应用real-time PCR定量检测小麦条锈菌潜伏侵染量方法的建立

 小麦条锈病是我国小麦主要病害之一。快速、及时地诊断与定量监测处于潜育状态下的病叶,对准确估计越冬、越夏后的病情,制定正确的防治方案具有重要的意义。根据小麦条锈菌Puccinia striiformis的β-tubilin基因序列设计对该病原菌的种具有特异性的引物betaf/betar,并分别在普通PCR和real-time PCR扩增时对该引物的特异性和灵敏性进行了测定。结果表明该引物对小麦条锈菌特异性高,可稳定扩增出243 bp的目标条带。Real-time PCR的灵敏度为普通PCR的100倍。应用此特异性引物,建立了real-time PCR测定系统,定量测定了条锈菌在小麦叶片接种后组织内的DNA随时间的变化。结果表明,在接种后12 h,可在小麦叶片内检测到条锈菌,且条锈菌在小麦叶片内潜育期间随时间呈指数增长。接种第6 d后叶片内的菌量有明显的增加。建立的小麦条锈菌的real-time PCR早期定量测定方法,为及时、快速监测小麦条锈病在潜育期间的发病规律以及为该病的预测、防治提供依据。     

陇南和阿坝地区小麦条锈菌传播的研究

 研究结果表明,甘肃陇南越夏区条锈菌源的主要传播路线是沿康县至略阳的近正西气流轨道和文县至广元的西北气流轨道,主要对汉中地区和绵阳的大多数地区小麦秋苗吹送,并可波及陕西安康地区和商洛部分地区及四川达县和万县等地。四川阿坝州越夏区菌源的主要传播路线是沿汶川至灌县的西北偏北气流轨道。其菌源与成都平原麦区的关系密切,对绵阳地区的北川、平武和绵竹等地也很重要,并可波及南充、内江及江汉流域等地区。湖北秋苗的初始菌源多来自西北越夏区。     

双重real-time PCR定量测定小麦条锈菌潜伏侵染方法的建立与应用

 小麦条锈病是我国小麦生产上的重要病害之一。为高效准确地定量检测处于潜伏侵染阶段的小麦条锈菌,本研究根据已发表的小麦条锈菌和寄主小麦的引物,设计了各自的探针,建立了双重real-time PCR检测方法。为排除多个引物互作造成的干扰,对小麦条锈菌引物探针体系、小麦体系以及二者的双重real-time PCR体系进行了比较。CT值相关线性回归分析证明,引物之间互作很小,对定量检测无影响。已知浓度样品经梯度稀释,进行灵敏度检测,确定了双重real-time PCR对小麦条锈菌DNA和小麦DNA准确定量测定的最小检测限为0.4 pg和0.5 ng。同时建立了小麦条锈菌和小麦各自的标准曲线。用此方法检测来自两个不同地区的田间样本,得到的分子病情指数(MDI)与随后的发病趋势一致。本研究建立的双重real-time PCR分析方法可靠、高效、低误差,是对本实验室已有小麦条锈菌潜伏期分子检测方法的进一步优化。     

紫外线照射对小麦条锈菌生物学效应的研究

 以紫外线(UV)不同剂量照射小麦条锈菌夏孢子,结果表明,其严重削弱夏孢子的存活,剂量效应曲线无明显肩区和尾指数区,小表条锈菌夏孢子对低剂量照射较为敏感;经紫外线照射的夏孢子接种小麦感病品种,其致病性发生改变,潜伏期延长,反应型降低,严重度减少;亦降低了小麦条锈菌的繁殖能力,单叶孢子堆数量减少,孢子堆长度变短,产孢量降低,产孢期缩短。这些生物学效应随照射剂量增加而加强。     

粉锈宁药效与小麦品种对条锈菌感病性关系的研究

在田间小区试验条件下,粉锈宁喷雾防治小麦条锈病,每666.6m~28克用药量对各类型感病小麦品种的效应是有显著差异的,对慢锈品种和前期中感,但抽穗后有抗性的品种均可控制为害,而对极感品种则未能控制产量损失;等比系列用药量对各类型感病品种的效应也是有明显差异的,在 ED_(50)和 ED_(95)(克/666.6m~2)平均值上,极感品种为9.38和74.85,高感品种为4.53和25.43,中感品种为3.62和20.96,慢锈品种为1.82和15.30。根据这一结果,模拟出各类型感病品种大田防治的每666.6m~2适宜用药量,高感品种9—12克,中感品种7—9克,慢锈品种4—6克,并经实践证明,这一剂量标准是可行的。拌种对压低各类型感病品种的成株期病情也有明显差异,造成这种差异的主要原因是各类型品种流行速度不同。     

