水楊酸信號路徑在調(diào)控入侵生物煙粉虱誘導(dǎo)植物間接防御中的作用

俞曉平,何瑜晨,酈衛(wèi)弟,趙 嬋,趙 昭,張蓬軍

(1.中國計量大學 生命科學學院 浙江省生物計量及檢驗檢疫技術(shù)重點實驗室,浙江 杭州 310018;2.浙江省農(nóng)科院 植物保護與微生物研究所,浙江 杭州 310021)

水楊酸信號路徑在調(diào)控入侵生物煙粉虱誘導(dǎo)植物間接防御中的作用

俞曉平1,何瑜晨1,酈衛(wèi)弟2,趙 嬋1,趙 昭1,張蓬軍1

(1.中國計量大學 生命科學學院 浙江省生物計量及檢驗檢疫技術(shù)重點實驗室,浙江 杭州 310018;2.浙江省農(nóng)科院 植物保護與微生物研究所,浙江 杭州 310021)

當植物受到植食者為害后,能夠迅速合成并釋放出一些揮發(fā)性物質(zhì),進而吸引植食者天敵,達到控制蟲害的目的.大量的研究表明,茉莉酸信號路徑在調(diào)節(jié)植物釋放揮發(fā)物的過程中發(fā)揮了重要作用.然而,最近的研究表明，外來入侵刺吸式昆蟲如煙粉虱,能通過激活植物體內(nèi)的水楊酸信號路徑,同時還能抑制茉莉酸信號路徑.那么,水楊酸信號路徑是否在刺吸式昆蟲誘導(dǎo)植物釋放揮發(fā)物吸引天敵過程中發(fā)揮重要作用？我們分別以擬南芥和番茄作為模式植物,研究經(jīng)煙粉虱取食為害后的野生型植株以及不同信號路徑突變體植株對麗蚜小蜂的嗅覺行為反應(yīng).研究結(jié)果表明，煙粉虱為害120 h后的野生型擬南芥或番茄植株均能夠顯著吸引麗蚜小蜂.當茉莉酸路徑被抑制時,煙粉虱為害后的擬南芥(dde2-2)或番茄(def-1和spr-2)植株同樣顯著吸引麗蚜小蜂;但是當水楊酸路徑被抑制后,煙粉虱為害后的擬南芥(NahG和npr-1)或番茄(NahG)植株則并未吸引麗蚜小蜂.植物激素分析結(jié)果表明,煙粉虱能夠誘導(dǎo)擬南芥或番茄植株內(nèi)源水楊酸含量顯著增加,但內(nèi)源茉莉酸的含量則并未變化.因此,我們認為水楊酸信號路徑在調(diào)控煙粉虱誘導(dǎo)植物間接防御過程中發(fā)揮重要作用.

刺吸式昆蟲;煙粉虱;水楊酸信號路徑;麗蚜小蜂

自然界中許多植物在受到植食者為害時,會迅速釋放出一系列揮發(fā)性化合物(Herbivore-induced plant volatiles),并利用這些蟲害誘導(dǎo)產(chǎn)生的揮發(fā)物來吸引植食性昆蟲的天敵,達到控制蟲害的目的,這一行為稱為植物的間接防御[1-3].植物間接防御已在寄主植物與咀嚼式昆蟲及螨類的互作關(guān)系中有廣泛的報道[4-6].隨著相關(guān)研究的深入,大量數(shù)據(jù)表明:茉莉酸信號路徑(JA signaling pathway)在調(diào)控蟲害誘導(dǎo)植物釋放揮發(fā)物過程中發(fā)揮重要作[7-9].例如,阻斷茉莉酸路徑能夠?qū)е轮参飺]發(fā)物釋放量減少[10],進而降低蟲害植株對天敵昆蟲的吸引作用[11-13].盡管這一植物間接防御現(xiàn)象在刺吸式昆蟲-寄主植物-天敵的三級營養(yǎng)關(guān)系中也屢有報道,但關(guān)于刺吸式昆蟲誘導(dǎo)植物釋放揮發(fā)物的內(nèi)在分子機制目前尚不清楚[14].

