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磷是作物生长必需的大量营养元素。然而，在农业生态系统中磷的限制是一个全球性的问题[1]。为维持作物产量，在农业生产中常常大量施用磷肥。但是其大部分的磷素会被土壤吸附固定，无法被作物吸收利用，当季磷肥利用率只有10%～25%[2]，过低的磷肥利用率会导致土壤磷素的累积[3-4]。而当土壤中累积的磷素超过一定值时，会随雨水或灌溉向水体移动，增加了水体富营养化风险[5]，同时也造成不可再生磷矿资源的浪费[6]。可见，合理减施磷肥对维持农田生态系统可持续发展和避免有限磷矿资源的浪费具有重要意义。
土壤酶在土壤养分循环中具有重要作用，是评价土壤质量的重要指标之一，其中磷酸酶在土壤磷循环过程起着重要作用[7]。土壤有机磷占土壤总磷库的30%～80%[8]，磷酸酶可以将有机磷矿化为有效磷。磷脂脂肪酸是所有生物细胞膜的重要组成部分，不仅作为微生物丰度和组成的生物指标，而且也是评价土壤质量的重要指标之一[9]。Samaddar等[10]在水稻土上研究表明，磷肥减施会显著改变土壤微生物群落结构，而Shi等[11]在潮土上研究表明，磷肥减施并不影响土壤微生物群落结构。可见，磷肥减施对土壤微生物群落结构的影响存在不确定性。溶磷微生物广泛分布于土壤，是一类能够溶解土壤难溶磷、改善磷素固定的微生物类群，是廉价的、环保的土壤磷活化生物措施[12-13]。施用解磷菌剂可活化土壤难溶性磷，增加土壤有效磷含量，提高作物对磷肥的利用[14]。目前种肥同播技术在国内外也得到了广泛的应用，可有效解决农户对磷肥施用量把握不准的问题，提高磷肥利用率[15]。
磷肥用量与作物产量关系表现为作物产量随磷肥用量的增加而增加，但当超过一定量时，产量不再增加反而甚至减产[16-17]。马琴等[18]研究发现，在陕西黑垆土磷肥减施3年玉米产量均无显著性差异，而土壤有效磷含量显著降低。宋书会[19]的研究表明，在黑龙江黑土上磷肥减施5年，玉米产量均无显著性差异，土壤有效磷含量从第三年开始显著降低。小麦是我国三大粮食作物之一，长江流域是其主要生产区之一，其安全生产对保障我国粮食安全具有重要地位。王旭等[20]研究结果表明，与20世纪80年代初相比，长江中下游小麦磷肥农学效率下降了6.2%。陈善荣等[21]在2016—2019年分析了长江流域的水质变化表明，总磷对长江流域水污染的贡献率最大，且农业面源污染是河流中总磷主要来源之一。目前关于种肥同播下磷肥减施对长江中下游地区土壤酶活性及微生物群落结构的影响研究相对较少。因此，本研究于2018年在湖北和浙江布置磷肥减施的大田试验，研究种肥同播下磷肥减施对小麦产量、土壤化学性质、酶活性以及微生物群落结构的影响，为磷肥减施增效、保护农田生态环境提供科学依据。
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试验开始于2018年小麦季，试验地位于在湖北广水市 (113°53′E，31°32′N) 和浙江平湖市 (121°14′E，30°38′N)。湖北广水市地处亚热带湿润季风气候区，年均气温 15.5℃，年降雨量990 mm，无霜期约220 天，年日照时数2083 h，供试土壤为黄壤，土壤质地为粘土，种植模式为“小麦－水稻”轮作，供试小麦品种为‘郑麦1342’、水稻品种为‘晶两优华占’。浙江平湖市地处亚热带湿润季风气候区，年均气温 16.0℃，年降雨量1170 mm，无霜期约225天，年日照时数2000 h，供试土壤为黄斑田，土壤质地为粘壤土，种植模式为“小麦－玉米”轮作，供试小麦品种为‘扬麦20’、玉米品种为‘京科糯928’。试验开始前湖北和浙江试验基地0—20 cm土层基础理化性质如表1所示。
表 1 试验基地0—20 cm土层基础理化性质
Table 1. Basic physical and chemical properties of 0–20 cm soil layer
地点
SitepH
(1∶2.5)容重
Bulk density
(g/cm3)有机碳
Organic C
(g/kg)全氮
Total N
(g/kg)碱解氮
Available N
(mg/kg)全磷
Total P
(g/kg)有效磷
Available P
(mg/kg)速效钾
Available K
(mg/kg)湖北广水市
Guangshui city, Hubei6.30 1.32 12.56 1.16 91.12 0.32 7.34 162.32 浙江平湖市
Pinghu city, Zhejiang7.80 1.42 14.40 1.34 199.73 0.68 11.55 200.68 -
本试验采用随机区组设计，3个重复，湖北小区面积为20 m2，浙江小区面积为40 m2，设置5个处理：不施磷肥对照 (CK)、习惯施磷量 (FP)、80%习惯施磷量 (P80)、60%习惯施磷量 (P60)、60%习惯施磷量+解磷菌剂 (PB60)。氮磷钾肥品种分别为尿素、过磷酸钙和氯化钾，解磷菌剂为江苏纳克生物工程公司生产，使用方法为将菌剂与清洁凉水1∶0.5混合，然后与种子拌匀，阴干后播种。小麦季种肥同播技术：按肥料用量将各种肥料称好后均匀混合，采用沟施播种方式，一次性将所有肥料与麦种间隔埋入沟中，开沟深10 cm左右，沟宽7～10 cm，沟左侧放入肥料，覆盖约一半土，沟右侧放种子，沟与沟的水平距离对应当地株距，氮磷钾肥全部作为基肥一次性施用，不用再追肥；水稻或玉米季施肥方式与当地一致。
湖北小麦和水稻季，习惯施磷量均为P2O5 150 kg/hm2，各处理氮、钾肥施用量一样，均为N 150 kg/hm2、K2O 90 kg/hm2。小麦季氮磷钾肥作为基肥一次性施入；水稻季，磷钾肥一次性施入，氮肥80%用做基肥，20%用做穗肥。
在浙江小麦季，习惯施磷量为P2O5 57 kg/hm2，各处理氮、钾肥施用量一样，均为N 190 kg/hm2，K2O 57 kg/hm2；玉米季习惯施磷量为P2O5 77 kg/hm2，各处理氮、钾肥施用量为N 159 kg/hm2、K2O 77 kg/hm2。所有氮磷钾肥均作为基肥一次性施入。
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2019、2020年小麦成熟后对各小区进行测产，并于2020年小麦收获时采集0—20 cm耕层土壤样品。土壤样品的基本理化性质参考《土壤农化分析》进行测定[22]。土壤微生物量磷采用氯仿熏蒸－NaHCO3浸提方法[23]。土壤酶包括参与氮循环的乙酰氨基葡萄糖苷酶和亮氨酸氨基肽酶，参与磷循环的磷酸酶[24]，采用荧光微型板酶检测技术进行测定[25]。磷脂脂肪酸 (Phospholipid fatty acid，PLFA) 采用Wu等[26]方法进行测定，所得样品通过气相色谱仪 (Agilent 6890N，FID) 和MIDI微生物自动鉴定系统进行微生物PLFA组成鉴定 (v4.5，MIDI Inc.，DE，USA)。以加入的内标 (19∶0) 为参考，计算PLFAs浓度 (nmol/g，dry soil)。每个样品中PLFAs的丰度以mol%表示，通过之前已发表的PLFA生物标志物数据来分类[27-28]。细菌包括a13:0，i13:0，i14:0，i15:0，a15:0，i16:0，a17:0，i17:0，i18:0，14:1ω5c，16:1ω7c，17:1ω8c，18:1ω5c，18:1ω7c，，20:1ω9c，22:1ω9c，cy19:0，20:1ω9c，14:0，16:0，17:0和18:0；真菌包括18:1ω9c和18:2ω6c；丛枝菌根包括16:1ω5c。
