水稻与稻瘟病菌非亲和性互作中重要防御酶活性变化规律研究

 选以CO39为背景的水稻抗稻瘟病近等基因系,与稻瘟菌生理小种ZC₁₃(菌株97-151a)组成的3类典型非亲和性互作,以亲和性互作为对照,对各互作中过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、几丁质酶及β-1,3-葡聚糖酶的活性变化规律进行了系统研究。完全非亲和性互作C101A51/97-151a、高度非亲和性互作C101L AC/97-151a及中度非亲和性互作C104 PKT/97-151a,POD比活性接种后即开始明显升高,48h前达到高峰,升高趋势一直持续到7d完全显症时,幅度基本与各互作非亲和程度呈正相关;亲和性互作CO39/97-151a接种后40 h POD比活性才开始升高,4~6 d达到高峰,峰值也较大。3类非亲和性互作PAL比活性在接种后0 h或16 h开始较明显升高,整个互作中形成3~4个较明显的峰;亲和性互作中PAL比活性一直明显下降。3类非亲和性互作外切几丁质酶比活性接种后即开始升高,基本一直保持升高趋势,在40 h前幅度较大,并形成1~3个较高的峰;亲和性互作外切几丁质酶比活性接种后即开始大幅度升高直至完全显症,48h后幅度远高于非亲和性互作。3类非亲和性互作β-1,3-葡聚糖酶比活性在24 h内开始较明显升高,在48h前形成2~3个较明显的峰;亲和性互作在接种后β-1,3-葡聚糖酶比活性即开始升高,在48h后显著高于非亲和性互作。讨论了POD、PAL、几丁质酶及β-1,3-葡聚糖酶参与水稻抗稻瘟病的可能性。     

中生菌素对水稻白叶枯病的防治机制

对不同白叶枯病抗性水稻品种用200μg/ml中生菌素55℃温汤药液浸种，自然降温，秧田3—4叶期和移栽前5d各用30μg/ml中生菌素处理后，于成株期剪叶接种白叶枯病菌。结果表明中生菌素前期处理，于成株期接种白叶枯病菌时，高抗、中等抗性和感病品种中过氧化物酶、苯丙氨酸解氨酶和多酚氧化酶3种酶活性都有不同程度的提高，且以中等抗性品种酶活提高最多，接种24和48h，3种酶活性较清水对照分别增加26．92％、26．74％、24．06％和7．09％、1．31％、1．60％。盆栽试验表明，中生菌素对中抗品种的防治效果最好，达58．4％。说明中生菌素对水稻防御酶活性的诱激作用是其防治白叶枯病的机制之一。     

井冈霉素A对水稻纹枯病菌的毒力和作用机理研究

根据水稻纹枯病菌 Rhizoctonia solani AG- 1IA在含系列浓度井冈霉素 A马铃薯葡萄糖琼脂培养基 (PDA)上的菌丝生长速率 ,计算出药剂的毒力回归方程为:Y=3.2 30 9+0 .872 0 X,r=0 .9910 ,药剂的 EC50 =10 6 .9μg/ m L,EC75=6 34.5 μg/ m L。在含 5 0 0 μg/ m L井冈霉素 A的 PDA上水稻纹枯病菌的菌丝生长抑制率为 74 .1% ,而田间水稻植株上人工接菌的药效试验结果表明 ,5 0 μg/ m L药液喷洒后 7d和 14 d的防效即可分别达到 77.4 %和 76 .7% ,即药剂在 PDA平板上的理论抑制作用仅是田间活体植株上对病菌实际作用效果的 1/ 10。比较室内毒力和田间药效试验的结果可以看出 ,井冈霉素 A具有对病原菌和寄主植物双重作用的特性。研究首次发现 ,在室内培养基上不能有效抑制病菌生长的 1μg/ m L的井冈霉素 A可以在水稻植株未喷药部位产生防御水稻纹枯病的作用 ,且能够持续诱导植物防御反应相关酶——过氧化物酶 (PO)和苯丙氨酸解氨酶 (PAL)的活性增高,表明该药剂可以激发水稻抗性防卫反应的表达。     

Enzymatic Maceration of Witloof Chicory by the Soft Rot Bacteria Erwinia carotovora Subsp. carotovora: The Effect of Nitrogen and Calcium Treatments of the Plant on Pectic Enzyme Production and Disease Development

