有机肥和化肥对烟叶气体交换、叶绿素荧光特性及叶绿体超微结构的影响

2015-06-15王通明潘文杰韦克苏宗学凤王三根

植物营养与肥料学报 2015年2期

关键词：叶龄超微结构叶绿体

王通明，陈伟，潘文杰，姚娟，韦克苏，陈波，刘川，董博，宗学凤*，王三根*

(1 西南大学农学与生物科技学院，重庆 400716； 2 贵州省烟草科学研究院，贵州贵阳 550081；3 水城县农业局，贵州六盘水 553040)

有机肥和化肥对烟叶气体交换、叶绿素荧光特性及叶绿体超微结构的影响

王通明1，陈伟2，潘文杰2，姚娟3，韦克苏2，陈波1，刘川1，董博1，宗学凤1*，王三根1*

(1 西南大学农学与生物科技学院，重庆 400716； 2 贵州省烟草科学研究院，贵州贵阳 550081；3 水城县农业局，贵州六盘水 553040)

烟叶；有机肥；叶绿素荧光；叶绿体；超微结构

烟草(Nicotianatabacum)是典型的叶用作物，叶片中96%左右的干物质直接或间接来自光合作用[1]。烟草在生长发育过程中对矿质元素吸收量大，需要大量施肥，但过量施用化学肥料可导致环境污染[2]。有机肥不仅成本较低、污染小[3]，而且能显著促进作物生长，提高作物的产量和品质[4]。已有研究证明，有机肥具有促进烤烟生长、提高产量、改进烟叶品质等作用[5]，但对比较有机肥与化肥在生育期如何影响烤烟叶绿素荧光特性及叶绿体超微结构差异的报道并不多见。为此，本试验以烤烟K326为材料，探讨了不同叶龄时期有机肥与化肥处理对烟叶气体交换参数、叶绿素荧光特性、叶绿素含量及叶绿体超微结构影响等方面的差异，以期从光能利用(宏观)及叶绿体超微结构(微观)方面，揭示两者对烟叶光合作用的影响机制，同时为探索烟叶高产优质栽培模式提供理论依据。

1 材料与方法

1.1 供试材料

本试验于2012年在贵州特色优质烟叶基地(贵州贵阳)进行，试验地位于N24.88°，E107.10°，海拔约为1112.8 m。供试土壤为黄壤土，pH 6.6，土壤有机质21.1 g/kg、碱解氮61.5 mg/kg、速效磷48.7 mg/kg、速效钾159.2 mg/kg。

供试烤烟品种为K326 (NicotianatabacumL. K326)，烤烟种子由贵州省烟草科学研究院提供，消毒处理后工厂化育苗，育苗基质主要以草炭蛭石为主，烟苗于4月8日移栽到温室土壤中。

1.2 试验设计

选定两块试验区域，设两个肥料处理，1)施用烤烟专用有机肥(OF)，肥料由贵州博锐生态技术有限公司提供，其N ∶P2O5∶K2O为9 ∶9 ∶10，有机质含量≥70%，中、微量元素≥12%；2)施用普通化肥尿素(CF)。有机肥用量为750.0 kg/hm2，普通化肥尿素用量为700 kg/hm2，作为基肥。

烟草追肥用KNO3，分别在4月20日(施用量37.5 kg/hm2)和5月14日(施用量75 kg/hm2) 追施，KNO3的N ∶P2O5∶K2O为13 ∶0 ∶44，管理方式与大田相同。

1.3 测定项目和方法

1.3.2 叶绿素荧光参数的测定使用M系列调制叶绿素荧光成像系统Maxi-Imaging-PAM (WALZ，德国)测定。将充分暗适应好的叶片摘下，迅速固定在叶室内的x-y台上，通过软件Imaging Win V2.40b选择测定叶片测定区域，测定荧光动力学曲线，其中测量光强1 μmol/(m2·s)，饱和脉冲光量子强度3000 μmol/(m2·s)，光化光量子通量密度186 μmol/(m2·s)。测定时各处理测定5株取平均值。

