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Endomycorrhizal fungi enhance spotted knapweed (Centaurea maculosa) invasion in western Montana grasslands
Marilyn Marler The University of Montana
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Endomycorrhizal fiingi enhance spotted knapweed (Centaurea
maculosa) invasion in western Montana grasslands
Marilyn Marler
B.S. University of California, Davis 1994
Presented in partial fulfillment of the requirements
for the degree of
Master of Science
The University of Montana
1997
Approved by:
littee Chairperson
Dean, Graduate School
3-20-97 Date UMI Number; EP34525
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UMI EP34525 Published by ProQuest LLC (2012). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code
ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Marler, Marilyn J., M.S., May 1997 Organismal Biology and Ecology
Endomycorrhizal fungi enhance Centaurea maculosa invasion into grasslands of western Montana (50 pp)
Director: Ragan M. Callaway
Exotic plant introductions often result in rapid exclusion of native species. Little is known about changes in mycorrhizal associations that accompany invasions, or how mycorrhizae mediate plant interactions during invasions. I quantified arbuscular mycorrhizal (AM) fungal colonization of the native bunchgrasses Agropyron spicatum and Festuca idahoensis. and the exotic forb, Centaurea maculosa. which has invaded extensive areas of grasslands in Montana. I sampled roots at two sites, comparing phenology and levels of colonization among the three species. At each site, I compared colonization of each species in "native", "exotic" and "mixed" community types. Total colonization of Agropvron was lower than Festuca and Centaurea. which did not differ significantly from each other. Centaurea had the highest density of vesicles at both sites. Sampling date affected total colonization of all species. Sampling date affected vesicle density for Centaurea at one site, but did not affected vesicle density of the grasses. Community type was not corrblated with total colonization for any species, or with density of vesicles in the bunchgrasses. Community type correlated with vesicle density in Centaurea. but not consistently. Arbuscule density did not differ between community types for any species. These results do not indicate that native mycorrhizae are altered during Centaurea invasion. In the greenhouse, I tested the effect of AM fungi on Centuarea's: 1) intraspecific competition, 2) competition with either small or large Festuca. and 3)growth with available or unavailable phosphorus. When grovm with small Festuca. Centaurea was 83% larger than when grovm with a conspecific. Mycorrhizae increased Centaurea's negative effect on Festuca. When competing with Centaurea. nonmycorrhizal Festuca were 171% larger than the mycorrhizal treatment. In competion with large Festuca. Centaurea biomass was 66% greater in the presence of AM fungi. AM fungi had no effect on biomass of Festuca in this experiment. Centaurea biomass was not affected by different phosphorus sources or AM fungi, but the root-to- shoot ratio varied between treatments. The presence of AM fimgi always favored Centaurea during competition, and may thus strongly enhance the ability of Centaurea to invade.
ii Acknowledgements
My committee really helped to make this thesis an enjoyable process. I thank Ray
Callaway and Cathy Zabinski for their advice and tremendous enthusiasm. Arma Sala and
Jim Gannon made many helpful, critical comments during this work. My 1996 field work was funded by USPS grant INT-95126-RJVA, through the Intermountain Research
Station in Missoula. I also received funding from NSF EPSCoR grant OSR-955450.
I thank Lauren Quinn and Todd Wojtowitz profusely for many, many hours of excellent help in the field, lab, and greenhouse. Erik Aschehoug, Dave Broadhead, Laura
Feist, TJ Fontaine, Jodee Smith all helped me in different ways, and I appreciate their interest and time. Brad Cook loaned me a very nice compound microscope for root measurements. I thank Angie Evendon for her interest and help finding appropriate field sites.
I want to thank Monte Jewell for his friendship and for encouraging me to pursue graduate school. David Schmetterling read many versions of this thesis, and I thank him for countless helpful comments, and for his support.
iii Table of Contents
Abstract ii
Acknowledgements iii
Chapter 1: Centaurea maculosa invasion and variation in mycorrhizal colonization in
intermountain grasslands of western Montana
Introduction ...... 1
Methods...... 4
Results...... 6
Discussion ...... 7
Literature cited ...... 10
Tables and Figures ...... 13
CHAPTER 2: Mycorrhizae indirectly enhance the competitive effect of an exotic forb on
a native bunch grass
Introduction ...... 20
Methods ...... 23
Results...... 26
Discussion ...... 28
Literature cited ...... 32
Tables and Figures ...... 37 CHAPTER 1
Centaurea maculosa (spotted knapweed) invasion and variation in mycorrhizal
colonization in intermountain grasslands of the Northern Rockies
Introduction
The species composition of plant communities is known to affect the composition and function arbuscular mycorrhizal (AM) fungal communities (Johnson et al. 1992,
Bever et al. 1996). The presence of nonmycorrhizal plants can decrease fungal colonization of host plants by decreasing available inocula (Kovacic et al. 1984,
Harinkumar and Bagyaroaj 1988) , or interfering with colonization (Hanson 1991). Also, host plants influence species composition of the fungal community (Johnson et al. 1992,
Bever et al. 1996), apparently by affecting sporulation rates of the fungal symbiont
(Bever et al 1996). It is important to understand the effects of plants on fungal communities because the interactions are complex: plants respond differently to the presence of AM fungi and to different AM fungal species (Francis and Read 1994,
Johnson 1993).
AM fungi can affect community composition, succession and diversity by mediating plant competition (Ocampo 1986, Grime et al. 1987, Allen and Allen 1988,
Hetrick et al. 1989, Hartnett et al. 1993, Francis and Read 1994, Moora and Zobel 1996).
