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《山东大学学报(理学版)》 ›› 2019, Vol. 54 ›› Issue (1): 36-46.doi: 10.6040/j.issn.1671-9352.3.2018.385

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基于宏基因组测序技术分析连作对杨树人工林土壤微生物群落的影响

张瑛(),马雪松,敬如岩,马风云,郭建曜,王延平,王华田*()   

  1. 山东省高校森林培育重点实验室,山东农业大学林学院,山东 泰安 271018
  • 收稿日期:2018-07-10 出版日期:2019-01-20 发布日期:2019-01-23
  • 通讯作者: 王华田 E-mail:cherrycedar@163.com;sdwht@sdau.edu.cn
  • 作者简介:张瑛(1992—),女,硕士研究生,研究方向为森林培育学. E-mail: cherrycedar@163.com
  • 基金资助:
    国家自然科学基金资助项目(31270670);国家自然科学基金资助项目(31070550);国家重点基础研究发展计划资助项目(2012CB416904/zgc)

Effects of successive-planting poplar plantation on soil microbial community

Ying ZHANG(),Xue-song MA,Ru-yan JING,Feng-yun MA,Jian-yao GUO,Yan-ping WANG,Hua-tian WANG*()   

  1. Key Laboratory of Silviculture in Shandong, Forestry College of Shandong Agricultural University, Taian 271018, Shandong, China
  • Received:2018-07-10 Online:2019-01-20 Published:2019-01-23
  • Contact: Hua-tian WANG E-mail:cherrycedar@163.com;sdwht@sdau.edu.cn
  • Supported by:
    国家自然科学基金资助项目(31270670);国家自然科学基金资助项目(31070550);国家重点基础研究发展计划资助项目(2012CB416904/zgc)

摘要:

为了了解连作人工林土壤微生物群落的演变动态,以Ⅰ代和连作Ⅱ代杨树人工林为研究对象,提取土壤微生物基因组DNA并进行宏基因组测序分析,结合土壤酚酸含量测定,分析阐明连作杨树人工林土壤微生物群落演变动态及其与土壤酚酸类化感物质累积的相关性。结果表明:杨树人工林土壤中共检测到细菌域微生物类群8个门63属,其中芽孢杆菌属(Bacillus)为优势菌群,Ⅰ代和连作Ⅱ代林之间土壤微生物群落多样性和均匀性没有显著变化。连作Ⅱ代林土壤中9属微生物丰度显著升高(P < 0.05),7属微生物丰度显著下降(P < 0.05),连作对杨树人工林土壤微生物群落组成和丰度有显著影响(P < 0.05);参与DNA修复与蛋白重组,次生代谢物的合成、转运与代谢等代谢功能的基因丰度显著下降(P < 0.05)。连作Ⅱ代林土壤酚酸总量显著升高(P < 0.05),并与土壤微生物群落组成和丰度显著相关。

关键词: 杨树人工林, 连作, 宏基因组测序, 微生物, 酚酸

Abstract:

The metagenome sequencing was used to analyze the changes of microbial community between the first and the second continuous cropping of poplar plantations for understanding the effect of successive-planting on soil microbial community, meanwhile the content of phenolic acid in soil was analyzed. The results showed that there were no significant differences in soil microbial community diversity and uniformity while significant differences (P < 0.05) were found in the composition and abundance of microbial community after successive-planting of poplar plantation. Microorganisms from eight categories and sixty-three genera were found with Bacillus being the dominant. The continuous cropping had significant effects on soil microbial community composition and abundance in poplar plantations. The relative abundance of nine genera microbes was significantly increased(P < 0.05) and the relative abundance of seven genera was significantly decreased (P < 0.05) in the soil of second generation forest. The relative abundance of genes related to metabolism such as DNA repair and protein recombination, secondary metabolites biosynthesis, transport and catabolism was decreased. The total content of phenolic acids in soil was significantly increased (P < 0.05), and there was a general correlation between the variation of microbial diversity and phenolic acid content.

