Mycorrhizal dependence and growth habit of warm ...

Mycorrhizal dependence and growth habit of warm-season and cool-season tallgrass prairie plants1 B. A. DANIELSHE TRICK,^ D. GERSCHEFSKE KITT, A N D G. THOMPSON WILSON Departmer~tof Plant Pathology, Kansas State Universi~,Manhattan, KS 66506, U.S.A.

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Received July 22, 1987 HETRICK, B. A. D., KITT,D. G., and WILSON,G. T. 1988. Mycorrhizal dependence and growth habit of warn-season and cool-season tallgrass prairie plants. Can. J. Bot. 66: 1376- 1380. Warn-season (C,) and cool-season (C,) mycorrhizal grasses were 63-215 and 0.12-4.1 times larger in dry weight than noninoculated controls, respectively. Nonmycorrhizal warm-season plants did not grow and frequently died, while cool-season plants grew moderately well in the absence of mycorrhizal symbiosis. Like warm-season grasses, tallgrass prairie forbs were highly dependent on mycorrhizal symbiosis, even though they are not known to employ the C, photosynthetic pathway. Thus, phenology may be more critical than photosynthetic pathway in determining mycorrhizal dependence. Warm-season grasses and forbs had coarser, less frequently branched root systems than cool-season grasses, supporting the hypothesis that mycorrhizal dependence is related to root morphology. Cool-season grasses may have developed more fibrous root systems because mycorrhizal nutrient uptake was not effective in the colder temperate environment in which they evolved. In contrast, warmseason plants and dependence on mycorrhizal fungi may have coevolved, because both symbionts are of tropical origin. HETRICK, B. A. D., KITT, D. G., et WILSON,G. T. 1988. Mycorrhizal dependence and growth habit of warn-season and cool-season tallgrass prairie plants. Can. J. Bot. 66 : 1376- 1380. Des herbes mycorhiziennes de climats chaud (C,) et de climats frais (C,) montrent respectivement des accroissements de 63-215 et de 0,12-4,l fois supCrieurs B ceux des tCmoins non inoculCs. Les herbes de climats chauds non mycorhizCcs ne poussent pas et m&memeurent souvent, alors que celles de climats frais montrent une croissance modCree en absence de symbiotes mycorhiziens. Tout comme les herbes de climats chauds, les arbustes des prairies B hautes herbes sont fortement debendants de la symbiose mycorhizienne, bien qu'on ne leur reconnaisse pas la capacitC d'utiliser le sentier mCtabolique de la photosynthkse en C,. I1 semble donc que la phinologie jouerait un r6le plus dkterminant que le senlier photosynthCtique, dans la ditermination de la dCpendance mycorhizienne. Les herbes et les arbustes de climats chauds posskdent des systkmes racinaires plus robustes et moins ramifiCes que les herbes de climats frais, ce qui supporte l'hypothkse que la dipendance mycorhizienne serait like B la morphologie racinaire. Les herbes de climats frais pourrait avoir dCveloppC des systkmes racinaires plus fibreux parce que l'accumulation mycorhizienne des nutriments serait moins efficace dans l'environnement plus froid des rCgions tempCrCes oh elles ont CvoluC. Par contre, les plantes de climats chauds ainsi que les champignons mycorhiziens dont elles dependent pourraient avoir co-CvoluC, puisque les deux symbiotes sont d'origine tropicale. [Traduit par la revue]

