稻瘟病菌分生孢子的诱发及保存方法

稻瘟病菌分生孢子的诱发及保存方法孔秀英（中国农业科学院品种资源研究所北京１０００８１）稻瘟病是水稻的主要病害之一,分布广,危害大,因此广大科研工作者对稻瘟病菌的致病性,生理生化特性及品种的抗病性等方面进行了广泛而深入细致的研究。但无论哪一方面的研究工...     

稻瘟病菌单克隆抗体的研制及其对稻瘟病菌附着胞形成的影响

稻温病菌的分生孢子、芽管、附着胞的混合物作为抗原免疫BALB/c小鼠,取免疫小鼠的脾细胞与SP2/0骨髓瘤细胞在50%PEG下融合成杂交瘤细胞,用间接ELISA筛选阳性孔,获11株单克隆抗体。间接免疫荧光试验表明其中4株单克隆抗体2B4、4A1、1D1和2H4分别与孢子、芽管或附着胞有特异性结合;Western blotting分析发现2B4、4A1、1D1单克隆抗体分别与孢子、芽管表面的提取物有不同的结合带;此四株单克隆抗体均干扰稻温病菌附着胞形成,并抑制稻温病菌在叶表的致病性。     

稻瘟病菌重复顺序PoR6和稻瘟病菌的分型

POR6是一具有高度多态性的稻瘟病菌(Pyricularia oryzae)重复顺序。利用脉冲电泳技术和Southern分析,表明它是非均匀地散布于基因组中的。经测定POR6的拷贝数约为30—40,序列测定未发现在内部有更小的重复单位。用POR6作探针对44株稻瘟病菌进行DNA指纹分析,分析的中国北方地区的22个菌株可根据相似率归并成8个谱系。对一些转管培养中致病型发生变化的菌株用POR6进行指纹分析,发现这些菌株在转管过程中基因组DNA是有变化的。     

稻瘟病菌侵染水稻的机理

深入了解水稻与稻瘟病菌相互作用的分子机理对稻瘟病防治是重要的,本文对这方面的研究进展作了阐述。     

西梭霉素产生菌斜面孢子培养条件研究

采用正交法设计斜面培养基,对西梭霉素产生菌的孢子生长所需要的碳源、氮源、生长因子、无机盐等作全面考察,确定了最佳斜面配方,其生物效价比原配方提高２６％。并考察了斜面孢子培养时间,冰箱保藏时间对孢子量和生物效价的影响。     

阿魏菇菌糠提取液对四种食用菌菌丝生长的影响

目的:解决菌糠再利用技术问题.方法:采用平板培养法,对在PDA培养基中加入不同体积比的阿魏菇菌糠提取液对鸡腿菇、香菇、杏鲍菇和金针菇菌丝生长的影响进行了研究.结果:当PDA培养基中菌糠提取液加入量为10%～50%时,对香菇和金针菇菌丝生长有促进或明显促进作用;而加入量为10%的菌糠提取液对杏鲍菇菌丝生长无显著影响,但当阿魏菇菌糠提取液加入量大于10%时,明显抑制杏鲍菇和鸡腿菇的菌丝生长.结论:可利用阿魏菇菌糠作为栽培香菇和金针菇的培养料.     

孢子丝菌病

孢子丝菌病（sporotrichosis）是由孢子丝菌感染所致,主要引起皮肤感染,也可引起黏膜、骨骼甚或系统性病变。     

稻瘟病菌微波诱发突变体的分析

通过诱变获得突变体是研究稻瘟病菌变异机制的基础。本文用微波炉对稻瘟病菌分生孢子进行低强度短时间处理获得了一批形态发育和致病性突变体,并对它们进行了分析。突变体1-40-271菌落呈白色,产孢与萌发均正常,但萌发后即便在人工疏水表面上也不能形成附着胞,且丧失了致病性;突变体2-20-6菌落呈黄色,孢子萌发率为1％,萌发的孢子其附着胞形成率仅为0．01％,致病性减弱;突变体2-30-3菌落呈黄色,形成的附着胞大部分不正常,但致病性正常。Rep-PCR指纹分析发现,突变体2-20-6和2-30-3比其相应野生型少1条带,而突变体1-40-271与其野生型比较没有变化,说明微波可能造成稻瘟病菌基因组DNA缺失或点突变而发生变异。继代分析表明微波处理获得的稻瘟病菌形态和致病性突变体是稳定的。     

影响水稻稻瘟病菌侵染过程的生态因子研究

通过田间和室内模拟等方法研究了水稻生长阶段,品种抗性,温度,降雨量和施N量等因子对稻瘟病菌侵染过程的影响。结果表明,水稻叶表单位面积病菌孢子附着量与水稻不同生长阶段呈负相关关系。稻瘟病潜育期与温度关系密切,在10℃-33℃范围内,以28℃条件下潜育期最短,小于℃,大于28℃的潜育期相应延长;病菌孢子侵染比值与水稻生长阶段呈负相关关系;稻叶表病菌孢子附着率在孢子与叶表接触后的5h内,与降水强度和降水持续时间密切相关,5h后影响变小;在一定条件下,稻瘟病扩展性病斑与非扩展性病的比值,扩展性病斑扩展的最大面积与水稻品种抗性和当时病斑所处叶位有关。     

