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
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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.
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(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
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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].
harbors Turmeric abundantly rhizosphere
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
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(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.
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After harvesting, turmeric rhizomes were sliced and dried. turmeric samples were extracted by an Curcuminoids from 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
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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
Statistical Analysis:
(PCA) and Principle Coordinate Analysis (PCoA) were conducted by applying R software (v 3.1.2, R Core Team 2014). Rarefaction curve 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].
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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 Institute of Seed and (supplied by lanceolata
Plantago Biotechnology - Vietnam Academy of Forest Science).
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PGPR fermentation and biomass harvesting
AMF spores harvesting
Preparation of PGPR and AMF inocula: Biomass mixture
of PGPR strains and spore mixture of AMFs were blended in 1:1 ratio (w/w).
Case study on the effect of biofertilizer in turmeric plant
Experimental design
(i) Microbial
Experiment was conducted at the Ministry of Health’s Botanical garden in Thanhtri, Hanoi from May to November 2018. The 12 m2 units were designed in a total area of 30 m2 belonging to 2 turmeric inocula applied experiment groups: (symbolized as CP), and (ii) Control (symbolized as DC).
Determination of growth and productivity parameters
The growth parameters are monitored periodically. Productivity was determined based on fresh biomass at the end of the experiment.
III. RESULTS AND DISCUSSION
3.1. Investigation of relationship between nitrogen fertilizer regimes and turmeric productivity
Environmental parameters of turmeric farming site
Initial edaphic conditions prior to experimental plotting were determined by standard methods according to TCVN 7373:2004, 7374:2004 and 7375:2004. Results showed that the parameters corresponded sandy loam characteristics with relatively good soil quality.
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Turmeric productivity and curcuminoids content in response to N fertilizer rates
Turmeric productivity of different fertilizing regimes was determined in terms of fresh rhizome yield and curcuminoids content. The averaged fresh rhizome yield after harvesting was recorded in form of mean±SD as following: 21038±5013 kg.ha-1.y-1 (N0), 27003±4703 kg.ha-1.y-1 (N150), 30902±1642 kg.ha-1.y-1 (N350); 20578±2306 kg.ha-1.y-1 (N500).
From these results, it is obvious that obtained turmeric yield in regimes N0 and N500 was lower than in N150 and N350. Most likely, either excessive or inadequate nitrogen inputs had negative effect on turmeric plant’s vigor and nutrient accumulation. Farming regimes with nitrogen fertilization levels ranging from 150 to 350 kg.ha-1 were considered optimal for fresh rhizome yield of turmeric.
Figure 3. 2. Averaged fresh turmeric rhizome yield and curcuminoids content in experimental regimes.
Curcuminoids content in response to N fertilizer rates
The averaged curcuminoids content of each farming regime was calculated from available data after determination of dry rhizome weight and analysis of rhizome extracts. Results revealed
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the highest curcuminoids yield in the experimental plots of N150 farming regime. Figure 3.2 summarizes averaged turmeric yield and curcuminoids content in relation to N fertilizer inputs. As illustrated in the figure, with N fertilizers ranging from 150 to 350 kg.ha-1.y-1, the turmeric yield and curcuminoid content were both recorded at higher levels than in either non-fertilized N0 or over- fertilized N500.
Effects of N, P and K chemical fertilizers on turmeric yield and curcumin content have been intensively investigated in various geoecological areas of the global, but research results were inconsistent. The present results have confirmed the hypothesis of N fertilizer’s effect on yield and curcumin content in C. longa. On the other hand, the research has supported fundamental data for designation of appropriated fertilizing practices for turmeric plant in Vietnam.
3.2. Investigation of effective microbial groups in turmeric rhizosphere
The investigation of effective microbial groups in turmeric rhizosphere was oriented by published research results and reviews concerning symbiotic microorganisms of C. longa. Accordingly, we focused on two main rhizosphere microbial groups, namely growth promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF), thereby aiming to screening for a collection of microbial candidates for preparation of effective biofertilizer.
3.2.1. Investigation of plant growth promoting rhizobacteria (PGPR) in turmeric rhizosphere
3.2.1.1. Isolation and screening of PGPR
The isolation of PGPR strains from turmeric rhizosphere was performed with following criteria: (i) Dissolve inorganic phosphate;
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(ii) Production of indole aceteic acid (IAA); (iii) Nitrogen fixation ability and (iv) Antagonism to test pathogenic microorganisms. Screening results for PGPR strains from turmeric rhizosphere soil samples are shown in Table 3.6.
