施肥模式对设施土壤抗生素及抗性基因的影响

刘肖应,李 莹,耿家亘,汪 杰,陈 清,李 思,2*

施肥模式对设施土壤抗生素及抗性基因的影响

刘肖应1,李 莹1,耿家亘1,汪 杰1,陈 清1,李 思1,2*

(1.中国农业大学资源与环境学院,农田土壤污染防控与修复北京市重点实验室,北京 100193；2.中国农业大学烟台研究院,山东 烟台 264670)

采集不同施肥模式下设施菜地表层土壤(0～20cm),对83种抗生素和203种抗性基因(ARGs)进行检测.结果表明,各处理组土壤中共检出14种抗生素、129种ARGs亚型和10种可移动遗传元件(MGEs).四环素类(TCs)是土壤中残留的主要抗生素,β-内酰胺类和多重耐药类抗性基因是主要的ARGs,放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、绿弯菌门(Chloroflexi)和厚壁菌门(Firmicutes)是主要的细菌门.施肥增加了土壤中抗生素残留和ARGs的多样性及丰度,并且提高了土壤细菌群落多样性.施用常量有机肥组抗生素、ARGs和MGEs相对丰度最大,有机肥减量可有效降低抗生素和ARGs污染程度,稻壳还田对控制抗生素和ARGs污染具有积极作用.设施菜地土壤中抗生素选择压力、细菌群落结构变化和MGEs是影响ARGs分布的驱动因素.

抗生素；抗生素抗性基因；抗性机制；微生物多样性；设施土壤

抗生素污染以及抗生素抗性基因(ARGs)的形成和传播是21世纪威胁人类健康的首要因素之一[1].中国是全球抗生素生产和使用大国,我国兽用抗生素使用量约占抗生素使用总量的52%[2].然而,抗生素被畜禽摄入后无法被完全吸收,约有30%～90%的抗生素以原药和代谢物的形式通过粪便或尿液排出体外[3],导致环境中抗生素和ARGs的积累.

畜禽粪肥施用是设施土壤中抗生素和ARGs的重要来源.与露天菜地相比,设施菜地单位种植面积的施肥量显著提高,造成设施土壤中抗生素[3-5]和ARGs[4,6]污染的加剧.与露天土壤相比,我国20个省份51个设施土壤中抗生素和ARGs的污染水平显著升高,设施土壤中四环素类(TCs)、喹诺酮类(QNs)和磺胺类(SAs)抗生素的平均浓度分别是露天土壤的2.28倍、2.15倍和0.25倍,设施菜地土壤中ARGs相对丰度提高了近一倍[4].

施肥模式可显著影响设施菜地土壤中抗生素和ARGs的赋存.与不施肥相比,施用化肥和有机肥可改变土壤理化性质[7],影响土壤细菌群落结构[8],显著增加土壤中抗生素[9]和ARGs的丰度[7,10].作物秸秆作为土壤改良剂,秸秆还田可降低土壤中ARGs的相对丰度[10-11].土壤中抗生素抗性菌(ARB)携带的ARGs可通过质粒等可移动遗传元件(MGEs)水平转移到土著细菌中[12-13],对土壤生态系统和人类健康构成潜在威胁.

目前,有关不同施肥模式下设施土壤中抗生素和ARGs赋存情况的研究相对较少.因此,本文以设施土壤为研究对象,考察不同施肥模式对土壤中抗生素、细菌群落、ARGs和MGEs的影响,为设施土壤中抗生素和ARGs的污染控制提供理论支撑.

1 材料与方法

1.1 样品采集与处理

试验地点位于山东省寿光市蔬菜标准中心基地(36°55'N,118°45'E),种植作物为番茄.棚高为6.5m,总面积为960m2,日平均温度为25℃,日平均湿度为65%,日平均二氧化碳浓度为650mL/m3,实验开始于2020年8月,2021年1月番茄收获后采集土壤样品.共设6个处理组,分别为:(1)不施肥(CK);(2)单施化肥(F):氮(N)、磷(P2O5)和钾(K2O)投入量分别为300,120和550kg/hm2;(3)单施有机肥(M):腐熟鸡粪,用量为20t/hm2;(4)化肥配施(FM):F+M;(5)化肥配施有机肥减量(FLM):在F处理组基础上,鸡粪用量减少为M组的1/6;(6)化肥配施有机肥减量增碳(FLMS):在FLM处理组基础上增施20t/hm2稻壳.6个处理组面积均为88.2m2,每个处理设置3个重复,采用随机区组排列.栽培模式为传统的双行垄栽,垄宽80cm,垄间距50cm,番茄株距40cm.鸡粪和稻壳在作物种植前均匀撒施后翻耕作为基肥,化肥在作物灌溉季随灌溉水冲施,浇水情况根据设置的灌溉下限(田间持水量的70%)进行自动灌溉.番茄收获后其作物残茬全部移出温室.

