Effects of reclaimed water irrigation and nitrogen fertilization on the chemical properties and microbial community of soil

Journal of Integrative Agriculture 2017, 16(3): 679–690 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Effects of reclaimed water irrigation and nitrogen fertilization on the chemical properties and microbial community of soil GUO Wei1, 2, Mathias N Andersen3, QI Xue-bin1, 4, LI Ping1, 4, LI Zhong-yang1, 4, FAN Xiang-yang1, 4, ZHOU Yuan1, 2 1

Farmland Irrigation Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Xinxiang 453003, P.R.China

2

Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China Department of Agroecology, Faculty of Science and Technology, Aarhus University, Tjele 8830, Denmark 4 Key Laboratory of High-efficient and Safe Utilization of Agriculture Water Resources of CAAS, Xinxiang 453003, P.R.China 3

Abstract The ecological effect of reclaimed water irrigation and fertilizer application on the soil environment is receiving more attention. Soil microbial activity and nitrogen (N) levels are important indicators of the effect of reclaimed water irrigation on environment. This study evaluated soil physicochemical properties and microbial community structure in soils irrigated with reclaimed water and receiving varied amounts of N fertilizer. The results indicated that the reclaimed water irrigation increased soil electrical conductivity (EC) and soil water content (SWC). The N treatment has highly significant effect on the ACE, Chao, Shannon (H) and Coverage indices. Based on a 16S ribosomal RNA (16S rRNA) sequence analysis, the Proteobacteria, Gemmatimonadetes and Bacteroidetes were more abundant in soil irrigated with reclaimed water than in soil irrigated with clean water. Stronger clustering of microbial communities using either clean or reclaimed water for irrigation indicated that the type of irrigation water may have a greater influence on the structure of soil microbial community than N fertilizer treatment. Based on a canonical correspondence analysis (CCA) between the species of soil microbes and the chemical properties of the soil, which indicated that nitrate N (NO3–-N) and total phosphorus (TP) had significant impact on abundance of Verrucomicrobia and Gemmatimonadetes, meanwhile the pH and organic matter (OM) had impact on abundance of Firmicutes and Actinobacteria significantly. It was beneficial to the improvement of soil bacterial activity and fertility under 120 mg kg–1 N with reclaimed water irrigation. Keywords: reclaimed water, nitrogen, soil chemical properties, 16S rRNA sequence, soil microbe community

1. Introduction Received 6 January, 2016 Accepted 29 April, 2016 GUO Wei, Mobile: +86-18790592328, E-mail: guowei1124@163. com; Correspondence QI Xue-bin, Tel: +86-373-3393277, Fax: +86-373-3393308, E-mail: [email protected] © 2017, CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) doi: 10.1016/S2095-3119(16)61391-6

In urban areas, the demand for water has sharply increased due to increased population. Reclaimed water is a major resource for the augmentation of inadequate water supplies, especially in arid zones and urban areas. Irrigation with reclaimed water is one of the principal alternatives for the maintenance of existing water resources, and it has been encouraged by governments and official entities worldwide

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(Biggs and Jiang 2009; Becerra-Castro et al. 2015). Irrigation with reclaimed water can improve soil health when using suitable management practices (Martinez et al. 2011; Chen et al. 2013; Nicolás et al. 2016) and changes in the availability of soil N have the potential to drastically alter the soil carbon (SC) cycle (Cox et al. 2000; Cramer et al. 2001). However, the potential health risks and environmental concerns related to irrigation with reclaimed water cannot be ignored (Chen et al. 2015). The impact on the environment of irrigation with reclaimed water and the long-term ecological effects has been the focus of people’s attention for a long time. The research on ecological effect of irrigation with reclaimed water has mainly focused on investigating soil pollution (Blanchard et al. 2001; Hidri 2014), leaching of N and phosphorus (P) to ground and surface waters (Katz et al. 2009) as well as the diversity of the soil ammonifiers community (Li et al. 2005) and soil microbial functional groups (Thayanukul et al. 2013). Nevertheless, research on the structure of the soil microbial community and related soil properties is still relatively weak with respect to irrigation with reclaimed water with different N levels. The reclaimed water contains high salts, potentially hazardous compounds like heavy metals and pharmaceutically active chemicals and pathogens (Liu et al. 2005; Xu et al. 2010). The microbial diversity may have been markedly changed following pesticide application despite unaltered metabolism, and such changes may affect soil fertility and the balance of the soil ecosystem (Johnsen et al. 2001). N is one of the major essential macronutrients for the biological growth and development of plants. Microorganisms play a central role in the natural biological cycle and for instance convert N2 in the atmosphere to available N in soil. The introduction of bacteria involved in N cycling or other bacteria with the ability to remediate soil contaminants (such as pesticide residue, inorganic fertilizer or pathogens) may lead to an amendment in soil quality (Oved et al. 2001; He et al. 2007; Hanjra et al. 2012). Recently, genetic and molecular methods have greatly enhanced the possibilities of gaining information on the diversity and structure of the soil microbial community compared to the cultivation of isolated microbes (Caporaso et al. 2011; Li et al. 2015). The hereditary characteristics of the soil microbial community in forests, grasslands, reclaimed mining areas and farmlands were reported in the literature (Dimitriu et al. 2010; Li et al. 2013). However the study of the microbial community diversity in soil with reclaimed water irrigation through high-throughput technology is still rare. After reviewing the past research work, we conclude that the study of the bacterial community structure model and the corresponding dynamic response characteristics

under irrigation by reclaimed water with a different range of N levels is necessary. Thus, the present article aims to explore the characteristics of microorganisms involved in the regulation of the soil micro-environment and the increase in N bio-availability, which are important aspects in relation to rational fertilization and minimization of agriculture’s environmental footprint when using reclaimed water for irrigation. In this study, we investigated the effects of irrigation using different types of water and N treatments on the diversity and composition of the microbial community. We hypothesized that both the water quality and the application of N would produce significant effects on the structure of the soil microbial community. N was applied only once just prior to planting in this study, therefore we further predicted that the type of water used for irrigation would have a larger impact on microbial properties than the N treatment.

