Characterization and identification of actinomycetes capable of antagonism with fungus colletotrichum gloeosporioides cause anthracnose disease in plants (khóa luận tốt nghiệp)

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS CHARACTERIZATION AND IDENTIFICATION OF ACTINOMYCETES CAPABLE OF ANTAGONISM WITH FUNGUS COLLETOTRICHUM GLOEOSPORIOIDES CAUSE ANTHRACNOSE DISEASE IN PLANTS HÀ NỘI - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS CHARACTERIZATION AND IDENTIFICATION OF ACTINOMYCETES CAPABLE OF ANTAGONISM WITH FUNGUS COLLETOTRICHUM GLOEOSPORIOIDES CAUSE ANTHRACNOSE DISEASE IN PLANTS Student name Class Student 's code Supervisor Department : NGUYEN MAI ANH : K62CNSHE : 620453 : Dr TRINH XUAN HOAT Assoc Prof Dr NGUYEN XUAN CANH : MICROBIAL TECHNOLOGY HÀ NỘI - 2022 COMMITMENT I hereby commit that the thesis is completely done by me under the guidance of Assoc Prof Dr Nguyen Xuan Canh, Trinh Xuan Hoat, PhD All the data and results that I have provided in this study are true, accurate, and not used in any other report I also assure that the literatures cited in the thesis indicated the origin and all help was thankful Hanoi, March 2022 Student Nguyen Mai Anh i ACKNOWLEDGEMENTS First of all, I want to express my sincere thanks to the Microbial Technology Department, Faculty of Biotechnology, and Vietnam National University of Agriculture because this thesis would not have been possible without the involvement and support of the assistance and guidance of dedicated teachers at the laboratories of the Department First and foremost, I pay my deep sense of gratitude to my principal supervisors Assoc Prof Dr Nguyen Xuan Canh, Trinh Xuan Hoat, PhD who encouraged me to complete my thesis and gave me lots of advice I would like to thank all staff and students at the Laboratory of the Department of Microbial Technology, especially Mrs Nguyen Thi Thu for helping and creating favorable conditions for me during the graduation thesis They were all very friendly and helpful to me, I was lucky to study and work in a great environment Last but not the least, my family is also an important inspiration for me So with due regards, I express my gratitude to them Thank you sincerely! Hanoi, March 2022 Student Nguyen Mai Anh ii INDEX COMMITMENT i ACKNOWLEDGEMENTS ii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ABSTRACT viii PART INTRODUCTION 1.1 Introduction 1.2 Purposes and requirements 1.2.1 Purposes 1.2.2 Requirements PART II LITERATURE REVIEW 2.1 Overview of Actinomycetes 2.1.1 Introduction and distribution of Actinomycetes 2.1.2 Classification of Actinomycetes 2.1.3 Morphological characteristics of Actinomycetes 2.1.4 Structure of Actinomycetes 12 2.1.5 The roles and applications of Actinomycetes 12 2.2 Overview of Colletotrichum gloeosporioides 19 2.2.1 Introduction of Colletotrichum gloeosporioides 19 2.2.2 Biology of Colletotrichum gloeosporioides infection process 21 2.2.3 Damage of Anthracnose disease caused by Colletotrichum gloeosporioides in agriculture 23 PART III MATERIALS AND METHODS 26 3.1 Materials 26 3.1.1 Materials 26 3.1.2 Chemicals, instruments and equipment 26 3.1.3 Experiment location and time 28 3.2 Methods 28 iii 3.2.1 Activate Actinomycetes strains preserved in glycerol 28 3.2.2 Screening of Actinomycetes with antifungal activity 28 3.2.3 Morphological characterizations of Actinomycetes 29 3.2.4 Ability to assimilate carbon sources 30 3.2.5 Ability to produce extracellular enzymes 30 3.2.6 Effects of temperature, pH and NaCl concentrations on the growth of Actinomycetes 31 3.2.7 Ability to utilize citrate 31 3.2.8 Ability to hydrolyze gelatin 32 3.2.9 Ability to decompose urea 32 3.2.10 Classification of the Actinomycetes based on 16S rRNA sequences 33 PART