田沛，張光明，南志標(biāo)

(1.草地農(nóng)業(yè)生態(tài)系統(tǒng)國(guó)家重點(diǎn)實(shí)驗(yàn)室，蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院， 甘肅 蘭州 730020；2.廣東粵明電力工程有限公司，廣東 珠海 519000 )

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禾草內(nèi)生真菌研究及應(yīng)用進(jìn)展

田沛1*，張光明2，南志標(biāo)1

(1.草地農(nóng)業(yè)生態(tài)系統(tǒng)國(guó)家重點(diǎn)實(shí)驗(yàn)室，蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院， 甘肅 蘭州 730020；2.廣東粵明電力工程有限公司，廣東 珠海 519000 )

內(nèi)生真菌感染大部分禾草形成互惠共生體，提高共生體對(duì)環(huán)境適應(yīng)性，并進(jìn)而對(duì)動(dòng)物、微生物以及整個(gè)生態(tài)系統(tǒng)產(chǎn)生廣泛的影響，使該領(lǐng)域成為近年來(lái)研究的熱點(diǎn)問(wèn)題。分子生物學(xué)技術(shù)、基因組學(xué)、蛋白組學(xué)和代謝組學(xué)以及相應(yīng)的信息生物學(xué)技術(shù)的應(yīng)用加快了禾草內(nèi)生真菌的研究，尤其是內(nèi)生真菌全基因組序列的測(cè)定，明確了次生代謝物多樣性及其與基因的關(guān)系，鑒定相關(guān)的功能基因和蛋白，基因敲除技術(shù)闡釋了維持內(nèi)生真菌與寄主動(dòng)態(tài)平衡的分子機(jī)制。利用基因組學(xué)等技術(shù)篩選對(duì)家畜無(wú)毒的內(nèi)生真菌菌株, 通過(guò)接種技術(shù)建立新的既具有抗逆性，又對(duì)家畜無(wú)毒的禾草-內(nèi)生真菌共生體，提高禾草的品質(zhì)并確保對(duì)動(dòng)物的安全性，在牧草及草坪草育種上獲得了巨大成功。但是內(nèi)生真菌的寄主特異性限制了內(nèi)生真菌可利用的范圍，因此后續(xù)應(yīng)繼續(xù)利用基因組學(xué)和代謝組學(xué)新技術(shù)深入研究?jī)?nèi)生真菌與寄主相互作用的機(jī)制，利用基因工程技術(shù)人工創(chuàng)造無(wú)毒菌株，克服共生體創(chuàng)制的瓶頸。本研究旨在對(duì)以上內(nèi)容進(jìn)行綜述，以期為更廣泛利用內(nèi)生真菌進(jìn)行牧草和草坪草育種奠定基礎(chǔ)。

內(nèi)生真菌；食物鏈；生態(tài)系統(tǒng)；微生物；基因組；牧草育種

禾草內(nèi)生真菌指在禾草體內(nèi)渡過(guò)全部或大部分生活周期，而禾草本身不顯示任何外部癥狀的一大類真菌[1]。廣泛研究的內(nèi)生真菌主要是子囊菌門(Ascomycota)麥角科(Clavicipitaceae)的有性世代Epichloё和其所對(duì)應(yīng)無(wú)性世代Neotyphodium屬內(nèi)生真菌。自1898年，從毒麥(Loliumtemulentum)種子內(nèi)分離出第一株內(nèi)生真菌至今，對(duì)內(nèi)生真菌的研究經(jīng)歷了由淺入深的過(guò)程，尤其是在20世紀(jì)70年代以后，Bacon等[2]揭示了高羊茅(Festucaarundinacea)內(nèi)生真菌與牛的狐茅中毒癥(fescue toxicosis)的密切關(guān)系，隨后Fletcher等[3]發(fā)現(xiàn)了多年生黑麥草(Loliumperenne)內(nèi)生真菌與新西蘭綿羊黑麥草蹣跚病(ryegrass staggers)的關(guān)系，禾草內(nèi)生真菌的研究得到了突飛猛進(jìn)的發(fā)展，對(duì)其分類、分布、生理、生態(tài)、代謝、遺傳以及與寄主植物的相互作用等方面開展了大量的研究工作[4-6]。最近，Leuchtmann 等[7]根據(jù)內(nèi)生真菌的基因序列和形態(tài)特征，依據(jù) “一種真菌一個(gè)名稱”的原則，將已發(fā)表鑒定的18種有性世代(Epichloё)和25種無(wú)性世代(Neotyphodium)內(nèi)生真菌均劃分為Epichloё 屬內(nèi)生真菌，原本Neotyphodium屬認(rèn)為暫時(shí)沒(méi)有發(fā)現(xiàn)其有性世代，也被冠以Epichloё屬。近年來(lái)，隨著分子生物學(xué)、基因組學(xué)、代謝組學(xué)、生物信息學(xué)的快速發(fā)展以及在植物和真菌學(xué)領(lǐng)域內(nèi)的廣泛應(yīng)用，內(nèi)生真菌的研究方向主要集中在內(nèi)生真菌與寄主相互作用的分子機(jī)制以及利用內(nèi)生真菌進(jìn)行牧草和草坪草育種等方面，本研究擬對(duì)近年來(lái)這方面的進(jìn)展進(jìn)行綜述。

