DNA extraction, PCR amplification and sequencing

Total DNA was extracted with an E.Z.N.A.^® Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany), according to the manufacturer’s instructions. The nuclear ribosomal internal transcribed spacer (ITS) region was amplified using ITS1 and ITS4 primers (White et al. 1990). The primers TUB2Rd and TUB4Fd (Aveskamp et al. 2009) were used to amplify part of the β-tubulin (tub2) gene. The partial translation elongation factor 1-α (tef1) gene was amplified with EF1-728F (Carbone & Kohn 1999) and EF2 (O’Donnell et al. 1998) primers. The amplification mixtures and cycling conditions for all three loci were followed as described in each of the cited references. Both strands of the PCR products were sequenced by Eurofins Genomics Service (Ebersberg, Germany). The sequences generated were analysed using Geneious v. 11.1.5 (Kearse et al. 2012, Auckland, New Zealand) and consensus sequences were processed.

Phylogenetic analyses

The newly generated sequences were analysed using BLAST search on the NCBIs GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi) database to achieve a taxonomic framework by determining the closest relatives. The MAFFT v. 7 online program (http://mafft.cbrc.jp/alignment/server/index.html) (Katoh & Standley 2013) was used to align each gene region of the sequences obtained from this study and sequences downloaded from GenBank. Alignments were then manually adjusted by MEGA v. 7 (Kumar et al. 2016). The analyses were conducted individually for each locus (data not shown) and as multi-locus analysis, with the aim of identifying the isolates at species level. Reference sequences were selected based on recent studies on Paraphoma species (Moslemi et al. 2016, Cao et al. 2020, Gomzhina et al. 2020, Magaña-Dueñas et al. 2021). The phylogeny was developed based on Maximum Parsimony (MP) approach for all individual loci, and on both MP and Bayesian Inference (BI) methods for the concatenated multilocus analyses. For BI, the best evolutionary model for each partition was selected with MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses. MrBayes v. 3.2.5 (Ronquist et al. 2012) was used to generate phylogenetic trees under optimal criteria per partition. The Markov Chain Monte Carlo (MCMC) analysis used four chains and started from a random tree topology. The heating parameter was established at 0.2 and trees were sampled every 1 000 generations. The analyses were considered done when the average standard deviation of split frequencies was less than 0.01. The MP analyses were conducted using PAUP (Swofford 2003). Phylogenetic relationships were estimated by heuristic searches with 100 random additional sequences. Tree bisection-reconnection was adopted, with the branch swapping option set at ‘best trees’ only with all characters weighted equally and alignment gaps treated as fifth state. Tree length (TL), consistency index (CI), retention index (RI) and re-scaled consistency index (RC) were calculated, and the parsimony and the bootstrap analyses (Hillis & Bull 1993) were based on 1 000 replications. Sequences generated in this study were deposited in GenBank (Table 1), and the alignments in TreeBASE (www.treebase.org; study number S29045).

Morphology

Slide preparations were mounted in lactic acid from colonies sporulating on sterilised pine needles placed on 2 % tap water agar (PNA) (Smith et al. 1996). Observations were performed under a Nikon SMZ25 dissection-microscope, and a Zeiss Axio Imager 2 bright field microscope using differential interference contrast (DIC) illumination, and images recorded on a Nikon DS-Ri2 camera with associated software. Colony features and pigment production were described on 2 % malt extract agar (MEA), PDA and oatmeal agar (OA; Crous et al. 2019) after 2 wk at 25 °C. Colony colours were scored using the colour charts of Rayner (1970). The taxonomic novelty was registered in MycoBank (www.MycoBank.org; Crous et al. 2004).

Taxonomy

Paraphoma garibaldii Guarnaccia, M.L. Gullino & Crous, sp. nov. MycoBank MB 842029. Fig. 1.

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Fig. 1.

