Origin,Migration Routes and Worldwide Population Genetic Structure of the Wheat Yellow Rust Pathogen Puccinia striiformis f.sp. tritici

Analyses of large-scale population structure of pathogens enable the identification of migration patterns, diversity reservoirs or longevity of populations, the understanding of current evolutionary trajectories and the anticipation of future ones. This is particularly important for long-distance migrating fungal pathogens such as Puccinia striiformis f.sp. tritici (PST), capable of rapid spread to new regions and crop varieties. Although a range of recent PST invasions at continental scales are well documented, the worldwide population structure and the center of origin of the pathogen were still unknown. In this study, we used multilocus microsatellite genotyping to infer worldwide population structure of PST and the origin of new invasions based on 409 isolates representative of distribution of the fungus on six continents. Bayesian and multivariate clustering methods partitioned the set of multilocus genotypes into six distinct genetic groups associated with their geographical origin. Analyses of linkage disequilibrium and genotypic diversity indicated a strong regional heterogeneity in levels of recombination, with clear signatures of recombination in the Himalayan (Nepal and Pakistan) and near-Himalayan regions (China) and a predominant clonal population structure in other regions. The higher genotypic diversity, recombinant population structure and high sexual reproduction ability in the Himalayan and neighboring regions suggests this area as the putative center of origin of PST. We used clustering methods and approximate Bayesian computation (ABC) to compare different competing scenarios describing ancestral relationship among ancestral populations and more recently founded populations. Our analyses confirmed the Middle East-East Africa as the most likely source of newly spreading, high-temperature-adapted strains; Europe as the source of South American, North American and Australian populations; and Mediterranean-Central Asian populations as the origin of South African populations. Although most geographic populations are not markedly affected by recent dispersal events, this study emphasizes the influence of human activities on recent long-distance spread of the pathogen.     

Phenotypic and Molecular Characterization of Wheat for Slow Rusting Resistance against Puccinia striiformis Westend. f.sp. tritici

Race‐specific resistance of wheat (Triticum aestivum L.) to yellow rust caused by Puccinia striiformis Westend. f.sp. tritici is often short‐lived. Slow‐rusting resistance has been reported to be a more durable type of resistance. A set of sixteen bread wheat varieties along with a susceptible control Morocco was tested during 2004–05 to 2006–07 in field plots at Peshawar (Pakistan) to identify slow rusting genotypes through epidemiological variables including final rust severity (FRS), apparent infection rate (r), area under disease progress curve (AUDPC), average coefficients of infection (ACI) and leaf tip necrosis (LTN). Epidemiological parameters of resistance were significantly (P < 0.01) different for years/varieties in three seasons, while variety × year interactions remained non‐significant. Sequence tagged site (STS) marker, csLV34 analyses revealed that cultivars Faisalabad‐83, Bahawalpur‐95, Suleman‐96, Punjab‐96, Bakhtawar‐93, Faisalabad‐85, Shahkar‐95 and Kohsar‐95 possessed Yr18 linked allele. Faisalabad‐83, Bahawalpur‐95, Suleman‐96, Punjab‐96, Bakhtawar‐93 and Faisalabad‐85 were relatively more stable over 3‐years where FRS, AUDPC and r values reduced by 80, 84 and 70% respectively compared to control Morocco. These six varieties therefore could be exploited for the deployment of Yr18 in breeding for slow rusting in wheat. Both FRS and ACI are suitable parameters for phenotypic selection.     

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%.     

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.     

小麦条锈菌分子生态学研究进展

小麦条锈病是危害最严重的小麦流行性病害之一,小麦条锈菌的生态学研究对制定合理的防治策略和抗锈育种具有重要意义.近十几年来,DNA分子标记技术被应用于小麦条锈菌的群体遗传学研究,推动了小麦条锈菌分子生态学研究的快速发展,为揭示小麦条锈菌的群体生态特性开辟了一个新的途径.本文系统介绍小麦条锈菌分子生态学研究的主要进展,并就我国当前研究的局限性和发展趋势进行了分析.     

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).     

