浙江大学学报（农业与生命科学版）

浙江大学学报（农业与生命科学版）, 2024, 50(1): 98-108 doi: 10.3785/j.issn.1008-9209.2023.03.012

资源利用与环境保护

施肥旱作农田土壤中人类病原菌群落组成及动态变化特征

齐明慧,,1, 程建华,,1,2, 唐翔宇1,2

1.浙江农林大学林业与生物技术学院，省部共建亚热带森林培育国家重点实验室，浙江 杭州 311300

2.中国科学院、水利部成都山地灾害与环境研究所，山地表生过程与生态调控重点实验室，四川 成都 610041

Composition and dynamic change characteristics of human pathogenic communities in dryland farmland with manure application

QI Minghui,,1, CHENG Jianhua,,1,2, TANG Xiangyu1,2

1.State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

2.Key Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, Chengdu 610041, Sichuan, China

通讯作者: 程建华（https://orcid.org/0000-0002-2954-1738），E-mail：chengjh@zafu.edu.cn

收稿日期: 2023-03-01   接受日期: 2023-05-16  

基金资助: 国家自然科学基金项目.  42007361
浙江农林大学科研发展基金项目.  2020FR040.  2021LFR045
浙江省自然科学基金项目.  LD21D010001

Received: 2023-03-01   Accepted: 2023-05-16  

作者简介 About authors

齐明慧（https://orcid.org/0009-0004-9190-284X），E-mail：2020102022012@stu.zafu.edu.cn , E-mail：2020102022012@stu.zafu.edu.cn

摘要

采集浙江省杭州、嘉兴、衢州、金华、龙游5个地区长期施用鸡粪、猪粪或化肥的农田土壤进行室内培养实验，然后采用高通量测序及序列比对方法分析供试土壤细菌和人类病原菌（human pathogenic bacteria, HPB）群落组成，旨在进一步探究施用粪肥后土壤中HPB群落组成差异及动态变化特征。结果表明：在160个供试土壤样品和2种粪肥样品中共比对到75种HPB，其中优势HPB为巨大芽孢杆菌（Bacillus_megaterium_QM_B1551，24.2%）和拜氏梭菌（Clostridium_beijerinckii_NCIMB_8052，23.1%）；施用猪粪处理的土壤中细菌和HPB的香农（Shannon）指数均有所降低，而施用鸡粪或化肥处理的土壤中细菌和HPB多样性无明显变化。主坐标分析结果表明，施用粪肥处理的土壤细菌群落组成与未施肥对照之间具有显著差异，尤其是施用猪粪处理差异最显著（P＜0.001）；土壤样品间共有HPB占所有HPB的22.7%；施用粪肥处理的土壤中大部分HPB的相对丰度高于未施肥对照，并随培养时间的延长而不断下降。方差分解分析结果表明，土壤理化性质、细菌群落及两者交互作用是土壤中HPB变异的重要因素。综上所述，施用粪肥处理的土壤中HPB的变化特征主要受肥料类型、土壤类型、土壤理化性质及固有细菌群落的影响。

