Soil pH Determines Microbial Diversity and ...

Microb Ecol DOI 10.1007/s00248-014-0530-2

SOIL MICROBIOLOGY

Soil pH Determines Microbial Diversity and Composition in the Park Grass Experiment Kateryna Zhalnina & Raquel Dias & Patricia Dörr de Quadros & Austin Davis-Richardson & Flavio A. O. Camargo & Ian M. Clark & Steve P. McGrath & Penny R. Hirsch & Eric W. Triplett

Received: 13 February 2014 / Accepted: 29 October 2014 # Springer Science+Business Media New York 2014

Abstract The Park Grass experiment (PGE) in the UK has been ongoing since 1856. Its purpose is to study the response of biological communities to the long-term treatments and associated changes in soil parameters, particularly soil pH. In this study, soil samples were collected across pH gradient (pH 3.6–7) and a range of fertilizers (nitrogen as ammonium sulfate, nitrogen as sodium nitrate, phosphorous) to evaluate the effects nutrients have on soil parameters and microbial community structure. Illumina 16S ribosomal RNA (rRNA) amplicon sequencing was used to determine the relative abundances and diversity of bacterial and archaeal taxa. Relationships between treatments, measured soil parameters, and microbial communities were evaluated. Clostridium, B a c t e ro i d e s , B r a d y r h i z o b i u m , M y c o b a c t e r i u m , Ruminococcus, Paenibacillus, and Rhodoplanes were the most abundant genera found at the PGE. The main soil parameter that determined microbial composition, diversity, and biomass in the PGE soil was pH. The most probable mechanism of the pH impact on microbial community may include

mediation of nutrient availability in the soil. Addition of nitrogen to the PGE plots as ammonium sulfate decreases soil pH through increased nitrification, which causes buildup of soil carbon, and hence increases C/N ratio. Plant species richness and plant productivity did not reveal significant relationships with microbial diversity; however, plant species richness was positively correlated with soil microbial biomass. Plants responded to the nitrogen treatments with an increase in productivity and a decrease in the species richness. Keywords Microbial community . Archaea . Bacteria . Shannon diversity . Plant species richness . Biomass

Introduction Soil is a structured and heterogeneous system with complex trophic interactions that provide an enormous diversity of microbial populations [1]. Torsvik et al. [2] estimated that

Electronic supplementary material The online version of this article (doi:10.1007/s00248-014-0530-2) contains supplementary material, which is available to authorized users. K. Zhalnina : R. Dias : P. D. de Quadros : A. Davis-Richardson : E. W. Triplett (*) Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, 1052 Museum Road, 32611-0700 Gainesville, FL, USA e-mail: [email protected]

P. D. de Quadros : F. A. O. Camargo Department of Soil Science, Federal University of Rio Grande do Sul, Porto Alegre, Brazil F. A. O. Camargo e-mail: [email protected]

