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.     

高温调控对黄瓜霜霉病菌侵染的影响

采取温湿度相结合的方法来研究高温处理对黄瓜霜霉病菌（Pseudoperonospora cubensis Rostov)侵染的影响,比较研究了35－50℃4个温度梯度、50％－90％5个湿度组合处理对病菌致病性的影响。同一相对湿度条件下,随着温度的上升病菌致病性降低;40－50℃的高温范围内,在同一温度下随着湿度的不断升高,受处理病菌的致病能力逐渐下降。在RH80％以上、温度40℃以上时,病菌的致病力随着处理时间的延长而变弱;45℃以下的高温高湿处理病菌超过1h,病菌基本上没有致病性。通过高温控制苗期黄瓜霜霉病的研究,确定高温高湿防治苗期黄瓜霜霉病的最佳温湿度为45℃1h(RH80%)。通过高温致死菌诱导植株抗性的研究,初步明确高温处理致死的病菌可以短期诱导植株的抗性。     

虎尾兰水提取物对黄瓜霜霉病的防治研究

明确虎尾兰水提取物对黄瓜霜霉病菌无性阶段的抑制作用及对黄瓜霜霉病的防治效果。采用凹玻片法和离体叶五点接种法分别测定该提取物对游动孢子的释放和游动、休止孢的萌发和芽管伸长以及菌丝生长和孢子囊产生的抑制作用,采用离体叶五点接种法测定该提取物的保护作用、治疗作用和持效期,并采用盆栽试验测定该提取物的保护作用和治疗作用。一系列试验结果显示,虎尾兰水提取物对黄瓜霜霉病菌游动孢子的释放和游动,休止孢的萌发和芽管伸长以及菌丝生长和孢子囊产生的EC₅₀值分别为1.19、0.77、0.70、2.13、9.09和13.15 mg/mL。在离体叶片条件下,虎尾兰水提取物对黄瓜霜霉病的保护和治疗作用的EC₅₀值分别为3.69 和14.57 mg/mL,持效期达14 d以上。盆栽条件下,该提取物对黄瓜霜霉病保护作用和治疗作用的防效分别为68.38%和21.33%。虎尾兰水提取物对黄瓜霜霉病具有很好的保护作用和较长的持效期。     

影响活体内马铃薯晚疫病菌卵孢子产生因素的研究*

研究了不同菌株组合,马铃薯植株茎、叶及接种物中A1和A2菌株孢子囊比例、温度、湿度对卵孢子产生的影响。不同菌株组合产生卵孢子的数量有显著差异;在离体接种情况下,叶片中产生卵孢子数量大于茎中产生卵孢子数量;A1和A2菌株中孢子囊不同比例对卵孢子产生影响很大,当比值为1∶1时卵孢子产生量最大;15℃光照条件下培养,并给侵染叶片持续的水分供应才能产生大量卵孢子;寄主的抗性水平对卵孢子产生有明显的影响,中抗品种上产生卵孢子量最多,高抗品种上产生卵孢子量最少,感病品种上产生卵孢子量居中。     

影响活体内马铃薯晚疫病菌卵孢子产生因素的研究

研究了不同菌株组合,马铃薯植株茎、叶及接种物中A1和A2菌株孢子囊比例、温度、湿度对卵孢子产生的影响。不同菌株组合产生卵孢子的数量有显著差异;在离体接种情况下,叶片中产生卵孢子数量大于茎中产生卵孢子数量;A1和A2菌株中孢子囊不同比例对卵孢子产生影响很大,当比例为1：1时卵孢子产生量最大;15℃光照条件下培养,并给侵染叶片持续的水分供应才能产生大量卵孢子;寄主的抗性水平对卵孢子产生有明显的影响,中抗品种上产生卵孢子量最多,高抗品种上产生卵孢子量最少,感病品种上产生卵孢子量居中。     

枯草芽孢杆菌ZH-8对黄瓜霜霉病防治效果的研究

枯草芽孢杆菌ZH-8分离于黄瓜根际,将该菌株发酵后,添加助剂制成液体菌剂。该液体菌剂在温室试验和田间试验中表现出较好的防治效果,对黄瓜霜霉病的防效分别达75．61％和69．12％。该菌剂可以有效抑制黄瓜霜霉病菌孢子囊的萌发,并且能够在黄瓜叶片上形成一层保护膜,阻止病原菌孢子的侵入。同时对其抗利福平标记菌株进行了在黄瓜叶片上定殖的研究,研究表明,该生防菌株在黄瓜叶片上具有较强的定殖能力,定殖时间达21d。     

黄瓜RGA基因的半定量RT-PCR表达分析

以黄瓜(Cucumis sativusL.)抗霜霉病品种东农129为材料,利用RT-PCR半定量法研究了接种霜霉病菌(Pseudoperonospora cubensisRostow)、喷施水杨酸(SA)和氯化钙(CaCl2)等不同处理对黄瓜抗病基因类似序列(RGA)表达的影响.结果表明:CsRGA1和CsRGA5基因的表达受霜霉病菌的侵染而启动或加强,外施SA和CaCl2都能够增强其表达;CsRGA4和CsRGA8属于组成型表达基因,其表达可能与霜霉病菌的侵染无关;CsRGA2的表达与外施SA和CaCl2缺乏密切关联.     

