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Brazilian Journal of Microbiology

Print version ISSN 1517-8382

Braz. J. Microbiol. vol.44 no.2 São Paulo  2013  Epub Aug 16, 2013

https://doi.org/10.1590/S1517-83822013005000035 

Molecular approach to characterize ectomycorrhizae fungi from Mediterranean pine stands in Portugal

 

 

Carla RagoneziI; A. Teresa CaldeiraII, III; M. Rosário MartinsI, II; Cátia SalvadorIII; Celeste Santos-SilvaI, IV; Elsa GanhãoIV; Krystyna KlimaszewskaV; Amely ZavattieriI, IV

IInstituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Évora, Portugal
IIDepartamento de Química, Universidade de Évora, Évora, Portugal
IIICentro de Química de Évora, Universidade de Évora, Évora, Portugal
IVDepartamento de Biologia, Universidade de Évora, Évora, Portugal
VNatural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Stn. Sainte-Foy, Québec, Canada

Correspondence

 

 


ABSTRACT

Stone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have beneficial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root growth cessation not only in vitro but also to improve root development during acclimation phase. Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were collected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P. arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the collected material, ITS amplification was applied using the pure cultures derived from the sporocarps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effectiveness of the techniques used to amplify DNA polymorphic sequences, which enhances the ­characte­rization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity at any stage of plant mycorrhization.

Key words: Pisolithus arhizus, Lactarius deliciosus, Pinus pinea, M13-PCR, ITS.


 

 

Introduction

Ectomycorrhizal fungi (ECM) are major components of the soil fungal communities in most forests around the world and, are ecologically and economically important (Mello et al. 2006). Plants in Betulaceae, Pinaceae and Fagaceae families form obligate association with ECM (Smith and Read, 1997). ECM fungi include species from multiple families in the Basidiomycetes, Ascomycetes and some from the Zygo­mycetes (Bruns et al., 2002). Globally, as many as 10,000 fungus species and 8,000 plant species maybe involved in ECM associations (Taylor and Alexander, 2005).

Development and growth of pine (Pinus spp.) roots are regulated in nature by ECM (Smith and Read, 1997). Inoculation with specific fungi can enhance pine root formation and/or subsequent root branching of cuttings (Nor­mand et al., 1996; Karabaghli et al., 1998; Niemi et al., 2000). Some research results demonstrated the potential use of ECM fungi in the vegetative propagation of conifers (Gay, 1990; Niemi et al., 2005) and during in vitro rooting of pine shoots (Zavattieri et al., 2009; Ragonezi et al., 20010a). Inoculations enhanced plant performance and contributed to alleviation of stress related with acclimation in a nursery and the subsequent growth in the field.

Stone pine (Pinus pinea L.) is one of the most important pines economically (due to the valued edible nut production) in the Mediterranean basin and it forms ecto­mycorrhizas. Rincón et al. (1999) reported that at least eight genera of ECM were associated with P. pinea seedlings in the nursery (Amanita, Hebeloma, Laccaria, Lactarius, Pisolithus, Rhizopogon, Scleroderma, and Suillus). Two species of fungi are commonly used for inoculation in controlled mycorrhization programs associated with P. pinea: Pisolithus arhizus (Scop.) Rauschert, (Marx et al., 1982; Burgess et al., 1995) a cosmopolitan fungus which grows in warm temperate regions of the world and is easy to propagate in vitro (Marx et al., 1982; Cline et al., 1987) and Lactarius deliciosus (L. ex Fr.) S.F. Gray, typically a Basidiomycetes which produces high commercially valuable edible fruiting bodies (Singer, 1986; Hutchison, 1999; FAO, 2004; Hortal et al., 2006).

In nature, and also in controlled inoculations, genetically distinct mycelia of the same ECM species were found on the root system of a single tree (Guidot et al., 1999). This was also demonstrated by other studies with Pinus banksiana (De La Bastide et al., 1995) and Pinus pinaster (Gryta et al., 1997). Even in cases where the in vitro inoculation was controlled, genetic diversity has been found in ex vitro phases caused by the lack of effective sterilization of the mixed substrates, contamination from the environment in the growth chamber and in some cases from the irrigation source. On the other hand, ECM fungi are relatively selective of host plant species (Allen et al., 1995) and host responses could be partially attributable to variation between different fungus taxa and strains. For all these reasons, accurate characterization and identification of the ECM fungi are fundamental requirements for in vivo or in vitro mycor­rhization programs.