Plant dependence on mycorrhizae varies from weakly facultative to obligate, and host plants do not benefit equally from all fungal partners (Allen and Allen 1990). Thus, feedback mechanisms between plant and fungal communities may affect the balance of
1 2
plant competition directly and indirectly (Johnson et al. 1992, Johnson 1993).
The roles of mycorrhizae in exotic plant invasions are virtually unknown.
Invasions often result in dramatic changes in native plant communities, including great
losses in species diversity (D'Antonio and Vitousek 1992). Plant invasive success
results from a complex network of factors, including biotic and abiotic interactions.
Mycorrhizal associations may also have strong effects on invasion processes (Goodwin
1992). Since mycorrhizae mediate many plant interactions, they potentially have an
important role in the competitive exclusion of native plants by exotics. Little is known
about changes in mycorrhizae that accompany an exotic plant invasion. If invasion
affects functions of mycorrhizae, available fungal symbionts, or causes a shift in fungal
community composition to less mutualistic species, the competitive ability of native
plants could be decreased.
Centuarea maculosa Lam. (Asteraceae), or spotted knapweed, is an abundant
invasive forb that was introduced to southwestern Montana in the 1920's from eastern
Europe. It is now one of the most important rangeland weeds in western North America
(Lackschewitz 1991, Watson and Renney 1974). I quantified hyphae, vesicle, and
arbuscule densities in the roots of Centaurea maculosa and two bunchgrasses Agropyron. spicatum and Festuca idahoensis. which are native to western Montana. Arbuscules are sites of nutrient exchange between the host plant and fungus, and precede vesicle formation which are the fungal storage organs (Koide and Schreider 1993). Since these fungal structures have different physiological roles, the abundance of vesicles and arbuscules over the growing season can provide insight into the functional relationship of 3 the mycorrhizae (Mullen and Schmidt 1993, Johnson 1993). Specifically, I investigated whether Centaurea was colonized with AM fungi, whether colonization differed among plant species or varied seasonally, and if colonization of any species differed between invaded and uninvaded community types. Because Centaurea has a ruderal life history strategy and thrives in disturbed areas, and has been shown to have allelopathic effects on native grasses (Ridenour 1996), I predicted that Centaurea would be nonmycorrhizal, and would have measurable effects on the mycorrhizal associations of native plant species. 4
Methods
Plant species and study areas. Centaurea maculosa is a deeply taprooted, peremaial plant that produces a prolific number of seeds and can form nearly monocultural stands after the rapid exclusion of native species (Lackschewitz 1991). Agropyron spicatum (Pursh)
Scribn. & Smith (bluebunch wheatgrass), and Festuca idahoensis Elmer (Idaho fescue), are two of the most common native bunch- forming grasses in grasslands of foothill and
montane zones in western Montana (Lackshewitz 1991).
In 1995 I sampled the southwest-facing slope of Mount Sentinel, adjacent to the
University of Montana campus in Missoula (elevation 1294m, T13N R19W, sec 34), and
in 1996, a site similar in aspect and elevation 4.2 km east of Hamilton, Montana
(elevation 1334m, T5N R17W, sec 21-22). Both sites have been invaded by several
exotic species including Linaria dalmatica (L.) Mill, (dalmatian toadflax). Euphorbia
esula L. (leafy spurge), and Bromus tectorum L. (cheatgrass), but retain remnant patches
of native prairie in good- condition, having very few exotic plants. At both sites, the
dominant natives were Agropvron spicatum. Balsamorhiza sagittata (Pursh) Nutt. and
Lupinus sericeus Pursh, while Centaurea maculosa was the most abundant invader.
Festuca idahoensis was common at the Missoula site but rare at the Hamilton site (where
F, scaberella was more common), and therefore was not included in 1996.
Sampling and scoring of roots. At each site, I identified adjacent areas as either intact
native Palouse prairie ( 0-10% Centaurea cover, "native"), invaded by Centaurea (20-
40% Centaurea cover, "mixed"), or dominated by Centaurea (>50% cover, "exotic"). I 5
collected 15 randomly selected individuals of each grass species from the native and
mixed communities. I also collected at least 15 Centaurea individuals from the mixed and exotic communities at each site. Sampling was repeated at six week intervals (mid
May, early July, and mid August) over the summers.
Root systems and surrounding intact soil were collected to a depth of about 20
cm. Roots were washed, then cleared in 2.5% KOH for 48 hours, acidified in 5% HCl for
12 hours, and stained in trypan blue for 12 hours. Colonization was scored using a
magnified intersections method (McGonigle et al. 1990) which allows scoring of vesicles,
arbuscules, and hyphae separately, as well as total colonization. For each individual, I
scored four transects through 36 one-cm root sections, for a total of apprt>ximately 140
random intersections per individual.
Analysis. Data for total colonization and colonization by vesicle- were transformed with
either natural log or arcsin of square root functions prior to analysis. For Centaurea. a two
way analysis of variance was used to determine the effect of community type and
sampling date on these two variables. For Agropvron and Festuca, an analysis of
variance was used to test the effect of host species, community and sampling date on total
and vesicle colonization. Comparisons between species were made with T- tests. Data
for arbuscule colonization was not normal even after transformation, and comparisons
between species and community types were made with the Mann-Whitney U test for
rank. 6
Results
AM fungi colonized all species. Total mycorrhizal colonization did not differ
between Centaurea and Festuca. but both had higher total colonization than Agropvron
(Table 1). At both sites, vesicle density was higher in Centaurea roots than in either grass species (Table 1). Festuca had the highest density of arbuscules at the Missoula site, and
Centaurea and Agropyron did not differ significantly. At the Hamilton site, Centaurea
had more arbuscules than Agropyron (Table 1).
