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Effectsof ozone and simulated acid rain on birch seedling growth and formation of ectomycorrhizae
Environmental Pollution 52 (1988) 55-65
Effects of Ozone and Simulated Acid Rain on Birch Seedling Growth and Formation of Ectomycorrhizae Kevin D. Keane & William J. Manning Department of Plant Pathology, University of Massachusetts, Amherst, Massachusetts 01003, USA (Received 1 September 1987; revised version received 15 October 1987: accepted 24 November 1987)
ABSTRACT Four- week-old paper birch (Bet ula papyrifera Marsh.) seedlings, inoculated or non-inoculated with the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker & Couch and grown in steamed or non-steamed soil, were exposed to ozone ( 0 3 ) and/or simulated acid rain ( S A R ) . Plants were exposed to 0 3 for 7h per day on 5days per week for 12 weeks. 0 3 concentrations were maintained between 0"06 and 0.08 ppm. SA R was applied lOmin per day on 2days per week. 0 3, SAR, soil regime and mycorrhizal treatment did not significantly affect any o f the measured variables. Interactions between 03 and SAR, S A R and mycorrhizal treatment, soil regime and mycorrhizal treatment and ozone and soil regime had significant effects. Treatment of seedlings with p H 3.5 S A R caused increases in growth which were more apparent in birch exposed to 03. Mycorrhizal treatment caused increased growth in non-steamed soil, while growth appeared to decrease in steamed soil. Birch seedlings grew much better in steamed soil. The implications of increased growth in steamed soil may demonstrate the impo'rtance of looking at the secondary effects of pollutants on soilborne organisms.
INTRODUCTION There have been m a n y reports on the effects of gaseous air pollutants and simulated acid rain on trees, particularly conifers (Costonis, 1970; Abrahamsen et al., 1977; W o o d & Bormann, 1977). Fewer studies have 55 Environ. Pollut. 0269-7491/88/$03.50 (~ 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain
56
Kevin D. Keane, William J. Manning
focused on the effects of these air pollutants on deciduous trees (Freer-Smith, 1985; Chappelka et al., 1985; Chappelka & Chevone, 1986; Elliott et al., 1987). Paper birch (Betulapapyrifera Marsh.), a deciduous tree, comprises an integral part of spruce-fir forests in the northeast region of the US (Bartuska & Medlarz, 1986). Red spruce (Picea rubens Sarg.) in the region is in a widespread state of decline. Some effects on balsam fir (Abies balsamea Mill.) and paper birch have also been noted at certain locations (Johnson, 1983). Ozone (03) and acidic precipitation are thought to be contributing factors to tree decline problems (Siccama et al., 1982). The effects of ozone and simulated acid rain have been studied by several researchers (Norby & Luxmore, 1983; Chappelka et al., 1985; Reich & Amundson, 1985; Chappelka & Chevone, 1986; Elliott et al., 1987). Most of this research, however, has been concerned with the above-ground plant parts. An equally important aspect, that has only recently received attention, are the effects of air pollutants and simulated acid rain on roots and mycorrhizae. Mycorrhizae, symbiotic fungal-root associations, are beneficial to the growth and development of trees (Marx, 1972; Sylvia & Sinclair, 1983; Beckjord et al., 1984). Mycorrhizae have also been shown to alter some of the effects of air pollutants through promotion of shoot and root growth (Carney et al., 1978; Garrett et al., 1982; Mahoney et al., 1985). However, only Reich and co-workers (1985) have presented information concerning the effects of gaseous pollutants and acidic precipitation upon mycorrhizal infection. Red oak (Quercus rubra L.) mycorrhizae were found to be adversely affected by ozone, sulphur dioxide and acidic precipitation. Our objective was to determine the impact of ozone and/or acidic precipitation upon the growth and development of paper birch seedlings and their mycorrhizal associations.
MATERIALS AND METHODS Preparation of inoeulum The ectomycorrhizal fungus, Pisolithus tinctorius (Pers.) Coker & Couch, strain 288 (obtained from D. H. Marx) was maintained on modified Melin-Norkrans medium (MMN) (Molina & Palmer, 1982; Smith, 1982). Liquid cultures of Pt were prepared by placing 15-20 agar discs of actively growing mycelium into flasks of MMN broth (Danielson et al., 1984). These cultures were then placed on a rotary shaker and allowed to grow for 2 weeks. Once the liquid cultures had grown sufficiently, they were aseptically inoculated into a solid substrate composed of vermiculite and peat moss
Effects o f ozone and acid rain on birch seedling growth
57
(28:1 v/v) in 500 ml Erlenmeyer flasks (Marx & Kenney, 1982). These fungal cultures were then allowed to grow until they had thoroughly colonised the substrate. Cultures were then placed in cold storage at 3°C until used.
Soil preparation Soil for the experiments was obtained from an indigenous stand of paper birch in western Massachusetts. Leaf litter was removed from the site and soil was collected primarily from the O, A and B horizons. This soil was mixed with sand (2 soil:l sand v/v) and divided into two lots. One lot was steam sterilised twice (30 min at 82°C). The other lot was not treated. Using vermiculite-peat mycorrhizal inoculum, half of each soil type was inoculated with Pisolithus tinctorius (Pt). Inoculation was carried out prior to potting in an 8:1 v/v ratio (soil:inoculum) using a broadcast method (Riffle & Maronek, 1982). All remaining soil received an equivalent amount of untreated vermiculite. Four-week-old seedlings of paper birch, grown from seed were transplanted into the four soil regimes. Seedlings had been germinated in Redi-Earth potting mixture (W. R. Grace and Co., Cambridge, MA) and were selected for relative uniformity.
