【作者】 黄任娥；

【导师】 叶建仁；

【作者基本信息】 南京林业大学 ， 森林保护学， 2007， 博士

【摘要】 线虫是动物王国中多样性最丰富的类群,地球上大约有4/5的动物是线虫。自从Brenner(1965)提出把秀丽隐杆线虫Caenorhabditis elegans当作动物发育学和行为学研究的模式生物以来,线虫学得到了跨跃式发展,并且成为了生物学中最令人关注和兴奋的学科之一。而植物线虫学虽然起步迟,但作为线虫学的一个分支,而且在整个农业生态系统中占据着重要的地位,因此具有广阔的发展空间。随着模式生物秀丽隐杆线虫C. elegans在植物线虫学应用中的不断推进,21世纪将可能是植物线虫学飞速发展的时代。松材线虫病Bursaphelenchus xylophilus(Steiner & Bubrer 1934)Nickle 1970是松树上的一种毁灭性病害,是当前世界上重大森林病害之一,但该病害的防治工作却还没有得到突破性的进展。基于松材线虫隶属于滑刃目Aphelenchida,本研究在全国范围内调查了感染松材线虫病松树体内滑刃目线虫的群落组成,研究松材线虫与其它滑刃线虫的发生关系,希望能为松材线虫病的综合治理工作提供理论依据。在滑刃目线虫的调查过程中,作者发现了两个新种,于是对两个新种的形态展开了全面的研究、描述和报道工作。基于两个新种在形态上很相似,进而设计了一组杂交试验进一步探讨它们之间的生殖关系。此外,作者在线虫形态学研究的基础上,继续深入到细胞遗传学水平,选择其中的两对秭妹线虫作为研究对象,试图从细胞遗传学的角度来探讨线虫种类之间的相互关系。1感染松材线虫病松树滑刃目线虫调查首次在全国范围内调查感染松材线虫病松树体内滑刃目线虫的种群组成,包括种类记述和数量组成分析。结果表明:在300份样品中共分离得到10种滑刃目线虫,隶属于3科、4属,从中发现有两个新种和两个国内新记录种;它们是:李氏长尾线虫新种Seinura lii n. sp.,吴氏长尾线虫新种S. wuae n. sp.,小麦长尾线虫S. tritica Bajaj & Bhatti 1982(国内新记录种),爱尔密那长尾线虫S. elmiraensis (van de Linde, 1938) J. B. Goodey, 1960(国内新记录种),松材线虫B. xylophilus,拟松材线虫B. mucronatus Mamiya & Enda 1979,霍夫曼尼伞滑刃线虫B. hofmanni Braasch 1998,薄荷滑刃线虫Aphelenchoides menthae Lisetzkaya 1971,大核滑刃线虫A. macronucleatus Baranovskaya 1963和外滑刃科线虫一种Ektaphelenchidae sp.;本章描述并图示了除新种以外的8种滑刃线虫的形态特征,同时比较10种滑刃目线虫的主要区别特征。种群数量组成分析表明:松材线虫在感病松树体内占据绝对的优势,削弱了松树线虫群落组成的多样性,即线虫群落结构趋向单一化。研究还表明,利用长尾属线虫的捕食习性来控制中国松材线虫病的流行与危害值得进一步探索与研究。2李氏长尾线虫与吴氏长尾线虫二新种报道鉴定、描述并且在国际上报道了从江苏省溧阳县和仪征县的感染松材线虫病松树样品中发现的两个新种:李氏长尾线虫新种Seinura lii n. sp.和吴氏长尾线虫新种S. wuae n. sp.。李氏长尾线虫新种的主要鉴别特征为:虫体相对较长,雌雄体长分别为810-1146μm和865-1068μm;口针长16-20μm,无基部球,但口针基部有时稍膨大;生殖细胞(卵母细胞和精母细胞)在生殖管前端呈双行排列;后阴子宫囊很发达,长约126-182μm,其内通常含有大量的精子;侧区有三条侧线;雌虫尾部呈长锥形;雄虫尾部有4对亚腹乳突。吴氏长尾线虫新种的主要鉴别特征为:虫体相对较长,雌雄体长分别为819-1179μm和838-1062μm;口针长15-18μm,基部球很小;侧区有3条侧线;卵母细胞呈2-3行排列;后阴子宫囊长123-172μm;雌虫尾部呈长至中锥形;雄虫尾部有2对亚腹乳突。本文除了对两个新种的形态进行全面描述以外,还用墨线图、光学图片和扫描电镜图片等一系列图片形象而全面地展示了它们的形态。两个新种的模式标本除了保存于南京林业大学森林病理实验室以外,还保存于美国农业部线虫标本收藏室(USDANC)。3李氏长尾线虫与吴氏长尾线虫的杂交试验研究基于李氏长尾线虫S. lii与吴氏长尾线虫S. wuae二新种之间在形态上很相似,我们设计了一组杂交试验来进一步探讨它们之间的生殖关系。结果表明,李氏长尾线虫与吴氏长尾线虫在生殖上相互隔离,并且它们都为有性生殖。研究还表明,将细胞遗传学的方法运用于线虫分类学值得我们的思考和进一步研究。4两对秭妹滑刃目线虫生殖系细胞遗传学特征分析采用DAPI染色和荧光显微观察的方法,对松材线虫Bursaphelenchus xylophilus、拟松材线虫B. mucronatus、李氏长尾线虫Seinura lii和吴氏长尾线虫S. wuae等两对秭妹滑刃目线虫的生殖系细胞遗传学特征(包括生殖细胞排列方式和染色体的变化行为)进行了比较研究,同时图示了它们的生殖系细胞核形态和染色体形态。研究表明,线虫生殖系细胞遗传学特征分析有助于人们更好地理解线虫种类之间的亲缘关系,这对促进线虫分类学的发展和研究线虫的进化与系统发育历程将可能发挥举足轻重的作用。研究表明,基于DAPI染色和荧光显微观察的生殖系细胞排列模式可作为有效而重要的线虫分类特征之一。此外,研究还表明,四种线虫雌雄两性均存在着6个二价体(单倍体n = 6),且相互之间都具有相似的形态,它们的性别决定机制可能为XX-XY型。总之,本研究首次在全国范围内调查了感染松材线虫病松树体内的滑刃目线虫群落组成,描述并且比较了各滑刃目线虫的形态分类特征,提出‘利用长尾属线虫Seinura spp.的捕食习性来控制中国松材线虫病的流行与危害值得进一步研究’的观点,为松材线虫病的综合治理工作提供了参考依据。在调查过程中,作者发现了两个新种和两个国内新记录种,对两个新种的形态进行了全面的研究和描述,并发表在国际性的线虫学杂志Nematology上,这是中国首次在国际上报道长尾线虫属Seinura的新种,将可能大大推进中国长尾线虫属的形态学和分类学研究。配模标本被国际上最大的线虫标本收藏室(USDANC)收藏,这对促进国际交流和提高中国线虫学研究的国际地位将可能发挥一定作用。接着,作者设计了一组杂交试验,进一步探讨了二新种之间的生殖关系,为后文的研究提供了不可缺少的理论依据。最后,作者在线虫形态学研究的基础上,进一步深入到细胞遗传学水平,研究与比较两对秭妹滑刃目线虫生殖系的细胞遗传学特征,提出‘线虫生殖系的细胞遗传学特征分析有助于人们更好地理解线虫种类之间的亲缘关系’这一观点,为促进线虫分类学研究的发展和更好地解释线虫的进化和系统发育过程将可能发挥举足轻重的作用;另外还提出一个观点,即‘基于DAPI染色与荧光显微观察的生殖细胞排列模式可作为线虫分类的有效而重要的分类特征之一’;最后作者还对松材线虫、拟松材线虫、李氏长尾线虫与吴氏长尾线虫的染色体行为和数目进行研究与讨论,为植物线虫的染色体研究奠定了一定的理论基础。更多还原

