VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY MAI EI NGWE ZIN THE IMPACT OF CLIMATE CHANGE ON THE SYMBIOSIS BETWEEN DARK SEPTATE ENDOPHYTIC FUNGI AND RICE PLANT MASTER’S THESIS VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY MAI EI NGWE ZIN THE IMPACT OF CLIMATE CHANGE ON THE SYMBIOSIS BETWEEN DARK SEPTATE ENDOPHYTIC FUNGI AND RICE PLANT MAJOR: CLIMATE CHANGE AND DEVELOPMENT CODE: 8900201.02QTD RESEARCH SUPERVISORS: PROF NARISAWA KAZUHIKO DR HOANG THI THU DUYEN Hanoi, 2020 PLEDGE I assure that this thesis is my own research and has not been published The used of result of other research and other documents must comply with regulations The citations and references to documents, books, research papers and websites must be in the list of references of the thesis Author of thesis MAI EI NGWE ZIN i TABLE OF CONTENTS PLEDGE i LIST OF TABLES iv LIST OF FIGURES iv LIST OF ABBREVIATIONS vi ACKNOWLEDGMENT vii FOREWORD viii CHAPTER INTRODUCTION 1.1 The Role of Rice Production in Asia 1.2 Vulnerable Rice Production under Climate Change (High Temperature) 1.3 The Concept of Endophytic fungi as Biofertilizers 1.4 Japonica Rice Species 1.5 Symbiosis Relationship between Plant and Fungi 1.5.1 Endophytic Fungi 1.6 The Impact of Climatic Change on the Symbiosis Relationship between Plant and Fungi 14 1.6.1 The Impact of temperature on the Soil Microbial Communities 14 1.6.2 The impact of Climate Factors on the Symbiosis between plant and Fungal Endophyte 15 1.6.3 The Effect of Seasonal Variation to Fungal Endophytes Communities 16 1.7 The Role of Fungal Endophytes in Sustainable Agriculture 17 1.7.1 Role of Fungal Endophytes in Controlling Environmental Contamination 18 1.7.2 The Role of Fungal Endophytes in Biotic and Abiotic stress resistance 18 1.7.3 The Role of Fungal Endophyte in Promoting the Plant Growth 21 1.7.4 Dark Septate Endophytic Fungi (DSE) and their abilities 22 1.8 Research Question and Hypothesis 24 1.9 Objective of the Research 24 CHAPTER MATERIAL AND METHODS 27 2.1 Experiment Design 27 2.1.1 Research Parameter 27 2.2 Materials and Methods 27 2.2.1 Rice Seed Germination 27 2.2.2 Transplantation 29 2.2.3 The effect of high temperature on the symbiosis 30 2.3 The Second Experiment 31 2.4 Detection of DSE fungi in the root of the Koshihikari rice plant and identification of bacteria in the first experiment 31 2.4.1 Isolation and Identification of Bacteria 31 2.4.2 Checking the root colonization capacity of DSE endophyte in the roots of the Koshihikari rice plant 35 2.5 Identification of DSE fungi and Measurement of plant growth parameter of Koshihikari seedlings under high temperature in the second experiment 36 ii 2.5.1 Measurement of plant growth parameters 36 2.5.2 Identification of DSE fungi in the roots of the 10 days old Koshihikari Seedling 36 2.6 Statistical Analysis 37 CHAPTER RESULTS AND DISCUSSION 39 3.1 Colonization capacity of DSE fungi in the root of the Koshihikari rice plant in the presence of bacteria 39 3.1.1 Identification of Bacteria from three isolates 39 3.1.2 Root colonization capacity of DSE Fungi in the roots of the Koshihikari rice plant in the presence of bacteria 41 3.2 Capacity of DSE fungi colonization and plant performance response to symbiosis under high-temperature treatment 44 3.2.1 Effect of DSE fungi symbiosis in plant growth 44 3.2.2 Identification of DSE fungi in the root of 10 days old Koshihikari rice seedling 45 3.3 The impact of high temperature on the symbiosis between DSE fungi and root of the Koshihikari rice plant 48 3.4 Proposed solutions for sustainable agriculture practice in the context of climate change 50 3.4.1 Recommendation for sustainable agriculture practice for Myanmar (future Research orientation) 51 REFERENCES 57 LISTS OF PUBLICATION BY THE AUTHOR 62 iii LIST OF TABLES Table 1.1 Ideal temperatures for various development stages of the rice plant Table 1.2 The Characterization of functional classes of endophytic fungi…………10 Table 2.1 Equipment and chemical used in rice seed germination 28 Table 2.2 Equipment and chemical used in transplantation 29 Table 2.3 Materials used in Bacteria DNA Extraction, PCR, and DNA sequencing 32 Table 2.4 Equipment used in Identification of DSE fungi 35 Table 3.1 The Percent Identify of the DNA sequence from the selected three isolates of the first experiment based on the NCBI BLAST