Differentiations between Microbes in Cultivated and Wild Paris Polyphylla Field Soils
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摘要:
目的 探明栽培与野生七叶一枝花土壤细菌群落组成的差异。 方法 采用illumina miseq 2×300 bp高通量测序对栽培与野生七叶一枝花土壤的细菌16S rRNA基因序列进行测序分析；同时，利用LDA Effect Size（LEfSE）软件对组间群落微生物丰富度的差异分析，比较栽培与野生七叶一枝花土壤细菌群落组成的差异，明确影响2种栽培模式的重要微生物门类。 结果 七叶一枝花土壤中的微生物组成中，野生七叶一枝花土壤细菌种类优于栽培七叶一枝花土壤。丰富度指数（Ace和Chao）和多样性指数（Shannon和Simpson）分析表明，野生七叶一枝花土壤细菌群落更具有更高的丰富性和多样性。在门水平，栽培和野生七叶一枝花土壤细菌群落具有显著差异的门包括Firmicutes、硝化螺旋菌门（Nitrospirae）和螺旋菌门（Spirochaetae）；显著差异的属包括芽孢杆菌（Bacillus）、纤线杆菌属（Ktedonobacter）和类芽孢杆菌（Paenibacillus）等。利用LEfSE软件对组间群落微生物丰富度的差异分析发现，厚壁菌门（Firmicutes）和硝化螺旋菌门（Nitrospirae）是野生七叶一枝花土壤样本中的优势菌门，优势属包括芽孢杆菌（Bacillus）、类芽孢杆菌（Paenibacillus）、Tumebacillus、Mucilaginibacter、硝化螺菌属（Nitrospira）、Shimazuella和Singulisphaera；栽培七叶一枝花土壤样本中起到重要作用的门水平细菌群落是装甲菌门（Armatimonadetes），属水平细菌群落是Bryobacter、Aquicella和纤线杆菌属（Ktedonobacter）。相关性分析结果表明，土壤pH与土壤全钾是影响七叶一枝花土壤微生物群落多样性的主要因素。 结论 不同栽培模式和土壤养分影响七叶一枝花土壤微生物多样性，为七叶一枝花的栽培管理与维护提供参考。 Abstract:Objective Diversity of the microbial communities in soils of cultivated and wild Paris polyphylla var. chinensis fields were compared. Method Total DNA of the microbes on cultivated land grown P. polyphylla plants or at field of the plants in the wild were sequenced using high throughput Illumina Miseq (2×300 bp). Structure and abundance of the microbial communities in soils of the fields were comparatively analyzed by LDA Effect Size. Result The microbial diversity of the wild P. polyphylla lots was richer than the cultivated land. The Chao, Ace, Shannon, and Simpson indexes of the wildP. polyphylla soil were higher than the cultivated counterparts. The communities significantly differed on the abundant phyla of Firmicutes, Nitrospirae, and Spirochaetae, and on the genera of Bacillus, Ktedonobacter, and Paenibacillus. LDA Effect Size showed Firmicutes and Nitrospirae to be the predominant phyla, while Bacillus, Paenibacillus, Tumebacillus,Mucilaginibacter, Nitrospira, Shimazuella, and Singulisphaera the dominant genera in the wild. In the cultivated soil, the Armatimonadetes phylum and the Bryobacter, Aquicella, and Ktedonobacter genera predominated the community. pH and total potassium content of soil were the critical factors affecting the diversity of a microbial community. Conclusion Cultivation and soil nutrients significantly differentiated the microbial composition at a P. polyphylla field. -
Key words:
- Paris polyphylla var. chinensis /
- rhizosphere /
- soil /
- metagenomics /
- diversity
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图 1 栽培和野生七叶一枝花土壤细菌物种数量16S rRNA 相似水平为 97%的稀释性曲线
Figure 1. Rarefaction curves on counts of microbes with 97% 16S rRNA similarity in cultivated and wild P. polyphylla soils
图 2 栽培和野生七叶一枝花土壤细菌物种（OTU）组成的韦恩图分析
Figure 2. Venn analysis on microbial OTUs of cultivated and wild P. polyphylla soils
图 3 门（A）和属（B）水平的微生物组成、相对丰度分析
Figure 3. Composition and relative abundance of dominant microbes at phylum level (A) and genus level (B)
