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Effects of Bio-Ash Amendments on the Metabolism of Ectomycorrhizal Fungi: A Method Development and Metabolomic Study
Mid Sweden University, Faculty of Science, Technology and Media, Department of Natural Sciences. (Analytisk kemi)
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Forest ecosystems have played a fundamental role in the development of our society. Since the beginning of the civilization, forest have provided us with wood as a product for construction, tools, furniture and domestic heating. The well-being of the forest is therefore fundamental to our existence. Today, our growing societies have increased energy needs; the resulting depletion of fossil reserves and the effects of their use has again shown how the forest is among the most important alternatives for sustainability of our ecosystem. In order to responsibly make this resource a key part of our energy and material supply, we need to understand how forestry practices influence the different processes taking place in the forest ecosystems.

The use of raw material from forest as energy source produces huge amounts of ash. The ash contains the base cations that have once been translocated from soil to the upper parts of the trees. Ash recycling has therefore been suggested as a measure to counteract soil acidification due to extensive harvest. Since spreading of ash can have great effects on the forest, it is important to understand which these effects are and how big they might be.

This thesis focuses on the effects that such an ash recycling may have on the metabolism of ectomycorrhizal fungi; that is, fungi that are able to colonize root of trees, and contribute to the acquisition of nutrients and water from soil. The work presented here utilized an in vitro metabolomic approach on eight species of ectomycorrhizal fungi normally found in boreal forests. A targeted metabolomic study addressed the effects of ash amendments on growth, external pH and the exudation of low molecular mass organic acids, amino acids and hydroxamate siderophores. This was complemented by an untargeted metabolomic study to address the effects of ash amendment on the general metabolism of the fungal species.

Analyses were performed with well-established chemical methods, and some that had to be developed specifically for this thesis work. A method for the analysis of amino acids without derivatization and yet compatible with mass spectrometry had to be developed and validated. The result was a robust method that works well with external calibration, shows good long-term stability, relatively low detections limits and high sample throughput. A screening protocol for the determination of siderophores from mass spectrometry data was also established.

The metabolomic studies showed that bio-ash amendment increased the exudation of low molecular mass organic compounds from all the studied species. This means that the species tended to exude more of the same compounds compared to the controls without ash. In some cases, the bio-ash also triggered the exudation of new compounds. There was some exceptions, though; bio-ash amendment had negative effects on the exudation of certain metabolites, but these negative effects were of lower magnitude compared to the positive effects.

Both metabolomics studies showed a differentiation between the ascomycetes and the basidiomycetes species. The targeted metabolomic study, indicated a trade-off in the utilization of carbon for accumulation of biomass or for the exudation of low molecular mass organic compounds, in which the ascomycetes accumulated more carbon as biomass compared to the basidiomycetes. According to the untargeted metabolomic study, the ascomycetes species presented the greatest number of metabolites that were influenced significantly by ash treatment, either as increase or decrease.

Adding extracted ash to the culturing medium at the beginning of the experiment increased the pH, but this was counteracted by species metabolism as exudation of organic acids correlated with a drop in external pH. Ash treatment triggered the total exudation of low molecular mass organic acids in five of the eight studied species and especially in Cortinarius glaucopus. Ash treatment also triggered the exudation of amino acids from Tomentellopsis submollis and ferricrocin from Hymenoscyphus ericae.

Of note was that no metabolite significantly influenced by ash was found to be common to all species, indicating that the ash amendments mainly affected the secondary metabolism under the culturing conditions used. Additionally, the ascomycetes Hymenoscyphus ericae exuded the greatest number of metabolites affected by ash that were exuded only by a single species. Conversely, Piloderma olivaceum exuded the largest number of unique metabolites not influenced by ash.

Abstract [sv]

Skogsekosystem har genom alla tider spelat en central roll i utvecklingen av vårt samhälle. Trä för konstruktioner, möbler och hushållsuppvärmning har varit tjänster från skogar sedan civilisationens början. Skogens välbefinnande kan därför anses grundläggande för vår existens. Idag har vårt samhälle utvecklats och behovet av skogsråvara ökar kontinuerligt. Detta i kombination  med minskande fossila reserver och den inverkan som användningen av dessa reserver har haft på vår miljö har återigen gjort skogen till ett av de viktigaste alternativen för att åstadkomma en hållbar utveckling. Detta innebär att vi måste öka vår kunskap om hur olika former av skogsbruk påverkar miljön i skogsekosystemen.

