VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** GRADUATION THESIS TITLE: “EFFECTS OF CELL-FREE FILTRATE OF BACILLUS SUBTILIS H14 STRAIN ON SCLEROTIUM ROLFSII CAUSING WHITE MOLD ROOT ROT DISEASE IN THE PEANUT” Hanoi-2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY *** GRADUATION THESIS TITLE: “EFFECTS OF CELL-FREE FILTRATE OF BACILLUS SUBTILIS H14 STRAIN ON SCLEROTIUM ROLFSII CAUSING WHITE MOLD ROOT ROT DISEASE IN THE PEANUT” Student’s name : NGUYEN THI KHANH LINH Class : K63CNSHE Student’s code : 637332 Supervisor : Dr NGO THU HUONG MSc TRAN THI HONG HANH Major : MICROBIAL TECHNOLOGY Hanoi-2022 COMMITMENT I hereby declare that this is a scientific research work carried out by me during the period from August 2022 to December 2022 under the guidance of Dr Ngo Thu Huong and MSc Tran Thi Hong Hanh, Laboratory of Microbiology Faculty of Biotechnology - Vietnam National University of Agriculture The data and research results in this thesis are honest and have never been published by anyone in other studies The documents cited in the thesis are listed in the references section Hanoi, 5th December, 2022 Sincerely, Nguyen Thi Khanh Linh i ACKNOWLEDGEMENT First of all, I would like to thank the Academy's Board of Directors, lecturers, and staff who are teaching and working at the Academy I am extremely grateful to the teachers of the Faculty of Biotechnology for teaching, guiding, and creating conditions for me to complete my study program, professional internship, and graduation thesis More specifically, I would like to express my deep respect and gratitude to Dr Ngo Thu Huong and MSc Tran Thi Hong Hanh for guiding my research and guiding me wholeheartedly during the course of this thesis I would like to thank the professors of Microbiology Technology, Assoc Prof Dr Nguyen Xuan Canh, Prof Dr Nguyen Van Giang, Ms Tran Thi Dao, and fellow researchers: Ms Nguyen Thi Thu, Mr Duong Van Hoan helped me during the thesis work Finally, I would like to thank my friends and my family for their encouragement and help so that I could complete my graduation thesis Thank you from the bottom of my heart for everything! Hanoi, 5th Decembr , 2022 Sincerely, Nguyen Thi Khanh Linh ii CONTENTS COMMITMENT i ACKNOWLEDGEMENT .ii CONTENTS iii LIST OF TABLES vi LIST OF FIGURES .vii LIST OF ABBREVIATIONS viii ABSTRACT ix PART INTRODUCTION PART LITERATURE REVIEW 2.1 Overview of peanuts 2.1.1 The history of peanuts 2.1.2 The situation of peanut production in the world and Vietnam 2.1.2.1 The situation of peanut production in the world 2.1.2.2 The situation of peanut production in Vietnam 2.1.3 The role of peanuts 2.1.3.1 The role of peanuts in the economy 2.1.3.2 The role of peanuts in agriculture 2.1.3.3 The role of peanuts in human life 10 2.1.4 White mold root rot disease on the peanuts by Sclerotium rolfsii 11 2.2 Overview of Sclerotium rolfsii 12 2.3 Overview of bacteria 16 2.3.1 Overview of Bacillus subtilis 17 2.3.2 Overview of Bacillus subtilis H14 22 2.3.3 The research situation in the world and Vietnam 23 2.3.3.1 The research situation in the world 23 2.3.3.2 The research situation in Vietnam 25 PART MATERIALS AND METHODS 26 3.1 Materials 26 iii 3.1.1 Research location and time 26 3.1.2 Research subjects 26 3.1.3 Equipments 26 3.1.4 Medium 26 3.2 Methods 27 3.2.1 The dual culture method for testing antagonistic activity 28 3.2.2 Methods for testing the effects of cell-free filtrate from B subtilis H14 strain on the mycelial growth and sclerotial germination of Sclerotium rolfsii 29 3.2.2.1 Method of filtration through sterile filter paper 29 3.2.2.2 Method of filtration through sterile Cellulose filter membrane with pore size 0.45 µm 29 3.2.2.3 Method of filtration through sterile Syringe filter membrane with pore size 0.22 μm 30 3.2.2.4 