陳振江，魏學(xué)凱，曹 瑩，田 沛，趙曉靜，李春杰

(草地農(nóng)業(yè)生態(tài)系統(tǒng)國家重點實驗室 蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院,甘肅 蘭州 730020)

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禾草內(nèi)生真菌檢測方法研究進(jìn)展

陳振江，魏學(xué)凱，曹 瑩，田 沛，趙曉靜，李春杰

(草地農(nóng)業(yè)生態(tài)系統(tǒng)國家重點實驗室 蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院,甘肅 蘭州 730020)

從最初發(fā)現(xiàn)黑麥草(Loliumperenne)和高羊茅(Festucaarundinacea)引起家畜中毒到后來其攜帶的內(nèi)生真菌被發(fā)現(xiàn)，以及進(jìn)一步的研究證實，內(nèi)生真菌的存在不但導(dǎo)致家畜中毒而且能顯著提高宿主在群落中的競爭力，也因禾草內(nèi)生真菌的這種重要生理和生態(tài)作用，使其逐步成為國內(nèi)外研究的熱點之一，這也給內(nèi)生真菌檢測技術(shù)的發(fā)展提供了契機(jī)。初期主要是借鑒病原真菌等微生物檢測的一些較成熟的方法，對禾草內(nèi)生真菌進(jìn)行染色鏡檢或分離檢測，但容易受宿主種類、物候期、檢測部位等因素的影響，造成檢測結(jié)果的不準(zhǔn)確。隨著分子生物學(xué)、遺傳學(xué)等學(xué)科的快速發(fā)展，酶聯(lián)免疫吸附測定法(enzyme linked immunosorbent assay,ELISA)、聚合酶鏈?zhǔn)椒磻?yīng)法(polymerase chain reaction，PCR)、實時熒光PCR法(realtime PCR)等現(xiàn)代分子技術(shù)在禾草內(nèi)生真菌方面的應(yīng)用和改進(jìn)，使得內(nèi)生真菌的檢測方法不斷推陳出新，這些方法能在一定程度上對傳統(tǒng)檢測方法給予有效的補充。快速有效的確定禾草內(nèi)生真菌的存在、分布規(guī)律、分類地位等，需要準(zhǔn)確合理地選擇特異性較強(qiáng)的檢測方法，例如定性或定量檢測，并結(jié)合經(jīng)典的染色鏡檢和分離法對禾草內(nèi)生真菌進(jìn)行確定。本研究對以上內(nèi)容進(jìn)行了綜述，以期更深層次的借鑒和發(fā)展關(guān)于其它微生物的經(jīng)典檢測方法，同時開發(fā)具有禾草內(nèi)生真菌特異性的檢測技術(shù)，這些技術(shù)不僅能夠鑒定內(nèi)生真菌存在與否，而且能對內(nèi)生真菌進(jìn)行定量和活性的檢測,是發(fā)展的重要方向和熱點。對內(nèi)生真菌定性、定量和活性的檢測是未來值得研究的重點。

禾草內(nèi)生真菌；分離檢測；顯微觀察法；酶聯(lián)免疫吸附法；生物堿高效液相色譜法；分子檢測法

禾草內(nèi)生真菌(grass endophyte)是指在禾草體內(nèi)度過大部分或者全部生命周期，但卻不會引起禾草外部顯示任何病害癥狀的一大類真菌[1]。目前，關(guān)于禾草內(nèi)生真菌的研究主要集中在與冷季型早熟禾亞科(Pooideae)植物共生的Epichloё 屬[2]。禾草與內(nèi)生真菌形成的共生體對宿主和內(nèi)生真菌均具有一定增益作用：提高了宿主的生物抗性[3-5]和非生物抗性[6-10],促進(jìn)禾草的根系發(fā)育、分蘗能力等[11-13],提高宿主植物的競爭力[14-15]；宿主禾草為內(nèi)生真菌的傳播提供了營養(yǎng)及繁殖場所,增加了草地的壽命,提高了生態(tài)服務(wù)價值[16]；但此共生體也會產(chǎn)生有毒物質(zhì)，家畜采食后會引起家畜中毒，對其生長性能、受孕率、神經(jīng)系統(tǒng)和免疫系統(tǒng)等會產(chǎn)生影響[17-19]，從而對草地畜牧業(yè)產(chǎn)生了不利的影響[17]。

除部分內(nèi)生真菌有廣泛的宿主范圍和部分寄主能被多種內(nèi)生真菌感染外，絕大多數(shù)禾草常被一種真菌感染，導(dǎo)致內(nèi)生真菌與宿主之間有著比較嚴(yán)格的宿主特異性[18]，因此分離自不同宿主的禾草內(nèi)生真菌數(shù)量和種類不同。而且由于禾草的種類和基因型以及禾草生長的環(huán)境因素影響，與之共生的禾草內(nèi)生真菌在種類和數(shù)量上具有很大的差異性和多樣性[20]，已經(jīng)發(fā)表的內(nèi)生真菌有43種，尚有部分菌株未鑒定[2,21-22]或未發(fā)表[23]。