Introduction Tallgrass prairie is dominated by warm-season (C,) grasses such as Andropogon gerardi, Sorghastrum nutans, and Panicum virgatum. These plants are believed to be of tropical origin, grow most rapidly during warmer summer months, have high photosynthetic capabilities, and are highly efficient in their use of nitrogen and water (Black 1971). Presumably, aridity and high soil temperatures have exerted a selective pressure which encouraged these special adaptations in warmseason plants (Black 1971). In contrast, cool-season (C,) grasses such as Poa pratensis, Koeleria cristata, and Elymus cinereus, also common in tallgrass prairie, show lessdeveloped mechanisms for survival in hot, dry, or nutrientlimited environments (Waller and Lewis 1979). These species of temperate origin grow best in cool, moist environments, and usually grow in s p i n g or fall in warm climates. Differences in phenology and growth habits of warm- and cool-season plants have probably fostered their coexistence in tallgrass prairie (Williams and Markley 1973), although other mechanisms (divergence in rooting depth or use of pollinators) are also known to reduce or prevent interspecific competition. Symbiotic mycorrhizal fungi, which aid in plant nutrient acquisition, are common in tallgrass prairie soils (Hetrick and 'Contribution No. 88-24-5, from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS 66506, U.S.A. 'Author to whom all correspondence should be addressed. Prinled in Canada i Imprim6 au Canada

Bloom 1983; Liberta and Anderson 1986). Attempts to determine the importance of this symbiosis to grassland plants have yielded mixed results. For example, tallgrass prairie grasses such as big bluestem or Indian grass fail to grow or fail to survive in prairie soils from which mycorrhizal fungi have been eliminated by pasteurization (Heterick et al. 1986), while forage crops such as perennial rye-grass and fescue may not benefit noticeably from the mycorrhizal association (Sparling and Tinker 1978; Powell 1977; Hall et al. 1984). This general lack of response of some grasses to mycorrhizal fungi led Baylis (1972) to hypothesize that grasses, because of their fibrous, highly branched root systems, are only weakly dependent on mycorrhizal symbiosis for nutrient uptake. The present study was undertaken to determine whether native C, and C, prairie plants differed in dependence on mycorrhizae. The relationship between mycorrhizal dependence and root morphology was also investigated.

Materials and methods Seed of six cool-season grasses (prairie junegrass (Koeleria cristata L.), awnless brome-grass (Bromus inermis Leyss.), reed fescue (Festuca arundinacea Schreb.), perennial rye-grass (Lolium perenne L.), western wheatgrass (Agropyron smithii Rydb.), and basin wild rye (Elynzus cinereus Scribn. & Merr.)), four warm-season grasses (big bluestem (Andropogon gerardi Vitm.), blackwell switchgrass (Panicum virgatum L.), Indian grass (Sorghastrum nutans L. (Nash)), and tall grama-grass (Bouteloua curtipendula Michx.)) and three forbs (rough gayfeather (Liatris aspera Michx.), "Kanib"

HETRICK ET AL

TABLEI. Mycorrhizal root colonization, growth response, and dependence of warm- and cool-season grasses and forbs Root colonization (inoculated)"

Total dry weight Inoculated

Noninoculated

Species' dependence, %

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Warm-season grasses (C,) Big bluestem Osage Indian grass Switchgrass Tall grama-grass Mean Cool-season grasses (C,) Basin wild rye Awnless brome-grass Western wheatgrass Perennial rye-grass Reed fescue Prairie junegrass Mean Forbs (C,) Gayfeather Purple prairie clover Prairie false indigo Mean *All noninoculated plants were nonmycorrhizal at harvest. Percent colonization (first number): 0, no colonization; I , 1-5%; 2, 6-25%; 3, 26-50%; 4, 51-75%: 5, 75-100%. Intensity of colonization (second number): 0, no colonization I, small or widely scattered colonization sites; 2, larger or uniformly distributed colonization sites; 3, feeder roots almost entirely colonized. tInoculeted plants significantly ( P = 0.05) different from noninoculated control using lcast significant difference test. Percent dependence calculated as [(dry weight inoculated - dry weight noninoculated)/dry weight inoculated] x 100. Means followed by the same letter are not significantly ( P = 0.05) different as detemiined by Kruskal -Wallis test. Inoculated with 400 spores of Glorr~lrserrrrricc~rrrrr~.

