Effects of rhizobium, arbuscular mycorrhizal fungi and anion content of simulated rain on subterranean clover

Environmental Pollution, Vol. 92, No. 1, pp. 55-66, 1996 Published by Elsevier Science Ltd Printed in Great Britain ELSEVIER

0269-7491(95)00081-X

EFFECTS OF RHIZOBIUM, A R B U S C U L A R MYCORRHIZAL FUNGI A N D ANION CONTENT OF SIMULATED RAIN ON S U B T E R R A N E A N CLOVER S. R. Shafer, aae M. M. Schoeneberger, b S. J. Horton, d C. B. Davey, ef & J. E. Miller ac aUS Department of Agriculture-Agricultural Research Service, 1509 Varsity Drive, Raleigh, NC 27606, USA bUS Department of Agriculture-Forest Service, University of Nebraska, Lincoln, NE 68583, USA Departments of cCrop Science, dPlant Pathology, eSoil Science, f Forestry, North Carolina State University, Raleigh, NC 27695, USA (Received 22 March 1995; accepted 12 September 1995)

plants suggest that conclusions concerning the impact of acid deposition on plants in the environment cannot be considered reliable because most experiments on which such assessments are based have not tested confounding influences of microorganisms and precipitation characteristics.

Abstract

An experiment was conducted to determine the extent to which rhizobia, mycorrhizal fungi, and anions in simulated rain affect plant growth response to acid deposition. Germinating subterranean clover seeds were planted in steam-pasteurized soil in pots and inoculated with Rhizobium leguminosarum, G l o m u s intraradices, Glomus etunicatum, R. legnminosarnm+G, intraradices, R. leguminosarum + G. etunicatum, or no microbial symbionts. Beginning 3 weeks later, plants and the soil surface were exposed to simulated rain in a greenhouse on 3 days week -1 for 12 weeks. Rain solutions were deionized water amended with background ions only (pH 5.0) or also adjusted to p H 3.0 with HN03 only, H2S04 only, or a 50/50 mixture of the two acids. Glomus intraradices colonized plant roots poorly, and G. intraradices-inoculated plants responded like nonmycorrhizal plants to rhizobia and rain treatments. Variation in plant biomass attributable to different rain formulations was strongest for G. etunicatum-inoculated plants, and the effect of rain formulation differed with respect to nodulation by rhizobia. The smallest plants at the end of the experiment were noninoculated plants exposed to rains (0.38 g mean dry weight total for 3 plants pot-l). Among nonnodulated plants infected by G. etunicatum, those exposed to HN03 rain were largest, followed by plants exposed to HNOz + H2S04, p H 5.0, and H2S04 rain, in that order. Among plants inoculated with both R. leguminosarum + G. etunicatum, however, the greatest biomass occurred with p H 5.0 rains, resulting in the largest plants in the stud), (1.00 g/3 plants). Treatmentrelated variation among root and shoot biomass data reflected those for whole-plant biomass. Based on quantification of biomass and N concentrations in shoot and root tissues, total N content of plants inoculated with G. etunicatum alone and exposed to the HNO3 + H2S04 rains was approximately the same as plants inoculated with R. leguminosarum + G. entunicatum and exposed to p H 5 rains. Thus, the acid-mixture rains and rhizobia under no acid deposition provided approximately equal amounts of N in biomass. The significant interactions among rain formulation and the symbiotic status of the

INTRODUCTION

The extent of any impact of ambient acid deposition on vegetation remains uncertain despite nearly 30 years of research. Essentially all that is known has been demonstrated with applications of simulated rain, mist, or fog to plants in controlled experiments, and the relevance of these studies to plants in the environment is a continuing concern. The many complex interactions among the chemical composition of precipitation, inherent physical and chemical characteristics of plants, and the physical, chemical, and biological properties of soil contribute to experimental inconsistencies and broad speculation about effects of acid deposition on vegetation in natural and agricultural systems. Even though the complexity of this situation has been recognized and discussed, major conclusions continue to be drawn from studies in which plant responses are judged solely in the context of the acidity of ambient precipitation or a rain simulant (Shriner et al., 1990). The vast available information on the mineral nutrition of plants suggests that the overall ionic composition of simulated precipitation, aspects of the environment that affect uptake of minerals by roots, and leaching of minerals from foliage can affect the outcome and conclusions drawn from experiments concerning acid deposition effects on plants. Nonetheless, early experiments with simulated acidic rain emphasized the impact of simulant acidity per se as adjusted with sulfuric acid (e.g. Fairfax & Lepp, 1975; Wood & Bormann, 1975; Hindawi et al., 1980; Cohen et al., 1981; Lee et al., 1981; Raynal et al., 1982; Proctor, 1983). By the mid-1980s, however, the occurrence and potential importance of nitric acid deposition was 55

56

S . R . Shafer et al.

