Effects of long-term ozone fumigations on growth and gas exchange of fraser fir seedlings

Environmental Pollution 85 (1994) 265-269

EFFECTS OF LONG-TERM OZONE FUMIGATIONS ON GROWTH A N D GAS E X C H A N G E OF FRASER FIR SEEDLINGS J. R. Seiler, a P. B. T y s z k o a & B. I. C h e v o n e b "Department of Forestry and bDepartment of Plant Pathology, Physiology and Weed Science. Virg#lia Teeh, Blaeksburg, Virginia 24061, USA

(Received 5 January 1993; accepted 1 June 1993)

characteristics of forest trees, but the effects are often minimal and sometimes contradictory (Pye, 1988). The research outcomes have been found to depend heavily on species (Reich & Amundson, 1985) and even family (Hanson et al., 1988). Another possible source of discrepancies in the literature is the variable and often short length of pollutant exposure. Significant ozone effects may result from long-term exposure rather than from fumigations lasting no longer than one vegetative growth period. This would be particularly true for species such as Fraser fir, which experience a fixed growth pattern, where only preformed over-wintered, primordia elongate rapidly in a given growing season. Because elongation and bud set in general are the most sensitive periods for tree growth, timing of ozone exposure may also be crucial for detecting effects. Heck et al. (1984) noted that changes in the growth rate of agricultural crops subjected to ozone fumigations were most likely to occur when the plants were exposed to pollutants during the early vegetative growth phase. In addition to the problems mentioned above, there are many species that have not been investigated sufficiently, including Fraser fir. Relatively few laboratory studies examining the effects of ozone on growth and gas exchange of Fraser fir have been published. A study investigating the influence of ozone exposure on growth and gas exchange of Fraser fir seedlings by Tseng et al. (1987) did not report any significant effects after 10 weeks of noncontinuous fumigations. However, unpublished thesis data (Tseng, 1987) suggested that fumigating seedlings for more than one growth cycle may be needed before a significant growth effect occurs. The present study involved fumigating Fraser fir seedlings with ozone during bud break, elongation and bud set for five growth cycles to determine if multiple exposures at various phenological stages are necessary for effects to develop. The seedlings were grown using an accelerated-growth regime allowing the completion of one growth cycle in about six to seven months. The influence of ozone exposure on the growth and physiology of the seedlings was examined after the completion of each growth cycle. Seedlings from two seed sources were examined: Mt Mitchell and Mt Rogers, both located in the Southern Appalachians. The spruce-fir

Abstract Fraser fir seedlings from two seed sources in the Southern Appalachians ( M t Mitchell, North Carolina, a declining population; and Mt Rogers, Virginia, a relatively healthy population) were subjected to long-term (2.5 years) intermittent ozone fumigations (0.025, 0.070, and 0.150 ppm) while being grown through five growth cycles in an accelerated-growing regime. Fumigations took place during bud break, stem elongation and bud set. Following each growing cycle, gas exchange parameters and dr)' weights were determined The ozone fumigations did not produce any effect on seedling growth. The ozone fumigation effects on gas exchange parameters were inconsistent, and generally not statistically different, with no differences occurring between seed sources. There was no correlation between photosynthetic rates and seedling growth. These results provide no evidence that ozone may be contributing to the differences in decline noted between the Mt Rogers and Mt Mitchell populations of Fraser fir. INTRODUCTION

The high-elevation red spruce (Picea rubens Sarg.) and Fraser fir [Abies fraseri (Pursh.) Poir.] forests in the Southern Appalachians have been experiencing growth decline (Adams et al., 1985) and considerable mortality (Dull et al., 1988) over the past few decades. Causes of the decline are often unclear and may vary from site to site. The factors implicated include pests and pathogens (Mielke, 1988; USDA, 1989; White & Cogbill, 1992), natural processes of aging (Zedaker et al., 1987), longterm climatic changes (Hamburg & Cogbill, 1988), extremely cold winters (Johnson et al., 1988), past disturbances (Pielke, 1980), and air pollution, notably ozone (Chevone & Linzon, 1988; USDA, 1989). Although ozone is thought to have the potential for damaging red spruce and Fraser fir forests in the Southern Appalachians, little is known about susceptibility of Fraser fir to ozone damage. Many studies have shown that ozone in ambient concentrations can modify growth and gas exchange Environ. Pollut. 0269-7491/94/$07.00 © 1994 Elsevier Science Limited, England. Printed in Great Britain

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J. R. Seiler, P. B. Tyszko, B. L Chevone

forest on Mt Mitchell (North Carolina) is in a state of decline apparently caused by infestation of Fraser fir trees by balsam woolly adelgid (Adelges piceae; USDA, 1989). Fir trees on Mt Rogers (Southwest Virginia) appear to be in good health (Zedaker et al., 1987; White & Cogbill, 1992). A further objective of this research was to determine whether a variation in ozone sensitivity between the two locations might be a contributor to the differences in their health.

METHODS Plant Material Half-sib (wind-pollinated) Fraser fir seeds from two sources (Mt Mitchell and Mt Rogers) were stratified for 1 month and sown into 160 cm 3 tubes (Cone-Tainer Nursery, Canby, Oregon) filled with soil collected from Mt Rogers (loamy-skeletal, mixed, frigid Typic Haplumbrept). After emergence, some seedlings were transplanted or removed so that the number of seedlings per tube did not exceed three. After the first growth cycle (see below), only one seedling per tube was left. Growing conditions Seedlings were grown in an accelerated-growing regime (Seiler & Kreh, 1986, 1987), which allows the completion of one growth cycle in approximately six to seven months. During bud break and elongation, seedlings were kept under a 16-h photoperiod in a green-house environment with charcoal-filtered air. The ozone fumigations began when the seedlings started breaking bud and continued under the long-day conditions for six weeks. Seedlings were then subjected to an additional four weeks of intermittent ozone exposure while subjected to an 8-h photoperiod and decreasing temperatures in order to induce bud formation. After bud set (approximately one month), the seedlings were transferred to a dark cold room (2°C) or to a shadehouse (in winter) for six weeks. After the chilling treatment, seedlings were moved back to the greenhouse under a 16-h photoperiod and following bud break were subjected to the next cycle of ozone fumigation. Beginning with the second growth cycle, seedlings were fertilized every week during the long day regime with 10 ml of 200 ppm N, 87 ppm P and 166 ppm K, supplied as 20-20-20. The completion of five growth cycles required 30 months. Treatment exposure Seedlings were exposed to three ozone levels (0-025 p p m - control, 0.07 ppm, 0.15 ppm) in Continuously Stirred Tank Reactors (CSTR's; Heck et al., 1978), three times a week, for four consecutive hours. Ozone was generated from oxygen using a UV ozone generator (Model T408, Welsbach Ozone System~ Corp., Philadelphia). All chambers were supplied with charcoal-filtered air. Fumigations lasted ten weeks per growing cycle [six weeks during bud break and elongation (long days) and four weeks during bud set (short days)]. Air

temperature and light levels in the CSTRs averaged 25°C and 550 /zM/m 2 s photosynthetically active radiation respectively. Relative humidity was held as constant as possible and typically fell between 45 and 55%. Only three CSTRs were used for the experiment. However, at every fumigation period, a particular ozone level was schematically reassigned to different chambers. This was done to avoid confounding the effects of ozone treatments with any inherent differences among individual chambers. The 0.07 ppm ozone concentration approximated ozone levels occurring in the Southern Appalachians during warm and dry summers (USDA, 1989). The 0.15 ppm ozone treatment roughly corresponded to the ozone concentrations occurring in the cities of the eastern United States during severe episodes. Over the entire experiment the Fraser fir seedlings received 19.4 ppm-h (0.025 ppm treatment--vontrol), 44.5 ppm-h (0.07 ppm treatment), and 89.1 ppm-h (0.15 ppm treatment). Measurements Two days after the last fumigation in each growth cycle, a sample of seedlings was harvested. Gas exchange parameters were measured using an LI-6200 portable photosynthesis system (LI-COR Inc., Lincoln, Nebraska). Seedlings were clipped at the root collar and immediately put into a 0.25-1 cuvette. Due to their initial small size, five replicates of a two- to threeseedlings subsample were taken for the first three harvests. Beginning with the fourth harvest, only one plant per replicate was used. Average temperature, photosynthetically active radiation, and relative humidity in the leaf cuvette were recorded for each harvest (Table 1). Leaf area of fir seedlings (except for the fourth harvest) was determined using a LI-COR 3000 portable area meter. Dry weights of roots, stems, and needles were determined after drying samples for 48 h at 65°C. Data analysis The experiment was analyzed as a completely randomized, two-way factorial design with two seed sources (Mt Mitchell and Mt Rogers) and three ozone levels (0.025, 0.07, 0.15 ppm). Individual chamber effects were averaged by rotating the treatments among the Table 1. Temperature, photosynthetically active radiation (PAR) and relative humidity (RH) in the leaf cuvette during consecutive harvests in the multiple-cycle ozone fumigation study"

