APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 291-2950099-2240/92/010291-05$02.00/0Copyright ©D 1992, American Society for Microbiology

Specific Amplification of 18S Fungal Ribosomal Genes fromVesicular-Arbuscular Endomycorrhizal Fungi Colonizing Roots

LUC SIMON,'* MAURICE LALONDE,1 AND THOMAS D. BRUNS2Centre de Recherche en Biologie Forestiere, Faculte de Foresterie et de Geomatique, Universite Laval, Sainte-Foy,Qmuebec GIK 7P4, Canada,' and Department of Plant Pathology, University of California, Berkeley, California 947202

Received 16 July 1991/Accepted 15 September 1991

The first DNA sequences obtained from arbuscular endomycorrhizal fungi are reported. They were obtainedby directly sequencing overlapping amplified fragments of the nuclear genes coding for the small subunitrRNA. These sequences were used to develop a polymerase chain reaction primer (VANSI) that enables thespecific amplification of a portion of the vesicular-arbuscular endomycorrhizal fungus small subunit rRNAdirectly from a mixture of plant and fungal tissues. The specificity of this primer for arbuscular endomycor-rhizal fungi was demonstrated by testing it on a number of organisms and by sequencing the fragment amplifiedfrom colonized leek (Allium porum) roots. This approach, coupled with other molecular techniques, willfacilitate rapid detection, identification, and possibly quantitation of arbuscular endomycorrhizal fungi.

Of the many symbioses that evolved between vascularplants and soil microorganisms, the arbuscular endomycor-rhizae are certainly the most ubiquitous (10, 15, 19, 24). Forthe plants, colonization results in improved acquisition ofmineral nutrients and improved growth and survival, butrecognizing, identifying, and quantifying the vesicular-ar-buscular endomycorrhizal (VAM) fungi involved are at bestdifficult and time-consuming and often not feasible. Further-more, these fungi have never been successfully grown inculture, and so all stocks must be maintained as cocultureson colonized roots. Although they are one of the fewfunguslike groups with a fossil record (21, 23), little iscurrently known about the evolution of these organisms.As the first gene to be analyzed in this group of organisms,

the small subunit rRNA (SSU) was chosen because priorstudies have shown that variable portions of the gene pro-vide abundant phylogenetic information (5, 8, 11, 13, 18, 26)as well as target regions for the design of taxon-specificprobes (1, 6). Furthermore, highly conserved regions of thegene enable it to be sequenced rapidly either directly fromrRNA or from DNA amplified by polymerase chain reaction(PCR) with universal primers (3, 7, 12, 22, 25). Our goal was

to design an oligonucleotide primer that would exclusivelyamplify a portion of the arbuscular endomycorrhizal fungusSSU gene from a mixture of plant and other fungal DNAs.Achievement of this goal would allow PCR-based detectionmethods similar to those described for ectomycorrhizal fungi(9) to be developed for arbuscular endomycorrhizal fungi.

MATERIALS AND METHODS

Sample preparation. DNAs of Glomius intraradicesShenck and Smith DAOM 197198 and Gigaspora mnargaritaBecker & Hall DAOM 194757 were isolated from a smallnumber of spores (60 and 20, respectively) as describedbelow, while DNA of Endogone pisiformis Link.: Fr. CRBF0001 was isolated from mycelia grown in axenic culture bystandard methods. Spores of the VAM isolates were pro-duced in vitro by a dual culture system with transformedcarrot roots (2). The spores were collected with forceps,

* Corresponding author.

