SciELO - Scientific Electronic Library Online

 
vol.43Methane emission induced by short-chain organic acids in lowland soilSummer Cover Crops Shoot Decomposition and Nitrogen Release in a No-Tilled Sandy Soil author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Revista Brasileira de Ciência do Solo

On-line version ISSN 1806-9657

Rev. Bras. Ciênc. Solo vol.43  Viçosa  2019  Epub Oct 14, 2019

http://dx.doi.org/10.1590/18069657rbcs20190044 

Division – Soil Processes and Properties

Commission – Soil Biology

Impact of agro-farming activities on microbial diversity of acidic red soils in a Camellia Oleifera Forest

1Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees, Ministry of Education, Central South University of Forestry and Technology, Changsha, Hunan 410004, China.

ABSTRACT

The production of Camellia oleifera (oil tea), typically planted in acidic red soils in southern China, is limited by low soil fertility. Agro-farming is one way to promote soil fertility by increasing organic matter and microbial communities. To understand the impact of agro-farming activity on soil fertility, three types of agro-farming, namely, raising laying hens under forest (RLH), cultivating Lolium perenne grass under forest (LPG), and maintenance of native grass (MNG), were employed in an oil tea farm with acidic red soil in Changsha, China. Soil samples were collected from the farm to estimate microbial communities, pH, and total organic carbon (TOC) in different seasons. The results indicated that TOC and temperature were the dominant factors influencing the variations of bacterial communities, while temperature and pH affected the fungal communities in the soil. The most abundant bacterial phyla were Acidobacteria, Proteobacteria, Actinobacteria, and Chloroflexi , while the most abundant fungal phyla were Ascomycota, Basidiomycota, and Zygomycota . Regardless of treatment, the bacterial richness and diversity were both low in spring, and the fungal richness and diversity in summer and autumn were higher than in spring and winter. The TOC content and pH in LPG were significantly higher than in other treatments. Microbial communities in LPG and MNG were more stable than in RLH. In summary, cultivating grass under forest treatment was the best way to improve the microenvironment with the highest TOC content and fewer pathogenic microorganisms.

Keywords: oil tea; season; soil microbial community; high throughput sequencing

INTRODUCTION

Camellia oleifera Abel. (oil tea) is an important woody oilseed plant which is mainly cultivated in Hunan, Jiangxi, and Guangxi provinces and is endemic to hilly areas of southern China with acidic red soil, which is classified as Oxisols in Soil Taxonomy or as Lixisols in World Reference Base for Soil Resources ( Ma et al., 2011 ; Hu et al., 2016 ; Liu et al., 2017a ). The low yield is the primary factor limiting the expansion and development of C. oleifera ( Liu et al., 2017 , 2018 ). One critical reason for low yield is infertile soil. Most regions of C. oleifera plantations have acidic red soils with low levels of pH, microorganisms, organic carbon, and phosphorus compounds ( He et al., 2011 ). Increasing soil nutrients and improving soil microbial communities are necessary to improve soil physical and biochemical properties. The conventional approaches to improving soil are through application of fertilizers and plowing ( Balasubramanian et al., 1999 ; Xu and Liang, 2006 ; Nayak et al., 2009 ). However, in recent years, excessive use of chemical fertilizers and farming activities have caused soil compaction and loss of organic matter which can lead to further degradation of the soil and reductions of microbial activity in croplands ( Liang et al., 2010 ; Motounu, 2010 ; Hegde et al., 2015 ; Wang et al., 2017 ).

Microorganism plays a significant role in the decomposition process of soil organic matter, as well as litter and wood residues ( Progar et al., 2000 ; Hamilton III and Frank, 2001 ; Wood et al., 2012 ). Microbial community structure reflects the condition of soil nutrients and health. Some bacteria phyla only proliferate in copiotrophic environments, including Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes , while others in oligotrophic environments, such as Acidobacteria ( Martin and Macleod, 1984 ; Fierer et al., 2007 ; Nemergut et al., 2010 ). Some fungi are interdependent with the plant rhizosphere, like arbuscular mycorrhizal fungi (AMF), while others can cause plant disease, like Venturia inaequalis ( Smith and Read, 1997 ; Islam et al., 2014 ; Masny, 2017 ). Microorganisms are sensitive to many factors, such as fertilization, cultivation, irrigation, soil coverage, and cropping patterns ( Carpenter-Boggs et al., 2003 ; Rabary et al., 2008 ; Singh et al., 2008 ; Meriles et al., 2009 ; Heidari et al., 2016 ; Nivelle et al., 2016 ). These factors can further affect the soil microenvironment and vegetation growth. Thus, environmentally friendly and sustainable farming practices, which may improve microbial community structure and soil nutrient condition, have been attempted over croplands and forest lands ( Montanaro et al., 2007 ; Larkin et al., 2011 ; Liu et al., 2016b; Wang et al., 2016a , 2016b , 2016c , 2017 ). For instance, raising chickens improved soil structure and increased organic matter ( Wang et al., 2016b ); sowing grass increased soil coverage and reduced soil erosion ( Tebrügge and Düring, 1999 ). All of these activities not only reduced plowing time and application rate of chemical fertilizers but also increased microbial diversity and abundance ( Hodge et al., 2001 ; Leff et al., 2015 ). However, there are little researches on the investigation of new biological farming activities for oil tea plantation. Also, there are no established guidelines regarding fertilization and management to maintain soil health and crop productivity for farmers ( Liu et al., 2018 ).

Understanding the microbial community structure is essential to make scientifically sound recommendations to cropping systems in red soil regions for farmers. This study aimed to investigate the impact of three agro-farming practices on the microbial diversity in an oil-tea-tree forest with: (a) raising laying hens (RLH); (b) cultivating of Lolium perenne grass (LPG); and (c) maintenance of native grass (MNG). We assumed that (1) the RLH treatment could bring more abundant and various microorganisms; (2) the MNG treatment could stabilize soil environment and microbial community structure.

MATERIALS AND METHODS

Study site and sample treatment

The experimental field is located at Wangcheng, Changsha, China (112° 03′ E, 20° 58′ N) with 15-year old C. oleifera ‘Xianglin210′. The climate is subtropical monsoon with mean annual total rainfall of 1,370 mm. The mean annual temperature is 17 °C. The average lowest and highest temperatures are 4.4 °C in January and 30 °C in July, respectively. The soil at the experimental site is a Quaternary red clay with a pH of 5.3.