Total AM colonization of Centaurea and Agropyron roots changed over the
growing seasons (Table 2 and 3). Total colonization of Agropyron decreased over the
growing season at the Missoula site, but did .not change the Hamilton site (Figure 1).
Total colonization of Centaurea increased over the season at Missoula site, and peaked in
July at the Hamilton site (Figure 1).
Seasonal variation in vesicle density was not consistent between sites. Sampling
date had a significant effect on vesicle density of Centaurea at the Missoula site (Table 2),
but not at the Hamilton site (Table 3). Vesicle density of the grasses was not affected by
sampling date at either site (Tables 2 and 3).
Total colonization did not differ between community types for any species at
either site (Tables 2 and 3). Arbuscule density did not differ between community types at
either site (Table 4). Vesicle density of Centaurea differed between community types at
both sites (Table 2 and 3), but not consistently. Vesicle density was significantly higher
in the mixed community at the Missoula site, but significantly lower in the mixed
community at the Hamilton site (Figure 2). 7
Discussion
I found no evidence for functional changes in native plant mycorrhizae in
Centaurea-invaded communities. Invasion did not correspond with a decrease in mycorrhizal colonization of either native grass species. Community type was correlated with a difference in vesicle density for Centaurea. However, patterns of vesicle density
between community types were opposite at the two sites (Figure 2).
Seasonal variation in colonization by is not surprising; fungal phenology is
known to change over a plant's growing season. Arbuscules usually precede vesicles, and correspond to times of low resource availability or high demand by the host plant
(Mullen and Schmidt 1993).
Across community types and sampling dates, Centaurea had higher levels of total
mycorrhizal colonization than Agropvron. and more vesicles than both native species.
This asymmetry of mycorrhizal associations may be an important factor in cornpetition
between these species. The presence of mycorrhizae can favor replacement of
nonmycorrhizal plants with mycorrhizal species (Allen and Allen 1990, Francis and Read
1994). Allen and Allen (1984, 1988) and Allen et al. (1988) found that the presence of
AM fungi favored replacement of the nonmycotrophic plant Salsola kali by a
facultatively mycorrhizal Agropvron species by causing a one-half to one-third decrease
in ^ kali survivorship. Mycorrhizae may have a similar effect in grasslands being
invaded by Centaurea: fungal symbionts may provide a competitive advantage to the
invader over less mycorrhizal species such as Agropvron spicatum. Greenhouse studies
of competition between Centaurea maculosa and Festuca idahoensis indicate that the 8 presence of AM fungi strongly favor Centaurea (Marler et al. 1996) even though these plants have similar levels of colonization by AM fungi in the field. Mycorrhizal fungi can connect plants of different taxa and translocate nutrients or photosynthate between them along source- sink gradients (Chiarello et al. 1982, Grime et al. 1987). Since
Centaurea grows more quickly than Festuca and Agropyron. it could act as a strong sink for resources from these species.
It is becoming more common for restoration attempts to include mycorrhizal inoculum with seeds or transplants. The argument for providing AM fungi assumes that native (or desirable) plants will benefit from the mycorrhizae, and show improved growth and survivorship after inoculation. My results suggest that this strategy may not always be advisable, since greenhouse studies also indicate that AM fungi in this system provide more benefit to the invader (Marler et al. 1996) and would not help a restoration attempt.
Grime et al. (1987) found that AM fungi strongly favor Centaurea nigra over Festuca ovina, and it is expected that these results would extend to other members of the genus
Centaurea. which includes several other invasive species (C. solstitialis. C. diffusa) in the western United States. Before using resources to provide mycorrhizal inoculum, managers should consider the mycorrhizal dependence of their target plant species, and which species of invasive plants, if any, are likely to be present at the resoration site.
Centaurea had a higher density of vesicles than both native species. Since vesicles are the fungal organs where fixed carbon is stored, the higher density of vesicles in Centaurea could reflect higher carbon allocation by this host species to its fungal symbiont. Vesicle density can also vary depending on soil type (Klironomos 1995), 9 which might help explain differences between sites in this study.
The higher vesicle formation found in Centaurea roots may also mean that different AM fungal species are associated with this invader. Recent research by Bever et al. (1996) suggests the possibility that invasive species can alter the native fungal community by changing fungal sporulation rates, and hence indirectly affect the fitness of native plants. Future studies should determine whether AM fungal species associated with Centaurea stands are different from those associated with native prairie communities, and whether Centaurea brings exotic fungal species with it during invasion, or if it associates with species that are already present in intermountain prairie soils. 10
Literature cited
Allen, E.B. and M.F. Allen. 1984. Competition between plants of different successional
stages: mycorrhizae as regulators. Canadian Journal of Botany 62; 2625-2629.
Allen, E.B. and M.F. Allen. 1988. Facilitation of succession by the nonmycotrophic
colonizer Salsola kali (Chenopodiaceae) on a harsh site; effects of mycorrhizal
fungi. American Journal of Botany 75: 257-266.
Allen, M.F., E.B. Allen, and C. F. Friese. 1988. Responses of the non-mycotrophic plant
Salsola kali to invasion by vesicular- arbuscular mycorrhizal fungi. New
Phytologist 111: 45-49.
Allen, E.B. and M.F. Allen. 1990. The mediation of competition by mycorrhizae in
successional and patchy envirormients. In Perspectives in Plant Competition.
Grace, J.B. and D. Tilman (editors). Academic Press. New York.
BeverJ.D., J.B.Morton, J. Antonovics, and P.A. Schultz. 1996. Host dependent
sporulation and species diversity of arbuscular mycorrhizal fungi in a mown
grassland. Journal of Ecology. 84:71-82.
