NO和H2O2提高葡萄抗霜霉病的生理作用机制

 以对霜霉病具有不同抗性的3个葡萄品种Fredonia、西拉和赤霞珠为材料,研究一氧化氮（NO）和过氧化氢（H₂O₂）在调控葡萄抵御霜霉病菌感染过程中的生理机制。结果表明：接种葡萄霜霉病菌后3个葡萄品种叶片中NO和H₂O₂含量均有猝发现象,H₂O₂猝发早于NO,抗性强的品种Fredonia的变化快而显著;外施一定浓度的NO供体硝普钠（SNP）和H₂O₂均可减缓霜霉病菌侵染过程,降低感病率和平均病情指数,并且能够不同程度地提高抗性弱的葡萄品种赤霞珠叶片的过氧化物酶（POD）、苯丙氨酸解氨酶（PAL）、β-1,3-葡聚糖酶（Glu）和几丁质酶（Cht）活性,增强葡萄过敏性坏死反应;而NO和H₂O₂的清除剂2-4,4,5,5-苯-四甲基咪唑-1-氧-3-氧化物（cPTIO）和抗坏血酸（AsA）在一定程度上能够提高感病率和平均病情指数。推测NO和H₂O₂可以通过提高POD、PAL、Glu和Cht病程相关蛋白活性,进而增强葡萄对霜霉病的抗性。     

外源Ca2＋和H2O2对NaCl 胁迫下枸杞离体叶片叶绿体中活性氧代谢的影响研究

以枸杞离体叶片为材料,研究NaCl胁迫下外源Ca2+和H2O2对枸杞离体叶片叶绿体中O2·产生速率、丙二醛(MDA)含量及抗坏血酸过氧化物酶(APX)、过氧化物酶(POD)和过氧化氢酶(CAT)活性的影响.结果表明:在NaCl胁迫下O2·产生速率呈上升趋势,丙二醛含量增大,保护酶APX、CAT、POD活性较对照升高.在NaCl胁迫下,加入外源Ca2+和H2O2时丙二醛(MDA)含量和O2·产生速率较单纯NaCl胁迫下降低;保护酶APX、CAT、POD活性较单纯NaCl胁迫下升高.表明外源Ca2+和H2O2在一定程度上可以缓解NaCl胁迫对植物体造成的伤害.     

HarpinXoo诱发烟草过敏反应早期H2O2变化及有关基因表达

 Harpin_Xoo是水稻黄单胞细菌(Xanthomonas oryzae pv.oryzae)产生的蛋白类激发子,用harpin_Xoo注射烟草最早在处理后20h出现肉眼可见的细胞死亡(macro-HR),分别经埃文斯蓝(Evans blue)和台盼兰(Trypan blue)染色观察,发现诱导后4-8h即开始有少量细胞死亡,表明在macro-HR发生前烟草细胞已陆续发生微细胞死亡(micro-HR);对二氨基联苯胺(diamino benzidine,DAB)染色显示,在烟草细胞死亡早期,伴有H₂O₂的明显积累;定量RT-PCR法对H₂O₂代谢相关基因的表达进行了分析,发现harpin_Xoo诱导后1h编码NADPH氧化酶的基因rboh即开始表达,3~7h表达较强,10h开始减弱,表达趋势与H₂O₂积累趋势一致;编码交替氧化酶的基因aoxl表达较迟,sodA基因则在诱导后表达减弱。这些基因的不同表达模式说明,它们在harpin_Xoo诱导的H₂O₂代谢过程中可能具有不同的作用。     

H2S位于H2O2下游介导葡萄抗霜霉病过程

 以霜霉病抗性葡萄品种左优红和感病品种霞多丽为材料,研究硫化氢(H₂S)在葡萄应答霜霉病过程中的作用及其与过氧化氢(H₂O₂)的相互关系。结果表明,接种霜霉病菌后,抗性品种左优红和感病品种霞多丽叶片均出现H₂S含量的猝发、H₂S合成酶基因表达量增加、抗病相关蛋白多酚氧化酶(PPO)和β-1,3葡聚糖酶(Glu)活性显著升高现象;且H₂S清除剂次牛磺酸(HT)可以显著抑制霜霉病菌诱导的PPO和Glu活性升高,并导致葡萄的感病率和病情指数显著升高;同时外源H₂S可降低两个葡萄品种的感病率和病情指数。说明,H₂S是参与葡萄抗霜霉病过程的新的信号物质。受霜霉病菌侵染后,葡萄叶片H₂O₂含量猝发早于H₂S,并且H₂O₂清除剂抗坏血酸(AsA)可显著抑制霜霉病菌所诱导的H₂S含量的上升,而H₂S清除剂HT对霜霉病菌诱导的H₂O₂含量变化影响不显著,推测,H₂S作用于H₂O₂的下游参与葡萄抵御霜霉病过程。     

