Advances in Research on Phyllosphere Microorganisms and Their Interaction with Plants
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中国耕地重金属污染形势严峻，根据2014年发布的《全国土壤污染状况调查公报》，中国耕地土壤点位超标率达19.4%；从污染物超标情况来看，镉 (Cd) 的点位超标率为7.0%，铜 (Cu) 的点位超标率为2.1%，在所有污染因子中分别位居第一和第四[1]。Cd和Cu等重金属在农田土壤中不断积累，会影响农作物生长，导致农产品中重金属含量超标，严重影响食品安全和消费者健康[2-3]。因此，土壤重金属污染修复技术及其应用一直是国内外研究的热点[4-5]。
土壤重金属污染修复主要有物理修复、化学修复、生物修复和农业生态修复等方法[6]。对于中轻度受污染农田，应尽量在不影响农业生产的前提下，选择环境友好型方式修复重金属污染，同时综合考虑修复效果和修复成本，以便应用于实际生产并能大面积推广。原位化学钝化法具有经济、高效和简单易行等优点，在农田重金属污染修复中运用广泛[7]，常用的钝化剂包括石灰性物质、炭材料、粘土矿物、有机肥和农业废弃物等。碳酸钙是一种来源广泛、成本低廉和性质稳定的石灰类钝化剂，它能与重金属发生反应生成沉淀，并通过调节土壤pH使土壤中的重金属生成氢氧化物沉淀，具有较好的钝化效果[8-9]。有机物料是国内外常用于重金属污染钝化修复的材料[10-11]，研究表明：施用有机肥不仅能够降低土壤Cd活性，减少农作物对重金属的吸收积累，还能改善土壤理化性质，增加土壤肥力，是一种较为经济有效的农田土壤重金属污染修复方法[12]。
赵家印等[13]通过盆栽试验研究发现：碳酸钙与有机肥配施对降低云南重金属污染农田土壤中有效态Cd和Cu及生菜中Cd和Cu的含量效果显著。为进一步探究在实际生产条件下降低该地区土壤及生菜中Cd和Cu含量的方法，本研究以云南重金属污染农田土壤为研究对象，通过大田试验研究不同肥料及其与碳酸钙石粉配施对土壤pH和有效态Cd、Cu含量动态变化以及生菜产量和生菜可食用部位中重金属含量的影响，以寻求高效低成本并符合当地农民生产习惯的重金属污染农田安全利用技术，同时为降低类似地区农田重金属污染风险、改善土壤质量和提高蔬菜安全品质提供科学依据。
1. 材料与方法
1.1 试验地概况及供试材料
试验于2019年10—12月在云南省昆明市盘龙区某蔬菜种植基地开展 (N25.05°，E102.7°，海拔1 975 m) 。试验区属中亚热带高原季风气候区，全年平均气温14.9 ℃，年平均降水量1000.5 mm，月最大降水量208.3 mm。
供试土壤为红壤，参考文献[14]的方法测定大田基础土样基本理化性质为：pH 5.86，全氮含量2.3 g/kg，有机质含量48.32 g/kg，速效磷含量41.96 mg/kg，阳离子交换量18.55 cmol/kg；全Cd含量0.70 mg/kg，有效态Cd含量0.26 mg/kg，全Cu含量193.27 mg/kg，有效态Cu含量11.10 mg/kg。该地区土壤重金属Cu和Cd含量超过《农用地土壤污染风险管控标准 (试行) 》[15]的土壤风险筛选值 (5.5＜pH≤6.5时，全Cu≤50 mg/kg、全Cd≤0.3 mg/kg)
试验蔬菜品种为结球生菜 (Lactuca sativa var. capitata L.) ，苗龄20 d后移栽至大田。试验所用有机肥为农场利用羊粪和鸽粪等原料充分发酵腐熟所生产，其基本理化性质为：pH 7.67，总氮含量24.70 g/kg，总磷含量17.45 g/kg，总钾含量32.60 g/kg，有机质含量505.00 g/kg，全Cd含量1.05 mg/kg，全Cu含量59.00 mg/kg。有机肥中Cd含量符合NY/T 525—2021[16]的规定 (总Cd≤3 mg/kg) 。供试碳酸钙石粉由石灰石 (购自云南昆钢钙镁熔剂有限公司) 磨制过250目筛后所得，其Cd和Cu含量均低于检出限，pH值为10.18。
1.2 试验设计
试验共设置5个处理 (表1) ，每个处理3次重复；共设置随机排列试验小区15个，每个小区规格为4 m×5 m。为防止串水，每个小区之间设置宽50 cm、高25 cm的田埂；地块四周设保护行，保护行各边宽度不少于1 m。有机肥和化肥作为基肥撒施，不进行追肥，施用量根据农民施肥经验及生菜需氮量确定。结球生菜移栽前1 d 进行土壤翻耕，同时根据各试验小区的施肥方案撒施基肥，碳酸钙石粉与基肥同时撒施。2019年10月15日移栽结球生菜，行距30 cm，株距20 cm。在其生长过程中，各小区采取相同的浇水、除草和病虫害防治等田间管理方式。