Key words: poplar plantation, successive-planting, metagenome sequencing, microbes, phenolic acid

中图分类号: 

  • S718.8

表1

杨树人工林主要土壤因子"

土样 pH 有机质含量/(mg·g-1) 全磷含量/(mg·g-1) 有效磷含量/(μg·g-1) 有效钾含量/(μg·g-1) 有效氮含量/(mg·g-1)
P1 6.17±0.01b 12.18±0.78b 0.10±0.15c 25.56±1.42a 78.36±2.07b 40.65±1.04b
P2 6.12±0.01c 15.42±1.38a 0.14±0.01ab 22.84±1.19a 75.89±4.42b 40.14±2.71b

图1

杨树人工林微生物多样性指数"

图2

微生物群落主成分分析二维排序图"

表2

杨树人工林土壤微生物在门和属水平上的丰度变化"

分类 相对丰度
Ⅰ代林P1 Ⅱ代林P2
厚壁菌门Firmicutes 62.42±1.22a 63.28±1.25a
芽孢杆菌属Bacillus 62.09±1.22a 62.78±1.09a
杆菌属Lysinibacillus 0.01±0.00a 0.00±0.00b
类芽孢杆菌属Paenibacillus 0.00±0.00b 0.01±0.01a
明串球菌属Leuconostoc 0.03±0.01a 0.04±0.03a
链球菌属Streptococcus 0.24±0.03a 0.36±0.13a
乳球菌属Lactococcus 0.06±0.02a 0.09±0.01a
变形菌门Proteobacteria 19.69±0.72a 19.31±0.41a
假单胞菌属Pseudomonas 5.37±0.07b 5.58±0.05a
不动杆菌属Acinetobacter 0.48±0.16a 0.41±0.12a
水栖菌属Enhydrobacter 0.66±0.10a 0.73±0.10a
Thioalkalivibrio 0.47±0.18a 0.22±0.07a
柄杆菌属Caulobacter 0.06±0.04a 0.04±0.01a
军团菌属Legionella 0.08±0.01a 0.04±0.01b
寡养单胞菌属Stenotrophomonas 0.07±0.01a 0.04±0.01a
亚硝化螺菌属Nitrosospira 0.02±0.01a 0.04±0.01a
堆囊菌属Sorangium 0.01±0.00a 0.04±0.02a
Desulfobulbaceae_unclassified 0.00±0.00b 0.02±0.01a
泛生菌属Pantoea 0.01±0.01a 0.00±0.00b
苯基杆菌属Phenylobacterium 0.04±0.01a 0.01±0.01a
Aurantimonadaceae_unclassified 0.19±0.06a 0.10±0.01a
Beijerinckiaceae_unclassified 0.36±0.01a 0.36±0.05a
慢生根瘤菌属Bradyrhizobium 0.68±0.10b 1.06±0.03a
阿菲波菌属Afipia 0.05±0.01a 0.05±0.03a
红假单胞菌属Rhodopseudomonas 1.10±0.20b 2.01±0.10a
硝化菌属Nitrobacter 0.13±0.02a 0.11±0.02a
甲基杆菌属Methylobacterium 0.57±0.11a 0.69±0.21a
Methylocystaceae_unclassified 1.85±0.39a 1.75±0.54a
中慢生根瘤菌属Mesorhizobium 0.26±0.08a 0.31±0.16a
根瘤菌属Rhizobium 0.05±0.02b 0.12±0.02a
Xanthobacteraceae_unclassified 0.09±0.02a 0.08±0.05a
Acetobacteraceae_unclassified 1.01±0.01a 0.67±0.04b
Erythrobacteraceae_unclassified 0.02±0.01a 0.00±0.00b
波氏杆菌属Bordetella 0.06±0.01a 0.04±0.01b
鞘脂单胞菌属Sphingopyxis 0.04±0.02a 0.00±0.00b
伯克氏菌属Burkholderia 5.54±0.79a 4.61±0.24a
贪噬菌属Variovorax 0.16±0.06a 0.16±0.05a
罗尔斯通菌属Ralstonia 0.04±0.01a 0.02±0.01a
贪铜菌属Cupriavidus 0.24±0.06a 0.00±0.00b
放线菌门Actinobacteria 13.62±0.27a 13.07±0.52a
Micromonosporaceae_unclassified 3.96±0.63a 3.22±0.21b
Catenulispora 0.27±0.05a 0.50±0.03a
弗兰克氏菌属Frankia 0.23±0.04b 0.47±0.04a
地嗜皮菌属Geodermatophilus 0.05±0.02a 0.04±0.02a
赖氏菌属Leifsonia 0.02±0.01a 0.01±0.01a
小单孢菌属Micromonospora 0.07±0.02a 0.01±0.01b
节细菌属Arthrobacter 1.06±0.06b 1.69±0.08a
盐孢菌属Salinispora 0.002±0.001a 0.00±0.000b
分歧杆菌属Mycobacterium 7.07±0.07a 5.70±0.14b
Nakamurella 0.00±0.00b 0.02±0.01a
Kribbella 0.03±0.01a 0.04±0.01a
类诺卡氏菌属Nocardioides 0.004±0.001a 0.00±0.000b
丙酸杆菌属Propionibacterium 0.01±0.01a 0.00±0.00b
拟无枝酸菌属Amycolatopsis 0.27±0.11b 0.72±0.16a
链霉菌属Streptomyces 0.41±0.10a 0.58±0.13a
链孢子囊菌属Streptosporangium 0.09±0.04a 0.00±0.00b
双歧杆菌属Bifidobacterium 0.01±0.01a 0.00±0.00b
Conexibacter 0.06±0.04a 0.08±0.04a
酸杆菌门Acidobacteri 1.88±0.53a 1.31±0.44a
Granulicella 1.88±0.53a 1.31±0.44a
Korebacter 0.01±0.01a 0.00±0.00b
拟杆菌门Bacteroidetes 1.49±0.41a 2.01±0.20a
Sphingobacteriaceae_unclassified 1.43±0.42a 1.84±0.19a
黄杆菌属Flavobacterium 0.07±0.02b 0.17±0.04a
绿弯菌门Chloroflexi 0.51±0.02b 0.84±0.01a
纤线杆菌属Ktedonobacter 0.06±0.02b 0.16±0.01a
疣微菌门Verrucomicrobia 0.37±0.05a 0.19±0.03b
Chthoniobacter 0.36±0.05a 0.19±0.03b
丰佑菌属Opitutus 0.01±0.00a 0.00±0.00b
浮霉菌门Planctomycetes 0.01±0.01a 0.01±0.01a
出芽菌属Gemmata 0.01±0.01a 0.01±0.01a

