MYCODIVERSITY · Using next generation DNA sequencing to link soil fungal diversity to ecosystem function

Using next generation DNA sequencing to link soil fungal diversity to ecosystem function

Background : This Marie Curie International Outgoing Fellowship consisted of two phases 1) an outgoing phase (18 months) in the University of California, Berkeley (UCB) and 2) a return phase (12 months) in Newcastle University (NU), UK. Research skills developed in the outgoing phase were applied to research carried out in the return phase and the large amount of data generated in the outgoing phase was also assessed in the return phase. Outgoing phase (UCB): The work focussed on the use of next generation DNA sequencing (NGS) to gain a better understanding of the diversity of active ectomycorrhizal fungi (EMF) in a unique Californian pine forest ecosystem. EMF are essential for productive forest ecosystems as they transfer nutrients to plants via their close association with plant roots. The use of new molecular techniques such as NGS offers the potential to fully assess the true diversity of fungi present in an ecosystem and therefore assist in conservation and re-establishment of forests on degraded land. Work carried out : A method to sample for growing (and therefore active) fungal hyphae in soil involving ‘in growth’ bags was used throughout the work. This involved the ‘in situ’ planting in soil around pine trees of an ‘in growth’ bag containing sterile acid washed sand; fungal hyphae grow into this bag from the soil and DNA can extracted from the hyphae inside bags for subsequent species identification. This allowed the diversity of active fungi in the pine forest soil to be assessed both spatially and over time. The research focussed on the Bishop Pine forest ecosystem at Point Reyes National Seashore (PRNS : approximately 1 and a half hours drive north of Berkeley, CA) and an effective DNA extraction technique was developed for the in growth bag samples. The DNA extracts obtained were examined for their suitability to use in next generation DNA sequencing for fungal diversity estimation. In this work fungal specific PCR products were obtained using fungal ITS primers (developed in the USA hosts laboratory) and sequenced using NGS (specifically a technique called pyrosequencing). A major part of the research involved the analysis of the large amount of DNA sequence data produced (e.g. typically 100,000 DNA sequences were obtained for each sample time) and a software analysis package (QIIME) was used to analyse the data. The project also took advantage of specialist fungal databases available in the Fellows host laboratory to enable accurate identification of the fungal species present and hence obtain diversity estimates. Interestingly, the fungal ITS primers used also provided an assessment of the active saprophytic fungal diversity in samples giving an additional dimension to the results. A field site was established during the initial stages of the project and 20 trees (10 young and 10 old) studied extensively to determine the spatial distribution of active fungi around each tree and how the diversity of the active fungi varied with time. Altogether 160 individual sample sites (8 per tree) were established and sampled at each time period. Overall, the field site was monitored for 12 months (samples taken every 2-3 months depending on season). The resulting fungal population data was analysed with appropriate statistical methods using the freely available R software. Soil analyses including total Bishop Pine root density, organic carbon, pH, total nitrogen and available phosphorous were also undertaken in order to determine if any of these factors were linked to the fungal population distributions observed. Extra objective : to develop more skills in the study of ectomycorrhizal fungi and to take advantage of the hosts laboratory expertise and facilities the Fellow established a laboratory based experiment to assess the effect of pollution (in this case, silver nanoparticles) on the diversity of ectomycorrhizal fungi found on Bishop Pine. This novel work enabled the applicant to study ECM fungi developing on plant roots and establish methodologies to extract fungal DNA from plant roots for subsequent molecular analysis allowing identification of the species present. Main results (outgoing phase): • Hyphal in growth bags are not entirely specific for EMF. This result contrasts with previous work but also enhances the potential of the use of in growth bags to study overall fungal diversity (symbiotic and saprophytic fungi) in soil. • there are no significant differences EMF population structures associated with distance from the trees used in this study but old and young tress were found to host distinct EMF communities. • Significant spatial diversity of the saprophytic fungal population in pine forest soil is evident and linked to distance from the tree trunk. This indicates that different environmental factors govern population structures of different fungal guilds. • Soil contamination with silver nanoparticles affects pine root growth and ectomycorrhizal species found on pine roots. Return phase (12 months : Newcastle University, UK). The techniques developed during the outgoing phase were used to assess the effect of predicted (Year 2050) atmospheric ozone levels on soil fungal (and bacterial) communities in typical grassland soils. The work took advantage of a unique long term (9 year) ozone exposure experiment at Newcastle University where elevated ozone has been shown to alter plant communities – we were interested to see if this plant community change was linked to any below ground changes in soil microbial communities and activities. Quadruplicate soil samples were taken from grassland mesocosms that had been exposed to current (control) and predicted atmospheric ozone levels for 9 years. DNA was extracted from the samples and subjected to either fungal or bacterial specific PCR and the PCR products analysed by two techniques : denaturing gradient gel electrophoresis and NGS (pyrosequencing). This allowed comparison of these molecular techniques to determine changes in microbial populations. Additional soil samples were taken to examine soil function using microrespirometry – essentially a method to determine the ability of the soil microbial community to transform a variety of carbon sources. The effect of ozone exposure on plant litter transformation was also studied and microbial diversity studies on the degraded plant material (6 month and 11 month incubations) are being carried out (ongoing). Further detailed statistical analysis of the Point Reyes data obtained during the outgoing phase was also performed. Main results (return phase) • Further statistical analysis from the Point Reyes field study shows that C:N ratios in soil appear to have a role in structuring active EMF communities • Continued ozone exposure of grassland mesocosms has small but significant effects on soil microbial communities and their function (change in the rate of carbon source utilisation) • Exposure of plants to predicted atmospheric ozone levels reduces subsequent plant litter mass loss. • Ozone exposed plant litter has a different microbial community to non-ozone exposed plant litter Expected final results and potential impact and use : The microbial (fungal and bacterial) communities involved in plant litter transformation are currently being assessed (the final samples were processed just before the end of the return phase). Initial results indicate that the bacterial community of ozone exposed plant litter is different to that of non-ozone exposed litter. We suspect that the microbial community results may offer insights as to why exposure to elevated ozone levels reduces rates of plant litter transformation – an essential nutrient recycling process. Overall, the results provide fundamental information of importance to the health of forest and grassland ecosystems. From a practical perspective, ultimately the information and techniques learned will be useful to improve and assess the success of regeneration of degraded land and improve current forest and grassland techniques. The technology is also applicable to many types of agriculture e.g. assessing soil health in organic agriculture and arid lands and so provides long-term research benefits. The Fellow has already been awarded further research funding (70,000 pounds) and is involved in several large grant applications (total application value of 2 million pounds) to study soil microbial communities.

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