Genome assembly of Ramulariopsis pseudoglycines, a fungal pathogen responsible for areolate mildew on cotton

TL;DR

This paper presents the first genome assembly of Ramulariopsis pseudoglycines, a fungal pathogen that causes areolate mildew on cotton.

Contribution

The study provides the first high-quality genome assembly of Ramulariopsis pseudoglycines using nanopore sequencing.

Findings

The genome size is approximately 77.9 Mb with an N50 of ~2.5 Mb.

The genome has a GC content of 52.74% and a BUSCO completeness of 99.5%.

Abstract

The fungal species Ramulariopsis pseudoglycines was recently reported as a pathogen of cotton in the United States. Here, we report the first genome of this pathogen assembled using nanopore sequencing. The genome size is ~77.9 Mb, with an N50 of ~2.5 Mb, GC content of 52.74%, and Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness of 99.5%.

Tables

TABLE 1. Genome sequencing, assembly statistics, and genomic features of Ramulariopsis pseudoglycines isolate AJ029-2023 (FELCC 511)

Sequencing/genomic features
No. of reads	12.71 million
Bases called	23.43 Gb
Contigs	148
Longest contig	8,117,343 bp
Genome size	77,925,435 bp
N 50	2,570,697 bp
GC content	52.74%
BUSCO completeness (ascomycota_odb10)	99.5%
CDS	76,268
Coding genes	24,936
Ribosomal RNAs	63
Transfer RNAs	141

Full text

ANNOUNCEMENT

The causal agent of areolate mildew of cotton (Gossypium hirsutum L.) was originally described as Ramularia areola (1), but the species was reclassified to Ramulariopsis gossypii (2). Multi-locus DNA sequencing revealed that Ramularia pseudoglycines is also associated with this disease (3). Currently, R. gossypii and R. pseudoglycines are both responsible for areolate mildew, and the latter is the most widespread species in Brazil affecting yield and fiber quality (4, 5). R. pseudoglycines was reported in the United States for the first time in 2023 (6), and it is becoming prevalent in the Southeastern United States. Given its importance, a genome assembly is needed to further investigate its pathogenicity, host-pathogen interactions, and ecology.

In August 2023, commercial cotton plants in Georgia (Colquitt County) showed yellow lesions in the adaxial leaf surface and white powdery fungal sporulation in the abaxial surface. Conidia observed from the leaf surface was scraped with a sterile scalpel onto V8 medium amended with chloramphenicol (75 ppm) and streptomycin sulfate (125 ppm) and incubated in the dark at 25°C. Fungal colonies developed after 6 days, and they were consistent with previous Ramulariopsis spp. descriptions (3, 6, 7). These colonies were transferred to V8-antibiotic-amended medium to obtain pure cultures. Isolate AJ029-2023 (culture collection: FELCC 511) was selected for whole-genome sequencing. Single colonies were restored from 30% glycerol stocks, and fungal biomass was grown in liquid V8 media at room temperature under shaking conditions (150 rpm) for 10 days.

High molecular weight genomic DNA was isolated using a modified CTAB protocol (8) and adjusted to 30 ng µL^−1^ in nuclease-free water. Genomic DNA was prepared for sequencing by first applying a DNA repair and end-prep step, then adapter ligation and clean up using the Ligation Sequencing Kit V14 (SQK-LSK114) without shearing or size selection, as specified in the manufacturer’s protocols. DNA was sequenced using the Oxford Nanopore MinION Mk1C platform and the R10.4.1 Flow Cell (FLO-MIN114). Sequencing quality was monitored using the MinKNOW version 22.10.5 GUI interface. Sequences were base called and demultiplexed using Guppy version 6.3.8 (9). The total number of reads was 12.71 million, and the estimated read N50 was 3.36 kb. The genome was assembled with Flye version 2.9.1 (10) and polished with Medaka version 1.7.2 (11), using a de novo assembly approach. Assembly quality was assessed with Quast version 5.0.2 (12) and completeness with the Benchmarking Universal Single-Copy Orthologs (BUSCO) software version 5.4.3 (13). Gene prediction and annotation were performed with the COMprehensive Parasite ANnotatION version 2.2.8 database (14), using Zymoseptoria tritici IPO323 version 2.0 (15, 16) as a reference. Default parameters were used for all software unless otherwise specified. The genome size is 77,925,435 bp, with 148 contigs, N50 of 2,570,697 bp, GC content of 52.74%, and BUSCO completeness (ascomycota_odb10) of 99.5% (Table 1).