Structure analysis of the ribosomal intergenic spacer region of Phaeoacremonium italicum as a study model

Copyright: © 2021 M. Laidani, M.L. Raimondo, A.M. D’Onghia, A. Carlucci. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/pm) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


INTRODUCTION
The genus Phaeoacremonium (Togniniales, Togniniaceae) was originally described in 1996, containing only six species (Crous et al., 1996). Ten years later, Mostert et al. (2006) described Togninia as the sexual morph of Phaeoacremonium, and defined 22 Phaeoacremonium and ten Togninia species. Gramaje et al. (2015) included Togninia in Phaeoacremonium genus, according to the change to single nomenclature for fungi (Hawksworth et al., 2011). The number of described Phaeoacremonium species has continued to increase to the present 63 (Ariyawansa et al., 2015;Crous et al., 2016;Da Silva et al., 2017;Spies et al., 2018), which also includes the report of P. thailandense from freshwater in Thailand (Calabon et al., 2021).
Phaeoacremonium has a wide host range and worldwide distribution. The host range includes woody plants, insect larvae, freshwater and humans, and species have been reported from South, Central and North America, Asia, Europe, the Middle East, the Far East, Oceania and Africa Spies et al., 2018). Many reports have associated Phaeoacremonium species with vascular wood diseases of several plants (Damm et al., 2008;Nigro et al., 2013;Raimondo et al., 2014;Carlucci et al., 2015;Olmo et al., 2015;Spies et al., 2018), where the grapevine trunk diseases, such as Esca and Petri diseases, are considered to be the most destructive and severe. To date, 36 Phaeoacremonium species have been isolated abundantly from necrotic wood of grapevines showing Esca and Petri diseases in vineyards, of which 22 species were in Europe and Mediterranean countries (Essakhi et al., 2008;Raimondo et al., 2014;Gramaje et al., 2015;Jayawardena et al., 2018;Spies et al., 2018). Phaeoacremonium species have also been isolated from different woody hosts, including olive and other fruit trees that show wilt, decline, dieback and cankers. In particular, 13 Phaeoacremonium species have been associated with olive trees, and 34 with fruit trees, of which ten were on olive and 18 were on fruit trees in Europe and Mediterranean countries (Crous and Gams, 2000;Mostert et al., 2006;Nigro et al., 2013;Carlucci et al., 2015;Gramaje et al., 2015;Soltaninejad et al., 2017;Spies et al., 2018;Sohrabi et al., 2020).
The recent taxonomic reassignment and the increasing recognition of novel Phaeoacremonium species associated with various woody host plants has highlighted the intraspecific genetic variation and paraphyly within several species-level clades, and also incongruence or lack of resolution for some Phaeoacremonium species using β-tubulin and actin sequences Spies et al., 2018). For example, in the Phaeoacremonium italicum/ Phaeoacremonium alvesii group, the phylogenetic position of some strains (e.g., CBS 113590) was incongruent among the combined and individual phylogenies. Spies et al. (2018) also reported that the morphological differences described for these closely related species did not correspond to the intraspecific genetic variation observed, and for this reason, morphology cannot be useful to clarify species identity of strains that have unresolved phylogenetic identities.
To improve the resolution of some Phaeoacremonium clades, further phylogenetic markers need to be investigated. To date, few studies have attempted to use other genes as putative markers for molecular identification of Phaeoacremonium species. Mostert et al. (2005) sequenced the calmodulin gene of 19 Phaeoacremonium strains that belonged to 11 species to provide greater clarity on the status of the taxa closely related to Phaeoacremonium rubrigenum, although the calmodulin gene did not distinguish between P. alvesii and P. rubrigenum (the closest species), as the sequences were identical. Together with the internal transcribed spacer (ITS) and the β-tubulin and actin genes, Úrbez-Torres et al. (2014) sequenced elongation factor (EF)1-α from 56 strains that belonged to 31 Phaeoacremonium species, and performed multilocus analyses. The tree obtained by inclusion of the EF1-α DNA marker improved the molecular characterisation and increased the phylogenetic resolution within Phaeoacremonium.
