譙天敏, 張靜, 麻文建, 朱天輝, 鄭磊

(四川農(nóng)業(yè)大學(xué)林學(xué)院,四川 雅安625014)

桉樹焦枯病菌巢式聚合酶鏈反應(yīng)快速檢測(cè)方法的建立與應(yīng)用

譙天敏, 張靜, 麻文建, 朱天輝*, 鄭磊

(四川農(nóng)業(yè)大學(xué)林學(xué)院,四川 雅安625014)

桉樹焦枯病是威脅桉樹生長(zhǎng)的首要病害,建立準(zhǔn)確、有效的桉樹焦枯病巢式聚合酶鏈反應(yīng)(polymerase chain reaction,PCR)快速檢測(cè)技術(shù)是桉樹焦枯病前期診斷的必要手段。本研究針對(duì)柱枝雙孢霉(Cylindrocladiumscoparium)菌株的β-微管蛋白(beta-tubulin)基因上保守序列極強(qiáng)的靶基因區(qū)域序列進(jìn)行特異性引物BT-S-1/BT-A-1的設(shè)計(jì)和通用引物BT-T1-S/BT-CYLTUBIR-A的合成,分別利用常規(guī)PCR和巢式PCR技術(shù)對(duì)引物特異性和靈敏度進(jìn)行檢測(cè)和比較,同時(shí)本研究也對(duì)所建立的巢式PCR用于桉樹焦枯病原菌快速檢測(cè)的田間時(shí)效性進(jìn)行驗(yàn)證。試驗(yàn)結(jié)果表明:利用beta-tubulin基因序列通用引物BT-T1-S/BT-CYLTUBIR-A對(duì)全部供試菌株進(jìn)行擴(kuò)增,所有參試菌株均可擴(kuò)增出1條約500 bp的條帶；而單獨(dú)使用特異性引物BT-S-1/BT-A-1進(jìn)行常規(guī)PCR擴(kuò)增時(shí)僅病原菌能夠擴(kuò)增出1條148 bp的明亮條帶；當(dāng)利用通用引物作為第1輪引物,以稀釋10倍后的第1輪PCR產(chǎn)物作為第2輪PCR模板,利用特異性引物進(jìn)行巢式PCR擴(kuò)增時(shí),也可擴(kuò)增出上述148 bp大小的明亮條帶,且巢式PCR的擴(kuò)增效果較常規(guī)PCR具有明顯的視覺優(yōu)越性；靈敏度檢測(cè)試驗(yàn)表明,巢式PCR的靈敏度可檢測(cè)到5 fg/μL,較常規(guī)PCR可以擴(kuò)增出的極限(DNA質(zhì)量濃度為5 pg/μL)至少提高了103倍；田間時(shí)效檢測(cè)試驗(yàn)也說明巢式PCR較常規(guī)PCR具有更高的準(zhǔn)確度和靈敏性,可達(dá)到田間檢測(cè)的要求。本研究建立的巢式PCR檢測(cè)技術(shù)可有效應(yīng)用于桉樹焦枯病的早期診斷。