关键词： 人类病原菌 ; 粪肥 ; 细菌群落 ; 土壤 ; 动态变化

Abstract

To understand the community structure composition and dynamic change characteristics of human pathogenic bacteria (HPB) in soils after manure application, laboratory cultivation experiments were conducted on agricultural soils with long-term application of chicken manure, pig manure, or chemical fertilizer in five regions of Hangzhou, Jiaxing, Quzhou, Jinhua, and Longyou in Zhejiang Province, and the community compositions of the soil bacteria and HPB were analyzed by high-throughput sequencing and sequence alignment methods. The results showed that a total of 75 HPB were detected in 160 soil samples and two manure samples, and the dominant HPB were Bacillus_megaterium_QM_B1551 (24.2%) and Clostridium_beijerinckii_NCIMB_8052 (23.1%). The Shannon indexes of bacteria and HPB in the soils decreased after the application of pig manure, while the diversities of bacteria and HPB in the soils with the application of chicken manure or chemical fertilizer had no significant changes. The results of the principal coordinate analysis showed that there was a significant difference in the bacterial community composition of soils between the manure treatment and the unfertilized control, especially in the pig manure treatment (P＜0.001); 22.7% of all HPB were shared among the soil samples; and the relative abundance of most HPB in the soils treated with manure was higher than that in the unfertilized control, and it decreased continuously with the extension of cultivation time. The results of the variance partitioning analysis showed that soil physicochemical properties, bacterial communities, and their interactions were important factors contributing to the variation of HPB in the soils. In summary, the HPB variation characteristics in soils treated with manure are influenced mainly by manure types, soil types, soil physicochemical properties, and inherent bacterial communities.

Keywords： human pathogenic bacteria ; manure ; bacterial community ; soil ; dynamic change

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齐明慧, 程建华, 唐翔宇. 施肥旱作农田土壤中人类病原菌群落组成及动态变化特征. 浙江大学学报（农业与生命科学版）[J]. 2024, 50(1): 98-108 doi:10.3785/j.issn.1008-9209.2023.03.012

QI Minghui, CHENG Jianhua, TANG Xiangyu. Composition and dynamic change characteristics of human pathogenic communities in dryland farmland with manure application. Journal of Zhejiang University （Agriculture & Life Sciences）[J]. 2024, 50(1): 98-108 doi:10.3785/j.issn.1008-9209.2023.03.012

土壤生态系统作为地球重要的生态系统之一，其服务功能的可持续性与人类栖息和粮食安全息息相关[1]。施用粪肥作为农田土壤中常见的施肥措施，能够提高土壤有机质、氮、磷等养分含量，从而显著增加作物产量[2-3]。但畜禽粪便中携带的多种人类病原菌（human pathogenic bacteria, HPB）也会通过施肥进到农田土壤、周围水体甚至作物体内，从而威胁人体健康[4-7]。FANG等[8]研究发现，HPB通过污水排放及粪肥施用从养猪场传播至河流及农田土壤。LI等[6]研究发现，施用粪肥导致表层土壤与小白菜根部的HPB丰度增加，甚至发生垂直迁移，传播到更深层土壤。土壤中HPB的存活受多种因素影响，包括粪肥类型[9]、施用量[10]、土壤理化性质[6]、固有微生物[11]等。

目前，相关研究主要关注施用一种或多种粪肥的某种土壤中HPB的赋存特征和变化。ZHU等[12]研究表明，化肥、猪粪、鸡粪、牛粪、蚕粪等不同肥料源HPB均会向土壤扩散，且其多样性和丰度存在显著差异。LI等[13]探究了HPB在2种施用量的猪粪、鸡粪和化肥处理土壤中的动态变化特征，结果表明，高施用量的猪粪显著提高了土壤中HPB丰度，且粪源HPB丰度随培养时间的延长而下降。但系统阐述施用不同肥料后土壤中不同类型HPB群落组成与动态变化特征的研究鲜有报道。尽管已有多种数学模型可以预测病原菌在农业环境中的运输和分布[14-15]，但仍需大量的室内和大田数据作为支撑。浙江省土壤类型多样，包括红壤、黄壤、石灰（岩）土、潮土、水稻土等10个土类[16]。此外，浙江省畜禽养殖业发达，年产值占全省总产值的1/4，畜禽粪尿产生量为1×107～2×107 t[17]。浙江省11个地市中畜禽粪尿产生量以杭州、嘉兴、衢州三地最高，占总量的48.3%[18]。对畜禽粪便的处理以农用储存和有机肥生产为主，占比为65%～75%[19]。因此，本研究选取浙江省杭州、嘉兴、衢州、金华、龙游5个地区长期施用粪肥和化肥的旱作农田土壤进行为期120 d的室内培养实验，并采用高通量测序技术分析施用不同肥料对土壤中细菌和HPB群落组成及其动态变化特征的影响，旨在为粪肥处理和合理施用提供科学参考。

