# Complete genome sequence of Metarhizium marquandii strain ISA501

Jun Zhou, Maria Elena Antinori, Gabriele Bellotti, Edoardo Puglisi, Michel Chalot

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Jun Zhou, Maria Elena Antinori, Gabriele Bellotti, Edoardo Puglisi, Michel Chalot. Complete genome sequence of Metarhizium marquandii strain ISA501. Microbiology Resource Announcements, 2025. hal-05123729

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# Complete genome sequence of *Metarhizium marquandii* strain ISA501

Jun Zhou,¹ Maria Elena Antinori,² Gabriele Bellotti,² Edoardo Puglisi,²,³ Michel Chalot¹

**AUTHOR AFFILIATIONS** See affiliation list on p. 3.

**ABSTRACT** We report the complete genome sequence of *Metarhizium marquandii* strain ISA501, isolated from maize rhizosphere soil. The 42.8 Mb genome, assembled with hybrid sequencing, comprises eight chromosomes with 51.65% GC content. This high-quality resource provides insights into its plant growth-promoting potential, secondary metabolism, and ecological roles in sustainable agriculture.

**KEYWORDS** *Metarhizium marquandii*, genome, rhizosphere

The genus *Metarhizium* comprises fungi known for their entomopathogenic properties and use as biocontrol agents (1). Some species also promote plant growth by associating with roots (2). Among them, *Metarhizium marquandii* has been reported as a saprophyte with plant growth-promoting abilities and roles in improving soil health (3, 4). However, no genome sequence has been made publicly available to date. Here, we report the chromosome-level genome assembly of strain ISA501, isolated from maize rhizosphere soil in Brescia, Italy (N 45.333096, E 10.438125).

Isolation was performed using a modified protocol based on Barillot et al. (5). Rhizospheric soil was suspended in 0.9% NaCl and agitated for 1 hour. Dilutions were plated on PDA with 0.1 mg/mL chloramphenicol. Colonies were transferred to antibiotic-free PDA, and single spores were isolated. Cultures were grown at 28°C for 7 days before DNA extraction (Fig. 1). Species identity was initially determined by ITS sequencing (primers ITS1 and ITS4) (6), followed by BLAST comparison against GenBank. The top hit was *M. marquandii* CBS 182.27 (NR_131994). For genome sequencing, fungal mycelia were grown in potato dextrose broth at 28°C for 7 days, harvested, washed, freeze-dried, and used for genomic DNA extraction (HiPure Universal DNA Kit, Genepioneer Biotechnologies).

A hybrid sequencing strategy was employed to ensure the accuracy and completeness of the genome assembly. DNA libraries for short-read sequencing were constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 ND607 (Vazyme). Sequencing was conducted on the Illumina NovaSeq 6000 platform in paired-end 150 bp mode, generating 21,053,627 reads, amounting to 6.32 Gb of data, with an average depth of coverage of approximately 144×. For Nanopore sequencing, DNA was purified using 0.4× magnetic beads to remove fragments <2 kb. Long-read sequencing libraries were prepared using the SQK-LSK110 ligation kit (Nanopore) and sequenced on the PromethION 48 platform (R9.4.3 flow cells). This yielded 992,019 reads with a total output of 9.94 Gb, achieving an average coverage depth of 214×. The N₅₀ read length was 18,680 bp. Guppy (high-accuracy mode) was used for baseCALLing. Quality control for both data sets involved removing low-quality reads and adapters using fastp (version 0.20.0) (7) and Filtlong (version 0.2.1). Genome assembly was performed by integrating short and long reads. Nanopore reads were corrected using Canu (version 2.1.1) (8) and LoRDEC (version 0.9) (9). Primary assembly was achieved using NextDenovo (version 2.5) (10), and potential misassemblies were resolved by aligning corrected reads to the draft

**Editor** Vincent Michael Bruno, University of Maryland School of Medicine, Baltimore, Maryland, USA

Address correspondence to Jun Zhou,
jun.zhou@univ-fcomte.fr.

The authors declare no conflict of interest.

**Received** 18 February 2025

**Accepted** 4 June 2025

**Published** 20 June 2025

Copyright © 2025 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
<figure><img src="image_3.png" /><figcaption>**FIG 1** *Metharizium marquandii* ISA501 grown 10 days on PDA. (A) Upper side of the plate. (B) Bottom of the plate. The medium becomes yellowish, as in the picture, as soon as the strain starts growing, probably due to the production of some metabolites.</figcaption></figure>

assembly with minimap2 (version 2.1) (11). Final polishing was conducted using three iterations of NextPolish (version 1.4.0) (12) with Nanopore reads and Pilon (version 1.23) (13) with Illumina reads. Default parameters were used except where otherwise noted.

