叶面喷施镁肥对缺镁番茄养分吸收和分配的影响-本科毕业论文

叶面喷施镁肥对缺镁番茄养分吸收和分配的影响-本科毕业论文 学号:2011011772 2015届本科生毕业论文,, 题 目:叶面喷施镁肥对缺镁番茄养分 吸收和分配的影响 学院(系): 资源环境学院 专业年级: 资源环境科学11级 学生姓名: 靳小勇 指导教师: 陈竹君 副教授 完成日期: 2015.6 目 录 前言 ............................................................................................................................................ 1 1 与方法 ............................................................................................................................ 2 1.1 供试材料 ...................................................................................................................... 2 1.2试验处理 ....................................................................................................................... 3 1.3样品的采集 ................................................................................................................... 3 ...................................................................................... 3 1.4 测定 ................................ 1.5 数据处理 ...................................................................................................................... 4 2 结果与讨论 ............................................................................................................................ 4 2.1不同生育期不同处理番茄诊断叶片中养分含量的变化趋势 ................................... 4 2.1.1不同生育期不同处理番茄诊断叶片中钾含量变化趋势 ................................. 4 2.1.2不同生育期不同处理番茄诊断叶片中钙含量变化趋势 ................................. 4 2.1.3不同生育期不同处理番茄诊断叶片中镁含量变化趋势 ................................. 5 2.2喷施镁肥对缺镁番茄养分含量、养分携出量及产量的影响 ................................... 6 2.2.1喷施镁肥对缺镁番茄不同部位养分含量和养分携出量的影响 ..................... 6 2.2.2喷施镁肥对缺镁番茄产量的影响 ..................................................................... 7 3 结 论 ...................................................................................................................................... 8 参 考 文 献 .............................................................................................................................. 9 附录:英文文献翻译 .............................................................................................................. 10 致谢 .......................................................................................................................................... 27 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 作者姓名:靳小勇 指导老师:陈竹君 副教授 (陕西 杨凌 西北农林科技大学 712100) 摘 要:石灰性土壤日光温室栽培番茄普遍出现典型缺镁症状且日益严重，而且土壤施肥改善效果甚微，针对上述问题研究了叶面喷施不同镁肥及其施用量对缺镁番茄K、Ca、Mg吸收和分配的影响，以其为番茄缺镁的矫正提供科学依据。研究结果表明:叶面喷施镁肥可以提高缺镁番茄在开花坐果期、第二茬果膨大期、第三茬果膨大期、红熟期诊断叶片中镁的含量，尤其喷施含镁0.4%的MgSO?7HO效果最佳，其次为喷施含镁 0.2%Mg(NO)?6HO。对叶片K和Ca的含量无影响。42322 对于收获期番茄不同器官养分含量与养分携出量而言，叶面喷施镁肥番茄叶片Mg含量和携出量均显著增加，产量也均提高，但产量差异未达显著水平。从缺镁番茄外部形态看，喷施镁肥对缺镁症状的改善效果不明显，可能与镁的吸收量及转运有关。 关键词:日光温室;番茄缺镁;喷施镁肥;养分吸收;产量 Effect of foliar application of Magnesium on magnesium tomato nutrient uptake and distribution Author:Jin Xiaoyong Instructor:Chen Zhujun (Shanxi Province Yangling NWSUAF 712100) Abstract:Solar greenhouse tomato production in limy soil always show magnesium deficiency symptoms and become more and more serious. It has little effect even with the fetilization.With those previous question,we researched on the absorption and distribution of magnesium after spraying magnesium fertilizer on the leaves of magnesium deficiency tomato in order to provide scientific basis for making correction of magnesium deficiency tomato.The result shows that this way will improve the magnesium content in sample leaves in the period of blossom and fruit, second stubble fruit enlargement stage, third stubble fruit enlargement stage and red ripe stage.MgSO?7HO(0.4%) spraying provides the best effect, 42 Mg(NO)?6HO(0.2%) the second,however they did not affect a lot on the content of K and Ga.To the 322 nutrition content and nutrition uptake in different organs of harvest stage tomato,after sparing magnesium fertilizer, both the content of magnesium and uptake increased significantly, so did the production. But there is no great difference in production of these two groups.From the external conditons of magnesium deficiency tomato,sparing magnesium fertilizer is not very effective to the magnesium deficiency symptom. It may relates to the absorpton and distribution of magnesium. KEY WORDS:Solar Greenhouse; Magnesium deficiency tomato; Spraying magnesium fertilizer; Nutrient absorption;Yield 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 前言 作为我国设施农业的主体产业，日光温室近30年来已成为农业生产中效益最高的产业之一。它为解决长期困扰我国北方地区在冬季的蔬菜供应问题、节约资源、改善农业产业结构、带动相关产业发展、提高农民收入、促进城乡居民的生活水平的发展与社 [1]会和谐等做出了重大贡献。据有关资料，全国设施蔬菜人均占有量:1980~1981年度只有0.2kg;1999~2000年度增加到58kg，增长了290多倍，平均每年增加3.11kg;2001年全国设施蔬菜生产总量达8700多万吨，人均占有量66kg，其中，有近40%是由日光温室提供的，尤其冬季设施蔬菜的95%以上是日光温室生产的。这些蔬菜不仅满足了我国北方地区蔬菜市场，解决了长期困扰我国北方地区的蔬菜淡季供应问题，而且已经有部分出口国外。同时，日光温室还提供了部分花卉、水果及水产品，使城乡的菜篮子更加丰富。 日光温室是我国北方地区蔬菜设施栽培的主要方式之一，近年来的发展十分迅速。 [2]2以陕西省为例，目前栽培面积已达约10. 75万hm。为了提高产量，增加收入，农户通过施用大量有机肥和化肥来为作物提供充足的养分，投入的养分量大大超过作物需求量。然而，调查发现，近年来陕西关中地区的日光温室频繁出现番茄出现植株矮小，生长缓慢，先在叶脉间失绿，而叶脉仍保持绿色;以后失绿部分逐步由淡绿色转变为黄色或白色，还会出现大小不一的褐色或紫红色的斑点或条纹。症状在老叶、特别是在老叶尖先出现;随着缺镁症状的发展，逐渐危及老叶的基部和嫩叶。 是什么原因导致温室番茄的这些症状,镁是植物叶片中叶绿素成分, 缺镁时,植物 [3]叶片失绿。对于网状脉的植物，失绿呈斑点状，严重时整片叶片干枯。但是，土壤测 ,,4定结果显示，缺镁番茄的温室土壤交换性镁含量并未降低，还略有增加。植物对镁 +2+3+2++的吸收不仅取决于土壤中有效镁的含量，阳离子如K、Ca 、NH、Al与Mg的拮4 +[5]抗作用也会引起植物缺镁。生产实践中由 K 诱导缺镁的现象较为普遍。据研究，橡 +胶缺镁的原因一方面是由于土壤镁素养分含量低，另一方面则是由于 K 的拮抗作用。 2+钾对镁的拮抗作用不仅表现在抑制根系对镁的吸收，而且还妨碍Mg由根系向地上部 [6][2]2+运输。据研究，从盐分含量、 离子组成及离子活度变化程度看，盐分累积使 Mg 活 +2+度大幅降低以及 K 富集对植物吸收 Mg 的拮抗作用是石灰性土壤上番茄缺镁的主要诱因。钾、钙、镁元素间的相互关系比较复杂。已有研究表明，钾、钙、镁元素间存在着明显的互作效应。曾有试验表明，钾、钙、镁元素间互为拮抗，其中的任何一种元 [7][8]素含量过多都会减少烟草对另外两种元的吸收。而晋艳等通过水培试验表明，钾、 2++2+钙、镁间表现的拮抗作用并不是相互的。培养液中存在Mg，对烟株吸收K和Ca都 2+2++产生一定的抑制作用，Ca对烟株吸收Mg产生抑制作用，而对K的吸收具有促进作 +2+2+用，培养液中存在大量的K时，对烟株吸收Ca和Mg都起到抑制作用。而谭红等人[7]认为，植物吸收钙、镁量随着土壤施钾量增加到0.4 kg/小区时，白三叶草中钾与钙、 1 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 镁之间产生拮抗作用。而低钾水平时，低量的钙、镁能促进白三叶草对钾的吸收, 而高 [9]量的钙、镁则抑制白三叶草对钾的吸收。而游有文等的研究则认为在钾不足的条件下，施用钙、镁肥会降低玉米营养体中钾含量，抑制玉米对土壤钾的利用;在钾充足的条件下, [10]施用钙、镁都表现正作用。陈际型认为，钾、钙、镁肥一起施用其联合效应大于各个别效应之和，显示钾、钙、镁是一种协同作用。钙、钾对玉米镁的吸收有抑制作用，但钾对镁的抑制作用大于钙对镁的抑制作用。因此，通过土壤施肥来改善温室番茄缺镁很难取得显著效果。叶是植物最重要的根外营养器官，叶面营养就是指植物通过叶片表面 [11]吸收利用各种养分。