环境科学  2024, Vol. 45 Issue (11): 6745-6755   PDF    
白膜覆盖下增效氮肥对春玉米土壤质量、微生物代谢和生态功能多样性的影响
杨睿哲1,2, 杨泽1, 徐灵颖3, 张璟玥4, 王菊4, 张绪成5, 王红丽5, 李廷亮6, 王朝辉1, 李紫燕1     
1. 西北农林科技大学资源环境学院, 农业农村部西北植物营养与农业环境重点实验室, 杨凌 712100;
2. 南京大学地理与海洋学院, 南京 210033;
3. 中国科学院南京土壤研究所, 江苏常熟农田生态系统国家野外科学观测研究站, 南京 210008;
4. 西北农林科技大学草业与草原学院, 杨凌 712100;
5. 甘肃省农业科学院旱地农业研究所, 兰州 730070;
6. 山西农业大学资源与环境学院, 太谷 030801
摘要: 过量施用氮肥是威胁土壤健康和降低土壤多种功能的主要驱动力.增效氮肥(EENFs)[例如脲酶抑制剂(NBPT)、硝化抑制剂(DCD)和树脂包膜尿素(RCN)]已被证明是减少氮肥施用的有效措施.然而, 增效氮肥对土壤质量(SQI)、微生物代谢特征和土壤生态功能多样性(EMF)的影响以及其内在关系还不清楚.因此, 基于宁夏回族自治区固原市彭阳试验站于2019年开始的田间定位试验, 研究了白膜覆盖下不同施肥策略[不施氮肥(N0)、矿物氮肥(N200)、DCD、NBPT和RCN]对SQI、酶化学计量比特征和EMF的影响.结果表明:①相比N0, N200和EENFs增加了TN、MBC和MBN含量;相比N0和N200, NBPT和DCD的SQI提高了59.97%~104.78%和43.28%~83.42%, 而RCN的SQI无显著变化.②EENFs比N200能更好地缓解微生物碳和氮限制, 并提高土壤生态功能多样性21.97%~51.53%.③土壤中的MBC、MBN、AN、AP和SWC是影响微生物碳限制、氮限制和土壤生态功能多样性的共同主要因素, 且提高土壤质量和缓解微生物碳和氮限制有利于提高土壤生态功能多样性.综合来看, 白膜覆盖下施用NBPT和DCD可短期内实现土壤健康和生态功能多样性的双赢, 这可为当地优化施肥管理措施提供参考.
关键词: 覆膜      增效氮肥(EENFs)      土壤质量(SQI)      微生物代谢特征      土壤生态功能多样性(EMF)     
Effects of Enhanced-efficiency Nitrogen Fertilizers on Soil Quality, Microbial Metabolism, and Soil Ecosystem Multifunctionality of Spring Maize Under White Plastic Film Mulching
YANG Rui-zhe1,2 , YANG Ze1 , XU Ling-ying3 , ZHANG Jing-yue4 , WANG Ju4 , ZHANG Xu-cheng5 , WANG Hong-li5 , LI Ting-liang6 , WANG Zhao-hui1 , LI Zi-yan1     
1. Key Laboratory of Plant Nutrition and the Agro-Environment in Northwest China, Ministry of Agriculture and Rural Affairs, College of Natural Resources and Environment, Northwest A & F University, Yangling 712100, China;
2. School of Geography and Ocean Science, Nanjing University, Nanjing 210033, China;
3. Changshu National Agro-Ecosystem Observation and Research Station, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China;
4. College of Grassland Agriculture, Northwest A & F University, Yangling 712100, China;
5. Institute of Dryland Farming, Gansu Academy of Agricultural Sciences, Lanzhou 730070, China;
6. College of Resources and Environment, Shanxi Agricultural University, Taigu 030801, China
Abstract: Excessive nitrogen fertilizer application is the main driving force threatening soil health and reducing multiple soil functions. The enhanced-efficiency nitrogen fertilizers (EENFs), such as urease inhibitors (NBPT), nitrification inhibitors (DCD), and coated controlled-release urea (RCN), have been proven to be effective measures for reducing nitrogen fertilizer application. However, the effects of EENFs on soil quality (SQI), microbial metabolic characteristics, and soil ecosystem multifunctionality (EMF) and their internal relationships are still unclear. Therefore, based on the field positioning experiment started in 2019 by Pengyang Experimental Station of Guyuan City, Ningxia Hui Autonomous Region, we studied the effects of different fertilization strategies (no nitrogen fertilizer (N0), mineral nitrogen fertilizer (N200), DCD, NBPT, and RCN) on SQI, soil enzyme stoichiometry, and EMF under white plastic film mulching. The results revealed that: ① Compared with that under N0, N200 and EENFs increased soil total nitrogen (TN), microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) contents. Compared to the SQI of N0 and N200, that of NBPT and DCD significantly increased by 59.97%-104.78% and 43.28%-83.42%, respectively, while RCN showed no significant change. ②EENFs can alleviate microbial carbon and nitrogen limitations better than N200 and increase soil EMF by 21.97% -51.53%. ③ The MBC, MBN, available nitrogen (AN), available phosphorus (AP), and water content (SWC) of soil factors were the common main factors affecting microbial C limitation, N limitation, and soil EMF. Moreover, the improvement in soil quality and alleviating microbial C and N limitation were conducive to improving soil EMF. Overall, the NBPT and DCD application under white plastic film mulching can achieve a win-win situation of soil health and EMF in the short term, which can provide references for optimizing local fertilization management measures.
Key words: plastic filming      enhanced-efficiency nitrogen fertilizer(EENFs)      soil quality(SQI)      microbial metabolic characteristics      soil ecosystem multifunctionality (EMF)     

