溶磷细菌及控释肥料对土壤磷有效性的影响
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摘要
磷是植物必需的三大营养元素之一，施入土壤中的大部分无机磷易被钙、镁、铁、铝、锰离子固定而难以被植物体利用。磷肥的过量施用导致土壤中的磷大量盈余，针对这种情况本研究通过土壤肥力调查、模拟降雨淋溶试验、缓控释肥料养分释放模拟试验、溶磷细菌的筛选鉴定评价试验、溶磷细菌在不同条件及不同培养基下的溶磷试验以及温室条件下番茄盆栽试验等一系列试验手段，深入探讨了溶磷细菌在不同环境下对难溶磷组分的溶解特征及施用缓控释肥料对土壤磷活性的影响。主要研究结果如下
1)太湖直湖港地区97.8%（n=93）的耕层土壤有机质高达20~69.5g/kg，具有极强的保肥能力；调查区域98%（n=98）的耕层土壤全氮含量高达1.5~4.4g/kg，达到丰富水平；该地区89.5%（n=95）的耕层土壤总磷含量在0.15~1.34g/kg之间，92.5%（n=94）有效磷含量在30~719mg/kg，属于丰富水平；该地区土壤pH主要（54%;n=92）集中在4.5~5.5，土壤酸化严重。
2)模拟淋溶中的淋溶液的电导率、全氮浓度和可溶性磷浓度均在模拟灌溉期达到峰值，施用控释肥料的处理显著低于其它处理。不同施肥处理氮素的淋失量和淋失率有一定的差异性，其中农民习惯施肥处理（FFP）氮淋失率最高，为68.98%，施用缓控释肥的处理（CRF）最低，仅为24.46%。缓控释肥处理（CRF）和传统化肥处理（CF）淋失的氮以硝态氮为主，其它处理的氮主要以酰胺态氮或有机氮的形式淋失。施用畜禽有机肥料能显著增加淋溶液中的磷含量，其中农民习惯施肥处理（FFP）的全磷淋失量是施用缓控释肥处理（CRF）的16.73倍。直湖港地区推广施用缓控释肥可明显降低氮磷淋失量，减少施肥对地下水质的影响。
3)缓控释肥在土壤中的实际释放规律与理想状态（25℃；净水浸提）有较大差异；施用缓控释肥时，田间耕层土壤同步埋瓶做静水释放能够反映肥料在土壤中的真实释放规律。
4)对番茄蔬菜地、热带水果果园和未经人类活动扰动过的根际土壤样品进行了溶磷菌的分离和筛选，获得了具有较强溶磷作用的20株溶磷菌，通过形态特征和16SrRNA鉴定后将所得溶磷菌归类为巨大芽孢杆菌[Bacillus megaterium (B. aryabhattai)]、枯草芽孢杆菌（Bacillus subtilis）、绿脓杆菌（Pseudomonas aeruginosa）、根瘤菌（Rhizobium sp.）、不动杆菌（Acinetobacter sp.）和栖稻假单胞菌（Pseudomonasoryzihabitans）。
5)平板法观测到的溶磷圈直径值仅可作为参考，具有明显聚落生长的细菌也应归为溶磷菌；同一株菌在三种基质测得溶磷参数各不相同，液体培养基中添加脓杆菌Krome3，测得增溶的磷523.69mg/l，土壤中仅有增溶17.02mg/l；砂土配以营养液时，添加溶磷菌的处理水溶性磷相比对照显著增高，M3-P有下降的趋势。
6)不同温度和含水量条件下石灰性土壤接种溶磷菌模拟培养45天后，相同土壤间磷组分无显著性差异；不同土壤利用方式，无机磷组分差异较大，未耕作的土壤（松林地土壤）的水溶性磷、可浸提磷、铝铁结合磷、钙镁结合磷、有机磷和残渣态磷各项指标都低于耕作土壤（番茄地土壤和热带水果果园土壤），且以残渣态磷为主要磷组分（96.72%），而耕作石灰性土壤以钙镁结合磷为主（63.70~75.60%），残渣态磷次之。
7)智能温室内盆栽条件下添加溶磷菌后番茄长势无显著差异，终止时土壤Olsen-P含量无显著性差异。
Phosphorus (P) is one of three major nutrients in crop productivity for its involvement inmany essential processes. Most of the soils, phosphate was present in unavailable forms dueto complex formation with Ca, Mg, Fe, Al or Mn. The continuing application of large amountof soluble P fertilizer to soil results in accumulation of phosphorus in most of cultivated soils.Controlled release fertilizer can release phosphate nutrient slowly, making P more available toplant. Phosphate-solubilizing bacteria can release special matters to make fixed phosphateavailable for plants. Controlled released fertilizer and phosphate-solubilizing bacteria shouldbe used together to reduce the costs of fertilization. The research results were as follows:
1) In Tai Lake Zhihugang Region, organic matters of soil samples were as high as20-69.5g/kg (97.8%of93samples) with great fertilities; The total nitrogen contents of89.5%of95soil samples were as high as1.5-4.4g/kg; Olsen-P contents were30-719mg/kg (92.5%,n=94); The main values of pH were4.5-5.5with54%of92samples.
