【作者】 贾凤姣；

【导师】 黄俊斌；

【作者基本信息】 华中农业大学 ， 植物病理学， 2007， 硕士

【摘要】 叶斑病是发生在高尔夫球场草坪上的一种重要病害，主要引起多种草坪草的叶斑和叶枯，严重影响草坪景观，破坏草坪质量。目前对该病的研究仍仅限于病害的调查和初步的化学防治，国内外还未见有较系统的研究报道。本研究旨在确定引起狗牙根叶斑病的病原，了解病菌的寄主范围、生物学特性，以及进行杀菌剂的初步筛选和病菌对四种草坪草的侵入过程观察。研究结果如下：从鄂州红莲湖高尔夫球场的狗牙根发病植株叶片上分离菌株Curvularia sp.，用菌丝块接种狗牙根叶片进行致病性测定，证实Curvularia sp.为致病菌。该菌在PDA培养基上培养5d后，菌落呈墨绿色，菌丝褐色，有隔，分枝，分生孢子平均大小为11.07×25.18μm，褐色至暗褐色，椭圆形，具有3个隔膜，主隔膜明显加粗，中间两细胞较大。rDNA-ITS测序结果与GeneBank中的序列进行同源性比较发现菌株与Curvularia verruculosus的同源性高达99％。根据形态学特征和分子鉴定结果，确认该病原菌为Curvularia verruculosus。寄主范围测定结果表明，该菌可侵染供试的四种草坪草(狗牙根、黑麦草、高羊茅、早熟禾)、水稻、小麦和玉米等禾本科植物。病原菌生物学特性研究结果表明，在有无光照或连续光照的条件下病菌均可生长和产孢，光暗交替最有利于菌丝的生长，黑暗条件下促进病菌产孢。该菌株在15-35℃的范围内均能生长和产孢，30℃为菌丝生长最快温度，最适产孢温度为25℃。病菌在pH值为4-12的PDA培养基上菌丝均能生长，其中以pH7生长最快；病原菌在pH 5-12范围内均可产孢，产孢最适pH值为6。病菌能够有效利用多种碳源和氮源，甚至在无碳或无氮条件下都能生长，但不同碳源和氮源对弯孢霉病菌菌丝生长和产孢有极显著的影响。病原菌分别以淀粉为碳源以及以酵母膏为氮源时生长最好，半乳糖和硝酸钙有利于该菌分生孢子的产生。试验证明55℃的温水处理10min可杀死菌丝体、抑制孢子萌发。PDA平板抑菌实验结果表明，供试六种药剂在设置的不同浓度下对病原菌的生长都有一定的抑制作用，其中10％世高可分散粒剂和30％爱苗乳油对菌丝生长有很好的抑制效果，10％多抗霉素B粉剂抑制效果最差。喷雾病原菌分生孢子悬浮液于狗牙根、黑麦草、高羊茅、早熟禾等几种草坪草活体叶片上并保湿培养，在接种后不同时间点分别从每个寄主上取样，对样品进行组织透明、染色，观察病原菌对寄主植物的侵染过程。结果表明，在四种寄主上接种后3h内均可以观察到分生孢子的萌发，分生孢子可以从一端或两端萌发，产生单根或2-4根芽管。接种后3h时分生孢子在不同寄主叶片上的萌发率没有差异性，而6h后分生孢子在不同寄主上的萌发率有微小差异，12h时，病原菌分生孢子在狗牙根和早熟禾叶片上的萌发率分别为96％和92％，略高于其在黑麦草和高羊茅上的萌发率。弯孢霉分生孢子在四种不同的草坪草上都较易萌发，24h时萌发率均超过90％，48h时在狗牙根叶片上萌发率达100％。病原菌分生孢子在寄主表面萌发后，可以从叶片气孔或表皮直接侵入，其中以气孔侵入为主。接种后6h便可以观察到有病菌从气孔侵入，病原菌分生孢子在四种草坪草寄主上从气孔侵入的百分率有所差异，24h时萌发的分生孢子在狗牙根和早熟禾上侵入的百分率较在黑麦草和高羊茅上的侵入百分率高。菌丝直接侵入寄主组织的情况比较少，接种后12h可观察到部分菌丝分支产生类似侵染钉的结构从同一部位或不同部位直接侵入寄主细胞。更多还原

