1. Introduction
Phlebiopsis gigantea (Fr.) Jülich has widely been used as a biological control of the fungus
Heterobasidion annosum (Fr.) Bref., the causative factor of conifer root rot infections [
1,
2,
3,
4,
5,
6,
7,
8,
9,
10,
11,
12]. In the literature this species is described with multiple synonymous names, e.g.,
Corticium giganteum (Fr.) Fr.,
Peniophora gigantea (Fr.) Massee,
Peniophora gigantea f. pruinosa Pilát,
Phanerochaete gigantea (Fr.) S.S. Rattan,
Phlebia gigantea (Fr.) Donk. The fungus, after the taxonomic revision made by Jülich (1978) In: Parmasto and Hallenberg [
13], was finally placed in the new genus
Phlebiopsis, which is now commonly accepted. The fungus is a typical saprotroph, regularly colonizing dead parts of coniferous woods, occurring mostly in the Northern Hemisphere, but it has also has a worldwide distribution; for example, few strains have been retrieved from South Africa, New Zealand and Canada [
9,
14].
According to the Index Fungorum database (
www.indexfungorum.org) the taxonomic status of
P. gigantea is well recognized and there are at least 23 species included in the genus
Phlebiopsis, based on morphological characteristics. A number of techniques have been employed to identify
P. gigantea and traditionally, the morphological characters of the spores were used for this purpose. Although the classical methods are reasonably easy and fast to apply [
15], molecular techniques confirm identification of this fungus and are very useful for identification of species [
16,
17].
Various molecular markers used in barcoding, like ITS 1/2, β-tubulin, histone H3 and elongation factor α (syn.
EF1α), are the most commonly applied in fungal taxonomy. Their application allows determination of fungal genotypes at a species level [
18,
19,
20]. Comparisons of DNA sequence data are increasingly being used in order to gain knowledge concerning the phylogenetic relationships among
P. gigantea isolates [
7,
10,
11,
21,
22]. Many studies have utilised DNA sequence data of the
EF1α gene for phylogenetic analyses including a wide range species of fungi [
23,
24].
The objectives of this study were to obtain DNA partial sequences for
EF1α for
P. gigantea strains, and to compare them with other some
Basidiomycota species from the NCBI database. This gene is a highly conserved ubiquitous protein involved in translation that has been suggested to have desirable properties for phylogenetic inference [
25]. It has been successfully used in phylogenetic studies as a phylogenetic marker for
Eukaryotes,
Acomycetes and
Basidiomycota [
23,
24,
25] Additionally, the partial sequences (intron and exon partial regions) obtained in the study of
EF1α gene isolates of
P. gigantea from Poland, Finland, Sweden and Great Britain, and partial genome sequence (gi:752829739) deposited in GenBank by Hori et al. [
26] were investigated. To date, in studies on the differentiation of chosen
P. gigantea isolates on the known activity of linear growth and wood decay [
3,
4,
11,
27] the
EF1α gene has not been studied.
Our study provides an additional gene region useful for testing taxonomic groupings and phylogenetic relationships, previously identified based on the other gene regions like ITS [
2,
3]. Several factors can affect biological activity of the fungus. Grossbard [
28] reported, that the presence of some fungi in soil can modify biological traits of co-occurring taxons. Schardl and Craven [
29] described that the variation in enzyme and decay activity of fungal isolates in time may suggested risks in lost or change the molecular and biochemical characteristics. The cause is showed in possible hybridisation from the mating of clearly homozygous individuals. Żółciak et al. [
3] and Sierota et al. [
27] suggested the changes in the activity of different
P. gigantea isolates with time, its origin, and wood density. For the effective use of competitive fungi used in biopreparations against pathogens in biological control (e.g.,
P. gigantea), there is a need for periodic exchange of strains for more effective ones [
4]. Checking the utility of the
EF1α region can be a valuable clue and can help in making decision regarding the selection of the most effective
P. gigantea isolates as a competitor of
Heterobasidion spp.
