Responses of some Asian and European barley cultivars to UK and Chinese isolates of soil-borne barley mosaic viruses

In field plots at Yancheng, Jiangsu, China, a range of European and Asian barley cultivars was grown in soil from three sites in China infested with barley yellow mosaic virus (BaYMV). Most of the cultivars resistant to the common European strain of BaYMV were susceptible to the Chinese isolates but cv. Energy remained disease-free. Barley mild mosaic virus (BaMMV) was also detected in one of these soils but affected only one Chinese cultivar and not those susceptible to BaMMV in Europe. This is the first report of BaMMV in China. Inoculation experiments confirmed the different cultivar response to UK and Chinese isolates of BaYMV and showed that resistance was to the virus and not to the vector. A range of Chinese cultivars selected for resistance to BaYMV were also resistant to a UK isolate of BaMMV.     

The susceptibility of barley cultivars to barley yellow mosaic virus (BaYMV) and its fungal vector, Polymyxa graminis

In glasshouse experiments, barley seedlings were inoculated with barley yellow mosaic virus (BaYMV) either mechanically or by using zoospores or cystosori of a viruliferous isolate of the vector, Polymyxa graminis, maintained on barley in sand culture. Experiments using mechanical inoculation showed that seedlings became more resistant with age. Consistent cultivar differences were obtained: cvs Maris Otter and Halcyon were very susceptible and cv. Athene seemed immune. Symptoms developed more rapidly at 23 than at 17 or 11 ^oC. After vector inoculation, symptoms developed more slowly than after mechanical inoculation but cultivar ranking was similar. Cultivars did not differ in susceptibility to the vector, as measured by zoospore production on their roots. Spring barley cultivars supported the growth of the vector which remained viruliferous and some showed symptoms although, in the field, symptoms do not appear on spring-sown crops.     

Confirmation of the transmission of barley yellow mosaic virus (BaYMV) by the fungus Polymyxa graminis

Resting spores (cystosori) of Polymyxa graminis, selected from roots of barley plants infected with barley yellow mosaic virus (BaYMV), were used to start mono-fungal sand cultures. Out of 20 attempts using over 800 cystosori, P. graminis became established in 12, and in two of these BaYMV symptoms also occurred. BaYMV was detected by ELISA in extracts of dried roots heavily infected with cystosori and in zoospores of P. graminis. Calculations suggested that, on average, each zoospore carried less than 100 virus particles. In two virus acquisition experiments, non-viruliferous isolates of P. graminis failed to acquire BaYMV from roots of mechanically-inoculated plants. In two further experiments, non-viruliferous isolates were grown on rooted tillers produced from healthy plants and those infected with BaYMV by either vector or mechanical inoculation. Zoospores and cystosori of P. graminis subsequently transmitted the virus, but only from plants where it had been introduced by the vector. Repeated mechanical transmission appeared to have selected a strain of virus that could not be acquired and/or transmitted by the vector. The results provide convincing evidence that P. graminis is a vector of BaYMV but suggest that, in natural populations, only a small proportion of spores may be viruliferous.     

Molecular analysis of the capsid protein gene of a german isolate of barley mild mosaic virus

Summary Barley mild mosaic virus (BaMMV) is one of the agents causing the barley yellow mosaic disease. The sequence corresponding to the 3end of the BaMMV RNA1 of a German isolate was sequenced and the coding sequence for the 251 amino acid containing capsid protein was determined. Comparison of this sequence to other potyviral sequences and to the corresponding sequence of two Japanese isolates of BaMMV was done. The three different isolates of BaMMV show a high degree of similarity.Abbrevations BaMMV barley mild mosaic virus - BaYMV barley yellow mosaic virus; bp: base pair - IPTG isopropyl -D thiogalactopyranoside - kb kilo base - NTR nontranslated region - ORF open reading frame - PVDF polyvinylidene difluoride     

Association of two barley yellow mosaic virus (RNA 2) encoded proteins with cytoplasmic inclusion bodies revealed by immunogold localisation

