The analytical methods were as previously described11 (link). To determine total soil C, N, and S contents, 2-g samples of homogenized soil from both bulk soil and tuberosphere were dried, milled, and analyzed using Vario MAX CNS analyzer (Elementar Analysensysteme, Hanau, Germany). To determine all other elements, soil subsamples were leached with boiling nitro-hydrochloric acid (aqua regia) and assessed by optical emission spectroscopy with inductively coupled plasma (ICP-OES) by Aquatest Inc. (Prague, Czech Republic). Analyses of potato periderm were performed by service laboratory of the Institute of Botany (Trebon, Czech Republic). For total nitrogen analysis, 1–3 mg dried periderm was mineralized by modified Kjeldahl method in H2SO4 with catalyzer at 360 °C. For total phosphorus, 20 mg of dried periderm was sequentially decomposed by HNO3 and HClO4. In mineralized samples, both N and P were determined by flow injection analysis with spectrophotometric detection using FIA Lachat QC 8500 analyzer (Lachat Instruments, Hach Company, Loveland, CO). Cation contents in periderm were determined by atomic absorption spectrometry using AAS spectrometer ContrAA 700 (Analytik Jena, Jena, Germany) after mineralization with nitro-hydrochloric acid.
Vario max cns analyzer
Manufactured by Elementar
Sourced in Germany
The Vario MAX CNS analyzer is a laboratory instrument designed for the determination of carbon, nitrogen, and sulfur content in a wide range of solid and liquid samples. It utilizes the combustion and detection principles to provide accurate and reliable elemental analysis.
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8 protocols using vario max cns analyzer
1
Soil and Potato Periderm Analysis
2
Plant Nutrient Uptake Analysis
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Total N in the roots and shoot biomass was separately determined by Elemental analysis (Variomax CNS analyzer, Elementar, Germany) as explained in Gebremikael et al.24 (link). N uptake was calculated as %N in the plant sample (shoot and root) multiplied by the total dry plant biomass collected at each sampling time and reported as mg N per tube.
Total P in the plant samples was extracted by a combination of ashing and wet digestion with 1N HNO362 . Total P concentration was determined according to the colorimetric Nitro-vanado-molybdate method of Hogue et al.65 using a Varian Cary 50 spectrophotometer at 425 nm. P uptake was also calculated in the same way as N uptake, but from the shoot biomass only, as the root biomass was not sufficient for P analysis.
Total P in the plant samples was extracted by a combination of ashing and wet digestion with 1N HNO362 . Total P concentration was determined according to the colorimetric Nitro-vanado-molybdate method of Hogue et al.65 using a Varian Cary 50 spectrophotometer at 425 nm. P uptake was also calculated in the same way as N uptake, but from the shoot biomass only, as the root biomass was not sufficient for P analysis.
3
Litter Decomposition Rates in Invaded Areas
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An in situ litterbag approach was used to test the litter decomposition rate of both focal species. At the end of the 2010 growing season, we hand collected newly produced litter from standing Eragrostis and Alternanthera at the Nanjing Agricultural University experimental station. Neither plant species is endangered or protected. All litter samples were air dried and carefully processed (cut into a size of litter length at 3∼6 cm) to fit into the litterbag. For each litter treatment, 10 g of litter was put into 10×15 cm litterbags (mesh size of 1 mm2) [29] , [30] . Single species bags received 10 g of a single litter type. For the mixed-species treatment, 5 g of each litter type was put in the bag. All litterbags were deployed simultaneously in invaded areas where both species co-existed in March 2011 and were retrieved after in situ decomposition for 3, 6 or 8 months. A subsample of litter of each species was oven dried for water, C and N measurement. Samples used for chemical properties measurement were ground to pass through a 2 mm sieve, decarbonized with HCl, and then analyzed for C and N content by a CNS elemental analyzer (Variomax CNS Analyzer, Elementar GmbH, Hanau, Germany) [31] .
4
Measuring TOC and Hg/TOC in Sediment Samples
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For samples from Stenlille-1 and Stenlille-4, the TOC [weight % (wt %)] was measured on approximately 50 mg of bulk dry samples using Rock-EVal 6 analysis at the Geological Survey of Canada with a detection limit for TOC of 0.24%. No TOC values below the detection limit were used for assessing Hg/TOC. For Rødby-1 and Norra Albert/Albert-1, sample powders were first treated with 10% HCl to dissolve any carbonates and centrifuged followed by decanting the supernatant. The remnants were washed with ultrapure Milli-Q water five to six times sufficient to neutralize the supernatant. Slurries were then dried for 36 to 48 hours at 50°C, reground in an agate mortar, and returned to the drying oven for several hours at 110°C. Powder (250 to 500 mg) was loaded in ceramic crucibles, and total (organic) carbon was analyzed with an Elementar Vario MAX CNS analyzer using the “Soil40” method in the Department of Geoscience, Aarhus University. Blanks, the sulfadiazine calibration standard, and working standard [Aarhus University (AU) 780523] were run at the beginning and end of each session and after every 10 samples. Blanks were <0.035 wt % C, while the AU standard was reproducible within 0.3% at 1.94 wt % C. For Rødby-1 and Norra Albert/Albert-1, no TOC values below 0.5% were used for assessing Hg/TOC. All TOC data are listed in table S9.
