2019
Elevated O3 alters soil bacterial and fungal communities and the dynamics of carbon and nitrogen
Chen, Z
Elsevier BV
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http://dx.doi.org/10.1016/j.scitotenv.2019.04.310
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dynamics of carbon and nitrogen
Zhan Chen, Mia R. Maltz, Jixin Cao, Hao Yu, He Shang, Emma Aronson
PII: S0048-9697(19)31845-5
DOI: https://doi.org/10.1016/j.scitotenv.2019.04.310
Reference: STOTEN 32018
To appear in: Science of the Total Environment Received date: 5 February 2019
Revised date: 17 April 2019 Accepted date: 20 April 2019
Please cite this article as: Z. Chen, M.R. Maltz, J. Cao, et al., Elevated O3 alters soil bacterial and fungal communities and the dynamics of carbon and nitrogen, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.04.310
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Elevated O
3alters soil bacterial and fungal communities and the dynamics of carbon and nitrogen
Zhan Chen
1, Mia R. Maltz
2, Jixin Cao
1, Hao Yu
3, He Shang
1, Emma Aronson
2,41. Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry; Key Laboratory of Forest Ecology and Environment, State Forestry Administration, Beijing 100091, China
2. Center for Conservation Biology, University of California, Riverside, Riverside, CA, 92521, USA
3. Department of Environmental and Biological Sciences, University of Eastern Finland, P.O. Box 1627，FI-70211 KUOPIO, Finland
4. Department of Microbiology and Plant Pathology, University of California, Riverside, Riverside, CA, 92521, USA
Corresponding author
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Abstract
Although many studies have reported the negative effects of elevated O3 on plant physiological characteristics, the influence of elevated O3 on below-ground processes and soil microbial functioning is less studied. In this study, we examined the effects of elevated O3 on soil properties, soil microbial biomass, as well as microbial community composition using high-throughput sequencing. Throughout one growing season, one-year old seedlings of two important endemic trees in subtropical China: Taxus chinensis (Pilger) Rehd. var. chinensis, and Machilus ichangensis Rehd. Et Wils, were exposed to charcoal-filtered air (CF as control), 100 nl l-1 (E100) or 150 nl l-1 (E150) O3-enriched air, in open top chambers (OTCs). We found that only higher O3 exposure (E150) significantly decreased soil microbial biomass carbon and nitrogen in M. ichangensis, and the contents of organic matter were significantly decreased by E150 in both tree species. Although both levels of O3 exposure decreased NO3-N in T. chinensis, only E150 increased NO3-N in M.
ichangensis, and there were no effects of O3 on NH4-N. Moreover, elevated O3 elicited changes in soil microbial community structure and decreased fungal diversity in both M. ichangensis and T.
chinensis. However, even though O3 exposure reduced bacterial diversity in M. ichangensis, no effect of O3 exposure on bacterial diversity was detected in soil grown with T. chinensis. Our results showed that elevated O3 altered the abundance of bacteria and fungi in general, and in particular reduced nitrifiers and increased the relative abundance of some fungal taxa capable of denitrification, which may stimulate N2O emissions. Overall, our findings indicate that elevated O3 not only impacts the soil microbial community structure, but may also exert an influence on the functioning of microbial communities.
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Keywords: Elevated O3, M. ichangensis, T. chinensis, Soil microbial communities
Introduction
In industrialized nations around the world, rising levels of tropospheric ozone (O3) are threatening public health, an environmental issue that has garnered increased attention. High levels of O3 remain a significant source of air pollution, exposing many people around the world to elevated O3. In many Chinese cities, such as Beijing, Xi’an, and Nanjing, O3 has replaced particulate matter as the major air pollutant, particularly on sunny, calm days. According to the Chinese ambient air quality standards, the level 1 standard (i.e., the lowest level of concern) refers to the 8-h mean O3 concentrations ranging from 50 to 80 nl l-1 (Yu et al., 2017). However, O3
concentrations routinely exceed the national standards. For instance, in Taihe County, Jiangxi Province in subtropical China in October, the maximum 8-h mean and peak O3 concentrations were 72.3 and 97 nl l-1, respectively (Chen et al., 2015). In addition, peak O3 concentrations in Beijing was recorded at ~120 nl l-1 in May 2016 (Beijing Municipal Environmental Monitoring Center). These recorded O3 levels are beyond the acceptable standards for safe concentrations of O3 in air, and could be toxic, causing damage to ecosystems and inhabitants of the region.
Ozone is a powerful oxidant, and it could lead to species losses and reductions in biodiversity within natural ecosystems (Feng et al., 2008; Hooper et al., 2012; Fuhrer et al., 2016). As a highly phytotoxic pollutant, ozone exposure could decrease plant growth, yield, and tissue quality by suppressing photosynthesis, inducing foliar damage and accelerating leaf senescence ( Dermody et al., 2006; Alonso et al., 2018; Moura et al., 2018; Oliver et al., 2018; Araminienė et al., 2019).
