Linking N2O Emissions from Biofertilizer-Amended Soil of Tea

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Linking N2O emissions from biofertilizer-amended soil of tea plantations to the abundance and structure of N2O-reducing microbial communities Shengjun Xu, Shugeng Feng, Haishu Sun, Shanghua Wu, Guoqiang Zhuang, Ye Deng, Zhihui Bai, Chuanyong Jing, and Xuliang Zhuang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04935 • Publication Date (Web): 10 Sep 2018 Downloaded from http://pubs.acs.org on September 11, 2018

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Environmental Science & Technology

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Linking N2 O emissions from biofertilizer-amended soil of tea plantations to the abundance and structure of N2O-reducing microbial communities

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Shengjun Xu, 1,3 Shugeng Feng, 1,3 Haishu Sun, 1,3 Shanghua Wu, 1,3 Guoqiang Zhuang,

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1,3

7

1

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Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

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2

1 2 3

Ye Deng, 1,3 Zhihui Bai, 1,3 Chuanyong Jing 2,3 and Xuliang Zhuang 1,3*

Key

Laboratory

of

Environmental

Biotechnology,

Research

Center

for

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research

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Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing

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100085, China

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3

13

Beijing 100049, China

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* S.X. and S.F. contributed equally to this paper.

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*Corresponding author: Professor Xuliang Zhuang

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(E-mail address: [email protected],

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KEYWORDS. nitrous oxide, biofertilizer, Trichoderma viride, denitrification,

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denitrifying genes, N2O-reducing microbial community

College of Resources and Environment, University of Chinese Academy of Sciences,

Tel. : +86 010 62849163)

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ACS Paragon Plus Environment


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ABSTRACT. Nitrous oxide (N2O) contributes up to 8% of global greenhouse gas

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emissions, with approximately 70% from terrestrial sources; over one-third of this

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terrestrial emission has been linked to increased agricultural fertilizer use. Much of

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the nitrogen in fertilizers is converted to N2O by microbial processes in soil. However,

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the potential mechanism of biofertilizers and the role of microbial communities in

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mitigating soil N2O emissions are not fully understood. Here, we used a

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greenhouse-based pot experiment with tea plantation soil to investigate the effect of

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Trichoderma viride biofertilizer on N2O emission. The addition of biofertilizer

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reduced N2O emissions from fertilized soil by 67.6%. Quantitative PCR (qPCR)

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analysis of key functional genes involved in N2O generation and reduction (amoA,

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nirK, nirS and nosZ) showed an increased abundance of nirS and nosZ genes linked

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to the pronounced reduction in N2O emissions. High-throughput sequencing of nosZ

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showed enhanced relative abundance of nosZ-harboring denitrifiers in the T. viride

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biofertilizer treatments, thus linking greater N2O reduction capacity to the reduced

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emissions. Our findings showed that biofertilizers can affect the microbial nitrogen

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transformation process and reduce N2O emissions from agroecosystems.

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INTRODUCTION

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China, the world’s largest tea producer, has approximately 1.3 million ha of tea

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plantations, and nitrogen fertilization of these plantations has led to substantial nitrous

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oxide (N2O) emissions (at least 3,000 tons annually)1,

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approximately 24% of the anthropogenic agricultural N2O emissions in China.2 The

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N2O generation in soil is linked to a series of microbial processes, including ammonia

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oxidization, nitrification and denitrification,4, 5 and each process could serve as the

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main source of N2O production under certain oxygen conditions.6,

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processes involved in nitrous oxide generation and the key functional genes that are

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key during the corresponding processes (amoA, nirK, nirS and nosZ)8-13 are

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summarized in Figure S1. Based on current understanding, microbial communities

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capable of reducing N2O to N2 are the only biotic sink for N2O.14

2, 3

that have accounted for

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The different

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Reducing N2O emissions by enhancing the activity of the soil N2O-reducing

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community has emerged as a central challenge. Several studies showed that N2O

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emission and the functional community were indeed affected by approaches such as

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the application of biofertilizers,

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including bacteria and fungi, that not only promote plant growth but also facilitate

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natural nitrogen cycling and mitigate N2O emission. 18-22 Because of these advantages,

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eco-friendly biofertilizers are preferred and expected to lessen the usage of chemical

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fertilizers, and the International Federation of Organic Agricultural Movements

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(IFOAM) has acknowledged the application of biofertilizers made from various

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biomasses and beneficial microorganisms can be one of the essential approaches to

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which contain beneficial microorganisms,



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regulate nitrogen cycling in soil and mitigate N2O emissions.16

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In a previous study, the plant growth-promoting fungus Trichoderma viride was

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cultivated and applied as biofertilizer, which, compared with chemical fertilizers,

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resulted in a 33.3 - 71.8% reduction in N2O emission.23 However, the underlying

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microbial mechanisms of N2O emission mitigation remain unclear. Hence, the

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motivation of this study is to investigate the potential mechanisms behind the

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reduction in N2O emission that occur with T. viride application, especially alterations

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in the functional microbial community and the regulated expression of key functional

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genes related to N2O production and reduction through denitrification.

