Isolation of 12 microsatellite loci,using an enrichment protocol,in the phytopathogenic fungus Puccinia triticina

We report the characterization of 12 polymorphic microsatellite markers in the biotrophic fungus Puccinia triticina, the causal agent of leaf rust on wheat. An enrichment protocol was used to isolate microsatellite loci and polymorphism was explored with 15 European isolates. Significant level of cross‐amplification (44% of the loci) was found in P. striiformis.     

Early molecular diagnosis and detection of Puccinia striiformis f. sp. tritici in China

Aims: Wheat stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most important foliar disease on wheat in China. Early molecular diagnosis and detection of stripe rust will provide a useful aid to the accurate forecast and seasonal control of this destructive disease. Our objective was to develop PCR assays for the rapid identification and detection of P. striiformis. Methods and Results: The genomic DNA of P. striiformis and P. triticina were amplified by a pair of primers derived from conserved β‐tubulin gene sequence. A 235‐bp specific DNA fragment of P. striiformis was isolated and purified. Based on its sequence, another two primer sets were designed successfully to obtain new sequence‐characterized amplified region (SCAR) markers of P. striiformis, which could be amplified in all test isolates of P. striiformis, whereas no DNA fragment was obtained in other nontarget wheat pathogens. The detection limit of the primer set YR (f)/YR (r1) was 2·20 pg μl^?1. The new SCAR markers of P. striiformis can also be detected in Pst‐infected wheat leaves postinoculated for 2 days. Conclusions: Our assays are significantly faster than the conventional methods used in the identification of P. striiformis. Significance and Impact of the Study: Development of a simple, high‐throughput assay kit for the rapid diagnosis and detection of wheat stripe rust would be anticipated in a further study.     

Virulence variation of Puccinia striiformis Westend. f. sp. tritici in Pakistan

Yellow rust populations of Pakistan were characterised for their virulence pathotypes/races and pathogenetic variation using seedling evaluation of differential genotypes under glasshouse conditions in Murree (6000 feet above sea level). Differential genotypes comprised a world set, an European set, near isogenic lines and the universally susceptible bread wheat cultivar “Morocco”. Over the two-year study a total of 18 race groups were identified. Out of these 18 race groups, several (68E0, 64E0, 66E0, 70E0, 6E0, 71E0, 6E0, 2E0, 67E0, and 68E16) were found previously. The new race group 70E32 found probably evolved because of mutation from the previously existing 70E16. Virulence frequencies of yellow rust (Yr) resistance genes were also determined on near isogenic lines. The highest virulence frequencies (%) were found for Yr7 (88%), Yr9 (57%), Yr18 (70%), and Yr24 (69%). Virulence frequencies were low for Yr 1 (4%), Yr5 (7%), Yr10 (10%) and Yr15 (4%). Our studies indicated that virulence existed for almost all yr genes, necessitating regular monitoring of the yellow rust populations and intensifying efforts to identify new sources of resistance to this pathogen.     

Isolation of microsatellite loci from expressed sequence tag library of Puccinia striiformis f. sp. tritici

We described twenty polymorphic microsatellite loci derived from the expressed sequence tags of Puccinia striiformis f. sp. tritici, which causes yellow rust disease on wheat. The numbers of alleles range from two to six and eight microsatellite loci show significant similarities to known genes. Observed and expected heterozygosities ranged from 0.12 to 0.78 and from 0.24 to 0.87, respectively.     

小麦抗条锈病近等基因系感染条锈病后丁布含量变化

选用2套遗传背景不同的抗条锈病近等基因系作为供试寄主材料,研究了不同抗条锈病近等基因系丁布的含量及其在感病过程中丁布含量的动态变化.结果表明,在未受病菌侵染情况下,2套分别含有Yr2、Yr9和YrSpP基因的近等基因系（抗病系）与其轮回亲本Taichung 29、铭贤169(感病系)间丁布含量没有显著差异（P＞0.05）.接种条锈病菌后,感病系在病菌侵染初期丁布含量下降,而抗病系在病菌侵染初期丁布含量迅速大幅度上升.感染条锈病最终导致感病植株丁布含量比未接种的植株明显减少, 感病系的减少幅度明显高于抗病系.在整个病程中,抗病系丁布的含量始终高于感病系,表明接种条件下小麦植株体内丁布含量变化与小麦抗条锈近等基因系的抗性有关.     

中国小麦条锈菌转主寄主小檗的鉴定

用萌发的小麦条锈菌冬孢子接种采自陕西省境内的陕西小檗、少齿小檗和长穗小檗,3种小檗均产生了性孢子器和锈孢子器。用人工接种小麦条锈菌冬孢子在陕西小檗上产生的锈孢子器接种小麦铭贤169产生了典型的条锈菌夏孢子堆症状。特异性PCR和DNA序列分析表明,人工接种产生于小檗上的锈孢子、接种锈孢子于小麦上产生的夏孢子堆与小麦条锈菌DNA的ITS区序列完全一致。更为重要的是,用采自田间受锈菌侵染的小檗叶片产生的锈孢子接种小麦铭贤169,经培养在小麦铭贤169叶片上产生了典型的条锈病症状。从而证实,在自然条件下,在中国,小檗不仅可作为小麦条锈菌的转主寄主,而且小麦条锈菌可在小檗上完成其有性繁殖过程。这一发现对进一步揭示我国小麦条锈菌高度的群体遗传多样性与毒性变异机理、完善小麦条锈病的防治策略具有十分重要的理论和实际意义。     

Isolation of eight polymorphic microsatellite loci,using an enrichment protocol,in the phytopathogenic fungus Fusarium culmorum

We report the development of eight microsatellite markers in the haploid filamentous fungus Fusarium culmorum, a pathogen of numerous cereal crops. An enrichment protocol was used to isolate microsatellite loci, and polymorphism was explored with isolates of Fusarium culmorum and F. graminearum from natural populations collected from several French locations.     

条形柄锈菌小麦专化型葡萄糖-6-磷酸脱氢酶基因PsG6PDH1的表达特征及功能分析

葡萄糖-6-磷酸脱氢酶是磷酸戊糖途径中的关键限速酶。基于已测序的条形柄锈菌小麦专化型基因组序列,利用RT-PCR方法克隆了该病菌葡萄糖-6-磷酸脱氢酶PsG6PDH1的全长cDNA序列（1 497bp）,编码498个氨基酸的蛋白。编码蛋白含有葡萄糖-6-磷酸脱氢酶的保守功能域。系统进化分析发现,PsG6PDH1与禾柄锈菌小麦专化型的G6PDH聚为一簇。qRT-PCR分析表明,PsG6PDH1在病菌侵染早期的表达明显上调,其中侵染24h时表达量最高,为对照夏孢子的30倍。将PsG6PDH1导入酿酒酵母G6PDH缺失突变体中成功表达,并表现出较强的葡萄糖-6-磷酸脱氢酶活性,明显酵母增强了菌株对过氧化氢的耐受性。由此推测,PsG6PDH1可能参与了条形柄锈菌小麦专化型在侵染寄主时抵御寄主的氧化胁迫反应。研究结果为进一步研究该病菌基础代谢及侵染机理奠定一定的理论基础。     

四川省小麦条锈菌群体遗传多样性的SSR分析

利用TP-M13-SSR自动荧光检测技术,对四川省小麦条锈菌群体遗传多样性水平进行了分析。研究结果表明,四川省小麦条锈菌群体遗传多样性比较丰富,地区之间存在明显的差异,川西北和四川盆地的种群遗传多样性相对较高,而四川西南部和四川东南部的种群遗传多样性较低。四川小麦条锈菌群体存在一定的遗传分化,地区间的遗传变异仅占14.92%,群体间的遗传变异占总变异的23.06%,群体内遗传变异占60.02%,遗传变异主要存在于群体内部。基因流和共享基因型从分子水平证实了四川小麦条锈菌在地区间的传播,且川西北和四川盆地之间的菌源交流最为广泛。     

Isolation of ten microsatellite loci in an EST library of the phytopathogenic fungus Puccinia striiformis f.sp. tritici

We report the characterization of ten microsatellite markers in the fungus Puccinia striiformis f.sp. tritici, responsible for yellow rust disease on wheat. A published EST library was scanned for microsatellite motives, and over 15 selected EST sequences, 13 were successfully amplified and ten exhibited polymorphism over an international collection of 43 isolates. These new microsatellites, added to the few previously published ones, provide a sufficient set of markers to perform population genetic studies.     