considered in simulated rain formulations (e.g. Troiano et al., 1982; Heagle et al., 1983; Irving, 1983; Shafer et al., 1985; Evans et al., 1989; Kuja & Dixon, 1989; Porter et al., 1989). The actual importance of the ionic composition of simulated precipitation in determining plant responses remains unclear because results from experiments that examine this have been contradictory: effects of simulated precipitation prepared exclusively with sulfuric acid or with a high sulfuric:nitric acid ratio on plants have been characterized as more damaging (Johnston et al., 1982; Ownby & Dees, 1985; Jacobson et al., 1990), less damaging (Irving, 1985), or not different (Rebbeck & Brennan, 1984; Jacobson et al., 1986) than that of simulant with a high nitric acid component. Variation in plant nutrient status has been suggested as an explanation for the inconsistencies among these studies (Shriner et al., 1990), but these experiments also differed with respect to deposition of simulated precipitation to foliage alone or to foliage and soil. This procedural difference alone could contribute to inconsistencies in results among experiments (Shafer, 1992). Another factor that contributes to uncertainties in acid deposition research is related to the many constant biological interactions to which plants are subject; among the most noteworthy are the symbiotic relations of plant roots with soilborne microorganisms that obtain metabolites from the roots and affect the mineral nutrition of the host. Roots of most vascular land plants in the field are infected by certain fungi, resulting in the formation of mycorrhizae. Most mycorrhiza-forming plant species are infected by fungi in the order Glomales, which form arbuscular mycorrhizae (AM) and enhance uptake of phosphorus and certain metals from soil (Marschner & Dell, 1993). Roots of leguminous plants usually are infected by AM fungi and bacteria in the genus Rhizobium or Bradyrhizobium, which form root nodules and fix N2 from air. Plant responses to acid deposition could be conditioned in part by the interactions among these microorganisms and the roots. Inclusion of leguminous plants into agricultural and forestry management systems provides an alternative to the application of manufactured fertilizers because of the biological fixation of atmospheric N (Postgate, 1978). Much research has focused on selection and development of leguminous plant-biological nitrogen fixing (LP-BNF) systems (Monson, 1978) composed of the tripartite association of plant, rhizobia, and AM fungi. Subterranean clover (Trifolium subterraneum L.), a cool-season annual, is an easily-established, widelyused crop that is more competitive than other winter clovers (Evers, 1982). This species can be useful in forestry systems because of increases in soil N and organic matter and control of erosion and weeds (Haines, 1978). The utility of subterranean clover as a forage or cover crop depends in part of the ability of the plant to flourish under a range of environmental conditions. Information on effects of environmental stresses on this and other LP-BNF systems is needed. Mycorrhizae are particularly important to LP-BNF systems in soils that are low in P (Barea & Azcon-

Aguilar, 1983; Linderman, 1992). The LP-BNF system has a high demand for P, and plants may not nodulate unless a minimum P concentration is reached in the tissues (Mosse et al., 1976). Furthermore, the N-fixation process itself has a high demand for P (Bergersen, 1971). Thus, supply of P to the host plant and to the nodules is a vital function of AM. Experiments with several species of leguminous plants demonstrated that AM plants had more nodular tissue, higher concentrations of leghemoglobin, and greater rates of acetylene reduction than nonmycorrhizal plants (e.g. Daft & E1-Giahmi, 1974, 1975, 1976). Such factors contribute to the greater fixation of N by mycorrhizal plants (Kucey & Paul, 1982; Ganry et al., 1982, 1986; Barea et al., 1987). Clearly, environmental factors and the physiological condition of the host plant influence the development and efficiency of the tripartite symbiosis. Acidic conditions in soil greatly affect all three symbionts and their interactions. Different isolates of the rhizobial symbiont of subterranean clover, Rhizobium leguminosarum biovar trifolii, differ in tolerance to acidic, high-aluminum conditions (Thornton & Davey, 1983a,b, 1984). Furthermore, effects of soil acidity on plants can vary with the species of AM fungus associated with the host roots (e.g. Hayman & Tavares, 1985). In experiments with leguminous plants, best growth and nodulation (nodule number) has varied with AM fungal species and soil acidity (Abbott & Robson, 1977; Skipper & Smith, 1979; Green et al., 1983; Huang et al., 1983). Thus, interactions among leguminous plant, nitrogen-fixing bacterium, AM fungus, and environmental characteristics such as soil acidity are quite complex. Evidence that soil acidity affects the tripartite association suggests that acid deposition could alter relationships among the three organisms. In controlled experiments, simulated acidic rain has inhibited N fixation or nodulation of leguminous plants by rhizobia (Porter & Sheridan, 1981; Shriner & Johnston, 1981; Chang & Alexander, 1983), suppressed sporulation of an AM fungus on soybean (Brewer & Heagle, 1983), but enhanced uptake of heavy metals by a mycorrhizal grass (Killham & Firestone, 1983). Experiments designed to examine effects of acid deposition on various combinations of the three organisms, however, have not been described. The objective of the experiment described here was to determine if effects of simulated acidic rain on subterranean clover are altered by rhizobia and AM fungi. The consistency of the impact of acid deposition on the tripartite association was evaluated with two species of AM fungi and variation in both acidity and anion content of the simulated rain.

MATERIALS AND METHODS Altavista sandy loam soil (fine-loamy, siliceous, thermic, Typic Paleudult; 0-10 cm depth) was collected from the Croatan National Forest in eastern North Carolina, sieved (6-mm mesh), and steamed (4 h at 80°C on 3 days

