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。     

Polygenic powdery mildew disease resistance in Arabidopsis thaliana: quantitative trait analysis of the accession Warschau-1

To further the understanding of the natural genetic diversity for disease resistance to powdery mildew ( Erysiphe cichoracearum ) in Arabidopsis thaliana , quantitative trait loci analysis was undertaken on recombinant inbred lines derived from a cross between the resistant accession Warschau-1 and the susceptible Columbia-0. Powdery mildew grew less well on Warschau-1, but the resistance was not associated with a specific block in the infection sequence. Two potential powdery mildew disease-resistance loci were identified and mapped, one with a major effect and one with a minor effect on disease resistance. The two loci acted in an additive manner to confer resistance, and together they explained 65% of the variation in resistance. In addition, the major powdery mildew disease-resistance locus was genetically mapped to the bottom of chromosome III, a region containing the powdery mildew resistance loci RPW7 , RPW8 and RPW10 . Unlike resistance mediated by the RPW8 locus in the accession Moscow-1, resistance in Warschau-1 was not correlated with the hypersensitive response, highlighting the influence of genetic background or environmental factors on the expression of disease resistance. Together with the powdery mildew resistance loci described in other studies, these results suggest that A. thaliana is a useful source of natural powdery mildew disease resistance, which potentially can be utilized in fundamental studies and as a tool for applied studies.     

EDS1-mediated basal defense and SA-signaling contribute to post-invasion resistance against tobacco powdery mildew in Arabidopsis

Arabidopsis-powdery mildew is a unique pathosystem in dissecting the molecular mechanism of plant-biotrophic fungus interactions. To increase the diversity of powdery mildew pathogens, we identified a tobacco powdery mildew, designated Gc SICAU1, which belonged to Golovinomyces cichoracearum (Gc) based on the internal transcribed spacer (ITS) rDNA fragment sequences. Inoculation test in laboratory conditions revealed that Gc SICAU1 could colonize on wild type Arabidopsis Col-0 leaves, but failed to grow as abundantly on tobacco leaves, indicating that Gc SICAU1 has completely overcome the pre-invasion resistance and its colonization is hindered by the post-invasion resistance in Arabidopsis. The growth of Gc SICAU1 in the fls2, efr-1 and pen1-1 mutants displayed no obvious difference from that of wild type plants. In contrast, salicylic acid (SA)-signaling deficient mutants such as pad4-1 and eds5-1, but not ethylene-signaling mutants, exhibited increased susceptibility to Gc SICAU1. Moreover, eds1-2 and pad4-1/sid2-1 were fully susceptible to Gc SICAU1 and the disease symptom was comparable to Gc SICAU1 on tobacco leaves. These data indicate that EDS1-mediated basal defense and SA-signaling play critical roles for post-invasion resistance against this pathogen. Taken together, our data demonstrated that Gc SICAU1 is a pathogen weakly virulent to Arabidopsis and can be used as a tool to finely dissect post-invasion resistance mechanisms.     

Gene expression profile and physiological and biochemical characterization of hexaploid wheat inoculated with Blumeria graminis f. sp. tritici

Wheat (Triticum aestivum L.) powdery mildew has direct effects on photosynthesis- and energy-related pathways. Because it is prevalent in Sichuan Province, China, knowledge concerning Pm40-mediated wheat powdery mildew resistance is important for understanding host–pathogen interactions. The aim of this study was to understand physiological changes during the host–pathogen interaction, including gene expression, photosynthetic and chlorophyll fluorescence parameters, chlorophyll content, and antioxidant activity, specific to the resistance response to powdery mildew. The Pm40-expressing L693 and Pm40-deficient L1095 wheat lines were employed for comparison analyses. Fifty-eight and 26 expressed sequence tags (ESTs) were obtained from forward and reverse suppression subtractive hybridization cDNA libraries constructed from Bgt-infected L693 and L1095 leaves, respectively, and 69% of the ESTs in the forward library were related to photosynthesis- and energy metabolism-related pathways. Our data indicate that photosynthesis and carbon metabolism as well as nitrogen metabolism related to photosynthesis are the main pathways involved in Pm40-mediated wheat powdery mildew resistance and that an acetyl CoA-like gene might be a key regulatory factor in these pathways. This study provides new insight into the physiological and genetic mechanisms of resistance to wheat powdery mildew.     