巢式聚合酶鏈反應(yīng); 桉樹焦枯病; 快速檢測(cè); 麗赤殼屬; 柱枝雙孢霉

桉樹與楊樹、松樹并稱為世界三大速生造林樹種,可見桉樹在我國(guó)乃至世界林業(yè)生產(chǎn)建設(shè)中占據(jù)著不可替代的作用[1-2]。近年來,由于桉樹的適應(yīng)性極強(qiáng)、生長(zhǎng)迅速、材質(zhì)優(yōu)良、用途廣泛、輪伐周期短等優(yōu)點(diǎn),在我國(guó)西南及東南各個(gè)地區(qū)廣泛引種和栽培[3]。國(guó)內(nèi)外對(duì)桉樹病害的報(bào)道較多,主要以桉樹花斑病(Aureobasidiumsp.)、炭疽病(Colletotrichumgloeosporioides)、紫斑病(Septoriamortarlensis)、灰霉病(Botrytiscinerea)和桉樹焦枯病(Eucalyptus)等葉部真菌性病害發(fā)生較為普遍,其中由麗赤殼屬(Calonectria)真菌引起的桉樹焦枯病為害最為嚴(yán)重,分布也最為廣泛[4]。桉樹焦枯病不同程度地威脅著桉樹幼苗、葉片、樹干、根部以及生理代謝,而被病原菌侵染的桉樹輕則出現(xiàn)植株葉片脫落,苗圃與幼林落葉枯稍、枝條萎蔫；嚴(yán)重時(shí)會(huì)出現(xiàn)感病苗木葉片全部脫落、頂枯、植株枝條全部枯死等現(xiàn)象[5]。目前,在澳大利亞、美國(guó)、中國(guó)、巴西、日本、印度尼西亞、泰國(guó)、越南等幾十個(gè)桉樹種植較為密集的國(guó)家均有桉樹焦枯病的報(bào)道,桉樹焦枯病從其分布范圍上來說已經(jīng)被定義為一種世界性病害[1]。而我國(guó)早在1996年1月5日便將之列入為國(guó)內(nèi)森林植物檢疫對(duì)象。

對(duì)林木病害快速、準(zhǔn)確的早期診斷是減少林木損失的有效途徑[6]。傳統(tǒng)病原菌的診斷方法主要以形態(tài)學(xué)及生理生化指標(biāo)鑒定為主,該方法不僅費(fèi)時(shí)費(fèi)力、準(zhǔn)確度也較低,而且對(duì)鑒定者需具有很高的專業(yè)知識(shí)。此外,肉眼鑒定病害的過程中會(huì)受到多種因素干擾,如分離培養(yǎng)的病原菌是否純化、培養(yǎng)條件的影響、生理指標(biāo)的不穩(wěn)定性等,因此基于傳統(tǒng)方法檢測(cè)病害在一定程度上仍存在大量弊端。隨著林木病害的普遍發(fā)生,診斷技術(shù)也隨之進(jìn)步,從早期的田間傳統(tǒng)鑒定和診斷技術(shù),過渡到免疫性診斷階段,再到分子生物學(xué)水平診斷。而分子生物學(xué)診斷技術(shù)也從早先的基礎(chǔ)聚合酶鏈反應(yīng)(polymerase chain reaction,PCR)技術(shù),到基于PCR的巢式PCR、雙重PCR、多重PCR以及最近幾年發(fā)展起來的循環(huán)介導(dǎo)等溫?cái)U(kuò)增、核酸快速擴(kuò)增和指紋檢測(cè)技術(shù),這些進(jìn)步使得分子生物學(xué)水平上的病害檢測(cè)具有更高的準(zhǔn)確性、可靠性、靈敏性以及時(shí)效性[7-8]。巢式PCR較一般PCR具有更高的靈敏度和準(zhǔn)確性,被廣泛地應(yīng)用于玉米葉斑病[9]、甘蔗白葉病[10]、梨黑星病[11]等多種植株病害的檢測(cè)。本試驗(yàn)以四川桉樹焦枯病的病原菌(Cylindrocladiumscoparium)為對(duì)象,構(gòu)建了C.scoparium的巢式PCR快速檢測(cè)體系,并對(duì)其田間野外進(jìn)行時(shí)效檢測(cè)應(yīng)用,為桉樹焦枯病的早期預(yù)判和提前防治提供重要的技術(shù)支持。

1 材料與方法

1.1 供試菌株

本實(shí)驗(yàn)使用的供試菌株均分離自桉樹病株,其分離采用常規(guī)組織分離法,具體參照韓永超等[12]的方法；鑒定采用形態(tài)學(xué)與分子生物學(xué)手段相結(jié)合,具體參照王俊麗等[13]的方法。其中菌株編號(hào)1～6為桉樹焦枯病原菌,菌株編號(hào)1和2購(gòu)自中國(guó)林業(yè)科學(xué)研究院森林生態(tài)環(huán)境與環(huán)境保護(hù)研究所,菌株編號(hào)3～6分離自各地方桉樹病株,除菌株編號(hào)8外的各菌株均分離自桉樹病株,供試菌株采集地、編號(hào)、菌株種屬詳見表1。

