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Journal of Microbiological Methods
Vol. 125, 2016, Pages: 91–97

Novel oligonucleotide primers reveal a high diversity of microbes which drive phosphorous turnover in soil

Fabian Bergkemper, Susanne Kublik, Friederike Lang, Jaane Krüger, Gisle Vestergaard, Michael Schloter, Stefanie Schulz

Research Unit Environmental Genomics, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.

Abstract

Phosphorus (P) is of central importance for cellular life but likewise a limiting macronutrient in numerous environments. Certainly microorganisms have proven their ability to increase the phosphorus bioavailability by mineralization of organic-P and solubilization of inorganic-P. On the other hand they efficiently take up P and compete with other biota for phosphorus. However the actual microbial community that is associated to the turnover of this crucial macronutrient in different ecosystems remains largely anonymous especially taking effects of seasonality and spatial heterogeneity into account. In this study seven oligonucleotide primers are presented which target genes coding for microbial acid and alkaline phosphatases (phoNphoD), phytases (appA), phosphonatases (phnX) as well as the quinoprotein glucose dehydrogenase (gcd) and different P transporters (pitApstS). Illumina amplicon sequencing of soil genomic DNA underlined the high rate of primer specificity towards the respective target gene which usually ranged between 98% and 100% (phoN: 87%). As expected the primers amplified genes from a broad diversity of distinct microorganisms. Using DNA from a beech dominated forest soil, the highest microbial diversity was detected for the alkaline phosphatase (phoD) gene which was amplified from 15 distinct phyla respectively 81 families. Noteworthy the primers also allowed amplification of phoD from 6 fungal orders. The genes coding for acid phosphatase (phoN) and the quinoprotein glucose dehydrogenase (gcd) were amplified from 20 respectively 17 different microbial orders. In comparison the phytase and phosphonatase (appAphnX) primers covered 13 bacterial orders from 2 different phyla respectively. Although the amplified microbial diversity was apparently limited both primers reliably detected all orders that contributed to the P turnover in the investigated soil as revealed by a previous metagenomic approach. Genes that code for microbial P transporter (pitApstS) were amplified from 13 respectively 9 distinct microbial orders. Accordingly the introduced primers represent a valuable tool for further analysis of the microbial community involved in the turnover of phosphorus in soils but most likely also in other environments.

Keywords: Phosphorus turnover; Forest soil; phoD; phoN; appA; pitA; pstS.

 
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