Seiridium cupressi (Nattrass, C. Booth & B. Sutton) Bonthond, Sand.-Den. & Crous, is an Ascomycete fungus belonging to the family Sporocadaceae. Together with other six Seiridium species, including S. cardinale (W. Wagener) B. Sutton & I.A.S. Gibson and S. unicorne (Cooke & Ellis) B. Sutton among them, S. cupressi is one of the causal agents of the Cypress Canker Disease pandemic (CCD) (Graniti 1998; Danti et al. 2013; Bonthond et al. 2018). The phytotoxic activity of S. cupressi has been studied, including the toxins that this species is able to produce, such as Cyclopaldic acid (Graniti et al. 1992; Sparapano & Evidente 1995) and Seiricuprolide (Ballio et al. 1988; Bartolucci et al. 1992). Nevertheless, genomic-level information that may enable us to better understand gene expression and the pathogenicity of CCD causal agents is still missing. In this study, two isolates of S. cupressi, BM-138–000515 and BM-138–000234, were retrieved from the Italian National Research Council of Sesto Fiorentino (Florence, Italy) CNR-IPSP-FTFC mycological collection (https://biomemory.cnr.it/collections/CNR-IPSP-FTFC/). These two isolates have been cultured from symptomatic tissue of Cupressus sempervirens located in Kos island (Grece) in 1984.

Each isolate was grown onto a cellophane disk placed on Petri dishes filled with Potato Dextrose Agar (PDA) for 14 days, at a constant temperature of 20 °C. Mycelia were collected, lyophilized and ground in liquid nitrogen using a sterile mortar and pestle. DNA was extracted using the Qiagen DNeasy plant kit, following the protocol provided by the manufacturer (Griffin et al. 2002). Quality and quantity of genomic DNA were assessed by gel electrophoresis and nanodrop (Desjardins & Conklin 2010). Short read sequencing was performed by Genartis SRL (Verona, Italy) using Illumina MiSeq (Caporaso et al. 2012). DNA libraries for these two samples were prepared with a KAPA Hyper Prep kit with a PCR-free protocol according to the manufacturer’s instruction (Whitehorn et al. 2018). A total of 500 ng of DNA was fragmented by sonication using a Covaris S220 focused-ultrasonicator (Covaris, Woburn, MA). A Whole Genome Sequencing DNA library was generated according to the manufacturer’s instruction (Roche), using 0.7X volume of AMPureXP beads (Jackson 2016) for the final size selection. Quality and size of WGS libraries were assessed by capillary electrophoretic analysis using the Agilent 4200 Tape station (Agilent Technologies). Libraries were preliminarily quantified using the Qubit BR dsDNA assay kit (Thermofisher) and then they were further quantified by Real-Time PCR, using the standard curve of the KAPA Library Quantification Kit (Kapa Biosystems). Libraries were pooled at equimolar concentrations and sequenced on a NovaSeq 6000 Illumina platform. DNA purity was measured using a NanoDrop Spectrophotometer, while DNA integrity was assessed using the Tape Station 4150 (Agilent Technologies). DNA quantity was measured using the Qubit dsDNA BR assay (ThermoFisher). DNA reads quality was determined using FastQC v0.11.7 (Andrews, 2010).  Illumina reads were assembled using SPAdes v3.15.5 (Bankevich et al. 2012). The first draft assembled for isolate BM-138–000515 consisted of 795 contigs (N50 = 289,630 bp and N75 = 146,648 bp), while isolate BM-138–000234 had 970 contigs (N50 = 359,772 bp and N75 = 196,592). In order to assess the genome assembly and integrity, BUSCO v.3.0.2 (Seppey et al. 2019) was utilized using the Sordariomycetes odb10 model dataset (Manni et al. 2021). Assembly statistics were produced using QUAST v5.2.0 (Gurevich et al. 2013) and results are reported in Table 1. Gene prediction and genome annotation have been performed with MAKER3 v3.01.03 (Cantarel et al. 2008). A total of 13,446 protein coding genes were obtained for the isolate BM-138–000234, while a total number of 13,376 protein coding genes were predicted for isolate BM-138–000515.

Table 1 Summary statistics for Seiridium cupressi, isolates BM-138–000234 and BM-138–000515
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SignalP v5.0 (Almagro Armenteros et al. 2019), designed for the prediction of signal peptides, predicted 1,504 secreted proteins for BM-138–000515 isolate and 1,504 secreted proteins for BM-138–000234 isolate. Effector prediction analyses, carried out with EffectorP-fungi v3.0 (Sperschneider & Dodds 2022), predicted 528 secreted proteins may be effectors in BM-138–000234, including 175 cytoplasmic effectors and 353 apoplastic effectors. Regarding isolate BM-138–000515, 529 candidate effectors were predicted, including 177 cytoplasmic effectors and 352 apoplastic effectors. A phylogenomic comparative analysis was performed following the methods described in Baroncelli et al. (2022). Both isolates of S. cupressi were compared to phylogenetically related species belonging to the Sporocadaceae family and to the order Xylariales  (Fig. 1). The genome of S. cupressi is the first Seiridium genome published, as well as the first genomic resource published regarding the CCD pathosystem, and will assist the scientific community in furthering its understanding of the mechanisms that make this disease a true world pandemic.

Fig. 1
figure 1

Phylogenetic tree comparing the newly sequenced isolates of S. cupressi BM-138–000515 and BM-138–000234 with those of closely available fungi belonging to Xylariales order and Sporocadaceae family. Botrytis cinerea and Diplodia corticola have been selected as outgroups. Asterisks identify the isolates whose genomes have been sequenced in this study

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GenBank accession number

This whole-genome shotgun project has been deposited in GenBank. Genomic sequences of S. cupressi isolate BM-138–000234 have been deposited under the accession number JARVKI000000000 (BioProject: PRJNA953536; BioSample: SAMN34119420). Genomic sequences of S. cupressi isolate BM-138–000515 have been deposited under the accession number JARVKH000000000 (BioProject: PRJNA953536; BioSample: SAMN34119421).