条形柄锈菌34号生理小种的分子标记

条形柄锈菌Puccinia striiformis f. sp. tritici 34号生理小种(CYR34)是目前我国毒性谱最宽、毒性最强的生理小种,对小麦生产和抗病品种选育造成了极大的影响。本研究采用RAPD-SCAR分子标记技术,从300条RAPD随机引物中筛选到CYR34的特异引物,通过特异性片段回收、克隆和测序(GenBank登录号为OL907303),依据序列设计出了S2008F34/S2008R34特异性引物,能够从CYR34及接种CYR34的小麦发病叶片总DNA中都扩增出417 bp的目标片段。采用该特异性引物检测2021年陕西渭南、咸阳和宝鸡地区小麦条锈菌CYR34的流行频率分别为8.6%、6.0%和10.8%。该项研究为小麦条锈菌CYR34号生理小种的快速检测提供了技术支撑。     

小麦条锈菌冬孢子发生的组织学和超微结构研究

采用冷冻切片技术、光学显微镜和电子显微镜技术,系统研究了小麦条锈菌冬孢子的个体发生过程和超微结构特征。结果表明,小麦条锈菌冬孢子由排列在冬孢子堆基部的双核产孢细胞产生。在发育初期,产孢细胞一端产生突起形成冬孢子芽,随后冬孢子芽经延伸并形成隔膜,依次分化形成冬孢子原基、柄细胞和冬孢子原体。冬孢子原体经有丝分裂后产生隔膜发育形成双核双细胞冬孢子。成熟的冬孢子表面光滑,具有明显加厚的细胞壁,双核融合,原生质密度增加,富含脂肪粒和糖原类物质。在部分冬孢子堆周围还可观察到莲花状包被结构。     

小麦条锈菌CY32夏孢子萌发研究

研究夏孢子萌发过程的分子机制对于从分子水平上理解条锈菌的侵染过程及其与寄主互作的关系具有重要的理论意义。本研究以小麦条锈菌Pucciniastriiformisf.sptritici32号生理小种(CY32)为材料,研究了用水培方法萌发夏孢子的适宜条件。结果显示,CY32夏孢子萌发的最适温度为9℃,最适宜的孢子量是6mg/200mL水,适宜的溶液是无菌蒸馏水。水化能促进夏孢子的萌发,新鲜夏孢子和干燥容器中放置2d的夏孢子经水化15h后,萌发率显著提高。此方法获得的萌发夏孢子提取的RNA可以满足cDNA文库构建和基因表达分析等分子生物学研究的要求,并为小麦条锈菌的分子生物学研究奠定了物质基础。     

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.     

A Novel Fungal Hyperparasite of Puccinia striiformis f. sp. tritici,the Causal Agent of Wheat Stripe Rust

Puccinia striiformis f. sp. tritici (Pst), the causal fungus of wheat stripe rust, was previously reported to be infected by Lecanicillium lecanii, Microdochium nivale and Typhula idahoensis. Here, we report a novel hyperparasite on Pst. This hyperparasitic fungus was identified as Cladosporium cladosporioides (Fresen.) GA de Vries based on morphological characteristics observed by light and scanning electron microscopy together with molecular data. The hyperparasite reduced the production and viability of urediniospores and, therefore, could potentially be used for biological control of wheat stripe rust.     

Effect of Low Temperature and Wheat Winter-Hardiness on Survival of Puccinia striiformis f. sp. tritici under Controlled Conditions

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. Understanding the survival of Pst during the overwintering period is critical for predicting Pst epidemics in the spring. Real-time quantitative PCR (qPCR) methods quantifying Pst DNA and RNA (cDNA) were developed and compared for the ability to quantify viable Pst in leaf tissues. Both qPCR of DNA and RNA can provide reliable measurement of viable Pst in plant tissues prior to the late sporulation stage for which qPCR of DNA gave a much higher estimate of fungal biomass than qPCR of RNA. The percentage of Pst biomass that was viable in detached and attached leaves under low temperatures decreased over time. Pst survived longer on attached leaves than on detached leaves. The survival of Pst in cultivars with strong winter-hardiness at 0°C and -5°C was greater than those with weak winter-hardiness. However, such differences in Pst survival among cultivars were negligible at -10, -15 and -20°C. Results indicated that Pst mycelia inside green leaves can also be killed by low temperatures rather than through death of green leaves under low temperatures. The relationship of Pst survival in attached leaves with temperature and winter-hardiness was well described by logistic models. Further field evaluation is necessary to assess whether inclusion of other factors such as moisture and snow cover could improve the model performance in predicting Pst overwintering potential, and hence the epidemic in spring.     