瑞拉菌素产生菌S-5120菌株防治黄瓜霜霉病害的研究初报

经黄瓜霜霉病孢子萌发试验和离体叶片抑菌试验表明,瑞拉菌素产生菌S-5120菌株和瑞拉菌素均对黄瓜霜霉病有较强抑制作用。温室防病试验表明,瑞拉菌素产生菌S-5120发酵液对黄瓜霜霉病有明显的保护和治疗作用。相对保护效果达75．17％;治疗效果达69．69％,且高于农抗-120的相对防效62．65％。     

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.     

黄瓜叶片胞间隙几丁质酶与抗霜霉菌侵染关系的研究

采用高抗霜霉病的‘津春4号’和易感霜霉病的‘长春密刺’黄瓜的温室穴盘幼苗为材料,研究了接种霜霉菌对黄瓜叶片胞间隙几丁质酶累积变化及幼苗生长的影响.结果显示:(1)与易感品种相比,抗病品种叶片胞间隙液蛋白浓度升高快、含量高;同时几丁质酶活性上升的速度快、幅度大,于接种后第2天达到峰值,且高活性维持至第7天;(2) SDS-PAGE电泳分析发现,抗病品种有一种27 kD的酸性几丁质酶在接种后第2天迅速积累,并在接种后第4、7天呈现出较高的表达量;Western blotting的结果也证实了上述胞间隙几丁质酶积累的变化.(3)与未接种对照相比,接种处理后第4、7天,易感黄瓜幼苗鲜重、干物质积累量均出现显著降低,但在接种后第7天抗病品种的上述生长指标要显著高于易感病品种.研究表明,接种霜霉菌后,抗病黄瓜叶片胞间隙几丁质酶迅速累积且活性快速升高,以减轻霜霉病对幼苗生长的侵害,这可能是黄瓜抗霜霉菌侵染的一种防御机制.     

Thermal imaging of cucumber leaves affected by downy mildew and environmental conditions

Pathogenesis of Pseudoperonospora cubensis causing downy mildew of cucumber resulted in changes in the metabolic processes within cucumber leaves including the transpiration rate. Due to the negative correlation between transpiration rate and leaf temperature, digital infrared thermography permitted a non-invasive monitoring and an indirect visualization of downy mildew development. Depending on the stage of pathogenesis and the topology of chloroses and necroses, infection resulted in a typical temperature pattern. Spatial heterogeneity of the leaf temperature could be quantified by the maximum temperature difference (MTD) within a leaf. The MTD increased during pathogenesis with the formation of necrotic tissue and was related to disease severity as described by linear and quadratic regression curves. Under controlled conditions, changes in temperature of infected leaves allowed the discrimination between healthy and infected areas in thermograms, even before visible symptoms of downy mildew appeared. Environmental conditions during thermographic measurement, in particular air temperature and humidity, as well as water content and age of the leaf influenced the temperature of its surface. Conditions enhancing the transpiration rate facilitated the detection of changes in leaf temperature of infected leaves at early stages of infection. As modified by environmental conditions, MTD alone is not suitable for the quantification of downy mildew severity in the field.     

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.     

Control of downy mildew (Pseudoperonospora cubensis) of greenhouse grown cucumbers with alternative biological agents

In organic cucumber production infection with downy mildew (Pseudoperonospora cubensis) is a major problem. Plant extracts from Glycyrrhiza glabra L. (licorice), a plant belonging to the family Fabaceae, and Salvia officinalis (sage) as well as cultures of the bacterium Aneurinibacillus migulanus were investigated for efficacy of disease control under commercial growing conditions. Contrary to bioassays, where sage extract and the microorganism showed highest activity, in the trials of 2008 G. glabra extract was more effective than sage extract or A. migulanus against P. cubensis. Parameters such as concentrations of the preparations or application intervals could have been the reason for this. In the following year's trial (2009) the concentration of these agents was therefore increased somewhat and plants were either treated in seven day application intervals or in ten day application intervals. In the semi-commercial trials of 2009 all alternative biological agents showed good efficacies up to around 80% against infection with downy mildew. The application interval seemed to have a marginal effect only. Again, the licorice extract tended to be the best agent.     