不同于咀嚼式昆蟲,刺吸式昆蟲利用其細長的口針直接吸食植物韌皮部營養(yǎng),并不會造成明顯的植物組織損傷[15-16].因此,其誘導(dǎo)的植物防御反應(yīng)也不同于咀嚼式昆蟲誘導(dǎo)的植物防御反應(yīng).研究表明,刺吸式昆蟲主要誘導(dǎo)植物體內(nèi)的水楊酸信號路徑(SA signaling pathway),同時抑制茉莉酸或乙烯信號路徑[17-19].例如,外來入侵昆蟲煙粉虱誘導(dǎo)擬南芥植物體內(nèi)水楊酸調(diào)控基因及蛋白的表達量顯著增加,而茉莉酸或乙烯調(diào)控的部分基因或蛋白表達量下降[19-20].值得注意的是,越來越多的試驗證據(jù)表明,刺吸式昆蟲通過抑制寄主植物茉莉酸防御反應(yīng),進而促進其生長發(fā)育[19,21].然而,從植物-植食者-天敵長期協(xié)同進化的角度分析,許多學者推測寄主植物應(yīng)該能夠識別刺吸式昆蟲的取食因子,并已經(jīng)發(fā)展相應(yīng)的有效防御機制[22-23];同樣,天敵昆蟲也應(yīng)該能夠利用刺吸式昆蟲誘導(dǎo)的特異性揮發(fā)物進行寄主定位.據(jù)此,我們推測水楊酸信號路徑在調(diào)節(jié)刺吸式昆蟲誘導(dǎo)植物釋放揮發(fā)物吸引天敵的過程中發(fā)揮重要作用.這一推測也陸續(xù)得到一些相關(guān)試驗證據(jù)的支持:水楊酸信號路徑在調(diào)控菜粉蝶幼蟲及二斑葉螨誘導(dǎo)的植物揮發(fā)物的釋放中發(fā)揮一定作用[4,24,25];水楊酸甲酯是吸引蚜蟲捕食者七星瓢蟲的有效成分[26].

在本研究中,我們以外來入侵昆蟲煙粉虱為研究對象,利用擬南芥或番茄-煙粉虱-麗蚜小蜂作為研究模式系統(tǒng),首先測定麗蚜小蜂對煙粉虱為害植株的嗅覺行為反應(yīng),包括擬南芥野生型、茉莉酸沉默突變體dde2-2、水楊酸沉默突變體NahG和npr-1,以及番茄野生型、茉莉酸沉默突變體def-1和spr-2、水楊酸沉默突變體NahG;其次,測定煙粉虱取食對植物內(nèi)源激素(茉莉酸和水楊酸)的影響.通過以上研究我們希望探明水楊酸信號路徑在調(diào)控煙粉虱誘導(dǎo)植物間接防御中的作用,為未來煙粉虱的可持續(xù)治理提供新思路.

1 材料方法

1.1 植物與昆蟲

擬南芥野生型Col-0和轉(zhuǎn)基因突變體dde2-2,NahG和npr-1均種植在人工氣候室里(22±2 ℃,R.H.60～70%,8 L:16D),6～8周后的植株用于試驗.番茄野生型(Lycopersiconesculentum, cv Moneymaker)及其突變體spr-2,def-1和NahG種植在人工氣候室里(25±2 ℃,R.H.60～70%,8 L:16D),5～6周后的植株用于試驗.

煙粉虱(半翅類:粉虱科),生物型為B型,其試驗種群在人工溫室內(nèi)(25±2 ℃,R.H.50～70%,14 L:10D)由野生型番茄植株飼養(yǎng).

麗蚜小蜂在人工溫室(25±2 ℃,R.H.50～70%,14 L:10D)由野生型番茄飼養(yǎng)的煙粉虱若蟲維持其種群.在擬南芥試驗組中,試驗前將麗蚜小蜂放置在經(jīng)煙粉虱為害的擬南芥野生型植株上24 h,使其獲得產(chǎn)卵經(jīng)歷.因為以往的研究表明,成功的產(chǎn)卵經(jīng)歷能夠幫助寄生蜂獲得對植物揮發(fā)物因子的顯著偏好性[27-28].2～5日齡的麗蚜小蜂用于嗅覺試驗.嗅覺試驗時間為每天12:00到15:00,因為這期間麗蚜小蜂表現(xiàn)最活躍.