Although recent studies have documented the potential of O3 to inflict harm upon plants (Feng et
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al., 2014; Bagard et al., 2015; Chen et al., 2015; Zhang et al., 2015; Aguiar-Silva et al., 2016;
Braun et al., 2017), most of the research documenting the impact of O3 on terrestrial ecosystems has thus far been limited to effects observed aboveground (Felzer et al., 2007; Paoletti, 2007;
Braun et al., 2010; Ismail et al., 2014; Aguiar-Silva et al., 2016). However, the effects of O3 on below-ground processes are poorly understood (Andersen, 2003; Chen et al., 2009; Schrader et al., 2009), particularly for natural ecosystems vegetated with native plant species. Few studies have reported the effects of O3 on soil microbial communities associated with agricultural systems and food crops (Dohrmann and Tebbe, 2005; Chen et al., 2010; Li et al., 2012; Bao et al., 2015; Chen et al., 2015; Agathokleous et al., 2016; Wang et al., 2016). However, few studies have examined the impact of O3 on soil microbial communities and below-ground processes in forested ecosystems (Phillips et al., 2002; Kasurinen et al., 2005; Matyssek et al., 2016). Yet, belowground processes may be sensitive to tropospheric O3 elevation (Agathokleous et al., 2016). In fact, exposure to O3 may alter below-ground microbial processes even before symptoms of plant exposure become detectable (Andersen, 2003). Given that aboveground and below-ground processes are inextricably linked, the effects of O3 on below-ground processes and soil microbial communities may have proximate effects on aboveground processes and plant communities.
In Chinese forests, O3 phytotoxicity may affect the structure and functioning of soil microbial communities while concurrently damaging endemic tree species. The two important endemic trees in China used in the study were Taxus chinensis (Pilger) Rehd. var. chinensis, a threatened tree species listed in the ICUN Red List, and Machilus ichangensis Rehd. Et Wils, a nationally protected tree species. Both are representative tree species of subtropical forests in China and widely used in urban landscaping. However, the effects of ozone exposure on T. chinensis and M.
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ichangensis in China may eventually alter the performance of these tree species both in natural
ecosystems and urban areas. Ostensibly, microbes play essential roles in T. chinensis and M.
ichangensis forests as plant mutualists and by recycling organic debris. If microbes provide
benefits for these tree species in pristine ecosystems, and the effects of elevated O3 alter below-ground communities and influence microbial interactions, then the consequences of elevated O3 may indirectly alter the performance of either T. chinensis or M. ichangensis. Further, the degree of O3 toxicity could subsequently affect the structure and functioning of China’s subtropical forest ecosystems.
In order to investigate the effects of O3 on the below-ground processes of forest ecosystems, we exposed the seedlings of T. chinensis and M. ichangensis to elevated O3 and examined the effects on soil microbial communities. We aimed to determine whether elevated O3 decreased soil microbial diversity and affected soil microbial structure and/or shifts in soil bacterial and fungal communities would affect soil C and N cycling. Moreover, we predict that elevated O3
would enhance N2O emission through stimulating microbial taxa capable of denitrification.
Further, since we detected greater negative effects of O3 treatment on M. ichangensis than on T.
chinensis (Yu, et al. 2017), we hypothesized that the changes in the soil microbial community
associated with M. ichangensis under ozone treatment would be more pronounced than those associated with T. chinensis.
1. Materials and methods 1.1 Experimental site
We conducted our experiment at the Qianyanzhou ecological station (115°03′29.2″E, 26°44′
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29.1″N) of the Chinese Academy of Sciences, located in the subtropical monsoon climatic zone.
The study site is a typical red earth, hilly region in the mid-subtropical monsoon landscape zone of Taihe county, Jiangxi Province, China. The elevation ranges from 60 m to 150 m.a.s.l., and relative altitudinal differences are between 20 m and 50 m. During the experiment, the annual mean temperature at this site was 17.8 C; annual precipitation was 1471.2 mm; annual evaporation was 259.9 mm; and mean relative humidity was 83%. Soil of the region, weathered from red sandstone and mudstone, is classified as Typic Dystrudepts Udepts Inceptisols according to the USDA system (Wang et al. 2012). Prior to the study, the soil pH was 5.36, organic matter content was 11.4 g_kg-1, and total nitrogen was 650.7 mg_kg-1.