Ozone and simulated rain exposures Seedlings were placed in a set of eight rectangular Plexiglas chambers (72 cm × 45 cm × 45 cm). These chambers are part of a single pass-through air system. All incoming air was filtered through two particulate filters and 10 in of activated charcoal. Air entered the chambers as carbon filtered air (0-00 ppm O3) or as CF plus O 3. Four chambers received carbon filtered air (CF). The remaining four received CF plus O 3. Each chamber contained 40 seedlings which received a 16 h photoperiod and were rotated between chambers on a weekly basis. Ozone was produced by an O R E C ozone generator (Ozone Research and Equipment Corp., Phoenix, AR) and constantly monitored using a Dasibi 1003H monitor (Dasibi Corp., Glendale, CA) which was calibrated to EPA standards. Ozone concentrations were maintained between 0.06 and 0"08ppm. Birch seedlings were fumigated with 03 for 7 h d a y -~ 5 days week- 1 for 12 weeks. Paper birch seedlings were also exposed to simulated acid rain (SAR) of pH 3"5 or pH5"6. The pH3"5 treatment was acidified with nitric and sulphuric acids (1:1 v/v). Rain treatments were composed of deionised water and appropriate concentrations of background ions (Table 1), common to precipitation in western Massachusetts. (H. Hemond, Massachusetts
58
Kevin D. Keane, William J. Manning
Institute of Technology, pers. comm.). Seedlings were removed from the chambers and treated with S A R o f p H 3 . 5 or p H 5 . 6 for 1 0 m i n d a y -1, 2 days w e e k - 1 through the use o f a pressurised system utilising special rain nozzles (Spray Systems Inc., Wheaton, IL). This provided each seedling with 30 ml o f SAR, comparable to normal rainfall. All seedlings were watered with appropriate rain simulants, as needed between rain treatments. N o additional nutrients were supplied during the experiment. The experiment was terminated after 12 weeks. Measurements of shoot height; root, shoot and leaf dry weights; leaf area and leaf number were
TABLE 1 Ion Concentrations of Rain Simulants (pH 3-5 and 5"6) Ion
H÷ C a +2
Mg +2 K+ Na + NH4 + C1SO,~2 NO 3
Concentration (peq litre- a)
Concentration (mg litre 1)
59"9 6-1 2"9 1"9 8"5 7"7 14.5 40-3 20"6
0"0604 0-1222 0"0352 0-0749 0-1954 0-1389 0-5141 1-9356 1.2773
Data from Precipitation Analysis at Bickford Reservoir, Massachusetts, July 1981 to June 1983. obtained. A quantitative analysis of total per cent mycorrhizal infection (natural and inoculated) was determined for each individual birch seedling (Grand & Harvey, 1982). A scale of 1-4 (1 = < 1%, 2 = < 2 5 % , 3 = < 5 0 % , 4 = > 75%) was utilised to describe per cent infection. A minimum of nine seedlings were evaluated for each treatment.
Experimental design and data analysis A randomised complete block design was used for all experiments. Within this design was a factorial consisting o f two levels of 0 3 x two levels of S A R x two soil regimes x two mycorrhizal treatments. All data were analysed with an A N O V A (analysis of variance). Further data analysis was carried out using a D u n c a n - W a l l e r multiple comparison procedure ( D a m o n & Harvey, 1987).
Effects of ozone and acid rain on birch seedling growth
59
RESULTS Chronic 0 3 injury was apparent on some birch seedlings. This injury consisted of chlorosis and in some cases was accompanied by yellow flecking. SAR did not cause visible symptoms. Ozone, SAR, soil and mycorrhizae did not significantly affect shoot height, number of leaves, leaf area, per cent mycorrhizal infection or root, shoot and leaf weights. There were several two-way interactions which were significant (Table 2). An 03 × pH interaction significantly influenced leaf area, per cent mycorrhizal infection and root, shoot and leaf weights (Table 3). Treatment of seedlings with SAR o f p H 3"5 caused increases in leaf area and root, shoot and leaf weights regardless of the 03 treatment involved. However, these growth increases were much greater for those seedlings exposed to 03 . The largest values for these parameters occurred among those birch treated with 03 and SAR of pH 3-5. Per cent mycorrhizal infection responded differently to the interaction of 03 and SAR. Treatment of seedlings with pH 3.5 SAR resulted in significant decreases of mycorrhizal infection in CF. Birch exposed to 03, however, did not show any effects due to pH 3-5 SAR. The greatest mycorrhizal infection occurred in seedlings treated with CF and pH 5"6 SAR. A two-way interaction between pH and mycorrhizal treatment significantly affected shoot height, leaf area, number of leaves and shoot and leaf weights (Table 4). These growth variables were all greater in seedlings noninoculated with Pt exposed to pH 5"6 SAR. In contrast, seedlings inoculated with Pt had greater shoot height, leaf area and shoot and leaf weights when TABLE 2 S u m m a r y o f I n t e r a c t i o n s for P a p e r B i r c h S e e d l i n g s . S i g n i f i c a n c e D e t e r m i n e d by S t a n d a r d Analysis of Variance (ANOVA)
Shoot height
Number of leaves
Leaf area
(%)
Mycorrhizal infection
Biomass Shoot
Root
Leaf
Interactions 0 3 x Soil
ns
ns
ns
ns
ns
*
ns
03 x pH Soil x Pt p H × Pt
ns ** **
ns ns *
** ** **
* ** ns
* ** **
* ns ns
** ** **
Pt = Pisolithus tinctorius. * S i g n i f i c a n c e at t h e 0-05 level. ** S i g n i f i c a n c e at t h e 0-01 level. ns, N o t s i g n i f i c a n t .
Kevin D. Keane, William J. Manning
60
TABLE 3 Effect o f the I n t e r a c t i o n Between O z o n e a n d p H o n Leaf A r e a %, Mycorrhizal Infection, a n d Shoot, R o o t a n d Leaf Weights of P a p e r Birch Seedlings. Each Value Represents the M e a n of 36 Plants
Leaf area (cm2) r % M y c o r r h i z a l infection z S h o o t weight (g) R o o t weight (g) Leaf weight (g)
CF
03
pH5"6 pH3"5
pH5"6 pH3"5
67"86 b 3" 1b 0'44 ~ 0"32 b 0"33 b
60-49 a 71"43 b 2.9ab 2.9~b 0'37 a 0"48 b 0"27 a 0"34 b 0"28 ~ 0-37 c
69"95 b 2.6 a 0"45 ab 0"34 b 0"33 b
Significance level
** * * * **
* Significance at the 0.05 level. ** Significance at the 0'01 level. " Mycorrhizal infection index (1 < 1%, 2 < 25%, 3 < 5 0 % a n d 4 > 75%). Y M e a n separation within rows by D u n c a n - W a l l e r multiple c o m p a r i s o n procedure at significance level indicated. C F = c a r b o n filtered air ( 0 . 0 0 p p m O3). O3 = C F plus ozone (0'06-0.08 ppm). 5"6 = control pH. 3"5 = simulated acid rain.
TABLE 4 Effect of the I n t e r a c t i o n Between p H a n d the Presence or Absence o f Pisolithus tinctorius o n S h o o t Height, L e a f Area, N u m b e r o f Leaves a n d S h o o t a n d Leaf Weights of Paper Birch Seedlings. Each Value Represents the M e a n o f 36 Plants
pH 5"6 w/Pt S h o o t height (cm) y Leaf area (cm 2) N u m b e r of leaves S h o o t weight (g) Leaf weight (g)
w/oPt
7.1" 8-0 b 59.53 ~ 68"82 ~b 6.44 7.1 b 0.38 a 0-44 b 0.28 a 0.32 b
pH 3"5 w/Pt 8.1 b 71.61b 7.5 b 0.47 b 0.35 b
Significance level
w/oPt 7-7 "b 69.77ab 9.4 ~ 0.46 b 0.34 b
** ** * ** **
* Significance at the 0'05 level. ** Significance at the 0'01 level. Y M e a n separation within rows by D u n c a n - W a l l e r multiple c o m p a r i s o n procedure at significance level indicated. 5.6 = control pH. 3.5 = simulated acid rain. Pt = Pisolithus tinctorius.
