usda national institute of food and agriculture (nifa ... · compendium of rose diseases and pests:...

Combating Rose Rosette DiseaseUSDA National Institute of Food and Agriculture (NIFA) Specialty Crop Research Initiative project,

“Combating Rose Rosette Disease: Short Term and Long Term Approaches” (2014-51181-22644/SCRI)

Jordan & Hammond, USDA-ARS, US National Arboretum

National Clean Plant Network – Roses

Safe Movement of Rose Germplasm

Virus and RRV Detection in Roses

Ramon Jordan, USDA, ARS, USNAJohn Hammond, USDA, ARS, USNADimitre Mollov, USDA, ARS, NGRL

Ben Lockhart, University of MinnesotaJen Olson, Oklahoma State University

Kevin Ong, Texas A&M UniversityMaher Al Rwanhnih, Foundation Plant Services

Jordan & Hammond, USDA-ARS, US National ArboretumApril 11-12, 2019 – College Station, TX


Combating Rose Rosette Disease: Project’s Diagnostic Objectives

Mathews Paret, Francisco Ochoa-Corona, John Hammond, Ramon Jordan, Jen Olson, Kevin Ong

Key to this effort to detect the virus and to control the disease is the development of efficient diagnostic tools to enable rapid, easy-to-use, accurate and affordable detection of the virus. Specifically –

Reagents will be designed and developed RRV-specific primers/probes (for nucleic acid-based assays) Monoclonal and/or polyclonal antibodies (for serological antibody-based assays)

Techniques & Assays will be developed and refined for lab and field use ELISA and immuno-dipstick tests RT-LAMP (Reverse transcription-Loop mediated isothermal amplification) Self-quenched primer (SqP) technologies (for laboratory detection systems) Lateral flow devices (LFD; both Ab & NA-based detection assays)


Combating Rose Rosette Disease: Project’s Diagnostic Objectives

Actual project output Developing reliable user friendly serological assays:

• ELISAs and Immuno lateral flow device for rapid and user friendly detection• To include monoclonal & polyclonal antibodies in TAS-ELISA (& IC-RT-PCR).

Developing reliable user friendly molecular assays:• Reference method based on Endpoint RT-PCR; SYBR Green RT-qPCR• RT-qPCR + Direct RRV trapping in plastic tubes• Artificial Positive control RRV + other 5 virus infecting ornamentals• Exploring user-friendly isothermal methods: Loop mediated amplification of DNA (LAMP) Helicase dependent amplification (HAD) with self quenched primers Recombinase Polymerase Amplification (RPA) Exploring a broad detection & discrimination for Emaravirus (RT-qPCR+HRM) EDNA-Rose (Electronic Diagnostic Nucleic-acid Analysis)


Progress Toward Development of Reliable, Efficient, Cost-effective, User-Friendly Diagnostic Assays for the

Detection of Rose Viruses

Table. Comparative analysis of the different potential diagnostic methods

Assay SensitivitySkill

requiredEquipment

neededHigh

throughputTime

requiredCost

Nucleic acid-based Assays

RT-PCR (& IC-PCR) High High Yes Low 6-8 hrs Med

RT-LAMP High Med Yes Med 1-3 hrs Med

RT-exoRPA High Med/Low Yes Med 20-30 min Med

NGS-HTS Med High Yes Low Months High

EDNA Low? Med/Low Yes [Laptop] High 10 min To be determined

Antibody-based Assays

ELISA (Tissue Print) Med Med Yes/No High 4-18 hrs Low

Immunostrip Low/Med Low No Med 10-30 min Med

Grafting - New from Francisco’s kitchenRose Microbudding

a faster alternative for assessment RRV Rose varietal resistance

Microbudding was Developed by Dr. M. Skaria (Texas A&M) during the late 90’s for high throughput propagation of citrus

Rose Microbudding stepsfor RRV screening of germplasm resistance

RRV +rootstock

Tested Germplasm

Micro-pipetttetip

2 months12 hours light/dark20°C/18°C79-80 RH

RRV+ controlKnock out®

RRV- healthy controlKnock out®/Knock out®Root callus initiation

RRV- Knock out®/ RRV+ Sweet® drift

TWO MONTHS AFTER MICROBUDDING-INOCULATION

RRV+ ‘Sweet drift®’ in Oklahoma

• Potentially a 3-4 months assayto assess varietal resistance of rose progenies

• Will require an infected blockin a infested area for sourcing ofinfected canes

• Easy to combine with a serological or molecular method of choice

Is this of interest for rose breeders?