如何改善温室番茄缺镁症状，提高温室番茄产量与品质的问题值得探究，本研究以陕西杨凌设施栽培基地DD175 号日光温室为对象，通过叶面喷施镁肥，测定番茄不同生育时期、不同部位K、Ca、Mg含量，为日光温室栽培养分管理及可持续发展提供科学依据。 1 材料与方法 1.1 供试材料 试验区位于陕西省杨凌农业高新技术产业示范区，地处陕西关中平原腹地，海拔520 ，年均降水量620 mm左右，主要集中在7~9月，年均蒸发量m左右，年平均气温13? 950~1000 mm，属半湿润易旱区。土壤属褐土类，塿土亚类，红油土属(系统分类为土垫旱耕人为土)。温室大多数自2009年陕西省政府提出建设百万亩设施蔬菜计划之后建立，截止2011年，杨凌已发展设施农业面积1 733.33公顷,其中建成日光温室和塑料大棚分别为800公顷和933.33公顷。主要栽培作物为番茄和黄瓜，一年一熟或一年两熟制。 田间试验在杨凌大寨镇温室栽培基地 DD175 号日光温室进行，该温室建于2009年，温室长57 m，宽7 m，约合0.6亩。2014年8月10日定植番茄，采用宽窄行栽培, 行距分别为90 cm及60 cm，行株数为20株，株距为33cm。品种为从荷兰引进的“铁观音”，栽培作物期间地面全覆盖聚乙烯薄膜。土壤耕层养分含量为有机质22.1g/kg，全氮 番茄基肥仅施用1.47g/kg，硝态氮77.4mg/kg，有效磷190.0mg/kg，速效钾302.7mg/k。拉多美复合肥(N-PO-KO含量为20-10-8)30kg，折合N、PO、KO含量分别为252252 -2-2-2150kg?hm、75kg?hm和60kg?hm。供试温室土壤理化性质见表1。 表1 供试温室种植前土壤理化性质 有机质硝态氮铵态氮有效磷速效钾棚号 (g/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) DD175 22.1 77.4 12.66 190.0 302.7 2 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 1.2试验处理 试验共设五个处理，即喷清水处理，喷施2.05% MgSO?7HO处理，喷施4.10% 42 MgSO?7HO处理，喷施2.13% Mg(NO)?6HO N1处理，喷施4.26% Mg(NO)2HO N24232232处理。各处理分别简写为:ck、 S1、 S2、 N1、 N2。每处理重复3次，采用完全随 2机区组排列，共15个小区，小区面积为3.0×7 m。每小区栽植四行,每行栽植20株，宽窄行栽培， 行距分别为90 cm及60 cm。番茄株距为33 cm，每小区共计80株。处理见表2。 表2试验处理方案(按小区喷) 处理 肥料 Mg浓度 施肥时期 清水 CK 0 分别在开花期、第一茬果膨大S1 0.2% MgSO•7HO 期、第二茬果膨大期、第三茬42S2 0.4% 果膨大期和红熟期进行叶面N1 0.2% 喷施镁肥，共计喷施5次。 Mg(NO)•6HO 322N2 0.4% 1.3样品的采集 土壤样品采集:在换茬休闲期采试验地土壤表层样品。 诊断叶片的采集:分别在番茄开花坐果期、第二茬果膨大期、第三茬果膨大期、红熟期采取诊断叶片，每个生育期每个小区采集5个样，每个处理采集一个样，即每个生育期采集15个样品，共计60个样品。 收获期番茄不同器官样品的采集:在盛果期采番茄果实样品;在番茄收获期每采集四株植株样品，并按根、茎、叶分开。每种器官每个小区采集5个样，每个处理采集一个样，即种器官采集15个样品，共计60个样品。 1.4 分析测定 土壤样品采集后，取鲜样测定土壤水分、硝态氮、铵态氮，剩余土壤样品风干，研磨，过筛，保存，以测定有机质、有效磷、速效钾、交换性钾。植物样品采集后烘干，粉碎，保存，以备测定全钾、全钙、全镁。有机质用重铬酸钾-外加热法测定;硝态氮、铵态氮以1mol/L氯化钾溶液浸提，流动分析仪测定;有效磷用Olsen法测定;速效钾用1mol/L醋酸铵浸提，火焰光度法测定。植物样品经干灰化后，用火焰光度计测钾，原子 [12]吸收光谱仪测钙、镁。在番茄收获期时分小区计产，最后统计总产量，在收获后每小区随机采5株番茄植株，并将根、茎、叶分开，分别测定根、茎、叶、的生物量。 3 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 1.5 数据处理 采用Excel 2010和SAS V8对试验数据进行统计分析。 2 结果与讨论 2.1不同生育期不同处理番茄诊断叶片中养分含量的变化趋势 2.1.1不同生育期不同处理番茄诊断叶片中钾含量变化趋势 [17]据研究叶面上的养分，首先以扩散方式通过蜡质层和角质层，然后进入叶肉细胞被吸收利用。随着番茄的生长，叶片以及根部等吸收的养分不断地传输给果实。从图1可以看出，在开花坐果期5个处理K含量基本一致，从开花坐果期到第三茬果膨大期，喷施MgSO?7HO(2.05%) S1、MgSO?7HO(4.10%)S2、Mg(NO)? 6HO(2.13%)N1、4242322 Mg(NO)?6HO(4.26%)N2四个处理K含量平稳下降(以下称CK、S1、S2、N1、N2)，322 CK在开花坐果期到第二茬果膨大期K含量下降缓慢，之后下降较快。处理N1在第三茬果膨大期到红熟期K含量基本无变化。总体来看，5个处理番茄诊断叶片K的含量随着生育期变化大体都呈下降趋势。不同处理对缺镁番茄对K的吸收与利用影响不大。 图1番茄诊断叶片不同生育期钾含量 叶面喷施镁肥对番茄在开花坐果期、第二茬果膨大期、第三茬果膨大期、红熟期K的含量影响不明显，每一个生育期的处理之间都不显著。 S1、S2、 N1、 N2与空白对照喷水处理CK没有明显差异。即叶面喷施镁肥对缺镁番茄对K的吸收没有显著影响。 2.1.2不同生育期不同处理番茄诊断叶片中钙含量变化趋势 从图2可以看出，5个处理番茄诊断叶片中Ca的含量变化趋势基本一致，即开花坐果期到第二茬果膨大期下降，第二茬果膨大期到红熟期缓慢上升。钙是构成灰分的主要成 [18]++份之一 。钙与钾之间的关系比较复杂, 一般认为, K与Ca两个阳离子在吸收上表现 4 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 [19]+为拮抗作用。在钾不存在时会有利于钙的吸收。在有钾的情况下, 当K进入细胞多时, 2+[20]2++阻碍了Ca的吸收 。而当供试土壤含有丰富的钙时, 作物吸收了大量的Ca, 对K吸 [21]收有拮抗作用 。 图2番茄诊断叶片不同生育期钙含量 对Ca而言，叶面喷施镁肥对缺镁番茄不同生育期对Ca的吸收也基本无影响，只有在第二茬果膨大期时S1、S2、N1、N2与CK比较 Ca的含量均有所下降，S1不显著，S2、N1、N2显著。推测浓度越大的镁肥对缺镁番茄Ca的吸收抑制越明显。 2.1.3不同生育期不同处理番茄诊断叶片中镁含量变化趋势 由图3可以看出，在对番茄进行5种不同处理的过程中，番茄中镁含量呈现总体类似的变化趋势。从开花坐果期到第二茬果膨大期，番茄中诊断叶片镁的含量全部为下降 [22]曲线，植物在这一阶段为快速生长时期，需要大量合成必需物质以及为植物本身的果实生殖提供各种所需的含镁的营养物质储备，这些含镁营养物质从叶片中陆续转移到果实中，造成诊断叶片中镁含量的大量下降，在这一时期的造成镁含量差异主要由于于外界人为的镁元素补给量。在第二茬果膨大期和第三茬果膨大期，植物本身镁的消耗量与自身的获取量趋向于一种平衡，镁含量趋向于一种平衡，这时人为的外界镁元素的补给对叶片中的镁含量影响不大，CK、S2、N1、N2明显印证了这种说法，但是在S1的处理情况下，诊断叶片中镁的含量持续下降，说明这段时期叶片依旧向果实中输送含镁的营养物质，证明在该浓度水平的镁元素的人为补给情况下，有利于镁含量在果实中的积累。在第三茬果膨大期到红熟期，诊断叶片中的镁再次下降，这是果实成熟的最终时期，需要合成相关含镁元素的物质，造成诊断叶片中的镁含量再次下降。 [13]据吴洵报道，由于离子间的拮抗作用, 茶树对镁的吸收常常受到钙的极大影响。 2+2+植物含镁量下降, 即Ca在植株体内的过量积累, 抑制了对Mg的吸收, 从而降低了植 [5][14] 2+2+物镁含量 。试验表明, 随着培养液中Ca含量的增加, 对Mg的吸收起着明显的 5 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 [15]抑制作用。试验表明, 在营养液培养下, 钾对大豆镁吸收有抑制作用。一些国外的研 +究资料表明, 钾肥抑制了各种作物对镁的吸收。有人观察到高含量K会导致苹果叶中 2++2+[16]Mg不足, 当营养液中无K时, 大豆对Mg的吸收特别快 。 图3显示，叶面喷施镁肥对缺镁番茄不同生育期对Mg的吸收有很大的影响，每一个生育期S1、S2、N1、N2与CK相比叶片镁含量均有所增加。 图3番茄诊断叶片不同生育期镁含量 综上，叶面喷施镁肥可以提高缺镁番茄诊断叶片中镁的含量，其中S2效果最明显，其次是N2。然而对Ca和K的吸收无明显影响。5各处理K都是随着生育期的变化呈现下降趋势，Ca则是先下降，之后略有回升。 2.2喷施镁肥对缺镁番茄养分含量、养分携出量及产量的影响 2.2.1喷施镁肥对缺镁番茄不同部位养分含量和养分携出量的影响 由表3可以看出，番茄吸收K、Ca、Mg养分以果实和叶片为主。对于养分含量，不同处理缺镁番茄根中K、Ca、Mg的含量有差异，但差异不显著;茎中Ca和Mg含量差异不显著，而K的含量差异显著，S2处理K 含量最高，N2处理K含量最低;叶片中K的含量差异不显著，Ca和Mg的含量差异显著，尤其Mg在叶片中的含量差异显著明显，S1、S2、N1、N2均高于CK，其中S2含量最高;果实中K、Ca、Mg的含量有差异，但差异不显著，但5个处理中果实K、Ca、Mg含量S2最高。 对于养分携出量，K、Ca、Mg在根中分别占2.28%、2.51%和1.51%;在茎中分别占12.90%、15.00%、14.25%;在叶中分别占29.19%、76.48%、72.27%;在果实中分别占55.64%、6.01%、11.97%。根和茎中K、Ca、Mg含量差别不大，叶片中Ca和Mg的含量远远大于K的含量，果实中K的含量远远大于Ca和Mg的含量。不同处理番茄根、茎、叶、果实干物质有所差异，其中根、叶和果实差异显著，茎差异不显著。不同处理番茄根、茎、叶、果实养分含量有所差异，叶片中差异显著，而根、茎、叶差异不 6 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 显著。不同处理缺镁番茄根中K、Mg的携出量有差异，且差异显著，Ca携出量差异不显著;茎中K和Ca含量差异显著，而Mg的含量差异不显著，但都是S2携出量最高;叶片中Ca的含量差异不显著，K和Mg的含量差异显著，S1、S2、N1、N2均高于CK，其中S2处理Mg的携出量最高;果实中K、Ca、Mg的含量有差异，且差异显著，同时5个处理中果实K、Ca、Mg携出量S2最高。 表 3不同处理番茄干物质量与养分携出量 2养分含量养分携出量 干物质g/kg kg/hm植株部位处理 kg/hm2 K Ca Mg K Ca Mg CK 71.85 b 26.22 a 31.48 a 3.72 a 1.88 d 2.26 a 0.27 b S1 93.45 a 27.96 a 29.1 a 4.13 a 2.61 a 2.72 a 0.39 a 根 S2 78.55 ab 29.13 a 25.23 a 3.39 a 2.29 bc 1.98 a 0.27 b N1 87.74 ab 27.3 a 26.75 a 3.32 a 2.4 ab 2.35 a 0.29 ab N2 81.31 ab 25.72 a 27.78 a 3.65 a 2.09 cd 2.26 a 0.3 ab CK 368.11 a 32.82 ab 32.03 a 7.41 a 12.08 bc 11.79 b 2.73 a S1 416.47 a 32.65 ab 35.17 a 7.1 a 13.6 a 14.65 a 2.96 a 茎 S2 405.17 a 33.47 a 35.01 a 7.53 a 13.56 a 14.18 a 3.05 a N1 410.12 a 32.73 ab 34.89 a 7.13 a 13.42 ab 14.31 a 2.92 a N2 379.4 a 29.26 b 37.7 a 7.17 a 11.1 c 14.3 a 2.72 a CK 992.31 ab 27.03 a 68.88 a 10.21 c 26.82 bc 68.35 a 10.13 d S1 1239.68 a 26.85 a 59.58 b 13.07 b 33.29 a 73.85 a 16.2 ab 叶 S2 1136.41 ab 26.07 a 63.49 ab 15.87 a 29.62 ab 72.15 a 18.03 a N1 1056.55 ab 27.79 a 69.08 a 14.12 b 29.36 b 72.98 a 14.92 bc N2 972.19 b 25.95 a 67.59 a 14.02 b 25.22 c 65.71 a 13.63 c CK 1367.4 ab 39.29 a 3.95 a 1.62 a 53.72 bc 5.41 ab 2.21 b S1 1477.07 ab 38.1 a 3.86 a 1.64 a 56.28 ab 5.7 ab 2.43 ab 果实 S2 1489.59 a 39.41 a 4.09 a 1.79 a 58.71 a 6.1 a 2.67 a N1 1440.55 ab 38.76 a 3.97 a 1.72 a 55.84 ab 5.71 ab 2.48 ab N2 1286.05 b 39.29 a 3.76 a 1.78 a 50.53 c 4.84 b 2.29 ab CK 2799.67 94.5 87.81 15.34 S1 3226.68 105.78 96.92 21.98 总量 S2 3109.73 104.18 94.41 24.02 N1 2994.96 101.02 95.35 20.61 N2 2718.94 88.94 87.11 18.94 2.2.2喷施镁肥对缺镁番茄产量的影响 由图4可以看出S1、S2、N1处理的番茄产量高于CK，S2产量最高，N2产量低于CK。