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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.
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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
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• 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
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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
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RBDV -positive RBDV -negative
‘Cascade Bounty’
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RBDV +
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RBDV with RLMV and RpLVPhoto courtesy of Diego Quito, pnwhandbooks.org
Crumbly Fruit Disease
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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
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Time Course Experiment
• ‘Meeker’ (S) and ‘Willamette’ (R)
• Inoculum plants tested RBDV+ for 3 consecutive years
• Natural pollination• Sampling after onset of
bloom
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Tissue Collection
•Receptacle•Pedicle
For each lateral, receptacles were bulked into one sample and pedicles were bulked into one sample.
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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
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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
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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
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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’?
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Breeding Plot Survey
• 49 plants sampled• 16 resistant
cultivars• 8 susceptible
cultivars• ELISA, RT-PCR and
Real-Time RT-PCR
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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 - - -
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New Question
Is there a resistance breaking strain of RBDV in the commercial growing regions in Washington State?
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Preliminary Results of a survey for the resistance-breaking strain of Raspberry bushy dwarf virus in Lynden, WA
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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
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Results
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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
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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
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Using Undergraduate Research Opportunities as a Plant Science Recruitment tool
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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
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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
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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
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Presented in November 2016 at the Murdock Undergraduate Research Symposium
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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
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Thank you! Thank you!