biosecurity built on science Virus and viroid testing of solanaceous and cucurbit seed shipments to Australia (2148) AgVic: Fiona Constable (Plant Virologist) AgVic: Brendan Rodoni NSW DPI: Mary Ann Terras, Andrew Daly DAWR: Mark Gibbs, Kevin Davis Plant Biosecurity Cooperative Research Centre
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Virus and viroid testing of solanaceous and cucurbit seed shipments to Australia
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biosecurity built on science
Virus and viroid testing of solanaceous and cucurbit seed shipments to Australia (2148)
AgVic: Fiona Constable (Plant Virologist)
AgVic: Brendan Rodoni
NSW DPI: Mary Ann Terras, Andrew Daly
DAWR: Mark Gibbs, Kevin Davis
Plant Biosecurity Cooperative Research Centre
biosecurity built on science
230+ seed-borne viruses and viroids
Internationally traded botanical seed is a pathway for introduction
Australia imports most of its tomato, capsicum and cucurbit seed
Tomato and Capsicum- Pospiviroids- Pepino mosaic virus
Cucurbits- Cucumber green mottle mosaic virus, Melon necrotic spot virus
Seed-borne plant virus and viroids
Reingold et al 2015. CGMMV on the seed surface (top left) and in the endosperm of seed (bottom right).
Matsushida and Tsuda et al 2016. PSTVd in tomato seed (left).
biosecurity built on science
Establish Australian developed seed testing protocols as an international standard for detection of viroids and CGMMV in seed
Reduce the risks presented by contaminated traded seed.
Objectives:
1. Validate and establish cost-effective molecular tools for testing for viroids and CGMMV in seed.
2. Measure infection rates and assess the risks posed by contaminated seed.
3. International harmonisation of the seed testing diagnostic protocols for viroids and CGMMV .
Aim
biosecurity built on science
Cucumber green mottle mosaicVirgaviridae; Tobamovirus
• May 2016: Western Australia – cucumber and watermelon
• March 2017: Queensland - cucumber
Host range: Primarily infects cucurbit crops
Transmission: Mechanical, pollen, insects, seed
Reingold et al. 2013.
Cucumber leaf symptoms and fruit symptoms; A. Dombrovsky
Affected watermelon fruit –intercepted in Victoria
biosecurity built on science
CGMMV interceptions in seed
Imported seed: October 2014 – December 2016
• >1100 quarantine seed submissions (EAMI and CHS)• Tested by ELISA
• 9400 or 20%
• 30 (2.3%) submissions positive
• Proficiency test: • ELISA is less sensitive than PCR• Seed type affects ELISA and PCR results• False negative results? Watermelon, squash, cucumber and pumpkin seed
• Only two PSTVd isolates from seed matched Australian PSTVd isolates
• Some viroids may be present in regions where they have not been previously reported
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PSTVd CLVd PCFVd TASVd TCDVd TPMVd CEVd
Number of tomato seed lots intercepted between March 2008 and December 2016 carrying a viroid species
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Afr
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Asia
East
Asia
Sou
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Euro
pe
Mid
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Ea
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merica
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rth
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South
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Un
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ow
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The number of positive seed lots from each seed producing region
nu
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f p
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d lo
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nu
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biosecurity built on science
The Australian interception rate for viroids in seed is greater than elsewhere
Critical differences in methodology affect detection rates:
• Extraction methods
• Seed sample and sub-samples size
• PCR tests
Laboratory
Seed sample dilutions A B C D
1000 infected seed 4/4 4/4 4/4 4/4
100 infected seed in 900 0/4 0/4 4/4 4/4
10 infected seed in 990 0/11 0/11 10/11 11/11
0 infected seed 0/5 0/5 0/5 0/5
Proficiency Test: Detection of PSTVd in tomato seeds December 2015
biosecurity built on science
Front end impacts for PCR: Nucleic acid extraction efficiency/quality in tomato and squash
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Australia
Naktuinbouw
ISHI-veg
Tomato seed
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Australia
Naktuinbouw
ISHI-veg
Squash seed
Ave
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Spike in dilution (72.3ng to 7.23pg)
• Australian and Naktuinbouw RNA extraction protocols were most sensitive and reliable
• Naktuinbouw RNA extraction method was more consistent
• Proportion of RNA detected in squash seed was much less than tomato seed• squash seed substrate has more inhibitors
biosecurity built on science
Seed test rates: biology and assumptions• Risk and seed sample size:
• Transmission rates and subsequent rate of spread• The number of seed producing plants• Size of the seed lot
• International assumptions (viroids):• Seed lot consists of 3,300,000 seed (10kg) produced from 1000 plants• Rate of transmission is low• More than one seed production plant is infected• Viroids will disperse to all seed in all fruits of an infected plant – nutritional sinks• Viroids may contaminate uninfected seed during extraction
• Biology, interception rates and sporadic incursions may not support assumptions• Time of infection• Point of infection• Plant host/cultivar• Virus/viroid species and strain• Virus/viroid concentration• Environment• One sub-sample may be positive• Outbreaks occur even when tested seed is used• Contaminated seed may not always lead to an outbreak
biosecurity built on science
Detection thresholds and sample size
Thresholds and sample size are difficult to determine:
• Larger sample sizes → Lower percentage of seed infections in commercial seed• CGMMV 20% or 9400 seed
• DAWR PRA – detecting a 0.05% infection rate in 100,000 seed • 99% confidence/9400 seed cf. 63% confidence/2000 seed
• Viroids 20% or 20,000 seed• A single sub-sample in 50 sub-samples (400 seed each) can be positive • Variable concentrations in individual seeds → smaller sub-sample sizes
• The low level of contamination in seed and the observation of out breaks in Australia justify the requirement to test larger samples, in smaller sub-samples sizes.
biosecurity built on science
Recommendations:CGMMV:• Retain a 9400/20% sample size
• Molecular testing for CGMMV and other viruses in cucurbit seed
• It may be possible to increase sub-sample size for some seed types
Viroids:• Retain a 20,000/20% sample size
• It may be possible to increase sub-sample size
Seed testing:• Seed sample and sub-sample size needs to be practical for laboratory testing
• Cost effective – labour, equipment and consumables
• Timely
• Accept both the Australian and Naktiuinbouw extraction methods
• Accept the current Pospiviroid conventional PCR, adopt the RT-qPCR for PSTVd and CLVd
• RT-qPCR for CGMMV and a backup conventional PCR test
• Incorporation into national and international protocols
biosecurity built on science
International workshop September 2017:
- Consider the different testing protocols and relevant regulations in the context of the scientific outcomes of the project.
- Facilitate discussion and acceptance of Australian testing standards
Australian and international regulators and scientists
Australian Seed Federation
ISHI-Veg
Seed company representatives
Outcomes:
- Improved international biosecurity
- Harmonized testing
Outlook:
biosecurity built on science
Acknowledgments
• The Plant Micro Team(AgriBio - AgVic)
• Chris Bottcher, Geoff Kelly, Cliff Kinoti, Jason Shiller, Ruvinda Perera, Brendan Rodoni, Daniel Timblin, Linda Zheng Joanne