Summary Antisera were raised against the RNA 2-encoded proteins of 28 kDa and 70 kDa of barley yellow mosaic virus (BaYMV) by using the corresponding cDNA sequences of a German isolate for protein overexpression inEscherichia coli BL 21 and subsequent purification. The proposed processing of a 98 kDa precursor polyprotein encoded by the long open reading frame of RNA 2 to two proteins of 28 kDa and 70 kDa could be confirmed by immunoprecipitation of the in vitro transcribed and translated cDNA-clone of RNA 2 and Western blot analysis of fragmentated protein extracts of BaYMV-infected winter barley plants. In situ localisation studies of infected leaf tissue using immunogold labeling techniques for electron microscopy revealed that both viral proteins of BaYMV (RNA 2) were associated with the crystal-like cytoplasmic inclusion bodies. No other parts of the cells and no other inclusions (pinwheelstructures or aggregated virus particles) showed any gold labeling when the 28 kDa and 70 kDa antisera were used. We suppose that both RNA 2-encoded proteins take part in the formation of the crystal-like cytoplasmic inclusion bodies which are the most dominant structures in the cytoplasm of BaYMV-infected tissue. Possible functions of the 28 kDa and 70 kDa protein of BaYMV (RNA 2) are discussed.Abbreviations PBS phosphate-buffered saline - CEA chicken egg albumin - BaYMV barley yellow mosaic virus - BaMMV barley mild mosaic virus     

The effect of cultivar used as host for Polymyxa gmminis on the multiplication and transmission of barley yellow mosaic virus (BaYMV)

A viruliferous isolate of the fungal vector Polymyxa graminis was grown on roots of barley cultivars immune or susceptible to barley yellow mosaic virus (BaYMV). Zoospores or resting spores of the vector produced on different cultivars were then inoculated to a virus-susceptible test cultivar. Although the vector established in all treatments, transmission of BaYMV was rare and usually nil from immune cultivars; amounts of virus detected serologically in their roots were very low, thus showing that resistance was to virus multiplication. If immune cultivars decrease the virus content of vector populations in the field, this would have important implications for disease control.     

Barley mild mosaic virus inside its fungal vector, Polymyxa graminis

In an electron microscope study to investigate the association of barley mild mosaic virus (BaMMV) with its fungal vector, Polymyxa graminis, thin sections were made of zoospores of the vector and of barley roots containing different stages in the life cycle of the fungus. Immunogold labelling was used to identify the virus in sections. Labelled bundles of presumed virus particles were seen in c. 1% of zoospores liberated from plant roots and in zoospores inside zoosporangia. A few zoosporangial plasmodia had localised labelling but no bundles were seen. No virus particles were seen in sections of resting spores.     

The effect of the weight of the seedling-derived tuber on subsequent clonal generations in a potato breeding programme

Cultures of Polymyxa graminis were maintained in roots of barley plants grown in sand at different temperatures using Wisconsin soil temperature tanks. At 17 – 20°C, the minimum time from inoculation with cystosori to the production of zoospores from the inoculated roots was 2 – 3 wk. At 11 – 20°C many zoospores were produced but the incubation period was longer at the lower temperatures. Above 20°C little fungal development occurred. The duration of motility of zoospores ranged from c. 1 h to > 24 h. Bovine serum albumen (BSA) prolonged motility but glycine and glucose had no effect or, at higher concentrations, were toxic. Zoospores were rapidly immobilised by zinc ions in solution at or above 10μg/ml. In some experiments BSA added to the zoospore suspension greatly increased transmission of barley yellow mosaic virus (BaYMV) while glucose, glycine and ovalbumen decreased it. When seedlings were incubated with zoospore suspensions for 24 h at different temperatures, BaYMV transmission was high (> 60%) at 10, 15 and 20°C but there was little at 5 or 25°C. In experiments to determine the time taken for zoospore penetration, seedlings were incubated in suspension for different periods of time and then rinsed in zinc sulphate solution to kill free zoospores. Between 3 and 3·5 h was needed for zoospores to establish infection. Transmission occurred equally to plants of various ages between 3 days and 7·5 wk.     

山东烟台小麦土传病毒病由小麦黄花叶病毒和土传小麦花叶病毒相关病毒复合侵染所致

在山东省烟台地区的小麦上发生一种由土壤中禾谷多粘菌Polymyxa graminis传播的病毒病,感病小麦植株表现矮化褪绿和花叶症状.我们于1997年4月从病区采集感病小麦植株,进行了病毒种类鉴定.直接电镜观察发现有二种病毒粒子,一种粒子呈棒状,占大多数,其长度约为300nm和150nm; 另一种粒子呈线状,数量较少,长度为500nm～700nm.免疫电镜结果表明,棒状病毒粒子仅与土传小麦花叶病毒(soil-borne wheat mosaic virus, SBWMV)抗血清反应,而不与小麦黄花叶病毒(wheat yellow mosaic virus,WYMV)抗血清和小麦梭条斑花叶病毒(wheat spindle streat mosaic virus,WSSMV)抗血清反应;反之,线状病毒仅与WYMV、WSSMV抗血清反应,而不与SBWMV抗血清反应.用WYMV和SBWMV两种抗血清同时进行修饰时,线状病毒粒子和棒状病毒粒子均发生反应.     

