19-22 november 2012 hanmer springs, new zealand

10TH AUSTRALASIAN PLANT VIROLOGY WORKSHOP

ABSTRACT AND PROGRAMME BOOK

19-22 November 2012Hanmer Springs,

New Zealand

CRC PLANTbiosecurity

Thank you to our conference sponsors

WWW.APVW2012.COM


APVW 2012 | Hanmer Springs, New Zealand page 3

Haere maiWelcome to the 10th Australasian Plant Virology Workshop (APVW) and to Hanmer Springs in New Zealand. This wonderful alpine town provides an ideal setting for us to maintain and nurture the interactive and collegial tradition of past APVW workshops for participants to present and discuss plant virus and virus-like organism research relevant to Australasia.

Building on the foundation of preceding workshops we have taken the opportunity at this meeting to integrate virus-like organism research into the program based on research theme, rather than by taxa. Posters are an important method of conveying research results and outcomes. To further unify the science being presented by research theme, we have incorporated the presentation of a poster précis into the oral sessions, rather than run a separate poster session. We have been delighted by the quality and breadth of the submissions, and hope we had done justice to all in our assembly of the science program for our meeting.

This meeting is latest in the series convened by the Plant Virology Working Group, a Special Interest Group of the Australasian Plant Pathology Society. We are indebted to APPS for their ongoing support for our meeting. We also wish to thank and acknowledge the support of Plant & Food Research and the Plant Biosecurity CRC. The generosity of all our sponsors has made this workshop possible.

We especially thank Yvonne McDiarmid, Rebecca Swaney and Donna Gibson, whose work ‘behind the scenes’ has made everything possible.

It’s our pleasure to welcome you, and be your host, at the 10th APVW in this superb alpine town in the Hurunui district of Canterbury, New Zealand.

Hei kona mai

Organising Committee 10th APVW 2012Grant Smith, Robin MacDiarmid, John Fletcher, Ian Scott, Arvind Varsani, Mike Pearson

APVW 2012 | Hanmer Springs, New Zealandpage 4

ContentsOrganising Committee ...................................................................................................................6

Social Programme .........................................................................................................................7

Summary programme ....................................................................................................................8

Programme ..................................................................................................................................10

Abstracts ....................................................................................................................................15


10th Australasian Plant Virology Workshop

Plant & Food Research and the Australasian Plant Pathology Society are proud to present the 10th Australasian Plant Virology Workshop.

Venue

Heritage Hotel 1 Conical Hill Road Hanmer Springs Canterbury New Zealand

Opening Reception

Tuesday 20th November at 7pm at the Heritage Hotel

Workshop Open

Wednesday 21st November at 9 am

Workshop Close

Thursday 22nd November at 1 pm


Grant Smith Plant & Food Research, Lincoln

Robin MacDiarmid Plant & Food Research, Mt Albert

John Fletcher Plant & Food Research, Lincoln

Ian Scott Plant & Food Research, Lincoln

Arvind Varsani University of Canterbury, Christchurch

Mike Pearson University of Auckland, Auckland

Organising Committee


Social Programme

Welcome Reception Monday 19th November | 7pm – 9pmRegistration desk will open from 6pm – 7pm.

Drinks and canapés will be served in the conference room following registrations.

Hanmer Springs Thermal Pools Tuesday 20th November | 9 - 11pm onwardsDelegates have the opportunity to make full use of the thermal pools opposite the Heritage Hotel. Change into your bathers/togs, don the Heritage robe and saunter across to the pools.

A poolside bar service will be available (cost of drinks will be on your own account). Pools close at 11.30pm.

Conference Dinner Wednesday 21st November | 7pm onwardsPre-dinner drinks will be served at Isobels bar from 7 – 7.30pm with seated dinner following in the ballroom.


Summary programmeMonday 19th November 20123:00 pm Bus departs Christchurch Airport Hotels for Hanmer Springs

5:30 pm Arrive Hanmer Springs

6:00 pm Accommodation check-in and Workshop registration

7:00 pm Welcome function at the Heritage Hotel

Tuesday 20th November 20129:00 am Open Workshop

9:15 am R.E.F Matthews’ Memorial Lecture | Chair: Grant Smith

10:15 am Morning tea

10:45 am Session One: New tools and technologies | Chair: Arvind Varsani

1:00 pm Lunch

1:45 pm Session Two: New and Emerging Viruses and VLOs | Chair: Mike Pearson

3:15 pm Afternoon Tea

3:45 pm Session Three: New and Emerging Viruses and VLOs | Chair: Mike Pearson

5:15 pm End of Presentations

9:00 pm Hanmer Thermal Pools Experience


Summary programmeWednesday 21st November 20129:00 am Session Four: Control and Eradication | Chair: John Fletcher

9:30 am Session Five: Interactions with Hosts and Vectors | Chair: John Fletcher


11:00 am Session Six: Interactions with Hosts and Vectors | Chair: Colleen Higgins

1:00 pm Lunch

1:45 pm Session Seven: Interactions with Hosts and Vectors | Chair: Robin MacDiarmid

3:30 pm Afternoon Tea

4:00 pm Session Eight: Interactions with Hosts and Vectors | Chair: Kieren Arthur

6:00 pm End of Presentations

7:00 pm Pre-dinner drinks

7:30 pm Workshop Dinner

Thursday 22nd November 20129:00 am Session Nine: Genomes and Genetics | Chair: Ian Scott


10:50 pm Session Ten: Genomes and Genetics | Chair: Ian Scott

12:15 pm APVW Meeting and Workshop Close | Chair: Grant Smith

1:00 pm Lunch

1:45 pm Buses depart from Heritage Hotel for Christchurch


ProgrammeMonday 19th November 20123:00 pm Buses depart Christchurch Airport Hotels for Hanmer Springs

5:30 pm Arrive Hanmer Springs

6:00 pm Accommodation check-in and Workshop registration

7:00 pm Welcome function at the Heritage Hotel

Tuesday 20th November 20129:00 am Open Workshop

9:15 am REFM R.E.F Matthews’ Memorial Lecture | Chair: Grant Smith James Dale Viruses: so many roles to play in the genetic improvement of bananas in East Africa


SESSION ONE: NEW TOOLS AND TECHNOLOGIES | Chair: Arvind Varsani

10:45 am S1-O1 Michael Jones Tailoring high-throughput sequencing approaches to next-generation plant virology in south-west Australia

11:05 am S1-O2 Lisa Ward Diagnosis of viruses and virus-like organisms using 454 sequencing

11:25 am S1-O3 Smriti Nair Universal primers for the amplification of EF1α and F-box and their usefulness as reference genes in taro and N. benthamiana

11:45 am S1-O4 Fiona Constable Molecular diagnostics for the reliable detection of strawberry viruses in Australia

12:05 pm S1-P1 Subuhi KhanRecommendations for the use of generic primers for detection of Dasheen mosaic virus

12:10 pm S1-O5 Daniel Cohen Grapevine leafroll-associated virus 3 strains infecting grapevines in New Zealand

12:30 pm S1-O6 Jenny VoCharacterisation of the Banana streak virus coat protein and linear epitope analysis using a pepscan approach

12:50 pm S1-P2 Roger Jones Development of a quantitative, bulk seedling test for seed transmission of Wheat streak mosaic virus in wheat seed samples using real time RT- PCR

12:55 pm Session One Close

1:00 pm Lunch

APVW 2012 | Hanmer Springs, New Zealand page 11page 11

Programme continuedSESSION TWO: NEW AND EMERGING VIRUSES AND VLOS | Chair: Mike Pearson

1:45 pm S2-O1 Fiona ConstablePhytoplasma associated diseases of potatoes in Australia

2:05 pm S2-O2 Arnaud Blouin A review of viruses infecting Actinidia

2:25 pm S2-O3 John Randles Detection of Fig mosaic virus in Australia, and development of tools to monitor its epidemiology

2:45 pm S2-O4 Murray Sharman A distinct polerovirus infecting crop and weed legumes from eastern Australia is transmitted by cowpea aphid

3:05 pm S2-P1 Heiko Ziebell A virus survey of Pisum sativum crops in Germany, 2012

3:10 pm S2-P2 John Fletcher A virus survey of Vicia faba crops in Canterbury, New Zealand, 2011-12

3:15 pm Session Two CloseAfternoon Tea

SESSION THREE: NEW AND EMERGING VIRUSES AND VLOS | Chair: Mike Pearson

3:45 pm S3-P1 Anisha Dayaram Novel Cassava associated single stranded DNA virus

3:50 pm S3-P2 Kathy Parmenter Barley yellow dwarf and Cereal yellow dwarf viruses detected in oats and barley grown in Queensland and New South Wales

3:55 pm S3-O1 Joe Tang The diversity of Strawberry latent ringspot virus in New Zealand

4:15 pm S3-O2 Simona Kraberger Diversity of monocot-infecting mastrevirus in Australia rivals that in Africa

4:35 pm S3-O3 Mahmoud Khalifa Molecular characterisation of three mitoviruses infecting a hypovirulent isolate of the phytopathogenic fungus Sclerotinia sclerotiorum

4:55 pm S3-O4 Robin MacDiarmid Plant virus discovery and the implications for biosecurity

5:15 pm Session Three Close

9:00 pm Hanmer Thermal Pools Experience


Programme continuedWednesday 21st November 2012

SESSION FOUR: CONTROL AND ERADICATION | Chair: John Fletcher

9:00 am S4-O1 Roger Jones Pathogenicity and epidemiology of Potato spindle tuber viroid in Australia

9:20 am S4-P1 Mohammad Aftab Assessment and validation of DAFWA canola model for predicting BWYV outbreaks in pulses and canola in Victorian conditions

9:25 am Session Four Close

SESSION FIVE: INTERACTIONS WITH HOSTS AND VECTORS | Chair: John Fletcher

9:30 am S5-O1 Brenda Coutts Zucchini yellow mosaic virus in cucurbits: transmission by contact, stability on surfaces, and inactivation with disinfectants

9:50 am S5-O2 Wee-Leong Chang Molecular variation in Dasheen mosaic virus: dynamics of virus population structure in evolutionary space and time

10:10 am S5-O3 Subuhi Khan Genetic diversity within and between different strains of Dasheen mosaic virus and its potential impact on diagnosis

10:30 am Session Five CloseMorning tea

SESSION SIX: INTERACTIONS WITH HOSTS AND VECTORS | Chair: Colleen Higgins

11:00 am S6-O1 Monica Kehoe Unravelling the cause of Black Pod Syndrome of narrow-leafed lupin

11:20 am S6-O2 Robin MacDiarmid Identification of a naturally occurring, mild isolate of Tamarillo mosaic virus

11:40 am S6-O3 Ron van Toor Aphid species feeding behaviour affects acquisition of potato potyviruses

12:00 pm S6-O4 Simon Bulman Detection of the brassica-infecting viruses TuMV and TuYV in aphids

12:20 pm S6-O5 Melanie Davidson Virus surveillance by proxy; monitoring insect vectors to detect plant viruses

12:40 pm S6-O6 Ralf Dietzgen Localization and interactions of Impatiens necrotic spot tospovirus proteins in Nicotiana benthamiana

1:00 pm Session Six CloseLunch


Programme continuedSESSION SEVEN: INTERACTIONS WITH HOSTS AND VECTORS | Chair: Robin MacDiarmid

1:45 pm S7-O1 Elizabeth Woo Molecular detection and characterisation of Cherry leaf roll virus in New Zealand

2:05 pm S7-O2 Roger Jones Influence of climate change on plant virus disease infections and epidemics

2:25 pm S7-O3 Narelle Nancarrow The effect of elevated temperature on the titre of Barley yellow dwarf virus-PAV in wheat cv. Yitpi using a multiplex normalised RT-qPCR assay

2:45 pm S7-O4 Mary Horner The effect of mineral nutrient availability to herbaceous indicators influences the disease expression of Apple stem grooving virus (ASGV) and Tobacco ringspot virus (TRSV)

3:05 pm S7-O5 Gardette Valmonte Is the most conserved calcium-dependent protein kinase important in plant viral responses?

3:25 pm S7-P1 Kieren Arthur Identifying Calcium dependent protein kinases that respond to virus infection

3:30 pm Session Seven CloseAfternoon Tea

SESSION EIGHT: INTERACTIONS WITH HOSTS AND VECTORS | Chair: Kieren Arthur

4:00 pm S8-O1 Tracey Immanuel A Novel Antivirus Defence in Plants? Characterization of CPK

4:20 pm S8-O2 Mary Horner Optimisation of herbaceous indicator screening for virus infection

4:40 pm S8-O3 Mike Pearson A re-evaluation of commonly held views on mycoviruses and their interaction with their fungal hosts

5:00 pm S8-O4 John Randles Spread of Grapevine leafroll-associated virus 1 and Grapevine virus A in a Cabernet Sauvignon vineyard in South Australia

5:20 pm S8-P1 Mahjoub Ejmal Characterisation and biological properties of isometric viruses infecting Aspergillus

5:25 pm S8-P2 Roger Jones Detection of the virus vector Polymyxa graminis infecting wheat roots in Western Australia

5:30 pm S8-P3 Brendan Rodoni Breeding for PVY resistance in Australian potato germplasm

5:35 pm S8-O5 Muhammad Shafiq The role of Cauliflower mosaic virus (CAMV) defence and silencing suppressor protein 6 (P6) in modulating auxin signalling

5:55 pm Session Eight Close

7:00 pm Pre-dinner drinks

7:30 pm Workshop Dinner


Programme continuedThursday 22nd November 2012

SESSION NINE: GENOMES AND GENETICS | Chair: Ian Scott

9:00 am S9-O1 Arvind Varsani Geminivirus phylogeography

9:20 am S9-O2 Daisy Stainton Recombination and re-assortment events detected in the multi-component Banana bunchy top virus (BBTV)

9:40 am S9-O3 Sarah Thompson Provisional assembly and analysis of two Candidatus Liberibacter solanacearum genomes derived from independent New Zealand sources

10:00 am S9-O4 Arvind Varsani A top-down approach: Discovery of a novel mastrevirus and alphasatellite-like circular DNA in the Caribbean using dragonflies

10:20 am Session Nine CloseMorning Tea

SESSION TEN: GENOMES AND GENETICS | Chair: Ian Scott

10:50 am S10-O1 Colleen Higgins Genome organisation of Vanilla mosaic virus as determined by deep sequencing

11:10 am S10-O2 Linda Zheng Genome sequencing of Australian Potato virus Y isolates

11:30 am S10-O3 Dan Cohen Molecular characterisation of two divergent strains of Grapevine leafroll-associated virus 3 in New Zealand

11:50 am S10-P1 Mike Pearson The molecular variability of Zucchini yellow mosaic virus and Papaya ringspot virus in the Pacific Islands

11:55 am S10-O4 Murray Sharman Complete genome sequences and genetic diversity of subgroup 1 Ilarviruses from eastern Australia

12:15 pm Session Ten Close

12:15 pm APVW Meeting | Chair: Grant Smith

12:55 pm Workshop Close

1:00 pm Lunch

1:45 pm Buses depart from Heritage Hotel for Christchurch


ABSTRACTS

APVW 2012 | Hamner Springs, New Zealandpage 16

Notes

REFM

Viruses: so many roles to play in the genetic improvement of bananas in East Africa

James DaleQueensland University of [email protected]

James Dale(1), Jerome Kubira(2), Charles Changa(2), Laura Karanja(3), Julius Mugini(4), Misek Soko(5), Geofrey Arinaitwe(2), Wilberforce Tushemeirwe(2), Jean-Yves Paul(1), Bulu Mlazazi(1), Robert Harding(1), Harjeet Khanna(1), Anthony James(1)

(1)Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Queensland, Australia(2)National Agricultural Research Organisation, Kampapa, Uganda (3)Kenyan Agricultural Research Institute, Njoro, Kenya(4)Mikocheni Agricultural Research Institute, Dar es Salaam, Tanzania(5)Bvumbe Agricultural Research Station, Limbe, Malawi

Bananas are an incredibly important crop in East Africa. In Uganda, they are the staple food with consumption of cooking bananas up around 1kg per person per day. They are also a major food source in Rwanda, Burundi, Democratic Republic of Congo and to a lesser extent in Kenya and Tanzania. In West Africa, plantains are a major food crop and, in southern Africa, bananas an important dietary component and cash crop but primarily as a dessert fruit. Unfortunately, these bananas are essentially sterile which has two negative impacts: conventional breeding is nearly impossible and they are vegetatively propagated with the result that banana viruses are widespread and often devastating. In 2005, we started a project funded by the Bill and Melinda Gates Foundation to enhance the bananas of East Africa, particularly East African Highland bananas and Sukali Ndizi, with elevated levels of pro-vitamin A and iron to assist in overcoming the widespread and severe effects of micronutrient deficiencies. Over the past seven years of the project, banana viruses have played key roles from very different aspects. It was important to have robust and appropriate virus diagnostics for two reasons: firstly, we were planning on genetically modifying the bananas and it was essential that our starting material was virus free; secondly, ultimately these micronutrient enhanced bananas would need to be distributed to farmers and again it was essential to have a system for distributing virus tested GM banana planting material. We decided upon DNA based diagnostics to ensure consumables would be available to national labs and we surveyed widely to gain an estimate of the genetic variability particularly within banana bunchy top virus (BBTV) and banana streak virus (BSV). Out of this came a new virus diagnostic method for BSV and an insight into the restrictions of geographic distribution of these two viruses. However, the impact of these viruses was not only negative. We had previously characterised the promoters in each of the six BBTV DNA components and the effect of intron enhancement. We have used one of these promoters, BT4 enhanced with the rice actin intron, to drive the expression of phytoene synthase, one of the key enzymes we are overexpressing to generate bananas with elevated pro-vitamin A.

