Epiphytic microbiota in Swedish grass-clover herbage and the effect of silage additives on fermentation profiles and bacterial community compositions of the resulting silages.

Abstract

To investigate the epiphytic microbiota in grass-clover herbage harvested at different sites and occasions; and to explore the effect of different silage additives on the resulting silage microbiota. Herbage was harvested from grass-clover leys at geographically distributed sites in a long-term field experiment in Sweden, in early and late season of two consecutive years. Different silages were made from the herbage using; 1) no additive, 2) acid-treatment, and 3) inoculation by starter culture. Herbages were analyzed for botanical and chemical composition, and the resulting silages for products of fermentation. Bacterial DNA was extracted from herbage and silage samples, followed by sequencing using Illumina 16S rRNA amplicon sequencing. Herbage microbiota showed no clear correlation to site or harvesting time. Silage additives had a major effect on the ensiling process; inoculation resulted in well fermented silages comprising a homogenous microbiota dominated by the genera Lactobacillus and Pediococcus. A minor effect of harvest time was also observed, with generally a more diverse microbiota in second harvest silages. Untreated silages showed a higher relative abundance from non-lactic acid bacteria compared to acid treated silages. In most silages, only a few bacterial amplicon sequence variants contributed to most of the relative abundance. The epiphytic microbiota in grass-clover herbage were found to be random and not dependent on site. From a microbial point of view, the most predictable and preferable silage outcome was obtained by inoculation with a starter culture. Acid-treatment with formic- and propionic acid surprisingly resulted in a less preferable silage. Silage making without additives cannot be recommended based on our results.