Here, we report the draft genome sequences of three Fusarium oxysporum f. sp. niveum isolates that were used to design markers for molecular race differentiation. The isolates were collected from watermelon fields in Georgia (USA) and were determined to be different races of F. oxysporum f. sp. niveum using a traditional bioassay.
ABSTRACT
Here, we report the draft genome sequences of three Fusarium oxysporum f. sp. niveum isolates that were used to design markers for molecular race differentiation. The isolates were collected from watermelon fields in Georgia (USA) and were determined to be different races of F. oxysporum f. sp. niveum using a traditional bioassay.
ANNOUNCEMENT
Fusarium wilt in watermelon is caused by the fungal pathogen Fusarium oxysporum f. sp. niveum, which is one of the most impactful pathogens for watermelon production worldwide (https://edis.ifas.ufl.edu/PP352) (1–3). Three isolates of F. oxysporum f. sp. niveum were obtained from infected watermelon plants (Citrullus lanatus) with typical symptoms of Fusarium wilt in commercial fields in Georgia. Traditional diagnostic methods are unable to identify the pathogen as F. oxysporum f. sp. niveum with 100% confidence, so molecular assays are required to confirm the identity of an isolate (4–6). Additionally, it is currently recognized that F. oxysporum f. sp. niveum has 4 races (R0, R1, R2, and R3) which require a bioassay for differentiation, as at the date of this writing, only race 2 may be distinguished molecularly (https://edis.ifas.ufl.edu/PP352) (1, 3, 7).
The three isolates that were chosen for sequencing were isolated from the lower stem (hypocotyl region) of diseased watermelon plants. Samples were surface-disinfested in 0.6% NaOCl, rinsed in sterile distilled water, and cultured on semiselective peptone pentachloronitrobenzene agar plates (8). Fungal cultures grown from the samples were identified based on the morphological characteristics, and single-spore isolates were grown on potato dextrose agar (PDA) plates and incubated at 25°C for 7 days. The races of the isolates were identified by inoculating differential watermelon plants and evaluating the disease development as reported previously (5, 7). Three isolates, representing three races of F. oxysporum f. sp. niveum, were grown on PDA for 10 days; next, 100 mg of mycelia was used for DNA extraction. DNA was extracted using the DNeasy plant minikit (Qiagen) and was concentrated and purified using Quantum Prep PCR Kleen Spin columns (Bio-Rad) according to the manufacturer’s instructions. PCR was performed on all isolates using FON-1/FON-2, primers specific to F. oxysporum f. sp. niveum, to confirm the identity of all F. oxysporum f. sp. niveum isolates, and the race 2-specific primer set FONSIX6F/R to confirm the race 2 isolate (9, 10). DNA was standardized at 200 ng/µL for each extraction and submitted to Novogene Co., Ltd. (Beijing, China) for whole-genome sequencing.
Libraries were prepped using an NEB Ultra II kit and sequenced using the paired-end strategy PE150 on an Illumina NovaSeq 6000 platform. The original optic data obtained by high-throughput sequencing were transformed into raw sequenced reads using Casava v.1.8 base calling and stored in FASTQ (fq) format. Quality control was performed using readfq v.10 (https://github.com/lh3/readfq) using default parameters, and low-quality sequences were removed (11, 12). Raw reads were assembled into scaffolds using SPAdes v.3.14.1 with the k-mer values 21, 33, 55, and 77 (13). Bowtie 2 v.2.4.1 was used to align the paired-end reads against the scaffolds produced by SPAdes, producing the SAM alignment files (14). SAMtools v.1.10 was used to convert the alignment files to BAM format and then sort and index the BAM files. Pilon v.1.23 (using - -frags mode) was used to polish the BAM files, yielding the final output of FASTA files (15, 16). Contigs shorter than 200 bp were removed from polished FASTA files using a novel Python script for contig filtration (17). The genome characteristics and accession numbers are given in Table 1.
TABLE 1.
Genome assembly data and accession numbers for sequenced Fusarium oxysporum f. sp. niveum isolates
| Isolate | Genome size (bp) | No. of contigs>50,000 bp | N50 (bp) | Avg coverage (×) | G+C content (%) | GenBank BioSample accession no. | GenBank BioProject accession no. | SRA accession no. |
|---|---|---|---|---|---|---|---|---|
| FONR1 | 61,207,430 | 245 | 154,443 | 28.65 | 48.79 | SAMN15791673 | PRJNA656528 | SRR12492378 |
| FONR2 | 54,074,873 | 217 | 161,737 | 22.92 | 47.53 | SAMN15791674 | PRJNA656528 | SRR12492379 |
| FONR3 | 55,220,015 | 220 | 158,709 | 22.38 | 47.54 | SAMN15791675 | PRJNA656528 | SRR12492380 |
Data availability.
All data for this whole-genome sequencing project were deposited under the GenBank BioProject accession number PRJNA656528. The raw reads of the genomic data for the isolates were deposited under the following SRA accession numbers: SRR12492378, SRR12492379, and SRR12492380. The accession numbers for each isolate are as follows: SAMN15791673, SAMN15791674, and SAMN15791675.
ACKNOWLEDGMENT
This work was supported by funds from the Georgia Department of Agriculture Specialty Crop Block Grant (grant number AWD00011227).
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Associated Data
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Data Availability Statement
All data for this whole-genome sequencing project were deposited under the GenBank BioProject accession number PRJNA656528. The raw reads of the genomic data for the isolates were deposited under the following SRA accession numbers: SRR12492378, SRR12492379, and SRR12492380. The accession numbers for each isolate are as follows: SAMN15791673, SAMN15791674, and SAMN15791675.