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 I assure that all research contents, conclusions, and information in my graduation thesis are entirely honest and unpublished.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 6 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. 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 do 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. iii LIST OF ABBREVIATIONS. v LIST OF TABLES.
vi LIST OF FIGURES. Objectives and requirements. Introduction of Bacillus thuringiensis species. History of Bacillus thuringiensis Discovery and Their Classification.
Biological toxin and toxicity mechanisms. Application of BACILLUS THURINGIENSIS biological products. In the world. Methods currently used for identification of bacteria.
Introduction of biological product “Delfin®WG”. MATERIALS AND METHODS. Time and place of the study. Research contents and methods.
Isolation of Bacillus thuringiensis from the sample. Identify Bt based on morphological and biochemical characteristics. Determining effective techniques for DNA extraction from Bt. Media and chemicals.
RESULTS AND DISCUSSION. Isolation of Bacillus thuringiensis from “Delfin®WG”. Identification Bt isolates based on the morphological characteristics. Spore and crystal morphology.
Identification Bt isolates based on the biochemical characteristics. Identification Bt at molecular level. Quantification of Bt bacterial CFU in “Delfin®WG”. CONCLUSION AND SUGGESTION.
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. variety Βme Beta-Mercaptoethanol v LIST OF TABLES Table 3. PCR primers used in the study. Morphology characterizations of Bt strains.
Results of biochemical testing. Purity and yield of genomic DNA extracted from Bt. 41 vi LIST OF FIGURES Figure 2. Colonies of Bacillus thuringiensis on Nutrient agar (NA) medium (Majdoub N, 2016).
Colony morphology of Bt on NA medium. Morphology of insecticidal crystals produced by the IS5056 isolate of B. Mode of action of Bacillus thuringiensis cry toxins. Insecticidal activity of Cry and Cyt δ-endotoxins against the orders Diptera, Coleoptera, Lepidoptera, Hemiptera, and Hymenoptera.
Product packaging images Delfin®WG. Biological control product “Delfin®WG”. Colony image of microbial strains in Delfin WG preparation. Colony morphology of DT isolate on T3 medium.
Gram staining of the isolates at 1000X magnification. Vegetative form of Bacillus (rod shaped and thin), gram- positive (violet). Coomassie staining of the isolates at 100X magnification. Lecithinase reaction results.
Catalase reaction results. Fermentation reaction results. Total DNA extraction using three different extraction techniques on n agarose gel electrophoresis. 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. PREFACE The use of chemical crop protection measures is a key component of integrated pest and disease management of crops.
Along with the benefits, this category of compounds also showed a lot of drawbacks. Chemical pesticides cause the ecosystem to become out of balance by killing beneficial insects as well as reducing the effectiveness of target insects by causing resistance. Additionally, using chemical pesticides over an extended period of time causes poisons to build up in the environment, harming both human health and the environment. In addition, the use of insecticide use in densely populated metropolitan areas will have an impact on public health (Dang Bao Ha, 2015).
Finding a bio-friendly substitute is thus urgently needed. The creation of bio- pesticides that are efficient, biodegradable, and ecologically benign is an appropriate course to take. The practice of gradually replacing chemical pesticides with biological ones is becoming more popular. The use of biopesticides in agriculture and public health has many advantages, including the absence of residue on agricultural products, which worries customers, especially for produce like vegetables and fruit (Dang Bao Ha, 2015).
From insects, plants, soil, and water, more than 90 species of specialist insecticidal bacteria have been discovered (Vos et al. However, only a few number of species—such as the bacterium Bacillus thuringiensis—have received much study and attention to yet (Bt). Because of their benefits, including their great specificity for target insects, safety, and especially environmental friendliness, biological insecticides from Bt are believed to be able to replace or enhance chemical medications. A gram-positive, aerobic soil bacterium called Bacillus thuringiensis has cry genes for spore manufacturing and toxin crystal protein, which may be used to kill a variety of squamous, 1 beetle, and bipedal insect species (Das et al.