database 39 Table 3.2 Result of the measurements of the physical parameters of the plant growth for the 2nd experiment after 10 days of transplantation 455 Table 3.3 Flow Chart of Problem in Wet Tel Gu Groups of Villages, Myanmar 53 iv LIST OF FIGURES Figure 1.1 The World’s Leading rice Producer during 2016-2019 Figure 2.1 Appearance of Bacteria Found in the first experiment during 24 hr of transplantation 31 Figure 2.2 Isolation of single colony bacteria for DNA extraction 33 Figure 3.1 Colonization of Veronaeopsis simplex (Y34) DSE in the roots of 17 days old Koshihikari rice seedling 42 Figure 3.2 Colonization of Cladophialophora chaetospira (OGR3) DSE in the roots of 17 days old Koshihikari rice seedling 42 Figure 3.3 Colonization of Meliniomyces variabilis (J1PC1) DSE in the roots of 17 days old Koshihikari rice seedling showing an early developmental stage of an intracellular microsclerotia (pointed with black arrow) and an early developmental stage of an intracellular microsclerotia (pointed with white arrow) 42 Figure 3.4 Colonization of Phialocephala fortinii (LtPE2) DSE in the roots of 17 days old Koshihikari rice seedling 43 Figure 3.5 Colonization of Veronaeopsis simplex (Y34) DSE in the roots of 10 days old Koshihikari rice seedling 46 Figure 3.6 Colonization of Cladophialophora Chaetospira (OGR3) DSE in the roots of 10 days old Koshihikari rice seedling 47 Figure 3.7 Colonization of Meliniomyces variabilis (J1PC1) DSE in the roots of 10 days old Koshihikari rice seedling 47 Figure 3.8 Map of the Republic of the Union of Myanmar 51 v LIST OF ABBREVIATIONS ADB AMF BGA BLAST DSE FAO FAOSTAT GCMs IPCC IRRI J1PC1 LtPE2 NCBI OGR3 Y34 Asian Development Bank Arbuscular mycorrhizal fungi Blue Green Algae Basic Local Alignment Search Tool Dark Septate Endophytic Fungi Food And Agriculture Organization Food and Agriculture Data General Circulation Models Intergovernmental Panel on Climate Change International Rice Research Institute Meliniomyces variabilis Phialocephala fortinii National Center for Biotechnology Information Cladophialophora chaetospira Veronaeopsis simplex vi ACKNOWLEDGMENT First and foremost, thanks to God, the Almighty, for His mercy and blessings throughout my two years of study of the master program including research work to complete the master’s program and research successfully This research was implemented during my internship in Ibaraki University, Japan I am grateful to Japan International Cooperation Agency (JICA) for their sponsorship during my internship under master program of Climate Change and Development, Vietnam-Japan University I am especially grateful to Prof Kazuhiko Narisawa for accepting me as his student and allowing me to conduct research in his laboratory under his supervision and sincere guidance I also would to express my appreciation to Dr Duyen Thi Thu Hoang for her advice, support, and encouragement till the end of my thesis I would like to thank the laboratory members of the College of Agriculture, Ibaraki University, Japan, especially Ms Wiwiek Harsonowati from the Department of Symbiotic Science of Environment and Natural Resources, United Graduate School of Agriculture Science, Tokyo University of Agriculture and Technology, Tokyo, Japan, for her help during the research I also would like to express my gratitude to all members of the Department of Climate Change and Development, Vietnam Japan University, especially Dr Ahiko Kotera and Ms Bui Thi Hoa, as well as staffs from ICAS (Ibaraki University) as for their support and guidance during the entire my master program Finally, a thank is not enough for my family, especially Miss Mai Ei Shwe Zin and Mr Gabriel for their support to continue my study vii C Figure 3.6 Colonization of Cladophialophora Chaetospira (OGR3) DSE in the roots of 10 days old Koshihikari rice seedling (A) Formation of the vesicle-like structure pointed with a black arrow and blue-stained hyphae with a white arrow; (B) intercellular blue-stained hyphae forming structures resembling anastomoses; (C) intracellular microsclerotia like structure with white arrow B A Figure 3.7 Colonization of Meliniomyces variabilis (J1PC1) DSE in the roots of 10 days old Koshihikari rice seedling (A) Formation of hyphal coils pointed with a black arrow; (B) blue-stained hyphae forming structures resembling anastomoses The second experiment