Peribacillus castrilensis sp. nov.: A Plant-Growth-Promoting and Biocontrol Species Isolated From a River Otter in Castril, Granada, Southern Spain

Abstract

A strictly aerobic, chemoheterotrophic, endospore-forming, Gram-positive, rod-shaped bacterial strain N3^T was isolated from the feces of a river otter in Castril (Granada, southern Spain). It is halotolerant, motile, and catalase-, oxidase-, ACC deaminase-, and C4- and C8-lipase-positive. It promotes tomato plant growth and can reduce virulence in Erwinia amylovora CECT 222^T and Dickeya solani LMG 25993^T through interference in their quorum-sensing systems, although other antagonistic mechanisms could also occur. A phylogenetic analysis of the 16S rRNA gene sequence as well as the phenotypic and phylogenomic analyses indicated that the strain N3^T is a novel species of the genus Peribacillus, with the highest 16S rRNA sequence similar to that of Bacillus frigoritolerans DSM 8801^T (99.93%) and Peribacillus simplex DSM 1321^T (99.80%). Genomic digital DNA-DNA hybridization (dDDH) between the strain N3^T and Bacillus frigoritolerans DSM 8801^T and Peribacillus simplex was 12.8 and 69.1%, respectively, and the average nucleotide identity (ANIb) of strain N3^T and Bacillus frigoritolerans DSM 8801^T and Peribacillus simplex was 67.84 and 93.21%, respectively. The genomic G + C content was 40.3 mol%. Its main cellular fatty acids were anteiso-C_15:0 and iso-C_15:0. Using 16S rRNA phylogenetic and in silico phylogenomic analyses, together with the chemotaxonomic and phenotypic data, we demonstrated that the type strain N3^T (=CECT 30509^T = LMG 32505^T) is a novel species of the genus Peribacillus and the name Peribacillus castrilensis sp. nov. is proposed.

Keywords: Peribacillus castrilensis; new species; phytopathogen; plant-growth promoting species; quorum quenching; river otter.

Figure 1

Phylogenetic position of the strain N3^T the 16S rRNA gene sequence (bold) and its relationship with other related species by using the maximum-likelihood algorithm based on the Kimura 2-parameter model. A discrete Gamma distribution was used to model evolutionary rate differences among sites [five categories (+G, parameter = 0.2703)]. The GenBank/EMBL/DDBJ accession number of each sequence is shown in parenthesis. Bootstrap values are expressed as percentages of 1,000 replications, and those >60% are shown at branch points. Bar shows sequence divergence. Bar−0.01 substitutions per nucleotide position. B. brevis DSM 30^T sequence was used as an outgroup.

Figure 2

Tree constructed according to the maximum-likelihood method based on 803 core orthologous proteins of the strain N3^T (bold) and the available genomes of Peribacillus species and representative strains of the related genera. Bootstrap values are expressed as percentages of 1,000 replications, and those over 60% are shown at branch points. Bar−0.05 substitutions per nucleotide position.

Figure 3

Determination of N3^T quorum quenching activity on synthetic AHLs. C. violaceum CV026, C. violaceum VIR07, and A. tumefaciens NTL4 (pZLR4) were used as biosensors in LB and AB-Xgal media, respectively.

Figure 4

Plant growth-promoting activity of the strain N3^T in tomato plants with respect to control plants treated with water. *statistically significant difference (p < 0.01).

Figure 5

N3^T strain interference in D. solani and E. amylovora virulence on potato slices and pears (A). Determination of N3^T quorum quenching activity on AHLs of D. solani and E. amylovora in AB-Xgal medium using A. tumefaciens NTL4 as biosensor (B).