1 內(nèi)生真菌對(duì)寄主的影響

大量以高羊茅-Epichloёcoenophiala和多年生黑麥草-Epichloёfestucaevar.lolii共生體為代表的研究形成了Epichloё屬內(nèi)生真菌與禾草形成互惠共生體，提高共生體對(duì)環(huán)境適應(yīng)性的結(jié)論[8-9]。已證實(shí)內(nèi)生真菌可以提高寄主的生長(zhǎng)以及對(duì)食草動(dòng)物[10-15]、昆蟲[8，16]、線蟲[16-17]、病原真菌、細(xì)菌[18-23]和競(jìng)爭(zhēng)性植物[14，16，24]等生物脅迫的抗性和對(duì)干旱[20，24-25]、營(yíng)養(yǎng)物質(zhì)缺乏[25]和化學(xué)他感作用[26]等非生物脅迫的抗性。有關(guān)內(nèi)生真菌對(duì)寄主的影響以及機(jī)理已有多篇文章和綜述發(fā)表[1，4-5，27-30]，筆者在這里不做贅述。隨著對(duì)內(nèi)生真菌資源調(diào)查的深入，發(fā)現(xiàn)大量新的禾草-內(nèi)生真菌共生體[7，31-35]，內(nèi)生真菌與寄主植物共生關(guān)系表現(xiàn)出明顯的不確定性，有證據(jù)表明Epichloё 屬內(nèi)生真菌并不能提高某些寄主尤其是野生禾草的適應(yīng)性，比如內(nèi)生真菌并沒(méi)有提高寄主亞利桑那羊茅(Festucaarizonica)的生物量[15]、競(jìng)爭(zhēng)力[36]和降低動(dòng)物的采食[37]。這些不同的結(jié)果可能是由于高羊茅和多年生黑麥草等栽培牧草經(jīng)長(zhǎng)期馴化，遺傳多樣性遠(yuǎn)遠(yuǎn)低于野生禾草，內(nèi)生真菌與寄主長(zhǎng)期共生并協(xié)同進(jìn)化，在演化過(guò)程中向更有利于寄主的方向進(jìn)化。而新的內(nèi)生真菌共生體，寄主植物遺傳多樣性高，其遺傳背景足以影響植物的生長(zhǎng)并超越內(nèi)生真菌的作用，同時(shí)由于真菌繁殖模式、傳播方式和環(huán)境的不同造成了不同的選擇壓力，使共生關(guān)系復(fù)雜化，因而對(duì)寄主造成了不同的影響[15，38-39]，降低了在栽培牧草上得出結(jié)論的可靠性。內(nèi)生真菌的傳播方式也顯著影響其對(duì)寄主的作用。水平傳播的有性階段內(nèi)生真菌能在宿主生殖枝上產(chǎn)生子座，抑制宿主的開花和結(jié)實(shí)，對(duì)宿主的有益影響可能小于嚴(yán)格傳播的無(wú)性階段內(nèi)生真菌[40]。有可能在自然群落中，內(nèi)生真菌與寄主形成穩(wěn)定的關(guān)系，但在溫室或試驗(yàn)田中開展的試驗(yàn)改變了共生體的生存環(huán)境，因而進(jìn)一步改變內(nèi)生真菌與寄主的關(guān)系。這些都是在研究?jī)?nèi)生真菌與寄主相互作用時(shí)需要關(guān)注和思考的問(wèn)題。

2 內(nèi)生真菌對(duì)動(dòng)物及食物鏈的影響

高羊茅內(nèi)生真菌引起的狐茅中毒癥和多年生黑麥草內(nèi)生真菌引起的黑麥草蹣跚病給畜牧業(yè)造成了巨大的經(jīng)濟(jì)損失[41-42]，內(nèi)生真菌對(duì)動(dòng)物尤其是家畜的影響已經(jīng)開展了大量研究。已經(jīng)明確內(nèi)生真菌與禾草共生時(shí)，能產(chǎn)生多種次生代謝物，其中以吲哚雙萜類(indolditerpene，以lolitrem B為代表)，吡咯并吡嗪類(pyrrolopyrazine，以peramine為代表)，麥角堿類(ergot，以ergovaline為代表)和飽和吡咯化合物(pyrrolizidine，以loline為代表)這四大類為代表[43]。除peramine以外的其余3類生物堿均對(duì)哺乳動(dòng)物有毒，其中毒性最強(qiáng)的是lolitrem B和ergovaline，分別是引致狐茅中毒癥和黑麥草蹣跚病的主要原因[10-12，44]。后又陸續(xù)發(fā)現(xiàn)禾草內(nèi)生真菌共生體對(duì)多種家畜如家兔[45]，鹿[46]，駱駝[47]，羊駝[48]，山羊[49]，馬[41]，驢[50]等的毒性。自黑麥草蹣跚癥與內(nèi)生真菌的關(guān)系確立不久，Prestidge等[51]發(fā)現(xiàn)內(nèi)生真菌能提高多年生黑麥草對(duì)阿根廷莖象甲(Listronotusbonariensis)的抗性，這種保護(hù)作用是因?yàn)閮?nèi)生真菌產(chǎn)生對(duì)昆蟲有強(qiáng)烈毒性的生物堿peramine和loline[52-53]，這些生物堿可直接導(dǎo)致昆蟲神經(jīng)系統(tǒng)紊亂而死亡，或者在體內(nèi)累積，影響對(duì)食物的消化利用，延緩生長(zhǎng)發(fā)育，降低存活率。據(jù)不完全統(tǒng)計(jì)，內(nèi)生真菌可對(duì)鞘翅目(Coleoptera)，鱗翅目(Lepidoptera)，異翅目(Heteroptera)，同翅目(Homoptera)，直翅目(Orthoptera)，蜱螨目(Acarina)等目昆蟲產(chǎn)生抗性[1，29，44，54]。近年來(lái)，逐漸發(fā)現(xiàn)一些新的生物堿，比如黑麥草內(nèi)生真菌菌株AR37產(chǎn)生janthitrems[55]，雖然尚未深入了解，但發(fā)現(xiàn)其可以提高寄主抗蟲性[56]，已經(jīng)在育種中得到了重視。

共生體對(duì)動(dòng)物的毒性是由內(nèi)生真菌產(chǎn)生的次生代謝物引起的，這些次生代謝物可在采食者體內(nèi)累積，幫助其防御捕食者，進(jìn)而對(duì)上一級(jí)營(yíng)養(yǎng)層甚至生態(tài)系統(tǒng)中多級(jí)營(yíng)養(yǎng)層產(chǎn)生影響。比如內(nèi)生真菌降低了寄主上蚜蟲(Rhopalosiphumpadi)的密度，共生體產(chǎn)生的生物堿在蚜蟲體內(nèi)累積[57]，經(jīng)蚜蟲食物鏈傳遞進(jìn)而降低了蚜蟲的寄生性天敵-寄生蜂和捕食性天敵-瓢蟲的密度，并降低這些天敵的產(chǎn)卵量和生存率，延長(zhǎng)發(fā)育期，其幼蟲和成蟲適應(yīng)度顯著降低，后代總數(shù)顯著降低，這種級(jí)聯(lián)作用能長(zhǎng)期影響蚜蟲天敵的種群動(dòng)態(tài)[58]。同時(shí)也發(fā)現(xiàn)內(nèi)生真菌對(duì)食物鏈的影響還包括蚜蟲的采食促使內(nèi)生真菌影響寄主分泌的揮發(fā)性有機(jī)物，這些有機(jī)物可降低其他昆蟲的采食[59]。除了對(duì)蚜蟲及食物鏈的應(yīng)用，內(nèi)生真菌也降低了阿根廷莖象甲和粘蟲(Spodopterafrugiperda)的寄生蜂(Microctonushyperodae)的生長(zhǎng)和存活率[60-61]。同時(shí)也發(fā)現(xiàn)采食被內(nèi)生真菌感染(E+)植株的昆蟲對(duì)線蟲及其寄生細(xì)菌具有更強(qiáng)的抵抗力。比如多年生黑麥草和高羊茅E+植株上的小地虎(Agrostisipsilon)不易受到昆蟲病原線蟲(Steinernemacarpocapse)的感染[54，62]。這些例子說(shuō)明禾草-內(nèi)生真菌共生體與食草動(dòng)物關(guān)系非常復(fù)雜，內(nèi)生真菌的存在改變食草動(dòng)物的取食行為，影響到植食性昆蟲物種多樣性和種群結(jié)構(gòu)，對(duì)采食者的捕食性和寄生性天敵種類和數(shù)量產(chǎn)生影響，影響捕食者的捕食、結(jié)網(wǎng)(或寄生)和繁殖能力，從而影響食物鏈的能量流動(dòng)和食物網(wǎng)結(jié)構(gòu)，對(duì)生態(tài)系統(tǒng)產(chǎn)生不可低估的作用。在田間條件下，這種作用受到環(huán)境及共生體的基因型、生物量等各種因素影響，生物堿對(duì)食物鏈的影響可能會(huì)弱化。值得一提的是，雖有研究表明吲哚雙萜類化合物lolitrem B和janthitrem在牛和羊的脂肪和牛奶中微量殘留[63-64]，但并不會(huì)對(duì)人類健康造成威脅[64-65]。

