Influence of Heavy Metal Leaf Contaminants on the in vitro Growth of Urban-Tree Phylloplane-Fungi WILLIAM I-I. SMITH School of Forestry and Environmental Studies Yale University New Haven, Connecticut 06511

Abstract. The surfaces of urban woody vegetation are contaminated with varying amounts of numerous metallic compounds, including Cd, Cu, Mn, AI, Cr, Ni, Fe, Pb, Na, and Zn. To examine the possibility that these metals may affect phylloplane fungi, the abovecations were tested in vitro for their ability to influence the growth of numerous saprophytic and parasitic fungi isolated from the leaves of London plane trees. Considerable variation in growth inhibition by the metals was observed. Generally Aureobasidium pullulans, Epicoccum sp., and Phialophora verrucosa were relatively tolerant; Gnomonia platani, Cladsporium sp., and Pleurophomella sp. were intermediate; and Pestalotiopsis and Chaetomium sp. were relatively sensitive to the incorporation of certain metals into solid and liquid media. [f similar growth inhibitions occur in nature, competitive abilities or population structures of plant surface microbes may be altered by surface metal contamination. Metals causing the greatest and broadest spectrum growth suppression included Ni, Zn, Pb, A1, Fe, and Mn.

Introduction The relationship b e t w e e n air pollution and m i c r o o r g a n i s m s is an important and i n c o m p l e t e l y appreciated topic [ 1 , 7 , 12, 16, 17]. O r g a n i s m s that exist in the p h y l l o p l a n e for all or part o f their life-cycle are particularly vulnerable to the influence o f air contaminants [21]. Since the leaves and t w i g s o f urban and roadside plants are superficially c o n t a m i n a t e d with numerous trace metals [ 1 8 - 2 0 , 22] and because several o f these metals are essential or toxic to microbes under certain c o n d i t i o n s [15], it is appropriate to e x a m i n e the effects o f particulate metal contaminants on plant surface fungi. This study e x a m i n e d the influence o f ten metals, all found to be c o m m o n c o n t a m i n a n t s o f urban tree leaf and twig tissue [20] on the g r o w t h o f selected saprophytic and parasitic fungi isolated from foliar surfaces o f streetside L o n d o n plane trees in d o w n t o w n N e w H a v e n . O f particular interest was an assessment o f differential h e a v y metal response o f n o n - p a t h o g e n i c and p a t h o g e n i c fungi.

Materials and Methods Leaf washing and impression techniques [3] were employed to isolate phylloplane fungi form the leaves of mature London plane (Platanas acerifolia [Ait.] Willd.) growing in downtown New Microbial Ecology 3, 231-239 (1977) 1977 by Springer-Verlag New York Inc.

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Haven. The following fungi were consistently isolated from various crown positions and at different times during the growing season. Those existing primarily saprophytically included; Aureobasidium pullulans (deBary) Arn., Caetomium sp., Cladosporium sp., Epicoccum sp., and Phialophora verrucosa Medlar. Those existing primarily parasitically included Gnomonia platani Kleb., Pestalotiposis sp., and Pleurophomella sp. These organisms were maintained in plate culture and evaluated in vitro to determine their tolerance to several metals known to be superficial contaminants of urban tree-leaf surfaces. The metals included in this investigation were Cd, Cu, Mn, AI, Cr, Ni, Fe, Pb, Na, and Zn. In every test a nitrate salt was used with the cation concentration equivalent to the average or maximum leaf burden (dry weight basis) of deciduous tree species sampled in the fall in New Haven, Connecticut [20]. The average concentrations in parts per million (/xg/gm) were Cd 1.5, Cu 8.7, Mn 469, AI 503, Cr 2.8, Ni 10, Fe 404, Pb 110, Na 373, and Zn 130. The maximum concentrations in parts per million (/xg/gm) were Cd 2.2, Cu 18, Mn 1311, AI 783, Cr 7.4, Ni 19, Fe 791, Pb 275, Na 977, and Zn 265. The in vitro tests performed included linear extension in plate culture and dry weight in liquid culture. No dry weight determinations were made with P. verrucosa, Pleurophomella sp., or Cladosporium sp. Media were amended with appropriate concentrations of the metal nitrates. Incubation was at 20~ The pH of all growth media with all metal amendments was within the range 5.0 to 5.8. Linear extension was determined by placing inverted agar plugs of the fungi in the center of Petri plates containing 15 ml of potato dextrose agar (Difco). Several defined media were tested, but none was acceptable for all fungi. Mean colony diameters were calculated from four replicate plates after 7 days growth. Treatments consisted of plates amended with both average and maximum concentrations of all metals. One treatment involved all metals combined at both concentrations. Control plates did not receive nitrate salt amendment. Dry weight values were obtained following inoculation of 250-ml Erlenmeyer flasks containing 50 ml of potato dextrose broth. Treatments consisted of medium amended with average concentrations only. Control media were amended with potassium nitrate in appropriate amounts to provide the same nitrate concentration as the various test-metal flasks. Four replicat e flasks were shaken at 175 rpm for 7 days. The mycelium was harvested, dried at 80~ and weighed.

