Effects of different leaf litters on the physicochemical properties and bacterial communities in Panax ginseng-growing soil

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Applied Soil Ecology xxx (2016) xxx–xxx

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Effects of different leaf litters on the physicochemical properties and bacterial communities in Panax ginseng-growing soil Hai Sun, Qiu-xia Wang, Ning Liu, Le Li, Chun-ge Zhang, Zheng-bo Liu, Ya-yu Zhang* Institute of Special Animal and Plant Science of Chinese Academy of Agricultural Science, 130112, Changchun, China

A R T I C L E I N F O

Article history: Received 1 August 2016 Received in revised form 14 November 2016 Accepted 14 November 2016 Available online xxx Keywords: Soil microbial community 16S rDNA Soil characteristics Leaf litter Panax ginseng

A B S T R A C T

Leaf litters play a very significant role in determining soil physicochemical properties and shaping soil microbial communities in forest ecosystems, but their impact on understory wild ginseng soil is unknown. In order to study that, different leaf litters from five tree species ((A) Acer mono. Maxim. var. mono; (B) Pinus densiflora Sieb. et Zucc.; (C) Juglans mandshurica Maxim.; (D) Tilia amurensis Rupr.; (E) Quercus mongolica Fisch. ex Ledeb) were added to Panax ginseng-growing soil. Our results indicated that the physicochemical properties of soil were significantly affected by all the leaf litter treatments. Soil total nitrogen, available NPK, and soil microbial biomass (carbon and nitrogen) significantly (P < 0.05) increased across all treatments. In addition, we found that the soil bulk density and C/N ratio was lower following all treatments than in the control (no addition of leaf litter). Although the different kinds of added leaf litter had few effects on bacterial diversity and abundance, significant changes in the bacterial community composition could be identified in all soils; specifically, the relative abundance of Proteobacteria was higher in treatments than in the control. In addition, the bacterial communities of Bacteroidetes were fewer in treatments with coniferous leaf litter than those with broad leaf litter (P < 0.05). Canonical discriminant analysis (CDA) ascertained that the shift of bacterial community composition and diversity were closely related with the changes in soil microbial biomass carbon and available nitrogen in all treatment soils. Our experiment results suggest that addition of leaf litter has a significant impact on soil bacterial community development, and it can lead to higher soil nutrients and soil microbial biomass, as well as a different bacterial community composition. ã 2016 Elsevier B.V. All rights reserved.

1. Introduction Panax ginseng C. A. Meyer, a perennial herb, is one of the most well-known Chinese traditional medicines. It has been consumed orally to promote general health such as immunity enhancement, in addition to its anti-aging, anti-inflammatory, and anti-fatigue effects (Kennedy and Scholey, 2003; Gu et al., 2009; Kim et al., 2011). Due to its extremely high medicinal value, ginseng has been artificially cultivated in large areas. There are two kinds of cultivation models in China, understory wild ginseng (UWG) and farmland cultivated ginseng (FCG) (Sun et al., 2015). UWG is a cultivation model without any human interference, and has developed rapidly owing to the high quality of products and the growing mindset of “returning to the true nature.” Previous studies

* Corresponding author. E-mail address: [email protected] (Y.-y. Zhang).

demonstrated that the seedling survival rate, red-skin root disease, and the quality and morphogenesis of ginseng root are affected by the forest soil ecological environment (Gao et al., 2014; Jang et al., 2015; Lee et al., 2016a, 2016b; Zhang et al., 2011). Hence, it is very critical to choose a suitable site for P. ginseng planting in a forest environment. Several factors (soil-physical and chemical, associated trees species, the mountain slope, attitude and so on) that affect ginseng growth and ginsenoside accumulation need to be considered. On top of that, tree species are vital to soil fertility and ginseng root morphogenesis (Li et al., 2013). Firstly, dry branches and fallen leaves are a major supplemental source of organic matter and nutrients; they constitutes a major step in carbon and nitrogen cycling between plants and soil (Suthar and Gairola, 2014; Li et al., 2015). Secondly, leaf litters contain a large amount of carbon and nitrogen, and serve as a nutrient source for microbial activities in soil (Wang et al., 2014). Adding leaf letter to soil has a positive effect on the soil microbial community (SMC), owing to an increase

http://dx.doi.org/10.1016/j.apsoil.2016.11.008 0929-1393/ã 2016 Elsevier B.V. All rights reserved.

