Preliminary results from a survey for a resistance-breaking strain of Raspberry bushy dwarf virus in Lynden, WA.

Rebecca Crust, Adrienne Scarcella, and Kara Lanning, Ph.D.

Presentation Agenda

• Summary of my research background• Preliminary results from a 2016 survey for a

resistance-breaking strain of RBDV in Lynden• Use of undergraduate research programs to

increase vocational interest in agriculture and plant science

• Ph.D. in Horticulture from Washington State University (2014)

• Advisor: Pat MooreResearch focus: Understanding the time course of RBDV infection in Red Raspberry from pollination to systemic infection

Research Background

Raspberry bushy dwarf virus (RBDV)

• Present wherever Rubus is grown• Pollen and seed-transmitted• Control: Remove plants, plant

resistant cultivars• Multiple strains identified

– Common D-type – Resistance-breaking strain (Europe)

• Common Diagnostic: ELISA

talk.ictvonline.org

Symptoms: leaf yellowing, stunted growth, crumbly fruit due to drupelet abortion

RBDV -positive RBDV -negative

‘Cascade Bounty’

RBDV +

RBDV with RLMV and RpLVPhoto courtesy of Diego Quito, pnwhandbooks.org

Crumbly Fruit Disease

Time Course Study

• Primary Objective: – Determine the time course of RBDV viral infection

from pollination to systemic infection in Red Raspberry

Disease-freeplants

Transmit Virus

Destructive Tissue

Sampling

Detect Virus Confirm Results

Analyze

Time Course Experiment

• ‘Meeker’ (S) and ‘Willamette’ (R)

• Inoculum plants tested RBDV+ for 3 consecutive years

• Natural pollination• Sampling after onset of

bloom

Tissue Collection

•Receptacle•Pedicle

For each lateral, receptacles were bulked into one sample and pedicles were bulked into one sample.

Mid

-frui

ting

late

ral

Base

-and

mid

-flor

ican

e

After collection, all samples were evaluated for the presence of RBDV using RT-PCR, and Real-Time RT-PCR

Pollination Experiment Results (2013)Cultivar No. of Samples RdRp (RNA-1) CP (RNA-2)

Meeker (S) 14 + +

Meeker 26 + -

Willamette(R)

7 + +

Willamette 33 + -

• Samples collected from 7 ‘Willamette’ and 9 ‘Meeker’ plants.Experiment was repeated in 2014 with a tighter collection timeline

Days Post-Onset of Bloom

Tissue Type Number of Samples Average Ct Value

1Receptacle 6/6 30Pedicle 5/6 30Fruiting Lateral 2/6 39Mid-floricane 1/2 34Base of Floricane 0/2 Undetected

2

Receptacle 2/2 27.5Pedicle 2/2 29Fruiting Lateral 2/2 35Mid-floricane 0/1 UndetectedBase of Floricane 0/1 Undetected

3

Receptacle 2/2 27Pedicle 2/2 29.5Fruiting Lateral 2/2 34Mid-floricane 1/1 38Base of Floricane 1/1 37

Conclusions from Pollination Experiment

• RBDV enters the vascular system of the plant within 24 hours of bloom

• A novel RBDV strain was identified that is able to replicate in cultivars that are genetically resistant to the common strain of RBDV

New Question: Is this new virus able to infect other raspberry cultivars that are genetically resistant to the common strain of RBDV, or just ‘Willamette’?

Breeding Plot Survey

• 49 plants sampled• 16 resistant

cultivars• 8 susceptible

cultivars• ELISA, RT-PCR and

Real-Time RT-PCR

Breeding Plot Results

•

ELISA (S/R) to Common RBDV

StrainNo. of Plants ELISA RT-PCR

(RNA-2)

Real-Time RT-PCR (RNA-

1)

Susceptible 11 + + +

Resistant 1 + + +

Susceptible 3 + - +

Resistant 3 + - +

Susceptible 3 - - +

Resistant 26 - - +

Resistant 2 - - -

New Question

Is there a resistance breaking strain of RBDV in the commercial growing regions in Washington State?

Preliminary Results of a survey for the resistance-breaking strain of Raspberry bushy dwarf virus in Lynden, WA

Experimental DesignLeaf samples were collected from 8 fields of ‘Meeker’ from Whatcom County. Field aged ranged between 4-7 years.

Grind up leaf samples RNA Extraction Real-Time RT-PCR with primer sets that amplify the coat protein of the common strain and the RB-strain

ELISA RT-PCR

Results

Conclusions

• RBDV was detected in all sampled fields using the three diagnostic methods

• The primers designed to detect the common strain of RBDV did not amplify viral RNA in any of the samples

• The primers designed to detect the resistance-breaking strain did amplify viral RNA in many of the samples

• Further work is needed to confirm that these data

Future Directions

• Graft tissue collected from selected field onto a resistant cultivar

• Perform genetic analysis of the isolated virus to compare with the known common isolate and the resistance-breaking isolates.

• Expand survey to Oregon• Assess the impact of the virus on

symptom development

Summer ‘17

Using Undergraduate Research Opportunities as a Plant Science Recruitment tool

The number of scientists involved in life science research has increased overall, but has growth has been stagnant for agricultural sciences

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Chart generated using statistical data collected by the National Science Foundationhttps://www.nsf.gov/statistics/2016/nsf16300/data-tables.cfm

Undergraduate Research has High Impact on Student Academic Success

• Students report many benefits from participating in undergraduate research, including gains in :– Scientific literacy– Collaborative skills– Data collection and analysis– Communication of scientific results– Finding connections between the impact of scientific

research and their greater community

Exposure to the plant sciences via undergraduate research experiences may help with increasing recruitment to agricultural graduate programs

Small Fruit Research is ideal for Undergraduate Programs

• Scope of work asks applied research questions that directly benefit community

• Provides exposure to the multidisciplinary practice of agricultural research broadened perspective of potential vocational options

• Logistical nature of small fruit research is ideal for operating a summer research program

• Undergrads are happy to collaborate with higher level research projects, and are happy to run routine methods that require low levels of training

Presented in November 2016 at the Murdock Undergraduate Research Symposium

Acknowledgements

• M. J. Murdock Charitable Trust• WSU Extension Center

• Puyallup- Dr. Patrick Moore• Mt. Vernon- Dr. Lisa Wasko DeVetter

• USDA-ARS Horticultural Crops Institute-• Bob Martin• Karen Keller

• Washington Red Raspberry Commission• Washington State Red RaspberryGrowers Cooperative

PACIFIC LUTHERAN UNIVERSITY

Thank you! Thank you!