Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: Effects of Escherichia coli O157:H7 and silage additives
Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: Effects of Escherichia coli O157:H7 and silage additives
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DOI：
10.3168/jds.2017-12876
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发表时间：
2018-03-01
影响因子：
3.5
通讯作者：
Adesogan, A. T.
中科院分区：
文献类型：
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作者：
Ogunade, I. M.;Jiang, Y.;Adesogan, A. T.
本研究的第一个目的是研究添加或不添加化学或微生物添加剂的大肠杆菌O 157：H7对苜蓿青贮饲料中细菌多样性和组成的影响。第二个目的是研究已知和未知细菌物种的相对丰度和青贮饲料发酵质量指数之间的关联。收获干物质含量为54%的苜蓿草，切碎至理论长度为19 mm，在实验室青贮窖中进行四倍量青贮，并进行以下处理：（1）蒸馏水（对照）;（2）1 × 10（5）cfu/g E.大肠杆菌O 157：H7（EC）;（3）EC和1 × 106 cfu/g植物乳杆菌（EC+LP）;（4）EC和1 × 106 cfu/g布氏乳杆菌（EC+LB）;和（5）EC和0.22%丙酸（EC+PA）。青贮100天后，通过Illumina MiSeq平台（Illumina Inc.，San Diego，CA）并进行化学表征。总体而言，厚壁菌门（74.1 +/- 4.86%）是最主要的门，其次是变形菌门（20.4 +/- 3.80%）。相对于对照，添加E.大肠杆菌O 157：在青贮时单独的H7不影响细菌多样性或组成，但添加EC+LP或EC+LB降低了香农指数，多样性的量度（分别为3.21对2.63或2.80）。与对照组（79.5%和16.5%）和EC+LB（77.4%和18.5%）相比，EC+LP和EC+PA处理降低了厚壁菌门（69.2%和68.8%）的相对丰度，而增加了变形菌门（24.0%和24.9%）的相对丰度。与对照相比，EC+LP处理使乳酸菌、鞘氨醇单胞菌、泛菌、假单胞菌和欧文氏菌的相对丰度分别增加了426、157、200、194和163%，而片球菌、魏氏菌和甲基杆菌的相对丰度分别减少了5、436、763和250%。与对照相比，EC+LB青贮饲料中的魏氏菌（9.19%）和甲基杆菌（0.94%）的相对丰度（分别为29.7%和1.50%）也有所降低。丙酸的应用并不影响乳酸杆菌，魏氏菌，或片球菌的相对丰度。乳酸浓度与乳酸菌相对丰度呈正相关（r = 0.56），与片球菌相对丰度呈负相关（r = -0.41）。氨态氮浓度与鞘氨醇单胞菌属（r = -0.51）、泛菌属（r = -0.46）、假单胞菌属（r = -0.45）和寡养单胞菌属（r = -0.38）的相对丰度呈负相关。青贮料pH值与乳酸菌（r = -0.59）、鞘氨醇单胞菌（r = -0.66）、泛菌（r = -0.69）、假单胞菌（r =-0.69）和寡养单胞菌（r = -0.50）的相对丰度呈负相关。未来的研究应针对本研究中检测到的未知细菌进行形态鉴定、培养和功能测定，以阐明其在青贮发酵中的作用。
The first objective of this study was to examine effects of adding Escherichia coli O157:H7 with or without chemical or microbial additives on the bacterial diversity and composition of alfalfa silage. The second objective was to examine associations between the relative abundance of known and unknown bacterial species and indices of silage fermentation quality. Alfalfa forage was harvested at 54% dry matter, chopped to a theoretical length of cut of 19 mm, and ensiled in quadruplicate in laboratory silos for 100 d after the following treatments were applied: (1) distilled water (control); (2) 1 x 10(5) cfu/g of E. coli O157:H7 (EC); (3) EC and 1 x 10(6) cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1 x 10(6) cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 0.22% propionic acid (EC+PA). After 100 d of ensiling, the silage samples were analyzed for bacterial diversity and composition via the Illumina MiSeq platform (Illumina Inc., San Diego, CA) and chemically characterized. Overall, Firmicutes (74.1 +/- 4.86%) was the most predominant phylum followed by Proteobacteria (20.4 +/- 3.80%). Relative to the control, adding E. coli O157: H7 alone at ensiling did not affect bacterial diversity or composition but adding EC+LP or EC+LB reduced the Shannon index, a measure of diversity (3.21 vs. 2.63 or 2.80, respectively). The relative abundance of Firmicutes (69.2 and 68.8%) was reduced, whereas that of Proteobacteria (24.0 and 24.9%) was increased by EC+LP and EC+PA treatments, relative to those of the control (79.5 and 16.5%) and EC+LB (77.4 and 18.5%) silages, respectively. Compared with the control, treatment with EC+LP increased the relative abundance of Lactobacillus, Sphingomonas, Pantoea, Pseudomonas, and Erwinia by 426, 157, 200, 194, and 163%, respectively, but reduced those of Pediococcus, Weissella, and Methylobacterium by 5,436, 763, and 250%, respectively. Relative abundance of Weissella (9.19%) and Methylobacterium (0.94%) were also reduced in the EC+LB silage compared with the control (29.7 and 1.50%, respectively). Application of propionic acid did not affect the relative abundance of Lactobacillus, Weissella, or Pediococcus. Lactate concentration correlated positively (r = 0.56) with relative abundance of Lactobacillus and negatively (r = -0.41) with relative abundance of Pediococcus. Negative correlations were detected between ammonia-N concentration and relative abundance of Sphingomonas (r = -0.51), Pantoea (r = -0.46), Pseudomonas (r = -0.45), and Stenotrophomonas (r = -0.38). Silage pH was negatively correlated with relative abundance of Lactobacillus (r = -0.59), Sphingomonas (r = -0.66), Pantoea (r = -0.69), Pseudomonas (r = -0.69), and Stenotrophomonas (r = -0.50). Future studies should aim to speciate, culture, and determine the functions of the unknown bacteria detected in this study to elucidate their roles in silage fermentation.