Plant and Soil 233: 203–211, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
203
Differences in above- and below-ground responses to ozone between two populations of a perennial grass Lidia C. Yoshida1∗ , John A. Gamon1 & Christian P. Andersen2 1 Department
of Biology and Microbiology, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032, USA, 2 US Environmental Protection Agency, Western Ecology Division, National Health and Environmental Effects Research Laboratory, Corvallis, OR 97333, USA ∗ Corresponding author: School of Biological Sciences, University of California, Irvine, Irvine, CA, 92697-2527, USA. Tel: +1-949-824-8185; FAX: +1-949-824-6599; E-mail:
[email protected] Received 3 April 2000. Accepted in revised form 1 March 2001
Key words: arbuscular mycorrhizae, Elymus glaucus (blue wildrye), Glomus intraradices, ozone, perennial grass
Abstract Our study examined the influence of elevated ozone levels on the growth and mycorrhizal colonization of two populations of Elymus glaucus L. (blue wildrye). We hypothesized that ozone would reduce carbon availability to the plants, particularly below ground, and would affect mycorrhizal colonization. Because of the wide geographic range of E. glaucus, two populations of plants were selected from areas of contrasting ozone histories to examine intraspecies variation in response to ozone. Two populations of E. glaucus (southern California versus northern California) exposed in a factorial experiment involving ozone, mycorrhizal inoculation with Glomus intraradices Schenck and Smith, and plant source population. Ozone had a subtle effect on leaf area and number of tillers but did not affect overall root:shoot ratio in either population. The impact of ozone on above-ground growth characteristics was most pronounced in the southern population that came from a high-ozone environment, while below-ground responses such as reduced arbuscular colonization was most pronounced in the northern population which originated in a low-ozone environment. Further analysis of soil characteristics from the northern population of plants revealed a significant reduction in active soil bacterial biomass and an increase in total fungi per gram dry weight soil, suggesting a possible role for ozone in altering soil processes. Whether or not population differences in response to ozone were due to genetic shifts resulting from prior ozone remains to be determined. However, these subtle but important differences in population response to ozone above- and below-ground have significant implications in any attempt to generalize plant response, even within a species. Future research efforts need to include better characterization of intraspecific variation in response to ozone as well as possible adaptive strategies that may result from chronic ozone exposure.
Introduction Several studies have focused on the impact of ozone on the aboveground growth of plants in natural ecosys∗ The information in this document has been funded wholly or in part by the US Environmental Protection Agency under Educational Cooperative Assistance # NG902628 to California State University, Los Angeles. It has been subjected to the Agency’s peer and administrative review, and it has been approved for publication as an EPA document. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
tems, and these have recently been reviewed (Davison and Barnes, 1998). A remaining challenge is to understand below-ground effects of ozone, and, in turn, to consider the impact of altered rhizosphere processes on biogeochemical cycles. Recent studies suggest interactive effects among air pollution, mycorrhizal fungi and plant growth (Cherfas, 1991). Forests in polluted sections of Europe have been declining, associated with changes in plant nutrition and soil chemical characteristics (acidification, cation imbalance and NO3 − saturation) (Durka
204 et al., 1994; Schulze, 1989) with apparently long-term consequences. Although similar processes may be occurring in certain eastern US forests (Cherfas, 1991), these processes may not apply to southern California conditions with its relatively low rainfall, high atmospheric concentrations of NO3 − and relatively low levels of SO2 (Bytnerowicz and Fenn, 1996; Cohanim et al., 1991). The Los Angeles Basin, with its unique combination of pollutant sources, weather conditions and geography has had a history of very high ozone levels (Heck, 1989; Treshow and Anderson, 1989). Agriculturalists in the Los Angeles Basin first observed ozone damage to crop plants in the 1940s and, since then, ozone has been recognized as the principal photochemical oxidant