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土壤磷活化系数 (phosphorus activation coefficient，PAC)(%) = Olsen-P (mg/kg) / [全磷 (g/kg) × 1000] × 100
利用Excel 2019对数据进行整理计算，Origin 2020b 进行基于Bray-Curtis距离非度量多维尺度分析 (Non-metric multidimensional scaling，NMDS) 及绘图。采用SPSS 24.0进行方差分析，LSD最小显著差数法进行多重比较。Canoco 5.0进行冗余分析。
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从图1可知，与FP处理相比，2019年湖北和浙江麦区CK处理小麦产量显著降低，P80、P60和PB60处理无显著差异；2020年湖北麦区CK、P80、P60和PB60处理的小麦产量分别显著降低了63.95%、31.53%、21.51%和26.44%，浙江麦区P80和PB60处理的小麦产量无显著差异，CK和P60处理显著降低了9.76%和10.29%。同时发现，湖北麦区2019和2020年，PB60处理的小麦产量比P60显著降低了13.80%和6.29%；浙江麦区2019年处理与PB60处理的小麦产量与P60无显著差异，2020年比P60显著增产9.80%。
图 1 磷肥减施对小麦产量的影响
Figure 1. Wheat yield under different phosphorous application rates
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由表2可知，与FP处理相比，磷肥减施对湖北和浙江麦区土壤pH、SOC、全氮以及速效钾含量均无显著性影响，但显著增加土壤NH4+-N和NO3--N含量。湖北麦区P80和P60处理NH4+-N含量分别提高61.18%和55.29%，NO3--N含量提高18.11%和7.64%；浙江麦区P80和P60处理NH4+-N含量分别提高25.19%和22.22%，NO3--N含量提高3.35%和3.88%。同时研究发现，在湖北和浙江麦区，与P60处理相比，PB60处理NH4+-N含量分别提高14.39%和5.45%；PB60处理NO3--N含量分别提高24.54%和1.31%。
表 2 小麦收获后不同磷肥减施处理土壤理化性质
Table 2. Physic-chemical properties of soils as affected by different phosphorous rates at wheat maturity
处理
TreatmentpH
(1∶2.5)有机碳
SOC
(g/kg)总氮
Total N
(g/kg)铵态氮
NH4+-N
(mg/kg)硝态氮
NO3--N
(mg/kg)总磷
Total P
(g/kg)有效磷
Available P
(mg/kg)速效钾
Available K
(mg/kg)微生物量磷
MBP
(mg/kg)湖北 Hubei CK 6.60 ± 0.12 a 11.38 ± 0.36 a 1.12 ± 0.04 a 0.85 ± 0.07 b 6.12 ± 0.25 c 0.30 ± 0.01 c 3.59 ± 0.40 d 151.55 ± 1.09 a 6.87 ± 0.51 d FP 6.11 ± 0.09 a 11.40 ± 0.82 a 1.12 ± 0.08 a 0.85 ± 0.05 b 6.02 ± 0.44 c 0.36 ± 0.01 a 12.05 ± 1.73 a 148.29 ± 5.32 a 11.56 ± 2.45 c P80 6.65 ± 0.45 a 12.10 ± 0.30 a 1.13 ± 0.09 a 1.37 ± 0.05 a 7.11 ± 0.48 b 0.35 ± 0.02 ab 9.64 ± 0.82 b 164.03 ± 9.70 a 15.89 ± 1.61 ab P60 6.48 ± 0.41 a 11.49 ± 0.03 a 1.11 ± 0.03 a 1.32 ± 0.26 a 6.48 ± 0.28 bc 0.32 ± 0.01 bc 7.37 ± 0.63 c 144.99 ± 12.69 a 13.55 ± 1.38 bc PB60 6.17 ± 0.11 a 11.82 ± 0.90 a 1.08 ± 0.03 a 1.51 ± 0.15 a 8.07 ± 0.31 a 0.33 ± 0.02 bc 9.59 ± 0.55 b 154.45 ± 14.74 a 16.71 ± 1.40 a 浙江 Zhejiang CK 7.63 ± 0.13 a 14.61 ± 0.46 a 1.34 ± 0.10 a 1.29 ± 0.02 b 9.43 ± 0.49 a 0.67 ± 0.02 a 10.32 ± 0.43 d 224.35 ± 9.53 a 7.72 ± 2.77 b FP 7.71 ± 0.18 a 13.76 ± 0.41 a 1.30 ± 0.04 a 1.35 ± 0.10 b 9.54 ± 0.33 a 0.70 ± 0.03 a 16.84 ± 0.67 ab 225.33 ± 10.65 a 17.49 ± 1.97 a P80 7.71 ± 0.25 a 13.60 ± 1.23 a 1.29 ± 0.10 a 1.69 ± 0.06 a 9.86 ± 0.22 a 0.68 ± 0.04 a 14.47 ± 1.59 bc 212.99 ± 7.11 a 15.55 ± 3.08 a P60 7.75 ± 0.20 a 14.44 ± 0.89 a 1.31 ± 0.02 a 1.65 ± 0.04 a 9.91 ± 0.79 a 0.69 ± 0.03 a 13.74 ± 0.59 c 252.8 ± 14.88 a 15.46 ± 3.60 a PB60 7.61 ± 0.15 a 15.05 ± 0.85 a 1.4 ± 0.07 a 1.74 ± 0.05 a 10.04 ± 0.58 a 0.70 ± 0.02 a 17.53 ± 2.67 a 233.65 ± 9.09 a 15.17 ± 3.11 a 注（Note）：施肥方法为种肥同播 All fertilizers