Composition and diversity of rhizosphere fungal community in Coptis chinensis Franch. continuous cropping fields

In this study, effects of continuous cropping on soil properties, enzyme activities, and relative abundance, community composition and diversity of fungal taxa were investigated. Rhizosphere soil from field continuously cropped for one-year, three-year and five-year by Coptis chinensis Franch. was collected and analyzed. Illumina high-throughput sequencing analysis showed that continuous cropping of C. chinensis resulted in a significant and continuous decline in the richness and diversity of soil fungal population. Ascomycota, Zygomycota, Basidiomycota, and Glomeromycota were the dominant phyla of fungi detected in rhizosphere soil. Fungal genera such as Phoma, Volutella, Pachycudonia, Heterodermia, Gibberella, Cladosporium, Trichocladium, and Sporothrix, were more dominant in continuously cropped samples for three-year and five-year compared to that for one-year. By contrast, genera, such as Zygosaccharomyces, Pseudotaeniolina, Hydnum, Umbelopsis, Humicola, Crustoderma, Psilocybe, Coralloidiomyces, Mortierella, Polyporus, Pyrenula, and Monographella showed higher relative abundance in one-year samples than that in three-year and five-year samples. Cluster analysis of the fungal communities from three samples of rhizosphere soil from C. chinensis field revealed that the fungal community composition, diversity, and structure were significantly affected by the continuous cropping. Continuous cropping of C. chinensis also led to significant declines in soil pH, urease, and catalase activities. Redundancy analysis showed that the soil pH had the most significant effect on soil fungal population under continuous cropping of C. chinensis.


Introduction
Coptis chinensis Franch. is one of the most important traditional medicinal plants in the family Ranunculaceae. The root of C. chinensis is regularly used for medicinal purposes, and has been prescribed alone or in combination with other traditional herbs for treating diabetes, PLOS  area are 1,667 m, 1,200 mm, and 16˚C, respectively. The soil type of the local area is sandy loam soil. The rhizosphere soil samples collected from the fields, continuously cropped with C. chinensis for one-year, three-year and five-year have been denoted as RMS1, RMS3 and RMS5, respectively. All the three treatments were arranged in the same area and conducted under uniform field conditions and management. Each treatment had the three replicate plots selected randomly. Each plot was 20 m 2 and contained 780 plants. These plants were kept 15 cm apart from each other. On August 5, 2015, rhizosphere soil samples were collected from 30 randomly selected plants in one plot. The roots were shaken gently to remove the loosely adhering soil. Then, the rhizosphere soil that clung to the roots and rhizomes of C. chinensis was brushed off and collected. The collected rhizosphere soil from the three replicate plots was thoroughly mixed as a composite sample. A part of each soil sample was stored in an ultralow temperature freezer at −80˚C for DNA extraction, and the remaining soil was air-dried for soil characteristic and enzyme activity analyses [27,38].

Total DNA extraction from rhizosphere soil
The total genomic DNA from the samples was extracted from 1 g soil using the Fast DNA Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer's instructions. DNA extracted for 30 times from every treated soil sample was mixed as one DNA mixed sample. Then, the three mixed DNA samples were used for the fungal internal transcribed spacer (ITS) gene amplification. DNA concentration and purity were monitored on a 1% agarose gel. Based on the concentration, the DNA was diluted to 1 ng.μL −1 and stored at −80˚C until use.

Fungal ITS gene amplification and Illumina sequencing
We consulted the protocol described by Caporaso et al. [33] to analyze the diversity and composition of the fungal communities in rhizosphere soil under continuous cropping. Primers ITS5-1737F and ITS2-2043R were selected to amplify the ITS1 region of the fungal genes. DNA was amplified following a previously described protocol [39].

Rhizosphere soil enzyme activities and physiochemical properties
The soil pH was tested with a Mettler-Toledo TE 20 (Mettler-Toledo Instruments Co., Ltd., Shanghai, China) using soil suspension with deionized distilled water (1:20 w/v). The rhizosphere soil enzyme activities were determined using an enzyme analysis kit (Suzhou Comin Biotechnology Co., Ltd., Jiangsu, China). The soil physiochemical properties were analyzed according to Bao [40] and are described briefly as follows. The soil organic matter was measured with the potassium dichromate internal heating method. The soil alkaline-hydrolyzable nitrogen was measured with the alkaline-hydrolyzable diffusion method. The available phosphorus was extracted with 0.05 mol L −1 HCl-0.025 mol L −1 (1/2 H 2 SO 4 ) and determined by molybdenum and antimony analyses. The available potassium was extracted with neutral NH 4 Ac and then measured by flame photometry.

