Morphological and molecular characterization of Pisolithus in soil under eucalyptus plantations in Brazil

Dezoito basidiocarpos de Pisolithus foram coletados em plantacoes comerciais de Eucalyptus no Estado de Minas Gerais, no Brasil. Esses basidiocarpos foram caracterizados morfologica e molecularmente. Eles variaram na forma, cor e tamanho. Alem disso, um deles foi encontrado abaixo do solo, sugerindo que seja um hipogeo. A principal caracteristica morfologica distintiva foi a ornamentacao dos esporos, que foram separados em dois grupos: um apresentou espiculas curtas e eretas, e o outro, espiculas longas e curvas. Eles foram identificados, respectivamente, como Pisolithus microcarpus e Pisolithus marmoratus, depois da analise do cladograma obtido pela relacao filogenetica baseada no espacador interno transcrito (ITS) do DNA ribossomal nuclear desses isolados.


PLANTATIONS IN BRAZIL (1) Maria Catarina Megumi Kasuya (2) , Irene da Silva Coelho (3) , Daniela Tiago da Silva Campos (4) , Elza Fernandes de Araújo (2) , Yutaka Tamai (5) & Toshizumi
Miyamoto (5) SUMMARY Eighteen Pisolithus basidiomes were collected from Eucalyptus plantations in the state of Minas Gerais, Brazil. These basidiomes were characterized morphologically and molecularly. The basidiomes varied in shape, color and size. One of them was found underground, indicating a hypogeous fungus. The main morphological distinctive characteristic was spore ornamentation, which distinguished two groups. One group with short and erect spines was identified as Pisolithus microcarpus, and the other with long and curved spines as Pisolithus marmoratus, after analyzing the cladogram obtained by phylogenetic relationship based on internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA of these isolates.

INTRODUCTION
Pisolithus is a group of gasteromycetes with worldwide distribution that form ectomycorrhizas with a wide range of angiosperm and gymnosperm tree species (Marx, 1977).
The dispersal of ectomycorrhizal trees, such as eucalyptus and pine, outside their natural range, has resulted in the introduction of a narrow range of exotic ectomycorrhizal fungi into forest plantations in many countries of Europe, South America, Africa and Asia (Garbaye et al., 1988;Martin et al., 1998;Gomes et al., 2000;Díez et al., 2001;Singla et al., 2004;Reddy et al., 2005). Whether the distribution of these fungi reaches beyond these areas depends on the host range of the fungi and their ability to compete with indigenous species.
Since Pisolithus was described, several taxa have been proposed based on distinctive carpophore and basidiospore morphology (Marx, 1977). There is considerable polymorphism in terms of basidiomes, spore and culture morphology in Pisolithus strains (Burgess et al., 1995;Watling et al., 1995;Anderson et al., 1998;Kasuya et al., 2008). Large variations in colony growth rates, enzyme activity, polypeptide patterns, and mycorrhizal ability have been reported Lamhamedi et al., 1990;Burgess et al., 1994Burgess et al., , 1995. Based on basidiospore morphology and mating incompatibility tests,  proposed that the genus Pisolithus comprises several biological species.
DNA-based methods have provided further support for this hypothesis. Junghans et al. (1998) reported that RAPD analysis (Random Amplification of Polymorphic DNA) clustered Pisolithus isolates in two main groups, according to host and geographical origin. Using the same isolates, these data were confirmed by Restriction Fragment Length Polymorphism (RFLP) in the internal transcribed spacer (ITS) regions amplified by Polymerase Chain Reaction (PCR) and on mitochondrial DNA (Gomes et al., 1999(Gomes et al., , 2000. Based on RFLP analysis of the ITS sequences of several Pisolithus isolates, Farmer & Sylvia (1998) suggested that this taxon represents a complex species. At least 11 different clades have been reported, of which the major were recognized as phylogenetic species (Martin et al., 2002).
Studies based on DNA sequences serve to reevaluate earlier classifications and delimit species more accurately (Taylor et al., 2000). The progress of phylogeny studies and their implications for understanding the biogeography of ectomycorrhizal fungi has been significant (Jarosh & Bresinsky, 1999;Wu et al., 2000;Kanchanaprayudh et al., 2003;Moyersoen et al., 2003;Singla et al., 2004).
Although inoculation is not a rule in tree nurseries in Brazil, it is rather common to find Pisolithus basidiomes under commercial eucalyptus plantations. However, with exception of the Southern region, where P. microcarpus was found (Giachini et al., 2000), Pisolithus basidiomes in other areas have not been identified at the species level. The objective of this study was to identify the Pisolithus species found in eucalyptus plantations in the state of Minas Gerais.

