邱華龍,趙丹陽(yáng),莫 羨,徐金柱,揭育澤,秦長(zhǎng)生*
(1. 廣東省林業(yè)科學(xué)研究院,廣東省森林培育與保護(hù)利用重點(diǎn)實(shí)驗(yàn)室,廣州 510520;2. 中山市林業(yè)有害生物防治檢疫站,廣東中山 528400)
肉桂雙瓣卷蛾為害對(duì)樟樹(shù)葉片揮發(fā)物的影響
邱華龍1,趙丹陽(yáng)1,莫 羨2,徐金柱1,揭育澤1,秦長(zhǎng)生1*
(1. 廣東省林業(yè)科學(xué)研究院,廣東省森林培育與保護(hù)利用重點(diǎn)實(shí)驗(yàn)室,廣州 510520;2. 中山市林業(yè)有害生物防治檢疫站,廣東中山 528400)
采用頂空固相微萃取(SPME)及氣相色譜-質(zhì)譜聯(lián)用儀(GC-MS)技術(shù)分析肉桂雙瓣卷蛾為害前后的樟樹(shù)葉片揮發(fā)物變化。結(jié)果表明,健康和蟲(chóng)傷的樟樹(shù)葉片中均含有26種相同的揮發(fā)物成份,但是肉桂雙瓣卷蛾為害后揮發(fā)物的相對(duì)含量發(fā)生顯著變化。肉桂雙瓣卷蛾危害后,葉片中的黃樟素含量顯著增高,健康葉片中黃樟素僅占0.6%,而蟲(chóng)傷葉片中為2.7%;但是反-水合倍半香檜烯、順-橙花叔醇、反-橙花叔醇及α-紅沒(méi)藥烯含量均顯著降低,健康葉中分別含1.9%、4.5%、11.6%和1.2%,而蟲(chóng)傷葉片中分別占0.6%、2.1%、7.0%和0.7%。揮發(fā)物組分含量變化可能在調(diào)控害蟲(chóng)取食、天敵對(duì)獵物的搜尋等行為過(guò)程中發(fā)揮作用。本研究為探索植物揮發(fā)物對(duì)害蟲(chóng)及天敵行為的影響奠定基礎(chǔ),為了解寄主-昆蟲(chóng)-天敵三層營(yíng)養(yǎng)關(guān)系的相互作用、利用天然活性化合物防治害蟲(chóng)提供理論依據(jù)。
肉桂雙瓣卷蛾;葉片;頂空固相微萃?。粴庀笊V-質(zhì)譜;揮發(fā)物
肉桂雙瓣卷蛾P(guān)olylophacassiicola隸屬于鱗翅目Lepidoptera卷蛾科Tortricidae,主要寄主為肉桂Cinnamomumcassia、樟樹(shù)C.camphora和黃樟C.Porrecium(彭石冰等,1992)。該蟲(chóng)以幼蟲(chóng)鉆食寄主植物的嫩梢,造成新梢大量死亡,主梢不斷枯死,側(cè)梢叢生(圖1)。肉桂雙瓣卷蛾在我國(guó)1年發(fā)生6-7代,第1代幼蟲(chóng)于5月下旬開(kāi)始出現(xiàn),第2-4代世代重疊明顯,每個(gè)世代約30 d(趙丹陽(yáng)等,2016)。該蟲(chóng)具趨嫩產(chǎn)卵習(xí)性,只選擇在新抽嫩梢上產(chǎn)卵,嫩梢長(zhǎng)達(dá)2 cm以上時(shí)是其大量產(chǎn)卵危害期。