桉树焦枯病菌巢式聚合酶链反应快速检测方法的建立与应用

谯天敏, 张静, 麻文建, 朱天辉, 郑磊

(四川农业大学林学院,四川 雅安625014)

桉树焦枯病菌巢式聚合酶链反应快速检测方法的建立与应用

谯天敏, 张静, 麻文建, 朱天辉*, 郑磊

(四川农业大学林学院,四川 雅安625014)

桉树焦枯病是威胁桉树生长的首要病害,建立准确、有效的桉树焦枯病巢式聚合酶链反应(polymerase chain reaction,PCR)快速检测技术是桉树焦枯病前期诊断的必要手段。本研究针对柱枝双孢霉(Cylindrocladiumscoparium)菌株的β-微管蛋白(beta-tubulin)基因上保守序列极强的靶基因区域序列进行特异性引物BT-S-1/BT-A-1的设计和通用引物BT-T1-S/BT-CYLTUBIR-A的合成,分别利用常规PCR和巢式PCR技术对引物特异性和灵敏度进行检测和比较,同时本研究也对所建立的巢式PCR用于桉树焦枯病原菌快速检测的田间时效性进行验证。试验结果表明:利用beta-tubulin基因序列通用引物BT-T1-S/BT-CYLTUBIR-A对全部供试菌株进行扩增,所有参试菌株均可扩增出1条约500 bp的条带；而单独使用特异性引物BT-S-1/BT-A-1进行常规PCR扩增时仅病原菌能够扩增出1条148 bp的明亮条带；当利用通用引物作为第1轮引物,以稀释10倍后的第1轮PCR产物作为第2轮PCR模板,利用特异性引物进行巢式PCR扩增时,也可扩增出上述148 bp大小的明亮条带,且巢式PCR的扩增效果较常规PCR具有明显的视觉优越性；灵敏度检测试验表明,巢式PCR的灵敏度可检测到5 fg/μL,较常规PCR可以扩增出的极限(DNA质量浓度为5 pg/μL)至少提高了103倍；田间时效检测试验也说明巢式PCR较常规PCR具有更高的准确度和灵敏性,可达到田间检测的要求。本研究建立的巢式PCR检测技术可有效应用于桉树焦枯病的早期诊断。

巢式聚合酶链反应; 桉树焦枯病; 快速检测; 丽赤壳属; 柱枝双孢霉

桉树与杨树、松树并称为世界三大速生造林树种,可见桉树在我国乃至世界林业生产建设中占据着不可替代的作用[1-2]。近年来,由于桉树的适应性极强、生长迅速、材质优良、用途广泛、轮伐周期短等优点,在我国西南及东南各个地区广泛引种和栽培[3]。国内外对桉树病害的报道较多,主要以桉树花斑病(Aureobasidiumsp.)、炭疽病(Colletotrichumgloeosporioides)、紫斑病(Septoriamortarlensis)、灰霉病(Botrytiscinerea)和桉树焦枯病(Eucalyptus)等叶部真菌性病害发生较为普遍,其中由丽赤壳属(Calonectria)真菌引起的桉树焦枯病为害最为严重,分布也最为广泛[4]。桉树焦枯病不同程度地威胁着桉树幼苗、叶片、树干、根部以及生理代谢,而被病原菌侵染的桉树轻则出现植株叶片脱落,苗圃与幼林落叶枯稍、枝条萎蔫；严重时会出现感病苗木叶片全部脱落、顶枯、植株枝条全部枯死等现象[5]。目前,在澳大利亚、美国、中国、巴西、日本、印度尼西亚、泰国、越南等几十个桉树种植较为密集的国家均有桉树焦枯病的报道,桉树焦枯病从其分布范围上来说已经被定义为一种世界性病害[1]。而我国早在1996年1月5日便将之列入为国内森林植物检疫对象。

对林木病害快速、准确的早期诊断是减少林木损失的有效途径[6]。传统病原菌的诊断方法主要以形态学及生理生化指标鉴定为主,该方法不仅费时费力、准确度也较低,而且对鉴定者需具有很高的专业知识。此外,肉眼鉴定病害的过程中会受到多种因素干扰,如分离培养的病原菌是否纯化、培养条件的影响、生理指标的不稳定性等,因此基于传统方法检测病害在一定程度上仍存在大量弊端。随着林木病害的普遍发生,诊断技术也随之进步,从早期的田间传统鉴定和诊断技术,过渡到免疫性诊断阶段,再到分子生物学水平诊断。而分子生物学诊断技术也从早先的基础聚合酶链反应(polymerase chain reaction,PCR)技术,到基于PCR的巢式PCR、双重PCR、多重PCR以及最近几年发展起来的循环介导等温扩增、核酸快速扩增和指纹检测技术,这些进步使得分子生物学水平上的病害检测具有更高的准确性、可靠性、灵敏性以及时效性[7-8]。巢式PCR较一般PCR具有更高的灵敏度和准确性,被广泛地应用于玉米叶斑病[9]、甘蔗白叶病[10]、梨黑星病[11]等多种植株病害的检测。本试验以四川桉树焦枯病的病原菌(Cylindrocladiumscoparium)为对象,构建了C.scoparium的巢式PCR快速检测体系,并对其田间野外进行时效检测应用,为桉树焦枯病的早期预判和提前防治提供重要的技术支持。

