【摘要】：自1873年Metchnkov首次使用金龜子綠僵菌(Metarhizium anisopliae)成功防治奧地利金龜(Anisoplia austriaca)后,綠僵菌作為真菌殺蟲劑被廣泛研究。隨后在1883年Sorokin以金龜子綠僵菌為模式種建立綠僵菌屬M(fèi)etarhizium,綠僵菌的研究步入以形態(tài)特征為主要分類依據(jù)的時(shí)期。隨著分子技術(shù)和基因工程的發(fā)展,分子數(shù)據(jù)便被廣泛用于系統(tǒng)發(fā)育研究。尤其是PCR技術(shù)的出現(xiàn)及DNA測(cè)序技術(shù)的不斷完善,使得系統(tǒng)發(fā)育分析成為可能。近年來系統(tǒng)發(fā)育分析的方法日益完善,多基因系統(tǒng)發(fā)育關(guān)系分析也被廣泛應(yīng)用于反映物種的進(jìn)化歷史。本文主要研究了中國綠僵菌的分類情況。首先將采集的僵蟲用僵蟲分離法分離綠僵菌,即用細(xì)針從僵蟲上挑取綠色孢子于SDAY培養(yǎng)基上劃線培養(yǎng)篩選綠僵菌。對(duì)采集的土壤用多果定選擇性培養(yǎng)基篩選綠僵菌。待這兩種培養(yǎng)基上的菌落產(chǎn)孢后,分別觀察菌落形態(tài),即菌絲和孢子的顏色,并在光學(xué)顯微鏡下觀察孢子形態(tài)大小,待初步鑒定為綠僵菌后,將目標(biāo)菌株轉(zhuǎn)移至SDAY培養(yǎng)基上純化。結(jié)合安徽農(nóng)業(yè)大學(xué)微生物防治重點(diǎn)實(shí)驗(yàn)室菌種庫(RCEF)中的綠僵菌種源,對(duì)其進(jìn)行活化培養(yǎng)。通過分子學(xué)方法和形態(tài)學(xué)方法對(duì)得到的共134株綠僵菌菌株進(jìn)行鑒定。根據(jù)KeplerRehner[1]的研究表明,Mz IGS3基因能夠有效鑒定金龜子綠僵菌復(fù)合種。為了驗(yàn)證Mz IGS3基因?qū)瘕斪泳G僵菌復(fù)合種的鑒定效果,實(shí)驗(yàn)通過Mz IGS3基因?qū)?34株綠僵菌進(jìn)行初步鑒定,結(jié)合EF-1a基因?qū)ζ渌G僵菌進(jìn)行補(bǔ)充性初步鑒定,再通過三基因測(cè)序?qū)z IGS3基因和EF-1a基因初步鑒定中的模糊種進(jìn)一步鑒定。1、實(shí)驗(yàn)通過MzIGS3基因?qū)?34株綠僵菌進(jìn)行初步鑒定。通過Mz IGS3基因擴(kuò)增,最終得到100株菌株的序列。根據(jù)這100株菌株序列構(gòu)建的單基因系統(tǒng)發(fā)育樹,可將這100株綠僵菌劃分為Clade I~Clade VIII共8個(gè)大分支。依據(jù)Mz IGS3系統(tǒng)發(fā)育樹可知,分支A、B、C構(gòu)成Clade I。分支C鑒定為平沙綠僵菌Metarhizium pingshaense,分支A、B中的菌株為模糊種。Clade II鑒定為洛泊斯綠僵菌Metarhizium robertsii。Clade III鑒定為金龜子綠僵菌Metarhizium anisopliae。Clade IV可能為棕色綠僵菌Metarhizium brunneum。Clade V由分支G、H、I組成,分支中的菌株均為模糊種,需進(jìn)一步鑒定。Clade VI鑒定為大孢綠僵菌Metarhizium majus。Clade VII鑒定為鱗腮綠僵菌Metarhizium lepidiotae。Clade VIII鑒定為蚱蜢綠僵菌Metarhizium acridum。2、EF-1a基因初步鑒定中10株鑒定為金龜子綠僵菌復(fù)合種中的菌株,4株鑒定為柱孢綠僵菌Metarhizium cylindrospora,13株鑒定為癭綿蚜綠僵菌Metarhizium pemphigi,4株為模糊菌株,1株為低溫綠僵菌Metarhizium frigidum,1株為萊氏綠僵菌Metarhizium rileyi,1株未知菌株Ma123。3、多基因分析能準(zhǔn)確的鑒定不同的種,利用多基因構(gòu)建的系統(tǒng)發(fā)育樹更接近真實(shí)的物種樹,并準(zhǔn)確的反映不同物種的分類地位。結(jié)合三基因發(fā)育分析,對(duì)于Mz IGS3基因系統(tǒng)發(fā)育樹中模糊菌株進(jìn)行進(jìn)一步鑒定。Clade I中的Ma106和Ma141鑒定為金龜子綠僵菌,其他菌株均鑒定為平沙綠僵菌。Clade IV鑒定為棕色綠僵菌。Clade V中分支G中的Mf03鑒定為平沙綠僵菌,其他菌株鑒定為貴州綠僵菌Metarhizium guizhouense,分支H鑒定為大孢綠僵菌,分支I鑒定為柱孢綠僵菌。對(duì)于EF-1a基因系統(tǒng)發(fā)育樹中的模糊菌株進(jìn)一步鑒定,4株為模糊菌株鑒定為癭綿蚜綠僵菌,未知菌株Ma123鑒定為一新種——安徽綠僵菌Metarhizium anhuiense。根據(jù)三基因分析并結(jié)合形態(tài)特征,可知本文的134株菌株共可分為13個(gè)種,即金龜子綠僵菌、棕色綠僵菌、平沙綠僵菌、洛泊斯綠僵菌、貴州綠僵菌、大孢綠僵菌、鱗腮綠僵菌、蚱蜢綠僵菌、柱孢綠僵菌、癭綿蚜綠僵菌、低溫綠僵菌、萊氏綠僵菌、安徽綠僵菌。這13個(gè)種包括3個(gè)中國新記錄種和1個(gè)新種。3個(gè)中國新記錄種分別是棕色綠僵菌、洛泊斯綠僵菌和低溫綠僵菌。新種為安徽綠僵菌。4、菌株Ma123鑒定為一新種,即安徽綠僵菌Metarhizium anhuiense。Ma123在SDAY/4培養(yǎng)基上25℃培養(yǎng),7天菌落直徑約15-17mm,14天菌落直徑達(dá)到33-52mm。菌落絨毛狀,最初白色,產(chǎn)孢時(shí)從中心部位開始產(chǎn)孢,向外圍擴(kuò)散,開始為黃色,后逐漸成綠色。培養(yǎng)時(shí)有時(shí)會(huì)有紫色的代謝產(chǎn)物液滴產(chǎn)生。邊緣規(guī)則平整。菌絲有隔、透明、光滑,直徑1.2-2.6μm。分生孢子梗上多1-3個(gè)瓶梗,瓶梗柱形,6.4-18.3×2.0-3.5μm。分生孢子單細(xì)胞,柱狀6.6-4.8×3.7-1.5μm,較短。孢子一般在瓶梗上向基性排列成分生孢子長鏈。
[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.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S476.12

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