UNIVERSITY OF WISCONSIN-LA CROSSE Graduate Studies MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF MYCORRHIZAL FUNGI ASSOCIATED WITH A DISJUNCT STAND OF AMERiCAN CHESTNUT (Casianea dentata) IN WISCONSIN A Chapter Style Thesis Submitted in Partial Fulfillment ofthe Requirements for the Degree ofMaster ofScience in Biology Jonathan M. Palmer College ofScience and Health December, 2006 MORPHOLOGICAL AND MOLECULA~RCHARACTERIZATION OF MYCORRHIZAL FUNGI ASSOCIATED WITH A DISJUNCT STAND OF A:MERICAN CHESTNUT (Castanea dentata) IN \VISCONSlN By Jonathan M. Palmer V.le recommend acceptance ofthis thesis in partial fulfillment ofthe candidate's requirements for the degree of Master of Science in Biology The candidate has complewd the oral defense of lht~ thesis. .1 . I ­ <J ~ ~ ::J/;' n '1· /./} Ii ,iii ~!".-/ .i.J i / ~.) __ (",;'~.) .." e.:>i .~~.-~,.:,l-L~:!--.-~g.Q __/-;-+y-<',,,,--::r.;:;.L...><t?:-e:..·_ .... _ Thomas J. VolkV !Yate Thesis Cornmittee Chairperson /--.,.) /f ./v I :'.~~;J2: ~---------. {lldt)/~ .. .t...: Daniel L. Lindner f ---, Date Thesis CO.ITl.lTlittee Member ;1/I ' I -;813 !-"'?,)/}'" __.__. .!+_ ,~. 'UI ,;t, v ...' k Dah.~ l ~ , i~~r-l /t:\1L~ A.·S'ri1.~')(J,"tA !\ ----"'1 t') ~'/ (~Oi /' (",:),A -Yv"l Y~, \n.. .. __ _. \ '--'C., v ~ l' Meredith Thomsen Date Thesis Committee Member Ibe~is accepted Vijendra K. Agarwal, Ph,D. Date Director, University Graduate Studies and Associate Vice Chancellor ABSTRACT Palmer. JJvL Morphological and molecular charactcrization ofmycorrhizal [\UJgi associated with a disjunct stand ofAmerican chestnut (Castanea dentata) in \\7isconsin. 1'.15 in Biology, December 2006. 77 pages (TJ Volk) Circa 1900 a farmer from the eastern U.S. planted eleven American chestnut (Castanea dentata) seeds on a newly established farm near West SaleHI in western Wis(:onsin. Thesc tre(~s were very suci.x~ssful, producing a large stand of ov(~r 6000 trees. Since this area was weil outside of the natur-ai range of chestnut, these trees remained frec from chestmlt blight., caused by Cryphonectria parasitica, until 1987. Because chestnut tre($ v,Jere almost entirely diminated from their native range by the early 1950's, no modem studies have exarnined mycorrhizae of a mature chestnut forest. To identify putative mycorrhizal associates ofchestnut, our approach was two-f()ld: (1) an exterlsive fruiting body survey was conducted for 3 seasons that yielded over 400 collections, of \vhich approximately 99 were putatively mycorrhizal, and (2) a belovl-ground molecular approach was used to generate DNA sequences ofthe ITS region from rnycorrhizae. To date, 100 root tip sequelwes have been generated. These seqm.'nces are phylogenetically diverse, although all are basidiomycetes or ascornycetes fallingjnto 11 families. In addition, 84 of the 99 fruiting body collections have been sequenced. From these data a website was created, \\'Vv'"\v.chestnutfimgi.com, which contains a BLAST searchable databa...;;e ofiTS sequences coupled with pictures ofboth root tips and fruiting bodies. ACKNO'WLEDGEMENTS First ofall, I would like to thank my family, Joseph, Kathleen, Matthc\v, and Sarah Palmer (also my dog, Layla), for their love and unwavering support throughout rny academic career. \Vithout their guidance I would not be who ram today, nor \vould this work have ever heen completed. I would also like to thank my committee members, Tom Volk, Dan Lindner, Mike Abler, and Men.'dith Thomsen for their constructive criticism ofthis manuscript Most importantly I would like to thank Tom for bringing me into the field ofmycology. He refers to me as an accidental mycologist, having taken his mycology course simply because it was the only elective that fit into my schedule. But his passion for mycology and specifically teaching mycology grew on my as the course progressed, and before long I was hooked. Torn has been an exceptional mentor, an exceptional teacher, but most importantly has become an exceptional friend. He has taught me the intricacies of mycology, but the lessons that will remain ingrained in my mind are more about life itseH: His kindness, generoslty, and passion tor life are some of his superlative virtues that will leave a lasting impression. rwould also I1ke to thank the Dolores Rhyme and Ron Brockenhauer families tor unl1mited access to the \Vest Salem chestnut site. In addtion, Da.niel T. Kinney, Sean Westmoreland, Nik Zitomer, Adarn Gusse, Kelsea Jc\vel, Bernadette O'Reilly. participants ofthe 2001 Smith foray, and 2005 NA11A foray for their help \vith collections. it TABLE OF CONTENTS EAQE .A:SSTR/\.<..-::-r'", , , , , , , , i ACK,1\JO\VLEDGEMENTS , , ii lJS'r ()F 'rAI3LES.. , , v LIS'1' ()F FICilJRf:S , vi LIST OF AI.PPENDICES , ,.., viii CHA.PTER 1: BACKGROUND AND INTRODUCTORY iNFORMATION Introduction " . lVlycorrhlzae ' .. 