SOIL RESOURCE ACQUISITION MECHANISMS, NUTRIENT CONCENTRATIONS AND GROWTH IN PERENNIAL GRASSES

Carolina Saint Pierre, Carlos A. Busso, Oscar A. Montenegro, Gustavo D. Rodríguez, Hugo D. Giorgetti, TomÁs Montani and Oscar A. Bravo

Carolina Saint Pierre. Agronomic Engineer and M.S., Universidad Nacional del Sur (UNS), Argentina. Ph.D. student, Oregon State University, USA.

Carlos Alberto Busso. Agronomic Engineer and M.S., UNS, Argentina. Ph.D. in Ecology, Utah State University, Logan, USA. Professor, Agronomy Department, UNS, Argentina. Temporary Research Assistant Professor, Department of Animal Biotechnology, University of Nevada, Reno, USA. Address: Departamento de Agronomía-CERZOS. UNS, 8000-Bahía Blanca, Argentina. e-mail: cebusso@criba.edu.ar

Oscar Montenegro. Agronomic Engineer, UNS, Argentina. Technician, Patagones Experimental Farm, Patagones, Buenos Aires. Ministerio de Asuntos Agrarios. Argentina.

Gustavo D. Rodríguez. Agronomic Technician. Patagones Experimental Farm, Patagones, Buenos Aires. Ministerio de Asuntos Agrarios. Argentina.

Hugo D. Giorgetti. Agronomic Engineer, UNS, Argentina. Director, Patagones Experimental Farm, Patagones, Buenos Aires. Ministerio de Asuntos Agrarios. Argentina.

Tomás Montani. Agronomic Engineer and M.S., UNS, Argentina. Professor, Agronomy Department, UNS, Argentina.

Oscar Abel Bravo. Agronomic Engineer, Universidad Nacional de la Plata, Argentina. M.S. and Doctoral student, UNS, Argentina.

Resumen

Se estudiaron las relaciones entre los mecanismos de adquisición de recursos del suelo y los componentes de producción de tejido foliar en la especie de etapas serales tardías, tolerante al pastoreo y competitiva Stipa clarazii Ball y en las especies de etapas serales más tempranas, menos tolerantes al pastoreo y competitivas S. tenuis Phil. y S. ambigua Speg. La historia de pastoreo y/o fuego determina la abundancia de estas gramíneas perennes cespitosas C₃ en los pastizales templados semiáridos de Argentina. La hipótesis de trabajo fue que las plantas defoliadas tendrían menor densidad de longitud de raíces (DLR) y porcentaje de colonización por micorrizas vesiculares-arbusculares (%MAV) que aquellas no defoliadas, y también que las plantas defoliadas y no defoliadas de S. clarazii tendrían mayores valores de DLR y %MAV que aquellas de las otras especies, por su mayor capacidad competitiva y tolerancia a la defoliación. Se condujo un estudio bajo condiciones de campo en un área excluída al pastoreo por herbívoros domésticos durante 2 años. Un grupo de plantas fue defoliado una vez a 5cm del suelo a principios de primavera, mientras que otro grupo fue defoliado dos veces, a principios y mediados de primavera. Un tercer grupo permaneció no defoliado. Las mediciones se condujeron 6-10 días después de cada defoliación, y al final de la estación de crecimiento. DLR y %MAV fueron similares en plantas defoliadas y no defoliadas en las tres especies. La mayor producción de rebrote en S. clarazii no estuvo asociado con mayores DLR y %MAV en la especie más competitiva. La relación positiva entre DLR y producción de peso seco, o la concentración de N y P en el tejido en estas especies, sugiere que su actividad radical debe mantenerse tras la defoliación para reestablecer una superficie fotosintética y mantener la distribución de recursos en equilibrio. Todas las especies mostraron valores consistentemente altos de % MAV, sugeriendo que pueden tener una asociación mutualística obligada con las micorrizas. La correlación negativa entre %MAV y DLR o P disponible del suelo para S. clarazii podría representar un mecanismo para evitar cambios de asociación mutualística a parasítica en la relación planta-micorriza.

