王曉燕,彭禮瓊,金則新,*

1 臺(tái)州學(xué)院生態(tài)研究所,臺(tái)州　318000 2 浙江省植物進(jìn)化生態(tài)學(xué)與保護(hù)重點(diǎn)實(shí)驗(yàn)室,臺(tái)州　318000 3 湖州市梁希森林公園管理處,湖州　313000

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模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗生長(zhǎng)與光合生理特性的影響

王曉燕1,2,彭禮瓊1,3,金則新1,2,*

1 臺(tái)州學(xué)院生態(tài)研究所,臺(tái)州318000 2 浙江省植物進(jìn)化生態(tài)學(xué)與保護(hù)重點(diǎn)實(shí)驗(yàn)室,臺(tái)州318000 3 湖州市梁希森林公園管理處,湖州313000

全球變暖已對(duì)植物尤其是珍稀瀕危植物產(chǎn)生重要影響。AMF對(duì)植物應(yīng)對(duì)氣候變化具有重要意義,但是在瀕危植物應(yīng)對(duì)氣候變暖過(guò)程中發(fā)揮的作用并不清楚。以瀕危植物夏蠟梅(Sinocalycanthuschinensis)一年生幼苗為對(duì)象,研究人工模擬增溫條件下接種AMF對(duì)其生長(zhǎng)、形態(tài)建成、光合生理、抗氧化酶活性和膜脂過(guò)氧化程度、營(yíng)養(yǎng)物質(zhì)積累和葉綠素相對(duì)含量的影響。實(shí)驗(yàn)共4個(gè)處理：模擬增溫條件下添加AMF(AMF+SW)、添加AMF(AMF)、模擬增溫(SW)和對(duì)照(CK)。結(jié)果表明：(1)接種AMF對(duì)幼苗株高、葉寬長(zhǎng)比、總根長(zhǎng)、根平均直徑、根尖總數(shù)、比根長(zhǎng)和比根表面積有顯著影響。(2)AMF+SW條件下幼苗光合日進(jìn)程呈現(xiàn)出明顯“雙峰”曲線(xiàn),AMF顯著提高葉片日均凈光合速率(Pn);光合有效輻射大于50mol m-2s-1時(shí),AMF+SW和AMF處理的Pn、最大凈光合速率Pnmax和呼吸速率Rd顯著高于CK;胞間CO2濃度大于100mol CO2/mol時(shí),AMF+SW與AMF處理的Pn、AMF+SW處理的初始羧化效率(α)及AMF處理的光合能力(Amax)顯著高于CK,而AMF+SW和AMF處理的CO2補(bǔ)償點(diǎn)均低于CK。(3)AMF處理的葉可溶性糖顯著高于其它處理,AMF+SW和AMF處理的葉可溶性蛋白顯著高于CK。因此,AMF能顯著促進(jìn)夏蠟梅幼苗的形態(tài)建成和光合作用;在模擬增溫條件下,接種AMF對(duì)夏蠟梅光合生理具有顯著影響。

夏蠟梅; 模擬增溫; 叢枝菌根真菌(AMF); 生長(zhǎng); 光合生理

全球氣候變化對(duì)自然生態(tài)系統(tǒng)的影響越來(lái)越受到人類(lèi)關(guān)注[1- 4]。到2100年,全球平均氣溫將升高1.8—4.0℃[5]。氣候變暖對(duì)植物形態(tài)指標(biāo)、生物量積累、光合生理及抗氧化酶活性有重要影響[6- 8]。這種影響將會(huì)持續(xù)作用于全球生物。對(duì)于分布范圍狹小的瀕危植物,通過(guò)生長(zhǎng)生理的調(diào)節(jié)應(yīng)對(duì)氣溫升高對(duì)于個(gè)體存活和種群維持至關(guān)重要。土壤真菌與植物相互作用對(duì)植物生長(zhǎng)生理具有重要影響,進(jìn)而對(duì)于植物適應(yīng)氣候變暖具有重要意義[9- 10]。其中叢枝菌根真菌(AMF)可與約80%的陸生植物形成菌根[11],促進(jìn)宿主植物對(duì)土壤中P,N,K等礦質(zhì)養(yǎng)分的吸收[12- 13],提高植物凈光合速率[14- 15]和生物量[16],通過(guò)對(duì)植物根系形態(tài)和生物量分配的影響使植物更有效的利用資源[17]。AMF還可緩解植物對(duì)干旱[18]、鹽分[19- 21]、溫度[22- 23]、病蟲(chóng)害[24]和重金屬[25]等環(huán)境脅迫的影響,但是,AMF在瀕危植物應(yīng)對(duì)氣候變暖過(guò)程中發(fā)揮怎樣的作用并不清楚。

夏蠟梅(Sinocalycanthuschinensis)為蠟梅科(Calycanthaceae)夏蠟梅屬,中國(guó)特有第三紀(jì)孑遺物種和國(guó)家二級(jí)重點(diǎn)保護(hù)植物,具有藥用價(jià)值和較高的觀賞價(jià)值[26]。歷史時(shí)期的氣候變遷導(dǎo)致夏蠟梅種群大幅減少,而自然生境的人為破壞和對(duì)種子大量采集導(dǎo)致的種群更新受阻使該物種資源十分有限[26],目前分布范圍狹小,極度瀕危[27]。前期研究表明,現(xiàn)存自然種群夏蠟梅具有較高的AMF侵染率(> 80%)。目前,有關(guān)夏蠟梅能否通過(guò)與AMF共生適應(yīng)氣候變暖的研究尚未見(jiàn)報(bào)道。