3 內(nèi)生真菌與其他微生物的關(guān)系

在自然界中，植物同時(shí)與多種微生物類群共生，內(nèi)生真菌對(duì)共生體-其他微生物的相互作用也產(chǎn)生顯著影響。研究較多的是對(duì)共生體與地上部分病原真菌和細(xì)菌以及地下部分菌根真菌和土壤微生物群落的相互作用的影響。

通過(guò)離體培養(yǎng)平板對(duì)峙、離體葉片和活體植株接種以及田間發(fā)病率調(diào)查等方法已發(fā)現(xiàn)禾草內(nèi)生真菌對(duì)多種病原真菌及其引起的植物病害產(chǎn)生影響[27，66]。內(nèi)生真菌通過(guò)抵抗病原菌的侵入、抑制病原菌的生長(zhǎng)、抑制孢子的萌發(fā)、抑制病斑的擴(kuò)展和阻止傳毒介體昆蟲等機(jī)制提高寄主對(duì)22種牧草和草坪草病害的抗性[1，27]。但是由于植物抗病本身就是一個(gè)非常復(fù)雜的過(guò)程，受病原物種，寄主基因型，環(huán)境條件如植物營(yíng)養(yǎng)供應(yīng)(尤其是氮的供應(yīng))、溫度、土壤濕度和水分條件和其他生物之間綜合作用的影響[24，67]，尤其田間條件下，內(nèi)生真菌對(duì)寄主的抗病性影響出現(xiàn)不太一致，相對(duì)于內(nèi)生真菌其他方面的研究，此方面研究涉及不多并較難得到一致的結(jié)論。

內(nèi)生真菌能提高寄主對(duì)一些植物病毒，如黑麥草花葉病毒和大麥黃矮病毒的抗性[68-69]。但是對(duì)感染大多數(shù)真菌類群且不產(chǎn)生明顯表現(xiàn)的真菌病毒關(guān)注較少。Marquez等[70]在Science雜志上報(bào)道了真菌Curvularia耐熱病毒決定了內(nèi)生真菌(Cryphonectriaprotuberata)與熱帶稗草(Dichantheliumlanuginosum)形成共生體的耐熱性，只有被病毒感染的內(nèi)生真菌才能提高寄主植物的耐熱性，反之則不能。這就揭示了真菌病毒能夠調(diào)整植物與內(nèi)生真菌形成的共生體系，因而部分學(xué)者開始關(guān)注Epichloё屬內(nèi)生真菌與真菌病毒之間的關(guān)系。從草地羊茅(Festucarubra)分離的內(nèi)生真菌E.festucae大部分被雙鏈RNA病毒或者裸露RNA病毒感染，這些病毒對(duì)真菌的表現(xiàn)無(wú)明顯影響，對(duì)共生體的影響也未知[71-72]。以后對(duì)內(nèi)生真菌的研究應(yīng)關(guān)注這方面的問(wèn)題。

菌根真菌與植物根系建立互惠共生關(guān)系，增加宿主植物對(duì)土壤中營(yíng)養(yǎng)元素特別是氮、磷的吸收，促進(jìn)水分吸收和利用,碳水化合物代謝和提高光合速率等[73-74]。而Epichloё內(nèi)生真菌與植物地上組織建立互惠共生關(guān)系。已開展了大量研究闡明單一共生真菌與寄主植物的相互關(guān)系，而菌根真菌和內(nèi)生真菌雙重感染時(shí)對(duì)寄主植物影響由于三者之間的互作受土壤營(yíng)養(yǎng)水平、共生真菌的基因型以及不同的草地管理方式(單作或者混作，施肥，灌溉等)等多種因素的影響，尚不能得出統(tǒng)一的結(jié)論。有研究表明內(nèi)生真菌與菌根真菌存在拮抗作用，內(nèi)生真菌降低菌根真菌的侵染和擴(kuò)展[18-19，75]，但是在某些情況下促進(jìn)菌根真菌的侵染[76]。比如內(nèi)生真菌提高一種AM真菌對(duì)其寄主披堿草(Elymushystrix)的侵染，卻抑制了另外一種AM真菌的侵染[77]；同時(shí)也發(fā)現(xiàn)，AM真菌共寄生提高多年生黑麥草-E.festucaevar.lolii共生體的冠根比，卻降低了多年生黑麥草-E.typhina共生體的冠根比[75]，這些研究都強(qiáng)調(diào)了基因型的強(qiáng)烈影響。雖然二者單獨(dú)寄生時(shí)均形成互惠共生體，有研究發(fā)現(xiàn)二者共同作用時(shí)，降低寄主生物堿含量和抗蟲性[78-79]，因此，如何同時(shí)利用這兩種真菌以形成超級(jí)共生體對(duì)寄主提供全方面的保護(hù)，還需要更多深入的研究。

內(nèi)生真菌除了影響與寄主共生的微生物，還能夠改變土壤的營(yíng)養(yǎng)元素水平，影響土壤微生物群落結(jié)構(gòu)和功能[80]。初步證明了內(nèi)生真菌直接影響根部生物量、形態(tài)和分泌物從而影響根部食物鏈和營(yíng)養(yǎng)循環(huán)，增加輸入到土壤中的有機(jī)營(yíng)養(yǎng)，提高根際營(yíng)養(yǎng)循環(huán)和土壤微生物活性，影響土壤微生物群落的代謝多樣性[81-84]；并可通過(guò)對(duì)共生體地上部分比如生物量，種群結(jié)構(gòu)，周圍微環(huán)境的綜合作用和家畜采食間接影響地下部分。共生體產(chǎn)生生物堿或者其他化感作用物質(zhì)可滲入到土壤中對(duì)土壤微生物和動(dòng)物群落產(chǎn)生影響。但是也有不同的結(jié)果，有研究表明禾草內(nèi)生真菌降低土壤微生物量和土壤呼吸作用，抑制土壤微生物活性，對(duì)土壤微生物群落的穩(wěn)定性存在一定的副作用[85-86]。

