Molecular Ecology (1995) 4,6148

Generation of RAPD-PCR primers for the identification of isolates of Glomus mosseae, an arbuscular mycorrhizal fungus L. LANFRANCO, P. WYSS, C. MARZACHkand P. BONFANTE Centro di Studio sulla Micologia del Terreno del CNR, and Dipartimento di Bwlogia Vegetale, Univmitd - V i d e Mattioli 25, 10125 Tmino and *Zstituto di Fitm'ro~ogia upplicutu del CNR, Strada delle Cacce 73,10135 Torino, Italy

Abstract

Mycorrhizal fungi are usually identified on the basis of the morphological characters shown by fruit bodies, spores, vegetative mycelia or symbiotic structures. The develop- ment of molecular techniques provides a valuable and alternative approach to identify mycorrhizal fungi, especially when it is difficult to gather a sufficient number of data on morphological features. Short arbitrary oligonucleotides were used as primers for the amplification of genomic DNA extracted from spores of arbuscular fungi. The RAPD fingerprints showed banding patterns which allowed us to distinguish between species and even isolates within Glomales. In order to identify mycorrhizal fungi during their symbiotic phase, a nonpolymorphic RAPD band identified as marker for some isolates of Glumus mosseae was purified from rrgarose gels and cloned in a bluescript vector. The fragmentwas sequenced and specific primers (PGM3) were designed for the mycorrhizal fungus. They specifically and successfdly amplified the DNA not only from G. rnosseae spores, but also from roots of pea, clover, leek and onion plants when they were colonized by G. musseae isolates.

Keywords: arbuscular mycorrhizal fungi, DNA probes, Glornus rnosseae, PCR, RAPD, specific primers

Received 14 May 1993; revision r e c e i d 8 July 1994; accepted 13 July 1994

Introduction

The roots of most land plants join with soil fungi to form mycorrhizas which play a central role in the capture of nutrients from soil (Harley 1989). Root-fungus interac- tions give rise to an underground network of impressive complexity, since about 6OOo fungal species have been SO

far identified as mycorrhizal together with 240 OOO plant species. Among the mycorrhizal fungi, a reduced number of Zygomycetes belonging to the order of Glomales (130 species according to Walker & Trappe' 1993) colonize about 80% of plants. They are obligate biotrophic fungi and are called arbuscular mycorrhizal (AM) fungi, on the basis of the infection structures they produce inside the host plants (Bonfante 1984). Due to their widespread range of hosts (from Pteridophytes to Gymnosperms and Angiosperms), they represent one of the most important components of the soil microbe populations. However,

Correspondence: Paola Bonfante. Fax 39 11 655839.

studies on the ecological relationships between AM fungi and their host plants, as well as many applicative projects, are hampered by the difficulty to identify them at the genus and species level. In the absence of known sexual stages, their identification relies on the structure of their spores or sporocarps (Morton & Benny 1990; Walker 1992). However, many problems are still open, since de- scriptions of many, especially earlier, species are inad- equate and the same term of 'species' has been-questioned as not appropriate for asexual organisms (Stuessy 1992; Walker 1992).

The development of molecular techniques based on the polymerase chain reaction (PCR) has recently pro- vided a valuable and alternative approach to morphol- ogy, which represents the primary criterion to define the taxonomic position of fungi (Kohn 1992). Bruns (1991) reviewed such molecular methods and matched them with taxonomic levels for which they are appropriate. He distinguished between probes useful to study phylo- genetic relationships (i.e. nuclear rDNA genes) from oth-

62 L . L A N F R A N C 0 e t a l .

ers useful to analyse population genetics (RFLPs and RAPD). Such molecular approaches are particularly at- tractive for mycorrhizal fungi, where many morphologi-

Materials and methods

Selection of AM species cal features which are diagnostic parameters (spore size and wall, fruit-body structure) are lost during the sym- biosis. The different symbiotic structures, i.e. the fungal mantle and Hartig net in ectomycorrhizae, inter-intracel- lular hyphae, arbuscules or coils in endomycorrhizae (Harley 19891, do not gather in fact a sufficient number of data on morphological features to guarantee their identi- fication.

