W(1). IBB8, pp. 579&&%. e loss ty n.2 Nclv h* Botaslsll &&,l, &en*, m tarriW1B

Monoxdc cuttnne.of the intramdid forms of Il=tomus sp. isolated from a tropical =system a proposed rnethodoIcqgy for gemplasm coI1Xection

d r L G t w i n , w d i Cmir du Sud. 1948 h&

Christian P1tnch.m Iwrirur h&lod& I. &%&&E +WGWJU*,

&Ydvnd'HgmoMe, 17raraSt+, 21334Dymi ccdag &%arm

A&ra& F I a F a u W myaxtfllarl [AM) fungi, iBotared h m the rhbmphc~e of banana and sugar wu, weres~~c&lIy hllarrevl in *Q in agnockdan with generically transbw~d roots of m m t The in- mmdicat form of &e fungi as mycorrhizal root piec- m and hgle imlakd vcsiclcs cowdmted excenenr sources of inmlurn for the esrabUsbmcnt of in ntro cliltures and for the lx~tinuous culture of b e 3pe- cia. Srreral thousand fungal propagub Here ob- rained for the nvo G. YJ&J%W species, G. mh-amt- tm and to a l&er extent for G. f b l h l a h a l ~ w h ~ ~ e a ~ few ~ p e s mm obtained k r G+ m ~ n a This wculture Wrn appeared suitmbfc for tbe aablish- men€ ofof incm ccSI&ns of AM fungal mains.

#slY Wbmh a r b ~ ~ ~ rnycarrhld fund, contin- uous culture, Fungal Eibrmy, Ri T-DM msfo~acd root, v&h

wmaoucnm A r b u W mycorrhi (AM) fuagi ;arc mil micro- otpnims whicb appmntly caa pmrhrce spocts and complete rheir lite ryck only upon the ~crloaistioa d a sw~cp'blc h m root Thq. are rhuf coddtred obligate biompbs. Many anemp to thue o e gdsmii in uitw w e fmplcmented h rhe km d c cad% mm wulw king abnrined 4th as starring mtdaT (Muam and Hepper, hatl@er and M a s s 1987; 3 6 d and Fartin, 1988; B b r d and Echt, 19991; Sf. Arnaud ct al.. XW9B). It appears, bwmer, that h+ae and &ctw wirhin rows abo

Aurp4hd For prrkkraiun Mmhg, SS8 ' M c k c l ~ P . € f n i M s ~ u t ~ b +

play a ky rde in the s o r n ~ n of the lift cydt of AM fungi [Eiarley and Smith 19831, fhUs represent- ing pmrialiygxmd inoEula.

Smllu uld R o m d /l%, 1887) demonstrated thaf the fungm vhmm roar pieces and aingIt b la td vcsidcs wns a& !%I geminate and form new rypirnl m y c o r r h in plao when issm'uaed wirh n swepdble host root This t~hniguc a l l m d the monorcnic (x1hm of Ghwm irrlpuandices Schenck and Smith isnd G. rrsm~mm (&mm) Be& (Diop 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 achieved (Declmk et at, 199th) and their entrap mcnr in a t p ' i be& offered a new kind of h i h q* tnoculmn, tree of pathqem- (Z)PCIerck et al., 1996b). This c d m n reehiique was M e r swc c d f k y cmadtd to qxt3-m originaung Gom the rhkwphere af a d net m -1 (Diup. 1995). Nowirhpranding these red& md the numee ow oppmmnitics &cd by @e inharadical brms of the fungus, this tcrhniqilt received Einle attention m date. The aim of tht prscnt W& vvar to whether

the mhamdical brm ofAM fan& could be ustd for the monoxenic culrivadpn oT specks oriel- naring f?um a vopical mogratea W t 8 are report- ed and potential u x s of this monoxenic adm~zion qsmm aa a tcol for germplasm collectians ia d k EUSIL~

