VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -  - GRADUATION THESIS TITLE: ISOLATION AND CHARACTERIZATION OF BACTERIA WITH ANTIFUNGAL ACTIVITY AGAINST PLANT PATHOGENS Hanoi, December 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  GRADUATION THESIS TOPIC: “ISOLATION AND CHARACTERIZATION OF BACTERIA WITH ANTIFUNGAL ACTIVITY AGAINST PLANT PATHOGENS” Student name : Nguyen Thi Hong Nhung Student code : 637162 Class : K63CNSHE Supervisor : Dr Dang Thi Thanh Tam Faculty : Biotechnology Execution time : 07/2022 – 12/2022 Hanoi – 10/11/2022 DECLARATION OF AUTHORSHIP I hereby declare that this is my research work The data, results and reports mentioned herein are honest and have not been used to be published in previous theses, dissertations, and scientific research works I hereby certify that the citation information used in the thesis is clearly stated in the source, ensuring that the citation is correct as prescribed I take full responsibility for this promise! Hanoi, November 2022 Student Nguyen Thi Hong Nhung i ACKNOWLEDGMENT During the process of completing this report, in addition to my efforts, I received a lot of dedicated help from teachers, family, and friends First of all, I would like to express my sincere thanks to Dr Dang Thi Thanh Tam - Lecturer in the Department of Botany, Faculty of Biotechnology, Vietnam National University of Agriculture - is the person who directly guided and imparted a lot of valuable knowledge during the completion of the graduation thesis , creating all conditions for facilities and equipment to help me complete the assigned topic At the same time, I would also like to express my deep gratitude to the teachers at the Faculty of Biotechnology, the Vietnam National University of Agriculture for their enthusiastic help and knowledge transfer during my time studying at the school Finally, I would like to thank my family and friends who wholeheartedly supported and encouraged me in the process of studying and completing this report Hanoi, November 2022 Student Nguyen Thi Hong Nhung ii ABSTRACT This study aim to screen the potential bacterial isolates with antifungal activity against plant pathogens from 86 isolates From six different soil samples in Northern regions (Ha Tay, Tuyen Quang, Quang Ninh, Hung Yen, Tue Vien, Thai Binh) screened 86 bacteria isolates with antifungal activity on screening plates From 86 bacteria isolates were evaluated the antifungal activity of the Fusarium oxysporum Continue, selected twenty isolates displaying strong antagonism against Fusarium oxysporum Twenty potential isolates were evaluated with antagonistic activity against Fusarium oxysporum after 3,5,7 days In addition, evaluated the antifungal activity of twenty isolates against other fungi such as Fusarium solani and Alternaria alternata after 3,5,7 days Since then, the selected 20 isolates were determined morphological, molecular, physiological and biochemical characteristics (Gram staining, Catalase test, IAA and morphology) Finally, some isolates showed strong antifungal activity against plant pathogens: HT4(52%), HT2(58%), QN3(53%), TV2.5(60%), TV2.11(54%), TV2.12(56%) and IAA production iii CONTENT DECLARATION OF AUTHORSHIP i ACKNOWLEDGMENT ii ABSTRACT iii CONTENT iv LIST OF TABLES vi LIST OF FIGURES vii PART 1: INTRODUCTION 1.1 Introduction 1.2 Aim and requirements 1.2.1 Aim 1.2.2 Requirements PART 2: LITERATURE REVIEW 2.1 Bacteria with antifungal activity against plant pathogens 2.1.1 Bacillus sp 2.2 Fungal pathogens 2.2.1 Fusarium oxysporum 2.2.2 Fusarium solani 10 2.2.3 Alternaria alternata 11 