2. Materials and methods 2.1. Test materials and design The study was conducted at the Agriculture Water and Soil Environment Field Science Research Station, China (35°19´N, 113°53´E), at an altitude of 73.2 m, in the continental monsoon climate area of the temperate zone. A greenhouse pot culture experiment was used to study the effects of reclaimed water and N fertilization on the composition of the soil microbial flora and soil chemical properties with clean water as the control. The tested soil was taken from the surface layer (0–20 cm) of a sandy loam from the experimental station. The air-dried soil samples were sieved to pass through a 2-mm sieve, then 6 kg air-dried soil was placed in each pot. The soil chemical properties of total nitrogen (TN), total phosphorus (TP) and organic matter (OM) were 1.20, 0.83 and 32.85 g kg–1, respectively. pH was 8.26, electrical conductivity (EC) was 0.39 ds m–1. The standard recommended dose of P (44 mg kg–1 of soil) and K (249 mg kg–1 of soil) was applied before planting of cabbage (Brassica campestris L. ssp. chinensis Makino) on 6 April. There were five fertilizer treatments: N0 (0 mg kg–1), N1 (80 mg kg–1), N2 (100 mg kg–1), N3 (120 mg kg–1), N4 (180 mg kg–1). Two types of water, clean water (C) and reclaimed water (R) were used for irrigation. The resulting 10 treatments had 6 replicates each, and thus a total of 60 pots were used. The reclaimed test water was taken from the Camel Bay sewage treatment plants after secondary treatment, a source of city sewage, and the water quality indicators were shown in Table 1. A total of 12.9 L water, corresponding to 226 mm, was applied to each pot during the experiment by irrigation every second day. The cabbage was harvested on 6 June by cutting the stem at the soil surface.

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Table 1 Quality characteristics of the two irrigation waters used in the experiment1)

Clean water Reclaimed water 1)

NO3–-N (mg L–1) 5.82 14.54

NH4+-N (mg L–1) 0 5.64

TN (mg L–1) 8.73 30.89

TP (mg L–1) 0 0.83

pH 7.56 7.45

EC (ds m–1) 1 2.23

Cu (mg L–1) 0.001 0.003

Cd (mg L–1) 0.007 0.003

Pb (mg L–1) 0.001 0.005

Zn Cr CODMn (mg L–1) (mg L–1) (mg L–1) 0.002 0.001 6.9 0.008 0.003 17.3

NO3–-N, nitrate nitrogen; NH4+-N, ammonia nitrogen; TN, total nitrogen; TP, total phosphorus; EC, electrical conductivity; pH, water pH value; CODMn, permanganate index.

2.2. Soil sampling In June 2014, just after the green vegetables were harvested, a soil sample representing the whole pot was collected and homogenized manually. After removing residual roots from the soil sample, the sample was put in sterilized fluoroethylene plastic bag, sealed and placed in a refrigerator at 4°C, and soon after taken to the laboratory. The soil samples were divided into two parts: sub-samples for microbial analysis were stored at –20°C until DNA extraction (<14 d), while other sub-samples were air dried for chemical analyses. All soil samples were sieved to pass through a 2-mm sieve.

2.3. Chemical analyses of water and soil The chemical indices of irrigation water sample were measured. The nitrate nitrogen (NO3–-N) and ammonia nitrogen (NH4+-N) was measured by AA3 flow analyzer (Bran Luebbe Gmbh, Germany); pH value was determined by the a LeiciShanghai PHS-1 pH meter, EC was measured by a LeiciShanghai DDB-303A conductivity meter, permanganate index (CODMn) was measured by chemical oxygen demand (COD) analyzer. Cu, Cd, Pb, Zn and Cr were measured by microwave digestion-atomic absorption spectrophotometry. Based on results from Adrover et al. (2012) and Lyu and Chen (2016), a total of 6 attributes reflecting soil health conditions were measured, including: (1) the soil chemical characteristics of soil pH (pH), OM, TN, and TP. (2) Soil salinity related attributes of EC. The soil pH was determined in distilled water at a soil-to-solution mass ratio of 1:5 by the Leici-Shanghai PHS-1 pH meter. The soil organic matter was determined based on Bao (2000) by the oxidation volumetric method for the determination of potassium dichromate. Soil TN and TP were determined by a flow analyzer. The electrical conductivity of the soil extracts at a soil and water ratio of 1:5 (EC 1:5) was measured by a Leici-Shanghai DDB-303A conductivity meter. (3) SWC was measured by aluminum boxes weighted method.

2.4. DNA extraction and PCR amplification of 16S rDNA and Illumina MiSeq high-throughput sequencing analysis DNA was isolated immediately in triplicate from 0.5 g

soil from each homogenized sample with the Power Soil DNA Isolation Kit (MO BIO Laboratories, USA) according to the manufacturer’s instructions. The recovered DNA was amplified using barcoded 16S rRNA Illumina MiSeq high-throughput sequencing tags to achieve parallel sequencing of samples. 16S V3–V4 primers were used for PCR amplification, and in this way all samples striped tested and qualified. To identify the bacterial species contained in each sample, a PCR was performed to amplify the 16S rRNA using the universal primers 16S V3–V4 (5´-TACG GRAGGCAGCAG-3´) and (5´-AGGGTATCTAATCCT-3´) with the appropriate Illumina Miseq Life Sciences adaptor sequence. All PCR reactions were quality-controlled for amplicon saturation by agarose gel electrophoresis. The representative sequences of the operational taxonomic units (OTUs) were acquired by the OTUs analysis method of Mothur default, namely the computation of the uncorrected pair-wise distance between sequence, and data from each sample were clustered based on the OTUs, using a threshold of 97% sequence similarity (Edgar et al. 2010). The ribosomal datebase project (RDP) classifier assigned a taxonomic rank to sequence reads by matching the distributions of nucleotide substrings to a model defined from the sequences of known microorganisms. Sequences were separated by soil type (clean water and reclaimed water treatments) and aligned using the RDP aligner, then were clustered using RDP complete linkage clustering at a maximum distance of 3% (corresponding to 97% sequence similarity). Rarefaction curves for each soil type were created based on the aligned sequences. We investigated the composition of the soil microbial community to evaluate changes in the microbial community structure with water quality, soil type and N level, as an index of viable microbial biomass (Federle et al. 1986). In this study, the 97% sequence identity threshold was chosen because it is commonly used to define bacterial species (Drancourt and Raoult 2005; Janda and Abbott 2007). The OTUs defined from sequenced data were analysed using a RDP classifier. A matrix of environmental values were also established, which were used to analyse the relationships between soil chemical properties and microbial community factors using a canonical correspondence analysis (CCA). These calculations were carried out using

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CANOCO 4.5 (Ter Braak and Smilauer 2002).

