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W(1). IBB8, pp. 579&&%.

e loss ty n
. Nclv h*Botaslsll &
2 &en*,
&,l, m tarriW1B

Monoxdc cuttnne.ofthe intramdid forms of Il=tomus sp. isolated from a


tropical =system a proposed rnethodoIcqgy for gemplasm coI1Xection

play a ky rde in the s o r n ~ onf the lift cydt of


drLGtwin,wdi AM fungi [Eiarley and Smith 19831, fhUs represent-
Cmir du Sud. 1948 h& ing pmrialiygxmd inoEula.
Smllu uld R o m d /l%, 1887) demonstrated
thaf the fungm vhmm roar pieces and aingIt b
l a t d vcsidcs wns a& %I! geminate and form new
rypirnl m y c o r r h in plao when issm'uaed wirh n
Christian P1tnch.m
swepdble host root This t~hniguca l l m d the
Iwrirur h & l o d & I.&%&&E +WGWJU*,
monorcnic (x1hm of Ghwm irrlpuandices Schenck
&Ydvnd'HgmoMe, 17raraSt+, 21334Dymi ccdag and Smith isnd G. rrsm~mm(&mm) Be& (Diop
&%arm et al, 1994a. b) and resulted rewnlly in a propcwcd
life cycle of G I o m spp. [Smrllu cl d.. 1997). MW
pduction ofsewrid thouwad V M I C prcpagdcswas
A&ra& F Ia F a u W myaxtfllarl [AM) fungi, achieved (Declmk et at, 199th) and their entrap
iBotared h m the rhbmphc~eof banana and sugar mcnr in a t p ' i be& offered a new kind of h i h
wu,weres~~c&lIy hllarrevl in *Q in agnockdan q* tnoculmn, tree of pathqem- (Z)PCIerck et al.,
with generically transbw~droots of m m t The in- 1996b). This c d m n reehiique was M e r swc
mmdicat form of &e fungi as mycorrhizal root piec- c d f k y cmadtd to qxt3-m originaung
m and hgle imlakd vcsiclcs cowdmted excenenr Gom the rhkwphere af a d net m -1 (Diup.
sources of inmlurn for the esrabUsbmcnt of in ntro 1995). Nowirhpranding these r e d &md the numee
cliltures and for the lx~tinuousculture of b e 3pe- ow oppmmnitics &cd by @e inharadical brms
cia. Srreral thousand fungal propagub Here ob- of the fungus, this tcrhniqilt received Einle attention
rained for the nvo G. YJ&J%W species, G. mh-amt- m date.
tm and to a l&er extent for G.f b l h l a h a l ~ w h ~ ~ e a ~ The aim of tht prscnt W& vvar to whether
few ~ p e m sm obtained k r G+m ~ n This a the mhamdical brm ofAM fan& could be ustd for
wculture Wrn appeared suitmbfc for tbe aablish- the monoxenic culrivadpn oT specks oriel-
men€ofof incm ccSI&ns of AM fungal mains. naring f?um a vopical mogratea W t 8 are report-
#slY Wbmh a r b rnycarrhld ~ ~ ~ fund, contin- ed and potential u x s of this monoxenic adm~zion
uous culture, Fungal Eibrmy, Ri T-DM msfo~acd qsmm aa a tcol for germplasm collectians ia d k
root, v&h EUSIL~

wmaoucnm Sworgwr a - T h & w r n e d mmt ID- MF


A r b u W mycorrhi (AM) fuagi ;arc mil micro- W L)mob wrrc provided by X F iche (CRBF, Univ-
otpnims whicb appmntly caa pmrhrce spocts and ersit4 M, Q.i:bec). Rautiae maiatblance d tht
complete rheir lite ryck only upon the ~crloaistioa m u m made m the mddificd Suullu-Romand
d a sw~cp'blc h m root Thq. are rhuf coddtred (MSK) medium (Diop, 1995) h aa tmrCrted pQpiacm
obligate biompbs. Many a n e m p to thue o e at '27 C ip the dark The medhrm cmtaincd in
gdsmii in uitw w e fmplcmented h rhe km d c L-8 W e d mter:q s 4 . m ~75% . 76;
cad% m m wulw king abnrined 4th as KHpPO,, 4.1; Ca[N0~,.4Npo, m; NalrtEOTA, 8;
starring mtdaT (Muam and Hepper, hatl@er KCL, 65; Mn50,.4Elz0, 2.4% ZnW4.7&0, 0.29;
and M a s s 1987; 3 6 d and Fartin, 1988; B b r d W% 1.88;,
C 1 6S O5,w ,
DDQP: tW),WH.4H,O,
and Echt, 19991;Sf. Arnaud ct al.. XW9B).It appears, 0.W N@&Cl,Si&. 0.004?4;&C-, 1: pyridan-
bwmer, that h+ae and &ctw wirhin rows abo be, o.9: nicarlnic add, l: calcium panrho~natc.8.9;
c p n a ~ W m e 0.4:, W o h , 0.9 X 10-%;dlrw~
Aurp4hd For prrkkraiun Mmhg, SS8 I O W and -5 W ,W.+%G pH wps adjusted m
'M c k c l ~ P . € f n i M s ~ u t ~ b + 6.5 before sterilidon at I21 C for 15 6
m@ftW&Kn*-Tht AM fungal atmina mcol-
l e d in Mnrtincquc, the l a r g r ~of t&
W i n Mb
W in the eamm Wbbeart, Mnrtinque is &m!-
d c ori* wirh trnpmnt soil and climate whbiL
hy. Soil war m p k d in tbc root lnnc of barrana and f. h vrm d n m and sqbminut p c a a a i the
sugarcane toadepthot25 cm,air dried and p a d hrrnrPdiePl~d~rrpA.Potcdt~~!dCtwRw
thmugb a Z mm s i w . Bulk cultum were then eseab-
LiaRed on Ieck plantr [AIliu= porraun L) g m on
enndaffned ~~~
nparatna;&mprrhlr.al~~~p~C.~~~wQ
root; E lperra
rfnn procceh; 9: wktion of vodc14: 9!
3: ddnfec-
of h
auwclav*d (121 C for two sepaate I h psrkdlp) Ter- gemdmrcd pmpsgwhwith n w m e h r n d a r m t mac; 4:
ragrecn* under gracshoupe mndittona After Q m% wbruhureaf~t x i d m m d rqco&hpt rmtpiawiah-a
aporea were mtracted Erom thc bulk cultures by slew n m ParuMrmcdroot
ing and thorn d the same r n o ' p h o ~gently
nwd with a mkrepipcm. Gmups Of S10 apms
were &m piposud on dre roots atpung A. tion untn use. PM &C & r e Dthcr &S i.e. G
seedlii and grown for another 5 ma in a ~ t m h v d ~ 1 l a . G . ~ a W T C . u g s fTPr 1 ~ ~ 2 ~
Terragrem* &r penhouslt codidans
a s I), the dlstnfrmed rcboo wm gm* lacuTltcd
were €in* exancrtd by ritving nnd s p i m idcad- with needles ka so exnsruhitlck C3uates-a of attached
Bed by P Daw (4&ricultm h a & , Omwn) using miclcr wetc then separated with andlea under bin-
the clapid morph10gW spona chamcuiirticr.
Fiw G b w sp, [G.fd&a Wter)CR
mhr m i ~ ~ ~ and ~ o rnsdmkhsd
p e in the audMMic
sotution.
h n n & T r a p . G, m m(I) and (9, G. AILP.
-'puns Thhme & lUa.sne, and a aain of G irr- Mmdc mlhm ~ - - B o t LPDcUlum
b
~raruhcajoriginaring h m a banmm A& g m un- m aseptidp tradkred an the MSR rnedilrm in
der tmpical cllmPtt in the tsnuy Island. ? mlcank the at 87 C, using repmheb fWChpip for the
laland in thE Mantic ocean mmc lOil km From Ihc root piemu a d a micmpip~urfar the wwitlck Aflcr
c m of nMthvmc AMcll] were considered h the fryphal *CC d sgm =p-g the g-
presmt $tildy CTAQLE11. Afmr wntlmic idmttfim m U & d pmpgtk, ii.t,, qcarrh&aI root piezed or
don and purity mmsrnent orthe c u l m leek roc*s isolated vesictw was m& wirh a 15mm cark
w e q l c d and q c o r r h i i Kgmenm aeltcxcdnm bdrer inm h c czpuimmtat P& G&. A ace ofagslr
dcr binocular micrascope for h vim culntre csab spas abruDwedfmm thbtxprbmtntal & w i t h
Ibminat. tbe cork borer W that the prcpaple an the agar alIce
m eo n BpDcsr.-haymm mrrr piecer wue could be inrerted in the hale. An &ly (gwrirtg
disinfected iO an ulQa~nicapparanrs under it M- tmmFarmod ~arrotroot W then ph6td in h e itear
PPT. hOdd h t s war auec&1y naked in a mlu- vicinlrg of the prpppdc. The r u l w wan &m in-
don c& cthanolQ$R (vit) foe IQ a, ealclum h* cubaredin w in~dpCa:dcrninthcdarkat27 C,
cblodte 6% (W/* fm B min, ckloramine T 2% [W! M m 5 ma dud cultwe, vanafonned mat piece
v) &&cl with some dropr o € l h t n 20 for 10 m h hawing heany m p c b d*velopmea~on their W-
and antibiatics (SueptcMIycinc 200 mg L-land p facc w e cut d t b a acdpd and gently removed from
tMlycint 100 mg L-l) for 10 min Aftcr wch dish- the M§R medium witit f o n e p Phc root p i e wrrt
fedon titcp. mts w e r i n d rhroc times in MC r e w b e d with a mn&tmFd mot of c a m s to hi-
&tilled W. For G. Jma.m.&&m aud G* wm75mxe date a urn culhnz. Tht fa vim whirr and arbcuC
{I ) (TABU I), the diiinkmd mats were chopped in rwc prmw of IbE h-mt tiwms of G h w
0.5 cm picccs and rnzinoined in the a n d b i h soh*. saaint is skeutrcbcd in RC.1,
TO our knowledge, 5 G&mw species are actually maintcnanfc of the species within collections and to
maintained and subcultured wing the inuaradical constitute a pool of in vivo material able to inidarc
f o r m of the fungi (Dip, 1995; Smllu et al.. 1997). new in viao and in vivo culturea
In this way, it is pDaaible to create in v h o germplasm
collcctiona and keep AM fungi under standard
growth conditiom. In the present study, five novel
G l o w strains were su~cessfullycultured in vitro. The wthon are &mLdul to Y. Dalpt (Agriculture C a d &
Two suains [G. fPrdeuhtum and G. wrrrfmnrs (l)] Onawa) for IdcntiFy3ng the GIonarr @CS. Thia work was
were p d u e t d with rnycorrRad root pieces as start- supponcd by a p n t hOCe STD3 N a 92104.
ing inoculum and thm s&ains [C. m&m (2). G
m.mm$um and C. inkramdim] with single isolated
veddcs. The spores obtained in these culu1m were
homogenous and able m rcinfsct leek plants under Waji. 1, M.J. Pouli, H.Vierhcilig, and Y PicM. 1995.
greenhouse condjtioru. The s p r u produced in Ueqmnrer of an ubuscular m y c m r h i i fungrrs G p
these greenhouse c u l m wm further similar ta the rpwa magrm'a to emdam and wlatile from rht RI
TDNA-Tmdwrntd roots of nonmycorrhfsal and m)-
spores identified initially, demonstrating the purity of c w h i d mutanm d &urn rduum L Sprklc. Exp.
the in vivo cutt~redstrdm. Thc long term maint* 1% 2 7 . W .
nancc of species through regular sulxulturea under B a d . G.,and J. A Fonin. 1W. &artr mnrr ofvrsicular-
controlStd s m d a d u e d conditions should cnable the a h ~ ~ I mycorrhiml
a r forrnhon on Ri T-DNA vana
propagation,throughout the scientific communityof rwmcd m m Nmplhlld. 106:411-218.
reference species For fundamental and prxtical r e -, and Y. Picht. 1989. h& gmwrh mimuladon by
scarch. Questions remain. however, concerning the CO, and m exudates in rPrlcularmbumh mycw
infectiviey and cffoftiwneas OF the species maintained r h i i aymbhh. AppL M i m h l 5 6 : 45-
for long periods in nuo. For ectomymrrhizal hngi,
a r i r a l k t i o n via host pawge every 4 yr was pr*
23-25.
md - 1992 hbllshmcnt of VA m&orrhb
zac in toor organ culuuc. rcvierr and p r o m mcth-
posed by Manr (1981) W maintain the symbiotic p p
d*. Pp %1M. Iw in mftLIc&Dkw,Eda,
centials of the hngi. Plenchettc et al. (1996) o b k Vsuma J. R Norris and D.J. Read. Academic Prm.
erved also a decrease in infectivity of succaiws c& New K o h
cures d a G. mm spccicr and arc& the im -, L P. Taylw, D. D. Douds, P. E PfcfFer, and L W.
portance of maintaining both trap pot cul- and h r . 1985. Flavonoids an nor necessuy plant signal
in v i t culturts
~ *AM fungal species. each mllecrion compounds in a r b u l a r mprrhiml a y m b i h . M&.
serving as a p w l for the other collection. In our P I - M M Ismua 6: Z2-m.
study, no thorough obmwtion W made on the in- Chrbot, S , R &l Rhlid, R. Q l h m r r and Y. Pichl.. 1922.
fective potentialof the species mainrained in vitro for Hyphal growth promotion in d m ofthe VA mycOn?ib
long periods. Hwvewr, each species gmwn in vitro f i m g u a , m q m l u & c k r r & HaH, by h e
was able to reinfect kck plants under grcenhollse dvig of sirucrurally @ l c flawmid compounds
under COflnriched mdirma h h Pkllol. 122: 461-
conditions and typical i n W d and extraradical
467.
AM fungal smcturcs wm observed. M n succesivc Dalp, Y. 1987. S p ~ viabiiiry
c of swnc Endqgmacrae a u b
subcultures of G. i*&s were recently obFained mitted W a single SW IpphiliPPtion. P. 279. IRI
by Saullu (pc= comm.) using mycorrhizal root seg- tladiqy q r l t s SmmIhNorlh A m A c s t l h # m ~ ~ r n
ments and spore p d u c t i o n rrmaincd constant dur- Eds. D. M. Sjhia, L L Hung and J. H.
ing the successive cultures, demonstrating the high Craham Gainsvillc. Florida.
potential of the inamdical form to maintain spe- Deckrck S. D. G. SlrulIu, and C Plcnchtuc. 1- In
cies w c r long pcriods. Smck ccnrers, bgsed on v viva ~ m d u c t i o mof thc axbu4cular mymrrhiial
cies propagated on host plant growing in disinfected fungus. G h n u wr@m~,agociattd with Rl T-DNA
substrate, already exist, and should be broadened W wansformed carrot m o w h 4 y d k 1M): 1267-1242.
in vim culture collections. In n w o culture is l a b + --- , ,and T. CuiUemette. 1996b. Entrap
mcnt of in vim produccds p ~ m of C h i u am@m#
~
ous and time consuming. Therefore a method for
in alginatt btads: in vim and in vivo inocuIum poten-
long-term swrage is m n l i a l . Actually, pot c u l m in- tial~.J. Ea~&lDd 48:51-57.
acula are successfully stored for sewrat mo tither at Diop. T.A 1985. Erophysiologiedes charnpigncna mycor
4 C and through cryoprescrvation (Douds and hiziena IvEsiculcr t t arbusculesarsoci& a A& &-h
Schenck, 1940) and single sragc Iyophilimion har deL dans k s zones Sahkimnts et SoudPooCuintmne
been c k r i v e for some AM fungal species (DalpP. du StnLgaL Pt. D.U, UniKFsity ofAngers. FM-.
1987). Further rmarch is needed to adapt Ifie 151 PP
methods W in vim cu1runs to ensure the long term -, G. Ecard, and Y Piht. l$%!. Long4crm in vim
~EZLF.RCKE
T Al..: MONOXI

m l ~ of e an endomyeorrhiral fungus, mrrhizal infections in root organ culture. PL


golit&, on Ri T-DNA t~~ +
roots of -L PaMd P 215229.
Y& 12: 249-259. Mugnicr. J., and B. MW. 1987. Vmkdar arbuscubx
-, C. Plmhrttc, and D.G. S h h . 1- Dual awe fecdona in tm=bmcd Ri T-DNA grown mmi-
nit culrurc of rheanbroot I n& of v & ~ ~ l a ~ r b W-.AP 77: l(H61(KIO.
cuhr mymrrbual &ngi assocked with tomato m m . Nair, Y C . , G.R W,andJ. 0. Siquwra. 1991. Isolahn
My
- 5: 17-22, md idtneiion of vesicular-arburcuht mycomhiep
-, -, and-. -1 In vim culmrt of srimularopy c c m p u n d s from dmcr (-m rcpllor)
m
s h d mycorrhkd a l r o o t k 17: 217-m. mofs.AQpLEtfBitDRtRMU57:W.
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