61
Effeets o f ozone and acid rain on birch seedling growth
e x p o s e d to S A R o f p H 3.5. N u m b e r o f leaves was greatest in n o n - i n o c u l a t e d seedlings regardless o f the S A R p H . T h e greatest values for s h o o t height, leaf area, a n d s h o o t a n d leaf weights o c c u r r e d in birch i n o c u l a t e d with Pt a n d e x p o s e d to p H 3.5 S A R . N u m b e r o f leaves was greatest in n o n - i n o c u l a t e d seedlings e x p o s e d to the l o w e r pH. A n i n t e r a c t i o n b e t w e e n soil regime a n d m y c o r r h i z a l t r e a t m e n t signific a n t l y affected s h o o t height, leaf area, per cent m y c o r r h i z a l infection a n d s h o o t a n d leaf weights (Table 5). N o significant g r o w t h differences o c c u r r e d for a n y o f the m e a s u r e d p a r a m e t e r s as a result o f m y c o r r h i z a l t r e a t m e n t within n o n - s t e a m e d soil. H o w e v e r , n o n - i n o c u l a t e d seedlings g r o w n in s t e a m e d soil h a d significantly larger s h o o t height, leaf area a n d s h o o t and leaf weights t h a n those which were inoculated. T h e greatest values for these g r o w t h variables also o c c u r r e d in birch g r o w n in s t e a m e d soil w i t h o u t Pt. In c o n t r a s t to o t h e r g r o w t h variables, per cent m y c o r r h i z a l infection was u n i q u e in its response to the i n t e r a c t i o n b e t w e e n soil regime a n d m y c o r r h i z a l t r e a t m e n t (Table 5). Per cent m y c o r r h i z a l infection was greatest in n o n s t e a m e d soil, a l t h o u g h n o significant difference was a p p a r e n t b e t w e e n seedlings i n o c u l a t e d with Pt a n d those n o n - i n o c u l a t e d seedlings. Significant differences were o b s e r v e d b e t w e e n m y c o r r h i z a l t r e a t m e n t s in s t e a m e d with n o n - i n o c u l a t e d birch h a v i n g significantly lower per cent m y c o r r h i z a l infection. A final t w o - w a y i n t e r a c t i o n , soil regime × m y c o r r h i z a l t r e a t m e n t , significantly influences r o o t weight (Table 6). O z o n e t r e a t m e n t resulted in n o TABLE 5
Effect of the Interaction Between Soil Treatment and the Presence or Absence of Pisolithus tinctorius on Shoot Height, Leaf Area, % Mycorrhizal Infection and Shoot and Root Weights of Paper Birch Seedlings. Each Value Represents the Mean of 36 Plants Non-steamed soil w/Pt
Shoot height (cm)y Leaf area (cm 2) % Mycorrhizal infection~ Shoot weight (g) Leaf weight (g)
w/o Pt
7.3ab 6.8" 61.95~ 61.42a 3"5be 3"6~ 0.36" 0.36" 0.28a 0.27"
Steamed soil w/Pt
Sign(/J'eanee level
w/o Pt
7.8b 9.0c 69-200 77.17c 3.1 h 1-2° 0.48b 0-53c 0.35h 0.39c
** ** ** ** **
* Significance at the 0-05 level. ** Significance at the 0-01 level. Mycorrhizal infection index (1 < 1%, 2 < 25%, 3 < 50% and 4 > 75%). ~' Mean separation within rows by Duncan-Waller multiple comparison procedure at significance level indicated. Pt = Pisolithus tinetorius.
62
Kevin D. Keane, William J. Manning TABLE 6 Effect of the Interaction Between Ozone and Soil Treatment on R o o t Weight of Paper Birch Seedlings. Each Value Represents the Mean of 36 Plants
CF
R o o t weight (g)Y
Significance level
0 3
NS
SS
NS
SS
0-28 a
0.38 c
0-28 a
0-33 b
*
*Significance at the 0"05 level. r Mean separation within rows by D u n c a n - W a l l e r multiple comparison procedure at significance level indicated. C F = carbon filtered air (0.00 ppm 03). 0 3 = C F plus ozone (0.06~.08 ppm). NS = non-steamed soil. SS = steamed soil.
significant differences in non-steamed soil. However, treatment of seedlings with 0 3 caused decreases in root weight when compared to CF in steamed soil. The greatest root weights occurred in birch treated with 0 3 and grown in steamed soil. DISCUSSION Previous work on the combined effects of gaseous pollutants and acidic precipitation has shown that significant interactions did not occur (Norby & Luxmoore, 1983; Norby et al., 1985; Reich et al., 1985). We have shown a significant interaction between 0 3 and SAR. Treatment of birch seedlings with pH 3"5 SAR caused increases in growth. These increases, however, were much greater for birch also exposed to 0 3 as opposed to those exposed to CF. Increased nutrient input from acidic precipitation has been shown to result in increased plant growth (Irving, 1983). Similarly, 0 3 at the concentrations used in our research, has also been shown to stimulate growth (Jensen & Masters, 1975). Exposure of paper birch to 03 and SAR could result in additive or synergistic growth increases. Several studies (Carney et al., 1978; Garrett et al., 1982; Mahoney et al., 1985) have indicated that mycorrhizae may provide some degree of protection from injury caused by gaseous pollutants. These results were observed with loblolly (Pinus taeda L.) and white pine (Pinus strobus L.) seedlings. In contrast to these observations, neither natural mycorrhizal, nor Pt appeared to have any protective influence on birch seedlings exposed to 03 or SAR. Reich and co-workers (1985), working with red oak seedlings, also found no direct or indirect protection due to mycorrhizae. In our research, total mycorrhizal infection was significantly decreased by
Effects of ozone and acid rain on birch seedling growth
63
treatment with pH 3-5 SAR seedlings in CF. A concomitant decrease due to 3.5 pH SAR was, however, not apparent in birch exposed to 0 3. Decreases due to SARs alone have been previously reported (Shafer et al., 1985; Stroo & Alexander, 1985). The interaction of soil regime and mycorrhizal treatment (Table 5) displayed significant differences in the manner in which Pt exerted its influence. The addition of Pt caused increased growth in non-steamed soil. In contrast, seedlings in steamed soil inoculated with Pt appeared to have decreased growth. However, perhaps a more important aspect of this interaction can be seen in a comparison of growth in steamed and nonsteamed soil (Table 5). Seedlings grown in steamed soil had significantly increased growth, than those in non-steamed soil. The majority of air pollution studies are conducted utilising commercial potting mixtures or sterilised field soil, with supplemental nutrients provided on a regular basis. Our results indicate that seedlings behave differently in steamed and nonsteamed soil. This is most likely a function of the presence of active soil microfauna and microflora in non-steamed soil. The secondary effects of air pollutants on ectomycorrhizae and other microbial organisms integral to tree growth have been largely ignored. These effects could be more deleterious to the growth of the trees than the direct effects of pollutants. More intensive and long-term research into the secondary effects of air pollutants on mycorrhizae and other important soilborne organisms is necessary. REFERENCES Abrahamsen, G., Horntvedt, R. & Tveite, B. (1977). Impacts of acid precipitation on coniferous forest ecosystems. Water Air Soil Pollut., 8, 57 73. Bartuska, A. M. & Medlarz, S. A. (1986). Spruce fir decline Air pollution related'? In Atmospheric deposition and Jorest productivity, Proceedings ~[ the Fourth Regional Technical Conference of the Appalachian SocieO' ~[ the American Foresters. Raleigh, N.C., 55 73. Beckjord, P. R., Smith, D. W. & Mclntosh, M. S. (1984). Effects of nitrogen fertilization and Pisolithus tinctorius on Quercus ruhra seedling root and top development. For. Sci., 30, 124-8. Carney, J. L., Garrett, H. E. & Hedrick, H. G. (1978). Influence of air pollutant gases on oxygen uptake of pine roots with selected ectomycorrhizae. Phytopatholo~y, 68, 1160 3.