Sworgwr a - T h & w r n e d mmt ID- MF W L) mob wrrc provided by X Fiche (CRBF, Univ- ersit4 M, Q.i:bec). Rautiae maiatblance d tht m u m made m the mddificd Suullu-Romand (MSK) medium (Diop, 1995) h aa tmrCrted pQpiacm at '27 C ip the dark The medhrm cmtaincd in

L-8 W e d mter: qs4.m~. 75% 76; KHpPO,, 4.1; Ca[N0~,.4Npo, m; NalrtEOTA, 8; KCL, 65; Mn50,.4Elz0, 2.4% ZnW4.7&0, 0.29; W%, 1.88; C16SO,5w, DDQP: tW),WH.4H,O, 0.W N@&Cl,Si&. 0.004?4; &-C, 1: pyridan- 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~ I O W and -5 W, W. +%G pH wps adjusted m 6.5 before sterilidon at I21 C for 15 6

m@ ftW&Kn*-Tht AM fungal atmina m col- l e d in Mnrtincquc, the l a r g r ~ of t& W i n M b 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 sugarcane toadepthot25 cm, air dried and p a d thmugb a Z mm s i w . Bulk cultum were then eseab- LiaRed on Ieck plantr [AIliu= porraun L) g m on auwclav*d (121 C for two sepaate I h psrkdlp) Ter- ragrecn* under gracshoupe mndittona After Q m% aporea were mtracted Erom thc bulk cultures by slew 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. seedlii and grown for another 5 ma in a ~ t m h v d Terragrem* &r penhouslt codidans were €in* exancrtd by ritving nnd s p i m idcad- Bed by P Daw (4&ricultm h a & , Omwn) using the clapid morph10gW spona chamcuiirticr. Fiw G b w sp, [G. fd&a Wter) CR h n n &Trap. G, mm (I) and (9, G. AILP.

-'puns Thhme & lUa.sne, and a aain of G irr- ~raruhcaj originaring h m a banmm A& g m un- der tmpical cllmPtt in the tsnuy Island. ? mlcank laland in thE Mantic ocean mmc l O i l km From Ihc c m of nMthvmc AMcll] were considered h the presmt $tildy CTAQLE 11. Afmr wntlmic idmttfim don and purity mmsrnent orthe c u l m leek roc*s w e q l c d and q c o r r h i i Kgmenm aeltcxcd nm dcr binocular micrascope for h vim culntre csab Ibminat. meon BpDcsr.-haymm mrrr piecer wue disinfected iO an ulQa~nic apparanrs under it M- PPT. hOdd h t s war auec&1y naked in a mlu- don c& cthanolQ$R (vit) foe IQ a, ealclum h* cblodte 6% (W/* fm B min, ckloramine T 2% [W!

v) &&cl with some dropr o € l h t n 20 for 10 mh and antibiatics (SueptcMIycinc 200 mg L-l and p tMlycint 100 mg L-l) for 10 min Aftcr wch dish- fedon titcp. mts w e r i n d rhroc times in MC &tilled W. For G. Jma.m.&&m aud G* wm75mxe {I ) (TABU I), the diiinkmd mats were chopped in 0.5 cm picccs and rnzinoined in the a n d b i h soh*.

f. h vrm dnm and sqbminut p c a a a i the h r r n r P d i e P l ~ d ~ r r p A . P o t c d t ~ ~ ! d C t w R w n p a r a t n a ; & m p r r h l r . a l ~ ~ ~ p ~ C . ~ ~ ~ w Q . enndaffned root; E lperra ~~~ 3: ddnfec- rfnn procceh; 9: wktion of vodc14: 9! of h gemdmrcd pmpsgwh with n w m e h r n d a r m t mac; 4: wbruhureaf~ t x i d m m d rqco&hpt rmtpiawiah-a n m ParuMrmcd root

tion untn use. PM &C & r e Dthcr &S i.e. G ~ 1 l a . G . ~ a W T C . u g s 1 ~ ~ 2 ~ fTPr a s I), the dlstnfrmed rcboo wm gm* lacuTltcd with needles ka so exnsruhitlck C3uates-a of attached miclcr wetc then separated with andlea under bin- mhr m i ~ ~ ~ ~ o p e and rnsdmkhsd in the audMMic sotution.

