Please wait a minute...
Journal of Integrative Agriculture  2023, Vol. 22 Issue (6): 1763-1781    DOI: 10.1016/j.jia.2022.08.126
Plant Protection Advanced Online Publication | Current Issue | Archive | Adv Search |

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-xia1#, PENG De-liang4#

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

Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      

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,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单倍型分化,并通过ITS-rDNA28S D2-D3系统发育关系、ITS-RFLRITS特异性引物PCR扩增进一步验证不同群体单倍型分化。甘肃、青海、陕西、内蒙和黑龙江等5个省采集当归、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。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。H9螺旋结构的有无及其差异,43群体的ITS-rDNA序列可划分10个单倍型与已知单倍型(A-G)对比分析,发现其中3单倍型分别AB和C单倍型一致,而另外7单倍型与已知单倍型不同,将其依次命名为HIJKLMN单倍型,7单倍来源于中药材。综合本研究和已知单倍型分体系,腐烂茎线虫中发现A-N 14个单倍型。ITS28S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单倍型为一支。对比ITS28S系统发育,发现A单倍型均单独聚为一支,但B-N分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,其单倍型群体间明显差异。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的识别和防治具有指导意义。


The potato rot nematode (Ditylenchus destructor) is a very economically important nematode in agronomic and horticultural plants worldwide.  In this study, 43 populations of Ddestructor 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 Ddestructor 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 Cpilosula 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 Ddestructor.  Phylogenetic analyses of ITS-rDNA and D2–D3 showed that all populations of Ddestructor 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 Ddestructor populations on Chinese medicinal materials are very different from those on other hosts and this work provides a paradigm for relevant researches.

Keywords:  Ditylenchus destructor        minisatellites        ITS-RFLP        phylogeny        RNA secondary structure  
Received: 15 May 2022   Online: 27 August 2022   Accepted: 13 June 2022
Fund: This work was supported by the National Natural Science Foundation of China (31760507) and the National Key R&D Program of China (2018YFC1706301).
About author:  NI Chun-hui, Mobile: +86-18893816153, E-mail:; #Correspondence LI Hui-xia, Tel: +86-931-7645441, E-mail:; PENG De-liang, Tel: +86-10-62815576, E-mail:

Cite this article: 

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. 2023.

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

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. Mycologia96, 102–118.

Basson S, Waele D D, Meyer A. 1993. Survival of Ditylenchus destructor in soil, hulls and seeds of groundnut. Fundamental and Applied Nematology16, 79–86.

Benson G. 1999. Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Research27, 573–580.

Biémont C, Vieira C. 2006. Junk DNA as an evolutionary force. Nature443, 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 Evolution68, 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 Parasitology28, 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 Evolution50, 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. Protist149, 135–146.

Darty K, Denise A, Ponty Y. 2009. VARNA: Interactive drawing and editing of the RNA secondary structure. Bioinformatics25, 1974–1975.

Faulkner L R, Darling H. 1961. Pathological histology, hosts, and culture of the potato rot nematode. Phytopathology51, 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 Evolution21, 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 Biology3, 1488–1500.

Goodey J. 1962. Taxonomic relatedness in nematology. Annals of Applied Biology50, 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 destructorActa Phytopathologica Sinica39, 125–131. (in Chinese)

Huelsenbeck J P, Ronquist F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics17, 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 dipsaciDitylenchus destructor and Ditylenchus gigas populations occurring in Poland. Journal of Phytopathology162, 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 DitylenchusPest Management Science71, 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 University29, 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 Research27, 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 Sinica43, 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 Research22, 1–12.

Liu B, Mei Y Y, Zheng J W. 2007. Species-specific detection of inter-populations of Ditylenchus destructorJournal 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 & Evolution8, 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 Industries15, 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 Evolution44, 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 DitylenchusPlant Pathology59, 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 Research27, 2271–2282.

Morgan J A, Blair D. 1998. Trematode and monogenean rRNA ITS2 secondary structures support a four-domain model. Journal of Molecular Evolution47, 406–419.

Müller T, Philippi N, Dandekar T, Schultz J, Wolf M. 2007. Distinguishing species. RNA13, 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 destructorBiotechnology Bulletin37, 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 Nematology52, 1–2.

Nylander J A A. 2004. MrModeltest V2. Program distributed by the author. Bioinformatics24, 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 Disease101, 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 Biology2, 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-Sinica23, 234–237. (in Chinese)

Rambaut A. 2016. FigTree version 1.4.3. [2019-11-30].

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 Evolution18, 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. Trees34, 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). Nematology13, 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. Phytopathology95, 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. Nematology8, 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 Evolution21, 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 Evolution56, 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. PeerJournal9, 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 Washington12, 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 Helminthology90, 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 Nematology15, 563–573.

De Waele D, Jones B, Bolton C, Van den Berg E. 1989. Ditylenchus destructor in hulls and seeds of peanut. Journal of Nematology21, 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 Sinica38, 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 destructorActa Agriculturae Jiangxi23, 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 destructorJournal 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-Sinica26, 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 Sinica20, 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 Research97, 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. RNA11, 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 Zoology38, 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 Mediterranea37, 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 Sinica39, 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 Sinica49, 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 Sinica38, 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 Bulletin37, 45. (in Chinese)

Zuker M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research31, 3406–3415.

No related articles found!
No Suggested Reading articles found!