VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ---------- ---------- THESIS TITLE: ISOLATION AND CHARACTERIZATION OF FUNGUS ASSOCIATED WITH LEAF SPOT OF GRAPEVINES IN VIET NAM HANOI – 2022 1 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ---------- ---------- UNDERGRADUATE THESIS ISOLATION AND CHARACTERIZATION OF FUNGUS ASSOCIATED WITH LEAF SPOT OF GRAPEVINES IN VIET NAM Student’s name : Giap Thi Thuy Chi Student’s code : 620429 Class : K62-CNSHE Supervisor : Dr. PHAM HONG HIEN MSc. NGUYEN THANH HUYEN HA NOI – 2022 COMMITMENT I hereby declare that all the data and the results that I have provided in this thesis are truthful, accurate, and not used in any other reports. I also assure you that any help in this thesis has been greatly appreciated thankful and the information cited has been acknowledged.
Hanoi, May, 2022 Student Giap Thi Thuy Chi i ACKNOWLEDGEMENTS First and foremost, I have to thank the Vietnam National University of Agriculture for giving me a chance to study in a professional environment that provided me the sufficient knowledge throughout four and a half academic years. In doing my thesis, many people helped me bring this work into existence, but the most influential and inspiring person I would like to extend thanks to is my supervisor, MSc. Nguyen Thanh Huyen without her assistance and dedicated involvement in every step throughout the thesis, this thesis would have never been accomplished. I would like to express my deep gratitude to all teachers from the Faculty of Biotechnology, who encouraged me to enhance my knowledge and help me get orientation for my thesis.
Getting through my undergraduate thesis required more than academic support, and I have many, many people to thank for listening to and, at times, having to tolerate me, thank all my friends, who were with me up until this point, simultaneously motivate me to accomplish more. Last but not least, I would like to thank myself, and my parents who endured this long process, for always supporting and assisting me during the process of completed graduate thesis. Ha Noi, May, 2022 Student Giap Thi Thuy Chi ii ABSTRACT Grapevine (Vitis spp.) is among the most widely grown fruit crops worldwide and plays a significant role in its commercial value in Vietnam as well as having an excellent source of nutrition. However, recently many diseases appeared and affected the yield and quality such as downy mildew (Plasmopara viticola), powdery mildew (Uncinula necator), and leaf spot (Phomopsis viticola).
Among them, leaf spot disease is a fairly common disease and causes a significant reduction in the yield of Grapevines. The thesis was conducted in leaf spots of grapevines collected from different places in Ha Noi and Bac Giang, Vietnam in 2021. The fungus was Isolated from leaf spot disease on grapevines, and the fungus was selected and then used for artificial infection into healthy grapevine leaves. The fungal strains causing the grape leaf spots will be selected to study the morphological and biochemical characteristics, as well as the conditions affecting the growth of the fungus.
Then, these fungal strains are identified by molecular tools. Research results show that 2 strains (SM and HV) of fungi have been isolated, but after re-infection, only one strain (SM) is capable of causing leaf spot disease on grape leaves, SM strain has mycelium that is profusely branched (monopodial branching). Studying the growth conditions of fungal strains proves that SM grows well on MEA and PDA, at around the temperature range of 20-30°C with pH from 5. SM strain has the ability to produce chitinase, cellulase, and pectinase.
The causal fungus strain (SM) was identified as Phomopsis viticola. iii INDEX COMMITMENT. iii LIST OF ABBREVIATIONS. vi LIST OF TABLES.
vii LIST OF FIGURES. General introduction of grapevines. Description of morphological characteristics of the grapevine. MATERIALS AND METHODS.
Chemicals and medium components. Laboratory apparatuses and equipment. Research location and time. Isolation of fungus causing leaf spot disease on grapes (Chomnunti et al.
Artificial infection (Jayawardena, Ruvishika S. The effects of some factors on fungal growth (culture medium, temperature, and pH ). Extracellular enzyme production of isolated fungal strain. DNA extraction, PCR amplification and Sequencing.
RESULTS AND DISCUSSION. Isolation of fungal pathogens causing leaf spots from grapevines leaf. Morphological characteristics of SM strain. The effects of culture medium on SM strain growth.
Effect of different temperatures on the growth of SM strain. Effect of different pH on the growth of SM strain. Testing extracellular enzyme activity. DNA extraction, PCR amplification and Sequencing and Phylogenetic analysis.
DNA extraction, PCR amplification of SM strain. Phylogenetic analysis of SM strain. CONCLUSION AND SUGGESTION. 37 v LIST OF ABBREVIATIONS Abbreviations Explanations PDA Potato Dextrose Agar SDA Sabouraud Dextrose Agar MEA Malt Extract Agar CDA Czapek Dox Agar WA Water Agar ISP International Streptomyces Project et al Et alia CWDEs cell wall-degrading enzymes CMC Carboxymethyl cellulose vi LIST OF TABLES Table 2.
Nutrition of Grapes. Experimental materials for detecting extracellular enzymes. Morphological characterization of two isolated fungal strains. The ability of extracellular enzyme production of SM.
31 vii LIST OF FIGURES Figure 2. One grapevine garden in Ninh Thuan. Symptoms of powny mildew disease. Symptoms of powdery mildew disease.
Symptoms of leaf spot disease. Leaf samples with leaf spot disease. Fungal strains isolated from infected grape leaves. Re-infection of isolated fungal strains (HV, SM) in grapevine leaf and control sample on the 3rd days.
Microscopic observation of the selected fungus strain-SM. Growth of SM strain in different media on the 5th days. Growth of SM strain in different temperatures on the 5th days. Growth of SM strain in different pH on the 5th days.
