玉米灰斑病抗性机制中活性氧代谢的作用

研究了玉米灰斑病菌侵染四个抗病和感病的玉米品种时，叶片内部活性氧代谢酶及细胞过氧化产物含量的动力学变化。结果表明，抗、感病品种的SOD、CAT及POD酶活性在病茵侵染后都变化显著，抗病品种各酶活性变化幅度比感病品种大。叶片内过氧化产物MDA含量则相反，抗病品种沈试29在接种第13天时叶片MDA含量只为18．46nmol／g，而感病品种铁单9为23．14nmol／g，抗病品种比感病品种增加幅度小。说明活性氧代谢在植物抗病机制中起着重要作用，抗病品种对活性氧代谢的酶调节能力强，病菌侵染后细胞过氧化程度低，在痛菌侵染时活性氧清除酶活性最大增加值与发病程度呈显著正相关。     

豇豆与锈菌互作中的活性氧代谢研究

 本文分析了不同抗性水平的豇豆(Vigna sesquipdalis Wight)品种与锈菌(Uromyces vignae Barcl)互作中活性氧(ac-tive oxygen species,AOS)的发生、防御酶活性及膜脂过氧化水平的变化规律及其与豇豆抗锈病性的关系。结果表明,在接种后,免疫品种(益农)和感病品种(揭上和金迪)的超氧物歧化酶(superoxide dismutase,SOD)比活性均升高,且升高幅度都在12 h出现高峰;抗病品种(金山和特青)开始时降低,至12h时回升,在24 h(高抗)和48 h(中抗)出现高峰;在24 h时,免疫和抗病品种的SOD比活性高于感病品种。在接种后12 h内,免疫和抗病品种的过氧化氢酶(catalase,CAT)比活性都下降;而感病品种均上升,并在12 h出现第1个高峰。受锈菌侵染后,豇豆各品种都产生超氧阴离子(superoxide anion,O₂^·),在多数测试时段中,免疫和抗病品种的O₂^·产率净变化值低于感病品种。丙二醛(malondialdehyde,MDA)含量变化幅度与品种抗性呈负相关,与O₂^·产率的变化趋势基本相吻合。这些结果表明AOS代谢在豇豆与锈菌互作中起重要作用。     

豇豆与锈菌互作中的多酚氧化酶和过氧化物酶活性及其与抗性的关系

测定了具有不同抗性水平的5个豇豆Vigna sesquipdalis Wight品种在受锈菌Uromyces vignae Barcl侵染前和侵染后的若干阶段中的多酚氧化酶(PPO)和过氧化物酶(POD)活性,并分析其与抗性的关系.结果表明,在接种后24h内,免疫和抗病品种的PPO比活性及其变化率均高于感病品种,且前者PPO比活性变化率高峰出现早,后者出现迟.在接种后,各品种的POD比活性及其变化率均上升,但中抗和感病品种的高峰出现早,免疫和高抗品种出现晚.此外,中抗和感病品种的POD比活性及其变化率在接种12h左右出现高峰后立即下降,而高抗品种的则持续上升至24h左右出现高峰,免疫品种的POD比活性也在24h左右出现高峰,但其POD比活性变化率则持续到48h左右达到高峰,且免疫和抗病品种的峰值明显大于感病品种.     

抗感枯萎病黄瓜品种的病理组织结构学研究

选取不同抗性黄瓜品种中农13(抗)和中农6号(感)为试材,采用蘸根法接种,利用电镜技术和石蜡切片方法观察了病菌侵染前后根茎组织结构的变化。结果表明,抗病品种有环纹和网纹两种导管类型,有较厚的角质层,感病品种有环纹导管一种类型,无角质层。抗病品种在接种后细胞壁加厚、导管腔内出现褐色物、胞壁覆盖物以及侵填体。感病品种仅出现胞壁加厚和胼胝体。对于病原菌侵染,抗感品种具有不同程度的反应,抗病品种反应程度早于且强于感病品种。     

