The impact of climate change on the symbiosis between dark septate endophytic fungi and rice plant

Plant roots play the main role in maintaining the resistant capacity to biotic and abiotic stress by forming a symbiotic relationship with endophytic fungi under [r]

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

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

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

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

2.5.1 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

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

Table3.1 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

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

Figure3.6 Colonization of Cladophialophora Chaetospira (OGR3) DSE in the roots

LIST OF ABBREVIATIONS

ADB Asian Development Bank AMF Arbuscular mycorrhizal fungi BGA Blue Green Algae

BLAST Basic Local Alignment Search Tool DSE Dark Septate Endophytic Fungi FAO Food And Agriculture Organization FAOSTAT Food and Agriculture Data

GCMs General Circulation Models

IPCC Intergovernmental Panel on Climate Change IRRI International Rice Research Institute

J1PC1 Meliniomyces variabilis LtPE2 Phialocephala fortinii

NCBI National Center for Biotechnology Information OGR3 Cladophialophora chaetospira

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

FOREWORD

Rice production in South East Asia is under the threat of climate change such as

global warming, which is projected to increase 2ºC or more in the late- 20th –Century

(IPCC AR5) In that case, endophytes can be used as the natural-based adaptation tools since they are found ubiquitously in different ecosystems However, symbiosis activity between the endophytes and their host plant under high temperature is not well known, requiring more elucidation The purpose of this research is to clarify the symbiotic features of endophytic fungi in the early stage of rice growth and to evaluate the impact of high temperature on rice-endophytic symbiosis through the observation of the fungi present in the roots The symbiosis activity between the dark septate endophytes (DSE) and Koshihikari rice plant under high temperature was tested by comparing morphophysiology of rice colonize with Cladophialophora

chaetospira (OGR3), Meliniomyces variabilis (J1PC1), Phialocephala fortinii (LtPE2), Veronaeopsis simplex (Y34) and without colonization under continuous high

temperature of 35°C Root colonization was investigated under the microscope and

CHAPTER INTRODUCTION

1.1 The Role of Rice Production in Asia

More than 2.2 billion individuals in Asia depend on agribusiness (Asia Development Bank 2009) and 90 percent of the world's paddy cultivation and utilization are from Asia Rice production in the monsoon land that is known for Asia begins from the Islamic Republic of Pakistan to Japan, where the populace is densely conveyed and a significant consumer of rice in the world China and India are recorded as the world's biggest rice producers by a wide margin The harvested region in India is bigger than China, however, the yield in China is higher than India since about all of the harvested area in China is irrigated, while practically 50% of India's rice zone is inundated Southeast Asia nations, for example, Indonesia, Bangladesh, Vietnam, Myanmar, and Thailand trails China and India During 2008-2010, the normal rice production from these seven nations was in excess of 30 billion tons of paddy according to IRRI 2013 Furthermore, as indicated by the source from the United States Department of Agriculture, China was driving as the world's first rice producer At that point, India, Indonesia, and Bangladesh followed in the year 2016/2017 to 2018/2019

Figure 1.1 The World’s Leading rice Producer during 2016-2019 (source: United States Department of Agriculture, 2019)

the most recent decade (FAO, 2018) As indicated by India involved the most elevated fare volume of rice for around the world, by 12.5 million metric tons starting in 2018/2019 Thailand followed the second-biggest rice exporter, with about 10.3 million metric tons of rice worldwide in that year Concerning the Southeast Asia nations, Vietnam, Burma (The Republic of the Union of Myanmar) and Cambodia were among the world's driving rice trading nations with 7, 3, and 1.3 million metric tons for the year 2018/2019 respectively

Even though China is the world's greatest cultivation and consumer country of rice, from the year 2015/2016 to 2017/2018, according to the United States Department of Agriculture, 2019, China turned into the top rice bringing in the nation over the world As per the "Exploration Report on Paddy and Rice Import in China, 2019-2023", Dublin, Jan 18, 2019 (GLOBE NEWSWIRE), the explanation might be that because of the quick urbanization and monetary development, many work power from the rural area moved to the modern segment since horticultural part contributes little pay in China, which lead to slow-moving in agrarian yield and because of the significant expense of What's more, the subsequent factor is that the cost of domestic rice is higher than imported rice China's rice imports are predominantly from Vietnam and Thailand Their costs are just 80% of those of locally developed rice at a similar quality level or lower as indicated by the examination report on paddy and rice import in China, 2019-2023

gradient in both temperature and precipitation (Yanai and Li 1994, Qiu 2008, Yao et al 2012, Sharma et al 2016) As indicated by the IPCC AR5 report, in tropical and

temperate areas, the local temperature increment of 2֯C or increasingly above in

late-twentieth-century levels will negatively affect the significant yield production without adjustment Besides, as per the most models, utilizing a scope of General Circulation Models (GCMs) and Special Report on Emission Scenarios (SRES) situations, high temperature will prompt the lower measure of rice production because of the shorter developing time frame

1.2 Vulnerable Rice Production under Climate Change (High Temperature)

Rice is delicate to extreme climate stress, for example, high temperature will affect the basic development stages, floods cause halfway or complete submergence, additionally, it is exceptionally sensitive to salinity identified with sea-level rise and dry season spells by rain-fed paddy Rice is ordinarily developing during mid-year (summer) in tropical and sub-tropical locales and heat stress is a typical compel during anthesis and grain-filling stages (Kobata and Uemuki, 2004) The ideal temperatures for various development and advancement phases of rice are as follow:

Table 1.1 Ideal temperatures for various development stages of the rice plant

Development Stages Optimum Temperature Reference

Seed Germination 25 - 35ºC Ueno and Miyoshi, 2005

Rate of Leaf Emergence 26 ºC Ellis et al, 1993

Days of Heading 27 – 30 ºC Horie, 1994

Rate of flowering 30 ºC Nakagawa et.al, 2005

Seed set 29 ºC Ziska and Manalo, 1996

photosynthesis and plant respiration Halford, 2009 reported that when the plant is under prolonged high-temperature, electrolytic leakage may occur from leaves While another examination expressed that temperatures past the basic limits will decrease the development length of the rice crop just as expanded the spikelet sterility, diminishing grain-filling term, and upgrading respiratory losses, prompting lower yield and lower quality rice grain (Fitzgerald and Resurreccion 2009, Kim et al., 2011) It is projected that worldwide rice production, grain quality, and healthful advantages will be seriously diminished by increasing mean air temperature, (Teixeira et al., 2013, Lin et al., 2010, Wang et al., 2011)

According to IPCC AR4, it is projected that the world’s temperature will be increased

by 2-4°C at the end of the 21st century Meanwhile, it is anticipated that expansion in

1ºC over the basic temperature (>24 ºC) will prompt a 10% decrease in both grain yield and biomass (S Mohanty, R Wassmann, A Nelson, P Moya, and S.V.K Jagadish, 2012) Moreover, IPCC anticipated that that "the quantities of cold days and nights have diminished and the quantities of warm days and nights have expanded across the majority of Asia since around 1950, and heatwave recurrence has expanded since the center of the twentieth century in enormous pieces of Asia" (IPCCAR5, Chapter 24) Endophytes as biofertilizers can be used as one of the natural-based adaptation tools since they are found obviously in different ecosystems

1.3 The Concept of Endophytic fungi as Biofertilizers

Agriculture is one of the most vulnerable sectors under climate change, which in turn contributes to climate change through land degradation, methane production, and so on In Asia, extreme use of chemical fertilizer reasons soil pollution, nutrient imbalance, and soil erosion and that they lead to unfavorable effects on soil fertility (Zhang et al., 1996 and Hedlund et al., 2003) Therefore, the utility of biofertilizers promising in sustainable and ecofriendly agriculture cultivation while ensuring food demand for the world population