purple prairie clover (Dalea purpurea Vent.), and prairie false indigo (Baptisia leucarztha Nutt.)) were germinated in vermiculite. After 3 weeks, 14 seedlings of each species were individually transplanted into 6 x 25 cm plastic pots containing 420 g (dry weight) freshly collected, unamended prairie soil (Chase silty clay loam, with 10 ppm plant-available P (Bray test), from Konza Prairie Research Natural Area, Manhattan, KS. Before seedling transplanting, soil was steam pasteurized at 100°C for 2 h and allowed to cool for 48 h. For each species, seven pots were each inoculated with 400 spores of Glornus etunicaturn Becker & Gerd. which were harvested from Sudan grass (Sorghum vulgare var. sudarzese (Piper) Hitch) pot cultures. Pot cultures were initiated in 1982 from G. eturzicaturn spores collected from Konza Prairie Research Natural Area, and maintained since then at 15-25°C in a greenhouse. The spores were recovered by wet sieving, decanting, and centrifuging in a 20-40-60% sucrose density gradient (Daniels and Skipper 1982). Spores were pipeted onto roots of each seedling at transplanting. The remaining pots were not inoculated. This experiment was arranged in a completely randomized design and plants were maintained at 15-25°C in a greenhouse. Plants were watered daily and fertilized biweekly with 0.0625 g Peter's No-Phos Special fertilizer solution (25-0-25) (Peter's Fertilizer Products, Fogelsville, PA 18051) dissolved in 25 mL H,O. Therefore, approximately 35 ppm N and 30 ppm K were added biweekly to each pot. After 12 weeks, plants were harvested and shoot and root dry weights were recorded. Subsamples of dried roots were stained in trypan blue (Phillips and Hayman 1970) and percent root colonization and colonization intensity were determined microscopically (Kormanik and McGraw 1982). A simple one-way analysis of variance (P = 0.05) was performed on dry weights and root colonization, using Duncan's multi~le-rangetest for mean se~aration.Mvcorrhizal de~endencies wereca1cula;ed for each plant splcies as folldws: percent Aycorrhizal dependency = [(dry weight inoculated - dry weight noninocu-

1ated)ldry weight inoculated] X 100. Primary root diameters of each plant species were measured microscopically using an ocular micrometer. In addition, root system coarseness and branching and number of primary roots were rated on a scale of 1-8: 1, one primary root (taproot), few or no lateral branches; 2, few primary roots, moderately to highly branched; 3, moderate number of primary roots (10-30), sparingly branched, coarse; 4, moderate number of primary roots (10-30), moderately branched, moderately coarse to fine; 5, moderate number of primary roots (10-30) highly branched, abundant fine roots; 6, large number of primary roots (>30), sparingly branched, coarse; 7, large number of primary roots (>30), moderately branched, moderately coarse to fine; 8, large number of primary roots ( >30), highly branched, abundant fine roots. Primary roots are defined here as the large adventitious roots emerging directly from the crown, corm, or woody caudex of the plant. Each root system was rated and the relationship between root morphology and plant species' dependence on mycorrhizae ascertained using polynomial curve fitting. A simple analysis of variance and calculation of least significant difference were performed using dry weight, primary root diameter, and rooting habit. Differences between C, and C, grasses and forbs were determined using the nonparametric Kruskal-Wallis test. To determine whether mycorrhizal dependence of C, and C, grasses and forbs differed, treatment means of each plant group (C,, C,, or forbs) rather than individual plant species were compared statistically.

