D. Hariri

Biological and sequence analysis of a novel European isolate of Barley mild mosaic virus that overcomes the barley rym5 resistance gene

Summary.A Barley mild mosaic virus (BaMMV) isolate from France (BaMMV-Sil) capable of overcoming rym5-controlled resistance was inoculated to barley genotypes carrying various genes for resistance to the barley mosaic viruses. BaMMV-Sil was unable to infect genotypes carrying rym1, rym4, rym8, rym9, or rym11 but genotypes carrying rym3, rym5, rym6 or no known bymovirus resistance gene were susceptible. Plants carrying rym7 or rym10 showed partial resistance with delayed virus accumulation. The two genomic RNAs of BaMMV-Sil were sequenced and compared to published sequences and those of a further common strain isolate from the UK. Four amino acid differences were observed between BaMMV-Sil and European common strain isolates in the polypeptide encoded by RNA1, the RNA species which determines pathogenicity on the rym5 genotypes. Only two of these differences are likely to be functionally important (His rather than Gln at position1217 in the VPg cistron; His rather than Asp at position 1776 in the NIb cistron). Comparisons with related viruses in the genera Bymovirus and Potyvirus suggest that the change in the VPg, which occurs within a motif conserved amongst all viruses within the family Potyviridae, is the more likely cause of rym5 resistance-breaking.

Characterization of a New Barley Mild Mosaic Virus Pathotype in France

In March 2002 in a French field, severe mosaic symptoms appeared on plants of the barley cultivar Tokyo with the rym5 locus controlling resistance to all European strains of barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV). Electron microscopic examination revealed that the disease symptoms were associated with the presence of flexuous particles which resemble bymoviruses. From these observations and after enzyme-linked immunosorbent assay analysis it was first determined that the plants could be infected by BaMMV and BaYMV. Mechanical transmission of these viruses to the barley cultivar Magie susceptible to both viruses was only possible for BaMMV. This new pathotype (BaMMV-Sil) from Sillery (Marne Department, 51, France), in contrast to another mechanically transmitted French BaMMV isolate (BaMMV-MF), could be transmitted mechanically to two barley cultivars (Tokyo, Misato Golden), Arachis hypogaea, Datura stramonium and Lactuca sativa. BaMMV-Sil was indistinguishable from three BaMMV isolates from Germany (G), Japan (Ka1) and France (PF) by monoclonal antibodies in ELISA while the Japanese isolate (Na1) and BaMMV-MF were distinguishable from all. The sequence of the 3′-terminal region of BaMMV-Sil RNA1 was determined. Comparison with previously published sequence data of capsid proteins indicated that BaMMV-Sil was closely related to BaMMV-Ka1, BaMMV-G and another German isolate (BaMMV-ASL1). Resistance-breaking BaMMV strains able to infect cultivars carrying the rym5 locus have also been described in Japan (BaMMV-Na1) and Korea (BaMMV-Kor). No specific amino acid differences were detected between the capsid proteins of BaMMV-Sil, BaMMV-Na1, BaMMV-Kor and those BaMMV isolates that do not overcome the rym5 resistance gene. These results indicate that BaMMV-Sil is a new pathotype of BaMMV in France and suggests that the capsid protein is not the determining factor of the pathogenicity towards the resistance gene rym5.

A new furovirus infecting barley in France closely related to the Japanese soil-borne wheat mosaic virus

In April 2001, stunted barley plants bearing mosaic symptoms were observed in a field in France (Marne Department, 51). Rod-shaped and flexuous particles were visualized by electron microscopy and positive serological reactions were detected by ELISA with Barley yellow mosaic virus (BaYMV) and Soil-borne cereal mosaic virus (SBCMV) polyclonal antisera. The tubular virus which was soil transmissible to barley cv. Esterel was separated from BaYMV by serial mechanical inoculations to barley cv. Esterel. This furo-like virus, in contrast to a French isolate of SBCMV, could be transmitted to Hordeum vulgare, Avena sativa, Beta vulgaris and Datura stramonium. RT-PCR was used to amplify the 3′-terminal 1500 nucleotides of RNA1 and the almost complete sequence of RNA2. Nucleotide and amino acid sequence analyses revealed that the French virus infecting barley is closely related to a Japanese isolate of Soil-borne wheat mosaic virus (SBWMV-JT) which was originally isolated from barley. This French isolate was named SBWMV-Mar. The 3′ UTRs of both RNAs can be folded into tRNA-like structures which are preceded by a predicted upstream pseudoknot domain with seven and four pseudoknots for RNA1 and RNA2, respectively. The four pseudoknots strongly conserved in RNAs 1 and 2 of SBWMV-Mar show strong similarities to those described earlier in SBWMV RNA2 and were also found in the 3′ UTR of Oat golden stripe virus RNAs 1 and 2 and Chinese wheat mosaic virus RNA2. Sequence analyses revealed that the RNAs 2 of SBWMV-Mar and -JT are likely to be the product of a recombination event between the 3′ UTRs of the RNAs 2 of SBWMV and SBCMV. This is the first report of the occurrence of an isolate closely related to SBWMV-JT outside of Japan.

