基于线粒体DNA-COI序列的禾谷孢囊线虫和菲利普孢囊线虫双重PCR检测

doi:10.3864/j.issn.0578-1752.2016.08.007

摘要/Abstract

摘要： 【目的】设计禾谷孢囊线虫（Heterodera avenae）和菲利普孢囊线虫（Heterodera filipjevi）特异性引物，建立同步快速检测这两种线虫的双重PCR体系，为中国小麦孢囊线虫（cereal cyst nematodes，CCN）的田间快速诊断与综合治理提供技术支持。【方法】通过比对分析10种植物寄生线虫24个群体的线粒体DNA（mtDNA）COI序列，分别设计并筛选禾谷孢囊线虫和菲利普孢囊线虫的上游特异性引物HaF8和HfF9，以及下游通用引物HafR8；通过引物浓度比和退火温度的优化，建立针对这两种CCN的双重PCR检测体系；利用该体系对中国黄淮麦区部分CCN样品进行种类鉴定。【结果】筛选并获得的特异性引物HaF8/HafR8及HfF9/HafR8，可在一步PCR反应中实现对禾谷孢囊线虫和菲利普孢囊线虫的特异性检测。引物HaF8/HafR8对禾谷孢囊线虫的特异性扩增产物为200 bp，而HfF9/HafR8对菲利普孢囊线虫的特异性扩增产物为320 bp，条带区分较明显。对双重PCR检测体系的优化发现，引物HaF8、HfF9、HafR8的退火温度为58℃时，该检测体系具有较高的特异性和扩增效率；引物HaF8﹕HfF9﹕HafR8浓度比为0.24﹕0.16﹕0.4 μ·L^-1时，该检测体系能从不同供试线虫中同时特异性地检测出禾谷孢囊线虫和菲利普孢囊线虫，且扩增效率较高。该检测体系对禾谷孢囊线虫和菲利普孢囊线虫单孢囊的检测灵敏度为1/2 000 000个孢囊，而对2龄幼虫的检测灵敏度则分别为1/640条线虫和1/1 280条线虫。对采集自中国黄淮麦区CCN病田的14份土样进行双重PCR检测，发现有8个样本只扩增出200 bp的条带，表明这些样本的CCN种类为禾谷孢囊线虫，有4个样本只扩增出320 bp的条带，表明CCN种类为菲利普孢囊线虫，2个样本同时扩增出200和320 bp两个条带，表明样品为禾谷孢囊线虫和菲利普孢囊线虫的混合发生。以上结果表明，该检测体系可以成功用于两种小麦孢囊线虫田间复合侵染情况的快速诊断。【结论】基于mtDNA-COI序列开发的特异性引物以及建立的双重PCR检测体系，可用于禾谷孢囊线虫和菲利普孢囊线虫田间单一和混合发生样本的同步快速检测。

关键词: 禾谷孢囊线虫, 菲利普孢囊线虫, 双重PCR, COI, 特异性

Abstract: 【Objective】 The specific primers for the duplex-PCR reaction of Heterodera avenae and H. filipjevi were designed and a synchronous detection system was established for these two species. The work will provide technical supports for rapid and accurate diagnosis of cereal cyst nematodes (CCN) in wheat field of China and benefit to the integrate management strategies.【Method】The forward primers HaF8 and HfF9 respectively specific to H. avenae and H. filipjevi together with the common reverse primer HafR8 were designed according to the comparisons of mtDNA-COI sequences from 24 populations of 10 plant parasitic nematode species. The concentrations of primers and the annealing temperatures for duplex-PCR reaction were optimized and a high efficient detection system for these two CCN species was developed. The established system was applied to identify the CCN species of samples collected from wheat fields in Huanghuai region of China. 【Result】 The specific primers HaF8/HafR8 and HfF9/HafR8 obtained in this study could rapidly detect H. avenae and H. filipjevi in one PCR reaction with high specificity. The PCR product for H. avenae amplified with primers HaF8/HafR8 was 200 bp, for H. filipjevi with primers HfF9/HafR8 was 320 bp. The band sizes were easily distinguished. The optimization showed that the duplex-PCR detection system had a high specificity and amplification efficiency with the annealing temperature of primers HaF8/HfF9/HafR8 at 58℃. In addition, the detection system could identify H. avenae and H. filipjevi simultaneously from all detected samples with a high efficiency, when the concentration of HaF8﹕HfF9﹕HafR8 was 0.24﹕0.16﹕0.4 μmol·L^-1. The sensitivities of the duplex-PCR system detection for single cyst of H. avenae and H. filipjevi were both 1/2 000 000, but for single second stage juvenile were 1/640 and 1/1 280, respectively. The duplex-PCR system was used to identify the species of 14 CCN samples collected from wheat fields in Huanghuai region of China. The results showed that the 200 bp band was amplified from 8 samples, indicating the species is H. avenae. The 320 bp band was amplified from 4 samples, indicating the species is H. filipjevi. However, both the band 200 and 320 bp were only amplified from 2 samples assuming a mixture of H. avenae and H. filipjevi occurred. The results demonstrated that the duplex-PCR detection system could be successfully applied in rapid diagnosis of the complex infection situation of CCN in fields. 【Conclusion】 The established duplex-PCR system with the specific primers designed on basis of mtDNA-COI sequences is a reliable and simple technique for simultaneously rapid detecting H. avenae and H. filipjevi in field samples on wheat.

