Alstroemeria yellow spot virus (AYSV): a new orthotospovirus species within a growing Eurasian clade.

An orthotospovirus distinct from all other orthotospoviruses was isolated from naturally infected alstroemeria plants. Disease symptoms caused by this virus mainly consisted of yellow spots on the leaves based on which the name alstroemeria yellow spot virus (AYSV) was coined. A host range analysis was performed and a polyclonal antiserum was produced against purified AYSV ribonucleoproteins which only reacted with the homologous antigen and not with any other (established or tentative) orthotospovirus from a selection of American and Asian species. Upon thrips transmission assays the virus was successfully transmitted by a population of Thrips tabaci. The entire nucleotide sequence of the M and S RNA segments was elucidated by a conventional cloning and sequencing strategy, and contained 4797 respectively 2734 nucleotides (nt). Simultaneously, a next generation sequencing (NGS) approach (RNAseq) was employed and generated contigs covering the entire viral tripartite RNA genome. In addition to the M and S RNA nucleotide sequences, the L RNA (8865 nt) was obtained. The nucleocapsid (N) gene encoded by the S RNA of this virus consisted of 819 nucleotides with a deduced N protein of 272 amino acids and by comparative sequence alignments to other established orthotospovirus species showed highest homology (69.5% identity) to the N protein of polygonum ringspot virus. The data altogether support the proposal of AYSV as a new orthotospovirus species within a growing clade of orthotospoviruses that seem to share the Middle East basin as a region of origin.

Generic RT-PCR tests for detection and identification of tospoviruses.

A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5' upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a non-described tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories.

First Report of a 16SrIX Group ('Candidatus Phytoplasma phoenicium'-Related) Phytoplasma Associated with a Chrysanthemum Disease.

In November 2010, approximately 2% of the chrysanthemum (Chrysanthemum morifolium) cv. Paniz plants showed numerous small leaves in the top and stunting in a field collection of the National Research Center of Ornamental Plants in Mahallat, Iran. Next to these plants, some plants of the same collection showed leaves with a reddish and/or chlorotic discoloration around the veins. The observed symptoms were believed to represent infection by a phytoplasma and/or a viroid. Two plants with each type of the symptoms were individually analyzed. Using a total RNA extract from diseased leaves, RT-PCR with primer pairs targeting all known pospiviroids, including Chrysanthemum stunt viroid (CSVd) (3), were negative. Purified DNA was examined for the highly conserved phytoplasma 16S rRNA gene by nested-PCR using the universal primer sets P1/P7 and R16F2n/R16R2 (2). Fragments of 1.2 kb, obtained only from the plants with the small leaves and stunting, were sequenced and one of these sequences, which were identical, was deposited in GenBank (Accession No. KC176800). BLAST analysis of the chrysanthemum phytoplasma sequence exhibited 99% identity to Candidatus Phytoplasma phoenicium (Ca. P. phoenicium) species of the 16SrIX group. Subsequently, in silico RFLP analysis of the nested PCR product with the pDRAW32 program using AluI and TaqI restriction sites used for 16SrIX subgroups A, B, C, D, and E indicated that the 16SrIX chrysanthemum isolate belonged to subgroup D (1). Recently, based on GenBank sequences, several strains of Ca. P. phoenicium have been isolated and identified from diverse host species like Lactuca serriola, L. sativa, Solanum lycopersicon, Sonchus sp. [16SrIX-E], Carthamus tinctorius, and Prunus amygdalus [16SrIX-B] (4) in Iran. The vector species transmitting Ca. P. phoenicium to C. morifolium still needs to be identified. The leafhopper Neoaliturus fenestratus may be a potential vector as it is an often encountered efficient transmitter vector of 16SrIX group phytoplasmas in Iran (2). Next to the susceptibility of chrysanthemum to members of aster yellows, stolbur, and Ca. P. aurantifolia phytoplasma groups, this is, to our knowledge, the first report of a 16SrIX group member infecting chrysanthemum. The detection of this phytoplasma in chrysanthemum can form a new threat to this crop and other ornamentals in the Mahallat flower production region. References: (1) R. E. Davis et al. New Dis. Rep. 20:35, 2010. (2) M. Salehi et al. Plant Pathol. 56:669, 2007. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) M. G. Zamharir. Afr. J. Microbiol. Res. 5:6013, 2011.