1 材料与方法

1.1 供试菌株

本实验使用的供试菌株均分离自桉树病株,其分离采用常规组织分离法,具体参照韩永超等[12]的方法；鉴定采用形态学与分子生物学手段相结合,具体参照王俊丽等[13]的方法。其中菌株编号1～6为桉树焦枯病原菌,菌株编号1和2购自中国林业科学研究院森林生态环境与环境保护研究所,菌株编号3～6分离自各地方桉树病株,除菌株编号8外的各菌株均分离自桉树病株,供试菌株采集地、编号、菌株种属详见表1。

表1 供试菌株及其来源

1.2 供试菌株DNA提取

1.2.1 菌丝的收集 采用液体摇床振荡的方法进行供试菌株培养,参照Wang等[14]的方法进行菌丝提取,稍加改动。分别接种供试菌株于马铃薯葡萄糖琼脂(potato dextrose agar,PDA)培养基上,室温下连续培养5 d后,取菌落边缘菌丝少许,转接于瓶装量为40-mL的马铃薯葡萄糖肉汤(potato dextrose broth,PDB)液体培养锥形瓶(100-mL)中,25 ℃,pH 7,在125 r/min条件下振荡培养5～7 d。用无菌水冲洗，纱布过滤后收集到的菌丝置于-20 ℃条件下保存备用。

1.2.2 供试菌丝DNA的提取 称取0.2 g冷冻保存的菌丝,利用液氮研磨法破解细胞壁,然后按天根植物基因组DNA提取试剂盒(plant genomic DNA kit)的操作步骤进行供试菌丝DNA的提取,具体参照韩振云等[15]的方法改进使用。所提取的DNA溶液分别在紫外分光光度计(UV-3600)下检测浓度,并作适当稀释后于-20 ℃保存、备用。

1.2.3 发病组织DNA的提取 2013年对桉树焦枯病发病时期进行野外样品的采集,焦枯病害的感病程度共分5级,分别用0级(健康叶片)；一级(叶片感病面积在0～25%之间)；二级(叶片感病面积在25%～50%之间)；三级(叶片感病面积在50%～75%之间),四级(叶片感病面积在75%～100%之间)；五级(叶片感病面积达到100%)表示。分别随机选择10片不同感病程度的桉树叶片放入冰壶内,标号、带回实验室备用,同时采集健康叶片作为对照。首先对采集的样品用70%乙醇表面消毒,蒸馏水冲洗3次,吸水纸吸干后,将叶片样品用刀片切成面积为1.0 cm×1.0 cm的小块,每感病程度取20个,置于研钵中用液氮研磨成粉末,参照天根植物基因组DNA的提取方法操作。

1.3 通用引物PCR扩增

利用Cylindrocladium(Calonectria)属真菌β-微管蛋白(beta-tubulin)基因区通用引物[16]BT-T1-S(5′-AACATGCGTGAGATTGTAAGT-3′),BT-CYLTUBIR-A(5′-AGTTGTCGGGACGGAAGAG-3′)对桉树焦枯病菌(C.scoparium)进行常规PCR扩增。PCR扩增体系(50 μL):10×PCR缓冲液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,基因组DNA提取液4.0μL,反应条件:94 ℃热启动 5 min;94 ℃变性 45 s;58 ℃退火 45 s;72 ℃延伸40 s;循环32次,72 ℃延伸8 min。以ddH2O代替模版DNA作阴性对照。PCR产物检测在1.5%琼脂糖凝胶电泳上进行,经EB染色后,在凝胶成像系统上拍照、观察。PCR扩增产物委托上海生物工程有限公司完成测序。

1.4 病原菌特异性引物设计与合成

根据供试菌株beta-tubulin基因序列的测序结果,结合GeneBank中Cylindrocladium属种的差异进行C.scoparium病原菌特异性引物的设计,所设计的引物对为BT-S-1(5′-GGCTCCAAGAACT ATGTGA-3′)/BT-A-1(5′-CCTAACCACGAATG TCAGT-3′),扩增片段大小148 bp。引物由上海生工生物工程有限公司合成。