模擬增溫已成為當(dāng)前生態(tài)學(xué)家研究氣候變暖對(duì)植物影響的主要手段[28- 29]。以夏蠟梅一年生幼苗為對(duì)象,研究模擬增溫條件下接種AMF對(duì)夏蠟梅生長(zhǎng)、光合生理特性、保護(hù)酶活性及營(yíng)養(yǎng)物質(zhì)積累的影響。試圖闡明接種AMF在夏蠟梅響應(yīng)氣候變暖過(guò)程中發(fā)揮的作用,為夏蠟梅的繁衍和復(fù)壯以及在人工繁育中如何利用AMF促進(jìn)植株生長(zhǎng)提供理論依據(jù)。

1　材料與方法

1.1實(shí)驗(yàn)材料

夏蠟梅種子采自浙江省天臺(tái)縣大雷山野生種群。對(duì)種子萌發(fā)和幼苗培養(yǎng)基質(zhì)(有機(jī)土∶河沙=1∶2,有機(jī)土為浙江省天臺(tái)山森林表層至20 cm深土壤,過(guò)0.5 cm篩網(wǎng)去除石塊)高壓蒸汽滅菌(121 ℃,2 h)。2012年9月2日,對(duì)夏蠟梅種子預(yù)處理(消毒,種皮軟化及水浴吸脹)后,播于裝有滅菌基質(zhì)的塑料筐(50 cm×40 cm×30 cm)中,于遮陰條件下萌發(fā)。待夏蠟梅長(zhǎng)至4葉苗齡,于2012年11月15日取相似大小的幼苗移栽到花盆(上口徑13 cm,底徑9 cm,高11 cm)中,每盆1棵。

1.2實(shí)驗(yàn)設(shè)計(jì)

本研究設(shè)置模擬溫度(增溫、不增溫)和AMF(接種、不接種)兩因素兩水平,共4個(gè)處理：模擬增溫(Simulated Warming,SW)+AMF(SW+AMF)、AMF、模擬增溫(SW)、和對(duì)照(CK)。增溫處理采用紅外線(xiàn)輻射加熱管(Electric radiant infrared heater,Model MR- 2420,Kalglo Electronics,USA),調(diào)整加熱管高度使植株頂部溫度增加2℃;AMF接種物為天臺(tái)大雷山野生夏蠟梅的根際土(平均每毫升土壤28.3個(gè)孢子),接種AMF采用5%野外采集的土壤與95%基質(zhì)(體積比),混勻后待用[30- 31]。不接種AMF處理則添加5%(體積比)接種物的濾液(濾網(wǎng)孔徑45m以去除AMF)[22],以保證其它微生物一致。 每個(gè)處理15個(gè)重復(fù),在遮陰(37%全光照)條件下進(jìn)行。實(shí)驗(yàn)處理時(shí)間為2013年3月4日—2013年7月25日。據(jù)2013年7月統(tǒng)計(jì),SW+AMF、AMF、SW和CK處理的AMF侵染率分別達(dá)到71.11%、70.00%、5.56%和3.33%。

1.3測(cè)定指標(biāo)及測(cè)定方法1.3.1生長(zhǎng)指標(biāo)的測(cè)定

于2013年7月25日—7月31日對(duì)所有處理中夏蠟梅幼苗生長(zhǎng)指標(biāo)進(jìn)行測(cè)定。株高和基徑分別用直尺與游標(biāo)卡尺測(cè)定。葉片形態(tài)指標(biāo)(葉面積、葉長(zhǎng)、葉寬、葉周長(zhǎng)和寬長(zhǎng)比)用掃描儀(Epson 1680)與WinFOLIA葉片分析系統(tǒng)(Regent Instruments Inc., Quebec, Canada)測(cè)定;根系形態(tài)指標(biāo)(總根長(zhǎng)、總根表面積、總根體積、根平均直徑和根尖總數(shù))用掃描儀(Epson 1680)與WinRHIZO根系分析系統(tǒng)(Regent Instruments Inc., Quebec, Canada)測(cè)定。測(cè)定時(shí)將收獲的每株幼苗的葉片和根系取下分別置于掃描儀中掃描,葉片和根系分析系統(tǒng)對(duì)掃描的圖片進(jìn)行識(shí)別和定量化分析獲得相應(yīng)數(shù)據(jù)。根、莖、葉分別于105 ℃殺青后70 ℃烘至恒重。同時(shí)計(jì)算獲得：比根長(zhǎng) = 總根長(zhǎng) / 根干重;比根表面積 = 根總表面積 / 根干重。

1.3.2光合生理指標(biāo)的測(cè)定

光合日進(jìn)程、光響應(yīng)曲線(xiàn)和CO2響應(yīng)曲線(xiàn)于2013年7月25日—7月31日,采用Li- 6400便攜式光合作用測(cè)定系統(tǒng)(LI-COR, Lincoln, USA)進(jìn)行測(cè)定。每個(gè)處理隨機(jī)選取3株,每株取1片健康、葉位一致的功能葉為樣葉,每個(gè)樣葉重復(fù)測(cè)3次,結(jié)果取平均值。光合日進(jìn)程的測(cè)定選擇晴朗少云的天氣,測(cè)定時(shí)間為6:00—18:00,每隔2 h測(cè)定1次。測(cè)定參數(shù)包括葉片凈光合速率(Pn)、蒸騰速率(Tr)、胞間CO2濃度(Ci)、氣孔導(dǎo)度(Gs)等。

光響應(yīng)曲線(xiàn)在9: 00—11: 00測(cè)定,采用LED光源控制光合有效輻射依次為2000、1500、1200、1000、800、600、400、200、150、100、50、20、0 μmol m-2s-1。光響應(yīng)曲線(xiàn)模型用直角雙曲線(xiàn)修正模型[32],同時(shí)用光合4.1.1軟件對(duì)響應(yīng)曲線(xiàn)擬合,得到初始量子效率(a),最大凈光合速率(Pnmax),光補(bǔ)償點(diǎn)(LCP),光飽和點(diǎn)(LSP) 和暗呼吸速率(Rd)。