禾草內(nèi)生真菌主要分布在植株的莖髓、葉鞘、葉片和種子等地上部分，與宿主植物協(xié)同生長；內(nèi)生真菌菌絲在細(xì)胞間隙分布不規(guī)則且在禾草不同組織中的菌絲形態(tài)存在一定差異[22,24]。Epichloё屬內(nèi)生真菌種類和數(shù)量較多，其不同內(nèi)生真菌種類的菌落顏色、質(zhì)地、分生孢子及分生孢子梗的形態(tài)和大小存在差異，明顯顯示出豐富的形態(tài)多樣性[19，25]。禾草內(nèi)生真菌分子系統(tǒng)發(fā)育的研究表明,Epichloё的起源有3種途徑[26]:直接起源于Epichloё的某個種，Epichloё的種間雜交，Epichloё與其無性態(tài)Neotyphodium屬之間雜交；通過雜交重組產(chǎn)生了新的遺傳特性，從而出現(xiàn)了許多新的種、亞種及變種[2]。因此,檢測和鑒定禾草中是否存在內(nèi)生真菌，確定禾草內(nèi)生真菌的種屬對研究禾草內(nèi)生真菌共生體多樣性、內(nèi)生真菌提高禾草抗逆性及篩選有益內(nèi)生真菌從而培育優(yōu)良品種十分重要。

內(nèi)生真菌的研究最基礎(chǔ)的方法是分離檢測，從形態(tài)學(xué)上鑒定內(nèi)生真菌的形態(tài)；但其檢測周期長，且無法控制環(huán)境等因素對菌種的保存和鑒定產(chǎn)生的影響[16]。鏡檢法與分離檢測法常相結(jié)合應(yīng)用于禾草內(nèi)生真菌的分離鑒定上，在顯微鏡下通過苯胺藍(lán)染色快速、直觀地檢測禾草內(nèi)生真菌的存在，對分離菌落的分生孢子等進(jìn)行觀測，彌補了分離檢測法的不足。對高羊茅(Festucaarundinacea)內(nèi)生真菌的亞顯微結(jié)構(gòu)進(jìn)行觀察發(fā)現(xiàn)，禾草的葉片、葉鞘、莖髓和種子中普遍存在內(nèi)生真菌，葉鞘中帶菌率最高[27]。免疫學(xué)上檢測內(nèi)生真菌的方法有免疫印跡法、酶聯(lián)免疫吸附法(enzyme linked immunosorbent assay,ELISA)等，通過酶的高效催化作用與底物發(fā)生顯色反應(yīng)，從而定性或者定量鑒定不同禾草的內(nèi)生真菌，Musgrave應(yīng)用ELISA法檢測出多年生黑麥草(Loliumperenne)內(nèi)生真菌，其菌絲量濃度為 0.1 mg·mL-1。相對于鏡檢測法，此法特異性強(qiáng)，在一定程度上提高了檢測的速度和靈敏性[28]。此外普通PCR標(biāo)記和實時熒光PCR標(biāo)記等分子生物學(xué)技術(shù)也被用作檢測禾草內(nèi)生真菌，利用Tub-2基因可設(shè)計出Epichloё 及無性態(tài)通用Taqman探針及引物，建立雙色熒光實時定量PCR檢測醉馬草(Achnatheruminebrians)內(nèi)生真菌的方法，可準(zhǔn)確快速地檢測出醉馬草內(nèi)生真菌DNA片段的含量，進(jìn)而計算出醉馬草內(nèi)生真菌的含量[29]。因此，正確合理地選擇定性或定量檢測禾草內(nèi)生真菌的方法(形態(tài)學(xué)法、鏡檢法、分子法、免疫學(xué)法等)對確定禾草內(nèi)生真菌的有無、分布規(guī)律、種屬等至關(guān)重要。

1 鏡檢和分離檢測

禾草內(nèi)生真菌研究初期以鏡檢和分離檢測兩種檢測方法結(jié)合使用為主，通過對組織進(jìn)行染色，觀察內(nèi)生真菌在宿主中的形態(tài)特征和分離培養(yǎng)的菌落形態(tài)來鑒定種間的區(qū)別。