1.5 常规和巢式PCR特异性验证

常规PCR:利用特异性引物BT-S-1/BT-A-1对所有参试菌株PCR扩增。PCR扩增体系(50 μL):10×PCR缓冲液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。反应条件同1.3节通用引物PCR扩增,退火温度为60 ℃。以灭菌水代替模版DNA设置阴性对照。

巢式PCR:分别以真菌通用引物BT-T1-S/BT-CYLTUBIR-A作为第1轮引物进行PCR扩增,反应体系和程序同1.3节,取第1轮PCR产物,稀释10倍,作为第2轮PCR模板,第2轮引物为BT-S-1/BT-A-1,反应体系和程序同常规PCR。

1.6 灵敏度检测

将提取的发病组织叶片病原菌基因组DNA溶液以10的倍数进行稀释,分别得到质量浓度为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作为PCR反应模版,分别用于常规PCR和巢式PCR,比较二者灵敏度。常规PCR反应体系和反应程序同1.5节,巢式PCR反应程序和反应体系分别同1.3节和1.5节。

1.7 田间时效检测

按发病组织方法提取DNA，按1.5节所述的方法进行巢式PCR扩增,同时以ddH2O代替DNA模板组和健康组做阴性对照。根据电泳检测结果判定桉树初期发病植株、中期发病植株、严重发病植株、具有典型症状植株和健康植株的样品中是否携带Calonectriamorganii致病菌。

2 结果与分析

2.1 通用引物PCR扩增结果

利用真菌通用引物BT-T1-S/BT-CYLTUBIR-A可以将全部供试菌株扩增出1条500 bp大小条带,而阴性空白对照无条带(图1),表明提取菌株的DNA较纯,可以保障后续试验。

2.2 常规PCR特异性引物BT-S-1/BT-A-1扩增结果

扩增反应结果(图2)显示:桉树焦枯病菌(C.scoparium)可扩增出明亮条带,大小为148 bp,而其他同属和非同属参试菌、对照组都没有扩增出条带。通过片段回收测序及与现有的C.scoparium序列比对发现同源率为100%,证明该引物可以从混合DNA样中准确地检测出桉树焦枯病菌(C.scoparium),准确率为100%。因此,说明特异性引物BT-S-1/BT-A-1可有效地用于桉树焦枯病菌(C.scoparium)林木病害基因组DNA的PCR扩增。

2.3 巢式PCR扩增反应结果

巢式PCR检测结果(图3)显示:桉树焦枯病菌(C.scoparium)可扩增出明亮条带,大小为148 bp,而其他同属和非同属参试菌、对照组都没有扩增出条带；相对常规PCR特异性检验而言,巢式PCR特异性检验的条带更为明亮。与常规PCR扩增相比,巢式PCR具有一定的视觉优越性。

2.4 常规PCR与巢式PCR灵敏度检测

对不同质量浓度桉树焦枯病菌(C.scoparium)基因组DNA进行常规PCR扩增(图4)和巢式PCR扩增(图5),结果表明巢式PCR具有更高的灵敏度。在50 μL反应体系中,常规PCR检测基因组浓度极限为5 pg/μL,低于5 pg/μL则不能检测到条带,而巢式PCR可达到5 fg/μL,检测灵敏度至少提高了103倍,可满足田间检测要求。

2.5 巢式PCR野外时效性检测

利用巢式PCR快速试剂盒进行检测,结果见6,并通过片段回收测序与现有C.scoparium序列比对发现同源率为100%,证明该引物可以从混合DNA样中准确地检测出桉树焦枯病菌(C.scoparium),准确率为100%。常规PCR扩增只能检测出新枯死样和典型感病组织内病原菌；而巢式PCR能够有效检测出新枯死样、典型感病组织、感病初期和部分感染后未显症样品(泳道10、11、12),可见其灵敏度明显高于常规PCR扩增。巢式PCR之所以只能检测出部分未显症样品,可能是因为未检测出的部分未感病样品组织不携带有病原菌或其携带的病原菌的数量极少,达到了巢式PCR检测的极限。

M:DL2000标志物;CK:阴性对照;(1～22):通用引物BT-T1-S/BT-CYLTUBIR-A对表1中所对应的供试菌株DNA溶液PCR扩增。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.图1 常规PCR通用引物BT-T1-S/BT-CYLTUBIR-A扩增反应结果Fig.1 Results of PCR amplification using universal primers BT-T1-S/BT-CYLTUBIR-A

M: DL2000标志物;CK:阴性对照;(1～22):引物BT-S-1/BT-A-1直接对表1中所对应的供试菌株DNA溶液PCR扩增.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.图2 已知供试菌PCR的BT-S-1/BT-A-1特异性扩增结果Fig.2 Results of PCR amplification using specific primers BT-S-1/BT-A-1