4 內(nèi)生真菌對(duì)生態(tài)系統(tǒng)的影響

上述內(nèi)生真菌與寄主、草食動(dòng)物和微生物的研究屬于個(gè)體或種群水平的研究，隨著內(nèi)生真菌研究的深入，其已拓寬到內(nèi)生真菌與寄主所在的整個(gè)生態(tài)系統(tǒng)的影響。通過(guò)上述提到的直接或間接影響，內(nèi)生真菌在生物群落和整個(gè)生態(tài)系統(tǒng)中發(fā)揮重要作用[87]。比如內(nèi)生真菌能提高寄主對(duì)環(huán)境的適應(yīng)性，使具有較強(qiáng)抗逆性的帶菌禾草能迅速占據(jù)生態(tài)位，提高其在群落中的競(jìng)爭(zhēng)力，降低周圍群落雜草的多樣性，影響了植物群落和生態(tài)系統(tǒng)的多樣性[88]。而生物堿等次生代謝物對(duì)草食動(dòng)物及食物鏈的影響，改變自然群落中競(jìng)爭(zhēng)和捕食關(guān)系，進(jìn)而改變?nèi)郝渲袆?dòng)物的多樣性[87]，比如內(nèi)生真菌導(dǎo)致高羊茅草地中小型哺乳動(dòng)物、節(jié)肢動(dòng)物多樣性的降低[89-90]，也有共生體為其他草類植物和節(jié)肢動(dòng)物提供了庇護(hù)，從而保護(hù)生物多樣性的報(bào)道[91]。內(nèi)生真菌對(duì)寄主地下部分的影響包括對(duì)根際和土壤微生物兩方面的作用，改變根系分泌物和根際代謝物[92]，影響土壤真菌和細(xì)菌群落[93]，進(jìn)而影響地下部分生態(tài)系統(tǒng)營(yíng)養(yǎng)循環(huán)；內(nèi)生真菌對(duì)地上部分生物量及營(yíng)養(yǎng)品質(zhì)的影響，會(huì)進(jìn)一步影響寄主枯葉的質(zhì)量和降解速率，影響土壤碳釋放和溫室氣體的排放，這些作用反過(guò)來(lái)影響地上和地下部分群落結(jié)構(gòu)[94](圖1)。由此可以看出內(nèi)生真菌雖然作為微生物在寄主體內(nèi)生存，但是對(duì)草原植物群落多樣性、穩(wěn)定性和草原生態(tài)系統(tǒng)均有非常重要的影響，微生物已經(jīng)成為影響生態(tài)系統(tǒng)不可忽略的因素。雖然內(nèi)生真菌對(duì)生態(tài)系統(tǒng)的研究相對(duì)較少，但卻已取得了具有國(guó)際影響力的成果， Clay等[14]表明雖然內(nèi)生真菌沒(méi)有提高高羊茅地上部分的生物量，但卻提高了寄主在群落中的優(yōu)勢(shì)地位，降低物種多樣性，導(dǎo)致群落結(jié)構(gòu)的改變。Omacini等[58]通過(guò)對(duì)內(nèi)生真菌-蚜蟲食物鏈的研究表明內(nèi)生真菌干擾能量從植物向更高級(jí)營(yíng)養(yǎng)水平的傳遞，從而影響生物鏈的能量傳遞，進(jìn)而改變生物群落的多樣性。這些成果分別在頂級(jí)學(xué)術(shù)期刊Science和Nature上發(fā)表，從而使內(nèi)生真菌對(duì)生態(tài)系統(tǒng)的重要性在國(guó)際范圍內(nèi)引起關(guān)注。

5 禾草內(nèi)生真菌的基因組學(xué)和代謝組學(xué)

隨著分子生物學(xué)技術(shù)、基因組學(xué)、蛋白組學(xué)和代謝組學(xué)以及相應(yīng)的生物信息學(xué)技術(shù)的迅速發(fā)展和日益成熟，內(nèi)生真菌的研究也得到了空前的發(fā)展[4-6，96-97]，相對(duì)于上述提到的幾個(gè)方面，基因組學(xué)得到最多的關(guān)注和迅速的發(fā)展，開展了內(nèi)生真菌及其寄主植物的全基因組序列測(cè)定，明確次生代謝物多樣性及其與基因的關(guān)系，并鑒定相關(guān)的功能基因和蛋白。代表性工作有美國(guó)肯塔基大學(xué)Schardl實(shí)驗(yàn)室圍繞生物堿合成的比較基因組學(xué)研究，現(xiàn)已完成26株麥角菌其中包括20個(gè)不同的Epichloё內(nèi)生真菌的全基因組測(cè)序，其序列在http://www.endophyte.uky.edu/上公布，有助于全球科學(xué)家共同開展基因圖譜構(gòu)建等工作[6]。另外，澳大利亞維多利亞農(nóng)業(yè)生物研究中心(AgriBio, Victoria)Spangenberg實(shí)驗(yàn)室也完成了16株羊茅屬內(nèi)生真菌和19株黑麥草屬內(nèi)生真菌的全基因組測(cè)序[98-99]。這些全基因組序列的獲得為研究?jī)?nèi)生真菌的系統(tǒng)進(jìn)化，深入分析生物堿合成相關(guān)基因，尋找真菌與寄主相互作用的基因奠定了重要的基礎(chǔ)。比如以前廣泛利用DNA分子標(biāo)記技術(shù)如SSR等[100-101]和特定DNA片段如tubB、tefA 序列對(duì)內(nèi)生真菌進(jìn)行遺傳多樣性、系統(tǒng)發(fā)育、起源和進(jìn)化研究[7，102]。但這些基因片段往往小于整個(gè)基因組的千分之一，數(shù)據(jù)不足以反映整個(gè)基因組的特點(diǎn)。而全基因組序列的獲得，可以用更多的長(zhǎng)片段基因比如線粒體基因進(jìn)行真菌系統(tǒng)發(fā)育的研究，更清楚地闡明基因的結(jié)構(gòu)變異、拷貝數(shù)目、堿基缺失等與真菌生態(tài)功能的關(guān)系，加速分子標(biāo)記的開發(fā)，更準(zhǔn)確地追溯真菌起源，對(duì)以前分類模糊的種進(jìn)行精確的定性[34，98-99]。內(nèi)生真菌產(chǎn)生的四大類生物堿的合成途徑及調(diào)控基因已通過(guò)基因克隆和基因沉默等技術(shù)闡明[88，103-105]，可根據(jù)生物堿合成途徑基因出現(xiàn)與否推斷內(nèi)生真菌的產(chǎn)堿類型[97，106-108]。而全基因組序列的獲得能克服基因克隆的片面性和AT富集區(qū)干擾片段的出現(xiàn)等問(wèn)題，精確分析具有不同生活史和生態(tài)功能的生物堿合成相關(guān)基因的結(jié)構(gòu)變化，轉(zhuǎn)座子、微型反向重復(fù)轉(zhuǎn)座元件、端粒以及基因內(nèi)部堿基缺失對(duì)基因功能的影響，并發(fā)現(xiàn)了一些新的與生物堿合成相關(guān)的基因，闡明基因與內(nèi)生真菌次生代謝物多樣性的關(guān)系[5-6，109]。