The present study aimed to investigate the structure of the IGS rDNA regions of a representative collection of P. italicum strains as a study model, and to compare them with strains of the closely related species P. alvesii and P. rubrigenum, to emphasise intraspecific and interspecific genetic variations. For these purposes, the entire IGS regions of 31 P. italicum, two P. alvesii and two P. rubrigenum strains were amplified and sequenced to define the IGS structures. To determine whether morphological features corresponded with putative intraspecific and interspecific genetic variation expressed by the IGS rDNA region, a detailed microscopy study of P. italicum strains was also carried out. To verify that IGS rDNA could be used to discriminate the Phaeoacremonium species, it was also amplified and sequenced in another 12 Phaeoacremonium species. These data were used to obtain a phylogenetic tree and to compare this with that obtained by the markers presently used for molecular identification of Phaeoacremonium species.

Fungal isolates
This study included a total of 57 isolates of Phaeoacremonium that were obtained from different hosts and localities, with 49 isolates from the collection of the Department of Agriculture, Food, Natural resources and Engineering (DAFNE), University of Foggia (Italy), one isolate from Instituto de Ciencias de la Vid y del Vino (ICVV) (Spain), and seven isolates from Westerdijk Fungal Biodiversity Institute (CBS, Utrecht, The Netherlands). Two isolates of Pleurostoma richardsiae from DAFNE were included in this study as outgroups (Table 1).

DNA extraction, PCR and sequencing
Genomic DNA was extracted from fresh mycelia for each isolate, using the methods of Carlucci et al. (2013). Partial sequences of the β-tubulin and actin genes of each of the strains were amplified according to the protocols and conditions described by Raimondo et al. (2014). The IGS rDNA region flanking 28S and 18S of each of the strains was amplified using the universal primers LR12R (5'-GAACGCCTCTAA-GTCAGAATCC-3'; anchored in the 3' of the LSU gene) and invSR1R (5'-ACTGGCAGAATCAACCAGGTA -3'; anchored in the 5' of SSU of the RNA gene) (Durkin et al., 2015).
The PCR reactions were each performed in a thermal cycler (C-1000 Touch; BioRad) in a final volume of 25 µL. The reaction mixture contained 1× PCR buffer, 3 mM MgCl 2 , 200 μM deoxynucleotide triphosphates, 0.2 μM of each primer, 1.25 U Taq polymerase, and 30 ng template DNA. The Taq polymerase (LA Taq long-PCR), nucleotides, MgCl 2 and buffer were supplied by TaKaRa Bio Europe. The PCR protocol and conditions were optimised for P. italicum, which included 95°C denaturation for 10 min, followed by 25 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2.5 min; with a final extension step at 72°C for 10 min. The same PCR conditions were used for all of the Phaeoacremonium species, except for P. parasiticum and P. croatiense, for which the annealing temperatures were 58°C for P. parasiticum and 62°C for P. croatiense. For amplifications of the two P. richardsiae strains were used PCR conditions of 30 cycles, with annealing temperature of 58°C.
The sequencing was performed in both directions for all of the fungal isolates by Eurofins Genomics. As the complete fragments of the IGS regions were large, the design of additional internal primers was needed (Table 2). The sequence of each locus was assembled and manually corrected (BioEdit version 7.0.9; http://www. mbio.ncsu.edu/BioEdit). The taxonomic identification of the Italian Phaeoacremonium strains through BLAST searches was carried out considering threshold similarity values of 98 to 100%, and as comparisons of the β-tubulin and actin sequences with the reference strains as the ex-types. All of the novel DNA sequences generated in this study were deposited to GenBank (Table 1).

Structure analysis of the intergenic spacer region of P. italicum
The IGS sequences obtained from each of the strains were aligned using the online multiple alignment programme MAFFT v.7 (http://mafft.cbrc.jp/alignment/ server/) (Katoh and Frith, 2012;Katoh and Standley, 2013), with the iterative refinement method E-INS-I, as recommended for <200 sequences with multiple conserved domains and long gaps. The alignment was checked visually and adjusted manually where necessary. As the sequences were very large, to determine the distribution of polymorphisms among the P. italicum strains and those that belonged to the closest species (i.e., P. alvesii, P. rubrigenum), the GeneQuest program of the Lasergene 6 software package was used (DNAstar). The pairwise identities of the IGS sequences were evaluated using MegAlign version 15 (DNAstar, Madison, WI, USA).