Key words: Heterodera avenae, Heterodera filipjevi, duplex-PCR, COI, specificity

牛雯雯，王暄，李红梅，鞠玉亮，万文文. 基于线粒体DNA-COI序列的禾谷孢囊线虫和菲利普孢囊线虫双重PCR检测[J]. 中国农业科学, 2016, 49(8): 1499-1509.

NIU Wen-wen, WANG Xuan, LI Hong-mei, JU Yu-liang, WAN Wen-wen. Duplex-PCR Detection for Heterodera avenae and Heterodera filipjevi Based on mtDNA-COI Sequences[J]. Scientia Agricultura Sinica, 2016, 49(8): 1499-1509.

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参考文献

[1] Subbotin S A, Sturhan D, Rumpenhorst H J, Moens M. Molecular and morphological characterization of the Heterodera avenae species complex (Tylenchida: Heteroderidae). Nematology, 2003, 5(4): 515-538.

[2] Nicol J M, Rivoal R, Taylor S, Zaharieva M. Global importance of cyst (Heterodera spp.) and lesion nematodes (Pratylenchus spp.) on cereals: yield loss, population dynamics, use of host resistance and integration of molecular tools. Nematology Monographs and Perspectives, 2004, 2: 1-19.

[3] Rivoal R, Nicol J M. Past research on the cereal cyst nematode complex and future needs//Riley I T, Nicol J M, Dababat A A, eds. Cereal Cyst Nematodes: Status, Research and Outlook. Ankara, Turkey: CIMMYT Press, 2009: 3-10.

[4] 陈品三, 王明祖, 彭德良. 我国小麦禾谷孢囊线虫 (Heterodera avenae Wollenweber) 的发现与鉴定初报. 中国农业科学, 1991, 24(5): 89-91.

Chen P S, Wang M Z, Peng D L. Preliminary report for identification of Heterodera avenae Wollenweber on wheat in China. Scientia Agricultura Sinica, 1991, 24(5): 89-91. (in Chinese)

[5] 李惠霞, 柳永娥, 魏庄, 李敏权. 新疆和西藏发现禾谷孢囊线虫. 中国线虫学研究, 2012, 4: 164-165.

Li H X, Liu Y E, Wei Z, Li M Q. The detection of Heterodera avenae from the cereal field in autonomous regions of Tibet and Xinjiang. Nematology Research in China, 2012, 4: 164-165. (in Chinese)

[6] 王振跃, 李洪连, 袁虹霞. 小麦孢囊线虫病的发生危害与防治对策. 河南农业科学, 2005(12): 54-55.

Wang Z Y, Li H L, Yuan H X. Occurrence and damage of wheat cyst nematode disease and its control countermeasures. Journal of Henan Agricultural Sciences, 2005(12): 54-55. (in Chinese)

[7] 侯生英, 王爱玲, 张贵, 黄丽丽. 小麦禾谷孢囊线虫病危害损失研究. 农学学报, 2012, 2(8): 33-36.

Hou S Y, Wang A L, Zhang G, Huang L L. Effect of cereal cyst nematode on agronomic characteristics and yield losses of spring wheat. Journal of Agriculture, 2012, 2(8): 33-36. (in Chinese)

[8] Peng D L, Ye W X, Peng H, Gu X C. First report of the cyst nematode (Heterodera filipjevi) on wheat in Henan Province, China. Plant Disease, 2010, 94(10): 1262.

