Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1996 The oler of arbuscular mycorrhizal fungi in prairie wetlands Paul Robert Wetzel Iowa State University
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The role of arbuscular mycorrhizal fiingi in prairie wetlands
by
Paul Robert Wetzel
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major; Ecology and Evolutionary Biology
Major Professor: Arnold G. van der Valk
Iowa State University
Ames, Iowa
1996 UMI Niimber: 9712619
UMI Microform 9712619 Copyright 1997, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 ii
Graduate College Iowa State University
This is to certify that the Doctor's dissertation of
Paul Robert Wetzel has met the dissertation requirements of Iowa State University
Signature was redacted for privacy. Major Professor
Signature was redacted for privacy. or the Major Program
Signature was redacted for privacy. iii
TABLE OF CONTENTS
ABSTRACT v
CHAPTER 1. GENERAL INTRODUCTION 1 Plant-Fungus Symbiosis 1 Arbuscular mycorrhizal fiingi 1 Host plant and mycorrhizal fungi interaction 2 Environmental factors 5 General ecology of prairie wetlands 10 Arbuscular mycorrhizal fiingi in wetlands 11 Mycorrhizal continuum model 12 Research Questions 15 Dissertation Organization 16 References 17
CHAPTER 2. VESICULAR-ARBUSCULAR MYCORRHIZAE IN PRAIRIE POTHOLE WETLAND VEGETATION IN IOWA AND NORTH DAKOTA 24 Abstract 24 Introduction 25 Materials and Methods 27 VAM colonization determination 28 Fungus indentification 29 Soil measurements 29 Statistical analysis 31 Results 32 Discussion 39 Acknowledgments 43 References 43
CHAPTER 3. THE EFFECTS OF ARBUSCULAR MYCORRHIZAL AND NON- MYCORRHIZAL FUNGI ON WETLAND PLANTS DURING DIFFERENT REDOX POTENTIALS AND HYDROPERIODS 47 Abstract 47 Introduction 48 Methods 51 Experimental design 51 Measurement of redox potential 53 Data collection 54 Results 55 Discussion 63 Acknowledgments 68 References 68 iv
CHAPTER 4. MYCORRHIZAE AND THE COMPETITIVE ABILITY OF THE FACULTATIVE MYCOTROPH CAREXSTRICTA IN DIFFERENT ENVIRONMENTAL CONDITIONS 71 Abstract 71 Introduction 72 Methods 74 Experimental design 74 Data collection 76 Statistical analysis 78 Results 79 Principal components analysis 82 Treatment effects on plant productivity and macronutrient concentration 84 Treatment effects on interspecific competition 86 Discussion 91 Acknowledgments 95 References 95
CHAPTER 5. GENERAL CONCLUSIONS 98 Greneral Summary 98 References 104
ACKNOWLEDGEMENTS 106 V
ABSTRACT
Research on arbuscular mycorrhizal fungi (AMF) suggests that their effect on plant growth is a continuum that changes from positive to negative along environmental gradients as conditions for plant growth improve. Prairie wetlands were used to test this mycorrhizal continuum model. First, three saline and three freshwater wetlands were surveyed for AMF, to investigate the hypothesis that AMF colonization would be greater in wetlands with high soil salinity, lower nutrient levels, and during periods of drought. Second, a greenhouse experiment on Carex striata and Calamagrostis canadensis tested whether AMF improved plant growth and nutrient uptake under five hydrologic regimes. Third, another greenhouse experiment tested whether AMF improved the competitive ability, productivity, and macronutrient uptake of the facultative mycotroph, C striata, along nutrient and soil moisture gradients while growing with Phalaris arundinacea and Typha latifolia.
AMF colonization in Iowa and North Dakota ranged from 0.2 to 72% among all plant species and was strongly dependent on the plant host regardless of environmental factors or the plant's life stage. Colonization was greatest at the peak growing and fiiiiting period (June) on many early season grasses and sedges. AMF colonization was also greater in plants growing in the poor soil conditions found in North Dakota—low phosphorus and high soil pH.
Soil moisture did not have a significant effect on AMF colonization. However, the survey was done in a wet year and may have underestimated the importance of soil moisture on AMF colonization. AMF colonization levels measured in the field supported the mycorrhizal continuum model. vi
High soil phosphorus levels in the hydrology experiment probably prevented AMF colonization of C. stricta and C. canadensis. The lack of AMF colonization suggests that mycorrhizae are not important to these plants growing in natural wetlands with high soil phosphorus levels, even in dry conditions. Non-mycorrhizal fungi reduced biomass of C. canadensis, but not C stricta, indicating that C stricta may have antifungal properties.
Nutrient levels and interspecific competition explained 35% of the total variation on C stricta productivity and macronutrient concentration, whereas soil moisture explained only 7% and AMF 0% of the variation. AMF did not increase the competitive ability, productivity, or nutrient uptake of C. stricta. 1
CHAPTER 1. GENERAL INTRODUCTION
Plant-Fungus Symbiosis
Arbuscular mycorrhiz/d fungi
Mycorrhizae, a symbiotic association between fungi and plants, occur with most species of angiosperms, all gymnosperms, all pteridophytes, and some bryophytes. Five classes of mycorrhizal associations occur, with arbuscular mycorrhizal fungi (Zygomycetes) or endomycorrhizae, being the most common. This association includes the plant root, the fungal structures within the root cells, and the mycelium in the soil. Fungal structures within the plant consist of intercellular mycellium, vesicles, and arbuscules. Vesicles are formed from hyphal expansions between the plant cells and range from 10-100 nm in diameter. They are usually filled with lipids, suggesting a storage role, but their exact function is not known.
Arbuscules consist of finely branched hyphae within the cortical cells of the plant. They persist up to 15 days, until they are digested by the plant. Nutrient transfer to the plant and carbon transfer from the plant is believed to occur between the finely branched hyphae and plant cell membranes. Fungal spore formation occurs outside of the plant root except for one species. Glomus intradices. Spores range from 40-500 |im in diameter and it is by spores that these flingi may be spread by wind, water, or animals.
The mycorrhizal association has three tightly intertwined components; the host plant, fiingi, and environmental factors. This dissertation considers the effects of all three components on plant nutrient uptake, biomass development, and competitive ability using prairie wetlands as a model system. Each component of the mycorrhizal association will be discussed, followed by an introduction to prairie wetlands, a review of mycorrhizal fiingi in 2 wetland and floodplain systems, the mycorrhizal continuum model and the use of prairie wetlands as a model system, and the hypotheses and objectives of each part of the dissertation project.
Host plant and mycorrhizal fungi interaction
Arbuscular mycorrhizal fungi (AMF) are considered obligate symbionts since they have never been observed in nature without a host and all attempts to culture AMF in the laboratory without a host have failed (Allen, 1991; Harley and Smith, 1983). AMF have a broad host range, with individual AMF species associating with diverse plant groups (Molina et al., 1992). Conversely, plants also associate with many species of AMF (Molina, et al.,
1978). Festuca spp. growing in a variety of habitats throughout the western United States and British Columbia were colonized with eleven species of AMF, often with two or more fungal species colonizing a single plant.
AMF improve nutrient uptake for most plants, particularly in non-productive soil conditions, by increasing the assimilation rate of phosphorus, certain micronutrients, and water from the soil (Allen, 1991; Brundrett, 1991; Douds and Chaney, 1986; Harley and
Smith, 1983; Hayman, 1983). Nitrogen absorption is also improved, but this may be a secondary consequence of increased phosphorus uptake (Harley and Smith, 1983). AMF fiingi may also be involved with the reduction of nitrate (Harley and Smith, 1983; Khan,
1975). AMF fungi increase plant water uptake and reduce wilting during water stress (Harley and Smith, 1983). Water and nutrient assimilation is believed to be improved because the fungal mycelium extends the plant's root system, making a greater pool of water and nutrients available to the plant. 3
AMF colonization of various plant communities—sand dunes (Koske and Halvorson,
1981), sugar maple forest (Brundrett and Kendrick, 1988), tallgrass prairie (Hartnett et al.,
1993; Hetrick et al., 1988), xeric shrublands (Allen, 1989; Bethlenfalvay et al., 1984), and tropical forests (Janos, 1980; Koske et al., 1992)—suggest that plants have a wide range of dependence on AMF and can be classified as obligate, facultative, and non-mycorrhizal.
Determining the mycorrhizal status of a plant is difficult because AMF colonization changes over the course of the plant's life cycle or in response to environmental factors. Presence of
AMF colonization also does not indicate whether the mycorrhizal association is positive, negative, or neutral. Obligate mycorrhizal plants need mycorrhizae sometime during their lifetime to reproduce successfully in their natural habitat. Facultative mycorrhizal plants benefit fi'om mycorrhizae in certain environments or have inconsistent or low levels of colonization (usually < 25%). Non-mycorrhizal plants resist mycorrhizal colonization
(Brundrett, 1991).
Non-mycorrhizal plant species are found in many plant families (see Brundrett, 1991;
Molina et al., 1992), and the mycorrhizal status of plants is best understood in a succession fi-amework. Tropical pioneer species are non-mycorrhizal and facultatively mycorrhizal whereas mature forest species are obligately mycorrhizal plants (Janos, 1980). Janos (1980) speculated that non-mycorrhizal plants can attain maximum growth rates in habitats where
AMF inoculum is limited and may perpetuate their own existence by not supporting AMF and reducing inoculum available to obligate mycotrophs. Facultative mycotrophs use mycorrhizae in infertile soils, but rely on fine roots in fertile soils where AMF would be a detriment. This model has been further refined with the inclusion of nutrient and moisture gradients (Allen and 4
Allen, 1990). Non-mycotrophic plants are most important in nutrient rich habitats. In
nutrient-poor and low-moisture habitats, facultatively mycorrhizal plants dominate early
succession. During late succession, non-mycotrophic species diminish in importance and are
replaced by &cultative and obligate species. Habitats with high nutrients and moisture are also
dominated by facultative or obligate plant species because of the highly competitive nature of
these productive environments.
Evidence for non-mycorrhizal and facultatively mycorrhizal plants is suggested by
studies in which plants colonized with AMF have depressed leaf areas (Koide, 1985), a
reduction of biomass (Buwalda and Goh, 1982), and a greater translocation of fixed carbon
than plants not colonized by AMF (Snellgrove et al., 1982). Snellgrove ei al. (1982) found
that AMF increased the transport of the total photosynthate by 7%. They suggested that
plants with AMF compensate for this loss of photosynthate by lowering the percentage of dry
matter in the leaves, increasing the assimilation rate per unit dry weight of leaf, but
maintaining the same net assimilation rate as a plant without AMF. The result is a lower dry
weight for plants with AMF than plants without AMF grown with no nutrient stress. The
affect of high nutrient environments on AMF is discussed below.
It is expected that a reduction in the photosynthetic capacity of the host plant would
reduce available carbohydrates supplied to the plant roots and in turn affect the AMF.
Hayman (1974) determined that low light levels reduced AMF colonization in onions
compared to AMF colonization of plants grown at nearly twice the light level. In contrast,
Coprosma robusta tree seedlings became more mycorrhizal in one-third of the daylight than in
full light (Baylis, 1967). The biomass of both test plants colonized with AMF was greater at 5
high light levels than at low light levels, suggesting that AMF were strongly beneficial when adequate photosynthate was available.
Photosynthate also is transferred fi-om one plant species to another via AMF. Using
"CO2 isotopes. Read et al. (1985) injected the shoots of a donor plant, Plcmtago lanceolata, and found that "CO2 was transferred to the roots and shoots of a receiver plant, Festuca ovina, via AMF mycelium. In an interesting experiment, F. ovina was shaded to half of fiill light and to complete darkness. "CO2 activity in the roots increased 51% in the half-shaded treatment and 84% in the complete darkness treatment. There was no statistical difference in
^"*002 activity in the shoot between the three treatments. Read et al. suggest that interplant transfer of carbon by AMF allows seedlings of woodland and grassland species to survive prolonged periods of deep shade.
Environmental factors
Soil fertility is a major factor in governing the level of AMF colonization and the level of benefit received by the plant. Often, an inverse relationship exists between the level of
AMF colonization and the amount of available phosphate in the soil (Douds and Chaney,
1986; Harley and Smith, 1983; Hayman, 1983). AMF have decreased the biomass of some plant species in high nutrient soils compared to plants without AMF, suggesting that AMF can act as pathogenic parasites (Crush, 1973; Hayman, 1983; Koide, 1985). Phosphorus additions can substitute for the positive impact of AMF on the growth of mycorrhizal plants. The addition of phosphorus to obligate AMF prairie plants without AMF growing in a low phosphorus soil enabled them to grow as well as plants with AMF and no phosphorus
(Hetrick et al., 1986). Non-mycorrhizal plants with added phosphorus also had intra- and 6
interspecific competitive abilities at varying plant densities similar to those of mycorrhizal
plants (Elartnett et al., 1993). Additions of phosphorus also overcame the growth response
due to mycorrhizae in plants growing in shaded conditions (Elayman, 1974).
The studies discussed above related soil phosphorus to mycorrhizal activity, but Menge
et al. (1978) found that AMF colonization is controlled by the concentration of phosphorus
within the plant root and not the soil. Phosphorus deficiency in the roots causes the roots to
release organic carbon into the rhizosphere, and it is believed that AMF respond to this release
of root exudates (Marschner, 1992). However, It should be noted that Tester et al. (1987)
have proposed that mycorrhizal fungal penetration is controlled by the chemical composition
of the host cell walls and middle lamella.
Little is known about the effects of soil pH on AMF colonization, although AMF are
found in the low pH bogs and openings of boreal forests and in higher pH chaparral
environments (Allen, 1991). AMF colonization in field studies remained constant over the pH
range 4.2-7.0 (Read et al., 1976; Wang et al., 1985). Certain fungal species tolerate lower
pH levels than others (Wang et al., 1985). Soil pH was negatively correlated with the number
of AMF spores (-0.82) and the number of AMF species (-0.61) in a tallgrass prairie wetland
(Anderson et al., 1984). The effect of soil pH can be confounded by other soil faaors such as
organic matter and elemental soil composition, making the impact of pH on AMF difBcult to
determine.
AMF are found in saline soils and have been studied along salinity gradients found in coastal and iiJand wetlands (Ho, 1987; Hoefhagels et al., 1993; Johnson-Green et al., 1995).
Generally, AMF colonization declines with increasing soil salinity, disappearing when total 7 salts range from 55-78 mg/ml ^Cim and Weber, 1985; Johnson-Green et al., 1995).
Halophytic grasses located in the Alvord Desert, southwestern Oregon, were found to be heavily colonized with AMF. Spore counts were inversely correlated with soil sodium concentration (Ho, 1987). AMF improved growth of some plants under salinity stress in salt water marshes by increasing water uptake (Rozema et al., 1986), but AMF produced no effect on the growth of Disiichlis spicata, a salt tolerant plant, when grown at 0, 1000, and 2000 mg
Na/kg soil (Allen and Cunningham, 1983). Like soil pH, the effect of soil salinity is confounded by changing ion concentrations with rainfall, groundwater flow, and tides as well as by changes in soil oxygen levels associated with water level changes.
AMF colonization is affected by temperature, soil moisture, and light levels.
Envirorunental conditions may directly impact the host plant and thereby indirectly affect the
AMF, making it difBcuh to determine the total effect of environmental factors. For example, temperature affects both the host plant, the fungi, and the symbiosis, since an effect on either will indirectly affect both. Spore germination of fungal species collected from the same soil was tested at temperatures between 10-30°C (Tommerup, 1983). Spore germination declined at both temperature extremes, but was not affected in the middle range of temperatures for each fungal species. The results were the same whether plants were present or not. In another experiment, the number of fiingal entry points on the main roots ofMedicago truncatula and Trifolium subterraneum increased with increasing temperature when tested at
12°, 16°, 20°, and 25°C (Smith and Bowen, 1979). The largest increase in entry points occurred between 12° and 16° with no statistical difference among the 16°, 20°, and 25°C treatments. Absorption and translocation of phosphorus by AMF does not occur below 7°C 8
(Hayman, 1983). On a landscape level, average AMF species richness increased from north to south along a 355 km latitudinal transect and was positively correlated with growing-degree days and frost-free days (Koske, 1987).
Field studies of principal plant species in a community or entire communities have documented a seasonal pattern of AMF colonization that closely matches the growth and development of the host plant. Overall AMF colonization in a sugar maple forest community did not fluctuate seasonally in plants with long-lived roots. AMF colonization of herbaceous species with short-lived roots mcreased when the roots grew and declined with root senescence (Brundrett and Kendrick, 1988). Boemer (1986) also found no seasonal AMF variation in two perennial deciduous forest herbs. AMF density of bienniel species in the chalklands of southern England was highest during the period of maximum accumulation of foliar nitrogen, phosphorus, and potassium (Gay et al., 1982). In the perennial species studied, AMF activity was lowest during the period of maximum accumulation of foliar nitrogen, phosphorus, and potassium (Gay et al., 1982). Study of cool- and warm-season tallgrass prairie grasses found that AMF activity was greatest at the temperature that favored growth of the host (Bentivenga and Hetrick, 1992). Cool-season tallgrass prairie grasses had greater AMF activity than warm-season tallgrass prairie grasses in April, May, and December, while AMF activity in warm-season grasses was greater than cool-season grasses in July and
August. These differences were measured despite the fact that cool-season grasses appear to be facultative mycotrophs and warm-season grasses appear to be obligate mycotrophs
(Hetrick etal., 1988). Measurements of in pot experiments found that approximately 100 times more was observed in the cool-season grass Bromus inermis at 18°C than at 29°C. 9
The warm-season grass, Andropogon gerardii, had four times more at 29°C than at 18°C
(Hetrick a/., 1994).