was started on the 24th of November and transplantation was done on the 28th of November, 2019 The harvesting took place on the 8th of December, 2019 All the selected dark septate endophytic fungi can well colonize with Koshihikari rice plant under a continuous high temperature of 35°C within 10 days because the characteristics of DSE fungi colonization were recognized in the roots of the 47 Koshihikari rice plant in all treatments such as the occurrence of DSE fungal hyphae and micro-sclerotium (Fig.3.5, 3.6, and 3.7) Meanwhile, according to the table (3.2), measurements of the physical parameters of plant growth, treatments with Cladophialophora Chaetospira (OGR3) DSE, and Veronaeopsis simplex (Y34) DSE seem to be a better symbiosis than treatment with Meliniomyces variabilis (J1PC1) DSE These results suggest that Cladophialophora Chaetospira and Veronaeopsis simplex could promote the Koshihikari rice plant under high-temperature stress Previous research has defined that a few species of Cladophialophora DSE can predominant at tropical and subtropical areas and produce septate, brown hyphae and unicellular conidia, meanwhile, Veronaeopsis simplex (Y34) DSE can be isolated from subtropical regions along with Yaku Island in Japan In the existing research, we can prove that the subtropical DSE fungi: Cladophialophora Chaetospira (OGR3) and Veronaeopsis simplex (Y34) can colonize with temperate Japonica rice plant beneath a continuous temperature of 35ºC The Meliniomyces variabilis (J1PC1) DSE species can be determined abundantly in temperate areas And within the present study, J1PC1 DSE can colonize with temperate Japonica rice under 35ºC in step with the formation of hyphal coils and hyphae in the root of Koshihikari rice plant (Fig 3.7) 3.3 The impact of high temperature on the symbiosis between DSE fungi and root of the Koshihikari rice plant In this study, constant 35°C was used as high-temperature stress in order to elucidate the impact of high temperature stress on the symbiosis between the dark septate endoophytic fungi and Koshihikari rice plant All the selected DSE fungi could colonize to the roots of Koshihikari rice plant at constant 35°C and rice plant with DSE fungi treatment showed higher root/shoot ratio than those without DSE symbiosis Excessive temperatures is one of the key elements that set off abiotic pressure to plants including osmotic and oxidative pressure that lead to physiological and 48 biochemical metabolism modifications to plants and result in yield loss and even to the loss of life Rice seedlings are reported to sensitive to heat stress and Shah Fahad et al., 2018 reported that 70% of the rice plant development is upheld by enzymatic degradation of seed reserve and development rate increments with expanding the temperature from 22°C to 31°C during the first principal week after germination And it is reported that the optimal temperature for seedling growth is 35°C and the growth declined sharply beyond the optimal temperature Tillering stage of rice plant is reported that severely affected by temperatures exceeding 33°C (Chaudhary and Ghildyal, 1970) Threshold temperature for grain yield of rice plant is 34°C, and temperatures range from 32°C to 36°C induce high spikelet sterility, (Satake and Yoshida, 1978, Morita et al., 2004) Koshihikari, popular cultivar of japonica rice, can be grown at the latitudes range between 40°N and 31°N of Japan including Tochigi, Ibaraki, Chiba, Tokyo, Niigata, Hokuriku region, Toyama and Fukui (Asako Kobayashi et al., 2018) The environment adaptability of Koshihikari is high and it was reported that strong tolerance to cold weather, Ozeki et al., 1995 and Hosoi, 1989 On the other hand, the japonica species of Koshihikari rice plant is reported to a moderate level of heat tolerance (Prasad et al., 2006; Matsui et al., 2005) Another research reported that Koshihikari commonly produces higher shoot biomass at 31ºC and 34 ºC (Estela M Pasuquin, et al., 2013) Meanwhile, it is projected that temperature will increase by 2.4 °C for temperate Asia regions at 2070 according to CSIRO 2006 Moreover, Japan is reported that it will be very likely longer-length, more intense, and greater frequent heatwaves or warm spells in summer and a very probably decrease inside the frequency of very cold days and the temperature will be extended by 