2) There was no significant difference in pH values of leachate in different treatmentsafter late stage of experiment, but the pH value in leachate of two organic fertilizer (madefrom digested livestock manure) treatments were lower than other treatments during prophase.The peaks of electrical conductivity (EC), total nitrogen, and water-soluble phosphorusconcentration in leachate were occurred on the simulated irrigation period, and that oftreatments treated by controlled release fertilizer (CRF) were lower than other treatments. Therate of N loss of farmers' fertilization practice (FFP) was highest among other treatments as68.98%, and that of treatments treated with controlled released fertilizer were significantlylower than other treatments, the lowest of that was24.46%, respectively. The forms of N inleachate were mainly nitrate for CRF and common fertilizer (CF) treatment, and amide-nitrogen or organic nitrogen for other treatments. Compared with other no-organic fertilizertreatments, adding organic fertilizer significantly increased the total phosphorus leachingamount and water-soluble phosphorus concentration. Total phosphorus loss from thetreatment of farmer’s traditional fertilization was16.73times of that from the CRF treatment.The promotion of CRF in Zhihugang Region of Tai Lake could decrease leaching loss of Nand P, and reduce pollution of ground water.
3) There were significantly differences on the release of nitrogen and phosphorus fromcontrolled release fertilizer in the field compared with laboratory conditions (water as matrix;25℃). Nutrient release in water at the temperature of field is more comparable than25℃.
4) Twenty phosphate-solubilizing bacteria (PSB) were isolated from rhizospheres ofcalcareous soils. These isolates were identified by sequence analysis of16S rRNA genes asbacterial species of Bacillus megaterium (B. aryabhattai), Bacillus subtilis, Pseudomonasaeruginosa, Rhizobium sp., Acinetobacter sp., and Pseudomonas oryzihabitans.
5) Seven of these isolates were evaluated with the National Botanical ResearchInstitute’s Phosphate (NBRIP) plate culture, NBRIP liquid culture, and real soil incubation.Results showed that halo zone formation by PSB on NBRIP plates was a good indicator forscreening PSB, but not good enough to quantify capability of P solubilization because of poorcorrelation between sizes of halo zone and water soluble P (WS-P). The NBRIP liquidmedium culture showed four PSB strains lowered medium pH (<4.3) and released WS-P up to523.69mg/l with three days incubation and Pseudomonas aeruginosa Krome3straindissolved95.3%tricalcium phosphate added after35days incubation. Incubation of PSB in asandy soil showed that PSB increased WS-P, but no Mehlich-3P (M3-P). Therefore, each ofthree culture practices has strength and weakness for charactering PSB and performing allthree tests provide the better understanding of PSB.
6) Soil samples from the Pineland and nearby farmlands (vegetable fields and tropicalfruit groves) were sequentially extracted for water soluble P; exchangeable P; Al-and Fe-bound P; Ca-and Mg-bound P; NaOH-extractable organic P; and residual P. Phosphorusconcentrations were higher in each fraction of the farmed soils than in corresponding fractionsof the unfarmed soils. The unfarmed sites contained96.72%residual P. In the farmed sites,the Ca-and Mg-bound P fraction was the largest, ranging from63.70to75.60%of total P,and the residual P fraction was the second largest.
