PPRI 11 19Prepared by: Prof. G Pietersen Tel: 012 420 3265 or 082 647 5326 Email:...

This document is confidential and any unauthorised disclosure is prohibited Version 2015

Industry allocated project number

PHI allocated project number

.

FINAL REPORT 2017

AGRICULTURAL RESEARCH COUNCIL

PLANT PROTECTION RESEARCH INSTITUTE P/Bag X134, Queenswood, Pretoria 0121

FINAL REPORT Ref: PPRI-11/19

Improved virus detection and identification for the Wine Grape Certification Scheme

Prepared by: Prof. G Pietersen

Tel: 012 420 3265 or 082 647 5326 Email: [email protected]

Prepared for: Client: Winetech Contact person: Anel Andrag

Tel: (021) 276 0499 Fax: 086 611 7817 Email: [email protected]

JULY 2017

Researcher surname 2


SATI

CFPA

SAAPPA/SASPA

DFTS

Winetech

[email protected] [email protected] [email protected] [email protected] [email protected]

Tel: 021 863-0366 Tel: 021 872-1501 Tel: 021 882-8470 Tel: 021 870 2900 Tel: 021 276 0499 X

FINAL REPORT

(2017)

1. PROGRAMME AND PROJECT LEADER INFORMATION Research

Organisation Programme leader

Research Team Manager Project leader

Title, initials, surname

Dr. Isabel Rong Prof.GerhardPietersen Prof.GerhardPietersen

Present position

Programme Manager SpecialistScientistandExtra-OrdinaryProfessor

SpecialistScientistandExtra-OrdinaryProfessor

Address ARC-PPRI Private bag X134 Queenswood 0121

ARC-PPRI,c/oDept.ofMicrobiologyandPlantPathology,UniversityofPretoria,0002

ARC-PPRI,c/oDept.ofMicrobiologyandPlantPathology,UniversityofPretoria,0002

Tel. / Cell no.

012 808-8000 0124203265/0826475326 0124203265/0826475326

Fax 0124203266 0124203266E-mail [email protected] [email protected] [email protected] Co-worker Student Title, initials, surname

Dr. AEC Jooste Jennifer Wayland

Present position

Researcher MSc student

Address ARC-PPR Private bag X134 Queenswood, 0121

Dept. of Microbiology & Plant Pathology, University of Pretoria, Pretoria, 0002

Tel. / Cell no.

0128088197 012 420-3279

Fax 0128088299 012 420 3266 E-mail [email protected] [email protected]



2. PROJECT INFORMATION Research Organisation Project number

PPRI 11-19

Project title Improved virus detection and identification for the Wine Grape certification scheme

Short title Grapevine virus detection by NGS Fruit kind(s) Wine grapes Start date (mm/yyyy) 01-04-2012 End date (mm/yyyy) 31-03-2017