were applied during sowing; 所有处理 N、K 使用量相同，CK 为不施磷，FP 为习惯施磷量，P80、P60 表示施磷量为 FP 处理的 80% 和 60%, PB60 表示 P60 + 解磷菌剂 All treatments applied the same amount of N and K fertilizer, CK treatment represents no P application, FP treatment represents farmers’ conventional phosphorus, P80 and P60 treatments represent P application rate of 60% and 80% of that in FP, PB60 treatments represents P60+phosphate-solubilizing bacteria; SOC—有机碳 Soil organic carbon; MBP—微生物量磷 Microbial biomass P; 同列数据后不同小写字母表示同一试验点不同处理间差异显著 (P < 0.05) Values followed by different small letters mean significant differences among treatments at same experimental site (P < 0.05). 在湖北麦区种肥同播下，与FP处理相比，CK、P80和P60处理土壤全磷含量降低，其中CK和P60处理土壤全磷含量分别显著降低16.67%和11.11%；与FP处理相比，CK、P80和P60处理土壤有效磷含量分别显著降低70.21%、20.00%和38.84%；与FP处理相比，CK处理土壤MBP含量显著降低40.57%，P80和P60处理土壤MBP含量分别增加37.46%和17.21%。在浙江麦区，种肥同播下，与FP处理相比，磷肥减施对土壤全磷含量无显著性影响；与FP处理相比，P80处理土壤有效磷含量无显著性差异，CK和P60处理土壤有效磷含量分别显著降低38.72%和18.41%；与FP处理相比，种肥同播下P80和P60处理土壤MBP含量无显著性差异，CK处理土壤MBP含量显著降低55.86%。同时，可以发现，在湖北和浙江麦区，与P60处理相比，PB60处理的土壤有效磷含量分别显著增加30.12%和27.58%，特别是浙江麦区PB60处理土壤有效磷含量与FP处理相当。而PB60处理的MBP含量较P60处理在湖北麦区显著增加了23.32%，在浙江麦区无显著性差异。
从图2可知，在湖北和浙江麦区种肥同播下，与FP处理相比，CK、P60和P80处理均显著降低土壤磷活化系数 (PAC)。与P60处理相比，PB60处理的PAC在湖北和浙江麦区分别显著增加16.37%和22.98%。湖北麦区施磷处理的土壤磷活化系数高于浙江麦区。
图 2 不同磷肥施用量下土壤磷活化系数
Figure 2. Phosphorus activation coefficient of soil under different phosphorus rates
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图3显示，与FP处理相比，P80处理提高了乙酰氨基葡萄糖苷酶、亮氨酸氨基肽酶以及磷酸酶活性，在湖北麦区分别提高了13.22%、4.93%和1.37%，在浙江麦区分别提高了14.43%、175.42%和5.58%；P60处理提高了亮氨酸氨基肽酶活性，湖北和浙江麦区分别提高了15.78%和157.38%，降低了乙酰氨基葡萄糖苷酶活性，湖北和浙江麦区分别降低了17.16%和16.09%。与P60处理相比，湖北麦区PB60处理的土壤磷酸酶活性无显著性差异，浙江麦区显著提高了28.73%。
图 3 小麦收获期不同施磷处理土壤酶活性
Figure 3. Soil enzyme activity as affected by different phosphorus rates at wheat harvest
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表3可知，与FP处理相比，湖北麦区CK、P80和P60处理的土壤总磷脂脂肪酸含量分别显著降低了42.64%、21.23%和26.04%，浙江麦区CK处理无显著差异，P80和P60处理分别显著提高了126.82%和290.23%。与P60相比，湖北麦区PB60处理的总磷脂脂肪酸含量显著增加，而浙江麦区显著降低。与FP处理相比，湖北和浙江麦区CK、P80和P60处理的细菌相对丰度均无显著性差异，真菌相对丰度显著增加。与FP处理相比，湖北麦区PB60处理的丛枝菌根的相对丰度无显著差异，而浙江麦区显著提高了40.34%。与FP处理相比，CK、P80和P60处理均显著降低了真菌与细菌的比值，湖北麦区分别显著降低了35.82%、26.44%和21.93%，浙江麦区分别显著降低了8.67%、7.86%和19.72%。
表 3 不同施磷处理土壤中总磷脂脂肪酸含量及微生物相对丰度
Table 3. Total phospholipid fatty acid content and the relative abundance of microorganisms affected by differentphosphorous treatments
地点
Site处理
Treatment总磷脂脂肪酸
Total PLFAs
(nmol/g, dry soil)相对丰度 (mol%)
The relative abundance PLFAs细菌/真菌
Bacteria/Fungi细菌
Bacteria真菌
Fungi丛枝菌根
Arbuscular mycorrhizal fungi湖北 Hubei CK 14.10 ± 2.59 c 59.87 ± 2.00 a 2.98 ± 0.08 a 1.91 ± 0.30 a 20.10 ± 1.16 d FP 24.58 ± 0.09 b 59.75 ± 1.10 a 1.91 ± 0.04 d 1.95 ± 0.39 a 31.32 ± 0.69 a P80 19.36 ± 1.99 c 58.42 ± 0.78 a 2.54 ± 0.07 b 1.62 ± 0.35 a 23.04 ± 0.82 c P60 18.18 ± 4.13 c 57.88 ± 0.48 ab 2.37 ± 0.08 c 1.40 ± 0.11 a 24.45 ± 0.96 c PB60 31.48 ± 3.41 a 55.84 ± 0.96 b 2.05 ± 0.13 d 1.95 ± 0.47 a 27.26 ± 2.05 b 浙江 Zhejiang CK 16.00 ± 0.93 c 58.56 ± 0.55 a 2.24 ± 0.03 b 2.10 ± 0.21 b 26.11 ± 0.32 b FP 15.77 ± 0.65 c 58.60 ± 1.50 a 2.05 ± 0.01 c 2.33 ± 0.30 b 28.59 ± 0.66 a P80 35.77 ± 9.69 b 58.21 ± 1.21 a 2.21 ± 0.08 b 2.78 ± 0.23 ab 26.34 ± 1.42 b P60 61.54 ± 3.27 a 58.85 ± 1.16 a 2.56 ± 0.06 a 2.69 ± 0.50 ab 22.95 ± 0.22 b PB60 17.98 ± 0.36 c 58.80 ± 0.67 a 2.33 ± 0.10 b 3.27 ± 0.49 a 25.27 ± 1.25 c 注（Note）：施肥方法为种肥同播，所有处理 N、K 使用量相同，CK 为不施磷，FP 为习惯施磷量，P80、P60 表示施磷量为 FP 处理的 80% 和 60%, PB60 表示 P60 + 解磷菌剂。同列数据后不同小写字母表示同一试验点不同处理间差异显著 (P < 0.05)。All fertilizers were applied during sowing; All treatments applied the same amount of N and K fertilizer, CK treatment represents no P application, FP treatment represents farmers’ conventional phosphorus, P80 and P60 treatments represent P application rate of 60% and 80% of that in FP, PB60 treatments represents P60+phosphate-solubilizing bacteria. Values followed by different small letters mean significant differences among treatments in same experimental site (P < 0.05). NMDS分析 (图4A和B) 显示，磷肥减施显著影响了土壤微生物群落结构。通过冗余分析可知，在湖北麦区土壤微生物量磷MBP显著影响了土壤微生物群落结构的变化 (F = 4.1，P = 0.002)，可解释24.1%的群落结构变化；浙江麦区土壤NH4+-N显著影响了土壤微生物群落结构的变化 (F = 6.8，P = 0.002)，可解释34.4%的群落结构变化 (图4C和D)。
图 4 土壤微生物群落组成的非度量多维尺度分析 (A和B) 及与土壤理化性质的冗余分析 (C和D)
Figure 4. Non-metricmulti dimensional scaling (NMDS) of microbial community composition (A and B)and redundancy analysis with soil properties (C and D)
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磷是影响作物产量的重要营养元素之一。冯媛媛等[17]研究结果表明，在基础土壤有效磷含量0—10 mg/kg和10—20 mg/kg条件下，小麦产量随磷肥用量的增加呈直线上升趋势，但0—10 mg/kg的直线斜率大于10—20 mg/kg，而土壤有效磷含量 > 20 mg/kg，小麦产量在磷肥用量达到一定水平后不再上升。孙慧敏等[29]研究发现，在初始有效磷含量为15.94和30.44 mg/kg的棕壤上，磷肥减施对小麦产量无显著影响。本研究中，湖北麦区基础有效磷含量较低，减少习惯施磷量的20%或40% (P80、P60)，第一年小麦产量下降不显著，第二年显著降低；浙江麦区基础有效磷含量相对较高，连续两年减少20%的施磷量 (P80)，小麦均未减产，土壤基础有效磷含量的差异可能是两个麦区产量结果不同的原因[17,29]。吴良全等[30]研究认为，长江流域小麦季的推荐施磷量为P2O5 70 kg/hm2。黄晓萌等[31]于2019年在长江流域布置共计50个试验表明，农民习惯施磷量的80%为湖北和浙江麦区的推荐施磷量。在本研究中，湖北麦区农民习惯施磷量80%是P2O5 120 kg/hm2，高于吴良全等[30]推荐施磷量，而浙江麦区农民习惯施磷量80%是P2O5 46 kg/hm2，低于吴良全推荐施磷量。上述差异性结论主要是因为不同地区施磷量是基于目标产量、土壤有效磷含量及当地农民习惯施磷量进行调整而造成的[30]。在本研究中，湖北麦区的小麦产量要高于浙江麦区，黄晓萌[32]等调查了长江流域各省 (市) 小麦产量也得到类似研究结论，这可能与种植制度、土壤类型及气候条件等有关[32-33]。
本试验中，与习惯施磷量相比，连续2年不施磷和减施40%磷肥量后 (CK、P60)，湖北麦区土壤总磷含量显著下降，浙江麦区无显著性变化，这可能与湖北土壤全磷含量相对较低有关。土壤有效磷含量反映了土壤的供磷能力，不施磷肥土壤的有效磷含量显著降低[34-36]。陈磊等[37]在河北潮土上连续3年的试验结果表明，减施20%～40%的磷肥用量会导致土壤有效磷含量的降低。本研究中，在湖北和浙江麦区，减少习惯施磷量的20%、40%(P80、P60) 也会降低土壤的有效磷含量。在湖北和浙江麦区，减少40%的习惯施磷量配合解磷菌剂 (PB60)，可显著降低湖北麦区土壤有效磷含量的下降幅度，在浙江麦区则可避免土壤有效磷含量的下降，前人也有类似的报道[38]，表明解磷菌剂在水旱轮作和旱旱轮作种植体系下均能活化土壤难溶性磷，增加土壤有效磷含量。土壤磷活化系数 (PAC) 表征土壤磷的供应能力，当PAC > 2% 时，土壤全磷的转化率高，有机磷容量、供给强度较大[39]。本研究中所有施磷处理的土壤PAC > 2%，而不施磷处理的土壤PAC < 2%，表明湖北、浙江2个麦区只要有磷肥施入，就会提高土壤磷的供应能力。同时PAC受磷肥投入的影响，磷肥投入越大，PAC越大[40-41]。湖北麦区施磷土壤的PAC大于浙江麦区，这是因为湖北磷肥习惯施用量 (P2O5 150 kg/hm2) 大于浙江施用量 (P2O5 57 kg/hm2) 造成的。但是，PAC越大，磷的淋失风险也就越大。因此PAC也需要控制在一个合理的范围内。
在本研究中，两麦区不施磷处理的土壤总碳、总氮、铵态氮以及硝态氮含量较习惯施磷量均无显著性差异，而土壤有效磷和微生物量磷含量均显著降低，Shi等[11]也发现在碳氮丰富而有效磷较低的情况的下，土壤微生物量磷主要受土壤有效磷含量的限制。本研究还发现，湖北麦区80%习惯施磷量处理土壤微生物量磷含量较习惯施磷量显著增加，而浙江麦区差异变化不大，可能与土壤磷素平衡状况相关[43]。Liu等[42]研究显示，在深灰色潜育土上，施用P2O5 40 kg/hm2时土壤微生物量磷含量最大，此时刚好处于磷平衡状态。土壤酶是土壤氮、磷转化的重要驱动因子，其活性是评价土壤质量和氮磷转化能力的重要指标[43]。本研究发现，在湖北和浙江麦区，与习惯施磷量相比，80%和60%习惯施磷量处理均提高土壤铵态氮和硝态氮含量及参与氮循环的亮氨酸氨基肽酶活性，说明亮氨酸氨基肽酶活性对土壤铵态氮和硝态氮含量的变化具有正向调控作用。有研究表明，磷肥减施能促进土壤磷酸酶活性[44]，也有研究认为，磷肥减施对其活性没有影响[45]或降低其活性[46]。本研究结果显示，在湖北和浙江麦区，磷肥减施并没有显著影响土壤磷酸酶活性，与马垒[45]在安徽砂姜黑土上的研究结果相似。
