Developing molecular diagnostics for the detection of strawberry viruses, following BS07003 (BS10002)
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What was it all about?
Certified strawberry runners were supplied through the Victorian and Queensland strawberry certification schemes to most strawberry fruit growers across Australia. These strawberry runners were certified as high health planting material and were derived from high quality nucleus plants. The nucleus collection was indexed annually for the major strawberry viruses including Strawberry mottle sadwavirus (SMoV), Strawberry crinkle cytorhabdovirus (SCV), Strawberry mild yellow edge potexvirus (SMYEV), Strawberry vein banding caulimovirus (SVBV), Beet pseudos yellows crinivirus (BPYV), and Strawberry pallidosis associated crinivirus (SPaV) and Strawberry necrotic shock virus (formally thought to be Tobacco streak virus), as well fungal diseases. It was a fundamental requirement of the certification scheme that these pathogens were not detected in any strawberry plant within the nucleus collection. A strawberry nucleus collection had been maintained for the Victorian strawberry industry for nearly 50 years and these high health plants contributed greatly to increased yields for strawberry growers due to the exclusion of the major pathogens from industry.
Scientists from DPI-Knoxfield developed rapid and sensitive molecular diagnostic tests for the detection of endemic viruses of strawberries that were tested for in Australian certification schemes. Molecular indexing via the polymerase chain reaction (PCR) offered the Australian strawberry industry a more rapid and cost effective method of indexing the strawberry nucleus collection.
In the past, detection of these viruses relied on the use of the biological indexing method of petiole grafting onto sensitive indicator species. While this method was reliable and sensitive it was labour intensive, expensive and was time consuming taking 6-8 weeks for results based on symptom expression. Additionally, it could only be reliably done in the spring and early summer months of each year. Adoption of the molecular based tests, such as PCR offered the Australian strawberry industry a more rapid and cost effective method of indexing the strawberry nucleus collection. PCR returned a diagnosis in 1-2 days resulting in a much reduced cost to industry for the annual indexing of the nucleus collection.
A “dummy nucleus” of strawberry cultivars that were inoculated with a range of viruses was established and maintained under the same conditions as the Victorian strawberry nucleus plants. These plants were tested monthly for viruses during three growing seasons to determine the reliability of the PCR tests that were developed by DPI. The results indicated that there was a seasonal effect on detection of some viruses in strawberries and spring (October-December) and autumn (March-May) were the best times for virus detection in Victoria.
In November 2010 a replicated “dummy nucleus” was also established in Queensland and the plants were tested monthly until July 2011. The results indicated that virus detection was variable under these conditions and there was no single month in which all viruses were reliably detected. It was therefore recommended that under these climatic conditions that two PCR tests were conducted each year in December - January and again in July.
Molecular tests were validated for eight high priority pests and diseases that posed a quarantine risk to the local Strawberry industry and must be tested for during post entry quarantine (PEQ) including: Xanthomonas fragariae (angular leafspot), Phytophthora fragariae var. fragariae (Strawberry red stele), Arabis mosaic nepovirus (ArMV), Raspberry ringspot nepovirus (RpRSV), Tomato ringspot nepovirus (ToRSV), Tomato black ring nepovirus (TBRV) Strawberry latent ringspot sadwavirus (SLRV) and Tomato bushy stunt tombusvirus, (TBSV).
The protocols for both endemic viruses and quarantine pathogens were incorporated into a pathogen-testing manual that could be used by pathologists and industry in Australia. The protocols combined traditional biological indexing with the new molecular methods to improve the stringency of strawberry virus testing for the Australian strawberry industry. The molecular tests were also incorporated into DPI-Knoxfield’s fee for service unit (Crop Hygiene – Crop Health Services) and supported the Australian strawberry certification schemes. The technology was also transferred to Queensland pathologists.
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This project was funded by Hort Innovation (then Horticulture Australia Limited) with the financial support of the Department of Employment, Economic Development and the Victorian Strawberry Industry Certification Authority.
Copyright © Horticulture Innovation Australia Limited 2011. The Final Research Report (in part or as whole) cannot be reproduced, published, communicated or adapted without the prior written consent of Hort Innovation (except as may be permitted under the Copyright Act 1968 (Cth)).