小麦条锈菌异核新菌系的筛选及核游离试验研究

 采用生物学和细胞学方法研究小麦条锈菌的异核作用。不同小种的单孢菌系间的混合接种繁殖筛选出三个新菌系。新菌系的毒性明显不同于相应的原始菌系,新菌系夏孢于芽管的多核率较原始菌系高,这表明三个新菌系是经异核作用产生的。异核体的多核率随继代接种而降低,但有的菌系的毒性仍保持稳定。在采自陇南自然条件的条锈菌的夏孢子群体中,存在有3核和4核体,感病品种上夏孢子的多核率明显高于抗病品种。这一结果表明小麦条锈菌异核作用在感病品种上的发生机率较在抗病品种上的为高。     

基于GIS的新疆小麦条锈菌越夏区划研究

为明确新疆小麦条锈菌越夏区分布情况及其与实际病情间的关系,基于地理信息系统(GIS)以2010—2020年42个气象站点最热旬(7月下旬或8月上旬)平均温度作为越夏区划指标进行了相关研究。结果表明,新疆小麦条锈菌易越夏区和可越夏区范围分布广泛,面积可达7 217万hm²,新疆北部的小麦种植区易越夏区和可越夏区面积占比较高。同时,各年小麦条锈菌越夏区划分布与条锈病实际发病情况具有相关性,相关系数为0.82,模型预测准确度为81.9%。以上研究结果可为新疆小麦条锈病预测预报提供重要依据。     

小麦条锈菌国际鉴别寄主Lee对中国条锈菌系CY23的抗性遗传分析

采用常规杂交方法,以国际小麦条锈菌鉴别寄主Lee与感病品种铭贤169杂交、自交、测交,获正、反交的F1、F2和BC1代。根据条锈菌小种的毒性谱,选用CY23单孢菌系,对其双亲及其杂交后代进行苗期抗性鉴定和统计分析,明确抗性基因数目、显隐性、互作方式和抗性特点。结果表明,在正交情况下,Lee对CY23菌系的抗性由1对显性基因和1对隐性基因互补控制,反交情况下也由1对显性基因和1对隐性基因互补控制,正反交分析结果一致,说明Lee对CY23的抗性属核遗传,由1对显性基因和1对隐性基因互补控制。通过对CY23毒性分析和对Yr7、Yr22和Yr23遗传特点研究,表明Lee对CY23抗性的1对显性基因和1对隐性基因分别为Yr7和Yr23,两者互补控制对CY23的抗性。在抗性鉴定中可用CY23区别Yr22与Yr23,并作为标准菌系用于基因推导分析。     

林芝地区小麦条锈菌与气象因子的关系及流行动态模型的构建

为了解西藏林芝地区气象因子对小麦条锈病的影响及其流行动态,2016年采用五点取样法对林芝地区小麦条锈病的发病情况进行监测,通过相关性分析、逐步回归C(p)统计法和线性回归等方法,分析了病情指数与气象因子的关系,并结合时间和病情指数建立了病害预测模型。结果表明,在林芝地区,温度X_1、湿度X_2均与小麦条锈病病情指数Y呈极显著相关,降雨量X_3与病情指数Y呈显著相关;线性回归方程为:Y=-482.5991+19.7494X1+3.7974X2-0.8439X3。根据模拟情况选择的病害流行动态方程为Y=1/e~((0.914t+0.385)),决定系数为0.952,模型的拟合效果较好,表明该模型能够为林芝地区小麦条锈病的预测预报提供有效的参考依据。     