AMF do not have a clear effect on the water relations of plants. Maize plants colonized with AMF had increased leaf water potential and transpiration rates and lower stomatal resistance than plants without AMF (Subramanian et al., 1995). However, transpiration rates and water uptake per root length of maize fertilized with phosphorus was not different between mycorrhizal and non-mycorrhizal plants (George etaL, 1992). AMF also increased transpiration rates and reduced stomatal conductance compared to non-mycorrhizal plants in clover (Hardie and Leyton, 1981), Bouteloua gracilis (Allen et al., 1979), and citrus trees during recovery from water stress (Levy and Krikun, 1980).
In other studies, AMF have either little or no effect on water relations (Allen and Allen,
1986; Fitter, 1988). The biomass oiAndropogen gerardii inoculated with AMF was 27% greater than non-inoculated plants in a severely water stressed treatment and nearly the same amount as produced in the adequately watered treatment (Hetrick et al., 1986). The biomasses of inoculated and noninoculated plants exposed to moderate soil moisture stress were not significantly different.
Despite the mixed results of whole plant studies, a physiological mechanism of how
AMF alter water relations has emerged. Safir and Nelsen (1985) found that mycorrhizal soybeans recovered from water stress faster than non-mycorrhizal soybeans. They noted that roots with AMF had higher hydraulic conductivities. AMF also were found to increase the symplastic water and thus increase the turgor in roots of droughted Rosa plants (Auge and
Stodola, 1990). Such benefits to plants may be limited. The small diameter of AMF hyphae 10
and measurements of water loss in soil compartments containing only fungal hyphae (George
et al., 1992) suggest that AMF could not transport substantial amounts of water to a plant.
Many water relations studies found that mycorrhizal plants also had higher phosphorus levels. Water relations were not different between mycorrhizal and non-mycorrhizal onion
plants in high phosphorus conditions (Nelsen and Safir, 1982). In dry conditions, phosphorus mobility greatly decreases, which led Safir and Nelsen (1985) to conclude that reported effects of AMF colonization on plant water relationships are related to improved nutrition of the mycorrhizal plants. The effects of AMF on plants in well-watered or flooded conditions have not been as thoroughly investigated, but the level of phosphorus is important in this condition too. AMF colonization in a high soil moisture and phosphorus environment produced no effect on plant water relations (Nelsen and Safir, 1982).
General ecology of prairie wetlands
Moisture is considered the single most important environmental gradient in prairie wetland habitats, playing a principal role in determining wetland vegetation composition and zonation. Prairie wetlands have a dynamic seasonal hydrology that leaves them with either greatly lowered water levels or completely dry (drawdown) in late summer as well as interannual water-level cycles that can leave them dry for one or more years out of every 10
(Duvick and Biasing, 1981; Kantrud et al, 1989; Winter, 1989). Drawdowns caused by periodic droughts can be very stressfiil for wetland species and significantly reduce plant growth, although they do not seem to cause any noticeable mortality for wetland species. It can take two years for the standing crop of emergent species to recover its biomass from a 11 prolonged drawdown (van der Valk and Davis, 1980). Salinity also increases during drought periods in the saline wetlands of North Dakota (Swanson et ai, 1988).
Vegetation zonation develops along the soil moisture gradient in concentric rings and this zonation forms the basis of prairie wetland classification (Stewart and Kantrud, 1971).
An area with high soil moisture, but generally without standing water, that contains a mixture of upland prairie and wetland plants is called the low prairie vegetation zone. An area inundated with water a few weeks in the early spring that contains perennials, forbs, sedges, and grasses is called the wet meadow vegetation zone. Two other zones found in deeper basins are the shallow emergent zone, which contains water fi-om spring to mid-summer or early fall, and the deep emergent vegetation zone, which contains water throughout the year except during drought years (Kantrud et al., 1989).
Arbuscular mycorrhizal fungi in wetlands
The presence of mycorrhizae in aquatic and wetland habitats has been documented, but the available information is limited and often contradictory. Growth and development of
AMF are believed to be limited in wet habitats because the fiingi cannot survive in anaerobic environments (Allen, 1991). Many researchers report no AMF colonization in aquatic macrophytes, particularly in species in the Cyperaceae and Juncaceae (Allen, 1991; Anderson et al., 1984; Harley and Smith 1983; Khan, 1974) or only mycelial colonization in the rhizosphere and occasional lodgements in the root epidermal cells (Powell, 1975). However,
Sondergaard and Laegaard (1977) reported that fi-om 0 up to 96 % of the roots examined on seven aquatic plant species in four oligotrophic lakes had mycorrhizal colonization. Tanner and Clayton (1985) found that a submerged aquatic plant colonized with AMF contained 20% 12 more phosphorus on a diy weight basis than non-mycorrhizai plants. Read et al. (1976) reported that 0-21 % of the root segments examined from six wetland plant species were colonized with AMF during one growing season. Anderson et al. (1984) observed no
"functional mycorrhizae" (presence of arbuscules) in plants growing in the wettest habitats
(shallow emergent zone) along a soil moisture gradient, but some plants growing in moist to wet habitats were mycorrhizal. AMF colonization has been dociunented in rice (Dhillion and
Ampompan, 1992) and in Populus, Salix, and Nyssa spp. growing in flooded soils (Keeley,
1980; Lodge, 1989). AMF also have been reported in both fresh (Bagyaraj etal., 1979;
Sengupta and Chaudhuri, 1990) and saline (Ragupathy et al., 1990) tropical wetlands.
Mycorrhizal continuum model
The studies discussed above clearly suggest that the effect of AMF on a host plant ranges from positive to negative depending on the plant's environment and life stage. AMF cause a carbon drain on the plant under high phosphorus conditions, while the nutrient harvesting capabilities of AMF can provide a net benefit to the plant under low phosphorus conditions. The nutrient harvesting capabilities of AMF become apparent when the addition of phosphorus to tallgrass prairie plants eliminates the benefit of AMF on biomass production and intra- and interspecific competition. A similar pattern, although not as well studied, emerges during drought experiments in the field (Allen and Allen, 1986). AMF mediated the effects of temperature, as onions colonized with AMF had greater relative growth rates except when exposed to 5°C temperatures (Fairweather and Parbery, 1982). AMF can create a lag in seedling growth (Buwalda and Goh, 1982), be useful in the initial stages of soybean development, but not during flowering and fhiit development (Bethlenfalvay et al., 1982), or 13 seedling growth (Buwalda and Goh, 1982), be useful in the initial stages of soybean development, but not during flowering and fruit development (Bethlenfalvay et al., 1982), or develop a mycelial network that provides carbon and nutrients that seedlings can exploit
(Allen, 1991). Hayman (1983) called the change of the mycorrhizal relationship from a mutualistic symbiosis to pathogenic parasitism a 'symbiotic imbalance'. The results of experiments by Allen and Allen (1986) suggest that mycorrhizae are primarily important during an 'ecological crunch'. These patterns suggest that the mycorrhizal symbiosis is best considered as a continuum controlled by environmental factors and the plant life stage (Figure
1). On the mycorrhizal axis of the model, AMF range from having a mutual symbiosis with the plant host to being a parasitic pathogen. The symbiosis changes depending on the
Mycorrhizal Effect on q Plant Growth
Environmental Plant Life Stage Factors
Figure 1. Mycorrhizal continuum model. The effect of arbuscular mycorrhizal fiingi on plant growth ranges from positive (symbiotic) to negative (parasitic pathogen) depending on the environmental conditions and stage of the plant life cycle. L=Low, M=Moderate, H=High, and SU=Surplus, exceeds what plant needs for optimal growth; S=Seedling, V=Vegetative, and R=Reproductive. environmental conditions experienced by the plant and the life stage of the plant. Mycorrhizal effect is positive when nutrients, water, or temperature, levels are below those required for optimal plant growth. The effect of mycorrhizae becomes negative at high environmental levels, and mycorrfiizae do not become established when nutrients, water, or temperature exceed levels required for optimal plant growth. Such changes could happen rapidly, with a sharp drop in temperature, or seasonally as with a drought. Mycorrhizae could be beneficial only during a certain life stage, e.g. for seedlings or finiting plants. For some plants, the effect of mycorrhizae on plant growth is positive at the seedling stage, may become negative during the vegetative stage, and then is positive during the reproductive stage. Finally, mycorrhizae may be beneficial only during a combination of stressful environmental factors at a certain point in the plant's life cycle. On a community level, mycorrhizae could extend the range of some plant species and affect plant succession, community composition, and diversity
(Chanway e? a/., 1991; Grime a/., 1987).
The dynamic annual and interannual hydrology and variable soil environment of prairie wetlands make them an ideal system to test the mycorrhizal continuum model. Recent studies found that AMF colonization increased on certain wetland plant species during dry conditions.
This pattern was observed in three wetland species in two prairie wetlands located in South
Dakota (Rickerl et al., 1994). Arbuscules were observed on wetland plants in drier, lower- nutrient sites but not on the same plants growing in more fertile sites with greater soil moisture (Anderson et al., 1984), although Lodge (1989) found that AMF colonization was greater in moist soil than in very dry or flooded soils in a greenhouse moisture gradient experiment. The literature suggests that AMF may be advantageous to wetland plants during 15
periods of low soil moisture associated with annual and interannual drawdowns, in wetlands
with low soil phosphorus, and in wetlands with high salinity.
Research Questions
This project had three parts. The first part was a field survey for AMF in the dominant
plants of the low prairie, wet meadow, and shallow emergent vegetation zones of three
fi^eshwater prairie wetlands located in the southern part of the prairie pothole region and three
saline wetlands located in the central part of the prairie pothole region (North Dakota). The
objective of the field survey was to determine the extent of AMF colonization in northern
prairie wetlands and examine whether environmental factors affect the AMF colonization of
wetland vegetation. The hypotheses of this part of the projea were that AMF colonization
will be higher (i) in wetlands with high soil salinity, (ii) in wetlands with lower nutrient levels,
and (iii) during short-term periods of low soil moisture, such as during late summer
drawdowns and in the drier or upper wetland vegetation zones. The extent of AMF
colonization was measured in June and August in three vegetation zones of all six wetlands.
Environmental factors examined included soil moisture (soil matric potential), soil pH,
available soil phosphorus, and soil specific conductance.
The second part of the project consisted of a greenhouse experiment that tested the
environmental factors continuum axis by determining whether AMF improved the growth and
nutrient uptake of two wet meadow plants, Carex striata and Calamagrostis canadensis, under conditions of variable hydrology. It was hypothesized that AMF would improve plant growth and nutrient uptake of both plants in the driest hydrologic regimes and reduce plant 16 growth in the hydrologic regimes that resulted in greater soil moisture. Comparing plants growing under different anaerobic/aerobic regimes also tested whether AMF mitigate the phytotoxic effects of anaerobic end products.
The third part of the project involved another greenhouse experiment that tested whether AMF improved the competitive ability of the facultative mycrotroph, Carex striata, in high/low nutrient and soil moisture levels. It was hypothesized that colonization by AMF, by itself or in interaction with an environmental factor, would alter the relative competitive ability, measured by relative yield, productivity, and macronutrient uptake, of C. striata. The second objective of part three was to measure the response of AMF to the high/low nutrient and soil moisture levels and interspecific plant competition environments. It was hypothesized that percentage AMF colonization would be greater in the low nutrient and soil moisture treatments.
Dissertation Organization
This dissertation is composed of three papers prepared for publication. Chapter 2 has been published in the Can. J. Bot. and authorization for copyright transfer has been given by the publishing company. In this chapter arbuscular mycorrhizal fungi are referred to as vesicular arbuscular mycorrhizal (VAM) fungi, reflecting the terminology at the time of writing. Chapter 3 is a manuscript intended for submission to the Canadian Journal of Botany.
Chapter 4 is a manuscript intended for submission to Vegetatio. The references cited in each chapter are given at the end of each chapter. The research presented in these chapters was 17 performed by myself under the supervision of Dr. Arnold G. van der Valk in the Department of Botany at Iowa State University, between January 1992 and December 1996.
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CHAPTER 2. VESICULAR-ARBUSCULAR MYCORRHIZAE IN PRAIRIE POTHOLE WETLAND VEGETATION IN IOWA AND NORTH DAKOTA
A paper published in the Canadian Journal of Botany 1996, 74:883-890
Paul R. Wetzel and Arnold G. van der Valk
Abstract
Vesicular-arbuscular mycorrhizae (VAM) fungal colonization was measured in June and August 1993 for 19 plant species in several vegetation zones in three wetlands in central
Iowa and in three wetlands in North Dakota, U.S.A. Percent VAM fungal colonization in all the plants sampled varied greatly with species, ranging from 0.2 to 52.1% (mean = 13, SE =
2.4) in lowan wetlands and 7.3 to 71.8% (mean = 32, SE = 4.7) in North Dakotan wetlands.
The lowan wetlands had more fertile soils (higher organic matter, higher phosphorus, and lower pH) than the North Dakotan wetlands. Soil salinity was higher in the North Dakotan wetlands. A principal components analysis of environmental factors (soil water matric potential, soil pH, available soil phosphorus, soil specific conductance, and season) indicated that when data for all plant species were pooled, there were only three significant components; location (which reflected differences between Iowa and North Dakota in soil pH, phosphorus, specific conductance, and season), vegetation zone Gow prairie, wet meadow, shallow emergent), and soil matric potential. Multiple regression analysis of these principal components and plant species as independent variables and percent VAM fungal colonization as the dependent variable were performed. Regression analyses were also done with VAM 25 fungal colonization as the independent variable and the principal components as the dependent variables on three individual species, Poa pratensis, Spartina pectinata, and Scirpus acutus, which are common to both geographic regions. For the pooled plant species, only plant species and the location principal component were statistically significant. For Poa pratensis,
Spartina pectinata, and Scirpus acutus, only the first principal component was statistically significant. The results of this study are consistent with previous studies that suggest plant host species, soil phosphorus, and soil pH influence VAM fungal colonization. Because this study was done during an exceptionally wet year, seasonal changes in mycorrhizal colonization caused by a seasonal decline in soil moisture did not occur as predicted.
Key words: vesicular-arbuscular mycorrhizae, wetland vegetation, environmental gradients,
Iowa, North Dakota
Introduction
The presence of mycorrhizae in aquatic and wetland habitats has been documented, but the available information is limited and often contradictory. Many researchers report no vesicular-arbuscular mycorrhizae (VAM) fungal colonization in aquatic macrophytes, particularly in species in the families Cyperaceae and Juncaceae (Allen 1991; Anderson et al.,
1984; Harley and Smith 1983; Khan 1974) or only mycelial colonization in the rhizosphere and occasional lodgments in the root epidermal cells (Powell 1975). However, other investigators report VAM fungal colonization in temperate wetland plants (Read et al. 1976), in rice (Dhillion and Ampompan 1992), in halophytic wetland grasses (Ho 1987), and in 26 aquatic plants (Sondergaard and Laggard 1977; Tanner and Clayton 1985). Anderson al.
(1984) observed no "functional mycorrhizae" (presence of arbuscules) in plants growing in the wettest habitats (shallow emergent zone) along a soil moisture gradient, but some plants growing in moist to wet habitats were mycorrhizal. A similar colonization pattern was found by Lodge (1989).
The presence of mycorrhizae in wetlands suggests that they are ecologically significant, but their function is not well understood. Numerous studies found that VAM fiingi benefit plants, particularly in stressed environments. Colonized plants frequently contain higher concentrations of phosphorus and nitrogen. VAM fiingi were also found to increase plant water uptake and reduce wilting due to water stress (Harley and Smith 1983) or high salinity
(Rozema et al. 1986). Prairie wetlands have a seasonal hydrology that leaves them either with greatly lowered water levels or completely dry (drawdown) in late summer. They also have interannual water-level cycles that can leave them dry for 1 or 2 years of every 10 (Duvick and
Biasing 1981; Kantrud et al 1989). Salinity also increases during drought periods in the saline wetlands of North Dakota (Swanson et al. 1988). Drawdowns caused by periodic droughts can significantly reduce the growth of wetland species, and it can require 2 years for the aboveground biomass of emergent species to recover fi-om a drawdown (van der Valk and
Davis 1980). Nevertheless, prolonged drawdowns do not seem to cause any noticeable mortality for wetland species. Recent studies indicate that certain wetland plant species increased VAM fungal colonization during dry conditions. This pattern was observed in three wetland species in two prairie wetlands located in South Dakota (Rickerl et al. 1994).
However, Lodge (1989) found that VAM colonization was greater in moist soil than in very 27
dry or flooded soils in a greenhouse moisture gradient experiment. In short, the literature
suggests that VAM fiingi may be advantageous to wetland plants during periods of low soil
moisture associated with annual and interannual drawdowns, in wetlands with low soil
phosphorus, and in wetlands with high salinity.