表 1 田间试验处理设计Table 1. Design for treatments of the field experiment处理
treatment施肥模式及施用量
fertilization pattern and amountCK 不施肥 no fertilization OM 有机肥 22.5 t/hm2 organic fertilizer 22.5 t/hm2 OMC 有机肥 22.5 t/hm2+碳酸钙石粉1.5 t/hm2
organic fertilizer 22.5 t/hm2+calcium carbonate powder 1.5 t/hm2CF 化肥 (尿素1.2 t/hm2+过磷酸钙 2.95 t/hm2+硫酸钾0.38 t/hm2)
chemical fertilizer (urea 1.2 t/hm2+calcium superphosphate 2.95 t/hm2+potassium sulfate 0.38 t/hm2)CFC 化肥+碳酸钙石粉1.5 t/hm2
chemical fertilizer+calcium carbonate powder 1.5 t/hm21.3 样品采集与处理
考虑到土壤的异质性，各处理在翻耕与施肥前都采集土样，测定土壤有效态Cd和Cu含量及pH值；在试验开始后第15、30、45、60、75天时采集各小区0~20 cm表层土壤样品，经风干和粉碎后过100目筛用于土壤参数分析。约60 d 结球生菜成熟后，在各小区随机采集10颗测其鲜质量，计算各小区生菜的平均鲜质量，估算产量；随后用去离子水清洗干净，烘干和粉碎后过100目筛，待测。
土壤Cd含量的测定采用石墨炉原子吸收分光光度法[17]，土壤Cu含量的测定采用火焰原子吸收分光光度法[18]；土壤重金属有效态Cd和Cu含量的测定采用二乙烯三胺五乙酸浸提—电感耦合等离子体发射光谱法[19]；生菜总Cd和总Cu含量的测定采用原子吸收光谱法[20-21]。
1.4 数据处理
采用Microsoft Excel 2010和Origin 2019进行数据处理和制图；采用SPSS 22.0软件进行显著性检验，检验方法采用Duncan法，显著水平为P<0.05。
2. 结果与分析
2.1 不同施肥模式下土壤pH值的动态变化
由图1可知：单施有机肥 (OM) 和有机肥与碳酸钙石粉配施 (OMC) 处理的土壤pH值总体呈增加趋势，在试验第15天时出现第1次较为明显的增加，pH值提高0.49~0.91；OM处理在第75天时pH值达最大值，比处理前增加1.01，OMC处理在第60天时pH值达最大值，比处理前增加1.04。单施化肥 (CF) 处理的土壤pH值总体呈先增加后降低的趋势，在第15~30天降幅明显，pH值降低1.21；化肥与碳酸钙石粉配施 (CFC) 处理的土壤pH值呈总体先降低后增加的趋势，在第0~15天pH值短暂下降，随后波动升高。
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图 1 不同施肥模式下土壤pH值动态变化趋势注：CK. 不施肥；OM. 单施有机肥；OMC. 有机肥+碳酸钙石粉；CF. 单施化肥；CFC. 化肥+碳酸钙石粉；下同。Figure 1. Dynamic change trend of soil pH under different fertilization patternsNote: CK. no fertilization; OM. organic fertilizer; OMC. organic fertilizer+calcium carbonate powder; CF. chemical fertilizer; CFC. chemical fertilizer+calcium carbonate powder; the same as below.2.2 不同施肥模式下土壤中有效态重金属含量的动态变化