2.3 Pathogenicity of Fusarium oxysporum 11 2.4 Control of Fusarium diseases 12 2.5 Studies of bacteria with antifungal activity against plant pathogens 14 PART 3: MATERIALS AND METHODS 16 3.1 Materials 16 3.1.1 The culture medium was used as follows 16 3.1.2 Fungal pathogens 16 3.1.3 Soil samples 18 iv 3.1.4 Tools, chemicals 19 3.2 Methods 19 3.2.1 Dilution Plate Method 19 3.2.2 Gram Staining 20 3.2.3 Catalase test 21 3.2.4 Determination of indole -3-acetic acid (IAA) synthesis capability 22 3.2.5 Screening of potential bacteria isolates with antagonistic activity against fungal pathogens 24 PART RESULTS AND DISCUSSION 25 4.1 Results 25 4.1.1 Experiment 1: Isolating and screening bacteria isolates with antifungal activity 25 4.1.2 Experiment 2: Screening of potential bacteria isolates with antagonistic activity against fungal pathogens 26 4.1.3 Experiment 3: Evaluation of 20 potential isolates with antifungal activity against fungal pathogens 27 4.1.4 Experiment 4: Characterization of the potential bacterial strains 34 4.2.Discussion 37 PART CONCLUSION AND PROPOSAL 38 5.1 Conclusion 38 5.2 Recommendations 38 REFERENCES 39 v LIST OF TABLES Table 4.1: Summarization of potential bacteria isolates with antifungal 25 Table 4.2: Effects of bacteria strains on the mycelial growth inhibition to Fusarium oxysporum (3-7 days) 28 Table 4.3: Effects of bacteria strains on the mycelial growth inhibition to Fusarium solani (3-7 days) 30 Table 4.4: Effects of bacteria strains on the mycelial growth inhibition to Alternaria alternata (3-7 days) 32 Table 4.5: Characteristic of the potential bacterial strains 34 Table 4.6: IAA production produced by potential isolates 35 vi LIST OF FIGURES Figure 2.1: Some probiotics are applied from bacillus strains with antifungal 14 Figure 3.1: Fusarium oxysporum 17 Figure 3.2: Fusarium solani 17 Figure 3.3 Alternaria alternata 18 Figure 3.4: soil samples 18 Figure 3.5: Soil sample dilution and inoculation 20 Figure 3.6: Gram staining steps 21 Figure 3.7: Catalase test 21 Figure 3.8: Standard curve 23 Figure 3.9: Experimental design images 24 Figure 4.1: The number of bacteria selected isolates from soil samples 26 Figure 4.2: Experimental design images 26 Figure 4.3: Antagonistic activity of screening isolates against Fusarium oxysporum 27 Figure 4.4: Ten isolates showed strongest antifungal activity against Fusarium oxysporum 29 Figure 4.5: Ten isolates showed strongest antifungal activity against Fusarium solani 31 Figure 4.6: Ten isolates showed strongest antifungal activity against Alternaria alternata 33 Figure 4.7: Catalase test of 20 potential bacterial strains 36 Figure 4.8: Colonial morphology of 20 potential isolates with antifungal activity against fungal pathogens 36 vii PART 1: INTRODUCTION 1.1 Introduction Plants often suffer from diseases caused by fungi, bacteria, protists, nematodes, and viruses Plant pathogens are of scientist interest for some reasons, ranging from concerns about fragile ecosystems to the desire to protect the food supply Plant pathogens that cause plant diseases reduce a grower’s ability to produce crops and can infect almost all types of plants Plant pathogens cause disease in plants and cause losses in food and other necessary items The losses may be light or very severe, sometimes destroying all the plants and causing hunger, starvation and famines Some common plant diseases such as Black spots, other leaf spots, powdery mildew, downy mildew, blight, etc In Vietnam, plant diseases caused by fungi are very common and seriously affect agriculture and the economy One of them is