Mycorrhizal activity in warm- and cool-season grasses ...

Mycorrhizal activity in warm- and cool-season grasses: variation in nutrient-uptake strategies1 B. A.D. HE TRICK^

AND

G.W.T. WILSON

Department of Plant Pathology, Kansas Stare Universiq, Manhattan, KS 66506, U.S.A. AND

A.P. SCHWAB Department of Agronomy, Kansas State Universiq, Manhattan, KS 66506, U.S.A.

Can. J. Bot. Downloaded from www.nrcresearchpress.com by Renmin University of China on 06/04/13 For personal use only.

Received December 7 , 1993 HETRICK,B.A.D., WILSON,G.W.T., and SCHWAB,A.P. 1994. Mycorrhizal activity in Warm- and cool-season grasses: variation in nutrient-uptake strategies. Can. J. Bot. 72: 1002- 1008. Because cool-season grasses display little or no mycorrhizal responsiveness in prairie soil, it is unclear whether the high levels of mycorrhizal activity observed previously in these grasses represent nutrient uptake by external hyphae or simply metabolism of stored fungal reserves in roots. To distinguish between these hypotheses, a warm-season grass, Andropogon gerardii, or a cool-season grass, Bromus inermis, were grown at two temperatures on one side of a pot divided by a 43-pm al was assessed by measuring the amount of 32Ptranslocated from one side of the pot nylon root barrier. ~ ~ c o r r h i zfunction to plants on the other side. As a control, mycorrhizal hyphae crossing the barrier were severed manually. Approximately 100 times more 32P was observed in mycorrhizal B. inermis grown at 18OC versus 29OC, and in B. inermis with intact versus severed hyphae at the cooler temperature. In contrast, A. gerardii accumulated approximately 4 times more 32P at 29°C than at 18OC, and approximately 100 times more with intact versus severed hyphae at the warmer temperature. Thus, it appears that mycorrhizal hyphae are highly active in both plant species regardless of the host's mycorrhizal responsiveness. Furthermore, mycorrhizal activity is highest at the temperature that favors growth of each species. The considerable activity of mycorrhizae in B. inermis is enigmatic since it usually has no biomass response. To further clarify the relationship between nutrient uptake and biomass response, both plant species were fertilized with a range of P levels and grown at a neutral temperature that supported the growth of both species. Although the concentration of P in B. inermis plant tissue increased in response to fertilization, there was no corresponding increase in biomass. In contrast, for A. gerardii, there was a direct and positive relationship between P fertilization and biomass produced, but tissue P concentrations remained relatively stable. Mycorrhizal symbiosis had no overall effect on biomass of B. inermis but significantly improved the growth of A. gerardii. These experiments showed clear differences in the growth strategies used by these two plant species. It is unclear whether these are differences that can be attributed to warm- and cool-season grasses in general. Short-term biomass responses as a measure of a plant's reliance on the symbiosis may not entirely reflect the contribution of the symbiosis if plants store nutrients with subsequent and perhaps delayed effects on fecundity, offspring performance, or even biomass. However, if the stored nutrient merely represents luxury consumption, this could still affect competitive ability because luxury consumption preempts the availability of nutrients for competitors. Key words: mycorrhizal responsiveness, mycorrhizal dependence, big bluestem, smooth bromegrass. HETRICK,B. A.D., WILSON,G.W.T., et SCHWAB,A.P. 1994. Mycorrhizal activity in warm- and cool-season grasses: variation in nutrient-uptake strategies. Can. J. Bot. 72 : 1002- 1008. Puisque les herbacCes de saison froide montrent peu ou pas de reaction au mycorhizes dans les sols de prairie, on peut se demander si les importantes activitCs mycorhiziennes observkes prCcCdemment chez ces herbactes peuvent s'expliquer par l'absorption par les hyphes externes ou simplement par le mCtabolisme de reserves fongiques conservtes dans les racines. Pour Cvaluer ces hypothbses, une herbacte de saison chaude, 1'Andropogon gerarHii, et une herbade de saison froide, le Bromus inermis, ont CtC cultivCes sous deux temperatures, dans un c8tC de pot comportant une barrikre de nylon de 43 pm. Le fonctionnement des mycorhizes a CtC CvaluC en mesurant la quantitt de 32PtransloquCe d'un c6tt du pot a la plante situCe de l'autre c8tC. On observe environ 100 fois plus de 32Pchez le B. inermis cultivC ?i 18°C qu'a 29°C et chez le B. inermis ayant des hyphes intacts versus des hyphes coupCs, la tempkrature froide. Au contraire, 1'A. gerardii accumule environ 4 fois plus de 32P5 2g0C qu'8 18OC, et approximativement 100 fois plus avec des hyphes intacts qu'avec des hyphes coupCs a la temperature la plus chaude. Ainsi il apparait que les hyphes mycorhiziens sont fortement actifs chez les deux espbces de plantes, indkpendament de la reaction de l'h8te mycorhizien. De plus 1'activitC mycorhizienne est plus importante a la tempkrature qui favorise la croissance de chacune des espbces. L'importante activite observke chez le B. inermis est tnigmatique puisque sa biomasse ne montre gCnCralement pas de reaction aux mycorhizes. Afin de mieux comprendre la relation entre l'absorption des nutriments et le dCveloppement de la biomasse, les deux espkces de plantes ont CtC fertilisCes avec un Cventail de concentrations de P et cultivCes h une temptrature neutre qui permet la croissance des deux espbces. Bien que la teneur en P des plants de B. inermis augmente en fonction de la fertilisation, on observe une relation positive directe avec la fertilisation en P et la biomasse produite, mais les teneurs en P dans les tissus demeurent relativement constantes. La symbiose mycorhizienne n'a pas d'effet gCnCral sur la biomasse du B. inermis, mais augmente significativement la croissance de 1'A. gerardii. Ces experiences montrent qu'il existe des differences marquCes dans les strattgies utilisCes par ces deux espbces de plantes. Il n'est pas Cvident s'il s'agit de diffkrences qui peuvent &re attribuCes de f a ~ o ngCntrale aux herbacCes de saison froide. I1 se pourrait que les rtactions de croissance sur de courtes pCriodes ne constituent pas une bonne mesure de l'utilisation de la symbiose par la plante, ne permettant pas de rendre totalement compte de sa contribution, si la plante