圖1 內(nèi)生真菌對(duì)地上和地下生態(tài)系統(tǒng)的影響[4，95]Fig.1 The influence of endophytes on ecosystem of both above and belowground[4，95]

內(nèi)生真菌與寄主相互作用非常復(fù)雜，其相互作用機(jī)制仍不清楚。研究者曾試圖分析內(nèi)生真菌及寄主的轉(zhuǎn)錄組以闡明內(nèi)生真菌如何調(diào)控寄主。比如利用基因芯片技術(shù)分析了固體和液體培養(yǎng)基中黑麥草內(nèi)生真菌[110]以及E+和E-高羊茅植株的差異表達(dá)基因[111]；利用抑制消減雜交方法尋找寄主體內(nèi)受內(nèi)生真菌影響的基因[112]。但是這些技術(shù)需要已知基因序列，并且受探針數(shù)量的限制，不能覆蓋全部基因組。而高通量轉(zhuǎn)錄組測(cè)序技術(shù)無(wú)需全基因組信息，直接對(duì)mRNA反轉(zhuǎn)錄生成的cDNA進(jìn)行測(cè)序，根據(jù)轉(zhuǎn)錄組差異表達(dá)基因?qū)ふ覂?nèi)生真菌調(diào)控基因。尤其是內(nèi)生真菌全基因譜圖的構(gòu)建，可快速闡明內(nèi)生真菌轉(zhuǎn)錄組差異表達(dá)基因的功能，以期解釋這些基因如何在寄主中發(fā)揮調(diào)控作用。比較E+和E-植株轉(zhuǎn)錄組的差異揭示了內(nèi)生真菌通過(guò)改變寄主的代謝產(chǎn)物以調(diào)控內(nèi)生真菌與寄主之間的信號(hào)和化學(xué)物質(zhì)轉(zhuǎn)導(dǎo)，提高寄主的生長(zhǎng)和競(jìng)爭(zhēng)能力[113-115]；而通過(guò)比較某些基因缺失菌株和野生型菌株對(duì)寄主轉(zhuǎn)錄組的影響，發(fā)現(xiàn)突變菌株的水解酶類、轉(zhuǎn)運(yùn)蛋白和代謝物的基因表達(dá)顯著上調(diào)，對(duì)寄主表現(xiàn)顯著的致病作用，寄主植物抗病，轉(zhuǎn)座子活性、激素合成及應(yīng)答的基因表達(dá)上調(diào)，由互惠共生關(guān)系轉(zhuǎn)變?yōu)橹虏￡P(guān)系[114]。同樣的，分析不同逆境條件下共生體轉(zhuǎn)錄組表達(dá)差異，也發(fā)現(xiàn)了多種基因簇參與調(diào)控寄主體內(nèi)代謝途徑的改變，從而引起抗逆性的變化[115]。由于高通量測(cè)序信息量較大，受內(nèi)生真菌調(diào)控影響的基因數(shù)目較多，經(jīng)常有數(shù)百個(gè)在植物生理生化過(guò)程中起作用的功能蛋白及化合物的基因受到調(diào)控，通過(guò)基因功能分析可以推斷內(nèi)生真菌與寄主之間的物質(zhì)轉(zhuǎn)導(dǎo)，但是闡明這些基因間的關(guān)聯(lián)以及如何受到信號(hào)調(diào)控還是現(xiàn)在的難點(diǎn)，隨著生物信息的發(fā)展，網(wǎng)絡(luò)譜圖構(gòu)建加快，相信很快會(huì)克服這些困難，闡明內(nèi)生真菌與寄主相互作用的分子機(jī)制。

而以前基于基因敲除技術(shù)開展的單個(gè)目標(biāo)基因的研究，也已獲得了突破性的成果，闡釋了維持內(nèi)生真菌與寄主動(dòng)態(tài)平衡的分子機(jī)制。首先發(fā)現(xiàn)內(nèi)生真菌產(chǎn)生活性氧(reactive oxygen species，ROS)以阻止寄主的保護(hù)反應(yīng)，NADPH氧化復(fù)合體調(diào)控胞間合成活性氧ROS，Nox復(fù)合體任何基因(NoxA，NoxR，RacA和BemA)缺失或突變都會(huì)導(dǎo)致寄主感病和矮化，菌絲無(wú)序生長(zhǎng)，喪失互惠共生關(guān)系[116-119]。MAP激酶也調(diào)控Nox復(fù)合體的活性，編碼該酶的SakA基因的缺失也會(huì)導(dǎo)致互惠共生關(guān)系破壞，寄主矮化和早熟[113]。另外也發(fā)現(xiàn)菌絲融合相關(guān)基因soft(so)和轉(zhuǎn)錄因子proA基因?qū)S持內(nèi)生真菌與寄主互惠共生關(guān)系的重要作用[102，120]。進(jìn)行鐵離子吸收和貯存的嗜鐵蛋白-胞外嗜鐵素A對(duì)維系內(nèi)生真菌與多年生黑麥草互惠共生也是必需的。內(nèi)生真菌利用鐵通透酶介導(dǎo)的還原性鐵吸收系統(tǒng)或直接通過(guò)嗜鐵蛋白進(jìn)行鐵離子吸收[5，121]。非核糖體多肽合成酶是合成嗜鐵蛋白的關(guān)鍵酶，其相關(guān)調(diào)控基因sidN的缺失，抑制內(nèi)生真菌分泌鐵載體，從而改變了共生鐵離子的動(dòng)態(tài)平衡，最終導(dǎo)致其互惠性向拮抗性轉(zhuǎn)換[5]。這些機(jī)制的研究有助于闡明植物與真菌之間信號(hào)轉(zhuǎn)導(dǎo)，物質(zhì)交流過(guò)程，從而解決利用新內(nèi)生真菌創(chuàng)制共生體時(shí)出現(xiàn)的不融合性，克服共生體創(chuàng)制的瓶頸。

