Mycorrhizae in HydrothermallyAltered Soils of Yellowstone National Park

Mycorrhizae in Hydrothermally-Altered Soils of Yellowstone National Park R. A. Bunn and C. A. Zabinski Department of Land Resources and Environmental Science, Montana State University, Bozeman ABSTRACT Our research focuses on arbuscular mycorrhizae in the hydrothermally-altered soils of Yellowstone National Park. Sparse vegetation cover, acidic to alkaline soils, elevated rooting zone temperatures, low nutrient levels, and phytotoxic elements characterize these sites. Mycorrhizae are thought to be important in hydrothermal soils and we have documented their presence at these sites. We conducted a greenhouse experiment testing the hypothesis that arbuscular mycorrhizae increase plant growth in soils with elevated temperatures. Seedlings were grown for 4 weeks in rhizosphere temperatures of ambient (20 -25ºC) or elevated (40ºC). Soil treatments were sterilized, sterilized with a non-mycorrhizal microbial wash added, and non-sterilized (mycorrhizal). Plant biomass was analyzed with a 3 -way ANOVA to test for plant species, soil microbial treatment and soil temperature effects. All treatments significantly affected plant biomass, and all 2 -way interactions were significant. With the exception of D. lanuginosum at elevated temperatures, plants receiving the microbial wash were larger than either mycorrhizal plants or plants grown in sterilized soil, most likely due to non-mycorrhizal microbial interactions or hormones in the soil. Root length decreased in mycorrhizal plants and elevated temperatures. Furthermore, root mass ratio (root mass: total biomass) decreased for mycorrhizal versus non-mycorrhizal plants across both temperatures, though the decrease was much greater in the elevated temperature treatments. The formation of significantly shorter and less massive roots by mycorrhizal plants (a phenomenon more pronounced at elevated temperatures) may indicate that mycorrhizal associations compensate for reduced root length and mass. If environmental conditions restrict root growth (elevated soil temperatures), this effect is beneficial. In elevated temperatures, mycorrhizal D. lanuginosum maintained total biomass and increased shoot biomass relative to non-mycorrhizal plants, which may ultimately increase plant fitness. The mycorrhizal symbiosis appeared mutualistic for D. lanuginosum growing in elevated temperatures. In contrast, mycorrhizae was parasitic for A. scabra under both ambient and elevated temperatures. However, because the A. scabra seed was from a non-thermal area, the A. scabra data are difficult to interpret. BACKGROUND Mycorrhizal Infectivity Potential Field Specimens Mycorrhizal infectivity potential is a quantification of mycorrhizal propagules in the soil. Thermal and background soils were planted with Deschampsia cespitosa in the greenhouse. Plants were harvested after 10 weeks, and AM colonization levels determined. In June 2000, we collected plants from five thermal basins across Yellowstone. Rhizosphere soils varied from acidic to neutral (p. H 3. 4 to 6. 5) with rooting zonetemperatures from 21 to 53°C. Plants were mycorrhizal with colonization levels up to 54% in soil temperatures up to 48 o. C and p. H levels down to 3. 7. Thermal – 26% colonization Background – 46% colonization AM fungi vesicles in fine root INTRODUCTION Site Description The existence of arbuscular mycorrhizae (AM) in the first land plants (Blackwell 2000, Redecker et al. 2000) and their current ubiquity suggest that understanding the nature of the mycorrhizal symbiosis is critical for elucidating plant response to environmental stresses. Our research focuses on mycorrhizal ecology in the hydrothermally-altered (thermal) environments of Yellowstone National Park (Yellowstone). Thermal areas present a myriad of potential environmental stresses for plant growth including acidic to alkaline soils, low nutrient levels, phytotoxic elements, low water availability, and extreme above- and below-ground temperatures. While mycorrhizae have been hypothesized to be important in thermal sites because of limiting nutrient levels (Burns 1997), we are the first to document the presence of both AM fungal propagules and AM fungi in plants living in thermal soils (Zabinski and Bunn, in review). The role of AM in thermal areas is difficult to predict, especially across the range of existing environmental stresses. Although mycorrhizae can augment plant growth in a broad range of environments for numerous host and endosymbiont species, they have also been shown to decrease growth in situations where the cost of carbon allocation to the fungus exceeds the benefits of the symbiosis (Johnson et al. 1997). In these high stress environments where carbon fixation and biomass accumulation are limited, the mycorrhizal symbiosis may function differently than in more amenable habitats. Because thermal sites are ancient on the scale of plant-microbe interactions, we hypothesize that the mycorrhizal symbiosis has evolved adaptations to life in thermal areas, resulting in changes in both the plant and the fungus. In Yellowstone’s thermal areas, plants grow in soils that are heated from below with temperatures up to 64°C in the rooting zone, and rooting systems are shallow to avoid higher temperatures (Stout et al. 1997). We found these plants to be mycorrhizal (Zabinski and Bunn, in review) and conducted a greenhouse experiment testing the hypothesis that AM fungi increase plant growth in soils with elevated temperatures. Yellowstone’s hydrothermal sites are located on a 6, 500 km 2 volcanic region that has been intermittently active for at least 2. 2 million years Thermal soils range (Christiansen 1984). from alkaline chloride to acid sulfate chemistry. In the latter, acidification and acid leaching are major processes removing plant nutrients relative to non-hydrothermal soils. Arbuscular Mycorrhizae Arbuscular mycorrhizae (AM) are an ancient symbiosis between plants and fungi (Remy et al. 1994, Redecker et al. 2000) that is nearly ubiquitous among plant species (Smith and Read 1997). AM fungi are obligate biotrophs: their spores can germinate without a host plant, but hyphal growth depends on plant root exudates (Bécard and Fortin 1988, Giovannetti et al. 1993). The host plant provides carbon for the fungus, while the fungus may increase plant fitness by increasing nutrient uptake (Johansen et al 1993, Smith and Read 1997, Subramanian and Charest 1999), increasing water acquisition (Stahl et al. 1998), and providing protection from pathogens (Newsham et al. 1995), potentially increasing Arbuscules host plant fitness (Heppell et al. 1998, Stanley et al. 1993). and vesicle in fine root Soil Chemistry In September 2000, we collected rhizosphere soils adjacent to thermal features (thermal) and in areas approximately 10 m from thermal features (background) near Rabbit Creek in Midway Geyser Basin. Table 1. Thermal and background soil parameters were Thermal Background significantly Characteristics different (t(n=8) (n=4) test, p<0. 05) o Temp C 35 26 for all mmhos cm 1 measured EC 2. 22 0. 49 1 parameters p. H 1 3. 78 5. 42 except available Cu 2 mg Kg-1 0. 81 0. 62 copper. Fe 2 mg Kg-1 181. 3 48. 5 Mn 2 mg Kg-1 7. 2 Zn 2 mg Kg-1 2. 3 P 3 mg Kg-1 – 4 Notes Vegetative Cover <25% 1. 2: 1 ratio of deionized water to soil 98. 2 4. 5 12. 8 75– 100% 2. Available concentrations by DTPA-TEA extraction, and ICP analysis 3. Olsen method 4. Not measured GREENHOUSE EXPERIMENT Results Hypothesis: AM relations will increase plant growth at elevated soil temperatures Experimental Design Complete factorial : • 2 plant species o o Dichanthelium lanuginosum Agrostis scabra • 3 soil treatments o Sterilized with non-mycorrhizal microbial wash o Not sterilized (mycorrhizal) • 2 soil temperatures o Ambient (20 -25°C) o Elevated (40°C) • 10 replicates Methods • Plants were grown in 3: 1 mixture of sterilized silica sand to field soil. • Microbial wash prepared by removing mycorrhizal propagules (filtration to 8μm) from a 1: 5 soil: water slurry (modified from Johnson 1993). Ten m. L were added to each pot. • Soil moisture was maintained within the range of plant-available water through daily replacement of consumed water. • Plants were dried at 60°C for 48 hours prior to weighing. • Presence/absence of mycorrhizae was confirmed by clearing and staining roots (Phillips and Hayman 1970), and quantifying AM colonization levels (Mc. Gonigle et al. 1990). After 4 weeks of seedling establishment, plants were transferred to water baths for an additional 4 weeks. (hot springs panic grass) seeds from thermal sites Shoot biomass Plant Biomass Figure 1. Plants receiving the microbial wash were larger than mycorrhizal plants and plants grown in sterilized soil 0. 3 0. 2 0. 1 0. 0 Agrostis scabra (rough bentgrass) seeds from non-thermal, metal-contaminated site Plants were grown in containers placed inside sand-filled plastic bags to allow drainage from the root chamber and efficient heat transfer to the roots. Figure 2. Root mass ratio (root mass: total biomass) decreased for mycorrhizal plants across both temperatures, though the decrease was much greater in the elevated temperature treatments 0. 6 0. 4 0. 2 0. 0 Sterile Microbial Wash Mycorrhizal Plants grown from thermal seeds (D. lanuginosum) performed better at elevated root temperatures than those not previously exposed to thermal soils (A. scabra), though with this preliminary data there is no way to distinguish species' differences from ecotypic variation. Mycorrhizal D. lanuginosum had significantly greater shoot biomass than plants with the sterile treatment (post hoc LSD tests, p=0. 002), and close to significantly greater than plants with the microbial wash treatment (post hoc LSD tests, p=0. 088). Root Length Root length was Table 4. Roots were shorter in analyzed with a 2 -way elevated temperatures ANOVA to test for and mycorrhizal plants. temperature effects and Treatment Mean mycorrhizal versus non. Root mycorrhizal effects. Lengt Table 2. Total biomass was significantly affected by all • Roots were shorter in h (cm) parameters and all 2 -way interactions were elevated temperatures significant (3 -way ANOVA). Elevated 11. 5 (F 1, 116=116, p < 0. 001) Temperature Parameters df F p • Roots were shorter in 19. 6 Soil microbial treatment 2 46. 22<0. 001 mycorrhizal plants Ambient Temperature (F 1, 116=42, p < 0. 001) Soil temperature 1 131. 0<0. 001 Mycorrhizal 11. 9 6 Plant species 1 51. 62<0. 001 Treatment x Temperature 2 10. 84<0. 001 Treatment x Species 2 6. 88 0. 002 Temperature x Species 1 42. 51<0. 001 Treatment x Temperature x 2 1. 81 0. 169 Species Mycorrhizal Effectiveness Error 108 The benefit or cost of mycorrhizae can be expressed as mycorrhizal effectiveness (ME). When total biomass is used as a proxy of plant fitness, then: ME=(biomassmycorrhizal /biomassmicrobial-wash) Table 3. Mycorrhizae decreased plant biomass except for D. lanuginosum at Temperature Species ME elevated temperatures. Ambient D. lanuginosum 0. 61 A. scabra 0. 45 Elevated D. lanuginosum 0. 96 A. scabra 0. 26 Non-mycorrhizal 17. 4 Figure 4. Root length decreased in elevated temperatures and in mycorrhizal plants