There are various varieties of biofertilizer including:

(b) Phosphorous mobilizing biofertilizers: (i) Phosphate solubilizer, (ii) Phosphate absorber

(c) Organic matter decomposer biofertilizers: (i) Cellylolytic, (ii) Lignolytic All those styles of biofertilizers may be bacteria, fungi, and cyanobacteria

Nitrogen is one of the important nutrients for plant life and crop yields because it's far a primary component of chlorophyll, by which plants utilize sunlight and convert sugars from water and carbon dioxide from the air called photosynthesis Moreover, Nitrogen is also a basic component of amino acids, which are building blocks of proteins Although nitrogen is abundant in the air, plants cannot use the gaseous form

of nitrogen (N2) directly from the air Plants use nitrogen in the form of “mixed” by

fixation The nitrogen fixation can occur in ways:

Biologically- organisms in the soil convert the gaseous state of nitrogen into

ammonium iron (NH4) which can be used by plants;

Lighting- lighting process convert N2 into ammonia and nitrate (NaNO3); and

Industrially- by applying nitrogen fertilizer

Nitrogen fixation can be also formed through symbiosis relationships between soil microbes and plants In the process, bacteria colonize plant roots, they reside and multiply After that, the association of plant and bacteria synchronize and stimulates plant growth processes such as the formation of root nodules in which they convert free nitrogen to ammonia, enlargements of plant cells, etc Blue-green algae (BGA) called cyanobacteria can be found on land and in water They also fix nitrogen and are used as biofertilizers for cereal crops and non-legumes Oscillatoria, Nostoc, and

Anabaena, etc are examples of cyanobacteria The symbiotic association between the

aquatic fern Azolla and Anabaena is very important for paddy and in this microbial association, Anabaena receives carbon and nitrogen from the plant in exchange for fixed nitrogen

infections By which phosphorous supports the growth of the whole life cycle of the plant The symbiosis association can also be found between plant and fungi and are classified as "mycorrhiza" The fungi affiliation serves the host plant by retaining the phosphorus from the soil and bolster the host plant Also, through the fungi affiliation, the host plant can resilience to biotic and abiotic stress, protection from the pathogen, and generally increment the plant development

It was reported that soil-infertility is the major constrain for low productivity especially in developing countries, Khosro and Yousef, 2012 Therefore, to maintain

the soil quality is important Meanwhile, it was reported that the utilization of

biofertilizers enhances soil particles and continues soil fruitfulness and upgrades root expansion by producing the plant growth-promoting hormones (Subba Rao, 1993) Application of biofertilizers is known to improve soil fruitfulness and harvest efficiency in several crops, for example, improving the rice production by applying the high yielding assortments and through advancing the agrarian practices, for example, organic manures and biofertilizers application (Zaki et al., 2009) High yield of different vegetables after immunization with nitrogen-fixing microorganism allow fixed nitrogen to plants as well as the Nitrogen status of soil (Zaidi et al., 2003) Field execution of excrement from biogas plants has been found to builds the yield just as soil microbial activity in maize (Mehetre and Kale, 2007) Biofertilizers are fit for providing significant supplements as well as micronutrients for the successful development of plants through the association of microorganisms

1.4 Japonica Rice Species

as the most cultivation area in Japan since 1979 Koshihikari has a highly adaptive capacity to the climate that they can be cultivated at altitudes ranging from 40°N to 31°N The vegetative phase of Koshihikari was 35.2 days if they are grown under short-day conditions after sprouting at 28 °C (Mimoto et al 1989) and are highly sensitive to temperature (Hosoi 1979) Meanwhile, tropical Japonica can be found in the Central Dry Zone, Delta Region, and Coastal Region of Myanmar (21.9162° N, 95.9560° E), Ohm Saw, Genetic Diversity of Myanmar rice Cultivar The global mean temperature has risen and the meteorological research projects an increase of one or two degrees all over Japan in 2081 to 2100, which may cause threats to rice productivity and growth Therefore, the symbiosis relationship with the fungal endophyte in rice roots is promising to enhance the resistant capacity of plants in

coping with water stress and soil nutrient deficiency due to climate change impacts

1.5 Symbiosis Relationship between Plant and Fungi

Symbiosis: “Any association between two species populations that live together is

symbiotic, whether the species benefit, harm, or not affect one another”, Heinrich

Anton de Bary 1879 All the plant that lives in the natural ecosystem has a symbiosis

relationship microbes including mycorrhizal fungi and fungal endophytic (R J.Rodriguez, et al, 2009 and Petrini, 1986) Therefore, mycorrhizas and fungal endophytes are the two main groups of symbiotic association with plants and their symbiosis relationship impact the structure and function of the ecosystem (Smith SE and Read, 1997)

1.5.1 Endophytic Fungi

artificial medium Endophytic fungi have huge biodiversity, particularly in the tropical and temperate rainforest Unlike AMF, the endophytic fungi colonize only through the root of the host, and live inside roots, stems, leaves, and develop during host senescence (Saikkonen et al., 1998) Their symbiosis benefit is that endophytic fungi receive protection and nutrients from the host, whereas the host plants may benefit from the endophytic fungi through increased resistance to biotic stress such as herbivores and pathogens, and also various abiotic stresses and enhanced competitive abilities (Wilson, 1995; Rodriguez and Redman, 1997; Lehtonen et al., 2005)

1.5.1.1 Endophytic Fungi Classification and host range

Fungal endophytes can be classified into two basic groups according to their taxonomy, host range, colonization transmission design, tissue characteristics and ecological function, clavic-endophytes (C-endophytes), which are connected with grasses and non-Clavic-endophytes (NC-endophytes) Table 1.4 describes the characterization of the functional class of four classes of endophytic fungi

Class I Clavicipitaceous Endophytes: these are a relatively modest number of species that are limited to a few monocot hosts and contain animal-harming alkaloids The Clavicipitaceous endophytes symbiosis with grasses was first noticed by European specialists (Guerin, 1898; Vogl, 1898) Bacon et al., (1977) responded with the hypothesis that animals that consume contaminated tissue from endophytic fungi are related to the poisonous disorder Clavicipitaceous Endophytes have many benefits in hosting plants such as:

Insect deterrence: insect feeding resistance of mushrooms to plants through the release of loline and peramine-mychotoxin alkaloidis (Clay 1990; Patterson et al 1991) has been observed to resist insect feeding to host plants (Rowan and Gaynor 1986)

Nematode decrease: Endophytic fungus Acremonium coenophialum is described to be resistant to infection by decreasing the number of nematode populations in fields of Festuca arundinacea by an infection in high fescus (Kimmons et al., 1990) Increased host disease resistance: Certain Clavicipitaceous Endophytes increase their resistance to host plant disease The release, in symbioses’ with Epichloë festucae (cool-seaon-grease), 2010 of the indole supplies, such as the secretion of sesquiterpene and the diacetamide compound, will resist the growth of other pathogenic fungi

Boost the ecological physiology of host plants: Arechavaleta et al., 1989 have indicated that some Clavicipitaceous Endophytes can withstand abiotic stress, such as drought and contamination by chemicals The host plant can be encouraged by certain endophytes to extend the hair of the root, through which 'phenolic compounds' fill the rhizo The host plant will assimilate higher quantities of soil phosphorus and improve tolerance to aluminum by chelation according to this mechanism (Malinowski and Belesky 2000)