Notes

APVW 2012 | Hamner Springs, New Zealand page 17

S1 - O1

Tailoring high-throughput sequencing approaches to next-generation plant virology in south-west Australia.

Michael JonesMurdoch [email protected]

Stephen J. Wylie (1), Hua Li (1), Jamie Ong(1), Kingsley Dixon (2), Muhammad Saqib (1) and Michael G.K. Jones (1).(1)Plant Virology Group, Plant Biotehcnology Research Group, School of Biological Sciences and Biotechnology, Western

Australian State Agricultural Biotechnology Centre, Murdoch University, WA 6150, Australia(2)Botanic Gardens and Parks Authority, Kings Park and Botanic Garden, West Perth, WA 6005, Australia.

The south-west of Australia, part of an ancient landmass, is recognised for the many plants endemic to this region. It comprises a series of ecosystems with different species adapted to them, which have been geographically isolated for many millenia. Despite the many endemic species, very little research has been undertaken to study its indigenous viral flora. In addition to native plants, over the past two centuries many exotic plants and virus vectors have become established in Australia, and in addition to the likely presence of indigenous viruses, it seemed probable that the indigenous flora could be invaded by aggressive exotic viruses inadvertently imported in these new species. We are testing these ideas for a range of indigenous and exotic plant groups, with a focus on terrestrial orchids, a group of major conservation concern. Approaches we have used to identify RNA viruses are based on ‘Next Generation Sequencing’ (NGS) of RNA – extracted from single plants; sequencing RNA pooled from multiple plants coupled with subsequent matching of plants and viruses found; sequencing small RNA species generated by plants in defense of viruses; labeling RNA from individual plants before pooling and sequencing; as well as trying various methods to enrich plant RNA for viral transcripts before sequencing. The pros and cons of the various approaches tested will be discussed, together with summarising examples of a series of new viruses found, and implications of this work on conservation management.


Notes

S1 - O2

Diagnosis of viruses and virus-like organisms using 454 sequencing

Lisa WardMinistry for Primary [email protected]

Lia Liefting, Lisa Ward

Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140

MPI’s Plant Health and Environment Laboratory is responsible for the identification of new pests and diseases affecting plants, plant products and the environment. In some cases identification can take an extended period, especially for new pathogens and emerging diseases. Metagenomic analysis using next-generation sequencing has the potential to detect the full spectrum of pathogenic organisms in a single test and is therefore being implemented in our laboratory for plant pathogen diagnosis. Our strategy is to use total RNA in order to detect all pathogen types. The processes involved in preparing the RNA for 454 sequencing will be described. The method was validated on at least one representative from each of the major groups of plant pathogens and genome types, including RNA and DNA viruses, viroids and a liberibacter. The method also successfully diagnosed a previously uncharacterised virus.

Notes


S1 - O3

Universal primers for the amplification of EF1α and F-box and their usefulness as reference genes in taro and N. benthamiana

Smriti NairAuckland University of [email protected]

Smriti Nair, Colleen M. Higgins

School of Applied Sciences, Auckland University of Technology, New Zealand

Changes in the environment often induce variation in gene expression levels of an organism; for example, changes in host mRNA accumulation may occur in response to viral infection. Quantitative reverse transcriptase PCR (RT-qPCR) is currently the method of choice for measuring these changes; however, the use of appropriate reference genes is critical for accurate quantification of mRNA. For gene expression analysis to be valid, the mRNA level of the reference gene must remain stable under given experimental conditions, for example between healthy and infected plants. Housekeeping genes such as β-actin and 18S rRNA have been used as reference genes; however, many studies have shown significant variation in the expression stability of these genes under various experimental conditions, indicating these genes are not suitable as reference genes in many instances. Although immense efforts have been made to analyse host responses to viral infection, very few suitable reference genes have been identified that could be used for such studies. Lilly et al (2011) identified four genes namely, elongation factor α (EF1α), F-box, SAND family protein (SAND) and protodermal factor 2 (PDF2) as being suitable reference genes for studying changes in host mRNA accumulation in virus infected Arabidopsis thaliana. For these genes to be widely useful for virus studies, they must be validated in a range of hosts. This study assessed EF1α and F-box genes as reference genes in taro (a monocot) and Nicotiana benthamiana (a dicot) using RT-qPCR. N.benthamiana is of special interest as it is a widely used model host for virus studies while taro is an important staple crop in the South Pacific. Universal primers for the amplification of these genes from dicots and monocots were designed and tested on taro, A.thaliana and N.benthamiana using RT-PCR. It is expected that these primers will amplify these genes from any plant species. Further, these genes have been validated for use in virus infected taro and N.benthamiana widening their applicability for virus studies.

References: Lilly, S.T., Drummond, R.S.M., Pearson, M.N., & MacDiarmid, R.M. (2011). Identification and validation of reference genes for normalization of transcripts from virus-infected Arabidopsis thaliana. Molecular Plant-Microbe Interactions, 24 (3), 294-304


Notes

S1 - O4

Molecular diagnostics for the reliable detection of strawberry viruses in Australia

Fiona ConstableDepartment of Primary [email protected]

Fiona Constable(1), Lien Ko (2), Chris Bottcher (1), Geoff Kelly (1), Narelle Nancarrow (1), Mirko Milinkovic (1), Denis Persley (3), Brendan Rodoni (1)

(1)Department of Primary Industries - Knoxfield, Private Bag 15,Ferntree Gully Delivery Centre, Victoria 3156, AUSTRALIA(2)Department of Agriculture, Fisheries and Forestry, Maroochy Research Facility, 47 Mayers Road, Nambour, Queensland

4560, AUSTRALIA(3)Department of Agriculture, Fisheries and Forestry, Ecociences Precinct, 41 Boggo Road, Dutton Park, Brisbane,

Queensland 4102, AUSTRALIA

In recent years PCR assays have been developed for the detection of 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). To assist the integration and implementation of the PCR assays into pathogen testing protocols for production of certified strawberry runners in Australia, a virus inoculated “dummy nucleus” (“DN”) of two strawberry varieties was created in December 2008. The “DN” was maintained in a temperate climate in a similar manner to the Victorian nucleus collection and used to validate In 2010/11 the “DN” was also replicated in a subtropical climate in Queensland. The “DN” plants were tested once each year by biological indexing and were tested monthly PCR. PCR testing of the 2008/09 Victorian “DN” showed that viruses were not reliably detected in strawberry plants during the first six months post-inoculation. Reliable detection was only achieved during the following seasons and the rate of virus transmission from mother to daughter plants can reach 100%. Virus testing during spring (October-December) of the Victorian “DN” plants during the 2009/10 and 2010/11 was the most reliable time for virus detection. In some years autumn (March-May) may also be a reliable time for virus detection by PCR. The results for the Queensland “DN” collection during the 2010/11 season indicated that there may not be a trend for the timing of detection of all virus species and no single month or season appears was more reliable than another for virus detection. The results from Victoria and Queensland indicated that biological indexing is less reliable for virus detection than PCR techniques as many of the inoculated indicators that were expected to show symptoms were symptomless even though viruses could be detected by PCR. However, the PCR tests were also not 100% reliable because there were only a few months of the year in which viruses were detected in all known infected plants. This highlights the importance of timing for testing and using a combination of molecular and biological tests for certification.

Notes


S1 - P1

Genetic diversity within and between different strains of Dasheen mosaic virus and its potential impact on diagnosis

Subuhi KhanAuckland University of [email protected]

Subuhi Khan, Wee-Leong Chang , Gardette Valmonte , Smriti Nair, Colleen M. Higgins


The genus Potyvirus is one of the largest of plant viruses with around 180 definitive and possible members i.e. 30% of all known plant viruses. Therefore, precise and accurate identification of the potyviruses that infect commercially important crops is required to monitor disease incidence, to identify virus reservoirs and to recommend appropriate control mechanisms. The development of better anti-viral strategies for potyviruses is difficult because potyviruses have RNA genomes and RNA viruses live as quasi-species within the host plant. The diffuse ‘cloud-like’ nature of potyviral populations allows them to adapt rapidly to changing replicative environments by selecting pre-existing variants with better fitness. Therefore, several important virus properties cannot be explained by a single consensus sequence, but require knowledge about the microvariants present in viral populations within and between host plants. These sequence variants may be critically relevant to viral diagnostics, evolution, spread and virulence. With the advent of deep sequencing technologies, it is now possible to determine the full extent of genetic diversity of a virus within a host. We used Illumina technology to determine the diversity of the potyvirus Dasheen mosaic virus (DsMV) within and between host taro plants. The aims of this work were three-fold: firstly, to determine if different strains of the virus show the same degree of diversity; secondly, to identify conserved genome regions that may have functional importance, and determine if these are the same between different virus strains, and thirdly, determine if improved universal primers for potyvirus diagnostics can be designed. Parent plants infected with DsMV strains NZ or B were propagated into two daughter plants giving rise to isolates DsMV-NZ1.1 and NZ1.2, B1.1 and B1.2. Transcriptomes from these six plants were sequenced and the virus genomes assembled against the publicly available DsMV complete genome. Analysis of the consensus sequences for each assembly supported the placement of DsMV in the Bean common moaic virus group and showed that the B strain is more variable than the NZ strain. Variation within each genome assembly was similar to other assemblies, indicating that the degree of sequence plasticity was common between strains and isolates, and finite. The sequence diversity varied along the length of the genomes, with small regions of conservation. For example, the 5’ and 3’UTRs showed 27% and 28% variation overall, respectively. Within these regions, short stretches of nucleotides were conserved, which may have significant roles in translation and/or replication. Identification of such conserved regions and potential improvement of universal primer design is ongoing.


Notes

S1 - O5

Grapevine leafroll-associated virus 3 strains infecting grapevines in New Zealand

Daniel CohenThe New Zealand Institute for Plant & Food Research [email protected]

Daniel Cohen(1) , Arnaud Blouin(1), Kar Mun Chooi(3), Vaughn Bell(2), Michael Pearson(3), Robin MacDiarmid(1), (1)The New Zealand Institute for Plant & Food Research Ltd, Auckland, New Zealand(2)The New Zealand Institute for Plant & Food Research Ltd, Hastings, New Zealand(3)School of Biological Sciences, The University of Auckland, Auckland, New Zealand

Enzyme-linked immunosorbent assay (ELISA) is a very sensitive, robust, cost-effective diagnostic technique that is used widely to detect Grapevine leafroll-associated virus 3 (GLRaV-3), the most damaging grapevine virus in New Zealand. Commercial reagents from Bioreba detect GLRaV-3 with very high sensitivity. Analysis of test results from different vineyards showed that in some blocks all, or most, samples infected with GLRaV-3 appeared to be low titre, whereas other blocks showed a wide range of titre from high to very low. The conjugated antibody used in the Bioreba reagents is a monoclonal antibody raised against the type strain of GLRaV-3, NY1. To determine whether the apparent low titre in some blocks resulted from lower avidity to some strains of GLRaV-3 a second test was developed using polyclonal antibodies in a modified TAS-ELISA format. This test gave a much narrower range of apparent titre. The ratio of the reaction rates obtained using these two reagent sets is proving useful to distinguish vines infected with the novel GLRaV-3 strains, NZ1 and/or NZ2, from vines infected with the type strain NY1, against which the Bioreba reagents react strongly. As part of a continuing programme to control the spread of GLRaV-3 by annual roguing of symptomatic grapevines, cane samples were collected from 224 symptomatic vines and 11 asymptomatic vines of Cabernet Sauvignon in a Hawke’s Bay vineyard to confirm GLRaV-3 infection. Extracts of phloem scrapings were ELISA tested using two sets of antibodies and the reaction rate ratio of polyclonal to Bioreba tests was calculated from all positive samples. A selection (159) of these samples was also tested by RT-PCR using a set of strain specific and generic primers (see Chooi et al., this conference). High reaction rate ratios correlated with the presence of NZ1 and/or NZ2 and low ratios indicated the presence of NY1. Of the samples tested by RT-PCR, 53% were infected with NZ2, 40% with NY1, 5% with NZ1, and only 2% were infected with two strains, indicating that newly infected vines were predominantly infected with a single strain of GLRaV-3. In contrast, tests on vines from a heavily infected germplasm block found that most vines were infected with multiple strains of GLRaV-3. Our results demonstrate the complementarities of ELISA and RT-PCR in the detection and analysis of GLRaV-3 variants.

Notes


S1 - O6

Characterisation of the Banana streak virus coat protein and linear epitope analysis using a pepscan approach

Jenny VoThe University of [email protected]; [email protected]

Jenny Vo(1)(2) , Paul Campbell(4) , Ross Barnard(2)(3) , John Thomas(1)(3)(4) , Andrew Geering(1)(3)(4)

(1)CRC for National Plant Biosecurity, Innovation Centre, University of Canberra, Bruce, ACT, Australia(2)School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland, Australia(3)Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, Queensland, Australia(4)Queensland Department of Agriculture, Forestry and Fisheries, GPO Box 267, Brisbane, Queensland 4001, Australia

Banana streak disease is caused by a cryptic species complex of four officially recognized and several more tentative badnavirus species. These viruses are collectively known as banana streak virus (BSV). BSV is a plant pararetrovirus which uses reverse transcriptase to replicate its genome. The BSV genome consists of a non-covalently closed, circular, double-stranded DNA molecule of c. 7.2-7.8 kbp and contains three open reading frames (ORFs). ORF1 encodes a protein of unknown function, ORF2, a minor and probably multifunctional virion-associated protein and ORF3, a polyprotein that contains the precursors of the movement protein, coat protein, aspartic protease and reverse transcriptase with associated RNaseH1 activity. The mature coat protein is released from the polyprotein through the action of the aspartic protease but unlike other viral proteases, it is not possible to predict the cleavage sites by sequence analysis alone. In this study, the putative coat protein (CP) of BSMYV has been determined using a combination of N-terminal sequencing and mass spectroscopy techniques. In order to map the distribution of linear epitopes on the coat protein, a library of 16-mer biotinylated peptides spanning the length of the predicted coat protein was designed and used to react with BSMYV-specific rabbit polyclonal antibodies in an enzyme-linked immunosorbent assay. The epitope mapping data in combination with our predicted 3-D model of the coat protein shows that major linear epitopes are located mainly at the protruding N-terminus of the BSMYV coat protein. Two antigenic regions are located in the coil region and one epitope is located at a conserved helix-loop-beta motif of putative badnavirus coat proteins. Epitope-containing peptides identified in this study are currently being used to raise new antisera and could potentially be used to affinity purify specific antibodies from available antisera. Information obtained from this study can play an important role in developing new diagnostic assays for BSV, in better defining virion structure and possibly identifying new BSV serotypes.