CO2響應(yīng)曲線(xiàn)的測(cè)定采用LED光源將光強(qiáng)控制在1200 μmol m-2s-1(接近葉片飽和光強(qiáng)),以小鋼瓶?jī)?nèi)液態(tài)CO2為氣源,控制CO2濃度梯度依次為1500、1200、1000、800、600、400、200、150、120、100、80 μmol/mol。CO2響應(yīng)曲線(xiàn)模型用直角雙曲線(xiàn)修正模型[33],同時(shí)用光合4.1.1軟件對(duì)CO2響應(yīng)曲線(xiàn)擬合,得到初始羧化效率(α),光合能力(Amax),飽和胞間CO2濃度(Cisat),CO2補(bǔ)償點(diǎn)和光呼吸速率(Rp)。

1.3.3抗氧化酶與膜脂過(guò)氧化程度的測(cè)定

超氧化物歧化酶(SOD)活性的測(cè)定采用氮藍(lán)四唑(NBT)光化還原法,以抑制NBT光化還原50%的酶量為1個(gè)酶活力單位[34]。過(guò)氧化物酶(POD)和過(guò)氧化氫酶(CAT)活性的測(cè)定采取愈創(chuàng)木酚法,分別以每分鐘A470增加0.01和每分鐘A240減少0.01為1個(gè)酶活力單位[34]。丙二醛(MDA)含量的測(cè)定采用硫代巴比妥酸(TBA)法[34]。

1.3.4營(yíng)養(yǎng)物質(zhì)及葉綠素相對(duì)含量的測(cè)定

葉可溶性糖的測(cè)定采取蒽酮比色法[35]。葉可溶性蛋白的測(cè)定采取考馬斯亮藍(lán)染色法[34]。葉綠素相對(duì)含量用CCM- 200手持式葉綠素測(cè)定儀(OPTI-SCIENCES,USA)測(cè)定。

1.4數(shù)據(jù)分析

用SPSS16.0軟件對(duì)不同處理生長(zhǎng)和生理指標(biāo)進(jìn)行方差分析(P< 0.05),對(duì)符合方差齊性的參數(shù)用LSD 法對(duì)各參數(shù)平均數(shù)進(jìn)行顯著性檢驗(yàn)和多重比較,對(duì)不符合方差齊性的參數(shù)用Dunnett T3法對(duì)各參數(shù)平均數(shù)進(jìn)行顯著性檢驗(yàn)和多重比較。同時(shí),以模擬增溫和接種AMF作為固定因子,對(duì)各指標(biāo)進(jìn)行雙因素方差分析。此外,還對(duì)夏蠟梅幼苗光合生理生態(tài)指標(biāo)進(jìn)行Pearson相關(guān)性分析。

2　結(jié)果與分析

2.1模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗生長(zhǎng)與形態(tài)建成的影響 2.1.1模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗生長(zhǎng)的影響

對(duì)夏蠟梅幼苗生長(zhǎng)指標(biāo)分析表明,模擬增溫條件下添加AMF(AMF+SW)和AMF處理的株高均顯著高于CK(表1)。模擬增溫(SW)對(duì)株高沒(méi)有顯著影響。AMF處理的莖生物量比也顯著高于CK(P= 0.027)。模擬增溫和添加AMF對(duì)夏蠟梅幼苗基徑、總生物量、葉生物量比及根生物量比沒(méi)有顯著影響。雙因素方差分析表明(表1)：AMF對(duì)夏蠟梅幼苗株高有極顯著影響。SW對(duì)夏蠟梅幼苗的生長(zhǎng)指標(biāo)雖高于CK,但差異不顯著。AMF與SW的交互作用對(duì)夏蠟梅幼苗莖生物量比的影響達(dá)到顯著,而對(duì)其它生長(zhǎng)指標(biāo)均沒(méi)有影響。

表1　模擬增溫條件下接種叢枝菌根真菌對(duì)夏蠟梅幼苗生長(zhǎng)的影響

AMF：添加叢枝菌根真菌 Arbuscular Mycorrhizal Fungi;SW：模擬增溫 Simulated Warming;AMF+SW：既添加AMF又模擬增溫;數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤,同列數(shù)據(jù)不同小寫(xiě)字母表示不同處理間存在顯著差異(P< 0.05);*表示P< 0.05; **表示P< 0.01

2.1.2模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗葉片形態(tài)建成的影響

由表2可知,AMF+SW處理?xiàng)l件下夏蠟梅幼苗總?cè)~片數(shù)顯著高于SW處理。與CK相比,AMF處理的葉平均長(zhǎng)度高出18.48%,且差異達(dá)到顯著。同時(shí),CK處理的葉寬長(zhǎng)比比AMF高出16.36%。AMF+SW處理的總?cè)~片數(shù)、總?cè)~面積、葉平均寬度、葉平均長(zhǎng)度、葉平均周長(zhǎng)均高于CK,但未達(dá)到顯著差異。雙因素方差分析表明(表2)：AMF對(duì)夏蠟梅幼苗葉寬長(zhǎng)比有顯著影響。

表2模擬增溫條件下接種叢枝菌根真菌對(duì)夏蠟梅幼苗葉片形態(tài)的影響

Table 2Effects of arbuscular mycorrhizal fungi inoculation on the leaf morphology ofSinocalycanthuschinensisseedlings under simulated warming

處理Treatment總?cè)~片數(shù)Totalleafnumber總?cè)~面積/cm2Totalleafarea葉平均寬度/cmAverageleafbreadth葉平均長(zhǎng)度/cmAverageleaflength葉平均周長(zhǎng)/cmAverageleafperimeter葉寬長(zhǎng)比Breadth/lengthAMF+SW7.22±0.57a118.19±10.57a3.75±0.24a6.38±0.29ab16.44±0.94a0.59±0.01aAMF6.33±0.50ab108.53±9.64a3.63±0.22a6.86±0.41a17.22±1.05a0.55±0.02aSW5.56±0.53b97.29±7.82a3.96±0.20a6.51±0.39ab17.45±0.94a0.63±0.05aCK6.44±0.44ab96.25±6.75a3.61±0.11a5.79±0.33b15.84±0.65a0.64±0.04aFAMF1.9422.7400.2201.7570.0494.459*SW0.0060.6591.3510.0610.1720.197AMF×SW3.1030.4820.2952.7841.7060.350

