作物杂志, 2019, 35(6): 168-176 doi: 10.16035/j.issn.1001-7283.2019.06.027

植物保护

基于qPCR和LAMP技术的马铃薯晚疫病菌快速检测方法

祝菊澧1,2,3, 梁静思1,2,3, 张佩1, 王伟伟1,2,3, 林桐司骐1, 谢欣娱1, 苏瑞1, 唐唯1,2,3

1云南师范大学生命科学学院,650500,云南昆明

2云南师范大学马铃薯科学研究院,650500,云南昆明

3云南省马铃薯生物学重点实验室,650500,云南昆明

A Rapid Detection Method for Potato Late Blight Caused by Phytophthora infestans Based on qPCR and LAMP Assays

Zhu Juli1,2,3, Liang Jingsi1,2,3, Zhang Pei1, Wang Weiwei1,2,3, Lin Tongsiqi1, Xie Xinyu1, Su Rui1, Tang Wei1,2,3

1School of Life Science, Yunnan Normal University, Kunming 650500, Yunnan, China

2Joint Academy of Potato Science, Yunnan Normal University, Kunming 650500, Yunnan, China

3Key Laboratory of Potato Biology of Yunnan Province, Kunming 650500, Yunnan, China

通讯作者: 唐唯,副教授,主要从事马铃薯致病疫霉有性生殖发生机制研究

收稿日期: 2019-05-24   修回日期: 2019-08-26   网络出版日期: 2019-12-15

基金资助: 国家自然科学基金(31660503)
云南师范大学2019年度研究生科研创新基金(ysdy js2019132)

Received: 2019-05-24   Revised: 2019-08-26   Online: 2019-12-15

作者简介 About authors

祝菊澧,硕士研究生,主要从事马铃薯病原菌快速检测研究; 。

梁静思为并列第一作者,硕士研究生,主要从事马铃薯病原物基因组学研究 。

摘要

马铃薯晚疫病菌(Phytophthora infestans)能侵染多种茄科植物,它引起的马铃薯晚疫病,是马铃薯生产中的第一大病害。为了开发能在田间快速检测马铃薯晚疫病病原的方法,利用P. infestans T30-4基因组测序数据的contig 1.18131,设计qPCR和LAMP引物,优化扩增条件后得到引物的特异性和灵敏度,最后通过检测田间收获薯块,比较形态学传统方法、qPCR及LAMP的差异。特异性检测结果发现,qPCR和LAMP仅在含有P. infestans DNA模板的体系有阳性扩增,在寄主和其他微生物DNA中均无扩增;在优化的条件下,qPCR和LAMP的检测下限可达1×10 -6ng/μL,在有寄主和其他微生物DNA存在的条件下,引物的灵敏度没有显著差异。利用两种快速方法对在大理、丽江及昆明3个地区田间收获薯块上检测发现,qPCR和LAMP方法得到的检出率差异极为不显著(P=0.420),两种快速检测方法和形态学鉴定方法检出率差异极显著(P=0.009)。在大理、丽江及昆明3个地区的薯块中,两种分子检测方法检出率均比形态学方法高。其中,qPCR检测方法比形态学方法分别提高了12.00%、2.00%、8.70%;LAMP检测方法比形态学方法分别提高了11.30%、2.00%、8.70%。

关键词: 定量PCR(qPCR) ; 环介导等温扩增(LAMP) ; 马铃薯晚疫病菌 ; 潜伏侵染 ; 检测

Abstract

Phytophthora infestans can infect many kinds of Solanaceae plants. Potato late blight (PLB) caused by P. infestans is a most severe disease in the potato production. To better and fast diagnose PLB in the fields, quantitative PCR (qPCR) and Loop-mediated isothermal amplification (LAMP) primers were designed based on the P. infestans T30-4 whole genome shotgun contig 1.18131. After amplification conditions optimized, both specificity and sensitivity were detected. Finally, developed qPCR and LAMP methods were used to detect PLB incidence and compared with traditional isolation method on harvested potato tubers in the fields. Specific detection showed that qPCR and LAMP were positive amplification only in the presence of P. infestans DNA template, and were no amplification in the presence of host and other microorganism DNA. Moreover, as low as 1×10 -6ng/μL gDNA was detected using both qPCR and LAMP methods, while the sensitivity of primers showed no significant difference in the presence of P. infestans DNA template, host and other microorganism DNA. Detecting harvested tubers in the field using two methods in Dali, Lijiang and Kunming, qPCR and LAMP showed no significant difference but extremely significant difference between two fast detection methods and morphology based identification. Additionally, in Dali, Lijiang and Kunming, PLB incidence of the two molecular detection methods were both higher than morphology based identification method, in which qPCR detection method was 12.00%, 2.00% and 8.70% higher than morphology based identification method, and LAMP detection method was 11.30%, 2.00% and 8.70% higher than morphology based identification method, respectively.

Keywords: Quantitative PCR (qPCR) ; Loop-mediated isothermal amplification (LAMP) ; Phytophthora infestans ; Latent infection ; Detection

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本文引用格式

祝菊澧, 梁静思, 张佩, 王伟伟, 林桐司骐, 谢欣娱, 苏瑞, 唐唯. 基于qPCR和LAMP技术的马铃薯晚疫病菌快速检测方法[J]. 作物杂志, 2019, 35(6): 168-176 doi:10.16035/j.issn.1001-7283.2019.06.027

Zhu Juli, Liang Jingsi, Zhang Pei, Wang Weiwei, Lin Tongsiqi, Xie Xinyu, Su Rui, Tang Wei. A Rapid Detection Method for Potato Late Blight Caused by Phytophthora infestans Based on qPCR and LAMP Assays[J]. Crops, 2019, 35(6): 168-176 doi:10.16035/j.issn.1001-7283.2019.06.027

由致病疫霉[Phytophthora infestans (Mont. de Bary)]引起的马铃薯晚疫病(potato late blight,PLB)是世界范围内马铃薯的第一大病害。低温和较高的湿度、昆虫取食、农药的滥用及病原菌有性生殖的发生均可以加速病害的发生[1,2]。在适合的条件下,如果从发现第一个病斑开始不采取任何防治措施,PLB在病害中心形成后蔓延速度极快,3~4周就可以毁掉整块种植地。PLB在田间主要危害马铃薯叶片,也可侵染任何发育时期的块茎和储藏期的薯块,症状为褐色病斑,有凹陷,切开后病部皮下薯肉呈褐色,温度适合条件下向四周扩大形成烂薯。此外,在马铃薯残枝上和土壤中越冬的卵孢子可以在早期侵染下一个种植季的种薯及幼苗,使得病害的发生提前并加重[3]。传统的PLB病原检测主要依靠症状观察和病原菌分离鉴定。然而,马铃薯晚疫病菌存在潜伏侵染特点,在没有症状发生时,很难利用传统方法来诊断[4]。此外,由于马铃薯晚疫病在田间植株间和在收获的薯块中扩展速度极快,当利用传统的方法能检测到时,病害已发展到了中期或后期,此时防治已经失去实际意义。目前主要通过预测预报系统[5]和监测空气中孢子的密度[6]来预测PLB病害,但此类方法过于依赖气候条件,对病害发生和流行的预测并不准确。据此,开发PLB病原的早期监测技术,对PLB的化学防治具有重要意义。

