Tomato Brown Rugose Fruit Virus: Survival and Disinfection Efficacy on Common Glasshouse Surfaces.

Tomato brown rugose fruit virus (ToBRFV) is a contact-transmitted tobamovirus affecting many tomato growing regions of the world. This study investigated the effects of different glasshouse surfaces on the survival of the virus; the efficacy of different disinfectants; and heat treatment against ToBRFV (surfaces included steel, aluminium, hard plastic, polythene, glass and concrete). A bioassay followed by ELISA was used to check virus viability. ToBRFV survived for at least 7 days on all surfaces tested and on some for at least 6 months. The virus survived for over two hours on hands and gloves. Hand washing was shown to be unreliable for the removal of the virus. Glutaraldehyde and quaternary ammonium compound disinfectants were effective at one hour on all surfaces. Some other disinfectants were effective at one hour of contact time, on all surfaces except concrete. Sodium hypochlorite was partially effective against ToBRFV, even on concrete. A 5 min soak of plastic trays in water at 90 °C was effective at denaturing ToBRFV; however, 5 min at 70 °C was not. Heating infected sap showed the thermal inactivation point to be 90 °C, confirming the hot water treatment results and showing that deactivation was due to the heat treatment and not a washing effect of the water.

Integrating High throughput Sequencing into Survey Design Reveals Turnip Yellows Virus and Soybean Dwarf Virus in Pea (Pisum Sativum) in the United Kingdom.

There is only limited knowledge of the presence and incidence of viruses in peas within the United Kingdom, therefore high-throughput sequencing (HTS) in combination with a bulk sampling strategy and targeted testing was used to determine the virome in cultivated pea crops. Bulks of 120 leaves collected from twenty fields from around the UK were initially tested by HTS, and presence and incidence of virus was then determined using specific real-time reverse-transcription PCR assays by testing smaller mixed-bulk size samples. This study presents the first finding of turnip yellows virus (TuYV) in peas in the UK and the first finding of soybean dwarf virus (SbDV) in the UK. While TuYV was not previously known to be present in UK peas, it was found in 13 of the 20 sites tested and was present at incidences up to 100%. Pea enation mosaic virus-1, pea enation mosaic virus-2, pea seed-borne mosaic virus, bean yellow mosaic virus, pea enation mosaic virus satellite RNA and turnip yellows virus associated RNA were also identified by HTS. Additionally, a subset of bulked samples were re-sequenced at greater depth to ascertain whether the relatively low depth of sequencing had missed any infections. In each case the same viruses were identified as had been identified using the lower sequencing depth. Sequencing of an isolate of pea seed-borne mosaic virus from 2007 also revealed the presence of TuYV and SbDV, showing that both viruses have been present in the UK for at least a decade, and represents the earliest whole genome of SbDV from Europe. This study demonstrates the potential of HTS to be used as a surveillance tool, or for crop-specific field survey, using a bulk sampling strategy combined with HTS and targeted diagnostics to indicate both presence and incidence of viruses in a crop.

Direct detection of plant viruses in potato tubers using real-time PCR.

Virus indexing of seed potatoes can be carried out by growing eye plugs to produce small plants and then testing them by ELISA, but this method is time consuming. Direct testing of the eye plugs by ELISA is not reliable, and so a method has been developed for the routine testing of seed potatoes for virus by PCR.

A study of crop-to-crop gene flow using farm scale sites of fodder maize (Zea mays L.) in the UK.

From 2000 to 2003 a range of Farm Scale Evaluation (FSE) trials were established in the UK to assess the effect of the release and management of herbicide tolerant (HT) crops on arable weeds and invertebrates. The FSE trials for maize were also used to investigate crop-to-crop gene flow and to develop a statistical model for the prediction of gene flow frequency that can be used to evaluate current separation distance guidelines for GM crops. Seed samples were collected from the non-GM half of 55 trial sites and 1,055 were tested for evidence of gene flow from the GM HT halves using a quantitative PCR assay specific to the HT (pat) gene. Rates of gene flow were found to decrease rapidly with increasing distance from the GM source. Gene flow was detected in 30% of the samples (40 out of 135) at 150 m from the GM source and events of GM to non-GM gene flow were detected at distances up to and including 200 m from the GM source. The quantitative data were subjected to statistical analysis and a two-step model was found to provide the best fit for the data. A dynamic whole field model predicted that a square field (150 m x 150 m in size) of grain maize would require a separation distance of 3 m for the adjacent crop to be below a 0.9% threshold (with <2% probability of exceeding the threshold). The data and models presented here are discussed in the context of necessary separation distances to achieve various possible thresholds for adventitious presence of GM in maize.

Crop-to-crop gene flow using farm scale sites of oilseed rape (Brassica napus) in the UK.

From 2000-2003 a range of Farm Scale Evaluation (FSE) trials were established in the UK to assess the effect of the release and management of herbicide tolerant (HT) crops on the abundance and diversity of farmland wildlife compared with their conventionally managed non-GM-equivalents. The objective of this research project was to investigate gene flow within the winter (WOSR) and spring oilseed rape (SOSR) FSE trials and to develop a statistical model for the prediction of cross-pollination frequency that can be used to evaluate current separation distance guidelines. Seed samples were collected from the non-GM half of the trial sites and were tested for evidence of cross-pollination from the GM HT halves using a quantitative PCR assay specific to the HT (bar) gene. Rates of cross-pollination were found to decrease rapidly with increasing distance from the GM source. The quantitative data were subjected to statistical analysis and a two-step model was found to provide the best fit for the data. Significant differences were found between the results for WOSR, SOSR and varietal association (VA) crops. The model predicted that the %GM content (including upper 95% confidence limits) of a sample taken at a distance of 50 m away from the GM source would be 0.04% (0.84%) for WOSR, 0.02% (0.39%) for SOSR, 0.77% (21.72%) for WOSR VA and 0.37% (5.18%) for SOSR VA. The data and models presented here are discussed in the context of necessary separation distances to meet various possible thresholds for adventitious presence of GM in OSR.

Identification of Agrobacterium spp. present within Brassica napus seed by TaqMan PCR--implications for GM screening procedures.

A fluorogenic probe (fliG-P), designed within a chromosomal DNA sequence, was used in a TaqMan PCR assay to identify Agrobacterium spp. The TaqMan assay detected 58 of 59 Agrobacterium strains tested, but did not detect 13 other Rhizobiaceae strains. Seedlings were grown from seven lots of surface-sterilised Brassica napus seed. Seedlings from these samples were placed in phosphate buffer and the resulting suspensions used to inoculate broth media selective for Agrobacterium biovars 1 and 2. Lysed broths (after 48 h incubation) were used as template in the fliG TaqMan PCR to detect Agrobacterium sp. in one of the seed samples. Individual Agrobacterium strains were isolated from this sample and tested by three Ti-plasmid conventional PCR assays. None of the strains possessed a plasmid. This is the first report of Agrobacterium sp. present within the seed of B. napus, a crop routinely screened for genetically modified DNA contamination using PCR assays with Agrobacterium sequences as targets.