responsible for crop damage in the western United States (Heck, 1989). Visible signs of ozone damage include tip burn, stippling and flecking, foliar chlorosis and necrosis, and premature leaf abscission (Heagle et al., 1991; Wellburn, 1994; Woodbury et al., 1994). A summary of studies comparing ozone concentration, exposure period and plant growth indicate that plant growth decreases after prolonged exposure to ozone at levels as low as 50 ppb (MacKenzie and El-Ashry, 1989). While the majority of air pollution research has focused on visible, above-ground aspects of plant growth, a number of studies have focused on belowground processes, examining interactions between pollution and carbon allocation to roots of both AM (arbuscular mycorrhizal) and ECM (ectomycorrhizal) plants (Adams and O’Neill, 1991; Andersen and Rygiewicz, 1995; McCool and Menge, 1983, 1984). Studies on mycorrhizal tomatoes indicate that ozone may delay the onset of and reduce AM fungal colonization and influence the flow of carbon to the roots (McCool and Menge, 1983, 1984; McCool et al., 1982). Arbuscular mycorrhizal fungi in clover suppress growth under low ozone conditions (Miller et al., 1997). However, sugar maple saplings treated with ozone had greater AMF colonization than trees in ambient conditions (Duckmanton and Widden, 1994). The apparently contradictory results of these studies suggest no consistent effect of ozone on below-ground processes. More work is needed to resolve this, particularly in the case of wild species in natural ecosystems, where multiple factors may be important (Watkinson, 1998). Recent research has begun examining the possibility that chronic air pollution is leading to long-term changes in species composition, population structure
or vegetation type. Grasslands are an important vegetation type in southern California and in much of the world. However, to our knowledge, no studies of ozone effects on native California mycorrhizal grasses have been reported. We chose to examine Elymus glaucus L. (blue wildrye), a native California bunchgrass, which grows over a wide range in California (Crampton, 1974) and is colonized by AMF. Our study examined the impact of ozone on growth and mycorrhizal colonization. We expected that ozone would reduce carbon fixation by the plants, decrease overall plant growth, and alter plant biomass allocation. We hypothesized that reduced carbon availability would also affect mycorrhizal colonization and possibly soil microorganisms. Because of the wide geographic range of E. glaucus and the prospect of evolutionary change in response to contrasting histories of ozone exposure, we also examined the possibility of population-level differences in ozone responses by comparing the responses of plant populations with different histories of ozone exposure.
Materials and Methods Elymus glaucus is a native California perennial bunchgrass found throughout the state below 2500 m, often growing in association with oaks and conifers (Crampton, 1974). E. glaucus was chosen because it is a fast-growing perennial, colonized by AMF, and because the two source populations (Sky Forest, ‘high ozone’; and Stanislaus, ‘ambient ozone’) had been exposed to many years of contrasting ozone regimes. During the 10-year period prior to seed collection, the highest hourly ozone concentrations at Lake Gregory, near Skyforest, were almost double that in the vicinity of Stanislaus Forest, sometimes reaching 330 ppb (California Air Resources Board, 1978–1992). One population of seeds, designated ‘northern’, was originally collected in 1990 in the Stanislaus National Forest in Tuolomne County (on the western slopes of the Sierra Nevada Mountains), northern California, elevation 1524 m. From these seeds, plants were grown for two generations near Rio Vista, California by S & S Seeds (Carpinteria, CA). The northern seeds used in this study were collected from these second-generation stock plants. A second population of seeds, designated ‘southern’, was collected in 1988 from plants in Skyforest in the San Bernardino National Forest (34◦ 14 30 N, 117◦ 10 30 W), San