1.2 植物處理

取煙粉虱成蟲100頭,將其轉(zhuǎn)移到提前放有植物的籠子里(高21 cm,直徑為13.5 cm),讓其在植物上自由取食24、72、120 h后,用于嗅覺儀試驗;健康植株作為煙粉虱處理組的對照植物.另外,健康植株或煙粉虱為害120 h后的蟲害植株均要用于內(nèi)源激素檢測.

1.3 嗅覺儀試驗

寄生蜂對于植株揮發(fā)物的行為反應(yīng)在Y型管嗅覺儀中進行測定.凈化后的空氣(經(jīng)活性炭)分別進入2.5 L玻璃容器,然后進入到嗅覺儀的兩臂,氣流速度為100 mL/min.在Y型管底部釋放單頭寄生蜂,每頭寄生蜂觀察時間為10 min.當寄生蜂選擇任一氣味源,并在該氣味源的管壁停留超過15 s,則記錄為選擇該氣味源;當寄生蜂在10 min內(nèi)沒有做出選擇,則記錄為“未選擇”,每頭寄生蜂僅測試一次.每4棵植株作為一組氣味源.該試驗重復(fù)3～4次,每次測試15頭寄生蜂.

1.4 內(nèi)源茉莉酸及水楊酸的測定

內(nèi)源茉莉酸與水楊酸的提取與定量主要參考SCHULZE等[29](2006)整理的方法.簡單地說,取250～300 mg植物材料,在液氮中進行冷凍,研磨至粉末.樣品定量時,加入[9,10-2H2]-9,10-dihydro-JA(15 ng)和[3,4,5,6-2H4]-SA(30 ng)作為內(nèi)標,樣品使用Finnigan GCQ離子阱質(zhì)譜儀(Thermoelectron, Bremen, Germany)進行分析,每個處理設(shè)置3個生物學重復(fù).

1.5 試驗數(shù)據(jù)統(tǒng)計學分析

嗅覺儀試驗數(shù)據(jù)進行二項式分布檢測分析;植物激素數(shù)據(jù)均進行單因素方差分析.

2 結(jié)果與分析

2.1 寄生蜂嗅覺行為反應(yīng)

在擬南芥處理組中,與健康植株釋放的揮發(fā)物相比,麗蚜小蜂對煙粉虱為害24 h的植株釋放的揮發(fā)物并未表現(xiàn)出偏好性(P=0.32);但對煙粉虱為害72 h、120 h的植株釋放的揮發(fā)物均表現(xiàn)出顯著的偏好性(72 h:P=0.02;120 h:P=0.003;圖1A).

在番茄處理組中,與健康植株釋放的揮發(fā)物相比,麗蚜小蜂對煙粉虱為害24 h、72 h的植株釋放的揮發(fā)物并未表現(xiàn)出偏好性(24 h:P=0.61;72 h:P=0.024);但對煙粉虱為害120 h的植株釋放的揮發(fā)物均表現(xiàn)出顯著的偏好(120 h:P<0.001;圖1B).

以上結(jié)果表明當煙粉虱為害擬南芥或番茄120 h后,蟲害植株對麗蚜小蜂均有顯著的吸引作用,說明寄主植物表現(xiàn)出了間接防御反應(yīng).因此,在接下來關(guān)于擬南芥或番茄突變體植株的相關(guān)行為生測及內(nèi)源激素測定試驗中,煙粉虱取食時間均為120 h.