'Contribution No. 94-255-5 from the Kansas Agricultural Experiment Station, Kansas State University, 2Author to whom all correspondence should be addressed. Printed in Canada 1 lmprirn6 au Canada

anh hat tan,

Kans.

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HETRICK ET AL.


met en reserve des nutriments qui peuvent subsCquemment et peut Ctre tardivement avoir des effets sur la fCconditC, la performance des descendants et m&mela biomasse. Si cependant les nutriments accumulCs ne reprksentent qu'une consommation de luxe, ceci pourrait encore affecter la compCtitivitC parce que cette consommation de luxe aurait pour effet de rCduire la disponibilite des nutriments pour les compCtiteurs. Mots clis : :&action aux mycorhizes, dkpendance mycorhizienne, barbon de gCrard, brome inerme. [Traduit par la redaction]

Introduction Although mycorrhizal fungi are abundant in grasslands (Khan 1974; Stahl and Christensen 1982; Hetrick and Bloom 1983), their contribution to grasses has not been widely elucidated. In earlier studies (Hetrick et al. 1988, 1990), we observed distinct differences in the mycorrhizal responsiveness of the warm- and cool-season grasses that dominate the tallgrass prairie. Warm-season grasses appear to be obligate mycotrophs and cannot grow to maturity in native prairie soil without the additional nutrient supplied by the symbiosis. In contrast, the cool-season grasses show relatively small biomass increases or in certain cases biomass decreases in response to the symbiosis. The latter appear to be facultative mycotrophs and only respond to the symbiosis below some threshold level of nutrients. Based on these observations, we proposed that coolseason grasses do not rely as heavily on the symbiosis and have developed alternative strategies for the acquisition of nutrients (i.e., more fibrous root systems) because they grow when soil temperatures are relatively low and inhibitory to mycorrhizal fungi (Hetrick et al. 1988, 1990). Low temperatures are believed to adversely affect mycorrhizal fungi. Both metabolic activity of mycorrhizal fungi (Hayman 1983) and spore germination and germ-tube extension (Tommerup 1983) are reduced or slower at cool temperatures. Because nutrient transport from the fungus to the host plant is ATPase dependent (Gianinazzi-Pearson and Gianninazzi 1989) and enzyme activity is slower in cool temperatures, temperature may also regulate mycorrhizal fungus acquisition and transfer of nutrients to the plant. These inhibitory effects of cool temperatures have been proposed to explain the lower colonization of crops sown in autumn (Asai 1934; Hetrick and Bloom 1984) and the lower mycorrhizal responsiveness of C3, cool-season grasses (Hetrick et al. 1990). In opposition to this hypothesis, studies of plants with differing phenologies have indicated that mycorrhizal fungus activity is tied more closely to host metabolism than to the phenology of the host. Allen et al. (1984) observed arbuscules in the coolseason grass Agropyron smithii in March, while they were first observed in the warm-season grass Bouteloua gracilis in June. Similarly, Bentivenga and Hetrick (1992) reported higher mycorrhizal activity (metabolically active arbuscules) in coolseason grasses in late fall and early spring than occurred at warmer temperatures. A slight increase in biomass of mycorrhizal plants was observed in some cool-season grasses at lower temperatures, while none was observed at warmer temperatures. It was unclear, however, whether the mycorrhizal activity observed at cool temperatures represented a metabolic use of plant resources by the fungus (such as conversion of carbohydrates into stored lipids), or whether hyphal elongation, exploration of soil, nutrient uptake, and translocation to the plant still occurred at cool temperatures. The present study extends our understanding of differences in mycorrhizal symbiotic function in warm-season (C4) and cool-season (C3) plants by assessing the relationship between

