摘要:【目的】探索施用氮肥对提高植物抗旱性的影响。【方法】以干旱区重要造林树种头状沙拐枣(Calligonum caput-medusae)幼苗为研究对象,采用盆栽试验,设置两组水分处理(亏缺供水和正常供水)和两组氮肥处理(未施氮和施氮),测定幼苗同化枝的生理生化指标。【结果】(1)同正常供水相比,亏缺供水下的幼苗同化枝相对含水量(RWC)、正午同化枝水势(Ψm)、最大气孔导度(gs)、叶绿素含量(Chl)、表观量子效率(Φ)和可溶性糖(SS)含量显著降低;而脯氨酸(Pro)含量显著增加;(2)同未施氮组相比,施氮减缓了亏缺供水和正常供水下头状沙拐枣幼苗同化枝相对含水量的下降,但不显著;同正常供水比,亏缺供水下施氮降低了头状沙拐枣幼苗的同化枝水势;(3)正常供水下,施氮提高了幼苗Pro、SS含量,而丙二醛(MDA)含量显著降低(Plt;0.05);亏缺供水下,施氮提高了超氧化物歧化酶(SOD)含量、Pro含量和SS含量(Plt;0.05),而MDA含量显著降低(Plt;0.05)。
【结论】沙拐枣幼苗生理活性受土壤水分有效性的影响,而施氮有助于降低干旱胁迫造成的影响。施氮提高了亏缺供水和正常供水下头状沙拐枣幼苗的抗氧化酶活性和细胞溶质浓度,有助于头状沙拐枣幼苗降低氧化应激反应并减少损伤,增加渗透调节物质。
关键词:头状沙拐枣;施氮;抗旱性;生理生化
中图分类号:S511.2文献标志码:A文章编号:1001-4330(2024)09-2330-11
0引 言
【研究意义】植树造林是干旱、半干旱区防治荒漠化的一种重要手段[1]。但幼苗在造林后,容易暴露于缺水环境中,出现干旱胁迫或死亡[2,3]。探讨氮肥在提高幼苗抗旱性和维持存活中的作用,对幼苗抚育管理有重要意义。【前人研究进展】由于干旱地区蒸发量高降水量低,水分通常限制植物的初级生产力。干旱胁迫引发的植被退化将进一步导致土地沙化和荒漠化。植树造林有助于生态恢复和提供多样的生态系统服务[1],干旱荒漠区土壤贫瘠,植物生长经常受到养分限制[4]。土壤缺氮会使叶片的叶绿素含量降低,光合作用能力下降,抑制植物的生产力[5]。此外,氮受限可能会显著影响植物的抗旱性[6]。而人工施加氮肥可以促进幼苗早期的生长,防止细胞膜损伤和增强渗透调节,增加叶绿素含量、净光合速率,缓解养分胁迫,从而提高水分利用效率[4]。由此可提高植物对贫瘠土壤的适应能力,增强植物对干旱胁迫的耐受性。在干旱胁迫下植物的光合作用受损、渗透调节物质可溶性糖(soluble sugar, SS)和脯氨酸(proline, Pro)积累增加[4]。此外,植物受到干旱胁迫时,活性氧(reactive oxygen species, ROS)会过量产生,如超氧离子(superoxide anion, O2-·)和丙二醛(malonic dialdehyde, MDA),会导致脂质过氧化,对植物细胞蛋白质、脱氧核糖酸(desoxyribonucleic acid, DNA)和脂质造成损伤,进而抑制植物生长[7]。但植物细胞可通过增加超氧化物歧化酶(superoxide dismutase, SOD)来应对干旱胁迫,保护其不受ROS的伤害[8-10]。
【本研究切入点】蓼科沙拐枣属(Calligonum mongolicum Turcz)是干旱荒漠地区防风固沙主要造林树种[11]。头状沙拐枣(Calligonum caput-medusae)是一种典型的旱生小灌木,具有耐干旱、高温、瘠薄和适应流沙的特性,在我国西北干旱、半干旱地区被广泛用于防风固沙和生态修复[12-13]。有关沙拐枣属植物抗旱性的研究较多,大多数集中在不同品种抗旱性评价[14-17],以及不同水分条件下生理生态响应方面[18-20],但有关施加氮肥能否提高沙拐枣抗旱性的研究较少。【拟解决的关键问题】以干旱区重要造林树种头状沙拐枣(Calligonum caput-medusae)幼苗为研究对象,采用盆栽实验,设置四组处理:亏缺供水下未施氮(N0)、亏缺供水下施氮(N1)、正常供水下未施氮(N0)、正常供水下施氮(N1),测定幼苗同化枝的生理生化指标,探讨施加氮肥对沙拐枣抗旱性的影响。
1材料与方法
