Enhanced specificity of Bacillus metataxonomics using a tuf-targeted amplicon sequencing approach

Abstract

Bacillus species are ubiquitous in nature and have tremendous application potential in agriculture, medicine, and industry. However, the individual species of this genus vary widely in both ecological niches and functional phenotypes, which, hence, requires accurate classification of these bacteria when selecting them for specific purposes. Although analysis of the 16S rRNA gene has been widely used to disseminate the taxonomy of most bacterial species, this gene fails proper classification of Bacillus species. To circumvent this restriction, we designed novel primers and optimized them to allow exact species resolution of Bacillus species in both synthetic and natural communities using high-throughput amplicon sequencing. The primers designed for the tuf gene were not only specific for the Bacillus genus but also sufficiently discriminated species both in silico and in vitro in a mixture of 11 distinct Bacillus species. Investigating the primers using a natural soil sample, 13 dominant species were detected including Bacillus badius, Bacillus velezensis, and Bacillus mycoides as primary members, neither of which could be distinguished with 16S rRNA sequencing. In conclusion, a set of high-throughput primers were developed which allows unprecedented species-level identification of Bacillus species and aids the description of the ecological distribution of Bacilli in various natural environment.

How to identify and quantify the members of the Bacillus genus?

“…For a primary delineation, Dunlap (2019a) suggested gyrA or gyrB to be used for the B. subtilis group, and pycA for the B. cereus group. Recently, we thoroughly analysed primer sets frequently employed in literature and revealed that gyrA and rpoB-based primers have high intra-species specificity within the B. subtilis group (Xu et al, 2023). Surprisingly, gyrB-based degenerated primers had no amplification of certain Bacillus genomes which prompted the doubts about phylogenetic discrimination capacity of gyrB.…”

“…It is challenging to find genes that contain a highly variable region that can be used universally for Bacillus species or sub-species identification and flanked by highly conserved regions that can serve as binding sites for amplicon primers. Recently, a tuf-targeted amplicon sequencing approach was developed which exhibits the highest coverage of Bacillus species diversity reported to date with high specificity (Xu et al, 2023). It allows precise species resolution of the Bacillus community in natural soil communities and could be potentially applied to track the persistence of Bacillus inoculant in the field.…”

Antagonistic activity of two Bacillus strains against Fusarium oxysporum f. sp. capsici (FOC-1) causing Fusarium wilt and growth promotion activity of chili plant

“…Biological control is an alternative method that promotes sustainable and environmentally friendly agricultural practices (Iqbal et al, 2023b). Trichoderma, Paecilomyces, Bacillus, and Pseudomonas are most four acceptable genera, comprising more than 200 species which have exhibited remarkable abilities to control a large number of plant diseases while promoting plant growth (Peix et al, 2009;Rivas et al, 2009;Cai et al, 2022;Xu et al, 2023). Out of these, Bacillus is one of the most promising biocontrol genus in the agriculture field, controlling soil-borne diseases in many crops (Yuan et al, 2012;Sun et al, 2023), which is also confirmed as plant growth-promoting bacteria (PGPB).…”