图 4 栽培和野生七叶一枝花土壤细菌物种的Alpha多样性指数
Figure 4. Alpha diversity of microbes in cultivated and wild P. polyphylla soils
图 5 LDA Effect Size组间群落的差异分析
Figure 5. LDA Effect Size on differential abundant microbes
图 6 基于OTU水平的Principal co-ordinates analysis分析
Figure 6. Principal coordinates analysis (PCoA) based on OTU of 8 samples
图 7 不同栽培方式七叶一枝花土壤的unweighted.unifrac差异性矩阵热图（相似水平97%）
Figure 7. Heatmap of unweighted.unifrac dissimilarity matrix on microbes in cultivated and wild P. polyphylla soils (Similar level 97%)
表 1 栽培和野生七叶一枝花土壤样本信息
Table 1. Information on cultivated and wild P. polyphylla soils
编号
Code采集地
Locality栽培模式
Transplanting modes栽培年限
Transplanting period/aYR1 福建省南平市建瓯吉阳镇郭岩山双龙庙 野生 — YNR1 福建省南平市建瓯吉阳镇郭岩山双龙庙 野生 — YR2 福建省南平市建瓯吉阳镇郭岩山双龙庙 野生 — YNR2 福建省南平市建瓯吉阳镇郭岩山双龙庙 野生 — ZR1 福建省南平市建瓯吉阳镇郭岩山双龙庙 栽培 5 ZNR1 福建省南平市建瓯吉阳镇郭岩山双龙庙 栽培 5 ZR2 福建省南平市政和县官湖村王坑陇 栽培 2 ZNR2 福建省南平市政和县官湖村王坑陇 栽培 2 YR和ZR分别代表野生和栽培的根际土壤，YNR和ZNR分别代表野生和栽培的非根际土壤。
YR and ZR represent wild and cultivated rhizosphere soils, respectively; YNR and ZNR represent wild and cultivated non-rhizosphere soils, respectively.
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表 2 栽培和野生七叶一枝花土壤养分含量
Table 2. Nutrient contents of cultivated and wild P. polyphylla soils
编号
CodepH 有机质
Organic matter/(g·kg−1)全氮
Total nitrogen/(g·kg−1)全磷
Total phosphorus/(g·kg−1)全钾
Total potassium/(g·kg−1)YR1 5.20±0.30ab 118.00±6.81a 2.60±0.15ab 0.70±0.04a 3.20±0.18a YNR1 5.30±0.31ab 105.30±6.08a 1.90±0.11de 0.50±0.03b 3.30±0.19a YR2 5.90±0.34ab 110.10±6.36a 1.40±0.08f 0.80±0.05a 1.50±0.09c YNR2 5.70±0.33ab 107.10±6.18a 1.40±0.08f 0.70±0.04a 1.90±0.11b ZR1 6.30±0.36a 60.70±3.50b 2.30±0.13bc 0.30±0.02c 1.50±0.09c ZNR1 6.30±0.36a 56.80±3.28b 2.70±0.16a 0.40±0.02bc 1.70±0.10bc ZR2 5.10±0.29b 70.30±4.06b 2.20±0.13cd 0.50±0.03b 1.00±0.06d ZNR2 5.30±0.31ab 39.70±2.29c 1.80±0.10e 0.40±0.02bc 1.10±0.06d 同列数据后不同小写字母代表处理间差异显著（P＜0.05）。
Data with different lowercase letters on same column indicate significant differences ( P＜0.05).
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表 3 栽培和野生七叶一枝花土壤的细菌分离数量
Table 3. Plate counts of cultivated and wild P. polyphylla soils
样品名称
Sample name细菌含量
Bacterial content /
(×106 cfu·g−1)样品名称
Sample name细菌含量
Bacterial content /
(×106 cfu·g−1)YR1 5.05 ZR1 3.50 YNR1 4.70 ZNR1 6.85 YR2 8.10 ZR2 5.50 YNR2 8.55 ZNR2 1.75
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表 4 栽培和野生七叶一枝花土壤样本不同分类阶元细菌物种（OTU）数量
Table 4. Number of microbes (OTU) at different taxonomical levels of cultivated and wild P. polyphylla soils
样本
SampleOTU 门水平
Phylum level纲水平
Class level目水平
Order level科水平
Family level属水平
Genus level种水平
Species levelYR1 1173 19 31 44 73 114 125 YNR1 1049 21 30 46 77 123 131 YR2 1122 20 28 42 73 114 130 YNR2 1173 19 29 40 70 117 128 ZR1 1198 21 31 47 78 142 140 ZNR1 1417 21 31 48 81 138 149 ZR2 630 20 27 34 62 88 85 ZNR2 904 20 27 36 64 108 104
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表 5 栽培和野生七叶一枝花土壤细菌物种的Alpha多样性指数
Table 5. Alpha diversity of microbes in cultivated and wild P. polyphylla soils