This study provides a survey of the extent of VAM fungal colonization in northern
prairie wetlands and examines whether environmental factors affect the VAM fiingal
colonization of wetland vegetation. It is our hypothesis that VAM fungal colonization will be
higher (i) in wetlands with high soil salinity, (ii) in wetlands with lower nutrient levels, and
(iii) during periods of low soil moisture, in the short-term during late summer drawdowns and in the drier or upper wetland vegetation zones. To test these hypotheses, the extent of VAM fungal colonization was measured in June and August in three vegetation zones in wetlands in
Iowa and North Dakota. lowan wetlands have more fertile soils and lower salinities than
North Dakotan wetlands. Environmental factors examined in this study included soil matric
potential, soil pH, available soil phosphorus, and soil specific conductance.
Materials and Methods
The three fi^eshwater wetlands sampled in Iowa were located in the central (Hamilton and Story counties) and northwest parts (Dickinson County) of the state (Table I). These wetlands ranged fi'om 1 to 5 ha with a water depth less than 2 m. The three wetlands sampled in North Dakota were located in the central part of the state (Stutsman County) in the
NCssouri Coteau region (Table I). These wetlands were larger in area, 5 to 40 ha, with depths less than 3 m. The spring and summer of 1993 were wet, causing water levels in both Iowa 28
and North Dakota to remain high throughout the summer and preventing the usual end-of-
summer drawdown. In each wetland, populations of three dominant plant species from the
low prairie, the wet meadow, and the shallow emergent vegetation zones (as defined by
Stewart and Kantrud 1971) were sampled in June and August along five random transects.
Soil cores were taken in the area along the transect dominated by each species, resulting in a
total of 45 cores collected from each wetland. The aboveground portions of the plants were
clipped ofiF and plant roots were sampled with a soil core extractor to a depth of 15-20 cm
(core diameter =10 cm) and double wrapped in plastic bags. Root cores were refiigerated
(4°C) until they could be washed. A total of490 root cores were collected. Water depth and soil temperature were measured at each collection point.
VAM colonization determination
All root cores were washed with water to remove soil particles. Root cores containing large amounts of clay were shaken in a 3% (w/v) Electrasol™ detergent solution for 4-8 h prior to washing. Washed roots were stored in 70% ethanol (Koske and Gemma 1989). An arbitrary sample of secondary roots with attached tertiary roots from each plant were stained to detect VAM fiingi following Koske and Genuna (1989). The roots were autoclaved in a
2.5% KOH solution for 3 nunutes and then bleached by soaking 10-30 minutes in a 1% hydrogen peroxide solution. Darkly stained roots, such as Scirpus acutus roots, were bleached overnight. After rinsing with water, the roots were then autoclaved in 0.05% acid
fiichsin in acidic glycerol solution for 3 minutes. The roots were destained at room temperature. Percent VAM fungal colonization was determined with the root-gridline intersection method described by Giovannetti and Mosse (1980). Approximately 100 root 29 segments (1.0-1.5 g fresh weight) were laid out on a plastic petri dish marked with a 13.5 mm grid. The initial measurement of VAM fungal colonization for each plant species was viewed at lOOx magnification to ensure the identification of mycorrhizal fiingi in the roots. Additional
VAM fungal colonization counts were done with 40x magnification. The presence or absence of colonization was recorded for the first 200 root-gridline intersections.
Fungus identification
Common VAM fungal species were identified from Iowa and North Dakota. Soil was removed from each root core prior to washing and allowed to air dry. The soil was then ground with a mortar and pestle to pass through a 1 mm sieve and sieved for fungal spores with a mechanical sieving apparatus (elutriator) (Byrd et al. 1976). Spores were then isolated from the sievings by centrifugation, resuspending the pellet in a 1.8 A/sucrose solution, and centrifiiging the suspension (Allen et al. 1979). Spores in the supernatant were identified with a key adapted by S.P. Bentivenga and B.A.D. Hetrick (unpublished) from Trappe (1982) and
Schenck and Perez (1987).
Soil measurements
Soil used for the soil analyses was removed from each root core prior to washing and allowed to air dry. The soil was then ground with a mortar and pestle to a granular powder.
Percent organic matter, nitrate (extracted with 0.025 M aluminum sulfate solution), and available potassium (extracted with 0.14 M ammonium acetate solution) were measured on composite samples (10 from each zone) for each zone within a wetland by the Soil Testing
Laboratory at Iowa State University. Soil pH and available phosphorus (also measured by the
Soil Testing Laboratory, Olsen test) were determined with 15 samples (5 from each zone) 30 from each wetland. Soil pH was measured on 10 g of dry soil allowed to equilibrate with 10 ml of 0.01 A/CaCl2 solution for 30 min.
Soil specific conductance was measured on all soil cores (490) following procedures in
Rhoades (1982). A 1;1 soil to deionized water paste was mechanically shaken for 1 h. The paste was filtered through a Whatman No.1 filter. Turbid samples were filtered a second time with GF/C glass microfibre filters (pore size 1.2 nm). Specific conductivity was measured with a YSI (model 31A) conductance bridge.
Soil water tension or matric potential measures the amount of water available to the plant and allows soils from different wetlands to be more realistically compared than when using gravimetric soil moisture measurments. Soil matric potential was determined on all the soil cores by calculating a standard curve of pressure (kPa) versus percent soil moisture for the soil from each vegetation zone in each wetland. Percent soil moisture was measured on each soil core collected and soil matric potential was determined from the standard curve
(Klute 1986). To determine the standard curve, soil matric potential was measured on two undisturbed cores (7.8 cm long by 7.2 cm diameter) taken from the middle of the low prairie, wet meadow, and shallow emergent vegetation zones in each wetland. These cores were wrapped in plastic bags, and stored at 4°C until analysis. Soil water retention was determined corresponding to soil water matric potentials of 0, -1.2, -2.5, -5.0, -7.5, -10.0, -12.5, -25.0, and -50.0 kPa (BClute 1986). The cores were weighed at each pressure level and percent soil moisture was calculated. A linear regression model was calculated from the logarithm of pressure versus logarithm of percent soil moisture. Thirty percent of the plant cores had percent soil moisture values outside the range of matric potential pressures used to calculate 31 the standard curves (most of these cores were from Iowa). However, the standard curves fit a straight line model very closely, and therefore soil matric potentials were extrapolated beyond the range of the standard curve for these values.
Statistical analysis
Statistical analyses were conducted on four class variables: pooled plant species sampled from both regions (19 total), location of the wetland based on the township line
(latitude), vegetation zone, and Julian date of sampling (152-163 and 230-237).
Environmental variables included in the model were soil specific conductance, soil matric potential, soil pH, and available soil phosphorus. Recognizing that environmental variables, location, and season were highly correlated with each other, a principal components analysis was performed on them with VAM fungal colonization as the dependent variable. Because of the high cost of soil analyses, a stratified random subset of 120 samples (including all wetlands, both sample dates, and all vegetation zones) was used for the principal components analysis. Initially, all percentage data were transformed using the arcsine square root transformation. However, the statistical analysis of the transformed data made little difference when compared with the analysis of the original data. To prevent values in the tails of the distribution from having an inflated influence, the percentage data were not transformed.
The object of the principal components analysis is to find combinations or indices of the variables that explain the largest amount of variance. The indices or principal components are by definition independent (orthogonal) of each other (Manly 1994). The sum of the variances of the principal components is equal to the sum of the variances of the original variables. To prevent one variable from influencing the principal components, the data were 32
normalized to have means of zero and variances of one (Ivlanly 1994). Because the original
data began with a variance of one, a principal component with an eigenvalue less than one
explains less variance than the original data and was not used in further analyses. The percent
eigenvalues are the percentage of the variation described by that particular principal
component.
Multiple regression analyses were then performed on the principal components with an
eigenvalue of one or greater and plant species as independent variables and VAM fungal
colonization as the dependent variable. Poa pratemis, Spartina pectinata, and Scirpus acutus
were common to both regions and a similar regression analysis on the principal components
was also done for each plant species.
Results
Soil pH in the lowan wetlands ranged from 5.1 to 7.2, and soil specific conductance was between 22.1 and 90.3 mS m"'. Both parameters varied little between sampling periods
(Table 1). Soil pH in the North Dakotan wetlands ranged fi'om 7.3 to 7.9. Soil specific conductance ranged from 33.8 to 371.1 mS m"' and varied between sampling periods.
Consistent with greater salinity, the North Dakotan wetlands had higher concentrations of available potassium than the lowan wetlands (Table 1).
Percent organic matter ranged from 6.8 to 9.4% in Iowa compared with 1.2 to 6.3% in
North Dakota (Table I). Available phosphorus was much greater in the lowan wetlands (6.0 to 52.0ng g'^) than in North Dakota (0.1 to 5.0 jig g*'). In the lowan wetlands, phosphorus concentrations were lowest in the low prairie zone and increased downslope, with the highest Table 1. Location and soil parameters (SE) for tiie wetlands sampled in this study.
Soil Specific Organic Available Available Wetland Location Vegetation Conductance (mS m"')'' Matter Nitrate-N Phosphorus Potassium 2U)ne* Soil pH June August (%) (ng/g) (ng/g) (ng/g)
Iowa
Hamilton Co. LP 6.2 29.2 (2,8) 30.8 (2.8) 6.8 3,6 6.8 120 T86N R26W S30 NWI/4 WM 6.9 58.2 (7.1) 56.8 (8.3) 8.9 5,3 8.3 97 SEM 7.2 90.3 (27.3) 56.5(1.1) 9.4 3.9 24.5 94 Dickinson Co. LP 5.6 23.2(1.0) 25.3(1.5) 6.9 2.2 6.2 149 T99N R37WSI7NWI/4 WM 5.2 28.7(3.3) 28.6(2.5) 8.2 1.4 36.4 146 SEM 5.1 22.1 (1.5) 24.2(1.9) 9.0 2.0 52.0 173 Stoiy Co. LP 6.2 42.6(3.8) 39.1 (3.4) 8.4 2,8 6.0 185 T85N R24W S24 SEl/4 WM 6.6 37.6(1.9) 43.5(3.2) 8.1 2.1 12.5 202 SEM 6.1 28.6 (0.9) 26.0(1.0) 7.7 1.8 22.7 215
Regional Means 6.1 (0.2) 40.1 (7.3) 36.8(4.3) 8.2 (0.3) 2.8 (0.4) 19.5(5.4) 153(15) North Dakota
Stutsman Co. LP 7.4 33.8(1.3) 62.7(29.6) 3.8 2.6 2.0 121 T140N R69W S22 NWl/4 WM 7.5 35.6 (2.5) 82.5 (17.7) 3.8 3.5 1.6 188 SEM 7.5 371.1 (90.0) 182.7(33.2) 6.3 8.0 5.0 417 Stutsman Co. LP 7.4 76.8 (7.5) 114.5(15.9) 5.2 3.9 4,4 331 T142N R67W S22 NWl/4 WM 7.6 222.5(33.1) 162.2(34.9) 2.6 3.2 1,2 375 SEM 7.8 260.5 (53.8) 149.2(19.2) 2.1 5.7 3.8 610 Stutsman Co. LP 7.3 105.8(32.8) 335.4 (62.9) 4.0 2.9 2.0 336 T139NR69WS4SWI/4 WM 7.4 257.8 (36.9) 299.1 (30.3) 3.0 3.7 1.7 314 SEM 7.9 222.5(19.9) 233.2(10.7) 1.2 2.5 0.1 281
Regional Means 7.5(0.1) 176.3 (39.3) 180.2 (93.5) 3.6 (0.5) 4.0 (0.6) 2.4 (0.5) 330 (46)
* LP = Low Prairie, WM=Wel Meadow, SEM=ShaIIow Emergent. mS m ' = millisiemens per meter. 34
concentrations found in the shallow emergent zone (Table 1). Nitrate concentrations varied
little between the wetlands in Iowa and North Dakota (Table 1).
VAM colonization was observed on all the plant species sampled (Fig. 1); colonization
varied from 0.2 to 52.1% in lowan wetlands (June mean=14.8, SE=4.0; August mean=11.9,
SE=2.7) and from 1.7 to 71.8% in North Dakotan wetlands (June mean=37.0, SE=7.6;
August mean=26.0, SE=5.6) (Table 2).
O Iowa North Dakota 80 r
70
60 c o 50
"c 40 "o U 30 1 20 J
10 o 0 O I Low Prairie Wet Meadow Shallow Emergent Vegetation Zone
Figure 1. Comparison of percent VAM fungal colonization of plant species along the hydrologic gradient in lowan and North Dakotan wetlands. VAM colonization values were grouped by the vegetation zone from which their plant hosts were sampled, [empty circle], Iowa; [filled circle] North Dakota. 35
Table 2. Plant species sampled and the percent vesicular-arbuscular mycorrhizal fungal colonization measured in June and August.
Mean % VAM Plant Species Vegetation Avg. water % colonization (SE) zone* Depth (cm) June Augus Iowa Cirsium vulgare (Savi.) Tenore LP 0.5 8.3 (6.6) 24.5 (6.7) Panicum virgatum L. LP 0.0 2.2 (0.6) 9.3 (2.6) Poa pratensis L. LP 0.0 19.1 (2.4) 16.6 (2.4) Solidago canadensis L. LP 0.0 52.1 (7.8) 14.3 (3.3) Mean 0.1 (0.1) 20.4(11.1) 16.2(3.2) Calamagrostis canadensis WM 4.8 41.4 (2.0) 27.8 (3.6) (Michx.) P. Beauv. Carex lasiocarpa Ehrh. WM 5.8 3.7 (0.9) 8.5 (1.8) Carex striata Lam. WM 15.7 21.9 (6.9) 5.9 (1.2) Carex vesicaria L. WM 15.2 12.6 (3.3) 4.4 (4.4) Polygonum pesicaria L. WM 8.3 7.6 (1.2) 9.6 (4.8) Scirpus atrovirens Willd. WM 1.4 5.9 (3.4) 14.3 (3.2) Spartina pectinata Link. WM 9.1 39.3 (2.9) 39.6 (3.5) Mean 8.6 (2.0) 18.9 (6.0) 15.7 (5.0) Alisma subcordatum Raf. SEM 27.7 3.6 (1.5) 2.6 (1.2) Polygonum amphibium L. SEM 43.0 0.3 (0.2) 0.4 (0.2) Sagitaria latifolia Willd. SEM 28.7 11.3(7.6) 4.4(1.7) Scirpus acutus Muhl. SEM 41.6 0.2(0.1) 0.7(0.3) Sparganium eurycarpum Engelm. SEM 27.2 7.2 (2.7) 7.3 (1.6) Mean 33.6 (3.6) 4.5 (2.1) 3.1 (1.3)
Regional Mean 14.3 (3.7) 14.8 (4.0) 11.9(2.7) North Dakota Poa pratensis L. LP 0.3 52.3 (4.6) 40.2 (5.3) Potentilla anserina L. LP 1.4 17.3 (4.5) 18.5 (4.2) Solidago canadensis L. LP 0.4 33.1 (7.5) 7.3 (1.5) Mean 0.7 (0.4) 34.2(10.1) 22.0 (9.7) Carex atherodes Sprengel. WM 1.0 56.4 (3.2) 28.0 (3.7) Distichlis striata (Torr.) Rydb. WM 4.7 71.8 (3.9) 33.3 (6.8) Hordeum jubatum L. WM 5.7 34.2 (3.8) 20.2 (3.8) Spartina pectinata Link. WM 3.1 70.0 (2.7) 67.5 (3.8) Mean 3.6 (1.2) 58.1 (8.7) 37.3 (10.4) Puccinellia nuttalliana SEM 17.1 16.0 (4.5) 17.8 (4.7) (Schultes) AJIitchc. Scirpus acutus Muhl. SEM 17.4 9.7 (2.4) 11.8 (3.3) Scirpus maritimus L. SEM 18.7 9.6 (3.2) 15.2 (3.5) Mean 17.3 (0.5) 11.8(2.1) 14.9(1.7)
Regional Mean 6.9 (2.4) 37.0 (7.6) 26.0 (5.6) ' LP = Low Prairie, WM=Wet Meadow, SEM=Shallow Emergent. 36
Plants growing in the wettest zone, the shallow emergent zone, consistently had the lowest
VAM colonization in both regions (Fig. 1). Percent VAM fungal colonization of plants found in the other vegetation zones varied greatly, but species growing in North Dakotan wetlands generally had higher VAM fungal colonization. VAM fungal colonization was statistically higher in North Dakota for all three plant species common to both regions (Table 2).
Principal components analysis of the data from the pooled plant species resulted in a first component with a high loading of soil pH, location, season, and soil phosphorus factors.
This component explained 44% of the variance in VAM fungal colonization (Table 3). The second principal component had high loadings of vegetation zone, soil specific conductance, and soil phosphorus factors and explained an additional 19% of the variance. The third principal component was dominated by soil matric potential and explained 14% of the variance. Multiple regression analysis of the three principal components and plant species as independent variables resulted in only species and principal component I as statistically significant variables (Table 4).
Principal components analysis of the plant species common to both regions varied with individual plant species, although location and soil pH had high loadings on the first principal component of all three species (Table 3). For Poa pratensis, season was an additional important loading on principal component I. Soil specific conductance and matric potential had high loadings on principal component n. Cumulatively, these two components explained
70% of the variance in VAM fungal colonization (Table 3). Only the first principal component was statistically significant in the multiple regression analysis (Table 4).
For Spartina pectinata, the first principal component had high loadings for location. 37
Table 3. Results of principal components analysis for all plants sampled and species common to both regions, with VAM fungal colonization as the dependent variable.
