Indian Journ al or Ex perimental Biology Vol. 40, Septcmber 2002, pp. 1087- I 09 I

Monoxenic in vitro production and colonization potential of AM fungus Glomus intraradices

A Mohammad' & A G Khan **

School or Science, Food & Horticulture, Coll ege or Science, Technology & En vironment. Uni versity of Western Sydney, Loc ked Bag 1797, Penrith South DC NSW 1797 , Australia

Received 15 Jalluary 2002, rel'ised 15 Mav 2002

The paper reports the estab li shmcill or mycorrhi za l inrecti on or a non-mycorrhi za l Ri-T-DN A trans rormcd carrot root when co-c ultured with a su rrace sterili zcd swcd potato root scgment coloni zcd by arbusc ular mycorrhi zal (A M) rungus C. ill/rami/ices on minimal M medi um. Extensivc rungal hypha I cmergence rrom eac h cut end of the mycorrhi zal swcet potato root piece was observed in one weck old cullUres. Thes\: hyphae causcd infecti on on contacting the transrormed-carrot- root segment and produced many hyphae and spores both inside and outside the zone of the root aft er 6 week or growth. Axeni ­call y produced rungal propagules prolirerated on the surface of fresh minimal M medium when sub-cu ltured without any root seg ment. On repeated sub-culturing, thesc propagules did not lose their ability to grow and produced man y juveni le small spore-likc ves icles durin g thc non-sy mbiotic phase. Although these spores were morphologicall y and anatomical ly si milar to their soi l borne counter part s, they were much smaller. When pl aced in the vieinity or a fresh hairy root on the minimal medium or a Sudan grass seedling in sand eu llllre, thc axenica ll y produced AM runga l propagu les caused root in ­rec ti on, but the inrection characteri stics we re signiri cantl y dirfe rent to the ori ginal culture in terills o r shape (spheri cal vs oval) anc! sizc (20 ~lIn vs 45 pm) or thc intraradical ves icles. and absence or 'H' branches. Sudan grass seedlings inocu lated with the axcni call y cultured rungus showed signiri cantly (P <: 0.05) higher dry weights plant i

. When compared to the plants inocu lated wi th sand cultures. the growt h paranlcters and thc pcrcentage inrec ti on werc not signifi cant ly dilTerent. However, when bot h sources o r inoc l'la werc uscd together, a sy nergistic cl'fcct on plant growth as we ll as root infection was obscrvcd.

Glomalean fun gi are universal sy mbionts , which form arbuscular mycorrhizal CA M) associations with most plants 1.2 and are importan t in various ecological condi ­tions. 111 \'ilro culturing of AM fu ngi is difficult due to their obligate biotropic nature and thi s limits their use for com mercia l utili zation. AMF inocululll has been produced mainly by pot cultures, soil-free methods' ,

. ~·5 d I I . (, aeroponlc systems , an lye roponlcs ,

Axenic cultures of AMF with living roots7 or sus­pension cultured plant cells as symbiont partnerX are reported. Diop et al.~ reported ill vitro culture of AMF on Ri-T-DNA transformed tomato roots producing ex tensive ex ternal hyphae with spores and typical AMF infecti on of the roots. Plenchette el 01. to main­tained AM fun gus GIOIllIlS versifol'llle under axenic in vitro cond iti o ll s on modifi ed medium over three gen­eration s without loss of infectiv ity . 10licoer et 0/.

11

produced propagules of AMr GIOIllIlS intraradices in an airlift bioreactor by culturing transformed carrot hairy roots.

*Prescnt address: Post Doctoral Fell ow, Departmcnt de Biolog ia , Av Univcrsiclad 2001. Col ChalTlilpa CP62250, Cuclllnvaca, Morc!os, MEXICO **Correspondcllt author

The purpose of this study is to report the produc­tion of AMF inoc ulum by co-culturing AMF colo­ni zed root segment with a non-mycorrhi zal hairy (Ri­T-DNA transfonned) root on a minimal M medium to result in producing ex tra-matri ca l AMF hyphae and spores and then sub-culturing the fungus so produced in the absence of the root. Infecrivity of the monox­enic subculture of the AMF so produced is also tested by MPN method.