Notes

S1 - P2

Development of a quantitative, bulk seedling test for seed transmission of Wheat streak mosaic virus in wheat seed samples using real time RT-PCR

Roger JonesUniversity of western [email protected]

Cox, Belinda A Luo, Hao and Jones, Roger AC(1)School of Plant Biology, University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA 6009, Australia(2)Department of Agriculture and Food, Locked Bag No.4, Bentley Delivery Centre, WA 6983, Australia

Wheat streak mosaic virus (WSMV) causes severe yield losses in wheat when a source of infection in present and weather conditions are conducive to build up of its wheat curl mite vector. WSMV is seed-borne in low levels in wheat. The virus is also present in the seed coat, so grow out testing is required to quantify the level of seed-borne infection in bulk wheat seed samples. Currently WSMV seed testing to determine levels of seed transmission to seedlings is carried out by ELISA. Seedlings are tested in groups of 10 or 20, which is a time consuming process. Usually, only 1000-2000 seeds are tested per seed lot, so lower levels of infection will not be detected. A bulk leaf RNA extraction method has been developed, where samples from up to 5000 wheat seedlings are tested as a single sample. As few as one WSMV infected seedling in a 5,000 seedling bulk sample can be detected reliably by RT-PCR. To accurately determine the number of WSMV infected seedlings present in a sample, a specific one-step, quantitative, real time reverse transcription PCR (RT-qPCR) using a dual-labelled probe has been developed. The assay has been optimised for sensitive detection, and has been tested on bulk wheat samples simulating different levels of seedling infection from 0.02% up to 1%. It has also been validated by testing seedlings from bulk WSWV infected seed samples by ELISA in addition to real time RT-qPCR. Currently the test is specific for WSMV but has the potential to be multiplexed for detection and quantification of other seed-borne wheat viruses in a single tube assay.

Notes


S2 - O1

Phytoplasma associated diseases of potatoes in Australia

Fiona ConstableDepartment of Primary [email protected]

Fiona Constable, Alan Yen, Jo Luck, Brendan Rodoni

Biosciences Research, Department of Primary Industries - Knoxfield, Private Bag 15 , Ferntree Gully Delivery Centre, Victoria 3156, AUSTRALIA

Potato purple top, potato purple top wilt, haywire disease and stolbur of potato are disease syndromes that occur in Mexico, USA, Europe, South America, Asia, Iran, New Zealand and Australia and can have a significant impact on plant growth and tuber quality and production. The diseases are associated with phytoplasma species representing seven taxonomically distinct phytoplasma groups (16SrI, 16SrII, 16SrIII, 16SrVI, 16SrXII, 16SrXIII and 16SrXVIII). These diseases can have similar symptoms to zebra chip disease which is associated with Candidatus Liberibacter solanacearum, a bacterium that is not known to occur in Australia and is considered a significant biosecurity threat. Phytoplasmas and Liberibacter species are un-culturable and require sensitive molecular diagnostic tests for their detection. A survey of 188 potato samples and three nightshade samples, collected from Victoria, Tasmania and Western Australia was conducted to determine the incidence of phytoplasmas and Ca. L. solanacearum in Australian potatoes and to validate molecular diagnostic tests for the detection of these pathogens under Australian conditions. Six of the 188 potato samples had symptoms of purple top and witches’ broom disease and phytoplasmas were detected by PCR in these samples. Sequence analysis of the 16S rRNA gene amplified by PCR indicated that the phytoplasmas detected in 4/6 potato samples, from cvs. Nadine and Russet Burbank, had 99% sequence similarity with Tomato big bud phytoplasma (TBBp; 16SrII group). Before the development of molecular tools it was assumed that TBBp was associated with these diseases in Australia and these results confirm that association. A previously uncharacterised phytoplasma was detected in 2/6 potato samples, cv. White Lady. Sequence analysis of the 16SrRNA gene, the 16S-23S intergenic spacer region (Genbank accession JQ740643) and the rp gene region (GenBank accession JQ740642), amplified by PCR from one of these samples, indicated that these regions had only 90-92%, 76% and between 69-71%sequence similarity respectively with several known phytoplasma species. The detection of a potential previously uncharacterized phytoplasma highlights the value of universal tests to detect genera of pathogens such as bacteria rather than a specific test to detect individual species. Ca. L. solanacearum was not detected in any of the 188 samples providing evidence of absence from the zebra chip associated pathogen for the states of WA, Victoria and Tasmania and demonstrates, in part, area freedom for this organism in Australia.


Notes

S2 - O2

A review of viruses infecting Actinidia

Arnaud BlouinThe New Zealand Institute for Plant & Food Research [email protected]

Arnaud G Blouin(1) , Ramesh R Chavan2(2), Michael N. Pearson(2), Robin M. MacDiarmid(1)(2), Daniel Cohen(1)

(1) The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland, New Zealand(2) School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand

Kiwifruit (Actinidia deliciosa) was introduced in New Zealand in 1904 from seeds imported from China. Since then seeds and scionwood has been imported to New Zealand from China, to increase the germpool for breeding purposes. In one importation, the first virus naturally infecting kiwifruit, Apple stem grooving virus, was identified following symptoms observed in quarantined plants (2003). Three novel viruses, two vitiviruses, and a citrivirus closely related to Citrus leaf blotch virus, have since been identified from the same importation. All belong to the family Betaflexivirdae. Members of the family Bromoviridae, Alfalfa mosaic virus and Cucumber mosaic virus (CMV), have been detected in the field. In New Zealand, these were mostly limited to Actinidia guilinensis and A. glaucophylla seedlings, while CMV was detected in A. chinensis in Italy. These viruses are cosmopolitan, and weeds provide a reservoir for infection. From the same family, Pelargonium zonate spot virus (PZSV) has been detected in Italy associated with severe symptoms on leaves and fruit. Four viruses appear to have limited effect on kiwifruit. Two tobamoviruses, Ribgrass mosaic virus and Turnip vein clearing virus are probably present worldwide in kiwifruit and are also present in Plantago spp., a common weed in kiwifruit orchards. Cucumber necrosis virus family Tombusviridae has been detected in kiwifruit at very low titre without apparent symptoms. Additionally, a novel potexvirus has been transmitted to herbaceous indicators from three kiwifruit plants, but direct detection from the original plants was unsuccessful. Finally, Cherry leaf roll virus (CLRV), family Secoviridae, has been detected in New Zealand on kiwifruit and also in Rumex spp growing below the infected vines. Symptoms observed on kiwifruit included leaf spots, fruit malformation, reduction in yield, bark cracking and cane wilting. Most of these symptoms have also been observed in Italy from PZSV infection.

Notes


S2 - O3

Detection of Fig mosaic virus in Australia, and development of tools to monitor its epidemiology.

John RandlesThe University of [email protected]

Suhair Hassan(1), Qi Wu(1), Richard Glatz(1,2), Nuredin Habili(1), Tamara Cooper(2), John Randles(1)

(1)School of Agriculture, Food & Wine, University of Adelaide, Urrbrae, SA, Australia(2)Entomology, South Australian Research and Development Institute, GPO Box 397, Adelaide, SA, Australia

Evidence for association of virus particles with transmissible symptoms on edible fig (Ficus carica), such as mosaic and mottling, has been available for decades. In addition, it had been established that symptoms are transmitted by an eriophyid mite (Aceria ficus). However, it was only in 2009 that a specific RNA virus was conclusively associated with these symptoms, and given the name Fig mosaic virus (FMV) (Elbeaino et al.,2009a). The FMV genome was first thought to consist of four RNA molecules that showed high similarity with several other mite-vectored plant RNA viruses. More recently, RNA5 and RNA6 of FMV have been characterised (Ishikawa et al., 2012) although they have not been detected in closely related viruses as yet, except for RNA5 from Raspberry leaf blotch virus. It has recently been proposed that these currently taxonomically unassigned viruses be grouped within a new bunyavirid genus (Emaravirus) (Elbeaino et al., 2009b). Here, we report the first conclusive detection of FMV in Australia, utilising an extraction protocol developed by Waite Diagnostics, The University of Adelaide, and an RT-PCR assay. Using this approach, RNA polymerase, capsid and RNA6 from FMV were detected in commercial and “wild” edible fig varieties as well as a range of other Ficus spp. growing in the Waite Arboretum, Adelaide. Sequence analysis of these amplicons shows that the isolate we detected in F. carica most closely resembles a Canadian isolate. This suggests that our isolate may have originated from a country where FMV is not endemic, rather than directly from countries in which edible figs are endemic (southwest Asia) or have been naturalised over the long term (Mediterranean region). Additionally, we have cloned, expressed and purified the full capsid protein from the Australian FMV, in order to develop FMV antibodies. Fig mosaic disease affects the quality of fruit and we now have the diagnostic tools to evaluate the effects of FMV on commercial production and devise management options.

References: Elbeaino et al.2009a. J. Gen.Virol.90:1281-1288 Elbeaino et al.2009b. Arch. Virol.154:1719-1727 Ishikawa et al. 2012. J. Gen.Virol.93:1612-1619


Notes

S2 - O4

A distinct polerovirus infecting crop and weed legumes from eastern Australia is transmitted by cowpea aphid

Murray SharmanQueensland Department of Agriculture, Fisheries and [email protected]

Murray Sharman(1), Calum Wilson(2), John Thomas(3)

(1)Department of Agriculture, Fisheries and Forestry, Ecosciences Precinct, GPO Box 267, Brisbane, Queensland, 4001, Australia

(2)Tasmanian Institute of Agriculture, New Town Research Laboratories, University of Tasmania, 13 St. Johns Ave., New Town, Tasmania 7008, Australia

(3)Centre for Plant Science, The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, Ecosciences Precinct, GPO Box 267, Brisbane, Queensland, 4001, Australia

A novel polerovirus was identified independently from the Fabaceae plants Field peas (Pisum sativum) from Tasmania and Phasey bean (Macroptilium lathyroides) from Queensland. Partial genome sequence and preliminary host range studies suggest it is genetically distinct from, but most closely related to Cucurbit aphid-borne yellows virus (CABYV) with only about 80% nt identity in the coat protein gene. Although the samples from Tasmania and Queensland share approximately 95% nt identity over 585 nt of the CP gene, the exact relationship between these isolates remains uncertain. It is hereafter referred to as Phasey bean virus (PhBV) but further characterization is required for a more appropriate name. PhBV reacts with a general luteovirus (5G4) monoclonal antibody in TBIA. Results indicate TBIA alone can not distinguish it from other poleroviruses and PhBV-specific RT-PCR is needed to confirm the virus species. PhBV was found in natural field infections of chickpeas from New South Wales (NSW) and was transmitted into chickpeas and other pulse crops using the cowpea aphid (Aphis craccivora). PhBV was transmitted to five out of 33 chickpea test plants, nine out of 11 Faba beans and at least three out of five Field peas. The infected Faba bean and Field pea plants displayed general yellowing symptoms while the chickpeas displayed reddening and dwarfing symptoms. Symptoms appear to consist of indistinct yellowing of leaves on Phasey bean. These results suggest that PhBV may be present as natural infections in chickpeas, Faba beans and Field peas, and possibly other pulse crops. To determine the geographical distribution of PhBV in Phasey bean, samples were tested by either PhBV-specific RT-PCR or luteovirus TBIA. PhBV was detected in Phasey bean in Central Queensland, the northern Darling Downs, SE Queensland and NE NSW, from 12 of 15 locations. PhBV was also detected from Macroptilium atropurpureum in Queensland, and Medicago polymorpha and Physalis ixiocarpa from Narrabri, NSW. These field survey results and the recent report of the same polerovirus from Tasmania indicate that this newly identified polerovirus has a very wide geographical range, may be either endemic or have been present in eastern Australia for some time, and may be commonly infecting leguminous species including vegetables and pulse crops throughout these areas.

Notes


S2 - P1

A virus survey of Pisum sativum crops in Germany, 2012

Heiko ZiebellJulius Kü[email protected]

Heiko Ziebell (1), Arnaud Blouin (2), Robin MacDiarmid (2)

(1)Julius Kühn-Institut, Messeweg 11-12, 38126 Braunschweig, Germany(2)The New Zealand Institute for Plant & Food Research Limited, Mount Albert, Private Bag 92169, Auckland 1142,

New Zealand

Fresh peas (Pisum sativum) for human consumption are an important crop grown in Germany. They are produced conventionally and organically on more than 4000 ha. In 2010, a new disease was discovered on pea crops in Saxony-Anhalt. It was found that the plants were infected with a novel nanovirus. So far, nanoviruses had been known only to occur in Asia, Australia, North African countries and the Middle East but had not been known to occur in Europe (apart from a sporadic outbreak of faba bean necrotic yellows virus in Spain). The nanovirus found in Germany was genetically distinct to other nanoviruses so that it was proposed as a new species with the name, Pea necrotic yellow dwarf virus (PNYDV). Nanoviruses have isometric particles of approximately 20 nm diameter, are phloem-limited and aphid-transmitted in a persistent matter. They cannot be transmitted mechanically and seed transmission also has not been observed. Only few aphid species are vectors of these viruses. The experimental host range included peas, faba beans and chick peas but excluded alfalfa, bean, cowpea and other legumes. Although PNYDV and other novel nanoviruses were subsequently found in Sweden, Austria, Hungary and Serbia, no information about the distribution of PNYDV in Germany was available at the time. In the growing season of 2012, a virus survey in the major pea crop regions of Germany (Saxony, Saxony-Anhalt, Schleswig-Holstein) was carried out. The primary aim of the survey was the description of occurring viruses on peas as well as an investigation of PNYDV incidence including the molecular characterisation of PNYDV isolates from geographically distinct locations. Preliminary results indicated that Pea enation mosaic virus was the predominant virus in all geographical locations followed by polero- and luteoviruses. However, to a lesser extent, PNYDV was found in Saxony and Saxony-Anhalt in conventionally grown crops but not in Schleswig-Holstein in organic crops. The molecular characterisation of the isolates is currently being carried out.


Notes

S2 - P2

A virus survey of Vicia faba crops in canterbury, New Zealand 2011–12

John FletcherThe New Zealand Institute for Plant & Food [email protected]

John Fletcher (1), Heiko Ziebell (2)

(1)The New Zealand Institute for Plant & Food Research Limited, Lincoln, New Zealand(2)Julius Kühn-Institut, Braunschweig, Germany

Broad bean (Vicia faba L.) is a well-established vegetable crop grown in Canterbury. Recently there has been interest in growing related field bean or tick bean crops for both human and animal consumption. These crops can form a useful break crop for cereals, brassica, pasture and other cropping systems. Previous research has shown that V. faba can suffer from a number of virus and other plant diseases when grown in the South Island. The last such virus survey of V.faba was completed in 1991 when we detected the following viruses: Soybean dwarf virus (SDV) and Beet western yellows virus (BWYV), now classified as Turnip yellows virus (TuYV) which cause ‘bean leafroll’; Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV); and the potyviruses Pea seed-borne mosaic (PSbMV)and Bean yellow mosaic virus (BYMV).In 2011, 16 broad, faba and tick bean crops throughout mid- and South Canterbury were selected and surveyed for viruses known and not known to be present in New Zealand. Overall, virus incidences were low, with only a few crops noticeably damaged, largely by ‘bean leaf roll’. When compared with previous surveys, only TuYV appears to have become more widespread but with a similar incidence (0–7%). The other component of ‘bean leaf roll’ SDV, while being less widespread, had a higher incidence (0–25%). A single Faba bean necrotic yellows virus (FBNYV) positive specimen indicates a need to more closely examine plants with ‘bean leaf roll’ symptoms. The incidences of other viruses seem similar to the previous survey: AMV (incidence 0–9%), PSbMV (0–3.5%), BYMV (0–5%), CMV (not detected). Broad bean wilt virus 1 (BBWV-1), Broad bean wilt virus 2 (BBWV-2), and Broad bean true mosaic (BBTMV) were not detected. It may be significant that we have detected Red clover vein mosaic virus (RCVMV) which has not been described for New Zealand before and found it to be reasonably widespread and at high incidences within some crops.