1.2 O
3exposure
Our experimental plants were exposed to O3 for about eight months; exposure began in March and ended in November 2015. We used octagonal open-top chambers (OTCs) with aluminum frames covered by transparent film to conduct our experiment. Our nine OTCs were 2 m in diameter and 2.2 m in height, with three replicates of each treatment, including untreated controls. Our chambers were 4 m apart from each other. Each chamber was ventilated twice per minute.
In each OTC, a rotatable transparent perforated pipe with many small holes (diameter of 10 mm at intervals of 10 cm) released either charcoal-filtered air (control, hereafter CF), or charcoal-filtered air with O3 added (O3 treatment). We fumigated O3 into our OTCs to reach levels above first criteria, reaching and surpassing O3 concentration ranges commonly recorded for the region. For low ozone treatment (hereafter E100) and high ozone treatment (hereafter E150), the
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OTCs were fumigated from 09:00 to 17:00 with a mean 8 h O3 concentration of 100 and 150 nl l , respectively. The average 8 h O3 concentrations observed during the study period for E100 and E150 were actually 96.3 and 147.06 nl l-1, respectively. Cumulative O3 exposure was expressed as AOT40 ( accumulated exposure over a concentration threshold of 40 nl l-1 O3 , based on hourly means) (Fuhrer et al., 1997), which was on 0.64, 75.25 and 145.04 ppm·h respectively in the CF, E100, and E150 treatments. The charcoal-filtered air or the O3 + charcoal-filtered air was driven by a fan inside the chamber. Ozone was generated from pure oxygen by high-voltage electric discharge (Jinan Sankang Envi-tech Co., LTD, Shandong, China). Ozone concentrations in the OTCs were regulated by mass flowmeters by controlling the oxygen volume, and monitored by an O3 analyzer (49i, Thermo Fisher Scientific Inc., Waltham, MA USA).
All OTCs were equally divided into two parts with a plastic plate, and one of the two experimental tree species was planted in each part. For sample analysis, the two charcoal-filtered controls are referred to herein as CFY for M. ichangensis and CFH for T. chinensis, according to the Chinese name of each plant species. Likewise, sample analyses on plants exposed at elevated O3 concentrations of 100 nl l-1 and 150 nl l-1 are herein referred to as E100Y and E150Y for M.
ichangensis, and E100H and E150H for T. chinensis, respectively.
1.3 Growth conditions
In early March 2015, we transported one-year-old container seedlings of M.
ichangensis and T. chinensis from the nursery garden of Qianyanzhou ecological
station to our study site under ambient air conditions. On March 13
th, 2015, 10
seedlings of similar height and basal diameter were selected of each species and
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directly transplanted into bottomless pots of 15 cm diameter and 20 cm height, placed on the natural soil of each of the nine OTCs. The bottomless pots were used to avoid root growth limitation, as well as the intermingling of roots between plants. The OTCs were built on the land of the study site with a small area of approximately 250 m
2prior to the onset of this experiment, where the soil adopted by the study was naturally developed, therefore, the soil was extremely homogeneous within each OTC and among all the OTCs. Seedlings were adequately watered with tap water to avoid drought stress during the experiment.
1.4 Soil collection and analyses
At the end of the experiment (Nov. 20th , 2015), four soil cores (10cm depth, 3.5cm diameter) were randomly collected within each 15 cm diameter pot, around each plant, which was thoroughly mixed into a composite representational soil sample, and used for both physicochemical and microbiological analyses. Soil organic matter was tested by potassium dichromate oxidation titration. Soil NO3-N and NH4-N content was measured as followed: 12 g of fresh soil was placed in a 250 ml flask, then 100 ml CaCl2 of 0.01 mol/L was added. The flask was oscillated for 1 hour on a shaker, then filtered with filter paper. The filtrate was analyzed by a flow analyzer (AA3, Seal, Germany). Soil microbial biomass carbon (MBC) and nitrogen were analyzed using standard fumigation-extraction methods, as per (Vance et al., 1987). Three of each of six replicates 10 g was extracted with 40 ml of 0.5M K2SO4, agitated at 150 rev min-1 and 20 ℃ for 30 min and filtered through 5.5 cm dia Whatman 41 filter papers. The other three replicates were fumigated for 18 h with CHCl3 in bell jars at room temperature. After fumigation, each
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sample was extracted as described for the unfumigated replicates. The extraction was measured by a total organic carbon analyzer (vario TOC, Elementar, Germany). And for pH measuring, 5 g of soil (dry weight) passing through a 2 mm sieve hole was weighed into a 50 mL high-type beaker, and 25 mL of distilled water was added. The mixture was vigorously stirred with a glass rod for 1.5 min, and then measured with a pH meter after 30 min. Next, we extracted DNA from these soil aliquots and targeted hypervariable portions of both the V3 and V4 region of the bacterial/archaeal 16S rRNA gene and the fungal internal transcribed spacer (fungal ITS1) for Polymerase chain reactions (PCR) amplification. We purified and quantified our PCR products, prior to pooling in equimolar concentrations for Illumina MiSeq PE300 sequencing (Illumina, Inc., CA, USA; S1 Molecular methods).