2009—2010年河南省小麦白粉菌群体毒性及其遗传多样性分析

利用40个鉴别寄主对河南省5个小麦产区的44个单孢子堆纯化分离的小麦白粉病菌进行毒性鉴定分析,并采用多基因家系法进行了遗传多样性分析。结果显示,供试群体对Pm8、Pm1、Pm3a、Pm3b、Pm3c、Pm3f、Pm3e、Pm5、Pm6、Pm7、Pm17、Pm19、Pm34等抗病基因的毒性频率分别达到90%以上,已不能再作为抗源利用;对Pm12、Pm16、Pm21、Pm18、Pm2、Pm2＋6、Pm2＋MLD、Pm5（Mli）、Pm23、Pm30、Pm4a等基因的毒性频率低于15%,仍可以在抗病育种中加以利用;河南省的小麦白粉病菌存在地理之间的差异,同时病菌在地区之间有自西南向东北/东方向的传播。表明省内群体中存在着丰富的遗传多样性。     

The role of jasmonic acid signalling in wheat (Triticum aestivum L.) powdery mildew resistance reaction

Jasmonic acid (JA) signalling plays an important role in plant resistance to pathogens. Previously, JA has been found to play a role in induced disease resistance to necrotrophic pathogens in various plant species, but current researches showed that JA also enhanced resistance to biotrophic pathogens. However, its role in wheat (Triticum aestivum L.) powdery mildew (Blumeria graminis f. sp. tritici, Bgt) resistance reaction is largely unknown. To settle this issue, several typical powdery mildew resistant and susceptible wheat varieties were employed. The sensitivity to exogenous methyl jasmonate (MeJA) to wheat powdery mildew resistance, the concentration fluctuation of endogenous JAs after Bgt inoculation, and the expression profiles of nine pathogenesis-related protein genes (PR genes) after MeJA and Bgt treatments were studied systematically. Exogenous MeJA significantly enhanced the powdery mildew resistance of the susceptible varieties. After inoculation with Bgt, endogenous JAs accumulated rapidly, reached the maxima at 2 to 5 h post-inoculation (hpi), then decreased rapidly, and the concentration was almost the same as that of un-inoculated control at 96 hpi. The expression levels of the nine PRs were measured by real time quantitative RT-PCR (qRT-PCR) at different time points after MeJA application or Bgt inoculation respectively. The MeJA and Bgt strongly activated PR1, PR2, PR3, PR4, PR5, PR9, PR10 and Ta-JA2, but almost didn’t affect Ta-GLP2a. The induced powdery mildew resistance was positively correlated with the activated PR genes. JA plays a positive role in defence against Bgt. JA is a signalling molecule in wheat powdery mildew resistance and future manipulation of this pathway may improve powdery mildew resistance in wheat breeding.     

Quantitative trait Loci mapping for adult-plant resistance to powdery mildew in bread wheat

ABSTRACT Powdery mildew, caused by Blumeria graminis f. sp. tritici, is a major disease to wheat (Triticum aestivum) worldwide. Use of adult-plant resistance (APR) is an effective method to develop wheat cultivars with durable resistance to powdery mildew. In the present study, 432 molecular markers were used to map quantitative trait loci (QTL) for APR to powdery mildew in a doubled haploid (DH) population with 107 lines derived from the cross Fukuho-komugi x Oligoculm. Field trials were conducted in Beijing and Anyang, China during 2003-2004 and 2004-2005 cropping seasons, respectively. The DH lines were planted in a randomized complete block design with three replicates. Artificial inoculation was carried out in Beijing with highly virulent isolate E20 of B. graminis f. sp. tritici and the powdery mildew severity on penultimate leaf was evaluated four times, and the maximum disease severity (MDS) on penultimate leaf was investigated in Anyang under natural inoculation in May 2004 and 2005. The heritability of resistance to powdery mildew for MDS in 2 years and two locations ranged from 0.82 to 0.93, while the heritability for area under the disease progress curve was between 0.84 and 0.91. With the method of composite interval mapping, four QTL for APR to powdery mildew were detected on chromosomes 1AS, 2BL, 4BL, and 7DS, explaining 5.7 to 26.6% of the phenotypic variance. Three QTL on chromosomes 1AS, 2BL, and 7DS were derived from the female, Fukuho-komugi, while the one on chromosome 4BL was from the male, Oligoculm. The QTL on chromosome 1AS showed high genetic effect on powdery mildew resistance, accounting for 19.5 to 26.6% of phenotypic variance across two environments. The QTL on 7DS associated with the locus Lr34/Yr18, flanked by microsatellite Xgwm295.1 and Ltn (leaf tip necrosis). These results will benefit for improving powdery mildew resistance in wheat breeding programs.     