1 材料与方法

1.1 材料

土壤样品取自浙江省嘉兴市（JX）、金华市婺城区汤溪镇（TX）、衢州市开化县塘坞（TW）、杭州市临安区昌化镇（CH）、龙游市（LY）5个地区，对应的土壤类型分别为潮土、红壤、石灰土、黄壤、红壤。在每个地区采集相邻的长期（5年以上）施用猪粪、鸡粪和化肥的旱作农田耕作层（0～20 cm）土壤，分别记作PM_Soil、CM_Soil、NPK_Soil。因在龙游地区未找到3种相邻的土壤，故分别采集与施用猪粪或鸡粪相邻的施用化肥的农田土壤（1_NPK_Soil和2_NPK_Soil）。表1为原始土壤样品的基本理化性质。将所有土壤样品风干后，研磨并过2 mm筛，备用。新鲜鸡粪与猪粪取自农户，风干后过2 mm筛，备用。化肥为复合肥[m（N）∶m（P）∶m（K）=16∶16∶16]。

表1   原始土壤样品的基本理化性质

Table 1  Basic physicochemical properties of the original soil samples

原始土壤样品

Original soil sample

pH值

pH value

电导率

EC/

(µS/cm)

铵态氮

NH4＋-N/

(mg/kg)

硝态氮

NO3－-N/

(mg/kg)

有效磷

AP/

(mg/kg)

总有机碳

TOC/

(mg/g)

全磷

TP/

(mg/g)

全氮

TN/

(mg/g)

全钾

TK/

(mg/g)

嘉兴市

JX

CM_Soil7.54103.5512.2724.9171.2920.762.501.686.89
PM_Soil8.08132.2519.8042.11129.5626.641.341.816.92
NPK_Soil6.8694.3016.7340.9799.1623.011.301.686.60

汤溪镇

（金华市）

TX

CM_Soil5.5763.7515.0528.98750.8822.230.991.324.96
PM_Soil6.45120.9019.1062.231 457.0424.080.601.425.25
NPK_Soil4.5856.556.8227.0938.3313.400.491.294.49

塘坞

（衢州市）

TW

CM_Soil5.8382.3516.7346.7845.9418.812.771.156.26
PM_Soil5.16103.3522.4471.23305.4419.882.281.186.51
NPK_Soil7.96142.504.9990.9430.0923.324.441.115.72

昌化镇

（杭州市）

CH

CM_Soil7.13704.5012.45520.70540.9952.083.808.5110.27
PM_Soil7.93190.005.81108.5144.0616.740.121.076.32
NPK_Soil5.3366.959.0125.82201.1915.010.110.986.84

龙游市

LY

CM_Soil5.60283.0037.59119.82134.4428.350.111.196.16
PM_Soil6.79166.3525.0568.62587.5529.380.101.355.97
1_NPK_Soil4.8938.9514.7313.65136.326.650.550.577.46
2_NPK_Soil4.7963.2010.8523.34112.7915.590.480.647.25

CM_Soil：长期施用鸡粪的土壤；PM_Soil：长期施用猪粪的土壤；NPK_Soil：长期施用化肥的土壤；1_NPK_Soil：龙游地区长期施用化肥的土壤1（LY1）；2_NPK_Soil：龙游地区长期施用化肥的土壤2（LY2）。下同。

CM_Soil: Soil with long-term application of chicken manure; PM_Soil: Soil with long-term application of pig manure; NPK_Soil: Soil with long-term application of chemical fertilizer; 1_NPK_Soil: Soil 1 with long-term application of chemical fertilizer in Longyou area (LY1); 2_NPK_Soil: Soil 2 with long-term application of chemical fertilizer in Longyou area (LY2). EC: Electrical conductivity; AP: Available phosphorus; TOC: Total organic carbon; TP: Total phosphorus; TN: Total nitrogen; TK: Total potassium. The same as below.

