA with an OD260/280 value of 1.96 (4T1) and 2.1 (DT) and an OD260/230 value of 2.31 (4T1) and 1.91 (DT) demonstrating that the 41 DNA genome meets purity standards The DNA access has an OD260/280 value ranging from 1.05 to 1.45 (1.6) and an OD260/230 value ranging from 0.76 to 1.05, respectively, based on rapid boiling and CTAB method could indicate that the extracted DNA did not meet the purity standards In order to evaluate whether the total DNA was sufficient for the PCR reactions, we also run total genomic DNA obtained from three methods on 1% gel agarose The results of the electrophoresis were shown in the following figure 4.8 Figure 4.8 Total DNA extraction using three different extraction techniques on an agarose gel electrophoresis Notes: M: Marker; 4T1: reference strain; DT: putative Bt isolate Figure 4.8 demonstrates the indistinct or even missing bands in the DNA products obtained from the quick boiling method and CTAB method, demonstrating that the entire DNA product was not qualified for the PCR reactions The results, on the other hand, demonstrate that the DNA products obtained from lyzozyme and CTAB-based technique have distinct and straight bands indicating that the DNA was qualified for the PCR reactions The boiling process is quick, simple, cheap, and requires no chemical reagents, whereas the other procedures are more involved and employ 42 numerous chemical solutions, some of which are dangerous and require a high level of skill However, it makes the most sense to select the methodology that produces the most amount of extraction product if it can produce large amounts of DNA at a greater cost compared to other procedures On the other hand, the DNA bands extracted by using the lyzozyme and CTAB-based method are brighter and more clear, demonstrating that this method is more efficient, produces higher quality and concentration DNA products for the PCR reactions In conclusion, the best method for extracting DNA from Bt is one based on lyzozyme and CTAB 4.4 IDENTIFICATION OF BACILLUS THURINGIENSIS AT MOLECULAR LEVEL Chelliah et al (2019) showed that the presence of B thuringiensis is very similar to B cereus group in both biochemical and genetic properties Hence, new biomarkers were developed based on a molecular and genetic approach to replace the existing biomarkers with low detectability and false positives The housekeeping genes GroEL and GyrB, as well as the transcriptional crystal protein regulator (XRE), were found to be effective in identifying B thuringiensis (Chelliah et al., 2019) Additionally, according to Truong et al (2019), in Vietnam, the amplified products belonged to Cry2A type genes of B thuringiensis with an identity rate above 90% when compared with Cry2A sequences released in the Gen Bank sequence database Electrophoresis on a gel of agarose was used to identify the outcomes of PCR reactions using the specific genes GroEL, GyrB, XRE, and Cry2 and are displayed in the figures below 43 Figure 4.9 Amplification of PCR results of GroEL, GyrB, XRE and Cry2 from Bt strains on agarose gel electrophoresis Notes: M: Marker; DW: distilled water; (+) Positive controls: Reference strains 4T1; 4T4 and 4D4, DT: putative Bt isolate; (-) Negative controls: Bacillus subtilis (ATCC79530), Bacillus amyloliquefaciens (NT 1.8), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 85922) 44 Figure 4.9 show that the reference strain and DT isolate showed expected PCR band sizes as in three reference strains including 4T1, 4T4 and 4D4 These bands included bands with sizes of 700 bp for Cry2 gene, 600 bp for GroEL gene, 221 bp for GryB gene, and 246 bp for the XRE gene These results indicated that DT isolate were positive for the presence of four particular genes On the other hand, there were no distinct bands in the water control or the negative controls utilizing genomic DNA as a template from Bacillus subtilis (ATCC79530), Bacillus amyloliquefaciens (NT 1.8), Staphylococcus aureus (ATCC 25923), or Escherichia coli (ATCC 85922) In conclusion, it is possible to confirm that the isolated bacterial strain is Bacillus thuringiensis by combining the findings of analyses of morphological and biochemical properties and analyses of the presence of particular genes of Bt bacteria by PCR method 4.5 Quantification of Bt bacterial CFU in “Delfin®WG” After performing morphological, biochemical and molecular analyzes of the DT isolate, we found that the characteristics of DT isolate were similar to those of the reference strain 4T1 From this we can conclude that DT isolate is Bacillus thuringiensis The majority of microbiological research depend heavily on the quantification of bacterial cells The amount of bacteria found using this methodology was comparable to that found using the counting method, which was one of the accepted techniques for sample analysis A CFU is an acronym for colony-forming units in microbiology We measure the amount of live bacteria in a sample using this unit The original CFU/g (or CFU/mL) is mathematically determined by deducing the CFU/plate from a plate in the linear range and accounting for the amount plated and its dilution factor (Schug et al., 2020) Based on the findings of experiment (Table 4.2), we suggested the number of Bt spores in "Delfin®WG" in this 45 investigation, utilizing 10¯⁴ and 10¯⁵ dilution levels The total number of Bt spores contained in the sample (CFU/ml) was determined using the following formula with an initial sample dilution ratio of 1:39 (v/v): - In which: + = 461+409 = 870 (colonies) + V= 0.01 (ml) + d = 10-4 + a = 39 As a result, the “Delfin®WG” sample may have a total spore count determined of 3.163x1010 CFU/ml (>106) in accordance with national standards for biopesticides as well as the notification of manufacturer with specified concentrations 8.5x10² CFU/kg and maximum 6x10¹³ CFU/kg 46 PART V CONCLUSION AND SUGGESTION 5.1 CONCLUSION - The amount of spores in the sample was calculated after dilution it at a ratio of 1:39, incubating it at 80 °C for minutes, and then using 10 µl of that solution to culture on LB medium The results show that the amount of spores in the biological product DelfinđWG is 3.163x10ạ CFU/ml (>10), hence in accordance with the manufacturer's claim - The morphological traits of the isolated DT strain were similar as the reference strain 4T1 such as: white colonies, round shape, gram-positive status, and spore production It is possible to base on morphological characteristics to select a putative Bt strain from which to conduct further validation experiments - The ability of DT strain to produce toxic crystals is a characteristic feature of Bt bacteria, which helps researchers to quickly eliminate non-Bt bacterial strains - DT strain were positive for biochemical tests such as: lethicinase, sucrose and catalase - The best yield and purest genomic DNA products were obtained from Bt using the lyzozyme and CTAB-based extraction technique - Four genes, GroEL, GyrB, XRE, and Cry2, produced unique PCR results for putative Bt isolates and the reference Bt strains, but not for non-Bt bacteria, indicating that these primers can be used to detect Bt bacteria -The findings of this study will be an important source of information for identification of Bt bacteria in biopesticide products in Vietnam 5.2 SUGGESTION - More research needs to be done on the techniques for identifying Bt bacteria using biochemical tests 47 - Bt bacteria can be recognized using the multiplex PCR technique - The methods to identify and measure Bacillus thuringiensis in other biological insecticides containing Bt in Vietnam must be developed 48 REFERENCES Adang MJ, Crickmore N, Jurat-Fuentes JL Diversity of Bacillus thuringiensis crystal toxins and mechanism of action Advances in Insect Physiology 2014;47:39-87 Alaeddinolu Fasang (1998) A rapid and simple method for staining of the crystal protein of Bacillus thuringiensis Journal of Industrial Microbiology Vol pp 227-229 Arthur I Aronson, William Beckman & Peter Dunn (1986) Bacillus thuringiensis and Related Insect Pathogens American 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