Chappelka, A. H. & Chevone, B. I. (1986). White ash seedling growth response to ozone and simulated acid rain. Can. J. For. Res., 16, 786 90. Chappelka, A. H., Chevone, B. I. & Burk, T. E. (1985). Growth response of yellowpoplar (Liriodendron tulipifera L.) seedlings to ozone, sulfur dioxide, and simulated acidic precipitation, alone and in combination. Environ. Exp. Bot., 25, 233-44.
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Costonis, A. C. (1970). Acute foliar injury of eastern white pine induced by sulfur dioxide and ozone. Phytopathology, 60, 994-9. Damon, R. A. & Harvey, W. R. (1987). Experimental Design, ANOVA, and Regression. Harper and Row, New York. Danielson, R. M., Visser, S. & Parkinson, D. (1984). The effectiveness of mycelial slurries of mycorrhizal fungi for the inoculation of container grown jack pine seedlings. Can. J. For. Res., 14, 140-2. Elliott, C. L., Eberhardt, J. C. & Brennan, E. G. (1987). The effect of ambient ozone pollution and acidic rain on the growth and chlorophyll content of green and white ash. Environ. Pollut., 44, 61-70. Freer-Smith, P. H. (1985). The influence of SO2 and NO2 on the growth, development and gas exchange of Betula pendula Roth. New Phytologist, 99, 417-30. Garrett, H. E., Carney, J. L. & Hedrick, H. G. (1982). The effects of ozone and sulfur dioxide on the respiration of ectomycorrhizal fungi. Can. J. For. Res., 12, 141-5. Grand, L. F. & Harvey, A. E. (1982). Quantitative measurement of ectomycorrhizae on plant roots. In Methods and principles ofmycorrhizal research, ed. by N. C. Schenck. APS Press, St Paul. 157-64. Irving, P. M. (1983). Acidic precipitation effects on crops: A review and analysis of research. J. Environ. Qual., 12, 442-53. Jensen, K. F. & Masters, R. G. (1975). Growth of six woody species fumigated with ozone. Plant Dis. Reptr., 59, 760-2. Johnson, A. H. (1983). Red spruce decline in the northeastern US: Hypothesis regarding the role of acid rain. J. Air Pollut. Control Assoc., 33, 1049-54. Mahoney, M. J., Chevone, B. I., Skelly, J. M. & Moore, L. D. (1985). Influence of mycorrhizae on the growth of loblolly pine seedlings exposed to ozone and sulfur dioxide. Phytopathology, 75, 679-82. Marx, D. H. (1972). Ectomycorrhizae as biological deterrents to pathogenic root infections. Ann. Rev. Phytopath., 10, 429-54. Marx, D. H. & Kenney, D. S. (1982). Production of ectomycorrhizal inoculum. In Methods and principles ofmycorrhizal research, ed. by N. C. Schenck, APS Press, St. Paul. 131-46. Molina, R. & Palmer, J. G. (1982). Isolation, maintenance and pure culture manipulation of ectomycorrhizal fungi. In Methods and principles in "mycorrhizal research. (Schenck, N. C. (Ed.)), APS Press, St. Paul. 115-29. Norby, R. J. & Luxmoore, R. J. (1983). Growth analysis of soybean exposed to simulated acid rain and gaseous air pollutants. Phytologist, 95, 277-87. Norby, R. J., Richter, D. D. & Luxmoore, R. J. (1985). Physiological processes in soybean inhibited by gaseous pollutants but not by acid rain. New Phytologist, 100, 79-85. Reich, P. B. & Amundson, R. G. (1985). Ambient levels of ozone reduce net photosynthesis in tree and crop species. Science, 230, 566-70. Reich, P. B., Schoettle, A. W., Stroo, H. F., Troiano, J. & Amundson, R. G. (1985). Effects of O3, SO 2 and acidic rain on mycorrhizal infection in northern red oak. Can. J. Bot., 63, 2049-55. Riffle, J. W. & Maronek, M. (1982). Ectomycorrhizal inoculation procedures for greenhouse and nursery studies. In Methods and principles of mycorrhizal research, ed. by N. C. Schenck, APS Press, St. Paul. 147-55. Shafer, S. R., Grand, L. F., Bruck, R. I. & Heagle, A. S. (1985). Formation of
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ectomycorrhizae on Pinus taeda seedlings exposed to simulated acidic rain. Can. J. For. Res., 15, 66-71. Siccama, T. G., Bliss, M. & Vogelmann, H. W. (1982). Decline of red spruce in the Green Mountains of Vermont. Bull. Torrey Bot. Club., 109, 162-8. Smith, R. A. (1982). Nutritional study of Pisolithus tinctorius. Mycologica, 74, 54-58. Stroo, H. F. & Alexander M. (1985). Effect of simulated acidic rain on mycorrhizal infection of Pinus strobus L. Water Air Soil Pollut., 25, 107-14. Sylvia, D. M. & Sinclair, W. A. (1983). Suppressive influence of Laccaria laccata on Fusarium oxysporum and on Douglas-fir seedlings, Phytopathology, 73, 384-- 9. Wood, T. & Bormann, F. H. (1977). Short-term effects of a simulated acid rain upon the growth and nutrient relations of Pinus strobus, L. Water Air Soil Pollut., 7, 479-88.
