Elevated Atmospheric Carbon Dioxide Concentrations Amplify Alternaria alternata Sporulation and Total Antigen Production

Julie Wolf¹, Nichole R. O’Neill², Christine A. Rogers³, Michael L. Muilenberg³, Lewis H. Ziska²

¹Department of Environmental Science and Technology, University of Maryland, College Park, Maryland, USA; ²U.S. Department of Agriculture–Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, Maryland, USA; ³Department of Public Health, University of Massachusetts, Amherst, Massachusetts, USA

---

Environ Health Perspect 118:1223-1228 (2010). http://dx.doi.org/10.1289/ehp.0901867 [online 13 May 2010]

---

Abstract

Background: Although the effect of elevated carbon dioxide (CO₂) concentration on pollen production has been established in some plant species, impacts on fungal sporulation and antigen production have not been elucidated.

Objective: Our purpose was to examine the effects of rising atmospheric CO₂ concentrations on the quantity and quality of fungal spores produced on timothy (Phleum pratense) leaves.

Methods: Timothy plants were grown at four CO₂ concentrations (300, 400, 500, and 600 µmol/mol). 
Leaves were used as growth substrate for Alternaria alternata and Cladosporium phlei. The spore abundance produced by both fungi, as well as the size (microscopy) and antigenic protein content (ELISA) of A. alternata, were quantified.

Results: Leaf carbon-to-nitrogen ratio was greater at 500 and 600 µmol/mol, and leaf biomass was greater at 600 µmol/mol than at the lower CO₂ concentrations. Leaf carbon-to-nitrogen ratio was positively correlated with A. alternata spore production per gram of leaf but negatively correlated with antigenic protein content per spore. At 500 and 600 µmol/mol CO₂ concentrations, A. alternata produced nearly three times the number of spores and more than twice the total antigenic protein per plant than at lower concentrations. C. phlei spore production was positively correlated with leaf carbon-to-nitrogen ratio, but overall spore production was much lower than in A. alternata, and total per-plant production did not vary among CO₂ concentrations.

Conclusions: Elevated CO₂ concentrations often increase plant leaf biomass and carbon-to-nitrogen ratio. Here we demonstrate for the first time that these leaf changes are associated with increased spore production by A. alternata, a ubiquitous allergenic fungus. This response may contribute to the increasing prevalence of allergies and asthma.

Key words: allergic rhinitis, Alternaria alternata, asthma, Cladosporium phlei, elevated atmospheric carbon dioxide (CO2), fungal antigenic protein, fungal sporulation, global climate change, plant carbon-to-nitrogen ratio (C:N), timothy grass (Phleum pratense)

Address correspondence to J. Wolf, University of Maryland Department of Environmental Science and Technology, 1109 HJ Patterson Hall, College Park, MD 20742 USA. Telephone: (301) 504-6184. Fax: (301) 504-5062. E-mail: thejuliewolf@gmail.com

The authors declare they have no actual or potential competing financial interests.

We thank A. Thomasson (University of Pennsylvania School of Medicine, Division of Biostatistics) and M. Kramer (USDA-ARS Biometrical Consulting Service) for help with statistical analyses and the R program, and M. Tomecek (USDA-ARS Crop Systems and Global Change Laboratory), who provided invaluable assistance throughout the experiment.

Received 22 December 2009; accepted 22 April 2010; online 13 May 2010.

Anthropogenic increases in global atmospheric carbon dioxide (CO₂) concentration have been shown to stimulate earlier and greater production of allergenic pollen (LaDeau and Clark 2006; Rogers et al. 2006; Ziska et al. 2003), as have warming temperatures (Wan et al. 2002; Yli-Panula et al. 2009). The effects of anthropogenic climate change on the production of airborne fungal spores are not as well documented, but the implications for allergic disease are no less important. As with pollen, exposure to fungal spores is associated with allergy and asthma symptoms [Institute of Medicine (IOM) 2000, 2004; Salo et al. 2006], although the specifics of the relationship are not completely understood (Portnoy et al. 2008). Among patients with asthma from six regions of the world, 11.9%, on average, were sensitized to Alternaria alternata, with the proportion as high as 28.2% in Portland, Oregon; in addition, sensitivity to A. alternata was more prevalent among patients with more severe asthma (Zureik et al. 2002). Allergenic fungal spores may be increasingly abundant in some areas of the globe as well. In Derby, United Kingdom, mean seasonal airborne spore concentrations of the genus Alternaria have increased over the years 1970–1998, as have the number of days with spore counts > 50 per cubic meter of air, and the start of the Alternaria spore season has advanced from the end to the beginning of June (Corden and Millington 2001).

