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 The vegetation of the Tallgrass Prairie Preserve (unpublished report).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