样本 Sample Chao指数
Chao indexAce指数
Ace indexShannon指数
Shannon indexSimpson 指数
Simpson index覆盖率
CoverageYR1 1383.4930 1353.6621 5.2116 0.0200 0.9929 YNR1 1252.7638 1229.8781 5.1894 0.0166 0.9933 YR2 1266.0820 1239.7667 5.4007 0.0143 0.9943 YNR2 1347.2803 1314.3759 5.1740 0.0303 0.9937 ZR1 1318.3147 1313.4214 5.5856 0.0112 0.9944 ZNR1 1544.8750 1515.2990 5.9857 0.0070 0.9944 ZR2 758.2692 708.8108 4.1124 0.0588 0.9967 ZNR2 1013.1720 995.5008 5.0457 0.0211 0.9956
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表 6 门和属水平的微生物组间群落显著性的差异分析
Table 6. Differential abundant microbes at phylum and genus levels
分类水平
Classification level细菌名称
Bacterial name丰富度 Abundance P值
P value栽培 Transplanting 野生 Wild 门
Phylum厚壁菌门 Firmicutes 0.0047 ±0.0009b 0.0713 ±0.0066a 0.0001 硝化螺旋菌门 Nitrospirae 0.0071 ±0.0039a 0.0309 ±0.0052a 0.0107 绿弯菌门 Chloroflexi 0.0617 ±0.0045a 0.0446 ±0.0062a 0.0659 螺旋菌门 Spirochaetae 0.0002 ±0.0002b 0.0009 ±0.0001a 0.0133 属
Genus芽孢杆菌属 Bacillus 0.0015 ±0.0004b 0.0337 ±0.0022a 0.0000 Dyella 0.0022 ±0.0013a 0.0106 ±0.0038a 0.0797 类芽孢杆菌属 Paenibacillus 0.0015 ±0.0005b 0.0245 ±0.0049a 0.0033 Tumebacillus 0.0017 ±0.001b0.0101 ±0.0024a 0.0186 Shimazuella 0.0000 ±0.0000b 0.0028 ±0.0007a 0.0052 Mucilaginibacter 0.0016 ±0.0008a 0.0069 ±0.0023a 0.0689 Bryobacter 0.0195 ±0.0026a 0.0082 ±0.0008b 0.0064 硝化螺旋菌属 Nitrospira 0.0022 ±0.0013b 0.0068 ±0.001a0.0322 分枝杆菌属 Mycobacterium 0.0007 ±0.0001b 0.0013 ±0.0001a 0.0115 丰佑菌属 Opitutus 0.0030 ±0.0007a 0.0009 ±0.0002b 0.0315 中华单胞菌属 Sinomonas 0.0003 ±0.0002a 0.0000 ±0.0000a 0.0826 Dokdonella 0.0001 ±0.0001a 0.0004 ±0.0001a 0.0692 Candidatus_Entotheonella 0.0001 ±0.0001a 0.0005 ±0.0001a 0.0615 Bryocella 0.0006 ±0.0002a 0.0000 ±0.0000b 0.0400 Singulisphaera 0.0001 ±0.0000b 0.0002 ±0.0000a 0.0148 纤线杆菌属 Ktedonobacter 0.0003 ±0.0000a 0.0000 ±0.0000b 0.0015
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表 7 七叶一枝花土壤微生物多样性与土壤理化性质的Pearson相关性分析
Table 7. Pearson correlation between microbial diversity and physicochemical properties of P. polyphylla soil
项目
ItemspH 有机质
Organic
matte全氮
Total
nitrogen全磷
Total
phosphorus全钾
Total
potassium细菌含量
Bacterial
contentChao指数
Chao
indexAce指数
Ace
indexShannon指数
Shannon
indexSimpson 指数
Simpson
index覆盖度
coveragepH 1 有机质 Organic matter 0.043 1 全氮 Total nitrogen 0.718 0.509 1 全磷 Total phosphorus −0.765 0.307 −0.110 1 全钾 Total potassium 0.969* 0.011 0.805 −0.618 1 细菌含量 Bacterial content 0.309 0.660 0.884 0.365 0.451 1 Chao指数 Chao index 0.946 −0.174 0.699 −0.664 0.983* 0.323 1 Ace指数 Ace index 0.950 −0.196 0.671 −0.696 0.978* 0.282 0.999** 1 Shannon 指数 Shannon index 0.912 −0.339 0.552 −0.742 0.934 0.145 0.982* 0.988* 1 Simpson 指数 Simpson index −0.832 0.519 −0.333 0.823 −0.824 0.100 −0.907 −0.923 −0.970* 1 覆盖度 Coverage −0.958* 0.242 −0.534 0.849 −0.928 −0.088 −0.959* −0.970* −0.976* 0.954* 1 *和**分别表示在0.05和0.01水平上相关性显著。
* and * * indicate significant correlation at 0.05 and 0.01 levels, respectively.
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