Rising atmospheric CO₂ concentration is well documented [Intergovernmental Panel on Climate Change (IPCC) 2007]. Large increases in atmospheric CO₂ concentration (e.g., doubled or greater increases in concentration) have been shown to alter both plant biomass and chemistry in many plant species (Ainsworth and Long 2005; Taub et al. 2008). The responses of individual plant species to large increases in atmospheric CO₂ concentration are variable but often include greater total biomass production (de Graaf et al. 2006) and greater carbon-to-nitrogen ratios (C:N) of plant tissues (Taub and Wang 2008). Primary consumers, including allergenic fungi that grow on living or dead plant materials, are therefore likely to encounter changes in substrate quality or quantity as global atmospheric CO₂ concentration increases (Reich et al. 2006).

The responses of plant–fungal interactions to increased atmospheric CO₂ concentration may be more complex and difficult to elucidate than the observed changes in pollen production and allergenicity. Fungi play several different roles in plant biology, acting as plant pathogens, saprobes (decomposers of dead plant material), or plant symbionts, with neutral, beneficial, or negative impacts on living plant hosts. There may be a number of indirect effects on fungal growth and reproduction as a result of plant changes under increased atmospheric CO₂ concentration as well as feedback and interactions among plants, fungi, and abiotic (e.g., environmental, nonorganismal) factors (Rillig 2007; Stiling and Cornelissen 2007).

In a field experiment using open-top chambers to grow individual poplar trees in ambient or doubled atmospheric CO₂ concentration, Klironomos et al. (1997) found that air and leaf litter in chambers with doubled CO₂ concentration contained significantly more fungal spores than those in ambient chambers. An indirect effect of doubled atmospheric CO₂ concentration on sporulation, mediated by changes in plant chemistry, was suggested by their findings; however, a direct effect of atmospheric CO₂ concentration or of related changes such as soil moisture and relative humidity could not be ruled out.

To directly assess the quantitative and qualitative impacts of rising atmospheric CO₂ concentration on allergenic fungi, we grew the perennial C₃ monocot timothy grass (Phleum pratense), a common fodder used for hay, at four levels of atmospheric CO₂ concentration approximating preindustrial, current, and potential/projected future concentrations (300, 400, 500, and 600 µmol/mol, respectively). We used leaves from these plants to separately grow two fungal species. The first species, A. alternata, is a ubiquitous, facultatively plant-pathogenic or saprobic species known to produce allergenic airborne conidial spores (Sanchez and Bush 2001). The second species, Cladosporium phlei (syn. Heterosporium phlei), is a specific pathogen of timothy that has not been specifically shown to produce allergenic spores; other species within the genus Cladosporium are known to cause allergies in humans (Kurup 2003; Poll et al. 2009). We also examined the sporulation of A. alternata 
on clippings from field-grown grasses to see if responses were similar on other grass 
species. Our objective was to quantify changes in sporulation on plant leaves grown at varying atmospheric CO₂ concentrations and elucidate the mechanism that drives such changes.

Methods

General approach. We used two growth chambers to provide atmospheric CO₂ concentration at 300, 400, 500, or 600 µmol/mol to experimental timothy plants for 60 days of growth. These approximate atmospheric CO₂ concentrations at the beginning of the 19th century, current ambient levels, and those projected by the years 2025 and 2040, respectively (IPCC 2007). During repeated runs over time, each of the two growth chambers was used for each level of CO₂ concentration twice. During each run, each chamber contained 10 timothy plants. After 60 days of growth, leaf subsamples from each plant were used to grow A. alternata and C. phlei separately for 7 days inside loosely capped media bottles in an incubator. Media bottles were then volumetrically flooded with water, shaken, and subsampled for spore counts and, for A. alternata only, for antigenic protein extraction and quantification via ELISA using polyclonal antibodies.

Growth, harvest, and C:N quantification of timothy grass. The study was conducted using two controlled environment chambers (EGC Corp., Chagrin Falls, OH) at Beltsville, Maryland, with a given chamber set at one of four atmospheric CO₂ concentration set points (300, 400, 500, and 600 µmol/mol) for 24 hr/day. The atmospheric CO₂ concentration of the air within each chamber was controlled by adding either CO₂ or CO₂-free air to maintain the set concentration. Actual average 24-hr atmospheric CO₂ concentration values (± SD) were 315 ± 19, 395 ± 11, 491 ± 10, and 589 ± 12 µmol/mol. Injection of CO₂ and CO₂-free air was controlled by a TC-2 controller using input from an absolute infrared gas analyzer (WMA-2, PP Systems, Haverhill, MA). Chamber temperatures were altered diurnally (in steps) from an overnight low of 20°C to a maximum afternoon value of 30°C. Photosynthetically active radiation (PAR) was altered concurrently with temperature, with the highest PAR value (900–1,000 µmol/m² per sec) occurring during the afternoon (1,200–1,500). The duration of daily PAR was 14 hr, which was supplied by a mixture of high-pressure sodium and metal halide lamps.