Summary

Relationships among soil resource acquisition mechanisms and components of shoot tissue production were studied in the late-seral, grazing tolerant and competitive Stipa clarazii Ball and in the earlier-seral, less grazing tolerant and competitive S. tenuis Phil and S. ambigua Speg. History of grazing and/or fire determines abundance of these C₃ perennial tussock grasses in temperate, semiarid rangelands of Argentina. We hypothesized lower values for root length density (RLD) and percentage vesicular-arbuscular mycorrhizal (%VAM) colonization on defoliated than on undefoliated plants, and also greater values for RLD and %VAM colonization on defoliated and undefoliated plants of S. clarazii than on those of the other two species, because of its greater competitive ability and defoliation tolerance. Field study was conducted within a 2-year exclosure to domestic herbivory. One group of plants was defoliated once to 5cm stubble height in early spring, while another group was defoliated twice, in early and mid-spring. A third group remained undefoliated (control). Measurements were conducted 6-10 days after each defoliation, and at the end of the growing season. RLD and %VAM colonization were similar on defoliated plants and on undefoliated controls in all three species. Greater regrowth production in S. clarazii was not associated with higher RLD and %VAM colonization in the more competitive species. The positive relationship between RLD and either dry weight production or tissue N and P concentration in these species suggests that their root activity must be maintained after defoliation to rapidly reestablish a photosynthetic canopy and keep resource supply at equilibrium. All perennial grasses showed consistently high %VAM colonization, suggesting they may function as obligate mycotrophs. The negative correlation between %VAM colonization and RLD or soil available P for S. clarazii could represent a mechanism to avoid shifts from mutualism to parasitism in the plant-mycorrhizae relationship.

Resumo

Estudaram-se as relações entre os mecanismos de aquisição de recursos do solo e os componentes de produção de tecido foliar na espécie de etapas serais tardias, tolerante ao pastoreio e competitiva Stipa clarazii Ball e nas espécies de etapas serais mais prematuras, menos tolerantes ao pastoreio e competitivas S. tenuis Phil. e S. ambigua Speg. A história de pastoreio e/ou fogo determina a abundância destas gramíneas perenes cespitosas C₃ nos pastizais temperados semi-áridos da Argentina. A hipótese de trabalho foi de que as plantas desfolhadas teriam menor densidade de longitude de raízes (DLR) e porcentagem de colonização por micorrizas vesiculares-arbusculares (%MAV) que aquelas não desfolhadas, e também que as plantas desfolhadas e não desfolhadas de S. clarazii teríam maiores valores de DLR y %MAV que aquelas das outras espécies, por sua maior capacidade competitiva e tolerância à desfolhação. Se conduziu um estudo sob condições de campo em uma área excluída ao pastoreio por herbívoros domésticos durante 2 anos. Um grupo de plantas foi desfolhado uma vez a 5 cm do solo no início da primavera, enquanto que outro grupo foi desfolhado duas vezes, no início e na metade da primavera. Um terceiro grupo permaneceu não desfolhado. As medições se conduziram 6-10 dias depois de cada desfolhação, e no final da estação de crescimento. DLR y %MAV foram similares em plantas desfolhadas e não desfolhadas nas três espécies. A maior produção de rebrote em S. clarazii não esteve associado com maiores DLR y %MAV na espécie mais competitiva. A relação positiva entre DLR e produção de peso seco, ou a concentração de N e P no tecido nestas espécies, sugere que sua atividade radical deve se manter após a desfolhação para restabelecer uma superficie fotossintética e manter a distribuição de recursos em equilíbrio. Todas as espécies mostraram valores consistentemente altos de % MAV, sugerindo que podem ter uma associação mutualística obrigada com as micorrizas. A correlação negativa entre %MAV e DLR ou P disponível do solo para S. clarazii poderia representar um mecanismo para evitar mudanças de associação mutualística a parasítica na relação planta-micorriza.