The cucurbit downy mildew pathogen Pseudoperonospora cubensis

Pseudoperonospora cubensis[(Berkeley & M. A. Curtis) Rostovzev], the causal agent of cucurbit downy mildew, is responsible for devastating losses worldwide of cucumber, cantaloupe, pumpkin, watermelon and squash. Although downy mildew has been a major issue in Europe since the mid-1980s, in the USA, downy mildew on cucumber has been successfully controlled for many years through host resistance. However, since the 2004 growing season, host resistance has been effective no longer and, as a result, the control of downy mildew on cucurbits now requires an intensive fungicide programme. Chemical control is not always feasible because of the high costs associated with fungicides and their application. Moreover, the presence of pathogen populations resistant to commonly used fungicides limits the long-term viability of chemical control. This review summarizes the current knowledge of taxonomy, disease development, virulence, pathogenicity and control of Ps. cubensis. In addition, topics for future research that aim to develop both short- and long-term control measures of cucurbit downy mildew are discussed. TAXONOMY: Kingdom Straminipila; Phylum Oomycota; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Pseudoperonospora; Species Pseudoperonospora cubensis. DISEASE SYMPTOMS: Angular chlorotic lesions bound by leaf veins on the foliage of cucumber. Symptoms vary on different cucurbit species and varieties, specifically in terms of lesion development, shape and size. Infection of cucurbits by Ps. cubensis impacts fruit yield and overall plant health. INFECTION PROCESS: Sporulation on the underside of leaves results in the production of sporangia that are dispersed by wind. On arrival on a susceptible host, sporangia germinate in free water on the leaf surface, producing biflagellate zoospores that swim to and encyst on stomata, where they form germ tubes. An appressorium is produced and forms a penetration hypha, which enters the leaf tissue through the stomata. Hyphae grow through the mesophyll and establish haustoria, specialized structures for the transfer of nutrients and signals between host and pathogen. CONTROL: Management of downy mildew in Europe requires the use of tolerant cucurbit cultivars in conjunction with fungicide applications. In the USA, an aggressive fungicide programme, with sprays every 5-7 days for cucumber and every 7-10 days for other cucurbits, has been necessary to control outbreaks and to prevent crop loss. USEFUL WEBSITES: http://www.daylab.plp.msu.edu/pseudoperonospora-cubensis/ (Day Laboratory website with research advances in downy mildew); http://veggies.msu.edu/ (Hausbeck Laboratory website with downy mildew news for growers); http://cdm.ipmpipe.org/ (Cucurbit downy mildew forecasting homepage); http://ipm.msu.edu/downymildew.htm (Downy mildew information for Michigan's vegetable growers).     

β-Aminobutyric Acid-induced Resistance in Cucumber against Biotrophic and Necrotrophic Pathogens

The non-protein amino acid β-aminobutyric acid (BABA) protects plants against a wide range of pathogens. Protection of cucumber plants by BABA depends on the potentiation of pathogen specific defence responses. To contribute to the analyses of the mode of action of BABA, we established a protocol for a fast and reproducible leaf disc assay to evaluate the effect of this chemical compound on cucumbers infected with either the biotrophic downy mildew fungus Pseudoperonospora cubensis, or the necrotrophic microbial pathogen Colletotrichum lagenarium . Accumulation of callose could be found in interactions with both pathogens after BABA-treatment. Furthermore, a localized rapid cell death and the production of reactive oxygen intermediates were detected after downy mildew attack. In contrast to this, degenerated primary hyphae were found in BABA induced tissue after inoculation with C. lagenarium .     

Zoosporogenesis in Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew

During sporulation of Pseudoperonospora cubensis on cucumber leaves ( Cucumis saliva ) zoosporangia are formed on the dichotomously branched sporangiophore. The mature zoosporangium has a preformed discharge papilla and the cytoplasm is uncleaved. The zoosporangium wall is decorated and the outer layer of the wall is electron opaque in ultrathin sections. As the zoosporangium is able to survive freezing (- 18°C) for prolonged periods of time (3–4 months) the zoosporangium may serve as the "resting" structure which survives overwintering in Northern latitudes in the absence of oospore formation.
Zoospore cleavage can be synchronized by placing freshly harvested zoosporangia in distilled water. Cleavage of the zoosporangial cytoplasm is by means of the fusion of small vesicles apparently derived from dictyosomes which become highly active after zoosporogenesis is induced.
Vesicles with an osmiophilic electron opaque content are the dominant type of vesicle found in the zoosporangia. The content of these vesicles undergoes dynamic changes during zoosporogenesis and during the late stages of sporogenesis the content becomes finely striated as is typical of these vesicles when observed in the zoospore. On the basis of the results presented here it is suggested that zoosporangium formation and zoosporogenesis in P. cubensis could serve as a model system for assays with obligate oomycetous plant pathogens, also in relation to fungicide mode of action studies.