Chemistry of root exudates and rhizosphere soils of prairie plants

Chemistry of root exudates and rhizosphere soils of prairie plants U. G. BOKHARI,' D. C. COLEMAN, A N D AMYRUBINK Ntrt~~rtrl Resource, Ecology Labortrtor:~, Colo~.crdoSttrte Urri~vrsity, Fort Collin.s, C O . U . S . A .80523

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Received September 5, 1978 , A. R U B I N K .1979. Chemistry of root exudates and BOKHARI,U. G., D. C. C O L E M A Niind rhizosphere soils of prairie plants. Can. J . Bot. 57: 1473-1477. Root exudates may have important effects on microbial growth and nutrient cycling in many ecosystems. We examined exudation patterns of two dominant plants in the shortgrass prairie. Root exudates of mature blue grama plants contained greater amounts of carbohydrates than younger plants, whereas the latter exuded slightly more amino N than the older plants. Axenic seedlings exuded larger amounts of sugars than normal seedlings; however, there was no difference in the amounts of amino N exuded. Among the amino acids exuded by axenic seedlings the levels of proline, methionine, and glutamic acids were higher than those of the otheramino acids. The rhizosphere soil of both blue grama and fringed sagewort had more soluble sugars than the nonrhizosphere soil while the latter had more nonsoluble sugars. Both soluble and nonsoluble amino N were in greater concentrations in the nonrhizosphere soil of blue grama and fringed sagewort than in the rhizosphere soil of the same species. Rhizosphere soil of the above two species contained more polyphenols than the nonrhizosphere soil. Trace amounts of terpenes were found only in the soil associated with fringed sagewort. BOKHARI, U. G.. D. C . C O L E M A N et A. R U B I N K1979. . Chemistry of root exudates and rhizosphere soils of prairie plants. Can. J . Bot. 57: 1473-1477. Les exsudats ~xcinairespeuvent avoir des effets importants sur la croissance microbienne et le recyclage des elements dans plusieurs ecosysttmes. Les auteurs ont etudie les patrons d'exsudation d e deux plantes dominantes de la prairie. Les exsudats racinaires d'individus matures de Boutclorrtr grtrcilis contiennent de plus fortes quantites d e glucides que les individus plus jeunes, tandis que ces derniers exsudent Iegkrement plus d'amino N que les plantes plus ;gees. Les plantules cultivees en conditions axlniques exsudent plus de sucres que les plantules normales; cependant, il n'y a pas d e differences dans les quantites d'amino N exsude. Parmi les acides amines exsudes par les plantules axeniques, la proline, la methionine et I'acide glutamique sont plus abondants que les autres acides amines. Dans le sol d e la rhizosphtre d e B . grtrciliset du Artc~rnisic1f,igitl(1 il y a plus de sucres solubres que dans le sol hors de la rhizosphtre. tandis que c e derniercontient plus de sucres non-solubres. Les composes amino-azotes solubles aussi bien que non-solubles sont en plus forte concentration dans le sol non-rhizospherique d e B. gl.clcilis et de A. ji.igiclc~que dans la rhizosphkre d e ces m h e s espkces. L e sol d e la rhizosphtre de ces deux esptces contient plus d e polyphlnols que le sol non-rhizospherique. Des traces d e terpknes ont I t e trouvees dans le sol associe au A . ji.i!iclc~. [Traduit par le journal]

cells (Griffin et 01. 1976; Martin 1977; Hale et ril. 1978). Soluble exudates released by plants may exert a 'priming effect' (i.e., an enhancement of immobilization and remineralization by microfauna of inorganic N and P) on microbial growth (Coleman et rrl. 1977). This enhanced nutrient regeneration may play an important role in the establishment and growth of plants, particularly in nutrient-poor situations. The allelopathic effect, i.e., the influence of one plant on another through chemical substances released by plant roots or leached from the foliage or the decomposition by-products of litter and dead roots, has been extensively studied in cultivated 'Present address: United States Department of Agriculture. Science and Education Administration, Southwestern Live- plant communities (Whittaker and Feeny 1971) and stock and Forage, Research Station, Route 3 El Reno, O K , natural plant communities (Rice 1974; Newman U.S.A. 73036. and Rovira 1976; Bokhari 1978). The 'rhizosphere

Introduction Root exudation and the release of organic and inorganic substances from the roots of intact plants occur under various environmental conditions (Richter et rrl. 1968; Rovira 1969; McDougall 1970, Rovira and Davey 1974; Smith 1976; Barber and Martin 1976). Root exudation and the contents of the exudates are influenced by both the aboveground and belowground plant environment and by rhizosphere microorganisms (Rovira 1969; Martin 1971, 1977). The rhizosphere effect is the composite of the interaction of soil microflora with root exudates, root lysates, mucigel, and intact plant