处理间CK、S1、N1差异不显著，S2、N2与CK、S1、N1都差异显著，S2与N2 7 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 差异显著。由此可以看出叶面喷施镁肥在一定程度上可以提高日光温室缺镁番茄的产量。且S2效果最佳。对于N2处理，可能由于Mg(NO)?6HO浓度过高，对番茄植株322 造成伤害，使产量下降。 图4不同处理番茄产量 3 结 论 从缺镁番茄外部形态来看，喷施镁肥对缺镁番茄叶片黄化症状改善不明显;但是，养分含量与养分携出量在处理间有所差异，尤其是Mg在叶片中的含量与携出量均差异显著，5个处理中CK最低，S2最高;再从番茄产量来看，S1、S2、N1的产量都比CK高，其中S2最高。 综合来看，相对于土壤施肥，通过叶面喷施镁肥在一定程度上更能够改善北方石灰性土壤日光温室番茄缺镁症状。同一浓度MgSO?7HO效果比Mg(NO)?6HO效果显著。42322但是更适合石灰性土壤日光温室缺镁番茄镁肥的浓度还有待探究。 8 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 参 考 文 献 [1]李天来.论我国设施蔬菜产业可持续发展中应注意的几个问题[J].沈阳农业大学学报,2000,31 (1):9~14. [2]白新禄, 高佳佳, 雷金繁, 陈竹君, 周建斌. 2013. 杨凌新建日光温室番茄施肥现状调查及分析.西 北农业学报, 22(2): 148~151 [3]中国农业科学院土壤肥料研究所主编.中国肥料.上海:上海科学技术出版社.1994 [4]陈竹君，王益权，周建斌，等(日光温室栽培对土壤养分累积及交换性养分含量和比例的影响(水 土保持学报，2007，21(1):5~8( [5]许能醌 ,肖召民 ,梁福露 ,等 . 从粤西植胶区三种土壤看矿质肥料对胶苗组分、缺素症状和生长的 影响[ J].热带作物学报 , 1985, 7( 2): 47~55. [6]OHNO T, Grunes D L. Potassium-M agnesium interactions affecting nutrient uptake by w heat forage[ J ].Soil Sci Soc Amer J, 1985, 49( 3) : 685~690. [7]谭红, 何锦林.钾、钙、镁营养水平对白三叶养分吸收的影响[ J] .四川草原, 1994(2):15~16,22. [8]晋艳, 雷永和.烟草中钾钙镁相互关系研究初报[ J] .云南农业科技, 1999(3):6~9,47. [9]游有文, 黄鸿翔, 王伯仁, 等.湘南地区几种土壤钾钙镁施用效果研究[ J] .湖南农业科学, 1999(1):39~41 [10]陈际型, 宣家祥.低盐基土壤K、Ca、Mg的交互作用对水稻生长与养分吸收的影响[J] .土壤学报, 1999, 36(4):433~439. [11]Peuke A D, Jeschke W D, Dietz K J, Schreiber L, Hartung W. Foliar application of nitrate or ammonium as sole nitrogen supply in Ricinus communis?. Carbon and nitrogen uptake and inflows. New Phytologist, 1998, 138: 675~687. [12]鲍士旦. 2000. 土壤农化分析. 北京:中国农业出版社 [13]吴洵.茶树的钙镁营养及土壤调控[ J] .茶叶科学,1994, 14(2):115 ~121. [14]周卫, 林葆.植物钙素营养机理研究进展[ J] .土壤学进展, 1995, 23(2):12 ~17. [15]汪洪, 褚天铎.缺镁对菜豆幼苗膜脂过氧化及体内活性氧清除酶系统的影响[ J] .植物营养与肥料 学报, 1998,4(4):368 ~392. [16]邵岩.镁在烟草生产中的作用[ J] .云南农业大学学报,1992, 7(2):105 ~109. [17]Franke W. Mechanism of foliar penetration of solutions. Annual Review of Plant Physiology, 1967, 18: 281~300. [18]訾大镇, 郭月清.烟草栽培[ M] .北京:中国农业出版社, 1996:113. [19]胡国松, 陈江华, 曹志洪, 等.田间状况下烤烟养分吸收动力学及其在平衡施肥中的应用[ J] .中国 烟草学报,1996, 3(2):14 ~21. [20]鲁如坤.土壤-植物营养学原理和施肥[ M] .北京:化学工业出版社, 1998:228 ~230. [21]赵鹏, 谭金芳, 介晓磊, 等.施钾条件下烟草钾与钙镁相互关系的研究[ J] .中国烟草学报, 2000, 6(1):23 ~25 [22]汪洪, 褚天铎.植物镁素营养的研究进展[ J] .植物学通报, 1999, 16(3):245 ~250 9 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 附录:英文文献翻译 Release of carboxylic anions and protons by tomato roots in response to ammonium nitrate ratio and pH in nutrient solution 1121Patricia Imas, B. Bar-Yosef , U. Kafkafi and Ruth Ganmore-Neumann 1 Agricultural Research Organization, Institute of Soils and Water, Bet-Dagan 50250, Israel and 2Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel Abstract:The exudation of certain organic anions and protons by roots which may affect solubility of metals and P and uptake by plants, is affected by nitrogen form and pH. The objective of this work was to study exudation of carboxylates and H +/OH by tomato plants in response to NH4/NO3 ratio and pH in nutrient solution. Four NH4/(NH4+NO3) ratios (R= 0, 0.33, 0.67 and 1) and constant vs. variable solution pH treatments were investigated. The sum of the exudation rates of all carboxylates tended to decline with increasing R, particularly tri- and dicarboxylates. The molar fraction of the exuded tri- and dicarboxylates, averaged over all treatments and plant ages, increased in the order tartarate ( 2%), malate ( 6%), succinate ( 15%), citrate ( 26%) and fumarate ( 46%). At R=1 the solution pH dropped from 5.2 to 3 and at R=0 increased to 8. The R corresponding to the pH stat of tomato plant was 0.3. For the constant solution pH treatment, the effect of solution pH on carboxylate exudation rate was small as compared to the effect of R. The exudation of citrate and H + efflux which were initiated when NO3 and NH4 uptake rates per plant exceeded certain threshold values, increased with plant age. +4Key words: carboxylic anions, NH /NO , pH, roots, tomato 3 10 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 Introduction Carboxylic anions serve to balance charge and regulate pH within plants in response to uneven cation and anion uptake and to nitrate reduction in leaves (Gerendas and Sattelmacher, 1990; Kraffczyk et al., 1984; Mengel and Kirkby, 1987; Touraine et al., 1990; Troelstra et al., 1990). In the shoots of tomato the predominant carboxylates are malate and citrate (Kirkby and Mengel, 1967); in the roots, malate, citrate and succinate (Miller et al., 1990); and in the xylem exudates, citrate, malate and malonate (Senden et al., 1992). While NO3-N nutrition stimulates malic acid synthesis in tomato shoots and roots, NH4-N nutrition enhances the rate of citrate production (Kirkby and Mengel, 1967). The total carboxylate contents in tomato plants was fivefold greater for NO3 than for NH4 nutrition (Kirkby and Mengel, 1967). High K concentration in nutrient solutions enhanced organic acid content in tomato roots (Miller et al., 1990), presumably in response to increased K+ uptake and the cation-anion imbalance. In one of the few experiments reporting quantities of organic acids exuded by tomato roots, Mozafar et al. (1992) found mainly fumaric and citric acid, followed by oxalic, malic and succinic acids. No data were found in the literature on the effect of N nutrition on the rate and composition of carboxylate exudation in tomato. One can assume that since N form and concentration in solution affect carboxylate contents and compositions in plants, carboxylate exudation will also be affected by these factors. The influence of N nutrition on organic anion exudation is of considerable importance, as citrate and oxalate can increase P availability to plants growing in P-sorbing substrates (Bar-Yosef, 1996). The objective of this study was to relate the release of organic ions and H + by tomato plants to the NH4/(NO3+NH4) ratio and pH in the culture solution. Knowledge of composition and exudation rate of carboxylates will clarify previous reports on N nutrition-P availability