K. Zhalnina e-mail: [email protected]

I. M. Clark : S. P. McGrath : P. R. Hirsch Rothamsted Research, Harpenden, Hertfordshire, UK

R. Dias e-mail: [email protected]

I. M. Clark e-mail: [email protected]

P. D. de Quadros e-mail: [email protected]

S. P. McGrath e-mail: [email protected]

A. Davis-Richardson e-mail: [email protected]

P. R. Hirsch e-mail: [email protected]

K. Zhalnina et al.

about 10,000 different microbial species are present in 1 g of boreal forest soil. Gans et al. [3], using a computational approach, predicted existence of more than 1×107 microbial species in each gram of soil. The diversity and richness of soil microorganisms has been a fascinating subject for scientists over the years. Recent improvements in technologies, from culture-dependent techniques to molecular analysis, have allowed scientists to obtain a higher resolution of taxonomic diversity of soil microorganisms and to evaluate edaphoclimatic parameters that impact the abundance and diversity of particular groups in soil, as well as providing information on the microbial community on local and global scales. Recent studies have suggested that soil pH and nitrogen input are the main predictors of the microbial diversity in soils [4–7]. The highest bacterial diversity is often found in neutral soils, and it was significantly lower in acidic soils [4, 5, 7]. On the other hand, Roesch et al. [8] found higher microbial diversity in forest soil with a relatively low pH than in three other agricultural soils with a higher pH. Agricultural soils are commonly fertilized with nitrogen, and this could be another possible factor regulating the diversity of microorganisms. In many studies, nitrogen additions resulted in a significant decrease in the diversity of a soil microbial community [9]. There are also studies that show the close relationships between plant diversity and soil microorganisms [10]. However, other studies did not find a clear correlation between plants and microbial diversity [4]. Whether the main triggers of microbial diversity are pH, nitrogen, vegetation, or other factors remains largely undetermined. Many studies compared physically, chemically, and geographically different soils in attempts to find general trends in the drivers of microbial diversity, but to date, there is a lack of systematic analysis of the effect of changes in soil parameters, due to management, on soil microbial diversity. Multiple studies of nitrogen fertilization effects on microbial diversity frequently omitted reporting changes in soil properties, such as pH, that are likely to have a primary impact on the diversity of microorganisms. Thus, it is unclear how the application of different types of nutrients may affect microbial diversity in soils. Established in 1856, the Park Grass Experiment (PGE), the oldest ecological experiment in the world, has allowed scientists to study the influence of nutrient additions and different pH levels on biodiversity and ecology for many years [11]. A broad range of fertilizers (nitrogen as ammonium sulfate, nitrogen as sodium nitrate, phosphorous, potassium) has been applied to different treatments over the years. Also, a gradient of pH has been created within each nitrogen treatment (maximally pH 3.6–7) [11]. It has been shown, by analyzing plant communities from the PGE, that plant species richness is highly controlled by pH, and it is the greatest at relatively high pH levels. Also, nutrient input reduced the species

richness of plants at the PGE [12]. Additionally, Silvertown et al. [13] studied the distribution of invertebrates and their relationship with the plant communities. However, only a few studies have investigated microbial community structure and its relationship with the plant community at this site. In one study, at the PGE, soil bacterial and fungal growth was measured by leucine and acetate incorporation [14]. This method revealed a decrease in bacterial and an increase in fungal growth with low pH levels. The phospholipid fatty acid (PLFA) composition changed only when the pH level was below 4.5. Also, the input of nitrogen fertilizers did not affect bacterial growth, but it did reduce microbial biomass [14]. At the Hoosfield acid strip on the same farm as the PGE, quantitative PCR and pyrosequencing across a similar pH gradient found a strong positive correlation between the abundance of bacterial operational taxonomic units (OTUs) and soil pH [5]. The relationships between the relative abundances of the most dominant bacterial groups and soil pH levels were shown in the Hoosfield experiment. However, the taxonomic resolution of all of the above experiments did not allow an assessment of the abundance and distribution of some groups that are important contributors to nutrient cycles in soil, such as diazotrophs, ammonia-oxidizing archaea, and ammoniaoxidizing bacteria. Our objectives for this study were (i) to determine bacterial and archaeal community composition at the PGE, (ii) to compare the changes in microbial community composition and diversity in the different nutrient treatments and soil parameters associated with these changes, and (iii) to determine the relationships between microbial diversity and plant species richness, as well as productivity. To achieve our goals, we analyzed the microbial communities in PGE plots with different pH gradients and nitrogen inputs using 16S ribosomal RNA (rRNA) Illumina sequencing. This work presents a detailed analysis of microbial community composition at the PGE. Previous studies have described an impact of the long-term agricultural treatments on plant community structure and the diversity of invertebrates. Also, this work shows that pH is the main driver of microbial community in the Park Grass soil. Moreover, pH changes in soils are the result of nutrient management, and these pH changes affect nutrient availability to both plant and microbial communities.

Materials and Methods Soil Characterization, Sample Collection, and DNA Extraction The soil from the PGE at Rothamsted Research in the UK is classified as Typic Paleudalf (USDA, 1992). It is acidic, well drained, and developed in a relatively silty superficial deposit

Soil pH Determines Microbial Diversity and Composition

overlay mixed with flints. The top 23 cm of soil is a flinty, silty clay loam with 18–27 % clay. The PGE started in 1856 with the purpose of studying the impact of fertilizers on plant productivity [13]. The site of the experiment is about 2.8 ha. The soil originally had a pH 5.6–5.8. Various combinations of inorganic fertilizers (P, K, Mg, Na, nitrate-N, and ammoniumN) and organic manures (farmyard manure and fishmeal) have been tested since the start. The application of fertilizers caused soil acidification. Each fertilizer treatment was subdivided into four subplots, and three subplots were limed to maintain pH levels of 5, 6, and 7. The fourth subplot received no lime, and its pH ranges from 3.5 to 5.7, depending on the fertilizer treatment (Supplementary material, Table S1 and S2). In the present study, we analyzed soil samples from the following five treatments (Table 1). In May, July, and September 2009, 60 samples in total were collected from five treatments using a soil corer (2–10 cm depth, 2 cm diameter). Soil samples were sieved (0.01 % of total analyzed microbial community