Mycelial growth inhibition test 30 3.2.2.5 Hyphal morphology inhibition test 31 3.2.2.6 Sclerotial formation inhibition test 31 3.2.2.7 Sclerotial germination inhibition test 31 3.2.3 Sclerotial morphology inhibition test 33 3.2.4 Biocontrol of S rolfsii on peanuts in vivo condition 33 PART RESULTS AND DISCUSSION 34 4.1 Antagonistic activity of B subtilis H14 strain on S rolfsii 34 4.2 Antifungal ability of cell-free filtrate from B subtilis H14 strain on the mycelial growth and sclerotial germination of Sclerotium rolfsii 34 4.2.1 Antifungal ability of cell-free filtrate by using sterile filter paper 35 4.2.2 Antifungal ability of cell-free filtrate by using sterile Cellulose filter membrane with pore size 0.45µm 36 4.2.3 Antifungal ability of cell-free filtrate by using sterile Syringe filter membrane with pore size 0.22 μm 37 4.2.3.1 Inhibitory ability of cell-free filtrate on mycelial growth 37 4.2.3.2 Inhibitory ability of cell-free filtrate on hyphal morphology 41 iv 4.2.3.3 Inhibitory ability of cell-free filtrate on sclerotial formation 41 4.2.3.1 Inhibitory ability of cell-free filtrate on sclerotial germination 44 4.2.3.2 Inhibitory ability of cell-free filtrate on sclerotial morphology 48 4.2.4 Biocontrol ability of cell-free filtrate on S rolfsii on peanuts in vivo condition 49 PART CONCLUSIONS AND PROPOSAL 52 5.1 Conclusions 52 5.2 Proposal 52 REFERENCES 53 v LIST OF TABLES Table 2.1 The situation of peanut production in Vietnam in the period from 2010 to 2020 Table 2.2 Colony, morphological, physiological, and biochemical characteristics of Bacillus subtilis 19 Table 2.3 Biological properties of Bacillus subtilis H14 22 Table 2.4 Studies investigating the effective mechanism of Bacillus subtilis against plant diseases 24 Table 4.1 The diameter of zone inhibition and antifungal ability of cell-free filtrate by using Cellulose membrane 37 Table 4.2 Effect of cell-free filtrate on the mycelium of S rolfsii 39 Table 4.3 Number of sclerotia and ratio of inhibition after 20 days of culture 43 Table 4.4 Radius and inhibitory ability of mycelium germinating from sclerotia 44 Table 4.5 Radius and inhibitory ability of mycelium germinating from sclerotia 46 Table 4.6 Radius and inhibitory ability of mycelium germinating from sclerotia 47 vi LIST OF FIGURES Figure 2.1 The situation of peanut production in the world in the period 2010 - 2020 Figure 2.2 The situation of peanut production of the largest producing countries (China, India, Nigeria) in 2020 Figure 2.3 The situation of peanut production in Vietnam in the period 2010 – 2020 Figure 4.1 The antifungal ability of S Rolfsii of B subtilis H14 after 96 h of culture 34 Figure 4.2 Effect of filtrate from methods on the growth of S rolfsii after 72 h 35 Figure 4.3 Effect of cell-free filtrate on S rolfsii mycelia after 24 h of culture 38 Figure 4.4 Effect of cell-free filtrate on S rolfsii mycelium after 48h of culture 38 Figure 4.5 Effect of cell-free filtrate on S rolfsii mycelium after 72h of culture 39 Figure 4.6 The graph shows the effect of filtrate with different dilution concentrations on the mycelium of S rolfsii 40 Figure 4.7 Mycelial morphology of S rolfsii under 100X Microscope Objective Lens 41 Figure 4.8 Effect of cell-free filtrate on the number of fungal sclerotia 42 Figure 4.9 Effect of cell-free filtrate on the number of sclerotia 43 Figure 4.10 Mycelium germinated from sclerotia after days 45 Figure 4.11 Inhibition of the germination of the mycelium of S rolfsii 46 Figure 4.12 Effect of cell-free filtrate on the germination of sclerotia under in vivo condition 47 Figure 4.13 Effect of cell-free filtrate on sclerotia morphology 48 Figure 4.14 Biocontrol effects of H14 cell-free filtrate with different concentrations on suppression of S rolfsii on peanut 49 