1.1材 料
1.1.1试验区概况
试验地位于新疆阜康荒漠生态系统国家野外科学观测研究站 (87°56′E,44°17′N, 475 m asl)。试验区属温带大陆性气候,年平均气温6.6 ℃,年均降水量约164 mm,年蒸发量为2 000 mm。土壤质地为风沙土,沙土粒径为0~500 mm,粒径组成为:1.3%粘土13.7%壤土和85%砂[21],土壤pH值为9.5[22],土壤容重为1.53 g/cm-3[23],土壤饱和含水率为42.8%[24]。
1.1.2沙拐枣幼苗
试验材料选自中国科学院吐鲁番沙漠植物园(42°51′N,89°11′E,80 m asl)2年生头状沙拐枣幼苗,并于2019年4月移栽至阜康荒漠生态系统国家野外科学观测研究站遮雨棚下。将头状沙拐枣幼苗种植于装有荒漠土的花盆中(盆高50 cm,上径34 cm,下径26 cm)。每盆移植2~3棵幼苗,成活后每盆留1株。
1.2方 法
1.2.1试验设计
幼苗经过一个半月生长后,施加水、氮肥处理,处理20 d后开始试验数据采集。水分处理分为正常供水和亏缺供水,正常供水是以土壤含水量2%作为参考基准,利用花盆容积和土壤容重计算花盆正常供水量为1 000 mL[21],亏缺供水量为正常供水量的一半,即500 mL。每周浇水2~3次。每组水分处理设2种施肥处理:N0(未施氮);N1(施氮,使用含氮量为35%硫磺包膜尿素[25];施肥量参考中国北方大气氮沉降量3.5 gN/(m2·a),根据花盆的上表面积换算出每盆需要供应0.91 g 硫磺包膜尿素。共4组处理,每种处理10盆,共40盆。
1.2.2指标测定
1.2.2.1土壤含水量
在花盆表土往下15 cm处,取土15~20 g装入已知重量的铝盒中,迅速封膜,到室内称重,记录土样原土重。置于105℃烘箱中烘干至恒重,测定烘干土重[26]。
土壤含水量=[(原土重-烘干土重)/(烘干土重-铝盒重量)]×100%。(1)
1.2.2.2同化枝相对含水量和同化枝水势
每组随机摘取同化枝后立即称重(FW)。将同化枝放入温度为4℃的蒸馏水中浸泡12 h,吸水纸吸取表面水分,测量同化枝饱和重(TW),80 ℃烘干24 h,得到同化枝干重(DW)。计算同化枝相对含水量(RWC)[27]:
RWC=[(FW–DW)/(TW–DW)]×100%."(2)
在黎明前和正午测定同化枝水势(Ψ)。每个处理随机选取5株,分别剪取其同化枝,迅速装入带有湿纸球的自封袋置于冰盒中。在实验室中用压力室水势仪(Model 3005,PMS Instrument Company, Albany, NY, USA)测定同化枝水势。[28,29]
1.2.3生理指标测量
1.2.3.1光合速率和气孔导度
处理20 d后,每株选取3枝向阳且健康生长的同化枝挂牌标记,于晴天9:00-13:00利用便携式光合仪Li-6400(Li-Cor, Lincoln, NE, USA)测量幼苗同化枝的光响应曲线。使用非直角双曲线模型[30]拟合光响应曲线,其表达式为:
Pn(I)=αI+Pmax-(αI+Pmax)2-4θαIPmax2θ-Rd.(3)
式中,Pn(I)为净光合速率;I为光强;θ为曲线的曲率;α为植物光合作用对光响应曲线在I=0时的斜率,即光响应曲线的初始斜率,也称为初始量子效率;Pmax为最大净光合速率;Rd为暗呼吸速率[31]。
利用SC-1稳态气孔计(Model SC-1,Decagon,USA)测定头状沙拐枣同化枝的气孔导度。
1.2.3.2叶绿素含量和最大光化学量子产量
于7月中旬在上午11:00~12:00,分别选取长势良好的头状沙拐枣同化枝,利用便携式叶绿素测定仪(SPAD-502Plus)测量其叶绿素含量[32,33]。使用便携式脉冲幅度调制荧光计(Pocket PEA, PE 32 1JL, Hansatech Instruments Ltd, King′s Lynn Norfolk, UK)测定叶绿素荧光参数。暗适应20 min后,在0.8 s以上调制光的弱脉冲下测量初始荧光(F0),其中最大荧光(Fm)由0.7 s上施加的饱和光脉冲(5 000 mmol/(m2·s))诱导所测得。计算出同化枝的光系统Ⅱ的最大光化学量子产量(maximal quantum yield of PSⅡ):Fv/Fm= (Fm-F0)/Fm,其中Fv是Fm和F0之间的差值[34]。