成蟲(chóng)產(chǎn)卵于樟樹(shù)葉背面,5-8 d后卵孵化為幼蟲(chóng),初孵幼蟲(chóng)沿葉柄爬至嫩梢、嫩葉,從嫩葉主脈或芽頂直接鉆蛀入髓心為害(圖1a),4-5 d后葉芽出現(xiàn)枯萎狀(圖1b),此時(shí)為1齡幼蟲(chóng);2齡幼蟲(chóng)蛀入嫩梢為害,當(dāng)蛀道達(dá)2 cm左右時(shí)嫩梢有萎蔫狀變化;3齡幼蟲(chóng)大量取食,蛀道約4-7 cm,梢部完全枯萎(圖1c);老熟幼蟲(chóng)掉頭回到嫩梢上方咬1小孔吐絲下垂落地,尋找合適的地方化蛹(鄭寶榮,2007)。
目前在農(nóng)林生產(chǎn)上主要是利用化學(xué)藥劑防治肉桂雙瓣卷蛾,而尚未有利用寄主植物釋放的化學(xué)信息素監(jiān)測(cè)和防治肉桂雙瓣卷蛾的相關(guān)報(bào)道。植物產(chǎn)生并釋放的揮發(fā)性物質(zhì)是形成植物氣味特征的主體,其組成成分與比例構(gòu)成了該種植物的化學(xué)指紋圖(Dicke, 2016)。這些氣味物質(zhì)調(diào)控著昆蟲(chóng)的多種行為,諸如寄主定向、產(chǎn)卵、逃避、取食、聚集和傳粉等(劉芳等,2003;郭祥令等,2011)。因此,研究這些物質(zhì),對(duì)于研究植物揮發(fā)物對(duì)昆蟲(chóng)行為的影響有著非常重要的意義,可以探索昆蟲(chóng)各種行為的內(nèi)在機(jī)理,更好地了解寄主-昆蟲(chóng)-天敵三層營(yíng)養(yǎng)關(guān)系的相互作用,為利用天然活性化合物防治害蟲(chóng)及生物防治提供理論依據(jù)。目前植物揮發(fā)物提取的方法主要有溶劑提取法、水蒸氣蒸餾法、抽氣吸附法、動(dòng)態(tài)頂空采集法和固相微萃取法(SPME)。其中SPME具有無(wú)需有機(jī)溶劑、靈敏度高、操作簡(jiǎn)單、重現(xiàn)性好、防止二次污染等優(yōu)點(diǎn),集采樣、萃取、濃縮、進(jìn)樣、分析于一體,可以直接與氣相色譜或液相色譜相聯(lián)(孫凡等,2008)。文中采用SPME技術(shù)對(duì)健康及肉桂雙瓣卷蛾為害的剛抽出的樟樹(shù)嫩葉的揮發(fā)物進(jìn)行了采集和分析,然后采用氣譜質(zhì)譜聯(lián)用技術(shù)對(duì)其揮發(fā)物進(jìn)行分離與鑒定,以期為肉桂雙瓣卷蛾的監(jiān)測(cè)和防治提供理論依據(jù)。
圖1 肉桂雙瓣卷蛾為害樟樹(shù)狀Fig.1 Cinnamum camphora was damaged by larvae of Polylopha cassiicola注:a,肉桂雙瓣卷蛾幼蟲(chóng);b,肉桂雙瓣卷蛾危害后初期癥狀;c,肉桂雙瓣卷蛾危害后后期癥狀。Note: a, Larva of Polylopha cassiicola; (b), Damage symptom of P. cassiicola at early satge;c, Damage symptom of P. cassiicola at late satge.
