Genome Sequence Resource of Bacillus velezensis Strain ML61, a Potential Biocontrol Bacterium Isolated from the Rhizosphere of Bok Choy (Brassica rapa var. chinensis)

Bacillus velezensis is widely known as a biocontrol agent against various plant diseases. Here, we report on the genome sequence of Bacillus velezensis strain ML61, which was isolated from the rhizosphere of bok choy (Brassica rapa var. chinensis) in Hangzhou, China, in 2020. ABSTRACT Bacillus velezensis is widely known as a biocontrol agent against various plant diseases. Here, we report on the genome sequence of Bacillus velezensis strain ML61, which was isolated from the rhizosphere of bok choy (Brassica rapa var. chinensis) in Hangzhou, China, in 2020.

B acillus velezensis is well known for its ability to suppress microbial pathogens and promote plant growth (1,2). Rhizosphere microorganisms can be exploited to promote plant growth and protect plants from pathogen attack (3). In this study, rhizosphere soil of bok choy (Brassica rapa var. chinensis), a common and important leafy vegetable in China (4), was collected and dissolved in sterile pure water. The suspension was diluted and spread onto potato dextrose agar (PDA) plates, which were seeded with Fusarium oxysporum spores (10 6 /mL, final concentration). After incubation for 4 days at 26°C, emergent bacterial colonies showing inhibitory halos were isolated and further purified by streaking onto a Luria-Bertani (LB) agar plate. Among them, a colony exhibiting a significant inhibitory halo was identified as Bacillus velezensis by analyzing its 16S ribosomal DNA sequence using BLASTn searches in the NCBI databases (5, 6), and we named this bacterial strain Bacillus velezensis ML61 (Fig. 1).
For genome sequencing, strain ML61 was propagated in LB liquid medium on a rotary shaker at 30°C for 24 h. Genomic DNA for PacBio sequencing was extracted using the Qiagen Genomic-tip 20/G kit. After that, the genomic DNA was fragmented using a Covaris g-TUBE device and concentrated using AMPure PB magnetic beads. Following DNA damage repair and end repair, the adapters were ligated. The treated DNA was purified and size selected using the BluePippin system (Sage Science) with cutoffs of 8 to 20 kb. The constructed library was bound with primers and polymerases using a PacBio binding kit. The final reaction products were sequenced using the Sequel II system (PacBio) with the Hi-Fi sequencing method, since it yields accurate long-read sequencing data sets (7,8). The circular consensus sequencing (CCS) analysis workflow (SMRT Link software; PacBio) was used to trim adapters and compute consensus sequences from multiple passes around a single circularized DNA molecule. After filtering the low-quality and short reads (,2,000 bp), PacBio sequencing yielded 38,646 subreads, including 374,544,534 bp, with an N 50 read length of 10,243 bp. The filtered subreads were assembled using Canu v1.5 software (9), and Circlator v1.5.5 was then used to circularize the genome assemblies (10). In the meantime, bacterial genomic DNA was extracted using the cetyltrimethylammonium bromide method (11), and a library was prepared using the Hieff next-generation sequencing (NGS) OnePot Pro DNA library prep kit for Illumina (Yeasen Biotech, Shanghai, China). Sequencing was performed on the Illumina NovaSeq 6000 platform with 2 Â 150-bp paired-end sequencing. Adapters were removed from the sequence reads and low-quality reads were filtered using fastp (parameters, -q 10 -u 50 -y -g -Y 10 -e 20 - l 100 -b 150 -B 150). For quality control, the reads were filtered when more than 50% of bases had a Phred score lower than 10 or a read length of less than 100 bp. Illumina sequencing yielded a total of 1,274,611,406 clean bases with a Q30 value of 93.35%, and these data were used to correct the PacBio genome sequence using Pilon v1.22 (12).
A polished gapless circular chromosome of 4,035,016 bp with a GC content of 46.56% was finalized. Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (13), and it predicted 3,804 protein-coding genes, 27 rRNA genes, 86 tRNA genes, and 5 noncoding RNAs (ncRNAs). Using antiSMASH v5.0.0 (14), 13 secondary metabolite biosynthetic gene clusters were predicted. The whole-genome sequencing data set will provide us with a deeper understanding of the molecular basis for ML61 as a beneficial microorganism.
Data availability. The genome sequence of B. velezensis ML61 has been deposited at GenBank under the accession number CP103769. The associated BioProject and BioSample accession numbers for this project are PRJNA873517 and SAMN30498090, respectively.

ACKNOWLEDGMENTS
This project was supported by the Open Research Program of the State Key Laboratory for Conservation and Utilization of Bio-Resource in Yunnan (2021KF012) and start-up funding from Zhejiang A&F University (2018FR059).