Table 3. 6. Phosphate solubilizing ability, development in nitrogen- free medium and IAA production of PGPR strains.
Strain
N o.
1 2 3 4 5 6 7 8 9
PGP-V2 PGP-V4 PGP-V5 PGP-V15 PGP-V18 PGP-V20 PGP-V21 PGP-V22 PGP-V24
Phosphate solubilizing ability (D-d, mm) 3 5 3 5 3 4 6 4 4
Development in nitrogen-free medium + - + + + + + + -
IAA production (ppm) 24.80±2.21 9.66±1.72 67.51±2.11 35.47±3.05 26.82±1.46 77.87±2.78 63.11±2.09 11.61±1.85 45.81±2.89
* D: diameter of halos around bacterial colony; d: diameter of
bacterial colony on agar plate.
The indirect plant growth promoting effect of PGPR strains was evaluated basing on in vitro antagonism to plant pathogenic fungi Aspegillus niger and Fusarium oxysporum on agar plates. The result revealed inhibitory activity against both tested fungi A. niger and F. oxysporum of Bacillus sp. PGP-V21 with halo diameters of 9 and 3 mm, respectively.
characteristics, and
PGP-V5, PGP-V20 and PGP-V21 emerged as promising effective bacteria with typical PGP characteristics in terms of relatively potent P solubility, nitrogen fixation and IAA production. The bacterial strains were taxonomical characterized by determining in physio-biochemical morphological combination to partial 16S rRNA gene sequences analysis.
3.2.1.2. Morphological and taxonomical characteristics
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Figure 3.3. Phylogenetic tree of PGP-V5, PGP-V20, PGP-V21 and
published bacterial species basing on parial 16S rRNA gene sequences (Maximum likelihood method, 100 bootstrap replicates, consensus tree). Methylobacterium populi AP014809 was the out group.
Morphological characteristics of bacterial colonies on agar plates containing LB medium and under microscope (magnification x1000) were observed. The morphological, physiological and biochemical features of the strains were compared with Bergey's classification key [141] [142]. Based on published sequences in GenBank and subsequent sequence analysis, a classification tree of strains PGP-V5, PGP-V20 and PGP-V21 has been constructed (Figure 3.3). As a result, the PGP bacterial strains were determined as Bacillus sp. PGP-V5, Enterobacter sp. PGP-V20 and Bacillus sp. PGP-V21.
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In conclusion, four PGPR strains were
isolated and biologically characterized from rhizosphere of turmeric plant in the course of the investigation.
3.2.2. Investigation of arbuscular mycorrhizal fungi (AMF) in turmeric rhizosphere
3.2.2.1. Isolation of AMF
Besides PGPR, AMF has been intensively mentioned as effective symbionts of turmeric rhizosphere in earlier reports, especially those in Indian turmeric varieties [160, 161], however, there has not been any similar research hitherto on the indigenous turmeric sample of Vietnam.
By wet decanting and filtrating [147], the presence of AMF spores in rhizosphere soil samples of turmeric in the study area at three different investigated periods (2, 5 and 8 months after planting) was determined. The results showed that investigated AMF spore numbers increased from 23.6±5.5 spores/100 g soil two months after planting to 66.5±7.5 spores/100 g at 8 months old turmeric.
3.2.2.2. Morphological and taxonomical characterization of AMF strains isolated from turmeric rhizosphere
Depending on AMF’s spores’ microscopic characteristics, including spore sizes, shapes and color, three most popular morphological groups were selected to preserve and culture, namely AM-N1, AM-N2 and AM-N3. The AMF groups were identified to belong to three distinct fungal groups of genus Glomus.
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Figure 3. 5. Phylogenetic tree of AM-N1, AM-N2, AM-N3 and
published fungal species basing on parial 18S rRNA gene sequences (Maximum likelihood method, 100 bootstrap replicates). Gigaspora margarita BEG152 was the out group.
By analyzing partial 18S gene sequences after DNA isolation and Nested-PCR amplification, the AMF strains were taxonomically identified. The results unraveled relationship between selected AMF strains and published species on GenBank, most of which were found to belong to the genus Glomus. The phylogenetic tree of strains AM-N1, AM-N2 and AM-N3 was constructed and depicted in Figure 3.5.
Taken these above results together, the isolated AMF strains from turmeric rhizosphere samples were identified as Glomus sp. AM-N1, Glomus intraradices AM-N2 and Glomus mosseae AM-N3.
