(Luận văn tốt nghiệp) the effect of soil samples on growth, phenotype, and the root microbiome of tubtim chum phae rice (oryza sativa)

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN THI MY DUYEN TOPIC TITLE: THE EFFECT OF SOIL SAMPLES ON GROWTH, PHENOTYPE, AND THE ROOT MICROBIOME OF TUBTIM CHUM PHAE RICE (ORYZA SATIVA) BACHELOR THESIS Study Mode : Full – time Major : Biotechnology Faculty : Biotechnology and Food Technology Batch : 2014 – 2018 Thai Nguyen, 05/06/2018 Luan van Luan van THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN THI MY DUYEN TOPIC TITLE: THE EFFECT OF SOIL SAMPLES ON GROWTH, PHENOTYPE, AND THE ROOT MICROBIOME OF TUBTIM CHUM PHAE RICE (ORYZA SATIVA) BACHELOR THESIS Study Mode : Full - time Major : Biotechnology Faculty : Biotechnology and Food Technology Batch : 2014 – 2018 Supervisors : Dr Songsak Wattanachaisaereekul Dr Nguyen Xuan Vu MSc Phimrak Khaokhajorn Thai Nguyen, 05/06/2018 i Luan van DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Major Biotechnology Student name Nguyen Thi My Duyen Student ID DTN1451150003 The effect of soil samples on growth, phenotype and the root microbiome of Tubtim Chum Phae rice Thesis Title (Oryza Sativa) Dr Songsak Wattanachaisaereekul Supervisors Dr Nguyen Xuan Vu MSc Phimrak Khaokhajorn Abstract: Tubtim Chum Phae rice is rice from hybridization between Hom Mali rice and Sung Yod rice It can be grown all year round and in all regions of Thailand It is known that rice contain a number of nutrients and bioactive compounds, including dietary fiber and phytochemicals with potent antioxidant activities Hypertension appears to have a complex association with endothelial dysfunction and oxidative stress When people regularly eat Tubtim Chum Phae rice, it will reduce myocardial infarction risk, as for dyslipidemic subjects, it has been reported that can improve low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-cholesterol concentrations and enhance cellular cholesterol efflux to serum and anti-oxidant effects, anti-inflammatory effects, cancer risk The purpose of this study is to study and point out the difference of soil sample groups to growth, phenotype and microbiome system of root in the revolution of Tubtim Chum Phae rice Keywords: Tubtim Chum Phae, growth and phenotype of rice plant, microbiome system Number of pages: Date of Submission: ii Luan van ACKNOWLEDGMENTS First and foremost, I would like to express my sincere and deepest gratitude to my supervisor, Dr Songsak Wattanachaisaereekul from the School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (KMUTT), Thailand, who providing me the opportunity to conduct research in his lab and giving me endless support during the internship since December 15th, 2017 to May 30th, 2018 and then special thank and highly thankful goes to my advisors Msc Phimrak Khaokhajorn from the School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (KMUTT), Thailand, who cared so much about my work, and who responded to my questions and queries so promptly, for their patience, motivation, enthusiasm, and immense knowledge I have been extremely lucky to have a supervisor and one advisor like them I would also like to thank P’Phan, one staff from Fungal Biotechnology, the School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (KMUTT), Thailand and Dr Nguyen Xuan Vu from the Biotechnology and Food Department of Thai Nguyen University of Agriculture and Forestry (TUAF), Thai Nguyen City, Vietnam, who used to help, support and give me encouragements during this thesis implementation Last but not the least; I would like to thank my family: my Mother, for giving birth to me at the first place and supporting me spiritually throughout my life Many thank you and best regards Student Nguyen Thi My Duyen iii Luan van CONTENT DOCUMENTATION PAGE WITH ABSTRACT ACKNOWLEDGMENTS iii CONTENT iv LIST OF ABBREVIATIONS vi LIST OF TABLE vii LIST OF FIGURE viii PART INTRODUCTION 1.1 Tubtim Chum Phae rice 1.1.1 Important of Tubtim Chum Phae rice 1.1.2 Life cycle of Tubtim Chum Phae rice 1.2 Microbiota 1.2.1 Plant microbiota 1.2.2 Rice microbiota 1.2.3 Microbiome engineering 1.2.4 Microbiota application for plant growth promote 1.3 Types of soil sample 1.3.1 Chemical soil 1.3.2 Intensive Chemical soil 1.3.3 Transition soil 10 1.4 Objectives 10 PART METHODOLOGY 11 2.1 Equipment and materials 11 2.1.1 Equipment 11 2.1.2 Materials 11 2.2 Methods 13 2.2.1 Soil sample preparation 13 2.2.2 Rice germination 14 2.2.3 Rice growing 14 iv Luan van 2.2.4 Rice phenotype observation 