Please cite this article in press as: H. Sun, et al., Effects of different leaf litters on the physicochemical properties and bacterial communities in Panax ginseng-growing soil, Appl. Soil Ecol. (2016), http://dx.doi.org/10.1016/j.apsoil.2016.11.008

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in carbon and nutrient input alternate soil microclimate. (Fanin and Bertrand, 2016). Many studies have indicated that fallen leaves were a supplement for soil carbon and nitrogen, and the decomposition of tree leaves played a critical role in the releasing of nutrients for plant growth as well as carbon cycling (Hristovski et al., 2014). Microorganisms are highly diverse and ubiquitous in soil ecosystems; they participate in a variety of key ecosystem functions such as nitrogen cycling, mineralization, contribution and preservation of soil organic matter, feeding back responses to climate change and biomass production (Puissant et al., 2015; Wassila et al., 2015; Nguyen et al., 2016). A stable microbial community contributes essentially to stabilizing soil structure and maintaining soil ecosystem services (Nazaries et al., 2015). Therefore, several investigations on discontinuous cultivation in FCG, disease resistance in UWG, and ginseng root morphogenesis have focused on the evaluation of soil microbial community (Ying et al., 2012; Li et al., 2014). Interestingly, several findings from previous studies have illustrated that continuous planting changed the microbial diversity in the rhizosphere soil and the soil of UWG enhanced the proportion of anti-pathogenic microorganisms (You et al., 2012; Xiao et al., 2016). Despite the widely accepted fact that the accumulation of fallen tree leaves could change the soil properties and affect microbial activities, most experiments performed in the field had different kinds of leaf mixtures (Wang et al., 2014; Zhou et al., 2015). Leaves from different trees release different compounds and different quantities as the leaves are different in their chemical composition (Bayarsaikhan et al., 2016). The litter decomposition is a key channel which affects the nutrients availability, and the extracellular enzyme secreted by soil microbes are important to decomposition and nutrient cycling, closely related to the biogeochemical and microbiological properties of ecosystems (Allison et al., 2006; DeForest, 2009; Ge et al., 2010; Chaparro et al., 2012). Several experimental studies have indicated that external substrate addition can alter soil microbial community structure (de Vries et al., 2006; Moore-Kucera and Dick, 2008; Denef et al., 2009; Dungait et al., 2011), which may consequently affect the magnitude and direction of SOC mineralization and change C flow within the soil microbial community (Williams et al., 2006; Garcia-Pausas and Paterson, 2011; Yao et al., 2012). However, specific tree leaves might be key factors for affecting change in the soil microbial community, so it is necessary to study whether the microbial community changes after adding tree leaves of different species. The aim of this study was to understand the changes in soil property, soil bacterial abundance, diversity, and community composition by adding tree leaves of different species, followed by investigation of the relationship between soil properties and soil microbial community. Thus, a single-factor pot culture experiment was performed in a greenhouse to test the effects of fallen tree leaves on soil microorganisms. The species were chosen based on the common distribution of trees in the fields where UWG is practiced. We hypothesized that: (1) physical and chemical properties of the soil are affected by added tree leaves (Acer mono. Maxim. var. mono, Pinus densiflora Sieb. et Zucc., Juglans mandshurica, Tilia amurensis Rupr., Quercus mongolica Fisch. ex Ledeb). Specifically, P. ginseng should grow faster on leaves with lower bulk density, higher TC, TN, SMBC, SMBN, and available NPK; (2) the bacterial abundance, diversity, and community composition might be affected by different leaves, and the changes might influence the soil microbial biomass carbon (SMBC) and soil microbial biomass nitrogen (SMBN); (3) because the soil nutrients increment are different by adding different leaf litter, the size and structure of the microbial community would be the key factors in determining the leaf-litter decomposition and wild understory ginseng management.