Disease incidence of bacterial soft rot caused by Erwinia carotovora subsp. carotovora and activities of bacterial pectolytic enzymes were studied in witloof chicory. Disease incidence after forcing of the chicory heads was enhanced by the nitrogen treatment and reduced by the calcium treatment of the chicory plants prior to forcing. Significant differences in susceptibility to bacterial soft rot were found for the tested chicory cultivars. Disease severity after 96h ranged from 6% in Clause R2 to 100% in Bea and Tabor. Chicory cultivars Rumba and Salsa showed a final average severity of 65–70%. Activity of the pectolytic enzymes, polygalacturonase and pectate lyase increased in artificially inoculated chicory heads of cultivar Rumba and was not affected by calcium and nitrogen treatments of the plants. Polygalacturonase showed highest activity 48h after inoculation while pectate lyase activity increased throughout 72h of incubation. Maceration of the chicory tissue and bacterial growth increased continuously until 96h after inoculation, when more than 60% of the chicory heads was macerated by pectolytic enzymes of the bacteria. Enzyme activity of Erwinia carotovora subsp. carotovora grown on chicory cell wall extracts was influenced by nitrogen and calcium treatments of the chicory plants. The activity of polygalacturonase reached its highest level at 48h after incubation and pectate lyase activity peaked at 24h followed by a continuous decrease until 72h after inoculation.     

Influence of Abscisic Acid and the Abscisic Acid Biosynthesis Inhibitor, Norflurazon, on Interactions Between Phytophthora sojae and Soybean (Glycine max)

A comparison was made of the effects of abscisic acid (ABA) and the ABA biosynthesis inhibitor, norflurazon on the interaction between soybean leaves and Phytophthora sojae. Inoculation of leaves of cv. Harosoy resulted in a compatible interaction typified by the presence of spreading, water soaked lesions with ill-defined margins while inoculation of cv. Haro 1272 resulted in an incompatible interaction with lesions restricted to the inoculation site. Activity of phenylalanine ammonia lyase (PAL) slowly increased in the compatible interaction but in the incompatible interaction there was a rapid rise in activity within 8h after inoculation. When Haro 1272 plants were treated with ABA the normally incompatible interaction with race 1 was changed to what resembled a compatible interaction and activity of PAL was reduced to control levels. There was no visible effect on the compatible combination. In contrast when plants of cv. Harosoy were treated with norflurazon the normally compatible interaction with race 1 was changed to that which resembled an incompatible interaction and PAL activity increased to high levels rapidly. There was no effect of norflurazon on the incompatible interaction of cv. Haro 1272 with race 1. Stomata on leaves of cv. Harosoy treated with norflurazon closed within 2h of inoculation resembling the response of stomata in normal incompatible interactions but not compatible interactions where stomata remained open. On leaves of cv. Harosoy treated with norflurazon at sites 3 and 20mm from the inoculation point stomata also closed. These results extend and confirm the idea that ABA is a molecule that may regulate the outcome of the interaction between soybeans and P. sojae.     

Some compatible Rhizobium leguminosarum strains in peas decrease infections when parasitised by Orobanche crenata

Orobanche crenata (crenate broomrape) produces serious damage to many legume crops and particularly becomes a limiting factor for pea production in the Mediterranean basin. Nodulation effects on pea–broomrape relationships were studied using the commercial pea cultivar Douce de Provence and different Rhizobium strains using pot and Petri dish experiments. First, the benefit of bacterial inoculation on plant growth and efficiency of N incorporation were demonstrated for two isolates, P.SOM and P.1236. These isolates did not influence parasite germination induced by the artificial stimulant, GR24. In contrast, pea root inoculation with P.SOM and P.1236 isolates led to a reduced root infection by O. crenata , resulting from a lower Orobanche germination rate close to pea roots and a limited capacity of the parasitic seedlings to develop tubercles. Broomrape necrosis was observed both before and after parasite attachment to inoculated pea roots. Concomitantly, reduction in infection was accompanied by enhanced peroxidase activity and constantly high phenylalanine ammonia lyase activity in pea roots. These data suggest the involvement of these enzymes in pea resistance to crenate broomrape induced by the compatible rhizobia. Management of Orobanche via crop selection based on these enzyme systems is a viable option.     

皱纹盘鲍内脏酶的酶学性质及褐藻胶裂解酶的分离纯化

采用(NH4)2SO4分段盐析、透析、阴离子(DEAE-52)交换柱层析、SephadexG-200凝胶柱层析等分离纯化技术，通过SDS-PAGE电泳分析了皱纹盘鲍内脏酶的组成，结果表明鲍内脏酶主要含有两种褐藻胶裂解酶Ⅰ, Ⅱ，一种纤维素酶，一种琼脂酶。对酶的酶学性质分析结果表明两种褐藻胶裂解酶Ⅰ, Ⅱ的最适pH分别为8.6, 7.2，最适温度为35 ℃，分子量分别为35.2 ku, 67 ku；两种褐藻胶裂解酶的热稳定性比较差，且易受金属离子影响；纤维素酶的最适pH为5.0，最适温度为40 ℃。并确定了皱纹盘鲍内脏酶分离纯化的方法及参数，为进一步研究鲍内脏复合酶的性能提供了基础参数。图14表3参12 关键词：皱纹盘鲍； 褐藻胶裂解酶； 纤维素酶； 纯化 E-mail：wqk320@dlfu.edu.cn     

Characterization of an Alginate Lyase,FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.