Regulation of resistance and susceptibility in wheat–powdery mildew pathosystem with exogenous cytokinins

Dose–response relationship between resistance of wheat seedlings (Triticum aestivum, cultivar Zarya) to Erysiphe graminis f. sp. tritici Marchal. (Syn. Blumeria graminis), a causal organism of wheat powdery mildew and exogenous zeatin has been investigated. Two-week-old seedlings were inoculated with the pathogen. Zeatin or zeatinriboside were added to the nutrient solution immediately after inoculation. The dose–response curve of cytokinin in the most cases was multiphasic, with peaks of increased susceptibility occurring at 0.25–1.5 and 1.5–9 μM cytokinin, separated by a region of increased resistance at 0.5–3 μM cytokinin. The change in mineral nutrition or simultaneous treatment with thidiazuron revealed alterations of the dose–response curve ranging from a curve with maximum of resistance to a curve with maximum of susceptibility. Both multiphase nature of dose–response and its variability were proposed as possible explanations for earlier observed discrepancies in experimental data on modification of disease resistance by cytokinins. A mathematical model for two metabolic processes with substrate inhibition connected in-series was suggested to explain the multiphase dose–response. In this model, the product of the first reaction was used as substrate for the second reaction. Numerical experiments showed the changes in the shape of dose–response curve with changes in parameters dependent of cytokinin metabolism.     

小麦抗白粉病相关基因的转化

利用玉米花青素苷合成调节基因C1-Lc作为报告基因, 通过瞬间表达后愈伤组织表面红色斑点的统计分析, 优化了小麦幼胚愈伤组织的基因枪转化参数。小麦Beclin1类似基因TaTBL和硫代硫酸硫转移酶基因TaTST是2个在白粉菌诱导条件下具有增强表达特性的抗病相关基因。本实验进一步利用基因枪将ubi强启动子控制下的2个基因导入到小麦品种扬麦158的幼胚愈伤组织细胞中, 使用除草剂经两轮选择培养基上的筛选和再生获得抗性植株, 进一步通过抗性植株的PCR分析获得转TaTBL基因植株5株, 转TaTST基因植株6株。转基因植株离体叶片的人工接种实验表明, 外源基因的导入不同程度上增强了植株的白粉病抗性, 表现为延缓了白粉菌的发育。利用玉米花青素苷合成调节基因C1-Lc作为报告基因,通过瞬间表达后愈伤组织表面红色斑点的统计分析,优化了小麦幼胚愈伤组织的基因枪转化参数。小麦Beclin1类似基因TaTBL和硫代硫酸硫转移酶基因TaTST是两个在白粉菌诱导条件下具有增强表达特性的抗病相关基因。本实验进一步利用基因枪将ubi强启动子控制下的两个基因导入到小麦品种扬麦158的幼胚愈伤组织细胞中,使用除草剂经两轮选择培养基上的筛选和再生获得抗性植株,进一步通过抗性植株的PCR分析获得转TaTBL基因植株5株,转TaTST基因植株6株。转基因植株离体叶片的人工接种实验表明,外源基因的导入不同程度上增强了植株的白粉病抗性,表现为延缓了白粉菌的发育。     

Development of wheat near-isogenic lines for powdery mildew resistance

Using three Chinese wheat cultivars, Bainong 3217, Beijing 837 and Laizhou 953, as recurrent parents, 33 near-isogenic lines (NILs) carrying 22 powdery mildew resistance genes (Pm1c, Pm2, Pm4b, Pm12, Pm13, Pm16, Pm20, Pm21, Pm23, and 13 undocumented genes) were developed. All NILs had no significant difference to their recurrent parents in the investigated traits of agronomic importance. The results of AFLP analysis indicated Jaccards genetic similarity of the NILs with their recurrent parents varied from 0.96 to 0.98, and confirmed that the NILs had high genetic similarity with their recurrent parents. The resistance to powdery mildew was stably expressed by the relevant NILs. Eleven of the NILs were tested using molecular markers linked to the resistance genes Pm1c, Pm4b, Pm13, Pm21, PmP, PmE, PmPS5A, PmPS5B, PmY39, PmY150, and PmH, and they were all found to carry the targeted genes. The potential application of these NILs in gene discovery is discussed.     

Competitive performance of transgenic wheat resistant to powdery mildew

Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes.     