Variable PC I PC H PC m All plant species Location 0.50 0.00 -0.01 Season 0.42 -0.06 -0.19 Zone 0.11 0.77 0.10 Soil conductivity 0.34 0.44 -0.02 Soil phosphonis -0.40 0.44 0.06 Soil pH 0.53 -0.04 0.00 Matric potential 0.11 -0.11 0.97 Eigenvalue 3.07 1.35 1.00 % Eigenvalue 44 19 14 Cumulative % eigenvalue 44 63 77 Common plant species Poa pratensis Location 0.51 0.03 - Season 0.45 0.36 -
Soil conductivity 0.21 -0.72 -
Soil phosphorus -0.35 0.27 -
Soil pH 0.53 -0.16 -
Matric potential 0.30 0.49 - Eigenvalue 3.02 1.20 - % Eigenvalue 50 20 -
Cumulative % eigenvalue 50 70 -
Spartina pectinata Location 0.47 0.05 -
Soil conductivity 0.25 -0.66 -
Soil phosphorus -0.47 0.07 - Soil pH 0.54 0.00 -
Matric potential 0.18 0.74 - Eigenvalue 2.96 1.08 - % Eigenvalue 49 18 -
Cumulative % eigenvalue 49 67 -
Scirpus acutus Location 0.49 -0.11 - Season 0.33 -0.58 - Soil conductivity 0.36 0.45 -
Soil phosphorus -0.48 0.09 -
Soil pH 0.49 -0.02 -
Matric potential 0.20 0.67 - Eigenvalue 4.08 1.12 - % Eigenvalue 68 19 -
Cumulative % eigenvalue 68 87 - 38
Table 4. Multiple regression analysis with VAM flingal colonization as the dependent variable for all plant species and plants common to both regions. The principal components are independent variables.
Dominant &ctors in principal Variable component F value p value
All plant species
Plant species 16.07 0.0001 PCI Location, season, 17.44 0.0001 soil phosphorus, soilpH pen Zone, soil conductivity, 0.77 0.38 soil phosphorus pcm Matric potential 0.83 0.36 Common plant species
Poa pratensis PCI Location, season, soil 46.10 0.0001 pH pen Soil conductivity, 0.54 0.47 matric potential Spartina pectinata PCI Location, soil pH, 33.12 0.0001 soil phosphorus pcn Matric potential, soil 0.03 0.87 conductivity Scirpus acutus PCI Location, soil pH, 9.64 0.01 soil phosphonis pcn Matric potential, season, 2.23 0.16 soil conductivity
The prindpal components are independent variables. F and p values from type HI sums of squares. 39 soil pH, and phosphorus. For principal component II, matric potential and soil specific conductance had high loadings. Cumulatively both components explained 67% of the variation in VAM fungal colonization (Table 3). As with Poapratensis, only the first principal component was statistically significant in the multiple regression analysis (Table 4).
Location, soil pH, and soil phosphorus had high loadings on the first principal component for Scirpus acutus. Matric potential, season, and soil specific conductance had high loading on the second principal component. Both principal components explained 87% of the variation in VAM fungal colonization (Table 3). Only principal component I was significant in the multiple regression analysis (Table 4).
A limited number of fungal species were identified from the soil of approximately 20 root cores. Those vesicular-arbuscular mycorrhizae identified were dominated by Glomus species in both regions. Glomus intraradices Schenck&Smith (a species that produces at least some of its spores inside the host root) was observed on plant species sampled in both regions. Other flingal species identified from lowan wetland soils included: Glomus aggregatum Schenck&Smith emend. Koske (sporocarps were also observed attached to some root samples). Glomus claroideum Schenck&Smith, and Gigaspora albida Schenck&Smith.
Glomus claroideum. Glomus aggregatum, and Glomus manihotis Schenck&Smith were identified in wetland soils firom North Dakota.
Discussion
The colonization levels reported in this study are based on the presence of either vesicles, mycelium, or spores observed at 40x magnification; hyphal coils and arbuscules were 40 rarely observed. The results indicated that all of the wetland species examined were colonized by VAM fiingi and that the amount of VAM fungal colonization in a prairie wetland varies significantly among plant species (Fig. 1). Members of the Poaceae generally had the highest levels of colonization and species in the Cyperaceae and Juncaceae had lower rates of colonization; this trend is consistent with previous reports. However, colonization levels were generally higher on the plants sampled in this study. For example, Carex stricta was reported as having no colonization by Anderson et al. (1984) compared with 6 - 22% in this study
(Table 2). The technique used by Anderson et al. to determine colonization was not reported, and it is possible that differences in measurement technique may be the reason for the discrepancy in VAM colonization levels between the two studies. Rickerl et al. (1994), using the same measurement technique as we did to determine colonization, reported no colonization for Carex atherodes. However, we observed between 28 - 56% colonization on
Carex atherodes in a North Dakotan wetland (Table 2). Similar colonization levels in
Polygonum amphibium were observed in this study as were reported by Rickerl et al. (1994).
Spores of five mycorrhizal fungi were identified in the limited survey done for this study: three of these species occurred in both lowan and North Dakotan wetlands. Gigaspora albida spores were found only in lowan wetlands. Anderson et al. (1984) also found fungal spores belonging to the genus Gigaspora at wet sites in Illinois. Spores of Glomus manihotis were found only in North Dakotan wetlands. This species was not previously recorded in wetlands. However, other species of Glomus have been found in wetlands (Khan 1993;
Stenlund and Charvat 1994). The significance of different fungal floras in different wetlands is unknown and needs further study. 41
As hypothesized, VAM fungal colonization was greater on plants growing in wetlands with higher salinities and lower nutrient levels; i.e. it was higher in North Dakotan than in lowan wetlands (Table 2). Environmental conditions were markedly different between Iowa and North Dakotan wetlands; soil phosphorus was higher in Iowa, whereas soil pH and soil specific conductance were higher in North Dakota. Whether the higher VAM colonization in
North Dakota was primarily due to soil pH, phosphorus levels, soil specific conductance, or a combination of these factors is impossible to establish from these data. Micro- or meso-scale variations of these parameters in the soil may also influence the conclusions of the statistical analysis. The lack of saline wetlands in Iowa or identical plant and fungal populations between the two regions limits the conclusions that can be made from this type of a field study.
Further research must be done under controlled experimental conditions to determine the relative importance of these environmental factors. It is also possible that other environmental factors not measured in this study may have been important, for example, the length of growing season or differences in soil micronutrients (Harley and Smith 1983; Menge et al.
1982; Young and Jarrell 1985). Different fungal species may also contribute to the different levels of mycorrhizal colonization observed between Iowa and North Dakota.
Soil moisture, and the resulting reduction-oxidation levels, is the single most important environmental gradient in a prairie pothole wetland and has a principal role in determining wetland vegetation composition and zonation. The hypothesis that VAM fungal colonization would be higher in drier zones or during seasonal drawdowns was not supported by the data.
The principal component with soil matric potential as the highest loading factor was not significant in the multiple regressions. This was also true with the principal component for 42
which the highest loading was vegetation zone, a long-term measure of soil moisture.
Previously, researchers found a negative correlation between mycorrhizal colonization or
spores and soil moisture in non-arid regions (Anderson et al. 1984; Khalil and Loynachan
1994; Rickerl et al. 1994). As tne results of the present study were undoubtedly influenced by
the wet growing season in 1993, the importance of soil moisture may be underestimated for
wetland plants. However, the fact that VAM fungal colonization was present in wetland
vegetation during a wet year also suggests that soil moisture may not be an important factor
regulating VAM fungal colonization. The exact function of mycorrhizae during seasonal
fluctuations in soil moisture and during periods of prolonged drought in prairie wetlands needs further study.
Many of the early season grasses and sedges (Poa pratensis, Calamagrostis canadensis, Distichlis stricta, Hordum jubatum, Carex stricta, Carex \esicaria, and Carex atherodes) had a higher level of VAM fungal colonization in June than in August (Table 2). A
pattern of increasing VAM fungal colonization of prairie grasses during the growing season, with a decline after plant fiuiting, was observed by Hetrick et al. (1989). Carex lasiocarpa and Puccinellia nuttalliana (early season) were exceptions to this trend. In this study,
Panicum virgatum, a warm season grass, had an increase in mycorrhizal colonization between the two seasons, while mycorrhizal colonization remained constant between sample periods for Spartina pectinata (warm season grass) and many broad leafed plants. Seasonal variation of VAM fungal colonization was also found to reflect temporary changes in available phosphorus or percent soil moisture (Rabatin 1979). Because little change in seasonal soil 43 moisture occurred during this study, seasonal changes in VAM fungal colonization seem to reflect changes in the phenology of the the plant species.
In summary, the percentage of VAM fungal colonization varied significantly with host plant species in prairie pothole wetland ecosystems. VAM fungal colonization also was influenced by location, reflecting differences in soil nutrients and soil specific conductance in
Iowa and North Dakota. The lack of saline wetlands in Iowa or identical plant and fungal populations between the two regions limits the conclusions that can be made from this field study. Further research must be done under controlled experimental conditions to determine which environmental factors influence VAM fungal colonization and whether mycorrhizae have a positive benefit on plant growth and survival. Mycorrhizal colonization may also be affected by environmental gradients not measured in this study, as well as by biotic factors such as plant competition or different fungal populations found in the two regions.
Acknowiedgments
We thank Todd Cheever for help in washing roots and measuring soil parameters and
Paul Hinz and Eric Seabloom for helpful statistical discussions. This research was flmded by
Wetlands Research Incorporated and by a Grant-in-Aid of Research from the National
Academy of Sciences, through Sigma Xi, The Research Society.
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CHAPTER 3. THE EFFECTS OF ARBUSCULAR MYCORRHIZAL AND NON-MYCORRHIZAL FUNGI ON WETLAND PLANTS DURING DIFFERENT REDOX POTENTIALS AND HYDROPERIODS
A paper submitted to Canadian Journal of Botany
Paul R. Wetzel and Arnold G. van der Valk
Abstract
We believe that the effect of arbuscular mycorrhizal fungi (AMF) on the growth of a host plant falls along a continuum from beneficial symbiont, when environmental conditions are suboptimal for plant growth, to pathogenic parasite, when environmental conditions are optimal. To test this model, we hypothesized that AMF would increase plant biomass and foliar nutrient uptake of Carex stricta and Calamagrostis canadensis, two wet meadow prairie wetland species, when soils are drier and have higher redox potentials. Soil moisture treatments consisted of continuously flooded (cumulative redox potential -6817 mV); 32 day cycle (16 days flooded-16 days dry, -3909 mV); 6 day cycle (3 days flooded-3 days dry, 1555 mV); saturated, well drained (0.5 1/day, 2053 mV); and low soil moisture (0.2 1/day, 2026 mV). Benomyl fungicide was applied monthly to non-AMF treatments to reduce fungal contamination. Biomass of non-inoculated C. stricta was 47% greater than that of inoculated plants in the 6 day cycle, otherwise biomass was not significantly different between inoculated and non-inoculated plants. Non-inoculated C canadensis had 29 to 41% more biomass than inoculated plants in the flooded, 6 day cycle and the saturated, well-drained hydrologic regimes. Our results suggested that AMF were pathogenic parasites in these treatments. 48
However, analysis of entire root samples determined that total root length colonized by AMF was never greater than 1%. The lack of AMF colonization was due to high soil phosphorus levels in this study. High phosphorus levels prevented AMF colonization even in the hydrologic regimes with the lowest soil moisture (32 day cycle and 0.21/day), indicating that
AMF are not important to C canadensis and C. striata in the low soil moisture environments that occur in prairie wetlands with high nutrient levels.
Application of benomyl fungicide to non-inoculated AMF plants reduced or eliminated parasitic non-mycorrhizal fiingi and increased plant biomass. Non-mycorrhizal fungi that grew on agar plates from root samples of non-mycorrhizal treatments were identified as Fusarium spp., Aspergillus niger and Helminthosporium spp., and Altemaria spp. Non-mycorrhizal fiingi had less impact on the biomass of C. striata, suggesting that C. striata has antifungal properties and these properties may be the reason members of the Cyperaceae have been considered non-mycorrhizal or facultatively mycorrhizal.
Introduction
The effect of arbuscular mycorrhizal fungi (AMF) on a host plant can range from positive to negative depending on the plant's environment. AMF have been found to cause a carbon drain on the host plant under high soil phosphorus conditions (Crush, 1973; Hayman,
1983; Koide, 1985), while the nutrient-harvesting capabilities of AMF can provide a net benefit to the plant under low phosphorus conditions (Allen, 1991; Harley and Smith, 1983).
The addition of phosphorus to tallgrass prairie plants eliminated the benefit of AMF on biomass production under intra- and interspecific competition (Hetrick et al., 1986). A similar 49
pattern, although not as well studied, emerges during drought experiments in the field (Allen
and Allen, 1986). Hayman (1983) called the change of the mycorrhizal relationship from a
mutualistic symbiosis to pathogenic parasitism a 'symbiotic imbalance'. The results of
experiments by Allen and Allen (1986) suggest that mycorrhizae are primarily important during an 'ecological crunch'. These patterns indicate that the mycorrhizal symbiosis be considered as a continuum, controlled in part by environmental factors (Figure 1). A
Mycorrhizal Effect on q Plant Growth
t
Low ffigh Environmental Factor
Figure 1. Mycorrhizal continuum model. The benefit a plant receives from arbuscular mycorrhizal fiingi ranges from positive (symbiotic) to negative (parasitic pathogen) depending on the environmental conditions. 50
The dynamic annual and interannual hydrology of prairie wetlands make them an ideal system for applying the mycorrhizal continuum model. Prairie wetlands experience a pulsed hydrology with varying wet-dry cycles during the growing season. When the soil is flooded and highly reduced (low redox potential), pH will decrease and inorganic phosphorus bound to iron will become more soluble. Phosphorus becomes less mobile as redox potential increases and soil moisture decreases. It is in these dynamic systems that AMF could go from being parasitic pathogens during wet periods with low redox potentials to beneficial symbionts during dry periods with high redox potentials.
Evidence for this pattern is suggested in the study of AMF colonization of prairie wetland plants. Total AMF colonization of prairie wetland plants in Iowa generally decreased with increasing water depth (Wetzel and van der Valk, 1996). Several wetland plant species have been found to have functional AMF (presence of arbuscules) in lower-nutrient, drier sites, while the same plant species growing in wet, higher nutrient envbonments are colonized with AMF, but lack arbuscules (Anderson et al., 1984). Total AMF colonization of four wetland plants was greater on individuals in a dry environment compared to a wet one
(Rickerl et al., 1994). Arbuscules were found on two of these species, but were not correlated with higher shoot phosphorus concentrations. No one has investigated the effect of
AMF on plant growth in low redox potential environments, and it is not clear whether AMF affect the growth of plants during the pulsed wet-dry hydrologic cycles characteristic of prauie wetlands.
This experiment tested whether the effect of AMF on plant growth depends on water regimes as predicted by the mycorrhizal continuum model, for two common wet meadow 51
This experiment tested whether the efifect of AMF on plant growth depends on water
regimes as predicted by the mycorrhizal continuum model, for two common wet meadow
wetland species, Carex stricta and Calamagrostis canadensis. We hypothesized that AMF
will improve plant biomass and foliar nutrient uptake in drier, high redox potential
environments and reduce it in flooded hydrologic regimes (low redox potential) when the
AMF are believed to act as parasitic pathogens.
Methods
Experimented design
Two common sedge meadow plants, Calamagrostis canadensis and Carex stricta, were grown in five different soil moisture treatments, with and without arbuscular mycorrhizal fimgal (AMF) inoculation. The soil moisture treatments were: constantly flooded; 32 day wet/dry cycle (flooded 16 days, dry 16 days); a 6 day wet/dry cycle (flooded 3 days, dry 3 days); saturated, well-drained (0.5 I water/d); and dry (0.21 water/d). Lengths of flooded periods were chosen to minuc prairie wetland hydrologies (Kantrud et al., 1989). During the flooded periods of each treatment, the water was 5 cm deep.
Plants were grown in a 60:40 mixture of sand:wetland soil, to which granular phosphorus fertilizer (0-45-0) was added at the beginning of the experiment to mimic natural wetland phosphorus levels (Wetzel and van der Valk, 1996). This soil mixture contained organic matter 1.6%; available phosphorus 17 |ig/g; potassium 86 |ig/g; nitrate 14 (ig/g (Iowa
State University Soil Testing Laboratory). The wetland soil used in this study, Webster clay loam, was obtained from a pothole in Story County, Iowa. It was necessary to add sand to 52 the wetland soil to facilitate root cleaning at the end of the experiment. The soil mixtures were steamed for 2 hours.
Seeds of both species were washed with a 0.5% NaOCl solution and rinsed three times with distilled water. The seeds were spread into petri dishes containing a paper filter disk.
The seeds were stratified at 4°C for 48 hours and germinated in a greenhouse. Seedlings were transplanted to community trays filled with sterilized potting soil (peat moss and perlite) when they were two centimeters tall.
After three weeks, seedlings were transplanted to deep trays (32 x 28.5 x 17.8 cm) filled with 0.013 m^ of steam sterilized soil (13.6 kg dry soil of the 60:40 mixture). Trays were lined with 50:50 cotton:polyester fabric to prevent soil loss and root growth through the drainage holes. Four plants were planted in a cross in each tray. All seedlings survived and no replacements were necessary. The plants grew for seven weeks in the trays before the water treatments began. Greenhouse temperatures ranged fi"om 18 to 30°C and plants were grown under high intensity lights (average sunny day photosynthetically active radiation at tray height of420 ^mol/m^/s) with a 14 hour photoperiod. A randomized block design was used, with each treatment replicated five times. The overall experimental design contained five hydroperiods x two mycorrhizal treatments (+/- AMF) x two plant species (C. striata and
C. canadensis), x five replicates for a total of 100 trays.
Treatments with arbuscular mycorrhizal fungi (AMF) were inoculated with spores of
Glomus intraradices (Schenck & Smith), Glomus aggregatum (Schneck & Smith emend.