Paraphoma garibaldii (CBS 148459). A. Pycnidia with setae forming on PNA. B–D. Brown setae arising from outer pycnidial wall. E, F. Conidiogenous cells giving rise to conidia. G. Aseptate, guttulate conidia. Scale bars: A = 300 μm; All others = 10 μm.

Etymology: Named after Prof. Angelo Garibaldi, in recognition of his contribution to research on ornamental plant diseases.

Conidiomata pycnidial, erumpent to superficial on PNA, wall of 3-4 layers of brown, thin-walled textura angularis, globose, 200–300 μm diam, covered by brown, septate, thick-walled, subcylindrical setae, 30–70 × 3–4 μm, with obtuse ends. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner cavity, phialidic, hyaline, smooth-walled, ampulliform to doliiform, 5–8 × 4–6 μm, with prominent periclinal thickening. Conidia solitary, aseptate, hyaline, smooth-walled, guttulate, subcylindrical, obtuse at the apex and truncate at the base, (4–)5–6(–7) × (2–)2.5(–3) μm.

Culture characteristics: On MEA, PDA and OA, colonies erumpent, spreading with moderate aerial mycelium and even lobate margins, up to 70 mm diam after 2 wk, surface and reverse red.

Typus: Italy, Piedmont, Biella, on leaf spots of Campanula rapunculoides (Campanulaceae), May 2021, A. Garibaldi (holotype CBS H-24894, culture ex-type CBS 148459).

Additional material examined: Italy, Piedmont, Biella, on leaf spots of Ca. rapunculoides, May 2021, A. Garibaldi, CBS 148460.

Notes: Paraphoma garibaldii is phylogenetically distinct from all 14 species of the genus. Morphologically, its conidia are similar to those of P. variabilis (4–8 × 2–3 μm, from dung, Spain; Crous et al. 2021), but distinct in that the latter has greyish colonies and shorter (7–25 × 2.5–3 μm), subhyaline setae.

Phylogenetic analyses

Based on the results by BLAST search, all the sequences obtained in this study showed high similarity (around 96 %) with species included in the Paraphoma genus, however they were identical with no particular species. Three alignments representing single locus analyses of ITS, tub2, tef1 (data not shown), and a combined alignment of the three loci were analysed. The single phylogenetic analysis generated by each locus produced a similar tree topology. The strains of Paraphoma garibaldii formed a well-supported monophyletic clade in the ITS, tub2 and tef1 single-locus trees, with maximum bootstrap values, respectively. The multi-locus phylogeny consisted of 30 sequences, including Setophoma terrestris (CBS 335.29, Gomzhina et al. 2020) as outgroup. A total of 1 172 characters (ITS: 1–502, tub2: 509–777, tef1: 784–1 172) were included in the phylogenetic analysis, 456 characters were parsimony-informative, 239 were variable and parsimony-uninformative, and 464 were constant. A maximum of 1 000 equally MP trees were saved (Tree length = 1 899, CI = 0.656, RI = 0.737 and RC = 0.483). Bootstrap support values from the MP analysis are included on the Bayesian tree in Fig. 2. For the BI, MrModeltest suggested that all partitions should be analysed with dirichlet state frequency distributions. The following models were recommended by MrModeltest and used: GTR+I+G for ITS, K80+G for tub2 and HKY+G for tef1. In the BI, the ITS partition had 219 unique site patterns, the tub2 partition had 161 unique site patterns, the tef1 partition had 245 unique site patterns and the analysis ran for 675 000 generations, resulting in 1 352 trees of which 534 trees were used to calculate the posterior probabilities.

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Fig. 2.

Consensus phylogram of 1352 trees resulting from BI of the combined ITS, tub2 and tef1 datasets. Bayesian posterior probability values and bootstrap support values are indicated at the nodes. The tree was rooted with Setophoma terestris (CBS 335.29).

This work was funded by the Ministry of Education, Universities and Research (MIUR), Local research (ex 60 %).