磷脂脂肪酸被广泛用于指示土壤微生物的生物标记。土壤磷脂脂肪酸总量能够反映土壤总微生物量，施用氮肥、磷肥导致土壤理化性质变化进而影响土壤总微生物量，其中细菌、真菌等特征脂肪酸含量的响应特征受土壤磷水平的影响[47]。本研究结果显示，在湖北麦区磷肥减施 (CK、P80和P60) 土壤总磷脂脂肪酸含量显著下降，在浙江麦区磷肥减施提高了土壤总磷脂脂肪酸含量，可能由于湖北麦区基础磷含量较低，磷肥减施后土壤磷含量显著降低从而抑制了土壤微生物的生长[45]。丛枝菌根在植物根吸收土壤磷素过程中具有重要的作用[48]。本研究中，在浙江麦区，与习惯施磷量 (FP) 相比，减少磷肥用量 (P80和P60) 可增加土壤中的丛枝菌根相对丰度，配施解磷菌剂 (PB60) 后，丛枝菌根相对丰度的增加达到了显著水平，促进了植物对土壤磷吸收，并且配施解磷菌剂 (PB60) 后可避免土壤有效磷含量下降，这也解释了为什么浙江麦区减施磷肥配合解磷菌剂处理能够避免作物产量下降的原因。本研究中，在湖北、浙江两麦区，与习惯施磷量相比，磷肥减施 (CK、P80和P60) 对细菌相对丰度无显著性影响，而显著提高真菌相对丰度，进而显著降低细菌与真菌的比值，说明磷肥减施条件下有利于真菌的生长，这主要由于真菌偏好生存于低营养，有机物难分解环境；细菌偏好生存于营养丰富，有机物易分解环境[49]。目前微生物群落结构变化是否与磷肥施用相关也存在较大争议，Shi等[11]研究表明，土壤微生物群落结构不受磷肥施用影响，而在本研究中微生物群落结构受磷肥施用的影响，与Wang等[50]在江苏冬麦区的研究结果一致。根据资源配比理论，微生物群落结构随着系统内潜在的生长限制资源的供应和比率的变化而变化[51]，本研究RDA分析也证实了这点 (图4)，在湖北麦区，土壤微生物量磷是影响微生物群落结构变化的主控因子；而在浙江麦区，土壤铵态氮是影响微生物群落结构变化的主控因子。
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在土壤基础磷含量较低的湖北麦区，磷肥减施土壤有效磷含量显著降低，在减施第二年出现减产，种肥同播下减量施磷的方法不宜推荐；在土壤基础磷含量较高浙江麦区，减少20%的习惯施磷量对小麦产量和土壤有效磷含量均无不利影响，提出种肥同播下习惯施磷量80%是较为理想的施肥策略。此外，在湖北麦区，磷肥减施下施用解磷菌剂能减缓土壤有效磷下降幅度，但未能减缓产量下降幅度，而在浙江麦区，施用解磷菌剂后土壤有效磷含量和小麦产量均未显著下降。磷肥减施不会改变土壤磷酸酶活性，但显著影响土壤微生物群落结构，增加了土壤真菌相对丰度以及降低了细菌/真菌比。在湖北麦区，土壤微生物量磷是影响微生物群落结构变化的主控因子；而在浙江麦区，土壤铵态氮是影响微生物群落结构变化的主控因子。
磷肥减施对长江中下游小麦产量、土壤酶活性及微生物群落结构的影响
Effects of reduced phosphate fertilizer application on wheat yield, soil enzyme activity and microbial community structure in the middle and lower reaches of the Yangtze River
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摘要:
【目的】 在“化肥零增长”背景下，探究磷肥减施对小麦产量及土壤质量的影响，为长江流域中下游地区磷肥减量提供科学依据。 【方法】 在湖北和浙江麦区采用种肥同播技术进行田间试验，设置5个处理：1) 不施磷肥 (CK)；2) 习惯施磷量 (FP)；3) 习惯施磷量80% (P80)；4) 习惯施磷量60% (P60)；5) 习惯施磷量60%+解磷菌剂 (PB60)。于2019、2020年对小麦连续2年进行测产，于2020年小麦收获时测定0—20 cm耕层土壤基础理化性质，微生物量磷、酶活性及磷脂脂肪酸含量。 【结果】 种肥同播下，与FP处理相比，湖北麦区磷肥减施处理 (P80、P60) 第一年小麦产量无显著性差异，而第二年产量分别显著降低31.53%和21.51%；浙江麦区P80处理较FP处理连续两年均未减产，且施用解磷菌剂可避免小麦产量下降。与FP处理相比，湖北麦区CK、P80和P60处理土壤有效磷含量显著降低，P80和P60处理土壤微生物量磷 (MBP) 含量分别增加37.46%和17.21%。在浙江麦区，与FP处理相比，P80处理土壤有效磷含量无显著性差异，P60和CK处理土壤有效磷含量显著降低；P80和P60处理较FP处理土壤MBP含量无显著性差异。两个麦区采用种肥同播技术还发现，与FP处理相比，P80和P60处理土壤磷活化系数和细菌与真菌的比值显著降低，土壤铵态氮和硝态氮含量、参与氮循环的土壤酶活性及真菌相对丰度增加，而土壤pH值、有机碳含量、总氮含量、速效钾含量、土壤磷酸酶活性、细菌相对丰度及丛枝菌根相对丰度均无显著变化；与P60处理相比，PB60处理增加了土壤有效磷含量、土壤磷活化系数及丛枝菌根的相对丰度。通过冗余分析发现，MBP是影响湖北麦区土壤微生物群落结构变化的主控因子；土壤铵态氮是影响浙江麦区土壤微生物群落结构变化的主控因子。 【结论】 不同肥力麦区采用种肥同播，土壤有效磷、微生物量磷和小麦产量对磷肥减施的响应各异。在基础磷含量较低的湖北麦区，连续减施磷肥易减产，不推荐种肥同播下减量施磷的方法；而基础磷含量较高的浙江麦区，在保证小麦产量和土壤质量不降低的情况下，较为理想的施肥策略是种肥同播 + 80%习惯施磷量。此外，磷肥减施没有显著影响土壤磷酸酶活性，但显著影响土壤微生物群落结构，增加了土壤真菌相对丰度以及降低了细菌/真菌比。解磷菌剂在浙江麦区的应用效果优于湖北麦区。 Abstract:【Objectives】 Under the background of “zero growth of fertilizer”, this study aimed to explore the effect of phosphorus fertilizer reduction on wheat yield and soil quality. It may provide scientific basis for phosphorus fertilizer reduction in the middle and lower reaches of the Yangtze River Basin. 