Cloning and Phylogenetic Analysis of Coat Protein of Barley yellow dwarf virus Isolates from Different Regions of Pakistan

Barley yellow dwarf virus (BYDVs) is an emerging threat for wheat and may seriously threaten its production, especially as climate change may result in increased infestation by aphids, the insect vectors of the virus. To assess the possibility of using pathogen‐derived resistance against the virus, the genetic diversity of BYDVs originating from different wheat‐growing areas of Pakistan where its incidence has been higher was investigated. Wheat samples with suspected symptoms of BYDVs were screened for the presence of Barley yellow dwarf and Cereal yellow dwarf viruses (B/CYDVs) subgroup 1 (Barley yellow dwarf virus‐PAV, BYDV‐MAV, BYDV‐SGV) and subgroup II (BYDV‐RPV, CYDV‐RPV, BYDV‐GPV) by PCR using basic multiplex oligonucleotides designed on coat protein (CP) of the virus. Of 37 samples tested, 13 were positive for BYDV subgroup I and only one sample was positive for BYDV subgroup II. Samples positive for subgroup I were further tested by PCR, and results showed that 10 samples were positive for BYDV‐PAV and three for BYDV‐MAV. DNA sequences of CP region of nine isolates (BYDV‐PAV) were determined and compared with available sequences in databases. Sequence analysis showed that three isolates (from Fatehjang, Nowshera and Attock districts) had maximum identity (92.8–94.6%) to BYDV‐PAS, and six isolates (from Peshawar, Islamabad Swabi and Faisalabad districts) had maximum identity (99.3–99.7%) to BYDV‐PAV. Thus BYDV‐PAV species may be dominant in northern wheat‐growing areas of Pakistan. The conserved nature of the BYDVs suggests that pathogen‐derived resistance strategies targeting the coat protein of the virus are likely to provide protection under field conditions.     

Phylogenetic analysis of coat protein gene of BYDV-MAV strain from wheat

Barley yellow dwarf disease is globally the most important viral disease of wheat. The full-length nucleotide sequence of coat protein (CP) gene of 12 isolates revealed the presence of three distinct clusters. Pakistani isolate of MAV (MAV-PK) has maximum similarity of 99.23% with MAV isolate of Morocco and PAV-Australia following 99.22 and 99.22% with PAV-France. Similar degree of similarity was found in comparison of amino acid sequence. The finding of this study is that MAV-PK has similarity with both MAV-France and PAV-Australia, which is due to the reason that both MAV and PAV belong to the same group and both share maximum nucleotide homology. Low genetic diversity was found not only between MAV isolates but also between MAV and PAV isolates because phylogenetic analysis was done on the CP gene which is highly conserved region in genome of Barley yellow dwarf viruses (BYDVs). Divergence in MAV-PK was due to this recombination which is now most prevalent in Pakistan. MAV-PK has maximum similarity with MAV-Morocco followed by MAV-Sweden and MAV-Cz, which seems to indicate that Pakistani isolate of MAV evolved as the result of recombination between MAV isolates of the USA and PAV isolates of Australia and France. At the same time, recombination of MAV-CZ and MAV-Sweden also occur. This work can be successfully utilised in epidemiological studies of MAV isolate in Pakistan. Further analysis of variation level in these isolates will help scientists to formulate appropriate management strategies like incorporation of BdV 2 gene in wheat against BYDVs.     

Non‐cultivated grass hosts of yellow dwarf viruses in Ethiopia and their epidemiological consequences on cultivated cereals