Key words Grapevine leafroll disease, technology transfer, presentations

Approved by Research Organisation Programme leader (tick box) 3. EXECUTIVE SUMMARY The South African Wine Grape Certification Scheme only tests for the most important of the total range of viruses infecting Vitis worldwide. In this project, we intended expanding the number of viruses that can be tested within the certification scheme. We developed a protocol in which vines can be tested via multiple parallel PCR systems, each capable of detecting a range of viral species within a specific genus, along with some virus specific systems. Should a requirement exist for the identity of the individual virus species to be determined from genus wide PCR system this will be done by Illumina sequencing of pooled preparations of the amplicons from the PCR’s of any given vine, and by doing a number of such vines in parallel by indexing such amplicon pools separately. We have implemented PCR to the following genera to achieve this: Tombusviruses, Maculaviruses, Marafiviruses, Geminiviruses, Reoviruses, Nepoviruses (clades A, B, and C), Vitiviruses, Foveaviruses, Trichoviruses, Vealriviruses, Ampeloviruses, Closteroviruses, Ilarviruses, Alfamoviruses, Cucumoviruses, Potexviruses, Badnaviruses, Potyviruses, Tobamoviruses and Luteoviruses. While the protocol was not be evaluated against all viruses of grapevines, it is theoretically capable of detecting 41 viruses reported to infect this host. Because of the unexpected phenomenon of index-leaching during Illumina sequencing, it was necessary to develop a method of determining the positive/negative threshold for the systems along with a protocol for read analysis. We tested the protocol successfully on a number of vines from nuclear material to symptomatic field-collected material. The protocol and appropriate reagents was transferred to the Virus diagnostic unit of the ARC-PPRI for routine testing of viruses of grapevines. It is anticipated that utilisation of this system by the Wine industry to test nuclear material, will lead to the production of vine planting material of the highest virus-free status in the world, and will have a positive impact on South African winegrape production which unfortunately will be difficult to quantify. 4. PROBLEM IDENTIFICATION AND OBJECTIVES . Within the South African Wine Grape Certification Scheme, only few of the total range of viruses infecting Vitis worldwide, are tested for in nuclear-, foundation- and mother-block planting material. In addition, a number of virus-like diseases of unknown aetiology are also tested using biological indexing. In this project, we intend expanding the number of tests that could be used in the certification scheme on primarily nuclear plants. To achieve this we will:

X



1) Assess the ability of massively parallel sequencing (MPS) also known as next generation sequencing (NGS) to serve as a means of improving routine virus detection within the South African Wine Grape Certification Scheme of nuclear material in a non-virus-specific way for known and unknown viruses of Vitis. 2) Assess the usefulness of MPS/NGS to replace IEM and PCR tests that are currently outsourced at great cost to the industry, 3) Assess the use of MPS/NGS on highly multiplexed samples for the detection of viruses in foundation block and mother-block planting material for viruses other than those associated with leafroll. As a second aspect, we intended evaluation of routine application of GLRaV-3 LAMP, developed in project PPRI-GP2, and comparison with industry performed ELISA tests (as a finalisation project PPRI-GP2), to detect GLRaV-3 in rootstocks, and to assess the usefulness of a published LAMP protocol for the detection of Aster yellows phytoplasma in the field 5. WORKPLAN (MATERIALS AND METHODS) In this project a protocol for routine, non-specific detection of viruses in Vitis plants was developed using massively parallel sequencing and multiplexing to replace current IEM and PCR (and possibly indexing) -based techniques done within the industry. To achieve this, the following objectives were pursued.

1) Initially a protocol was developed as a proof-of concept study using six genus wide primer pairs in RT-PCR systems to amplify members of the Clostero-, Viti-, Fovea- , GLRaV-4 like and Nepoviruses (two primer sets).

2) Various polymerase, reverse transcriptase, and buffer systems were evaluated in PCR

to obtain a standardised system that could be performed with multiple primer systems.

3) Using this, a known, multiple virus infected vine, which had been characterised in the past (Black Spanish; PPRI Accession 90-0246) was tested for the presence of the viral genera mentioned. Amplicons obtained were pooled and subjected to Illumina sequencing in order to identify the viruses present.

4) In subsequent years additional primer sets directed at other viral genera were assessed

for their usefulness in parallel with the previously established RT-PCR systems. Some DNA virus systems were also included for use in PCR.

5) Methods of data analysis of the Illumina reads obtained against the amplicons of the

PCRs tests were developed to streamline the search for virus within the Vitis host background.

6) The multiplexing of samples was evaluated using indexed pools of cloned amplicons as

templates, in order to determine the number of samples that can be sequenced in parallel (a critical component in reducing costs), without compromising the coverage of the sequence data and accuracy of sequence identification.

7) Using the established template or template preparations and multiplexing conditions a

number of nuclear plants from the certification scheme were be selected and the virus status determined by MPS.

8) Symptomatic vines from foundation or mother-block vineyards were analysed using the

developed MPS protocol.