1.5 Statistical analysis
Treatment means were statistically compared using the statistical package SPSS (SPSS Inc., Chicago, IL, USA). Pairwise post-hoc testing of the one-way ANOVA, Tukey's HSD was used to determine statistically significant differences in soil microbial biomass, soil properties, microbial diversity and taxa abundance among treatments. A two-way ANOVA was applied to analyze the effects of tree species and O3 exposure treatment on microbial communities. Differences between the treatments were compared using the average of the relative abundance of the OTUs (Operational Taxonomic Units) of each treatment. For the OTU-based analysis, the diversity within each individual sample was estimated using OUT richness and the Shannon diversity index, and pairwise post-hoc testing of the one-way ANOVA, Tukey's HSD was used to compare the relative levels of bacterial OTU diversity across all soil samples (Zhao et al. 2014). To compare
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soil bacterial/archaeal community structures across all samples based on the OTU composition and examine the relationship between relative abundances of abundant phyla/classes and environmental variables, a Bray-Curtis distance matrix was constructed. This was used for unweighted Principal Coordinates Analysis (PCoA), Redundancy Analysis (RDA), and PERMANOVA analysis of microbial community data using Bray–Curtis dissimilarity and the adonis function in the vegan package of R (Anderson et al. 2008; Oksanen et al. 2012). To explore the specific difference of microbial composition among treatments, the relative abundance of the bacterial and fungal phylum and class were analyzed using one-way ANOVA, Tukey's HSD.
Furthermore, the relative abundance of the nitrifiers and N2O producer were analyzed using student’s t test to determine the potential effects of elevated O3 on nitrification and N2O emission.
AOT40 and soil properties were used to identify the abiotic factors that are most frequently related to fungal community composition by using RDA and PERMANOVA analysis.
2. Results
2.1 Soil physicochemical properties and soil microbial biomass
Soil microbial biomass and most soil physicochemical parameters were similar
in control (charcoal-filtered) treatments for both tree species. E150 treatment
significantly reduced MBC and MBN, and increased the contents of soil DN and
NO
3-N in M. ichangensis. While there were no effects of elevated O
3on soil MBC,
MBN and DN, but the content of NO
3-N was significantly decreased in T. chinensis
by elevated O
3. Elevated O
3decreased pH in both species. Total soil organic matter
content decreased only in E150 treatments compared with CF respectively in both
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species. (Table1). There were no effects of elevated O
3on DOC (data not shown).
2.2 Soil microbial diversity
Bacterial and fungal communities in soils associated with T. chinensis were more diverse than those associated with M. ichangensis. Elevated O3 significantly reduced microbial diversity in soils associated of M. ichangensis relative to the controls. Yet, there was no significant effect of elevated O3 on soil bacterial diversity associated with T. chinensis. We detected lower bacterial taxa richness in bacterial communities associated with M. ichangensis; the E100 and E150 levels of O3 reduced diversity metrics, including observed OTU richness by 20.4% (p=0.028) and 38.0%
(p=0.001) and the Shannon–Wiener Index by 10.8% (p＞0.05) and 21.3% (p=0.016), respectively,
compared to untreated controls. Elevated O3 significantly decreased fungal observed OTU richness by 7.11% and 8.70% (p=0.041) respectively in soils associated with M. ichangensis, and significantly reduced total fungal observed OTU richness by 8.19% (p=0.02) and 10.14%
(p=0.007) and didn’t affect Shannon–Wiener Index in soils associated with T. chinensis compared to CF treatments (Table 2).
2.3 Soil microbial structure and composition
For both tree species, the Principal Coordinates Analysis (PCoA) depicted variation in both soil bacterial and archaeal (Fig. 1A) and fungal community composition (Fig. 1B) among different O3 treatments. Although the soil bacterial and fungal community structures were similar for M.
ichangensis and T. chinensis in the CF treatment, they clustered based on different O3 treatments and species. For both tree species, the first axis divided the communities by O3 treatment, while
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the second axis divided them by species. For the 16S locus, O3 levels was the primary driver of M.
ichangensis associated bacterial community composition (Fig 1). Although the same pattern was
observed for O3 treated T. chinensis, these bacterial communities from were distinct from those from M. ichangensis. For fungal ITS, both control M. ichangensis and T. chinensis (non-O3 treated) were loosely clustered together. Likewise, although fungal communities from E100Y grouped together with the fungal communities from E100H, and fungal communities from E150Y were similar to those from E150H, these fungal communities each clustered together by tree species and with other samples from within the same treatment group.