Seeds of timothy grass (P. pratense, var. Climax) were obtained from Pawnee Buttes Seed (Greeley, CO). We sowed three seeds in each 4-inch pot used and thinned to one seedling per pot 6–8 days after emergence. Pots were watered to the drip point daily with a complete nutrient solution containing 14.5 mmol/L nitrogen. Ten pots were grown in each of the two chambers during each run. In eight runs over time, each level of CO₂ concentration was run a total of four times, twice in each chamber. Plants from the third run were stunted and grew poorly for unknown reasons; this run was omitted, resulting in three runs for CO₂ concentration levels of 300 and 500 and four runs for CO₂ concentration levels of 400 and 600.

Plants were harvested 60 days after sowing. All leaves were separated from stems above the collar and weighed. Ten leaves from each plant were used for determination of total area, mass, and nitrogen and carbon content, which was determined using a PerkinElmer 2400 Series II CHNS/O analyzer (PerkinElmer, Waltham, MA). Leaf subsamples of approximately 2 g fresh weight were removed from each plant for fungal growth, refrigerated, and processed within 3 hr of harvest.

All solutions, suspensions, and rinses were made using sterilized, deionized, distilled water (ddw) with Tween 80 (Fisher Scientific, Fairlawn, NJ) added at the rate of 5 drops/L (ddw+T). Leaves were surface-sterilized by immersion and agitation in 1 L of 0.3% sodium hypochlorite solution for 3 min, followed by three successive 1-min rinses. All solutions and rinses were changed after leaves from all plants in a chamber were immersed to avoid transfer of any leaf material among treatments. Surface-sterilized leaves were oven-dried at 68°C for 48 hr.

Inoculum preparation, leaf inoculation, and fungal growth. The fungal isolates grown were A. alternata (Fries) Keissler, isolate EGS 34-016, obtained from the collection of E. Simmons, and C. phlei (C.T. Greg.) G.A. de Vries, isolate CBS 306.5, obtained from the Fungal Biodiversity Centre of the Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands). Isolates were cultured on standard petri plates with half-strength V8 juice agar (100 mL V8 juice; Campbell’s Soup Company, Camden, NJ), 900 mL ddw, 2 g CaCO₃, 15 g agar powder, autoclaved for 20 min at 121°C).

Fungal inocula were prepared immediately before inoculating leaves and prepared the same way for both fungal species. Two Petri plates with conidia covering most of the agar surface were selected. Conidia were removed from these plates by pouring small aliquots of ddw+T onto the surface of the agar in each plate, gently scraping the agar surface with a ceramic scraper, pouring the water with removed conidia through four layers of sterilized cheesecloth (Fisher Scientific) into a sterilized 1-L beaker, and repeating three more times. The resulting spore suspensions were vortexed (Vortex Genie2, Daigger, Vernon Hills, IL) at maximum speed for 30 sec to separate clusters of spores and subtending hyphae. The total volume was brought up to 1 L with additional ddw+T. Inoculum strength was standardized visually: A 10-mL subsample was removed with a glass wide-mouth pipette, filtered with a 47-mm diameter, 0.45-µm pore size, gridded mixed-cellulose filter (Fisher Scientific) in a glass vacuum filtration apparatus, and examined at 5× magnification under a dissecting microscope (SMZ 1500; Nikon Precision Inc., Belmont, CA). If spores were more than one layer deep on the filter, ddw+T was added to the inoculums suspension in 200-mL increments until subsamples resulted in filters densely covered with a single layer of spores. Five hundred milliliters of the final inoculum suspension was placed into two sterilized 1-L beakers; each beaker was used to inoculate leaves from a single chamber.

Weighed leaf subsamples (0.5 ± 0.075 g dry weight) from each single plant were inoculated separately by immersion and agitation in the inoculum suspension for 1 min. Inoculated leaves were placed in a sterilized media bottle (250-mL size used for A. alternata, 500-mL size for C. phlei) (VWR, West Chester, PA) with caps placed loosely, then incubated for 1 week in an environmental controller (Percival, Perry, IA) set at 21°C with a 12-hr light/dark cycle and ambient CO₂ concentration.