a plant disease caused by Fusarium oxysporum, Fusarium solani, and Alternaria alternata Fusarium oxysporum is also a ubiquitous fungus widely distributed in the environment Fusarium oxysporum diseases are significant hindrances to food plant production and are very difficult to control, especially soilborne diseases caused by Fusarium oxysporum Soilborne plant diseases are those caused by the infection of pathogens in soil via the roots Fusarium oxysporum is a representative of soilborne pathogens It inhabits the soil for a long time in the form of chlamydospores It penetrates the roots, extends in the tissues, colonizes and metastasizes in xylem vessels, and causes systemic yellowing, wilting, and death in plants (Arie, 2019) In Vietnam, Fusarium oxysporum causes diseases in bananas, cabbage, tomatoes, peppers etc Fusarium oxysporum has strong growth at soil temperatures above 24°C and can live indefinitely in soil without access to living host plants Infected plants are usually stunted; their leaves turn pale green to golden yellow and later wilt, wither, die, and drop off progressively Figure 4.3: Antagonistic activity of screening isolates against Fusarium oxysporum 4.1.3 Experiment 3: Evaluation of 20 potential isolates with antifungal activity against fungal pathogens 27 Table 4.2: Effects of bacteria strains on the mycelial growth inhibition to Fusarium oxysporum (3-7 days) No Isolates 10 11 12 13 14 15 16 17 18 19 20 Control HT1 HT2 HT4 QN1 QN3 QN5 M1 M8 M10 DL1 DL10 NA6 NA8 NA10 NA15 TV2.1 TV2.5 TV2.9 TV2.11 TV2.12 Mycelial growth inhibition (%) After days After days After days 0 21.72±10.59 18.75±8.57 37.04±5.75 16.21±1.18 40.75±4.22 54.88±1.11 10.68±7.47 23.69±6.57 35.81±6.35 17.65±5.93 20.70±9.97 21.92±1.15 11.14±8.85 29.65±11.37 36.28±9.09 12.26±3.43 21.45±1.78 34.48±4.28 0.39±6.51 6.4±6.78 25.65±14.29 5.9±8.06 33.07±4.61 0 14.06±2.19 9.7±10.66 4.88±6.85 19.27±4.84 17.55±4.05 12.55±11.32 21.81±16.07 13.72±8.49 21.96±5.17 32.33±11.47 3.34±5.59 16.62±4.23 32.74±2.76 18.67±7.62 25.11±4.57 26.81±4.69 12.53±8.09 27.71±5.59 36.51±13.57 12.5±13.24 11.58±2.71 36.78±6.09 7.95±8.13 17.8±4.48 49.30±2.38 21.13±4.72 6.29±6.44 36.73±0.92 24.25±17.5 24.39±12.77 45.19±7.72 18.79±0.63 30.84±8.49 52.45±3.64 The results showed that the percentage of mycelial growth inhibition ranged from 14% to 54 % (Table 4.2) We can see that isolates: HT1, HT2, TV2.1, TV2.5, TV2.11, TV2.12 have the strongest antifungal activity against Fusarium oxysporum (35.81% - 54.88%) Moreover, the strain HT2 presented the highest activity, causing more than 52% inhibition of mycelial growth among the 20 evaluated isolates For HT2 strain, the ability to inhibit Fusarium oxysporum after days of dual culture was 16% The rate of inhibition 28 increased gradually after 5-7 days reaching 54% In contrast, the strain M10 showed the lowest effect, less than 15 % inhibition of colony growth in two of the tested fungi In the research of Rania Aydi Ben Abdallah showed Alcaligenes faecalis (S18) and Bacillus cereus (S42) have the ability to inhibit the Fusarium oxysporum Pathogen mycelial growth was inhibited by 44.1% and 42.1% with S42 and S18 isolates, respectively (Abdallah et al., 2016) Thus, HT2, TV2.11 and TV2.12 strains have higher antifungal activity against Fusarium oxysporum than the two strains S18 and S42 (>45%) Figure 4.4: Ten isolates showed strongest antifungal activity against Fusarium oxysporum 29 Table 4.3: Effects of bacteria strains on the mycelial growth inhibition to Fusarium solani (3-7 days) No Isolate Control HT1 HT2 HT4 Mycelial growth inhibition (%) After