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/(odnośnik otworzy się w nowym oknie)) 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 results
Pilot-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.