1.2.4生化指标测量
于8月中旬取头状沙拐枣同化枝鲜样用于生化指标测定。采用氮蓝四唑(NBT)法[9,35,36]测定超氧化物歧化酶(SOD)活性;采用硫代巴比妥酸(TBA)法测定丙二醛[3,37-38];采用羟胺氧化法测定超氧阴离子[27,39];采用苯酚硫酸比色法测定可溶性糖[40];采用磺基水杨酸法测定脯氨酸[41,42]。
1.3数据处理
使用Microsoft Excel 2007整理数据,用均数±标准差(SD)表示。用SPSS 26.0(SPSS Inc., Chicago, IL, USA)进行单因素和双因素方差分析(ANOVA),用邓肯检验法对试验结果进行显著性差异分析(P<0.05)。用Origin pro 8.5(Origin Lab Corp., Northampton, MA, USA)进行绘图。主成分分析使用R的基础函数 Prcomp分析,使用 ggbiplot 包展示结果。
2结果与分析
2.1水肥处理下土壤和植物的水分状况
研究表明,同正常供水处理相比,亏缺供水下SWC、同化枝相对含水量显著下降 (P<0.05),施氮使同化枝相对含水量维持在较高水平。在不同水分条件下施氮与未施氮对其同化枝水势的影响亦有不同,正常供水下,施氮植株的黎明同化枝水势(Ψpd)比未施氮植株的Ψpd高0.13 MPa,施氮植株的Ψmd比未施氮植株的Ψmd高0.12 MPa;亏缺供水下,施氮植株正午同化枝水势(Ψmd)比未施氮植株低0.34 MPa。表1
2.2水肥处理下植物生理指标变化
研究表明,同亏缺供水处理相比,正常供水下表观量子效率(Φ)、最大净光合速率(Pmax)、暗呼吸速率(Rd)、最大气孔导度(gs)叶绿素SPAD值、最大光化学量子产量(Fv/Fm)均有所提升。其中最大气孔导度和叶绿素含量均呈现显著性差异(Plt;0.05),表现为施加氮肥显著增加最大气孔导度和叶绿素含量。正常供水下,施氮植株的最大气孔导度比未施氮植株高11.8%;亏缺供水下,施氮植株的最大气孔导度比未施氮植株高11.9%;正常供水下未施氮植株的叶绿素含量比亏缺供水下未施氮植株的叶绿素含量高19%。施氮肥后,提高了同一水分处理下的植株叶绿素含量。正常供水下施氮植株的叶绿素含量增加了11%,亏缺供水下施氮植株的叶绿素含量增加了16%。施氮还能缩小正常供水植株与亏缺供水植株的叶绿素含量差异,两者仅相差13.8%。表2
2.3水肥处理下植物生化指标变化
研究表明,不同处理条件下,MDA、Pro和SS差异显著,表现为施氮显著降低MDA(Plt;0.05),显著增加Pro和SS(Plt;0.05),而SOD仅在亏缺供水条件下呈现显著性差异,表现为施氮显著增加SOD(Plt;0.05),而O2-·无显著差异(Pgt;0.05);
亏缺供水下,施氮植株的MDA比未施氮植株的MDA低3.5%;正常供水下,施氮植株的MDA比未施氮植株的MDA低4%;亏缺供水下,施氮植株的SOD是4组处理中最高值,为78.44 μmol/(g·FW),比未施氮植株的SOD活性高15.9%;
亏缺供水下,施氮植株的Pro含量比未施氮植株多20%。正常供水下,施氮植株的Pro含量比未施氮的Pro含量高100.9%,未施氮植株的Pro含量为4组的最低值60.42μg/mol。在未施氮条件下,亏缺供水的Pro含量比正常供水高148%,而在施氮条件下,亏缺供水下的Pro含量比正常供水高49%。亏缺供水下,施氮植株的SS含量比未施氮植株多6%。正常供水施氮植株的SS含量与其他3组的SS含量差异显著,为4组中的最高值273.74μg/ mL,比正常供水未施氮植株的SS含量高21.6%。图1~3
2.4水肥处理对植物生理生化指标的综合影响
研究表明,浇水对沙拐枣幼苗的SWC、RWC、Ψmd、Φ、gs、SS均有极显著性影响(Plt;0.01),对Ψpd有显著影响(Plt;0.05)。施肥对PRO有显著影响(Plt;0.05)。水×肥对Ψmd有显著影响(Plt;0.05)。表3