Class II Nonclavicipitaceous Endophytes (NC Endophytes): According to Rodriguez et al., (2009), the class II NC-endophytes represent three distinct functional groups The host range of NC endophyts is very wide and includes both monocots and dicots (Rodriguez and Redman, 2004) and tropical leaves (Lodge et al., 1996; Fröhlich and Hyde, 1999; Arnold et al., 2000; Gamboa and Bayman, 2001) In biomes ranging from tropically woodland to boreal including Arctic-Antarctic communities (Carroll & Carroll, 1978; Petrini, 1986; Stone, 1988), they can grow in both the above and lower ground tissues of the non-vascular plants, seedless vascular plants, conifers, woody, and herbaceous angiosperms And it looks like tolerance to stress adapted to the climate (Rodriguez et al 2008) Symbiosis with class II NC endophytes has many advantages for hosting plants:

habitat-adjusted habitat (Redman et al 1999a, 2001, 2002; Rodriguez and Redman 2007)

Increase of biomass: Tudzynski and Sharon (2002) expressed that class II NC endophytes are able to develop shoot and additionally root biomass because of the enlistment of plant hormones by the host or through biosynthesis of plant hormones by the fungi

Protection from fungal pathogen: Class NC endophytes can resist fungal pathogens by their various strategies including the production of secondary metabolites (Danielsen et Jensen, 1999; Narisawa et al., 2002; Campanile et al., 2007) (Schulz et al., 1999)

Table 1.2 The Characterization of functional classes of endophytic fungi

Criteria Clavicipitaceous

Endophytes

Non- Clavicipitaceous Endophytes

Class I Class II Class III Class IV

Host range Narrow Broad Broad Broad

Plant tissue colonize

Shoot, rhizosphere Shoot, root,

rhizosphere

Shoot Root

In-plant colonization

intensity

Extensive Extensive Limited Extensive

In-plant biodiversity

intensity

Low Low High Unknown

Mode of transmission

Fitness benefits Non-habitat adapted Non-habitat adapted and

habitat-adapted

Non-habitat adapted

Non-habitat adapted

1.5.1.2 Bioactive compounds produced by fungal endophytes

Protein synthesis and respiration associated fungal metabolites are diverse, and several secondary metabolites have been isolated frequently and are chemically defined, which include waste products as well as pigments, toxins, and antibiotics, and have biological functions Schulz et al., 2002 reported that at least a research organism of antibacterial, fungicidal, algicide, and herbicide activities comprised 83% of the algal isolates and 80% of the endophytic fungi isolated from plants and only 64% of soil operation

Diverse classes of natural compounds produced by Fungal Endophytes

Alkaloids: A chemical compound containing basic nitrogen atoms and produced as common secondary metabolites by fungal endophytes occurs naturally The endophytic fungal-producing alkaloids have antimicrobial activity (Souza et al., 2004) Chaetomium globosum fungal endophyte isolated from Ginkgo biloba and the species produces chaetoglobosins A, G, V, Vb, and C, some of which have been shown to be cytototoxic (Li et al., 2014) Vincristine is an alkaloid from Catharanthus roseus originally isolated from vinca (Zhang et al., 2000) Chaetoglobosin U is an alkaloid dependent on cytochalasin, isolated from Chaetomium globosum sp of the stem of Imperata cylindrica (Ding et al., 2006)

Steroids: Fungal endophytes produce steroids and most of the isolated compounds have moderate antimicrobial activities The liquid culture of fungal endophyte

Colletotrichum sp isolated from Artemisia annua produce

3β,5α,6β-trihydroxyergosta-7,22-diene; 3β-hydroxyergosta-5-ene;

3-oxoergosta-4,6,8,22-tetraene; 3β-hydroxy-5α,8α-epidioxyergosta-6,22-diene;

3β-hydroxy-5α,8α-epidioxyergosta-6,9,22-triene and 3-oxoergosta-4-ene, two new steroids,

3β,5α-dihydroxy-6β-acetoxyergosta-7,22-diene and 3β,5α-dihydroxy-6β-phenyl-

acetoxyergosta-7,22-diene along with ergosterol Some of them have antifungal activity and have resistant to Gaeumannomyces graminis var tritici, Rhizoctonia

cerealis, Helminthosporium sativum, and Phytophthora capisici (Lu et al., 2000; and

Yu et al., 2010)

Terpenoids: Fungal endophytes produce large terpenoids such as Sesquiterpenes, Diterpenoids, and Triterpenoids (Yu et al., 2010) Sixty-five sesquiterpenes, forty-five diterpenes, forty-five monoterpenes and twelve other terpenes were classified as isolates from fungal endophytes, out of a total of 127 terpenoid compounds, all of which have anti-microbial, anti-cancer and anti-protozoan activity (Souza et al., 2011)

Quinones: Some fungal endophytes produce quinones that significantly inhibit pathogenic development Edenia gomzpompae, the endophytic fungi that contain quinones such as spiroketals (Wiyakrutta et al., 2004) The Pestalotiopsis microspora endophytic fungi, isolated from Torreya taxifolia, develop Torreyanic acid which is an unusual dimeric quinone (Lee et al., 1996) Hormonema dematioides, fungal endophyte isolated from balsam fir produce rugulosin with an insecticide (Findlay et al 1997) Coniothyrium sp the endophytic fungus, produce the new ras farnesyl-protein transferase inhibitors Preussomerin N1, palmarumycin CP4a and palmarumycin CP5 ((Tan and Zou, 2001)

Peptides: Endophytic fungi develop peptides and some have significant antimicrobial activity Acremonium sp endophyte sp Produce di-O-b-glucoside leucinostatin A and leucinostatin A when grown in liquid culture (Strobel et al., 1997a and Kharwar et al., 2011) The endophyte fungal Aspergillus rugulosus, and A Varies nidulans

fungi isolated from Redwood produces cryptocandin, which has an antifungal activity (Tan and Zou, 2001)

Polyketides: Fungal endophyte Periconia sp F-31 isolated from the medicinal plant

Annona muricata, produce a new polyketide synthase-nonribosomal peptide

synthetase hybrid pericoannosin B (Zhang et al, 2016) A tryptophan−polyketide hybrid, Codinaeopsin, isolated from the fungal endophyte CR127A that was symbiosis with a white yemeri tree Vochysia Guatemalensis in Costa Rica, which has the ability against Plasmodium falciparum, the causative agent of the most lethal form of malaria (Kontnik and Clardy, 2008) Endophytic fungi Chaetomium globosum, which was found in the leaves of Ginkgo biloba produce chaetoglobosins A and C, which were isolated from the EtOAc These compounds have the activity against the growth of brine shrimp Artemia salina and Mucor miehei (Qin et al., 2009) The three polyoxygenated polyketides, epicolactone, epicoccolides A and B were shown to have antimicrobial activities and significant inhibitory effects on the mycelia growth of two peronosporomycete phytopathogens such as Pythium ultimum and Aphanomyces cochlioides, and the basidiomycetous fungus Rhizoctonia solani, were isolated from the fungal endophyte of Epicoccum sp CAFTBO, symbiosis with Theobroma cacao (Talontsi et al., 2013)

Acids: The fungal endophyte Salvia miltiorrhiza produces salvianolic acid C (Li et al., 2016) New acid of 3,7,11,15-Tetrahydroxy-18-hydroxymethyl-14,16,20,22,24-pentamethyl- hexacosa-4E,8E,12E,16,18-pentaenoic acid were isolated from the endophytic fungi Phoma glomerata D14 (Khiralla ,2015) The fungal endophyte

Curvularia papendorfii symbiosis with Vernonia amygdalina produce Khair acid,

protease with values of IC50 = 43µM and IC50 =11µM respectively (Guo et al., 2000) The cryptocin was isolated from Cryptosporiopsis cf quercinain, which has antimycotic activity against several plant pathogenic fungal strains including Pythium ultimum, Pyricularia oryzae, with MIC values 0.39 and 0.78 μg mL-1 respectively (Li et al., 2000)

