【作者】 刘骁蒨；

【导师】 张小平； 涂仕华；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 土壤肥力对于保证粮食安全及农业的可持续发展具有重要意义,施肥是影响作物产量的关健因子,找到施肥的临界点可为土壤肥力的保持和避免因过量施肥造成土壤污染提供科学依据。为研究秸秆还田方式和施肥对水稻土土壤质量的影响,四川省农业科学院于2002年以来在四川广汉市西高镇建立了秸秆覆盖还田试验以及在连山镇建立了秸秆翻埋还田试验,并对秸秆还田方式和施肥下土壤的理化性质以及作物产量进行了研究,为深入认识秸秆还田方式和施肥对水稻土质量的影响奠定了基础。本文在前人已有研究基础上,运用平板菌落计数对不同施肥制度下的土壤可培养微生物数量进行了测定,利用化学分析法对土壤微生物生物量碳、氮和土壤酶活进行了分析,并且应用变性梯度凝胶电泳(DGGE)分子标记技术对秸秆还田方式和施肥下细菌、真菌、固氮菌、氨氧化细菌和氨氧化古菌等微生物群落结构特征进行了研究。文章结果总结如下：(1)秸秆覆盖还田和施肥对土壤微生物数量、微生物量及土壤酶活的影响结果表明：在水稻幼穗分化期和水稻收获后,与对照只施肥而没有秸秆还田处理(CK)相比,秸秆覆盖还田配施足量氮磷钾肥处理(SCK)能显著增加土壤微生物数量、土壤微生物量碳和量氮、土壤脲酶、蔗糖酶和磷酸酶酶活,而显著降低土壤多酚氧化酶活性(P≤0.01)；同时秸秆覆盖后不施用钾肥的处理(SNP)、低氮肥处理(SNL)以及不施用磷肥的处理(SNK)的土壤微生物数量、微生物量碳和量氮、脲酶、转化酶和磷酸酶酶活均显著低于秸秆覆盖还田配施适量氮磷钾的处理,而多酚氧化酶活性则显著相反(P≤0.01)。(2)秸秆覆盖还田和施肥对土壤细菌群落结构及多样性的研究结果表明：在水稻幼穗分化期和水稻收获期,0～10cm的土层中,秸秆覆盖还田配施足量氮磷钾肥的处理细菌多样性显著高于单施肥而无秸秆还田的处理、秸秆覆盖还田只配施低氮肥和不施磷肥的处理(P≤0.01),但它与秸秆覆盖还田不配施钾肥的处理没有显著差异(P≥0.05)。在10-20cm的土层中,秸秆覆盖还田配施足量氮磷钾的处理细菌多样性显著高于单施肥无秸秆处理、秸秆还田低氮处理、秸秆还田不施钾肥处理和秸秆还田不施磷肥处理(P≤0.01)。DGGE优势条带测序表明：秸秆还田处理土壤的优势菌群包括,β变形菌纲(Beta proteobacteria)的杜擀氏属(Duganella sp.)、α-变形菌纲(Alpha proteobacteria)红游动菌属(Rhodoplanes sp.)和磁螺菌属(Magnetospirillum sp.)、绿弯菌门(Chloroflexi)、地杆菌属(Geobacter)、绿弯菌门(Chloroflexi)、暖绳菌科(Caldilineaceae)、厌氧绳菌科(Anaerolineaceae)、芽单胞菌门(Gemmatimonadetes)、酸杆菌门(Acidobacteria)、硝化螺旋菌门(Nitrospirae)。对照处理土壤的优势细菌为,厚壁菌门(Firmicutes)、β变形菌纲(Beta proteobacteria)的杜擀氏属(Duganella)、绿弯菌门(Chloroflexi)、α-变形菌纲(Alpha proteobacteria)的红游动菌属(Rhodoplanes)和磁螺菌属(Magnetospirillum)。(3)秸秆覆盖还田和施肥对土壤真菌群落结构和多样性研究表明：水稻幼穗分化期,在0～10cm和10～20cm土层中,秸秆覆盖还田配施足量氮磷钾的处理真菌多样性都明显高于无秸秆覆盖的对照组(P≤0.01)。水稻收获后,在0～10cm土层中,秸秆覆盖还田配施足量氮磷钾肥的处理真菌多样性与无秸秆对照相比没有明显差异(P≥0.05)。在10～20cm土层中,秸秆覆盖配施足量氮磷钾肥的处理真菌多样性显著低于无秸秆对照组(P≤0.01)。DGGE条带测序发现,秸秆还田和施肥的优势真菌为,半知菌纲的枝顶孢属(Acremonium sp.)、子囊菌纲的翅孢壳属(Emericellopsis sp.)、半知菌亚门的曲霉属(Aspergillus sp.)、枝孢属(Cladosporium sp.)、红酵母属(Rhodotorula sp.)、侧耳属Pleurotus sp.)、被孢霉属(Mortierella sp.)。而对照土壤的优势菌为子囊菌门(Ascomycota)和sordariomyceta。表明秸秆还田增加了土壤真菌的多样性改变了土壤真菌的群落结构。(4)秸秆覆盖还田和施肥对固氮基因nifH群落结构和多样性研究表明：在水稻幼穗分化期和水稻收获后,0～10cm和10～20cm土层中,秸秆覆盖还田配施足量氮磷钾肥处理固氮基因n归多样性显著高于仅施肥无秸秆覆盖的对照组(P≤0.07)。DGGE条带测序得知,14个条带的近缘种大部分为非培养细菌nifH基因片段,主要优势菌群其归属于变形菌门(Proteobacteria)的p-变形菌纲(Betaproteobacteria)。秸秆还田和施肥的土壤优势固氮微生物为,非培养伯克氏菌属、假食酸菌属(Pseudacidovorax sp.)、 Azospira属、Ideonella属。而对照土壤优势菌为假食酸菌属(Pseudacidovorax sp.)、 Azospira属以及未确定归属的固氮细菌。(5)秸秆覆盖还田和施肥对氨氧化细菌群落结构和多样性的影响研究显示,在水稻幼穗分化期,0～10cm土层中,秸秆覆盖还田后施用足量氮磷钾肥处理的氨氧化细菌多样性指数明显高于单施化肥对照处理以及秸秆还田后低氮肥、无钾肥的处理(P≤0.01),但它与秸秆覆盖还田后不施钾肥的处理没有显著差异(P≥0.05)。在10～20cm土层中,秸秆覆盖还田配施足量氮磷钾肥处理的氨氧化细菌多样性显著高于单施肥而没有秸秆覆盖的对照组(P≤0.01)。水稻收获后,在0～10cm和10～20cm土层中,秸秆覆盖还田配施足量氮磷钾肥的处理氨氧化细菌多样性显著高于无秸秆还田的处理(P≤0.01)。从DGGE条带测序分析得出,所有的优势氨氧化细菌的系统发育都比较单一。秸秆覆盖还田和施肥处理与对照土壤的优势氨氧化细菌均为β-变形菌纲(Betaproteobacteria)的亚硝化螺菌属(Nitrosospira)和亚硝化单胞菌属(Nitrosomonas)。(6)秸秆覆盖还田和施肥对氨氧化古菌群落结构和多样性的影响研究表明：水稻幼穗分化期,在0～10cm和10-20cm土层中,秸秆覆盖配施足量氮磷钾肥处理氨氧化古菌多样性均显著高于单施肥无秸秆覆盖还田处理(P≤0.01)。水稻收获之后,在0-10cm和10-20cm土层中,秸秆覆盖配施足量氮磷钾处理多样性显著高于处理单施肥无秸秆覆盖还田的处理(P,≤0.01),但它与秸秆覆盖后施用低氮肥、不施磷肥和不施钾肥的处理没有显著差异(P≥0.05)。通过对氨氧化古菌优势条带测序结果表明,秸秆覆盖还田和对照土壤得到的全部氨氧化古菌均归属于非培养泉古菌门(Crenarchaeota)。(7)秸秆翻埋还田和施肥对土壤微生物量、土壤酶活研究表明：在水稻幼穗分化期和水稻收获后,与对照无肥处理(CK)相比,施用氮磷钾肥处理(NPK)和秸秆翻埋配施适量氮磷钾处理(SNPK)都能显著增加土壤微生物量碳和量氮(P≤0.01)。同时秸秆翻埋还田和施肥可以明显增加土壤中脲酶、转化酶、磷酸酶以及多酚氧化酶活性(P≤0.01)。(8)秸秆翻埋还田和施肥对土壤氨氧化细菌群落结构和多样性研究表明：在水稻幼穗分化期,0～10cm和10～20cm土层中,不施肥处理(CK)土壤氨氧化细菌多样性均显著低于单施氮磷钾肥处理(NPK)。施用氮磷钾肥的处理氨氧化细菌多样性显著低于秸秆翻埋配施适量氮磷钾肥的处理(SNPK)。在水稻收获后,0～10crm和10～20cm土层中,无肥处理氨氧化细菌多样性显著低于单施氮磷钾肥的处理。单施氮磷钾肥处理多样性与秸秆翻埋后配施氮磷钾处理没有显著差异。