禾草內(nèi)生真菌鏡檢法根據(jù)宿主不同組織特異性常采用不同的染色試劑(表1)，主要使葉鞘的內(nèi)表皮、莖的髓質(zhì)和種子著色，然后在光學(xué)顯微鏡下檢測禾草內(nèi)生真菌菌絲，統(tǒng)計禾草帶菌率。不同的染劑可使不同的共生體著色：乳酚-棉藍(lán)使Danthoniaspicata和多年生黑麥草的種子著色[37-38]；乳酚-酞酚藍(lán)可染色多年生黑麥草內(nèi)生真菌[39]；苯胺藍(lán)使高羊茅和黑麥草的菌絲著色[40-41]；龍膽紫溶液和革蘭氏染劑也可使菌絲染色[42]；玫瑰紅染劑可將牧草和草坪草的莖髓、種子等組織中的內(nèi)生真菌染成紅色在顯微鏡下觀察菌絲形態(tài)[43];苯胺藍(lán)染色高羊茅種子后在硝酸中浸泡過夜使其軟化，可在顯微鏡下觀察到糊粉層中被染成藍(lán)色的菌絲[44]。除了試劑染色鏡檢法外，熒光素雙醋酸鹽(FDA)溶液作為染劑，在熒光顯微鏡下觀察禾草內(nèi)生真菌，鏡下亮藍(lán)色的絲狀體就是具有活性的禾草內(nèi)生真菌菌絲體。熒光素雙醋酸鹽(FDA)溶液也可以作為載體在熒光顯微鏡下觀察多年生黑麥草內(nèi)生真菌并檢測其菌絲體的活性[45](表2)。目前禾草內(nèi)生真菌鏡檢法主要使用的是苯胺藍(lán)染色劑，多使用1%的苯胺藍(lán)染液進(jìn)行染色效果最佳[16]。此方法只能檢測禾草是否帶菌，但無法準(zhǔn)確鑒定禾草內(nèi)生真菌的活性。李春杰等對醉馬草不同生育期和不同組織內(nèi)生真菌菌絲體檢測對比，建立了適合醉馬草內(nèi)生真菌檢測的方法[45-46]。

除了光學(xué)顯微鏡的使用外，一些更加先進(jìn)的顯微鏡也被應(yīng)用到內(nèi)生真菌的檢測中。如利用光學(xué)顯微鏡、電子顯微鏡和熒光顯微鏡對黑麥草葉片細(xì)胞間隙的Epichloё 及無性態(tài)內(nèi)生真菌進(jìn)行觀察，發(fā)現(xiàn)內(nèi)生真菌的侵染寄主是沿著禾草葉片延伸生長的方向分裂和擴(kuò)展，同時證明了禾草內(nèi)生真菌具有居間生長的特點[47]。透射電子顯微鏡只能觀察禾草組織超薄切片標(biāo)本，而有可能禾草內(nèi)生真菌表面的結(jié)構(gòu)與內(nèi)生真菌內(nèi)部的結(jié)構(gòu)不同，使檢測復(fù)雜化；超薄切片標(biāo)本，制樣過程復(fù)雜，制作標(biāo)本有損傷；此外電子顯微鏡購買和維護(hù)的價格都比較高[16]。鏡檢法與分離檢測法常相結(jié)合應(yīng)用于禾草內(nèi)生真菌的分離鑒定上，在顯微鏡下觀察禾草顯微結(jié)構(gòu)快速、直觀，彌補了分離檢測法僅依靠形態(tài)學(xué)特征鑒定菌種的不足。Christensen在E.festucae真菌和寄主基因型對高羊茅內(nèi)生真菌共生體兼容性和維管束影響的研究中使用光學(xué)顯微鏡和透射電子顯微鏡觀察寄主細(xì)胞的變化以及葉片維管束胞間菌絲，并通過觀察母體和后代禾草內(nèi)生真菌共生體分離培養(yǎng)的菌落形態(tài)學(xué)特征，發(fā)現(xiàn)維管束胞間菌絲濃度最高[48]。

通過顯微鏡鏡檢，發(fā)現(xiàn)內(nèi)生真菌的菌絲后進(jìn)行分離培養(yǎng)，根據(jù)菌落形態(tài)來鑒定種間的差異。禾草內(nèi)生真菌分離培養(yǎng)(形態(tài)學(xué)鑒定)的步驟一般是：首先對禾草表面進(jìn)行消毒滅菌處理，然后將禾草組織切成1 cm小節(jié)放在培養(yǎng)基中培養(yǎng)，最后通過分離培養(yǎng)的菌株形態(tài)特征來鑒定菌種[16]。但Epichloё 屬內(nèi)生真菌在體外分離培養(yǎng)時生長緩慢，容易受到培養(yǎng)條件、培養(yǎng)基類型及自身遺傳組成的影響，且有的菌株在常規(guī)培養(yǎng)基上不易產(chǎn)生孢子，而孢子是禾草內(nèi)生真菌形態(tài)學(xué)鑒定的一個重要因子[49]。不產(chǎn)孢的菌株一般無法用形態(tài)學(xué)特征來鑒定，需要和其它方法相結(jié)合來檢測；對夏季采自黃山景區(qū)海拔500～1 600 m處的23個屬631株禾草鏡檢發(fā)現(xiàn)，Epichloё 屬內(nèi)生真菌的確存在，并得出禾草內(nèi)生真菌的分布與寄主植物的種類、寄主植物的地理分布類型等有關(guān)，通過分離培養(yǎng)觀察發(fā)現(xiàn)其子座等形態(tài)特征與歐洲菌株有較大的差異性[50]。將禾草組織中的內(nèi)生真菌在不同的培養(yǎng)基中進(jìn)行分離培養(yǎng)[51]，通過觀察其菌落形態(tài)特征(顏色、形狀)、菌落生長速率及產(chǎn)孢量、產(chǎn)孢特征、孢子特性等鑒定指標(biāo)，并通過顯微鏡下觀察菌絲及分生孢子的形態(tài)學(xué)特征[52]，從而鑒定禾草內(nèi)生真菌的種、屬[53-54]。已知的內(nèi)生真菌通常采用鏡檢和分離結(jié)合的方法進(jìn)行鑒定，如分離培養(yǎng)多年生黑麥草內(nèi)生真菌獲得了多年生黑麥草植株Gliocladium-like和Acremoniumloliae兩個內(nèi)生真菌菌株[55-56]；通過觀察亞利桑那羊茅(Festucaarizonica)內(nèi)生真菌分離培養(yǎng)的菌株孢子和菌落的形態(tài)學(xué)特征，建立了新種E.typhinavar.huerfanum[57]；通過比較醉馬草分生孢子大小、分生孢子梗長寬及醉馬草內(nèi)生真菌菌落的形態(tài)，確定了新種E.gansuensis[58]。

