Abstract
Relative quantification 16S-seq (RQS) has drawn deeper insights into bacterial community compositions in silage. However, it provides no information on dynamics of the total amount of bacterial DNA through the ensiling process and across different treatments. In this study, bacterial compositions in alfalfa silage with and without Lactobacillus plantarum inoculation after 10 and 60days of ensiling were investigated using absolute quantification 16S-seq (AQS), and bacterial composition and its interaction with fermentation properties of silage indicated by AQS and RQS were compared. Variation in total bacterial DNA amounts across different treatments and ensiling periods was illustrated by AQS. AQS indicated higher bacterial richness indices and closer correlations of these indices with fermentation properties than RQS via spearman’s correlation analyses, as well as more taxa with significance on bacterial abundance via lefse analyses. In conclusion, AQS effectively illustrated the dynamics of bacterial communities during the ensiling process.
Highlights
Ensiling has become a global practice for forage preservation (Eikmeyer et al, 2013)
Alfalfa was harvested at early bloom stage on May 9, 2019 from Zhengzhou, Henan Province, and wilted to a dry matter (DM) of 371.57±2.39 g/kg fresh weight (FW)
Alfalfa was harvested at early bloom stage and wilted to a DM of 372 g/kg FW
Summary
Ensiling has become a global practice for forage preservation (Eikmeyer et al, 2013) It is an anaerobic microbial-based fermentation process, during which lactic acid bacteria (LAB) dominate the bacterial community and produce lactic acid (LA) for pH decline and undesirable microorganism inhibition. The development of PCR-based techniques enables us to define the microbial communities more accurately, and recent studies applying high through put sequencing technology have illustrated the dynamics and compositions of relative abundance of microorganism groups in alfalfa silage during ensiling process (Guo et al, 2018; Ogunade et al, 2018; Yang et al, 2019, 2020). Disregarding of the absolute abundance of microbial community in conventional 16S amplicon sequencing technology based on relative quantitative analyses may lead to incomprehensive interpretations (Props et al, 2017; Vandeputte et al, 2017). Humic acids in the silage samples will inhibit polymerase chain reaction by inhibiting the activity of enzymes, thereby affecting the accuracy of qPCR quantitative results of bacterial copy number
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