The dominant bacterial phyla across all soil samples from both tree species were Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria and Chloroflexi (Fig. 2A). For M.
ichangensis, ozone treatments had the opposite effects on two of the most abundant phyla. Indeed,
when exposed to both low and high ozone treatments, the relative abundance of Proteobacteria associated with M. ichangensis significantly decreased, while that of Firmicutes increased two and three-fold, in E100Y and E150Y respectively, as compared to CFY (SI Table 1). Yet, in T.
chinensis there was no effect of ozone treatment on either Proteobacteria or Firmicutes, and only
E150H increased the relative abundance of Acidobacteria, while elevated O3 had no significant effects on other dominant phyla. Both elevated O3 treatments were characterized by lower relative abundances of Beta- and Delta-proteobacteria, Nitrospira and Gemmatimonadetes (Fig. 2B).
However, in M. ichangensis, the most severe O3 treatment (i.e., E150) influenced microbial taxa by diminishing the relative abundances of Alphaproteobactria, Thermoleophilia, Actinobacteria and Acidimicrobiia within the total microbial community. Also, in the M. ichangensis treatments, the relative abundance of Bacilli increased in the E150 treatment plots, compared to untreated
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controls (i.e., CF). In contrast, for T. chinensis, the relative abundances of microbial taxa from Nitrospira and both Beta- and Delta-proteobacteria decreased in the E150 treatment, while both
Gammaproteobacteria and Acidimicrobiia increased in abundance in the moderate (E100) O3
treatment (SI Table 2).
Across all tree species and O3 treatments, we found that the Ascomycota dominated the fungal communities. In fact, the relative abundance of fungal taxa from Ascomycota accounted for more than 80% of all fungal taxa detected in all of our samples. In the soil from both tree species, the second dominant fungal phylum, Mucoromycota was more sensitive to elevated O3 than ascomycotan fungi. The third most abundant fungal phylum: Basidiomycota, was increased in relative abundance within plots of M. ichangensis exposed to elevated O3 (Fig. 3A and SI Table 3).
We found Sordariomycetes to be the most abundant fungal class in all soil samples. Although the relative abundance of Sordariomycete fungi was lower in E150Y plots of M. ichangensis, as compared to charcoal-filtered controls of the same tree species, in T. chinensis, the relative abundance of Sordariomycete fungi increased in E100H, as compared to control plots of T.
chinensis (CFH). The highest ozone treatments (E150) significantly increased the relative
abundance of Eurotiomycetan fungal, which was the second most abundant fungal class detected in the soil fungal communities. In both M. ichangensis and T. chinensis, the relative abundance of Dothideomycetes increased with O3 concentration (SI Table 4).
Some nitrifiers’ relative abundances were found to be decreased by elevated O3, including Alpha-, Beta-, Delta-, Gamma-proteobacteria and Nitrospira. Both elevated O3 significantly decreased the relative abundance of Beta-, Delta-, Gamma-proteobacteria and Nitrospira, while
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only E150 treatments demonstrated significantly lower relative abundance of Alphaproteobacteria in soils associated with M. ichangensis. However only E150 significantly decreased the relative abundance of Beta-, Delta- proteobacteria and Nitrospira in soils associated with T. chinensis (SI-Table 3). We also detected the effects of elevated O3 on N2O-producing fungal and bacterial taxa. Although E100 treatment had no detectable effects on the relative abundance of some N2O-producers, E150 demonstrated significant increases in relative abundances of Eurotialean fungi, such as Penicillium, and Hypocrealean fungi, including Acremonium, as well as bacterial taxa, such as Bacillus; these aforementioned N2O-producing taxa significantly increased at least
60% when M. ichangensis and T. chinensis were exposed to severe E150 treatments compared to CF (Table 3).”
2.4 Soil microbial community links to O
3fumigation and soil properties
For both soil fungal and bacterial communities, results from our PERMANOVA analyses showed significant main effects of ozone (p<0.001), tree species (p<0.001), organic matter (p<0.001), and soil pH (p<0.001), with significant interactions between ozone and tree species (p<0.001). A significant interaction among soil pH (p<0.001) and NH
4+/NO
3-(p<0.021) were found for bacterial communities, when both tree species and ozone treatment were included in the model.
The Redundancy Analysis (RDA) plots of the bacterial and fungal community structure indicated that AOT40 and soil pH are strong drivers of bacterial and fungal community structure (Fig. 4).3 Discussion
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Soil processes, such as organic matter decomposition and nutrient cycling are driven by soil organisms and their interactions with plants and soil abiotic conditions (Wardle et al., 2004).