Rhizobia, mycorrhizal fungi and anions in simulated rain in a 5-day period) to eliminate indigenous rhizobia and mycorrhizal fungi. The soil was spread on a greenhouse bench, misted with double-labeled urea (15NH2CO15NH2, 99% atom excess; equivalent to 10 kg 15N ha -1) in aqueous 0.1% sucrose, and covered with a polyethylene tarp. Soil was mixed by hand several times during the 3 weeks prior to planting so that the labeled urea was well-incorporated. Soil properties (determined by the North Carolina Department of Agriculture--Agronomic Division, Soil Testing Laboratory, Raleigh, NC, USA) after incorporation of the urea were: 1.3% organic matter by weight; bulk density 1.24 g cm-3; pH 4.7; cation exchange capacity 4.7 meq 100 cm-3; base saturation 34%; exchangeable acidity 3.1 meq 100 cm-3; potassium 0.1 meq 100 cm-3; calcium 1.24 meq 100 cm-3; magnesium 0.27 meq 100 cm-3; phosphorus 7.2 mg liter-l; manganese 6.2 mg liter-l; zinc 3.4 mg liter-l; copper 0.7 mg liter -1. Soil nitrogen concentration was 0.06% as determined with a Carlo-Erba N analyzer. Polyethylene pots (11.4-cm-diam) with drain holes covered by fiberglass screen were filled with 0.5 liter of labeled soil, and each was set inside another pot of the same size. Seven planting holes approx. 5 mm diam x 5 mm deep were made in the surface of the soil in each pot. Seeds of subterranean clover cultivar 'Mt. Barker' were surface-disinfested in 80% ethanol for 10 min, rinsed five times in sterile deionized water, soaked in sterile water for 30 rain, rinsed in sterile deionized water, and placed on sterile moist filter paper in foilcovered Petri dishes for 24 h. One seedling with a 1-2mm-long radicle was transplanted into each of the seven holes in the soil in each pot. Rhizobium leguminosarum biovar trifolii isolate RO39 (obtained from A. G. Wollum, Department of Soil Science, North Carolina State University, Raleigh, NC, USA) was grown at room temperature in yeast extractmannitol broth (YEM; Vincent, 1970) cultures on a wrist-action shaker for 4 days. Half the cultures were bulked for live inoculum, and the others were autoclaved (20 min at 121°C) and bulked for rhizobium controls. Spores of Glomus etunicatum Becker and Gerdemann and Glomus intraradices Schenck and Smith (obtained from Native Plants, Inc., Salt Lake City, UT) were surface-disinfested in 10% chlorine bleach for 2 min, rinsed on a sieve under running tap water for 5 min, and diluted to 125 spores mliter-1 in sterile dilute water agar (1 g agar liter-1 water). Inoculum for a mycorrhiza control treatment was sterile water agar. Each of the fungal inocula were combined 1:1 (by volume) with live or dead (control) rhizobial inocula. For the six inoculum combinations (noninoculated controls, abbreviated R - G - ; inoculated with live rhizobia only, R + G - ; G. intraradices only, R - G i + ; G. etunicatum only, R - G e + ; rhizobia and G. intraradices, R + G i + ; or rhizobia and G. etunicatum, R + Ge + ), 500 mliter each of the appropriate inocula were combined (62 spores mliter -1 final concentration for R - G i + , R - G e + , R + G i + , or R + G e + ) , and inoculum was pipetted onto the seedling in each plant-

57

ing hole (4 mliter per pot). The soil was then closed over the germinated seeds. Plants were maintained on a bench in a non-shaded greenhouse cooled by passing charcoal-filtered air through evaporative pads. Soil was moistened several times daily with a fine mist of deionized water. One week after planting, the soil surface in each pot was covered by a 1-cm-deep layer of autoclaved perlite to insulate the soil from heat and provide some protection against soil splash. Twelve days after planting, plants were re-inoculated with fresh YEM cultures of rhizobia to ensure nodulation; 4 mliter of broth and cells from live or autoclaved cultures, corresponding to the first inoculation, were pipetted onto the perlite in each pot and rinsed into the soil with several milliliters of deionized water. Fourteen days after planting, plants were thinned to three per pot. Pots of each inoculum combination were separated into three size groups based on a visual classification of the amount of foliage in each pot; thereafter, these three size groups were the three blocks of the experiment. Plants and soil were first exposed to simulated rains 18 days after planting. Exposures occurred each Monday, Wednesday, and Friday during 12 consecutive weeks (28 January-19 April) in a greenhouse room (also cooled by charcoal-filtered air) adjacent to the plant maintenance room. The rain simulation room was covered by 30% shade cloth so that exposures never occurred under full sunlight. Immediately before each exposure, plants were moved from benches in the plant maintenance room onto rotating turntables beneath spray nozzles (Sharer, 1988) in the exposure room. Following exposure, plants were allowed to air-dry for approx. 1 h, then each was returned to its original position on a bench in the maintenance room. For each exposure, fresh simulated rain solutions were prepared from deionized water amended with stock solutions of background ions composed primarily of all sulfate or all nitrate salts (Table 1). The control rain solution contained a mixture of these background stock solutions in a 50 meq SO4-2:50 meq NO3- ratio to approximate rain chemistry typical of the area near Raleigh, North Carolina (NADP, 1985, 1986, 1987); control rain was pH 5.0 (abbreviated rain5) and was not amended further. The other three rain solutions contained only the SO4-2 background ions and was adjusted to pH 3.0 with sulfuric acid (rain3S); only the NO3- background ions and was adjusted to pH 3.0 with nitric acid (rain3N); or the NO3 -~-504 -2 background mixture and was adjusted to pH 3.0 with a 50 meq NO3-:50 meq SO4-2 mixture of nitric and sufuric acids (rain3NS). All rain solutions were adjusted within 0.1 pH unit of the nominal values. Rain deposition per exposure ranged from 0.49 cm in 12 min (first exposure) to 1.10 cm in 26 min; most exposures were 0.8-1.0 cm (Fig. 1) applied in 20-25 min. Although early exposures were short in duration and deposition to avoid thoroughly saturating the soil while plant demand for water was low, proportional accumulation was relatively constant throughout the experiment (Fig. 1). Total deposition was 32.2 cm

S . R . Shafer et al.