7) We didn’t find any differences on plant growth conditions and Olsen-P content in soils,during tomato growing, even the phosphate-solubilizing bacterial strains were inoculated torhizosphere soils every week.
1)太湖直湖港地区97.8%（n=93）的耕层土壤有机质高达20~69.5g/kg，具有极强的保肥能力；调查区域98%（n=98）的耕层土壤全氮含量高达1.5~4.4g/kg，达到丰富水平；该地区89.5%（n=95）的耕层土壤总磷含量在0.15~1.34g/kg之间，92.5%（n=94）有效磷含量在30~719mg/kg，属于丰富水平；该地区土壤pH主要（54%;n=92）集中在4.5~5.5，土壤酸化严重。
2)模拟淋溶中的淋溶液的电导率、全氮浓度和可溶性磷浓度均在模拟灌溉期达到峰值，施用控释肥料的处理显著低于其它处理。不同施肥处理氮素的淋失量和淋失率有一定的差异性，其中农民习惯施肥处理（FFP）氮淋失率最高，为68.98%，施用缓控释肥的处理（CRF）最低，仅为24.46%。缓控释肥处理（CRF）和传统化肥处理（CF）淋失的氮以硝态氮为主，其它处理的氮主要以酰胺态氮或有机氮的形式淋失。施用畜禽有机肥料能显著增加淋溶液中的磷含量，其中农民习惯施肥处理（FFP）的全磷淋失量是施用缓控释肥处理（CRF）的16.73倍。直湖港地区推广施用缓控释肥可明显降低氮磷淋失量，减少施肥对地下水质的影响。
3)缓控释肥在土壤中的实际释放规律与理想状态（25℃；净水浸提）有较大差异；施用缓控释肥时，田间耕层土壤同步埋瓶做静水释放能够反映肥料在土壤中的真实释放规律。
4)对番茄蔬菜地、热带水果果园和未经人类活动扰动过的根际土壤样品进行了溶磷菌的分离和筛选，获得了具有较强溶磷作用的20株溶磷菌，通过形态特征和16SrRNA鉴定后将所得溶磷菌归类为巨大芽孢杆菌[Bacillus megaterium (B. aryabhattai)]、枯草芽孢杆菌（Bacillus subtilis）、绿脓杆菌（Pseudomonas aeruginosa）、根瘤菌（Rhizobium sp.）、不动杆菌（Acinetobacter sp.）和栖稻假单胞菌（Pseudomonasoryzihabitans）。
5)平板法观测到的溶磷圈直径值仅可作为参考，具有明显聚落生长的细菌也应归为溶磷菌；同一株菌在三种基质测得溶磷参数各不相同，液体培养基中添加脓杆菌Krome3，测得增溶的磷523.69mg/l，土壤中仅有增溶17.02mg/l；砂土配以营养液时，添加溶磷菌的处理水溶性磷相比对照显著增高，M3-P有下降的趋势。
6)不同温度和含水量条件下石灰性土壤接种溶磷菌模拟培养45天后，相同土壤间磷组分无显著性差异；不同土壤利用方式，无机磷组分差异较大，未耕作的土壤（松林地土壤）的水溶性磷、可浸提磷、铝铁结合磷、钙镁结合磷、有机磷和残渣态磷各项指标都低于耕作土壤（番茄地土壤和热带水果果园土壤），且以残渣态磷为主要磷组分（96.72%），而耕作石灰性土壤以钙镁结合磷为主（63.70~75.60%），残渣态磷次之。
7)智能温室内盆栽条件下添加溶磷菌后番茄长势无显著差异，终止时土壤Olsen-P含量无显著性差异。
Phosphorus (P) is one of three major nutrients in crop productivity for its involvement inmany essential processes. Most of the soils, phosphate was present in unavailable forms dueto complex formation with Ca, Mg, Fe, Al or Mn. The continuing application of large amountof soluble P fertilizer to soil results in accumulation of phosphorus in most of cultivated soils.Controlled release fertilizer can release phosphate nutrient slowly, making P more available toplant. Phosphate-solubilizing bacteria can release special matters to make fixed phosphateavailable for plants. Controlled released fertilizer and phosphate-solubilizing bacteria shouldbe used together to reduce the costs of fertilization. The research results were as follows:
1) In Tai Lake Zhihugang Region, organic matters of soil samples were as high as20-69.5g/kg (97.8%of93samples) with great fertilities; The total nitrogen contents of89.5%of95soil samples were as high as1.5-4.4g/kg; Olsen-P contents were30-719mg/kg (92.5%,n=94); The main values of pH were4.5-5.5with54%of92samples.
