Root-associated fungi within a semi-natural dry grassland. MallnowITS

The root-associated fungal community of Festuca brevipila was surveyed within a semi-natural dry grassland located in a natural reserve in Mallnow, eastern Germany (Mallnow Lebus, Brandenburg, Germany, 52°27.7780 N, 14°29.3490 E). The region is influenced by sub-continental climate with a mean annual precipitation of below 500mm. The area is managed by sheep grazing twice a year. The plant community consists of Sileno otitae-Festucetum-brevipilae and Festuco psammophilae-Koelerietum glaucae. Festuca brevipila R. Tracey is the most abundant species at the sampling site (coverage >60% and in some cases >80%) as well as throughout the grassland of the study area. The sampling design consists of a hierarchical nesting of macroplots and plots, with three replicated macroplots of 15 x 15, 12 x 12 and 12 x 12 m in extent, located on the slopes of hills in an area of about 5 ha. Each macroplot was divided into 3 x 3 m plots, at each corner of the macroplots. In each plot, soil cores (5cm radius, 15 cm deep) centered on 5 randomly chosen F. brevipila individuals per plot were excavated, resulting in 60 (3 macroplots x 4 plots x 5 plants) samples in total. The sampling was done at the end of June 2011. Roots were washed and root DNA was extracted from 0.25 g using the PowerSoil DNA isolation kit. The soil fungal community was analyzed with 300 bp paired-end Illumina MiSeq sequencing of ITS2 sequences (primers fITS7: 5′‐GTGARTCATCGAATCTTTG‐3′ / ITS4: 5′‐TCCTCCGCTTATTGATATGC‐3′). Sequences were processed using usearch and 97% sequence similarity OTUs were produced using UPARSE. Taxonomic annotations were performed using the Naive Bayesian Classifier against UNITE (version of 04.02.2020). Non-fungal OTUs were removed keeping 914 OTUs with an annotation at least at the phylum level at a 80% bootstrap support for subsequent analyses. OTUs with unaccepted taxonomy at high ranks were however deposited as “uncultured soil fungus”.