Results

1
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 masstotal
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.
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 the 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 the
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 Yellowstones
thermal areas, plants grow in soils that are
heated from below with temperatures up to 64C 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.
Yellowstones hydrothermal sites are located on a
6,500 km2 volcanic region that has been
intermittently active for at least 2.2 million
years (Christiansen 1984).
Thermal soils range 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
BACKGROUND
Mycorrhizal Infectivity Potential Mycorrhizal
infectivity potential is a quantification of
mycorrhizal propagules in the soil. Thermal and
background soils were planted
Field Specimens In June 2000, we collected plants
from five thermal basins across Yellowstone.
Rhizosphere soils varied from acidic to neutral
(pH 3.4 to 6.5) with rooting zonetemperatures
from 21 to 53C. Plants were mycorrhizal with
colonization levels up to 54 in soil
temperatures up to 48oC and pH levels down to
3.7.
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 host plant fitness (Heppell et al.
1998, Stanley et al. 1993).
with Deschampsia cespitosa in the greenhouse.
Plants were harvested after 10 weeks, and AM
colonization levels determined. Thermal 26
colonization Background 46 colonization
Arbuscules 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.
GREENHOUSE EXPERIMENT Hypothesis AM relations
will increase plant growth at elevated soil
temperatures
Results Plant Biomass
Shoot biomass Mycorrhizal D. lanuginosum had
significantly greater shoot biomass than plants
with the sterile treatment (post hoc LSD tests,
p0.002), and close to significantly greater than
plants with the microbial wash treatment (post
hoc LSD tests, p0.088).
Root Length Root length was analyzed with a 2-way
ANOVA to test for temperature effects and
mycorrhizal versus non-mycorrhizal effects.
Table 4. Roots were shorter in elevated
temperatures and mycorrhizal plants.
Figure 1. Plants receiving the microbial wash
were larger than mycorrhizal plants and plants
grown in sterilized soil
Table 1. Thermal and background soil parameters
were significantly different (t-test, plt0.05) for
all measured parameters except available copper.

• Experimental Design
• Complete factorial
• 2 plant species
• Dichanthelium lanuginosum
• Agrostis scabra
• 3 soil treatments
• Sterilized
• Sterilized with non-mycorrhizal microbial wash
• Not sterilized (mycorrhizal)
• 2 soil temperatures
• Ambient (20-25C)
• Elevated (40C)
• 10 replicates

0.3 0.2 0.1 0.0
0.3 0.2 0.1 0.0
Dichanthelium lanuginosum (hot springs panic
grass) seeds from thermal sites
Table 2. Total biomass was significantly
affected by all parameters and all 2-way
interactions were significant (3-way ANOVA).

• Roots were shorter in elevated temperatures (F1,
  116116, p lt 0.001)
• Roots were shorter in mycorrhizal plants
  (F1, 11642, p lt 0.001)

Agrostis scabra (rough bentgrass) seeds from
non-thermal, metal-contaminated site
Figure 2. Root mass ratio (root masstotal
biomass) decreased for mycorrhizal plants across
both temperatures, though the decrease was much
greater in the elevated temperature treatments

• Notes
• 1. 21 ratio of deionized water to soil
• 2. Available concentrations by DTPA-TEA
  extraction, and ICP analysis
• Olsen method
• Not measured

0.6 0.4 0.2 0.0
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.
0.6 0.4 0.2 0.0
Mycorrhizal Effectiveness 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)
Sterile Microbial Wash Mycorrhizal

• Methods
• Plants were grown in 31 mixture of sterilized
  silica sand to field soil.
• Microbial wash prepared by removing mycorrhizal
  propagules (filtration to 8µm) from a 15
  soilwater slurry (modified from Johnson 1993).
  Ten mL 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 60C 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
  (McGonigle et al. 1990).

Table 3. Mycorrhizae decreased plant biomass
except for D. lanuginosum at elevated
temperatures.
Figure 4. Root length decreased in elevated
temperatures and in mycorrhizal plants
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.
After 4 weeks of seedling establishment, plants
were transferred to water baths for an additional
4 weeks.