Three different farming managements were employed in plots of oil-tea woodlands with an area of 0.67 ha each: raising 500 laying hens under the forest (RLH); cultivating Lolium perenne grass under the forest (LPG) at a density of 85 kg seeds per hectare in October 2013 and cultivating again in June 2014 and February 2015; and control field plots consisting of the maintenance of native grass (MNG) by removing the most troublesome weeds ( Amaranthus retroflexus, Chenopodium glaucum, Datura stramonium , and Cirsium setosum ) and keeping the benignant weeds ( Digitaria sanguinalis, Setaria viridis, Chloris virgata, Eleusine indica ). The height of the grasses in MNG was kept below 0.10 m by mowing.

For each treatment, three sampling plots with a dimension of 10 × 10 m were selected on December 29, 2014, May 14, 2015, September 9, 2015, and October 20, 2015, respectively. In each plot, soils were sampled four times at five different points between 0.00 and 0.10 m depth from December 2014 to October 2015 ( Table 1 ). After removing debris and roots, soil samples were well mixed, ground, and sieved (<2 mm). Approximately 200 g of each soil was placed into a sterile bag and stored at −80 °C for microbial analyses ( Gao et al., 2015 ).

Table 1 Temperature, total organic content (TOC), and pH of soil samples subjected to the following treatments: raising laying hens under forest (RLH), cultivating Lolium perenne grass under forest (LPG), and maintenance of native grass under forest (MNG) collected at four times during the year 

Sample time Seasons Average temperature Treatments TOC pH(H2O)
°C g kg−1
2015/05/14 Spring 24 RLH1 11.14±1.52 Cb 4.8±0.2 Ab
LPG1 22.30±2.11 Ba 5.2±0.1 ABa
MNG1 5.80±2.10 Dc 4.5±0.2 Bc
2015/09/09 Summer 31 RLH2 12.05±3.11 Cb 4.8±0.1 Ab
LPG2 23.20±1.52 Ba 5.0±0.2 Ca
MNG2 13.06±2.00 Cb 4.6±0.3 ABb
2015/10/20 Autumn 20 RLH3 16.45±0.42 Bb 4.6±0.2 Bc
LPG3 21.00±2.62 Ba 5.3±0.2 Aa
MNG3 15.22±1.80 Bb 4.8±0.2 Abc
2014/12/29 Winter 4 RLH4 18.23±2.51 Ab 4.7±0.3 BCb
LPG4 31.02±1.11 Aa 5.4±0.2 Aa
MNG4 17.85±1.02 Ab 4.5±0.1 Bc

TOC of soil samples was determined using an elemental analyzer (Vario EL III, Germany). pH(H2O) at a soil:water ratio of 1:2.5 v/v. Data presented are mean ± SD. Different capital letters in the same column indicate significant difference among different seasons within same treatments at p≤0.05 (Duncan's multiple range tests); different lowercase letters in the same column indicate significant difference among different treatments within same seasons at p≤0.05. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter.

Determination of temperature and soil physical-chemical index

Soil temperatures at each sampling time were recorded by a mercurial thermometer. The total organic carbon (TOC) of soil samples was determined using an elemental analyzer (Vario EL III, Germany). Soil pH (soil:distilled water = 1:2.5) of each sample was determined by a PHS-3C pH meter (INESA Scientific Instrument Co. Ltd).

Bacterial and fungal community assessment

Total DNA (0.5 g wet weight) was extracted using an E.Z.N.A Soil DNA kit (OMEGA, USA) according to the manufacturer's instruction. The extracted DNA was diluted in a TE buffer (Tris-HCl 10 mmol L−1, EDTA 1 mmol L−1, pH 8.0) and stored at −20 °C until analysis ( Liu et al., 2014 ).

An aliquot of the extracted DNA from each sample was used as a template for amplification. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified with primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) ( Peng et al., 2017 ). The hypervariable regions of the fungal ITS1 gene were amplified with primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) ( White, 1990 ; Gardes et al., 1993 ). The following thermal program was used for amplification: at an initial denaturation at 98 °C for 30 s, followed by 10 cycles of 98 °C for 10 s, 65 °C for 30 s, and 72 °C for 30 s, followed by an extension at 72 °C for 5 min. Each sample was amplified in triplicate, and the PCR products were pooled and purified using the Agarose Gel DNA purification kit (TaKaRa). An equal amount of the PCR product from each sample was combined in a single tube to be run on an Illumina MiSeq PE300 platform at Biomarker Technologies Co., Beijing, China. After sequencing, paired-end reads were assembled with a minimum overlap of 10 bp using FLASH (version 1.2.7). Sequences with an average quality score <20 over a 50-bp sliding window were truncated using Trimmomatic (version 0.33). Chimeras were identified and removed using UCHIME (version 4.2).

Statistical and bioinformatics analysis

Effective sequences were clustered into operational taxonomic units (OTUs), with a similarity cutoff of 97 % using QIIME (version 1.8.0). Finally, the taxonomy of bacterial and fungal sequences were annotated by Ribosomal Database Project RDP classifier (version 2.2) based on Silva (Release 119) and Unite (Release 7.0) databases, respectively. Alpha diversity indexes, including OTUs and abundance-based coverage estimator (ACE), which both mean the microbial richness, and Shannon diversity, which means the microbial diversity, were estimated by MOTHUR (version 1.30). Classification heatmaps, Venn diagrams, and redundancy analysis of environmental factors and microorganisms were drawn by the R programming language platform. The variance analysis of environmental factors was performed by Duncan's multiple range tests using SPSS for Windows (version 17.0)

RESULTS

Physicochemical properties of soil

The TOC content increased gradually over time in all treatments during 2015 ( Table 1 ). The TOC content of all treatments was significantly higher in winter than in any other seasons. The highest TOC content was always detected in LPG in every season. The TOC content in RLH, LPG, and MNG treatments ranged from 11-19 %, 20-32 %, and 5-32 %, respectively. The pH ranged from 4.5 to 5.4. The highest pH was detected in LPG across all seasons ( Table 1 ).

Microbial diversity and richness

In the RLH and LPG treatments, the highest bacterial richness and diversity were both observed in autumn ( Table 2 ). However, in the MNG treatment, the highest bacterial richness was in winter and the highest bacterial diversity was in summer. According to table 3 , in the RLH and LPG treatments, the fungal richness in summer and autumn were both higher than in spring and winter. In the MNG treatment, the highest fungal richness and diversity were both in summer and the lowest both in winter.

