甘肃天祝草原孢囊线虫种类鉴定和生态分布研究

发布时间：2018-06-26 03:45

本文选题：天祝草原 + 禾谷孢囊线虫　；参考：《甘肃农业大学》2015年硕士论文

【摘要】：禾谷孢囊线虫(Cereal cyst nematode,CCN Heterodera SPP.)是一类危害小麦(Triticum aestivum)、大麦(Hordeum vulgare)、燕麦(Avena sativa)等禾谷类作物的世界性重要病原线虫。禾谷孢囊线虫除为害小麦、大麦和燕麦等禾谷类作物外,一些多年生的禾本科植物科也可作为禾谷孢囊线虫的中间寄主或桥梁寄主。本研究根据甘肃省天祝草原农牧区的分布特征,系统的对禾谷孢囊线虫在天祝高寒草甸草原的发生、生态分布、孢囊线虫种类及优势种群进行了研究。2013-2014年,采用随机采样法,对甘肃省天祝高寒草甸草原主要牧区的禾谷孢囊线虫进行了较系统的调查,调查范围覆盖了天祝县主要牧区和半农半牧区的13个乡镇。采集样共计本248份,其中56份样品中检出有孢囊线虫,占22.6%。通过镜检,所调查的13个乡镇,7个乡镇土样有孢囊线虫检出,检出率由高到低分别为打柴沟镇47.4%、塞拉隆乡46.7%、抓喜秀龙乡44.9%、石门镇40.0%、安远镇34.4%、大红沟乡11.1%和旦马乡9.8%。炭山岭镇、塞什斯镇、天堂镇、松山镇、祁连乡和哈溪镇土样中孢囊线虫检出率为0。抓喜秀龙乡一采样地点孢囊基数最高为54个/100 g土样。通过以0?10 cm、10?20 cm、20?30 cm、30?40 cm为垂直采样标准,在已发现有孢囊线虫的40个样点进行分层采样研究天祝草原孢囊线虫的垂直分布情况,结果表明,孢囊线虫均布在0?10 cm和10?20 cm土层中,30 cm以下土样中未检出孢囊线虫,且所有土样中0?10 cm孢囊线虫密度均大于对应土样的10?20 cm中孢囊密度。本研究应用形态学结合分子生物学鉴定,鉴定结果表明,甘肃天祝高寒草甸草原孢囊线虫优势种群为禾谷孢囊线虫(Heteroder avenae)。其形态学特征为孢囊浅褐色,阔柠檬形,阴门锥双膜孔,未见阴门下桥,泡状突明显。利用通用引物AB28和TW81,通过PCR技术对禾谷孢囊线虫群体的核糖体基因的内转录间隔区(rDNA-ITS)序列的扩增,后经PCR产物纯化回收、克隆、测序,得到ITS区序列长度为1045 bp,序列中无碱基插入和缺失,均有碱基替换。将所测得序列在Genbank和Blast上比对,下载近缘种序列,在MEGA5.2中运用UPGMA法构建系统发育树,结果显示,此禾谷孢囊线虫群体与H.avena群体相似度达99%~100%,进一步证实寄生于甘肃天祝高寒草甸草原牧草孢囊线虫均为H.avenae。ITS构建的系统发育树表明,本研究线虫群体与中国的禾谷孢囊线虫、俄罗斯的早熟禾孢囊线虫(H.pratensis)和德国的早熟禾孢囊线虫(H.pratensis)群体同时聚为一个小的分支,揭示了它们之间具有较高的亲缘关系。为了测定天祝牧草禾谷孢囊线虫(H.avenae)群体对小麦的致病力,采用室内人工接种的方法,将分离自嵩草和矮嵩草的2个禾谷孢囊线虫孢囊经低温处理后,将线虫卵接种至小麦“矮抗58”,每株3000粒卵,将接种的小麦苗置于15±2℃的培养室中,用镝灯灯管补光,每天光照14 h。1周后,温度调至18±2℃。定期浇水,使PVC管内的土壤保持一定的湿度,75天后观察统计小麦根部和根际土壤孢囊数。结果表明,天祝牧草禾谷孢囊线虫群体可以侵染小麦。
[Abstract]:Cereal cyst nematode (CCN Heterodera SPP.) is a class of worldwide important pathogenic nematodes that are harmful to wheat (Triticum aestivum), barley (Hordeum vulgare), oat (Avena sativa) and other cereal crops. The cereal cyst nematode is a perennial grasses except for wheat, barley, and oats. This study, based on the distribution characteristics of the farm and pastoral areas of Tianzhu grassland in Gansu Province, studied the occurrence, ecological distribution, the species of cyst nematode and the dominant population of the cyst nematode in Tianzhu meadow grassland in Gansu province. The random sampling method was applied to Gansu. A systematic investigation was conducted in the main pastoral areas of the main pasture pasture of Tianzhu alpine meadow, which covered 13 villages and towns in the main pastoral areas and half agricultural and semi pastoral areas of Tianzhu County. There were 248 samples collected in the main pastoral areas of Tianzhu County. Among them, 56 samples were found with cyst nematode, accounting for 22.6%. through microscopic examination, 13 villages and towns and 7 villages and towns. The detection rate of cyst nematode from high to low were 47.4% in Chai Gou Town, 46.7% in saailon Township, 44.9% in Xi Xiu long Township, 40% in Shimen Town, 34.4% in Anyuan Town, 11.1% in Dagu Township, 9.8%. in denaten Township, sash Town, Paradise Town, Songshan Town, Qilian Township and Haxi Town, 0. The highest cysts were 54 /100 g soil samples. By using 0? 