[1]宋雅婷,刘任军,江奕,等.中国辽宁咽滑刃线虫的形态与分子系统学描述[J].南京林业大学学报(自然科学版),2020,44(4):093-101.[doi:10.3969/j.issn.1000-2006.201911059]
 SONG Yating,LIU Renjun,JIANG Yi,et al.Molecular and morphological characterization of Laimaphelenchus liaoningensis n. sp. (Nematoda: Aphelenchoididae) in China[J].Journal of Nanjing Forestry University(Natural Science Edition),2020,44(4):093-101.[doi:10.3969/j.issn.1000-2006.201911059]
点击复制

中国辽宁咽滑刃线虫的形态与分子系统学描述/HTML
分享到:

《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
44
期数:
2020年4期
页码:
093-101
栏目:
研究论文
出版日期:
2020-09-01

文章信息/Info

Title:
Molecular and morphological characterization of Laimaphelenchus liaoningensis n. sp. (Nematoda: Aphelenchoididae) in China
文章编号:
1000-2006(2020)04-0093-09
作者:
宋雅婷 刘任军 江奕 周立峰 谈家金 孙守慧 陈凤毛
作者单位:南京林业大学,南方现代林业协同创新中心,南京林业大学林学院,江苏 南京 210037; 沈阳市森林资源监测中心,辽宁 沈阳 110136; 沈阳农业大学林学院,辽宁 沈阳 110866
Author(s):
SONG Yating1 LIU Renjun2 JIANG Yi1 ZHOU Lifeng1 TAN Jiajin1 SUN Shouhui3 CHEN Fengmao1
(1.Co?Innovation Center for the Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China; 2.Shenyang Forest Resources Monitoring Center, Shenyang 110136, China; 3.College of Forestry, Shenyang Agricul
关键词:
线虫 形态学 系统发育 核糖体RNA 新种 分类鉴定 咽滑刃属
Keywords:
nematode morphology phylogeny ribosomal RNA new species taxonomy genus
分类号:
S763
DOI:
10.3969/j.issn.1000-2006.201911059
文献标志码:
A
摘要:
目的 2018年7月自辽宁省沈阳市枯死华山松(Pinus armandii Franch.)体内分离得到一种非常见的线虫,经光学显微镜初步观察到该线虫在枯死松树内种群单一且数量较多,为研究其与松树枯萎的关系,从形态学和分子生物学特征对该线虫进行系统的描述说明,以确定该线虫的种属,更好地进行线虫的鉴定与分类,并对咽滑刃属线虫的数据库进行补充,便以期为松树枯萎病的相关研究提供理论参考。 方法 将采集到的华山松枯死木样品劈成1 cm(长)×1 cm(宽)的薄片,采用贝尔曼漏斗法在室温条件下分离8~12 h后收集线虫液,于光学显微镜下观察。同时将分离到的线虫用4%的甲醛溶液浸泡法杀死并立即固定,制作永久玻片。将一定数量的线虫用无菌水漂洗3 次后移入长满灰葡萄孢(Botrytis cinerea)的马铃薯葡萄糖琼脂(potato dextrose agar)培养基中,置于培养箱25℃暗培养7 d后可得到大量线虫。利用光学显微镜观察拍摄雌成虫和雄成虫的形态特征,采用De Man 公式进行形态测计,并依据特征绘制出墨线图。扫描电子显微镜则用于观察线虫的侧区、唇区、阴门区、尾尖突等部位处的细微差别。提取线虫DNA并对其18S和28S D2—D3进行片段扩增、克隆和测序,多基因串联构建进化树,通过Bootstrap 值分析聚类可靠性。经系统的形态学观察、测计以及分子生物学系统发育分析对该线虫进行种类鉴定。 结果 该线虫能以真菌为食,主要形态特征为雌虫体长1 252~1 722 μm,角质层具环纹;唇区圆形,缢缩明显;口针长12~16 μm,具有明显的基部球;中食道球长卵圆形近圆形,瓣膜清晰;排泄孔通常位于中食道球后方;食道腺长叶状,从背面覆盖肠;卵巢前伸,卵母细胞呈单行排列;侧线数量为4,具阴门盖;尾部具单个茎状末端,附两个梗节,分别具4~6个端缘指状突起;雄虫体长略短,为972~1 383 μm,体前部特征与雌虫相似;单精巢前伸,交合刺呈玫瑰刺状,长24~30 μm,具2对尾乳突;尾部形态与雌虫相似,但更向腹面弯曲。系统发育分析基于核糖体RNA(rRNA)的18S和28S D2—D3基因序列,多基因串联序列构建的系统进化树也表明该线虫种群与基因库中具有可用序列的其他咽滑刃属线虫物种分开聚类。 结论 该线虫鉴定为辽宁咽滑刃线虫(Laimaphelenchus liaoningensis n. sp.),在形态特征方面通过具有阴门盖、4条侧线和尾端梗节的特征将其与其他类似种区分开来,在分子特征方面则通过与该属中其他种类分开聚类进行区分。这是中国关于咽滑刃属线虫的首次报道,同时华山松也为该属线虫的一新寄主。在实验室条件下用灰葡萄孢成功培养了该线虫,表明该咽滑刃线虫能以真菌为食。咽滑刃属线虫的尾端结构在不同种间存在差异,只有借助扫描电镜才能观察到特征上的细微差别,说明仅基于光学显微镜的形态分类学鉴定面临一定困难,扫描电镜是观察线虫细微的尾尖形状和结构所必需的。
Abstract:
Objective A nematode species associated with wood samples from dead pine trees (Pinus armandii Franch.) was collected from Shenyang, Liaoning Province, China, during July 2018. It was preliminarily observed by light microscope that the nematode population in the dead pine was sizeable and singular. The nematode was described and illustrated based on its morphological and molecular characteristics in order to identify the species and study its relationship with pine wilt disease. The continuous development of molecular biology allows us to supplement the database of Laimaphelenchus species, leading to the improved disease diagnosis and taxonomy, and providing a reference for pine wilt research. Method Wood samples were sliced into approximately 1 cm × 1 cm slices, and nematodes were collected using the Baermann funnel method at room temperature for 8-12 h. Nematodes to be prepared for permanent slides were killed by immersion in 4% formaldehyde solution and immediately fixed. A certain number of nematodes were rinsed with sterile water three times and then transferred into a potato dextrose agar medium containing Botrytis cinerea. After being placed in the incubator for dark culture at 25℃ for 7 days, a large number of nematodes were obtained. Measurements, descriptions and light micrographs of nematodes were carried out and De Man’s formula was used to measure morphological traits and the line was drawn based on these characteristics. Scanning electron microscopy was used to observe the nuances of the lateral field, lip region, vulva and mucronatus. Nematode DNA was extracted and its 18S and 28S D2-D3 regions were amplified, cloned and sequenced. The phylogenetic tree construction used Neighbor-Joining (NJ) methods and an bootstrap analysis was performed to test the support of each node in the tree. The nematode was identified by systematic morphological observation, morphometric values and the comparative analysis of molecular biological sequences. Result The nematode was cultured successfully on B. cinerea in the laboratory, indicating that it can feed on fungi. Females are characterized by a 1 252-1 722 μm body length, a slender stylet of 12-16 μm, four incisures in the lateral field, a vulva with a flap, and a tail-tip structure comprising a single stalk-like terminus and two tubercles with four to six finger-like protrusions. Males have spicules 24-30 μm in length, two pairs of caudal papillae, and a tail morphologically similar to that of the female but more ventrally curved. Phylogenetic analyses were based on the D2-D3 expansion segments of the 28S and 18S rRNA gene. Construction of the phylogenetic tree used multi-gene tandem sequences of Laimaphelenchus liaoningensis n. sp. clustered separately from all Laimaphelenchus species with available sequences in Genbank. Conclusion The L. liaoningensis n. sp. is the first report of a Laimaphelenchus species in China, and P. armandii is a new host for the genus.