D'Antonio, C.M. and P.M. Vitousek. 1992. Biological invasions by exotic grasses, the
grass/fire cycle, and global change. Annual Review of Ecology and Systematics
23; 63-87.
Demars, B.G. and R.E.J. Boerner. 1994. Vesicular-arbuscular mycorrhizal fungi
colonization in Capsella bursa-t)astoris (Brassicaceae). American Midland
NaturaUst 132: 377-380.
Francis, R. and D. J. Read. 1994. The contributions of mycorrhizal fungi to the 11
determination of plant community structure. Plant and Soil 159: 11-25.
Grime, J.P., J.M.L. Mackey, S.H. Hillier and D.G. Read. 1987. Floristic diversity in a
model system using experimental microcosms. Nature 328: 420- 422.
Goodwin, J. 1994. The role of mycorrhizal fungi in competitiv e interactions among
native bunch grasses and alien weeds: a review and synthesis. Northwest Science
66:251-260.
Hanson, D. E. 1991. Russian knapweed interference with corn VA mycorrhiza, western
wheatgrass, and smooth brome. Thesis, Colorado State University. Fort Collins,
Colorado, USA.
Harinkumar, K.M. and D.J. Bagyaraj. 1988. Effect of crop rotation on native vesicular
arbuscular mycorrhizal propagules. Plant and Soil 110: 77-80.
Johnson, N.C. 1993. Can fertilization of soil select less mutualistic mycorrhizae?
Ecological Applications 3: 749-757.
Johnson, N C., D. Tilman, and D. Wedin. 1992. Plant and soil controls on mycorrhizal
fungal communities. Ecology 73:2034-2042.
Klironomos, J. 1995. Arbuscular mycorrhizae of Acer saccarum in different soil types.
Canadian Journal of Botany 73:1824-1830.
Kovacic, D.A., T.V. St John, and M.I. Dyer. 1984. Lack of vesicular-arbuscular
inoculum in a Ponderosa pine forest. Ecology 65:1755-1759.
Lackschewitz, K. 1991. Vascular Plants of West- Central Montana— Identification
Guidebook. USDA Forest Service GTR INT-277. Ogden, Utah, USA.
Marler, M.J., C.A. Zabinski, and R.M. Callaway. 1996. Mycorrhizal mediation of 12
competition between an exotic forb and a native bunchgrass. Program and
Abstracts of the First International Conference on Mycorrhizae. Szaro, T.S. and T.
D- Bruns, eds. University of Califonia, Berkeley, USA.
McGonigle, T.P., M.H. Miller, D.G. Evans, G.L. Fairchild, and J.A. Swan. 1990. A new
method which gives an objective measure of colonization of roots by vesicular-
arbuscular mycorrhizal fungi. New Phytologist 115:495-501.
Moora, M. and M. Zobel. 1996. Effect of arbuscular mycorrhiza on inter- and
intraspecific competition of two grassland species. Oecologia 108; 79-84.
Mullen, R.B. and S.K. Schmidt. 1993. Mycorrhizal infection, phosphorus uptake and
phenology in Ranunculus adoneus: implications for the functioning of
mycorrhizae in alpine systems. Oecologia 94: 229-234.
Ocampo, J. A. Vesicular-arbuscular mycorrhizal infection of "host" and "non-host"
plants: effect on the growth responses of the plants and competition between
them. Soil Biology and Biochemisty 18; 607-610.
Ridenour, W. L. 1996. Mechanisms of Centaurea maculosa invasion to Palouse
prairie of western Montana and possible biocontrols. Thesis. University of
Montana. Missoula, Montana, USA.
Watson, A.K. and A.J. Renney. 1974. The biology of Canadian weeds. 6. Centaurea
diffusa and C, maculosa. Canadian Journal of Botany. 54: 687-701. 13
Figure Legends
Figure 1. Mycorrhizal colonization of Centaurea maculosa. Agropyron spicatum. and
Festuca idahoensis at each site. Bars are average of both community types for each species. Total colonization is divided into colonization by hyphae (grey), arbuscules
(black) and vesicles (diagonal stripes). Bars within a graph that do not share letters are different at P<0.05.
Figure 2. Vesicle density of Centaurea roots in mixed and exotic community types at both sites. Error bars represent one standard error of the mean. Table 1, Comparisons between species. Vesicle density and total colonization were compared with T tests. Arbuscule density was compared with the Mann-Whitney U test for rank. Values in a column at each site that are followed by different letters are significant at P<0.05. Values in parentheses are the standard error of the mean.
Species Total Vesicle Arbuscule
colonization colonization colonization
Missoula site
Agropyron .5269 (.023)a .1161 (.012)a .008 (.002)a
Festuca .7213 (.020)b .1380 (.011)b .043 (.009)b
Centaurea .7067 (.021)b .1853 (.014)c .004 (.001)a
Hamilton site
Agropyron .1569 (.013)a .0106 (002)a .002 (.001)a
Centaurea .5546 (.021)b .0383 (.004)b .006 (.001)b 15
Table 2. F ratios from ANOVAs for the Missoula site in 1995. ANOVAs were used to test the effect of sampling date, community type and host plant species on vesicle density and on total colonization of Agropvron and Festuca. Separate ANOVAs were used to test the effect of sampling date and community type on vesicle density and total colonization of Centaurea. (* P<
0.05, ** P<0.01, *** P<0.001)
Source of Variation
Date X Date X
Host X
Date Community Host Community Host
Community 3 way
(2 df) (1 df) (Idf)
Response variable
Agropvron and Festuca
Vesicle density 1.338 0.433 1.836 0.383 0.650
3.012* 2.36*
Total colonization 2.506* 0.003 45.970*** 2.401 1 478
0.177 0.329
Centaurea
Vesicle density 3.509** 6.228** 2.253
Total colonization 7-435** 0.244 0.860 Table 3. F ratios from ANOVAs for the Hamilton site in 1996. ANOVAs were used to test the effect of sampling date and community type on vesicle density and total colonization of Agropyron.. Separate ANOVAs were used to test effects of these factors on vesicle density and total colonization of Centaurea. (* P< 0.05, ** P<0.01, ***
P<0.001)
Source of Variation
Date Community Date x Community
Source of variation 2 df 1 df 2 df
Agropyron
Vesicle density 0.460 3.111 2.089
Total colonization 15119*** 3.485 0.461
Centaurea
Vesicles 1.961 4.932** 1.975
Total colonization 5.613** 0.658 0.874 17
Table 4. Mann- Whitney U test comparisons of arbuscule density of each species across community types. Values in parentheses are the standard error of the mean, (ns = not significant at P<0.05).
