环境科学  2023, Vol. 44 Issue (5): 2936-2944   PDF    
引用本文
张济世, 刘春增, 郑春风, 张琳, 张香凝, 吕玉虎, 曹卫东, 张玉亭. 紫云英还田与化肥减量配施对稻田土壤细菌群落组成和功能的影响[J]. 环境科学, 2023, 44(5): 2936-2944.
ZHANG Ji-shi, LIU Chun-zeng, ZHENG Chun-feng, ZHANG Lin, ZHANG Xiang-ning, LÜ Yu-hu, CAO Wei-dong, ZHANG Yu-ting. Effects of Chinese Milk Vetch Returning Incorporated with Chemical Fertilizer Reduction on the Composition and Function of Soil Bacterial Communities in Paddy Fields[J]. Environmental Science, 2023, 44(5): 2936-2944.
紫云英还田与化肥减量配施对稻田土壤细菌群落组成和功能的影响
张济世1,2, 刘春增1,2, 郑春风1,2, 张琳3, 张香凝1,2, 吕玉虎3, 曹卫东4, 张玉亭1,2     
1. 河南省农业科学院植物营养与资源环境研究所, 郑州 450002;
2. 河南省农业生态环境重点实验室, 郑州 450002;
3. 信阳市农业科学院, 信阳 464000;
4. 中国农业科学院农业资源与农业区划研究所, 北京 100081
收稿日期: 2022-07-23; 修订日期: 2022-08-08
基金项目: 河南省农业科学院科技创新团队项目（2022TD37）；国家重点研发计划项目（2021YFD01700203）；国家绿肥产业技术体系项目（CARS-22-G-06）
作者简介: 张济世(1991~), 男, 博士研究生, 助理研究员, 主要研究方向为养分综合管理与土壤质量调控, E-mail: jishizhang1227@163.com
通信作者: 刘春增, E-mail: liuczhnnky@126.com; 张玉亭, E-mail: 15600660918@163.com
摘要: 紫云英是豫南稻区土壤培肥的重要有机肥资源，探究紫云英还田与化肥减量配施对稻田土壤理化性质和细菌群落特性的影响，旨在为该区域的土壤培肥和化肥减量提供依据.开展连续12 a田间定位试验，设置了6个施肥处理（空白对照，CK；单施化肥，F100；80%化肥和22.5t·hm-2紫云英还田量配施，MV1F80；80%化肥和45 t·hm-2紫云英还田量配施，MV2F80；60%化肥和22.5 t·hm-2紫云英还田量配施，MV1F60；60%化肥和45 t·hm-2紫云英还田量配施，MV2F60）.通过采用高通量测序方法比较不同施肥处理对土壤细菌群落多样性、组成和结构特性的影响；采用FAPROTAX功能预测的方法分析不同施肥处理间功能类群的丰度差异，并结合土壤理化性质，探究改变土壤细菌群落结构与功能特性的关键土壤环境因子.结果表明，紫云英还田与化肥减量配施降低了土壤容重，提高了土壤有机碳（SOC）、全氮（TN）、全磷（TP）和全钾（TK）含量，较CK提高的范围分别为12.7%~35.5%、38.2%~65.7%、66.7%~95.2%和20.3%~31.6%.紫云英还田与化肥减量配施处理较F100处理降低了细菌Sobs指数和Shannon多样性指数，且Sobs指数和Shannon多样性指数均与容重（BD）呈显著正相关（P＜0.05），而与SOC和TN呈显著负相关（P＜0.05）.MV1F80和MV2F60处理的厚壁菌门（Firmicutes）的相对丰度较F100处理分别提高了82.2%和67.4%（P＜0.05），而酸杆菌门（Acidobacteria）的相对丰度分别减少了32.6%和40.5%（P＜0.05），仅MV2F60处理放线菌门（Actinobacteria）的相对丰度提高了30.0%（P＜0.05）.RDA分析表明，土壤SOC、TN和TK是显著影响细菌群落结构变化的主要土壤环境因子（P＜0.05）.紫云英还田与化肥减量配施处理还提高了化能异养类、固氮类、发酵类和尿素分解类功能类群丰度，降低了动物寄生或共生类、人类所有病原体和人类病原体肺炎类功能类群丰度，其中MV1F80和MV2F60处理效果较好.综上所述，长期的紫云英还田与化肥减量配施改善了土壤理化性质，进而改变了土壤细菌群落结构与功能特性，有助于稻田土壤肥力的提升、微生态系统的稳定和健康，进而保障区域农业的绿色可持续发展.
关键词: 化肥减量      紫云英还田      土壤理化性质      细菌群落      功能预测     
Effects of Chinese Milk Vetch Returning Incorporated with Chemical Fertilizer Reduction on the Composition and Function of Soil Bacterial Communities in Paddy Fields
ZHANG Ji-shi1,2 , LIU Chun-zeng1,2 , ZHENG Chun-feng1,2 , ZHANG Lin3 , ZHANG Xiang-ning1,2 , LÜ Yu-hu3 , CAO Wei-dong4 , ZHANG Yu-ting1,2     
1. Institute of Plant Nutrition, Agricultural Resources and Environmental Sciences, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
2. Henan Key Laboratory of Agricultural Eco-Environment, Zhengzhou 450002, China;
3. Xinyang Academy of Agricultural Sciences, Xinyang 464000, China;
4. Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Abstract: Chinese milk vetch (Astragalus sinicus L.) is an important organic nutrient resource in the southern Henan rice-growing area. Thus, the effects of Chinese milk vetch (MV) returning incorporated with reduced chemical fertilizer on the physicochemical properties and bacterial community characteristics in paddy soil were studied. These results can provide a certain theoretical basis for the improvement of soil fertility and reduction of chemical fertilizer in this area. A field experiment was conducted for 12 consecutive years, involving six fertilization treatments (blank control, CK; 100% chemical fertilizer, F100; 80% chemical fertilizer+22.5 t·hm-2 MV, MV1F80; 80% chemical fertilizer+45 t·hm-2 MV, MV2F80; 60% chemical fertilizer+22.5 t·hm-2 MV, MV1F60; and 60% chemical fertilizer+45 t·hm-2 MV, MV2F60). The high-throughput sequencing method was used to compare the effects of different fertilization treatments on soil bacterial community diversity, composition, and structural characteristics. The FAPROTAX function prediction method was used to analyze the abundance differences of functional groups between different fertilization treatments. Additionally, combined with soil physicochemical properties and bacterial community characteristics, we explored the key soil environmental factors that changed the structure and functional characteristics of the soil bacterial community. Compared with that under CK, the soil bulk density (BD) under the MV returning incorporated with reduced chemical fertilizer treatment was decreased, whereas soil organic carbon (SOC), total nitrogen (TN), total phosphorus (TP), and total potassium (TK) were increased by 12.7%-35.5%, 38.2%-65.7%, 66.7%-95.2%, and 20.3%-31.6%, respectively. Compared with that under the F100 treatment, the Sobs index and Shannon diversity index of the bacterial community under the MV returning incorporated with reduced chemical fertilizer were decreased, and the Sobs index and Shannon diversity index were significantly positively correlated with BD (P < 0.05) but significantly negatively correlated with SOC and TN (P < 0.05). Compared with that under the F100 treatment, the relative abundances of Firmicutes under the MV1F80 and MV2F60 treatments were significantly increased by 82.2% and 67.4% (P < 0.05), but the relative abundances of Acidobacteria were significantly reduced by 32.6% and 40.5% (P < 0.05), respectively. The relative abundance of Actinobacteria under the MV2F60 treatment was significantly increased by 30.0% (P < 0.05) compared with that under the F100 treatment. According to RDA analysis, soil SOC, TN, and TK were the main soil environmental factors that significantly affected bacterial community (P < 0.05). Compared with that under CK and the F100 treatment, the abundance of functional groups of chemoheterotrophy, nitrogen fixation, fermentation, and ureolysis under the MV returning incorporated with reduced chemical fertilizer treatment were improved, whereas the abundance of functional groups of animal parasites or symbionts, all human pathogens, and human pathogen pneumonia were reduced, particularly under MV1F80 and MV2F60. To summarize, the long-term MV returning to the field incorporated with reduced chemical fertilizer improved the soil physical and chemical properties, thus changing the structure and functional characteristics of the soil bacterial communities, contributing to the improvement in the soil fertility, stability, and health of micro-ecosystems in paddy fields, thus ensuring the green and sustainable development of regional agriculture.
Key words: fertilizer reduction      Chinese milk vetch returning      soil physicochemical properties      bacterial community      function prediction     