棉疫病菌90kD胞外蛋白激发子诱导烟草过敏性反应的研究

 就棉疫病菌90 kD胞外蛋白激发子诱导烟草过敏反应(HR)过程中细胞死亡和防卫反应酶系活性变化及病程相关蛋白PR5的诱导进行研究。结果是,以10 nmol/L激发子溶液注射处理W38烟草叶片,HR枯斑周围5 mm宽组织在UV光下呈现蓝色荧光,对处理部位进行Evans blue染色测定结果是至20 h处理部位细胞全部死亡;激发子可诱导烟草防卫反应中苯丙氨酸解氨酶(PAL)的活性提高;可快速诱导PR5基因的转录。上述结果表明90 kD蛋白激发子可诱发烟草的细胞死亡、苯丙烷代谢和PR基因的表达等多条信号途径。     

katExoo基因缺失突变对水稻白叶枯病菌H2O2抗性和致病性的影响

 为了揭示过氧化氢酶基因katExoo在水稻白叶枯病菌(Xanthomonasoryzaepv.oryzae,Xoo)过氧化氢(H₂O₂)抗性和致病性中的功能,本研究构建了基因缺失突变体ΔkatExoo,测定了突变体的H₂O₂抗性、过氧化氢酶(CAT)活性、在离体培养条件下的生长速率以及对水稻的致病性。用标记基因置换法获得了ΔkatExoo突变体,其保守的CAT结构域(GATase1_catalase和catalase_clade_2)被Gm^R片段所替换。katExoo基因缺失突变并不导致病菌的H₂O₂抗性和CAT活性降低或丧失,反而在一定程度上使之增强和升高。ΔkatExoo离体生长量显著降低,水稻接种叶片病斑明显缩短、在叶组织内的种群量下降。表明基因缺失突变显著地影响了病菌的生长、定殖和致病性。本研究结果为“KatExoo可能是Xoo的一个毒性因子”的假设提供了遗传学证据。     

H2O2对水稻白叶枯病菌过氧化氢酶相关基因crg表达的诱导作用

 为了阐明H₂O₂对水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,Xoo)过氧化氢酶(CAT)相关基因(crg)表达的诱导作用,本研究定量分析了在水稻细胞-Xoo互作体系及其加入H₂O₂清除剂CAT后H₂O₂产量和crg表达;外源添加H₂O₂后的病菌生长和crg表达。结果表明:在互作条件下,H₂O₂含量稳定增加,10 h可达到峰值;在互作6 h时crg显著地被诱导表达;加入CAT显著地降低了H₂O₂含量和crg表达;在外源H₂O₂胁迫条件下,H₂O₂以浓度效应的方式影响病菌增殖,显著地诱导了catB和srpA表达。因此,Xoo-水稻互作导致了H₂O₂的发生。无论是互作产生的还是外源的H₂O₂均显著地诱导了Xoocrg表达,从而活化了H₂O₂降解途径。     

双氧木脂素E对东方粘虫幼虫体壁肌超微结构及Na+-K+-ATPase、Ca2+-ATPase的影响

双氧木脂素E（haedoxane E）是从杀虫植物透骨草Phryma leptostachya L.中分离出的一种对多种农林害虫和卫生害虫具有杀虫活性的化合物。本研究以东方粘虫Mythimna separata为供试昆虫,对双氧木脂素E对其幼虫杀虫作用部位进行了初步研究。电镜观察表明,双氧木脂素E对东方粘虫幼虫体壁肌具有致毒作用,能使肌细胞发生明显病变,细胞核皱缩变形,染色质浓缩,线粒体肿胀、大面积空泡化,肌质网扩张,肌原纤维排列紊乱,胞质内出现大量多泡体。酶活力测定结果也显示,双氧木脂素E能不同程度抑制Na+-K+-ATPase和Ca2+-ATPase活力。因此推测双氧木脂素E是一种作用于东方粘虫肌肉组织的肌肉毒剂。     

疫霉蛋白激发子PB90对病原菌生长与致病力的影响

 激发子PB90是由棉疫病菌分泌的可诱发非寄主植物系统获得抗病性的一类新型蛋白激发子。本文就该激发子在棉疫病菌生长发育及致病过程中的作用进行了研究。采用胶体金标记发现该激发子主要分布在棉疫病菌的细胞壁上,该激发子可以分泌到细胞外;在离体条件下,PB90的多克隆抗体并不抑制棉疫病菌游动孢子的萌发和孢子萌发后形成菌落的能力,而且抗体包埋的游动孢子仍能激发非寄主植物烟草的过敏性坏死反应;但是,抗体的包埋处理可以使棉疫病菌的游动孢子丧失对棉花的致病力。结果表明,特异性存在于细胞壁上的疫霉蛋白激发子PB90是棉疫病菌的一种重要的毒性因子,在病菌对棉花的致病过程中具有重要作用;此外本研究结果还提示PB90并不是惟一能诱导非寄主烟草过敏性反应的棉疫病菌激发子,除了PB90之外,棉疫病菌还能产生其它的激发子诱导烟草的过敏性坏死反应。     

稻瘟菌GP66激发子诱导的水稻膜脂过氧化及其保护酶活性变化

 以抗稻瘟病水稻近等基因系C101LAC及背景品系CO39为材料,研究稻瘟菌(Magnaporthe grisea)来源的GP66激发子诱导的水稻膜脂过氧化及保护酶活性变化.结果表明,激发子诱导非亲和性互作水稻超氧阴离子(O₂^·)积累和脂氧合酶(LOX)活性在早期明显高于亲和性互作水稻;O₂^·积累和LOX活性的升高进而导致了非亲和性互作水稻的膜脂过氧化,其相对电导率及丙二醛(MDA)含量出现的高峰期和强度也明显要早和高于亲和性互作水稻.超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性均趋于下降,不同亲和性互作水稻间的变化则不明显;非亲和性互作水稻过氧化物酶(POD)活性则在激发子诱导早期明显高于亲和性互作水稻,可能与其参与其它抗性有关.这些结果表明膜脂过氧化的发生是激发子诱导水稻抗性的主要生理机制之一.     