Morphology
The growth rates of the P. italicum isolates were determined over 8 d and 16 d on malt extract agar (MEA; 50 g malt extract agar (Oxoid), 1 L water), potato dextrose agar (PDA; 39 g potato dextrose agar (Oxoid), 1 L water), and oatmeal agar (OA; 30 g oats, 8 g agar (Oxoid), 1 L water), with incubations at 23±2°C. The cardinal temperatures for growth of each isolate were determined on MEA, with incubations in the dark at temperatures from 5°C to 40°C, at 5°C intervals, and including 37°C. Colony morphologies were determined on MEA, PDA and OA after 21 d at 23±2°C, and colony colours were determined using the colour charts of Rayner (1970).
Micromorphological characterisation of each isolate was performed according to Raimondo et al. (2014). The dimensions and morphologies of the conidiophore structures, and the sizes, phialide types and shapes, presence of bundles, and conidium shapes and sizes were measured from 100% lactic acid mounts as 30 measurements (×100 magnification), using a measurement module (Application Suite; Leica Microsystem GmbH). The phialide types and shapes were determined according to Mostert et al. (2006). Photomicrographs were recorded with a digital camera (DFC320; Leica) on a DMR microscope (Leica) fitted with Nomarski differential interference contrast optics. The 5th and 95th percentiles were calculated for all of the measurements.

Phylogenetic analyses
The sequences of the β-tubulin and actin genes were aligned using MAFFT v.7 (http://mafft.cbrc.jp/ alignment/server/) (Katoh and Standley, 2013), with the default parameters. The alignment was checked visually and improved manually where necessary. Model Finder Plus (Kalyaanamoorthy et al., 2017) implemented in the IQTREE software (Nguyen et al., 2015) was used to select the best-fit DNA substitution models using Bayesian information criteria. The sequences were concatenated in one dataset to perform multilocus analyses. A partitioned model with two segments was created, each with a different model of evolution estimated previously by Model Finder Plus (Kalyaanamoorthy et al., 2017). Phylogenetic analyses were carried out based on maximum likelihood and maximum parsimony. Before performing the phylogenetic analyses, the presence of different phylogenetic hypotheses in a partitioned dataset was assessed by a partition homogeneity test (i.e., an incongruence length difference test) using PAUP version 4.0b10 (Swofford, 2003). In this test, 100 datasets were artificially created by random sampling among all of the observed sites of genotypes, and then swapping sites among the loci, to obtain a P value. If the P value was >0.01, there was no incongruence among the loci, and so combination of the data improves or does not reduce the phylogenetic accuracy (Cunningham, 1997). Maximum likelihood analysis was performed using the IQTREE software (Nguyen et al., 2015), with 1000 ultrafast bootstrap replicates. The ultrafast bootstrap approximation (Minh et al., 2013) assesses branch supports from 10 to 40 times faster than the RAxML rapid bootstrap, and obtains less biased support values (Minh et al., 2013;Hoang et al., 2018). Unlike the non-parametric bootstrap, which considers bootstrap values ≥50% as statistically significant, the ultrafast bootstrap considers ≥80% as statistically significant. The maximum parsimony analysis was performed using PAUP version 4.0b10 (Swofford, 2003), with the heuristic search option, 10,000 random taxa additions, and tree bisection and reconstruction as the branch swapping algorithm. Branches of zero length were collapsed and all of the multiple, equally parsimonious trees were saved. The gaps were treated as missing data. Bootstrap support values were calculated from 1000 heuristic search replicates and 1000 random taxon additions. The tree lengths (TL), consistency index (CI), retention index (RI), homoplasy index (HI) and rescaled consistency index (RC) were calculated, and the resulting trees were visualised with TreeView version 1.6.6 (Page, 1996). The final tree was selected among the suboptimal trees from each run by comparison of the likelihood and bootstrap scores.
The aligned dataset of the IGS sequences was subjected to maximum likelihood and maximum parsimony analyses, as described above. Before performing the maximum likelihood analysis, the IGS dataset was first analysed with Model Finder Plus (Kalyaanamoorthy et al., 2017), as described above. Two strains of P. richardsiae (Pl3, Pl29) were used as outgroups in all of the phylogenetic analyses.