由图2可知：各处理土壤有效态Cd含量随处理时间的延长呈先降低后升高的趋势。在45 d时，OM和OMC处理的土壤有效态Cd含量降至最低，与处理前相比降幅分别为38.41%和43.12%，同期CK处理的降幅为21.99%；CF处理的土壤有效态Cd含量也在45 d时降至最低，降幅为12.98%；CFC处理的土壤有效态Cd含量在30 d时最低，降幅为24.46%，同期CK处理的降幅为14.45%。OM和OMC处理的土壤有效态Cd含量在0~15和30~45 d 时降幅较大，其中OM处理的降幅分别为16.05%和21.10%，OMC处理的降幅分别为23.67%和23.46%；CF和CFC处理的土壤有效态Cd含量降低主要发生在15~30 d，分别降低7.54%和21.43%。到75 d时，与处理前相比，OM和OMC处理的土壤有效态Cd含量分别降低12.50%和16.67%，CFC处理的土壤有效态Cd含量升高13.04%，CF处理的土壤有效态Cd含量基本不变。可见，有机肥以及化肥配施碳酸钙石粉均能在短时间内促进土壤有效态Cd含量降低。
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图 2 不同施肥模式下土壤中有效态Cd和Cu含量的动态变化Figure 2. Dynamic changes of available Cd and Cu content in soil under different fertilization patterns由图2还可知：土壤中有效态Cu含量变化趋势与有效态Cd基本一致，均呈先降低后升高的趋势。OM和OMC处理的土壤有效态Cu含量在45 d时降至最低，较处理前分别降低54.79%和53.79%，同期CK处理的降幅为40.03%。CF和CFC处理的土壤有效态Cu含量在30 d时降至最低，降幅分别达28.51%和38.46%，同期CK处理的降幅为31.67%。OM和OMC处理在0~15和30~45 d降幅较大，OM处理降幅分别为32.17%和31.69%，OMC处理降幅分别为27.86%和34.10%；CF和CFC处理则在15~30 d时降幅最大，降幅分别为22.39%和31.90%。到75 d时，与处理前相比，OM和OMC处理的土壤有效态Cu含量分别降低36.61%和35.43%，而CF和CFC处理土壤有效态Cu含量分别升高17.66%和28.30%。可见，长期施用化肥不利于土壤有效态Cu的降低，化肥配施碳酸钙石粉可以在短期内促进土壤有效态Cu含量降低。
2.3 不同施肥模式对结球生菜产量及其重金属含量的影响
由表2可知：OM处理的结球生菜产量最高，其次是OMC处理，两者分别比对照增产184.33%和153.31%；CFC处理的结球生菜产量显著高于CF处理，CF与CK处理的结球生菜产量无显著差异。各处理的结球生菜可食用部位Cd含量顺序为CFC>CF>CK>OMC>OM，Cd含量均未超过食品安全国家标准限值 (0.2 mg/kg)[22]。CFC处理结球生菜中Cd含量显著高于CK处理，与CF处理差异不显著；OM处理生菜Cd含量显著低于CFC、CF和CK处理。与CK相比，OM和OMC处理生菜Cd含量分别降低60.17%和36.40%。各处理的结球生菜可食用部位Cu含量顺序为CF>CFC>CK>OMC>OM，且OM处理的Cu含量显著低于其他处理，仅为CF处理的49.89%。与CK相比，OM和OMC处理的生菜Cu含量分别降低47.27%和19.35%。
表 2 各处理下结球生菜的产量及其Cd和Cu含量Table 2. Yield and Cd and Cu content of head lettuce under different treatments处理
treatment产量/(kg·667 m−2)
yieldCd含量/(mg·kg−1)
Cd contentCu含量/(mg·kg−1)
Cu contentCK 930.35±108.96 c 0.08±0.01 bc 0.86±0.08 a OM 2645.25±291.11 a 0.03±0.01 d 0.46±0.04 b OMC 2356.65±140.81 ab 0.05±0.02 cd 0.70±0.07 a CF 1273.48±129.54 c 0.09±0.01 ab 0.91±0.20 a CFC 2141.23±241.81 b 0.11±0.00 a 0.89±0.00 a 注：各处理施肥方式见表1；同列数据后不同小写字母表示在0.05水平差异显著。
Note: Fertilization patterns of each treatment list in Tab.1; different lowercase letters indicate significant differences at 0.05 level.3. 讨论