IV RESULTS AND DISCUSSION 36 4.1 Screening and selection of fungal antagonist Actinomycetes 36 4.2 Morphological characteristics 37 4.3 Ability to assimilate carbon sources 40 4.4 Ability to produce extracellular enzymes 42 4.5 Effects of temperature, pH and NaCl concentrations on the growth of Actinomycetes 44 4.6 Ability to utilize citrate 46 4.7 Ability to hydrolyze gelatin 47 4.8 Ability to decompose urea 48 4.9 Classification of the Actinomycetes based on 16S rRNA sequences 49 4.9.1 Total DNA extraction and concentration 49 4.9.2 Amplification of 16S rRNA sequences 50 4.9.3 Sequencing PCR products and building a phylogenetic tree 51 PART V CONCLUSIONS AND PROPOSALS 53 5.1 Conclusions 53 5.2 Proposals 53 REFERENCES 54 APPENDIX 59 iv LIST OF TABLES Table Primers for PCR 34 Table Components of PCR reaction 35 Table Colony morphology of Actinomycetes strain VNUA48 in ISP field 38 Table Assimilation of different carbon sources of Actinomycetes strain VNUA48 42 Table Effects of environmental conditions on the growth of Actinomycetes strain VNUA48 44 v LIST OF FIGURES Figure Single spore production in short chains .8 Figure 2 Morphological features of spores 10 Figure Spore production within sporangia 11 Figure Screening fungal antagonist Actinomycetes: Control fungus (A), Dual cultured Actinomycetes (B) after days of culture 36 Figure Colony morphological characteristics of Actinomycetes VNUA48 in ISP field after days of culture 37 Figure Mycelia and spore chains morphology after 50 hours of culture (A), Spore chain and spore morphology after 72 hours of culture (B) 39 Figure 4 Actinomycetes strain VNUA48 on ISP medium after days of culture 40 Figure Growth of Actinomycetes strain VNUA48 on: positive control (A), negative control (B), and basal medium containing starch (C) 41 Figure Ability to produce Amylase, Cellulase, Chitinase, Pectinase, Protease, Catalase of strain VNUA48 after days of culture 43 Figure Citrate utilization test of strain VNUA48: Negative control tube (A), test tube containing strain VNUA48 (B) 46 Figure Gelatin hydrolysis test of Actinomycetes strain VNUA48: Negative control tube (A), test tube containing Actinomycetes strain VNUA48 (B) 47 Figure Urea decomposition test: Negative control tube (A), test tube containing Actinomycetes strain VNUA48 (B) 49 Figure 10 Gel electrophoresis of total DNA extraction off Actinomycetes strain VNUA48 50 Figure 11 Gel electrophoresis of PCR product of Actinomycetes strain VNUA48 50 Figure 12 Phylogenetic tree of Actinomycetes strain VNUA48 51 vi LIST OF ABBREVIATIONS Abbreviation C gloeosporioides Meaning Colletotrichum gloeosporioides cAMP Cyclic Adenosine Monophosphate ISP International Streptomyces Project PDA Potato Dextrose Agar DNA Deoxyribonucleic acid NCBI National Center for Biotechnology Information PCR Polymerase Chain Reaction rRNA Ribosome RNA pH Power of hydrogen/potential of hydrogen R Reverse F Forward vii ABSTRACT The main object of this study is to screening and characterization Actinomycetes capable of antagoním with pathogenic fungus Colletotrichum gloeosporioides causing anthracnose disease in plants Thirty Actinomycetes were activated from preservation at the Department of Microbial Technology, Faculty of Biotechnology, Vietnam National University of Agriculture Dual culture method was used to screen and select potential antagonistic Actinomycetes The Actinomycetes strain VNUA48 with the inhibition rate was 51.11% was selected to study morphological and biochemical characteristics Then, this Actinomycetes strain was evaluated that it has ability to utilize carbon sources, produce melanin pigment, grow on different culture conditions, utilize citrate, hydrolyze gelatin, and decompose urea This