3°C at the end of 21st Century (The Global Warming Projection Vol Eight, JMA) Accordingly, constant 35ºC was used in the present research to elucidate the high-temperature stress to DSE fungi and Koshihikari symbiosis by creating high-temperature stress to the Koshihikari rice plant meanwhile, colonization capacity of the selected DSE fungi under constantly high temperature was elucidated And the results suggest that the Koshihikari rice plant with the DSE fungi symbiosis has a higher root/shoot ratio than without DSE fungi symbiosis under high-temperature stress 49 The class IV fungal endophytes, DSE fungi, are reported to confer habitat-specific stress tolerance to host plant plants, Redman and Rodriguez, 2007 The results of the present study confirm this report that Cladophialophora Chaetospira and Veronaeopsis simplex DSE fungi can well colonize to temperate japonica rice and could promote plant growth under high temperature condition Most research showed that environmental factors such as rainfall, altitude, and forest types as well as seasonal variations and climate change such as high temperature, drought have an impact on the diversity and occurrence of the fungal endophytes symbiosis However, research about the impact of high temperature on the DSE fungi colonization capacity is still limited to my knowledge The findings of the present study suggest that the selected DSE fungi can colonize the root of Koshihikari rice plant at the seedling stage under continuously high temperatures IPCC AR4 projected that the global atmospheric temperature will increase to 2.0 – 4.5ºC at the end of the 21st Century The high-temperature adaptation practices under the threat of climate change in the rice production system are keen to set up and the present study help in understanding the potential of applying the selected DSE fungi as biofertilizers, one of nature and ecosystem-based adaptation practices for rice production under the threat of climate change Due to the time limitation, the research could not continue until the growing stages of the Koshihikari rice plant, as well as the experiment, could not compare with other temperatures such as the normal and the low temperature Therefore, further research is needed to continue to get more proof 3.4 Proposed solutions for sustainable agriculture practice in the context of climate change The result of the current research suggests that the DSE fungi could promote the growth of rice plants under high-temperature condition of 35°C and they have the potential to be used as biofertilizers Alternatively, the current research demonstrates that DSE fungi play an important role in providing ecosystem services such as supporting and provisioning services Therefore, this hypothesis can be applied as one of the sustainable agriculture practices by using DSE fungi as biofertilizers 50 3.4.1 Recommendation for sustainable agriculture practice for Myanmar (future Research orientation) Republic of the Union of Myanmar (Burma) is situated in Southeast Asia region, between latitudes 09º 32′ N and 28º 31′ N and longitudes 92º 10′ E and 101º 11′ E And it is bordered with China on the north and northeast, Laos and Thailand on the east and southeast The Andaman Sea and the Bay of Bengal arrange in the south of the country and the west by Bangladesh and India The country’s total area is 676 590 km2 The country is comprising of the seven states, mainly covering the hill regions: Chin, Kachin, Kayah, Kayin, Mon, Rakhine and Shan States; and seven divisions, covering the plains: Ayeyarwady, Bago, Magway, Mandalay, Sagaing, Tanintharyi, and Yangon A B Figure 3.8 Map of the Republic of the Union of Myanmar A Rice growing hectare across Myanmar B, Republic of the Union of Myanmar displaying the Seven States and Seven Divisions According to the geographic position of the country, Myanmar has influenced by the seven Koppen climates with three distinct seasons: the monsoon season or wet season 51 from June to September, cold season from October to February, and the dry or hot season from March to May Myanmar has already experienced climate change and the average daily temperature over the country has increased by 0.25°C and the maximum daily temperature has risen with a rate of 0.4°C during 1981-2010 The temperature in Myanmar is projected to rise by 0.7-1.1°C during 2011-2040 