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 : NGUYEN MAI ANH Class : K62CNSHE Student 's code : 620453 Supervisor : Dr. TRINH XUAN HOAT Assoc. NGUYEN XUAN CANH Department : MICROBIAL TECHNOLOGY HÀ NỘI - 2022 COMMITMENT I hereby commit that the thesis is completely done by me under the guidance of Assoc. 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.
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. ii LIST OF TABLES.
v LIST OF FIGURES. vi LIST OF ABBREVIATIONS. Purposes and requirements. Overview of Actinomycetes.
Introduction and distribution of Actinomycetes. Classification of Actinomycetes. Morphological characteristics of Actinomycetes. Structure of Actinomycetes.
The roles and applications of Actinomycetes. Overview of Colletotrichum gloeosporioides. Introduction of Colletotrichum gloeosporioides. Biology of Colletotrichum gloeosporioides infection process.
Damage of Anthracnose disease caused by Colletotrichum gloeosporioides in agriculture. MATERIALS AND METHODS. Chemicals, instruments and equipment. Experiment location and time.
Activate Actinomycetes strains preserved in glycerol. Screening of Actinomycetes with antifungal activity. Morphological characterizations of Actinomycetes. Ability to assimilate carbon sources.
Ability to produce extracellular enzymes. Effects of temperature, pH and NaCl concentrations on the growth of Actinomycetes. Ability to utilize citrate. Ability to hydrolyze gelatin.
Ability to decompose urea. Classification of the Actinomycetes based on 16S rRNA sequences. RESULTS AND DISCUSSION. Screening and selection of fungal antagonist Actinomycetes.
Ability to assimilate carbon sources. Ability to produce extracellular enzymes. Effects of temperature, pH and NaCl concentrations on the growth of Actinomycetes. Ability to utilize citrate.
Ability to hydrolyze gelatin. Ability to decompose urea. Classification of the Actinomycetes based on 16S rRNA sequences. Total DNA extraction and concentration.
Amplification of 16S rRNA sequences. Sequencing PCR products and building a phylogenetic tree. CONCLUSIONS AND PROPOSALS. 59 iv LIST OF TABLES Table 3.
Primers for PCR. Components of PCR reaction. Colony morphology of Actinomycetes strain VNUA48 in ISP field 38 Table 4. Assimilation of different carbon sources of Actinomycetes strain VNUA48.
Effects of environmental conditions on the growth of Actinomycetes strain VNUA48. 44 v LIST OF FIGURES Figure 2. Single spore production in short chains. Morphological features of spores.
Spore production within sporangia. Screening fungal antagonist Actinomycetes: Control fungus (A), Dual cultured Actinomycetes (B) after 7 days of culture. Colony morphological characteristics of Actinomycetes VNUA48 in ISP field after 7 days of culture. Mycelia and spore chains morphology after 50 hours of culture (A), Spore chain and spore morphology after 72 hours of culture (B).
Actinomycetes strain VNUA48 on ISP 6 medium after 7 days of culture. Growth of Actinomycetes strain VNUA48 on: positive control (A), negative control (B), and basal medium containing starch (C). Ability to produce Amylase, Cellulase, Chitinase, Pectinase, Protease, Catalase of strain VNUA48 after 7 days of culture. Citrate utilization test of strain VNUA48: Negative control tube (A), test tube containing strain VNUA48 (B).
Gelatin hydrolysis test of Actinomycetes strain VNUA48: Negative control tube (A), test tube containing Actinomycetes strain VNUA48 (B). Urea decomposition test: Negative control tube (A), test tube containing Actinomycetes strain VNUA48 (B). Gel electrophoresis of total DNA extraction off Actinomycetes strain VNUA48. Gel electrophoresis of PCR product of Actinomycetes strain VNUA48.
Phylogenetic tree of Actinomycetes strain VNUA48. 51 vi LIST OF ABBREVIATIONS Abbreviation Meaning C. gloeosporioides 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. Introduction Colletotrichum is a large genus including a plenty of important species that are among the most popular fungal pathogens causing diseases in various fruits and crops. A majority of every crop grown throughout the world is susceptible to one or more species of Colletotrichum. Recently, the genus Colletotrichum was the eighth most significant group of phytopathogenic fungi in the world (Dean 2012).
Colletotrichum gloeosporioides and the anthracnose disease cause mounting threat to global agriculture. This fungus causes bitter rot in variety of crops worldwide, from monocotyledons to higher dicothyledons (Bailey, 1992). Colletotrichum species lead to losses of more than 50% fresh fruit and vegetables productivities (Paull, 1997). Some of the important host plants include citrus, yam, papaya, avocado, coffee, eggplant, sweet pepper, and tomato.