Spore harvest and A. alternata antigen extraction. After 1 week, 200 mL sterilized ddw+T was added to each media bottle. Bottles were vortexed at maximum speed for 30 sec to dislodge spores from leaves and subtending hyphae. For A. alternata only, antigen was then extracted: media bottles were left at room temperature for 2 hr; bottles were then inverted several times by hand. With a sterile syringe, 20 mL water with suspended spores was removed from each bottle and passed through a syringe filter (0.22-µm pore size, 33-mm diameter, polyethersulfone; Millipore, Billerica, MA). The resulting spore-free extract was frozen inside a 50-mL plastic centrifuge tube (Corning Inc., Corning, NY) and lyophilized on a Freezone 4.5 freeze dry system (Labconco, Kansas City, MO). For both fungal species, media bottles were then inverted several times by hand; 10-mL subsamples were removed using glass wide-mouth pipettes, placed on filters, and examined as described above. If spore density on the filter was too sparse (or too crowded) for accurate counting, a larger (or smaller) subsample was obtained, a new filter was made, and spore density was rechecked. The final volume used for each subsample ranged from 1 to 100 mL and was recorded for extrapolation of the total number of spores produced per gram of dried leaf tissue.

Spore quantification. Filters were rewetted from below with several drops of sterilized ddw and placed on a compound light microscope stage (Axioplan2 Imaging Microscope; Carl Zeiss Microimaging Inc., Thornwood, NY). Eight 300 × 400 µm fields of view were digitally photographed (Axiocam digital camera and Axiovision imaging software; Carl Zeiss Microimaging, Inc.) at random positions over one half of each wetted filter. Spores within each of the eight fields were counted manually from the photographs; all whole, uncollapsed spores were counted regardless of size. For A. alternata only, the first 10 spores laying flat on the filter were also measured for length and width.

Antigen quantification. Lyophilized extracts were reconstituted in 3 mL ddw and assayed for A. alternata antigen using a competitive inhibition ELISA described by Salo et al. (2005). The assay kit consisted of a rabbit polyclonal antibody (lot ZA4-4; Greer Laboratories, Lenoir, NC) produced using whole mycelial extracts of A. alternata (lot XPM1-X10); the A. alternata mycelial extract was also used as the standard in the assay. This assay has been shown to detect multiple A. alternata antigens including the allergen Alt a 1 (Portnoy et al. 1993; Salo et al. 2006). Briefly, the standard A. alternata extract was bound to plastic microtiter plate wells, excess was washed away, and unbound sites were blocked with 1% bovine serum albumin solution. Dilution series of the sample extracts were pipetted into the wells and followed immediately by the rabbit anti-A. alternata antibody. After incubation, we aspirated solutions, washed the wells, and added a peroxidase-labeled goat anti-rabbit antibody solution (Sigma-Aldrich, St. Louis, MO). Finally, we added substrate and measured the color change at 405 nm using a microtiter plate reader (Molecular Devices, Sunnyvale, CA). We compared the resulting reaction rates against a dilution series of standard A. alternata antigen to determine antigen concentration. Results are reported as micrograms A. alternata antigen per spore or per plant.

Data analysis. All statistical analyses were conducted using R version 2.8.1 (R Development Core Team 2008). The R package “lme4” (Bates et al. 2008) was used to fit and evaluate generalized linear mixed models of the following effects: atmospheric CO₂ concentration level (fixed at 300, 400, 500, or 600 µmol/mol), run (random effect of seven runs over time), and chamber (random effect of the two growth chambers used). All fungal responses were also modeled with the covariate of leaf C:N of each plant. Factors were sequentially removed from models and model fit was evaluated using Akaike’s information criterion; for each response, the simplest statistical model with good fit was used. We used the R package “languageR” (Baayen 2008) to estimate p-values for the fixed effect of atmospheric CO₂ concentration level in each model using Markov Chain Monte Carlo resampling and to generate highest posterior density confidence intervals (CIs) for means at each atmospheric CO₂ concentration level. We considered p-values ≤ 0.05 to be statistically significant. The responses evaluated were the following: a) A. alternata spore counts (natural log transformed to meet statistical model assumptions that variance does not increase with group mean); b) mean length and width of A. alternata spores; c) quantity of antigenic protein produced (square-root transformed to linearize relationships with model factors and to meet statistical model variance assumptions); and d) C. phlei spore counts (natural log transformed, as above).

The random effect of chamber was neither significant nor needed to improve model fit for any responses and is not discussed further. The random effect of run was very important for modeling all plant and fungal responses; model-adjusted means, which account for variation due to run, are presented for responses at individual levels of CO₂ concentration. When the fixed effect of CO₂ concentration was determined to be statistically significant, the R package “multcomp” (Hothorn et al. 2008) was used to run pairwise Tukey’s comparisons of CO₂ concentration level means. The contribution of each factor toward explaining response variance was calculated as described by Kramer (2005).