Table 2 Bacterial diversity (Shannon) and richness, number of operational taxonomic units (OTUs), and abundance-based coverage estimator (ACE) of soil samples subjected to the following treatments: RLH, LPG, and MNG 

Treatments Effective reads OTUs ACE Shannon
RLH1 168177 1255 1388.7 5.55
RLH2 193436 1591 1661.2 5.89
RLH3 188959 1613 1671.9 5.93
RLH4 201415 1328 1412.0 5.03
LPG1 92925 1416 1480.6 5.56
LPG2 81368 1433 1490.1 5.62
LPG3 173387 1535 1580.2 5.77
LPG4 197364 1477 1540.9 5.76
MNG1 118779 1340 1468.5 5.61
MNG2 119741 1526 1597.1 5.78
MNG3 114777 1510 1552.6 5.73
MNG4 84372 1609 1696.1 5.78

RLH: raising laying hens under forest; LPG: cultivating Lolium perenne grass under forest; MNG: maintenance of native grass under forest. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter.

Table 3 Fungal diversity (Shannon) and richness (OTUs, ACE) of soil samples subjected to the following treatments: RLH, LPG, and MNG 

Treatments Effective reads OTUs ACE Shannon
RLH1 263867 451 544.42 3.059
RLH2 227738 753 763.77 4.473
RLH3 270666 685 693.93 4.213
RLH4 270622 461 492.43 3.207
LPG1 121577 594 631.44 4.110
LPG2 103082 635 657.78 3.871
LPG3 271820 772 819.98 4.328
LPG4 288909 594 628.89 3.939
MNG1 96504 727 748.37 4.883
MNG2 118700 775 780.61 5.224
MNG3 113592 744 745.95 5.084
MNG4 133504 703 730.66 4.485

RLH: raising laying hens under forest; LPG: cultivating Lolium perenne grass under forest; MNG: maintenance of native grass under forest. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter.

In spring, the rank of bacterial richness and diversity was LPG>MNG>RLH and MNG>LPG>RLH, respectively; in summer, the ranks of OTUs and Shannon were both RLH>LPG>MNG; in autumn, the ranks of bacterial richness and diversity were both RLH>LPG>MNG, but they were MNG>LPG>RLH in winter ( Table 2 ). Except in autumn, the highest fungal richness and diversity were observed in the MNG treatment in other three seasons; in autumn, the lowest fungal richness and diversity was detected in the RLH treatment ( Table 3 ).

Distribution of microorganisms

Distribution of bacteria

Acidobacteria, Proteobacteria, Actinobacteria , and Chloroflexi were the dominant phyla in every sample ( Figure 1 ). At the class level, Acidobacteria, Alphaproteobacteria, Gammaproteobacteria , and Actinobacteria were dominant in all treatments and seasons. However, the abundance of phyla and classes of the bacterial community were significantly more diverse among treatments and seasons.

Figure 1 Relative abundance at the bacterial taxonomic levels of different treatments. Note: Bacteria phylum (a); Bacteria class (b). RLH: raising laying hens under forest; LPG: cultivating Lolium perenne grass under forest; MNG: maintenance of native grass under forest. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter. 

At the phylum level, Actinobacteria gradually increased with time and the annual dynamics in MNG had a similar pattern to that in LPG and RLH ( Figure 1a ). The distribution of Proteobacteria showed similar relative abundance in every sample. Annual dynamics of Chloroflexi , which gradually increased with time and then decreased in autumn in MNG, was significantly different from that in LPG and RLH which were distributed evenly in each season.

The major classes ( Acidobacteria, Alphaproteobacteria , and Gammaproteobacteria ) decreased gradually with time which was contrary to Actinobacteria ( Figure 1b ). Most of the classes in LPG remained relatively stable in spring and autumn; yet the minor classes, like Actinobacteria and Bacilli , increased significantly in summer and winter while Acidobacteria decreased significantly. In RLH, there were significant differences between the four seasons. Acidobacteria in spring was much lower than in summer and winter that lead to a more homogenous bacterial community. It is noteworthy that Alphaproteobacteria always remained in relatively stable abundance ranging from 15 to 25 % in all samples.

Distribution of fungus

At the phylum level ( Figure 2a ), Ascomycota were higher than other phyla in most samples. In MNG, Ascomycota and Basidiomycota in summer and autumn were higher than in spring and winter whereas Zygomycota was lower in summer and autumn than in spring and winter; Ascomycota remained in relatively stable abundance in four quarters. The variation of phyla, except Ascomycota , in LPG was similar to that in MNG, but the ranges of variation were greater in LPG than in MNG. In LPG treatment, Ascomycota in spring and winter was much higher than other phyla and lower in summer and autumn. In RLH, the Ascomycota gradually increased with time; Basidiomycota in summer was much higher than that in any other seasons and Zygomycota was highest in autumn.

Figure 2 Relative abundance at the microbial taxonomic levels of different treatments. Note: a: fungus phylum; b: fungus class. RLH: raising laying hens under forest; LPG: cultivating Lolium perenne grass under forest; MNG: maintenance of native grass under forest. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter. 

At the fungal class level ( Figure 2b ), in MNG, Leotiomycetes were higher in summer and winter than in spring and winter, and Eurotiomycetes increased along with time. Agaricomycetes and Dothideomycetes in LPG were both significantly higher than that in MNG and RLH in every season. During the whole year, variation of Agaricomycetes in LPG was the same as in RLH. Eurotiomycetes in RLH increased along with time which was the same as that in MNG. In MNG, LPG, and RLH treatment, there were still many unknown fungi, which were between 30-50, 18-40, and 45-80 %, respectively at phylum level as well as between 37-65, 25-50, and 56-83 %, respectively at class level.

Relationships between microbial communities

Venn diagrams were used to show shared and unique communities in various seasons ( Figure 3 ). For bacterial communities ( Figure 3a ), four seasons shared 1230, 984, and 1174 OTUs, accounting for 73.35, 55.06, and 67.43 % of each total reads in LPG, RLH, and MNG, respectively. The variation of bacterial communities in LPG was more stable than in RLH and MNG because of the high ratio of shared OTUs and the lower difference in unique communities.

Figure 3 Bacterial (a) and fungal Venn (b) diagrams of OTUs within same methods in different seasons. Note: RLH: raising laying hens under forest; LPG: cultivating Lolium perenne grass under forest; MNG: maintenance of native grass under forest. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter. 

For fungal communities ( Figure 3b ), four seasons shared 397, 240, and 426 OTUs, accounting for 42.78, 24.19, and 45.61 % of each total reads, in LPG, RLH, and MNG, respectively. The variation of fungal communities in MNG was more stable than in LPG and RLH because of the high ratio of sharing OTUs and the less difference of unique communities.

