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MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ---------------------------- Hoang Kim Chi STUDY ON RHIZOSPHERE MICROBIAL COMMUNITIES OF MEDICINAL PLANT Curcuma longa L. TO ENHANCE TURMERIC YIELD AND QUALITY Major: Microbiology Code: 9 42 01 07 SUMMARY OF DOCTORAL THESIS Hanoi - 2020 The thesis was accomplished at: Graduate University of Science and Technology, Vietnam Academy of Science and Technology. First supervisor: Prof. Dr. Le Mai Huong Second supervisor: Dr. Tran Thi Nhu Hang First reviewer: Second reviewer: Third reviewer: The thesis defence will be held on …………….……………….. at Graduate University of Science and Technology, Vietnam Academy of Science and Technology. The thesis will be documented at: - Library of Graduate University of Science and Technology - National Library of Vietnam INTRODUCTION 1. Relevance of the research topic Curcuminoids are main bioactive ingredients of turmeric Curcuma longa L.. As a result of the recent growing demand for these compounds for pharmaceutical industrial application, the development of high quality turmeric production has become an urgent issue. To solve the problem, amendments in agricultural practices, post-harvesting processing techniques, and biotechnological methods have been highlighted. From another perspective, reducing chemical fertilizer while remaining crop yield is a trend of modern environmental friendly agronomy these years. Developing microbial inoculations from effective microorganisms for particular agricultural plants has thus been considered a practical focus. On the other hand, several rhizosphere soil microorganisms have been reported to play important roles in promoting biosynthesis of secondary metabolites, including curcuminoids. The entitled “Study on rhizosphere microbial communities of medicinal plant Curcuma longa L. to enhance turmeric yield and quality” was conducted to apply microbiology in sustainable development of agricultural productivity in Vietnam and worldwide. 2. Thesis objectives The purpose of this thesis was to exploit beneficial aspects of microbial rhizosphere communities, especially effective bacteria and fungi of turmeric plant C. longa. The results were expected to 1 support fundamental documents for proposing a suitable integrated nutrient management for turmeric plant in Vietnam. 3. Thesis contents Major contents of the research include: (i) Study on the relationship between varied nitrogen fertilizing managements and turmeric productivity; (ii) Isolation and biological assessments of rhizobacteria and fungi from turmeric plant; (iii) Study on genetic diversity of turmeric rhizosphere microbiomes in relation to high productivity nitrogen fertilizing managements; (iv) Preparation of biofertilizer from selected turmeric rhizosphere microbial candidates and case study in turmeric plant. CHAPTER 1. OVERVIEW 1.1. Turmeric Curcuma longa L. and curcuminoids component Turmeric (Curcuma longa L.) is a medicinal plant of family Zingiberaceae that distributes widely in South- and Southeast Asia, most abundantly in India and Thailand, and followed by Bangladesh, Indonesia, Myanmar and Vietnam [38] [39]. Chemical composition The rhizome of turmeric C. longa was determined to compose of 6,3-7% protein, 5,1-7,5% fat, 3,5-5% minerals, 69,4% carbohydrate and 9,5-13,1% water [43]. Major bioactive components of turmeric rhizome comprise curcuminoids [including curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC)], aromatic turmerone (ar-turmerone), α-turmerone and β-turmerone. Commercialized curcumin mix was known to composed of 77% pure (Cur), 17% DMC and 3% BDMC [44]. According to Naama et al. 2 (2013), curcumin and DMC are less stable than BDMC [45]. In terms of antioxidant and tumor inhibiting active intensities, curcumin was considered the most potent, followed by DMC and BDMC, respectively [46] [44]. Biological and pharmaceutical activities of curcuminoids from turmeric The biological and pharmaceutical activities of turmeric and curcuminoids in particular have been well studied. As estimation until 2011, more than 7000 published articles have shed light on various aspects of curcumin including its antioxidant, hypoglycemic, anti-inflammatory and anti-cancer activities. Also, this natural compound exerts its beneficial effects by modulating different signaling molecules including transcription factors, chemokines, cytokines, tumor suppressor genes, adhesion molecules, microRNAs, etc. [47]. 