Results and discussion Warm-season (C4) grasses appeared to benefit significantly from mycorrhizal inoculation (Table 1). Dry weight of mycorrhizal plants was 63 -215 times greater than that of nonmycorrhizal plants, and dependency values of mycorrhizal plants

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were 98 -99 % . Without inoculation, warm-season grasses did not grow and often died. In contrast, cool-season (C,) grasses benefitted only moderately from mycorrhizal symbiosis, i.e., in only half the species tested was the difference between mycorrhizal and nonmycorrhizal plant dry weight significant. Mycorrhizal plants were only 0.12 -4.1 times larger than nonmycorrhizal plants. Dry weights of inoculated C, grasses were generally lower than those of inoculated C4 plants, while dry weights of noninoculated C, grasses were generally much larger than those of noninoculated C, plants. In other words, coolseason (C,) grasses, with dependency values of - 15 to 75 % , were able to grow normally in the absence of mycorrhizae (although dry weight was not always as great as that achieved following inoculation), while warm-season plants did not. Thus, warm and cool-season grasses differed significantly in mycorrhizal dependence, with cool-season grasses displaying approximately one-third the dependence of warm-season grasses. Although only three forbs were examined, it is interesting to note that while all are thought to be C, plants, optimum growth of these forbs occurs during warmer summer months. These forbs might therefore be considered warm-season plants despite their C, photosynthetic pathway. Among the forbs, dependence on mycorrhizal symbiosis was 91 -97%, significantly higher than in C, grasses ( - 15 to 75 %) but still lower than in C, grasses (98 -99 %). As with the C, grasses, noninoculated forbs failed to grow beyond the size they had achieved at transplanting. The plant species used in this experiment differ in nutrient requirements, seed source, life strategy, and origin. For example, the genera Festuca, Bromus, and Lolium are of Eurasian origin, while Koeleria, Agropyron, and Elymus evolved in North American tallgrass prairie (Weaver 1954). Despite these differences, these cool-season plants responded to mycorrhizal fungus inoculation in a similar manner to each other, and very differently from warm-season plants. The environment in which the cool-season plants used in this experiment developed may have been similar, and may have resulted in low mycorrhizal dependence, regardless of plant origin. Also, the plants used in this experiment differ widely in temperature requirements for optimal growth. While a 1 5 2 5 ° C greenhouse supports growth of both warm- and cool-season species, cool-season plants are favored slightly at these temperatures. However, this experiment was repeated with similar results during the summer months, when greenhouse temperatures regularly exceeded 35°C and C, species would have been favored. Thus, the observed differences in mycorrhizal dependence appear to be genetically fixed rather than a response to current environment. The fact that C, forbs and C4 grasses respond similarly to mycorrhizal symbiosis suggests that warm- and cool-season phenology and growth habit may be more critical in determining mycorrhizal dependence than is photosynthetic pathway alone. Recently, the existence of interspecific mycorrhizal hyphal connections and their role in stabilizing nutrient availability within communities of plants have been realized (Chiariello et al. 1982; Read 1984). This phenomenon may play a major role in community ecology. Therefore, it is not surprising that plants with similar temporal growth habits have developed similar dependencies on mycorrhizal symbiosis. Berch et al. (1985) have suggested that endomycorrhizal fungi are of tropical origin. This may also be inferred from the failure of fall-seeded crops, such as winter wheat, to become mycorrhizal until late in the growing season, when soil

TABLE2. Primary root diameter and rooting habit of warm- and coolseason prairie grasses Primary root diameter (mm)

Rooting habit

Warm-season grasses Big bluestem Osage Indian grass Switchgrass Tall grama-grass Mean Cool-season grasses Basin wild rye Awnless brome-grass Western wheatgrass Perennial rye-grass Reed fescue Prairie junegrass Mean Forbs Gayfeather Purple prairie clover Prairie false indigo Mean NOTE:Only root systems of mycorrhizal plants were measured and rdted because many nonmycorrhizal plants did not grow normally and could not be regarded as representative. Values for individual species within the same column followed by the same letter are not significantly ( P = 0.05) different as determined by least significant difference test. Means followed by the same letter are not significantly ( P = 0.05) different as determined by Kruskal-Wallis test. See text for explanation of scale used to n t e rooting habit.