表1 供試菌株及其來源

1.2 供試菌株DNA提取

1.2.1 菌絲的收集 采用液體搖床振蕩的方法進(jìn)行供試菌株培養(yǎng),參照Wang等[14]的方法進(jìn)行菌絲提取,稍加改動(dòng)。分別接種供試菌株于馬鈴薯葡萄糖瓊脂(potato dextrose agar,PDA)培養(yǎng)基上,室溫下連續(xù)培養(yǎng)5 d后,取菌落邊緣菌絲少許,轉(zhuǎn)接于瓶裝量為40-mL的馬鈴薯葡萄糖肉湯(potato dextrose broth,PDB)液體培養(yǎng)錐形瓶(100-mL)中,25 ℃,pH 7,在125 r/min條件下振蕩培養(yǎng)5～7 d。用無菌水沖洗，紗布過濾后收集到的菌絲置于-20 ℃條件下保存?zhèn)溆谩?/p>

1.2.2 供試菌絲DNA的提取 稱取0.2 g冷凍保存的菌絲,利用液氮研磨法破解細(xì)胞壁,然后按天根植物基因組DNA提取試劑盒(plant genomic DNA kit)的操作步驟進(jìn)行供試菌絲DNA的提取,具體參照韓振云等[15]的方法改進(jìn)使用。所提取的DNA溶液分別在紫外分光光度計(jì)(UV-3600)下檢測(cè)濃度,并作適當(dāng)稀釋后于-20 ℃保存、備用。

1.2.3 發(fā)病組織DNA的提取 2013年對(duì)桉樹焦枯病發(fā)病時(shí)期進(jìn)行野外樣品的采集,焦枯病害的感病程度共分5級(jí),分別用0級(jí)(健康葉片)；一級(jí)(葉片感病面積在0～25%之間)；二級(jí)(葉片感病面積在25%～50%之間)；三級(jí)(葉片感病面積在50%～75%之間),四級(jí)(葉片感病面積在75%～100%之間)；五級(jí)(葉片感病面積達(dá)到100%)表示。分別隨機(jī)選擇10片不同感病程度的桉樹葉片放入冰壺內(nèi),標(biāo)號(hào)、帶回實(shí)驗(yàn)室備用,同時(shí)采集健康葉片作為對(duì)照。首先對(duì)采集的樣品用70%乙醇表面消毒,蒸餾水沖洗3次,吸水紙吸干后,將葉片樣品用刀片切成面積為1.0 cm×1.0 cm的小塊,每感病程度取20個(gè),置于研缽中用液氮研磨成粉末,參照天根植物基因組DNA的提取方法操作。

1.3 通用引物PCR擴(kuò)增

利用Cylindrocladium(Calonectria)屬真菌β-微管蛋白(beta-tubulin)基因區(qū)通用引物[16]BT-T1-S(5′-AACATGCGTGAGATTGTAAGT-3′),BT-CYLTUBIR-A(5′-AGTTGTCGGGACGGAAGAG-3′)對(duì)桉樹焦枯病菌(C.scoparium)進(jìn)行常規(guī)PCR擴(kuò)增。PCR擴(kuò)增體系(50 μL):10×PCR緩沖液5.0 μL,Mg2+3 μL,10 mmol/L dNTP 1.5 μL,BT-T1-S (10 pmol/μL) 1.5 μL,BT-CYLTUBIR-A (10 pmol/μL) 1.5 μL,Tag聚合酶(5 U/μL) 0.3 μL,ddH2O 33.2 μL,基因組DNA提取液4.0μL,反應(yīng)條件:94 ℃熱啟動(dòng) 5 min;94 ℃變性 45 s;58 ℃退火 45 s;72 ℃延伸40 s;循環(huán)32次,72 ℃延伸8 min。以ddH2O代替模版DNA作陰性對(duì)照。PCR產(chǎn)物檢測(cè)在1.5%瓊脂糖凝膠電泳上進(jìn)行,經(jīng)EB染色后,在凝膠成像系統(tǒng)上拍照、觀察。PCR擴(kuò)增產(chǎn)物委托上海生物工程有限公司完成測(cè)序。