BTH处理对甜瓜叶片活性氧代谢的诱导作用

为了明确活性氧（reactive oxygen species,ROS）代谢在甜瓜抗病性诱导中的作用,以抗白粉病甜瓜品种Tam Dew和感病品种卡拉克赛幼苗为材料,通过盆栽试验研究了苯丙噻二唑（BTH）喷雾或白粉菌接种后甜瓜叶片超氧阴离子（O2.-）产生速率、过氧化氢（H2O2）含量及超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、苯丙氨酸解氨酶（PAL）活性的变化。BTH处理或白粉菌接种均可诱导甜瓜叶片SOD、PAL活性升高,抑制CAT活性,导致叶组织O2.-产生速率和H2O2含量增加,BTH喷雾＋白粉菌接种比二者单独处理效果更好。结果表明,BTH处理后叶片O2.-产生速率提高和H2O2积累是甜瓜抗白粉病能力提高的重要机制,BTH通过诱导ROS代谢酶活性调节H2O2含量,且BTH诱导的甜瓜抗病性与品种的基础抗性有关。     

南方根结线虫侵染对黄瓜植株生长和抗氧化系统的影响

为探明野生黄瓜对南方根结线虫的抗性机制,采用温室盆栽苗期人工接种技术,研究了线虫侵染对黄瓜抗感品种植株生长和抗氧化系统的影响。南方根结线虫的侵染显著降低了感病品种津春4号的株高、叶面积、地上部鲜重与干重及根系活力,并显著增加其根系鲜重与干重,而对抗病品种西印度瓜的各项生长指标均未造成明显影响。西印度瓜中超氧阴离子(O.2-)、过氧化氢(H2O2)、电导率和丙二醛(MDA)含量增加幅度均小于津春4号,超氧化物歧化酶(SOD)活性降低,过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)活性增加幅度均显著高于津春4号。研究表明西印度瓜遭受的氧化胁迫较小,其抗氧化系统有效清除了氧化损伤。     

枯萎病菌对不同抗性黄瓜品种几憾活性的影响

用枯萎病菌接种不同抗性黄瓜品种,研究不同抗性黄瓜品种的过氧化物酶（POD）、多酚氧化酶（PPO）和几丁质酶的活性变化。结果表明,接种后抗感品种POD、PPO和几丁质酶活性基本都呈现先升后降再升（再降）的趋势。抗感品种均在接种后12h时出现第1次POD活性峰值,抗病品种中农13号、津优3号分别在接种后60、72h、感病品种在84h出现第2次POD活性峰值;接种后24h时抗感品种均达到第1次PPO活性峰值,抗病品种在48h、感病品种在60h时达到第2次PPO活性峰值;接种后抗病品种在48h时达到第1次几丁质酶活性峰值,72h时达到第2次峰值,而感病品种只在60h时出现1次几丁质酶活性峰值。抗感品种的POD、PPO、几丁质酶活性的2次培值都显著或极显著地高于各自的对照,在接种后的早期阶段,感病品种的POD、几丁质酶活性的第1次峰值都显著或极显著地高于抗病品种,PPO活性的第1次峰值极显著地低于抗病品种。     

枯萎病菌对不同抗性黄瓜品种几种酶活性的影响

用枯萎病菌接种不同抗性黄瓜品种,研究不同抗性黄瓜品种的过氧化物酶（POD）、多酚氧化酶（PPO）和几丁质酶的活性变化。结果表明,接种后抗感品种POD、PPO和几丁质酶活性基本都呈现先升后降再升（再降）的趋势。抗感品种均在接种后12 h时出现第1次POD活性峰值,抗病品种中农13号、津优3号分别在接种后60、72 h、感病品种在84 h出现第2次POD活性峰值;接种后24 h时抗感品种均达到第1次PPO活性峰值,抗病品种在48 h、感病品种在60 h时达到第2次PPO活性峰值;接种后抗病品种在48 h时达到第1次几丁质酶活性峰值,72 h时达到第2次峰值,而感病品种只在60 h时出现1次几丁质酶活性峰值。抗感品种的POD、PPO、几丁质酶活性的2次峰值都显著或极显著地高于各自的对照,在接种后的早期阶段,感病品种的POD、几丁质酶活性的第1次峰值都显著或极显著地高于抗病品种,PPO活性的第1次峰值极显著地低于抗病品种。     