PCR amplification and sequencing
The β-tubulin and actin genes were amplified for all of the 61 isolates and produced the expected fragments of 700 bp and 300 bp, respectively. The BLAST search and the comparison of the β-tubulin and actin sequenc- Table 2. Internal sequencing primers designed to obtain the complete sequences of the intergenic spacer rDNAs.
es with the reference strains as ex-type allowed the 49 Italian strains to be attributed to 11 Phaeoacremonium spp., as follows: P. amygdalinum, P. croatiense, P. fraxinopennsylvanicum (one strain each), P. iranianum (two strains), P. italicum (30), P. minimum (three), P. oleae (two), P. parasiticum (two), P. scolyti (three), P. sicilianum (two) and P. viticola (two strains) ( Table 1). The β-tubulin and actin sequences of the remaining Phaeoacremonium strains (seven from CBS, one from Spain) were compared with their sequences already deposited in BLAST to confirm the taxonomic identities. Therefore, the newly generated β-tubulin and actin sequences of the above-mentioned strains were not re-submitted to GenBank, except for the β-tubulin sequences of P. rubrigenum strains, as those present in GenBank were incomplete ( Table 1). The consensus IGS sequences obtained from 57 different strains of Phaeoacremonium spp. and for two P. richardsiae strains showed different nucleotide lengths, which ranged from 2088 bp to 2427 bp. The IGS sequence lengths among the P. italicum strains were also variable, and ranged from 2264 bp to 2300 bp. Analysis of the complete IGS sequences showed that the 5S rDNA gene was not found, and thus the IGS consists of a single uninterrupted region between the end of the large subunit (LSU) region and the beginning of the next small subunit (SSU) region.

Structural analysis of the intergenic spacer region of Phaeoacremonium italicum
The alignment obtained from all of the 59 strains (i.e., including the outgroup taxa) consisted of 3451 characters, including gaps. Based on the differences observed among the aligned P. italicum sequences, seven different sub-groups were identified, and are indicated as sub-groups #1, #2, #3, #4, #5, #6 and #7, where, respectively, the representative strains used to analyse the structures of the IGS regions were defined as CBS 137763 (ex-type), Pm31M, Pm50M, Pm45, Pm17, Pm59 and CBS 113590 (Table 1). Three structural regions were defined: one central polymorphic region (PR, positions 231-2807) that is flanked by two conserved regions (CR1, positions 1-230; CR2, positions 2808-3451) (Figure 1a). CR1 is adjacent to the 28S rDNA region, and it is a conserved sequence that ranges from 99% to 100% for pairwise identities. CR2 is adjacent to the 18S rRNA region, and is also highly conserved (99.6%-100% for pairwise identities). The polymorphisms in these two conserved regions consisted of single base substitutions (i.e., transitions and/or transversions) and small (1 or 2 bp) insertions and deletions (indels).
In contrast, PR is less conserved, with pairwise identities of 84.1%, which has resulted from variable indels and point mutations. Detailed analysis of the IGS structures of the representative P. italicum strains of the seven sub-groups revealed the presence of five short repeat elements that are organised in five different patterns, which are here indicated as elements A, B, C, P and R (Figure 2a, b). Element A consists of a pair of perfect repeats of 12 bp; element B consists of an imperfect direct repeat of 15 bp; element C consists of a perfect consecutive repeat of 12 bp; element P consists of a single palindromic sequence of 14 bp located before the first copy of element C; and element R is the most commonly encountered and consists of an imperfect direct repeat of 15 bp (Figure 1b and c). Based on the distributions of the short repeat elements, the central PR can be subdivided into two sub-regions, as PR-a (positions 231-1643) and PR-b (positions 1644-2807) (Figure 1b and c). Sub-region PR-a shares a common structural organisation of three short repeat elements (A, B, R) in all of the isolates.