1.4 病原菌特異性引物設(shè)計(jì)與合成

根據(jù)供試菌株beta-tubulin基因序列的測(cè)序結(jié)果,結(jié)合GeneBank中Cylindrocladium屬種的差異進(jìn)行C.scoparium病原菌特異性引物的設(shè)計(jì),所設(shè)計(jì)的引物對(duì)為BT-S-1(5′-GGCTCCAAGAACT ATGTGA-3′)/BT-A-1(5′-CCTAACCACGAATG TCAGT-3′),擴(kuò)增片段大小148 bp。引物由上海生工生物工程有限公司合成。

1.5 常規(guī)和巢式PCR特異性驗(yàn)證

常規(guī)PCR:利用特異性引物BT-S-1/BT-A-1對(duì)所有參試菌株P(guān)CR擴(kuò)增。PCR擴(kuò)增體系(50 μL):10×PCR緩沖液5.0 μL,Mg2+3 μL,10 mmol/L dNTP 1.5 μL,BT-S-1(10 pmol/μL) 1.5 μL,BT-A-1 (10 pmol/μL) 1.5 μL,Tag聚合酶(5 U/μL) 0.3 μL,ddH2O 33.2 μL,DNA 4.0 μL。反應(yīng)條件同1.3節(jié)通用引物PCR擴(kuò)增,退火溫度為60 ℃。以滅菌水代替模版DNA設(shè)置陰性對(duì)照。

巢式PCR:分別以真菌通用引物BT-T1-S/BT-CYLTUBIR-A作為第1輪引物進(jìn)行PCR擴(kuò)增,反應(yīng)體系和程序同1.3節(jié),取第1輪PCR產(chǎn)物,稀釋10倍,作為第2輪PCR模板,第2輪引物為BT-S-1/BT-A-1,反應(yīng)體系和程序同常規(guī)PCR。

1.6 靈敏度檢測(cè)

將提取的發(fā)病組織葉片病原菌基因組DNA溶液以10的倍數(shù)進(jìn)行稀釋,分別得到質(zhì)量濃度為50 ng/μL、5 ng/μL,500 pg/μL 50 pg/μL、5 pg/μL、500 fg/μL、50 fg/μL、5 fg/μL和500 ag/μL。每次取1 μL作為PCR反應(yīng)模版,分別用于常規(guī)PCR和巢式PCR,比較二者靈敏度。常規(guī)PCR反應(yīng)體系和反應(yīng)程序同1.5節(jié),巢式PCR反應(yīng)程序和反應(yīng)體系分別同1.3節(jié)和1.5節(jié)。

1.7 田間時(shí)效檢測(cè)

按發(fā)病組織方法提取DNA，按1.5節(jié)所述的方法進(jìn)行巢式PCR擴(kuò)增,同時(shí)以ddH2O代替DNA模板組和健康組做陰性對(duì)照。根據(jù)電泳檢測(cè)結(jié)果判定桉樹初期發(fā)病植株、中期發(fā)病植株、嚴(yán)重發(fā)病植株、具有典型癥狀植株和健康植株的樣品中是否攜帶Calonectriamorganii致病菌。

2 結(jié)果與分析

2.1 通用引物PCR擴(kuò)增結(jié)果

利用真菌通用引物BT-T1-S/BT-CYLTUBIR-A可以將全部供試菌株擴(kuò)增出1條500 bp大小條帶,而陰性空白對(duì)照無條帶(圖1),表明提取菌株的DNA較純,可以保障后續(xù)試驗(yàn)。