Statistical analysis
The paired-end reads from the original DNA fragments were merged using FLASH [36] and assigned to each sample according to the unique barcodes. Sequences were analyzed using the QIIME (version 7.0) [33] software package, and in-house Perl scripts were used to analyze alpha diversity. Sequences with !97% similarity were assigned to the same operational taxonomic unit (OTU). We selected representative sequences for each OTU and used the RDP classifier [41] to annotate the taxonomic information for each representative sequence. The observed species richness, Shannon diversity index, ACE, and Good's coverage index were used to calculate the alpha diversity with QIIME and were displayed with R software (version 2.15.3). Rarefaction curves were generated based on the observed species richness. The Venn diagram was used to display the common and unique OTUs among the three soil samples. Spearman rank analysis, redundancy analysis (RDA), and the generation of a heat map of the 35 most abundant fungal genera were performed using the R software. The soil physiochemical properties and rhizosphere soil enzyme activities were analyzed by performing a one-way ANOVA as implemented with using the SPSS statistical software (version 16.0; IBM, Armonk, NY, USA).

Sequence accession numbers
All raw sequence data are accessible in the NCBI Sequence Read Archive (SRA) database under accession number SRR5248582.

Physicochemical characteristics and enzyme activities of rhizosphere soil
The physiochemical characteristics of soil during the continuous cropping of C. chinensis are shown in Table 1. Overall, the soil pH declined continuously as the number of planted years increased. The soil organic matter, alkaline-hydrolyzable nitrogen, available phosphorus and available potassium all increased with the increase in the number of continuous planted years.
The soil enzyme activities of sucrase, urease, cellulase, polyphenol oxidase, and catalase were determined to measure the potential turnover rates of nitrogen and carbon ( Table 2). The enzyme activities of urease and catalase showed a sustained significant reduction with the increase in the number of planting years (P < 0.05). The activities of sucrase, cellulase and polyphenol oxidase in RMS5 were significantly higher than those in RMS1 or RMS3 (P < 0.05). Values are means ± standard error (n = 3). Means followed by the same letter for a given factor are not significantly different (P < 0.05; LSD test).

Fungal species richness and alpha diversity
The results of rarefaction curves can directly reflect the ordering of the volume of sequencing data and indirectly reflect the richness of species across all samples. Fig 1a shows that the curve tends to flatten out, indicating that the sequencing data volume is reasonably ordered and that more data would not result in more OTUs. More than 477,898 effective tags were obtained (i.e., 154,043 for RMS1, 160,253 for RMS3, and 163,606 for RMS5) (S1 Table). The tags effective for all the samples were clustered at 97% sequence consistency (i.e., identity) to analyze the species composition and diversity of the samples. The sequence cluster was considered the OTUs. A total of 2,502, 2,064, and 1,720 OTUs were obtained from RMS1, RMS3, and RMS5, respectively (Fig 1b). The alpha diversity of the three samples is presented in Table 3. The observed species richness, Shannon diversity index and ACE declined significantly with the increase in the number of continuous cropping years (P < 0.05). This result indicated that a decrease in fungal species richness and diversity with increasing planting years.

Fungal taxa and relative abundance in continuous cropping rhizosphere soil of C. chinensis
In the rhizosphere soil samples, all the sequences were classified into six fungal phyla using the Mothur program. The overall fungal composition of the three treatments was similar; Ascomycota, Zygomycota, Basidiomycota, and Glomeromycota were the dominant phyla (Fig 2) and had relative abundances > 0.01. ANOVA showed that the relative abundance of Basidiomycota in RMS3 was significantly lower than in RMS1 and in RMS5 (P < 0.05). The relative abundance of Glomeromycota in RMS1 was significantly higher than that in RMS3 and RMS5 (P < 0.05). No significant differences in the relative abundances of Ascomycota or Zygomycota were observed among the three samples (P > 0.05) (S2 Table). For a better and direct illustration of the results, particularly the similarity, variation and relative abundance of fungal composition among the three samples a diagram was produced. Among the fungal samples, the 35 most abundant fungal genera were plotted as a heat map diagram (Fig 3).
As shown in Fig 3, Table).