However, exposure to elevated levels of O3 pollution may influence aboveground plant performance, as well as soil microbial communities and processes. We found that across soils from M. ichangensis, E150reduced soil microbial biomass, and organic matter, while increasing DN and NO3
-N. While there was no effect of elevated O3 on microbial biomass and DN, but
elevated O3 decreased NO3-N in T. chinensis. Additionally, although tree species differed in their overall microbial diversity, effects of elevated O3 on soil microbial diversity were only evident in soils from M. ichangensis plots. Even though soils from O3 treated T. chinensis were equivalent in microbial diversity to untreated control plots of the sample plant species, the microbial community composition significantly shifted in O3 exposed T. chinensis and M. ichangensis plots. These findings showed that elevated O3 not only affects plants and soil properties, but also sensitizes soil microbial communities to it, which could have a lasting impact on the function of forest ecosystem.
Soil organisms are responsible for recycling nutrients and maintaining soil properties which may be affected by altered carbon allocation patterns in plants exposed to O3 (Andersen 2003).
Elevated O3 decreased MBC and MBN, and also led to decreased alpha diversity of bacteria and fungi in M. ichangensis. These findings highlight how O3 may not only constrain microbial diversity in M. ichangensis forests, but may also exert limits on soil microbial functioning and activity in these ecosystems. In contrast, elevated O3 only affected fungal diversity in T. chinensis, without impacting bacterial diversity; these findings suggested that bacterial diversity in soils of M.
ichangensis was more sensitive to elevated O3 than those of T. chinensis. This is consistent with
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our hypothesis that microbial communities associated with M. ichangensis would be more strongly affected by exposure to O3, as the impacts of exposure were more extreme in the growth of this tree species (Yu et al., 2017). Furthermore, leaf dry mass per unit leaf area (LMA) of M.
ichangensis was lower than that of T. chinensis (SI Table 5). This may indicate a greater sensitivity
of M. ichangensis to O3 than T. chinensis, putatively because species with high LMA may be less susceptible to O3 toxicity (at a given external ozone exposure) (Feng et al., 2018).
Overall, the shifts observed in the relative abundance of bacterial taxa showed that elevated O3 had greater, and more variable, effects on the bacterial structure and composition of soils associated with M. ichangensis than that of T. chinensis (SI Table 1). The most abundant phylum of bacteria was Proteobacteria, which was decreased by elevated O3 in M. ichangensis, but not in T. chinensis. Betaproteobacteria, a diverse group that includes several species involved in nutrient
cycling, were less represented in microbial communities exposed to elevated O3 in both M.
ichangensis and T. chinensis than in control treatments (SI Table 1). Acidobacteria, the second most abundant phylum in our study, were found to be enriched in soils with low resource availability, and often associated with lower SOC mineralization rate (Fierer et al., 2007). Notably, their preference for soil organic matter and the ability of several groups of Acidobacteria to decompose organic carbon has been reported in many previous studies (Cleveland et al., 2007;
Rawat et al., 2012; Tveit et al., 2014). In this study, only E150 treatment significantly impacted the relative abundance of Acidobacteria in T. chinensis. Gram-positive bacteria from the phylum Actinobacteria are known to play an important role in decomposition of organic materials (Kramer and Gleixner, 2008), such as cellulose and chitin. The prevalence of Actinobacteria diminished in M. ichangensis plots in E150, which may correlate with reduced organic matter
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decomposition. Many Firmicutes bacteria are able to utilize xylose, an abundant carbon source in natural ecosystems (Zhao et al., 2009), the relative abundance of which was increased by elevated O3 in M. ichangensis. The majority of the known members of Bacteroidetes exhibit copiotrophic attributes and are abundant in soils with high C availability (Fierer et al., 2007). The abundance of Bacteroidetes decreased in elevated O3 treated M. ichangensis plots, which may indicate that the soil microorganisms in association with this plant species experienced a lower C availability with elevated O3.
Carbon cycling is a complex process, involving many groups of bacteria and fungi, some of which were increased while others were decreased by elevated O3 in this study. We observed a reduction in organic matter in soils associated with M. ichangensis and T. chinensis after exposure to E150, relative to CF treatment. Other studies have shown that elevated O3 decreased soil dissolved organic carbon and changed the carbon cycle in numerous ways (Jones et al., 2009, Chen et al., 2015; Lu et al., 2015). The indirect effects of elevated O3 on soil organic carbon was likely via reductions in plant biomass, and associated implications for hindering photosynthesis.
