View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Edinburgh Research Explorer Edinburgh Research Explorer Evaluation of the microbiome of decaying alder nodules by next generation sequencing Citation for published version: McEwan, NR, Wilkinson, T, Girdwood, SE, Snelling, TJ, Collins, T, Dougal, K, Jones, DL & Godbold, DL 2017, 'Evaluation of the microbiome of decaying alder nodules by next generation sequencing' Endocytobiosis and Cell Research, vol 28, no. 1, pp. 14-19. Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Endocytobiosis and Cell Research General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 30. Jun. 2018 Endocytobiosis and Cell Research (2017) VOL 28 | Issue 1 | 14–19 | International Society of Endocytobiology Endocytobiosis and zs.thulb.uni-jena.de/content/main/journals/ecb/info.xml Cell Research Evaluation of the microbiome of decaying alder nodules by next generation sequencing Neil R. McEwan1,4*, Toby Wilkinson1, Susan Introduction E. Girdwood1, Tim J. Snelling1, Tatiana Collins1, Kirsty Dougal1, David L. Jones2 and Members of the genus Frankia are actinomycetes which fix Douglas L. Godbold3 atmospheric nitrogen in symbiotic association with the 1Aberystwyth University, Institute of Biological, roots of a range of trees and woody shrubs, including alders. Environmental and Rural Sciences, Aberystwyth, SY23 3DD, In this partnership Frankia acts as the microsymbiont, with Wales, United Kingdom; 2Bangor University, School of the host plants acting as the macrosymbiont. As a result of Environment, Natural Resources and Geography, Bangor, this relationship the trees show an improved growth due to LL57 2UW, Wales, United Kingdom; 3University of Natural the provision of fixed nitrogen. In addition, other organisms Resources and Life Sciences, Institute of Forest Ecology, also present in the immediate environment may also gain Peter-Jordan-Straße 82, A–1190 Vienna, Austria; 4Robert benefits in terms of access to nitrogenous compounds, which Gordon University, School of Pharmacy and Life Sciences, is the reason that underpins the use of alder trees to help Garthdee Campus, Aberdeen, AB10 7GJ, Scotland, United facilitate growth of other trees in forestry practices. By way Kingdom; *correspondence to: [email protected] of illustration, many species of alders (e.g. Alnus glutinosa, A. rubra) have been planted in forestry plots to facilitate growth of the trees planted with them (Wheeler and Miller This work investigated the microbial content of 1990) due to fixation of atmospheric nitrogen taking place decaying nodules from alders. The 16S rDNA in their root systems. composition of the microbiome of six senescent alder Mature alder trees will commonly have several nodules nodules was investigated by 454 sequencing. All associated with their root systems, and it is known that nodules still had some Frankia sequences present, but actinorhizal plants can have more than one strain of Frankia in each case it was only detected at minor levels, with in the various nodules around their roots (Dobritsa and other organisms predominating. Although organisms Stupar 1989). However the level of diversity within an alder from three different phyla (Bacteroidetes, single nodule is low (Kennedy et al. 2010), with each nodule Proteobacteria and Actinobacteria) constituted almost containing one predominant strain (Dai et al. 2004; McEwan all (98% or more) of all sequences, Bacteroidetes were et al. 2015). most abundant in four nodules with Proteobacteria There has been considerable research carried out into being most abundant in the other two. In addition a few the nodulation process (e.g. Berry et al. 1986; Van Ghelue et families were represented at a level of 10% or more of al. 1997) and also the events within the nodule during the the total sequences: Sphingobacteriaceae (all 6 process of nitrogen fixation (e.g. Vikman et al. 1990) nodules); Chitinophagaceae (5 of 6); non-Frankia including analysis of genes involved in nitrogen-fixation (e.g. Actinomycetales (2 of 6); Caulobacteraceae (2 of 6); Normand et al. 1988). However, although there has been Flavobacteriaceae (2 of 6); Oxalobacteraceae (1 of 6); some work done to examine what happens after nodule and Xanthomoadaceae (1 of 6). Analysis at the genus senescence, the level of information is relatively limited. To level showed a diverse range of organisms, with date, it is known that a