橡胶树白粉菌侵染过程转录组学分析

为明确橡胶树白粉菌Erysiphe quercicola参与致病过程相关基因的表达情况,基于RNA-Seq测序技术对橡胶树白粉菌侵染过程进行转录调控研究,通过对病原菌孢子（0 h）及3个侵染时期（接种1、3和30 d）的转录组进行比较,筛选差异表达基因并对其进行功能注释分析,同时对不同侵染阶段的基因表达趋势进行聚类分析。结果表明,相比于病原菌孢子,3个侵染时期（接种后1、3和30 d）分别有198、458和27个差异表达基因。基因功能富集分析发现氧化还原酶相关基因在侵染1 d阶段显著富集,可能参与病原菌侵染前期对活性氧的防御。基因表达趋势聚类分析显示不同侵染阶段的基因共分为51种表达类型,其中编码候选效应蛋白基因集中分布在侵染1 d后上调表达的6个类型当中。表明橡胶树白粉菌侵染过程相关基因具有明显的功能倾向性和表达趋势特征。     

刺槐白粉病病原菌鉴定

<正>刺槐(Robinia pseudoacacia L.)又名洋槐,属蝶形花科刺槐属的落叶乔木。原生于北美洲,现被广泛引种到亚洲、欧洲等地。刺槐白粉病是刺槐叶部、嫩枝病害,能降低叶片光合作用,严重的引起叶部畸形、茎尖和枝梢枯死。Braun最早记载中国刺槐白粉病病原菌为Microsphaera subtrichotoma~([1,2])。刺槐白粉病在日本也有报道,引起白粉病的病原菌为Erysiphe     

Isolation of genes expressed during compatible interactions between powdery mildew (Blumeria graminis) and wheat

Powdery mildew of wheat is an important disease caused by the obligately biotrophic fungus Blumeria graminis. In compatible interactions, the powdery mildew fungus undergoes a series of developmental stages to form haustoria within host cells through which nutrients are obtained. In this study, we utilized the cDNA-AFLP technique to isolate wheat genes expressed at 5 days' post-inoculation. The expression patterns of several sequences identified in the cDNA-AFLP profiling were further investigated by Northern hybridization and RT-PCR analyses. Genes with sequence similarity to GenBank accessions AAR91119, P20076, BT009372, ABA99697, BAD81963 or AAO72574 showed greater expression in susceptible rather than resistant or mock-inoculated leaves. In addition, several sequences with similarity to CAD27894, XM_466672, AAT79487 or AAM64566 were expressed only in the compatible interactions. Possible involvement of these genes in susceptibility of host wheat upon powdery mildew infection is further discussed.     

Responses of Erysiphe graminis germlings to contact with artificial and host surfaces

Germling development by Erysiphe graminis f. sp. hordei was compared between conidia held in a simulated air-borne state on microthreads constructed from safety-line threads produced by orb--weaving spiders (Araneus diadematus), and conidia inoculated onto glass, agar, or living or dead barley coleoptile epidermes. Suspended conidia germinated but generally produced only multiple short germ tubes. Conidia on living or dead coleoptiles, bathed from beneath with 0.01 Ca(NO₃)₂ solution, generally produced one short germ tube and a second germ tube which elongated and formed a normal appressorium. On glass and agar, multiple short germ tubes were sometimes formed but long germ tubes were formed less frequently than on host epidermis. When conidia with short germ tubes were transferred from microthreads to coleoptiles, they produced a long germ tube which differentiated an appressorium. Conidia with a single short germ tube were also transferred from microthreads so that only the tip of the short germ tube was in contact with a leaf epidermal strip layed on agar, whilst the conidium rested on the agar. Long germ tubes were formed more frequently by such conidia than by controls which had no contact with the leaf epidermis. This suggested that a stimulus causing elongation of the second tube was perceived through the short germ tube in contact with the epidermal strip. Where long germ tubes made contact with the epidermal strip, normal appressoria were formed more frequently than where the long tube made contact with the agar surface alone. The results indicate that germlings develop through distinct stages in response to particular stimuli.     