58 Table 1. Formulation of simulated rain

Chemical

g/10 liter H20 (Stock solution)

All N

Mg(NO3)2 Ca(NO3)2.4H20 NaCI NH4NO3 KNO3 NaNO3 H3PO4

3.97 5.90 4.28 6.69 0.45 2.65 0. l 0

All S

MgSO4.2H20 CaSO4-2H20 NaCI

4.16 4.30 4.28 l 1.09 0.78 2.22 0.10

Stock type

(NH4)2SO 4

K2SO4 Na2SO4 H3PO4

pH 5 rain: 0.6 mliter 'all S' stock+ 0.6 mliter 'all N' stock per liter H20. pH 3, all N rain: 1.2 mliter 'all N' stock per liter H20, acidity adjusted with HNO3. pH 3, all S rain: 1.2 mliter 'all S' stock per liter H20, acidity adjusted with H2SO4. pH 3, N + S rain: 0.6 mliter 'all S' stock + 0.6 mliter 'all N' stock per liter H20, acidity adjusted with H2SO4+HNO3 (50 meq: 50 meq).

applied in 36 exposures. The cumulative proportion of total deposition of ions throughout the experiment was proportional to water deposition. The experimental design was three randomized complete blocks of split-split plots. The main-plot treatments were the four rain formulations (one turntable per formulation per block); sub-plot treatments were the two rhizobium inocula (live or dead); and the subsub-plot treatments were the three fungal inocula (control, G. etunicatum, or G. intraradices). Three duplicate pots were placed in each sub-sub-plot. As plants were maintained in the growth room or exposed to simulated rain, pots were kept together in their assigned block, main-plot, sub-plot, and sub-sub-plot group. Pots in the six sub-sub-plots on each rain table were protected from

1.2 ~

i

100

l . O

rs -g

0.8

to

o~ 0.6

60

t- 0.4 "~CC E 0.2

0.~ ¢" ,"J

I .......

pS#S

0.0

I

I

cm Rain I

I

I

I

I

% of Total I

I

I

I

25

I

I

° 0

0 7 1421283542495663707784 Days Fig. 1. Deposition of simulated rain in individual exposures and accumulation over the experiment. Total deposition ffi 32.2 era.

cross-contamination by placing each pot on an inverted saucer on the table surface and separating sub-sub-plots radially by 41 x 56-cm plexiglass dividers mounted vertically on the rain tables. Hereafter, for ease of reference in the text, treatment combinations will be identified with the rain/inoculum abbreviations used above. Three examples of the 24 abbreviations (four rain formulations x two rhizobial inocula × three fungal inocula) are: r a i n 5 / R - G - (the treatment combination for plants exposed to rains at pH 5 and given the rhizobial and fungal control inocula); rain3NS/R + Ge + (simulated rain adjusted to pH 3 with the nitric+sulfuric acid mixture and inoculated with rhizobia and G. etunicatum); and rain3S/R-Gi+ (the treatment combination of plants exposed to simulated rain adjusted to pH 3 with sulfuric acid only and grown without rhizobia but inoculated with G. intraradices). Plants were harvested beginning three days after the last exposure to simulated rain. For each pot, shoots were severed at the soil line, rinsed with deionized water, placed in a paper bag, dried at 70°C for 48 h, and weighed. Loose soil was shaken from the roots, and the soil from the three pots in each sub-sub-plot was bulked, mixed, and air-dried. The pH of bulked soil from each sub-sub-plot was determined by suspending soil in deionized water (1:1, by volume), letting the suspension settle for 1 h, and then immersing a glass combination electrode into the suspension while it was stirred. This method was consistent with the method used to determine the pre-plant soil pH values. Roots were washed under running tap water, placed in glass pans, and cleaned thoroughly with forceps and artist's brushes under a dissecting microscope. Cleaned roots were rinsed in deionized water, blotted between clean paper towels, and weighed. The root system was cut into strips approx. 1 cm wide, and root fragments were selected at random until a subsample representing 3050% of the entire root fresh mass was collected. This subsample was preserved in formalin-alcohol-acetic acid (FAA; Kormanik & McGraw, 1982) and stored for assessment of mycorrhizal infection; the remainder of the roots were bagged and handled like the shoots. The fresh weight of each portion of the root system was recorded so that the fresh weight-dry weight relationship for each root system could be calculated for estimating the dry weight of the portion preserved in FAA. Preserved roots were cleared in 10% KOH and stained in lactoglycerine-Trypan Blue (Hoefnagels et al., 1993). Rhizobium nodules on the stained roots were counted, and an estimate of the number of nodules per pot was derived from the proportion of the total root mass that was represented by the stained roots. The percentage of the root length infected by mycorrhizal fungi was estimated by the gridline-intersect method (Giovannetti & Mosse, 1980). Dried tissues were weighed and ground in a stainlesssteel Wiley mill to 20-mesh size. Total N in root and shoot tissues was determined with a Carlo Erba N analyzer. Total S and P were determined from tissue digested in nitric acid and analyzed by inductively

Rhizobia, mycorrhizal fungi and anions in simulated rain

59

Table 2. Sums-of-squaru from ~ of vmtance for effects of rain formulation, rlgzobia, arlmscular mycorrhizal hmgi, and interactions on dry weight of subterranean clover after exposure to s/mulated rain for 12 weeks Source of variation

df

Shoot

Root

Total

Block Rain (R) All pH 3 versus 5 (Con 1) Rain3N versus rain3S (Con 2) Rain3NS versus rain3S (Con 3)