Barley yellow mosaic virus is overcoming RYM4 resistance in Belgium

Barley yellow mosaic virus (BaYMV) is the causal agent of a soil-borne systemic mosaic disease on barley. It has been reported in Belgium since the 1980s. The control of this disease is managed almost exclusively through the use of resistant varieties. The resistance of most commercial barley cultivars grown in Europe is conferred mainly by a single recessive gene, rym4. This monogenic resistance provides immunity against BaYMV pathotype 1 and has been mapped on barley chromosome 3HL and shown to be caused by mutations in the translation initiation factor eIF4E. Another pathotype, BaYMV pathotype 2, which appeared in the late 1980s (in Belgium, in the early 1990s), is able to overcome the rym4-controlled resistance. Until recently, this pathotype remained confined to specific locations. During a systematic survey in 2003, mosaic symptoms were observed only on susceptible barley cultivars collected in Belgian fields. BaYMV was detected by ELISA and RT-PCR on the susceptible cultivars and only by RT-PCR on the resistant cultivars. In 2004, mosaic symptoms were observed on susceptible and resistant cultivars. BaYMV was detected by ELISA and RT-PCR on both cultivars. In addition to developing RT-PCR methods for detecting and identifying BaYMV and Barley mild mosaic virus (BaMMV), an RT-PCR targeting the VPg/NIa viral protein part of the genome, known to discriminate the two BaYMV pathotypes, was set up to accurately identify the pathotype(s) now present in Belgium. The sequences from the generated amplicons revealed the single nucleotide substitution resulting in an amino acid change from lysine to asparagine specific to BaYMV pathotype 2. The possible reasons for the change in the BaYMV pathotype situation in Belgium, such as climatic change or a progressive build-up of soil inoculum potential, will be discussed, as well as the use of eIF4E-based resistance.     

Specific Isozyme Marker of a Virus Resistant Barley Derived from Interspecies Cross Between Hordeum vulgare and H. bulbosum

A diploid barley cultivar "Supi 1" was crossed with a tetraploid Hordeum bulbosum “GBC141” to transfer the disease resistant traits. Eleven viable triploid F1 plants were produced by means of embryo rescue technique. The resulting triploid hybrids were backcrossed to diploid barley, and seven BC1 plants were obtained. One of the BC1 plants exhibited barley yellow mosaic virus (BaYMV) resistance when grown in the diseased nursery. Isozyme analysis of H. vulgate, H. bulbosum and their backcross hybrids were made via slab polyacrylamide gel electrophoresis technique. The primary results showed that zymogram variation could be obviously found between diploid barley "Supi 1' and tetraploid H. bulbosurn "GBC141”. A peroxidase isozyme (Rf=0.47) from H. bulbosum was detected in the peroxidase isozyme zymogram of young roots of backcross hybrid BC1-2. This peroxidase isozyme was related to the BaYMV resistance but the linkage relation will be determined by the genetic analysis of the F2 population in the future. The BaYMV resistant line of the backcross with isozyme marker is the important resource of barley disease-resistant breeding.     

Effects of barley yellow mosaic disease resistant gene rym1 on the infection by strains of Barley yellow mosaic virus and Barley mild mosaic virus

Although a Chinese landrace of barley, Mokusekko 3, is completely resistant to all strains of Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), and is known to have at least two resistant genes, rym1 and rym5, only rym5 has been utilized for BaYMV resistant barley breeding in Japan. In order to clarify the effect of rym1 on BaYMV and BaMMV, and to utilize the gene for resistant barley breeding, the susceptibilities of only rym1 carrying breeding lines against BaYMV and BaMMV were investigated. In the assessment of resistance to BaYMV-I, 341 F(2) populations derived from a cross between the resistant line Y4 with only rym1 and the susceptible cv Haruna Nijo shows that the segregation loosely fits a 1R:3S ratio (0.05 > P > 0.01), suggesting that the resistance is controlled by a single recessive gene, rym1. Further, none of the F(3) lines derived from the nine resistant F(2) plants showed any disease symptoms in the field infected by BaYMV-I. The same nine F(3) lines showed almost the same agronomic characters in the field infected by BaYMV-III as those in the uninfected field, apart from the symptom of showing numerous mosaics. This result indicates that the gene rym1 has an acceptable level of resistance to BaYMV-III. In the assessment of resistance to BaYMV-II, BaMMV-Ka1 and -Na1, an artificial infection method was adopted and the susceptibilities to those viruses were investigated. Although the control varieties, Ko A and Haruna Nijo, were infected with all of them, the rym1 gene carrying BC(2)F(3) lines were completely resistant to all strains. In summary, rym1 is completely resistant to BaYMV-I, -II, BaMMV-Ka1 and -Na1, and has an acceptable level of resistance to BaYMV-III. This study concludes with a discussion of the reason why the important resistance gene rym1 was eliminated along with resistant cultivars during breeding for resistance to BaYMV.     