In the second aspect of the project (primarily a finalisation of project PPRI-GP2), we evaluated 1) the routine application of GLRaV-3 LAMP, developed in project PPRI-GP2, and comparison with industry performed ELISA tests. (Finalisation project PPRI-GP2) 2) the GLRaV-3 LAMP developed in project PPRI-GP2, for the detection of GLRaV-3 in rootstocks. And, 3) the usefulness of a published LAMP protocol for the detection of Aster yellows phytoplasma in the field. 6. RESULTS AND DISCUSSION We searched the literature for viruses infecting grapevines worldwide. Lists were prepared according to their respective genus or family. Published degenerate or universal (genus-wide) PCR primers were identified and synthesized locally. Grapevine plants were identified within the virus collection of PPRI that could serve as positive controls in the various PCR tests. Collaborators worldwide were approached to provide appropriate positive controls where none were available locally. Initially PCR’s were established to detect Closteroviruses, Ampeloviruses, Velariviruses, Vitiviruses, Foveaviruses Trichoviruses, and Nepoviruses of grapevines. A generic (with regards PCR buffers and reagents barring the primers) RT-PCR protocol based on GoScript reverse transcriptase and Gotaq polymerase enzymes, was developed in order to standardise an amplification protocol for the different primer sets. The various PCR systems were utilised within the generic master mix, in a proof of concept test on a single grapevine plant previously shown to harbour multiple viruses. Amplicons obtained were pooled and subjected to Illumina sequencing in order to identify the individual viruses obtained. Illumina reads obtained were analysed for virus identity. The results confirmed the presence of grapevine leafroll associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3 and grapevine fleck virus (GFkV) all previously found in the vine using immune electron microscopy (ISEM). The plant however also contained GLRaV-4, GLRaV-6, grapevine virus A (GVA), grapevine virus F (GVF) and Rupestris stem pitting associated virus (RSPaV) as determined by using the polyspecific PCR and Illumina sequencing protocol. At that stage grapevine virus F had not previously been reported in South Africa nor had it been reported to be detectable with the specific primers utilised. This confirmed the undoubted potential of the technique for the routine detection and identification of multiple viruses of grapevines (either as mixed- or single infections), including some related non-target viruses, within the certification scheme. To assess the usefulness of tagging/indexing different samples for parallel sequencing it was utilised to detect members of the viral genera listed above in a number of grapevine samples. During these tests we obtained some false positives suggestive of index leaching or cross sample contamination during the protocol. This required a considerable re-prioritisation of the project to identify the steps at which contamination occurs, to minimize this, and to account for this through the establishment of positive/negative thresholds in the analysis of the data. Regions cognate to the amplicons from the PCR’s were cloned to serve as positive controls for all systems developed and were used to create defined templates in order to establish an analysis pipeline for Illumina data interpretation (Figure 1). During this process we assessed the: 1) variability in results obtained, introduced by various steps of the protocol; 2) the limitations that pooling a number of numbers of amplicons from different tests to serve as NGS template may have on the ability to detect each of the systems;



