{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001805","ANALYSIS_ID":"AN002927","VERSION":"1","CREATED_ON":"May 24, 2021, 8:55 am"},

"PROJECT":{"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","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","PROJECT_TITLE":"Metabolic responses of two pioneer wood decay fungi to diurnally cycling temperature","LAST_NAME":"Rawlings"},

"STUDY":{"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","NUM_GROUPS":"4","TOTAL_SUBJECTS":"2","PHONE":"01792513003"},

"SUBJECT":{"SUBJECT_TYPE":"Fungi","SUBJECT_SPECIES":"Exidia glandulosa;Mucidula mucida"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Mucidula mucida",
"Sample ID":"SAMP01_MS1",
"Factors":{"Treatment":"Stable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP01_MS1"}
},
{
"Subject ID":"Mucidula mucida",
"Sample ID":"SAMP02_MS2",
"Factors":{"Treatment":"Stable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP02_MS2"}
},
{
"Subject ID":"Mucidula mucida",
"Sample ID":"SAMP03_MS3",
"Factors":{"Treatment":"Stable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP03_MS3"}
},
{
"Subject ID":"Mucidula mucida",
"Sample ID":"SAMP04_MV1",
"Factors":{"Treatment":"Variable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP04_MV1"}
},
{
"Subject ID":"Mucidula mucida",
"Sample ID":"SAMP05_MV2",
"Factors":{"Treatment":"Variable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP05_MV2"}
},
{
"Subject ID":"Mucidula mucida",
"Sample ID":"SAMP06_MV3",
"Factors":{"Treatment":"Variable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP06_MV3"}
},
{
"Subject ID":"Exidia glandulosa",
"Sample ID":"SAMP07_ES1",
"Factors":{"Treatment":"Stable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP07_ES1"}
},
{
"Subject ID":"Exidia glandulosa",
"Sample ID":"SAMP08_ES2",
"Factors":{"Treatment":"Stable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP08_ES2"}
},
{
"Subject ID":"Exidia glandulosa",
"Sample ID":"SAMP09_ES3",
"Factors":{"Treatment":"Stable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP09_ES3"}
},
{
"Subject ID":"Exidia glandulosa",
"Sample ID":"SAMP10_EV1",
"Factors":{"Treatment":"Variable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP10_EV1"}
},
{
"Subject ID":"Exidia glandulosa",
"Sample ID":"SAMP11_EV2",
"Factors":{"Treatment":"Variable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP11_EV2"}
},
{
"Subject ID":"Exidia glandulosa",
"Sample ID":"SAMP12_EV3",
"Factors":{"Treatment":"Variable Temp"},
"Additional sample data":{"RAW_FILE_NAME":"SAMP12_EV3"}
}
],
"COLLECTION":{"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_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)."},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"GC","INSTRUMENT_NAME":"Agilent 5975","COLUMN_NAME":"Phenomenex Zebron ZB-WAXplus (30m x 0.25mm, 0.25um)","FLOW_RATE":"1ml/min","COLUMN_TEMPERATURE":"250","INTERNAL_STANDARD":"Tetracosane"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Agilent 5975","INSTRUMENT_TYPE":"Single quadrupole","MS_TYPE":"EI","ION_MODE":"POSITIVE","MS_COMMENTS":"-"},

