ACTIVITIES OF THE NCPN-ROSES & THE ROSE PROGRAM AT FPSAPRIL 11-12, 2018

NCPN-Rose Annual Tier II Meeting, College Station, Texas

MAHER AL RWAHNIH, LABORATORY DIRECTOR

DEBORAH GOLINO, DIRECTOR

FOUNDATION PLANT SERVICES

Foundation Plant Services Mission

• Produce, test, maintain and distribute elite disease-tested plant propagation material.

• Provide plant importation and quarantine services, virus testing and virus elimination.

• Coordinate release of UC-patented varieties.

• Link researchers, nurseries, and producers.

FPS Crop Programs

• Grape

• Strawberry

• Fruit and Nut Trees

• Rose

• Sweet potato

Disease – testedProfessionally – identifiedTrue-to-variety$1.00/cane

Premier Rose Planting Stock

GOH Block

Main Building –Offices, Labs, Greenhouses

MPP Greenhouse and Shadehouse

NYL Block

BKN Block

BKS Block

Classic Foundation Vineyard

Rose scion and roostock removed from Nyland Block

New Brooks South Block

Removal of Nyland Block

• Plantings dated back to 1995

FPS Virus-Indexed Rose Collection – Brooks South (BKS)

• ~10 acres• Planted 2015–2018 (replaced Nyland Block)• 711 cultivars/3,482 plants

FPS Virus-Indexed Rose Collection – Brooks North (BKN)

8 rootstock varieties:

Burr multifloraDe La GrifferaieDr. HueyRosa fortunianaIXLManettiOdorataNatal Briar

• Planted in 2013-2014• ~2 acres

Virus Testing

• Breeder Selections• Proprietary Selections• Heritage Cultivars

Foundation Collection New Introductions

Virus Testing

• Lab Tests – qPCR• PNRSV – ELISA also

• ApMV – ELISA also

• RRV

• RSDaV

• BCRV

• RYVV

• Field Tests - Graft indexing • Shirofugen cherry

• Rosa multiflora ‘Burr’

Shirofugen Graft Index – 30 days

Virus-Infected

Healthy

Multiflora Graft Index– 2 years

Candidate bud T-budded into multiflora rose

Virus symptoms

Multiflora cut back to force new growth:Symptoms observed for two years

Queen Elizabeth biological indexing

Only for foreign importsSymptoms of rose color breaking pathogen

High Throughput Sequencing (HTS)

• This new technology involves high-throughput DNA sequencing

• HTS gives a comprehensive picture of the entire microbial profile in a sample

NextSeq 500 MinION SmidgION

Process Equipment Kit Timeline

TNA Extraction KingFisher™ Flex Purification System

MagMAX™ Plant RNA Isolation Kit

½ day

Full large scale sample processing

Biomek 4000 AutomatedWorkstation

TruSeqStranded Total RNA Kit with Ribo-Zero Plant

2 days

HTS NexSeq® 500 NextSeq500/550 High Output Kit v2

1 day

Bioinformatic Analysis

Bioinformatician/software In house Pipeline

2 days

24 samples can be processed in approximately one week

HTS testing at FPS

Study diseases of unknown etiology

Metagenomic virome analysis

Re-sequencing of known viruses

Host–pathogen interactions

Post entry quarantine

HTS Current Applications: Grapevine

High Throughput Sequencing (HTS)

• Used if other laboratory tests are inconclusive

• Rose side-by-side is underway

• 33 roses tested by HTS in the last year

Tests required by APHIS and CDFA for certification

• Standard detection methods for a range of suspected ‘known’ viruses using a panel of specific tests: ELISA RT-PCR RT-qPCR

Herbaceous Index Woody Index• Biological Indexing: A broader techniques

Al Rwahnih et al. 2015, Phytopathology 105:758-763

Side-by-side comparison HTS vs. biological indexing in grapevine

Can High Throughput Sequencing (HTS) replace the field indexing

requirement ??

What about other crops?

• Comparison of HTS vs. biological indexing in fruit trees

• 178 infected trees

• Comparable results were obtained for 170 of 178 specimens (discrepancies were observed in less than 5% of the specimens)

• Most useful when screening for a large number of viruses or to identify a novel cause of the disease

• HTS is as sensitive as PCR Currently working on:• Fruit trees• Roses• Sweet potato

Rott, M., et al. 2017 http://dx.doi.org/10.1094/PDIS-03-17-0306-RE

HTS analysis has advantages over the standard bioassay in:

Detection of viruses of agronomic significance (including low titer viruses)

Comprehensiveness

Speed of analysis

Discovery of novel, uncharacterized viruses

Summary

Estimated cost for conventional virus testing

Minimum release time: 2 – 3 years

Assay cost/selection

PCR assays (37 pathogens) $1,200

ELISA (4 pathogens) $250

Herbaceous host indexing $100

Woody host indexing $350

Total $1,900

With HTS testing option

• Cost: $350 per selection

• Total testing time: 1 – 2 months

Main advantage: • Detects ALL known and unknown viruses (all strains)

Collection of Rose Virus Symptom Photos

Thank you