图3

eggNOG功能注释结果在各土壤样本中相对丰度变化 A: RNA加工和修饰; B:染色质结构和动力学; C:能源生产和转换; D:细胞周期控制,细胞分裂,染色体分割; E:氨基酸转运和代谢; F:核苷酸转运和代谢; G:碳水化合物的运输和代谢; H:辅酶转运和代谢; I:脂质运输和代谢; J:翻译,核糖体结构和生源; K:转录; L:复制,重组和修复; M:细胞壁/膜/包膜生源; N:细胞运动; O:翻译后修饰,蛋白质周转,伴侣蛋白; P:无机离子转运和代谢; Q:次生代谢产物的生物合成,转运和分解代谢; R:仅通用功能预测; S:不详; T:信号转导机制; U:细胞内运输,分泌和囊泡运输; V:防御机制; W:细胞外结构; Y:核结构; Z:骨架。*表示差异显著,下同。"

图4

KEGG代谢通路第2层次注释结果在各土壤样本中相对丰度变化 L2_01:碳水化合物代谢; L2_02:脂类代谢; L2_03:氨基酸代谢; L2_04:辅酶因子和维生素代谢; L2_05:外源性物质降解和代谢; L2_06:其他次生代谢产物的生物合成; L2_07:能量代谢; L2_08:多酮类化合物和萜类化合物的代谢; L2_09:消化系统; L2_10:信号转导; L2_11:其他氨基酸代谢; L2_12:运输和分解代谢; L2_13:代谢; L2_14:核苷酸代谢; L2_15:多糖生物合成和代谢; L2_16:神经退行性疾病; L2_17:内分泌系统; L2_18:不详; L2_19:排泄系统; L2_20:转录; L2_21:循环系统; L2_22:细胞过程和信号; L2_23:遗传信息加工; L2_24:感染病; L2_25:复制和修复; L2_26:翻译; L2_27:折叠、排序和退化; L2_28:细胞运动性; L2_29:酶家族; L2_30:信号分子与互动; L2_31:癌症; L2_32:代谢疾病; L2_33:免疫系统; L2_34:环境适应; L2_35:细胞生长和死亡; L2_36:心血管疾病; L2_37:膜运输; L2_38:免疫系统疾病; L2_39:细胞交流; L2_40:感觉系统。"