Genomics-based high-resolution mapping of the BaMMV/BaYMV resistance gene rym11 in barley (Hordeum vulgare L.)

Soil-borne barley yellow mosaic virus disease, caused by different strains of Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), is one of the most important diseases of winter barley (Hordeum vulgare L.) in Europe and East Asia. The recessive resistance gene rym11 located in the centromeric region of chromosome 4HL is effective against all so far known strains of BaMMV and BaYMV in Germany. In order to isolate this gene, a high-resolution mapping population (10,204 meiotic events) has been constructed. F₂ plants were screened with co-dominant flanking markers and segmental recombinant inbred lines (RILs) were tested for resistance to BaMMV under growth chamber and field conditions. Tightly linked markers were developed by exploiting (1) publicly available barley EST sequences, (2) employing barley synteny to rice, Brachypodium distachyon and sorghum and (3) using next-generation sequencing data of barley. Using this approach, the genetic interval was efficiently narrowed down from the initial 10.72 % recombination to 0.074 % recombination. A marker co-segregating with rym11 was developed providing the basis for gene isolation and efficient marker-assisted selection.     

两株黄瓜花叶病毒卫星RNA的竞争与共存研究

通过体外转录方法 ,将大小分别为 36 9nt和 385nt的 2个黄瓜花叶病毒 (Cucumbermosaicvirus,CMV)的卫星RNAYi和Yns共同与不含卫星的辅助病毒株CMV_CNa进行假重组 ,接种CMV系统寄主心叶烟。结果表明 :在接种5d的接种叶上同时检测到卫星RNA_Yi和卫星RNA_Yns;在系统叶上 ,接种 5d和 10d亦可同时检测到 2株卫星 ;但接种 15d ,在系统叶组织中只检测到卫星RNA_Yi。再将接种 5d的接种叶扩大接种到几种不同的指示植物后 ,经dsRNA抽提 ,也只获得 1条与卫星RNA_Yi大小相符的条带。通过假重组病毒株中分别获得卫星RNA并测序 ,确定2个卫星RNA的序列没有变化。卫星RNA_Yns和Yi在辅助病毒CMV_CNa作用下 ,表现出明显的竞争性 ,它们在辅助病毒中不能形成稳定的共存关系。     

Rapid production of recombinant barley yellow mosaic virus resistant Hordeum vulgare lines by anther culture

Summary In a winter barley breeding program for barley yellow mosaic virus (BaYMV) resistance, the resistant six-rowed cv. Franka was crossed to 17 susceptible and two resistant cultivars, three of which were tworowed. A total of 233,445 anthers of the 19 hybrids and their parents were cultured and 831 green plants regenerated. Anther culture responsiveness varied greatly between genotypes, and the responsiveness of F₁hybrids was generally related to that of the more responsive (high) parent. On average, 3.6 green plants were recovered from 1,000 cultured anthers, almost twice as many as in comparable spring barley experiments. Androgenetic green plants were tested for their reaction to mechanical inoculation of BaYMV. In crosses of resistant parents, all the cross progeny proved to be resistant, which indicates that both parents carry identical gene(s). In the crosses of the resistant cv. Franka to susceptible parents, an average of 62% of the androgenetic progenies were resistant, which indicates that probably more than one gene is responsible for Franka's BaYMV-resistance. From the crosses of Franka to two-rowed cultivars, 282 androgenetic plants were produced. When 132 of these were tested for their reaction to BaYMV, 79 (59.8%) were resistant, and 30 of the latter were shown to be two-rowed recombinant lines. Doubled haploid lines are field-tested for other agronomic characters including grain yield and its components.     

