VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - - -  - - - - GRADUATION THESIS SCREENING AND CHARACTERIZATION OF STREPTOMYCES CORCHORUSII STRAIN L72 CAPABLE OF ANTAGONISM WITH FUNGUS SCLEROTIUM ROLFSII HANOI - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - - -  - - - - GRADUATION THESIS SCREENING AND CHARACTERIZATION OF STREPTOMYCES CORCHORUSII STRAIN L72 CAPABLE OF ANTAGONISM WITH FUNGUS SCLEROTIUM ROLFSII Student nam : NGO VAN ANH Student code : 637404 Class : K63CNSHE Supervisor : Dr PHAM HONG HIEN : Assoc.Prof NGUYEN XUAN CANH Department : MICROBIAL TECHNOLOGY HANOI – 2022 DECLARATION I hereby commit that the thesis is completely done by me under the guidance of Assoc Prof Dr Nguyen Xuan Canh, Dr Pham Hong Hien 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, 5𝑡ℎ Deceber 2022 Student Ngo Van Anh i ACKNOWLEDEGE During the process of implementing my graduation project, I have received a lot of attention and help from individuals and groups First of all, I would like to express my respect and deep gratitude to Dr Pham Hong Hien and Assoc Prof Nguyen Xuan Canh for giving me the opportunity to carry out this work, and their huge efforts, enthusiasm, and support throughout the duration of the undergraduate thesis Secondly, I would like to thank the teachers in the Faculty of Biotechnology have helped and taught me during my training at university Especially the teachers of the Faculty of Biotechnology who gave me advice during carrying out Finally, I would like to sincerely thank my family members and friends who always trust, support and encourage me to complete this report Sincerely thank! HaNoi, 5𝑡ℎ December 2022 Student Ngo Van Anh ii CONTENTS DECLARATION i ACKNOWLEDEGE ii CONTENTS iii LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS ix ABSTRACT x PART I INTRODUCTION PART II LITERATURE REVIEW 2.1 Overview of peanuts 2.1.1 History 2.1.2 Biological characteristics 2.1.3 The role of peanuts 2.2 Some common harmful diseases in peanuts 2.2.1 White mold root rot disease 2.2.2 Rust disease in peanuts 2.2.3 Germ rot disease in peanuts 2.3 Overview of the Sclerotium rolfsii 10 2.3.1 General introduction the Sclerotium rolfsii 10 2.3.2 Growth characteristics of the Sclerotium rolfsii 11 2.3.3 Disease-causing characteristics 12 2.4 Overview of actinomycetes 12 2.4.1 General introduction to actinomycetes 12 2.4.2 Morphological characteristics of actinomycetes 15 2.4.3 Biochemical characteristics 17 2.4.4 The role of actinomycetes 17 2.5 Research situation in Vietnam and the world 18 iii 2.5.1 Research situation in the world 18 2.5.2 Research situation in Vietnam 19 PART III MATERIAL AND METHODS 21 3.1 Material 21 3.1.1 Location and time of the study 21 3.1.2 Material 21 3.1.3 Equipment and Chemicals 21 3.1.4 Medium 22 3.2 Methods 23 3.2.1 Screening and selection of fungal antagonist Actinomycetes 23 3.2.2 Morphological characteristics of colonies and spores 24 3.2.3 Research on cultural characteristics 24 3.2.4 Evaluation of the influence of cultural conditions 25 3.2.5 Investigation of biochemical characteristics 26 3.2.6 Classification of the Actinomycetes based on 16S rRNA sequences 31 PART IV RESULTS AND DISCUSSION 35 4.1 Screening and selection of fungal antagonist Actinomycetes 35 4.2 Morphological characteristics of colonies and spores 36 4.3 Cultural characteristics 37 4.4 Evaluation of the influence of cultural conditions 40 4.4.1 Effect of temperature 41 4.4.2 Effect of pH 42 4.4.3 Salt tolerance 43 4.4.4 The ability to assimilate the source of Carbon 45 4.4.5 Ability to assimilate Nitrogen source 46 4.5 Biochemical characteristics of actinomycete strain L72 47 4.5.1 Extracellular enzyme production 47 4.5.2 Ability to produce IAA 48 iv 4.5.3 Ability to solubilize insoluble phosphate 50 4.5.4 VP Test ( Voges – Proskauser test) 53 4.5.5 MR Test (Methy red test) 53 4.5.6 Test for catalase activity 54 4.6 Classification of the actinomycetes based on 16S rRNA sequences 55 4.6.1 Amplification of 16S rRNA sequences 55 4.6.2 Sequencing PCR products and building a phylogenetic tree 56 PART V: CONCLUSIONS AND PROPOSAL 58 5.1 Conclusions 58 5.2 Proposals 58 REFERENCES 59 v LIST OF TABLES Table 3.1 Instructions for preparing the working standard solution IAA 28 Table 3.2 Construct the concentration range of the standard curve 30 Table 3.3 Primers for PCR 33 Table 3.4 Components of PCR reaction 34 Table 4.1 Rate of antifungal resistance of selected actinomycete strains 35 Table 4.2 Growth characteristics of actinomycetes L72 on different media after days of culture 38 Table 4.3 Biochemical characterization of actinomycete strain L72 55 vi LIST OF FIGURES Figure 2.1 White mold root rot