1.1 實(shí)驗(yàn)材料
供試植物為樟樹(shù)新抽嫩梢上的嫩葉,實(shí)驗(yàn)樣品采自廣州市天河區(qū)廣東林科院苗圃?xún)?nèi)。采集剛受肉桂雙瓣卷蛾為害的嫩梢上的新鮮葉片,設(shè)為蟲(chóng)傷組;采集健康無(wú)損的嫩梢葉作為健康對(duì)照組,每組各重復(fù)4次。
1.2 試驗(yàn)方法
實(shí)驗(yàn)儀器為頂空固相微萃取裝置收集揮發(fā)物,固相微萃取采用PDMS(100 μm)涂層的萃取頭(Supelco公司,美國(guó))。將剛采集的健康和蟲(chóng)傷的2-3個(gè)嫩梢(每個(gè)嫩梢含3-6片嫩葉,每片嫩葉長(zhǎng)約1-2 cm)放入15 mL樣品瓶中,瓶蓋上還有硅膠隔墊,將萃取頭從隔墊插入瓶中,伸出萃取頭,使萃取頭在葉片的正上方,然后置于恒溫培養(yǎng)箱中,35℃條件下萃取40 min,將收集的揮發(fā)物用于后續(xù)分析。
利用島津氣象色譜-質(zhì)譜聯(lián)用儀(GC-MS,島津QP2010 Ultra)對(duì)收集到的揮發(fā)物進(jìn)行分析。色譜柱為島津SH-RXI-5SIL MS毛細(xì)管柱(30 m × 0.25 μm × 0.25 mm),載氣為99. 99%高純氦氣,流速1.2 mL/min。進(jìn)樣口溫度250℃,質(zhì)譜采用EI電離方式,離子源溫度250℃,四級(jí)桿溫度150℃,接口溫度280℃。柱升溫程序?yàn)槌跏紲囟?0℃保留3 min,以5℃/min升至200℃,再以30℃/min升至250℃,保留4 min。采用不分流進(jìn)樣,溶劑延遲3 min。
1.3 數(shù)據(jù)分析
利用島津GC-MS工作站(GC-MS solution 2.7)對(duì)揮發(fā)物的離子譜圖的峰進(jìn)行定性和相對(duì)定量分析。采用SIMCA-P Version 15軟件包(Umetrics Umea, Sweden)對(duì)數(shù)據(jù)進(jìn)行分析。將每個(gè)揮發(fā)性化合物峰的相對(duì)百分含量進(jìn)行反正弦轉(zhuǎn)換,用于主成分PCA建模分析。利用SPSS(V22, IBM)軟件一般線性模型(General linear model: GLM)中的多元方差分析(SPSS-MANOVA)檢驗(yàn)蟲(chóng)傷和健康葉片揮發(fā)性化合物的物質(zhì)及含量差異。利用R(Version 3.3.1)軟件中的vegan軟件包進(jìn)行聚類(lèi)和變量之間的相關(guān)性分析。
健康葉片和蟲(chóng)傷葉片的揮發(fā)性物質(zhì)的GC-MS總離子流圖如圖2所示。通過(guò)NIST譜庫(kù)檢索,從健康葉片和蟲(chóng)傷葉片的離子色譜圖中均鑒定出26種揮發(fā)性物質(zhì)(表1)。健康葉片中,含量最高的前5種揮發(fā)物為異丁香烯、反-橙花叔醇、β-芹子烯、(反)3,7-二甲基-3,6-辛二烯醛和芳樟醇。
圖2 健康葉片和蟲(chóng)傷葉片揮發(fā)物的GC-MS總離子流圖Fig.2 Gas-chromatogram of volatiles of normal and insect damaged leaves
編號(hào)Number出峰時(shí)間Retentiontime化合物名稱(chēng)CompoundnameCAS號(hào)CASNumber相對(duì)百分含量(%)Relativecontent健康葉Healthy蟲(chóng)傷葉InsectdamagedP值Pvalue11267反?β?羅勒烯Beta?trans?Ocimene3779?61?119±0716±09053621301順?β?羅勒烯Bbeta?cis?Ocimene3338?55?442±1236±19049131475芳樟醇Linalool78?70?659±1579±24028141659(順)3,7?二甲基?3,6?辛二烯醛Isoneral106?26?321±0219±0404751722(反)3,7?二甲基?3,6?辛二烯醛Isogeranial1754?00?374±1179±41081261783異環(huán)檸檬醛 2,4,6?Trimethyl?4?cyclohexene?1?carboxaldehyde1335?66?626±1116±06008771840順馬鞭草烯醇Cis?Verbenol1845?30?316±0409±05009681884順式檸檬醛Neral5392?40?514±0308±04015592026黃樟素Safrole94?59?706±0327±220042?102268蒎烯Copaene3856?25?516±0119±160954112353異丁香烯Isocaryophyllene118?65?013±2816±230117122383石竹烯Caryophyllene87?44?510±0515±090526132437雙環(huán)吉馬烯Beta?Cyclogermacrane37839?63?723±1013±0500951424674?