"MS_METABOLITE_DATA":{
"Units":"Integrated signal (SpectConnect)",

"Data":[{"Metabolite":"T04","SAMP01_MS1":"NaN","SAMP02_MS2":"NaN","SAMP03_MS3":"NaN","SAMP04_MV1":"NaN","SAMP05_MV2":"NaN","SAMP06_MV3":"NaN","SAMP07_ES1":"NaN","SAMP08_ES2":"NaN","SAMP09_ES3":"0.001216425","SAMP10_EV1":"0.001373892","SAMP11_EV2":"0.001769793","SAMP12_EV3":"NaN"},{"Metabolite":"T06","SAMP01_MS1":"0.004429462","SAMP02_MS2":"0.028531077","SAMP03_MS3":"NaN","SAMP04_MV1":"NaN","SAMP05_MV2":"NaN","SAMP06_MV3":"0.003900163","SAMP07_ES1":"NaN","SAMP08_ES2":"0.0018575","SAMP09_ES3":"NaN","SAMP10_EV1":"0.001040573","SAMP11_EV2":"NaN","SAMP12_EV3":"0.005061961"},{"Metabolite":"T07","SAMP01_MS1":"0.021098945","SAMP02_MS2":"0.14205732","SAMP03_MS3":"0.023246388","SAMP04_MV1":"0.025701747","SAMP05_MV2":"0.029325833","SAMP06_MV3":"0.037227418","SAMP07_ES1":"0.012927115","SAMP08_ES2":"0.019098136","SAMP09_ES3":"0.035469105","SAMP10_EV1":"0.03598819","SAMP11_EV2":"0.014626237","SAMP12_EV3":"0.015766159"},{"Metabolite":"T08","SAMP01_MS1":"0.50383124","SAMP02_MS2":"2.660251576","SAMP03_MS3":"0.570493919","SAMP04_MV1":"0.459227335","SAMP05_MV2":"0.484569819","SAMP06_MV3":"0.469852527","SAMP07_ES1":"0.333094699","SAMP08_ES2":"0.309687042","SAMP09_ES3":"0.756936967","SAMP10_EV1":"0.405185937","SAMP11_EV2":"0.354041017","SAMP12_EV3":"0.937104822"},{"Metabolite":"T09","SAMP01_MS1":"0.027503897","SAMP02_MS2":"0.137209852","SAMP03_MS3":"0.026909037","SAMP04_MV1":"0.032254876","SAMP05_MV2":"0.049914889","SAMP06_MV3":"0.045656021","SAMP07_ES1":"0.086445807","SAMP08_ES2":"0.132818605","SAMP09_ES3":"0.412324694","SAMP10_EV1":"0.197606736","SAMP11_EV2":"0.230677672","SAMP12_EV3":"0.190745421"},{"Metabolite":"T10","SAMP01_MS1":"0.013857848","SAMP02_MS2":"0.057379456","SAMP03_MS3":"0.013392928","SAMP04_MV1":"0.014208529","SAMP05_MV2":"0.02751219","SAMP06_MV3":"0.016882587","SAMP07_ES1":"0.004987985","SAMP08_ES2":"0.00342397","SAMP09_ES3":"0.013784162","SAMP10_EV1":"0.005400672","SAMP11_EV2":"0.01067546","SAMP12_EV3":"0.020043211"},{"Metabolite":"T11","SAMP01_MS1":"0.056541729","SAMP02_MS2":"0.367962194","SAMP03_MS3":"0.061281859","SAMP04_MV1":"0.051994505","SAMP05_MV2":"0.055809216","SAMP06_MV3":"0.049285874","SAMP07_ES1":"0.068700243","SAMP08_ES2":"0.070832817","SAMP09_ES3":"0.13696439","SAMP10_EV1":"0.110548211","SAMP11_EV2":"0.091769626","SAMP12_EV3":"0.127261217"},{"Metabolite":"T12","SAMP01_MS1":"0.003700206","SAMP02_MS2":"0.0190244","SAMP03_MS3":"0.003902816","SAMP04_MV1":"NaN","SAMP05_MV2":"0.002751718","SAMP06_MV3":"0.003275725","SAMP07_ES1":"NaN","SAMP08