腐烂茎线虫中药材群体ITS单倍型分析

doi:10.1016/j.jia.2022.08.126

Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (6): 1763-1781.DOI: 10.1016/j.jia.2022.08.126

腐烂茎线虫中药材群体ITS单倍型分析

收稿日期:2022-05-15 修回日期:2022-08-27 接受日期:2022-06-13 出版日期:2023-06-20 发布日期:2022-06-13

Diagnosis and characterization of the ribosomal DNA-ITS of potato rot nematode (Ditylenchus destructor) populations from Chinese medicinal herbs

NI Chun-hui¹, HAN Bian¹, LIU Yong-gang², Maria MUNAWAR³, LIU Shi-ming⁴, LI Wen-hao¹, SHI Ming-ming¹, LI Hui-xia^1#, PENG De-liang^4#

¹ College of Plant Protection, Gansu Agricultural University/Biocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Lanzhou 730070, P.R.China
² Institute of Plant Protection, Gansu Academy of Agricultural Sciences, Lanzhou 730070, P.R.China
³ Department of Biological Sciences, University of Lethbridge, Lethbridge 4401, Canada
⁴ State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

Received:2022-05-15 Revised:2022-08-27 Accepted:2022-06-13 Online:2023-06-20 Published:2022-06-13
About author:NI Chun-hui, Mobile: +86-18893816153, E-mail: 2719723977@qq.com; #Correspondence LI Hui-xia, Tel: +86-931-7645441, E-mail: lihx@gsau.edu.cn; PENG De-liang, Tel: +86-10-62815576, E-mail: pengdeliang@caas.cn
Supported by:
This work was supported by the National Natural Science Foundation of China (31760507) and the National Key R&D Program of China (2018YFC1706301).

摘要/Abstract

摘要：

腐烂茎线虫（Ditylenchus destructor）是一种重要的检疫性病原线虫，严重危害甘薯、马铃薯和中药材等根茎类作物。该线虫的种内分化明显，根据ITS-rDNA序列差异，国外的研究将其分为A-G 7个单倍型，但主要集中于马铃薯和甘薯的线虫群体。本研究对腐烂茎线虫中药材群体ITS-rDNA序列及其RNA二级结构进行分析，以明确其单倍型分化，并通过ITS-rDNA与28S D2-D3系统发育关系、ITS-RFLR和ITS特异性引物PCR扩增进一步验证不同群体单倍型的分化。在甘肃、青海、陕西、内蒙和黑龙江等5个省，共采集当归、党参、马铃薯和甘薯的腐烂茎线虫群体43个，其中中药材群体37个。线虫群体的ITS-rDNA序列长度为727 bp-969 bp，长度差异主要表现在ITS1区串联重复序列的数量不同，且串联重复序列在ITS1二级结构中形成了稳定的茎环，即H9螺旋。依据H9螺旋结构的有无及其差异，43个群体的ITS-rDNA序列可划分为10个单倍型。与已知单倍型（A-G）对比分析，发现其中3个单倍型分别与A、B和C单倍型一致，而另外7个单倍型与已知单倍型不同，将其依次命名为H、I、J、K、L、M和N单倍型，这7个新单倍型均来源于中药材。综合本研究和已知单倍型分类体系，腐烂茎线虫中发现A-N 14个单倍型。ITS和28S系统发育分析显示，所有单倍型群体分化为两支：A单倍型为一支，B-N单倍型为另一支。对比ITS和28S系统发育树，发现A单倍型均单独聚为一支，但B-N分支不太一致，且不同单倍型系统发育关系较为混乱。ITS-RFLP和特异性引物PCR结果显示，H和A单倍型酶切图谱和特异性片段长度相同， B和C单倍型特异性片段长度相同，但其它单倍型间存在明显差异。除K单倍型不同群体间有差异外，其它单倍型的群体间无明显差异。本研究发现了腐烂茎线虫中药材群体中存在新的单倍型，并明确了不同单倍型的差异，该结果将推动茎线虫生物学的研究进展，且对中药材腐烂茎线虫病的识别和防治具有指导意义。

Abstract:

The potato rot nematode (Ditylenchus destructor) is a very economically important nematode in agronomic and horticultural plants worldwide. In this study, 43 populations of D. destructor were collected from different hosts across China, including 37 populations from Chinese herbal medicine plants. Obtained sequences of ITS-rDNA and D2–D3 of 28S-rDNA genes of D. destructor were compared and analyzed. Nine types of significant length variations in ITS sequences were observed among all populations. The differences in ITS1 length were mainly caused by the presence of repetitive elements with substantial base substitutions. Reconstructions of ITS1 secondary structures showed that the minisatellites formed a stem structure. Ten haplotypes were observed in all populations based on mutations and variations of helix H9. Among them, 3 known haplotypes (A–C) were found in 7 populations isolated from potato, sweet potato, and Codonopsis pilosula, and 7 unique haplotypes were found in other 36 populations collected from C. pilosula and Angelica sinensis compared with 7 haplotypes (A–G) according to Subbotin’ system. These unique haplotypes were different from haplotypes A–G, and we named them as haplotypes H–N. The present results showed that a total of 14 haplotypes (A–N) of ITS-rDNA have been found in D. destructor. Phylogenetic analyses of ITS-rDNA and D2–D3 showed that all populations of D. destructor were clustered into two major clades: one clade only containing haplotype A from sweet potato and the other containing haplotypes B–N from other plants. For further verification, PCR-ITS-RFLP profiles were conducted on 7 new haplotypes. Collectively, our study suggests that D. destructor populations on Chinese medicinal materials are very different from those on other hosts and this work provides a paradigm for relevant researches.

Key words: Ditylenchus destructor , minisatellites , ITS-RFLP , phylogeny , RNA secondary structure

NI Chun-hui, HAN Bian, LIU Yong-gang, Maria MUNAWAR, LIU Shi-ming, LI Wen-hao, SHI Ming-ming, LI Hui-xia, PENG De-liang. 腐烂茎线虫中药材群体ITS单倍型分析[J]. Journal of Integrative Agriculture, 2023, 22(6): 1763-1781.

NI Chun-hui, HAN Bian, LIU Yong-gang, Maria MUNAWAR, LIU Shi-ming, LI Wen-hao, SHI Ming-ming, LI Hui-xia, PENG De-liang.

Diagnosis and characterization of the ribosomal DNA-ITS of potato rot nematode (Ditylenchus destructor) populations from Chinese medicinal herbs [J]. Journal of Integrative Agriculture, 2023, 22(6): 1763-1781.

参考文献

Den Bakker H C, Gravendeel B, Kuyper T W. 2004. An ITS phylogeny of Leccinum and an analysis of the evolution of minisatellite-like sequences within ITS1. Mycologia, 96, 102–118.

Basson S, Waele D D, Meyer A. 1993. Survival of Ditylenchus destructor in soil, hulls and seeds of groundnut. Fundamental and Applied Nematology, 16, 79–86.

Benson G. 1999. Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Research, 27, 573–580.

Biémont C, Vieira C. 2006. Junk DNA as an evolutionary force. Nature, 443, 521–524.

Bower J E, Cooper R D, Beebe N W. 2009. Internal repetition and intraindividual variation in the rDNA ITS1 of the Anopheles punctulatus group (Diptera: Culicidae): multiple units and rates of turnover. Journal of Molecular Evolution, 68, 66–79.

Chilton N B, Hoste H, Newton L A, Beveridge I, Gasser R B. 1998. Common secondary structures for the second internal transcribed spacer pre-rRNA of two subfamilies of trichostrongylid nematodes. International Journal for Parasitology, 28, 1765–1773.

Coleman A W. 2009. Is there a molecular key to the level of “biological species” in eukaryotes A DNA guide. Molecular Phylogenetics and Evolution, 50, 197–203.

Coleman A W, Preparata R M, Mehrotra B, Mai J C. 1998. Derivation of the secondary structure of the ITS-1 transcript in Volvocales and its taxonomic correlations. Protist, 149, 135–146.

Darty K, Denise A, Ponty Y. 2009. VARNA: Interactive drawing and editing of the RNA secondary structure. Bioinformatics, 25, 1974–1975.

Faulkner L R, Darling H. 1961. Pathological histology, hosts, and culture of the potato rot nematode. Phytopathology, 51, 778–786.

Gernandt D S, Liston A, Piñero D. 2001. Variation in the nrDNA ITS of Pinus subsection Cembroides: Implications for molecular systematic studies of pine species complexes. Molecular Phylogenetics and Evolution, 21, 449–467.

Goldman W E, Goldberg G, Bowman L, Steinmetz D, Schlessinger D. 1983. Mouse rDNA: Sequences and evolutionary analysis of spacer and mature RNA regions. Molecular and Cellular Biology, 3, 1488–1500.

Goodey J. 1962. Taxonomic relatedness in nematology. Annals of Applied Biology, 50, 175–177.

Hall T, Biosciences I, Carlsbad C. 2011. BioEdit: An important software for molecular biology. GERF Bulletin of Bioscience, 2, 60–61.

Hooper D J. 1990. Extraction and processing of plant and soil nematodes. In: Luc M, Sikora R A, Bridge J, eds., Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. Commonwealth Agricultural Bureaux International, Wallingford. pp. 45–68.

Huang J, Qi L J, Wang J C, Li H M, Song S Y, Wang H B, Lin M S. 2009. Morphological and genetic analysis on different populations of Ditylenchus destructor. Acta Phytopathologica Sinica, 39, 125–131. (in Chinese)

Huelsenbeck J P, Ronquist F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17, 754–755.

Jeszke A, Budziszewska M, Dobosz R, Stachowiak A, Protasewicz D, Wieczorek P, Obrępalska-Stęplowska A. 2014. A comparative and phylogenetic study of the Ditylenchus dipsaci, Ditylenchus destructor and Ditylenchus gigas populations occurring in Poland. Journal of Phytopathology, 162, 61–67.

Jeszke A, Dobosz R, Obrępalska-Stęplowska A. 2015. A fast and sensitive method for the simultaneous identification of three important nematode species of the genus Ditylenchus. Pest Management Science, 71, 243–249.

Ji L, Wang J C, Yang X L, Huang G M, Lin M S. 2006. PCR-RFLP patterns for differentiation of three Ditylenchus species. Journal of Nanjing Agricultural University, 29, 39–43. (in Chinese)

Joseph N, Krauskopf E, Vera M, Michot B. 1999. Ribosomal internal transcribed spacer 2 (ITS2) exhibits a common core of secondary structure in vertebrates and yeast. Nucleic Acids Research, 27, 4533–4540.

Li H X, Xu P G, Li J R, Jian J Z, Zhao P, Peng D L. 2016. Identification of the pathogenic nematodes from potatoes in Dingxi of Gansu Province. Acta Phytophylacica Sinica, 43, 580–587. (in Chinese)

Li Y Q, Huang L Q, Jiang R, Han S M, Chnag Q, Li Y M, Chen Z J, Peng H, Huang W K, Guo J M, Li H X, Liu S M, Peng D L. 2022. Molecular characterization of internal transcribed spacer (ITS) of ribosomal RNA gene, haplotypes and pathogenicity of potato rot nematode Ditylenchus destructor in China. Phytopathology Research, 22, 1–12.

Liu B, Mei Y Y, Zheng J W. 2007. Species-specific detection of inter-populations of Ditylenchus destructor. Journal of Zhejiang University (Agriculture and Life Sciences), 33, 490–496. (in Chinese)

Ma H, Overstreet R M, Subbotin S A. 2008. ITS2 secondary structure and phylogeny of cyst-forming nematodes of the genus Heterodera (Tylenchida: Heteroderidae). Organisms Diversity & Evolution, 8, 182–193.

Mahmoudi N, Pakina E N, Limantceva L A, Ivanov A V. 2020. Diagnosis of potato rot nematode Ditylenchus destructor using PCR-RFLP. RUDN Journal of Agronomy and Animal Industries, 15, 353–362.

Mai J C, Coleman A W. 1997. The internal transcribed spacer 2 exhibits a common secondary structure in green algae and flowering plants. Journal of Molecular Evolution, 44, 258–271.

Marek M, Zouhar M, Douda O, Mazakova J, Rysanek P. 2010. Bioinformatics-assisted characterization of the ITS1-5.8S-ITS2 segments of nuclear rRNA gene clusters, and its exploitation in molecular diagnostics of European crop–parasitic nematodes of the genus Ditylenchus. Plant Pathology, 59, 931–943.

Michot B, Joseph N, Mazan S, Bachellerie J. 1999. Evolutionarily conserved structural features in the ITS2 of mammalian pre-rRNAs and potential interactions with the snoRNA U8 detected by comparative analysis of new mouse sequences. Nucleic Acids Research, 27, 2271–2282.

Morgan J A, Blair D. 1998. Trematode and monogenean rRNA ITS2 secondary structures support a four-domain model. Journal of Molecular Evolution, 47, 406–419.