(1,5?二甲基?1,4?己二烯基)?1?甲基環(huán)己烯4?[(1E)?1,5?Dimethyl?1,4?hexadienyl]?1?methyl?1?cyclohexene29837?07?813±0708±0304941524831,5,9,9四?甲基?1,4,7環(huán)?十一碳三烯1,5,9,9?Tetramethyl?1,4,7?cycloundecatriene不詳U(kuò)nknow83±00478±310583162516gamma?姜黃烯Gamma?Curcumene451?55?815±0209±04014172528衣蘭烯Ylangene14912?44?819±0311±090203182571β?芹子烯Beta?Selinene17066?67?0103±08105±080692192585α?芹子烯Alpha?Selinene473?13?249±0548±010728202626反?水合倍半香檜烯Cis?Sesquisabinene119238?96?919±0606±020005?212660順?橙花叔醇Nerolidol7212?44?445±1021±060005?222742反?橙花叔醇E?Nerolidol40716?66?3116±2170±24004?232778α?紅沒(méi)藥烯Alpha?Bisabolene495?62?512±0407±020039?242815(E)?beta?金合歡烯 (E)?beta??Famesene18794?84?817±0506±030065253020庚?2?基酯外型3?甲基丁酸?1,7,7?三甲基二環(huán)[221]庚?2?基酯3?Methyl?but?2?enoicacid,1,7,7?trimethyl?bicyclo[221]hept?2?ylester不詳U(kuò)nknow12±0408±0603032631133?甲基?2?丁烯酸苯酯3?Methyl?2?butenoicacid,tridec?2?ynylester不詳U(kuò)nknow34±2228±150639
注:相對(duì)百分含量為平均值±標(biāo)準(zhǔn)差;P值是健康和蟲(chóng)傷葉片間的差異顯著性,P≤0.05表明差異顯著。Note: The relative contents of volatiles were indicated by means ± SD;Pvalues represent the level of difference of each volatile between normal and insect damaged leaves,P≤0.05 means significant difference.
多元方差分析結(jié)果表明,蟲(chóng)傷葉片和健康葉片之間的揮發(fā)物區(qū)別較明顯,接近顯著性水平(Pillai’s Trace=0.999,F(xiàn)1, 6=187.64,P=0.056)。單變量組間比較結(jié)果表明,蟲(chóng)傷葉片和健康葉片之間的黃樟素、反-水合倍半香檜烯、順-橙花叔醇、反-橙花叔醇和α-紅沒(méi)藥烯相對(duì)含量具有顯著差異(P≤0.05,P值分別為0.042,0.005,0.005,0.04和0.039)(表1)。
通過(guò)主成分分析對(duì)健康葉片和蟲(chóng)傷葉片的揮發(fā)性物質(zhì)進(jìn)行二維和三維PCA建模分析,表明蟲(chóng)傷葉片和健康葉片樣本可以在得分圖中明顯區(qū)分開(kāi)來(lái),并且三個(gè)主成分對(duì)模型的解釋率分別為RX[1]=39.8%、RX[2]=22.4%和RX[3]=18.3%,累積的解釋率R2Xcum=80.4%(圖3a, b)。Cluster聚類(lèi)分析表明,所有對(duì)照樣本均聚為一支,蟲(chóng)傷葉片三個(gè)樣本(T1-T3)聚為一支,而樣本4(T4)單獨(dú)為一分支(圖3c)。
圖3 主成分和聚類(lèi)分析比較蟲(chóng)傷和健康葉片的揮發(fā)物差異Fig.3 Analysis of volatiles between normal and insect damaged leaves with PCA and cluster methods注:a,二維PCA得分圖;b,三維PCA得分圖模型,其中黑色代表健康葉片,紅色代表蟲(chóng)傷葉片;c,健康(CK-CK4)和蟲(chóng)傷(T1-T4)葉片樣本聚類(lèi)分析圖。Note: a, 2-D scores plot;b, 3-D scatter plot of PCA model, in which black and red symbles represent normal and insect damaged leaves, respectively; c, cluster plot of four normal leaves samples(CK1-CK4) and four insect damaged leaves samples (T1-T4).
