小麦根腐病菌索氏平脐蠕孢SYBR Green I实时荧光定量PCR检测技术研究

由索氏平脐蠕孢Bipolaris sorokiniana引起的小麦根腐病,常和其他土传真菌病害混合发生,传统的症状鉴别方法很难区分,导致病害防控难度增加。为建立病菌实时荧光定量检测体系,根据ITS序列设计引物,筛选出1对特异性引物BS‐F/R,扩增片段大小为280bp。以菌丝DNA为标准品构建实时荧光定量标准曲线,并对其灵敏度、特异性、可重复性进行评价。结果表明,建立的实时荧光定量PCR检测方法速度快,灵敏度高,特异性强,重复性好。构建的荧光定量PCR标准曲线循环阈值与模板浓度呈良好的线性关系,溶解曲线的吸收峰单一,扩增效率良好。利用该定量检测体系,可以检测出田间小麦样品中52.8fg/μL的病菌DNA。     

Mapping of resistance to spot blotch disease caused by Bipolaris sorokiniana in spring wheat

Spot blotch caused by Bipolaris sorokiniana is a destructive disease of wheat in warm and humid wheat growing regions of the world. The development of disease resistant cultivars is considered as the most effective control strategy for spot blotch. An intervarietal mapping population in the form of recombinant inbred lines (RILs) was developed from a cross ‘Yangmai 6’ (a Chinese source of resistance) × ‘Sonalika’ (a spot blotch susceptible cultivar). The 139 single seed descent (SSD) derived F₆, F₇, F₈ lines of ‘Yangmai 6’ × ‘Sonalika’ were evaluated for resistance to spot blotch in three blocks in each of the 3 years. Joint and/or single year analysis by composite interval mapping (CIM) and likelihood of odd ratio (LOD) >2.2, identified four quantitative trait loci (QTL) on the chromosomes 2AL, 2BS, 5BL and 6DL. These QTLs were designated as QSb.bhu-2A, QSb.bhu-2B, QSb.bhu-5B and QSb.bhu-6D, respectively. A total of 63.10% of phenotypic variation was explained by these QTLs based on the mean over years. Two QTLs on chromosomes 2B and 5B with major effects were consistent over 3 years. All QTL alleles for resistance were derived from the resistant parent ‘Yangmai 6’.     

TaRPP13-3, a CC-NBS-LRR-like gene located on chr 7D,promotes disease resistance to wheat powdery mildew in Brock

Disease resistance (R) gene, RPP13, plays an important role in the resistance of plants to pathogen infections; its function in resistance of wheat to powdery mildew remains unknown. In this study, a RNA-Seq technique was used to monitor expression of genes in susceptible wheat ‘Jing411’ and resistant near-isogenic line ‘BJ-1’ in response to powdery mildew infection. Overall, 413 differential expression genes were observed and identified as involved in disease resistance. RPP13 homologous gene on wheat chromosome 7D was preliminarily identified using the wheat 660K SNP chip. RPP13 was highly expressed in ‘BJ-1’ and encodes 1,027 amino acids, including CC, NB and LRR domain, termed TaRPP13-3. After inoculation with powdery mildew, expression of TaRPP13-3 in resistant wheat changed with time, but average expression was higher when compared to susceptible variety, thus indicating that TaRPP13-3 is involved in resistance to powdery mildew. Virus-induced gene silencing (VIGS) was used to inhibit expression of TaRPP13-3 in resistant parent ‘Brock’. Results indicated that silencing of TaRPP13-3 led to decreased disease resistance in ‘Brock’. Overall results of this study indicate that TaRPP13-3 gene is involved in the defence response of wheat to powdery mildew and plays a positive role in wheat powdery mildew interactions.     

Identification of resistance source in wheat germplasm against spot blotch disease caused by Bipolaris sorokiniana

Four hundred and twenty-two spring wheat germplasm (Triticum aestivum L.) lines belonging to Indian, CIMMYT and Chinese wheat programme were evaluated for their tolerance against natural epiphytotic conditions of spot blotch caused by Bipolaris sorokiniana at the hot spot location, Pusa, Bihar, India. Of the 422 entries screened, none of the genotype showed immunity to the disease, whereas 52 were resistant, 180 moderately susceptible, 171 susceptible and 19 highly susceptible. Indian germplasm lines tended to be more susceptible than lines originated from CIMMYT and China. Chirya 3, Chirya 7 and Mayoor from CIMMYT showed high degree of resistance to the disease both under field and polyhouse conditions. On the basis of the disease severity under field conditions, 20 promising resistant genotypes and 10 highly susceptible lines were isolated for further testing under artificial epiphytotic conditions in polyhouse for genetic analysis and their potential for spot blotch resistance breeding.     