圖1 麗蚜小蜂對煙粉虱為害后的野生型植株的嗅覺行為生測Figure 1 Bioassays of olfactory response of Encarsia Formosa towards the odors emitted from whitefly-infested wild-type plants

(A)麗蚜小蜂對健康及煙粉虱處理的野生型擬南芥(WT)植株的嗅覺選擇反應(yīng);(B)麗蚜小蜂對健康及煙粉虱處理的野生型番茄(MM)植株的嗅覺選擇反應(yīng).條形圖右邊數(shù)字(n)代表做出選擇反應(yīng)的麗蚜小蜂的數(shù)量.圖中星號代表寄生蜂的選擇比例與50∶50的分布比例差異顯著(*,P<0.05,**,P<0.01;***,P<0.001;ns,差異不顯著).WF代表煙粉虱.

2.2 水楊酸或茉莉酸信號路徑沉默對麗蚜小蜂嗅覺行為的影響

在擬南芥處理組中,當水楊酸路徑被沉默時,麗蚜小蜂對煙粉虱為害后的NahG或npr-1植株并未表現(xiàn)出顯著的偏好性(NahG,P=0.16;npr-1,P=0.59);相反,當茉莉酸路徑被沉默時,麗蚜小蜂對煙粉虱為害后的dde2-2植株表現(xiàn)出顯著的偏好性(dde2-2,P=0.002;圖2A).

同樣的,在番茄處理組中,當水楊酸路徑被沉默時,麗蚜小蜂對煙粉虱為害后的NahG植株并未表現(xiàn)出顯著的偏好性(P=0.41);當茉莉酸路徑被沉默時,麗蚜小蜂對煙粉虱為害后的def-1或spr-2植株表現(xiàn)出顯著的偏好性(def-1,P=0.003;spr-2,P=0.008;圖2B).

以上結(jié)果說明水楊酸信號路徑在調(diào)控煙粉虱誘導(dǎo)的植物揮發(fā)物吸引麗蚜小蜂過程中發(fā)揮重要作用.

圖2 麗蚜小蜂對煙粉虱為害后的突變體植株的嗅覺行為生測Figure 2 Bioassays of olfactory responses of Encarsia Formosa towards the odors emitted from whitefly-infested mutants

(A)麗蚜小蜂對擬南芥不同突變體植株的嗅覺選擇反應(yīng);(B)麗蚜小蜂對番茄不同突變體植株的嗅覺選擇反應(yīng).條形圖右邊數(shù)字(n)代表做出選擇反應(yīng)的麗蚜小蜂的數(shù)量.圖中星號代表寄生蜂的選擇比例與50∶50的分布比例差異顯著(*,P<0.05,**,P<0.01;***,P<0.001;ns,差異不顯著).WF代表煙粉虱.

2.3 內(nèi)源茉莉酸及水楊酸定量分析

在擬南芥處理組中,與健康植株相比,經(jīng)煙粉虱蟲害取食的植株內(nèi)源水楊酸含量顯著增加(P=0.02;圖3A).但是,健康及蟲害植株樣品均未檢測到內(nèi)源茉莉酸,其原因可能有二:一,受脅迫條件下,植物體內(nèi)內(nèi)源茉莉酸會在較短時間內(nèi)呈現(xiàn)一個瞬間的高峰,本試驗的檢測時間錯過了這一時間點;二,樣本中內(nèi)源茉莉酸含量低于儀器檢測極限.

在番茄處理組中,與健康植株相比,煙粉虱蟲害植株內(nèi)源水楊酸含量顯著增加(P<0.001);但是,內(nèi)源茉莉酸含量并未明顯變化(P=0.37;圖3B).

圖3 煙粉虱取食對內(nèi)源水楊酸(SA)及茉莉酸(JA)的影響Figure 3 Effects of whitefly infestation on the levels of endogenous SA and JA.

煙粉虱(100頭/株)取食5d后,擬南芥(A)或番茄(B)植株內(nèi)源水楊酸及茉莉酸的變化情況.圖中星號代表煙粉虱蟲害處理組與對照組間內(nèi)源水楊酸或茉莉酸含量差異顯著(**,P<0.01;***,P<0.001;ns,差異不顯著).