随着人口的不断增长, 预计2050年全球粮食产量需要在2005年的基础上增加60% ~110%才能满足需求[1].施用化肥是提高作物产量不可或缺的农业措施[2], 然而长期大量的化肥投入不仅降低了资源利用效率, 还会带来负面影响, 如土壤质量退化和环境污染等一系列生态环境问题, 进而影响作物生产系统的可持续性[3].而有机无机配施技术模式已被大量研究证明, 不仅可以减少化肥的投入, 还比单施化肥模式有更好的增产效应[4, 5], 然而有机无机配施技术模式也存在一定的不确定性, 源于有机肥(如牛粪、猪粪等)可能包含抗生素和重金属等, 增加二次污染的风险[6, 7].因此, 绿肥作为一种清洁有机肥源得到了广泛的关注.在我国南方稻田生产体系中, 种植和翻压豆科绿肥(如紫云英, Astragalus sinicus L.)已成为主要可持续生产模式, 不仅可以为水稻生长提供充足养分, 减少化肥投入, 降低环境风险, 还可以培肥土壤, 改善土壤理化性质, 同时影响土壤微生物学特性[8~10].

土壤微生物参与了多种农业生态过程, 如营养循环和有机物料腐解等, 是衡量土壤肥力和土壤健康程度的重要指标[11].土壤细菌是微生物中丰度最高的群体, 同时也是养分活化的驱动者, 其群落组成和功能关系到土壤肥力和农业可持续性[12].有研究指出, 施肥能够提高土壤肥力, 改变细菌的丰富度和功能多样性, 而不合理的施肥可能降低某些功能基因丰度[13, 14].绿肥还田可以为细菌的生长提供充足的新鲜有机质和氮钾养分资源, 有助于营养循环和土壤肥力的提升[15].Stark等[16]研究发现绿肥还田提高了微生物的生物量和活性, 进而改变了土壤微生物群落组成.一个31 a不同轮作体系的试验发现, 种植冬绿肥改变了微生物群落结构, 提高了有益细菌丰度[17]. Tao等[18]研究发现紫云英还田较蚕豆和野豌豆还田显著增加了酸杆菌门(Acidobacteria)和疣微菌门(Verrucomicrobia)的相对丰度, 进而提高了玉米产量.

前人关于绿肥还田的研究主要集中在土壤培肥、资源利用效率和作物产量方面[8, 15], 针对微生物群落特性也有一部分研究[17, 18], 不过主要集中在不同绿肥轮作体系中探究种植和翻压绿肥对细菌群落特性的影响, 而紫云英还田与化肥减量配施对细菌群落特性的影响和驱动细菌群落结构与功能变化的主控因子的研究较少, 且紫云英还田与化肥减量配施对细菌群落功能特性的影响也尚缺少研究.另外, 不同的土壤类型、作物类型和施肥措施等都会影响土壤微生物学特性[19].因此, 本文基于12 a的长期定位试验, 通过高通量测序方法, 探究细菌群落组成和功能对长期紫云英还田与化肥减量配施的响应差异及其与土壤理化性质的关系, 以期为进一步优化施肥提供理论依据, 助推我国南方稻田绿色可持续生产.