利用特异性引物,结合卵菌形态学特征,已经可以把P. infestans从疫霉属(Phytophthora)的许多种间区分开来[7,8,9]。用于鉴定和快速检测疫霉属下各个种的特异性引物,主要基于ITS序列[7-8,10-11]、高度重复序列[12]、β-tubulin和Ras基因[13]、EF1-α[14]以及Ypt1基因[15]。另外,基于SYBR Green的实时定量PCR技术已经被用于检测土壤和植物组织中卵菌的孢子量[16,17]。除PCR技术外,2000年环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)被开发出来[18,19],该技术方法最大的特点就是仅需一个等温(60℃~65℃)的反应过程,不需要复杂的变温设备,因此可以用于田间病害的快速检测。LAMP技术已经成功用于疫霉属各个种[20],如P. ramorum[21]和P. infestans[22]。2017年又开发出基于荧光染料的实时LAMP技术,大幅度提高了检测灵敏度并可对病原物DNA进行相对定量检测[17]。上述研究的不足之处在于,利用ITS区域的多拷贝序列在疫霉属各个系统发育种之间不易完全区分[8];Ypt1基因被认为在基因组中是单拷贝的,因此可能会降低检测灵敏度[23];薯块作为马铃薯晚疫病的主要初侵染部位,上述研究没有对田间收获的薯块进行过LAMP检测及与qPCR和传统形态学方法比较。因此,本研究利用P. infestans基因组上特异片段设计qPCR、LAMP特异性和高灵敏度引物,用两种方法对田间收获的薯块进行检测,确立马铃薯晚疫病菌快速检测方法。

1 材料与方法

1.1 试验材料

供试P. infestans于2016-2017年采集自云南马铃薯各主产区,另有3株来自于国外,其中DN111、80029由中国农业大学国立耘教授赠送,88069由华中农业大学田振东教授赠送。菌株信息见表1,菌株分离纯化后于18℃条件下培养于Rye B培养基[24]。疫霉属其他种和马铃薯其他病原菌于2012年分离自云南各地区(表1),分离纯化后接在PDA或LA培养基上,于22℃恒温箱中培养。450个马铃薯“大西洋”薯块分别于2017年7-8月收获于大理白族自治州农业科学推广研究院、丽江市农业科学研究所和昆明市农业科学研究院3地(市)马铃薯育种基地。

表1   用于qPCR和LAMP检测卵菌及其他马铃薯相关病原菌菌株信息

Table 1  Strain information of oomycetes and other potato related pathogens for qPCR and LAMP detection

分类
Clossify
编号
No.
菌株
Strain

Species
寄主(分离部位)
Hosts (Isolation source)
来源
Source
ITS序列
ITS sequence
卵菌1110PPhytophthora infestansSolanum tuberosum (leaf)中国云南LS479123
Oomycetes2XD1213P. infestansS. tuberosum(leaf)中国云南LS479124
3XD15P. infestansS. tuberosum(leaf)中国云南LS479193
4JC1207P. infestansS. tuberosum(leaf)中国云南LS479126
5XD1314P. infestansS. tuberosum(leaf)中国云南LS479127
6XA-4P. infestansS. tuberosum(tuber)中国云南LS479171
7DN111P. infestansS. tuberosum(unknown)日本北海道LS479169
880029P. infestansS. tuberosum(unknown)荷兰瓦格宁根LS479173
988069
P. infestans
Lycopersicon esculentum(leaf)荷兰瓦格宁根LS479172
10Pds13P. sojaeGlycine max(leaf)中国云南LS479895
11Ddr1231P. parasiticaNicotiana tabacum (leaf)中国云南LS479896
12LL2480P. capsicaCapsicum annuum(fruit)中国云南LS479897
13Fuz37Pythium acanthicumSaccharum officiarum L. (root)中国云南LS479899
真菌14AT1289Alternaria alternateS. tuberosum(leaf)中国云南LS479189
Fungus15Hz1177Synchytrium endobioticumS. tuberosum(root)中国云南LS479191
16Hz1608Fusarium solaniS. tuberosum(leaf)中国云南LS479188
细菌17Hh1503
Clavibacter michiganensissubsp. SepedonicusS. tuberosum(tuber)中国云南LS479190
Bacteria18Hhrf1
Pectobacterium carotovorum subsp.CarotovorumS. tuberosum(tuber)
中国云南LS479898
19Hhqk1Ralstonia solanacearumS. tuberosum(stem)中国云南LS479192

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1.2 试验方法

1.2.1 引物设计 利用P. infestans T30-4全基因组的contig 1.18131(NCBI accession No. AATU01018131.1)与不同致病疫霉在NCBI中进行Blast分析后,发现该区域与致病疫霉的ITS区有99%以上的相似性,具有代表性。随后利用该序列在Vector NTI(Version 11.5,Invitrogen Inc.)中设计qPCR引物。同样利用contig 1.18131片段在线设计LAMP引物(http://primerexplorer.jp/e/v4_manual/index.html.),引物信息见表2

表2   LAMP与实时荧光定量PCR的引物

Table 2  LAMP and real-time fluorescent qPCR primers

扩增类型PCR type引物名称Primer name序列Sequence (5'-3')目标长度Target length (bp)
qPCRinfnew1-FACCACACCTAAAAACTTTCCACGT241
infnew1-RCATCCACTGCTGAAAGTTGC
LAMPF3ACCCAATAGTTGGGGGTCTT
B3AGCGTTCTTCATCGATGTGC
BIPCTGTGGGGACGAAAGTCTCTGCGC
CTAGACATCCACTGCTGA
FIPCGAAGTCCAAACGCTCGCCTTGCT
TTATTGCTGGCGGCTA