Each of the P. italicum strains that belong to the first six of the sub-groups (i.e., #1 to #6) show the same structural organisation of PR-a, and contain: one perfect repeat copy of element A; nine copies of imperfect repeats of element R (R1-R9); one truncated imperfect repeat indicated here as Rt1; and one imperfect repeat of element B, indicated here as B1 (Figure 1b). Instead, PR-b starts with the element P and shows characteristic differences across the IGS. The main feature of this sub-region consists of a variable number of copies of perfect consecutive repeats of element C (2-10), and of elements R (8-9). For elements R, three (R4, R5, R8) were also contained in the PR-a region, and the other three (indicated as R10, R11 and R12) were newly encountered. In addition, two truncated imperfect repeats (Rt2 and R8t) were identified. The P. italicum strains of sub-groups #1, #2 and #3 show eight R elements located in the same positions, while those of sub-groups #4 and #5 show nine R elements (Figure 1b). The P. italicum strain of sub-group #6 shows seven R elements and lacks elements R5 and R12 (Figure 1b). Two imperfect repeats of element B, indicated here as B2 and B3, were found for PR-b, and one perfect repeat copy of element A, was found for CR2 ( Figure 1b). The strain CBS 113590, representative of sub-group #7, renamed as P. italicum sensu lato by Spies et al. (2018), showed some differences inside the IGS structure. In particular, for this strain, PR-a includes the same elements of PR-a of P. italicum (sub-groups #1 to #6), except for the absence of element R8 (position 1088), and for its modified (*) element R, here indicated as R12* (position 1130). The PR-b has a single copy of element C, and nine elements R in the same positions as sub-groups #4 and #5 (Figure 1b).
In addition, structure analysis was carried out for the closest species to P. italicum, as P. alvesii and P. rubrigenum. The same repeated elements seen for the P. italicum sub-groups were also present in P. alvesii and P. rubrigenum, although the positioning and composition of some of these elements were different (Figure  2b, c). In particular, P. alvesii strain CBS 408.78 shows the same IGS structure as P. italicum sensu lato (CBS 113590; sub-group #7) (Figure 1c).

Morphology
The morphological features of the representative isolates of each sub-group of P. italicum in culture are outlined in Table 3. The strains that belonged to sub-groups #1 to #6 (respectively, CBS 137763, Pm31, Pm50M, Pm45, Pm17, Pm59) have similar morphologies and culture features. The mycelia consisted of branched and septate hyphae that occurred singly or in bundles, and rarely had tuberculate hyphae, with sub-hyaline to pale brown, verruculose to smooth warts. Unbranched conidiophores were frequently observed, occasionally narrower at the bases, erect, with up to nine septate (generally three or four septate) and each ending in a single terminal phialide, which often had one or two lateral phialides of type II next to the terminal phialide. Percurrent rejuvenation was frequently observed (Figure 3).
Branched conidiophores with different levels of branching were also observed. Phialides were integrated into the terminal or lateral conidiophores, and were monophialidic, and rarely polyphialidic. Phialides arising directly from the mycelia were mainly monophialidic, often polyphialidic; type I phialides were cylindrical, frequently widened at the bases; type II phialides were predominant, one-septate, elongate-ampulliform and attenuated at the bases to navicular, tapering towards the apices; type III phialides were one-septate, mainly cylindrical, often subulate or navicular. Hyphal coils were observed. Conidia were hyaline and mostly oblong-ellipsoidal to reniform or allantoid, biguttulate, and occasionally ovoid (Figure 3). No perithecium formation was observed under laboratory conditions. The colonies reached a radius of 25-33 mm after 16 d at 23±2 °C. Minimum temperature for growth was 15°C, with optimum of 25 to 30 °C, and maximum of 35°C. The strains belonging to sub-groups #4, #5 and #6 reached a colony radius of 5 mm at 37°C. The morphology and colour of the colonies were variable among the isolates that belonged to the six subgroups (Table 3; Figure 4). Yellow pigment was observed on OA for strains that belonged to sub-groups #4 and #5 (Figure 4).
