苹果茎沟病毒部分分离物的生物学特性与分子鉴定

通过昆诺藜接种鉴定和ELISA检测,从梨和苹果上分离获得苹果茎沟病毒(Apple stem grooving virus,ASGV)23个分离物.采用TC-RT-PCR对这些分离物进行扩增,均获得特异的扩增片段,PCR产物经5%PAGE电泳,出现大小约500、530和600bp的3种迁移率不同的泳动带型.根据PCR产物电泳迁移率的差异,选取3个来源于梨的分离物P-L4、P-6-1-17和P-3-2-67的PCR产物进行克隆与序列测定.经BLAST搜索,3个分离物的扩增片段与苹果分离物P-209的CP基因3′端核苷酸序列同源性分别为92.2%、90.4%和88.4%.3个分离物间的核苷酸序列也有较大差异,P-L4/P-6-1-17为95.5%、P-L4/P-3-2-67为90.4%、P-6-1-17/P-3-2-67为88.6%.     

大豆疫霉菌对大豆下胚轴侵染过程的细胞学研究

 接种后1.5~24h,用光镜和电镜研究了2个大豆品种与大豆疫霉菌Ps411的亲和性和非亲和性互作。观察结果表明,大豆疫霉菌对大豆下胚轴的侵染过程可分为侵入前、侵入、皮层组织中的扩展和进入维管束组织4个连续阶段。大豆下胚轴接种后在25℃保湿培养,1.5h后游动孢子即形成休止孢并萌发产生附着孢,3h后侵入表皮细胞,6h后进入皮层组织,24h后进入维管束组织。病原菌主要以侵染菌丝直接侵入表皮,表皮细胞间隙是主要侵入部位。皮层细胞是病原菌定殖和发展的主要场所,胞间菌丝侵入皮层细胞并形成吸器。在菌丝与寄主细胞接触部位的寄主细胞壁与质膜之间常有胞壁沉积物的形成。在抗病品种上病菌的侵染事件与感病品种基本一致,但不能形成正常的吸器,胞壁沉积物明显多于感病品种,菌丝在寄主组织内的扩展明显受到抑制。利用β-1,3-葡聚糖免疫金标记单克隆抗体进行的免疫细胞化学的研究表明,胞壁沉积物内含有大量的β-1,3-葡聚糖,在大豆疫霉菌菌丝壁中也存在β-1,3-葡聚糖。以上结果表明,病原菌的侵染可诱导抗病寄主细胞内β-1,3-葡聚糖迅速的合成与积累、并形成胞壁沉积物,以抵御病菌的侵染与扩展。     

螺旋毛壳ND35 β-1,3-葡聚糖酶的诱导、性质及其抑菌作用

 以病原菌Rhizoctonia solani的细胞壁为诱导物,模拟毛壳菌自然的重寄生过程,研究了内生真菌螺旋毛壳(Chaetomium spirale) ND35 β-1,3-葡聚糖酶的产酶条件、性质,尤其是不同碳源的调控作用。结果表明,不同种类的真菌细胞壁及几丁质和昆布多糖,均可诱导产生β-1,3-葡聚糖酶,而作为分解代谢产物的葡萄糖则抑制产酶。经硫酸铵沉淀、DEAE-Sepharose阴离子交换层析及Phenyl-Sepharose疏水层析,并通过SDS-PAGE鉴定,纯化了一种分子量约为73 kDa的内切β-1,3-葡聚糖酶GLUC73。其最适反应温度为55℃,在40℃以下较稳定;最适pH值为5.5,在pH 5-9范围内均很稳定;酶活性受Hg²⁺、Fe³⁺、Zn²⁺、Mg²⁺等金属离子不同程度的抑制,Mn²⁺和Co²⁺对酶有激活作用;以昆布多糖为底物时,该酶的米氏常数K_m为0.412 mg·mL^-1,最大反直速度V_max为3.876 U·mL^-1。粗酶液同时具有β-1,3-葡聚糖酶和几丁质酶活性,离体抑菌试验表明,对苹果炭疽病菌(Glomerella cingulata)、杨树腐烂病菌(Valsa sordida)、苹果树腐烂病菌(Valsa mali)的菌丝生长和孢子萌发有明显的抑制作用。通过对β-1,3-葡聚糖进行免疫细胞化学标记和超微结构观察,间接证明了β-1,3-葡聚糖酶在螺旋毛壳重寄生过程中的作用。     

桃潜隐花叶类病毒RT-PCR和分子杂交检测技术的建立

本研究从武汉周边采集表现典型花叶症状的桃样品,提取总RNA为模板,采用RT-PCR方法对这些样品进了分析,结果显示所分析的5个样品(P1～P5)均获得了预期大小约为337bp的目标扩增条带,表明样品均带有PLMVd.通过回收RT-PCR产物,用生物素标记制备探针,分别采用DNA斑点杂交、RNA斑点杂交和组织印迹杂交3种杂交方法对这些样品进行检测比较,3种杂交方法中,组织印迹杂交操作步骤相对简单快捷,适合于对PLMVd进行大田快速检测.     