隨著蛋白組學(xué)和代謝組學(xué)的發(fā)展，尤其是色譜-質(zhì)譜連用，核磁共振等技術(shù)的應(yīng)用，可以對(duì)共生體次生代謝物進(jìn)行更加細(xì)致深入的分析，對(duì)未知化合物定性也變得更加容易。結(jié)合蛋白組及代謝組的比較分析技術(shù)，同時(shí)分析共生體整體代謝物的變化，闡述多種代謝產(chǎn)物和代謝途徑的動(dòng)態(tài)變化過(guò)程，有助于闡明轉(zhuǎn)錄組分析識(shí)別的重要基因，闡明基因的功能以及基因組之間的相互作用。此類研究剛剛起步，但是已發(fā)現(xiàn)內(nèi)生真菌感染能降低共生體中含氮化合物、氨基酸和鎂含量，提高可溶性碳水化合物、脂類和一些有機(jī)酸的含量[122]；提高寄主的葡萄糖、果糖、海藻糖、糖醇、脯氨酸和谷氨酸含量以及內(nèi)生真菌次生代謝物甘露醇和生物堿loline含量以提高寄主抗旱性[123]；提高與糖酵解、三羧酸循環(huán)和氨基酸合成途徑的相關(guān)代謝物水平以提高寄主抗寒能力[124]；全面影響寄主根系的分泌代謝物以改變植物地下部分以及土壤微生物群落[92]。結(jié)合上述提到的轉(zhuǎn)錄組學(xué)技術(shù)，同時(shí)分析轉(zhuǎn)錄組基因差異和代謝產(chǎn)物差異的一致性可能會(huì)更快的揭示內(nèi)生真菌調(diào)控寄主的機(jī)制。

同時(shí)，由于技術(shù)的發(fā)展，對(duì)內(nèi)生真菌產(chǎn)生的四大類生物堿合成途徑中的化合物及前體物質(zhì)的毒性及獨(dú)特的藥理作用展開了更深入的研究。以麥角堿為例，所有前體物質(zhì)現(xiàn)都可進(jìn)行定量檢測(cè)以確定各代謝產(chǎn)物的關(guān)系以及對(duì)家畜的毒性[125]。其合成途徑包含多種化合物，他們顯著抑制了依賴于Na+/K+和Mg2+離子通道的兩種ATP酶，或與D2多巴胺相結(jié)合而抑制AMP循環(huán)[126-128]，從而使動(dòng)物體溫升高，采食量減少，體重、催乳激素及牛奶產(chǎn)量降低[9]。通過(guò)對(duì)前體物質(zhì)進(jìn)行分離純化和毒性檢測(cè)，發(fā)現(xiàn)ergoamide及衍生物主要對(duì)動(dòng)物神經(jīng)產(chǎn)生影響，ergopetines主要進(jìn)行血管收縮和減少催乳激素分泌，其中ergovaline 和 ergotamine具有最強(qiáng)的血管收縮和與D2多巴胺結(jié)合的能力，而麥角酸收縮血管能力較弱。這些不同的毒性物質(zhì)有不同的毒性動(dòng)力學(xué)，與家畜體內(nèi)的藥物代謝酶作用能力也不盡相同[129]。這些研究證明了麥角堿類化合物功能非常復(fù)雜，其純品化合物可用于不同的醫(yī)療用途，而共生體代謝途徑的任何微小改變都可能引起共生體毒性及對(duì)家畜影響的變化。隨著對(duì)化合物研究的深入，在以后利用內(nèi)生真菌進(jìn)行牧草育種時(shí)，不能只考慮終產(chǎn)物的影響，必須借助代謝組學(xué)篩選不含有任何有毒物質(zhì)的菌株。

除了以生物堿為代表的活性物質(zhì)，內(nèi)生真菌還產(chǎn)生其他化學(xué)結(jié)構(gòu)和功能多樣的次生代謝物，De Battista等[130]很早就發(fā)現(xiàn)高羊茅內(nèi)生真菌在離體培養(yǎng)條件下能產(chǎn)生植物生長(zhǎng)素-吲哚乙酸。隨后，自梯牧草內(nèi)生真菌E.typhina中分離出數(shù)種抗真菌活性化合物后，陸續(xù)有吲哚類化合物[131]，倍半萜烯等多種抗真菌化合物被分離鑒定[132-133]。Song 等[133]還在披堿草內(nèi)生真菌中分離純化出具有類似除草劑活性化合物，對(duì)多年生黑麥草和早熟禾幼苗生長(zhǎng)具有明顯的抑制作用。從這些研究可以看出，內(nèi)生真菌是豐富的天然產(chǎn)物資源庫(kù)，孕育著多種新型化合物，根據(jù)這些資源創(chuàng)制具有新分子骨架、新作用機(jī)制的新型殺蟲劑、殺菌劑并在生產(chǎn)上進(jìn)行開發(fā)利用還任重道遠(yuǎn)。

6 利用新內(nèi)生真菌菌株進(jìn)行新品種選育

由于內(nèi)生真菌對(duì)家畜的毒性，曾嘗試建立不帶內(nèi)生真菌的高羊茅(美國(guó))和黑麥草(新西蘭)草地以防治家畜中毒，但是不帶內(nèi)生真菌的牧草建植困難、生長(zhǎng)緩慢、產(chǎn)量下降、易受蟲害危害，難以管理[1，42]，在某些逆境條件下甚至不能存活[25]，證明內(nèi)生真菌對(duì)寄主有極其重要的作用，建立不帶內(nèi)生真菌的草地并不可行。減少家畜病害的其他措施還包括E+牧草與三葉草(Trifoliumsp.)、苜蓿(Medicagosp.)等豆科牧草混播，與谷類混飼，制作青貯飼料等降低家畜采食的毒素[134]；或者補(bǔ)飼藥物或添加劑；加強(qiáng)放牧管理比如輪牧減少家畜對(duì)生物堿含量高的莖基部采食[10，135]。但是這些措施都不能從根本解決家畜中毒問(wèn)題，而對(duì)家畜無(wú)毒的內(nèi)生真菌菌株的發(fā)現(xiàn)為解決這一問(wèn)題提出了新途徑。從禾草內(nèi)生真菌種質(zhì)資源中篩選出不產(chǎn)生對(duì)家畜有毒生物堿的無(wú)毒菌株，通過(guò)接種技術(shù)建立新的既具有抗逆性又對(duì)家畜無(wú)毒的禾草-內(nèi)生真菌共生體，提高禾草的品質(zhì)并確保對(duì)動(dòng)物的安全性，在育種上已獲得了巨大成功[4-5，136]。表1列出了部分廣泛用于多年生黑麥草和高羊茅育種的新內(nèi)生真菌菌株。目前已有5種內(nèi)生真菌用于黑麥草育種(Endosafe、AR1、NEA2、Endo5和AR37)，尤其是AR1，投放市場(chǎng)3年內(nèi)占據(jù)了新西蘭黑麥草種子市場(chǎng)的60%[137]。還有澳大利亞維多利亞農(nóng)業(yè)生物研究中心和新西蘭Agriseeds聯(lián)合發(fā)現(xiàn)了多株無(wú)毒菌株正擬投放市場(chǎng)[138]。高羊茅內(nèi)生真菌E.coenophiala菌株AR542只產(chǎn)生對(duì)昆蟲有毒的loline和peramine生物堿，將其接種到一些優(yōu)質(zhì)高羊茅中開發(fā)了MaxQ和 MaxP商用品種。這些共生體既不影響采食家畜的健康，又同時(shí)保持了植株的生長(zhǎng)及抗逆優(yōu)勢(shì)，在畜牧業(yè)生產(chǎn)中取得了巨大的經(jīng)濟(jì)價(jià)值[136，139]。而進(jìn)行草坪草育種時(shí)，不需要考慮內(nèi)生真菌毒素引致的家畜中毒問(wèn)題，其為寄主帶來(lái)的抗逆、抗蟲、耐踐踏等特性正是草坪草業(yè)所需要的[1，67]。因此根據(jù)內(nèi)生真菌的不同表現(xiàn)可以將其利用在不同方面，比如新西蘭利用對(duì)家畜和昆蟲有毒生物堿含量極高的內(nèi)生真菌菌系A(chǔ)R95和AR601，分別接入到多年生黑麥草和高羊茅草坪草品種中，成功培育機(jī)場(chǎng)防鳥草Avanex品種[140]。

