南京林业大学学报(自然科学版) ›› 2020, Vol. 44 ›› Issue (3): 105-110.doi: 10.3969/j.issn.1000-2006.201909046

• 研究论文 • 上一篇    下一篇

滑刃属线虫1个中国新记录种

宋雅婷1(), 王立超1, 孙守慧2, 陈凤毛1   

  1. 1.南京林业大学林学院,南方现代林业协同创新中心,江苏 南京 210037
    2.沈阳农业大学林学院,辽宁 沈阳 110866
  • 收稿日期:2019-09-24 修回日期:2020-02-22 出版日期:2020-05-30 发布日期:2020-06-11
  • 通讯作者: 陈凤毛
  • 作者简介:宋雅婷(137368923@qq.com)。
  • 基金资助:
    国家重点研发计划(2017YFD0600104);沈阳市科技计划项目(18-400-3-03)

First report of Aphelenchoides rotundicaudatus in China

SONG Yating1(), WANG Lichao1, SUN Shouhui2, CHEN Fengmao1   

  1. 1.Co -Innovation Center for the Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
    2.College of Forestry, Shenyang Agricultural University, Shenyang 110866, China
  • Received:2019-09-24 Revised:2020-02-22 Online:2020-05-30 Published:2020-06-11
  • Contact: CHEN Fengmao

摘要: 目的

2017年在安徽省黄山博村林场枯死的马尾松(Pinus massoniana)树体内分离得到一种不常见的线虫。经光学显微镜初步观察,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描述,确定该线虫的种属并对线虫数据库进行补充,以便于更好进行线虫的鉴定与分类,为松枯萎病的相关研究提供参考。

方法

将采集到的马尾松枯死木样品劈成长、宽均为1 cm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。将一定数量的线虫用无菌水漂洗后移入长满灰葡萄孢(Botrytis cinerea)的马铃薯葡萄糖琼脂培养基中,置于培养箱25 ℃暗培养7 d后得到大量线虫。利用光学显微镜观察拍摄雌成虫和雄成虫的形态特征并进行形态测计。提取线虫DNA并分别对其ITS、18S和28S D2?D3区进行片段扩增、克隆和测序,构建进化树进行分子系统发育分析。综合系统形态学观察、测计及分子生物学系统发育分析对该线虫进行了种类鉴定。

结果

该线虫种群主要形态特征为:雌虫体长约642.0 μm,角质层有微弱环纹;唇区圆形,缢缩明显,口针长约10.7 μm,具有明显的小基部球;排泄孔通常位于中食道球前部,距体前端32.9 μm;中食道球近卵圆形,瓣膜清晰,位于中心处;食道腺长叶状,从背面覆盖肠;侧线数量4条,生殖系统发达,卵巢前伸,卵母细胞呈单行排列,后阴子宫囊发育良好,一般长80.9 μm,阴门横裂,无阴门盖;尾圆锥形,稍向腹面弯曲,末端钝圆,带有小的栓状突起。雄虫体长稍短,约为590.0 μm,体前部与雌虫相似,单精巢前伸,通常延伸至食道腺末端附近;尾圆锥形,明显向腹面弯曲,尾长约为泄殖腔直径的3.0倍,带有1个小的栓状凸起;尾部具3对尾乳突,交合刺成对,呈玫瑰刺状,基顶和喙突明显;无交合伞和引带。基于ITS、18S和28S D2–D3区基因DNA序列分别构建的NJ系统进化树均表明该线虫与圆尾滑刃线虫(Aphelenchoides rotundicaudatus)以100%的置信度聚在同一进化分支。

结论

结合形态学与分子生物学特征,该线虫鉴定为圆尾滑刃线虫(A. rotundicaudatus),是自原始文献之后在中国的实树寄生首次发现,为新记录种。原始文献中的韩国种群无法在灰葡萄孢上繁殖,且仅有46.0%的雄成虫和97.0%的雌成虫尾部具有短的栓状凸起;此次中国种群线虫在灰葡萄孢上发育完好,几乎所有成虫均具有栓状凸起的尾部特征。形态学测计中,中国种群的数值略大于原始文献中韩国种群的,但形态特征基本一致,这可能是不同地理种群间的差异或种内变异造成的。