2.5. Statistical analysis The effects of the type of irrigation water and fertilizer treatments on the chemical properties and microbe characteristics of the soil were examined with separate one-way analyses of variance (ANOVA) using SPSS 17.0. A two-way ANOVA was applied to analyze the effects of irrigation water type and N treatments on chemical properties and microbial communities of soil. The significance of all statistical analyses was accepted at P=0.05. The genetic diversity index of soil bacteria (Nei 1978), the Shannon index and the genetic similarity parameters were analysed by the software Popgene32 and MEGA6.0.

3. Results 3.1. The impact of reclaimed water irrigation and N fertilization on soil chemical, physical and biological characteristics Reclaimed water (Table 1) typically delivers high amounts of N, P, organic matter and other nutrients to the soil compared to tap water. Table 2 illustrates the differences in eight parameters related to soil nutrient conditions after the soil grown with cabbage which received tap water or reclaimed water by irrigation for 2 mon. Irrigation with reclaimed water significantly increased (P<0.05) pH of soil at

N2 and N3 levels (Table 2). Irrigation with reclaimed water significantly increased (P<0.05) soil water content (SWC) (except N2) and EC (except N4) of the soil. The SWC and EC were considerably increased compared to the clean water treatment (P<0.05). Average soil EC in the pots irrigated with reclaimed water was about 20% higher than that in those irrigated with tap water. The N treatments also had a highly significant effect on the pH and furthermore on OM and C/N of the soil (P<0.01). A decreasing trend of OM with higher N-level was found which was especially pronounced going from N0 to N1 and occurred during irrigation with both types of water. It has to be bore in mind that the soil had an OM content of 32.85 g kg–1 before filling into pots and received additional input of organic material with the reclaimed water and therefore the breakdown of OM was substantially increased by N fertilization. The soil C/N ratio decreased with the decrease of OM, which was shown by a tight correlation r2=0.93** between OM and C/N. Irrigation water type and N level had a significant interaction effect on TP (P<0.05) as irrigation with reclaimed water resulted in a slight increase or decreas in the soil TP in some of the N treatments. Also with respect to EC an interaction was found, which was smaller than the effect of irrigation water type. Irrigation with reclaimed water tended to reduce the soil TN content at some N fertilizer levels. Soil NO3–-N increased significantly (P<0.05) with N level and in general was higher when irrigating with reclaimed water than with tap water apart from the N4-level, which gave rise to an interaction effect between irrigation water type and N level

Table 2 The chemical properties of the soil after 2-mon treatments with clean (C) or reclaimed (R) water in combination with 5 nitrogen levels (N0–N4) Treatments1) pH2) SWC (%)3) EC (ds m–1) 8.24 abcd 11.5 c 0.63 e CN0 8.31 a 15.8 ab 1.18 ab RN0 8.18 cdfe 11.2 c 0.72 de CN1 8.20 cdf 15.3 b 1.14 ab RN1 8.11 fg 14.0 b 0.82 de CN2 8.22 bcd 15.7 ab 1.10 b RN2 8.06 g 11.1 c 0.83 de CN3 8.16 dfe 14.7 b 1.32 a RN3 8.29 ab 12.0 c 0.89 cd CN4 8.26 abc 14.5 b 1.04 bc RN4 Significance based on two-way ANOVA (F value) 57.297** 88.038** W (water) 9.458** 12.926** 2.548 2.148 N (nitrogen) 2.237 1.381 3.193* W×N

OM (g kg–1) 30.0 ab 32.3 a 26.2 c 27.2 bc 25.9 c 26.1 c 24.9 c 24.5 c 26.2 c 24.3 c 0.176 11.466** 1.015

C/N4) 18.6 a 20.0 a 15.1 bc 15.6 bc 15.1 bc 16.4 b 14.6 bc 14.6 bc 15.3 bc 14.7 bc 1.1882 9.764** 0.460

TP (g kg–1) 0.82 bc 0.81 bc 0.82 bc 0.84 abc 0.82 bc 0.84 abc 0.87 a 0.80 c 0.83 bc 0.83 abc

TN (g kg–1) 0.94 0.94 1.00 1.01 0.99 0.93 0.99 0.92 1.00 0.97

1.166 0.471 3.194*

3.406 2.087 0.876

NO3–-N (mg kg–1) 2.93 e 2.90 e 38.62 d 45.60 d 99.21 c 102.15 c 86.15 c 146.62 a 152.04 a 120.65 a 0.276 11.197* 18.268**

C, clean water irrigation; R, reclaimed water irrigation; N0, 0 mg kg–1 nitrogen addition; N1, 80 mg kg–1 nitrogen; N2, 100 mg kg–1 nitrogen; N3, 120 mg kg–1 nitrogen; N4, 180 mg kg–1 nitrogen. The same as below. 2) pH, soil pH value. 3) SWC, soil water content. 4) C/N, soil carbon and nitrogen ratio. Different letters in the same column indicated significant differences (P<0.05) among the treatments (Duncan’s test). *, significance at the 5% (P=0.05) level; **, significance at the 1% (P=0.01) level. The same as below. 1)

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on soil NO3–-N (P<0.01).