从DGGE条带测序分析得出,秸秆翻埋还田与对照土壤所有优势条带没有区别,均为β-变形菌纲(Betaproteobacteria)的亚硝化螺菌属(Nitrosospira)和亚硝化单胞菌属(Nitrosomonas)。(9)秸秆翻埋还田和施肥对氨氧化古菌群落结构和多样性的影响研究表明；在水稻幼穗分化期和水稻收获后,0～10cm和10～20cm土层中,不施肥处理(CK)氨氧化古菌多样性最低。秸秆翻埋还田配施氮磷钾的处理多样性显著高于单施氮磷钾肥料的处理。对系统进化树研究发现,所测序列相近菌株的来源分成水和土壤两大部分,其中相似菌株有来自高原、湿地、耕作土壤、保护性耕地以及湖水沉积物等。氨氧化古菌的序列结果分析表明,秸秆翻埋的处理与CK氨氧化古菌优势菌群并无明显区别,得到的全部氨氧化古菌均属于非培养泉古菌门(Crenarchaeota)的古菌。综上研究结果表明：秸秆还田和施肥能显著增加水稻土土壤微生物数量、微生物量碳和量氮及土壤脲酶、蔗糖酶和磷酸酶活性；丰富水稻土土壤细菌、真菌、固氮菌、氨氧化细菌和氨氧化古菌的多样性和群落结构,有利于土壤中物质的转化和肥力形成,为水稻土秸秆还田这一保护性耕作措施提供了科学依据。更多还原

【Abstract】 The soil fertility plays important role in the food safety and the sustainable development of agriculture. Fertilization is the key factor of influencing crop yields. Therefore, it is necessary to find out the critical value of fertilization for maintaining the soil fertility and avoiding soil contamination from over use of fertilizers. In order to address and monitor the effect of straw returning and different fertilizer combination on the soil quality, the Soil and Fertilizer Institute of Sichuan Academy of Agricultural Sciences established straw returning and fertilization combination experiments on paddy soil in Xigao town and Lianshan town, Guanghan city, Sichuan province, China, since2002. The impact of the different fertilizer treatments with or without straw returning on soil physiochemical properties and crop yields were studied extensively. Based on the previous works, the pour plate count method was used to study the impact of different fertilizer combination and straw returning on the soil viable microbial counts. Chemical analysis was employed to determine the influence of straw returning and different fertilizer combination on soil microbial biomass carbon and nitrogen and soil enzyme activities. Then, the denaturing gradient gel electrophoresis (DGGE) molecule fingerprint method was applied to study the community structure of bacteria, fungi, nitrogen fixation genes nif H, ammonium oxidizing bacterial and ammonia oxidizing archaea. The results are summarized as the following.(1) The results of different fertilizer combination and straw mulching on soil microbial counts, biomass and soil enzyme activities showed that straw mulching with additional adequate NPK significantly increased the number of soil microbes, SMBC, SMBN, invertases, urease and phosphatase, but decrease the activities of polyphenoloxidase compared with CK (no straw mulching)(P≤0.01) at rice ear initiation stage and after rice harvest; then these indicators in the treatments of no potash fertilizer plus straw mulching(SNP), low nitrogen fertilizer plus straw mulching(SNL) and no phosphate fertilizer plus straw mulching were lower than that introduced straw mulching plus adequate NPK, however, the polyphenol oxidase activity were significantly opposite. (2) The study on soil bacterial community and diversity affected by straw mulching and fertilization indicated that at rice ear initiation stage and after rice harvest, the bacterial diversity index of straw mulching plus adequate NPK treatment was significantly higher than the groups of CK without straw, low N plus straw mulching and no P plus straw mulching (P≤0.01), but there was not significant difference compared with treatment of no K plus straw mulching (P>0.05) at0-10cm soil depth; soil bacterial diversity was higher of straw mulching