THE EFFECTS OF ACID PRECIPITATION AND OZONE ON THE ECTOMYCORRHIZAE OF RED SPRUCE SAPLINGS

DIANA R. ROTH and TIMOTHY J. FAHEY 

Department of Natural Resources, Fernow Hall, Cornell University, Ithaca, NY 14853, U.S.A. E-mail: tjf5@cornell.edu (Received 22 March, 1995; accepted in nal form 8 January, 1997)

Abstract. The effects of acid precipitation and ozone on the composition of the ectomycorrhizal community of red spruce saplings were evaluated. In 1986, saplings were excavated from a site in Maine that had been clear-cut in 1979. Saplings were then potted in native soil and transported to Ithaca, New York. With the exception of an ambient control treatment, trees were grown in opentop chambers. Saplings were exposed to ve levels of ozone and three levels of acid precipitation beginning in July 1987. Ectomycorrhizae were sampled in 1988 and 1991 after one and four years of treatment, respectively. Although the percentage of root tips infected by ectomycorrhizal fungi was not affected by treatments, a shift in the composition of the ectomycorrhizal community occurred in response to acid precipitation treatments for both sampling years. Among the seven ectomycorrhizal morphotypes identied, the percent composition of one morphotype increased and another decreased in response to higher rain acidity. Alone, ozone treatments did not inuence ectomycorrhizal composition; however, a signicant interactive response to ozone and acid precipitation was observed in the organic soil horizon in 1988. Such shifts in the composition of the ectomycorrhizal community may indicate that the experimental trees were stressed by pollution treatments. Key words: air pollution, acid rain, forest decline, morphotypes, Picea rubens

1. Introduction Airborne pollutants have been implicated in the unusual decline of red spruce populations (Picea rubens Sarg.) that has occurred in recent years at high elevations in the northeastern United States (Johnson and Siccama, 1989). Among the pollutants of concern are ozone and acid precipitation (Johnson and Siccama, 1983; McLaughlin, 1985). The objective of this study was to quantify the response of the ectomycorrhizae of red spruce saplings to treatments of acid precipitation, ozone or the interaction of the two. A shift in the composition of ectomycorrhizae in response to treatments might result from differences in abilities to adapt to environmental stresses, which in turn may affect forest health. This research was part of a larger study to assess the effects of acid precipitation and ozone on the growth, physiology, and nutrition of red spruce (Kohut et al., 1987) called ROPIS (Response of Plants to Interacting Stresses). While there is concern that air pollution may be adversely affecting the mycorrhizae of forest trees, experimental studies of various tree species have given conicting results regarding cause and effect relationships. Several investigators have
 Author for all correspondence.

Water, Air, and Soil Pollution 103: 263276, 1998. c 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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established that acid rain decreases mycorrhizal colonization (Stroo and Alexander, 1985; Reich et al., 1986; Esher et al., 1992), whereas Adams and ONeill (1991) observed no effect of acid rain on mycorrhizal colonization. Moreover, Shafer et al. (1985) found that acid rain enhanced the formation of ectomycorrhizae. Similarly, in some studies ozone has been shown to suppress mycorrhizal colonization in pine seedlings (Meier et al., 1990; McQuattie and Schier, 1992), whereas no effect of ozone was observed in a study by Mahoney et al. (1985). Several studies have addressed the effects of both acid deposition and ozone on ectomycorrhizae. Blaschke and Weiss (1990) did not detect a response to ozone or acid mist on ectomycorrhizae. In contrast, Stroo et al. (1988) and Reich et al. (1985) observed effects of acid rain and ozone on mycorrhizal infection. As a given tree species may be associated with many different fungal species (Barbour et al., 1980), some recent studies have addressed whether or not a change in the occurrence of ectomycorrhizal morphotypes is observed in response to acid rain or ozone treatments. In one study (Dighton and Skefngton, 1987), the growth of two of six morphotypes of Scots pine (Pinus sylvestris L.) was suppressed as rain pH decreased. In a study of loblolly pine (Pinus taeda L.), Edwards and Kelly (1992) reported a shift in ectomycorrhizal morphotypes in response to ozone treatments, whereas acid precipitation treatments did not have an effect. Meier et al. (1989) found that of ve morphotypes observed on red spruce, four were not affected by short term acid rain and the number of ectomycorrhizal tips of one type (Cenococcum geophilum) increased with rain acidity. The results of that experiment suggested that there may be changes in the relative occurrence of specic morphotypes or fungal species in response to acid rain. In general, most past studies addressing the effects of acid deposition and ozone on mycorrhizae have quantied the total number of infected tips, total percent infection or the number of short roots per gram ne root biomass (or per root length). In order to assess potential treatment effects on the composition of the ectomycorrhizal community, the present study quanties the percent contribution of the different morphotypes for each red spruce sapling.

