Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA167620

Local Sample ID:	SAMP05_MV2
Subject ID:	SU001882
Subject Type:	Fungi
Subject Species:	Exidia glandulosa;Mucidula mucida
Taxonomy ID:	5219;139077

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Subject:

Subject ID:	SU001882
Subject Type:	Fungi
Subject Species:	Exidia glandulosa;Mucidula mucida
Taxonomy ID:	5219;139077

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
SAMP05_MV2	SA167620	FL019929	Variable Temp	Treatment

Collection:

Collection ID:	CO001875
Collection Summary:	The two fungal strains M. mucida and E. glandulosa were isolated from dead branches attached to standing beech (F. sylvatica) trees at Clyne Valley Woodlands, a minimally managed woodland in Swansea, South Wales, UK (Lat 51.6063, Long -4.0068). Isolates were collected from different but closely positioned trees within the same woodland stand.
Sample Type:	new
Collection Method:	Woodchip isolation
Collection Location:	Clyne Valley Woodlands, Swansea, United Kingdom, Lat 51.6063, Long -4.0068

Treatment:

Treatment ID:	TR001895
Treatment Summary:	F. sylvatica blocks (John Harrison, Wrexham, UK) with dimensions 2 cm3 were autoclaved three times and then pre-colonised by placing them on 0.5% malt-extract agar (MEA; 0.5% malt, 1.5% agar w/v; Sigma-Aldrich, Dorset, UK) cultures of the appropriate fungal strain and incubated in the dark at 20°C. Following 8-weeks pre-colonisation, experimental treatments consisting of 2 x 2 block matrices colonised with one of the two fungal strains per microcosm. Microcosms were subjected to a diurnally cycling temperature sequence of 5, 15, 25 and 15°C per 24 hours so that temperature changed by 10°C every 6 hours with a mean of 15 ± 10°C. Temperatures were chosen to represent the minima and maxima canopy-dwelling ligninolytic fungi encounter during the growing season in a temperate woodland ecosystem. Control microcosms were kept at a constant temperature of 15°C for the duration of the experiment. These methods were also chosen to be comparable with those of Toljander Lindahl, Holmer & Högberg (Toljander et al., 2006). All microcosms were incubated in the dark for 8 weeks with three replicates per species per treatment (n = 12).

Treatment ID:

TR001895

Treatment Summary:

F. sylvatica blocks (John Harrison, Wrexham, UK) with dimensions 2 cm3 were autoclaved three times and then pre-colonised by placing them on 0.5% malt-extract agar (MEA; 0.5% malt, 1.5% agar w/v; Sigma-Aldrich, Dorset, UK) cultures of the appropriate fungal strain and incubated in the dark at 20°C. Following 8-weeks pre-colonisation, experimental treatments consisting of 2 x 2 block matrices colonised with one of the two fungal strains per microcosm. Microcosms were subjected to a diurnally cycling temperature sequence of 5, 15, 25 and 15°C per 24 hours so that temperature changed by 10°C every 6 hours with a mean of 15 ± 10°C. Temperatures were chosen to represent the minima and maxima canopy-dwelling ligninolytic fungi encounter during the growing season in a temperate woodland ecosystem. Control microcosms were kept at a constant temperature of 15°C for the duration of the experiment. These methods were also chosen to be comparable with those of Toljander Lindahl, Holmer & Högberg (Toljander et al., 2006). All microcosms were incubated in the dark for 8 weeks with three replicates per species per treatment (n = 12).

Sample Preparation:

Sampleprep ID:	SP001888
Sampleprep Summary:	At the end of the experiment, each block was split into three segments from top to bottom using a sterile chisel. The top and middle sections were quenched in liquid nitrogen, lyophilised and stored at -20°C. From the bottom third of each block, four wood chips (~0.5 mm2) were extracted, one from each quadrant, and placed onto 2% (w/v) MEA. Cultures were incubated at 20°C for 7 – 10 days and mycelial morphology confirmed that no woodblocks had become contaminated during the experiment. One third of the frozen woodblock sections from each treatment group were each weighed and manually chipped, added to ≤ 10 ml acetonitrile : methanol : H2O (2:2:1) and shaken at 180 rpm at room temperature for 1 h. Extracts were filtered through glass wool and Whatman filter paper (no. 1) and centrifuged at 3,500 g for 30 min. The supernatant was dried in vacuo (Eppendorf, Hamburg, Germany) overnight. Dried samples were derivitized by addition of 30 μl methoxylamine hydrochloride (15 mg ml-1 in pyridine; Sigma, UK) followed by incubation at 60°C for 90 min. Subsequently 50 μl MSTFA+ 1% (v/v) TMCS (Thermo Scientific) was added to the sample and incubated at 40°C for 60 min. Derivatized samples were transferred to autosampler vials and 10 μl tetracosane (5 mg ml-1 in heptane; Sigma-Aldrich) added as an internal standard.

Sampleprep ID:

SP001888

Sampleprep Summary:

At the end of the experiment, each block was split into three segments from top to bottom using a sterile chisel. The top and middle sections were quenched in liquid nitrogen, lyophilised and stored at -20°C. From the bottom third of each block, four wood chips (~0.5 mm2) were extracted, one from each quadrant, and placed onto 2% (w/v) MEA. Cultures were incubated at 20°C for 7 – 10 days and mycelial morphology confirmed that no woodblocks had become contaminated during the experiment. One third of the frozen woodblock sections from each treatment group were each weighed and manually chipped, added to ≤ 10 ml acetonitrile : methanol : H2O (2:2:1) and shaken at 180 rpm at room temperature for 1 h. Extracts were filtered through glass wool and Whatman filter paper (no. 1) and centrifuged at 3,500 g for 30 min. The supernatant was dried in vacuo (Eppendorf, Hamburg, Germany) overnight. Dried samples were derivitized by addition of 30 μl methoxylamine hydrochloride (15 mg ml-1 in pyridine; Sigma, UK) followed by incubation at 60°C for 90 min. Subsequently 50 μl MSTFA+ 1% (v/v) TMCS (Thermo Scientific) was added to the sample and incubated at 40°C for 60 min. Derivatized samples were transferred to autosampler vials and 10 μl tetracosane (5 mg ml-1 in heptane; Sigma-Aldrich) added as an internal standard.

Combined analysis:

Analysis ID	AN002927
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 5975
Column	Phenomenex Zebron ZB-WAXplus (30m x 0.25mm,0.25um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5975
Ion Mode	POSITIVE
Units	Integrated signal (SpectConnect)

Chromatography:

Chromatography ID:	CH002169
Instrument Name:	Agilent 5975
Column Name:	Phenomenex Zebron ZB-WAXplus (30m x 0.25mm,0.25um)
Column Temperature:	250
Flow Rate:	1ml/min
Internal Standard:	Tetracosane
Chromatography Type:	GC

MS:

MS ID:	MS002718
Analysis ID:	AN002927
Instrument Name:	Agilent 5975
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	-
Ion Mode:	POSITIVE