关键词: 滑刃属, 圆尾滑刃线虫, 分类鉴定, 新记录种

Abstract: Objective

A nematode specie with a rare morphology was isolated from dead Pinus massoniana found in the Bocun Forest Farm, Yellow Mountain, Anhui Province, in 2017. A general observation using light microscope revealed that the population of the nematode was dominant and unique. The nematode was described and illustrated based on its morphological and molecular characteristics in order to identify its relationship with pine wilt symptoms. Besides, this identification of new nematode species would not only enhance the database of Aphelenchoides species, but also provide theoretical references for research on pine wilt disease.

Method

Wood samples were sliced into 1 cm × 1 cm slices, followed by isolation of nematodes by the Baermann funnel method at room temperature for 8-12 h. A certain number of nematodes were rinsed with sterile water and then transferred into the Potato Dextrose Agar medium inoculated with Botrytis cinerea to acquire sufficient nematode population after incubation for 7 days at 25°C. Measurements, descriptions, and micrographs of nematodes were obtained using a light microscope and the data was statistically analyzed. The DNA of the nematodes was extracted and corresponding ITS, 18S and 28S D2-D3 regions were amplified, cloned and sequenced. A phylogenetic tree was constructed for subsequent molecular development analysis. The nematode species was identified by systematic morphological observation and statistical and comparative analysis of the molecular biological sequences.

Result

The female nematode population was characterized by a body length of 642.0 μm, weakly annulated cuticle, rounded and offset lip region, and 10.7 μm long stylet with obvious small basal knobs. Additionally, the excretory pore was located anterior to the median bulb, 32.9 μm from the anterior end. The median bulb was rounded to oval, with conspicuous valve plates situated more or less centrally. The pharyngeal glands were long lobed, overlapping the intestine dorsally. The female reproductive system was highly developed and monodelphic, with the ovary outstretched forward and oocytes arranged into a single-row layout. Post-uterine sac was well-developed and 80.9 μm long in a pouch shape. The vulva was located in the posterior part of the body, with a simple slit visible in ventral view, and without a vulval flap. The tail was cylindrical and slightly ventrally arcuate, and the terminus was broadly rounded with a blunt peg. The general characteristics of the male nematode population were similar to those of the female; however, the males were usually shorter in length at 590.0 μm. The genital system of the males was monorchic, with the testis stretched forward, often extending to the vicinity of the posterior edge of the esophageal gland. The tail was conical, curved to the ventral surface, with a length that was about 3.0 times the diameter of cloaca. Three pairs of subventral caudal papillae were present in the male tail. Moreover, the males had paired spicules with rose-thorn shape and rounded apex and rostrum. Bursa and gubernaculum were absent. The neighbor-joining tree method based on ITS, 18S and 28S DNA sequences demonstrated that the nematode and A. rotundicaudatus are clustered in the same branch with 100% confidence.

Conclusion

Morphological and molecular analyses revealed the nematode species as A. rotundicaudatus. This is the first report of A. rotundicaudatus in China after its first introduction in the scientific literature. The Korean population mentioned in the original literature cannot reproduce on B. cinerea and only 46.0% males and 97.0% females were observed to have tails terminating in a blunt peg. In the Chinese population, nematodes could reproduce well on B. cinerea, and almost all the adults had their tails terminating in a blunt peg. Based on morphological and statistical analyses, the data of Chinese population is slightly larger than Korean population. However, the morphological characteristics are basically the same, and the variations detected could have been due to different geographic origins or intraspecific mutations.

Key words: Aphelenchoides, Aphelenchoides rotundicaudatus, identification, new record species

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