It results in a dramatic amount of pre- and post-harvest losses in these crops worldwide. This fungus acts as a secondary invader of injured tissue or survives as a saprophyte. Colletotrichum gloeosporioides involve various symptoms depending on the host species and tissue infected. There are black or brown lesions in the infected fruits, while blight, necrosis, and lesions with flecks and streaks appear in the inflorescence.
Abnormal colors and patterns with dark, necrotic, angular, or irregular areas are observed in the infected leaves. Infection on the stems produces dieback and discoloration with gummosis and resinosis. Many solutions are applied in plant protection to decrease the harm caused by fungal diseases. However, chemical measures have their limitations such as drug resistance in harmful agents, increase in environmental pollution, ecological imbalance, and chemical residues in the soil, endanger human health.
1 Therefore, biological-origin fungicides are a good way to overcome the limitations of chemical measures in the protection of plants from harmful fungi. Actinomycetes are prominent for their ability to produce secondary metabolites, many of which have antibacterial and antifungal properties (Sabaratnam & Traquair, 2002; Minuto et al. Actinomycetes have the ability to manufacture an abundance of extracellular hydrolytic enzymes (Mccarthy & Williams, 1992; Schrempf, 2001), particularly in the soil, where they are responsible for the breakdown of dead plants, animals, and fungal materials. They also play an important role in soil biodegradation and humus formation because of their ability to recycle the nutrients associated with recalcitrant polymers, such as chitin, keratin, and lignocelluloses (Goodfellow & Williams, 1983; Mccarthy & Williams, 1992; Stach & Bull, 2005).
The bioactive secondary metabolites produced by microorganisms is reported to be around 23,000 of which 10,000 are produced by Actinomycetes, thus representing 45% of all bioactive microbial metabolites discovered (Berdy, 2005). Among Actinomycetes, approximately 7,600 compounds are produced by Streptomyces species (Berdy, 2005). Consequently, there has been an upward trend from actively pursuing research centers that have made their screening programs more intensive, resulting in a growing number of reports of novel active compounds. Their characterizations are based on morphological and physiological criteria revealed by many laboratory tests.
In this research, I study about the topic: "Characterization and identification of Actinomycetes capable of antagonism with fungus Colletotrichum gloeosporioides cause Anthracnose disease in plants". Purposes and requirements 1. Purposes - Characterization and identification of Actinomycetes capable of antagonism with fungi Colletotrichum gloeosporioides cause Anthracnose disease in plants. Requirements - Screen Actinomycetes strain with ability of resistant to fungus Colletotrichum gloeosporioides cause anthracnose disease in plants.
- Study morphological and biochemical characteristics of selected Actinomycetes train. - Classify and identify Actinomycetes strain based on 16S rRNA sequences. Overview of Actinomycetes 2. Introduction and distribution of Actinomycetes Streptothrix foersteri, described by Ferdinand Cohn in 1875, and Actinomyces bovis, described by Carl Otto Harz in 1877, were two of the first Actinobacteria described.
Harz described Actinomycetes bovis, which causes the 'lumpy jaw' sickness in cattle, as having thin filaments that ended in club-shaped structures resembling those of fungi, hence the name Actinomyces (Latin for ray fungus). The 'gonidia,' on the other hand, very definitely housed cells, and so the resemblance to ordinary fungi was misleading. Several other bacteria, including the causative agents of leprosy and tuberculosis, were identified with some of the same features and were assumed to belong to the same group as the ones named above. Although their phylogenetic position as actual bacteria was not proven until the 1960s, this group of organisms was officially classified as Actinomycetales in 1916, and it became clear that they formed a big diverse group with a wide range of physiological and biochemical features.
The Actinobacteria are today regarded one of the bacterial kingdom's largest phyla. Some corynebacteria have a GC value of 54%, while Streptomycetes have a GC content of more than 70%. The causal agent of Whipple's disease, Tropheryma whipplei, has a GC content of 46.3%, which is the lowest known for Actinobacteria. The Actinomycetes are a well-defined group of bacteria with a high GC content and a diverse variety of habitats.
The group's members can be found in a variety of habitats, including soil, rhizosphere, marine, and freshwater systems. They are more common in soil than in other habitats, especially in alkaline and organic-rich soils, where they make up a large portion of the microbial community. Several are major human and animal pathogens, with only a few 4 being plant infections. The population density of Actinomycetes depends on the living habitat and climatic circumstances; 1 gram of soil contains between 10 6 and 109 cells, most of which are streptomycetes (accounting for more than 95% Actinomycetes in the soil).
The great majority of Actinomycetes are crucial saprophytes that can decompose a variety of plant and animal waste. Antibiotics, enzymes, enzyme inhibitors, signaling molecules, and immunomodulators are all produced prolifically by some genera, such as Streptomyces and Micromonospora. Actinomycetes, like other soil bacteria, are mostly thermophilic, with a growth temperature of 25-30°C.