15 2.2.5 Measure pH of soil sample 15 2.3 Inspection of quantity microbiome in Chemical, Intensive Chemical, Transition soil by DNA extraction 16 2.4 Measure Nanodrop 17 PART RESULTS AND DISCUSSIONS 19 3.1 The effect of the soil on the revolution of a rice plant with each Chemical, Intensive Chemical and Transition soil group 19 3.1.1 Comparison the different of rice plant about the color of rice leaves and the tiller number of rice plant in Chemical soil group 20 3.1.2 Comparison the different of rice plant about the color of rice leaves and the tiller number of rice plant in Intensive Chemical soil group 22 3.1.3 Comparison the different of rice plant about the color of rice leaves and tiller number of rice plant in Transition soil group 23 3.1.4 Comparison the different of rice plant between Chemical soil, Intensive Chemical soil, Transition soil groups 26 3.1.5 Observation of the change of pH in three types of soil sample 28 3.2 The result of Nanodrop 29 3.3 The result of DNA extraction for check microbiome from soil samples 30 PART CONCLUSIONS AND SUGESTIONS 32 REFERENCES 33 v Luan van LIST OF ABBREVIATIONS G Gram Mg Milligram Kg Kilogram µl Microliter Min Minute Hrs Hour mL Milliliter C Chemical soil I Intensive Chemical soil T Transition soil Rpm Revolution per DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid TAE Tris- acetate-EDTA Ng Nano gram Kb Kilo base °C Degree Celsius DI Deionized NaCl Sodium Chloride NaOH Sodium hydroxide SDS Sodium dodecyl sulfate PEG Polyethylene glycol vi Luan van LIST OF TABLE Table 2.1 Equipment used for studies 11 Table 2.2 DNA extraction buffer 12 Table 2.3 Data of soil sample collected from somewhere in Thailand 13 Table 2.4 Weight amount of the soil sample in growth rice plant 14 Table 3.1 Variation in the phenotype of rice plant in Chemical soil group after every week from week until week 20 Table 3.2 Variation in the phenotype of rice plant in Intensive Chemical soil after every week from week until week 22 Table 3.3 Variation in the phenotype of rice plant in Intensive Chemical soil after every week from week until week 24 Table 3.4 Variation in the phenotype of rice plant between three soil groups after every week from week until week 26 Table 3.5 Calculation the average of pH all of soil sample to observe the different of soil to rice plant 28 Table 3.6 The result of Nanodrop after DNA extraction 29 vii Luan van LIST OF FIGURE Figure 1.1 Growth stages of the rice plant Part of the image collection of the International Rice Research Institute (IRRI) Figure 1.2 Plant-growth promoting microbes are capable of conferring benefits to multiple species of plant hosts, and of offering improved tolerance to multiple stresses simultaneously (Image from Coleman-Derr at al Front Microbiol., 06 June 2014) Figure 3.1 Comparison the average of the tiller number of rice plant in Chemical soil group from week until week 21 Figure 3.2 Comparison the average of the tiller number of rice plant in Intensive Chemical soil group from week until week 23 Figure 3.3 Compared average of the tiller number of rice plant in Transition soil group from week until week 25 Figure 3.4 Comparison the average of the tiller number of rice plant in Chemical soil, Intensive Chemical soil, Transition soil group from week until week 27 Figure 3.5 The average of pH of soil sample during growth rice progress and compare the differences between each sample 28 Figure 3.6 The result of electrophoresis of DNA from soil samples of the best rice plant Land M: Kb DNA ladder, land 2: C2/1, land 2: C2/2, land 3: C2/3, land 4: I2/1, land 5: I2/2, land 6: I2/3, land 7: O3/1, land 8: O3/2, land 9: O3/3 30 viii Luan van 3.1.3 Comparison the different of rice plant about the color of rice leaves and tiller number of rice plant in Transition soil group Table 3.3 Variation in the phenotype of rice plant in Transition soil after every week from week until week Time The best rice plant The worst rice plant 35 days T1/2 T2/1 T3/3 T1/3 T2/3 T3/2 42 days T1/2 T2/1 T3/3 T1/3 T2/2 T3/1 49 days T1/2 T2/1 T3/3 T1/3 T2/2 T3/1 56 days T1/3 T2/3 T3/3 T1/2 T2/2 T3/1 24 Luan van Comparison the tiller number of rice plant in Transition group Transition Transition Transition AVERAGE OF TILLER NUMBER 60.00 50.00 40.00 30.00 20.00 10.00 00 Week Week Week Week Week Week TIME Figure 3.3 Compared average of the tiller number of rice plant in Transition soil group from week until week Figure 3.3 Shows the tiller number of rice plant in Transition soil Calculation error bar and saw that error bar on