VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ******* GRADUATION THESIS CHARACTERIZATION AND IDENTIFICATION OF AN ACTINOMYCES VNUA30 STRAIN WITH BIOACTIVITY AGAINST FUSARIUM OXYSPORUM CAUSING THE PANAMA DISEASE ON BANANA Student’s name : Hoang Ngoc Anh Student’s code : 610585 Class : K61CNSHE Supervisor : Nguyen Xuan Canh, PhD Ha Noi, 2021 GUARANTEE I hereby commit that the thesis is completely done by me under the guidance of Nguyen Xuan Canh, PhD All the data and results that I have provided in this study are true, accurate and not used in any other report To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made I take full responsibility for my commitment to the Academy Hanoi, January 2020 Student i ACKNOWLEDGEMENTS I have finished my thesis after six months working in Microbial Technology Department, with the assistance and guidance of dedicated teachers at the laboratories of the Department, with my effort to learn own pratice First and foremost, I gratefully give acknowledgement to Vietnam National University of Agriculture for giving us a good environment to study and a chance of completing my thesis Second, my sincere thanks to my principle supervisor Nguyen Xuan Canh He guided and supported me so wholehertedly during the time of the thesis He gave me lots of advice so I could complete my thesis I would like to thanks to all staffs and students at the Laboratory of Department of Microbial Technology for helping and creating favorable conditions for me during graduation exercise They were all very friendly and helpful to me, I was lucky to study and work in a great enviroment Last but not least, I am very approciated to my family and friends for all their help, encouragement and for supporting me during the process of learning and research Thank you sincerely ii CONTENTS GUARANTEE i ACKNOWLEDGEMENTS ii LIST OF TABLE iv LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ABSTRACT viii PART INTRODUCTION 1.1 Introduction 1.2 Purpose and requirements 1.2.1.Purpose 1.2.2.Request PART LITERATURE REVIEW 2.1 Overview of banana and diseases on banana 2.1.1 Overview of banana 2.1.2 Diseases on Banana 2.2 Overview of Fusarium oxysporum and Panama disease 2.2.1 Overview of Fusarium oxysporum 2.2.2 Overview of Panama disease 2.3 Overview of Actinomycetes 10 2.3.1 Definition, classification and distribution of Actinomycetes 10 2.3.2 Morphological characteristics of actinomycetes 11 2.4 Application of actinomycetes 14 3.1 Materials 15 3.1.1 Materials 15 3.1.2 Chemicals, instruments and equipments 15 3.1.3 Medium 16 3.1.4 Location and time studies 17 iii 3.2 Research methods 17 3.2.1 Selection of actinomycetes capable of antagonisms with pathogenic fungi - Actinomycetes culture method 17 3.2.2 Research on biological characteristics and identified actinomycetes 18 PART IV RESULT AND DISCUSSION 20 4.1 Screening actinomycetes capable of antagonism with fungi 20 4.2 Characterization of VNUA30 strain 21 4.3 Identification of actinomyces VNUA30 based on 16S rRNA sequence 29 4.3.1 DNA extraction 29 4.3.2 PCR amplification 30 4.3.3 Phylogenetic tree 31 PART V CONCLUSION – SUGGESTION 32 5.1 Conclusion 32 5.2 Suggestion 32 REFERENCES 33 APPENDIX 37 iv LIST OF TABLE Table 2.1 Diseases on banana Table 4.1 Some characteristics of VNUA30 strain after cultured days at 30° 22 Table 4.2 Results of testing the effect of temperature 28 Table 4.3 The ability to assimilate different carbon sources of VNUA30 strain after cultured days 29 v LIST OF FIGURES Figure 2.1 Banana trees afflicted with Panama disease Figure 3.1 Morphology of Fusarium oxysporum - GT1 on PDA medium 15 Figure 4.1 The results of testing the antifungal activity of actinomyces VNUA30 strain by dual cultured method after cultured days 20 Figure 4.2 Morphology of VNUA30 strain on the Gause I medium after cultured days at 30°C 21 Figure 4.3 Conidiophore morphology of VNUA30 strain under optical microscope at 1000 times magnification after 48 cultured hours 22 Figure 4.4 Salt concentration affect the growth and development of actinomyces VNUA30 strain 24 Figure 4.5 Testing the effect of pH 27 Figure 4.6 The result of total DNA electrophoresis 30 Figure 4.7 The result of PCR reaction marker 1500bp 30 Figure 4.8 The taxonomic tree based on the sequence of 16S rRNA gene coding 31 32 vi LIST OF ABBREVIATIONS Abbreviation Meaning DNA Deoxyribonucleic acid NCBI National Center for Biotechnology Information PCR Polymerase Chain Reaction Deoxylribose nucleic acid DNA rDNA Ribosome DNA vii ABSTRACT For the purpose of this research is to characterize and identify the actinomyces VNUA30 strain with bioactivity against