2 3 1 1 1

0.012"* 0.052** 0.009** 0.042"* 0.038**

0.015"* 0.046"* 0.045"* 0.001 0.000

0.050"* 0.149"* 0.093"* 0.055"* 0.047**

Error a

6

0.003

0.002

0.007

Rhizobium (Rh) R x Rh Con 1xRh Con 2xRh Con 3 x Rh

1 3 1 1 1

0.025** 0.035"* 0.032** 0.002 0.003

0.000 0.005 0.004* 0.001 0.000

0.026** 0.061"* 0.060** 0.001 0.001

Error b

8

0.009

0.004

0.015

Mycorrhizae (M)

2

0.219"*

0.244**

0.925**

RxM Con Con Con Con Con Con

6 1 1 1 1 1 1

0.064** 0.000 0.000 0.000 0.010"* 0.053** 0.033**

0.029** 0.000 0.000 0.000 0.006** 0.022** 0.021 **

0.175** 0.000 0.000 0.000 0.030** 0.144** 0.107**

RhxM

2

0.010"

0.002

0.018"

R x Rh x M Con I x R h x G - versus Gi + Con 2 x Rh x G - versus CA+ Con3 x Rh x G - versus Gi + Con I x Rh x (G-,CA + ) versus Ge + Con 2 x Rhx (G-,CA + ) versus Ge + Con 3 x Rh x (G-,CA + ) versus Ge +

6 1 1 1 1 1 1

0.064** 0.002 0.000 0.000 0.061"* 0.000 0.001

0.036** 0.000 0.000 0.000 0.035** 0.000 0.000

0.195** 0.003 0.000 0.000 0.187** 0.000 0.002

Error c

32

0.025

0.020

0.064

1x G - versus Gi + 2 x G - versus Gi + 3 x G - versus Gi + 1x ( G - , G i +) versus Ge + 2 x (G-,Gi + ) versus Ge + 3 x (G-,Gi + ) versus Ge +

Sums of squares values followed by * or ** are significant at p<0.05 or p <0.01, respectively, in an F-test. Rain formulation abbreviated rain3S=pH 3, sulfuric acid only; rain3N=pH 3, nitric acid only; rain3NS=pH 3, nitric-sulfuric acid mixture. Mycorrhizal inocula abbreviated G - = non-inoculated control, Gi + = G. intraradices, Ge + = G. etunicatum.

coupled plasma spectrophotometry (Wallace & Barnett, 1981). Results o f ~SN and total non-structural carbohydrate analyses will not be presented here. D a t a were transformed when necessary based on tests for stability of variances (Box & Cox, 1964); values for root N concentrations and number of nodules per pot required log-transformation, but transformations were not required for other data. D a t a were analyzed by analyses of variance (ANOVA) to determine significance of treatment main-effects (rain formulation, inocula) and their interactions. The sums-of-squares for each main-effect and interaction were partitioned into preplanned single-degree-of-freedom contrasts to examine relationships a m o n g treatment means. In this experiment with an acidic soil and simulated acidic rain, we anticipated that clover growth would be stimulated more by G. e t u n i c a t u m than by G. intraradices because the former was isolated originally from an acidic soil, whereas the latter was obtained from a near-neutral soil (Reese Nelson, Native Plants, Inc., personal communication). Therefore, the pre-planned contrasts involving fungal inoculum effects included one contrast in which sums-of-squares for G - and Gi + plants were

pooled and contrasted with that for Ge + plants (e.g. Table 2).

RESULTS Estimated total deposition of SO4 -2 from acid in rain3S was equivalent to 155 kg SO4 -2 h a - l ; estimated total deposition of N O 3 - from acid in rain3N was equivalent to 200 kg N O 3 - h a - l ; and estimated total deposition of SO4 -2 and N O 3 - from acids in rain3NS was equivalent to 77 and 100 kg ha -1, respectively. In comparison, wet deposition of SO4 -2 and N O 3 - approached 50 and 35 kg ha -~, respectively, at certain locations in eastern N o r t h America in 1980 (Linthurst & Altshuller, 1984). Free acidity in soil after exposure for 12 weeks to rain5, rain3N, rain3NS, or rain3S averaged p H 4.87, 4.72, 4.64, or 4.57, respectively. Thus, deposition of S contributed to acidification of soil beyond that attributable solely to the H ÷ content of simulated rain. N o foliar injury was visible on leaves of plants exposed to any of the simulated rains, but different formulations of simulated rain affected the microbial

60

S . R . Shafer et al.

Table 3. Main-effects of rain formulation, R. leguminosarum biovar trifolii, or arbuscular mycorrhizai fungi on biomass and root:shoot dry weight ratio (R:S) of subterranean clover exposed to s~aulated rain

Independent variable

Shoot

Root

Whole-plant

Root/shoot

(g pot -1) Rain formulation pH 5 pH 3, all N pH 3, N+ S pH 3, all S SE

0.332 0.338 0.314 0.270 0.008

0.239 0.187 0.180 0.177 0.006

0.571 0.525 0.494 0.447 0.012

0.721 0.535 0.558 0.654 0.015

Rhizobial inoculum Autoclaved (R-) Live (R + ) SE

0.295 0.332 0.002

0.195 0.196 0.005

0.490 0.528 0.010

0.658 0.576 0.018

0.269 0.280 0.391 0.008

0.147 0.162 0.277 0.007

0.416 0.442 0.668 0.013

0.549 0.580 0.722 0.017

Fungal inoculum

Control (G-) G. intraradices (Gi + ) G. etunicatum (Ge +) SE

Each pot contained three plants. SE, standard error of the differencebetween two means.