2. Materials and methods 2.1. Collection of potting soil and tree leaves The soil and leaf samples were collected from the base of a UWG site (E: 127 75’78”, N: 42 81’12”), which is a major ginseng producing base in the Jilin province of Northeastern China during October 2014. Soil from a depth of 20 cm was collected from a farmland that had been abandoned for 3 years, and passed through a 0.9 mm soil sieve. Tree leaves were collected from under the trees of Acer mono. Maxim. var. mono, Pinus densiflora Sieb. et Zucc., Juglans mandshurica, Tilia amurensis Rupr., and Quercus mongolica Fisch. ex Ledeb in the same zone during the withering season. The leaves were sorted by species and dried in a forced-air oven for one week at 35  C, then smashed and passed through a 0.01 mm sieve. 2.2. Experimental design and soil sampling 2.2.1. Content of the leaves The experiment was conducted at the base of UWG to obtain the amount of litter leaves per unit area, according to the method of Zhang et al. (2008) with slight modification. Five sites were marked and leaves within a 10 m2 area were collected in the month of October from 2011 to 2013. The leaves were dried in a forced air oven for one week at 35  C and weighed after they achieved constant weight. The amount of leaves within one square meter was then calculated. The amount of added leaves to the soil was 5.0 g leaves per pot, according to the calculated results. 2.2.2. Experimental design and soil sample preparation A pot culture experiment based on completely randomized design(CRD), and conducted from October 2013 to March 2014 at the Institute of Special Animal and Plant Sciences of CAAS, Changchun, China. Black PVC pots of 120 mm diameter and 180 mm height were used, and five three-year-old ginseng seedlings, which were consistent in appearance, size, shape, and weight, were planted per pot. The ginseng seedlings which were covered with 1.5 cm depth soil were cultivated in pot. Five tree species were included in this experiment: (A) Acer mono. Maxim. var. mono, (B) Pinus densiflora Sieb. et Zucc., (C) Juglans mandshurica Maxim., (D) Tilia amurensis Rupr., (E) Quercus mongolica Fisch. ex Ledeb. Leaf litter (dry weight: 5.0 g) from five tree species was added to the soil of each pot. Pots with no added leaf litter were used as controls (F). Each pot was considered a replicate, and each treatment consisted of 3 replicates. The mean minimum air temperature and maximum air temperature during the experiment were 17  C and 28  C respectively, and the relative humidity maintained 70%-80% in greenhouse during the experiment. All the treatments were watered 1L distilled water every week. Twenty grams of soil from each ginseng root zone was collected in March 2014 after a reproductive cycle. Five ginseng root zone soils from one pot were mixed to yield a composite sample. All soil samples were passed through a 2-mm sieve. Samples for chemical property analysis were air-dried, whereas the others were stored at 80  C for subsequent DNA extraction. 2.3. Physiochemical analysis of soil Soil chemical properties were determined according to the method described by the Analysis Method of Soil Agricultural Chemistry (Lu, 2000). Briefly, soil bulk density was determined using general methods after drying at 105  C; the soil pH (1:5 soil: water suspension) was measured using a glass electrode (SK220, Switzerland). Soil organic carbon (SOC) was measured using the K2Cr2O7 oxidation-reduction colorimetric method. Total nitrogen

Please cite this article in press as: H. Sun, et al., Effects of different leaf litters on the physicochemical properties and bacterial communities in Panax ginseng-growing soil, Appl. Soil Ecol. (2016), http://dx.doi.org/10.1016/j.apsoil.2016.11.008

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(TN) and soil organic carbon to nitrogen ratio (C/N) were determined via a dry combustion method using an Element Analyzer (Vario EL, Germany). Ammonium nitrogen (NH4+-N) was extracted with 0.01 M CaCl2, and the concentrations were determined with an Auto Analyzer (Auto Analyzer 3, Germany). Available potassium (K) in the soil was extracted with ammonium acetate and its concentration was determined by flame photometry. Available phosphorus (P) in the soil was extracted with sodium bicarbonate and its concentration was determined using the molybdenum blue method. Soil microbial biomass carbon (SMBC) and nitrogen (SMBN) were determined by fumigation-extraction (Vance et al., 1987) and it was calculated using a KC factor of 0.38 and a KN factor of 0.45, respectively (Joergensen and Mueller, 1996). 2.4. Bacterial DNA extraction and 16S rDNA amplification Total soil genomic DNA was extracted using the Power Soil DNA isolation Kit (MoBio laboratories, Carlsbad, CA, USA), following the manufacturer's instructions. DNA concentration and purity were checked on 1% agarose gels, and DNA was diluted to a 1 ng/mL working stock. The quantity and quality of extracted DNA were checked photometrically using a NanoDrop ND-1000 UVeVis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The V3-V4 region of 16S rDNA gene (300–350 bp) was amplified with a broadly conserved primer set, 341F (CCT AYG GGR BGC ASC AG) and 806R (GGA CTA CNN GGG TAT CTA AT) (Michelsen et al., 2014). The reverse primer contained a 6-bp error-correcting barcode unique to each sample. All PCR reactions were carried out in 30 mL reaction volumes containing 15 mL of Phusion HighFidelity PCR Master Mix New England Biolabs, Ipswich, MA, USA), 0.2 mM forward and reverse primers, and 10 ng template DNA. Thermal cycling conditions were: 98  C for 1 min, followed by 30 cycles of 98  C for 10 s, 50  C for 30 s, and 72  C for 30 s, with a final extension at 72  C for 5 min. Resulting amplicons were confirmed on 2% agarose gels containing ethidium bromide. PCR reactions were performed following the protocol described in Charlotte et al. (2014). Amplicon sequencing was conducted on an Illumina MiSeq platform at Novogene co., Beijing, China. All amplicons were in the size range of 400–450 bp, and were purified using a GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). Following quantitation, equal concentrations of the purified amplicons were combined into a single tube. Sequencing libraries were generated using a NEB Next Ultra DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) following manufacturer’s recommendations, and index codes were added. The library quality was assessed on a Qubit 2.0