Supplemental experiment with field-
collected plant material. Nine samples of plant material were collected during routine mowing from three different sites at each of three local golf courses. A mixture of perennial ryegrass (Lolium perenne L.) and annual poa (Poa annua), both cool season grasses with C₃ photosynthetic metabolism, is grown at all sites, but fertilizer application rates vary. The samples were collected on 8 January 2008 and refrigerated within 3 hr of mowing. Leaves were treated and used as growth substrate for the two fungal species as described above. Resulting C:N and spore counts were not statistically analyzed because of small sample size but are shown for visual comparison (Figure 1).

Results

Plant responses to atmospheric CO₂ concentration levels. Leaf C:N was significantly higher in plants grown at 500 and 600 µmol/mol 
atmospheric CO₂ concentrations than at the two lower concentrations (p = 0.017) (Table 1). Leaf dry weight per plant was significantly greater at 600 µmol/mol atmospheric CO₂ concentration than at the three lower concentrations (p < 0.001) (Table 1). Specific leaf area was not significantly affected by atmospheric CO₂ concentration (not shown). Plant responses were variable among individual plants and runs (Table 2). Among individual plants from all treatment levels, leaf C:N ranged from a minimum of 7.7 to a maximum of 16.2, which corresponds to total leaf nitrogen contents of 5.6% and 2.8%.

General fungal growth. Inoculations of all experimental plants were successful for both fungal species. No spores other than those of the inoculated fungal species, and no other contaminants, were observed in any measurements of fungal growth. All antigen extracts were above the lower limit of antigen detection (LLOD) of the inhibition assay; the LLOD was always < 0.050 µg/mL and was < 0.011 µg/mL for most samples. Fungal responses also varied across individual plants and runs (Table 2).

Fungal sporulation on leaves grown at four atmospheric CO₂ concentration levels. The log-transformed counts of A. alternata spores produced per gram leaf were positively correlated with leaf C:N (p < 0.001; adjusted R² = 0.25; Figure 1), with an order-of-magnitude increase across the range of leaf C:N of experimental plants. A. alternata sporulation on field-
collected grass leaves (supplemental experiment) shows a similar relationship (Figure 1). Mean length and width of A. alternata spores did not change significantly with leaf C:N (not shown). In contrast to the positive association with spore numbers, the quantity of A. alternata antigen per spore (square-root transformed) decreased as leaf C:N increased (p < 0.001; adjusted R² = 0.34; Figure 2). Despite the decreased antigen content per spore at higher C:N, the quantity of A. alternata spores as well as the total antigen produced on a per-plant basis were positively associated with atmospheric CO₂ concentration levels beyond the variation attributed to leaf C:N, with nearly three times the number of spores and twice the amount of antigen produced on plants grown at 500 and 600 µmol/mol atmospheric CO₂ concentrations (p < 0.001; Table 1) than at the lower two concentrations.

The log-transformed counts of C. phlei spores produced per gram leaf also increased with C:N (p < 0.001; adjusted R² = 0.22; Figure 3). Spores of C. phlei were produced in much lower numbers than A. alternata spores, although the slopes of the relationships with leaf C:N were not significantly different between the two fungi (T = 1.21; p > 0.20). In contrast with A. alternata spores, however, the mean number of C. phlei spores produced on a per-plant basis was not significantly different among plants grown at different CO₂ concentration levels (p = 0.78; Table 1).

Discussion

Climate change and urbanization are expected to increase the prevalence of asthma and allergies (Schmier and Ebi 2009). Concomitant rises in temperature, atmospheric CO₂ concentration, and pollen abundances over recent decades have been suggested as causes of increasingly prevalent and severe asthma and allergy symptoms observed over the same time period (Beggs and Bambrick 2005; Shea et al. 2008). Our findings indicate that, as with pollen production, the sporulation of allergenic fungi is likely to be amplified as atmospheric CO₂ concentration increases and therefore is also likely to contribute to increasing prevalence and severity of asthma and allergies.

We found significant positive relationships between leaf C:N and A. alternata and C. phlei spore production per gram leaf, although only A. alternata sporulation was associated with CO₂ concentration level after accounting for changes in leaf C:N. These relationships suggest that considerable increases in sporulation of both species will occur, and may already be occurring, if rising atmospheric CO₂ concentration increases plant C:N in the field. The response of C. phlei, however, may not be as strong as that of A. alternata. More study of this species is needed.

