Lacticaseibacillus parahuelsenbergensis sp. nov., Lacticaseibacillus styriensis sp. nov. and Lacticaseibacillus zeae subsp. silagei subsp. nov., isolated from different grass and corn silage.

Four rod-shaped, non-motile, non-spore-forming, facultative anaerobic, Gram-stain-positive lactic acid bacteria, designated as EB0058(T), SCR0080, LD0937(T) and SCR0063(T), were isolated from different corn and grass silage samples. The isolated strains were characterized using a polyphasic approach and EB0058(T) and SCR0080 were identified as Lacticaseibacillus zeae by 16S rRNA gene sequence analysis. Based on whole-genome sequence-based characterization, EB0058(T) and SCR0080 were separated into a distinct clade from Lacticaseibacillus zeae DSM 20178(T), together with CECT9104 and UD2202, whose genomic sequences are available from NCBI GenBank. The average nucleotide identity (ANI) values within the new subgroup are 99.9 % and the digital DNA-DNA hybridization (dDDH) values are 99.3-99.9 %, respectively. In contrast, comparison of the new subgroup with publicly available genomic sequences of L. zeae strains, including the type strain DSM 20178(T), revealed dDDH values of 70.2-72.5 % and ANI values of 96.2-96.6 %. Based on their chemotaxonomic, phenotypic and phylogenetic characteristics, EB0058(T) and SCR0080 represent a new subspecies of L. zeae. The name Lacticaseibacillus zeae subsp. silagei subsp. nov. is proposed with the type strain EB0058(T) (=DSM 116376(T)=NCIMB 15474(T)). According to the results of 16S rRNA gene sequencing, LD0937(T) and SCR0063(T) are members of the Lacticaseibacillus group. The dDDH value between the isolates LD0937(T) and SCR0063(T) was 67.6 %, which is below the species threshold of 70 %, clearly showing that these two isolates belong to different species. For both strains, whole genome-sequencing revealed that the closest relatives within the Lacticaseibacillus group were Lacticaseibacillus huelsenbergensis DSM 115425 (dDDH 66.5 and 65.9 %) and Lacticaseibacillus casei DSM 20011(T) (dDDH 64.1 and 64.9 %). Based on the genomic, chemotaxonomic and morphological data obtained in this study, two novel species, Lacticaseibacillus parahuelsenbergensis sp. nov. and Lacticaseibacillus styriensis sp. nov. are proposed and the type strains are LD0937(T) (=DSM 116105(T)=NCIMB 15471(T)) and SCR0063(T) (=DSM 116297(T)=NCIMB 15473(T)), respectively.