Harvest 1 2 3 4 5

Date 25 Mar. 1988 28Sept. 1988 23 May 1989 23 Nov. 1989 2 July 1990

Temperature PAR (°C) (/zE m 2 s 1) 30.6+0.3 30.4+0.5 36.3 _+0.2 25.9 + 0.6 29.5 + 1.0

328+4 508+11 553 _+ 10 363 + 15 460 + 7

Results are given as mean _+standard deviation.

RH (%) 41.3_+2.4 34.3+4-7 38.2 + 1.9 42-8 + 5-9 29-3 + 4-2

Ozone effects on Fraser fir chambers at each fumigation. In this way, all seedlings spent equal time in all chambers and any inherent chamber differences were not confounded with ozone effects. Therefore, individual seedlings and not chambers served as the experimental unit. Five measurement replicates were taken for each ozone and seed source treatment combination. The following variables were analyzed: needle, shoot, root, and total dry weight, r o o t : s h o o t ratio, net photosynthesis, transpiration, needle conductance and water use efficiency (WUE). The last of these was calculated as a ratio of net photosynthesis to needle conductance. Data were analyzed using analysis of variance to detect significant differences among the responses of seed sources to ozone treatments. Duncan's multiple range test at the 0-05 probability level was used to separate the response means. Most of the measured variables were not affected by the seed source. Therefore, where appropriate, the seed sources were pooled for the analysis of ozone effects.

RESULTS Growth None of the intermediate harvests produced significant differences among the ozone treatments. Although the highest ozone treatment had substantially more biomass at the final harvest, this result was not significant due to considerable random variability (Table 2). At the final harvest, dry weights of foliage, stems and roots and r o o t : s h o o t ratio were unaffected by the ozone exposure, and no apparent trends were evident. Seed source had no effect on the total dry weights; however, at the last harvest r o o t : s h o o t ratio was significantly different between provenances of Fraser fir seedlings (Mt Rogers, 0-64; Mt Mitchell, 0.73). N o visible injury or premature senescence was evident at any time during the experiment. Gas exchange Seed source had no effect on measured gas exchange parameters. Therefore, seed sources were pooled for the analysis of ozone effects on gas exchange. Photosynthetic rate patterns shown by Fraser fir

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Table 2. Total dry weights of Fraser fir seedlings subjected to multiple-cycle ozone fumigations a Total dry weight (g) Harvest

0.025ppm 03

0.070 ppm 03

0.150 ppm 03

1 2 3 4 5

0-049 + 0.120 0.101 + 0.031 0.125 + 0.020 0-305 + 0.278 0.785 + 0.471

0.050+ 0.088+ 0.142+ 0.221 + 0.650+

0.045+ 0.083+ 0-135+ 0.329+ 0.904+

0.009 0.010 0.029 0.089 0.224

0-012 0-012 0-016 0.178 0.623

There were no statistically significant differences due to ozone exposure at any harvest (P = 0.05). Means and standard deviations at intermediate and final harvest. seedlings during consecutive harvests were erratic (Table 3). At the first harvest the highest rates were found in the control treatment, whereas at the second and fourth harvests, the highest rates were observed in the intermediate treatment (0.07 ppm). At the third and fifth harvests, the photosynthetic rates were not significantly different. The unusually low photosynthesis of the Fraser fir seedlings at the second harvest was probably linked to nutrient depletion of the soil. Fertilization started during the second needle flush. Apparently the application was too late to prevent occurrence of moderate foliar chlorosis. The seedlings fully recovered during later growth cycles, as shown by both the photosynthetic and growth rates (Tables 2 and 3). The needle conductances of Fraser fir seedlings were not significantly different among ozone treatments (Table 4). Conductances tended to be lower on the harvest dates where the seedlings showed the lowest photosynthetic rates (harvests 2 and 3), while higher photosynthetic rates accompanied higher conductances (harvests 1, 4 and 5). Water use efficiency of fir seedlings differed significantly among treatments only during the first two harvests. The pattern was similar to that of the photosynthetic rate, with the 0.15 ppm ozone treatment showing the lowest W U E value. The differences in photosynthesis largely determined the W U E pattern because the needle conductance was similar across treatments.

Table 3. Net photosynthesis of Fraser fir seedlings subjected to multiple-cycle ozone fumigations, expressed on dry weight and area basis a Harvest

Photosynthesis/unit dry weight (nmol COjs g) 0-025 ppm 03

1 2 3 4 5

52-0 + 14.7 + 26-2 + 48.0 + 43.1 +

5.5 6.3 8-5 18.8 12.5

0.070 ppm 03 45.2 + 18.3 + 24.0 + 57-0 + 46.4 +

6.2 6.5 10.1 10.8 8.7

0.150 ppm 03 44.1 + 11.7 + 29.9 + 43.9 + 53.6 +

7.5 *b 2.8§ 6.8 7.6§ 30.3

Photosynthesis/unit area (nmol C02/s m 2) 0.025ppm 03 816 + 429 + 536 + -1 120 +

121 222 323 393

0.070ppm 03 704 + 9.9 541 + 246 429 + 182 -1 153 + 230

0.150ppm 03 721 + 118"* 311 + 75§ 498 + 89 1435 _+968

a Means and standard deviations in ozone treatments at consecutive harvests. b. indicates a significant linear response (P < 0-01); ** indicates a significant linear response (P < 0.05); § indicates a significant quadratic response (P < 0.01).

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J. R. Seiler, P. B. Tyszko, B. L Chevone Table 4. Needle conductance and water use efficiency in Fraser fir seedlings subjected to multiple-cycle ozone fumigations a

Harvest

1 2 3 4 5

Water use efficiency (mmol CO2/tool H20 )

Needle conductance (mmol H20/s g) 0.025 ppm 03

0.070 ppm 03

0-150ppm 03

0.025 ppm 03

0.070 03

0.923 + 0.130 0.314 + 0.078 0.280 + 0.099 0.871 +0.468 0.556+0.160

0.768 + 0.169 0.372 + 0.072 0.269 + 0.048 1.00_+0.348 0-593_+0.115

0.882 + 0-147 0.309 + 0.042 0.314 + 0.084 0.774+0.218 0.674+0.309

2-97 + 0-31 1.38 + 0.29 4.76 + 1.24 3.22+ 1 . 1 1 2.73_+0.27

2.88 + 0-47 1.49 + 0.43 3.96 + 1.32 3.03-+0.71 2-63_+0.14

0.150 ppm 03 2.33 + 0.080 *h 1.14 _+0.29** 4.50 + 0.44 3.15+0.92 2.76-+0.26

"Means and standard deviations in ozone treatments at consecutive harvests. * indicates a significant linear response (P < 0-01); ** indicates a significant linear response (P < 0.01).