3.2. The diversity and structure of microbial community in soils irrigated with clean or reclaimed water Table 3 illustrates the changes in the diversity of the soil microbial community with increasing N-level under both clean and reclaimed water irrigation. With an increase in the N level, the number of the bacterial representative sequences of the OTUs gradually increased (except N0), while the type of irrigation water had no significant effect (P>0.05) on the diversity of the soil microbial community. Chao and ACE indicated the community richness. The N treatments significantly influenced the ACE, Chao, Shannon (H) and Coverage index (P<0.01). Community richness of ACE and the Chao index were the highest at CN4 and the lowest at RN1 and generally increased with N-level. The H index of CN4 and CN3 was significantly higher than that of CN0, RN0, CN1, RN1 (P<0.01), but there was no significant difference in the H index among the N2, N3 and N4 levels (P>0.05). The coverage was at its lowest at the medium N-level (N2) and the highest at N3 under reclaimed water irrigation. The irrigation water type and N treatments showed significant interaction effects on the ACE, Simpson, and the Coverage index (P<0.05). These indices were all higher under clean water than under reclaimed water irrigation at the low N-level (N0), then they became less in clean water than in reclaimed water at the medium N-levels (N2 and/or N3), and finally they become higher again at high N-level (N4). Rarefaction curves of OTUs defined at 97% sequence similarity through the RDP pipeline showed significantly greater richness in the clean water treatment (Fig. 1). A principal coordinates analysis (PCoA) of weighted and unweighted UniFrac results showed that samples from the reclaimed water and clean water treatment clustered

more tightly than the N fertilizer control, although several samples of the N treatments from the clean water treatment were clustered in the reclaimed water irrigation group when weighted (Fig. 2-A and B). On the first principal component (PC1) axis, the sample point of irrigation with reclaimed water was mainly distributed in the negative direction, while the sample point of clean water irrigation was distributed mostly in the positive direction by the weighted method (Fig. 2-A). On the second principal component (PC2) axis, the sample point of irrigation with reclaimed water was mainly distributed in the positive direction, while the sample point of clean water irrigation distributed mostly in the negative direction by the unweighted method (Fig. 2-B). The distribution of the differences in the two different irrigation types showed that the soil microbial community had different metabolic functions in soil irrigated with either reclaimed or clean water. The contribution ratios of PC1 were 38.86 and 41.67% while the contribution ratios of PC2 were 24.54 and 20.1% for the reclaimed water and clean water treatments, respectively (Fig. 2-C and D). On the PC1 axis, the Verrucomicrobia, Actinobacteria, Bacteroidetes and Firmicutes were distributed in the positive direction, while the Proteobacteria, Acidobacteria, Other, and Gemmatimonadetes were distributed in the negative direction partly under the reclaimed water treatment. On the PC2 axis, the Verrucomicrobia, Actinobacteria, Acidobacteria, Other and Gemmatimonadetes were distributed in the positive direction, and the Bacteroidetes, Firmicutes and Proteobacteria were distributed in negative direction partly under the reclaimed water treatment (Fig. 2-C). On the PC1 axis, the Gemmatimonadetes, Other, Bacteroidetes, Acidobacteria and Proteobacteria were distributed in the positive direction, but the Actinobacteria, Firmicutes and Verrucomicrobia were distributed in the negative direction partly under the clean water treatment; on the PC2 axis, the Gemmatimonadetes, Other, Firmicutes and

Table 3 The effect of irrigation with reclaimed water on the diversity of the soil microbial community1) Treatments OTUs ACE 4 120 5 451 de CN0 3 551 4 876 de RN0 3 937 5 564 de CN1 3 262 4 530 e RN1 4 032 5 981 cd CN2 4 170 7 154 ab RN2 4 390 6 174 bcd CN3 5 040 6 824 abc RN3 5 507 7 368 a CN4 5 014 7 028 abc RN4 Significance based on two-way ANOVA (F value) W 0.013 N 14.723** 3.413* W×N 1)

Chao 5 756 cd 5 142 de 5 728 cd 4 785 e 6 044 cd 6 533 bc 6 347 bc 7 006 ab 7 546 a 7 153 ab 0.744 17.393** 2.840

Shannon (H´) 7.36 bc 7.36 bc 7.37 b 7.26 c 7.39 ab 7.45 ab 7.50 a 7.47 ab 7.50 a 7.46 ab 1.363 9.715** 1.652

Simpson 0.00156 ab 0.00132 bc 0.00160 a 0.00160 a 0.00172 a 0.00150 abc 0.00127 c 0.00159 a 0.00155 ab 0.00162 a 0.071 2.614 4.268*

Coverage 0.966 a 0.944 abcd 0.936 cd 0.939 bcd 0.921 de 0.901 e 0.937 cd 0.957 abc 0.963 ab 0.947 abc 2.041 12.087** 2.931*

OTUs, operational taxonomic unites; ACE and Chao represent the richness of bacterial community; Shannon, Simpson and Coverage represent the diversity of bacterial community.

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3 000

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Fig. 1 Rarefaction curves of the bacterial operational taxonomic units (OTUs) in soils irrigated with reclaimed water (A) and clean water (B) in combination with 5 nitrogen levels (N0–N4). R, reclaimed water irrigation; C, clean water irrigation; N0, 0 mg kg–1 nitrogen addition; N1, 80 mg kg–1 nitrogen; N2, 100 mg kg–1 nitrogen; N3, 120 mg kg–1 nitrogen; N4, 180 mg kg–1 nitrogen. All data were reported as the arithmetic mean (n=3). The same as below.

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Fig. 2 The analysis of soil bacterial communities in soils irrigated with two types of water based on 16S rRNA amplicons. The differences of bacterial community structure in soils irrigated with reclaimed water and clean water were analyzed by the principal coordinates analysis (PCoA) of weighted UniFrac (A) and unweighted UniFrac (B) based on distance matrix. The differences of bacterial community in soils irrigated with reclaimed water (C) and clean water (D) were analyzed by the principal component analysis (PCA) based on similarity coefficient matrix.

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Verrucorricrobia were distributed in the positive direction, while the Bacteroidetes, Acidobacteria, Proteobacteria and Actinobacteria were distributed in the negative direction partly under the clean water treatment (Fig. 2-D).