Sampling and sites
Basidiomes of 18 Pisolithus isolates, collected from the soil of commercial plantations of Eucalyptus spp., between February/2002 and February/2004, in different areas of CENIBRA -Celulose Nipo-Brasileira S.A., in Minas Gerais, Brazil, were used in this study.

Electron microscopic observation of basidiospores
Basidiospores from mature basidiomes were airdried and sputter-coated with gold+palladium before examination under a JSM5310 scanning electron microscope, low vacuum, at an accelerating voltage of 15 kV. Mean basidiospore diameter (n = 20) was determined for each isolate using scanning electron micrographs and basidiospore spine morphology classified by the terminology of .

DNA manipulations
Samples of Pisolithus spores or internal parts of dried mushrooms were used for DNA extraction. DNA was isolated using DNAeasy Kit (Qiagen, Düsseldorf, Germany). The ITS region of rDNA (ITS1-5.8S-ITS2) was amplified with ITS1f (Gardes & Bruns, 1993) and ITS4 primers (White et al., 1990). Amplifications were performed on a GeneAmp PCR System 2400 (Perkin-Elmer). Samples were initially denatured for 30 s at 94 ºC, followed by 40 cycles of denaturation for 1 min at 94 ºC, annealing for 1 min at 55 ºC, and extension for 2 min at 72 ºC.
Samples of PCR products were purified using QIAquick® PCR purification kit (Qiagen, Düsseldorf, Germany) and sequenced by the ByeDyeTM Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, USA), according to the manufacturer's instructions. Sequences were generated for both strands using the ITS1f and ITS4 primers for comparison and to ensure reliability.

Sequence analysis
The sequences were deposited in a GenBank database (Table 1), and compared with other sequences ( Table 2). The sequences were imported into MEGA, version 3.1 (Kumar et al., 2004), and aligned using the program Clustal W (Higgins et al., 1994). The resulting multiple alignments were optimized visually. The final alignment of this study is available on request. ITS sequences of Paxillus involutus (AF167700) and Suillus luteus (L54110) were used as outgroup taxa. A neighbor joining (NJ) algorithm method was used for phylogenetic analysis, applying a p-distance model with software MEGA (Kumar et al., 2004). The robustness of each branch was determined using the nonparametric bootstrap test (Felsenstein, 1985) with 1000 replicates.

RESULTS
The basidiomes shape and size varied (Table 1). Furthermore, isolate VIC 30600 was found underground, indicating a hypogeous fungus. Two distinctive spore ornamentations were observed, forming one group with the isolates VIC 30596, VIC 30597 and VIC 30600, with long and curved spines, and spore diameter from 7.24 to 7.91 μm (Figure 1, Table 1). The other isolates had short and erect spines and a spore diameter from 6.2 to 8.62 μm (Figure 1, Table 1). As many as eight basidiospores per basidium were observed in all cases (data not shown). PCR amplification with the specific primers for the ITS region generated only band with 700 bp. In the phylogenetic analysis, all sequences in the Table 2
cladogram were divided into three major clades. The structures were similar to the phylogenetic cladogram in Martin et al. (2002). The sequences of all isolates used in this study were distributed in clades 1 and 2, with strong bootstrap support (99 %) (Figure 2).
Clade 1 consisted of four species groups. The first species included isolates VIC 30596, VIC 30597 and VIC 30600 that grouped with MU98/5A and MU98/9, identified as Pisolithus marmoratus, isolated from Eucalyptus globulus in a forest of Australia (Martin et al., 2002). The second species group was the hypogeneous Pisolithus called P. hypogaeus. The isolates MARX270 and PT301 formed a species group identified as Pisolithus tinctorius, both from the United States and isolates from Pinus forests. The last species group contained other species, all from Japan ( Figure 2, Table 2).

