捕食线虫性真菌的分离鉴定与主要生物学特性的研究

发布时间：2018-09-03 08:21

【摘要】：捕食线虫性真菌作为自然界中动物或植物寄生线虫的天敌,是世界广布性的一类微小真菌。利用捕食线虫性真菌对感染性幼虫的生物防治,在控制家畜寄生线虫病的替代措施中是一种理想选择。鉴于此,本研究目的是调查与牛羊有关的不同样品中捕食线虫性真菌的种类和出现频率,探寻可用于生物防治家畜寄生线虫的潜在菌种；并通过光学显微镜及扫描电镜观察研究鞭式达丁屯氏菌(Duddingtonia flagrans)与捻转血矛线虫(Haemonchus contortus)感染性幼虫(L3)、自由生活线虫秀丽隐杆线虫(Caenorhabditis elegans)相互作用的动态过程：也观察了培养温度和pH值对捕食线虫性真菌分离株一长孢隔指孢菌(Dactylella leptospora)(IS-307)生长的影响。(1)本研究自2013年4月至2014年12月,从中国20省(自治区、直辖市)43个市、县89个采样点,采集样品共计1408份,其中粪便1 050份,草场上的土壤108份,粪堆149份及厩舍周围的土壤101份。从3094个检测平皿中分离到捕食线虫性真菌614株,鉴定为4属20种,包括节丛孢属(Arthrobotrys)13种,其中11个已定种和2个未定种；单顶孢属(Monacrosporium)4种；隔指孢属(Dactylella)2种；达丁屯氏属(Duddingtonia)1种。捕食线虫性真菌在所有样品中的检出率为19.82%；在粪便、粪堆、厩舍周围土壤、草场上土壤中的检出率分别为：11.04%、33.42%、39.48%、52.57%。此次调查的所有样品中,主要种类为少孢节丛孢少孢变种(Arthrobotrys oligospora var. oligospora),圆锥节丛孢(Arthrobotry conoides),指状节丛孢(Arthrobotry dactyloides),弯孢节丛孢(Arthrobotry musiformis),多孢节丛孢(Arthrobotry superba),奇妙单顶孢(Monacrosporium thaumasiun)和D. flagrans,检出率分别为：13.06%、0.52%、0.68%、0.61%、1.10%、0.48%、1.20%,其余种类的检出率小于0.4%。在已记录的这些捕食线虫性真菌中,17种产生黏性网,出现频率为94.46%,仅2种产生其它捕食结构,如收缩环或者黏性球与非收缩环,所占比例为3.74%。A. oligospora是最常见的种,在每个采样地区都己分离到,而A.conoides,A. dactyloides, A. musiformis, A. superba及M. thaumasiun分别在不同地区出现。D. flagrans是动物寄生线虫生物防治中重要的候选菌种,此次调查中,从宁夏、甘肃、黑龙江、山东、贵州都有检出,在粪便及粪堆中的检出率分别为0.87%、4.35%。(2)将D. flagrans培养于0.2g/L玉米粉琼脂培养基(CAM)上,用于扫描电镜观察时则接种于覆盖有纤维素膜的CAM上。培养7 d后,加入试验线虫,随之在相互作用后的0、1、2、3、4、6、8、12、16、20、24、36和48 h取含有虫体与真菌的琼脂块观察,当虫体刚被真菌捕捉即开始计为0h。结果表明,该菌在未加入试验线虫培养36 h后可自发形成捕食结构；在加虫相互作用30min后,可诱导产生捕食器,此时也有虫体被捕捉；捕捉后4 h,菌丝侵入秀丽隐杆线虫导致其快速死亡,而L3被菌丝侵入致死则需要20-24 h；捕捉后24 h,整个秀丽隐杆线虫被菌丝侵占并完全消化；而L3则需要36 h被菌丝完全侵占,48 h被完全消化仅留表皮层。(3)在玉米粉琼脂培养基上,观察捕食线虫性真菌分离株D. leptospora(IS-307)在4、5、6、7、8、9、10、11、12不同pH值下以及在15℃、20℃、25℃、30℃不同温度下的生长情况。结果显示,该菌在pH值为4-12,温度为15℃-30℃之间的环境下均能生长,最佳培养条件pH值为11.0,温度为25℃。
[Abstract]:Nematode-trapping fungi, as natural enemies of parasitic nematodes of animals and plants in nature, are a kind of microfungi widely distributed in the world. The biological control of infectious larvae by nematode-trapping fungi is an ideal alternative to control parasitic nematodes of livestock. In view of this, the purpose of this study is to investigate the relationship between cattle and sheep. Species and frequencies of nematode-trapping fungi in different samples were studied to explore potential fungi for biological control of parasitic nematodes in livestock. Infectious larvae of Duddingtonia flagrans and Haemonchus contortus (L3), free-living lines were observed by light microscope and scanning electron microscope. Dynamics of interaction between Caenorhabditis elegans: Effects of culture temperature and pH value on the growth of nematode-trapping fungal isolate Dactylella leptospora (IS-307) were also observed. (1) This study was conducted from April 2013 to December 2014 in 43 cities and counties of 20 provinces (autonomous regions, municipalities directly under the Central Government) in China. A total of 1 408 samples were collected from 1 050 feces, 108 grassland soils, 149 fecal heaps and 101 surrounding stables. 