Mmdc mlhm ~ - - B o t b LPDcUlum m aseptidp tradkred an the MSR rnedilrm in the at 87 C, using repmheb fWChpip for the root p iemu a d a micmpip~ur far the wwitlck Aflcr fryphal *CC d sgm =p-g the g- m U & d pmpgtk, ii.t,, qcarrh&aI root piezed or isolated vesictw was m& wirh a 15mm cark bdrer inm h c czpuimmtat P& G&. A ace ofagslr spas abruDwedfmm thbtxprbmtntal & w i t h tbe cork borer W that the prcpaple an the agar alIce could be inrerted in the hale. An &ly (gwrirtg tmmFarmod ~arrot root W then ph6td in h e itear vicinlrg of the prpppdc. The rulw wan &m in- cubaredin w in~dpCa:dcrninthc darkat27 C, M m 5 ma dud cultwe, vanafonned mat piece hawing heany m p c b d*velopmea~ on their W- facc w e cut d t b a acdpd and gently removed from the M§R medium witit f o n e p Phc root p i e wrrt

r e w b e d with a mn&tmFd mot of c a m s to hi- date a urn culhnz. Tht fa vim whirr and arbcuC rwc prmw of IbE h-mt tiwms of G h w saaint is skeutrcbcd in RC. 1,

TO our knowledge, 5 G&mw species are actually maintained and subcultured wing the inuaradical f o r m of the fungi (Dip, 1995; Smllu et al.. 1997). In this way, it is pDaaible to create in vho germplasm collcctiona and keep AM fungi under standard growth conditiom. In the present study, five novel G l o w strains were su~cessfully cultured in vitro. Two suains [G. fPrdeuhtum and G. wrrrfmnrs (l)] were p d u e t d with rnycorrRad root pieces as start- 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 greenhouse condjtioru. The s p r u produced in these greenhouse c u l m wm further similar ta the spores identified initially, demonstrating the purity of the in vivo cutt~red strdm. Thc long term maint* nancc of species through regular sulxulturea under controlStd s m d a d u e d conditions should cnable the propagation, throughout the scientific community of reference species For fundamental and prxtical r e scarch. Questions remain. however, concerning the infectiviey and cffoftiwneas OF the species maintained for long periods in nuo. For ectomymrrhizal hngi, a r ira lkt ion via host pawge every 4 yr was pr* posed by Manr (1981) W maintain the symbiotic p p centials of the hngi. Plenchettc et al. (1996) o b erved also a decrease in infectivity of succaiws c& cures d a G. mm spccicr and arc& the im portance of maintaining both trap pot cul- and in v i t ~ culturts *AM fungal species. each mllecrion serving as a p w l for the other collection. In our study, no thorough obmwtion W made on the in- fective potential of the species mainrained in vitro for long periods. Hwvewr, each species gmwn in vitro was able to reinfect kck plants under grcenhollse conditions and typical i n W d and extraradical AM fungal smcturcs wm observed. M n succesivc subcultures of G. i*&s were recently obFained by Saullu (pc= comm.) using mycorrhizal root seg- ments and spore pduction rrmaincd constant dur- ing the successive cultures, demonstrating the high potential of the inamdical form to maintain spe- cies wcr long pcriods. Smck ccnrers, bgsed on v cies propagated on host plant growing in disinfected substrate, already exist, and should be broadened W in vim culture collections. In nwo culture is lab+ ous and time consuming. Therefore a method for long-term swrage is mnlial . Actually, pot c u l m in- acula are successfully stored for sewrat mo tither at 4 C and through cryoprescrvation (Douds and Schenck, 1940) and single sragc Iyophilimion har been c k r i v e for some AM fungal species (DalpP. 1987). Further rmarch is needed to adapt Ifie methods W in vim cu1runs to ensure the long term

maintcnanfc of the species within collections and to constitute a pool of in vivo material able to inidarc new in viao and in vivo culturea

The wthon are &mLdul to Y. Dalpt (Agriculture C a d & Onawa) for IdcntiFy3ng the GIonarr @CS. Thia work was supponcd by a p n t h OCe STD3 Na 92104.