_ES2":"0.001352617","SAMP09_ES3":"0.008450562","SAMP10_EV1":"0.002454794","SAMP11_EV2":"0.002105853","SAMP12_EV3":"0.005582127"},{"Metabolite":"T13","SAMP01_MS1":"0.003017669","SAMP02_MS2":"0.011082389","SAMP03_MS3":"NaN","SAMP04_MV1":"0.001825487","SAMP05_MV2":"0.002073492","SAMP06_MV3":"0.001802132","SAMP07_ES1":"0.00135893","SAMP08_ES2":"0.0015917","SAMP09_ES3":"0.006490459","SAMP10_EV1":"0.001399803","SAMP11_EV2":"NaN","SAMP12_EV3":"0.006178728"},{"Metabolite":"T14","SAMP01_MS1":"0.012224528","SAMP02_MS2":"0.054312747","SAMP03_MS3":"0.009871189","SAMP04_MV1":"0.010068497","SAMP05_MV2":"0.008877249","SAMP06_MV3":"0.011217143","SAMP07_ES1":"0.008896733","SAMP08_ES2":"0.007367578","SAMP09_ES3":"0.031021526","SAMP10_EV1":"0.014736218","SAMP11_EV2":"0.013739485","SAMP12_EV3":"0.014425843"},{"Metabolite":"T15","SAMP01_MS1":"NaN","SAMP02_MS2":"0.003855903","SAMP03_MS3":"0.001204441","SAMP04_MV1":"0.001425445","SAMP05_MV2":"0.00081194","SAMP06_MV3":"NaN","SAMP07_ES1":"NaN","SAMP08_ES2":"0.000527094","SAMP09_ES3":"0.00135754","SAMP10_EV1":"0.000982073","SAMP11_EV2":"0.001288906","SAMP12_EV3":"NaN"},{"Metabolite":"T16","SAMP01_MS1":"NaN","SAMP02_MS2":"NaN","SAMP03_MS3":"0.003270451","SAMP04_MV1":"0.002601101","SAMP05_MV2":"NaN","SAMP06_MV3":"0.003231073","SAMP07_ES1":"0.011065098","SAMP08_ES2":"0.007905788","SAMP09_ES3":"0.023410988","SAMP10_EV1":"0.015563818","SAMP11_EV2":"0.006120794","SAMP12_EV3":"NaN"},{"Metabolite":"T17","SAMP01_MS1":"0.034482512","SAMP02_MS2":"0.036142939","SAMP03_MS3":"0.016340005","SAMP04_MV1":"0.017311736","SAMP05_MV2":"0.01458706","SAMP06_MV3":"0.009465191","SAMP07_ES1":"NaN","SAMP08_ES2":"NaN","SAMP09_ES3":"0.017205194","SAMP10_EV1":"0.010160357","SAMP11_EV2":"NaN","SAMP12_EV3":"0.017273721"},{"Metabolite":"T18","SAMP01_MS1":"NaN","SAMP02_MS2":"NaN","SAMP03_MS3":"0.010351112","SAMP04_MV1":"NaN","SAMP05_MV2":"NaN","SAMP06_MV3":"0.011731995","SAMP07_ES1":"NaN","SAMP08_ES2":"0.009296001","SAMP09_ES3":"NaN","SAMP10_EV1":"0.010268299","SAMP11_EV2":"0.007994542","SAMP12_EV3":"0.014000246"},{"Metabolite":"T19","SAMP01_MS1":"NaN","SAMP02_MS2":"NaN","SAMP03_MS3":"0.001770216","SAMP04_MV1":"0.003877263","SAMP05_MV2":"0.003912203","SAMP06_MV3":"0.003843984","SAMP07_ES1":"0.000641091","SAMP08_ES2":"0.000439894","SAMP09_ES3":"0.004521374","SAMP10_EV1":"0.00125156","SAMP11_EV2":"NaN","SAMP12_EV3":"NaN"},{"Metabolite":"T20","SAMP01_MS1":"NaN","SAMP02_MS2":"NaN","SAMP03_MS3":"NaN","SAMP04_MV1":"NaN","SAMP05_MV2":"NaN","SAMP06_MV3":"NaN","SAMP07_ES1":"NaN","S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