especially from November to February and March to May The temperature during June to October is projected to increase by 1.1-2.4°C, which is the rice-growing season in Myanmar According to World Bank 2018, agriculture in Myanmar is significantly vulnerable to climate change and it is projected that agriculture in Myanmar will have negative impacts due to the risen temperature Wet Te Ku group of villages, comprising with three sub-villages (Thit Taw Village, Wet Te Ku Village, and Naung Pin Thar Village), located in the Lewe Township, Dekkhina District, Nay Pyi Taw, Myanmar There are 804 households and the total population is 3,240 according to the 2014 census information for Lewe Township by the Republic of the Union of Myanmar Agriculture is the major livelihood for the local people and rice contributes as the major crop Most of the local people are Burmese and Asho Chin ethnic and Buddhism, Christianity and Hinduism are the major religion in the community This group of villages is characterized by low income and migrant workers Agriculture is the major livelihood for the local people and unsustainable livelihood contributed by a climate-related problem such as high temperature, shifting the monsoon rainfall patterns, the non-climatic related problem such as the decline in soil quality, high expenditures for rice production and lack of irrigation water supply system are key facts that lead to low productivity and unsustainable livelihood A livelihood survey was conducted from 19th – 23rd February 2020 to get detail information about how climate change impact on Myanmar’s agriculture and livelihood of rural people Thirty farmers, including female-headed households, were selected randomly within the three villages for the interview Types of livelihood, income contribution from their livelihood, the practice of chemical fertilizer, experiences of climate change impact in their farms, and farmers’ impressions on 52 their soil quality are targeted According to the interview result, the monsoon paddy is the major cultivar and black gram, groundnut and sesame are cultivated after harvesting the paddy According to the survey, almost all of the interviewees encountered harvest losses for the previous season of 2019/2020 due to the earlier leaving of monsoon rain and some farmers still could not harvest till February of this year, 2020 And they said the frequency of shifting in raining patterns has become frequent during the previous five years but this season was the worst Another problem is a technical problem The expenditure of rice production through transplanting practice is very high and some farmers, especially female-headed households changed their practice of rice growing They changed into direct seeding from the transplanting and these farmers encountered low productivity Due to lower productivity, some of the youths and men have to migrate to the nearby city and abroad to find jobs, and this leads to increasing woman-headed households Declining the soil quality due to a high dose of fertilizer augment the problem These problems drive rural poverty and therefore sustainable agriculture practices are keen to set up for the community development Table 3.3 Flow Chart of Problem in Wet Tel Gu Groups of Villages, Myanmar Climate Change Factor Shifting Monsoon Rain Non-Climate Change Factor Unavailable Irrigation system High expenditure Technical Problem Soil Quality Result Low Productivity Migration Crop Establishment in Wet Tel Gu Groups of villages Rice is typically grown by transplanting or direct wet seeding in Myanmar as well as the Wet Tel Groups of Villages Transplanting is the most widely recognized strategy and rice seedlings developed in a nursery stage grown in the field are pulled and transplanted again into puddled and leveled fields 15 to 70 days in the stage of seeding This activity is done physically Also, some farmers wet seeding and this 53 technique includes the planting of pre‐germinated seeds onto a puddled soil and the seed might be communicated by hand Future Research Direction To apply the DSE fungi as biofertilizer for Myanmar’s agriculture, the following scientific research is needed to conduct 1) The fundamental step is to isolate the fungal endophytes to elucidate diversity and the occurrence of fungal endophytes in Myanmar 2) Since high temperature and water shortage