ProdllClioll of l1Iycorrhizal rools -Mycorrhizal sweet potato roots were produced aeroponically using Glomlls illlraradices Schenk & Smith (lNVAM iso­late WV 994A-l) as inoculum in an ultrasonic nebu­li ser system under gree n house conditions5

. Mycorrhi ­za l root pieces harboured AM fungal structures such as non-septate inter- and intra-ce llul ar hyphae averag­ing 8.9 J.Hn di ameter, oval to e lliptical intra-radical vesicles (20-42 ~lm in diam.) and arbuscules. AMF coloni zed root pi eces were surface sterili zed with 3% H20 2 for 15 min followed by 3-4 washings with steril­ized di still ed water, and then aseptically transferred to Petri dishes containing minimal M medium l 2 with 1% sucrose. The dishes were stored at 4°C until used.

1088 INDI AN J EXP SIOl, SEPTEMBER 2002

Producliol1 of hairy rools -Agrobacleriul1l rhizogenes (UNSW) was used to produce Ri-T-DNA transformed carrot roots. The root cultures were rou­tinely propagated on minimal M medium as per Sharp and Doran 13.

Dual cultures of mycorrhizal alld nOIl-lIIycorrhizal roots -A 6-7 cm Ri T-DNA transformed non­mycorrh izal carrot root ex plant and a surface steril­ized heavily AMF infected 0.5 cm piece of sweet po­tato root produced by improved aeroponi c culture techniqueS were pl aced in close proximity onto the minimal M medium, and incubated at 25 "c in the dark. For the contro l, on ly mycorrhi zal sweet potato root pieces were allowed to grow on the minimal M medium.

Viabi lily of 1/ IOI10xellic subcultures of the AMF­The viability of the spores and hyphae produced on minimal M medium in dual cultures was tested by transfe rring (a) an infected transformed carrot root piece, (b) a minimal M medium agar block with fun­gal hyphae but without transformed root piece, and (c) an AMF infes ted minimal M medium agar block and a non-transformed root onto the fresh minimal IV! me­dium and incubating the plates in dark at 25°C.

Illfec li vity of sub cuI lures of the AMF- fn order to compare the infectivity of the sub-cultures of the AM fungus so produced, fi ve day old Sudan grass seed­lings grown from surface sterili zed seeds (3 % H20 2

for 3 min followed by 3 washing with sterili zed di s­tilled water) on moistened filter paper, were trans­ferred to 500 ml plastic pots fill ed with steamed sand watered to fi eld capacity and inoculated by adding (i) 10 9 of the original sand culture of AM fun gus (INYAM 994A-I ) from the USA, (ii ) lO x I cm pieces of the transformed and mycorrhi zal carrot root, (iii) I cm2 minimal M medium block infested with the AM fungus, and (iv) a combination of (i) and (iii), into a 5 cm deep hole before inserting the radical s of 5 pre-germinated Sudan grass seed lings per pot. The pots were maintained in a glass house (temperature range 13-2SoC and photoperiod 14 h). After 6 weeks, the Sudan grass seed lings were harvested and their fresh and dry weights (80°C unti I constant) were de­termined. Some fres h root segments from each treat­ment were cleared (2 .5% KOH) and stai ned (acid glycerol aniline blue) before measuring the amount of AM fungal coloni zations. The number of viable AM funga l propagules per g of inoculum was determined by the most probable number (MPN) technique as per Khan l 4

.

Continuous cp lture of G. introradices were main­tained over a peri od of 1 year. Carrot di scs, infected with A. rhizogenes, produced 2-3 hairy roo ts at the infection sites afte r 3-4 weeks of incubation. The ex­cised root tips grew well on hormone-free minimal M medium and produced a few lateral branches.