土壤是人类赖以生存的重要基础, 具有粮食生产、环境服务和维持气候稳定等多种生态系统服务功能(即生态功能多样性, EMF)[1, 2].因此, 维持土壤健康是保证农作物生产力和生态功能多样性的基础[3].然而, 过量矿质氮肥施用是威胁土壤健康和生态功能多样性的主要原因[4 ~ 6], 因为矿质氮肥会极大地影响土壤物理性状和生物化学过程[7], 进而威胁土壤健康并改变土壤功能[8].增效氮肥(EENFs)[例如脲酶抑制剂(NBPT)、硝化抑制剂(DCD)和树脂包膜尿素(RCN)]是减少氮肥施用、提高作物产量和作物氮素利用率的有效方法之一[9 ~ 11].目前, 关于增效氮肥的研究多集中在作物氮素利用率、氮淋溶和温室气体排放等方面[12 ~ 14], 而增效氮肥对土壤健康和土壤功能的研究还鲜有报道.此外, 增效氮肥的有效性高度依赖农业管理措施(例如覆膜)[15], 因为不同农业管理措施通过改变环境条件(例如温度或湿度)影响EENFs作用效果的持续时间[16 ~ 18], 导致其在农业生产实践中的推广应用效果差异较大.因此, 有必要进一步研究特定农业管理措施(白膜覆盖)下施用增效氮肥对土壤健康和生态功能多样性的影响, 为实现农业的可持续化发展提供基础.

土壤质量(SQI)是体现土壤的农业生产力和维持生态环境健康的综合指标[19], 已被广泛运用于土壤健康评价体系.此外, 土壤酶活性对几乎所有的生化反应都至关重要, 被用作土壤健康和生产力的敏感指标[20].酶化学计量学可以反映微生物代谢需求与土壤养分供应的关系[21], 被广泛用于揭示微生物碳氮磷限制差异[22]. Sinsabaugh等[23]研究表明, 表层土壤的碳循环酶(C-acqing)、氮循环酶(N-acqing)和磷循环酶(P-acqing)的比例通常等于1∶1∶1, 如果土壤酶化学计量比偏离1∶1∶1则表明微生物整体面临某种养分(碳、氮或磷)限制[24].一般来说, 土壤微生物比作物在获取土壤养分方面更具有优势[25].在微生物养分限制的情况下, 作物也可能面临相同的养分限制[26], 所以研究土壤微生物的代谢特征对确定增效氮肥是否有利于土壤健康和作物生产力非常重要.

