|Project Title: Molecular methods for virus detection in fruit plants
Project Number: SF/HNS 87: CSA 4363: HortLINK 37: Ref HL0117
Project Leader: A N Adams, HRI-East Malling
Report: Final Report, December 2000
Previous Reports: Annual Reports December 1998, December 1999
Key Workers: A N Adams (Plant Virologist), M J Kirby (Molecular Biologist), Y Hong (Molecular Virologist), C M Guise (Plant Pathologist), K Posthuma (PhD Student)
Location of Project: HRI-East Malling
Kent ME19 6BJ
Tel: 01732 843833 Fax: 01732 849067
Project Co-ordinator: A N Adams
Government Sponsor: MAFF
Consortium Members: HRI; HDC, Bradbourne House, The Stable Block, East Malling, Kent ME19 6DZ; The East Malling Trust for Horticultural Research, Bradbourne House, East Malling, West Malling, Kent ME19 6DZ; Nuclear Stock Association, P O Box 126, Terrington St Clement, King’s Lynn, Norfolk PE34 4EP
Start Date: September 1997
Completion Date: 31 August 2000 (Final Report Due 1 January 2001)
Key Words: Apple, pear, strawberry, virus, detection, PCR, ELISA
2000 Horticulture Research International
The contents of this publication are strictly private to Horticulture Research International. No part of this publication may be copied or reproduced in any form or by any means without prior written permission of Horticulture Research International.
Intellectual Property Rights are invested in Horticulture Research International on behalf of the Consortium members for HortLINK project 37
The following are members of the Consortium for HortLINK project 37:
Horticultural Development Council
East Malling Trust for Horticultural Research
Nuclear Stock Association
PRACTICAL SECTION FOR GROWERS 4
Commercial benefits 4
Background and objectives 4
Specific targets 5
Summary of results 5
Action points for growers 8
Anticipated practical and financial benefits 8
Year 1 9
Year 2 10
Year 3 11
SCIENCE SECTION 14
MATERIALS AND METHODS 17
Objective 1 – Devise PCR-based assay for Apple stem pitting virus 33
Objective 2 - Comparison of assays for Apple stem grooving virus 40
Objective 3 – Expression of coat proteins of Apple stem pitting virus and
Apple stem grooving virus in yeast for antiserum production 47
Objective 4 – Development of a PCR based method for the detection of
Strawberry crinkle virus 56
INDUSTRIAL RELEVANCE AND EXPLOITATION 68
TECHNOLOGY TRANSFER 70
PRACTICAL SECTION FOR GROWERS
Commercial benefits of the project
The project has provided a firm basis for the development of laboratory assays for two fruit viruses (one affecting apple and pear, the other affecting strawberry). The current tests for these viruses entail grafting to indicator plants and such tests are slow and expensive. The cost of such tests for tree fruit has led to severe cuts in the EMLA scheme for provision of virus free fruit trees. We have made a comparison of assays for one fruit tree virus and have demonstrated the reliability of laboratory tests for this virus (apple stem grooving) and the value, therefore, of continuing the development and comparison of such assays for other viruses. This will strengthen the machinery for providing healthy planting material at all levels from Nuclear Stock to Certified plants.
Background and objectives
The importance of viruses to the quality of propagation material and to the establishment and performance of fruiting plantations has been recognised for many years; it was a major reason for developing schemes for producing healthy planting material of strawberry and fruit trees.
The success of such schemes depends upon reliable tests for viruses. These have, until recently, consisted of bioassays conducted in the glasshouse or the field. Bioassays are used as standard throughout the world for testing the highest quality planting material (Nuclear Stock or their equivalent) but such tests are not always reliable. They are also expensive and take a long time to provide results (months for strawberries and years for fruit trees).
There are clear advantages to using laboratory tests for detecting viruses but they have to be thoroughly tested with a range of isolates and compared against the best, established test. This has been done for apple stem grooving virus in this project.