When a mycorrhi zal and aeroponically produced surface sterilized sweet potato root segmen t was placed in close proximity of carrot hairy root on the same minimal M medium plate, fu ngal hyphae ap­peared both laterally and from the ends of the root cuttings from the sweet potato mycorrhizal root pieces. Extensive hyphal growth emerging from ends of mycorrhi zal root pieces was observed in one week old cultures. These hyphae caused infection on con­tacting the non-mycorrhi zal transformed carrot root segment placed in the close proximity on the same plate. Sporulation started after 6 day of incubati on of Ri-T-DNA transformed roots. Spores were found both inside and outside the zones of root after 6 weeks of growth. External hyphae of the fungu s possessed all the characteristics typical of AMF, i.c. non-septate hyphae with angular projections and term inal spores, but the spores formed on the minimal M medium were much smaller than the ori ginal fungus, ( 15-47.2 pm compared wi th 90 ~lm ). Intern all y, sta ined trans­formed carrot roots showed relat ively strai ght dark stained hyphae whi ch produced '1-/' branches and nu­merous intraradi ca l oval vesicles. Arb uscules were rare l y observed.

Axenically produced fun gal propagul es proliferated on the surface of minimal M medium and covered it 4 weeks after incubation. On repeated sub-culturing, these propagules did not lose their ability to sporulate on fresh minimal M medium. No sporocarps were observed in the present ill vitro systems. When pl aced in the vicinity of a fresh hairy root on minimal M me­dium or a Sudan grass seed ling in sand culture, these ax inically produced AMF propagules caused root in­fection, but the infec ti on charac teristics were signifi­cantly different to the origi nal axeni c culture in terms of intraradical vesicles shape (spherical) and size (18 .5-22 pm) and absence of '1-/' branches.

Sudan grass seed lings inocul ated wi th the axeni­cally cultured fungal inoculum sources, i. e. mycorrhi­zal transformed roots or minimal medium block in­fested with axenic propagules of G. intraradices, showed significant (P < 0.05) differences in plant dry weights (Table I ). Microscop ic examination revealed numerous intrarad ical vesicles and hyphae in the root

NOTES 1089

Table 1- Dry we ight ·1 plant , percen tage coloni zat ion of Sudan grass roots by axe ni c and sand cultures of AM fungi and thei r

inoc ulum potenti als (MPN)

I Valucs are mcans of 4 rcpli cates l

Inoculum DW·1plant(g) % infection MPN indcx· l g

Cont ro l O. IS" 0.00 0.00 Sand culturc 0.5 1 b 33.00" 1,353" Transfonncd Mycorrhizal roots 0.46b 360" I,S63b

M medium bl ock Infested with ax enic AMF propagulcs 0.461> 35.5" 2,14 1' Transformed Mycorrhi zal roots + sa nd cu lture O.S' 39.4h 4,264"

Mean va lues in a column foll owed by different letters arc signifi ­cantl y different at P <0.05 (n= IO)

cortices and numerous terminal spores on the ex­tramatrical mycelium on the root surfaces of Sudan grass seedlings. Root colonization values for seed­lings inocul ated with mycorrhi zal transformed roots or with axenic propagu les of G. int raradices raised on minimal M medium, were not statistically different (Table I). When compared to the seedlings inoculated with the sand cultures of G. intraradices, the OW and the percentage root infecti on values were also not sig­nificantly different. However, when transformed my­corrhizal roots and sand culture of G. illtraradices, were used together, a sy nergistic effect on plant growth as well as root infection was observed (Table I). The infectivity of the subcultures of the axenic AMF was not red uced, as ev ident from the MPN data (Table 1). The MPN assay also showed that the ax­enic AM F inoculum had a significantl y hi gher num­ber of infec ti ve propagules than those produced by sand cultures (Table I).