205 Bernardino County, southern California, elevation 1753 m (P.R. Miller, pers. comm.). To study growth effects of ozone and mycorrhizal infection in two populations, this study involved a three-factor factorial design including ozone level (control and high), mycorrhizal treatment (mycorrhizal and nonmycorrhizal) and source population (northern and southern). Replicates were evenly divided between two growth chambers, which were treated as a random block effect. Situated within each growth chamber there was a control and EP120 (high ozone) chamber, resulting in two ozone and two control chambers for the experiment. Nine plants were used for each treatment-block combination totaling 144 plants (two ozone exposures × two mycorrhizal treatments × two source populations × two chambers × nine replicates = 144). The growth chambers were located in the Terrestrial Ecological Research Facility (TERF) at the Western Ecology Division of the US Environmental Protection Agency in Corvallis, OR. Housed inside the TERF was the Controlled Environment Exposure System (CEES), consisting of two plant growth chambers, with a computerized ozone exposure system (Andersen and Rygiewicz, 1995). Photosynthetic photon flux density (PPFD) was totaled hourly and stored daily. Day/night temperature averaged 28/18◦C. Average daily light levels inside the exposure chambers were 350 µmol m−2 s−1 . Day length was 16 h. Daily humidity ranged from 25 to 50%. In order to focus on population differences in response to ozone, a common soil and inoculum source was used. The soil was a native sandy loam, with a mean phosphorus content of 13.7 mg kg−1 collected from the banks of the Willamette River and autoclaved at 104◦ C for 20 min. Sterilized containers, 550 cm3 (Deepots, Steuwe and Sons, Corvallis, OR), were filled with sterile soil to 7–8 cm from the rim of the pot. Sterile clay pellets were placed in the controls, and 3.3 g of inoculum plus clay pellets were placed in each of the mycorrhizal treatments. The inoculum contained celery roots (T. St. John, pers. comm.), infected with Glomus intraradices Schenck and Smith (Tree of Life Wholesale Nursery, San Juan Capistrano, CA). This species was used because it is ubiquitous and provided more readily describable and repeatable treatments than applications of fungal mixtures from field soils from the different sites. Seeds were sterilized for 5 min in 10% bleach solution and then germinated in vermiculite on a mist bench. After 5 days, seedlings of average height were selected
and transplanted into the pots, placed in the ozone exposure and watered as needed with no fertilization. Ozone was generated inside a custom-built system consisting of an air compressor, reaction tank, dilution tank and UV lamps; delivery was controlled by an HP9816 computer which collected environmental data continuously (Hendricks, 1994). Ozone treatment profiles were episodic in nature with varying daily peak concentrations. Ozone was low in the morning and increased to a daily peak at approximately 14:00 h each day, followed by decreasing concentrations into the afternoon and evening. Ozone peaks (1 h) ranged from 1 to 25 ppb in ambient exposure chambers and from 30 to 215 ppb in the high ozone chambers. Adding the hourly average concentration, 24 h per day, over the experiment provided cumulative ozone exposure over the entire experiment. Total ozone exposure, using this summing approach resulted in 14.9 and 16.2 ppm-h in the ambient chambers and 120.3 and 124.6 ppm-h in the ozone treatments. The episodic profile was favored over the more commonly used daily peak exposure pattern because it reflects more realistic exposure patterns based on actual monitored data, and because previous studies demonstrated that the episodic profile elicited a greater plant growth response to ozone (Hogsett et al., 1985; Lefohn et al., 1986). After 16 weeks, all plants were harvested, and plant growth responses were measured: leaf mass, tiller mass, root mass, dead plant material mass, number of leaves, number of tillers, leaf area and tiller area. Leaves, tillers, dead plant material and roots were dried at 60◦ for 24 h and weighed. Due to significant ozone effects on mycorrhizae in the northern population (discussed below), that population was selected for further soil microbial analyses. Soil samples from the northern population were collected and assessed for total hyphal length, active fungal length and active bacterial biomass (Ingham, 1992). The AMF staining procedure of Koske and Gemma (1989) was followed for root staining except for the addition of a water rinse between the staining and destaining steps. The extra rinse was necessary because the fine roots absorbed so much stain that it was difficult to distinguish mycorrhizal structures from those of the root. The roots were stained using a modification of the containerized syringes described by Claassen and Zasoski (1992). Root samples were examined under a light microscope with a magnification of ×200. Vesicular, arbuscular and hyphal colonization was evaluated using a modified version of the magnified intersections method of McGonigle et al. (1990).