Changes in leaf gas exchange,chlorophyll a fluorescence and antioxidant metabolism within wheat leaves infected by Bipolaris sorokiniana

The photosynthetic performance (leaf gas exchange and chlorophyll a (Chla) fluorescence), activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX)] and the concentrations of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) in the flag leaves of plants from two wheat cultivars with contrasting levels of resistance to spot blotch was assessed. Spot blotch severity was significantly lower in plants from cv. BR‐18 compared to cv. Guamirim. Net carbon assimilation rate, stomatal conductance and concentrations of Chla, Chlab and carotenoids were significantly decreased from fungal infection. In contrast, internal CO₂ concentration was significantly increased from fungal infection in comparison to their non‐inoculated counterparts. Similarly, inoculation significantly reduced photochemical performance in the inoculated flag leaves in comparison to their non‐inoculated counterparts. However, plants from cv. BR‐18 were able to sustain greater functionality of the photosynthetic apparatus during fungal infection process compared to cv. Guamirim. The activities of SOD, POX, APX and CAT increased in inoculated flag leaves from both cultivars compared to non‐inoculated plants, and the highest increases were measured in cv. BR‐18. The greater activities of these enzymes were associated with a reduced H₂O₂ concentration in the inoculated flag leaves from cv. BR‐18, resulting, therefore, in a lower MDA concentration. Thus, a more efficient antioxidative system in flag leaves from cv. BR‐18 plays a pivotal role in removing the excess reactive oxygen species that were generated during the infection process of Bipolaris sorokiniana, therefore limiting cellular damage and largely preserving the photosynthetic efficiency of the infected flag leaves.     

The expression of a bean PGIP in transgenic wheat confers increased resistance to the fungal pathogen Bipolaris sorokiniana

A possible strategy to control plant pathogens is the improvement of natural plant defense mechanisms against the tools that pathogens commonly use to penetrate and colonize the host tissue. One of these mechanisms is represented by the host plant's ability to inhibit the pathogen's capacity to degrade plant cell wall polysaccharides. Polygalacturonase-inhibiting proteins (PGIP) are plant defense cell wall glycoproteins that inhibit the activity of fungal endopolygalacturonases (endo-PGs). To assess the effectiveness of these proteins in protecting wheat from fungal pathogens, we produced a number of transgenic wheat lines expressing a bean PGIP (PvPGIP2) having a wide spectrum of specificities against fungal PGs. Three independent transgenic lines were characterized in detail, including determination of the levels of PvPGIP2 accumulation and its subcellular localization and inhibitory activity. Results show that the transgene-encoded protein is correctly secreted into the apoplast, maintains its characteristic recognition specificities, and endows the transgenic wheat with new PG recognition capabilities. As a consequence, transgenic wheat tissue showed increased resistance to digestion by the PG of Fusarium moniliforme. These new properties also were confirmed at the plant level during interactions with the fungal pathogen Bipolaris sorokiniana. All three lines showed significant reductions in symptom progression (46 to 50%) through the leaves following infection with this pathogen. Our results illustrate the feasibility of improving wheat's defenses against pathogens by expression of proteins with new capabilities to counteract those produced by the pathogens.     

The chloroplast protein RPH1 plays a role in the immune response of Arabidopsis to Phytophthora brassicae

Plant immune responses to pathogens are often associated with enhanced production of reactive oxygen species (ROS), known as the oxidative burst, and with rapid hypersensitive host cell death (the hypersensitive response, HR) at sites of attempted infection. It is generally accepted that the oxidative burst acts as a promotive signal for HR, and that HR is highly correlated with efficient disease resistance. We have identified the Arabidopsis mutant rph1 ( resistance to Phytophthora 1 ), which is susceptible to the oomycete pathogen Phytophthora brassicae despite rapid induction of HR. The susceptibility of rph1 was specific for P. brassicae and coincided with a reduced oxidative burst, a runaway cell-death response, and failure to properly activate the expression of defence-related genes. From these results, we conclude that, in the immune response to P. brassicae , (i) HR is not sufficient to stop the pathogen, (ii) HR initiation can occur in the absence of a major oxidative burst, (iii) the oxidative burst plays a role in limiting the spread of cell death, and (iv) RPH1 is a positive regulator of the P. brassicae -induced oxidative burst and enhanced expression of defence-related genes. Surprisingly, RPH1 encodes an evolutionary highly conserved chloroplast protein, indicating a function of this organelle in activation of a subset of immune reactions in response to P. brassicae . The disease resistance-related role of RPH1 was not limited to the Arabidopsis model system. Silencing of the potato homolog StRPH1 in a resistant potato cultivar caused susceptibility to the late blight pathogen Phytophthora infestans .     

