高温处理防治黄瓜霜霉病的生理生化机制

以黄瓜感病品种‘长春密刺’为试材,通过室内盆栽试验研究高温对已感染霜霉病菌的黄瓜幼苗生理生化特征的影响.结果显示:(1)在黄瓜幼苗接种霜霉菌后8h,用45℃高温处理90min防治效果最明显.(2)与只接种霜霉病菌的黄瓜幼苗相比,接种霜霉菌后进行高温处理可显著提高叶片叶绿素含量,降低丙二醛含量;与对照相比,接种霜霉菌后进行高温处理可显著提高叶片几丁质酶活性,Western blotting验证了几丁质酶的活性变化.(3)SDS-PAGE结果表明,霜霉菌侵染可诱导一种28kD蛋白表达,高温处理后28kD蛋白的表达量降低.研究结果表明高温处理可能在杀死病原菌的同时,还诱导黄瓜幼苗对霜霉病产生了部分抗性.     

霜霉菌或水分胁迫诱导的黄瓜叶片胞间隙27kD蛋白为几丁质酶

蛋白含量测定和十二烷基硫酸钠．聚丙烯酰胺凝胶电泳（SDS-PAGE）分析结果表明,水分胁迫或霜霉菌接种处理均使黄瓜（Cucumis sativus L.）叶片胞间隙总蛋白含量升高,27kD蛋白积累。通过基体辅助激光解析电离飞行时间质谱0VIALDI-TOFMS）分析纯化的27kD蛋白,将所得的PMF（肽指纹图谱）在NCBInr蛋白质数据库中比对,发现水分胁迫和霜霉菌接种所诱导的27kD蛋白是同一种蛋白,均为一种酸性的几丁质酶。其酶活性测定结果表明,水分胁迫或霜霉菌接种处理的叶片胞间隙液几丁质酶活性均高于对照。     

草酸诱导黄瓜幼苗对霜霉病的抗性与H2O2的关系

以长春密刺黄瓜幼苗为材料,对经草酸处理或霜霉菌接种后黄瓜叶片的过氧化物酶(POD)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性及H2O2含量的变化进行了研究.结果表明:草酸处理或霜霉菌接种均可诱导黄瓜幼苗叶片H2O2含量显著增加,且草酸预处理后接种的叶片比相应对照叶片能更快地积累H2O2;草酸处理后叶片SOD和POD活性均升高,而CAT活性却受到一定程度的抑制.研究发现,H2O2参与了幼苗对霜霉病的抗性诱导;叶片H2O2含量的增加与其SOD、POD活性升高、CAT活性下降有关;通过调节黄瓜叶片H2O2的含量来调控有关黄瓜霜霉病抗性的防御基因表达是草酸诱导抗性的机制之一.     

黄瓜叶片胞间隙蛋白质双向电泳体系的建立

以黄瓜种质‘PI088’幼苗为材料,提取胞间隙液,制备蛋白样品,通过对不同IPG胶条、等电聚焦条件、分离胶浓度、上样量等条件的探索,建立适合黄瓜叶片胞间隙蛋白质组的双向电泳体系.结果显示:(1)用pH 3～10的非线性IPG胶条,等电聚焦时间为70 000 Vh,分离胶浓度为10％,上样量为800 μg时,能够得到较好的2-DE图谱.(2)利用所建立的双向电泳体系找到了对照及接种霜霉菌后2d的黄瓜叶片胞间隙差异蛋白,其中的12个上调表达点和10个下调表达点的表达量变化在1.5倍以上.并选取一个差异点成功进行了质谱分析.(3)质谱分析结果显示,所找的差异点为一种酸性的几丁质酶,等电点为4.27.可见,采用所建立的双向电泳体系可获得分辨率高、重复性好的2-DE图谱并能很好地用于质谱分析.     

植物源诱导剂对甜瓜叶片防卫酶活性的影响

以甜瓜主栽品种‘银帝'(抗病)和‘卡拉克赛'(感病)幼苗为材料,通过盆栽试验研究了几种植物源诱导剂对其接种古巴假霜霉菌叶片过氧化物酶(POD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)、几丁质酶(CHI)和葡聚糖酶(GLU)活性的影响,以阐释植物源诱导剂提高甜瓜抗病性的生化机制.结果显示:苯丙噻二唑(BTH)、侧柏叶提取液(CB)和中草药制剂(ZY)处理对甜瓜霜霉菌具有显著的预防效果;诱导处理或接菌后甜瓜叶片中上述抗病相关酶活性都显著上升,抗病品种‘银帝'的PPO、PAL、CHI、GLU活性增幅大于感病品种‘卡拉克赛',‘卡拉克赛'的POD活性增幅则较‘银帝'大.研究表明,叶面喷施两种植物源诱抗剂对甜瓜叶片防卫酶活性增强具有系统诱导作用,而叶片防卫酶活性增强可能是诱导剂处理后甜瓜对霜霉抗性提高的重要生化机制.     