Identification of new sources of resistance to powdery mildew in oat

Powdery mildew caused by Blumeria graminis DC. f. sp. avenae Em. Marchal. is a deleterious foliar disease of common oat which occurs in many parts of the world. Resistance existing in oat cultivars is broken down by new races of this pathogen and because of this fact there is necessity to look for new and effective sources of resistance to powdery mildew in oat. To identify new potential resistance sources to powdery mildew, a total of 67 oat genotypes from five different species Avena sterilis, A. fatua, A. sativa, A. maroccana and A. murphyi and 20 powdery mildew isolates were investigated for disease reactions. Among tested oat accessions the highest level of resistance was associated with A. maroccana genotypes. Genotypes belonging to species of A. murphi and A. sterilis also showed a good level of resistance. Identified genotypes could be used in oat breeding programmes to improve levels of resistance to powdery mildew.     

苦瓜白粉病病原菌和生理小种的鉴定及苦瓜对白粉病的抗性遗传分析

为明确海南省苦瓜白粉病的病原菌、生理小种及苦瓜对白粉病的抗性遗传规律,结合形态学鉴定和分子鉴定解析白粉病菌及生理小种种类,通过显微镜观察白粉病菌侵染过程,并应用主基因+多基因混合遗传模型分析法探讨苦瓜对白粉病的主要抗性遗传规律。结果表明:采集自海南省6个市(县)的苦瓜白粉病病原菌均为单囊壳白粉菌Sphaerotheca fuliginea,属生理小种2F,该菌在侵染苦瓜叶片时有4个关键时期:接种后4 h为分生孢子萌发高峰期,8 h为附着孢形成高峰期,16～24 h为次生菌丝形成高峰期,5 d为分生孢子梗形成高峰期。将其接种于苦瓜抗、感品系,对白粉病的抗性符合2对加性-显性-上位性主基因+加性-显性多基因模型,主基因和多基因共同控制苦瓜对白粉病的抗性,其中以主基因遗传为主,且会受到环境变异的影响。根据苦瓜抗性遗传规律,F2代主基因遗传率最高,受环境影响最小,在苦瓜的白粉病抗性育种中,以早期世代F2代作为有效选择世代。研究表明白粉病菌侵染叶片的前2 d是白粉病防治的最佳时期,所以在白粉病易发的物候期,可将防治时间提前1～2 d。     

Defining the resistance risk of the new powdery mildew fungicide quinoxyfen

The new powdery mildew fungicide quinoxyfen belongs to the novel quinoline class of chemistry. Although its biochemical mode of action is unknown, quinoxyfen does not act in the same way as other cereal fungicides. It is a systemic protectant which inhibits the early stages of mildew infection on a wide range of crops, and provides season-long protection from a single early-season spray applied around GS 31. The base-line sensitivity profile of quinoxyfen was defined for barley powdery mildew (Erysiphe graminis f.sp. hordei) from over 340 field isolates collected from different parts of the UK from 1991 onwards. Sensitivities ranged from <0·0001→0·16 mg litre^-1 with a mean of 0·003 mg litre^-1. Current work is extending the base-line sensitivity studies to wheat powdery mildew (E. graminis f.sp. tritici), and includes isolates from European trials, but so far this new data set has shown no differences from barley powdery mildew. Quinoxyfen-resistant mutants were generated in the laboratory, and some similar resistant strains were obtained from treated field crops. These laboratory and field strains were always defective, in some way, for sporulation and, curiously, all required the presence of quinoxyfen for survival in culture. Attempts to generate resistant mutants that sporulated normally were unsuccessful. These studies suggested that the resistance risk for quinoxyfen is low. The recommended anti-resistance strategy accompanying introduction of quinoxyfen avoids seed treatments and late-season applications. Instead, a single early (GS 31) treatment using either pre-formulated mixtures or alternating with a fungicide with different mode of action is recommended. This strategy will be supported by continued monitoring of wheat and barley powdery mildew. ©1997 SCI     

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.     

Virulence structure of the <Emphasis Type="Italic">Blumeria graminis</Emphasis> DC.f. sp. <Emphasis Type="Italic">avenae</Emphasis> populations occurring in Poland across 2010–2013

The aim of the present study was to determine the virulence structure of powdery mildew of oats (Blumeria graminis DC.f. sp. avena) in Poland in the years 2010–2013. For this purpose, powdery mildew isolates were collected from three experimental stations in Poland. To assess the virulence of the isolates, eight oat varieties with different responses to the pathogen were used. The results showed that a significant proportion of powdery mildew isolates found in Poland overcame the resistance genes of varieties Bruno (Pm6), Jumbo (Pm1) and Mostyn (Pm3). In contrast, lines Av1860 (Pm4), Am27 (Pm5) and Cc3678 (Pm2) were completely resistant to all pathogen isolates involved in the experiment. Changes constantly occurring in the powdery mildew population perfectly reflect diversity indexes, which were the smallest in the first year of observation, where in the following years these parameters were significantly higher. It is worth noting that the presence of powdery mildew is seasonal and local, which is reflected in the prevalence of the disease in a defined area of the country.     