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In this study, a microcosm experiment was conducted by applying T. viride

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biofertilizer to a plant-soil system with soils collected from a tea plantation orchard.

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Considering that tea trees are technically difficult to transfer and cultivate in the pot

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experiment, the plant Ipomoea aquatica Forsk, was introduced to mimic the tea

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plantation.24 The effects of T. viride culture on the microbial community and the key

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functional genes involved in N2O generation and reduction were analyzed through

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quantitative PCR and high-throughput sequencing. The main objectives of this study

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were to investigate a) the effect of T. viride culture on soil N2O mitigation and

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transformation and b) whether the responsive changes in the microbial community

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and corresponding key genes could explain the reduction in soil N2O emission.

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MATERIALS AND METHODS

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Soil Sampling and Biofertilizer Preparation. The experiment site was located in a

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tea orchard of the Changsha Environmental Observation Station of the Chinese

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Academy of Sciences (CAS) in Hunan Province, China (113°20' E, 28°34' N). This

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plantation has been cultivating Camellia sinensis Baekho for years, and due to the

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massive use of chemical fertilizer, the soil shows a substantial N2O emission rate (data

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not shown). The topsoil (0-20 cm) was randomly collected, passed through a 4-mm

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sieve and mixed evenly for experimental use. The basic properties of the soil,

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including its pH, total nitrogen (TN), total phosphorus (TP), dissolved organic carbon

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(DOC), and sand and clay content, were determined accordingly (Table S1).

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The T. viride culture biofertilizer was prepared as follows. A frozen stock of T.

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viride was grown on a potato dextrose plate for 24 h, and the T. viride mycelia were

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then transferred into a potato dextrose liquid culture using a sterile inoculating loop.

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The cultures were grown in conical flasks shaking at 150 rpm and 25°C for 48 h. The

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growth of the culture was monitored until the spore density reached 3.0×108

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CFU/mL.23

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Experimental Preparation. A set of pots (30×40 cm, diameter by height), each of

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which contained five kilograms of soil and for growing I. aquatica Forsk., was used

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for the experiment. Three I. aquatica Forsk. seedings, with four leaves and a root

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length of 8 cm, were planted in each pot. Three treatments were performed: control

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(CK) samples were irrigated with 400 mL of DI water, urea treatments (UTs) samples

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were applied with 400 mL of chemical nutrient (2 g/L nitrogen, 0.8 g/L phosphate and



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0.4 g/L potassium), and biofertilizer treatments (BTs) were with 400 mL of

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biofertilizer (2 g/L nitrogen, 0.8 g/L phosphate and 0.4 g/L potassium, with 25 mL of

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T. viride culture). Treatments were irrigated with only nutrients at the beginning and

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with DI water (400 mL every three days) subsequently. Each treatment (CK, UT and

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BT) was performed with three replicates, and all the treatments were placed in a

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greenhouse with relatively constant temperature (30 ± 2°C daytime, 25°C at night)

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and humidity (80%).

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Measurement of Nitrous Oxide Flux. Nitrous oxide fluxes were determined using

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a method adopted from a previous study with modifications.25 The gas emission was

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sampled immediately after the gas chambers (30×40 cm, diameter by height)15 had

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been placed on the pots. A 20-mL syringe was used to sample the gas fluxes within

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the chamber at 0, 30, 60, 90 and 120 min. Prior to gas collection, the gas in the

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chamber was evenly mixed through pumping the syringe several times. Each 20-mL

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gas sample was stored in a vacuum tube for subsequent analysis.

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The gas samples were analyzed using gas chromatography (Agilent 7890A,

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ThermoTM, CA, USA) equipped with a 63Ni electron capture detector (ECD) as well as

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a precolumn (1000 mm by 2 mm) and an analytical column (3000 mm by 2 mm), both

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packed with Porapak Q. The working temperature of the ECD was calibrated to

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330°C, and the oven temperature was maintained at 55°C. Pure nitrogen gas (99.99%)

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was used as the carrier gas with a flow rate of 30 cm3 per minute, and the retention

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time of the target peak was 3.5 min on average. N2O flux was measured as

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previously.25 Briefly, samples were collected and analyzed at 30-min intervals for 2 h,


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fluxes were determined for each interval, the mean of these fluxes was used to

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determine the N2O flux, and flux was collected and determined on a weekly base.

