Creation of a new genus in the family Secoviridae substantiated by sequence variation of newly identified strawberry latent ringspot virus isolates.

To obtain insight into the sequence diversity of strawberry latent ringspot virus (SLRSV), isolates from collections and diagnostic samples were sequenced by high-throughput sequencing. For five SLRSV isolates, the complete genome sequences were determined, and for 18 other isolates nearly complete genome sequences were determined. The sequence data were analysed in relation to sequences of SLRSV and related virus isolates available in the NCBI GenBank database. The genome sequences were annotated, and sequences of the protease-polymerase (Pro-Pol) region and coat proteins (CPs) (large and small CP together) were used for phylogenetic analysis. The amino acid sequences of the Pro-Pol region were very similar, whereas the nucleotide sequences of this region were more variable. The amino acid sequences of the CPs were less similar, which was corroborated by the results of a serological comparison performed using antisera raised against different isolates of SLRSV. Based on these results, we propose that SLRSV and related unassigned viruses be assigned to a new genus within the family Secoviridae, named "Stralarivirus". Based on the phylogenetic analysis, this genus should include at least three viruses, i.e., SLRSV-A, SLRSV-B and lychnis mottle virus. The newly generated sequence data provide a basis for designing molecular tests to screen for SLRSV.

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.

Development and validation of a real-time RT-PCR assay for generic detection of pospiviroids.

In many countries phytosanitary regulations apply to Potato spindle tuber viroid, because it can cause serious diseases in potato and tomato crops. Other pospiviroids, some of which are distributed widely in ornamental crops, can cause similar diseases. Consequently, there is a need for a reliable and cost-effective generic testing method. An assay was developed that detects all known species of the genus Pospiviroid, using real-time RT-PCR based on TaqMan technology. This GenPospi assay consists of two reactions running in parallel, the first targeting all pospiviroids, except Columnea latent viroid, the second specifically targeting the latter viroid (already published). To monitor the RNA extraction a nad5 internal control was included. Method validation on tomato leaves showed that the GenPospi assay detects all pospiviroids up to a relative infection rate of 0.13% (equals 770 times dilution). The assay was specific because no cross reactivity was observed with other viroids, viruses or nucleic acid from plant hosts. Repeatability and reproducibility were 100% and the assay appeared robust in an inter-laboratory comparison. The GenPospi assay has been shown to be a suitable tool for large-scale screening for all known pospiviroids. Although it has been validated for tomato leaves it can potentially be used for any crop.

First Report of Iresine viroid 1 in Celosia plumosa in the Netherlands.

In 2008, in the framework of surveying for pospiviroids, nine symptomless clones of Celosia plumosa (Voss) Burv. (Amaranthaceae) from a Dutch breeding company were tested by reverse transcription (RT)-PCR with primer sets Pospi1-RE/FW and Vid-RE/FW (4). In four samples, amplicons of 227 nt were obtained with primers Pospi1-RE/FW. Sequencing of the amplicons showed identities of more than 99% to the partial sequence of Iresine viroid 1 (IrVd-1) from Alternanthera sessilis, NCBI GenBank Accession No DQ846886 (2). Subsequently, a set of primers was designed to amplify the complete viroid genome, i.e., IrVd-FW1 5'-GCG GAA GAA ACA GGA GCT CGW CT-3' and IrVd-RE1 5'-CGC GWG GAG TTC TCC GGT CTT TA-3' - identical to nt 168 to 190 and 145 to 167 of the complete IrVd-1 sequences in the NCBI GenBank (Nos. DQ094293, DQ094294, NC_003613, and X95734). One isolate from C. plumosa was amplified with this primer pair and amplicons were cloned into the pGEM-T Easy Vector System II. Sequencing of one individual cDNA clone (GenBank Accession No. GU911350) revealed a genome size of 370 nt and 98.1% sequence identity to the IrVd-1 isolate from Vinca major, GenBank Accession No. DQ094293 (1). Hence, the viroid was identified as IrVd-1. The isolate from C. plumosa was also mechanically inoculated to 10 healthy plants of C. plumosa, chrysanthemum (Chrysanthemum × morifolium) cv. White Delianne, potato (Solanum tuberosum) cv. Nicola, and tomato (Solanum lycopersicum) cv. Moneymaker. No symptoms were observed over a 6-week period, and RT-PCR with primers Pospi1-RE/FW on bulked samples of five plants per species only identified IrVd-1 in both samples of C. plumosa. For tomato, these results confirm those of Spieker (3). Therefore, in contrast to the other pospiviroids, it seems unlikely that IrVd-1 poses a threat to potato and tomato. References: (1) X. Nie et al. Can. J. Plant Pathol. 27:592, 2005. (2) R. P. Singh et al. Plant Dis. 90:1457, 2006. (3) R. L. Spieker. J. Gen. Virol. 77:2631, 1996. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