as drought stress are the major constrain factors for Myanmar agriculture, research related to elucidate the effect of fungal endophytes in promoting the growth of plant under high-temperature conditions and drought stress Experiment Design The factorial experiment will be conducted in a greenhouse The first factor will be the treatments of plant: without endophytic fungi symbiosis, with endophytic fungi The second factor will be water content The water content will be given by three conditions such as 100% of field capacity for wet condition, 50% of field capacity for moderate wet condition, and 25% of field capacity for the dry condition And the last factor will be temperature Different constant temperature conditions will be given to elucidate the effect of endophytic fungi in different temperature conditions Research parameter: Colonization capacity of the DSE fungi will be check by reisolating the fungal endophytes from the roots, stems, and leaf of the plant, Leaf color, tiller numbers, panicle size alternatively spikelet per panicles, root/shoot ratio will be calculated to estimate biomass Conclusions In this research, the effect of high temperature on the seedling stage of Koshihikari rice was elucidated in the meantime, the symbiosis capacity between the dark septate endophytic fungi and Koshihikari rice plant was clarified at constant 35ºC The 54 results of the measurement of plant growth parameters show that the Koshihikari plant without symbiosis with DSE fungi have a lower root/shoot ratio than those with DSE symbiosis And through the identification of DSE fungi colonization in the roots, the selected tropical DSE fungi can well colonize to the roots of Koshihikari rice plant under high-temperature stress Therefore, the present study suggests that Koshihikari with the selected tropical DSE fungi symbiosis can tolerance the hightemperature condition than those without DSE fungi symbiosis Moreover, according to the result of first experiment, the selected DSE fungi can colonize to the roots in the presence of seed-endophytic bacteria However, difference between the plantgrowth parameters between the presence and absence of bacteria could not observe since the data from second experiment was collected after 10 days of transplantation due to the limitation of time where the results from first experiment was collected after 17 days of transplantation Accordingly, the selected DSE fungi have the potential to be used as biofertilizers IPCC AR4 projected that the global atmospheric temperature will increase to 2.0 – 4.5ºC at the end of the 21st Century The high-temperature adaptation practices under the threat of climate change in the rice production system are keen to set up And the present study could use as one of nature and ecosystem-based adaptation practices for rice production under the threat of climate change However, the whole life cycle of the Koshihikari rice plant with DSE fungi treatment as well as in comparison with different temperature conditions could not conduct due to the limited time Further research 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from Six Halophytes in Suncheon Bay pp 1549–1556, 2012 Yuspradana.R, Wiyono.S and Widyastuti.R (2017) Rice Resistance-Treated with Endophyte Fungi Against Drought Stress Basic and Applied Research (IJSBAR) ISSN2307-453 61 LISTS OF PUBLICATION BY THE AUTHOR (1) “The Impact of Climate Change on the Symbiosis between the Dark Septate Endophytic Fungi and Koshihikari Rice Plant”, 2020 10th International Conference on Asia Agriculture and Animal (ICAAA 2020), Bangkok, Thailand (2) “The Impact of Climate Change on the Symbiosis between the Dark Septate Endophytic Fungi and Koshihikari Rice Plant”, Journal of Advanced Agricultural Technologies, Vol: 8, No.1, 2020 62 ... NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY MAI EI NGWE ZIN THE IMPACT OF CLIMATE CHANGE ON THE SYMBIOSIS BETWEEN DARK SEPTATE ENDOPHYTIC FUNGI AND RICE PLANT MAJOR: CLIMATE CHANGE AND. .. 1.5 Symbiosis Relationship between Plant and Fungi 1.5.1 Endophytic Fungi 1.6 The Impact of Climatic Change on the Symbiosis Relationship between Plant and Fungi ... colonization capacity of DSE Fungi in the roots of the Koshihikari rice plant in the presence of bacteria 41 3.2 Capacity of DSE fungi colonization and plant performance response to symbiosis