3.3. The composition of microbial community and canonical correspondence analysis (CCA) in soils irrigated with clean or reclaimed water There was no obvious difference in the abundance of bacteria phylum in the RDP-analysed sequence data between the two types of irrigation water (Fig. 3). The total number of unique sequences of the soil was 43 023, which was divided into eight phyla, while Proteobacteria, Gemmatimonadetes, Bacteroidetes, Actinobacteria and Acidobacteria were predominant constitutent parts of phyla (Fig. 3). Of the top five most abundant classes, the followings were more abundant in the soil of the reclaimed water treatment than in the soil of the clean water treatment (values indicated the percent of amplicons in the reclaimed water soil vs. the clean water soil): Proteobacteria (36.91% vs. 36.24%), Gemmatimonadetes (19.40% vs. 16.85%), Bacteroidetes (17.52% vs. 17.02%). The followings were less abundant in the soil of the reclaimed water treatment than in the soil of the clean water treatment: Actinobacteria (15.51 % vs. 18.51%) and Acidobacteria (6.15% vs. 6.28%). In the RDP-analysed sequence data, the 24th most abundant class Acidobacteria-6, were only found in the clean water treatments (Appendix A), and the 34th most abundant order, Pseudomonadales, was only found in the clean water treatments (Appendix B). In the RDP-analyzed sequence

data, the 31st, 35th, 36th and 37th most abundant genera, Erythrobacteraceae_unclassified, Micrococcaceae_unclassified, iii1−15_unclassified and Pseudomona were only found in the clean water treatments (Fig. 4), while the 32nd and 34th most abundant genera Sphingobacteriales_unclassified and Xanthomonadaceae_unclassified were only found in the reclaimed water treatments (Fig. 4). A Venn diagram was used to display the shared characteristics between different samples and peculiar OTU, which can be compared directly based on the performance of the similarity degreeof OTU between various environmental samples. The Venn diagram in Fig. 5 illustrates the numbers of shared and unique OTUs among the sample types. The clustering was complemented by an analysis of bacterial richness using the number of shared and unique OTUs in the five groups (Fig. 5), indicating that a core microbiome existed in the clean water and reclaimed water treatments. Based on Monte Carlo permutation test, it was shown that the significant environmental factors was TP (P=0. 012, F=2.629, permutation number=499). Based on CCA between species of soil microbes and soil chemical properties (Fig. 6), we found that NO3–-N, TP, and EC had a greater influence on the structure of the soil microbial community in the reclaimed water treatment, while TP, pH and organic material in the clean water treatment had a large effect on soil microorganisms.

4. Discussion Irrigation with wastewater altered the physicochemical and

Verrucomicrobia

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Fig. 3 Relative abundance of the bacterial phyla in soils irrigated with two types of water based on 16S rRNA amplicons.

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A

B 3

Other Gemmatimonadetes_unclassified Gemm−5_unclassified Cytophagaceae_unclassified Pontibacter Kaistobacter Acidimicrobiales_unclassified Gemm−3_unclassified Gemm−1_unclassified Chitinophagaceae_unclassified Gaiellaceae_unclassified Myxococcales_unclassified 0319−7L14_unclassified Solirubrobacterales_unclassified Flavisolibacter Rhodospirillaceae_unclassified Syntrophobacteraceae_unclassified Betaproteobacteria_unclassified Solibacterales_unclassified MND1_unclassified C114_unclassified Nocardioidaceae_unclassified Sinobacteraceae_unclassified Saprospiraceae_unclassified Rhodospirillales_unclassified DS−18_unclassified Sva0725_unclassified Adhaeribacter Steroidobacter Sphingomonas Erythrobacteraceae_unclassified Haliangiaceae_unclassified Thermomonas Salinimicrobium Micrococcaceae_unclassified iii1−15_unclassified Pseudomonas

2 1 0 −1 −2 −3

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CN0.1 CN0.2 CN0.3 CN1.1 CN1.2 CN1.3 CN2.1 CN2.2 CN2.3 CN3.1 CN3.2 CN3.3 CN4.1 CN4.2 CN4.3

RN0.1 RN0.2 RN0.3 RN1.1 RN1.2 RN1.3 RN2.1 RN2.2 RN2.3 RN3.1 RN3.2 RN3.3 RN4.1 RN4.2 RN4.3

Other Gemm−5_unclassified Gemmatimonadetes_unclassified Kaistobacter Cytophagaceae_unclassified Gemm−3_unclassified Pontibacter Chitinophagaceae_unclassified Acidimicrobiales_unclassified Flavisolibacter Gemm−1_unclassified C114_unclassified Gaiellaceae_unclassified Myxococcales_unclassified MND1_unclassified Rhodospirillaceae_unclassified Syntrophobacteraceae_unclassified Solibacterales_unclassified Saprospiraceae_unclassified Betaproteobacteria_unclassified 0319−7L14_unclassified Solirubrobacterales_unclassified Sinobacteraceae_unclassified Rhodospirillales_unclassified DS−18_unclassified Nocardioidaceae_unclassified Sva0725_unclassified Adhaeribacter Steroidobacter Sphingomonas Haliangiaceae_unclassified Sphingobacteriales_unclassified Thermomonas Xanthomonadaceae_unclassified Salinimicrobium

Fig. 4 Relative abundance of bacterial taxonomic groups (genera) in soils irrigated with reclaimed water (A) and clean water (B) based on 16S rRNA amplicons. The percent of all amplicons in a soil type that was classified to each group is indicated by the stacked bars. Each column is labeled with the sample name with numbers representing field replicates.

A

RN0

CN0

B

1285

1 016

200

RN1

69

71

129 468

190 821

223

1 666

RN4

337

69 2 881

145 85

171

161 208

384

97

89 49

114 120

136

88

115

CN1

1 076

276

3 123 336

160 235

187

128

150

2 107

223

159

86

1 757

69

90 141

287

146

303

225

430

CN4

280 208

332

84

144

338 172

134

1 470

1 133

1 191

RN2

74

RN3

CN2

CN3

Fig. 5 The samples of the common and unique OTU numbers in soils irrigated with reclaimed water (A) and clean water (B) in the case of cut-off=0.03.

microbiological properties of the soil (Tables 2 and 3) which was also found in previous studies (Hidri et al. 2010; BecerraCastro et al. 2015). Earlier studies used biochemical means (Friedel et al. 2000) and gene clone library (Guo et al. 2015) to analyse changes in microbial biomass caused by

irrigation with reclaimed water. N and soil microbial activity are the major factors of soil biochemical process, which are important indicators of reclaimed water irrigation on the soil environment. The analyses of the effect of irrigation with reclaimed water with varied N levels on the soil bacterial

