Increased levels of airborne fungal spores in ...

Increased levels of airborne fungal spores in response to Populus tremuloides grown under elevated atmospheric COP

Can. J. Bot. Downloaded from www.nrcresearchpress.com by Renmin University of China on 06/05/13 For personal use only.

John N. Klironomos, Matthias C. Rillig, Michael F. Allen, Donald R. Zak, Kurt S. Pregitzer, and Mark E. Kubiske

Abstract: Soil fungi are important components of terrestrial ecosystems. They function as decomposers, pathogens, parasites, and mutualistic symbionts. Their main mode of dispersal is to liberate spores into the atmosphere. In this study we tested the hypothesis that a higher atmospheric CO, concentration will induce greater sporulation in common soil fungi, leading to higher concentrations of fungal propagules in the atmosphere. In our field experiment, the concentration of airborne fungal propagules, mostly spores, increased fourfold under twice-ambient CO, concentrations. Analysis of decomposing leaf litter (likely the main source of airborne fungal propagules) indicated that the fungi produced fivefold more spores under elevated C 0 2 . Our results provide evidence that elevations in atmospheric CO, concentration can directly affect microbial function, which may have important implications for litter decay, fungal dispersal, and human respiratory health. Key words: atmospheric CO?, fungal spores, global change, Populus tremuloides.

RCsurnC : Les champignons du sols sont d'importants constituants des Ccosystbmes terrestres. I1 jouent un r6le comme dCcomposeurs, pathogknes, parasites, et symbiontes mutualistes. Leur dispersion s'effectue principalement par la IibCration de spores dans I'atmosphbre. Dans cette Ctude, nous avons vCrifiC I'hypothkse qu'une teneur accrue de l'atmosphkre en CO, conduirait i une augmentation de la sporulation de champignons communs du sol. Les rCsultats obtenus au champ montrent que la teneur de l'atmosphbre en propagules fongiques, surtout des spores, a quadruplC lorsque la teneur en CO, de l'air ambiant a CtC doublCe. Une analyse des champignons dCcomposeurs de litibre (vraisemblablement la source principale de propagules fongiques aCroportCes) indique que ces champignons produisent cinq fois plus de spores en prCsence de CO, accrue. Ces rCsultats dkrnontrent que les ClCvations des teneurs atmosphCriques en CO? peuvent directement affecter les fonctions microbiennes, ce qui pourrait avoir d'importantes implications pour la dCcomposition des litikres, pour la dispersion des champignons ainsi que sur la santC respiratoire des humains. Mots elks : CO, atmosphCrique, spores fongiques, changement globa, Populus tremrc.loides. [Traduit par la rCdaction]

Introduction The increasing global atmospheric C 0 2 concentration (Houghton et al. 1990) and its influence on primary production, have led to a wide variety of investigations into plantenvironment responses, ranging from plant biochemistry to ecosystem function (Bazzaz 1990; Mooney et al. 1991; Bowes 1993; Vitousek 1994; Koch and Mooney 1996). However, studies focusing on the potential for an elevated Received January 28, 1997.

J.N. Klironornos.' Department of Botany, University of Guelph, Guelph, ON N1G 2W1, Canada. M.C. Rillig and M.F. Allen. Department of Biology, San Diego State University, San Diego, CA 92182, U.S.A. D.R. Zak. School of Natural Resources and Environment, University of Michigan, Ann Arbor, MI 48109, U.S.A. K.S. Pregitzer and M.E. Kubiske. School of Forestry and Wood Products, Michigan Technological University, Houghton, MI 4993 I, U.S.A.

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Author to whom all correspondence should be addressed. e-mail: [email protected]

Can. J. Bot. 75: 1670-1673 (1997)

C 0 2 atmosphere to affect microbial functioning have not been well addressed. Fungi are ubiquitous microorganisms and can function as decomposers, pathogens, parasites, and mutualistic symbionts (Kendrick 1992). Their main mode of dispersal is to liberate sexually or asexually produced spores into the atmosphere. These spores can travel great distances, so they are important vectors by which fungi colonize new substrates. These airborne propagules have also been linked with human respiratory allergies (Burge 1990; Lacey 1990). Atmosphericspore concentrations in the order of lo5 spores . m-3 air are common and are influenced by a wide array of environmental and biological factors &eluding rain, wind, humidity, temperature, substrate quality, and various interspecies and intraspecies interactions (Lacey 1990). Until now, fungal sporulation, under the context of a globally changing CO, environment, has not been investigated. Fungal growth is greatly influenced by the chemical composition of plant litter. Since C:N ratios of plant tissue typically increase in response to CO, fertilization, we expect that the fungi that utilize this plant material will also be affected. Fungi in soil that decompose this material are

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Fig. 1. Fungal spore levels in the air (0) and on the Pop~il~is trern~iloidesleaf litter Values are means f SE (11 = 8 per group).