Production,survival and evaluation of solid-substrate inocula ofConiothyrium minitans againstSclerotinia sclerotiorum

Coniothyrium minitans grew on all ten solid-substrates (barley, barley-rye-sunflower, bran-vermiculite, bran-sand, maizemed-perlite, millet, oats, peat-bran, rice and wheat) tested, producing high numbers of germinable pycnidiospores (1.9–9.3×10⁸ g^–1 air dry inocula). All solid substrate inocula survived better in the laboratory at 5 and 15 °C than at 30 °C for at least 64 weeks.In pot bioassays carried out in the glasshouse and field, soil incorporations of each inoculum almost completely inhibited carpogenic germination ofS. sclerotiorum. In the field bioassay, no sclerotia were recovered after 38 weeks fromC. minitans-treated pots compared to 56% from control pots. In the glasshouse bioassay, 9–30% of sclerotia were recovered after 20 weeks fromC. minitans-treated pots, but 88–100% of these were infected by the antagonist. The antagonist also spread to infect sclerotia in control pots.In larger scale glasshouse trials, single preplanting soil-incorporations of five inocula (barley-ryesunflower, maizemeal-perlite, peat-bran, rice and wheat) controlled Sclerotinia disease in a sequence of lettuce crops, with only small differences between the types of inocula tested. At harvest,C. minitans reduced sclerotial populations on the soil surface and over 74% of sclerotia recovered fromC. minitans-treated plots were infected by the antagonist.C. minitans survived in soil in all solid-substrate inocula-treated plots for at least 39 weeks at levels of 10⁴–10⁵ colony forming units cm^–3 soil and spread to infect over 36% of sclerotia recovered from control plots.     

Screening for Sclerotinia resistance in Helianthus tuberosus L. (Jerusalem artichoke) varieties, lines and somaclones, in the field and in vitro

Yields in large-scale 'continuous' cultivation of H. tuberosus (Jerusalem artichoke) can be greatly reduced by Sclerotinia sclerotiorum , which causes a basal stem and tuber rot. In 34 varieties/lines of H. tuberosus from European, Russian and Canadian breeding programmes, responses to the pathogen varied from field resistance to 34% stem rot, cultivars Nahodka and Columbia showing c. 25% stem rot after three seasons of cultivation on the same site. Attempts to develop an in-vitro selection for S. sclerotiorum resistance based on culture of H. tuberosus nodes on media precolonized with Sclerotina were unsuccessful. There was a strong positive correlation between capacity of nodes to grow in vitro on calcium-free medium and field resistance. Somaclones of'Nahodka' selected on the latter basis showed no field infection when grown on land that was heavily contaminated with the pathogen, and they were resistant to artificial inoculation with the pathogen.     

Effect of Coniothyrium minitans on sclerotial survival and apothecial production of Sclerotinia sclerotiorum in field-grown oilseed rape

In two field trials with oilseed rape, Coniothyrium minitans was applied to soil as a maizemeal-perlite preparation in order to determine its effect on sclerotial survival and apothecial production of Sclerotinia selerotiorum. The mycoparasite infected sclerotia and decreased sclerotial survival, carpogenic germination and production of apothecia. Effects were greatest when inoculum of C. minitans was applied in autumn, at the time of sowing, rather than when it was applied in spring. C. minitans survived in soil for 2 years and spread to adjacent control plots and infected sclerotia within those plots. However, despite the fact that the inoculum potential of S. selerotiorum was reduced by C. minitans treatment, no disease control was obtained either in trial 1, where disease levels were low (0-20% of plant stems affected), or in trial 2, where disease levels were high (up to 70% of plant stems affected). Possible reasons for this failure of C minitans to control sclerotinia disease in oilseed rape, and strategies to improve its efficacy in the field, are discussed.     

小麦离体叶段鉴定白粉病抗性方法的研究

１９９０年４月至１９９４年４月对小麦离体叶段鉴定白粉病抗性方法进行了研究。结果表明：（１）在灭菌条件下，以在培养皿中垫两层滤纸，用６ＢＡ作保绿主，超过人主至进行鉴定效果较好。（２）保绿剂的浓度低，发病快、退绿早，反之反病慢、退绿晚。其最适浓度是：苗期６ＢＡ为６０－１００ｐｐｍ、苯咪唑为６０ｐｐｍ ；成株期６ＢＡ以８０－１５０ｐｐｍ为佳。（３）份病快、退绿迟的角度出发，鉴定材料以株龄小、叶龄短的初展     

Studies of clomazone mode of action

Intact spinach chloroplasts were used to determine if clomazone, 5-OH clomazone, and/or 5-keto clomazone inhibited the chloroplastic isoprenoid pathway. When isopentenyl pyrophosphate was used as a precursor, neither clomazone nor the clomazone metabolites (5-OH clomazone and 5-keto clomazone) inhibited the formation of products separated by HPLC in the organic phase. However, when pyruvate, a substrate for the first committed step of the pathway, was used as a precursor, both 5-keto clomazone and fosmidomycin reduced the formation of a non-polar product and increased the formation of a polar product in the organic phase. Only 5-keto clomazone, not 5-OH clomazone or clomazone, inhibited the formation of an additional product other than fosmidomycin in the aqueous phase from pyruvate incorporation. In an in vitro assay, 5-keto clomazone inhibited DXP synthase, the enzyme catalyzing the first committed step of the chloroplastic isoprenoid pathway. Therefore, our studies show that neither clomazone nor 5-OH clomazone inhibits the chloroplastic isoprenoid pathway, only 5-keto clomazone does.