利用GFP荧光标记比较大丽轮枝菌对抗/感马铃薯品种的侵染过程

为了对马铃薯黄萎病菌的系统侵染进行研究，本研究首先建立了用于观察大丽轮枝菌侵染马铃薯过程的培养皿滤纸体系，在此基础上利用带有GFP标记的大丽轮枝菌对不同抗性水平马铃薯根系侵染过程进行了比较研究，结果表明：马铃薯根系在浓度为10⁷个·mL^-1的大丽轮枝菌分生孢子液中浸泡处理2 h后，分生孢子可附着在抗/感马铃薯根系的根毛上，单个视野中感病品种根系上分生孢子附着平均数量为65个，显著高于抗病品种根系上附着的分生孢子数量(23个);接种12 hpi,根系上附着的分生孢子开始萌发，但抗/感品种根系表面孢子的萌发率无显著差异；接种3 dpi后，菌丝体能够突破根系表皮，进入细胞间隙，但感病品种根系中菌丝的扩展速度更快，并在7 d后到达根系的维管束内部；而抗病品种则在接种9 dpi,在维管束内才能够观察到绿色荧光信号；接种10 dpi,感病品种根系中可见新的分生孢子梗和分生孢子形成，而抗病品种中未见有分生孢子的形成。接种30 dpi,感病品种地上部茎杆中定殖的病原菌量是抗病品种的5倍以上；同时，感病品种叶柄中的病原菌数量也显著高于抗病品种。     

黄瓜黑星病菌致病机理的研究Ⅱ细胞壁降解酶及其在致病中的作用

 本文通过活体内外黄瓜黑星病菌(Cladosporium cucumerinum)产生的细胞壁降解酶活性分析,初步明确了Cx-酶(羧甲基纤维素酶)、β-葡萄糖苷酶和PMG(聚甲基半乳糖醛酸酶)、PMTE(果胶甲基反式消除酶)在该菌侵染黄瓜中的作用。瓜条感病的各部位,以病健交界处细胞壁降解酶活性最高;在检测的各类酶中,活性最高的为PMG、PMTE,Cx-酶、β-葡萄糖苷酶和FPA(滤纸酶)活性较低。抗感病黄瓜品种染病后,细胞壁降解酶活性均迅速增强。感病品种染病后,果胶酶(PMG、PMTE)活性远远高于抗病品种,其高峰值是抗病品种的2.72~7.52倍。感病品种接种后纤维素酶(Cx-酶、β-葡萄糖苷酶)活性一直增强;抗病品种接种后β-葡萄糖苷酶随着接种天数的增加酶活性一直增强,Cx-酶在染病后初期活性迅速增强,增至高峰后逐渐降低。     

Histochemical studies on the accumulation of reactive oxygen species (O2 and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.     

Histochemical studies on the accumulation of reactive oxygen species (O2− and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.     

Microscopic detection of reactive oxygen species generation in the compatible and incompatible interactions of Alternaria alternata Japanese pear pathotype and host plants

 Reactive oxygen species (ROS) generation was examined in the interaction of Alternaria alternata Japanese pear pathotype and host plants using three methods: nitro blue tetrazolium (NBT) method for microscopic detection of O₂ ⁻, diaminobenzidine (DAB) methods for microscopic detection of H₂O₂, and cerium chloride methods for ultrastructural detection of H₂O₂. ROS generation was detected by NBT and DAB methods at appressoria on leaves of susceptible cultivars and heat-shocked leaves of resistant cultivars but not in leaves of resistant cultivars. Ultrastructural detection by the cerium chloride method identified ROS generation at cell walls of appressoria and penetration pegs in susceptible, resistant leaves and heat-shocked leaves. These differences in the ultrastructural and microscopic data in resistant areas were due to the restriction of ROS generation in limited areas, the side facing the plant surface, of appressoria and penetration pegs. Therefore, ROS generation was apparently induced regardless of the resistance or susceptibility of the cultivar with the difference being in the volumes generated. After evaluating the pathological role of ROS generation in fungal structures, such generation was found to be associated with early penetration of cell walls in pear plants. Additionally, ROS generation in plants was also found in degrading pectin layers near infected hyphae and in plasma membrane modification sites in susceptible leaves but not in resistant leaves. ROS generation in susceptible leaves might be accompanied with plasma membrane damage, although the role of ROS generation in the pectin layers is not clear. ROS generation in both fungal and plant cells during their interaction was likely associated with the expression of susceptibility. Received: June 3, 2002 / Accepted: July 31, 2002     