本研究結(jié)果表明,樟樹(shù)嫩葉受肉桂雙瓣卷蛾幼蟲(chóng)為害后,葉片中的揮發(fā)物的相對(duì)含量發(fā)生變化,其中黃樟素的含量顯著升高。據(jù)研究報(bào)道,植物在遭受植食性昆蟲(chóng)或病原菌危害時(shí),揮發(fā)物組分的質(zhì)和量會(huì)發(fā)生改變,從而影響害蟲(chóng)和媒介昆蟲(chóng)取食、天敵對(duì)寄主或獵物的搜尋等行為(Cusumanoetal., 2015;楊玉枝等,2015)。害蟲(chóng)取食寄主植物葉片后,誘導(dǎo)植物釋放揮發(fā)性化合物驅(qū)避其它植食性昆蟲(chóng)。其它昆蟲(chóng)通過(guò)感應(yīng)寄主植物揮發(fā)物含量變化而得知植物上已存在競(jìng)爭(zhēng)者、食物質(zhì)量降低或植物已啟動(dòng)化學(xué)防御等而遠(yuǎn)離受害植物(Dicke, 2016)。例如,經(jīng)過(guò)斜紋夜蛾Spodopteralittoralis取食誘導(dǎo)的水稻揮發(fā)物對(duì)褐飛虱具有明顯的拒避效果(Xuetal., 2002);菜粉蝶Pierisrapae更喜歡在健康且未受其它昆蟲(chóng)取食為害的寄主植物上產(chǎn)下后代(嚴(yán)善春等,2007)。此外,研究報(bào)道捕食性天敵和寄生性天敵在和害蟲(chóng)的協(xié)同進(jìn)化過(guò)程中,可以利用害蟲(chóng)或其寄主植物釋放的化學(xué)信號(hào)物質(zhì)對(duì)獵物進(jìn)行遠(yuǎn)距離定位(Steinbergetal., 1993)。比如,小黑瓢蟲(chóng)Delphastuscatalinae對(duì)健康、機(jī)械損傷、煙粉虱卵危害和煙粉虱若蟲(chóng)危害的花菜植株所產(chǎn)生的揮發(fā)性物質(zhì)選擇性實(shí)驗(yàn)表明,蟲(chóng)害誘導(dǎo)后,花菜植株的揮發(fā)物對(duì)小黑瓢蟲(chóng)的引誘效果顯著增強(qiáng)(徐桂萍,2011)。因此,推測(cè)樟樹(shù)被肉桂雙瓣卷蛾為害后,可能通過(guò)釋放黃樟素驅(qū)避其它害蟲(chóng)繼續(xù)為害,或者引誘天敵如螟黃赤眼蜂Trichogrammachilonis控制肉桂雙瓣卷蛾的種群數(shù)量。然而,在植物和昆蟲(chóng)的協(xié)同進(jìn)化中,有些蟲(chóng)害誘導(dǎo)的植物揮發(fā)物可以讓害蟲(chóng)受益。例如三葉楊葉甲Chrysomelapopuli(Kendrick and Raffa, 2006)和白楊葉甲Chrysomelapopuli(Brillietal., 2009)取食誘導(dǎo)的植物揮發(fā)物均對(duì)同種昆蟲(chóng)具有較強(qiáng)的引誘作用。因此,受肉桂雙瓣卷蛾為害后,黃樟素含量升高,其具體的生態(tài)功能還有待于進(jìn)一步研究。
在本研究中,反-水合倍半香檜烯、順-橙花叔醇、反-橙花叔醇及α-紅沒(méi)藥烯受肉桂雙瓣卷蛾為害后相對(duì)含量均顯著降低,推測(cè)可能是嫩葉受蟲(chóng)害后,光合作用及代謝能力減弱,影響物質(zhì)的合成和釋放。研究報(bào)道寄主植物受害蟲(chóng)危害后,將更多的能量用于自身的免疫防御從而減少揮發(fā)物的合成和釋放(Kaloshian and Walling, 2016)。此外,研究報(bào)道蟲(chóng)口密度和危害時(shí)間等因素會(huì)影響寄主植物揮發(fā)物的釋放,茶麗紋象甲Myllocerinusaurolineatus、假眼小綠葉蟬Empoascavitis、茶尺蠖Ectropisobliquehypulina危害誘導(dǎo)茶樹(shù)產(chǎn)生的吲哚在達(dá)到釋放頂峰后,其釋放量會(huì)隨著危害時(shí)間的延長(zhǎng)而逐步減少(蔡曉明,2009)。由于樟樹(shù)葉片揮發(fā)物的組分種類(lèi)較多,這些揮發(fā)物釋放時(shí)空動(dòng)態(tài)節(jié)律、對(duì)肉桂雙瓣卷蛾的作用方式及各組分之間的相互協(xié)同作用等問(wèn)題還需要深入研究,才有可能將這些植物揮發(fā)物更好地應(yīng)用于肉桂雙瓣卷蛾的生物防治中。
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The impact ofPolylophacassiicoladamage on the volatiles ofCinnamomumcamphoraleaves
QIU Hua-Long1, ZHAO Dan-Yang1, MO Xian2, XU Jin-Zhu1, JIE Yu-Ze1, QIN Chang-Sheng1*