Koske), and Glomus claroideum (Schenck & Smith), three species found in prairie pothole wetlands in Iowa (Wetzel and van der Valk, 1996). Spores were recovered with wet sieving 53 and sucrose centrifugation (Daniels and Skipper, 1982) irom Andropogon geradii (Vitman),
Schizachyrium scoparium (Michx.) Nash, and Trifolium repense (L) growing in pots in the greenhouse. Spores of each fungal species were suspended in water and approximately 170 spores of G. claroidivm, 140 spores of G. aggregattm, and 600 spores of G. intraradices were pipeted into a small depression in the soil immediately prior to planting each seedling. A total of approximately 3600 fungal spores were added to each tray in the plus AMF treatments. Benomyl fungicide was applied to non-mycorrhizal treatments every four weeks
(50 mg/kg dry weight of soil) to reduce potential AMF contamination.
Measurement of redox potential
Redox potential was measured using platinum wire soldered directly to 12-gauge copper wire following the construction instructions of Faulkner et al. (1989). Probes were permanently installed to a depth of 6 cm in fifteen trays. Redox measurements were made in all five hydroperiods of three randomly selected replicates. Five probes were placed in the selected replicates in a stratified random manner to include all hydrologic regimes. Probes were calibrated with a ferrous-ferric reference solution following ASTM Committee D-19 on
Water (1988). Measurements were taken with a portable pH meter set on millivolt and referenced with a calomel probe. Redox measurements were initially made once a week and then every two days during the last four weeks of the experiment. The cumulative redox potential (the addition of all redox measurements in each hydrologic regime for the last 30 days of the experiment) was calculated for each treatment to characterize its overall redox condition. 54
Data collection
Eleven weeks from the start of water treatments (when plants were a total of 20 weeks old), the four plants in each tray were harvested. Above- and below-ground biomass samples were oven-dried for 3-5 days at 65°C to minimize nitrogen loss (Chapin and Van Cleve, 1989) and then weighed. Above-ground biomass of all four plants in a tray was ground with a Wiley mill to a 0.5-mm (#40) mesh size. Percent foliar total nitrogen and carbon were determined on each treatment replicate by combustion with a Carlo-Erba NA 1500 Carbon-Nitrogen-
Sulfiir analyzer. Total foliar phosphorus was measured with a sodium hypobromite oxidation extraction and colorimetric determination with a heteropoly blue method (Dick and Tabatabai,
1982).
Percent AMF colonization was measured to establish the extent of colonization in each treatment. After drying, an arbitrary sample of secondary roots with attached tertiary roots from each plant was stained to detect AMF, following Koske and Gemma (1989). The roots were autoclaved in a 2.5% KOH solution for 3 minutes and then bleached by soaking 10-30 minutes in a 1% hydrogen peroxide solution. After rinsing with water, the roots were autoclaved in 0.05% acid fiichsin in acidic glycerol solution for 10 minutes and then destained at room temperature. Percent AMF colonization was determined following McGonigle et al.
(1990). AMF colonization counts were done with 200x magnification and the presence or absence of colonization was recorded for the first 100 root segments spread on a glass slide.
Colonization also was determined on the roots of entire plants. The root samples were initially scanned at 40x magnification and once colonization was found confirmed at 200x magnification. Total area occupied by colonized roots was recorded. 55
Approxiniately 0.5 g of dry root samples of the with and without AMF treatments of each hydrologic regime were placed on sterile 2% agar plates. The plates were stored at room temperature. After two weeks, non-mycorrhizal fiingi genera were identified.
Lacunae were measured in selected, robust green stems approximately 30 cm tall. The first 5 cm of the stem fi-om soil level was blotted dry and weighed, then cut on an angle into 2-
4 mm pieces and placed into 0.05% Tween 20 solution. Cut stem pieces were placed in a vacuum chamber evacuated to -186 kPa for 10 minutes. The cut stem pieces were then blotted dry and reweighed. The difference between initial and final weights was used as a measure of lacunae.
Analysis of variance was performed on above- and below-ground biomass, total foliar phosphorus, foliar nitrogen, and lacunae for each plant species. All proportional data were arcsine square root transformed to reduce heteroscedasticity and improve normality.
Results
Initial analysis of root subsamples fi^om all treatments indicated no AMF colonization.
Consequently, roots of entire plants were stained and sporadic AMF colonization (mycelium, vesicles, and spores) was found in the treatments with AMF inoculation, but total percent of root length colonized was never greater than 1%. Non-mycorrhizal fungi were identified as predominately Fusarium spp., including Fusarium moniliforme. Aspergillus niger,
Helminthosporium spp., and Altemaria spp. were also identified.
Mean redox potential ranged fi"om a high of -247 to a low of -349 mV for the constantly flooded treatment to a high/low of 208 / 7 mV for the dry treatment (Table 1). 56
Cumulative redox potentials ranged from -6817 to 2053 mV and increased as time of flooding decreased, with the saturated, well-drained and dry treatments having nearly the same cumulative redox potential (Table 1). It was diflBcult to separate the effects of low redox potential from low soil moisture, but comparison of the constantly flooded and saturated, well-drained hydrologic regimes indicated that redox potential was not an important factor for
Table 1. Average daily water amount, high/low redox range, and cumulative redox potentials measured for each hydrologic regime.
Average Redox Potential Range (mV) Cumulative Daily Water Last 30 days of Experiment Redox Hydrologic Regime (1/day) Average High/Low Potential (mV)
32 day cycle (16 day dry/wet) 0.1 192/-417 -3909 Dry 0.2 208/7 2026 Saturated Well Drained 0.5 155 /19 2053 6 day cycle (3 day dry/wet) 0.7 240 / -244 1555 Continuously flooded 2.0 -247/-349 -6817
the above- and below-ground biomass of either plant. There was no statistical difference between the two hydrologic regimes for all the parameters measured on C striata. Only below-ground biomass and nitrogen were slightly lower for C. canadensis in the constantly flooded hydrologic regime. Periodic flooding experienced in the 6 day cycle also had little effect on the parameters measured when compared to the saturated, well-drained hydroperiod.
None of the biomass or nutrient parameters except C. stricta below-ground biomass were statistically different between these hydrologic regimes.
The data for both plant species produced two general patterns. First, except for lacunae, if there was a statistical difference between the inoculated and non-inoculated plants then the non-inoculated plant had a higher biomass or nutrient level, regardless of hydrologic
regime or cumulative redox potential (Figures 2 and 3). Lacunae had the opposite pattern
(Figure 2). Second, biomass and nutrient acquisition were statistically greater in the
constantly flooded, 6 day cycle and the saturated, well-drained hydrologic regimes than in the
32 day cycle and dry hydrologic regimes (Figures 2 and 3).
Above- and below-ground biomass for C. canadensis were lowest in the dry and 32
day cycle hydrologic regimes and were greatest in the constantly flooded, 6 day cycle and
saturated, well-drained hydrologic regimes (Figure 2). There was no statistical difference
between the biomass of plants without AMF (with fungicide) and plants with AMF (without fimgicide) in the dry and 32 day cycle treatments. For plants in the other hydrologic regimes,
biomass of plants without AMF was 26 to 45% greater in these treatments than biomass of
plants with AMF (Table 2, Figure 2). C. striata above- and below-ground biomass was
reduced 52 and 44% respectively, only during the 6 day cycle. Above-ground biomass
remained fairly constant across all other hydrologic regimes, while below-ground biomass followed a pattern similar to that of C. canadensis (Table 2).
The above-ground:beIow-ground biomass ratio for C. canadensis was greater than
one for the constantly flooded, 6 day cycle, saturated, well-drained, and 32 day cycle
hydrologic regimes. However, more root biomass was produced than above-ground biomass in the dry treatment. The biomass ratio remained below one for C. stricta and was fairly constant across all hydrologic regimes (Figure 2). 58
Calamagrostis canadensis Carex striata
y"
5 — J
J I J L
J L J L
J I I L J I I 1 L
WithAMF Without AMF
& J I I L 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 Hydrologic Regime (I water/day) Hydrologic Regime (1 water/day)
Figure 2. Mean biomass and stem lacunae levels (±SEM) at each hydrologic regime. Open symbols, dotted line=without arbuscular mycorrhizal fungi, closed symbols, solid line= with arbuscular mycorrhizal fimgi. 59
Calamagrostis canadensis Carex striata
r I
J I 1 L J I I I L
J I I I L With AMF Without AMF
tf £>,• 6. o X I j_ I 0.0 0.5 l.O 1.5 2.0 0.0 0.5 1.0 1.5 2.0 HydroJogic Regime (I water/day) Hydrologic Regime (I water/day)
Figure 3. Mean foliar phosphorus, nitrogen, and carbonrnitrogen ratios (±SEM) at each hydrologic regime. Open symbols, dotted line=without arbuscular mycorrhizal fungi, closed symbols, solid line= with arbuscular mycorrhizal fungi. Table 2. Percent change in biomass and nutrients of plants with AMF (without fungicide) compared to plants without AMF (with fungicide) of statistically significant differences.
Average Daily Above-ground Below-ground Foliar Foliar Stem Plant Species Water (1/day) Biomass Biomass Phosphorus Nitrogen Lacunae
Calamagrostis 0.1 ns ns -20 -43 +48 canadensis 0.2 ns ns ns -39 ns 0.5 -45 -37 +33 -23 ns 0.7 -31 -26 ns ns 55 2.0 -34 -27 ns ns ns
Carex stricta 0,1 ns ns ns -23 ns 0.2 ns ns ns -29 ns 0.5 ns ns ns -27 ns 0.7 -52 -44 ns ns +40 2.0 ns ns ns ns ns 61
Concentrations of foliar phosphorus were not statistically different between the with and without AMF treatments for either plant species (Table 3). Between hydrologic regimes, phosphorus levels were highest in the 32 day cycle for both C. canadensis (p<0.0004) and C. striata (p<0.0001). Phosphorus concentrations at all other treatments and between plants were not statistically different (Figure 2).
Foliar nitrogen of plants without AMF (with fungicide) was between 23-43% greater in C. canadensis and 23-29% greater in C. stricta than in plants with AMF (without fungicide) growing in the 32 day cycle, saturated, well-drained, and dry hydrologic regimes
(Table 2). Treatments with AMF produced a greater foliar carboninitrogen ratio for both plants across all hydrologic regimes (Figure 2). Foliar carbon remained constant across all treatments, and changes in the ratio resulted from changes in the foliar nitrogen. This pattern indicated a greater efiBciency of cell building per nitrogen molecule available when AMF and non-mycorrhizal fimgi were present, but less total nitrogen uptake than plants without fungi. Table 3. Analysis of variance for both plant species tested. AMF=Arbuscular Mycorrhizal Fungi.
Above-ground Below-ground Foliar Foliar Plant Species Source of Variation Biomass Biomass Phosphorus Nitrogen Lacunae df p = p = P = P = P =
Calamagrostis Block 4 0.000 0.000 0.113 0.040 0.693 canadensis Hydropcriod 4 0.000 0.000 0.000 0.000 0.002 AMF/Fungicide 1 0.000 0.000 0.978 0.000 0.012 Hydroperiod x AMF 4 0.001 0.024 0.003 0.003 0.160 Error 49
Carex stricta Block 4 0.041 0.000 0.007 0.001 0.440 Hydropcriod 4 0.002 0.000 0.000 0.000 0.164 AMF/Fungicide 1 0.000 0.010 0.214 0.000 0.264 Hydroperiod x AMF 4 0.003 0.015 0.964 0.446 0.382 Error 49 63
Discussion
It was hypothesized that AMF would have a positive efifect on plant growth in hydrologic regimes with low soil moisture and a negative effect on plant growth in hydrologic regimes with high soil moisture. The overall pattern of greater biomass and nutrients in treatments without AMF and with greater soil moisture (continuously flooded, 6 day cycle, and saturated, well-drained) suggests that AMF were pathogenic parasites during these conditions. Both Crush (1973) and Koide (1985) found AMF to be pathogenic parasites in soils with 8 |ig/g and 15 jig/g of phosphorus, respectively, less than the 17 |ig/g of phosphorus in the soil used in this experiment. Prairie wetland soils in Iowa range from 6.0-
52.0 ng/g phosphorus, and C. striata and C. canadensis have been found with AMF colonization of 6-22% and 28-42%, respectively, in prairie wetlands in Iowa (Wetzel and van der Valk, 1996).
However, AMF colonization is controlled by the concentration of phosphorus within the plant root and not the soil (Menge et al., 1978). Phosphorus deficiency in the roots causes the roots to release organic carbon into the rhizosphere, and it is believed that AMF respond to this release of root exudates (Marschner, 1992). Thus, high levels of phosphorus can prevent AMF colonization of plants (Harley and Smith, 1983). The high levels of soil and foliar phosphorus in this experiment and the sporadic and negligible AMF root colonization indicate that the phosphorus concentration in the plant roots was high enough to prevent mycorrhizal colonization in our treatments with AMF. 64
The lack of mycorrhizal colonization on the plant roots implied that the statistically
significant increase in above- and below-ground biomass and nitrogen in the high soil moisture
hydrologic regimes without AMF was caused by the application of benomyl fungicide and consequent reduction or elimination of non-mycorrhizal fungi. Several species of Fusarium,
Aspergillus, and Helminthosporium are known to cause plant vascular wilts, root and stem
rots, and rotting of tubers of various crop plants (Agrios, 1988). Wilts and rots were not
observed in this experiment, but have been found in other studies of aquatic plants (Andrews and Hecht, 1981; Tanaka etal., 1993). The impacts of ho\h Fusarium spp. and Aspergillus spp. are proportional to the amount of soil moisture and are greatest near the saturation point
(Agrios, 1988). Non-mycorrhizal fiingi had the largest impact on the above- and below- ground biomass of C. canadensis in the continuously flooded, 6 day cycle, and saturated, well-drained hydrologic regimes (Figure 2). In the benomyl treatments, increased foliar
phosphorus (32 day cycle) and nitrogen were found in the 32 day cycle and dry hydrologic
regimes, indicating that the plants were concentrating nutrients under these conditions. For C. canadensis, non-benomyl treatments in the wetter hydrologic treatments caused the same decrease in above- and below-ground biomass as the extended periods of low soil moisture experienced in the 32 day cycle and dry hydrologic regimes (Figure 2). Interestingly, foliar
phosphorus of C canadensis with AMF (without fungicide) increased 33% above that of
plants without AMF in the saturated, well-drained hydrologic regime, while nitrogen
decreased 23% (Table 2). Non-mycorrhizal fiingi significantly increased shoot phosphorus concentrations in two alpine Carex species, Carex firma and Carex sempervirens 65
(Haselvandter and Read, 1982), but no increase of foliar phosphorus was observed in C stricta in this study (Table 2).
The benomyl treatments had little impact on the above- and below-ground biomass of
C. stricta, which were not statistically different in any of the hydroperiods except the 6 day cycle (Fig^re 2). These results suggest that C. stricta has some antifungal properties and may be the reason members of the Cyperaceae have been considered non-mycorrhizal (Brundrett,
1991; Harley and Smith, 1983) or facultatively mycorrhizal under certain environmental conditions (Read and Haselwandter, 1981). AMF have been found to increase plant defenses against pathogenic parasites, including Fusarium spp. (Benhamou et ai, 1994). The lack of
AMF colonization on C. stricta roots suggests that this species has evolved an antifungal defense system adapted for water logged soil conditions. Other wetland plants have antifimgal properties, for example, Lotus (Nelumbo nucifera) rhizomes had potent antifungal activity on numerous fungi, including Aspergillus niger (Matthews and Haas, 1993).
Although C. canadensis and C. stricta naturally grow together along the same portion of the soil moisture gradient in wetlands, they responded differently to the hydrologic regimes and the elimination of non-mycorrhizal fungi. C. canadensis produced more above-ground biomass than beiow-ground biomass in all hydrologic regimes with flooding. The reduction or elimination of flingi also increased the above-ground:below-ground biomass ratio in every hydrologic regime (Figure 2). C. stricta put more resources into root development, and the amount of resources put into roots remained consistent across all hydrologic regimes.
Patterns of above- and below-ground biomass for both plants across the hydrologic gradient (Figure 2) did not match the predicted model relationship on plant growth along the 66 environmental continuum (Figure 1). High phosphorus levels in the plant roots, and for C. stricia, possible anti-fongal properties, prevented AMF colonization even in the hydrologic regimes with the lowest soil moisture (32 day cycle and dry). High phosphorus levels also prevented AMF colonization and any consequent negative (parasitic pathogenic) effect on plant growth. The results from this study suggest that soil phosphorus has an interactive effect on other environmental factors. The predicted model relationship on plant growth diagrammed in Figure 1 is expected to occur in low phosphorus environments that allow the establishment of AMF. In a surplus phosphorus environment, AMF do not become established because they are unable to colonize plant hosts. Therefore, the predicted model relationship on plant growth in a surplus phosphorus environment is a straight line (Figure 4).