2 结果与分析

2.1 两种肥料对烟叶气体交换特性的影响

由图1可知，随着叶龄的增加，有机肥处理烟叶净光合速率(Pn)在生长前期逐渐增加，在后期逐渐降低，与化肥处理烟叶Pn有着相似的变化规律，两者在叶龄30 d时达到最大值，有机肥处理为CO224.13 μmol/(m2·s)，化肥处理为CO225.07 μmol/(m2·s)；在生长前期，两者之间差异不明显，在叶龄45 d以后，随着叶龄的增加，有机肥处理下烟叶Pn明显高于化肥处理，两者差异显著。

图1 两种肥料对烟叶气体交换参数影响的差异比较Fig.1 Differences of gas exchange parameters of tobacco leaves under two fertilizer patterns[注(Note)： OF—有机肥 Organic fertilizer; CF—化肥 Chemical fertilizer. 不同字母表示同一采样期处理间差异达到5%显著水平Different letters at the same sampling date mean significant at the 5% level.]

2.2 两种肥料对烟叶叶绿素荧光特性的影响

图2 两种肥料对烟叶叶绿素荧光参数影响的差异比较Fig.2 Differences of chlorophyll fluorescence parameters of tobacco leaves under two fertilizer patterns[注(Note)： OF—有机肥 Organic fertilizer; CF—化肥 Chemical fertilizer. 不同字母表示同一采样期处理间差异达到5%显著水平 Different letters at the same sampling date mean significant at the 5% level.]

2.3 两种肥料对烟叶叶绿素含量的影响

2.4 两种肥料对烟叶叶绿体超微结构的影响

表1 两种肥料对烤烟叶片叶绿素含量的影响

注(Note): OF—有机肥 Organic fertilizer; CF—化肥 Chemical fertilizer.不同字母表示同一采样期处理间差异达到5%显著水平 Different letters at the same sampling date mean significant at the 5% level.

表2 两种肥料对烤烟叶片叶绿素超微结构的影响

图3 叶龄15 d时烟草叶肉细胞的叶绿体超微结构Fig.3 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 15 days[注(Note)： ①—有机肥 Organic fertilizer; ②—化肥 Chemical fertilizer. Ch—叶绿体Chloroplast; S—淀粉颗粒Starch grain.]

图4 叶龄30 d时烟草叶肉细胞的叶绿体超微结构Fig.4 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 30 days[注(Note)： ①—有机肥 Organic fertilizer; ②—化肥 Chemical fertilizer. Ch—叶绿体Chloroplast; S—淀粉颗粒Starch grain.]

图5 叶龄45 d时烟草叶肉细胞的叶绿体超微结构Fig.5 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 45 days[注(Note)： ①—有机肥 Organic fertilizer; ②—化肥 Chemical fertilizer. Ch—叶绿体Chloroplast; S—淀粉颗粒Starch grain.]

图6 叶龄60 d时烟草叶肉细胞的叶绿体超微结构Fig.6 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 60 days[注(Note)： ①—有机肥 Organic fertilizer; ②—化肥 Chemical fertilizer. Ch—叶绿体Chloroplast; S—淀粉颗粒Starch grain.]

图7 叶龄75 d时烟草叶肉细胞的叶绿体超微结构Fig.7 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 75 days[注(Note)： ①—有机肥 Organic fertilizer; ②—化肥 Chemical fertilizer. Ch—叶绿体Chloroplast; S—淀粉颗粒Starch grain.]

图9 叶龄75 d时烟草叶肉细胞的叶绿体超微结构Fig.9 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 75 days[注(Note)： ①—有机肥 Organic fertilizer; ②—化肥 Chemical fertilizer. 图中箭头所示为叶绿体片层结构The chloroplast lamellae is marked in the FIG.]

