Distribution of Neotyphodium lolii-endophyte metabolic activity in perennial ryegrass

226 Pages·2011·4.37 MB·English

by  Spiering, Martin Jürgen

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NEOTYPHODIUM
DISTRIBUTIOOFN LOLII-ENHDOYPTE
(LOLIUM
METABOLIACC TIVITIYNP ERENNIARLY EGRASS
PERENLN.E),
ANDI TSI MPLICIAOTNSF ORA LKALOID
DISTRIBUTIAONNDP HOTOSYNTHESIS
A thesis preisnpe anrtteida l fulfilment
oft her equiremfeonrtt hse d egreoef
DoctoorfP hilosophy
inM icrobiology
atM asseUyn iversPiatlym,e rsNtoornt h,
New Zealand
MartiJnii rgeSnp iering
2000
11
Abstract
Neotyphodiluomli sia ifim g ale ndophoyftp ee rennriyaelg r(aLsosl pieurnmen e).
Int hissy mbioasliksa,l aoripedr so ductehdas ti gnificiamnptalocynt t hpee rformoafn ce
farmaendi maLlist.tw laesp reviokunsolwyan b outth ep hysiolocgoincdailtf iootrnh se
endophyitnte h ep lanlte aditnotg h epirro duc.t Ai osntraoifnN. lolpirie,v iously
transfowrimtethdh eB -D-glucur(oGnUiSdg)ae sneoe f E. colunid etrh ec ontroofal
constitfimugtailpv reo motwears,u sefdo ri nvestigianttitooh neis np lanmteat abolic
activoift yte hned ophyte; doifs trtaihlbeku atlieooringd osv apleirnarninee,, a ndl olitrem
B;a ndp hotosynthesis.
Inv itsrtou dwiietsh ttrhainss foN.r mleodls itria dienm onstrtahteue tdi loift y
constitGuUtSei xvper esfsoiaros ns estshimene gt abosltiaoct fet hee ndophyBtyeu .s ing
constitGuUtSei xvper esasnidao m ne thofdoq ru atnitaotfi otnih npe l anbtiao maosfs the
endophyteen,d otphhemy ettea bosltiac(t EeM Si)n t heg rapslsa nwta sd etermiTnheed .
EMS wash igahn du nifoinr mp lantti ssuaensdg enotydpieffse riin negn dophyte
concentriantdiiocnat,th iapntrg o lifeorfat thieeon nd ophiyntt eh pel anitsn otc ontrolled
vitah EeM S.
Ergovalpienrea,m inel,o laintBdr eexmh ibietaecadh c haractewriitshtiinc- tiller
distribmuatiinotna iancerdo ss dipfflearngeten nto typNeosn.eo ft hea lkalowiadss
distribiunet xeadcp tr oporttioto hned istribouftm ieotna boliaccatlilevyne d ophyte
mycel.iD uimffereninc tehsae c cumuloatfti hoean l kalpoeirdm sy celiwuemr oeb served
betwepelna tnits saunedps l agnetn otyspuegsg,e sdtiifnfge rreanttoeifssa yln theasnids/ or
degradaotfti hoaenl koaildisnt hmey celiaunmdt ranslowciatthitiohngne r atsisl .l er
III
Rateosfn epth otosynatthh eisglihis g ihntt enswietrileeo sw eirnp lanitnsf ected
byN loliinidi,c aftoiarnn eg ff ecotnp hotosyntchaeptaciicHt oyw.e vetrh iesff ecwta s
plant-adgeep endePnlta.ng tr owtwha sn ots tronagffleyc tbeyd t hee ndophybtuet,
infectedh apdcl oannstiss tleonwtelry e lleoanfg artaitoeTnsh .ec hangeisnl eaefl ongation
andp hotosynctahpeatciiicnit nyf ecptleadn mtisg hsti gnaflo arn e ffecotfN loloint ih e
nitromgeetna boliisthmso sotf.
Thee xperimceonntcse rningt hei np lanEtMaS ,a lkaldoiisdt ribpultaionnt,
growtahn,dp hotosynwtehreceso insd ucitnae c do ntroelnlveidr ontmheeens tt asbhlmient
ofw hicwha sp arotft hisst udIyna. d ditfiootrnh ,de e taimlaepdp ionfge rgovawliitnhei n
theg rastsi lal meert hofdo qru antitativeo fet xhtriaaslc ktailwooanis dd eveloapnedd
optimised.
N
Acknowledgements
Il itkoee xprmeyss si ncegrrteaistttu omd yefI rsstu pervDirJs aoSnrc ,h mfiodr,
givinmget hoep porttuonw iotroykn t hipsr ojIeh citg.ha lpyp rectihsaeut pep aonrdt
encouragteoem mebnaotrn ka l als peocftt hssi tsu adnyfid n ancsiuaplp tohrrto ugthhoeu t
projtehcaattl lomweet do a ttecnodn ferienNn ecweZ se alaannddo versIea amsa .l so
gratteofum lyc o-supeDrrvG iesooLfraf,n a etA gReseGarrascslh a ,nf dosdr i scussions,
angdu idainnac laels peocftt hspi orsj epcatr,t ictuhlaeal rklaywl oorikda, n di mmediate
feedbtahcrko ugthhoweuh to le pIra omjt ehcankfult .t om y co-superDvrPi asuolr ,
O'Toofloaerl, sp or ovisduiepnrgv iasnisdou ng gestions.
MikCeh ristaetAn gsReens eGarracshs lparnodvsia dnie ndv aliunapboulfite d eas
ansdu ggestthiwaoetnr aseg reat inasnpidin rcaetnfitooimrnve te o u ndertthaiskste u dy.
Thanyko uM ikeT!h ew orokn tphheo tosynitnth heitsshi essw iosu lndo hta vbee en
possiwbilteh DourDt e nnGirse aetrH ortReswehaorp crho,v iidntrumesde ntatainodn
mucht echnaincdoa tlh aedrcv eif owrh icIh a ma lsgor ateLfuilz. Dafrvoimet sh e
EndophTyotxeLi anab t A gRseearIlc ihk teh tafnook r hearn vdga rleuaahtbe llapen ldo ts
ofa dviocnte h ael kaalnoaildy Tsheasn.ik ns trheigsagr od alDsroB rtioTa anp paenrd,
focro ntrimbauntsyiu ngggi eosnttsh rougthhaoelu kta wloorikDd.r O llBiaelI lw istho
thafnoklr e ttmiehn agv aeg oa tE LISIAa .m a lsion debttoe dCD hrir stMionoan f or
commenotnms y i ntroduacntdti oDo rnC ,h rSicsh ardld ifsocru sasnidlo intse rature
adviocnce l opnraolp agIaa tliswooin st.ho t hatnhkve a ripoeuospi lnte h lea abtM assey,
YonTga na ndDr T ahAal -Samfarortrae ic hnaiscsails teasnpceec,wi iatlthlh ype l ant
maintenaanndNc iec ovloenM altzfaohmrnak intgh lea ab l ivepllyaa cned tfhoer
enjoycaobffleaeen l du nbcrhe aks.
v
A spectihaalng koset sot hpee opaltMe a sseAyu,s tGeann lfeoygir v inmget he
chantcope e rfdoirlme titnca rnitclakyne adtl t lh mee mbeorfts h wee eksloyc cgearma en d
thfeo rmDeerp otf.M icrobiaonlGdoe gnye tAi bcistg.h antkofrs i endsp eaonptdlh ea t
madmey s tay iZne aNlevawne dre yn joyaanbdml eem oraibnml aen yw aysM:i kki,
Henni&n Lgi se, &N CihcroilseSt hoeprha&,l Aelea n, STthemoffaes&n A,n keA,n drew,
Delwyn, PMeatLreeirwa ai,ns Sd t uaanradtl frlo mM anawaHtaukp idPoe,t Secro atntd,
fmalEllyi zaabneGdtr he gfoorber i ng l(o?va)in nldgo vaflbaltet Miaensyt. h antkoys o u
all!!!
A bitgh aynokug oes taoml ysfr oi enbdasch ko mee,s pecKiaa&il Hleyi kaen d
Palflocero mianltglh l eo nwga yP,e t&e S usafnoncrea tchuiprn egg ulaanradll yst,oom y
parefnottrsh eciorn tinsuuopupsAo rstp.e ctihaalng koset soA mirBai anca.
Thiwso rwka ss uppobryta eM da ssDeoyc toSrcahlo laarnsbdhy iA pg Research
GrasslPaanldmse,rN sotrotnh .
VI
TabloefC ontents
Abstract. .ii
............................................................................... .............................................
Acknowledg.e..m..e.n..t..s.. ............................................................................i..v... . ............
.
TableC oonft ents .. . vi
............................. ............................. ............................ ....................
LisotfT abl..e.s.. .................................................................x..i..i.. .........................................
LisotfF igures . . xiv
................................ ......... ........................................................................
RelaPtuebdla itci.o.n.s .................................................................x.v.i..i. ................................
1.0I NTRODUCTI.O.N. .. . .. 1
... ................ ........................................................... .............
1.1 TheF ung-iA n ImportaGnrto upo fO rganisms . 1
........................... ................
1.2 FungaEln dophytoefGs r asses .. .. 2
.................... ... .............................................. .
12..1 EndophGyrtoew tihnt hGer asPsl aanntdP lant-EndIonptheyrtaeca ttti hoen s
Cellu.Lleavr.e l. . . .. ..4
....................................................... .... .......... ... .......................... ............ .
12..2 EffeocftE sn doph-yItnefecotnPi loanGn rto wtahn dP hotosynth.es7i s
...... .....
12..3 TheP roducotfSi eocno ndMaertya bolbiyGt reassE sn dophyatnedTs h eir
EffecotnsA nimHaela latnhdP laPnetr sisten.ce. . . . 8
......... ..... .... ...... .................................. ....
1.3 BiosynthAensailsy,s IinPs ,l anDtisat ribuatnidPo hny,s ioloofgA yl kaloid
Product.ion. ... . . .12
........ . . ...................... .................................. .... ........................................ .
1.3.1 TheB iosyntohfEe rsgiosv aLloilniet,Br aenmdP erarnine .. .... 12
.... .. ..... .... ......
1.23 . AnalytMiectahlof dostr h Qeu antitDaettievrem inoaftt hiAeol nk aloid1s5
.......
13.3. AlkalDoiisdt ribwuittihtoihnnPe l a.n.t 18
............................ .............................
13.4. EndophCyotnec entraantdAi lokna lPoriodd ucatsIi noflnu enbcyet dh e
EnvironmanednP tu,t atRiovleeo sfP hysioallo gPiacrafmoert erAsl kSaylnotihd.e s.i.1s.9 . ..
1.4 TheG USR eportGeern eA: T ooflo Ars sesstihnegM etaboSltiacto ef
EndophyMtyec eliu..m ..... ... ..21
.. . ... ..... .. .. ............................................... ........................ ..
1.5 Aimsa ndO bjectiovfte hsiS st udy .. ..23
................... ... ................................... ... ..
2.0M ATERIAALNSD M ETHODS ...26
.................................................................. ... ...
2.1 FungaSlt raiannsdP lanLti nes ..... ............. 26
............. .. .. .. ..... . .... ...... .. .. ... . ...........
2.2 Media . . . .2 6
................... .... ............ ................................................ ............................
2.12 . PotaDteox trAogsaear n dP otaDteox trBorsoet( hP DAa ndP DB). ..2.6. ......
Vll
2.2.2 YeasEtx tract GMleudciou(smYe E GM ediu.m.) ............2.6
.. .................. ... . .. . .
2.3 Buffearnsd Soluti.ons . ............ 26
..... .................... .................... . .... . .......................
2.3.1 GUSE xtracBtuifofn.e. r . . . ............ .2 8
... ........... ..... ......... .............. . . . . .................
2.3.2 GUSA ssaByu ffer. . ....... . 28
...... ........................ ........................ ... . .............. .....
2.3.3 GUSS toBpu ff.e.r ........... .... .............28.
.. . ........ ... . . . . . . ..... ...................... .. . . . . .. .. ..
2.3.4 GUSC alibrSaotliuotn.i ..o.n. ............ .. .............28
. .. . . . . ...... . .. ...... .. .............. .. . . . . .
2.3.5 SaliSnoel ution 28
......................................................... .....................................
2.3.6 Lacto-GlSyocleurtoilo n .......... 28
................................................ . . ....................
2.3.7 DecoloriSsoaltuito.in.o .n ................. .............2.9
. .. ...... .. . . . . ... .. .. .................. . .. . ..
2.3.8 BleachSionlgu tion ......... 29
............................................ ... ... . ...... ......................
2.3.9 MountiSnogl ution ........... 29
..................................................... .. . . .. ..................
2.3.10 AnilBilnueSe o lut...io.n ............ ......... ..............2.9.
. .. ...... . . . ... .... .. . ............. . . .. . . ..
2.3.11 lOxF ungNaulc leSatra inSionlgu t.i.o.n.. ........... ..........2. 9. .
. .. . . .. .. . .. ... ............ . . .
2.3.12 lOFxu ngSaelp tSat ainSionlgu tion . ...............2. 9
................................ ..... . ... . .. ..
2.3.13 ChloriBnlee ach . .. ......... ... .........2.9.
......... ...... .. ..... .. . . . ... ...... ... .. ................... . .....
2.4 Growtahn dM aintenaonfcF eu ngaClu lturaensdP lan.t..s 30
. . ......................
2.4.1 FungIaslo lafrtoimoP nl aTnits su.e.s. .........................3.0
.. ........ . . . ... ................
2.4.2P reparaotfIi noonc ulanudmG rowtihnL iquCiudl tu.r.e...s.. .... .30
.. . . .. . .............. ..
2.4.3D eterminoafFt uinognBa ilo maassDs r yW eigihntL iquCiudl tures 31
...............
2.4.4 PlaCnutl taunredM aintenoafSn ycmeb io.s..e.s. ........ . 32
. .... . . ... .. . . ................ ...
2.4.5 InoculoaftP iloannw tistE hn dophyte My..c.e..l..i.um 35
. . . .... . ..........................
2.4.6 GeneraotfiE nngd ophyteC-lFornPealela nbtyFs u ngicTirdeea tment3 5
.........
2.5H arvestoifnP gl antIss,o latoifo nE ndopahnydtP el anTti ssEuxet racti3o6n s
...
2.5.1 PlaanntdT illDeirs secftoiAron na lyosfeG sU SA ctivaintdAy l kalo.i3d6s
... ..
2.5.1.1 PlaDnits sec..t.i.o..n.. ........................... ......... 36
... .. . . . . . . . ... . . . . ............ ... . . . ......
2.5.1.2 TillDeirs sec.t.io.n.. .... .....................3.6
....... . . .................................. .. . . .. . . . . . . .
2.5.2 Simultaneous AosfGs UeSsA scmteinvatin tdAy l kaloiinPd lsa Tnits s.u3 e8s
2.5.3 ExtracotfEi nodno peh-yLtinakuesdA ctivfirtoFymu ngMaylc eliaunmd
PlaTnits s3u8e
2.6 fS-Glucuirdoans(eG USA)s says . ..39
............................ ....................................... .
2.6.1 QuantitEaxttriavcetA isvsea y.. .........................................39
.... . . .. . .. .... ... . . . . .. . .. ..
2.6.2 Semi-quantMiitcartoitPvileta GrtUeeS A ssay .... 41
............................ ... ... .......
2.6.3 QualitMaitcirvoetP iltarGteUe S A ssa.y. ..... .. .... 42
. .. .. ... .. . .... .. ... .. .. ...................
Vlll
2.7 DeterminaotfHi yopnh aBli omaCsos ncentraitnPi loann Tti ssu.e.s.. ..42
.. ..
2.8 AnalysoefAs l kaloiindP sl anTti ssu.e.s. ...............4.6. .......................................
2.8.1 ExtractiQouna natnidto afPt eiroanm. i.n.e. .............4.6. ....................................
2.8.1.1 ExtracwtiitIohsn o propanoAlc-i.Ld.a .c.t.i.c. .......................................... 46
2.8.1.2 SeparaatniQdou na ntitoafPt eiroanm biynH eP LC. .........4.7. ....................
2.8.2 ExtracatniQdou na ntitoafLt oilointB r .e..m. ... ...............4..8.
. .. . .. ............ . . . .. . ...
2.8.2.1 ExtracwtiitCohhn l oroform-.M.e.t.h.a.n.o.l. .......4.8. ..................................
2.8.2.2 Extracwtiit1oh,n D2 i-chloro1e0t%hM aenteho,al .n .........4.8. ...................
2.8.2.3 SeparaatnidQo una ntitoafLt oilointB r .e.m. .............. . 49
. ... .. . . .. .. .. . .. ....... ....
2.8.3 ExtracatniQdou na ntitoafEt rigoonv a.l.i.n.e. ............4.9. ..................................
2.8.3.1 Chlofroorm-MethanolE-xAtmrmao.cn.ti.ia.o .n. ........4.9. ............................
2.8.3.2 AcetAicci Edx trac.t.i.o.n. ..................................5.0. ....................................
2.8.3.3 IsopropanoAlc-iELdxa tcrtai.cc.t .i.o.n. ............5.0. .....................................
2.8.3.4 Sepaornaa tniQdu atnitaotfEi rogno va.l.i.n.e. ..........5.0. ..............................
2.9 FluoresMciecnrto scoofpy Nuclei anidnF uSnegpaMtlya c elium. .51
...... ....
2.10O bservatdiuornisnP gl anGtr owth ................. . 52
...... . . . .. . ... .... . .. .. .... ..................
2.10.1 TilNluemrb earn Tdi lleRrait.ne.g . ...............................5..2.. ............................
2.10.2 uafE xtenRsaito.en. s. .........................................5..2. .....................................
2.11A ssessmeonfLt e aNfe t-Photosyn.th.es.i..s. .......5.2
. .... ...... . .. ................. . ... . .....
2.11.1 Princoiftp hlOeep eInn fraGraAesnd a lySsyisst .e.m. .........5.2. ......................
2.11.2 Configuroaftt ihAoenn a lySsyisst feomMr e asuremoefun atfNs e t
Photyonsthesis 53
.............................................................. .....................................................
2.11.3 Measuernetmosfu afN et-Photos.y.n.t.h.e.s.i.s. .......5.4. ................................
2.11.3.1 DeterminoafLt iigoRhnet s po.n.s.e. .............5.4. .....................................
2.11.3.2 DeterminoafTt eimopne raRteusrpeo. n.s.e. ..........5.5. ............................
3.0 RESULTS.. ........... .............................. ....5 6
. . . . . ...... .. ......... .. ... .. .. . . .. .... .. . .. .. .. .. .. .......... . .
3.1 MethoDde velopm.e.n..t. ... . .... . 56
. . ... ...... ... ...................... ........ .. .. ................... .....
3.1.1 Developomfae M nett hfoodQr u antiEtxattriaveco tfGi UoSAn c tivfriotmy
FungMaylc el.i.u.m. .......................................................5...7... ................................. .............
IX
3.1.2 Developomfe Snaet l ecStyisotnfe omOr b taiGneinnegt iHcoamlolgye neous
EnodphyItseo lfraotmeN so n-SporuMlyacteilnigu m . 59
................................................ ......
3.1.3 Establiosfha Cm eonntt roElnlveidr onfmoeSrnt tu doifEe nsd ophPyltaen-t
Interactions 63
..................... ............................................... ..................................................
3.1.3.1 PlaGnrto wthi nt hGer wothC abinet.. 63
........................... ..........................
3.1.3.2 Assessmoeftn htIe m paocfFt u ngicidIen saencdtA ipcpicldaiet ioonIn n
PlanGtUaS A ctivity 64
....................................................................................... ..................
3.1.4 Develoenpatmn Odp timisoafat M ieotnh foodEr x tracotfEi rogno vafrloimn e
SmalQlu antiotfEi nedso phIyntfee-cPtleaMdna tt erial .. 66
......................... ..........................
3.1.4.1 SampMlaet erfioaMrle thDoedv elopment.. .. 67
...................... ............ .. .....
3.1.4.2 ExtracwtiitSohon l veCnotnsti aniIgns oproapnaLdna oclt ic Aci7d0
............
3.1.4.3 ExtractainoRdne covoefrE yr gvoa lianneId n ternSataln dwairtdh A caent ic
AciSdo lvent. . . .. 74
...... .......... ............................................ ................................ ............ . ....
3.1.4.4 TimCeo urosfEe r govaElxitrnaec tainoodn fR ecovoefryI nternSatla ndard
...................................................................7....6.... ...................... .................
3.1.4.5 TheI nflueonfSc aem pDlreyW eigihnEt x tracotfEi rogno vaalnidn e
Recovoefr yI ntSetrnaanld ard .... 78
..................................................... ................. ....... .. ........
3.1.4S.u6m maarnyCd o nclusions ..8 0
............................. .................... ............................ .
3.1.5 Comparoifsto hQneu antiEtxattriavceGt UiSAv ses ay awS ietmhi -Quantitative
MicrotPiltaArtsees ay .. .. .. ... . 81
................... ... ...... .. ................... .. .......... . ....... .........................
3.1.6 DetermiiononafI t nP laanE-tndophMyytcee lCioanlc entbryaM tiicosrnco opy
....................................................8..3.. ..............................................................
3.1.7 AssesmsenotfF resh Weight-CDoreyf fiWceifieogPnrhlt tas Tn its su.e8 s6
...
3.1.8 PositSieclvetei foonGr U SR eporterA-cGteinvieinE t nyod phyMtyec eli-um
SegregoaftG iUoSEn-x presasniNdno gnE -xpresMsyicnegl iiunmtG or aTsis lle.r.8s 8
.... ..
3.91 . EstimaotfIi nohni bitGiUoSAn c toifvi inPt lya Tnits sEuxet r.ac.t.s 91
.... . ........
3.2 EndophyMteet aboAlcitci vaintdyA lkaloSiydn theins iN.sl oElnidio phyte-
InfecPteerde nnial Ryegrass . . 93
.................................. ............................. ................... .....
3.2.1 Assessmoeftn hCteo rreloafGt UiSoA nc tivwiittEyhn dophMyettea bolic
ActiviinFt uyn gTarla fnosrmants . . . ..93
........................ .................... ......... ............... ......... .....