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1.2.2 DNA提取及病原物种类鉴定 真菌和卵菌基因组DNA用Plant DNA Extraction kit [DP320,天根生化科技(北京)有限公司]提取。提取的DNA用NanoDrop-1 000(Thermo Fisher Scientific,美国)测定浓度。将DNA统一稀释至2ng/µL后保存于-20℃备用。为了对表1中分离的菌株进行系统发育种鉴定,提取基因组DNA后,引物ITS1(5′-TCCGTAGGTGAACCTGCGG-3′)和ITS4(5′-TCCTCCGCTTATTGATATGC-3′)[25],以及27F(5′-TCCGTAGGTGAACCTGCGG-3′)和1492R(5′-TCCGTAGGTGAACCTGCGG-3′)[26]分别用于扩增卵菌(真菌)和细菌。PCR卵菌扩增体系:10mmol/L正、反向引物各1µL,0.5µL (2U/µL) Taq DNA聚合酶(TaKaRa Inc.日本),5µL 10× PCR buffer,4µL dNTPs(2.5mmol/L),1µL(10~20ng/µL)DNA模板,38.5µL ddH2O。细菌扩增体系:94℃3min;94℃30s,55℃30s,72℃90s,35个循环;72℃10min。PCR产物纯化后用ABI 3500测序仪(ABI Life Science,美国)两端测序。序列拼接后在EMBL数据库(https://www.ebi.ac.uk/)中比对并上传。参照文献[27]和[28]进行形态学种的鉴定。1.2.3 qPCR和 LAMP 条件的优化 为了优化qPCR扩增条件,把引物分别稀释到终浓度为0.1、0.2、0.4、0.6、0.8、1.0μmol/L。引物的退火温度为57℃、59℃、61℃、63℃。qPCR采用3步法,扩增程序为95℃ 20s;95℃ 10s,55℃ 30s,72℃ 30s,40个循环。qPCR扩增体系为25μL,包含12.5μL SYBR Premix Ex Taq II (2×)(TaKaRa Inc.日本),0.5μL Rox Reference Dye (50×),正反向引物各1μL,DNA模板15ng,补足ddH2O至25μL。在ABI StepOne Plus(ABI Life Science,美国)中进行PCR,试验重复3次。LAMP反应体系为25μL,包含20ng基因组DNA,终浓度5pmol/μL的F3和B3引物,终浓度40pmol/μL的FIP和BIP引物,2× reaction mix 12.5μL,Bst DNA polymerase 1.5U,反应完后加入1μL SYBR Green I fluorescent(Eiken Chemical,日本)显色观察。设置梯度等温孵育时间(30、40、50、60min)和梯度等温温度(60℃、61℃、62℃、63℃、64℃、65℃)。所有LAMP反应在ABI 9700 PCR system(ABI Life Science,美国)中进行,每一个反应均重复3次。1.2.4 引物特异性检测 为了验证引物的特异性,利用19个分离的菌株(表1)为模板,以ddH2O为阴性对照,在qPCR和LAMP优化的条件下进行扩增。同时,为了验证引物特异性在多种病害共存以及有寄主DNA存在的情况下是否会受到影响,取表1中除P. infestans外的10株病原物已提取的基因组DNA健康寄主叶片DNA各1μL组成混合液,混匀后取混合液2μL,加入2ng/μL P. infestans DNA 1μL用作qPCR和LAMP待测模板,以ddH2O为阴性对照1,不含P. infestans的混合液为阴性对照2,以单一的P. infestans DNA为阳性对照。所有反应重复3次。1.2.5 引物灵敏度的测定 为了建立qPCR标准曲线和检测LAMP反应极限,将P. infestans基因组DNA稀释10倍(1×10-7~1×10-1ng/μL)后作为模板,以ddH2O为阴性对照,每个反应3个重复处理,建立标准曲线。为了验证在寄主DNA存在的条件下检测引物灵敏度是否会受到显著影响,提取健康马铃薯叶片基因组DNA后稀释至2ng/μL,取1μL分别混合于梯度稀释的P. infestans模板DNA中,在优化的扩增条件下,进行qPCR和LAMP反应。每个处理重复3次。1.2.6 薯块中P. infestans的检测 为了评估qPCR和LAMP体系,2017年3月在昆明、丽江和大理选取3个地区的种植地块(约0.27hm2/个,种植品种为大西洋),从出齐苗后第10天采用5点随机取样法开始调查PLB发病情况,每个点调查相邻3株植株,统计3株植株叶片PLB发病率(发病率=发病叶片数/总叶片数,以发病率≥10%记为发病),每隔15d调查一次,直至收获期为止。7-8月从3个地区的种植地块各随机收集150个“大西洋”薯块(薯块需无晚疫病明显症状),采集后薯块表面用75%酒精浸泡2min,用清水冲洗后在薯块表面随机选5个点,每个点取1cm2的薯皮,混合后用于DNA的提取,分别用优化好的qPCR和LAMP进行检测反应。同时,把薯块剩下部分置于18℃培养箱保湿培养20d后,记录发病情况。在发病位置取1cm2表皮放置于PDA培养基,继续培养直至长出菌落,利用参考菌株80029进行形态学比较和种类鉴定[27],利用引物infnew1-F/infnew1-R进行SYBR Green qPCR扩增,以ddH2O为阴性对照模板,以CT<30为阳性扩增。试验重复3次。

2 结果与分析

2.1 晚疫病菌和其他微生物的分离和鉴定

表1中的晚疫病菌和其他相关微生物,得到每一个单株的纯培养后代,经形态学鉴定和Blast同源分析,鉴定到种并同时将扩增序列上传到EMBL数据库,ITS序列号和种见表1

2.2 qPCR引物的特异性和灵敏度

Blast分析结果表明,P. infestans T30-4全基因组的contig 1.18131与致病疫霉的ITS区有99%以上的相似性,利用该序列设计的引物具有代表性与特异性(图1)。在同一DNA模板量(2ng)的条件下,引物梯度稀释及温度梯度的优化结果表明,引物浓度为0.1μmol/L,且退火温度在61℃时,CT阈值最低(22.47±0.84);qPCR特异性检测表明,全部9株P. infestans为阳性扩增(CT<25),其他10株马铃薯相关病原物、健康马铃薯叶片组织和阴性对照均无扩增(CT>35)(图2A),引物的熔解曲线为单峰(图2B)。在有其他病原物和寄主DNA干扰的条件下,扩增发现在含有P. infestans的混合液作为模板的时候有阳性扩增,且qPCR的特异性没有受到显著影响(图3A)。灵敏度检测发现,因1×10-7ng/μL浓度下CT阈值大于35,因此引物的检测浓度极限为1×10-6ng/μL。随后用1×10-6~1×10-1ng/μL模板DNA浓度梯度建立了qPCR扩增标准曲线,y=3.0845x+14.238(R2=0.9939),E=103%,x为模板DNA浓度,y为CT阈值(图3B)。在有寄主DNA存在的情况下,引物的灵敏度下限为1×10-6ng/μL,y=3.0867x+13.720(R2=0.9929),E=108%(图3C)。

图1

图1   利用Neighbor-joining分析Blast结果

中括号位置为contig 1.18131所在位置

Fig.1   Neighbor-joining analysis of Blast results

Middle brackets represent the position of contig 1.18131


图2

图2   利用优化qPCR测得的致病疫霉种特异性检测结果

Fig.2   P. infestans species-specific test results by optimized qPCR


图3

图3   qPCR特异性和灵敏度检测

A:qPCR特异性检测,1:P. infestans gDNA;2:加入P. infestans gDNA的混合溶液;3:不加P. infestans gDNA的混合溶液;4:ddH2O。B:灵敏度检测,利用P. infestans(1×10-6~1×10-1ng/μL)DNA模板,建立标准曲线;E:扩增效率(103%)。C:在有寄主存在条件下的灵敏度检测,在有寄主存在条件下,利用引物infnew1,以P. infestans DNA浓度梯度(1×10-6~1×10-1ng/μL)建立标准曲线,E:扩增效率(108%)