5
Biomass Composition Analysis Protocol
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Dry weight was determined by oven drying (105°C, 3 h) followed by ashing in a muffle furnace (600°C, 5 h). Crude protein was determined by the Dumas combustion method using a Vario Max C/N/S-Analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). Cyanophycin was measured as described in the section “CGP quantification”. AA (without Trp) were determined by HPLC (Shimadzu, Kyoto, Japan) using a cation column (LC K06, Alltech-Grom GmbH, Rottenburg-Hailfingen, Germany). AA (without Trp) were determined by HPLC (Shimadzu, Kyoto, Japan) using a cation column (LC K06, Alltech-Grom GmbH, Rottenburg-Hailfingen, Germany) according to Hackl et al. (2010) (link). The temperature program of the column was set between 57°C and 74°C, and the pH gradient from 3.45 to 10.85. The buffer flow rate was 0.45 mL/min. AA were mixed with ninhydrin at a flow rate of 0.25 mL/min for tinting at 128°C and determined with an UV-detector at 570 nm (proline at 440 nm).”
6
Elemental Analysis of Gall-Infected Leaves
7
Soil Physicochemical Characterization
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Soil pH was measured using a 1:2.5 soil to water ratio. The organic matter content (%) was determined using the loss-on-ignition (LOI) method by heating 5.0 g of soil at 550 °C for seven hours following the method of Ostrowska et al. (1991 ).
The soil concentration of heavy metals (Cd, Mn, Zn, Fe, Pb) and macronutrients (K, Mg, Na, Ca, P, S) were estimated in air-dried soil samples that had been sieved through a 2 mm sieve according to Zheljazkov et al. (2008 (link)) and Wójcik et al. (2014 (link)). The metals and macronutrients were extracted from the samples with concentrated HNO3 (65%) (acid extracted elements) or with 0.01 M CaCl2 (potentially bioavailable elements—only metals). For the HNO3-extractable fraction, the soil samples (0.5 g) were placed in digestion tubes, soaked overnight in 5 ml of concentrated HNO3 at room temperature, then decomposed further on an aluminum digestion block at 150 °C for 8 h, filtered and diluted to 25 ml with deionized water. For the CaCl2 extraction, 5 g of soil with a 50 ml 0.01 M CaCl2 solution was mechanically shaken for 2 h at room temperature. The levels of the metals were measured in the filtered extracts using inductively coupled plasma-atomic emission spectroscopy (Spectro Analytical Instruments).
Total carbon, total nitrogen and the C/N ratio were measured in an Elementar Vario MAX CNS Analyzer.
The soil concentration of heavy metals (Cd, Mn, Zn, Fe, Pb) and macronutrients (K, Mg, Na, Ca, P, S) were estimated in air-dried soil samples that had been sieved through a 2 mm sieve according to Zheljazkov et al. (2008 (link)) and Wójcik et al. (2014 (link)). The metals and macronutrients were extracted from the samples with concentrated HNO3 (65%) (acid extracted elements) or with 0.01 M CaCl2 (potentially bioavailable elements—only metals). For the HNO3-extractable fraction, the soil samples (0.5 g) were placed in digestion tubes, soaked overnight in 5 ml of concentrated HNO3 at room temperature, then decomposed further on an aluminum digestion block at 150 °C for 8 h, filtered and diluted to 25 ml with deionized water. For the CaCl2 extraction, 5 g of soil with a 50 ml 0.01 M CaCl2 solution was mechanically shaken for 2 h at room temperature. The levels of the metals were measured in the filtered extracts using inductively coupled plasma-atomic emission spectroscopy (Spectro Analytical Instruments).
Total carbon, total nitrogen and the C/N ratio were measured in an Elementar Vario MAX CNS Analyzer.
8
Soil Nutrient Analysis Protocol
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The total nitrogen (N tot ) content was determined with the Kjeldahl method [25] . The ammonium lactate (AL) method [26] was used to measure the contents of plant-available: (i) phosphorous (P A ), (ii) potassium (K A ), (iii) calcium (Ca A ) and (iv) magnesium (Mg A ). The total carbon content was measured by Dumas combustion method using a VarioMax CNS analyzer (ELEMENTAR, Germany). Considering that the average soil pH KCl was 6.0, and therefore carbonates were absent, it was assumed that the soil total carbon content was equivalent to that of the soil organic carbon (SOC) content.
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