【作者】 王建书；

【导师】 强胜；

【作者基本信息】 南京农业大学 ， 植物学， 2013， 博士

【摘要】 马唐属(Digitaria)杂草广泛分布于世界的热带和温带地区,是危害玉米、棉花、大豆、高粱、花生、甘蔗、烟草等秋熟旱作田的世界性恶性杂草。目前对马唐属杂草主要通过人工、机械和化学的方法进行防除,但效果并不十分理想,并且带来很大的负面影响。画眉草弯孢霉(Curvularia eragrostidis J. A. Meyer)属于真菌界、半知菌类、腔菌目、腔菌科、弯孢霉属(Curvularia Boedijn.),画眉草弯孢霉菌株QZ-2000作为新开发的生物除草剂——敌散克(Disancu)的马唐致病菌,对马唐具有较强的致病性。此前的相关研究已经涉及到菌株QZ-2000的生物学、大批量生产工艺、剂型研制、寄主范围、室内和田间除草剂效果评价等方面。但是,有关该真菌与寄主间的相互作用的研究较少,这种互作关系可以揭示画眉草弯孢霉菌株QZ-2000对马唐防控的机制,而这正关系到最终的防除效果和产品的稳定性。因此,本文围绕画眉草弯孢霉菌株QZ-2000与防治对象马唐的相互作用,开展侵染过程中防除对象的反应机制和大批量生产方法以及孢子贮藏对产品效果影响的研究,为研发效果稳定的敌散克产品提供理论基础。本文首先介绍了画眉草弯孢霉菌株QZ-2000与马唐互作的生理生化、叶绿素荧光动力学参数的变化和基因差异表达,揭示了两者的互作机制；最后讨论了画眉草弯孢霉菌株QZ-2000孢子活力的影响因素,提出了保持和提高孢子致病力的方式,为发挥画眉草弯孢霉菌株QZ-2000在与马唐互作中的效力、开发生物除草剂奠定基础。使用画眉草弯孢霉菌株QZ-2000孢子悬浮液喷施马唐幼苗,从处理后第一天开始,连续测定马唐叶绿素、可溶性蛋白和丙二醛的含量；并利用聚丙烯酰胺凝胶电泳适时测定酯酶同工酶。与对照相比,各处理马唐叶片的叶绿素和可溶性蛋白含量呈下降趋势,丙二醛含量呈上升趋势；酯酶同工酶带也有差异。孢子悬浮液与低剂量化学除草剂复配处理也有类似效果,马唐叶绿素和可溶性蛋白含量下降幅度和丙二醛含量上升幅度最大,低剂量化学除草剂处理的三种指标变化幅度最小。真菌孢子在与杂草互作中的强弱是相对的,与低剂量化学除草剂复配可以增强画眉草弯孢霉菌株QZ-2000孢子的致病力,使其在与马唐互作中占有优势。表明画眉草弯孢霉菌株QZ-2000孢子以及与低剂量化学除草剂复配,对马唐幼苗的生长抑制效果最佳。荧光动力学研究显示,画眉草弯孢霉菌株QZ-2000与化学除草剂复配或使用画眉草弯孢霉菌株QZ-2000悬浮液单独处理,均可降低马唐光合系统原初光化学反应的最大量子速率、光合系统Ⅱ光化学最大效率、阻碍光合作用过程中的电子传递,进而降低马唐的光合作用。利用cDNA-AFLP技术对画眉草弯孢霉菌株QZ-20005×105,1×106两种浓度的孢子液和12h,24h两种保湿时间条件下侵染马唐的基因表达谱进行分析。结果表明,接种高浓度的孢子对马唐的致病性更强。利用256对选择性扩增扩引物组合共获得了差异条带1214条,其中有518个上调表达的转录衍生片段。对其中的46条进行克隆,测序,得到35个TDF,其中25个序列在Blastx GenBank非冗余的公共序列数据库检索出同源序列,有6个是上调基因,参与了信号转导、能量代谢、细胞生长和发育、应激反应、脱落酸生物合成和应激。这些反应是马唐被画眉草弯孢霉菌株QZ-2000侵染后致病的重要途径。研究结果为进一步研究画眉草弯孢霉菌株QZ-2000侵染马唐的发病机理和作为生物除草剂的致病菌防除马唐提供了基础。保持和提高孢子的活力可以增强其致病力,探讨影响孢子活力的因素,明确保持孢子致病力的方式是成功开发生物除草剂的关键之一。研究了画眉草弯孢霉孢子的传代次数和核酸含量与贮藏时间的关系以及对孢子活力的影响。在4℃冰箱保藏期1-8年间,随着保藏时间的延长,RNA含量的变化幅度大于DNA,两者的含量均呈现下降趋势,其萌发率和致病力也随着贮藏时间的延长和核酸含量的下降而降低。贮藏3年,孢子萌发率不足20%；贮藏4年,萌发率降至10%以下,致病力极低,基本失去致病作用；贮藏5年失去致病力；贮藏6年孢子失去活力,不能萌发。在20代以内,画眉草弯孢霉菌株QZ-2000随着传代次数增加,孢子萌发率、萌发芽管长度和致病力呈现下降趋势,但差异不明显。研究了杂粮作为固体基质以及改进生产方式对提高孢子活力的影响。与麦麸等固体培养基比较,所供试验的杂粮克产孢量普遍较高,其中以小米的克产孢量最高；改进孢子培养方式,对孢子产量、萌发芽管长度等有明显促进作用,但对孢子萌发率的促进效果不显著。综合产孢量、萌发率及萌发芽管长度三个方面的因素,小米是马唐生防菌株QZ-2000产孢最适培养基组分。为了了解孢子的形态与萌发的关系,观察了画眉草弯孢霉菌株QZ-2000的孢子,用苯胺蓝染色后进行显微观察,每个孢子由四个部分组成,中部两部分较大,染色较浅,表明具有丰富的原生质；两端的两部分小而圆尖,染色较深,表明原生质含量较少。两者之间由通道相连,孢子的两端部各有一个孔口,芽管由此萌发孔伸出。中部原生质通过通道经两端向外伸出萌发芽管。这种结构对理解孢子的形态特点具有一定意义。更多还原