GUO Wei et al. Journal of Integrative Agriculture 2017, 16(3): 679–690

CN0 RN0

CN1 RN1

CN2 RN2

0.8

CN3 RN3

CN4 RN4

NO3– TN

TP Bacteroid pH

OM

Verrucom Gemmatim Other Proteobc Acidobac

Eirmiat

Actionobc

EC

–0.8 –1.0

1.0

Fig. 6 The soil bacteria samples, species and environmental factors from a canonical correspondence analysis (CCA). TN, total nitrogen; OM, soil organic matter; NO3–, nitrate nitrogen; TP, total phosphorus; EC, electrical conductivity; pH, soil pH value.

community are poorly understood. Based on Illumina MiSeq sequencing technique, we found significant differences between soil-related taxonomic and phylogenetic in the structure of the bacterial communities in soil treated with either fresh or reclaimed waste water. These differences were also significantly correlated with the chemical properties of the soil after the two treatments.

4.1. The effects of reclaimed water irrigation on the chemical properties of soil Effects of reclaimed water irrigation on soil chemical porperties is evidenced by the strong relationship among SC, microbial biomass and enzyme activities. Some researchers reported that irrigation with reclaimed water could increase soil pH due to its high content of HCO3–, salt and plant nutrients (Mancino and Pepper 1992; Qian and Mecham 2005). In the present study, the soil pH values in both irrigation treatments were approximately 8.0 with only small variation among treatments. This may be due to high soil buffer capacity. TN was slightly reduced in the soil under reclaimed water treatment, which may be attributed to the fact that most of the N in the reclaimed water was in forms that could be easily taken up by plants (Duncan et al. 2009). A decreasing trend of OM with higher N-level was likely related to the increased soil respiration with N topdressing (Resh et al. 2002; Micks et al. 2004), N as substrate and energy source, the addition of mineral-N fertilizers possibly caused the acceleration of OM mineralization due to priming effects (Kuzyakov et al. 2000). N-fertilizers provided substrates for

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soil microbial communities, the rate of soil respiration was enhanced accordingly, the content of OM was decreased (Shaver et al. 2000; Zhang et al. 2007). The accumulation of salts in the soil was affected by many factors including the quality of irrigation water, irrigation practices, soil properties and plant uptake characteristics. In addition, it increased along with the number of irrigation cycles, which added more ions to the soil. Overall, large differences were found in the EC between plots irrigated with clean water and reclaimed water. C/N ratio is the index measuring SC and N nutrition balance, which is considered to be an indicator of soil nitrogen mineralization capacity. Higher carbon content promotes denitrification (Mohan et al. 2016), while it is generally believed that when C/N<15, nitrogen mineralization surmount the assimilation capacity of microorganism making N available to plants. The soil C/N ratio under irrigation with clean water was higher than that under irrigation with reclaimed water. The C/N ratio of soil under irrigation with reclaimed water was 14.6 and 14.7 in N3 and N4 treatments. This was conducive to the plants’ absorption of nutrients under reclaimed water irrigation.

4.2. Ecological inferences from community structure differences under reclaimed water irrigation Rarefied Illumina MiSeq sequencing data revealed that bacterial OTUs at a medium N level were richer in the reclaimed water treatment than in the clean water treatment. The N treatments significantly influenced the diversity of microbial communities. The higher richness in the reclaimed water treatment with middle N treatments could be forecasted owing to the suitable soil micro-environment for microorganisms growth provided by the reclaimed water and fertilizer. The activity of heterotrophic bacteria was obviously suppressed by low and high concentrations of N under reclaimed water irrigation. In our study, soil bacterial community clustering was better correlated with irrigation water type than with N fertilizer treatments (Fig. 3-A and B). These results showed that irrigation water type have greater impacts on the structure of microbial communities than fertilizer treatments. Reclaimed water was rich in nutrients, which was beneficial to the growth of microorganisms, and the rate of nutrient circulation and energy flow in the root layer soil was enhanced, the change of soil micro ecological environment was conducive to microbial growth (Brussaard 1994; Oved et al. 2001). The soil irrigated with reclaimed water was dominated by the Proteobacteria (36.91% of amplicons). The soil irrigated with clean water, with a slower decomposition rate of soil carbon, was dominated by the Acidobacteria (6.28% of amplicons), and the Acidobacteria had an obvious inhibitory effect on organic matter mineralization, which was similar to

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the study of Guo et al. (2015). The reclaimed water tended to increase the growth of Proteobacteria, Gemmatimonadetes, and Bacteroidetes, which is in agreement with previous studies (Ahn et al. 2012; Qiu et al. 2012; Becerra-Castro et al. 2015). Gemmatimonadetes had a strong denitrification function, and Bacteroidetes was the main contributor to the mineralization of organic matter (Guo et al. 2015). The microbial populations were intrinsically able to cope with the reclaimed water irrigation. Bacteria such as Acidobacteria-6 and Pseudomonadales were abundant in soil under clean water irrigation, while Pseudomonas can cause human infections by colonization of human organs and tissues (Berg et al. 2005). Acidobacteria are capable of degrading structural carbon (Russo et al. 2012).

4.3. Soil-related differences in phylogenetic community structure The bacterial community structure in the soil was significantly related to the soil chemical properties, indicating that the soil chemical composition affects the soil microbiome. In this study, NO3–-N, TP and EC influenced the structure of the soil microbial community in the reclaimed water treatment, and the TP, pH and OM had a large effect on the soil microorganisms in the clean water treatment. The structure of the soil microbial community showed a certain spatial difference with the change in chemical indicators: NO3–-N and TP had a larger effect on Verrucomicrobia and Gemmatimonadetes, while pH and OM had a larger influence on Firmicutes and Actinobacteria. The characteristics of the structure of the soil microbial community were strongly influenced by soil quality. Although this study cannot disentangle these complex SC-N feedbacks at different N levels under reclaimed water irrigation, the measurement and monitoring of soil microbial communities might allow us to better understand their composition and function in ecosystem processes. Meanwhile, it provides an initial theoretical basis for interpreting the mechanisms affecting the pedologic and biogeochemical variance in the bacterial community structure. Furthermore, the soil microbial community also were affected by the water type used for irrigation. Therefore, a change in the origin or composition of the reclaimed water could influence and give rise to a different microbial community structure.