temperature increases (Asai 1934; Hetrick and Bloom, 1984; Hetrick et al. 1984). In addition, no cold-tolerant endomycorrhizal fungi have been identified. Therefore, cool-season (C,) grasses may have evolved alternative strategies for nutrient acquisition that minimize or reduce dependence on mycorrhizal symbiosis. Development of a fine, diffuse root system would be such an alternative strategy. As previously mentioned, Baylis (1972) hypothesized that mycorrhizal dependence was related to root morphology; plants with fibrous, highly branched root systems were believed to function more independently of mycorrhizal symbiosis than plants with coarser root systems. To explain the differences in mycorrhizal dependence of warm- and coolseason grasses, two morphological characteristics of roots, primary root diameter and rooting habit, were examined (Table 2). Primary root diameter of warm-season grasses was significantly greater than that of cool-season grasses. Only tall grama-grass (C,) and basin wild rye (C,) had similar primary root diameters. Primary root diameters of forbs were highly variable. Primary roots of gayfeather, purple prairie clover, and prairie false indigo developed from a corm or woody caudex and differed significantly in diameter. Thus, primary root diameter was not as indicative of mycorrhizal dependence for forbs as it was for grasses. The rooting-habit scale was developed to assess the overall architecture (i.e., coarseness or fineness) of root systems because primary root diameter only described morphological differences between C, and C4 grasses, and did not assess branching frequencies. Warm-season grasses had coarser, less highly branched root systems as compared with the finer, more heavily matted and interwoven root systems of the cool-season grasses (Table 2). Again, tall grama-grass and basin wild rye were somewhat

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dence on mycorrhizal fungi is also an adaptive advantage, which could explain the abundance of C4 plants in prairie soils low in available nutrients or in areas where plant competition for nutrients is great. Determining whether enhanced nutrient uptake by mycorrhizae contributes to more efficient nitrogen used by C4 plants will require further research.

y=

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Acknowledgement This research was partially supported by the National Science Foundation Long Term Ecological Research Program (grant BSR-85 14 327). ASAI, T. 1934. ~ b e rdas Vorkommen und die Bedeutung der Wurzelpilze in den Landpflanzen. Jpn. J. Bot. 7: 107-150.

BAYLIS, G. T. S. 1972. Fungi, phosphorus, and the evolution of root systems. Search, 3: 257-258.

BERCH,S. M., MILLER, K., and THIERS, H. D. 1985. Evolution of

o warm-season grasses I

cool-season grasses

ROOTING HABIT

FIG. 1. Relationship between mycorrhizal dependence and rooting habit of prairie plants. The scale used to assess rooting habit is explained in text.

different from the other C4 and C3 grasses because both were intermediate in rooting habit, having root systems that were either not as coarse or not as fine as other C4 and C, plants, respectively. Forbs, despite significant differences in $mav root diameter, were of similar rooting habit. While forb root systems were somewhat coarser than those of C4 grasses, these differences were not significant. Thus, while many forbs are C, plants physiologically, their coarser root system probably contributes to the high level of mycorrhizal dependence exhibited by these plants. When polynomial curve fitting was used to describe the relationshk-between mvcorrhizal de~endence and rooting habit (Fig. I), a second-degree polynomial curve with R2 = 0.759 and P = 0.0034 best described the data. Basin wild rye is absent from this curve because it appeared to be not representative; R2 and significance level were reduced to 0.630 and 0.01 1, respectively. This second-order equation was superior to first- and third-order models which were also examined. It appears, as Baylis (1972) suggested, that rooting habit is strongly related to dependence on mycorrhizal symbiosis. It seems likely that cool-season (C,) grasses developed more fibrous root systems because mycorrhizal nutrient uptake was not effective for them in the environment in which they evolved. Brown (1978) suggested that more efficient use of nitrogen by C4 plants confers an adaptive advantage in lownitrogen soils, and he further suggested that this adaptive advantage may explain the abundance of C4 plant species in low-nitrogen rangeland soils. This study suggests that depen-

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