Strain CBS 113590, in sub-group #7 had mycelium with branched septate hyphae occurring singly or in bundles; hyphae were tuberculate with sub-hyaline to pale brown, verruculose to smooth warts. Unbranched conidiophores predominated, and were generally one-or two-septate (up to four-) septate and each ending in a single terminal phialide, often bearing one lateral phialide (mainly of type II) next to the terminal phialide; percurrent rejuvenation was rarely observed ( Figure 5). Branched conidiophores were less frequently observed, with few levels of ramification. Phialides were integrated into the conidiophores terminally and laterally, and arose directly from the mycelia. These phialides were mainly monophialidic but occasionally polyphialidic. Type I phialides were cylindrical, occasionally widened at the base. Type II phialides were one-septate, subcylindrical to navicular, tapering towards the apex. Type III phialides predominated, and were one-septate, mainly navicular to subcylindrical, and occasionally subulate. Hyphal coils were observed. Conidia were hyaline and mostly oblong ellipsoidal to obovoid, frequently biguttulate, and occasionally reniform to allantoid ( Figure 5). No perithecium formation was observed under laboratory conditions. The colonies reached a radius of 38-43 mm after 16 d at 23±2°C. The minimum temperature for growth was 15°C, with optimum growth at 30°C, and the maximum temperature for growth was of 37°C. Yellow pigment was produced in OA cultures ( Figure 5).
The optimum model selected for the IGS fragment was the Transversion model with empirical base frequencies and gamma distribution (TVM+F+G4) (lnL = -22064.046). The calculated parameters were: assumed nucleotide frequencies, A = 0.186, G = 0.288, T = 0.234, and C = 0.292; substitution rate matrix with A→C substi-    (1.   tution = 1.014, A→G = 4.371, A→T = 2.087, C→G = 1.273, C→T = 4.371 and G→T = 1.000; rates for variable sites were assumed to follow a gamma distribution (G4) with shape parameter = 0.513. The partition homogeneity test for the β-tubulin and actin sequences of Phaeoacremonium spp. produced a P-value of 0.61, which indicated that the dataset was congruent and the two genes could be combined. The combined β-tubulin and actin dataset consisted of 59 taxa, which included the outgroup taxon P. richardsiae, and contained 864 characters (β-tubulin, 594; actin, 270; including alignment gaps), of which 416 were constant (β-tubulin, 282; actin, 134), while 27 were variable and parsimony uninformative. Maximum likelihood analysis of the remaining 421 parsimony informative characters (β-tubulin, 296; actin, 125) produced a consensus tree with a Log-likelihood (lnL) = -5742.620763. Maximum parsimony analysis resulted in one most-parsimonious tree (TL = 1071; CI = 0.675; RI = 0.889; RC = 0.600; HI = 0.325) with similar topology to the maximum likelihood shown in Figure 6 (TreeBASE S28462). The phylogenetic tree obtained by combined analysis of the β-tubulin and actin genes clustered the Phaeoacremonium strains into 15 distinct clades, confirming the BLAST identification.
The P. italicum strains that belonged to sub-groups #1 to #6 clustered in a single clade that showed intraspecific genetic variation, with bootstrap values ranging from 80 to 98%. Strain CBS 113590 that belonged to sub-group #7 clustered in a sister clade of P. italicum (as already reported by Spies et al. (2018), with bootstrap support of 80% for maximum parsimony and 98% for maximum likelihood (Figure 6).
The IGS dataset alignment consisted of 59 taxa that included the outgroup taxon P. richardsiae and contained 3451 characters (including alignment gaps), of which 1547 were constant, while 202 were variable and parsimony uninformative. Maximum likelihood analysis of the remaining 1702 parsimony informative characters produced a consensus tree with lnL = -20723.212183. Maximum parsimony analysis resulted in 100 mostparsimonious trees (TL = 4170; CI = 0.720; RI = 0.884; RC = 0.637; HI = 0.280) with similar topology to maximum likelihood, one of which is shown in Figure 7 (TreeBASE S28463). In the comparison of the tree from the phylogenetic analysis of IGS sequences with the phylogenetic tree obtained in the combined analysis of the β-tubulin and actin sequences, the species segregation and tree topology were similar. Although the topology of the trees is not identical, each species was always well separated, and the clusters and segregation of the species were identical to the β-tubulin and actin phylogenetic tree, except for strain CBS 113590 that belonged to    P. italicum (sub-group #7) (Figures 6 and 7). In the IGS tree, this strain clustered in the clade of P. alvesii, with the strains CBS 408.78 and CBS 729.97.