Although C:N was correlated with increased sporulation, a large proportion of the variability in spore production remains unexplained. Some of this variability in sporulation may result from inherent variation in growth among individual plants, which can be of a magnitude large enough to obscure responses to elevated atmospheric CO₂ concentration treatments (Poorter and Navas 2003). Other aspects of environmental variability, which deserve additional scrutiny, may also contribute to variation in spore numbers and quality. Temperature was kept constant during fungal growth in this experiment, but relative humidity was not controlled. Additional plant attributes such as leaf tissue concentrations of mineral elements (other than nitrogen), stomatal density, and other leaf surface properties, and the chemistry of leaf carbon compounds were not measured. All of these factors may contribute to the variation in sporulation, and all are also likely to be altered by anthropogenic climate change (Denman et al. 2007). For example, plant transpiration is likely to decrease under elevated atmospheric CO₂ concentration, which often leads to increased moisture in soils and overlying litter (Johnson et al. 2003); warming temperatures and altered precipitation patterns will further impact moisture in soil and plant litter layers. Global climate change factors encompass many direct and indirect effects on plants and fungi, as well as complex interactions and feedbacks. The net effects of global changes are difficult to capture with a single study. Long-term (multidecadal) observational studies are needed to distinguish the impact of multiple abiotic changes on allergenic spore production in toto.

Our results corroborate the findings of Klironomos et al. (1997), who found large increases (~ 100–150%) in Alternaria spore abundance in chambers with poplar trees growing in elevated atmospheric CO₂ concentration. Our results also suggest the potential ubiquity, across plant species, of increased spore production as a function of increasing atmospheric CO₂ concentration. Klironomos et al. (1997) also found a large increase in the abundance of spores from the genus Cladosporium at elevated atmospheric CO₂ concentration (~ 225–350% increases), which was not apparent in our study. A strong response to atmospheric CO₂ concentration may relate to a saprobic (growing on dead plant tissues) lifestyle. A. alternata is facultatively pathogenic or saprobic. Although C. phlei can grow on dead plant tissues, as it did in this experiment, it is described primarily as a pathogen of living timothy grass. Other species within Cladosporium, however, are saprobic (Zalar et al. 2007). Specializations that allow fungal species to grow on specific host plants, such as unique metabolic pathways, may cause these fungi to be limited more by other plant characteristics than by carbon content alone.

Although only a few studies have examined fungal sporulation on plants grown at elevated atmospheric CO₂ concentration, there has been extensive work examining sporulation on artificial growth media and on plants with different levels of nitrogen content. In some pathogenic fungal species, sporulation on living leaves is greatest at medium C:N (Walters and Bingham 2007), but other species sporulate maximally at high C:N. The C:N that promotes the most sporulation varies among fungal species and even strains (Elson et al. 1998; Gao et al. 2007). These varying trends among fungal taxa may reflect different reproductive strategies, specializations, and functional tradeoffs needed to thrive in different environmental and host conditions (Pariaud et al. 2009). There is experimental evidence that while the greatest numbers of spores may be produced at high C:N in some fungal taxa, spore protein content, germination success, and host-plant infectivity are greater when more nitrogen is available (Jackson and Schisler 1992; Yu et al. 1998). In our study, the decreasing content of antigenic protein in A. alternata spores grown at higher C:N suggests increasing nitrogen limitation of the fungus. It is not yet clear if this nitrogen limitation is associated with compromised germination, growth, or allergenicity of individual A. alternata spores.

Meta-analyses of plant responses to elevated atmospheric CO₂ concentration reveal clear trends despite variation among plant species and experimental conditions. The mean increase in C:N across many studies of grass species grown at doubled atmospheric CO₂ concentration was 24.4% (Taub and Wang 2008). The responses of plants to less-than-doubled increases in atmospheric CO₂ concentration have not been well studied, so expectations for C:N of plant tissues grown in real-world, gradually increasing atmospheric CO₂ concentration cannot be predicted with certainty. However, an observational study of herbarium leaf specimens found that leaf nitrogen content decreased by a mean of 17% across samples from the early, mid, and late 20th century for 11 C₃ plant species (Penuelas and Estiarte 1997) [recorded global levels of atmospheric CO₂ concentration increased from < 320 in 1958 to 388 µmol/mol in July 2009 (National Oceanic and Atmospheric Administration 2009)]. The authors attribute the decreased leaf nitrogen content to the combination of direct and indirect (warming) effects of rising atmospheric CO₂ concentration, and their findings are corroborated by other observational and experimental measurements (Denman et al. 2007; Gifford et al. 2000; Taub et al. 2008; Taub and Wang 2008). Thus, this evidence of increased plant C:N associated with rising atmospheric CO₂ concentration and climatic change is consistent with increased sporulation of allergenic fungi, as observed in the current study, and has implications for increasing the prevalence and severity of allergy and asthma symptoms (Beggs and Bambrick 2005; D’Amato et al. 2005).

