Effects of Heat Shock on Photosynthetic Properties,Antioxidant Enzyme Activity,and Downy Mildew of Cucumber (Cucumis sativus L.)

Heat shock is considered an abiotic stress for plant growth, but the effects of heat shock on physiological responses of cucumber plant leaves with and without downy mildew disease are still not clear. In this study, cucumber seedlings were exposed to heat shock in greenhouses, and the responses of photosynthetic properties, carbohydrate metabolism, antioxidant enzyme activity, osmolytes, and disease severity index of leaves with or without the downy mildew disease were measured. Results showed that heat shock significantly decreased the net photosynthetic rate, actual photochemical efficiency, photochemical quenching coefficient, and starch content. Heat shock caused an increase in the stomatal conductance, transpiration rate, antioxidant enzyme activities, total soluble sugar content, sucrose content, soluble protein content and proline content for both healthy leaves and downy mildew infected leaves. These results demonstrate that heat shock activated the transpiration pathway to protect the photosystem from damage due to excess energy in cucumber leaves. Potential resistance mechanisms of plants exposed to heat stress may involve higher osmotic regulation capacity related to an increase of total accumulations of soluble sugar, proline and soluble protein, as well as higher antioxidant enzymes activity in stressed leaves. Heat shock reduced downy mildew disease severity index by more than 50%, and clearly alleviated downy mildew development in the greenhouses. These findings indicate that cucumber may have a complex physiological change to resist short-term heat shock, and suppress the development of the downy mildew disease.     

基于冠层温湿度模型的日光温室黄瓜霜霉病预警方法

利用温室环境参数构建室内微环境模拟模型,并结合温室病害模型进行预警,便于开展病害生态防治,以减少农药使用,从而保护温室生态环境和保证农产品质量安全.本文利用温室内能量守恒原理和水分平衡原理,构建了日光温室冠层叶片温度和空气相对湿度模拟模型.叶片温度模拟模型考虑了温室内植物与墙体、土壤、覆盖物之间的辐射热交换,以及室内净辐射、叶片蒸腾作用引起的能量变化;相对湿度模拟模型综合了温室内叶片蒸腾、土壤蒸发、覆盖物与叶面的水汽凝结引起的水分变化.将温湿度估计模型输出值作为参数,输入黄瓜霜霉病初侵染和潜育期预警模型中,估计黄瓜霜霉病发病日期,并与田间观测的实际发病日期比较.试验选取2014年9月和10月的温湿度监测数据进行模型验证,冠层叶片温度实际值与模拟值的均方根偏差（RMSD)分别为0.016和0.024 ℃,空气相对湿度实际值与模拟值的RMSD分别为0.15%和0.13%.结合温湿度估计模型结果表明,黄瓜病害预警系统预测黄瓜霜霉病发病日期与田间调查发病日期相吻合.本研究可为黄瓜日光温室病害预警模型及系统构建提供微环境数据支持.     

黄瓜霜霉病菌侵染若干因子的研究

研究了温、湿度条件对黄瓜霜霉病菌致病性的影响,结果表明,25～35℃最适宜黄瓜霜霉病的发生,15/35℃的交替温度变化最有利于霜霉病菌的侵染,但35℃以上的高温对霜霉病菌具有杀伤作用;2 h的湿度条件就足以引起侵染,一旦侵入寄主,环境湿度条件对病害的发展影响不大.-20℃低温冷冻保存10个月和干燥放置7 d的霜霉菌种仍具致病力.发病的黄瓜叶片可以连续产生孢子囊,但随着发病时间的延长产生孢子囊量逐渐减少.活体叶片单位面积上产生的孢子囊量比离体叶片大,且显症天数与叶片单位面积产生孢囊量呈抛物线型关系.     