1.6 The Impact of Climatic Change on the Symbiosis Relationship between Plant and Fungi

1.6.1 The Impact of temperature on the Soil Microbial Communities

Temperature influenced the physical and biochemical mechanisms of soil as well as regulating the soil microbial activities The increased surface temperature corresponds with decreased soil moisture, Venkat Lakshmi et al., 2003 Soil moisture controls the soil microorganism as well as biochemical activities of soil (A Borowik and J Wyszkowska, 2016) It is due to the fact that soil microorganism can rapidly equilibrate to the osmotic conditions of their environment and remain hydrated when the soil near them are dried by accumulating solutes to retain water within their cells

(Joshua P Schimel, 2018) As well as indirectly by changing the moisture supply to

the substrates of microbes by dissolution, diffusion, and transportation

The Mechanism

erode hillsides and scour stream channels Therefore water always flows downhill due to the gravitational potential gradient And in the process of osmosis, water on the freshwater side of a membrane moves across to the salty side because the solute potential on the salty side is lower The total water potential gradient made water to transfer from soil through plants to the atmosphere

For the soil microbes, water potential is a basic in controlling their endurance and capacity since they are in micro size and cozy contact with their environment and are along these lines essentially equilibrate with the water potential in the soil around them At the point when the soil got dried, the total water potential drops and the organisms must gather solutes to bring down their inside solute potential to coordinate the water capability of the encompassing soil to abstain from losing water to their condition and drying out When the cellular water potential of microbes drops, it might prompt a loss of cell turgor (Harris 1981), practically equivalent to shrinking in plants This process interferes with microbial physiological capacities and prompts decreasing the metabolic rates and this can even lead their cell capacities to fall flat, slaughtering the microbes

The global mean surface temperature has risen during the previous century (Jones et al 2012, Stocker et al 2013, Sun et al 2017) and according to IPCC AR5, it is projected that heat waves will prone to be progressively extreme, successive and cold scenes are anticipated in a diminishing later on The daily minimum temperature is projected to increase than the daily maximum temperature

1.6.2 The impact of Climate Factors on the Symbiosis between plant and Fungal Endophyte

abundance in the southeastern Queensland plantation The researcher argued that "this variation is probably a reflection of the range of environmental conditions sampled in Hong Kong and northern Queensland Sites differ primarily from rainfall, altitude, and forest types; these are all factors that may influence the abundance of fungal species as they affect moisture, temperature, and potential inoculum sources” The impact of climate on symbiotic fungal endophyte diversity and performance was further explained by Hannah Qiauque and Christine V Hawkes, 2013 In this research, endophytes in grasses across the steep precipitation gradient of Edwards Plateau in Central Texas were collected to elucidate the relative importance of environmental and spatial factors in the structuring of endophyte communities Annual rainfall across the Plateau varies from ~40 to 90 km west to east, with an average annual precipitation change of ~10 cm every 40–50 km The 20 endophyte isolates in symbiosis with grass seedlings were also sampled from drier and wetter regions with high and low soil moisture in the greenhouse Environmental factors related to historical and current precipitation have been the most important predictors of endophyte communities in the field due to the possibility of past drought patterns that create legacies that constrain the current community and ecosystem properties The historical mean annual and current spring rainfall also affected around 35% of the variation in the endophyte community composition For the performance of fungal endophytic fungi, the reduction of plant water loss in the greenhouse was reduced for endophytic fungi in western sites compared to fungi in eastern sites They concluded that although historical and current climatic factors have an impact on the endophyte community, their symbiotic function could not be predicted by the local environmental condition

1.6.3 The Effect of Seasonal Variation to Fungal Endophytes Communities

The impact of seasonal variation and environmental factors on endophytes transmission in tall fescue was studied by Ho J Ju, 2003 In this study, the impact of seasonal variation on endophytes in tall fescue was investigated by collecting the

samples by 1st July 2000 and continued monthly until June 2002 in two sites;

January to May in Oregon The temperature impact was tested by Jesup Max Q seeds in the greenhouse and four temperature treatment regimen were established: (1) 12/6 ° C day/night temperature for weeks, 25/19 ° C day/night for weeks, (2) 25/19 ° C day/night temperature for weeks, 12/6 ° C day/night for weeks, (3) 12/6 ° C day /night temperature for weeks, and (4) 25/19 ° C day /night temperature for weeks And it has resulted that plants grown at higher temperature regimes showed higher growth and greatest dry matter throughout the experiment than those grown at the cooler temperature regime And the endophyte concentration was increased at the 25/19ºC regime, throughout the experiment

J Collodo et al., 1999 investigated the impact of geographical and seasonal influences on the distribution of fungal endophytes, Quercus ilex samples were collected from four sites in central Spain, one of which was sampled twice in autumn and spring After the insulation of the fungal strain, a total of 2921 fungal strains were identified as the 10 dominant species with an insulation frequency of > 1.5 percent And research has shown that the colonization capacity and diversity of fungal species were significantly higher in the spring, except for C Quercina, Acremonium sclerotigenum (F & V Moreau ex Valenta) Gam and that's D mutila species

The above theories and previous research discussed the impact of climate factors on the occurrence and diversity of fungal endophytes However, to my knowledge, how the colonization capacity of fungal endophyte will reflect under the high-temperature condition is limited

1.7 The Role of Fungal Endophytes in Sustainable Agriculture

1.7.1 Role of Fungal Endophytes in Controlling Environmental Contamination

Previous research elucidated studied the role of endophytic fungi in heavy metal resistant and related to the phytoremediation mechanism which is a low cost, environmentally friendly, and effective method to remove toxicants from contaminated soils In this research, it is described that the fungal endophytes related to hyperaccumulators plants can tolerance metal accumulated in the roots of their host plant due to the long term adaptation

Iqbal Ahmad., Mohd Ikram Ansari, Farruk Aqil, 2016 studied the role of fungal endophytes Aspergillus niger and Penicillium sp in Cr, Ni, and Cd bioabsorption capacity Endophytic fungi have been isolated from long-term municipal wastewater treatment soil mixed with untreated mechanical profluence By applying alkali-treated dried and powdered mycelium, Aspergillus niger, and Penicillium sp Bioabsorption of Cr, Ni, and Cd was tested for their potential And it was concluded that “Aspergillus niger and Penicillium sp have a promising bioabsorption capacity of Cr, Ni, and Cd from single and multi-metal solutions”

Another study examined the role of dark septate endophytic (DSE) fungi in the control of contaminated environments Ousmane Diene et al., 2014 studied the role of DSE fungi in Cesium management following the Fukushima Daiichi Nuclear Power Plant accident in 2011 The result showed that P ibarakiensis isolates I.4-2-1, unidentified taxon isolates 312-6, and V simplex Y34 isolates in symbiosis with Chinese cabbage seedlings showed improved plant biomass of 49 percent, 64 percent, and 82% respectively under Cs of 5ppm, but there was no increase in biomass below 10ppm Symbiosis with tomato seedlings showed an increase in biomass of 96 percent and 122% under Cs [5ppm] Under Cs [10ppm], a symbiotic tomato plant And it was concluded that the selected DSE fungi are likely to function in radionuclide-polluted environments by regulating the bioactivities of Cs

1.7.2 The Role of Fungal Endophytes in Biotic and Abiotic stress resistance

Some symbiotic relationships with fungi are benefited to the host plant that they provide nutritional support or contribute to defense against damage from herbivores, but other symbioses with fungi are pathogens or parasites (Atkinson and Urwin 2012) Environmental factors such as temperature, humidity, light intensity, as well as water, mineral, and CO2 availability are the abiotic stress to plants These abiotic factors influence plant growth parameters and resources Plants need an optimum level of biotic and abiotic factors to achieve maximum growth and yield

