VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------- UNDERGRADUATE THESIS TITLE: SCREENING THE ANTAGONISTIC ABILITY TO PATHOGENIC MICROORGANISMS AND RESEARCHING BIOCHEMICAL CHARACTERISTICS OF STREPTOMYCES SP. VNUA27 HANOI- 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ---------- ---------- UNDERGRADUATE THESIS SCREENING THE ANTAGONISTIC ABILITY TO PATHOGENIC MICROORGANISMS AND RESEARCHING BIOCHEMICAL CHARACTERISTICS OF STREPTOMYCES SP. VNUA27 Student : TRINH MINH PHUONG Class : K62CNSHE Student’s code : 620408 Supervisor : Dr. TRINH XUAN HOAT Assoc.
NGUYEN XUAN CANH Hanoi – 2022 COMMITMENT I assure that this is my research. The data and experimental results presented in the thesis are honest and have never been published in any other works. The figures in the table serving the analysis, comment and assessments were collected by the author itself from different sources specified in the references. If there is any fraud, I am fully responsible for the content of the thesis.
Sincerely Trinh Minh Phuong i ACKNOWLEDGEMENT In fact, there are no successes without associated with support or assistance, whether more or less, directly or indirectly by others. This project consumed a huge amount of work, enthusiasm and dedication. Still, implementation would not have been possible if I did not have the support of many people and organizations. Therefore, I would like to extend our sincere gratitude to all of them.
Firsts of all, I would like to express sincere thanks to the School Board, the Dean of Biotechnology Faculty, and all teachers who have imparted to me the knowledge that is advantageous and valuable during time learning, training and implementation thesis. Through working, I did not only gain much knowledge but more importantly, I also had a great chance to sharpen my skills in a professional working environment. I have developed myself both academically, professionally and socially. I would like to express my deep and sincere gratitude to my supervisor, Assoc.
Nguyen Xuan Canh, PhD, Chief of Faculty of Biotechnology, Viet Nam National University of Agriculture for giving me the opportunity to complete this thesis and providing invaluable guidance through this thesis. His dynamism, vision, sincerity and motivation have deeply inspired us. He has taught me the methodology to contribute a thesis and to present that as much as possible. It was a great privilege and honour to work and study under his guidance.
I would also like to thank Dr. Trinh Xuan Hoat for guiding and giving me advice so that I can complete this thesis well. Besides my instructor, I express my special thanks to Assoc. Nguyen Van Giang; M.S Tran Thi Dao; Mrs.
Nguyen Thi Thu. I am extremely grateful to my family for their love, prayers, caring and sacrifices for educating and preparing me for my future. Finally, our thanks go to all the people who have supported me to complete the project directly or indirectly. Hanoi, February 2022 Sincerely Trinh Minh Phuong ii INDEX COMMITMENT.
iii LIST OF TABLES. vi LIST OF FIGURES. vii LIST OF ABBREVIATIONS. ix PART I: INTRODUCTION.
Research purpose and content. The purpose of the study. 2 PART II: LITERATURE REVIEWS. Overview of Actinomycetes, genus Streptomyces, Streptomyces sp.
Overview of pathogenic strains. Overview of pathogenic bacteria. Overview of bacteria Xanthomonas axonopodis. Overview of the bacterium Ralstonia solanacearum.
Overview of the bacteria Clavibacter michiganensis. Overview of pathogenic fungal strains. Overview of the fungus Fusarium solanin. Overview of the fungus Colletotrichum gloeosporioides.
Review of recent studies on the antagonism of Actinomycetes against bacteria and fungi. 29 PART III: MATERIALS AND METHODS. Location and time of the study. Screening the antagonistic ability to pathogenic microorganisms of Streptomyces sp.
Biochemical characteristics of Streptomyces sp. The ability to produce extracellular enzymes. The ability to produce IAA. The ability to produce Hydro sulfide (H2S).
The ability to produce indole. The ability to utilize citrate. The ability to hydrolyze gelatin. The ability to solubilize phosphate.
The ability to decompose ureases. The ability to produce siderophores. 39 PART I V: RESULT AND DISCUSSION. Antagonistic ability of Streptomyces sp.