the first weeks but change not much but on week Transition change quite much and Transition and Transition change also on week until week Especially Transition but this is the worst plant with the others With Transition the first time growth rice the tiller number of rice plant always stable the revolution after every week but in week until week it did not change Because in week until week it was fast developed and rice plant quite big in the pot so in week it did not enough space well and fertilizer for rice so tiller did not develop continue With Transition was the worst rice plant when collected soil they just used photosynthetic bacteria with time Therefore, soil really very a little bit nutrient inside soil so when collect for grows rice also did not add any fertilizer, only added water so did not nutrient for rice develop About Transition rice always develop well after every week but on week and low develop but still develop more because this soil still got a lot of fertilizer inside soil with the others Before collected soil, the farmer used organic fertilizer quite regular with chicken dung and fermented banana shoot and photosynthetic bacteria every month 25 Luan van 3.1.4 Comparison the different of rice plant between Chemical soil, Intensive Chemical soil, Transition soil groups Table 3.4 Variation in the phenotype of rice plant between three soil groups after every week from week until week Time The best rice plant The worst rice plant 35 days C2/3 I3/2 T3/3 C3/2 I3/3 T2/3 42 days C2/3 I2/1 T3/3 C3/2 I3/3 T2/2 49 days C1/3 I2/1 T3/3 C3/1 I3/3 T2/2 56 days C3/3 I1/1 T3/3 C1/1 I3/3 T2/2 26 Luan van Comparison the tiller number of rice plant in three soil groups AVERAGE OF TILLER NUMBER Chemical Intensive Transition 60.00 50.00 40.00 30.00 20.00 10.00 00 21 days 28 days 35 days 42 days 49 days 56 days TIME Figure 3.4 Comparison the average of the tiller number of rice plant in Chemical soil, Intensive Chemical soil, Transition soil group from week until week Figure 3.4 Shows the tiller number of rice plant between Chemical group, Intensive Chemical group, Transition group The first time growth rice in week until week the tiller of rice plant did change non-significant because this time the rainy season so rice plant always stays in situation flood with rice got a lot of water they can be "lazy" and need not grow very much So the tiller number of rice plant of three soil group same together and they only change a lot in week Observation the develop of tiller number can saw that Chemical and Intensive Chemical soil group best but when growing rice plant saw Intensive Chemical got pathogen but in about the tiller number is equal to Chemical but when put two group and take a photo to compare can observe that Chemical better than because the tiller number of rice plant of Intensive Chemical group much but they were so small, a lot of yellow leaves, body of rice plant so thin About Transition, this is the worst plant with the others because during growth rice plant progress i did not add fertilizer but inside Chemical, Intensive Chemical they got Chemicals for example Chemical fertilizer N-P-K, Liquid bio-fertilizer…, already so they had nutrient for rice develop but with Transition group inside soil did not fertilizer because Transition soil group is mean soil did not use synthetic fertilizers, pesticides, and growth regulators Instead, they 27 Luan van rely on crop rotations, crop residues, animal manures, legumes, green manures, offfarm wastes and biological pest control to maintain soil health, supply plant nutrients, and minimize insects, weeds, and other pests Use this fertilizer need to add regular three months once tim and used too little soil sample and so much soil standard so they did not enough nutrient for rice 3.1.5 Observation of the change of pH in three types of soil sample Table 3.5 Calculation the average of pH all of soil sample to observe the different of soil to rice plant Average pH of soil sample pH of soil Before rice plant Week 5.04 6.25 4.76 6.10 4.77 6.12 4.85 5.69 4.89 6.13 4.46 5.90 6.15 6.12 5.90 6.18 5.59 6.19 Soil sample C1 C2 C3 I1 I2 I3 T1 T2 T3 Week 5.29 5.58 5.49 5.50 5.34 5.22 5.59 6.00 6.19 Measure pH soil from rice field soil from pot after weeks soil from pot after weeks 8.00 7.00 6.00 PH 5.00 4.00 3.00 2.00 1.00 0.00 C1 C2 C3 I1 I2 I3 T1 T2 T3 NAME OF SOIL Figure 3.5 The average of pH of soil sample during growth rice progress and compare the differences between each sample 28 Luan van Figure 3.5 Shows a pH of soil from grow