The final genome assembly comprises eight chromosomes, totaling 42,805,715 bp, with a GC content of 51.65%. The largest chromosome is 7,777,640 bp. The $N_{50}$ value is 7,303,840 bp. Completeness was assessed using BUSCO (version 5.2.1) (14), revealing 98.0% complete BUSCOs (96.7% single copy and 1.3% duplicated), with 0.3% fragmented and 1.7% missing (Table 1). Coverage analysis with minimap2 confirmed even read distribution. Telomeric repeats (CCCTAA)n were detected at both ends of each contig, supporting telomere-to-telomere completeness.

<table><caption>**TABLE 1** Genome sequencing statistics</caption><thead><tr><th>Parameter</th><th>Value</th></tr></thead><tbody><tr><td><strong>Illumina sequencing</strong></td><td></td></tr><tr><td>Average depth of coverage, Illumina short reads</td><td>~144x</td></tr><tr><td>Number of reads</td><td>21,053,627</td></tr><tr><td>Total bases (bp)</td><td>6,316,088,100</td></tr><tr><td><strong>Nanopore sequencing</strong></td><td></td></tr><tr><td>Average depth of coverage, ONT long reads</td><td>~214x</td></tr><tr><td>Number of reads</td><td>992,019</td></tr><tr><td>Total bases (bp)</td><td>9,944,606,976</td></tr><tr><td>N<sub>50</sub> read length (bp)</td><td>18,680</td></tr><tr><td><strong>Genome assembly</strong></td><td></td></tr><tr><td>No. of chromosomes</td><td>8</td></tr><tr><td>Total length (bp)</td><td>42,805,715</td></tr><tr><td>Largest chromosome</td><td>7,777,640</td></tr><tr><td>GC (%)</td><td>51.65</td></tr><tr><td>N<sub>50</sub> (bp)</td><td>7,303,840</td></tr><tr><td>L<sub>50</sub></td><td>3</td></tr><tr><td><strong>Genome assembly assessment</strong></td><td></td></tr><tr><td>Complete BUSCOs</td><td>98.0%</td></tr><tr><td>Complete and single-copy BUSCOs</td><td>96.7%</td></tr><tr><td>Complete and duplicated BUSCOs</td><td>1.3%</td></tr><tr><td>Fragmented BUSCOs</td><td>0.3%</td></tr><tr><td>Missing BUSCOs</td><td>1.7%</td></tr></tbody></table>
ACKNOWLEDGMENTS

Financial support has been provided to SIRAM by PRIMA, a program supported by H2020, the European Programme for Research and Innovation, by the French ANR under ANR-22-PRIM-0001-05, and by the Italian MUR (SIRAM PRIMA CALL 2021 SECTION 2 CUP J33C22000510008).

AUTHOR AFFILIATIONS

¹Chrono-environnement (UMR 6249), Université Marie et Louis Pasteur, CNRS, Montbéliard, France

²Department for Sustainable Food Process, Università Cattolica del Sacro Cuore, Piacenza, Italy

³ISA Innovations for Sustainable Agriculture R&D, srl, Piacenza, Italy

AUTHOR ORCIDs

Jun Zhou ID http://orcid.org/0000-0003-3618-1037

Maria Elena Antinori ID http://orcid.org/0000-0002-2258-6204

Gabriele Bellotti ID https://orcid.org/0000-0001-8521-3256

Edoardo Puglisi ID http://orcid.org/0000-0001-5051-0971

Michel Chalot ID http://orcid.org/0000-0001-8614-0917

AUTHOR CONTRIBUTIONS

Jun Zhou, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review and editing | Maria Elena Antinori, Conceptualization, Investigation, Methodology, Visualization, Writing – review and editing | Gabriele Bellotti, Conceptualization, Investigation, Methodology, Writing – review and editing | Edoardo Puglisi, Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review and editing | Michel Chalot, Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review and editing

DATA AVAILABILITY

The genome and sequencing data have been deposited at NCBI under BioProject accession PRJNA1215054. The BioSample accession is SAMN46395144. The Illumina sequencing reads are available in the NCBI Sequence Read Archive under accession SRR32309563, while the nanopore sequencing reads are accessible under accession SRR32316793. This Whole Genome Shotgun project has been deposited in DDBJ/ENA/ GenBank under the accession no. JBLFEU000000000. The version described in this paper is the first version, JBLFEU010000000.

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