The yellow dwarf (YD) disease complex epidemics in cultivated cereals grown in a specific period of the year mainly depend on the presence of potential reservoir alternative hosts harbouring both the viruses and the vectors over the off‐season and serve as a source of inoculum in subsequent cropping season, further spread being supported by efficient aphid vectors. As such, an extensive and intensive exploration to generate base line information on the identity and prevalence of YD viruses [barley yellow dwarf virus (BYDV)‐PAV, BYDV‐MAV and BYDV‐SGV; cereal yellow dwarf virus (CYDV)‐RPV; and maize yellow dwarf virus (MYDV)‐RMV] on wild annual and perennial grasses and forage cereals alternative hosts was conducted consecutively during 2013–2015 main‐ and short‐rainy seasons in cereals growing belts of Ethiopia. Random sampling was employed to collect the samples that were tested by the tissue blot immunoassay (TBIA) to identify the YDVs associated with the hosts using a battery of virus‐specific polyclonal antibodies. Of 13,604 samples analysed, YDVs were detected in 392 (2.9%) samples, which consisted of various wild grasses, forage cereals and three cultivated crops. YDVs were identified from at least 26 grass species and forage cereals, some of them are new records, and some are previously documented hosts. To our knowledge, this is the first report of YDV infection of Andropogon abyssinicus (FresenR.Br. ex Fresen.) (BYDV‐PAV), Avena abyssinica Hochst (BYDV‐PAV), Bromus pectinatus Thunb. (BYDV‐PAV and BYDV‐MAV), Eragrostis tef (Zuccagni) Trotter (BYDV‐PAV), Eragrostis sp. (BYDV‐PAV), Hyparrhenia anthistrioides Stapf. (BYDV‐PAV), Panicum coloratum L. (BYDV‐PAV), Polypogon monspeliensis (L.) Desf. (BYDV‐PAV), Setaria pumila (Poir.) Roem & Schult (BYDV‐PAV, BYDV‐SGV and MYDV‐RMV), Setaria australiensis (Scribn. & Merrill) Vickery (BYDV‐PAV, BYDV‐MAV and CYDV‐RPV) and Snowdenia polystachya (Fresen.) Pilg (BYDV‐PAV, BYDV‐MAV, BYDV‐SGV, CYDV‐RPV and MYDV‐RMV).     

The occurrence of barley yellow dwarf viruses in CIMMYT bread wheat nurseries and associated cereal crops during 1988–1990

Enzyme-linked immunosorbent assay (ELISA)-based surveys of the occurrence of five barley yellow dwarf virus (BYDV) serotypes (MAV, PAV and SGV in “Group 1”; RPV and RMV in “Group 2”) in CIMMYT bread wheat nurseries and other small grain crops in various locations world-wide were undertaken in 1988, 1989 and 1990. The objective was to investigate the relative occurrence of BYDV serotypes in areas relevant to CIMMYT cereal breeding programs. Overall, MAV and PAV serotypes predominated in the samples collected, though their relative frequencies depended on the location. SGV serotypes were uncommon in most locations. Group 2 serotypes occurred widely, but RMV serotypes were more common than RPV serotypes.     

大麦黄矮病毒GAV株系外壳蛋白基因的克隆和序列分析

The Barley yellow dwarf virus (BYDV) GAV isolate was preserved at the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences. The cDNA of BYDV GAV coat protein (CP) gene was amplified from the extracted RNA of BYDV GAV by using the polymerase chain reaction (PCR), and cloned into pGEM-7zf(+). Its complete nucleotide sequence has been determined by means of Sanger's dideoxy-mediated chain-termination method. The result showed that BYDV GAV CP gene has 600nt. It shares 97.5% and 96.5% identity with CP gene of BYDV MAV-PS1 in terms of nucleotide and amino acid sequences respectively.     

Occurrence of barley yellow dwarf virus (BYDV) isolates in different farmland habitats in western France and south-west England

The incidence and distribution of the three principal isolates of barley yellow dwarf virus (PAV, RPV and MAV) are described in winter cereal crops, cereal (stubble) regrowth and grasses from 11 sites in western France and south-west England during 1987 and 1988. Isolates were identified by indirect ‘sandwich’ ELISA using the monoclonal antibodies MAC91, MAC92 and MAFF2. More virus infection occurred in all localities and in most of the plant communities sampled, with the exception of perennial grass leys, in 1987 than in 1988. All three isolates were widespread. MAV was associated more with sites further north and PAV more with those further south. The geographical distribution of RPV was less variable. Underlying these trends, the relative abundance of isolates differed considerably between habitats. RPV always predominated in perennial grass leys and MAV in most cereal crops, although in the latter MAV was less prevalent in 1987 than in 1988. The greatest regional difference was found in stubble regrowth where PAV predominated in France but MAV predominated in England. Grasses from field margins (only sampled in England) were mainly infected by MAV and RPV. The implications of these findings for the epidemiology of BYDV are discussed, especially the roles of different host plant communities or habitats in the annual infection cycle of small-grain cereals.     