Effects of Ozone and Simulated Acid Rain on Birch Seedling Growth and Formation of Ectomycorrhizae Kevin D. Keane & William J. Manning Department of Plant Pathology, University of Massachusetts, Amherst, Massachusetts 01003, USA (Received 1 September 1987; revised version received 15 October 1987: accepted 24 November 1987)
ABSTRACT Four- week-old paper birch (Bet ula papyrifera Marsh.) seedlings, inoculated or non-inoculated with the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker & Couch and grown in steamed or non-steamed soil, were exposed to ozone ( 0 3 ) and/or simulated acid rain ( S A R ) . Plants were exposed to 0 3 for 7h per day on 5days per week for 12 weeks. 0 3 concentrations were maintained between 0"06 and 0.08 ppm. SA R was applied lOmin per day on 2days per week. 0 3, SAR, soil regime and mycorrhizal treatment did not significantly affect any o f the measured variables. Interactions between 03 and SAR, S A R and mycorrhizal treatment, soil regime and mycorrhizal treatment and ozone and soil regime had significant effects. Treatment of seedlings with p H 3.5 S A R caused increases in growth which were more apparent in birch exposed to 03. Mycorrhizal treatment caused increased growth in non-steamed soil, while growth appeared to decrease in steamed soil. Birch seedlings grew much better in steamed soil. The implications of increased growth in steamed soil may demonstrate the impo'rtance of looking at the secondary effects of pollutants on soilborne organisms.
INTRODUCTION There have been m a n y reports on the effects of gaseous air pollutants and simulated acid rain on trees, particularly conifers (Costonis, 1970; Abrahamsen et al., 1977; W o o d & Bormann, 1977). Fewer studies have 55 Environ. Pollut. 0269-7491/88/$03.50 (~ 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain
56
Kevin D. Keane, William J. Manning
focused on the effects of these air pollutants on deciduous trees (Freer-Smith, 1985; Chappelka et al., 1985; Chappelka & Chevone, 1986; Elliott et al., 1987). Paper birch (Betulapapyrifera Marsh.), a deciduous tree, comprises an integral part of spruce-fir forests in the northeast region of the US (Bartuska & Medlarz, 1986). Red spruce (Picea rubens Sarg.) in the region is in a widespread state of decline. Some effects on balsam fir (Abies balsamea Mill.) and paper birch have also been noted at certain locations (Johnson, 1983). Ozone (03) and acidic precipitation are thought to be contributing factors to tree decline problems (Siccama et al., 1982). The effects of ozone and simulated acid rain have been studied by several researchers (Norby & Luxmore, 1983; Chappelka et al., 1985; Reich & Amundson, 1985; Chappelka & Chevone, 1986; Elliott et al., 1987). Most of this research, however, has been concerned with the above-ground plant parts. An equally important aspect, that has only recently received attention, are the effects of air pollutants and simulated acid rain on roots and mycorrhizae. Mycorrhizae, symbiotic fungal-root associations, are beneficial to the growth and development of trees (Marx, 1972; Sylvia & Sinclair, 1983; Beckjord et al., 1984). Mycorrhizae have also been shown to alter some of the effects of air pollutants through promotion of shoot and root growth (Carney et al., 1978; Garrett et al., 1982; Mahoney et al., 1985). However, only Reich and co-workers (1985) have presented information concerning the effects of gaseous pollutants and acidic precipitation upon mycorrhizal infection. Red oak (Quercus rubra L.) mycorrhizae were found to be adversely affected by ozone, sulphur dioxide and acidic precipitation. Our objective was to determine the impact of ozone and/or acidic precipitation upon the growth and development of paper birch seedlings and their mycorrhizal associations.
MATERIALS AND METHODS Preparation of inoeulum The ectomycorrhizal fungus, Pisolithus tinctorius (Pers.) Coker & Couch, strain 288 (obtained from D. H. Marx) was maintained on modified Melin-Norkrans medium (MMN) (Molina & Palmer, 1982; Smith, 1982). Liquid cultures of Pt were prepared by placing 15-20 agar discs of actively growing mycelium into flasks of MMN broth (Danielson et al., 1984). These cultures were then placed on a rotary shaker and allowed to grow for 2 weeks. Once the liquid cultures had grown sufficiently, they were aseptically inoculated into a solid substrate composed of vermiculite and peat moss
Effects o f ozone and acid rain on birch seedling growth
57
(28:1 v/v) in 500 ml Erlenmeyer flasks (Marx & Kenney, 1982). These fungal cultures were then allowed to grow until they had thoroughly colonised the substrate. Cultures were then placed in cold storage at 3°C until used.
Soil preparation Soil for the experiments was obtained from an indigenous stand of paper birch in western Massachusetts. Leaf litter was removed from the site and soil was collected primarily from the O, A and B horizons. This soil was mixed with sand (2 soil:l sand v/v) and divided into two lots. One lot was steam sterilised twice (30 min at 82°C). The other lot was not treated. Using vermiculite-peat mycorrhizal inoculum, half of each soil type was inoculated with Pisolithus tinctorius (Pt). Inoculation was carried out prior to potting in an 8:1 v/v ratio (soil:inoculum) using a broadcast method (Riffle & Maronek, 1982). All remaining soil received an equivalent amount of untreated vermiculite. Four-week-old seedlings of paper birch, grown from seed were transplanted into the four soil regimes. Seedlings had been germinated in Redi-Earth potting mixture (W. R. Grace and Co., Cambridge, MA) and were selected for relative uniformity.