Results Bacterial and Archaeal Community Composition A total of 2.7 million barcoded reads was obtained from sequencing. Of these, 97.5 % sequences were classified as Bacteria, 0.3 % as Archaea, and 2.2 % remained unclassified; 65.5 % of all sequences were classified at the phylum level. The 12 most represented phyla (16S rRNA >0.1 % of total reads) were determined (Fig. 1a). Proteobacteria, Firmicutes, A c i d o b a c t e r i a , A c t i n o b a c t e r i a , B a c t e ro i d e t e s , Verrucomicrobia, and Planctomycetes comprised 98 % of the classified community. One archaeal genus and 36 bacterial genera occurred with a relative abundance of 16S rRNA sequences greater than 0.1 % (Fig. 1b). Clostridium and Bacteroides were the most abundant genera representing the

Soil pH Determines Microbial Diversity and Composition Fig. 2 Spearman correlations of the abundance of OTUs and the five nutrient treatments. Only taxa significantly correlated with one of the treatments are represented. *Values are significant (Pvalue ≤0.01)

bacterial community, and Candidatus Nitrososphaera was the only representative of the archaeal community among the abundant genera at the PGE. Approximately 20 % of all sequences were classified at genus level and only 16 % at species level. Majority of the most represented species (0.1– 0.7 %) remained unclassified and belonged to the most abundant Park Grass genera. Responses of the Microbial Community to the Nutrient Treatments The relationships between five different nutrient treatments and the microbial community were analyzed in this study (Fig. 2a, b, Table S3). Nil treatment was positively correlated with the abundance of Bradyrhizobium and Paenibacillus. The treatment receiving P only did not reveal any correlations with classified OTUs. The N-treatment in the form of ammonium sulfate revealed negative correlations with Verrucomicrobia, and Chloroflexi phyla (Fig. 2a), as well as the genera Fig. 3 NMDS scaling/ordination plot of the Bray-Curtis similarities in community composition at a phylum and b genus level with different pH gradients. Each point represents an individual sample

Bradyrhizobium, Paenibacillus, and Geobacter (Fig. 2b). The addition of N as sodium nitrate showed a positive correlation with two phyla: Thaumarchaeota and Nitrospirae, as well as four genera: Geobacter, Ca. Nitrososphaera, Nitrospira, and Methylibium. The remaining OTUs were not significantly correlated with the treatments analyzed. Plots where sodium nitrate was added had the highest pH, moisture, and the lowest C/N ratio compared to other treatments (Supplementary material, Table S1). N-treatments in the form of ammonium sulfate had lowest pH, moisture, the highest C/N ratio, and the highest concentration of ammonium. N-treatment, in the form of ammonium sulfate with phosphorous, had the highest carbon and nitrogen content. Spearman correlations and NMDS were used to identify the relationships between the most abundant phyla and genera with soil parameters. NMDS determined that pH was the strongest soil factor that impacted on the microbial community across all samples at phylum and genus levels (Fig. 3).

K. Zhalnina et al.

Positive Spearman correlation of pH with the relative abundance of Proteobacteria, Gemmatimonadetes, Nitrospira, and Thaumarchaeota was revealed in this study; acidic pH favored the increase in abundance of Chlamydiae (Table 2). The percentage of moisture and concentration of ammonium did not show any significant correlations. Concentration of nitrate was significantly and positively correlated with Thaumarchaeota and Nitrospirae abundance. Total carbon (TC) and total nitrogen (TN) was negatively correlated with the abundance of Firmicutes, Verrucomicrobia, and Chloroflexi. The C/N ratio negatively c o r r e l a t e d w i t h t h e a b u n d a n c e o f N i t ro s p i r a , Thaumarchaeota, and Gemmatimonadetes, but it was positively correlated with the abundance of Acidobacteria and Chlamydiae. Soil pH was positively correlated with 14 of the 37 most abundant soil genera: Mycobacterium, Flavobacterium, Sphingomonas, Ca. Nitrososphaera, Steroidobacter, N o c a rd i o i d e s , S t re p t o m y c e s , M i c ro m o n o s p o r a , Pseudomonas, Mezorhizobium, Solirubrobacter, Nitrospira, Paenibacillus, and Methylibium, and pH was negatively correlated with Ca. Solibacter, Clostridium, and Actinomadura (Supplementary Material, Table S4). Correlation-based principal component analysis (PCA) also showed that pH and C/N ratio were the main factors that influenced microbial community composition. Calcium carbonate and ammonium had less effect on microbial population at genus level (Fig. 4). Although PCA indicated a relationship b etween se vera l groups, including Pseudomonas, and nitrate concentration (Fig. 4), this was significantly and positively correlated only with the abundances of Ca. Nitrososphaera and Nitrospira (Supplementary Material, Table S4). The percentage of moisture did not