Rose Microbuddinga faster alternative for assessment RRV

Rose varietal resistance


National Clean Plant Network – RosesSafe Movement of Rose Germplasm


Suggested Discussion Points:

1. How the plant is handled before it (or some budwood from it) is moved [Discussed this morning]

2. What options do we have for testing for the viruses (and the cost of these) OK - Which viruses?

3. How should plant material be handled once it arrives at its destination (isolation, further testing, time of observation, how maintained etc.) [Discussed this morning]


National Clean Plant Network – RosesSafe Movement of Rose Germplasm


3rd Edition to APS Compendium of Rose Diseases and PestsJohn Hammond, Dimitre Mollov, Ramon Jordan (eds)

- Rose Parentage/Hybrids/Breeding – to include information on traits potentially conferred by different rose species (David Byrne and David Zlesak) - Cultivar Classification (David Byrne and David Zlesak)- General Maintenance/Production Practices (Mark Windham and Alan Windham) - National Clean Plant Network – Rose, and the Rose Collection and Repository

Deborah Golino- Rose Diseases & Pests - Multiple contributors from different areas [Infectious diseases

(Fungi, Bacteria, Viruses); Insect & Mite Pests; Noninfectious diseases/disorders]- Disease and Pest Management (Mark Windham and Alan Windham)


27 viruses reported• 24 Complete genomes sequenced• 3 No sequence available• 26 2 DNA / 24 RNA genomes [1 unknown]

Taxa distribution at a glance• 4 Ilarvirus• 4 (+1) Nepovirus• 4 Tospovirus• 3 virus species (3 Genera in Betaflexiviridae)• 10 virus species in different genera/family

Source: List of Rose Viruses 6_16/RRV projectDimitri/Maher/Kevin


Diseases caused by Viruses (new APS Rose Compendium)

1. Apple chlorotic leaf spot virus2. Apple mosaic virus

(1 of 2 called ‘Rose mosaic virus’)3. Apple stem grooving virus4. Arabis mosaic virus5. Blackberry chlorotic ringspot virus6. Cherry necrotic rusty mottle virus7. Impatiens necrotic spot virus8. Iris yellow spot virus9. Prunus necrotic ringspot virus

(2 of 2 called ‘Rose mosaic virus’)10. Raspberry ringspot virus11. Rosa rugosa leaf distortion virus

(syn. Rose yellow leaf virus)12. Rose color break virus

(syn. Rose tobamovirus)13. Rose cryptic virus 1

(syn. Rosa multiflora cryptic virus& Rose transient mosaic virus)

1. Rose leaf curl virus2. Rose leaf rosette-associated virus3. Rose necrotic mosaic virus

[only a single report to date]4. Rose rosette virus5. Rose spring dwarf-associated virus6. Rose wilt ‘virus’ [unidentified]7. Rose yellow mosaic virus8. Rose yellow vein virus

(syn. Rose chlorotic ringspot virus)9. Strawberry latent ringspot virus10. Tobacco ringspot virus11. Tobacco streak virus12. Tomato ringspot virus13. Tomato spotted wilt virus14. Tomato yellow ring virus

(syn. Tomato Varamin virus)


COMMERCIALLY AVAILABLE ASSAYS:ELISA: 13Lateral flow devices : 4RT-PCR: 1

RESEARCH/PUBLISHED ASSAYS (MOLECULAR)RT-PCR: 22

2016Rose antivirus

arsenal

• Minimize the number of assays• Minimize assay testing time• Minimize complexity of assays• Transfer friendly assays to Diagnostic networks & Industry*

… and we should:

Which ones most important to concentrate on? -- Available Diagnostics?


Apple mosaic virus (ApMV), family BromoviridaeBlackberry chlorotic ringspot virus (BCRV), family BromoviridaePrunus necrotic ringspot virus (PNRSV), family BromoviridaeRosa multiflora cryptic virus (RMCV), family PartitiviridaeRosa rugosa leaf distortion virus (RrLDV), family TombusviridaeRose rosette emaravirus (RRV), family FimoviridaeRose spring dwarf associated virus (RSDaV), family LuteoviridaeRose yellow leaf virus (RoYLV), family TombusviridaeRose yellow mosaic virus (RoYMV), family PotyviridaeRose yellow vein virus (RYVV), family CaulimoviridaeStrawberry latent ringspot virus (SLRSV), family SecoviridaeTobacco streak virus (TSV), family Bromoviridae

Which ones most important to concentrate on? -- ‘The Chosen 12’ (Dimitre/Maher/Kevin)


OK – Let’s Talk About This

What criterion?

• Presence in the US• Available (and reliable) Diagnostics

o Molecular toolso Serological toolso Grafting


OK – Let’s Talk About This

THANKS !!

Combating Rose Rosette DiseaseUSDA National Institute of Food and Agriculture (NIFA) Specialty Crop Research Initiative project,

“Combating Rose Rosette Disease: Short Term and Long Term Approaches” (2014-51181-22644/SCRI)

usda national institute of food and agriculture (nifa ... · compendium of rose diseases and pests:...

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