1 材料与方法 1.1 研究地点

试验区位于河南省信阳市农业科学院试验园区(北纬32°06′10″, 东经114°03′27″), 该区属于亚热带向暖温带过渡区, 年均降雨900~1 400 mm, 年均气温15℃.田间试验始于2009年, 试验土壤为黄棕壤性潜育型水稻土, 基础土壤(0~20 cm)特性为：土壤有机碳ω(SOC) 14.7 g·kg-1, 碱解氮ω(AN) 81.5 mg·kg-1, 有效磷ω(AP) 10.5 mg·kg-1, 速效钾ω(AK) 58.2 mg·kg-1.

1.2 试验设计与管理

供试材料：本试验紫云英选用信紫1号, 在水稻收获后原地种植, 于次年4月上旬紫云英盛花期翻压还田(根据不同小区试验要求翻压), 多余部分移出小区, 少的补充, 其中紫云英鲜草含水量90%, 干草中含氮37.5 g·kg-1、含磷3.4 g·kg-1和含钾35.0 g·kg-1.供试水稻品种为扬两优013, 于每年5月下旬划行移栽, 移栽密度为16.7 cm×20 cm, 每穴2棵基本苗.

试验设计：本试验采用随机区组试验设计, 包括6个处理：①不施肥对照(CK); ②100%化肥(F100); ③紫云英还田22.5 t·hm-2+80%化肥(MV1F80); ④紫云英还田45 t·hm-2+80%化肥(MV2F80); ⑤紫云英还田22.5 t·hm-2+60%化肥(MV1F60); ⑥紫云英还田45 t·hm-2+60%化肥(MV2F60).每个处理包括3次重复, 共计18个小区.每个小区面积为6.66 m2(3.33 m×2.0 m), 小区间筑田埂并覆膜防止串水串肥, 埂宽30 cm.

肥料管理：施肥类型为尿素(46% N)、过磷酸钙(12% P2O5)和氯化钾(60%K2O).100%化肥为氮肥165 kg·hm-2(以N计)、磷肥112.5 kg·hm-2(以P2O5计)和钾肥112.5 kg·hm-2(以K2O计), 80%和60%化肥分别减少氮和钾的用量, 磷的用量不减少, 其中氮肥分3次施肥, 基肥占50%, 分蘖肥占30%, 孕穗肥占20%, 磷钾全部基施, 其他田间管理和当地管理方式一致.

1.3 测定项目与方法

样品采集：于2020年9月水稻收获前, 采集表层土壤(0~20 cm)样品, 混匀后分成3份, 放入保温箱带回实验室, 一份土壤样品风干, 用于测试土壤养分含量; 一份土壤样品保留鲜土, 用于测试土壤无机氮; 另一份样品即刻保存于-80℃, 用于细菌群落特性分析.

土壤理化性质测定[20, 21]：土壤容重采用环刀法测定; 土壤SOC和TN采用碳氮元素分析仪(Elementar, Langenselbold, Germany)进行测试; 土壤无机氮(Nmin)经1 mol·L-1KCl溶液浸提后采用连续流动分析仪(AA3, SEAL Analytical, Germany) 测定; 全磷(TP)采用H2SO4-HClO4消煮和钼锑抗比色法测定; 有效磷(AP)采用0.5 mol·L-1 NaHCO3浸提-钼锑抗比色法测定; 全钾(TK)和速效钾(AK)分别采用NaOH熔融-火焰光度法和1 mol·L-1 NH4OAc浸提-火焰光度法.

土壤样品微生物的DNA抽提和PCR扩增：根据E.Z.N.A.® soil DNA kit (Omega Bio-tek, Norcross, GA, U.S.)说明书进行微生物群落总DNA抽提, 使用1%的琼脂糖凝胶电泳检测DNA的提取质量, 使用NanoDrop2000测定DNA浓度和纯度; 使用338F (5′-ACTCCTACGGGAGGCAGCAG-3′)和806R (5′-GGACTACHVGGGTWTCTAAT-3′) 对16S rRNA基因V3-V4可变区进行PCR扩增.每个样本3个重复.

Illumina MiSeq测序：将同一样本的PCR产物混合后使用2%琼脂糖凝胶回收PCR产物, 利用AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) 进行回收产物纯化, 2%琼脂糖凝胶电泳检测, 并用QuantusTM Fluorometer (Promega, USA) 对回收产物进行检测定量.通过Illumina公司的MiSeq PE300平台进行测序, 由上海美吉生物医药科技有限公司提供技术支持.

1.4 数据统计与分析

采用Microsoft Excel 2019软件整理试验数据.采用SAS软件进行单因素方差分析(one-way ANOVA)和Duncan多重比较法分析不同施肥处理间土壤理化性质、群落α多样性(丰富度指数：Sobs; 多样性指数：Shannon; 均匀度指数：Shannoneven; 覆盖度指数：Coverage)、群落组成和功能的差异显著性(P＜0.05), 采用SPSS 20.0软件进行各个指标的斯皮尔曼(Spearman)相关性分析; 通过美吉生物云平台研究土壤理化性质对细菌群落结构的影响(冗余分析RDA); 采用FAPROTAX数据库进行细菌功能预测.采用Origin 2021和Sigmaplot 12.5软件进行数据作图.

2 结果与分析 2.1 土壤理化性质

不同的施肥处理下土壤的理化性质如表 1所示.不同的施肥处理显著影响了SOC、TN、TP和TK.与CK相比, MV2F80、MV1F80和MV2F60处理显著提高了SOC, 增幅分别为35.5%、26.4%和25.5%; 仅MV2F80处理的SOC较F100处理显著提高19.2%; 而紫云英还田与化肥减量配施处理均显著提高了TN, 增幅为38.2% ~65.7%, 仅MV2F60处理的TN显著高于F100处理, 增幅为28.3%; 对于TP和TK来说, 施肥处理均显著高于CK, 但是施肥处理间无显著差异.MV2F60处理的BD较CK显著降低了12.1%, 土壤Nmin提高了45.8%, 其他处理与CK均为显著差异; 而对于AP和AK来说, 施肥处理均高于CK, 但是各处理间差异未达到显著水平.