References

1. Ainsworth EA, Long SP. 2005. What have we learned from 15 years of free-air CO₂ enrichment (FACE)? A meta- analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO₂. New Phytol 165:351–372.
2. Baayen RH. 2008. languageR: Data sets and functions with “Analyzing Linguistic Data: A practical introduction to statistics.” R package version 0.955. Available: http://CRAN.R-project.org/package=langua​geR [accessed 8 June 2009].
3. Bates D, Maechler M, Dai B. 2008. lme4: Linear mixed-effects models using S4 classes. R package version 0.999375-28. Available: http://lme4.r-forge.r-project.org/ [accessed 8 June 2009].
4. Beggs PJ, Bambrick HJ. 2005. Is the global rise of asthma an early impact of anthropogenic climate change? Environ Health Perspect 113:915–919.
5. Corden JM, Millington WM. 2001. The long-term trends and seasonal variation of the aeroallergen Alternaria in Derby, UK. Aerobiologia 17:127–136.
6. D’Amato D, Liccardi G, D’Amato M, Holgate S. 2005. Environmental risk factors and allergic bronchial asthma. Clin Exp Allergy 35:1113–1124.
7. de Graaf M-A, van Groenigen K-J, Six J, Hungate B, van Kessel C. 2006. Interactions between plant growth and soil nutrient cycling under elevated CO₂: a meta-analysis. Glob Chang Biol 12:2077–2091.
8. Denman KL, Brasseur G, Chidthaisong A, Ciais P, Cox PM, Dickinson RE, et al. 2007. Couplings between changes in the climate system and biogeochemistry. In: Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change (Solomon S, Qin D, Manning M, et al., eds). New York:Cambridge University Press. pp. 499–587.
9. Elson MK, Schisler DA, Jackson MA. 1998. Carbon-to-nitrogen ratio, carbon concentration, and amino acid composition of growth media influence conidiation of Helminthosporium solani. Mycologia 90:406–413.
10. Gao L, Sun MH, Liu XZ, Che YS. 2007. Effects of carbon concentration and carbon to nitrogen ratio on the growth and sporulation of several biocontrol fungi. Mycol Res 111:87–92.
11. Gifford RM, Barrett DJ, Lutze JL. 2000. The effects of elevated [CO₂] on the C:N and C:P mass ratios of plant tissues. Plant Soil 224:1–14.
12. Hothorn T, Bretz F, Westfall P. 2008. Simultaneous inference in general parametric models. Biom J 50:346–353.
13. IOM (Institute of Medicine). 2000. Clearing the Air: Asthma and Indoor Air Exposures. Washington, DC:IOM.
14. IOM (Institute of Medicine). 2004. Damp Indoor Spaces and Health. Washington, DC:IOM.
15. IPCC (Intergovernmental Panel on Climate Change). 2007. Climate Change 2007: Synthesis Report. Contribution of Working Groups I, II and III to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change (Core Writing Team, Pachauri RK Reisinger A, eds). Geneva:IPCC.
16. Jackson MA, Schisler DA. 1992. The composition and attributes of Colletotrichum truncatum spores are altered by the nutritional environment. Appl Environ Microbiol 58:2260–2265.
17. Johnson NC, Wolf J, Koch GW. 2003. Interactions among mycorrhizae, atmospheric CO₂ and soil N impact plant community composition. Ecol Lett 6:532–540.
18. Klironomos JN, Rillig MC, Allen MF, Zak DR, Pregitzer KS, Kubiske ME. 1997. Increased levels of airborne fungal spores in response to Populus tremuloides grown under elevated atmospheric CO₂. Can J Bot 75:1670–1673.
19. Kramer M. R-squared statistics for mixed models. In: 17th Annual Kansas State University Conference on Applied Statistics in Agriculture. Manhattan, KS:Kansas State University Department of Statistics. 2005, 148–169.
20. Kurup VP. 2003. Fungal allergens. Curr Allergy Asthma Rep 3:416–423.
21. LaDeau S, Clark J. 2006. Pollen production by Pinus taeda growing in elevated atmospheric CO₂. Funct Ecol 10:1365–1371.
22. National Oceanic and Atmospheric Administration. 2009. Trends in Atmospheric Carbon Dioxide: Recent Mauna Loa CO₂. Available: http://www.esrl.noaa.gov/gmd/ccgg/trends​/#mlo_full [accessed 1 August 2009].
23. Pariaud B, Robert C, Goyeau H, Lannou C. 2009. Aggressiveness components and adaptation to a host cultivar in wheat leaf rust. Phytopathology 99:869–878.
24. Penuelas J, Estiarte M. 1997. Trends in plant carbon concentration and plant demand for N throughout this century. Oecologia 109:69–73.