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Materials and methods Our study was located at the University of Michigan Biological Station, near Pellston, Michigan (45"34'N, 84"401W). Eight opentop chambers (Curtis and Teeri 1992) were used for each of the four treatments of a 2 x 2 factorial experiment (total 32 chambers). The two factors were atmospheric CO, (350 and 700 ppm) and soil N availability (high and low). Two Populus trernuloides saplings were grown in open-bottom root boxes (Zak et al. 1993). Two soil fertility levels were established by filling the boxes with either 100% locally excavated Kalkaska series topsoil (Typic Haplorthod, high-N treatment) or a homogenized mixture of 20% topsoil, 80% native Rubicon sand (Entic Haplorthod, low treatment). Net N mineralization was significantly higher in the high-N treatment (348 mg N . g-' . d-I) than in the low-N treatment (45 mg N . g-' . d-I) (Pregitzer et al. 1995). These rates of N mineralization are typical of the range that occurs in the upper Great Lakes forest ecosystems (Zak and Pregitzer 1990). After the experiment ran for 14 months, we measured aerial- and litter-fungal propagule levels in the chambers. This was done in the month of July, the peak time for airborne spores. Ten-minute air samples were taken using a Samplair-MK1 particle sampler (supplied by Allergenco, 403-7834 Broadway, San Antonio, Tex.). The sampler drew 9 L of a i r . min-I, and fungal spores were counted and identified at 4 0 0 ~or 1 0 0 0 ~magnification (Li and Kendrick 1995a, 1995b). A 300-mg sample of P. tremuloides leaf litter was collected from each chamber and subjected to washing (Harley and Waid 1955), and the spores were counted and identified.

response to doubling of CO, concentration

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largely N limited (Coleman and Crossley 1996), so any changes in the nutrient quality and quantity of the substrate may change fungal behaviour, and alter carbon allocation from vegetative growth to reproduction. increase in airborne fungal spores will have great implications for ecosystem functioning,and human health. The objective of this was to test the that C02 will increase sporulation by common litter fungi, leading to higher airborne concentrations of spores in the atmosphere.

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Two 2 x 2 multivariate analyses of variance (MANOVA) with the Wilk's criterion were performed on the dependent variables. The independent variables were N availability and C 0 2 concentration. and the dependent variables were the different fungal taxa. All variables, except for (i) Aspergill~ls-Pet~icillium,and (ii) unidentified spores. were logarithm transformed to satisfy assumptions of normality. The variables were also analyzed further using univariate factorial ANOVA to help determine which variables contribute to significant differences observed in the multivariate analyses. The Tukey post-hoc test was subsequently used to test differences among means.

Results The combined fungal taxa were significantly affected by both C 0 2 (MANOVA, p < 0.0001) and nitrogen availability (MANOVA, p < 0.001) and by their interaction ( p < 0.001). In the chambers, total airborne spore concentrations were highest ( p < 0.001) under elevated C 0 2 conditions (Fig. l), but the highest levels were found under the combined treatments of elevated C 0 2 and low soil nutrients ( p < 0.0001). The source of these spores was most likely from decomposing leaf litter on the ground, as the fungi infecting those tissues showed similar responses to C 0 2 and N (Fig. 1). The majority of fungal types were stimulated to sporulate under elevated C 0 2 conditions, but it was species of Aspergillus ( p < 0.0001), Penicillium ( p < 0.0001), and Cladosporiunt.r(p - < 0.000 1) that showed greatest stimulation (Figs. 2, 3). In air (Fig. 2) and litter (Fig. 3) elevated C 0 2 concentrations increased spore numbers by 222-499%. These were collectively the most common spores of fungal genera in the air in all treatments in the air (44-73 %) and from the litter (48 -69%). Nonetheless, some less frequently found spores (for example, species of Fusarium ( p < 0.0001) and Alternaria ( p < 0.0001)) also increased from 30 to 195%. Interestingly, spore production by some fungal genera did decrease, suggesting that the @ 1997 NRC Canada

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Fig. 2. Percent change in aerial spore concentration of different fungal genera in response to doubling of C02 concentration. The patterned bars denote under low soil nutrients, and the solid bars under high soil nutrients. Bars with an asterisk denote significant differences ( p < 0.05) compared with ambient CO, controls. Data were analyzed using a two-way factorial ANOVA followed by Tukey post-hoc tests. hyphal fragments


hyphal fragments

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unidentified spores

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unidentified spores Pithomyces

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Fig. 3. Percent change in spore levels on decaying litter of fungal genera in response to doubling of CO, concentration. The patterned bars denote under low soil nutrients, and the solid bars under high soil nutrients. Bars with an asterisk denote significant differences ( p < 0.05) compared with ambient CO? controls. Data were analyzed using a two-way factorial ANOVA followed by Tukey post-hoc tests.

* Epicoccum

Curvularia

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Cladosporium

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unidentified Basidiospores

unidentified Basidiospores

unidentified Ascospores

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AspergillusPenicillium

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Agrocybe Alternaria

Alternaria

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various fungi responded differently to atmospheric C 0 2 and soil N.