disease on peanut and its causative agent Figure 2.2 Peanut leaves infected with the fungus causing rust disease Figure 2.3 Germ rot disease in peanuts Figure 2.4 The fungus Rhizopus arrhizus 10 Figure 2.5 Schematic drawings of the different types of spore chains produced by actinomycetes 15 Figure 2.6 Diversity of structural characteristics of Actinomycetes 16 Figure 4.1 Antifungal activity of actinomycete strain L72 against Sclerotium rolfsii after days of follow-up 36 Figure 4.2 Morphology of strain L72 37 Figure 4.3 Investigation of heat resistance of actinomycete strain L72 41 Figure 4.4 Growth ability of actinomycete strain L72 at different pH levels 43 Figure 4.5 Investigation of salt concentration of strain L72 44 Figure 4.6 Assimilation of carbon sources of Actinomycetes strain L72 45 Figure 4.7 Assimilation of nitrogen sources of Actinomycetes strain L72 47 Figure 4.9 IAA standard curve 49 Figure 4.10 Experiment to generate IAA of actinomycete strain L72 49 Figure 4.11 Phosphate standard curve 51 Figure 4.12 Insoluble phosphate decomposition experiment 51 Figure 4.13 The results of the VP Test 53 Figure 4.14 MR test results of strain L72 54 Figure 4.15 Catalase activity of actinomycete strain L72 54 vii Figure 4.16 PCR product of actinomycete strain L72 56 Figure 4.17 Phylogenetic tree of Actinomycete strain L72 57 viii This result is similar to that reported by (Khamna et al., 2009), thirty-six actinomycetes were isolated producing IAA, and 30 of these were Streptomyces sp The product range for IAA is 5.5–144µg/ml Streptomyces CMU-H009 isolated from lemongrass (Cymbopogon citrates) showed high IAA production capacity Meanwhile, Datta et al., (2000) isolated and selected Enterobacteria, Azotobacteria, and Pseudomonas-producing siderophore and IAA from Colombian rice rhizomes, IAA-producing bacteria were localized in Colombian rice rhizomes from 3.5 µg/ml to 32.2 µg/ml 4.5.3 Ability to solubilize insoluble phosphate In nature, phosphate is an extremely important element for plant growth, especially in the early stages of growth, but most of it exists in the insoluble form of phosphate An essential plant nutrient, it is a non-renewable resource whose availability depends entirely on mined rock phosphate Phosphate has very important physiological roles in photosynthesis, metabolism, and cell division Therefore, actinomycetes have a particularly important role in breaking down insoluble phosphates into absorbable forms through acidification treatment and in reducing the pH due to organic acids produced by bacteria due to the presence of bacteria presence of many 𝐻 + ions Phosphate has very important physiological roles in photosynthesis, metabolism, and cell division Phosphate has a great influence on root development, stem and stem strength, flower and seed formation, plant growth and development, nitrogen fixation capacity of legumes, and resistance to various pathogens pathogens (Khan et al., 2009) Phosphate deficiency in the soil leads to reduced food production because all plants need an adequate supply of phosphorus to grow and develop Although synthetic phosphate fertilizers have played several important roles in improving crop yields, overuse of fertilizers also has its dark side as it is damaging to the environment, destroy the fertility of the soil and through the food chain seriously affect human 50 health Therefore, an experiment to test the ability to degrade insoluble phosphate of actinomycete strain L72 was conducted After liquid culture of actinomycetes in liquid NBRIP medium for days, cultured in a shaking cabinet with a temperature of 30oC, 1ml of sample solution was aspirated and centrifuged at 6000 rpm for 20 minutes The content of PO43released into the medium was determined by the molybdate green method Measuring the OD at 820 nm gives a true sample result of 0.735 Phosphate standard curve 0.25 y = 0.0432x + 0.0003 R² = 0.9963 0.2 0.211 OD 820 nm 0.18 0.15 0.133 0.1 0.081 0.05 0.045 0 3PO4 concentration mg/l Figure 4.11 Phosphate standard curve Control L72 Figure 4.12 Insoluble phosphate decomposition experiment 51 The phosphate calibration curve has the form y = 0.0432x + 0.0003 (𝑅2 = 0.9963) The content of PO43- (mg/l) released by L72 is: ~ 17.01 mg/l The potential application of L72 strains to both produce IAA and dissolve phosphate in biofertilizer production to increase crop yield is very high 52 4.5.4 VP Test ( Voges – Proskauser test) The VP reaction detects the presence of acetoin in the medium The microorganisms were grown in Clark-Lubs medium under static conditions for days The culture medium is then added with reagents If the culture turns redbrown, it means that