温度与条锈病菌双胁迫下小麦内参基因的选择

 选择合适的内参基因是采用实时定量PCR研究基因表达获得可信数据的关键。以小麦在条锈病菌及温度双因素胁迫为研究对象,采用实时荧光定量PCR评价了CDC、RLI、ACTIN和26S基因的表达情况,采用geNorm和NormFinder软件进行了比较分析。结果表明,小偃6号小麦在常温(15℃±1℃)未接种条锈病菌,以及接种条锈病菌并培育至花斑期分别在高温(20℃±1℃)、变温(20℃到15℃)条件处理中,基因RLI表达不稳定,基因ACTIN在常温条件下以及在条锈病菌及高温处理条件下的表达量相对稳定,但在变温条件下波动较大。表达最稳定的基因是26S,其次是CDC基因,它们可以作为小偃6号-CYR32-温度互作体系中基因表达实时荧光定量PCR研究的内参基因,26S和CDC是最佳内参基因组合。     

小麦条锈菌冬孢子生物学特性研究进展

由小麦条锈菌引起的小麦条锈病是我国广大麦区的严重病害,研究其冬孢子产生原因与作用对于解析该病菌的生活史及遗传变异机制至关重要.通过对国内外相关研究成果的整理,综述了小麦条锈菌冬孢子的形态特征、生物学特性、寄主范围及其作用与功能等方面的研究进展.     

Thermal generalist behaviour of invasive Puccinia striiformis f. sp. tritici strains under current and future climate conditions

Yellow rust is a devastating wheat disease. Since 2000, Puccinia striiformis f. sp. tritici strains PstS1 and PstS2 have become adapted to high temperatures and have spread worldwide. By 2011, Warrior strains had invaded both warm and cold areas of Europe. This study questioned whether thermal aptitude promoted the spread of Warrior strains, similar to PstS1/PstS2, by comparing infection efficiency (IE) at five temperatures and latent period (LP) under warm and cold regimes for Warrior isolates and pre‐2011 reference strains on two susceptible wheat varieties. The Warrior isolates showed a range of IE and LP responses to temperature that was intermediate between the northern reference isolates adapted to cold conditions and both the southern and invasive PstS2 isolates adapted to warm conditions. Warrior isolates had the highest IE under optimal temperatures of 10 and 15 °C, and displayed reduced infectivity under the warmest (20 °C) and coldest (5 °C) temperatures. Warrior strains acted as thermal generalists and the reference isolates acted as specialists. An IE thermal response was used to simulate the development of each isolate under future climate scenarios in a temperate and Mediterranean region. Isolates had the same ranking for yearly IE over the three 30‐year periods (1971–2000, 2021–2050, 2071–2100) and both locations, with a slight infection increase in the future. However, in the future IEs increased in earlier months. The thermal generalist profile of Warrior isolates for IE was confirmed, with an intermediate capacity to tolerate warming climate, whereas the southern isolates are better adapted to warm conditions, but do not have the virulences necessary to develop on current varieties.     

The recent history of Puccinia striiformis f.sp. tritici in Denmark as revealed by disease incidence and AFLP markers

Disease observations and amplified fragment length polymorphism (AFLP) markers were used to study recent developments in the Puccinia striiformis f.sp. tritici population in Denmark. The fungus appeared spontaneously at 10 locations in Denmark in 1997 after it was not observed under natural conditions in 1996. The pattern of disease development and prevailing winds suggested that the fungus reappeared by airborne spores from the south or west. In 1998, disease incidence was more evenly distributed throughout the country. Forty-eight single lesion isolates were collected from most crops where the disease was observed in these years; all except one from 1997 belonged to two pathotypes that were not previously detected in the country, and both possessed the newly discovered Yr17 virulence. The isolates were characterized with AFLP markers together with 28 isolates representing eight of 13 pathotypes observed prior to 1996. Initial screening of 240 Pst I/ Mse I AFLP primer combinations on four isolates showed that a primer combination, on average, revealed 0·4 polymorphisms between any isolate pair. A selection of 21 primer combinations resulted in 28 AFLP markers, which revealed 16 AFLP phenotypes among all 76 isolates. The two Yr17- virulent pathotypes consisted of three AFLP phenotypes, which were observed in both 1997 and 1998; the two most frequent AFLP phenotypes occurred at most sampling locations and often within the same crop. AFLP diversity was larger among samples collected prior to 1996, and also in this period most AFLP phenotypes were observed at different sampling locations. These results are consistent with the features of an entirely asexually reproducing pathogen dispersed by aerial spores across large areas.     