Discussion This study clearly shows that common litter fungi can be greatly stimulated to sporulate under elevated concentrations of atmospheric C 0 2 . These results point to interactions between changes in atmospheric C 0 2 and fungal dynamics which have not been previously considered, and which can have great implications in terrestrial biogeochemistry, plant pathology, and human respiratory health. Although it is dangerous to extrapolate from any one study, these results form part of a general pattern. T h e rising atmospheric C 0 2 concentration is known to alter the chemistry of plant tissues (Mooney et al. 1991), a response that is modified by soil N availability (Pregitzer et al. 1995). Increases in atmospheric C 0 2 lowers the nutrient quality of plant tissues in most plant species. The general trend is higher levels of C , particularly in nonstructural forms, and lower levels of N in plant tissues, resulting in tissues that have higher C:N ratios (Mooney et al. 1991). Fungi in soil which decompose this material are largely N limited (Coleman and Crossley 1996), so any changes in the nutrient quality and quantity of the substrate will modify fungal behaviour, and alter carbon allocation from vegetative growth to reproduction. An alternative mechanism is that the fungal responses observed here are a result of direct C 0 2 effects on the fungi rather than indirect effects via changes in litter quality. ~ i g h atmospheric C 0 2 concentrations under laboratory conditions are known to directly increase spore production in some fungal taxa (Barnett and Lilly 1955; Cotty 1987). Elevated C 0 2 concentrations in laboratory Petri dishes cause this gas to accumulate and O2 levels to decrease, altering fungal metabolism, and resulting in modified growth patterns.

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0

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Sporulation and other forms of fungal reproduction are either stimulated or inhibited under these conditions, depending on the requirements of the fungal species. However, even though C 0 2 levels in the atmosphere are expected to rise, they will not get so high as to affect O2 levels, so it is unlikely that the rising atmospheric C 0 2 concentration of the Earth could produce a similar response. Furthermore, the concentration of C 0 2 in the soil is typically ten times greater than that of the bulk atmosphere (La1 et al. 1995), and raising the concentration in the latter does not significantly affect soil C 0 2 levels. The fungi are so successful, partly because of their ability to produce and disperse large numbers of spores (Kendrick 1992). By sheer numbers, the fungi make sure that, whenever and wherever a new food substrate becomes available, they will be present to exploit it. It is difficult to predict the consequences of an increase in spore production on the functioning of terrestrial ecosystems. How will this affect litter decomposition rates? Chemical changes associated with C 0 2 enrichment are remarkably similar across plant species (Mooney et al. 1991), but decomposition studies report responses ranging from no change to reduced decomposition rates for plants grown under enriched C 0 2 environments (O'Neill 1994). Will increased sporulation lead to a rise in pathogen loads that plants will encounter? The present results indicate a high potential fd:an increase in the incidence of plant diseases as C 0 2 concentrations rise in our atmosphere. For example, spore levels of Alternaria spp. and Fusarium spp. were stimulated under high C 0 2 concentrations. Both of these genera have representative species that can act as decomposers, weak parasites, and pathogens (Domsch et al. 1980). If so, is an increase in pathogen load offset by increased disease resistance? Considering the wide variety of functions performed by the fungi and the lack of knowledge on environmental change and fungal responses, it is very important that this line of research be given more attention.

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Klironornos et al The implications of our findings are also striking for human health. Species of Aspergillus, in particular A. &migatus, appear to be etiological agents in various lung diseases (Burge 1990; Wallenbeck et al. 1991). Inhalation of low doses may induce sensitization and asthma in Aspergillussensitive patients, while inhalation of high doses may trigger symptoms of alveolitis and farmer's lung. T h e genus Cladosporium is believed to be the most common cause of mould allergy (Malling et al. 1985), and Claclasporium herbarium can cause allergenic asthma and rhinitis. Fusarium is not considered a strong allergen, but a common species of Alternaria, A. altertzata, and other related fungi a r e considered to b e the most important mould allergens in North America (Hoffman 1984: O'Hollaren et al. 1991). About 2 0 % of the population is easily sensitized by concentrations usual in the air (Lacey 1981). These people react immediately on exposure in the upper airways with hay-fever-like symptoms o r asthma o r may become sensitive to several of the allergens to which they are exposed. T h e remainder of the population requires exposure greater than l o 6 spores . m P 3 for sensitization (Lacey 1981). Although in this study spore levels did not reach such levels, they are high enough to indicate a need for further research, particularly since allergies and other human respiratory-related diseases are on the rise (O'Hollaren et al. 1991). Almost all children and adults with asthma are allergic to common aeroallergens, and there is increasing evidence for a direct causal relationship between exposure to aeroallergens in childhood and development of asthma (O'Hollaren et al. 1991; Jones 1991). In conclusion, our research shows that the rising atmospheric C 0 2 concentration could also have significant impacts on levels of airborne fungal propagules. This will affect decomposition, plant pathogen dynamics, and air quality as it affects humans, and shows the need to study fungal epidemiology in the context of a globally changing environment.

Acknowledgements W e thank A. Harizanos, S. Harney, and T. Zink for technical assistance, and H. Burge, H . Mooney, J . Seltzer, G. O'Neill, D. Larson, and B. Husband for comments on the manuscript. This work was supported by the U.S. Department of Energy and the Natural Sciences and Engineering Research Council of Canada.

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