Mapping genes for resistance to barley stripe rust (Puccinia striiformis f. sp. hordei)

Two genes conferring resistance to the barley stripe rust found in Mexico and South America, previously identified as race 24, were mapped to the M arms of barley chromosomes 7 and 4 in a doubled haploid population using molecular markers and the quantitative trait loci (QTL) mapping approach. The resistance gene on chromosome 7 had a major effect, accounting for 57% of the variation in disease severity. The resistance gene on chromosome 4 had a minor effect, accounting for 10% of the variation in trait expression. Two pairs of restriction fragment length polymorphism markers are being used to introgress the resistance genes to North American spring barley using molecular marker-assisted backcrossing.Ore. Agric Exp Stn J no. 10283     

小麦条锈菌胞质游离钙离子动态检测方法的建立

胞质游离钙离子变化与植物病原真菌侵染寄主的动态过程具有重要的关联性。本研究以侵染小麦叶片的条锈菌31号生理小种(CYR31)为材料,以孵育法将Ca2+荧光探针Fluo-3-AM载入到小麦条锈菌细胞中,并结合激光共聚焦扫描显微技术,建立了测定侵染过程中条锈菌胞质游离Ca2+分布的试验方法。结果表明,采用10μmol/L Fluo-3-AM装载顺次进行低温4℃孵育1h,25℃孵育1h,可获得较为理想的条锈菌胞质游离Ca2+染色结果。该方法可用于检测不同侵染阶段的小麦条锈菌细胞质游离钙离子的分布变化,为进一步研究锈菌胞内钙离子动态与侵染寄主的关联性提供了技术支撑。     

小麦条锈菌钙调素依赖蛋白激酶基因Pscamk的功能

【目的】克隆小麦条锈菌钙调素依赖蛋白激酶基因Pscamk,并分析其在条锈菌侵染小麦过程中的表达特征及初步功能。【方法】基于本实验室已测序的小麦条锈菌基因组序列,利用RT-PCR方法,从小麦条锈菌生理小种CYR32中克隆Pscamk基因的cDNA序列,并利用网络数据库和生物信息学工具预测该基因编码蛋白的基本特征和保守结构;运用qRT-PCR技术分析Pscamk在不同发育及侵染阶段的表达水平,进一步通过钙调素依赖蛋白激酶(CaMK)的免疫抑制剂KN-93处理小麦条锈菌夏孢子,观察其萌发状况。【结果】获得1个1620 bp的小麦条锈菌CaMK基因Pscamk;序列分析发现,Pscamk编码蛋白包含CaMK蛋白的保守结构域,并与小麦杆锈菌该类蛋白序列相似性最高。qRT-PCR分析表明,Pscamk在条锈菌侵染初期过程中的芽管发育、初生菌丝侵染及吸器形成时期呈显著上调表达,且在条锈菌接种6 h时表达量最高,为对照夏孢子的20.74倍。在专一性免疫抑制剂KN-93处理后,随着KN-93施加浓度的增加,条锈菌夏孢子萌发率逐渐降低,当浓度为1.4μmol/L时夏孢子萌发率为8.02%,仅为对照的12%。【讨论】推测Pscamk基因参与了小麦条锈菌夏孢子萌发、芽管发育以及初期侵染结构的形成。本研究为进一步探索条锈菌细胞钙信号传导机理和致病机制奠定了基础。     

Development and characterization of novel microsatellite markers in Puccinia striiformis f.sp. tritici and their transferability in Puccinia species

Puccinia striiformis f.sp. tritici (Pst) and P. striiformis f.sp. hordei (Psh) causing stripe rust disease in wheat and barley, respectively, are two devastating phytopathogens. Microsatellite/simple sequence repeat (SSR) markers are increasingly being utilized for analysis of genetic diversity, diagnosis, population structure and possible migratory routes of plant pathogens. In the current study, novel polymorphic SSR markers were designed for Pst using the genomic sequences of PST-78 isolate. A total of 1,191 SSR motifs, comprising 30% each of di- and tri-nucleotide type of repeats, 17% of penta-nucleotide, 15% of tetra-nucleotide and 8% of hexa-nucleotide repeats, were detected through in silico scanning of PST-78 genomic sequences. Polymorphism was detected by nine of the 50 designed SSRs (PsSSRs) in seven stripe rust pathotypes of wheat and barley. The mean number of alleles per SSR locus, mean polymorphism information content (PIC), mean heterozygosity, mean major allele frequency (MAF) and mean gene diversity were 2.33, 0.34, 0.33, 0.71 and 0.40, respectively. The dendrogram analysis suggested that newly developed PsSSR markers could distinguish stripe rust pathotypes based on their virulence phenotype. Further, the cross-genera and cross-species amplification test of these markers in 14 different rust pathotypes revealed that 9 PsSSRs are capable of amplification in Pst species infecting wild grass, followed by 6 PsSSRs in Pt, 3 PsSSRs in Pgt, 1 PsSSRs in Puccinia species on barberry and Melampsora lini. Thus, the transferability of PsSSRs to other species reduced with increasing genetic distance of target species. These newly designed SSR markers expand the available Pst SSR marker resources and allow better genetic studies.