Comparisons of diazotrophic communities in native and agricultural desert ecosystems reveal plants as important drivers in diversity

FEMS Microbiology Ecology, 92, 2016, fiv166 doi: 10.1093/femsec/fiv166 Advance Access Publication Date: 24 December 2015 Research Article RESEARCH ARTICLE Martina Köberl1,∗,† , Armin Erlacher1 , Elshahat M. Ramadan2,3 , Tarek F. El-Arabi2,3 , Henry Müller1 , Anastasia Bragina1 and Gabriele Berg1 1 Institute of Environmental Biotechnology, Graz University of Technology, 8010 Graz, Austria, 2 Faculty of Agriculture, Ain Shams University, 11566 Cairo, Egypt and 3 Biotechnology Laboratory, Heliopolis University, 11777 Cairo, Egypt ∗ Corresponding author: Petersgasse 12/I, 8010 Graz, Austria. Tel: +43 316 873-8423; Fax: +43 316 873-8819; E-mail: Present address: Pacific Northwest National Laboratory, Biological Sciences Division, Richland, WA 99354, USA. Tel: +1 509 371-6925 One sentence summary: The diazotrophic microbiome of desert ecosystems is characterized by a high diversity and abundance and specific for each plant rhizosphere. Editor: Angela Sessitsch † ABSTRACT Diazotrophs provide the only biological source of fixed atmospheric nitrogen in the biosphere. Although they are the key player for plant-available nitrogen, less is known about their diversity and potential importance in arid ecosystems. We investigated the nitrogenase gene diversity in native and agricultural desert soil as well as within root-associated microbiota of medicinal plants grown in Egypt through the combination of nifH-specific qPCR, fingerprints, amplicon pyrosequencing and fluorescence in situ hybridization–confocal laser scanning microscopy. Although the diazotrophic microbiota were characterized by generally high abundances and diversity, statistically significant differences were found between both soils, the different microhabitats, and between the investigated plants (Matricaria chamomilla L., Calendula officinalis L. and Solanum distichum Schumach. and Thonn.). We observed a considerable community shift from desert to agriculturally used soil that demonstrated a higher abundance and diversity in the agro-ecosystem. The endorhiza was characterized by lower abundances and only a subset of species when compared to the rhizosphere. While the microbiomes of the Asteraceae were similar and dominated by potential root-nodulating rhizobia acquired primarily from soil, the perennial S. distichum generally formed associations with free-living nitrogen fixers. These results underline the importance of diazotrophs in desert ecosystems and additionally identify plants as important drivers in functional gene pool diversity. Keywords: desert farming; diazotrophs; medicinal plants; nitrogen-fixing communities; organic agriculture; Rhizobiales INTRODUCTION Nitrogen is one of the most yield-limiting factors in agricultural production systems throughout the world and an essential macronutrient for plants. Nitrogen-fixing microorganisms provide the only natural source of fixed atmospheric nitrogen in the biosphere (Gaby and Buckley 2012), and the capability for nitrogen fixation is widely dispersed among prokaryotic taxa including very divergent, distantly related bacteria and archaea (Zehr and Turner 2001; Zehr et al. 2003). Biological nitrogen fixation by diazotrophic bacteria together with the input of recycled organic waste, such as manure or compost, is considered a sustainable Received: 17 August 2015; Accepted: 17 December 2015  C FEMS 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Comparisons of diazotrophic communities in native and agricultural desert ecosystems reveal plants as important drivers in diversity 2 FEMS Microbiology Ecology, 2016, Vol. 92, No. 2 microscopy (FISH–CLSM) analyses targeting the most dominant diazotrophic taxa were performed in order to reveal their habitat preferences and colonization type. MATERIALS AND METHODS Experimental design and sampling Nitrogen-fixing communities were studied at the organically managed Sekem farm Adleya (www.sekem.com) located in the north-eastern desert region of Egypt near Bilbeis (30◦ 13 44 N, 31◦ 23 39 E) at Sharqia governorate. Physicochemical data of the soil is provided in Luske and van der Kamp (2009). The diazotrophic community of bulk agricultural soil was also investigated in comparison to the community of native desert soil from the Sinai Peninsula, Egypt (30◦ 21 00 N, 32◦ 15 18 E). At each site, four composite samples of soil in a horizon of 10–30 cm depth were collected. Profiles of the nifH gene in communities associated with the rhizosphere and endorhiza of three different species of medicinal plants (M. chamomilla L., C. officinalis L., and S. distichum Schumach. and Thonn.) cultivated on Adleya farm were studied and compared. From each plant species, four independent composite samples consisting of 5–10 plant roots with adhering soil were taken. The detailed sampling strategy is described by Köberl et al. (2011). To isolate total community DNA from the soil and rhizosphere, 5 g of soil or roots with adhering soil were added to 45 mL of sterile 0.85% NaCl and vortexed. For isolation from the endorhiza, 5 g of roots were surface-sterilized with 4% NaOCl for 5 min. The roots were washed three times with sterile distilled water, then 10 mL sterile 0.85% NaCl were added and further homogenized using mortar and pestle. For isolation of total DNA from the rhizosphere, endorhiza and soil, 4 mL of the suspensions were centrifuged (16,000 × g, 4◦ C) for 20 min and the resulting microbial pellets were stored at –70◦ C. In the desert soil, a lower concentration of DNA was expected. Therefore, the pellets of 10 mL suspension were used for the isolation of total DNA. Total community DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA). Quantification of microbial nifH genes by qPCR To determine nifH gene abundances, quantitative PCRs were performed according to Hai et al. (2009) with following modifications. Reactions were conducted in a total volume of 10 μL containing 1× KAPA SYBR FAST qPCR MasterMix Universal (PEQLAB, Polling, Austria), 0.6 mg mL−1 BSA, 0.125 μM of primers nifH-F and nifH-R (Rösch, Mergel and Bothe 2002), and 0.8 μL template DNA dilutions with a concentration of ∼1 ng μL−1 (95◦ C, 10 min; 39 cycles of 95◦ C, 45 s; 55◦ C, 45 s; 72◦ C, 45 s; and melt from 72 to 95◦ C). Rotor-Gene 6000 real-time rotary analyzer (Corbett Research, Sydney, Australia) was used for fluorescence quantification. For absolute quantification, the PCR amplified nifH gene fragment from Pectobacterium atrosepticum SCRI1043 was ligated into the pGEM-T Easy Vector (Promega, Mannheim, Germany) and transformed into Escherichia coli DH5α. Serial dilutions of PCR fragments generated with the vector-specific primers USP and RSP (Köberl et al. 2011) were u (...truncated)