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1.2 实验设计

实验开始前，调节土壤样品含水率为田间最大持水量的60%，25 ℃预培养2周。称取50 g（干土质量）预培养后的土壤于培养皿内，置于培养箱中进行培养实验，培养温度为25 ℃。每个地区共设6个处理：在长期施用鸡粪的土壤上进行鸡粪处理（CMs）与不施肥对照处理（CMs_CK）、在长期施用猪粪的土壤上进行猪粪处理（PMs）与不施肥对照处理（PMs_CK）、在长期施用化肥的土壤上进行化肥处理（NPKs）与不施肥对照处理（NPKs_CK）。每个处理设置3个重复。参考常规施肥量，粪肥施用量为40 mg/g（相当于田间施用量30 m3/hm2），化肥施用量为600 kg/hm2，均一次性施入。在培养期间，根据失水情况每隔3～5 d补足水分至田间最大持水量的60%。取样时间为第1、7、30、60、120天。每次取样后，另将同一处理的3个重复样品混合，作为该处理该天的代表样品。实验共获得160个土壤样品，将其冷冻干燥后置于－80 ℃冰箱中保存，备用。

1.3 土壤理化指标测定

采用pH计（瑞士Mettler-Toledo公司）测定土壤上清液的pH值（土水质量体积比为1∶5），并使用DDS-608电导仪测定土壤上清液的电导率（EC）。采用2 mol/L氯化钾提取-分光光度法测定土壤铵态氮（NH4＋-N）和硝态氮（NO3－-N）含量。根据土壤pH值，分别采用碳酸氢钠法（pH值＞6.5）与盐酸-氟化铵法（pH值≤6.5）测定土壤有效磷（AP）含量。利用重铬酸钾氧化法测定土壤总有机碳（TOC）含量。采用浓硫酸-高氯酸消煮和钼锑抗比色法测定土壤全磷（TP）含量。采用凯氏定氮法测定土壤全氮（TN）含量。

1.4 土壤DNA提取、16S rRNA测序与HPB比对

采用DNeasy PowerSoil Pro试剂盒（德国Qiagen公司）提取土壤、鸡粪（chicken manure, CM）和猪粪（pig manure, PM）DNA。使用引物对（515F/907R）对DNA样品16S rRNA的V4～V5区进行聚合酶链反应（polymerase chain reaction, PCR）扩增。将扩增产物回收并纯化后进行文库构建，然后利用Illumina NovaSeq PE250平台进行测序（上海美吉生物医药科技有限公司）。原始序列数据已上传至组学原始数据归档库（Genome Sequence Archive, GSA），序列号为CRA008761。

采用QIIME2软件对原始序列进行处理，并使用Fastp[20]（v0.19.6）软件（https://github.com/OpenGene/fastp）对双端原始测序序列进行质控，然后使用FLASH[21]（v1.2.11）软件（http://www.cbcb.umd.edu/software/flash）进行拼接。基于默认参数，使用QIIME2软件中的DADA2方法对质控后的拼接序列进行降噪处理，获得扩增子序列变体（amplicon sequence variants, ASVs）表格。参照Silva138数据库，采用朴素贝叶斯（Naive Bayes）方法进行物种注释。根据样品的最小序列数，对所有样品序列进行抽平处理。后续统计分析均基于抽平后的ASVs丰度信息进行。

从美国国家生物技术信息中心（https://www.ncbi.nlm.nih.gov/）下载HPB数据库（557个序列），采用BLASTN软件将样品16S rRNA序列与HPB数据库进行比对，以E值＜1×10－10、同一性值≥99%为阈值[22]。样品中HPB的相对丰度为比对得到的HPB序列数与总序列数（16S rRNA）的比值。

