Determinants of Taxonomic Composition of Plant Viruses at the Nature Conservancy’S Tallgrass Prairie Preserve, Oklahoma Vaskar Thapa,1,2 Daniel J
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Virus Evolution, 2015, 1(1): vev007 doi: 10.1093/ve/vev007 Research article Determinants of taxonomic composition of plant viruses at the Nature Conservancy’s Tallgrass Prairie Preserve, Oklahoma Vaskar Thapa,1,2 Daniel J. McGlinn,2,† Ulrich Melcher,3 Michael W. Palmer,2 and Marilyn J. Roossinck1,*,†,‡ 1Department of Plant Pathology and Environmental Microbiology, Center for Infectious Disease Dynamics, Pennsylvania State University, University Park, PA 16802, USA, 2Department of Botany, Oklahoma State University, Stillwater, OK 74078, USA and 3Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA *Corresponding author: E-mail: [email protected] †Present address: Biology Department, College of Charleston, Charleston, SC, USA. ‡http://orcid.org/0000-0002-1743-0627 Abstract The role of biotic and abiotic factors in shaping the diversity and composition of communities of plant viruses remain understudied, particularly in natural settings. In this study, we test the effects of host identity, location, and sampling year on the taxonomic composition of plant viruses in six native plant species [Ambrosia psilostachya (Asteraceae), Vernonia baldwinii (Asteraceae), Asclepias viridis (Asclepiadaceae), Ruellia humilis (Acanthaceae), Panicum virgatum (Poaceae) and Sorghastrum nutans (Poaceae)] from the Nature Conservancy’s Tallgrass Prairie Preserve in northeastern Oklahoma. We sampled over 400 specimens of the target host plants from twenty sites (plots) in the Tallgrass Prairie Preserve over 4 years and tested them for the presence of plant viruses applying virus-like particle and double-stranded RNA enrichment meth- ods. Many of the viral sequences identified could not be readily assigned to species, either due to their novelty or the short- ness of the sequence. We thus grouped our putative viruses into operational viral taxonomic units for further analysis. Partial canonical correspondence analysis revealed that the taxonomic composition of plant viruses in the target species had a significant relationship with host species (P value: 0.001) but no clear relation with sampling site or year. Variation partitioning further showed that host identity explained about 2–5 per cent of the variation in plant virus composition. We could not interpret the significant relationship between virus composition and host plants with respect to host taxonomy or ecology. Only six operational viral taxonomic units had over 5 per cent incidence over a 4-year period, while the remainder exhibited sporadic infection of the target hosts. This study is the first of its kind to document the dynamics of the entire range of viruses in multiple plant species in a natural setting. Key words: wild plant viruses; plant virus composition. VC The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 1 2|Virus Evolution, 2015, Vol. 1, No. 1 1 Introduction also provides a unique opportunity to document the diversity of plant viruses in a natural state. Studies to uncover patterns in the diversity and composition of viruses of wild plants have only been initiated recently (Cooper and Jones 2006; Wren et al. 2006; Melcher et al. 2008; Roossinck, 2 Materials and Methods Martin, and Roumagnac 2015). Understanding patterns of diver- sity and species composition with respect to environmental 2.1 Field methods properties is a widely used approach in ecology. It is meaningful Our study area is The Nature Conservancy’s TGPP, Osage to determine how strongly a given set of environmental vari- County, Oklahoma (Allen et al. 2009). TGPP vegetation consists ables can explain the distribution of a set of species and mainly of tallgrass prairie and cross timber forest that provides their evolutionary relationships. For plant viruses, our current habitat for 763 species of vascular plants (Palmer 2007). We col- knowledge on the topic is limited to interactions of single lected our samples within 10 m of twenty semi-randomly se- viruses, primarily from cultivated or experimental sources. lected 10 m Â10 m permanent plots (Fig. 1) in tallgrass prairie The environmental factors determining virus composition in vegetation (plant species composition and details of plot loca- natural plant communities across ecosystems are mostly un- tion are given in McGlinn, Earls, and Palmer 2010). At each sam- known. Recent reviews emphasize the influence of both biotic pling area, we collected at least one sample each of Ambrosia and abiotic factors on the distribution of plant viruses in nature psilostachya (Asteraceae), Vernonia baldwinii (Asteraceae), (Malmstrom, Melcher, and Bosque-Perez 2011; Roossinck 2015). Asclepias viridis (Asclepiadaceae), Ruellia humilis (Acanthaceae), Here, we focus on three attributes in determining the distribu- Panicum virgatum (Poaceae), and Sorghastrum nutans (Poaceae). tion of plant viruses: host identity; location; and time. These six target plant species are readily identifiable in the veg- etative state and represent some of the most frequent vascular 1.1 Host identity effects plants of the TGPP. The samples were collected in the month of June from 2005 to 8. Lovisolo, Hull, and Rosler (2003) argued that plant hosts influ- At least 10 g of leaf tissue sample were collected from top ence the evolutionary divergence of plant viruses. In general, leaves of each host plant. The samples were collected irrespec- many plant viruses are host generalists rather than host spe- tive of any disease symptoms. Proper sanitation protocol was cialists (Power and Flecker 2003). However, host ranges (defined followed while collecting the samples to avoid contamination. by virologists as the set of plant species a virus can infect) of Collected samples were immediately transferred to an ice- closely related plant viruses do not overlap completely (Dawson cooled chest in the field and stored at À80 C in the laboratory and Hilf 1992). For example, Tomato chlorosis virus and Tomato in- before further processing for plant virus assays. fectious chlorosis virus are closely related taxonomically and have similar influence on hosts, but they differ in host range (Wintermantle and Wisler 2006). Host identity also influences 2.2 Laboratory methods the incidence of plant viruses (Lavina, Aramburu, and Moriones 1996; Sacrista´n, Fraile, and Garcı´a-Arenal 2004). Plant virus-like particles (VLPs) and double-stranded RNA (dsRNA) were isolated from the plant samples collected for plant virus assay. VLPs were obtained by differential centrifuga- 1.2 Location effects tion as described in detail in Melcher et al. (2008). For dsRNA, to- A study on Barley/Cereal yellow dwarf viruses suggests a latitudi- tal nucleic acid was obtained from 5 g of plant leaf samples by nal gradient in species composition (Seabloom et al. 2010). performing phenol-chloroform extraction followed by dsRNA Harrison (1981) reported biogeographic patterns in plant virus enrichment using CF11 cellulose chromatography (Roossinck distribution. Environmental differences among sites appear to et al. 2010). The VLP method has the limitation that plant vi- have strong influences on plant virus species composition at ruses that do not produce encapsidated forms and those that broad scales (Atiri, Njukeng, and Ekpo 2000). Spatial genetic make unstable particles will not be captured (Melcher et al. structure is also reported at the local scale (Skotnicki, 2008). The dsRNA method is based on the premise that unin- Mackenzie, and Gibbs 1996; Pinel et al. 2000; Hall 2006) and the fected plants normally do not contain detectable amount of regional scale (Stenger, Seifers, and French 2002; Ahmad et al. high molecular weight dsRNA and, when present, dsRNA is con- 2006). sidered as an indicator of presence of ssRNA, ambi-sense, or dsRNA viruses (Dodds, Morris, and Jordan 1984). Isolated VLPs were subjected to polymerase chain reaction (PCR) or reverse 1.3 Time effects transcriptase-PCR (RT-PCR). Similarly, dsRNAs were subjected Many plant viruses exhibit variation in incidence over time to RT-PCR as described (Roossinck et al. 2010). The PCR products (Coutts and Jones 2002). It might be expected that large fluctua- were finally sequenced by a massively parallel sequencing tech- tions in plant virus incidence over time likely influence the nique using a Roche 454 pyrosequencer (Roossinck et al. 2010). overall virus species composition. Rates of evolutionary diversi- In this study, a total of 184 and 445 samples of target species fication in plant viruses may also be temporally variable (Gibbs were analyzed with VLP and dsRNA methods, respectively. et al. 2010). The putative plant virus species were identified by perform- In this study, we determined the effect of host identity, sam- ing BLAST searches of obtained nucleic acid sequences. pling site, and year on the composition of plant viruses isolated Although the 454 sequence procedure produced virus-related from six frequent native plants samples over a 4-year period reads that did not assemble into contigs within reads from from their natural populations in the Tallgrass Prairie Preserve many plant samples (singletons), these were disregarded for the (TGPP) in northeastern Oklahoma. In addition,