Fig.3   Specific test and sensitive test by qPCR for species

A: P. infestans species specific test by qPCR, 1: P. infestans gDNA; 2: Mixed solution with P. infestans gDNA; 3: Mixed solution but without P. infestans gDNA; 4: ddH2O. B: Sensitive test, sensitive test profiles of 10-fold serial dilutions of P. infestans (1×10-6~1×10-1ng/μL) to create standard curve; E: Reaction efficiency (103%). C: Sensitive test with the presentation of host DNA, quantitative real-time PCR (qPCR) amplification profiles of 10-fold serial dilutions of P. infestans (1×10-6~1×10-1ng/μL) using infnew1 primers with host gDNA; E: Reaction efficiency (108%)


2.3 LAMP引物的特异性和灵敏度

在61℃、1h的恒温条件下,9个P. infestans提取的DNA有阳性扩增,其余10个病原物DNA均无扩增(图4A)。LAMP引物灵敏度检测结果表明,利用紫外灯激发,在加入SYBR Green I dye反应后的体系中,在浓度为1×10-6ng/μL的模板DNA中,能检测到亮度发生变化(图4B),同时在2.0%琼脂糖凝胶电泳上能检测到条带(图4C)。在有其余病原物和寄主DNA干扰的检测中发现,LAMP的特异性没有受到显著影响,灵敏度为1×10-6ng/μL。

图4

图4   LAMP特异性及灵敏度检测

A:19株菌株的LAMP反应;B:SYBR Green染色后在紫外线下观察阳性扩增的亮度变化;C:2.0%琼脂糖凝胶电泳检测LAMP产物

Fig.4   Specific test and sensitive test by LAMP for species

A: LAMP using 19 isolates; B: Observation of intensity change of positive amplification by SYBR Green staining under ultraviolet light; C: LAMP products analyzed by 2.0% agarose gel


2.4 利用qPCR和LAMP在寄主中检测P. infestans

对田间收获的薯块利用两种快速检测方法qPCR和LAMP,以及形态学鉴定方法检测P. infestans(表3),结果发现两种快速检测方法得到的检出率差异不显著(P=0.420),两种快速检测方法和形态学鉴定方法检出率差异极显著(P=0.009),两种快速检测方法的检出率均比传统形态学检测方法高。在大理、丽江、昆明3个地区收获的薯块中,利用qPCR检测的检出率比形态学检测分别提高了12.00%、2.00%、8.70%,利用LAMP检测的检出率比形态学检测分别提高了11.30%、2.00%、8.70%。试验还发现,两种快速检测方法的检出率和实际发病率差异极不显著(P=0.809)。此外,对田间收获薯块储藏20d后观察其PLB症状,结果表明,在大理收获的150个薯块储藏20d后,73个为0级(无PLB病斑),其余77个有PLB症状,其中19个薯块病害症状为1级(PLB病斑面积小于25%),35个为2级(PLB病斑面积小于50%),16个为3级(PLB病斑面积小于75%),7个为4级(PLB病斑面积小于100%);对于丽江收获的150个薯块储藏20d后,129个为0级,其余21个有PLB症状,其中6个薯块的病害症状为1级,9个为2级,2个为3级,4个为4级;对于昆明收获的150个薯块储藏20d后,102个为0级,有48个观察到PLB症状,其中11个薯块的病害症状为1级,19个为2级,15个为3级,3个为4级(表3)。

表3   LAMP,qPCR及形态学方法检测薯块PLB检出率

Table 3  PLB incidence of potato tubers caused by P. infestans determined by LAMP, qPCR and morphology observation

地区
Region
PLB发病率a (%)
PLB incidencea
样品数
Number of samples
症状b
Disease symptomsb
LAMPqPCR形态学c
Morphologyc
大理Dali32.50±10.8015077 (51.30%)78 (52.00%)79 (52.70%)61 (40.70%)
丽江Lijiang12.66±4.2515021 (14.00%)21 (14.00%)21 (14.00%)18 (12.00%)
昆明Kunming22.71±11.6215048 (32.00%)46 (30.70%)46 (30.70%)33 (22.00%)

Note: a. PLB incidence of leaves in the field; b. PLB symptoms of harvested potato tubers after stored 20 days; c. Morphology and ITS sequence blast analysis to determine P. infestans in tubers after isolation from tubers

注:a为田间调查叶片的PLB发病率;b为收获薯块储藏20d后观察PLB症状;c为菌株分离后通过形态学观察及ITS序列比对确定薯块中的致病疫霉

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结果还发现,将450个薯块的薯皮经过培养后,除P. infestans外,共分离、鉴定出了5个属的微生物(表4)。

表4   样品中分离出微生物数量情况

Table 4  Number of various microbe isolated from tuber samples

地点Site致病疫霉
P. infestans
腐皮镰孢霉菌
Fusarium solani
枯草芽孢杆菌
Bacillus subtilis
赖氏菌属
Leifsoniasp.
类芽孢杆菌属
Paenibacillussp.
副球菌属
Paracoccussp.
大理Dali6171331113
丽江Lijiang18010015
昆明Kunming335187510

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3 讨论

由于致病疫霉在侵染马铃薯时具有多重侵染和潜伏性感染的特性,在田间感染初期,很难用常规方法检测出PLB侵染,而症状出现后病害发生十分迅速,当观察到了PLB病叶,则已经失去了最佳用药机会。因此,对于PLB的早期快速检测具有重要意义。本研究根据致病疫霉T30-4全基因组contig 1.18113序列区域,分别设计了qPCR和LAMP检测的特异性PCR引物。Blast分析发现,contig 1.18113序列共有863个DNA碱基对,核苷酸序列与致病疫霉的ITS区有99%以上的相似性。在真菌和卵菌中,ITS区域易于扩增和测序,被广泛利用于系统发育研究。在致病疫霉中,相比核糖体DNA其他区域和核基因组大多数区域,ITS区拥有较好的种特异性[25,29]。但有研究也表明,疫霉属部分种的菌株ITS序列相似性较高,因此加大了设计种特异性引物的难度,并在相近种中存在交叉污染[6,29]