Relative tolerances of wild and cultivated barley to infection by Blumeria graminis f.sp. hordei (Syn. Erysiphe graminis f.sp. hordei). II—the effects of infection on photosynthesis and respiration

The relative levels of tolerance of two wild barley lines (Hordeum spontaneum), B19909 and I-17-40, and one cultivated barley (Hordeum vulgare), cv. Prisma, to Blumeria graminis f.sp. hordei were determined by comparing the effects of different levels of infection on the photosynthesis and respiration rates of the third leaf. Infection caused the early onset of senescence in all three lines, and in particular in cv. Prisma, and was accompanied by decreases in gross and net photosynthesis rates, increases in respiration rates, and loss of chlorophyll. The onset of senescence occurred at approximately the same time in infected leaves of the two wild lines, but once triggered, photosynthesis rates and chlorophyll levels declined more rapidly in I-17-40 than in B19909. A burst of respiratory activity accompanied the onset of senescence, and this was greatest in cv. Prisma. Conidial production was higher in B19909, indicating a higher level of tolerance in this line.     

大麦白粉菌种群毒性监测及抗性材料鉴定

2005和2006年从我国冬大麦区采集和分离大麦白粉菌单胞菌株729个,利用Pallas近等基因系进行致病型鉴定和群体毒性频率分析.同时,利用分离得到的不同致病型菌株,通过抗谱分析的方法鉴定了328份大麦品种(系)的白粉病抗性和抗病基因.结果显示:大麦白粉菌群体对抗病基因Mlal Mla(A12)、Mla3、Mla6 Mla14、Mla7 Mla(No3)、Mla7 Ml(Lg2)、Mla9 Mlk、Mla9、Mlal3 MlaRu3、Mlpl、Mlg(Cp)和mlo5的毒性频率为0;对Mla12 MlaEm2、Mla7 Mlk、Mlat Mla8、Mla10MlaDu2和Mlk1的毒性频率很低,分别为0.1%、0.4%、0.9%、2.8%和4.2%.两年共鉴定出不同的致病型21个,致病型000、001和003在两个年度皆为优势致病型.所鉴定的328份材料绝大多数感病,仅37份抗病材料,能明确推导出抗白粉病基因的品种(品系)很少,这些品种(品系)含有的抗白粉病基因为Mr(Bw)Mla8、Mlg、Mira Mla8、Mla9 Mla1、Mla Mla(A12)和mlo5.     

Apparent calcium mediation of resistance of an ml-o barley mutant to powdery mildew

Resistance to Erysiphe graminis f. sp. hordei conditioned by the ml-o gene in barley was inhibited with treatments that were expected to lower the concentration of cytoplasmic, ionized calcium in the host cells. When partially dissected coleoptiles were incubated on 10 m Ca(NO₃)₂ or CaCl₂, resistance was very high, whereas lowering the exogenous calcium concentration to c. 0.25 m decreased resistance by 50%. Adding indoleacetic acid to the low levels of calcium enhanced this effect, and resistance was almost totally inhibited. Treatments with the calcium ion chelators, chlortetracycline and anhydrochlortetracycline, also inhibited resistance, whereas treatment with the nonchelating analogue, isochlortetracycline, did not. This inhibition could be overcome by adding calcium to the tetracycline solution. These results lead us to hypothesize that calcium is required for activation of the resistance mechanism and that the ml-o mutation affects calcium regulation in the cell, resulting in an elevated cytosolic calcium ion level in the resistant isoline.     

白粉菌侵染后的拟南芥酵母双杂交cDNA文库构建及其利用

 拟南芥广谱抗病基因RPW8对拟南芥白粉病菌、霜霉病菌和烟草花叶病毒等均具有抗性。为了深入研究其广谱抗病机制,筛选鉴定与RPW8具有直接相互作用的蛋白,我们以含有RPW8的拟南芥纯合转基因系S5为材料,接种拟南芥白粉菌系UCSC1,36 h后取样,构建了白粉菌侵染初期的拟南芥cDNA文库。为了提高文库对长片段基因5′端的覆盖率,分别使用含有oligo(dT)和oligo(dN)的接头引物反转录cDNA第一链,PCR扩增双链cDNA。将纯化后的双链cDNA与线性化载体pGADT7-Rec混合,利用同源重组技术在酵母菌株Y187中构建cDNA文库。经检测,文库转化效率为5.0×10⁶/3 μg pGADT7-Rec,滴度为2.5×10⁸ CFU·mL^-1,插入片段长度在350～2 000 bp之间,平均插入片段大小为750 bp。用RPW8.1和RPW8.2构建诱饵载体,分别获得11和12个候选互作蛋白。结果表明此cDNA文库质量较好,适用于互作蛋白的筛选。     