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Fluorometer (Thermo Fisher Scientific, Carlsbad, CA, USA) and Agilent Bioanalyzer 2100 system. Sequencing was conducted on an Illumina MiSeq platform, which generated 300-bp paired-end reads. 2.5. Data analysis and statistics The soil physicochemical data were analyzed using SAS version 9.1 (SAS institute Inc., Cary, NC, USA), the significant difference at 0.05 level among different treatment was performed by one-way analysis of variance (ANOVA). Sequencing reads from the data set were trimmed, quality-controlled, and aligned. Operational taxonomic units (OTUs) were clustered at 97% identity using the UPARSE software (UPARSE v7.0.1001, http://drive5.com/uparse/) (Burns et al., 2015).). Taxonomic classification was conducted using the Ribosomal Database Project. The phylogenetic measure of beta diversity, i.e., weighted UniFrac distance, was calculated using Quantitative Insights into Microbial Ecology (QIIME) (Version 1.7.0) (Chang et al., 2011). Principal Component analysis (PCA) based on weighted UniFrac metric matrices was performed to explore the differences in bacterial across all the soil samples. Canonical discriminant analysis (CDA) was performed using CANOCO 5.0 for windows, to examine the relationships between the frequencies of abundant phyla and soil environmental parameters (Drenovsky et al., 2010; Heikkinen et al., 2004). 3. Results 3.1. Variations in soil characteristics The physicochemical characteristics of soil are presented in Table 1. After one reproductive cycle of 3-year-old ginseng, the five treatments (A, B, C, D, and E) had soil pH values greater than 6, except for F, which had a pH value of 5.91; the values for treatments B, C, and D were significantly (P < 0.05) higher than the control (F). Soil bulk density decreased from 0.96 g soil cm 3 in the control (F) to 0.90, 0.95, 0.94, and 0.95 g soil cm 3 in the treatments A, C, D, and E, respectively. The average soil SMBC, SMBN, and available NPK increased significantly (P < 0.05) in all the leaf-added treatments. The average soil SMBC were significantly (P < 0.05) higher in treatments A, B, and C than that of treatments D, and E. The average soil SMBN was most highest in treatment E, followed by treatments A, D, C, and B, and significantly different at 0.05 level in treatments A, B, and E. Soil SOC and TN in the leaf-added treatments varied from 20.20 to 28.90 g kg 1 soil and from 2.66 to 4.11 g kg 1 soil, respectively, and were higher than that of control. Soil microbial biomass carbon (SMBC), which serves as an important indicator for soil microbial biomass accumulation, was

Table 1 Soil characteristic of adding different tree leaves. Treatment

A

B

C

D

E

F

pH BD (g cm 3) SOC(g/kg) TN(g/kg) SMBC mg/kg SMBN mg/kg C/N AN mg/kg AP mg/kg AK mg/kg

6.24  0.07 cd 0.90  0.01 21.27  0.20 b 3.20  0.03 bc 145.90  1.90 a 16.14  0.48 b 6.65  0.04 d 188.38  0.59 a 29.33  0.38 d 206.86  1.12 b

6.68  0.14 a 0.96  0.04 20.20  0.09 ab 2.66  0.14 cd 142.89  1.27 a 8.83  0.72 d 7.64  0.40 cd 164.87  1.12 d 32.07  0.52 c 140.71  2.59 d