[9] McDonald A H, Nicol J M. Nematode parasites of cereals//Luc M, Sikora R A, Bridge J. Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. Wallingford, UK: CABI Publishing, 2005: 131-191.

[10] 邢小萍, 袁虹霞, 孙君伟, 张洁, 孙炳剑, 李洪连. 河南省小麦主推品种对2种禾谷孢囊线虫的抗性及其评价方法. 作物学报, 2014, 40(5): 805-815.

Xing X P, Yuan H X, Sun J W, Zhang J, Sun B J, Li H L. Resistance to two species of cereal cyst nematode and evaluation methods in major wheat cultivars from Henan Province, China. Acta Agronomica Sinica, 2014, 40(5): 805-815. (in Chinese)

[11] Abidou H, Valette S, Gauthier J P, Rivoal R, El-Ahmed A, Yahyaoui A. Molecular polymorphism and morphometrics of species of the Heterodera avenae group in Syria and Turkey. Journal of Nematology, 2005, 37(2): 146-154.

[12] Yan G P, Smiley R W. Distinguishing Heterodera filipjevi and H. avenae using polymerase chain reaction-restriction fragment length polymorphism and cyst morphology. Phytopathology, 2010, 100(3): 216-224.

[13] Subbotin S A, Vierstraete A, De Ley P, Rowe J, Waeyenberge L, Moens M, Vanfleteren J R. Phylogenetic relationships within the cyst-forming nematodes (Nematoda: Heteroderidae) based on analysis of sequences from the ITS regions of ribosomal DNA. Molecular Phylogenetics and Evolution, 2001, 21(1): 1-16.

[14] Rivoal R, Valette S, Bekal S, Gauthier J P, Yahyaoui A. Genetic and phenotypic diversity in the graminaceous cyst nematode complex, inferred from PCR-RFLP of ribosomal DNA and morphometric analysis. European Journal of Plant Pathology, 2003, 109: 227-241.

[15] 亓晓莉, 彭德良, 彭焕, 龙海波, 黄文坤, 贺文婷. 基于SCAR标记的小麦禾谷孢囊线虫快速分子检测技术. 中国农业科学, 2012, 45(21): 4388-4395.

Qi X L, Peng D L, Peng H, Long H B, Huang W K, He W T. Rapid molecular diagnosis based on scar marker system for cereal cyst nematode. Scientia Agricultura Sinica, 2012, 45(21): 4388-4395. (in Chinese)

[16] Peng H, Qi X L, Peng D L, Long H B, He X F, Huang W K, He W T. Sensitive and direct detection of Heterodera filipjevi in soil and wheat roots by species-specific SCAR-PCR assays. Plant Disease, 2013, 97(10): 1288-1294.

[17] Yan G P, Smiley R W. Species-specific PCR assays for differentiating Heterodera filipjevi and H. avenae. Plant Disease, 2013, 97(12): 1611-1619.

[18] Toumi F, Waeyenberge L, Viaene N, Dababat A, Nicol J M, Ogbonnaya F, Moens M. Development of two species-specific primer sets to detect the cereal cyst nematodes Heterodera avenae and Heterodera filipjevi. European Journal of Plant Pathology, 2013, 136(3): 613-624.

[19] 彭德良, 徐小琴, 彭焕, 亓晓莉, 黄文坤, 贺文婷, 姜道宏. 一种禾谷孢囊线虫LAMP快速检测方法及应用. 中国发明专利, ZL201310020383.4[P]. 2014-04-09.

Peng D L, Xu X Q, Peng H, Qi X L, Huang W K, He W T, Jiang D H. A rapid detection of Heterodera avenae by LAMP and its application. China Patent, ZL201310020383.4[P]. 2014-04-09. (in Chinese)

[20] 彭德良, 徐小琴, 彭焕, 黄文坤, 亓晓莉, 姜道宏. 一种菲利普孢囊线虫LAMP快速检测方法及应用. 中国发明专利, ZL201310053055.4[P]. 2014-02-26.

Peng D L, Xu X Q, Peng H, Huang W K, Qi X L, Jiang D H. A rapid detection of Heterodera filipjevi by LAMP and its application. China Patent, ZL201310053055.4[P]. 2014-02-26. (in Chinese)

[21] Subbotin S A, Peng D L, Moens M. A rapid method for the identification of the soybean cyst nematode Heterodera glycines using duplex PCR. Nematology, 2001, 3(4): 365-371.