Serological and biological characterisation of isolates of barley yellow dwarf virus in Sweden in 1983

In 1983, cereal plants showing symptoms of barley yellow dwarf virus (BYDV), collected from 15 localities in Sweden, were tested for BYDV using enzyme-linked immunosorbent assay (ELISA). Antisera against two Swedish isolates of BYDV were used, a mild isolate (27/77) transmitted specifically by Sitobion avenae and a severe one (39/78) transmitted mainly by Rhopalosiphum padi. No virus was detected in 57 of 607 plants of oats and barley tested. Of the 550 plants in which virus was detected, 366 were infected with viruses similar to isolate 27/77, 116 with viruses similar to 39/78 and the remaining 68 reacted strongly with both antisera. When tested, the latter isolates were shown to be mixtures. Thirty-nine selected samples were also tested with antisera against the USA isolates RPV, RMV, MAV and PAV, and for transmission by S. avenae and R. padi. Twenty-six of these samples were transmitted specifically by S. avenae, one was transmitted only by R. padi and the remaining 12 samples were shown to be infected with a mixture of an S. avenae-specific isolate and one transmitted mainly by R. padi. Antisera against PAV and MAV each detected all isolates tested and the results were very similar to those with the antisera to the 39/78 and 27/77 isolates, respectively. None of the field isolates reacted with antisera against RMV or RPV. It was concluded that 1983 was an epidemic year for BYDV in Sweden and that isolates specifically transmitted by S. avenae predominated. Symptoms of infection by these isolates on oat plants ranged from mild to severe.     

Nucleotide sequence of beet western yellows virus RNA.

The nucleotide sequence of the genomic RNA (5641 nt) of beet western yellow virus (BWYV) isolated from lettuce has been determined and its genetic organization deduced. The sequence of the 3'terminal 2208 nt of RNA of a second BWYV isolate, obtained from sugarbeet, was also determined and was found to be very similar but not identical to that of the lettuce isolate. The complete sequence of BWYV RNA contains six long open reading frames (ORFs). A cluster of three of these ORFs, including the coat protein cistron, display extensive amino acid sequence homology with corresponding ORFs of a second luteovirus, the PAV isolate of barley yellow dwarf virus (BYDV) (1,2). The ORF corresponding to the putative viral RNA-dependant RNA polymerase, on the other hand, resembles that of southern bean mosaic virus. There is circumstantial evidence that expression of the BWYV RNA polymerase ORF may involve a translational frameshift mechanism. The ORF immediately following the coat protein cistron may be translated by in-frame readthrough of the coat protein cistron amber termination codon. Similar mechanisms have been proposed for expression of the corresponding ORFs of BYDV(PAV) (1).     

Distribution of Cereal Luteoviruses and Molecular Diversity of BYDV‐PAV Isolates in Central and Southern Iran: Proposal of a New Species in the Genus Luteovirus

During a survey , 148 wheat, 70 barley and 24 wild grass samples of plants showing symptoms of yellowing or reddening of leaves and general stunting were collected in central and southern provinces of Iran and tested for Barley yellow dwarf virus (BYDV) and Cereal yellow dwarf virus (CYDV) infection by enzyme‐linked immunosorbent assay (ELISA) and tissue print immunoassay (TPIA). The results showed the presence of the viruses in most regions. Positive reactions to BYDV‐PAV, BYDV‐MAV, CYDV‐RPV and BYDV‐SGV antisera were recorded. BYDV‐PAV was the most prevalent virus. The genetic diversity of BYDV‐PAV isolates in central and southern provinces was studied by analysing ORF1 (903 nt) and read through domain (RTD) (575 nt) of 13 and nine isolates respectively. Sequence analysis of RTD at nucleotide and amino acid levels revealed a high identity (91.8–97.2% and 91.4–100% respectively) between Iranian and other available isolates in the GenBank. However, in regards to ORF1, a high genetic diversity among Iranian and other known PAV isolates at both amino acid (2–16.9%) and nucleotide (4.1–16.5%) levels were detected. Based on phylogenetic analysis of ORF1, two major groups of BYDV‐PAV isolates were distinguished. The Iranian isolates were divided between the two clusters. Our results suggest that the occurrence of two genetically distinct groups of PAV isolates in central and southern Iran, from which according to the ICTV criteria for species demarcation in the family Luteoviridae, four isolates from central parts of the country, qualify for designation as new species.     