寡雄腐霉施用时期对设施黄瓜霜霉病的防治试验

寡雄腐霉是一种对多种植物病害有很好防治效果的新型生物农药。本文通过田间试验明确了寡雄腐霉在黄瓜霜霉病不同发病时期使用时的防治效果。结果表明,寡雄腐霉对黄瓜霜霉病预防效果达到68.9%,发病初期防治效果达到61.9%,发病后期防治效果只有23.5%。因此,在使用寡雄腐霉防治黄瓜霜霉病时一定要在未发病或发病早期使用才能达到理想的防病效果。     

黄瓜霜霉病及寄主抗性机制研究进展

在黄瓜生产中,由古巴假霜霉菌(Pseudoperonospora cubensis)引起的霜霉病危害严重,影响叶、茎和花序生长发育,导致黄瓜产量及品质降低。通过对黄瓜霜霉病的病原菌检测和防御途径、影响及调控因素、抗病原菌候选基因发掘、蛋白质组和基因组分析、黄瓜霜霉病QTL连锁标记开发及其抗病育种等多方面的最新进展进行综述,以期为今后进一步揭示黄瓜乃至农作物对霜霉病的抗性机制研究提供借鉴和参考。     

Endogenous levels of polyamines in the organs of cucumber plant (Cucumis sativus) and factors affecting leaf polyamine contents

Polyamine compositions of various organs from hydroponically cultivated cucumber plants ( Cucumis sativus L. cv. Sharp-1) and factors affecting the leaf polyamine content were examined. Diamine putrescine was found most abundantly in the root, while a relatively large amount of spermine was detected in the reproductive organs such as the immature fruit and the calyx (+stamen). Spermidine was present at the highest level in rapidly growing tissues such as newly expanded leaf and fruit at an early developing stage, implying the possible involvement of spermidine in the growth and development of these young tissues. Polyamine content of cucumber leaves changed during the day. Especially, the putrescine content of upper leaves showed a striking decrease from the morning to the night. Alterations of leaf Ca or Mg content did not significantly affect leaf polyamine composition. On the other hand, abnormal cucumber leaves showed altered polyamine composition. Yellowing of the leaf intervein resulted in a striking decrease in spermidine content without a significant change in putrescine and spermine content. By contrast, the leaves infected with the phytopathogen, powdery mildew, showed decreased putrescine and increased spermine content in response to the degree of fungi infection. The possible usefulness of polyamines as a diagnostic marker of plant development and physiological disorder is discussed.     

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).     

Leaf Disk Inoculation — a Useful Tool for Selecting Infections of Sunflower Downy Mildew at Low Inoculum Concentration, but Inappropriate to Pathotype Characterization

A modified technique for leaf disk inoculation of sunflower with zoosporangia of the downy mildew pathogen Plasmopara halstedii was developed. Infection with low concentrations of inoculum was obtained down to the level of single sporangia. No significant difference in the infection rate was seen between disks from cotyledons and true leaves. This makes leaf disk inoculation particularly suitable for infections at low sporangium density and for the investigation on plant tissue which cannot be infected with whole seedling inoculation. The technique seems, however, to be inappropriate for pathotype characterization using differential host lines.     

Analysis of expressed sequence tags derived from pea leaves infected by Peronospora viciae f. sp. pisi

A cDNA library was constructed from field pea leaves infected by the downy mildew pathogen, Peronospora viciae f. sp. pisi, using a suppression subtractive hybridisation approach. The library consists of 399 expressed sequence tags, from which 207 unisequences were obtained after sequence assembly. Of the unisequences, six were shown to be of Peronospora viciae f. sp. pisi origin. The remaining unisequences were subjected to gene ontology analysis and their functions were predicted in silico. Eleven of these unisequences (representing 24 clones) shared significant sequence similarities with Arabidopsis genes known to be involved in downy mildew resistance, including the well‐characterised genes RPP5, RPP6 and RPP27. Expression analysis of five selected unisequences by real‐time PCR indicated that all five were up‐regulated during downy mildew pathogenesis, suggesting a significant role for these genes in the host response to downy mildew infection.     