2. Materials and Methods 2.1. EXPERIMENTAL DESIGN Red spruce saplings were collected from a site in northern Maine (60 km north of Millinocket) in May of 1986. The plot was clear-cut in 1979, and soils at the site are Typic Haplorthods. Tree height at the time of sampling averaged 1.3 m. Trees were trenched to the base of the major rooting zone (about 15 cm) and then removed so as to keep the soil around the roots intact. There was little ground vegetation other than mosses at the site and the few herbaceous plants were removed from the samples. Each sapling was then placed into a 1 m diameter, 360 l plastic pot

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which had been backlled with B horizon soil from the site. The saplings were then transported overnight to a eld site in Ithaca, New York. Pots were placed below ground, with one-half resting on weighing lysimeters (to allow for continuous weight measurements of the water-soil-pot system) (Sherman and Fahey, 1994). Based on similarities in photosynthetic rate and crown morphology, 90 trees were chosen for use in the full experiment (Sherman and Fahey, 1990/91). All trees in the experiment were healthy and vigorous at the initiation of treatments. Four ozone levels were applied to open-top fumigation chambers as follows: 0.5 times ambient (charcoal-ltered air = CF), ambient (air not ltered = NF), 1.5 times ambient (1.5x), and 2.0 times ambient (2x). An ambient treatment (amb) without a chamber was used as a control to assess chamber effects. The trees in the ambient treatment were exposed to ambient conditions, except during natural and simulated rain events, when the trees were covered with a sliding chamber. As the experiment had three blocks, each ozone chamber was replicated three times. Each ozone chamber was split into three subplots containing two spruce trees that received simulated rain of different pH (3.1, 4.1 and 5.1). All ambient precipitation was excluded from the chambers during the ozone fumigation period (i.e., April to October), and simulated rain events were scheduled based on the soil water status of the pots (Sherman and Fahey, 1994). Simulated precipitation treatments were prepared in batch using deionized water, distributed to chambers, and then applied to each subplot via a nozzle located above the trees (Kohut et al., 1990). All acid precipitation treatments had a sulfate to nitrate ratio of 2 to 1 and contained background ion concentrations typical of rain in Ithaca (Kohut et al., 1987, Lauver et al., 1990). Simulated rain events averaged approximately 2.5 hours and average deposition per simulated rain event for the entire period was 28 mm (Sherman and Fahey, 1994). Annual addition of strong acids varied across years within treatments; nitrate deposition ranged from 6.73 to 9.04 g/m2 -yr in the pH 3.1 treatment, 1.02 to 1.37 in pH 4.1, and 0.58 to 0.71 in pH 5.1. Analogous values for sulfate were 13.5 to 18.1 g/m2 -yr in the pH 3.1 treatment, 2.0 to 2.7 in pH 4.1 and 1.2 to 1.4 in pH 5.1. Ozone and simulated rain treatments began in July of 1987, and soil cores were collected in the fall of 1988 and the early spring of 1991 after one and four years of treatment, respectively. As there were two trees per acid rain subplot, one of the two trees was sampled in 1988, and the other in 1991. Because sampling protocols differed for the two dates, the procedures and results are described separately below. 2.2. SAMPLING PROCEDURES FOR 1988 Samples were taken from saplings within the three pH treatments (3.1, 4.1, and 5.1) and the ve ozone treatments (amb, CF, NF, 1.5x and 2x). A 2-cm diameter metal soil corer was used to randomly sample both the organic (Oa) and upper mineral (Bs) soil horizons to a depth not greater than 25 cm from a total of 45 potted trees (from 3 rain pH levels, 5 ozone levels and 3 blocks). Two cores were taken from the