Fusarium oxysporum causing the Panama disease on Banana I conducted testing capabilities antagonism between actinomyces and fungi, then researched the morphology, physiological- biochemical characteristics and identified the actinomyces with 16s rRNA gene sequencing From 30 actinomycete strains were stored at the laboratory, VNUA30 strain had capable of strong resistance to pathogenic fungi I conducted to studied the morphology characteristics and physiology- biochemical characteristics VNUA30 strain had off- white colonies, bulging center and globose shape Optimal temperature of VNUA30 strain is 30°C, VNUA30 strain belong to alkaline actinomycetes, it grew very well at pH 7-10 Beside, VNUA30 strain could survice at medium with low salt concentration and had capable of assimilating different sources from various sugar sources Based on morphological characteristics, physiological and biochemical characteristics, VNUA30 strain was similar to Streptomyces actinomycetes Moever, by 16s rRNA gene sequencing the actinomyces VNUA30 strain was identified as Streptomyces VNUA30 viii PART INTRODUCTION 1.1 Introduction Banana is one of the important crops in Vietnam's agriculture The constituents of banana are now using widly in the food industry Ripe bananas are known as a nutritious fruit Beside they may also be used to flavour muffins, cakes, or breads Banana not only is known as nutritious fruit but also is medicinal fruit The active principles of banana are beneficial to cure skin diseases, to help in diarrhoea and it is so good for stomatch Nowadays, in Vietnam, the cultivation of banana supplies a large amount as nutritious fruit for consumers demand, bring high economic value to rural economic development However, diseases have caused great damage in yield and quality of banana One of the main disease in banana is Panama The disease is caused by fungiFusarium oxysporum But, the use of chemical substance in the treatment of diseases was not optimal and one of the best ways to prevent and treat is that using of bioproducts to inhibit and destroy pathogenic fungi Actinomycetes are the most widely- distributed group of microorganisms in nature, which primarily inhabit the soil (Oskay et al., 2004) They are known to have the capacity to synthesize bioactive secondary metabolites, which include enzymes, herbicides, pesticides and antibiotics Therefore, the study and use of actinomycetes as well as biologically active substance that they generate to inhibit pathogenic organisms is a method efficiently and enviromentally friendly From this practice, I conducted the theme :”Characterization and Identification of an Actinomyces VNUA30 strain with bioactivity against Fusarium oxysporum causing the Panama disease on Banana” 1.2 Purpose and requirements 1.2.1.Purpose Characterization and identification of an Actinomyces VNUA30 strain with bioactivity against Fusarium oxysporum causing the Panama disease on Banana pH 10 pH 11 pH 12 Figure 4.5 Testing the effect of pH Result showed that VNUA30 strain had capable of growth on the medium with wide pH range They can live and grow at pH fom 4-11 At the pH from 710, VNUA30 strain can grow perfectly, so the pH from to 10 is their optimum growth pH VNUA30 strain can not grow at the pH lower than and higher than 12 4.2.2.3 Testing the effect of temparature on the growth capacity There are lots of microbial species and each types of them also effected by the various temperature conditions The frequent changes of temperature affect growth, development and synthesis of compounds of actinomycetes 27 Similar to the pH, each type of microorganism has temperature limitations: Lowest temperature, optimal temperature and the highest temperature To find the optimal temperature that VNUA30 strain can grow perfectly, I conducted culturing VNUA30 strain on the Gause I medium and placed at different temperatures 15°C, 20°C, 30°C, 37°C, 40°C, 50°C After 5- cultured days, I observed the result Results are shown in the table below: Table 4.2 Results of testing the effect of temperature Temperature 15°C 20°C 30°C 37°C 45°C 50°C Result - + ++ + - - Note: (-): No growth; (+): Grow; (++): Good growth Results showed that VNUA30 strain can grow well in the teamperature range from 20°C to 40°C The optimal temperature of VNUA30 strain is 30°C The temperature less than 15°C and more than 40°C not suitable for strain 30 to survice 4.2.2.4 The ability to assimilate carbon sources As we know, Carbon compounds were in the leading meaningful growth and formation of antibiotics in actinomycetes For many actinomycetes, appropriate carbon source was starch