Actinomycetes strain was further extracted with total DNA, ran a 16S rRNA gene sequence reaction (PCR) using primers 27F and 1492R, then sequenced and built a phylogenetic tree Based on the morphological, biochemical and 16S rRNA gene sequences, Actinomycetes strain VNUA48 which antagonizes the fungus Colletotrichum gloeosporioides has close relative to Streptomyces sp strain PSKA49 viii 4.9.3 Sequencing PCR products and building a phylogenetic tree The PCR product was purified and sequenced in Singapore After receiving the sequence, compared the obtained sequence with other sequences on the gene bank by Blast tool, build a classification tree for strain VNUA48 using MEGA11 software The results are shown in Figure 4.12 Figure 12 Phylogenetic tree of Actinomycetes strain VNUA48 Based on this classification tree, it can be seen that Actinomycetes strain VNUA48 belongs to the same clade as Streptomyces sp strain PSKA49 with a bootstrap value of 82 Besides, the results of nucleotide sequencing showed a high level of the similarity of 16S rRNA of Actinomycetes strain VNUA48 and Streptomyces sp strain PSKA49 was 99.31% In terms of reliability and similarity, these two strains are similar In addition to the morphological, physiological and biochemical characteristics studied, I found that the studied Actinomycetes strain VNUA48 has many similarities with Streptomyces sp on the gene bank Therefore, combining biological characteristics and molecular methods, I conclude that strain VNUA48 is closely related to Streptomyces sp strain PSKA49 and I named this strain as Streptomyces sp strain VNUA48 51 Streptomyces sp strains were reported that they were efficient in inhibiting mycelium growth of Colletotrichum gloeosporioids and Curvularia eragrostides fungi cause diseases in yam (Soares et al., 2006) They also antagonize some pathogenic fungi such as Sclerotinia sclerotiorum FW43, Rhizoctonia solani FW408, Fusarium oxysporum f.sp lactucae L74, Pythium ultimum FW407, Thielaviopsis basicola FW406 and Phytophthora sp FW409 (Kunova et al., 2016) 52 PART V CONCLUSIONS AND PROPOSALS 5.1 Conclusions - Only one Actinomycetes strain screened and selected from 30 strains preserved in Department of Microbial Technology, Faculty of Biotechnology, Vietnam National University of Agriculture has ability to antagonize to pathogen fungus Colletotrichum gloeosporioides - Actinomycetes strain VNUA48 has 51.11% inhibition rate with fungus Colletotrichum gloeosporioides - Actinomycetes strain VNUA48 is able to assimilate carbon sources, produce extracellular enzymes, utilize citrate, hydrolyze gelatin, and decompose urea - Actinomycetes strain VNUA48 can growth in the range of 20-40oC, pH 4-12, and 0-4% of NaCl concentration - Depending on morphological, biological characteristics and molecular methods of Actinomycetes strain VNUA48, I conclude that the VNUA48 strain belongs to genus Streptomyces and is closely related to Streptomyces sp strain PSKA49 5.2 Proposals - Evaluation of the effect of antagonistic Actinomycetes on mycelial morphology and germination of fungal spores - Secondary screening for antifungal metabolites - Evaluation of the stability of activity of Actinomycetes extracts under the influence of different environmental conditions - Researching biochemical characteristics of Actinomycetes such as the ability to produce IAA, H2S, etc - Evaluation of the effectiveness of in vivo biocontrol of Actinomycetes against C.gloeosporioides 53 REFERENCES Ahmad M S., El-Gendy A O., Ahmed R R., Hassan H M., El-Kabbany H M & Merdash A G (2017) Exploring the Antimicrobial and Antitumor Potentials of Streptomyces sp AGM12-1 Isolated from Egyptian Soil Front 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