20 ^ Car ex striata w 15 • Calamagrostis canadensis Low Phosphorus Model CO CQ 10 * " Surplus Phosphorus Model a o 5 (5 0
O -5 E- -10 co o -15 -20 -25 0.0 0.5 1.0 1.5 2.0 Soil Moisture (l*day'^)
Figure 4. Revised mycorrhizal continuum model that includes the interaction of soil phosphorus level over a soil moisture gradient. The effect on total biomass was the difference in biomass between plants grown with AMF and plants grown without. The predictive models were adjusted to the experimental soil moisture levels. 67
Total biomass for both species with AMF plotted across the experimental hydrologic regimes produced straight lines (Figure 4). Neither slope was statistically different from zero
(p=0.5) and these results matched the surplus phosphorus environment model. Removing the soil phosphorus interaction from the original mycorrhizal continuum model clearly indicates that AMF do not affect plant growth along a soil moisture gradient in a surplus phosphorus environment. This finding suggests that AMF are not important to C canadensis and C. stricta in low soil moisture environments produced by hydrologic regimes similar to those observed in prairie wetlands.
In summary, AMF did not find adequate host sites on C. canadensis or C stricta in a wide range of hydrologic regimes. Lack of colonization may have resulted from high root and soil phosphorus levels, or in the case of C. stricta, a strong antifungal defense. AMF were not important to C. canadensis and C. stricta in low soil moisture environments produced by hydrologic regimes similar to those observed in prairie wetlands. The reduction or elimination of non-mycorrhizal fixngi naturally found in the experimental trays significantly reduced the above- and below-ground biomass and nitrogen uptake of C. canadensis in hydroperiods that produced high soil moisture. Non-mycorrhizal fiingi only significantly reduced the biomass of
C. stricta in one hydroperiod, the 6 day cycle. These results indicated that C. stricta normally has a strong antifungal defense and that fungi play a significant role in wetland plant development and perhaps competition and reproduction during certain environmental conditions. 68
Acknowledgements
We thank Marilynn Bartels for help harvesting plants and measuring foliar phosphorus and Lois Tiffany for identifying non-mycorrhizal fungi. This research was funded by Wetlands
Research Inc. and by a Grant-in-Aid of Research from the National Academy of Sciences, through Sigma Xi, The Research Society.
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Tanaka, T. H.K. Abbas, and S.O. Duke. 1993. Structure-dependent phytotoxicity of fiimonisins and related compounds in a duckweed bioassay. Phytochem. 33:779-785.
Wetzel, P.R. and A.G. van der Valk. 1996. Vesicular-arbuscular mycorrhizae in prairie pothole wetland vegetation in Iowa and North Dakota. Can. J. Bot. 74:883-890. 71
CHAPTER 4. MYCORRHIZAE AND THE COMPETITIVE ABILITY OF THE FACULTATIVE MYCOTROPH CAREX STRICTA IN DIFFERENT ENVIRONMENTAL CONDITIONS
A paper submitted to Vegetatio
Paul R. Wetzel and Arnold G. van der Valk
Abstract
We hypothesized that arbuscular mycorrhizal fiingi (AMF) would affect the interspecific competitive ability of the facultative mycotroph Carex striata when it is growing in low nutrient or low soil moisture levels. Specifically, we predicted that AMF under these conditions would increase the relative yield, productivity, and foliar macronutrient concentration of C. striata when grown in a factoral experiment with high-low nutrient levels, high-low soil moisture levels, with and without AMF inoculation, and with or without either
Typha latifolia or Phalaris arundinacea. AMF did not increase the competitive ability of C. striata under any of the environmental conditions tested. Nor did AMF improve the productivity or nutrient uptake of C. striata. Nutrient levels and interspecific competition explained 35% of the total variation in C. striata productivity and foliar macronutrient concentration, compared to soil moisture (7%) and AMF (0%). None of the total variation in
P. arundinaaea productivity and foliar macronutrient concentration was explained by interspecfic competition. The morphological characteristics of P. arundinaaea—growth rate, tall leafy shoots, and extensive canopy—enabled it to exert a dominating competitive effect on C. striata. The physiological status of the host plant depended on nutrient and 72 competition levels and affected AMF colonization. Total root colonization on C striata and
T latifolia showed the greatest decline in a low nutrient environment or with competition from P. arundinacea.
Introduction
Experimental evidence suggests that arbuscular mycorrhizal fungi (AMF) can afifea competition between plant species that respond differently to mycorrhizal colonization. AMF increased the competitive ability of an obligate mycorrhizal (C4 warm season) prairie grass when grown with a co-occurring facultative mycorrhizal (C3 cool season) grass (Hartnett et al., 1993; Hetrick et al., 1989). In these studies, a high level of phosphorus substituted fimctionally for mycorrhizae in the obligate mycotrophs. According to a model described by
Allen (1991), AMF mcrease the survival of facultative mycotrophs in non-productive conditions. Mycorrhizae have been found to increase the competitive ability of facultative mycotrophs during early stages of succession and cause shifts in plant communities when nutrient levels are low (Allen and Allen, 1990; Grime et al., 1987).
Members of the Cyperaceae generally have been considered nonmycorrhizal or rarely mycorrhizal (Anderson etal., 1984; Gerdemann, 1968; Powell, 1975), but Carex spp. have been found to be colonized with AMF in grasslands (Davidson and Christensen, 1977) and in alpine ecosystems (Allen et al., 1987; Lesica and Antibus, 1985; Read and Haselwandter,
1981). Colonization of Carex spp. may be related to poor soil conditions in alpine ecosystems, suggesting that Carex spp. actually are facultatively mycorrhizal. Carex striata was chosen for this study because it is a common sedge meadow plant that has 6-22% AMF 73 colonization in natural prairie wetlands (Wetzel and van der Valk, 1996). Because of inter- annual and seasonal fluctuations in water levels and potential resource depletion from other competing plants, we hypothesized that AMF could be an important factor influencing the competitive ability of C. stricta.
Within native and restored sedge meadow wetland communities, severe competition from the weedy species Phalaris arundinacea and Typha spp. can result in the displacement of characteristic wet meadow species such as Carex stricta (Galatowitsch and van der Valk,
1995; Reinartz and Wame, 1993). Both P. arundinacea and T. latifolia have growth forms that maximize the capture of available resources in productive environments and are considered to be competitively selected (C-selected) plants, sensu Grime (1979). C. stricta is moderately productive and can tolerate low intensity stress and is classified as a stress tolerant competitor (Grime, 1979).
The first objective of this study was to determine if AMF would improve the competitive ability of the facuhative mycotroph C. stricta in low nutrient and low soil moisture levels when competing with T. latifolia and P. arundinacea. It was hypothesized that AMF, by itself or in interaction with an environmental factor, would alter the relative competitive ability, as measured by relative yield, productivity, and macronutrient uptake of C. stricta.
The second objective of the study was to measure the response of AMF to low nutrient and soil moisture levels and interspecific plant competition. AMF response was measured by determining percent root colonization on each plant species. It was hypothesized 74 that percent AMF colonization would be greater in the low nutrient and low soil moisture treatments for all species and greater for C. stricta when growing with competitors.
Methods
Experimental design
A common sedge meadow plant, Carex stricta, and two invasive sedge meadow species, Phalaris arundinacea and Typha latifolia, were grown in a factorial experiment with four treatments: high-low nutrient levels, high-low soil moisture, plus-minus arbuscular mycorrhi2aI fungal (AMF) inoculation, and inter- or intraspecific competition. A randomized block design was used with each treatment replicated three times. The overall experimental design involved two nutrient treatments (40:60 and 90:10 sand:wetland soil) x two water treatments (0.5 1 and 1.01 every two days) x two mycorrhizal treatments (+/- AMF) x sbc competition treatments (C stricta with P. arundinacea, C. stricta with T. latifolia, P. arundinacea with T. latifolia and only C stricta, P. arundinacea, or T. latifolia) x three replicates for a total of 144 trays.
The high nutrient treatment consisted of a 40:60 mixture of sand:wetland soil, organic matter, 2.9%; available phosphorus, 20 |ig/g; potassium, 120 |ag/g; nitrate, 7 ng/g (Iowa State
University Soil Testing Laboratory). The low nutrient treatment was a 90:10 mixture of sand:wetland soil with an organic matter, 0.5%; available phosphorus, 5 ng/g; potassium, 30
|ig/g; nitrate, 2 ng/g. The wetland soil used in this study, Webster clay loam, was obtained from a prairie pothole wetland in Story County, Iowa. The soil mixtures were steamed for 2 hours. IS
Seeds of all three species were washed with a 0.5% NaOCl solution and rinsed three times with distilled water. C. striata and P. arundinacea seeds were spread into petri dishes containing a filter paper disk on the bottom, stratified at 4°C for 48 hours, and then placed in a greenhouse to germinate. Seedlings were transplanted to community trays filled with sterilized potting soil (peat moss and perlite) when they were 2 cm tall. T. latifolia seeds were mixed directly with sterilized potting soil, stratified at 4°C for 48 hours and placed in the greenhouse to germinate.
After two weeks, seedlings were transplanted to deep trays (32 x 28.5 x 17.8 cm) filled with approximately 0.012 m^ of steamed soil (19.4 kg dry soil of the 90; 10 mixture and
13.6 kg dry soil of the 40:60 mixture). Drainage holes in the bottom of the trays were covered with fiberglass mesh (1 mm) to prevent soil loss. Six plants were planted in two rows of three in each tray. The same arrangement was used in the competition treatments, where two plant species were planted together and three individuals of each species were planted in positions alternating with each other. The few seedlings that died were replaced with same age seedlings only during the first four weeks after the initial planting. The greenhouse temperature ranged fi-om 18 to 30°C and plants were grown under high intensity lights
(average sunny day photosynthetically active radiation, 420 lamol m'^ s"^) with a 14 hour photoperiod.
All plants were grown for six weeks in trays before the soil moisture treatments were begun. The low soil moisture trays received 0.5 I of water every four days for the first two weeks then 0.5 1 every two days for the remainder of the experiment. Trays in the high soil moisture treatment received I.O 1 of water every two days. 76
Treatments with arbuscular mycorrhizal fiingi (AMF) were inoculated with spores of
Glomus intraradices (Schenck & Smith) and Glomus claroideum (Schenck & Smith)
(courtesy ofB. Hetrick, University of Northern Iowa), two species found in prairie pothole
wetlands in Iowa (Wetzel and van der Valk, 1996). Spores of each fungal species were
suspended in water, and approximately 40 spores of both species were pipetted into a small
depression in the soil immediately prior to planting a seedling. A total of approximately 480 fungal spores was added to each tray in the plus AMF treatments.
Data collection
This experiment ran eight weeks from the start of water treatments. Above and below- ground biomass was harvested, oven dried for 3-5 days at 65°C to minimize nitrogen loss
(Chapin and Van Cleve, 1989), and weighed. Above-ground biomass of each species in the
entire tray was ground with a Wiley mill to a 0.5-mm (#40) mesh size. Percent foliar total
nitrogen and carbon were determined on each treatment replicate with a Carlo-Erba NA 1500
Carbon-Nitrogen-Sulphur analyzer. Total foliar phosphorus was measured with a sodium hypobromite oxidation extraction and colorimetric determination using a heteropoly blue method (Dick and Tabatabai, 1982).
Percent AMF colonization was measured to establish the extent of colonization in each treatment. After drying, an arbitrary sample of secondary roots with attached tertiary roots
from each tray was stained to detect AMF, following the procedure of Koske and Gemma
(1989). The roots were autoclaved in a 2.5% KOH solution for 3 minutes and then bleached by soaking 10-30 minutes in a 1% hydrogen peroxide solution. After rinsing with water, the roots were autoclaved in 0.05% acid flichsin in acidic glycerol solution for 3 minutes and then 77 destained at room temperature. Percent AMF colonization was determined with the magnified intersections method described by McGonigle et al. (1990). Root segments were placed on a glass slide and viewed at 200x magnification. The presence or absence of mycelium, vesicles, arbuscules, or spores was recorded for 100 root-microscope crosshair intersections, providing percentage of roots colonized with AMF for each tray.
The relative growth rate (RGR) of each plant species in each treatment was calculated as;
RGR. — Pn(M2/Mi)]/t2-ti where Mi is initial biomass, M2 is final biomass, and t2-ti is the length of the growth period in days (Harper, 1977). The RGR was calculated for the last twelve weeks (87 days) of the experiment. Regression relationships between plant height and dry above-ground biomass of control plants were used to determine the initial biomass of the plants of each species. Green height of the longest leaf of the experimental plants was measured biweekly. Additional plants of each species were grown under the same light and temperature conditions in the greenhouse as the experimental plants. These plants were grown in trays containing a 40:60 sandisoil mixture, with six individuals per tray, and were watered to saturation regularly.
Plant height and biomass were measured biweekly by harvesting one tray of each species. The regression equation for each species was:
Carexstriata In(ABIO) = ln(HT) * 2.607 - 10.863 ^ = 0.74
Phalaris arundinacea ABIO = HT * 0.0896 - 2.964 r^ = 0.54
Typha latifolia In(ABIO) = ln(HT) • 2.514 - 10.233 = 0.87 where ABIO=above-ground biomass and HT=length of longest leaf 78
The competitive efifects of T. latifolia and P. arundinacea were examined by calculating the relative yield of individuals of a species when grown with another species
(interspecific competition) compared to the relative yield of the species grown alone
(intraspecific competition) while maintaining the same overall density. Relative Yield (RY) was calculated as
RY = Yij/Yi. where Yy is the mean biomass yield of individuals of species i grown with individuals of species j and Yi is the mean biomass yield of individuals of species i grown alone (Harper,
1977). ARY value less than one indicated that interspecific competition exerted a greater influence on plant biomass than intraspecific competition.
Statistical analysis
Analysis of variance was performed on total root, mycelium, vesicle, and spore AMF colonization and the relative yield for each plant species. Only the main treatment efifects
(block, nutrient level, water level, AMF inoculation, and competition) and the first order interactions were included in the analysis of variance models. All proportional data were arcsine square root transformed to reduce heteroscedasticity and improve normality.
To determine the relative importance of competition to C. striata in diflferent environmental conditions, a principal components analysis was conducted on six measured variables: above- and below-ground biomass, relative growth rate, percent total foliar nitrogen, percent foliar carbon, and total foliar phosphorus for each plant species. The object of principal components analysis is to find linear combinations of the variables that explain the largest amount of variance. The combinations or principal components are by definition 79 independent (orthogonal) of each other (Manly, 1994). The sum of the variances of the principal components is equal to the sum of the variances of the original variables. To prevent one variable from unduly influencing the principal components, the data were normalized to have means of zero and variances of one (Manly, 1994). Because the normalized data began with a variance of one, a principal component with an eigenvalue less than one explains less variance than the original data and was not used in further analyses. Analysis of variance by the same models described above was performed on the principal components with an eigenvalue of one or greater. The proportion of variance described by the model was calculated and plotted for all statistically significant main treatment effects and first order interactions.
