Fungi Saltmarsh

Assessment of fungal diversity of Gulf Coast saltmarshes,

with implications for coastal restoration.

Allison Kennedy and Jinx Campbell

Department of Coastal Sciences, University of Southern Mississippi

703 East Beach Drive, Ocean Springs, MS 39564 allison.kennedy@usm.edu

DISCUSSION

Five species of saprophytic marine fungi were detected morphologically on S. alterniflora from both natural and created sites (Fig. 2). A significantly greater number of fruit bodies were produced by the dominant species P. spartinicola. Morphological detection is limited to those species which are fruiting. ITS T-RFLP analysis detected additional species and indicated a higher species richness on S. alterniflora from the created island (Fig. 4). T-RFLP analysis did not detect Mycosphaerella sp. 2 in the Fall 2005 natural site collection, while direct microscopy did. Also, the restriction digest of Mycosphaerella sp. 1 did not produce fragments of usable size for molecular site profiling, indicating that T-RFLP studies can only provide conservative diversity estimates. T-RFLPs can detect the presence of a species, but cannot prove the absence of one (Dickie et al 2002). Correlating fragment size with species helps to avoid biases introduced by sampling, fungal genomic structure and restriction enzyme characteristics (Avis et al 2006). A combination of methods is needed to fully characterize the fungal community present.

ITS T-RFLP Analysis

Cultures of dominant marine saprotrophic fungi were generated from single-spore isolates and DNA was extracted using DNeasy Plant Mini Kit (Qiagen). Bulk community DNA was extracted from leaves of S. alterniflora and J. roemerianus using FastDNA SPIN Kit for Soil (QBiogene). The ITS region was amplified using the ascomycete specific primers ITS 1F and ITS 4A (Larena et al 1999). The ITS 1F primer was fluorescently labeled on the 5' end with FAM (Invitrogen). Products were purified using Qiaquick PCR Purification Kit (Qiagen) and digested with 10 U of the restriction enzyme HaeIII (Roche Diagnostics) in a 10 ml total volume containing either 10 ng (isolate) or 100 ng (community) purified PCR product. Digests were carried out at 37 C for 3 h. Terminal restriction fragment lengths were determined on an ABI PRISM 3730xl Analyzer using ABI GeneMapper software.