Periodic Reporting for period 1 - NITRIPHYLL (A novel approach to determine canopy nitrification in the phyllosphere of European forests: combining multiple isotope tracers and proteogenomic techniques) Reporting period: 2016-06-06 to 2018-06-05 Summary of the context and overall objectives of the project Forest canopies play a significant role in regulating carbon and water exchanges with the atmosphere, with profound effects on our climate. However, their role in altering the chemical composition of precipitation and, consequently, the nutrient input to the soil has been less investigated. This is particular relevant for nitrogen (N)-limited forests in the Northern hemisphere, which have been exposed to a rapid human-induced increase in emissions of nitrogen compounds over the last century, due to increase in industrial activities, traffic and use of fertilizer in agriculture, to enhance food production and sustain the increase in growth population [1]. These reactive N compounds, however, do not remain in the atmosphere, but are deposited on terrestrial and aquatic ecosystems as N deposition, thus altering global N cycling. Significant differences for the nitrogen fluxes between precipitation in the open field (rainfall, RF) and below the forest canopies (throughfall, TF) have been observed at many forests [2]. This suggests that N deposition is substantially altered through its path into the canopy. How do these changes take place? By applying a multiple isotope approach in nitrate collected in RF and TF water we showed for the first time that biological nitrification (microbial conversion of ammonium/ammonia in nitrate) was responsible for the large changes in the amount of nitrate from RF to TF across two UK forests at high N deposition level [3]. Nevertheless, a number of studies have pointed out that tree canopies are habitat (i.e. phyllosphere) for incredible diverse microbial communities, with bacteria being the most abundant [4]. NITRIPHYLL aimed to prove that the microbial communities harbored in forest canopies carry out processes hitherto unrecognized for their significance, i.e. nitrification in the canopy, thereby helping cycling N before litter is returned to the soil. To achieve these objectives NITRIPHYLL for the first time merged two separate research avenues, i.e. the investigation of N transformation by forest canopies (through stable isotopes) with the study of abundance and diversity of microbes involved in nutrient cycling (through qPCR and NGS). [1] Galloway et al. (2004). Biogeochemistry 70: 153–226. [2] Ferretti et al. (2016). Global Change Biology 20: 3423–3438[3] Guerrieri et al. (2015). Global Change Biology 21: 4613-4626[4] Vacher et al. (2016). Annu. Rev. Ecol. Evol. Syst. 47:1-24 Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The three multidisciplinary objectives of NITRIPHYLL were:1) To use the Δ17O tracer to depict the occurrence of canopy nitrification along broader N deposition and climate gradients in EU.2) To characterize microbial communities harbored in tree canopies for two of the most dominant species in EU (beech and Scots pine) across the EU gradient and for one of the most important tree species in the Mediterranean area (holm oak). 3) To quantify Bacterial and Archaeal species responsible for nitrification in tree canopies across all sites.To achieve these objectives I considered forests within the European ICP forests network (http://icp-forests.net/) going from Fennoscandia to the Mediterranean area (Figure 1). An overview of samples and methods considered is provided in Figure 2. Across all the sites, foliar, filters (used to process RF and TF water, Figure 3) and soil samples were used to extract microbial DNA and then carry out meta-barcoding and qPCR analyses, to characterize bacterial communities and quantify functional genes related to nitrification, respectively (objective 2 and 3). Moreover, filtered RF and TF water samples were passed through anion resins (Figure 3) to trap nitrate and then measure oxygen and nitrogen isotope ratios by mass spectrometer to quantify the proportion of microbiologically vs. atmospheric derived nitrate (objective 1). NITRIPHYLL was successfully completed thanks to the tremendous support from collaborators i) at all the sites across EU (Figure 1) and ii) at the Servei de genomica (UAB), CEAB and CREAF.Overview of resultsPilot-study at LC. We found isotopic evidence of biologically derived nitrate in holm oak canopy only in August and September, after a significant drought, while atmospheric deposition was the dominant source of nitrate through October-December (objective 1). This seasonal partitioning between biologically and atmospherically derived nitrate in TF inferred from oxygen isotope data was also reflected in the temporal trend of Archaeal amoA gene copies, the functional gene that is involved in the nitrification (objective 3). Finally, we found that holm oak tree canopies host a highly diverse community of bacteria (objective 2). A paper including these results is currently in preparation and will be submitted by September 2018. Study at the EU sites. Preliminary results showed that the structure and composition of bacterial communities are different across the three sample types, i.e. phyllosphere, water and soil (objective 2) and within the phyllosphere, between Scots pine and beech. Nitrifying bacteria and Archaea were present across all the samples, including the phyllosphere (objective 3). Completing stable isotope analyses will allow us to test, as we did at LC, whether the two independent methods (stable isotopes and qPCR) agree in supporting both presence and activity of nitrifiers. Observations of bacteria on some of the samples were carried out in collaboration with colleagues at the Centre of Advanced Studies of Blanes (CEAB, Spain) (Figure 4).Results from NITRIPHYLL were i) presented at several international conferences/workshop/seminars in EU and in the USA (at some of them invited as speaker); ii) included in the ICP Forests 2017 Executive Report for policy makers and general public. Finally, several outreach activities were organized to foster participation of general public to science. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) NITRIPHYLL gained the attention of communities working at the interface between research and policy makers (ICP forests community). Moreover, the project has been received with great interest and support from scientific communities working in different fields (forest ecology, biogeochemistry, soil science, atmospheric chemistry, microbiology). There are still uncertainties in our understanding on the effects of N deposition on forest N cycling, e.g. where does atmospheric N go and how and where is it cycled within forests? NITRIPHYLL moved the discussion forward by proving that 1) tree canopies host a highly diverse community of bacteria, which includes species related to N cycling. Moreover, 2) atmospheric N is bio-transformed when interacting with tree canopies, as we found evidences of nitrification occurring at our sites, even under low level of N deposition. NITRIPHYLL has certainly increased the awareness of 1) N deposition (related to human activities) and its consequences on forests worldwide, and 2) the hidden biological diversity in tree canopies and its role in cycling N. Reducing the gap between general public and academic “world” is very important to develop a more scientifically literate and environmentally-conscious population, thus contributing to one of the priorities of the Horizon 2020. Last but not least, by adopting a combination of novel methodological approach, NITRIPHYLL has undoubtedly contributed to an important 2020 Horizon objective, i.e. strengthen the EU global position in research. First page Figure 3. Filters used to process TF water (left). Water passing through anion resins (right) Figure 1. Map showing the sites included in the study. Figure 4. Image obtained by epifluorescence microscopy showing bacteria (white dots) on filters Last page Figure 2. Overview of samples collected and methods used to accomplish the three goals of NITRIPHYLL