【Methods】 Field experiments were conducted in Hubei and Zhejiang Province using the technology of sowing and fertilizering simultaneously. The treatments included non-phosphorus control (CK), farmers’ conventional phosphorus (FP), 80% of farmers’ conventional phosphorus (P80), 60% of farmers’ conventional phosphorus (P60), and P60+phosphate-solubilizing bacteria (PB60). The wheat yieldswere investigated in 2019 and 2020. When wheat was harvested in 2020, the basic physical and chemical properties, microbial phosphorus content, enzyme activity and phospholipid fatty acid content in 0-20 cm horizon in cultivated soils were determined. 【Results】 In Hubei wheat region, under sowing and fertilizering simultaneously, relative to FP treatment, P80 and P60 treatments did not lead to any significant changes in wheat yield in the first year of phosphorus fertilizer reduction, while in the second year of phosphorus fertilizer reduction, P80 and P60 treatments significantly decreased wheat yield by 31.53% and 21.51%, respectively. However, in Zhejiang wheat region, relative to FP treatment, P80 treatment did not significantly affect wheat yield after two years of phosphorus fertilizer reduction, and the application of phosphate-solubilizing bacteria could avoid the decline of wheat yield. In Hubei wheat region, relative to FP treatment, CK, P80 and P60 treatments significantly reduced available P content, and P80 and P60 treatments increased soil microbial biomass P (MBP) content by 37.46% and 17.21%, respectively. In Zhejiang wheat region, relative to FP treatment, P80 treatments did not cause any significant changes in soil available P content, while P60 and CK treatments significantly decreased soil available P content, and P80 and P60 treatments did not result in any significant changes in MBP content. In Hubei and Zhejiang wheat region under sowing and fertilizering simultaneously, relative to FP treatment, P80 and P60 treatments linked to significantly lower soil phosphorus activation coefficientandthe ratio of bacteria to fungi, promoted soil NH4+-N content, NO3--N content, soil N-cycling enzyme activitiesand the relative abundance fungi, and did not result in any significant changes in soil pH value, organic carbon content, total N content, available K content, soil phosphatase activity, the relative abundance of bacteriaand the relative abundance of arbuscular mycorrhizal fungi.Relative to P60 treatment, PB60 treatment elevated soil available P content, soil phosphorus activation coefficient, and the relative abundance of arbuscular mycorrhizal fungi. Through redundancy analysis, soil microbial biomass P was considered as a dominant factor affecting soil microbial community structure change in Hubei wheat region; while soil NH4+-N was considered as a dominant factor affecting the microbial community structure change in Zhejiang wheat region. 