plus adequate NPK treatment than groups of CK without straw, low N, no P or no K plus straw mulching at10-20cm soil depth (P≤0.01). Sequencing results showed that the dominant bacteria in groups with treatment of straw mulching including Duganella of Betaproteobacteria, Rhodoplanes and Magnetospirillum of Alphaproteobacteria, Chloroflexi, Caldilineaceae, Anaerolineaceae Gemmatimonadetes, Acidobacteria and Nitrospirae. The dominant bands of CK were falling in the class Duganella of Betaproteobacteria, Chloroflexi, Rhodoplanes and Magnetospirillum of Alphaproteobacteria, Firmicutes.(3) The results about the impact of straw mulch and fertilization on the soil fungi community and diversity showed that at rice ear initiation stage straw mulching with addition of adequate NPK treatment had higher diversity index than CK without straw at0-10cm and10-20cm soil depth (P≤0.01). After rice harvest the diversity index of straw mulching with addition of adequate NPK treatment was not significantly different with CK at0-10cm soil depth (P≥0.05). The diversity index of straw mulching with addition of adequate NPK treatment was significantly lower than CK at10-20cm soil depth (P≤0.01). The dominant bands of DGGE patterns were sequenced and the results showed that the dominant species of straw mulching treatment were falling in class of Acremonium, Emericellopsis, Aspergillus, Cladosporium, Rhodotorula, Pleurotus and Mortierella. While the dominant species of CK were Ascomycota, Sordariomyceta. It indicated that the measure of straw returning can increase the diversity and change the community structure of soil fungi. (4) The results about the impact of straw mulch and fertilization on the soil nifH gene diversity showed that at rice ear initiation stage and after rice harvest the straw mulching plus adequate NPK treatment had higher diversity index of nifH gene than the CK both at0-10cm and10-20cm soil depth (P≤0.01). DGGE bands sequencing further revealed that the14closely related species were niJH gene fragment of uncultured bacteria with the dominant community falling in the class of Proteobacteria in Betaproteobacteria. The dominant nitrogen-fixing microbial of straw mulching treatment were uncultured Burkholderia, Pseudacidovorax, Azospira and Ideonella. The dominant species of CK were Pseudacidovorax and Azospira.(5) The results about the impact of straw mulching and fertilization on the soil ammonia-oxidizing bacteria community and diversity showed that at rice ear initiation stage the straw mulching plus adequate NPK treatment had the higher diversity index of ammonia-oxidizing bacteria than the CK, the low N or no P plus straw mulching treatments (P≤0.01), but it had no significant difference with no K plus straw mulching treatment (P≥0.05)at0-10cm soil depth. The straw mulching plus adequate NPK treatment had the higher diversity index of ammonia-oxidizing bacteria than the CK at10-20cm soil depth (P≤0.01). After rice harvest the diversity index of straw mulching with addition of adequate NPK treatment was significantly higher than CK at0-10cm and10-20soil depth. The analysis of sequencing derived from DGGE showed very simple phylogeny of the advantageous ammonia oxidizing bacteria. The dominant ammonia-oxidizing bacteria of straw mulching treatment and CK both were Nitrosospira and Nitrosomonas of beta-Proteobacteria.