1.5 数据处理与分析

使用Microsoft Excel 2010进行数据整理，利用SPSS 26.0进行单因素方差分析，显著性水平为0.05。利用美吉生物云平台（https://cloud.majorbio.com）计算香农（Shannon）指数。采用R软件的vegan包进行主坐标分析（principal coordinate analysis, PCoA）、置换多元方差分析（ADONIS）、冗余分析（redundancy analysis, RDA）和方差分解分析（variance parti-tioning analysis, VPA）。利用R软件的UpsetR包和ComplexHeatmap包绘制Upset图和热图。

2 结果与分析

2.1 土壤理化性质

整体而言，与不施肥对照相比，施用粪肥的土壤pH值更接近中性，而施用化肥使土壤pH值降低；施肥处理的土壤其他理化指标含量与不施肥对照相比有所增加，其中施用粪肥处理的土壤与对照的差异更为显著（表2）。

表2   培养土壤样品的基本理化性质

Table 2  Basic physicochemical properties of the cultured soil samples

采样地

Sampling site

处理

Treatment

pH值pH value

电导率

EC/

(µS/cm)

铵态氮

NH4＋-N/

(mg/kg)

硝态氮

NO3－-N/

(mg/kg)

总有机碳

TOC/

(mg/g)

有效磷

AP/

(mg/kg)

全磷

TP/

(mg/g)

全氮

TN/

(mg/g)

JXCMs6.89bc543.60a21.63a292.04ab32.00b219.94b1.79a1.32a
CMs_CK7.22ab194.70b4.60b121.82b27.25c101.28c1.58b1.08a
PMs7.37a603.80a11.69ab415.64a35.39a369.72a1.56b1.59a
PMs_CK7.16ab267.00b3.86b190.39b31.12b163.64bc1.38c1.44a
NPKs6.69c224.20b10.24ab164.40b26.71c137.86c1.55b1.20a
NPKs_CK6.83bc156.32b3.56b114.81b27.07c133.73c1.59b1.08a
TXCMs5.78c386.40b65.02a122.35b27.42b886.57c2.74c1.09bc
CMs_CK4.97d153.96d14.97bc123.80b24.30c756.81c2.34d0.82cd
PMs6.54a531.40a26.11b342.07a32.57a1 251.15b5.88a2.03a
PMs_CK6.21b251.18c4.11c191.70b28.92b1 529.93a5.40b1.54b
NPKs4.30e146.48d27.34b80.17b12.71d29.95d0.77e0.41d
NPKs_CK4.46e77.50e2.54c46.27b13.23d23.47d0.80e0.49d
TWCMs6.38b460.60a70.32a146.86a24.92b223.50c1.76c1.45a
CMs_CK5.60c139.84d8.57c122.18a19.22c72.01d1.32e0.89b
PMs6.00bc372.80ab36.93b219.59a26.63b589.09a2.40a0.70b
PMs_CK5.00d171.34d15.13c150.84a20.41c303.85b1.98b0.65b
NPKs7.30a285.62bc8.41c201.68a29.52a60.72d1.46d0.61b
NPKs_CK7.46a209.54cd3.75c146.36a28.98a63.51d1.45d0.62b
CHCMs7.05b1 357.60a686.60b807.62a223.17a748.78a5.41a10.16a
CMs_CK7.05b1 087.00a1 087.00a816.84a213.78a630.11b4.82b8.95b
PMs7.36ab536.80b465.09bc315.12b28.66b223.51c1.35c0.86c
PMs_CK7.59a248.60bc248.60c180.99b25.78b56.53d1.03c0.75c
NPKs4.94c203.00bc203.00c127.35b17.48b202.02c1.37c0.69c
NPKs_CK5.10c126.46c126.46c85.41b15.75b192.44c1.35c0.58c
LYCMs5.78b846.20a66.41a363.84a34.76b307.26b2.30b1.25b
CMs_CK4.74c389.80b5.10c295.64ab25.55c83.95b1.55c1.03bc
PMs6.82a493.40b25.74bc302.12ab38.86a1 611.15a6.79a1.99a
PMs_CK6.55a272.26c14.78bc183.70bc33.65b1 362.30a6.73a1.86a
1_NPKs4.68c137.56d39.32ab92.86c10.00f135.69b0.87d0.36d
1_NPKs_CK4.85c91.20d13.59bc61.58c9.99f125.05b0.82d0.33d
2_NPKs4.58c163.08cd29.10bc89.95c20.72d98.09b0.94d0.39d
2_NPKs_CK4.68c104.88d18.54bc65.86c17.24e88.90b0.99d0.54cd