Results
Total AMF colonization ranged fi^om 0-25% in C stricta, 0-15% in P. arundinacea, and 0-14% for T. latifolia across all treatments. AMF colonization was greater in mycorrhizal treatments than in non-mycorrhizal treatments for P. arundinacea roots (p<0.05), but not for
C. stricta and T. latifolia (p=0.56 and p=0.61, respectively). A second analysis of variance that included data only from trays inoculated with spores indicated that total root colonization was greater in the high nutrient treatments than in the low nutrient treatments for C. stricta
(Table 1). Spores were the greatest component of total AMF colonization under these conditions and AMF in C. stricta had a higher sporulation at the high nutrient level than the low nutrient level. Total root colonization was greater in the high soil moisture than in the low soil moisture treatments for both P. arundinacea and T. latifolia (Figure 1, Table 1). 80
C. stricta P. arundinacea 7 r T. latifolia
ab
ab ab ab ab
^ a
ac I Low High Low High No With No With No With
Nutrients Nutrients Water Water Competitor T. latifolia Competitor T. latifolia Competitori'. arundinacea With With With
P. arundinacea C. stricta C. stricta Treatments
Figure 1. Percent (± SEM) AMF colonization for nutrient and competition treatments for C. stricta, P. arundinacea, and T. latifolia. Within each environmental treatment and plant species, bars with different letters indicate the means are significantly different (p <0.05).Arbuscules were not observed in the root segments of any of the species tested and were not included in the analysis of variance. Table 1. Results of analysis of variance of total percent AMF root, mycelium, vesicular, and spore colonization of inoculated plants. Only statistically significant (p Type of AMF Colonization Plant Species Source of Variation Total Root Mycelium Vesicle Spore df P = P = P = P = Carex stricta Block 2 0.575 0.372 0.764 0.944 Nutrients 1 0.026 0.098 0.833 0.039 Soil Moisture 1 0.884 0.999 0.353 0.970 Competition I 0.055 0.101 0.304 0.016 Error 24 Phalaris Block 2 0.181 0.618 0.201 0.246 anmdinacea Nutrients 1 0.306 0.246 0.943 0.458 Soil Moisture 1 0.039 0.095 0.166 0.266 Competition 1 0.285 0.461 0.772 0.156 Error 21 Typha latifolia Block 2 0.126 0.378 0.767 0.063 Nutrients 1 0,173 0.217 0.146 0.644 Soil Moisture 1 0.026 0.003 0.817 0.204 Competition 1 0.014 0.006 0.992 0.028 Soil Moisture 2 0.007 0.010 0.069 0.437 Competition Error 23 82 Total AMF colonization of C stricta was greatest without a competitor, slightly less when growing with T. latifolia, and lowest when growing with P. arundinacea (Pigure 1). The level of spore colonization followed a similar pattern. Interspecific competition of T. latifolia with P. arundinacea also reduced total AMF colonization (Figure 1). Mycelium and spore colonization followed a similar pattern. Principal components analysis The first principal component of each plant species accounted for variation (loadings) fi"om above-ground, below-ground, and relative growth rate factors, all representing a measure of plant productivity (Table 2). The productivity component explained 38% of the experimental variation of the C. stricta data, for P. arundinacea 49% of the variation, and 41% of the variation for T. latifolia (Table 2). As expected, the above-ground, below- ground, and relative growth rate factors within each plant species were positively correlated with each other. The second principal component of each plant species contained high loadings of two macronutrients, total foliar nitrogen, and phosphorus (Table 2). The macronutrient component of C stricta also included a total carbon loading and explained 29% of the experimental variation. The macronutrient component of P. arundinacea explained 18% of the variation. For T. latifolia, the macronutrient component explained 26% of the variation (Table 2). Total foliar nitrogen and phosphorus were positively correlated with each other in all plant species. 83 Table 2. Loading factors contributing to the principal components of C stricta, P. anmdinacea, and T. latifoUa. The first principal component consisted of two productivity variables, biomass and relative growth rate. The second component consisted of two macronutrient variables, nitrogen and phosphorus. Prindpal Principal Component I Component n Variables (Productivity) (Macronutrients) Carex stricta Above-ground Biomass 0.63 0.05 Below-ground Biomass 0.62 0.00 Relative Growth Rate 0.45 0.08 Total Nitrogen -0.03 0.57 Total Caibon -0.14 0.52 Total Phosphorus 0.02 0.63 Eigenvalue 2.30 1.73 % Eigenvalues 0.38 0.29 Cumulative % Eigenvalues 0.38 0.67 Phalaris Above-ground Biomass 0.55 -0.14 arundinacea Below-ground Biomass 0.52 -0.21 Relative Growth Rate 0.51 -0.11 Total Nitrogen 0.20 0.57 Total Caibon 0.34 0.20 Total Phosphorus 0.08 0.75 Eigenvalue 2.91 1.10 % Eigenvalues 0.49 0.18 Cumulative % Eigenvalues 0.49 0.67 Typha latifolia Above-ground Biomass 0.57 0.34 Below-ground Biomass 0.56 0.31 Relative Growth Rate 0.47 -0.25 Total Nitrogen -0.17 0.56 Total Carbon 0.28 -0.10 Total Phosphorus -0.20 0.64 Eigenvalue 2.44 1.54 % Eigenvalues 0.41 0.26 Cumulative % Eigenvalues 0.41 0.67 84 Treatment effects on plant productivity and macronutrient concentration Nutrient level, soil moisture level, interspecific competition, and the nutrient x competition interaction all had statistically significant effects on the productivity of C. striata (Table 3, Figure 2). When interspecific competition was present, productivity of C. stricta declined as nutrient levels increased. Nutrient level and the nutrient x interspecific competition interaction also had statistically significant effects on macronutrient uptake (Table 3). Macronutrient uptake increased as nutrients increased when interspecific competition was present. Conversely, macronutrient uptake was higher at the low nutrient level without interspecific competition. C. stricta had greater nutrient concentration under stressfiil conditions (low nutrient or interspecific competition). Table 3. Analysis of variance of the principal components productivity and foliar macronutrients for Carex stricta. Nut. = Nutrient level; AMF = Arbuscular Mycorrhizal Fungi. Productivity Macronutrients Source of (Principal (Principal Plant Species Variation Component I) Component II) df P = P = Carex stricta Block 2 0.212 0.000 Nutrients 1 0.000 0.030 Water 1 0.005 0.811 AMF 1 0.794 0.126 Competition 2 0.000 0.949 NuL X Water 1 0.381 0.151 NuL X AMF 1 0.354 0.361 NuL X Competition 2 0.001 0.028 Water x AMF 1 0.226 0.880 Water x Competition 2 0.484 0.057 AMF X Competition 2 0.762 0.383 Error 54 85 0.5 C. striata H P. arundinacea T. latifolia 0.4 CJ •§ 0.3 S3 > O oC '€ o 0.2 oo. 0.1 - 0.0 I Nutrient Interspecific Water Nutrients x Nutrients x Water x Level Compkition Level Competition Water Competition Figure 2. Proportion of variation explained by the productivity and foliar macronutrient concentration principal components for all three experimental plants, C. stricta, P. arundinacea, and T. latifolia. All factors and interactions plotted are statistically significant. 86 The proportion of the variance attributable to each treatment was calculated to estimate the effect that a treatment or treatment interaction had on each plant species, allowing the comparison of C. stricta with the two other species. No significant amount of the variation in productivity and macronutrient acquisition for any of the species was attributable to AMF. The effect of interspecific competition on the productivity and macronutrient uptake of C stricta explained the largest amount of variation (18%) (Pigure 2). Nearly 24% of the variation in productivity and nutrient acquisition of T. latifolia was attributable to soil moisture, followed by interspecific competition and nutrient level (Figure 2). The experimental water treatments created an unproductive environment for this shallow emergent macrophyte. Nutrient level had the most impact on P. arundinacea (42% of the variation) and explained three times the amount of variation of its productivity and macronutrient concentration compared to T. latifolia and four times that of C stricta figure 2). Nutrient levels had the least effect on C stricta. Except for a small impact of water level, none of the other experimental treatments or main interactions had an impact on the productivity or macronutrient concentration of P. arundinacea. Treatment effects on interspecific competition Competition with P. arundinacea had a greater influence on above- and below-ground relative yield (RY) of C. stricta than competition with T. latifolia. The above- and below- ground RY values were less than one, which indicated that interspecific competition was greater than intraspecific competition (Figures 3 and 4). This pattern was true over all the experimental treatments (nutrient levels, soil moisture levels, or mycorrhizal colonization), but 87 Treatments N W M + + + + + - + - + O C. stricta with P. arundinacea + - - (H) # C. stricta with - + + T. latifolia - + - 0 - - + 0 + + + + + - + - + A P. arundinacea with C. stricta + - - A P. arundinacea - + + with T. latifolia - + - - - + I A A I + + + + + - l-BH + - + B • T latifolia + - - B- with C. stricta - + + -a- • T. latifolia with P. arundinacea - + - l-M - - + m e h- -B- _L 0 1 Relative Yield Figure 3. Above-ground relative competitive yield (± SEM) by treatment for C. stricta, P. arundinacea, and T. latifolia. A value greater than one indicates that interspecific competition < intraspeclfic competition; a value less than one indicates that interspecific competition > intraspecific competition; and a value of one indicates that inter- and intraspecific competitive effects were similar. N=nutrient level, W=water level, and M=mycorrhiza inoculation level. S8 Treatments N W M + + + + + O C. stricta with + - + P. arundinacea + - 0 # C. stricta with - + T. latifolia - + + + + + AA I I + + + - i I I A P. arundinacea + - with C. stricta + + Irai A I 'A I A P. arundinacea + - kAh i with T latifolia r^3r**i» A > A I i + + + o + + - f-BH + - + -Q—I • T. latifolia with C. stricta h-BH - + + f-BH • T. latifolia-mih P. arundinacea - + - l-aiD - - + • B -B-H 0 1 Relative Yield Figure 4. Below-ground relative competitive yield (± SEM) by treatment for C. stricta, P. arundinacea, and T. latifolia. A value greater than one indicates that interspecific competition < intraspecific competition; a value less than one indicates that interspecific competition > intraspecific competition; and a value of one indicates that inter- and intraspecific competitive effects were similar. N=nutrient level, W=water level, and M=mycorrhizal inoculation level. 89 there were no statistically significant differences in RY among treatments figures 3 and 4). When C stricta and T. latifolia were grown together, above- and below-ground RY values were near or slightly below one. Relative yield values were not statistically different for any of the treatment combinations (Figures 3 and 4). The tall, leafy stature and rapid lateral spread of P. arumUnacea enhanced its competitive ability, a pattern evident in relative yield figures 3 and 4) and biomass yield per plant (Figure 5). Only 0.5% of the variation in productivity and macronutrient concentration of P. arundinacea was explained by competition with the other plant species, compared to 18% for C. stricta (Figure 2). For P. arundinacea growing with C. stricta, the above- and below-ground relative yield (RY) was at or near one for all treatments (Figures 3 and 4). Analysis of variance of both the above- and below-ground RY produced no statistically significant effects. 90 High Nutrient Low Nutrient 00 30 25 20 2 U 15 > : X es 10 oE 5 ffi 0 ii Wi* AbiiiJuii W* r Wi* T.k o»tr rtavuta (Mr r.MVM* o>)r f-' (My rw«e OMly iNKaak C-iatamw* C.jatemWin CJMetBWi* C«J High Soil Moisture Low Soil Moisture 30 25 20 2 U 15 en escn 10 1 5 i 1 o 5 « m 0 A h (My r.toUMhtWMi A (Mr T.kti/baaWi* r (My Widi wi* WMi CJVMB Plus AMF Minus AMF Li n CjMeia(My rteUbfaWidi f (My Widi CjMrta Cartx striata Phalaris arundinacea Typha latifolia Figure 5. Effects of different competitive treatments and environmental conditions on biomass yield per individual plant (± SEM) for C. striata, P. arundinacea, and T. latifolia. AMF=arbuscular mycorrhizal fungi. 91 Discussion The hypothesis that arbuscular mycorrhizal fungi (AMF) would improve the competitive ability of C. stricta in low nutrient and soil moisture levels was not supported by our data. AMF were not a statistically significant factor in the productivity and macronutrient acquisition of C. stricta under any of the experimental conditions. This result was reflected in the relative yield (RY) and biomass per plant values (Figures 3-5). An abundant literature exists on the benefits of mycorrhizae to plant productivity and phosphorus uptake, particularly on plants in low nutrient and soil moisture environments. There are several possible reasons why the productivity or macronutrient acquisition of C. stricta was not improved with AMF, even without interspecific competition (Table 3, Figure 5). The level of AMF colonization in C. stricta may not have been high enough to make a difference in productivity or macronutrient acquisition, since there was not a statistical difference between the AMF colonization between the plus-minus treatments. Lack of difference between AMF treatments may be the result of contamination, but could also be because C stricta may be nonmycorrhizal as suggested by Powell (1975). Forty-four percent of the plus AMF treated trays had 1% AMF colonization or less. The low nutrient conditions also may have been poor enough to prevent vigorous AMF colonization on C. stricta. C. stricta also may form symbiotic relationships with fungi other than arbuscular mycorrhizal fungi. Haselwandter and Read (1982) found that fungi fi'om the genus Rhizoctonia or Phialophora improved shoot phosphorus concentration in two alpine sedges, Carex sempervirens and Carex firma. The presence of AMF in the roots of C. stricta growing in prairie wetlands suggests that AMF also may be functioning as pathogenic parasites. Any 92 benefit provided by AMF to C. stricta is counterbalanced by the demands of the fungi on its host. In addition, Hetrick et al. (1994) have suggested that short-term biomass responses as a measure of a plant's reliance on AMF may not entirely reflect the contribution of the symbiosis and that AMF may have an impact on later plant stages or future fecundity. Most AMF competition studies involve neighboring plant species with di£ferent responses to mycorrhizae or plants in different successional seres (Allen and Allen, 1990; Hartnett er a/., 1993; Hetricke/a/., 1989, 1994). Few studies have considered the impact of AMF on competition between nonmycorrhizal or facultatively mycorrhizal plant species. Competition experiments with a nonmycorrhizal annual (Salsola kali L. var. tenuifolia) and a facultative mycorrhizal grass, Agropyron smithii Rydb., found that AMF had no more influence on crowding coefBcients than temperature or moisture regimes (Allen and Allen, 1990). A facuhatively mycorrhizal, cool season grass accumulated phosphorus when available with no corresponding increase in biomass, independently of mycorrhizae (Hetrick et al., 1994). The significant nutrient x competition interaction of macronutrient concentration in C. stricta in the present study also suggests that macronutrients are accumulated in highly competitive environments with P. arundinacea and T. latifolia, while productivity is severely depressed (Table 3). This pattern occurred whether C. stricta, a cool season sedge, was mycorrhizal or not. The results of this study suggest that soil nutrients and competitor morphology have the greatest influence on C. stricta. The proportion of variance of productivity and macronutrient concentration of P. arundinacea attributable to nutrient level was nearly four times greater than for the other two species (Figure 2). Nevertheless, despite the high impact 93 of nutrient levels on P. arundinacea, its competitive effect on C. stricta was overwhelming under low nutrient or soil moisture conditions (Figures 3-5). The results of this study indicate that P. arundinacea is a strong competitor regardless of the environmental conditions, contrary to findings of other studies of emergent macrophytes across nutrient gradients (Shipley and Keddy, 1988). This growth response of a C-selected plant to productive or unproductive environments was predicted by Grime (1979). Differences in morphology between C stricta and P. arundinacea increased the competitive effect on C. stricta. P. arundinacea had growth rates twice as high as C. stricta for both above- and below-ground biomass, had tall leafy shoots, and had rapid lateral spread of ramets, compared to the lower biomass production, shorter stature, and thinner leaves of C. stricta. Under a single set of environmental conditions, Gaudet and Keddy (1988) found that high production of above- and below-ground biomass, tall shoots, leaf shape, and large canopy diameter were morphological characteristics that were significantly correlated with increased competitive ability in wetland plants. For C. stricta, the effect of competition with P. arundinacea was 13 times greater at high nutrient levels than low nutrient levels. At low nutrient levels, the biomass and lateral spread of P. arundinacea were reduced, decreasing, but not eliminating its competitive effect on C. stricta. At high nutrient levels, increased biomass and canopy production of P. arundinacea suggested that competition for light rather than nutrients, water, or AMF occurred. Competition between C. stricta and T. latifolia was much less intense over all experimental conditions (Figure 5) and resulted in RY values slightly less than one (Figures 3 and 4). Both of these plants have morphologies that reduced competition for light. We 94 suspected that competition between T. latifolia and C. stricta would have intensified if the experiment had extended over a longer period. The level of total AMF colonization of C stricta and T. latifolia was lowest when these species were competing with P. arundinacea, regardless of nutrient or soil moisture environments (Figure 1). Competition with P. arundinacea reduced the productivity and macronutrient level of C. stricta and T. latifolia, and the results of this study suggested that AMF colonization is influenced by the physiological state of the host plant. Links between root colonization and the physiological state of the host plant have been observed in competing grassland plants where greater AMF colonization also was correlated with greater biomass and nutrient uptake (Christie et al., 1978) and when growth of cool-season and warm-season grasses was greatest (Bentivenga and Hetrick, 1992). The height and extensive canopy of P. arundinacea severely shaded both T. latifolia and C. stricta. A reduction of light to the host plant has been found to reduce the development of AMF (Hayman, 1974). Competition also reduced the incidence of spores found on the roots of C. stricta, but had no effect on mycelium or vesicle colonization (Table 1). Nutrient level also may have influenced the physiological state of the host plant. The low nutrient environment in this experiment had a much greater impact on the AMF colonization of C. stricta (p<0.03) than did differences in soil moisture (p=0.88). In summary, the resuhs of this experiment indicated that AMF did not improve the competitive ability of C stricta in low nutrient and soil moisture levels, nor did AMF have an effect on the productivity or foliar macronutrient concentration. Nutrient levels and interspecific competition had the greatest effects on productivity and macronutrient 95 concentration. The morphological characteristics of P. arundinacea—n.'giA growth rate, tall leafy shoots, and extensive lateral spread of the canopy and ramets—enabled it to exert a doniinating competitive effect on C. stricta. AMF colonization was linked to the physiological status of the host plant; total root colonization on C. stricta and T. latifolia had the greatest decline during competition with P. anmdimcea. Acknowledgements We thank Lisa Kraus and Bonny Meier for help harvesting plants and washing roots, Marilynn Bartels for measuring foliar phosphorus, and Eric Seabloom and Paul Hinz for helpful statistical discussions. This research was funded by Wetlands Research Inc. and by a Grant-in-Aid of Research from the National Academy of Sciences, through Sigma Xi, The Research Society. References Allen, E.B. and M.F. Allen. 