表1 不同染色劑溶液的比較Table 1 Comparison of different stain solutions

染色劑Stain方法Method染色過程Dyingprocess實驗對象 Subject 效果Result乳酚?棉藍(lán)Lactophenolcot?tonblue種子檢測法methodforseeddetection1)10%的NaOH浸泡過夜overnightsoakwith10%NaOH；2)除去種稃種皮removelemmaandseedcoat；3)擠扁，染色flat，dyeing；4)鏡檢microscopicexamination．多年生黑麥草種子perennialryegrassseedsDanthoniaspicata．較好better龍膽紫溶液Gentianvioletso?lution種子檢測法methodforseeddetection1)除去種稃種皮removelemmaandseedcoat；2)染色dyeing；3)鏡檢microscopicexamination．多年生黑麥草種子perennialryegrassseeds好good革蘭氏Gram種子檢測法methodforseeddetection1)除去種稃種皮removelemmaandseedcoat；2)染色dyeing；3)鏡檢microscopicexamination．多年生黑麥草種子perennialryegrassseeds好good玫瑰紅Rosebengal莖髓、種子、葉鞘檢測法methodforstemmarrow，seed，andleafsheathdetection1)NaOH浸泡過夜overnightsoakwithNaOH；2)刮取髓質(zhì)和除去種稃種皮scrapethemedullaandremovelemmaandseedcoat；3)染色dyeing；4)鏡檢microscopicexamination．牧草和草坪草的莖髓、種子等組織stemmarrowandseedsofforageandturfgrass良fine苯胺藍(lán)Anilineblue莖髓、種子、葉鞘檢測法methodforstemmarrow，seed，andleafsheathdetection1)硝酸浸泡過夜overnightsoakwithHNO3；2)除去種稃種皮removelemmaandseedcoat；3)擠扁，染色flat，dyeing；4)鏡檢microscopicexamination．醉馬草莖髓、葉鞘、種子和高羊茅種子stems，leafsheaths，seedsofdrunkhorsegrass，andtallfes?cueseeds良fine熒光素雙醋酸鹽Fluoresceindiace?tate葉鞘檢測法methodforleafsheathdetection1)撕取葉鞘薄皮tearthinskinofleafsheath；2)染色dyeing；3)鏡檢microscopicexamination．多年生黑麥草和醉馬草葉鞘leafsheathsofperennialryegrassanddrunkhorsegrass優(yōu)excellent

2 免疫檢測法

酶聯(lián)免疫吸附法(ELISA)是采用抗原與抗體的特異反應(yīng)將待測物與酶連接然后通過酶的高效催化作用與底物發(fā)生顯色反應(yīng)，對受檢物質(zhì)進(jìn)行定性或定量分析的一種檢測方法[27]。因ELISA技術(shù)準(zhǔn)確度高、特異性強(qiáng)、適用范圍廣、檢測樣本多速度快，并且ELISA技術(shù)的條件要求比較低，方便操作，所以ELISA技術(shù)經(jīng)常被制成試劑盒的形式，被廣泛運用在禾草內(nèi)生真菌的檢測分析上[59-62]。