Mining wild barley for powdery mildew resistance

Powdery mildew, caused by Blumeria graminis f. sp. hordei (Bgh), is a worldwide disease problem on barley (Hordeum vulgare) with potentially severe impact on yield. Historically, resistance genes have been identified chiefly from cultivated lines and landraces; however, wild barley (H. vulgare subsp. spontaneum) accessions have proven to be extraordinarily rich sources of powdery mildew resistance. This study describes the characterization of a collection of 316 wild barley accessions, known as the Wild Barley Diversity Collection (WBDC), for resistance to powdery mildew and the genetic location of powdery mildew resistance loci. The WBDC was phenotyped for reaction to 40 different Bgh isolates at the seedling stage and genotyped with 10 508 molecular markers. Accessions resistant to all 40 isolates of Bgh were not found; however, three accessions (WBDC 053, 085 and 089) exhibited resistance to 38 of the isolates. Gene postulation analyses revealed that many accessions, while resistant, contained none of the 12 genes present in the Pallas near‐isogenic lines Mla1, Mla3, Mla6, Mla7, Mla9, Mla12, Mla13, Mlk1, MlLa, Mlg, Mlat and Ml(Ru2), suggesting that the accessions carry novel genes or gene combinations. A genome‐wide association study of powdery mildew resistance in the WBDC identified 21 significant marker‐trait associations that resolved into 15 quantitative trait loci. Seven of these loci have not been previously associated with powdery mildew resistance. Taken together, these results demonstrate that the WBDC is a rich source of powdery mildew resistance, and provide genetic tools for incorporating the resistance into barley breeding programmes.     

Powdery mildew resistance in selections from moroccan barley landraces

Thirty barley landraces collected from Morocco in 1985 and 1989, and held in the Polish Gene Bank, IHAR, Radzików, Poland, were screened for resistance to powdery mildew. Fifteen tested landraces (50%) showed powdery mildew resistance reactions and 24 single plant lines were selected. Eighteen lines originating from 13 landraces were tested with 17 isolates of powdery mildew and another six lines originating from six landraces were tested with 23; the isolates were chosen according to their virulence spectra observed on the ‘Pallas’ isolines differential set. Three lines (E 1090-2-2, E 1110-3-2 and E 1077-1-1) showed resistance to all powdery mildew virulence genes prevalent in Europe. In 21 lines, unknown genes alone or in combination with specific ones were detected. Five different resistance alleles(Mlat, Mlal, Mla3, Mlg andMl(CP)) were postulated to be present in the tested lines, alone or in combination:Mlat was postulated to be present in nine (~38%) lines;Mlg andMl(CP) in two lines, andMla1 andMla3 in one tested line each. The use of newly identified sources of resistance in barley breeding as a means of controlling powdery mildew is discussed.     

Mechanisms of powdery mildew resistance of wheat – a review of molecular breeding

Powdery mildew (Blumeria graminis f. sp. tritici) results in serious economic loss in wheat production. Exploration of plant resistance to wheat powdery mildew over several decades has led to the discovery of a wealth of resistance genes and quantitative trait loci (QTLs). We have provided a comprehensive summary of over 200 powdery mildew genes (permanently and temporarily designated genes) and QTLs reported in common bread wheat. This highlights the diverse and rich resistance sources that exist across all 21 chromosomes. To manage different data for breeders, here we also present a bridged mapping result from previously reported powdery mildew resistance genes and QTLs with the application of a published integrated wheat map. This will provide important insights to empower further breeding of powdery mildew resistant wheat via marker-assisted selection (MAS).     

普通小麦“兰考90(6)”品系对白粉病抗性的遗传研究

 普通小麦(Triticum aestivum L.)"兰考90(6)"系列品系是以六倍体小黑麦(X Triticosecale Wittmack;AABBRR)为白粉病抗源培育的新的小麦-黑麦1BL/1RS异易位系。这些品系高抗白粉病。小麦白粉病抗性基因推导试验证明,"豫麦66"携带的抗病基因与大多数已经报道的小麦抗白粉病基因不同。用白粉菌[Blumeria graminis (DC.) E. O. Speer f. sp. tritici]单孢堆分离物进行的遗传分析表明,"兰考90(6)"品系携带一个小种专化的隐性抗白粉病基因。对"中国春"和"兰考90(6)21-12"杂交F₂分离群体进行1RS染色体检测,结果证明该抗白粉病基因不在1RS染色体臂上。本研究为有效利用"兰考90(6)"系列品系中的抗白粉病基因提供了科学依据。     