2.2 常規(guī)PCR特異性引物BT-S-1/BT-A-1擴(kuò)增結(jié)果

擴(kuò)增反應(yīng)結(jié)果(圖2)顯示:桉樹焦枯病菌(C.scoparium)可擴(kuò)增出明亮條帶,大小為148 bp,而其他同屬和非同屬參試菌、對(duì)照組都沒有擴(kuò)增出條帶。通過片段回收測(cè)序及與現(xiàn)有的C.scoparium序列比對(duì)發(fā)現(xiàn)同源率為100%,證明該引物可以從混合DNA樣中準(zhǔn)確地檢測(cè)出桉樹焦枯病菌(C.scoparium),準(zhǔn)確率為100%。因此,說明特異性引物BT-S-1/BT-A-1可有效地用于桉樹焦枯病菌(C.scoparium)林木病害基因組DNA的PCR擴(kuò)增。

2.3 巢式PCR擴(kuò)增反應(yīng)結(jié)果

巢式PCR檢測(cè)結(jié)果(圖3)顯示:桉樹焦枯病菌(C.scoparium)可擴(kuò)增出明亮條帶,大小為148 bp,而其他同屬和非同屬參試菌、對(duì)照組都沒有擴(kuò)增出條帶；相對(duì)常規(guī)PCR特異性檢驗(yàn)而言,巢式PCR特異性檢驗(yàn)的條帶更為明亮。與常規(guī)PCR擴(kuò)增相比,巢式PCR具有一定的視覺優(yōu)越性。

2.4 常規(guī)PCR與巢式PCR靈敏度檢測(cè)

對(duì)不同質(zhì)量濃度桉樹焦枯病菌(C.scoparium)基因組DNA進(jìn)行常規(guī)PCR擴(kuò)增(圖4)和巢式PCR擴(kuò)增(圖5),結(jié)果表明巢式PCR具有更高的靈敏度。在50 μL反應(yīng)體系中,常規(guī)PCR檢測(cè)基因組濃度極限為5 pg/μL,低于5 pg/μL則不能檢測(cè)到條帶,而巢式PCR可達(dá)到5 fg/μL,檢測(cè)靈敏度至少提高了103倍,可滿足田間檢測(cè)要求。

2.5 巢式PCR野外時(shí)效性檢測(cè)

利用巢式PCR快速試劑盒進(jìn)行檢測(cè),結(jié)果見6,并通過片段回收測(cè)序與現(xiàn)有C.scoparium序列比對(duì)發(fā)現(xiàn)同源率為100%,證明該引物可以從混合DNA樣中準(zhǔn)確地檢測(cè)出桉樹焦枯病菌(C.scoparium),準(zhǔn)確率為100%。常規(guī)PCR擴(kuò)增只能檢測(cè)出新枯死樣和典型感病組織內(nèi)病原菌；而巢式PCR能夠有效檢測(cè)出新枯死樣、典型感病組織、感病初期和部分感染后未顯癥樣品(泳道10、11、12),可見其靈敏度明顯高于常規(guī)PCR擴(kuò)增。巢式PCR之所以只能檢測(cè)出部分未顯癥樣品,可能是因?yàn)槲礄z測(cè)出的部分未感病樣品組織不攜帶有病原菌或其攜帶的病原菌的數(shù)量極少,達(dá)到了巢式PCR檢測(cè)的極限。

M:DL2000標(biāo)志物;CK:陰性對(duì)照;(1～22):通用引物BT-T1-S/BT-CYLTUBIR-A對(duì)表1中所對(duì)應(yīng)的供試菌株DNA溶液PCR擴(kuò)增。M: DL2 000 marker; CK: Negative control; (1-22): Strain numbers listed in Table 1 for PCR amplification using primers BT-T1-S/BT-CYLTUBIR-A.圖1 常規(guī)PCR通用引物BT-T1-S/BT-CYLTUBIR-A擴(kuò)增反應(yīng)結(jié)果Fig.1 Results of PCR amplification using universal primers BT-T1-S/BT-CYLTUBIR-A

