A novel high-throughput sequencing approach reveals the presence of a new virus infecting Rosa: rosa ilarvirus-1 (RIV-1).

Roses are one of the most valuable ornamental flowering shrubs grown worldwide. Despite the widespread of rose viruses and their impact on cultivation, they have not been studied in detail in the United Kingdom (UK) since the 1980's. As part of a survey of rose viruses entering the UK, 35 samples were collected at Heathrow Airport (London, UK) and were tested by RT-qPCR for different common rose viruses. Of the 35 samples tested using RT-qPCR for prunus necrotic ringspot virus (PNRSV; genus Ilarvirus), 10 were positive. Confirmatory testing was performed using RT-PCR with both PNRSV-specific and ilarvirus-generic primers, and diverse results were obtained: One sample was exclusively positive when using the ilarvirus-generic primers, and subsequent sequencing of the RT-PCR product revealed homology to other ilarviruses but not PNRSV. Further work to characterise the virus was performed using high throughput sequencing, both the MinION Flongle and Illumina MiSeq. The sequencing confirmed the presence of a new virus within group 2 of the genus Ilarvirus and we propose the name "rosa ilarvirus-1″ (RIV-1). Here, we describe the identification of a novel virus using the low-cost Flongle flow cell and discuss its potential as a front-line diagnostic tool.

Susceptibility of Garden Plants to Phytophthora Root Rot.

Phytophthora root rot (PRR) is a serious disease of horticultural, forest, and ornamental plant species caused by species of the oomycete genus Phytophthora. Their wide host range makes the choice of resistant plants in the management of the disease difficult. We used the Royal Horticultural Society diagnostic dataset of PRR records from U.K. gardens to compare the susceptibility of different host genera to the disease. The dataset was compared with existing reports of plants recorded as notably resistant or notably susceptible to PRR. An index-based approach was used to separate 177 genera of woody plants into three categories: 85 were low index (<0.10: rarely affected), 34 were medium index (0.10 to 0.20: sometimes affected) and 58 were high index (>0.20: frequently affected). Similarly, genera of nonwoody plants were separated into: 45 low index (<0.22), 16 medium index (0.22 to 0.44), and 18 high index (>0.44). Taxus was the genus with the highest index, while most genera in the Malvales and Ericales were in the high-index group. Most genera in the Myrtales, Fabales, and Monocotyledons were low index. While 30 Phytophthora species were recorded in our study, the wide host range spp., P. plurivora, P. cryptogea, and P. cinnamomi, represented 63% of identifications. P. plurivora was the most common species on woody plants and P. cryptogea on nonwoody plants. These results provide confidence in the use of host resistance as part of the integrated management of PRR.

Susceptibility of Garden Trees and Shrubs to Armillaria Root Rot.

Armillaria root rot (ARR) is a serious disease of woody plants caused by several species of Armillaria. Armillaria isolates from diagnostic samples received in 2017 were identified by genus- and species-specific PCR and compared with isolates from an earlier survey (2004 to 2007). The results were comparable and, therefore, were combined for further analysis. Three species were identified: Armillaria mellea (83%), A. gallica (15%), and A. ostoyae (2%). Their wide host range makes choice of resistant plants in management of the disease difficult. We used the Royal Horticultural Society diagnostic dataset of ARR records from U.K. gardens to compare the susceptibility of different host genera to the disease. The dataset was compared with an earlier experiment at the University of California. An index-based approach was used to separate genera into three categories: 77 low-index (<0.99), 37 medium-index (0.99 to 1.76), and 56 high-index (>1.76) genera were recorded. All three species were associated with both angiosperms and gymnosperms; moreover, A. ostoyae did not show the host preference for gymnosperms that has been reported elsewhere. A. gallica was particularly common on herbaceous perennials and showed a trend to occur on resistant hosts that may be under other stress, supporting its description as an opportunistic pathogen. Four monocotyledons grown as trees or shrubs in U.K. gardens had a very low ARR index according to indices associated with A. mellea and A. ostoyae. Genera in the order Myrtales were almost always low index, while those in the Saxifragales and Fagales were mostly high index. These results provide confidence in the use of host resistance as part of the integrated management of ARR.

Associations Between Armillaria Species and Host Plants in U.K. Gardens.

