【作者】 宁红；

【导师】 刘世贵；

【作者基本信息】 四川大学 ， 遗传学， 2002， 博士

【摘要】 玉米弯孢菌叶斑病是近几年在我国发展起来的一种对玉米危害很大的新病害，关于病菌致病性分化及遗传方面的报道较少，目前还不清楚病菌的生理分化情况，也少有关于病原菌遗传背景分析的研究，更缺乏病原菌快速鉴定和带菌种子检测技术的研究，本研究以病菌孢子形态学和可溶蛋白质多样性比较为基础，建立了两种免疫学的鉴定技术，并将该技术用于带菌玉米种子和幼苗的检测和诊断；同时，在分子水平用AFLP标记技术对该菌的遗传多样性用进行了分析，同时与多种相似病菌的AFLP分子标记进行了比较研究，确定了代表Curvularia lunata的特异性分子标记，为下一步的克隆、测序和建立分子生物学鉴定方法提供了捷径，也为植物病原真菌鉴定的分子标记筛选提供了范例。 首先，搜集国内外和分离四川主要玉米产区的玉米弯孢菌叶斑病样品，共获得73个纯化的相关菌株，其中包括C. lunata菌株34个，Cochlibolus carbonum菌株3个，Co. heterostrophus菌株13个，Alternaria alternata菌株8个。对其中的12个C. lunata菌株和6个相似或分离过程中容易混淆的菌株在分生孢子形态、菌落型态和可溶性蛋白质电泳图谱进行了比较研究。结果表明分生孢子形态是病原菌形态鉴定的主要依据，但在C. lunata和C. inaequalis之间孢子形态并不易区分；菌落型态在不同种之间存在明显的差异，同种之间也存在一定差异，但不稳定和存在变异。18个菌株SDS聚丙烯酰胺凝胶电泳图谱在种间存在明显的差异，C. lunata种内蛋白带也存在多样性。图谱显示Curvularia在Rf值为0.177处有一条该属的特征蛋白带，C. lunata在Rf值为0.225处有一条该种的特征蛋白带。聚类分析将未能确定种的13号菌株确定为C. inaequalis。 在以上蛋白质分析的基础上建立了C. lunata菌株鉴定和种子的免疫检 四川大学博土学位论文 测方法。用于 I．ELISA、ABC－ELISA和 DOt－ELISA三种方法比较研究了不 同抗血清的检测灵敏度和特异世，结果IELISA方便快捷，首选使用； D。t－ELISA灵敏度稍低适用于基层检验。用 C 菌株混合的可溶性蛋白 质作为免疫抗原，获得一抗兔抗 C Iunata抗血清（PAhs）；抗血清经近似菌 可溶性蛋白质吸附后获得对C lunata菌株可溶性蛋白质具有特异性的抗血 清（APAbs卜 可应用于 C lunata菌株的鉴定；用玉米种子可溶性蛋白质对 APAs进行进一步吸附纯化，获得了与玉米种子蛋白质无交叉反应的特异性 抗血清（DAPAbS人可用于对带菌玉米种子的检测鉴定，并制定了相应的应 用程序。几种抗血清分别用于间接ELISA和ABC＊LISA兔疫分析，以比较 其检测灵敏度和特异性。 在免疫检测的基础上，为了从分子水平进一步了解新月弯抱菌的遗传背 景和多样性，以寻找能够作为 C Iunata种的分子标记，从而为建立该菌的分 子检测技术奠定基础，本研究首次用 AFLP指纹技术对C Iunata不同菌株及 相似菌株进行了遗传多样性研究。用改进的氯化书法获得了高质量的供试菌 株DNA，并优化了AFLP各步的试验条件。利用AFLP技术，分析了5个 C lunata菌株和5个不同种的近似菌株AFLP的DNA指纹特征，并进行了 不同引物的聚类分析。分别采用两对具有E．ZM．l 的选择性引物 EcoRJ－AC／Mel．A和EcoAI－AC／Mel．C，以及两对具有E－ZM－3的选择性引 物EcoXi叭 cTG和EcORI叭 （TC对供试菌进行选择性扩增。 结果供试菌株的AFLP指纹图谱具有很高的多态性，反映了菌株间存在丰富 的遗传多样性；四对引物共得到 11 69条扩增条带，其中多态性条带 778条， 占67％。根据不同引物的不同指纹图谱得到的聚类结果反映了 C lunata菌株 间以及近似菌株间种、属的遗传距离和亲缘关系；四对引物分别得到了 944hp、229hp、254hp、130hp、1129hp、296hp六条属于 C lunata的稳定的 特异性片段，这些特异性片段都有希望成为鉴定 C Iunata有效的分子标记， 为进一步确定用于鉴定c1U——的有效的分子标记奠定了基础。研究表明， 利用 AFLP不同的引物组合，不仅能稳定地检出 C Iunata和近似菌株丰富的 多态性，同时也是寻找特异性分子标记的理想方法。更多还原