6.19  0.03 c 0.95  0.04 25.19  0.14 ab 3.18  0.01 bc 143.62  3.59 a 10.77  0.27 cd 7.91  0.08 ab 185.46  1.14 a 30.66  0.35 cd 185.07  3.70 c

6.44  0.03 bc 0.94  0.05 24.38  0.91 ab 3.27  0.02 b 135.37  3.70 b 11.68  0.30 c 7.46  0.29 bc 173.05  0.71b 37.59  0.09 a 142.51  3.43 d

6.57  0.02 ab 0.95  0.05 28.90  0.54 a 4.11  0.06 a 126.48  0.49 c 31.32  0.47 a 7.04  0.21 cd 169.75  0.98 c 52.80  1.26 a 128.67  0.76 e

5.91  0.24 d 0.96  0.01 20.66  0.32 b 2.48  0.11 d 115.08  0.76 d 7.40  0.11 e 8.35  0.31 a 156.21  1.16 e 24.66  0.43 e 216.23  1.31 a

Note: Data are means  standard error (n = 3), * means between the treatment of added leaves (A,B,C,D or E) with control (F) are significant at P < 0.05. pH: pH value in water (1:2.5) BD: bulk density; BD: bulk density (g dry soil per 200 cm 3 volume); SOC: soil organic carbon(g kg 1 dry soil); TN: total nitrogen(g kg 1 dry soil); SMBC: soil microbial biomass carbon (mg kg 1 dry soil); SMBN: soil microbial biomass nitrogen (mg kg 1 dry soil); C/N: carbon to nitrogen ratio; AN: available nitrogen (mg kg 1 dry soil); AP: available phosphorus (mg kg 1 dry soil); AK: available potassium (mg kg 1 dry soil).

Please cite this article in press as: H. Sun, et al., Effects of different leaf litters on the physicochemical properties and bacterial communities in Panax ginseng-growing soil, Appl. Soil Ecol. (2016), http://dx.doi.org/10.1016/j.apsoil.2016.11.008

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obviously affected by the addition of leaf litter (Table 2). The average SMBC increased significantly (P < 0.05) from 115.08 mg C kg 1 soil in the control (F) to 145.90, 142.89, 143.62, 135.37, and 126.48 mg C kg 1 soil in the treatments A, B, C, D, and E, respectively. A similar increase was observed in SMBN as well; the average SMBN increased significantly (P < 0.05) from 7.40 mg C kg 1 soil in the control (F) to 16.14, 8.49, 10.77, 11.68, and 31.32 mg C kg 1 soil in the treatments A, B, C, D, and E, respectively. The average of soil available NPK were remarkable (P < 0.05) different in all treatments. The average soil available nitrogen were most highest in treatment A, followed by the treatments C, D, E, B, and F, and the soil available phosphorus were most highest in treatment E, followed by the treatments D, B, C, A, and F. However, the average soil available potassium was significantly (P < 0.05) decreased in all the leaf-added treatments, and it decreased from 216.23 mg K kg 1 soil in the control (F) to 208.86, 140.71, 185.07, 142.51, and 128.67 mg K kg 1 soil in the treatments A, B, C, D, E, and F, respectively. 3.2. Differentiations in bacterial abundance and diversity 3.2.1. Richness, observed species, and chao 1 and shannon indices for bacterial abundance In total, we obtained 1478922 DNA sequences from 18 samples, with a median read length of 418 bp. After quality filtering, 1367082 sequences remained, with 29572–236044 sequences obtained per sample (average = 75949 sequences). All valid reads were classified from phylum to genus according to QIIME, using the default settings. The taxonomic distribution at phylum level is summarized in Fig. 1. These sequences were assigned to 36 phyla as shown, and Proteobacteria was the most abundant phylum across all samples, accounting for 35.02–45.44% of the total valid reads in all samples, with an average relative abundance of 40.88%. Acidobacteria was the second most abundant phylum across all samples, accounting for 6.26–58.68% of the total valid reads in all samples, with an average relative abundance of 15.59%. The other dominant phyla were Actinobacteria (4.66–36.58%, averaging at 11.55%), Gemmatimonadetes (2.56–30.86%, averaging at 7.36%), Chloroflexi (1.44–15.61%, averaging at 4.17%), Crenarchaeota (0.39– 8.28%, averaging at 2.42%), and Nitrospirae (0.90–7.77%, averaging at 2.23%). At the class level, a wide range of classes dominated. Based on the average relative abundance, the most abundant classes were Alphaproteobacteria, Acidobacteria, Betaproteobacteria, Deltaproteobacteria, and Actinobacteria. At the order level, a total of 18 orders were dominant (>1% across all soil samples). Based on the average abundance, iii1-15 (8.45%) was the most abundant relative order, followed by Rhizobiales (5.95%), Actinomycetales (5.39%), Myxococcales (4.70%), Sphingomonadales (4.45%), Rhodospirillales

Fig. 1. Taxonomic classification of bacterial reads of soil samples with different leaf addition treatments at the phylum level, using RDP classifier. Error bars represent standard errors (n = 3).