Abiotic Stress: Drought stress, a combination of reduced water content and

diminished water potential, is one of the major constrain to crops growth and yield which lead to turgor reduction, increased stomatal closure, and thus reduced cell growth and development, (Jaleel et al.,2009) Water deficiency can inhibit the photosynthesis and metabolic processes including respiration, translocation, ion uptake, carbohydrate metabolism, and nutrient uptake in plants and lead to plant cell death (Jaleel et al 2009 and Farooq et al., 2009)

Mechanism of abiotic stress tolerance by fungal endophytes can be formed by three types, (Malinowaki and Belesky, 2000): 1) Accumulation and translocation of assimilates, 2) Maintenance of cell wall elasticity and 3) Osmotic adjustment Through the first mechanism of accumulation and translocation of assimilates, when the plant is under the abiotic stress, for example, changes during the time spent photosynthesis, adjustments in the carbon metabolism and level of starches (sugar) are seen, (Gonzalez et al., 2009) The evidence of this mechanism was confirmed by the process that the plants with fungal endophytes symbiosis produce a higher amount of soluble sugars, for example, glucose and fructose in the leaf blades, (Richardson et al., 1992) The fungal endophytes may provoke the host plant during the metabolic process to secrete soluble sugars through which the plant gives out wall elasticity and osmotic adjustments during the condition

Rangga Yuspradana et al, 2017, studied the role of endophytic fungi in promoting rice growth under drought stress conditions They proved that endophytic fungi for drought-resistant in rice plant by testing Acremonium sp., Penicillium sp., Nigrospora

colonization capability under the three different water availability conditions (wet, moderate, and dry conditions by giving water 100, 50 and 25% of field capacity, respectively) and the result proved that these selected species of endophytic fungi are drought resistant according to the three parameters

Kumkum Azad, 2016, tested the role of fungal endophyte in drought and salt conditions-resistant by applying the symbiosis between tomato (Solanum Lycopersicum var Rutgers) and Saskatchewan saline endophyte strains For the endophyte isolation, samples were collected during summer, and for the salt-resistant test, NaCl and Hoagland’s solution was used and applied to two-week-old plants for 20 days And for the drought stress test, plants were rehydrated for days after each round of drought stress As a result, following the NaCl test, the plant with endophyte colonization tended to result in a 30-50 % higher shoot biomass than non-colonized plants For the drought stress test, treatment with SK isolates Hz613, 419, and 414, Hz613 and 414 had significantly greater fresh root biomass than non-symbiosis plants during the drought test And it was concluded that treatment with Saskatchewan saline endophyte strains was able to tolerate salt or drought stress than the control treatments

Biotic Stress: “Stress that is caused in plants due to damage instigated by other living

organisms, including fungi, bacteria, viruses, parasites, weeds, insects, and other native or cultivated plants” can be defined as biotic stress (Newton et al., 2011) Biotic stress is also a major constraint that contributes to a significant loss in crop production Plants respond with the protection system to biotic stress and the mechanism can be categorized into an innate and systemic response According to Atkinson and Urwin 2012, when infection occurred, plants produce reactive oxygen species (ROS) and oxidative bursts that control the spread of pathogens Plants increase cell lignification and block the invasion of parasites to attack pathogens Also the production of natural bioactive compounds, plant protection against biotic stress

suppress pathogens Fungal endophytes promote plant pathogen resistance by systematically acquired (SAR) and systemically induced (ISR) resistance SAR proteins are designed to fight plant pathogens through Salicylic acid (SA) and Pathogenesis-related (PR) proteins

According to Gunatilaka, 2006, numerous fungal endophytes produce secondary metabolites, for example, antifungal and antibacterial metabolites compounds, which emphatically hinder the development of different microorganisms including plant pathogens It was reported that a group of biocontrol strains of fungal endophytes can create antibiotics such as terpenoids, alkaloids, aromatic compounds, and polypeptides and are reported that plant pathogens are sensitive to them Hellwig et al., 2003, also proved that Alkaloids also strongly suppressed microbes In this research, fungal endophytes Alternaria spp produced a new alkaloid of alteration isolate, which was elucidated for having antibacterial activity against several pathogenic gram-positive bacteria Volatile oil also is one of the antibiotic compounds produced by fungal endophytes This hypothesis was proved by Atmosukarto et al., 2005 This research elucidated that the fungal endophytes

Muscodor albus, isolated from the tropical tree species, produce many volatile

organic compounds including tetrahydrofuran, 2-methyl furan, 2-butanone, and aciphyllene which have antibiotic activities

Research on the role of DSE fungi, Veronaeopsix Simplex Y34 isolated from Yaku Island, Japan, in suppressing the disease of Fusarium, was conducted by Rida O Khastini et al., 2012 Veronaeopsix Simplex Y3 symbiosis with Chinese chicken showed that the disease with Fusarium wilt decreased to 71 percent and remained good The research has defined the production of mechanical resistance created by the V simplex Y34-Hyphae network that colonized the host root and indirectly induced a mechanism of resistance in the plant for this disease suppression activities

1.7.3 The Role of Fungal Endophyte in Promoting the Plant Growth

Increment accessibility of nutrients: the fungal endophytes can fix, solubilize, assemble different kinds of nutrients such as in micro or macro, and made them accessible for the host plants And thus the application of fungal endophytes can reduce the practice of chemical fertilizers It was reported that the endophytes isolated from the soybean able to solubilize phosphate, Kuklinsky-Sobral, J et al., 2004 Some endophytes isolated from the grasses grown under nitrogen-deficient soil can fix atmospheric nitrogen into ammonia (K B Rakholiya, and M D Khunt, 2015) Production of Phyto-hormone: the endophytic microorganism can produce signal molecules called phytohormones They can produce auxins, gibberellins, principally nodule-3-acetic acid (IAA), etc., which are the plant growth promoters Auxins can against ethylene and therefore, a high level of IAA with a low level of ethylene promotes plant growth and root development (Witzel et al., 2012)

Toxic molecules degradation: endophytes can degrade toxic and recalcitrant molecules at the rhizosphere as per their genetic machinery

The gibberellins (GAs) were reported to enhance crop growth and alleviate the harmful effects of abiotic stresses (Khan et al 2011, Crozier 2000; Davies 2010; King and Evans 2003; Pharis and King 1985) Lubna Bilal et al., 2018 researched to elucidate the mechanism of fungal endophytes in promoting plant growth of

Asprgillus fumigatus TS1 and Fusarium proliferatum BRL1 Fungal endophytes were

screened for IAA production and the strains that produce IAA were selected And concluded that the selected endophytic fungi can synthesize bioactive compounds, and assume that these compounds are fundamental in promoting plant growth

1.7.4 Dark Septate Endophytic Fungi (DSE) and their abilities

1.8 Research Question and Hypothesis

Plant roots play the main role in maintaining the resistant capacity to biotic and abiotic stress by forming a symbiotic relationship with endophytic fungi under climate change However, how the fungal endophyte symbiosis with the rice plant reflects under high-temperature conditions is still unclear

Accordingly, an experiment was conducted with “Koshihikari” rice to test two hypotheses:

i) The selected Dark Septate Endophytic (DSE) fungi can be well colonized with temperate rice plants under high continuous temperature

ii) The colonization DSE promotes temperate rice growth under the high-temperature condition and, thus being potential for as biofertilizers

1.9 Objective of the Research

The research was implemented to aim:

i) To elucidate the symbiosis capacity of endophytic fungi in the early stage of rice growth

ii) To evaluate the impact of high temperature on rice-endophytic symbiosis through the observation of fungi presence in the roots

