Fungal Diversity: 130 isolates sequenced and at least 12 genera

To understand disease we also must understand the lack of disease. Almost all viruses that have been studied in depth are pathogenic to humans or their domesticated plants and animals. We have been studying plant viruses from wild plants that usually do not have any disease symptoms. We first became interested in fungal viruses because often they do not induce any known disease. In addition, they are often persistent (i.e. they remain associated with their hosts for a long time, sometimes for generations).

Plant virus biodiversity: Studies of pathogenic plant viruses have probably left out the vast majority of plant viruses. We are working to understanding the diversity and ecology of plant viruses in two natural ecosystems. The Area Conservacion Guanacaste in northwestern Costa Rica is a biodiversity hotspot, containing almost 3% of the world species in only 160,000 hectares. The Tall Grass Prairie Preserve (TGPP) is a Nature Conservancy owned preserve of 30,000 acres in northeastern Oklahoma with relatively low plant diversity. We use double-stranded RNA (dsRNA), a hallmark of RNA virus infection, to detect and analyze RNA viruses. Plant samples are collected systematically, plants are photographed and a GPS reading is taken. In the lab, total nucleic acid is isolated from the plant tissue, and enriched for dsRNA. We are using an adaptation of 454 Life Science's genome sequencing technology to rapidly determine the sequences of the dsRNA from these samples. The sequence data will be used to assess diversity, incidence, and movement of the viruses. The data will also be deposited in a unique Taxonomy Node in GenBank.

Fungal virus biodiversity: Virtually all plant (and perhaps animal) species harbor pathogenic or mutualistic fungi in their tissues. Viruses of these fungi (mycoviruses) can affect parasitism by modulating the virulence of fungal pathogens. Mycoviruses can alter host tolerance to environmental stress (Márquez et al., 2007). However, almost nothing is known about fungal viruses in an ecological context. Metagenomic approaches to environmental samples have illuminated extremely diverse and previously unknown fungi, bacteria and viruses, but it is not possible to determine the associations between individual species from these data. As part of a large NSF-EPSCoR funded project on plant virus biodiversity and ecology we are conducting a study of endophytic fungal virus prevalence and diversity in TGPP. Fungi in diverse genera are hosts to viruses from more than 8 families. All of the 18 viruses encountered so far, from a sample of 125 plant-associated fungi, are novel, and some are unrelated to any known viruses. We are working to develop a system to study effects of some of these fungal viruses on their fungal hosts (Alternaria alternata and Stemphylium solani), and on plants associated with these fungi: the parasitic plant Cuscuta cuspidata and one of its host plants, Ambrosia psilostachya.

Felipe Chavarria measures the soil temperature at a plant in a fumerol at Volcán Rincón. This plant was growing at 58 C.

Viruses as mutualists: In a survey of fungal endophytes from Yellowstone National Park we noticed an interesting correlation: Curvularia protuberata endophytes from plants growing in geothermal soils always had two distinct dsRNAs that were not seen in the fungus from other plants in Yellowstone. Our colleagues Rodriguez, Redman and Henson had already shown that the fungus was required for thermal tolerance of the plants. This led us to explore the virus in depth. We found that a three-way mutualism between the virus, the fungus and the plant was required for thermal tolerance, a story that was published in 2007 in Science. We are exploring the mechanism of this thermal tolerance by expressing each of the viral genes independently in the fungus, and by looking at the physiological and transcriptional changes that are induced in the plant and the fungus when all three are present.

We are also exploring other plant-endophyte-virus interactions that are involved in adaptation to other extreme environments such as high salt, and in other geothermal areas, such as the Volcán Rincón in Costa Rica. This area has the advantage of much more plant diversity in geothermal soils as well as non-geothermal soils.