抗稻瘟病体细胞突变体的抗性遗传分析

以ZA_(15)和ZB_(11)等2个致病小种对6个抗稻瘟病体细胞突变体进行了抗性遗传分析。结果表明,86-S1、88-86、88-42和88-40等4个突变体对ZA_15、ZB_(11)小种的抗性分别由1个显性基因控制,同时这2个抗性基因还存在紧密的连锁关系。88-127和88-145对ZA_15的抗病性受2个重复显性基因控制;而对ZB_(11)的抗性则分别受2个互补显性基因和1个显性基因控制。等位性测定表明,86-S1、88-42和8B-86等3个突变体具有的抗性基因是等位的,可能是Pi-(?)或与之等位的抗性基因。     

二个粳稻品种抗稻瘟病遗传特性的分析

本文用累积分布曲线法对东农 363及农东415两个粳稻品种进行了抗稻瘟遗传分析,结果表明东农363对Hokul菌株的抗性是由一对显性抗性基因控制的,东农415对Ken53-33菌株的抗性是由两对互补的显性基因控制的;对Ina72菌株的抗性是由两对显性基因控制的,其中一对控制高抗反应,另一对控制中抗反应。两个杂交组合的正反交分析结果表明,水稻对稻瘟病菌的抗性遗传是由细胞核控制的,细胞质在抗瘟遗传中的作用在本试验的测试品种中并没有表现出来。 Abstract:By means of cumulative distribution curve methods,two Japonica varieties Dongnong 363 and Dongnong 415 were analysed for the inheritance of blast resistance.The results showed that the resistance of Dongnong 363 variety to Hokul blast strain was controlled by one dominant gene.The resistance of Dongnong 415 to Ken53-33 strain was controlled by two complementary dominant genes,to Ina72 strain was controlled by two dominant genes,one dominated over the high-resistant and the other over the middle-resistant.Genetic analysis of F3 plants of two reciprocal crosses showed that the resistance to the rice blast disease was controlled by nuclear gene,no cytoplasmic effect was found in the tested varieties.     

四霉素与噁霉灵混配制剂对水稻立枯病的防治效果

水稻是我国第一大粮食作物,为有效防治水稻立枯病并减少化学农药的使用量,进行了农药混配制剂的筛选。通过室内不同比例的噁霉灵与四霉素混配抑菌试验及田间防治试验,筛选出能有效防治水稻立枯病的四霉素与噁霉灵混配制剂,四霉素与噁霉灵有效成分比例为0.152.5,共毒系数为139.03,增效作用明显;当田间使用剂量为33.13~39.75 g/hm2时,田间防效达到73.30%。试验结果表明,四霉素与噁霉灵按一定比例混配可以有效防治水稻立枯病,有效减少化学农药的使用量。     

水稻病程相关PR1家族蛋白质在叶片生长及与白叶枯病菌互作反应中的表达

病程相关(PR)蛋白质经常被用作抗病反应的分子标记。利用免疫印迹(WB)技术检测了7个PR1家族蛋白质在水稻(Oryza sativa)叶片生长及与白叶枯病菌互作反应过程中的表达,发现6个PR1家族蛋白质在叶片生长中有表达。检测PR1蛋白质在Xa21介导的抗白叶枯病过程中的表达,结果显示PR1#052、PR1#072、PR1#073和PR1#121四个蛋白质在抗病反应后期呈上调或诱导表达,PR1#071则表达下调。进一步比较它们在抗病、感病和对照(Mock)反应中的表达丰度,发现在抗病和感病反应中的变化幅度均明显大于对照反应,推测这些PR蛋白质在水稻-白叶枯病菌互作反应中发挥作用。另外,对PR1基因上游启动子区的cis元件进行了分析。该研究初步揭示了水稻PR1家族蛋白质的表达谱,为进一步了解PR1蛋白质的功能提供了线索。     

稻瘟病拮抗菌的筛选及抑菌活性的研究

利用对峙法和牛津杯法从大连近海海域分离得到1株对稻瘟病菌有拮抗作用的菌株BCHN-15,经生理生化实验和16S r DNA方法鉴定,该菌株为解淀粉芽胞杆菌。实验结果显示,BCHN-15能显著抑制稻瘟病菌的生长,与稻瘟病菌作用48 h后,稻瘟病菌的菌丝干重与对照组相比减少了74.36%。该菌产生的抑菌成分可破坏稻瘟病菌的细胞膜,且对热稳定,其活性成分能被盐酸沉淀。结果表明,BCHN-15可通过破坏稻瘟病菌的细胞膜,引起菌丝畸形和断裂来发挥其抑菌作用。     

