Summary of Study ST001805

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001140. The data can be accessed directly via it's Project DOI: 10.21228/M8BQ3C This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST001805
Study Title	Metabolic responses of two pioneer wood decay fungi to diurnally cycling temperature
Study Summary	1. Decomposition of lignin-rich wood by fungi drives nutrient recycling in woodland ecosystems. Fluctuating abiotic conditions are known to promote the functioning of ecological communities and ecosystems. In the context of wood decay, fluctuating temperature increases decomposition rates. Metabolomics, in tandem with other ‘omics tools, can highlight the metabolic processes affected by experimental treatments, even in the absence of genome sequences and annotations. Globally, natural wood decay communities are dominated by the phylum Basidiomycota. We examined the metabolic responses of Mucidula mucida, a dominant constituent of pioneer communities in beech branches in British woodlands, and Exidia glandulosa, a stress-selected constituent of the same communities, in response to constant and diurnally cycling temperature. 2. We applied untargeted metabolomics and proteomics to beech wood blocks, colonised by M. mucida or E. glandulosa and exposed to either diurnally cycling (mean 15 ± 10°C) or constant (15°C) temperature, in a fully factorial design. 3. Metabolites and proteins linked to lignin breakdown, the citric acid cycle, pentose phosphate pathway, carbohydrate metabolism, fatty acid metabolism and protein biosynthesis and turnover were under-enriched in fluctuating, compared to stable temperatures, in the generalist M. mucida. Conversely E. glandulosa showed little differential response to the experimental treatments. 4. Synthesis. By demonstrating temperature dependant metabolic signatures related to nutrient acquisition in a generalist wood decay fungus, we provide new insights into how abiotic conditions can affect community-mediated decomposition and carbon turnover in forests. We show that mechanisms underpinning important biogeochemical processes can be highlighted using untargeted metabolomics and proteomics in the absence of well-annotated genomes.
Institute	Swansea University
Department	Biosciences
Laboratory	Fungal Molecular Ecology
Last Name	Eastwood
First Name	Daniel
Address	Wallace 102, Biosciences, College of Science, Swansea University, Swansea, SA2 8PP
Email	d.c.eastwood@swansea.ac.uk
Phone	01792513003
Submit Date	2021-05-17
Num Groups	4
Total Subjects	2
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	GC-MS
Release Date	2021-06-02
Release Version	1

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Project:

Project ID:	PR001140
Project DOI:	doi: 10.21228/M8BQ3C
Project Title:	Metabolic responses of two pioneer wood decay fungi to diurnally cycling temperature
Project Summary:	1. Decomposition of lignin-rich wood by fungi drives nutrient recycling in woodland ecosystems. Fluctuating abiotic conditions are known to promote the functioning of ecological communities and ecosystems. In the context of wood decay, fluctuating temperature increases decomposition rates. Metabolomics, in tandem with other ‘omics tools, can highlight the metabolic processes affected by experimental treatments, even in the absence of genome sequences and annotations. Globally, natural wood decay communities are dominated by the phylum Basidiomycota. We examined the metabolic responses of Mucidula mucida, a dominant constituent of pioneer communities in beech branches in British woodlands, and Exidia glandulosa, a stress-selected constituent of the same communities, in response to constant and diurnally cycling temperature. 2. We applied untargeted metabolomics and proteomics to beech wood blocks, colonised by M. mucida or E. glandulosa and exposed to either diurnally cycling (mean 15 ± 10°C) or constant (15°C) temperature, in a fully factorial design. 3. Metabolites and proteins linked to lignin breakdown, the citric acid cycle, pentose phosphate pathway, carbohydrate metabolism, fatty acid metabolism and protein biosynthesis and turnover were under-enriched in fluctuating, compared to stable temperatures, in the generalist M. mucida. Conversely E. glandulosa showed little differential response to the experimental treatments. 4. Synthesis. By demonstrating temperature dependant metabolic signatures related to nutrient acquisition in a generalist wood decay fungus, we provide new insights into how abiotic conditions can affect community-mediated decomposition and carbon turnover in forests. We show that mechanisms underpinning important biogeochemical processes can be highlighted using untargeted metabolomics and proteomics in the absence of well-annotated genomes.
Institute:	Swansea University
Department:	Biosciences
Laboratory:	Fungal Molecular Ecology
Last Name:	Rawlings
First Name:	Anna
Address:	Floor 2, Data Science Swansea Medical School, Swansea University, Swansea, Swansea, SA2 8PP, United Kingdom
Email:	a.rawlings@swansea.ac.uk
Phone:	07810847321
Funding Source:	NERC grants NE/K011588/1 and NE/K011383/1

Subject:

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

Factors:

Subject type: Fungi; Subject species: Exidia glandulosa;Mucidula mucida (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA167613	SAMP08_ES2	Stable Temp
SA167614	SAMP09_ES3	Stable Temp
SA167615	SAMP01_MS1	Stable Temp
SA167616	SAMP07_ES1	Stable Temp
SA167617	SAMP02_MS2	Stable Temp
SA167618	SAMP03_MS3	Stable Temp
SA167619	SAMP12_EV3	Variable Temp
SA167620	SAMP05_MV2	Variable Temp
SA167621	SAMP11_EV2	Variable Temp
SA167622	SAMP10_EV1	Variable Temp
SA167623	SAMP06_MV3	Variable Temp
SA167624	SAMP04_MV1	Variable Temp

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

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