Effects of Fire on Pathogens and Mycorrhizae Associated with Forest Trees and Woody Vegetation in Fire Damages Areas of Great Smoky Mountains Using Illumina Sequencing and Field Plot Collecting of Fleshy Fungi.

Specific Plot Design: Selection of specific sites will include those recently established by Dr. Karen Hughes (lead PI) at Twin Creeks (mixed burn), Baskins Creek Trail (mid to heavy burn), Balsom Point quiet walkway (mixed burn), Cove Hardwood Trail out of the Chimneys Picnic Area (mixed burn), yet to be established is the severe burn plots at Bullshead Trail and at severe burned heath area. Final plot establishment will be completed in April 2017 with guidance by Park Service personnel familiar with the fire severities at different sites in park. I will select the specific sites based on at least three treatments: control or health plots (4 replicate plots), lightly damaged sites (4 replicate plots) litter surface layer burned, and heavy or severe fire 4 replicate plots). This would account for 12 plots/forest type. I hope to locate two forest types to replicate the experiment and treatments. Levels of fire severity based on soil surface and sublayer damage will be defined in more detail before plots are established. Fire damaged plot areas will be measured for slope and aspect using four randomly selected points per area and averaged. Grids (subplots- 4 m × 4 m) will be established within 20 × 20 m plots to enable collections of fleshy fungi to assess and monitor sporophore formation of ectomycorrhizal (EMC), wood decay and possible parasitic soil fungi. For the Illumina Whole-Community Study, the soils are time=zero soils collected as part of the Nitrogen Mineralization measurements, at 0-10 cm depth from each of the 20 plots. First soil sampling will be completed in May, 2017 with another later during the same year to evaluate microbial recovery potential (October). The samples are composited soils from each plot, made up from 10 soil cores per plot; PVC cores are 4.8 cm diameter. Soils will be used for both nutrient analysis (total C, total N, pH, Ca, Mg, K, ECEC, % base saturation) and for microbial community analysis using next generation sequencing. Genomic DNA will be extracted from the soils for evaluating spatial distributions of below ground mycelium (eg stipitate hydnums) compared with data from sporophore formation following methods by Van der Linde et al. (2008). In addition, information on abiotic climatic factors will be recorded by a climate stations and macronutrients especially C and N (eg. total N) measurements will be collected from soil and forest floor N pools (Knoepp et al. 2008). Identification of fungi from soil samples: Sporophore forming ECM and other fungi will be characterized to species (as possible), as will species richness, diversity, and abundance (evenness) of each species using standard ecological methods (Baird et al. 2007, 2009). To identify species, community composition and abundance Illumina sequencing will be employed using primers concentration on ITS 2 gene region for sequencing. Potentially for each sample, Illumina sequencing can identify up to 1500 fungal species (Gihring et al. 2011). DNA will be extracted from these soil-litter samples using several protocol including Mo Bio Power® kits and CTAB as needed. The variable region ITS2 rDNA of fungi as stated above will be PCR amplified and analyzed using Illumina sequencing (Illumina Technology, Eurofin MWG). In order to reduce costs proven published strategies allowing multiple samples to be sequenced in a single tube will be used to identify fungi from soil-litter samples. Sporphore (mushroom) collections: Adjacent to each rhodendron (riparian) plot at each location, all sporophores present will be collected monthly over a three-year period (April-November) or 21 sampling dates for 2017 – 2019. Collections will be made for all sporophores and documented as per 4 × 4 m subplot, and returned to laboratory for specimen labeling, photographic documentation, morphological data collection and dried for preservation and herbarium storage. Computation of fungal richness and diversity will be compared to nutrient data

Tags: aphn, appalachian-highlands-network, chimney-tops-2-fire, chimney-tops-two, ct2, ecological-framework-landscapes-extreme-disturbance-events-extreme-disturbance-events, ecological-framework-landscapes-fire-and-fuel-dynamics-fire-and-fuel-dynamics, fire, great-smoky-mountains-national-park, grsm, origin-external, ser, southeast-region, studyid-grsm-01299
Organization: Department of the Interior