Waji. 1, M. J. Pouli, H. Vierhcilig, and Y PicM. 1995. Ueqmnrer of an ubuscular mycmrhi i fungrrs Gp rpwa magrm'a to emdam and wlatile from rht RI TDNA-Tmdwrntd roots of nonmycorrhfsal and m)- c w h i d mutanm d &urn rduum L Sprklc. Exp.

1% 2 7 . W . B a d . G., and J. A Fonin. 1W. &artr mnrr ofvrsicular-

a h ~ ~ I a r mycorrhiml forrnhon on Ri T-DNA vana rwmcd m m Nmplhlld. 106: 411-218.

-, and Y. Picht . 1989. h& gmwrh mimuladon by CO, and m exudates in rPrlcularmbumh mycw r h i i aymbhh. AppL M i m h l 5 6 : 45- 23-25.

md - 1992 hbllshmcnt of VA m&orrhb zac in toor organ culuuc. rcvierr and p r o m mcth- d*. Pp %1M. Iw in mftLIc&Dkw, Eda, k Vsuma J. R Norris and D. J. Read. Academic Prm. New K o h

-, L P. Taylw, D. D. Douds, P. E PfcfFer, and L W. h r . 1985. Flavonoids an nor necessuy plant signal compounds in arbular mprrhiml aymbih . M&. P I - M M Ismua 6: Z2-m.

Chrbot, S , R &l Rhlid, R. Q l h m r r and Y. Pichl.. 1922. Hyphal growth promotion in d m of the VA mycOn?ib

f i m g u a , m q m l u & c k r r & HaH, by h e dvig of sirucrurally @ l c flawmid compounds under COflnriched mdirma h h Pkllol. 122: 461- 467.

Dalp, Y. 1987. S p ~ c viabiiiry of swnc Endqgmacrae a u b mitted W a single SW IpphiliPPtion. P. 279. IRI tladiqy q r l t s SmmIhNorlh AmAcst l h # m ~ ~ r n

Eds. D. M. Sjhia, L L Hung and J. H. Craham Gainsvillc. Florida.

Deckrck S. D. G. SlrulIu, and C Plcnchtuc. 1- In viva ~ m d u c t i o m of thc axbu4cular mymrrhiial fungus. G h n u wr@m~, agociattd with Rl T-DNA wansformed carrot m o w h4yd k 1M): 1267-1242. --- , , and T. CuiUemette. 1996b. Entrap

mcnt of in vim produccd s p ~ m of C h i u ~ am@m# in alginatt btads: in vim and in vivo inocuIum poten- tial~. J. Ea~&lDd 48: 51-57.

Diop. T. A 1985. Erophysiologie des charnpigncna mycor hiziena I vEsiculcr t t arbuscules arsoci& a A& &-h deL dans k s zones Sahkimnts et SoudPooCuintmne du StnLgaL Pt. D. U, UniKFsity of Angers. FM-. 151 PP

-, G. Ecard, and Y Piht. l$%!. Long4crm in vim

~EZLF.RCK ET Al..: MONOXI

m l ~ e of an endomyeorrhiral fungus, golit&, on Ri T-DNA t~~ roots of -L + Y& 12: 249-259.

-, C. Plmhrttc, and D. G. S h h . 1- Dual awe nit culrurc of rheanbroot In& of v & ~ ~ l a ~ r b cuhr mymrrbual &ngi assocked with tomato m m . My- 5: 17-22,

-, -, and -. 1- In vim culmrt of s h d mycorrhkd a l r o o t k m 17: 217-m.

Douda, D. D., and N. C. Schenck 1990. Cryopresemtiwr of spore ofrcs icul~~~~busedar m~orrhiral h@. hkw Phytd 11% 667474.

ELias, K. S., and G. R. W. 1987. Hyphal clongation of GbtPlw jas&&huAin response to tmt exudates. Appl Bwimnm, Mimbici 59: 19%1933.