relationships (Cole et al., 1963) and enable us to alleviate P deficiency by adjusting ammonium to nitrate ratios in soil and irrigation solutions. Materials and methods Experimental Tomato (Lycopercicon esculentum L. cv. Daniela) seedlings, germinated in vermiculite, were transferred 15 d after germination to 8-L containers filled with well aerated half-strength Hoagland solution (Hoagland and Arnon, 1938). Eight days later, when roots had started to entangle, single plants were transferred to 1-L conical flasks containing the same solution. These solutions were well aerated and replaced every second day until the beginning of each experimental period when the uniform solutions were replaced with treatment solutions. Then, after a 24-h adjustment period, the plants were transferred to 100-mL flasks containing fresh treatment solutions. Two antibiotics, rifampicin and tetracycline at concentrations of 50 and 25 mg L 1, respectively, were added to the flasks in order to prevent bacterial interference (Schwab et al., 1983). Periodically, microbial contamination was tested by counting colonies after incubation of solution samples on nutrient agar plates. The results showed no bacterial contamination of the solutions 11 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 treated with antibiotics. After 6-12 h of uptake the plants were taken for chemical analysis and the remaining solutions were measured for volume and analyzed for organic anions, pH and nutrient concentrations. The base solution of all treatments contained a total N concentration of 7.5 m M; K, P, Ca and Mg concentrations were 3, 0.5, 2.5 and 1.0 m M, respectively, and Fe, Zn, Mn, Cu, Mo and B concentrations were 2.5, 0.03, 0.4, 0.025, 0.018 and 0.25 mg L 1 , respectively. Treatment solutions were titrated with HCl and NaOH to determine the buffer capacity of the systems to variations in pH. The plants grew throughout the experiment in a growth chamber under 450 E m 2s 1 light intensity and 13/11-h day/night photoperiod. Experiment I: NH4/(NO3+NH4) ratio effects Treatments included four NH4/(NO3+NH4) molar ratios (R) (0, 0.33, 0.67 and 1), each in the presence and absence of antibiotics. The experiment was performed at three plant ages: 30, 37 and 44 days (R=1 minus antibiotics treatment was not tested at d 44). Flask solutions were collected in the three experiments after11, 6.7 and 6 h of uptake, respectively. The growthchamber temperature was kept at 26/22 C day/night. Experiment II: solution pH effects Four treatments were studied: two NH4/(NO3+NH4) ratios (1 and 0), each under two pH regimes. In the first pH regime the solution pH was not corrected during the uptake period; in the second one the solution pH was maintained at 5.0 by adding 0.1 M HCl or NaOH every 30 minutes during the test period. The plant age was 33 days, and the uptake period was 6 h.The growth chamber temperature was kept at 27/25 C day/night.The experimental design of Experiments I and II was a randomized complete block with 3 or 4 replicates. Upon sampling, plant roots were rinsed with water, and the fresh and dry weights of the shoots and roots were determined. Nitrate and NH4 concentrations at the beginning and end of the uptake period were determined by steam distillation (Bremner and Keeney, 1965) Carboxylate determination in test solutions A subsample of the test solution was filtered through Whatman no. 41 paper, and adjusted to pH 8.0 with 0.1 M NaOH. Bond elute (Varian) strong anion exchange (SAX) cartridges (quaternary amines on silica) containing 100 mg sorbent mass per 1.0 mL column were used for solid-phase extraction (SPE) of the organic anions from solution. The cartridge was conditioned by rinsing it with two 1-mL quantities of methanol (absolute HPLC) followed by two 1-mL quantities of bidistilled H2O. A known sample volume was then vacuum extracted through a 0.22- m Millipore celluloseacetate filter into the cartridge via a VacElut-20 manifold. The cartridge was then washed with two 1-mL quantities of bidistilled water. Elution of the carboxylates from the cartridge was performed by two 0.5-mL doses of 0.5 M H2SO4. The HPLC apparatus used a Perkin-Elmer 410LC pump and a 300 7.8 mm ionexclusion column (Bio-Rad). The column contained Aminex HX-87H resin, a strong cation exchange agent which separates organic anions by ion exclusion and partition chromatography. An ion-exclusion microguard pre-column (Bio-Rad) was connected to the analytical column to remove contaminants from the sample. A 100 L sample was injected into the HPLC instrument. The column elution was done with 0.005 M H2SO4 as mobile phase, at a flow rate of 0.4 mL min 1 at room temperature. Peaks were detected with a UV detector (Perk in-Elmer diode-array detector LC-235C) at 210 nm wave-length and identified by comparing retention times of the unknowns with those of standard organic anion mixture (Bio-Rad organic acid analysis standard 125-0586). 12 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 A fumarate standard was prepared from fumaric acid (Sigma). Peak areas and retention times were calculated with a V4 Turbochrom processor (PE Nelson Systems). In a preliminary experiment the recovery of organic anions subjected to the new technique of solid-phase extraction (SPE) was determined. The recovery percentages were: citrate 85%; tartarate 100%; malate 91%; succinate 99%; formate 100% and acetate 100%. The SPE pretreatment caused the oxalate peak in the HPLC to coincide with the large salt peak and, therefore, oxalate could not be detected in this study. The chromatograms (not presented) demonstrate that the SPE technique caused no shifts in ion-retention time, that good separation of organic anions was obtained and that, under characteristic conditions, adequate peak areas were obtained, allowing accurate determination of carboxylate concentration in test solutions. Statistical analysis Analysis of variance and fitting of model parameters were carried out by means of the GLM (General Linear Model) and