Native Mixed Exotic Sig
Missoula site
Agropvron .0098 (.004) .0071 (.003) ns
Festuca .0340 (.007) .0540 (.018) ns
Centaurea .0039 (.002) .0046 (.002) ns
Hamilton site
Agropvron .0024 (.001) .0010 (.001) ns
Centaurea — .0070 (.001) .0050 (.001) ns 18
Centaurea Agropyron Festuca Missoula site Missoula site Missoula site 0.7 0,7 0.7
0.6 i 0.6 0.6
0.5 0,5 0.5
0.4 0.4 0.4
0.3 0.3 ab 0.3
0.2 0.2 0.2
0.1 0.1 0.1
0 0 May July August May July August May July August
Centaurea Agropyron Hamilton site Hamilton site b a Vesicles 0.7 0.7 B Arbuscuies • Hyphae 0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 May July August May July August
Figure 1. 19
0.08
0.07 - (/) _g) I mixed community o D exotic community CO 0.06 - Q) > 0.05 - •D 0 N c 0.04 o o o 0.03 oc o Q. 0.02 o
0.01
0.00 Missoula site Hamilton site
Figure 2. CHAPTER 2
Mycorrhizae indirectly enhance the competitive effect of an exotic forb on a native
bunchgrass
Introduction
Interactions between pairs of species are often mediated by one or more intermediate species (Kareiva 1994, Miller 1994). Indirect interactions include those between sea otters, kelp and sea urchins (Estes 1978), starfish, molluscs and algae (Paine
1966, 1974), parasitic and autotrophic plants (Pennings and Callaway 1996), plants and soil microflora, (Bever 1994, van der Putten 1993) and plants and mycorrhizal fiingi
(Grime 1987, Hetrick et al. 1989, Hartnett et al. 1993, Moora and Zobel 1996). An indirect interaction may result in a reduction in the competitive ability of one of the directly interacting species, altering the outcome of the pairwise interaction. Thus some species in a community, either within or between trophic levels, can have a disproportional effect on community structure by decreasing the abundance of dominant species, promoting less abundant species and maintaining species diversity. Arbuscular mycorrhizal (AM) fungi can affect plant community structure by altering the balance of competition (Hetrick et al. 1989, Allen and Allen 1990, Hartnett et al. 1993, Moora and
Zobel 1996). This occurs because AM fungi enhance resource acquisition of some host plant species better than others (Allen and Allen 1990), and possibly mediate direct transfer of resources or fixed carbon between individuals (Chiarello et al. 1982, Grime et al. 1987, Walter et al. 1996).
20 21
Differential plant responses to mycorrhizae have been shown to contribute to
coexistence of species (Grime et al. 1987, Hetrick et al. 1989 Hartnett et al. 1993, Moora
and Zobel 1996) and to successional replacement of nonmycorrhizal plants by
mycorrhizal species (Allen and Allen 1984, 1988, Francis and Read 1994). In both cases
competitive hierarchies among plants are altered. The ability of AM fungi to mediate
interactions depends, in part, on abiotic factors. For example, phosphorus availability can significantly affect the host plant's relationship with mycorrhizae. When phosphorus is directly available for uptake by the plant, the plant may not need a symbiont for its acquisition. In this situation, the fungus may be rejected (Koide and Schreider 1993,
DeLucia et al. in press). If the plant is unable to regulate colonization, the symbiont can
present an unnecessary drain on the host's carbon balance (Hartnett et al. 1993, Koide
and Schreider 1993). While AM fungi have been shown to promote species coexistence,
they may also contribute to competitive exclusion and losses in species diversity if a
superior competitor is favored.
Exotic plant invasions provide clear examples of competitive exclusion and
dominance. However, the role of mycorrhizae in mediating interactions between invaders
and native species are still not well understood (Goodwin 1992). Exotic invasive plants
include both mycorrhizal and nonmycorrhizal species, and very different mechanisms
have been reported by which AM fungi influence interactions. Increased nutrient
acquisition relative to native species (Caldwell et al. 1985) and reduction in fungal
propagules available to native plants as a result of invasion (Goodwin 1992) have both
been suggested. Only one controlled study of the effect of AM fungi on competition 22 between an exotic and a native species has been reported (Allen and Fisher 1985).
Centaurea maculosa Lam. (Asteraceae) and others in its genus are among the most economically destructive range weeds in western North America (Watson and Renney
1974, Griffith and Lucey 1991). Centaurea forms persistent, nearly monocultural stands after displacing native grasses and forbes (Lackshewitz 1991). Despite extensive efforts to eradicate it, Centaurea species are spreading at prodigious rates (Muller-Scharer and
Schroeder 1993). Field populations of Centaurea maculosa in western Montana are heavily colonized by AM fungi, and colonization levels are higher than some species of native bunchgrasses which it frequently displaces (Marler et al. 1996).