291

placed in a microcentrifuge tube, and crushed with a minia-ture pestle, and 40 pI of chelex resin (20% in sterile water)was added, and then sterile water was added to a finalvolume of 200 ,u1. After four freeze-thaw cycles, the tubewas centrifuged for 2 min, and the supernatant was treatedwith RNase. This crude extract was diluted 1/50 and used as

template for PCR.Leeks (Alliium porum var. Musselburg) colonized with

Glomus v,esiculiferum were maintained in standard growthcabinet conditions. Their roots were harvested, and DNAwas extracted as previously described (3).PCR conditions. Two overlapping fragments were ampli-

fied in separate reactions by using the primer pairs NS1-NS6and NS3-NS8. The reaction mixture consisted of a templatesolution and a master mix (20 mM Tris-HCl [pH 8.4], 100mM KCl, 3 mM MgCl2, 0.02% gelatin, 60 mM [each]nucleotide) (1:1, vol/vol) containing 0.5 U of Taq DNApolymerase and 1 pmol of two appropriate primers in a 20-,ultotal reaction volume, overlaid with light mineral oil. Thetemperature profile was programmed to repeat 40 times a

cycle of denaturation (2 min at 95°C for the first cycle, 35 s

at 96°C afterward), annealing (55 s at 53°C), and polymeriza-tion (35 s at 72°C for the first 14 cycles, 2 min at 72°C for thenext 11 cycles, 3 min at 72°C for the last 15 cycles), and the

SS38 t NS31

NS1t VANS1 NS3 t--* --lo ---

NS51

NS5t NS7t

NS2 NS4 NS6 NS8

NS21 NS41 NS61 SS1492'

FIG. 1. Diagram of the relative locations of the SSU primers.Locations of the universal primers and taxon-specific primer(VANS1) are shown on a schematic representation of the nucleargene coding for the SSU. Arrows indicate direction of primerextension. Thin lines represent flanking intergenic spacers. t and t,primer sequences described in references 3 and 25, respectively.The remaining primer sequences are as follows: VANS1, GTCTAGTATAATCGTTATACAGG; NS21, AATATACGCTATTGGAGCTGG; NS31, TTGGAGGGCAAGTCTGGTGCC; NS41, CCCGTGTTGAGTCAAATTA; NS51, GGGGGAGTATGGTCGCAAGGC;NS61, TCAGTGTAGCGCGCGTGCGGC.

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88 8*****8T***** GRRTGG8888 TT*RRRTCRG ****T **** ****** 888 ********GlIomus AG CCRTGCRTGT CTRGTRTRRT CGTTRTACAG GTGRRRCTGC GRRTGGCTCR TTRRRTCAGT TRTARTTTAT TTGRTRGTRC -CTTRCTACTGigaspora RAARGRTTRRG CCRTGCRTGT CTRGTRTRRT CGTTRTRCRG GTGRRRCTGC GRRTGGCTCR TTAARTCRGT TRTRGTTTRT TTGRTRGTRC RRTTRCTRCTEndogone TTTGTRCT- GTGRRRCTGC GRRTGGCTCR TTRRRTCRGT TATRGTTTRT TTGRTRRTRC CTTTRCTRCT

-------- URNS.I----------.

********s** s*********s ********** ********** *8***** 888*** *** *8******* ***** **** *8***Glous TGGRTRRCCG TGGTRRTTCT RGRGCTRRTR CRTGCTRARR CCTCCGRCTT CTGGRRGGGG GTGTRTTTRT TRGRTRRRRR RCCRRTR-TC GGGCRRCCGRGigOspora TGGRTRRCCG TGGTRRTTCT RGRGCTRRTR CRTGCTRARA ATCCCGRCTT CTGGRR-GGG RTGTRTTTRT TRGRT-RRRR RCCRRTRRCC TTCGG-----Endogone TGGRTRACCG TGGTRRTTCT RGRGCTRRTR CRTGCTRRRR RTCCCGRCTT CTGGRR-GGG RTGTRTTTRT TRGRTRRRRR RCCRRCGT-- GGGCRRCC-R

** 88 888888888 *8*8**88** 8888888888 8 * * 8888888 8 8888888888 ********** *8******** **********Glomus --TTCCCTTG GTGATTCRTR RTRRCTTTTC GRATCGTRTG RCTTTRCGTC GRCGRTGRRT CRTTCRRATT TCTGCCCTRT CRRCTTTCGR TGGTRGGRTRGigospora GTTTCCCTTG GTGRTTCRTG RTRRCTTTTC GRATCGTRTG GCCTTGTGCT GRCGRTGTRT CRTTCRRRTT TCTGCCCTAT CRRCTTTCGR TGGTRGGRTREndogone --CTCRTCTG GTGRTTCRTR RTRRCTTTTC GRRTCGTRTG GCCTRGTGCC GRCGRTGRTT CRTTCRRRTT TCTGCCCTRT CRRCTTTCGR TGGTRGGRTR