However, there were some strains using well simple sugars such as glucose, fructose, lactose and some strains used well doubles sugars such as maltose To determine the ability to assimilate different carbon sources, we conducted culturing strain 30 on the Gause I medium with addition of various carbon sources at the concentration of 1% ISP-9 medium added with 1% D-glucose sugar was considered positive control Check the resuls after days of culture The results are showed in the table: 28 Table 4.3 The ability to assimilate different carbon sources of VNUA30 strain after cultured days Carbon sources Ability to assimilate Raffinose +++ Starch +++ Glyxerol +++ Cellulose + D- Galactose + D- Inositol ++ D- Manitol +++ Note (+): It can assimilate carbon sources; (++): Assimilate strongly sources of carbon; (+++): Assimilate very strongly sources of carbon Look at the table, results showed that VNUA30 strain had capable of assimilating different sources from various sugar sources VNUA30 strain had strongly assimilating raffinose, starch, glyxerol and D- Manitol and it growed poorly in the Cellulose and D- Inositol 4.3 Identification of actinomyces VNUA30 based on 16S rRNA sequence 4.3.1 DNA extraction Total DNA was extracted from VNUA30 strain After extraction, total DNA was loaded in Agarose gel 0.8% 29 Figure 4.6 The result of total DNA electrophoresis 4.3.2 PCR amplification Total DNA were enough quality for PCR reaction.We use a pair of primers: 27F and 1249R Two primers 27F and 1429 R have respective sequences: Forward primer 27F: 5’- TAACACATGCAAGTCGAACG-3’ Reverse primer 1249R: 5’ GGTGTGACGGGCGGTGTGTA-3’ Reaction was operated according to the following amplification process: 95°C (5min) following by 30 cycles of 95°C (30 second), 53°C (30 second), and 72°C (1 min) and followed by a final extention step of mins at 72°C Figure 4.7 The result of PCR reaction marker 1500bp 30 4.3.3 Phylogenetic tree Figure 4.8 The taxonomic tree based on the sequence of 16S rRNA gene coding The tree is construted according to the neighbor- joining method The analysis involved 11 nucleotide sequences Evolutionary analyses were conducted in MEGA X I compared with corresponding 16S rRNA gene sequence published on Genbank, the 16S rRNA gene of VNUA30 strain was strongly homologous to corresponding 16S rRNA gene of genus Streptomyces neyagawaensis The low genetic difference belong to Streptomyces neyagawaensis strain with confidence is 76% Moever, based on the study results of morphological, physiological, biochemical characteristics of VNUA30 strain so similar to the Streptomyces neyagawaensis strain Based on all of them, the VNUA30 strain belongs to the genus Streptomyces.So, VNUA30 strain is identified as Streptomyces VNUA30 31 PART V CONCLUSION – SUGGESTION 5.1 Conclusion We found VNUA30 strain had capable of strong resistance to fungi cause disease in banana Research morphological and biochemical, physiological characteristics of VNUA30 strain: Optimal temperature, optimal pH, suitable salt concentration for growth of VNUA30 strain - Morphological: Colonies of VNUA30 strain belong to white group, bulging center and globose shape - Optimal temperature: VNUA30 strain had temperature range from 20°C -40°C, It can grow well from 30°C-40°C and the optimal teperature of VNUA30 strain is 30°C - Optimal pH: pH range of VNUA30 strain from 5- 11 VNUA30 strain grow well from 7-10 Therefore, VNUA30 strain belong to alkaline actinomycetes - Suitable salt concentration: VNUA30 strain can survice at the medium add salt NaCl at concentration from 0%-3% It can not survice and grow at higher concentration VNUA30 strain belong to low salt concentration group - The ability to assimilate carbon sources: VNUA30 strain had capable of assimilating different sources from various sugar sources VNUA30 strain had strongly assimilating raffinose, starch, glyxerol and D- Manitol - VNUA30 strain was identified as Streptomyces VNUA30 5.2 Suggestion After identified the Streptomycetes VNUA30 I suggest to research the biocontrol and plant growth promoting effect of Streptomyces VNUA30 32 REFERENCES Vietnamese literature Nguyễn Lân Dũng(2000) Vi sinh vật học, Nhà xuất giáo dục Nguyễn Lân Dũng, Đoàn Xuân Mượn, Nguyễn Phùng Tiến, Đặng Đức Trạch, Phạm Văn Ty( 1972) Một số phương pháp nghiên cứu vi sinh vật học, Tập I, NXBKHKT Hà Nội Lê Thị Hiền, Đinh Văn Lợi, Vũ Thị Vân, Nguyễn Văn Giang(2014) Phân lập tuyển chọn chủng xạ khuẩn (Streptomyces spp.) đối kháng bệnh nấm cây, Tạp chí Khoa học Phát triển 5:656-664 Lương Đức Phẩm (2004) Công nghệ vi sinh vật.NXB Nông 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