1.2. Turmeric rhizosphere associated microorganisms Rhizosphere is defined as the area around a plant root that is inhabited by a unique population of microorganisms influenced by the chemicals released from plant roots [70, 71, 72]. The special conditions shape rhizosphere a desirable niche for microbial communities and one of the most biodiverse and dynamic habitat on the earth. Rhizosphere microorganisms have received attention since the intimate plant-microbe relationship being mentioned and evidenced. About 2–5% of rhizosphere microorganisms have been known to positively affect plant growth, and plants in turn are able to control these beneficial microorganisms [73, 74]. Accordingly, the rhizosphere microbiome plays an important role in improving soil fertility, plant metabolisms and ultimately enhancing plant 3 productivity. Plant growth promoting rhizobacteria (PGPR) for instance, could directly or indirectly control the plant nutrient pools by releasing phytohormones (e.g. auxins or cytokinins), improving plant nutrient availability (e.g. N, P and Fe), and increase plant resistance via synthesis of antibiotics or secondary metabolites [75, 76]. Turmeric rhizosphere harbors abundantly diverse microorganisms. Several PGPR genera such as Pseudomonas, Bacillus, Klebsiella, Agrobacterium, Azotobacter and Burkholderia have been found to be dominant species [103, 104]. The association of AM fungi with different cultivars of turmeric C. longa was assessed and characterized to belong to genera of Glomus, Gigaspora and Sclerocystis, wherein Glomus dominated the population [109, 110]. Several scientists have inoculated PGPR and AMF inoculums with C. longa’s rhizomes and demonstrated their beneficial effects on the growth and productivity [116, 119]. Due to the fact that certain secondary metabolite pathways in plant are induced by microorganisms, it is therefore necessary to focus on rhizosphere microorganisms and soil health in order to improve turmeric productivity. The study will contribute to exploit the repository of biotechnologically potential microorganisms and eventually to a sustainable production of the novel bioactive metabolites. CHAPTER 2. MATERIALS AND METHODS 2.1. Materials 2.1.1. Turmeric cultivar and crop This study was conducted at a turmeric growing area located in Dai-Tap commune, Khoai-Chau district of Hung-Yen province 4 (20°47′35″ N, 105°56′42″ E) using local seed rhizome of Curcuma longa. 2.1.2. Chemicals, oligonucleotides, media and microorganisms 2.2. Methods 2.2.1. Soil sampling and determination of soil physiochemical parameters Sampling: Soil samples were taken randomly from turmeric rhizome soil (10-15 cm depth from the top soil). Samples of each plot were then bulked, homogenized and grouped together to one sample set, followed by storing at 4oC prior to DNA extraction. Determination of soil physiochemical parameters 2.2.2. Study on impacts of chemical N fertilizing rates to turmeric productivity Experimental design Field study was conducted from April to December 2016 and replicated in 2017. The experiment was laid out in a randomized complete block design and three replicates. Experimental units consist of 10 m2 plots each with one fertilizer regime, resulting in a total of 16 plots in a total area of 160 m2. The treatments were four N fertilizer rates (0, 150, 350 and 500 kgN.ha-1.y-1) incorporated with K and P fertilizers (400:200 kg.ha-1.y-1), resulting in 5 fertilizer regimes: N0, N150, N350 and N500, respectively. Soil samples were taken randomly from turmeric rhizome soil (10-15 cm depth from the top soil) at five points of each plot following a W-pattern (Thomas 1985) [115]. Plant growth and productivity parameters: Plant height (cm); Number of leaves/plant; Fresh rhizome yield (kg/ha); Fresh rhizome yield/dry rhizome yield ratio (%); Curcuminoids content. 