Cellular characteristics of temporary partial breakdown of mlo- resistance in barley to powdery mildew

When water-stress is relieved, powdery mildew (Erysiphe graminis f.sp. hordei) infection increases on both Mlo -susceptible and mlo -resistant spring barley cultivars. The breakdown is temporary and is determined by the genetic background rather than the specific resistance allele. The relief of water-stress time-point for maximum mildew infection frequency on mlo -resistant barley is approximately 7 h post-inoculation. The degree of breakdown varies with epidermal cell type; increased infection frequency is greatest on short followed by long and adjacent epidermal cell types, rather than on the stomatal subsidiary cells with which occasional colonies are typically associated. Infection frequency on the short cells of mlo -resistant cv. Atem increases from less than 1% under a non-stressed watering regime to more than 10% after a relief of water-stress at 7 h post-inoculation. Following haustorium formation, colonies develop and reach sporulation within 7 days post-inoculation. In host epidermal cells, the extent of cross-linked protein at interaction sites is reduced under conditions of water-stress or stress-relief. Cross-linked protein is reduced in terms of the frequency of occurrence at primary germ tube interaction sites 7 h post-inoculation and the deposition size at appressorial germ tube interaction sites 24 h post-inoculation. This indicates a delayed or reduced defence response during the recovery period. These data demonstrate a differential cellular response to powdery mildew in barley genotypes prone to resistance expression breakdown following relief of water-stress.     

Resistance levels of cucumber to <Emphasis Type="Italic">Podosphaera xanthii</Emphasis> in a growth chamber are related to haustorial formation and hyphal branching frequency

To clarify relationships between powdery mildew resistance of cucumber and hyphal developmental state of the pathogen, haustorial formation and the hyphal branching frequency were compared among cucumber varieties that differ in resistance levels to powdery mildew pathogen Podosphaera xanthii. Cotyledons of four cultivars were inoculated with P. xanthii. By 2 days after inoculation, secondary haustoria had developed from the first hyphal cell that formed beside the conidium in susceptible cultivars. The fungus on susceptible cultivars also tended to have hyphal branches just after the hyphal cell producing haustoria.     

甘肃小麦白粉病抗源材料的筛选及抗病基因库的组建

2002-2005年,对收集到的30份已知抗白粉病基因载体品种在甘肃省的不同生态区进行了抗病性监测,结果表明:Pm1、Pm3 a、Pm3 b、Pm3 c、Pm3 f、Pm4 a、Pm5、Pm6、Pm7、Pm8、Pm17在田间抗病性丧失,失去利用价值;Pm2、Pm19、Pm4+8、Pm4、Pm5+6、Pm13在田间抗病性较低,不宜单独作为亲本利用;Pm1+2+9、Pm2+6、Pm2+mld、Pm2+talent、Pm4+2 x、Pm4 b、Pm4 b+5、Pm20、Pm21、Pmxbd在田间抗性表现良好,在今后的育种工作中应充分加以利用。同时经过4年抗病性监测,从省内外2 638份小麦品种(系)材料中筛选出了抗病性强、综合农艺性状优良的92R137、贵农21等优异材料10余份,初步组建了抗白粉病基因库。文中还对抗病基因现状和利用及今后如何避免由于抗源单一化带来的白粉病流行进行了初步探讨。     

The Hordeum vulgare subsp. spontaneum–Blumeria graminis f. sp. hordei pathosystem: its position in resistance research and breeding applications

Barley (Hordeum vulgare L.) is an important cereal crop and powdery mildew, caused by Blumeria graminis f. sp. hordei, is one of the most serious diseases that occurs on barley throughout the world. In the Middle East, which is the centre of diversity for barley and its pathogens, the wild barley–powdery mildew pathosystem co-evolves resulting in many specific resistances in the host as well as corresponding virulences in the pathogen. Many specific resistances have been used in European breeding programmes and a centre of pathogen diversity has arisen also especially in central Europe. This short review briefly summarizes the use of host resistances derived from wild barley and land races including the durable resistance gene mlo. The use of powdery mildew pathogenicity for studying new and unknown specific resistances and for identifying resistances in commercial varieties is described. However, highly heterogeneous wild barley is also characterized as a valuable source of minor genes for powdery mildew resistance. These might be exploited by barley breeders especially for winter barley improvement where the non-specific resistance gene mlo cannot be used.