Screening the antagonistic ability to pathogenic microorganisms and researching biochemical characteristics of streptomyces sp vnua27

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.Prof Ph.D 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 Hanoi, February 2022 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 Prof 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 Prof PhD 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 i ACKNOWLEDGEMENT ii INDEX iii LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS viii ABSTRACT ix PART I: INTRODUCTION 1.1 Introduction 1.2 1.2.1 1.2.2 Research purpose and content The purpose of the study Research content PART II: LITERATURE REVIEWS 2.1 Overview of Actinomycetes, genus Streptomyces, Streptomyces sp VNUA27 2.2 2.2.1 2.2.1.1 2.2.1.2 2.2.1.3 Overview of pathogenic strains 11 Overview of pathogenic bacteria 11 Overview of bacteria Xanthomonas axonopodis 14 Overview of the bacterium Ralstonia solanacearum 19 Overview of the bacteria Clavibacter michiganensis 21 2.3 2.3.1 2.3.2 Overview of pathogenic fungal strains 23 Overview of the fungus Fusarium solanin 24 Overview of the fungus Colletotrichum gloeosporioides 25 2.4 Review of recent studies on the antagonism of Actinomycetes against bacteria and fungi 29 PART III: MATERIALS AND METHODS 30 3.1 3.1.1 3.1.2 3.1.3 3.1.4 Materials 30 Location and time of the study 30 Material 30 Equiments 30 Medium 30 iii 3.2 Research methods 30 3.2.1 Screening the antagonistic ability to pathogenic microorganisms of Streptomyces sp VNUA27 31 3.2.1.1 Antibacterial 31 3.2.1.2 Antifungal 32 3.2.2 Biochemical characteristics of Streptomyces sp VNUA27 33 3.2.2.1 The ability to produce extracellular enzymes 33 3.2.2.2 The ability to produce IAA 34 3.2.2.3 The ability to produce Hydro sulfide (H2S) 36 3.2.2.4 The ability to produce indole 36 3.2.2.5 MR Test (Methyl red test) 37 3.2.2.6 VP Test (Voges-Proskauer test) 37 3.2.2.7 The ability to utilize citrate 38 3.2.2.8 The ability to hydrolyze gelatin 38 3.2.2.9 The ability to solubilize phosphate 39 3.2.2.10 The ability to decompose ureases 39 3.2.2.11 The ability to produce siderophores 39 PART I V: RESULT AND DISCUSSION 42 4.1 4.1.1 4.1.2 Antagonistic ability of Streptomyces sp VNUA27 42 Antibacterial 42 Antifungal 43 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 4.2.8 4.2.9 4.2.10 4.2.11 Biochemical characteristics of Streptomyces sp VNUA27 44 The ability to produce extracellular enzymes 44 The ability to produce IAA 48 The ability to produce Hydrosulfide (H2S) 50 The ability to produce indole 51 MR Test (Methyl red test) 52 VP Test (Voges-Proskauer test) 53 The ability to utilize citrate 54 The ability to liquefaction gelatin 55 The ability to solubilize phosphate 56 The ability to decompose ureases 58 The ability to produce siderophores 58 PART V: CONCLUSION AND SUGGESTION 61 5.1 Conclusion 61 5.2 Suggestion 62 iv REFERENCES 63 v LIST OF TABLES Table 1: Families and genera of Actinomycetales Table 2: Classification of genus Streptomyces based on morphologic, chemotaxonomix and molecular studies 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 VNUA27 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 sepedonicum 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 VNUA27 42 Figure 6: Antagonistic activity of Streptomyces sp VNUA27 against C gloeosporioides strain (HD01) 43 Figure 7: Antagonistic activity of Streptomyces sp VNUA27 against F solanin strain (HT39) 44 Figure 8: Extracellular enzyme production ability of Streptomyces sp VNUA27 46 Figure 9: IAA Standard curve plot from to µg/ml 49 Figure 10: IAA production of Streptomyces sp VNUA27 50 Figure 11: H2S production ability of Streptomyces sp VNUA27 51 Figure 12: Indole production ability of Streptomyces sp VNUA27 52 Figure 13: MR test results of Streptomyces sp VNUA27 53 Figure 14: V-P test results 54 Figure 15: Citrate utilization ability of Streptomyce sp VNUA27 55 Figure 16: Gelatin liquefaction ability of Streptomyces sp VNUA27 56 Figure 17: Testing phosphate solubilizing ability of Streptomyces sp VNUA27 57 Figure 18: Testing the urease-producing ability of Streptomyces sp VNUA27 58 Figure 19: Testing the sideropho production ability of Streptomyce sp VNUA27 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 catabolism raise the pH of the medium to above 7.6 causing the bromothymol blue to change from the original green colour to blue Control VNUA27 Figure 15: Citrate utilization ability of Streptomyces sp VNUA27 This