Key words / Defoliation / Mycorrhiza / Nitrogen / Phosphorus / Root Length Density / Shoot Growth /

Received: 01/23/2004. Modified: 05/10/2004. Accepted: 05/12/2004.

Species historically heavily grazed may have been selected for grazing-resistance traits associated with improved nutrient use efficiency (Chapin and Slack, 1979). Higher root length density (RLD) and vesicular-arbuscular mycorrhizae (VAM) colonization have been associated with greater nutrient acquisition in perennial grasses, thus contributing to competitive ability (Jackson and Caldwell, 1996). This is especially true in unproductive environments where competition for belowground resources is strong (Jackson and Caldwell, 1996). Mycorrhizal fungi can affect competitive interactions among plants by improving soil nutrient uptake (Marschner and Dell, 1994; Mohammad et al., 1998). However, lack of or even negative responses have been obtained under increased levels of mycorrhizal colonization (Fitter and Hay, 1983).

Belowground competition is the major form of competition in semiarid and arid environments (Fowler, 1986). Occupation of soil space, which is of primary importance in belowground competition, depends on root characters such as length, which is of primary importance to acquire nutrients (Casper and Jackson, 1997). Species with less root length per soil volume, however, may be more dependent on mycorrhizal colonization for P acquisition than species with greater root length densities (Koide and Li, 1991). Greater root length densities and association with mycorrhizal-forming fungi can contribute to plant nutrient increase. However, these are often alternative strategies for nutrient acquisition by plants (Kothari et al., 1990). Determination of the relative contribution of these mechanisms to plant competitive ability constitutes an interesting and difficult research challenge (Derner and Briske, 1999). Studies of correlations among RLD, VAM colonization, plant biomass, soil P concentration, and concentration of P and N in plant tissues increase our understanding of the integration of plant shoot and root functions (Hetrick et al., 1989).

The role of grazing by domestic livestock must be considered when evaluating strategies that contribute to competitive ability of rangeland vegetation. Factors that reduce photosynthetic capacity will also reduce soil resource acquisition mechanisms because these are dependent upon plant C fixation (Briske and Richards, 1995). Some grazing tolerant, competitive grass species (like Agropyron desertorum; Briske and Richards, 1995) are able to preferentially allocate C to regrowing shoot sinks while curtailing root growth after defoliation which allows for rapid photosynthetic canopy reestablishment. In contrast, root growth has remained unabated after defoliation in other grazing sensitive, less competitive grasses (like Pseudoroegneria spicata Syn. A. spicatum; Briske and Richards, 1995) thus delaying reestablishment of a shoot/root equilibrium. Fungal simbiont biomass was doubled in A. spicatum and reduced by 50% in A. desertorum after severe defoliation (Allen et al., 1989). However, Allsopp (1998) suggested that any defoliation treatment would reduce percentage VAM colonization and external fungal hyphae in grass species with low competitive ability.

Root length density (RLD) and percent VAM colonization were evaluated in this study for Stipa clarazii, S. tenuis y S. ambigua after plants of these species were defoliated or remained undefoliated. These perennial tussock grasses have shown a different response to continuous, long-term grazing in rangelands of central Argentina (Fernández and Busso, 1999). S. clarazii is palatable and dominant under exclosure or light grazing conditions (Cano, 1988; Busso, 1997). Fernández and Distel (1999) suggested that S. clarazii may have been one of the most abundant species in the herbaceous layer of the pristine vegetation in rangelands of central Argentina. This suggests that S. clarazii is a late-seral species (Saint Pierre et al., 2004). Because of selective herbivory, however, this species is replaced by S. tenuis, another palatable grass, under continuous, moderate grazing (Busso, 1997). At sites where grazing has been continuous and severe, however, S. clarazii and S. tenuis have been replaced by undesirable perennial grasses like S. ambigua, of earlier successional stages (Giorgetti et al., 1997), which are grazed only when a better forage is not available (Cano, 1988). S. clarazii has shown a greater competitive ability and grazing tolerance than S. tenuis and S. ambigua when root proliferation and dry matter production of these species were determined in plants growing in isolation or in various neighborhoods exposed to different defoliation patterns (Saint Pierre et al., 2000a, 2002; Saint Pierre, 2002). In these studies, a neighborhood consisted of a central plant of one species surrounded by five neighboring plants of a different species. However, mechanisms contributing to its greater competitive ability are largely unexplored.