plant phenology (warm- versus cool-season grasses) and ability of the plant to symbiotically acquire nutrients via mycorrhizal hyphae at two temperatures.

Materials and methods Tracer acquisition study To assess acquisition and translocation of nutrients in tallgrass prairie grasses, seedlings of a warm-season (big bluestem, ~ n d r o pogon gerardii Vitm.) and a cool-season grass (smooth bromegrass, Brolnus inermis Leyss.) were planted into divided pots. The pots were modeled after those of Faber et al. (1991) but were constructed by cutting plastic pots (10.5 cm in diameter x 18 cm in height) in half, covering each open face of the pots with nylon mesh screen (43 pm), and refitting the pots together with duct tape. The double layers of mesh screen allowed passage of fungal hyphae from side A to side B (Fig. I), while excluding passage by plant roots. Therefore, nutrient acquired by side B was attributed to hyphal uptake rather than direct uptake by plant roots. When soil was added, the two layers of mesh were closely appressed to each other and were not a barrier to mass flow. An additional removable plastic divider was placed in side B (Fig. 1). For each pot, 725 g steamed (dry wt.) of prairie soil was added to the nondivided side A and 300 g of steamed soil was added to the area between the mesh and the plastic divider on side B. The soil was a native prairie soil (a Chase silty clay loam, fine montmorillonitic mesic Aquic Arguidoll), freshly collected from Konza Prairie Research Natural Area, (Manhattan, Kans.), and steamed at 80°C for 2 h and allowed to cool and equilibrate for 72 h thereafter with no measurable change in soil chemistry. This soil had a pH of 6.4 and contained 6.0 pglg plant available P (Bray test l), 319 mglkg K, 15 mglkg NO,, 13 mglkg NH,, 1 mglkg DTPA-extractable Zn, 35 mglkg extractable Fe, and 3.9% organic matter, as determined by the Kansas State University Soil Testing Laboratory (Manhattan, Kans.). Seeds were germinated in vermiculite in a 23°C greenhouse. Two weeks after germination, one seedling per pot was transplanted into side A of each pot. Each seedling was inoculated with 400 spores of Glomus etunicatum Becker & Gerdemann (originally isolated from Konza Prairie and maintained on Schizachyrium scoparium), an efficient colonizer of prairie grasses (Hetrick and Wilson 1990). The spores were collected from pot culture by wet-sieving, decanting, and centrifuging in a 20:40:60% sucrose density gradient (Daniels and Skipper 1982). Spores were pipetted onto roots of each seedling at transplant. One-half of the plants of each species were grown in a growth chamber (Conviron model E- 15) maintained at 18OC, with the remainder in a 29°C chamber. There were 16 replicates of each plant species for each temperature. The temperature of each chamber was monitored throughout the experiment using maximum-minimum thermometers and did not deviate more than k 2 " C from the set point. All plants were maintained in a 14-h photoperiod and a photosynthetically active photon flux density of 280 pmol , mP2 . s-' (measured at shoot tops), as delivered by a combination of cool white fluorescent and incandescent lights. Plants were fertilized every 14 days with a dilute solution of Peter's No-Phos Special Fertilizer solution (25:0:25, N-P-K; Robert B. Peter's Co., Inc., Allentown, Pa.) providing approximately 35 mglkg N and 35 mglkg K to each pot. After 12 weeks, the plastic divider was removed from side B and the remaining space in the pot was filled with 425 g (dry wt.) steamed prairie soil amended with 10 mL of a 2.22 MBq 32Pand 6 rnM RbCl solution. As an additional control upon which background levels of 32Pand Rb could be established, 425 g (dry wt.) of nonamended soil