have a profound effect on the microbial community. TN and TC were negatively correlated with Geobacter and Ca. Solibacter. The group of bacteria that commonly inhabits human and/or animal gut, skin, and oral cavities, such as Ruminococcus, Streptococcus, and Enterococcus, formed a separate cluster (Fig. 4). In the PGE, the soil microbial diversity of OTUs, clustered at the 80, 90, and 95 % of sequence similarity, was measured by using the Shannon diversity index (H′) and varied across the five different treatments and established pH gradients (Figs. 5 and 6). Nitrogen treatments did not show any correlation with microbial diversity (Fig. 6, Table S5). Each of the five treatments had four different pH levels, and the combined effect of treatment and pH had a significant influence on the Shannon diversity index at phylum, family, and genus levels (Fig. 5, Table 3). The lowest diversity was observed in the plots with low pH levels and addition of ammonium sulfate (Fig. 5). Compared to other soil parameters correlated with diversity of the microorganisms in soil (C/N ratio, CaCO3, NH4+), pH has demonstrated the most profound impact on microbial diversity measured at 80, 90, and 95 % sequence similarity (Table 3). Changes in Microbial Biomass Under Different Treatments The input of phosphorous only or nitrogen as nitrate did not change microbial biomass. However, plots where nitrogen as ammonium sulfate was added showed negative correlation with microbial biomass (Fig. 6, Table S5). The most significant correlations between measured soil parameters and microbial biomass were pH and C/N ratio. Microbial biomass increased with pH and plant species number but decreased with higher C/N ratios (Table 4).

Table 2 Spearman correlations for the Park Grass Experiment between 16S rRNA relative abundance of main phyla and soil properties (TN—total nitrogen, TC—total carbon, C/N ratio, NH4-N—ammonium, NO3−—nitrate, moisture, CaCO3, and pH) Phyla

pH

Moisture

TN

TC

C/N ratio

CaCO3

NO3−N

NH4-N

Proteobacteria Firmicutes Acidobacteria

0.49a −0.05 −0.25

0.11 −0.19 0.19

−0.10 −0.40 0.27

−0.17 −0.35 0.30

−0.21 −0.24 0.55

0.30 −0.29 0.21

0.22 −0.07 −0.10

−0.23 −0.14 0.34

Actinobacteria Bacteroidetes Verrucomicrobia Planctomycetes Gemmatimonadetes Thaumarchaeota Chlamydiae Chloroflexi Nitrospirae

0.01 0.07 0.16 −0.01 0.38 0.56 −0.55 −0.09 0.73

−0.26 0.06 0.02 0.06 0.05 0.29 −0.17 −0.28 0.32

0.29 −0.33 −0.58 −0.02 −0.29 0.21 0.13 −0.50 0.14

0.28 −0.33 −0.64 −0.01 −0.38 0.10 0.12 −0.51 0.03

0.19 −0.32 −0.32 0.23 −0.51 −0.62 0.49 −0.19 −0.66

0.17 −0.15 −0.09 0.17 0.04 0.34 −0.28 −0.19 0.48

−0.01 0.03 0.04 −0.18 0.27 0.56 −0.26 −0.07 0.54

0.02 −0.12 −0.42 −0.09 −0.36 −0.14 0.11 −0.30 −0.28

a

Values in bold are significant (Pvalue ≤0.001)


Fig. 4 Principal component analysis (correlation-based PCA) of soil parameters—supplementary variables (pH, CaCO3, moisture, TC—total carbon, TN—total nitrogen, C/N ratio, NH3, NO3-N) on microbial com-

munity structure—active variables at genus level at Park Grass Experiment. Numbers in brackets indicate the percent of the total variance explained by each axis

Changes in Plant Productivity and Plant Species Richness Under Different Treatments, Relations Between Plant and Microbial Community Composition

Discussion

Plant biomass measured in June significantly increased in plots where N was added together with P (Fig. 6). The number of plant species at the PGE ranged from 4 to 39 depending on treatment [12]. Addition of either form of nitrogen significantly decreased plant species richness. Plant biomass revealed a strong negative correlation with plant species richness (Table 4). Although plant species richness did not show a statistically significant relationship with microbial diversity, it was strongly positively correlated with microbial biomass (Table 4).