Our objectives were to examine: i) the effect of defoliation on shoot dry weight production and root traits and ii) the relationships between shoot and root characteristics. Since defoliation can diminish RLD and % VAM colonization by compromising carbohydrate supply to the fungi as an energy source, we hypothesized lower values for these parameters for defoliated than for intact control plants. We also hypothesized greater values for RLD and % VAM colonization on defoliated and undefoliated plants of S. clarazii than on those of the other two species, because of its greater competitive ability and defoliation tolerance.

Materials and Methods

Study site

The study was conducted within a 2-year exclosure to domestic herbivory during 1998 at the Chacra Experimental de Patagones, south of the Provincia de Buenos Aires (40º39'S, 62º54'W), in the Phytogeographical Province of El Monte (Fernández and Busso, 1999). The community is characterized by an open shrubby layer, which also includes herbaceous species of different desirability for livestock production (Fernández and Busso, 1999). Dominance of a particular grass species within the herbaceous layer at any site is determined, at least in part, by the previous grazing and fire history of that site (Fernández and Busso, 1999). The tree layer may be composed of occasional individuals of Prosopis caldenia Burkart. The soil was classified as a typical haplocalcid with an A horizon having a loamy-clay-sandy texture; 0.20m deep; 16.9g·kg^-1 organic C; 28.7ppm available P and 1.23g·kg^-1 total N. A B_w horizon was found below 0.20m of soil depth followed by a BC_k horizon between 0.28 and 0.43m of depth. A C_k horizon existed below 0.43m with very scarce roots. Average pH was 7. Long-term (1901-1950) mean annual values for rainfall were 331mm; 14.6ºC for air temperature; -7.6ºC (Aug) and 43ºC (Jan) for absolute minimum and maximum air temperatures, respectively; 60% for relative humidity and 13km·h^-1 for wind speed. Rainfall was 295.6mm in 1998. A meteorological station located at the Chacra Experimental de Patagones provided evapotranspiration and rainfall data during the study period (May to December, 1998; Figure 1).

Treatments and measurements on plants

A total of 72 plants of S. clarazii, S. tenuis and S. ambigua which had no neighbors within a radius of more than 0.5m were randomly selected. Plant circumference was measured at soil surface. Half of the plants were defoliated to 5cm stubble height on Sept 17 (apical meristems were not removed from the plants) and Oct 12 (apical meristems at the reproductive stage of development were removed), while the other half remained undefoliated (controls). Shoot dry weight production was measured at the time of the defoliation treatments (C, clipping). At the end of the growing cycle, the amount of dry weight produced by undefoliated control plants during the whole study period, and that produced by defoliated plants from the time of treatment to the end of the growing cycle (R, regrowth) were measured. Total dry weight produced by defoliated plants was calculated as C+R. All plant tissues obtained above clipping height were oven-dried at 60ºC and then weighed. Current-year live and recently dead material were separated from older, previous-year tissues which had a grey color, weighed and ground to pass a 40-mesh screen. This separation based on leaf color was clear and reliable and it has been reported in several studies on perennial grasses (Busso et al., 2001). Shoot P and N concentrations were determined following Chapman and Pratt (1961) and Nelson and Sommers (1980), respectively.