部分小麦近缘物种材料的白粉病抗性鉴定

鉴定了170份小麦近缘物种材料苗期对北京地区流行的小麦白粉菌小种的抗性表现,包括引自美国和欧洲的斯卑尔脱小麦81份,密穗小麦27份,中国的西藏半野生小麦4份,和引自 CIMMYT 的人工合成六倍体小麦58份。结果表明,3份斯卑尔脱小麦表现抗病,它们是瑞士品种 Hubel 和 Lueg 以及德国的原始品种69Z6.245(编号 PI348085)。人工合成六倍体小麦中有19份材料表现高抗至免疫。密穗小麦材料中有2份(即美国材料 DN-2263和 Coda)表现抗病。4份西藏半野生小麦苗期都不抗小麦白粉病。     

对CIMMYT小麦品种抗白粉病的鉴定简报

1999～2001年连续2年,对 CIMMYT 提供的165份小麦品种进行成株期田间人工诱发抗白粉病鉴定。鉴定病圃设在本院试验农场,面积为200m~2。人工诱发鉴定圃的接种菌源,以上年度采集并保存的当地白粉病菌菌株,先在温室的感病品种8017-2上繁殖,2月下旬将带有大量分生孢子堆的病株移植到病圃的二个诱发行中间,每距33cm 种植2株病株。由于病株上有大量分生孢子,使诱发行麦株迅速受侵染且严重发病,形成充分接种菌源,保证对供鉴品种的重复侵染,以诱发供试小麦材料成株期感染发病。     

Inheritance of partial resistance to powdery mildew in spring wheat

Summary Four spring wheat (Triticum aestivum L.) cultivars exhibiting partial resistance to powdery mildew induced by Erysiphe graminis f.sp. tritici were crossed to a common susceptible cultivar to study the inheritance of resistance. The genetic parameters contributing to resistance were estimated by generation means analyses. Additive gene action was the most important genetic component of variation among generation means in all four crosses. Additive by additive effects were significant in one cross and both additive by additive and additive by dominance effects were significant in another. Dominance effects were not significant. The F2/F3 correlations in three crosses ranged from 0.27 to 0.43. Three additional crosses among resistant cultivars were employed to study the effectiveness of selection in improving resistance. By selecting the most resistant plants from the F2 and evaluating the progenies in the F4, increases in resistance ranging from 21% to 31% were obtained. In all crosses, there was transgressive segregation in both directions indicating that the genes conferring resistance to these cultivars differ and exhibit additive effects.     

Susceptibility to powdery mildew of wheat plants deficient in copper

Summary Wheat plants were grown in deep pots at three levels of copper nutrition in an open-sided glasshouse where they were subject to natural infection by powdery mildew. Plant growth and the degree of infection of each leaf were assessed weekly throughout the life of the plants.During the middle phase of growth especially — from tillering to anthesis — severity of infection of leaves was decreased by increasing the level of copper supply. Stems of copper-deficient plants were also severely infected. In these plants serious disease was sustained until final harvest. The results are discussed in relation to known and speculative roles of copper in plants.     

Glucose, and not sucrose, is transported from wheat to wheat powdery mildew

The main host carbon energy source transferred from wheat leaves (Triticum aestivum L.) to wheat powdery mildew (Erysiphe graminis f.sp. tritici) has been investigated in three ways. When the uptake of sugars by isolated mycelial suspensions was examined, the uptake rate for glucose was considerably higher than that for a range of other solutes. Analysis by high-performance liquid chromatography of leaf and mycelial extracts following uptake of sugars into infected leaf pieces confirmed that sucrose was rapidly hydrolyzed in the leaf; no sucrose or fructose could be detected in mycelial extracts. Furthermore, studies of the uptake of asymmetrically labelled sucrose indicated that this sugar is cleaved prior to uptake by the pathogen. Thus several lines of evidence show that glucose, and not sucrose, is the major carbon energy source transferred from host to fungal mycelium. Received: 11 November 1998 / Accepted: 18 January 1999     