当前, 以生物化学过程之间相互作用为特征的多种土壤功能越来越成为人们关注的焦点[3].土壤EMF方法的基础是对参与土壤养分循环过程中的相关酶的评估[27], 是评价土壤碳氮循环过程和土壤生物状况的主要指标[28, 29].然而, 先前的大量研究主要集中在某些土壤养分变化改变了微生物多样性和群落组成, 进而影响了特定的土壤生态功能[30, 31], 而关于土壤的综合状况(即土壤质量)和微生物代谢特征与土壤生态功能多样性之间的关系仍不清楚[32 ~ 34].

中国西北干旱区是春玉米的主要产地, 因该地域光热资源丰富, 也是我国重要的玉米制种基地[35].因此, 保持中国西北半干区的玉米种植健康发展是直接关系到西北地区的生态环境建设、群众的经济收入以及乡村振兴的关键.基于此, 本研究依托宁夏回族自治区固原市彭阳试验站, 旨在探明∶①不同增效氮肥对土壤质量和养分含量影响的差异;②不同增效氮肥对微生物代谢特征和生态功能多样性的影响以及其驱动因子;③土壤质量、微生物碳限制、微生物氮限制与土壤生态功能多样性之间的关系.

1 材料与方法 1.1 研究区概况

田间试验于2019年设立于宁夏回族自治区固原市彭阳试验站(35°45'N, 106°40'E), 为温带半干旱大陆性季风气候, 属于典型的旱作雨养农业区.本试验地土壤类型为轻壤质浅黑垆土, 试验前0~20 cm的土壤有机碳[ω(SOC)]为16.64 g·kg-1, 全氮[ω(TN)]为0.81 g·kg-1, 全钾[ω(TK)]为17.95 g·kg-1, pH值为8.02.

1.2 试验设计

本试验采用随机区组设计, 每组3个重复, 包括5个处理:不施氮肥(N0), 普通尿素(施氮量200 kg·hm-2, 以N计, N200), N200+DCD(施N量的1%), N200 +NBPT(施N量的1%), 尿素(200 kg·hm-2的70%, 以N计)+ RCN(200 kg·hm-2的30%, 以N计).在每个处理中, 施用等量的磷肥和钾肥, 用量为90 kg·hm-2的过磷酸钙(以P2O5计), 45 kg·hm-2的硫酸钾(以K2O计).所有肥料在春玉米播种前作为基肥在土壤表面施用一次.此外, 为保证施肥均匀, 在施肥前将所有肥料提前混合.

试验地的田间试验小区面积为16.5 m2, 种植作物为玉米(Zea mays L.), 品种为“先玉335”, 种植密度为65 000株·hm-2, 种植深度为5 cm, 于2021年4月下旬播种, 2021年10月中旬收获.农田用水均来源于自然降雨, 并采用白膜覆盖.

1.3 样品采集和测定

本研究于2021年玉米吐丝期(R1)分别采集土壤和植株样品.在去除地表植物凋落物, 细根和塑料碎片后, 用直径为5 cm的土钻采集表层(0~20 cm)土壤样品.在每个小区随机采集10个样点土壤再均匀混合为一个土壤样品后分为两份:一部分风干过筛后用于SOC、TN、TK、全磷(TP)、有效氮(AN)、有效磷(AP)、有效钾(AK)和pH等指标测定;另一部分放置于4℃冰箱用于土壤含水量(SWC)、微生物量碳氮和碳氮磷循环酶活等指标测定. SOC通过重铬酸钾外加热法测定[36]. TN通过凯氏定氮法测定[37]. AN通过碱解还原扩散法测定[38]. TP和AP通过钼蓝法测定[38] .TK和AK通过火焰光度法测定[38].土壤pH通过探针在土水混合液(1∶2.5)中测定(Mettler Toledo FE28标准).SWC通过烘箱105℃干燥24 h测定.土壤微生物量碳(MBC)和微生物量氮(MBN)通过氯仿熏蒸萃取法测定[39].采用微孔板荧光法测定土壤酶活性[β-1, 4-葡萄糖苷酶(BG)]、纤维二糖苷酶(CBH)、亮氨酸氨基肽酶(LAP)、1, 4-β-N-乙酰氨基葡萄糖苷酶(NAG)和碱性磷酸酶(AKP)活性[40, 41].

