Final Report Summary - ERICARB (Does plant C regulate the decomposition of soil organic matter by ericoid mycorrhizal fungi?)
To account for inherent spatial heterogeneity, we increased our sampling effort in Tongariro National Park (TNP, in New Zealand North Island) from the originally planned 66 to 104 root systems. The sampling protocol was designed to test for differences in fungal communities between plant species (invasive versus native), for spatial patterns at the meter to kilometre scale within each species, and for significant impact of immediate presence / absence of invasive plant species (C. Vulgaris) on the fungal community hosted in the roots of native plant species (D. Subulatum).
By the end of the reporting period, all root samples have been processed all the way through to TRFLP analysis of fungal ITS-rDNA sequences (including processing for microscopic observations). Reference fungal cultures were also processed and analysed by Terminal Restriction Fragment Length Polymorphism (TRFLP) to provide fragments database to aid statistical analyses. Results from TRFLP analyses suggest possible co-invasion between Calluna vulgaris and the ericoid mycorrhizal fungus Rhizoscyphus ericae.
The first consequence of our results was to engage with New Zealand's Environmental Protection Authority to modify the fungal species list present in New Zealand and request the inclusion of Rhizoscyphus ericae. The second consequence was that, to back-up our conclusion of fungus-plant invasion, we needed to assess whether this Northern fungal species could also been found in areas not yet invaded by European heathers. To do so, we conducted an additional sampling campaign (not described in Annexe I) including 29 sites covering most part of South New Zealand, and collected a total of 140 additional root samples from 2 native plant genera capable of forming ericoid mycorrhizal associations. All additional samples were processed as described above, and analysed by TRFLP and NGS (next generation sequencing). NGS analyses were contracted to IMGM Laboratories GmbH in Germany. This is a deviation from the original proposal, which was required because the original proposed sequencing facilities (collaborator UWS) were unable to conduct analyses on 454-FLX titanium platform.
Both field-sampling campaigns were financially supported by Landcare Research (capability fund) to cover travel and subsidence cost of student and technical assistance.
Built into objective 3 was the investigation of the fate of soil C released by the decomposing activity of mycorrhizal fungi, but not respired as CO2. We aimed to test the hypothesis that mycorrhizal fungi can take up C released from SOM and transport it from the site of decomposition to the site of mycorrhizal structures located within the plant roots. To address this hypothesis, we planned to develop a magnetic bead capture of fungal rRNA, in collaboration with Drs Pringle & Pearson from Harvard University (Cambridge, USA). The method development was delayed because of the disruptions caused by the 2010 and 2011 Christchurch earthquakes, and was therefore postponed until relocation to European host premises (Aberdeen). Once relocated to Aberdeen, the technical challenges of such methods could not be overcome. Parallel method development has been attempted in the laboratory of our collaborator Professor Ann Pearson (Harvard), but has so far not been taken to a stage where biological applications can be envisaged. This is a late major deviation from Annexe I, with repercussion both research and training objectives. To counter-balance the impact of this technological failure on the science excellence of this project, corrective measures were taken and are described in section 3.4.
To complement the results of the magnetic bead capture of fungal RNA, we collected soil samples from all soil cores labelled with 13C from all plants included in the pot experiment (110 cores in total). Soil samples were destined to provide material for extraction of PLFA, and trace labelled C (13C) within the rapidly turning over cell membrane of the soil microbiota (13C-SIP-PLFA). All soil samples were therefore freeze-dried, milled and shipped to our collaborators in James Hutton Institute, Aberdeen. At the onset of this research fellowship, our collaborator had generously offered to subsidise the cost of the 13C-SIP-PLFA analyses. Because of the delays incurred in the implementation of the experiment and the collection of soil samples, changes in the financial forecast on our collaborator’s side prevented from going ahead with the analyses in the academic year 2012-2013. The samples are currently stored in Aberdeen and awaiting analyses in the coming academic year, but the results will not be available within the scope of the reporting period for this fellowship. Marie Curie funding evidently be acknowledged in any subsequent putative publications including data obtained from these soil samples.
Significant findings and outcomes:
• 1st record of fungus Rhizoscyphus ericae in New Zealand, likely imported into New Zealand with plant host Calluna vulgaris in the mid-1920s.
• Development of species-specific PCR primers for detection of Rhizoscyphus ericae.
• Development of high-throughput gas sampling and isotopic analysis protocol used for measuring 13/12 C isotopic content of soil CO2 efflux.
• Invasion by Calluna vulgaris causes ecosystem degradation in Tongariro National Park: changes in vegetation composition, fungal communities and soil C cycle.
• Native ericoid mycorrhizal communities are host-dependent and spatially structured.