Results L i n e a r e x t e n s i o n in plate culture s h o w e d that A. pullulans g r o w t h was significantly r e d u c e d by a v e r a g e and m a x i m u m c o n c e n t r a t i o n s o f Fe, AI, M n , and Ni and by m a x i m u m c o n c e n t r a t i o n s o f Na and C d (Fig. 1A). L i n e a r g r o w t h o f

C h a e t o m i u m sp. w a s r e d u c e d by all m e t a l s e x c e p t Ni and N a at the a v e r a g e c o n c e n t r a t i o n and by all c a t i o n s at the m a x i m u m c o n c e n t r a t i o n (Fig. 1B). L i n e a r ext e n s i o n o f Cladosporium

sp. w a s r e d u c e d by a v e r a g e and m a x i m u m c o n c e n -

trations o f Fe, AI, Z n , and Ni and by m a x i m u m c o n c e n t r a t i o n s o f Na, M n , and Pb (Fig. IC). L i n e a r g r o w t h o f E p i c o c c u m sp. w a s s u p p r e s s e d by a v e r a g e and m a x i m u m c o n c e n t r a t i o n s o f Fe, Al, and M n and m a x i m u m a d d i t i o n s o f C u , Ni, and Zn (Fig. ID). P. verrucosa linear d e v e l o p m e n t w a s very t o l e r a n t o f trace metal a m e n d m e n t and w a s significantly r e d u c e d o n l y by a v e r a g e and m a x i m u m c o n c e n t r a t i o n s o f Fe and AI (Fig. 1E). I n c o r p o r a t i o n o f all m e t a l s t o g e t h e r in potato d e x t r o s e agar plates in e i t h e r a v e r a g e o r m a x i m u m c o n c e n t r a t i o n corn-

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pletely inhibited the growth of all saprophytic fungi. The addition of certain metal nitrates significantly stimulated the linear extension of all saprophytic fungi except Chaetomium sp. and P. verrucosa. Maximum additions of Pb (NOz)= significantly stimulated the linear growth of A. pullulans and Epicoccum sp. The growth of Epicoccum sp. in particular was increased by the incorporation of several metal nitrates. 9.0

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Fig. 1. Linear extension of saprophytic fungi isolated from the leaf surfaces of urban Platanus acerifolia and grown on potato dextrose agar amended with various trace metals in average and maximum concentrations equivalent to urban-tree leaf-contamination. Data represent four replicate plates grown at 20~ for 7 days. Means - 95% confidence intervals. A. AureobasMium pullulans. B. Chaetomium sp. C. Cladosporiurn sp. D. Epicoccum sp. E. Phialophora verrucosa.

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Linear extension of G. platani was reduced by average and maximum concentrations of Fe, AI, Ni, and Zn and by maximum concentrations of Mn, Na, and Pb (Fig. 2A). Mycelial extension of Pestalotiopsis sp. was lessened by average and maximum levels of Fe, Al, and Pb and maximum levels of Cd and Ni (Fig. 2B). Linear extension ofPleurophomella sp. was reduced by average levels of Fe and Al and by eight metals (Fe, A1, Cd, Zn, Pb, Ni, Cu, and Mn) at the maximum concentration (Fig. 2C). Dry weight data obtained from the shake culture experiment show significant growth suppression of A. pullulans only by Fe (Fig. 3A); Chaetomium sp. by all metals except Na, Cu, and Cr (Fig. 3B); and Epicoccum sp. by Fe, A1, and Zn (Fig. 3C). Dry weight data for parasitic species show growth suppression of G. platani by Zn, Al, and Ni (Fig. 4A) and ofPestalotiopsis sp. by Zn, Al, Fe, Cd, and 9.0

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Fig. 2. Linear extension of parasitic fungi isolated from the leaf surfaces of urban Platanus acerifolia and grown on potato dextrose agar amended with various trace metals in average and maximum concentrations equivalent to urban-tree leaf-contamination. Data represent four replicate plates grown at 20~ for 7 days. Means • 95% confidence intervals. A. Gnomoniaplatani. B. Pestalotiopsis sp. C. Pleurophomella sp.