Ozone and simulated rain exposures Seedlings were placed in a set of eight rectangular Plexiglas chambers (72 cm × 45 cm × 45 cm). These chambers are part of a single pass-through air system. All incoming air was filtered through two particulate filters and 10 in of activated charcoal. Air entered the chambers as carbon filtered air (0-00 ppm O3) or as CF plus O 3. Four chambers received carbon filtered air (CF). The remaining four received CF plus O 3. Each chamber contained 40 seedlings which received a 16 h photoperiod and were rotated between chambers on a weekly basis. Ozone was produced by an O R E C ozone generator (Ozone Research and Equipment Corp., Phoenix, AR) and constantly monitored using a Dasibi 1003H monitor (Dasibi Corp., Glendale, CA) which was calibrated to EPA standards. Ozone concentrations were maintained between 0.06 and 0"08ppm. Birch seedlings were fumigated with 03 for 7 h d a y -~ 5 days week- 1 for 12 weeks. Paper birch seedlings were also exposed to simulated acid rain (SAR) of pH 3"5 or pH5"6. The pH3"5 treatment was acidified with nitric and sulphuric acids (1:1 v/v). Rain treatments were composed of deionised water and appropriate concentrations of background ions (Table 1), common to precipitation in western Massachusetts. (H. Hemond, Massachusetts
58
Kevin D. Keane, William J. Manning
Institute of Technology, pers. comm.). Seedlings were removed from the chambers and treated with S A R o f p H 3 . 5 or p H 5 . 6 for 1 0 m i n d a y -1, 2 days w e e k - 1 through the use o f a pressurised system utilising special rain nozzles (Spray Systems Inc., Wheaton, IL). This provided each seedling with 30 ml o f SAR, comparable to normal rainfall. All seedlings were watered with appropriate rain simulants, as needed between rain treatments. N o additional nutrients were supplied during the experiment. The experiment was terminated after 12 weeks. Measurements of shoot height; root, shoot and leaf dry weights; leaf area and leaf number were
TABLE 1 Ion Concentrations of Rain Simulants (pH 3-5 and 5"6) Ion
H÷ C a +2
Mg +2 K+ Na + NH4 + C1SO,~2 NO 3
Concentration (peq litre- a)
Concentration (mg litre 1)
59"9 6-1 2"9 1"9 8"5 7"7 14.5 40-3 20"6
0"0604 0-1222 0"0352 0-0749 0-1954 0-1389 0-5141 1-9356 1.2773
Data from Precipitation Analysis at Bickford Reservoir, Massachusetts, July 1981 to June 1983. obtained. A quantitative analysis of total per cent mycorrhizal infection (natural and inoculated) was determined for each individual birch seedling (Grand & Harvey, 1982). A scale of 1-4 (1 = < 1%, 2 = < 2 5 % , 3 = < 5 0 % , 4 = > 75%) was utilised to describe per cent infection. A minimum of nine seedlings were evaluated for each treatment.
Experimental design and data analysis A randomised complete block design was used for all experiments. Within this design was a factorial consisting o f two levels of 0 3 x two levels of S A R x two soil regimes x two mycorrhizal treatments. All data were analysed with an A N O V A (analysis of variance). Further data analysis was carried out using a D u n c a n - W a l l e r multiple comparison procedure ( D a m o n & Harvey, 1987).
Effects of ozone and acid rain on birch seedling growth
59
RESULTS Chronic 0 3 injury was apparent on some birch seedlings. This injury consisted of chlorosis and in some cases was accompanied by yellow flecking. SAR did not cause visible symptoms. Ozone, SAR, soil and mycorrhizae did not significantly affect shoot height, number of leaves, leaf area, per cent mycorrhizal infection or root, shoot and leaf weights. There were several two-way interactions which were significant (Table 2). An 03 × pH interaction significantly influenced leaf area, per cent mycorrhizal infection and root, shoot and leaf weights (Table 3). Treatment of seedlings with SAR o f p H 3"5 caused increases in leaf area and root, shoot and leaf weights regardless of the 03 treatment involved. However, these growth increases were much greater for those seedlings exposed to 03 . The largest values for these parameters occurred among those birch treated with 03 and SAR of pH 3-5. Per cent mycorrhizal infection responded differently to the interaction of 03 and SAR. Treatment of seedlings with pH 3.5 SAR resulted in significant decreases of mycorrhizal infection in CF. Birch exposed to 03, however, did not show any effects due to pH 3-5 SAR. The greatest mycorrhizal infection occurred in seedlings treated with CF and pH 5"6 SAR. A two-way interaction between pH and mycorrhizal treatment significantly affected shoot height, leaf area, number of leaves and shoot and leaf weights (Table 4). These growth variables were all greater in seedlings noninoculated with Pt exposed to pH 5"6 SAR. In contrast, seedlings inoculated with Pt had greater shoot height, leaf area and shoot and leaf weights when TABLE 2 S u m m a r y o f I n t e r a c t i o n s for P a p e r B i r c h S e e d l i n g s . S i g n i f i c a n c e D e t e r m i n e d by S t a n d a r d Analysis of Variance (ANOVA)
Shoot height
Number of leaves
Leaf area
(%)
Mycorrhizal infection
Biomass Shoot
Root
Leaf
Interactions 0 3 x Soil
ns
ns
ns
ns
ns
*
ns
03 x pH Soil x Pt p H × Pt
ns ** **
ns ns *
** ** **
* ** ns
* ** **
* ns ns
** ** **
Pt = Pisolithus tinctorius. * S i g n i f i c a n c e at t h e 0-05 level. ** S i g n i f i c a n c e at t h e 0-01 level. ns, N o t s i g n i f i c a n t .
Kevin D. Keane, William J. Manning
60
TABLE 3 Effect o f the I n t e r a c t i o n Between O z o n e a n d p H o n Leaf A r e a %, Mycorrhizal Infection, a n d Shoot, R o o t a n d Leaf Weights of P a p e r Birch Seedlings. Each Value Represents the M e a n of 36 Plants
Leaf area (cm2) r % M y c o r r h i z a l infection z S h o o t weight (g) R o o t weight (g) Leaf weight (g)
CF
03
pH5"6 pH3"5
pH5"6 pH3"5
67"86 b 3" 1b 0'44 ~ 0"32 b 0"33 b
60-49 a 71"43 b 2.9ab 2.9~b 0'37 a 0"48 b 0"27 a 0"34 b 0"28 ~ 0-37 c
69"95 b 2.6 a 0"45 ab 0"34 b 0"33 b
Significance level
** * * * **
* Significance at the 0.05 level. ** Significance at the 0'01 level. " Mycorrhizal infection index (1 < 1%, 2 < 25%, 3 < 5 0 % a n d 4 > 75%). Y M e a n separation within rows by D u n c a n - W a l l e r multiple c o m p a r i s o n procedure at significance level indicated. C F = c a r b o n filtered air ( 0 . 0 0 p p m O3). O3 = C F plus ozone (0'06-0.08 ppm). 5"6 = control pH. 3"5 = simulated acid rain.
TABLE 4 Effect of the I n t e r a c t i o n Between p H a n d the Presence or Absence o f Pisolithus tinctorius o n S h o o t Height, L e a f Area, N u m b e r o f Leaves a n d S h o o t a n d Leaf Weights of Paper Birch Seedlings. Each Value Represents the M e a n o f 36 Plants
pH 5"6 w/Pt S h o o t height (cm) y Leaf area (cm 2) N u m b e r of leaves S h o o t weight (g) Leaf weight (g)
w/oPt
7.1" 8-0 b 59.53 ~ 68"82 ~b 6.44 7.1 b 0.38 a 0-44 b 0.28 a 0.32 b
pH 3"5 w/Pt 8.1 b 71.61b 7.5 b 0.47 b 0.35 b
Significance level
w/oPt 7-7 "b 69.77ab 9.4 ~ 0.46 b 0.34 b
** ** * ** **
* Significance at the 0'05 level. ** Significance at the 0'01 level. Y M e a n separation within rows by D u n c a n - W a l l e r multiple c o m p a r i s o n procedure at significance level indicated. 5.6 = control pH. 3.5 = simulated acid rain. Pt = Pisolithus tinctorius.