Biochemical characteristics of Streptomyces sp. The ability to produce extracellular enzymes. The ability to produce IAA. The ability to produce Hydrosulfide (H2S).
The ability to produce indole. The ability to utilize citrate. The ability to liquefaction gelatin. The ability to solubilize phosphate.
The ability to decompose ureases. The ability to produce siderophores. 58 PART V: CONCLUSION AND SUGGESTION. 63 v LIST OF TABLES Table 1: Families and genera of Actinomycetales.
6 Table 2: Classification of genus Streptomyces based on morphologic, chemotaxonomix and molecular studies. 8 Table 3: Some common plant diseases caused by Xanthomonas campestris. 17 Table 4: Preharvest diseases of fruits and vegetables caused by Xanthomonas campestris. 17 Table 5: Ingredients for medium.
34 Table 6: Chemical composition of standard IAA solution. 35 Table 7: The ability to produce extracellular enzymes of Streptomyces sp. 45 vi LIST OF FIGURES Figure 1: Morphology of Streptomyces on starch casein agar and in Gram’s staining. 10 Figure 2: Xanthomonas colonies on starch plate.
14 Figure 3: Potato ring rot disease caused by Clavibacter michiganense sub sp. 22 Figure 4: Conidial morphology of Colletotrichum gloeosporioides isolated from dragon fruit under light microscope, arrow indicates the length of the conidia. 26 Figure 5: Results on the ability to antagonize three pathogenic bacteria strains of Streptomyces sp. 42 Figure 6: Antagonistic activity of Streptomyces sp.
43 Figure 7: Antagonistic activity of Streptomyces sp. 44 Figure 8: Extracellular enzyme production ability of Streptomyces sp. 46 Figure 9: IAA Standard curve plot from 0 to 8 µg/ml. 49 Figure 10: IAA production of Streptomyces sp.
50 Figure 11: H2S production ability of Streptomyces sp. 51 Figure 12: Indole production ability of Streptomyces sp. 52 Figure 13: MR test results of Streptomyces sp. 53 Figure 14: V-P test results.
54 Figure 15: Citrate utilization ability of Streptomyce sp. 55 Figure 16: Gelatin liquefaction ability of Streptomyces sp. 56 Figure 17: Testing phosphate solubilizing ability of Streptomyces sp. 57 Figure 18: Testing the urease-producing ability of Streptomyces sp.
58 Figure 19: Testing the sideropho production ability of Streptomyce sp. 60 vii LIST OF ABBREVIATIONS IAA Indole-3-acetic acid DNA Deoxyribonucleic acid PDA Potato dextrose agar IPM Integrated Pest Management PGP Plant growth promotion PGPR Plant growth-promoting rhizobacteria viii ABSTRACT Bacteria that cause plant diseases are the cause of serious damage to yield and product quality not only in Vietnam but also in many other countries. Currently, biological control is being considered as a necessary solution to replace the use of chemical drugs. With the aim of discovering more sources of biological materials that can antagonize plant pathogens.
Based on studies on Streptomyces sp. VNUA27: capable of antagonizing the fungus Fusarium oxysporum; grows well at temperatures 30oC and 37oC; good use of different sugar sources (Maltose, Sucrose, Cellulose and D-Manitol); growth well in salt concentration from 0% - 3%; pH 4 -11. I continued to investigate other characteristics of Streptomyces sp. VNUA27: resistance to three strains of bacteria and two strains of fungi; capable of producing extracellular enzymes (amylase, protease, cellulase, chitinase, catalase); citrate utilization; H2S production; gelatin liquefaction; produce urease enzyme; IAA production; phosphate solubilizing, produce siderophores.
ix PART I: INTRODUCTION 1.Introduction Vietnam is an agricultural country located in the hot and humid tropics. These are favorable conditions for the growth of microorganisms, especially plant pathogens. These microorganisms have caused great damage to crop yields. In the development trend of world agriculture, in recent years our country's agriculture has achieved great achievements.