NLIN (Non-Linear Model) procedures of SAS (SAS, 1985). Results Experiment I Root and shoot weights and N uptake characteristics Shoot and root weights increased exponentially with time during the experimental period (Figure 1). The exposure time of plants to the treatment solutions ( 30h) was too short to cause significant differences in plant weight. Treatments had no significant effect on dry matter as a percentage of fresh weight, and the dry matter percentages of shoots and roots averaged over all treatments and all times were 7.1 0.7 and 4.2 0.6, respectively. Nitrate and NH4 uptake rates per unit root weight declined as plant age increased from 30 to 37 and to 44 days (Figure 2). For each age, uptake rates could be significantly expressed by the simple Michaelis-Menten equation (Figure 2). The high Km values found for both Figure 1. Shoot and root weights (SW, RW) as a function of plant age(t). Broken and solid lines correspond, respectively, to the eqs. RW= 0.127 e 0 : 121t (R2=0.91); and SW = 0.570 e 0 : 115t (R2=0.95).All coefficients were significant at the p=0.001 level. Differencesin SW and RW between treatments at given plant ages were notsignificant ( p=0.05). Circles, triangles, squares and diamonds standfor mean NH4/(NH4+NO3 ) ratios of 1, 0.67, 0.33 and 0, respectively.Encircled points 13 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 belong to Experiment II. NO3 and NH4 suggest a two-component kinetics, but a dual uptake model (Epstein, 1972) could not be fitted to the experimental results. The encircled points in Figure 2b were excluded from the Michaelis-Menten model as they were probably subject to an alytical error. NH4/NO3 ratio effects on root exudation Organic anions. The sum of the studied di- and tricarboxylic anion exudation rates, averaged across treatments, significantly increased as plant age advanced from 30 to 37 days, but then decreased as plant age increased further to 44 days (Table 1). For each age, the sum was maximal when nitrate was the sole N source in solution (R=0, time-averaged value of 1.1 mol plant1 6 h 1). Monocarboxylic anions (formate and acetate) were detected in the solutions, and concentrations were maximal at R=0.67 (Table 1). The monocarboxylate rates were not included in the sum because formate and acetate have not been reported to exist in tomato plant tissue or root exudates, and they might have degraded from other aliphatic acids (Fox and Comerford, 1990). The carbon in all the exuded organic anions (Table 1) constituted less than 0.2% of the total C in dry matter which was added to the plant during the same period. The mean molar fraction of the exuded di- and tricarboxylates, averaged across the NH4/NO3 treatments and ages, increased in the order tartarate ( 2%), malate ( 6%), succinate ( 15%), citrate ( 26%) and fumarate ( 46%) (Table1). Figure 2. Uptake rate per unit fresh root weight (F) as a function of time-averaged concentration in solution (C) and plant age (DAP). Curves correspond to the best fitted Michaelis-Menten equation. a. Nitrate. Values of FmaxNO3 for 30-, 37- and 44-day old plants are 31.0(***), 11.6(***) and 6.0(***) mol (g fw root) 1 6 h 1 , of KmNO3 0.70(**), 0.56(***) and 0.56(*) m M, and of model R2 0.92, 0.97 and 0.97, respectively. b. Ammonium. Values of FmaxNH4 are 84(***), 70(n.s.) and 8.9(***) mol (g fw root) 1 6 h 1 , of KmNH4 2.13(*), 7.3(n.s.), and 1.09(n.s.) m M, and of model R2 0.95, 0.95 and 0.92, respectively. ***, ** and * stand for significance at p=0.001, 0.01 and 0.05, respectively; n.s.=not significant at p=0.05. Bounded points were excluded from the calculation. The effect of antibiotics on the total carboxylate exudation rate and on the proportion of the individual 14 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 exudates was non-significant (data not presented). Only the malate and succinate fractions tended to be reduced in the presence, compared with the absence of antibiotics. The opposite trend was observed in fumarate. The exudation rates of citrate and of tartarate+malate+succinate+fumarate declined exponentially with increasing R in the nutrient solution (Figures3a, b). A plant age of 30 days was chosen in Figure3, as root activity at this stage was maximal (Figure2), and shoot activity was increasing. Calculations for 37- and 44-day old plants were performed according to data in Table 1 and Figure 1, and the trends obtained were similar (results not presented). Table 1. Sum of dicarboxylic (tartarate, malate, succinate, fumarate), tricarboxylic (citrate) and monocarboxylic (formate, acetate) anionexudation rates as a function of NH4/(NH4+NO3 ) ratio in solution (R) and plant age (DAP). Data are mean of the data obtained with andwithout antibiotics. Individual di- and tricarboxylates are expressed as molar fractions of the sum To determine whether the observed increase in pH stemmed from exudation of the organic anions, as proposed by Landsberg (1981), Na-citrate corresponding to the maximal amount of citrate exuded by the plant in Experiment I was added to 20 mL of the initial test solution, which represented the solution volume left at the end of the uptake period. The pH obtained was 6, which indicates that H + influx into the roots increased in response to the NO3 uptake, thus resulting in an alkalinization of the solution up to the experimentally observed pH value of 8 (Figure 4). At a R value of 1 (100% NH4), the decrease in solution pH (Figure 4) stemmed primarily from H + efflux in response to NH4 uptake by the roots - since addition H3citrate in a quantity corresponding to the amount exuded by plant had no significant effect on solution of pH. Plants decreased the solution pH in the presence of NH4 more strongly at the age of 30 days than at ages of 37 or 44 days. In the sole NO3 treatment the variation in pH was unaffected by plant age. Antibiotics had no significant effect on solution pH, except at R=0.33, where the addition resulted in a pH 15 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 