the medium contains acetoin This is the color of the diacetylguanidine complex because diacetyl combines with guanidine in the peptone L72 Control Figure 4.13 The results of the VP Test The test results in Figure 4.13 show that the actinomycete strain L72 is capable of producing acetoin 4.5.5 MR Test (Methy red test) The MR reaction test is used to detect certain bacteria that can produce and maintain stable acids from glucose fermentation Initially, bacteria convert glucose into pyruvic acid and then convert it further to form stable acids such as lactic acid and acetic acid The acid produced lowers the pH of the medium from 4.5 or less and changes the color of methyl red If the medium is acidic, the red methyl red when added to the culture solution remains red Strain L72 was cultured in Clark-Lubs medium after days of culture and tested for results as shown in Figure 4.14 53 Control L72 Figure 4.14 MR test results of strain L72 The test results showed that the fluid of strain L72 was yellow, which was a negative result This indicates that this strain is incapable of converting Glucose into stable acids and maintaining it 4.5.6 Test for catalase activity Catalase is an enzyme that is ubiquitous or present in most living organisms The strains can convert energy by respiration with oxygen to produce H2O2 Catalase will hydrolyze H2O2 into H2O and O2 Hydrolysis of H2O2 will release O2 and cause air bubbles After experimenting, the results were obtained: Actinomycete strain L72 has the ability to degrade catalase enzyme Figure 4.15 Catalase activity of actinomycete strain L72 54 Table 4.3 Biochemical characterization of actinomycete strain L72 Biological properties L72 VP Test + MR Test - Catalase activity + Pectinase + Amylase - Protease - Xylanase + Cellulase + Ability to produce IAA + Ability to solubilize insoluble phosphate + 4.6 Classification of the actinomycetes based on 16S rRNA sequences 4.6.1 Amplification of 16S rRNA sequences The total DNA of actinomycete strain L72 was extracted according to the method of (Weisburg et al., 1991), then electrophoresed on 1% agarose gel for 45 at 80V potential of 5µl DNA mixed with 2µl loading dye to check the purity and integrity of the DNA DNA of this strain was amplified with primers 27F and 1492R (Weisburg et al., 1991) The obtained PCR product has a size of about 1,500 bp 55 Ladder Blank L72 Figure 4.16 PCR product of actinomycete strain L72 4.6.2 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 L72 using MEGA11 software The results are shown in Figure 4.17 56 Figure 4.17 Phylogenetic tree of Actinomycete strain L72 Based on the taxonomic tree, the actinomycete strain L72 belongs to the same clade as Streptomyces corchorusii with a bootstrap value of 91 The results of nucleotide sequencing showed that the similarity density of the two 16S rRNA sequences of L72 and Streptomyces corchorusii was 98.99% Therefore, combining biological characteristics and molecular methods, we conclude that strain L72 is closely related to Streptomyces corchorusii strain AUH-1 and we named this strain as Streptomyces corchorusii strain L72 Streptomyces corchorusii 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) 57 PART V: CONCLUSIONS AND PROPOSAL 5.1 Conclusions Based on research results on morphological, physiological, biochemical and 16S rRNA gene sequence analysis, the actinomycete strain L72 with the strongest antagonist activity with S.rolfssi was identified as Streptomyces corchorusii strain L72 It has the ability to grow well on ISP2, ISP3 and ISP4 medium with a temperature range from 30oC - 45oC, pH 7-9, NaCl concentration from 0% - 4% In addition, actinomycete strain L72 also has the ability to assimilate carbon sources in the form of double and single sugars Peptone is the best nitrogen source for the strain This strain can produce IAA and the ability to solubilize insoluble phosphate 5.2 Proposals Continue to evaluate the effect of actinomycete strain L72 on peanut growth 58 REFERENCES VIETNAMESE Cảnh N X., Cường H T., Định N T & Hiếu P T (2016) Nghiên cứu chủng xạ khuẩn có khả đối kháng Tạp chí Khoa học Nơng nghiệp Việt Nam, 14(11): 1809-1816 Lê Như Cương, Nguyễn Thị Nhung & Nguyễn Thị Diễm (2018) Khả kháng nấm hạn chế bệnh héo rũ gốc mốc trắng lạc (Sclerotium rolfsii) dung dịch nano bạc Hue University Journal of Science: Agriculture and Rural Development, 127(3A), 161–171–161–171 Đoàn Thị Thanh Nhàn (1996) Giáo trình cơng nghiệp, NXB Nơng nghiệp Hà Nội Đỗ Tấn Dũng (2006) Nghiên cứu bệnh héo rũ gốc mốc trắng (Sclerotium rolfsii sacc.) hại số trồng cạn vùng Hà Nội phụ cận năm 20052006 Tạp chí BVTV, số 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