Bacterial communities in vetch-oat silages with the addition of microbial inoculants

Abstract

Metagenomic approaches by means of direct DNA extraction and subsequent analyses provide the opportunity to better understand the processes underlying silage formation and estimate the effects of applied starter cultures on microbial population shifts. Consequently, our objective was to acquire comprehensive insights into the bacterial communities within vetch-oat silages by utilising 16S gene data. Vetch-oat forage was ensiled in the experimental field of EEA INTA Rafaela without microbial inoculant as control; with a native spray-dried inoculant containing Lactiplantibacillus plantarum Hv75, Pediococcus acidilactici 3903, and Lentilactobacillus buchneri B463; or with a commercial freeze-dried inoculant. Bucket silos were compacted, sealed and stored at 25 °C. DNA isolation from silage samples was made in duplicates for the raw forage (day 0), after 30 and 60 days of fermentation, and for vetch-oat samples subjected to an aerobic stability evaluation. Total DNA was extracted using a ZymoBIOMICS™ DNA Miniprep kit and the PCR was conducted using the 16S barcoding kit 1-24. Sequencing was carried out with the MinION Mk1C instrument. Last, R software v 4.2.2 was employed to assess composition and bacterial diversity through the treatments. Statistical significance was established at P ≤ 0.05. Pseudomonas was the predominant genus in fresh samples (P < 0.001). At day 30, 60 and after exposure to aerobic conditions, the abundance of Lactobacillus in untreated silage was lower than that in inoculated samples (P < 0.001). Moreover, the uninoculated silage was characterised by the emergence of Staphylococcus (P < 0.001). Besides, inoculation significantly reduced bacterial alpha diversity (P < 0.05).