花椰菜幼苗抗黑腐病的生理机制研究

以对黑腐病抗性不同的2个花椰菜(B rassica oleracea var.botry tis)品种为材料,研究了花椰菜苗期抵抗黑腐病的生理机制。结果表明:接种7 d后,黑腐病菌(X anthom onas camp estris pv.C amp estris)的侵入导致花椰菜幼苗干物质积累下降,抗病品种‘雪峰’的下降幅度明显低于感病品种‘2003X-106’,这与接种3 d后雪峰的净光合速率、气孔导度、蒸腾速率和叶绿素含量等下降较慢有关。病原菌侵染后,2个品种叶片的可溶性糖含量均有增加,但是抗病品种‘雪峰’的增幅较感病品种‘2003X-106’低;‘雪峰’叶片的可溶性蛋白质含量在接种后逐渐降低,而‘2003X-106’却逐渐增加;接种后,‘雪峰’叶片的IAA和M e-JA含量均上升,ABA则显著降低,而‘2003X-106’的IAA含量降低,ABA显著增加,M e-JA则在接种后的不同时期有增有减。     

亚精胺诱导黄瓜幼苗对白粉病抗性的研究

以黄瓜品种‘长春密刺’幼苗为材料,研究了亚精氨(Spd)诱导黄瓜幼苗对白粉病的抗性,并测定Spd处理和白粉菌接种对黄瓜叶片4种防御酶活性及3种防卫基因表达的影响。结果显示:(1)0.2～1.0mmol.L-1 Spd对黄瓜幼苗白粉病抗性均有不同程度的诱抗效果,并以0.8mmol.L-1 Spd处理效果最明显,诱导效率可达55.3%。(2)喷施Spd或接种白粉菌均可提高黄瓜叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、几丁质酶和β-1,3-葡聚糖酶的活性,且诱导并接种处理的植株叶片上述酶活性均比只诱导不接种处理的上升速度更快;同时,Spd处理和接种白粉菌可以提高植株叶片中POX、PAL、PR-1a基因的表达量。研究表明,Spd处理可以诱导防卫基因表达的增强,提高防御酶活性,显著降低病情指数,增强黄瓜幼苗对白粉病的抗性。     

高温诱导黄瓜抗霜霉病机理

研究了高温对黄瓜霜霉病菌致病力的影响以及高温控制霜霉病发生的效果.结果表明,40 ℃高温处理2 h和45 ℃处理1 h对黄瓜霜霉病的诱导抗病性作用最明显,其在接种后4 d时的防效分别为58.40%和45.81%,到接种后6 d时,防效分别下降为39.35%和37.65%.经高温诱导后,过氧化物酶（POD）、苯丙氨酸解氨酶(PAL)、几丁质酶（Cht）、β-1,3-葡聚糖酶（Glu）活性均显著高于对照,与未诱导植株相比,高温诱导后叶片组织的细胞壁表面有大量木质素沉积,表明高温处理后黄瓜表现出对霜霉病的抗病性.     

水稻品种过氧化物酶活性和木质素含量与抗稻瘟病菌的关系(简报)

8个水稻品种的幼苗接种稻瘟病菌后,第3d抗病品种比感病品种过氧化物酶活性增加得快,5～7d后则相反。未接菌的抗病品种比感病品种含有较多木质素。接菌后第7d感病品种较抗病品种诱导了更多木质素。表明稻瘟病菌侵染早期,过氧化物酶活性的增加与抗病性有一定相关性,出现病斑症状后相关性则不明显。     

列当寄生对不同品种向日葵幼苗生长及抗氧化酶活性的影响

采用连作重茬含向日葵列当的土壤,进行不同列当抗性向日葵品种苗期盆栽试验,调查播种后第30、50和70天向日葵幼苗根系列当的寄生率、幼苗地上部和地下部干物质量及株高,并测定其叶片丙二醛(MDA)、苯丙氨酸解氨酶(PAL)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性。结果表明:不同向日葵品种苗期对列当抗性明显不同,其中油葵品种‘T012244’和‘MGS’对向日葵列当寄生免疫,‘S31’和‘白杂9号’为高抗,而‘星火大白边’和‘白杂6号’为敏感。列当寄生后明显抑制敏感品种向日葵幼苗生长,而对免疫品种和高抗品种的影响较小。列当寄生后的敏感性品种‘星火大白边’幼苗叶片MDA含量迅速上升,PAL活性也大幅度增加,列当对幼苗生长伤害加重;而苗期免疫品种"T012244"和高抗品种"S31"MDA含量下降,PAL活性则变化不大。随接种时间延长,不同抗性向日葵品种苗期SOD、POD和CAT活性均呈现先升高后降低的变化趋势,且免疫品种的保护酶活性变化幅度大于敏感品种,说明列当寄生对敏感向日葵品种造成了逆境胁迫,诱导其幼苗叶片保护酶系统产生防御响应。     