3 討 論

我們的研究結(jié)果表明,煙粉虱為害擬南芥或番茄植株120 h后,蟲害植株均顯著吸引麗蚜小蜂(圖1).這一結(jié)果與以前的研究結(jié)果相一致:煙粉虱為害3～5 d后才能夠顯著誘導(dǎo)寄主植物釋放更多的揮發(fā)物[30],并吸引麗蚜小蜂[31].另外,當擬南芥或番茄的茉莉酸信號路徑被阻斷時,麗蚜小蜂仍顯著偏好經(jīng)煙粉虱蟲害的植株;但是當擬南芥或番茄的水楊酸信號路徑被阻斷時,麗蚜小蜂并未對蟲害植株表現(xiàn)出偏好性,甚至對蟲害處理的NahG和npr-1植株表現(xiàn)出了一定的排斥反應(yīng)(圖2),這說明當煙粉虱誘導(dǎo)的特異性揮發(fā)物減少時,其他非特異性揮發(fā)物能夠排斥寄生蜂.植物激素的結(jié)果進一步證實煙粉虱取食能夠激活擬南芥或番茄植株的水楊酸信號路徑,但并未激活茉莉酸信號路徑(圖3).綜上所述,我們認為水楊酸信號路徑在調(diào)控煙粉虱為害植株吸引麗蚜小蜂的過程中發(fā)揮關(guān)鍵作用.

目前為止,關(guān)于寄主植物調(diào)控刺吸式昆蟲誘導(dǎo)的植物揮發(fā)物吸引寄生蜂的信號傳導(dǎo)途徑的研究仍然較少[14].僅有的少數(shù)研究表明:茉莉酸信號路徑可能在蚜蟲-寄主植物-寄生蜂的三級營養(yǎng)關(guān)系中發(fā)揮一定作用[31].例如,外源茉莉酸酮(cis-jasmone)能夠顯著上調(diào)擬南芥植株體內(nèi)兩個P450基因,包括CYP81D11和CYP72A13;其中CYP81D11基因負責誘導(dǎo)揮發(fā)物(Z)-3-hexen-1-ol的釋放,而該揮發(fā)物能夠吸引蚜蟲寄生蜂Aphidiuservi[32].而另一種蚜蟲寄生蜂Diaeretiellarapae則對擬南芥突變體Cev1(茉莉酸信號路徑組成性表達)表現(xiàn)出明顯的偏好性,但對于蚜蟲為害的coi1-16(茉莉酸信號路徑被阻斷)植株則并未表現(xiàn)出明顯的偏好性[33].結(jié)合本研究結(jié)果,很顯然植物應(yīng)對煙粉虱及蚜蟲取食誘導(dǎo)產(chǎn)生揮發(fā)物吸引寄生蜂的調(diào)控機制可能是不同的.因此,接下來的工作有必要進一步研究:1)水楊酸路徑調(diào)控的植物揮發(fā)物是否同樣吸引蚜蟲寄生蜂？2)蚜蟲及煙粉虱的取食策略是否存在本質(zhì)的不同？

值得注意的是,由于煙粉虱在擬南芥野生型及不同突變體上的發(fā)育速率不同[16],因此很有可能煙粉虱在不同突變體植株上的取食強度不同.而刺吸式昆蟲取食強度的差異可能影響其誘導(dǎo)植物揮發(fā)物的釋放量[34].我們在另外一個試驗中發(fā)現(xiàn),煙粉虱在擬南芥或番茄植株野生型及不同突變體上連續(xù)取食7 d后,其產(chǎn)卵量在植物組間差異不顯著(未發(fā)表數(shù)據(jù)),這說明煙粉虱在不同突變體植株間取食強度相似.因此,我們認為蟲害處理的擬南芥dde2-2、番茄def-1和spr-2植株對寄生蜂的吸引作用,以及蟲害處理擬南芥NahG和npr-1及番茄NahG植株，對寄生蜂的不吸引作用,并不是由煙粉虱取食強度變化而引起的.