【Abstract】 Nematodes are the most abundant and diversified group in the animal kingdom and four out of every five metazoan on earth is a nematode. Since Sydney Brenner settled on Caenorhabditis elegans as a model organism to study animal development and behavior in 1965, nematology has made unparalleled advances and become one of the most dynamic and exciting disciplines in the biological sciences. Plant-parasitic nematology owns a brilliant future although it is a young discipline, for the development of nematology is largely attributed to the discovery of the importance of nematodes in agricultural ecosystem and their impact on society. The 21st century might be an era of plant-parasitic nematology with Caenorhabditis elegans as a model system being applied in every aspect of nematology.The pine wood nematode, Bursaphelenchus xylophilus(Steiner & Bubrer 1934)Nickle 1970, is the causal agent of pine wilt disease in East Asia and destroys millions of pine trees every year. However, effective methods of controlling this disease have not yet been developed. Due to the pine wood nematode belongs to the order of Aphelenchida, a nation-wide survey for the presence of Aphelenchida spp. was conducted in China. From which we could learn much about the occurrence of pine wood nematode accompanied by other Aphelenchida species. It might provide some guides for integrated management of the pine wilt disease. Because two new species were discovered during this survey, they were described and illustrated thereafter, and a following hybridization test had been taken to further confirm that they were reproductively isolated. In addition, studies were continued into cytogenetic level to try to understand relationships between nematode species. The main contents are described as follows:1 Investigation on Aphelenchida from pine wood infested with B. xylophilusA nationwide survey for Aphelenchida from pine wood infected with Bursaphelenchus xylophilus (Steiner & Bubrer 1934) Nickle 1970 was conducted in China.Results showed that ten species were found from 300 samples and they belonged to four genera and three families based on morphological identification. From which two new species and two new records from ChinaWhich were Bursaphelenchus xylophilus, B. mucronatus Mamiya & Enda 1979, B. hofmanni Braasch 1998, Aphelenchoides menthae Lisetzkaya 1971, A. macronucleatus Baranovskaya 1963, Seinura tritica Bajaj & Bhatti 1982, S. elmiraensis (van de Linde, 1938) J. B. Goodey, 1960, S. lii Huang & Ye 2006, S. wuae Huang & Ye 2006 and Ektaphelenchidae sp., respectively. Among which S. tritica and S. elmiraensis were new records from China. Besides descriptions, illustrations were provided in the paper. Population variations of these ten species suggested that the pine wood nematode was the dominate species and its occurrence would lead to a significant weakness in diversities of nematodes within pine wood. Results also indicated that biological control potentials of Seinura spp. to pine wood nematode need further studies in China.2 Description of Seinura lii n. sp. and S. wuae n. sp. (Nematoda: Seinuridae)Seinura lii n. sp. and S. wuae n. sp. discovered from dying Pinus massoniana infested with B. xylophilus in Liyang county and Yizheng county, respectively, were described and illustrated in this chaper. S. lii n. sp. is characterised by relatively long female and male bodies (810-1146 and 865-1068μm, respectively), stylet 16-20μm long and lacking knobs, two rows of developing oocytes in the anterior gonad, well-developed postuterine sac (126-182μm long), three lines or incisures in the lateral field, four pairs of subventral papillae on the male tail region, and an elongate-conoid female tail. S. wuae n. sp. is characterised by relatively long female and male bodies (819-1179 and 838-1062μm, respectively), stylet 15-18μm