PC1 和 PC2 分别解释了 40.4%和 15.6%的性状变异。沿 PC1 轴的正向,代表了沙拐枣的脯氨酸,而与其相反的是植物的土壤含水量和正午叶水势,植株脯氨酸与植株水分状况负相关。正常供水组及亏缺供水组沿 PC1 轴分离,独立样本T检验的结果表明,缺供水组的 PC1 得分要显著高于正常供水组,供水量的减少导致水分状况下降引起了干旱胁迫,但植株通过增加渗透调节物质(PRO)来应对干旱胁迫。施氮组及未施氮组沿 PC1 轴相交,施肥与不施肥处理之间无显著性差异。图4
3讨 论
3.1水肥处理对植株水分状况的影响
一般认为在外界环境水分减少的情况下,反映植物水分状况的指标会随着SWC的降低而减弱[43],例如在对干旱胁迫影响润楠幼苗生理生长的研究中[44],发现植株的叶片相对含水量显著降低。
试验研究表明,浇水对沙拐枣幼苗的RWC有极显著性影响,随着浇水量的减少,同化枝的相对含水量变化较大,亏缺供水下的同化枝相对含水量均低于正常供水;同化枝相对含水量越高表示植株的抗旱能力越强。陈少瑜等[45]的研究中提到干旱胁迫下,抗旱性强的植株与抗旱性弱的植株相比叶片含水量下降更缓慢。试验研究中发现不同补水量下,未施氮组的同化枝相对含水量差异大于施氮组。具体表现为施氮后头状沙拐枣幼苗同化枝相对含水量的下降速度相对缓慢,其抗旱性有所增强。
在试验中,亏缺供水下同化枝水势均低于正常供水。具体表现为:在正常供水下,施加氮肥植株比未施氮植株的Ψpd和Ψm高。未施氮植株的同化枝水势在不同水分处理下无显著差异。但施氮使得同一水分处理下植株的同化枝水势呈显著差异。植株的同化枝水势可反映植株组织水分状况和植株受水分条件的制约程度[46],其Ψpd的高低代表植株的水分恢复状况,而植物经受的最强胁迫程度则可以通过Ψm反映[47]。试验研究发现,水分处理对沙拐枣幼苗的水分状况有显著影响,主成分分析进一步表明了供水量的减少导致水分状况下降引起了干旱胁迫。同时发现了施氮提高了正常供水下头状沙拐枣幼苗的水分恢复状况,减缓了正午头状沙拐枣幼苗受到的胁迫程度。在水分条件较差的情况下(亏缺供水),施氮植株正午遭受的胁迫程度比未施氮植株略高。
3.2水肥处理对幼苗生理性状的影响
通过光响应曲线可计算最大光合速率(Pmax)、光补偿点(LCP)、暗呼吸速率(Rd)等光合参数[48]。试验研究中,在不同氮肥处理下,植株Pmax虽无显著差异,但表现为水分较好的情况下Pmax会有所提高。在不同水分处理下,植株的最大气孔导度(gs)在施氮处理下也有所增长。同时我们发现施氮植株的叶绿素含量均显著增加。最大光化学量子产量能够表示植株在环境中受到的胁迫程度[49],在试验研究中,施肥处理对植株最大光化学量子产值有显著影响。
荒漠灌木能够在土壤水有效性降低的情况下,生物量会更多的分配到地下部分,增加根系吸水能力,保证原有的生理特征和正常的光合作用[21]。头状沙拐枣在荒漠区分布广泛[13],荒漠灌木倾向于优化同化器官的密度,以适应土壤水分的有效性[21]。头状沙拐枣的这种特性决定了其在干旱胁迫发生时,会通过加大落叶量来减弱代谢活动,并且优化其同化器官的密度,利用肉质茎内贮藏水分与绿色茎秆的光合性能维持代谢,可能是导致最大净光合速率在试验研究中的水分差异下未发生显著变化的主要原因。
3.3水肥处理对幼苗抗旱性的影响
植物在进行光合作用时,无法避免地产生对细胞有损伤的ROS,但正常情况下会被各类抗氧化防护机制清除,达到活性氧产生和清除的平衡,倘若生长环境受到干旱胁迫等外界刺激,则会打破这种平衡[7]。植物可以通过酶类和非酶类的防御系统保护细胞免受或减少氧化伤害[50]。SOD在植物的抗氧化胁迫中有重要作用,主要是将O2-·歧化为H2O2[51]。试验研究发现亏缺供水下施氮植株的SOD活性最高,将显著地提升其抗氧化能力,与未施氮植株相比施氮植株的抗旱能力明显增强[52]。