Rice production not only meets domestic demand but also exports to major countries and tightly controlled markets such as Europe. In addition to food crops, industrial crops, vegetable crops also flourished in production. However, agricultural production also encountered many difficulties such as adverse weather, pests and diseases caused by pests, diseases, weeds, rats, snails, etc…, which reduced the productivity and quality of agricultural products. According to statistics of FAO (1984), annual plant diseases not only reduce the yield and quality of crops but also increase production costs.
Therefore, to protect production, we must apply a series of farming methods, physical mechanics., especially chemical measures to protect plants, which have long been widely popular in Vietnam. Pesticides not only destroy ecosystems but also remain in soil and agricultural products, affecting water quality and especially human health. Moreover, in recent years, plant pests such as fungi and bacteria have caused a lot of diseases on agricultural crops in Vietnam. There have also been a number of research works on using beneficial microorganisms as B.
thuringiensis and Beauveria bacteria to kill pests, and Metarhizium fungi to kill insects such as some beetle type. However, there are not many studies on applying microorganisms to inhibit bacteria and fungi that cause plant diseases. Furthermore, the 1992 FAO Regional Consultative Conference for Asia and the Pacific confirmed biological warfare as the foundation of an IPM 1 (Integrated Pest Management) program with a strategy of using biological agents to limit the growth of parasitic populations. One of the research directions following this trend is the use of biological agents to limit pathogenic microorganisms.
Among the biological agents commonly used to inhibit pathogenic microorganisms, Actinomycetes are known to be a special group of bacteria. They are widely distributed and play an important role in the natural cycle of matter. During life, Actinomycetes secrete many highly biologically active substances that are resistant to various microbial species including fungi and bacteria. Of the 23,000 bioactive compounds produced by microorganisms, more than 10,000 were isolated from Actinomycetes (Watve et al.
Over 80% of these compounds are different antibiotics. The researchers showed that for every 1,000 Actinomycetes isolated at random, about 10 would produce streptomycin and four would produce tetracycline. Therefore, Actinomycetes are one of the potential sources of bioactive substances (Mitra et al. Therefore, in order to have a more realistic and clearer view, I conducted research on the topic: “Screening the antagonistic ability to pathogenic microorganism and researching biochemical characteristics of the Streptomyces sp.
The research results will serve to provide the basis for the creation of biological products to replace the previous ineffective treatment methods. There by improving crop productivity, meeting the demand for clean food, saying no to pesticides, improving lives and bringing economic benefits to the people. Research purpose and content 1. The purpose of the study Screening the antagonistic ability to pathogenic microorganism and researching biochemical characteristics of the Streptomyces sp.
Research content - Collect some strains of pathogenic bacteria: 2 • The bacterium Xanthomonas axonopodis causes infections and lesions on the leaves, stems and fruits of citrus trees. • The bacterium Ralstonia solanacearum causes severe leaf wilt in many vegetables and crops. • The bacterium Clavibacter michiganensis causes diseases in tomato and potato plants. - Collect some strains of pathogenic fungi: • Fusarium solani strain (HT39).
• Colletotrichum gloeosporioides strain (HD01). - Testing the antagonistic ability of Streptomyces sp. - Study on biochemical characteristics of Streptomyces sp. 3 PART II: LITERATURE REVIEWS 2.
Overview of Actinomycetes, genus Streptomyces, Streptomyces sp. VNUA27 The Actinomycetes are known as particularly variable organisms. Actinomycetes were originally thought of like bacteria in the guise of fungi or vice versa. It was indeed a challenge to classify them.
Some scientists considered them as Eubacteriales or higher bacteria, while some thought them to belong to Hyphomycetes or lower fungi. Selman Waksman opined that these microorganisms could be grouped separately. Actinomycetes, like true bacteria (Eubacteria) are prokaryotes. Their growth (prothallus) is characterized by the formation of normally branching threads and rods, frequently giving rise to a typical mycelium, which is unicellular, especially during the early stages of growth (Experiment et al.
Generally non-septate, the hyphae may turn septate under special conditions (Experiment et al. The mycelium is prostrate, vegetative and growing in the substrate, or aerial, when a special mycelium is produced above the vegetative growth.