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upper Bs horizon of each pot. For the Oa, at least one core was taken from each pot. Additional Oa cores were taken in conjunction with a ne root study which was conducted at the same time. Cores were taken from randomly-chosen locations, avoiding the areas within a few cm of the pot edges and the tree bole. Soil was gently removed from roots immediately after coring, and root samples were stored in glass vials in a 1.2% glutaraldehyde solution. 2.3. SAMPLING PROCEDURES FOR 1991 Ectomycorrhizae were sampled in conjunction with a study of the soil rhizosphere chemistry. Therefore, it was important to use a corer that would allow removal of the soil sample from the corer without disturbing the root environment. Corers were made with 1.5-inch diameter PVC pipe, split in half along the length of the pipe, and held together during coring with cable ties. Cores were randomly sampled from pots following the same protocol as for 1988. Whereas in 1988 all ozone and simulated rain treatments were sampled, only three ozone levels (NF, 1.5x, 2x) and two rain pHs (3.1 and 5.1) were sampled in 1991. Eighteen potted trees (from 2 rain pH levels, 3 ozone levels and 3 blocks) were sampled from both the organic (Oa) and upper mineral (Bs) soil horizons. Two cores per pot were randomly taken just as the soil was beginning to thaw (i.e., late March and early April). These cores were split into an organic sample (surface to E horizon) and the upper Bs horizon (approximately 10 to 25 cm in depth). If the organic horizon was very thin, it was sometimes necessary to take additional Oa cores in order to obtain sufcient root material. Soil cores were immediately stored in two sealed polyethylene bags (to form a double-layer) at 4  C. A randomly chosen, intact portion of the core was later taken from cold storage and roots were then carefully separated from the soil. All root samples were preserved for future analysis. Small root fragments (< 2 cm in length) were not included in the sample because of time constraints and problems with dessication during protracted sample processing. The samples were stored in glass vials in a solution of 95% methanol and 5% glycerol. In order to monitor any potential changes in color that might occur during storage in alcohol, one sample of each morphotype was set aside, and the color was noted using color chart codes at the beginning and end of storage (Royal Horticultural Society, 1966). No color changes were observed during the storage period. 2.4. MORPHOTYPE CLASSIFICATION In 1988, morphotype classications were determined in conjunction with Dr. J. Dighton at the Institute of Terrestrial Ecology, Merlewood Research Station in Cumbria, United Kingdom. In preparation for the 1991 study, written descriptions, sketches and photographs from the 1988 analysis were studied in detail. In order to assure that morphotype classications were the same in both years, the person who

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conducted the 1988 analysis was consulted in 1991 to clarify any uncertainties in the classication scheme (S. Wapner, personal communication). The preserved roots were removed from vials and put into a petri dish (i.e., lled with storage solution) for viewing under a dissecting stereoscope. A 9  9 cm (81 squares) grid was placed under the petri dish. Fifteen grid cells were randomly selected for counting. Root tips in these cells were classied into the following categories of ectomycorrhizal morphotypes based on Hartig net presence, color, texture, shape and mantle characteristics: type A, type H, type B, type E, type F, type J, type X, non-ectomycorrhizal (NON), and broken-dead-unidentiable (BDU) (e.g., as in Glenn et al., 1991). In 1988, approximately 34 000 total root tips were examined (sampled from 45 trees) and in 1991, approximately 14 000 total root tips were examined (sampled from 18 trees). In brief, type A is characterized by a black mantle and black hyphae, type E corresponds to a golden-brown ectomycorrhizae, type B corresponds to a wooly white ectomycorrhizae, and type X corresponds to a silvery gray ectomycorrhizae. Because mantle formation did not appear to be complete for types J and F, they were categorized as potentially ectomycorrhizal; these two morphotypes may be developmental stages of other morphotypes. Type H is similar to type A, but differs in that the tip of the root is not encompassed by the fungus and the root tip appears to have emerged from what was previously type A. These categories are described in more detail elsewhere (Roth, 1993), and are similar to morphotype categories that have been described by others (e.g., Meier et al. 1989; Glenn et al. 1991). 2.5. STATISTICAL ANALYSIS Although there were 90 possible observations (2 soil horizons x 3 rain pH levels x 5 ozone levels x 3 blocks) in 1988, 7 observations were missing (i.e., samples were lost), and a total of 83 observations were used in the nal analysis. In 1991, all of the 36 possible observations (2 soil horizons x 2 rain pH levels x 3 ozone levels x 3 blocks) were analyzed. To correct for the non-normal distribution of the data, the proportions (number of tips of a morphotype per total tips per tree) were transformed by the arcsine of the square-root (Sokal and Rohlf, 1981; Snedecor and Cochran, 1989), resulting in a normal distribution. While the transformed data were used for all statistical analyses, means presented in this paper are actual means. Statistical analyses were carried out using SAS on an IBM mainframe. A splitsplit plot analysis of variance was conducted using the SAS GLM procedure to determine whether the percent composition of mycorrhizal morphotypes responded to treatments (SAS Institute, 1985; 1991). Post-ANOVA analysis included checking for signicant effects in the model and then calculating orthogonal contrasts (i.e., when more than two treatments) with SAS to better analyze the effects of (1) simulated rain pH treatments, (2) ozone treatments, and (3) chamber effects (in