参考文献/References:

1 MALEITA C M N, COSTA S R, ABRANTES I. Laimaphelenchus suberensis sp. nov. associated with Quercus suber in Portugal[J]. European Journal of Plant Pathology, 2018, 150(3):747-758. DOI: 10.1007/s10658-017-1324-4.
2 HUNT D J. Aphelenchida, Longidoridae and Trichodoridae: their systematics and bionomics[M]. Wallingford UK: CABI Publishing, 1993.
3 HUNT D J. A checklist of the Aphelenchoidea (Nematoda: Tylenchina)[J]. Journal of Nematode Morphology and Systematics, 2007, 10(2):99-135.
4 ZHAO Z Q, DAVIES K A, RILEY I T, et al. Laimaphelenchus heidelbergi sp. nov. (Nematoda: Aphelenchina) from Victoria, Australia, and emendment of the diagnosis of the genus[J]. Trans R Soc S Aust, 2007, 131(2):182-191. DOI: 10.1080/03721426.2007.10887082.
5 MASSEY C L. The nematode parasites and associates of Dendroctonus adjunctus (Coleoptera: Scolytidae) in New Mexico[J]. Annals of the Entomological Society of America, 1966, 59(3): 424-440. DOI: 10.1093/aesa/59.3.424.
6 ELMILIGY I, GERAERT E. Laimaphelenchus deconincki n. sp. (Nematoda: Tylenchida) [J]. Biologisch Jaarboek, 1971(39):145-149.
7 BAUJARD P. Revue taxonomic du genre Laimaphelenchus Fuchs, 1937 et description de Laimaphelenchus pini n. sp. [J]. Revue de Nématologie, 1981(4):75-92.
8 DOUCET M E. A new species of the genus Laimaphelenchus Fuchs, 1937 (Nemata: Aphelenchina)[J]. Fundamental & Applied Nematology, 1992, 15(1):1-6.
9 SWART A. Description of Laimaphelenchus patulus sp. n. (Nematoda: Aphelenchoididae) from Pinus pinaster Ait. in South Africa[J]. African Plant Protection, 1997, 3(1):23-28.
10 PENEVA V, CHIPEV N. Laimaphelenchus helicosoma (Maslen, 1979) n. comb. (Nematoda: Aphelenchida) from the Livingston Island (the Antarctic)[J]. Bulgarian Antarctic Research Life Sciences,1999(2):57-61.
11 BAJAJ H K, WALIA K K. A new species of Laimaphelenchus Fuchs, 1937 (Nematoda: Aphelenchina) from Kalesar forest, Haryana, India[J]. Indian Journal of Nematology, 2000(30):86-110.
12 NEGI S, KALIA C D, WALIA K K, et al. New species of Aphelenchoides Fischer and Laimaphelenchus Fuchs (Nematoda: Aphelenchida) from pine twigs, Himachal Pradesh, India[J]. Indian Journal of Nematology, 2009, 39(2):192-197.
13 ZHAO Z Q, DAVIES K A, RILEY I T, et al. Laimaphelenchus australis sp. nov. (Nematoda: Aphelenchina) from exotic pines, Pinus radiata and P. pinaster, in Australia[J]. Zootaxa, 2006(1248):35-44. DOI: 10.5281/zenodo.172959.
14 ZHAO Z Q, DAVIES K A, RILEY I T, et al. Laimaphelenchus preissii sp. nov. (Nematoda: Aphelenchina) from native pine Callitris preissii in South Australia[J]. Transactions of the Royal Society of South Australia, 2006, 130(1):10-16. DOI: 10.1080/3721426.2006.10887044.
15 ASGHARI R, POURJAM E, HEYDARI R, et al. Laimaphelenchus persicus n. sp. (Nematoda: Aphelenchoididae) from Iran[J]. Zootaxa, 2012, 9(3325):59-67. DOI: 10.1186/1742-9994-9-10.
16 ASGHARI R, ESKANDARI A. Morphological and molecular characterization of Laimaphelenchus deconincki (Nematoda: Aphelenchoid before the last authoridae) based on specimens from Iran[J]. Biologia, 2014, 69(9):1172-1178. DOI: 10.2478/s11756-014-0425-1.
17 ESMAEIL M, RAMIN H, TANHA M Z, et al. Laimaphelenchus hyrcanus n. sp. (Nematoda: Aphelenchoididae), a new species from northern Iran[J]. Zootaxa, 2015, 3915(4):591-600. DOI: 10.11646/zootaxa.3915.4.9.