表 1 不同施肥处理对土壤理化特性的影响1) Table 1 Effect of different fertilization treatments on soil physicochemical properties

2.2 土壤细菌群落多样性

不同施肥处理下土壤细菌群落α多样性指数如图 1所示.紫云英还田与化肥减量配施处理的Sobs指数均低于CK和F100处理, 尤其是MV2F80和MV2F60处理.从Shannon指数来看, 各施肥处理均低于CK处理, 尤其是MV2F60; 对于Shannoneven指数来说, 各处理间均无显著差异.而各样本的Coverage指数均高于96.1%, 说明测序深度能够比较真实地反映土壤样本中细菌群落的多样性.

1.CK, 2.F100, 3.MV1F80, 4.MV2F80, 5.MV1F60, 6.MV2F60; 不同小写字母表示处理间差异显著(P＜0.05) 图 1 不同施肥处理对土壤细菌群落α多样性的影响 Fig. 1 Effect of different fertilization treatments on alpha diversity of soil bacterial communities

各处理间的细菌群落α多样性与土壤理化特性的Spearman相关性如表 2所示.细菌Sobs指数和Shannon指数与BD均呈显著正相关(P＜0.05), 与SOC呈显著负相关(P＜0.01和P＜0.05); TN与细菌Sobs指数(P＜0.01)、Shannon指数(P＜0.05)和Shannoneven指数(P＜0.05)也呈显著负相关; AK与细菌Sobs指数呈显著负相关(P＜0.05).

表 2 细菌群落α多样性与土壤理化特性之间的Spearman相关性1) Table 2 Spearman correlation between alpha diversity of bacterial communities and soil physicochemical properties

2.3 土壤细菌群落组成与差异

不同施肥处理下细菌群落结构在门水平的物种组成如图 2所示.绿弯菌门(Chloroflexi)、放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、酸杆菌门(Acidobacteria)和厚壁菌门(Firmicutes)是主要细菌菌群, 在18个样本中平均相对丰度分别为22.9%、16.5%、15.9%、13.1%和7.12%, 约占总丰度的75.5%, 另有11类菌门相对丰度大于1.00%, 约占总丰度的21.3%, 而相对丰度小于1.00%的菌门约占3.20%.

1.CK, 2.F100, 3.MV1F80, 4.MV2F80, 5.MV1F60, 6.MV2F60; a1.Chloroflexi, a2.Actinobacteria, a3.Proteobacteria, a4.Acidobacteria, a5.Firmicutes, a6.Myxococcota, a7.Myxococcota, a8.Nitrospirae, a9.Gemmatimonadetes, a10.Bacteroidota, a11.MBNT15, a12.Planctomycetota, a13.Methylomirabilota, a14.Verrucomicrobia, a15.Latescibacteria, a16.Cyanobacteria, a17.others; 将所有样本中相对丰度均小于1%的物种归为others 图 2 不同施肥处理对土壤细菌群落组成的影响 Fig. 2 Effect of different fertilization treatments on soil bacterial community compositions

如图 3所示, 在细菌主要菌门中, 不同施肥处理显著影响了厚壁菌门(Firmicutes)的相对丰度(P＜0.05), 其中MV1F80和MV2F60的厚壁菌门(Firmicutes)的相对丰度较CK显著增加了48.0%和35.9%(P＜0.05), 较F100处理显著增加了82.2%和67.4%(P＜0.05), 其他处理间无显著差异; 对于放线菌门(Actinobacteria)来说, MV2F60处理的相对丰度显著高于其他处理(P＜0.05).与F100相比, 所有处理均降低了酸杆菌门(Acidobacteria)的相对丰度, 尤其是MV1F80和MV2F60显著降低了32.6%和40.5%(P＜0.05).

不同小写字母表示处理间差异显著(P＜0.05) 图 3 不同施肥处理对土壤细菌群落主要菌门丰度的影响 Fig. 3 Effect of different fertilization treatments on abundance of dominant phyla in soil bacteria

细菌群落结构与土壤理化性质的冗余分析(RDA)如图 4所示.结果表明, RDA1和RDA2分别解释了土壤细菌群落变异的29.0%和8.75%, 前两轴共解释了37.8%的细菌群落总变异, 其中, SOC(R2=0.450 2, P=0.015)、TN(R2=0.390 8, P=0.016)和TK(R2=0.406 5, P=0.013)是影响细菌群落的主要土壤环境因子.

图 4 土壤细菌群落与土壤化学特性间的冗余分析 Fig. 4 Redundancy analysis (RDA) of soil bacterial community and soil physicochemical properties

2.4 土壤细菌功能预测

本研究采用FAPROTAX对土壤细菌群落进行功能预测和注释, 共获得50种功能分组.其中, 相对丰度大于1%的功能类型有22个, 共占总丰度的94.5% ~96.0%, 如表 3所示.将该22种功能类型进行单因素方差分析, 筛选出在处理间有显著差异的8项功能类群.MV2F60处理的化能异养类种群相对丰度显著高于CK和F100(P＜0.05), 其他处理间无显著差异; 4个紫云英还田与化肥减量配施处理的发酵作用种群相对丰度均显著高于CK和F100(P＜0.05); 与CK和F100相比, MV1F80、MV1F60和MV2F60显著提高了固氮作用和尿素分解种群的相对丰度(P＜0.05), 降低了动物寄生或共生类和人类病原体类的种群相对丰度(P＜0.05); 与CK相比, 4个紫云英还田与化肥减量配施处理显著降低了硫酸盐呼吸的种群相对丰度(P＜0.05).