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3.3. Impact of N fertilizer rates to turmeric rhizosphere microbial communities
3.3.1. Abundance of turmeric rhizosphere bacterial communities under varied N fertilizer rates
indices of turmeric rhizosphere bacterial
3.3.1.1. Diversity communities
Achieved bacterial OTUs from turmeric rhizosphere samples of four fertilization regimes (N0, N150, N350 and N500) were analyzed using bioinformatic softwares and Unite database. The average number of fungal species in samples of the N0, N150, N350 and N500 regimes were 3105, 2286, 2760 and 2843, respectively (Table 3.16).
Table 3. 1. 16S amplicons metagenome sequencing data and analyzed diversity indices.
N0 (n=3) N150 (n=3) N350 (n=3) N500 (n=3)
3105
2286
2760
2843
Sample name Averaged species number
Simpson index
0.703 ± 0.11
0.712 ± 0.10 0.733 ± 0.12
0.665 ± 0.08
ACE index Chao1 index
2012 ± 354 1893 ± 196 2068 ± 244 1992 ± 259 1765 ± 103 1994 ± 262 1549 ± 306 1266 ± 220 Shannon-Weaver 3.77 ± 0.50 2.95 ± 0.25 4.04 ± 0.71 3.73 ± 0.56
Coordinate analysis (PCoA) based on biodiversity indices (Figure 3.10) allowed a separation of group L (blue) from group H (red). In other words, there was a difference between the bacterial communities from the high yielding group H (regimes N150 and N350) and the low yields L (regimes N0, N500).
3.3.1.2. Taxonomic composition of turmeric rhizosphere bacterial communities
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The analysis results showed relatively uniform compositions of turmeric rhizosphere bacterial communities at phylum and class levels of groups L and H. Accordingly, predominant proportions of Alpha-proteobacteria belonging to Proteobacteria were observed in both groups, accounting for over 40% of the total identified OTUs (p <0.05). Similarly, class Gemm-1 of Gemmatimonadetes appeared most analyzed samples with over 5% of total OTUs (p<0.05). The analysed 16S amplicons metagenome data suggested on one hand an indigenous soil bacterial communities structure in the research area, on the other hand a specific composition of rhizosphere bacterial communities of turmeric plant.
Noteworthy, the Gram negative bacteria of Proteobacteria, whose representers such as free-living nitrogen-fixing and poor nutritional conditions adapters [171], were abundantly found in rhizosphere samples of regime N0. Most likely, the N nutritional poverty condition in regime N0 inhibited other microbial groups, promoting competitive advantage and increased distribution of Proteobacteria, especially those belong to Alphaproteobacteria.
Firmicutes Crenarchaeota
the dynamic of microbial taxonomical groups
In general, after analyzing NGS sequencing data of ITS and 16S metagenome amplicons of C. longa rhizosphere samples, a total of 4776 OTUs of bacteria and 1760 OTUs of fungi were obtained. Bacterial OTUs are identified mainly to belong to Proteobacteria (40-60%), (1-15%), (5-20%), Gemmatimonadetes (5-10%), Acidobacteria (5-10%), Actinobacteria (3-10%) and Chloroflexi (3-8%). The majority of identified fungal OTUs was Ascomycota (40-85%), Basidiomycota (10-60%) and about 1-12% of the remaining OTUs belonged to Mortierellomycota, results Glomeromycota and Chytridiomycota. The obtained suggested in rhizosphere soils under the influence of changing farming conditions (especially N fertilizers) and in addition proposed a relationship
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the structure of rhizosphere soil microbiota with
between productivity and quality of plants.
3.3.2. Abundance of turmeric rhizosphere fungal communities under varied N fertilizer rates
3.3.2.1. Diversity indices of turmeric rhizosphere fungal communities
The fungal composition of turmeric rhizosphere at four fertilization regimes (N0, N150, N350 and N500) was determined by sequence analysis of ITS amplicons from total DNA samples. After analyzing the sequence using Uparse software, comparing with Unite database, OTUs of rhizosphere samples were identified (Table 3.17).
Table 3. 2. ITS amplicons metagenome sequencing data and analyzed diversity indices.