(4.05%), Xanthomonadales (3.71%), Burkholderiales (2.18%), Acidimicrobiales (2.70%), Nitrososphaerales (2.38%), and Nitrospirales (2.23%), in the descending order. The average number of observed species ranged from 2422 to 2660, based on the numbers at 97% similarity. Chao 1 richness estimate and the Shannon index ranged from 3030 to 3602 and from 9.845 to 9.994, respectively, across all soil samples (Fig. 2). The average number of observed species, and Chao 1 and Shannon indices in the leaf-added treatments were slightly higher than control, although the differences between them were not significant (P > 0.05). 3.2.2. Community profiling of the bacterial communities by illumine sequencing The similarities among the microbial communities in the six leaf-added treatment soils were evaluated using cluster analysis (Fig. 3a). Cluster analysis indicated that the bacterial communities could be clustered into four groups: Group I contained samples E and F, Group II contained samples A and C, Group III contained sample D, and Group IV contained sample B. Additionally, samples A, C, and D were grouped together with high support. Meanwhile, samples A, C, D, and B were also relatively close. This grouping was further confirmed by PCA analysis (Fig. 3b). This pattern indicated that bacterial community structure might be direct correlated with trees species.

Table 2 Pearson’s correlation coefficients between soil characteristics and abundant (dominant phylum, relative abundance >1%).

Proteobacteria Acidobacteria Actinobacteria Gemmatimonadetes Firmicutes Crenarchaeota Chloroflexi Verrucomicrobia Nitrospirae Bacteroidetes Note:

***

AN

AP

AK

pH

BD

SMBC

SMBN

TN

TC

0.778*** 0.594** 0.270 0.306 0.217 0.2560 0.460 0.032 0.171 0.002

0.077 0.090 0.286 0.026 0.314 0.117 0.502* 0.061 0.058 0.310

0.101 0.093 0.385 0.106 0.099 0.109 0.237 0.016 0.051 0.011

0.297 0.067 0.506 0.0137 0.284 0.033 0.061 0.152 0.205 0.088

0.265 0.317 0.015 0.144 0.159 0.030 0.183 0.276 0.032 0.021

0.967*** 0.732** 0.379 0.327 0.504 0.171 0.515* 0.284 0.375 0.018

0.089 0.214 0.190 0.189 0.252 0.117 0.488 0.158 0.247 0.499*

0.103 0.004 0.339 0.106 0.257 0.228 0.371 0.212 0.259 0.191

0.109 0.075 0.182 0.047 0.466 0.265 0.283 0.381 0.270 0.330

correlation is significant at the 0.001 level,

**

correlation is significant at the 0.01 level,

*

correlation is significant at the 0.05 level.

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determine the relationship between environment parameters and community structure (Fig. 4). The length of an environmental parameter arrow indicated the strength of the environmental parameter to the overall microbial communities. As such, SMBC, SMBN, and AP concentrations appear to be the most important environmental parameters. As indicated by the CDA analysis of relative abundance at phylum level (average abundance >1%) and environmental parameters, Proteobacteria, Acidobacteria, Gemmatimonadetes, and Firmicutes were significantly affected by SMBC, SMBN, TC, and AP, while Crenarchaeota, Chloroflexi, Bacteroidetes, Actinobacteria, and Verrucomicrobia were significantly affected by AN, pH, and C/N. 4. Discussion 4.1. The effects of adding different leaves on the physicochemical properties of soil

Fig. 2. The number of observed species and the Chao1 and Shannon indices, error bars represent standards errors (n = 3).