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CAN. J. BOT. VOL. 57, 1979

effect' on plant growth and soil microorganisms has been studied in cultivated crops. Blue grama (Boureloua g~.cr~.ilis (H.B.K.) Lag. ex Steud) is a major constituent of the producer subsystem in the shortgrass prairie ecosystem; and fringed sagewort, a potential source of volatile substances, is closely associated with blue grama vegetation. Thus, we investigated root exudates of blue gl-ama ( B O L ~ ~ ~ grnc-ilis I O I I N (H.B. K.) Lag. ex Steud) and the rhizosphere and nonrhizosphere soils of blue grama and fringed sagewort (Arremisicr .f,-igidcr Willd.). The objectives were (1) to understand the amount and quality of root exudates and rhizosphel-e soils as a source of nutrients or- phytotoxicity for soil microorganisms colonizing the rhizosphere and rhizoplane of prairie plants, and (2) to assess the effect of microorganisms on root exudation in a study consisting of axenic and nonaxenic plants. Methods and Materials Blue gmma plants were raised from seeds and were grown in environment-controlled growth chambers at 30 and 24°C (day and night) temperatures alternating with 12-h photoperiods at 90 Wlm2. Seeds were surface sterilized with 15% commercial Chlorox for 20 minutes and then rinsed with sterile water. Seeds were germinated in a 25-mL Erlenmeyer flask previously autoclaved and containing I to 2 m L of sterile water. Germinated seedlings were transferred to test tubes containing agar medium (0.5% agar, 0.2% Bacto-peptone, 0.3% yeast extract. 0.5% glucose) to test for contamination. Those found to be surface sterile were transferred to a modified Berzelius beaker (Corning glassware 1040) containing 6 strength sterile complete Hoagland's solution. Fresh nutrient solution was added to each plant at 5-7 day intervals a s needed. The flat base of the beaker was drawn out to a conical funnel which extended into a glass tube for withdrawing samples. Beakers were kept in growth chambers and were not aerated. Root exudates were collected once at the end of 30 days from seedlings, at five-leaf stage from plants, and at maturity. The bulk root exudates were first filtered through Millipore filters (0.45 and 0.27 pm) and then concentrated in a rotary evaporator. The dried exudates were redissolved in 10 m L of 10% isopropanol. For the study of rhizosphere soils, blue grama and fringed sagewort sods were excavated from the native shortgrass prairie site and immediately transferred to growth chambers maintained at conditions stated above. These plants were allowed to establish themselves in prairie soil in plastic buckets for 2 months, at which time the rhizosphere and nonrhizosphere soil was collected for chemical analyses. Plants of blue grama and fringed sagewort were hand pulled and gently shaken to remove most of the loose soil. The soil that remained firmly adhering to the roots following shaking was removed carefully from all the roots and was considered rhizosphere soil (Louw and Webley 1959). Total soluble sugars in root exudates of blue grama were determined by the anthrone method (Morris 1948) and values were expressed as glucose equivalents. Free amino N was determined as ninhydrin-positive substances (Rosen 1957) and values were expressed a s tyrosine equivalents. Root exudates were first fractionated into sugars, neutral compounds, organic acids, and amino acids o n exchange resins. Analytical-grade cation exchange resin AG-50W-X8, hydrogen

form, 20-50 mesh. was used to separate amino acids from other compounds by passing root exudates through the column (13 cm) at the rate of 3 m L min-', followed by rapid washing with 35-50 mL of deionized water. The eluate contained sugars, neutral compounds, and organic acids while amino acids were retained by the column and were removed by passing 75 mL of 2 N HCI at a rate of 3 m L min-I, followed by washing with 50 m L of deionized water. The fractions containing the sugars, neutral compounds, and organic acids were passed through anion exchange resin, AG-I-X8, chloride form. 20-50 mesh. Organic acids from the anion exchange column (13 cm) were removed with 100 mL of 8 N formic acid followed by a rapid wash with 50 m L of water. All the fractions were dried using a rotary evaporator at a temperature not exceeding 40°C. These were redissolved in 10 m L of 10% isopropanol and an aliquot from each fraction was taken fordeterminationofc:u.bohydrates and free amino N. Amino acids were analysed by an automatic amino acid analyser. Soluble sugars in the rhizosphere soils were extracted from a 5-g air-dried soil sample with 50 m L of hot water for 48 h. Nonsoluble sugars (cellulose, lignin) were extracted from the soil by hydrolyzing a 5-g sample with 50 m L of 6 N HCI for 24 h. Sugar content was determined by the anthrone method and is expressed as glucose equivalents. The amino acids in the soil were extracted with hot water (soluble amino N) and with 8 N HISO, (Wang e / trl. 1967) and determined a s ninhydrin-positive substances (Rosen 1957). Phenolic compounds in the soil were extracted by the method of Guenzi and McCalla (1966) and were determined colorimetrically a s polyphenols (King and Heath 1967). For the extraction of polyphenols, a sample of250g of soil was shaken with250 m L of 2 N NaOH for 4 h, centrifuged at 20 400 x g for 10 rnin, and filtered through a pad of glass wool. The filtrate was acidified to pH 2 with HCI and recentrifuged. The clear filtrate was extracted with diethyl ether, washed with 5% NaOH, and again acidified and reextracted with ether. Volatile substances (terpenes) were extracted from the soil by incubating a 50-g soil sample with 200 m L of anhydrous ethyl ether for 48 h and an aliquot of the above extract was analysed by gas chromatography (Muller and Muller 1964). Individual terpenes were identified by comparison with the profiles of known standards. Sloughed-off root materials were measured by filtering the root exudates onto a filter paper and weighing the residues retained on filter paper. Data were analysed by one-way factorial analysis of variance and by Tukey's Q Test ( p < 0.01). All results mentioning significant differences are at p