Factors affecting aphid acquisition and transmission of soybean mosaic virus

Myzus persicae transmitted soybean mosaic virus (SMV) most efficiently following 30 or 60 s acquisition probes on infected plants. There were no differences in susceptibility to SMV infection of soybean plants 1 to 12 wk old, but symptoms were more severe in plants inoculated when young than when old. Soybeans inoculated between developmental stages R3 and R6 only showed yellowish-brown blotching on one or more leaves. There were no observable differences in the time of appearance or type of symptoms shown by soybean seedlings inoculated either by sap or by aphids; infected plants became acquisition hosts for aphids 5–6 days after inoculation. There was no change in the efficiency with which M. persicae transmitted SMV from source plants up to 18 wk after inoculation. M. persicae transmitted SMV from leaves of field-grown soybeans when plants were inoculated at developmental stages V6, R2, and R3 and tested as sources 57–74 days after inoculation but not from plants inoculated at R5 and tested as sources 14 to 32 days after inoculation. M. persicae acquired SMV from soybean buds, flowers, green bean pods, and unifoliolate, trifoliolate, and senescent leaves. Middle-aged and deformed leaves were better sources of the virus than buds, unfolding and old symptomless leaves. The results are being incorporated into a computer model of SMV epidemiology.     

Novel resistance to the Bymovirus BaMMV established by targeted mutagenesis of the PDIL5-1 susceptibility gene in barley

The Potyviridae are the largest family of plant-pathogenic viruses. Members of this family are the soil-borne bymoviruses barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV), which, upon infection of young winter barley seedlings in autumn, can cause yield losses as high as 50%. Resistance breeding plays a major role in coping with these pathogens. However, some viral strains have overcome the most widely used resistance. Thus, there is a need for novel sources of resistance. In ancient landraces and wild relatives of cultivated barley, alleles of the susceptibility factor PROTEIN DISULFIDE ISOMERASE LIKE 5–1 (PDIL5-1) were identified to confer resistance to all known strains of BaYMV and BaMMV. Although the gene is highly conserved throughout all eukaryotes, barley is thus far the only species for which PDIL5-1-based virus resistance has been reported. Whereas introgression by crossing to the European winter barley breeding pool is tedious, time-consuming and additionally associated with unwanted linkage drag, the present study exemplifies an approach to targeted mutagenesis of two barley cultivars employing CRISPR-associated endonuclease technology to induce site-directed mutations similar to those described for PDIL5-1 alleles that render certain landraces resistant. Homozygous primary mutants were produced in winter barley, and transgene-free homozygous M₂ mutants were produced in spring barley. A variety of mutants carrying novel PDIL5-1 alleles were mechanically inoculated with BaMMV, by which all frameshift mutations and certain in-frame mutations were demonstrated to confer resistance to this virus. Under greenhouse conditions, virus-resistant mutants showed no adverse effects in terms of growth and yield.     

Mechanical Transmission of Barley Mild Mosaic Virus (BaMMV) to Rye (Secale cereale L.)

After mechanical spraygun inoculation barley mild mosaic virus (BaMMV) was detected in barley cv. ‘Gerbel’ (control) as well as in rye cv. ‘Somro’, but not in wheat cv. ‘Kanzler’ and oat cv. ‘Alfred’. ELISA values of infected barley and rye were similar. Furthermore, infected rye plants developed symptoms typical for barley yellow mosaic virus infection.     

Concentration of virus and ultrastructural changes in oats at various stages of barley yellow dwarf virus infection

Enzyme-linked immunosorbent assay was used to monitor the concentration of barley yellow dwarf virus (BYDV) in roots and leaves of oats inoculated at the 1 - 2 leaf stage and at the 4 - 5 leaf stage, respectively. Virus was detectable 20 h after inoculation in the roots and after 48 h in the leaves of plants inoculated at the 1 - 2 leaf stage. The virus concentration reached a plateau in the roots after 7–8 days, and was 3–4 times higher than in the leaves. In plants inoculated at the 4 - 5 leaf stage virus was detectable in roots and leaves after 3 and 5 days, respectively. The concentration reached a maximum after 10 days in the roots and after 18 days in the leaves; the concentration in the leaves was 2–3 times higher than in the roots. Virus was readily detectable in seeds from infected plants, both fresh and old dried seeds. However, seed transmission could not be demonstrated. Virus-like particles were first observed in phloem cells of roots 4 days after inoculation, but no ultrastructural changes were detected at this stage. After 5–6 days, disintegrated nuclei and virus-induced vesicles were observed in many cells and abnormal production of callose was found after 10 days. Necrotic phloem cells were observed from day 13, shortly after the appearance of external symptoms.