Chitinase activity of the SM strain. Cellulase activity of the SM strain. Pectinase activity of the SM strain. PCR product of SM strain on gel agarose.
Phylogenetic tree of SM strain. Introduction Grapevine (Vitis sp., Vitaceae) is one of the most important fruit crops cultivated around the globe and has a high nutritional value in many countries. According to FAO data (2014), the global grape-growing area has 8,485,000 hectares and is increasing at a pace of 2% per year, with yearly production reaching 60,473 million tons, accounting for 17.5 percent of total global fruit production. Approximately 71% of grapes are utilized as raw materials for wine production, 27% are consumed fresh, and 2% are dried (seedless grapes).
Currently, vines are grown on all five continents in climates that are conducive to their growth. Spain (1,175,000 ha), Russia, Italy, France, Turkey, Portugal, and the United States are the countries with the largest grape areas in the world. Over 60% of the world's grape output has been produced in Europe. In recent years, grape acreage and production have expanded in Asia.
The entire area is currently around 1. China, India, Japan, Korea, Pakistan, Thailand, and Vietnam are Asian countries having substantial grape acreage (FAO, 2014). According to the conference on grape production in Asia Pacific countries, which included eight countries: China, India, Korea, Japan, Myanmar, Thailand, and Vietnam, the grape area of the participating countries is 393,725 ha, with a yield of 5.432 million tons and an average grape yield of 13.6 tons per ha per year. The grape output in Vietnam is lower than the regional average, at only 11.2 tons per hectare per year (caytrong, 2019).
In Viet Nam, there are also many grape growing regions, of which Ninh Thuan is the most planted with about 2,400 hectares in Ninh Thuan (accounting for 90%) and 250 hectares in Binh Thuan (accounting for 9% of the total grape growing area of the country) (Le Quang Quyen et al. The results of a random survey of 50 vineyards with different planting ages in 2014 showed that 1 the average grape yield in Ninh Thuan was over 30tons/ha/year. Growing grapes can make a profit of over 200-300 million VND/ha/year (Tran Ngoc Tu et al. Although it has great economic value, currently the yield and quality of vines are reduced due to several diseases such as downy mildew (Plasmopara viticola (Berk.
& MA Curtis) Berl. Currently, there are many measures to control diseases on grapevines, but measures Using chemical drugs are widely applied because of their quick effect. However, the long-term use of chemical drugs causes serious health consequences. For the above reasons, the topic " Isolation and characterization of fungus associated with leaf spot of grapevines in Viet Nam " was carried out.
This study was undertaken for the evaluation of the characteristics of pathogenic fungal strains and will provide useful information to find solutions. Effective disease prevention while maintaining the nutritional value of grapes without affecting the health of the user. Research purpose - Isolation of fungal strains causing leaf spot disease on grapes in Vietnam. - Determination of the morphological, and biochemical characteristics of the pathogenic fungus causing grape leaf spot spots Vietnam.
Requirement - Isolation and selection of fungus associated with leaf spots in grapevine leaves - Pathogenicity test on healthy grapevine leaves. - Characterization of fungal strain causing leaf spot in grapevine leaves: morphology colony, mycelium, and spore of fungus. General introduction of grapevines The grapevine (Vitis vinifera) belongs to the Vitaceae family, which includes approximately 60 inter-fertile wild Vitis species found in Asia, North America, and Europe under subtropical, Mediterranean, and continental– temperate climatic conditions. It is the single Vitis species that has gained significant economic interest and is a commercially valuable plant over time, which can be used as a rich source of nutrients in multiple industries (Xu et al.
2017; Chitarrini et al., 2017), for example, the North American, are used as breeding rootstock due to their resistance against grapevine pathogens, such as Phylloxera, Oidium and mildews. Indeed, a great majority of cultivars are widely cultivated for fruit, juice, and mainly for wine which was classified as Vitis vinifera L. Vinifera (or Sativa), derive from wild forms (Vitis vinifera L. sylvestris (Gmelin) Hegi] ((Rossetto, McNally et al., 2002); (Sefc, Steinkellner et al., 2003); (Crespan, 2004, This, Jung et al.
According to Nikolai Ivanovich Vavilov (1926), a Russian botanist work of prominent, the highest biodiversity of a crop is usually its center of origin. In 1938, his disciple Aleksandr Mikhailovich Negrul observed that the highest biodiversity in cultivated grapes was in today’s Georgia, Armenia, Azerbaijan, Turkey, and Iran, which makes them the best candidates for being the cradle(s) of viticulture. Analysis of morphological similarities between wild and cultivated grapes from all over Eurasia generally supports a Near Eastern origin for grape domestication. The closest genetic relationship was discovered between local wild grapevines and traditionally cultivated grape varieties from southern Anatolia, Armenia, and Georgia.
This suggests that the headwaters of the Tigris and Euphrates rivers in the Taurus Mountains are the most likely location for the first domestication of the grapevine (Arroyo‐García, R. 2 Grapevine was domesticated (Vitis vinifera, sometimes called V. sativa) and was one of the most important fruit species in the classic Mediterranean world, and it is the most important economic fruit species in the modern world today. As in the ancient past, sun-loving grapevines are today cultivated to produce fruits, which are eaten fresh (as table grapes) or dried (as raisins), and, most especially, to make wine, a drink of great economic, cultural, and symbolic value (K.
The Vitis family consists of about 60 inter-fertile species that exist almost exclusively in the Northern Hemisphere: of those, V. vinifera is the only one extensively used in the global wine industry. Approximately 10,000 cultivars of V. vinifera exist today, although the market for wine production is dominated by only a handful of them.
Cultivars are typically classified according to whether they produce wine grapes, table grapes, or raisins (K. Domesticated grapevine (Vitis vinifera, also known as V.