Identification of eight effector candidate genes involved in early aggressiveness of the barley powdery mildew fungus

The barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), expresses 491 candidate for secreted effector proteins (CSEPs), identified based on presence of a predicted signal peptide and relative sequence uniqueness. Virtually nothing is known about the mechanism of action of CSEPs. The present study used host‐induced gene silencing (HIGS) to identify genes involved in early fungal aggressiveness. Screening of 22 selected CSEP‐encoding genes suggested that CSEP0007, CSEP0025, CSEP0128, CSEP0211, CSEP0247, CSEP0345, CSEP0420 and CSEP0422 are individually required for normal levels of aggressiveness of Bgh. Expression data showed that these genes were up‐regulated at initial stages of infection, suggesting their involvement in early fungal aggressiveness. Altogether, the findings expand the current knowledge of genes contributing to Bgh pathogenesis and provide the basis for future functional characterization.     

公主岭霉素对草莓的防病诱抗和促生效应

为明确公主岭霉素在草莓生产中的防病诱抗效应,于大田采用五点取样法调查公主岭霉素对草莓白粉病的防控效果及对植株生长性状的影响,并于室内采用生测法测定其对草莓叶片防御酶活性和根际土壤酶活性的影响。结果显示,以公主岭霉素水浸提液100倍稀释液进行灌根并协同叶面喷雾对草莓白粉病的预防效果为71.90%;单独叶面喷雾对草莓白粉病的治疗效果为69.90%;灌根协同叶面喷雾预防与叶面喷雾治疗的综合防控处理对草莓白粉病的防治效果可达85.37%。公主岭霉素水浸提液100倍稀释液灌根后草莓幼苗的株高、叶柄长、叶长、叶宽、叶数、径冠等方面均优于清水对照;叶面喷施可提升叶片中多酚氧化酶和超氧化物歧化酶的活性,喷雾后1 d内即较清水对照显著提高;5 d后过氧化氢酶活性显著高于清水对照。灌根后根际土壤中蔗糖酶的活性在用药后15 d内均显著高于清水对照。表明合理施用公主岭霉素可实现对草莓白粉病的有效防控,并诱导提升草莓植株的抗病性,改善草莓生长环境,促进其生长。     

Impact of proquinazid on appressorial development of the barley powdery mildew fungus Blumeria graminis f.sp hordei

The effect of a novel mildew-specific quinazolinone fungicide, proquinazid, on the barley powdery mildew fungus, Blumeria graminis f.sp. hordei, has been studied using scanning electron microscopy and quantitative RT-PCR. Proquinazid has previously been shown to perturb conidial morphogenesis, similar to quinoxyfen, a currently widely used mildewicide. In this study, we confirm an effect of proquinazid on appressorial differentiation. By comparison to quinoxyfen, however, proquinazid affects this highly coordinated process differently, with more deformed appressorial germ tubes observed, often growing away from the leaf surface. Comparison of the expression of genes involved in the transduction of signals directing conidial development has also suggested differences in the affects of proquinazid and quinoxyfen. In particular, the expression of the Ras-type GTPase activating gene, previously implicated in quinoxyfen resistance, is distinctly affected by proquinazid treatment at time points critical to normal conidial morphogenesis. Together, these data indicate differences in the mechanisms by which proquinazid perturbs appressorial differentiation in comparison with quinoxyfen.     