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1988 only, when the ambient treatment was sampled) (Neter et al., 1985). Contrasts were constructed to include the assessment of trend type (e.g., linear, quadratic) when relevant (i.e., more than two treatments per model effect). Multiple contrasts were analyzed using the Bonneferonni method: a given statement is rejected if p > (0.10  n), where n equals the total number of contrasts (Mason et al., 1989). In one case, evaluation of a three way interaction term (horizonpHozone) for the 1988 data required the use of the Scheff multiple comparison test (at alpha = e 0.10) because Bonneferonni comparisons were non-estimable using SAS. 3. Results 3.1. GENERAL In 1988, nine ectomycorrhizal morphotypes were described initially. However, a number of the original 1988 categories did not appear to be distinctly different and were combined for the nal data analysis to make a total of six categories. Overall, ve morphotypes (types A, H, E, B, and F) were observed both in 1988 and 1991. Type X was observed only in 1988, and three categories, type J, nonectomycorrhizal (NON), and broken-dead-unidentiable (BDU), were described only in 1991. Two morphotypes, type X in 1988 and type B in 1991, were not analyzed using ANOVA statistics because they were rarely observed and data points were not normally distributed. Although not statistically signicant, in both cases relative abundance was greatest at high ozone (2x) and low rain pH (3.1) (Tables I and III). 3.2. RESPONSES AFTER ONE YEAR OF TREATMENT (1988) Of the six mycorrhizal morphotypes examined, only type B appeared to be affected by the presence of chambers (i.e., the amb versus the NF treatment). There was a signicantly greater percentage (p = 0.003) of morphotype B in the pots outside of the chambers (amb) than inside chambers (Table I). Ozone treatments did not have an effect on any of the morphotypes. Analysis of the response of morphotype A to pH indicated that both a linear (p = 0.025) (i.e., as pH increases, the proportion of type A decreases) and quadratic trend (p = 0.006) were signicant. As there is not a clear biological explanation for the presence of a quadratic trend, the linear trend appears most plausible. Several morphotypes exhibited a horizon response. Type A (p < 0.001) and type F (p = 0.097) contributed more to the mycorrhizal composition of the organic than the mineral horizon. Conversely, type E (p = 0.015) contributed less in the organic than the mineral horizon. Types A and E both exhibited a horizonpHozone interaction (type A, p = 0.033; type E, p = 0.056). For both the NF and CF treatments in the organic horizon, there was a greater percentage of type A in the pH 3.1 treatment than

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Table I Mean percent composition (and standard error) by ectomycorrhizal morphotype at different ozone levels, rain pH levels and horizons after one year of treatment (1988). Morphotypes with signicant model effects are denoted with asterisks, where  = (p-value 0.10), and  = (p-value 0.05)

<

<

Ectomycorrhizal morphotypes Ozone amb CF(0.5x) NF(1x) 1.5x 2x A 15.4 (3.3) 14.1 (4.9) 12.9 (3.4) 16.3 (5.9) 13.0 (2.9) A 15.8c (3.9) 18.5c (3.1) 8.4c (1.8) A 21.0 (2.9) 8.3 (1.9) H 22.4 (4.4) 20.2 (4.7) 19.0 (4.7) 17.7 (3.4) 21.3 (4.9) H 15.8 (3.8) 22.9 (3.4) 21.8 (2.9) H 21.4 (2.5) 19.1 (3.0) B 10.4a (3.6) 3.6b (1.8) 2.9b (1.2) 1.9b (0.7) 3.9b (1.3) B 3.7 (1.1) 5.1 (2.0) 4.8 (1.5) B 3.1 (0.9) 5.8 (1.5) E 32.6 (5.3) 36.3 (5.7) 39.0 (8.2) 37.6 (6.1) 31.9 (5.4) E 36.7 (5.6) 29.3 (4.1) 40.5 (4.4) E 25.8 (2.2) 44.0 (4.4) F 16.8 (3.6) 24.8 (3.8) 23.1 (4.5) 24.1 (3.9) 22.5 (4.0) FX 21.4 (3.1) 22.0 (3.0) 23.5 (3.2) F 25.8 (2.5) 19.1 (2.5) X 2.4 (1.4) 1.0 (0.6) 3.2 (2.9) 2.3 (0.9) 7.5 (5.1) # obs 6.6 (3.4) 2.4 (1.6) 1.1 (0.5) X 3.0 (2.2) 3.7 (2.2) n = 28 n = 28 n = 27 # obs n = 16 n = 16 n = 17 n = 16 n = 18

Rain pH 3.1 4.1 5.1

Horizon Oa upper Bs
a;b

# obs n = 39 n = 44

Mean values with different letters for a given ozone treatment differ signicantly from each other, whereas those with the same letters do not. c Tests for linear and quadratic trends were both signicant.

in pH 4.1 and 5.1 (Table II). For type E in the CF treatment, there was a smaller proportion of type E in the organic horizon in the pH 3.1 treatment than in pH 4.1 and 5.1. Although this rain pH response was signicant only for the ozone levels given above in the Oa (i.e., NF and CF for type A, and CF for type E), the data were analyzed to see if there was a similar trend for non-signicant ozone levels in the Oa. The contribution of type A increased as rain pH decreased for all but the highest

