【作者】 李奕林；

【导师】 沈其荣；

【作者基本信息】 南京农业大学 ， 植物营养学， 2007， 博士

【摘要】 越来越多的研究表明，水稻能有效的吸收利用硝态氮（NO₃^--N），水培试验结果表明，与单一的铵（NH₄⁺）营养相比，在增NO₃^-营养下水稻能获得更大的生物产量和经济产量，且氮素利用率（NUE）有所提高。由于淹水条件下水稻硝化作用被强烈抑制，所以以往人们在对水稻氮素营养的研究中，重点放在NH₄⁺-N营养的研究上，但水稻通过贯穿于植株茎部和根部的通气组织将氧气从地上部向根部运输，并将其中一部分氧气释放到根际土壤中，硝化作用在根表和根际立即发生。因此，尽管在淹水条件下，水稻田土体土壤氮素形式以NH₄⁺-N为主，但水稻根际土壤和根表存在着一定数量的NO₃^-，水稻根系实际上是处于NH₄⁺、NO₃^-混合营养（又称增NO₃^-营养）中。那么不同水稻品种根际硝化强度是否存在差异?水稻根际硝化作用对水稻氮素营养的影响如何?施氮肥是否能够影响水稻根际土壤硝化作用?水稻根表、根际和土体土壤硝化强度是否存在差异，究竟那个发生部位对水稻氮素营养影响更大?以及是什么原因引起不同水稻品种间根际土壤硝化强度的差异?揭示这些问题不仅具有重要的理论意义，还具有重要的实际意义。本文选择了两个生长及氮素吸收利用差异较大的籼稻（扬稻6号）和粳稻（农垦57）品种，利用根际培养箱研究其苗期根际土壤硝化作用及其与氮素营养的关系。同时研究不同水稻品种苗期根系通气组织差异是否是引起根际土壤硝化强度差异的原因。通过田间试验研究了以往田间试验筛选出的氮高效品种（4007和武运粳7号）和氮低效品种（Elio）在无肥(0 kg N hm^-2)、中肥(180 kg N hm^-2)和高肥(300 kg N hm^-2)水平下籽粒产量、吸N量、N肥利用率、根际土壤铵态氮（NH₄⁺-N）和硝态氮（NO₃^--N）含量、硝化强度和氨氧化细菌（AOB）数量。阐明施氮对不同水稻品种氮肥利用率及根际硝化作用和硝化微生物的影响。最后在田间条件下研究了氮高效（4007）和氮低效（Elio）品种在NO(0 kgN hm^-2)、N180(180 kgN hm^-2)和N300(300 kgN hm^-2)水平下根表、根际和土体土壤pH值、NH₄⁺-N和NO₃^--N含量、硝化强度和AOB数量。阐明不同氮效率水稻生育后期根表、根际和土体土壤硝化作用和硝化微生物特征。主要结果如下：1．籼稻在苗期鲜重和干重均显著高于粳稻，主要表现为根干重和鲜重的差异，而且籼稻根冠比显著大于粳稻。两个品种水稻氮积累量均随生育期延长而增加，而NUE随时间延长而下降。其中籼稻氮积累量和氮利用率显著高于粳稻。无论籼稻还是粳稻叶片谷氨酰胺合成酶活性（GSA）和硝酸还原酶活性（NRA）均显著高于根的GSA和NRA，籼稻叶片的GSA和NRA均显著高于粳稻，而籼稻根GSA和NRA与粳稻无差异．2．在淹水条件下，土壤矿质态氮主要为NH₄⁺-N，NH₄⁺含量随水稻生育期的推进变化不大，但随着距根表的距离增加其含量随之增加，两个水稻品种之间差异不显著；而NO₃^-的变化趋势与NH₄⁺不一致，NO₃^-含量随水稻生育期的延长而显著下降，在培养58天时其平均含量约为0.05 mg kg^-1，同时在整个土体内呈均匀分布，两个水稻品种之间差异显著．土壤的硝化强度随水稻的生长而增强，且两种水稻的硝化强度均为根际土壤最高，然后依次为土体土壤和根表土壤。扬稻6号和农垦57硝化强度最大值分别出现在距根6 mm和2 mm处，最大值分别为0.88和0.73 mg kg^-1h^-1。土壤AOB数量随水稻生长时间的增加而增加，且其水平变异趋势与土壤的硝化强度一致，根际土壤AOB数量最多，土体土壤次之，根表土壤最少。相关分析结果表明，硝化强度和AOB数量呈显著正相关关系（r=0.86，p<0.01）．种植扬稻6号的土壤NO₃^-浓度、硝化强度以及AOB数量总是高于农垦57．因此，发生在水稻根际的硝化作用对水稻N素营养起到很重要的作用，尤其是对籼稻。3．扬稻6号在水稻播种后44、51和58天根系生物量（鲜重和干重）、根孔隙度（POR）和根际硝化强度均显著高于农垦57，相关性分析结果表明POR和根际土壤硝化强度呈显著正相关关系（r=0.763，p<0.01）。扫描电镜（SEM）结果表明水稻播种后51天，在距根尖0-5mm范围内，通气组织尚未形成，在距根尖10mm处，扬稻6号根横切面中已经能明显观察到有通气组织形成；而农垦57尚未形成明显的通气组织；在距根尖15mm处，扬稻6号和农垦57根横切面中已可以观察到发育完好的通气组织。扬稻6号和农垦57通气组织分别属于径向溶生型和切线溶生型。不同水稻品种POR和根际土壤硝化强度均随水稻生育期延长显著增加。归根结底，具有发达的根系（表现为根系生物量大），且通气组织较发达（表现为POR大），相应的径向泌氧量就大，因此根际AOB数量和活性增强，最终导致根际土壤硝化作用更强。4．不同水稻品种的籽粒产量在3个N处理中差异极显著，4007在中肥处理中获得最高产量11117 kg hm^-2，而Eilo在所有处理中籽粒产量均最低。各品种地上部吸N量随施N量增加而增加，但各品种之间差异不显著．不同水稻品种N肥利用率差异显著，4007显著高于武运粳7号和Elio。本试验根据不同品种水稻在不施N肥水平下的籽粒产量与N肥利用率的大小，将三个品种分为N肥高效敏感型（4007）、N肥高效不敏感型（武运粳7号）和N肥低效不敏感型（Elio）。在水稻中后期干湿交替的水分管理条件下，无肥和中肥区的水稻根际土壤以NO₃^--N为主；而在高肥区则以NH₄⁺-N为主。随着施N量增加，水稻根际土壤铵、硝态N含量也随之增加。NH₄⁺-N含量在无肥、中肥和高肥水平下分别为0.88、0.94 mg kg^-1和13.5 mg kg^-1，而NO₃^--N含量分别为1.61、1.73 mg kg^-1和2.33 mg kg^-1。不同水稻品种根际土壤硝化强度之间差异极显著，在三个施N水平下均表现为4007>武运粳7号>Elio。其平均值分别为6.94、5.46μg kg^-1 h^-1和2.42μg kg^-1 h^-1。在三个施N水平下，Elio根际土壤AOB数量均显著低于4007和武运粳7号。4007根际土壤AOB数量在高肥水平下达最大值2.02×10⁶个g^-1土，而最小值为中肥水平下Elio的根际土壤(1.89×10⁵个g^-1土)。相关性分析表明，水稻根际土壤硝化强度在无肥、中肥和高肥条件下与产量呈极显著正相关关系(r=0.799^**，0.877^**，0.934^**)，而且在中肥条件下与水稻N肥利用率显著相关（r=0.735^*）。水稻根际土壤AOB数量分别和硝化强度以及水稻籽粒产量呈极显著正相关关系。水稻根际的硝化作用较大程度上决定着水稻籽粒产量或水稻N肥利用率，深入开展这方面的研究是一项理论和实践意义均很重要的工作。5．无论是齐穗期、灌浆期还是成熟期，根表土壤pH值均显著低于根际和土体土壤。水稻土壤pH值范围在5.95至6.84之间变化。土壤NH₄⁺-N含量随水稻生长显著下降，且随施氮量增加，水稻根表、根际和土体土壤NH₄⁺-N含量显著增加。根表土壤NH₄⁺-N有明显亏缺区，且随距水稻根距离增加，NH₄⁺-N含量逐渐升高。土壤NO₃^--N含量随水稻生长显著增加，施氮处理均显著高于不施氮处理，但N180和N300处理差异不显著。根际土壤NO₃^--N含量最高，其次为土体土壤，根表土壤NO₃^--N含量最低。水稻根表和根际土壤硝化强度随水稻生长显著下降，而土体土壤随时间延长小幅增加．施氮显著提高4007水稻根表土壤在齐穗和收获期硝化强度以及Elio在齐穗期根际硝化强度，但在施氮处理N180和N300中无显著差异。水稻在整个采样期间，土壤硝化强度均表现为根际>根表>土体。水稻根表和根际AOB数量随水稻生长而显著降低，而土体土壤无显著变化。例如，根表土壤AOB数量在齐穗期、灌浆期和收获期分别为16.7、8.77和8.01×10⁵个g^-1 dry soil。根表和根际土壤AOB数量无显著差异，但二者显著高于土体土壤AOB数量。就两个氮效率水稻品种间差异而言，土壤pH值基本无差异。4007 NH₄⁺-N含量均显著高于Elio。在齐穗期水稻根表、根际和土体土壤NO₃^--N含量在N180水平下均表现为Elio显著高于4007。而在灌浆期和收获期，水稻根表、根际和土体土壤则表现为4007显著高于Elio。在所有采样期，两个水稻品种土体土壤硝化强度和AOB数量在三个施氮量下均无显著差异。Elio根表和根际土壤硝化强度和AOB数量在水稻灌浆期之前一直显著高于4007，而在灌浆期之后则显著低于4007，且最终产量和NUE显著低于4007，这可能是由于4007灌浆期后硝化作用强，根际产生的NO₃^--N含量高，从而4007根吸收NO₃^--N的量也高造成的。因此水稻灌浆期和收获期根表和根际硝化作用以及AOB与水稻高产及氮素高效利用有密切关系。更多还原

