Applied Microbiology and Biotechnology

, Volume 101, Issue 4, pp 1385–1394 | Cite as

Comparative microbiota assessment of wilted Italian ryegrass, whole crop corn, and wilted alfalfa silage using denaturing gradient gel electrophoresis and next-generation sequencing

  • Kuikui Ni
  • Tang Thuy Minh
  • Tran Thi Minh Tu
  • Takeshi Tsuruta
  • Huili Pang
  • Naoki NishinoEmail author
Biotechnological products and process engineering


The microbiota of pre-ensiled crop and silage were examined using denaturing gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS). Wilted Italian ryegrass (IR), whole crop corn (WC), and wilted alfalfa (AL) silages stored for 2 months were examined. All silages contained lactic acid as a predominant fermentation product. Across the three crop species, DGGE detected 36 and 28 bands, and NGS identified 253 and 259 genera in the pre-ensiled crops and silages, respectively. The NGS demonstrated that, although lactic acid bacteria (LAB) became prevalent in all silages after 2 months of storage, the major groups were different between crops: Leuconostoc spp. and Pediococcus spp. for IR silage, Lactobacillus spp. for WC silage, and Enterococcus spp. for AL silage. The predominant silage LAB genera were also detected by DGGE, but the presence of diverse non-LAB species in pre-ensiled crops was far better detected by NGS. Likewise, good survival of Agrobacterium spp., Methylobacterium spp., and Sphingomonas spp. in IR and AL silages was demonstrated by NGS. The diversity of the microbiota described by principal coordinate analysis was similar between DGGE and NGS. Our finding that analysis of pre-ensiled crop microbiota did not help predict silage microbiota was true for both DGGE and NGS.


Denaturing gradient gel electrophoresis Fermentation Microbiota Next-generation sequencing Silage 


Compliance with ethical standards


This work was supported by the Ministry of Agriculture, Forestry, and Fisheries of Japan (Grant No. 26081C).

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Department of Animal Science, Graduate School of Environmental and Life ScienceOkayama UniversityOkayamaJapan
  2. 2.Henan Provincial Key Laboratory of Ion Beam Bio-engineeringZhengzhou UniversityZhengzhouChina

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