MDA常是细胞脂质过氧化水平的判断指标[53]。在试验中不同水肥处理下,各组脂质过氧化水平由小到大为正常供水N1lt;正常供水N0lt;亏缺供水N1lt;亏缺供水N0。试验证明水分胁迫是影响植株脂质过氧化水平的主要因素;同时施氮可以减少MDA的产生,即未施氮植株的脂质过氧化水平均高于施氮植株。MDA属于脂质过氧化反应产生的强毒力的脂质过氧化终产物[54];与施氮植株相比,MDA含量较高的未施氮植株的细胞膜受损程度较高,更多量的MDA造成细胞膜的流动性和通透性改变加大,甚至导致细胞结构和功能变化[55]。
植物在干旱胁迫下常通过主动增加细胞内的Pro、SS等可溶性物质调节渗透势,保持水分,维持膨压[56],其可溶性物质积累的浓度亦能体现植株的抗旱性水平[57]。在试验中,主成分分析结果揭示了供水量的减少会导致水分状况下降,引起了干旱胁迫时植株会通过增加渗透调节物质(PRO)来应对干旱胁迫。在试验中,施肥处理对PRO的影响显著(Plt;0.05),施氮植株的Pro、SS均比同一水分处理下的植株水平高,明显促进了植株的渗透调节能力。C、N代谢的产物多为渗透调节物质[58],适当的施氮可能有利于N的代谢和C的同化,能够促进Pro大量积累。同时,施氮可能提高了淀粉酶的活性,加快了淀粉的分解,使得SS含量显著增加。Pro除了参与渗透调节以外,能够作为抗氧化剂,可有效清除O2-·、·OH等ROS,其积累还能诱导谷胱甘肽(GSH)的上升,进一步加强植株的抗氧化能力[56]。施氮植株较高的Pro含量以及亏缺供水下施氮植株较高的SOD活性很有可能是导致施氮植株MDA含量降低、脂质过氧化水平降低的原因之一。也有研究表明,Pro与叶绿素的合成的有关,在试验中施氮植株大量积累的Pro极有可能促进了施氮植株的叶绿素合成。
4结 论
水肥处理对幼苗的水分生理和抗旱性状都产生了一定的影响。在干旱胁迫条件下,随着浇水量的减少,植株的相对含水量降低,而施氮可以增强植物的抗旱能力。此外,施氮还提高了正常供水下植株的水分恢复状况,并减缓了幼苗在正午受到的胁迫程度。光合特性方面,施氮处理有助于提升植株的最大光合速率和叶绿素含量。在抗氧化能力方面,施氮处理显著提高了植物的SOD活性,并降低了脂质过氧化水平,进一步增强了植物的抗旱能力。此外,施氮还促进了可溶性物质如Pro和SS的积累,从而提高了植物的渗透调节能力和抗氧化能力。因此,适量水肥处理有利于改善植物的水分状况、幼苗生理性状和抗旱性能,提高植物对干旱胁迫的适应能力。
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Study on the effect of N fertilization on drought resistance of Calligonum caput-medusae seedlings
LI Jinyao1,XU Guiqing2,WANG Lisheng3,LYU Ping3,SHI Dongfang4,ZHENG Weihua5
(1. ""College of Forestry and Landscape Architecture, Xinjiang Agricultural University, Urumqi 830052, China; 2. Fukang Desert Ecological Experimental Station, Chinese Academy of Sciences, Fukang Xinjiang 831505, China; 3. General Work Station of Forestry and Grassland, XPCC, Urumqi 830013, China; 4. Xinjiang Ruiyixin Ecological Garden Technology Co., Ltd., Urumqi 830000, China; 5. Institute of Agricultural Quality Standards and Testing Technology, Xinjiang Academy of Agricultural Sciences, Urumqi 830002, China)
Abstract:【Objective】 An important afforestation tree in arid region.
