Identification of barley yellow mosaic virus by immuno-electron microscopy in barley but not in Polymyxa graminis or Lagena radicicola

Samenvatting Gerstegeelmozaïekvirus (BaYMV) werd in een perceel wintergerst (cv. Igri) gevonden by Wittem in Limburg, Nederland. Het virus werd met immuno-elektronen-microscopie geïdentificeerd in bladweefsel als BaYMV. In de wortels van geïnfecteerde planten werden alle vormen die van de schimmel-vector (Polymyxa graminis Led.) bekend zijn aangetroffen nl. cystosori, plasmodia en zoosporangia. Inwendig kon geen BaYMV worden aangetoond of waargenomen inPolymyxa ofLagena spp.Cooperative investigation of the ARS, USDA, the Nebraska Agricultural Experiment Station, and the Plant Protection Service, Wageningen. Research conducted under Project 21-12. Published as Paper No. 7787, Journal Series, Nebraska Agricultural experiment Station. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

大麦黄花叶病的分子生物学与基因工程研究——Ⅱ.大麦黄花叶病毒抗性突破株系的分子分析

大麦山黄花叶病毒抗性突破株系的出现克服了已有大麦抗性基因的作用。本研究通过对大麦黄花叶病毒原株系和抗性突破株系RNA1中包含NIa蛋白酶基因的1Kb区段进行克隆和序列分析,比较两株系的序列差异。表明两者间核苷酸水平的同源性为97.2％,氨基酸水平的同源性为97.3％。根据存在的这些微小差异,人工合成具有株系特异性的聚合酶链式反应(PCR)引物,建立PCR检测系统,以期在大田黄花叶病毒株系鉴定中应用。     

Localisation and combination of resistance genes against soil-borne viruses of barley (BaMMV,BaYMV) using doubled haploids and molecular markers

Barley yellow mosaic virus disease caused by different strains of BaYMV and BaMMV is a major threat to winter barley cultivation in Europe. Different resistance genes against these viruses have been mapped and suitable PCR-based markers have been developed. In this respect doubled haploid (DH) populations proved to be advantageous as they facilitate a repeated test for resistance against all agents of the barley yellow mosaic virus complex and besides this, dominant marker systems are as informative as co-dominant ones in DHs due to the lack of heterozygous genotypes. Using DH populations resistance genes rym4, rym5, rym11, rym13, rym15 and the BaYMV/BaYMV-2 resistance of the barley cultivar ‘Chikurin Ibaraki 1’ have been mapped. DHs are also well suited to pyramiding resistance genes against BaMMV and BaYMV. Since homozygous recessive genotypes are more frequent in DHs than in segregating F₂ populations, DHs can be efficiently used to create broad-spectrum resistance and to extend the usability of partly overcome resistance genes. Results from employing two different strategies for pyramiding, based on one and two DH-steps, respectively, combining three recessive resistance genes, i.e. rym4/rym5, rym9 and rym11, are presented. The faster strategy based on one haploidy step resulted in the identification of all three and two-way combinations of the respective resistance genes.     

应用单克隆抗体检测大麦黄花叶病毒

作者应用已建立的大麦黄花叶病毒(BaYMV)的4F_(10)单克隆抗体杂交瘤细胞株,经小白鼠腹水生产单克隆抗体,用过碘酸钠法制备了辣根过氧化物酶标记的酶标单克隆抗体。并由此建立了检测大麦黄花叶病毒的标准的双抗体夹心酶联免疫吸附法(DAS-ELISA)。其检测提纯病毒的灵敏度为3.0μg/ml左右,感病大麦叶片汁液的最大稀释度为1:2560,病茎稀释度为1:160。对采自我国7个主要BaYMV发病区感病的样品进行了检测,绝大多数均显示强阳性,只有宝山样品例外。本项试验为BaYMV诊断的标准试剂盒的建立进行了有益的尝试。     

大麦黄花叶病抗源的转育与利用

 用多棱高抗大麦黄花叶病（BaYMV）品种，二棱耐BaYMV品种作抗源，配制成5个类型（Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ）79个组合，选择700个品系，1982-1988年先后在病圃进行抗BaYMV鉴定。结果表明，培育抗BaYMV二棱品种，除利用免疫或高抗六棱品种外，尚可用耐病二棱品种作抗源。以“8-2”六棱作抗源的Ⅰ型组合中育成了沪麦8号，以77-130、如东早3选盐选③两个耐病二棱品种作抗源的V型组合中，育成了沪麦10号，在生产上获得了大面积推广。从育种上支持了BaYMV存在多基因数量遗传的论点。转育成的42个抗病品系的穗数、粒数、粒重分别比感病对照种增加17.6%、12.3%、9.7%，平均增产49%。由于生产上病田严重度的差异，为使抗病品种广泛应用，既要提高抗病性，又要在丰产性上超过对照种，在病田种植需增产60%以上。11月份降雨量少，下旬温度在9℃以下，不利于BaYMV发生。在上海地区播种期提早到10月底或11月初，播后增加田间水分，有利于转育和获得可靠的鉴定结果。     

