DNA probes for species and strain identification in the ectomycorrhizal fungus Hebeloma

Mycol. Res. 96 (3): 161-165 (1992)

161

Printed in Great Britain

DNA probes for species and strain identification in the ectoinycorrhizal fungus Hebeloma

R. MARMEISSP'Z

J. C. DEBAUDZ,'f AND L. A. CASSEL TONI

Department of Biological Sciences, Queen Mary and Westfield College, Mile End Road, London E1 4NS, UK Laboratoire d'Ecologie Microbienne du Sol associi au C.N.R.5., Universiti Lyon I, Bat. 405, 43 Boulevard du 11 Novembre F-69622, Villeurbanne Cidex, France

1 2

Randomly cloned genomic sequences of the ectomycorrhizal fungus Hebeloma cylindrosporum have been used as hybridization probes to digests of genomic DNAs of eleven Hebeloma species and ten independently isolated strains of H. cylindrosporum. Two sequences identified unique RFLP patterns in all species tested and could thus be used for species differentiation. A third sequence only hybridized to H. cylindrosporum DNA and was strain specific in that it identified a unique RFLP pattern in all strains examined.

Many fungal species of both ascomycete and basidiomycete genera form ectomycorrhizal symbioses with the roots of gymnosperm and angiosperm trees in forest soils. The symbiosis offers several advantages to the host tree such as improved mineral nutrition and protection against water stress and pathogens (Harley & Smith, 1983; Harley, 1989). Although studies have been made on the in vitro physiology and biochemistry of fungal partners, ecological studies have proved more difficult. Most of the information regarding fungal communities has, of necessity, been based largely on a record of sporocarp development. This has led to the recognition of pioneer species and late stage fungi (Last et al., 1983; Deacon et aI., 1983; Last et al., 1984 a; Mason et al., 1987) and the importance of such factors as host genotype (Mason, 1975; Last et aI., 1984b) and substrate composition (Mason, Last & Wilson, 1986; Gibson & Deacon, 1990) or the age of the subtending root region of the mycorrhiza (Gibson & Deacon, 1988). Sporocarp sampling is probably not always representative of the actual variety and distribution of species present on the root system but more a reflection of environmental conditions. Moreover, at the species level, sporocarps cannot be used to differentiate between individual strains and little is, therefore, known about the population genetics of individual ectomycorrhizal species. If it were possible to identify fungal strains at the mycelial stage, it would allow analysis of the genetic stability and propagation of individual genotypes in a natural environment. It would also provide information on the spacial and timedependent modifications of symbiotic fungal communities. This information is particularly relevant in forestry, where better use could be made of species which improve tree growth or survival in poor soils. It is important to be able to

• To whom all correspondence should be addressed. 11

follow the fate of selected strains after introduction into the soil. Mating behaviour, enzyme polymorphisms or somatic incompatibility have all been used to identify strains of the mycorrhizal fungi Suillus tomentosus and S. luteus (Fries, 1987; Zhu et aI., 1988; Fries & Neumann, 1990), S. bovinus (Dahlberg & Stenlid, 1990; Sen, 1990) and Laccaria laccata (Fries, 1983, 1984). More recently, DNA techniques have been introduced and these promise a rapid and accurate method of strain and species identification. Mitochondrial DNA polymorphisms have been used to characterize different species of mycorrhizal Laccaria (Gardes et aI., 1991). Ribosomal DNA (rDNA) has been shown to be remarkably conserved between different organisms and the coding sequences cloned from one species generally hybridize even under full stringency conditions to the homologous sequences of other related species (De Long et al., 1989). The observed polymorphisms result mainly from divergences in the non-coding spacer sequences in each repeat and have been used to make interesting phylogenetic comparisons between species. Restriction fragment length polymorphisms (RFLPs) in the DNA sequence encoding the rDNA have been demonstrated for several mycorrhizal species (Armstrong, Fowles & Rygiewicz, 1989; Rogers et al., 1989). However, within genera, the restriction patterns can be very similar, and more specific probes are required for strain and species identification. In this paper we describe the isolation of DNA probes which have allowed us to use RFLPs to characterize ectomycorrhizal species belonging to the genus Hebeloma and to identify individual strains of Hebeloma cylindrosporum. H. cylindrosporum is a model system for studies on ectomycorrhizal species. It is known to have a bifactorial mating type system (Debaud, Gay & Bruchet, 1986) and controlled crosses between haploid monokaryons can be made. Dikaryotic mycelia can form sporocarps in vitro in association with its MYC 96

DNA probes for Hebeloma

162

habitual host plant Pinus pinaster (Debaud & Gay, 1987) making genetic analysis possible. Genetic variation within populations of wild and laboratory construded strains has been analysed for enzyme adivities involved in auxin synthesis (Gay & Debaud, 1987) and nitrogen assimilation (Wagner, Gay & Debaud, 1988, 1989). The results presented in this paper concern 11 edomycorrhizal species of the genus Hebeloma and 10 dikaryotic strains of H. cylindrosporum from different geographical origins.

MATERIALS AND METHODS Fungal strains Fungal strains are listed in Table 1. With one exception, all strains were dikaryons obtained form sporophores colleded in the wild (Bruchet, 1970, 1980). Strain hI of Hebeloma cylindrosporum Romagnesi is a monokaryon derived as a single-spore culture from HC1.

Media and culture conditions Cultures were routinely maintained on the yeast malt glucose medium of Rao & Niederpruem (1969). For DNA extradions, mycelium was grown for 15-21 days on cellophane placed over solid medium in 9 em Petri dishes. After harvesting, the mycelium was freeze dried and stored until required. All cultures were grown at 21°C.

Small-scale preparations of genomic DNAs were made using the miniprep method of Zolan & Pukkila (1986). For cloning purposes, DNA from H. cylindrosporum strain hI was prepared by the method of Wu et ai. (1983) and purified by CsClTable 1. Fungal strains used Strain number"

3 4 5 6 7 8

BR 63-19 BR 63-10 BR 64-28 SA 60 BR 65-25 BR 63-29 GG 74-1 BR 65-5

9

BR 63-21

1 2

10

802

A B

HCl (= BR 70-60)

C

HC2 HD HC5 HC6 HC8 HC9 HCII HC12 HC15

E G H I

J K

Species Hebeloma anthracophilum Maire circinans Qu
A first attempt to charaderize RFLPs between different Hebeloma species was made using the cloned rDNA repeat from another basidiomycete species, Coprinus cinereus (Cassidy et ai., 1984). Genomic DNAs from eleven different Hebeloma species were digested with both EcoRl and BamHl and, following Southern bloHing, were probed with the C. cinereus rDNA. The results of this analysis are illustrated in Fig. 1. Seven different restridion patterns were clearly deteded 2 6·6 "

3

4

5

8910 7 6 ...... _---.,...-...

2·3"

2·0"

hI

D

F

RESUL TS AND DISCUSSION Polymorphisms within the ribosomal DNA repeat

DNA manipulations

Designation

bisbenzimide gradient centrifugation to remove mitochondrial DNA (Raeder & Broda, 1985). DNA was digested to completion with BamHl and fragments separated in a 0'5 % agarose gel. DNA fragments of 12-20 kb in size were recovered by eledroelution and ligated to the arms of the lambda vedor EMBU (Stratagene, Cambridge, UK.). Recombinant lambda clones were seleded by infedion of host E. coli strain WL95 (metE, supE, supF, hsdRk, tonA, trpR, P2). Subcloning was into Bluescript plasmids (Stratagene, Cambridge, U.K.) using the host strain DH5a (cp80lacZM 15) li(lacZYArgf) U196 recA, endA 1, hsdR 17 r-m+supE44, thil, gyrA, reIAl). Standard techniques were used for baderiophage and plasmid amplification and purification (Sambrook et ai., 1989) Southern transfer and DNA labelling were carried out as described by Mellon et ai. (1987) using Hybond N membrane (Amersham, UK.) and nick translation kit from BRL (Maryland, US.A.). All filters were hybridized for 24 h at 65° in an aqueous buffer and then washed at the same temperature in successively 3 x SSC (1 x SSC = 0'15 M-NaCl, 0'015 M Na citrate, pH 7'4), 0'1 % SDS, 5 mM Na phosphate buffer, pH 7; then in 1 x SSe 0'1 % SDS, 5 mM Na phosphate buffer; and finally in 0'2 x SSC, 0'1 % SDS, 5 mM Na phosphate buffer to ensure that the probes only hybridized to homologous genomic sequences. peel, containing the cloned Coprinus cinereus rDNA repeat, was provided by Dr P. Pukkila.

Hebeloma cylindrosporum Romagnesi

"Refers to the culture collection of the University of Lyon.

Fig. 1. Southern blot analysis of genomic ONAs of different Hebeloma species to show RFLPs in the rONA repeat. Genomic ONAs were digested with EcoR1 and BamH1 and fragments separated by electrophoresis through 0'8 % agarose. The peel plasmid containing the Coprinus cinereus rONA was used as the hybridization probe. A Hind/II digest was used as size markers (in kb). Lanes are numbered for different species as listed in Table 1.

R. Manneisse,

J.

c. Debaud and L. A. Casselton

AI23456789

163

A 1 2 3 456 7 8 9

6.6·~'_4"

· 6·6· ·4·4·

Fig. 6. Segregation of two distinct RFLP patterns in the monokaryotic progeny of dikaryon HCI. Genomic DNAs were digested with EeaRI and probed with the entire genomic sequence present in the recombinant lambda clone ARCI Size markers in kb.

·2·3· • 2·0'

·0·6 .

2

3

Figs 2 and 3. Identification of Hebeloma species using probes derived from Hebelama cylindrasparnm genomic DNA. Genomic DNAs from ten species were digested with EeoRI and probed with the nonspecific sequence in ARC3 (Fig. 2) or the species-specific sequence ARCI (Fig. 3). Size markers in kb. Lanes are numbered for different species as listed in Table I.

A

BCDEFGH

K

J

6·6· 44·

Fig. 4. Southern blot analysis of genomic DNAs of different strains of Hebelama eylindrosporum digested with SaIl and probed with the recombinant lambda clone ARC3. Size markers in kb. Lanes are labelled for strains as listed in Table I.

ABCDEFGH

23-\

94

• 94·

K

J

94· 6·6· 44· 2·3· 2·0·

Fig. 5. Southern blot analysis of genomic DNAs of different strains of Hebeloma eylindraspornm digested with SaIl and probed with the recombinant lambda clone ARCI. Size markers and strains as in Fig. 4.

amongst fragments in the 1-6 kb size range. This was not, however, sufficient to distinguish all the species. H. cireinans and H. subsaponaeeum for example had identical profiles (lanes 2 and 3) as did H. hiemale and H. longicaudum (lanes 7 and 8).

The rDNA can clearly be used to distinguish between Hebeloma species but to optimize its effectiveness, several different enzymic digests of the genomic DNAs would need to be compared.

Hebeloma cylindrosporum DNA probes Species identification. From the point of view of rapid species identification, it is more useful to be able to digest genomic DNAs with just one enzyme and have single probes which differentiate species or strains. More specific probes were obtained by cloning random fragments of the genomic DNA of H. eylindrosporum strain hI. DNA was cut to completion with BamHI and fragments of 12-20 kb cloned into the lambda vector EMBL3. Three clones were picked at random and used as hybridization probes to genomic DNA digests of ten different Hebeloma species. Clones ARC3 and ARCH, having inserts of 14 kb and 15 kb respectively, both hybridized to DNAs of all ten species tested. The hybridization patterns were unique for each species using either clone as probe and using either BamHI or EeoRI to digest the DNAs. The results of probing EeoRI cut DNAs with ARC3 are shown in Fig. 2. The third clone ARCl, contained an 18 kb insert which hybridized significantly only to H. cylindrosporum DNA as shown in Fig. 3. In order to detect hybridization of this cloned sequence to DNAs from other species a much longer exposure of the autoradiographs was required and only then were very weak hybridization signals detectable. This sequence can, therefore, be considered to be species-specific since it only recognizes H. cylindrosporum and not the most closely related species. Strain identification. Genomic DNAs from the monokaryotic strain hI and ten different wild-type dikaryons of H. cylindrosporum were separately digested with BamHl, EeoRI, HindIII and SaIl and Southern blots probed with DNA from all three recombinant lambda clones. Clones ARCI and ARC3 contained sequences which were clearly hypervariable and unique restriction patterns were evident for most strains tested following digestion of DNAs with all four enzymes. This is illustrated for SaIl digests in Fig. 4 using the ARC3 probe and in Fig. 5 using the ARCI probe. It is interesting to note that dikaryotic strain HCI (lane B) and its monokaryotic progeny hI (lane A) had different hybridization patterns showing that the two parental nuclei in the dikaryon were polymorphic for both these sequences. This was confinned by analysing 17 monokaryotic progeny from the HCI dikaryon. As illustrated in Fig. 6, the hI RFLP pattern segregated 1: 1 with a second RFLP pattern (seven monokaryons of one type 11-2

DNA probes for Hebeloma SBS YI

H ,

E I

, ,

-

pRCll 2kb

164

H

...

S

strains tested. This is illustrated for the pRClS probe in Fig.

BS

8.

,

pRCl2 p~""_ _..... pRCl4 "'pRClS

to

Fig. 7. Physical map of the 18 kb Hebe/oma cylindrosporum genomic sequence present in the recombinant lambda clone ARCI. Restriction sites are designated; B, BamHl, E, EcoRl, H, HindIII and S, Sail. The insert was subcloned as five smaller fragments to give pRCl1 to pRCIS as indicated.

ABC

0

E

F

G

H

K

94.

B· 2·0·

Fig. 8. Southern blot analysis of genomic DNAs of different strains of Hebeloma cylindrosporum digested with Sail and probed with pRCIS. Size markers and strains as in Fig. 4.

and ten of the other). We conclude that the complex banding patterns observed with some dikaryotic strains might result from heteroallely between the two parental haploid nuclei.

Analysis of the hypervariable sequence in clone AXel Clone "RC1 contained an 18 kb sequence which was both species-specific and hypervariable and therefore of most interest for developing probes for H. cylindrosporum population studies. A restriction map of this 18 kb BamH1 fragment and five smaller fragments subc10ned to determine the extent of the hypervariable region is illustrated in Fig. 7. All fragments could be used to detect extensive RFLP between the strains. These data are summarized in Table 2. Two of the subcloned fragments, pRC12 and pRC1S, could be used independently to provide a unique hybridization pattern to separate all ten

RFLP can result from small chromosome rearrangements involving insertions and deletions and point mutations within non-coding sequences of the genome. Wu et al. (1983) showed that such variability was common in the genome of another basidiomycete species, Coprinus cinereus, and could readily be detected by cloning random genomic fragments. RFLPs can also be generated where mobile elements are present in the genome. These will be randomly distributed but likely to be present in multiple copies like the Ty element of yeast (Boeke, 1989). Two arguments can be put forward to clarify the nature of the "RC1 insert. Firstly, the banding patterns observed with digested genomic DNA from strain hI are more complex than expected from the restriction map of the "RC1 insert. For instance there are two Sail sites (Fig. 7) but more than three bands (some very faint) are clearly visible on the genomic digest (Fig. 5, lane A). This implies that the probe shares some homologies with other genomic DNA sequences. Secondly, we showed that only two patterns segregated among the haploid progeny of the HCl dikaryon (Fig. 7, the first lane is a digest of hI genomic DNA). Therefore these homologous regions did not recombine following meiosis and are tightly linked. The high degree of polymorphism present in the randomly cloned sequences from H. cylindrosporum was unexpected since the strains used were all collected within a restricted geographical area of some 100 km in S.W. France. This must reflect the fact that the population is strongly outbreeding and that variations within the genome are rapidly recombined. In contrast, the population is too small to have picked up any variability within the rDNA repeat. This was found to be completely homogeneous in the samples tested. The results show that the hypervariable sequences cloned from H. cylindrosporum can be used to characterize individual strains from a natural population. We are now in a position to address important questions relating to the ecology of this fungus and its development of mycorrhizal associations in nature. The availability of simple DNA methods for strain identification will now make it possible for us to correlate physiological characteristics identified in the laboratory with ability to survive in the competitive natural environment.

Table 2. Number of RFLP patterns obtained following digestion of ten Hebeloma cylindrospornm strains with either HindIll or Sail and probed with five different subclones of a hypervariable genomic sequence isolated in the recombinant lambda clone ARCI

Banding pattern obtained for each dikaryotic strain Probe pRCll

Genomic digest

HindIll Sail

pRCll

HindIll Sail

pRC13

Hindlll Sail

pRCI4

Hindlll Sail

pRCI5

Hindlll SaW

"lllustrated in Fig. 8.

HCl

HC2

HC3

HC5

HC6

HCs

HC9

HCII

HC12

HCl5

a a a a a a a a a a

b b b b b b a b b b

c

d d d d d b c d d d

e e e e e d d e e e

a

f b g b e f f g g g

f g h g e g g h h h

f h

g

c

b c

e e e

Number of restriction patterns observed 7

9 10

h e e

9

f h h

6 8 s 9

10 10

R. Marmeisse,

J. c. Debaud

and L. A. Casselton

This work was supported by a European Community BRIDGE and 'Biotechnology Action Programme' grant (0512 U.K. CH) to R.M. The authors wish to thank Professor G. Bruchet and Dr G. Gay (Universite Lyon I) for determination and isolation of Hebeloma strains and Colette Raffier for her valuable technical assistance.

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(Accepled 11 September 1991)

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