本研究设计的特异性引物在疫霉属中的其他3个种中没有非特异性扩增,但未分离到更多的种进行特异性测定,因为除P. infestans外,疫霉属中其他种目前还未发现能侵染马铃薯。利用多个种间特异性引物进行高灵敏度的病原物检测也有报道,如rDNA的ITS区[7,30]、IGS区[31]、cox2基因[32]和PDN基因[33]。在对P. infestans检测的相关研究中,检测极限最低为1×10-6ng/μL。本研究也探索了不同的退火温度(59℃~63℃)对qPCR扩增效率及特异性的影响,发现61℃为最佳qPCR退火温度,能特异检测出P. infestans。当退火温度升高为63℃时,部分致病疫霉扩增重复性不好,9个P. infestans作为模板时有2个无阳性扩增。在前人的研究报道[15]中,利用受PLB感染的块茎和叶片,基于Ypt1基因开发的qPCR的检测极限为1.28×10-2ng/μL,以及基于ITS1和ITS2区域,同样在基于叶片和块茎的qPCR检测极限为1×10-4ng/μL[34]。本研究qPCR检测极限较上述报道更低。较长的qPCR目标产物长度可能是制约灵敏度的一个原因,基于SYBR Green的qPCR扩增产物建议范围为100~150bp,本研究设计的扩增产物长度超过200bp,因此要想进一步提高检测下限,必须设计用于扩增更短目标片段的引物。

LAMP技术使用Bst DNA聚合酶和4条或6条引物扩增环状DNA,扩增时间通常少于1h,在LAMP反应后加入一些荧光染料(如SYBR Green或钙黄绿素)可使得试验结果直接可视化。目前这种检测技术已经被广泛应用于田间病害诊断。前人研究[12]表明,用于检测P. infestans的常规PCR反应检测极限在10fg/μL,巢式PCR极限可达5fg/μL,可检测到2个孢子囊和4个游动孢子的DNA量[16]。在本研究中用于设计引物的模板序列来源于ITS区域,在基因组上是多拷贝的,因此通过优化反应条件,使LAMP在实验室条件下检测极限在1×10-6ng/μL,低于基于Ypt1基因检测的1×10-4ng/μL[15],以及基于ITS2区域设计的50×10-6ng/μL[17]和200pg/μL[22],检测下限和巢式PCR相近,但检测时间可大幅缩短。另外,通过混合DNA模板的qPCR和LAMP引物灵敏度测试,发现检测下限没有显著变化。

本研究利用LAMP和qPCR技术,通过采集3个地区的薯块,检测了块茎表皮PLB感染率,发现两种方法检出率差异极不显著,但均明显高于症状观察和传统分离鉴定的检出率。据此,两种方法可用于薯块收获期、储藏期和播种期的PLB快速检测。因为PLB在田间也可侵染叶片和茎,且叶片和茎部病样的DNA提取方法和薯皮DNA提取方法相同,因此,推测LAMP和qPCR技术也适用于检测PLB在田间马铃薯生长期各组织中的早期潜伏侵染。

4 结论

薯块是田间PLB发生的重要初侵染源,因此早期诊断非常重要,本研究开发的LAMP和qPCR引物具有针对P. infestans的特异性,检测灵敏度能达到1×10-6ng/μL,两种方法在有寄主DNA干扰的情况下能保证检测效率,并且,利用SYBR Green或钙黄绿素的可视化LAMP反应,操作简单、成本低,灵敏度高,如结合简易和快速提取DNA的方法和移动式等温孵育设备,可在1h内检测出是否有PLB感染。因此本研究结果也有助于储藏期病害发生和流行的动态变化研究,以及马铃薯生产中的种薯质量检测。

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DOI:10.1016/S1087-1845(03)00052-5      URL     [本文引用: 1]

Abstract

Single-strand-conformation polymorphism (SSCP) of ribosomal DNA of 29 species (282 isolates) of Phytophthora was characterized in this study. Phytophthora boehmeriae, Phytophthora botryosa, Phytophthora cactorum, Phytophthora cambivora, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora colocasiae, Phytophthora fragariae, Phytophthora heveae, Phytophthora hibernalis, Phytophthora ilicis, Phytophthora infestans, Phytophthora katsurae, Phytophthora lateralis, Phytophthora meadii, Phytophthora medicaginis, Phytophthora megakarya, Phytophthora nicotianae, Phytophthora palmivora, Phytophthora phaseoli, Phytophthora pseudotsugae, Phytophthora sojae, Phytophthora syringae, and Phytophthora tropicalis each showed a unique SSCP pattern. Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, and Phytophthora megasperma each had more than one distinct pattern. A single-stranded DNA ladder also was developed, which facilitates comparison of SSCP patterns within and between gels. With a single DNA fingerprint, 277 isolates of Phytophthora recovered from irrigation water and plant tissues in Virginia were all correctly identified into eight species at substantially reduced time, labor, and cost. The SSCP analysis presented in this work will aid in studies on taxonomy, genetics, and ecology of the genus Phytophthora.

Tooley P W, Bunyard B A, Carras M M , et al.

Development of PCR primers from internal transcribed spacer region 2 for detection of Phytophthora species infecting potatoes

Applied and Environmental Microbiology, 1997,63(4):1467-1475.

URL     PMID:9097445      [本文引用: 1]

We developed PCR primers and assay methods to detect and differentiate three Phytophthora species which infect potatoes and cause late blight (Phytophthora infestans) and pink rot (P. erythroseptica and P. nicotianae) diseases. Primers based on sequence analysis of internal transcribed spacer region 2 of ribosomal DNA produced PCR products of 456 bp (P. infestans), 136 bp (P. erythroseptica), and 455 bp (P. nicotianae) and were used to detect the pathogens in potato leaf (P. infestans) and tuber (P. infestans, P. erythroseptica, and P. nicotianae) tissue with a sensitivity of 1 to 10 pg of DNA. Leaf and tuber tissue were processed for PCR by a rapid NaOH method as well as a method based on the use of commercially available ion-exchange columns of P. infestans primers and the rapid NaOH extraction method were used to detect late blight in artificially and naturally infected tubers of potato cultivar Red LaSoda. In sampling studies, P. infestans was detected by PCR from artificially infected tubers at 4 days postinoculation, before any visible symptoms were present. The PCR assay and direct tissue extraction methods provide tools which may be used to detect Phytophthora pathogens in potato seedlots and storages and thus limit the transmission and spread of new, aggressive strains of P. infestans in U.S. potato-growing regions.

Hussain T, Sharma S, Singh B P , et al.

Detection of latent infection of Phytophthora infestans in potato seed tubers

Potato Journal, 2013,40(2):142-148.

DOI:10.1007/BF02849142      URL     [本文引用: 1]

Judelson H S, Tooley P W .

Enhanced polymerase chain reaction methods for detecting and quantifying Phytophthora infestans in plants

Phytopathology, 2000,90(10):1112-1119.

DOI:10.1094/PHYTO.2000.90.10.1112      URL     PMID:18944474      [本文引用: 2]

Sensitive and specific primer sets for polymerase chain reaction (PCR) for Phytophthora infestans, the oomycete that causes late blight of potato and tomato, were developed based on families of highly repeated DNA. The performance of these primers was compared to those developed previously for the internal transcribed spacer (ITS) of ribosomal DNA. The detection limit using the new primers is 10 fg of P. infestans DNA, or 0.02 nuclei. This is about 100 times more sensitive than ITS-directed primers. Nested polymerase chain reaction (PCR) allows the measurement of down to 0.1 fg of DNA using the new primers. To enhance the reliability of diagnostic assays, an internal positive control was developed using an amplification mimic. The mimic also served as a competitor for quantitative PCR, which was used to assess the growth of P. infestans in resistant and susceptible tomato. A key dimension of this study was that two laboratories independently checked the specificity and sensitivity of each primer set; differences were noted that should be considered when PCR is adopted for diagnostic applications in any system.