【Abstract】 The Leaf spot is one of important diseases in golf courses. It causes leaf spot and leaf blight on many species of grasses, and can influence sights and destroy quality of grasses. At present there are only reports about the disease investigation and primary chemical control. The aim of this study is to indentify the pathogen of bermudagrass Leaf spot, to understand host range, biological characteristics, and the infection process of the pathogen on four species of grasses. The results showed as follow:Curvularia sp. was isolated from the diseased leaf of Cynodon hybrid in Red Lotus Lake golf courses in E-zhou. Pathogenicity was tested by inoculating the leaf with mycelium plug of Curvularia sp., the result indicated that Curvularia sp. isolate was the pathogen. The morphological characteristic showed that the colonies were black green and the hyphae were septum, brown, Spore size was 11.07×25.18μm, mostly ellipse, with three septate , two central cells were larger than both terminal cells , brown to dark brown , terminal cells subhyaline to light Brown after cultured on PDA medium at 5d. The DNA sequence of ribosomal ITS of the isolates was found to match 99% with an ITS sequence of Curvularia verruculosus in GenBank. According to the morphological characteristics, and the DNA sequence of ribosomal ITS, the pathogen was identified as C.verruculosus. The pathogen can infected four turfgrass species （Cynodon hybrid, Lolium perenne, Agrostis plustris, Poa pratensis） and other gramineous plants （ Oryzae sativa, Blumeria tristic, Zea mays）.The studies on biological characteristic revealed that this fungus could grow and sporulate under dark or continuous illumination, the optimal illumination condition for growth of mycelium was alternant illumination and darkness facilitated spotulation.The mycelium could grow under the temperature from 15-35℃, the optimal temperature for the mycelial growth and sporulation was 30℃and 25℃, respectively. pH value range for mycelium growth were 4-12 and pH7 was optimal. pH value range for sporulation were 5-12 and pH6 was optimal. The isolate could utilize various carbon and nitrogen sources, but different carbon and nitrogen sources had different influence for mycelial growth and sporulation. Among carbon and nitrogen sources, starch and yeast extract were best for mycelial growth, D-galctose and CaNO₃ were best for sporulation.The lethal temperature for mycelial growth and spores gemination were 55℃for 10 min, respectively.The result of determining of the toxicity of fungicides to C.verruculosus showed that the six fundicides choosed could inhibit mycelial growth at different concentration. 10% Score GR and 30% Armure EC showed best inhibiting to mycelial growth, but the 10% polyoxin B DP was the worst.Leaves of four turfgrass species （Cynodon hybrid, Lolium perenne, Agrostis plustris, Poa pratensis） were inoculated with conidial suspensions of C.verruculosus. Inoculated leaves were incubated at 25℃under high humidity.Samples were collected from inoculated leaves at different timing intervals,and then decolorated and stained. Observe the infection process on four species of grasses with fluorescence microscope under bright field.. The result indicated that the conidia began to germinate after 3 hours of inoculation, and formed one to four primary germtubes from one or two ends. The rate of conidial germination of C.verruculosus on leaves of four turfgrass species after 3h of inoculation showed no significant differences. After 12 hours of inoculation, the percentage of germination were significantly larger on the Cynodon hybrid and Poa pratensis than on the other two hosts.The pathogen C.verruculosus was able to penetrate host through two paths such as leaf stomata and epidermis.The penetration from leaf stomata was considered as a major way, and can be observed after 6h of inoculation.The rate of pathogen infecting from leaf stomata of four turfgrass species showed significant differences. After 24 hours of inoculation, the percentage of infecting were significantly larger on the Cynodon hybrid and Poa pratensis than the other two hosts. Direct penetration into epidermis was uncommon observed.更多还原