Spatiotemporal patterns of oxidative burst and micronecrosis in resistance of wheat to brown rust infection

The accumulation of H₂O₂ (oxidative burst) and the progress of pathogen development were studied in compatible and incompatible wheat‐brown rust interactions. The accumulation of H₂O₂ was detected in 98·7% of guard cells with appressoria 8 h post inoculation (hpi). The reaction in both susceptible and resistant plants declined 2–3 days post inoculation (dpi). The second phase of the oxidative burst was observed in the mesophyll and/or epidermis. In susceptible plants it began 4–5 dpi and was detected only in the epidermis. In resistant plants the response was observed in the mesophyll. In moderately resistant plants it was induced 1–3 dpi, and the percentage of infection units reached 80–90% 8 dpi. This corresponded with severe necrotic symptoms. In highly resistant plants, the oxidative burst was short and transient. The percentage of infection units with H₂O₂ accumulation reached its highest level (60–70%) 2 dpi, and decreased thereafter. Four days later, the low percentage and weak DAB staining indicated very low H₂O₂ accumulation. The localization and the time‐course changes of the oxidative burst correlated with the profiles of the micronecrotic response, haustorium mother cell formation and pathogen development termination. An early and localized induction of oxidative burst followed by its rapid quenching correlated with high resistance and almost no disease symptoms. The possible correlation of the oxidative burst and pathogen development patterns with the level and durability of resistance conferred by Lr genes are discussed.     

Avirulent strain of Colletotrichum induces a systemic resistance in strawberry

Strawberry plants exposed to an avirulent isolate of Colletotrichum fragariae acquired strong resistance against a virulent strain of C. acutatum. Biochemical, morphological and molecular markers indicated that the strong defence response was associated with an oxidative burst and a transient accumulation of salicylic acid (SA). A maximum accumulation of H₂O₂ and O₂ ^? was observed 8 h after inoculation (hai), callose was detected 48 hai, and a peak of SA was observed 48 hai. Biochemical and phytopathogenic analyses carried out in non-treated tissues revealed that the defence response was systemic and remained fully active 60 days after the first inoculation. Experiments also showed that the resistance acquired by mother plants after the inoculation with the avirulent isolate could be passed to daughter plants through runners. Further characterization of the induced systemic resistance showed that the resistance was not only effective against a virulent strain of C. acutatum but also against Botrytis cinerea.     

Comparative analysis of early H2O2 accumulation in compatible and incompatible wheat–powdery mildew interactions