小麦条锈菌水源11类群的RAPD分析及SCAR标记的建立

鉴于水源11类群近年来一直处于优势地位,为简化其检测手段,本研究利用RAPD技术对该类群的8个主要致病类型进行了多态性分析,以寻找其中主要流行类型的特异性分子标记。结果如下:共筛选出10个碱基随机引物190条,其中94条可得到稳定清晰的扩增图谱,用该94条引物进行RAPD分析,发现各致病类型间遗传变异丰富;以引物S1410扩增得到了水源11-4的特异性DNA条带;以引物S1412和S1304扩增得到了水源11-14的特异性DNA条带;对引物S1304扩增得到的特异性DNA条带回收、克隆和测序,设计了1对19bp/18bp的引物,并成功地将其转化为对水源11-14特异的SCAR标记。以上结果表明,通过规模筛选来寻找小麦条锈菌生理小种的特异性DNA片段,并将其转化为稳定的SCAR标记,有可能建立起中国小麦条锈菌流行生理小种的快速分子鉴定体系。     

四川省小麦条锈菌耐高温性鉴定及其cDNA文库构建

近年条锈菌越夏调查发现,小麦条锈菌耐高温性增强,越夏海拔下限有所降低。本研究通过在高温(21±0.2)℃条件下接种,评价了119个来自四川省的条锈菌菌株耐高温性,并构建了耐高温菌株SC-PX1-4-3萌发夏孢子的cDNA文库。结果发现来自四川的条锈菌群体中耐高温菌株占35.3%,主要分布在成都市、凉山市、简阳市和广元市。夏孢子萌发率统计分析表明,耐高温菌株在高温条件下比温度敏感菌株具有更高的萌发率。采用SMART技术构建了耐高温菌株萌发夏孢子cDNA文库,库容为2.1×106cfu,插入片段平均多大于1.0kb。本研究结果初步明确了四川省小麦条锈菌群体中耐高温菌株组成和分布区域,不同菌株在夏孢子萌发阶段对高温的耐受性存在显著差异,为进一步研究条锈菌耐高温机制奠定基础。     

Support for a stepwise mutation model for pathogen evolution in Australasian Puccinia striiformis f.sp. tritici by use of molecular markers

Since its initial detection in Australia in 1979, wheat yellow (stripe) rust ( Puccinia striiformis f.sp. tritici ) has evolved in Australia and New Zealand into more than 20 pathotypes with assorted virulence characteristics. This evolution is believed to have occurred in a stepwise fashion from an original single pathotype, with no subsequent new introductions. A combination of random amplified polymorphic DNA (RAPDs) and amplified fragment length polymorphisms (AFLPs) was used to examine the level of molecular variation in Australian and New Zealand isolates, and to compare this with variation amongst other isolates of P. striiformis . Using 60 RAPD primers on seven Australian isolates representing seven different pathotypes collected between 1979 and 1991, more than 300 potentially polymorphic loci were analysed and no polymorphisms were detected. Using the same primers on two UK isolates, 3% of loci showed a polymorphism. A similar level of polymorphism was found between UK isolates using AFLP primers, and between 5 and 15% of fragments were polymorphic between an isolate from the UK, an isolate from Denmark, and one from Colombia. However, no AFLP polymorphisms were found amongst 14 Australian and New Zealand isolates tested, at over 100 potentially polymorphic loci. The lack of molecular variation in the Australian and New Zealand collection is consistent with the stepwise mutation theory of pathotype evolution from a single introduction.     

小麦条锈菌鉴别寄主和小种命名现状

 中国是世界上小麦条锈病最大的流行区之一,也是限制中国小麦生产的最严重病害之一,特别是当条件适于其发生时会造成巨大的产量损失.小麦条锈菌小种生理专化性研究及小种监测是掌握小麦条锈菌及其毒性变化动态、抗病基因有效性及病害流行预测预报的重要依据,是治理由此病原菌引起的小麦条锈病的基本环节之一.由于地域、条锈菌群体结构、研究水平和历史等的差异,目前国际上不同国家和地区采用相应的小麦条锈菌鉴别寄主和小种命名方法.中国、美国和印度均使用了各自不同的小麦条锈菌鉴别寄主,并采用了相应的小种命名体系,例如从1957至2002年,中国利用10多个鉴别寄主已鉴定和命名了32个小种和30多个致病类型,美国利用10多个鉴别寄主已鉴定和命名了80个小种;另一类是采用1972年Johnson提出的二进制国际小种命名法,这种体系包括了国际鉴别寄主和欧洲鉴别寄主,象一套鉴别寄主被整体使用,欧洲、中东、亚洲、非洲和南美国家及澳大利亚和新西兰均采用此体系监测小麦条锈菌变化,是一套被最为广泛接受和使用的小麦条锈菌小种鉴别方法和命名系统,但随着研究深入和各国实际情况差异等原因,不同研究人员加入了不同的辅助鉴别寄主,以更能准确和实际地反映本国和地区的现实情况,为生产服务,本文对此现状进行了分析和比较.由于此病菌具有长距离气传和扩散的特点(地区间、国家间和洲际间),因而也讨论了未来小麦条锈菌鉴别寄主组成、小种命名方法和研究结果的交流和比较等实际问题.     