M: DL2000標(biāo)志物;CK:陰性對(duì)照;(1～22):引物BT-S-1/BT-A-1直接對(duì)表1中所對(duì)應(yīng)的供試菌株DNA溶液PCR擴(kuò)增.M: DL2000 marker; CK: Negative control; (1-22): Strain numbers listed in Table 1 for PCR amplification using primers BT-S-1/BT-A-1.圖2 已知供試菌PCR的BT-S-1/BT-A-1特異性擴(kuò)增結(jié)果Fig.2 Results of PCR amplification using specific primers BT-S-1/BT-A-1

M: DL2000標(biāo)志物;CK:陰性對(duì)照;(1～22):引物BT-T1-S/BT-CYLTUBIR-A和BT-S-1/BT-A-1對(duì)表1中所對(duì)應(yīng)的供試菌株DNA溶液巢式PCR擴(kuò)增。M: DL2000 marker; CK: Negative control; (1-22): Strain numbers listed in Table 1 for nested-PCR amplification using primers BT-T1-S/BT-CYLTUBIR-A and BT-S-1/BT-A-1.圖3 β-微管蛋白基因區(qū)特異性引物巢式PCR擴(kuò)增結(jié)果圖Fig.3 Results of nested-PCR amplification using specific primers in beta-tubulin gene

M:DL2000標(biāo)志物;(1～6):桉樹焦枯病原菌C. scoparium基因組DNA濃度分別為50 ng/μL,5 ng/μL,500 pg/μL,50 pg/μL,5 pg/μL,500 fg/μL. M: DL2000 marker; (1-6): Different quantity of DNA: 50 ng/μL, 5 ng/μL, 500 pg/μL， 50 pg/μL, 5 pg/μL, 500 fg/μL, respectively.圖4 常規(guī)PCR靈敏度檢測(cè)結(jié)果Fig.4 Sensitivity detection of universal PCR

M:DL2000標(biāo)志物;(1～9):桉樹焦枯病原菌C. scoparium基因組DNA濃度分別為50 ng/μL,5 ng/μL,500 pg/μL,50 pg/μL,5 pg/μL,500 fg/μL,50 fg/μL,5 fg/μL,500 ag/μL。M: DL2000 marker; (1-9): Different quantity of DNA: 50 ng/μL, 5 ng/μL, 500 pg/μL, 50 pg/μL, 5 pg/μL, 500 fg/μL, 50 fg/μL, 5 fg/μL, 500 ag/μL, respectively.圖5 巢式PCR靈敏度檢測(cè)結(jié)果Fig.5 Sensitivity detection of nested-PCR

M:DL2000標(biāo)志物；(1～7):BT-T1-S/BT-CYLTUBIR-A第1輪擴(kuò)增結(jié)果；(8～14):巢式PCR第2輪擴(kuò)增結(jié)果;(15～21):常規(guī)PCR擴(kuò)增結(jié)果；22:陽(yáng)性對(duì)照(C. scoparium).其中1,8,15為新枯死組織；2,9,16為典型發(fā)病組織；3,10,17為感病初期；4,11,18為未感病組；5,12,19為殘?bào)w病株；6,13,20為健康組；7,14,21為陰性對(duì)照。M: DL2000 marker; (1-7): PCR with primers BT-T1-S /BT-CYLTUBIR-A for the first round amplification; (8-14): Nested-PCR with primers BT-S-1/BT-A-1 for the second round amplification; (15-21): Conventional PCR with primers BT-S-1/BT-A-1; 22: Positive control (C. scoparium). Including: Lane 1, 8, 15: New dead tissues; Lane 2, 9, 16: Diseased tissues with typical symptoms; Lane 3, 10, 17: Latent infected tissues; Lane 4, 11, 18: Infected tissues; Lane 5, 12, 19: Residues of dead plant; Lane 6, 13, 20: Healthy tissues; Lane 7, 14, 21: Negative control (ddH2O).圖6 巢式PCR田間檢測(cè)桉樹焦枯病菌Fig.6 Field test on C. scoparium of nested-PCR