另外, 在每个小区内选择长势均匀的植株样品, 并在根部位置切断, 分别采取地上植物样品和地下根部样品.最终将采集各部分的玉米样品带回后晾晒、杀青、烘干, 最终运用称量法计算玉米地上(APB)和地下干物质量(UPB).

1.4 数据分析

为了评估不同施肥类型对土壤质量的影响, 本文将不同土壤指标运用隶属函数与因子分析结合的方式进行计算[42], 计算公式如下:

(1)
(2)

式中, XAB表示第A个土壤样本的第B个土壤指标.XmaxXmin分别表示B土壤指标中所有土壤样本的最大值和最小值.F(XAB)表示第A个土壤样品的第B个土壤指数的隶属函数值.如果土壤指数与SQI呈正相关, 则使用公式(1).与之相反, 使用公式(2).

根据土壤指标因子分析结果的公因子方差, 得到各项指标权重值并计算土壤质量指数, 计算公式如下:

(3)

式中, W(XAB)表示第A个土壤样品的第B个土壤指标的权重值, n表示土壤样本的数量.

为了评估不同施肥类型对不同土壤碳, 氮和磷循环酶过程的相关酶活性, 用平均土壤酶活性(EEAs)分别统一表征碳循环相关酶活性(BG和CBH, C-acq), 氮循环相关酶活性(NAG和LAP, N-acq), 磷循环相关酶活性(AKP, P-acq), 计算公式如下[43]

(4)
(5)

通过酶化学计量比来表征不同施肥策略对微生物代谢特征的影响[44], 计算公式如下:

(6)
(7)
(8)
(9)

式中, Vector length表示土壤微生物相对碳的限制程度, Vector length越大表明微生物的代谢受碳限制越大. Vector angle表示土壤微生物相对氮(或磷)的限制程度.当向量角度大于45°表示微生物代谢受磷限制, 而向量角度小于45°表示微生物受土壤氮限制, 且越小表明氮限制程度越高[44].

土壤EMF是基于土壤酶活性和作物参数(地上和地下作物生物量)[8, 45]计算得到, 公式如下:

(10)

式中, PiP分别表示不同施肥处理的测定值和最大值, n表示指标个数.

1.5 统计分析

所有变量均采用Levene和Shapiro-WilK检验进行正态性和齐性检验, 之后进行统计学分析.采用单因素方差分析确定不同施肥措施对土壤养分、SQI、向量长度、向量角度和EMF的影响, 不同处理间差异性通过邓肯多重范围检验(Duncan multiple-range test)进行评估(P < 0.05).柱状图主要通过R语言软件(版本4.1.3)中的“ggplot2”包绘制.运用冗余分析(RDA)确定土壤酶、向量长度和角度与微生物量和土壤基本理化性质之间的关系, 并通过“Vegan”包进行分析.通过“random Forest”包构建向量长度和角度的随机森林模型, 以判断哪些土壤质量因子为重要影响因素.同时, 通过使用“rf Utilities”包来确定模型的整体显著性.通过R语言软件(版本4.1.3)中的“ggcor”包绘制土壤理化性质、微生物量之间的相关关系热图, 以及与EMF的Mantel-test检验组合图.采用线性回归检验了SQI、向量长度、向量角度与EMF之间的关系.通过的“corrplot”软件包计算土壤理化性质、微生物量、SQI、C-acq、N-acq、P-acq、地上生物量、地下生物量、向量长度、向量角度和EMF之间的Pearson相关性.

