Characterization of intact polar lipids in soils for assessing their origin

Soil and root samples were taken at the Jena Experiment, a large grassland biodiversity experiment located in the Saale valley near Jena (east Thuringia, Germany, 50°55'N, 11°35'E, 130 m above sea level). In 2002, the experiment was established with a total number of 81 grassland plots of 20 × 20 m (Roscher et al., 2004). The soil type is Eutric Fluvisol and the soil texture changes from sandy loam to silty clay with increasing distance to the Saale river (FAO-Unesco, 1997; Fischer et al., 2014). In June 2016, three surface soil samples (0-10 cm) from each plot were collected, combined to reduce the spatial heterogeneity, and homogenized. The soil samples were sieved (2 mm mesh size). Fine roots (if present) were picked using steel tweezers and stored at −20 ℃. The root samples were taken separately from six plots with different combinations of four functional groups (grass, legume, tall herb, small herb; Table S1). The roots were washed, freeze-dried and frozen. All fungal strains used originate from Jena Microbial Resource Collection (JRMC), University of Jena and HKI, Germany. The saprotrophic fungi Schizophyllum commune FSU:3214xFSU:2896 and Mucor plumbeus JMRC:SF:013709 were cultivated in Petri dishes on solid complex yeast medium (CYM; Schwalb and Miles, 1967) and the mycorrhizal fungi Tricholoma vaccinum JMRC:FSU:4731 and Pisolithus tinctorius FSU:10019 on modified Melin Norkrans b (MMNb) medium (Kottke et al., 1987) at room temperature for 2 and 5 days for the fast growing M. plumbeus and S. commune and 2 and 3 weeks for the slow growing P. tinctorius and T. vaccinum (Table S1). Further six bacterial strains were chosen for this study: Streptomyces acidiscabies E13 (JMRC:ST:033552 from JRMC), Streptomyces mirabilis P16B-1 (Schmidt et al., 2009), Bacillus subtilis DSM-10, Agrobacterium tumefaciens DSM-30150, Pseudomonas fluorescens DSM-50090 (DSMZ, Braunschweig, Germany), and Acetobacter xylinum NQ5 (ATCC 53582; ATCC, USA European Office at Wesel, Germany). The strains were cultivated for 2 to 5 days in Petri dishes on minimal medium (MM; Schmidt et al. 2009) at 28 °C. The collembolans species Heteromurus nitidus (Templeton, 1835) and Folsomia candida Willem, 1902 were taken from laboratory cultures fed with baker's yeast (Saccharomyces cerevisiae; Table S1). Laboratory cultures were maintained in glass jars filled with moist potting soil at 15 °C in darkness and kept moist with distilled water. Before analysis, collembolans were starved for three days to empty their guts; subsequently they were frozen and stored in methanol. Polysphondylium pallidum strain was from the Stallforth Lab at Leibniz Institute for Natural Product Research and Infection Biology in Jena (Germany). Amoebae were cultured (xenically) in the presence of the bacterium Klebsiella aerogenes as food. Briefly, amoebal spore suspension (from previously collected sori) was added to the surface of SM/5 agar plate seeded with 1 X 108 CFU/ml food bacterium K. aerogenes. Plates were incubated at 22°C for 7 to 10 days for mature fruiting bodies to appear. The entire cell mass of amoebal fruiting bodies was carefully collected using a sterile inoculation loop and suspended in KK2 buffer. This cell mass was washed clean of any attached bacteria using the same buffer. Resulting amoebal cells were then subjected to further analysis. Before analyses, roots, fungi, collembolans and amoebae were frozen in liquid nitrogen. They were ground into fine powder and extracted using the same protocol as for the soil samples. Cultured bacteria were collected from Petri dish plates, weighed and extracted using the same protocol.