由于內(nèi)生真菌的寄主特異性，利用人工接種建立新的禾草-內(nèi)生真菌共生體時(shí)，會(huì)發(fā)生一些不融合反應(yīng)(incompatability)，這就限制了內(nèi)生真菌可利用的范圍。其次寄主和內(nèi)生真菌的相互關(guān)系受植物和內(nèi)生真菌基因型的強(qiáng)烈影響[13，15]。同一內(nèi)生真菌接種到不同寄主中后其生物堿種類和含量會(huì)發(fā)生改變。有時(shí)候新的共生體產(chǎn)生的生物堿與期待并不一致，一個(gè)很明顯的例子就是Endosafe，Endosafe是只產(chǎn)生吡咯并吡嗪類化合物(peramine)卻不產(chǎn)生吲哚雙萜類化合物(lolitrem B)的E.festucaevar.lolii(N.lolii)菌株，當(dāng)它接種到黑麥草中卻產(chǎn)生了麥角堿(ergovaline)[149]，因此該菌株后來(lái)被AR1取代，并提醒育種者后續(xù)利用內(nèi)生真菌育種時(shí)必須開展長(zhǎng)期嚴(yán)格評(píng)估才能投放市場(chǎng)[5]。研究也發(fā)現(xiàn)不產(chǎn)生動(dòng)物毒素的內(nèi)生真菌對(duì)寄主的保護(hù)作用有所降低，使其在實(shí)際生產(chǎn)中的應(yīng)用有所下降[150]。比如接種了新內(nèi)生真菌的黑麥草，產(chǎn)生的吡咯并吡嗪類化合物(peramine)含量降低，因而更容易受阿根廷莖象甲的危害[147，150]，這些都是利用內(nèi)生真菌育種時(shí)需要解決的問(wèn)題。基于這些問(wèn)題，一方面正在利用基因組學(xué)和代謝組學(xué)新技術(shù)深入研究?jī)?nèi)生真菌與寄主相互作用的機(jī)制，從而解決利用內(nèi)生真菌創(chuàng)制共生體時(shí)出現(xiàn)的不融合性，克服共生體創(chuàng)制的瓶頸。另一方面利用基因工程技術(shù)人工創(chuàng)造無(wú)毒菌株，而生物堿合成途徑及相關(guān)調(diào)控基因的闡明也為采用生物工程方法創(chuàng)制對(duì)家畜無(wú)毒菌株奠定了基礎(chǔ)[6，102，113]。比如澳大利亞Spangenberg實(shí)驗(yàn)室將菌株進(jìn)行基因突變，定向篩選缺少lolitrem B合成基因的菌株，獲得了不產(chǎn)生lolitrems的內(nèi)生真菌菌株；或者利用CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)基因編輯技術(shù)，切除有毒生物堿的基因以獲得無(wú)毒菌株；利用根癌農(nóng)桿菌誘導(dǎo)轉(zhuǎn)化將抗蟲peramine基因?qū)氲街划a(chǎn)生有益生物堿janthitrems的菌株中，獲得了可以同時(shí)產(chǎn)生這兩種抗蟲生物堿的菌株(Spangenberg G，個(gè)人通訊)。通過(guò)生物技術(shù)創(chuàng)制的菌株接回原始分離寄主則可以減少內(nèi)生真菌與寄主不融合性的出現(xiàn)。