【Abstract】 More and more evidence shows that rice plants may take up more nitrate （NO3） than we expected. Hydroponic experiments show that rice growth, yield and nitrogen （N） use efficiency （NUE） can be much improved when the plant is provided with both NO3" and ammonium （NH₄⁺） compared with that fed solely on NH₄⁺. Rice plants have aerenchyma in their shoots and roots that allow oxygen to diffuse down into the rice roots, and partial oxygen is released into the soil, and thus nitrification occurs immediately in niche of the rhizosphere or on the surface of the roots. Therefore, even in a flooded paddy soil, rice roots are actually exposed to a mixed N supply （NH₄⁺+ NO_<sup>-）, although the predominant species of mineral N in bulk soil in paddy rice field is likely to be NH₄⁺. This raised several questions such as whether there is a difference of nitrification activity happened in rhizosphere soil growing with different rice cultivars, or whether there is a relationship between the NUE and the nitrification activity in the rhizosphere of different rice cultivars; what is the effect on nitrification in the root rhizosphere to N nutrition of rice plants in soil culture condition; whether there has an effect of N fertilizer application on nitrification; whether there is difference of nitrification activities happened in different place, such as root surface, rhizosphere and bulk soil, or which place is more important to rice N nutrition; what is the reason to cause the difference of nitrification activities among different rice cultivar. To answer these questions, a rhizobox with three compartments and subsequent soil-slicing after quick freezing was used to measure simultaneously the spatiotemporal variations of mineral nitrogen, nitrification and AOB in the rhizosphere soil of two group of rice cultivars Yangdao 6 （Indica） and Nongken 57 （Japonica）. Rice growth, N accumulation, NUE and root porosity （POR） were also examined to clarify the importance of nitrification in rhizosphere soil to rice N nutrition Three Japonica rice cultivars （4007, Wuyunjing7 and Elio） with different NUE were used to study rice grain yields, total N accumulation and the nitrification characteristics under three N treatments, such as zero N level (0 kg N hm^-2), moderate N level (180 kg N hm^-2) and high N level (300 kg N hm^-2) in field conditions. This is to further examine the effect of N application on fertilizer-NUE, nitrification and nitrifying microorganisms in rhizosphere soil growing with different rice cultivars. Finally, the high NUE cultivar （4007） and low NUE cultivar （Elio） were used to illustrate the relationship between characteristics of nitrification and NUE in field condition. The main results obtained were as followed.1. The fresh and dry weights of Indica were significantly higher than those of Japonica at the whole incubation periods. The variation trends of the root/shoot ratio of dry