2 结果与分析 2.1 施肥措施对土壤质量和养分的影响

本研究发现, 不同施肥类型几乎对所有土壤理化性质、微生物量和作物生物量都有影响(表 1).结果表明, 施用氮肥(尤其EENFs)降低土壤SOC、AP和SWC含量(部分处理不显著), 提高土壤TN、AN、MBC和MBN含量, 以及地上和地下生物量.同时, 本研究发现, 相比N200, 施用NBPT和DCD可显著提高AN、MBC和MBN含量, 而RCN无显著差异.此外, 相比N200, 施用NBPT和DCD可显著提高地下和地上生物量, RCN可显著提高地下生物量.

表 1 不同施肥类型对土壤理化性质、微生物量、土壤酶活性和玉米生物量的影响1) Table 1 Effects of different fertilization types on soil physicochemical properties, microbial biomass, soil enzyme activity, and maize biomass

图 1表明, 相比N0, 施用N200和RCN的SQI无显著变化.相比N0和N200, 施用NBPT的SQI提高了59.97%~104.78%, DCD的SQI提高了43.28%~83.42%(P < 0.05).

误差棒表示数值±标准误差(n=3);不同小写字母表示处理间差异显著(P < 0.05);SQI表示土壤质量指数 图 1 不同施肥处理对春玉米土壤质量的影响 Fig. 1 Response of soil quality (SQI) to different fertilization treatments of spring maize

2.2 施肥措施对土壤酶活性和微生物代谢特征的影响

施肥处理对不同土壤酶活性有不同影响(表 1).本研究发现, 相比N0, 施用氮肥可以不同程度地提高土壤C-acq、N-acq和P-acq酶活性.同时, 相比N200, EENFs可显著提高P-acq酶活性, NBPT、DCD和RCN分别提高45.58%、37.59%和30.55%.

酶化学计量学的结果表明, 本研究中的N0和N200数据点均在1∶1线以下[图 2(a)], 表明微生物代谢存在较强的氮限制, 而EENFs更有利于微生物养分平衡.图 2(b)2(c)表明, EENFs比N200可更好地缓解微生物碳和氮限制.回归分析表明, 向量长度与向量角度负相关[R2=0.84, P < 0.05;图 2(d)].

误差棒表示数值±标准误差(n=3);不同小写字母表示处理间差异显著(P < 0.05);灰色阴影表示95%的置信区间;BG表示β-1, 4-葡萄糖苷酶, CBH表示纤维二糖苷酶, NAG表示β-1, 4-N-乙酰氨基葡萄糖苷酶, LAP表示L-亮氨酸氨基肽酶, AKP表示碱性磷酸酶 图 2 不同施肥处理的春玉米土壤酶化学计量比和矢量特征 Fig. 2 Soil enzyme stoichiometry and vector characteristics of spring maize under different fertilization treatments

2.3 土壤微生物代谢特征与土壤环境因子的关系

RDA分析表明, 几乎所有土壤环境因子(除TP)对C-acq、N-acq、P-acq、向量长度和向量角度有显著影响[(P < 0.05;图 3(a)].随机森林模型表明, MBC、MBN、AN、AP和SWC是影响向量长度和向量角度的主要土壤因子[图 3(b)3(c)].同时, 向量长度与MBC、MBN和AN均为显著负相关, 而向量角度与其均为显著正相关(图 7).此外, 回归分析表明, 向量角度随着SQI增加而提高[P < 0.05;图 4(a)], 向量长度随着SQI增加而下降[P < 0.01;图 4(b)], 这表明土壤质量的提高有利于缓解微生物氮限制和微生物碳限制.