7 Ectoluycon·hizae , > ••••• , •••• 9 Site Characterization ofthe West Salem Chestnut Stand . 14 Ectomvcorrhizal SIJeclficitv . 19 ~ . Myconhizal Colonization of the \Vest Salem Chestnut Stand . 21 Mycorrhizal Fungi and Species Concepts .. 2J DNA, the Molecular Tools for Identification, and The World Wide Web ... 26 Objectives , , '" , , , .. 31 ",,"' Experinlcntal Design " '" ." '" '" " . ..1 ...) C:onclusions . 37 H.eferences . 38 111 CHAPTER II: ECTOMYCORRHIZAL CHA.RACTERIZATION OF AN AMERICAN CHESTNUT (Castanea dentata) DOMINATED COMJ'vHJNiTY rN WESTERN \VISCONSrN. (prepared for submission to A{vcorrhiza) fNTRC)DUC'TI()N, <0 ••••• , , "" ••• ••••••••• 43 fviATERIALS AND METHODS , ,. 49 Above-ground Sampling.. , , >.. 49 Below-ground Sampling <0.... 49 DNA Extraction, peR, and Sequencing '" 50 Web-site• De.;.,1""gn ,..... ,,";',,"" RE.S lJL'rS , ,., , " , , " ,,.. 54 Above-ground Sampling , ".......................... 54 Below-ground Sampling ,., ,........... 54 fHS(::LJSS:!()N' , <0 "', ,. 6i ECM Composition and Diversity , , ,..................... 61 Plant Host Complexity , ,.. ... 63 (:onclusions , ,..... 66 HJ~FERE'NCES.« > ••••••••••••••••• , ••••••••• ", ••• '" ,., •••••••••••••••••••••••• 69 lV LIST OF TABLES Tl~J1LJ~ £~igE 1. Kev feamres ofthe two most common .f()rms ofmvcorrhizae , ,.. 1. {} . ~ 2. Summary oftree vegetation at the West Salem chestnut stand...... 20 3. Mycorrhizal status ofthe tree vegetation at the West Salem.."....................... 22 4. Reported mycorrhizal status of the tree vegetation at the West Salem stand..... 4 <i 5. Sequence accession numbers and rnolecular ldentifi,~ation ofECJ\,l mot tips collected at the West Salem chestnut stand , ., ,., 55 6. Molecular identifications ofputative ECM root tips based on DNA sequel1\.~es , " ,.................................. 59 v LIST OF FIGURES PJI1.tJ.RE PAGE 1, Aruerican chestnuts are borIH.~ 3 to a bur and the nuts are dropped in late fall at the largest remaining chestnut stand in West Salem" WI. Wisconsin's largest American chestnut tree has a dbh (diameter at breast height) of approximatdy 4 fcet and is over 100 years old. It \vas th ..., one ofthe original 11 planted around the turn ofthe 20 century .""... " .;;.. 2. /\ canker on an Amerkan chestnut tree that sprouts vigorously from its root slock afler being -jntilcted with Cryphonectrla. Sprouting from the roots is usually one ofthe first signs of infection. A close-up ofa chestnut blight canker shows orange stroma poking through the bark. Embedded in the stroma are reproductive structun.$ that f()fcibly eject spores from an infected tree " " 00 .. 4 3, My\.~orrhizal Hmgi wrap themselves around the roots of a suitable host tree and emanate out into the soil. The fungi then absorb water and nutrients for the plant and in turn the plant gives photosynthates to its fungal synlbiont " , , , < •••••••••••••• 8 4, Ectomycorrhizal fungi are members ofthe Basidiomycota and Ascomycota and their fruiting structures can take a variety ofshapes, colors and sizes. Some are choice edible fungi such as Boletus edulis and Cantharelius cibarius,whiJe others are inconspicuous like Tomentella cristata. The deadly poisonous AmaniiabL~porigerais also an ectomycorrhizal mushroom '" 00' >< >< ".. 11 5. Cross sectional drawing ofan ectomyccHThizal root tlp showing the mantle, the hyphae wrapped around the outside ofthe root. The Hartig net is the hyphae growing in between the cortical cells ofthe root, which is ".'here the exchange ofnutriems occurs...."",............................ 13 6. Examples ofmycorrhizal root tips ofAmerican chestnut (Castanea dentata) A and B are yet to be identified mycorrhizas, C is Boletus puiverulentus and Dis Byssocorticium atrovirens. Note the short and fat growth ofthe root tips as well as the branching patterns. Also some species exhibit extensive hyphaI growth. while others do not.. ..... .. ..... 15 7. Chestnut Hills in West Salem, \Visconsin is located well outside ofthe natural range of American chestnut.. " "........................17 VI 8. ITS primer map shmving the relative locations ofcommon primers used tl-,[ the amplification ofthe ITS region ofthe rONA... 28 9. Only one PHP template page is required to display the information about a given species. The Boletus pu!verulentus page and the Byssocorticium atrovirens page are simply different entries in the MySQL database. This saves server space and is easier to maint?in...._ 32 10. A two-fold experimental approach including a traditional above­ ground fungal biodiversity survey and a modem molecular-based below-ground survey yields a more a(.~curate depiction ofthe mycorrhizal associates ofchestnut. .. ... ... ... ... ... ..... 34 11 Distribution of fruiting bodies conected from