采用5点采样法采集6个处理组表层土壤(0～20cm),每个处理采集3个平行样,将平行样混合进行后续测试.去除植物根茎、砂砾石块等后装入铝箔袋,在干冰保护下尽快运回实验室.一部分土样经风干、磨碎过2mm筛,用于土壤基本理化性质的测定(结果如表1所示),包括土壤pH值和有机质含量;其余土样分别于-20和-80℃保存,用于抗生素测定和DNA提取.

表1 不同施肥模下土壤的理化性质

1.2 抗生素提取和分析检测

根据文献调研,土壤中抗生素提取和分析采用超声提取-固相萃取-超高效液相色谱-串联质谱法[14-17].目标抗生素共7类83种,包括22种SAs、16种QNs、13种TCs、15种β-Ls (β-内酰胺类)、10种MLs (大环内酯类)、5种PEs (聚醚类)和2种LMs (林可霉素类).称取5g土样,加入10mL提取液(柠檬酸缓冲液(pH=3)和乙腈,体积比为1:1)和50ng抗生素内标超声提取3次,合并上清液后用超纯水稀释至500mL,经HLB小柱(500mg, 6mL, Waters, USA)净化富集,然后用甲醇进行洗脱,氮吹至近干后,再用甲醇定容至1mL,过0.22μm滤膜后于-20℃保存待测.抗生素检测采用的流动相为甲醇和含0.1%(体积分数)甲酸的超纯水,进样量为5μL,质谱采用正离子模式.方法加标回收率为67.2%～129%,检出限为0.001～0.14ng/g[15].

1.3 土壤细菌群落结构分析

土壤DNA提取后,通过琼脂糖凝胶电泳和Nanodrop微量分光光度计对DNA进行质量和浓度评估,使用Illumina MiSeq平台测序,上述步骤均由上海美吉生物医药科技有限公司完成.生物信息学分析在美吉平台生物云(http://www.i-sanger.com)进行.

1.4 ARGs和MGEs的高通量定量检测

采用Wafergen智能芯片超高通量PCR系统定量检测203种ARGs(多重耐药类、大环内酯类-林可霉素类-链霉素类(MLS)、磺胺类、四环素类、β-内酰胺类、氯霉素类和多粘菌素类)和11种MGEs(8种转座酶基因和3种整合酶基因),共设置216对引物,其中包括1对16S rRNA内参引物.反应体系为1 ×LightCycler 480SYBR Gree I Master,DNA模板浓度2ng/μL,引物浓度为500nmol/L,反应体系100nL. PCR反应程序为:预变性95℃下10min,然后95℃下30s,60℃下30s共进行40个循环[10].

1.5 数据分析

采用Origin 2019b绘制柱状图;使用R软件(v4.1.2)pheatmap包绘制热图,ggplot2包绘制冗余分析图;igraph包构建共现网络,并利用Gephi(v0.9.2)可视化.

2 结果与讨论

2.1 不同施肥模式下土壤抗生素种类和浓度

不同施肥模式设施菜地土壤样品中共检出15种抗生素,包括3种QNs、6种TCs、4种MLs和2种PEs,它们的总浓度为2.95～12.70µg/kg(图1),与山东省不同种植年限设施菜地中抗生素浓度相当[18],远低于北京市11个温室土壤中的抗生素浓度(28～ 1051μg/kg)[3].从组成来看,主要的抗生素为强力霉素(1.78～8.81µg/kg)、脱水红霉素(0.44～1.15µg/kg)和噁喹酸(0.32～0.80µg/kg) (图1).这3种抗生素的浓度分别与珠三角地区某蔬菜生产基地土壤[19]、珠江口典型水产养殖区沉积物[20]以及河北石家庄土壤中抗生素的浓度水平相当[21].

施肥模式

与CK和F处理组相比,施用常量有机肥组(M和FM)和减量有机肥组(FLM和FLMS)中,TCs类抗生素浓度由0.86～1.31µg/kg分别增加至6.78～ 9.89µg/kg和2.27～3.13µg/kg,可见鸡粪施用增加了TCs浓度,这与其他研究结果一致[3,22],主要是由于TCs类抗生素是畜禽养殖中使用量最大的一类抗生素,并且它们在土壤颗粒上的吸附能力较强[23].鸡粪中TCs类的总浓度为43.47µg/kg,因此有机肥施用是土壤抗生素的重要来源[24],而减量施用有机肥可有效降低土壤中抗生素残留.从TCs类组成来看,金霉素在腐熟鸡粪中的含量最高(30.58µg/kg),但在处理组土壤中未检出,土壤中检出了3种金霉素的转化产物,包括脱水金霉素、差向脱水金霉素和4-差向金霉素,研究表明差向异构化是金霉素在环境中的主要降解过程[25].因此,除抗生素母体外,环境中抗生素降解产物的赋存和毒性也应引起关注.