long and with small knobs, three lines or incisures in the lateral field, two or three rows of developing oocytes in the anterior gonad, long postuterine sac (123-172μm long), two pairs of subventral papillae on the male tail region, and an elongate-conoid female tail. Type specimens were deposited in pathology laboratory of Nanjing Forestry University and USDA nematode collection.3 Interbreeding between Seinura lii and S. wuaeDue to Seinura lii was morphologically very similar to S. wuae, a following hybridization test was taken after they were originally described to learn more about the reproductive relationship between them. And the results showed that S. lii and S. wuae were reproductively isolated, additionally biological features of both species might vary considerably. Results also indicated that both Seinura species studied reproduced by amphimixis. It suggested that cytogenetic methods might provide some guides to learn more about the relationships between nematode species which would facilitate nematode taxonomy.4 Cytological features of adult germ-line in two pairs of sibling Aphelenchida speciesStructures of adult germ-line in two pairs of sibling species of Bursaphelenchus xylophilus and B. mucronatus and Seinura lii and S. wuae were characterized. Within and between the two pairs of sibling species, nuclear morphologies of the germ-lines were compared, including germ-cell arrangements and chromosomal behavior. Variance of germ-cell arrangement between species was well correlated with their taxonomic distance. It suggests that studies on cytogenetic features from germ lines might provide valuable information to understand the relationships between nematode species which provide important guides for nematode classification and taxonomy and for better interpreting the evolution of nematode groups and phylogenetic development of nematode populations. In addition, six bivalents of chromosomes were observed in both male and female germ cells in all four species studied. To sum up, a nation-wide survey for the presence of Aphelenchida spp. from pine wood infested with pine wood nematode was first conducted. Descriptions were provided and illustrations were compared in the paper. And an issue that biological control potentials of Seinura spp. to pine wood nematode needed further studies in China was presented. All of which might provide some guides to the management of pine wilt disease. Two new Seinura species and two new records from China were discovered during the survey. The two new species were described and reported in the international journal, Nematology. This is the first time to report new species in the genus Seinura from China, which will initiate studies on morphology and taxonomy of Seinura in China. Besides, paratype specimens were deposited in the well-recognised international nematode collection (USDANC), which might facilitate academic communication with international experts and enhance national prestige to some degree. In addition, a hybridization test was taken and reproductive relationship was discussed, which offered some guides for the later part of research. Finally, studies had been put into cytogenetic level. An idea of“cytogenetic features from germ lines might provide valuable information to understand the relationships between nematode species”was presented which might serve for nematode taxonomy and interpretation of phylogenetic development of nematode populations.更多还原