Honey fungus (Armillaria spp.) root rot is the disease most frequently inquired about by U.K. gardeners to the Royal Horticultural Society. Armillaria epidemiology has been studied within forestry and agriculture, but data are lacking within gardens, which have greater host plant diversity than orchards and vineyards and greater disturbance than woodlands. Which Armillaria species are responsible for garden disease, and how the broad range of susceptible ornamentals are differentially affected is not known. To address this, isolates of Armillaria were obtained from dead and dying plants from across the U.K. over a 4-year period (2004 to 2007). Species were identified by PCR-RFLP for IGS, and further verified by species-specific PCR for EF-1 α. Of the seven species known in the U.K., three were identified: A. mellea (83.1%), A. gallica (15.8%), and A. ostoyae (1.1%). Armillaria was isolated from trees, shrubs, and nonwoody plants including bulbs and vegetables, with newly recorded hosts listed herein. A. mellea was associated with infections of multiple hosts, and with all infections of the most common host, Ligustrum. In sites where more than one Armillaria species was found, the combination was of A. mellea and A. gallica, raising questions regarding the interactions of these species in U.K. gardens.

Novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in South-East Australia.

A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australia's grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus. Five sets of novel genus-specific primers were designed and tested in reverse-transcription polymerase chain reaction assays against a range of virus isolates in plant virus diagnostic laboratories in both Australia and New Zealand. Three of these assays were then chosen to screen samples in a three-year survey of cereal crops in western Victoria, Australia. Of the 8900 cereal plants screened in the survey, all were tested free of furoviruses, hordeiviruses and rymoviruses. To date, there were no published genus-specific primers available for the detection of furoviruses, hordeiviruses and rymoviruses. This study shows for the first time a broad-spectrum molecular test being used in a survey for exotic grain viruses in Australia. Results from this survey provide important evidence of the use of this method to demonstrate the absence of these viruses in Victoria, Australia. The primer pairs reported here are expected to detect a wide range of virus species within the three genera.

The Diversity of Strawberry latent ringspot virus in New Zealand.

Strawberry latent ringspot virus (SLRSV) is widespread in many countries, especially in Europe. The virus was thought to be uncommon in New Zealand, having only been recorded in Prunus spp. However, this study revealed that SLRSV infects a much wider range of hosts. From 1999 to 2009, SLRSV was isolated from anemone (Anemone × hybrida), blackberry (Rubus spp.), impatiens (Impatiens walleriana), pepino (Solanum muricatum), and tibouchina (Tibouchina sp.) in the North Island of New Zealand. These SLRSV isolates were identified using electron microscopy, mechanical inoculation, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction techniques. This is thought to be the first report of anemone, impatiens, pepino, and tibouchina as hosts of SLRSV. Phylogenetic analysis and host range suggest that the five newly identified New Zealand isolates belong to two distinct strains: blackberry and impatiens isolates represent one strain and the other three isolates, plus the flowering cherry isolate reported previously in New Zealand, represent another strain. Both these strains are distinct from isolates reported elsewhere in the world. The strain infecting blackberry and impatiens is especially different and produced an unusual reaction in mechanical inoculation tests on herbaceous indicators. It is postulated that SLRSV may have gone undetected on its wider host range in New Zealand due to the latent infection in some hosts. The relationship of SLRSV isolates between New Zealand and overseas and the transmission modes of this virus are also discussed.

Detection of Tomato black ring virus by real-time one-step RT-PCR.

A TaqMan-based real-time one-step RT-PCR assay was developed for the rapid detection of Tomato black ring virus (TBRV), a significant plant pathogen which infects a wide range of economically important crops. Primers and a probe were designed against existing genomic sequences to amplify a 72 bp fragment from RNA-2. The assay amplified all isolates of TBRV tested, but no amplification was observed from the RNA of other nepovirus species or healthy host plants. The detection limit of the assay was estimated to be around nine copies of the TBRV target region in total RNA. A comparison with conventional RT-PCR and ELISA, indicated that ELISA, the current standard test method, lacked specificity and reacted to all nepovirus species tested, while conventional RT-PCR was approximately ten-fold less sensitive than the real-time RT-PCR assay. Finally, the real-time RT-PCR assay was tested using five different RT-PCR reagent kits and was found to be robust and reliable, with no significant differences in sensitivity being found. The development of this rapid assay should aid in quarantine and post-border surveys for regulatory agencies.