【Conclusions】 It was found that soil available P, microbial biomass P and wheat yield had different responses to phosphorus fertilizer reduction in different soil fertility under sowing and fertilizering simultaneously. In Hubei wheat region with lowsoil basic P content, continuous reduction of phosphate fertilizer is easy to result in a decrease in wheat yield. Therefore, it was not recommended to reduce phosphorus fertilizer under sowing and fertilizering simultaneously. However, in Zhejiang wheat region with high soil basic P content, under the condition of no decrease in wheat yield and soil quality, it is suggested that 80% of farmers’ conventional phosphorusapplicationwith sowing and fertilizering simultaneously is an ideal fertilization strategy. In addition, reduction of phosphate fertilizer did not significantly affect soil phosphatase activity, but significantly affected soil microbial community structure, increased the relative abundance of fungi and decreased the bacterial/fungi ratio. The effect of phosphate-solubilizing bacteria application in Zhejiang wheat region is better than that in Hubei wheat region. -
图 2 不同磷肥施用量下土壤磷活化系数
Figure 2. Phosphorus activation coefficient of soil under different phosphorus rates
图 3 小麦收获期不同施磷处理土壤酶活性
Figure 3. Soil enzyme activity as affected by different phosphorus rates at wheat harvest
图 4 土壤微生物群落组成的非度量多维尺度分析 (A和B) 及与土壤理化性质的冗余分析 (C和D)
Figure 4. Non-metricmulti dimensional scaling (NMDS) of microbial community composition (A and B)and redundancy analysis with soil properties (C and D)
表 1 试验基地0—20 cm土层基础理化性质
Table 1. Basic physical and chemical properties of 0–20 cm soil layer
地点
SitepH
(1∶2.5)容重
Bulk density
(g/cm3)有机碳
Organic C
(g/kg)全氮
Total N
(g/kg)碱解氮
Available N
(mg/kg)全磷
Total P
(g/kg)有效磷
Available P
(mg/kg)速效钾
Available K
(mg/kg)湖北广水市
Guangshui city, Hubei6.30 1.32 12.56 1.16 91.12 0.32 7.34 162.32 浙江平湖市
Pinghu city, Zhejiang7.80 1.42 14.40 1.34 199.73 0.68 11.55 200.68 
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表 2 小麦收获后不同磷肥减施处理土壤理化性质
Table 2. Physic-chemical properties of soils as affected by different phosphorous rates at wheat maturity
处理
TreatmentpH
(1∶2.5)有机碳
SOC
(g/kg)总氮
Total N
(g/kg)铵态氮
NH4+-N
(mg/kg)硝态氮
NO3--N
(mg/kg)总磷
Total P
(g/kg)有效磷
Available P
(mg/kg)速效钾
Available K
(mg/kg)微生物量磷
MBP
(mg/kg)湖北 Hubei CK 6.60 ± 0.12 a 11.38 ± 0.36 a 1.12 ± 0.04 a 0.85 ± 0.07 b 6.12 ± 0.25 c 0.30 ± 0.01 c 3.59 ± 0.40 d 151.55 ± 1.09 a 6.87 ± 0.51 d FP 6.11 ± 0.09 a 11.40 ± 0.82 a 1.12 ± 0.08 a 0.85 ± 0.05 b 6.02 ± 0.44 c 0.36 ± 0.01 a 12.05 ± 1.73 a 148.29 ± 5.32 a 11.56 ± 2.45 c P80 6.65 ± 0.45 a 12.10 ± 0.30 a 1.13 ± 0.09 a 1.37 ± 0.05 a 7.11 ± 0.48 b 0.35 ± 0.02 ab 9.64 ± 0.82 b 164.03 ± 9.70 a 15.89 ± 1.61 ab P60 6.48 ± 0.41 a 11.49 ± 0.03 a 1.11 ± 0.03 a 1.32 ± 0.26 a 6.48 ± 0.28 bc 0.32 ± 0.01 bc 7.37 ± 0.63 c 144.99 ± 12.69 a 13.55 ± 1.38 bc PB60 6.17 ± 0.11 a 11.82 ± 0.90 a 1.08 ± 0.03 a 1.51 ± 0.15 a 8.07 ± 0.31 a 0.33 ± 0.02 bc 9.59 ± 0.55 b 154.45 ± 14.74 a 16.71 ± 1.40 a 浙江 Zhejiang CK 7.63 ± 0.13 a 14.61 ± 0.46 a 1.34 ± 0.10 a 1.29 ± 0.02 b 9.43 ± 0.49 a 0.67 ± 0.02 a 10.32 ± 0.43 d 224.35 ± 9.53 a 7.72 ± 2.77 b FP 7.71 ± 0.18 a 13.76 ± 0.41 a 1.30 ± 0.04 a 1.35 ± 0.10 b 9.54 ± 0.33 a 0.70 ± 0.03 a 16.84 ± 0.67 ab 225.33 ± 10.65 a 17.49 ± 1.97 a P80 7.71 ± 0.25 a 13.60 ± 1.23 a 1.29 ± 0.10 a 1.69 ± 0.06 a 9.86 ± 0.22 a 0.68 ± 0.04 a 14.47 ± 1.59 bc 212.99 ± 7.11 a 15.55 ± 3.08 a P60 7.75 ± 0.20 a 14.44 ± 0.89 a 1.31 ± 0.02 a 1.65 ± 0.04 a 9.91 ± 0.79 a 0.69 ± 0.03 a 13.74 ± 0.59 c 252.8 ± 14.88 a 15.46 ± 3.60 a PB60 7.61 ± 0.15 a 15.05 ± 0.85 a 1.4 ± 0.07 a 1.74 ± 0.05 a 10.04 ± 0.58 a 0.70 ± 0.02 a 17.53 ± 2.67 a 233.65 ± 9.09 a 15.17 ± 3.11 a 注（Note）：施肥方法为种肥同播 All fertilizers were applied during sowing; 所有处理 N、K 使用量相同，CK 为不施磷，FP 为习惯施磷量，P80、P60 表示施磷量为 FP 处理的 80% 和 60%, PB60 表示 P60 + 解磷菌剂 All treatments applied the same amount of N and K fertilizer, CK treatment represents no P application, FP treatment represents farmers’ conventional phosphorus, P80 and P60 treatments represent P application rate of 60% and 80% of that in FP, PB60 treatments represents P60+phosphate-solubilizing bacteria; SOC—有机碳 Soil organic carbon; MBP—微生物量磷 Microbial biomass P; 同列数据后不同小写字母表示同一试验点不同处理间差异显著 (P < 0.05) Values followed by different small letters mean significant differences among treatments at same experimental site (P < 0.05).
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表 3 不同施磷处理土壤中总磷脂脂肪酸含量及微生物相对丰度
Table 3. Total phospholipid fatty acid content and the relative abundance of microorganisms affected by differentphosphorous treatments
地点
Site处理
Treatment总磷脂脂肪酸
Total PLFAs
(nmol/g, dry soil)相对丰度 (mol%)
The relative abundance PLFAs细菌/真菌
Bacteria/Fungi细菌
Bacteria真菌
Fungi丛枝菌根
Arbuscular mycorrhizal fungi湖北 Hubei CK 14.10 ± 2.59 c 59.87 ± 2.00 a 2.98 ± 0.08 a 1.91 ± 0.30 a 20.10 ± 1.16 d FP 24.58 ± 0.09 b 59.75 ± 1.10 a 1.91 ± 0.04 d 1.95 ± 0.39 a 31.32 ± 0.69 a P80 19.36 ± 1.99 c 58.42 ± 0.78 a 2.54 ± 0.07 b 1.62 ± 0.35 a 23.04 ± 0.82 c P60 18.18 ± 4.13 c 57.88 ± 0.48 ab 2.37 ± 0.08 c 1.40 ± 0.11 a 24.45 ± 0.96 c PB60 31.48 ± 3.41 a 55.84 ± 0.96 b 2.05 ± 0.13 d 1.95 ± 0.47 a 27.26 ± 2.05 b 浙江 Zhejiang CK 16.00 ± 0.93 c 58.56 ± 0.55 a 2.24 ± 0.03 b 2.10 ± 0.21 b 26.11 ± 0.32 b FP 15.77 ± 0.65 c 58.60 ± 1.50 a 2.05 ± 0.01 c 2.33 ± 0.30 b 28.59 ± 0.66 a P80 35.77 ± 9.69 b 58.21 ± 1.21 a 2.21 ± 0.08 b 2.78 ± 0.23 ab 26.34 ± 1.42 b P60 61.54 ± 3.27 a 58.85 ± 1.16 a 2.56 ± 0.06 a 2.69 ± 0.50 ab 22.95 ± 0.22 b PB60 17.98 ± 0.36 c 58.80 ± 0.67 a 2.33 ± 0.10 b 3.27 ± 0.49 a 25.27 ± 1.25 c 注（Note）：施肥方法为种肥同播，所有处理 N、K 使用量相同，CK 为不施磷，FP 为习惯施磷量，P80、P60 表示施磷量为 FP 处理的 80% 和 60%, PB60 表示 P60 + 解磷菌剂。同列数据后不同小写字母表示同一试验点不同处理间差异显著 (P < 0.05)。All fertilizers were applied during sowing; All treatments applied the same amount of N and K fertilizer, CK treatment represents no P application, FP treatment represents farmers’ conventional phosphorus, P80 and P60 treatments represent P application rate of 60% and 80% of that in FP, PB60 treatments represents P60+phosphate-solubilizing bacteria. Values followed by different small letters mean significant differences among treatments in same experimental site (P < 0.05).
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