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.amended soil

P acquisition versus biomass study To further compare the relationship between P acquisition and biomass of big bluestem and smooth bromegrass, seeds of each species were germinated in vermiculite. Two weeks after emergence, seedlings were individually transplanted in 6 X 25 cm (diam. x ht.) plastic pots containing 450 g (dry wt.) steam-pasteurized prairie soil. The pots were divided into six subgroups of 10 pots each. Each subgroup was amended with 0, 25, 50, 100, 200, or 300 mg Plkg soil, applied as KH2P04 in 10 mL of water to the soil surface. Five pots of each subgroup were inoculated with G. etunicatum (400 sporeslpot) as previously described. As a control, the remaining five were not inoculated. These 12 treatments were arranged in a complete block design, maintained in a 22". greenhouse for 14 weeks, and fertilized with Peter's No-Phos Special Fertilizer Solution as described earlier. Plants were harvested, roots washed free of soil, and oven dried for 72 h for dry weight determination. Roots were subsampled, stained, and assessed for percent root colonization. Plant shoot and root tissue were digested and analyzed for P concentrations, as described earlier. Slope and y-intercept estimates were determined from linear regressions of total plant dry weight and plant P concentrations (pg gg-I) versus phosphorus amendment. Lg

-

SlDE A

rane-mesh . .. - .n. .b .. Root Barriers -

\\ SlDE B

Frc. 1. Design of pots used to assess hyphal uptake of 32Pand Rb. was added to eight pots of each species at each temperature. In half of the pots, a knife was passed between the two nylon barriers daily to sever hyphal extension between the two sides of the pots. Therefore, there were eight replicates of each species for each temperature and hyphal condition (eight intact and eight severed, of which four were amended with 32Pand four were not). Plants were harvested 14 days later and oven-dried at 80°C for 72 h for shoot, root, and total dry weight determination. Subsamples of dried roots were stained in trypan blue (Phillips and Hayman 1970) and examined microscopically to assess percentage root colonization using a Petri dish scored in 1-rnm squares (Daniels et al. 1981). Root subsamples were then destained in 0.5% sodium hypochlorite, rinsed in distilled water, and dried at 80°C for 24 h. Dried root and shoot samples were ground, passed through a 2-mm mesh sieve, and digested in double acid (HNO, -HC104 at a 1 :1 vlv ratio). Digested samples were analyzed for P concentrations (phosphomolybdate method with ascorbic acid reduction; Olsen and Sommers 1982) and Rb (atomic absorption spectroscopy). Digested samples were mixed with 5 mL of scintillation cocktail (1:l vlv for 32P quantification using a scintillation counter (Tricarb, United Technologies Packard, Downers Grove, nl.). Tissue samples from the plants receiving nonamended soil Sewed to estimate background Rb and 32P levels. An analysis of variance (ANOVA, p I0.05) with mean separation by Fisher's least significant difference (LSD) test was performed on shoot, root, and total dry weights, root colonization, root to shoot ratio, and root, shoot, and total plant P, Rb, and 32Pconcentrations and uptake (concentration X dry weight). For all the parameters measured, root and shoot measurements were highly correlated with total plant measurements; therefore, only total plant measurements are presented for simplification of data presentation. Concentration and uptake were also highly correlated for each parameter measured, and thus only uptake is presented.

