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%), TV2.12(56%) and IAA production. iii CONTENT DECLARATION OF AUTHORSHIP. iv LIST OF TABLES.
vi LIST OF FIGURES. vii PART 1: INTRODUCTION. Aim and requirements. 3 PART 2: LITERATURE REVIEW.
Bacteria with antifungal activity against plant pathogens. Pathogenicity of Fusarium oxysporum. Control of Fusarium diseases. Studies of bacteria with antifungal activity against plant pathogens.
14 PART 3: MATERIALS AND METHODS. The culture medium was used as follows. Dilution Plate Method. Determination of indole -3-acetic acid (IAA) synthesis capability.
Screening of potential bacteria isolates with antagonistic activity against fungal pathogens. RESULTS AND DISCUSSION .1 Experiment 1: Isolating and screening bacteria isolates with antifungal activity. Experiment 2: Screening of potential bacteria isolates with antagonistic activity against fungal pathogens. Experiment 3: Evaluation of 20 potential isolates with antifungal activity against fungal pathogens.
Experiment 4: Characterization of the potential bacterial strains. CONCLUSION AND PROPOSAL. 39 v LIST OF TABLES Table 4.1: Summarization of potential bacteria isolates with antifungal .2: Effects of bacteria strains on the mycelial growth inhibition to Fusarium oxysporum (3-7 days).3: Effects of bacteria strains on the mycelial growth inhibition to Fusarium solani (3-7 days) .4: Effects of bacteria strains on the mycelial growth inhibition to Alternaria alternata (3-7 days) .5: Characteristic of the potential bacterial strains.6: IAA production produced by potential isolates. 35 vi LIST OF FIGURES Figure 2.1: Some probiotics are applied from bacillus strains with antifungal .5: Soil sample dilution and inoculation .6: Gram staining steps .9: Experimental design images .1: The number of bacteria selected isolates from 6 soil samples .2: Experimental design images .3: Antagonistic activity of screening isolates against Fusarium oxysporum .4: Ten isolates showed strongest antifungal activity against Fusarium oxysporum .5: Ten isolates showed strongest antifungal activity against Fusarium solani .6: Ten isolates showed strongest antifungal activity against Alternaria alternata .7: Catalase test of 20 potential bacterial strains .8: Colonial morphology of 20 potential isolates with antifungal activity against fungal pathogens.
36 vii PART 1: INTRODUCTION 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 1 upward from the stem base. Dark streaks occur in the xylem vascular tissue of the roots and lower stem, and the roots may decay. Infected seedlings will wilt and die.
Currently, farmers rely heavily on pesticides, but toxicity hazards to farm workers, consumers, and non-target organisms as well as environmental contamination cause great concern. Using beneficial and antagonistic microorganisms for biological control offers a potential, environmentally friendly and effective approach to control Fusarium oxysporum. So, the objective of this study was to evaluate bacteria strains isolated with antagonistic activity against Fusarium oxysporum. These isolates inhibited Fusarium oxysporum mycelial growth in vitro and reduced Fusarium oxysporum disease severity in plants grown in Fusarium oxysporum infested.
The potential use of microorganisms in the treatment of plant fungal diseases is based on the antagonistic nature of microbes towards fungal pathogens. In addition, some bacteria have the ability to promote plant growth, such as the synthesis of plant auxin indole-3-acetic acid (IAA) and its analogs. The results of experimental studies of microbial antagonists against plant fungal pathogens are promising. In this study, we would like to explore the possibility of using natively isolated bacteria strains which were collected from 6 different soil samples in Northern regions for Fusarium oxysporum antagonism.
Then, we selected 20 isolates displaying strong antagonism against Fusarium oxysporum. In addition, these isolates also have the ability to stimulate plant growth. With these two characteristics, it will help plants easily resist plant pathogens. This study is helpful to initially assess the biocontrol potential of these antagonistic strains.
Aim and requirements 1. Aim Isolating and selecting potential bacterial strains with antagonistic activity against fungal pathogens. Requirements Isolation of potential bacterial strains can suppress fungal pathogens from six soil samples. Collected bacterial strains with antifungal activity against fungal pathogens.
Screening of potential bacterial isolates with antagonistic activity against fungal pathogens with Fusarium oxysporum, Fusarium solani, Alternaria alternata. Selecting potential bacterial strains have antifungal activity against fungal pathogens (Fusarium oxysporum, Fusarium solani, Alternaria alternata). Characteristic of the potential bacterial strains. 3 PART 2: LITERATURE REVIEW 2.
Bacteria with antifungal activity against plant pathogens Biological control is an environmentally-friendly alternative to chemical pesticides and it is an attractive method of protecting plants from pathogens because the wide usage of chemicals has a negative impact on the environment and human health. Soil-borne bacteria that are antagonistic to plant pathogens could make a substantial contribution to prevention of plant diseases, and therefore represent an alternative to the use of chemical pesticides in agriculture Many biocontrol agents were isolated by screening a large number of soil or plant-associated microorganisms for antagonism against phytopathogens in vitro or plants. Some bacterial species with antagonistic activity were isolated from the rhizosphere of different plants. Among those, bacilli and pseudomonads are the most common isolates.
In the world, various species of the Bacillus genus can stimulate plant growth. Plant growth can be promoted by bacteria through several mechanisms, including improved nutrition, systemic resistance, toxicity against pests, and antagonism against pathogens. Many Bacillus isolates were shown to have antifungal activity against phytopathogenic fungi which make them good biocontrol candidates (Mardanova et al. The antagonistic activity of Bacillus is associated with the synthesis of various antimicrobial peptides, secreted enzymes, proteins and volatile organic compounds (VOCs).
Many Bacillus isolates were shown to have antifungal activity against phytopathogenic fungi that make them good biocontrol candidates. form endospores and can tolerate extreme conditions such as pH, temperature, and osmotic pressure; they therefore offer several advantages over other types of organisms. was found to colonize the root surface, increase plant growth, and cause lysis of fungal mycelia. They 4 areregarded as safe biological agents and their potential is considered high (Islam et al.
Bacillus is a microbial strain 0.5 µm to 20 µm in size, rod- shaped, gram-positive, spore-forming, aerobic or facultative anaerobic. Bacillus frequently occur in chains. There are many strains of Bacillus used in agriculture, including: Bacillus thuringiensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis (Islam et al.1 Bacillus thuringiensis Bacillus thuringiensis is an aerobic, soil-dwelling bacterium, produce Gram-positive spores and produce proteins with powerful insecticidal properties. Bacillus thuringiensis is considered to be one of the most successful biopesticides ever used against insect pests of crops.
The activity of Bacillus thuringiensis subsp. thuringiensis delta-endotoxin against pathogenic fungi related to Phytophthora and Fusarium was investigated. A possible mechanism of delta-endotoxin's antifungal activity involves the uncoupling of oxidative phosphorylation and respiration. In the fungal cultures, the toxin increased respiratory activity.
This effect was comparable with processes that occurred in insect cells after contact with entomopathogenic bacteria. The antifungal activity of delta-endotoxin against phytopathogenic fungi was dependent on the concentration and time of contact with fungal cells. The protective effects of the endotoxin against Fusarium oxysporum f. Lycopersici as a causative agent of wilt and late blight disease was estimated throughout the storage of tomatoes.
Additionally, the delta-endotoxin has a cytostatic effect on several economically important phytopathogenic fungi related to Fusarium, Bipolaris, Phytophthora, Alternaria, and Rhizoctonia. A single cell structure of eukaryotic organisms demonstrated the universal mechanism of the Bacillus thuringiensis delta- endotoxin action to both insect and fungal cells (Tran et al. However, the mechanism of the antifungal action of delta-endotoxins remains scantly explored.