3.3. Correlation analysis between bacterial community and soil characteristics Microbial community structure might be correlated with environmental parameters in the different leaf-added treatments. Therefore, CDA (canonical discriminant analysis) was used to

Lower bulk density and higher soil pH were observed when different leaf litters were added (Table 1). The pH value is one of the most important indicators while choosing suitable cultivating land for ginseng production (Kim et al., 2015). Likewise, soil bulk density directly affects the morphogenesis of ginseng root (Li, 1991). Previous studies suggested that ideal ginseng-growing conditions were: soil bulk density lower than 1 g/cm3, pH slightly acidic, and high nutrient concentration (You et al., 2014; Yang and Wu, 2016). Previous studies verified that continuous cropping of ginseng resulted in lower soil pH, decreased soil nutrient levels, and disordered soil microbial community structure (Jian et al., 2011). The changes in soil pH and bulk density by the treatment of added leaf litters might benefit ginseng growth via alleviation of harsh environmental conditions. Addition of leaves resulted in a significant increase for the average SMBC, SMBN, and available NPK, and there were differences among different tree species treatments. The release of nutrients from decomposing leaf litter is an important pathway for mineral cycles in a forest ecosystem (Hobbie, 2015; Li et al., 2015; Schmidt et al., 2015). The quantity and quality of soil nutrients were affected by tree species by the outputs of different leaves litter (Liu et al., 2016). Leaf litter served as the main source of SOC

Fig. 3. (a) Weighted Unifrac UPGMA cluster of microbial communities associated with different soil samples from different leaf-added treatments. The figure was constructed on the basis of Illumina sequencing data. (b) Principal Component Analysis (PCA) plot based on the 16S rDNA sequencing genes from eighteen soil samples. The scatter plot is of principal component 1 (PC1) vs. principal component 2 (PC2).

Please cite this article in press as: H. Sun, et al., Effects of different leaf litters on the physicochemical properties and bacterial communities in Panax ginseng-growing soil, Appl. Soil Ecol. (2016), http://dx.doi.org/10.1016/j.apsoil.2016.11.008

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Fig. 4. Canonical discriminant analysis biplots to investigate the ecological correlation between abundance of microorganisms and soil characteristics. (a) Sequences of 16S rDNA and environmental parameters; (b) based on the relative abundance at phylum level (average abundance >1%) and environmental parameters.

and altered the native SOC mineralization rate. The content of SOC, TN, and available NPK were increased by different degrees; the SOC was especially higher in treatments C, D, and E. This result was in agreement with the past findings that nutrient levels improve with increasing leaf litters (Wang et al., 2014; Li et al., 2015; Ravindran and Yang, 2014; Deng et al., 2015). One possible mechanism is that the mineralization of leaf litter by soil microorganisms generated one of the greatest C fluxes in the global C cycle (Schlesinger and Andrews, 2000) and returned nutrients back to soil, making them available for plant growth. Furthermore, microbes themselves have nutrient demands for their energy- and growth-related metabolism, and thereby affect the decomposition process. After adding leaf litter, significant increase in soil microbial biomass carbon and nitrogen contents were found in the present study, which was consistent with changes in the soil microbial biomass and community structure in forest soil (Mukhopadhyay and Joy, 2010; Suthar and Gairola, 2014). The reason was that the application of leaf litter offered a wide range of carbon source materials for soil microbes, and promoted microorganism reproduction (Hawke and Vallance, 2015). It is widely accepted that soil C/N is a key indicator of soil quality change, and its value could represent the nutrient utilization efficiency, and closely associated with carbon and nitrogen circulation as well as nutrient absorption by plant (Blair et al., 1970). This is supported by the fact that a low ratio of C/N is conducive to nutrient release during the process of organic matter decomposition by soil microbial community (Hawke and Vallance, 2015). Our findings of higher contents for available NPK and SOC in leaf-added soil might be a result of low C/ N ratios (<8 in all leaf-added soils and 8.36 in control), which promoted leaf decomposition by soil microorganisms (Yu et al., 2003; Zhang et al., 2003). 4.2. Response of the bacterial communities toward adding leaf litter to the soil Although the effects of adding leaf litter on microbial communities have been reported in many studies (Joly et al., 2015; Wang et al., 2015), no firm conclusions about the responses of microbial communities and their functions in such scenarios have been drawn. Leaf litter is regarded as a key determinant of soil