N0 (n=3) N150 (n=3) N350 (n=3) N500 (n=3)
736
588
718
688
Sample name Averaged species number
Simpson index 0,927 ± 0,21 0,749 ± 0,09 0,823 ± 0,11 0,833 ± 0,16
ACE index Chao1 index Shannon-Weaver
770 ± 113 761 ± 83 5,76 ± 0,3
783 ± 75 755 ± 106 623 ± 91 788 ± 177 754 ± 98 645 ± 136 4,05 ± 0,70 4,43 ± 0,52 4,85 ± 0,48
The principle component analysis (PCA) for OTUs of rhizosphere soil samples showed a divergence of group H (N150 and N350) from low yield group L (N0, N500). This result suggested that the amount of N fertilizer inputs not only affect turmeric yield and curcuminoids content but also impact the rhizosphere fungal community structure.
Chemical fertilizers had been determined to be an alteration factor to microbiological system in different agricultural soils [169] [170], however this is the first time its impact on rhizosphere fungal community of turmeric plant C. longa being structurally analyzed and reported.
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3.3.2.2. Taxonomic composition of turmeric rhizosphere fungal communities
Rozellomycota Zygomycota,
The taxonomic composition at phylum and class levels of turmeric rhizosphere fungal communities in all investigated regimes appeared relatively uniformed. Accordingly, OTUs of Ascomycota were prevalent; especially in soil samples of regime N150 with 74.16±9.06% of total identified OTUs. Besides, the proportion of OTUs belonging to Basidiomycota tended to increase as the raising amount of N fertilizer. In addition, several fungal genera such as Zygomycota, Rozellomycota and Blastocladiomycota were found at higher rates in soil samples of L group (N0 and N500). However, statistical analysis showed no significant differences in abundance of Basidiomycota, and Blastocladiomycota between H and L groups (p>0.05).
At class level, OTUs of Sordariomycetes were distributed more intensively in samples of regimes N350 and N500, while Eurotiomycetes appeared with a significantly higher proportion in samples of N0 regime (p<0.05). Agaricomycetes was found as the most dominant fungal class of Basidiomycota with significant increased abundance in samples of H group (p<0.05). Findings of dominant fungal classes in the microbiota of turmeric rhizosphere as well as their dynamics under varying N fertilizing rates were unprecedented. These results have contributed to specify a fundamental reference for extensive studies on optimal fertilization dosages for turmeric C. longa in Vietnam on worldwide scale.
3.3.3. Differences in composition of selected effective microbial groups in turmeric rhizosphere
Based on characterized effective microorganisms in turmeric rhizosphere samples (Section 3.2), an oriented analysis in the rhizosphere microbial communities of turmeric was performed,
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focusing on the difference in abundances of PGPR belonging to Bacilliaceae and AMF belonging to Glomeromycota in four N fertilization regimes.
3.3.3.1. Bacteria of Bacilliaceae
rhizosphere samples, turmeric
Depending on the distribution of OTUs belonging to Bacilliaceae of three main taxonomical groups were determined: genus Bacillus, genus Geobacillus and unclassified bacteria.
Notably, the average proportion of OTUs belonging to Bacillus in the samples of N0 and N500 regimes (regimes of L group) accounted for 64 and 65% of the total OTUs of the Bacilliaceae family, respectively, and reached the peak at over 90% in samples under N150 and N350 regimes (H group). By statistical analysis, the difference in abundance of Bacillus-OTUs between two groups was determined to be significant, with p<0.05. These results suggest the role of some Bacillus bacteria in turmeric yield and quality. In particular, the finding may contribute to the orientation for preparation of biofertilizers from the PGPR strain belonging to Bacillus for turmeric farming in Vietnam.
3.3.3.2. Fungi of Glomeromycota
Statistical analysis results in taxonomic composition of the turmeric rhizosphere of groups L and H showed no difference in proportion of fungi belonging to Glomeromycota at phylum level. However, the difference between two groups was significant when considering at the genus level (p<0.05). In detail, the OTUs belonging to genus Glomus in total OTUs of Glomeromycota accounted for 13 and 10% in N0 and N500 regimes of L group, respectively, and for 59 and 36% in N150 and N350 of H group, respectively.
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In general, metagenome analysis at the genus level of Bacilliaceae and Glomeromycota revealed statistically significant differences between H and L groups, especially at the abundance of OTUs belonging to genera Bacillus and Glomus, of these effective representatives were isolated in the turmeric rhizosphere samples. These results have contributed confirming the prediction of effective microorganism groups correlating to turmeric yield and quality, thereby played a pivotal role for selection of indigenous microbial candidates for preparation of biofertilizer for turmeric plant C. longa.