PEs类抗生素是养鸡业中广泛用于治疗鸡球虫病的一种离子载体类药物[26],本研究鸡粪样品中检出2种PEs类抗生素,其中莫能菌素的浓度高达1081µg/kg,与牛粪中的值(1351µg/kg)[27]相当,低于肉鸡粪便中的值(5.60mg/kg)[28].与腐熟鸡粪相比,各处理组土壤中PEs浓度显著降低,可能是因为有氧条件下PEs在土壤中的降解速率较快[29].研究发现莫能菌素以1mg/kg初始浓度施入土壤时,一个月内便会消散,半衰期仅3.3d[30].此外,SAs类抗生素仅在鸡粪中检出,在土壤中未检出,这可能与磺胺类抗生素水溶性较大而在土壤中吸附较弱有关[31].

2.2 不同施肥模式对ARGs和MGEs的影响

各处理组土壤中共检出66～81种ARGs,其中MLS类和四环素类ARGs数目最多(图2(a)).ARGs和MGEs的相对丰度分别为9.23´10-2～1.61´10-1copies/16S rRNA gene copies和5.90×10-3～1.48× 10-2copies/16S rRNA gene copies(图2(b)).从ARGs组成来看,β-内酰胺类和多重耐药类丰度最大,占各处理组总丰度的51.13%～71.62%和20.60%～35.34%,与其他施肥研究结果相似[32-34].抗生素失活和外排泵是最主要的抗性机制(图2(c)),与其他研究结果一致[35-36].

不同施肥模式下,与CK和F处理组相比,有机肥施用增加了土壤中ARGs的多样性和丰度.CK组和F组土壤中ARGs的Shannon指数为1.26和1.15,而在M组和FM组分别升高至1.51和1.73与CK组相比,M组ARGs总丰度增加了68%,这与其他研究结果一致[6,37].主要是由于动物源有机肥中含有大量ARGs,施入设施菜地后,一方面向土壤中引入了外来ARGs,另一方面有机肥可以促进土壤细菌群落生长,促进ARGs在细菌群落之间的水平转移,致使土壤中内源ARGs富集[32,34].与CK相比,有机肥减量处理组(FLM和FLMS)中ARGs丰度分别增加了33.89%和30.81%,但低于M组,说明有机肥减量可以有效缓解ARGs污染.加入稻壳后,与FLM组比,FLMS组中ARGs相对丰度略有下降,这与已有研究结果相似[38].同时,与CK相比,施肥增加了土壤中MGEs的种类(图2(a))和丰度(图2(b)),因此,施肥增加了ARGs水平基因转移的风险[39].

施肥模式

6个处理组土壤中共检出129种ARGs,其中32种在各处理组均有检出(图3(a)),它们占ARGs总丰度的70.7%～80.6%.从组成来看,TEM和F是两种主要的ARGs(图3(b)),研究表明,这两种ARGs在鸡粪、猪粪、牛粪、土壤、城市废水及污泥、甚至饮用水中均有检出[30,40-42],可能意味着它们在环境中普遍存在.转座酶基因A-04和整合酶基因Ⅰ-1是两种主要的MGEs,在各处理组土壤均有检出(图3(b)).

不同施肥模式下,M组土壤中F、A1和L/H-02基因的相对丰度较CK组增加了12～19倍,同时新增1、C、A-02和A(图3(b)),表明有机肥施用促进了土壤中多种ARGs的富集[43].同时,与CK相比,施肥增加了A-04和Ⅰ-1的相对丰度,增加了ARGs水平转移的风险.与M和FM组相比,有机肥减量处理组(FLM和FLMS)中共有12种ARGs丰度降低,包括5种四环素类(G-02、L-02、G-01、L-01和Q)、3种MLS类(B-02、A和F)、3种多重耐药类(Edelta1-02、R和Edelta1-01)和1种磺胺类(2),表明有机肥减量处理有利于削减土壤中ARGs污染.与FLM组相比,FLMS组TCs类和MLS类ARGs相对丰度下降,同时,MGEs相对丰度下降,表明秸秆还田对ARGs风险传播具有一定的阻控作用,这可能与FLMS组中有机质含量较FLM组增加有关(表1),研究发现秸秆还田后有机质对ARGs消除有积极作用[44].

2.3 不同施肥模式下土壤细菌群落结构变化

抗生素的抗菌活性可抑制土壤微生物的生长,从而影响土壤微生物群落组成,这可能会导致土壤生态功能的改变[45].与CK相比,常规施肥处理组中Chao1指数和Shannon指数均升高,表明施肥增加了土壤细菌群落的多样性(表2).可能是由于施肥可以增加土壤中有机质含量,提高土壤养分水平,从而促进土壤微生物的生长[12].与FM组相比,FLM组中Chao1指数和Shannon指数降低,但施用稻壳后, FLMS组中Shannon指数上升,可能是由于稻壳还田能够为土壤微生物提供丰富的碳源[11,13],从而提高土壤细菌群落的多样性.