Giovsnneni, M., C Sbrma, k Citernesi, ancl L Avio. 1996. Analysis of factors Inwlvad in fungal rerognition reaponsea to hostderived rlgnab by subwular mycop him1 Fungi. Nm Phgrd 13% &7l.

Hacley,J. L., and S E. Smith. l l b . #. Academic Press, Inc. London. 485 pp

Heppcr, C M. 1979. Gmninption and growth of Ghnw e i u s spores: the &ecu Of inhibitors ancl nutri- ents. Sod Bid &chum. 11: 269977.

Lim, L. L, k L. J. Colc, and b A. FIntm. 1984. Ultra- uwture of thick-walled hyphac of Ghwjamiuhhm In d d m of c l w r (w&utn v@sia#). COR. j. B& BP: 262-W1.

M m , D. H. 1981. VariabiU~y In in ecwmymrrhieal dcucl- opmcnt and growth among hlates of Pimlithtu tine ~ m i u ~a afFccted by source, age and rc(solarion. Can J F m k 11: lE&I69.

Mossc, B. 1959. Regular gelminaion drrsring rpwes and swnc observations m the -h rcquircrmnts of an Endogoprr sp. causing vnkularabuscular raycorrh'oa. h w . Brit. M g d Soc. 4% 27SH6.

-. 1962. The csrablimmt of vtaiculawrbuscular mycorrhiza under ascptic condition& Gaa. WwW 27: 509-520.

-+ and C. M. Hcpper. 1975. Vcfilcular arbuscular mp

mrrhizal infections in root organ culture. PL PaMd P 215229.

Mugnicr. J., and B. MW. 1987. Vmkdar arbuscubx fecdona in tm=bmcd Ri T-DNA grown mmi- W. PA- 77: l(H61(KIO.

Nair, Y C. , G. R W, andJ. 0. Siquwra. 1991. Isolahn md idtneiion of vesicular-arburcuht mycomhiep srimularopy ccmpunds from dmcr (-m rcpllor) m o f s . A Q p L E t f B i t D R t R M U 5 7 : W .

Plcnchcue, C.. S Dcckrck. T. A Dip, aud D. G. S I X U ~ ~ . 1996. infKtivig of mnoaunic sukulrurts d the ar- hmdar myroclhkd fmgu C h w -M d with WT-DNA tradormtd carrot row M& mW h r s o k d 46: 5 M .

SL Amaud, M.. C h e l , B. M. Cam, End J. A. Fortin. 1% lhhancad hyphni p w b and spwt p duction of Ihc a r h h r r n ~ ~ fungur Ghms irurrmadiarinnoinviuogwcmintkabscnccdharr rWtk Ehr. 100: S2&33Z

Smith, S. E., and D. J. Rcad. 1996. m tprQdoai. 2nd td. m Re& Inc., Sen Diego. 5[15 pp.

M u , D. G., T. k Wwp, a d C P l m m . 1 W . R&& W o n dt colkcpiona in vim dc G h w m- (Schcnck et Smirh) ct dc Cbms - (Kaoun et Bmh) ct propmjiiion d'un cyck dt dk loppmmt C R A d . *, Pm* Sm 4147.

-, and C. Romand. 1986. MCthodt d'obrmtiw! d'tndomycorhizu h vMculcr et prbuscules en wndk umr ~ x b l q u e a C R A d S&, PBI* SOS: 245-450. - nnd -. 1987. Culture a*tnigue dc vtsicukr

i s o b A panir d ' c n d o v h ' i c l kmoclatiom tn viuo i dcr racinn de tomsue. C R A t d . Sd, BP: 1*1%

-, -, and C. PLcnchcDc 1I1. knit c u l m and cncagsuladon d rhc inparadid h of h W. WJ. Mirrobid E m k h d 7: Z92-W.

Tommcnrp, I. C, and L. K abbot^ IBBI. Prolonged wr- viva1 and vkbiliry d V A ~ o r r h i m l hyphae root death. SDil BioL E a c h 13: 431-433.

Wlliams, P. C. 1990. Disinfecting vesbhwhmtlar my- corrhisu .M@ &. 9C: -7.