weight were different between the two rice cultivars and the root/shoot ratio of Indica, for instance, decreased with the development of the incubation time, while the reverse was true for the Japonica. The root/shoot ratio of Indica was significantly higher than Japonica. N accumulations of the Indica and Japonica were significantly increased with the incubation time, while the NUE were significantly decreased with the time passed. The N accumulations and NUE of Indica were significantly higher than those of Japonica during the three sampling dates. GSA and NRA of leaves were always higher than those of roots for the two rice cultivars. The leaf GSA and NRA of Indica were significantly higher than those of Japonica, while there were no significant differences of the root GSA and NRA between Indica and Japonica.2. The main nitrogen form was NH₄⁺-N in flooded paddy soil and NH₄⁺-N concentrations in bulk soil showed almost no changes with incubation time, but NH₄⁺-N concentrations increased with the distance from root surface of both rice cultivars. The NH₄⁺-N concentration of both Yangdao 6 and Nongken 57 in the zone 40 mm from the root at 51 days after sowing, for example, achieved 13.8 and 14.6 mg kg^-1 soil, respectively. However, the NO₃^--N concentration decreased significantly with the development of the incubation time, although the distribution of NO₃^--N was even in the bulk soil. The average NO₃^--N concentration for both cultivars was 0.05 mg kg^-1 soil. When the two varieties were compared, the NH₄^--N concentration was almost the same, while NNO₃^--N concentration was significantly different at every sampling time.The nitrification activities of both rice cultivars increased with incubation time. Maximal nitrification activities were found in rhizosphere soil, followed by those in the bulk soil and in the root surface in every sampling. In the rhizosphere the nitrification activities decreased with increased distance from the root. The maximal nitrification activity measured at 44, 51 and 58 days after sowing of Yangdao 6 and Nongken 57 rice cultivars was at a distance of 6 mm and 2 mm from root surface, respectively. The maximal nitrification activity values measured were 0.88 and 0.73 mg kg^-1 h^-1, respectively. The AOB in the root surface, rhizosphere （0-4 mm away from root surface） and bulk soil （>4 mm away from root surface） for both rice cultivars increased during the growth periods. The AOB in the root surface was always the lowest, while that in the rhizosphere soil was the highest in both cultivars. In these experiments, the nitrification activities measured were significantly proportional to AOB （r=0.86, p<0.01）. The nitrate concentration, nitrification activities and AOB of Indica were always higher than those of Japonica rice. Therefore, nitrification in rhizosphere had more important significance for rice N nutrition, especially for the Indica rice cultivars.3. The root biomass （fresh and dry weights）, POR, and rhizosphere soil nitrification activity of Yangdao 6 were higher than Nongken 57 at 44, 51 and 58 days after sowing. The POR values were significantly and positively correlated with the nitrification activity （r=0.763, p<0.01）. Scanning electron microscope （SEM） results indicated that aerenchyma was not fully developed in the tissues close to the roots apex （0-5mm behind