drop from 5.2 to 4.5 (data not presented). On the basis of the mean of the data obtained with and without antibiotics, the pH stat R value was 0.29-0.30, which agrees well with the pH stat value reported for tomato by Feigin et al. (1980). Experiment II Effect of constant vs. variable solution pH on citrate exudation When the fraction of NO3 in the nutrient solution was raised (Experiment I), the solution pH increased as well (Figure 4). To distinguish between the effect of the nitrate ratio and solution pH on carboxylate exudation rates, solutions with 100% NO3 and 100% NH4 were either maintained at a constant pH of 5 or allowed to vary as in Experiment I. In the variable pH treatments, after 20 minutes of placing the plants in the test solutions, the pH of the 100% NH4 treatment decreased to 4.1 and the pH of the 100% NO3 treatment increased to 7.0. One hour later, the pH values were 3.6 and 7.1, and at the end of the 6 h-experiment, the pHs were 3.3 and 7.8 for the NH4 and the NO3 solutions respectively. When 100% NO3 solution was maintained at pH 5, carboxylic anion exudation rates per plant decreased relative to those whose solution was not corrected for pH, but the differences were not significant at p=0.05 (Figure 5). This indicates that the root-induced rise in solution pH perhaps stimulated exudation of carboxylates, but the pH effect was small relative to the direct NH4/(NH4+NO3) (R) effect.When maintaining the 100% NH4 solution at pH 5, as compared with the uncorrected final pH of 3.3, no significant effect on the exudation rate of the organic anions was observed (Figure 5). At a constant pH of 5, the exudation rate of all investigated carboxylates was considerably higher in the 100% NO3 than in the 100% NH4 solution, as obtained in the pH-uncorrected solutions (Figure 3), however, due to the large experimental variability in Experiment II, the differences between the two N forms were insignificant. A plausible reason for the larger variability in Experiment II than in Experiment I is that the titration to maintain the constant pH raised the solution EC from 0.8 to 2.0 dS m 1 at the end of the experimental uptake period. The different EC of the nutrient solutions could have affected plant growth and consequently root exudation. Discussion The presence of antibiotics did not affect the amount and composition of exuded carboxylates from roots, in agreement with the results of Kraffczyk et al. (1984), who reported no significant differences in the amounts of organic acids exuded by sterile and non-sterile maize roots. This suggests that either carboxylates were not exuded or utilized by bacteria, or that their production and consumption were in equilibrium (Kraffczyk et al., 1984; Mozafar et al., 1992). The inverse relationship between the NH4/(NH4+NO3) ratio (R) in solution and the exudation rates of triand dicarboxylic anions in tomato plants is similar to results obtained by Kraffczyk et al. (1984) for malate exudation by maize roots. As the fraction of NO3 in the nutrient solution increased, its uptake flux increased (Figure 2a). The excess of NO3 over cations uptake was compensated by H + influx, resulting in the observed increase in solution pH. Nitrate reduction in shoots results in the release of an equivalent amount of OH , that has to be neutralized in the plant cell via carboxylation and formation of phloem-mobile organic anions (Marschner, 1995; Touraine et al., 1990). Part of the carboxylates were indeed exuded to the root surrounding solution. Kirkby and Mengel (1967) reported that the predominant carboxylic anion in tomato leaves and roots was malate, followed by citrate and oxalate. The results in Table 1 show that among the exuded organic anions, citrate exudation exceeded malate exudation. This indicates that organic anion contents in plants and exudates are 16 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 not directly proportional, probably because of differences among carboxylates in phloem mobility and efflux across root membranes. Below a certain threshold NO3 uptake rate (55-95 mol plant1 6 h 1, depending on plant age) the citrate exudation rate was low (Figure 6).Above this threshold value, the citrate exudation rate sharply increased, and an incremental release of 0.4 mol citrate plant16 h 1 per incremental uptake rate of 10 mol NO3 plant1 6 h 1 for 37 d-old plants was observed (Figure6). It is hypothesized that at nitrate uptake rates below the threshold value, carboxylates are retained in root and shoot plant cells. At uptake rates surpassing the threshold, the carboxylates were released as described above. The threshold nitrate uptake rate increased with plant age (Figure 6), owing to the fact that the capacity of the growing plant to retain carboxylates increasedas well. The relationship between NH4 uptake rate and H +efflux from roots is presented in Figure 7. H + efflux was calculated from the differences in solution volumes and pH values at the beginning and end of the uptake period. The calculation is based on the fat that the titration curves of the 100% NO3 and 100% NH4 solutions (not presented) showed negligible pH buffering capacity. Under conditions of low NH4 (high NO3) concentrations, the exuded carboxylates probably increased the buffering capacity of the solutions,but under these conditions H + is expected to be negligible. 