Intercellular 27 kD protein is a chitinase induced by water stress or Pseudoperonospora cubensis in cucumber leaves]

Cucumber seedlings were drought-stressed or inoculated with Pseudoperonospora cubensis. After 3 or 6 d the intercellular fluids of treated cucumber leaves were extracted and analyzed. Protein contents increased after pathogen inoculation and a 27-kD protein was found in intercellular fluids (Figs.1, 7). Both 27 kD proteins were purified from the intercellular fluids of cucumber leaves after drought stress or pathogen inoculation by SDS-PAGE and electro-elution protocol respectively (Fig.2, 3). Purified proteins from drought-stressed and P. cubensis infected seedlings were analyzed by MALDI-TOF MS and their peptide mass fingerprinting (PMF) results were obtained (Figs.4, 5). The PMF results were compared with protein database using the software Profound. The results show that the 27 kD proteins from seedlings after drought stress and after P. cubensis infection were the same protein, i.e. an acidic chitinase (Tables 1, 2; Fig.6). The activities of chitinase in the intercellular fluids of cucumber leaves after pathogen inoculation and in those drought stress were also analyzed. Results showed that both treatments induced the increase in chitinase activity (Fig.8), which indicated that chitinase may be involved in the protection of cucumber plant against both pathogen attack and water stress.     

Differences in the Induction of Defence Responses in Resistant and Susceptible Muskmelon Plants Infected with Colletotrichum lagenarium

Defence reactions occurring in resistant (cv. Gankezaomi) and susceptible (cv. Ganmibao) muskmelon leaves were investigated after inoculating with Colletotrichum lagenarium. Lesion restriction in resistant cultivars was associated with the accumulation of hydrogen peroxide (H₂O₂). The activity of antioxidants catalase (CAT) and peroxidase (POD) significantly increased in both cultivars after inoculation, while levels of both CAT and POD activity were significantly higher in the resistant cultivar. Ascorbate peroxidase (APX) increased in both cultivars after inoculation, and level of APX activity was significantly higher in the resistant cultivar. Glutathione reductase (GR) activity significantly increased in both cultivars following inoculation, but was higher in the resistant cultivar, resulting in higher levels of ascorbic acid (AsA) and reduced glutathione (GSH). Phenylalanine ammonia lyase (PAL) significantly increased in inoculated leaves of both cultivars, resulting in higher levels of total phenolic compounds and flavonoids. The pathogenesis‐related proteins chitinase (CHT) and β‐1, 3‐glucanase (GLU) significantly increased following inoculation with higher activity in the resistant cultivar. These findings show that resistance of muskmelon plants against C. lagenarium is associated with the rapid accumulation of H₂O₂, resulting in altered cellular redox status, accumulation of pathogenesis‐related proteins, activation of phenylpropanoid pathway to accumulation of phenolic compounds and flavonoids.     

Expression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen.

The expression of PR protein encoding genes and genes involved in the phenylpropanoid metabolism was analysed on grapevine leaves of susceptible and resistant cvs. in response to inoculation with the host-pathogen Plasmopara viticola and the non-host pathogen Pseudoperonospora cubensis, the downy mildew pathogen of cucumber. These experiments were conducted to elucidate whether or not grapevine plants susceptible to downy mildew exhibit an identical defence response after inoculation with the non-host pathogen. Expression analysis of defence-related genes revealed marked differences between the susceptible cultivar "Riesling" (Vitis vinifera) and the resistant cultivar "Gloire de Montpellier" (Vitis riparia). Whereas some genes seem to be expressed constitutively in "Gloire" or induced after an inoculation with both pathogens, expression of defence-related genes in Riesling was influenced mainly after inoculation with the non-host pathogen: PR-2, PR-3, PR-4, a PGIP gene, and especially genes encoding enzymes involved in anthocyanin biosynthesis (DFR, F3H, LDOX) were affected. Therefore, the occurrence of the respective products (flavans and other phenolics) in inoculated leaves was investigated with appropriate histological staining techniques. These stainings revealed a production of catechins and related phenolic compounds within the first 48 hai (hours after inoculation) with Ps. cubensis but not with P. viticola in Riesling, whereas in Gloire no further production was seen, which may be due to the high content of polyphenolics as observed in control leaves. In addition to the staining procedures, sporulation intensity was monitored on leaf discs. Pretreatments of leaf discs with Ps. cubensis led to a reduced browning reaction (as a result of a hypersensitive reaction) in Gloire and significantly reduced the intensity of sporulation in Riesling after a subsequent inoculation with P. viticola.     