3.4. Case study for effect of microbial inocula in turmeric
As a combination of isolating result (section 3.2) and genetic analysis (section 3.3.3), a biofertilizer from strains of PGPR and AMF was prepared and tested in turmeric plants. The microbial inocula were composed of Bacillus sp. PGP-V21 and 3 AMF strains (Glomus sp. AM-N1, Glomus intraradices AM-N2 and Glomus mosseae AM-N3). These indigenous microbial strains were determined to be safe in animal models.
Table 3. 3. Growth and productivity parameters of turmeric plants in experimental plots. DC (control) CP
97.3 ± 4.3f 5-7e 38.66 ± 6.54d 0.59 ± 0.03b 95.2 ± 6.4f 5-7e 30.72 ± 8.21c 0.45 ± 0.05a
Growth and productivity parameters Plant height (cm) Number of leaves Root length (cm) Rhizome weight (kg/plant) Note: * Different letters indicate significant differences (Tukey’s HSD test, p<0.05).
Study on effects of microbiological inocula on turmeric plant was designed with 2 cultivation formulas as follows: Control formula (Without application of biofertilizer) and CP formula (Biofertilizer applied at dosage of 150 g/m2). After 6 months of cultivating under turmeric were commonly conditions, growth parameters of
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determined turmeric. The productivity of turmeric belonging to two tested formulas was determined after harvesting. The results were summarized in Table 3.19.
The case study has initially determined effects of the microbial inocula on the yield and growth parameters of tested turmeric. Notably, in terms of rhizome weight, the difference between control and CP cultivation formula was statistically significant (p<0.05). The result has partly demonstrated the positive impact of inoculants from indigenous microorganisms, especially PGPR and AMF, in improving turmeric productivity. Further studies with more replications at longer cultivation periods in extensive scales are though necessary to fully claim effects and potential of the microbial inocula in turmeric farming, particularly in Vietnam.
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CONCLUSION
The study has achieved following major results:
1. The impact of nitrogen (N) fertilizer rates to
turmeric productivity was investigated. Accordingly, both higher turmeric rhizome yields of 27003-30902 kg.ha-1.y-1 and elevated curcuminoids contents of 3,182-3,263% were recorded by applying 150 to 350 kg.ha-1.y-1 of N fertilizer.
2. Differences between
rhizosphere microbial turmeric communities under optimized (150-350 kg N.ha-1.y-1) and low- yield regimes (0 and 500 kg N.ha-1.y-1) in terms of biodiversity and abundance were analyzed. Prevalent abundances of Bacillus bacteria and fungi belonging to Glomus in optimized regimes arose as the most significant difference between these two groups at genus level.
3. Effective microbial groups of plant growth promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) were isolated from turmeric rhizosphere samples at farming site. As the result of the study, nine strains of PGPR were isolated, of these Bacillus sp. PGP-V21 exhibited phosphate-solubilizing, nitrogen-fixing, IAA-producing and antifungal activities. Additionally, three AMF strains were selected, namely (Glomus sp. AM-N1, Glomus intraradices AM-N2 and Glomus mosseae AM-N3).
4. A biofertilizer for turmeric plant in Vietnam was prepared from inocula of Bacillus sp. PGP-V21 and 3 AM fungal strains (Glomus sp. AM-N1, Glomus intraradices AM-N2 and Glomus mosseae AM-N3). Results of the case study in turmeric plants confirmed the inoculating potential and positive effect of experimental inoculants on turmeric yield. The study suggested applications of these inocula in appropriated integrated nutrient management system for turmeric in Vietnam.
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Further research for optimized preparation of biofertilizers, especially those from AMF and PGPR, to enhance turmeric yield and quality are recommended. In addition, studies on biologically activities of microbial strains associated with turmeric plant are considered prospective.
NEW CONTRIBUTIONS OF THE DISSERTATION
This research has brought following contributions:
The first data on effects of chemical nitrogen fertilization inputs
on yield of turmeric plant in Vietnam
The first data on the impact of chemical nitrogen fertilization rates on the rhizosphere microbiota of turmeric in Vietnam. This is also one of the first studies on the dynamic of microbial community of medicinal plant rhizosphere in response to chemical fertilizers.
The first data on turmeric rhizosphere growth promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) in Vietnam, as well as promising microbial strains with potential use as biofertilizer for indigenous turmeric plant.
The first case study on preparation of biofertilizer for turmeric in Vietnam, thereby confirming the potential of applying these microorganisms in effective and safe inoculants, not only for turmeric but also for other economic valued crops.
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