Sample clustering analysis
The samples were clustered according to their dissimilarity, whereby RMS3 and RMS5 grouped together and were separated from RMS1 (Fig 3), indicating the soil fungal community structure can be affected by continuous cropping of C. chinensis.

Correlation between fungal community composition and environmental factors
The results of the detrended correspondence analysis showed that the fungal community composition responded to the soil physicochemical factors following a linear model. This finding indicates that a further study of the fungal communities associated with the soil environment factors is necessary. Thus, a correlation based on the RDA was conducted (S3 Table). In Fig 4, RDA1 separated RMS1 from RMS3 and RMS5, whereas RDA2 separated RMS3 from RMS5. The pH value showed the most significant effects on the fungal community composition (Fig 4).

Discussion
Continuous cropping problem, also known as continuous cropping obstacle, involving roots, is common to the cultivation of Chinese medicinal herbs [18]. Studies of this problem focused on evaluating the decreasing trend of soil nutrients [42,43]. However, increasing studies have shown that the soil nutrient did not decrease under continuous cropping. In this study, we also observed that available nutrients, such as alkaline-hydrolyzable nitrogen, available phosphorus, and available potassium, all showed an increasing trend with the increase in continuous cropping years. Similar results were also obtained from cropping fields under long-term orchard cropping of black pepper [27], Pseudostellaria heterophylla [18] and wild Rehmannia glutinosa [44]. Moreover, several researchers believed that the accumulation of allelochemicals under field conditions is a cause of continuous cropping obstacle [45]. However, other researchers opposed this assumption [18]. At present, more studies focus on detecting the roles of soil microbial communities and their relationships with soil environmental factors [27].
The richness and variation of the microbial population plays key role in the sustainable development of soil quality, function and ecosystem [46]. In this study, Illumina HiSeq sequencing of ITS1 showed considerable change of the rhizosphere soil fungal community structure and diversity of C. chinensis under continuous cropping. The number of OTUs, Shannon diversity index and ACE declined continuously with the increase in the number of continuous cropping years. Zhang [45] also detected that the total microbial biomass and number of fungi decreased significantly with the increase in continuous cropping years by phospholipids fatty acid analysis.
Compared with the traditional 454 pyrosequencing technology, the Illumina HiSeq sequencing technology can detect species diversity efficiently and precisely [47,48]. In the present research, the fungal ITS1 region as analyzed by Illumina HiSeq sequencing provided detailed insights into fungal community patterns in rhizosphere soil of C. chinensis under continuous cropping. A total of 477,893 effective tags were detected from the three rhizosphere soil samples (S1 Table). The detection of a large number of effective tags indicates that this high-throughput sequencing technique is suitable for analyzing the fungal community composition and their evolution in rhizosphere soils of C. chinensis under continuous cropping.
Soil microbial communities play essential roles in soil organic matter dynamics and nutrient cycling in agro-ecosystems and have been used as soil quality indicators [25,37]. To some extent, changes in the composition of soil microbial communities (e.g., community types) or in microbial biomass can lead to changes in soil quality [49]. The results of the present study indicated that Ascomycota, Zygomycota, Basidiomycota, and Glomeromycota were the dominant fungal phyla in rhizosphere soil under continuous cropping of C. chinensis (Fig 2). At the genus level, this research also showed that continuous cropping of C. chinensis resulted in the increase in the relative abundances of Phoma, Volutella, Pachycudonia, Heterodermia, Cladosporium, Gibberella, Trichocladium, and Sporothrix and a decrease in the relative abundances of Zygosaccharomyces, Pseudotaeniolina, Hydnum, Umbelopsis, Humicola, Crustoderma, Psilocybe, Coralloidiomyces, Mortierella, Polyporus, Pyrenula, and Monographella.
The clustering analysis and RDA indicated a considerable variation in the fungal community structure across the three samples (Figs 3 and 4). This result consistent with the findings of Bai et al. [50] and Tan et al. [36], which showed that the rhizosphere soil fungal community composition and structure were significantly different based on the number of continuous cropping years.