CHAPTER MATERIAL AND METHODS

2.1 Experiment Design

All the experimental procedures were conducted at the College of Agriculture, Ibaraki University, Japan The five treatments of Koshihikari Rice plant: without endophytic symbiosis (control), with Cladophialophora chaetospira (OGR3), Meliniomyces

variabilis (J1PC1), Phialocephala fortinii (LtPE2), Veronaeopsis simplex (Y34) were

compared in the growth chamber at a constant temperature of 35 °C to elucidate the colonization capacity of dark septate endophytic fungi to their host plant under the high temperature

Plant growth conditions: the temperature of the growth chamber was controlled at 35ºC continuously for day and night while the light condition was given 12 hr of light as day time and 12 hr dark as night time

2.1.1 Research Parameter

Root colonization capacity: The capacity of DSE colonization to the root of the Koshihikari rice plant was checked by observing the typical characteristics of DSE symbiosis in the root such as the formation of fungal hyphae coils, vesicles, etc., in the roots under the microscope

Root’s response to DSE colonization: After 10 days of transplantation, plant growth parameters such as root length, root volume, length of root, and root shoot ratio were measured Root dry weight was measure by inserting the fresh root into the oven for

24 hours at 70֯ C and the root/shoot ratio was obtained by dividing the dry weight of

the root by the dry weight of the shoot

2.2 Materials and Methods 2.2.1 Rice Seed Germination

Table2 2.1 Equipment and chemical used in rice seed germination

Equipment/Chemical and

Consumables

Type Producer

Vortex mixer CM 1000 Tokyo Rikakikai

Pipet-Aid Drummond Scientific

Conical laboratory tube e.g As one

Incubator As one

70% Ethanol Wako

1% HCloNa Wako

Agar powder Wako

Plastic petri dish As one

Seeds were sterilized by vortex with 70% ethanol for min, followed by 1% sodium hypochlorite for and then washed with sterile distilled water for times, 5min each After that, the seeds were immersed into the sterile distilled water using the 50 ml conical laboratory tube and incubated at room temperature (19º C) for days After seed sterilization, the sterilized rice seeds were air-dried in a laminar airflow cabinet for 30 and transferred to the Petri dish containing water agar medium (500ml WA medium: agar powder 5g and sterile distilled water 500ml) for germination process and incubated in 30°C incubator for one day

2.2.2 Transplantation

Materials used in transplantation

Table 2.2 Equipment and chemical used in transplantation

Equipment/Chemical and

Consumables

Type Producer

Autoclave e.g Tomy

Plant growth chamber e.g LEEC

Cork borer e.g As one

Pipet-Aid Drummond Scientific

MgSO4.H2O Wako

KH2PO4 Wako

The experiment was conducted with treatments: Koshihikari rice plant without endophytic symbiosis (control), with Cladophialophora chaetospira (OGR3),

Meliniomyces variabilis (J1PC1), Phialocephala fortinii (LtPE2), Veronaeopsis simplex (Y34) Each treatment was replicated times

For the control treatment, the germinated rice seeds were transplanted into Petri dish of the oatmeal medium which was mixed with other mineral and nitrogen sources

(MgSO4, H2O, KH2PO4, NaNO3,Nature Aid (Sakata-no-tane), powdered oatmeal

and agar for plant cultivation) For DSE fungi treatment, the germinated rice seed was transplanted into the Petri dish of the oatmeal medium Then, each species of DSE fungi maintained in the Petri dish of 50% Corn Meal Malt Medium (derived from the culture collection of the laboratory of Microbial Ecology, Ibaraki University) was excised from an edge of an actively growing colony on culture medium into 5mm diameter circular shape by using autoclaved cork borer and placed near the root of the germinated rice seed Then the open Petri dishes were enclosed into the autoclaved plant culture PC square jar and transferred to the plant growth chamber

2.2.3 The effect of high temperature on the symbiosis

To elucidate the effect of high-temperature condition on the colonization capacity of DSE fungi, the temperature of the chamber was set up at 35°C constantly

After 24hr of transplantation, bacteria appeared in all treatments in the growth chamber Since the main purpose of this research was to elucidate the effect of high temperature on the symbiosis between DSE fungi and rice plants, the experiment was repeated by conducting the second experiment to suppress the bacteria

Nature aid (Sakata-no-tane) Wako

Plastic petri dish e.g As one

Figure 2.1 Appearance of Bacteria Found in the first experiment during 24 hr of

transplantation

2.3 The Second Experiment

The experiment was repeated as second experiment in order to eliminate the effect of bacteria in the research However, in this experiment, 2% streptomycin was added into the oatmeal medium to suppress the bacteria during transplantation, and the drying time after seed sterilization process before germination was changed into 24 hr instead of 30 to reduce the risk of bacteria growth from the vapor based on the preliminary test

In the second experiment, due to the limitation of stocks, only applied for three species of DSE fungi were used Therefore, only four treatments of Koshhikari Rice plants were compared in the second experiment: Control, Cladophialophora

chaetospira (OGR3) treatment, Meliniomyces variabilis (J1PC1) treatment, Veronaeopsis simplex (Y34) treatment, respectively

2.4 Detection of DSE fungi in the root of the Koshihikari rice plant and identification of bacteria in the first experiment

2.4.1 Isolation and Identification of Bacteria

Petri dished were incubated at 35ºC The procedure was repeated for 2times until the single colony of bacteria was isolated

Identification of Bacteria

Materials used in bacteria DNA extraction, PCR, and DNA sequencing

Table 2.3 Materials used in Bacteria DNA Extraction, PCR, and DNA sequencing

Equipment/Chemical and Consumables Producer

Autoclave e.g Tomy

Laboratory test tube dry block heater e.g Thermo Scientific

Laboratory centrifuge e.g ENOVA

Thermal cycler e.g Bio-Rad

DNA sequencer machine Applied Biosystems PRISM

Pipet-Aid Drummond Scientific

PrepMan Ultra Sample Preparation Reagent Wako

Gel running buffer Wako

AgrTherose Wako

Triple dye Wako

 DNA Extraction

Pipetted 50µl PrepMan Ultra Sample Preparation Reagent into each 1.5ml Eppendorf tube Then took the single colony of bacteria from three isolate samples and soak into the reagent in the tube and vortex The tubes were incubated in the heater at 100ºC for 10min Centrifuged at 13500 rmp by 3min at room temperature Pipetted the supernatant into a new 1.5ml Eppendorf tube And those supernatant are the DNA extraction solution

Figure 2.2 Isolation of single colony bacteria for DNA extraction  Polymerase chain reaction (PCR)

PCR is a method that is broadly utilized in atomic science to intensify the objective grouping from small quantities of DNA and RNA layouts (Van Tuinen et al., 1998; Schwarzott et al., 2001) The PCR process contains four stages: Denaturation, Annealing, Elongation, and Final elongation, which is performed to ensure any staying single-standard DNA is completely expanded (Sambrook and Russell, 2001) And the Primer pair of 27F and 1492R were used for the amplification of the objective

dNTP Wako

Ex Taq Wako

Primer forward/Primer Reverse Wako

sequence PCR reactions were performed in a final volume of 45μl containing PCR buffer (of primer 27F 0.32 μl, primer 1492R 0.32 μl, big dye 0.5 μl, 5x sequencing

buffer 1.5 μl, sterilized MilliQ H2O 6.68 μl and DNA template μl) Amplification

was performed according to the program: Initial denaturation cycle 95 °C (2 min), followed by 25 cycles of denaturation at 95 °C (30 s), annealing cycle 55 °C (2 min), elongation cycle 72 °C (2 min) The last cycle was followed by a post elongation cycle 72 °C (7 min) Then the cooling at 4ºC

 Urification of PCR products

1,174.5 μl of purification solution (3M Sodium Acetate (ph 4.8) 324 μl, 40%PEG 810