To get an overall view of the identified connections among the samples, hierarchically clustered heatmaps were generated. The closer the color was to the red, the more dominant microorganism was. There were differences among every treatment and every season.

According to the heatmaps, the fluctuation of bacterial communities in RLH was greater than in LPG and MNG ( Figure 4 and Figure 5 ). At the family level, MNG1 and MNG2, LPG1 and LPG2 clustered together, respectively. Clustering of MNG3 and LPG3 at family and order level showed that there were similar bacterial communities between MNG and LPG in autumn. In autumn, the activity of bacteria was the lowest during the whole year, and there were very little dominant bacteria at order and family level.

Figure 4 Heatmap analysis of the bacterial community of soil samples subjected to RLH, LPG, and MNG treatments collected at four times during the year and multiple samples similarity tree at the order level. The connecting lines on top describe the bacterial communities clustering of each sample. The connecting lines on the left side describe the clustering of each bacterium. The closer the color to red was, the more dominant microorganism was. Note: RLH: raising laying hens under forest; LPG: cultivating Lolium perenne grass under forest; MNG: maintenance of native grass under forest. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter. 

Figure 5 Heatmap analysis of the bacterial community of soil samples subjected to RLH, LPG, and MNG treatments collected at four times during the year and multiple samples similarity tree at the family level. The connecting lines on top describe the bacterial communities clustering of each sample. The connecting lines on the left side describe the clustering of each bacterium. The closer the color to red was, the more dominant microorganism was. Note: RLH: raising laying hens under forest; LPG: cultivating Lolium perenne grass under forest; MNG: maintenance of native grass under forest. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter. 

According to heatmaps of fungal communities ( Figure 6 and Figure 7 ), the fungal communities in LPG were more stable than in other treatments during the whole year. At the order level, the clustering of LPG1 and LPG4 assembled with the clustering of LPG2 and LPG3. The LPG1 and LPG4, MNG1 and MNG4, RLH1 and RLH4 clustered together respectively at the family level.

Figure 6 Heatmap analysis of the fungal community of soil samples subjected to RLH, LPG, and MNG treatments collected at four times during the year and multiple samples similarity tree at the order level. The connecting lines on top describe the fungal communities clustering of each sample. The connecting lines on the left side describe the clustering of each fungus. The closer the color close to red was, the more dominant microorganism was. Note: RLH: raising laying hens under forest; LPG: cultivating Lolium perenne grass under forest; MNG: maintenance of native grass under forest. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter. 

Figure 7 Heatmap analysis of the fungal community of soil samples subjected to RLH, LPG, and MNG treatments collected at four times during the year and multiple samples similarity tree at the family level. The connecting lines on top describe the fungal communities clustering of each sample. The connecting lines on the left side describe the clustering of each fungus. The closer the color close to red was, the more dominant microorganism was. Note: RLH: raising laying hens under forest; LPG: cultivating Lolium perenne grass under forest; MNG: maintenance of native grass under forest. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter. 

The influence factor on microbial communities

Redundancy analysis (RDA) indicated that temperature and TOC were the dominant factors for explaining the most variations in bacterial and fungal communities of four seasons and three treatments ( Figure 7 and Figure 9 ).

Figure 8 Redundancy analysis (RDA) of the relationship between the soil physicochemical properties and the relative abundance of each bacterial genus of the twelve soil samples (p<0.05). Different symbols in the graph represent soil samples from different treatments: Circle: MNG; Square: LPG; and Diamond: RLH. Arrows indicate the direction and magnitude of each variable. Note: RLH: raising laying hens under forest; LPG: cultivating Lolium perenne grass under forest; MNG: maintenance of native grass under forest. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter. Bottle green lines represent bacterial genus; red lines represent environmental factors. 

Figure 9 Redundancy analysis (RDA) of the relationship between the soil physicochemical properties and the relative abundance of fungal genus of the twelve soil samples (p<0.05). Different symbols in the graph represent soil samples from different treatments: Circle = MNG; Square = LPG; and Diamond = RLH. Arrows indicate the direction and magnitude of variables. Note: RLH: raising laying hens under forest; LPG: cultivating Lolium perenne grass under forest; MNG: maintenance of native grass under forest. The number after the treatment symbol means: 1 = spring; 2 = summer; 3 = autumn; 4 = winter. Bottle green lines represent fungal genus; red lines represent environmental factors. 

According to figure 8 , temperature positively influenced Bryobacter, Candidatus_Solibacter , and Bradyrhizobium while negatively influenced Acidothermus, Paenibacillus, Modestobacter , and Crossiella . The TOC and pH positively influenced Burkholderia while negatively influenced Rhizomicrobium, Flavobacterium, Stenotrophomonas, Phenylobacterium, Brevundimonas, Sorangium , and Sphingomonas .

According to figure 9 , temperature positively influenced Purpureocillim, Leucoagaricus , and Dictyophora while negatively influenced Gymnopilus, Penicillium , and Talaromyces . There was a significant positive influence of pH on Rasamsonia, Leucocoprinus, Gymnopus , and Hydropus while there was negative influence on Hyaloscypha, Scleroconidioma, Cephaliphora, Cladophialophora , and Cryptococcus . The TOC appeared to positively influence Gymnopus and Aspergillus while negatively influence Clitopilus, Agaricus, Scleroconidioma , and Conocybe .

DISCUSSION

The effect of farming patterns on microbial communities

Growing grass is a sustainable management practice that decreases soil bulk densities, increases soil porosity, soil moisture, and nutrient holding capacity ( Wang et al., 2013 ; Jia et al., 2014 ). This management practice has been adopted in many orchards ( Greenham, 1955 ). These factors can significantly change the microbial communities ( Du et al., 2015 ). There are two major ways to grow grass, including artificially cultivating grass and growing grass without tillage. Both ways have been identified to have beneficial effects on improving soil chemical properties and soil microbial communities ( Chalak et al., 2011 ; Yagioka et al., 2015 ; Boukhdoud et al., 2016 ). Several researchers have identified that poultries can contribute to improvement of soil fertility and soil structure, such as increasing soil nitrogen and phosphorus, and improving soil porosities and soil water content ( Wilkins, 2008 ; Lin et al., 2013 ; Xu et al., 2014 ; Wang et al., 2016b ). These changes are able to provide a more appropriate environment for microorganisms. In this study, the TOC and pH in LPG treatment were significantly higher than in other treatments. Xu et al. (2014) found that there were no remarkable changes in the soil during the first year of raising laying hens but the concentration of nutrients in the soil increased considerably when after 2 to 4 years. Wild grass was found to create more nutrients than artificially cultivated grass in an apple orchard ( Yan et al., 2014 ). The differences between Yan's research and ours might be caused by the differences in dominant cultivars, grass species, and soil properties. Although the TOC and pH in LPG were remarkably higher than in other treatments, the bacterial diversity and richness had no significant difference among the three treatments, which is in agreement with Tao et al. (2011) . However, the stability of bacterial richness in LPG was the best while that in RLH was the worst during whole year, indicating that the grass cover could provide a relatively stable environment for soil bacterial communities.