The project has targeted two important viruses, for which routine laboratory tests have not previously been available, (apple stem pitting virus and strawberry crinkle virus) with the objective of developing laboratory assays for them. Apple stem pitting virus is associated with at least four diseases of apple and pear and several indicator species are required for field tests, making this a very expensive virus to test for. Strawberry crinkle virus occurs world-wide and so far the only means of assay is by leaf grafting. The virus is difficult to purify and the information did not exist at the start of this project for devising any kind of laboratory assay.
A fourth major objective was to investigate the possibility of cloning virus coat protein in yeast as a route to obtaining high quality immunogen for ‘difficult’ viruses (for serology tests such as ELISA). ELISA is a simple and well-tried procedure and is the assay of choice, providing the reagents are of sufficient quality to achieve good results. Success in this objective would improve the chances of attaining good results with a wider range of viruses than is possible at the moment.
Specific targets for the project
Devise a PCR-based laboratory assay for apple stem pitting virus.
Refine and validate the detection methods for apple stem grooving virus.
Express apple stem grooving virus and apple stem pitting virus coat protein genes in yeast for antiserum production.
Develop a PCR-based method for the detection of strawberry crinkle virus.
Summary of results
Objective 1. Assay for apple stem pitting virus (ASPV)
In the first two years of the project the effectiveness of a newly published protocol for detecting ASPV by immunocapture PCR, was assessed. It detected the virus in many of the isolates of ASPV in the reference collection at East Malling but not all of them. Modifications to the test improved its performance but results were inconsistent, particularly with a group of virus isolates from pear. These isolates were from trees with stony pit disease, which is associated with this virus and may be caused by it.
The tests showed, surprisingly, that in extracts from dormant wood it was easier to detect ASPV by a combination of electron microscopy and serology than by the potentially highly sensitive PCR. Antiserum to ASPV is not commercially available and in the final year efforts were directed at a) making antiserum to ASPV and b) obtaining sequence information of an isolate of ASPV from pear with stony pit disease.
These final year objectives were partially achieved. The coat protein gene of an isolate of ASPV was cloned into a plasmid vector and the protein was expressed in the bacterium Escherichia coli. Sufficient protein has been purified from this to prepare antiserum. A small amount of sequence was obtained of the ASPV isolate from pear, but this was insufficient for meaningful comparison with other isolates.
At the end of the project there is: 1) a viable PCR assay for many, but not all, strains of apple stem pitting virus; 2) the material to prepare antiserum to the virus and 3) the knowledge that serology may provide a better assay for this virus (applicable to a wider range of isolates) than the more sensitive PCR-based assay.
Objective 2. Refine and validate detection protocols for apple stem grooving virus (ASGV)
Four assays for this virus were compared for their effectiveness in detecting ASGV in a sample of 105 apple and oriental pear trees. Twenty two of these trees were infected and all infections were detected by the serology assay (ELISA) and by the nucleic acid test (immunocapture PCR). Only eighteen infections were detected by the standard field test, which takes three years to complete and involves double budding to the sensitive Virginia Crab indicator. Eighteen infections were also detected by a glasshouse bioassay that involves double budding to the indicator Malus micromalus and takes only three months to obtain a result. There were inconsistencies between the two bioassay results and three infections detected by M. micromalus were undetected by the Virginia Crab test and vice versa.
It was surprising that the field test was less reliable than the laboratory tests. Double budding tests are recognised internationally as being the best assays and the standard by which all other tests are judged. These results strongly indicate that, for some viruses at least, this is not true. Provided that adequate comparisons are made, laboratory assays should be applied to a wide range of fruit viruses with confidence.
Objective 3 – Expression of coat proteins of apple stem pitting (ASPV) and apple stem grooving viruses in yeast for antiserum production.
This objective was only partially achieved. The coat protein gene of apple stem pitting virus was successfully cloned into plasmid vectors and expressed in the yeast Pichia pastoris. However, expression was inconsistent. This did not allow the conditions of expression to be optimised. Evidence was obtained that the problem could be resolved by using a different plasmid expression vector that is not sensitive to the methanol:cell density ratio.
