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One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control. To eliminate false negative results due to variation in the viral genome sequences, for each target virus two assays were developed on distinct conserved genomic regions. Specificity, PCR efficiency and compatibility of primers and probes were tested using gBlock constructions. Diagnostic samples were used to evaluate the strategy. High Throughput Sequencing of a set of the diagnostic samples, further verified the presence or absence of the viruses in the RNA samples and sequence variations in the target genes. This interchangeable multiplex panel strategy may be a valuable tool for the detection of viruses in certification, surveys and virus diagnostics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

The complete genome sequence of chrysanthemum stem necrosis virus (CSNV) was determined using Roche 454 next-generation sequencing. CSNV is a tentative member of the genus Tospovirus within the family Bunyaviridae, whose members are arthropod-borne. This is the first report of the entire RNA genome sequence of a CSNV isolate. The large RNA of CSNV is 8955 nucleotides (nt) in size and contains a single open reading frame of 8625 nt in the antisense arrangement, coding for the putative RNA-dependent RNA polymerase (L protein) of 2874 aa with a predicted Mr of 331 kDa. Two untranslated regions of 397 and 33 nt are present at the 5' and 3' termini, respectively. The medium (M) and small (S) RNAs are 4830 and 2947 nt in size, respectively, and show 99 % identity to the corresponding genomic segments of previously partially characterized CSNV genomes. Protein sequences for the precursor of the Gn/Gc proteins, N and NSs, are identical in length in all of the analysed CSNV isolates.

A new virus was isolated from a tomato plant from the state of Sinaloa in Mexico. This plant showed symptoms locally known as 'marchitez disease': severe leaf necrosis, beginning at the base of the leaflets, and necrotic rings on the fruits. A virus was isolated from the infected plant consisting of isometric particles with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7221 (RNA1) and 4898 nts (RNA2). The viral capsid contains three coat proteins of 35, 26 and 24 kDa, respectively. The abovementioned characteristics: symptoms, morphology, number and size of coat proteins, and number of RNAs are similar to those of the previously described tomato torrado virus (ToTV). Sequence analysis of the entire viral genome shows that this new virus is related to, but distinct from, ToTV and that these members of two obviously new virus species belong to the recently proposed plant virus genus Torradovirus. For this new virus, the name tomato marchitez virus (ToMarV) is proposed.

A new virus was isolated from tomato plants from the Murcia region in Spain which showed symptoms of 'torrado disease'; very distinct necrotic, almost burn-like symptoms on leaves of infected plants. The virus particles are isometric with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7793 (RNA1) and 5389 nts (RNA2). RNA1 contains one open reading frame (ORF) encoding a predicted polyprotein of 241 kDa that shows conserved regions with motifs typical for a protease-cofactor, a helicase, a protease and an RNA-dependent RNA polymerase. RNA2 contains two, partially overlapping ORFs potentially encoding proteins of 20 and 134 kDa. These viral RNAs are encapsidated by three proteins with estimated sizes of 35, 26 and 23 kDa. Direct protein sequencing mapped these coat proteins to ORF2 on RNA2. Phylogenetic analyses of nucleotide and derived amino acid sequences showed that the virus is related to but distinct from viruses belonging to the genera Sequivirus, Sadwavirus and Cheravirus. This new virus, for which the name tomato torrado virus is proposed, most likely represents a member of a new plant virus genus.

Previously, transmission of poleroviruses has relied solely on the use of their aphid vectors. Biolistic inoculation allowed for the first time the mechanical transmission of Beet western yellows virus (BWYV) and Potato leafroll virus (PLRV) to several host plants. Inoculation with purified preparations and viral RNA extracts of PLRV resulted in 30-50% systemically infected Nicotiana occidentalis P1 plants and 15-30% infected Nicotiana clevelandii plants. Particle bombardment was also used successfully to infect N. clevelandii plants with in vitro RNA transcripts of full-length cDNA of BWYV.

Rhizoctonia solani is a plant pathogenic fungus with a wide host range. Host plant specificity within R. solani was analysed on seedlings grown aseptically on agar, which allowed continuous observation of both the fungus and the whole plant without disturbing the interaction. Symptom development on cauliflower, Arabidopsis, eggplant. tomato and potato by 32 R. solani isolates, belonging to six different anastornosis groups (AGs), was studied. Host plant specificity of isolates, as analysed by similarity clustering, was similar to AG-related host plant specificity as observed in the field, with AG3 isolates (except two avirulent strains) separating from the other isolates. Two R. solani isolates with a reciprocal pathogenicity on cauliflower and tomato were selected for further studies. These showed that in the pathogenic combination, R. solani isolates grew over the plant, adhered and formed infection structures, while in the nonpathogenic combination isolates grew over the plant, but neither adhesion nor the formation of infection structures occurred. From these data, it was concluded that host plant specificity is mediated in the early steps of the infection process. [ABSTRACT FROM AUTHOR]

In October 2006, tomato plants with torrado disease were sampled in Spain. In a sample of cv. Mariana, originating from Gran Canaria, Tomato torrado virus (ToTV, genus Torradovirus) was detected (isolate GCN06; Alfaro-Fernández et al., 2010). In 2013, the sample was further analysed using next-generation sequencing (NGS). Total RNA, extracted from ca. 150 mg of leaf tissues, was used for an RNA library suitable for Illumina HiSeq paired end sequencing. A de novo assembly (using CLC Genomic Workbench v.6.5) resulted in 1380 sequence contigs. BLAST searches revealed the presence of ToTV and Tomato chlorosis virus (ToCV, genus Crinivirus). One contig (3427 nts, 8901 reads), showed 99.85% identity with Southern tomato virus (STV, genus Amalgavirus), isolate Mexico-1 (GenBank accession No. EF442780), a dsRNA seed-transmitted virus (Sabanadzovic et al., 2009). Reference assembly with the Mexico-1 sequence resulted in a partial sequence of 3420 nts (GenBank KJ174690). The presence of STV in sample GCN06 was verified in RT-PCR using the specific primers STV-fw and STV-rev (Candresse et al., 2013) and sequencing of the obtained amplicon. A new sampling in May 2014 in Gran Canaria revealed the presence of STV in six out of seven tomato samples of cv Mariana. All these samples were co-infected with Pepino mosaic virus (PepMV, genus Potexvirus), Tomato spotted wilt virus (TSWV, genus Tospovirus), and Tomato yellow leaf curl virus (TYLCV, genus Begomovirus). Because STV was generally found in samples co-infected with other viruses, its symptomatology in tomato remains unclear. To our knowledge, this is the first report of STV in the Canary Islands and Spain. [ABSTRACT FROM AUTHOR]