C. M. Henry

Sampling and modeling for the quantification of adventitious genetically modified presence in maize

The coexistence of genetically modified (GM) and non-GM crops is an important economic and political issue in the European Union. We examined the GM content in non-GM maize crops in Spain in 2005. Both the standing crop and the harvest were tested, and the %GM DNA was quantified by real-time polymerase chain reaction. We compared the level of GM as a function of distance from known GM source fields in a 1.2 km2 landscape. The distribution of GM was compared to predictions from previous studies, and good agreement was found. Control and monitoring of adventitious GM presence in non-GM crops can only be achieved by fit-for-purpose sampling and testing schemes. We used a GM dispersal function to simulate non-GM crops in the studied zone and tested the accuracy of five different sampling schemes. Random sampling was found to be the most accurate and least susceptible to bias by GM spatial structure or gradients. Simulations showed that to achieve greater than 95% confidence in a GM labeling decision of a harvest (when treated as a single marketed lot), 34 samples would be needed when the harvest was outside 50% of the GM threshold value. The number of samples required increased rapidly as the harvest approached the GM threshold, implying that accurate labeling when the harvest is within +/-17% of the threshold may not be possible with high confidence.

Management of plant health risks associated with processing of plant-based wastes: a review

The rise in international trade of plants and plant products has increased the risk of introduction and spread of plant pathogens and pests. In addition, new risks are arising from the implementation of more environmentally friendly methods of biodegradable waste disposal, such as composting and anaerobic digestion. As these disposal methods do not involve sterilisation, there is good evidence that certain plant pathogens and pests can survive these processes. The temperature/time profile of the disposal process is the most significant and easily defined factor in controlling plant pathogens and pests. In this review, the current evidence for temperature/time effects on plant pathogens and pests is summarised. The advantages and disadvantages of direct and indirect process validation for the verification of composting processes, to determine their efficacy in destroying plant pathogens and pests in biowaste, are discussed. The availability of detection technology and its appropriateness for assessing the survival of quarantine organisms is also reviewed.

Development of a highly sensitive nested RT-PCR method for Beet necrotic yellow vein virus detection.

A diagnostic test incorporating reverse-transcription polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (nPCR) was developed for the detection of Beet necrotic yellow vein virus (BNYVV). The RT-PCR used the primers designed by (Henry et al., J. Virol. Methods 54 (1995)15) but refinements were made to the protocol including simplification of the extraction method, the use of standard reagents and adoption of a one-step procedure. None of these changes impaired sensitivity or specificity. The RT-PCR could also be used to amplify immunocaptured virus but this was slightly less sensitive than amplification from purified RNA. In nPCR, a second round of amplification was performed using primers, which produce a specific 326 base-pair product. Both RT-PCR and nPCR detected a range of 21 isolates collected from Europe, America and Asia (including A, B and P pathotypes) isolated from either sugar beet or Chenopodium quinoa. Neither assay produced PCR products using total RNA extracted from the roots of healthy sugar beet or beet infected with Beet soil-borne virus. However, the sensitivity of the nPCR was 1000 times greater than the standard RT-PCR. The reliability of the standard RT-PCR and nPCR was demonstrated using a range of cultivars collected from an infected field site. The use of the nPCR assay is recommended for applications where its improved sensitivity over standard RT-PCR is necessary, for example in the early detection of infection from bait-test soils and for quarantine and breeding purposes.

Response of UK winter wheat cultivars to Soil-borne cereal mosaic and Wheat spindle streak mosaic viruses across Europe.

Twenty-one UK winter wheat cultivars were grown over three seasons at sites with natural inoculum sources of Soil-borne cereal mosaic virus (SBCMV) and Wheat spindle streak mosaic virus (WSSMV) located in France, Italy and the UK. Plants were assessed visually for virus symptoms and leaf extracts were tested for the presence of each virus using enzyme-linked immunosorbent assays (ELISA). Cultivars showing little or no foliar symptoms and low levels of virus in leaf tissue were classified as resistant to each virus. All the trials were taken to harvest and agronomic data collected. At the most heavily infected sites, severe symptoms of SBCMV were observed in all UK cultivars except Aardvark, Charger, Claire, Cockpit, Hereward and Xi 19. The latter cultivars exhibited either light or no symptoms and little or no SBCMV infection in leaves. In fields with WSSMV, the virus failed to develop in Italy, but was detected in the leaves of all the susceptible control cultivars at a site in France. However, no UK cultivar tested positive for WSSMV. Multi-site analysis indicated that the presence of WSSMV did not increase the impact of SBCMV on the height, thousand-grain weight or yield of UK cultivars. The wheat cultivars on test gave a similar response to SBCMV across three European countries. Possible sources of SBCMV resistance are discussed.

Detection of beet necrotic yellow vein virus using reverse transcription and polymerase chain reaction.

A diagnostic test based on reverse transcription followed by the polymerase chain reaction (RT-PCR) was developed for the detection of beet necrotic yellow vein virus (BNYVV). A specific 500-base-pair fragment was amplified from the read-through region of the coat protein gene located on RNA-2. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having 94.5% homology with published sequence data for BNYVV. The assay gave a sensitivity of 800 times that of a TAS-ELISA and 50 times that of an amplified TAS-ELISA method. A range of BNYVV isolates from the UK and worldwide could be detected by this test, either as mechanically inoculated Chenopodium quinoa leaves or infected sugar beet roots. Use of the assay in routine diagnostic tests allowed a reduction of time needed for the detection of Rhizomania in soils from 7 to 4 weeks.

The Characterization of a Subgenomic RNA and In Vitro Translation Products of Oat Chlorotic Stunt Virus

Oat chlorotic stunt virus (OCSV) is a 35 nm icosahedral plant virus comprising of a single capsid protein with a Mr of 48.2 kDa and a 4.1 kb single stranded, positive sense genomic RNA. Northern blot analysis detected a single 3′ terminal subgenomic RNA in extracts from infected plants, which was also found to be encapsidated. Virion RNA directs the synthesis of a 23 kDa polypeptide in a rabbit reticulocyte in vitro translation system. Primer extension analysis has been used to map the end of both the genomic and subgenomic RNA’s, and has shown the genomic size to be 4115 nucleotides in length. The results have enabled a model for the genome expression to be proposed.

Detection of European Isolates of Oat Mosaic Virus

Oat mosaic virus (OMV) is a fungally-transmitted virus which causes yield losses in winter oats in five European countries. Detection of the virus has depended upon the recognition of transient symptoms or electron microscopy. Recent research has confirmed that the virus is a Bymovirus, yet OMV could not be reliably detected by the enzyme-linked immunosorbent assay (ELISA) using a range of antisera raised against other members of the genus. Therefore, a reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed to specifically detect the virus. Using total RNA isolated from 16 field OMV isolates collected from throughout Europe, these primers were shown to reliably detect the virus in either one-step or two-step RT-PCR. The primers were specific and no PCR product was obtained with either Oat golden stripe virus (OGSV), which is frequently associated with OMV, or with other members of the Bymovirus genus. The two-step protocol was able to detect as little as 5 × 10−3μl (10ng) of total RNA isolated from an infected plant. Both protocols were as reliable as electron microscopy, but were more sensitive and were able to detect infection earlier than in mechanically-inoculated plants. However, this protocol did not detect three American isolates of the virus nor was amplification achieved using alternative primers raising the possibility that these isolates may represent a separate strain or virus. This protocol enables sensitive, rapid and reliable detection of OMV and will therefore assist management of the disease.