days After days After days 0 51.13±4.37 53.93±6.21 53.29±2.24 57.19±1.75 53.78±2.58 62.58±1.52 QN1 15.43±5.65 19.91±6.34 59.38±2.75 66.41±1.1 QN3 19.59±6 56.15±3.06 63.66±3.5 QN5 49.09±4.8 57.65±5.16 M1 50.6±2.07 56.78±1.32 M8 46.43±2.74 54.21±1.81 M10 10.04±4.54 18.26±5.84 51.17±3.76 58.28±5.34 10 DL1 16.25±4.52 48.56±2.76 56.94±3.79 11 DL10 19.42±6.75 49.5±4.56 55.67±3.38 12 NA6 19.55±4.71 51.2±2.88 60.36±0.41 13 NA8 20.6±7.29 47.92±4.43 57.84±1.09 14 NA10 16.91±5.61 45.47±1.54 52.61±3.57 15 NA15 26.76±2.08 50.79±2.75 60.59±1.51 16 TV2.1 35.33±3.77 45.34±3.42 57.51±2.22 17 TV2.5 44.72±1.38 55.09±1.37 67.37±2.16 18 TV2.9 31.97±5.61 44.77±3.81 49.48±2.42 19 TV2.11 26.5±3.95 41.54±4.03 54.73±1.99 20 TV2.12 50.92±10.54 48.81±8.59 60.87±7.10 9.31±7.99 24.71±4.01 13.29±16.1 15.67±6.11 The results showed that the percentage of mycelial growth inhibition ranged from 49% to 67% (Table 4.3) We can see that isolates: HT4, QN1, QN3, 30 TV2.5, TV2.12, NA15 have the strongest antifungal activity against Fusarium solani (60.59% - 67.37%) Moreover, the strain TV2.5 presented the highest activity, causing more than 67% inhibition of mycelial growth among the 20 evaluated isolates For TV2.5 strain, the ability to inhibit Fusarium oxysporum after days of dual culture was 44% The rate of inhibition increased gradually after 5-7 days reaching 67% In contrast, the strain TV2.9 showed the lowest effect, less than 49% inhibition of colony growth in two of the tested fungi However, 20 isolates were all able to strongly antifungal activity against Fusarium solani ( ˃49%) In the research of Nailea Báez-Vallejo, two bacterial strains, Bacillus sp CCeRi1-002 and Pseudomonas sp CCeRi5-020, significantly inhibited Fusarium solani mycelial growth, by 50.6 % and 64.7 % (Abdallah et al., 2016) Thus, HT4, QN1, QN3 and TV2.5 strains have higher antifungal activity against Fusarium solani than the two strains CCeRi1-002 and CCeRi5-020 (>62%) Figure 4.5: Ten isolates showed strongest antifungal activity against Fusarium solani 31 Table 4.4: Effects of bacteria strains on the mycelial growth inhibition to Alternaria alternata (3-7 days) No Isolates Mycelial growth inhibition (%) Control After days HT1 10.44±11.1 42.27±5.1 61.11±3.39 HT2 52.99±6.25 62.14±3.47 HT4 28.31±3.77 18.98±2.78 42.2±10.09 57.40±2.15 QN1 30.64±7.61 53.12±7.51 59.36±3.36 QN3 51.34±1.36 59.43±2.05 QN5 37.92±18.62 56.20±10.83 M1 34.88±12.19 56.13±7.48 M8 11.93±3.94 41.16±1.69 54.07±9.44 M10 10.8±8.35 35.04±1.78 51.22±1.56 10 DL1 12.63±3.75 46.69±0.53 60.17±1.36 11 DL10 19.23±6.5 48.97±2.98 62.31±1.87 12 NA6 21.01±9.11 44.44±3.67 61.91±1.31 13 NA8 13.31±9.66 45.14±7.08 57.03±2.99 14 NA10 11.46±9.42 41.98±2.61 55.19±3.89 15 NA15 19.02±8.35 41.16±6.18 54.35±3.78 16 TV2.1 20.29±1.23 47.65±2.87 58.91±1.03 17 TV2.5 26.5±3.45 48.99±2.5 61.96±3.88 18 TV2.9 26.55±8.96 48.08±4.35 59.89±2.58 19 TV2.11 1.67±6.83 40.79±6.03 62.68±2.83 20 TV2.12 17.04±13.66 40.83±5.48 54.70±5.42 26.91±2.32 13.29±19.11 After days After days The results showed that the percentage of mycelial growth inhibition ranged from 51% to 62.68% (Table 4.4) We can see that isolates: : HT1, HT2, 32 NA6, DL10, TV2.5, TV2.11 have the strongest antifungal activity against Alternaria alternata (61.11% - 62.68%) Moreover, the strain TV2.11 presented the highest activity, causing more than 62% inhibition of mycelial growth among the 20 evaluated isolates For TV2.11 strain, the ability to inhibit Fusarium oxysporum after days of dual culture was 2% The rate of inhibition increased gradually after 5-7 days reaching 62% In contrast, the strain M10 