不同施肥模式对土壤细菌群落结构的影响见图4.放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、绿弯菌门(Chloroflexi)和厚壁菌门(Firmicutes)为土壤中的优势菌门,分别占细菌总丰度的23.6%～45.1%、16.9%～28.4%、6.1%～19.0%和5.6%～13.8%(图4(a)),这些优势菌门在其他研究中也有报道[7-8,46-47].与CK组相比,施肥组土壤中放线菌门的相对丰度下降,而变形菌门的相对丰度升高,这与已有研究结果一致[7,48].变形菌门属于富营养型菌,施入土壤中的鸡粪易分解[49-50],为该类菌的生长提供了丰富的营养物质,从而促进了它们的生长[51].与FLM组相比,稻壳还田增加了FLMS组中绿弯菌门和厚壁菌门的相对丰度,这与以前的研究结果一致[8,46].绿弯菌门和厚壁菌门是有机物分解的重要参与者[52],稻壳还田可改善土壤结构,增加土壤有机质含量,提高养分利用率,从而促进这两类菌的生长[53].

图3 不同施肥处理土壤中ARGs亚型变化情况

选取相对丰度>0.1%的ARGs亚型和相对丰度前50%的MGEs

表2 不同施肥模式下土壤细菌Alpha多样性指数

在属水平上,相对丰度大于1.0%的微生物共检出19种,占细菌总丰度的46.6%～57.2%.它们主要来自上述4类优势菌门、酸杆菌门(Acidobacteriota)和芽单胞菌门(Gemmatimonadota)(图4(b)).其中,厚壁菌门中的芽孢杆菌属()在各处理组中相对丰度最高(占3.55%～8.76%).与CK组相比,芽孢杆菌属的相对丰度在施肥处理组中呈现不同程度的升高,FLMS组增幅最大为1.8倍,芽孢杆菌属能很好的适应环境条件,具有改善土壤养分状态和促进植物生长的作用[54].

2.4 施肥土壤中ARGs形成机制

土壤中抗生素和ARGs的冗余分析结果见图5,RDA1和RDA2两轴共解释了总变量的54.15%. TCs类抗生素是影响土壤中ARGs分布的主要因子,与四环素类ARGs、磺胺类ARGs以及MGEs正相关,表明设施菜地土壤中TCs浓度升高,对ARGs造成选择压力[55],MGEs通过对抗性基因的捕获、积累和扩散促进DNA在胞内或胞外的移动性[56],增加水平转移的风险.此外,MLs类抗生素与MLS类抗性基因呈现相关性,抗生素的选择压力以及它们所引起的细菌群落变化可能是影响ARGs组成的驱动因素[55,57].

ARGs、MGEs和细菌群落的共现网络关系见图6,该图由59个节点和117条边构成.ARGs与MGEs之间的共现意味着它们容易在环境中转移[37].例如,转座酶基因A-04与Edelta1-01、G-02、F和D等8种ARGs正相关,A-05与A1、G-02和A等6种ARGs正相关;插入序列IS613与R、Edelta1-02和G-01等5种ARGs正相关.这些结果表明MGEs可能促进多种ARGs在土壤中的扩散,从而增加了土壤的潜在生态风险[58].

图4 不同施肥模式下土壤细菌群落组成

图5 设施菜地土壤中抗生素和ARGs的冗余分析

ARGs与细菌的显著正相关关系可用于识别ARGs的潜在宿主[59].而当ARGs宿主为病原菌时,其生态风险应引起优先关注.例如,微枝形杆菌属()与2、Edelta1-02和R基因等7种ARGs具有正相关关系,说明该菌可能是多种ARGs的潜在宿主[32].同时,也是一种致病菌[60],并且可能通过A-04实现ARGs水平转移.芽孢杆菌)与A-02和C正相关,而某些芽孢杆菌属具有致病性[61].综上可知,除抗生素的选择压力外,细菌和MGEs也是影响土壤中ARGs分布的重要因素[10,32,59].

图6 ARGs、MGEs和细菌属水平之间的共现网络关系

选取平均相对丰度大于0.01%的ARGs、10种MGEs和平均相对丰度大于1%的细菌属绘制,节点大小代表相对丰度大小.Spearman相关系数>0.8,<0.05

本文对土壤混合样品的抗生素和ARGs进行了分析,有关不同处理组之间的差异有待进一步验证.此外,本文在土壤-番茄系统中研究了不同施肥模式下土壤中抗生素和ARGs的变化情况,但未关注植物中抗生素和ARGs.植物微生物组是ARGs的重要储库,可通过食物链进人体内,产生抗生素耐药问题,在未来研究中需重点关注.