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Table II Mean percent composition (and standard error) of ectomycorrhizal morphotypes A and E within ozone and rain pH levels for the Oa horizon after one year of treatment (1988). Morphotypes with signicant effects are denoted with asterisks, where  = (p-value 0.10),  = (p-value 0.05)

<

<

Rain pH level Ozone level Morphotype A amb CF (0.5x) NF (1x) 1.5x 2x 21.3 (3.1) 32.9a (19.5) 30.0a (4.1) 40.1 (24.4) 10.8 (4.3) 19.5 (3.0) 21.6b (16.3) 17.1b (3.7) 32.0 (18.6) 29.9 (11.3) 4.9 (0.0) 15.4b (14.3) 10.5b (0.9) 9.2 (1.8) 14.0 (7.2) pH 3.1 pH 4.1 pH 5.1

# obs

n=7 n=7 n=8 n=8 n=9

Morphotype E amb CF (0.5x) NF (1x) 1.5x 2x 20.6 (10.4) 12.3a (1.8) 13.0 (1.6) 17.3 (9.4) 42.4 (4.3) 30.0 (2.2) 24.2b (6.8) 23.4 (6.6) 20.1 (7.9) 17.6 (10.9) 34.8 (0.0) 25.0b (0.8) 30.7 (5.9) 37.7 (8.4) 36.5 (12.2) n=7 n=7 n=8 n=8 n=9

a;b Mean values with different letters for a given ozone treatment differ signicantly from each other, whereas those with the same letters do not.

ozone level (2x), where type A peaked at pH 4.1 (Table II). Thus, the pH trends for the ambient and 1.5x ozone treatments were analogous to the signicant results for the NF and CF ozone treatments, while means at the 2x ozone level did not exhibit a similar trend. The contribution of type E decreased as rain pH decreased for all but the highest ozone (2x) treatment, where type E was most abundant at pH 3.1 and 5.1 (Table II). Thus, the trends for the ambient, NF, and 1.5x ozone levels were analogous to the signicant CF response for type E, while the response at 2x

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Table III Mean percent composition (and standard error) by ectomycorrhizal morphotype at different ozone levels, rain pH levels and horizons after four years of treatment (1991). Morphotypes with significant effects are denoted with asterisks, where  = (p-value 0.10),  = (p-value 0.05), NON = non-ectomycorrhizal, and BDU = broken-dead-unidentiable

<

<

Ectomycorrhizal morphotypes Ozone NF(1x) 1.5x 2x A 14.6 (2.1) 25.9 (3.5) 19.3 (3.2) A 21.5 (3.1) 18.4 (2.1) A  24.8 (2.7) 15.1 (1.9) H 8.7 (2.0) 10.4 (1.9) 11.6 (2.3) H 8.3 (1.6) 12.2 (1.6) H 10.0 (1.1) 10.5 (2.1) B <0.1 (<0.1) 0.1 (0.1) 1.4 (1.1) B 1.0 (0.7) <0.1 (<0.1) B 0.8 (0.7) 0.2 (0.2) E 21.5 (3.8) 17.0 (4.7) 17.2 (3.9) E 21.3 (4.1) 15.9 (2.2) E 17.7 (3.1) 19.5 (3.6) F 9.6 (2.0) 4.4 (1.3) 10.1 (2.8) F 6.2 (1.6) 9.8 (1.9) F 11.0 (2.0) 5.0 (1.3) J 3.3 (1.0) 5.3 (1.0) 5.8 (2.1) J 4.7 (1.2) 4.9 (1.2) J 3.3 (0.8) 6.3 (1.5) NON 8.0 (1.9) 4.5 (1.4) 4.4 (1.5) NON 5.2 (1.3) 6.0 (1.4) NON 8.3 (1.6) 3.0 (0.6) BDU 34.2 (3.3) 32.5 (3.2) 30.2 (4.3) BDU 31.9 (3.0) 32.7 (2.9) BDU 24.2 (1.9) 40.4 (2.5) # obs n = 12 n = 12 n = 12

Rain pH 3.1 5.1

# obs n = 18 n = 18

Horizon Oa upper Bs

# obs n = 18 n = 18

differed. Similar trends were not observed in the upper Bs horizon for either types A or E. 3.3. RESPONSES AFTER FOUR YEARS OF TREATMENT (1991) Morphotype F exhibited an overall response to rain pH treatment as a signicantly higher percentage (p = 0.048) was observed in the pH 5.1 than the 3.1 treatment (Table III). Horizon responses were detected for type A (p = 0.046) and F (p = 0.003), both having a greater percentage in the organic than the mineral horizon, matching the result after one year of treatment. Additionally, a greater percentage (p = 0.001) of non-ectomycorrhizal roots occurred in the organic than in the mineral horizon, and a greater proportion (p < 0.001) of broken-dead-unidentiable roots were observed in the mineral than in the organic horizon.

