王通明， 陳 偉， 潘文杰， 姚 娟， 韋克蘇， 陳 波，劉 川， 董 博， 宗學(xué)鳳*， 王三根*

(1 西南大學(xué)農(nóng)學(xué)與生物科技學(xué)院，重慶 400716； 2 貴州省煙草科學(xué)研究院，貴州貴陽(yáng) 550081；3 水城縣農(nóng)業(yè)局，貴州六盤水 553040)

有機(jī)肥和化肥對(duì)煙葉氣體交換、葉綠素?zé)晒馓匦约叭~綠體超微結(jié)構(gòu)的影響

王通明1， 陳 偉2， 潘文杰2， 姚 娟3， 韋克蘇2， 陳 波1，劉 川1， 董 博1， 宗學(xué)鳳1*， 王三根1*

(1 西南大學(xué)農(nóng)學(xué)與生物科技學(xué)院，重慶 400716； 2 貴州省煙草科學(xué)研究院，貴州貴陽(yáng) 550081；3 水城縣農(nóng)業(yè)局，貴州六盤水 553040)

煙葉； 有機(jī)肥； 葉綠素?zé)晒猓?葉綠體； 超微結(jié)構(gòu)

煙草(Nicotianatabacum)是典型的葉用作物，葉片中96%左右的干物質(zhì)直接或間接來(lái)自光合作用[1]。煙草在生長(zhǎng)發(fā)育過(guò)程中對(duì)礦質(zhì)元素吸收量大，需要大量施肥，但過(guò)量施用化學(xué)肥料可導(dǎo)致環(huán)境污染[2]。有機(jī)肥不僅成本較低、污染小[3]，而且能顯著促進(jìn)作物生長(zhǎng)，提高作物的產(chǎn)量和品質(zhì)[4]。已有研究證明，有機(jī)肥具有促進(jìn)烤煙生長(zhǎng)、提高產(chǎn)量、改進(jìn)煙葉品質(zhì)等作用[5]，但對(duì)比較有機(jī)肥與化肥在生育期如何影響烤煙葉綠素?zé)晒馓匦约叭~綠體超微結(jié)構(gòu)差異的報(bào)道并不多見(jiàn)。為此，本試驗(yàn)以烤煙K326為材料，探討了不同葉齡時(shí)期有機(jī)肥與化肥處理對(duì)煙葉氣體交換參數(shù)、葉綠素?zé)晒馓匦?、葉綠素含量及葉綠體超微結(jié)構(gòu)影響等方面的差異，以期從光能利用(宏觀)及葉綠體超微結(jié)構(gòu)(微觀)方面，揭示兩者對(duì)煙葉光合作用的影響機(jī)制，同時(shí)為探索煙葉高產(chǎn)優(yōu)質(zhì)栽培模式提供理論依據(jù)。

1 材料與方法

1.1 供試材料

本試驗(yàn)于2012年在貴州特色優(yōu)質(zhì)煙葉基地(貴州貴陽(yáng))進(jìn)行，試驗(yàn)地位于N24.88°，E107.10°，海拔約為1112.8 m。供試土壤為黃壤土，pH 6.6，土壤有機(jī)質(zhì)21.1 g/kg、 堿解氮61.5 mg/kg、 速效磷48.7 mg/kg、 速效鉀159.2 mg/kg。

供試烤煙品種為K326 (NicotianatabacumL. K326)，烤煙種子由貴州省煙草科學(xué)研究院提供，消毒處理后工廠化育苗，育苗基質(zhì)主要以草炭蛭石為主，煙苗于4月8日移栽到溫室土壤中。

1.2 試驗(yàn)設(shè)計(jì)

選定兩塊試驗(yàn)區(qū)域，設(shè)兩個(gè)肥料處理，1)施用烤煙專用有機(jī)肥(OF)，肥料由貴州博銳生態(tài)技術(shù)有限公司提供，其N ∶P2O5∶K2O為9 ∶9 ∶10，有機(jī)質(zhì)含量≥70%，中、微量元素≥12%；2)施用普通化肥尿素(CF)。有機(jī)肥用量為750.0 kg/hm2，普通化肥尿素用量為700 kg/hm2，作為基肥。