61
Effeets o f ozone and acid rain on birch seedling growth
e x p o s e d to S A R o f p H 3.5. N u m b e r o f leaves was greatest in n o n - i n o c u l a t e d seedlings regardless o f the S A R p H . T h e greatest values for s h o o t height, leaf area, a n d s h o o t a n d leaf weights o c c u r r e d in birch i n o c u l a t e d with Pt a n d e x p o s e d to p H 3.5 S A R . N u m b e r o f leaves was greatest in n o n - i n o c u l a t e d seedlings e x p o s e d to the l o w e r pH. A n i n t e r a c t i o n b e t w e e n soil regime a n d m y c o r r h i z a l t r e a t m e n t signific a n t l y affected s h o o t height, leaf area, per cent m y c o r r h i z a l infection a n d s h o o t a n d leaf weights (Table 5). N o significant g r o w t h differences o c c u r r e d for a n y o f the m e a s u r e d p a r a m e t e r s as a result o f m y c o r r h i z a l t r e a t m e n t within n o n - s t e a m e d soil. H o w e v e r , n o n - i n o c u l a t e d seedlings g r o w n in s t e a m e d soil h a d significantly larger s h o o t height, leaf area a n d s h o o t and leaf weights t h a n those which were inoculated. T h e greatest values for these g r o w t h variables also o c c u r r e d in birch g r o w n in s t e a m e d soil w i t h o u t Pt. In c o n t r a s t to o t h e r g r o w t h variables, per cent m y c o r r h i z a l infection was u n i q u e in its response to the i n t e r a c t i o n b e t w e e n soil regime a n d m y c o r r h i z a l t r e a t m e n t (Table 5). Per cent m y c o r r h i z a l infection was greatest in n o n s t e a m e d soil, a l t h o u g h n o significant difference was a p p a r e n t b e t w e e n seedlings i n o c u l a t e d with Pt a n d those n o n - i n o c u l a t e d seedlings. Significant differences were o b s e r v e d b e t w e e n m y c o r r h i z a l t r e a t m e n t s in s t e a m e d with n o n - i n o c u l a t e d birch h a v i n g significantly lower per cent m y c o r r h i z a l infection. A final t w o - w a y i n t e r a c t i o n , soil regime × m y c o r r h i z a l t r e a t m e n t , significantly influences r o o t weight (Table 6). O z o n e t r e a t m e n t resulted in n o TABLE 5
Effect of the Interaction Between Soil Treatment and the Presence or Absence of Pisolithus tinctorius on Shoot Height, Leaf Area, % Mycorrhizal Infection and Shoot and Root Weights of Paper Birch Seedlings. Each Value Represents the Mean of 36 Plants Non-steamed soil w/Pt
Shoot height (cm)y Leaf area (cm 2) % Mycorrhizal infection~ Shoot weight (g) Leaf weight (g)
w/o Pt
7.3ab 6.8" 61.95~ 61.42a 3"5be 3"6~ 0.36" 0.36" 0.28a 0.27"
Steamed soil w/Pt
Sign(/J'eanee level
w/o Pt
7.8b 9.0c 69-200 77.17c 3.1 h 1-2° 0.48b 0-53c 0.35h 0.39c
** ** ** ** **
* Significance at the 0-05 level. ** Significance at the 0-01 level. Mycorrhizal infection index (1 < 1%, 2 < 25%, 3 < 50% and 4 > 75%). ~' Mean separation within rows by Duncan-Waller multiple comparison procedure at significance level indicated. Pt = Pisolithus tinetorius.
62
Kevin D. Keane, William J. Manning TABLE 6 Effect of the Interaction Between Ozone and Soil Treatment on R o o t Weight of Paper Birch Seedlings. Each Value Represents the Mean of 36 Plants
CF
R o o t weight (g)Y
Significance level
0 3
NS
SS
NS
SS
0-28 a
0.38 c
0-28 a
0-33 b
*
*Significance at the 0"05 level. r Mean separation within rows by D u n c a n - W a l l e r multiple comparison procedure at significance level indicated. C F = carbon filtered air (0.00 ppm 03). 0 3 = C F plus ozone (0.06~.08 ppm). NS = non-steamed soil. SS = steamed soil.
significant differences in non-steamed soil. However, treatment of seedlings with 0 3 caused decreases in root weight when compared to CF in steamed soil. The greatest root weights occurred in birch treated with 0 3 and grown in steamed soil. DISCUSSION Previous work on the combined effects of gaseous pollutants and acidic precipitation has shown that significant interactions did not occur (Norby & Luxmoore, 1983; Norby et al., 1985; Reich et al., 1985). We have shown a significant interaction between 0 3 and SAR. Treatment of birch seedlings with pH 3"5 SAR caused increases in growth. These increases, however, were much greater for birch also exposed to 0 3 as opposed to those exposed to CF. Increased nutrient input from acidic precipitation has been shown to result in increased plant growth (Irving, 1983). Similarly, 0 3 at the concentrations used in our research, has also been shown to stimulate growth (Jensen & Masters, 1975). Exposure of paper birch to 03 and SAR could result in additive or synergistic growth increases. Several studies (Carney et al., 1978; Garrett et al., 1982; Mahoney et al., 1985) have indicated that mycorrhizae may provide some degree of protection from injury caused by gaseous pollutants. These results were observed with loblolly (Pinus taeda L.) and white pine (Pinus strobus L.) seedlings. In contrast to these observations, neither natural mycorrhizal, nor Pt appeared to have any protective influence on birch seedlings exposed to 03 or SAR. Reich and co-workers (1985), working with red oak seedlings, also found no direct or indirect protection due to mycorrhizae. In our research, total mycorrhizal infection was significantly decreased by
Effects of ozone and acid rain on birch seedling growth
63
treatment with pH 3-5 SAR seedlings in CF. A concomitant decrease due to 3.5 pH SAR was, however, not apparent in birch exposed to 0 3. Decreases due to SARs alone have been previously reported (Shafer et al., 1985; Stroo & Alexander, 1985). The interaction of soil regime and mycorrhizal treatment (Table 5) displayed significant differences in the manner in which Pt exerted its influence. The addition of Pt caused increased growth in non-steamed soil. In contrast, seedlings in steamed soil inoculated with Pt appeared to have decreased growth. However, perhaps a more important aspect of this interaction can be seen in a comparison of growth in steamed and nonsteamed soil (Table 5). Seedlings grown in steamed soil had significantly increased growth, than those in non-steamed soil. The majority of air pollution studies are conducted utilising commercial potting mixtures or sterilised field soil, with supplemental nutrients provided on a regular basis. Our results indicate that seedlings behave differently in steamed and nonsteamed soil. This is most likely a function of the presence of active soil microfauna and microflora in non-steamed soil. The secondary effects of air pollutants on ectomycorrhizae and other microbial organisms integral to tree growth have been largely ignored. These effects could be more deleterious to the growth of the trees than the direct effects of pollutants. More intensive and long-term research into the secondary effects of air pollutants on mycorrhizae and other important soilborne organisms is necessary. REFERENCES Abrahamsen, G., Horntvedt, R. & Tveite, B. (1977). Impacts of acid precipitation on coniferous forest ecosystems. Water Air Soil Pollut., 8, 57 73. Bartuska, A. M. & Medlarz, S. A. (1986). Spruce fir decline Air pollution related'? In Atmospheric deposition and Jorest productivity, Proceedings ~[ the Fourth Regional Technical Conference of the Appalachian SocieO' ~[ the American Foresters. Raleigh, N.C., 55 73. Beckjord, P. R., Smith, D. W. & Mclntosh, M. S. (1984). Effects of nitrogen fertilization and Pisolithus tinctorius on Quercus ruhra seedling root and top development. For. Sci., 30, 124-8. Carney, J. L., Garrett, H. E. & Hedrick, H. G. (1978). Influence of air pollutant gases on oxygen uptake of pine roots with selected ectomycorrhizae. Phytopatholo~y, 68, 1160 3.
