the incidence and diversity of plant viruses in the tallgrass prairie preserve -vaskar thapa, ulrich...
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The Incidence and Diversity The Incidence and Diversity of Plant Viruses in of Plant Viruses in
the Tallgrass Prairie the Tallgrass Prairie PreservePreserve
-Vaskar Thapa, Ulrich Melcher, Daniel McGlinn, Marilyn Roossink, Drew Porter, Rita Marvelli, Tracy Feldman, and Michael Palmer
OutlineOutline
1) What is PVBE?
2) Methods
• Field methods
• Laboratory methods
3) Results
• Incidence of plant viruses
• Diversity of plant viruses
4) Summary
Background: What is PVBE?Background: What is PVBE?
• Plant Virus Biodiversity and Ecology is one of two scientific theme areas, awarded for research by the National Science Foundation (NSF)-Oklahoma Experimental Program to stimulate Competitive Research (EPSCoR).
• Funding provided 2005 to 2008.
ObjectiveObjective for PVBE for PVBE
To discover diversity and ecological functions of
plant viruses in natural systems.
Underlying hypothesisUnderlying hypothesis
The distribution and evolution of viruses are
determined by complex environmental interactions
among many factors including distributions of
hosts, vectors, other viruses and climate.
Working teamWorking team
University of Tulsa joining soon
Specializing in
Ecology
Virology
Molecular biology
Genomics
Structural biology and
Bioinformatics.
Research siteResearch site
The Nature Conservancy’s Tallgrass Prairie Preserve in Osage County, Oklahoma
•Representation of intact native Tallgrass Prairie landscape
• 15,000 hectares
• more than 700 plant species
•12 vegetation types
Palmer, M. W., P. Earls, and J. R. Arévalo. 2000. The vegetation of the Tallgrass Prairie Preserve (unpublished report).
Focus of this presentationFocus of this presentation
Preliminary results from plants collected since May 2005
Analysis of double-stranded (ds) RNA from the
plant collected
MethodsMethods
Field methods
Plants for voucher herbarium
Sample collection for dsRNA isolation
Plant samples for intensive study
Laboratory methods
ds RNA isolation
Field methodsField methods
Plant sample for voucher herbarium
• Sample from each species
• Collection irrespective of
symptoms
• Collection from sites with
abundance of target species
• Record of GPS location
• Habitat and individual plant photos
• Two repositories for herbarium –OSU and TGPP
Sample collection for ds RNA isolation
• 10 grams of young leaves
• Transported to the laboratory in a container
with ice packs
• Stored in cold room at 4 ْ C before processing
for dsRNA isolation
Field methods ..Field methods ..
Field methods ..Field methods ..
Plant samples for intensive study
• Six of the most frequent
plants in tallgrass prairie
vegetation
• Represent major taxonomic
groups
• Multiple samples from 20
random plots
Ambrosia psilostachya, Cuman ragweed
Asclepias viridis, green antelopehorn
Panicum virgatum, switchgrass
Sorghastrum nutans, Indiangrass
Ruellia humilis, fringeleaf wild petunia
Vernonia baldwini, baldwin's ironweed
Young leaves (5 g)
Grind in liquid
nitrogenTransfer into 50 ml tube
containing 10 ml extraction buffer and 10
ml Ph:Ch
Mix vigorously to form
emulsionCentrifuge
Transfer top phase into new tube
Repeat Ph:Ch extraction
Final aqueous phase
Total NA (for bar coding and making
hybridization target)
Add absolute proof ethanol
(16,5% of aqueous volume)
Pass through enocolumn containing
CF11powder cellulose binding
dsRNA (if ethanol concentration is
16,5%)
Wash in 6 time with application
buffer
Add elution buffer
Transfer eluate to a 15 ml tube
Precipitate with NaOAc and EtOH overnight at -20 ْ C
Centrifuge to pellet dsRNA
Decant and resuspend in 0,1 mM EDTA / 0.3 M NaOAC
Transfer to a microcentrifuge tube and fill with cold ethanol to
reprecipitate
Resuspend in 50 mkl 0.1 mM EDTA
Check the dsRNA by electrophoresis on a 1.5% agarose gel in
0.5X TBE II
1 2 3 4 Lad 1 kb
506,5
1
1,6212,2
396
Extraction buffer: 0.1 M NaCl50 mV Tris, pH 81 mV EDNA, pH 81% SDS
Application buffer:0.1 M NaCl50 mM Tris, pH80.5 mV EDTA, pH816.5% Ethanol
Elution buffer:0.1 M NaCl50 mV EDTA, pH 8
Buffers
Laboratory method for double-stranded RNA isolationLaboratory method for double-stranded RNA isolation
344298
bp
Vernonia baldwinii (line 1) and Flavoparmelia sp. (line 4) have no dsRNA.
Ambrosia psilostachya (line 2) and Parmelia sp1. (line 3) show bands for dsRNA
Protocol for ds RNA isolation adopted from M. Roossinck, 2005
ResultsResults
•635 specimen from 485
species, 307 genera and 91
families collected.
•592 specimens analyzed for
ds RNA
•gels of 592 these specimens
•308 of the are putatively
positive for dsRNA (i.e.
probable viruses)
48% 52%
Positive for dsRNA Negative for dsRNA
Distribution within the top plant familiesDistribution within the top plant families
0
50
100
150
200
250
300
Nu
mb
er o
f sp
ecim
en
Negative
Positive
2 = 37.39, p = 0.00
Double-stranded RNA in native and exotic speciesDouble-stranded RNA in native and exotic species
0%
20%
40%
60%
80%
100%
120%
Native Exotic
Nativity
Per
cen
tage
of
spec
imen
Negative
Positive
2 = 0.06, p = 0.8
Distribution of dsRNA in different life formsDistribution of dsRNA in different life forms
0%
20%
40%
60%
80%
100%
120%
Perennial Annual Tree Shurb Vine
Life forms
Per
cen
tage
of
spec
imen
Negative
Positive
2 = 1.23, p = 0.87
Distribution of dsRNA in different taxonomic groupsDistribution of dsRNA in different taxonomic groups
0%
20%
40%
60%
80%
100%
120%
Dicot Monocot Fern
Per
cen
tage
of
spec
imen
Negative
Positive
2 = 13.81, p = 0.00
Distribution of dsRNA in six selected speciesDistribution of dsRNA in six selected species
0% 20% 40% 60% 80% 100% 120%
Ambrosia psilostachya
Panicum virgatum
Sorghastrum nutans
Asclepias viridis
Vernonia baldwinii
Ruellia humilis
Positive
Negative
2 = 9.76, p = 0.08
Viral DiversityViral Diversity
• Too early to comment on plant
virus diversity in TGPP
• Gel analysis shows wide
variation in banding patterns
• Different banding patterns within
and across species.
CaveatsCaveats
• The results are preliminary, based on a limited
sample
• dsRNA is not unique for plant virus, it may be from
fungal or arthropod viruses
• Viruses of low titer may have been missed
• DNA viruses are not assessed.
• The reading of the gels has some subjectivity; this
will be resolved in the sequencing phase of PVBE
ConclusionsConclusions
• 50% of plant samples contain dsRNA, indicating
viruses are widespread in nature.
• Viruses are frequent in all growth forms, life
histories, and taxonomic groups.
AcknowledgmentsAcknowledgments
Following persons who help us in plant collection
Pete Earls Ray Moranz Fumiko Shirakura Josh LoftonM.Hara Mari Carmen Cobo Will Lowry Laxman KarkiShyam Thomas Katie Lewis
Rest of all team members of PVBE
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