μl, 200mM MgCl2 40.5 μl) was prepared and each 43.5 μl purification solution was

added into each PCR product tube and then vortex After that, the tubes were incubated at 4ºC overnight Then the tubes were centrifuged at 15000 rpm by 15min at room temperature Get rid of supernatant Then 180 μl of 80% ethanol was added, mixed, and centrifuge as previously then get rid of supernatant Then dried out the suspension in the clean drawer covered by tissue paper for 2hrs Then 15 μl of

sterilized MilliQ H2O and mixed by pipette and kept at -20°C

 Polymerase chain reaction (PCR) Sequencing

Pipette μl system solution (primer forward 0.32 μl, big dye 0.5 μl, 5x sequencing

buffer 1.5 μl, sterilized MilliQ H2O 6.68 μl) into 0.2ml PCR tube, and then pipette

μl each purified PCR solution Then sequencing the PCR according to the cycle: 96ºC at 2min, then 25 cycles of 96 ºC for 30sec, 50 ºC for 15 sec, and 60ºC for 3min

 Purification of Sequencing PCR products

Add 3X purification solution was added into each PCR product tube and then vortex and incubated at room temperature for 15min Then the tubes were centrifuged at 15000 rpm by 15min at room temperature Get rid of supernatant Then 150 μl of 70% ethanol was added, mixed, and centrifuge as previously then get rid of supernatant Then dried out the suspension in the clean drawer covered by tissue paper for 2hrs Then kept at -20°C

The purified DNA fragment was the sequence at Applied Biosystems PRISM The sequence data were BLAST searched against the GenBank database

2.4.2 Checking the root colonization capacity of DSE endophyte in the roots of the Koshihikari rice plant

Materials used in identification of DSE fungi in the roots of Koshihikari rice plant Table 2.4 Equipment used in Identification of DSE fungi

Toluidine blue or TBO stain method was used to verify the DSE fungal structures in the root of the plant and it was applied for both experiments: the first experiment and the bacteria control (second) experiment

TBO was used to check the presence of the DSE colony in the roots of the Koshihikari rice plant TBO staining help to show the cell wall lignification at the epidermis Therefore, DSE hypha penetration to plant cell can be checked by using TBO

Equipment/Chemical and

Consumables

Type Producer

Light microscope e.g Olympus

Vortex mixer CM-1000 T Tokyo Rikakikai

Pipet-Aid Drummond Scientific

50% Ethanol Wako

10%V/V KOH Wako

1%b HCl Wako

After 17 days of transplantation, the roots of the Koshihikari seedling were harvested The roots of the Koshihikari seedlings were cut and washed with sterile distilled water (SDW) for 3times Then the roots were soaked into 50% ethanol for 24 hr at room temperature As the second step, the roots were rinsed SDW for times and placed in 10% (v/v) KOH and heated at 80ºC by using tube heating block for 20 In the third step, the roots were rinsed with SDW and placed in a 1%HCl solution for at room temperature For the last step, the roots were quickly stained in 0.0005% cotton blue in 50% acetic acid

The DSE fungi structures such as the formation of DSE hyphae, hyphal coil, microsclerotia like structures, and septate in the hyphae were verified

To check these DSE fungi structures, the blue stained roots were cut into small pieces and placed on the microscope glass slide The glass slice was covered with microscope slice square coverslip and checked under the compound microscope through 40X and 100X magnification

2.5 Identification of DSE fungi and Measurement of plant growth parameter of Koshihikari seedlings under high temperature in the second experiment

2.5.1 Measurement of plant growth parameters

Plant growth parameters were measured to elucidate the effect of DSE fungi in Koshihikari rice plant seedlings

Shoot height and seminal root length were measured by using a cm ruler Root volume was measured through volume displacement by using measuring cup: simply subtract the water volumes of before and after inserting the roots in the cup

To measure the dry weight of the shoots and roots, the roots and shoots of the harvested Koshihikari seedlings were dried overnight at 72ºC in an oven Root/ shoot ratio was obtained by the ratio of the dry weight of root to dry weight of shoot

2.5.2 Identification of DSE fungi in the roots of the 10 days old Koshihikari Seedling

first experiment: using a blue stain method The criteria to verify the DSE fungi structures in the roots were also the same with the first experiment such as the

formation of DSE hyphae, hyphal coil, microsclerotia like structures, and septate in

the hyphae And check under the compound microscope through 40X and 100X

magnification

2.6 Statistical Analysis

39

CHAPTER RESULTS AND DISCUSSION

3.1 Colonization capacity of DSE fungi in the root of the Koshihikari rice plant in the presence of bacteria

3.1.1 Identification of Bacteria from three isolates

Isolation and identification of bacteria procedures were performed to know the origin of bacteria Table 3.1 shows the percent identity of the DNA sequence of the selected three isolates based on the NCBI BLAST database Max score refers to the highest

alignment score and the total score is the sum of alignment scores of all segments

from the same database sequence that match the query sequence Query cover means

a number that describes how much of the query sequence is covered by the target sequence where E value is how many times you would expect a match by chance in a database of that size, the lower E value means the match is more significant And the percent identity describes the percentage similarity of the query sequence with the targeted sequence

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

Isolate Description Max Scor e Total Scor e Quer y Cover E valu e Per Identit y Accession

Y34 Leclercia

adecarboxylata strain

L16 16S ribosomal RNA gene, partial sequence

1144 1481 89% 0.0 96.59 %

KT937143.1

Y34 Uncultured bacterium partial 16S rRNA gene, clone

SICC390_N11D2_16S _B

1144 1466 88% 0.0 96.59 %

40

Isolate Description Max Scor e Total Scor e Quer y Cover E valu e Per Identit y Accession

Y34 Unidentified marine bacterioplankton clone P5-4B_13 16S ribosomal RNA gene, partial sequence

1144 1437 86% 0.0 96.59 %

KC002238.1

OGR3 Uncultured bacterium clone SHCB0981 16S ribosomal RNA gene, partial sequence

1144 1479 89% 0.0 96.59 %

JN698091.1

OGR3 Uncultured bacterium clone BIGO569 16S ribosomal RNA gene, partial sequence

1144 1461 88% 0.0 96.59 %

HM558672

OGR3 Uncultured bacterium clone BICP525 16S ribosomal RNA gene, partial sequence

1144 1464 88% 0.0 96.59 %

HM557409

Control Uncultured bacterium clone BICP1507 16S ribosomal RNA gene, partial sequence

1144 1461 88% 0.0 96.59 %

HM557341

Control Uncultured bacterium clone BICP1483 16S ribosomal RNA gene, partial sequence

1144 1466 88% 0.0 96.59 %

41

Isolate Description Max Scor e Total Scor e Quer y Cover E valu e Per Identit y Accession

Control Uncultured bacterium clone BICP1350 16S ribosomal RNA gene, partial sequence

1144 1466 88% 0.0 96.59 %

HM557285

The above table is the result of Bacterial ribosomal (16S rRNA) gene, amplified by

using 27F and 1492R primer sets Uncultured bacteria was identified in the DNA

sequence because culture-independent microbial diversity analysis was used and it revealed the presence of unculturable bacteria in most environmental samples, Connon SA, Giovannoni SJ, 2002 and Rappé MS, Connon SA, Vergin KL, et al, 2002 According to table 3.1, the percent identity of the DNA sequence of the selected three isolates based on the NCBI BLAT database was about 96% similarity with the targeted sequence description And according to the description of each accession, the bacteria that appeared in the first experiment can be the seed-borne bacteria

3.1.2 Root colonization capacity of DSE Fungi in the roots of the Koshihikari rice plant in the presence of bacteria

Under the microscope, the colonization capacity of DSE fungi in the roots of the Koshihikari rice plant was demonstrated for all species According to the 3.1, 3.2, 3.3, and 3.4, we can see that the selected DSE fungi can colonize the roots of Koshihikari rice plant in the presence of bacteria