The 12 most abundant microbial phyla composed the core of the microbial community of the PGE soil (Fig. 1a). They included common representatives from copiotrophic groups (Proteobacteria, Firmicutes, Actinobacteria), microorganisms with mainly oligotrophic lifestyle (Acidobacteria, Verrucomicroba, Chloroflexi), and phyla with specific ecological traits. The microbial phyla determined at the PGE were found to be abundant in many other soils [5–7, 23–27]. Previous metagenomic studies of the PGE, confined to the unfertilized and unlimed plots [28], revealed that the most abundant taxa in this soil were Bradyrhizobium,

Analysis of Bacterial and Archaeal Community Composition

K. Zhalnina et al.

Fig. 5 The relationship between microbial diversity (80 % of sequence similarity) measured by the Shannon diversity index (H′), for five different nutrient treatments at different pH levels. The same letter above the

bars indicates no significant difference between means as measured by Duncan’s test at the 95 % confidence interval

Rhodopseudomonas, and Nitrobacter genera from Alphaproteobacteria; Ca. Solibacter and Acidobacteria genera from Acidobacteria; Pseudomonas from G a m m a p ro t e o b a c t e r i a ; a n d B u r k h o l d e r i a f r o m Betaproteobacteria. Our analysis of 16S rRNA genes in the PGE soil found similarly high abundances of Bradyrhizobium, Ca. Solibacter, and Pseudomonas, along with another 34 abundant genera (Fig. 1b).

we can suggest that both of these phyla demonstrated an oligotrohic lifestyle by decreasing in abundance with the addition of nutrients. Ramirez et al. [25] hypothesized that changes in community composition after nitrogen was added might be caused not only by the increase of nitrogen concentration in the soil but also by the alteration of soil pH or changes in plant composition. In our study, pH was the strongest factor that determines microbial community composition in the Park Grass soil at phylum and genus level (Fig. 3). This finding corroborates other studies that demonstrated similar effect of pH on microbial community in soils at global and local scales [4, 6, 7]. We have observed positive correlation of Ca. Solibacter (Acidobacteria) and Clostridium (Firmicutes) with acidic pH (Table S4). Similar preferences of these genera to acidic pH were found in other studies [31, 32]. Two main mechanisms were suggested to explain pH effect on microbial community [7, 33]. The first mechanism explains pH impact on the community by the narrow pH tolerance of some prokaryotic taxa. Acidification of the interior of a microbial cell inhibits the activity of most enzymes and of overall cell metabolism [34]. However, many soil microorganisms have developed adaptive responses to survive in acidic environments [34, 35]. The second mechanism includes pH mitigation of nutrient availability and ion toxicity in soils. Measured soil pH of the Park Grass soil correlated significantly with other soil parameters, such as C/N ratio and the concentrations of ammonia and nitrate (rho=−0.62, rho=0.91, rho=0.45, Pvalue ≤0.001, respectively) (Supplementary Material, Table S6). This may suggest pH regulation of the availability of these nutrients. While concentrations of ammonia

Microbial Community Composition and Soil Parameters We determined the increase in abundance of certain phyla and the decrease of other phyla in response to nitrogen treatments. Five different treatments were analyzed in our experiment, as well as the changes in soil parameters caused by these treatments. Verrucomicrobia responded negatively to the addition of nitrogen as a sulfate, and correlations with soil parameters revealed a negative relationship between the abundance of this phylum and total carbon and total nitrogen concentrations in soil (Fig. 2). This corroborates previous reports [29], which suggested that the phylum is specialized for oligotrophy. A similar decrease in abundance of Verrucomicrobia with nitrogen addition was reported by Ramirez et al. [30]. Chloroflexi also demonstrated a decrease in abundance with the input of nitrogen as ammonium sulfate. A similar trend was observed at the Kellogg Biological Station and Cedar Greek experiment [26], where the abundance of Chloroflexi decreased with medium and high levels of nitrogen input. Following the oligotroph-copiotroph concept [24], where groups of microorganisms with growth under low nutrient conditions, and slow growth rates, may decline after the addition of nitrogen,