the root tip） of both Yangdao 6 and Nongken 57. However, at the point of 10mm behind the root tip of Yangdao 6, developing aerenchyma was observed but not yet for Nongken 57. At the point of 15 mm behind the root tip, well-developed aerenchyma was found in both rice cultivars. There were two distinct patterns of lysigenous origin of lacunae between two rice cultivars: radial lysigeny （Yangdao 6） and tangential lysigeny （Nongken 57）. The POR and rhizosphere nitrification activity increased with the rice plant development. Therefore, rice cultivar with well-developed root system （having high root biomass） and aerenchyma （having high POR） lead to more root oxygen release to the soil, which resulted in more AOB survived in the rhizosphere soil and consequently led to stronger nitrification activity in the rhizosphere.4. There were significant differences of rice grain yields among the three rice cultivars under different N application rates. The maximal and minimal grain yields were obtained in 4007 in the moderate N level and in Elio in the zero N level, achieving 11117 kg hm^-2 and 5322 kg hm^-2, respectively. There were significant differences of the total N accumulation among the three N treatments, and the total N accumulation increased with the increase of the N fertilizer application rates. Significant differences were found in the fertilizer-NUE （FNUE） and rice grain yields among the three rice cultivars under different N application rates. For example, the FNUE of 4007 was always significantly higher than those of the Wuyunjing 7 and Elio in both moderate and high N level treatments, and the average FNUE in the high N level treatment was 42.2% lower than that of the moderate N level treatment. Based on the FNUE and grain yield at zero N fertilization level, the three rice cultivars could be classified into efficient and responsive （4007）, efficient and nonresponsive （Wuyunjing7） and nonefficient and nonresponsive （Elio） to N fertilizers.Under the water management of alternation of wetting and drying during the middle-late rice growing stages, the main N form in the rice growing rhizosphere soil was NO₃^--N in the zero and moderate N level treatments, while NH₄⁺-N was the main N form in the high N level treatment. The contents of NH₄⁺-N and NO₃^--N in the rhizosphere soil increased with the increase of the N fertilizer applications. For example, the average contents of NH₄⁺-N at the zero, moderate and high N level conditions were 0.88, 0.94 mg kg^-1 and 13.5 mg kg^-1, respectively, while those of NO₃--N were 1.61,1.73 mg kg^-1 and 2.33 mg kg^-1, respectively. The nitrification potential in the rice growing rhizosphere soil represented significant differences among the three rice cultivars, and the average values were 6.94, 5.46μg kg^-1 h^-1 and 2.42μg kg^-1 h^-1 for 4007, Wuyunjing 7 and Elio, respectively, under all the N application levels. The abundance of AOB in the rhizosphere soil of Elio was significantly lower than those of 4007 and Wuyunjing 7 under the different N application rates. The maximal abundance of AOB was 2.02×10⁶ g^-1 soil in the rhizosphere soil of 4007 at high N level, while the minimal one was 1.89×10⁶ g^-1 soil in the rhizosphere soil of Elio at moderate N level. The nitrification potentials in rhizosphere soil were significantly correlated with the rice grain yield at zero, moderate and high N levels (r=0.799^** 0.877^** and 0.934^**, respectively), and also they were significantly correlated with the N physiological efficiency at moderate N level （r=0.735^*）. Furthermore, the abundances of AOB in the rhizosphere soil were correlated with the nitrification potentials and the grain yields. These results inferred that there should be a relationship among rice yields, FNUE and nitrification potential in rhizosphere of rice plants. 