Establishment of Reverse-transcription Loopmediated Isothermal Amplification Method for Detection of Wheat Streak Mosaic Virus

A reverse-transcription loop-mediated isothermal amplification （RT-LAMP） method was established for the detection of wheat streak mosaic virus （WSMV）. Ac-cording to the conservative regions of the genes that encode the coat protein of WSMV, 2 pairs of primers were designed. Final y, the 1st pair of primers was select-ed through the specificity test. The sensitivity test showed the sensitivity of RT-LAMP method was 10 times higher than that of RT-PCR. In addition, the amplifica-tion of target gene could be judged visual y from the presence of fluorescence （cal-cein） in the final reaction system. The RT-LAMP method, established in this study, was rapid, easy, specific and sensitive. Moreover, it did not require sophisticated equip-ment. The RT-LAMP was suitable for the rapid detection of WSMV.

Chinese wheat mosaic virus: A long-term threat to wheat in China

In China, a soil-borne virus causing a disease of winter wheat and associated with Polymyxa graminis, has been reported for many years and is now recognized as a new species, Chinese wheat mosaic virus(CWMV). Since the determination of its genomic sequence, more progress has been made in understanding its genomic structure and functions. Molecular and serological methods have been developed to help survey the distribution of the virus and to provide the basic information needed for disease forecasting and control. At present, the best countermeasure is cultivation of resistant wheat varieties. In addition, development and application of some auxiliary countermeasures, such as rotation of non-host crops, delayed seed-sowing, reasonable application of nitrogen fertilizer, and treatment of imported seeds with fungicides before sowing, may be helpful for controlling the disease. The viral distribution and damage, virion properties, genome organization and spontaneous mutation, temperature sensitivity, and disease management options are here reviewed and/or discussed to help in developing more cost-effective countermeasures to control the disease in the future.

Remote Monitoring of Wheat Streak Mosaic Progression Using Sub-Pixel Classification of Landsat 5 TM Imagery for Site Specific Disease Management in Winter Wheat

Wheat streak mosaic (WSM), caused by Wheat streak mosaic virus is a viral disease that affects wheat (Triticum aestivum L.), other grains, and numerous grasses over large geographical areas around the world. To improve disease management and crop production, it is essential to have adequate methods for monitoring disease epidemics at various scales and multiple times. Remote sensing has become an essential tool for monitoring and quantifying crop stress due to biotic and abiotic factors. The objective of our study was to explore the utility of Landsat 5 TM imagery for detecting, quantifying, and mapping the occurrence of WSM in irrigated commercial wheat fields. The infection and progression of WSM was biweekly assessed in the Texas Panhandle during the 2007-2008 crop years. Diseased-wheat was separated from uninfected wheat on the images using a sub-pixel classifier. The overall classification accuracies were >91% with kappa coefficient between 0.80 and 0.94 for disease detection were achieved. Omission errors varied between 2% and 14%, while commission errors ranged from 1% to 21%. These results indicate that the TM image can be used to accurately detect and quantify disease for site-specific WSM management. Remote detection of WSM using geospatial imagery may substantially improve monitoring, planning, and management practices by overcoming some of the shortcomings of the ground-based surveys such as observer bias and inaccessibility. Remote sensing techniques for accurate disease mapping offer a unique set of advantages including repeatability, large area coverage, and cost-effectiveness over the ground-based methods. Hence, remote detection is particularly and practically critical for repeated disease mo- nitoring and mapping over time and space during the course of a growing season.