數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤,同列數(shù)據(jù)不同小寫(xiě)字母表示不同處理間存在顯著差異(P< 0.05); *表示P< 0.05; **表示P< 0.01

2.1.3模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗根系形態(tài)建成的影響

由表3可知,接種AMF顯著改變了夏蠟梅幼苗根系形態(tài)。CK處理的總根長(zhǎng)、總根表面積、根尖總數(shù)、比根長(zhǎng)和比根表面積分別比AMF處理高47.63%、21.79%、122.47%、46.74%、17.35%,而AMF處理的根平均直徑則比CK高12.36%。且AMF處理的總根長(zhǎng)、根平均直徑、根尖總數(shù)和比根長(zhǎng)與CK存在顯著差異。與CK相比,SW處理顯著降低了夏蠟梅幼苗的根尖總數(shù),對(duì)其它指標(biāo)均無(wú)顯著影響。與AMF類(lèi)似,AMF+SW處理顯著提高夏蠟梅幼苗根平均直徑和顯著降低根尖總數(shù)。雙因素方差分析也表明(表3)：AMF對(duì)總根長(zhǎng)、根平均直徑、根尖總數(shù)、比根長(zhǎng)和比根表面積均存在顯著影響。且AMF與SW對(duì)根平均直徑和根尖總數(shù)的影響存在顯著的交互作用。SW對(duì)根系形態(tài)各指標(biāo)無(wú)顯著影響。

表3模擬增溫條件下接種叢枝菌根真菌對(duì)夏蠟梅幼苗根系形態(tài)的影響

Table 3Effects of arbuscular mycorrhizal fungi inoculation on the root morphology ofSinocalycanthuschinensisseedlings under simulated warming

數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤,同列數(shù)據(jù)不同小寫(xiě)字母表示不同處理間存在顯著差異(P< 0.05);*表示P< 0.05;**表示P< 0.01

2.2模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗光合生理生態(tài)的影響2.2.1模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗光合日進(jìn)程的影響

由圖1可知,AMF+SW條件下夏蠟梅幼苗光合日進(jìn)程為“雙峰”曲線(xiàn)。Pn于10: 00出現(xiàn)第1個(gè)峰值,14:00左右出現(xiàn)第2個(gè)峰值,12：00存在明顯的光合午休現(xiàn)象。AMF+SW處理的Pn在14:00和16:00顯著高于CK,而在18:00顯著低于CK。模擬增溫處理?xiàng)l件下的Gs在大多數(shù)觀察時(shí)間高于非增溫處理。AMF+SW處理組的葉片Gs變化規(guī)律與Pn一致,也呈現(xiàn)出“雙峰”。葉片Tr的變化呈現(xiàn)“單峰”曲線(xiàn),而SW處理在12:00具有較高的Gs,這與正午較高的Tr相一致。

對(duì)各參數(shù)日均值的比較也發(fā)現(xiàn)(表4)：AMF+SW的日均Pn、Gs、Ci和Tr均高于CK,且AMF+SW的日均Pn顯著高于SW處理。雙因素方差分析也表明,AMF對(duì)夏蠟梅幼苗日均Pn有顯著影響。相關(guān)性分析表明,日均Pn與Gs和Tr呈現(xiàn)顯著正相關(guān)。

圖1　模擬增溫條件下接種叢枝菌根真菌對(duì)夏蠟梅幼苗葉片凈光合速率(Pn)、葉片氣孔導(dǎo)度(Gs)、胞間CO2濃度(Ci)和蒸騰速率(Tr)日變化的影響Fig.1　Effects of arbuscular mycorrhizal fungi inoculation on the diurnal variations of net photosynthetic rate, stomatal conductance, intercellular CO2 concentration and transpiration rate in the leaves of Sinocalycanthus chinensis seedlings under simulated warmingAMF(Arbuscular Mycorrhizal Fungi)表示添加叢枝菌根真菌;SW(Simulated Warming)表示模擬增溫;AMF+SW表示既添加AMF又模擬增溫;圖中數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤

2.2.2模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗光響應(yīng)的影響

用直角雙曲線(xiàn)修正模型對(duì)不同處理?xiàng)l件下夏蠟梅幼苗光響應(yīng)擬合發(fā)現(xiàn),擬合效果較好(表5)。由圖2可知,各處理?xiàng)l件下夏蠟梅幼苗葉片Pn均呈現(xiàn)出隨光合有效輻射先迅速增加然后逐漸變緩甚至略有下降的趨勢(shì)。其中,當(dāng)光合有效輻射大于50mol m-2s-1時(shí),AMF+SW和AMF處理的Pn顯著高于CK,但是與SW處理不存在顯著差異。雙因素方差分析也表明,光合有效輻射大于50mol m-2s-1時(shí),AMF對(duì)夏蠟梅幼苗葉片的Pn有顯著影響,而SW對(duì)Pn無(wú)顯著影響, 且AMF與SW在各光合有效輻射條件下對(duì)Pn的影響不存在顯著的交互作用。