Except for the unknown phyla, Ascomycota and Basidiomycota were the dominant fungal phyla in all treatments, which agreed with most previous studies ( Buée et al., 2009 ; Weber et al., 2013 ; Coats et al., 2014 ). Previous studies have found that most pathogenic microbes were fungi ( Agrios, 2005 ; Raaijmakers et al., 2009 ; Liu et al., 2016). Many researchers have reported that manure could dramatically increase bacterial activity while decrease fungal activities ( Bittman et al., 2005 ; Liu et al., 2013 ). Compared to the control, the relative abundance of Ascomycota , which was linked to many kinds of pathogenic fungi by previous studies ( Toledo et al., 2007 ; Vujanovic and Labrecque, 2008 ; Rodrigues et al., 2016 ), decreased under the laying hens treatment. The relative abundance of Agaricomycetes and Eurotiomycetes , which was similar to Zhou's research ( Zhou et al., 2016 ), in LPG were significantly higher than in other treatments. Agaricomycetes , known as the decomposer of lignin, could increase soil nutrients but could also lead to plant white rot or soft rot ( Morgenstern et al., 2008 ). Eurotiomycetes was found in high N content ( Zhou, et al., 2016 ). Therefore, we could speculate that there was more N in LPG than in other treatments, and the fungi in LPG could facilitate the decomposition of those nutrients whereas the saprophytic fungi also increased the risk of plant root disease.

Acidobacteria and Proteobacteria were the major phyla identified in soil bacterial communities in this study and as reported by previous studies ( Barns et al., 1999 ; Janssen et al., 2002 ; Jones et al., 2009 ). Acidobacteria was regarded as oligotrophic bacteria. Smit et al. (2001) found a high ratio of Acidobacteria to Proteobacteria when the soil nutrients were low. Surprisingly, in the RLH treatment, the relative abundance of Acidobacteria in spring and winter were remarkably lower than other treatments, indicating that the activities of laying hens might improve content of some other soil nutrients except TOC.

Seasonal variation of microbial communities

According to the redundancy analysis, temperature was the primary factor influencing microbial communities, which indicated that microbial communities were strongly affected by seasonal dynamics. Additionally, TOC was also influential on bacteria while pH was influential on fungi.

Regardless of treatment, the highest TOC content was found in winter, which was identical to the findings of Aanderud et al. (2010) . However, Laudon et al. (2004) found that the highest TOC content was observed during spring. In RLH, activity from the hens, such as scraping soil to find insects and producing manure, enhanced microbial activity but drastic seasonal variations in microbial communities were observed due to the absence of grass covering. In LPG, the fluctuation range of microbial communities was smaller than in other treatments throughout the year. The single-species grass covering provided relatively stable microbial communities. As temperature decreased, many grass residues provided nutrients to bacteria, however, further declining temperatures would inhibit microbial activity. In MNG, the diversity of weed species made the micro-environment unstable but the residues of these weeds lasted longer in comparison to single grass. In winter, cold-hardy grass would tolerate the low temperatures and could create relatively suitable micro-environments for microorganisms.

Proteobacteria and especially Alphaproteobacteria remained stable throughout the year while Acidobacteria had the lowest relative abundance during winter and Actinobacteria had the lowest relative abundance during spring and summer, which was similar to the observations of Lipson and Schmidt (2004). As indicated by Lazzaro et al. (2012) , seasonal changes had no effect on Alphaproteobacteria . The most dominant bacteria were found in spring and winter. Xanthomonadales and Burkholderiales were phytopathogens ( Campos et al., 2016 ) predominant in RLH1. This means the activity of the hens during the spring may not improve the soil nutrition but increase the risk of plant disease.

Similar to other studies ( Schmidt et al., 2013 ; Weber, et al., 2013 ), the relative abundance of Ascomycota and Basidiomycota in our study were the predominant phyla during all seasons. The relative abundance of Agaricomycetes in summer and autumn was higher than in spring and winter, indicating that the disease rate of infection of plants in summer and autumn was higher than in spring and winter.

CONCLUSION

Raising laying hens may provide manure to increase the soil organic matter; however, a stable micro-environment for microorganisms was not achieved without grass cover. Also, maintaining native grasses resulted in limited quantity of soil TOC. Cultivating grasses under oil tea trees was the best among all options in this study for the improvement of the microenvironment, such as increasing TOC content and pH, stabilizing microbial communities, and reducing pathogenic microorganisms. In addition, cultivating grasses increased the relative abundance of microorganisms. These changes in soil microenvironment may benefit the growth of oil tea trees and further study is needed to quantify the beneficial effect on the performance of oil tree trees.

ACKNOWLEDGMENTS

This study was funded by the Key Research and Development Program of Hunan, China (No. 2017NK2201).

REFERENCES

Aanderud ZT, Richards JH, Svejcar T, James JJ. A shift in seasonal rainfall reduces soil organic carbon storage in a cold desert. Ecosystems. 2010;13:673-82. https://doi.org/10.1007/s10021-010-9346-1Links ]

Agrios GN. Plant pathology. 5th ed. London: Academic Press; 2005. [ Links ]

Balasubramanian V, Morales AC, Cruz RT, Abdulrachman S. On-farm adaptation of knowledge-intensive nitrogen management technologies for rice systems. Nutr Cycl Agroecosyst. 1999;53:59-69. https://doi.org/10.1023/a:1009744605920Links ]

Barns SM, Takala SL, Kuske CR. Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment. Appl Environ Microb. 1999;65:1731-7. [ Links ]

Bittman S, Forge TA, Kowalenko CG. Responses of the bacterial and fungal biomass in a grassland soil to multi-year applications of dairy manure slurry and fertilizer. Soil Biol Biochem. 2005;37:613-23. https://doi.org/10.1016/j.soilbio.2004.07.038Links ]

Boukhdoud N, Gros R, Darwish T, Silva AMF. Effect of agricultural practices and coastal constraints on soil microbial functional properties in mediterranean olive orchards. Eur J Soil Sci. 2016;67:470-7. https://doi.org/10.1111/ejss.12347Links ]