3 討論與結(jié)論

桉樹焦枯病作為一種世界性病害,越來越受到人們的重視。加強(qiáng)病害的早期診斷是降低重大病害損失的有效手段。傳統(tǒng)病害的診斷方法主要以形態(tài)學(xué)及生理生化指標(biāo)鑒定為主,該方法不僅費(fèi)時(shí)費(fèi)力、往往準(zhǔn)確度也較低,而且對(duì)鑒定者需具有很高的專業(yè)知識(shí)。此外,肉眼鑒定病害的過程中會(huì)受到多種因素干擾,如分離培養(yǎng)的病原菌是否純化、培養(yǎng)條件的影響、生理指標(biāo)的不穩(wěn)定性等,因此基于傳統(tǒng)方法檢測(cè)病害在一定程度上仍存在大量弊端[17-18]。PCR及基于PCR的快速診斷技術(shù)在林業(yè)病害上的應(yīng)用彌補(bǔ)了傳統(tǒng)檢測(cè)的不足,同時(shí)較高的準(zhǔn)確性、精確性、時(shí)效性使得林業(yè)上多種重要病害的早期診斷成為可能,例如松材線蟲病[19]、楊樹潰瘍病[20]、桉樹青枯病[21]等的診斷。目前,對(duì)于桉樹焦枯病的診斷仍停留在傳統(tǒng)形態(tài)學(xué)檢測(cè)上,因此建立桉樹焦枯病的快速檢測(cè)技術(shù)十分必要。

巢式PCR是在PCR基礎(chǔ)上發(fā)展起來的一項(xiàng)更快速、更靈敏、精確度更高的快速檢測(cè)技術(shù)[22]。巢式PCR的原理是利用2對(duì)特異性引物先后共同擴(kuò)增一段目的區(qū)域,而第2次擴(kuò)增的目的區(qū)域位于第1次擴(kuò)增的目的區(qū)域內(nèi)部,能夠降低常規(guī)PCR經(jīng)常出現(xiàn)的非特異性擴(kuò)增概率,保證PCR的準(zhǔn)確性[23]。靈敏度檢測(cè)表明,巢式PCR具有比常規(guī)PCR更高的靈敏度。供試菌的選擇是PCR構(gòu)建的基礎(chǔ)。參考以往PCR體系構(gòu)建的供試菌選擇,主要來源有以下幾類:與病原菌同屬不同種的菌株,從發(fā)病寄主上分離得到的內(nèi)生菌、外生菌、寄生菌以及可能引起寄主感病的其他病原菌,資源豐富的供試菌株是該體系建立的基礎(chǔ)保障。

此外,保守性極強(qiáng)的特異序列是引物設(shè)計(jì)成功的關(guān)鍵因素。beta-tubulin基因在原核和真核生物中均有存在,且具有高度的特異性,常常被用于多種病原菌的分類鑒定、系統(tǒng)發(fā)育、地理小種的區(qū)分上[24]。本試驗(yàn)選擇了Cylindrocladium在其beta-tubulin基因區(qū)通用引物BT-T1-S/BT-CYLTUBIR-A作為第1輪引物,在此基礎(chǔ)上又在其beta-tubulin特異性保守序列上設(shè)計(jì)了巢式PCR第2對(duì)特異性引物BT-S-1/BT-A-1,尤其是第2對(duì)引物的特異性完全保證了桉樹焦枯病巢式PCR體系構(gòu)建的成功。此外,優(yōu)化的退火溫度是保證巢式PCR順利進(jìn)行的關(guān)鍵因素,研究中對(duì)該體系進(jìn)行溫度梯度優(yōu)化,得到第1輪通用引物的退火溫度為58 ℃,第2輪特異性引物的退火溫度為60 ℃,解決了其他溫度擴(kuò)增出現(xiàn)條帶不清晰和無條帶的問題。而田間病害的時(shí)效性檢測(cè)表明,該體系可用于桉樹焦枯病的野外發(fā)病檢測(cè),桉樹焦枯病巢式PCR快速檢測(cè)技術(shù)的構(gòu)建實(shí)現(xiàn)了桉樹焦枯病的早期診斷,為桉樹焦枯病的防治提供了必要的前提保障。

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Development and application of nested polymerase chain reaction for rapid detection ofCylindrocladiumscopariumonEucalyptus.