Urban-Tree Phylloplane-Fungi

235

Pb (Fig. 4B). Significant stimulation by metal nitrates was not observed in the dry weight experiment.

Discussion The evidence presented supports the suggestion that there is no correlation between saprophytic or parasitic activity and sensitivity to trace metals in vitro. Both linear e x t e n s i o n and dry weight data indicate that the saprophytic Chaetomium sp. is very sensitive to numerous metals. Aureobasidium pulhdans, Epicoccum sp. and especially P. verruosa, on the other hand, appear much more tolerant. Of the parasites, G. platani appears more tolerant than Pestalotiopsis sp, and Pleurophomella sp. The stimulation o f growth observed in the linear extension experiment was probably not a result o f cationic but rather o f the anionic fraction o f the metal nitrate amendment. The nitrate salts provided increased nitrogen substrate rela-

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Urban-Tree Phylloplane-Fungi

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tive to the non-nitrate controls. When comparable nitrate concentrations were supplied in the liquid culture experiment, none of the metal nitrate amendments was stimulatory. Another explanation of stimulation might involve the detoxification hypothesis advanced by Englander and Corden [4] and employed in their explanation of the ability of high concentration of Cu and Fe to stimulate the growth of Endothia parasitica (Murr.) And. Metals exhibiting the broadest spectrum growth suppression were Fe, AI, Ni, Zn, Mn, and Pb. The general fungitoxicity of metal cations is presumably related to the strength of their cell surface covalent binding power [24] or electronegativity [15]. Certain of the metals shown to have broad-spectrum toxicity in this experiment (Ni, Zn, and Pb) are members of standard lists of heavy metals exhibiting toxicity to a broad range of fungal species [8, 23, 24]. The other metals shown to be significant by these data (AI, Fe, and Mn) are presumably toxic because of their extremely high concentration. The numerous and varied particulate sources in urban atmospheres [2, 5] release quantities of particules high in AI, Fe and Mn [2, 6, 11, 14] and accounts for the high values we have recorded on leaves [20] and hence have employed in the in vitro tests. Unfortunately it is difficult to extrapolate from in vitro observations to the natural environment. The chemistry of the trace-element compounds on leaf surfaces in the field is unknown. Nitrate salts were employed to provide a common anion and completely soluble compounds. In nature the trace metals probably occur as less soluble oxides, halides, sulfates, or phosphates. The dosages employed were arbitrary and may be relatively high, as late season concentrations based on dry leaf weights were employed. Other concentrations would have been no less arbitrary, however, as the use of any natural product medium will cause alteration of available metal concentrations due to binding by media components [3]. Accurate determination of thresholds of fungal trace-metal influence will require the use of synthetic-defined media and dose-response curves prepared over a range of concentrations bracketing those known to occur in nature. Since the metals were reacted with the fungi individually, it is possible that important antagonistic, additive, or synergistic interactions were overlooked. Because the phylloplane has a complex microflora [9], with much interaction between pathogenic and nonpathogenic microbes [5, 10], it is possible that trace-element impact on organisms that influence the fungi examined in this study may be more significant in nature than direct metal impact on these test organisms. In spite of these limitations, we conclude that our data support the importance of determining more specifically the relationships between trace metals deposited on plant surfaces and microorganisms inhabiting these surfaces. The hypothesis that trace metals in urban, industrial, and roadside environments may alter species composition of foliar microbial ecosystems is worthy of investigation.

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Acknowledgements This research was supported by a grant from the Consortium for Environmental Forestry Studies through the Pinchot Institute for Environmental Forestry Research, U.S. Department of Agriculture, Forest Service. Appreciation is extended to Susan M. Bicknell and Brian J. Staskawicz, Yale University for technical assistance and to Professor Joseph L. Peterson, Department of Plant Biology, Rutgers University, New Brunswick, New Jersey for assistance in fungal identification.