5. Conclusion In this study, reclaimed water irrigation increased the soil EC. A decreasing trend of soil organic matter with higher N level was related to the increased soil respiration with N topdressing. Reclaimed water tended to increase the numbers of

Proteobacteria in soil, while clean water tended to increase the numbers of Acidobacteria in soil. Furthermore, the experiment indicated that the type of irrigation water had a greater influence on the structure of soil microbial community than N fertilizer treatment. Meanwhile, the characteristics of the bacterial community structure were strongly influenced by soil chemical properties and demonstrated a direct relationship between the soil properties and the soil microbiome. Especially, NO3–-N, TP and EC were the important factors which had a significant impact on soil microbial community structure under irrigation with reclaimed water.

Acknowledgements The authors would like to extend their sincere gratitude to the Agriculture Water and Soil Environment Field Science Research Station, China, for the permission to conduct the research. We would like to acknowledge the financial support for this research from the National High-Tech R&D Program of China (2012AA101404) and the National Natural Science Foundation of China (51209208, 51479201). Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

References Adrover M, Farrús E, Moyà G, Vadell J. 2012. Chemical properties and biological activity in soils of Mallorca following twenty years of treated wastewater irrigation. Journal of Environmental Management, 95, 188–192. Ahn J H, Song J, Kim B Y, Kim M S, Joa J H, Weon H Y. 2012. Characterization of the bacterial and archaeal communities in rice field soils subjected to long-term fertilization practices. Journal of Microbiology, 50, 754–765. Bao S D. 2000. The Soil Agrochemical Analysis. 3rd ed. China Agriculture Press, Beijing. pp. 25–38. (in Chinese) Becerra-Castro C, Lopes A R, Vaz-Moreira I, Silva E F, Manaia C M, Nunes O C. 2015. Wastewater reuse in irrigation, A microbiological perspective on implications in soil fertility and human and environmental health. Environment International, 75, 117–135. Berg G, Eberl L, Hartmann A. 2005. The rhizosphere as a reservoir for opportunistic human pathogenic bacteria. Environmental Microbiology, 7, 1673–1685. Biggs T W, Jiang B B. 2009. Soil salinity and exchangeable cations in a wastewater irrigated area, India. Journal of Environmental Quality, 38, 887–896. Blanchard M, Teil M J, Ollivon D, Garban B, Chestérikoff C, Chevreuil M. 2001. Origin and distribution of polyaromatic hydrocarbons and polychlorobiphenyls in urban effects to wastewater treatment plants of the Paris area (FRANCE). Water Research, 35, 3679–3687. Brussaard L. 1994. An appraisal of the dutch programme

GUO Wei et al. Journal of Integrative Agriculture 2017, 16(3): 679–690

on soil ecology of arable farming systems (1985–1992). Agriculture, Ecosystems & Environment, 51, 1–6. Caporaso J G, Lauber C L, Walters W A, Berg-Lyons D, Lozupone C A, Turnbaugh P J, Knight R. 2011. Global patterns of 16s rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences of the United States of America, 108 (Suppl 1), 4516–4522. Chen W P, Lu S D, Jiao W T, Wang M, Chang A C. 2013. Reclaimed water: A safe irrigation water source? Environmental Development, 8, 74-83. Chen W P, Lu S D, Pan N, Wang Y C, Wu L S. 2015. Impact of reclaimed water irrigation on soil health in urban green areas. Chemosphere, 119, 654–661. Cox P M, Betts RA, Jones C D, Spall S A, Totterdell I J. 2000. Acceleration of global warming due to carboncycle feedbacks in a coupled climate model. Nature, 408, 184–187. Cramer W, Bondeau A, Woodward F I, Prentice I C, Betts R A, Brovkin V, Young-Molling C. 2001. Global response of terrestrial ecosystem structure and function to CO2 and climate change, results from six dynamic global vegetation models. Global Change Biology, 7, 357–373. Dimitriu P A, Prescott C E, Quideau S A, Grayston S J. 2010. Impact of reclamation of surface-mined boreal forest soils on microbial community composition and function. Soil Biology Biochemistry, 42, 2289–2297. Drancourt M, Raoult D. 2005. Sequence-based identification of new bacteria, a proposition for creation of an orphan bacterium repository. Journal of Clinical Microbiology, 43, 4311–4315. Duncan R R, Carrow R N, Huck M T. 2009. Understanding irrigation water quality tests. In: Turfgrass and Landscape Irrigation Water Quality: Assessment and Management. CRC, Boca Raton, Florida. pp. 39–68. Edgar R C. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics, 26, 2460–2461. Federle T W, Dobbins D C, Thornton-Manning J R, Jones D D. 1986. Microbial biomass, activity, and community structure in subsurface soils. Ground Water, 24, 365–374. Friedel J K, Langer T, Siebe C, Stahr K. 2000. Effects of long-term waste water irrigation on soil organic matter,soil microbial biomass and its activities in central Mexico. Biology and Fertility of Soil, 31, 414–421. Guo Y H, Gong H L, Guo X Y. 2015. Rhizosphere bacterial community of Typha angustifolia L. and water quality in a river wetland supplied with reclaimed water. Applied Microbiology and Biotechnology, 99, 2883–2893. Hanjra M A, Blackwell J, Carr G, Zhang F, Jackson T M. 2012. Wastewater irrigation and environmental health, implications for water governance and public policy. International Journal of Hygiene and Environmental Health, 69, 215–255. He J Z, Shen J P, Zhang L M, Zhu Y G, Zheng Y M, Xu M G, Di H J. 2007. Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria and ammoniaoxidizing archaea of a Chinese upland red soil under long-

