ABSTRACT
Here, we report 10 distinct bacterial genomes from Amazonian dark earths, including six identified as Paenibacillus polymyxa, while the remaining four were unique representatives of Paenibacillus vini, Bacillus cereus, Brevibacillus agri, and Fictibacillus sp., respectively. Each strain exhibited antagonistic activity against Fusarium oxysporum, underscoring their potential as sustainable agriculture resources.
KEYWORDS: plant-microbe interaction, rhizosphere-inhabiting microbes, Fusarium, terra preta
ANNOUNCEMENT
Amazonian dark earths (ADEs), a fertile soil type formed by the management practices of pre-Columbian indigenous near Amazonian rivers over two millennia ago (1), is known for promoting plant growth and nutrient retention. Its diverse microbial communities play key roles in nutrient cycling and soil fertility, offering the potential for agricultural and ecological advancements (2). Microbial isolation from ADE allows for a detailed study of the soil’s distinctive chemical and biological properties. This understanding paves the way for insights into the ecological roles of resident microorganisms and their potential biotechnological applications in areas such as sustainable agriculture, waste management, and biofuel production (3).
The strains presented here were previously isolated in 2016 from an experiment using ADE soils and two different cultivars of common bean, exhibiting contrasting levels of resistance to the soil-borne pathogen Fusarium oxysporum. The experiment was conducted at the Center for Nuclear Energy in Agriculture of the University of São Paulo (22° 42' 27.60" S, 47° 38' 41.17" W) (4). Plants were collected at the R1 development stage (early flowering), and the roots were shaken to remove loosely adhering soil. The firmly attached soil was collected with sterile brushes and considered to be the rhizosphere soil. For microbial isolation, 1 g of rhizosphere soil was mixed with 9 mL of saline solution (8.5 g L-1 NaCl). Serial dilutions (10−1 to 10−6) were performed and then transferred onto KING medium plates (5). After incubating at 25°C for 48 h, colonies were isolated using the streak plate method. Total DNA from isolates was extracted using the DNeasy Ultraclean Microbial Kit (Qiagen Mobio, Hilden, Germany) following the manufacturer’s instructions.
Whole-genome sequencing was performed using the Illumina HiSeq 2500 platform. Genomic DNA was used for paired-end libraries (2 × 150 bp) with NEBNext Fast DNA Fragmentation and Library Preparation Kit (New England Biolabs Inc., MA, USA). The number of library reads is described in Table 1. We visualized the quality of raw sequences obtained from Illumina HiSeq sequencing by using FastQC v0.12.1 (6). Given that all raw reads exhibited a quality score exceeding Q30, trimming was not performed. Then, the sequences underwent de novo assembly using SPAdes v3.15.5 (7). We employed QUAST v4.4 (8) to measure the assembled genomes’ quality metrics. Paired-end reads were aligned to the assemblies using the Burrows-Wheeler Aligner tool v0.7.17 (9). The average sequencing coverage was then calculated from the resulting BAM (Binary Alignment Map) file using SAMtools v1.15 (10). Taxonomic classification was performed with GTDB-tk v.2.3.2 (11). The genome annotation was performed by NCBI Prokaryotic Genome Annotation Process v6.5 (12). An additional functional gene annotation was carried out using RAST-tk 1.3.0 (13). Default parameters were used for all software. Genome assembly statistics and information about the strains are summarized in Table 1.
TABLE 1.
