The rhizosphere microbiome plays an essential role in enhancing the growth of plants, raising the need for comprehension of their metabolic abilities. Here, we investigated rhizospheric and bulk soils of maize plants in Mafikeng, South Africa. Metagenome-assembled genomes containing plant growth-promoting genes were reconstructed.
ABSTRACT
The rhizosphere microbiome plays an essential role in enhancing the growth of plants, raising the need for comprehension of their metabolic abilities. Here, we investigated rhizospheric and bulk soils of maize plants in Mafikeng, South Africa. Metagenome-assembled genomes containing plant growth-promoting genes were reconstructed.
ANNOUNCEMENT
Zea mays (maize) has become one of the foremost staple crops in modern agriculture, with roughly 1.4 billion tonnes produced annually in 2017. Organic compounds, including amino acids, sugars, and mucilage, are exuded by the roots of maize plants, thereby attracting microbes from the bulk soil to the rhizosphere. Rhizosphere microbes can have significant influences on plant growth; plant growth-promoting rhizobacteria (PGPR) have been proposed as an ecological additive to enhance plant growth (1–3). It is therefore imperative to identify functional genes that typify the rhizospheric microbes of maize for a proper understanding of the metabolic capabilities of the rhizosphere.
Replicate soil samples were collected from the rhizosphere (F4R1 and F4R2) of maize plants and the bulk soil (F4B1 and F4B2) from a farm situated in Mafikeng, South Africa (25°85′S, 25°63′33″E). The rhizosphere soil samples were collected on 17 June 2019 at 8-cm diameter and 15-cm depth of the maize plants. The soil samples were transported to the laboratory on ice and stored until further use. Genomic DNA extraction was carried out using the DNeasy PowerSoil DNA isolation kit (MoBio Laboratories, Carlsbad, CA) in accordance with the manufacturer’s directions. Using 50 ng of DNA from each sample, libraries were prepared using the Nextera DNA Flex library preparation kit (Illumina) as instructed by the manufacturer. The determination of library average insert size was done using the Agilent 2100 bioanalyzer. The libraries were pooled, diluted (to 0.6 nM), and sequenced as paired ends for 300 cycles using the NovaSeq system (Illumina).
The sequencing results are shown in Table 1. The metagenomic sequences were analyzed using the DOE Systems Biology Knowledgebase (KBase) (4). Except where otherwise stated, default parameters were used for all the software employed. For the processing of the paired-end reads, the qualities of the read libraries were examined with FastQC v0.11.5 (5), and Trimmomatic v0.36 (6) was used for the elimination of barcode sequences and sections of truncated quality from the reads. Combined assemblies were generated (F4R1 and F4R2 formed F4R, while F4B1 and F4B2 formed F4B). The metagenomes were assembled into contigs using MEGAHIT v1.2.9 (7), and coverage information was determined using Bowtie 2 v2.3.2 (8). Metagenome-assembled genomes (MAGs) were reconstructed with MaxBin 2 v2.2.4 (9). The quality of the MAGs was determined using CheckM v1.0.18 (10). Using GTDB-Tk classification, the MAGs were classified taxonomically. For F4R, a MAG classified as a member of the Actinobacteriota was generated. One MAG assigned to an unclassified taxon was reconstructed for F4B. The RAST algorithm (11) was applied for annotating the genomes, and standard features were called using Glimmer3 and Prodigal. Using Kmers v2, Kmers v1, and protein similarity, the genome features were functionally annotated. The functional analysis showed that the resulting MAGs have genes involved in plant growth promotion. The MAG was classified as Actinobacteria, a class that has been reported to possess plant growth-promoting traits (12).
TABLE 1.
Genome statistics of the maize rhizosphere and bulk soil metagenomes
| Genome characteristic | Data for sample code: |
|||
|---|---|---|---|---|
| F4R1 | F4R2 | F4B1 | F4B2 | |
| SRA accession no. | SRR12285195 | SRR12285194 | SRR12285196 | SRR12285197 |
| Raw read counts | ||||
| No. of reads (paired-end) | 26,868,696 | 20,420,846 | 25,092,166 | 20,222,698 |
| Total no. of bases | 3,467,131,872 | 2,641,232,068 | 3,121,851,578 | 2,660,693,984 |
| GC content (%) | 64.53 | 64.23 | 65.04 | 64.93 |
| Counts post-QC (no. of reads retained) | 12,906,831 | 9,730,631 | 11,942,839 | 19,336,030 |
| F4R | F4B | |||
| Counts after combined assembly formation | ||||
| No. of contigs | 1,570 | 241 | ||
| Avg length (bp) | 2,471.75 | 2,388.99 | ||
| Coverage estimate | 7.54 ± 9.85 | 7.47 ± 12.06 | ||
| N50 (bp) | 2,390 | 2,280 | ||
Data availability.
The raw sequencing data are available at the NCBI Sequence Read Archive (SRA) under BioProject PRJNA647806 with accession numbers SRR12285195 (F4R1), SRR12285194 (F4R2), SRR12285196 (F4B1), and SRR12285197 (F4B2). The metagenome assemblies and the MAGs are available at the European Nucleotide Archive (ENA) under the project number PRJEB39783 with accession numbers SAMEA7159858 (F4R assembly), SAMEA7819642 (F4B assembly), SAMEA7819643 (F4R MAG), and SAMEA7819640 (F4B MAG).
ACKNOWLEDGMENTS
O.O.B. thanks the National Research Foundation of South Africa for a grant (UID123634) that has supported work in our laboratory. R.R.M. thanks the North-West University for the postgraduate bursary that was granted during her M.Sc. program. A.E.A. is grateful to the North-West University for postdoctoral bursary and research support.
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Associated Data
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Data Availability Statement
The raw sequencing data are available at the NCBI Sequence Read Archive (SRA) under BioProject PRJNA647806 with accession numbers SRR12285195 (F4R1), SRR12285194 (F4R2), SRR12285196 (F4B1), and SRR12285197 (F4B2). The metagenome assemblies and the MAGs are available at the European Nucleotide Archive (ENA) under the project number PRJEB39783 with accession numbers SAMEA7159858 (F4R assembly), SAMEA7819642 (F4B assembly), SAMEA7819643 (F4R MAG), and SAMEA7819640 (F4B MAG).