By the end of the project the ASPV coat protein gene had been cloned into a yeast vector that is not sensitive to methanol and will allow continuous expression of the protein. The project ended before this could be done. However, the ASPV coat protein gene was cloned into the bacterium E. coli and sufficient protein has been expressed for antiserum production (see objective 1). The antiserum will be of value for furthering this research, for the immunocapture phase of PCR tests and for electron microscope detection of the virus but probably not for the simple and robust ELISA.
Objective 4 – Development of a PCR based method for the detection of strawberry crinkle virus (SCV)
During the first two years of the project attempts were made to obtain sequence of SCV by a subtractive hybridisation procedure. All clones obtained by this method were plant associated and not from the virus, although several were from genes coding for proteins known to be involved with plant defence.
A different, and novel, approach was taken at the end of year two. The gene sequences of seven viruses in the same taxonomic group as SCV were compared. The viruses were from groups infecting mammals, fish, insects and plants but despite their diversity, similarities were found in the L (large) protein gene of these viruses. This information was used to design primers that successfully amplified part of the L protein gene of SCV. This is the first sequence to have been obtained of this virus.
Subsequent work obtained more of the L gene sequence and primers were designed that consistently detected SCV in the experimental host Physalis pubescens. They also detected the virus in the aphid vector but detection in strawberry was not always successful. A small amount of development work is now needed to provide, for the first time, a reliable laboratory diagnostic test for this damaging virus.
Action points for growers
The results from this project cannot be acted upon directly by growers. The aim of the work is to improve the monitoring of healthy planting material. Growers should always seek plants that are Certified and originate from virus tested material.
Anticipated practical and financial benefits from the study
The project has provided and validated a quicker, cheaper and more reliable assay for apple stem grooving virus than existed before the project began.
A small amount of development work should provide similar tests for two other viruses (apple stem pitting virus and strawberry crinkle virus).
The development and application of up to date methods for monitoring the health of planting material is vital to the success of the Nuclear Stock Association and the Plant Health Propagation Scheme for providing high quality plants to the fruit growing industry.
Milestones for year 1
1a Transfer of six isolates of apple stem pitting virus from fruit trees to Nicotiana occidentalis for maintenance in the glasshouse
1b,c Design and assessment primers that will amplify products from six isolates of apple stem pitting virus
2a Bud rootstocks in the field with 100 sources of budwood for apple stem grooving virus field tests
2b Transfer six isolates of apple stem grooving virus from apple and/or oriental pear to Chenopodium quinoa for maintenance in the glasshouse
2c i) Obtain graftwood of Malus micromalus (indicator species) and graft to M26 for use as budwood
ii) Bud seedlings raised in 1997/98 for a preliminary glasshouse assay
3a Design primers from published sequence to amplify the entire
coat protein region of apple stem grooving virus for cloning into a yeast expression vector
3b Devise a protocol for producing sufficient quantity and quality of RNA of apple stem pitting virus for amplification experiments
4a Establish six isolates of strawberry crinkle virus in Physalis by aphid and/or mechanical transfer from strawberry to use as working material. Isolates will be maintained in strawberry and (if possible) in Physalis throughout the project
4b Establish a protocol for obtaining extracts from Physalis enriched for strawberry crinkle virus RNA
These milestones were achieved with the following exceptions:
1a: six isolates of apple stem pitting were transferred to N. occidentalis but they were all lost during the summer. This had no adverse effect on the project as three isolates were re-established in N. occidentalis, and it was possible to use material directly from fruit trees for assessing the effectiveness of primers to amplify a wide range of isolates.
2c: the graftwood of Malus micromalus indicator was thin and insufficient buds took to provide material for the large-scale glasshouse assay for apple stem grooving virus that was planned for 1999. This was postponed for one year but completed during the project.