3 结论

3.1 设施菜地土壤中共检出14种抗生素,施用有机肥增加了土壤中抗生素残留,有机肥减量可有效降低抗生素污染,特别是TCs类.

3.2 设施菜地土壤中共检出129种ARGs,单施有机肥组ARGs相对丰度最大,减量施用有机肥可降低ARGs种类和丰度,稻壳还田对ARGs的形成和传播具有一定的阻控作用.

3.3 施用有机肥可增加土壤中微生物群落多样性,秸秆还田提高了土壤有机质含量,增加了绿弯菌门和厚壁菌门的丰度,促进芽孢杆菌属的生长.

3.4 土壤中TCs类抗生素与四环素类抗性基因及MGEs正相关,A-04A-05和IS613可能会促进ARGs的传播,致病菌可能是ARGs的潜在宿主,增加ARGs的生态风险.

3.5 设施蔬菜生产过程应合理施肥,适当减少粪肥的投入,增加作物回田以提高土壤养分含量.优化有机肥生产工艺,削减粪肥中抗生素和抗性基因含量.

[1] Shrivastava S R, Shrivastava P S, Ramasamy J, et al. World health organization releases global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics [J]. Journal of Medical Society, 2017,32:76-77.

[2] Zhang Q, Ying G, Pan C, et al. Comprehensive evaluation of antibiotics emission and fate in the river basins of China: source analysis, multimedia modeling, and linkage to bacterial resistance [J]. Environmental Science & Technology, 2015,49(11):6772-6782.

[3] Li C, Chen J, Wang J, et al. Occurrence of antibiotics in soils and manures from greenhouse vegetable production bases of Beijing, China and an associated risk assessment [J]. Science of the Total Environment, 2015,521-522:101-107.

[4] Zeng Q, Sun J, Zhu L, et al. Occurrence and distribution of antibiotics and resistance genes in greenhouse and open-field agricultural soils in China [J]. Chemosphere, 2019,224:900-909.

[5] Hu X, Zhou Q, Luo Y, et al. Occurrence and source analysis of typical veterinary antibiotics in manure, soil, vegetables and groundwater from organic vegetable bases, northern China [J]. Environmental Pollution, 2010,158(9):2992-2998.

[6] Li J, Xin Z, Zhang Y, et al. Long-term manure application increased the levels of antibiotics and antibiotic resistance genes in a greenhouse soil [J]. Applied Soil Ecology, 2017,121:193-200.

[7] Liu P, Jia S, He X, et al. Different impacts of manure and chemical fertilizers on bacterial community structure and antibiotic resistance genes in arable soils [J]. Chemosphere, 2017,188:455-464.

[8] 张翰林,白娜玲,郑宪清,等.秸秆还田与施肥方式对稻麦轮作土壤细菌和真菌群落结构与多样性的影响 [J]. 中国生态农业学报(中英文), 2021,29(3):531-539.

Zhang H L, Bai L N, Zheng X Q, et al. Effects of straw returning and fertilization on soil bacterial and fungal community structures and diversities in rice-wheat rotation soil [J].Chinese Journal of Eco- Agriculture, 2021,29(3):531-539.

[9] 鲍陈燕,顾国平,徐秋桐,等.施肥方式对蔬菜地土壤中8种抗生素残留的影响 [J]. 农业资源与环境学报, 2014,31(4):313-318.

Bao C Y, Gu G P, Xu Q T, et al. Residues of eight antibiotics in vegetable soils affected by fertilization methods [J]. Journal of Agricultural Resources and Environment, 2014,31(4):313-318.

[10] Chen Q, An X, Li H, et al. Do manure-borne or indigenous soil microorganisms influence the spread of antibiotic resistance genes in manured soil? [J]. Soil Biology and Biochemistry, 2017,114:229-237.

[11] Liu Z P, Zhou H P, Xie W Y, et al. Long-term effects of maize straw return and manure on the microbial community in cinnamon soil in Northern China using 16S rRNA sequencing [J]. Plos One, 2021,16(4):e249884-e249899.

[12] Hu X, Liu J, Wei D, et al. Soil bacterial communities under different long-term fertilization regimes in three locations across the black soil region of northeast China [J]. Pedosphere, 2018,28(5):751-763.

[13] 栾 璐,郑 洁,程梦华,等.不同秸秆还田方式对旱地红壤细菌多样性及群落结构的影响 [J]. 土壤, 2021,53(5):991-997.

Luan L, Zheng J, Cheng M H, et al. Effects of different types of straw returning on bacterial diversity and community structure in dryland red soil [J]. Soils, 2021,53(5):991-997.

[14] Li S, Shi W, You M, et al. Antibiotics in water and sediments of Danjiangkou Reservoir, China: spatiotemporal distribution and indicator screening [J]. Environmental Pollution, 2019,246:435-442.