Rapid and sensitive probes to identify ectomycorrhizal fungi during their interactions with the host plants, have been developed by using primers designed on the sp quences of (i) the 18s and 28s nuclear ribosomal genes which flank the internal transcribed spacer (ITS) region (Gardes et af. 1991; Gardes & Bruns 1993) or of (ii) the mitochondrial large subunit rRNA ( B N ~ & Gardes 1993). The analysis of the nuclear 185 ribosomal genes has

Spores of AM fungi were produced by fungal isolates of different origin (Table 1). When possible for each sample a specimen voucher was deposited in the Herbarium Cryptogamicum (HC) of the Dipartimento di Biologia Vegetale dell'Universita di Torino. Mycorrhizal roots were obtained by inoculating spores of Glornus versiforme, G. rnosseue (Torino isolates), Gigusporu ? t ~ q U n ' t U into seed- lings of leek, white clover or pea as host plants as de- scribed in detail in Spanu et al. (1989). Mycorrhizal roots were collected after two months, when the degree of in- fection was microscopically evaluated as ranging from 50 to 70% of the whole root length. Leek roots colonized by G. intramdices and onion roots colonized by G. mossme (Kent I1 isolate) were kindly provided by Dr M. Giovannetti and Dr J. Dodd, respectively.

been a successful approach for the identificatiin of AM fungi during their sporal phase. Parts of these genes have DNA extraction horn spores and plant material been amplified in A M &gi by using conserved universal sequences as binding s i t s for the oligonucleotide prim- ers; specific primers have then been designed on thc sequencing of the amplification products (Simon etal. 1992). Sequences from 12 endomycorrhizal fungal species and comparison with other 18s sequences indicated that Glomales originated about 400 million years ago (Simon et al. 1993b). When coupled to a fluorescent single-strand conformation polymorphism analysis, such sequences have also been demonstrated to provide some informa- tion on the fungal identity during the symbiotic phase (Simon et al. 1993a).

The aim of this study was to generate probes which allow to identify AM fungi during their sporal and sym- biotic phase by following an approach alternative to the ribosomal probes. We have used the random amplified polymorphic DNA (RAPD) analysis (Williams et a[. 1990) based on singleshort arbitrarily designed primers. This technique has been demonstrated to be a powerful tool to analyse genetic relationships and diversity in ecological and population biological studies (Hadrys e t d . 1992). When applied to AM fungi, i t revealed RAPD polymor- phic fragments among different AM species and RAPD :ionpolymorphic fragments among isolates of the same species (Wyss h Bonfante 1993). Here, we assess the pos-

Extraction was performed as described in Wyss & Bonfante (1993). Five to ten pooled spores were cleaned by sonication (4 x 15 s) and by four rinses with sterile bi- distilled water, and then crushed with sterile forceps in 100 pL water; 50 pL of 20% Chelex (Biorad) suspension was added immediately. After a 15-s pulse sonication and 4 times of freezing/thawing in liquid nitrogen, the prepa- ration was heated for 15 min at 95 "C. The supernatants were stored at -20 "C.

DNA was extracted from mycorrhizal roots, non- inoculated roots and leaves with a small-scale method, as described in Henrion etnl. (1992). Ten to forty micro- grams of material was ground in liquid nitrogen and lysed with 500 pL of buffer containing 5 0 - m ~ Tris-HQ (pH 9.0), 5 0 - m ~ EDTA (pH 8.0), 2 5 0 - m ~ NaCl, 0.3% SDS and 0.1 mg of proteinase K for 1 h at 65 "C. After extrac- tion with Tris-saturated phenol/chlomform/isoamyl al- cohol (24/24/2, v/v/v) and chloroform/isoamyl alcohol (24/1, v/v) (Sambrook et af. 1989) the aqueous phase was incubated with 10 units of RNAase A (Sigma) for 1 h at 37°C. DNA was then precipitated with 8OOuL of iso- propanol, washed with 70% (v/v) ethanol and dissolved in 50 pL of 10-KIM Tris-HC1 buffer (pH 8.0) containing 1- m~ EDTA. DNA was stored at 4 "C.

sibility of coupling the sensitivity of the RAPD analysis with the specificity offered by specific primers, designed R A P D analysis on the sequencing of a RAPD amplification product. A similar approach, based on the generation of novel spe- cific probes and called SCAR (sequence characterized amplified regons), has already helped to develop mark- e n for the identification of downy mildew resistance genes in lettuce (Paran & Michelmore 1993).

Amplification of DNA was carried out in one-primer re- actions. The primer OPA-9 (5'4GGTAACGCC-3') was obtained by Operon Technologies, Inc. (Alameda, Cali- fornia). The reaction mix consisted of 1 0 - m ~ Tris-HC1 (pH 9.0), 50-m~ KCl, 0.1% Triton X-100, 2 - m ~ MgCl,, 2 0 0 ~ ~ of each d", 15 ng of primer, 0.4 units of

RAPD-PCR PRIMERS A N D G L O M U S I D E N T I F I C A T I O N 63

Table 1 Isolates of arbuscular-mycorrhizal fungi. For %me of the fungal isolates, the numbers from the collections of origin is provided. Vouchers have been deposited in the Herbarium Cryptogarnicum (HC) of the Diparthento di Biologia Vegetale dell'universita di Torino.

Origin Obtained from Voucher

Glomus m o s m (Nicol. & Gerd.) Gerd. k Trappe

Glomus m i m r s t . ) Berch

Glomus ualedonium(NicoL k GeidJTrappe & Gerd.

Glomusfistulosum

Glomus intraradicesSchendc & Smith

Glomus sp.

Acaulospora 1aevisGerd. & Trappe

Scutellospra sp.(Schenck & NicolJWalker & Sanders

Gigasporn margnritaBecker & Hall

Rothamsted (UK)

unknown Kent I (UK) Kent I1 (UK) Riesenbeck (D) Ahnsen (D)

Rothamsted (UK)

Gryndler 23 A Czech Republic

unknown

Hotteln (D)

Rothamsted (UK)

Migliarino (I)

New Zealand (I. R Crush)

A. Schubert Uorino, I) M. Giovannetti (&a, I) H. Vierhedig (Basel, CH; Granada, E) C. teyval (Nancy, F) A. Trotta Uorino, I) J. Dodd (Canterbury, UK) J. Dodd (Canterbury, UK) H. von Alten (Hannover, D)(Ha-104) H. von Alten (Hannover, D)(Ha-136)

J. Trappe (Corvallis, USA)

V. Gianinaui-Pearson(Dipn, D LPA 12

C. Walker (Roslin, UK)

M. Ciovannetti (Pisa, I) IMA6

H. von Alten (Hannover, D)(Ha-114)

V. Gianinazzi-Pearson (Dipn, F) LPA 1

M. Giovannetti (Pisa, I)

V. Gianinazzi-Pearson (Dibn, F) LPA 2

HC / F-E05

HC /F-E12 HC/F-E15

HC/F-EOl

HC/F-E04

HC/F-E13

HC/F-E09

HC / F-El 1

HC / F-El0

SuperTaq-Polymerase (Stehelin, Basel, Switzerland) and 6 pL of the template diluted five to tenfold in a final vol- ume of 15 pL. Mineral oil (15 pL) was added on the top of the reaction. The amplification reaction was performed in a Perkin Elmer thermal cycler programmed as follows: 1 x 2.5 min at 94 "C, 45 x 30 s at 94 OC, 1 min at 36 OC, 2 min at 72 "C, 1 x 5 min dt 72 "C. Control experiments were performed without adding the template DNA. The amplification products were separated electrophoreti- cally on 1.4% agarose gels and stained with ethidium bro- mide (Williams et nl. 1990).

The 655-bp DNA fragment amplified with OPA-9 primer was isolated from the gel, treated with T4 DNA polymerase and T4 polynucleotide kinase and subcloned into SmI-digested and dephosphorylated pBluescript KS+ (Stratagene, La Jolla, CA). The nucleotide sequence was determined by the dideoxynucleotide method with Sequenase version 2.0 (United States Biochemicals, Cleve- land) using primers T7 (S'-AATACGACrCACrATAG- 33 and T3 (5'-Al'TAACCCTCACTAAAG33 following the supplier's recommendations.

Design of spec@ primers and PCR amplification

In order to design primers and to search for similarity with sequences listed in different databases, DNA se-

quence analysis was performed by using PCIgene pro- gram version 6.7 (IntelliGenetics, Inc. Mountain View, Ca, USA).

Specific primers PO (S'-AAGGAAATGA'I?TGCATA- GCG33 and M3 (S'-TATAACATAlTG"AACTGGAIT- -33 with a gel filtration degree of purity were ob- tained from Primm s.r.1. (Milan, Italy).

The reaction of FCR amplification was carried out in 5OpL of final volume containing: 1 0 - m ~ Tris-HC1 (pH 9.0), 5 0 - m ~ KCl, 0.1% Triton X-100,1.5-m~ MgCJ, 200 p~ of each dN"F', 1 p~ of each primer, 0.5 units of SuperTaq- Polymerase (Stehelin, B a d , Switzerland) and 1-10 pL of undiluted DNA extracted from spores or 0.1-2.5pL of DNA obtained from roots or leaves. To preveFt evapora- tion, the reaction mixtures were overlaid with 40 pL of mineral-oil (Sigma).

Control experiments were performed by using the universal primers NSl and NS2 (White et al. 1990). NSl and NS2 region may be represented by about 100-200 more copies than the RAPD region essayed. One might therefore be able to amplify the rDNA region without be- ing able to amplify the other. Notwithstanding the possi- ble difference in sensitivity of the two probes, the primers NSl and NS2 were run in parallel to control the quality of DNA extracted from spore and plant material. Primers were obtained from Primm s.r.1. (Milan, Italy).

64 L. L A N F R A N C O et al.

We camed out 40 cycles of amplification in a DNA thermal cycler (Perkin-Elmer-Cetus) by using a step pro- gram 94 "C for 60 s, 55 "C for 90 s, 72 "C for 90 s, followed by a I m i n final extension at 72 "C.

Southern bZot and hybridization conditions

The Glornus mosseae cloned DNA fragment was extracted from the gel with Glassmilk (Bio 101, San Diego, CAI, la- belled with a DIG DNA Labelling kit (Boehringer Mann- heim) and used as a probe in Southern blot hybridizations on specific-primers (w-M3) PCR amplification products. Transfer onto nylon positively charged membranes (Boehringer Mannheim) was performed in 2 x SSC (Sam- brook et 01. 1989). Detection was performed with the DIG Luminescent Detection kit m h r i n g e r Mannheim) fol- lowing the manufacturer's protocol.

Results

W D technique revealed a different degree of DNA polymorphism among isolates, species and genera of sev- eral arbuscular mycorrhizal fungi (Wyss & Bonfante 1993). The primer of the arbitrary sequence OPA-9 led to a constant pattern of amplification products on DNA ex- tracted from spores of different lines and isolates of Gfomus rnosme (Wyss L Bonfante 1993 and Fig. 11, where the word lines' indicates the cultures of the Rothamsted isolate propagated for more than 12 years at four different sites. A DNA band of about 650 bp (arrow in Fig. 1) was selected as possible marker for the G. mussme isolates, since it was constantly present with the exception of Ha- 136 isolate. In addition, OPA-9 did not lead to amplified fragments of the same size in the other species (Fig. 1). After cloning, the DNA fragment was sequenced (Fig. 2). The fragment displays the exact sequence of the OPA-9 primer next to the cloning site. In addition, the SmI rec- ognized site is reconstituted by the presence of the CCC (cloning site bases) followed by GGG (first bases of OPA- 9 primer).

From the preliminary data of the DNA sequence a pair of oligonucleotides were designed: PO and M3,21 and 24 bases long, respectively. The full-length sequence of the DNA band was completed by using the two internal primers which should give in PCR experiments an ex- pected product of about 550 bp.

To check the validity of these specific primers, FCR experiments were set up by using as templates DNA ex- tracted from spores of different isolates and species of Glomales (Fig. 3).

A DNA fragment of the expected size (compared with that obtained from the recombinant plasmid) was pro- duced by the PO and M3 amplification reaction from the G. rnmsene isolates: Rothamsted (all the four lines),

Fig. 1 R4PD electrophoretic pattern by using OPA-9 primer (5' GGaAACGCC 3') on spores DNA from: Lanes: 1, G. rnosseae Roth. Torino; 2, G. mossenc Roth. pisa, 3, G. mosscue Roth. Granada; 4, G. rnossac Roth. Nancy; 5, G. rnosseae Torino; 6, G. mossenc Kent I; 7, G.msencKentII;8, C. mosseaeHa-104;9,G.rnos~prHa-136;10, Glomus amiforme; 11, Glomus mledoniurn; 12, Clausfistulosurn; 13, Giomus sp. Ha-114; 14, Rcaulospora famis; 15, ScutcIlosporu sp.; 16, Gigusporn mnrganta. Arrow points to the 655-bp cloned fragment.

10 2 0 30 4 0 so

1 C G C T A A C G C C T G C A G A M A A G C C M G A C T C A G C C M A A ~ A ~ A CCCATn;CGGACGTCmTTCGGTCCI!GAGTCGGT"TMAATUCT

I I I I I

51

101

15 1

201

251

301

351

401

4 5 1

501

5 5 1

501

651

M ? m A T T A T M T T A C I \ T A T M T T G A T C T M ~ ~ C A G ~ G T ~ T T h l l l V \ T M T A T T M T G T A T A T T M ~ A G A T C A C ~ G T ~ ~ ~

MAATTTTAT@ATAACATATTGTAACTGGATT+TTATATATMCCGA T"AMATATATATTGTATACATTGACCTAAC~MTATATATTGGCT

AA~MMTGGTCGAAATGATTATATGATTGAAATGGTCGMATTGA'ITT T T M l T T T A C C A C C T T T A C T A T A T A C T M ~ A C C A G ~ M ~ M A

TGACTATTTGTTMAAA4TTTGAAAAGTCGAUTTMTTACGAGAGTCGA A C n ; A T A A A C A A m T T T A A A C ~ G C T T T ~ T T M ~ C T C T C A G C T

MTCMTTATIUGGGTCGAAATTAATTATGAGAGTCGMATACATMAGC TTAGTTAATATTCCCAGCTTTAATTMTACTCTCAGCTTTATGTATTTCG

TGGCUGAATTACGATTACGTGTTGTTACACMATCTAUAAAAGGAAAA ACCCCTCTThATGCTAATGCACAACMTGTGTITAGATTTTTITCCTTTT

TmTCGTCCTTGTGAAAATTGAMAGTATTACACCGAGGTGGTACMAA AAAAAGCAGGAACACITTTAACTTTTCATAAT-GTGGCTCCACCATGTTlT

TTCCTMAAAGGMATTTCGATATTGATCATTTMl"GGGCCGUATAA AAGGATTTTTCCTTTAAAGCTAThACl'AGTAAATTAAACCCGGCTTTAlT

TGAAATGCAAACAAAACGGTTGMTTTCACCAAAATTAAGCGAAACAAGT A C P T P A C C T T T G T T T T G C C A A C T T U A G T G G T T T T C G C A

AAMTTTAGCTGAAAAATTTAGTCTGACTAGATATGGACATA~TAAGATG TTTTAAATCGACTTTCAGACTGATCTATACCTGTATCATTCTAC

TGACGCAAATGAATCACACCCCACAAAGACTATGGTlTTlTATTCGTCAC ACTGCGTTTACTTP.GTGTGGGGTGTTTCTGATACCWTAAGCAGTG

CGTTTATATCACATATTACGCGCTATGCAAATCAlTTCCTTTTTGGGCGT GCAAATATAGTGTATAATGCECZEACGTTTAGTAAAGGAF~AAAC~ TACCC ATGGG -

Fig. 2 Nucleic acid sequence of the GIornus rnosseae OPA-9 primer cloned fragment. OPA-9 primer sequence (5'-GGGTAACGCC-3') is underlined and PO (S'-AAGGAAATGATlTGCATAGCG-3') and M3 (5'-TATAACATAlTGTAAff GGAlTGG-3') specific primers are boxed. These sequence data are available from EMBL/GenBank/DDBJ under accession number No. X79155.

RAPD-PCR P R I M E R S A N D G L O M U S I D E N T I F I C A T I O N 65

Fig. 3 Gel electrophoresis of PCR products using lWM3 primers pair on spores DNA. Lanes: 1, L DNA digested with HindIII-EcoRI; 2, recombinant plasmid; 3, G . mosseaeTorino;4, G . mosseae Roth. Torino; 5, G . mossear Roth. Granada; 6, G. mosscne Roth. Pisa; 7, G . mosseae Roth. Nancy;8,G.mossenrKent1;9,C. m~~eKent11;10,G.mossmeHa-l04;11,Glomussp. Ha-l14;l2,G.rnossmeHa-136;13,GIornusurledoniurn; 14, Scutellospora sp.; 15, Gigaspora margarita; 16, Aurulospora Iamis; 17, Glomus wrsijorme; 18, Glomusfistulosum; 19, no DNA. Lower band is given by the not incorporated primers.

Torino, Kent I and 11, and Ha-104. No amplification prod- ucts were revealed by using DNA from other isolates or species (Fig.3), with the exception of two of them (G. mossene Ha-136 and Scutellospoln sp.). The amplified frag- ments were, however, of different size. DNA quality of the nonamplified samples was checked by using a set of primers (NSI and NS21, which amplified a part of the 18s ribosomal genes in the same PCR conditions (data not shown).

The hybridization of the labelled cloned fragment to the PCR products confirmed the specificity of the amplifi- cation (Fig. 4). Table 2 summarizes the results provided by the three experiments: RAPD, PCR and Southern hy- bridiza tion.

To verify whether the primers might detect AM fungi during their symbiotic phase, the analysis was extended

Fig. 4 SouthernbIothybridizationofspecificprimers(PDandM3) PCR products from spores DNA, using the 655-bp cloned frag- ment as a probe. lanes: I, recombinant plasmid; 2, C. mossme Torino; 3, G. mossme Roth. Torino; 4, G. mosseae Roth. Granada; 5, C. nwssae Roth. P i ; 6, G. mosseae Roth. Nancy; 7, C. mossene Kent I; 8, G. mosseac Kent 11; 9, G . mosseae Ha-104; 10, Glornus sp. Ha-114.

to roots of different host plants (white clover, pea, leek and onion) colonized by different Glornus mossem isolates. They were subjected to a small-scale DNA extraction to- gether with the corresponding uninfected roots or leaves. An amplified DNA band of the expected size was ob- tained from the amplification of mycorrhizal roots with the specific primers PO-M3 (Fig.5). Nonmycorrhizal roots or leaves did not originated any band using the two primers, whereas a specific region of the ribosomal genes was amplified by using NS1 and NS2 primers. The ampli- fied products produced a band of about 550 bp, as ex- pected for the universal primers (White et a!. 1990).

The same experiments were performed on DNA ex- tracted from roots colonized by different AM fungi. Even though DNA was amplified by the ribosomal primers, no signals were revealed from PO-M3 PCR reactions (Fig. 6).

Discussion

The experiments performed on 17 strains of arbuscular mycorrhizal fungi demonstrate that a Combination of RAPD and PCR leads to the generation of specific prim- ers, which successfully identify GZornus rnosseae isolates not only during their sporal phase, but also during their colonization process of the roots.

The two-step approach consists of the identification and characterization of a sporespecific RAPD band, cho- sen as marker for some Glomus rnosseue isolates, followed by the design of a primer pair for PCR amplification. The procedure allows to overcome some drawbacks of the RAPD technique. This is a powerful technique which works remarkably well to distinguish genetic differences among populations as an alternative to RFLP (Foster et al. 1993; Williams et a / . 1991). Due to its high sensitivity (a single nucleotide difference is revealed by RAPD analy- sis), it offers a substantial contribution to the molecular

66 L. L A N F R A N C O el 01.

Species or isolate

GIomus mossclll (Roth.) Torino Glmus mossdlc (Roth.) Pisa GImus m ~ s ~ c ~ c (Roth.) Granada Glomus mossa~~ (Roth.) Nancy G l a u s mossaacTorlno G h u s ~OSJCIIC Kent I Glomus mossaPrKent I1 GIomus m m a e Ha-104 Glomus mosdaac Ha-136 Glomus amiforme

RAPD OPA-9 amplification

+ + + + + + + + + +

RAPD OPA-9 655-b~ fragments

+ + + + + + + +

Hybridization on P0-m PCR PO-h43 FCR products

+ + + + + + + + + + + + + + + + +* -t - -t

Glomus calcdonium + -t Gtomusfistulasum Glmus sp. Ha-1 14 Acaulospora laevis Scutellosporrc sp.

+ +

- + + -

-t -

- -t ++ -t

Gigaspora margarita + - - -t

Table2 Summary of the results on 17 AM strains obtained by us- ing three different ex- periments: RAPD, specific-primers PCR, Southern blot hybridi- zation

Amplification products different in size from the expected 55o-b~ DNA fragment.

t Data not shown.

Fig. 5 Gel electrophoresis of PCR products using primers W and M3 (lanes 2-11) or ribosomal primers NSI-NS2 (lanes 13-17) and the following templtes: recombinant plasmid (lane 2); Trifolium repms roots colonized by G. mosscne Torino (lane 3); T. r e p roots (lane 4 and 13);Pisrcm satiuum roots colonized by G. mosse~eTo~ino (lane5); P. sativum roots (lanes 6 and 14); Allium porrum roots colonized by G. massme Torino (lane 7); A. porrum leaves (lanes 8 and 15); AlIium wpa roots colonized by G. nwsseae Kent I1 (lane 9); A. cppa leaves (lanes 10 and 16); no DNA (lanes 11 and 17). Lanes 1 and 12: k DNA digested with HindIII-EcoRI. Lower band is given by the not incorporated primers.

Fik 6 Gel electrophoresis of PCR products using primers W and M3 (lanes 2-9) or ribosomal primers NSI-NS2 (lanes 11-14) and the following templates: recombinant plasmid (lane 2); Trifolium rrpms roots colonized by G. mossme Torino (lane 3); Alliurn cepn mots colonized by G. mossc~tc Kent 11 (lane 4); T. rcpens rootscolonized by Gigusporn mnrgaritn (lanes 5 and 11); T. repens roots colonized by Acaulospora inmis (lanes 6 and 12); Allium porrum roots colonized by Glomus i n h m a d i a (lanes 7 and 13); T. r e p s roots colonized by Scutellospom sp. (lanes 8 and 14); no DNA (lane 9). Lanes 1 and 1 0 L DNA digested with HindIII. Lower band is given by the not incorporated primers.

RAPD-PCR PRIMERS A N D GLOMUS I D E N T I F I C A T I O N 67

analysis of relatedness between genotypes (Hadrys et ul. 1992). Its application to mycorrhizal fungi has revealed polymorphism in the spores of AM fungi (Wyss & Bonfante 1993) or in the fruit bodies of seven species of ectomycorrhizal fungi belonging to the Tuber genus (Lanfranco et ul. 1993). However, the use of primers of arbitrary sequence requires axenic cultures, since the same short sequences may recognize complementary re- gions during the scanning of any genome. By contrast, PCR assay by using the specific primers designed after characterization of an anonymous 655-bp band can be performed on a mixture of plant and fungal templates.

Among the nine G. mossaae isolates, eight of them gave the same expected amplified fragment, spedfidty of which was confirmed by Southern blot hybridization. The patterns shown by the Hannover isolates rise some questions, since one of them (Ha-104) gave an amplifica- tion product similar to the other isolates, whereas Ha-136 led to faint bands of different size. Careful microscopic observations suggested that spores of Glmus mossem and another species were mixed in the pot cultures used in the experiments (C. Walker, personal communications). By contrast, the absence of any amplification with Ha-114 together with the microscopical observations (C. Walker, p e r ~ ~ ~ l communications) should suggest that this iso- late is not related to the species G. mossae. DNA from Scutellosporu spores also act as a template for the primers, since an amplified product of 400 bp was observed. It is evident that the primers target some portions of the Scufelluspuru genome, but the lacking of hybridization in Southern experiments (results not shown) suggests that there is no homology between the G. m o s w and Scutellospuru bands.

The results rise some questions on the nature of the cloned sequence. Computer analysis suggests that the 655 sequence could be part of the repetitive fungal DNA, since A and T bases were present in high percentage in the form of direct and inverted repeats. Fungi tend to have small genomes and AM fungi fit to this general rule, since their genome ranges from 0.25 to 0.75 pg of DNA per nu- cleus in G. aersifonne and Gigusporu m r g u d u , respec- tively (Bianciotto & Bonfante 1992). Due to this small genome, the fungal repeated DNA seems to be confined to some dispersed repeated gene families and to the well characterized gene family encoding the ribosomal RNAs (Selker 1991). However, data on the amount of repeated DNA are not available for AM hngi and therefore con- clusive information can not be achieved.

The two-step methodology, described in this paper and similar to that already reported for the genetic char- acterization of Fusarium strains (Ouellet & Seifert 1993), may represent a useful and powerful alternative to the sequences based on 18s ribosomal genes developed by Simon et ul. (1992 and 1993a,b). The generation of RAPD

markers which can be specific at species or isolate level seems of particular interest in ecological studies, in field experiments as well as in the characterization of markers linked to the symbiotic status. The role of AM fungi needs to be studied in natural conditions, where the plant-fun- gus interactions evolve and function depending on the population dynamics (Read 1992). An improved produc- tivity of ecosystems by inoculation with more effective fungal symbionk require to develop markers for effi- ciency and to follow the fate of the introduced symbiont within the endogenous microbial community. Both the goals may become more tractable with the availability of species or isolate-specific primers.

Acknowledgements We are grateful to Dr Chris Walker for his generous help in check- ing AM spores, to Dr Monique Gardes and Dr S i Perotto for their comments on the manuscript. We thank the following col- leagues of the European COST Action 8.21 for supplying different isolates: Andrea Schubert, Antonio Trotta. Manuela Giovannetti, John Dodd, Hont Vierheilig, Corinne Leyval, Henningvon Alten, Vivienne Gianinazzi-Pearson and Chris Walker. Funding was provided by the CNR special project RAISA, subproject 2, N Italy, and by the ECC Biotechnology Project (IMPACT Project, Contract Number BI02cT93-0053).

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