*8*8**8*** 88888 8 8 8888888888 88 888888 888 888 8888888888 8********* ********** ******* 88 **********Glous GRGGCCTRCC RTGGTGGTRR CGGGTRRCGG GGTGTTRGGG CRCGRCRCCG GRGRGGGRGC CTGRGRRRCG GCTRCCACRT CCRRGGRTGG CRGCRGGCGCGigaspora GRGGCCTRCC RTGGTTTTRR CGGGTRRCGG GGARTTAGGG TTCGRTTCCG GRGRGGGRGC CTGRGRRRCG GCTRCCRCRT CCRRGGRRGG CRGCRGGCGCEndogone GAGGCCTRCC RTGGTRTTTR CGGGTRRCGG GGRATTAGGG TTCGRTTCCG GRGRGGGRGC CTGRGRRRCG GCTRCCRCRT CCRRGGRRGG CRGCRGGCGC

8888888888 8888 8888 8888888888 8888888888 8888888888 8 888 88 88 888 8 ********** 8*8***88** 888888 *8**Glomus GCRRRTTRCC CRRT-CCGRC RCGGGGRGGT RGTGRCRRTR RRTRRCRRTR CGGGGTTCTT TRGGRTCTCG TRATTGGRRT GRGTRCRRTT TRRRTCTCTTGigasporo GCRRRTTRCC CRRTTCCGRT RCGGGGRGGT RGTGRCRRTR RRTRRCRRTR CRGGGCTCTT RTGGGTCTTG TRRTTGGRRT GRGTRCRRTt taaatcncttEndogone GCRRRTTRCC CRRT-CCGRT RCGGGGRGGT RGTGRCRRTR RRTRRCRRTR CRGGGCCTTT TTGGGTCTTG TRRTTGGRRT GRGTRCRRTT TRRRTCCCTT

8888888888 8888888888 8888888888 ********** 8888888888 8888888888 8888888888 ********** 8888888888 8**8******GloMus RRCGRGGARC RRTTGGRggg coogtctggt gecagcogcc gcggtoattc cagctccaat agcgtRTRTT RRRGTTGTTG CRGTTRRRRR GCTCGTRGTTGigospora oacgoggaac oattggoggg coogtctggt gccagcogcc gcggtoottc cagctccoat ogcgtRTRTT RRRGTTGTTG CRGTTRRARR GCTCGTRGTTEndogone RRCGRGGRAC RRTTGGRGGG Caagtctggt gccagcagcc gcggtoottc cagctccaot agcgTRTRTT RRRGTTGTTG CRGTTRRRRA GCTCGTRGTT

****** 8 8 8 8888*8 * ** ****8 **88*888 *888 8888 888 888 *8** **Glomus GRRTTTCGGG GTTRGTRGGT T-GGTCRTGC CTCT--GGTR TGTRCTGGTC TCRCTGR--T TCCTCCTTCC TTRTGRRCCG TRRTGCCRTT RRTTTGGTGTGIgaspora GRRTTTCGGG GTTCTRCCGT T-GGTCGGG- CRRT--TGTC TGTRCTGG-C GTGTGGRRTT TCTRCCTT-C TGGGGRRCCR TCRTGTTRTT RRTTTRGCGTEndogone GRRTTTTRGC TCTGGCTGGG C-GGTCCTGC CTTTRCGGTG GGTRCTRCTT TGGCTGGGGT TC-RCCTT-C TGGTGRGCTG GCRTGTTCTT RRCTGGRTGT

88 88 8888 88 88 8888888 8888888888 88 888888 88 888 8888888888 8888888888 888 ***** 888 88 8GIomus TGCGGGGRRT TTGGRCTGTT RCTTTGRRRR RRTTRGAGTG TTTRRRGCRR GCTRRC-GCT TGRRTRCRTT RGCRTGGRRT RRTGRRRTRG GRCGTT-CGRGigospora GGTGGGARRC CRGGRCCTTT RCCTTGRRRR RRTTRGRGTG TTCRRRGCRG GCTTRC-GTC TGRRTRCRTT RGCRTGGRRT RRTRRRRTRG GRCGGT--GGEndogone GTCAGGGRRC CRGGRCTTTT RCTTTGRRRR ARTTRGRGTG TTTRRRGCRG GCRTCC-GCT TGRRTRCRTT RGCRTGGRRT RRTRGRRTRG GRCTTT--GG

8 8 8****

GIo*us TCTTATTTTGGi gospora TCCTGTTTTGEndogone TTCTATTTTG

88888888 8

GIoMus GRCTAACTACG i gospora GACTAACTTCEndogone GRCTAACTTC

********* 8 * 8* 8******8** *8******** 8888888888 ***** 8**** ********** *8*8**8*** 8***** **TTGGTTTCTR GGRTTGRCGT RRTGRTTRRT RGGGRTRGTT GGGGGCRTTR GTRTTCRRTT GTCRGRGGTG RRRTTCTTGG RTTTRTTGRRTTGGTTTCT- GRRTCRCCGT RRTGRTTRRT RGGGRTRGTT GGGGGCRTTR GTRTTCRRTT GTCRGRGGTG RRRTTCTTGG RTTTATTGARTTGGTTTCTR GGRCCGRGGT RRTGRTTRRT RGGGRTRGTT GGGGGCRTTR GTRTTTRRTT GTCRGRGGTG RRRTTCTTGG RTTTRTGRRR

*8**8***** TT*TG*CC8R 8888888888 ********** *CG******* 8888888888 ********** *R*CC ***** *T*8T*8***TGCGRRRGCR TTTGCCRRGG RTGTTTTCRT TRRTCRRGRR CGRRRGTTRG GGGRTCGRRG RCGRTCRGRT RCCGTCGTRG TCTTRRCCRTTGCGRRRGCR TTTGCCRRGG RTGTTTTCRT TRRTCRRGRR CGRRRGTTRG GGGRTCGRRG RCGRTCRGRT RCCGTCGTRG TCTTRRCCRTTGCGAAAGCA TTTGCCRRGG ATGTTTTCAT TRRTCRRGRR CGRARGTTRG GGGRTCGRRG RCGRTCAGRT RCCGTCGTAG TCTTRRCCRT

8*8******* 8********* 8888 **** * *8 8 **** 88 *88* **** * **** 88 *8** 8*8*8*G6I us RRRCTATGCC GRCTAGGGRT CGGATGRTGT TRRTTTTT-R RTGACTCRTT CGGCG-CTTR CGGGRRRCCR RRGTGTTTGGGigospora RRRCTRTGCC GRCTRGGGRT CGGRCGRTGT TRRTTTTT-C TTGRCTCGTT CGGCG-CTTR CGGGRRRCCR RRGTGTTTGGEndogone RRRCTRTGCC GRCTRGGGRT CGGRCGRTGN T---NNTTRC -THRCTCGTT CGGCRCCTTR TGRGRRRTCR RRGTTTTTGG

8****8**** ********** 8888888888 88 ******* 8888 888 888888888 ********** 8***8****GIosus GCRRGGCTGR RRCTTRRRGG RRTTGRCGGR AGGGCRCCRC CRGGGGTGGR CCGTGCGGCT TRRTTTGACT CRRCRCGGGGGigospora GCRRGGCTGR RACTTRRRGG RRTTGRCGGR RGGGCACCRC CRGGGGTGGR CCGTGCGGCT TRRTTTGRCT CRRCRCGGGREndogone GCRRGGCTGR RRCTTRRRGG RATTGRCGGA AGCGCRCCRC CRGGRGTGCG RCGTGCGGCT TRRTTTGRCT CARCRCGGGG

8 *8****** ********** R*G******* C*TTGRTTCT TG88T***** TGCRTGGC** 8********* G***8*****Glomus TRGTRRGGRT TGRCRGRTTG RGRGCTCTTT CTTGRTTCTR TGGGTGGTGG TGCRTGGCCG TTCTTRGTTG GTGGRGTGRTGEingospora TAGTRRGGRT TGRCRGRTTG RGRGCTCTTT CTTGRTTCTR TGGGTGGTGG TGCRTGGCCG TTCTTRGTTG GTGGAGTGRTEndogone TGGTRRGGRT TGRCRGRTTG RGRGCTCTTT CTTGRTTCTR TGGGTGGTGG TGCRTGGCCG TTCTTRGTTG GTGGRGTGRT

GlomusG i gosporaEndogone

RCGRRCGAGR CCTTARCCTG CTRRRTRGCTRCGRACGRGR CCTTRACCTG CTRAATRGTCRCGRACGRGR CCTTAACCTG CTRRATRGTT

AGGCCTARC-AGGCTRTTCTRGGCC-RRC-

******8*8* G***8*88**GTTCCGGGGG GRGTRTGGTCGTTCCGGGGG GRGTRTGGTCGTTCCGGGGG GRGTRTGGTC*8****8*** *8**8*8*8*RRACTCACCR GGTCCRGRCRRRACTCACCA GGTCCAGRCARRRCTCRCCR GGTCCRGRCR

TTGTCTGGTT RATTCCGTTATTGTCTGGTT ARTTCCGRTRTTGTCTGGTT RRTTCCGTTR

888 8 88 *** 8 *** *8*8****** *8** * 8 *8 8 *8* **8*8*****ATTGTT-AGG TCGCCRGCTT CTTRGAGGGR CTRTCGGTGT TTRRCCGRTG GRAGTTTGRGTTTGAR-TGG TCGACGACTT CTTAGAGGGR CTATCGGCGT TTRGCCGATG GARGTTTGRGTTTGGT-TGG TCGTCARCTT CTTAGAGGGR CTATTGRCGA CTRGTCAATG GRRGTTTGRG

8*8******** ********** *888888*88 ********** *8**8***** ********** 88888 8 88 8 8 888 8 8**8** 8 8888*8*Gloaus GCARTRRCRG GTCTGTgatg cecttogatg ttctgggccg cacgcgcgct acactgatga aGTCRTCGRG TTCCTTTCCT TTRTCGGRRG ATATGGGTAAGigaspora GCARTRARCAG GTCTGTGRTG CCCTTRGRTG TTCTGGGCCG CACGCGCGCT RCACTGRTGR RGTCRTCGRG TRCATTTCCT TTRCCGGARG GTRTGGGTAAEndogone GCAATRRCRG GTCTGTGATG CCCTTAGATG TTCTGGGCCG CRCGCGCGCT RCACTGACGR RGTCRGCRAG TTTATRRCCT TGGTCGGARG ATCTGGGTRR

********8* **** 88888 ********* *8******** 888888888 * ******** ** ******* ** *8** * **** 8 8 ****8**8GIomus TCTTTTGRAR CTTCATCGTG CTGGGGRTRG AGCATTGCAR CTATTGCTCT TGRACGAGGR ATCCCTAGTA AGTRCRRGTC ACTAGCTTGT GCTGATTACGGigaspora TCTTTTGRAR CTTCRTCGTG RTGGGGATRG RGCRTTGCRA TTATTGCTCT TCRACGRGGR ATCCCTAGTR RGCRTGAGTC ATCAGCTCGT GCTGATTACGEndogone TCTTTTGRAR CTTCGTCGTG CTGGGGATRG RGCATTGCRR TTRTTGCTCT TCARCGAGGR RTTCCTRGTA RGCGTGAGTC ATCAGCTCGC GTTGRTTRCG

********** 8*8******8 8********* ********** ********** 888888 88 ****** 8 8 8 * 8GloMus TCCCTGCCCT TTGTRCRCAC CGCCCGTCGC TRCTRCCGRT TGRRTGGCTT RAGTGGGCCT TCGGRTTGAG RTTCGGAGA- CTGCRACRGA ----C-TCTTGigaspora TCCCTGCCCT TTGTRCRCAC CGCCCGTCGC TACTACCGRT TGARTGGCTT RGTGRGACCC TCGGRTCGRT -RAGTGGRAAR CCTTCACGGG TRTCCTTCTTEndogone TCCCTGCCCT TTGTRCACAC CGCCCGTCGC TACTACCGRT TGRRTGGCTT RGTGRGTCCC TCGGATTGGA -GCCCRGARG TTGGCRRCAR CRTCC-AGGT

8 8 8888 88 *8**** 8888888888 *8*888*8** 8*8*88***** 888888*8

GIomus GTT-TRRRGT TGG-CARRCT T-GGTCRTTT RGAGGRAGTA AARGTCGTAR CARGGTTTGigOspora GTTGRGRRGT TGGTCRAACT T-GGTCATTT RRGGGRRGTA RfRGTCGTfR CRRGGTTTEndogone GCTGRGRAGT CGGGCARACT T-GGTCATTT RGAGGARGTR Aaagtcgtoa caaggttt

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SPECIFIC AMPLIFICATION OF VAM FUNGUS RIBOSOMAL GENES 293

FIG. 2. Alignment of the SSU sequences. Sequences of the SSU from two arbuscular endomycorrhizal fungi, Glomus intraradices andGigaspora margarita, as well as a non-VAM fungus, E. pisiformis, begin approximately 40 bp from the 5' end of the gene and endapproximately 30 bp from the 3' termini. Gaps(-)were manually introduced to optimize the alignment. In some instances, the -sequence closeto the primers could not be read confidently and is reported in lowercase. Although these portions must be considered unsure, theycorrespond to highly conserved sequences among eukaryotes. Asterisks indicate positions of identity among the three taxa. The position ofthe VANS1 primer is indicated.

last cycle was followed by a final extension step of 10 min at720C.DNA sequencing. The gene fragments were gel purified,

diluted, and used as templates for subsequent rounds ofasymmetric amplifications (4) with various combinations ofthe internal primers illustrated in Fig. 1. The yield ofsingle-stranded DNA was estimated by analyzing the asym-

TABLE 1. Assessment of the specificity of VANS1 versusuniversal primers for the amplificationa of a

portion of the eukaryotic SSU

Result with primerSource of DNA pair:

Universalb VANSLC

PlantsAngiospermsAllium porum + -

Alnus crispa + -

Alnus glutinosa + -

Arabidopsis thaliana + -

Quercus agrifolia + -

GymnospermsAbies concolor +Pinus ponderosa +

Fungi and funguslike protistsOomycetePhytophthora cinnamomi a2423 +

ZygomycetesEndogone pisiformis CRBF 0001 +Mycotypha africana NRL 2978 +Phycomyces blakesleeanus NRL 1465 +Syncephalastrum racemosum NRL 2496 +Glomus mosseae INVAM-156 + +Glomus intraradices DAOM 197198 + +Gigaspora gigantea WV-932 + +Gigaspora margarita DAOM 194757 + +Entrophospora colombiana WV-877 + +Scutellospora pellucida WV-873 + +

AscomycetesCenococcum geophilum S166 + -

Dipodascus geotrichium ATCC 22600 + -

Neurospora tetrasperma FGSC 1271 + -

Tuber melanosporum S489 + -

Wilcoxina mikolae CSY 4 + -

BasidiomycetesBoletus satanas TDB 1000 +Dacrymyces palmatus SR449 + -

Peridermium harknessi RUR 152 + -

Russula laurocerasi TDB 1222 + -

Telephora americana S4938 + -

a Forty cycles of PCR under conditions as described in Materials andMethods, except for an annealing set at 55°C.

b NS1-NS2 or SS38-NS21 primer pair.c VANS1-NS2 or VANS1-NS21 primer pair.

metric PCR products by agarose electrophoresis. Excessprimers and nucleotides were removed by centrifugal ul-trafiltration, and the resulting purified DNA was sequencedwith a chain termination sequencing kit (T7 sequencing kit[Pharmacia] or Taquence [U.S. Biochemicals, Inc.]).

Nucleotide sequence accession numbers. Reported se-quences are deposited in the EMBL data bank. Accessionnumbers are X58724 (E. pisiformis), X58725 (Glomus in-traradices), X58726 (Gigaspora margarita).

RESULTS

In order to design an arbuscular endomycorrhizal-fungus-specific primer, we first needed to obtain SSU sequencesfrom those fungi and their relatives. This was accomplishedby amplification and direct sequencing for two species ofarbuscular endomycorrhizal fungi, Glomus intraradices andGigaspora margarita and for a non-VAM fungus that is aputative relative, E. pisiformis. For Glomus intraradices andGigaspora margarita, since no arbuscular endomycorrhizalfungi are available in pure culture, we took advantage of acoculture system in which plant roots, following transforma-tion by Agrobacterium rhizogenes, can be maintained andrapidly propagated in vitro (16, 17). These root cultures wereinoculated with a single surface-sterilized spore of an arbus-

M 1 2 3 4 5 6 7 8 9 10 11 12

603 bp l

FIG. 3. Taxon-specific amplification of a fragment of the SSUfrom VAM fungi. DNA samples were amplified as described inMaterials and Methods, except for the annealing set at 55°C. Both ataxon-specific primer pair (VANS1-NS21, odd-numbered lanes) anda universal primer pair (SS38-NS21, even-numbered lanes) wereused. Following agarose electrophoresis and ethidium bromidestaining, the gel was photographed under UV illumination. Samplesconsisted of purified SSU DNA of E. pisiformis (lanes 1 and 2) andGigaspora margarita (lanes 3 and 4), crude extracts of E. colombi-ana spores (lanes 5 and 6), purified SSU DNA of Glomus intrara-dices (lanes 7 and 8), crude extracts from leek roots colonized byGlomus vesiculiferum (lanes 9 and 10), and Alnus crispa totalgenomic DNA (lanes 11 and 12). Lane M, 4X174-HaeIII sizemarker, fragments of 1,353, 1,078, 872, 603, 310, 271 and 281, 234,and 194 bp.

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APPL. ENVIRON. MICROBIOL.

cular endomycorrhizal-fungus isolate (2). Viable, axenicspores were collected after a few months of incubation.DNA was extracted from them and used for PCR amplifica-tion with universal SSU primers (Fig. 1). Portions of theSSU were amplified from crude extracts and used for asym-metric PCR reactions that had been devised to producecontiguous single-stranded DNA fragments covering bothstrands of the gene. These DNA fragments were sequencedwith the same universal primers to yield the SSU sequencesof E. pisiformis, Gigaspora margarita, and Glomus intrara-dices shown in Fig. 2.The aligned sequences were compared with published

fungal SSU sequences to identify a region unique to the twoarbuscular endomycorrhizal fungi near the start of the SSU.This region was then chosen as the basis for the synthesis ofan arbuscular endomycorrhizal-fungus-specific PCR primer(VANS1 (Fig. 1 and 2). To examine the specificity of theVANS! primer, we paired it with a downstream universalprimer (NS21) and tested its ability to amplify a 550-bpportion of the SSU sequences from DNAs of other arbuscu-lar endomycorrhizal fungi as well as a variety of other fungiand plants. The fungi selected for testing spanned a broadphylogenetic range and included putative relatives of arbus-cular endomycorrhizal fungi as well as other rhizosphereassociates such as ectomycorrhizal species and root patho-gens. Among the organisms tested, only arbuscular endomy-corrhizal fungi yielded the expected SSU fragment. Otherorganisms tested yielded either no visible amplified productsor faintly visible bands of inappropriate sizes. Control ex-periments with either the NS1 or the SS38 universal primerdemonstrated that a larger overlapping region of the ex-pected size could be amplified from all of the samples (Fig. 3and Table 1). The general applicability of this primer for allarbuscular endomycorrhizal fungi is suggested by the findingthat the amplification succeeded not only with other speciesof Glomus and Gigaspora but also with species from twoother genera, Entrophospora and Scutellospora (Table 1).We have also found that VANS1 can be paired with otheruniversal primers (e.g., NS8 or SS1492') to yield virtuallyfull-length SSU genes from these species.Although the specificity of this primer could be tested

further against a larger number of organisms, a more func-tional test is to determine whether VANS1 can specificallyamplify arbuscular endomycorrhizal-fungus SSU directlyfrom colonized roots of a host plant grown in the presence ofmany natural contaminants. For this test we chose colonizedleek (A. porum) roots because leek pot cultures are com-monly used to maintain and produce endomycorrhizal fungi(20). Such colonized roots resemble field-collected materialbut have the advantage of containing a unique and knownVAM associate. Aliquots of DNA samples were amplifiedeither with the taxon-specific primer, VANS1-NS21, or withthe universal primers (Fig. 3). As expected from the locationof the primers used, products of two different sizes wereobtained: the overlapping 600-bp universal product wasamplified from all samples, while the 550-bp product wasamplified only from samples containing arbuscular endomy-corrhizal fungi. The 550-bp fragment obtained from thecolonized root sample was directly sequenced. A simplealignment with other corresponding sequences confirmedthat this specifically amplified fragment belonged to a Glo-mus SSU. It differed from the Glomus intraradices gene atonly 3 positions out of 447 sequenced, while Gigasporamargarita and E. pisiformis exhibited 45 and 46 differences,respectively.

DISCUSSION

The PCR allows the obtention of a specific gene or genefragment from a very limited amount of starting material.This obvious advantage has made this technique useful inmany fields, but care has to be taken since low levels ofcontamination can also be exponentially amplified. This isespecially important when new genes are amplified withuniversal primers. The SSU sequences reported here can beconfidently assigned to Glomuis intraradices and Gigasporamargarita because (i) axenic starting material was used, (ii)comparative analysis of the sequences shows that they arerelated fungal sequences (unpublished data), and (iii) aprimer designed with these sequence data could be used tospecifically amplify SSU fragments from many arbuscularendomycorrhizal fungi, even in the presence of plant tissues.Although the amplification of the SSU from a single spore ofarbuscular endomycorrhizal fungi was not attempted, thisshould be possible, since the actual portion of diluted crudeextracts used already corresponds to less than a spore.The ability to specifically amplify arbuscular endomycor-

rhizal fungi SSU sequences directly from DNA extractedfrom colonized plant tissue has two important applications:it will enable many more SSU sequences to be rapidlyacquired, and these data will make it possible to rigorouslytest the proposed phylogenetic hypothesis for these fungi(14); it also provides a new approach to rapid detection,identification, and quantification of arbuscular endomycor-rhizal fungi in plant roots. Our results from the colonizedleek root DNA extracts demonstrate that detection via directamplification is already feasible. The observed divergencewithin the SSU of the arbuscular endomycorrhizal fungi thatwe have sequenced is sufficient for the design of oligonucle-otide taxon-specific probes. These could be targeted eitherto filter-bound amplified fragments (9) or to rRNAs by in situhybridization (1, 6).

ACKNOWLEDGMENTS

Axenic spores of Gigaspora margarita and Glomus intraradiceshave been kindly provided by G. Becard and S. Chabot, respec-tively. Spores of Glomus mosseae, Gigaspora gigantea, Entrophos-porca colombiana, and Scutellospora pellucida were provided by J.Morton (INVAM, West Virginia University). We thank M. Gray foraccess to his data base and T. Szaro for technical assistance. We aregrateful to L. Bernier, J. Bousquet, J. Morton, and Y. Pichd forcomments on this work.

REFERENCES1. Amann, R., N. Springer, W. Ludwig, H.-D. Gortz, and K.-H.

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