1 investigated the effects of AM fungi on the competitive success of Centaurea maculosa in a series of greenhouse experiments in which the presence of AM fungi, the size of a bunchgrass competitor, and the form of phosphorus available to the plants were controlled. 23
Methods
All experiments were conducted in a naturally lit greenhouse supplemented with high pressure sodium lighting. PAR ranged from 700- 1200 mmol/m-2 /s-1.
Temperatures ranged from 18-22°C.
In the first experiment, I grew the native bunch grass Festuca idahoensis Elmer, and Centaurea in intra- and interspecific combinations, both with and without mycorrhizae. The plants were started at the same time from seed in 21.25 cm diameter,
27.5 cm deep pots filled with sterile, 20/30 grit silica sand. Inoculum was added as a 2 cm layer of whole field soil, collected from beneath many species of native plants and
Centaurea on Mt Sentinel in Missoula, MT. Nonmycorrhizal treatments received autoclaved field soil and 50 ml of a microbial wash. This solution was prepared by filtering a soil slurry through a 25 um filter paper, which is fine enough to remove mycorrhizal fungal propagules while allowing other soil microorganisms, including potential pathogenic bacteria and fungi to pass through (Johnson 1992). Plants received
250 ml of a 1/8-strength modified Hoagland's solution weekly. Phosphorus was provided as inositol hexaphosphate, a form that is not directly available to plants for uptake, but requires alteration by mycorrhizal fiingi or soil microbes. After three months, I measured total biomass of both species and checked for mycorrhizal infection under lOOx magnification. Biomass of Festuca was natural log transformed for normality prior to analysis with a 2 way ANOVA for effects of neighbor and AM fungal treatments.
In a second experiment, competition was established between Centaurea and a large, established Festuca neighbor. I grew a single Centaurea from seed in 30 pots, each 24 containing an eight- week old Festuca. In this experiment, final biomass of Festuca was
0.525 g, compared to 0.145 g in the previous experiment. The pots and growth medium were the same as described for the previous experiment. The grasses were provided live field soil inoculum during the establishment period, and thus had the potential to form mycorrhizae. At the time Centaurea seeds were planted, AM fungi were eliminated from half the pots with Benomyl applied in water at the rate of 50 mg Benomyl/ kg soil
(Hetrick et al. 1989). I watered and fertilized the plants as in the first experiment, and reapplied Benomyl every three weeks. After seven weeks, I compared mean total biomass and root-to-shoot ratio of both species in mycorrhizal and nonmycorrhizal treatments with T-tests for significance at P<0.05, and checked for mycorrhizal colonization.
In a final experiment, Centaurea was grown from seed without a competitor in pots identical in size and containing the same medium as the previous two experiments.
Plants were watered weekly with 250 ml 1/8 strength modified Hoagland's solution with phosphorous added as either inositol hexaphosphate (unavailable for direct plant uptake), or potassium phosphate which is directly available to the plant. In each phosphorus treatment there were both mycorrhizal and nonmycorrhizal treatments. It should be noted that the experimental pots were not sterile, and that bacteria may have been responsible for transforming the phosphorus even in the absence of AM fungi. This makes our measurements conservative, since such bacteria are present in field soils and would reduce differences between the phosphorus treatments. Fungi were eliminated from nonmycorrhizal pots with repeated Benomyl applications as described above. After 7 weeks I measured total biomass and root-to-shoot ratios for each plant, and compared the
Means with two way ANOVAs for the effects of phosphorus and AM fungi treatments. 26
Results
Mycorrhizal hyphae and vesicles were observed in roots of both species in the mycorrhizal treatment of the first experiment. No evidence of mycorrhizae was found in the autoclaved treatment. In the second and third experiments, Benomyl application reduced mycorrhizal colonization of roots from an average of 21% to 3.5% across all treatments.
In the first experiment, the final average total biomass of Festuca was significantly lower when grown with Centaurea than when grown with a conspecific
(Figure 1, Table 1). However, the competitive effect of Centaurea was much greater in the presence of AM fungi, indicated by a significant interaction term between competitor species and AM treatments (Table 1). When competing with Centaurea. average Festuca biomass was 171% greater when AM fungi where absent (Figure 1). AM fungi had no effect on Festuca biomass in the absence of Centaurea (Table 1, Figure 1), nor did AM fungi alone change the root to shoot ratio of Festuca (Figure 2, Table 2). AM fungi decreased root allocation during intraspecific competition, but increased root allocation during competition with Centaurea (Table 2, Figure 2).
Intraspecific competition had a greater effect on Centaurea biomass than did interspecific competition (Figure 3, Table 3). The presence of AM fungi alone had no effect on Centaurea biomass; however, the interaction between AM fungi treatment and neighbor species was significant (Table 3), indicating that the response of Centaurea to the presence of AM fungi depended on which competitor was present. AM fungi significantly depressed growth in the presence of conspecifics, but not during competition 27 with Festuca (Figure 3). AM fungi decreased the root to shoot ratio of Centaurea overall, indicating less allocation to roots in this treatment (Figure 4, Table 4).
The final biomass of Centaurea that were competing with established Festuca was
66% larger when AM fungi were present (T test, P=0.025, Figure 5). Festuca biomass was not affected by the presence of AM fungi in this experiment (T test, P<0.818, Figure
5). Root-to-shoot patterns of Centaurea and large Festuca were not altered by AM fungi
(Table 5a,b).