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4. Discussion Ectomycorrhizal responses to acid rain and ozone were evaluated over the short term (in 1988 after one year of treatment) and over the long term (in 1991 after four years of treatment). In addition to assessing air pollution effects, the composition of the ectomycorrhizal community was compared between two different soil horizons for each sampling year. There was a highly signicant chamber effect on the percentage of type B after one year of treatment: this morphotype was relatively more abundant in the absence of the open-top fumigation chambers. This was the only chamber effect observed in this study and is probably a result of different environmental conditions in the chambers as compared to the ambient treatments. One important difference may have been air temperature; it was not always possible to maintain air temperature in the chambers at ambient values on sunny days when temperatures were 1 to 4  C warmer inside than out (unpublished data). Kohut et al. (1990) noted that leader extension was higher outside the chambers. After one year, a rain pH treatment effect was observed for type A, which contributed signicantly less in the high versus the lower pH treatments. A similar, though non-signicant, response occurred after four years. In contrast to type A, the occurrence of type F was greater in the higher than the lower rain pH treatments. While this trend was observed after both one and four years of treatment, it was signicant only after four years. As morphotype F was identied as potentially ectomycorrhizal, and may have represented a developmental stage of other morphotypes (e.g., type B or E) (Roth, 1993), it is difcult to compare this response to other studies. In general, there appears to have been an overall shift in the relative contribution of different morphotypes to the ectomycorrhizal community, where type A increased and type F decreased as rain pH decreased. The horizonpHozone response after one year suggested that for all but the highest ozone level (2x), type E contributed more and type A contributed less as rain pH increased in the Oa horizon. The horizonpHozone interaction indicates that the overall response of type A to rain pH was most evident in the Oa horizon after one year. It is difcult to distinguish responses to rainfall pH itself from responses to changes in ecosystem factors resulting from acid rain additions. Soil chemistry responses to acid rain additions have been demonstrated for the present experimental system by Sherman and Fahey (1990/1991; 1994). In 1988, soil pH declined signicantly with acid rain additions. Soil solution concentrations of nitrate and hydrogen ions were greater in the rain pH 3.1 treatment for the Oa but not the upper Bs horizon. There were also rain pH effects on soil solution aluminum, where labile and total Al were greatest in the rain pH 3.1 treatment (Sherman and Fahey, 1990/1991). Soil chemistry was also impacted by simulated rain treatments in 1990 (the growing season before the early 1991 sampling): soil pH was signi-

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cantly lower for the rain pH 3.1 treatments. Additionally, soil solution nitrate and aluminum (total and labile fractions) were greater in the rain pH 3.1 than in the higher rain pH treatments (Sherman and Fahey, 1994). Because soil nitrogen, aluminum and acidity generally increased as rain pH decreased, it is possible that changes in soil chemistry played a role in the observed ectomycorrhizal responses to rain pH. The sensitivity of red spruce to soil acidication has been previously suggested (Shortle and Smith, 1987). Moreover, the addition of nitrogen fertilizer reduced mycorrhiza formation and altered the relative proportions of mycorrhizal morphotypes in Sitka spruce (Picea sitchensis) (Alexander and Fairley, 1983). In contrast, Gibson and Deacon (1990) indicated that low nitrogen suppressed ectomycorrhiza formation in asceptic culture. Dighton and Skefngton (1987) observed a decrease in the occurrence of two ectomycorrhizal morphotypes of Scots pine as soil solution Al increased and pH decreased. In the present study, soil solution aluminum concentration for the pH 3.1 treatment was as high as 100 m during 1987 to 1989 and reached a maximum of 200 m in 1990. In comparison to other studies (Thompson and Medve, 1984; Dighton and Skefngton, 1987), soil aluminum probably was not high enough to affect ectomycorrhizae after one year (1988), but may have been after four years (1991). Meier et al., (1989) evaluated the effect of rainfall pH treatments and the consequent changes in soil characteristics (i.e., lower base saturation, lower exchangeable Ca and Zn, and higher exchangeable acidity) on the total numbers of ectomycorrhizae of red spruce seedlings. Although one ectomycorrhizal species (Cenococcum geophilum) responded to rain pH, this response could not be attributed to changes in soil chemistry alone. Cenococcum responded the same way whether rain was applied to the foliage and stem only or to soil alone. They concluded that rain acidity may result in an environment that is favorable for acidiphilous fungi. In the present study, it is impossible to adequately establish whether the observed shift was due to changes in rain pH, changes in soil chemistry, or some other factor. Soil horizon effects for type A and F exhibited the same trend for both sampling years: a signicantly greater percentage of types A and F were observed in the Oa horizon as compared to the upper Bs. While there was proportionately more of type E in the upper Bs as compared to the Oa horizon after one and four years, this trend was signicant only after one year. As pH in the organic horizon was lower than the mineral horizon in the present study (Sherman and Fahey, 1990/1991), the horizon response for types A and E may be explained by pH differences between horizons; however, as the Oa and upper Bs horizons differ substantially in other respects, other factors may have caused the apparent horizon response. Type F tended to be more abundant in the more acidic Oa horizon, contrasting the trend for type F across rainfall pH treatments. Thus, there is likely some characteristic of the Oa horizon other than pH which is favorable for type F. There were signicantly more non-ectomycorrhizal root tips in the Oa than in the upper Bs horizon after four years of treatment. This difference in ectomycorrhizal colonization may be due to differences in soil characteristics between the two