3) effect of varying amounts of amplicon templates from individual tests on reads obtained; 4) the effect that amplicon G:C ratio and amplicon length on the NGS reads obtained, 5) effect introduced by primer degeneracy, and 6) requirement of de novo assembly for detection of novel viruses. The detailed establishment and requirements of this pipeline can be found in the 2015 and 2016 Progress reports of this project, but are summarized in Figure 1. The number of PCR systems were increased during the course of the project to detect an increasing number of virus genera (Table 1). In some instances, the importance of specific viruses or the poor performance of polyspecific PCR’s, resulted in virus specific PCR’s being utilised rather than genus-wide PCR’s (eg. GLRaV-1, -2 and -3). The usefulness of the optimised protocol was assessed on nuclear material and some field-grown material with abnormalities submitted by Mr. T. Oosthuizen (Vititec) and Mrs. M. Louw (Bosman Brothers). The samples from Vititec were also sent to Waite Diagnositic for parallel testing in order to validate those obtained at UP/PPRI. The samples tested negative for the majority of the PCR systems (Table 5 of 2016 Progress report). Four samples yielded very feint bands in the GLRaV-3 PCR possibly due to the presence of a GLRaV-3 variant or low concentration of GLRaV-3 or from a related, cross-reacting virus. Many samples analysed yielded feint amplicon bands within the Viti-Fovea virus PCR, and in two instances these bands were clear. It is evident that further studies on this group of viruses needs to be performed. A total of 56 wine grape samples were collected from the regions of Somerset Wes and Stellenbosch in the Western Cape, including samples of Cabernet Sauvignon, Affenthaler, Bacchus, Aleatico nero, Capes Donnes Seedling, Blaue Kadarka, Cabernet franc, Grand noir de la Calmette, Majestic, Merlot, Malbec and Shiraz. The wine grape samples were selected based on various virus-like abnormalities or particularly severe leafroll symptoms. With additional THRIP funding, a total of 49 table grape samples (table grapes selected as they would have a greater likelihood of “unusual” viruses) were collected from the Western Cape in the regions Hex river valley, Paarl and Wellington. Included were Vitis vinifera cv. Starlight, Crimson, La Rochelle, Red Globe, Prime, Melody, Autumn Royal, Sugra 19 and Waltham cross. The samples were selected based on virus like disease symptoms. All of these samples were subjected to virus analysis using the expanded grapevine virus PCR protocol (Table 2, 2016 Progress report). In a previous project (GP2) RT-LAMP was shown to be a viable alternative to ELISA for the detection of GLRaV-3 on site, however actual implementation in these environments were problematic due to either contamination of the LAMP reactions or to failure of the reaction when utilised by industry. In the current project we finalised the optimisation and assessment of the LAMP for the detection of GLRaV-3 in white cultivars and rootstocks in the laboratory and published this work. Attempts to modify the protocol for field use was not very successful due to the unavailability of suitable sealable reagent vessels, and as the desired reporter dye “Genefinder” (red/green colour change) could not be accessed from China. We intend using a microcrystalline wax-dye capsule according to Tao et al. (2011) to detect GLRaV-3 in rootstock samples in a recently approved Winetech project on rootstocks as a means of preventing the contamination n problems inherent in our previous protocol. The ability of RT-LAMP to detect GLRaV-3 in grapevine rootstocks was confirmed when 78% of samples of five different rootstocks (R99, R110, 101-14, SC, Ruggeri) inoculated with GLRaV-3 tested positive for GLRaV-3 using this method. ELISA on the same rootstocks had only detected GLRaV-3 in 28%. The lowest incidence of RT-LAMP detection was with R110 where 3



of 9 samples tested were negative while the highest was with Salt Creek where only one of 11 samples tested was negative. Detection of Aster yellows was successful against AY-infected grapevines as well as Mgenia fuscovaria individuals using the LAMP protocol of Tomlinson et al., 2010. 7. COMPLETE THE FOLLOWING TABLE

Milestone Target Date

Extension Date

Date completed Achievement

1. Develop multiple genus- wide PCR’s and NGS protocol as a proof-of concept study.

04-2013

04-2013

Use six genus wide primer pairs in RT-PCR systems to amplify members of the Clostero-, Viti-, Fovea- , GLRaV-4 like and Nepoviruses (two primer sets)

2. Standardise polymerase, reverse transcriptase, and buffer systems for multiple parallel PCR systems.

04-2013

04-2013

RT-PCR protocol based on GoScript reverse transcriptase and Gotaq polymerase enzymes developed for use in multiple PCR’s

3. Using the protocol developed in 1 and 2 test a previously characterised infected vine with multiple virus infections.

04-2014

04-2014

Test conducted on Black Spanish sample confirmed the presence of GLRaV-1, GLRaV-2, GLRaV-3 and GFkV. Additionally showed that the vine contained GLRaV-4, GLRaV-6, GVA, GVF and RSPaV. Demonstrated usefulness of the technique