Results and discussion Dry weight and total P uptake Analysis of variance for plant biomass and P uptake revealed significant interactions ( P < 0.001). However, there were no significant effects of severing on these parameters, presumably because severing occurred only during the final 2 weeks of the experiment. Therefore, only the data from plants with intact (not severed) hyphae are presented (Table i). For big bluestem plants, dry weight and total P uptake at 29°C exceeded that at 18"C, while higher growth and P uptake of smooth bromegrass plants was achieved at 18°C than at 29°C. Root colonization by mycorrhizal fingi For big bluestem with intact hyphae, significantly higher levels of root colonization (33.8 %) were observed in plants grown at 29°C than in plants grown at 18°C (17.3%). These levels were similar to those of plants for which hyphae were 'severed near the end of the experiment. There were no differences in percentage colonization of smooth bromegrass, i.e., colonization ranged from 10.5 to 15.5% whether or not hyphae were severed or plants were grown at warm or cool temperatures. * Uptake of 32P Big bluestem plants absorbed 600 - 80 000 times more 32P when hyphae were intact than when they were severed and approximately 3.7 times more isotope was translocated to the plant with intact hyphae at warm than at cool temperatures (Table 1). For smooth bromegrass, significant differences in uptake of the isotope occurred only between plants grown at 18 and 29°C with intact hyphae. There were no differences between temperatures for plants with severed hyphae, and for these plants 32P uptake was not different from that observed for bromegrass plants with intact hyphae grown at 29"C.,This suggests that in the cool-season grass, active uptake of nutrients by the mycorrhizae occurs only at cool temperatures. In contrast, for the warm-season grass, active mycorrhizal uptake of the isotope was greatest at warm temperatures but still occurred at the lower temperature. Uptake of rubidium For big bluestem, uptake of Rb paralleled uptake of 32P (Table 1). The greatest uptake by Rb occurred at the warmer

HETRICK ET AL.


TABLE1 . The influence of temperature on dry weight, P uptake, 32P uptake, and Rb uptake by the warm-season grass big bluestem and the cool-season grass smooth bromegrass Rb uptake (pglplant)

32P uptake (cprn)

Temp. ("C)

Dry wt. (g)

P uptake (rnglplant)

Severed

Big bluestem 29 18

4.19b 1.76~

32206 1589c

156d 158d

77 4076 20 716c

17.3d Od

504.66 171.6~

Brornegrass 29 18

0.77~ 8.91~

1003c 6253a

27d 556d

283d 217913~

6 . ld 47.7d.

4.4d 897.7~

Unsevered

Severed

Unsevered

NOTE:Means followed by the same letter are not significantly different (P < 0.05) as determined by Fischer's least significant difference (LSD) test.

TABLE2. Comparison of slopes and y-intercepts of regression lines for total plant P concentration and P fertilization amendment and total plant dry weight and P fertilization amendment of smooth bromegrass and big bluestem with or without mycorrhizal inoculation as shown in Fig. 2 y-intercept

Slope Bromegrass Total P (pg . g - I ) Mycorrhizal Nonrnycorrhizal Plant dry wt. (g) My corrhizal Nonrnycorrhizal

6.0~ 5.la 2.94 x 10-4b -3.72 x 1 0 - ~ b

Big bluestem 1.7b 2.2b 0.01~ 0.01~

Bromegrass 1491.3~ 1283.5~ 0.9~ 1.2~

R2

Big bluestern 1273.7~ 675.46 l.la 0.2b

Bromegrass

Big bluestern

0.83 0.82

0.61 0.57

0.00 0.05

0.79 0.76

NOTE:Slopes and intercepts followed by the same letter are not significantly different using paired I-tests (P < 0.05). Differences in slope indicate differences in response to P fertilization, while differences in y-intercepts indicate differences in plant dry weight at 0 pglg P amendment.