614 strains of nematode-trapping fungi were isolated from 3 094 detection dishes and identified as 20 species belonging to 4 genera, including 13 species of Arthrobotrys, of which 11 were identified and 2 were undetermined. There are 4 species of Monacrosporium, 2 species of Dactylella and 1 species of Duddingtonia. The detection rate of nematode-trapping fungi in all samples is 19.82%. The detection rates of nematode-trapping fungi in feces, manure heaps, soil around stables and grassland soil are 11.04%, 33.42%, 39.48% and 52.57% respectively. Arthrobotrys oligospora var. oligospora, Arthrobotrys conoides, Arthrobotrys dactyloides, Arthrobotrys musiformis, Arthrobotrys superba, Monacrosporium thaumasium and D. flagrans were detected. Among the recorded nematode-trapping fungi, 17 species produced viscous nets with a frequency of 94.46%. Only 2 species produced other predatory structures, such as contractile rings or viscous spheres and non-contractile rings, accounting for 3.74%. A. conoides, A. dactyloides, A. musiformis, A. superba and M. thaumasiun were found in different areas. D. flagrans is an important candidate strain for biological control of animal parasitic nematodes. In this investigation, it was found in Ningxia, Gansu, Heilongjiang, Shandong and Guizhou provinces, and in feces and dunghills. (2) D. flagrans were cultured on 0.2 g/L corn flour agar medium (CAM) and inoculated on CAM covered with cellulose membrane for scanning electron microscopy observation. After 7 days of culture, experimental nematodes were added, and then the agar containing worms and fungi was obtained at 0, 1, 2, 3, 4, 6, 8, 12, 20, 24, 36 and 48 hours after interaction. The results showed that the predatory structure could be formed spontaneously after 36 h incubation without the experimental nematode, the predator could be induced after 30 min interaction with the insect, and the parasite was also caught; 4 h after the capture, the mycelium invaded C. elegans and the L3 was killed quickly. It took 20-24 hours for the mycelium to invade and kill, 24 hours after capture, the whole C. elegans was invaded and digested completely by the mycelium, while L3 was invaded completely by the mycelium for 36 hours and digested completely for 48 hours, leaving only the epidermis. (3) The predatory fungal isolate D. leptospora (IS-307) was observed on corn flour agar medium at 4,5,6,7,8,9,10,11,12 hours. The results showed that the strain could grow at pH 4-12 and temperature 15-30, and the optimum culture conditions were pH 11.0 and temperature 25.
【学位授予单位】：西北民族大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.9

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