First Report of Moroccan pepper virus on Lisianthus in Iran and Worldwide.

During the last decade, lisianthus (Eustoma grandiflorum) has been introduced in Iran in the horticultural cut-flower industry. This crop is currently produced in more than 800 small greenhouses on a surface of an estimated 0.8 km2 in the Pakdasht region (southeast of Teheran Province). Plants exhibiting virus-like symptoms were observed in several greenhouses in 2010. The infected plants produced yellow and necrotic spots on the leaves and became severely deformed because of a strong leaf curling and the production of shorter internodes. Flower breaking has not been observed in the blue flowering plants. Approximately 85% of the plants were apparently infected in the inspected greenhouses. Extracts of infected material inoculated onto some indicator plant species induced mosaic and leaf malformation on Nicotiana benthamiana, mottling on Capsicum annuum, necrotic lesions on Datura stramonium, chlorotic local spots on Vigna unguiculata, systemic necrotic spots on Emilia sonchifolia, chlorotic local spots on Cucumis sativus, and necrotic local lesions on Petunia hybrida. Back-inoculation of infected material on lisianthus seedlings resulted in several chlorotic spots on the inoculated leaves and a severe downward curling of the systemic infected leaves. No symptoms were observed after inoculation of Pisum sativum, Phaseolus vulgaris, Vicia faba, and Chrysanthemum spp. The virus could also be transferred from infected to healthy N. benthamiana plants by pricking leaves with a Pasteur pipette. Spherical tombusvirus-like particles of approximately 29 nm were found by transmission electron microscopy in leaf-dip and partially-purified preparations of infected N. benthamiana. Since Tomato bushy stunt virus (TBSV; genus Tombusvirus, family Tombusviridae) and Moroccan pepper virus (MPV) have been found in Iran, we studied by using ELISA whether our samples matched with TBSV. Since a negative response was obtained, two primers were designed on the basis of the available sequences of the coat protein in the GenBank (Accession No. EU27780) of an MPV isolate from soil in Fars Province, Iran. A reverse transcription (RT)-PCR of total RNA extract from infected lisianthus and N. benthamiana with the primers MPV-R (5'-TTACAACAATGTGGCATCATTG-3') and MPV-F (5'-ATGGCAATGGTAGTAAG AAAC-3') resulted in a DNA fragment of 1,176 bp. This fragment from N. benthamiana was cloned, sequenced (Accession No. HQ663881), and showed a 96% nucleotide and 99% amino acid identity with the coat protein of the soil isolate. MPV was originally found in pepper (1), tomato and pelargonium (4), pear tree (3), and surface water (2). To our knowledge, this is the first report of MPV on lisianthus in Iran and worldwide. This virus, which persists in soil, water, and plant debris, can be considered as a substantial threat for the lisianthus industry in Iran because farmers do not apply strict crop rotation or other sanitation measures. References: (1) H. U. Fischer and B. E. L. Lockhart. Phytopathology 67:1352, 1977. (2) R. Koenig and D.-E. Lesemann. Phytopathol. Z. 112:105, 1985. (3) M. Russo et al. J. Plant Pathol. 84:161, 2002. (4) H. J. Vetten and R. Koenig. 108:215, 1983.

Identification of Iris yellow spot virus on Leek (Allium porrum) in Sri Lanka.