17 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 The relationship between NH4 uptake rate and the H + efflux also was plant-age- dependent (Figure 7).Below uptake rates of 150 and 300 mol NH4 plant1 6 h 1 (30 d-old plants and older plants, respectively) the H + efflux was very small. Above the threshold NH4 uptake values, H + efflux sharply increased, yielding a slope of 15 mol H + plant 1 6 h 1 per uptake rate increment of 50 mol NH4 plant1 6 h 1 for 30 dold plants (Figure 7). The threshold value is affected by the H + inflow and pH buffering capacity of the tomato roots, where the assimilation of NH4 into amino acids takes place (Marschner, 1995). As the root system grew with plant age (Figure 1), the threshold NH4 uptake rate also increased. The low solution pH which was obtained in the 100% NH4 solution (pH 3-4, Figure 4) approached a range which could cause root growth retardation and plasmalemma malfunctioning in various plants (Yanet al., 1992). Due to the short duration of the experiment and the relatively high Ca concentration in solution (2.5 m M), the adverse effect of the low pH on current root growth was insignificant (Figure 1).Using NO3 and NH4 uptake kinetics data (Figure2) and the known fresh root weight per plant (Figure1) enables us to estimate the NO3 and NH4 concentrations in nutrient solutions which were required to furnish the threshold NO3 and NH4 uptake rates at which organic anion and proton release by the roots were initiated. These limiting values are important as enhanced acidity and organic anions concentration in the rhizosphere can increase P availability to plants under P-deficiency conditions. Acidification and Ca2 + chelation by organic acids stimulate mineral-P solubilization, and citrate ions raise o-phosphate concentration in soil solution due to competition on common specific adsorption sites on soil clay minerals and iron oxide surfaces (Bar-Yosef, 1996). To obtain the threshold uptake rate of 55 mol NO3 plant1 6 h 1 for 30 day-old plant, the NO3 concentration in the solution should be 0.5 m M. The threshold value of 95 mol NO3 plant1 6 h 1 for 44-days-old plant requires 1.5 m M NO3; and to obtain uptake of 115 mol NH4 plant1 6 h 1 , which is the acidification threshold value for 30 day-old plants, the NH4 concentration in solution should be 1.4 m M. To obtain the threshold value for 44 days old plants (330 mol NH4 plant16 h 1), the corresponding concentration should be 8.3m M NH4. 18 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 +番茄根系分泌羧酸离子、H以响应营养液NH/NO、pH值变化 43 ++摘 要:根系分泌有机离子和H受氮素形态和pH值影响，且有机离子和H可以提高金属元素、磷的溶解性及植物吸收效率。本试验是为了研究营养液NH/NO、pH值变化对番茄分泌羧酸离子、43 +H/OH-的影响。试验设置4个NH/( NH+NO)比(R=0，0.33，0.67，1)，及恒定和可变pH值的处理。443 根系分泌各种羧酸盐的总量有随着R值增大而减小的趋势，尤其是三元和二元羧酸盐。所有处理及株龄中，三元和二元羧酸盐所占的平均摩尔比，酒石酸盐(,2%),苹果酸盐(,6%),琥珀酸盐(,15%),柠檬酸盐(,26%),延胡索酸盐(,46%)。当R=1时，营养液pH值从5.2下降到3左右;当R=0时，pH值增加到8左右。与R值相对应的番茄pH统计值约为0.3。对于pH值保持恒定的处理来说，根系分泌羧酸盐的量受溶液pH值的影响要小于受R值的影响。当植株吸收NH和NO超过某一临43 +界值后，柠檬酸盐和H会首先被分泌，且随着株龄的增加而增加。 +-关键词:羧酸离子; NH/NO; pH; 根系; 番茄 43 19 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 20 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 1 引言 羧酸离子有助于平衡植物体内电荷和调节pH值以适应植株吸收阴阳离子的不平衡及叶片对NO3的还原(Gerendas and Sattelmacher, 1990; Kraffczyk et al., 1984; Mengel and Kirkby, 1987; Touraine et al., 1990; Troelstra et al., 1990)。番茄地上部占主导地位的羧酸离子是苹果酸盐和柠檬酸盐(Kirkby and Mengel, 1967);根系中为苹果酸盐、柠檬酸盐和琥珀酸盐(Miller et al., 1990);木质部为柠檬酸盐、苹果酸盐和丙二酸盐(Senden et al., 1992)。NO-N刺激番茄地上部和根系合成苹果酸，而NH-N加快34柠檬酸盐的合成(Kirkby and Mengel, 1967)。培养在含NO营养液中的番茄植物羧酸盐含量是培养在3+NH中的5倍(Kirkby and Mengel, 1967)。营养液中K浓度高可以增加番茄根系有机酸的含量(Miller et 4 +al., 1990)，原因可能是增加了对K的吸收、阴阳离子吸收不平衡。 Mozafar等(1992)研究报导番茄根系分泌的有机酸主要是延胡索酸和柠檬酸，其次是苹果酸和琥珀酸。未有研究表明番茄分泌羧酸离子的速率和组成是否受N素形态的影响。假设营养液中N素形态和浓度影响植物体内羧酸离子的含量和组成，那么羧酸离子的分泌也将受到这些因素的影响。N对有机离子的分泌影响重大，如柠檬酸盐和草酸盐可以提高P吸附土壤上P的有效性(Bar-Yosef, 1996)。 +本研究的目的是为了揭示培养液中不同NH/( NH+NO)比及pH值对番茄分泌有机离子和H的443 影响。借助分泌羧酸离子的组成和速率方面的知识可以很好的对前人报导的有关N素营养与P有效性的关系进行理解(Cole et al., 1963)，也启发我们可以通过调节土壤NH/NO比及灌溉措施来缓解P缺乏43 的状况。 2 材料与方法 2.1 试验概况 将番茄种子萌发15 d后的幼苗移栽到通气良好、装有浓度减半的霍格兰营养液(Hoagland and Arnon, 1938)的8 L的容器中。8 d后，当根系开始交错生长，将每个植株转移到装有相同营养液的1 L锥形瓶中。培养液通气良好，每隔2 d更换一次，在试验正式开始时换成不同NH/NO比的营养液。43经过24 h的适应阶段，将植株转移到装有新鲜处理营养液的100 mL锥形瓶中。为防止细菌滋生，每个锥形瓶中分别加入50 mg/L的利福平和25 mg/L的四环素两种抗生素(Schwab et al., 1983)。定期使用琼脂培养基培养营养液样品，用以数微生物菌落来观测被微生物污染的状况。结果表明，营养液中加入抗生素后不会受到微生物的污染。 6-12 h后，取出番茄植株进行化学分析，营养液用来分析测定有机离子、pH值和元素含量。 初始营养液中N为7.5 mM;K，P，Ca，Mg浓度依次为3、0.5、2.5、1.0 mM;Fe，Zn，Mn，Cu， -1Mo，B浓度依次为2.5，0.03，0.4，0.025，0.018，0.25 mg L。用HCl或NaOH滴定营养液，以确定 -2-1系统对pH值变化的缓冲能力。试验期间，植株培养在生长箱中，光强为450 μE m s，光周期为13/11 h昼/夜。 2.1.1 试验1:NH/( NH+NO)比的影响 443 该试验设置4个NH/( NH+NO)摩尔比(R)(0，0.33，0.67，1)，每一个处理都分为加抗生素和不443 加两种情况。本试验测定3个株龄:30、37和44 d(R=1没加抗生素的处理没有测定第三个株龄)。在这三个株龄下，锥形瓶中的营养液分别于植株吸收11，6.7，6 h后收集。生长箱的温度保持在26/22?昼/夜。 2.1.2 试验2:营养液pH值的影响 21 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 该试验设置4个处理，在R=0、1两个NH/( NH+NO)比处理下，再设置两个pH值处理，其中第443 一个处理，在植株吸收营养过程中，不调节pH值;第二个处理是在测定阶段通过每隔30 min加入0.1 M的HCl或NaOH保持溶液的pH=5.0。株龄为33 d，吸收时间控制在6 h。生长箱温度保持在27/25?昼/夜。 试验1和2都采用完全随机区组设计，重复3-4次。采样时，用水冲洗植株根系，测定地上部和根系的鲜重和干重。培养前后的NO、NH含量采用蒸馏法测定(Bremner and Keeney, 1965)。 34 2.2 营养液中羧酸离子的测定 取部分待测液用Whatman NO. 41滤纸过滤，用0.1 M的NaOH调节pH=8.0。小柱(Varian) 强阴离子交换(SAX)盒(季胺硅胶)的每毫升萃取柱含有100 mg吸附剂用以溶液中有机酸的固相萃取(SPE)。该柱需先用1 mL的甲醇(HPLC级)、再每次1 mL的重蒸馏水洗两次进行活化。对已知体积的样品可通过0.22 μm微孔醋酸纤维过滤装置抽真空注入强阴离子交换盒中。洗脱出的羧酸离子用两个0.5 mL剂量的0.5 M HSO接收。HPLC(高效液相色谱仪)用的是Perkin-Elmer 410LC泵和300×7.8 mm的离子排24 斥柱(Bio-Rad)。该柱含有强阳离子交换剂Aminex HX-87H树脂，通过离子洗脱和分离色谱分离羧酸离子。把离子洗脱微型保护前柱(Bio-Rad)连接到分析柱上，用以清除样品中的杂质。将100 μL的样 -1品注入HPLC仪，用0.005 M HSO作为流动相，室温下控制流速为0.4 mL min。用紫外检测器24 (Perkin-Elmer二极管阵列检测器LC-235C)于210 nm波长处检测峰值，从分辨保留时间确定物质成份，并参照有机离子混合物的相关标准(Bio-Rad有机酸分析标准125-0586)。延胡索盐的标准是参照延胡索酸的标准得来的。用V4 Turbochrom处理机(PE Nelson系统)计算峰面积和保留时间。由于采用新的固相萃取技术(SPE)，所以在预试验中测定有机离子的回收率。回收率依次为:柠檬酸盐85%，酒石酸盐100%，苹果酸盐91%，琥珀酸盐99%，甲酸盐100%，乙酸盐100%。SPE预处理导致草酸盐的峰与其他盐的峰重合，所以该试验检测不到草酸盐。色谱显示SPE技术未导致离子保留时间漂移，可以很好的分离有机离子，且在该条件下，可以获得准确的峰面积，精确测定溶液中羧酸盐的含量。 2.3 数据分析 采用SAS软件(SAS, 1985)进行方差分析，并通过拟合GLM(一般线性模型)和NLIN(非线性模型)确定模型参数。 3 结果 地上部 根系 3.1 试验1 3.1.1 根系及地上部重量和N素吸收特 征 试验期间，地上部和根系的重量随着时 根 系 鲜 重 (g/株) 间的延长呈指数增加(图1)。植株光照时间太 短(,30 h)，使得植株重量存在显著差异。不 同处理间植株鲜重的干物质百分比无显著差 时 间 (d) 异，各处理及株龄地上部和根系干物质平均 百分比分别为7.1?0.7、4.2?0.6。 图1 株龄对地上部和根系鲜重的影响 22 地 上 部 鲜 重 (g/株) 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 ] ] -1 株龄 株龄 -1 6 h 6 h-1-1 [μmol (g 鲜根)[μmol (g 鲜根) 浓度(mM) NH浓度(mM) NO34 图2 营养液NO、NH浓度(C)及株龄(DAP)对根系吸收NO、NH速率的影响 3434 曲线用米氏方程拟合 -1-1a:NO 30 d、37 d、44 d植株Fmax 分别为31.0(***)、11.6(***)、6.0(***) μmol (g 鲜根) 6 h;Fm 3NO3NO32分别为0.70(**)、0.56(***)、0.56(*)mM;R分别为0.92、0.97、0.97 -1-1b:NH Fmax 分别为84(***)、70(n.s.)、89(***) μmol (g 鲜根) 6 h;Fm 分别为2.13(*)、7.3(n.s.)、4NH4NH4 21.09(n.s.)mM;R分别为0.95、0.95、0.92 ***、**、*分别代表在p=0.001、0.01、0.05水平上差异显著;n.s.