Buée M, Reich M, Murat C, Morin E, Nilsson RH, Uroz S, Martin F. 454 pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity. New Phytol. 2009;184:449-56. https://doi.org/10.1111/j.1469-8137.2009.03003.xLinks ]

Campos SB, Lisboa BB, Camargo FAO, Bayer C, Sczyrba A, Dirksen P, Albersmeier A, Kalinowski J, Beneduzi A, Costa PB, Passaglia LMP, Vargas LK, Wendisch VF. Soil suppressiveness and its relations with the microbial community in a Brazilian subtropical agroecosystem under different management systems. Soil Biol Biochem. 2016;96:191-7. https://doi.org/10.1016/j.soilbio.2016.02.010Links ]

Carpenter-Boggs L, Stahl PD, Lindstrom MJ, Schumacher TE. Soil microbial properties under permanent grass, conventional tillage, and no-till management in South Dakota. Soil Till Res. 2003;71:15-23. https://doi.org/10.1016/s0167-1987(02)00158-7Links ]

Chalak L, Noun J, El Haj S, Rizk H, Assi RR, Attieh J, Maalouf F, Antoun MA, Sabra N. Current status of agro-biodiversity in lebanon and future challenges. Geneconserve. 2011;10:23-41. [ Links ]

Coats VC, Pelletreau KN, Rumpho ME. Amplicon pyrosequencing reveals the soil microbial diversity associated with invasive japanese barberry ( Berberis thunbergii DC.). Mol Ecol. 2014;23:1318-32. https://doi.org/10.1111/mec.12544Links ]

Du YF, Fang KK, Wang ZK, Li HK, Mao PJ, Zhang XX, Wang J. Carbon source utilization characteristics of soil microbial community for apple orchard with interplanting herbage. Environm Sci. 2015;36:4260-7. https://doi.org/10.13227/j.hjkx.2015.11.042Links ]

Fierer N, Bradford MA, Jackson RB. Toward an ecological classification of soil bacteria. Ecology. 2007;88:1354-64. https://doi.org/10.1890/05-1839Links ]

Gao S-j, Zhang R-g, Cao W-d, Fan Y-y, Gao J-s, Jin H, Bai J-s, Zeng N-h, Chang D-n, Shimizu K-y. Long-term rice-rice-green manure rotation changing the microbial communities in typical red paddy soil in South China. J Integr Agr. 2015;14:2512-20. https://doi.org/10.1016/S2095-3119(15)61230-8Links ]

Gardes M, Bruns TD. ITS primers with enhanced specificity for Basidiomycetes - application to the identification of mycorrhizae and rusts. Mol Ecol. 1993;2:113-8. https://doi.org/10.1111/j.1365-294X.1993.tb00005.xLinks ]

Greenham DWP. The environment of the fruit tree-managing fruit soils. Sci Hortic. 1955;12:25-31. [ Links ]

Hamilton III EW, Frank DA. Can plants stimulate soil microbes and their own nutrient supply? Evidence from a grazing tolerant grass. Ecology. 2001;82:2397-402. https://doi.org/10.1890/0012-9658(2001)082[2397:CPSSMA]2.0.CO;2Links ]

He G, Zhang J, Hu X, Wu J. Effect of aluminum toxicity and phosphorus deficiency on the growth and photosynthesis of oil tea ( Camellia oleifera Abel.) seedlings in acidic red soils. Acta Physiol Plant. 2011;33:1285-92. https://doi.org/10.1007/s11738-010-0659-7Links ]

Hegde R, Natarajan A, Meena RS, Niranjana KV, Thayalan S, Singh SK. Status of soil degradation in an irrigated command area in Chikkarasinakere Hobli, Mandya district, Karnataka. Curr Sci India. 2015;108:1501-11. [ Links ]

Heidari G, Mohammadi K, Sohrabi Y. Responses of soil microbial biomass and enzyme activities to tillage and fertilization systems in soybean ( Glycine max L.) production. Front Plant Sci. 2016;7:1730. https://doi.org/10.3389/fpls.2016.01730Links ]

Hodge A, Campbell CD, Fitter AH. An arbuscular mycorrhizal fungus accelerates decomposition and acquires nitrogen directly from organic material. Nature. 2001;413:1-9. https://doi.org/10.1038/35095041Links ]

Hu JJ, Wu W, Cao ZH, Wen J, Shu QL, Fu SL. Morphological, physiological and biochemical responses of Camellia oleifera to low-temperature stress. Pak J Bot. 2016;48:899-905. [ Links ]

Islam S, Akanda AM, Sultana F, Hossain MM. Chilli rhizosphere fungus Aspergillus spp. PPA1 promotes vegetative growth of cucumber ( Cucumis sativus ) plants upon root colonisation. Arch Phytopathol Pfl. 2014;47:1231-8. https://doi.org/10.1080/03235408.2013.837633Links ]

Janssen PH, Yates PS, Grinton BE, Taylor PM, Sait M. Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria , and Verrucomicrobia . Appl Environ Microb. 2002;68:2391-6. https://doi.org/10.1128/AEM.68.5.2391-2396.2002Links ]

Jia ML, Guo H, Li HK. Mineralization of soil organic carbon and its relationship with soil enzyme activities in apple orchard in Weibei. Environm Sci. 2014;35:2777-84. https://doi.org/10.13227/j.hjkx.2014.07.048Links ]

Jones RT, Robeson MS, Lauber CL, Hamady M, Knight R, Fierer N. A comprehensive survey of soil acidobacterial diversity using pyrosequencing and clone library analyses. Isme J. 2009;3:442-53. https://doi.org/10.1038/ismej.2008.127Links ]

Larkin RP, Honeycutt CW, Griffin TS, Olanya OM, Halloran JM, He Z. Effects of different potato cropping system approaches and water management on soilborne diseases and soil microbial communities. Phytopathology. 2011;101:58-67. https://doi.org/10.1094/phyto-04-10-0100Links ]

Laudon H, Köhler S, Buffam I. Seasonal TOC export from seven boreal catchments in northern Sweden. Aquat Sci. 2004;66:223-30. https://doi.org/10.1007/s00027-004-0700-2Links ]

Lazzaro A, Brankatschk R, Zeyer J. Seasonal dynamics of nutrients and bacterial communities in unvegetated alpine glacier forefields. Appl Soil Ecol. 2012;53:10-22. https://doi.org/10.1016/j.apsoil.2011.10.013Links ]