表 3 不同施肥处理对土壤细菌群落功能的影响1) Table 3 Effect of different fertilization treatments on soil bacterial community function

将上述8项差异功能与土壤部分理化特性(BD、SOC和TN)、细菌α多样性指数和主要菌门相对丰度指标进行相关分析(表 4).化能异养类和发酵类功能与BD均呈显著负相关(r为-0.484和-0.525), 而SOC与固氮类和发酵类功能均呈显著正相关(r为0.492和0.560); 化能异养类、固氮类和发酵类功能均与TN呈显著正相关(r为0.493~0.586), 而与细菌Sobs指数呈显著负相关(r为-0.498~-0.593); 化能异养类功能与放线菌门、变形菌门和厚壁菌门呈显著正相关(r为0.470~0.713), 而与酸杆菌门呈极显著负相关(r=-0.666); 动物寄生或共生类、人类所有病原体和人类病原体肺炎类功能与放线菌门呈显著负相关(r为-0.486~-0.515), 与厚壁菌门呈极显著负相关(r为-0.787~-0.835), 而与酸杆菌门呈显著正相关(r为0.540~0.587); 另外, 发酵类功能与酸杆菌门呈显著负相关(r=-0.503), 固氮类、发酵类和尿素分解类功能与厚壁菌门均呈极显著正相关(r为0.612~0.839).

表 4 土壤细菌群落功能与土壤理化特性和主要菌门相对丰度之间的关系1) Table 4 Relationships of soil bacterial community function with soil physicochemical properties and abundance of dominant phyla

3 讨论 3.1 紫云英还田与化肥减量配施对土壤理化性质的影响

本研究发现, 紫云英还田与化肥减量配施处理降低了土壤容重, 其中在MV2F60处理下达到显著水平, 这与Zhou等[8]的研究结果类似.其原因可能是较高的紫云英还田量可以改善土壤孔隙结构, 提高大团聚体含量[22, 23].另外, 高的紫云英还田量与化肥减量配施均显著提高了土壤有机碳, 这主要是因为外源有机物料的添加激发了土壤微生物的活性, 促进了土壤中碳的积累[24, 25], 这与Chen等[26]的研究结果一致.与CK相比, 紫云英还田与化肥减量配施处理均显著增加了土壤全氮、全磷和全钾, 这可能是由于豆科绿肥作物的固氮作用和活化土壤磷钾的生物学特性, 致使紫云英秸秆本身富含氮磷钾养分[27, 28], 而化肥的投入促进了作物生长发育, 提高了秸秆和根系的还田量, 进一步提高了养分的投入量[29].此外, 紫云英还田与化肥减量配施处理中较低的化肥用量处理的土壤总氮较高, 且在MV2F60处理显著高于单施化肥处理, 这可能因为过多的化肥氮的投入降低土壤碳氮比, 同时为异养微生物提供了充足氮源, 促进了有机氮的矿化分解, 增加氮素损失[30, 31].

3.2 紫云英还田与化肥减量配施对土壤细菌群落多样性和群落结构的影响

不同的施肥措施会影响土壤细菌群落的α多样性[32].本研究发现, 与单施化肥相比, 紫云英还田与化肥减量配施降低了细菌群落的Sobs指数和Shannon指数, 紫云英还田量越高, 效果越明显, 其中以MV2F60处理效果最为明显, 这与Tao等[18]和Caban等[33]的报道一致.此外, 紫云英还田与化肥减量配施处理显著影响了土壤细菌的β多样性, 改变了细菌的群落结构, 这与Ren等[34]关于有机肥部分替代化肥对土壤细菌群落特性影响的研究结果一致.这可能是由于长期紫云英还田与化肥减量配施改变了土壤细菌群落组成, 促使一些特定的细菌类群增长, 增强了细菌群落之间对营养物质的竞争, 从而抑制了其他细菌类群的生长[17].

不同施肥处理中, 绿弯菌门、放线菌门、变形菌门、酸杆菌门和厚壁菌门是细菌群落的优势菌门, 这与Gao等[10]在多位点、多年的绿肥-水稻轮作试验中得到的细菌优势类群相似.但不同施肥处理间优势类群的相对丰度存在一定差异.与单施化肥相比, 紫云英还田与化肥减量配施增加了放线菌门的相对丰度, 降低了酸杆菌门的相对丰度, 尤其是MV2F60处理, 这是因为放线菌门作为富营养菌, 需要通过分解作物残渣获取碳氮等营养基质而快速生长, 紫云英还田可以为放线菌门的生长提供充足的营养基质[34]; 酸杆菌门易富集在低肥力土壤上[12], 而本研究也已证实, 紫云英还田与化肥减量配施提高了土壤有机碳和全氮.厚壁菌门可以通过分泌胞外酶, 降解有机物料和植物源多糖, 并参与各种碳氮代谢功能, 有效促进土壤碳氮循环[35, 36].本研究发现, 与CK和单施化肥相比, MV1F80和MV2F60处理显著提高了厚壁菌门的相对丰度, 而对于MV1F60和MV2F80处理来说, 厚壁菌门的相对丰度没有统计学差异, 这可能是因为优化的紫云英还田与化肥减量配施比例可以提供合适的养分供应量, 进而提高参与土壤碳氮代谢过程的厚壁菌门的相对丰度[37].