Figure 4.15 Biocontrol effects of H14 cell-free filtrate with different concentrations on suppression of S rolfsii on the peanut 50 vii LIST OF ABBREVIATIONS Abbreviation Full word B subtilis Bacillus subtilis S rolfsii Sclerotium rolfsii Spp Subspecies Sp Species P aeruginosa Pseudomonas aeruginosa S Marcescens Serratia marcescens L monocytogenes Listeria monocytogenes A hypogaea Arachis hypogaea CT Control EX Experiment viii Figure 4.11 Inhibition of the germination of the mycelium of S rolfsii CT: Control, 1:0, 1:4, 1:9: Experiment Table 4.5 Radius and inhibitory ability of mycelium germinating from sclerotia CT: Control, 1:0, 1:4, 1:9: Experiment From Table 4.5, it shows that the inhibitory ability of H14 cell-free filtrate is very high with 100% at 1:0 ratio, 76% at 1:4 ratio and 57% at 1:9 ratio This result is similar to the study of (Li el at, 2017): At 24 h of cultivation, sclerotial germination of S rolfsii was completely inhibited on PDA medium mixed with the cell-free cultures of actinomycetes (4:1 v/v), except for strain Act12 However, the rates of inhibition obtained with the actinomycete strains decreased overtime, except for strain 25 When the sclerotia were cultivated for 96h, strain 25 still mediated 100% inhibition of sclerotial germination, while the rates for the other four strains ranged from 75.0% to 91.7% Moreover, with prolonged observation, the ability to germinate was completely lost when the sclerotia were incubated in PDA medium mixed with strain 25 cell-free culture 46 iii The antagonistic effects of cell-free filtrate on sclerotia in the soil After 15 days, affected sclerotia from the cell-free filtrate with different dilutions were removed from the soil Figure 4.12 clearly demonstrates the strong influence of cell-free filtrate on the germination of sclerotia with 100% inhibition at 1:0 ratio Figure 4.12 Effect of cell-free filtrate on the germination of sclerotia under in vivo condition CT: Control, 1:0, 1:4, 1:9: Experiment Meanwhile, the fungus at the ratio 1:4 only germinated sclerotia after days of follow-up with an inhibitory ability about 84.34% ±2.24 The effect of H14 cell-free filtrate at 1:9 dilution was about times lower than that of the 1:4 ratio with 21.09% It indicates that the germination rate of the mycelium depends on the dilution concentration Table 4.6 Radius and inhibitory ability of mycelium germinating from sclerotia CT: Control, 1:0, 1:4, 1:9: Experiment 47 The sclerotia is a survival structure composed of a hard rind and cortex containing hyphae and is typically considered the primary inoculum Sclerotia are a sustainable reserve that allows pathogens to be transmitted in the soil After 3-5 days, the mycelium will knit together to produce hundreds of sclerotia Therefore, even if you switch to a new crop, the possibility of infection is very high The sclerotia persists in the soil and on the soil surface from year to year, so it is the source and cause of white mold root rot on peanuts Therefore, this study is very meaningful in helping to effectively limit pathogens Research by Liu el at., (2019) also shown that when sclerotia were dipped into the spore suspension of NEAU-S7GS2 and placed in potted field soil for 30 days, the viability of these sclerotia was reduced to ± 2% in comparison with 82 ± 3% of the untreated sclerotia 4.2.3.2 Sclerotial morphology inhibition test Sclerotia from cell-free filtrate in a ratio of 1:0 and the control treatment were collected and observed under a stereo microscope CT EX Figure 4.13 Effect of cell-free filtrate on sclerotia morphology CT: Control, EX: Experiment Observing the results in Figure 4.13 shows a clear difference in the external morphology of the sclerotia for different cell-free filtrate addition treatment In the control