Transmission Electron Microscopy of Susceptible and Resistant Tomato Leaves Following Infection with Pseudomonas syringae pv. tomato

Ultrastructural changes in tomato leaves of susceptible cv. Peto 95 and resistant cv. Ontario 7710 infected with Pseudomonas syringae pv. tomato were followed by transmission electron microscopy. Up to 48 hours from the inoculation host cells of both cultivars looked quite normal and no bacteria were visible in the intercellular spaces; bacterial cells were found only in the substomatal chambers. Afterwards, the leaf cells of cv. Peto 95 began to degenerate and bacteria invaded the intercellular spaces which seemed enlarged. After 15 days the disorganization was complete: tomato cells were plasmolyzed and the intercellular spaces were filled with bacteria. In the leaves of resistant cv. Ontario 7710 no bacteria were observed later than 48 hours and no visible modifications occurred up to 15 days after the inoculation.     

Intercellular chitinase and peroxidase activities associated with resistance conferred by geneLr35to leaf rust of wheat

The effect of leaf rust (Puccinia triticina) infection on intercellular chitinase (EC 3.2.1.14) and peroxidase (EC 1.11.1.7) activities was studied in resistant [RL 6082 (Thatcher/Lr35)] and susceptible (Thatcher) near isogenic wheat (Triticum aestivum L.) lines at seedling, stem elongation and flag leaf stages of plant growth. The levels of activity of these enzymes were low during the seedling and stem elongation stages. Resistant plants at the flag leaf stage, during which the Lr35 resistance gene was maximally expressed, exhibited high constitutive levels of chitinase and peroxidase activities, in contrast to the lower constitutive levels of susceptible plants. The results suggest that chitinase and peroxidase, constitutively present in the intercellular spaces of Thatcher/Lr35 wheat leaves, may play a role in Lr35 mediated resistance to leaf rust.     

Screening for Barley yellow dwarf virus-Resistant Barley Genotypes by Assessment of Virus Content in Inoculated Seedlings

The content of Barley yellow dwarf virus (BYDV) in roots and leaves of barley seedling plants differing in their level of resistance was assessed by quantitative ELISA 1–42 days after inoculation with the strain of BYDV (PAV). High virus accumulation in roots and low concentration in leaves was characteristic of the period 9–15 days after inoculation. In leaves, the differences in virus content between resistant and susceptible genotypes became significant after 15 days and resistance to virus accumulation was better expressed 30–39 days after inoculation. Roots of resistant materials exhibited evident retardation of virus accumulation and the greatest difference in virus content between resistant and susceptible plants was detected 9 days after inoculation. By these criteria, the selected winter and spring barley cultivars and lines (in total 44 materials) fell in to five groups according to field reactions and the presence or absence of the Yd2 resistance gene. There were highly significant and positive relations between ELISA values and 5‐year field data on symptomatic reactions and grain‐yield reductions due to infection. Using the described method, resistant and moderately resistant genotypes (both Yd2 and non‐Yd2) were significantly differentiated from susceptible genotypes. The possible use of this method in screening for BYDV resistance is discussed.     

Superoxide dismutase responses of strawberry cultivars to infection by Mycosphaerella fragariae

In controlled conditions, the effect of leaf infection by Mycosphaerella fragariae on total superoxide dismutase (SOD, EC 1.15.1.1) activity and induction of SOD isozymes was studied in three different strawberry cultivars, i.e. "Joliette" (resistant), "Honeoye" (partially resistant) and "Kent" (susceptible). Infection of the strawberry leaves with M. fragariae resulted in increase in SOD activities in all three cv. Total SOD increased 1d after inoculation in Joliette and Kent, and 2d after inoculation in Honeoye and reached the highest level in all three cv, at the 2nd day after inoculation, then slowly declined afterward. Total SODs in Joliette and Honeoye at the 2nd day after inoculation were 4516 and 4947Ug(-1) FW, respectively, which were significantly higher than that of Kent (3255Ug(-1)FW). Banding pattern of SOD isozymes in all three cv was also affected by infection. Electrophoresis profile of infected cv revealed two newly synthesized isozymes in Joliette and Honeoye, in which one band, i.e. R(f) = 0.53 was observed exclusively in inoculated Joliette and Honeoye. Therefore, it is considered to be associated with leaf spot resistance.     

Role of polyisoprenoids in tobacco resistance against biotic stresses

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria ( P.  syringae ) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.