Notes


S3 - P1

Novel cassava associated single stranded DNA virus

Anisha DayaramUniversity of [email protected]

Anisha Dayaram(1), Allen Opong(2), Anja Jäschke(1)(3), James Hadfield(1), Marianna Baschiera(4), Renwick Dobson(1)(6), Samuel Offei(7), Dionne Shepherd(4), Darren Martin(5), Arvind Varsani(1)(6)(8)

(1)School of Biological Sciences, University of Canterbury, Ilam, Christchurch 8140, New Zealand(2)Virology Section, CSIR-Crop Research Institute, Kumasi, Ghana(3)Department of Infectious Diseases, University of Heidelberg, D-69120 Heidelberg, Germany(4)Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, 7701 Cape Town, South Africa(5)Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape

Town, South Africa(6)Biomolecular Interaction Centre, University of Canterbury, Ilam, Christchurch 8140, New Zealand(7)School of Agriculture and Consumer Sciences, University of Ghana, Legon, Ghana(8)Electron Microscope Unit, University of Cape Town, Rondebosch, 7701 Cape Town, South Africa

Over the last couple of years our knowledge of single stranded DNA (ssDNA) viruses has increased tremendously as a result of new molecular techniques such as non-specific rolling circle amplification coupled with viral metagenomics. We recently isolated a putative novel ssDNA virus; cassava associated circular DNA virus (CasCV) from a cassava leaves infected with the fungi Collectotrichum and Plectosphaerella sp. CasCV has a circular ambisense genome that is 2220 nt. The genome has three major open reading frames, one encodes for a possible capsid protein (CP). The other two ORFs encode for a putative replication associated protein (Rep) and also contained an intron. When the intron was removed the Rep showed close similarity to other Rep encoding genes found in geminiviruses. This was further supported by the highly conserved rolling circle replication (RCR) related motifs that are conserved across many geminivirus, circovirus and nanovirus Reps. Overall the genome shows close similarities with the recently charaterised Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), Mosquito VEM virus SDBVL and Meles meles faecal virus (MmFV). Further analysis of the CasCV Rep and CP demonstrates that it shares 62.7% and 39.8% amino acid identity with the Rep and CP of SsHADV-1. Host range studies using infectious clones all still needed to demonstrate the origin and host of this virus.


Notes

S3 - P2

Barley yellow dwarf and Cereal yellow dwarf viruses detected in oats and barley grown in Queensland and New South Wales

Kathy ParmenterDAFF [email protected]

Mohammad Al-Mashhadani(1), Kathy Parmenter(2), Murray Sharman(2), Mark Schwinghamer(3), John Thomas(4), (1)School of Biological Sciences, The University of Queensland, St. Lucia campus, Queensland, 4072, Australia(2)Agri-Science Queensland, Department of Agriculture, Fisheries and Forestry, Ecosciences Precinct, Dutton Park,

Queensland, 4102, Australia(3)NSW Department of Primary Industries, Tamworth Agricultural Research, Tamworth, New South Wales, 2340, Australia(4)Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Ecosciences Precinct, Dutton

Park, Queensland, 4102, Australia

Serotypes of barley yellow dwarf viruses (BYDV-PAV, -MAV, -RMV) and cereal yellow dwarf virus (CYDV-RPV) (family Luteoviridae) were detected in symptomatic oats (Avena sativa) or barley (Hordeum vulgare) from the New South Wales (NSW) and Queensland (QLD) grain belt in 2010 and 2011. Symptomatic plants were assayed using TAS-ELISA for PAV, MAV and RPV and a duplex degenerate RT-PCR amplifying PAV/MAV/SGV and RPV/RMV/GPV (Malmstrong and Shu, 2004). Twenty-one of 26 were positive for at least one virus. CYDV-RPV was detected in oat samples (one NSW, three QLD) and a barley sample (NSW) by ELISA, PCR, and except for the NSW oat sample, also by partial sequence confirmation (95-96% nucleotide identity to CYDV-RPV, GenBank EF521848.1). The oat isolates were transmitted by Rhopalosiphum padi but not by R. maidis or Metopolophium dirhodum. This is a new record for QLD and the first record of RPV in barley and oats in NSW. Several symptomatic oat and barley samples that were negative for PAV, MAV and RPV by ELISA tested positive for RPV/RMV/GPV by degenerate RT-PCR. Amplicons from the QLD isolates had 95-96% nucleotide identity to BYDV-RMV (GenBank L12758.1). A barley isolate from NSW had similar ELISA and RT-PCR results and was transmitted by R. maidis, and appears to be the first record of RMV in NSW. Oat samples from QLD (4) and NSW (2) tested positive by ELISA for MAV but failed to amplify in the degenerate PAV/MAV/SGV RT-PCR. A second degenerate RT-PCR for PAV/MAV/PAS (Chomic et al. 2010) amplified the expected product. However, the 133 bp sequences obtained had only 79% nucleotide identity to the BYDV-MAV type sequence (GenBank NC_003680). These sequences were 96% identical to a BYDV-MAV-like sequence from New Zealand (GenBank EF408185), which is itself only 77% identical over 758 nt to the MAV type sequence. Additionally, two QLD isolates were transmitted by R. padi but the one tested not by R. maidis or M. dirhodum. Transmission by R. padi but not M. dirhodum fits with previous observations of MAV-serotype isolates from Victoria (Sward and Lister, 1988). Together, these data indicate the presence of a new Barley yellow dwarf virus in Australia and New Zealand which is genetically distinct, but reacts with the MAV antiserum.

References: Malmstrong and Shu, 2004. J. Virol. Methods 120: 69-78 Chomic et al., 2010. Plant Pathology 59: 429-442 Sward and Lister, 1988. Aust. J. Agric. Res. 39: 375-384

Notes


S3 - O1

The diversity of Strawberry latent ringspot virus in New Zealand

Joe TangPlant Health and Environment Laboratory, New [email protected]

Joe Tang , Lisa I. Ward , Gerard R. G. Clover

Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140 New Zealand

Strawberry latent ringspot virus (SLRSV) is widespread in many countries especially in Europe. The virus was thought uncommon in New Zealand, having only been recorded in Prunus species. However, this study revealed that SLRSV infects a much wider range of hosts. From 1999 to 2009, SLRSV was isolated from anemone (Anemone × hybrida), blackberry (Rubus spp.), impatiens (Impatiens walleriana), pepino (Solanum muricatum) and tibouchina (Tibouchina sp.) in the North Island of New Zealand. These SLRSV isolates were identified using electron microscopy, mechanical inoculation, ELISA and RT-PCR techniques. This is thought to be the first report of anemone, impatiens, pepino and tibouchina as hosts of SLRSV. Phylogenetic analysis and host range suggest that the five newly identified NZ isolates belong to two distinct strains: blackberry and impatiens isolates represent one strain and the other three isolates, plus the flowering cherry isolate reported previously in New Zealand, represent another strain. Both these strains are distant from isolates reported elsewhere in the world. The blackberry/impatiens strain is especially different and produced an unusual reaction in mechanical inoculation tests on herbaceous indicators. It is postulated that SLRSV may have gone undetected on its wider host range in New Zealand due to the latent infection in some hosts. The relationship of SLRSV isolates between New Zealand and overseas and the transmission modes of this virus are also discussed.


Notes

S3 - O2

Diversity of monocot-infecting mastrevirus in Australia rivals that in Africa

Simona KrabergerUniversity of [email protected]

Simona Kraberger(1), John Thomas(2) , Andrew Geering(2), Anisha Dayaram(1) , Daisy Stainton(1) , James Hadfield(1) , Matthew Walters(1), Kathleen Parmenter(3), Sharon van Brunschot(2) , David Collings(1)(5) , Darren Martin(4) , Arvind Varsani(1)

(5)(6)

(1)School of Biological Sciences, University of Canterbury, Christchurch, 8140, New Zealand(2)Centre for Plant Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland,

Ecosciences Precinct, GPO Box 247, Brisbane, QLD, 4001, Australia.(3)Agri-Sciences, Queensland Department of Agriculture, Forestry and Fisheries, Ecosciences Precinct, GPO Box 247,

Brisbane, QLD, 4001, Australia.(4)Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape

Town South Africa(5)Biomolecular Interaction Centre, University of Canterbury, Christchurch, 8140, New Zealand(6)Electron Microscope Unit, University of Cape Town, Rondebosch, 7701, Cape Town, South Africa

In order to shed light on the level of monocotyledon (monocot) infecting mastrevirus diversity in Australia we have sequenced 41 viral genomes isolated from endemic and native grasses. Members of the mastrevirus genus (family Geminiviridae) have been documented in Africa (including the south-west Indian Ocean islands), Eurasia, the Middle-East, Japan and Australasia. Previous studies have shown that the highest level of monocot-infecting mastrevirus diversity is found in Africa. This study however, shows that with more extensive sampling the diversity of mastreviruses in Australia may rival that found in Africa. From 40 symptomatic grasses we have identified four new mastrevirus species, two of which were isolated from a single Sporobolus grass and are highly divergent, sharing closest nucleotide identity to the African streak virus group. This knowledge, along with reports of high levels of diversity in dicotyledon (dicot) infecting mastrevirus from Australia, highlights the possibility that mastrevirus may have originated somewhere other than Africa. Our investigation into recombination hotspots and patterns of inter- and intra- species recombination within this Australian monocot-infecting mastrevirus group shows they reflect those previously identified in both the African monocot-infecting and Australian dicot-infecting groups.

Notes


S3 - O3

Molecular characterization of three mitoviruses infecting a hypovirulent isolate of the phytopathogenic fungus Sclerotinia sclerotiorum

Mahmoud KhalifaThe University of [email protected]

Mahmoud E. Khalifa, Michael N. Pearson

School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand

Sclerotinia sclerotiorum is a phytopathogenic fungus capable of causing significant yield losses in numerous crops in New Zealand and worldwide. It has a host range of over 400 plant species and is considered a serious threat to many economically important crops. Chemical control is increasingly fraught by fungicide resistance and chemical residues. An alternative innovative approach is to exploit mycoviruses as biocontrol agents. Mycoviruses have been described in diverse fungal species including S. sclerotiorum which is known to harbour mycoviruses from several different mycovirus families, the simplest being those assigned to the genus Mitovirus. Three double-stranded RNAs (dsRNAs), indicative of mycovirus presence, were detected in a single isolate of S. sclerotiorum and sequenced. The three sequences (A, 2438 nts; B, 2588 nts; C, 2744 nts) all showed similarity to mitoviruses, consisting of a single open reading frame (ORF) with the characteristic conserved motifs of RNA-dependent RNA polymerase (RdRp). Mitochondrial malformations and reduced virulence and growth were associated with the presence of the dsRNAs. The terminal sequences of the positive strand of the three dsRNAs could all be folded into stem-loop structures and the inverted terminal complimentary sequences of sequence A can potentially form a panhandle structure. Sequence A showed 91.6% aa similarity to the previously described Sclerotinia sclerotiorum mitovirus 2 and was tentatively assigned the acronym SsMV2/NZ. Sequences B and C showed only 16.4% similarity to each other and 15-48% aa similarity to the previously described mitoviruses and consequently appear to be new mitoviruses.


Notes

S3 - O4

Plant virus discovery and the implications for biosecurity

Robin MacDiarmidThe New Zealand Institute for Plant & Food Research [email protected]

Robin MacDiarmid (1), Brendan Rodoni (2), Francisco Ochoa-Corona (3), Marilyn Roossinck (4), Ulrich Melcher (5) (1)The New Zealand Institute for Plant & Food Research Limited, Auckland 1142, New Zealand. (2)Department of Primary Industries, Victoria 3156, Australia. (3)Institute for Microbial Forensics & Food and Agricultural Biosecurity, Department of Entomology & Plant Pathology,

Oklahoma State University, OK 74078, USA. (4)Plant Pathology and Biology, Center for Infectious Disease Dynamics, Pennsylvania State University, PA 16802, USA. (5)Department of Biochemistry & Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA.

Human activity is causing “new encounters” between viruses and plants. Anthropogenic interventions include changing land use, decreasing biodiversity, trade, the introduction of new plant and vector species to native landscapes, and changing atmosphere and climatic conditions. The discovery of thousands of new viruses, especially those associated with healthy native plants, is shifting the paradigm for their role within the ecosystem from foe to friend. Can the negative effects of globalization on the environment be deterred, or can global trading be less damaging on the environment? Can plant biosecurity strategies facilitate a more sustainable level of food production and at the same time protect our environment? The cost of new plant virus incursions is high and can result in the loss of trade and/or production for short or extended periods. We present and justify five recommendations for plant biosecurity to improve communication about plant viruses, assist with the identification of viruses and their impacts, and protect the high economic, social, environmental and cultural value of our respective nations’ unique flora: 1) A shared burden of proof for identifying the biosecurity risk continues to be endorsed, 2) Countries and jurisdictions should identify what pests already exist in, and which pests pose a risk to, their native flora, 3) Plant virus sequences not associated with a recognized virus infection are designated as “uncultured virus” and tentatively named using the host plant species of greatest known prevalence, the word ‘virus’, a general location identifier, and a serial number, 4) Improved tools to be adopted and further developed by biosecurity agencies to identify known and new plant viruses, 5) Research into the ecology of known and new viruses to determine their relationship within their existing and potential new plant hosts and vectors and as a basis for developing new predictive tools.

Notes


S4 - O1

Pathogenicity and epidemiology of Potato spindle tuber viroid in Australia

Roger JonesUniversity of Western [email protected]

Alison Mackie(1)(2)(3), Martin Barbetti(1)(2), Brendan Rodoni(1)(4), Simon McKirdy(1) and Roger Jones(1)(2)(3)

(1)Cooperative Research Centre for National Plant Biosecurity, (2)University of Western Australia, (3)Department of Agriculture and Food Western Australia, and (4)Department of Primary Industries Victoria.

Potato growing areas of Australia are currently considered free of Potato spindle tuber viroid (PSTVd) and this pathogen is classified an emergency plant pest (Category 3) under the Emergency Plant Pest Response Deed (EPPRD). However, there have been numerous emergency responses to PSTVd outbreaks in tomatoes in the last 10 years. Research was therefore undertaken to provide more information on the pathogenicity and epidemiology of PSTVd under Australian conditions. Pathogenicity studies were undertaken in a PC2 glasshouse using commonly grown and traditional PSTV indicator potato and tomato cultivars. A tomato PSTVd isolate from Carnarvon, Western Australia was inoculated to plants of potato cultivars Russet Burbank, Atlantic and Nadine, and tomato cultivars Rutgers, Petula and Swanson. All three potato cultivars showed symptoms of PSTVd infection with Russet Burbank the most severely affected. All three tomato cultivars also showed symptoms with Rutgers developing them most severely. Tubers from infected Russet Burbank plants were small and spindle shaped, infected Atlantic plants produced small, severely cracked tubers, and infected Nadine plants clusters of tiny tubers. None of the infected tomato cultivars produced any marketable fruit yield. Thus, the tomato PSTVd isolate was pathogenic on potato. In a PC2 glasshouse, retention of PSTVd infectivity on surfaces was investigated by pipetting PSTVd-contaminated leaf sap onto metal, cotton, glass, string, plastic, rubber, leather and wood surfaces. After 5 minutes, 1 hour, 6 hours and 24 hours, sap dried on the different surfaces was rehydrated with water and used to inoculate healthy tomato plants. Rehydrated sap contaminating metal was still infective after 1 hour while sap contaminating cotton was still infective after 6 hours. While the results for rubber were inconclusive, those for wood, plastic and leather revealed that sap was still infective after 24 hours. These results have implications regarding PSTVd transmission when contaminated surfaces brush against healthy tomato plants. Surveys in Carnarvon detected PSTVd in Blackberry Nightshade (Solanum nigrum), Thornapple (Datura stramonium), Annual Saltbush (Atriplex semilunaris), and volunteer Tomato and Capsicum plants, confirming that diverse solanaceous hosts and even non-solanaceous hosts can harbour PSTVd. In addition, an unidentified pospiviroid was detected in both Thorny Saltbush (Rhagodia eremeae) and Annual Saltbush. PSTVd specific nucleotide sequences were obtained for five isolates (181, 189, 209, 212 and 217). A further two isolates were weakly positive for the Pospiviroid RT-PCR test and negative for both the PSTVd specific RT-PCR tests, suggesting the likely presence of an additional Pospiviroid species.