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Measurement of Soil Physiochemical Properties. Soil samples were collected

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after the 4-week experiment. After removing the plants and debris, the soil of each pot

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was thoroughly mixed; from these pots several soils were sampled, mixed and divided

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into two parts. One part of the soil sample was passed through a 2-mm sieve and

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stored at 4°C for further measuring of the physiochemical properties. The other part of

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the sample was frozen in liquid nitrogen and stored at -80°C. Soil pH was determined

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through a soil suspension (1:2.5 w/v) using a regular pH meter according to a

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published standard method (NY/T 1377-2007, Chinese Standard). Briefly, pure water

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was boiled and cooled, vigorously shaken with soil samples for 10 min and then kept

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stationary for 1 h. The suspension was used for pH determination. The soil TN

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contents were determined using an elemental analyzer, and the soil NH4+ and NO3-

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contents were measured using an AA3 continuous flow analytical system (SEAL

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AutoAnalyzer 3 HR, SEAL Analytical Inc.).26, 27

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DNA Extraction and Real-Time Quantitative PCR. DNA was extracted from 0.5

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g of soil using the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA, USA)

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according to the manufacturer’s instructions. The DNA was dissolved in 80 µL of

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sterile, deionized, nuclease-free water and divided into two fractions: one fraction was

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used for quantitative PCR, while the other was stored at -20°C for downstream

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Illumina sequencing.

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Functional marker genes (amoA, nirK, nirS and nosZ) were amplified with Premix



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Ex Taq (TaKaRa®, Japan) and specific primer sets adopted from previous studies

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(Table 1).28-32 Each sample was quantified in triplicate using the CFX Connect™

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Real-Time PCR Detection System (Bio-Rad laboratories). Results with correlation

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coefficients and amplification efficiencies above 0.98 and 98%, respectively, were

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used for downstream analyses. All amplified samples were examined on an agarose

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gel after PCR to confirm successful amplification, and the specificity of the

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amplification products was confirmed by melting curve analysis.

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High-Throughput Sequencing of nosZ. The N2O-reducing bacterial community

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harboring the marker gene nosZ was investigated through high-throughput sequencing

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analysis by Allwegene Technology Inc. at Beijing. The nosZ gene was amplified using

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the primers nosZF (5’-CGYTGTTCMTCGACAGCCAG-3’) and nosZ1622R (5’-

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CGSACCTTSTTGCCSTYGCG -3’).33 Each pair of primers used to amplify a soil

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sample was tagged at the 5’-end with a sample-specific 12-bp barcode on both

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forward and reverse primers. A 50-µL PCR mixture contained 20 to 30 ng of DNA,

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1.5 µL of each primer (at a final concentration of 0.3 µM), and 25 µL of Premix Ex

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Taq (TaKaRa, Dalian, China). The PCR conditions, performed in a thermal cycler

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(EASTWIN, China), consisted of an initial denaturation step at 95°C for 1 min,

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followed by 35 cycles at 95°C for 10 s, 55°C for 30 s and 72°C for 1 min, and a final

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extension step at 72°C for 10 min. The PCR amplicons were purified with a Gel

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Extraction Kit (D2500-02, OMEGA BioTek), and concentrations were quantified

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using spectrometry (Thermo Scientific, Wilmington, USA). These PCR amplicons

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were combined in equal concentrations, and the DNA library was obtained according


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to the MiSeq Reagent Kit Preparation Guide (Illumina, San Diego, CA, USA). Then,

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the DNA library was sequenced using the Illumina MiSeq platform according to the

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manufacturer’s instructions. Each treatment was performed with three replicates, with

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each replicate from individual pots, for DNA extraction and library preparation.

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To analyze the sequencing data of the environmental samples, metabarcoding

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analysis pipelines were adapted from previous studies.34, 35 Illumina MiSeq reads were

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demultiplexed using CASAVA (version 1.9; Illumina Inc.). The UPARSE program

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was used to remove chimeras and classify the sequences into operational taxonomy

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units (OTUs) at the 97% similarity level.36, 37 The BLASTN algorithm (blast-2.2.17)

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was used to align reads with a GenBank-derived sequence library using the following

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settings: E-value cutoff 1e-5, number of alignments 1, output format 0, number of

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descriptions 1, and percent identity threshold 97%.38 Alignment outputs were then

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annotated in MEGAN (version 5.1.5) using the lowest common ancestor (LCA).35

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Statistical Analyses. A univariate analysis of variance (ANOVA) was performed

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and corrected with the Tukey test to investigate the statistical significance of

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fertilizers causing changes in soil properties and reducing N2O emissions. The

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significant changes in the functional gene copy numbers of CK, UT and BT groups

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were compared through one-way ANOVA analysis. The significant differences in the

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relative abundances of the N2O-reducing community was also analyzed through

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ANOVA. All analyses were performed using the SPSS software package (version 20.0,

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IBM, Armonk, NY, USA), and the statistical significance level was 0.05. Additionally,

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the changes in within-sample diversities (α-diversity, including the Chao1 estimator,



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observed species, PD whole tree and Shannon diversity) of the N2O-reducing

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community in different treatments were also determined.

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RESULTS AND DISCUSSION

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Change in Soil Physiological Properties. The effects of chemical (urea) and

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biological fertilizer (T. viride cultures) application on soil properties were investigated

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after the 4-week experimental period (Table 2). In general, the application of urea and

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biofertilizer significantly increased (P

Linking N2O Emissions from Biofertilizer-Amended Soil of Tea

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