Amanita Species Clarified Using ITS Region

Many weeks have been spent in the lab amplifying the DNA extracted from fungal specimens and sending off the PCR products for sequencing.  I switched to a new PCR kit, Taq-Pro Red Core Kit, manufactured by Denville Scientific, Inc.  The results have been promising, with amplifications of the ITS region using primer set ITS5 and ITS4 reaching an average of nearly 70 percent.  This is quite an increase from the average amplification of 30-60 percent for the same ITS region using stock solutions and the same primer set.  The sequence results of 60 specimens from the Hartley Wood collection have been edited using Sequencher v4.8 and a BLAST search performed.  One interesting sequence helped  clarify a pure white Amanita species originally identified by me as Amanita sp. cf. virosa.  The sequence data indicated that this specimen was A. bisporigera, with a 99 percent maximum identity match. The image to the right was taken of A. bisporigera in the field and the image below is a micrograph of the 10x10 micrometer spores.  The sequence result brought up the question of how often this deadly poisonous mushroom is misidentified in the field.  It would be interesting to sample across the numerous vouchers collected from area forays and determine how often this mistake has occurred.

Interesting Xylariaceae Found

During a collection event in June 2009, I came across a very interesting resupinate fungus.  The fruiting body was spreading across the barkless surface of a large downed tree.  I had seen many resupinate fruiting structures during my two years of research, however the gray center and white colored margin of this fungus caught my eyes from nearly ten meters away.  Upon closer examination, this fungus appeared to be in a state of active growth.  Later, this observation would be confirmed with images and descriptions of the lifecycle of this fungus provided through multiple resources.  You might be asking yourself what fungus I have been mentioning.  Kretzschmaria sp. cf. deusta  (Hoffm.) P.M.D. Martin is the proposed name for this collected specimen.  This species of fungus is common, but is often overlooked once the dark black stroma gives it's substrate a burnt appearance.  The fungus causes rot of trees and is often located at the base of infected trees.  Infected trees become quite brittle and should be monitored as they are prone to structural failure with little to no warning.  The upper right image was taken during the immature or actively growing state of this fungus.  The second image allows one to see the mature fruiting structure with the embedded perithecia creating a pimpling of the stroma surface.  A stereoscopic image of the stroma in cross section showcases the perithecia and the asci found within them.  A wet mount was prepared from tissues extracted from the opened perithecia and spores exhibiting classic Xylariaceae characteristics were observed.  The final image clearly shows the narrow, dark-brown to black spores.  The spore size was 32x8 micrometers with a smooth texture and central germ split.    DNA from the collected specimen will be extracted and amplified using PCR.  The PCR product will then be sequenced and this ITS sequence will be entered into a BLAST search to aid in its identification.  Keep an eye open for this very interesting fungus during your next hike through the forest.  Click on the title of this post to view other images of this fungus!

Happy New Year !

Hey folks.  Great to be back in the lab running PCR on my fungal samples and sending them off for sequencing.  I have picked a few interesting specimens from the collection to sequence at first, then will move on to the more common species and genera.  A few of the selected specimens may yet to be described in Pennsylvania and the thought of documenting novel species is exciting.  There is a species within the order Xylariales that is most common in Europe and has rarely been documented in the United States.  Teasing out the data for the past 2 years of collection from my research site will surely be interesting and I am optimistic that the DNA sequences will greatly aid in their identification.  For now I am concentrating on perfecting my techniques in the lab and ability to edit ITS sequence data for submission to GenBank.  Due to the heavy workload this fall, I will most likely be posting results on a weekly basis.  Still, please stop back and provide comments and suggestions.  Have a great year and hope you enjoy the new blog template...it will most likely change as the months progress.

Hartley Wood Research: Initial Findings

I have now spent the better part of two weeks looking through the research database.  As a whole, it includes 612 collected specimens encompassing the phyla of Ascomycota (17%), Basidiomycota (78%), and Myxomycota (5%).  There were 130 genera and 199 species identified, not taking into account any repeats  or  unknown specimens found within the collection.  DNA has been extracted from voucher material with nearly 78% confirmed through gel electrophoresis.  The next step will be presenting the findings to my adviser and determining what extracted DNA to sequence.  My hunch is that all of the identified species will be sequenced and submitted into GenBank.  This should include species found within my sampling plots and those found randomly within the research site.  Once their identification has been confirmed using the ITS region of the DNA, a more confident approach can be made toward describing the species richness within the site.  Average temperature, precipitation, and relative humidity at the site during this two year study was examined with no obvious conclusion as to its impacts on the fruiting frequency of observed fungi.  This came as no surprise because most mycologists understand the ephemeral nature of the fruiting bodies and this study was based on collection/documentation of those fruiting structures.  More sorting and sifting through the database should disclose interesting associations among the specimens within the collection.  I will be sure to keep you all up to date on these findings as they become available.  Have a safe and happy holiday season!

Visualizing Extracted DNA

I have spent the better part a week extracting DNA from dried tissue samples acquired during my research in the Hartley Wood.  There is over 600 specimens in the collection, of which 420 have had their DNA extracted.  Now begins the task of confirming the presence of DNA using gel electrophoresis.  This is not to say that if DNA is absent using this visual assay that it is physically absent, only that I could not observe the bands created during the electrophoresis process.  I have not standardized the concentration of extracted DNA before running the gels and therefore some bands are easy to see and others are quite faint.  Again, this assay is only being performed as a troubleshooting tool if my PCR products are not visible when run out on a gel.  Some of these terms might be foreign to some of you and I apologize, still the main point I am trying to express is the tedious nature of describing my collection through molecular techniques.  In today's field of mycology, these molecular characteristics (DNA sequences) are valuable sources of information used to identify and describe relationships between groups or species of fungi.  To the right is the image of a gel that has been stained with Ethidium bromide and photographed under UV illumination.  The "L" in the far left lanes are the 1Kb ladder used as a reference of size and the numbers above the remaining lanes indicate the extraction codes for the specimens.