Notes

S4 - P1

Assessment and validation of DAFWA canola model for predicting BWYV outbreaks in pulses and canola in Victorian conditions

Mohammad AftabDepartment of Primary Industries [email protected]

Mohammad Aftab(1), Moin Salam(2), Angela Freeman(1), Shane King(1)

(1)Department of Primary Industries, private bag 260 Horsham, VIC 3400(2)Department of Agriculture and Food Western Australia, Locked Bag 4, South Perth, WA 6983

Over the 2006-2009 period Beet western yellows virus (BWYV) emerged as a serious problem in Victorian pulse crops. In 2010, a project was initiated to assess management of BWYV through the use of a decision support system (DSS) developed by the Department of Agriculture and Food Western Australia (DAFWA). DAFWA has developed a DSS based on a model for forecasting outbreaks of BWYV in canola in WA. This model is being validated and assessed under Victorian conditions for predicting BWYV in canola and pulses. Validation involved fortnightly sampling of plants in canola and pulse plots to test for BWYV and counts of live aphids on the plants, as well as weekly trapping of alate aphids on yellow sticky traps for counting and identification. The validation data from the last two years showed that the DAFWA canola model was not quite capable of simulating BWYV incidence under Victorian conditions. The model algorithms were then modified to simulate dynamics of seasonal aphid population constrained by rainfall and low and high temperatures, using ten years of historical weather data. The modified model was then compared with the actual incidence of BWYV over the same ten year period, based on data from annual Victorian pulse virus surveys. The model largely simulated the pattern of BWYV incidence across southern Australia for the ten year period. This model is being further tested under Victorian conditions. The challenge will be to determine whether or not the predictions enable timely advice to be provided to growers to reduce virus impact in years where BWYV is likely to cause economic losses.

Notes


S5 - O1

Zucchini yellow mosaic virus in cucurbits: transmission by contact, stability on surfaces, and inactivation with disinfectants

Brenda CouttsUniversity of Western Australia [email protected]

Brenda Coutts (1)(2), Monica Kehoe (1)(2), Roger Jones (1)(2)

(1)Crop Protection Branch, Department of Agriculture and Food Western Australia, Locked Bag No. 4, Bentley Delivery Centre, Perth, WA 6983, Australia.

(2)School of Plant Biology, Faculty of Natural and Agricultural Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia.

Zucchini yellow mosaic virus (ZYMV) causes severe yield and quality losses in cucurbit crops worldwide. In northern Australia it is widespread in cucurbit crops and causes losses of up to 100%. ZYMV is non-persistently transmitted by aphids and is also seed-borne at low levels in cucurbits. Anecdotal evidence indicates ZYMV may also be contact-transmitted in cucurbit plantings. ZYMV was transmitted from infected to healthy zucchini (Cucurbita pepo) plants by direct contact when leaves were rubbed against each other, crushed or trampled, and also on blades contaminated by infective sap. When ZYMV infective sap from zucchini plants was kept at room temperature for up to 6 hr, it infected healthy plants readily. Also, when infective sap was applied to seven surfaces (cotton, plastic, leather, metal, rubber vehicle tyre, rubber soled footwear and human skin) and left for up to 48 hr before the contaminated surface was wiped onto healthy zucchini plants, ZYMV remained infective for 48 hr on tyre, 24 hr on plastic and leather, and up to 6 hr on cotton, metal and footwear. With human skin, ZYMV was infective for 5 min only. The effectiveness of nine disinfectants at inactivating ZYMV was evaluated by adding them to infective sap which was then inoculated to zucchini plants. No plants became infected when nonfat dried milk (20% w/v) or bleach (42g/l sodium hypochlorite, diluted 1:4) were used, but a varying amount of infection occurred with the other disinfectants. This demonstrates ZYMV can be transmitted by contact and highlights the need for on-farm inclusion of hygiene practices (de-contaminating tools, machinery, clothing, etc.) in integrated disease management tactics against ZYMV epidemics in cucurbit plantings.


Notes

S5 - O2

Molecular variation in Dasheen mosaic virus: dynamics of virus population structure in evolutionary space and time.

Wee-Leong ChangAuckland University of [email protected]

Wee-Leong Chang(1), Mary Cong(1), Annie Yuan(1), Nitish Anand(1), Michael N. Pearson(2), Colleen M. Higgins(1)

(1)School of Applied Sciences, Auckland University of Technology, New Zealand(2)School of Biological Sciences, The University of Auckland, New Zealand

Exploring the genetic diversity and evolutionary history of plant viruses is critical to understanding their ecology and epidemiology. Like many other plant RNA viruses, Dasheen mosaic virus (DsMV) is an important and conspicuous viral disease of ornamental and edible aroids throughout the South Pacific and worldwide, but its population diversity and variability are poorly understood. To further investigate this virus, phylogenetic and population genetics based methods were used to investigate the temporal and spatial dynamics of the evolutionary mechanism and genetic variability among the DsMV isolates. A selected region of the coat protein (CP) gene was amplified and sequenced to infer genetic relationships between viral isolates at the temporal and spatial scales. This study demonstrated that (i) genetic variation occurs between the DSMV isolates and (ii) the population structure of DsMV in individual plants consisted of a consensus sequence and a pool of similar but not identical sequences, consistent with the quasispecies concept described for many RNA viruses. The quasispecies-like nature of the DsMV population suggested that the virus is capable of rapid evolution and adaptation in response to changing ecological factors and agricultural practices. Analysis of DsMV isolates on a temporal scale suggested the role of stochastic and selection-fitness levels are the key determinants in the dynamics of plant virus population genetics and evolution. In contrast, spatial analysis suggested that diversification and spread of DsMV have been concomitant with an extension of human migration and taro/tannia cultivation in the South Pacific islands. The combined actions of genetic drift and selection pressure have continually remoulded this diversity, creating a geographic mosaic in the degrees of diversity found within and between geographic regions.

Notes


S5 - O3

Genetic diversity within and between different strains of Dasheen mosaic virus and its potential impact on diagnosis

Subuhi KhanAuckland University of [email protected]

Subuhi Khan, Wee-Leong Chang , Gardette Valmonte , Smriti Nair, Colleen M. Higgins


The genus Potyvirus is one of the largest of plant viruses with around 180 definitive and possible members i.e. 30% of all known plant viruses. Therefore, precise and accurate identification of the potyviruses that infect commercially important crops is required to monitor disease incidence, to identify virus reservoirs and to recommend appropriate control mechanisms. The development of better anti-viral strategies for potyviruses is difficult because potyviruses have RNA genomes and RNA viruses live as quasi-species within the host plant. The diffuse ‘cloud-like’ nature of potyviral populations allows them to adapt rapidly to changing replicative environments by selecting pre-existing variants with better fitness. Therefore, several important virus properties cannot be explained by a single consensus sequence, but require knowledge about the microvariants present in viral populations within and between host plants. These sequence variants may be critically relevant to viral diagnostics, evolution, spread and virulence. With the advent of deep sequencing technologies, it is now possible to determine the full extent of genetic diversity of a virus within a host. We used Illumina technology to determine the diversity of the potyvirus Dasheen mosaic virus (DsMV) within and between host taro plants. The aims of this work were three-fold: firstly, to determine if different strains of the virus show the same degree of diversity; secondly, to identify conserved genome regions that may have functional importance, and determine if these are the same between different virus strains, and thirdly, determine if improved universal primers for potyvirus diagnostics can be designed. Parent plants infected with DsMV strains NZ or B were propagated into two daughter plants giving rise to isolates DsMV-NZ1.1 and NZ1.2, B1.1 and B1.2. Transcriptomes from these six plants were sequenced and the virus genomes assembled against the publicly available DsMV complete genome. Analysis of the consensus sequences for each assembly supported the placement of DsMV in the Bean common moaic virus group and showed that the B strain is more variable than the NZ strain. Variation within each genome assembly was similar to other assemblies, indicating that the degree of sequence plasticity was common between strains and isolates, and finite. The sequence diversity varied along the length of the genomes, with small regions of conservation. For example, the 5’ and 3’UTRs showed 27% and 28% variation overall, respectively. Within these regions, short stretches of nucleotides were conserved, which may have significant roles in translation and/or replication. Identification of such conserved regions and potential improvement of universal primer design is ongoing.


Notes

S6 - O1

Unravelling the cause of Black Pod Syndrome of narrow-leafed lupin

Monica KehoeUniversity of Western [email protected]

Monica Kehoe (1)(2), Bevan Buirchell (1)(2), Roger Jones (1)(2), , (1)School of Plant Biology, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia(2)Department of Agriculture and Food Western Australia, 3 Baron-Hay Court, South Perth, WA 6151, Australia

Black pod syndrome (BPS) causes devastating losses in narrow-leafed lupin crops in south-west Australia. A number of possible causes have been studied in the past, including abiotic factors such as competition between pods on the main stem or nutrient deficiencies. However, late infection by biologically distinct strains of Bean yellow mosaic virus (BYMV) that cause necrotic symptoms in narrow-leafed lupin has for some time been generally accepted as the major cause of BPS. This project aims to establish whether BYMV is acting alone in causing this syndrome, or whether it might also have additional causes. One hypersensitive resistance gene to BYMV has already been identified in narrow leafed lupin (Nbm-1). In 2011, a survey was conducted during the growing season across seven sites (three at Esperance, two in Perth, and one each at Pingelly and Arthur River). Whole plant samples with BPS were collected, sub-sampled and tested for virus presence by both ELISA and RT-PCR. RT-PCR proved most reliable for virus detection in plants with BPS. Biological studies have successfully reproduced BPS symptoms in narrow-leafed lupin plants using late-stage sap inoculations with BYMV in the glasshouse. Thus, the results obtained so far are consistent with late BYMV-infection in the field being the primary cause of BPS. Partial sequencing of the coat protein gene confirmed the presence of phylogenetic strains from both specialist lupin and generalist groups of BYMV. Progress with analysis of whole virus genomes derived from next generation sequencing of virus isolates from symptomatic plants with BPS will be described.

Notes


S6 - O2

Identification of a naturally occurring, mild isolate of Tamarillo mosaic virus

Robin MacDiarmidThe New Zealand Institute for Plant & Food Research [email protected]

Katrin Pechinger (1,2), Daniel Cohen (1), Arnaud Blouin (1), Russell Joblin (3), Allan Laird (4) and Robin MacDiarmid (1).(1)The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand (2)Lion - Beer, Spirits & Wine (NZ) Limited, 55 Ormiston Road, Auckland 2016, New Zealand (3)526 Woodlands Rd Opotiki 3198, New Zealand (4)50 Mile Rd Bombay Pukekohe, New Zealand

Tamarillo mosaic virus (TamMV), a strain of Potato virus A (PVA), is the most damaging of the viruses that infect tamarillo, resulting in low plant vigour, poor fruit yield and discolouration of fruit. To date no method to protect tamarillo plants has been determined except for genetically engineered plants that are resistant to virus infection. In this study, a branch of a TamMV-infected tree that bore leaves and fruit that had no or few virus symptoms was identified in a commercial orchard; the branch tested positive for potyvirus infection by ELISA. Seedlings originating from that branch were tested for tolerance to a typical severe isolate of TamMV and were found susceptible, demonstrating that the genotype of the tamarillo plant did not confer resistance to TamMV. The virus infecting that branch was used as source of inoculation in a large-scale inoculation experiment. Most of the seedlings inoculated tested positive for the virus but remained symptomless, indicative of the presence of a mild strain of TamMV. The mild strain of TamMV is closely related to previously published sequences of TamMV and PVA but sequences derived from both targeted gene amplification and non-targeted double-stranded RNA preparations have revealed some differences in the HC-Pro gene. The discovery of this naturally occurring, mild isolate of TamMV was made possible by the close working relationship between orchardists and scientists. Future studies will determine the ability of the mild isolate of TamMV to cross-protect against severe isolates.


Notes

S6 - O3

Aphid species feeding behaviour affects acquisition of potato potyviruses

Ron van ToorThe New Zealand Institute for Plant & Food Research [email protected]

Ron van Toor(1) , Gaynor Malloch(2), Matthew Hooper(2), Brian Fenton(2)

(1)The New Zealand Institute for Plant & Food Research Limited, Lincoln, Canterbury, New Zealand(2)James Hutton Institute, Invergowrie, DD2 5DA, United Kingdom

How the behaviour of 2- or 24- hour-starved Myzus persicae, Macrosiphum euphorbiae and Metopolophium dirhodum alatae influenced the acquisition of the potato potyvirus A (PVA) and Y (PVY) was assessed on virus-infected excised potato leaves. Within 10 minutes after placement on the leaves, 83% of the M. dirhodum, 23% of M. persicae and 0% of M. euphorbia acquired PVA, while 14-38% of these aphid species acquired PVY. The difference in virus acquisition between the species was not related to the differences in the time the aphids spent inactive, walking, probing or feeding on the leaves. These findings have ramifications for insecticide management of aphid virus vectors on potatoes.

Notes


S6 - O4

Detection of the brassica-infecting viruses TUMV and TUYV in aphids

Simon BulmanThe New Zealand Institute for Plant & Food Research [email protected]

Sarah Thompson, John Fletcher, David Teulon, Ian Scott, Simon Bulman

The New Zealand Institute for Plant & Food Research, Private Bag 4704, Christchurch 8140, New Zealand

Insects are responsible for transmitting the majority of known plant infecting viruses. Of these vectors, aphids are especially prominent, transmitting close to 300 viruses. At present, the threat presented by aphid-transmitted viruses is usually assessed in New Zealand by trapping aphids in the vicinity of crops. However, counting and identification of the aphids is costly, and is only a proxy for virus prevalence. A more accurate, speedy and cost effective measure of virus and aphid numbers would be desirable. In this study, qPCR assays were developed for detection of the non-persistent Turnip mosaic virus (TuMV) and the persistent Turnip yellows virus (TuYV) when carried by the aphids Myzus persicae and Brevicoryne brassicae. We were able to detect both viruses in RNA from single aphids. Virus levels were higher for TuYV than for TuMV, both by relative and absolute measures. These assays were used to assess the suitability of propylene glycol (PG) as a field capture and storage medium for trapped aphids. RNA was extracted at weekly intervals up to 4 weeks from viruliferous aphids stored in PG. For TuYV, the measured level of virus reduced upon storage, but the virus remained detectable from all aphids after 4 weeks in PG. For TuMV, the virus was also detectable from aphids after 4 weeks, however the level of virus was at the detection limit and not all replicate reactions gave amplification. Internal control assays based upon the aphid elongation factor 1-alpha (EF1α) gene were used to confirm successful RNA extraction and RT-PCR procedures. These assays also provided a better comparative measure of virus levels in freshly captured individual aphids. In recent experiments, TuYV was successfully detected in wild, field captured aphids from a Lincoln brassica crop field.