Soil pH Determines Microbial Diversity and Composition Fig. 6 Changes in microbial biomass, microbial diversity, plant biomass, and plant species richness in five different nutrient treatments at different pH levels. To represent changes at different treatments in microbial biomass, Shannon diversity index, plant biomass, and plant species richness on the heatmap, each parameter was placed on a scale 0 to 100 (lowest to highest). *No data

Table 3 Spearman correlations for the Park Grass Experiment between microbial diversity (H′) at 80, 90, and 95 % of sequence similarity and soil features

a


Variables

H′80

H′90

H′95

pH Moisture TN TC C/N ratio CaCO3 NO3-N NH4-N

0.51a 0.09 −0.25 −0.32 −0.51 0.19 0.38 −0.47

0.52 0.17 −0.10 −0.18 −0.42 0.32 0.34 −0.42

0.64 0.20 0.00 −0.10 −0.38 0.48 0.33 −0.42

and nitrite showed direct effect on the abundance of nitrifiers (Thaumarchaeota and Nitrospirae), the C/N ratio provides a relative index of nutrient status in the soil and does not have a direct effect on the community. The addition of N as ammonium sulfate increased concentration of NH4+ in soil, which was positively correlated with C/N (rho=0.40, Pvalue ≤0.05). Nitrification leads to the soil acidification, and it causes buildup of organic C and hence the increase of C/N ratio. Recently, it was shown that at a population level, microbial C/N/P ratios are well conserved [36] and strongly interrelated with soil C/N/P ratios [37]. Any stoichiometric C/N imbalance between microbial element ratio and soil element ratio is regulated by

K. Zhalnina et al. Table 4 Spearman correlations between microbial, plant biomass, diversities, and soil parameters Variables

Microbial biomass (mg C/kg)

Microbial biomass Microbial diversity Plant biomass Plant sp. richness pH Moisture TN TC C/N ratio CaCO3 NO3-N NH4-N

0.44 −0.02 0.66a 0.61 0.37 −0.27 −0.36 −0.49 0.28 0.28 −0.41

a

Microbial diversity (H′) 0.44 0.24 0.33 0.51 0.09 −0.25 −0.32 −0.51 0.19 0.38 −0.47

Plant biomass (t/ha)

Plant sp. richness (number of species)

−0.02 0.24

0.66 0.33 −0.50

−0.50 0.30 0.51 0.44 0.32 −0.36 0.35 0.32 −0.21

0.47 0.13 −0.39 −0.39 −0.19 0.20 −0.04 −0.23


the changes in nitrogen and carbon use efficiencies in the terrestrial microbial communities in order to maintain microbial elemental homeostasis [37]. We could see in our study that each unlimed plot where NH4+ was added had low pH and elevated C/N ratio (Supplementary Material, Table S1), while NO3− amended plots (unlimed and limed) did not show soil acidification and hence any increase of C/N ratio. Recently, microbial functional traits were shown to predict biogeographical patterns of microorganisms [38]. PCA analysis of the Park Grass community revealed a clustering of the community based on the ecological functions of microorganisms in this soil (Fig. 4, Table S4). Genera that are closely correlated with ammonia are known for participation in the soil nitrogen cycle. Animal-associated microbiota (Ruminococcus, Eubacterium, Bacteroides, Lactobacillus, Akkermansia, Clostridium) clustered together and did not show any significant correlation with examined soil parameters. This group could have appeared in the soil because the Park Grass area was in a permanent pasture before the experiment was established [11]. Sheep last grazed the plots in 1875, and this could be an example of how a microbial profile can retain the land’s history of use for centuries. Alternatively, rabbits (although limited due to rabbit proof fencing) and other small mammals, birds, and soil-dwelling fauna are ubiquitous potential sources of animal-associated microorganisms.