Tissue expression map of a large number of expressed sequence tags and its application to in silico screening of stress response genes in common wheat

In order to assess global changes in gene expression patterns in stress-induced tissues, we conducted large-scale analysis of expressed sequence tags (ESTs) in common wheat. Twenty-one cDNA libraries derived from stress-induced tissues, such as callus, as well as liquid cultures and abiotic stress conditions (temperature treatment, desiccation, photoperiod, moisture and ABA) were constructed. Several thousand colonies were randomly selected from each of these 21 cDNA libraries and sequenced from both the 5′ and 3′ ends. By computing abundantly expressed ESTs, correlated expression patterns of genes across the tissues were monitored. Furthermore, the relationships between gene expression profiles among the stress-induced tissues were inferred from the gene expression patterns. Multi-dimensional analysis of EST data is analogous to microarray experiments. As an example, genes specifically induced and/or suppressed by cold acclimation and heat-shock treatments were selected in silico. Four hundred and ninety genes showing fivefold induction or 218 genes for suppression in comparison to the control expression level were selected. These selected genes were annotated with the BLAST search. Furthermore, gene ontology was conducted for these genes with the InterPro search. Because genes regulated in response to temperature treatment were successfully selected, this method can be applied to other stress-treated tissues. Then, the method was applied to screen genes in response to abiotic stresses such as drought and ABA treatments. In silico selection of screened genes from virtual display should provide a powerful tool for functional plant genomics.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Sources and donors of resistance to wheat spot blotch caused by Bipolaris sorokiniana Shoem. (Cochliobolus sativus Drechs. ex Dastur)

The resistance of wheat lines and cultivars from the Institute of Crop Breeding (Harbin, China) and synthetic, hexaploid wheat lines derived from T. durum and T. tauschii (CIMMYT) were screened for resistance to spot blotch Bipolaris sorokiniana Shoem. using field and laboratory tests. The highly and moderately resistant wheat samples were determined. The satisfactory coincidence of data obtained from evaluation of type reaction of seedlings and disease severity in adult plant stage was demonstrated. The genetics of resistance in Chinese lines Long 98-4554, Long 98-4546, Long mai 24, Long mai 23 and Canadian line 181-5 was studied using hybridological analysis. The resistance in these lines was inherited as quantitative traits and was conditioned by a few (one or two) genes. The absence of susceptible plants in F₂ in crosses of resistant lines Long 98-4554, Long 98-4546, Long mai 24 and 181-5 can testify to the presence of a common gene of resistance. Our data reveals the poor genetic diversity for spot blotch resistance in studying wheat genotypes.     

Candidate gene analysis of quantitative disease resistance in wheat

 Knowledge of the biological significance underlying quantitative trait loci (QTLs) for disease resistance is generally limited. In recent years, advances in plant-microbe interactions and genome mapping have lead to an increased understanding of the genes involved in plant defense and quantitative disease resistance. Here, we report on the application of the candidate-gene approach to the mapping of QTLs for disease resistance in a population of wheat recombinant inbreds. Over 50 loci, representing several classes of defense response (DR) genes, were placed on an existing linkage map and the genome was surveyed for QTLs associated with resistance to several diseases including tan spot, leaf rust, Karnal bunt, and stem rust. Analysis revealed QTLs with large effects in regions of putative resistance (R) genes, as previously reported. Several candidate genes, including oxalate oxidase, peroxidase, superoxide dismutase, chitinase and thaumatin, mapped within previously identified resistance QTLs and explained a greater amount of the phenotypic variation. A cluster of closely linked DR genes on the long arm of chromosome 7B, which included genes for catalase, chitinase, thaumatins and an ion channel regulator, had major effects for resistance to leaf rust of adult plants under conditions of natural infestation. The results of this study indicate that many minor resistance QTLs may be from the action of DR genes, and that the candidate-gene approach can be an efficient method of QTL identification. Received: 12 June 1998 / Accepted: 24 July 1998     

Silencing of copine genes confers common wheat enhanced resistance to powdery mildew

Powdery mildew, caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a major threat to the production of wheat (Triticum aestivum). It is of great importance to identify new resistance genes for the generation of Bgt‐resistant or Bgt‐tolerant wheat varieties. Here, we show that the wheat copine genes TaBON1 and TaBON3 negatively regulate wheat disease resistance to Bgt. Two copies of TaBON1 and three copies of TaBON3, located on chromosomes 6AS, 6BL, 1AL, 1BL and 1DL, respectively, were identified from the current common wheat genome sequences. The expression of TaBON1 and TaBON3 is responsive to both pathogen infection and temperature changes. Knocking down of TaBON1 or TaBON3 by virus‐induced gene silencing (VIGS) induces the up‐regulation of defence responses in wheat. These TaBON1‐ or TaBON3‐silenced plants exhibit enhanced wheat disease resistance to Bgt, accompanied by greater accumulation of hydrogen peroxide and heightened cell death. In addition, high temperature has little effect on the up‐regulation of defence response genes conferred by the silencing of TaBON1 or TaBON3. Our study shows a conserved function of plant copine genes in plant immunity and provides new genetic resources for the improvement of resistance to powdery mildew in wheat.     