同列数据后不同小写字母表示同一采样地不同施肥处理间在P＜0.05水平差异有统计学意义；n=5（5个培养时间点的值作为重复值）。

Values within the same column followed by different lowercase letters indicate significant differences among different fertilization treatments in the same sampling site; n=5 (the values of five cultivation time points are used as repeated values).

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2.2 土壤微生物群落及HPB结构特征

2.2.1α多样性特征

本研究在土壤和粪肥样品中共获得优化序列22 145 265条，平均序列长度376 bp。这些序列可分为366 289个ASVs，分属54个门，1 923个属，5 418个种。如图1所示：施用猪粪后土壤细菌群落的香农指数（α多样性）低于对照组；与之相比，施用鸡粪和化肥后其香农指数未表现出明显变化。整体而言，培养期间土壤细菌群落的香农指数随时间的变化趋势不一致。

经序列比对，共得到75种HPB，主要分属厚壁菌门（Firmicutes）、放线菌门（Actinobacteria）、变形菌门（Proteobacteria）。鸡粪中的HPB种类数多于猪粪，但均低于土壤样品（图2）。不同处理土壤间共有的HPB有17种，占HPB总数的22.7%。这说明土壤是HPB的重要储存库之一，并且HPB的适应能力很强，可以在多种土壤中存活。整体而言，不同处理土壤中HPB的数量以施用鸡粪处理（CMs）为最高，施用化肥处理（NPKs）为最低。此外，施用粪肥的土壤中HPB的种类数高于其不施肥对照，而施用化肥处理低于其对照，说明施用粪肥增加了土壤中HPB的数量，施用化肥的作用则相反。由图1可知：与对照相比，施用猪粪后土壤中HPB香农指数（α多样性）明显降低，但施用鸡粪后则略有增加；与土壤细菌α多样性相似，施用化肥处理的HPB多样性与其对照土壤间无明显差异。整体而言，培养期间土壤中HPB多样性随时间变化并不明显。

图2

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图2   不同样品间属水平下共有和独有的HPB数量（A）及门水平下HPB组成（B）

点线图表示集合间不同交集，单个节点表示特有的HPB，连线节点表示共有的HPB。

Fig. 2   Numbers of shared and unique HPB among different samples at the genus level (A) and the composition of detected HPB at the phylum level (B)

The dotted line diagram represents different intersections between sets, with the single node representing the unique HPB and the connected nodes representing the shared HPB.


2.2.2β多样性特征

由图3A可以看出，3种施肥处理的土壤细菌群落沿着第一主坐标轴分开（P＜0.001），说明肥料类型是影响土壤细菌群落组成的重要因素。施用猪粪的土壤在排序图上与其对照样品明显分离，而施用鸡粪和化肥的土壤与对应的不施肥对照土壤几乎重叠。上述结果说明，与对照相比，施用猪粪改变了土壤细菌群落的组成，而施用鸡粪和化肥无显著影响。此外，所有地区不同采样时间点的样品间并没有明显分隔，说明在培养期间土壤细菌群落组成变化并不大。由图3B可以看出：粪肥施用能显著改变HPB的组成（P＜0.01），且施用猪粪与未施肥对照之间有显著差异，而鸡粪和化肥则无明显影响；此外，培养期间土壤中HPB组成无明显变化。