温室条件下条形柄锈菌体细胞重组的分子确证

为了获得温室条件下条形柄锈菌发生体细胞重组而导致毒性变异的直接证据,本研究选取7个美国条形柄锈菌小麦专化型菌系和2个美国条形柄锈菌大麦专化型菌系按照夏孢子颜色和专化型与毒性差异组成9对菌系组合,对于室内混合接种产生的子代菌系用具有不同抗性的小麦或大麦品种进行筛选,采用毒性分析及SSR分子标记技术对条形柄锈菌体细胞重组现象进行了研究。对获取的413个单孢子代菌系进行的毒性分析结果显示,有84个单孢子代菌系的毒性谱表现与亲本菌系不同,初步证明体细胞重组过程的存在。SSR标记分析结果显示,11对SSR引物中有6对引物在5对菌系组合的28个毒性谱不同的单孢子代菌系中,检测发现3个单孢菌系的扩增条带与其亲本菌系不同,且表现为亲本菌系扩增条带的重组,为体细胞重组菌系。这一结果从分子水平上证明了条形柄锈菌在室内接种条件下可以通过体细胞重组产生新小种而导致毒性变异。     

小麦条锈菌PsNCS1基因的克隆及转录表达特征

摘要：【目的】克隆小麦条锈菌神经钙离子感应蛋白基因PsNCS1,分析其在病菌不同发育时期的表达水平。【方法】利用文库筛选和RT-PCR技术克隆PsNCS1的cDNA序列,采用生物信息学技术预测分析该基因编码蛋白的保守结构域及基本特性,构建系统发育树;运用实时荧光定量RT-PCR技术分析PsNCS1在病菌不同生长发育时期的表达水平。【结果】PsNCS1全长cDNA为1007 bp（GenBank登录号GU134621）,开放阅读框为573 bp,编码190个氨基酸,分子量为22.17 kDa, 等电点为4.     

Race analysis of Puccinia striiformis f.sp. tritici in Iran

The wheat stripe (yellow) rust is one of the most important diseases in Iran. In this study, 41 races out of 104 isolates in greenhouse were determined from 2008 to 2010. Races 6E6A+, 6E10A+ and 6E0A+ were more common. Races 0E0A+ was less aggressive than races 166E158A+ and 134E158A+ with virulence on 11 known genes. Virulence on plant/s with gene/s Yr1, Yr2, Yr4, Yr6, Yr7, Yr8, Yr9, Yr10, Yr25, Yr27, YrSU, YrSD, YrND, Yr3, Yr2+, Yr6+, Yr9+, Yr7+, YrCV and YrA was detected. The majority of isolates with high frequency (more than 70%) showed virulence on plant/s with Yr2, Yr7, Yr9 and YrA genes. No virulence was detected on plant/s with Yr3, Yr5 and YrSP. In greenhouse test, frequency of virulence to wheat genotypes with Yr1, Yr4, Yr10, YrCV (32+) and YrSD gene was less than 7%. Frequency of virulence to other wheat genotypes was between 8 and 100%.     

TaADF4, an actin‐depolymerizing factor from wheat,is required for resistance to the stripe rust pathogen Puccinia striiformis f. sp. tritici

Actin filament assembly in plants is a dynamic process, requiring the activity of more than 75 actin‐binding proteins. Central to the regulation of filament assembly and stability is the activity of a conserved family of actin‐depolymerizing factors (ADFs), whose primarily function is to regulate the severing and depolymerization of actin filaments. In recent years, the activity of ADF proteins has been linked to a variety of cellular processes, including those associated with response to stress. Herein, a wheat ADF gene, TaADF4, was identified and characterized. TaADF4 encodes a 139‐amino‐acid protein containing five F‐actin‐binding sites and two G‐actin‐binding sites, and interacts with wheat (Triticum aestivum) Actin1 (TaACT1), in planta. Following treatment of wheat, separately, with jasmonic acid, abscisic acid or with the avirulent race, CYR23, of the stripe rust pathogen Puccinia striiformis f. sp. tritici, we observed a rapid induction in accumulation of TaADF4 mRNA. Interestingly, accumulation of TaADF4 mRNA was diminished in response to inoculation with a virulent race, CYR31. Silencing of TaADF4 resulted in enhanced susceptibility to CYR23, demonstrating a role for TaADF4 in defense signaling. Using a pharmacological‐based approach, coupled with an analysis of host response to pathogen infection, we observed that treatment of plants with the actin‐modifying agent latrunculin B enhanced resistance to CYR23, including increased production of reactive oxygen species and enhancement of localized hypersensitive cell death. Taken together, these data support the hypothesis that TaADF4 positively modulates plant immunity in wheat via the modulation of actin cytoskeletal organization.     