【Abstract】 Recenily there have been many reports all over the world about fungi diseasecaused by Curvularia lunata [Cochliobolus Iunana]. The economic importanceand the wide range of its hosts can be found from these reports. The biggestcomznedty of the host of C.lunata is gramineous plants such as rice, malze,wheat and sorghurn. Othedrise, cotton, cowpea,sunflower, onion etc could beinfected by C lunata too. C lunata is one of the importan seed-bome mycelialfungus pathogen. Microscopic examination showed C.lunata could be deectedin all components of maize seeds.Maize is the third importan food crop in addition to rice and wheat in China.Com northem leaf blight (Helminthosporium turcicum) and com southern leafblight (Cochliobolus heterostrophus) were the main maize diseases in China inthe past. But recentiy a new disease named maize cedaria leaf spot or malzeyellow spot caused by C.lunata (infrequentiy by C hoequais), is becomingprevalent in north China and is spreadin to south China.The disease ischaraterized by chlorotic spot symptoms on leaves: chlorotic pin points appearedin the beginning, then yellow spots with a white center and a chlorotic halofollowed,and finaly leaves bligh when a lot of spotS joined togetheL Size of thespot usually is 1 × 2mm-2mm. Maize cedaria leaf spot is easy to cothee withcom southem leaf bligh and com helrninthosporium leaf spot (Co. carbonum) insymptoms. C lunata can be found on the sdse of seeds, the spores andmycelium were also found on the buttrss roots, and on the soil surface but not at4dePths of 5 or 10crn. So isolation of the causal agent C lunata is the main methodfOr diagnoses at presen though there are variabilihes of cultural cforteristics ofC lUnata isolates and coWons by other sdriilar fungi occur frequently inthe Process of isolation. There are some reportS on the variabi1ity growh andsPOrulation of isolates from differen geograPhical locations, and on theulbetrUM of C IUnaha. So far there is still laCk of a raPid, sPecific andsensitive tecboque for the diagnosis of C lunata and there are few stUdies on thegenetic baskground of Ans pathogen.ln order to exPlore the differences betWeen C Iunata and otlier sbolar fungiand establish a specific and sensihve diagnostic method fOr C lunata, this stUdyhas compared the moboometrics including the fungi colony and in medium andthe sPores of C Iunata in maize and Othr swhlar forgi aPPearing bequently byisolation. We have compared their protein SDS polyacrytalde gelelectrophoresis pattems too. An immunological detection tecboque of C lunatafrOm cultUre and on the seed based on the assays of Proteins is established. Wealso study the genetic diversihes using amPlified fragYnent 1ength polyInorphismand comPared the molecular markers of C Iunata, the sbolar strains. Six specificPOlymorphic frgnents of AFLP have been found srochronously. They wiIl bethe twrtan molecular markers for idenopg C lunata. The main resultS arefollowed.First we collected 73 pure isolates of C IunaIa and their sindlar stains fromabroad, our councy and the main areas of maize in Sichuan, including 34 isolatesof C Iunata, 3 isolates of CO. Carbonum, l3 isolates of CO. heterosmphus, and 8isolates of A. alternata. TWlve isolates of C Iunata and six sbolar sPecie strainsfrom haze or other hoSt plants wer exaInined for the analysis of mycelioidcolony morphometrics, sPores morphometrics and SDS polyaCryamide geleleCtrOPhoresis pattemS of their soluble proteins. The diffeences of colonymorphometrics of l8 isolates existed arnong differen SPeCies and the same speciebu they wer no W and special. Th differences of SDS POlyaCrylndde geleleCtrphoresis pattems ekisted sighficantiy among differen Species and the5saxne sPecie. There was a sPecific protein band for CimIaria at relative mobilityvalue 0.l77 W0.177) and a sPeCial PrOtein band for C bo at Rf0.225.Second, we have set uP the bological detection tecboque to ide更多还原