temperature when hyphae were intact, and more uptake occurred when hyphae were intact at both temperatures than when hyphae were severed. For smooth bromegrass, Rb uptake also paralleled 32Puptake, with greater uptake of Rb at the cool than at warm temperature only when hyphae remained intact. When hyphae were severed, Rb uptake at the cool temperature was not significantly different from the uptake at the warm temeprature. Because Rb uptake so closely paralleled 32Puptake, only the latter will be discussed throughout the remainder of the manuscript. Given that uptake of 32P and Rb were higher in plants with intact mycorrhizal hyphae than in plants with severed hyphae, it can be inferred that in P-limiting soils, mycorrhizal fungi contribute to the nutrient acquisition of the cool-season grass under cool conditions when growth of the cool-season grass is most rapid. However, the extent of mycorrhizal activity (32P and Rb uptake) in the cool-season grass at the lower temperature seems inconsistent with the relatively small mycorrhizal growth response reported for this species by Bentivenga and Hetrick (1992) and generally observed in cool-season grasses (Hetrick et al. 1990). To further investigate the relationship between P uptake and growth response in warm- and coolseason grasses, a second experiment was initiated that examined plant P uptake and biomass produced by mycorrhizal and nonmycorrhizal plants under varied P amendments. Relationship between P uptake and biomass When big bluestem and smooth bromegrass were fertilized with varied amounts of P, considerable differences in root colonization by the mycorrhizal fungus were evident. At 0, 25, 50, 100, 200, and 300 pg/g P there was 67, 70, 71, 29,

12, and 11% colonization in inoculated big bluestem, and 27, 24, 17, 14, 7, and 5% colonization in smooth bromegrass, respectively. There was no colonization in noninoculated plants. Mycorrhizal bromegrass plants had similar total P concentrations to those of nonmycorrhizal plants (Fig. 2A; Table 2). For both mycorrhizal and nonmycorrhizal plants total P concentrations increased significantly as P fertilization level increased. In contrast, there was not a significant increase in biomass of bromegrass in response to increasing levels of P fertilization (slopes not significantly different from zero; Table 2). Furthermote, dry weight of mycorrhizal plants did not surpass that of nonmycorrhizal plants (Fig. 2B). The P concentration of big bluestem did not increase significantly in response to P fertilization (slopes not significantly different from zero; Table 2), although mycorrhizal plants did accumulate more P than their nonmycorrhizal counterparts (Fig. 2A; Table 2). In contrast, the biomass of big bluestem increased sharply with increasing levels of P amendment, and mycorrhizal big bluestem plants were significantly larger than nonmycorrhizal plants (Fig. 2B). The results presented here suggest two diverse strategies by which plants acquire nutrients. The warm-season grass, which is highly dependent on mycorrhizal symbiosis (Hetrick et al. 1988, 1990), maintains similar P concentrations in plant tissue and increases in biomass when additional P is available. In contrast, the cool-season grass, which is facultatively mycotrophic and does not display a biomass increase in response to the symbiosis, appears to accumulate P when it is available even though there was no corresponding increase in plant dry weight. It is also interesting that these responses of the coolseason grass occur whether or not it is mycorrhizal. The

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0

A

Smooth brome b i g bluestem

-mycorrhizal ---nonmycorrhizal


---

-------a _____-------- ,----______-----I

.

.

.

I

.

.

.

I

PHOSPHORUS AMENDMENT

.

.

.

I

(Uglgl

FIG.2. (A) Influence of phosphorus amendments on plant P concentration of mycorrhizal or nonmycorrhizal smooth bromegrass and mycorrhizal or nonmycorrhizal big bluestem. (B) Influence of phosphorus amendments on total dry weight of mycorrhizal or nonmycorrhizal smooth bromegrass and mycorrhizal or nonmycorrhizal big bluestem. Symbols differentiate regression lines and do not represent data points.

responses of the warm-season grass also occur whether or not it is mycorrhizal, but the dry weight and P concentration levels were greater when the were mycorrhizal. Why these differences in plant strategies occur is not clear, but they may be related to the intrinsic differences between obligate and facultative mycotrophs. In this study the obligate mycotroph appeared to be more responsive to P and produced biomass in proportion to the P accumulated. Since high internal P concentrations can inhibit colonization of roots (Graham et al. 1981), it may be critical that the obligate mycotroph not accumulate high concentrations of P in plant tissues (luxury consumption). As a result of this, Janos (1984) predicted that the P concentration of obligate mycotrophs would be relatively constant over a range of soil fertilities. This prediction was supported by the present studies. In contrast, luxury consumption of P by a facultative mycotroph may be beneficial because it allows the plant to inhibit mycorrhizal infection and reduce carbon loss to the fungal symbionts when nutrient availability is adequate. Janos (1984) therefore predicts that because facultative mycotrophs can accumulate P in plant tissues, mycorrhizal and nonrnycorrhizal plants may produce similar dry

