中国辽宁咽滑刃线虫的形态与分子系统学描述

doi:10.3969/j.issn.1000-2006.201911059

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南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (4): 93-101.doi: 10.3969/j.issn.1000-2006.201911059

中国辽宁咽滑刃线虫的形态与分子系统学描述

宋雅婷¹(), 刘任军², 江奕¹, 周立峰¹, 谈家金¹, 孙守慧³, 陈凤毛¹

^1.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
^2.沈阳市森林资源监测中心，辽宁沈阳 110136
^3.沈阳农业大学林学院，辽宁沈阳 110866

收稿日期:2019-11-29 修回日期:2020-03-08 出版日期:2020-07-22 发布日期:2020-08-13
通讯作者: 陈凤毛
作者简介:宋雅婷（137368923@qq.com）
基金资助:
沈阳市科技计划项目(18-400-3-03)

Molecular and morphological characterization of Laimaphelenchus liaoningensis n. sp. (Nematoda: Aphelenchoididae) in China

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 Agricultural University, Shenyang 110866, China

Received:2019-11-29 Revised:2020-03-08 Online:2020-07-22 Published:2020-08-13
Contact: CHEN Fengmao

摘要/Abstract

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。

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

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.

Key words: nematode, morphology, phylogeny, ribosomal RNA, new species, taxonomy, Laimaphelenchus genus

中图分类号:

S763

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): 93-101.

SONG Yating, LIU Renjun, JIANG Yi, ZHOU Lifeng, TAN Jiajin, SUN Shouhui, CHEN Fengmao. 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): 93-101.DOI: 10.3969/j.issn.1000-2006.201911059.

图/表 5

图1

图2

图3

表 1

中国辽宁咽滑刃线虫种群的形态测计值"

morphometric character 形态指标	holotype 正模标本	paratypes（range）副模标本（范围）
morphometric character 形态指标	holotype 正模标本	female 雌虫	male 雄虫
strain number线虫数	–	15	15
body length/μm虫体长度	1 520	1 462±139 (1 252–1 722)	1 206±117 (972–1 383)
the ratio of body length to width体长/体宽	42	39.0±3.3 (32.0–46.0)	44.0±4.5 (37.6–53.6)
the ratio of body length to distance from anterior end to junction of oesophagusand intestine(b) 体长/头端至食道与肠连接处长度	17	16.0±2.0 (12.0–20.0)	14.0±1.1 (12.0–16.0)
the ratio of body length to distance from anterior end to oesophagus end体长/头端至食道腺末端长	6.7	6.4±0.6 (5.2–7.1)	6.1±0.3 (5.6–6.6)
the ratio of body length to tail length体长/尾长	24	24.0±1.4 (21.0–26.0)	20.0±1.0 (19.0–22.0)
the ratio of tail length to anus body width尾长/肛门处体宽	3.3	3.6±0.3 (3.1–4.1)	3.0±0.3 (2.6–3.4)
the ratio of conus length to stylet length针锥长度/口针长	46	45.0±4.1 (37.0–54.0)	50.0±4.8 (41.0–56.0)
the ratio of distance of vulva from anterior end to body length阴门至头端的长度/体长 the ratio of distance of cloaca orifice from testis end to body length泄殖腔口至精巢末端的长度/体长	70	69.0±2.4 (61.0–72.0)	69.0±5.2 (57.0–75.0)
the ratio of distance from anterior end to center of median bulb to distance from anterior end to oesophagus end体前端至中食道球中心距离/体前端至食道腺末端距离	35	35.0±4.8 (29.0–44.0)	39.0±4.0 (33.0–44.0)
maximum body diameter/μm 最大体宽	36	37.0±4.5 (30.0–45.0)	28.0±3.9 (20.0–33.0)
lip region height /μm 唇区高度	3.4	3.6±0.4 (3.0–4.5)	3.4±0.3 (2.9–3.9)
lip region diameter /μm 唇区宽度	7.7	7.6±0.5 (6.1–8.1)	7.3±0.4 (6.8–8.0)
the ratio of median bulb length to diameter 中食道球长/宽	1.2	1.2±0.1 (1.0–1.5)	1.3±0.1 (1.1–1.5)
median bulb diameter /μm 中食道球宽度	17	17.0±2.4 (14.0–22.0)	14.0±1.1 (12.0–16.0)
median bulb length /μm 中食道球长度	20	21.0±2.5 (15.0–26.0)	18.0±1.0 (16.0–20.0)
stylet length /μm 口针长度	14	14.0±1.0 (12.0–16.0)	14.0±0.8 (12.0–16.0)
conus length /μm 口针针锥长度	6.7	6.4±0.7 (5.5–8.1)	6.8.0±0.9 (5.1–7.9)
excretory pore from anterior end /μm 排泄孔距体前端长度	107	116.0±14.2 (101.0–151.0)	108.0±10.0 (92.0–126.0)
anterior end to posterior of pharyngeal glands /μm 体前端距食道腺末端长度	226	232.0±21.0 (201.0–272.0)	202.0±17.0 (175.0–223.0)
post?uterine sac length /μm 后阴子宫囊长度	143	141.0±19.6 (102.0–184.0)	–
vulva to anus distance /μm 肛阴距	387	378.0±42.7 (307.0–442.0)	–
the ratio of post?uterine sac length to distance from vulva to anus 后阴子宫囊长/肛阴距	37	37.0±3.9 (31.0–43.0)	–
the ratio of anal dianeter to cloacal body diameter 肛门/泄殖腔处体宽	18	17.0±1.9 (13.0–20.0)	20.0±2.1 (17.0–25.0)
tail length /μm 尾长	59	62.0±5.8 (53.0-70.0)	61.0±6.4 (48.0-70.0)
spicule (chord) length /μm 交合刺弧长	–	–	21.0±2.0 (19.0-25.0)
spicule (dorsal limb)/ length /μm 交合刺背侧长	–	–	35.0±2.7 (29.0-39.0)
spicule (ventral limb) length /μm 交合刺腹侧长	–	–	23.0±2.2 (19.0-28.0)
spicule (curved median line) length /μm 交合刺弧中线长	–	–	28.0±1.7 (24.0-30.0)

表 1

图4

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中国辽宁咽滑刃线虫的形态与分子系统学描述

Molecular and morphological characterization of Laimaphelenchus liaoningensis n. sp. (Nematoda: Aphelenchoididae) in China

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