土壤特性是影响细菌群落特性的重要因子[38].相关性分析结果表明, 土壤细菌群落的多样性指数均与土壤容重呈显著正相关, 而与有机碳显著负相关, 这与Pan等[39]的研究结果类似.同时也有研究表明, 土壤细菌群落的多样性指数与土壤容重呈显著负相关, 而与有机碳显著正相关[34, 40].利用RDA分析土壤理化指标对土壤细菌群落结构特征的影响, 研究发现, 不同的施肥处理下显著影响土壤细菌群落结构的主要土壤环境因子是土壤有机碳、全氮和全钾, 这与Zhang等[17]的报道类似; 但在南方双季稻田的研究发现, 土壤有机碳、速效磷和pH值是影响土壤细菌群落结构的主要环境因子[41].研究结果的差异可能与生态区域、轮作方式和取样时间的不同有关.

3.3 紫云英与化肥配施对土壤细菌群落功能的影响

通过FAPROTAX功能预测揭示了土壤细菌群落功能对不同施肥措施的响应特性.紫云英还田与化肥减量配施提高了化能异养类、发酵类、固氮类和尿素分解类功能类群丰度, 这可能是由于紫云英作为豆科绿肥作物, 不仅可以充分用光、水、热等自然资源, 增加碳的固持, 还可以通过根瘤菌固定大气中的氮, 富集于紫云英秸秆中[8], 而土壤中大多数细菌均可以通过降解有机物料中的碳氮化合物, 进而促进自身的生长繁殖, 提高与碳氮循环相关的细菌功能类群丰度[42].相关分析也表明, 发酵类和固氮类功能与有机碳显著正相关, 化能异养类、发酵类、固氮类和尿素分解类功能与总氮显著正相关, 这也证实了前人的研究, 化能异养类和发酵类功能与碳循环相关, 而固氮类和尿素分解类功能与土壤氮循环相关[43, 44].细菌功能类群丰度与主要细菌门丰度的相关分析表明, 化能异养类功能类群丰度与放线菌门、变形菌门和酸杆菌门显著相关, 这与Rivett等[45]的研究结果类似, 可能是因为这3种细菌菌门中的部分微生物参与了土壤有机质的矿化, 执行了细菌的化能异养类功能种群的功能[46, 47].厚壁菌门相对丰度与化能异养类、发酵类、固氮类和尿素分解类功能类群丰度呈显著正相关, 这可能是因为厚壁菌门中存在一些固氮菌属, 可以利用紫云英秸秆中的碳氮等能量来源, 促进自身生长, 有利于土壤碳氮循环过程的发生[12].本研究还找到了3种病原菌功能, 分别是动物寄生或共生类、人类所有病原体和人类病原体肺炎.本研究发现, 紫云英还田与化肥减量配施处理均降低了这3种病原菌功能类群丰度, 且相关分析表明, 这3种病原菌功能类群丰度与厚壁菌门和放线菌门的相对丰度呈极显著负相关, 而与酸杆菌门的相对丰度呈极显著正相关.这说明长期的紫云英还田与化肥减量配施可以改善土壤微生态环境, 提高有益菌丰度, 降低致病菌丰度, 有益于土壤健康.

4 结论

紫云英还田与化肥减量配施降低了土壤容重、增加了有机碳和全氮含量, 在相同化肥减量的基础上, 较高紫云英还田量的效果更显著.土壤肥力特性的改善, 促进了土壤细菌群落的变化, 其中, 有机碳、全氮和全钾是影响细菌群落结构的主要环境因子.此外, 紫云英还田与化肥减量配施降低了细菌α多样性指数, 增加了厚壁菌门的相对丰度, 还提高了化能异养类、发酵类、固氮类和尿素分解类功能类群丰度, 降低与病原菌相关的功能类群丰度.综合以上研究结果, MV2F60是较为适宜的紫云英还田量与化肥减量的配施组合.