the microbial diversity in the Cedar Creek soil, and they suggested that N/C availability might play an important role in shifting community composition. However, Campbell et al. [9] reported that the microbial diversity of acidic tundra soils decreases under nitrogen input. Fertilizer input changes other soil parameters, and in many cases, it causes soil acidification [12]. In the PGE, some plots with the same treatment were limed to maintain neutral pH levels, while others acidified. Acidified plots within NH4+ treatments showed decreased diversity of microorganisms (Fig. 5). This leads to the conclusion that pH has an important impact on microbial diversity, and addition of fertilizers in the form of ammonium significantly decreases soil pH. Our findings agreed with other studies, where soil pH was a major factor in defining microbial diversity [4, 5] through impacting pH homeostasis of microbial cell or regulating availability of soil nutrients. Another soil parameter that significantly correlated with diversity was the C/N ratio (Table 4). While on a continental scale, the C/N ratio has not been found to play significant role in microbial diversity [4], we found a high interrelation between microbial diversity and the C/N ratio in the PGE. Hence, the C/N ratio may have a stronger effect on a local scale. The Relationship Between Microbial Biomass, Microbial Diversity, Plant Species Richness, and Plant Productivity

Microbial Diversity and Soil Parameters Microbial diversity did not respond to either phosphorous or the nitrogen-containing treatments (Fig. 6, Table S5). In other studies using pyrosequencing, similar results have been reported. For instance, low, medium, and high nitrogen inputs at the Kellogg Biological Station and Cedar Creek did not change phylotype diversity at these sites [26]. Ramirez et al. [25] showed that nitrogen additions did not directly change

Although a number of studies have examined relationships between microorganisms in soils and plants, it is still unclear whether bacterial diversity and biomass are linked to plant diversity, species richness, or plant productivity [10]. Microbial biomass decreased by the addition of nitrogen as ammonium sulfate, but not nitrate (Fig. 6). Recent studies have demonstrated that nitrogen fertilization decreases microbial biomass, estimated by PLFA, and by substrate-induced


respiration (SIR) methods [14, 30]. In our study, strong positive correlations between soil pH and plant species richness, and a negative correlation with C/N ratio, suggest that these parameters may have an impact on microbial biomass. As described above, soil microbial C/N/P ratios are constant [36] and significantly depend on soil C/N/P stoichiometry [37]. The positive correlation between microbial biomass and plant species richness was similar to the relationships found in other studies [39]. Microbial diversity and plant species richness were not related. Similar findings were shown in other studies. Fierer and Jackson [4] did not find any clear relationships between plant diversity and soil bacterial diversity at the continental scale. Xue et al. [40] has examined the contribution of soil parameters and vegetation in shaping the soil microbial composition; and crop variables explained only 11.9 % of the variation in the microbial communities, while soil parameters contributed 53.3 % to the variation. Another study found that the rhizosphere bacterial community composition largely depends on the bacterial community composition of the bulk soil rather than on plant composition [41]. This study suggested that plant community composition does not affect microbial community due to functional redundancy of microbial groups, in which it is common among a majority of microorganisms to utilize common root exudates. Moreover, Kowalchuk et al. [42] demonstrated lower microbial diversity in the rhizosphere compared to the bulk soil community, suggesting plant rhizosphere selection of specific microbial populations. Nitrogen amendments did not significantly affect microbial diversity, but they depressed plant species richness. These results add to the growing amount of evidence that plant diversity is more sensitive to the nitrogen treatments than microbial diversity [26]. It was previously shown that plant species richness in the PGE is the highest in the plots without nutrient input, and it is the lowest in the plots amended with ammonium sulfate. The main factor contributing to the changes in the plant diversities was determined to be pH [12]. When ammonium sulfate is added to soil, pH decreases, which causes a decrease in the number of plant species because most lack tolerance to acidic pH [12], while microorganisms have developed a variety of different adaptations to survive acidic pH [33, 34].

Conclusions Simultaneous analysis of the microbial communities at the PGE with nutrient treatments added to the plots over a hundred year time period and long-equilibrated soil parameters suggest that pH was the strongest predictor for microbial community composition, diversity, and microbial biomass in the PGE soil. The most likely effect that pH has on microbial

community structure is modulating the nutrient availability in soil. Future work is required to investigate whether relationships in other ecosystems between plant, microbial diversities, productivity, and fertilization will be similar to what we found in the grassland ecosystem. In addition, the environmental features that indicated a strong correlation to taxa relative abundances can be used to build statistical models to predict composition of microbial communities in soil. Acknowledgments This work was supported by the National Science Foundation (grant number MCB-0454030); and the US Department of Agriculture (grant numbers 2005-35319-16300, 00067345). Rothamsted Research receives strategic funding from the UK Biotechnology and Biological Sciences Research Council for research and support for the Long-Term Experiments National Capability.

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