Project Title: Molecular methods for virus detection in fruit plants
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ExpenditureApproved expenditure for year 3: £123,707Approved expenditure for project: £390,599 Actual expenditure for year 3: £125,837Actual expenditure for project: £390,594Staff effort (based on 223 working days per year)Band 4 20%Band 6 100%Band 7 15%Band 8 15%Band 5 2%Student 100%SCIENCE SECTIONINTRODUCTIONViruses are important pathogens of fruit plants, affecting the quality of propagation material as well as the establishment and performance of fruiting plantations. This has been recognised for many years and was a major reason for founding schemes for the production of healthy planting material of strawberry (Harris, 1937) and fruit trees (Cutting & Montgomery, 1973). Viruses were originally discovered in fruit plants because sensitive indicator species or cultivars were planted, usually unintentionally, and infection gave rise to severe symptoms. These coincidences gave rise to bioassays, which are recognised internationally as being the most reliable means of testing fruit planting material for viruses and are required by European Certification schemes (OEPP/EPPO, 1992) for the highest level of health status (Nuclear Stock). Despite this requirement, bioassays can be misinterpreted as symptom expression is dependent on growing conditions, particularly with strawberry indicators. In addition, bioassays are time-consuming and take months (strawberries) or years (fruit trees) to complete. The time factor is particularly important with tree fruit and prevents the rapid introduction of desirable new clones of accredited health status into industry. The cost is also high for conducting bioassays and has led to the virtual collapse of the original EMLA scheme for producing healthy planting material. Laboratory methods of virus assay have advantages over bioassays in speed, cost, the capability for large-scale testing and precise identification of virus strains or serotypes. The prospects of providing such methods for a significant spectrum of fruit viruses have not been good until recently. However, molecular methods provide new opportunities for characterising and diagnosing viruses and the object of this project is to apply such methods to two key virus pathogens of strawberry and tree fruit: apple stem pitting virus (ASPV) and strawberry crinkle virus (SCV). Apple stem pitting virus is associated with at least four disease syndromes in apple and pear, each requiring a separate woody indicator species and it is possibly also associated with stony pit in pear which requires a three year test in a fruiting tree for diagnosis (Leone et al., 1995). A strain of the virus was purified by Koganezawa & Yanase (1990) but the resulting antiserum was not suitable for ELISA (enzyme-linked immunosorbent assay) and other research groups have been unable to purify any other strains of the virus. Sequence data is available for two isolates of the virus (Jelkmann, 1994) and provided a starting point for designing primers for this project. Since the start of the project, methods and primers have been published for the detection of ASPV in extracts from the leaves of Nicotiana occidentalis, apple and pear (Jelkmann & Keim-Konrad, 1997) by ELISA and immunocapture PCR (IC-PCR). These methods have potential but need assessment with a wide range of virus isolates. ELISA is a less demanding process compared to assays for nucleic acid and is preferable to them provided that sensitivity is adequate. One strategy for providing the immunogen to obtain sero-reagents is to clone the virus coat protein gene(s) for expression in a prokaryote. Antisera produced in this way have usually not been useful for detection of fruit plant viruses by ELISA although they have been satisfactory for other experimental uses e.g. serologically specific electron microscopy. Recently, antiserum to ASPV was obtained by expressing the coat protein gene in E. coli as a chimaeric protein lacking four amino acids at the N-terminus and possessing a tail of six histidine residues for affinity purification. This antiserum detected ASPV in extracts from the leaves of N. occidentalis and pear but not from apple. Also, the antiserum worked in ‘plate-trapped’ ELISA but not in the more reliable antibody-sandwich types of assay (Jelkmann & Keim-Konrad, 1997). An alternative to expression of the coat protein in E. coli is to use the yeast Pichia pastoris for protein expression. High levels of easily purified protein can be obtained which are likely to be structurally more similar to the native protein than that expressed in a bacterial system. This alternative is being investigated. Strawberry crinkle virus (SCV) is one of the most damaging viruses of strawberry. There are numerous strains of varying pathogenicity and although it is often latent, the virus combines synergistically with other aphid-borne viruses to induce severe damage (Frazier et al., 1987). The standard leaf-grafting technique can often be unsuccessful in detecting virus, possibly because of erratic distribution in infected plants (Converse et al., 1988). The virus has been purified in small amounts only and no antisera have been produced that are effective for detecting SCV in strawberry plants (Schoen & Leone, 1995; Hunter et al., 1990). The results suggest that a more successful approach might be to use molecular methods to obtain sufficient sequence information to develop a diagnostic test. SCV is a plant rhabdovirus and a feature of the family Rhabdoviridae is the transcription of genes, from the negative sense virus RNA, via polyadenylated mRNAs (Murphy et al., 1995). This characteristic was utilised in the project to obtain RNA from infected plants for subtractive hybridisation and cloning. Information on the sensitivity of laboratory assays and their ability to detect a wide range of virus strains is essential for any decision on the usefulness and reliability of the assay in comparison with the best method available. There has been sufficient progress with apple stem grooving virus (ASGV) for several laboratory methods to be compared with the standard field assay and a glasshouse assay (Howell et al., 1996) and this is an aim of the project. The project has sought to exploit molecular methods to devise laboratory assays for two important viruses of fruit plants for which routine laboratory assays have not previously been possible (apple stem pitting and strawberry crinkle). It has investigated the possibility of cloning virus coat protein in yeast as a route to obtaining high quality immunogen for ‘difficult’ viruses. In addition, it has compared a range of field and laboratory assays for one virus (apple stem grooving) to provide a model for the acceptance of laboratory assays internationally to accredit the health status of high quality propagation material such as Nuclear Stock plants. MATERIALS AND METHODS |