symbionts (AM fungi versus rhizobia) differently. Rain formulation did not cause significant effects on colonization of roots by AM fungi. The G - plants were nonmycorrhizal, and only trace amounts of mycorrhizal infection occurred in G i + plants (< 10°/0 of the root length). Mean percentage of root length infected in G e + plants ranged 40-66% but was not affected significantly by simulated rain formulation or inoculation with Rhizobium. In contrast, nodulation by Rhizobium was affected by the composition of simulated rain. Anion content of simulated rain but not acidity per se affected nodulation because rain3N and rain 3NS suppressed nodule formation by nearly two-thirds (averages of < 15 nodules per pot) relative to the numbers observed on plants exposed to rain5 (42 nodules per pot), whereas rain3S increased nodule numbers (104 nodules per pot). Because of these different effects of pH 3.0 simulated rain with or without S, the 1-df contrast for rain5 versus others was not significant, but contrasts of anion contents (all nitric versus all sulfuric acid, and nitric-sulfuric mixture versus all sulfuric) were significant (p < 0.05). The mycorrhizal status of the plants did not affect nodule numbers. Main-effects of rain formulation, rhizobia, and mycorrhizal fungi each were significant for plant biomass by the end of the 12-week experiment (Table 2 and Table 3). Averaged over rhizobial and fungal inocula, plants exposed to rain5 had the greatest total biomass per pot, whereas those exposed to rain3S were smallest. Effect of rain on shoot biomass varied among formulations: when plants were exposed to rain3N, shoot biomass, was similar to that of plants exposed to rain5. Inclusion of sulfuric acid in the simulated rain suppressed final shoot biomass. Root biomass was affected by acidity per se but not by anion composition; root biomass per pot was suppressed 22-26°/0 by any pH 3.0 rain relative to that for plants exposed to rain5.

Effect of rain formulation on total biomass was similar to that for shoot biomass. Averaged over rain formulations and fungal inocula, inoculation with Rhizobium stimulated final shoot biomass by 13% over noninoculated controls, but root biomass was unaffected by rhizobia. Responses of total biomass and root:shoot ratio to rhizobia were consistent with the effect on shoots. Averaged over rain types and rhizobial inocula, G. etunicatum stimulated growth such that shoot, root, and total biomass was 45%, 88% and 61% greater, respectively, than that of nonmycorrhizal control plants. Plants inoculated with G. intraradices, however, were similar in size to non-inoculated controls. Some two-way interactions of rain formulation, rhizobial inoculum, and fungal inocula on biomass variables were significant, but the significant three-way interaction (Table 2) provides the focus for interpreting biomass data from this experiment. One particular contrast accounted for the significance of this interaction (Table 2; Fig. 2): variation in plant biomass attributable to different simulated rain solutions was strongest for Ge+ plants, and the rain formulation effect on the Ge+ plants differed with respect to nodulation. The greatest mean total biomass was 1.00 g per pot for plants given the rain5/R + Ge + treatment combination, and the least was 0.38 g per pot for those that received the r a i n 3 N / R - G - combination. Biomass of G - or Gi + plants averaged 0.38-0.44 g per pot (0.23-0.30 g shoot and 0.12-0.21 g root per pot) regardless of rain formulation or nodulation by rhizobia. For Ge+ plants, however, rain formulation and rhizobia altered biomass (total 0.50-1.00 g per pot). Among R - G e + plants, those exposed to rain3N were largest, and those exposed to rain5 were intermediate to those exposed to rain3NS and rain3S (smallest). Among R + Ge + plants, however, the largest were those exposed to rain5; R + Ge + plants exposed to rain3N were similar in size

Rhizobia, mycorrhizal fungi and anions in simulated rain to R - G e + plants exposed to rain3N. Biomass of individual plant components (shoots and roots) reflected the total biomass (Fig. 2). Concentrations of N in shoot tissues varied with each

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S. R. Shafer et al.

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Rhizobia had little effect on N concentration in shoots of plants exposed to either of the N-containing rain formulations. However, N concentration in shoots of R - plants exposed to simulated rain without N averaged 48% (rain3S) or 54% (rain5) less than that in rain3N-exposed plants, but infection by rhizobia decreased these differences to 34-35% (Fig. 3(A)). Shoot N concentrations in G - and G i + plants were similar regardless of rain formulation, but the growth stimulation provided by G. etunicatum caused a dilution effect that was strongest in association with rain formulations without N (Fig. 3(B)). The dilution effect in Ge + plants was less apparent for R + Ge + plants than for R - G e + plants probably because the nodules contributed to tissue N in the former group (Fig. 3(C)). Relative relationships among treatment means for root N concentration were identical to those for shoot N, and the same two-way interactions were significant (p < 0.01; data not shown). Total N contained in plant tissue appeared to be related to biomass despite the dilution effect on concentration, and N content varied with the three-way interaction among rain formulation, rhizobial inocula, and fungal inocula (Fig. 4). The greatest amount of N contained in plant tissues (29 mg pot -1) occurred for nodulated or non-nodulated G e + plants exposed to rain3N (from which 48 mg N per pot was deposited by the end of the experiment), even though these were not the largest plants. Thus, even though the growth stimulation provided by G. etunicatum diluted N in tissues, the quantity of tissue produced by Ge + plants exposed to the high-N rain resulted in the greatest quantity of