DISCUSSION Over five growing cycles, the growth of Fraser fir seedlings remained unaffected by ozone exposure. The suggestion of Tseng (1987) that Fraser fir seedlings could be susceptible to ozone exposure over several growth cycles was not confirmed. Most of the published research indicates that ozone exposure can increase retention of assimilate in leaves (Barnes, 1972; Tingey et al., 1976; McLaughlin et al., 1982). Interestingly, Reich et al. (1987) found that the lowest r o o t : s h o o t ratio occurred in an intermediate ozone treatment, while the lowest (0-02 ppm) and the highest (0.14 ppm) ozone treatments produced higher r o o t : s h o o t ratios. In this study, no effect of ozone exposure on assimilate allocation was found. Although negative effects seem to prevail (Pye, 1988), the air pollution literature concerning other tree species presents a wide range of photosynthetic responses to ozone exposure. Agreeing with the finding reported by Tseng et al. (1987), this study did not show any consistent significant effects of ozone exposure on net photosynthetic rates of the Fraser fir seedlings. Ozone was reported to both stimulate (Keller & Haesler, 1984) and suppress (Vogels et al., 1986) leaf conductance in Norway spruce. Tseng et al. (1987) did not detect any effects of ozone exposure on leaf conductance in Fraser fir seedlings. In the present study no clear effect of ozone on leaf conductance was shown. Also, water use efficiency was not consistently affected by treatments. The phenotypic appearance of Fraser fir on Mt Mitchell and Mt Rogers indicates varying degrees of environmental stress at both locations. Mt Mitchell coniferous stands are severely affected by an infestation of the balsam woolly adelgid, while Mt Rogers forests apparently remain healthy (USDA, 1989; White & Cogbill, 1992). Both ecosystems are subjected to relatively high concentrations of ozone which may have potential to increase their susceptibility to other stresses (Chevone & Linzon, 1988; Lefohn et al., 1990). The present study did not reveal any differences between seed sources in the response of Fraser fir seedlings to ozone. Thus, it seems that factors other than differential ozone susceptibility are contributing to the differences in forest health at both locations.

The results of this study indicated that Fraser fir seedlings were not susceptible to ambient-level ozone fumigations. The resistance of red spruce seedlings to ambient ozone injury has also been confirmed by open-top chamber studies conducted at the BoyceThompson Institute in Ithaca, New York (Laurence et al., 1989; Kohut et al., 1990), and on Whitetop Mountain, Virginia (Thornton et al., 1992). The abovementioned studies used continuous exposure regimes. In this experiment, because of the intermittent fumigation schedule, cumulative exposure was rather low when compared with the actual exposure in the field. The seedlings received a total exposure of from 19 ppm-h (control) to 89 (0.15 ppm) ppm-h, while in the natural habitat on Whitetop Mountain, trees typically receive 15-20 ppm-h in the daylight hours every month during the growing season (Lefohn et al., 1990). Nevertheless, this study provides information on the ambient ozone sensitivity of fir seedlings during their most succulent period (bud break) and during bud development for several growth cycles. On the other hand, our cumulative dose is quite high if compared with the numerous intermittent exposure studies of ten weeks or less where negative results often occur. In comparison with these studies one would have to conclude that Fraser fir is rather tolerant to ozone. The results of this study also draw attention to several problems associated with the methodology of air pollution research. First, they indicate that the conclusions based on a single growth cycle experiment may be misleading; different conclusions concerning the impact of ozone on photosynthesis of Fraser fir seedlings could have been drawn depending on the harvest date considered. An important argument in support of long-term, multiple-growth cycle experiments is the possibility of changes in ozone sensitivity with a plant's age and physiological status. Second, the statistical significance did not seem to reflect the biological significance; the former often showed an apparent randomness for a specific measured variable among the treatments on consecutive harvest dates. Third, the differences in photosynthetic rates did not translate into differences in growth. This uncoupling of growth and photosynthetic effects is fairly common in the literature (Reich et al., 1986, 1987). Lee et al. (1990) found decreased growth of red spruce seedlings subjected

Ozone effects on Fraser fir to o z o n e f u m i g a t i o n s , b u t no c h a n g e in p h o t o s y n t h e t i c rates. This leads us to a n o t h e r c o n c l u s i o n which this s t u d y c o n f i r m e d : i n s t a n t a n e o u s p h o t o s y n t h e t i c rates are n o t necessarily a reliable m e a s u r e o f a p l a n t ' s response to e n v i r o n m e n t a l factors. T h e g r o w t h p e r f o r m a n c e , a n d u l t i m a t e l y r e p r o d u c t i o n , is in any case the u l t i m a t e test o f a p l a n t ' s p e r f o r m a n c e .

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