5. The pH values in root surface soil were significantly lower than those in rhizosphere and bulk soil in Heading, filling and harvesting stages, ranging from 5.95 to 6.84. NH₄⁺-N concentration decreased but NO₃^--N increased with the time. N application increased NH₄⁺-N and NO₃^--N concentrations. Depletion sections of both NH₄⁺ and NO₃^- were found in root surface soil. The NH₄⁺-N concentration increased with increasing distance from the root surface. The maximal NO₃^- concentration was in rhizosphere soil, then the bulk soil and the lowest was in root surface soil. Nitrification activities in both root surface and rhizosphere soils significantly decreased with the incubation time, but the reverse was true with the bulk soil. N application improved nitrification activities in root surface soil growing with 4007 both at heading and harvesting stages, and also improved nitrification activity in rhizosphere soil growing with Elio at heading stage. But there was no significant difference between N180 and N300 treatments. The nitrification activity showed such order as rhizosphere>root surface>bulk soil in the whole sampling stages. AOB abundance in both root surface and rhizosphere soils significantly decreased with the incubation time, while those in the bulk soil indicated no difference as the time passed. For example, the AOB abundances in root surface soil at heading, filling and harvesting stages were 16.7, 8.77 and 8.01×10⁵ g^-1 dry soil, respectively. There was no significant difference of AOB abundance between root surface soil and rhizosphere soil, but they were all significantly higher than those in the bulk soil. As far as the two rice cultivars were concerned, there was no difference with the soil pH values. The 4007 growing soil NH₄⁺-N concentration was higher than Elio. NO₃^--N concentrations in root surface, rhizosphere and bulk soils for Elio growing treatment under N180 level at heading stage were higher than those for 4007. But NO₃^--N concentrations in root surface, rhizosphere and bulk soils for Elio growing treatment at filling and harvesting stages were significantly lower than those for 4007. Nitrification activities and AOB abundance in bulk had no difference among the three N treatments. Nitrification activity and AOB abundance in root surface and rhizosphere soil for Elio growing treatment were significantly higher than those for 4007 before filling stage, while the reverse was true after filling stage, and the rice yield and NUE for Elio were much lower than 4007. That might be due to the higher nitrification and higher NO₃^--N concentration in thizosphere soil for 4007 than Elio after filling stage, which caused more NO₃^--N absorption by 4007 than Elio. Thereby nitrification and AOB abundance in root surface and rhizosphere soil at filling and harvesting stages were important to a high rice yield and high NUE.更多还原