The present in vestigation reveals sy nthesis of AMF infection by G. intraradices in transformed carrot roots. The transformed carrot root was able to grow on the minimal M medium. These results are in agreement with those of previous researchers. Karan­dashov et al. ls raised dual cultures of AM fungi (in­cluding Gloll1us intra radices ) and carrot hairy roots and found well-developed mycorrhizae with typical characteristics. Pawlowska et al.16 reported estab li sh­ment of mon xenic cultures of Glomus etuJ/icatum in association with excised Ri T-ONA transformed car­rot roots and demonstrated that AMF can be propa­gated in vitro using monoxenically fo rmed resting spores and/or colonized root fragments . Abdul-Khaliq 'nd Bagyara/ 7 reported successful mycorrhizal infec-

tion of Ri T-ONA transformed tomato roots during co-cultivation wi th spores of Gigaspora margarita on minimal M medium. This technique prov ides a better way to visualize the full life cyc le and the events that occur during the pre- and post-colonization phases of Glomalean fungi.

Although Strullu and Romand lx reported regenera­tion of vigorous hyphae from three Glomus spp. from surface sterilized mycorrhizal root segment on mini­mal M medium, no mycorrhization fro m the fun gus so produced was tested. This is the first known report of mycorrhi zation of a Ri-T-ONA transformed non­mycorrhi zal root segment when co-cultured with a non-transformed mycorrhi za l root segemnt. Earlier . . 19··)J d d ' f AM . f In vesti gators - reporte pro uctlon 0 In ec-tions in the root organ cultures, but in the grow ing system used fungal hyphae in the rootless compart­ment were still physicall y connected with the parent root through the dividing wall. Many researchers found that growth of the AM fun gi ceases when the hyphallink to the root was broken. Various nutritional and genetic factors have been suggested to explai n the lack of growth of the extraradical phase of AM fun gi (for references see Oiop et al.9). In contrast, we were ab le to achieve the axenic growth of AMF in absence of a host root after the initi al stimulus has been pro­vided by the starter culture. Ishii et al. 22 reported suc­cessful axenic culture of Gigaspora ramisporophora in the absence of a plant partner but with the addi tion of methanol extract from bahi a grass roots to the cul ­ture medium. Recently , Karandashov et ai. 15 estab­lished monoxenic cu ltures of AM fungi including G. intra radices from a sterili zed root pi ece with my­corrhiza alongwith fungal spores.

Sudan grass seed lings inoculated with axenicall y produced AMF in the present study showed typical AM fungal colonization and had significantl y higher OW plant l. These results are consistent with those of Mathur and Vyas20 who found ill vitro produced G. diserticola significantly beneficial for biomass pro­ducti on and nutrient uptake of in vitro raised plantlets in pots. Vimard el al. D

, in contrast, found no di ffe r­ence in colonization potential of monoxeni c in vitro produced G. intra radices spores compared with a root-segment inoculum from open pot culture.

Although G. intra radices spores, formed in vitro on minimal M medium in the absence of any host root in the present study, exhibited general morphological and anatomical similarity to soil-borne spores, they were signi ficantl y smaller than their soi I borne coun­terparts. They may be juvenile spores as they pro-

1090 INDI AN J EXP BIOl, SEPTEMBER 2002

duced typical AMF infection in Sudan grass seedlings when used as inoculum in the present study. de Souza and Berbara2~ studied the ontogeny of G. clantll l in Ri-T-DNA transform ed clover and carrot roots during the non-symbiotic growth phase and after the coloni­zation of the transformed root and found that spore­like small vesicles during the non-symbiotic phase were juvenile spores . Pawlowska el al.16 observed similar differences between soil borne andmonoxenic cultures of G. elllllicallllli in association with exci sed Ri-T-DNA transformed carrot roots. The literature also reveals that the spore size and morphology of AMF differ greatl y, even within the same species25. Thi s cou ld be associated with the relief of root in­duced growth repression, as postulated by St Arnaud el ct!. 21. It may also be that the fla vinoids released by the non-transformed sweet potato root and trans­fo rmed carrot 1'0 01 in the present study had a differen­tial effect on the fun gal growth . Piche et 0 /.