Effects of sulfur dioxide on the development of powdery mildew of cucumber

Environment is a major factor that does influence host parasite relationships. Air pollution caused by SO₂ may directly alter the environment around the plant and pathogen. It is hypothesised that plants may respond differently to foliar pathogens in air polluted environments. To test this hypothesis, effects of intermittent exposures of SO₂ at 143, 286 and 571 μg m⁻³ were investigated on the development of powdery mildew of cucumber (Cucumis sativa) caused by Sphaerotheca fuliginea, using pre-, post- and concomitant-inoculation exposures in closed-top chambers. Sulfur dioxide (except 143 μg m⁻³) and the fungus acting alone caused chlorosis and/or necrosis, and mildew colonies on leaves, respectively and both reduced the plant growth and yield of cucumber. Fungus colonization was relatively greater on the plants exposed to 143 μg SO₂ m⁻³, but at the higher concentrations, the colonies were greatly suppressed. Gas injury on fungus-infected plants was also less in the other treatments. Conidia of S. fuliginea collected from exposed plants varied in size. Conidial germination was considerably greater at 143 μg SO₂ m⁻³. This concentration also promoted germination of the conidia exposed on glass slides. Higher concentrations (286 and 571 μg m⁻³), however, suppressed the germination of conidia from exposed plants or exposed on glass slides. The number of fibrosin bodies declined at all the concentrations. Synergistic effects of 143 μg SO₂ m⁻³ and S. fuliginea were recorded on plant growth and yield of cucumber. Sulfur dioxide at 571 μg m⁻³ and powdery mildew infection had an antagonistic effect on plant growth.     

Genetic engineering of wheat for increased resistance to powdery mildew disease

 Fungal wheat (Triticum aestivum) diseases greatly affect crop productivity and require the economically and ecologically undesirable application of fungicides in wheat agriculture. We have generated transgenic wheat plants constitutively expressing an antifungal barley-seed class II chitinase. The transgene was stably expressed and the chitinase properly localized in the apoplast of the transgenic lines. The engineered wheat plants showed increased resistance to infection with the powdery mildew-causing fungus Erysiphe graminis. Received: 20 October 1998 / Accepted: 26 October 1998     

Suppression of the powdery mildew pathogen by chitinase microinjected into barley coleoptile epidermal cells

An exogenous chitinase from Streptomyces griseus was introduced into coleoptile epidermal cells of barley (Hordeum vulgare) by microinjection, and the effect of injected chitinase on the growth or development of the powdery mildew pathogen (Erysiphe graminis f. sp. hordei) was examined. Prior to microinjection, an enzymatic degradation of fungal haustorium, the organ taking nutrients from host plant cells, was examined by treating fixed coleoptile epidermis harboring haustoria with this enzyme. The result showed that haustoria were effectively digested by chitinase, suggesting the effectiveness of chitinase treatment for suppressing the fungal development. Microinjection of chitinase was conducted using living coleoptile tissues inoculated with the pathogen. Epidermal cells in which the haustorial primordia had been formed, or in which the haustoria had matured, were selected as targets for injection. The result clearly indicated that injection at the stage of primordium formation was effective in completely digesting haustoria and suppressing the subsequent formation of secondary hyphae of the pathogen. In microinjection after haustorial maturation, hyphal elongation was considerably suppressed though there was no detectable morphological change in the haustoria. Thus, the present study provides the experimental basis for genetically manipulating barley to produce transgenic plants resistant to the powdery mildew disease.     

Assessment of partial resistance to powdery mildew in hexaploid wheat genotypes

In the present study the degree of partial resistance (PR) of eleven hexaploid wheat (Triticum aestivum L.) genotypes was evaluated in laboratory (ratio of infection units in stage of second germ tube elongation versus stage of appressorium formation — ESH/App) and field conditions (calculating area under the disease progress curve — AUDPC). Based on the obtained data, genotypes with high degree of PR (Estica, GK Csornoc and Lívia), middle-resistant genotypes (Sana, Mv Vilma and Folio), genotypes with low portion of PR (Barbara, Torysa and Proteinka), and supersensitive genotypes (Renesansa and Am22/99) were differentiated. Both approaches appeared to be suitable for PR measuring with a good discriminating capability between the given genotypes. The results were equivalent in both instances. In addition, a new statistical approach permitting comparison of the obtained data is described.     