2. Materials and Methods
2.1. Cultivation of Isolates
Twelve previously identified and tested isolates of
P. gigantea [
11] were used in the experiment: six from Poland (not registered as biocontrol agent) and one from Finland, one from Sweden and four from Great Britain (registered as biocontrol agent) (
Table 1). The number of Polish isolates was limited to six due to difficulties in obtaining homogeneous single-spore cultures, while the Finnish and British isolates were accepted as previously tested and approved. Isolates were grown on potato dextrose agar (PDA) medium (Difco
TM, Sparks, MD, USA) in Petri dishes for ten days at 20 °C according to Kwaśna et al. [
30].
2.2. DNA Extraction
Total fungal DNA of P. gigantea was extracted from mycelium grown on PDA by using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), according to the protocol. Quality of the DNA was checked with an Infinite 200 PRO multimode plate reader (Tecan, Group Ltd., Männedorf, Switzerland).
2.3. Primers and PCR Conditions
The PCR reactions were done in 25 μL volumes using a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Each reaction contained 1 unit (0.25 μL) of Taq DNA polymerase (recombinant) (Thermo Scientific, Life Technologies Inc., Carlsbad, CA, USA), 2.5 μL of 10 × Taq buffer, 2 mM of each dNTP (0.5 μL), 1.5 mM of MgCl2 (1.5 μL), 12.5 pmol of forward/reverse primers (0.125 μL), and 20 ng (1 μL) of DNA.
The primers and the touchdown PCR reaction conditions were used according to modified procedure of Rehner [
18]. Amplicons of the partial region of the
EF1& gene were generated using two overlapping primer combinations, 526F (5′ GTC GTY GTY ATY GGH CAY GT 3′) × 1567R (5′ AC HGT RCC RAT ACC ACC RAT CTT 3′) and EFdF (5′ AAG GAY GGN CAR ACY CGN GAR CAY GC 3′) × 2218R (5′ AT GAC ACC RAC RGC RAC RGT YTG 3′). For 526F × 1567R primers the touchdown PCR was as follows: 5 min at 94 °C (an initial denaturation), 30 s at 94 °C (denaturation) and 45 s at 60 °C (annealing) in the first cycle, successively reducing the Tm by 1 °C per cycle over the next 9 cycles to a final Tm 50 °C, which was used in the remaining 36 cycles. An extension step per cycle was 1 min 30 s at 72 °C. The final extension step was 1 cycle of 7 min at 72 °C.
PCR parameters for EFdF × 2218R primers were as follows: 5 min at 94 °C, 45 cycles of (30 s at 94 °C, 30 s at 63 °C, 1 min 30 s at 72 °C) and 7 min at 72 °C. Amplicons were run in 1.5% (w/v) ethidium bromide-stained agarose gels (Sigma-Aldrich, St. Louis, MO, USA) and the bands were visualized under UV illumination. PCR products were sequenced using Sanger’s sequencing method at the Polish Academy of Sciences Institute of Biochemistry and Biophysics (Warsaw, Poland).
2.4. DNA Sequence Analysis
Sequencing results were analyzed using the BLAST algorithm on The National Center for Biotechnology Information (NCBI,
https://blast.ncbi.nlm.nih.gov/) database and MEGA version 5 [
31]. The DNA sequences were aligned using Clustal W version 2.0. [
32]. Genetic variability was estimated for the sequences of
P. gigantea and realigned separately for intron and exon regions using FGENESH 2.6 [
33,
34].
The obtained sequences were analyzed using Tajima’s Neutrality Test. The number of sequences (m), number of segregating sites (S), mean number of segregating sites (Ps), nucleotide diversity (π) and estimates of Theta (Θ) per site as well as the Tajima test statistic (D) were estimated using MEGA5 program [
35,
36].
EF1α partial gene sequences of
P. gigantea and selected
Basidiomycota species from the NCBI database were analyzed using the maximum likelihood approach for phylogeny reconstruction and tested by bootstrapping with 1,000 replicates. Missing and ambiguous characters were excluded from the analysis. Phylogenetic trees were generated based on maximum likelihood method [
37]. Two sequences of
P. gigantea were deposited in GenBank NCBI (Accession numbers: KU886024 and KU886025,
www.ncbi.nlm.nih.gov.genbank/).
