Molecular characterization of Italian Soil-borne cereal mosaic virus isolates

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1 11 Parasitica, 2005, 61(1):11-16 Molecular characterization of Italian Soil-borne cereal mosaic virus isolates C. RATTI 1, A. PISI 1, V. VALLEGA 2 & C. RUBIES AUTONELL 1 1 Department of Agroenvironmental Science and Technology, University of Bologna Viale Fanin 44, I Bologna, Italy. crubies@agrsci.unibo.it 2 C.R.A. Experimental Institute for Cereal Research Via Cassia 176, I Rome, Italy Summary Soil-borne cereal mosaic virus (SBCMV) was reported to affect wheat crops in Italy in Subsequently in many other European countries too. SBCMV causes a serious disease of wheat and growing resistant varieties represents the only economical means of control. Several wheat samples showing typical Furovirus symptoms were collected in Italy during the 2003 and 2004 seasons. Molecular characterization of the viral coat protein gene was carried out to study the degree of genetic diversity of these viruses over a relatively wide geographic area. Sequence data analysis revealed a high degree of genetic identity among Italian isolates, and between Italian and others SBCMV accessions. Alignment of deduced CP amino acids sequences showed an homology close to 100% between European isolates. Our results confirm a low degree of genetic diversity between European Furoviruses infecting wheat. Introduction A mosaic disease of winter wheat was first described in the USA in 1919 (MCKINNEY, 1925) and subsequently in many other wheat growing countries. The disease was worldwide associated to Soil-borne wheat mosaic virus (SBWMV), transmitted by the soil-borne plasmodiophorid Polymyxa graminis. Recently, sequence analysis (KOENIG et al., 1999; DIAO et al., 1999; KOENIG AND HUTH, 2000; YANG et al., 2001), has resulted in the International Committee on Taxonomy of Viruses approving a taxonomic proposal to divide American, European and Chinese isolates into different species within the Furovirus genus. The novel species have been denominated Soil-borne wheat mosaic virus, Soil-borne cereal mosaic virus (SBCMV) and Chinese wheat mosaic virus (CWMV). On the basis of the new classification, the wheat mosaic disease is believed to be generally caused by SBWMV in United States, Brazil and Canada, by SBCMV in Europe, and by CWMV in Asia. In Italy, SBCMV was first reported in 1960 on cultivars of common wheat (Triticum aestivum) grown in the Po valley, and subse-

2 12 quently also on durum wheat (Triticum durum) in the Central and Southern regions. SBCMV has been shown to cause grain yield reductions of about 50-70% on the most susceptible varieties in Italy (RUBIES AUTONELL et al., 2003). Due to the persistent nature of the virus within the soil, the only practical and economically viable means of control is the use of resistant varieties. The SBWMV, SBCMV and CWMV genomes consist of two plus-sense strands of RNA. RNA1 (about 7 kb) contains three open reading which encode for the RNA polymerase, the methyltransferase-helicase and the cellto-cell movement proteins (AN et al., 2003) The RNA2 (about 3.5 kb) encodes for the coat protein (19 kda), the 84 kda protein, involved on plasmodiophorid transmission of the virus (AN et al., 2003), the 19 kda protein, a pathogenicity determinant and a suppressor of RNA silencing (TE et al., 2005) and the 25 kda protein with unknown function (VERCHOT et al., 2001). Analyses of the coding sequences of movement and coat protein (CP) genes are often used as a tool for phylogenetic analysis between SBWMV, SBCMV and CWMV isolates (SHIRAKO et al., 2000; KOENIG et al., 2002). In this work, using CP nucleotide and amino acids sequence analyses, we completed a molecular characterization of Italian SBCMV isolates. Materials and methods Twenty four wheat samples showing typical Furovirus symptoms were collected in Northern, Central and Southern Italy during 2003 and 2004 seasons SsRNA was extracted and the nucleotide sequence of a viral coat protein gene was determined for each sample. Nucleotide and amino acids sequences alignment between sequences obtained and published RNA2 CP sequences of SBCMV, SBWMV and CWMV isolates was generated applying the Clustal V method (MegAlign, DNAStar). Phylogenetic distance trees were also constructed from a data set.

3 13 CWMV IT4 - Ostra - An IT5 - Ostra - An IT9 - Castelfranco Emilia - Mo IT61 - Rome IT16 - San Felice SP - Mo AJ I IT63 - Rome IT13 - Arda Samoggia - Pc IT11 - Parma - Pr ITC1 - Foggia - Fg AJ F NC_ F IT17 - San Pancrazio - Pr IT24 - Granarolo - Bo AJ UK AJ UK IT6 - Cadriano - Bo IT10 - Minerbio - Bo IT14 - Fiorenzuola d Arda - Pc IT15 - Crevalcore - Bo IT32 - Ozzano - Bo IT7 - Ozzano - Bo IT56 - Idice - Bo IT18 - Ozzano (Triticale) - Bo IT2 - Case nuove di Osimo - An IT3 - Ascoli Piceno - Ap IT45 - Ozzano - Bo IT1 - Osimo stazione - An SBCMV - AF G SBCMV IT57 - Idice - Bo AF G AF G AB Japan NC_ USA SBWMV D USA X USA AF G AB USA NC_ China AJ China AJ China Figure 1. Phylogenetic tree of CP amino acid sequences of SBCMV, SBWMV and CWMV isolates.