Journal of Zhejiang University (Agric. & Life Sci.)， 2015，41(5):497-504

Qiao Tianmin, Zhang Jing, Ma Wenjian, Zhu Tianhui*, Zheng Lei

(CollegeofForestry,SichuanAgriculturalUniversity,Ya’an625014,Sichuan,China)

Eucalyptus, as one of the most important components of the forestry resources, was originated in Australia, and then was cultivated worldwide including China. Prior to the early twentieth century, with the increasing introduction of this tree species,Eucalyptusdieback, caused byCylindrocladiumscoparium(teleomorph:Calonectriamorganii), was frequently affected in almost regions of China. Also it has historically been an important disease ofEucalyptus, which was defined as a key national plant quarantine disease in China, on January 5, 1996. In Southern China, this disease affectedEucalyptusthroughout its range of cultivation and caused severe yield loss and tree mortality.Cylindrocladiumscoparium, especially in late infection, can cause a large damage to the growth ofEucalyptus, as well as dieback, even all branches withered severely. Even in early infection, it can easily lead to reddish-brown spots, wilting leafs, and droping branches. However, thus far, it is still a great constraint to the cultivation and production expansion ofEucalyptusin many provinces of China, such as Sichuan, Fujian, and Guangxi. Therefore, to establish a rapid and effective detection technology has an important significance for control and prevention ofEucalyptusdieback.

A total of 19 isolates of fungus, includingC.scopariumwere obtained from diseasedEucalyptusin Sichuan Province, and three isolates (C.scopariumandCylindrocladiumilicicola) were purchased from Chinese academy of Forestry. Based on its conserved domain beta-tubulin gene, two pairs of specific primers BT-S-1/BT-A-1 and universal primers BT-T1-S/BT-CYLTUBIR-A were severally designed by the aid of the software Premier 5.0, and then were synthesized respectively, and the specificities and sensitivities of these two pairs were tested with the method of universal polymerase chain reaction (PCR) and nested-PCR. Meanwhile, the field tests were conducted.

The results showed that a band of 500 bp with the universal primers BT-T1-S/BT-CYLTUBIR-A was amplified among these isolates, while only a band of 148 bp was amplified by these six isolates ofC.scoparium, and the nested-PCR with these two pairs of primers also amplified the band of 148 bp. The difference was that the sensitivity of nested-PCR was 5 fg/μL, which was significantly 103times higher than universal PCR detection. The field tests showed that nested-PCR was much more accurate and sensitive than universal PCR.

In conclusion, the detection technique of nested-PCR ofC.scopariumwas proved to be rapid and specific in monitoring ofC.scopariumand it has a great significance for the prevention and control ofEucalyptusdieback. Furthermore, the result of the nested-PCR can play an effective role in the early diagnosis ofEucalyptusdieback and also provide a new basis for the detection of other diseases.

nested polymerase chain reaction;Eucalyptusdieback; rapid detection;Calonectria;Cylindrocladiumscoparium

國(guó)家科技基礎(chǔ)條件平臺(tái)基金(2005DK21207-13)。

聯(lián)系方式:譙天敏(http://orcid.org/0000-0001-9970-6975)，E-mail:525636693@qq.com

2014-07-27;接受日期(Accepted):2015-02-13；網(wǎng)絡(luò)出版日期(Published online):2015-09-18

Q 939.5; S 432.4

A

*通信作者(Corresponding author):朱天輝(http://orcid.org/0000-0002-1082-5175)，E-mail:zhuth1227@126.com

URL:http://www.cnki.net/kcms/detail/33.1247.s.20150918.1739.002.html