中国绿僵菌分子分类的研究

发布时间：2018-09-12 15:01

【摘要】：自1873年Metchnkov首次使用金龟子绿僵菌(Metarhizium anisopliae)成功防治奥地利金龟(Anisoplia austriaca)后,绿僵菌作为真菌杀虫剂被广泛研究。随后在1883年Sorokin以金龟子绿僵菌为模式种建立绿僵菌属Metarhizium,绿僵菌的研究步入以形态特征为主要分类依据的时期。随着分子技术和基因工程的发展,分子数据便被广泛用于系统发育研究。尤其是PCR技术的出现及DNA测序技术的不断完善,使得系统发育分析成为可能。近年来系统发育分析的方法日益完善,多基因系统发育关系分析也被广泛应用于反映物种的进化历史。本文主要研究了中国绿僵菌的分类情况。首先将采集的僵虫用僵虫分离法分离绿僵菌,即用细针从僵虫上挑取绿色孢子于SDAY培养基上划线培养筛选绿僵菌。对采集的土壤用多果定选择性培养基筛选绿僵菌。待这两种培养基上的菌落产孢后,分别观察菌落形态,即菌丝和孢子的颜色,并在光学显微镜下观察孢子形态大小,待初步鉴定为绿僵菌后,将目标菌株转移至SDAY培养基上纯化。结合安徽农业大学微生物防治重点实验室菌种库(RCEF)中的绿僵菌种源,对其进行活化培养。通过分子学方法和形态学方法对得到的共134株绿僵菌菌株进行鉴定。根据KeplerRehner[1]的研究表明,Mz IGS3基因能够有效鉴定金龟子绿僵菌复合种。为了验证Mz IGS3基因对金龟子绿僵菌复合种的鉴定效果,实验通过Mz IGS3基因对134株绿僵菌进行初步鉴定,结合EF-1a基因对其他绿僵菌进行补充性初步鉴定,再通过三基因测序对Mz IGS3基因和EF-1a基因初步鉴定中的模糊种进一步鉴定。1、实验通过MzIGS3基因对134株绿僵菌进行初步鉴定。通过Mz IGS3基因扩增,最终得到100株菌株的序列。根据这100株菌株序列构建的单基因系统发育树,可将这100株绿僵菌划分为Clade I~Clade VIII共8个大分支。依据Mz IGS3系统发育树可知,分支A、B、C构成Clade I。分支C鉴定为平沙绿僵菌Metarhizium pingshaense,分支A、B中的菌株为模糊种。Clade II鉴定为洛泊斯绿僵菌Metarhizium robertsii。Clade III鉴定为金龟子绿僵菌Metarhizium anisopliae。Clade IV可能为棕色绿僵菌Metarhizium brunneum。Clade V由分支G、H、I组成,分支中的菌株均为模糊种,需进一步鉴定。Clade VI鉴定为大孢绿僵菌Metarhizium majus。Clade VII鉴定为鳞腮绿僵菌Metarhizium lepidiotae。Clade VIII鉴定为蚱蜢绿僵菌Metarhizium acridum。2、EF-1a基因初步鉴定中10株鉴定为金龟子绿僵菌复合种中的菌株,4株鉴定为柱孢绿僵菌Metarhizium cylindrospora,13株鉴定为瘿绵蚜绿僵菌Metarhizium pemphigi,4株为模糊菌株,1株为低温绿僵菌Metarhizium frigidum,1株为莱氏绿僵菌Metarhizium rileyi,1株未知菌株Ma123。3、多基因分析能准确的鉴定不同的种,利用多基因构建的系统发育树更接近真实的物种树,并准确的反映不同物种的分类地位。结合三基因发育分析,对于Mz IGS3基因系统发育树中模糊菌株进行进一步鉴定。Clade I中的Ma106和Ma141鉴定为金龟子绿僵菌,其他菌株均鉴定为平沙绿僵菌。Clade IV鉴定为棕色绿僵菌。Clade V中分支G中的Mf03鉴定为平沙绿僵菌,其他菌株鉴定为贵州绿僵菌Metarhizium guizhouense,分支H鉴定为大孢绿僵菌,分支I鉴定为柱孢绿僵菌。对于EF-1a基因系统发育树中的模糊菌株进一步鉴定,4株为模糊菌株鉴定为瘿绵蚜绿僵菌,未知菌株Ma123鉴定为一新种——安徽绿僵菌Metarhizium anhuiense。根据三基因分析并结合形态特征,可知本文的134株菌株共可分为13个种,即金龟子绿僵菌、棕色绿僵菌、平沙绿僵菌、洛泊斯绿僵菌、贵州绿僵菌、大孢绿僵菌、鳞腮绿僵菌、蚱蜢绿僵菌、柱孢绿僵菌、瘿绵蚜绿僵菌、低温绿僵菌、莱氏绿僵菌、安徽绿僵菌。这13个种包括3个中国新记录种和1个新种。3个中国新记录种分别是棕色绿僵菌、洛泊斯绿僵菌和低温绿僵菌。新种为安徽绿僵菌。4、菌株Ma123鉴定为一新种,即安徽绿僵菌Metarhizium anhuiense。Ma123在SDAY/4培养基上25℃培养,7天菌落直径约15-17mm,14天菌落直径达到33-52mm。菌落绒毛状,最初白色,产孢时从中心部位开始产孢,向外围扩散,开始为黄色,后逐渐成绿色。培养时有时会有紫色的代谢产物液滴产生。边缘规则平整。菌丝有隔、透明、光滑,直径1.2-2.6μm。分生孢子梗上多1-3个瓶梗,瓶梗柱形,6.4-18.3×2.0-3.5μm。分生孢子单细胞,柱状6.6-4.8×3.7-1.5μm,较短。孢子一般在瓶梗上向基性排列成分生孢子长链。
[Abstract]:Since Metchnkov first used Metarhizium anisopliae to control Austrian turtles (Anisoplia austriaca) in 1873, Metarhizium has been extensively studied as a fungal insecticide. Sorokin established Metarhizium in 1883, using Metarhizium anisopliae as a model species. Metarhizium has entered the field of morphological characterization. With the development of molecular technology and genetic engineering, molecular data have been widely used in phylogenetic studies. In particular, the emergence of PCR and the continuous improvement of DNA sequencing technology have made phylogenetic analysis possible. Metarhizium anisopliae is separated by the method of isolating Metarhizium anisopliae, i.e. using fine needles to select green spores from Metarhizium anisopliae and lined on SDAY medium for screening Metarhizium anisopliae. The colony morphology, namely the colour of mycelium and spore, was observed respectively after sporulation on the two media. The morphology and size of spore were observed under light microscope. After preliminary identification of Metarhizium anisopliae, the target strain was transferred to SDAY medium for purification. A total of 134 strains of Metarhizium anisopliae were identified by molecular and morphological methods. The results of Kepler Rehner [1] showed that Mz IGS3 gene could effectively identify Metarhizium anisopliae complex. To verify Mz IGS3 gene against Metarhizium anisopliae complex The results showed that 134 strains of Metarhizium anisopliae were preliminarily identified by Mz IGS3 gene, and the complementarity of other Metarhizium anisopliae was preliminarily identified by EF-1a gene. The fuzzy species in the preliminary identification of Mz IGS3 gene and EF-1a gene were further identified by three gene sequencing. 