University of Southern Queensland – BSc. (Hons) Thesis Jed Calvert U1098952 Su$ervisor% Dr John &earnaley 2017 1 Cover i(a)e sho*s hy$hae of an endophytic fungus (+$iognomonia s$., isolate -. 2.1.# cultured from roots of the native Australian orchid Bul/o$hyllu( e0iguu() gro*in) against the inside of an agar plate. Bar is 50μm. # +/stract Throughout their life cycles orchids are reliant on inorganic nutrients $rovided /y mutualistic orchid mycorrhi2al fungi (345), to *hich the $lants allocate sugars $roduced in $hotosynthesis. 3rchid seeds usually re6uire a fungal $artner to facilitate their germination, and sometimes a se6uence of fungal s$ecies to $romote their growth to maturity. 3rchid mycorrhi2al (3M) relationshi$s can /e very s$ecific, with e$i$hytic/lithophytic (tree/roc8 d*elling) orchids often associating *ith a narrow range of fungi. Additionally, closely-related grou$s of orchids tend to share common associations *ith the same lineages of fungi. The genus Bul/ophyllu( is the largest in the 3rchidaceae (:2000 s$$.) /ut no study to date has investigated mycorrhi2al associations of Bul/ophyllum in Australia. ;n this study, fungi *ere isolated from the roots of the native orchids B. e0i)uu(, B. /racteatu(, B. minutissimu(, B. elisae and B. she$herdii at 7 sites in south-east Queensland. 5ungi *ere identified /ased on internal transcri/ed s$acer (;TS) gene se6uences to determine *hether these congeneric orchids share 345 $artners. Analysis of orchid <uBisC3 large su/unit (rbcL) gene se6uences *as also $erformed to ascertain $hylogenetic relationshi$s, and sy(/iotic seed germination of B. e0iguum *as tested using 4 fungal inocula. ;n all, 90 fungal isolates *ere obtained. Molecular identification revealed a diversity of $utatively mycorrhi2al fungi from the 345 genera Tulasnella, Serendi$ita and Ceratobasidiu(, and dark se$tate endophytes (&S?s) from the ascomycete order Helotiales. Significantly, 3 orchid s$$. (B. e0iguu(, B. /racteatum and B. elisae) across 3 sites *ere found to harbour a single Tulasnella s$. that is li8ely ne* to science. This indicated narrow 345 s$ecificity and suggested that these orchids may /elong to a common su/-clade *ithin Bul/ophyllu(, an observation su$$orted /y $hylogenetic analysis of rbcL genes and /y taxonomic reassignments that have /een $roposed /ased solely on morphological features. B. e0iguum *as found to harbour an undescri/ed Serendi$ita s$. that *arrants investigation as a $otential agricultural inoculum. B. she$herdii harboured a Ceratobasidium s$. $reviously found in Norway, /ut the $lant sampled in this study had /een relocated from nearby *oodland and thus may not usually associate *ith this 345. ;solation of &S? helotialean fungi *ith highest BLAST matches to ericoid mycorrhi2al (?rM) se6uences $ointed to a $ossi/le 345 role for these isolates, however further confirmation is needed to establish *hether intracellular nutrient-e0change structures are $resent. The $resence of ?rM-li8e fungi associated *ith orchids su$$orts recent *ork suggesting a /lurring of functional /oundaries /et*een mycorrhi2al ty$es. B. e0iguum seed germination e0$eriments *ere i($eded /y overgrowth of fungal contaminants, *hich *ere li8ely endophytes from seed $od tissue, and /y $oorly-developed seeds that may have resulted from in/reeding. 