Assembly properties of 10 strains isolated from Amazonian dark earth, including contig statistics, genome size, GC content, and predicted gene count
| P. vini | P. polymyxa | P. polymyxa | Fictibacillus sp. | B. agri | P. polymyxa | B. cereus | P. polymyxa | P. polymyxa | P. polymyxa | |
|---|---|---|---|---|---|---|---|---|---|---|
| Isolate | CENA-BCM001 | CENA-BCM002 | CENA-BCM003 | CENA-BCM004 | CENA-BCM005 | CENA-BCM006 | CENA-BCM007 | CENA-BCM008 | CENA-BCM009 | CENA-BCM010 |
| Library size (million of reads) |
20.9 | 23.2 | 25.7 | 24 | 26.8 | 21.6 | 19.6 | 24.4 | 26.7 | 22.3 |
| Number of contigs | 65 | 57 | 32 | 16 | 111 | 34 | 22 | 34 | 31 | 30 |
| Largest contig (bp) | 343,800 | 689,511 | 1,524,845 | 1,557,022 | 1,557,022 | 371,701 | 1,100,025 | 1,517,151 | 1,524,846 | 1,445,065 |
| Total length (bp) | 5,712,199 | 5,635,190 | 5,849,366 | 4,967,627 | 5,280,964 | 5,280,964 | 5,569,359 | 5,726,433 | 5,840,294 | 5,711,641 |
| N50 (bp) | 188,024 | 202,973 | 686,293 | 1,390,346 | 1,390,346 | 79,882 | 668,640 | 1,443,580 | 686,293 | 690,213 |
| N75 (bp) | 101,676 | 128,558 | 230,621 | 407,875 | 407,875 | 47,434 | 362,608 | 207,366 | 186,286 | 206,180 |
| GC (%) | 48.88 | 45.28 | 45.36 | 43.65 | 43.65 | 53.62 | 34.96 | 45.40 | 45.36 | 45.38 |
| Genes | 5,274 | 4,975 | 5,169 | 5,118 | 5,255 | 5,446 | 5,635 | 5,075 | 5,162 | 5,011 |
| Protein coding | 5,159 | 4,913 | 5,044 | 4,882 | 5,060 | 5,310 | 5,459 | 4,950 | 5,033 | 4,907 |
| Non-coding | 72 | 22 | 75 | 64 | 72 | 75 | 58 | 79 | 79 | 61 |
| Coverage (×) | 1082.86 | 1082.86 | 1305.91 | 1437.35 | 1471.72 | 1041.85 | 1047.66 | 1269.73 | 1359.57 | 1160.26 |
| SRA identifiers | SRX20866903 | SRX20866904 | SRX20866905 | SRX20866906 | SRX20866907 | SRX20866908 | SRX20866909 | SRX20866910 | SRX20866911 | SRX20866912 |
| GenBank identifiers | GCA_030412165.1 | GCA_030412275.1 | GCA_030412235.1 | GCA_030412265.1 | GCA_030412255.1 | GCA_030412175.1 | GCA_030412285.1 | GCA_030412135.1 | GCA_030412145.1 | GCA_030412155.1 |
ACKNOWLEDGMENTS
This study was supported by grants from Fundação de Amparo à pesquisa do Estado de São Paulo (FAPESP 2014/03217-3 and 2015/00251–9), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES 88887.185941/2018–00), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 408191/2018–0), and PRPI-USP (22.1.08498.01.0). L.W.M. also thanks CNPq for the Productivity Research Grant (CNPq 307670/2021–0).
All authors meet the MRA authorship criteria. T.A.P., M.G.C., and L.W.M. were responsible for the organization and coordination of the analysis. L.W.M. was the chief investigator, and T.A.P. was responsible for the data analysis. All authors contributed to the writing of the final manuscript.
Contributor Information
Lucas William Mendes, Email: lwmendes@cena.usp.br.
Simon Roux, DOE Joint Genome Institute, Berkeley, California, USA .
DATA AVAILABILITY
This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the BioProject accession number PRJNA988090. The genome sequences are deposited under BioSample accession numbers SAMN35999008 to SAMN35999017 and raw sequences under SAMN36017836 to SAMN36017845. The SRA and GenBank identifiers are described in Table 1. RASTtk annotations are available in zenodo doi: 10.5281/zenodo.8087953.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the BioProject accession number PRJNA988090. The genome sequences are deposited under BioSample accession numbers SAMN35999008 to SAMN35999017 and raw sequences under SAMN36017836 to SAMN36017845. The SRA and GenBank identifiers are described in Table 1. RASTtk annotations are available in zenodo doi: 10.5281/zenodo.8087953.