18 ORO V. Description of Laimaphelenchus belgradiensis sp. nov. (Nematoda: Aphelenchoididae) and its phylogenetic and systematic position within Aphelenchoidoidea[J]. European Journal of Plant Pathology, 2005, 142(1):13-23. DOI: 10.1007/s10658-014-0585-4.
19 RYSS A Y. Express technique to prepare permanent collection slides of nematodes[J]. Zoosystematica Rossica, 2003(11): 257-260.
20 WERGIN W P, ORION D. Scanning electron microscope study of the root-knot nematode (Meloidogyne incognita) on tomato root[J]. Journal of Nematology, 1981, 13(3), 358-367. DOI: 10.2307/1380615.
21 CHEN F M, YE J R, TANG J, et al. Identification of Bursaphelenchus xylophilus and B. mucronatus by PCR-RFLP technique[J]. Journal of Nanjing Forestry University (Nature Sciences Edition), 2006, 30(4):5-9.
22 DE LEY, FELIX M A, FRISSE L M, et al. Molecular and morphological characterisation of two reproductively isolated species with mirror-image anatomy (Nematoda: Cephalobidae)[J]. Nematology, 1999, 2: 591-612. DOI: 10.1163/156854199508559.
23 CHIZHOV V N, CHUMAKOVA O A, SUBBOTIN S A, et al. Morphological and molecular characterization of foliar nematodes of the genus Aphelenchoides: A. fragariae and A. ritzemabosi (Nematoda: Aphelenchoididae) from the Main Botanical Garden of the Russian Academy of Sciences, Moscow [J]. Russian Journal of Nematology, 2006, 14(2):179-184. DOI: 10.1016/j.dsr2.2007.12.007.
24 HALL T A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[J]. Nucleic Acids Symposium Series, 1999, 41:95-98.
25 TAMURA K, NEI M, KUMAR S. Prospects for inferring very large phylogenies by using the neighbor-joining method[J]. Proceedings of the National Academy of Sciences, 2004, 101(30): 11030-11035. DOI: 10.1073/pnas.0404206101.
26 FELSENSTEIN J. Confidence limits on phylogenies: an approach using the bootstrap[J]. Evolution 1985, 39(4):783-791. DOI: 10.1111/j.1558-5646.1985.tb00420.x.
27 TAMURA K, STECHER G, PETERSON D, et al. Mega6: molecular evolutionary genetics analysis version 6.0[J]. Molecular Biology & Evolution, 2013, 30(12): 2725-2729. DOI: 10.1093/molbev/mst197.
28 BRAASCH H, CAROPPO S, AMBROGIONI L, et al. Pathogenicity of various Bursaphlenchus species to pines and implication to European forests[C]//Sustainability of pine forests in relation to pine wilt and decline. Tokyo: Proceedings of International Symposium, 1998.
29 ZHOU L F, CHEN F M, WANG J C, et al. Virulence of Bursaphelenchus mucronatus to pine seedlings and trees under field conditions[J]. Forest Pathology, 2016, 46(6): 643-651. DOI: 10.1111/efp.12285.

相似文献/References:

[1]侯森林,郭海涛,薛晓明.4种食肉动物直针毛的显微形态学观察[J].南京林业大学学报(自然科学版),2006,30(06):135.[doi:10.3969/j.jssn.1000-2006.2006.06.032]
 HOU Sen-lin,GUO Hai-tao,XUE Xiao-ming.A Survey on the Micro-morphologic Character of Four Carnivore’s Straight Guard Hair[J].Journal of Nanjing Forestry University(Natural Science Edition),2006,30(4):135.[doi:10.3969/j.jssn.1000-2006.2006.06.032]
[2]韩凌云,徐 振,王良桂*.多维视角下城市开放空间研究框架的建构[J].南京林业大学学报(自然科学版),2014,38(01):083.[doi:10.3969/j.issn.1000-2006.2014.01.015]
 HAN Lingyun,XU Zhen,WANG Lianggui*.Open space morphology and research framework with multi-perspectives[J].Journal of Nanjing Forestry University(Natural Science Edition),2014,38(4):083.[doi:10.3969/j.issn.1000-2006.2014.01.015]

备注/Memo

备注/Memo:
收稿日期:2019-11-29
更新日期/Last Update: 2020-08-13