從表5可以看出,和CK相比,AMF+SW處理的初始量子效率(a)、最大凈光合速率(Pnmax)、光補(bǔ)償點(diǎn)(LCP)、光飽和點(diǎn)(LSP)和暗呼吸速率(Rd)分別增加了23.21%、68.22%、17.16%、32.63%和47.63%,其中Pnmax和Rd的差異顯著。AMF處理與AMF+SW處理結(jié)果類(lèi)似,各光響應(yīng)參數(shù)均高于CK,其中Pnmax和Rd的差異顯著。SW處理的各光合參數(shù)指標(biāo)均高于CK,但都無(wú)顯著差異。雙因素方差分析表明(表5)：AMF處理對(duì)夏蠟梅幼苗的最大凈光合速率(Pnmax)的影響達(dá)到極顯著水平,對(duì)LSP和Rd的影響達(dá)到顯著水平。SW處理僅對(duì)夏蠟梅幼苗的LSP有顯著影響。

表4模擬增溫條件下接種叢枝菌根真菌對(duì)夏蠟梅幼苗葉片光合參數(shù)的影響

Table 4Effects of arbuscular mycorrhizal fungi inoculation on the photosynthetic parameters of leaves ofSinocalycanthuschinensisseedlings under simulated warming

處理日均凈光合速率(Pn)日均氣孔導(dǎo)度(Gs)日均胞間CO2濃度(Ci)日均蒸騰速率(Tr)TreatmentDailymeannetDailymeanstomatalDailymeanintercellularDailymeantranspirationphotosyntheticrateconductanceCO2concentrationrate/(μmolm-2s-1)/(mmolm-2s-1)/(μmol/mol)/(mmolm-2s-1)AMF+SW1.513±0.090a0.036±0.008a296.907±19.433a1.407±0.274aAMF1.334±0.064ab0.029±0.002a310.259±6.373a1.231±0.075aSW1.189±0.091b0.032±0.007a318.640±10.749a1.311±0.282aCK1.242±0.107ab0.023±0.005a279.943±11.457a0.911±0.182aFAMF5.447*0.7490.1110.895SW0.4991.8800.9661.721AMF×SW1.6900.0304.0740.260

數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤,同列數(shù)據(jù)不同小寫(xiě)字母表示不同處理間存在顯著差異(P< 0.05); *表示P< 0.05; **表示P< 0.01

表5模擬增溫條件下接種叢枝菌根真菌對(duì)夏蠟梅幼苗葉片光響應(yīng)參數(shù)的影響

Table 5Effects of arbuscular mycorrhizal fungi inoculation on the light response parameters of leaves ofSinocalycanthuschinensisseedlings under simulated warming

處理Treatment初始量子效率(a)Initialquantumefficiency最大凈光合速率(Pnmax)Maximumnetphotosynthesisrate/(μmolCO2×m-2×s-1)光補(bǔ)償點(diǎn)(LCP)Lightcompensationpoint/(μmolCO2m-2s-1)光飽和點(diǎn)(LSP)Lightsaturationpoint/(μmolCO2m-2s-1)暗呼吸速率(Rd)Darkrespirationrate/(μmolCO2m-2s-1)決定系數(shù)(R2)DeterminationcofficientAMF+SW0.069±0.005a3.213±0.147a7.769±0.406a933.297±34.179a0.468±0.004a0.997±0.001aAMF0.079±0.010a3.312±0.248a6.487±1.410a790.917±30.207a0.435±0.029a0.997±0.001aSW0.071±0.006a2.582±0.256ab6.458±0.944a801.554±4.122a0.396±0.041ab0.993±0.004aCK0.056±0.008a1.910±0.444b6.631±0.461a703.682±80.852a0.317±0.040b0.990±0.003aFAMF1.90311.940**0.4175.553*8.849*4.792SW0.1080.9480.3776.685*3.0780.164AMF×SW3.0091.7130.6500.2290.5240.475

數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤,同列數(shù)據(jù)不同小寫(xiě)字母表示不同處理間存在顯著差異(P< 0.05); *表示P< 0.05; **表示P< 0.01

2.2.3模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗CO2響應(yīng)的影響

對(duì)夏蠟梅幼苗葉片CO2響應(yīng)用直角雙曲線(xiàn)修正模型擬合發(fā)現(xiàn),擬合效果較好(表6)。如圖3所示,隨著胞間CO2濃度的增加,Pn隨之增加。與CK相比,當(dāng)胞間CO2濃度大于100mol CO2/mol時(shí),AMF+SW與AMF處理在各胞間CO2濃度下的Pn均顯著高于CK。雙因素方差分析表明,AMF在各胞間CO2濃度處理?xiàng)l件下均對(duì)Pn具有顯著影響;而SW處理及與AMF的交互作用在各胞間CO2濃度下對(duì)Pn均不存在顯著影響。由表6可知,與CK相比,AMF+SW處理的初始羧化效率(α)和光合能力(Amax)分別增加了80.00%和37.44%,CO2補(bǔ)償點(diǎn)降低了29.18%;AMF處理的Amax也比CK高出45.58%,CO2補(bǔ)償點(diǎn)比CK低24.20%。不同處理間飽和胞間CO2濃度(Cisat)和光呼吸速率(Rp)不存在顯著差異。說(shuō)明AMF+SW和AMF處理在較低的CO2濃度下即可進(jìn)行光合產(chǎn)物的凈積累,而不同處理間光呼吸速率沒(méi)有顯著差異,因此提高了夏蠟梅幼苗的光合能力。雙因素方差分析表明(表6)：AMF對(duì)夏蠟梅幼苗CO2補(bǔ)償點(diǎn)具有極顯著影響,而SW對(duì)夏蠟梅幼苗CO2響應(yīng)各參數(shù)影響均不顯著。

2.3模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗保護(hù)酶活性和膜脂過(guò)氧化程度的影響

模擬增溫條件下AMF對(duì)夏蠟梅幼苗保護(hù)酶SOD、POD和CAT活性及膜脂過(guò)氧化程度指標(biāo)MDA均無(wú)顯著影響(表7)。雙因素方差分析表明(表7)：AMF和SW及兩者交互作用對(duì)夏蠟梅幼苗保護(hù)酶和膜脂過(guò)氧化程度的影響均不顯著。

