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1252~1722μm,角质层具环纹;唇区圆形,缢缩明显;口针长12~16μm,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972~1383μm,体前部特征与雌虫相似;单精巢�
【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,
作者
宋雅婷
刘任军
江奕
周立峰
谈家金
孙守慧
陈凤毛
SONG Yating;LIU Renjun;JIANG Yi;ZHOU Lifeng;TAN Jiajin;SUN Shouhui;CHEN Fengmao(Co-Innovation Center for the Sustainable Forestry in Southern China,College of Forestry,Nanjing Forestry University,Nanjing 210037,China;Shenyang Forest Resources Monitoring Center,Shenyang 110136,China;College of Forestry,Shenyang Agricultural University,Shenyang 110866,China)
出处
《南京林业大学学报（自然科学版）》
CAS
CSCD
北大核心
2020年第4期93-101,共9页
Journal of Nanjing Forestry University：Natural Sciences Edition
基金
沈阳市科技计划项目(18-400-3-03)。