[22] Amiri S, Subbotin S A, Moens M. Identification of the beet cyst nematode Heterodera schachtii by PCR. European Journal of Plant Pathology, 2002, 108(6): 497-506.

[23] Jiang L Q, Zheng J W, Waeyenberge L, Subbotin S A, Moens M. Duplex PCR based identification of Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) Nickle, 1970. Russian Journal of Nematology, 2005, 13(2): 115-121.

[24] Bae C H, Robbins R T, Szalanski A L. Molecular identification of some Hoplolaimus species from the USA based on duplex PCR, multiplex PCR and PCR-RFLP analysis. Nematology, 2009, 11(3): 471-480.

[25] Waeyenberge L, Viaene N, Moens M. Species-specific duplex PCR for the detection of Pratylenchus penetrans. Nematology, 2009, 11(6): 847-857.

[26] 宋志强, 王暄, 林宇, 迟元凯, 鞠玉亮, 李红梅. 土壤中南方根结线虫的实时荧光PCR检测和定量. 植物保护学报, 2013, 40(3): 255-260.

Song Z Q, Wang X, Lin Y, Chi Y K, Ju Y L, Li H M. Detection and quantification of Meloidogyne incognita in soil sample using real-time PCR. Acta Phytophylacica Sinica, 2013, 40(3): 255-260. (in Chinese)

[27] 王婷婷, 王暄, 王金成, 李鸿, 李红梅. 食线虫真菌蠕虫埃斯特菌对植物寄生线虫的侵染特性. 植物保护学报, 2014, 41(5): 540-546.

Wang T T, Wang X, Wang J C, Li H, Li H M. Infection characteristics of nematophagous fungus Esteya vermicola to plant parasitic nematodes. Acta Phytophylacica Sinica, 2014, 41(5): 540-546. (in Chinese)

[28] 王暄, 梁中伟, 裴世安, 乐秀虎, 李红梅, 彭德良. 江苏省小麦禾谷孢囊线虫群体核糖体DNA-ITS区序列分析. 南京农业大学学报, 2010, 33(6): 55-62.

Wang X, Liang Z W, Pei S A, Le X H, Li H M, Peng D L. Sequences analysis of rDNA-ITS region of cereal cyst nematodes on wheat from Jiangsu Province. Journal of Nanjing Agricultural University, 2010, 33(6): 55-62. (in Chinese)

[29] Derycke S, Remerie T, Vierstraete A, Backeljau T, Vanfleteren J, Vincx M, Moens T. Mitochondrial DNA variation and cryptic speciation within the free-living marine nematode Pellioditis marina. Marine Ecology Progress Series, 2005, 300: 91-103.

[30] 李洪连, 张煜, 袁虹霞, 孙炳剑, 付博. 用于同时检测燕麦孢囊线虫和菲利普孢囊线虫的特异引物序列、探针序列及其方法. 中国发明专利, ZL201110408449.8[P]. 2013-10-09.

Li H L, Zhang Y, Yuan H X, Sun B J, Fu B. Specific primer sequence and probe sequence for simultaneous detection on Heterodera avenae and Heterodera filipjevi, and detection method with specific primer sequence and probe sequence. China Patent, ZL201110408449.8[P]. 2013-10-09. (in Chinese)

[31] Zheng J W, Subbotin S A, Waeyenberge L, Moens M. Molecular characterisation of Chinese Heterodera glycines and H. avenae populations based on RFLPs and sequences of rDNA-ITS regions. Russian Journal of Nematology, 2000, 8(2): 109-113.

[32] Hajibabaei M, Singer G A C, Hebert P D N, Hickey D A. DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. Trends in Genetics, 2007, 23(4): 167-172.

[33] Bhadury P, Austen M C, Bilton D T, Lambshead P J D, Rogers A D, Smerdon G R. Development and evaluation of a DNA-barcoding approach for the rapid identification of nematodes. Marine Ecology Progress Series,2006, 320: 1-9.

[34] Derycke S, Vanaverbeke J, Rigaux A, Backeljau T, Moens T. Exploring the use of cytochrome oxidase c subunit I (COI) for DNA barcoding of free-living marine nematodes. PLoS One, 2010, 5(10): e13716.

[35] Kiontke K C, Felix M A, Ailion M, Rockman M V, Braendle C, Pénigault J B, Fitch D H A. A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits. BMC Evolutionary Biology, 2011, 11: 339.