206 Statistical analyses were performed using the SYSTAT statistics program (Wilkinson, 1989). Because multiple variables were measured in this experiment, multivariate analysis of variance (MANOVA) was considered to be the most appropriate statistical approach for analysis. For an in-depth treatment of the appropriate use of MANOVA refer to Scheiner (1993). For our experiment, MANOVA was performed first on the plant growth responses (root mass; tiller and leaf mass; tiller and leaf areas; and tiller and leaf number) to examine the effects of ozone and mycorrhizae. An additional split plot analysis was performed to more accurately determine ozone effects. The chambers were treated as random block effects and ozone, mycorrhizae and population as fixed effects. Univariate F-tests (Wilks’ lambda, Pillai trace and HotellingLawley trace) were performed on each of the growth responses to determine how much each treatment contributed to a change in each of the growth responses. Since all three P values were identical, only the Pillai trace results are shown. MANOVA was applied separately to the mycorrhizal colonization and soil microbial biomass data and the appropriate univariate F-tests were performed. Since the chambers had no effect on the summary plant growth characteristics or on the mycorrhizal colonization of either population of E. glaucus, the statistical results for the chambers are not presented here. Root:shoot ratio, total aboveground mass (tillers + leaves + dead plant material) and the total plant mass (total aboveground mass + roots) were analyzed by performing four-way analysis of variance (ANOVA), including all possible interactions between chamber, mycorrhizae, ozone and population. Since there were no significant chamber interactions, the ANOVA was repeated with chamber as a block effect and the chamber effects were not shown in the results. P values between 0 and 0.1000 were regarded as significant.
Results Ozone Ozone had a significant effect on the summary plant growth characteristics with no specific interaction with population or mycorrhizae. High ozone levels slightly reduced leaf area and root mass but slightly increased the number of tillers (Fig 1b; Table 1a) and these effects appeared to be more pronounced in the southern population. Leaf mass, tiller mass, number of
Figure 1. (a) Leaf area, (b) number of tillers and (c) root mass of mycorrhizal and nonmycorrhizal E. glaucus.
leaves (Table 1a), tiller area, dead plant material mass (not shown), total plant mass, total aboveground mass and root:shoot ratio (Table 2) did not show any significant response to ozone treatments. High ozone treatment significantly reduced arbuscular colonization (Fig. 2a), particularly in the northern population. None of the exposed plants showed significant differences in vesicular colonization (Fig. 2b). Colonization by hyphae followed a similar trend to that of the vesicles (not shown). However, total colonization (percentage of arbuscules, vesicles and hyphae in the roots) was significantly lower by approxim-
207 Table 1. Multivariate and univariate F -test results of treatment effects; only factors having significant Pillai trace values are shown (a) Plant growth characteristics (n = 144, d.f. = 1, 135). Source
Ozone (split plot) Population Mycorrhizae
Factor Leaf mass P
Tiller mass P
Root mass P
Number of leaves P
Leaf area P
Number of tillers P
0.037 0.061 0.000
0.075 0.902 0.022
0.207 0.000 0.247
0.294 0.000 0.000
0.078 0.414 0.016
0.224 0.002 0.011
(b) Mycorrhizal colonization (n = 72, d.f. = 1, 64). Source Arbuscules Total colonization P P Ozone
0.019
0.119
(c) Soil fungi and bacteria. (n = 72, d.f. = 1, 64). Factor Source Active hyphae Total hyphae p p
Active bacterial biomass p
Chamber Ozone Mycorrhizae Ozone × mycorrhizae
0.000 0.250 0.833 0.034
0.849 0.075 0.000 0.889
0.005 0.352 0.000 0.045
ately 30% in the northern ozone-exposed plants but not in the southern ozone-exposed plants (Fig. 2c). Analyses of the microbial biomass indicated that, while there were significant differences between the chambers (Table 1c), total fungal hyphae/gdw soil increased significantly (Fig. 3a) while active bacterial biomass decreased significantly (Fig. 3b) in the ozone-exposed mycorrhizal plants. However, there was a significant effect of the chambers (P < 0.0001) on the bacterial biomass. Mycorrhizae Mycorrhizal inoculation had a strong effect on aboveground plant mass from both populations regardless of ozone exposure. Leaf area, leaf mass, number of tillers and number of leaves were reduced in the presence of mycorrhizae (Fig. 1a and b and Table 1a). Analysis of variance indicated that mycorrhizal inoculation significantly reduced total aboveground mass, which significantly raised the root:shoot ratio in most treatments. The effect on total plant mass was significant (P = 0.076), reducing total plant mass in most