Elevated O3 significantly reduced net photosynthesis in both species (Yu et al., 2017) and also influenced the growth and biomass of two species (SI Table 6). Further, cellulolytic fungi, including Sordariomycetes, Eurotiomycetes and Dothideomycetes in Ascomyceta have been shown to be significantly correlated with cellobiohydrolase activity (Fan et al., 2012), therefore changes to these fungal groups could influence the availability of both labile and recalcitrant carbon sources. In our study, the relative abundance of the most abundant fungal phylum: Ascomycota was unaffected by elevated O3 (SI Table 3). However, the relative abundance of some cellulolytic classes of Ascomycotan fungi, including Sordariomycetes and Eurotiomycetes, responded
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differently to elevated O3 (SI Table 4). There could be functional implications of the heightened relative abundances of Sordariomycetes, Eurotiomycetes and Dothideomycetes with exposure to both E100 and E150 treatments for both M. ichangensis and T. chinensis. For instance, increased abundance and activity of these fungal classes would likely promote cellulose decomposition. The Mucoromycota, another abundant fungal phylum in this study, was sensitive to elevated O3 in plots planted with both tree species (SI Table 3). Mucoromycota are an ecologically diverse group of fungi that function as mutualists, parasites, commensals and decomposers, thereby playing many important roles in the ecosystem. Coprophilic and saprotrophic fungi from Mucoromycota perform essential functions in the carbon cycle, decomposing substrates in soil, such as plant materials (Spatafora et al. 2016).
Nitrification is another key nutrient-cycling process affected by ozone exposure.
Nitrite-oxidizing bacteria (NOB) are widely distributed phylogenetically, among the Alpha-, Beta- and Delta- Proteobacteria and the Nitrospira phylum (Alawi et al., 2007; Attard et al., 2010).
Additionally, many processes in the nitrogen cycle, including nitrification, can be influenced by soil properties, such as dissolved organic carbon (Strauss and Lamberti, 2002), or atmospheric conditions. In our study, we found that NOB was sensitive to O3 in M. ichangensis plots (SI-Table 2). The relative abundance of Beta-, Delta- Proteobacteria and Nitrospira was decreased in soils associated with M. ichangensis plots by both elevated O3 exposure, while was only inhibited in soils associated T. chinensis by E150. Additionally, our findings were consistent with Huang et al.
(2010) and Wertz et al. (2012), which found no correlation between Nitrospira-like NOB and NO3 -
concentration. Elevated O3 reduced NOB including Alpha-, Beta-, Delta- Proteobacteria and Nitrospira, thus may have likely inhibited nitrification and influenced nitrogen cycling.
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Denitrification is a microbial-mediated process of stepwise reductions from nitrate (NO3) to dinitrogen gas (N2). Both nitrification and denitrification contribute to N2O emission. Previous in situ studies have shown that fungi often contribute >50% of total soil N2O emissions (Laughlin and Stevens, 2002; Yanai et al., 2007; Chen et al., 2014). Ninety percent of the fungi reported to produce N2O belong to the phylum Ascomycota, followed by Basidiomycota and Mucoromycota (Mothapo et al., 2015). The N2O-producing activity seems to be most widely dispersed across the subphylum Pezizomycotina, with Eurotiales (Eurotiomycetes) including most of the species
（Chen and Shi, 2017）and species belonging to the genera Penicillium, Acremonium (Jirout et al., 2013). Bacillus is also found to be the most potent N2O-producing bacteria (Sun et al., 2016). In our study, although the abundance of them were not affected by E100, the relative abundance of these microbes increased by ~60.55% -155.25% when M. ichangensis was exposed to severe E150 treatments. And the relative abundance of these increased 64.03-130.36% when T. chinensis was exposed to elevated O3. Because N2O is a potent greenhouse gas and O3 depleting substance (Mothapo et al., 2015), this observed increase in fungal taxa capable of denitrification in elevated O3 treatments could stimulate N2O emissions, which could potentially lead to broad, ecosystem-level ramifications.
In the soil of M. ichangensis, the concentrations of NO3
- were higher in the elevated O3
treatments.After O3 fumigation, leaf N content significantly decreased (SI Table 1), which led to more available nitrogen in soil. However, DN increased after O3 fumigation, while microbial nitrogen assimilation (MBN) decreased. Although nitrifiers were inhibited, our results suggest that denitrifying microbes increased, which resulted in a net reduction of NH4+
oxidation and an increased transformation of NO3
- to N2O or N2, leading to NO3
- reduction. Perhaps any observed
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reduction may have been related to differences between DN and MBN, and as a result, led to increased NO3
-. In contrast, when soils associated with T. chinensis were exposed to similar levels of elevated O3, NO3
- decreased. Elevated O3 had no effect on leaf N content (SI Table 6), soil DN
and MBN in T. chinensis, and the inhibition of nitrifiers may have contributed to our observed reductions in NO3-
. Additionally, we detected both reduced denitrifying bacteria and fungi in T.
chinensis exposed to elevated O3, which likely stimulated denitrification and resulted in decreased NO3
-.