Incidence of Soil-borne Wheat Mosaic Virus in Mixtures of Susceptible and Resistant Wheat Cultivars

The multiplication of Soil-borne wheat mosaic virus (SBWMV) was studied in mixtures of two winter wheat (Triticum aestivum) cultivars, one susceptible (Soissons) and the other resistant (Trémie). Two seed mixtures of susceptible and resistant varieties in ratios of 1 : 1 and 1 : 3 and their component pure stands, i.e. each variety grown separately, were grown in a field infected with SBWMV. The presence of the virus was detected using DAS-ELISA from January to May. The resistant cultivar Trémie showed no foliar symptoms nor could the virus be detected in the leaves or roots. In May, about 88% of plants of susceptible cultivar Soissons grown in pure stands were infected. At this time, the disease reduction relative to pure stands was 32.2% in the 1 : 1 mixture and 39.8% in the 1 : 3 mixture. Optical density (OD) values from ELISA of the infected plants in the two mixtures were consistently lower than that of the infected plants in cultivar Soissons in pure stands. The ELISA index (EI) calculated using three scales of OD values was 65.5% in the susceptible cultivar in pure stands. The value for this index was 19.1% in the 1 : 1 mixture and 7.9% in the 1 : 3 mixture. The plants of the resistant cultivar Trémie infected in the same field and transferred in January to a growth cabinet at 15 °C multiplied the virus and produced viruliferous zoospores. These results show that the resistant cultivar Trémie plays a role in disease reduction in the cultivar mixtures in field conditions. Possible reasons for this are discussed.

Comparison and differentiation of Wheat Yellow Mosaic Virus(WYMV), Wheat Spindle Streak Mosaic Virus (WSSMV) and Barley Yellow Mosaic Virus (BaYMV) isolates using WYMV monoclonal antibodies

Twelve monoclonal antibodies (MAbs) were obtained by immunizing mice with a French isolate (F1) of wheat yellow mosaic virus (WYMV). Three of these (3D12, 2C1, 6C3) belong to the IgM class and the nine others to the IgG class (3D8, 3H1, 2B8, 1F2, 3C10, 4F12, 3H9, 1G5, 54). In antigen-coated plate (ACP) ELISA and indirect double antibody sandwich (IDAS) ELISA, all MAbs recognize the WYMV (F1) both in the form of purified particles and in wheat leaf extract. The analysis of numerous French isolates of WYMV shows a variable reactivity with MAbs 3D8, 3H1, 2B8, 3C10, 3H9 and 1G5 in IDAS — and ACP-ELISA. The Japanese isolate of WYMV and United States isolates of wheat spindle streak mosaic virus (WSSMV) were detected in IDAS- and ACP-ELISA by ten of the MAbs tested showing that the wheat bymoviruses originating from the three locations share a high epitopic homology. French isolates of barley yellow mosaic virus (BaYMV; pathotypes 1 and 2) were only detected in ACP-ELISA with MAbs 6C3, 3D8, 3H1 and 2B8 whereas the two Japanese strains (I-1, II-1) of MaYMV were recognized with these and also with that of 3C10. In IDAS-ELISA, the two Japanese strains were clearly detected by MAbs, 6C3, 3D8, 3H1, 1F2, 3C10 and 1G5 and the British and Belgian (pathotype 2) isolates only by that of 6C3. Only the Japanese strain of BaYMV, 1-1 could be detected with MAb 3H9 in this ELISA system.

Characterisation of BaYMV and BaMMV pathotypes in France.

The reaction of thirty-four barley cultivars from European and Asiatic origin was analysed in six soils infected with barley yellow mosaic virus complex (BaYMV, BaMMV). These soils were selected from 16 sites for their differences in cultivar response. Amongst the six cultivars carrying the ym4 gene (Esterel, Express, Labéa, Majestic, Réjane, Vanoise), only cv Majestic was infected at one site with BaYMV and BaMMV. Concerning BaYMV, three cultivars were infected on all the soils and 19 on none of them. Twelve cultivars were differentially infected depending on the soil. In the case of BaMMV, four cultivars were infected on all the soils and 19 on none of them. Eleven cultivars were differentially infected depending on the soil. ELISA tests revealed the presence, in these soils, of variants of BaYMV and BaMMV that were able to overcome at least seven of the 12 known resistance genes (ym3, ym4, ym6, ym8, ym9, ym10, ym11) and the resistance of three varieties (Tosan Kawa 73, OU1 and Taihoku A) in which the genetic basis is unknown. Amplification by RT-PCR of the N-terminal region for three of BaYMV variants was performed. Nucleotide and amino acid sequences were determined and compared with the corresponding sequence of a common strain of BaYMV-G. A few nucleotide differences were detected between all the French isolates, but there were no strain specific amino acid differences.

Aubian wheat mosaic virus, a new soil-borne wheat virus emerging in France

The properties of Aubian wheat mosaic virus (AWMV), a new soil-borne wheat virus in France, were investigated. Symptoms include foliar mosaic and severe stunting of winter wheat. The vector of the disease is unknown but the plants infected carry Polymyxa graminis in the roots. AWMV was transmitted mechanically to wheat and to two dicotyledoneous species: Lactuca sativa and Vicia faba. This virus was transmitted by seed to three winter wheat cultivars tested. Purified preparations contained rod-shaped particles with a variable length of 150–700 nm. Certain particles are very long and appear flexible. Antiserum raised against AWMV reacted specifically with AWMV in both indirect and direct enzyme-linked immunosorbent assays (ELISA). The incidence of AWMV in 26 winter wheat cultivars was investigated in the field during the growing season of 1999–2000. AWMV was detected in roots and shoots of all cultivars regardless of the symptoms. Twelve virus species belonging to the genera Benyvirus, Bymovirus, Furovirus, Pecluvirus and Pomovirus did not react with the AWMV antisera. A new tubular virus described in winter wheat in Bedfordshire in England reacted strongly with AWMV in ELISA. It is concluded that AWMV and probably the Bedford-virus constitute a previously undescribed tubular virus biologically and serologically distinct from other soil-borne viruses of wheat.