1. 134 strains of Metarhizium anisopliae were preliminarily identified by Mz IGS3 gene. The sequence of 100 strains was obtained by amplification of Mz IGS3 gene. According to the single gene phylogenetic tree constructed by these 100 strains, the 100 strains of Metarhizium Pingsha could be divided into 8 branches: Clade I~Clade VIII. According to the phylogenetic tree of Mz IGS3, branches A, B and C constituted Clade I. Branch C was identified as Metarhizium pingsha. Clade II was identified as Metarhizium robertsii. Clade III was identified as Metarhizium anisopliae. Clade IV may be Metarhizium anisopliae. Clade V. Metarhizium brunneum. Clade V is composed of branch G, H, I. The strains in the branch are all fuzzy species and need further identification. Metarhizium majus. Clade VII was identified as Metarhizium lepidiotae. Clade VIII was identified as Metarhizium acridum. 2, EF-1a gene preliminary identification of 10 strains identified as Metarhizium cylindrospora, 13 strains identified as Metarhizium cylindrospora. For Metarhizium pemphigi, 4 strains were fuzzy, 1 strain was Metarhizium frigidum, 1 strain was Metarhizium frigidum, 1 strain was Metarhizium rileyi, and 1 unknown strain was Ma123.3. Polygenic analysis could accurately identify different species, and the phylogenetic tree constructed by polygenic method was closer to the real species tree, and could accurately reverse. Ma106 and Ma141 in Clade I were identified as Metarhizium anisopliae, and other strains were identified as Metarhizium anisopliae. Clade IV was identified as Metarhizium anisopliae. Mf03 in branch G of Clade V was identified as Metarhizium anisopliae. Metarhizium guizhouense from Guizhou, Metarhizium guizhouense from Guizhou, Metarhizium megasporum from H and Metarhizium guizhouense from I were identified as Metarhizium anisosporum, and Metarhizium guizhouense from EF-1a was identified as Metarhizium guizhouense from the phylogenetic tree. Metarhizium anhuiense. According to the analysis of three genes and morphological characteristics, 134 strains of Metarhizium anisopliae can be divided into 13 species: Metarhizium anisopliae, Metarhizium anisopliae, Metarhizium anisopliae, Metarhizium anisopliae, Metarhizium anisopliae, Metarhizium anisopliae, Metarhizium anisopliae, Metarhizium anisopliae, Metarhizium anisopliae, Metarhizium anisopliae, Metarhizium anisopliae, Metarhizium anisopliae Metarhizium anisopliae, Metarhizium anisopliae, and Metarhizium anisopliae. Ma123 were identified as a new species in SDAY/4 medium. Colony diameter was about 15-17 mm on the 7th day and 33-52 mm on the 14th day. Colony villous, initially white, sporulation began from the central part of sporulation, began to diffuse to the peripheral, began to be yellow, then gradually green. Sometimes there were purple metabolite droplets produced during culture. The edge was regular and smooth. The hyphae was septal, transparent, smooth, and 1.5 mm in diameter. 2-2.6 micron. There are 1-3 more pedicels on the conidia. The pedicels are columnar, 6.4-18.3 *2.0-3.5 micron. The conidia are single-celled, columnar, 6.6-4.8 *3.7-1.5 micron. The conidia are usually arranged in long chains of basal components on the pedicel.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.12

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