weights despite differences in tissue P concentrations. This prediction was also supported by the present study. The present study has confirmed these predictions in that (i) the obligate mycotroph A. gerardii was more highly colonized over a wider range of P fertilization levels than the facultative mycotroph B. inermis; (ii) as P fertilization increased, the tissue P concentration of the obligate mycotroph was constant, while that of the facultative mycotroph increased; (iii) the facultative mycotroph had similar dry weight despite differences in internal P concentration over the range of fertilities; and (iv) mycorrhizal and nonmycorrhizal plants with the same dry weight differed, little in tissue P concentration, suggesting that the observed differences in tissue P concentration are characteristics of the plant species rather than differences imposed by the mycorrhizal symbiosis. However, the latter is an exception to the general findings of Stribley et al. (1980) that shoots of infected plants commonly contain higher internal P concentrations than nonmycorrhizal plants of similar dry weight. They suggested that the loss of fixed carbon associated with the symbiosis could explain the failure of the plant to reach its potential growth rate despite its greater percent P concentration. While Stribley et al. (1980) concede that limitation of a nutrient(s) other than P could also explain the failure of plants to use excess P for biomass production, they do not believe the latter was the mechanism explaining their observations because the addition of P alone produced a large yield response. In the present study, however, it seems likely that the observed differences in the relationship between internal P concentration and biomass production of the obligate and facultative mycotrophs are related to the plant availability of other nutrients, particularly N. Using the logic of Stribley et al. (1980), P fertilization resulted in a yield increase only for the warm-season, obligate mycotroph. The failure of P fertilization to stimulate growth of the cool-season, facultative mycotroph suggests that P was not the most limiting nutrient. The tallgrass prairie soil is generally more nitrogen than P limited (Mader 1956; Moser and Anderson 1964; Rains et al. 1975; Woolfolk et al. 1975; Wallace 1981). The warm-season grasses are well-adapted to infertile soils and are able to reduce N concentrations in soil to a lower level than cool-season grasses (Wedin and Tilman 1992). They are consequently superior competitors for N (Tilman and Wedin 1991). The plant tissues of warm-season grasses are slower to decompose, thus immobilizing nutrients and maintaining the low N conditions under which they have a competitive advantage (Wedin and Tilman 1992). In the present study, the greater N scavenging ability of the warm-season grass would explain its ability to respond positively to P fertilization, while the cool-season grass could not. The cool-season grasses are N-limited in infertile soils (Wedin and Tilman 1992). Although N was supplied in the present experiments, it is possible that the rate of N fertilization was sufficient to elicit growth from only the warm-season grass. Further research would be necessary to confirm this. However, the present studies do indicate that plants continue to acquire P via mycorrhizae even when other nutrients such as N may be limiting to biomass production. In this way a coolseason grass may. engage in nutrient reduction, a competitive strategy'whereby P is immobilized and kept from competitors. When availability of a limiting nutrient increases, the coolseason grass may use stored P reserves to produce more -


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biomass. Alternatively, the competitive advantage associated with nutrient preemption may in itself justify the maintenance of supraoptimal nutrient concentrations in plant tissues. To the extent that nutrient accumulation and growth are not coupled in certain plant groups, estimates of mycorrhizal dependence or responsiveness (Janos 1993) based on short-term dry weight differences between mycorrhizal and nonmycorrhizal plants may not in all cases accurately reflect the entire contribution of the symbiosis. From these experiments, it also appears that mycorrhizal fungi function most intensively at temperatures that support the active growth of a particular plant species. Based on the differences in uptake of 32P observed for bromegrass plants with hyphae intact versus hyphae severed, it appears that mycorrhizal activity is considerable even in the facultative mycotroph at cool temperatures. While the amount of nutrient transported from soil to host is no doubt influenced by the proportion of metabolically active external hyphae and hyphal capacity for uptake (Jakobsen et al. 1992; O'Keefe and Sylvia 1992), the present study indicates that host carbon availability has the more profound effect on nutrient assimilation. The present studies have further clarified that the mycorrhizal activity that persists at cool temperatures in plants adapted to growth at those temperatures is not limited to metabolism of stored nutrients or pathogenic use of plant resources.

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