本研究OM和OMC处理土壤pH值总体呈增加趋势，可能是有机物料加入土壤后产生的铵离子导致pH值上升，而后期pH值短暂下降可能与铵的硝化作用、有机肥腐解及植物根系分泌的有机酸增多等因素有关[23-24]。CF处理土壤pH值总体呈先增加后降低的趋势，可能是尿素的水解作用使土壤pH值在短期内上升，之后的硝化作用又使pH值降低。曾清如等[25]研究发现：酸性红壤中施用尿素后，硝化作用主要集中在第2~4周；刘小燕等[26]研究发现：在砷污染土壤施入尿素，pH值先短暂上升至最大值然后下降，其中3~4周降幅最大，与本研究结果类似，从长期来看，施用尿素等化肥会导致土壤酸化。CFC处理因为碳酸钙的施入促进了土壤硝化作用[27]，再加上尿素的硝化作用使土壤pH值先短暂下降，随后由于碳酸钙溶解产生OH−使土壤pH值升高[28]。李明等[29]在重度酸性镉污染菜地 (Cd含量1.06 mg/kg) 施用4.5 t/hm2 CaCO3，9个月后仍保持较好的调酸效果；而本研究OMC和CFC处理在试验第75天还维持着降酸效果，其中第60天时降酸效果最佳，pH值分别较处理前增加1.04和0.67。
土壤有效态Cd和Cu含量降低主要发生在试验开始的45 d内，且OM和OMC处理的土壤有效态Cd和Cu含量降幅明显大于CK，表明有机肥单施和有机肥与碳酸钙石粉配施均有利于降低土壤有效态Cd和Cu含量，且有机肥与碳酸钙石粉配施对土壤有效态Cd的降低效果优于单施有机肥。施用有机肥能够提高土壤pH，增加有机质含量，有机质可通过与土壤重金属发生络合作用从而影响重金属的移动性及其植物有效性[30-31]，但不同有机肥对土壤重金属具有活化和固定双重影响。赵家印等[13]研究发现：有机肥的活化作用可明显提高土壤有效态Cd和Cu含量；JONES等[32]研究发现：生物炭和堆肥配施可显著降低Cu的移动能力；叶俊文[33]指出：随着土壤有机质增加，土壤Cu和Cd可交换态含量显著降低，施用有机肥可有效治理Cu和Cd污染土壤。导致上述研究产生差异的原因可能是有机肥中重金属含量[34]、有机质含量、有机质中胡敏酸和富里酸组成差异[35]以及土壤的理化性质和种植条件 (盆栽、大田和温室等) 不同。碳酸钙对土壤重金属Cd和Cu的钝化机理相似，一方面通过调节土壤pH使其生成氢氧化物沉淀，另一方面可与Cd和Cu发生反应生成碳酸盐沉淀，且一定范围内钝化效果随着碳酸钙添加剂量的增大而增强[36-38]。CF处理的土壤有效态Cd和Cu含量降幅均小于CK，CFC处理的降幅小于或略高于CK，表明施用化肥不利于降低土壤有效态Cd和Cu含量，配施碳酸钙的效果主要体现在试验初期，能在短期内促进土壤有效态Cd和Cu含量降低。
施用有机肥和碳酸钙石粉可显著增加结球生菜产量，而单施化肥对其增产效果不显著。施用化肥会导致土壤pH降低，进而引起土壤Al溶解导致铝毒伤害，抑制蔬菜生长[25]；而有机肥可提高土壤pH，增加有机质含量，优化土壤结构，提高作物产量，唐明灯等[39]研究证实：施用猪粪、牛粪、鸡粪和花生麸4种有机肥均可显著提高生菜生物量。有研究发现：对酸性土壤增施2.25~4.50 t/hm2 CaCO3可增加土壤微生物量及其活性，进而促进土壤溶解性有机碳增加及土壤氮素转化[26,40]，有利于作物生长[29]。施用化肥使pH值降低，土壤重金属生物有效性提高，蔬菜对重金属的吸收增加[41]；施用有机肥可提高土壤有机质含量，使土壤中易被植物吸收的重金属通过有机络合反应变为有机结合态；施用碳酸钙石粉能降低土壤有效态Cd和Cu含量，进而降低蔬菜重金属含量[42]。因此，本研究OM和OMC处理均有利于降低结球生菜可食用部位的重金属Cu和Cd含量，且前者效果略优。李明等[29]在Cd污染菜地施用CaCO3后，各类蔬菜Cd含量均降低，其中叶菜类蔬菜Cd含量降幅最大。唐希望等[43]研究发现：整个生长阶段生菜地上部Cd含量和Ca含量呈正相关，同时与生菜地上部生物量的变化高度相关，这可能是本研究CFC处理生菜Cd含量大于CF处理以及OM处理生菜Cd含量小于OMC处理的原因。
4. 结论
施用有机肥和碳酸钙石粉有利于提高土壤pH值，增加结球生菜产量，降低结球生菜可食用部位Cd和Cu的含量；施用化肥对结球生菜的增产效果不明显且不利于降低土壤及结球生菜中的Cd和Cu含量。可见，单施有机肥以及有机肥与碳酸钙石粉配施是降低酸性农田土壤重金属Cd和Cu污染风险、提高蔬菜产量及其安全品质的良好措施。
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