Three destructive harvests were conducted during spring on the following dates: 6-10 days after the first defoliation (Sept 26), 6-10 days after the second defoliation (Oct 22), and at the end of the growing season (Dec 2). Plants of the first harvest had been defoliated once while those of the second and third harvests had been defoliated twice. Four plants were harvested per species and defoliation treatment on each sampling date. After harvesting, two soil cores (0.08m diameter) were taken at the periphery of each plant up to 0.30m depth. Becker et al. (1997) found that root weight density of field-grown S. tenuis and Piptochaetium napostaense, another native perennial grass in central Argentina, was more than two times higher between 0-0.25m than at 0.25-0.50m of soil depth. One of the soil cores was used to determine available soil P (Olsen and Sommers, 1982). The roots in the other soil core were used for determinations of both root length and frequency of mycorrhizal colonization.

Roots were removed from soil cores by manual washing using a 60-mesh sieve. Thereafter, root length was estimated using Tennant´s method (1975). Since soil core volume was known (1507.96cm³), RLD (cm roots · cm^-3 soil) could be calculated.

For mycorrhizal assessment, roots were cleared and stained according to Phillips and Hayman (1970). Percentages of root intercepts (n=40 per root sample) containing hyphae, arbuscules and/or vesicules were determinet using a microscope (x100). Colonized root length density was calculated by multiplying percentage colonization by root length density (Allen et al., 1989).

Statistical analyses

A completely randomized design was used in all cases. Data were analyzed using a three-way analysis of variance (3 species x 2 defoliation treatments x 3 dates). When a significant (p<0.05) interaction or main treatment effect was found, mean values were compared using Fisher’s least significant difference (LSD) test. Treatment means were considered significantly different at p<0.05. Data for colonized root length density were arcsin square root transformed. Plant biomass [ln(x+2)] and root length density (square root) data were also transformed. The figures and tables show untransformed means.

Relationships between variables were examined using linear regression analysis. Significance of these relationships was accepted at p<0.05. Regression lines were compared using the procedure described by Neter et al. (1985). Within each species, regression lines for defoliated and undefoliated plants were first compared and the data pooled if they were not significantly different (p>0.05).

Results

Dry weight production

Plant basal area was highly variable within and among treatments for all species. Minimum and maximum values were 15.6 and 108.9cm2 in S. clarazii, 17.9 and 147.1cm² in S. tenuis, and 30.2 and 357.2cm² in S. ambigua. Differences in plant basal area between any species pair were the result of inherent size variation among species. Because of this, all dry weight production data are reported on a per surface plant basal area basis. The initial biomass (g·cm^-2) at the first clipping was 0.131 ±0.028 (mean ±1SE) in S. clarazii, 0.045 ±0.005 in S. tenuis and 0.304 ±0.017 in S. ambigua. Total dry weight production was more than 78% greater (p<0.05) on defoliated and undefoliated plants of S. ambigua than on those of the other two species (Tables I and II). However, the amount of regrowth was greater (p<0.05) in S. clarazii than in S. tenuis and S. ambigua, at the end of the growing season (Table I).

Total dry weight production by undefoliated plants of all three species was greater (p<0.05) for the first and second than the last sampling date (Table I). As a result, total dry weight production of all three species was greater (p<0.05) for defoliated than on undefoliated plants at the end of the study period (Table I).

Root length density and percentage VAM colonization

Root length density was greater (p<0.05) in S. ambigua than in S. clarazii and S. tenuis (p<0.05) by the end of the growing season (Table I). This parameter was similar (p>0.30) for defoliated and undefoliated plants of all three species (Table I).

Percentage VAM colonization was similar among defoliation treatments (p>0.90, Tables I and II). All three species showed similar (p>0.05) VAM colonization percentages, except at the second sampling date, when this variable was greater (p<0.05) in S. tenuis than in S. clarazii (Tables I and II). Values for this parameter varied from 61 to 81% in S. clarazii, S. tenuis and S. ambigua (Table I). Percentages of VAM colonization increased (p<0.05) from the second to the last sampling date in S. clarazii, and from the first to the second sampling in S. tenuis (Tables I and II).

