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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


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).


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


Cucumber green mottle mosaicVirgaviridae; Tobamovirus

UK, Europe, Asia, Middle East, Canada, USA• August 2014: Northern territory – watermelon, squash

• April 2015: Queensland - watermelon

• 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


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

http://provenancegrowers.blogspot.com.au/2010/07/sorting-vegie-seeds.html


Pospiviroids• Potato spindle tuber viroid (PSTVd)

• Pepper chat fruit viroid (PCFVd)

• Tomato chlorotic dwarf viroid (TCDVd)

• Tomato apical stunt viroid (TASVd)

• Tomato planta macho viroid (TPMVd)

• Columnea latent viroid (CLVd)

• Citrus exocortis viroid (CEVd)

• Host range: Tomato, Capsicum, Potato • Other solanaceous and non-solanaceous hosts

• Transmission: Seeds, mechanical, water, pollen

• 2008: Importation of tomato seed regulated - PSTVd testing• 2012/13: Testing seed for all viroids that infect Tomato and Capsicum

TCDVd

PSTVd


Viroid interception rates in seed

2008-2012: PSTVd testing only2012-2016: all Pospviroids tested

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2008 (11) 2009 (11) 2010 (8) 2011 (5) 2012 (117) 2013 (632) 2014 (330) 2014 (615) 2015 (672) 2016 (518) Total

Proportion (%) of viroid positive seed each year between March 2008 and December 2016 intercepted at the Australian border


Which viroid and which region?

• Six Pospiviroid species

• Significant genetic diversity amongst intercepted isolates

• 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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The number of positive seed lots from each seed producing region

nu

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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


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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e

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


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


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.


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


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:


Acknowledgments

• The Plant Micro Team(AgriBio - AgVic)

• Chris Bottcher, Geoff Kelly, Cliff Kinoti, Jason Shiller, Ruvinda Perera, Brendan Rodoni, Daniel Timblin, Linda Zheng Joanne

Mackie, Mark Gibbs, Kevin Davis (DAWR)

• Grant Chambers, Mary Ann Terras (NSW DPI EAMI)

• Lucy Tran Nguyen, DPIF, NT

• Trandos Hydroponic Growers

• ISHI Veg

• Seed companies

• Funding: DEDJTR, NSW DPI, DAWR, PBCRC, HIA Limited

• For more information, please email [email protected]

Download - Virus and viroid testing of solanaceous and cucurbit seed shipments to Australia

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