Müller T, Philippi N, Dandekar T, Schultz J, Wolf M. 2007. Distinguishing species. RNA, 13, 1469–1472.

Ni C H, Li H X, Li W H, Liu Y G, Xu X F, Han B. 2021. Comparison of molecular characteristics of the hybrid progenies from different haplotypes of Ditylenchus destructor. Biotechnology Bulletin, 37, 118. (in Chinese)

Ni C H, Zhang S L, Li H X, Liu Y G, Li W H, Xu X F, Xu Z P. 2020. First report of potato rot nematode, Ditylenchus destructor Thorne, 1945 infecting Codonopsis pilosula in Gansu Province, China. Journal of Nematology, 52, 1–2.

Nylander J A A. 2004. MrModeltest V2. Program distributed by the author. Bioinformatics, 24, 581–583.

Ou S Q, Wang Y W, Peng D L, Qiu H, Bai Q R, Shi S S. 2017. Discovery of potato rot nematode, Ditylenchus destructor, infesting potato in Inner Mongolia, China. Plant Disease, 101, 1554.

Paskewitz S M, Wesson D, Collins F. 1994. The internal transcribed spacers of ribosomal DNA in five members of the Anopheles gambiae species complex. Insect Molecular Biology, 2, 247–257.

Qi Y H, Li X H, Ma J, Li M Q, Chen S L. 2008. The infection and dynamics of Ditylenchus destructor in sweet potato. Acta Agricul Boreali-Sinica, 23, 234–237. (in Chinese)

Rambaut A. 2016. FigTree version 1.4.3. [2019-11-30]. http://tree.bio.ed.ac.uk/software/figtree

Von der Schulenburg J H G, Hancock J M, Pagnamenta A, Sloggett J J, Majerus M E, Hurst G D. 2001. Extreme length and length variation in the first ribosomal internal transcribed spacer of ladybird beetles (Coleoptera: Coccinellidae). Molecular Biology and Evolution, 18, 648–660.

Singewar K, Moschner C R, Hartung E, Fladung M. 2020. Species determination and phylogenetic relationships of the genus Betula inferred from multiple chloroplast and nuclear regions reveal the high methyl salicylate-producing ability of the ancestor. Trees, 34, 1131–1146.

Subbotin S A, Deimi A M, Zheng J W, Chizhov V N. 2011. Length variation and repetitive sequences of Internal Transcribed Spacer of ribosomal RNA gene, diagnostics and relationships of populations of potato rot nematode, Ditylenchus destructor Thorne, 1945 (Tylenchida: Anguinidae). Nematology, 13, 773–785.

Subbotin S A, Madani M, Krall E, Sturhan D, Moens M. 2005. Molecular diagnostics, taxonomy, and phylogeny of the stem nematode Ditylenchus dipsaci species complex based on the sequences of the internal transcribed spacer-rDNA. Phytopathology, 95, 1308–1315.

Subbotin S A, Sturhan D, Chizhov V N, Vovlas N, Baldwin J G. 2006. Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences. Nematology, 8, 455–474.

Subbotin S A, Vierstraete A, De Ley P, Rowe J, Waeyenberge L, Moens M, Vanfleteren J R. 2001. 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, 21, 1–16.

Sun P, Clamp J C, Xu D. 2010. Analysis of the secondary structure of ITS transcripts in peritrich ciliates (Ciliophora, Oligohymenophorea): Implications for structural evolution and phylogenetic reconstruction. Molecular Phylogenetics and Evolution, 56, 242–251.

Tarieiev A S, Gailing O, Krutovsky K V. 2021. ITS secondary structure reconstruction to resolve taxonomy and phylogeny of the Betula L. genus. PeerJournal, 9, e10889.

Thorne G. 1945. Ditylenchus destructor n. sp. the potato rot nematode, and Ditylenchus dipsaci (Kühn, 1857) Filipjev, 1936, the teasel nematode (Nematoda: Tylenchidae). Proceedings of the Helminthological Society of Washington, 12, 27–34.

Vovlas N, Troccoli A, Palomares-Rius J E, De Luca F, Cantalapiedra-Navarrete C, Liébanas G, Landa B B, Subbotin S A, Castillo P. 2016. A new stem nematode, Ditylenchus oncogenus n. sp. (Nematoda: Tylenchida), parasitizing sowthistle from Adriatic coast dunes in southern Italy. Journal of Helminthology, 90, 152–165.