已有的研究表明,煙粉虱取食行為能誘導(dǎo)擬南芥或番茄體內(nèi)的水楊酸防御反應(yīng),同時抑制茉莉酸或乙烯防御反應(yīng)[20,35].從煙粉虱角度分析,茉莉酸防御反應(yīng)的抑制對其種群發(fā)育是有利的,因為該防御反應(yīng)被證明是寄主植物防御煙粉虱的有效防御反應(yīng)[19].顯然,煙粉虱能夠有效的“瓦解”寄主植物的直接防御反應(yīng)[36].然而我們的結(jié)果暗示,麗蚜小蜂已在長期協(xié)同進化的過程中,適應(yīng)了水楊酸路徑調(diào)控的植物揮發(fā)物,并利用其作為煙粉虱搜尋的線索;從植物的角度來說,釋放特異性揮發(fā)物吸引麗蚜小蜂,是其在長期協(xié)同進化過程中應(yīng)對煙粉虱瓦解其直接防御的手段之一,即建立有效的間接防御.

茉莉酸信號路徑在調(diào)節(jié)植物間接防御,即調(diào)控植物釋放揮發(fā)物吸引寄生蜂或捕食者中的重要作用已有廣泛的報道[5,8].并且有研究表明,茉莉酸路徑調(diào)控的植物間接防御在田間條件下對農(nóng)作物有利[7].但是,其他信號路徑在調(diào)控植物釋放揮發(fā)物吸引天敵中的作用目前尚不清楚[2].因此,本研究首次報道了水楊酸路徑在調(diào)控刺吸式昆蟲煙粉虱誘導(dǎo)的植物間接防御中發(fā)揮重要作用.我們的結(jié)果再次證明,植食者的取食行為是促進植物揮發(fā)物這一信號因子進化為植物間接防御的選擇壓;另外,植食者取食策略的不同導(dǎo)致植物應(yīng)對其間接防御的機制不同.

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A study on the role of salicylic acid signaling pathways in mediating whitefly-induced indirect plant defense

YU Xiaoping1, HE Yuchen1, LI Weidi2, ZHAO Chan1, ZHAO Zhao1, ZHANG Pengjun1
(1. Zhejiang Provincial Key Laboratory of Biometrology, Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China; 2.Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China)

In response to herbivory, plants are able to initiate indirect defense by synthesizing and releasing complex blends of volatiles that attract natural enemies of the herbivore. It has been well-documented that the jasmonic acid(JA) signaling pathway plays an important role in mediating the plant volatile emission. Recently, a few studies have indicated that phloem-feeding insects, such as whiteflies and aphids, mainly induce the salicylic acid(SA) signaling pathway but suppress the JA signaling pathway. However, whether the SA signaling pathway plays a key role in mediating indirect plant defense induced by phloem-feeding insects remains unknown. In the present study, we usedArabidopsisthalianaand tomato as model plants and examined the olfactory responses of parasitoid waspEncarsiaformosatowards the odors released fromB.tabaci-infested wild-type(WT) plants and some mutants. ParasitoidsE.formosawere significantly attracted by the odors emitted from WTArabidopsisor tomato plants infested withB.tabacifor 120 h compared to uninfested plants. The attraction of parasitoids still occurred inB.tabaci-infested JA-deficientArabidopsis(dde2-2) or tomato(def-1 andspr-2) mutants, but not inB.tabaci-infested SA-defectiveArabidopsis(NahGandnpr-1) or tomato(NahG) mutants. Phytohormone analyses further demonstrated thatB.tabaciinfestation induced the accumulation of endogenous SA inArabidopsisor tomato, but not the accumulation of JA. Taken together, we conclude that the SA signaling pathway plays a key role in mediating whitefly-induced indirect plant defense.

phloem-feeding insects;Bemisiatabaci; SA signaling pathway;Encarsiaformosa

2096-2835(2017)01-0001-06

10.3969/j.issn.2096-2835.2017.01.001

2017-02-01 《中國計量大學學報》網(wǎng)址：zgjl.cbpt.cnki.net

國家自然科學基金資助項目(No.31301676, 31471779)，農(nóng)業(yè)部公益性行業(yè)項目(No.201303019).

俞曉平(1963- )，男，浙江省杭州人，研究員，主要研究方向為生物安全和生物計量.E-mail:yxp@cjlu.edu.cn

S433.3

A