Quantitative trait loci analysis for resistance to Cephalosporium stripe, a vascular wilt disease of wheat

Cephalosporium stripe, caused by Cephalosporium gramineum, can cause severe loss of wheat (Triticum aestivum L.) yield and grain quality and can be an important factor limiting adoption of conservation tillage practices. Selecting for resistance to Cephalosporium stripe is problematic; however, as optimum conditions for disease do not occur annually under natural conditions, inoculum levels can be spatially heterogeneous, and little is known about the inheritance of resistance. A population of 268 recombinant inbred lines (RILs) derived from a cross between two wheat cultivars was characterized using field screening and molecular markers to investigate the inheritance of resistance to Cephalosporium stripe. Whiteheads (sterile heads caused by pathogen infection) were measured on each RIL in three field environments under artificially inoculated conditions. A linkage map for this population was created based on 204 SSR and DArT markers. A total of 36 linkage groups were resolved, representing portions of all chromosomes except for chromosome 1D, which lacked a sufficient number of polymorphic markers. Quantitative trait locus (QTL) analysis identified seven regions associated with resistance to Cephalosporium stripe, with approximately equal additive effects. Four QTL derived from the more susceptible parent (Brundage) and three came from the more resistant parent (Coda), but the cumulative, additive effect of QTL from Coda was greater than that of Brundage. Additivity of QTL effects was confirmed through regression analysis and demonstrates the advantage of accumulating multiple QTL alleles to achieve high levels of resistance.     

水稻全基因组编码抗病基因同源序列分析

利用模糊搜索的方法,在TIGR水稻日本晴基因组数据库(TIGR Rice Genome Annotation-Release5)中识别出565个编码抗病蛋白质的同源序列;利用识别出565个编码抗病蛋白质序列分别与籼稻基因组数据库进行BLASTP联配,共确定320个对应的等位基因。通过在线生物信息学软件,识别了这565个抗病基因的保守结构域、保守模体和DNA序列内转座子元件,其中有14个抗病基因同源序列注释错误。同时绘出了这些基因的基因组分布,并基于这些基因的同源树分析和基因组物理分布,认为基因的原位和远程复制事件产生了抗病基因的现存分布和多样性,其中转座子在复制过程中扮演了重要角色。这些对抗病机制研究和抗病基因进化研究以及抗病基因的转育具有重要意义。     

Expression and immunological analysis of capsid protein precursor of swine vesicular disease virus HK/70

VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore,anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.     

Specific patterns of changes in wheat gene expression after treatment with three antifungal compounds

The two fungicides azoxystrobin and fenpropimorph are used against powdery mildew and rust diseases in wheat (Triticum aestivumL). Azoxystrobin, a strobilurin, inhibits fungal mitochondrial respiration and fenpropimorph, a morpholin, represses biosynthesis of ergosterol, the major sterol of fungal membranes. Although the fungitoxic activity of these compounds is well understood, their effects on plant metabolism remain unclear. In contrast to the fungicides which directly affect pathogen metabolism, benzo(1,2,3) thiadiazole-7-carbothioic acid S-methylester (BTH) induces resistance against wheat pathogens by the activation of systemic acquired resistance in the host plant. In this study, we monitored gene expression in spring wheat after treatment with each of these agrochemicals in a greenhouse trial using a microarray containing 600 barley cDNA clones. Defence-related genes were strongly induced after treatment with BTH, confirming the activation of a similar set of genes as in dicot plants following salicylic acid treatment. A similar gene expression pattern was observed after treatment with fenpropimorph and some defence-related genes were induced by azoxystrobin, demonstrating that these fungicides also activate a defence reaction. However, less intense responses were triggered than with BTH. The same experiments performed under field conditions gave dramatically different results. No gene showed differential expression after treatment and defence genes were already expressed at a high level before application of the agrochemicals. These differences in the expression patterns between the two environments demonstrate the importance of plant growth conditions for testing the impact of agrochemicals on plant metabolism.     

植物抗病基因克隆与功能研究进展

植物抗病基因（R基因）是分子植物病理学和植物基因工程研究的热点之一,R基因的克隆及其在抗病反应中的功能研究为揭示植物抗病机制和有效－控制植物病害奠定了基础,本文介绍了R基因的成功克隆方法和克隆新策略,对R基因编码产物的功能进行了分类分析,并对通过遗传工程途径发展R基因介导的抗病植物新品种进行了展望。