rice plant after collect from rice field and after grow rice some week with Chemical soil sample group, Intensive Chemical soil sample group, Transition soil sample group Soil pH provides various clues about soil properties and is easily determined About fluctuations, average pH of soil from rice field for calculated error bar can observation pH of rice plant change nonsignificant but with Intensive Chemical of soil from the pot on week error bar got the fluctuations quite large and this is rice plant observe have the pathogen in soil with a lot of yellow leaves Compared pH of rice plant after measure three-time can observe on week pH of rice plant best with a pH range of approximately promotes the readiest availability of plant nutrients and known that this week the good environment condition for growing rice and this week is the rainy season so rice plant always in situation flood so it can affect to pH of soil In week measured again and saw pH of soil decrease with pH 4-5 it means extremely and strongly acid soils can have high concentrations of soluble aluminum, iron, and manganese which may be toxic to the growth of some plants The pH scale is logarithmic, which means that a pH reading of is ten times more acidic than a reading of so if pH only changes a little bit it also factors affect to the rice plant 3.2 The result of Nanodrop After growing rice plant in twenty-seven different types of soil samples observe the revolution of rice plant after every week and compared the difference of the rice plant and choose the best rice plant in one group Each big group choose the best rice plant in one small group and collected soil for DNA extraction to determined concentration DNA inside the soil Table 3.6 The result of Nanodrop after DNA extraction C2/1 C2/2 C2/3 I2/1 I2/2 I2/3 T3/1 T3/2 T3/3 ng/µl 32.3 154.0 16.6 200.2 327.7 10.4 72.9 344.1 176.5 260/280 1.45 1.32 1.48 1.47 1.43 1.46 1.36 1.46 1.40 260/230 1.12 1.05 1.10 1.33 1.29 0.89 1.13 1.35 1.17 29 Luan van Table 3.2 Shows the result of Nanodrop about the concentration of DNA appeared inside the soil Observation of all of the soil samples got the concentration of DNA quite high but with C2/1, T3/1 samples concentration of DNA quite low and C2/3, I2/3 very low when measure nanodrop observe into chart saw that concentration of DNA almost did not have DNA Pure of DNA all of the samples in A260/280 and A260/230 ratios contamination with the others chemical 3.3 The result of DNA extraction for check microbiome from soil samples After checked concentration of DNA by Nanodrop technique continues with electrophoresis Product for extract DNA was been checked by gel electrophoresis is gel 0.8 % agarose in 50 ml 1X TAE buffer The result of electrophoresis showed in this picture 3.9 Figure 3.6 The result of electrophoresis of DNA from soil samples of the best rice plant Land M: Kb DNA ladder, land 2: C2/1, land 2: C2/2, land 3: C2/3, land 4: I2/1, land 5: I2/2, land 6: I2/3, land 7: O3/1, land 8: O3/2, land 9: O3/3 30 Luan van Figure 3.6 Shows the result of the concentration of DNA inside soil samples There was appeared light DNA on each run line of land 4, land 5, land 8, land so DNA extraction succeed and observed smear on land 2, land but with land 1, land and land did not observe anything because land 1, land is Chemical soil group inside this soil got a lot of chemicals so it difficult to extraction DNA and on land this is soil when growing rice and observation rice plant in this soil always the worst rice plant so in this soil poor about microbiome Did not enough time to proceed to carry out further molecular biology analysis 31 Luan van PART CONCLUSIONS AND SUGESTIONS CONCLUSIONS - No different of tiller number of rice but rice show different character about height and color of rice leaves in each group of soil - Observation some disease in Intensive Chemical - Rice plant of Chemical soil group is the best rice plant in three groups - Water, fertilizer and space very important of rice plant SUGGESTIONS Rice was observed only weeks and it should be observing to flower and ripening phase Repeat experiment with better environment condition for growing rice plant Should be develop the protocol for DNA extraction with Chemical soil group After success should be purify and 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Shows the result of Nanodrop about the concentration of DNA appeared inside the soil Observation of all of the soil samples got the concentration of DNA quite high but with C2/1, T3/1 samples concentration