10 cm, 10? 20 cm, 20? 30 cm, 30? 40 cm as vertical sampling criteria, the vertical distribution of the cyst nematode in Tianzhu steppe was studied by stratified sampling at 40 samples of the cyst nematode. The results showed that the cyst nematode was distributed in the 0? 10 cm and 10? 20 cm soil. No cyst nematode was detected, and the density of 0? 10 cm cyst nematode in all soil samples was greater than that of 10? 20 cm in the corresponding soil samples. The results showed that the dominant population of Gansu Tianzhu alpine meadow steppe was that the dominant population of the cyst nematode in the meadow was Heteroder avenae. The cysts were light brown, broad lemonade and double membrane holes in the cones of the vulva. There were no inferior pons and obvious alveolar processes. Using universal primers AB28 and TW81, the PCR technique was used to amplify the internal transcriptional interval (rDNA-ITS) sequence of the ribosome gene of the cyst nematode population, and then the PCR product was purified and sequenced, and the sequence length of the ITS region was 1045 BP, The sequences were inserted and missing with base substitutions. The sequences measured on Genbank and Blast were compared, the sequences were downloaded and the phylogenetic tree was constructed by UPGMA method in MEGA5.2. The results showed that the similarity of this colony of Cereal Cyst Nematode with H.avena population was 99%~100%, further confirmed parasitic in Gansu Tianzhu alpine meadow. The phylogenetic tree of the original herbage bursa nematode is H.avenae.ITS, which shows that the nematode population of this study conforms to a small branch of China's cereal cyst nematode, Russian precocious bursa nematode (H.pratensis) and German precocious bursa nematode (H.pratensis), which reveals a high affinity between them. In order to determine the pathogenicity of H.avenae population in Tianzhu grasses, 2 cereal cyst nematode cysts from Kobresia and Kobresia humilis were treated at low temperature by artificial inoculation, and the eggs were inoculated to "dwarf 58" and 3000 eggs per plant. The inoculated wheat seedlings were cultured at 15 + 2 centigrade. In the room, the light was supplemental with a dysprosium lamp tube. After 14 h.1 weeks of illumination, the temperature was adjusted to 18 + 2 degrees centigrade. The soil in the PVC tube was kept certain humidity regularly, and the number of the root and rhizosphere soil cysts in the root and rhizosphere of the wheat was observed after 75 days. The results showed that the Tianzhu grass cyst nematode population could infect the wheat.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S812.6

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