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

    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

  • 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

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

  • biosecurity built on science

    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

  • biosecurity built on science

    Viroid interception rates in seed

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

    % c

    on

    tam

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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

  • biosecurity built on science

    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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    45

    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

    ica

    Asia

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    Euro

    pe

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    Ea

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    al A

    merica

    No

    rth

    Am

    eri

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    South

    Am

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    Un

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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

  • 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

    0

    5

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    15

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    35

    40

    Australia

    Naktuinbouw

    ISHI-veg

    Squash seed

    Ave

    rage

    Ct

    valu

    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

  • 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

    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]