[15] Li S, Kuang Y, Hu J, et al. Enrichment of antibiotics in an inland lake water [J]. Environmental Research, 2020,190:110029-110037.

[16] Zhang R, Zhang R, Li J, et al. Occurrence and distribution of antibiotics in multiple environmental media of the East River (Dongjiang) catchment, South China [J]. Environmental Science and Pollution Research, 2017,24(10):9690-9701.

[17] Award Y M, Kim S, Abd EI-Azeem S A M, et al. Veterinary antibiotics contamination in water, sediment, and soil near a swine manure composting facility [J]. Environmental Earth Sciences, 2014, 71(3):1433-1440.

[18] 苑学霞,方丽萍,张太平,等.不同年限设施菜地土壤中重金属和抗生素污染特征 [J]. 生态环境学报, 2020,29(8):1669-1674.

Yuan X X, Fang L P, Zhang T P, et al. Characteristics of heavy metals and antibiotics pollution in vegetable greenhouses after different cultivating years [J]. Ecology and Environmental Sciences, 2020,29(8): 1669-1674.

[19] 邰义萍,莫测辉,李彦文,等.东莞市蔬菜基地土壤中四环素类抗生素的含量与分布 [J]. 中国环境科学,

Tai Y P, Mo C H,Li Y W, et al. Concentration and distribution of tetracycline antibiotics in soils from vegetable fields of dongguan City [J]. China Environmental Science, 2011,31(1):90-95.

[20] 梁惜梅,施 震,黄小平,等.珠江口典型水产养殖区抗生素的污染特征[J]. 生态环境学报, 2013,22(2):304-310.

Liang X M, Shi Z, Huang X P, et al. Occurrence of antibiotics in typical aquaculture of the Pearl River Estuary [J]. Ecology and Environmental Sciences, 2013,22(2):304-310.

[21] 赵鑫宇,剧泽佳,陈 慧,等.石家庄市土壤中喹诺酮类抗生素空间分布特征及其与微生物群落相关性 [J]. 环境科学, 2022,43(9):4684- 4696.

Zhao X Y, Ju Z J, Chen H, et al. Spatial distribution of quinolone antibiotics and its correlation relationship with microbial community in soil of Shijiazhuang City. [J] Environmental Science, 2022,43(9): 4684-4696.

[22] Wei R, Ge F, Zhang L, et al. Occurrence of 13veterinary drugs in animal manure-amended soils in Eastern China [J]. Chemosphere, 2016,144:2377-2383.

[23] Scaria J, Anupama K V, Nidheesh P V, et al. Tetracyclines in the environment: an overview on the occurrence, fate, toxicity, detection, removal methods, and sludge management [J]. Science of the Total Environment, 2021,771:145291-145313.

[24] Pan Z, Yang S, Zhao L, et al. Temporal and spatial variability of antibiotics in agricultural soils from Huang-Huai-Hai Plain, northern China [J]. Chemosphere, 2021,272:129803.

[25] Loftin K A, Adams C D, Meyer M T, et al. Effects of ionic strength, temperature, and pH on degradation of selected antibiotics [J]. Journal of Environmental Quality, 2008,37(2):378-386.

[26] 林雨鑫,孙明明,戴国俊,等.鸡抗球虫病的研究进展 [J]. 中国畜牧兽医, 2014,41(8):210-214.

Lin Y X, Sun M M, Dai G J, et al. Effects of Lipopolysaccharide on antiviral immune response induced by porcine reproductive and respiratory syndrome virus [J]. China Animal Husbandry&Veterinary Medicine, 2014,41(8):210-214.

[27] Hafner S C, Watanabe N, Harter T, et al. Effects of solid-liquid separation and storage on monensin attenuation in dairy waste management systems [J]. Journal of Environmental Management, 2017,190:28-34.

[28] Žižek S, Hrženjak R, Kalcher G T, et al. Does monensin in chicken manure from poultry farms pose a threat to soil invertebrates? [J]. Chemosphere, 2011,83(4):517-523.

[29] Sassman S A, Lee L S. Sorption and degradation in soils of veterinary ionophore antibiotics: Monensin and lasalocid [J]. Environmental Toxicology and Chemistry, 2007,26(8):1614-1621.

[30] Donoho A L. Biochemical studies on the fate of monensin in animals and in the environment [J]. Journal of Animal Science, 1984,58(6): 1528-1539.

[31] 陈 姗,许 凡,张 玮,等.磺胺类抗生素污染现状及其环境行为的研究进展 [J]. 环境化学, 2019,38(7):1557-1569.

Chen S, Xu F, Zhang W, et al. Research progress in pollution situation and environmental behavior of Sulfonamides [J]. Environmental Chemistry, 2019,38(7):1557-1569.