Responses of Some American, European and Japanese Wheat Cultivars to Soil-Borne Wheat Viruses in China

Wheat seeds of 109 cultivars from USA, Europe and Japan were sown in experiments at seven sites in different provinces of China for one or two seasons. Five of the sites were infested with the bymovirus wheat yellow mosaic virus (WYMV) and two jointly with WYMV and the furovirus Chinese wheat mosaic virus (CWMV). Disease symptoms were assessed visually and leaf samples were tested for virus (es) by ELISA. At least 29 cultivars were resistant to WYMV at the sites where only this virus was present but all the cultivars were severely infected at Rongcheng (Shandong Province) where CWMV was mixed with WYMV. There was evidence that the presence of CWMV assisted infection by WYMV and also resulted in more severe symptoms. At the mixed site in Yantai, Shandong Province, symptoms were mild and many cultivars had symptomless infection . Of the two strains of WYMV identified in Japan, the Chinese sites seem to be most similar to the type isolated, WYMV-T. Eleven cultivars seemed to be susceptible to WYMV only at Loutian (Hubei Province),suggesting that the virus at this site would be worth studying further.

Research Progress of Wheat Yellow Mosaic

Wheat yellow mosaic is a soil-borne virus disease,and now has become one of the main viral diseases endangering wheat production in China,posing serious threat to wheat production. The pathogen,transmission media,incidence regularity and performance of wheat yellow mosaic were introduced in the paper,and its molecular biology and breeding research were summarized,in order to provide a reference for resistance breeding and production practice of wheat yellow mosaic.

Mapping of a Wheat Resistance Gene to Yellow Mosaic Disease by Amplified Fragment Length Polymorphism and Simple Sequence Repeat Markers

Wheat （Triticum aestivum L.） yellow mosaic virus （WYMV） is transmitted by a fungal vector through soil and causes serious wheat yield losses due to yellow mosaic disease, with yellow-streaked leaves and stunted plants. In the present study, the amplified fragment length polymorphisms （AFLP） and simple sequence repeat （SSR） were used to identify the molecular linkages with the resistance gene against WYMV. Bulked segregant analysis was performed with an F2 population derived from the cross of cultivar Ningmai 9 （resistant） × cultivar Yangmai 10 （susceptible）. By screening among the resistant or susceptible parents, the F2 pools and the individuals in the F2 population with 64 combined selective AFLP primers （EcoRI/MseI） or 290 reported SSR primers, a polymorphic DNA segment （approximately 120 bp） was amplified using the primer pair E2/M5, and an SSR marker （approximately 180 bp） was located on wheat chromosome 2A using the primer Xgwm328. Analysis with MAPMAKER/Exp Version 3.0b （Whitehead institute for Biomedical Research, Cambridge, MA, USA） indicated that these two markers were dominantly associated with the resistance gene at distances of 5.4 cM or 17.6 cM, respectively. The resistance gene to WYMV derived from Ningmai 9, is temporarily named YmNM, and was mapped to wheat chromosome 2A.

The Empidemiology of Wheat Rosette Stunt in Hulunbeir District of Inner-Mongolia

The virus disease threatening wheat production in Hulunbcir District of Inner - Mongolia was identified as NCMV (Northern Cereal Mosaic Virus) or WRSV (Wheat Rosette Stunt Virus) by tests of host range, transmission, virus particle and scrology. The main vector for spreading the virus is planthopper, Laodelphax striatellus (Fallen), which overwinters in the stubbles of wheat underground. The overwintered virulifcrous nymphs emerged in late April is responsible for the early infection of the disease. Agropyron repens, an important perennial weed in cultivated regions, is also an important wild host of the virus and its vector. Severe loss is induced when wheat is infected before 3 - leaf stage. The disease incidence could be predicted by a model with the population and virulifcrous rate of overwintered vectors as independent variables.

Cloning and expression of the Chinese wheat mosaic virus RNA2 coat protein read-through and 19 ku cysteine-rich domains and localization of these proteins

The 5’-terminal (RTn) and 3’-terminal (RTc) halves of the coat protein readthrough domain and the 19 ku cysteine-rich protein of Chinese wheat mosaic virus (CWMV) were amplified by RT-PCR, cloned and expressed in E. coll. Antisera and monoclonal antibodies against these proteins were prepared by immunising these purified proteins to mice. Detection of RTn, RTc and 19 ku proteins in CWMV infected wheat sap and leaf tissue indicated that the RTn and RTc proteins were distributed on the surface of virus particles whereas the 19 ku protein was in the cytoplasm of the infected wheat cells.