【Methods】 To explore the effects of nitrogen fertilizer on drought resistance of Calligonum caput-medusae Schrenk seedlings, ""Two groups of water treatments (deficit water supply and normal water supply) and two groups of nitrogen treatments (no nitrogen application and nitrogen application) were set up in pot experiment to determine the physiological and biochemical indexes of assimilated branches of seedlings.
【Results】 "(1) Compared with normal water supply, the relative water content (RWC), midday water potential (Ψm), maximum stomatal conductance (gs), chlorophyll content (Chl), apparent quantum efficiency (Φ) and soluble sugar (SS) contents of assimilated branches were significantly decreased under deficit water supply."The content of proline (Pro) increased significantly."(2) Compared with no N application group, N application slowed down the relative water content of the assimilated branches of A."capulosa seedlings under deficit and normal water supply, but it was not significant; Compared with normal water supply, nitrogen application under deficit water supply reduced the assimilative branch water potential of A."capillata seedlings."(3) Under normal water supply, the contents of Pro and SS were increased by nitrogen application, while the contents of malondialdehyde (MDA) were significantly decreased (Plt;0.05)."Under deficient water supply, nitrogen application increased the content of superoxide dismutase (SOD), Pro and SS (Plt;0.05), but significantly decreased the content of MDA (Plt;0.05).
【Conclusion】 "The physiological activity of Jujube japonica seedlings is affected by the availability of soil water, and nitrogen application can reduce the effect of drought stress on them."Nitrogen application increases the activity of antioxidant enzymes and the concentration of cytosoles in the deficient and normal water supply, and helps the seedlings to overcome oxidative stress, reduce damage and increase osmoregulatory substances.
Key words:Calligonum caput-medusae; fertilization; drought resistance; physiology and biochemistry
Fund projects:Commissioned Project of Forestry and Grassland Work Station of Xinjiang Production and Construction Corps \"Evaluation of No-Irrigation Vegetation Restoration Effectiveness of Damaged Ecological Public Welfare Forests in the Southern Margin of Junggar Basin\" (E1400209);Project of National Natural Science Foundation of China (NSF), \"Groundwater Dependence and Future Prospects of Typical Scrub in Oasis Desert Transition Zone \"and\" Accumulation of Large Aggregates of Soil Water Stability and Its Driving Mechanism during Oasisization\" (32171874, 42271068)
Correspondence author: XU Guiqing (1976-), male, from Xinjiang, associate researcher, Ph. D., master's supervisor, research direction: plant physiological ecology in arid region.(E-mail)xugq@ms.xjb.ac.cn
收稿日期(Received):
2024-02-15
基金项目:
新疆生产建设兵团林业和草原工作总站委托项目“准噶尔盆地南缘兵团受损生态公益林免灌植被恢复成效评价”(E140020901);国家自然科学基金项目“绿洲荒漠过渡带典型灌丛的地下水依赖性及其存续前景”,“绿洲化过程中土壤水稳性大团聚体的累积与其驱动机制研究”(32171874,42271068)
作者简介:
李金瑶(1998-),女,河南人,在读硕士研究生,研究方向植物生理学。(E-mail)865268214@qq.com
通讯作者:
徐贵青(1976-),男,新疆人,副研究员,博士,硕士生导师,研究方向干旱区植物生理生态学,
(E-mail)xugq@ms.xjb.ac.cn