Meanwhile, investigations of the microbial community in rhizosphere soil could provide new opportunities to explore the potential of antagonistic microorganisms for use in the suppression of plant pathogens [35,51]. Fusarium is a pathogen causing root rot in traditional Chinese herbs [18,52]. Previous studies focused on Trichoderma because this genus includes species well known for their biological control properties [53][54][55]. In the present research, the relative abundances of Fusarium were 0.22%, 0.60%, and 0.07% in RMS1, RMS3, and RMS5, respectively. Moreover, the proportions of Trichoderma were 18.61%, 16.20%, and 22.97% in RMS1, RMS3, and RMS5, respectively (S4 Table). The relative abundances of Fusarium and Trichoderma indicated lower proportions of root rot in one-year and five-year continuous cropping of C. chinensis than that in three-year continuous cropping. Changes in Fusarium and Trichoderma levels in the rhizosphere soil may be associated with root rot in C. chinensis plants.
Luo et al [56] first reported that Fusarium solani was one of the fungal pathogens responsible for root rot in C. chinensis cultivated in Shizhu County. However, the dominant species in the present study was Fusarium oxysporum, which showed relative abundance percentages of 0.17%, 0.57%, and 0.07% in RMS1, RMS3, and RMS5, respectively. Furthermore, the relative abundances of F. solani were only 0.04%, 0.02%, and 0.0023% in RMS1, RMS3, and RMS5, respectively (S5 Table). F. oxysporum is a well-described soil-borne fungus [57] responsible for root rot in many traditional Chinese herbs [18,52]. The high relative abundance of F. oxysporum in the samples showed that it may be a potential pathogen contributing to the challenges experienced in C. chinensis continuous cropping system.
Furthermore, knowledge of the soil physicochemical properties and soil enzyme activities in C. chinensis fields under continuous cropping systems could lead to a better understanding of soil productivity and health. Moreover, detecting the connection between the diversity of soil fungal community and the soil environmental factors could provide direct insight into the mechanisms of continuous cropping obstacle [19,27]. Specific constituents of soil nutrients (e.g., C, N, P and K) and pH may impose physiological constraints on fungal survival and growth, thereby directly altering fungal community composition [58]. Of these constituents, soil pH has been proven to be the most influential factor [59]. In this study, the RDA between the soil physiochemical properties and the fungal community at the genus level showed that the pH of rhizosphere soil had the most significant effect on the fungal community composition. Correlation analysis showed that pH positively correlated with the relative abundance of Ascomycota, Glomeromycota, Chytridiomycota, and Neocallimastigomycota, but negatively correlated with the relative abundance of Basidiomycota and Zygomycota (S1 Fig). Soil enzymes are involved in the cycling of soil biological elements and the development of soil fertility [24]. Therefore, soil enzyme activity is an important index of soil quality and ecosystem stability [60]. Urease catalyzes the hydrolysis of urea to produce ammonia and carbamate and is an important indicator of soil health. In this study, the activity of urease showed a significant and continuous decline with the increase in continuous cropping years. This result is consistent with the results obtained for the continuous cropping of the pea (Pisum sativum L.) [61] and sweet wormwood (Artemisia annua L.) [38] and for the monocropping of peanut (Arachis hypogaea L.) [62]. Catalase can break down hydrogen peroxide into molecular oxygen and water and thus preventing reactive oxygen species from damaging the cells. In the present study, the catalase activity decreased significantly with the increase in continuous cropping years of C. chinensis (P < 0.05). The decline in catalase activity may cause the increase in the accumulation of toxic substances in the plant roots of C. chinensis as the number of years of continuous cropping year increases.

Conclusion
In this paper, Illumina high-through sequencing of ITS1 amplicon showed great change in fungal community composition and diversity of rhizosphere soil under different continuous cropping years of Coptis chinensis. pH, urease, and catalase activities of the rhizosphere soil showed a continuous significant decline with the increasing continuous cropping years. The results also indicated that fungal abundance and population diversity decreased continuously with the increase of continuous cropping years. The soil pH had the greatest impact on fungal community composition in rhizosphere soil under continuous cropping of C. chinensis. Whether Fusarium oxysporum is a potential pathogen, which cause the root rot of C. chinensis will depend on the prospective research.
Supporting information S1 Table. Effective tags from the three of C. chinensis rhizosphere soil samples.