Shoot P and N concentrations

Tissue P concentrations were more than 20% greater (p<0.05) on defoliated than on undefoliated plants in S. clarazii and S. ambigua (Table I). Tissue P concentrations of defoliated plants were similar (p>0.05) in S. clarazii, S. tenuis and S. ambigua (Table I). When plants remained undefoliated, however, tissue P concentrations were more than 38% lower (p<0.05) in S. ambigua than in S. clarazii and S. tenuis (Table I).

S. ambigua was the species that showed the lowest (p<0.05) tissue P concentrations in comparison to S. tenuis, S. tenuis and S. clarazii, or S. clarazii at the first, second or last sampling date, respectively (Tables I and II). Tissue P concentrations were similar (p<0.05) among sampling dates in S. clarazii and S. ambigua, and they decreased (p<0.05) as the growing season progressed for S. tenuis (Tables I and II).

Nitrogen concentrations for plants of all three species defoliated twice were more than 31% greater (p<0.05) than values of undefoliated controls (Tables I and II).

Available soil P concentrations

Soil P concentrations increased (p<0.05) when plants of S. clarazii and S. tenuis were defoliated in comparison to undefoliated controls, and the reverse was true in S. ambigua (Tables I and II). When plants remained undefoliated, soil P concentrations were greater (p<0.05) under shoots of S. tenuis and S. ambigua than under those of S. clarazii. However, soil under defoliated S. clarazii and S. tenuis showed higher (p<0.05) P concentrations than that under defoliated S. ambigua (Tables I and II).

Relationships between shoot and root variables

Only data demonstrating significant correlations are reported. Lack of more consistent associations in this field study may partly be the result of the small sample size (n=4), which is the minimum for detecting correlations. Root length density was positively correlated (p<0.05, r=0.65, n=14) with total dry weight production on plants which remained undefoliated or were defoliated twice in S. tenuis (Figure 2a). RLD and percentage VAM colonization showed a negative correlation (p<0.05, r=0.72, n=8) in S. clarazii plants that were defoliated twice during the growing season (Figure 2b).

Plants of S. tenuis that remained undefoliated or were defoliated once showed a positive correlation (p=0.05, r=0.70, n=8) between RLD and tissue P concentration (Figure 2c). A similar response between these two variables (p<0.05, r=0.79, n=8) was obtained on plants of S. ambigua that were defoliated once or twice in the study period (Figure 2d). Tissue N concentrations of S. clarazii plants that were defoliated twice (p<0.05, r=0.84, n=6; Figure 2e) and on those of S. ambigua that were defoliated once or twice (p<0.05, r=0.61, n=11; Figure 2f) were positively correlated with RLD.

Percentage VAM colonization and tissue P concentration were positively correlated (p<0.01, r=0.99, n=4) in S. tenuis after 6-10 days from the first defoliation (Figure 3a). These plants, however, showed a negative correlation (p=0.05, r=0.95, n=4) between percentage VAM colonization and total dry weight production (Figure 3b). This response was mostly the result of the negative correlation (p<0.05, r=0.91, n=6) between tissue P concentration and total dry weight production.

Available soil P was negatively correlated (p<0.01, r=0.84, n=8) with percentage VAM colonization on defoliated and undefoliated plants of S. clarazii at the first sampling date (Figure 4).

Discussion

Since dry weight accumulated at the time of the first defoliation was more than 75% of total dry weight production of defoliated plants of the three species, initial biomass appeared to be the major determinant of that total dry weight production. Total dry weight did not increase and sometimes decreased with age during spring for undefoliated plants of the three species (Table I). Standing crop declines have been reported in other perennial grasses during the growing season, and have been attributed to translocation, leaching, oxidation, leaf dropping and seed dissemination (Turner and Klipple, 1952; Pieper, 1978).