3 讨论

矿质元素参与植物光合作用、呼吸作用及物质合成等许多生理过程[13-15]。有机肥不仅含有植物生长所需的大量元素及微量元素，而且其中的微生物可对土壤中的有机物(质)进行降解和转化为土壤养分，刺激作物根系的生长，促进作物对水分和矿质营养的吸收[16]，因此，与普通化肥(尿素)相比，有机肥对烤烟K326叶片的气体交换参数、叶绿素荧光参数及叶绿素含量均产生了不同程度的影响，尽管烟株生长前期差异不明显，但随着叶龄的增加，有机肥处理的优势逐渐显现，尤其在叶龄75 d时，其烟叶净光合速率、气孔导度及蒸腾速率分别比化肥处理高135.3%、84.5%和51.3%，烟叶的叶绿素荧光参数Y[Ⅱ]和qL分别比化肥处理高22.2%和39.5%，Fv/Fm在叶龄60 d时比化肥处理高18.7%，而NPQ值则相反，在叶龄45 d时化肥处理比有机肥处理高23.3%(图2)。NPQ是过剩光能的指示计，植物主要通过跨膜质子梯度介导的叶黄素循环来耗散多余光能[7]。在后期，化肥处理的烟叶NPQ值较高，一方面说明随着烟叶衰老，光合能力下降，光能过剩，另一方面说明叶黄素积累增加了叶片对光的耗散能力，使烟株免于强光灼伤。在叶龄60 d时，有机肥处理的叶片叶绿素(Chl a+b)含量比普通化肥高136.1%(表2)，说明施用有机肥可显著提高叶绿素含量，延缓烟株衰老，这与前人的研究结果一致[17]。

两种施肥处理，烟叶栅栏细胞超微结构观察结果表明，叶绿体形态随着叶龄的增加而逐渐增大，随着细胞逐渐趋于衰老，基粒垛叠数小于10的片层占基粒垛叠片层总数的百分比(I值)逐渐增加，淀粉颗粒在叶绿体内所占的面积比例(Ra)也逐渐增大，最终叶绿体被膜破裂而释放出淀粉颗粒，这与王程栋等[18]的研究结果一致。叶绿体形态的变化主要原因可能是其内部孕育的淀粉粒膨大的缘故，两处理Ra值差异明显，尤其是在叶龄45 d时，化肥处理的烟叶Ra值比有机肥处理高31.8%，但这并不表明化肥栽培下烟叶合成淀粉的能力强。比较两种施肥处理的叶绿体形态，有机肥处理的烟叶叶绿体呈梭形，长宽比大，而化肥处理的叶绿体形态近圆形，长宽比小；与化肥处理相比，有机肥可显著提高叶绿体基粒垛叠数大于10的片层占基粒垛叠片层总数的百分比，这也是作物提高光合效率的关键所在。

4 结论

施用有机肥可显著提高烟叶叶绿素含量及光合速率，且随叶龄的增加，有机肥的作用效果日趋明显。叶绿素荧光参数的差异表明，增施生物有机肥可延缓烟株衰老、提高烟叶光能利用效率。两种施肥方式下，淀粉颗粒Ra值的差异不能说明施用化肥可提高烟叶合成淀粉的能力。与普通化肥处理下的烟叶相比，有机肥可显著提高叶绿体基粒垛叠数大于10的片层占基粒垛叠片层总数的百分比。

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Differences of leaf gas exchange traits, chlorophyll fluorescence characteristics and chloroplast ultrastructure ofNicotianatabacumL. K326 under organic fertilization and chemical fertilization

WANG Tong-ming1, CHEN Wei2, PAN Wen-jie2, YAO Juan3, WEI Ke-su2, CHEN Bo1, LIU Chuan1, DONG Bo1, ZONG Xue-feng1*, WANG San-gen1*

(1CollegeofAgronomyandBiotechnology,SouthwestUniversity,Chongqing400716,China; 2GuizhouTobaccoScienceResearchInstitute,Guiyang,Guizhou550081,China; 3AgriculturalBureauofShuichengCounty,Liupanshui,Guizhou553040,China)