黑色箭头表示土壤理化性质和微生物量, 红色箭头表示土壤酶活性和矢量特征 图 3 土壤酶活性和向量特征与土壤因子和微生物量的关系 Fig. 3 Relationship between soil enzyme activity and vector characteristics with soil factors and microbial biomass

灰色阴影表示95%的置信区间 图 4 土壤质量与向量长度和向量角度的回归分析 Fig. 4 Regression analysis between soil quality (SQI) and vector features

误差棒表示数值±标准误差(n=3);不同小写字母表示处理间差异显著(P < 0.05);EMF表示生态功能多样性 图 5 不同施肥处理对土壤生态功能多样性的影响 Fig. 5 Response of soil ecosystem multifunctionality (EMF) to different fertilization treatments

灰色阴影表示95%的置信区间 图 6 土壤生态功能多样性与土壤环境因子、土壤质量和向量特征的关系 Fig. 6 Relationship between soil ecosystem multifunctionality and soil environmental factors, soil quality, and vector features

1.SOC, 2.TN, 3.TP, 4.TK, 5.AN, 6.AP, 7.AK, 8.SWC, 9.MBC, 10.MBN, 11.pH, 12.ST, 13.SQI, 14.C-acq, 15.N-acq, 16.P-acq, 17.UPB, 18.APB, 19.向量长度, 20.向量角度, 21.EMF;数值为相关系数, 空值为指标之间不具有显著相关性;椭圆为蓝色表示正相关, 椭圆为红色表示负相关;颜色深浅和椭圆大小表示相关性强弱 图 7 土壤质量、向量特征、土壤生态功能多样性与土壤环境因子、微生物量、土壤酶活性和作物生物量的相关性分析 Fig. 7 Correlation analysis of soil quality(SQI), vector features, and soil ecosystem multifunctionality (EMF)with soil environmental factors, microbial biomass, soil enzyme activity, and crop biomass

2.4 施肥措施对土壤生态功能多样性的影响以及主要驱动力

相比N0, 施用氮肥可显著提高土壤EMF(图 5).相比N200, 施用NBPT、DCD和RCN的土壤EMF分别显著提高了51.53%、21.97%和30.67%.

Mantel-test分析表明, SOC、TN、AN、AP、SWC、MBC和MBN是影响土壤EMF的主要驱动力[r > 0.2, P < 0.01;图 6(a)].同时, 土壤EMF与TN、AN、MBC和MBN为显著正相关, 与SWC为显著负相关(图 7).回归分析表明, EMF随着SQI和向量角度增加而提高[图 6(b)6(d)], 而随着向量长度增加而降低[图 6(c)], 这表明土壤质量的提高以及缓解微生物氮和碳限制有利于提高土壤生态功能多样性.

3 讨论 3.1 增效氮肥对土壤质量和养分的影响

本研究发现, 施用氮肥(N200和EENFs)增加了TN、MBC和MBN含量, 但对SOC、AP和SWC含量有消极影响.一般而言, 施用氮肥会通过提高AN的含量, 进而对TN含量产生积极影响[46, 47].此外, 由于土壤C、N和P循环紧密耦合[48], 因此施用氮肥对土壤N调控的同时不可避免影响了土壤C和P的动态变化(图 7).相比N200, 施用NBPT和DCD可显著提高玉米吐丝期的土壤AN含量, 这与很多研究结果相似[10, 49, 50], 因为NBPT和DCD可通过调节土壤氮素转换过程减少玉米生育前期的土壤N损失(例如减少氮氧化物排放), 以保证玉米生长关键期的氮素供应[51].与之相反, 施用矿物氮肥(N200)后, 由于脲酶的作用将尿素快速转换为NH4+-N并最终通过硝化作用转变为NO3--N, 这会造成作物生长前期的矿质氮含量较高, 易导致更多的土壤N素通过淋溶等途径损失.同时, RCN的AN含量与N200无显著差异, 这与其它研究结果不同, 其原因可能是由于白膜覆盖下的温度较高导致RCN的涂层快速分解, 使RCN在玉米生育前期向土壤释放N氮素过快.此外, 施用氮肥(尤其EENFs)会不同程度地减少土壤SOC和AP含量并提高MBC和MBN含量, 同时SOC与AP有强正相关性(图 7).有研究表明, 氮肥施用通过提高土壤N含量, 导致微生物的数量增加和活性增强, 从而提高了SOC的矿化速率, 最终减少了SOC含量, 并增加了MBC和MBN含量[52, 53].同时, 根据吸附位置或较少可用表面的竞争关系原理, 有机碳含量减少可增加土壤对P的吸附潜力, 进而减少了AP含量[54, 55].另一方面, 氮肥施用会通过更好地促进作物对土壤P的吸收来减少AP含量[56].