DISCUSSION
The characteristic that distinguished the two species found in the studied area was spore ornamentation (Figure 1), as confirmed by molecular analysis (Figure 2). One group with short and erect spines was identified as Pisolithus microcarpus, and the other with longer and curved spines as Pisolithus marmoratus.
Pisolithus microcarpus and P. marmoratus were described by Bougher & Syme (1998). Although native to Australia, isolates of this species were collected in exotic plantations worldwide (e.g. Brazil, China, Portugal, Senegal, and South Africa). P. marmoratus is considered the southern hemisphere counterpart of P. tinctorius. They are morphologically very similar (Bougher & Syme, 1998), but unlike P. tinctorius, P. marmoratus is associated with eucalypts.
The clustering of all Pinus isolates together is consistent, from the point of view that some degree of host specificity may exist within the Pisolithus taxa (Martin et al., 1998). Several previous studies have suggested that Pisolithus isolated from basidiomes occurring under Pinus spp. are poor colonizers of Eucalyptus spp. (Burgess et al., 1994;Junghans et al., 1998;Pereira et al., 2005). Moreover, in a molecular study of Pisolithus in Kenya, three taxa were identified as occurring only in either pine plantations, eucalyptus plantations or native Afzelia vegetation (Martin et al., 1998).
Basidiome morphology has been used in Pisolithus taxonomy (Watling et al., 1995;Kanchanaprayudh et al., 2003;Thomas et al., 2003), but size and shape were not useful due to the possible influence of soil and environmental conditions and could not identify Pisolithus in Hokkaido (Kasuya et al., 2008). Although isolate VIC 30600 was found to be hypogaeous (Table 1), it was not included in the P. hypogaeus species, described occurring in Australia (Thomas et al., 2003). Instead, the isolate VIC 30600 presents similarity with the P. microcarpus spores and this date was confirmed by phylogenetic analysis.
Genetic diversity and host specificity of Pisolithus was reported in exotic Eucalyptus and native hosts in the western Mediterranean region. The ITS sequences suggested the occurrence of several ecological species adapted to exploit different soil types (basic, acid and clayey slate-derived soils), with specificity for particular indigenous hosts. Isolates from eucalypt plantations in Brazil, Kenya and the Mediterranean region grouped together with eucalypt-associated Australian isolates. No transfer to native hosts occurred; the host specificity range of these exotic strains may prevent out-competition and interbreeding with local species. The origin of Pisolithus spp. in eucalypt plantations in the Mediterranean basin is Australia; the co-introduction of the ectomycorrhizal fungi might explain the success of these exotic forest plantations (Díez et al., 2001).
In Thailand, the presence of at least three Pisolithus species was suggested. Pisolithus basidiomes collected in pine forests and under some Shorea roxburghii trees in a pine-dipterocarp forest corresponded to species 5, as previously described by Martin and collaborators in 2002. The basidiomes collected under S. roxburghii and Dipterocarp alatus trees in the dipterocarp forests did not match any previously reported species. Basidiomes collected from eucalyptus plantations were all identified as Pisolithus albus (Kanchanaprayudh et al., 2003).
Australian eucalypts was introduced into Southeast Asia, India, Africa, South America, South Africa, and southern Europe in the last 200 year in form of seeds and saplings, which facilitated the dispersal of Australian Pisolithus (Martin et al., 2002).
The ITS sequences of rDNA of Pisolithus isolates were also determined to provide information on the genetic variability among isolates and to compare the sequences to other isolates described in the literature. This study should provide a better understanding of the taxonomy and variability of Pisolithus in Brazil, underlying the selection of fungal genotypes for forestry inoculation programs. Such programs are of key importance, since forest management is an essential part of Brazilian agriculture.

CONCLUSIONS
1. The trait spore ornamentation is a criterion to distinguish the Pisolithus species.
2. Molecular analysis confirmed the presence of two Pisolithus species in the State of Minas Gerais.
3. The two Pisolithus species in the study area were Pisolithus microcarpus and Pisolithus marmoratus.