References 1.

Babich, H. W. and Stotzky, G. 1974. Air pollution and microbial ecology, Crit. Rev. Environ. Control 4: 353.

2.

Buchnea, D. and Buchnea, A. 1974. Air pollution by aluminium compounds resulting from corrosion of air conditioners, Environ. Sci. Technol. 8: 752.

3.

Dickinson, C. H. 1971. Cultural studies of leaf saprophytes. In: Ecology of Leaf Surface Microorganisms. T. F. Preece and C. H. Dickinson, (eds) pp. 129-137. Academic Press, New York.

4.

Englander, C. M. and Cordon, M.E. 1971. Stimulation of mycelial growth of Endothia parasitica by heavy metals, Appl. Microbial. 22" 1012.

5.

Fokkema, N. J. and Lorbeer, J. W. 1974. Interactions between Alternania porri and the saprophytic mycoflora of onion leaves, Phytopathology 64:1128.

6.

Gladney, E. S., Zoller, W. H., Jones, A. G. and Gordon, G. E. 1974. Composition and size distributions of atmospheric particulate matter in Boston area, Environ. Sci. Technol. 8:551.

7.

Heagle, A. S. 1973. Interactions between air pollutants and plant parasites, Aqn. Rev. Phytopathol. 11: 365. Horsfall, J. G. 1956. Principles of Fungicidal Action. Chronica Botanica Co., Waltham, Mass., 279 pp. Last, F.T. and Deighton, F. C. 1965. The non-parasitic microflora on the surfaces of living leaves, Trans. Br. Myco[. Soc. 48: 83.

10.

Last, F. T. and Warren, R. C. 1972. Non-parasitic microbes colonizing green leaves: Their form and functions, Endeavor 31: 143.

11.

Lee, R. E. Jr., Crist, H. L., Riley, A. E. and MacLeod, K. E. 1975. Concentration and size of trace metal emissions from a power plant, a steel plant and a cotton gin, Environ. Sci. Technol. 9: 643.

12.

Manning, W. J. 1975. Interactions between air pollutants and fungal, bacterial and viral plant pathogens, Environ. Pollut. 9: 87.

13.

Ramamoorthy, S. and Kushner, D. J. 1975. Binding of mercuric and other heavy metal ions by microbial growth media, Microbiol. Ecol. 2: 162.

14.

Ranweiler, L. E. and Mayers, J. L. 1974. Atomic absorption procedure for analysis of metals in atmospheric particulate matter, Environ. Sci. Technol. 8: 152.

15.

Ross, I. S. 1975. Some effects of heavy metals on fungal ceils, Trans. Br. Mycol. Soc. 64: 175.

Urban-Tree Phylloplane-Fungi

239

16.

Saunders, P. J. W. 1971. Modification of the leaf surface and its environment by pollution. In: Ecology of Leaf Surface Microorganisms. T. F. Preece and C. H. Dickinson (eds.) pp. 81-89. Academic Press, New York.

17.

Saunders, P. J. W. 1973. Effects of atmospheric pollution on leaf surface microflora, Pestic. Sci. 4: 589.

18.

Smith, W. H. 1971. Lead contamination of roadside white pine, For. Sci. 17: 195.

19.

Smith, W. H. 1972. Lead and mercury burden of urban woody plants, Science 176: 1237.

20.

Smith, W. H. 1973. Metal contamination of urban woody plants, Environ. Sci. Technol. 7: 631.

21.

Smith, W. H. 1976a. Air pollution-effects on the structure and function of plant-surface microbial ecosystems. In: Microbiology of Aerial Plant Surfaces. T. F. Prcecc and C. H. Dickinson (eds.) pp. 75-105. Academic Press, New York.

22.

Smith, W. H. 1976b. Lead contamination of the roadside ecosystem, J. Air Pollut. Contr. Assoc. 26:753-766.

23.

Somers, E. 1959. Fungitoxicity of metal ions, Nature 184" 475.

24.

Somers, E. 1961. The fungitoxicity of metal ions, Ann. Appl. Biol. 49" 246.

25.

Vandergrift, A. E., Shannon, L. J., Sallee, E. E., Gorman, P. G. and Park, W. R. 1971. Particulate air pollution in the United States, J. Air Pollut. Control Assoc. 21: 321.