【作者】 赵杏利；

【导师】 牛永春；

【作者基本信息】 中国农业科学院 ， 农业微生物， 2012， 博士

【摘要】 微生物除草剂因其环境影响小、杂草不易产生抗性、研发周期短、研发成本低等特点越来越受到重视。本研究在对自然发病的常见禾本科杂草样本采集的基础上，对病原真菌进行了分离、鉴定和筛选，并对除草潜力菌株的次级代谢产物进行了分离提取、结构鉴定和植物毒性分析，以期为杂草病原真菌资源的利用和微生物除草剂的研发提供基础和依据。于2009年7月下旬至8月上旬，从山东省的淄博、潍县、泰安、青岛、曲阜和江苏省的淮安、南京、常州、无锡、苏州以及上海等地的农田和草坪中采集狗尾草、马唐、牛筋草、稗草等常见禾本科杂草的病害样本99份，共分离获得206个真菌菌株，其中产孢菌株有160个。经形态学观察和对67个菌株的rDNA ITS区域序列测定和分析，共鉴定到11属21种真菌： Alternariacompacta、 Bipolaris cynodontis、 B. heveae、 B. sacchari、 B. sorokiniana、 B.setariae、 Cladosporium cladosporioides、 Colletotrichum dematium、Colletotrichum gloeosporioides、 Curvularia affini、 Curvularia intermedia、Curvularia lunata、 Exserohilum rostratum、 E. fusiforme、 Fusarium equiseti、F. lateritium、F. tricinctum、Nigrospora oryzae、N. sphaerica、Phoma glomerata、P. herbarum。其中平脐蠕孢属（Bipolaris）、弯孢属（Curvularia）和链格孢属（Alternaria）真菌是常见禾本科杂草上的主要真菌种类。采用孢子悬浮液接种法、菌种培养粗滤液离体叶片浸渍法、种子根和茎的抑制法，对从本研究分离的206个菌株和作者前期工作分离的689个菌株中选出的156个菌株进行了试验。筛选出的6个菌株NY1、ZXL09013a、ZXL09025a、ZXL09035a、ZXL09066d、ZXL09078a的粗滤液均能在狗尾草叶片上产生明显褐斑，且对狗尾草、马唐、稗草的根和茎都产生了明显抑制作用。室外接种试验表明，狗尾草平脐蠕孢菌株NY1和麦根腐平脐蠕孢菌株ZXL09078a能引起狗尾草严重发病，接种10天后，狗尾草叶片和茎秆上均布满褐色病斑，有的叶片病斑连片呈枯死状，整个植株的生长势明显减弱。对菌株NY1和菌株ZXL09078a的寄主范围测定表明，两菌株对禾本科植物均能引起不同程度的发病，尤其对虎尾草、马唐等能引起严重发病，而对非禾本科植物均只引起轻度发病或不引起发病，表现出一定的寄主选择性。从菌株NY1的大米发酵物中共分离和鉴定出7种化合物：邻苯二甲酸二异壬酯、3-脱羟基蛇孢菌素A、蛇孢菌素I、麦角甾-5,7,22-三烯-3β-醇、5α,8α-过氧化麦角甾-6,22-二烯-3β-醇、6-甲基苯-1,2,4-三酚、邻羟基苯甲醛。从菌株ZXL09078a的PD发酵液中分离和鉴定出化合物长蠕孢醇。对狗尾草的植物毒性测定表明，蛇孢菌素类化合物在狗尾草叶片上引起的褐色近圆形病斑与其产生菌B. setariae菌株NY1接种狗尾草时引起的病斑相同，因此蛇孢菌素类化合物是菌株NY1次级代谢产物的主要植物毒性成分。其中3-脱羟基蛇孢菌素A在狗尾草叶片上形成病斑最快，引起病斑所需浓度最低，因此其植物毒性最强。3-脱羟基蛇孢菌素A溶液、含有3-脱羟基蛇孢菌素A及其它2种蛇孢菌素的混合液对供试单子叶植物均有不同程度的毒性，尤其对狗尾草、大画眉草、马唐、稗草、谷子、甜高粱和鸭跖草的毒性较强。它们对双子叶植物的毒性则因植物种类不同有很大差异，如对藜、龙葵、马齿苋等植物毒性较强，对醴肠、山苦荬等植物毒性较弱，对草莓、花生、向日葵、绿豆等植物则无毒性。相同浓度条件下，混合物所引起的病斑明显大于3-脱羟基蛇孢菌素A单独使用时引起的病斑，说明不同的蛇孢菌素之间具有协同增效的作用。更多还原