Gómez-Alpizar L, Carbone I, Ristaino J B .

An Andean origin of Phytophthora infestans inferred from mitochondrial and nuclear gene genealogies

Proceedings of the National Academy of Sciences of the United States of America, 2007,104(9):3306-3311.

[本文引用: 1]

Llorente B, Bravoalmonacid F, Cvitanich C , et al.

A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

Letters in Applied Microbiology, 2010,51(6):603-610.

DOI:10.1111/j.1472-765X.2010.02942.x      URL     PMID:21039667      [本文引用: 1]

To establish a reliable and rapid protocol to simultaneously obtain high quality DNA from an infected host plant and the infecting pathogen. To develop an accurate and sensitive low-cost assay for the quantification and in planta monitoring of Phytophthora infestans growth.

Mehran K, Benjin L, Yue J , et al.

Evaluation of different PCR-based assays and LAMP method for rapid detection of Phytophthora infestans by targeting the Ypt1 gene

Frontiers in Microbiology, 2017,8:1920.

DOI:10.3389/fmicb.2017.01920      URL     PMID:29051751      [本文引用: 3]

Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases affecting potato and tomato worldwide. Early diagnosis of the P. infestans pathogen causing late blight should be the top priority for addressing disease epidemics and management. In this study, we performed a loop-mediated isothermal amplification (LAMP) assay, conventional polymerase chain reaction (PCR), nested PCR, and real-time PCR to verify and compare the sensitivity and specificity of the reaction based on the Ypt1 (Ras-related protein) gene of P. infestans. In comparison with the PCR-based assays, the LAMP technique led to higher specificity and sensitivity, using uncomplicated equipment with an equivalent time frame. All 43 P. infestans isolates, yielded positive detection results using LAMP assay showing no cross reaction with other Phytophthora spp., oomycetes or fungal pathogens. The LAMP assay yielded the lowest detectable DNA concentration (1.28 × 10-4 ng μL-1), being 10 times more sensitive than nested PCR (1.28 × 10-3 ng μL-1), 100 times more sensitive than real-time PCR (1.28 × 10-2 ng μL-1) and 103 times more sensitive than the conventional PCR assay (1.28 × 10-1 ng μL-1). In the field experiment, the LAMP assay outperformed the other tests by amplifying only diseased tissues (leaf and stem), and showing no positive reaction in healthy tissues. Overall, the LAMP assay developed in this study provides a specific, sensitive, simple, and effective visual method for detection of the P. infestans pathogen, and is therefore suitable for application in early prediction of the disease to reduce the risk of epidemics.

Hussain S, Lees A K, Duncan J M , et al.

Development of a species-specific and sensitive detection assay for Phytophthora infestans and its application for monitoring of inoculum in tubers and soil

Plant Pathology, 2005,54(3):373-382.

DOI:10.1111/ppa.2005.54.issue-3      URL     [本文引用: 2]

Ammour M S, Bilodeau G J, Tremblay D M , et al.

Development of real-time isothermal amplification assays for on-site detection of Phytophthora infestans in potato leaves

Plant Disease, 2017,101(7):1269-1277.

DOI:10.1094/PDIS-12-16-1780-RE      URL     PMID:30682973      [本文引用: 3]

Real-time loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays were developed targeting the internal transcribed spacer 2 region of the ribosomal DNA of Phytophthora infestans, the potato late blight causal agent. A rapid crude plant extract (CPE) preparation method from infected potato leaves was developed for on-site testing. The assay's specificity was tested using several species of Phytophthora and other potato fungal and oomycete pathogens. Both LAMP and RPA assays showed specificity to P. infestans but also to the closely related species P. andina, P. mirabilis, P. phaseoli, and P. ipomoeae, although the latter are not reported as potato pathogen species. No cross-reaction occurred with P. capsici or with the potato pathogens tested, including P. nicotianae and P. erythroseptica. The sensitivity was determined using P. infestans pure genomic DNA added into healthy CPE samples. Both LAMP and RPA assays detected DNA at 50 fg/μl and were insensitive to CPE inhibition. The isothermal assays were tested with artificially inoculated and naturally infected potato plants using a Smart-DART platform. The LAMP assay effectively detected P. infestans in symptomless potato leaves as soon as 24 h postinoculation. A rapid and accurate on-site detection of P. infestans in plant material using the LAMP assay will contribute to improved late blight diagnosis and early detection of infections and facilitate prompt management decisions.

Notomi T, Okayama H, Masubuchi H , et al.

Loop-mediated isothermal amplification of DNA

Nucleic Acids Research, 2000,28(12):e63.

DOI:10.1093/nar/28.12.e63      URL     PMID:10871386      [本文引用: 1]

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.

Nagamine K, Watanabe K, Ohtsuka K , et al.

Loop-mediated isothermal amplification reaction using a nondenatured template

Clinical Chemistry, 2001,47(9):1742-1743.

URL     PMID:11514425      [本文引用: 1]

Martin F N, Abad Z G, Balci Y , et al.

Identification and detection of Phytophthora:reviewing our progress,identifying our needs

Plant Disease, 2012,96(8):1080-1103.

DOI:10.1094/PDIS-12-11-1036-FE      URL     PMID:30727075      [本文引用: 1]

With the increased attention given to the genus Phytophthora in the last decade in response to the ecological and economic impact of several invasive species (such as P. ramorum, P. kernoviae, and P. alni), there has been a significant increase in the number of described species. In part, this is due to the extensive surveys in historically underexplored ecosystems (e.g., forest and stream ecosystems) undertaken to determine the spread of invasive species and the involvement of Phytophthora species in forest decline worldwide (e.g., oak decline). The past decade has seen an approximate doubling in the number of described species within the genus Phytophthora, and the number will likely continue to increase as more surveys are completed and greater attention is devoted to clarifying phylogenetic relationships and delineating boundaries in species complexes. The development of molecular resources, the availability of credible sequence databases to simplify identification of new species, and the sequencing of several genomes have provided a solid framework to gain a better understanding of the biology, diversity, and taxonomic relationships within the genus. This information is much needed considering the impact invasive or exotic Phytophthora species have had on natural ecosystems and the regulatory issues associated with their management. While this work is improving our ability to identify species based on phylogenetic grouping, it has also revealed that the genus has a much greater diversity than previously appreciated.

Tomlinson J A, Barker I, Boonham N .

Faster,simpler,more-specific methods for improved molecular detection of Phytophthora ramorum in the field

Applied and Environmental Microbiology, 2007,73(12):4040-4047.

DOI:10.1128/AEM.00161-07      URL     PMID:17449689      [本文引用: 1]

Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.

