Identification of Tuber spp and corresponding ectomycorrhizae through molecular markers

Abstract

Analyses, based on the structure of the rDNA region, were developed to examine the possibility of identifying and typing some of the most important Tuber spp at both the symbiotic and non-symbiotic levels. Internal transcribed spacers (ITS) were amplified by the polymerase chain reaction using specific primer pairs on genornic DNAs, isolated from ascocarps and ectomycorrhizae of various Tuber spp and from rootlets of uninoculated host plants. The variability in the lengths of ITS amplification products of DNA isolated from fruit bodies allowed a primary discrimination of the Tuber spp considered. At the ectomycorrhizal Level the same species showed a pattern which was the combination of those typical of Tuber plus the host plants. Southern analyses, performed using the ITS product of Tuber brumale as a probe, confirmed the previous results: each fungus species showed a specific pattern, as well as each Tuber-host plant combination. It is concluded that molecular markers from the rDNA region can be used to differentiate Tuber spp and that the method proposed could be used to check and certify mycorrhizally infected plants.

Polymorphism in the internal transcribed spacer (ITS) of the ribosomal DNA of 26 isolates of ectomycorrhizal fungi

“…Indeed, cluster analysis based on RAPD-PCR analysis grouped the Brazilian isolates, while ITS polymorphism of ectomycorrhizal fungi 481 isolates collected in the USA and France have been shown to be quite distinct (Junghans et al, 1998). The two bands observed by us on the PCR amplified ITS region of ectomycorrhizae correspond to plant and fungi patterns, this result confirming that ITS1/ITS4 primers are not fungi specific (Paolocci et al, 1995 andAmicucci et al, 1996). However, in some cases, heterologous DNA from the plant material did not interfere with PCR amplification (Gardes et al, 1991;Henrion et al, 1992;Erland, 1995).…”

Specific PCR‐primers as a reliable tool for the detection of white truffles in mycorrhizal roots

“…Molecular methods are therefore necessary. However, the use of primers designed for the amplification of the variable intergenic region (ITS) of ribosomal RNA genes could lead to amplification of the same region of the host plant (Paolocci et al, 1995 ;Mello et al, 1996), thus necessitating the designing of primers specific to T. borchii and T. magnatum.…”

“…The former is of little value and marketing of the latter is illegal. Since the morphological features of mycorrhizal roots do not allow positive differentiation of truffle species during their symbiotic phase, a range of PCR protocols, mostly based on amplification of the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes, have been developed (Henrion et al, 1994 ;Chevalier et al, 1995 ;Paolocci et al, 1995Paolocci et al, , 1997Mello et al, 1996 ;Gandeboeuf et al, 1997a ;Amicucci et al, 1998 ;Bertini et al, 1998).…”

Reevaluation of the Life Cycle of Tuber magnatum

“…The reproductive structures of these species in pure cultures have not been reported, and axenic spore germination remains an unresolved problem (26). Furthermore, the mating-type genes have never been characterized in truffles.Molecular markers are being developed to type each truffle species to overcome the difficulty of identifying these species solely on their morphological traits (1,8,11,17,18,19,22,23). By combining molecular markers with an appropriate sampling strategy, we may be able to critically evaluate the truffle reproductive system and life cycle even without reproducing the entire life cycle in the lab.…”