Biological Characterization of an Italian Isolate of Barley Yellow Dwarf Luteovirus from Barley

An isolate of BYDV (BYDV-OC), from barley in Northwest Italy with typical symptoms of yellowing and dwarfing, was transmitted by Rhopalosiphum padi, Sitobion fragariae. S. avenae, Metopolophium festucae, R. maidis and M. dirhodum , but not by Myzus persicae or Schizaphis graminum . It reacted in DAS-ELISA with monoclonal and polyclonal antisera to PAV, but not with antibodies to MAV, RPV and RMV. A polyclonal antiserum prepared to BYDV-OC did not react with MAV-like, RPV-like, or RMV-like isolates of BYDV in ELISA or in Western blots. The concentration of BYDVOC in Avena byzantina plants decreased from weeks 1 to 10 after inoculation, but the total virus content per plant increased up to weeks 7 to 8, following the increase of plant weight.     

Aerial flow of barley yellow dwarf viruses and of their vectors in western France

During the years 1989–1992 cereal aphids were caught alive in a low level (1.5 m high) suction trap operated in Le Rheu (Brittany, France) and tested for BYDV transmission. In most cases comparisons with data collected simultaneously by a 12.2 m suction trap operating in the same site resulted in good relationships between weekly catches at both heights. Results from transmission tests showed that: (i) the two main BYDV vectors were Rhopalosiphum padi and Metopolophium dirhodum during the years of experiment; (ii) PAV and MAV were the commonest viruses and RPV was relatively scarce; (iii) during spring M. dirhodum appeared to be the most important MAV vector and nearly as good a PAV vector as R. padi; (iv) during autumn R. padi was the only vector of the three viruses with mixed transmission allowing it to transmit also MAV probably by heteroencapsidation. To give an indication of the risk of infection, infectivity indices were calculated by multiplying the numbers of aphids caught by the 12.2 m suction trap by the proportion that were infective. These infectivity indices agreed with field records of primary infections.     

Some characteristics of monoclonal antibodies to a British M A V-like isolate of barley yellow dwarf virus

Rat monoclonal antibodies (MAbs) specific for a British F (MAV-like) isolate of barley yellow dwarf virus (BYDV) were produced and studied. In indirect ELISA using an antiserum to BYDV-F to trap virus from infected sap, the MAbs were shown to be specific for MAV-like isolates of BYDV from Britain, USA and Sweden but, in this test, they did not detect PAV-, RPV-, SGV- or RMV- like isolates of BYDV. In similar tests using homologous antisera to trap the viruses, the MAbs did not detect BYDV-PAV or -RPV or two other luteoviruses (potato leafroll and beet western yellows). One of the MAbs (MAFF 2) was partially purified from ascitic fluid, and used successfully in ELISA as a coating antibody and when conjugated to the enzyme alkaline phosphatase. Also, MAFF 2 successfully trapped BYDV-F particles when used to coat electron microscope grids. In indirect ELISA using three MAbs (MAFF 2, MAC 91 and MAC 92) it was possible to type the three major strain groups of BYDV, viz. MAV, PAV and RPV-like strains from Britain, USA and Europe.     

Positive and negative sense coat protein gene-mediated protection against poplar mosaic carlavirus in Nicotiana benthamiana

Plants of Nicotiana benthamiana were transformed with four constructs based on the coat protein gene of a poplar mosaic carlavirus (PMV) isolate from the UK. The four constructs were: the capsid protein coding sequence plus a portion of the adjacent sequence encoding a protein with a molecular mass of 14 kDa (CP14k); the capsid protein coding sequence in the positive sense (CPP); a mutated capsid protein coding sequence (CPM) and the capsid protein coding sequence in the negative sense (CPN). Forty-one regenerated plants, after selection for their kanamycin resistance, were confirmed by PCR to contain the appropriate sequences. Virus coat protein was detected in small amounts in 50% of the plants transformed with the CP14k or CPP constructs. Primary transformants showed a range of reactions to challenge with two isolates of PMV. These varied from apparently no infection in inoculated or in later-formed young leaves, as assessed by ELISA, to typical systemic symptoms associated with large amounts of serologically detected virus. There was no correlation between the level of protection against virus infection and the observed accumulation of transgene protein product. Plants were protected whether transformed with the coat protein coding sequence in the positive or negative sense.     