Generation of transgenic wheat resistant to wheat yellow mosaic virus and identification of gene silence induced by virus infection

The plasmid containing the promoter Actl, the coat protein (cp) gene of wheat yellow mosaic virus (WYMV) and the selectable bar gene, was delivered via particle bombardment, directly into immature embryos of a wheat culti-vars. PCR and PCR-RFLP were employed to screen the existence of the cp gene in T0 and T1 generations. Seeds from the positive T1 plants were sowed in fields heavily contaminated with WYMV to detect their resistance. In field trial of virus infection, one of the transgenic wheat lines, P8-T2, exhibited highly disease-resistance. Western blot and RT-PCR analysis showed that the expression level of cp gene in the resistant transgenic line was reduced greatly compared to those susceptible to WYMV infection. This provided evidence to presume that the resistance obtained by the transgenic wheat line was stimulated by the mechanism of the virus induced gene silencing.

Analysis of nucleotide sequence of wheat yellow mosaic virus genomic RNAs

Wheat yellow mosaic virus (WYMV) isolate HC was used for viral cDNA synthesis and sequencing. The results show that the viral RNA1 is 7 629 nucleotides encoding a polyprotein with 2 407 amino acids, from which seven putative proteins may be produced by an autolytic cleavage processing besides the viral coat protein. The RNA2 is 3 639 nucleotides and codes for a polyprotein of 903 amino acids, which may contain two putative non-structural proteins. Although WYMV shares a similarity in genetic organization to wheat spindle streak mosaic virus (WSSMV), the identities in their nucleotide sequences or deduced amino acid sequences are as low as 70% and 75% respectively. Based on this result, it is confirmed that WYMV and WSSMV are different species within Bymovirus .

Over-expression of 72 ku protein of wheat yellow mosaic virus in E. coli and preparation of its antiserum

By reverse transcription-polymerase chain reaction (RT-PCR), cDNA fragment of wheat yellow mosaic vir黶 (WYMV) RNA2 encoding 72 ku protein has been synthesized and cloned into plasmid pET30a(+) for overexpression in prokaryotic celis. BL21(DE3) pLys S of E.coli transformed with the recombinant plasmid pETP72 containing the fragment has been induced to express the 72 ku protein on high level. The produced protein has been purified from sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for its antiserum preparation. in western-blotting analysis, the antibodies reacted with the 72 ku protein expressed in E.coli.

小麦黄花叶病抗性研究及育种应用进展

小麦黄花叶病是一种全球性的土传病毒病,严重时会导致小麦产量大幅下降。小麦黄花叶病毒(WYMV)是主要的病原体之一,属于马铃薯Y病毒科(Potyviridae)大麦黄花叶病毒属(Bymovirus),以禾谷多黏菌(Polymyxa graminis)为媒介感染小麦。目前已鉴定出14个抗WYMV的基因位点,图位克隆了1个抗病基因,证实了3个基因具有抗病性调控功能,已育成多个抗病小麦品种(系)。本文从小麦黄花叶病毒的病原体特性、病害分布、小麦抗性遗传和抗病育种等方面,综述了WYMV及小麦抗病性的研究进展,并对WYMV抗性基因挖掘和小麦抗病育种应用进行了展望。