【Abstract】 Microbial herbicides are receiving more and more attention because of theirlittle effect on environment, difficulty for weeds to develop resistance, shortperiod and low cost of R&D. In this study, isolation, identification andscreening of pathogenic fungi were performed on the basis of sample collectionof diseased gramineous weeds in the field. The secondary metabolites from twoisolates with herbicidal activity were separated, structurally identified andphytotoxically tested. The results provided the basis and information forutilization of pathogenic fungal resources on weeds and development ofmicrobial herbicides.In grass growing season of2009, field survey and sample collection ofdiseases were carried out on common gramineous weeds, such as Setaria viridis,Digitaria sanguinalis, Eleusine indica, Echinochloa crusgalli etc. Two hundredand six fungal isolates were obtained from99samples collected from ten citiesand counties of Shandong and Jiangsu provinces and Shanghai. Based onmorphological characteristics of160spore-producing isolates and sequencealignments of rDNA ITS of67isolates,21species of11genera were identified:Alternaria compacta, Bipolaris cynodontis, B. heveae, B. sacchari, B.sorokiniana, B. setariae, Cladosporium cladosporioides, Colletotrichumdematium, Colletotrichum gloeosporioides, Curvularia affini, Curvulariaintermedia, Curvularia lunata, Exserohilum rostratum, E. fusiforme, Fusariumequiseti, F. lateritium, F. tricinctum, Nigrospora oryzae, N. sphaerica, Phomaglomerata, P. herbarum. Among them, the fungi belonging to Alternaria,Bipolaris, and Curvularia were the primary on the common gramineous weeds.One hundred and fifty-six isolates, selected from the206isolates obtainedin this study and the689isolates obtained in earlier study, were tested by usinginoculation with spore suspension, detached leaves, seminal root and ste mdipping with the crude culture filtrate of the fungal isolates. The crude culturefiltrates from six isolates NY1, ZXL09013a, ZXL09025a, ZXL09035a,ZXL09066d and ZXL09078a caused obvious brown spot on leaves of Setariaviridis, and remarkable inhibitive effect on the seminal roots and stems ofSetaria viridis, Digitaria sanguinalis and Echinochloa crusgalli. The results ofoutdoor inoculation showed that the isolate NY1of B. setariae and the isolateZXL09078a of B. sorokiniana caused severe disease on Setaria viridis. Ten daysafter inoculation, leaves and stems of the grass were covered with brown spots,and some spots merged and leaves wilted. Both of the two isolates causeddisease on the tested gramineous plants in different degrees, especially severe disease on Setaria viridis and Digitaria sanguinalis, but light or no disease onthe tested non-gramineous plants.Seven compounds were obtained from the ethyl acetate extract of ricefermentation of B. setariae and determined as diisononyl phthalate,3-anhydroophiobolin A, ophiobolin I, ergosta-5,7,22-trien-3β-ol,5α,8α-epidioxyergosta-6,22-diene-3β-ol,6-methylbenzene-1,2,4-triol, and2-hydroxybenzaldehyde, respectively, by extensive spectroscopic analysis.Bioassay on punctured and intact detached leaves of Setaria viridis indicatedthat ophiobolins produced obvious brown lesions, which were similar to thatcaused by the pathogen B. setariae. It suggested that ophiobolins were theprimary phytotoxic me tabolites of the isolate NY1of B. setariae. Among them,the formation of the lesions produced by3-anhydroophiobolin A was the fastestand the concentration of agent was the lowest. Therefore the phytotoxicity of3-anhydroophiobolin A was the highest.In bioassay on punctured detached leaves of43plant species of18families,both the3-anhydroophiobolin A and the mixture of three ophiobolins including3-anhydroophiobolin A showed phytotoxic to all of the monocotyledons tested indifferent degrees, especially strong phytotoxicity on Setaria viridis, Eragrostiscilianensis, Digitaria sanguinalis, Echinochloa crusgalli, Setaria italic,Sorghum dochna and Commelina communis. The phytotoxicity of the3-anhydroophiobolin A and the mixture to the tested dicotyledons varied greatlyon different plant species. There were strong toxicity on Chenopodium album,Solanum nigrum, Euphorbia humifusa, Oxalis corniculata and Portulacaoleracea and no phytotoxicity on Helianthus annuus, Lactuca sativa, Spinaciaoleracea, Potentilla supine, Fragaria ananassa, Arachis hypogaea andPhaseolus radiates. The results also indicated that the lesions produced by themixed solutions were larger than those produced by3-anhydroophiobolin A,which suggested the synergy between different ophiobolins.更多还原