To elucidate the mechanisms that drive changes in the soil microbial community in plots exposed to elevated O3, the relationships were examined among AOT40, soil properties, and soil microbial structure and composition. Certainly, AOT40 and soil pH were both related to observed shifts in soil microbial communities. Due to the high activity of O3 molecules and their tendency to react with moist surfaces, the direct effect of O3 on soil is negligible, but cannot be tested in this study. The effects of elevated O3 on below-ground processes could be regulated indirectly by the influence of elevated O3 on plant litter production (Uddling et al., 2006) and root exudates, which ostensibly provides substrates for microbial proliferation and decomposition (Andersen, 2003; Li et al., 2012; Li et al., 2013). A range of root exudates may alter the diversity and abundance of carbohydrate inputs into soil, as well as soil pH, which is an important driver of soil microbial community diversity and structure (Fierer and Jackson, 2006; Pietri and Brookes, 2009; Cheng et al., 2013). Soil pH decreased in plots treated with elevated O3 in this study, which was maybe caused by more organic acid in root exudates, and also the symbiotic ECM and AM produced organic acids when O3 elevated. Moreover, a significant positive correlation was detected between soil pH and AOT40. These results may indicate that elevated O3 affects soil pH by changing root
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exudates, further impacting the composition and function of soil bacterial and fungal communities.
Further studies on root exudates from plants at elevated levels of O3 would elucidate mechanisms underlying changes in tree-associated soil microbial communities in these exposed forested ecosystems. Findings from this study would inform and support approaches for the restoration of ecosystems exposed to elevated O3, including the use of soil treatments or microbial inoculants to ameliorate the impact of elevated O3 on above- and below-ground ecosystems (Wang et al. 2016).
4
ConclusionSoil microbial biomass and soil pH was lower in both M. ichangensis and T. chinensis soils after O3 exposure, as compared to CF. Although elevated O3 significantly shifted soil community structure in both tree species, it only reduced soil microbial diversity associated with M.
ichangensis, as well as fungal diversity, but not bacterial diversity in T. chinensis soils. This
suggests that soil microbial communities in M. ichangensis may be more sensitive than in T.
chinensis. Changes in soil microbial community structure and diversity after O3 exposure also affected both carbon and nitrogen cycling. In this study, as O3 increased, some groups of bacteria and fungi involved in the complex processes of the carbon cycle increased, while others decreased.
The nitrogen cycle was also affected by O3, such that elevated O3 reduced NOB including Alpha-, Beta-, Delta- Proteobacteria and Nitrospira, and likely inhibited nitrification and influenced the nitrogen cycle. Elevated O3 may have also affected denitrification processes by increasing the relative abundance of denitrifying fungal taxa, which may ostensibly lead to increases in N2O emissions. AOT40 and soil pH were strong drivers of bacterial and fungal community structure, which suggests that as elevated O3 affects soils, the combined effect of O3 and changes to soil pH
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may further impact soil microbial community composition and function.
Acknowledgements
This research was supported by the State Forestry Administration 948 project (2014-4-65),
and the National Public Benefit Special Fund of China for Forestry Research (No. 201304313), as well as NSF ICER-1541047 from EarthCube to MM and EA and the Translational Mycology Postdoctoral Research Award from the Mycological Society of America to MM. The authors wish to acknowledge the valuable assistance of the Qianyanzhou Ecological Station of Chinese Academy of Sciences for conducting this study. Our thanks also go to the staff of the station, especially to Mr. Shanyuan Yin, for their assistance in the field work.
Figure captions
Fig. 1 Principal Coordinates Analysis of soil bacterial and archaeal 16S (A) and fungal ITS (B) communities. For both soil fungal and bacterial communities, PERMANOVA analyses showed significant main effects of ozone (p<0.001), tree species (p<0.001), with significant interactions between ozone and tree species (p<0.001).
Fig. 2 Bacterial taxonomic classification at the (a) phylum and (b) class levels of dominant phylogenetic groups from different samples. Relative abundance was defined as the number of sequences affiliated with taxa divided by the total number of sequences per sample (%). Relative abundance of phyla and classes with 1% of total composition are combined and defined as
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“others”.
Fig. 3 Fungal taxonomic classification at the (a) phylum and (b) class levels of dominant phylogenetic groups from different samples. Relative abundance was defined as the number of sequences affiliated with taxa divided by the total number of sequences per sample (%). Relative
abundance of phyla and classes with 1% of total composition are combined and defined as
“others”.
Fig. 4 Redundancy analysis to reveal soil bacterial (a) and fungal (b) community responses to O
3fumigation treatment and correlations with edaphic properties.
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