Notes

S6 - O5

Virus surveillance by proxy; monitoring insect vectors to detect plant viruses

Melanie DavidsonThe New Zealand Institute for Plant & Food Research [email protected]

Melanie Davidson(1) , Suvi Viljanen-Rollinson(1) , John Fletcher(1)

(1)The New Zealand Institute for Plant & Food Research Limited, Private Bag 4704, Christchurch, New Zealand

Virus dispersal and transmission can rely on insect vectors. This may increase the probability of finding the virus in the environment, as the vector may be more likely to encounter the virus. For example, insect vectors have been shown to be more attracted to virus infected plants. The mobility and behavioural responses of insects to a variety of cues has been exploited through the development of behaviour-modifying traps (e.g. pheromone, kairomone, coloured traps, trap plants), to improve detection of the vector and thus, potentially the virus. However, the plant virus may alter the insect’s behaviour and/or biology. How this change in the vector may affect its response to, or likelihood of being captured in behaviour-modifying traps has not been well studied. We examined the response of virus-infected and virus-free western flower thrips females’ response to a known thrips lure in a Y-tube olfactometer. While we did not detect any difference between virus-infected and virus free thrips, we know that this lure can increase the trap capture of thrips capable of vectoring viruses, thus potentially increasing the likelihood of detecting the viruses. How such vector trapping data relates to the detection and epidemiology of the virus in the environment and whether vectors should be used as a proxy is discussed.

Notes


S6 - O6

Localization and interactions of Impatiens necrotic spot tospovirus proteins in Nicotiana benthamiana

Ralf DietzgenThe University of [email protected]

Ralf G Dietzgen(1,2), Kathleen M Martin(2), Gavin Anderson(2), Michael M Goodin(2)

(1)Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia Qld 4072, Australia(2)Department of Plant Pathology, University of Kentucky, Lexington KY 40546, USA

Impatiens necrotic spot tospovirus (INSV) is a significant pathogen of ornamentals. The tripartite negative- and ambi-sense RNA genome encodes six proteins that are involved in cytoplasmic replication, movement, assembly, insect transmission and defense. To gain insight into the associations of these viral proteins, we determined their intracellular localization and interactions in living plant cells. Nucleotide sequences encoding the nucleoprotein N, non-structural proteins NSs and NSm, and glycoproteins Gn and Gc of a Kentucky isolate of INSV were amplified by RT-PCR, cloned, sequenced and transiently expressed as fusions with autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana. All proteins accumulated at the cell periphery and co-localized with an endoplasmic reticulum marker. The Gc protein fusion also localized to the nucleus. N and NSm protein self-interactions and an NSm:N interaction were observed using bimolecular fluorescence complementation. This is the first report of a tospovirus NSm homotypic interaction.


Notes

S7 - O1

Molecular detection and characterisation of Cherry leaf roll virus in New Zealand

Elizabeth WooThe University of [email protected]

Elizabeth N.Y. Woo(1), Bénédicte S.M. Lebas(2), Stella Veerakone(2), Joe Tang(2), Lisa Ward(2), Michael N. Pearson(1), (1)School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.(2)Plant Health and Environment Laboratory, Ministry for Primary Industries, PO Box 2095, Auckland 1140, New Zealand.

Cherry leaf roll virus (CLRV) is an established species within the genus Nepovirus, family Secoviridae. The virus has a wide natural and experimental host range and infects species in more than 36 plant families. This includes a variety of wild and cultivated, herbaceous and woody plant species. The virus has been detected worldwide, including Europe, the former USSR, North America, Chile, Peru, China, Japan, Australia and New Zealand. CLRV was first reported in 1955 to cause leaf rolling in cherry in England. Subsequently, the virus has been reported to cause economically significant diseases in horticultural crops and is therefore of concern to phytosanitary authorities. In New Zealand, CLRV was first reported in 1978 in Rubus and more recently we have detected it in Actinidia, Malus, Hydrangea, Ribes, Rumex and Vaccinium. The presence of CLRV in Actinidia, Malus and Vaccinium are new host records for New Zealand and to the best of our knowledge, the world. CLRV has been classified into six major phylogenetic groups based on a 375 bp nucleotide sequence of the 3´ non-coding region (NCR) of isolates originating from different woody and herbaceous host plants and geographical regions. The existence of strains within CLRV has important consequences for biosecurity but many tests do not differentiate the strains and New Zealand isolates have not previously been identified to this level. Since CLRV is regulated at strain level it is important to know which strains are currently in New Zealand and have suitable tests to accurately identify strains that are intercepted at the border or in post entry quarantine. The current study tested a range of commercial crops and cultivar collections of Actinidia, Malus, Ribes, Rubus, Rumex and Vaccinium for CLRV, and compared the sequences of isolates from different hosts. The nucleotide sequences of RNA2 of isolates from Actinidia, Ribes, Rubus and Rumex of CLRV were determined and the variability of sequences of RNA2 of these isolates are compared and presented. Phylogenetic analysis of the 3´ NCR revealed clustering of the New Zealand isolates within two phylogenetic groups (B and C) of CLRV. Whilst these isolates were found to be clustered only in two clades, vigilance is required in terms of biosecurity and management of the disease as CLRV is known to have a wide host range.

Notes


S7 - O2

Influence of climate change on plant virus disease infections and epidemics


Roger Jones(1)School of Plant Biology, University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA 6009, Australia(2)Department of Agriculture and Food, Locked Bag No.4, Bentley Delivery Centre, WA 6983, Australia

An assessment of the influence of climate change on plant virus disease infections and epidemics was motivated by (i) the magnitude of the threat to world food security and diversity of natural vegetation posed by viral pathogens of plants at a time of accelerating climate change, and (ii) the inadequate attention given to this subject by researchers studying climate change and plant disease. Background information on critical features of viral pathosystems and the general influence of environmental factors upon them will be described briefly. Then, use of comprehensive climatic and biological frameworks to determine the likely influences of direct and indirect climate change parameters on the many different host, vector and pathogen parameters that represent the diversity of viral pathosystems will be described. This approach proved a powerful way to identify the relevant international research data available and many information gaps where research is needed in the future. The analysis suggested that climate change is likely to modify many critical viral epidemic components in different ways often resulting in epidemic enhancement but sometimes having the opposite effect, depending on the type of pathosystem and circumstances. With vector-borne pathosystems and new encounter scenarios, the complication of having to consider the effects climate change parameters on diverse types of vectors and the emergence of previously unknown pathogens added important additional variables. The increasing difficulties in controlling damaging plant viral epidemics predicted to arise from future climate instability warrants considerable research effort to safeguard world food security and biodiversity. Reference: Jones RAC, Barbetti MJ (2012). Influence of climate change on plant disease infections and epidemics caused by viruses and bacteria. CAB Reviews 7, No. 22, 1-32 (on-line publication)


Notes

S7 - O3

The effect of elevated temperature on the titre of Barley yellow dwarf virus-PAV in wheat cv. yitpi using a multiplex normalised rt-qPCR assay

Narelle NancarrowDepartment of Primary Industries, [email protected]

Narelle Nancarrow(1), Fiona Constable(1), Jo Luck(1), Kyla Finlay(1), Simone Vassiliades(2), Angela Freeman(3), Brendan Rodoni(1)

(1)Department of Primary Industries, Knoxfield, Victoria, Australia(2)Department of Primary Industries, Bundoora, Victoria, Australia(3)Department of Primary Industries, Horsham, Victoria, Australia

Barley yellow dwarf virus-PAV (BYDV-PAV) is a phloem- limited luteovirus (family Luteoviridae) that is primarily transmitted by Rhopalosiphum padi (the oat aphid) and Sitobion (Macrosiphum) avenae. BYDV-PAV is associated with yellow dwarf disease, one of the most economically important groups of diseases of cereals worldwide. The aim of this study was to quantify the titre of BYDV-PAV in wheat under current and future temperature conditions. Wheat plants were grown from seed in growth chambers at 20°C. All plants were inoculated with BYDV-PAV at 10-days old. The plants were then exposed to ambient (5.0-16.1°C, night-day) or elevated (10.0-21.1°C, night-day) temperature treatments. The ambient temperature treatment was designed to represent the average daily temperature cycle during the wheat-growing season in Horsham, Victoria, Australia. A constant 5°C was added to the ambient temperature regime to simulate predicted future/elevated temperature. Whole above-ground plant samples were collected from each temperature treatment at 0, 3, 6, 9, 12, 15, 18, 21 and 24 days after inoculation. Nucleic acid was extracted using a KingFisher 96 magnetic extractor and an Agencourt Chloropure magnetic extraction kit. The nucleic acid extracts were treated with DNase to remove all DNA. The RNA was analysed using a specific one-step multiplex normalised reverse transcription quantitative PCR (RT-qPCR) assay using dual-labelled fluorescent hydrolysis probes which was developed in this project to accurately measure BYDV-PAV titre in wheat. Physical measurements, including plant height, dry and wet weight and tiller number were also taken at each sampling point. The titre of BYDV-PAV in wheat plants grown at elevated temperature peaked at days 12-15, then decreased until day 21 and stabilised from day 21. The titre of BYDV-PAV in wheat plants grown at ambient temperature continued to increase until days 15-18 and stabilised from day 18. The titre of BYDV-PAV in plants grown at elevated temperature was significantly greater on days 6, 9, 12 and 15 than the titre of the virus in wheat plants grown at ambient temperature. Significant differences in plant height, fresh weight, dry weight and tiller number were observed between the ambient and elevated temperatures. The plants grown at the elevated temperature were bigger, developed at a faster rate and symptoms associated with BYDV-PAV were visible earlier compared to those plants grown at the ambient temperature. This information will be used to model the epidemiology of yellow dwarf diseases on wheat yield in future climates.

Notes


S7 - O4

The effect of mineral nutrient availability to herbaceous indicators influences the disease expression of Apple stem grooving virus (ASGV) and Tobacco ringspot virus (TRSV)

Mary HornerThe New Zealand Institute for Plant & Food Research [email protected]

Mary Horner(1), Roy van den Brink(2), Paul Austin(2)

(1)The New Zealand Institute for Plant and Food Research Limited, Private Bag 1401, Havelock North 4157, New Zealand(2)The New Zealand Institute for Plant and Food Research Limited, Private Bag 11600, Manawatu Mail Centre,

Palmerston North, 4442, New Zealand

The effect of mineral nutrient availability to herbaceous indicators influences the disease expression of Apple stem grooving virus (ASGV) and Tobacco ringspot virus (TRSV) infection in herbaceous indicators. TRSV infected plants express severe disease symptoms when plants are grown with low nutrient availability, and have a lower symptom severity scores when grown in soil with ample fertiliser. The reverse applies with ASGV, where disease symptom expression from ASGV infection increases with higher fertiliser availability. ASGV infection in herbaceous indicators did not decrease the plant height whereas TRSV infection in herbaceous indicators resulted in an average of 10% reduction in plant height. The amount of fertiliser did not affect the time to develop viral symptoms. Reduced light levels were associated with an increased level of virus infection.


Notes

S7 - O5

Is the most conserved calcium-dependent protein kinase important in plant viral responses?

Gardette ValmonteThe New Zealand Institute for Plant & Food Research Limited and AUT [email protected]

Gardette Valmonte(1)(2), Colleen M. Higgins(2), Robin MacDiarmid(1)(3)

(1)The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand(2)School of Applied Sciences, AUT University, New Zealand(3)School of Biological Sciences, University of Auckland, New Zealand

Calcium-dependent protein kinases (CPKs) are a large multigene family of cellular signalling proteins involved in plant development and stress response. CPKs are present in all plants studied to date, as well as in protists, algae, and oomycetes, but are not found in fungi and animals. We have investigated the evolutionary history of this gene family by carrying out a broadly sampled phylogenetic analysis and have identified that Group IIB CPKs are the most conserved members. Thus, these CPKs may also have the highest degree of functional conservation and therefore present good target genes for a study of stress tolerance that is applicable to a broad range of plant species. A group IIB CPK, Arabidopsis CPK3 (AtCPK3), appears to be important in salt and osmotic stress, stomatal closure and bacterial (flg22) responses. Hence, we aim to characterize the function of this CPK and its orthologues in response to various biotic and abiotic stresses. As part of this project, we aim to determine if AtCPK3 and its orthologues are important in plant viral responses. To determine this, we will examine the expression levels of AtCPK3 and its orthologues in response to common plant viruses, including Cauliflower mosaic virus (CaMV), Tobacco mosaic virus (TMV) and Tomato spotted-wilt virus (TSWV). The transcript accumulation of AtCPK3 in Arabidopsis and its orthologues in kiwifruit and rice will be monitored at 0, 2, 3, 7, 14, and 21 days post inoculation (dpi) using reverse-transcriptase quantitative PCR (RT-qPCR). Symptom severity and stress responsiveness among over-expressing and knockout Arabidopsis and kiwifruit lines upon viral infection will be assessed. Very little is known about CPK responses to viral infections. Only a small number of Arabidopsis CPKs (AtCPK14, 18, 28, 29 & 32) have been demonstrated to change transcript abundance in response to viruses such as Plum pox virus (PPV) and Cucumber mosaic virus (CMV). No virus response has yet been reported for AtCPK3 and its orthologues, despite its responsiveness to certain stresses and bacterial elicitors. This study would therefore establish the importance of CPKs in plant viral responses. This could be of enormous value to agriculture, providing potential opportunities to develop novel strategies to protect against viral diseases.

Notes


S7 - P1

Identifying calcium dependent protein kinases that respond to virus infection

Kieren ArthurThe New Zealand Institute for Plant & Food Research [email protected]

Kieren Arthur (1), Gardette Valmonte (1)(2), Colleen Higgins (2), Robin MacDiarmid (1)

(1) The New Zealand Institute for Plant & Food Research Limited(2) School of Applied Sciences, AUT University, New Zealand

Calcium dependent protein kinases (CPKs) are a diverse gene family that are part of plant signalling responses to environmental stimuli including biotic stresses induced by pathogens. However, studies with plant virus-responding CPKs are limited to two Gene Chip studies with Arabidopsis thaliana. In the first study, with A. thaliana (ecotype Col-24) AtCPK29 showed a 0.626 fold difference in transcript accumulation in a resistant interaction 18 hours after infection with Cucumber mosaic virus [Marathe et al., 2004] compared with uninfected control plants. In a second study, three AtCPKs (14, 32, 28) showed increased transcript accumulation (5.29x, 3.32x and 2.62x, respectively), while AtCPK18 showed decreased transcript accumulation (2.7x) compared with uninfected plants 17 days after infection of A. thaliana with Plum pox virus [Babu et al., 2008]. To complement this information, we will use RT-qPCR to measure the transcript accumulation of these five CPKs in response to both compatible and incompatible virus infections compared with uninfected control plants. As there are 34 CPKs in A. thaliana, it is also of interest to know if any of the other 29 CPKs respond to virus infection. Using RT-qPCR to monitor these remaining AtCPKs would be both time consuming and labour intensive. To hasten the process, we aim to pre-screen infected and healthy leaf material for virus responsive AtCPKs using degenerate primers targeted to CPKs, followed by massively parallel sequencing to quantify mRNA accumulation of specific CPKs. Analysis of the sequencing data will identify those CPK sequences which accumulate differently between the virus-challenged and mock-challenged leaf material. These differences will then be confirmed by RT-qPCR of specific CPK transcripts. After virus responsive AtCPKs have been identified, T-DNA knock out lines for these AtCPKs will be monitored for phenotypic differences following virus infection compared with the wild type. Our studies will be extended to determine if orthologous CPKs from other plant species have the same response to virus infection as monitored by RT-qPCR. These experiments will help us understand the role that CPKs play in signalling and defence upon virus infection, and whether these pathways are similar across species. Moreover, the use of CPK degenerate primers combined with deep sequencing may in the future provide a useful method to screen CPKs that respond to other types of pathogens as well as abiotic stresses.