689

term fertilization practices. Environmental Microbiology, 9, 2364–2374. Hidri Y, Bouziri L, Maron P A, Anane M, Jedidi N, Hassan A, Ranjard L. 2010. Soil DNA evidence for altered microbial diversity after long-term application of municipal wastewater. Agronomy for Sustainable Development, 30, 423–431. Hidri Y, Fourti O, Eturki S. Jedidi N, Charef A, Hassen A. 2014. Effects of 15-year application of municipal wastewater on microbial biomass, fecal pollution indicators, and heavy metals in a Tunisian calcareous soil. Journal of Soil and Sediments, 14, 155–163. Janda J M, Abbott S L. 2007. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory, pluses, perils, and pitfalls. Journal of Clinical Microbiology, 45, 2761–2764. Johnsen K, Jacobsen C S, Torsvik V, Sørensen J. 2001. Pesticide effects on bacterial diversity in agricultural soils - A review. Biology and Fertility of Soils, 33, 443–453. Katz B G, Griffin D W, Davis J H. 2009. Groundwater quality impacts from the land application of treated municipal wastewater in a large karstic spring basin: Chemical and microbiological indicators. Sciences of the Total Environment, 407, 2872–2886. Kuzyakov Y, Friedel J K, Stahr K. 2000. Review of mechanisms and quantification of priming effects. Soil Biology and Biochemistry, 32, 1485–1498. Li H, Zhang Y, Zhang C G, Chen G X. 2005. Effect of petroleum containing waste water irrigation on bacterial diversities and enzyme activities in a paddy soil irrigation area. Journal of Environmental Quality, 34, 1073–1080. Li J, Li Y T, Yang X D, Zhang J J, Lin Z A, Zhao B Q. 2015. Microbial community structure and functional metabolic diversity are associated with organic carbon availability in an agricultural soil. Journal of Integrative Agriculture, 14, 2500–2511. Li J J, Zheng Y M, Yan J X, Li H J, Wang X, He J Z, Ding G W. 2013. Effects of different regeneration scenarios and fertilizer treatments on soil microbial ecology in reclaimed opencast mining areas on the loess plateau, China. PLOS ONE, 8, doi: org/10.1371/journal.pone.0063275 Liu W H, Zhao J Z, Ouyang Z Y, Söderlund L, Liu G H. 2005. Impacts of sewage irrigation on heavy metal distribution and contamination in Beijing, China. Environment International, 31, 805–812. Lyu S D, Chen W P. 2016. Soil quality assessment of urban green space under long-term reclaimed water irrigation. Environmental Science and Pollution Research, 23, 4639–4649. Mancino C F, Pepper I L. 1992. Irrigation of turfgrass with secondary sewage effluent, soil quality. Agronomy Journal, 84, 650–654. Martinez C J, Clark M W, Toor G S, Hochmuth G J, Parsons L R. 2011. Accounting for the nutrients in reclaimed water for landscape irrigation. Agricultural and Biological Engineering, AE479, 1–8. Micks P, Aberb J D, Boone R D, Davidson E A. 2004. Short-

690

GUO Wei et al. Journal of Integrative Agriculture 2017, 16(3): 679–690

term soil respiration and nitrogen immobilization response to nitrogen applications in control and nitrogen-enriched temperature forests. Forest Ecology and Management, 196, 57–70. Mohan T V K, Nancharaiah Y V, Venugopalan V P, Sai P M S. 2016. Effect of C/N ratio on denitrification of high-strength nitrate wastewater in anoxic granular sludge sequencing batch reactors. Ecological Engineering, 91, 441–448. Nei M. 1978. Estimation of average heteroxygosity and genetic distance from a small number of individuals. Genetics, 19, 583–590. Nicolás E, Alarcón J J, Mounzer O, Pedrero F, Nortes P A, Alcobendas R, Romero-Trigueros C, Bayona J M, MaestreValero J F. 2016. Long-term physiological and agronomic responses of mandarin trees to irrigation with saline reclaimed water. Agricultural Water Management, 166, 1–8. Oved T, Shaviv A, Goldrath T, Mandelbaum R T, Minz D. 2001. Influence of effluent irrigation on community composition and function of ammonia-oxidizing bacteria in soil. Applied and Environmental Microbiology, 67, 3426–3433. Qian Y L, Mecham B. 2005. Long-term effects of recycled wastewater irrigation on soil chemical properties on golf course fairways. Agronomy Journal, 97, 717–721. Qiu M H, Zhang R F, Xue C, Zhang S S, Li S Q, Zhang N, Shen Q. 2012. Application of bio-organic fertilizer can control Fusarium wilt of cucumber plants by regulating microbial community of rhizosphere soil. Biology and Fertility of Soils, 48, 807–816. Resh S C, Binkley D, Parrotta J A. 2002. Greater soil carbon

sequestration under nitrogen-fixing trees compared with Eucalyptus species. Ecosystems, 5, 217–231. Russo S E, Legge R, Weber K A, Brodie E L, Goldfarb K C, Benson A K, Tan S. 2012. Bacterial community structure of contrasting soils underlying Bornean rain forests, Inferences from microarray and next-generation sequencing methods. Soil Biology and Biochemistry, 55, 48–59. Shaver G R, Canadell J, Chapin F S, Gurevitch J, Henry G, Ineson P, Jonasson S, Melillo J, Pitelka L, Ruatad L. 2000. Global warming and terrestrial ecosystems, a conceptual framework for analysis. Bioscience, 50, 871–882. Ter Braak C J F, Smilauer P. 2002. CANOCO Reference manual and canodraw for windows user’s guide software for canonical community ordination (version 45). In: Microcomputer Power. Wageningen Biometris, Ithaca NY, USA. Thayanukul P, Kurisu F, Kasuga I, Furumai H. 2013. Evaluation of microbial regrowth potential by assimilable organic carbon in various reclaimed water and distribution systems. Water Research, 47, 225–232. Xu J, Wu L S, Chang A C, Zhang Y. 2010. Impact of long-term reclaimed waste water irrigation on agricultural soils: a preliminary assessment. Journal of Hazardous Materials, 183, 780–786. Zhang Q, Wang G, Yao H. 2007. Phospholipid fatty acid patterns of microbial communities in paddy soil under different fertilizer treatments. Journal of Environmental Sciences, 19, 55–59. (Managing editor SUN Lu-juan)