cases (Table 2). Mycorrhizae had no conclusive effect on root mass, tiller area, dead plant material mass or total plant mass (Tables 1a and 2). ANOVA tests revealed no two-way interactive effects between ozone × mycorrhizae, ozone × population, and population × mycorrhizae, with the exception of population × mycorrhizae on dead plant material (not shown). No significant interaction between ozone and population was detected in plant mycorrhizal colonization. An analysis of the three-way interaction between ozone, mycorrhizae and population revealed a significant interaction between ozone, population and mycorrhizae for total aboveground mass but was not significant for total plant biomass (Table 2). Population The origin of population had a strong effect on the summary plant growth characteristics of E. glaucus (Table 1a). Root mass (Fig. 1c), number of tillers (Fig. 1b) and number of leaves were greater in northern E. glaucus, while mass of dead plant material was
208 Table 2. Analysis of variance (four-way) of the effects of ozone, population and mycorrhizae and their interactions on plant mass and root:shoot ratio on two populations of E. glaucus (n = 144, d.f. = 1, 135) Source
Total plant mass F -value P
Total above-ground mass F -value P
Root:shoot ratio F -value P
Ozone Population Mycorrhizae Ozone × population Ozone × mycorrhizae Population × mycorrhizae Ozone × population × mycorrhizae
2.9510 10.435 3.200 0.078 0.501 0.082 1.866
0.560 5.911 15.988 0.783 2.072 0.154 5.678
2.310 104.877 15.291 0.780 3.736 0.685 2.987
0.088 0.002 0.076 0.781 0.480 0.774 0.174
0.456 1.016 0.000 0.378 0.152 0.285 0.019
0.131 0.000 0.000 0.379 0.055 0.197 0.086
Figure 3. (a) Total fungal biomass (hyphae per gram dry weight) and (b) active bacterial biomass per gram dry weight of northern population of E. glaucus.
Figure 2. (a) Arbuscules, (b) vesicles and (c) total colonization of northern and southern populations of E. glaucus.
slightly elevated in the southern plants (not shown). Total plant mass and root:shoot ratio were significantly greater in northern E. glaucus while total aboveground mass was smaller (not shown). Leaf area, tiller area, leaf mass, and tiller mass, were not different between populations (Table 1a). No effect of population source on mycorrhizal colonization was detected.
209 Discussion Our results showed that the southern population had a more significant above-ground response to ozone exposure than the northern population (Figs. 1a and b), while the northern population had a more apparent below-ground response to ozone exposure, as evidenced in the mycorrhizal response (Fig. 2a, c). Since population differences in colonization were not observed in the absence of ozone, the fungal isolate used in this study appeared to be equally suitable for both populations; therefore, we conclude that differences in colonization between the two populations were due to inherent physiological differences in the way the populations responded to ozone stress. Whether or not the population differences were due to genetic shifts in response to ozone histories remains to be determined. However, these subtle but important differences in population response to ozone above- and belowground have significant implications in any attempt to generalize plant response, even within a species. Our results clearly suggest that additional research needs to be conducted to better understand intraspecific variation in response to ozone as well as possible adaptive strategies that may result from chronic ozone exposure. Total plant biomass was reduced by ozone exposure, although the reduction was not highly significant (0 < 0.088). Ozone was found to significantly increase tiller number, while reducing shoot mass, primarily in the southern population of plants. Similar reductions in leaf area in response to ozone stress have been reported for other species (Darrall, 1989; Held et al., 1991; Woodbury et al., 1994), and can result from reduced growth or early senescence of foliage or both (Miller et al., 1999; Woodbury et al., 1994). The increased tiller number in ozone-treated plants may be a sign of increased turnover and replacement of vegetative parts. A parallel phenomenon may have occurred in radish where ozone-treated plants had a greater accumulation of dry matter (Held et al., 1991). While the increase in tiller number has not been reported in other ozone investigations, increased tillering has been reported in response to other stresses, e.g., high UV-B exposure (Wellburn, 1994). Mycorrhizal colonization reduced the size of the grass plants in this experiment, possibly due to increased carbon demand of the fungus. The ‘parasitic’ response of plants to colonization by mycorrhizae has been widely reported, particularly under low light and during early stages of development. Although light