Detection of BMYV and BWYV isolates using monoclonal antibodies and radioactive RNA probes, and relationships among luteoviruses

In order to discriminate between sugar beet infecting beet mild yellowing virus (BMYV) and other isolates of beet western yellows virus (BWYV), monoclonal antibodies (MAbs) and radioactive riboprobes were used. With MAbs prepared against BMYV or potato leafroll virus (PLRV) no distinction could be established between BMYV and BWYV. Seven probes were synthesised from a lettuce infecting BWYV isolate; their localisation in the genome is known and they cover almost its entire length. Probes from the '3 part of the genome hybridised with all BMYV and BWYV isolates whereas those from the '5 part did not recognise BMYV isolates, showing that a divergent '5 region exists in the genomes of BMYV and BWYV. Probes also readily detected the virus in single aphids. The relevance of this finding for epidemiological studies is discussed.
MAbs and riboprobes were also tested against other luteoviruses (PLRV; barley yellow dwarf virus (BYDV) MAV, PAV and RPV strains). The serological relationship between BMYV and PLRV was confirmed and an epitope common to PLRV and BYDV-RPV was found. Using probes, PLRV and BYDV-RPV were found to share domains of homology with BWYV. BYDV-PAV showed weak homology with BWYV, while BYDV-MAV showed none.     

A single wheatgrass chromosome reduces the concentration of barley yellow dwarf virus in wheat

Seedlings of a series of addition or substitution lines of wheat containing different Thinopyrum intermedium chromosomes were inoculated with the PAV and RPV serotypes of barley yellow dwarf virus (BYDV). Reduced virus titres in infected plants were ascribed to a single pair of homoeologous group 7 chromosomes from Th. intermedium in the disomic addition lines L1 and TAF 2. The group 7 chromosome is associated with red pigmentation of coleoptiles, which was also observed in two lines ditelosomic for the α arm of the chromosome. However, when infected with the PAV serotype of BYDV, the ditelosomic lines had normal virus titres and it is concluded that potential determinants of BYDV resistance are located on the β arm of the Group 7 chromosome.     

Identification, Distribution and Vector Population Dynamics of Barley Yellow Dwarf Virus in Three Cereal-Producing Areas of Spain

ELISA-based surveys during 1985–87 in three major cereal-growing areas of Spain confirmed the presence of barley yellow dwarf virus (BYDV). Samples of small grain cereals and grasses with and without BYDV-like symptoms were collected in the central, southwestern, and northeastern Spain. Infections were found in all cereal species sampled and in some grasses. About 37 % of the samples collected in 1985 were infacted with isolates of the PAV serotype. Isolates of the RPV serotype were less common, and were detected only in samples from the central region at El Encin, Madrid. Only a single sample, collected from El Encin in 1987, was unequivocally diagnosed as containing an isolate of the MAV serotype. Aphid vector population dynamics was monitored during fall and winter of 1984–87 in the central region. Rhopalosiphum padi L. appeared to be the most abundant species during fall and winter months, infesting grasses and volunteer wheat. Other species present were Sitobion avenae (F.), Metopolophium dirhodum (Walker) and Rhopalosiphum maidis (Fitch). Both R. padi and S. avenae seem to be anholocyclic in the central region of Spain, and are able to remain and reproduce on wheat volunteers and grasses until the beginning of spring. S, avenae populations increase quickly on wheat volunteers in April, while populations of R. padi remain low. Therefore, spread of S. avenae-transmitted BYDV types to neighbouring cereal fields seem more likely to occur than spread of other types. Other possible virus reservoirs, such as maize, also need investigation for a better understanding of BYDV epidemiology in the central and other cercal-growing areas of Spain.     

RT-PCR-RFLP在大麦黄矮病毒检测中的应用

The universal primer of Luteovirus was designed and synthesized.An experimental system of RT PCR RFLP was developed in barley yellow dwarf virus (BYDVs).BYDVs can be distinguished qualitatively by RT PCR method.It was seen that different serotypes of BYDVs have critical different RFLP patters when the PCR producs were digested by restriction enzyme HinfI.The RFLP patterns of 7 isolates of PAV serotype were greatly different.These results indicate there existed sequence variations among different serotypes of BYDVs and vector phenotypes of PAV serotype.There is no difference between MAV serotypes in RFLP analysis.The slight distinction in the segment of coat protein gene of BYDVs can be revealed by RT PCR RFLP system.     

烟草花叶病毒蚕豆株系外壳蛋白基因及3‘端非编码区的克隆与序列分析

根据烟草花叶病毒Ｕ１株系序列,人工合成引物,用ＲＴ法合成了ｃＤＮＡ后,通过ＰＣＲ技术扩增并克隆了烟草花叶病毒蚕豆株系的外壳蛋白的基因和３‘端非编码区。ＤＮＡ序列测定结果表明,外壳蛋白基因全长４８０个碱基,编码１５８个氨基酸,３’端非编码区全长２０４个碱基,与ＴＭＶ－Ｕ１株系的同源率为１００％。