圖2　模擬增溫條件下接種叢枝菌根真菌對(duì)夏蠟梅幼苗葉片光響應(yīng)曲線(xiàn)的影響Fig.2　Effects of arbuscular mycorrhizal fungi inoculation on the light response curves of leave of Sinocalycanthus chinensis seedlings under simulated warming圖中數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤

圖3　模擬增溫條件下接種叢枝菌根真菌對(duì)夏蠟梅幼苗葉片CO2響應(yīng)曲線(xiàn)的影響Fig.3　Effects of arbuscular mycorrhizal fungi inoculation on leave CO2 response curves of Sinocalycanthus chinensis seedlings under simulated warming

Table 6Effects of arbuscular mycorrhizal fungi inoculation on the CO2response parameters of leaves ofSinocalycanthuschinensisseedlings under simulated warming

處理Treatment初始羧化效率(α)Initialcarboxylationefficiency/(molm-2s-1)光合能力(Amax)Photosytheticcapacity/(μmolCO2m-2s-1)飽和胞間CO2濃度(Cisat)SaturationintercellularCO2concentration/(μmol/mol)CO2補(bǔ)償點(diǎn)CO2compensationpoint/(μmol/mol光呼吸速率(Rp)Photorespirationrate/(μmolCO2m-2s-1)決定系數(shù)(R2)DeterminationcofficientAMF+SW0.018±0.003a10.205±0.498ab1502.248±217.340a118.489±7.183a1.994±0.248a0.998±0.001aAMF0.015±0.001ab10.809±0.969a1682.957±113.230a126.833±4.623ab1.832±0.117a0.998±0.001aSW0.013±0.003ab9.933±0.913ab2133.814±629.708a161.736±16.767bc1.919±0.402a0.993±0.003aCK0.010±0.002b7.425±1.451b1958.956±313.272a167.320±14.961c1.461±0.190a0.994±0.003aFAMF4.0263.2361.48512.132**0.7303.996SW1.5870.8790.0010.3361.4100.015AMF×SW0.0072.3460.2280.0130.3230.029

數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤,同列數(shù)據(jù)不同小寫(xiě)字母表示不同處理間存在顯著差異(P< 0.05); *表示P< 0.05; **表示P< 0.01

2.4模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗營(yíng)養(yǎng)物質(zhì)積累和葉綠素相對(duì)含量的影響

AMF處理的夏蠟梅葉可溶性糖分別比AMF+SW,SW和CK高出81.72%,35.98%和32.94%,且AMF+SW,SW和CK之間不存在顯著差異。AMF+SW和AMF處理的夏蠟梅葉可溶性蛋白分別比CK高出86.09%和30.60%,且AMF+SW處理顯著高于其它處理。雙因素方差分析表明(表8)：AMF和SW及兩者交互作用對(duì)葉綠素相對(duì)含量的影響均不顯著。SW對(duì)夏蠟梅葉可溶性糖具有顯著影響,且AMF和SW對(duì)葉可溶性糖的影響有顯著的交互作用。添加AMF和SW對(duì)夏蠟梅葉片可溶性蛋白含量的影響均達(dá)到極顯著,但兩者交互作用不顯著。

3　討論

模擬增溫條件下添加AMF對(duì)夏蠟梅幼苗光合生理的影響與AMF在夏蠟梅幼苗生長(zhǎng)過(guò)程中發(fā)揮的作用密不可分。AMF菌絲直徑較小(厚壁菌絲20—30m,薄壁菌絲2—7m)[36],僅為夏蠟梅根系平均直徑的百分之一,因此AMF能進(jìn)入夏蠟梅根系無(wú)法抵達(dá)的細(xì)小土壤孔隙吸收水分和養(yǎng)分。此外,氣候變暖還會(huì)通過(guò)土壤溫度對(duì)AMF的影響作用于共生植物。土壤溫度升高會(huì)提高AMF的拓殖率和菌絲長(zhǎng)度,從而促進(jìn)共生植物養(yǎng)分吸收和生長(zhǎng)[37- 38]其中AMF對(duì)于共生植物P元素的吸收貢獻(xiàn)最大。菌絲能分泌有機(jī)酸,磷酸酶,活化土壤中難溶性P進(jìn)而通過(guò)無(wú)隔菌絲供給宿主植物[12- 13]。在植物光合作用中,元素P是光反應(yīng)階段的輔酶,暗反應(yīng)階段固定CO2的1,5-二磷酸核酮糖羧化酶Rubisco,以及能量物質(zhì)ATP的重要組成元素。因此,AMF促進(jìn)夏蠟梅光合作用的原因之一可能是AMF對(duì)土壤中有限P元素吸收的增加。夏蠟梅對(duì)養(yǎng)分和水分吸收的增加將有利于光合作用和有機(jī)物積累。本研究表明接種AMF顯著提高了夏蠟梅最大凈光合速率Pnmax和光合能力Amax,其它研究也有類(lèi)似的結(jié)果[14- 15]。接種AMF還會(huì)影響氣孔行為,縮短氣孔關(guān)閉時(shí)間[36, 39- 40]。在光合日進(jìn)程中,夏蠟梅Pn與Gs呈現(xiàn)出一致的變化趨勢(shì),相關(guān)性分析也表明夏蠟梅幼苗日均Pn與Gs和Tr呈顯著的正相關(guān),說(shuō)明夏蠟梅光合作用主要以氣孔限制為主。添加AMF還提高了夏蠟梅幼苗葉片的LSP,說(shuō)明接種AMF可以提高夏蠟梅幼苗對(duì)強(qiáng)光的利用能力。夏蠟梅是典型的陰生植物,50%的全光照下夏蠟梅的Pnmax是100%全光照的2.6倍[26]。因此,對(duì)強(qiáng)光利用能力的提高有利于夏蠟梅開(kāi)拓新生境。在不同的CO2濃度下,接種AMF的Pn均高于CK,而CO2補(bǔ)償點(diǎn)則低于CK,說(shuō)明接種AMF可能有利于夏蠟梅幼苗光合產(chǎn)物的凈積累。