Chappelka, A. H. & Chevone, B. I. (1986). White ash seedling growth response to ozone and simulated acid rain. Can. J. For. Res., 16, 786 90. Chappelka, A. H., Chevone, B. I. & Burk, T. E. (1985). Growth response of yellowpoplar (Liriodendron tulipifera L.) seedlings to ozone, sulfur dioxide, and simulated acidic precipitation, alone and in combination. Environ. Exp. Bot., 25, 233-44.
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Kevin D. Keane, William J. Manning
Costonis, A. C. (1970). Acute foliar injury of eastern white pine induced by sulfur dioxide and ozone. Phytopathology, 60, 994-9. Damon, R. A. & Harvey, W. R. (1987). Experimental Design, ANOVA, and Regression. Harper and Row, New York. Danielson, R. M., Visser, S. & Parkinson, D. (1984). The effectiveness of mycelial slurries of mycorrhizal fungi for the inoculation of container grown jack pine seedlings. Can. J. For. Res., 14, 140-2. Elliott, C. L., Eberhardt, J. C. & Brennan, E. G. (1987). The effect of ambient ozone pollution and acidic rain on the growth and chlorophyll content of green and white ash. Environ. Pollut., 44, 61-70. Freer-Smith, P. H. (1985). The influence of SO2 and NO2 on the growth, development and gas exchange of Betula pendula Roth. New Phytologist, 99, 417-30. Garrett, H. E., Carney, J. L. & Hedrick, H. G. (1982). The effects of ozone and sulfur dioxide on the respiration of ectomycorrhizal fungi. Can. J. For. Res., 12, 141-5. Grand, L. F. & Harvey, A. E. (1982). Quantitative measurement of ectomycorrhizae on plant roots. In Methods and principles ofmycorrhizal research, ed. by N. C. Schenck. APS Press, St Paul. 157-64. Irving, P. M. (1983). Acidic precipitation effects on crops: A review and analysis of research. J. Environ. Qual., 12, 442-53. Jensen, K. F. & Masters, R. G. (1975). Growth of six woody species fumigated with ozone. Plant Dis. Reptr., 59, 760-2. Johnson, A. H. (1983). Red spruce decline in the northeastern US: Hypothesis regarding the role of acid rain. J. Air Pollut. Control Assoc., 33, 1049-54. Mahoney, M. J., Chevone, B. I., Skelly, J. M. & Moore, L. D. (1985). Influence of mycorrhizae on the growth of loblolly pine seedlings exposed to ozone and sulfur dioxide. Phytopathology, 75, 679-82. Marx, D. H. (1972). Ectomycorrhizae as biological deterrents to pathogenic root infections. Ann. Rev. Phytopath., 10, 429-54. Marx, D. H. & Kenney, D. S. (1982). Production of ectomycorrhizal inoculum. In Methods and principles ofmycorrhizal research, ed. by N. C. Schenck, APS Press, St. Paul. 131-46. Molina, R. & Palmer, J. G. (1982). Isolation, maintenance and pure culture manipulation of ectomycorrhizal fungi. In Methods and principles in "mycorrhizal research. (Schenck, N. C. (Ed.)), APS Press, St. Paul. 115-29. Norby, R. J. & Luxmoore, R. J. (1983). Growth analysis of soybean exposed to simulated acid rain and gaseous air pollutants. Phytologist, 95, 277-87. Norby, R. J., Richter, D. D. & Luxmoore, R. J. (1985). Physiological processes in soybean inhibited by gaseous pollutants but not by acid rain. New Phytologist, 100, 79-85. Reich, P. B. & Amundson, R. G. (1985). Ambient levels of ozone reduce net photosynthesis in tree and crop species. Science, 230, 566-70. Reich, P. B., Schoettle, A. W., Stroo, H. F., Troiano, J. & Amundson, R. G. (1985). Effects of O3, SO 2 and acidic rain on mycorrhizal infection in northern red oak. Can. J. Bot., 63, 2049-55. Riffle, J. W. & Maronek, M. (1982). Ectomycorrhizal inoculation procedures for greenhouse and nursery studies. In Methods and principles of mycorrhizal research, ed. by N. C. Schenck, APS Press, St. Paul. 147-55. Shafer, S. R., Grand, L. F., Bruck, R. I. & Heagle, A. S. (1985). Formation of
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ectomycorrhizae on Pinus taeda seedlings exposed to simulated acidic rain. Can. J. For. Res., 15, 66-71. Siccama, T. G., Bliss, M. & Vogelmann, H. W. (1982). Decline of red spruce in the Green Mountains of Vermont. Bull. Torrey Bot. Club., 109, 162-8. Smith, R. A. (1982). Nutritional study of Pisolithus tinctorius. Mycologica, 74, 54-58. Stroo, H. F. & Alexander M. (1985). Effect of simulated acidic rain on mycorrhizal infection of Pinus strobus L. Water Air Soil Pollut., 25, 107-14. Sylvia, D. M. & Sinclair, W. A. (1983). Suppressive influence of Laccaria laccata on Fusarium oxysporum and on Douglas-fir seedlings, Phytopathology, 73, 384-- 9. Wood, T. & Bormann, F. H. (1977). Short-term effects of a simulated acid rain upon the growth and nutrient relations of Pinus strobus, L. Water Air Soil Pollut., 7, 479-88.