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- Materials and Methods
The Mediterranean region forms a belt around the Mediterranean Sea in Southern Europe, the Levant and North Africa. The western region comprises the Iberian Peninsula and some areas of Morocco. The natural vegetation corresponds to forests of sclerophyllous oaks, adapted to a climate regime characterized by a dry summer and a cool moist winter (Archibold, 1995). In these regions, efficient pine germination after fire and extensive plantations account for today’s wide-spread pine woodlands. On siliceous soils, dense scrub formations, known as jaral (Cistus spp.), often dominate (Schultz, 1995). During the last two centuries extensive plantations of Eucalyptus have been established in the Mediterranean region to produce paper pulp. Such eucalypt species are of Australasian origin, and thus exotic to the Mediterranean region.
Most Mediterranean forest trees are obligate ectomycorrhizal plants, establishing mycorrhizal symbiosis with ascomycetes and basidiomycetes. Pisolithus is a common ectomycorrhizal fungus in native Quercus forests, Pinus woodlands and Cistus scrubs (Calonge & Demoulin, 1975). This fungus also occurs in plantations of eucalypt. Pisolithus has been reported on acid (siliceous or slate-derived) and basic (calcareous and marsh-gypsum) soils. Pisolithus is often regarded as a cosmopolitan ectomycorrhizal fungus with a wide host range, establishing mycorrhizas with angiosperms and gymnosperms (Marx, 1977). Moreover, Pisolithus isolates are commonly used as inoculants to enhance tree establishment and growth of pine and eucalypt plantations worldwide (Garbaye et al., 1988).
A great variation in the effects of inoculation with different strains of Pisolithus on forest trees has been reported (Burgess et al., 1994). Since fungal variability plays a major role in strain selection for mycorrhizal inoculation programmes, some work has been done on Pisolithus heterogeneity. There is considerable polymorphism in terms of carpophore, spore and culture morphology among Pisolithus strains. Large variations in colony growth rates, enzyme activity, polypeptide patterns and mycorrhizal ability have been reported (Ho, 1987; Kope & Fortin, 1990; Lamhamedi et al., 1990; Burgess et al., 1994, 1995).
Since Pisolithus was described, several taxa have been proposed based on distinctive carpophore and basidiospore morphology (Marx, 1977). Morphological differences were considered as nondiagnostic, and taxa within the genus Pisolithus were regarded as conspecific (Coker & Couch, 1928; Pilát, 1958). However, several new species have been described, that is Pisolithus microcarpus Coke & Mass, P. kisslingi E. Fisch, P. pussillum Pat, and P. auriantioscabrosum (Watling et al., 1995).
Information on the genetic polymorphism of Pisolithus is scattered in different locations, and is sometimes contradictory and hard to reconcile. For instance, in North America, Grand (1976) found no correlation between the basidiocarps and basidiospores of Pisolithus, their geographical location, habitat or plant association. However, others studies have stressed that host and world location may play a significant role in such variability (Marx, 1981; Malajczuk et al., 1990; Burgess et al., 1994). Burgess and co-workers (1995) reported a correlation between geographical origin, basidiospore morphology and polypeptide patterns in Australian isolates. In addition, host specificity among Pisolithus isolates has been revealed in mycorrhizal synthesis experiments (Malajczuk et al., 1990; Burgess et al., 1994).
Based on basidiospore morphology and incompatibility mating tests, Kope & Fortin (1990) proposed that the genus Pisolithus comprises several biological species. DNA-based methods have provided further support for this hypothesis (Cairney et al., 1999). Junghans et al. (1998) reported that RAPD analysis (Random Amplification of Polymorphic DNA) clustered isolates in two main groups according to their host and geographical origin. Gomes et al. (1999) confirmed these data on the same isolates by Restriction Fragment Length Polymorphism (RFLP) in the internal transcribed spacer (ITS) regions amplified by Polymerase Chain Reaction (PCR) and Gomes et al. (2000) on mitochondrial DNA. Farmer & Sylvia (1998) based on RFLP analysis of the ITS sequences from several isolates of Pisolithus, suggested that this taxon represents a species complex. Furthermore, Anderson et al. (1998) and Martin et al. (1998), after studying ITS sequences of Pisolithus isolates from a defined region in New South Wales (Australia) and in Kenya, respectively, suggested that Pisolithus may comprise several species.
In the Western Mediterranean region, it is envisaged that mycorrhizal inoculation with selected isolates of Pisolithus may become a common practice in eucalypt plantations, in order to improve seedling survival and growth after outplanting. Since mycorrhizal fungi of Australian origin may out-compete native fungal symbionts or even interbreed with native strains, environmental concerns arise about the introduction of symbiotic microorganisms together with these exotic trees. Little information on the behaviour of Pisolithus strains in natural ecosystems is available, particularly when plantations of exotic Pisolithus hosts co-exist with native forests (Martin et al., 1998). If natural ectomycorrhizal inocula are absent and mycorrhizal inoculation is not used, exotic forest plantations usually fail. However, most eucalypt plantations in Mediterranean regions have been successful without undertaking artificial inoculation. Two hypotheses can be proposed: either exotic ectomycorrhizal fungi were co-introduced with eucalypt seedlings, or some compatible Mediterranean ectomycorrhizal fungi were able to colonize seedlings of Eucalyptus. The aim of this work was to study the genetic variability of Pisolithus in the Mediterranean region and its correlation with ecological and host factors. In addition, it aimed to discover whether Pisolithus strains in eucalypt plantations are native or were introduced with eucalypt seedlings. Sequencing of the ITS regions from 17 isolates associated to Mediterranean hosts and Eucalyptus was conducted. These sequences were compared with those available in DNA database by using distance and parsimony methods. The ITS sequence analysis approach was chosen because its has previously been shown to be useful in identifying and grouping Pisolithus strains (Anderson et al., 1998; Martin et al., 1998).
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We thank Dr F. Martin (INRA-Nancy, France) for suggestions on an earlier version of the manuscript, and to Dr I. Alexander (University of Aberdeen, UK) and two anonymous reviewers for critical comments on the manuscript. We also thank Dr A. Casares (Oviedo University), Mr A. Hernández (Alborada Tree Nursery), the Curator of University Alcalá Herbarium (AH) and Dr Abourouh (DREF-Morocco) for providing some of the isolates. We also appreciate Dr G. Moreno’s taxonomic assistance. This work received financial support from the INIA (SC98-030) and the AECI. J. Díez is grateful to the EU for a postdoctoral fellowship (Contract HPMF-CT-1999–00174). The present study is a joint project between the Plant Biology Departments of the Universities of Alcalá and Murcia and Alborada Tree Nursery.
J. Díez and B. Anta contributed equally to this work.