长江中下游麦区279个品种(系)小麦黄花叶病抗性研究

小麦黄花叶病是影响长江中下游麦区小麦(Triticum aestivum L.)产量的重要病害之一。为了筛选抗小麦黄花叶病种质资源,本研究对近30年来长江中下游麦区选育的279个小麦品种(系)进行田间小麦黄花叶病抗性鉴定,同时利用与抗小麦黄花叶病主效数量性状基因座(QTL)QYm.nau-5A.1和QYm.nau-2D连锁的分子标记检测,以分析抗病QTL在品种(系)中的传递过程。结果显示,抗病材料为174个,占62.4%,其中仅含QYm.nau-5A.1和QYm.nau-2D的材料分别为30、98个,两个QTL均含的材料为9个,两个QTL均无的为37个,表明还存在其他抗病基因或QTL;感病材料为105个,占37.6%,其中仅含QYm.nau-5A.1和QYm.nau-2D的分别为6、25个,两个QTL均无的为74个。进一步分析小麦品种(系)系谱发现,长江中下游麦区小麦品种(系)的QYm.nau-5A.1主要来自于西风小麦,通过宁麦9号传递;QYm.nau-2D主要来自于苏麦6号、扬辐麦9311、郑麦9023,其中苏麦6号中的抗性QTL主要通过镇麦9号传递。本研究结果为长江中下游麦区小麦黄花叶病抗性分子育种及新抗病基因挖掘与利用提供了理论支撑。

中国小麦花叶病毒富含半胱氨酸蛋白多克隆抗体的制备与应用

中国小麦花叶病毒(Chinese wheat mosaic virus,CWMV)是小麦花叶病的重要病原体之一,长期威胁小麦的产量和品质;CWMV富含半胱氨酸蛋白(cysteine-rich protein,CRP)在病毒侵染过程中具有重要而复杂的功能。为了深入研究CRP的功能和CWMV侵染机制,本研究采用反转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)从CWMV侵染的小麦叶片中获得CRP基因编码区,将其克隆至原核表达载体pET-32a上,并将重组质粒pET-CRP转化至大肠埃希菌BL21(DE3)感受态细胞中进行诱导表达;通过镍柱亲和层析法纯化CRP重组蛋白,并用作抗原免疫新西兰白兔,制备多克隆抗体。蛋白质印迹法、间接酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和斑点ELISA分析结果显示:纯化的CRP抗体不仅具有高度的特异性,而且效价高达1∶4096000,是未纯化抗体效价的4倍;该抗体能识别0.5 ng抗原,显示出较高的灵敏度;在1∶120000稀释条件下,该抗体能特异且灵敏地识别天然CRP。综上所述,本研究所制备的CRP抗体不但可用于田间CWMV病株样品的精准诊断,还可用于植物体内瞬时表达CRP的检测分析,为后续CRP检测、定量分析及其亚细胞定位等研究提供了依据。

不同品种不同播期对小麦黄花叶病毒病的抗性及产量的影响

为了预防小麦黄花叶病毒病的发生及扩散,开展小麦黄花叶病毒病抗性试验研究,试验选择3个小麦品种:‘鲁原502’、‘鑫麦296’和‘齐民8号’,2个播期:2021年10月20日和2021年11月1日。结果表明,‘齐民8号’在2个播期下小麦黄花叶病毒病都是无症状;‘鲁原502’在11月1日的播期下小麦黄花叶病毒病无症状,但是在10月20日的播期下新叶出现褪绿条纹或黄花叶症状;而‘鑫麦296’在2个播期下多数叶片出现褪绿条纹或黄花叶症状,有时会出现新叶扭曲,植株矮化不明显。3个品种在10月20日的播期下,产量最高的是‘齐民8号’,为8960.4 kg/hm^(2);其次是‘鲁原502’,为8570.4 kg/hm^(2);产量最低的是‘鑫麦296’,为8330.4 kg/hm^(2)。3个品种在10月20日的播期下平均产量达8620.4 kg/hm^(2)。3个品种在11月1日的播期下,产量最高的是‘齐民8号’,为8120.4 kg/hm^(2);其次是‘鑫麦296’,为6260.25 kg/hm^(2);产量最低的是‘鲁原502’,为6190.35 kg/hm^(2)。3个品种在11月1日的播期下平均产量是6857 kg/hm^(2)。3个品种在10月20日播期的平均产量比在11月1日播期的平均产量高出1763.4 kg/hm^(2)。通过小麦品种选择、土壤改良技术、晚播技术、化肥减量增效技术多项技术进行融合和改进,可以达到降低小麦黄花叶病毒病发病的效果。