5uture studies *ith seed germination $rotocols optimised for very small $ods are needed to ascertain *hether fungal sy(/ionts can stimulate germination in these Bul/ophyllum s$$. These results $rovide evidence for narrow 345 s$ecificity for Tulasnella in some S? Queensland Bul/ophyllu( s$$. and further raise the intriguing possi/ility of DS? forming mycorrhi2as *ith orchids. &eclaration ; certify that the *ork re$orted in this thesis is *holly my own e0ce$t *here otherwise noted. ; also certify that this *ork is original and has not /een $reviously su/mitted for assessment in any other course of study at any other university. Jed Calvert Honours candidate Date ?ndorsed by &r John &.A. &earnaley Su$ervisor Date > +c8no*led)e(ents Than8 you to my su$ervisor, &r John &earnaley, *ho has given me the op$ortunity to learn about t*o fascinatin) grou$s of organisms and e0$lore some /eautiful forest landscapes in the $rocess, as *ell as to greatly i($rove my *ritin) and laboratory s8ills. You have /een $atient, 8nowledgeable, su$$ortive and a great guy to /e around. Than8 you also to Shirali. You hel$ me in a million different *ays, and your care and fle0i/ility have meant that my research never feels overwhelming or burdensome. At USQ ; *ould also li8e to than8 &r Ant Youn), *ho has $rovided some very interestin) conversations and made hel$ful suggestions to i($rove my *riting. Than8 you also to &r Levente Ciss, *hose insights into the *orld of Mycology and academic machinations have /een ins$iring and instructive. 5inally, ; *ould li8e to than8 my mu( and dad, *ho have al*ays /een full of love and su$$ort, never $ushin) me in any direction ; didnDt *ant to *al8 myself. ; canDt /elieve how luc8y I am to have t*o $eople as lovely as you as my $arents. " Ta/le of Contents +/stract EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE.EEEEE. &eclaration EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE.EE..E > Ac8nowledgements EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE..E.. " Table of Contents EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE..EEE F List of 5igures EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE 10 List of Tables EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE. 13 List of +//reviations EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE. 15 Glossary of Technical Terms EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE..E 15 1. ;ntroduction EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE.E 17 1.1 Orchid mycorrhi2al fungi (345) EEEEEEEEEEEEEEEEEEEEEEEEEE.. 17 1.# E$i$hytic orchids and O45 EEEEEEEEEEEEEEEEEEEEEEEEEEE.EE 21 1.. The genus Bul/o$hyllu( EEEEEEEEEEEEEEEEEEEEEEEEEEEE...EE #. 1.> O45 evolution, ecology and conservation EEEEEEEEEEEEEEEEEEEE.EE #> 1." Project hy$otheses and overvie* EEEEEEEEEEEEEEEEEEEEEEEE...EE #F #. 4aterials I 4ethods EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE..EEE #! 2.1 Identification of orchid mycorrhi2al fungi EEEEEEEEEEEEEEEEEEEE..EE #! 2.1.1 Collection sites EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE.EEE #! 2.1.# Root sampling EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE..EEE 30 2.1.. Isolation and culturing of mycorrhi2al fungi EEEEEEEEEEEEEEEEE.EEE 30 2.1.