普通小麦品种陕旱8675中抗条锈病基因的分子定位

 利用SSR标记技术对小麦品种陕旱8675的抗条锈病基因进行了分子标记定位。通过对290对微卫星引物的筛选,发现Xwmc170和Xcfa20432对引物在抗亲、感亲、抗池和感池之间均有多态。群体分析结果表明,Xwmc170和Xcfa2043与陕旱8675中抗病基因相连锁,遗传距离分别为9.8 cM和15.0 cM,基因和标记之间的顺序为着丝点-Xcfa2043-Xwmc170-YrSh,间隔距离分别为19.05、5.2和9.8 cM。根据作图的结果,将陕旱8675中所含有的抗病基因定位于2A染色体长臂上,根据该基因的作图位置与抗谱分析,认为该基因可能是1个新的抗条锈基因,暂定名为YrSh。     

Pathogenomic analyses of Puccinia striiformis f. sp. tritici supports a close genetic relationship between South and East Africa

Wheat stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst), occurs in all major wheat-growing regions worldwide and poses a constant threat to production. In South Africa, Pst first emerged in 1996 in the Western Cape and has since caused frequent epidemics with three further distinct races (pathotypes) recorded to date. Herein, we undertook detailed genomic-based analyses of four Pst isolates that represent the four dominant Pst races in South Africa recorded between 1996 and 2005. This analysis identified a number of polymorphic genes with features of known effector proteins and provided additional support of the likely stepwise changes in virulence profile of these South African Pst isolates. Next, we carried out comparative genomic-based analyses with 54 additional Pst isolates collected across wheat-growing regions within South Africa between 1996 and 2017 and 58 Pst isolates from East Africa, Pakistan, the UK, and France. This revealed a close genetic relationship between Pst isolates in South Africa and a number from East Africa. Furthermore, we found the South African Pst isolates also grouped closely with isolates identified in the UK in 2013 that were specifically found on triticale, illustrating long-distance transmission of Pst isolates either between these regions or from a common independent source area. This highlights the critical need for close monitoring of Pst. With wheat being the most planted winter cereal crop in South Africa, investment in continuous surveillance is essential to rapidly identify any future introductions that could quickly lead to rust epidemics.     

Disease severity and pathotype dynamics of Puccinia striiformis f.sp. tritici in Denmark

Disease severity of wheat yellow rust, Puccinia striiformis f.sp. tritici , was analysed in Denmark from 1985 to 1999 in relation to the effects of weather on winter survival, distribution of host cultivars and pathotype dynamics. Below-average temperatures in January and February (midwinter) reduced yellow rust on the susceptible cv. Anja, and in three of four growth seasons following cold winters no yellow rust was observed on any cultivar under natural conditions. The agronomic consequences of dispersal of yellow rust urediniospores from external sources to Denmark, in a period during which large areas were planted with relatively few wheat cultivars, were demonstrated in several cases, most evidently when the Yr9 and Yr17 resistance genes became ineffective. Yr9 was overcome by the pathogen in a period with severe yellow rust epidemics on commercial cultivars, while virulence for Yr17 was first observed in a year with almost no yellow rust. In contrast, the resistance in cv. Kraka ( Yr1, CV ) was increasingly effective in controlling yellow rust, because pathotypes with the matching combination of virulence declined in the pathogen population. Pathotype frequency dynamics were thus influenced by selection forces within the country, and by selection forces in areas where spores were spread to Denmark from outside. The importance of a sufficient level of partial resistance in the wheat germplasm to prevent too much damage by yellow rust epidemics, in the event that the resistance genes are overcome by the pathogen population, is emphasized.