References: Marathe R, Guan Z, Anandalakshmi R, Zhao H, Dinesh-Kumar S (2004) Study of Arabidopsis thaliana resistome in response to cucumber mosaic virus infection using whole genome microarray. Plant Molecular Biology 55:501-520 Babu M, Griffiths J, Huang T-S, Wang A (2008) Altered gene expression changes in Arabidopsis leaf tissues and protoplasts in response to Plum pox virus infection. BMC Genomics 9:325-346


Notes

S8 - O1

A novel antivirus defence in plants? Characterization of CPK

Tracey ImmanuelThe New Zealand Institute for Plant & Food Research [email protected]

Tracey Immanuel(1)(2), Elaine Chan (1), David Greenwood (1)(2), Robin MacDiarmid (1)(2)

(1) School of Biological Sciences, The University of Auckland, Auckland, New Zealand(2) BioProtection Technologies, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand

An antiviral mechanism that is well characterized in vertebrates is the inhibition of protein translation, a core function that viruses require from their cellular hosts. In mammals, inhibition of translation occurs when the translation initiation factor eIF2α is phosphorylated by the double-stranded RNA (dsRNA)-activated protein kinase PKR. Thus, PKR acts as an inhibitory switch for translation upon activation by virus dsRNA. Research questioning the existence of an analogous antiviral defence in plants has led to the identification of an Arabidopsis thaliana calcium-dependent protein kinase (CPK) as a candidate for a dsRNA-activated kinase that phosphorylates eIF2α. We have demonstrated that CPK phosphorylates eIF2α; however, this phosphorylation event does not occur on the conserved Serine residue common to other well-characterized eIF2α kinases, including PKR. It is yet to be determined if CPK-induced phosphorylation of eIF2α regulates protein translation in a similar manner to those of PKR and other eIF2α kinases.

Notes


S8 - O2

Optimisation of herbaceous indicator screening for virus infection

Mary HornerThe New Zealand Institute for Plant & Food Research [email protected]

Mary Horner (1), Paul Austin (2), Roy van den Brink (2)

(1)The New Zealand Institute for Plant and Food Research Limited, Private Bag 1401, Havelock North 4157, New Zealand(2)The New Zealand Institute for Plant and Food Research Limited, Private Bag 11600, Manawatu Mail Centre,

Palmerston North, 4442, New Zealand

Herbaceous indicator screening is still a preferred technique for detecting virus infection in plant germplasm in post entry quarantine. Experiments were carried out to determine optimal growing conditions of indicator plants and inoculation techniques to maximise the level of disease expression. Experiments were carried out using Apple stem grooving virus and Tobacco ringspot virus . Inoculum dilutions of 1:10, 1:100 and 1:1000 resulted in 97% , 83% and 11% infection rates respectively. Shading of the herbaceous indicator plants 24 hours prior to inoculation increased the infection rate by 76%. No significant differences were observed in the percentage of infected plants when diatomaceous earth was incorporated into the inoculum buffer or sprinkled directly onto the plants. However, the development of disease severity and symptoms was increased when the diatomaceous earth was sprinkled directly onto the plants. The use of a gloved finger, spun-bonded polyester cloth, or muslin to apply inoculums did not change in the percentage of infected plants. ASGV inoculated Chenopodium quinoa plants grown at 20, 23 or 26 °C resulted in 85%, 78% and 63% infection rates respectively. The number of local lesions per plant increased with temperature, while the infection rate decreased with temperature.


Notes

S8 - O3

A re-evaluation of commonly held views on mycoviruses and their interaction with their fungal hosts

Mike PearsonThe University of [email protected]

Michael Pearson

School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand.

Until relatively recently the mycovirus literature was dominated by reports of dsRNA viruses, either unencapsidated or with isometric particles, but it is becoming increasingly evident that there is a far greater variety of mycoviruses than previously thought. The predominant view of viruses, in genral, is that as obligate parasites they have a negative affect on their hosts. For mycoviruses it is clear that this view is too simplistic and their effects on their fungal hosts can range from clearly detrimental through symptomless to advantageous. In addition the effects on the fungal host may have either positive of negative flow on effects on crop production depending upon the role of the fungus in the crop ecosystem. For example, the fungus may be a plant pathogen, such as Botrytis or Sclerotinia, or an introduced biological control agent such as the entomopathogenic fungus Beauveria. While in most cases the effects of the viruses on in-vitro growth of their fungal host are small, results from Botrytis viruses have produced conflicting results from in-vitro growth assays and pathogenicity assays on plants. In addition we have observed substantial differences between the effects of viruses on different B. cinerea isolates. Since hyphal anastomosis is the only known mechanism of horizontal transmission of mycoviruses, vegetative incompatibility is often assumed to be a barrier to mycovirus spread. However, indirect evidence from the distribution and genetic variability of Botrytis mycoviruses and direct experimental evidence of spread between incompatible fungal isolates of other fungi is challenging this assumption.

Notes


S8 - O4

Spread of Grapevine leafroll-associated virus 1 and Grapevine virus A in a Cabernet Sauvignon vineyard in South Australia

John RandlesUniversity of [email protected]

Nuredin Habili, John Randles

Waite Diagnostics, School of Agriculture Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA5064, Australia

Leafroll is a serious virus disease of the grapevine which reduces the yield and negatively affects wine quality by reducing sugar and increasing acidity in the fruit. Grapevine leafroll viruses account for 41% yield reduction in Vitis vinifera cv. Muscat Gordo Blanco in South Australia (Nicholas 2004). To date up to 10 serologically distinct leafroll viruses from the Closteroviridae have been isolated from V. vinifera, of which Grapevine leafroll-associated virus 3 (GLRaV-3; Ampelovirus) has been reported to spread naturally in Australia, although no mealybug vectors have been found. GLRaV-1 is another ampelovirus which is also vectored by mealybugs, but to date there has been no report of its natural spread in Australia. Here, we report the natural spread of this virus in parallel with another unrelated virus, Grapevine virus A (GVA; Vitivirus, Betaflexiviridae) in a Cabernet Sauvignon vineyard at Robe, south eastern South Australia. Leafroll disease symptoms were first observed in the autumn of 2008 in three clones of V. vinifera, cv. Cabernet Sauvignon (clones G9V3, LC10 and SA125). Each clone was planted in 6 rows of 69 plants per row (a total of 414 vines per clone). These vines have since been monitored annually. The reddening symptoms in clone G9V3 increased from 19% (78 vines) in 2008 to 56% (232 vines) in 2012, and the respective increase in clone SA125 was from 5% to 36%. However, no symptoms were observed in clone LC10 until 2011 when an initial incidence of 36% was recorded and this has remained steady until 2012. The relative humidity in 2011 was high, coinciding with the increase in incidence of symptoms. Symptomatic vines were clustered in the block, consistent with spread by a slowly moving vector. Random samples from symptomatic and non-symptomatic vines were tested for 12 viruses using the standard protocol of Waite Diagnostics. A latent virus, Grapevine rupestris stem pitting-associated virus (GRSPaV) was present in all clones, while GLRaV-1 was detected only in the symptomatic vines. Over 60% of these symptomatic samples also tested positive for GVA. We conclude that the reddening is associated with mixed infection by GRSPaV and GLRaV-1, and possibly GVA. Spread seems likely to have occurred via insects such as mealybugs, and identification of vine virus vectors will be required to manage spread of viruses in Australian vineyards.


Notes

S8 - P1

Characterisation and biological properties of isometric viruses infecting Aspergillus

Mahjoub EjmalSchool of Biological Sciences, Auckland [email protected]

Mahjoub Ejmal(1), Mike Pearson(1), David Holland(2)

(1) Plant and fungal virology lab,Thomas Building, Level 3, Room 338B, School of Biological Sciences, Auckland University.(2) Middlemore Hospital,Hospital Road, Otahuhu, Auckland.

Mycoviruses have shown potential as biological control agents for plant pathogenic fungi which raises the question whether they might also be used to help treat human fungal diseases. Aspergillus is a common fungus which can sometimes infect humans, especially disease immunocompromised patients. Treatment of Aspergillosis by conventional chemical means is difficult and not always successful. A major attraction of mycoviruses as control agents is their high degree of specificity to fungi, making them safe to use on humans. Forty one clinical isolates and nine environmental isolates of Aspergillus spp. were screened for the presence of dsRNAs, which are indicative of the presence of mycoviruses, using CF-11 cellulose chromatography. Thirty four percent of the isolates contained dsRNAs, ranging between 200 and 6500 bp, which were present in various combinations. The in vitro growth rates of dsRNA positive and negative isolates were compared for A. fumigatus and A. niger. Statistically significant differences (P < 0.05) were observed between virus infected and virus-free isolates of A. niger, but not A. fumigatus. Isometric virus particles ranging between 15nm and 46nm were observed by transmission electron microscopy in most dsRNA containing isolates.

Notes


S8 - P2

Detection of the virus vector Polymyxa graminis infecting wheat roots in Western Australia


Belinda Cox(1)(2), Hao Luo(1)(2), Roger Jones(1)(2), (1)Crop Protection Branch, Department of Agriculture and Food Western Australia, Locked Bag No. 4, Bentley Delivery

Centre, Perth, WA 6983, Australia. (2)School of Plant Biology, Faculty of Natural and Agricultural Sciences, University of Western Australia, 35 Stirling Highway,

Crawley, WA, 6009, Australia.

The plasmodiophorid Polymyxa graminis is an obligate root parasite and a vector of soil-borne viruses that infect cereal crops. There are two groups of soil-borne viruses transmitted by P. graminis that severely reduce wheat and barley yields in many other parts of the world, the Furoviruses and Bymoviruses. The former include Soil-borne wheat mosaic virus (SBWMV), Soil-borne cereal mosaic virus (SBCMV) and Chinese wheat mosaic virus (CWMV). The latter include Wheat spindle streak mosaic virus (WSSMV), and Wheat yellow mosaic virus (WYMV). In New Zealand, both P. graminis and SBWMV have been found. In Australia, P. graminis was detected in the roots of a barley crop in Queensland in 2009. However, no soil borne-viral diseases of cereals have been recorded. In the 2011 broadacre growing season, a small-scale survey of wheat crops in the WA grainbelt was conducted as a component of another study. Random 100 leaf samples were collected from 22 wheat crops and tested for the presence of SBWMV, SBCMV and WSSMV by RT-PCR, but none of them were detected. Soil and wheat root samples were also collected from low lying areas at four sites. DNA extractions were carried out on root samples and tested for P. graminis by PCR. P. graminis was detected in wheat roots from Esperance and from DAFWA experimental field plots in South Perth. Presence of more than one variant of P. graminis was indicated, as two PCR bands of slightly different sizes were obtained. DNA sequencing was carried out across the 18S, ITS1 and 5.8S region of P. graminis, and the DNA sequences found were compared with sequences on genbank. The sequence from South Perth matched with P. graminis f. sp. temperata, and was almost identical to two isolates from Europe, and one from New Zealand. The sequence from an Esperance sample matched with a P. graminis isolate from garden soil from the UK. Soil-bait experiments were carried out in which wheat bait plants were grown in soil collected from Esperance and South Perth mixed with sand. Bait plants grown in sterilized soil served as a control. The plants were grown in growth cabinets under controlled environment conditions. P. graminis was detected in the roots of bait plants grown in soils from Esperance and South Perth by PCR, but none of the three soil-borne viruses were detected in their leaves.


Notes

S8 - P3

Breeding for PVY resistance in Australian potato germplasm

Brendan RodoniDepartment of Primary Industries, Victorian AgriBiosciences [email protected]

Rodoni, B.C. (1)(2), , Schultz, L. (1), , Cogan, N.O.I. (1), , Milinkovic, M. (1), , Forster, J.W. (1)(2), , Slater, A.T. (1)(2)

(1) Department of Primary Industries, Victorian AgriBiosciences Centre, Bundoora, Victoria 3083, Australia (2) LaTrobe University, Bundoora, Victoria 3086, Australia

Potato virus Y (PVY) incidence has dramatically increased in Australia over the past decade, requiring urgent development of locally adapted resistant cultivars. Reported yield losses due to the PVY infections are 10-80%, depending on cultivar, virus strain characteristics and the field and tuber storage conditions. Multiple strategies can be used to control PVY in potato production including reduction of PVY inoculum and aphid vector control, as well as the use of resistant cultivars. We report the screening of the main commercial cultivars that are available to Australian potato growers against PVYO and PVYNTN strains. Of the 74 cultivars that have been phenotyped for PVYNTN resistance, 61 were shown to be susceptible, while only 3 cultivars were considered resistant, with two additional cultivars testing negative twice and may also be resistant to both strains of PVY. Of the 69 cultivars that have been phenotyped for PVYO resistance, 6 tested negative on three or more occasions and were therefore considered resistant, a further 16 cultivars tested negative twice and require a third trial to confirm resistance and 28 cultivars were found to be susceptible to PVYO. The 74 cultivars that were phenotyped for PVY resistance were also genotyped with the markers RYSC3, M45 and STM0003. Only three cultivars amplified the RYSC3 marker, while the M45 marker was amplified in the same three cultivars and eight additional cultivars. The three cultivars that amplified both markers were Emma, Eva and PO3, while the M45 marker was also amplified in BC0894-2, Carlingford, Friar, Galil, KT3, Lady Christl, Melody and Royal Blue. These eleven cultivars all tested negative for the virus, when phenotyped for resistance to both strains of PVY, except for Emma, which was identified as susceptible to PVYNTN.

Notes


S8 - O5

The role of cauliflower mosaic virus (CAMV) defence and silencing suppressor protein 6 (P6) in modulating auxin signalling

Muhammad ShafiqInstitute of Agricultural Sciences University of the Punjab [email protected]

Muhammad Shafiq (1), Muhammad Ilyas (2), Muhammad Saleem Haider (3), Shaheen Asad(4)

Institute of Agricultural Sciences, University of the Punjab New Campus Lahore Pakistan

Expression of P6 protein of CaMV in Arabidopsis induced dwarfness in transgenic plants. It is reported that Arabidopsis plants with TIR3 gene mutated (tir3) are also dwarf. P6 transgenic (A7, B6) and tir3 Arabidopsis plants which were resistant to Auxin and ethylene also showed resitance to TIBA treatment.It indicates that P6 interacts with a pathway overlapped with TIR pathway. Symptoms appearance in Arabidopsis expressing P6 protein of CaMV is probably by disturbance of Auxin Response Factor 10 (ARF10), ARF16, and ARF17 also. As P6-expressing transgenic Arabidopsis plants showed reduced accumulation of miR160 which is known to regulate ARF10, ARF16 and ARF17.


Notes

S9 - O1

Geminivirus phylogeography

Arvind VarsaniUniversity of [email protected]

Pierre Lefeurve (1), Gordon Harkins (2), Adérito Monjane (3), Darren Martin (4), Arvind Varsani (5)(6)

(1)CIRAD, UMR 53 PVBMT CIRAD-Université de la Réunion, Pôle de Protection des Plantes, Ligne Paradis, 97410 Saint Pierre, La Réunion, France

(2)South African National Bioinformatics Institute, University of the Western Cape, Cape Town South Africa(3)Department of Molecular and Cell Biology, University of Cape Town, South Africa(4)Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa(5)School of Biological Sciences, University of Canterbury, New Zealand(6)Electron Microscope Unit, University of Cape Town, South Africa

Over the past five years an overwhelming diversity of circular single stranded DNA viruses (ssDNA), in particular those infecting plants and transmitted by insects, has been catalogued. This has primarily been attributed to new molecular tools for non-specific amplification of circular DNA coupled with cheap sequencing. The high resolution of data, for example for mastreviruses and begomoviruses, has enabled our team to address important aspects of viral evolution and spread using Bayesian phylogeographic analysis to reconstruct the plausible history and diversification of these viruses at a continental and global scale. For example, 1) Tomato yellow leaf curl virus (TYLCV) a pathogen crippling tomato production globally most probably arouse in the Middle East between 1930s and 1950s, with a global spread occurring in the 1980s and recombination playing a major role in its evolution; 2) Maize streak virus strain-A (MSV-A) which seems to have emerged in Southern Africa in the 1860s though recombination of mastreviruses infecting indigenous grasses, has spread transcontinentally at an average rate of 32.5 km/year.