The traditional method of fungal identification by colour, shape and other macroscopic features and microscopic characteristics (Agerer, 1987-2002) could be applied only to a limited number of fungal species (Iotti and Zambonelli, 2006). Neverthless, today a wide range of molecular techniques can be used to distinguish DNA sequence for the identification of ECM fungi (Gardes et al., 1991a, 1991b; Henrion et al., 1992; Hortal et al., 2006) and also to verify the genetic variation within a specific group (Alves et al., 2007; Caldeira et al., 2009).

Amplification of the internal transcribed spacer (ITS) regions in the ribosomal genes (rDNA) usually reveals interspecific variations (Bruns et al., 1991; Gomes et al., 2000; Horton 2002). This region has four primary advantages over other regions: 1 - it is multicopy, so the amount of sample material needed for successful amplification is low; 2 - it has well-conserved fungal specific priming sites directly adjacent to multiple highly variable regions; 3 - there are many sequences already available for comparison, which facilitates the identification of unknown samples; and 4 - it correlates well with morphologically defined species in many groups (Smith et al., 2007).

Genetic profiles and polymorphic sequences on the other hand, are important tools for rapid and effective ­cha­racterization of ECM species (Caldeira et al., 2009). The polymerase chain reaction (PCR) based genomic fingerprinting is a good alternative to methods that rely on specifically targeted primers. This technique, which analyzes the whole genome, has been shown to be relatively robust and discriminatory (Alves et al., 2007). PCR fingerprinting is also used in the study of genetic variability in yeast and filamentous fungi (Godoy et al., 2004; Alves et al., 2007; Lopes et al., 2007).

The goals of the present study were, first to identify ECM fungi associated with stone pine stands through PCR amplification of the ITS region of the ribosomal genes and to use them in in vitro mycorrhization experiments. Second goal was to test the applicability of the M13-PCR fingerprinting methodology for monitoring different species of Basidiomycetes and Ascomycetes which can be found in association between P. pinea and ECM fungi.

 

Materials and Methods

Collection of mushrooms from stone pine (Pinus pinea L.) stand

Fruiting bodies of Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray were collected from a pure stand of stone pine (N 38°25'; W 7°56') in January of 2010. Morphological identification was done in situ at the collection time. Specimens were stored at 4 °C prior to sterilization and isolation procedures. Voucher specimens of Pisolithus arhizus and Lactarius deliciosus were deposited at Évora University Herbarium with the numbers UEVH-FUNGI 2001610 and UEVH-FUNGI 2001712, respectively.

Mycelia isolation and fungal cultures

For the asepsis, the fruiting bodies were cut into large pieces, placed in running water for 10 min followed by 70% ethanol for 2 min. Then, pieces were rinsed with sterile distilled water in a laminar flow unit, placed in 20% (v/v) commercial bleach (< 5% active chlorine) for 10 min and rinsed four times with sterile water. The larger pieces were then cut in smaller pieces (50 mm3) for growth and subsequently DNA extraction or were stored at -20 °C. Isolates were cultured in Hagen medium (Modess, 1941). The formulation of modified Hagen per liter was: KH2PO4 0.5 g, NH4CL 0.5 g, MgSO4 7H2O 0.5 g, FeCL3 (1%) 0.5 mL, glucose 5 g, malt extract 5 g, thiamine HCL 50 μg and agar 15 g and the pH was adjusted to 4.5-5.0. With the purpose to avoid the contamination by bacteria, 100 mg/mL of Rifampicin (Sigma-Aldrich®) was added to the media after cooling. Pieces of sporocarps were kept in Petri dishes filled with Hagen medium, grown at 25 °C in the dark and subcultured at weekly intervals. Isolates have been growing in Hagen slants for 14 days at 25 °C and stored at 4 °C. Fungal isolates of Pisolithus arhizus and Lactarius deliciosus were deposited in the Culture Collection of the Biotechnology Laboratory of University of Évora and preserved at -80 °C in cryovials containing 10% glycerol.

DNA extraction

The extraction of the genomic DNA from the smaller fragments of sporocarps and from the mycelia (after 14 days of culture) was performed using the modified micro­sphere method (Martins, 2004; Guimarães et al., 2011). The quality and quantity of the obtained DNA was evaluated by agarose gel.