26 have shown that the Ri-T-DNA transformed and non­transformed carrot roots rel ease different flavinoids which are involved in a regulatory system which con­trols the hyphal growth of GIOIIIIlS lIlargarila . Further, after establi shing itse lf in side a root, AM fun gus changes the ex udation pattern of host roots both quan­ti tatively and qualitati ve ly27 . G I OIllIlS IIlOCroC(//PIIIiI

was shown to reduce thc ex udat ion or both sugars and amino acids rrom the fine roots 2~ . Perhaps, a ~tilllul a­tory substance produced in AM-coloni zed swet t po­tato root in association 'Vv ith Ri-T-O A transformed carrot root in the present st udy suppli ed its needs to grow and sporu late without any plant partner.

The root organ culture tec h;,ique employed in thi s study provides a better way to visua lize the full life cycle and the events that occur during the pre- and post-colonization phases of Gl ol11a lean fun gi. Fur­thermore, the use of molecular techniques and DNA technology will al so provide additi onal tool s for studying genetic variations between various AM­fun gi 2'1.32. Clapp el al. 33 reported that the ri bosomal small subunit sequence varied cons iderably within spores of SClIlel/ospora spp., an AM fun gus .

The two novel biotechnological approaches used in this study, i.e. the use of Ag rouaclerilll!l-tran sformed

plant roots and mycelial cultures of mycorrIli zal fungi , are considered as the research too ls in the envi­ronmental remediation research 3~. The contributions of mycorrhi zal symbionts to soil productivity and en­hanced heavy metal uptake during ph ytoremediation of contam inated sites has not yet been seriou sly con­sidered and require further exploitation35. Effo rts to

phytoremediate contaminated soil can be enhanced by inoculating hyperaccumulator plants with axenic AMF inocula most appropriate for the site.

The award of a postgraduate scholarshi p to the first author by the Australian Internati onal Development and Assistance Bureau is gratefull y acknowl edged. This project was partly funded by the UWSM Re­search Board. We wi sh to thank Prof. Pauline Doran, University of New South Wales, Sydney, for provid­ing culture of Ag rouaclerillJll r/tizogenes.

References I Jeffries P, Use of mycorrhi zae in agri cul lL: re. CRC Crit ReI'

/Jioteehl/ol. 5 ( 19X7) 3 19. 2 Khan A G & Belik M, Occ urrence and .:colog ica l sign iri­

cance of mycorrhi zal sy mbi os is in aqu;.I tic plants. in My­corrhi : a -stntc/llre. ji ,l/cliol/ .. Il lIileell lar iJiologv Ol/rl iJ iolech ­I/ology edit ed by A Varma and B I-lock (Spri nger, Heidd­berg) 1995, 427.

:; Sylvia 0 & Jarstfe r A G. Production or inucu lum with arbus­cular mycorrhi za l rungi, in M w/(/gt'lIl elll (){ IIIVCOITh i:o iI/ agriclIltllre. l /UrticlIltllre al/dF)resflT. edit ed by A 0 Robson. A K Abbotl & Mal ajczuk (Kl ower. DonJrec hl) 1994,23 1.

4 I-lung l l & Sylvia 0 M, Production or vesicul ar arbuscular mycorrhi zal rungus inoculum i:l aeroponic cultl\l"c. A/i/i I: I/\'i ­J"(iIl MicroiJiol, 54 ( 19118) 353 .

5 Mohammad A, Kh:1I1 A G & Kuck C, Improvcd aeroponi c culture or inocula or arbuscula r mycorrhi zal rungi . /v/r ­corrhiz,o , 9 (2000) 337 .

6 Elmes R P & Mosse 13 . Ves icular arhusclI iar cncJo mycorrhi­zal inocu lum production. II. Expe riments with maize (Lco Il/aI'S) and other hosls in nutri elll rIow cu:lUrc. COl/ J /Jot. 62 ( 1984) 153 1.

7 Chabot S, Becard G & Pi chc Y, lire cyel,· or Glo/ll llS illll"(l' radices in root organ cu llurc, Myeologio .R4 ( 1992) 3 15.