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horizons. Although studies of other tree species have concluded that acid rain or ozone can affect the percentage of uninfected tips (e.g., Meier et al., 1990; Esher et al., 1992), neither ozone nor acid rain treatments had an effect on ectomycorrhizal infection in the present study. There were signicantly more broken, dead, or unidentiable tips (BDU) in the upper Bs than the Oa after four years. This may be due to the fact that the upper Bs horizon tended to be wetter than the Oa (personal observation), and during the winter this may have caused relatively more root death due to freezing. In a study of red spruce in northwestern New Jersey, Glenn et al. (1991) reported that, on average, about one-third of the root tips analyzed were unidentiable. This is comparable to the percentage of root tips, 24% (Oa) and 40% (upper Bs), that were broken, dead or unidentiable in this study. Roots were preserved in glutaraldehyde immediately after coring in 1988 (after one year of treatment), whereas in 1991 (after four years) it was necessary to store intact soil cores for several weeks before the roots could be extracted and preserved. This may explain why broken, dead and unidentiable roots were present after four years, but not after one year. In addition, one important difference between the two different sampling dates was that the 1988 sampling took place in the early fall and the 1991 sampling occurred in the early spring. In general, it is likely that there would be similar morphotypes during the two seasons sampled (i.e., no new species would appear over the winter while the soil was frozen), but a higher percentage of dead, and thus unidentiable, roots in the early spring. This situation would be problematic if certain morphotypes were more prone to winter damage than others, and could explain why, for example, type X was not observed after four years. Unfortunately, there is no way to determine whether this was the case, making it difcult to adequately compare the fall 1988 data to the spring 1991 data. In conclusion, the results of this experiment indicate that the composition of the ectomycorrhizal community of red spruce may be affected by acid rain. Changes in the composition of the ectomycorrhizal community in response to air pollution may be an indication of ecosystem stress (Vogt et al., 1993). During the course of the experiment, tree growth and health were not signicantly affected by the treatments (Sherman and Fahey, 1994). A longer term experiment might have more visibly affected sapling health and produced a more signicant shift in ectomycorrhizal composition. Unfortunately, it is unknown how differences among species of mycorrhizal fungi, such as the quantity of extramatrical hyphae, relate to the nutrition and carbon drain of the host (Dighton and Jansen, 1991). Although changes in ectomycorrhizal morphotype composition appear to have resulted from environmental stress, further research is needed to elucidate the processes through which such changes may affect the host.