MBC和MBN是表征土壤微生物活动的指标[57].相比于其它土壤碳和氮库, 微生物量的周转速率更快, 是土壤养分循环和全球生物化学循环中的主要驱动力[58].有研究表明, MBC和MBN是土壤碳或氮素的一个重要的暂时储存库并可调控相关微生物在土壤养分循环中的作用, 是反映土壤健康状况的准确指标[59].在本研究中, MBC、MBN和AN与SQI为强显著正相关(图 7).由此可见, 相比N200和RCN, NBPT和DCD主要通过短时间内提高MBC、MBN和AN含量进而改善土壤质量.然而, 相比N0, N200和RCN的SQI无显著增加, 这说明施用N200和RCN虽可改变不同土壤养分含量, 但不能全面提高土壤质量.

3.2 增效氮肥对土壤酶活性和微生物代谢特征的影响

土壤酶活性已被广泛应用于土壤健康评价[60], 其土壤酶活性大小与土壤环境因子密切相关[8].本研究发现, 施用氮肥可以提高碳、氮和磷循环酶活性并改变微生物代谢特征(表 1图 2), 且大部分土壤因子对不同酶活性、向量长度和向量角度有显著影响[图 3(a)].本研究结果证实氮肥施用可通过改变土壤养分资源的可用性来刺激微生物营养循环过程, 并促使土壤微生物进行了生理的调整(微生物量的改变), 从而最终影响微生物资源限制的状况, 这与其他研究结果相似[26, 61].

酶化学计量学结果表明, N0和N200处理下的土壤微生物群落主要受到氮限制, 这与其它研究结果一致[62].Cui等[63]研究表明, 微生物代谢需求与土壤养分资源供应之间的不平衡会制约微生物代谢活动.因此, 土壤微生物可通过改变酶活性, 矿化有机化合物, 以便减轻营养物质限制, 从而维持微生物C∶N∶P平衡[64].同时, 相比N200, EENFs可更好地缓解微生物氮限制, 且向量角度与AN和AP分别为显著正相关和负相关(图 7), 这表明EENFs可有效通过提高AN含量来缓解微生物与作物的土壤氮素竞争, 有利于微生物的养分平衡[48].此外, 土壤氮素有效性的提高会增加微生物氮代谢对土壤P的需求[26], 而土壤AP含量下降导致微生物获取土壤P素的难度升高, 使微生物分泌更多磷循环酶来获得更多的土壤P元素(表 1)[65], 从而在元素化学计量比偏移情况下维持酶化学计量平衡, 最终使土壤C、N、P循环相关的酶活性更接近1∶1∶1.

微生物受某种营养物质的代谢限制也可能受到另一种营养物质代谢的影响, 因为微生物需要维持自身的内稳态[66].本研究发现, 向量长度和向量角度之间存在负相关关系[图 2(d)], 这表明缓解微生物N限制可减弱微生物C限制程度.同时, 相比N0或N200, 施用EENFs也可显著缓解微生物C限制[图 2(c)], 且地上和地下生物量的增加有利于缓解微生物碳限制(图 7).因此, 其原因可能是施用EENFs可更好地提高作物生物量(地上或地下生物量), 从而有利于作物通过更多的凋落物输入来缓解微生物C限制[15, 46].同时, EENFs通过提高AN含量刺激微生物分解SOC, 进而增加自身碳含量(即MBC;表 1), 最终缓解微生物碳限制(图 7).回归分析表明, 土壤质量的提高有利于缓解微生物碳和氮限制[图 4(a)4(b)], 这就是DCD和NBPT对缓解微生物碳和氮限制有更高的正向效应的原因.随机森林模型表明, SWC也是影响微生物碳和氮限制的主要因素之一, 其原因是SWC变化会通过改变土壤养分(AN、AP、MBC和MBN)含量和有效性(图 7), 导致微生物代谢过程改变(即N-acq和P-acq;图 7), 从而改变土壤微生物碳和氮限制程度[26, 67].