8 Carr G R, Hin kley F, l eTacon F, Heppe r eM, Jones M G K & Thomas E, Im proved hyphal growth of two specics or ve­siclIlar-arbu sc ul ar mycorrhi za l rungi in Ihe pn;scncc of sus­pension-c ultured plant cc ll s, Nell' Phyroi. I (J I ( 191\5 ) 417 .

9 Di op T A Plenchelle C & Slnillu 0 G, Dual axc ni c cu ltu re of sheared-root inocul a o f vcsicula r-arbll',c ular Ill )'corrhi; al fun gi assoc iatecI with tomato roots, Mycolog io . 5 ( 1994) 17.

10 Pl enchette C, Dec lerk S, Diop T A & Struilu D G. In fec tivit y or Illonoxeni c subc ul lures or the arbuscu lar mycurrh i/.al run­gus Glo/lllls l ·ersilorJl/e associa ted with RI-DNA-transf"onncd carrol root. A/ip M ierobiol /Jiolec/lllol . 46 (1096) 545.

II Jo li coer M, Will iams R 0 , Chavari e C. Fortin J A & Ar­chamb:lllit J, Producti on or Glolllus illt rumdices propagules, an arbu sc ular mycorrhi zal fun gus, in :111 airlift biorcaclOr. lJiotecll/ lUllJioellg ill , 63 ( 1999) 224 .

12 Becard G & Fortin J A. Earl y eve nt s ot vc, icul ar-arbuscular mycorrhi za formati on on Ri -T-DNA tr:lI1srormed roots. NI'II"

Pitytol, 108 ( 1988) 2 11. 13 Sharp J M & Doran PM. Characteri stic, of growth and 11'0 -

P:1I1 C alkal oids sy nthes is in Atrope bel/lli lollll a roots trans­formcd by Agrob{/cter i lllll rh i:ogeIl Cl·. .I i3 iote("it llol. 16 ( 1990) 171.

14 Khan A G, Inoculum densit y of GlolIIl/s iIIosse{/e and grow lh of oni on plants in un stcrili zcd bituminous coa l w:,stc. Soli IJ io / /Jiochelll 20 ( 19R8) 749.

NOTES 1091

15 Karandashov V E, Kuzovk in<l I N, Gcorge E & Marsc hner H, Monoxc ni c cu lturc of arbuscular mycorrhizal fun gi and plant hairy roots , Rllssiall J PI Phl'siol, 46 ( 1999) 87.

16 Pawlowska T E, Douds D D & Charvat I, III vilro propaga­ti on and life cyc le of the arbu sc ul ar mycorrhizal rungus GIOIIIIIS ellillic(I{lIl1l , Mycolog Res, 103 ( 1999) 1549.

17 Abdul-Khaliq & Bagyaraj D J, Coloni za ti on or arbusc ul ar­mycorrhi zal rungi on Ri-T-DN A transrormcd roots in sy n­thctic mcdium, Illdiall J E.rf1 /Jiol ,38 (2000) 1147.

18 Strulu D G & Romand C, Mct hodc d'obtenl.ion d'cndomy­corrhi zcs a' vc'siculcs ct arbusc ulcs cn conditions axe' niqucs. CR Acad Sci Paris, 303 (1996) 245 .

19 Mugnicr J & Mossc 13, Vcs icul ar arbuscular mycorrhi zal in­fections in transformcd Ri-T-DNA roots grow n axcnically , Pliysiof1lialliology, 77 ( 1987) 1045.

20 Mathur N & Vyus A, III vilro production or GIOIllIIS diseni­cola in assoc iati on with Zizif1 lills 1Il1l1l1l1l1laria. Plalll Cell Ref1,14(1995)735.

2 1 St Arnaud M, Haml ct C. Vimard B, Caron M & Fortin J A. Enhanccd hypha I growth and sporc production of thc arbu s­cular mycorrhi za l fungus GIOIIIIIS illlraradices in an in vi tro sys tcm in thc abscncc of host roo ts, Mycol Res, I 03( 1996) 328.

22 Ishii T, Mat sumota I, Shrcstha Y H, Murata H & Kadoya K, Axcnic cu lture of vcs icular arbuscuiar my~orrhizal rungi and thcir infccti vity to sevcra l plant roots, Jaf1all Soc. /-Ion Sci, 64 ( 1995) 190.

23 Vimard B, St Arnaud M. Furlan V & Fortin J A, Coloniza­tion potential or ill I'ilro produccd arbuscular mycorrhi zal fun gus spores comparcd with a root-scgmcnt inoc ulum rrom open pot cu lture, Mycorrliiza, 8 ( 1999) 335.

24 deSouza F A & Berbara R L L, Ontogeny or Glolllus clams in Ri-T-DNA transformed root , Mycologia, 91 ( 1999) 343 .

25 Neiderhofcr M A & Schcnck N C, Morpholog ical divcrsity within a single vcsicul ar arbuscul ar fungu s (V AMF) in a mi xed hardwood rorest in North Florida, in 7[h North Ameri­cal Conf on Mycorrhi zae: Mycorrhi zae in the next dccadc. Practical applications and prioriti es, edited by L L Hung &

Graham J H, Institute or Food and Agricu ltural Sc iences, Uni Florida, Gainesvill c, USA, 1987, 3 18.

26 Pi che Y, C~1lford J G, Simard J. Labrie F & Paul ini M J. Fla­vo noids: Fundamcntal growth ractors to AM rungi . Abstract I" Intern Co nI' Mycorrhi zae (ICOM). Uni vC1'sity of Caliror·· nia , Berkeley,USA . August 3-1 0, 1996.

27 Graham J H, Leonard R T & Menge J A, Mcmbranc­mcdiatcd dccrcase in root exudation is rcsponsiblc ror phos­phorus inhibiti on or vcs icular arbuscular mycorrhi zac form;)­ti on. Plalll Pliysiol 68 ( 198 1) 548.

28 Bansa l, Mukclji K G, Positivc correlation bctwccn VAM ­induccd changes in root cx udat ion and mycorrhi zosphcrc mycofl ora. Mycorrliiza, 5 ( 1994) 39.

29 Lanfranco L, Delpcro M & 130nfante P, Intrasporal va riabi ­lity or ribosomal scqucnccs in the endo mycorrhi zal fun gus Gigasf1o/'{/ lIIargarita, Mol Ecol 8 ( 1999) 37.

30 L1 oyed-Macg ilp S A, Chambcrs S M. Dodd J C, Fillcr A H, Walkcr C & Young J P W, Diversity or ribosomal internal transcribed spacers within and among isolatcs of GIOIIIIIS lIIosseae and related mycorrhi za l rungi. Nell' Phwol . 133 (1996) 103.

3 1 Vandcnkoornhuysc P & Leyval C, SSU RNA sequencing and PCR-fingcrprinting reveal ge neti c variation within GIOIIIIIS II/O.u eoe, Mycologia , 90 ( 1998) 79 1.

32 Chclius M K & Triplett E W, Rapid detecti on of arbuscu lar mycorrhizac in root and soil of an intensively managed turfgrass system by PCR amplification of small subunit rDN A, Mycorrhiza , 9 ( 1999) 6 1.

33 Clapp J P, Filler A H & Young J P W, Ribosomal small sub­unit sequence va ri ati on within spores or an arbuscular my­corrhi zal fungus, Sculel/oSf1ora sp ., Mol Ecol, 8 ( 1999) 9 15.

34 Pietsch M, deAraujo 13 S & Charlwood 13 V, Noval biotec h­nological approaches in environmental remedi ati on research. Biolechl/ol Adv, 17( 1999) 679.

35 Khan A G, Kue K C, Chaudhry T M, Khoo C S & Hayes W J, Role or plants, mycorrhi zae and phytoc helators in heavy metal contaminated land remed iation , Chelllosphere , 4 I (2000) 197.