小麦对黄花叶病的抗性鉴定及典型品种的遗传分析

本研究针对来自江苏、河南、四川、湖北、日本和美国的37个小麦品种,在进行小麦抗黄花叶病的抗病性鉴定、调查的基础上,结合分子生物学检测(RT-PCR和ELISA),证实小麦品种扬辐9311对小麦黄花叶病毒(WYMV)表现免疫。将该品种与表现高感的小麦品种进行杂交和回交,通过对其后代抗感分离的调查与分析,确定了小麦品种扬辐9311对黄花叶病的抗性受一对显性基因控制,并为寻找小麦抗黄花叶病基因的分子标记提供理论依据和实验材料。同时,本文对小麦抗黄花叶病表现的影响因素及宁麦9号的抗病性进行了分析和讨论。

中国小麦黄花叶病毒(WYMV)分布的RT-PCR鉴定

利用小麦黄花叶病毒（Ｗｈｅａｔｙｅｌｌｏｗｍｏｓａｉｃｖｉｒｕｓ，ＷＹＭＶ）和小麦梭条花叶病毒（ｗｈｅａｔｓｐｉｎｄｌｅｓｔｒｅａｋｍｏｓａｉｃｖｉｒｕｓ，ＷＳＳＭＶ）的特异性序列为引物，逆转录－ＰＣＲ方法并配合免疫电镜观察，对采自我国四川陕西、河南、安徽、江苏５省９个县、市的２７份感病田小麦样品进行检测。除２份样品未检测到病毒外，其余２５份样品均为小麦黄花叶病毒所侵染，没有发现小麦梭条花叶病毒，由此表明在中国长江中、下游、淮河及渭河流域严重危害小麦的真菌传线状病毒应主要为小麦黄花叶病毒。

禾谷多黏菌传小麦病毒病的分布及变化动态

为给小麦黄花叶病毒(Wheat yellow mosaic virus,WYMV)和中国小麦花叶病毒(Chinese wheatmosaic virus,CWMV)病的科学防控提供相关技术参考,于2008-2010年采用普查结合ELISA检测和电镜观察等方法,对这两种禾谷多黏菌传小麦病毒病的分布进行了研究。结果表明,WYMV重病区呈现向西、向北扩展的新趋势,发现山东泰安、河南商丘、湖北丹江口、襄樊、随州等新病区,四川省仅在内江发生。江苏省两种病毒复合侵染区正在扩大,浙江病区基本消失。

基因枪介导法向小麦导入黄花叶病毒复制酶基因的研究

筛选出适宜小麦转化的优良基因型扬麦 15 8和扬麦 10号。以扬麦 15 8幼胚愈伤组织为受体 ,利用基因枪介导法将含有小麦黄花叶病毒复制酶基因 (WYMV- N ib8)的 pubi Nib8质粒转入了小麦。T0 代经 PCR扩增分析 ,获得了 14株含 WYMV- N ib8基因的转化植株 ,转化率为 0 .4 3%。T1、T2 代跟踪分子检测和 T2 代田间病毒抗性鉴定结果表明 ,获得了 4个抗

小麦梭条花叶病抗性的遗传研究

本试验选用 17个高抗品种、 9个高感品种随机组合进行杂交 ,通过对 F1、 F2 和部分 BC1、 BC2的抗感鉴定结果以及 F3代的追踪分析 ,研究了小麦品种对小麦梭条花叶病的抗性遗传规律 .结果表明 ,小麦品种对小麦梭条花叶病的抗性为细胞核遗传 ,这一性状受多对显性基因的控制 ,不同品种含有其中的一对或两对或三对 ,大多数品种都含有一对抗性效应很强的抗病基因 .这些结果显示 ,通过对高产感病品种进行遗传改良 。