 Användningen av skogsråvara som energikälla ger upphov till stora mängder aska. Askan innehåller höga halter av de baskatjoner som en gång har transporteras från jorden till de övre delarna av träden. Återföring av aska har därför föreslagits som en skogsbrukspraxis för att motverka den försurning som en omfattande avverkning kan ge upphov till. Eftersom återföring av aska kan ha stora effekter på skogen är det viktigt att förstå vilka dessa effekter är och hur omfattande de kan vara.

Denna avhandling fokuserar på de effekter som askåterföring till skogsmark kan ha på metabolismen hos ektomykorrhizasvampar, det vill säga svampar som kan kolonisera rötter och bidra till trädens upptag av näringsämnen och vatten från jorden. Arbetet uppnåddes med ett in vitro metabolomiskt tillvägagångsätt på åtta arter av ektomykorrhizasvampar som förekommer normalt i boreala skogar. En riktad metabolomisk studie genomfördes för att studera effekterna av olika doser av aska på tillväxt, yttre pH och utsöndring av lågmolekylära organiska syror samt aminosyror och hydroxamatsideroforer. Denna studie kompletterades med en mer generell metabolomikstudie där effekterna av aska på svamparnas totala metabolism studerades.

I ett arbete som detta är det viktigt med tillförlitliga metoder. En stor del av detta arbete har varit att utveckla nya kemiska analysmetoder. En metod för analys av aminosyror utan derivatisering och samtidigt kompatibel med masspektrometrisk detektion behövde utvecklas och valideras. Detta resulterade i en robust metod som fungerade väl med extern kalibrering och uppvisade bra detektionsnivåer samt förhållandevis hög genomströmning. Vidare så utvecklades även en metod för att kunna upptäcka förekomst av låga halter av sideroforer i komplexa prover med stöd av masspektrometri.

De metabolomiska studierna visade att exponering for bioaska ledde till ökad utsöndring av lågmolekylära organiska föreningar hos alla studerade arter. Huvudsakligen tenderade svamparna till att utsöndra mer av samma föreningar som registrerades för kontrollerna utan tillförsel av aska. I vissa fall ledde exponeringen även till utsöndring av nya föreningar. Det fanns också undantag där askan snarare hade negativ inverkan på utsöndringen av vissa metaboliter. Generellt var dessa negativa effekter av lägre magnitud än de positiva effekterna. Båda metabolomikstudierna visade en differentiering mellan basidiomyceter och ascomyceter. Den riktade metabolomiska studien antydde en kompromiss mellan användningen av kol för ackumulering av biomassa respektive utsöndring av organiska föreningar med låg molekylvikt, där ascomyceterna tycktes ackumulera mer kol som biomassa jämfört med de basidiomyceter som ingick i studien. Den mer generella metabolomikstudien, visade också att ascomyceterna var de arter som utsöndrade flest antal metaboliter som påverkades av bioaska, antingen i positiv eller i negativ riktning.

Tillsats av extraherad bioaska till odlingsmediet vid experimentets början ledde till ökat pH, men detta motverkades med tiden av metabolismen genom utsöndringen av organiska syror. Askbehandlingen resulterade i ökad utsöndring av organiska syror med låg molekylvikt för fem av de åtta studerade arterna och speciellt för Cortinarius glaucopus. Behandlingen resulterade också i ökad utsöndring av aminosyror från Tomentellopsis submollis och av ferricrocin från Hymenoscyphus ericae.

Ingen av de metaboliter som påverkades signifikant av bioaska visade sig vara gemensam för alla studerade arter. Detta indikerar att under de odlingsbetingelser som användes så var det huvudsakligen den sekundära metabolismen som påverkades.

 

Place, publisher, year, edition, pages
Sundsvall: Mid Sweden University , 2019. , p. 74
Series
Mid Sweden University doctoral thesis, ISSN 1652-893X ; 290
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:miun:diva-35052ISBN: 978-91-88527-77-6 (print)OAI: oai:DiVA.org:miun-35052DiVA, id: diva2:1268188
Public defence
2019-01-11, O102, Holmgatan 10, Sundsvall, 10:15 (English)
Opponent
Supervisors
Note

Vid tidpunkten för disputationen var följande delarbeten opublicerade: delarbete 2 (inskickat), delarbete 4 (manuskript).

At the time of the doctoral defence the following papers were unpublished: paper 2 (submitted), paper 4 (manuscript).