Milestones for year 2
1 b,c Primers designed in year 1 will have been tested against the panel of isolates in herbaceous plants and modified as necessary so that they will amplify all isolates consistently (use may also be made of information from the sequencing in objective 3) Mch 99
1 d Obtain nucleotide sequence data for ASPV isolate 7/40 Aug 99
2 e i) Trial PCR and ELISA assays will have been conducted on material from herbaceous plants and on forced buds of apple and pear infected with ASGV to optimise assay conditions Feb 99
ii) PCR and ELISA assays will have been conducted on 100 cvs from Brogdale for comparison with bioassays Jun 99
3 a,b Sufficient sequence information will have been obtained for the coat protein gene of ASPV to have been cloned into one or more of the pPIC vectors
3 c i) Yeast will have been transformed with the Pichia vectors containing the coat protein region sequences of ASGV and ASPV Jun 99
ii) Recombinant yeasts that express the ASGV and ASPV coat proteins will have been selected Aug 99
4c i) A subtractive cDNA library will have been constructed from SCV template RNA Dec 98
ii) The library will have been screened by +/- differential hybridisation
4d Up to four of the longest SCV clones will have been selected and partially sequenced to confirm their viral origin Aug 99
These milestones were achieved with the following exceptions.
1d Clones derived from material infected with ASPV isolate 7/40 have so far contained only plant and not virus-related cDNA. This milestone was therefore carried forward to year 3.
3 Two yeast cell lines were transformed, via homologous recombination, with both ASGV and ASPV coat protein containing expression vectors. However, recombinant protein expression was not detected in initial small-scale expression studies. This milestone was therefore carried forward to year 3.
4 The milestones in objective 4 (strawberry crinkle virus – SCV) depended upon a positive outcome to milestones 4c ii) Screening the library and 4d Partial sequencing of SCV clones to confirm their viral origin. By the end of year 2 only plant related cDNA had been obtained. The milestones for objective 4 were re-scheduled in year 3 to take account of this.
Milestones for year 3
1 d Obtain nucleotide sequence data for ASPV isolate 7/40 Apr 00
1 e i) The PCR protocol will be tested for its ability to amplify ASPV in extracts from woody plants in the autumn Nov 99
ii) The protocol will be adjusted as necessary for use with extracts from trees, using experimental material obtained in the autumn and herbaceous extracts spiked with frozen leaf and bark from apple Mch 00
iii) The final protocol will have been assessed with samples from trees from a) the East Malling collection, b) any positives from 2a and c) key isolates from overseas Jun 00
2a The standard double-budding field assay for ASGV in 100 apple/oriental pear cvs will have been completed Nov 99
2d The glasshouse assay for ASGV on the same 100 cvs as used in 2a will have been completed Aug 00
3c i) Expression of the coat proteins by the yeast recombinants will have been optimised and scaled up Oct 99
ii) Protocols for purifying the expressed proteins will have been devised and used to produce immunogens Dec 99
iii) ASPV coat protein will be expressed and purified from E. coli Aug 00
3d i) Antisera will have been prepared by immunising rabbits Mch 00
ii) Antisera will have been evaluated for the detection of ASPV and ASGV in extracts from herbaceous and woody plants Jun 00
4c ii) Continue screening the cDNA libraries until SCV clones are found
4d Sequence up to four of the longest SCV clones to confirm their viral origin
i) SCV clones will have been analysed to produce a partial map of the viral genome Jun 00
ii) Amplification primers will have been designed, a range of SCV isolates amplified and the primers modified accordingly, to provide primers that satisfactorily amplify the range of isolates in Physalis Jun 00
4e The primers will be assessed for their use in amplifying SCV in extracts from stawberry plants and a protocol for field use will have been developed
These milestones were met with the following exceptions.
3 c) and 3 d) The objectives concerning expression of virus coat protein in yeast were not met due to problems with erratic expression, thus preventing optimisation of expression conditions and protein purification.
Medium scale production and native purification of the protein was achieved in E. coli. Sufficient protein has been purified for antiserum production but antiserum was not obtained before the project ended.