PCR assays for the detection of members of the genus Ilarvirus and family Bromoviridae.

A PCR assay was developed for the universal detection of ilarviruses using primers designed to the RNA-dependent RNA polymerase gene in RNA2. The assay detected 32 isolates of 15 definite and 2 tentative ilarvirus species using a one-step RT-PCR. The assay was more specific, and at least as sensitive as a commercial assay, and allowed direct sequencing of amplicons. No cross-reaction was observed with neither healthy plants of 15 host species nor from isolates in other genera of the Bromoviridae. A further PCR assay targeting the helicase motif of RNA1 was able to detect all species tested within the family Bromoviridae, including members of the Alfamovirus, Anulavirus, Bromovirus, Cucumovirus and Ilarvirus. The assays provide a sensitive and cost-effective way for detecting and characterising members of the Bromoviridae and can be used for quarantine and certification programmes.

A generic method to identify plant viruses by high-resolution tandem mass spectrometry of their coat proteins.

Although a number of protocols have been developed for detection of viruses at the genus or family level, universal approaches to detect and identify unknown viruses are still required. High-resolution tandem mass spectrometry was used to identify accurately peptide masses and their constituent sequences from partially purified plant virus preparations. Analysis of the peptide fragment masses against a virus database using pattern-matching algorithms identified sequences with homology to known virus peptides and also predicted peptides using de novo sequence analysis. This method provided sufficient information to confirm the identity of two known viruses that were included as controls (Cucumber mosaic virus and Tomato spotted wilt virus) and to identify unknown viruses in six viral isolates. The unknown viruses have been identified as four common viruses (Alfalfa mosaic virus, Tobacco streak virus, Citrus leaf blotch virus and Ribgrass mosaic virus), and two novel viruses (a potexvirus and a vitivirus). The identification of viruses from five distinct families by the tandem mass spectrometric determination of their coat protein demonstrates that this is a useful method for initial virus identification. This method, complemented with molecular or immunological procedures, provides a rapid and convenient way to identify both known and novel plant viruses.

Characterization of hydrangea chlorotic mottle virus, a new member of the genus Carlavirus.

An isolate of the tentative carlavirus species Hydrangea chlorotic mottle virus (HdCMV) was found in New Zealand (NZ) in 2007. The host range, serological properties and complete genome sequence of this isolate were determined in this study. While the NZ isolate shared 98% nucleotide sequence identity with the US isolate of HdCMV, differences in titre and host range were found. The HdCMV-NZ genome sequence of 8,433 nt possessed a typical carlavirus organisation with six open reading frames. HdCMV is most closely related (60.4% nt identity) to blueberry scorch virus, a relationship also suggested by serology. These data suggest that HdCMV is a new carlavirus species.

'Candidatus Liberibacter solanacearum', associated with plants in the family Solanaceae.

A liberibacter (isolate NZ082226) was detected in a symptomatic tomato plant and subsequently in five other members of the family Solanaceae: capsicum, potato, tamarillo, cape gooseberry and chilli. Phylogenetic analyses of the 16S rRNA gene sequence, the deduced amino acid sequence of the rplJ gene and a partial nucleotide sequence of the beta operon indicated that isolate NZ082226 represents a novel candidate species of 'Candidatus Liberibacter', for which the name 'Candidatus Liberibacter solanacearum' is proposed.

A New 'Candidatus Liberibacter' Species Associated with Diseases of Solanaceous Crops.

A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curling or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR) primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacterium shared high identity with 'Candidatus Liberibacter' species. Phylogenetic analysis showed that the bacterium is distinct from the three citrus liberibacter species previously described and has been named 'Candidatus Liberibacter solanacearum'. This is the first report of a liberibacter naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was developed for its detection.

Use of primers with 5' non-complementary sequences in RT-PCR for the detection of nepovirus subgroups A and B.