showed the lowest effect, less than 52 % inhibition of colony growth in two of the tested fungi However, 20 isolates were all able to strongly antifungal activity against Alternaria alternata ( ˃51%) In the research of Nailea Báez-Vallejo, Bacillus siamensis, strain LZ88, exhibited strong antifungal activity with an inhibition rate of 81.96% (Xie et al., 2021) Thus, isolated strains have lower antifungal activity against Alternaria alternata than strain LZ88 (49%, HT2 >54%, QN3 >36%) and with the high indole-3-acetic acid (IAA) synthesis capability TV2.11, TV2.12, HT4 trains are gram- positive, have the strong antifungal activity against fungal pathogens (HT4 >35%, TV2.11 >45%, TV2.12 >52%) and with the high indole-3-acetic acid (IAA) synthesis capability So, these strains can application in agriculture as bioinoculant for agriculturally important plants 4.2 Discussion The use of beneficial bacterias in agriculture and other distorted ecosystems can help to protect crops against phytopathogens It is known that bacterias associated with plants can promote their growth and development, e.g due to the growth inhibition of phytopathogenic microorganism Isolation of new antagonistic strains is necessary to improve biological control methods and restrain plant diseases Several bacterial strains have a suppressive effect on growth of certain phytopathogens and could be used as biocontrol agents In this study, 20 strains with high antagonistic activity against fungal pathogens were isolated from different soil samples Comparative analysis of antagonistic activity of isolated 20 isolates showed that strain TV2.12, HT2, TV2.5, TV2.11, QN3 inhibits the growth of various fungal pathogens more effectively than the other strains There were significant changes in the mycelium morphology caused by bacteria in comparison to the control: irregular, distorted, shrunken In addition, these strains with the highest indole3-acetic acid (IAA) synthesis capability 37 PART CONCLUSION AND PROPOSAL 5.1 Conclusion From eighty-six bacteria isolates were evaluated the antifungal activity of the Fusarium oxysporum Continue, selected twenty isolates displaying strong antagonism against Fusarium oxysporum Then, we evaluated the antifungal activity of twenty isolates against other fungi such as Fusarium oxysporum, Fusarium solani and Alternaria alternata after 3,5,7 days For Fusarium oxysporum, the strain HT2 presented the highest activity, causing more than 52% inhibition of mycelial growth among the 20 evaluated isolates For Fusarium solani, the strain TV2.5 presented the highest activity, causing more than 67% inhibition of mycelial growth among the 20 evaluated isolates For Alternaria alternate, the strain TV2.11 presented the highest activity, causing more than 62% inhibition of mycelial growth among the 20 evaluated isolates Most of isolated strains produced catalase and indole-3-acetic acid (IAA) synthesis capability We have isolated twenty new bacterial strains that differ in their antagonistic activity against a number of phytopathogenic fungi In addition, these strains of microorganisms that have the potential to stimulate plant growth (IAA) Thus, these strains can application in agriculture as bioinoculant for agriculturally important plants such as: Bio pesticides 5.2 Recommendations For agricultural applications, the evaluation of their activity in vivo need to study in the future 38 REFERENCES Abdallah R A B., Mokni-Tlili S., Nefzi A., Jabnoun-Khiareddine H & DaamiRemadi M (2016) Biocontrol of Fusarium 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