表7 模擬增溫條件下接種叢枝菌根真菌對(duì)夏蠟梅幼苗葉片保護(hù)酶活性和膜脂過(guò)氧化程度的影響

Table 7Effects of arbuscular mycorrhizal fungi inoculation on the protection enzyme activity and membrane lipid peroxide level of leavels ofSinocalycanthuschinensisseedlings under simulated warming

處理Treatment超氧化物歧化酶SOD/(U/g鮮重)過(guò)氧化物酶POD/(100Ug-1鮮重min-1)過(guò)氧化氫酶CAT/(100Ug-1鮮重min-1)丙二醛MDA/(μmol/μg鮮重)AMF+SW0.21±0.002a27.92±2.15a6.17±0.41a14.02±1.76aAMF0.21±0.01a31.10±4.96a4.63±0.36a14.12±2.21aSW0.21±0.003a28.94±2.17a4.80±0.54a14.37±1.40aCK0.21±0.01a31.10±5.56a6.50±0.98a10.54±0.55aFAMF0.0740.1040.0610.036SW0.0510.2520.1323.047AMF×SW0.1180.0243.0930.824

數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤,同列數(shù)據(jù)不同小寫(xiě)字母表示不同處理間存在顯著差異(P< 0.05); *表示P< 0.05; **表示P< 0.01

表8模擬增溫條件下接種叢枝菌根真菌對(duì)夏蠟梅幼苗葉片營(yíng)養(yǎng)物質(zhì)積累和葉綠素相對(duì)含量的影響

Table 8Effects of arbuscular mycorrhizal fungi inoculation on the nutrient accumulation and chlorophyll relative content of leaves ofSinocalycanthuschinensisseedlings under simulated warming

處理Treatment可溶性糖Solublesugar/(mg/g干重)可溶性蛋白Solubleprotein/(mg/g鮮重)葉綠素相對(duì)含量ChlorophyllrelativecontentAMF+SW22.92±4.11b12.04±0.40a14.15±1.43aAMF41.65±2.86a8.45±0.63b14.88±1.74aSW30.63±1.38b8.21±0.76bc17.27±1.56aCK31.33±2.17b6.47±0.65c14.88±1.28aFAMF0.02012.582**1.025SW8.847*13.638**0.291AMF×SW6.541*1.1001.034

數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤,同列數(shù)據(jù)不同小寫(xiě)字母表示不同處理間存在顯著差異(P< 0.05); *表示P< 0.05; **表示P< 0.01

AMF顯著提高可溶性糖的積累,而在增溫條件下添加AMF對(duì)可溶性糖的含量卻沒(méi)有影響。這可能與增溫條件下,AMF對(duì)植物糖類(lèi)的消耗增加有關(guān)。本研究表明接種AMF不僅顯著提高了夏蠟梅最大凈光合速率Pnmax和光合能力Amax,還顯著促進(jìn)了可溶性糖和可溶性蛋白的積累。AMF和模擬增溫均顯著提高葉可溶性蛋白,而模擬增溫條件下添加AMF卻對(duì)可溶性蛋白沒(méi)有影響。說(shuō)明AMF和模擬增溫對(duì)可溶性蛋白的積累具有相反的作用。

模擬增溫條件下接種AMF提高了夏蠟梅幼苗葉片的日均Pn和Pnmax,但是對(duì)生物量的積累沒(méi)有顯著影響。模擬增溫條件下對(duì)玉米接種AMF幼套球囊霉(Glomusetunicatum)后也發(fā)現(xiàn),不管增溫與否,接種AMF對(duì)玉米生物量沒(méi)有顯著影響[39]。原因可能有3個(gè)方面,首先,接種AMF提高夏蠟梅幼苗Pn的同時(shí)也提高了葉片的Rd和Rp,因此對(duì)生物量的積累沒(méi)有表現(xiàn)出顯著的影響;其次,AMF與植物共生過(guò)程中,植物光合產(chǎn)物中約有20%供給共生的AMF,而溫度升高可以通過(guò)提高菌根呼吸速率來(lái)加強(qiáng)養(yǎng)分從植物向AMF的流動(dòng),從而減少植物的有機(jī)物積累[22, 39, 41]。因此,模擬增溫條件下接種AMF雖然會(huì)提高夏蠟梅Pn,但是可能同時(shí)也提高了對(duì)有機(jī)物的消耗,其最終生物量的積累取決于兩者之差;此外,實(shí)驗(yàn)處理時(shí)間短也可能是一個(gè)原因,雖然從實(shí)驗(yàn)處理到指標(biāo)測(cè)定有8個(gè)多月,但是夏蠟梅為落葉植物,夏蠟梅有葉期僅有4—5個(gè)月,因此可能導(dǎo)致實(shí)驗(yàn)處理的效果未達(dá)到顯著。