The Effect of Ecological Factors on Spore Germination and the Viability of the Mycelial Fragments of Microscopic Fungi

Spore germination and the viability of the mycelial fragments of the microscopic fungi Alternaria alternata, Penicillium spinulosum, and Mucor hiemaliswere studied with respect to the action of certain ecological factors: sucrose concentration (0, 0.2, 2, 10, and 100 g/l), temperature (4, 20, 25, and 30°C), pH (3.5, 4.0, 5.0, 6.2, and 7.0), and cadmium concentration (0, 2, 10, and 100 mg/l). The spore germination and the viabilities of different mycelial fragments were found to reach their maxima at different values of the ecological factors studied. This finding suggests that the vegetative and asexual types of reproduction of microscopic fungi may have different ecological optima.     

稻曲病菌厚垣孢子不同破壁方法的比较

为了探究稻曲病菌[Ustiloginoidea virens(Cooke)Takahashi]厚垣孢子的最佳破壁方法,研究采用4种破壁法对该病菌黄色和黑色厚垣孢子进行破壁,血球计数板计算破壁效果,并用考马斯亮蓝法测定不同破壁方法中厚垣孢子壁内可溶性蛋白含量。结果表明,在普通光学显微镜下观察,破壁后厚垣孢子多数为碎片,少数为孢壁内空圆球。4种破壁方法中液氮研磨-超声破碎法破壁效果最好,黄色和黑色厚垣孢子的破壁率均可达98%以上,用该法破壁测得的黄色和黑色厚垣孢子壁内可溶性蛋白质含量也最高。由此可见,液氮研磨-超声波破碎法是一种稻曲病菌厚垣孢子破壁的有效、简便、适宜在实验室应用的方法。     

Disruption of actin motor function due to MoMyo5 mutation impairs host penetration and pathogenicity in Magnaporthe oryzae

Actin motor myosin proteins are the driving forces behind the active transport of vesicles, and more than 20 classes of myosin have been found to contribute to a wide range of cellular processes, including endocytosis and exocytosis, autophagy, cytokinesis and the actin cytoskeleton. In Saccharomyces cerevisiae, class V myosin Myo2 (ScMyo2p) is important for the transport of distinct sets of cargo to regions of the cell along the cytoskeleton for polarized growth. To study whether myosins play a role in the formation or function of the appressorium (infectious structure) of the rice blast fungus Magnaporthe oryzae, we identified MoMyo5 as an orthologue of ScMyo2p and characterized its function. Targeted gene disruption revealed that MoMyo5 is required for intracellular transport and is essential for hyphal growth and asexual reproduction. Although the ΔMomyo5 mutant could form appressorium‐like structures, the structures were unable to penetrate host cells and were therefore non‐pathogenic. We further found that MoMyo5 moves dynamically from the cytoplasm to the hyphal tip, where it interacts with MoSec4, a Rab GTPase involved in secretory transport, hyphal growth and fungal pathogenicity. Our studies indicate that class V myosin and its translocation are tightly coupled with hyphal growth, asexual reproduction, appressorium function and pathogenicity in the rice blast fungus.     

Genetic Analysis of Resistance to Rice Blast: A Study on the Inheritance of Resistance to the Blast Disease Pathogen in an F3 Population of Rice

Blast caused by the fungus Magnaporthae grisea (Herbert) Borr. (anamorphe Pyricularia oryza Cav.) is a serious disease of rice (Oryza sativa L.). One method to overcome this disease is to develop disease resistant cultivars. Due to the genetic plasticity in the pathogen genome, there is a continuous threat to the effectiveness of the developed cultivars. Additional studies of the genetics of resistance, virulence stability and functional genomics are required to accelerate research into understanding the molecular basis of blast disease resistance. In this study, individual plants of the F₃ population derived from Pongsu Seribu 2 and Mahsuri were used for pathogenesis assays and inheritance studies of blast resistance. The study was performed with two of the most virulent Malaysian M. grisea pathotypes: P7.2 and P5.0. For blast screening, plants were scored based on the IRRI Standard Evaluation System (SES). F₃ populations showed a segregation ratio of 3R:1S for pathotype P7.2, indicating that resistance to this pathotype is likely controlled by a single nuclear gene. Chi‐square analysis showed that the F₃ families segregated in a 15R:1S ratio for pathotype P5.0. Therefore, locus interactions or epitasis of blast resistance occur against pathotype P5.0 in the F₃ population derived from Pongsu Seribu 2 and Mahsuri. This can be explained by the presence of two independent dominant genes that when present simultaneously, provide resistance to the M. gresia pathotype P5.0. These results indicated that blast resistance in rice is due to the combined effects of multiple loci with major and minor effects. The genetic data generated here will be useful in the breeding of local cultivars for resistance to field blast. The methodology reported here will facilitate the mapping of genes and quantitative trait loci (QTLs) underlying the blast resistance trait.