Notes


S9 - O2

Recombination and reassortment events detected in the multi-component Banana bunchy top virus (BBTV)

Daisy StaintonUniversity of [email protected]

Daisy Stainton(1) , Simona Kraberger(1), Matthew Walters(1) , Elizabeth J Wiltshire(1) , Karyna Rosario(2) , Mana’ia Halafihi(3) , Samiuela Lolohea(4) , Ika Katoa(3) , Tu’amelie H Faitua(5) , Waikato Aholelei(3) , Luseane Taufa(3) , John E Thomas(6) , David A Collings(1)(7) , Darren P Martin(8) , Arvind Varsani(1)(7)(9)

(1)School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand(2)College of Marine Science, University of South Florida, St Petersburg, FL 33701, USA(3)Ministry of Agriculture and Food, Forests and Fisheries, Nuku’alofa, Tongatapu, Kingdom of Tonga(4)Tonga College, Nuku’alofa, Tongatapu, Kingdom of Tonga(5)Department of Education, Nuku’alofa, Tongatapu, Kingdom of Tonga(6)The University of Queensland, Centre for Plant Science, Queensland Alliance for Agriculture and Food Innovation,

Ecosciences Precinct, PO Box 46, Brisbane QLD 4001, Australia(7)Biomolecular Interaction Centre, University of Canterbury, Christchurch 8140, New Zealand(8)Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town,

Cape Town, South Africa(9)Electron Microscope Unit, University of Cape Town, Rondebosch 7701, Cape Town, South Africa

Banana bunchy top virus (BBTV), a member of the Nanoviridae family, is a ssDNA virus which infects banana plants throughout the banana growing regions of the world, causing plant stunting and severe crop loss. We sequenced all six components of this multi-component virus, which was isolated from 12 infected plants collected in the Kingdom of Tonga. These, along with full BBTV sequences sourced from Genbank, were analysed for evidence of recombination and reassortment. Evidence of 8 reassortment events and multiple intra- and inter-component recombination events were found within these genomes. Reassortment was found within and between the two phylogenetic groups, South Pacific and Asian. Inter- and intra-component recombination was seen within all components, with DNA-U3 displaying complex intra-component recombination and DNA-R showing a common intra-recombination event throughout all the South Pacific isolates. DNA-U3 and DNA-M showed more inter-component events than DNA-R, -S, -C and -N. The inter-component recombination breakpoint distributions across all components reveal a recombination hotspot around the common region major.


Notes

S9 - O3

Provisional assembly and analysis of two Candidatus Liberibacter solanacearum genomes derived from independent new zealand sources

Sarah ThompsonThe New Zealand Institute for Plant & Food Research [email protected]

Sarah Thompson, Mark Fiers, Andrew Pitman, Ian Scott, Grant SmithTheNew Zealand Institute for Plant & Food Research Ltd, Gerald Street, Lincoln 7608, New Zealand

The unculturable α-proteobacterium Candidatus Liberibacter solanacearum (CLso) is associated with Zebra Chip, an important emerging disease of potato (Solanum tuberosum). This phloem-limited bacterium, transmitted by the tomato-potato psyllid Bactericera cockerelli, was first identified in 2008 and is related to the Ca. Liberibacter species implicated in Huanglongbing (citrus greening), currently considered the most serious and destructive disease of citrus in the world. Two New Zealand CLso genomes have been provisionally assembled against the 1.26 Mbp genome of a USA isolate of CLso obtained from psyllids infesting potatoes (CLso-ZC1). The two independent New Zealand sources were a single psyllid from a colony which originated from insects collected from tamarillo (NZCLso-TPP), and a CLso infected tomato plant (NZCLso-Tom). Preliminary analysis of the genome alignments revealed that, despite being sourced from two different host organisms, the two NZ genomes are much more similar to each other than to the single USA genome. The first genome drafts identified 227 regions missing from the alignments. Many of these missing regions are intergenic. There are also four coding regions, containing 24 putative genes (approximately 2% of the putative coding sequences) which are currently missing. It is uncertain if these regions are missing from the NZ CLso genomes, or are not visible due to either genomic rearrangements or a high level of polymorphism. We have not been able to manually sequence these regions to date. One major inversion of 55 kB has been located in the NZCLso-Tom genome compared to CLso-ZC1, and both NZ CLso genomes contain two large putative prophage regions similar to the US CLso-ZC1 genome. The draft assemblies for the putative prophage regions confirm the similarity of the NZ genomes and the difference between the NZ genomes and the US genome. These findings suggest that there is significant genotypic variance in CLso.

Notes


S9 - O4

A top-down approach: Discovery of a novel mastrevirus and alphasatellite-like circular dna in the Caribbean using dragonflies

Arvind VarsaniUniversity of [email protected]

Karyna Rosario(1), Marco Padilla-Rodriguez(1), Simona Kraberger(2), Daisy Stainton(2), Darren P Martin(3), Mya Breitbart(1), Arvind Varsani(2)(4)(5)

(1)College of Marine Science, University of South Florida, St Petersburg, Florida, 33701, USA(2)School of Biological Sciences, University of Canterbury, Ilam, Christchurch, 8140, New Zealand(3)Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, 7925, Cape Town, South

Africa(4)Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand(5)Electron Microscope Unit, University of Cape Town, Rondebosch, Cape Town, 7701, South Africa

Members of the Geminiviridae family are as serious agricultural pathogens. Despite the numerous species that have been already discovered and catalogued, new molecular techniques continue to unravel the diversity and geographical ranges of these single-stranded DNA (ssDNA) viruses and their associated satellite molecules. Examination of insect vector populations through vector-enabled metagenomics (VEM) has been recently used to investigate the diversity of geminiviruses transmitted by Bemisia tabaci in Florida, USA. We adopt for top-down approach to sample plant viruses in ecosystems using top-end insect predators, dragonflies. Our ‘predator-enabled’ approach is not vector limited since dragonflies can accumulate a wide range of viruses within an ecosystem. As part of our ssDNA virus discovery program, analysis of six dragonflies collected from an agricultural field in Puerto Rico resulted in the discovery of the first mastrevirus (Dragonfly-associated mastrevirus; DfasMV) and alphasatellite molecule (Dragonfly-associated alphasatellite; Dfas-alphasatellite) from the Caribbean. Since DfasMV and Dfas-alphasatellite are divergent from the limited number of sequences that have been reported from the Americas, this study unequivocally demonstrates that there have been at least two independent past introductions of both mastreviruses and alphasatellites to the New World. Through the use of predacious insects as sampling tools, we have discovered a wealth of novel ssDNA viruses, some of which represent new viral families. Our top-down approach can profoundly alter our views of natural plant virus diversity and biogeography without a priori knowledge of the types of viruses or insect vectors in region.


Notes

S10 - O1

Genome organisation of Vanilla mosaic virus as determined by deep sequencing

Colleen HigginsAuckland University of [email protected]

Colleen M. Higgins, Subuhi Khan , Wee-Leong Chang


Vanilla mosaic virus (VanMV) is a potyvirus that infects only Orchidaceae Vanilla sp. It appears to be a strain of Dasheen mosaic virus (DsMV), a virus which only infects plants of a different family, namely the Araceae. It is of great interest that such closely related viruses have such different host ranges; understanding the molecular basis for this may provide insights into the determination of host ranges in general for this type of virus. To carry out such a study firstly requires comparison of the genome sequences of these viruses. Such a sequence is available for DsMV, but not for VanMV, thus, we have determined the full length sequence for VanMV using Illumina sequencing technology. Using the DsMV genome as the reference genome, the VanMV sequence was assembled, and compared with DsMV. The genome structure and variability of VanMV as well as its relationship with DsMV will be discussed.

Notes


S10 - O2

Genome sequencing of Australian Potato virus Y isolates

Linda ZhengDepartment of Primary [email protected]

Linda Zheng(1), Lee Schultz(2), Noel Cogan(2), Mirko Milinkovic(1), Anthony Slater(1)(3), John Thomas(4), Brendan Rodoni(1)

(1)Victorian Department of Primary Industries, Biosciences Research Division. Knoxfield Centre, Knoxfield, Victoria 3180, Australia.

(2)Victorian Department of Primary Industries, Biosciences Research Division.Victorian AgriBiosciences Centre, Bundoora, Victoria 3083, Australia.

(3)La Trobe University, Bundoora, Victoria 3086, Australia. (4)The University of Queensland QAAFI Institute, Centre for Plant Science, Dutton Park, Queensland 4102, Australia.

Potato virus Y (PVY) is found world-wide, and considered one of the most important virus diseases for seed and commercial potato growers. PVY exists as a complex group of strains and foliar symptoms vary from severe mottling and mild mosaic to symptomless depending on the host cultivar and virus strain. Until recently, PVY was a minor problem in Australia and the only detections were of the common PVYO strain. However, in 2003, the PVYN strain was first detected and tubers were found exhibiting tuber ringspot necrotic disease. Since this first detection of PVYNTN, the incidence of PVY has increased in south east Australia and effective management and control strategies are needed to minimise crop loses. One control strategy is breeding virus resistant cultivars, as it is often the most economical and environmentally acceptable way of controlling virus diseases of plants. The first step to breeding PVY resistant potatoes for Australia is to first identify the type isolates of PVY that currently pose a threat to the Australian potato industry, and then apply these type isolates in disease resistance trials. Sixteen isolates of PVY were collected from various parts of Australia and subjected to whole-genome sequencing and subsequent phylogenetic analysis. Included in the 16 isolates were PVYO and PVYC strains from reference collections. Preliminary results show that a majority of the isolates are PVYN strains, whilst a smaller clade of isolates belonged to the PVYO/PVYC subgroup. The full-length genomes of these PVY isolates have provided an insight into the diversity of PVY strains in Australia and the spread of the PVYN strain since its entrance into Australia in 2003.


Notes

S10 - O3

Molecular characterisation of two divergent strains of Grapevine leafroll-associated virus 3 in New Zealand

Dan CohenThe New Zealand Institute for Plant & Food Research [email protected]

Kar Mun Chooi(1), Daniel Cohen(2), Michael Pearson(1)

(1)School of Biological Sciences, University of Auckland, P.O. Box 92019, Auckland, New Zealand(2)The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland, New Zealand

Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically important virus that is found in all grapevine growing regions worldwide. A reliable GLRaV-3 detection method is important to any disease management programme. However, genetic variability within the virus population can compromise detection as was observed with the occasional false negatives by ELISA using the monoclonal Bioreba reagents and RT-PCR using common diagnostic primers. To date, at least six different GLRaV-3 phylogenetic variants have been identified in New Zealand, including two divergent variants, NZ1 and NZ2, which are more than 20% different at the nucleotide level to the previously published GLRaV-3 sequences, from phylogenetic groups 1 to 5. Subsequent testing found that samples, previously generating low ELISA readings using Bioreba reagents and testing negative by RT-PCR, contained only NZ1 and/or NZ2 variants. Therefore, a study was conducted to confirm the identity and better understand the genetic structure of these divergent GLRaV-3 strains. The partial genomic sequences of NZ1-B (an isolate of the previously identified divergent strain NZ1) and NZ2 were determined and analysed (11,827 nt and 7,612 nt, respectively). Both genetic variants show sequence variability throughout the genome sequence, particularly towards the 3’UTR resulting in changes to the open reading frames 11 and 12. Phylogenetic analysis indicated NZ2 is a new strain of GLRaV-3 and that there is a need for the classification of the GLRaV-3 phylogenetic groupings to be re-visited. In addition, amino acid analysis of the NZ1-B and NZ2 coat proteins indicate significant substitutions that are predicted to alter the coat protein structure, which potentially leads to the observed reduced immunological reactivity of both variants to the Bioreba anti-GLRaV-3 conjugated monoclonal antibody.

Notes


S10 - P1

The molecular variability of Zucchini yellow mosiac virus and Papaya ringspot virus in the Pacific Islands

Mike PearsonThe University of [email protected]

Karl Crosby (1), Colleen Higgins (2), Michael Pearson (1) , (1)School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.(2)School of Applied Sciences, Auckland University of Technology, New Zealand.

Potyvirus is an economically important virus genus and includes species that limit the production of cucurbit crops worldwide. Although considerable global effort has focused on characterizing and understanding these potyviruses very little research has focused on the Pacific. This project aimed to characterize the molecular variability of two important cucurbit potyviruses Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus (PRSV) in ten Pacific Island Countries and Territories through the molecular analysis of the coat protein. Their molecular variability was contextualized through comparing and contrasting them with worldwide samples via phylogenetic analyses and adaptive selection tests. Both ZYMV and PRSV sequences grouped according to geographic origin and had strong purifying selection that varied in its intensity along the codons of the coat protein, with a number of codons having neutral selection. The ZYMV sequences belonged to three of the sub-groups (I, II and IV) of group A. The Samoan and Tongan sequences grouped in A-IV with Japanese and New Zealand sequences which are cucurbit trade partners; Vanuatu and Nauru grouped in A-I; and New Caledonian and Federated States of Micronesia grouped in A-II. The PRSV sequences split into two groups; the Commonwealth of the Northern Marinara Islands with the Asia group, and all the remaining in the Americas group. Having established the identity of the ZYMV and PRSV strains in the major crops the next step would be to determine whether other hosts carry the same strains and are acting as reservoirs for crop infection or whether the infection cycle is limited to the crop hosts only (e.g. seed transmission). This information is essential in developing appropriate disease management strategies.


Notes

S10 - O4

Complete genome sequences and genetic diversity of subgroup 1 Ilarviruses from Eastern Australia

Murray SharmanQueensland Department of Agriculture, Fisheries and [email protected]

Murray Sharman(1)(2), John Thomas(3), Andre Drenth(3)

(1)Department of Agriculture, Fisheries and Forestry, Ecosciences Precinct, GPO Box 267, Brisbane, Queensland, 4001, Australia

(2)School of Agriculture and Food Sciences, University of Queensland, St. Lucia campus, Queensland, Australia(3)Centre for Plant Science, The University of Queensland, Queensland Alliance for Agriculture and Food Innovation,

Ecosciences Precinct, GPO Box 267, Brisbane, Queensland, 4001, Australia

Tobacco streak virus (TSV; genus ilarvirus, family Bromoviridae) has been one of the most damaging viruses in Australian oilseed and pulse crops in recent years. This is the first report of the genetic diversity of subgroup 1 ilarviruses from eastern Australia and has revealed the most divergent strains of TSV currently described worldwide, an isolate of Strawberry necrotic shock virus (SNSV) and a newly proposed subgroup 1 ilarvirus, Ageratum latent virus (AgLV). All were previously assumed to strains of TSV. Six separate phylogenetic analyses demonstrate that AgLV should be considered as a distinct ilarvirus subgroup 1 species. The closest amino acid identities for each of the putative proteins of AgLV are with Parietaria mottle virus for the replicase (p1) and RdRp (p2) proteins with 81% and 76% identity respectively, with Blackberry chlorotic ringspot virus for the 2b protein with 68% identity, and with TSV for the movement (3a) and coat (3b) proteins with 79% and 75% identity respectively. A multiplex RT-PCR showed that AgLV and the genetically distinct strains of TSV were commonly found as symptomless infections in virus-specific alternative weed hosts as symptomless infections from a wide geographical range in eastern Australia. The three strains were found to have wide geographical distributions, each spanning several hundred kilometres from north to south, apparently coinciding with, and possibly endemic throughout, the geographical range of their specific alternative host. The parthenium and crownbeard TSV strains were found in central Queensland, often occurring in the same locations but no natural mixed infections were identified. AgLV was found in coastal areas of south-east Queensland and northern NSW and its distribution was almost completely independent of the TSV strains.

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19-22 november 2012 hanmer springs, new zealand

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