总之, 施用EENFs可以缓解作物与土壤微生物对土壤N的竞争, 并最终缓解微生物碳和氮限制程度.

3.3 增效氮肥对土壤生态功能多样性的影响

本研究发现, EENFs相比N200和N0可以更好地提高土壤EMF.同时, 提高土壤质量和缓解微生物碳和氮限制更有利于提高土壤生态功能多样性.此外, SOC、TN、AN、AP、MBC和MBN是影响土壤EMF的主要原因[图 6(a)], 即土壤C、N和P有效性会改变多种微生物, 从而影响生物地球化学循环过程, 最终改变生态功能(土壤酶活性和作物生物量), 这与其它研究结果类似[68, 69].在本研究中, 提高土壤养分含量(例如TN、AN、MBC和MBN)或土壤质量有利于提高土壤生态功能多样性[图 76(b)], 这是因为较高的养分资源利用率和土壤质量有利于改善微生物代谢状况[68, 70], 并提高微生物多样性和改善群落组成[71], 从而驱动土壤多个功能[69, 72].类似的结果也被报道, 高土壤多功能性通常伴随着高土壤养分状况和较好的微生物代谢特征[62, 73].与之相反, 较差的土壤质量和较少的养分资源(N0)会导致微生物生态位分化, 微生物生存空间减少, 其资源分配压力增大(例如微生物受到较强的碳和氮限制)[74], 这可能对土壤EMF产生消极影响.此外, 本研究发现, 缓解微生物碳和氮限制有利于提高土壤生态功能多样性[图 6(c)6(d)], 这与Yang等[62]发现微生物碳限制与土壤生态功能多样性无显著关系的结果不一致.原因可能是白膜覆盖下的ST普遍高于黑膜, 而更高的ST有利于提高土壤放线菌等物种的丰度[75], 由于放线菌具有较高的土壤有机质分解能力, 最终增强了土壤C循环过程(更高的C-acq酶活性), 这可能预示着在特定环境条件下执行相关功能的微生物对单一土壤功能的重要性.

综上所述, 白膜覆盖下施用EENFs可以通过提高土壤质量和缓解微生物碳和氮限制来提高土壤功能多样性.同时, 在未来研究中, 有必要进一步探究生物群落在维持不同单一土壤功能的作用和贡献, 以及分析特定环境下的关键物种与土壤质量、微生物代谢和土壤生态功能多样性的关系.

4 结论

(1) 相比不施氮肥(N0), 施用氮肥(N200和EENFs)增加了TN、MBC和MBN含量, 但对SOC、AP和SWC含量有消极影响.NBPT和DCD主要通过短时间内提高MBC、MBN和AN含量进而改善土壤质量, 而N200和RCN虽可改变不同土壤养分含量, 但不能全面提高土壤质量.

(2) 相比N0和N200, EENFs可更好地缓解微生物碳和氮限制并提高土壤生态功能多样性.土壤中的MBC、MBN、AN、AP和SWC含量是影响微生物碳限制、微生物氮限制和生态功能多样性的共同主要因素.同时, 土壤质量的提高、微生物碳和氮限制的缓解有利于提高土壤生态功能多样性.

(3) 综合来看, 白膜覆盖下施用NBPT和DCD可短期内实现土壤健康和生态功能多样性的双赢.因此, 该结果可为当地优化施肥管理措施提供参考.

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