Fig. 5. Significant three-way interaction effects of rain formulation, rhizobia, and mycorrhizal fungi on phosphorus (A) and sulfur (B) concentrations in shoots of subterranean clover plants (three plants pot-1) after exposure to simulated rain for 12 weeks. Abbreviations: rain3N, simulated rain adjusted to pH 3.0 with HNO3; rain 3S, simulated rain adjusted to pH 3.0 with H2SO4; rain 3NS, simulated rain adjusted to pH 3.0 with a 50:50 mixture of HNO3+HzSO4;. rain5, simulated rain without acid, pH 5.0; R +, germinated seeds inoculated with R. leguminosarum biovar trifolii; R-, non-inoculated rhizobial controls; G+, germinated seeds inoculated with G. intraradices; Ge +, germinated seeds inoculated with G. etunicatum; G-, non-inoculated mycorrhizal controls.

tissue N. Total N of rain5/R + Ge + plants was nearly three times that of rain5/R-Ge + plants and indicated that approx. 15 mg N in plant tissue per pot was attributable to N fixation when plants were exposed to rain5 (total N deposition = 1 mg per pot). When Ge + plants were exposed to rain3S (total N deposition = 0.1 mg per pot), however, only 3-4 mg N pot -1 could be attributed to fi afion. The total N content of rain5/R + Ge +, rain 3NS/R-Ge + , and rain 3NS/R + Ge + plants were nearly identical (21-24 mg N pot-l), which suggests that, of the amount of N deposited in rain3NS (total N deposition =24 mg per pot), the amount allocated to biomass was similar to that fixed by rhizobia and allocated to biomass in plants exposed to rain5. Total N content was 29 mg pot -] for both rain3N/R-Ge + and r a i n 3 N / R + G e + plants, which was 5 mg pot -1 greater than that of r a i n 5 / R + G e + plants and indicates the amount of N deposited in rain3N that exceeded N fixation and was allocated to biomass.

Rhizobia, mycorrhizal fungi and anions in simulated rain Concentrations of P and S in shoot and root tissues varied according to the same three-way interaction among rain formulations and microbial inocula that affected biomass (all p < 0.01). Shoot P concentrations in G - and Gi + plants were nearly identical. G. etunicatum enhanced shoot P concentration, especially when plants were exposed to rain5; this latter group exhibited at least twice the shoot P concentration as all others (Fig. 5(A)). Although rain acidity did not affect mycorrhizal infection levels significantly, shoot P concentrations in plants exposed to any pH 3.0 formulation were less than half that in r a i n 5 / R - G e + plants. Infection of rain5/Ge+ plants by rhizobia apparently caused a growth stimulation in addition to that induced by G. etunicatum, resulting in a dilution of tissue P in shoots. Among both R + Ge + and R - G e + plants exposed to rain at pH 3.0, P concentration was greatest after exposure to rain3N, least for those exposed to rain3S, and intermediate for those exposed to rain3NS, but these differences were small. Treatment-related trends for P concentrations in root tissues were nearly identical to those for shoot tissues, and the same three-way interaction was significant (p < 0.01; data not shown). As a group, plants exposed to rain3S (total S deposition = 55 mg per pot over the entire experiment) had the greatest concentrations of S in shoots (Fig. 5(B)). Among plants exposed to rain5 (total S deposition = 1 mg per pot), infection by G. etunicatum in the absence of rhizobia resulted in the greatest shoot S concentration, and the growth stimulation induced by rhizobia again caused a tissue dilution effect for S. Plants exposed to rain3NS (total S deposition = 28 mg per pot) had shoot S concentrations intermediate to those exposed to rain3S or rain3N (total S deposition by the latter = 0 mg per pot). As in the case of P concentrations, the variation in S concentration in roots was similar to that in shoots, and the same three-way interaction was significant (p < 0.01; data not shown).

DISCUSSION This experiment demonstrates that published assessments of the impact of acid deposition on plants must be considered rudimentary despite a large and growing amount of information on the subject. Subterranean clover plants responded as expected to the individual experimental variables in this study, i.e. plants reacted differently to different inocula of mycorrhizal fungi; rhizobia stimulated plant growth, especially when adequate P nutrition (as supplied by an effective AM fungus) was apparent; nitrate deposition inhibited nodulation; growth of non-nodulated plants was stimulated by simulated precipitation that contained nitrate but was largely unchanged by simulant containing only sulfate; deposition of nitrate and sulfate in simulated rain increased the concentrations of N and S in plant tissues; microbial symbionts that stimulated plant growth caused a 'dilution effect' for N, P, and S concentrations in tissues. Although the role of rain

63

simulant formulation, rhizobia, or mycorrhizal fungi each has been investigated in acid deposition research, our study is the first to demonstrate the extent to which all these factors can interact and complicate interpretation. Many parameters must be set arbitrarily for any experiment, and the experiment described here has limitations in terms of study environment (plants in pots, greenhouse conditions), artificial soil conditions (sieved, steam-pasteurized), and the high rate of ion deposition. During the 12-week experiment, deposition of nitrate, sulfate and hydrogen ions exceeded annual deposition estimates for the natural environment. Thus, the experimental deposition rates represent exposures that are more 'acute' than those in natural conditions, so some effects may be accentuated in our data, making quantitative extrapolation to phenomena in the field unreliable. Nonetheless, our study demonstrated how very different conclusions could be drawn from other experiments, in which the composition of simulated rain and the choice of microbial symbionts (or choice not to include them) have been decided arbitrarily. For example, the simplest experiment (typical of some early work) conducted with subterranean clover plants grown in steamed soil and exposed to simulated rain at pH 5.0 or adjusted to pH 3.0 with H2SO4 only would lead to the observation that the acid deposition caused a 17% biomass suppression. Another possible experiment conducted only with an NS mixture would be typical of most research on acid deposition but would provide essentially the same result. With selection of clover as the test plant, a researcher might choose to inoculate all plants with rhizobia because plants in the field would be nodulated; in that case, biomass suppression in an experiment with S-only or NS-mixture rain would be only 1 or 2%, respectively, and suggest that subterranean clover grown as part of a forest management system or on an acidic soil would be largely unaffected by repeated exposure to acid deposition. In an attempt to provide perhaps the 'most realistic' conditions, a researcher might use a rain simulant containing both sulfuric and nitric acids and inoculate all plants with both rhizobia and spores of some AM fungus that might be available; however, inadvertent selection of a fungus that infects poorly and provides little or no growth stimulation (such as G. intraradices in this study) would result in less than a 7% whole-plant biomass suppression attributable to pH 3.0 rain. In contrast, selection of a different fungus (such as G. etunicatum in our study) for co-inoculation with rhizobia would result in a 40% suppression of biomass by the NS-mixture rain and lead to a radically different interpretation of the impact of acid deposition on subterranean clover that might at least prompt further investigation. Clearly, the inductive value of research to date on acid deposition effects on plants remains limited, and seemingly conclusive assessments must be judged with care. The demonstration that effects of acid deposition on clover plants vary with different AM fungi is consistent with the finding that response of tomato plants to ozone