煙草追肥用KNO3，分別在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為13 ∶0 ∶44， 管理方式與大田相同。

1.3 測(cè)定項(xiàng)目和方法

1.3.2 葉綠素?zé)晒鈪?shù)的測(cè)定 使用M系列調(diào)制葉綠素?zé)晒獬上裣到y(tǒng)Maxi-Imaging-PAM (WALZ，德國(guó))測(cè)定。將充分暗適應(yīng)好的葉片摘下，迅速固定在葉室內(nèi)的x-y臺(tái)上，通過(guò)軟件Imaging Win V2.40b選擇測(cè)定葉片測(cè)定區(qū)域，測(cè)定熒光動(dòng)力學(xué)曲線，其中測(cè)量光強(qiáng)1 μmol/(m2·s)，飽和脈沖光量子強(qiáng)度3000 μmol/(m2·s)，光化光量子通量密度186 μmol/(m2·s)。測(cè)定時(shí)各處理測(cè)定5株取平均值。

相關(guān)參數(shù)計(jì)算公式如下[6-8]：

Fv/Fm = (Fm - Fo)/Fm；

Y[Ⅱ]= (Fm’-Ft)/Fm’；

qL=(Fm’-F)/(Fm’-Fo’) XFo’/F；

NPQ=(Fm-Fm’)/Fm’。

1.4 數(shù)據(jù)處理

對(duì)各處理不同葉齡葉肉細(xì)胞柵欄組織內(nèi)的葉綠體長(zhǎng)、寬進(jìn)行統(tǒng)計(jì)，用軟件AutoCAD 2004計(jì)算淀粉顆粒在葉綠體中所占的相對(duì)面積比Ra[10]。葉綠體基粒片層的統(tǒng)計(jì)方法據(jù)Goodenough等改進(jìn)的Teichler-Zallen法[11]，對(duì)每個(gè)處理觀察100個(gè)基粒，按照公式 I=100nN/∑nN統(tǒng)計(jì)基粒片層數(shù)與基粒個(gè)數(shù)，計(jì)算出低基粒片層(≤10)所占的百分率。(公式中I代表基粒垛疊小于10片層占基粒垛疊片層總數(shù)的百分比)。

葉綠體超微結(jié)構(gòu)圖片用Photoshop 7.0處理；采用DPS 3.01軟件進(jìn)行數(shù)據(jù)統(tǒng)計(jì)分析，LSD法檢驗(yàn)差異顯著性；EXCEL 2013軟件作圖。

2 結(jié)果與分析

2.1 兩種肥料對(duì)煙葉氣體交換特性的影響

由圖1可知，隨著葉齡的增加，有機(jī)肥處理煙葉凈光合速率(Pn)在生長(zhǎng)前期逐漸增加，在后期逐漸降低，與化肥處理煙葉Pn有著相似的變化規(guī)律，兩者在葉齡30 d時(shí)達(dá)到最大值，有機(jī)肥處理為CO224.13 μmol/(m2·s)，化肥處理為CO225.07 μmol/(m2·s)；在生長(zhǎng)前期，兩者之間差異不明顯，在葉齡45 d以后，隨著葉齡的增加，有機(jī)肥處理下煙葉Pn明顯高于化肥處理，兩者差異顯著。

圖1 兩種肥料對(duì)煙葉氣體交換參數(shù)影響的差異比較Fig.1 Differences of gas exchange parameters of tobacco leaves under two fertilizer patterns[注(Note)： OF—有機(jī)肥 Organic fertilizer; CF—化肥 Chemical fertilizer. 不同字母表示同一采樣期處理間差異達(dá)到5%顯著水平Different letters at the same sampling date mean significant at the 5% level.]

2.2 兩種肥料對(duì)煙葉葉綠素?zé)晒馓匦缘挠绊?/p>

圖2 兩種肥料對(duì)煙葉葉綠素?zé)晒鈪?shù)影響的差異比較Fig.2 Differences of chlorophyll fluorescence parameters of tobacco leaves under two fertilizer patterns[注(Note)： OF—有機(jī)肥 Organic fertilizer; CF—化肥 Chemical fertilizer. 不同字母表示同一采樣期處理間差異達(dá)到5%顯著水平 Different letters at the same sampling date mean significant at the 5% level.]