ITS region amplification and sequencing

The region containing partial portions of the small subunit (18S), both internal transcribed spacers (ITS) and the 5.8S of the rDNA repeat unit was amplified using the oligonucleotides primers ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (Gardes and Bruns, 1993). PCR reactions were carried out on a MyCycler Thermal Cycler (BIO-RAD) and consisted of initial denaturing at 95 °C for 3 min followed by 30 cycles at 92 °C each 30 s, 55 °C for 30 s, and 72 °C for 1 min. The reaction was completed by a 10-min extension at 72 °C. PCR products were analyzed by agarose gel (1%) electrophoresis, purified with the NucleoSpin Extract II Kit (Macherey-Nagel) and sequenced by capillary electrophoresis using the ABI PRISM 3730 xl sequencer (Applied Biosystems) with the Kit BDT v1.1 (Applied Biosystems).

M13-PCR amplification

The M13 primer (5'- GAGGGTGGCGGTTCT-3') was used for the PCR. The PCR conditions consisted of an initial denaturing step of 5 min at 94 °C followed by 40 cycles of 1 min at 94 °C, 1 min at 50 °C and 2 min at 72 °C. The reaction was completed with a final extension at 72 °C for 5 min and then cooled at 4 °C. A sample of each PCR reaction product was electrophoresed in a 1.5% agarose gel and visualized, by staining with ethidium bromide, in a UV transilluminator (BIO-RAD). To evaluate the repro­ducibility of the assay, each sample was analyzed in at least three independent PCR reactions. A negative control (without DNA template) was included in every run. Subsequently, DNA sequence analysis was employed for confirmation of the fingerprint technique characterization.

Data analysis

The nucleotide sequences of the ITS region were aligned with those of related fungal species retrieved from the GenBank (National Center for Biotechnology Information - NCBI) databases for the homology analysis using the BLASTN 2.2.25+ program. The phylogenetic relationships between different species were inferred after multiple alignments using CLUSTAL W (Thompson et al., 1994). The distances of the DNA arrays were calculated with the option of Jukes-Cantor and from these matrixes, using the Neighbor-Joining method, the phylogenetic tree was constructed, using the program Mega 5 (Tamura, 2011).

For the M13-PCR analysis, the phylogenetic tree was generated by the Unweighted Pair Group Method with arithmetic Average (UPGMA), through the use of the Dice coefficient of similarity using Quantity One 1-D Analysis software (BIO-RAD).

 

Results and Discussion

Collection of fruiting bodies from stone pine stand

Representative voucher specimens of Pisolithus arhizus and Lactarius deliciosus fruiting bodies are shown in Figure 1a and 2a. Based on preliminary tests, we have selected Hagen medium as the most suitable for isolation and growth of the mycelia from sporocarps. The cultured mycelia were characterized by yellowish-ochraceous with paler margin in the case of P. arhizus (Figure 1b) and pinkish with paler margin for L. deliciosus (Figure 2b). The microscopic features showed the secondary mycelia at the septa of a Basidiomycota hypha (Figures 1c and 2c).

Fresh mycelia of each culture were used to inoculate stone pine microshoots at the rooting phase (Figure 3). The mycorrhization of the plants were confirmed in the acclimation phase (Figure 4a and b) revealing the typical ECM structure. The ECM fungi presence was monitored and confirmed during the mycorrhization process by applying two molecular complementary approaches: ITS sequencing and M13-PCR amplification.

 

 

 


 

Species identification of P. arhizus and L. deliciosus

In the past, the most common approach to evaluate fungal biodiversity in various plants/ systems has been the sample collection, fungal isolation and identification based on classical methods (Genilloud et al., 1994; De Jager et al., 2001; Moreira et al., 2001; Schmit and Lodge, 2005), but nowadays several molecular techniques can be used namely for ECM fungi identification (Rosling, 2003; Leake et al., 2004; Hortal et al., 2006; Caldeira et al., 2009).

Amplification of the ITS region is a common approach in molecular identification strategies (Hortal et al., 2006; Alves et al., 2007). PCR products of ITS4/ITS5 primers, corresponding to the ITS1, 5.8S and ITS2 regions of the rDNA were approximately 644 bp and 400 bp obtained from dikariontic isolates from Pisolithus sp. P1001 and Lactarius deliciosus UEZB1, respectively. Sequence alignments of P. arhizus showed identities that ranged from 99-100% among isolates belonging to P. arhizus. Sequences were aligned at the NCBI for isolates corresponded to other Pisolithus spp. In the case of L. deliciosus the homology was over 99%. The most similar sequences of P. arhizus and L. deliciosus are shown in Table 1. The phylogenetic tree (Figure 5) was obtained from the alignment of these sequences. We identified two different clusters, Pisolithus sp. P1001 and L. deliciosus isolate UEZB1 (Figure 5). Multiple alignment of Pisolithus sp. cluster corresponded to a partial sequence of 18S RNA gene and ITS1, 5.8S ribosomal RNA gene and ITS2, and partial sequence of 28S RNA ribosomal region. L. deliciosus UEZB1 corresponded to partial sequence of ITS1, 5.8S ribosomal RNA gene, ITS2 and partial sequence of 28S ribosomal RNA. Both sequences were published in GenBank with accession number HQ896485 and JQ066791, respectively.