(1. Guangdong Academy of Forestry, Guangdong Provincial Key Laboratory of Silviculture, Protection and Utilization, Guangzhou 510520, China; 2. Forest Disease and Insect Control and Quarantine Station of Zhongshan City, Zhongshan 528400, Guangdong Province, China)
Headspace solid phase micro extraction (SPME) and gas chromatography-mass spectrometry (GC-MS) technology were used to campare volatiles between normal andPolylophacassiicoladamaged leaves ofCinnamomumcamphora. Results showed that normal andP.cassiicolainjured leaves contained the same 26 volatiles, but relatively content of some volatiles were altered afterP.cassiicoladamage. Specifically, the content of safrole was significantly increased after insect damage. The relative content of safrole was only 0.6% in normal leaves, while it was increased up to 2.7% inP.cassiicoladamaged leaves. Neverrtheless, the relative contents of cis-Sesquisabinene hydrate, cis-nerolidol, anti-nerolidol and α-bisabolene were significantly reduced afterP.cassiicoladamage, they were decreased from 1.9%, 4.5%, 11.6% and 1.2% in normal leaves to 0.6%, 2.1%, 7.0% and 0.7% inP.cassiicoladamaged leaves, respectively. The alteration of content of volatiles might play a key role in regulating the insect feeding and prey searching behavior of natural enemy. This study lay the foundation for exploring the impact of plant volatiles on the behaviour of insect pests and their natural enemy, providing theoretical basis for understanding the three trophic relationships interaction among host plant, insect pest and natural enemy, as well as the use of natural active compounds for the control of insect pests.
Polylophacassiicola; leaf; SPME; GC-MS; volatile
廣東省林業(yè)科技創(chuàng)新專(zhuān)項(xiàng)(2013KJCX015-4,2015KJCX044)
邱華龍,男,1988年生,助理研究員,從事林業(yè)有害生物防治技術(shù)研究,E-mail: qiuhualong2008@163.com
*通訊作者Author for correspondence, E-mail: 919824595@qq.com
Received: 2016-10-24;接受日期Accepted: 2017-01-12
Q968.1;S433.4
A
1674-0858(2017)02-0464-07
邱華龍,趙丹陽(yáng),莫羨,等.肉桂雙瓣卷蛾為害對(duì)樟樹(shù)葉片揮發(fā)物的影響[J].環(huán)境昆蟲(chóng)學(xué)報(bào),2017,39(2):464-470.