(6) The results about the impact of straw mulch and fertilization on the soil ammonia-oxidizing archaea community and diversity showed that at rice ear initiation the straw mulching plus adequate NPK treatment had the higher diversity index of ammonia-oxidizing archaea than the CK without straw at0-10cm and10-20soil depth (P≤0.01). After rice harvest the straw mulching plus adequate NPK treatment had the higher diversity index of ammonia-oxidizing archaea than the CK without straw (P≤0.01), but it had no significant difference with no K, no P and low N plus straw mulching treatments. The dominant bands of DGGE patterns were sequenced and the results showed that all the ammonia oxidizing archaea belonged to the uncultured Crenarchaeota.(7) The results of different fertilizer combination and straw burying on soil biomass and soil enzyme activities showed that straw buried into the soil with addition of adequate NPK and only NPK treatments significantly increased the SMBC and SMBN compared with CK(no fertilizer treatment)at rice ear initiation stage and after rice harvest (P≤0.01). Then, the straw burying with addition of adequate NPK treatment significantly increased the activities of invertases, urease, phosphatase and polyphenoloxidase (P≤0.01)(8) The study on soil ammonia-oxidizing bacteria community and diversity affected by straw burying and fertilization indicated that at rice ear initiation stage the CK with no fertilization treatment had the lower diversity index of ammonia-oxidizing bacteria than the NPK fertilization treatment. The diversity index of NPK fertilization treatment was lower than straw burying with addition of adequate NPK treatment at0-10cm and10-20soil depth. After rice harvest diversity index of no fertilization treatment was significantly lower than NPK fertilization treatment at0-10cm and10-20soil depth. The diversity index of NPK fertilization treatment was not significantly different with straw burying with addition of adequate NPK treatment at0-10cm and10-20soil depth. The analysis of sequencing derived from DGGE showed that the dominant ammonia-oxidizing bacteria of straw burying treatment and CK both were Nitrosospira and Nitrosomonas of Betaproteobacteria.(9) The study on soil ammonia-oxidizing archaea community and diversity affected by straw burying and fertilization indicated that at rice ear initiation stage and after rice harvest CK without fertilization had the lowest diversity index of ammonia oxidizing archaea, while NPK with straw burying had higher diversity index than the NPK treatment0-10cm and10-20soil depth. The sequences were divided into two fractions:water and soil by phylogeny tree. Similar strains came from the plateau, wetlands, cultivated soil, protected arable land and lake sediments. The sequence of all dominant bands analysis showed that all the ammonia oxidizing archaea belonged to the uncultured Crenarchaeota.In conclusion, straw returning and fertilization can significantly increase the number of soil microbes, SMBC, SMBN, invertases, urease and phosphatase; enrich the diversity and community structure of bacteria, fungi, nifH gene, ammonia oxidizing bacteria and ammonia oxidizing archaea; further benefit substance transformation and formation of soil fertility. Thus the research provides a scientific basis for straw returning.更多还原