盡管模擬增溫和AMF對(duì)夏蠟梅幼苗生物量積累沒(méi)有顯著影響,但是對(duì)根系形態(tài)與生物量分配具有顯著影響。研究發(fā)現(xiàn),接種AMF對(duì)夏蠟梅根長(zhǎng)度、根表面積、根體積及根尖數(shù)均具有顯著的負(fù)效應(yīng),而對(duì)根平均直徑則表現(xiàn)出顯著的正效應(yīng)。AMF不同水分條件下接種AMF對(duì)顯著降低了濕地植物Bidensfrondosa的根系長(zhǎng)度與根系表面積[42]。而接種AMF對(duì)植物L(fēng)ythrumsalicaria和Panicumhemitomon的根系長(zhǎng)度則具有正效應(yīng)[43],但是另外的研究則發(fā)現(xiàn)接種AMF對(duì)L.salicaria的根長(zhǎng)和根表面積沒(méi)有影響[42]。因此,AMF對(duì)共生植物根系形態(tài)和生物量分配的影響與植物物種、AMF物種及實(shí)驗(yàn)條件有關(guān)。AMF對(duì)夏蠟梅根系結(jié)構(gòu)的影響與AMF的功能密不可分,AMF能使共生植物低成本高效率的吸收養(yǎng)分,從而使共生植物對(duì)根系系統(tǒng)生長(zhǎng)的投入比未接種植株減少[44- 45],因此,接種AMF有使夏蠟梅幼苗根系變短變粗的趨勢(shì)。但是,AMF+SW與SW的根系形態(tài)不存在顯著差異。說(shuō)明模擬增溫條件下添加AMF不會(huì)影響夏蠟梅根系,或者模擬增溫會(huì)減弱AMF對(duì)夏蠟梅根系形態(tài)的影響。這與模擬增溫與AMF對(duì)夏蠟梅根系的顯著的交互作用一致。

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Effects of AMF inoculation on growth and photosynthetic physiological characteristics ofSinocalycanthuschinensisunder conditions of simulated warming

WANG Xiaoyan1,2, PENG Liqiong1, 3, JIN Zexin1,2,*

1InstituteofEcology,TaizhouUniversity,Taizhou318000,China2ZhejiangProvincialKeyLaboratoryofPlantEvolutionaryEcologyandConservation,Taizhou318000,China3HuzhouLiangxiForestParkManagementOffice,Huzhou313000,China

The global climate warming is affecting ecosystems, especially endangered plant species whose distribution is mostly restricted to few areas. Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with many plant species, supplying mineral nutrients to their host plants.Sinocalycanthuschinensisis a national second-class protected plant species, distributed in only a few areas in Zhejiang and Anhui provinces in China. Preliminary investigation has revealed higher infection rate of natural populations ofS.chinensisby AMF. The role of AMF inS.chinensisunder global warming has remained unknown. To reveal the effects of AMF onS.chinensisunder the conditions of global climate warming, the pot experiment was conducted with one-year-oldS.chinensisseedlings inoculated with AMF (inoculation of rhizosphere soil at a rate of 5%, v/v; soil without AMF was used as control) and temperature increase was simulated by an electric radiant infrared heater (+ 2℃ measured around the leaves; normal temperature was used as control). Growth rate, morphogenesis, photosynthetic physiological characteristics, antioxidant activities, membrane lipid peroxide level, nutrient content ofS.chinensisseedlings were compared under four treatments: AMF, simulated warming (SW), both AMF inoculation and simulated warming (AMF + SW), and control (CK). The results showed that: (1) AMF inoculation had a significant positive effect on plant height and average root diameter, and a negative effect on the width-to-length ratio of mature leaves, total root length, total number of root tips, special root length (root length/root biomass), and special root surface area (root surface area/root biomass). Double-factor variance analysis showed that the average root diameter and total number of root tips was significantly correlated with AMF and SW. This showed that symbiotic association between AMF andS.chinensismay modify the nutrient absorption strategy ofS.chinensisthrough modification in root morphology. (2) The diurnal change in net photosynthetic rate (Pn) of leaves inS.chinensisseedlings under AMF + SW treatment had a typical bimodal curve that was determinately regulated by stomatal conductance. AMF affected significantly the daily meanPn. After fitting the photosynthesis light response using modified models of rectangular hyperbola, we found thatPnin plants with AMF + SW and AMF treatments were distinctly higher than that of CK when light intensity was more than 50mol m-2s-1. The double-factor variance analysis showed that AMF significantly affected the daily meanPn, maximum net photosynthetic rate, light saturation point (LSP), and dark respiration rate, whereas SW had a significant effect on LSP. Fitting of the CO2response by using modified models of rectangular hyperbola revealed thatPnin plants under AMF + SW and AMF treatments was significantly higher than that in the CK with increasing the concentration of CO2. Initial carboxylation efficiency under AMF + SW treatment and photosynthetic capacity under AMF treatment were significantly higher than that of the CK. The CO2compensation point after the treatment by AMF + SW or AMF was significantly lower than that of the CK. (3) The content of soluble sugars in leaves after the treatment with AMF was significantly higher than that in other treatments. The content of soluble proteins in leaves of plants treated with AMF + SW or AMF was distinctly higher than that of the CK. The double-factor variance analysis showed that SW and AMF + SW interaction had significant effect on the content of soluble sugars in leaves, and both AMF and SW had significant effect on the content of soluble proteins in leaves. The results provided the theoretical foundation for conservation and artificial cultivation ofS.chinensisunder conditions of global climate change.

Sinocalycanthuschinensis; simulated global warming; arbuscular mycorrhizal fungi(AMF); growth; photosynthetic physiology

國(guó)家自然科學(xué)基金(31400423);浙江省自然科學(xué)基金(LQ14C030001);臺(tái)州學(xué)院生態(tài)學(xué)浙江省重點(diǎn)學(xué)科開(kāi)放課題(EKD2013-07)

2015- 01- 22; 網(wǎng)絡(luò)出版日期:2015- 11- 30

Corresponding author.E-mail: jzx@tzc.edu.cn

10.5846/stxb201501220177

王曉燕,彭禮瓊,金則新.模擬增溫條件下接種AMF對(duì)夏蠟梅幼苗生長(zhǎng)與光合生理特性的影響.生態(tài)學(xué)報(bào),2016,36(16):5204- 5214.

Wang X Y, Peng L Q, Jin Z X.Effects of AMF inoculation on growth and photosynthetic physiological characteristics ofSinocalycanthuschinensisunder conditions of simulated warming.Acta Ecologica Sinica,2016,36(16):5204- 5214.