ELISA最初被應(yīng)用于定量檢測高羊茅種子和葉鞘組織中的內(nèi)生真菌[63-66]。使用ELISA法檢測高羊茅體內(nèi)的內(nèi)生真菌，發(fā)現(xiàn)每毫升冰凍干燥的菌絲體中至少含100 ng的量[67]。從兔子(Leporidae)體中提取多克隆抗血清來檢測高羊茅種子和組織中的內(nèi)生真菌，在種子檢測時需要讓種子吸水過夜保證其新鮮性，較費時[68]。鏡檢法和單克隆抗體免疫印跡試劑法對比檢測高羊茅內(nèi)生真菌的帶菌率，免疫印跡法在大樣品檢測中準(zhǔn)確率更高，更容易操作[69]。Koh和Hik借助免疫法驗證了內(nèi)生真菌在天然草地中的分布規(guī)律以及在生態(tài)環(huán)境中所起的作用[70]，之后免疫技術(shù)法被廣泛應(yīng)用到禾草內(nèi)生真菌的檢測及闡述禾草內(nèi)生真菌的分配模式[71-73]，禾草內(nèi)生真菌共生體動力學(xué)的季節(jié)性傾向[74-75]和食草動物、內(nèi)生真菌侵染禾草的動力學(xué)關(guān)系中[70,76-78]。但是由于各種禾草內(nèi)生真菌的理化性質(zhì)、形態(tài)特性等方面的不同，寄主植物不同，抗體生產(chǎn)、測定、酶標(biāo)記物的制備不能標(biāo)準(zhǔn)化，檢測時受到特異性和非特異性的干擾，使得ELISA技術(shù)有其局限性和不足。而且隨著分子生物技術(shù)的發(fā)展，測序等分子檢法方法成本的降低，ELISA使用率下降。

3 利用次生代謝物間接檢測

產(chǎn)生生物堿類次生代謝物是禾草與內(nèi)生真菌共生最重要的特征，也是采用這種方法檢測內(nèi)生真菌的重要依據(jù)。目前，已發(fā)現(xiàn)至少十余種與禾草內(nèi)生真菌相關(guān)的生物堿，主要有4種Epichloё 內(nèi)生真菌生成的不同植物保護(hù)性生物堿：吲哚雙萜類(indolditerpene)、吡咯并吡嗪類(pyrrolopyazine)、麥角堿類(ergot alkaloids)和飽和吡咯化合物(pyrroliziline)[5,79]。每種禾草內(nèi)生真菌共生體可以產(chǎn)生多種生物堿，同一種生物堿也可由不同的共生體產(chǎn)生[80]。一般在禾草內(nèi)生真菌共生體中可檢測到一種或多種生物堿，但絕大多數(shù)含有多種生物堿[81]。其中高羊茅和黑麥草中最常見的是吡咯并吡嗪類生物堿(peramine)，黑麥草堿(loline)和麥角堿次之，吲哚雙萜類生物堿(lolitrem)最少[82-83]。不同禾草內(nèi)生真菌共生體生成不同的生物堿，主要是由功能產(chǎn)堿基因在內(nèi)生真菌基因組中的有無決定，有時候也因基因表達(dá)水平的不同[84]。除生物堿類物質(zhì)之外，有關(guān)禾草內(nèi)生真菌共生體其它代謝產(chǎn)物的研究也越來越多[85-87]，如從麥賓草(Elymustangutorum)內(nèi)生真菌次生代謝產(chǎn)物中可分離得到17種化合物，其中cyclosporin T具有較強(qiáng)的滅菌活性[87]。

禾草內(nèi)生真菌生物堿研究的初期以簡單的顯色反應(yīng)和色譜法來檢測產(chǎn)堿量。Dimenna和Turner等曾用VanUrk反應(yīng)檢測麥角堿[88-89]。由于此檢測技術(shù)的靈敏度低，無法進(jìn)行定量檢測。生物堿檢測方法的不斷改進(jìn)，從最初的簡單比色法[90]、薄層析法(TLC)[91-92]發(fā)展為ELISA法[93]、氣相(GC)[94-95]、質(zhì)譜方法(MS)[96]以及高效液相色譜法(HPLC)[97-98](表3)。Najafabadi等利用HPLC-MS方法檢測高羊茅內(nèi)生真菌不同生長期麥角瓦靈的含量[108]。Roberts等建立了NIR檢測高羊茅內(nèi)生真菌中麥角堿含量的方法[109]。

近年來，國內(nèi)外開展了對禾草內(nèi)生真菌生物堿合成基因、基因組、比較基因組及分子系統(tǒng)發(fā)育關(guān)系等分子學(xué)的深入研究，證實了其實用性和精確性[110-113]。已有研究證實可以從分子層面修飾和敲除有毒生物堿基因，如從多年黑麥草分離出的Epichloёlolii和Epichloё sp.菌株中敲除dmaW基因使其共生體不產(chǎn)生麥角纈氨酸(ergovaline)[114]。陳麗通過對醉馬草內(nèi)生真菌Epichloёgansuensis(Eg)和Epichloёgansuensisvar.inebrians(Ei)菌株進(jìn)行了PCR分子檢測和鑒定，建立了適用于醉馬草內(nèi)生真菌的多基因 PCR 檢測體系，得到了醉馬草內(nèi)生真菌dmaW、idtG、mtA和mtB基因特異性引物[115]。多基因PCR檢測材料用菌株或共生體 DNA均可，既可檢測禾草內(nèi)生真菌共生體產(chǎn)生的生物堿，從而證實內(nèi)生真菌是否入侵禾草。除此之外，可以對生物堿進(jìn)行測序來間接地檢測內(nèi)生真菌，如對草地羊茅(Festucaelati)內(nèi)生真菌共生體中分離的麥角菌科(Clavicipitaceae)生物堿基因組通過測序分析，發(fā)現(xiàn)了Ergoline麥角菌科生物堿生物合成途徑[116]。這些禾草內(nèi)生真菌共生體次生代謝產(chǎn)物的檢測方法也間接證明了內(nèi)生真菌的存在。