2.3 兩種肥料對(duì)煙葉葉綠素含量的影響

2.4 兩種肥料對(duì)煙葉葉綠體超微結(jié)構(gòu)的影響

表1 兩種肥料對(duì)烤煙葉片葉綠素含量的影響

注(Note): OF—有機(jī)肥 Organic fertilizer; CF—化肥 Chemical fertilizer.不同字母表示同一采樣期處理間差異達(dá)到5%顯著水平 Different letters at the same sampling date mean significant at the 5% level.

表2 兩種肥料對(duì)烤煙葉片葉綠素超微結(jié)構(gòu)的影響

注(Note): OF—有機(jī)肥 Organic fertilizer; CF—化肥 Chemical fertilizer.不同字母表示同一采樣期處理間差異達(dá)到5%顯著水平 Different letters at the same sampling date mean significant at the 5% level.

圖3 葉齡15 d時(shí)煙草葉肉細(xì)胞的葉綠體超微結(jié)構(gòu)Fig.3 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 15 days[注(Note)： ①—有機(jī)肥 Organic fertilizer; ②—化肥 Chemical fertilizer. Ch—葉綠體Chloroplast; S—淀粉顆粒Starch grain.]

圖4 葉齡30 d時(shí)煙草葉肉細(xì)胞的葉綠體超微結(jié)構(gòu)Fig.4 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 30 days[注(Note)： ①—有機(jī)肥 Organic fertilizer; ②—化肥 Chemical fertilizer. Ch—葉綠體Chloroplast; S—淀粉顆粒Starch grain.]

圖5 葉齡45 d時(shí)煙草葉肉細(xì)胞的葉綠體超微結(jié)構(gòu)Fig.5 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 45 days[注(Note)： ①—有機(jī)肥 Organic fertilizer; ②—化肥 Chemical fertilizer. Ch—葉綠體Chloroplast; S—淀粉顆粒Starch grain.]

圖6 葉齡60 d時(shí)煙草葉肉細(xì)胞的葉綠體超微結(jié)構(gòu)Fig.6 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 60 days[注(Note)： ①—有機(jī)肥 Organic fertilizer; ②—化肥 Chemical fertilizer. Ch—葉綠體Chloroplast; S—淀粉顆粒Starch grain.]

圖7 葉齡75 d時(shí)煙草葉肉細(xì)胞的葉綠體超微結(jié)構(gòu)Fig.7 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 75 days[注(Note)： ①—有機(jī)肥 Organic fertilizer; ②—化肥 Chemical fertilizer. Ch—葉綠體Chloroplast; S—淀粉顆粒Starch grain.]

圖9 葉齡75 d時(shí)煙草葉肉細(xì)胞的葉綠體超微結(jié)構(gòu)Fig.9 The chloroplast ultrastructure illustrations of tobacco mesophyll cell in leaf age of 75 days[注(Note)： ①—有機(jī)肥 Organic fertilizer; ②—化肥 Chemical fertilizer. 圖中箭頭所示為葉綠體片層結(jié)構(gòu)The chloroplast lamellae is marked in the FIG.]

