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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 Outline What is PVBE? Methods Field methods Laboratory methods Results

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the incidence and diversity of plant viruses in the tallgrass prairie preserve

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

slide2

Outline

  • What is PVBE?
  • Methods
    • Field methods
    • Laboratory methods
  • Results
    • Incidence of plant viruses
    • Diversity of plant viruses
  • Summary
slide3

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.
slide4
Objective for PVBE To discover diversity and ecological functions of plant viruses in natural systems.
slide5

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.

slide6

Working team

  • University of Tulsa joining soon
  • Specializing in
    • Ecology
    • Virology
    • Molecular biology
    • Genomics
    • Structural biology and
    • Bioinformatics.
slide7
Research 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 The vegetation of the Tallgrass Prairie Preserve (unpublished report).

slide8
Focus of this presentation

 Preliminary results from plants collected since

May 2005 Analysis of double-stranded (ds) RNA from the

plant collected

slide9

Methods

  • Field methods
  • Plants for voucher herbarium
  • Sample collection for dsRNA isolation
  • Plant samples for intensive study
  • Laboratory methods
    • ds RNA isolation
slide10

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
field methods
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
field methods12
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
slide19

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 mlPh:Ch

Final aqueous phase

Repeat Ph:Ch 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%)

Precipitate with NaOAc and EtOH overnight at -20ْ C

Total NA (for bar coding and making hybridization target)

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 in50 mkl 0.1 mM EDTA

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

Elution buffer:

0.1 M NaCl

50 mV EDTA, pH 8

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

slide20

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)
slide26

Viral Diversity

    • Tooearly to comment on plant
    • virus diversity in TGPP
    • Gel analysis shows wide
    • variation in banding patterns
    • Different banding patterns within
    • and across species.
slide27

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
slide28

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.
slide29

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