5 After harvesting, turmeric rhizomes were sliced and dried. Curcuminoids from turmeric samples were extracted by an ultrasound assisted extraction method using ethanol/water (70:30, v/v) solvent as described by Mandal et al. (2009) [116]. The quantification of curcuminoids content was performed using high performance liquid chromatography (HPLC) following the method of Jayaprakasha et al. (2006) [46]. 2.2.3. Study on impacts of N fertilizing rates to diversity of turmeric microbial community Total DNA extraction from turmeric rhizosphere soil samples Total DNAs were extracted by using PowerSoil® DNA Isolation kit (Mo Bio Laboratories, Qiagen, USA) and quantified by Nano drop (Nanodrop 2000c, Thermo Fisher Scientific, USA) in combination with electrophoresis in gel agarose 1% and stored at -20oC. Metagenome amplicons sequencing Sequencing libraries were prepared from the PCR products using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA). The quality of libraries was assessed on Bioanalyzer 2100 system (Agilent, USA) before sequencing on Illumina HiSeq 2500 platform (Illumina, USA). Bioinformatic analysis The metagenome databases were analyzed following Qiime2 analyzing pipeline (https://qiime2.org/) [118]. The analysis process comprises 3 main steps, namely (i) Preprocessing; (ii) Taxonomy; and (iii) Diversity analysis and visualization.  Preprocessing: Using quality control tools of Qiime (V1.7.0, http://qiime.org/scripts/split_libraries_fastq.html) [146] and 6 UCHIME algorithm (http://www.drive5.com/usearch/ manual/uchime_algo.html) [148].  Taxonomy: Using Uparse software (Uparse v7.0.1001, http://drive5.com/uparse/) [149], Unite database (https://unite.ut.ee/) [150] and Silva database (https://www.arb-silva.de/) [151].  Diversity analysis and visualization  Microbial diversity indices Determination of Shannon-Weaver (H), Simpson (D1), Chao1 and ACE indices was performed by Qiime 2.  PCA & PCoA Principle Component Analysis (PCA) and Principle Coordinate Analysis (PCoA) were conducted by applying R software (v 3.1.2, R Core Team 2014).  Rarefaction curve  Statistical Analysis: Using ANOVA followed by Tukey’s Honest Significant Difference (HSD) post hoc tests. 2.2.4. Isolation of PGPR and plant growth promoting assays Isolation of PGPR strains with phosphate solubilizing ability: Rhizosphere soil samples were diluted and spread on IPA agar plates for bacterial colonies formation [138]. IAA producing assay: IAA assay was conducted following Salkowski’s method [139]. Antagonism to test pathogenic microorganisms: Agar diffusion test as described by Ahmad & cs. (1998) [140]. Determination of biochemical and physiological characteristics: According to Bergey’s Manual of Systematic Bacteriology [141, 142]. 7 Phylogenetic identification using partial 16S rDNA gene sequences: The partial 16S rDNA gene sequences of bacterial isolates were amplified using PCR with primers Pr16F-Pr16R and compared to published sequence in GenBank using BLASTn tool, and analyzed by BioEdit 7.0 [144] and MEGA X [145] [146] softwares. 2.2.5. Isolation and characterization of AMF Isolation of AMF spores from turmeric rhizophere soil samples: AMF spores from rhizosphere soil samples were isolated using wet sieving and decanting method (Gerdemann, Nicolson, 1963) [147]. Partial 18S rRNA gene amplification and phylogenetic inference Fragments of partial small subunit (SSU) rRNA gene from extracted genomic DNA samples were amplified using universal eukaryotic forward primer NS31 and reverse primers mixture AM containing AM1, AM2 and AM3 [148, 149, 150] to amplify AM fungal SSU sequences. Clones from each sample were tested for the PCR amplicons and sequenced on an ABI PRISM® 3100 Avant Genetic Analyzer (Applied Biosystems, USA) sequencer. 2.2.6. Preparation of biopfertilizer for turmeric and case study on turmeric productivity Preparation of biofertilizer from isolated turmeric rhizosphere effective microbial strains  Safety test: Safety tests for microbial strains were conducted in BALB/c mice as described by Carter et al. [151].  AMF AMF spores were preserved and inoculated in pot cultures of Plantago lanceolata (supplied by Institute of Seed and Biotechnology - Vietnam Academy of Forest Science). 8