> Fungal DNA e0traction, poly(erase chain reaction (-C<) and internal transcri/ed s$acer (;TS) gene se6uencing EE...EEEEEEEEEEEEEEEEEEEEEEEEEEE 31 F 2.1." Molecular identification of mycorrhi2al fungi EEEEEEEEEEEEEEEEEE..E .. 2.1.F Phylogenetic analysis of fungal ITS gene se6uences EEEEEEEEEEEEEEE..E .> 2.1.F.1 Phylogeny of Serendi$ita isolates EEEEEEEEEEEEEEEEEEEE..EE .> 2.1.F.2 Phylogeny of Tulasnella isolates EEEEEEEEEEEEEEEEEEEEEE.E ." 2.1.F.3 Phylogeny of Ceratobasidiu( isolate EEEEEEEEEEEEEEEEEE..EE ." 2.1.F.1 Phylogeny of Helotiales isolates EEEEEEEEEEEEEEEEEEEEE.EE .F 2.# Phylogenetic analysis of Bul/ophyllu( orchids EEEEEEEEEEEEEEEEEE..E .F 2.2.1 Leaf tissue collection EEEEEEEEEEEEEEEEEEEEEEEEEEE..EE. .F 2.2.# Plant DNA e0traction, polymerase chain reaction (-C<) and se6uencing E..E .' 2.2.. Phylogenetic analysis EEEEEEEEEEEEEEEEEEEEEEEEEEE..EE .' 2.. Bul/ophyllum e0iguu( mycorrhi2al seed germination EEEEEEEEEEEEEEEE . 2.3.1 Collection of seed pods EEEEEEEEEEEEEEEEEEEEEEEEEEE..E. 2.3.# Mycorrhi2al seed germination e0$eri(ents EEEEEEEEEEEEEEEE..EE . .. <esults EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE.EEE >. 3.1 Molecular identification of mycorrhi2al fungi EEEEEEEEEEEEEEEEEE..EE >. 3.1.1 Isolation and culturing of fungi EEEEEEEEEEEEEEEEEEEEEEE..E >. 3.1.# Fungal PC< of ITS gene re)ion EEEEEEEEEEEEEEEEEEEEE.EEE >" 3.1.. DNA se6uencing and identification using the basic local alignment search tool (BLAST) EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE >F 3.1.> Molecular identification of fungal isolates EEEEEEEEEEEEEEEEE.EE >' 3.1." Phylogenetic analysis of fungal isolates EEEEEEEEEEEEE..EEEEEEE "" 3.1.".1 Phylogeny of Serendi$ita isolates EEEEEEEEEEEEEEEEEE.EE "" 3.1.".2 Phylogeny of Tulasnella isolates EEEEEEEEEEEEEEEEEEEEE "' ' 3.1.".3 Phylogeny of Ceratobasidiu( isolate EEEEEEEEEEEEEEEEE..E " 3.1.".4 Phylogeny of Helotiales isolates EEEEEEEEEEEEEEEE.EEEEE F# 3.1.".5 Fungal phylogeny summary% taxonomic assignments EEEEEEEEE.E F" 3.# Phylogenetic analysis of Bul/ophyllu( orchids EEEEEEEEEEEEEEEEE..EE F" 3.2.1 Orchid PC< and ri/ulose-1,"9/is$hos$hate carboxylase/oxygenase (<uBisC3) large su/unit (rbcL) gene se6uencin) EEEEEEEEEEEEEEEE..EEEEEEE F" 3.2.# Phylogenetic analysis EEEEEEEEEEEEEEEEEEEEEEEEEE..EEE FF 3.. Bul/ophyllum e0iguu( mycorrhi2al seed germination EEEEEEEEEEEEEEEE 70 3.3.1 Collection and desiccation of seed pods EEEEEEEEEEEEEEEEEE.EE 70 3.3.# Mycorrhi2al seed germination e0$eri(ents EEEEEEEEEEEEEEEE..EE 71 >. &iscussion EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE..E '# 4.1 Roots of S? Q=& Bul/ophyllu( s$$. harbour a diverse range of $utatively mycorrhi2al fungi from three O45 genera and one ascomycete order EEEEEEE.EE '# 4.1.1 Tulasnella isolates EEEEEEEEEEEEEEEEEEEEEEEEEEEEE.EE '. 4.1.# Serendi$ita isolates EEEEEEEEEEEEEEEEEEEEEEEEEEEE..EE 'F 4.1.. Ceratobasidiu( isolate EEEEEEEEEEEEEEEEEEEEEEEEEEEEE '! 4.1.> Dark se$tate endophyte (Helotiales) isolates% functional overlap bet*een ericoid and orchid mycorrhi2asJ EEEEEEEEEEEEEEEEEEEEEEEEEEEE.EE 80 4.# Phylogeny of Bul/ophyllu( s$$. EEEEEEEEEEEEEEEEEEEEEEEEEEE !# 4.. Limitations and potential sources of error EEEEEEEEEEEEEEE.EEEEEEE !> 4.> Future directions and potential ap$lications of findings EEEEEEEEEEEEE.EE 87 ! ". Conclusions EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE