Testing Causes of Ring Formation in Blue Grama Grass in the Sevilleta
By Nora Dunkirk
August 10, 2013
Abstract:
Plants interact with their soil communities through root interactions with soil and
root-inhabiting microorganisms, also known as plant-soil community feedbacks. Plantsoil community feedbacks (PSFs) can shape how a plant grows in its habitat. Negative
PSFs, which result when a plant performs worse in association with its own soil
community than with the soil communities from other plant species, have been
hypothesized to explain why some plants, such as the grass blue grama (Bouteloua
gracilis), form patches in the shape of rings. The idea is that host-specific pathogens build
up in the center of the plant, causing plant dieback and the formation of a ring as the
plant grows outward toward pathogen-free soil. The mechanisms for grass ring formation
have been minimally studied in the field, and none, to our knowledge, have directly
tested for a role of PSFs in ring formation. This study tested the effect of negative PSFs on
ring formation by comparing the plant response of blue grama seedlings when grown in
soils taken from either inside or outside grass rings in the field to sterile controls. We
found higher rates of germination, survival, and growth for seedlings grown on live soil
from inside the ring than from live soil outside the ring, but no difference in plant
performance when soils were sterilized, suggesting that a seed would have higher fitness
if it landed in the center of a ring than at the outer edge. These results did not support
the hypothesis that negative PSFs cause ring formation in blue grama, but suggest instead
that pathogen loads may be highest on the outer edges of ring-forming plants.
Introduction:
As a plant grows and matures, it changes the composition of and interacts with the
soil microbial community around and inside its roots (Bever 1997). This interaction, or
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plant-soil community feedback (PSF) can be beneficial, detrimental, or neutral (Van der
Putten 2013). Feedbacks range in strength as well as in the direction of their effects on
plants (Kulmatiski et al. 2008). A positive plant soil feedback occurs when the plant
interacts with the soil organisms in a way that is beneficial to the growth of that plant and
its progeny relative to soil communities from other plant species. A negative plant soil
feedback occurs when the interaction inhibits the growth of the plant and/or its progeny.
PSFs can occur for one plant species (individual PSF) or for a community of species, and
can alter the survival, reproduction, and growth patterns of those species (Kulmatiski et
al. 2008).
Many grasses in differing ecosystems grow in ring shaped patches, such as Poa
bulbosa (Sheffer 2007), Sesleria appennina, Brachypodium rupestre, and Ampelodesmos
mauritanicus (Carteni et al 2012). Several studies link negative PSFs to ring formation in
clonal plants such as Scirpus holoschoenus (Bonanomi et al. 2005), the species studied by
Carteni et al. (2012), and Bouteloua gracilis, or blue grama, studied by Ravi et al. (2008).
The mechanism behind this has been a subject of past research because central dieback
implies a difference in soil community characteristics between the center and the outside
of the grass rings (Ravi et al. 2008). One study proposed the hypothesis that exogenous
factors, hydrologic patterns linked with aeolian deposits that cause plant death, can be a
cause for ring formation in blue grama, however, these mechanisms have received little
direct experimental testing (Ravi et al. 2008). One study suggests blue grama ring growth
is simply an “adaptation a plant has to maintain itself under unfavorable conditions,” yet
this does not explain the underlying mechanism for central dieback in these grasses
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(White 1989). The hypothesis that PSFs cause ring formation has only been studied in
conceptual models thus far and not empirically (Ravi et al. 2008). Therefore, this study
does the job of testing the theoretical model in a real-world experimental testing. This
project aims to increase our understanding of plant-soil feedbacks in grasses of the desert
environment at the Sevilleta National Wildlife Refuge (NWR). I hypothesize that negative
plant-soil feedbacks are causing the central dieback found in blue grama. This study
addresses the question: Is ring formation of blue grama caused by negative PSFs?
Methods:
Study species. Blue grama, Bouteloua gracilis, is an abundant, arid-land grass that
grows in rings. After reaching a certain size, the center of the patch of grass dies back
which grass leaves a ring-shaped patch. A prior field study by Ravi et al. (2008) found that
blue grama of a medium size (40-60 cm diameter) showed a larger edge vs. center
difference in soil qualities such as infiltration rate and hydraulic conductivity than did
plants of both smaller and larger ring sizes. Thus, we focused on plants in this size range
for our study.
Study site. The study site was a desert grassland dominated by blue grama located
in the McKenzie flats region of the Sevilleta NWR in New Mexico (106.6917W
34.3529N). The experiment was conducted in June - August 2013 and consisted of: soil
collection, soil sterilization, cone-tainer treatment, seed addition to pots, and data
collection.
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Plant observations. Forty mid-sized (40-60 cm diameter) blue grama plants were
chosen at random along two transects in a blue grama grassland in the Sevilleta NWR.
Each plant was a minimum of 1 m apart and formed an obvious single ring. For each
plant, we measured ring area by finding two perpendicular diameters of each the outer
and inner edges of the ring, and using the equation for the area of an ellipse (A = pi* r1*r2)
subtracted the inner area from the outer. We also collected roots from the inner and
outer edges of the ring for observations of fungal colonization. Approximately 60 cm
length of roots were collected from each plant July 12-31. Roots were stained using the ink
and vinegar method (Vierheilig et al. 1998) and fungal colonization was scored using the
grid-line intercept method (McGonigle et al. 1990).
Soil collection. At each of the 40 target plants, two soil samples (approx. 66 ml
each) were collected in the week of June 10 from each the center and the outer edge of the
ring using a soil corer for 0-10 cm depth (20 plants) or a hand trowel and soil corer for 1020 cm (20 plants). The soil corer and trowel were wiped clean with a towel between each
soil collection. One sample from each the center and the edge was placed directly into a
conetainer pot (2.5 cm diameter, 16 cm deep, Ray Leach Conetainers, Stuewe and Sons,
Corvalis, OR), and the other sample from each location was placed into a plastic
autoclave bag for sterilization. Pots were kept at room temperature for 6 days until seeds
were planted. The factorial experimental design is shown in Table 1.
Location on Ring
Inner
Outer
Live
40
40
Sterile
40
40
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TABLE 1. Experimental design showing soil origin X sterilization treatments, numbers
show sample sizes for each treatment combination.
Soil sterilization. All the soils collected in autoclave bags (half of the total soil
collected) were sterilized in an autoclave at the University of New Mexico. The bags were
autoclaved on a gravity cycle at a high temperature for 3 h, allowed to cool, then
autoclaved a second time for 3 h. Sterilization was used to kill soil microorganisms,
including the heat-tolerant microbes of the desert.
Pot set-up. Each pot, was scrubbed and sterilized in 10% bleach prior to soil
addition. Then the pots were separated by at least 6 cm in every direction in their pot
trays to reduce the possibility of cross-contamination among the treatments. Each pot
was filled with soil in the field so that approximately 2.5 cm remained empty in the top to
avoid overtopping during watering.
Planting. After the pots were filled with soil and tagged, blue grama seeds from a
local distributor were inserted onto each pot. Five seeds were added to each pot on June
18, 2013, and then each pot was supplemented with additional seeds on June 24 (after
some seeds had been lost due to wind) for a total of 6 seeds per pot. Pots were then
misted with water at least twice daily to keep the soil surface moist and optimal for
germination. After germination, the pots were given 5 ml of water two times daily and
allowed to grow for 4 weeks.
Response variables. Plant fitness was estimated by measuring germination rate,
survival, seedling height, and total biomass. Germination rate was determined by
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counting the total number of seeds that sprouted in each pot, and dividing that by the
total number of seeds planted. Survival was determined as the mean survival of a
germinated seedling at 20 days after seed addition. All but one seedling were pruned from
the pots after germination to avoid competition. After the seedlings grew for 4 weeks,
plant height was measured. Then the seedlings were harvested, soil was rinsed from roots,
and plants were dried at 60C in a convection oven for 3 days to measure above and
below-ground dry biomass.
Data analysis. The data were analyzed using ANOVA and General Linear Models
in the program R (version 3.0.0, R Core Team) to test for the effects of soil origin (inside
ring vs. outer edge), soil sterilization (live vs. sterile) and the origin X sterilization
interaction. Specifically, we predicted that if PSFs were responsible for ring formation, we
should detect a significant soil origin X sterilization interaction, with reduced plant
performance in live soils from inside the ring relative to live soils from the ring edge, but
no effect of soil origin when soils were sterilized. We additionally tested whether ring size
or depth of soil collection were significant covariates in the analyses.
Results:
Germination. In general, plants performed worse in soils from the outer ring edge
than in soils from the inside of rings. Soil origin had no effect when soils were sterilized,
implicating soil microbes in these differences. When measuring germination rate, we
found plants that grew on soil originating from the inside of the ring had a significantly
higher germination rate on average than plants growing on soils from the outside of the
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ring (F1,39=5.29, P=0.016).
Survival. The trend for live soils was higher survival on soil from inside the ring
than on soil from outside. Whether a plant survived better on soil from the inside the ring
depended on if it was sterile or live. The survival rate showed that the interaction of
treatments, soil origin X sterilization, was significant (F1,39=2.194, P=0.028).
Growth. The results for plant height showed marginally significant effects of ring
location (F1,39=4.041, P=0.053). For sterilized soil, the height average was very similar for
both inside and outside soils. But for live soil, there was a large trend for soil taken from
the inside of the ring to result in much greater average seedling height than soil from the
outer edges. The data taken for biomass had a very low sample size due to high rates of
seedling mortality, so statistical analyses could not be done. Nevertheless a trend can still
be found for live soil, which had on average higher biomass for above and belowground
on soil from the inside than from the outside of the ring. Another interesting find is that
seedlings grown on sterile soil all had greater biomass than seedlings grown on live soil
(inside and out) suggesting that live soils are largely pathogenic (rather than beneficial)
to seedlings.
Discussion:
We set out to find if negative PSFs were the cause of ring formation in blue
grama grass. This experiment found little evidence to support that hypothesis. Instead,
we found the opposite trend from natural occurrences, where we see plant death in the
center of the ring, a negative interaction, and growth and active tillers outside the ring, a
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positive interaction. Our results indicate that a seed in nature would do best if it landed
and grew inside the ring rather than outside it.
These results also imply that there is a higher abundance of soil pathogens located
outside the ring than inside it. Future studies can look at the roots from inside and
outside blue grama rings to evaluate the apparent fungal colonization and assess whether
there is a difference in abundance (inside vs. outside). This could help to determine
whether fungal parasites or mutualists affect grass ring formation.
The soil collected from inside the ring was taken from the approximate center,
where it is possible that few roots were present; whereas the soil collected from outside
the ring was taken as close to the plant as possible (about 5-10 cm away) where roots were
abundant. Soil pathogens that we would expect to cause negative PSFs, such as
pathogenic or parasitic fungi, are largely associated with plant roots. If no roots were
collected with the soil from the center of the ring, the pathogens could have been avoided
as well. This difference in collection could have had an effect on seedling growth. Future
experiments could sample soils at intervals inside and outside the ring to see if plant
growth patterns differ from those found in this experiment.
We conclude that negative PSFs do not appear to be a strong mechanism causing
ring formation in blue grama. In fact, plant growth was quantitatively better on soils from
inside the rings relative to soils from the outer edge of rings.
Acknowledgements:
This research was funded by the Research Experience for Undergraduates (REU)
program at the Sevilleta National Wildlife Refuge and the University of New Mexico. We
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would like to thank the Sevilleta Fish and Wildlife and Amaris Swann.
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