Available from: 2018-12-05 Created: 2018-12-05 Last updated: 2018-12-05Bibliographically approved
List of papers
1. Direct analysis of free amino acids by mixed-mode chromatography with tandem mass spectrometry
Open this publication in new window or tab >>Direct analysis of free amino acids by mixed-mode chromatography with tandem mass spectrometry
2017 (English)In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 40, no 7, p. 1482-1492Article in journal (Refereed) Published
Abstract [en]

We developed a straightforward, robust, and relatively fast method for the analysis of amino acids by mixed-mode high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The method does not involve derivatization and allows the detection of 21 amino acids, representing a wide range of isoelectric points, in less than 40 min. Chromatographic separation was governed by a silica-based mixed-mode column providing simultaneous hydrophobic and ion exchange separation mechanisms. The use of tandem mass spectrometry increased selectivity, reducing potential problems associated with poor selectivity in the chromatographic system. For an injection volume of 1 μL, we obtained detection limits <3 μM for the majority of analytes. For all analytes, a linearity of r > 0.99 was obtained, recovery in matrix was >86%, and the retention times were highly reproducible. The method was successfully applied to soil solution and fungal culture samples, demonstrating the advantages in successfully avoiding issues associated with high amounts of substances that may interfere with derivatization-based methods. This method represents an alternative to derivatization-based methods and can be applied in areas where sample matrices are highly complex.

Keywords
amino acids, fungi, mass spectrometry, mixed-mode chromatography, soil
National Category
Natural Sciences
Identifiers
urn:nbn:se:miun:diva-30655 (URN)10.1002/jssc.201601097 (DOI)000399782900006 ()2-s2.0-85014685988 (Scopus ID)
Available from: 2017-04-25 Created: 2017-04-25 Last updated: 2018-12-05Bibliographically approved
2. A siderophore candidate screening protocol based on gallium exchange and electrospray mass spectrometry
Open this publication in new window or tab >>A siderophore candidate screening protocol based on gallium exchange and electrospray mass spectrometry
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:miun:diva-35057 (URN)
Available from: 2018-12-05 Created: 2018-12-05 Last updated: 2018-12-05Bibliographically approved
3. Biofuel ash addition increases ectomycorrhizal fungal exudation in pure culture
Open this publication in new window or tab >>Biofuel ash addition increases ectomycorrhizal fungal exudation in pure culture
Show others...
2018 (English)In: Environmental Chemistry, ISSN 1448-2517, E-ISSN 1449-8979, Vol. 15, no 8, p. 481-492Article in journal (Refereed) Published
Abstract [en]

Environmental context. Spreading recycled wood ash in forests may counteract acidification and nutrient losses, but the process may also affect symbiotic fungi in these eco-systems. We show how fungal species react when exposed to ash solutions; for example, by an increased release of organic acids and other compounds. These effects can influence pH and metal availability in forest soils treated with ash.. Recycling of wood ash may counteract acidification and losses of base cations resulting from whole-tree harvesting in boreal forest ecosystems. The effects of ash treatment on growth and exudation of eight ectomycorrhizal fungal species were investigated in this study. Six basidiomycetes and two ascomycetes were grown in liquid pure culture with different levels of ash amendments. Biomass production, pH and the exudation of 17 low-molecular-mass organic acids (LMMOAs), 23 amino acids (AAs) and 9 hydroxamate siderophores (HSs) were recorded after 1, 2 and 4 weeks of incubation. Ash did not affect fungal growth, but resulted in higher exudation of the investigated compounds, in particular LMMOAs. Ash also influenced the composition of the exudates. We measured exudation of LMMOAs and AAs up to millimolar and micromolar concentrations respectively. For example, Rhizopogon roseolus mainly produced oxalic acid, whereas Lactarius rufus and Tomentellopsis submollis produced the highest concentrations of AAs. Ferricrocin, the only HS detected, was exuded at the nanomolar level. Exudation responses were also highly species-dependent, e.g. the ascomycetous isolates that produced the largest biomass released low amounts of exudates compared with the basidiomycetes, and were the only ones producing siderophores. This growth–exudation response to ash is likely a trade-off in carbon allocation whereby the mycorrhizal fungal species invest carbon in either higher biomass production or higher exudation.

Keywords
mass spectrometry, metabolomics, metal stress, soil acidification
National Category
Natural Sciences
Identifiers
urn:nbn:se:miun:diva-35059 (URN)10.1071/EN18146 (DOI)
Available from: 2018-12-05 Created: 2018-12-05 Last updated: 2018-12-05Bibliographically approved
4. Effects of bio-ash on the exudation pattern of eight ectomycorrhizal fungi - an untargeted metabolomic study
Open this publication in new window or tab >>Effects of bio-ash on the exudation pattern of eight ectomycorrhizal fungi - an untargeted metabolomic study
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:miun:diva-35060 (URN)
Available from: 2018-12-05 Created: 2018-12-05 Last updated: 2018-12-05Bibliographically approved

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