Exotic barley germplasms in breeding for resistance to soil-borne viruses

Summary Soil-borne mosaic inducing viruses, i.e., barley mild mosaic virus (BaMMV), barley yellow mosaic virus (BaYMV), and BaYMV-2, cause one of the most important diseases of winter barley in Western Europe. Since resistance of all commercial European barley cultivars is due to a single recessive gene (ym4) which is not effective against BaYMV-2, exotic barley germplasms (Hordeum vulgare L., H. spontaneum Koch) were screened for resistance to the different viruses and analyzed for genetic diversity concerning BaMMV resistance. In these studies it turned out that resistance to BaMMV is entirely inherited recessively and that a high degree of genetic diversity concerning resistance is present within the barley gene pool at least to BaMMV. Therefore, exotic barley germplasms are a very useful source for the incorporation of different resistance genes into barley breeding lines, thereby enabling the breeder to create cultivars adapted to cultivation in the growing area of fields infested by soil-borne viruses. Furthermore, in order to obtain more information on these germplasms they were evaluated for agronomic traits and isozyme, RFLP and RAPD analyses were carried out on these varieties to detect markers linked to the respective resistance genes and to obtain information on the genetic similarity between yellow mosaic resistant barley accessions derived from different parts of the world. Actual results of these studies are briefly reviewed.     

Combination of resistances to barley yellow mosaic virus and Rhynchosporium secalis by recurrent selection with repeated haploid steps

The aim of this investigation was the incorporation of resistance to BaYMV and Rhynchosporium secalis into German winter barley varieties together with the maintenance of their high agronomic characters. The agronomic value of DH-lines after two backcrosses and three haploid steps were compared with the original parents. Lines with similar or superior results could be selected for all the agronomic features evaluated. This method of recurrent selection with repeated haploid steps can therefore lead to the development of lines useful for practical purposes.     

大麦黄花叶病的分子生物学与基因工程研究:Ⅱ.大麦黄花叶病毒抗性突破株系的分子分析

 大麦山黄花叶病毒抗性突破株系的出现克服了已有大麦抗性基因的作用。本研究通过对大麦黄花叶病毒原株系和抗性突破株系RNA1中包含NIa蛋白酶基因的1Kb区段进行克隆和序列分析,比较两株系的序列差异。表明两者间核苷酸水平的同源性为97.2%,氨基酸水平的同源性为97.3%。根据存在的这些微小差异,人工合成具有株系特异性的聚合酶链式反应(PCR)引物,建立PCR检测系统,以期在大田黄花叶病毒株系鉴定中应用。     

Utility of a GFP-expressing Barley yellow mosaic virus for analyzing disease resistance genes

The soil-borne plasmodiophorid Polymyxa graminis is a vector for Barley yellow mosaic virus (BaYMV), which can severely damage barley plants. Although 22 disease resistance genes have been identified, only a few have been used for breeding virus-resistant cultivars. Recently, BaYMV strains capable of overcoming the effects of some of these genes have been detected. In this study, green fluorescent protein (GFP)-expressing BaYMV was constructed and used to examine viral dynamics in inoculated barley plants. Leaf inoculations resulted in higher infection rates than root or crown inoculations. Additionally, inoculations of some resistant cultivars produced infections that were similar to those observed in a field test. The results of this study indicate that the GFP-expressing virus is a useful tool for visualizing virus replication and dynamics, and for understanding resistance mechanisms.     

Molecular breeding for the BaMMV/BaYMV resistance gene ym4 in winter barley

The aim of this investigation was to develop a procedure for the largescale molecular breeding for ym4, allowing resistance to BaMMV/BaYMV to be fixed in early breeding generations of winter barley. A codominant STS marker derived from the restriction fragment length polymorphism marker MWG838 for the ym4 resistance gene was combined with a new and easy procedure for preparing leaf samples for polymerase chain reaction (PCR), theoretically allowing one person to extract DNA from 5000 samples in a single day. In the procedure for molecular breeding for ym4, all steps, including leaf sampling, DNA extraction, PCR amplification and digestion with restriction enzyme were assembled in microtitre plates allowing multipipetting throughout the procedure, including the loading of gels. The method is amenable to further automation with the aid of a robot arm. Double haploid (DH) lines, as well as F₂ and F₄ breeding lines were analysed and, based on markers, homozygous and heterozygous BaMMV/BaYMV resistant plants were identified for further breeding. The winter barley breeding programmes were modified to include marker-based selection for BaMMV/BaYMV resistance on DH or on F₂ individuals, which had been preselected for mildew and leaf rust resistance.