Rapid generation of new powdery mildew resistance genes after wheat domestication

Plant defence against pathogens is controlled by disease resistance (R) gene products that directly or indirectly detect specific pathogen effectors. Plant-pathogen interactions have been proposed to follow a co-evolutionary arms-race model where R genes are recent and evolve rapidly in response to structural changes in matching pathogen effectors. However, the longevity and extensive polymorphism of R genes studied were more consistent with balancing selection maintaining ancient and diverse R genes or alleles. In bread wheat (Triticum aestivum), the Pm3 locus confers race-specific resistance to wheat powdery mildew (Blumeria graminis f.sp. triticii). Here we describe recently generated Pm3 resistance alleles that all derive from one susceptible allele, Pm3CS, which is widespread among hexaploid bread-wheat lines. One group of four Pm3 resistance alleles shows few, clearly delimited, polymorphic sequence blocks of ancient origin, embedded in sequences identical to Pm3CS and possibly derived from gene conversion. A second group of three alleles differs from Pm3CS by only two to five mutations, all non-synonymous, and all in the leucine-rich repeat-encoding region. Transient transformation experiments confirmed that Pm3 resistance specificities are based on one or few amino acid changes. The Pm3CS allele was found in wild tetraploid wheat, the ancestor of hexaploid bread wheat, specifically from southern Turkey, a region proposed to be the site of wheat domestication. Based on these data, we propose that the Pm3 resistance alleles were generated in agricultural ecosystems after domestication of wheat 10,000 years ago. The evolution of Pm3 alleles in wheat is best described by the model of evolved recycling, where novel genetic variation is integrated in plant populations together with recycling of old variation.     

Genetic control of the wheat Triticum monococcum L. resistance to powdery mildew

Using hybrid analysis and test-clone method, 102 accessions of Triticum monococcum L. from the collection of the Vavilov All-Russia Institute of Plant Industry have been studied. This species of wheat has been found to by considerably polymorphic with respect to the resistance to the fungus Erysiphe graminis DC. f. sp. tritici Marchal. causing powdery mildew. The resistance of most accessions to the fungus population and clones is determined by dominant genes. In rare cases, the resistance was determined by recessive genes or one, two, or three oligogenes. A group of einkorn wheat accessions has been found in which the resistance to powdery mildew was determined by the same dominant factor or different but closely linked ones. Recessive resistance genes of T. monococcum differ from the recessive gene pm5 determining the resistance of T. aestivum plants. The genome of T. monococcum contains various genes of resistance to powdery mildew and is a potential source of effective genes to be used when selecting cultivated species of wheat for immunity.     

Intraspecies differentiation in the powdery mildew Erysiphe cichoracearum determined with rDNA RFLPs

The powdery mildew species Erysiphe cichoracearum has a described host range of over 300 plant species from among several families. Host-range testing indicates host-specialized subdivision within this taxonomic species. However, the extent of subdivision remains largely undetermined among host-limited forms. We have characterized diversity among field collections of E. cichoracearum from a variety of hosts, and from other powdery mildew species, with RFLPs from a PCR amplified ribosomal DNA (rDNA) segment The E. cichoracearum samples expressed six distinct RFLP haplotypes. Each haplotype was specific to either a single host or to a set of related host species. These haplotypes formed a continuum of divergence ranging from about 18–35% average pairwise distance from one another, while those from other mildew species clustered at consistently higher average pairwise distances from E. cichoracearum and from each other. Our findings support earlier suggestions, based on host-range and morphological characterizations, that E. cichoracearum is a complex of morphologically similar, but host-limited forms. Also, comparisons of rDNA haplotype distance between E. cichoracearum and Blumeria (Erysiphe) graminis were consistently greater than between E. cichoracearum and Sphaerotheca fulginea. This result supports earlier questions concerning the monophyletic nature of Erysiphe.     

Genetic control of the wheat Triticum monococcum L. resistance to powdery mildew

Using hybrid analysis and test-clone method, 102 accessions of Triticum monococcum L. from the collection of the Vavilov All-Russia Institute of Plant Industry have been studied. This species of wheat has been found to by considerably polymorphic with respect to the resistance to the fungus Erysiphe graminis DC. f. sp. tritici Marchal. causing powdery mildew. The resistance of most accessions to the fungus population and clones is determined by dominant genes. In rare cases, the resistance was determined by recessive genes or one, two, or three oligogenes. A group of einkorn wheat accessions has been found in which the resistance to powdery mildew was determined by the same dominant factor or different but closely linked ones. Recessive resistance genes of T. monococcum differ from the recessive gene pm5 determining the resistance of T. aestivum plants. The genome of T. monococcum contains various genes of resistance to powdery mildew and is a potential source of effective genes to be used when selecting cultivated species of wheat for immunity.