图3

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图3   不同采样地不同施肥处理下土壤细菌群落组成（A）和HPB组成（B）的差异

Fig. 3   Differences in the soil bacterial community composition (A) and HPB composition (B) under different fertilization treatments in different sampling sites


2.3 HPB的相对丰度

图4热图展示了不同施肥处理下主要HPB（总相对丰度前10）的相对丰度变化。其中，优势HPB为巨大芽孢杆菌（Bacillus_megaterium_QM_B1551，24.2%）和拜氏梭菌（Clostridium_beijerinckii_NCIMB_8052，23.1%）。巨大芽孢杆菌在施肥土壤与未施肥对照土壤中的相对丰度均随培养时间的延长而增加。炭疽杆菌（Bacillus_anthracis_strain_Sterne）在未施肥对照土壤中相对丰度逐渐增加。鸡粪中优势HPB为腐生葡萄球菌（Staphylococcus_saprophyticus_ATCC_15305_）、谷氨酸棒状杆菌（Corynebacterium_glutamicum_R）。在施用鸡粪的土壤中，腐生葡萄球菌和谷氨酸棒状杆菌的相对丰度在培养第1天高于对照土壤，但随培养时间的延长差异逐渐减小。猪粪中优势HPB有拜氏梭菌、解没食子酸链球菌（Streptococcus_gallolyticus_strain_ATCC_BAA-2069）。在施用猪粪的土壤中，拜氏梭菌的相对丰度随培养时间的延长呈增加趋势，而解没食子酸链球菌的相对丰度随培养时间的延长逐渐降低，直至背景水平。施用化肥与未施用化肥对照土壤中HPB丰度变化没有明显差异，其中：伯克霍尔德菌（Burkholderia_phytofirmans_PsJN）的相对丰度随培养时间的延长逐渐降低；结核分枝杆菌（Mycobacterium_tuber-culosis_str._Beijing/NITR203）在TX采样地和拜氏梭菌在TW采样地土壤中的相对丰度均高于其他采样地土壤。

图4

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图4   施用鸡粪（A）、猪粪（B）和化肥（C）土壤中HPB的相对丰度

Fig. 4   Relative abundance of HPB in soils treated with chicken manure (A), pig manure (B), and chemical fertilizer (C)


2.4 影响土壤中HPB组成变化的因素分析

从图5A～E中可以看出，土壤理化性质对HPB组成变化具有显著影响，并且施用粪肥处理与未施肥对照样点在排序图上明显分开，而施用化肥处理与未施肥对照样点几乎重叠。从图5F中可以看出，除LY外，其他4个地区土壤理化性质与细菌群落的交互作用对土壤中HPB变异贡献率为68.3%～75.9%。各主要因子对LY采样地土壤中HPB变异贡献率排序依次为细菌群落（45.6%）＞交互作用（28.1%）＞土壤理化性质（11.9%），而土壤理化性质、细菌群落及两者交互作用对JX采样地土壤中HPB变异贡献率最高，达95.5%。由此可见，土壤理化性质与细菌群落组成是影响土壤中HPB变异的重要因素，两者的交互作用更是不可忽视。

图5

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图5   土壤理化性质与细菌群落对HPB组成变化的影响

Fig. 5   Effects of soil physicochemical properties and bacterial communities on HPB composition changes