• Global ericoid mycorrhizal ITS-rDNA sequence database freely available to others via UNITE (http://unite.ut.ee/).
• Availability of 2 new whole genome sequences of ericoid mycorrhizal fungi (only 1 had been published so far).
For more information, please see pages 10-12 in the attached file.
By the end of the reporting period, all root samples have been processed all the way through to TRFLP analysis of fungal ITS-rDNA sequences (including processing for microscopic observations). Reference fungal cultures were also processed and analysed by Terminal Restriction Fragment Length Polymorphism (TRFLP) to provide fragments database to aid statistical analyses. Results from TRFLP analyses suggest possible co-invasion between Calluna vulgaris and the ericoid mycorrhizal fungus Rhizoscyphus ericae.
The first consequence of our results was to engage with New Zealand's Environmental Protection Authority to modify the fungal species list present in New Zealand and request the inclusion of Rhizoscyphus ericae. The second consequence was that, to back-up our conclusion of fungus-plant invasion, we needed to assess whether this Northern fungal species could also been found in areas not yet invaded by European heathers. To do so, we conducted an additional sampling campaign (not described in Annexe I) including 29 sites covering most part of South New Zealand, and collected a total of 140 additional root samples from 2 native plant genera capable of forming ericoid mycorrhizal associations. All additional samples were processed as described above, and analysed by TRFLP and NGS (next generation sequencing). NGS analyses were contracted to IMGM Laboratories GmbH in Germany. This is a deviation from the original proposal, which was required because the original proposed sequencing facilities (collaborator UWS) were unable to conduct analyses on 454-FLX titanium platform.
Both field-sampling campaigns were financially supported by Landcare Research (capability fund) to cover travel and subsidence cost of student and technical assistance.
Built into objective 3 was the investigation of the fate of soil C released by the decomposing activity of mycorrhizal fungi, but not respired as CO2. We aimed to test the hypothesis that mycorrhizal fungi can take up C released from SOM and transport it from the site of decomposition to the site of mycorrhizal structures located within the plant roots. To address this hypothesis, we planned to develop a magnetic bead capture of fungal rRNA, in collaboration with Drs Pringle & Pearson from Harvard University (Cambridge, USA). The method development was delayed because of the disruptions caused by the 2010 and 2011 Christchurch earthquakes, and was therefore postponed until relocation to European host premises (Aberdeen). Once relocated to Aberdeen, the technical challenges of such methods could not be overcome. Parallel method development has been attempted in the laboratory of our collaborator Professor Ann Pearson (Harvard), but has so far not been taken to a stage where biological applications can be envisaged. This is a late major deviation from Annexe I, with repercussion both research and training objectives. To counter-balance the impact of this technological failure on the science excellence of this project, corrective measures were taken and are described in section 3.4.
To complement the results of the magnetic bead capture of fungal RNA, we collected soil samples from all soil cores labelled with 13C from all plants included in the pot experiment (110 cores in total). Soil samples were destined to provide material for extraction of PLFA, and trace labelled C (13C) within the rapidly turning over cell membrane of the soil microbiota (13C-SIP-PLFA). All soil samples were therefore freeze-dried, milled and shipped to our collaborators in James Hutton Institute, Aberdeen. At the onset of this research fellowship, our collaborator had generously offered to subsidise the cost of the 13C-SIP-PLFA analyses. Because of the delays incurred in the implementation of the experiment and the collection of soil samples, changes in the financial forecast on our collaborator’s side prevented from going ahead with the analyses in the academic year 2012-2013. The samples are currently stored in Aberdeen and awaiting analyses in the coming academic year, but the results will not be available within the scope of the reporting period for this fellowship. Marie Curie funding evidently be acknowledged in any subsequent putative publications including data obtained from these soil samples.
Significant findings and outcomes:
• 1st record of fungus Rhizoscyphus ericae in New Zealand, likely imported into New Zealand with plant host Calluna vulgaris in the mid-1920s.
• Development of species-specific PCR primers for detection of Rhizoscyphus ericae.
• Development of high-throughput gas sampling and isotopic analysis protocol used for measuring 13/12 C isotopic content of soil CO2 efflux.
• Invasion by Calluna vulgaris causes ecosystem degradation in Tongariro National Park: changes in vegetation composition, fungal communities and soil C cycle.
• Native ericoid mycorrhizal communities are host-dependent and spatially structured.
• Global ericoid mycorrhizal ITS-rDNA sequence database freely available to others via UNITE (http://unite.ut.ee/).
• Availability of 2 new whole genome sequences of ericoid mycorrhizal fungi (only 1 had been published so far).
For more information, please see pages 10-12 in the attached file.