M: DL2000标志物;CK:阴性对照;(1～22):引物BT-T1-S/BT-CYLTUBIR-A和BT-S-1/BT-A-1对表1中所对应的供试菌株DNA溶液巢式PCR扩增。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.图3 β-微管蛋白基因区特异性引物巢式PCR扩增结果图Fig.3 Results of nested-PCR amplification using specific primers in beta-tubulin gene

M:DL2000标志物;(1～6):桉树焦枯病原菌C. scoparium基因组DNA浓度分别为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.图4 常规PCR灵敏度检测结果Fig.4 Sensitivity detection of universal PCR

M:DL2000标志物;(1～9):桉树焦枯病原菌C. scoparium基因组DNA浓度分别为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.图5 巢式PCR灵敏度检测结果Fig.5 Sensitivity detection of nested-PCR

M:DL2000标志物；(1～7):BT-T1-S/BT-CYLTUBIR-A第1轮扩增结果；(8～14):巢式PCR第2轮扩增结果;(15～21):常规PCR扩增结果；22:阳性对照(C. scoparium).其中1,8,15为新枯死组织；2,9,16为典型发病组织；3,10,17为感病初期；4,11,18为未感病组；5,12,19为残体病株；6,13,20为健康组；7,14,21为阴性对照。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).图6 巢式PCR田间检测桉树焦枯病菌Fig.6 Field test on C. scoparium of nested-PCR

3 讨论与结论

桉树焦枯病作为一种世界性病害,越来越受到人们的重视。加强病害的早期诊断是降低重大病害损失的有效手段。传统病害的诊断方法主要以形态学及生理生化指标鉴定为主,该方法不仅费时费力、往往准确度也较低,而且对鉴定者需具有很高的专业知识。此外,肉眼鉴定病害的过程中会受到多种因素干扰,如分离培养的病原菌是否纯化、培养条件的影响、生理指标的不稳定性等,因此基于传统方法检测病害在一定程度上仍存在大量弊端[17-18]。PCR及基于PCR的快速诊断技术在林业病害上的应用弥补了传统检测的不足,同时较高的准确性、精确性、时效性使得林业上多种重要病害的早期诊断成为可能,例如松材线虫病[19]、杨树溃疡病[20]、桉树青枯病[21]等的诊断。目前,对于桉树焦枯病的诊断仍停留在传统形态学检测上,因此建立桉树焦枯病的快速检测技术十分必要。

巢式PCR是在PCR基础上发展起来的一项更快速、更灵敏、精确度更高的快速检测技术[22]。巢式PCR的原理是利用2对特异性引物先后共同扩增一段目的区域,而第2次扩增的目的区域位于第1次扩增的目的区域内部,能够降低常规PCR经常出现的非特异性扩增概率,保证PCR的准确性[23]。灵敏度检测表明,巢式PCR具有比常规PCR更高的灵敏度。供试菌的选择是PCR构建的基础。参考以往PCR体系构建的供试菌选择,主要来源有以下几类:与病原菌同属不同种的菌株,从发病寄主上分离得到的内生菌、外生菌、寄生菌以及可能引起寄主感病的其他病原菌,资源丰富的供试菌株是该体系建立的基础保障。

此外,保守性极强的特异序列是引物设计成功的关键因素。beta-tubulin基因在原核和真核生物中均有存在,且具有高度的特异性,常常被用于多种病原菌的分类鉴定、系统发育、地理小种的区分上[24]。本试验选择了Cylindrocladium在其beta-tubulin基因区通用引物BT-T1-S/BT-CYLTUBIR-A作为第1轮引物,在此基础上又在其beta-tubulin特异性保守序列上设计了巢式PCR第2对特异性引物BT-S-1/BT-A-1,尤其是第2对引物的特异性完全保证了桉树焦枯病巢式PCR体系构建的成功。此外,优化的退火温度是保证巢式PCR顺利进行的关键因素,研究中对该体系进行温度梯度优化,得到第1轮通用引物的退火温度为58 ℃,第2轮特异性引物的退火温度为60 ℃,解决了其他温度扩增出现条带不清晰和无条带的问题。而田间病害的时效性检测表明,该体系可用于桉树焦枯病的野外发病检测,桉树焦枯病巢式PCR快速检测技术的构建实现了桉树焦枯病的早期诊断,为桉树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

国家科技基础条件平台基金(2005DK21207-13)。

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

2014-07-27;接受日期(Accepted):2015-02-13；网络出版日期(Published online):2015-09-18

Q 939.5; S 432.4

A

*通信作者(Corresponding author):朱天辉(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