3 討論

礦質(zhì)元素參與植物光合作用、呼吸作用及物質(zhì)合成等許多生理過(guò)程[13-15]。有機(jī)肥不僅含有植物生長(zhǎng)所需的大量元素及微量元素，而且其中的微生物可對(duì)土壤中的有機(jī)物(質(zhì))進(jìn)行降解和轉(zhuǎn)化為土壤養(yǎng)分，刺激作物根系的生長(zhǎng)，促進(jìn)作物對(duì)水分和礦質(zhì)營(yíng)養(yǎng)的吸收[16]，因此，與普通化肥(尿素)相比，有機(jī)肥對(duì)烤煙K326葉片的氣體交換參數(shù)、葉綠素?zé)晒鈪?shù)及葉綠素含量均產(chǎn)生了不同程度的影響，盡管煙株生長(zhǎng)前期差異不明顯，但隨著葉齡的增加，有機(jī)肥處理的優(yōu)勢(shì)逐漸顯現(xiàn)，尤其在葉齡75 d時(shí)，其煙葉凈光合速率、氣孔導(dǎo)度及蒸騰速率分別比化肥處理高135.3%、84.5%和51.3%，煙葉的葉綠素?zé)晒鈪?shù)Y[Ⅱ]和qL分別比化肥處理高22.2%和39.5%，F(xiàn)v/Fm在葉齡60 d時(shí)比化肥處理高18.7%，而NPQ值則相反，在葉齡45 d時(shí)化肥處理比有機(jī)肥處理高23.3%(圖2)。NPQ是過(guò)剩光能的指示計(jì)，植物主要通過(guò)跨膜質(zhì)子梯度介導(dǎo)的葉黃素循環(huán)來(lái)耗散多余光能[7]。在后期，化肥處理的煙葉NPQ值較高，一方面說(shuō)明隨著煙葉衰老，光合能力下降，光能過(guò)剩，另一方面說(shuō)明葉黃素積累增加了葉片對(duì)光的耗散能力，使煙株免于強(qiáng)光灼傷。在葉齡60 d時(shí)，有機(jī)肥處理的葉片葉綠素(Chl a+b)含量比普通化肥高136.1%(表2)，說(shuō)明施用有機(jī)肥可顯著提高葉綠素含量，延緩煙株衰老，這與前人的研究結(jié)果一致[17]。

兩種施肥處理，煙葉柵欄細(xì)胞超微結(jié)構(gòu)觀察結(jié)果表明，葉綠體形態(tài)隨著葉齡的增加而逐漸增大，隨著細(xì)胞逐漸趨于衰老，基粒垛疊數(shù)小于10的片層占基粒垛疊片層總數(shù)的百分比(I值)逐漸增加，淀粉顆粒在葉綠體內(nèi)所占的面積比例(Ra)也逐漸增大，最終葉綠體被膜破裂而釋放出淀粉顆粒，這與王程棟等[18]的研究結(jié)果一致。葉綠體形態(tài)的變化主要原因可能是其內(nèi)部孕育的淀粉粒膨大的緣故，兩處理Ra值差異明顯，尤其是在葉齡45 d時(shí)，化肥處理的煙葉Ra值比有機(jī)肥處理高31.8%，但這并不表明化肥栽培下煙葉合成淀粉的能力強(qiáng)。比較兩種施肥處理的葉綠體形態(tài)，有機(jī)肥處理的煙葉葉綠體呈梭形，長(zhǎng)寬比大，而化肥處理的葉綠體形態(tài)近圓形，長(zhǎng)寬比?。慌c化肥處理相比，有機(jī)肥可顯著提高葉綠體基粒垛疊數(shù)大于10的片層占基粒垛疊片層總數(shù)的百分比，這也是作物提高光合效率的關(guān)鍵所在。

4 結(jié)論

施用有機(jī)肥可顯著提高煙葉葉綠素含量及光合速率，且隨葉齡的增加，有機(jī)肥的作用效果日趨明顯。葉綠素?zé)晒鈪?shù)的差異表明，增施生物有機(jī)肥可延緩煙株衰老、提高煙葉光能利用效率。兩種施肥方式下，淀粉顆粒Ra值的差異不能說(shuō)明施用化肥可提高煙葉合成淀粉的能力。與普通化肥處理下的煙葉相比，有機(jī)肥可顯著提高葉綠體基粒垛疊數(shù)大于10的片層占基粒垛疊片層總數(shù)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

國(guó)家煙草專賣局科技重大專項(xiàng)(Ts-02-20110015)； 高等學(xué)校學(xué)科創(chuàng)新引智計(jì)劃項(xiàng)目 (B12006) 資助。

王通明(1986—)，男，甘肅省永靖縣人，碩士研究生，主要從事植物生理與生化方面的研究。E-mail: wtming@hotmail.com * 通信作者 E-mail: zxfeng@swu.edu.cn; wangsg@swu.edu.cn

S572.062.01

A

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