Comparative analysis was carried out for hydrogen peroxide (H₂O₂) accumulation in response to powdery mildew (Blumeria graminis[Erysiphe graminis] f.sp. tritici) primary germ tube (PGT) and appressorial germ tube (AGT) contact, and in papilla (cell wall apposition) and hypersensitive responses (HR) in wheat (Triticum aestivum). Using primary leaves of three susceptible wheat lines (Bainong 3217; Beijing 837; Jingshuang 16) and five resistant lines [Mardler (Pm2 + Pm6); Ulka/8 × Cc (Pm2); Mardler/7 × Bainong 3217 (Pm2); Pm16; Pm16/7 × Beijing 837 (Pm16)], early H₂O₂ accumulation between 10 and 48 h after inoculation was studied. Strong H₂O₂ accumulation was found in effective papillae and associated cytosolic vesicles in both susceptible and resistant wheat lines, suggesting the important role of H₂O₂ in effective papillae formed as a general plant defence against powdery mildew. High frequency of effective papilla formation was observed in all five resistant lines. Among resistant lines, hypersensitive cell death was detected earlier in Mardler and Ulka/8 × Cc than in Pm16 and Pm16/7 × Beijing 837. In all cases this was associated with H₂O₂ accumulation in attacked epidermal cells. Interestingly, penetration resistance but not HR appeared to be mediated by the Pm2 gene in the Mardler/7 × Bainong 3217 line, suggesting that Pm2 may govern an HR-independent defence pathway in this genetic background. As effective papillae and HR did not occur in the same cells, papilla deposition may be independent of the HR response, despite the fact that both defence mechanisms were associated with high H₂O₂ accumulation. In wheat powdery mildew-incompatible interactions, HR acts as a second line of defence to contain infection when the papilla defence fails.     

Mycorrhizal protection of chili plants challenged by <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Hydrogen peroxide (H₂O₂) has been implicated in many stress conditions. Control of H₂O₂ levels is complex and dissection of mechanisms generating and relieving H₂O₂ stress is difficult, particularly in intact plants. Here the role of the mycorrhizal inoculation in chili plants challenged with Phytophthora capsici was investigated to study the effect on hypersensitive response. In the treatment without mycorrhiza (treatment T3) and with mycorrhiza (considered treatment T4) visible disorders were detected two days after inoculation with P. capsici, but in the next days T3 plants rapidly developed 25% more necrotic lesions on the leaves than T4 plants. Leaf necrosis correlated with H₂O₂ accumulation and the greater damage observed in T3 plants coincided with larger accumulation of H₂O₂ after 12 h of inoculation accompanied with an increase in POX (peroxidase) and SOD (superoxide dismutase) activity. T4-infected and mycorrhizal plants exhibited an earlier accumulation of H₂O₂ starting 6 h after inoculation with lower levels compared to T3 plants. Correlated with observed damage, POX and SOD activity measured in T4 plants indirectly suggest a smaller accumulation of ROS (reactive oxygen species) leading to a decrease in the wounds observed and slightly diminishing the advance of the pathogen. According to these findings, we conclude that mycorrhizal colonization contributes significantly in maintaining the redox balance during oxidative stress, but the exact mechanism is still uncertain.     

Antioxidant,ethylene and membrane leakage responses to powdery mildew infection of near-isogenic barley lines with various types of resistance

Leaves of powdery mildew-susceptible barley (Hordeum vulgare cv. Ingrid) and related near-isogenic lines bearing various resistance genes (Mla12, Mlg or mlo5) were inoculated with Blumeria graminis f. sp. hordei race A6. Fungal attack induced several-fold increases in ethylene emission and electrolyte leakage in leaves of susceptible Ingrid beginning 3 days after inoculation. Activities of peroxidase, superoxide dismutase, glutathione S-transferase, ascorbate peroxidase and glutathione reductase enzymes were induced markedly in susceptible leaves 5–7 days after inoculation. Similar, but less pronounced pathogen-induced changes were detected in inoculated leaves of Mla-type resistant plants that show hypersensitive cell death upon inoculation, and, to an even lesser extent, in the Mlg and mlo lines, where no visible symptoms accompanied the incompatible interaction. Glutathione content increased only in susceptible barley 7 days after inoculation. Catalase activity, total ascorbate content and redox state were not influenced by inoculation in any of the genotypes. The activity of dehydroascorbate reductase was significantly reduced 3–5 days after inoculation in the susceptible parental plants and after 5 days in Mla and Mlg lines, while it was stable in the mlo barley. Slightly elevated levels of H₂O₂ were observed in the inoculated resistant plants. In contrast, H₂O₂ content decreased in the susceptible line 7 days after pathogen attack. These data indicate that high levels of antioxidants are involved in the compatible interaction of susceptible barley and powdery mildew by protecting the pathogen from oxidative damage.