[32] Liu W, Ling N, Guo J, et al. Dynamics of the antibiotic resistome in agricultural soils amended with different sources of animal manures over three consecutive years [J]. Journal of Hazardous Materials, 2021,401:123399-123409.

[33] Cheng J, Tang X, Cui J, et al. Effect of long-term manure slurry application on the occurrence of antibiotic resistance genes in arable purple soil (entisol) [J]. Science of the Total Environment, 2019,647: 853-861.

[34] Wang F, Han W, Chen S, et al. Fifteen-year application of manure and chemical fertilizers differently impacts soil ARGs and microbial community structure [J]. Frontiers in Microbiology, 2020,11(62):1-14.

[35] 余薇薇,杨 硕,杨 伦,等.养殖场沼液中雌激素排放特征及去除效果研究 [J]. 中国环境科学, 2020,40(5):2103-2109.

Yu W W, Yang S, Yang L, et al. Study of emission characteristics and removal efficiency of estrogens in biogas slurry at farm [J]., 2020,40(5):2103-2109.

[36] 罗芳林,刘 琛,唐翔宇,等.猪粪溶解性有机物对紫色土中抗生素迁移的影响 [J]. 罗芳林,刘 琛唐翔宇,等.

Luo F L, Liu C, Tang X Y, et al. Effects of pig manure-derived dissolved organic matter on the transport of antibiotics in purple soils [J]. China Environmental Science, 2020,40(9):3952-3961.

[37] Pu Q, Zhao L, Li Y, et al. Manure fertilization increase antibiotic resistance in soils from typical greenhouse vegetable production bases, China [J]. Journal of Hazardous Materials, 2020,391:122267-122274.

[38] Peng S, Feng Y, Wang Y, et al. Prevalence of antibiotic resistance genes in soils after continually applied with different manure for 30years [J]. Journal of Hazardous Materials, 2017,340:16-25.

[39] Zhang T, Zhang X, Ye L, et al. Plasmid metagenome reveals high levels of antibiotic resistance genes and mobile genetic elements in activated sludge [J]. Plos One, 2011,6(10):e26041-e26047.

[40] Munk P, Knudsen B E, Lukjancenko O, et al. Abundance and diversity of the faecal resistome in slaughter pigs and broilers in nine European countries [J]. Nature Microbiology, 2018,3(8):898-908.

[41] 李十盛,高 会,赵富强,等.水产养殖环境中抗生素抗性基因的研究进展 [J]. 中国环境科学, 2021,41(11):5314-5325.

Li S S, Gao H, Zhao F Q, et al. Research progress on the occurrence and influencing factors of antibiotic resistance genes in aquaculture environment [J] China Environmental Science, 2021,41(11):5314- 5325.

[42] Zhang K, Xin R, Zhao Z, et al. Antibiotic Resistance Genes in drinking water of China: occurrence, distribution and influencing factors [J]. Ecotoxicology and Environmental Safety, 2020,188: 109837-109844.

[43] Xie W, Yuan S, Xu M, et al. Long-term effects of manure and chemical fertilizers on soil antibiotic resistome [J]. Soil Biology and Biochemistry, 2018,122:111-119.

[44] Zhang Y, Zheng X, Xu X, et al. Straw return promoted the simultaneous elimination of sulfamethoxazole and related antibiotic resistance genes in the paddy soil [J]. Science of the Total Environment, 2022,806:150525.

[45] Cycoń M, Mrozik A, Piotrowska-Seget Z. Antibiotics in the soil environment—degradation and their impact on microbial activity and diversity [J]. Frontiers in Microbiology, 2019,338(10):338-382.

[46] Zhao S C, Qiu S J, Xu X P, et al. Change in straw decomposition rate and soil microbial community composition after straw addition in different long-term fertilization soils [J]. Applied Soil Ecology, 2019, 138:123-133.

[47] Kumar U, Kumar Nayak A, Shahid M, et al. Continuous application of inorganic and organic fertilizers over 47years in paddy soil alters the bacterial community structure and its influence on rice production [J]. Agriculture, Ecosystems & Environment, 2018,262:65-75.

[48] Calleja-Cervantes M E, Menéndez S, Fernández-González A J, et al. Changes in soil nutrient content and bacterial community after 12 years of organic amendment application to a vineyard [J]. European Journal of Soil Science, 2015,66(4):802-812.

[49] 陈莺燕,刘文深,丁铿博,等.有机改良剂及生物炭对离子型稀土矿尾砂地生态修复的改良探究 [J]. 环境科学学报, 2018,38(12):4769- 4778.

Chen Y Y, Liu W S, Ding K B, et al. Effects of organic amendments and biochar on ecological remediation of ionic rare earth mine tailings [J]. Acta Scientiae Circumstantiae, 2018,38(12):4769-4778.

[50] 张庆忠,吴文良,王明新,等.秸秆还田和施氮对农田土壤呼吸的影响[J]. 生态学报, 2005,25(11):91-95.