Leff JW, Jones SE, Prober SM, Barberán A, Borer ET, Firn JL, Harpole WS, Hobbie SE, Hofmockel KS, Knops JHM, McCulley RL, Pierre KL, Risch AC, Seabloom EW, Schütz M, Steenbock C, Stevens CJ. Consistent responses of soil microbial communities to elevated nutrient inputs in grasslands across the globe. PNAS. 2015;112:10967-72. https://doi.org/10.1073/pnas.1508382112Links ]

Liang Y, Li D, Lu X, Yang X, Pan X, Mu H, Shi D. Soil erosion changes over the past five decades in the red soil region of southern China. J Mt Sci. 2010;7:92-9. https://doi.org/10.1007/s11629-010-1052-0Links ]

Lin D, Zhong L-m, Zheng P, Wang J-y, Gong W, HU T-x. Effects of raising chicken on soil physical and chemical properties in pear orchard. Hubei Agricultural Sciences. 2013;18. [ Links ]

Liu FC, Xing S, Ma HL, Ding YQ, Chen B, Du BH. Effect of plant growth-promoting rhizobacteria (PGPR) on the microorganism population and bacterial diversity in ziziphus jujuba rhizosphere soil. Scientia Silvae Sinicae. 2013;49:75-80. https://doi.org/10.11707/j.1001-7488.20130811Links ]

Liu J, Li X, Jia Z, Zhang T, Wang X. Effect of benzoic acid on soil microbial communities associated with soilborne peanut diseases. Appl Soil Ecol. 2017b;110:34-42. https://doi.org/10.1016/j.apsoil.2016.11.001Links ]

Liu J, Sui Y, Yu Z, Shi Y, Chu Y, Jin J, Liu X, Wang G. High throughput sequencing analysis of biogeographical distribution of bacterial communities in the black soils of northeast China. Soil Biol Biochem. 2014;70:113-22. https://doi.org/10.1016/j.soilbio.2013.12.014Links ]

Liu J, Wu L, Chen D, Li M, Wei C. Soil quality assessment of different Camellia oleifera stands in mid-subtropical China. Appl Soil Ecol. 2017a;113:29-35. https://doi.org/10.1016/j.apsoil.2017.01.010Links ]

Liu J, Wu L, Chen D, Yu Z, Wei C. Development of a soil quality index for Camellia oleifera forestland yield under three different parent materials in southern China. Soil Till Res. 2018;176:45-50. https://doi.org/10.1016/j.still.2017.09.013Links ]

Ma J, Ye H, Rui Y, Chen G, Zhang N. Fatty acid composition of Camellia oleifera oil. J Verbr Lebensm. 2011;6:9-12. https://doi.org/10.1007/s00003-010-0581-3Links ]

Martin P, MacLeod RA. Observations on the distinction between oligotrophic and eutrophic marine bacteria. Appl Environ Microb. 1984;47:1017-22. [ Links ]

Masny S. Occurrence of Venturia inaequalis races in Poland able to overcome specific apple scab resistance genes. Eur J Plant Pathol. 2017;147:313-23. https://doi.org/10.1007/s10658-016-1003-xLinks ]

Meriles JM, Gil SV, Conforto C, Figoni G, Lovera E, March GJ, Guzmán CA. Soil microbial communities under different soybean cropping systems: characterization of microbial population dynamics, soil microbial activity, microbial biomass, and fatty acid profiles. Soil Till Res. 2009;103:271-81. https://doi.org/10.1016/j.still.2008.10.008Links ]

Montanaro G, Dichio B, Celano G, Xiloyannis C. Sustainable kiwifruit orchard management in semi-arid environments. Acta Hortic. 2007;753:591-8. https://doi.org/10.17660/ActaHortic.2007.753.78Links ]

Morgenstern I, Klopman S, Hibbett DS. Molecular evolution and diversity of lignin degrading heme peroxidases in the agaricomycetes. J Mol Evol. 2008;66:243-57. https://doi.org/10.1007/s00239-008-9079-3Links ]

Motounu M. Influence of alternative technologies for maintenance of soil on the vine hydric regime. Lucrări ştiinţifice. 2010;LIV:668-73. [ Links ]

Nayak P, Patel D, Ramakrishnan B, Mishra AK, Samantaray RN. Long-term application effects of chemical fertilizer and compost on soil carbon under intensive rice-rice cultivation. Nutr Cycl Agroecosyst. 2009;83:259-69. https://doi.org/10.1007/s10705-008-9217-8Links ]

Nemergut DR, Cleveland CC, Wieder WR, Washenberger CL, Townsend AR. Plot-scale manipulations of organic matter inputs to soils correlate with shifts in microbial community composition in a lowland tropical rain forest. Soil Biol Biochem. 2010;42:2153-60. https://doi.org/10.1016/j.soilbio.2010.08.011Links ]

Nivelle E, Verzeaux J, Habbib H, Kuzyakov Y, Decocq G, Roger D, Lacoux J, Duclercq J, Spicher F, Nava-Saucedo J-E, Catterou M, Dubois F, Tetu T. Functional response of soil microbial communities to tillage, cover crops and nitrogen fertilization. Appl Soil Ecol. 2016;108:147-55. https://doi.org/10.1016/j.apsoil.2016.08.004Links ]

Peng W, Bo C, Hua Z. High throughput sequencing analysis of bacterial communities in soils of a typical Poyang Lake wetland. Acta Ecologica Sinica. 2017;37:1650-8. https://doi.org/10.5846/stxb201510052000Links ]

Progar RA, Schowalter TD, Freitag CM, Morrell JJ. Respiration from coarse woody debris as affected by moisture and saprotroph functional diversity in Western Oregon. Oecologia. 2000;124:426-31. https://doi.org/10.1007/pl00008868Links ]

Raaijmakers JM, Paulitz TC, Steinberg C, Alabouvette C, Moënne-Loccoz Y. The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant Soil. 2009;321:341-61. https://doi.org/10.1007/s11104-008-9568-6Links ]

Rabary B, Sall S, Letourmy P, Husson O, Ralambofetra E, Moussa N, Chotte J-L. Effects of living mulches or residue amendments on soil microbial properties in direct seeded cropping systems of Madagascar. Appl Soil Ecol. 2008;39:236-43. https://doi.org/10.1016/j.apsoil.2007.12.012Links ]

Rodrigues AM, Cruz Choappa R, Fernandes GF, De Hoog GS, De Camargo ZP. Sporothrix chilensis sp. nov. (Ascomycota: Ophiostomatales), a soil-borne agent of human sporotrichosis with mild-pathogenic potential to mammals. Fungal Biol-UK. 2016;120:246-64. https://doi.org/10.1016/j.funbio.2015.05.006Links ]