4. Discussion
In this study, DNA partial sequence for the EF1α gene of Phlebiopsis gigantea was successfully identified and analyzed. All P. gigantea isolates yielded PCR products of similar size, indicating that the amplified partial gene region does not include large indels, and therefore is a suitable choice for phylogenetic studies. The aligned sequences showed considerable homology among P. gigantea but various species-specific nucleotide substitutions and indels were observed among all Basidiomycota species. No sequence variation was observed among all P. gigantea except of one isolate (GB Pg 16) from Great Britain and partial sequence of P. gigantea genome (gi: 752829739).
The remarkable variation was observed between different strains. This is consistent with previous studies employing ITS region and genetic fingerprinting using random amplified microsatellite (RAMS) markers in taxonomic studies of
P. gigantea [
21].
For the analyzed
P. gigantea EF1α partial gene the occurrence of exons and introns was identified. Introns represented the so-called 3rd introns group, spliced during the maturation of RNA with the participation of spliceosome [
38]. The size of introns ranged from 52 to 58 bp, at the average length of the gene from 0.75 to 1,000 bp of gene.
Among representatives of
Basidiomycota six introns in gene are observed average [
39]. Compared to higher
Eukaryota, introns occurring in fungi are relatively short [
40]. In the case of the analyzed gene, introns size corresponds to the average size of introns identified in representatives of the
Fungi kingdom (50–200 bp) [
41]. The length of introns of
EF1α gene was slightly smaller than the average for the
Puccinia graminis (0.65 kb, NCBI accession number: X73529.1) and
Neurospora crassa gene (0.81 kb) [
42].
Despite the randomness and variability of the structure of introns, in the case of I
5 of
EF1α partial gene, a larger number of segregating sites (six) was identified. In the sequences of other introns this number were from 1 to 3. Furthermore, the characteristic feature of the identified intron sequences was the presence of the sequence GT on the donor side (5’) and the sequence AG at the acceptor side (3’). They are essential in the process of identifying and splicing by spliceosome. Identified GT and AG sequences are considered as the most commonly occurring canonical dinucleotide fragments, respectively starting and ending the introns [
43].
Intraspecies analysis of EF1α partial gene showed small variability within the coding and non-coding regions. Introns of analyzed EF1α partial gene were well conserved among all tested isolates of P. gigantea. The evolution of the structure of all introns and E2–E6 exon regions of EF1α partial gene was neutral. Isolates representing P. gigantea from Poland, Great Britain, Finland and Sweden had similar partial sequences of EF1α gene and were grouped together. The only British isolate (GB Pg 16 = FOC PG B 20/5) of this fungus showed coupled single mutations like single nucleotide polymorphisms (SNPs) and was very similar to P. gigantea (gi: 752829739) with identity 99% and query cover 100%.
Results of the study using DNA-RAMS markers indicated genetic similarity among isolates collected in Finland and Great Britain [
10]. Additional studies using the previously mentioned markers showed that Polish isolate (PL 12) of
P. gigantea was genetically analogous to FC 16 from Great Britain [
11]. Vainio and Hantula [
21] also showed that European and North American ITS/A alleles of
P. gigantea were identical, while ITS/C alleles were different. The authors mentioned that the analysis of molecular variation and neighbor joining analysis using 28 RAMS markers revealed a considerable degree of differentiation between Europe and North America [
21].
The main advantage of this study is the phylogenetic analysis of
EF1α partial DNA sequence data for
P. gigantea in comparing it to species belonging to the same family—
Phanerochaetaceae and selected
Basidiomycota species. Phylogenetic trees showed that
P. gigantea is closely related to
Phanerochaete chrysosporium. EF1α is also the first protein-coding gene and first single-copy gene used for phylogenetic analysis of
P. gigantea. The whole genome sequence of
P. gigantea (gi: 752829739, accession number: AZAG01000080) was published by Hori et al. [
26]. The whole genome sequence size is approximately 30 Mbp and number of predicted genes (11,891). Sequence data from the majority of isolates belonging to the different species showed unique species-specific substitutions, allowing the isolates to be differentiated into clades representing the species.
The results of this study demonstrate that the EF1α region is useful for phylogenetic analysis and classification of Polyporales species. This is a large and taxonomically difficult order, which include several genera, for example: Phlebiopsis, Phanerochaete, Phlebia, Junghuhnia, Steccherinum, Androniella, Ganoderma, Coriolopsis, Perenniporiella and Trametes.