【Objectives】 Study of fertilizer effects on growth and development of tobacco mainly focuses on investigating tobacco’s macroscopic indexes (such as agronomic traits, photosynthetic efficiency, and etc.), but how fertilizers affect tobacco cell microstructure in different periods is seldom reported. In this experiment, using cultivarNicotianatabacumL. K326 as a material, we studied differences of tobacco photosynthesis, leaf gas exchange traits, chlorophyll fluorescence characteristics, chlorophyll content and chloroplast ultrastructure under two different fertilizer treatments, in order to reveal mechanism of fertilizer effects on photosynthesis in both photosynthetic efficiency and chloroplast ultrastructure aspects, and we hope these can provide a theoretical basis for exploring high yield and quality mode in tobacco cultivation.【Methods】 Vermiculite medium was used in pot experiment. Performed the flue-cured tobacco special organic fertilizer(N ∶P2O5∶K2O=9 ∶9 ∶10，organic matter≥70%，medium and trace elements≥12%)and common urea as the two treatments. Industrialized virus-free tobacco-plants were used for sampling or measurements to which the 6th-8thleaves taking superincumbent phyllotaxis during the leaf age of 15-days, 30-days, 45-days, 60-days and 75-days, and calculating the leaf age from the leaves up to around 2 cm length. The LI-6400 Portable Photosynthesis System (LICOR Inc., Lincoln, Nebraska, USA) was used to measure the net photosynthetic rate, stomatal conductance and transpiration. The Maxi-Imaging-PAM (WALZ, Germany) were used to measure the chlorophyll fluorescence parameters. A spectrophotometer was used to determine the absorbance values of the leaves pigments maceration extract, and calculate the content. The C-7000 Electron Microscope was used for chloroplast ultrastructure observation, the relative ratio of starch granules to chloroplasts (Ra value) was calculated by AutoCAD 2004 (Autodesk, Inc., USA) from digital pictures. Statistical methods of the chloroplast grana lamellae were used according to Teichler-Zallen (improved by Goodenough).【Results】 The results indicate that the organic fertilizer could increase the leaf chlorophyll content and photosynthetic rate, and the effects of the organic fertilizer are improved with the leaf age increasing. At the 60 day of leaves, the leaf chlorophyll content under the organic fertilizer application is 136.1% higher than which under the chemical fertilizer application. At the 75th day, compared to the chemical fertilizer application the photosynthetic rate, conductance and transpiration rate are 135.3%, 84.5% and 51.3% higher, respectively, and the chlorophyll fluorescence parameters, Y[Ⅱ] and qL, are 22.2% and 39.5% higher than the chemical fertilizer application and the leaf intrinsic photochemical efficiency (Fv/Fm) is 18.7% higher than that at the 60th day, while the non-photochemical quenching (NPQ) is 23.2% lower than the chemical fertilizer application during the 45 day growth stages. Starch grains increase with the leaf age gradually increasing, meanwhile, the area ratio of starch grain to chloroplast (Ra value) is increased. The difference of Ra values between the two different treatments are significant, especially in the 45 leaf-age days, and the leaf Ra value under the chemical fertilizer application is higher than that under the organic fertilizer application (31.8%). Comparing the chloroplast shapes under the two fertilization patterns, the organic fertilizer treatment appears fusiform with a higher aspect ratio, while the chemical fertilizer appears round shape with a lower aspect ratio. 【Conclusions】 The organic fertilizer can significantly increase the tobacco leaf chlorophyll content and photosynthetic rate, and these effects are gradually enhanced with the leaf age increasing. The differences of chlorophyll fluorescence parameters between two different treatments show that the organic fertilization could delay crop senescence and enhance light utilization efficiency of tobacco leaves. The difference of Ra values between two fertilization modes cannot represent if the ability of starch synthesis under the chemical fertilizer application is better. By contrast, the organic fertilization can significantly increase the ratio of chloroplast grana which is the number of lamellae >10 to the total grana lamellae.

tobacco leaf; organic fertilizer; chlorophyll fluorescence; chloroplast; ultrastructure

2013-09-03 接受日期： 2014-06-23

国家烟草专卖局科技重大专项(Ts-02-20110015)；高等学校学科创新引智计划项目 (B12006) 资助。

王通明(1986—)，男，甘肃省永靖县人，硕士研究生，主要从事植物生理与生化方面的研究。E-mail: wtming@hotmail.com * 通信作者 E-mail: zxfeng@swu.edu.cn; wangsg@swu.edu.cn

S572.062.01

1008-505X(2015)02-0517-10