distilled water 2.5 times that of the soil (5 g). Each soil physicochemical analysis was
replicated three times. For element analysis, air-dried soil samples were passed
through a 0.074-mm sieve and heated to 950
C in a muffle furnace. The element
composition of the soil samples was using wavelength dispersive X-ray fluorescence
(Axios
mAX , Malvern Panalytical Ltd., Almelo, Netherlands) under the following
measurement conditions: X-ray tube with Rh anode, 75 μm Be window, 60 kW max
voltage, 160 mA max current, O–Ni element flow detector, Ni–Ba scintillation
detector, and V–Zn and Ce–Ta Xe detectors. Analytical precision and accuracy
were calibrated against sandy soil (International Atomic Energy Agency, Vienna,
Austria), as certified reference material.
Sclerotia samples were carefully isolated from the soil using tweezers in the
laboratory. Approximately 30–90 grains were collected from each soil sample to
obtain enough sclerotia for elemental analysis. Each sclerotium grain was washed by
hand with distilled water in a microtube for 2–3 min and the procedure was repeated
five times. Surface-washed sclerotia were air-dried at 25
C, ground to a fine powder
using an agate mortar, and then weighed. For each sample, 20–25 mg of sclerotia
was weighed using a microbalance and placed in 2-mL glass tubes.
Powdered samples were placed in a beaker with 0.3 mL of ultra-pure nitric acid
on a hotplate at 120
C for approximately 5 min. Then 0.1 mL of a 30% hydrogen
peroxide solution was added to the residue and heated at <100
C for 2 min until
almost evaporated, whereupon 0.6 mL of 0.1 M nitric acid was added. Finally, the
solution was again evaporated to near dryness at 130–140
C. The residue was
dissolved in 1 mL of 0.1 M HNO 3 to produce a transparent solution. Quantitative
analyses of the concentrations of major and trace elements in the sclerotia grains
were carried out using an inductively coupled plasma optical emission spectrometer
(iCAP 6000, Thermo Fisher Scientific, Waltham, MA, USA) under the following
measurement conditions: 1150 W RF power, 50 rpm pump rate, 0.5 L min
À1
auxiliary gas flow, and normal purge gas flow. Inductively coupled plasma mass
spectrometry (XSERIES II, Thermo Fisher Scientific) was used under the following
measurement conditions: 1.9 bar neb pressure, 0.87 L min
À1 neb gas flow, 38 rpm
pump rate, 13 L min
À1 cool gas flow, 0.7 L min
À1 auxiliary gas flow, 1349 W fwd
power, 2 W ref, load 69, tune 153, 2.2 Â 10 mbar expansion pressure,
3.5 Â 10
À7 mbar analyzer chamber pressure, À90 extraction, and lens 1: À1110,
lens 2: À77.6, focus: 12.5, and D1: À42.4, D2: À140. A standard solution of
1000 ppm/matrix—2% HNO 3 (High-Purity Standards, USA) was used for instrument calibration.
11.2.3 Statistical Analysis
Data were recorded as the mean Æ the standard error (SE) and significant differences
between means were determined using a two sample t-test, where p values <0.05
were regarded as significant. Statistical analyses were performed using XLSTAT
(Version 2019.4.2.63677, Addinsoft).
11 Melanized Sclerotia Grains from Mongolian Steppe Forest Soils
197
replicated three times. For element analysis, air-dried soil samples were passed
through a 0.074-mm sieve and heated to 950
C in a muffle furnace. The element
composition of the soil samples was using wavelength dispersive X-ray fluorescence
(Axios
mAX , Malvern Panalytical Ltd., Almelo, Netherlands) under the following
measurement conditions: X-ray tube with Rh anode, 75 μm Be window, 60 kW max
voltage, 160 mA max current, O–Ni element flow detector, Ni–Ba scintillation
detector, and V–Zn and Ce–Ta Xe detectors. Analytical precision and accuracy
were calibrated against sandy soil (International Atomic Energy Agency, Vienna,
Austria), as certified reference material.
Sclerotia samples were carefully isolated from the soil using tweezers in the
laboratory. Approximately 30–90 grains were collected from each soil sample to
obtain enough sclerotia for elemental analysis. Each sclerotium grain was washed by
hand with distilled water in a microtube for 2–3 min and the procedure was repeated
five times. Surface-washed sclerotia were air-dried at 25
C, ground to a fine powder
using an agate mortar, and then weighed. For each sample, 20–25 mg of sclerotia
was weighed using a microbalance and placed in 2-mL glass tubes.
Powdered samples were placed in a beaker with 0.3 mL of ultra-pure nitric acid
on a hotplate at 120
C for approximately 5 min. Then 0.1 mL of a 30% hydrogen
peroxide solution was added to the residue and heated at <100
C for 2 min until
almost evaporated, whereupon 0.6 mL of 0.1 M nitric acid was added. Finally, the
solution was again evaporated to near dryness at 130–140
C. The residue was
dissolved in 1 mL of 0.1 M HNO 3 to produce a transparent solution. Quantitative
analyses of the concentrations of major and trace elements in the sclerotia grains
were carried out using an inductively coupled plasma optical emission spectrometer
(iCAP 6000, Thermo Fisher Scientific, Waltham, MA, USA) under the following
measurement conditions: 1150 W RF power, 50 rpm pump rate, 0.5 L min
À1
auxiliary gas flow, and normal purge gas flow. Inductively coupled plasma mass
spectrometry (XSERIES II, Thermo Fisher Scientific) was used under the following
measurement conditions: 1.9 bar neb pressure, 0.87 L min
À1 neb gas flow, 38 rpm
pump rate, 13 L min
À1 cool gas flow, 0.7 L min
À1 auxiliary gas flow, 1349 W fwd
power, 2 W ref, load 69, tune 153, 2.2 Â 10 mbar expansion pressure,
3.5 Â 10
À7 mbar analyzer chamber pressure, À90 extraction, and lens 1: À1110,
lens 2: À77.6, focus: 12.5, and D1: À42.4, D2: À140. A standard solution of
1000 ppm/matrix—2% HNO 3 (High-Purity Standards, USA) was used for instrument calibration.
11.2.3 Statistical Analysis
Data were recorded as the mean Æ the standard error (SE) and significant differences
between means were determined using a two sample t-test, where p values <0.05
were regarded as significant. Statistical analyses were performed using XLSTAT
(Version 2019.4.2.63677, Addinsoft).
11 Melanized Sclerotia Grains from Mongolian Steppe Forest Soils
197