AM fungi did not significantly affect final Centaurea biomass in either the available or unavailable phosphorus treatments of the third experiment (Table 6). Root to shoot ratio was not affected by AM fungi, but was affected by phosphorus availability
(Table 7, Figure 6). Centaurea responded to phosphorus deficiency by allocating significantly more biomass to roots (Figure 6). 28
Discussion.
AM fungi had no direct effect on the biomass of Centuarea or Festuca. but did substantially increase the competitive effect of Centaurea on Festuca. Furthermore, AM had a much stronger positive effect on Centaurea's growth when paired with large
Festuca than with small Festuca. Mycorrhizae favored Centaurea in both competition studies, but in different ways. When both species were started from seed, the presence of
AM fungi during interspecific competition had a large negative impact on Festuca and caused substantial reallocation of biomass from shoots to roots, while Centaurea showed marginal increase in growth. In the experiment with large Festuca. Festuca showed no significant response to the presence of AM fungi, while Centaurea biomass increased by more than half.
Mediation of competition by mycorrhizae has been attributed to differential responses of plant species to fungal colonization; that is, by mycorrhizae supplying nutrients or water to different host species at different rates (see Allen and Allen 1990).
AM fungi always favored Centaurea over Festuca during competition, but changes in
Centaurea biomass depended on the competitor species, and not directly on AM fungi
(Tables 3 and 6). The presence of mycorrhizae did not directly affect the biomass of
Centaurea growing alone and experiencing different forms of soil phosphorus. Centaurea was very plastic in its biomass allocation depending on presence of AM fungi and phosphorus availability. This trait probably decreases its reliance on AM fungi for nutrient uptake, and contributes to its competitive ability in the field. Festuca biomass and allocation patterns were also unaffected by AM fungi in the absence of Centaurea. 29
Thus, individual host plant response was not the main reason for the change in competitive ability.
Other mechanisms have been attributed to AM fungi's ability to mediate plant interactions, including the translocation of photosynthate or nutrients from one plant to another via mycorrhizal connections (Chiarello et al. 1982, Walter et al. 1996, Francis and Read 1984). By using radiolabelled carbon. Grime et al. (1987) found that the growth of Centaurea nigra was greatly increased at the expense of Festuca ovina in the presence of mycorrhizae, and that a large amount of '''C was transferred in the presence of mycorrhizae. Centaurea maculosa grows much faster than Festuca idahoensis. and the disparity in growth rates makes translocation of carbon between species along source- sink gradients a strong possibility.
Larger plants generally have stronger competitive effects on neighbors than smaller conspecifics (Keddy 1990). I found that the effects of size based competition were reversed by AM fungi. The effect of AM fungi on Centaurea biomass was greater in the presence of large Festuca competitors than small competitors. This too supports the hypothesis that fixed carbon is transferred from Festuca to Centaurea. as large Festuca could provide large sources of photosynthate. In contrast, small Festuca neighbors may be a small source of fixed carbon.
These results demonstrate strong indirect effects of AM fungi on competition between Centaurea and Festuca. but virtually no direct effects. This is unusual, as strong indirect effects are generally mediated by strong direct effects. For example, Miller
(1994) found that indirect positive effects among plants were strongest when a dominant 30 competitor disproportionally suppressed intermediate competitors, which reduced competitive pressure on the weakest competitors. Lubchenco (1978) attributed strong indirect effects of a herbivore on competition among algal species to its strong direct effects on the dominant competitor. This generality, which appears to be widespread both within and between trophic levels (Paine 1974, Hawkins and Hartnoll 1983, Hay et al
1983, Cottam et al. 1986, Brown and Heske 1990, Huntly 1991) was not supported by this study. This may be the first demonstration of a complex interaction with little or no direct effects but highly significant indirect effects.
The relationship between AM fungi, Centaurea and Festuca may parallel those between myco-heterotrophic plants and their hosts. Myco-heterotrophic plants and hemi- parasites are those that are photosynthetic but receive, via fungal symbionts, a portion of their photosynthates from neighboring plants (Leake 1994). It is possible that mycorrhizal fungi, typically regarded as mutualists, allow Centaurea to function as a facultative hemiparasite. If so, we should broaden our view of myco-heterotrophy and its role in structuring communities.
These results also emphasize the importance of studying mutualisms as dynamic interactions which can range from antangonism to true mutualism depending on the context of the interaction (Bronstein 1994, Thompson 1994). AM fungi may provide indirect benefits to Festuca in the absence of fast growing competitors; for example, AM fungi are believed to provide some protection to host roots from saprophytic fungi and other soil pathogens (Newsham et al. 1995).
Plant communities can influence fungal species (Bever et al. 1996, Johnson et al. 31
1992). Thus, Centaurea may alter the fungal community composition. If strong shifts in fungal species occur in the presence of Centaurea. then Centaurea's increased competitive ability could be due to fiingal species that are less mutualistic toward the native flora, and a decrease in native plant competitive ability. Mycorrhizal fungi in Centaurea roots form more and larger vesicles, which are fungal strucures for storage of carbohydrates and lipids, than native grass roots (M. Marler, personal observation). This may indicate that different fungal species are associated with Centaurea. or may simply reflect a different physiological function of the fungal symbiont when associated with different hosts.
Mycorrhizal fungi have the potential to strongly enhance invasion by Centaurea.
The results of these studies are as predicted by the Lotka- Volterra model of competition, which predicts that, for two species to coexist, the effects of intraspecific competition must be greater than the effects interspecific competition (Begon et al. 1990). Studies with coexisting plant species have found that AM fungi decrease interspecific competition and increase intraspecific competition (Moora and Zobel 1996, Hartnett et al.