2) There was no significant difference in pH values of leachate in different treatmentsafter late stage of experiment, but the pH value in leachate of two organic fertilizer (madefrom digested livestock manure) treatments were lower than other treatments during prophase.The peaks of electrical conductivity (EC), total nitrogen, and water-soluble phosphorusconcentration in leachate were occurred on the simulated irrigation period, and that oftreatments treated by controlled release fertilizer (CRF) were lower than other treatments. Therate of N loss of farmers' fertilization practice (FFP) was highest among other treatments as68.98%, and that of treatments treated with controlled released fertilizer were significantlylower than other treatments, the lowest of that was24.46%, respectively. The forms of N inleachate were mainly nitrate for CRF and common fertilizer (CF) treatment, and amide-nitrogen or organic nitrogen for other treatments. Compared with other no-organic fertilizertreatments, adding organic fertilizer significantly increased the total phosphorus leachingamount and water-soluble phosphorus concentration. Total phosphorus loss from thetreatment of farmer’s traditional fertilization was16.73times of that from the CRF treatment.The promotion of CRF in Zhihugang Region of Tai Lake could decrease leaching loss of Nand P, and reduce pollution of ground water.
3) There were significantly differences on the release of nitrogen and phosphorus fromcontrolled release fertilizer in the field compared with laboratory conditions (water as matrix;25℃). Nutrient release in water at the temperature of field is more comparable than25℃.
4) Twenty phosphate-solubilizing bacteria (PSB) were isolated from rhizospheres ofcalcareous soils. These isolates were identified by sequence analysis of16S rRNA genes asbacterial species of Bacillus megaterium (B. aryabhattai), Bacillus subtilis, Pseudomonasaeruginosa, Rhizobium sp., Acinetobacter sp., and Pseudomonas oryzihabitans.
5) Seven of these isolates were evaluated with the National Botanical ResearchInstitute’s Phosphate (NBRIP) plate culture, NBRIP liquid culture, and real soil incubation.Results showed that halo zone formation by PSB on NBRIP plates was a good indicator forscreening PSB, but not good enough to quantify capability of P solubilization because of poorcorrelation between sizes of halo zone and water soluble P (WS-P). The NBRIP liquidmedium culture showed four PSB strains lowered medium pH (<4.3) and released WS-P up to523.69mg/l with three days incubation and Pseudomonas aeruginosa Krome3straindissolved95.3%tricalcium phosphate added after35days incubation. Incubation of PSB in asandy soil showed that PSB increased WS-P, but no Mehlich-3P (M3-P). Therefore, each ofthree culture practices has strength and weakness for charactering PSB and performing allthree tests provide the better understanding of PSB.
6) Soil samples from the Pineland and nearby farmlands (vegetable fields and tropicalfruit groves) were sequentially extracted for water soluble P; exchangeable P; Al-and Fe-bound P; Ca-and Mg-bound P; NaOH-extractable organic P; and residual P. Phosphorusconcentrations were higher in each fraction of the farmed soils than in corresponding fractionsof the unfarmed soils. The unfarmed sites contained96.72%residual P. In the farmed sites,the Ca-and Mg-bound P fraction was the largest, ranging from63.70to75.60%of total P,and the residual P fraction was the second largest.
7) We didn’t find any differences on plant growth conditions and Olsen-P content in soils,during tomato growing, even the phosphate-solubilizing bacterial strains were inoculated torhizosphere soils every week.
引文
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