DISCUSSION
In the present analysis of the IGS regions of a population of P. italicum strains collected from different hosts and localities, the entire IGS rDNA regions were successfully amplified using standard primers anchored in the conserved 28S and 18S rDNA gene-coding regions. The same standard primers were used to amplify the closest species to P. italicum, as P. alvesii and P. rubrigenum, and also for 12 other Phaeoacremonium species.
As the analyses of the complete IGS rDNA sequences of Phaeoacremonium spp. revealed the absence of 5S rDNA, in agreement with Drouin and de Sà (1995), we considered that this region was not linked to the rRNA major transcription unit, but was distributed throughout the genomes, as has been reported for several other filamentous fungi (Lockington et al., 1982;Selker et al., 1986;Garber et al., 1988;Walker et al., 2011). Walker et al. (2011) attributed the variable gene linkages for 5S in the Eukaryotes to result from stochastic gains and losses of variant repeat units, where functional 5S rRNA had been transposed by the mechanisms that were responsible for the concerted evolution of tandemly repeated multigene families.
Variations were also seen in sequence lengths for the P. italicum strains. It is well known that the IGS region is highly variable, as it contains several repeat elements, indels, variable regions with nucleotide substitutions (Albee et al., 1996;James et al., 2001), and sequences called 'repeat motifs' that are essential for initiation of transcription, RNA processing, transcription termination and replication processes of ribosomal DNA ( Van't Hof and Lamm, 1991;Fernandez et al., 2000). In the present study, analysis of the distributions of the polymorphisms through the sequences highlighted the presence of five short repeat elements throughout the IGS rDNA region of the P. italicum strains, named here as elements A, B, C, P and R. Elements A, B and P and their locations were common to all of the P. italicum strains, while the other two elements, C and R showed different number of copies, compositions and distributions among the strains. The presence of these repeated elements and their distributions allowed identification of one polymorphic region (PR) that was flanked by two conserved regions (CR1; CR2). The presence of indels through the IGS sequences was restricted to only the PR. The nucleotide substitutions consisted of insertions and deletions, and transi-tions and transversions, with these conversely seen only in the CR1 and CR2 regions. A similar organisation was also reported by Pantou et al. (2003) for Metarhizium anisopliae, Papaioannou et al. (2013) for Verticillium dahliae, and Durkin et al. (2015) for Colletotrichum lentis. Papaioannou et al. (2013) reported that this organisation putatively reflected the different functions of these sub-regions, and suggested that the highly conserved sub-regions had functions related to rRNA production and processing. The polymorphic sub-region, PR, may be responsible for the promotion of unequal crossing-over events and the maintenance of homogeneity between rDNA complexes (Mirete et al., 2013).
The analysis of these sequences showed that element C was a 'repeat motif ', and the intraspecific variation observed among the P. italicum strains was mainly due to the number of copies of element C repeated in the IGS rDNA sequences, as well as to the absence of some elements R in the polymorphic region. Diaz et al. (2005) reported that repeat motifs in the transcribed region or upstream of the transcription start can act as promoter enhancers and regulators of transcription of rRNA. These could have originated from processes involved in duplication and amplification of short sequences, and from slippage of replication mechanisms. Many such repeat motifs have been described as highly conserved in different Eukaryote species (Diaz et al., 2005). For example, the repeat motif of CAAAAA has been described as a conserved motif in the promoter region of different crucifers, such as Brassica spp. (Bhatia et al., 1996), Raphanus spp. (Delcasso et al., 1988) and Arabidopsis (Gruendler et al., 1991). Other common repeat motifs, such as the TATA motif, are probably involved in assembly of the pre-initiation complex and selection of transcription sites (Melanè et al., 1998). These have been reported as common elements in fungi, including Schizophyllum commune (James et al., 2001), Laccaria bicolor (Martin et al., 1999) and Neurospora spp. (Selker et al., 1986). The high degree of sequence similarities seen for CR1 and CR2 among the P. italicum strains can be used in conjunction with the characteristic repeat motifs of the fungus, for the design of species or group-specific primers for intraspecific group detection (Diaz et al., 2005;Papaioannou et al., 2013).