characteristics and soil microbial communities (Akhtar et al., 2016), and differences in soil basic characteristics can induce different microbial assemblages (Shi et al., 2016). However, the differences in bacterial communities, including abundances and composition, led by adding different leaf litter might be attributed to the differences in soil properties, especially SMBC, SMBN, AN, and AP. A previous study has justified that soil microbial biomass carbon and nitrogen were the dominant determinants, although other factors might also affect the bacterial community structure (Chu et al., 2016) . Although the six treatment soils encompassed different bacterial communities, the bacterial community compositions displayed inconsistent changes in response to the addition of different leaf litter across soil samples (Fig. 3b). The result that the addition of leaf litter altered soil microbial community was consistent with our hypothesis and some previous findings (Spohn and Chodak, 2014; Lewandowski et al., 2016). Apart from the community composition, whether the soil microbial abundance, diversity, and function, all respond similarly to added leaf litter in ginseng-growing soils, remains unknown. Bacterial abundance did not show a significantly increasing tendency as did SMBC; this might be explained by the variation in fungal communities, which also contribute to the leaf litter treatment of biomass carbon. Proteobacteria was a dominant group across all soils in our study. Our results that the relative abundance of Proteobacteria was higher in leaf-litter-added soil suggested that this microbial community was favored under high SMBC (126.48 mg kg 1–145.90 mg kg 1) and AN (164.87 mg kg 1– 186.38 mg kg 1) conditions, which is in agreement with previous studies (Mander et al., 2012; Huang et al., 2016) (Fig. 2). In addition, we also found that the relative abundance of Bacteroidetes was higher in broad leaf litter treatment (A, C, D, and E) than in coniferous leaf litter treatment (B) (Fig. 5); this might be helpful for evaluating the effects of broad and coniferous leaf litter on soil characteristics. Bacteroidetes are well known for leaf litter decomposition and have been identified from soil environments such as agricultural soils (Sauvadet et al., 2016). The microbial biomass accumulation could provide important information about ecosystem functioning and could be regarded as a functional indicator (Awasthi et al., 2014). In our study, an

Please cite this article in press as: H. Sun, et al., Effects of different leaf litters on the physicochemical properties and bacterial communities in Panax ginseng-growing soil, Appl. Soil Ecol. (2016), http://dx.doi.org/10.1016/j.apsoil.2016.11.008

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Fig. 5. Dominant relative abundance of phylogenetic genera in different treatments. The bars followed by different letters are significantly different (P < 0.05) according to Tukey’s multiple comparison test.

apparent increase in SMBC and AN in adding leaf litter soil was observed, suggesting that both soil microbial community abundance and soil microbial biomass (carbon and nitrogen) were sensitive to leaf litter decomposition (He et al., 2016; Hu et al., 2016). 5. Conclusion In conclusion, the soil bacterial communities and soil physicochemical properties were significantly affected by the addition of leaf litter. Although adding leaf litter had few effects on the bacterial diversity and abundance, significant changes in bacterial community composition could be identified in all treatments. First, the relative abundance of Proteobacteria improved upon addition of leaf litter, and the Bacteroidetes might be helpful in evaluating the impact of broad and coniferous leaf litter on soil characteristics. These taxonomic changes might result in alternations in microbial biomass. In addition, we also found that adding different leaf litters increased the soil microbial biomass and available NPK, while changing the soil pH and bulk density. By further understanding the response of soil physicochemical properties and bacterial communities toward addition of leaf litter in ginseng-growing soil, we would be better able to model their distribution, spread, and abundance in forest soils, and the role of resultant soil bacterial communities in leaf-litter decomposition and wild understory ginseng management. Acknowledgments This work was supported by Jilin provincial Science and Technology Department (20140520157JH, 20150204053NY and 20160101350JC). References Akhtar, N., Goyal, D., Goyal, A., 2016. Physico-chemical characteristics of leaf litter biomass to delineate the chemistries involved in biofuel production. J. Taiwan Inst. Chem. Eng.. Allison, D.B., Cui, X., Page, G.P., Sabripour, M., 2006. Microarray data analysis: from disarray to consolidation and consensus. Nat. Rev. Genet. 7, 55–65. Awasthi, A., Singh, M., Soni, S.K., Singh, R., Kalra, A., 2014. Biodiversity acts as insurance of productivity of bacterial communities under abiotic perturbations. ISME J. 8, 2445–2452. Bayarsaikhan, U., Ruhl, A.S., Jekel, M., 2016. Characterization and quantification of dissolved organic carbon releases from suspended and sedimented leaf

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Please cite this article in press as: H. Sun, et al., Effects of different leaf litters on the physicochemical properties and bacterial communities in Panax ginseng-growing soil, Appl. Soil Ecol. (2016), http://dx.doi.org/10.1016/j.apsoil.2016.11.008