Veronaeopsis simplex (Y34) colonizing in the roots of 17 days old Koshihikari rice

42

Figure 3.1 Colonization of Veronaeopsis simplex (Y34) DSE in the roots of 17 days

old Koshihikari rice seedling (A) Intercellular blue-stained hyphae; (B) melanized

intracellular microsclerotia-like structure formed by (Y34)

Figure 3.2 Colonization of Cladophialophora chaetospira (OGR3) DSE in the

roots of 17 days old Koshihikari rice seedling.Showingformation of blue-stained

intracellular hyphae (A) and (B)

For J1PC1 DSE, the colonization demonstrated 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) (Fig 3.3) Similarly, the colonization of LtPE was well shown in blue-stained intracellular hyphae (Fig 3.4 A) demonstrating the occurrence of melanized intracellular microsclerotia- like structure (Fig 4B)

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

A B

43

intracellular microsclerotia (pointed with black arrow) and an early developmental stage of an intracellular microsclerotia (pointed with white arrow)

Figure 3.4 Colonization of Phialocephala fortinii (LtPE2) DSE in the roots of 17

days old Koshihikari rice seedling (A) Blue-stained intracellular hyphae; the (B) occurrence of melanized intracellular microsclerotia-like structure

The procedures of the first experiment had been started on 14th November and

harvested on 7th December 2019 Seed-borne bacteria regarded for the duration of 24

hr after the transplantation under the continuous temperature of 35ºC inside the growth chamber According to the result of the percent identity of the DNA sequence (Table.3.1), the percentage identity of the DNA sequence of the chosen three isolates was 96% similarity with the targeted sequence and through the description of each accession, the bacteria appeared in the first experiment could be seed-borne bacteria Previous research reported that seed endophytes can infect the next generation of the host plant through the ways or by the combination of individuals ways: (1) reside in the seed and transmitting the plant through the surfaces of other parts of the plant, and (2) remaining inside the seeds and transmit to the other parts of the plant via plant growth or move within the plant tissue, Kaga et al., 2009 Truyens et al 2014 reported that the internal seed endophytic bacteria which was inherited from past generation via seed and probably comprise of microbes and can tolerance desiccation and conditions of seed storage Therefore the bacteria that appeared in the first experiment can be the seed endophytic bacteria On the other hand, some characteristics of DSE fungi colonization were recognized in the roots of the Koshihikari rice plant in all treatments after 17 days of transplantation Such as the formation of DSE fungal hyphae and micro-sclerotium (Fig.3.1, 3.2, 3.3, and 3.4) were observed under the

44

compound microscope with the magnification of 40X and 100X accordingly Therefore the selected DSE endophytes can colonize the roots of the Koshihikari rice plant which was already in symbiosis with seed endophytic bacteria However, the effect of seed endophytic bacteria on the DSE fungi symbiosis with the Koshihikari rice plant is still unclear

A mass colony of single or multispecies of microbial cells adherent to the biotic or abiotic surface and in intimate contact with every other, encased in a self-produced matrix of intracellular polymeric substances are called biofilms (G Seneviratne et al., 2010) It was reported that most bacteria appeared to shape biofilms and this multicellular model of growth in all likelihood predominates in nature as a protection mechanism against adverse environmental situations (G Seneviratne et al., 2010) Previous research studied the rice plant with biofilms, with mixed culture without biofilms attachments and it has resulted that treatment with biofilms attachment produces higher indoleacetic acid-like substances (IAAS) that is critical for suppressing plant pathogen, than treatment with mixed cultures (W M M S Bandara et al., 2006) Therefore, the formation of biofilms by bacteria attaching the mycelium of fungal endophytes is efficient in promoting plant growth This mechanism can be applied to conduct future research to elucidate the effect of fungal bacteria communities in promoting plant growth

3.2 Capacity of DSE fungi colonization and plant performance response to symbiosis under high-temperature treatment

3.2.1 Effect of DSE fungi symbiosis in plant growth

45

Table 3.2 Result of the measurements of the physical parameters of the plant

growth for the 2nd experiment after 10 days of transplantation

Parameter Control OGR3 Y34 J1PC1

Shoot High (cm) 7.5 8.5 8.7 8.5

Seminal Root

Length(cm)

̴ 1.5 ̴ 2.5 ̴ 2.4 ̴ 2.2

Root Volume (cm3) 0.1 0.1 0.1 0.1

Total Dry Weight (mg) 7.18 8.5 7.825 6.95

Shoot Dry Weight (mg) 4.08 5.4 4.925 4.375

Root Dry Weight (mg) 3.1 3.1 2.9 2.575

Root/Shoot Ratio 3:4 3:5 3:5 1:2

Notes: The results of each parameter are the mean of the five replicates for each

treatment, p ≤ 0.05

According to the table 3.2, most of the four treatments, the treatment with OGR3 and Y34 DSE have the higher root/shoot ratio The root/shoot ratio is the “ratio of the amount of plant tissue which have supportive functions to the amount of these which have boom capabilities”, Oxford Reference This end result indicate that the symbiosis with Veronaeopsis simplex and Cladophialophora chaetospira have better symbiosis with the roots of the Koshihikari rice plant than Meliniomyces variabilis

3.2.2 Identification of DSE fungi in the root of 10 days old Koshihikari rice seedling

46

Figure 3.5 Colonization of Veronaeopsis simplex (Y34) DSE in the roots of 10 days old Koshihikari rice seedling (A) Strong colonization of blue-stained hyphae; (B) melanized hyphae (white arrow) and developing intracellular microsclerotium-like structures (black arrows); (C) Septate in the blue stained hyphae with white arrow The formation of the vesicle-like structure pointed with a black arrow and blue-stained hyphae with a white arrow are indies for the colonization of

Cladophialophora Chaetospira (OGR3) in the plant roots (Fig 3.6) Its intercellular

blue-stained hyphae formed structures resembling anastomoses While the colonization of Meliniomyces variabilis (J1PC1) DSE in the roots of 10 days old Koshihikari rice seedling indicated with the formation of hyphal coils pointed with a black arrow (Fig 3.7) and blue-stained hyphae forming structures resembling anastomoses

A

B A

C

A

47

Figure33.6 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

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

A B

48

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

49

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

50

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

51

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

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

52

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

53

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

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

Shifting Monsoon Rain

Climate Change Factor Non-Climate Change Factor

Unavailable Irrigation system

High expenditure Technical Problem Soil Quality

Low Productivity Migration

54

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

55

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 high-temperature 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 plant-growth 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

57

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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

ammonia, 1989) 1979) Max Scor Total Scor Query E valu Per Identit L16 16S ribosomal RNA KT937143.1 Uncultured bacterium partial 16S rRNA gene, LN561701.1 Unidentified marine KC002238.1 Uncultured bacterium clone SHCB0981 16S JN698091.1 Uncultured bacterium clone BIGO569 16S HM558672.1 Uncultured bacterium clone BICP525 16S HM557409.1 Uncultured bacterium clone BICP1507 16S HM557341.1 Uncultured bacterium clone BICP1483 16S HM557331.1 Uncultured bacterium clone BICP1350 16S HM557285.1 https://www.globenewswire.com/news-release/2019/01/18/1701949/0/en/China-Paddy-and-Rice-Import-Report-2019-2023.html https://pdfs.semanticscholar.org/12db/71ceb908ef7ee9d1b91d0f6f1012cb5affd4.pdf?_ga=2.228920088.546567381.1589540540-1080715982.1570249740. e: https://doi.org/10.1186/s40168-018 0497-1.