64

S . R . Shafer et al.

varied with endophyte species (McCool & Menge, 1984). We eliminated indigenous rhizobia and AM fungi by treating the soil with aerated steam, which kills many but not all species of microorganisms. Some noninfectious soilborne bacteria affect plant nutrition through activities such as P cycling. Others apparently influence infection of roots by mycorrhizal fungi, although this phenomenon has been described better for ectomycorrhizal fungi than for AM fungi (Garbaye, 1994). The extent to which the microbiological characteristics of the soil mix affected our results cannot be ascertained. Moreover, the information we obtained in trying to compare effects of different fungi is limited because one of the fungi infected roots poorly, and those plants responded to other treatments almost as if they were noninoculated. We did not evaluate the viability of the inocula before it was applied to the seedlings, but the trace level of infection obtained with G. intraradices (versus no infection in the controls) indicated that at least some portion of the inoculum was viable. Development of only a trace level of infection over a 3-month period suggests that something inhibited mycorrhizal development, but no claims for the cause can be made with certainty. Despite underlying causes, however, use of different inocula had considerable effect on plant response to rain formulations. If long-term acid deposition affects soil conditions, it might alter the species composition of the community of AM fungi in soil, and observed plant responses in a particular area might change. In a long-term experiment during which field plots were limed to different pH values, the proportion of oat or potato root length colonized by AM fungi did not differ over a range of pH 4.5-7.5. However, data on the occurrence of different spore types in the soil and morphological types of AM fungal hyphae in the roots indicated that changes in free acidity in soil altered the occurrence of different endophytes. Thus, a change in soil acidity caused shifts in species composition, but plant-related characteristics, not inoculum, controlled infection because actual infection levels did not vary among plots (Wang et al., 1993). Such shifts in fungal species available for AM development could interact with effects of acid deposition on the health of the host and effects on indigenous rhizobia in such a way that plant responses over years could change. These interactions could be modified further by other environmental characteristics, including fluctuations in precipitation chemistry. Although the proportion of root length infected by G. etunicatum was not affected significantly by rain composition, all pH 3.0 rain formulations suppressed the P concentration in shoots of these mycorrhizal plants. This suggests that P uptake by the fungus or transfer to the host was inhibited by H + deposition independently of the anionic characteristics of the solutions. Acidification of the soil could decrease the availability of the inorganic forms of P that are absorbed by hyphae in the soil and translocated to the roots because in acidic soils, relatively insoluble Fe and A1 phosphates are the

predominant forms of P (Tisdale & Nelson, 1985). Alternatively, acid deposition could have affected P translocation by some unknown mechanism. For example, the apparent difference of approx. 6 mg shoot N content per pot in rain5/R + Ge + plants versus that for rain3S/ R + Ge + plants may indicate either direct suppression of N fixation by rain3S or an interference with the interaction between the rhizobia and the P-supplying AM fungus. Such changes in the function of the AM system would constitute important indirect effects of acid deposition on plants. In most experiments on plant response to acid deposition, the anionic characteristics of the rain and the microbiological characteristics of the plant-soil system have not been major considerations. Our experiment, however, demonstrates that either can affect results and subsequent interpretation. Since the publication of Acidic Deposition: State of Science and Technology by the U. S. National Acid Precipitation Assessment Program (NAPAP) in 1990 (Irving, 1990), much of the research effort in the U. S. on plant responses to changes in air quality appears to have shifted from a focus on acid precipitation to wider issues involved in 'global climate change.' Results of the experiment reported here demonstrate that continued attention to acid deposition within the context of climate change is warranted; a reliable assessment of the impact of acid deposition alone on vegetation remains elusive.

ACKNOWLEDGEMENTS We acknowledge cooperative investigations of the USDA-ARS, USDA-Forest Service, and North Carolina State University. We thank Martha Bamford, Jeff Barton, Paula Bell, John Dunning, Patti Faulkner, Tommy Gray, Sharon Heagle, Julie Meyer, Gwen Palmer, Walt Pursley, Beth Sherrill, JoAnne Shyr, and Steve Vozzo for technical assistance; and Susan Spruill (Department of Statistics, North Carolina State University) for consultation on data analyses. This research was supported in part by a Special Research Grant from the US Department of Agriculture, Cooperative State Research Service. The use of trade names in this publication does not imply endorsement by the US Department of Agriculture or the North Carolina Agricultural Research Service of the products named or criticism of similar ones not mentioned.

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