Schmidt P-A, Bálint M, Greshake B, Bandow C, Römbke J, Schmitt I. Illumina metabarcoding of a soil fungal community. Soil Biol Biochem. 2013;65:128-32. https://doi.org/10.1016/j.soilbio.2013.05.014Links ]

Singh M, Tejo PN, Khanna S. Influence of diversified cropping pattern on microbial activity and population dynamics in agricultural soils. Res Crop. 2008;9:593-8. [ Links ]

Smit E, Leeflang P, Gommans S, Van Den Broek J, Van Mil S, Wernars K. Diversity and seasonal fluctuations of the dominant members of the bacterial soil community in a wheat field as determined by cultivation and molecular methods. Appl Environ Microb. 2001;67:2284-91. https://doi.org/10.1128/AEM.67.5.2284-2291.2001Links ]

Smith SE, Read D. Mycorrhizal symbioses. 2nd ed. London: Academic Press; 1997. [ Links ]

Tao S-t, Tian W-l, Liu-lin LI, Yang Y-n, Zhang S-l. Investigation on the distribution and amount of soil microorganisms in pear orchard under different soil managements. South China Fruits. 2011;06. [ Links ]

Tebrügge F, Düring R-A. Reducing tillage intensity - a review of results from a long-term study in Germany. Soil Till Res. 1999;53:15-28. https://doi.org/10.1016/s0167-1987(99)00073-2Links ]

Toledo AV, Lenicov AMMR, Lastra CCL. Pathogenicity of fungal isolates (Ascomycota: Hypocreales) against Peregrinus maidis, Delphacodes kuscheli (Hemiptera: Delphacidae), and Dalbulus maidis (Hemiptera: Cicadellidae), vectors of corn diseases. Mycopathologia. 2007;163:225-32. https://doi.org/10.1007/s11046-007-9006-yLinks ]

Vujanovic V, Labrecque M. Potentially pathogenic and biocontrol Ascomycota associated with green wall structures of basket willow ( Salix viminalis L.) revealed by phenotypic characters and ITS phylogeny. BioControl. 2008;53:413-26. https://doi.org/10.1007/s10526-007-9092-2Links ]

Wang J, Gong W, Xiangnan L. Effects of raising chicken on soil fertility and fruit quality in citrus orchard. J Fruit Science. 2016b;09. [ Links ]

Wang P, Wang Y, Wu QS. Effects of soil tillage and planting grass on arbuscular mycorrhizal fungal propagules and soil properties in citrus orchards in southeast China. Soil Till Res. 2016a;155:54-61. https://doi.org/10.1016/j.still.2015.07.009Links ]

Wang Y, Cao L, Fan J, Lu H, Zhu Y, Gu Y, Sun B, Liang Y. Modelling soil detachment of different management practices in the red soil region of China. Land Degrad Dev. 2017;28:1496-505. https://doi.org/10.1002/ldr.2658Links ]

Wang Y, Fan J, Cao L, Liang Y. Infiltration and runoff generation under various cropping patterns in the red soil region of China. Land Degrad Dev. 2016c;27:83-91. https://doi.org/10.1002/ldr.2460Links ]

Wang Y-j, Tong-Chuan LI, Zhang D-y, Jia M-l, Hui-ke LI, Cao W-d. Effects of intercropping white clover on soil aggregates and soil organic carbon of aggregates in apple-white clover intercropping system. Acta Agrestia Sinica. 2013;03. [ Links ]

Weber CF, Rytas V, Kuske CR. Changes in fungal community composition in response to elevated atmospheric CO2and nitrogen fertilization varies with soil horizon. Front Microbiol. 2013;4:78. https://doi.org/10.3389/fmicb.2013.00078Links ]

White TJ, Burns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego: Academic Press; 1990. p. 315-22. [ Links ]

Wilkins RJ. Eco-efficient approaches to land management: A case for increased integration of crop and animal production systems. Phil Trans R Soc B. 2008;363:517-25. https://doi.org/10.1098/rstb.2007.2167Links ]

Wood CT, Schlindwein CCD, Soares GLG, Araujo PB. Feeding rates of Balloniscus sellowii (Crustacea, Isopoda, Oniscidea): the effect of leaf litter decomposition and its relation to the phenolic and flavonoid content. ZooKeys. 2012;176:231-45. https://doi.org/10.3897/zookeys.176.1940Links ]

Xu H, Su H, Su B, Han X, Biswas DK, Li Y. Restoring the degraded grassland and improving sustainability of grassland ecosystem through chicken farming: a case study in northern China. Agr Ecosyst Environ. 2014;186:115-23. https://doi.org/10.1016/j.agee.2014.02.001Links ]

Xu MG, Liang G. The evolvement of soil fertility in China. Beijing: China Agricultural Science and Technology Press; 2006. [ Links ]

Yagioka A, Komatsuzaki M, Kaneko N, Ueno H. Effect of no-tillage with weed cover mulching versus conventional tillage on global warming potential and nitrate leaching. Agr Ecosyst Environ. 2015;200:42-53. https://doi.org/10.1016/j.agee.2014.09.011Links ]

Yan W-T, Qiu G-S, Zhang H-J, Sun L-N, Cheng C-G, Li Z-A, Zhao D-Y. Effects of three ground management models on soil physical-chemical properties and insect community in apple orchard of western Liaoning. J Fruit Science. 2014;31:801-8. [ Links ]

Zhou J, Jiang X, Zhou B, Zhao B, Ma M, Guan D, Li J, Chen S, Cao F, Shen D, Qin J. Thirty four years of nitrogen fertilization decreases fungal diversity and alters fungal community composition in black soil in northeast China. Soil Biol Biochem. 2016;95:135-43. https://doi.org/10.1016/j.soilbio.2015.12.012Links ]

Received: April 04, 2019; Accepted: August 05, 2019

*Corresponding author: E-mail: yuanjunchina@126.com

AUTHOR CONTRIBUTIONS

Conceptualization: Jun Yuan.

Methodology: Jun Yuan.

Validation: Jun Yuan.

Formal Analysis: Jun Li.

Investigation: Zelong Wu and Jun Li.

Resources: Zelong Wu and Jun Li.

Data Curation: Zelong Wu and Jun Li.

Writing – Original Draft: Jun Li.

Writing – Review & Editing: Jun Li and Jun Yuan.

Visualization: Jun Li.

Supervision: Jun Yuan.

Project Administration: Jun Yuan.

Funding Acquisition: Jun Yuan.

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.