4. Expand PCR systems for use in the detection and identification of grapevine viruses within the protocol.

04-2017

04-2017

The number of PCR systems for use in the protocol and their properties are listed in Table 1. These are theoretically capable of detecting 41 viruses of grapevines

5. Develop methods of data analysis of the Illumina reads obtained

04-2015

06-2016

Analysis Pipeline provided in Figure 1. Detailed description of the development process provided in Progress reports 2015 and 2016.



from parallel NGS sequencing of the amplicons of the PCR’s.

6. Assess the

multiplexing of samples using indexed pools of cloned amplicons.

04-2016

04-2016

Multiplexing protocol established along with positive/negative thresholds.

7. Using the PCR protocol and analysis pipeline developed test the virus status of nuclear plants from the certification scheme.

04-2016

04-2016

Test the optimised protocol using nuclear material from Mr. T. Oosthuizen (Vititec) and Mrs. M. Louw (Bosman Brothers).

8. Using the PCR protocol and analysis pipeline developed test the virus status of symptomatic vines from foundation or mother-block vineyards.

04-2016

04-2017

Test the optimised protocol using mother-block material and samples with various symptoms from mother-blocks (included table grape samples).

9. Evaluate the routine application of GLRaV-3 LAMP for field detection of GLRaV-3.

04-2013

04-2017

Finalise laboratory detection of GLRaV-3 and publish the protocol. Field detection could not be evaluated due to the unavailability of sealed reaction vessels. We will attempt the use of microcrystalline wax to seal the reaction



10. Evaluate the routine application of GLRaV-3 LAMP for the detection of GLRaV-3 in rootstocks

04-2013

04-2013

LAMP shown to be more useful than ELISA for the detection of GLRaV-3 in five rootstocks types

11. Evaluate the usefulness of a published LAMP protocol for the detection of Aster yellows phytoplasma.

04-2013

04-2013

Protocol shown to be useful for the detection of South African isolates aster yellows phytoplasma in both plants and leafhoppers

8. CONCLUSIONS We have developed multiple PCR systems for the routine parallel, theoretical detection of 41 known viruses of grapevines. These PCR’s can be utilised directly within the certification scheme, without consideration of the viral identity, as the primary aim is to have plants negative for these viruses. By utilizing a subsequent, optimised, next generation sequencing analysis pipeline the amplicon sequences derived from PCRs for any given sample can be pooled together for the specific identification of viruses found. Furthermore, multiple samples can be tested in parallel with this method utilising established positive/negative thresholds. This protocol has been handed over for routine utilisation as a service to the grapevine industry to the Virus Diagnostic unit of ARC-PPRI to provide this as a serve to the industry. 9. ACCUMULATED OUTPUTS a) TECHNOLOGY DEVELOPED, PRODUCTS AND PATENTS Develop genus wide, multi PCR systems for the parallel, theoretical detection of 41 known viruses of grapevines. Develop a next generation sequencing pipeline for the analysis of amplicon sequences derived from the amplicons for the identification of viruses found. b) SUGGESTIONS FOR TECHNOLOGY TRANSFER This protocol has been documented, and the protocol, primers and positive controls provided to Ms. Marika van der Merwe, ARC-PPRI for the provision of the detection of these viral genera as a service to the Wine Industry. Ms. E. Gagiano, SAPO, will also be trained in the use of this protocol to use in a PhD study to detect viruses of table grapes.



c) HUMAN RESOURCES DEVELOPMENT/TRAINING

Student Name and Surname Student Nationality

Degree (e.g. MSc Agric, MComm)

Level of studies in

final year of project

Graduation date

Total cost to industry

throughout the project

Honours students

J. Wayland South African BSc.(Hons) Microbiology

MSc. 2013 R10000 (2013)