Towards the end of the growing season, regrowth production was greater (p<0.05) in S. clarazii than in the other two species (Table I). In a parallel study on the same plants, S. clarazii showed more than 150% greater relative growth rates and more than 33% greater daughter tiller production than S. tenuis and S. ambigua (Saint Pierre et al., 2000b). Because greater growth rates are positively related to competitive ability (Grime, 1979), our results suggest that S. clarazii is a superior competitor when compared to S. tenuis and S. ambigua. Rapid growth rates after defoliation also contribute to herbivory tolerance (Briske and Richards, 1995). Similar to the finding of Anderson and Briske (1995), our study showed that late-seral species can have an either similar or greater herbivory tolerance than earlier-seral species. Selective defoliation of late-seral species, however, would be the dominant mechanism leading to their replacement by earlier-seral species in the community (Anderson and Briske, 1995).

Lack of significant differences in root length density between defoliated and undefoliated plants suggested that photosynthetic canopy reestablishment may be achieved without sacrificing RLD in all three species, at least as long as C reserves do not become limiting. An immediate root growth reduction after defoliation would allow preferential allocation of C resources to regrowing shoots, thus rapidly reestablishing a photosynthetic canopy and returning to a shoot/root equilibrium (Davidson, 1978; Briske and Richards, 1995). Root growth reductions after defoliation could then be an important determinant of tolerance to herbivory and competitive ability in grass species (Richards, 1984). In our study, RLD and total dry weight production were positively correlated for defoliated and undefoliated plants of S. tenuis (Figure 2a). Root length density also showed positive correlations with tissue N and P concentrations in S. clarazii, S. tenuis and S. ambigua (Figure 2c-f). Under local soil nutrient supply conditions, it appeared that root activity must be maintained after defoliation in these species to reestablish a green surface area rapidly and keep resource supply to the plant at equilibrium (Chapin and Slack, 1979). For example, high root densities allowed nutrient exploitation in Schizachyrium scoparium under very low soil nutrient concentrations (Wedin and Tilman, 1993). Past selection pressure on plants and the nature of grazing itself probably contribute to the ability of grasslands to sustain or increase root biomass under defoliation (McNaughton et al., 1998).  In particular, it seems likely that a long evolutionary history of co-occurrence of forages and grazers has led to feedback mechanisms that diminish the immediate detrimental effects of defoliation on soil resource acquisition (Milchunas et al., 1988).

Similar to the findings in previous studies (Allen et al., 1989; Busso et al., 2001), defoliation did not affect levels of VAM colonization in S. clarazii, S. tenuis or S. ambigua. Apparently, defoliation did not remove sufficient photosynthetic tissue to reduce substrate availability to fungi for sufficient periods to reduce infection rates (Trent et al., 1988). Other workers, however, have reported either decreases or increases in percentage colonization by VAM after defoliation (Bethlenfalvay and Dakessian, 1984). This plasticity in the response of VAM to defoliation might be a major reason for their persistence and importance in native ecosystems (Allen et al., 1989). Other factors such as water stress, timing of defoliation and species-specific physiological traits can also influence mycorrhizal symbiosis (Allen et al., 1989), and may thus have affected our observed plant responses.

Percentages of vesicular-arbuscular mycorrhizae in the three species were similar to values found for other C₃ and C₄ annual or perennial grasses (Busso et al., 2001). However, our results contrast with studies which report that C₃ plants are only weakly dependent on mycorrhizal symbiosis even under low levels of available P in native prairie soils (Hetrick et al., 1989). Diffusion rate of ions in the soil solution, rather than uptake kinetics, appears to be the limiting factor for nutrient absorption in nutrient-poor environments. This is because nutrient availability is low on average, and nutrients beyond depletion zones must diffuse towards the roots (Wedin and Tilman, 1993). Fungal hyphae can cross depletion zones and transport P to the roots for a much smaller biomass investment than roots (Fitter and Hay, 1983). Thus, species with high uptake kinetics face a disadvantage in these environments because their high C costs associated with construction and maintenance of absorption mechanisms do not translate into higher nutrient absorption (Aerts, 1999). Under these conditions, physiological mechanisms are likely to be less important than morphological mechanisms or those related to a greater exploration of the soil volume (Jackson and Caldwell, 1996). This may be one explanation for the high VAM colonization percentages in all three C₃ perennial grass species.