代表在p=0.05水平上差异不显著 被圈的点在计算时被剔除 单位根系干重吸收NO和NH的速率随株龄的增加而降低(图2)。每个株龄的吸收速率都可以很34 好的用米氏方程来拟合(图2)。NO和NH的Km值都大，说明这个趋势符合二元动力学方程，但二元34 吸收模型(Epstein, 1972)并不能很好的拟合本试验所得结果。图2b用虚线圈起来的点没有用米氏方程拟合，这些点可能是分析误差造成的。 3.1.2 NH/NO比对根系分泌物的影响 43 3.1.2.1 有机离子 二元和三元羧酸盐离子总量的平均分泌速率随着株龄的增加而显著的增加，但从37 d到44 d呈下 -1-1降趋势(表1)。每个株龄中，R=0时羧酸离子总分泌量出现最大值，平均值约为1.1 μmol 株 6 h。溶液中一元羧酸盐离子(甲酸盐、乙酸盐)最大浓度出现在R=0.67时(表1)。一元羧酸离子没有包括到总量中，是因为至今还没有研究表明番茄组织和根系分泌物中存在甲酸盐和乙酸盐，它们可能是其他脂肪酸分解的产物(Fox and Comerford, 1990)。分泌的有机离子含碳量占干物质总碳的比例小于0.2%。不同NH/NO比及株龄处理中，二元和三元羧酸盐平均摩尔分数，酒石酸盐(,2%),苹果酸盐(,6%)43 ,琥珀酸盐(,15%),柠檬酸盐(,26%),延胡索酸盐(,46%)(表1)。 抗生素对总羧酸盐分泌物及单一分泌物所占的百分比无显著影响(数据未给出)。与不加抗生素相比，加抗生素只会使苹果酸盐和琥珀酸盐的分泌呈现降低趋势，延胡索酸盐趋势正好相反。 柠檬酸盐和酒石酸盐+苹果酸盐+琥珀酸盐+延胡索酸盐分泌速率随着营养 23 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 )-1液中R值的增大，以指数形式减少(图3a, b)。 6 h-1由于株龄30 d的植株根系活力最大(图2)，地 上部活力增加，所以图3选择的这个阶段的植 株。由表1、图1分析株龄37 d、44 d的植株趋 势与此相类似(结果未给出)。 3.1.2.2 营养液pH值 R=0(100% NO)的营养液培养前，pH=5，3 柠檬酸盐分泌速率(μmol 株经过6-12 h的培养，溶液pH值增大到8左右。NH/( NH+NO)比 443当R=0.67、1(100% NH)，相同时间内，溶液4 )-1pH值分别下降到4.0?0.5、3.2_?0.4(图4)。 6 h-1为验证Landsberg (1981)提出的营养液 pH值变化是否是因为植株分泌有机离子的缘 故，我们依据试验1中植株分泌柠檬酸盐的最 大量，称取相同重量的柠檬酸钠添加到20 mL 的初始营养液中(试验中培养结束后溶液体 积为20 mL)。此时溶液pH值约为6，表明根系 +吸收NO的同时，伴随泵入了大量的H，使3二元羧酸盐分泌速率(μmol 株NH/( NH+NO)比 443得溶液碱化，正如试验中pH=8(图4)。 R=1(100% NH)溶液pH值降低是因为根系吸4 +/(NH+NO)比(R)对有机离子图3 营养液不同NH收NH的同时泵出了H的缘故，依据植株分4434 分泌速率(EX)的影响 泌量加入等量的柠檬酸对溶液的pH值没有显 株龄为30 d，NH与NO浓度之和为7.5 mM。图a为柠檬酸43著影响。 盐，图b为二元羧酸盐(酒石酸盐+苹果酸盐+琥珀酸盐+延胡株龄为30 d的植株溶液pH值下降的幅度索酸盐);平均变异系数(SD/平均值)分别为94%和99% 远大于37 d、44 d的植株。R=0(100% NO)处3 理中，溶液pH值的变化不受株龄的影响。 株龄 初始值 NH/( NH+NO)比 443 图4 不同NH/(NH+NO)比及株龄(DAP)对培养结443 束后营养液pH值的影响 NH与NO浓度之和为7.5 mM，株龄30、37、44 d的植株43 分别吸收11、6.7、6 h，各处理三种株龄对应的平均变异系 数依次为5.6、10.4、5.1% 24 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 表1 营养液不同 NH/(NH+NO)比(R)及株龄(DAP)对根系分泌羧酸离子速率的影响 443 以下数据是加和不加抗生素的平均值，二元和三元羧酸盐用占总和的摩尔百分数表示 二元和三元羧酸盐 一元羧酸盐 R 酒石酸盐 苹果酸盐 琥珀酸盐 延胡索酸盐 柠檬酸盐 和 甲酸盐 乙酸盐 -1-1(占总和的摩尔百分数) (μmol 株 6 h) 30 DAP 0 0.4 16.2 6.2 47.1 30.0 0.97 0.00 0.07 0.33 1.5 14.4 9.5 40.2 34.5 0.18 0.05 0.06 0.67 0.4 4.6 16.6 57.4 20.9 0.06 0.30 0.21 1 8.4 15.4 16.3 41.7 18.2 0.03 0.07 0.11 37 DAP 0 0.0 5.4 4.1 63.6 26.9 1.44 0.03 0.00 0.33 1.3 2.2 13.0 56.1 27.3 0.17 0.01 0.01 0.67 3.2 6.7 25.7 48.5 16.0 0.11 0.28 1.48 1 1.1 0.5 18.6 71.1 8.7 0.24 0.24 0.52 44 DAP 0 1.2 5.4 18.1 43.4 31.9 0.87 0.01 0.01 0.33 0.2 0.7 18.9 30.6 49.6 0.17 0.00 0.00 0.67 6.1 4.6 24.7 35.6 29.0 0.07 0.00 0.00 1 0.0 4.7 10.8 49.3 35.2 0.11 0.00 0.00 分泌离子速率的F检验 R 1.09 n.s. 4.35** 0.29 n.s. 2.54* 4.87*** 15.36*** 4.32** 4.84** 时间 0.50 n.s. 1.55 n.s. 6.41*** 0.93 n.s. 6.35*** 8.41*** 2.10 n.s. 2.97 n.s. DAP代表株龄 ***、**、*分别代表在p=0.001、0.01、0.05水平上差异显著;n.s.代表差异不显著 除R=0.33处理使溶液pH值从5.2下降到4.5外(数据未给出)，其他处理抗生素对溶液pH值变化无显著影响。在加和不加抗生素所得数据平均值的基础之上，得出与R值相对应的番茄pH统计值为0.29-0.30，与Feigin等(1980)研究结果一致。 3.2 试验2 3.2.1 恒定或可变营养液pH值对柠檬酸盐分泌的影响 溶液pH值随着营养液中NO比例的增加而增加(图4)。为区分营养液不同NH/NO比及pH值对羧343酸离子分泌速率的影响，试验1中100% NO和100% NH均设置维持营养液pH值恒为5或可变两个处34 理。在pH值可变处理中，幼苗移栽到营养液20 min后100% NH处理pH值下降到4.1，而100% NO处43理pH值上升到7.0。1 h后，pH值分别为3.6和7.1;6 h培养结束后，pH值分别为3.3和7.8。 25 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 100% NOpH值恒为5的处理，羧酸离子分泌速率相对于可变pH值处理有所降低，但差异不显著3 (p=0.05,图5)。表明根系导致溶液pH值上升是由分泌的羧酸离子累积所造成的，但pH值的影响较R值的直接影响小。 100% NH pH值恒为5的处理，与可变pH值处理的最终pH值为3.3相比，对有机离子的分泌速率4 没有显著影响(图5)。pH值恒为5的处理中，100% NO羧酸离子分泌速率明显高于100% NH的分泌速34率，与pH值可变处理中结果相类似(图3)。由于试验2中试验变异大，所以两种N素形态中无显著差异。 -1试验2较试验1变异大，可能是通过不断滴定维持溶液pH值恒定，使得溶液的EC从0.8上升到2.0 ds m的缘故。营养液中不同的EC值可能会影响植株生长，进而影响根系分泌。 4 讨论 Kraffczyk等(1984)研究表明，玉米根系在灭菌或不灭菌条件下，分泌的有机酸浓度无显著差异;本试验番茄根系也得到类似结果，添加抗生素不会影响根系分泌羧酸离子的浓度和组成。表明细菌不分泌或消耗羧酸盐，或分泌和消耗处于平衡状态(Kraffczyk et al., 1984; Mozafar et al., 1992)。 营养液不同NH/( NH+NO)比与番茄植株分泌三元和二元羧酸离子速率呈负相关关系，与443 Kraffczyk等(1984)研究的玉米根系分泌苹果酸盐的结果相类似。随着营养液中NO的浓度增加，其吸3 +收通量也增加(图2a)。根系对NO的奢侈吸收，伴随H的吸收，导致溶液pH值上升。地上部NO的还33 -原导致释放等当量的OH，通过羧化作用和木质部形成可移动的有机离子进入植株细胞进行中和(Marschner, 1995; Touraine et al., 1990)。部分羧酸盐确实分泌到根系周围的营养液中。Kirkby和Mengel (1967)报导，番茄叶片和根系中羧酸离子主要是苹果酸盐，其次是柠檬酸盐和草酸盐。表1结果显示在分泌的有机离子中，柠檬酸盐大于苹果酸，这表明植株中有机离子的含量和分泌量不成正比关系，可能是木质部可移动的羧酸离子与根表皮细胞膜所分泌的存在差异。 -1-1在NO的吸收速率阈值 (55-95 μmol 株 6 h，与株龄有关) 以下，柠檬酸盐分泌速率降低。超3 -1-1过阈值，37 d株龄的植株分泌柠檬酸盐的速率明显增加，每多吸收10 μmol 株 6 h的NO，植株约3-1-1释放0.4 μmol 株 6 h的柠檬酸盐(图6)。 假设植株吸收NO速率在阈值以下，羧酸离子会贮存在根系和地上部细胞中。超过阈值，羧酸3 离子被释放。该阈值随着株龄的增大而增大(图6)，因为随着植株的增长，贮存羧酸离子的能力相应增强。 最终pH值 )可变 -1 6 h恒定 -1 可变 恒定 羧酸盐分泌速率(μmol 株 柠檬酸盐 二元柠檬酸盐 26 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 图5 100% NO或100% NH营养液pH值恒定或可变处理对有机离子分泌速率的影响 34 两种营养液最终pH值分别为7.8和3.3 二元羧酸盐为酒石酸盐、苹果酸盐、琥珀酸盐、延胡索酸盐之和;不同字母标记为在p=0.05水平上差异显 著;误差棒为平均值的标准偏差 ) )-1-1株龄 株龄 6 h 6 h-1-1 分泌速率(μmol 株+H -1-1柠檬酸盐分泌速率(μmol 株吸收速率(μmol 株 6 h) NH4-1-1NO吸收速率(μmol 株 6 h) 3 图6 株龄(DAP)和NO吸收速率对柠檬酸盐图7 株龄(DAP)和NH吸收速率对番茄根系34+分泌速率的影响 分泌H的影响 ++图7要说明的是根系吸收NH的速率与分泌H的关系。H分泌是根据溶液体积及培养初始和结束4 时的pH值计算的。100% NO和100% NH的滴定曲线(未给出)表明其对溶液的pH值变化几乎没有缓34 +冲能力，H分泌的计算方法是在这个基础之上进行的。当营养液中NH浓度较低(即NO浓度较高)时，43 +分泌的羧酸离子可以提高溶液的缓冲能力，但在这种情况下，忽略了H的作用。 +-1株龄也会影响植株对NH的吸收和H的泵出(图7)。当吸收NH的速率小于150和300 μmol 株 6 44 -1++h(分别为30 d和株龄更大的植株吸收速率)时，H分泌量很少。超过吸收NH的阈值，H分泌量明显4 -1-1+增加，对于30 d的植株来说，吸收NH的速率每增加约50 μmol 株 6 h，H的释放速率迅速增加15 4-1-1+μmol 株 6 h，形成一个转折点(图7)。阈值影响番茄根系对H的吸收和对pH值变化的缓冲能力，同时番茄根系又是NH同化成氨基酸的场所(Marschner, 1995)。根系随着株龄的增大，吸收NH的阈值44也会增大。 100% NH处理营养液的pH值较低(pH 3-4, 图4)使得多种植物根系生长受阻，质膜受到破坏(Yan 4 2+et al., 1992)。由于是短期培养试验，再加上溶液中Ca浓度相对较高(2.5 mM)，使得低pH值对目前根系生长的不良影响并不显著(图1)。用NO和NH的吸收方程数据(图2)，及已知根系的鲜重(图1)，可34 以估测出营养液中NO和NH的浓度，从根系开始分泌有机离子和质子时，计算其吸收NO和NH速3434率的阈值。得到这些阈值很重要，如增加根际土壤酸度和有机离子浓度，可以提高缺磷土壤磷的有 2+效性。酸化和Ca被有机酸络合，可促使矿物态磷溶解，柠檬酸离子由于对土壤黏土矿物及铁氧化物表面专性吸附位点的竞争，可提高土壤溶液中有效磷的浓度(Bar-Yosef, 1996)。对30 d植株来说， -1-1为获得NO的55 μmol 株 6 h吸收速率阈值，需控制溶液NO的浓度为0.5 mM左右。对44 d植株来33 -1-1说，为获得NO的95 μmol 株 6 h吸收速率阈值，需控制溶液NO的浓度为1.5 mM左右;要使NH334-1-1吸收速率为115 μmol 株 6 h，需控制溶液NH的浓度为1.4 mM，这个值恰好是30 d植株的酸化阈值。4 -1-1为获得44 d植株的吸收NH的阈值(330 μmol 株 6 h)，溶液中相对应的NH浓度应为8.3 mM。 44 27 叶面喷施镁肥对缺镁番茄养分吸收和分配的影响 致谢 毕业之际，回想求学的这四年经历，有许多值得我记忆一生。 首先，衷心感谢导师陈竹君副教授的悉心指导，从选题、试验、实施、到最终的论文撰写，反复斟酌与修改，注入了老师大量的心血。导师渊博的知识、敏捷的思维，积极进取的科研精神，值得我学习，在导师的感染下，我也会在今后的学习生活中，锻炼自己的动手能力，凡事认真去做，要让自己在细节中不断进步，不断成长。 感谢感谢师兄王辉民在试验过程和论文撰写中给予的帮助。感谢同学在实验时的帮助，分担了大量的实验任务，感谢我的父母、哥哥对我学业的理解和支持。 感谢参加答辩的各位老师对本研究提出的宝贵和建议。感谢西北农林科技大学四年来对我的培养，在此向所有关心、支持和帮助过我的各位亲朋好友致以我最由衷的感谢～ 28