参考文献
[1] Cui Z L, Zhang H Y, Chen X P, et al. Pursuing sustainable productivity with millions of smallholder farmers[J]. Nature, 2018, 555(7696): 363-366. DOI:10.1038/nature25785
[2] Xiang X J, Liu J, Zhang J, et al. Divergence in fungal abundance and community structure between soils under long-term mineral and organic fertilization[J]. Soil and Tillage Research, 2020, 196. DOI:10.1016/j.still.2019.104491
[3] Yu D L, Wen Z G, Li X M, et al. Effects of straw return on bacterial communities in a wheat-maize rotation system in the North China plain[J]. PLoS One, 2018, 13(6). DOI:10.1371/journal.pone.0198087
[4] Han X M, Hu C, Chen Y F, et al. Crop yield stability and sustainability in a rice-wheat cropping system based on 34-year field experiment[J]. European Journal of Agronomy, 2020, 113. DOI:10.1016/j.eja.2019.125965
[5] 李文涛, 于春晓, 张丽莉, 等. 有机无机配施对水稻产量及氮肥残效的影响[J]. 中国土壤与肥料, 2022(1): 63-72.
Li W T, Yu C X, Zhang L L, et al. Effect of organic and inorganic fertilizer on rice yield and nitrogen residual effect[J]. Soil and Fertilizer Sciences in China, 2022(1): 63-72.
[6] Qaswar M, Liu Y R, Huang J, et al. Soil nutrients and heavy metal availability under long-term combined application of swine manure and synthetic fertilizers in acidic paddy soil[J]. Journal of Soils and Sediments, 2020, 20(15): 2093-2106.
[7] Li Y H, Bai N, Tao Z K, et al. Rethinking application of animal manure for wheat production in China[J]. Journal of Cleaner Production, 2021, 318. DOI:10.1016/j.jclepro.2021.128473
[8] Zhou X, Lu Y H, Liao Y L, et al. Substitution of chemical fertilizer by Chinese milk vetch improves the sustainability of yield and accumulation of soil organic carbon in a double-rice cropping system[J]. Journal of Integrative Agriculture, 2019, 18(10): 2381-2392. DOI:10.1016/S2095-3119(18)62096-9
[9] 曹卫东, 包兴国, 徐昌旭, 等. 中国绿肥科研60年回顾与未来展望[J]. 植物营养与肥料学报, 2017, 23(6): 1450-1461.
Cao W D, Bao X G, Xu C X, et al. Reviews and prospects on science and technology of green manure in China[J]. Journal of Plant Nutrition and Fertilizer, 2017, 23(6): 1450-1461.
[10] Gao S J, Cao W D, Zhou G P, et al. Bacterial communities in paddy soils changed by milk vetch as green manure: a study conducted across six provinces in South China[J]. Pedosphere, 2021, 31(4): 521-530. DOI:10.1016/S1002-0160(21)60002-4
[11] Pan H, Chen M M, Feng H J, et al. Organic and inorganic fertilizers respectively drive bacterial and fungal community compositions in a fluvo-aquic soil in Northern China[J]. Soil and Tillage Research, 2020, 198. DOI:10.1016/j.still.2019.104540
[12] 练金山, 王慧颖, 徐明岗, 等. 长期施用有机肥潮土细菌的多样性及功能预测[J]. 植物营养与肥料学报, 2021, 27(12): 2073-2082.
Lian J S, Wang H Y, Xu M G, et al. Diversity and function prediction of bacteria community in fluvo-aquic soils as affected by long-term organic fertilization[J]. Journal of Plant Nutrition and Fertilizer, 2021, 27(12): 2073-2082. DOI:10.11674/zwyf.2021225
[13] Wang H, Liu S R, Zhang X, et al. Nitrogen addition reduces soil bacterial richness, while phosphorus addition alters community composition in an old-growth N-rich tropical forest in southern China[J]. Soil Biology and Biochemistry, 2018, 127: 22-30. DOI:10.1016/j.soilbio.2018.08.022
[14] 刘志平, 周怀平, 解文艳, 等. 长期氮磷配施对褐土细菌多样性及土壤酶活性的影响[J]. 干旱地区农业研究, 2022, 40(2): 163-171.
Liu Z P, Zhou H P, Xie W Y, et al. Effects of long-term combined application of nitrogen and phosphorus on bacterial diversity and soil enzyme activities in cinnamon soil[J]. Agricultural Research in the Arid Areas, 2022, 40(2): 163-171.
[15] Li Z Q, Zhang X, Xu J, et al. Green manure incorporation with reductions in chemical fertilizer inputs improves rice yield and soil organic matter accumulation[J]. Journal of Soils and Sediments, 2020, 20(7): 2784-2793. DOI:10.1007/s11368-020-02622-2
[16] Stark C, Condron L M, Stewart A, et al. Influence of organic and mineral amendments on microbial soil properties and processes[J]. Applied Soil Ecology, 2007, 35(1): 79-93. DOI:10.1016/j.apsoil.2006.05.001
[17] Zhang X X, Zhang R J, Gao J S, et al. Thirty-one years of rice-rice-green manure rotations shape the rhizosphere microbial community and enrich beneficial bacteria[J]. Soil Biology and Biochemistry, 2017, 104: 208-217. DOI:10.1016/j.soilbio.2016.10.023
[18] Tao J M, Liu X D, Liang Y L, et al. Maize growth responses to soil microbes and soil properties after fertilization with different green manures[J]. Applied Microbiology and Biotechnology, 2017, 101(3): 1289-1299. DOI:10.1007/s00253-016-7938-1
[19] Liu J A, Shu A P, Song W F, et al. Long-term organic fertilizer substitution increases rice yield by improving soil properties and regulating soil bacteria[J]. Geoderma, 2021, 404. DOI:10.1016/j.geoderma.2021.115287
[20] 鲍士旦. 土壤农化分析[M]. (第三版). 北京: 中国农业出版社, 2000.
[21] Zhang J S, Miao Q, Xue Y F, et al. Managing soils and crops for sustainable agricultural intensification in coastal saline zones[J]. Agronomy Journal, 2020, 112(4): 3076-3088. DOI:10.1002/agj2.20249
[22] Wang L L, Sun X Y, Li S Y, et al. Application of organic amendments to a coastal saline soil in north China: effects on soil physical and chemical properties and tree growth[J]. PLoS One, 2014, 9(2). DOI:10.1371/journal.pone.0089185
[23] Xiu L, Zhang W M, Sun Y Y, et al. Effects of biochar and straw returning on the key cultivation limitations of albic soil and soybean growth over 2 years[J]. Catena, 2019, 173: 481-493. DOI:10.1016/j.catena.2018.10.041
[24] Liu X, Zhou F, Hu G Q, et al. Dynamic contribution of microbial residues to soil organic matter accumulation influenced by maize straw mulching[J]. Geoderma, 2019, 333: 35-42. DOI:10.1016/j.geoderma.2018.07.017