Tea polyphenol is a potential antifungal agent for the control of obligate biotrophic fungus in plants

Tea polyphenol (TP) exhibits broad‐spectrum antimicrobial properties. In this study, the in vitro and in vivo antifungal activities of TP on Puccinia striiformis f.sp. tritici (Pst), which is an obligate biotrophic fungus that causes severe wheat stripe rust disease, were evaluated to investigate the control efficacy of TP. In vitro experiments showed that, at a concentration of 1.0 mg/ml, TP significantly suppressed urediniospore germination and caused the aberrant growth of germ tubes. The inhibition ratio reached 100% by increasing the TP concentration. In vivo experiments showed that TP reduced incidence rate and the uredia coverage rate in a dose‐ and application time‐dependent manner. TP treatment also induced the aberrant differentiation of Pst on wheat leaves. Results suggest that the ideal TP concentration range is 20–40 mg/ml, and TP may be a potential antifungal agent for the control of obligate biotrophic fungus in plants.     

Quantitative resistance of some Elite wheat lines to Puccinia striiformis f. sp. tritici

Host resistance is the most economical way to manage wheat stripe rust caused by Puccinia striiformis f. sp. tritici. Slow rusting, a type of quantitative resistance, has been reported to last for a long time. Quantitative resistance, in terms of slow rusting parameters including final rust severity (FRS), apparent infection rate (r), relative area under disease progress curve (rAUDPC) and coefficient of infection (CI), was evaluated in a set of 29 wheat genotypes along with susceptible control during 2008–2009 and 2009–2010 cropping seasons. This study was conducted in field plots at Ardabil Agricultural Research Station (Iran) under natural infection conditions with two times artificial inoculation. Artificial inoculation was carried out by yellow rust inoculum having virulent genes against Yr2, Yr6, Yr7, Yr9, Yr22, Yr23, Yr24, Yr25, Yr26, Yr27, YrA and YrSU. Results of mean comparison for resistance parameters showed that lines C-86-1, C-86-2, C-87-1 and C-87-3 along with susceptible had the highest values of FRS, CI, r and rAUDPC, therefore were selected as susceptible lines. The lines C-86-3, C-86-9, C-87-2, C-87-6, C-87-8, C-87-11 and C-87-18 were susceptible at the seedling stage and had low level infection at adult plant stage. Consequently, these lines with low different parameters most probably have slow rusting resistance. The remaining lines had no infection or were at low level of infection. Thus, they were selected as resistant or moderately resistant lines. In this study, correlation coefficient between different parameters of slow rusting was significantly high (r = 0.92–0.99).     

Microscopic Observation on the Development of Puccinia striiformis in the Spike and Stem of Wheat

The majority of germ tubes of the pathotype CYR32 of Puccinia striiformis f.sp. tritici formed on the surface of spike organs of the susceptible wheat cv. Suwon 11 penetrated through the stomatal pore, only a few germ tubes formed small appressoria over the stomata. In the lemma, palea and glume, the stripe rust fungus spread between the parenchyma cells close to the inner epidermal layer, but the fungus did not develop between the thick‐walled cells near the outer epidermal layer of these organs. In the awn and stem, spread of the stripe rust was confined to the intercellular spaces of the chlorophyll parenchyma, beneath the invaded stomatal pore of the epidermis and the urediniospores to be released disrupted the epidermis. In the caryopsis, the spread of hyphae was restricted to the intercellular spaces of the pericarp cells.