Zhang Q Z, Wu W L, Wang M X, et al. The effects of crop residue amendment and N rate on soil respiration [J]. Acta Ecologica Sinica, 2005,25(11):91-95.

[51] Pascault N, Ranjard L, Kaisermann A, et al. Stimulation of different functional groups of bacteria by various plant residues as a driver of soil priming effect [J]. Ecosystems, 2013,16(5):810-822.

[52] Guo Z, Wan S, Hua K, et al. Fertilization regime has a greater effect on soil microbial community structure than crop rotation and growth stage in an agroecosystem [J]. Applied Soil Ecology, 2020,149: 103510.

[53] Wang X, Bian Q, Jiang Y, et al. Organic amendments drive shifts in microbial community structure and keystone taxa which increase C mineralization across aggregate size classes [J]. Soil Biology and Biochemistry, 2021,153:108062.

[54] Saxena A K, Kumar M, Chakdar H, et al. Bacillus species in soil as a natural resource for plant health and nutrition [J]. Journal of Applied Mircobiology, 2020,128(6):1583-1594.

[55] Yan M, Xu C, Huang Y, et al. Tetracyclines, sulfonamides and quinolones and their corresponding resistance genes in the Three Gorges Reservoir, China [J]. Science of the Total Environment, 2018, 631-632:840-848.

[56] Partridge S R, Kwong S M, Firth N, et al. Mobile genetic elements associated with antimicrobial resistance [J]. Clinical Microbiology Reviews, 2018,31(4):e17-e88.

[57] Wang B, Yan J, Li G, et al. Risk of penicillin fermentation dreg: increase of antibiotic resistance genes after soil discharge [J]. Environmental Pollution, 2020,259:113956-113963.

[58] 蔡天贵,张 龙,张晋东,等.抗生素抗性基因的生态风险研究进展 [J]. 应用生态学报, 2022,33(5):1435-1440.

Cai T G, Zhang L, Zhang J D, et al. Research advances in ecological risk of antibiotic resistance genes [J]. Chinese Journal of Applied Ecology, 2022,33(5):1435-1440.

[59] Zhu B, Chen Q, Chen S, et al. Does organically produced lettuce harbor higher abundance of antibiotic resistance genes than conventionally produced? [J]. Environment International, 2017,98: 152-159.

[60] Noguera J C, Aira M, Perez-Losada M, et al. Glucocorticoids modulate gastrointestinal microbiome in a wild bird [J]. Royal Society Open Science, 2018,5(4):171743-171750.

[61] Coburn P S, Miller F C, Enty M A, et al. Expression ofvirulence-related genes in an ocular infection-related environment [J]. Microorganisms, 2020,8(4):607.

Effects of fertilization regimes on antibiotics and antibiotic resistance genes in greenhouse soil.

LIU Xiao-ying1, LI Ying1, GENG Jia-gen1, WANG Jie1, CHEN Qing1, LI Si1,2*

(1.Beijing Key Laboratory of Farmland Soil Pollution Prevention and Remediation, College of Resource and Environmental Science, China Agricultural University, Beijing 100193, China；2.Yantai Institute of China Agricultural University, Yantai 264670, China)., 2023,43(2)：772～780

In this study, the surface soil (0～20cm) under different fertilization regimes was collected from a vegetable greenhouse and 83 antibiotics and 203 antibiotic resistance genes (ARGs) were analyzed. The results showed that a total of 14 antibiotics, 129 ARGs subtypes and 10 mobile genetic elements (MGEs) were detected in the soil. Tetracyclines (TCs) were the dominant antibiotics in soil, and β-lactams and multi-drug resistance genes were the dominant ARG types. Actinobacteria, Proteobacteria, Chloroflexi and Firmicutes were the main bacterial phyla in soil under different treatments. Fertilization increased the diversity and abundance of antibiotic residues and ARGs in soil, and increased the-diversity of soil bacterial communities. The relative abundances of antibiotics, ARGs and MGEs were the highest in the treatments of manure alone and manure plus chemical fertilizer, and they were decreased by applying reduced manure. The return of rice husk to the field was helpful in controlling the pollution of antibiotics and ARGs. The selection pressure from antibiotics, changes in bacteria community and MGEs were the driving factors affecting the distribution of ARGs in greenhouse vegetable soil.

antibiotics；antibiotic resistance genes；antibiotic resistance mechanism；microbial diversity；greenhouse soil

X53

A

1000-6923(2023)02-0772-09

刘肖应(1997-),女,四川自贡人,中国农业大学硕士研究生,从事新污染物治理研究.

2022-06-07

山东省自然科学基金资助项目(ZR2020QD132);北京高校卓越青年科学家计划项目(BJJWZYJH01201910004016)

* 责任作者, 副教授, sili@cau.edu.cn