【Abstract】 Curvularia eragrostidis J. A. Meyer, a species of fungi, belongs to family Dematiaceae, Moniliales, Deuteromycetes. Its strain QZ-2000as a key component of Disancu, a newly-developed bioherbicide, has a virulent pathogenicity to Large crabgrass (Digitaria sanguinalis (L.) Scop.).Large crabgrass, one of the worst weeds in dryland agricultural areas, is distributed throughout tropical and temperate regions of the world and is ranked fourth on a list of serious weeds in these regions. The manual, mechanical and chemical control techniques have been developed for this weed, but the successful results was not made yet. The previous studies have involved in biological characteristics, mass-production techniques, formulation, host range and effectiveness bioassay in greenhouse and fields for the strain QZ-2000. The interaction between the strain and host has not been fully studied, which may link to improve the stability and efficacy of this bioherbicide product. Therefore, the interaction of the strain QZ-2000and the target Large crabgrass is studied to provide theoretical basis for a development of Disancu product. Those studies include influencing factors in conidial viability through improvement of the mass-production of conidia of C. eragrostidis strain QZ-2000and storage, and synergy of low-rate herbicide and responses of the host to the inoculum infection.The physiological indexes such as chlorophyll content, soluble protein and MDA and enzymes in large crabgrass were tested after treatment of conidial suspension of C. eragrostidis, low dosage chemical herbicide alone and their mixture. The treatment of C. eragrostidis strain QZ-2000mixtured with low dosage chemical herbicide caused the maximum reduction of chlorophyll content and soluble protein and maximum increase of MDA content. The esterase isozymes demonstrated various activities in treated large crabgrass plants by polyacrylamide gel electrophoresis. The results indicate that C. eragrostidis strain QZ-2000working with low dosage chemical herbicide enhanced inhibition of large crabgrass seedling growth. The fluorescence kinetics parameters demonstrated that treatment of conidial suspension of C. eragrostidis alone or combination with low-rate herbicides caused Large crabgrass to reduce the PHI(PO) of PS Ⅱ and its activity and impede electron transfer and finally to decrease photosynthesis rate.Gene expression profiles of large crabgrass infected by C. eragrostidis strain QZ-2000at the treatment regime at two concentrations of conidia and two dew durations were analyzed by cDNA amplified fragment length polymorphisms (cDNA-AFLP). Inoculum strength was more determinant of gene expression than dew duration. A total of256primer combinations were used for selective amplification and1214transcript-derived fragments (TDFs) were selected for their differential expression. Of these,518up-regulated differentially expressed TDFs were identified. Forty-six differential cDNA fragments were chosen to cloned and35of them were successfully cloned and sequenced, of which25were homologous to genes of known function according to the GenBank database. Only6genes were up-regulated in C. eragrostidis-inoculated large crabgrass, with functions involved in signal transduction, energy metabolism, cell growth and development, stress responses, abscisic acid biosynthesis and response. It appears that a few pathways may be important parts of the pathogenic strategy of C. eragrostidis strain QZ-2000on large crabgrass. Our study provides the fundamentals to further study the pathogenic mechanism, screen for optimal C. eragrostidis strains as potential mycoherbicide and apply this product to control Digitaria sanguinalis.Fungal spore has the advantages of highly virulence and easy to storage. Ingredient of solid substrate had a great effect on the spore quantity of Curvularia eragrostidis strain QZ-2000. Coarse cereals were evaluated as better solid substrates for sporulation of C. eragrostidis compared to sideline products. Sporulation, length of gemma of conidia was increased obviously and germination rate unobviously with improved culture mode. The millet seed as substrate improved the sporulation. Germination rate, length of gemma and pathogenicity of conidia was gradually decreased with the serial passage generation increased in20generation, but the difference was insignificant. The relationship between the DNA and RNA content of C. eragrostidis conidium and its storage duration was studied. During1-8years’ storage, the DNA and RNA content of C. eragrostidis conidium decreased with storage duration extension. The bigger reduction happened in the RNA content than in the DNA content. In20generations of successive transfers of culture, there was a reducing trend in germination rate, tube length and pathogenicity of conidia with a serial passage generation extension despite insignificant difference.Conidia of C. eragrostidis strain QZ-2000stained with aniline blue were observed under a light microscope. The results demonstrated that a conidium was composed of four cells. The bigger two cells with full of protoplasm located in the middle. One cell each pole had a germ pore at end respectively. The protoplast within the bigger cells connected to that with the smaller cells through a thin channel. Germination tube grew out of one or both of the small cells.更多还原