loss of nitrogenase activity in the members of the genus Pedobacter being found at an microsymbiont is one of the first signs of nodule senescence abundant level within most nodules. in both alders (Vikman et al. 1990) and legumes (Swaraj et al. 1993). In terms of the macrosymbionts it has been shown that the alder protein ag13 may act as a plant marker for Endocytobiosis and Cell Research (2017) 28(1):14–19 senescence (Guan et al. 1997) and that in the case of Category: Research article soybeans cystatins increase during the onset of senescence Keywords: Alnus glutinosa, microbiome, Frankia, senescent (van Wyk et al. 2014). nodules, 454 sequencing However there has been no work done to investigate the bacterial species which invade a senescing alder nodule, and Accepted: May 18, 2017 presumably obtain the benefits of nutrients from a niche rich Published online: June 02, 2017 in fixed nitrogen. The work presented here is the first Endocytobiosis and Cell Research VOL 28 | Issue 1 | 2017 14 Microbial diversity in senescent alder nodules, McEwan NR et al. application of next generation sequencing to investigate the (McEwan and Wheeler 1995; McEwan et al. 2015) with the microbiome of the bacterial community within senescing 95°C incubation rupturing even relatively robust organisms. nodules from A. glutinosa roots by analysing amplicons The following PCR conditions were performed in keeping produced from PCR using 16S rDNA primers. with the guidelines of the manufacturer (Roche) for FastStart PCR for 454 sequencing of 16S rDNA amplicons: 95°C, 2 min followed by 30 cycles of 95°C for 30 s, 55°C for Materials and Methods 30 s and 72°C for 2 min and a final extension of 72°C for 7 min. Primers used were barcoded prokaryote primers Collection of nodules (Caporaso et al. 2010) and were used to target the V1-V3 region of the bacterial 16S rRNA genes. One primer was used Root nodules were collected from A. glutinosa plants which as the forward primer in all reactions had been growing at the Henfaes Experimental Station, (CCTATCCCCTGTGTGCCTTGGCAGTCTCAGAGAGTTTGATCM Abergwyngregyn, Gwynedd, Wales (53°14’N, 4°01’W) for 3 TGGCTCAG), corresponding to Escherichia coli position 27. years. The nodules were harvested from approximately 5 cm Details of the reverse primers used are contained in Table 1. below the soil surface and only those already showing early By using a different reverse primer for each of the six signs of senescence were used for the current analysis. samples allowed identification of the source nodule. In all Following collection, nodules were stored in a freezer at - cases these corresponded to E. coli position 357. A final 80°C until ready for DNA analysis. primer concentration of 400 nM was used with a reaction volume of 25 μl. After PCR was completed, amplicons were checked by electrophoresis on a 1% TAE agarose gel to Performing PCR on nodule material verify successful amplification and that amplicons were around the approximate size predicted when using these Thawed nodules were surface-washed to remove the primers (i.e. 300 bp). nodule’s surface-associated bacteria, taking care to avoid Sample quantification was carried out using the Quant- damaging the internal regions of the nodular structure. The iT™ PicoGreen® dsDNA reagent (Invitrogen) and a CFX 96™ periderm was peeled to remove the outer layer from six real-time system (Bio-rad) to measure relative fluorescence. nodules, each from a different tree. An individual lobe from Concentrations were calculated from a standard curve and each nodule was used for extraction of DNA and subsequent reactions were normalized and pooled. Samples were analysis. normalized by mixing the six PCR products in equimolar Lobes were placed in sterile molecular-grade water and concentrations. All replicates within the mix went through a then ground in microfuge tubes with mini-pestles, followed final concentration measurement and samples were stored by a 15 min incubation at 95°C and centrifugation at 13000 at -20ºC until ready for 454 sequencing. Unincorporated g for 10 min. Supernatants were removed and 1 µl of it was dNTPs and primer dimers were removed using Agencourt® added directly to a FastStart high fidelity PCR system master AMPure® XP beads (Beckman Coulter). Following this step, mix. This approach of performing PCR directly on extracted the pooled and purified libraries were re-quantified and material