[25] Li Z Q, Li D D, Ma L, et al. Effects of straw management and nitrogen application rate on soil organic matter fractions and microbial properties in North China plain[J]. Journal of Soils and Sediments, 2019, 19(2): 618-628. DOI:10.1007/s11368-018-2102-4
[26] Chen J R, Qin W J, Chen X F, et al. Application of Chinese milk vetch affects rice yield and soil productivity in a subtropical double-rice cropping system[J]. Journal of Integrative Agriculture, 2020, 19(8): 2116-2126. DOI:10.1016/S2095-3119(19)62858-3
[27] Qaswar M, Huang J, Ahmed W, et al. Substitution of inorganic nitrogen fertilizer with green manure (GM) increased yield stability by improving C input and nitrogen recovery efficiency in rice based cropping system[J]. Agronomy, 2019, 9(10). DOI:10.3390/agronomy9100609
[28] Solangi F, Bai J S, Gao S J, et al. Improved accumulation capabilities of phosphorus and potassium in green manures and its relationship with soil properties and enzymatic activities[J]. Agronomy, 2019, 9(11). DOI:10.3390/agronomy9110708
[29] 段晨骁. 有机无机肥配施对关中地区土壤肥力及冬小麦产量的影响[D]. 杨凌: 西北农林科技大学, 2021.
Duan C X. Effects of combined application of organic and inorganic fertilizers on soil fertility and winter wheat yield in Guanzhong area[D]. Yangling: Northwest A&F University, 2021.
[30] 张电学, 韩志卿, 吴素霞, 等. 不同施肥制度对褐土有机氮及其组分的影响[J]. 华北农学报, 2017, 32(3): 201-206.
Zhang D X, Han Z Q, Wu S X, et al. Effect of different fertilization regimes on organic nitrogen and its fractions in cinnamon soil[J]. Acta Agriculturae Boreali-Sinica, 2017, 32(3): 201-206.
[31] 李玲玲. 有机肥氮素有效性和替代化肥氮比例研究[D]. 北京: 中国农业科学院, 2011.
Li L L. Manure nitrogen availability and its substitution ratio for chemical fertilizer nitrogen[D]. Beijing: Chinese Academy of Agricultural Sciences, 2011.
[32] Fu H P, Li H, Yin P, et al. Integrated application of rapeseed cake and green manure enhances soil nutrients and microbial communities in tea garden soil[J]. Sustainability, 2021, 13(5). DOI:10.3390/su13052967
[33] Caban J R, Kuppusamy S, Kim J H, et al. Green manure amendment enhances microbial activity and diversity in antibiotic-contaminated soil[J]. Applied Soil Ecology, 2018, 129: 72-76. DOI:10.1016/j.apsoil.2018.04.013
[34] Ren J H, Liu X L, Yang W P, et al. Rhizosphere soil properties, microbial community, and enzyme activities: Short-term responses to partial substitution of chemical fertilizer with organic manure[J]. Journal of Environmental Management, 2021, 299. DOI:10.1016/j.jenvman.2021.113650
[35] He H B, Li W X, Zhang Y W, et al. Effects of Italian ryegrass residues as green manure on soil properties and bacterial communities under an Italian ryegrass (Lolium multiflorum L.)-rice (Oryza sativa L.) rotation[J]. Soil and Tillage Research, 2020, 196. DOI:10.1016/j.still.2019.104487
[36] 任敏. 塔里木盆地微生物群落结构及其在碳氮元素循环中的作用[D]. 武汉: 华中农业大学, 2018.
Ren M. Microbial communities in the Tarim Basin soil: diversity and their roles in carbon and nitrogen cycle[D]. Wuhan: Huazhong Agricultural University, 2018.
[37] Kong J Q, He Z B, Chen L F, et al. Efficiency of biochar, nitrogen addition, and microbial agent amendments in remediation of soil properties and microbial community in Qilian Mountains mine soils[J]. Ecology and Evolution, 2021, 11(14): 9318-9331. DOI:10.1002/ece3.7715
[38] Liu Y M, Cao W Q, Chen X X, et al. The responses of soil enzyme activities, microbial biomass and microbial community structure to nine years of varied zinc application rates[J]. Science of the Total Environment, 2020, 737. DOI:10.1016/j.scitotenv.2020.140245
[39] Pan J X, Shang Y W, Zhang W J, et al. Improving soil quality for higher grain yields in Chinese wheat and maize production[J]. Land Degradation & Development, 2020, 31(9): 1125-1137.
[40] Zhou J, Jiang X, Zhou B K, et al. Thirty four years of nitrogen fertilization decreases fungal diversity and alters fungal community composition in black soil in Northeast China[J]. Soil Biology and Biochemistry, 2016, 95: 135-143. DOI:10.1016/j.soilbio.2015.12.012
[41] Zhou G P, Gao S J, Lu Y H, et al. Co-incorporation of green manure and rice straw improves rice production, soil chemical, biochemical and microbiological properties in a typical paddy field in southern China[J]. Soil and Tillage Research, 2020, 197. DOI:10.1016/j.still.2019.104499
[42] 蒋梦芸. 农田土壤微生物群落对长期玉米秸秆还田的响应研究[D]. 重庆: 重庆三峡学院, 2021.
[43] Liang Z S, Yu Y, Ye Z K, et al. Pollution profiles of antibiotic resistance genes associated with airborne opportunistic pathogens from typical area, Pearl River estuary and their exposure risk to human[J]. Environment International, 2020, 143. DOI:10.1016/j.envint.2020.105934
[44] 赵文慧, 马垒, 徐基胜, 等. 秸秆与木本泥炭短期施用对潮土有机质及微生物群落组成和功能的影响[J]. 土壤学报, 2020, 57(1): 153-164.
Zhao W H, Ma L, Xu J S, et al. Effect of application of straw and wood peat for a short period on soil organic matter and microbial community in composition and function in fluvo-aquic soil[J]. Acta Pedologica Sinica, 2020, 57(1): 153-164.
[45] Rivett D W, Bell T. Abundance determines the functional role of bacterial phylotypes in complex communities[J]. Nature Microbiology, 2018, 3(7): 767-772. DOI:10.1038/s41564-018-0180-0
[46] 丛微, 喻海茫, 于晶晶, 等. 人参种植对林地土壤细菌群落结构和代谢功能的影响[J]. 生态学报, 2021, 41(1): 162-171.
Cong W, Yu H M, Yu J J, et al. Effects of ginseng cultivation on soil microbial community structure and metabolic functions in forest land[J]. Acta Ecologica Sinica, 2021, 41(1): 162-171.
[47] 翟亚萍, 王绍明, 刘鸯, 等. 不同种植地苜蓿根际土壤细菌群落结构多样性差异分析[J]. 新疆农业科学, 2021, 58(5): 955-964.
Zhai Y P, Wang S M, Liu Y, et al. Study on structural diversity of bacterial community in rhizosphere soil of alfalfa in parts of northern foot of Tianshan Mountains[J]. Xinjiang Agricultural Sciences, 2021, 58(5): 955-964.