Hansen Z R, Knaus B J, Tabima J F , et al.

Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen,Phytophthora infestans

Journal of Applied Microbiology, 2016,120(4):1010-1020.

DOI:10.1111/jam.13079      URL     PMID:26820117      [本文引用: 2]

To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA.

Chen Y, Roxby R .

Characterization of a Phytophthora infestans gene involved in vesicle transport

Gene, 1996,181(1/2):89-94.

DOI:10.1016/s0378-1119(96)00469-6      URL     PMID:8973313      [本文引用: 1]

Members of the Ras superfamily of monomeric GTP-binding proteins have been shown to be essential in specific steps of vesicle transport and secretion in widely divergent organisms. We report here the characterization of a gene from Phytophthora infestans encoding a deduced amino acid (aa) sequence belonging to the Ypt class of monomeric GTP-binding proteins, products shown in other organisms to be essential for vesicle transport between the endoplasmic reticulum and the cis-Golgi compartments. Analysis of genomic and cDNA sequences of this gene, Piypt1, indicates that it contains five introns, one in the 5'-untranslated region. All introns are typical in beginning with GT and ending with AG. The region of the transcription start point displays a number of features characteristic of fungi and other eukaryotes, but it does not contain TATA or CAAT motifs. A single transcript is produced from the gene, which is polyadenylated, but the gene does not contain a recognizable polyadenylation signal. Genomic DNA blots indicate that Piypt1 is a single-copy gene. Comparisons of Ypt1 aa sequences indicate that P. infestans is more closely related to algae and higher plants than to the true fungi. The protein product of the Piypt1 gene, expressed in Escherichia coli, cross-reacts with antiserum against yeast Ypt1 protein and binds GTP. Furthermore, the Piypt1 gene is able to functionally complement a mutant ypt1 gene in Saccharomyces cerevisiae. The aa sequence similarity, immunological cross-reactivity and functional attributes of Piypt1 make it likely that it is an authentic ypt1 gene which participates in vesicle transport in Phytophthora infestans.

Caten C E, Jinks J L .

Spontaneous variability of single isolates of Phytophthora infestans. I. cultural variation

Canadian Journal of Botany, 1968,46:329-348.

DOI:10.1139/b68-055      URL     [本文引用: 1]

Álvarez I, Wendel J F .

Ribosomal ITS sequences and plant phylogenetic inference

Molecular Phylogenetics and Evolution, 2003,29(3):417-434.

DOI:10.1016/s1055-7903(03)00208-2      URL     PMID:14615184      [本文引用: 2]

One of the most popular sequences for phylogenetic inference at the generic and infrageneric levels in plants is the internal transcribed spacer (ITS) region of the 18S-5.8S-26S nuclear ribosomal cistron. The prominence of this source of nuclear DNA sequence data is underscored by a survey of phylogenetic publications involving comparisons at the genus level or below, which reveals that of 244 papers published over the last five years, 66% included ITS sequence data. Perhaps even more striking is the fact that 34% of all published phylogenetic hypothesis have been based exclusively on ITS sequences. Notwithstanding the many important contributions of ITS sequence data to phylogenetic understanding and knowledge of genome relationships, a number of molecular genetic processes impact ITS sequences in ways that may mislead phylogenetic inference. These molecular genetic processes are reviewed here, drawing attention to both underlying mechanism and phylogenetic implications. Among the most prevalent complications for phylogenetic inference is the existence in many plant genomes of extensive sequence variation, arising from ancient or recent array duplication events, genomic harboring of pseudogenes in various states of decay, and/or incomplete intra- or inter-array homogenization. These phenomena separately and collectively create a network of paralogous sequence relationships potentially confounding accurate phylogenetic reconstruction. Homoplasy is shown to be higher in ITS than in other DNA sequence data sets, most likely because of orthology/paralogy conflation, compensatory base changes, problems in alignment due to indel accumulation, sequencing errors, or some combination of these phenomena. Despite the near-universal usage of ITS sequence data in plant phylogenetic studies, its complex and unpredictable evolutionary behavior reduce its utility for phylogenetic analysis. It is suggested that more robust insights are likely to emerge from the use of single-copy or low-copy nuclear genes.

Lane D J .

Nucleic acid techniques in bacterial systematics

New York:John Wiley and Sons, 1991.

DOI:10.3760/cma.j.issn.0253-9624.2019.11.011      URL     PMID:31683400      [本文引用: 1]

Objective: To understand the situation and genotype distribution of spotted fever group rickettsia (SFGR) in the border area of Tumen River Basin in free ticks in Yanbian Korean Autonomous Prefecture (Yanbian Prefecture), Jilin Province. Methods: From April to September, 2017, ticks were collected using flagging method from Hunchun, Tumen, Helong and Longjing cities in the Tumen River basin of Yanbian Prefecture. Outer membrane protein A (ompA) was detected by Polymerase Chain Reaction (PCR), then, the species were identified by gene sequencing and analyzed systematically. The positive rate of pools and MIR(minimum infection rate per 100 ticks,MIR) of SFGR were calculated, and the difference of positive rate of pools among ticks with different characteristics was compared by Chi-square test. Results: A total of 3 079 ticks were collected and divided into 536 pools. The positive rate of pools of SFGR nucleic acid was 39.7% (213 pools). The MIR of SFGR was 6.9%.The positive rate of pools of SFGR in Dermacentor silvarum, Haemaphysalis concinna, Haemaphysalis japonica, Haemaphysalis longicornis and Ixodes persulcatus were 80.4% (41/51), 14.0% (25/179), 20.2% (18/89), 78.9% (101/128) and 25.9% (21/81), and the difference was statistically significant (P&lt;0.001). There was statistical difference in the positive rate of pools of SFGR in developmental stages of ticks (P&lt;0.001); the positive rate of pools of female adults, male adults, nymph and larvae were 36.4% (95/261), 34.2% (67/196), 56.3% (40/71) and 7/8, and the MIR was 7.9%, 7.7%, 4.9% and 3.5%. The five genotype was detected which was Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis, Candidatus Rickettsia tarasevichiae,Rickettsia monacensis and have 98%-100% homology with known gene sequences. Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis and Candidatus Rickettsia tarasevichiae showed close evolutionary relationship with known specie (have 98%-100% homology with known gene sequences); Rickettsia monacensis showed Far from evolutionary relationship with known species (have 98% homology with known gene sequences). Conclusion: SFGR infection of ticks is common in the border areas of the Tumen River Basin. There was high diversity in SFGR species and tick species in the areas surveyed.

魏景超 . 真菌鉴定手册. 上海: 上海科学技术出版社, 1979.

[本文引用: 2]

Boone D R, Castenholz R W .

Bergey′s manual of systematic bacteriology

New York:Springer, 2001.

Kroon L P, Brouwer H, de Cock A W ,et al.

The genus Phytophthora anno 2012

Phytopathology, 2012,102(4):348.

DOI:10.1094/PHYTO-01-11-0025      URL     [本文引用: 2]

Plant diseases caused by Phytophthora species will remain an ever increasing threat to agriculture and natural ecosystems. Phytophthora literally means plant destroyer, a name coined in the 19th century by Anton de Bary when he investigated the potato disease that set the stage for the Great Irish Famine. Phytophthora infestans, the causal agent of potato late blight, was the first species in a genus that at present has over 100 recognized members. In the last decade, the number of recognized Phytophthora species has nearly doubled and new species are added almost on a monthly basis. Here we present an overview of the 10 clades that are currently distinguished within the genus Phytophthora with special emphasis on new species that have been described since 1996 when Erwin and Ribeiro published the valuable monograph 'Phytophthora diseases worldwide' (35).

Blancomeneses M, Ristaino J B .

Detection and quantification of Peronospora tabacina using a real-time polymerase chain reaction assay

Plant Disease, 2011,95(6):673-682.

DOI:10.1094/PDIS-05-10-0333      URL     [本文引用: 1]

Peronospora tabacina is an obligate plant pathogen that causes blue mold of tobacco. The disease is difficult to diagnose before the appearance of symptoms and can be easily spread in nonsymptomatic tobacco seedlings. We developed a real-time polymerase chain reaction (PCR) assay for P. tabacina that uses 5' fluorogenic exonuclease (TaqMan) chemistry to detect and quantify pathogen DNA from diseased tissue. The primers and probe were designed using 5.8S ribosomal DNA sequences from 12 fungal and oomycete tobacco pathogens and 24 Peronospora spp. The PtabBM Taq Man assay was optimized and performed with a final concentration of 450 nM primers and 125 nM probe. The real-time Taq Man assay was assessed for sensitivity and the lower detection limit was 1 fg of DNA. The assay was specific for P tabacina. None of the DNA from other tobacco pathogens, nonpathogens, or the host were amplified. The PtabBM TaqMan assay was useful for detection of P tabacina in field samples, artificially inoculated leaves, roots, and systemically infected tobacco seedlings. The assay was used to quantify host resistance and it was possible to detect the pathogen 4 days postinoculation in both medium-resistant and susceptible tobacco cultivars. The real-time PCR assay for P. tabacina will be a valuable tool for the detection of the pathogen and of use to regulatory agencies interested in preventing the spread of blue mold.

Diguta C F, Rousseaux S, Weidmann S , et al.

Development of a qPCR assay for specific quantification of Botrytis cinereal on grapes

Fems Microbiology Letters, 2010,313(1):81-87.

DOI:10.1111/j.1574-6968.2010.02127.x      URL     PMID:20946385      [本文引用: 1]

The aim of this study was to develop a system for rapid and accurate real-time quantitative PCR (qPCR) identification and quantification of Botrytis cinerea, one of the major pathogens present on grapes. The intergenic spacer (IGS) region of the nuclear ribosomal DNA was used to specifically detect and quantify B. cinerea. A standard curve was established to quantify this fungus. The qPCR reaction was based on the simultaneous detection of a specific IGS sequence and also contained an internal amplification control to compensate for variations in DNA extraction and the various compounds from grapes that inhibit PCR. In these conditions, the assay had high efficiency (97%), and the limit of detection was estimated to be 6.3 pg DNA (corresponding to 540 spores). Our method was applied to assess the effects of various treatment strategies against Botrytis in the vineyard. Our qPCR assay proved to be rapid, selective and sensitive and may be used to monitor Botrytis infection in vineyards.

Spring O, Marco T, Wolf S , et al.

PCR-based detection of sunflower white blister rust (Pustula helianthicola C. Rost and Thines) in soil samples and asymptomatic host tissue

Netherlands Journal of Plant Pathology, 2011,131(3):519-527.

[本文引用: 1]

Eshraghi L, Anderson J, Aryamanesh N , et al.

Phosphite primed defence responses and enhanced expression of defence genes in Arabidopsis thaliana infected with Phytophthora cinnamomic

Plant Pathology, 2011,60(6):1086-1095.

DOI:10.1111/j.1365-3059.2011.02471.x      URL     [本文引用: 1]

This paper describes the effect of phosphite (Phi), a systemic chemical, on the induction of defence responses in Phytophthora cinnamomi-infected Arabidopsis thaliana accessions Ler and Col-0. Application of Phi to non-inoculated A. thaliana seedlings of accession Ler elevated transcription of defence genes in the salicylic acid (PR1 and PR5) and jasmonic acid/ethylene (THI2.1 and PDF1.2) pathways. Furthermore, a systemic increase in the expression of the PR1 gene was demonstrated in Phi-treated seedlings using the transgenic line PR1::GUS in the presence/absence of the pathogen by 72 h after inoculation. The cells of Phi-treated A. thaliana Ler leaves responded to P. cinnamomi zoospore inoculation with a rapid increase in callose deposition and hydrogen peroxide (H(2)O(2)) production. Phi treatment resulted in the production of callose papillae 6 h earlier than in non-Phi-treated inoculated seedlings and enhanced the production of H(2)O(2) in the leaves of A. thaliana at the site of hyphal penetration and in cells away from the inoculation point. By 24 h after infection, clear differences in the amount of H(2O)2 production were observed between the Phi-treated and non-Phi-treated plants. These rapid host responses did not occur in non-Phi-treated inoculated seedlings. There was also a significant (P < 0.001) decrease in lesion size in Phi-treated plants. These results indicate that Phi primes the plant for a rapid and intense response to infection involving heightened activation of a range of defence responses.

Lees A K, Sullivan L, Lynott J S , et al.

Development of a quantitative real-time PCR assay for Phytophthora infestans and its applicability to leaf,tuber and soil samples

Plant Pathology, 2012,61(5):867-876.

DOI:10.1111/j.1365-3059.2011.02574.x      URL     [本文引用: 1]

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Phytophthora infestans, the cause of foliar and tuber late blight in potato. A primer pair (PinfTQF/PinfTQR) and a fluorogenic probe (PinfTQPR) were designed to perform a quantitative assay for the detection of P. infestans in leaves, tubers and soils. The assay was shown to be specific to P. infestans and the very closely taxonomically related non-potato pathogen species P. mirabilis, P. phaseoli and P. ipomoea, but did not detect the potato pathogens P. erythroseptica and P. nicotianae. The assay was able to reliably detect P. infestans DNA at 100 fg per reaction and was effective in quantifying P. infestans in infected leaf tissue from 24 h after inoculation and also in infected symptomless tubers and diseased tubers. Attempts to detect oospores of P. infestans in naturally and artificially infested soil samples are described and compared with baiting tests and previous literature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores themselves. However, the assay was shown to detect even very low levels of asexual inoculum (sporangia and mycelium) in soil. This work assembles all the necessary features of a quantitative P. infestans assay, which have previously been somewhat disparate: the sensitivity, specificity and quantitation are fully validated, the assay is shown to work in common applications in leaf and tuber tissue and the problems with P. infestans oospore detection are explored and tested experimentally.

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