25. Poll V, Denk U, Shen HD, Panzani RC, Dissertori O, Lackner P, et al. 2009. The vacuolar serine protease, a cross-reactive allergen from Cladosporium herbarum. Mol Immunol 46:1360–1373.
26. Poorter H, Navas M-L. 2003. Plant growth and competition at elevated CO₂: on winners, losers, and functional groups. New Phytol 157:175–198.
27. Portnoy J, Pacheco F, Barnes C, Upadrashta B, Crenshaw R, Esch R. 1993. Selection of representative Alternaria strain groups on the basis of morphology, enzyme profile, and allergen content. J Allergy Clin Immunol 91:773–782.
28. Portnoy JM, Barnes CS, Kennedy K. 2008. Importance of mold allergy in asthma. Curr Allergy Asthma Rep 8:71–78.
29. R Development Core Team. 2008. R: A Language and Environment for Statistical Computing. Vienna, Austria:R Foundation for Statistical Computing.
30. Reich PB, Hungate BA, Luo YQ. 2006. Carbon-nitrogen interactions in terrestrial ecosystems in response to rising atmospheric carbon dioxide. Annu Rev Ecol Evol Syst 37:611–636.
31. Rillig MC. 2007. Climate change effects on fungi in agroecosystems. In: Agroecosystems in a Changing Climate (Newton PCD, Carran RA, Edwards GR, Niklaus PA, eds). Boca Raton, FL:CRC Press, 211–230.
32. Rogers C, Wayne P, Macklin E, Muilenberg M, Wagner C, Epstein P, et al. 2006. Interaction of the onset of spring and elevated atmospheric CO₂ on ragweed (Ambrosia artemisiifolia L.) pollen production. Environ Health Perspect 114:865–869.
33. Salo PM, Arbes J, Samuel J, Sever M, Jaramillo R, Cohn RD, et al. 2006. Exposure to Alternaria alternata in US homes is associated with asthma symptoms. J Allergy Clin Immunol 118:892–898.
34. Salo PM, Yin M, Arbes J, Samuel J, Cohn RD, Sever M, et al. 2005. Dustborne Alternaria alternata antigens in US homes: results from the National Survey of Lead and Allergens in Housing. J Allergy Clin Immunol 116:623–629.
35. Sanchez H, Bush RK. 2001. A review of Alternaria alternata sensitivity. Rev Iberoam Micol 18:56–59.
36. Schmier J, Ebi K. 2009. The impact of climate change and aeroallergens on children’s health. Allergy Asthma Proc 30:229–237.
37. Shea KM, Truckner RT, Weber RW, Peden DB. 2008. Climate change and allergic disease. J Allergy Clin Immunol 122:443–453.
38. Stiling P, Cornelissen T. 2007. How does elevated carbon dioxide (CO₂) affect plant-herbivore interactions? A field experiment and meta-analysis of CO₂-mediated changes on plant chemistry and herbivore performance. Glob Chang Biol 13:1823–1842.
39. Taub DR, Miller B, Allen H. 2008. Effects of elevated CO₂ on the protein concentration of food crops: a meta-analysis. Glob Chang Biol 14:565–575.
40. Taub DR, Wang X. 2008. Why are nitrogen concentrations in plant tissues lower under elevated CO₂? A critical examination of the hypotheses. J Integr Plant Biol 50:1365–1374.
41. Walters DR, Bingham IJ. 2007. Influence of nutrition on disease development caused by fungal pathogens: implications for plant disease control. Ann Appl Biol 151:307–324.
42. Wan S, Yuan T, Bowdish S, Wallace L, Russell SD, Luo Y. 2002. Response of an allergenic species, Ambrosia psilostachya (Asteraceae), to experimental warming and clipping: implications for public health. Am J Bot 89:1843–1846.
43. Yli-Panula E, Fekedulegn DB, Green BG, Ranta H. 2009. Analysis of airborne Betula pollen in Finland; a 31-year perspective. Int J Environ Res Public Health 6:1706–1723.
44. Yu X, Hallett SG, Sheppard J, Watson AK. 1998. Effects of carbon concentration and carbon-to-nitrogen ratio on growth, conidiation, spore germination and efficacy of the potential bioherbicide Colletotrichum coccodes. J Ind Microbiol Biotechnol 20:333–338.
45. Zalar P, de Hoog GS, Schroers HJ, Crous PW, Groenewald JZ, Gunde-Cimerman N. 2007. Phylogeny and ecology of the ubiquitous saprobe Cladosporium sphaerospermum, with descriptions of seven new species from hypersaline environments. Stud Mycol 58(1):157–183.
46. Ziska LH, Gebhard DE, Frenz DA, Faulkner S, Singer BD, Straka JG. 2003. Cities as harbingers of climate change: common ragweed, urbanization, and public health. J Allergy Clin Immunol 111:290–295.
47. Zureik M, Neukirch C, Leynaert B, Liard R, Bousquet L, Neukirch F. 2002. Sensitisation to airborne moulds and severity of asthma: cross sectional study from European Community respiratory health survey. BMJ 325:411–414.