表1 用于多年生黑麥草和高羊茅育種的新內(nèi)生真菌菌株

Table 1 Novel endophytes strains and their outcomes

內(nèi)生真菌Endophyticfungi菌株Strain產(chǎn)堿特性Alkaliproperty生產(chǎn)中表現(xiàn)Performanceinproduction參考文獻(xiàn)Reference多年生黑麥草Perennialryegrass,內(nèi)生真菌E.festucaevar.loliiAR1吡咯并吡嗪類化合物Peramine無(wú)黑麥草蹣跚癥,提高家畜體重。Noryegrassstaggers,improvelivestockbodyweight.[141]提高種子產(chǎn)量和萌發(fā)率。Improveseedsproductionandgermination.[142]無(wú)黑麥草蹣跚癥,并提高牧草產(chǎn)量和家畜奶產(chǎn)量。Noryegrassstaggers,improveforageandlivestockmilkproduction.[143]AR37Janthitrems無(wú)黑麥草蹣跚癥,提高寄主對(duì)害蟲Wiseanacervinata的抗性。Noryegrassstaggers,improvehostresistancetopest-Wiseanacervinata.[144]提高牧草產(chǎn)量和草地持久性。Improveforageproductionandgrasslandpersistence.[145]NEA2較少的吲哚雙萜類,麥角堿類和吡咯并吡嗪類化合物lowlevelsofLolitremB+Ergot+Peramine提高寄主對(duì)阿根廷莖象甲的抗性。ImprovehostresistancetoListronotusbonariensis.[146]接種到不同黑麥草品種中,生物堿含量有變化。Alkaloidscontentsvariedinoculatedintodifferenthostcultivar.[100]Endosafe麥角堿類和吡咯并吡嗪類化合物Ergot+Peramine無(wú)黑麥草蹣跚癥,提高對(duì)阿根廷莖象甲的抗性。Noryegrassstaggers,im-provehostresistancetoListronotusbonariensis.[141]Endo5麥角堿類和吡咯并吡嗪類化合物Ergot+Peramine提高寄主抗蟲性。Improvehostresistancetopest.[137]AR6麥角堿類和吡咯并吡嗪類化合物Ergot+Peramine無(wú)黑麥草蹣跚癥,提高對(duì)阿根廷莖象甲的抗性。Noryegrassstaggers,im-provehostresistancetoListronotusbonariensis.[147]AR95吲哚雙萜類,麥角堿類和吡咯并吡嗪類化合物L(fēng)olitremB+Ergot+Peramine在機(jī)場(chǎng)草坪中,減少昆蟲和鳥的采食。Birdandwildlifedeterrentinryegrasssportfields.[140]高羊茅Tallfescue,內(nèi)生真菌E.coenophi-alaAR542,AR548吡咯并吡嗪類和飽和吡咯化合物Peramine+Lolines無(wú)狐茅中毒癥,提高牧草產(chǎn)量,草地持久性,提高家畜體重。Nofescuetoxico-sis,improveforageproduction,grasslandpersistenceandlivestockbodyweight.[139]提高寄主對(duì)蚜蟲的抗性。ImprovehostresistancetoRhopalosiphumpadi.[136]ArkPlus吡咯并吡嗪類和飽和吡咯化合物Peramine+Lolines無(wú)狐茅中毒癥,提高家畜體重。Nofescuetoxicosis,improvelivestockbodyweight.[148]AR601麥角堿類和飽和吡咯化合物Ergot+Lolines在機(jī)場(chǎng)草坪中,減少昆蟲和鳥的采食。Birdandwildlifedeterrentinryegrasssportfields.[140]

7 展望

經(jīng)過(guò)多年的努力，內(nèi)生真菌的研究已經(jīng)獲得了顯著的成果，發(fā)現(xiàn)了越來(lái)越多的Epichloё屬內(nèi)生真菌，這將有助于整體認(rèn)識(shí)和理解內(nèi)生真菌的系統(tǒng)發(fā)育過(guò)程，進(jìn)行優(yōu)良禾草的選育和改良，加強(qiáng)對(duì)內(nèi)生真菌資源的保護(hù)。但是除了高羊茅和黑麥草內(nèi)生真菌，其余菌株還需深入研究其進(jìn)化起源以及與寄主植物的關(guān)系。有關(guān)內(nèi)生真菌在生態(tài)系統(tǒng)水平對(duì)植物群落結(jié)構(gòu)、生產(chǎn)力和多樣性影響的研究還較少，為更好利用內(nèi)生真菌保護(hù)草原生態(tài)系統(tǒng)的穩(wěn)定性，需要加強(qiáng)了解內(nèi)生真菌在植物和真菌進(jìn)化過(guò)程中的作用和在天然草地群落中的作用。

由于禾本科植物內(nèi)生真菌對(duì)寄主植物品質(zhì)及抗逆性的影響，內(nèi)生真菌已經(jīng)廣泛應(yīng)用到牧草和草坪草育種中，而小麥(Triticumaestivum)、水稻(Oryzasativa)等糧食作物也屬于禾本科植物，新西蘭研究者嘗試通過(guò)人工接種，將小麥族植物中分離的內(nèi)生真菌轉(zhuǎn)接到小麥、大麥(Hordeumvulgare)等麥類作物中，但是由于寄主特異性和不融合性，尚未獲得成功，不能直接用于糧食作物中[151]。因此，應(yīng)進(jìn)一步擴(kuò)大內(nèi)生真菌資源調(diào)查的范圍，尤其是從冷季型草坪草擴(kuò)寬到暖季型植物，以期發(fā)現(xiàn)更多可以在糧食生產(chǎn)中應(yīng)用的內(nèi)生真菌。同時(shí)，應(yīng)盡快解決內(nèi)生真菌與寄主不融合性的問(wèn)題，借助基因組學(xué)和代謝組學(xué)闡明真菌與植物之間信號(hào)轉(zhuǎn)導(dǎo)和物質(zhì)交流過(guò)程，從而利用基因組編輯技術(shù)創(chuàng)制兼容性無(wú)毒菌株，為內(nèi)生真菌應(yīng)用到糧食作物育種，保障糧食安全奠定基礎(chǔ)。

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Advances in research on grass endophytes in agricultural systems and applications in forage breeding

TIAN Pei1*, ZHANG Guang-Ming2, NAN Zhi-Biao1

1.StateKeyLaboratoryofGrasslandAgro-ecosystems,CollegeofPastoralAgricultureScienceandTechnology,LanzhouUniversity,Lanzhou730020,China; 2.GuangdongYuemingElectricPowerEngineeringCo.Ltd,Zhuhai519000,China

TheEpichloё endophyte forms mutually beneficial associations with its hosts, which enhance their survival under abiotic and biotic stresses. Modern techniques in molecular biology, genomics, proteomics, metabolomics, and bioinformatics have accelerated research on endophytes. The diversity of secondary metabolites and the genes related to their biosynthesis have been identified and the molecular mechanisms of mutualism between theEpichloё endophyte and its hosts have been clarified. These endophytes are widely utilized in agriculture as they are animal-safe grass endophytes that improve the growth and stress resistance of their hosts, leading to increased pasture persistence, sustainability, and production. Thus, severalEpichloё endophytes have been developed and commercialized. In this paper, we review recent research on the interactions between grasses and endophytes, and the effects of endophytes on livestock and on microbial and pasture ecosystems. We also discuss the selection of endophyte strains and forage breeding based on multidisciplinary research.

endophyte; trophic cascade; ecosystem; microbe; genomics; forage breeding

10.11686/cyxb2016049

http://cyxb.lzu.edu.cn

2016-01-25；改回日期：2016-03-08

國(guó)家基礎(chǔ)研究發(fā)展規(guī)劃“973”(2014CB138702)，國(guó)家自然科學(xué)基金項(xiàng)目(31502001)和企事業(yè)單位委托科技項(xiàng)目[(15)0065]資助。

田沛(1979-)，女，河南新鄭人，副教授，博士。 E-mail: tianp@lzu.edu.cn*通信作者Corresponding author.

田沛， 張光明， 南志標(biāo). 禾草內(nèi)生真菌研究及應(yīng)用進(jìn)展. 草業(yè)學(xué)報(bào), 2016, 25(12): 206-220.

TIAN Pei, ZHANG Guang-Ming, NAN Zhi-Biao. Advances in research on grass endophytes in agricultural systems and applications in forage breeding. Acta Prataculturae Sinica, 2016, 25(12): 206-220.