3 讨论

3.1 施用粪肥对土壤细菌群落多样性的影响

本实验结果表明，施用猪粪导致土壤细菌α多样性低于相应对照组。TIAN等[23]研究也发现，施用粪肥会使细菌多样性显著降低。但也有研究表明，施用粪肥使细菌多样性显著提升[22]。这可能是畜禽粪便的添加量不同或其携带的微生物不同，导致通过粪肥引入的微生物对土壤微生物群落结构的促进或抑制作用不同。此外，本研究发现，相较于施用化肥，施用猪粪的土壤细菌群落组成与相应对照组的差异最为明显，施用鸡粪的土壤细菌群落组成次之。HAN等[24]研究也表明，施用粪肥的土壤在培养期内细菌群落组成发生明显变化。在本研究中，除CH采样地因原始施用鸡粪土壤的腐殖质较多，TOC异常高外，其他采样地中施用猪粪处理组的TOC总体上高于施用鸡粪处理组和施用化肥处理组，说明富含有机质的猪粪施入土壤后，改变了土壤碳源，从而影响了细菌群落组成。不同类型肥料中的碳氮比不同，所能提供的碳源也不相同。HU等[25]的研究结果表明，施用粪肥会提高受碳源限制的细菌丰度，从而改变细菌群落组成。由此可见，施肥类型是土壤细菌群落组成改变的重要影响因素，其中粪肥处理对土壤细菌群落组成的影响强于化肥处理。LI等[13]也发现，不同施肥类型对土壤细菌群落组成的影响具有显著差异（P＜0.001）。

3.2 施用粪肥对土壤中HPB多样性及丰度的影响

多项研究表明，施用粪肥会引入粪源病原菌，导致土壤中HPB多样性发生变化[12,26-27]。本研究发现，与对应的不施肥对照相比，施用鸡粪后土壤中HPB的α多样性没有明显变化，而施用猪粪后土壤中HPB的α多样性有所降低，可能是猪粪携带的微生物过多，改变了微生物群落组成，导致某种病原菌密集增殖，从而减少了多样性。本研究发现，施用粪肥后土壤中粪源HPB相对丰度显著高于对照，其中解没食子酸链球菌是最具临床相关性的病原菌之一，可见引入粪源HPB对土壤具有一定的风险[28]。此外，本研究发现，TW采样地未施肥对照土壤中巨大芽孢杆菌相对丰度较高，而施用粪肥后其相对丰度明显降低，施用化肥则无明显变化。已有研究表明，芽孢杆菌形成芽孢，能够抵抗干旱等极端环境[29]，说明TW采样地对极端环境的耐受性高，而施用粪肥后这种耐受性降低了。同时，大部分粪源HPB相对丰度随着培养时间的延长逐渐降低，可能是因为粪源HPB不适应土壤环境，这与施肥土壤中肠道沙门菌（Salmonella enterica）的变化结果相似[30]。WANG等[31]研究也发现，粪源HPB如大肠埃希菌O157:H7进入土壤后丰度会急速下降，存活时间为2.1～3.6 d。

本研究表明，施用粪肥后土壤中HPB的存活主要受土壤理化性质和固有微生物群落的影响。MOYNIHAN等[32]对12种不同类型土壤进行研究表明，微生物群落结构和土壤理化性质对肠道病原菌存活起到重要的抑制作用。XING等[33]研究表明，土壤pH会改变大肠埃希菌的存活时间，pH值越高，其存活时间越长。此外，TOC作为HPB的碳源，对HPB的存活具有显著影响[34]。朱永官等[35]研究表明，土壤固有微生物适应性较强，其活动可以使外来病原菌减少到正常水平。施用粪肥后土壤中HPB丰度总体呈下降趋势，一部分原因是土壤环境不利于粪源HPB的存活，另一部分原因是土壤中固有HPB的竞争能力比粪源HPB更强。

4 结论

1）与对照相比，施用猪粪的土壤中细菌和HPB群落的α多样性均有所降低。不同施肥处理对土壤微生物群落组成差异的影响程度不同，以施用猪粪最为显著，而施用化肥无明显影响。

2）施用粪肥后土壤中粪源HPB丰度显著高于相应的对照，但随培养时间的延长总体呈下降趋势。

3）方差分解分析显示，土壤理化性质、细菌群落及两者交互作用对土壤中HPB变异贡献率可高达95.5%。

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