橡胶树白粉菌Oidium heveae侵染寄主拟南芥的筛选

为筛选出橡胶树白粉菌Oidium heveae Steimnann新的寄主载体,通过接种野生型拟南芥Col-0、突变体Sr1-4D及eds1,观察了橡胶树白粉菌侵染不同拟南芥突变体的过程,PCR扩增测序法验证相关致病基因,构建橡胶树白粉菌-拟南芥互作体系。结果表明,在Col-0、Sr1-4D叶片上面,橡胶树白粉菌产生部分菌丝后停止生长,不能形成典型的橡胶树白粉病症状;但能成功侵染eds1叶片,在叶片的正、背面有银白色辐射状菌丝,后期在病斑上出现一层粉层,表现橡胶树白粉病的典型症状。组织染色和显微观察结果显示,在突变体eds1叶片上橡胶树白粉菌完成了侵染过程。PCR扩增测序结果表明接种后突变体eds1叶片上及组织中病菌均为橡胶树白粉菌;使用橡胶树白粉菌3个致病相关基因作为靶标,验证了eds1和橡胶树上白粉菌基因组中的3个致病相关基因相似度均达到99%~100%。表明橡胶树白粉菌可侵染拟南芥突变体eds1。     

L-methionine induced inhibition of powdery mildew and its reversal by folic acid

L-methionine inhibits development of powdery mildew on cucumber leaf discs, floating on a 8×10^–4 M solution of this compound. All other natural amino acids are ineffective in this way. This effect was reversed by 10^–4 M folic acid. The possible interference of L-methionine with folic acid metabolism, and its importance for powdery mildew development are discussed.Samenvatting L-methionine verhindert de ontwikkeling van meeldauw op schijfjes komkommerblad, die op een 8×10^–4 M oplossing van deze verbinding drijven. Alle andere naturrlijke aminozuren zijn in dit opzicht onwerkzaam. Een antagonering van dit effect werd verkregen door 10^–4 M folinezuur aan de oplossing toe te voegen. De mogelijke invloed van L-methionine op de folinezuurstofwisseling en de gevolgen hiervan voor de ontwikkeling van meeldauw worden besproken.     

解淀粉芽孢杆菌LJ1诱导黄瓜抗白粉病的研究

解淀粉芽孢杆菌LJ1是从土壤中分离得到的一株对黄瓜白粉病具有较好防效的生防细菌。田间试验发现,用LJ1发酵上清100倍稀释液喷施黄瓜幼苗,在施药后14 d时其对黄瓜白粉病的防效可达83.45%。为研究LJ1防治病害的作用机制,用LJ1发酵上清100倍稀释液喷施黄瓜幼苗,测定黄瓜叶片中的超氧化物歧化酶(SOD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)等与诱导抗病性相关的酶活性和信号分子水杨酸含量的变化,并检测了苗期根围土壤中真菌的动态。结果显示,经过LJ1发酵液处理后3种酶的活性和水杨酸的含量在不同时间点均有一个骤增的过程,其活性显著高于对照,并且7 d后土壤中的可培养真菌数量急剧减少。说明LJ1发酵液中有诱导黄瓜产生抗病性的物质,并且诱导后分泌的抗性物质对真菌具有广谱性。     

A mechanistic model simulating ascosporic infections by Erysiphe necator,the powdery mildew fungus of grapevine

A new dynamic model for Erysiphe necator ascosporic infections on grapevine was developed. Between budbreak of vines and the time when the pool of ascospores is depleted, the model uses weather data for calculating, at daily intervals: curve of ascospore maturation; ascospore discharge events and relative proportion of the discharged ascospores; infection periods and their relative infection severity; and progress of latency period and time when secondary infections should begin. The model was validated over a 4‐year period (2005–2008) in 26 vineyards in Italy by comparing model predictions with actual observations of the first seasonal symptoms of powdery mildew. The model showed high sensitivity, specificity and accuracy. Proportions of true and false positive predictions were TPP = 0·94 and FPP = 0·26, respectively. Because a proportion of predicted infection periods did not result in actual disease onset, confidence was higher for prediction of non‐infections than for prediction of infections. Most of the false positive predictions occurred in the earlier growth stages of the host, when the surface area of susceptible tissue may be very small so that the probability that ejected ascospores land on susceptible tissue is low. An equation was then developed to describe the probability that a predicted infection period results in disease onset as a function of the growth stage of vines at the time of prediction. The new model should improve early season powdery mildew management by helping vineyard managers schedule fungicide sprays or schedule the scouting of the vineyard for detection of first disease signs.