Fluxes of methane,carbon dioxide and nitrous oxide in an alpine wetland and an alpine grassland of the Tianshan Mountains,China

Methane(CH4), carbon dioxide(CO2) and nitrous oxide(N2O) are known to be major greenhouse gases that contribute to global warming. To identify the flux dynamics of these greenhouse gases is, therefore, of great significance. In this paper, we conducted a comparative study on an alpine grassland and alpine wetland at the Bayinbuluk Grassland Eco-system Research Station, Chinese Academy of Sciences. By using opaque, static, manual stainless steel chambers and gas chromatography, we measured the fluxes of CH4, N2O and CO2 from the grassland and wetland through an in situ monitoring study from May 2010 to October 2012. The mean flux rates of CH4, N2O and CO2 for the experimental alpine wetland in the growing season(from May to October) were estimated at 322.4 μg/(m2?h), 16.7 μg/(m2?h) and 76.7 mg/(m2?h), respectively; and the values for the alpine grassland were –88.2 μg/(m2?h), 12.7 μg/(m2?h), 57.3 mg/(m2?h), respectively. The gas fluxes showed large seasonal and annual variations, suggesting weak fluxes in the non-growing season. The relationships between these gas fluxes and environmental factors were analyzed for the two alpine ecosystems. The results showed that air temperature, precipitation, soil temperature and soil moisture can greatly influence the fluxes of CH4, N2O and CO2, but the alpine grassland and alpine wetland showed different feedback mechanisms under the same climate and environmental conditions.     

Induction of peroxidase, scopoletin, phenolic compounds and resistance in Hevea brasiliensis by elicitin and a novel protein elicitor purified from Phytophthora palmivora

Elicitin and a new protein 75 kDa elicitor were purified from the culture filtrate of Phytophthora palmivora, a pathogen of Hevea brasiliensis (rubber plant). Elicitin was obtained by using a one step of DEAE cellulose chromatography and the new elicitor was obtained by two steps of chromatography: a DEAE cellulose column followed by a hydrophobic column. Both elicitors were stable to heat and a wide range of pH values, but were sensitive to ProteaseK. Both elicitors induced scopoletin, peroxidase isozymes (with substrate o-dianisidine and scopoletin) and total phenolic compounds in cell suspension of H. brasiliensis with similar kinetics. In addition, both elicitors induced peroxidase enzyme (o-dianisidine), total phenolic compounds and enhanced local resistance against P. palmivora on young rubber tree seedlings. However, the increase of peroxidase enzyme and total phenolic compounds in rubber tree seedlings was different from those in cell suspension. Furthermore, during the expression of local resistance the zoospore of P. palmivora induced the peroxidase enzyme (o-dianisidine) more rapidly and with higher level than the control plants. H. brasiliensis is more responsive to the new elicitor than elicitin in triggering defense responses. That is the new elicitor was active at a concentration lower than those required for elicitin, about a 30-fold decrease for activation defense responses in cell suspension. For induction of peroxidase enzyme (o-dianisidine), phenolic compounds and local resistance of rubber plants against P. palmivora, the 75 kDa protein was active at about a 2-fold lower concentration when compared to elicitin.     

Dominance of a single clonal lineage in the Phytophthora infestans population from northern Shaanxi,China revealed by genetic and phenotypic diversity analysis

Late blight caused by Phytophthora infestans is the most serious disease of potato worldwide. To understand the P. infestans population structure in northern Shaanxi, an emerging potato production region in China, 125 single‐lesion isolates were randomly collected from farmers' fields in 2009 and characterized phenotypically and genotypically. A mating type assay showed that 94 isolates were A1 mating type. Virulence determination of selected isolates on a set of differential potato lines containing R1 to R11, respectively, showed the presence of two pathotypes, of which the pathotype lacking avirulence genes Avr3, Avr4 and Avr10 was dominant. Isolates lacking all avirulence factors Avr1 to Avr11 were detected but at lower frequency (13·6%). Analysis for mtDNA haplotype showed all 61 examined isolates were IIa. A total of seven multilocus genotypes were distinguished among 125 isolates, as determined with seven polymorphic microsatellite markers. The genotype SG‐1 was dominant in the population with a frequency of 75·2% and was present throughout the region. Analysis of the phenotypic and genotypic structures of P. infestans populations indicated strict clonal reproduction of the pathogen and suggested that sexual reproduction probably does not occur. Potential implications for disease management are discussed.