1990. The mediation of competition by mycorrhizae in successional and patchy environments. In, Perspectives on Plant Competition. Edited by J.B. Grace and D. Tilman. Academic Press, San Diego, CA. Allen, E.B., J.C. Chambers, K.F. Connor, M.F. Allen, and R.W. Brown. 1987. Natural reestablishment of mycorrhizae in disturbed alpine ecosystems. Artie Alp. Res 19:11- 20. Allen, M.F. 1991. The ecology of mycorrhizae. Cambridge University Press, Cambridge, England. Anderson, R.C., A.E. Liberia, and L.A. Dickman. 1984. Interaction of vascular plants and vesicular-arbuscular mycorrhizal fijngi across a soil moisture-nutrient gradient. Oecologia 64:111-117. 96 Bentivenga, S.P. and B.A.D. Hetrick. 1992. Seasonal and temperature effects on mycorrhizal activity and dependence of cool- and warm-season tallgrass prairie grasses. Can. J. Hot. 70:1596-1602. Chapin III, F.S. and K. Van Cleve. 1989. Approaches to studying nutrient uptake, use and loss in plants. In, Plant Physiological Ecologv. Edited R.W. Pearcy, J. Ehleringer, H.A. Mooney, and P.W. Rundel.Chapman & Hall, London. Christie, P., E.I. Newman, and R. Campbell. 1978. The influence of neighbouring grassland plants on each others' endomycorrhizas and root-surface microorganisms. Soil Biol. Biochem. 10:521-527. Davidson, D.E. and M. Christensen. 1977. Root-microflingal and mycorrhizal associations in a shortgrass prairie. In, The Belowpround Ecosystem: A Synthesis of Plant- Associated Processes. Edited by J.K. Marshall. Range Science Department Science Series No. 26, Colorado State University, Fort Collins. Dick, W.A. and M.A. Tabatabai. 1982. Sodium hypobromite oxidation method for determination of total phosphorus in plant materials. Agron. J. 74:59-62. Galatowitsch, SM. and A.G. van der Valk. 1995. Natural revegetation during restoration of wetlands in ihe southern prairie pothole region of North America, pp. 129-142. In, Restoration of Temperate Wetlands. Edited by B.D. Wheeler, S.C. Shaw, W.J. Fojt and R.A. Robertson. Wiley & Sons, Chichester, England. Gaudet, C.L. and P. A. Keddy. 1988. A comparative approach to predicting competitive ability from plant traits. Nature 334:242-243. Gerdemann, J.W. 1968. Vesicular-arbuscular mycorrhiza and plant growth. Ann. Rev. Phytopathol. 6:397-418. Grime, J J. 1979. Plant strategies and vegetation processes. John Wiley & Sons, Chichester, England. Grime, J.P., J.MX. Mackey, S.H. Hillier, and D.J. Read. 1987. Mechanisms of floristic diversity: Evidence from microcosms. Nature 328:420-422. Harper, J.L. 1977. Population Biology of Plants. Academic Press, London. Hartnett, D.C. Hetrick, B.A.D., Wilson, G.W.T., and Gibson, D.J. 1993. Mycorrhizal influence on intra- and interspecific neighbour interactions among co-occurring prairie grasses. J. of Ecology 81:787-795. 97 Haselwandter, K. and DJ. Read. 1982. The significance of a root-fungus association in two Carex species of high-alpine plant communities. Oecologia 53:352-354. Hayman, D.S. 1974. Plant growth responses to vesicular-arbuscular mycorrhiza. VI. Effect of light and temperature. New Phytol. 73:71-80. Hetrick, B.A.D., G.W.T. Wilson, and D.C. Hartnett. 1989. Relationship between mycorrhizal dependence and competitive ability of two tallgrass prairie grasses. Can. J. Bot. 67:2608-2615. Hetrick, B.A.D., G.W.T. Wilson, and A.P. Schwab. 1994. Mycorrhizal activity in warm- and cool-season grasses: variation in nutrient-uptake strategies. Can. J. Bot. 72:1002- 1008. Koske, RE. and J.N. Gemma. 1989. A modified procedure for staining roots to detect VA mycorrhizas. Mycol. Res. 92:486-488. Lesica, P. and RK. Antibus. 1985. Mycorrhizae of alpine fell-field communities on soils derived fi-om crystalline and calcareous parent materials. Can. J. Bot. 64:1691-1697. Manly, B.F.J. 1994. Multivariate statistical methods. A primer. Second Edition. Chapman & Hall, London. 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 fijngi. New Phytol. 115:495-501. Powell, C.L. 1975. Rushes and sedges are non-mycotrophic. Plant and Soil 42:481-484. Read, D.J. and K. Haselwandter. 1981. Observations on the mycorrhizal status of some alpine plant communities. New Phytol. 88:341-353. Reinartz, J.A. and E.L. Wame. 1993. Development of vegetation in small created wetlands in southeastern Wisconsin. Wetlands 13(3):153-164. Shipley, B. and P.A. Keddy. 1988. The relationship between relative growth rate and sensitivity to nutrient stress in twenty-eight species of emergent macrophytes. J. of Ecology 76:1101-1110. Wetzel, P.R. and A. G. van der Vaik. 1996. Vesicular arbuscular mycorrhizae in prairie pothole wetland vegetation in Iowa and North Dakota. Can. J. Bot. 74:883-890. 98 CHAPTERS. GENERAL CONCLUSIONS General Summary The general resevch goal of this project was to document arbuscular mycorrhizal fungi (AMF) in prairie wetlands and determine their function in prairie wetland ecosystems using the mycorrhizal continuum model as a fiamework. Few mycorrhizal surveys have been done in saline or freshwater prairie wetlands. The dynamic annual and interannual hydrology and variable soil environment found throughout the prairie pothole region suggests that AMF are ecologically significant. AMF may have a role in plant survival, in inter- and intraspecific competition, and may increase the niche breadth of individual plant species along a moisture gradient. The research presented here reports the extent of AMF colonization in freshwater and saline wetlands and whether AMF improves the growth and nutrient uptake of wetland plants under multiple environmental and competitive stresses. Traditionally, AMF were believed not to occur in aquatic or semi-aquatic ecosystems because of'insufBcient oxygen' (Allen and St. John, 1982; Hayman, 1983), but very few researchers had actually investigated AMF in aquatic habitats. The insufBcient oxygen hypothesis reflects a lack of understanding of plant growth in wetland and aquatic environments because all plants growing in these environments require oxygen for cell respiration. Wetland plants in particular have well-adapted morphologies to bring oxygen into their roots (Blom and Voesenek, 1996). The continued belief that AMF were absent in aquatic and wetland habitats was further surprising because salt marsh plants were long ago known to be mycorrhizal (Mason, 1928), and Gerdemann (1968) reported that plants that 99 were non-mycorrhizal in waterlogged soils became mycorrhizal when the water table became lower or the plants were transplanted into well-drained soils. The survey of three vegetation zones in three saline and three freshwater prairie wetlands indicated the presence of AMF in prairie wetlands. Percent AMF colonization was significantly correlated to plant species, season, and the soil parameters pH and soil phosphorus, which supported the mycorrhizal continuum model. AMF colonization ranged from 0.2 to 72 % between plant species. Although these plant species were growing at different positions along nutrient and soil moisture gradients, such a wide colonization level suggests that mycorrhizal effect depended on the plant host, regardless of environmental factors or the plant's life stage. Considering the plant life cycle axis of the continuum model, many of the early season grasses and sedges had a higher level of AMF colonization in June than in August, indicating that colonization was greatest at the peak growing and fruiting period of the plant life cycle. AMF colonization also was greater in the plants growing in poorer soil conditions—low phosphorus and high soil pH, as predicted by the environmental factors axis of the model. AMF colonization was greatest in plants growing in the low prairie and wet meadow vegetation zones, two zones that would be expected to experience the widest fluctuation of water levels. However, matric potential was not statistically significant in either the analysis of all plants collected or the three plants common to both North Dakota and Iowa. It was expected that AMF colonization would be correlated with matric potential. However, the 1993 growing season was one of the wettest on record and water levels did not decline at the end of the summer. The high water levels may have caused the importance of mycorrhizae to 100 be underestimated for the wetland plants sampled, but the fact that AMF colonization was present in wetiand vegetation during a wet year also suggests that soil moisture may not be an important factor regulating AMF colonization. The ambiguity of the soil moisture field data and the possibility that AMF would be important to plant survival in an ecosystem with pulsed hydrology led to a greenhouse experiment that tested the impact of AMF on plants grown under variable hydrologic regimes and the corresponding changes in redox levels created by those water regimes. AMF were expected to reduce the effects of temporary or long-term drought by increasing phosphorus uptake. Growing plants in different anaerobic/aerobic regimes also tested whether AMF mitigate the phytotoxic effects of anaerobic end products. Phosphorus levels similar to those found in wetiands in Iowa were simulated in the greenhouse experiment. The high levels of soil phosphorus prevented meaningful AMF colonization of the experimental species Calamagrostis canadensis and Carex stricta. The lack of AMF colonization suggests that mycorrhizae may not be important to plants growing in wetiands with high phosphorus inputs during dry periods. This result, and the fact that AMF colonization was greater in the less productive soils in the wetiands of North Dakota than the productive soils in the wetiands of Iowa, suggests a greater mycorrhizal role in North Dakota wetlands. It is not clear whether this difference in colonization is due to differences in soil nutrients, soil salinity, or another factor not measured. In the three plant species common to both regions, soil conductivity was not correlated with AMF colonization, but location was statistically significant. Soil salinity could represent a portion of the location factor or, more probably, the interaction of many variables differing between the wetlands of Iowa and North 101 Dakota, of which soil salinity is one. AMF colonization has been correlated with salinity in saline prairie wetlands; colonization is greatest in the vegetation zones with the lowest salinity and nearly absent in vegetation zones with consistently greater soil salinity (Johnson-Green et al., 1995). Inclusion of soil salinity as an environmental factor in the mycorrhizal continuum model needs further study. The application of benomyl, a general fungicide, to all non-mycorrhizal treatments in the variable hydrology greenhouse experiment indicated that non-mycorrhizal fungi have a negative impact on plant biomass. Non-mycorrhizal fungi reduced total biomass of C. canadensis between 29-41% in hydrologic treatments with high soil moisture. When soil moisture was low, non-mycorrhizal fungi produced no effect on biomass and treatments with and without benomyl were not statistically different. Non-mycorrhizal fungi reduced total biomass of C. stricta 47% only in the three days flooded/dry water treatment. Non- mycorrhizal fungi produced no effect on above- or below-ground biomass in any of the other water treatments. C. stricta either possesses antifungal properties or the non-mycorrhizal fungi that would attack C. stricta were not present in the soil during the experiment. Carex spp. have traditionally been reported non-mycorrhizal (Brundrett, 1991; Gerdemann, 1968; Powell, 1975), and the results of this experiment suggest that Carex spp. may be resistant to all fungal species that invade roots. However, Carex spp. have been reported to have AMF (Wetzel and van der Valk, 1996), particularly under certain environmental conditions (Read and Haselwandter, 1981; Tester et al., 1987). Such a pattern suggests that C stricta could be facultatively mycorrhizal at certain points along the environmental factor continuum axis. 102 The third part of this project tested the importance of AMF to the biomass and nutrient uptake of C. stricta on an environmental factor continuum axis with multiple &ctors. C stricta and two potential competitors, Phalaris arundinacea and Typha latifolia, were subjected to two levels of nutrients, soil moisture, and intra- and interspecific competition. Nutrient level and intense above- and below-ground competition from P. arundinacea had the greatest effect on biomass accumulation and nutrient uptake. AMF were not important in improving C. stricta biomass, nutrient uptake, or competitive ability under any of the experimental conditions. These results suggest that C. stricta is a non-mycorrhizal plant. Under the experimental conditions tested, nutrients, competition, and water availability are much more important growth factors than AMF for the three plant species tested. Comparison of AMF colonization levels in this experiment also clearly indicated that there is a mmimum level of plant host health necessary to sustain AMF colonization. The mycorrhizae may improve plant nutrient uptake during non-productive environmental conditions, but if conditions threaten the survival of the plant host, carbon allocation to the mycorrhizal fungi is presumed to be insufiBcient to enable the mycorrhizae to benefit the plant host. The level at which the plant is no longer a viable host for the fiingi is not known and needs fiirther investigation. All of the research presented here assumes that the effect of the mycorrhizal fungi is directly proportional to the level of total AMF colonization. Although it is reasonable to assume that the greater the root colonization, the greater the mycorrhizal impact on the host plant, this may not be a valid assumption. Even if true, the mycorrhizal continuum model 103 suggests that the effect of AMF on the host plant is not linear. The probable lack of linearity of the model axes adds another layer of complexity to the continuum model. Interpretations of total colonization values are further complicated because that measure says nothing about whether AMF have a positive or negative effect on the host plant. Nutrient transfer to the plant and carbon transfer from the plant are believed to occur in arbuscules, and many researchers accept only the presence of arbuscules as an indication of functional AMF (Anderson et al., 1984). Other forms of AMF colonization may be present, but not forming an active symbioses. For example, vesicular and mycelium colonization of characteristically non-mycorrhizal plants has been reported in experimental studies only when grown in the presence of a mycorrhizal companion plant (Hirrel et al., 1978). The disadvantage of relying completely on arbuscule colonization as an indicator is that arbuscule levels are constantly changing, since arbuscules persist only up to 15 days. The most reliable measure of AMF effect is comparisons of biomass (but see Hetrick et al., 1994) and "C or isotope tracing in the host plant. The mycorrhizal symbiosis is complex because it depends on multiple factors that are constantly changing. Future research must simultaneously consider as many of these factors as possible, recognizing that there is probably a hierarchy of importance among the factors. Mycorrhizal effect may be plant species or fungal species specific, making it necessary to organize mycorrhizal effect at a guild level. The relationship is further complicated by the lack of an easily measured, reliable indicator of mycorrhizal effect. Such an indicator, particularly if usable under field conditions, could have a profound effect on understanding the importance of arbuscular mycorrhizal fungi. 104 References Allen, MJ. and T.V. St. John. 1982. Dual culture of endomycorrhlzae. In, Methods and Principles of Mvcorrhi»>l Research Edited by N.C. Schneck. American Phytopathological Society, St. Paul, Minnesota U.S.A. Anderson, R.C., A.E. Liberta, and L.A. Dickman. 1984. Interaction of vascular plants and vesicular-arbuscular mycorrhizal fungi across a soil moisture-nutrient gradient. Oecologia, 64:111-117. Blom, C.W.PM. and L.A.C.J. Voesenek. 1996. Flooding: the survival strategies of plants. TREE 11:290-295. Brundrett, M.C. 1991. Mycorrhizas in natural ecosystems. In, Advances in Ecological Research. Volume 21. Edited by M. Begon, A.H. Fitter, and A. Macfadyen. Academic Press, London. Gerdemann, J.W. 1968. Vesicular-arbuscular mycorrhiza and plant growth. Ann. Rev. Phytopathol. 6:397-418. Hayman, D.S. 1983. The physiology of vesicular-arbuscular endomycorrhizal symbiosis. Can. J. Bot. 61:944-963. Hetrick, B.A.D., G.W.T. Wilson, and A.P. Schwab. 1994. Mycorrhizal activity in warm- and cool-season grasses: variation in nutrient-uptake strategies. Can. J. Bot. 72:1002- 1008. Krrel, M.C., H. Mehravaran, and J.W. Gerdemann. 1978. Vesicular-arbuscular mycorrhizae in the Chenopodiaceae and Cruciferae: do they occur? Can. J. Bot. 56:2813-2817. Johnson-Green, P.C., N.C. Kenkel, and T. Booth. 1995. The distribution and phenology of arbuscular mycorrhizae along an inland salinity gradient. Can. J. Bot. 73:1318-1327. Mason, E. 1928. Note on the presence of mycorrhiza in the roots of salt marsh plants. New Phytol. 27:193-195. Powell, C.L. 1975. Rushes and sedges are non-mycotrophic. Plant and Soil 42:481-484. Read, D.J. and K. Haselwandter. 1981. Observations on the mycorrhizal status of some alpine plant communities. New Phytol. 88:341-353. Tester, M., S.E. Smith, and F.A. Smith. 1987. The phenomenon of "nonmycorrhizal" plants. Can. J. Bot. 65:419-431. 105 Wetzel, P.R. and A. G. van der Valk. 1996. Vesicular arbuscular mycorrhizae in prairie pothole wetland vegetation in Iowa and North Dakota. Can. J. Bot. 74:883-890. 106 ACKNOWLEDGEMENTS No project is done alone, and this project was no exception. Many people not listed here helped me in a variety of ways during the course of this project. I am thankful for their interest, talents, and time. I appreciate Arnold van der Valk's willingness to allow me to develop a project in an area unfamiliar to both of us. That willingness, enabled me to pursue a project of intense interest to me for the pure joy of discovery. Arnold is also an excellent editor, and this dissertation bears the mark of those skills. Members of my committee, Barbara Hetrick (University of Northern Iowa), Thomas Jurik, Thomas Loynachan, James Raich, Lois Tiffany, and Gregory Tylka, gave freely of their time and laboratory resources. Such cooperation is not present at all universities, and my project, and Iowa State, are better because of it. The help of my undergraduate workers is also greatly appreciated. Todd Cheever, Marilytm Bartels, Lisa Kraus, Adrian van der Valk, and Bonny Meier all went beyond what was expected and not only made the project easier, but more fun for me too. Although geographically distant, the shadow of my parents, Carol and Robert Wetzel, looms large over this project. They instilled in me a sense of wonder about the world, a love of plants, and the values of hard work, organization, and common sense. The early laboratory training I received from my Dad has proven itself useful again and again throughout my career, and particularly for this project. Fellow graduate student, Eric Seabloom, knows as much about my project as anyone. From its conception, Eric provided suggestions, statistical advice, and challenging intellectual 107 discussions. His presence enhanced both my project and my graduate career at Iowa State. I am thankfiil that our paths crossed in Iowa. Numerous good suggestions and intellectual challenges also came from Carolyn, my wife. I fondly recall spending countless hours at the dinner table discussing the ups and downs of this project, as well as science in general. We experienced sadness and joy in Ames and our stay was longer than either of us expected. Her love and support inspired me more than she knows, and I am thankful to have such an endearing friend. Finally, I wish to acknowledge our daughter, Georgina. Bom in September 1996, and so innocent of what a motivating force she was for me to finish.