Complete Genome Sequence of Bacillus amyloliquefaciens EA19, an Endophytic Bacterium with Biocontrol Potential Isolated from Erigeron annuus

ABSTRACT Bacillus amyloliquefaciens strain EA19 is an endophyte isolated from Erigeron annuus with antifungal activity against Blumeria graminis f. sp. tritici, Magnaporthe oryzae, and Fusarium graminearum. The genome sequence of this strain is 3.96 Mb and contains 3,421 coding sequences, which will facilitate an understanding of the mechanisms of biocontrol.

amyloliquefaciens, a plant-associated bacterium, can be found in plant organs and exhibits antifungal activities against plant pathogens (2)(3)(4). Genome analysis helps to elucidate the gene clusters involved in the biosynthesis of active secondary metabolites of B. amyloliquefaciens (4).
Strain EA19 was isolated from the healthy stems of annual fleabane (Erigeron annuus) plants collected at the flowering stage in 2010 through the surface sterilization method (5). A single isolated colony was cultured on Luria-Bertani (LB) solid medium at 30°C for 24 h. Bacterial identification was performed using 16S rRNA and gyrA gene sequencing (6). Phylogenetic analysis of the two genes identified EA19 as a B. amyloliquefaciens subsp. strain ( Fig. S1; https://figshare.com/search?q=10.6084%2Fm9.figshare.15145227). The culture supernatant from a single colony was incubated in LB liquid at 30°C and 180 rpm for 48 h. The fermentation broth was centrifuged at 10,000 Â g for 5 min and then filtered through 0.22-mm sterile syringe filters (Durapore polyvinylidene fluoride [PVDF] membrane; Millipore). Antagonism tests against Blumeria graminis f. sp. tritici isolate W14 in planta and against Magnaporthe oryzae Guy11 and Fusarium graminearum PH-1 in plates were performed using the cell-free culture filtrate with detached leaf method and agar dilution method, respectively (7-9). These fungi are among the top 10 fungal pathogens in plant pathology and cause wheat powdery mildew, rice blast, and Fusarium head blight, respectively (10). The filtrate has inhibitory effects on the growth of the three pathogens (Fig. 1). Here, we report the complete genome sequence of B. amyloliquefaciens strain EA19 and its annotation.
The pellet from the fermentation broth above was harvested and genomic DNA was extracted using a QIAamp DNA extraction kit (Qiagen). The DNA library was prepared using a rapid Oxford Nanopore Technologies (ONT) sequencing kit (SQK-RAD004) and then sequenced using FLO-MIN106 flow cells on a GridION instrument (ONT, UK). Adaptor trimming and data filtering were conducted using Porechop v0.2.3 and Filtlong v0.2.0 software. Meanwhile, an Illumina library was constructed using the TruSeq Nano DNA low-throughput (LT) library prep kit (Illumina) and yielded high-quality short paired-end reads (150 bp) on the NovaSeq 6000 sequencing platform. The ONT library contained 588,025 reads with a total length of 1,884,340,507 bp, a subread N 50 value of 5,804 bp, and an average coverage of 475Â. Sequencing of the Illumina library generated 3,382,798 reads with a total length of 1,014,839,400 bp and an average coverage of 256Â. De novo assembly of the genome sequence was performed using the Hierarchical Genome Assembly Process (HGAP4) workflow (11), followed by polishing with the Illumina reads. The assembled genome sequence was circularized using Minimus2 (12). NCBI PGAP v5.2 software was used for the genome annotation (13). Default parameters were used for all software unless otherwise specified.
Data availability. The genome sequence has been deposited in GenBank under the accession number CP079834. The GenBank accession number for the plasmid is CP079835. The sequence data can be found in the Sequence Read Archive under the accession numbers SRX11361551 and SRX11361552.

ACKNOWLEDGMENTS
This study was financially supported by grants from the China Agriculture Research System (project CARS-3-1-2) and the Youth Foundation of Institute of Plant Protection and Soil Fertilizer, Hubei Academy of Agricultural Sciences (project 2021ZTSQJ12).