They offer a broad spectrum of actions that can oppose pathogenic fungus while being safe for people and plants. However, Bacillus also contributes to the process of turning complex organic compounds into simple organic compounds that plants can utilize readily, improving soil, and regulating and eradicating some hazardous microbes plant illnesses as a result of their unique biological roles (Le Duc Manh et al. The bacteria of the genus Bacillus are used in agriculture to improve soil, boost production, combat certain fungi and bacteria in the root zone of plants, enrich the soil microbiota, and restore soil fertility. The development of a technique to identify and assess Bacillus thuringiensis in samples of microbial products under Vietnamese conditions requires research and has both scientific and practical value.
Vietnam currently produces a large number of Bt pesticide products, however there are still few ways to identify and measure them. Additionally, the current baseline standards in Vietnam cannot subjectively or quantitatively measure Bt in pesticides. This study intends to establish a method to detect and quantify Bacillus thuringiensis in biological insecticides containing Bt in Vietnam. It is based on that practice and current research trends in the globe and the nation.
OBJECTIVES AND REQUIREMENTS 1. Objectives Identification and characterization of Bacillus thuringiensis in the biological product “DELFIN® WG”. Requirements - Isolate putative Bacillus thuringiensis from the sample - Characterize putative Bt isolates based on morphological and biochemical characteristics. - Identify the appropriate method for DNA extraction from Bt.
- Characterize putative Bt isolates by PCR amplification of specific genes of Bt. INTRODUCTION OF BACILLUS THURINGIENSIS SPECIES Bacillus thuringiensis (Bt) is a soil bacterium that forms spores during the stationary phase of its growth cycle. The crystals found in the spores are mostly made up of one or more Cry and/or Cyt proteins, commonly known as d- endotoxins, which have strong and targeted insecticidal activity. Different Bt strains create unique poisons, each of which affects a specific subset of insects.
Since the 1920s, pesticides containing the Bt toxin have been used extensively in organic farming. Additionally, Bt is a source of the genes that are used to genetically modify a variety of food crops so that they create poisons to fend against different insect pests. Lepidoptera (butterflies, moths, and bugs), Diptera (flies), and Coleoptera (beetles) are among the insect species for which the poison can be fatal; however, several strains of Bt are available for use in more targeted pest control (Mullaney et al. History of Bacillus thuringiensis Discovery and Their Classification The insecticidal properties of Bt were recognized many years before the bacteria were identified, with some records suggesting that Bt spores may have been used in ancient Egypt.
In modern times, this bacterium was isolated by Japanese biologist Shigetane Ishiwatari in 1901 while investigating silkworm wilt and he named it Bacillus sotto. Ten years later, Ernst Berliner isolated the same bacteria from a diseased Mediterranean butterfly (Ephestia kuehniella) in the German province of Thuringia, and it was named Bacillus thuringiensis (Siegel, 2000). The defining feature of Bt is its ability to produce protein crystals during sporulation. Bt is a member of the Bacillus cereus group of spore- forming gram-positive soil bacteria, which sometimes lose their ability to form crystals and are subsequently indistinguishable from B.
cereus can be transformed into Bt, and research into the transformation 3 mechanism has led to the discovery that crystal formation is caused by genes carried on the plasmid. The genes, which code for the Cry/Cyt proteins, become active during spore formation because they are controlled by a specialized RNA polymerase that is also specifically synthesized during spore formation. Up to 20% of the protein content of spores is represented by these Cry/Cyt toxins (Aronson, 2002). The insecticidal properties of crystals were discovered when dead pollen moths were found filled with spores and crystals.
Direct contact between spores / crystals and healthy caterpillars has no effect, but when the spores and crystals coat the leaves, the caterpillars stop feeding and die. After realizing the potential of Bt as an insecticide, Mattes (1927) isolated the Bt strain discovered by Ernst and subsequent field tests against the European stem borer (Ostrinia nubilalis) showed promising results (Husz, 1930). This work eventually led to the development of Sporeine, a commercial Bt insecticide, first used in 1938.