treatment, the morphology of the sclerotia was mostly spherical, round, with an average diameter of 1-1.5mm Meanwhile, sclerotia on the medium 48 supplemented with cell-free filtrate appeared unusually shaped like long rods, pea-shaped, and smaller than the control 4.2.4 Biocontrol ability of cell-free filtrate on S rolfsii on peanuts in vivo condition Given the notab inhibitory effect on S rolfsii, the biocontrol ability of H14 cellfree filtrate in sclerotinia stem rot of peanut was determined in a pot experiment under in vivo condition Compared to the control that only inoculated S rolfsii, the addition of filtrate reduced the disease symptom (Figure 4.14) Figure 4.14 Biocontrol effects of H14 cell-free filtrate with different concentrations on suppression of S rolfsii on peanut A 1:1, B 1:3, C.1:5 (v/v : S rolfsii culture solution/ H14 cell-free filtrate), D Negative control (only H14 cell-free filtrate), E Control (only sterile distilled water), F Positive control (only S rolfsii) The remarkable effect was observed at concentrations of 1:0, 1:2, 1:4 (v/v : H14 cell-free filtrate/sterile distilled water) The sclerotinia stem rot disease symptoms on peanut, such as cottony growth of white mold in stems and appearance of yellow leaves, became visible at days after inoculation of S 49 rolfsii In contrast, the pre-treatment of soils with the addition of filtrate could decrease sclerotinia stem rot disease symptom development Specifically, at all ratios, peanuts did not have white mold root rot but even thrived in height (Figure 4.15) After days, the control plants infected with S rolfsii grew 3cm and after root rot, the plants were unable to grow in height but instead wilt The mycelium adheres tightly and penetrates the roots causing damage, the sclerotia are produced a lot and stick around the lower stem 1:2 of H14 cell-free filtrate with different Figure 4.15 Biocontrol effects concentrations on suppression of S rolfsii on the peanut A 1:1, B 1:3, C.1:5 (v/v : S rolfsii culture solution/ H14 cell-free filtrate), 1:0, 1:2, 1:4 (v/v : H14 cell-free filtrate/sterile distilled water), D Negative control Negative control (only H14 cell-free filtrate), E Control (only sterile distilled water), F Positive control (only S rolfsii) At a ratio of 1: must stimulate the strongest growth for plant but because the concentration is too high sometimes create the opposite 50 The study by Liu el at., (2017) also gave similar results: With a remarkable inhibitory effect on S rolfsii, the biocontrol potential of the peanut strain NEAUS7GS2 was further determined in a pot experiment under greenhouse conditions Compared with the control experiment with only S rolfsii inoculation, the addition of NEAU-S7GS2 significantly reduced the incidence and disease index However, no significant difference was observed at high concentrations of NEAU-S7GS2 A significant effect was observed at the concentration of 10 CFU/ml, the morbidity and disease index were 12 and 29%, respectively, 77 and 38% lower than the control 51 PART CONCLUSIONS AND PROPOSAL 5.1 Conclusions Researching the most optimal method of filtration of bacterial culture is using a sterile Syringe filter with a pore size of 0.22 μm The presence of bacteria on the surface of the PDA medium was no longer observed The highest ratio of H14 cell-free filtrate was 1:0 Besides, cell-free filtrate also significantly affects mycelial morphology, ability to form sclerotia, sclerotia morphology and sclerotia germination When reinfected on peanuts, while the control had wilted and rotted, the experimental plants in all ratios were very healthy, without any sign of pathogens 5.2 Proposal Due to the limited time to carry out the topic, I have not yet delved into 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