 

 

 

 

Intraspecies identification by M13-PCR

The amplification using the M13 primer has generated a profile with 7-14 DNA fragments ranging from 100 to 2700 bp in the Basidiomycetes sporocarps species (P. arhizus, L. deliciosus and R. roseolus), Pisolithus sp. isolated P1001 and select Ascomycetes (P. brevicompactum, A. niger, Cladosporium sp.1, and F. oxysporum). These Ascomycetes species could live in association with ECM fungi and were commonly found in the isolation process. Reproducibility of the M13-PCR fingerprinting techniques was checked by comparing the banding profiles resulting from independent extractions and amplifications of the same fungus strains. The different samples have generated distinct patterns in the electrophoresis analysis (Figure 6). The M13-PCR band profile generated in each fingerprint varied according to the species included in this study: 10 fragments ranging from 200-900 bp for P. arhizus, 7 fragments for L. deliciosus (from 200-550 bp) and 9 fragments for R. roseolus (from 200-750 bp). The Ascomycetes, P. brevicompactum presented 9 fragments (from 200-1100 bp), A. niger 10 fragments (from 200-1850 bp), Cladosporium sp.1 presented 13 fragments (from 200-1850 bp) and F. oxysporum 14 fragments (from 200- 2700 bp). The Ascomycetes group presented larger DNA fragments (ranging from 950-2700 bp) than the Basi­dio­mycetes with fragments from 150-900 bp. Figure 7 shows a phylogenetic tree based on M13-PCR fingerprinting. In the analysis of the phylogenetic tree, P. arhizus formed a cluster of 41% similarity with R. roseolus, which formed a cluster with L. deliciosus with a homology of 34%. This approach also distinguished between Basidio­mycetes and Ascomycetes group, which formed a cluster with 15% of similarity for P. brevicompactum, 8% for A. niger, 5% for F. oxysporum and 2% for Cladosporium sp.1.

 

 

 

 

These results demonstrated that M13-PCR discriminated between species and taxonomic groups. Based on the specific PCR fingerprints and the high interspecies variation of these banding patterns, a clear distinction among all species was feasible. M13-PCR highlighted differentiation at the species and strain level (Caldeira et al., 2009). In this study, the M13-PCR approach was a rapid method to amplify DNA polymorphic sequences, with a high level of similarity for the same species, which enhances the characterization of the genetic profile of sporocarps such as L. deliciosus, P. arhizus and R. roseolus.

The advantages of this DNA amplification method are the technique simplicity, universal availability of PCR primers, reproducibility and amenability to the computer database analysis. Using only a single primer M13-PCR, it was possible to achieve high levels of resolution. This makes the procedure much faster and easier, and greatly reduces the cost (Alves et al., 2009). Hence, PCR fingerprinting offers a simple and reliable alternative method to resolve taxonomic problems and to "label" strains of filamentous fungi (Meyer et al., 1991).

 

Conclusion

Results of this study demonstrate that the combined use of sequence analysis of the ITS regions of the rDNA and the PCR fingerprinting technique can be successfully applied as an excellent tool to examine the species collected in the field associated with Pinus pinea and also as a methodology to monitor the fungus species involved in all the steps in a mycorrhization program. The applied molecular techniques accurately characterized field collected sporocarps and confirmed the presence of the fungus in inoculated plants.

Owing to its low cost and rapidity, the M13-PCR has a wide application in applied mycology as was demonstrated in this study. Also, we confirmed that the M13-PCR technique has a high level of reproducibility because the fungal samples amplified in independent PCRs displayed similar banding pattern profiles.

 

Acknowledgments

This research was supported by the Portuguese Foundation for Science and Technology (FCT) through the project PTDC/AGR-CFL/71437/2006) and by FEDER Funds through the Operational Programme for Competitiveness Factors - COMPETE and National Funds through FCT - Foundation for Science and Technology under the Strategic Project PEst-C/AGR/UI0115/2011.

 

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Correspondence:
M.R. Martins
Instituto de Ciências Agrárias e Ambientais Mediterrânicas
Universidade de Évora, Núcleo da Mitra
Ap. 94 7002-554 Évora, Portugal
E-mail: mrm@uevora.pt

Submitted: March 07, 2012;
Approved: July 23, 2012.

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