First Report of Tomato apical stunt viroid in the Symptomless Hosts Lycianthes rantonnetii and Streptosolen jamesonii in the Netherlands.

In 2009, in the framework of surveying for pospiviroids, samples of various ornamental plants from the Netherlands were tested by reverse transcription (RT)-PCR with the primer pairs Pospi1-RE/FW and Vid-RE/FW (2). With primer pair Pospi1-RE/FW, amplicons of the expected size were obtained in two samples of symptomless plants of Lycianthes rantonnetii and Streptosolen jamesonii. Sequencing of the amplicons, which were expected to correspond with partial pospiviroid genomes, showed identities of 100 and 98% to the sequence of Tomato apical stunt viroid (TASVd), NCBI GenBank Accession No. AM777161 (3). For the amplification of the complete viroid genomes, RT-PCRs were performed with primer pair CEVd-FW/RE (1). Sequencing of these amplicons yielded sequences of 364 nt and identities to TASVd AM777161 of 100 and 98.1%, respectively. Therefore, both isolates were identified as TASVd. The sequence variant from S. jamesonii was submitted to the NCBI GenBank as No. GU911351. In addition, both isolates were mechanically inoculated to four tomato plants (Solanum lycopersicum) of cv. Moneymaker. All inoculated plants developed chlorosis and growth reduction after 4 weeks and TASVd infections were confirmed in a bulked sample by RT-PCR with primer pair CEVd-FW/RE after 6 weeks. Hence, two more ornamental host plant species have been identified that may act as symptomless sources of pospiviroid inoculum. References: (1) N. Önelge. Turk. J. Agric. For. 21:419, 1997. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (3) J. Th. J. Verhoeven et al. Plant Dis. 92:973, 2008.

First Report of Tomato yellow leaf curl virus in Tomato in the Netherlands.

Tomato yellow leaf curl virus (TYLCV) is an economically important virus with tomato (Solanum lycopersicum L.) as its main host. The virus is widely distributed in subtropical areas and is transmitted by the tobacco whitefly (Bemisia tabaci) in a persistent manner. TYLCV has a quarantine status (IIAII) in the European Union (EU directive 2000/29/EC). It was not previously recorded in the Netherlands. In September 2007, symptoms were observed in tomato crops in a few greenhouses located in close proximity from each other in the western part of the Netherlands. Infected plants showed TYLCV-like symptoms, i.e., stunting, leaf curl, and marginal and interveinal chlorosis. Similar symptoms were evoked after grafting symptomatic tips onto healthy tomato seedlings, whereas no viruses were transmitted by mechanical inoculation to herbaceous test plants. Extracted DNA from symptomatic leaves was used in PCR with two sets of primers for universal detection of begomoviruses (1,2). Analysis of the overlapping amplified products revealed the highest identity to isolate TYLCV-Alm (GenBank Accession No. AJ489258) from Almeria, Spain. To amplify the remaining 60% of the virus genome, three additional primer sets were designed: TYLCV965F 5'-GGCAGCCAAGTACGAGAACC-3' and TYLCV1736R 5'-CCACTATCTTCCTCTGCAATCC-3'; TYLCV1598F 5'-TACTTGCGAACAGTGGCTCG-3' and TYLCV2282R 5'-TCCAAATCGATGGCAGATCAG-3'; TYLCV2229F 5'-ATGCGTCGTTGGCAGATTG-3' and TYLCV68R 5'-CAGTGACGTCTGTGGAACCCT-3'. Analysis of the five overlapping PCR products of one isolate revealed a total virus genome of 2,781 nucleotides. The complete sequence of the Netherlands Isolate (GenBank Accession No. FJ439569) showed 99.3% nucleotide identity to isolate TYLCV-Alm (AJ489258), and therefore, the virus was identified as TYLCV-Alm. After the initial identification, a survey was conducted in all tomato crops in a surrounding area of approximately 40 km2. TYLCV was found in 19 of 27 cultivations. The identity of one isolate per cultivation was confirmed by sequence analysis of the products obtained with the Wyatt and Brown primers (2) occasionally in combination with the Deng primers (with 99.1 to 100% and 99.2 to 100% nucleotide identity to the Netherlands isolate [FJ439569], respectively) (1). As many as 25 symptomatic plants were recorded per greenhouse. A subsequent survey of 34 randomly selected tomato growers in other areas of the country revealed no further infections. Results of the sequence analyses and surveys suggested that the outbreak resulted from a single introduction of the virus, whereas the insect vector B. tabaci accounted for local spread. Measures taken to eliminate the virus included the removal and subsequent destruction of infected tomato plants as well as eradication of B. tabaci. No TYLCV infections were found during surveys in 2008, and therefore, it is believed that the virus was eradicated effectively. References: (1) D. Deng et al. Ann. Appl. Biol. 125:327, 1994. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

First Report of Potato spindle tuber viroid in Cape Gooseberry (Physalis peruviana) from Turkey and Germany.

Since the recent identification of Potato spindle tuber viroid (PSTVd) in vegetatively propagated ornamental plant species (4), many growers have asked to have their mother plants tested for this viroid. In December of 2007, a grower from Turkey submitted cuttings of cape gooseberry (Physalis peruviana) to be tested for PSTVd. Initial testing by real-time reverse transcription (RT)-PCR according to Boonham et al. (1) indicated the presence of either Mexican papita viroid, PSTVd, or Tomato chlorotic dwarf viroid in four samples. To identify the viroid(s) present, isolated RNA from these samples was used for RT-PCR (2), and products of the expected full genome size for the three viroids were amplified from each sample. One of the PCR products was sequenced (GenBank Accession No. EU862230) and analysis of the 357 nt sequence indicated it was most related to PSTVd sequences belonging to the so-called 'Oceanian' strain of the viroid (3), with 99.7% identity to GenBank Accession No. AY962324. Therefore, the viroid was identified as PSTVd. Pathogenicity of this PSTVd genotype was demonstrated when 4 weeks after mechanical inoculation with sap extracts seedlings of tomato cv. Money-maker showed the expected viroid symptoms of chlorosis and stunting, and the presence of the viroid in these plants was confirmed by RT-PCR (2). In March of 2008, by use of RT-PCR (2) and sequencing of the PCR product (GenBank Accession No. EU862231), PSTVd was identified in young seedlings of P. peruviana from a German grower. The German isolate differed at only three nucleotide positions from the Turkish isolate. The identification of PSTVd in young seedlings indicates that seeds had been source of infection, whereas in the case of the PSTVd infected cuttings from Turkey, the infection originated from infected mother plants. To our knowledge, these are the first reports of PSTVd in P. peruviana. Although infected P. peruviana plants did not show symptoms, they might act as sources of inoculum for crops like potato and tomato, which may suffer serious damage. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) J. Th. J. Verhoeven et al. Plant Pathol. 57:399, 2008.