表3 禾草內(nèi)生真菌生物堿含量的主要檢測方法Table 3 Main methods of alkaloid detection of grass endophytes

4 分子檢測法

從1996年起Glenn和Bacon利用分子生物學(xué)技術(shù)建立真菌屬以來[117]，利用分子生物學(xué)進(jìn)行內(nèi)生真菌檢測的研究不斷發(fā)展進(jìn)步。

4.1 保守基因測序進(jìn)行內(nèi)生真菌種類鑒定

將基因序列分析等高識別率的方法用于內(nèi)生真菌親緣關(guān)系的比較，將更加準(zhǔn)確地鑒定禾草內(nèi)生真菌的種類。常用的多基因拷貝技術(shù)有同功酶、微衛(wèi)星等。在內(nèi)生真菌分類初期用核糖體DNA內(nèi)轉(zhuǎn)錄間隔區(qū)rDNA-ITS的多態(tài)性的序列進(jìn)行分析，檢測此序列是否可以用來分析遺傳關(guān)系，如ITS1-4的4條通用引物可用于內(nèi)生真菌的ITS序列檢測[118]。除了分析內(nèi)轉(zhuǎn)錄間隔區(qū)(ITS)序列外，肌動蛋白(act1)、β-微管蛋白(tub2)和翻譯延伸因子1-α(tef1)特異基因序列也可用于內(nèi)生真菌DNA的擴(kuò)增，如利用tub2、tef1和act1基因擴(kuò)增，建立了E.aotearoae、E.melicicola、E.australiensis[119]，也可以跟系統(tǒng)發(fā)育分析結(jié)合進(jìn)行禾草內(nèi)生真菌的鑒定[120]。

4.2 各種分子標(biāo)記技術(shù)進(jìn)行內(nèi)生真菌基因型分析

微衛(wèi)星(microsatellite)也稱為簡單序列重復(fù)(simple sequences repeats,SSR)，因數(shù)量多、分布廣、多態(tài)性豐富、檢測快速方便等優(yōu)點而被應(yīng)用于內(nèi)生真菌個體識別和遺傳多樣性分析等[121-122]。Jong等利用該技術(shù)對寄生宿主禾草體內(nèi)的Neptyphodiumcoenophialum和Neotyphodiumlolii進(jìn)行了檢測并分析了二者之間的遺傳變異特征[123]。對宿主和內(nèi)生真菌DNA用SSR引物進(jìn)行擴(kuò)增可以得到區(qū)別二者DNA的條帶，從而可以檢測禾草是否被內(nèi)生真菌侵染[124]。盡管微衛(wèi)星技術(shù)在禾草內(nèi)生真菌的檢測方面具有較明顯的優(yōu)勢，但仍有部分缺陷。在進(jìn)行微衛(wèi)星標(biāo)記應(yīng)用時，需要設(shè)計出特異性較強(qiáng)的引物進(jìn)行擴(kuò)增，然而這項工作通常費時費力，給微衛(wèi)星技術(shù)的廣泛應(yīng)用帶來一定的困難[125]。

實時PCR(real-time quantitative polymerase chain reaction,Rt-PCR)又稱為實時熒光定量PCR，實時熒光 PCR 標(biāo)記法靈敏度高、特異性強(qiáng)，能快速準(zhǔn)確地定量檢測禾草內(nèi)生真菌[126-128]。采用實時熒光PCR方法可以快速檢測E.coenophiala和E.festucaevar.lolii內(nèi)生真菌，并可檢測到單粒種子上[129]。定量PCR技術(shù)可以用于闡述不同禾草內(nèi)生真菌和寄主基因型在禾草內(nèi)生真菌共生體相互作用中的影響[130]，但實時熒光定量PCR因檢測光源和熒光素種類的局限及高成本限制了其被廣泛的應(yīng)用。