Two generic PCR protocols were developed to detect nepoviruses in subgroups A and B using degenerate primers designed to amplify part of the RNA-dependent RNA polymerase (RdRp) gene. It was observed that detection sensitivity and specificity could be improved by adding a 12-bp non-complementary sequence to the 5' termini of the forward, but not the reverse, primers. The optimized PCR protocols amplified a specific product ( approximately 340bp and approximately 250bp with subgroups A and B, respectively) from all 17 isolates of the 5 virus species in subgroup A and 3 species in subgroup B tested. The primers detect conserved protein motifs in the RdRp gene and it is anticipated that they have the potential to detect unreported or uncharacterised nepoviruses in subgroups A and B.

Novel approaches to mitigate primer interaction and eliminate inhibitors in multiplex PCR, demonstrated using an assay for detection of three strawberry viruses.

Multiplex PCR is an important technique for detecting a variety of pathogens simultaneously in a single assay. Previous research has focused on optimising the factors affecting reliable multiplex PCR, including primer design, PCR components and conditions, and inhibitors in samples. In this study, the interaction of primers to form complex secondary structures including visible dimers and invisible "primer clusters", a novel form of primer secondary structure found during this research, were shown to be the most important factors affecting successful multiplex PCR. Approaches to mitigate primer interaction and eliminate inhibitors were tested, including: reduction of primer concentrations especially those with preferential amplification; decrease of PCR extension temperature; increase of extension time and PCR cycles; and addition of bovine serum albumin. Based on these approaches, a multiplex RT-PCR with sensitivity comparable to the simplex PCR for individual viruses was developed for the detection of Raspberry ringspot virus, Strawberry latent ringspot virus and Tomato bushy stunt virus. A plant internal amplification control was also included. These approaches may be useful as a guideline for the development of multiplex PCR protocols for the detection of other pathogens or organisms associated with plants, humans, animals and the environment.

A multiplex RT-PCR assay capable of distinguishing beet necrotic yellow vein virus types A and B.

A multiplex reverse-transcription polymerase chain assay (mRT-PCR) was developed, based on primers designed to distinguish the A and B types of beet necrotic yellow vein virus (BNYVV). RNA was extracted from 72 BNYVV isolates from Asia, Europe and North America, and the type of each isolate determined using an established detection method based on single strand conformation polymorphisms (SSCPs). An area of the 'triple gene block' region on RNA 2 was amplified and sequenced from 16 isolates of the A and B types. These sequences were aligned and two sets of PCR primers were designed to amplify unique areas common to each type. The A type assay produced a single 324 base-pair RT-PCR fragment when positive samples were amplified. The B type assay produced a 178 base-pair product from positive samples. No amplification was observed from healthy Chenopodium quinoa or sugar beet plants and from plants infected by others sugar beet soil-borne viruses. Fragment length differed sufficiently to allow both assays to be run in a single PCR tube. The results obtained using the new multiplex RT-PCR assay were consistent with those from the established SSCP method for all 72 reference samples.

Detection and relative quantitation of Soil-borne cereal mosaic virus (SBCMV) and Polymyxa graminis in winter wheat using real-time PCR (TaqMan).

Soil-borne cereal mosaic virus (SBCMV) was first reported affecting wheat crops in Italy in 1960 and has spread subsequently to many other European countries, including the UK. SBCMV causes a serious disease of wheat, reducing yield by up to 70%; growing resistant varieties represents the only economical means of control. Real-time RT-PCR and PCR assays based on TaqMan chemistry were developed for the detection and quantitation of SBCMV and its vector, Polymyxa graminis. Each assay incorporated an RNA or DNA specific internal control to facilitate quantitation. Nucleic acid extracts from SBCMV-infected plants were diluted in a nucleic acid extract from a healthy plant and amplified by real-time PCR to produce a standard curve. The standard curve was used to quantify the amount of SBCMV and P. graminis in plant samples. The sensitivity of the real-time assays were compared to established serological quantitation and conventional PCR methods by testing a range of SBCMV-infected wheat varieties. The results indicate that real-time assays were a 1000 times more sensitive than ELISA for the quantitation of SBCMV, and a 100 times more sensitive than conventional PCR for the quantitation of P. graminis. Real-time assays enabled sensitive, reproducible and specific detection of both virus and vector in wheat tissues. The real-time assays are potentially useful tools for determining variations in virus and vector concentrations in plant tissue from wheat varieties differing in resistance to SBCMV.