Vrain T, Wakarchuk D, Levesque A, Hamilton R. 1992. Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fundamental and Applied Nematology, 15, 563–573.

De Waele D, Jones B, Bolton C, Van den Berg E. 1989. Ditylenchus destructor in hulls and seeds of peanut. Journal of Nematology, 21, 10–15.

Wan F, Peng D L, Yang Y W, He Y Q. 2008. Species specific molecular diagnosis of Ditylenchus destructor populations occurring in China. Acta Phytopathologica Sinica, 38, 263–270. (in Chinese)

Wang H B, Li R, Wei L H, Chen X H, Zhao G D, Lin M S. 2011a. Current situation of population differentiation of Ditylenchus destructor. Acta Agriculturae Jiangxi, 23, 110–112. (in Chinese)

Wang H B, Mao J, Cao K G, Fu Y S, Wu X P. 2019. Construction and verification of biological hybridization system of different genetic background Ditylenchus destructor. Journal of Yangtze University (Natural Science Edition), 16, 56–59. (in Chinese)

Wang H B, Mao J, Li R, Zhao G D, Lin M S. 2011b. The pathogenicity study of hybrids populations of Ditylenchus destructor from different geographical origin. Acta Agriculturae Boreali-Sinica, 26, 212–216. (in Chinese)

Wang H B, Qi L, Wang J, Song S, Lin M. 2009. Research on isozyme phenotypes and pathogenicity of Ditylenchus destructor in sweet potato. Journal of Zhejiang University (Agriculture and Life Sciences), 35, 425–432. (in Chinese)

Wang Y J, Sheng X L, Sun Z, Huo K C, Jing X L, Liu F Z, Miao X C. 1990. Study on hemp mouth disease of angelica sinensis. Acta Phytopathologica Sinica, 20, 13–19. (in Chinese)

Warberg R, Jensen K T, Frydenberg J. 2005. Repetitive sequences in the ITS1 region of ribosomal DNA in congeneric microphallid species (Trematoda: Digenea). Parasitology Research, 97, 420–423.

Wolf M, Achtziger M, Schultz J, Dandekar T, Müller T. 2005. Homology modeling revealed more than 20,000 rRNA internal transcribed spacer 2 (ITS2) secondary structures. RNA, 11, 1616–1623.

Wu L Y. 1960. Comparative study of Ditylenchus destructor Thorne, 1945 (Nematoda: Tylenchidae), from potato, bulbous iris, and dahlia, with a discussion of De Man’s ratios. Canadian Journal of Zoology, 38, 1175–1187.

Xu C, Xie H, Wang J, Wu Y, Zhang C, Jin X. 2009. Morphological and molecular identification of the nematodes parasitizing the roots and the stems of Astragalas membranaceus and Dioscorea opposite from China. Nematologia Mediterranea, 37, 39–44.

Yi G D, Zhang Y M. 1983. Correction of the pathogenic nematode of stem nematode disease in sweet potato. Journal of Shandong University (Natural Science Edition), 4, 122–132. (in Chinese)

Yu H Y, Peng D L, Hu X Q, Huang W K. 2009. Mulecular cloning and sequence analysis of 28S rDNA-D2/D3 regions of Ditylenchus destructor on sweet potato in China. Acta Phytopathologica Sinica, 39, 254–261. (in Chinese)

Zhang S L, Li H X, Xu P G, Liu Y G, Yu H, Liu X. 2019. Isolation and identification of Ditylenchus destructor from Heilongjiang. Acta Phytopathologica Sinica, 49, 756–762. (in Chinese)

Zhang S L, Zhang S S. 2008. PCR and sequence analyse of rDNA-ITS1 region of sweet potato stem nematode. Acta Phytopathologica Sinica, 38, 132–135. (in Chinese)

Zhao H H, Liang C, Zhang Y, Duan F M, Song W W, Shi Q Q, Huang W K, Peng D L. 2021. Research advances of biology in Ditylenchus destructor Thorne, 1945. Biotechnology Bulletin, 37, 45. (in Chinese)

Zuker M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 31, 3406–3415.

腐烂茎线虫中药材群体ITS单倍型分析

Diagnosis and characterization of the ribosomal DNA-ITS of potato rot nematode (Ditylenchus destructor) populations from Chinese medicinal herbs

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