finding is consistent with that of Rafiq et al (2018), who found that eight isolates had enough antibacterial efficacy against test organisms The ones that delivered satisfactory results were A9, A12, B7, C1, D5, E6, E8, and E9 The majority of the isolates produced results, but they were inconsistent The findings also suggest that the isolates exhibit broad spectrum antibacterial action in the environments where they were tested That strain was reported positive with citrate utilization 4.2.8 The ability to liquefaction gelatin Extracellular proteolytic enzymes of bacteria hydrolyse proteins into polypeptides, peptides and amino acids Gelatin is an unusual kind of protein, which in aqueous solution forms a solid gel at room temperature but changes to a liquid above 25-28°C It tolerates heating at 100-121oC without being coagulated, and therefore, unlike other proteins, can be sterilized by 55 autoclaving Upon exposure to formalin, its structure changes such that it remains solid on heating up to 100°C without losing its sensitivity to gelatinase By observing that the culture medium has changed to liquid form, compared with the negative control, Actinomycetes have grown The experiment on gelatin liquefaction of Streptomyces sp VNUA27 showed positive results, Actinomycetes have gelatinase production Control VNUA27 cccococcccontrol control Figure 16: Gelatin liquefaction ability of Streptomyces sp VNUA27 Gelatinase can hydrolyze gelatine into peptides and amino acids which is called hydrolyzed gelatin and this is used in industries Aghamirian & Amir Ghiasian (2009) was showed that N brasiliensis, Nocardiopsis dassonvillei, Streptomyces griseus and Streptomyces somaliensis were the species capable of liquefying gelatin, have reported a similar subject to my thesis 4.2.9 The ability to solubilize phosphate In nature, phosphorus is an extremely important element for plant growth, especially in the early stages of growth, but most of it exists in the insoluble phosphate form Therefore, bacteria play a particularly important role in breaking down insoluble phosphate into an easily absorbable form through acidification 56 processes and reducing pH due to organic acids produced by bacteria due to the presence of many ions H + Meanwhile, there are many soil microorganisms capable of decomposing phosphate Some studies are showing that some IAAproducing bacteria also have the ability to degrade insoluble phosphate Therefore, the experiment to test the ability to degrade insoluble phosphate to be VNUA27 was conducted Streptomyces sp VNUA27 cultured in Pikovskaya medium after 21 days, the results are shown in Fig.17 the appearance of a transparent ring of light around the colony The potential application of strains that both produce IAA and solubilize phosphate in the production of microbial fertilizers to increase crop yield is very high Figure 17: Testing phosphate solubilizing ability of Streptomyces sp VNUA27 This result is similar with a report by Nandimath et al (2017) In Pikovskaya’s broth maximum soluble phosphate was released by S longisporoflavus AN-27 at 28°C followed by S fulvissimus AN-12 and S olivoverticillatum AN-20 Relatively low amount of phosphate was solubilized by S cellulosae AN-31 and S nogalater At 50°C phosphate solubilization was highest for S olivoverticillatum AN-20 (Nandimath et al., n.d.) 57 As a result, using it as a bioinoculant will enhance accessible P in soil while lowering the need for P fertilizers, reducing pollution and promoting long-term sustainability 4.2.10 The ability to decompose ureases The enzyme urease is responsible for the breakdown of urea, which increases soil pH by producing NH , which is weakly basic, and CO Streptomyces sp VNUA27 is able to produce urease enzyme to break down urea into CO2 and NH3 An increase in pH due to the production of ammonia results in a colour change from yellow (pH 6.8) to bright pink (pH 8.2) Figure 18: Testing the urease-producing ability of Streptomyces sp VNUA27 4.2.11 The ability to produce siderophores Almost all microorganisms use iron (Fe) to build cytochromes and many enzymes Iron is difficult to absorb because iron ions (Fe3+) and their derivatives are difficult to dissolve, in the environment, there are usually very few soluble iron compounds that can be transported into cells The absorption of iron by microorganisms is extremely difficult To sequester and solubilize ferric iron, 58 many microorganisms utilize an efficient system consisting of low-molecular mass (