levels in the experiment were relatively low, they were in a range typical of understory conditions. E. glaucus occurs on a broad range of sites, and it is uncertain whether similar responses would be observed under greater light levels, characteristic of more open-grown conditions. Although we were not able to examine soluble carbon movement directly, the results suggest that there may have been a reduction in carbon availability from the root to the AM fungus, especially in the northern population of plants. Both populations showed similar colonization frequency in the absence of ozone. Reduced arbuscular numbers in the plants exposed to ozone may have resulted from reduced translocation of soluble carbon or specific carbon compounds to the roots in the ozone treatments, even though biomass was not significantly altered. As noted above, the response was most significant in the northern population of plants, raising the possibility of greater sensitivity to ozone below-ground than in the southern population. Because of the apparent sensitivity below-ground of the northern population, additional measures were performed to further examine possible changes in soil resulting from ozone stress (Table 1c and Fig. 3). Changes in soil resulting from ozone are thought to be indirect, since ozone does not penetrate the soil beyond a few centimeters (Blum and Tingey, 1977; Turner, 1973). The results of the soil analyses indicated that total soil fungal biomass increased and active bacteria decreased (approx. 15%) with ozone exposure. A similar increase in fungal biomass was reported in roots of container-grown ponderosa pine exposed to high levels of ozone (Scagel and Andersen, 1997). Shafer (1988) found that ozone increased the number of fungal propagules in the rhizosphere of Sorghum. Increased soil fungal biomass could have been due to a change in soluble carbon products through the roots, possibly increasing the distribution of external hyphae at the expense of internal hyphae. Another possibility is that saprophytic fungi from decaying roots increased, since our preliminary analyses did not distinguish between AM and saprotrophic hyphae. The microbial response parallels the behavior of soil microorganisms in previous studies that indicate that ozone may alter soil CO2 flux, change the composition of the soil bacterial populations, and influence enzyme production (Edwards, 1991; Reddy et al., 1991; Scagel and Andersen, 1997; Shafer, 1988). One possibility is that ozone alters soluble carbon release from the roots. Recent results with wheat suggest that root exudation is increased with short-term
210 exposure to ozone (McCrady and Andersen, 2000), however other studies showed decreased root exudation of amino acids in AM tomatoes exposed to ozone (McCool and Menge, 1983). Conflicting results, presumably due in part to species differences and contrasting ozone exposure protocols, suggest that more research is necessary to clarify these rhizosphere interactions. Our results show the importance of including intraspecies variation in response to ozone. The different responses observed between the northern and southern population indicate that making generalizations about ozone response is difficult. The fact that significant differences in below-ground response were observed suggests that soil processes may be impacted by ozone exposure. Besides providing nutritional benefits to plants, AMF play an important role in vital soil cycles. Carbon from hyphae and root exudation is important in maintaining other soil microorganisms (Allen, 1991; Brundrett, 1991; Finlay and Söderström, 1992). Thus, environmental stresses that affect mycorrhizal colonization could indirectly influence soil processes in the rhizosphere (Allen, 1991) and consequently influence plant competition and therefore species composition (St. John and Coleman, 1983). Long-term field studies are needed to understand the consequences of changes in fungal and microbial species composition in response to ozone and other stresses.
Acknowledgments We thank the technicians at Mantech and G. Neely at the United States Environmental Protection Agency Environmental Research Laboratory in Corvallis, Oregon for their valuable assistance, to P.R. Miller at the United States Department of Agriculture Forest Fire Laboratory for the Skyforest seeds, and to E. Ingham for the soil microbial analyses. We thank Dr. Henry Lee for assistance with the statistical analysis. A special thanks to E.B. Allen, J.A. Menge, R. Nakamura and R. Desharnais for their helpful comments and advice. This study was supported in part by the United States Environmental Protection Agency3 under Educational Cooperative Assistance # NG902628 to California State University, Los Angeles.
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