Based on the presence of repeated elements, on the number of copies of some of these elements, and on their composition, the P. italicum strains were grouped into seven different sub-groups (#1 to #7). Comparisons of the results of the polymorphic region PR sequences of all of the P. italicum strains analysed in this study showed that these were structurally similar across all of the sub-groups, although some differences were seen among the different P. italicum sub-groups. In particular, sub-groups #1 to #6 all shared the same initial layout of R elements in the first part of the polymorphic regions (PR-a), while they varied mainly in the numbers of copies of element C and for the absence of elements R5, R8 and R12 in the second part of polymorphic region (PR-b). Sub-group #7 contained strain CBS 113590, identified as P. alvesii by White et al. (2011) and Moyo et al. (2014), and more recently classified as P. italicum sensu lato by Spies et al. (2018); here it showed differences in the disposition and composition of the repeated elements.
Comparing the IGS structures of the sub-groups of P. italicum with the closest species of P. alvesii and P. rubrigenum, it was possible to confirm the same kinds of repeated elements, although they were differently organised and sometimes modified. The IGS structure of P. alvesii strain CBS 408.78 was identical to that of strain CBS 113590 of sub-group #7. Micromorphological analyses of the representative isolates of each of the P. italicum sub-groups showed similarities among sub-groups #1 to #6, and differences for sub-group #7. In comparison with the strains of sub-groups #1 to #6, CBS 113590 had longer and less septate unbranched conidiophores, rarely branched conidiophores, phialides predominantly of type III, ovoid conidia, more rapid growth rate, and maximum cardinal temperature for growth of 37°C. These features resemble those described by Moster et al. (2006) for P. alvesii. Based on the observations carried out on the strains of P. italicum sub-groups #1 to #6, the present study provides an upgrade of the morphological descriptions reported by Raimondo et al. (2014). For instance, compared to the original descriptions: the temperature minimum for growth (15°C) and growth optimum (25-30°C) were slightly different; and the morphological traits, such as branched conidiophores, were frequently, rather than occasionally, encountered. The frequent presence of polyphialides was another trait encountered. Based on the detailed examination carried out on this collection of P. italicum and P. alvesii strains in the present study, yellow pigment production was not to be a reliable feature for distinguishing these two species according to Spies et al. (2018). The strains that belonged to P. italicum subgroups #4 and #5, as well as CBS 113590 of sub-group #7 and P. alvesii (CBS 408.78,CBS 729.97), produced yellow pigment on OA. Therefore, the presence of abundant branched conidiophores, the high numbers of septa of the unbranched conidiophores, the predominance of type II phialides, the smaller sizes of type II and III phialides, the colour of the cultures on different media, and the minimum and maximum cardinal temperatures for growth, differentiate P. italicum from P. alvesii. Comparisons of the phylogenetic trees obtained for the combined analysis of β-tubulin and actin sequences with the phylogenetic analysis of the IGS sequences showed identical clustering and segregation of the Phaeoacremonium spp., except for strain CBS 113590. The combined analysis of the β-tubulin and actin sequences segregated this strain into a sister clade of P. italicum, while the IGS phylogenetic tree clustered it with P. alvesii.
The IGS rDNA region has been successfully used to study relationships in other fungi at intraspecific levels, including for Fusarium oxysporum (Appel and Gordon, 1996), Metarhizium anisopliae (Pipe et al., 1995), Microdochium nivale (Mahuku et al., 1998), Hebeloma cylindrosporium (Guidot et al., 1999), Cryptococcus neoformans (Diaz et al., 2005), Phomopsis helianthi (Pecchia et al., 2004), and Verticillium albo-atrum (Mahuku and Platt, 2002). The IGS region is one of the most rapidly evolving regions and provides large datasets that are considered to be phylogenetically useful, and these have provided large numbers of informative characters to delineate the relationships within and between species (Hillis and Dixon, 1991).
Based on micromorphological and molecular data, we conclude that the strain CBS 113590 belongs to P. alvesii and not to P. italicum sensu lato. Further molecular studies on phylogenetic signals and presence of barcoding gap, and multilocus analyses (β-tubulin, actin, and IGS sequences) using a large number of Phaeoacremonium strains will be carried out to determine whether the IGS rDNA region is a suitable marker for phylogenetic resolution of Phaeoacremonium species, to be used alone or in combination with β-tubulin and actin markers, to avoid misidentifications and introduction of vague species boundaries.