Erika Bruck South African BSc(Hons) Microbiology

Hons

2014 R10000 (2014)

Kersitn Kenchenten South African BSc(Hons) Microbiology

Hons 2014 R10000 (2014)

Masters Students

H. Walsh South African MSc. Microbiology

MSc. 2013 R36000

J. Wayland South African MSc. Microbiology

MSc. 2017 R153300

PhD students

Postdocs

Support Personnel

d) PUBLICATIONS (POPULAR, PRESS RELEASES, SEMI-SCIENTIFIC, SCIENTIFIC) WALSH,H.A.,ANDPIETERSEN,G.,2013.RapiddetectionofGrapevineleafroll-associatedvirustype3usingareversetranscriptionloop-mediatedamplificationmethod.JournalofVirologicalMethods194:308–316In Preparation: Development and optimization of a diagnostic system based on Illumina MiSeqsequencingofgenuswidePCRampliconsforthedetectionofvirusesofgrapevinesJenniferWayland,AnnaE.C.JoosteandGerhardPietersen. e) PRESENTATIONS/PAPERS DELIVERED Wayland,J.,Jooste,A.E.C.,andPietersen,G.,2015TheOptimizationofaHigh-ThroughputSequencing-basedDiagnosticSystemfortheDetectionofVirusesofGrapevines.VirologyAfrica2015,CapeTown,30November–3December2015



10. BUDGET a) TOTAL COST SUMMARY OF THE PROJECT

YEAR

CFPA DFTS Deciduous SATI Winetech THRIP OTHER TOTAL

2012

258300 258300

2013

274300 274300

2014

302000 112800 414800

2015

339124 169562 508686

2016

365948 365948

1539672 282362 1822034

b) FINAL BUDGET/FINANCIALS OF PROJECT

Project duration Proposed

budget Actual cost

incurred Variance Notes

TOTAL INCOME 1539672.00 1539672.00 0

Industry Funding

PHI Funding

Other Funding (THRIP) 282362 282362 0

TOTAL EXPENDITURE 1822034 1822034 0

Running Expenses

General operating costs (printing, communication, etc.)

25220 25220 0

Local Travel 65350 65350 0

Publication costs

Lab Analysis

Lab Consumables 431589 431589 0

Other 0

Running expenses SUB-TOTAL

522159 522159 0

HR Administration and Project Management



Project duration Proposed

budget Actual cost

incurred Variance Notes

HR Technical

HR Research 1080595 1080595 0

Student Bursaries 219280 219280 0

HR SUB-TOTAL 1299875 1299875 0

OTHER EXPENSES

Overheads

SURPLUS / DEFICIT 0 0 0



EVALUATION BY INDUSTRY This section is for office use only

Project number

Project name

Name of Sub-Committee*

Comments on project

Committee’s recommendation

• Accepted.

• Accepted provisionally if the sub-committee’s comments are also addressed. Resubmit this final report by___________________________________

• Unacceptable. Must resubmit final report. Chairperson__________________________________________ Date___________________ *SUB-COMMITTEES Winetech Viticulture: Cultivation; Soil Science; Plant Biotechnology; Plant Protection; Plant Improvement; Oenology: Vinification Technology; Bottling, Packaging and Distribution; Environmental Impact; Brandy and Distilling; Microbiology Deciduous Fruit Technical Advisory Committees: Post-Harvest; Crop Production; Crop Protection; Technology Transfer Peer Work Groups: Post-Harvest; Horticulture; Soil Science; Breeding and Evaluation; Pathology; Entomology



Figure 1: Schematic of genus-wide multiple PCR, Illumina read analysis for viruses of grapevine:



Tabel 1: PCR systems and their properties developed for the routine detection of viruses of grapevines in the South African wine Grape Certification Scheme.

PPRI 11 19Prepared by: Prof. G Pietersen Tel: 012 420 3265 or 082 647 5326 Email:...

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