4 14 Table 1. CP nucleotide sequence similarity (upper triangle) and divergence (lower triangle) percentage of different SBCMV, SBWMV and CWMV isolates. AJ F NC_ F AF G AF G AF G AJ UK AJ UK AJ I IT1 - Osimo Stazione - An IT2 - Case Nuove di Osimo - An IT3 - Ascoli Piceno - Ap IT4 - Ostra - An IT5 - Ostra - An IT6 - Cadriano - Bo IT9 - Castelfranco Emilia - Mo IT10 - Minerbio - Bo IT11 - Parma - Pr IT13 - Arda Samoggia - Pc IT14 - Fiorenzuola d'arda - Pc IT15 - Crevalcore - Bo IT16 - San Felice SP - Mo IT17 - San Pancrazio - Pr IT18 - Ozzano (Triticale) - Bo IT45 - Ozzano - Bo IT32 - Ozzano - Bo IT7 - Ozzano - Bo IT24 - Granarolo - Bo IT56 - Idice - Bo IT57 - Idice - Bo IT61 - Rome IT63 - Rome ITC1 - Foggia - Fg AJ China NC_ China AJ China AB Japan D USA NC_ USA X USA AB USA AF G AJ F NC_ F AF G AF G AF G AJ UK AJ UK AJ I IT1 - Osimo Stazione - An IT2 - Case Nuove di Osimo - An IT3 - Ascoli Piceno - Ap IT4 - Ostra - An IT5 - Ostra - An IT6 - Cadriano - Bo IT9 - Castelfranco Emilia - Mo IT10 - Minerbio - Bo IT11 - Parma - Pr IT13 - Arda Samoggia - Pc IT14 - Fiorenzuola d'arda - Pc IT15 - Crevalcore - Bo IT16 - San Felice SP - Mo IT17 - San Pancrazio - Pr IT18 - Ozzano (Triticale) - Bo IT45 - Ozzano - Bo IT32 - Ozzano - Bo IT7 - Ozzano - Bo IT24 - Granarolo - Bo IT56 - Idice - Bo IT57 - Idice - Bo IT61 - Rome IT63 - Rome ITC1 - Foggia - Fg AJ China NC_ China AJ China AB Japan D USA NC_ USA X USA AB USA AF G

5 Results and discussion 15 The branching orders resolved by this analysis identified three major phylogenetic groups. In the first group, all the Italian isolates clustered closely together along with French, English and German isolates (SBCMV species). To the second group belong American isolates (SBWMV species), and the third ascribes the Asian isolates (CWMV) (Figure 1). Sequence data analysis revealed high degree of genetic identity among Italian isolates (96.6 to %), and between Italian and SBCMV accessions (88.7 to 99.6 %). Moreover, sequence divergences between 29.6 to 45.9 % were observed among Italian and SBWMV or CWMV isolates (Table 1). Amino acid sequence analysis of CP cistron revealed only few not specific exchanges and sequence identity between 97.7 and 100% among Italian and others European isolates from France, Germany and United Kingdom. In addition, amino acids sequences similarity between Italian and Asian or American isolates range from 71.0 to 81.2 %. Our results confirm a low degree of genetic diversity between European Furoviruses infecting wheat and highlight that all Italian isolates so far analyzed belong to the novel SBCMV species. References AN H., MELCHER U., DOSS P., PAYTON M., GUENZI A.C. & VERCHOT-LUBICZ J., Evidence that the 37 kda protein of Soil-borne wheat mosaic virus is a virus movement protein. Journal of General Virology 84: DIAO A.P., CHEN J.P., YE R., ZHENG T., YU S.Q., ANTONIW J.F. & ADAMS M.J., Complete sequence and genome properties of Chinese wheat mosaic virus, a new furovirus from China. Journal of General Virology 80: KOENIG R. & HUTH W., Soil-borne rye mosaic and European wheat mosaic virus: two names for a furovirus with variable genome properties which is widely distributed in several cereal crops in Europe. Archives of Virology 145: KOENIG R., PLEIJ C.W.A. & HUTH W., Molecular characterization of a new furovirus mainly infecting rye. Archives of Virology 144: KOENIG R., BERGSTROM G.C., GRAY S.M. & LOSS S., A New York isolate of Soil-borne wheat mosaic virus differs considerably from the Nebraska type strain in the nucleotide sequences of various coding regions but not in the deduced amino acid sequences. Archives of Virology 147: MCKINNEY H.H., A mosaic disease of winter wheat and winter rye. Bulletin No Washington, DC, USA: United States Department of Agriculture. RUBIES-AUTONELL C., VALLEGA V. & RATTI C., Reactions of cultivars of common wheat (Triticum aestivum L.) to Soil-borne wheat mosaic virus in northern Italy. Journal of Plant Disease and Protection 110: SHIRAKO Y., SUZUKI N. & FRENCH R.C., Similarity and divergence among viruses in the genus Furovirus. Virology 270: TE J., MELCHER U., HOWARD A. & VERCHOT-LUBICZ J., Soilborne wheat mosaic virus (SBWMV) 19K protein belongs to a class of cysteine rich proteins that suppress RNA silencing. Virology Journal 2:18.

6 16 VERCHOT J., DRISKEL B.A., ZHU Y., HUNGER R.M. & LITTLEFIELD L.J., Evidence that soilborne wheat mosaic virus moves long distance through the xylem in wheat. Protoplasma 218: YANG J., CHEN J., CHENG Y. & ADAMS M.J., Sequence analysis of a soil-borne wheat mosaic virus isolate from Italy shows that it is the same virus as European wheat mosaic virus and Soil-borne rye mosaic virus. Science in China 44: