Typing truffle species by PCR amplification of the ribosomal DNA spacers

Henrion, B.; Chevalier, G.; Martin, Francis

doi:10.1016/s0953-7562(09)80333-x

Cited by 240 publications

(148 citation statements)

References 14 publications

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“…The IGS is known to be strain specific for certain ectomycorrhizal fungal symbionts (Henrion et al, 1993) and within strain variability (ie. dikaryotic and monokaryotic condition) has been identified in Laccaria bicolor (Selosse et al 1996).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the host genotype and fungal diversity in Scots pine ectomycorrhiza from natural humus microcosms using isozyme and PCR‐RFLP analyses

1997

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SUMMARYThe early development of indigenous and introduced Scots pine {Pinus sylvestris L.) ectomycorrhiza in natural forest humus was examined using molecular fingerprinting techniques. Non-mycorrhizal or mycorrhizal (Suillus bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch ex Fr.) Fr.) seedlings were placed in transparent twodimensional microcosms on a thin layer of sieved humus to allow observation of ectomycorrhizal development and fungal growth. Twelve ectomycorrhizal morphotypes, mainly based on colour, gross morphology and outer mantle structure, were identified over a 3-month period. No successional trends in mycorrhiza formation were observed. For further characterization, individual ectomycorrhiza representing the different morphotypes were subjected to analyses of esterase (FST) isozyme profiles and RFLPs of the internal transcribed spacer (ITS) from fungal rRNA genes following amplification using PCR. All 10 morphotypes analysed displayed different esterase isozyme profiles, and the characteristic 5. bovinus species diagnostic band {S. b. EST8) was detected in two white morphotypes. Ten Pink and Black ectomycorrhiza were all separated into one and three groups, respectively. A fast-running plant-specific polymorphic locus (Z) was also confirmed in most ectomycorrhizal morphotypes. Diagnostic species-specific {S. bovinus and Pink) EST bands were detected in intact external mycelium colonizing soil. Successful amplification of the ITS from individual ectomycorrhiza, of eight different morphotypes, was found to be mainly infiuenced by the DNA template concentration. Different RFLPs {Hini I, Mbo I and Hha I) of the ITS placed the white morphotypes into two groups, one corresponding to S. bovinus, in agreement with the esterase fingerprinting. By contrast. Pink and Beige morphotypes displayed different EST profiles but nearly identical RFLP fingerprints. The ITS amplification success rate of standardized DNA template concentrations from 10 individual Pink and Black ectomycorrhiza was 90 and 50%, respectively. In all successful amplifications, the black morphotype yielded the smallest ITS fragment, similar to earlier reports for Cenococcum geophilum, that gave identical RFLPs. The ecological significance of the observed mycorrhizal diversity and combined application of these two identification methods is discussed.

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Section: Discussionmentioning

confidence: 99%

Characterization of the host genotype and fungal diversity in Scots pine ectomycorrhiza from natural humus microcosms using isozyme and PCR‐RFLP analyses

1997

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“…DNA was routinely extracted by the cetyltrimethylammonium bromide (CTAB) protocol according to Henrion, Chevalier & Martin (1994). For recalcitrant samples giving no amplification or low amplification yield, DNA was extracted using a guanidinium buffer followed by DNA purification using GeneClean2 glassbeads (Grube et al, 1995 ;Martin et al, 1997).…”

Section: Dna Extractionmentioning

confidence: 99%

“…The ITS and IGS1 regions of the rDNA were amplified in duplicate by PCR using the primers ITS1\ITS4 and CNL12\5SA (Henrion et al, 1992) respectively, Taq DNA polymerase, and the buffer provided by the manufacturer (Applige ' ne-Oncor, Illkirch, France) according to Henrion et al (1994). Amplifications were carried out on a GeneAmp2 PCR System 9600 (Perkin Elmer).…”

Section: Pcr Amplification and Rflpmentioning

confidence: 99%

“…The basidiocarps were further examined by PCR\ RFLP of the rDNA IGS, a rDNA region known as highly variable in fungi (Henrion et al, 1992(Henrion et al, , 1994Apel & Gordon, 1996 ;Selosse et al, 1996). All basidiocarps of the Pinus-, Eucalyptus-and Afzeliamorphotypes produced a single IGS band of 450 bp (Fig.…”

Section: Rflp and Sequence Of Igsmentioning

confidence: 99%

“…Various methods based on the PCR have been proposed to characterize ectomycorrhizal fungi (Gardes et al, 1991 ;Henrion et al, 1992) and to examine phylogenetic relationships in the various taxa (Bruns, White & Taylor, 1991). Direct sequencing of genes coding for nuclear and mitochondrial rRNA genes and intergenic spacers amplified by PCR (Bruns et al, 1989 ;Bruns & Szaro, 1992 ;Kretzer et al, 1996 ;Selosse et al, 1996) and RFLP analysis of these PCR-amplified sequences (Gardes et al, 1991 ;Henrion et al, 1992Henrion et al, , 1994 have been used to characterize ectomycorrhizal fungi at the intraspecific and interspecific phylogenetic levels. In the present investigation, we have used this approach to investigate genetic and phylogenetic relationships between the morphotypes of Pisolithus found in plantations and woodlands of the Coast Province of Kenya.…”

Section: mentioning

confidence: 99%

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Genetic variability in intergenic spacers of ribosomal DNA in Pisolithus isolates associated with pine, eucalyptus and Afzelia in lowland Kenyan forests

Martin

Delaruelle

Ivory³

1998

New Phytologist

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Basidiocarps of Pisolithus associated with indigenous (Afzelia quanzensis Welw.) and introduced (Pinus caribaea Mor. and Eucalyptus camaldulensis Dehnh.) hosts in the lowland forests of the Coast Province of Kenya are morphologically distinct. Genetic variability among 52 Pisolithus basidiocarps, collected beneath the various host plants, was examined based on sequence polymorphism within the internal transcribed spacer (ITS) and intergenic spacer (IGS1) of ribosomal DNA genes. Variability in ITS and IGS1 sequences indicated that the three host-associated morphotypes were genetically different. Consensus trees generated by bootstrap analysis of sequence data of Pisolithus isolates from Australia and Kenya are polyphyletic and strongly suggest that the three different morphotypes\genotypes present in Kenya represent separate biological species. In addition, our data indicate that little genetic exchange occurs in silva between these species.

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ChemInform Abstract: Preparation of Fungal Specimens for Direct Molecular Applications

et al. 2010

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Typing truffle species by PCR amplification of the ribosomal DNA spacers

Cited by 240 publications

References 14 publications

Characterization of the host genotype and fungal diversity in Scots pine ectomycorrhiza from natural humus microcosms using isozyme and PCR‐RFLP analyses

Characterization of the host genotype and fungal diversity in Scots pine ectomycorrhiza from natural humus microcosms using isozyme and PCR‐RFLP analyses

Genetic variability in intergenic spacers of ribosomal DNA in Pisolithus isolates associated with pine, eucalyptus and Afzelia in lowland Kenyan forests

ChemInform Abstract: Preparation of Fungal Specimens for Direct Molecular Applications

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