图5

KEGG代谢通路第3层次注释结果在各土壤样本中相对丰度变化 ko03400: DNA修复和蛋白重组; ko02000:转运; ko00230:嘌呤代谢; ko00240:嘧啶代谢; L3_118:仅通用功能预测; ko02010: ATP结合盒转运蛋白; ko01007:氨基酸相关酶; ko01002:肽酶; ko02020:双组分系统; ko00190:氧化磷酸化; ko00720:原核生物中的碳固定途径; ko00620:丙酮酸代谢; ko00970:氨酰基-tRNA生物合成; ko00020: TCA循环; ko00250:丙氨酸,天冬氨酸和谷氨酸代谢; ko00010:糖酵解/糖异生; ko00680:甲烷代谢; ko00330:精氨酸和脯氨酸代谢; ko00640:丙酸代谢; ko02044:分泌系统; ko03032: DNA复制蛋白; ko00650:丁酸代谢; ko00280:缬氨酸,亮氨酸和异亮氨酸降解; ko03110:分子伴侣和折叠催化剂; ko00290:缬氨酸,亮氨酸和异亮氨酸的生物合成; ko00260:甘氨酸,丝氨酸和苏氨酸代谢; ko03036:染色体; L3_111:其他离子耦合转运蛋白; ko00630:乙醛酸和二羧酸代谢; ko00270:半胱氨酸和蛋氨酸代谢; ko00520:氨基糖和核苷酸糖代谢; ko03020: RNA聚合酶; ko03010:核糖体。"

表3

杨树人工林土壤微生物基因预测统计"

土样 编码蛋白数(106) KEGG同源基因数 eggNOG同源基因数
P1 4.70±0.16a 6 534±112a 43 580±219a
P2 4.58±0.16ab 6 520±58a 43 182±576a

表4

不同土壤样本酚酸含量变化"

土样 对羟基苯甲酸 香草醛 阿魏酸 苯甲酸 肉桂酸 总量
P1 13.31±0.03b 3.16±0.01a 5.89±0.33a 6.73±0.15b 0.53±0.02b 29.63±0.23b
P2 15.93±0.14a 2.12±0.09b 5.11±0.18b 7.15±0.15a 0.81±0.07a 31.12±0.03a

表5

土壤微生物与酚酸含量之间的相关系数"

对羟基苯甲酸 香草醛 阿魏酸 苯甲酸 肉桂酸 酚酸总量
假单胞菌属Pseudomonas 0.92* -0.92** -0.82* 0.78 0.87* 0.87*
红假单胞菌属Rhodopseudomonas 0.97** -0.96** -0.74 0.75 0.94** 0.99**
节细菌属Arthrobacter 0.98** -0.97** -0.87* 0.88* 0.97** 0.97**
慢生根瘤菌属Bradyrhizobium 0.96** -0.95** -0.76 0.77 0.94** 0.98**
拟无枝酸菌属Amycolatopsis 0.91* -0.91* -0.64 0.63 0.83* 0.93**
弗兰克氏菌属Frankia 0.96** -0.97** -0.91** 0.88* 0.90* 0.92**
根瘤菌属Rhizobium 0.92* -0.91* -0.76 0.78 0.92** 0.94**
黄杆菌属Flavobacterium 0.87* -0.87* -0.82* 0.82* 0.88* 0.86*
纤线杆菌属Ktedonobacter 0.97** -0.98** -0.89* 0.86* 0.88* 0.93**
分歧杆菌属Mycobacterium -0.99** 0.99** 0.86* -0.84* -0.93** -0.97**
Micromonosporaceae_unclassified -0.94** 0.94** 0.80 -0.76 -0.82* -0.97**
Acetobacteraceae_unclassified -0.99** 0.99** 0.85* -0.83* -0.93** -0.98**
Chthoniobacter -0.93** 0.92** 0.75 -0.76 -0.92** -0.95**
小单孢菌属Micromonospora -0.87* 0.87* 0.99** -0.97** -0.86* -0.80
军团菌属Legionella -0.89* 0.88* 0.86* -0.88* -0.94** -0.89*
波氏杆菌属Bordetella -0.96** 0.95** 0.95** -0.94** -0.94** -0.92*
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