Leek (Allium porrum) has become one of the major leafy vegetable export crops in Sri Lanka during last few years. This year-round crop is cultivated in open fields at elevations between 1,000 and 2,000 m on approximately 1,600 ha with a production of 27,000 t per year (2). In August 2009, straw-colored spots (2 to 3 mm in diameter), surrounded by a greenish halo and a necrotic area, resembling symptoms to those caused by Iris yellow spot virus (IYSV) were observed on leek in Kandapola in the Nuwara Eliya District. Additional thrips damage consisting of silver-colored spots was observed on all plants. IYSV (family Bunyaviridae, genus Tospovirus) was first described and characterized in the Netherlands in 1998 (1). During the last few years, this virus was reported from Australia, Brazil, Chile, France, Germany, Guatemala, India, Israel, New Zealand, Peru, Reunion Island, Serbia, South Africa, Spain, the United States (4), and Japan. Collected samples were initially analyzed for IYSV infections using antisera raised against nucleocapsid (N) protein in a double-antibody sandwich (DAS)-ELISA. The presence of IYSV was confirmed by a reverse transcription (RT)-PCR using IYSV-F-373 (5'CTGCGGGCTTCTCTGG3') and IYSV-R-779 (5'GACTCACCAATGTCTTCAAC3') primers that amplify a 400-bp fragment of the N gene. The entire N gene was not obtained when specific primers were used to retrieve the complete N gene. Four nucleotides of the reverse primer GAAAGATAGATATAATTAA (indicated in bold) did not match with sequence at the 3'end of the N gene. Hence, to obtain the remaining parts of the N gene, the primers UHP (5'CACTGGATCCTTTTGTTTTTGTTTTTTG3') and Asian Termini (5'CCCGGATCCAGAGCAATCGAGGY3') (3) were combined with IYSV-F and IYSV-R. The obtained amplicons were cloned into pGEM-T easy vector and sequenced. The N gene sequence has been deposited at the NCBI/GenBank (Accession No. GU901211). The deduced N protein sequence(s) were compared with other IYSV N protein sequences available in the GenBank and showed a 92% protein identity with the Brazilian strain (IYSV-BR) and 97% with the Dutch strain (IYSV-NL) with Accession Nos. AAF04199 and AAB61923, respectively. No data on the thrips vector species or on the disease incidence have been collected. The presence of IYSV in Sri Lanka can potentially be considered as a threat for the export of leek. To our knowledge, this is the first report that IYSV occurs in Sri Lanka. References: (1) I. Cortêz et al. Phytopathology 88:1276, 1998. (2) Department of Census and Statistics Sri Lanka. Retrieved from http://www.statistics.gov.lk , 2009. (3) A. Hassani-Mehraban et al. Phytopathology 95:852, 2005. (4) H. R. Pappu et al. Virus Res. 141:219, 2009.

Molecular and biological comparison of two Tomato yellow ring virus (TYRV) isolates: challenging the Tospovirus species concept.

Two strains of Tomato yellow ring virus (TYRV, genus Tospovirus), one from tomato (referred to as TYRV-t) and the other from soybean and potato (denoted TYRV-s), collected from different geographical regions in Iran, were compared. Their genomic S RNA segments differed in size by 55 nucleotides. Comparison of the S RNA intergenic regions revealed the absence of a stretch of 115 nucleotides within the S RNA segment of TYRV-s and, conversely, of 56 nts in that of TYRV-t, apparently a stable genetic difference as it was also found in another isolate of TYRV-s collected from potato. Sequence comparison of the N protein ORFs revealed an identity of 92% between the N proteins of both strains, and the observed strong cross-reaction of TYRV-s in DAS-ELISA with a polyclonal antiserum directed against the TYRV-t N protein confirmed this high identity. Host range analysis revealed several differences, e.g. TYRV-s, but not TYRV-t, being able to systemically infect Nicotiana species, and TYRV-s being localised in tomato. The observed molecular and biological differences of both viruses call into question the currently used criteria for Tospovirus species demarcation.