Identification and characterization of potential NBS-encoding resistance genes and induction kinetics of a putative candidate gene associated with downy mildew resistance in <Emphasis Type="Italic">Cucumis</Emphasis>

Background  

Due to the variation and mutation of the races of Pseudoperonospora cubensis, downy mildew has in recent years become the most devastating leaf disease of cucumber worldwide. Novel resistance to downy mildew has been identified in the wild Cucumis species, C. hystrix Chakr. After the successful hybridization between C. hystrix and cultivated cucumber (C. sativus L.), an introgression line (IL5211S) was identified as highly resistant to downy mildew. Nucleotide-binding site and leucine-rich repeat (NBS-LRR) genes are the largest class of disease resistance genes cloned from plant with highly conserved domains, which can be used to facilitate the isolation of candidate genes associated with downy mildew resistance in IL5211S.     

Leaf growth characteristics of Vitis vinifera and prediction of final lamina length for studies of powdery mildew

Total functional leaf area is a key factor in determining crop yield. A nonlinear mixed‐effects model was employed to estimate growth responses for individual leaves using repeated measures of lamina length ≥30 mm, in the absence of disease. Resulting growth curves make allowances for, and allow assessment of, leaf to leaf variability. The major source of variability in leaf growth was identified as differences in thermal time required to reach half final lamina length. Juvenile leaves of Vitis vinifera are susceptible to infection by the powdery mildew fungus (Erysiphe necator) which impairs leaf function. The model was used to predict unobserved final lamina length for a subset of leaves inoculated with E. necator immediately after observations ceased. The severity of infection by E. necator varies among infected leaves. A previous study identified which of the inoculated leaves developed symptoms of severe powdery mildew. Maximum severity of infection was found to occur when individual leaves were at 85.3–97.9% of predicted final lamina length.     

Management of powdery mildew and gray mold of cucumber by Trichoderma harzianum T39 and Ampelomyces quisqualis AQ10

Two biocontrol preparations were tested for their ability to control Sphaerotheca fusca and Botrytis cinerea on greenhouse cucumber. Trichoderma harzianum T39 (TRICHODEX) spray reduced powdery mildew severity by up to 97% but its efficacy declined to 18–55% control as the epidemic progressed. Unlike on young leaves, on older leaves the control of powdery mildew by T. harzianum T39 was poor. Ampelomyces quisqualis (AQ10) was very effective against powdery mildew, achieving up to 98% of control. Its effectiveness declined with the progress of the epidemic but unlike the other biocontrol agent it retained significant control capability on older leaves. Two aliphatic petroleum distillate oil products improved the efficacy of both biocontrol agents. The co-application of T. harzianum T39 and A. quisqualis AQ10 was tested on cucumber plants infected with powdery mildew followed by fruit gray mold infection. It resulted in no improvement of the control of powdery mildew, and in an improvement of gray mold control, the latter probably because of the use of additive oil (ADDQ) along with the second biocontrol preparation. There was no significant interference between the biocontrol agents in the co-application treatment as compared with the application of each agent alone; the level of population of T. harzianum T39 remained similar and the parasitism of S. fusca by A. quisqualis was not nullified. The application of T. harzianum T39 to soil instead of spraying it resulted in 75–90% lower powdery mildew coverage on the leaves. It was concluded that the mode of action of T. harzianum T39 in powdery mildew control is induced resistance, not mycoparasitism or antibiotic action.     

The Effect of Downy and Powdery Mildew on Grapevine (Vitis vinifera L.) Leaf Gas Exchange

The impact of powdery (Uncinula necator) and downy mildew (Plasmopara viticola) on grapevine leaf gas exchange was analysed. Gas exchange measurements (assimilation A, transpiration E, stomatal conductance g_s, intercellular concentration of CO₂C_i) were made on three different leaf materials: (i) healthy tissue of diseased leaves, (ii) infected tissue of diseased leaves, (iii) healthy tissue of healthy leaves (control treatment). Using the same source of leaf tissue, photosynthetic pigment concentration (chlorophyll a, b) and fluorescence levels (minimal fluorescence F₀, maximal fluorescence F_m and the optimal quantum yield [F_m ? F₀]/F_m) were determined to explain the mechanism of action of the two diseases on leaf assimilation. The results indicated that powdery and downy mildew reduced the assimilation rates, not only through a reduction in green leaf area (visual lesions), but also through an influence on gas exchange of the remaining green leaf tissues, determining a ‘virtual lesion’. The ratios between virtual and visual lesions were higher in powdery mildewed leaves than in the downy mildewed leaves. The photosynthetic fluorescence level (F_v/F_m) was affected by neither of the two pathogens. The reduction in intercellular concentration of CO₂ and photosynthetic pigment may explain the lower assimilation rates in the healthy tissues of powdery and downy mildewed leaves respectively.     

Glasshouse Experiments on Biocontrol of Cucumber Powdery Mildew (Sphaerotheca fuliginea) by the MycoparasitesVerticillium lecaniiandSporothrix rugulosa

Two potential biological control agents of cucumber powdery mildew (Sphaerotheca fuliginea),Verticillium lecaniiandSporothrix rugulosa,were tested under glasshouse conditions. Two experiments were carried out. In the first experiment, two cucumber varieties with different levels of resistance, cv Corona (susceptible) and cv Flamingo (partially resistant), were used.Verticillium lecaniicontrolled the mildew better thanS. rugulosa.On cv Flamingo,V. lecaniicould keep the mildew severity below 15% infected leaf area for 9 weeks after inoculation withS. fuliginea.Treatment by Hora Oleo 11E, alone or as an additive toV. lecanii,was as good as a fungicide treatment. In the second experiment, weekly and biweekly treatments withV. lecaniiwere compared on cv Flamingo. Weekly treatments withV. lecaniikept mildew severity at a level below 20% infected leaf area during 10 weeks after inoculation withS. fuliginea.If applied to a partially resistant cucumber cultivar,V. lecaniiis an effective candidate for biological control ofS. fuliginea.     

Grape downy mildew spread and mite seasonal abundance in vineyards: effects on Tydeus caudatus and its predators

Tydeus caudatus (Acari: Tydeidae) can prey upon grape eriophyoid mites but little is known about its alternative foods. Observations carried out during 1999–2003 in a commercial vineyard located in northeastern Italy showed that densities of T. caudatus were often correlated to downy mildew spread on the vegetation. Densities of T. caudatus increased in late summer when downy mildew symptoms occurred on a high number of leaves. The predatory mite Paraseiulus talbii (Acari: Phytoseiidae) increased in late season following the increase of tydeids. On several sampling dates, T. caudatus populations were significantly higher on leaves with downy mildew symptoms than on leaves without symptoms. Tydeid densities were often positively correlated to the extent of leaf surface showing symptoms. These relationships were sometimes found regarding P. talbii. In two experimental vineyards colonised by T. caudatus, untreated plots or plots treated with different fungicides to control downy mildew were monitored during 2002. Downy mildew infections rapidly spread in the control plots while they were effectively controlled on fungicide-treated plots. In both vineyards, T. caudatus densities reached significantly higher densities in the control than on fungicide-treated plots. A field study showed that most of these fungicides did not reduce tydeid populations when downy mildew was virtually absent. Isoelectric-focusing electrophoresis (IEF) was used to detect downy mildew in mites. Glucose phosphate isomerase (GPI) was selected from among different enzymes. A high proportion of T. caudatus females, collected from infected leaves, and analysed under IEF showed a GPI isozyme allele corresponding to the downy mildew isozyme in addition to the tydeid intrinsic alleles. This phenomenon was also observed for P. talbii but with a lower incidence. All T. caudatus females confined on symptomatic leaves became positive by IEF after few hours but the same did not occur with P. talbii. A high proportion of P. talbii females became positive after preying on tydeids collected from symptomatic leaves or reared on pollen and then confined on symptomatic leaves.     

Passive heat treatment of sweet basil crops suppresses Peronospora belbahrii downy mildew

Sweet basil (Ocimum basilicum) is an annual herb crop grown in polyethylene‐covered structures in Israel. It is Israel's leading herb crop, grown in warm regions of the country. Downy mildew (caused by Peronospora belbahrii) is a severe disease in Israel and in many other crop‐growing regions worldwide. Experiments were carried out to identify potential climate‐management techniques for suppression of this disease on basil in non‐heated greenhouses. Disease severity was evaluated under commercial‐like conditions in three experiments, with 8–10 walk‐in tunnels at each location. Pathogen inoculum was introduced into all walk‐in tunnels. Regression analysis was performed between the disease values and air temperature, relative humidity (RH) and soil temperature. Downy mildew severity was negatively related to high (>25°C) air temperature, RH in the range of 65–85% and high (>21°C) soil temperature. The increase in air temperature did not result in a significant increase in leaf temperature; canopy surface median temperatures only reached 30°C. Symptomless plants from relatively warmer tunnels (peak temperatures of 45–48°C) that were transferred to conditions that promote downy mildew (22 ± 2°C, RH > 95%) became severely diseased, showing sporulation of P. belbahrii, suggesting that infection occurred but at the high temperatures symptom expression/tissue colonisation was suppressed. Pot experiments in which aerial and subterranean plant organs were differentially heated revealed that treating the roots with a high temperature (26–31°C), similar to the soil temperatures in the warmer greenhouses, while maintaining the upper plant parts at ambient temperature (20°C), suppresses canopy downy mildew. The effect lasted for 1–2 weeks after the plants were removed from the heated soil treatments and maintained under optimal conditions for pathogen development. Furthermore, oospores were found in the symptomatic leaves. Oospores are minimally affected by high temperature, and therefore the high temperature presumably did not affect pathogen survival. In conclusion, the effect of high greenhouse temperature on basil downy mildew may not result from a direct negative effect of high temperature on the pathogen but from an indirect high‐temperature effect on the host, rendering it less susceptible to pathogen development.     

Elevated atmospheric carbon dioxide and ozone alter soybean diseases at SoyFACE

Human driven changes in the Earth's atmospheric composition are likely to alter plant disease in the future. We evaluated the effects of elevated carbon dioxide (CO₂) and ozone (O₃) on three economically important soybean diseases (downy mildew, Septoria brown spot and sudden death syndrome‐SDS) under natural field conditions at the soybean free air concentration enrichment (SoyFACE) facility. Disease incidence and/or severity were quantified from 2005 to 2007 using visual surveys and digital image analysis, and changes were related to microclimatic variability and to structural and chemical changes in soybean host plants. Changes in atmospheric composition altered disease expression, but responses of the three pathosystems varied considerably. Elevated CO₂ alone or in combination with O₃ significantly reduced downy mildew disease severity (measured as area under the disease progress curve‐AUDPC) by 39–66% across the 3 years of the study. In contrast, elevated CO₂ alone or in combination with O₃ significantly increased brown spot severity in all 3 years, but the increase was small in magnitude. When brown spot severity was assessed in relation to differences in canopy height induced by the atmospheric treatments, disease severity increased under combined elevated CO₂ and O₃ treatment in only one of the 3 years. The atmospheric treatments had no effect on the incidence of SDS or brown spot throughout the study. Higher precipitation during the 2006 growing season was associated with increased AUDPC severity across all treatments by 2.7 and 1.4 times for downy mildew and brown spot, respectively, compared with drought conditions in 2005. In the 2 years with similar precipitation, the higher daily temperatures in the late spring of 2007 were associated with increased severity of downy mildew and brown spot. Elevated CO₂ and O₃ induced changes in the soybean canopy density and leaf age likely contributed to the disease expression modifications.     

Enzyme Changes Associated with Host-Parasite Interactions between Pearl Millet and Downy Mildew Fungus

During the pathogenesis of pearl millet by the downy mildew (Sclerospora graminicola [Sacc.] Schroet.), the activities of peroxidase (PO) and indoleacetic acid oxidase (IAAO) and their isozyme pattern determined by isoelectric focusing on polyacrylamide gels, were observed in extracts of leaves and ears at different stages of development. The PO activity in extracts from infected plants of a susceptible cultivar was found to be higher than in healthy plants. The higher activity was most probably due to the acceleration of host senescence by the pathogen. Quantitative differences in the isozyme patterns of PO and IAAO were found. In inoculated plants of the resistant cultivar, no symptoms developed under the conditions used for infection of the susceptible cultivar and the changes in enzyme activities after inoculation were not significant. The results indicated that different proteins are synthesized in the two cultivars.     

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 .     

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.