Shoot nutrient concentrations were greater for defoliated than undefoliated plants of all three species (Table I). It has been widely reported that concentrations of P and N are higher in younger than older plant tissues (Mengel and Kirkby, 1982). Undefoliated plants of the unpalatable S. ambigua showed the lowest P and N concentrations in comparison to S. clarazii and S. tenuis (Table I). Other studies have already reported lower nutrient concentrations in unpalatable than in palatable perennial grasses (Cano, 1988; Wedin, 1995). Poorer litter quality in the unpalatable grasses may reduce nutrient turnover and soil nutrient availability (Berendse, 1994; Wedin, 1995). In agreement with these findings, soil under defoliated S. clarazii and S. tenuis canopies had greater available P concentrations than under S. ambigua canopies (Table I). Greater nutrient uptake rates on undefoliated plants of S. clarazii than on those of S. tenuis and S. ambigua (Giorgetti et al., 2000), however, may help explain the lower available soil P concentrations under S. clarazii than under S. tenuis and S. ambigua canopies when plants remained undefoliated.

Mycorrhizal dependency appears to be negatively related to root parameters such as root length and surface area at least for P uptake (Kothari et al., 1990). The negative correlation between percentage VAM colonization and root length density on plants of S. clarazii defoliated twice (Figure 2b) could represent a mechanism that allows this species to reduce C loss to the fungal symbiont when nutrient supply to the plant is adequate. Total length of the root system was reduced on plants of Schizachyrium scoparium after it was colonized by VAM (Anderson and Liberta, 1992). Similarly, Kothari et al. (1990) found that the most profound effect of VA mycorrhizal fungi in Zea mays was on root growth and morphology; root dry weight decreased by 16%, root length by 31% and root hair density and length by 41 and 43%, respectively.

There was a negative correlation between percentage VAM colonization and the P content of soil for plants of S. clarazii which remained undefoliated or those that were defoliated once during the growing season (Figure 4). This result, which confirms other studies demonstrating that VAM colonization is reduced in soils of higher P availability (Mohammad et al., 1998), suggests a strategy allowing S. clarazii to avoid shifts from mutualism to parasitism in the plant-mycorrhizae relationship. If soil P is not limiting, investment in a mycorrhizal association may not result in enhanced plant growth (Anderson and Liberta, 1992). However, several authors have reported that tissue P concentration of the host plant can also control VAM colonization levels (Tawaraya et al., 1994). As a result, the inhibition mechanism of VAM colonization because of the P content in the plant-soil system has not yet been fully documented (Tawaraya et al., 1996). It has even been suggested that depression of VAM colonization under high soil P contents is influenced by soil N availability (Marschner and Dell, 1994).

Except on plants of S. tenuis defoliated once, where percentage of VAM colonization and tissue P concentration showed a positive relationship (Figure 3a), percentages of VAM colonization did not correlate with shoot N or P concentrations in any other treatment. The negative correlation between tissue P concentration and total dry weight production in S. tenuis most likely reflected a dilution effect resulting from dry weight increases (Mengel and Kirkby, 1982). Experiments carried out in the field or under controlled environmental conditions have shown that mycorrhizal plants of several perennial grass species accumulated more P or N and/or had greater P or N concentrations than their non-mycorrhizal counterparts (Call and Davies, 1988; Hetrick et al., 1993; Trent et al., 1993; Noyd et al., 1995). However, mycorrhizal and non-mycorrhizal plants of Bromus inermis had similar total P concentrations (Hetrick et al., 1994).

Greater regrowth production in the more grazing tolerant and competitive species, S. clarazii, than in the less grazing tolerant and competitive S. tenuis and S. ambigua could not be explained by a greater RLD or percentage VAM colonization in our study. These variables were similar on defoliated and undefoliated plants in all three species. Saint Pierre et al. (2002) reported that both greater root length and dry weight increases in S. clarazii after defoliation appear determinant in contributing to explain its greater competitive ability and defoliation tolerance when compared with the other two species.

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