Abstract

1. Class Figure 1 Order Figure 3 Figure 6 Order Figure 5 Figures 5, 6 and 7 show differences in the populations of the eukaryotes isolated from the poplar rhizosphere under ambient and elevated CO2. Figure 7 Figure 2 Figure 4 N-235Population Dynamics in the Rhizosphere: How Elevated CO2 Transforms Microbial Community Composition C. Lesaulnier 1,3*, D. Papamichail 2, S. McCorkle 1, D. Zak 4, B. Ollivier 3, S. Skiena 2, S. Taghavi 1, D. van der Lelie 1 1Brookhaven National Laboratory, Upton, NY, USA. 2Stony Brook University, Stony Brook, NY, USA. 3Institut de Recherche pour le Developpement, Marseille, FRANCE. Abstract Results No significant variation in community composition among the delta-Proteobacteria, which comprises 10% of the prokaryotic community, was found under elevated CO2. This is not surprising given that they have previously been reported to tolerate microaerophillic conditions. Verrucomicrobia - This population has been suggested to be negatively impacted by soil moisture which has been reported to increase under elevated CO2 and therefore it is not surprising that we note a decrease in this aerobic heterotrophic population. Prokaryotic Community Scientists from all disciplines are collaborating to address the issues of anticipating the effects of anthropogenic activity and environmental change, for the purpose of predictive ecological modeling. Though no globally accepted model exists on plant responses to atmospheric carbon dioxide (CO2), little work has been done to characterize the forest flora rhizosphere to better determine their overall potential to counterbalance the effects of this greenhouse gas. Our findings show that though total soil biomass remained unchanged for prokaryotes and eukaryotes, microbial species richness decreased and noticeable shifts in dominant genera were observed. Archaea populations were noted to decrease under elevated CO2 and we hypothesize that oxidative stress due to increased fine root production and improved rhizosphere aeration inhibits their growth. This was further supported by similar observations in strictly anaerobic bacterial populations. Our data, combined with previously documented microbial activities, provides the foundation to better interpret observed changes in plant biomass, element cycling and the sustainability of terrestrial ecosystem productivity under elevated CO2. Eukaryotic Community Table 6 – Archaea representatives and their respective importance in each library Conclusion Objectives Figure 8 – Hypothesis on how cycles are affected by elevated CO2. Carbon cycle Though total reported fungal community biomass remained constant, as determined by qPCR, significant populations changes were observed. The most significant being a shift towards the Agaricales ectomycorrhizal fungi, predominantly belonging to the genus Inocybe. These are commonly associated with fast developing plant root systems and characteristic for poplar growing under conditions of elevated CO2. Basidomycete - The Basidomycete population importance remained stable; however a decrease in overall population diversity was observed especially among the Homobasidomycetes and included thedisappearance of Heterobasidomycetes, the Tremellomycetidae family most importantly, under elevated CO2. It has been reported that stimulated plant growth results in the inhibition of fungal plant pathogens. We note the disappearance of the Urediniomycetes under elevated CO2 which is very interesting given that they are predominantly represented and comprised of plant pathogens. Zygomycetes - Representatives of this phylum were observed to decrease in population importance, the most significant change occurring to representatives affiliated with Mortierella. This population was observed to decrease by 75%. Ascomycete – Though the Ascomycete population importance remained stable (i.e. number of reported clones stayed the same), we notice the disappearance of the dominant species Cazia under ambient conditions and the incurring dominance of representatives belonging to Pachyphloeus under conditions of elevated CO2. Chytridiomycetes - Representatives belonging to Chytridiomycetes appear to have been unaffected by the increase in CO2. In an attempt to better understand the importance and impact plant associated microbes play in this role, we aimed to provide the first large scale detailed analysis on microbial population changes due to elevated CO2 in all three domains of life present in the rhizosphere of Populus tremuloides. Actinobacteria - The most significant changes included their doubling, with Arthrobacter representatives increasing 15 fold (P <0.0001). These gram-positive chemoheterotrophs have been demonstrated to play a role in the degradation of many recalcitrant forms of soil carbon and are noted to play an important role in nitrification. A 50% decrease in the Streptomyces population was observed and representatives within this group are known to have denitrifying characteristics. A marked increase of 30% in Sporichthya was observed, though as yet we do not know what this could be due to. Acidobacteria - Members of the phylum Acidobacteria halved in population importance. The physiological properties