1993). In this study, AM fungi significantly increased the effect of Centaurea on Festuca. tipping the outcome of competition towards competitive exclusion, which has been the result of Centuarea maculosa introduction since the 1920s. Although field experiments are necessary to show that mycorrhizae are a significant factor, it appears that in situ soil fiangi have very weak direct effects, but substantial indirect effects that increase the ability of an exotic invader to suppress a native competitor. 32
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180-184. Figure Legends
Figure 1. Total biomass of Festuca when grown with either a conspecific or a
Centaurea. and with and without AM fungi. Error bars represent one standard error of
the mean. Bars that share letters were not different at P<0.05 (post- ANOVA Tukey
test).
Figure 2. Root to shoot ratio of Festuca when grown with either a conspecific or a
Centaurea. and with and without AM fungi. Error bars represent one standard error of
the mean. Bars that share letters were not different at P<0.05 (post- ANOVA Tukey
test).
Figure 3. Total biomass of Centaurea when grown with either a conspecific or a small
Festuca. and with and without AM fungi. Error bars represent one standard error of the
mean. Bars that share letters were not different at P<0.05 (post- ANOVA Tukey test).
Figure 4- Root to shoot ratio of Centaurea when grown with either a conspecific or a
Festuca. and with and without AM fungi. Error bars represent one standard error of the
mean. Bars that share letters were not different at P<0.05 (post- ANOVA Tukey test).
Figure 5. Total biomass of Centaurea and large Festuca when grown in competition
with one another, and with and without AM fungi. Error bars represent one standard
error of the mean.
Figure 6. Root to shoot ratio of Centaurea when grown alone, with either available or
unavailable phosphorus, with and without AM fungi. 38
Table 1. ANOVA table for natural log transformed biomass of Festuca grown in competition with either a conspecific or Centaurea. and with two AM treatments (present or absent) (Experiment 1). Mean Sig
Source of Variation DF Square F ofF
Competitor 1 3.286 4.954 .031
AM 1 1.955 2.948 .092
Competitor x AM 1 4.844 7.304 .009
Total 52 0.846 Table 2. ANOVA table for the effect of AM fungi and competition with either a conspecific or Centaurea on root to shoot ratio of Festuca.
Mean Sig
DF Square F ofF
Neighbor 1 4.449 6.610 .013
AM fungi 1 1.909 2.836 .099
Neighbor
X AM fungi 1 9.039 13.432 .001
Total 52 0.970 Table 3. ANOVA for biomass of Centaurea grown in competition with either a conspecific or Festuca, with two AM treatments (present or absent).
Mean Sig
Source of Variation DF Square F ofF
Competitor 1 45.196 42.521 .000
AM 1 0.617 0.581 .449
Competitor x AM 1 7.232 6.804 .012
Total 57 1.933 41
Table 4 ANOVA table for the effect of AM fungi and competition with either a conspecific or a small Festuca on the root to shoot ratio of Centaurea.
Mean Sig
DF Square F ofF
Neighbor 1 .20396 .406 .527
AM fungi 1 4.242 8.476 .005,
Neighbor x AM fungi 1 1.912 3.821 .056
Total 57 0.595 42
Table 5a.
T test comparisons of root to shoot ratio of large Festuca competing with Centaurea in
the presence and absence of AM fungi.
mean std error df t- value 2-tailed sig
with AM fungi 0.696 0.029 28 1.79 0.084
without AM fungi 0.595 0.048
Table 5b.
T- test comparisons of root to shoot ratio of Centaurea competing with Festuca in
the presence and absence of AM fungi.
mean std error df t-value 2-tailed sig
with AM fungi 0.292 0.027 27 -0.33 0.741
without AM fungi 0.307 0.038 Table 6. ANOVA table for effects of phosphorus source (available or unavailable) and presence of AM fungi on Centaurea biomass.
Mean Sig
Source of Variation DF Square F of F
AM 1 .100 2.351 .134
Phosphorous 1 .015 .364 .550
AM X Phosphorous 1 .003 .075 .786
Total 39 .042 Table 7. The ANOVA table for the effects of AM fungi and phosphorus availability on the root to shoot ratio of Centaurea.
Mean Sig
DF Square F ofF
Main effects 2 .050 5.097 .011
AM fungi 1 .029 3.022 .091
P source 1 .072 7.420 .010
AM fungi X
P source 1 .005 483 .492
Total 38 .012 45
0.35
+ AM fungi 0.30 - - AM fungi
0.25 -I CO (f) 03 ^ 0.20 g !q Ct3 O
CO 0.10 -
0.05
0.00 Festuca Centaurea Neighbor
Figure 1. 46
CD ••1 +AM fungi O I I - Ai\/I fungi SiM3 CO CD U. o g ro L_ •*-> o X SI Festuca Centaurea Neighbor Figure 2. 47 Festuca Centaurea Neighbor Figure 3. 48 CT3 =3 + AM fungi 3 - iS - AM fungi c: CD o M—o g 2 - ab X. "co L_ o t/) 1 - o o (H Festuca Centaurea Neighbor Figure 4. 49 0.8 n 0.7 + AM fungi I I - AM fungi 0.6 - 0.5 - U) (/) 0.4 CO CO ^ 0.3 g CQ 0.2 0.1 ^ 0.0 Festuca Centaurea Figure 5. 50 0.50 CD ^ 0.45 available P :3 H unavailable P ^ 0.40 - c ab ^ 0.35 ab 0.30 0.25 0.20 ^ o O 0.15 4-j O 0.05 a: 0.00 + AM fungi - AM fungi Figure 6.