隨機(jī)擴(kuò)增多態(tài)DNA(RAPD)標(biāo)記法不受環(huán)境、發(fā)育、數(shù)量性狀遺傳等的影響，能夠客觀地提示供試材料之間DNA的差異，可以檢測出RFLP標(biāo)記不能檢測的重復(fù)順序區(qū)。對E.festucaevar.lolii和E.Coenophiala用不同的引物擴(kuò)增，發(fā)現(xiàn)引物5′-CCGAGGGGGGGTGAC-3′可以區(qū)分二者[131]。RAPD技術(shù)可用來驗證兩種禾草內(nèi)生真菌是否可以同時侵染同一種宿主植物[132]。用RAPD技術(shù)對雀麥屬(Bromus)植物中的Epichloёtyphina進(jìn)行標(biāo)記檢測，發(fā)現(xiàn)在遺傳過程中存在著變異[133]。該技術(shù)使用的效果因生物種類而定，但因其具有快速、簡便、分辨率高和樣本少等特點，被廣泛應(yīng)用在禾草內(nèi)生真菌的檢測中[134-135]。利用分子生物學(xué)技術(shù)檢測禾草內(nèi)生真菌，可以彌補傳統(tǒng)禾草內(nèi)生真菌檢測方法的不足，并結(jié)合遺傳學(xué)確定了禾草內(nèi)生真菌的種屬。

5 問題與展望

Petrini 對內(nèi)生真菌與宿主的專一性進(jìn)行分析，得出每種宿主平均被4～5種專性內(nèi)生真菌侵染，按地球上目前已知的25萬種植物估算，全世界內(nèi)生真菌種類在100萬種以上[136]。目前，人們只發(fā)現(xiàn)80屬290種禾本科植物中含有內(nèi)生真菌；而我國幅員遼闊，生物物種資源豐富，有190余屬禾本科植物，目前只在早熟禾屬、羊茅屬、披堿草屬等14屬43種(種群)天然草地禾草中發(fā)現(xiàn)內(nèi)生真菌[17]，且明確其分類地位及命名的很少。傳統(tǒng)方法容易受主觀因素的影響,單憑傳統(tǒng)方法分類鑒定的結(jié)果存在一定的誤差，需要結(jié)合其分子手段進(jìn)一步鑒定[137]。普通的分子標(biāo)記需要特定的特異性引物，且成本高，目前已很難滿足一般的分子檢測的需要。

隨著生物技術(shù)和生物信息學(xué)的快速發(fā)展，真菌的檢測技術(shù)也在不斷進(jìn)步。新一代測序技術(shù)的發(fā)展和推廣，如全基因組測序技術(shù)的引進(jìn)，對內(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)。美國肯塔基大學(xué)植物病理學(xué)系和澳大利亞維多利亞農(nóng)業(yè)生物研究中心(AgriBio, Victoria) German Spangenberg實驗室相繼完成了對不同禾草內(nèi)生真菌的全基因組測序，有助于全球科學(xué)家共同開展禾草內(nèi)生真菌的基因圖譜構(gòu)建等工作[116,138-139]。也可以用更多的長片段基因(比如線粒體基因)進(jìn)行真菌系統(tǒng)發(fā)育的研究，更清楚地闡明了基因的結(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，對以前分類模糊的種進(jìn)行精確的定性[140]。

生物芯片是近十幾年迅速發(fā)展起來的一項基于基因表達(dá)和基因功能研究的高新技術(shù)，綜合了分子生物學(xué)、化學(xué)染料、激光、半導(dǎo)體微電子等領(lǐng)域的最新科學(xué)技術(shù)，它具有高通量、多參數(shù)同步分析，全自動、快速分析，高準(zhǔn)確度、靈敏度分析的優(yōu)點[141]，該方法在真菌毒素監(jiān)測等其它真菌及植物病原真菌檢測中已被廣泛應(yīng)用[142-143]，而在禾草內(nèi)生真菌檢測中的應(yīng)用仍是空白，若將此方法用于禾草內(nèi)生真菌田間檢測，可大大縮短天然禾草內(nèi)生真菌共生體篩選和培育含有高密度內(nèi)生真菌草坪草的工作量，提高工作效率。總之，不同領(lǐng)域新科學(xué)技術(shù)的不斷成熟和融合，更加成熟的新技術(shù)將會應(yīng)用到禾草內(nèi)生真菌的檢測中，為開發(fā)和挖掘新的禾草內(nèi)生真菌共生體提供基礎(chǔ)。

隨著內(nèi)生真菌在育種等領(lǐng)域的不斷深入，其在子代中的穩(wěn)定性成為困擾研究者的一個主要問題。結(jié)合傳統(tǒng)和分子生物學(xué)技術(shù)建立針對種子中內(nèi)生真菌存在及活力的高效檢測方法勢在必行。越來越多的研究發(fā)現(xiàn)，內(nèi)生真菌基因組中存在較多的對家畜等其它動物的毒性基因，這給內(nèi)生真菌的檢測帶了來新的挑戰(zhàn)。內(nèi)生真菌的檢測不應(yīng)僅停留在表面的帶菌率方面的檢測，深入檢測其基因組的產(chǎn)堿基因特征，分析其對家畜和食物鏈中其它動物及天敵甚至人類的潛在威脅，并進(jìn)行較全面的風(fēng)險評估，以期利用內(nèi)生真菌更好地為人類服務(wù)。傳統(tǒng)的內(nèi)生真菌檢測方法只是針對其在宿主禾草體內(nèi)的存在性進(jìn)行分析，但是并不能對內(nèi)生真菌的活力狀況進(jìn)行準(zhǔn)確判定，因此發(fā)展針對這一方面的檢測技術(shù)具有較大的潛力。目前新的真菌檢測技術(shù)不斷涌現(xiàn)，給內(nèi)生真菌的檢測提供了較好的借鑒，但是也給使用者的選擇帶來不少的困惑。因此，應(yīng)結(jié)合傳統(tǒng)和新一代的檢測方法，制定出一套切實可行的檢測技術(shù)體系；針對共生體的生長時期、形態(tài)特征和具體的物種特異性，分別選擇出較為適用的技術(shù)方法進(jìn)行檢測，這樣才能快速有效地對禾草內(nèi)生真菌進(jìn)行檢測。

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(責(zé)任編輯 王芳)

2017年第7期《草業(yè)科學(xué)》審稿專家

艾 辛 曹文俠 曹陽春 柴 琦 陳先江 崔 霞 丁路明 段廷玉
方強(qiáng)恩 馮琦勝 干友民 郭 鈮 郭正剛 何學(xué)青 侯扶江 胡 靖
胡龍興 胡小文 黃曉東 姜孝成 雷趙民 李 飛 李彥忠 劉文獻(xiàn)
劉學(xué)錄 龍明秀 婁燕宏 馬紅媛 馬偉強(qiáng) 尚占環(huán) 唐德富 田 沛
王虎成 王召鋒 王志偉 翁秀秀 伍國強(qiáng) 武高林 謝 燕 許立新
于應(yīng)文 魚小軍 袁明龍 張建全 張新慧 張興旭
承蒙以上專家對《草業(yè)科學(xué)》期刊稿件的審閱，特此表示衷心的感謝！

Research progress of methods on grass fungal endophyte detection

Chen Zhen-jiang, Wei Xue-kai, Cao Ying, Tian Pei, Zhao Xiao-jing, Li Chun-jie
(State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou 730020, China)

After the initial discovery of perennial ryegrass and tall fescue lead poisoning of farm animals, the responsible endophytic fungi in the grass was identified; further study confirmed the presence of endophytic fungi not only led to livestock poisoning, but significantly improved the competitive ability of the host in the community. The ecological and physiological functions of grass endophytic fungi have established this field as a popular research topic worldwide, which has provided opportunities for the development of endophytic fungi detection technology. Generally, well-established detection methods for pathogenic fungi have been used for the detection grass endophytic fungi, such as microscopic detection with staining. However, the accuracy of results was easily influenced by different host species, phenophases, and tissues. The rapid development and application of molecular biology, genetics, enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), real-time PCR methods, and other modern molecular techniques has enabled the continuous improvement of the detection methods of endophytic fungi, which can compensate for the shortage of traditional methods. The rapid and efficient determination of the existence, distribution, classification, and position of the endophytic fungi, requires an accurate and reasonable selection of specific detection methods, such as qualitative or quantitative detection combined with classical microscopy staining to determine the endophytic fungi. In this paper, we reported recent research on the detection methods of endophytic fungi and also discuss the development of classical detection method of other microbes, which are important for the development of specific detection technologies for endophytic fungi and can quantitatively determine the existence and the activity of endophytic fungi. Specific endophytic fungi detection technologies, which are no longer limited to only the identification of the presence of endophytic fungi, but can simultaneous quantify the activity of endophytic fungi detection, are an important focus for the technological development.

endophyte; isolation; microscopic observation; ELISA; HPLC; molecular biology

Li Chun-jie E-mail:chunjie@lzu.edu.cn

2016-07-17 接受日期：2016-12-29

國家“973”項目(2014CB138702);國家自然科學(xué)基金項目(31372366);教育部創(chuàng)新團(tuán)隊發(fā)展計劃項目(IRT13019)

陳振江(1991-),男,甘肅定西人,在讀博士生,研究方向為禾草內(nèi)生真菌共生體。E-mail:chenzj15@163.com

李春杰(1968-),男,甘肅鎮(zhèn)原人,教授,博士,研究方向為禾草內(nèi)生真菌共生體。E-mail:chunjie@lzu.edu.cn

10.11829/j.issn.1001-0629.2016-0386

S432.4+4;Q949.32

A

1001-0629(2017)07-1419-15

陳振江，魏學(xué)凱，曹瑩，田沛，趙曉靜，李春杰.禾草內(nèi)生真菌檢測方法研究進(jìn)展.草業(yè)科學(xué),2017,34(7)：1419-1433.

Chen Z J,Wei X K,Cao Y,Tian P,Zhao X J,Li C J.Research progress of methods on grass fungal endophyte detection.Pratacultural Science，2017,34(7)：1419-1433.