Ribosomal RNA gene detection and targeted culture of novel nitrogen-responsive fungal taxa from temperate pine forest soil.
Soil fungal communities are responsible for carbon and nitrogen (N) cycling. The high complexity of the soil fungal community and the high proportion of taxonomically unidentifiable sequences confound ecological interpretations in field studies because physiological information is lacking for many organisms known only by their rRNA sequences. This situation forces experimental comparisons to be made at broader taxonomic racks where functions become difficult to infer. The objective of this study was to determine OTU (operational taxonomic units) level responses of the soil fungal community to N enrichment in a temperate pine forest experiment and to use the sequencing data to guide culture efforts of novel N-responsive fungal taxa. Replicate samples from four soil horizons (up to 10 cm depth) were obtained from ambient, enriched CO2and N-fertilization plots. Through a fungal large subunit rRNA gene (LSU) sequencing survey, we identified two novel fungal clades that were abundant in our soil sampling (representing up to 27% of the sequences in some samples) and responsive to changes in soil N. The two N-responsive taxa with no predicted taxonomic association were targeted for isolation and culturing from specific soil samples where their sequences were abundant. Representatives of both OTUs were successfully cultured using a filtration approach. One taxon (OTU6) was most closely related to Saccharomycotina; the second taxon (OTU69) was most closely related to Mucoromycotina. Both taxa likely represent novel species. This study shows how observation of specific OTUs level responses to altered N status in a large rRNA gene field survey provided the impetus to design targeted culture approaches for isolation of novel N-responsive fungal taxa.
a Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545.
Cedar N
CN
Hesse
b Department of Biological Sciences, Western Illinois University, Macomb, Illinois 61455.
Terry J
TJ
Torres-Cruz
b Department of Biological Sciences, Western Illinois University, Macomb, Illinois 61455.
Terri Billingsley
TB
Tobias
b Department of Biological Sciences, Western Illinois University, Macomb, Illinois 61455.
Maryam
M
Al-Matruk
b Department of Biological Sciences, Western Illinois University, Macomb, Illinois 61455.
Andrea
A
Porras-Alfaro
a Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545.
Cheryl R
CR
Kuske
D016000:Cluster Analysis; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D065928:Forests; D005658:Fungi / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification* / Q000378:metabolism*; D008828:Microbiological Techniques; D009584:Nitrogen / Q000378:metabolism*; D010802:Phylogeny; D028223:Pinus / Q000254:growth & development; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D012339:RNA, Ribosomal, 28S; D009584:Nitrogen
Duke Forest; Endogone; Mucoromycotina; Saccharomycotina; soil nitrogen