of these organisms are unclear, though this observation is inconsistent with recently published results under conditions of elevated CO2. Bacteroidetes - A 30% increase in Chitinophaga representatives was observed and these Sphingobacteria have known chitinolytic properties. A 40% increase in Flavobacterium was also observed though we do not yet know why this could be. Firmicutes - Our results for Bacillus are surprising given that their metabolic capabilities are diverse and include the degradation of almost all substrates derived from plant sources. Their capacity to carry out nitrification, nitrogen fixation and denitrification has also been noted. Results for Clostridia are due to a significant decrease in representatives clustering with Desulfotomaculum species. These obligate anaerobes seem to be inhibited under elevated CO2. Obligate organisms may cease to function competitively as their environment segues to an incompatible redox state. Furthermore, they may quickly drop out of a soil community due to the effects of O2 toxicity as the environment is forced to become aerobic. Though this result is preliminary, its importance due to the effects of elevated CO2 has, to date, been overlooked. Proteobacteria - The importance of Proteobacteria is consistent with other soil studies whereby they have been noted to make up approximately 40% of libraries derived from soil bacterial communities. Total soil microbial biomass has been noted to be unaffected by elevated atmospheric CO2. These results are confirmed by our study; however, we do observe significant population rearrangements within the lower taxonomic levels. Any conclusions on the metabolic capabilities within this phylum are hindered by its extent of physiological diversity. Interestingly we noted no change in the populations of Beta-Proteobacteria involved in nitrification, predominantly among the Nitrosospira. ↑ CO2 ↓ denitrifying bacteria ↑ respiration N2 plants ↑ photosynthesis ≈ N fixing bacteria NO3 ↑ plant biomass ≈ nitrate bacteria Methods ↑ soil oxygenation NO2 ≈ nitrite bacteria ↑ fine root turnover • Anaerobic populations • Archaea • Clostridia • Desulfotomaculum ↑ in decomposers NH4+ A total of 3000 prokaryote, 3000 archaea 16S and 2000 18S ribosomal rRNA sequences were obtained from soil extracted DNA of the poplar rhizosphere, split equally in number from under both ambient and elevated (550ppm) atmospheric CO2 concentrations. These were then taxonomically assigned using an optimized blast classifier built from a curated data set and comparisons were then made between the two populations. Nitrogen cycle This is the first detailed analysis on how the rhizosphere microbial community of Populus tremuloides is affected by elevated CO2. Due to the observed changes in the poplar microbial community composition we postulate that that rhizosphere conditions under elevated CO2 alters the interaction between and the microorganisms in its rhizosphere. The microbial community adapts so as to sustain uptake of nitrogen and other microelements in favor of the plant. This increased soil oxygenation hinders anaerobic processes and results in the out competition of denitrifiers and the abundance of ectomychorrizal fungi. Though this may further assist the host plant in the uptake of essential nutrients such as nitrogen and phosphorus, the potential exists to significantly alter S cycling in the rhizosphere. Our results suggest that the increase in fine root biomass production and turnover under elevated CO2 favorizes the symbiotic relationship between poplar and ectomychorrizal fungi, resulting in the out-competing of saprophytic and other ectomychorrizal fungi. The effect of oxidative stress was further addressed in the archaea population where a large decrease in population importance under elevated CO2 was observed. How much is enough? Table 1 – DIMITRIS Archaea Community Upon examination of the 1500 cloned representatives belonging to each library we determined that both populations were predominantly comprised of uncultivated non-thermophillic Crenarchaeota. Non-thermophillic members of the archaeal domain Crenarchaeota have previously been documented in soils with some specifically associated with plant roots, suggesting that these organisms might play a significant role in the ecology of the rhizosphere. The difference in population importance between the two archaeal populations, as determined by qPCR, was surprising with representatives clustering within this domain decreasing significantly under elevated CO2 . Though it is not possible at present to extrapolate potential roles for these organisms, we notice significant rearrangements in the importance of represented species. Acknowledgements Table 2 – Quantification of domain specific soil DNA concentrations under ambient and elevated CO2. This work was supported by the US DOE, Office of Science, BER, project entitled New Genomic Strategies and Technologies and under Laboratory Directed Research and Development project number LDRD04-060. We would especially like to thank the JGI for all of their sequencing and Donald Zak at The University of Michigan for providing us with the rhizosphere soil samples. * lesaulnier@bnl.gov