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Acknowledgements We thank J. Dighton, J. Lardner, K. Mudge, R. Sherman, S. Wapner, and X. Zhihua for their assistance and advice during this study. We also thank the many people at the Boyce Thompson Institute for Plant Research who were involved with this project. This research was subcontracted through the Boyce Thompson Institute for Plant Research as part of the ROPIS Northeast project funded by the Electric Power Research Institute, the Empire State Electric Research Corporation, Albany, NY, and the Niagara Mohawk Power Corporation, Syracuse, NY. References
Adams, M. B. and ONeill, E. G.: 1991, Forest Science 37, 5. Alexander, I. J. and Fairley, R. E.: 1983, Plant and Soil 71, 49. Barbour, M. G., Burk, J. H. and Pitts, W. D.: 1980, Terrestrial Plant Ecology, The Benjamin/Cummings Publishing Company, Inc., California. Blaschke, H. and Weiss, M.: 1990, Environmental Pollution 64, 255. Dighton, J. and Skefngton, R. A.: 1987, New Phytologist 107, 191. Dighton, J. and Jansen, A. E.: 1991, Environmental Pollution 73, 179. Edwards, G. S. and Kelly, J. M.: 1992, Environmental Pollution 76, 71. Esher, R. J., Marx, D. H., Ursic, S. J., Baker, R. L., Brown, L. R. and Coleman, D. C.: 1992, Water, Air and Soil Pollut. 61, 269. Gibson, F. and Deacon, J. W.: 1990, Mycological Research 94, 166. Glenn, M. G., Wagner, W. S. and Webb, S. L.: 1991, Canadian Journal of Forest Research 21, 741. Johnson, A. H. and Siccama, T. G.: 1983, Environmental Science and Technology 17, 294A. Johnson, A. H. and Siccama, T. G.: 1989, Decline of Red Spruce in the High-Elevation Forests of the Northeastern United States, in J. J. and M. T. El-Ashry (eds.), Air Pollutions Toll on Forests and Crops, MacKenzie, Yale University Press, pp. 191234. Kohut, R. J., Laurence, J. A. and Amundson, R. G.: 1987, Evaluation of the Effects of Ozone and Acidic Precipitation, Alone and in Combination, on the Photosynthesis, Nutrition, and Growth of Red Spruce and Sugar Maple, in Perry, R., Harrison, R. M., Bell, J. N. B. and Lester, J. N. (eds.), Acid Rain: Scientic and Technical Advances. Selper Ltd., London, pp. 588595. Kohut, R. J., Laurence, J. A., Amundson, R. G., Raba, R. M. and Melkonian, J. J.: 1990, Water, Air, and Soil Pollut. 51, 277. Lauver, T. L., Laurence, J. A. and Kohut, R. J.: 1990, Archives of Environmental Contamination and Toxicology 19, 629. Mahoney, M. J., Chevone, B. I., Skelly, J. M. and Moore, L. D.: 1985, Phytopathology 75, 679. Mason, R. L., Gunst, R. F. and Hess, J. L.: 1989, Statistical Design and Analysis of Experiments, John Wiley & Sons, Inc. McLaughlin, S. B.: 1985, Journal of the Air Pollution Control Association 35, 512. McQuattie, C. J. and Schier, G. A.: 1992, Canadian Journal of Forest Research 22, 1901. Meier, S., Robarge, W. P., Bruck, R. I. and Grand, L. F.: 1989, Environmental Pollution 59, 315. Meier, S., Grand, L. F., Schoeneberger, M. M., Reinert, R. A. and Bruck, R. I.: 1990, Environmental Pollution 64, 11. Neter, J. and Wasserman, W. and Kunter,M. H.: 1985, Applied Linear Statistical Models, second edition. Irwin Publishers, Homewood, Illinois. Reich, P. B., Schoettle, A. W., Stroo, H. F., Troiano, J. and Amundson, R. G.: 1985, Canadian Journal of Botany 63, 2049. Reich, P. B., Schoettle, A. W., Stroo, H. F. and Admundson, R. G.: 1986, Journal of the Air Pollution Control Association 36(6), 724. Roth, D. R: 1993, Effects of acid precipitation and ozone on the ectomycorrhizae and rhizosphere chemistry of red spruce saplings, M.S. Thesis, Cornell University, Ithaca, New York.

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Royal Horticultural Society: 1966, R.H.S. Colour Chart, The Royal Horticultural Society, London. SAS Institute, Inc: 1991, SAS System for Linear Models, third edition. SAS Institute, Inc., Cary, North Carolina. SAS Institute, Inc: 1985, SAS Users Guide: Statistics, Version 5 edition. SAS Institute, Inc., Cary, North Carolina. Shafer, S. R., Grand, L. F., Bruck, R. I. and Heagle, A. S.: 1985, Canadian Journal of Forest Research 15, 66. Sherman, R. E. and Fahey, T. J.: 1990/91, Water, Air, and Soil Pollut. 54, 155. Sherman, R. E. and Fahey, T. J.: 1994, Biogeochemistry 24, 85. Shortle, W. C. and Smith, K. T.: 1988, Science 240, 1017. Snedecor, G. W. and Cochran, W. G.: 1989, Statistical Methods, eighth edition. Iowa State University Press. Sokal, R. R. and Rohlf, F. J.: 1981, Biometry, second edition. W. H. Freeman and Co., New York. Stroo, H. F. and Alexander, M.: 1985, Water, Air, and Soil Pollut. 25, 107. Stroo, H. F., Reich, P. B., Schoettle, A. W. and Amundson, R. G.: 1988, Canadian Journal of Botany 66, 1510. Thompson, G. W. and Medve, R. J.: 1984, Applied and Environmental Microbiology 48, 556. Vogt, K. A., Publicover, D. A., Bloomeld, J., Perez, J. M., Vogt, D. J. and Silver, W. L.: 1993, Environmental and Experimental Botany 33, 189.

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