The Incidence and Diversity of Plant Viruses in the Tallgrass Prairie Preserve

1
The Incidence and Diversity of Plant Viruses in
the Tallgrass Prairie Preserve
-Vaskar Thapa, Ulrich Melcher, Daniel McGlinn,
Marilyn Roossink, Drew Porter, Rita Marvelli,
Tracy Feldman, and Michael Palmer
2

• Outline
• What is PVBE?
• Methods
• Field methods
• Laboratory methods
• Results
• Incidence of plant viruses
• Diversity of plant viruses
• Summary

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• 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.

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Objective for PVBE? To discover
diversity and ecological functions of plant
viruses in natural systems.
5
Underlying hypothesisThe distribution and
evolution of viruses are determined by complex
environmental interactions among many factors
including distributions of hosts, vectors, other
viruses and climate.
6
Working team University of Tulsa joining
soon Specializing in Ecology Virology Molecular
biology Genomics Structural biology and
Bioinformatics.
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• Research site
• The Nature Conservancys 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  (unpublished report).

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• Focus of this presentation
• ? Preliminary results from plants collected since
• May 2005 ? Analysis of double-stranded (ds)
  RNA from the
• plant collected

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Methods Field methods Plants for voucher
herbarium Sample collection for dsRNA
isolation Plant samples for intensive
study Laboratory methods ds RNA isolation
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• Field 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

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Field methods ..

• 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

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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

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Ambrosia psilostachya, Cuman ragweed
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Asclepias viridis, green antelopehorn
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Panicum virgatum, switchgrass
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Sorghastrum nutans, Indiangrass
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Ruellia humilis, fringeleaf wild petunia
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Vernonia baldwini, baldwin's ironweed
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Laboratory method for double-stranded RNA
isolation
Mix vigorously to form emulsion
Young leaves (5 g)
Centrifuge
Grind in liquid nitrogen
Transfer top phase into new tube
Transfer into 50 ml tube containing 10 ml
extraction buffer and 10 ml PhCh
Final aqueous phase
Repeat PhCh extraction
Centrifuge to pellet dsRNA
Add absolute proof ethanol (16,5 of aqueous
volume)
Add elution buffer
Decant and resuspend in 0,1 mM EDTA / 0.3 M NaOAC
Wash in 6 time with application buffer
Pass through enocolumn containing CF11powder
cellulose binding dsRNA (if ethanol concentration
is 16,5)
Total NA (for bar coding and making hybridization
target)
Precipitate with NaOAc and EtOH overnight at -20?
C
Transfer eluate to a 15 ml tube
1
2
3
4
Lad 1 kb
Buffers
Transfer to a microcentrifuge tube and fill with
cold ethanol to reprecipitate
Extraction buffer 0.1 M NaCl 50 mV Tris, pH 8 1
mV EDNA, pH 8 1 SDS
Application buffer 0.1 M NaCl 50 mM Tris,
pH8 0.5 mV EDTA, pH8 16.5 Ethanol
Resuspend in 50 mkl 0.1 mM EDTA
bp
Elution buffer 0.1 M NaCl 50 mV EDTA, pH 8
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
12,2
2
1,6
Check the dsRNA by electrophoresis on a 1.5
agarose gel in 0.5X TBE II
1
506,5
396
344
298
Protocol for ds RNA isolation adopted from M.
Roossinck, 2005
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Results

• 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)

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Distribution within the top plant families
?2 37.39, p 0.00
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Double-stranded RNA in native and exotic species
?2 0.06, p 0.8
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Distribution of dsRNA in different life forms
?2 1.23, p 0.87
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Distribution of dsRNA in different taxonomic
groups
?2 13.81, p 0.00
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Distribution of dsRNA in six selected species
?2 9.76, p 0.08
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• Viral 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.

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• Caveats
• 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

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• Conclusions
• 50 of plant samples contain dsRNA, indicating
  viruses are widespread in nature.
• Viruses are frequent in all growth forms, life
  histories, and taxonomic groups.

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• Acknowledgments
• Following persons who help us in plant collection
• Pete Earls Ray Moranz
• Fumiko Shirakura Josh Lofton
• M.Hara Mari Carmen Cobo
• Will Lowry Laxman Karki
• Shyam Thomas Katie Lewis
• Rest of all team members of PVBE