基于宏基因组学技术检测全株玉米青贮期间和暴露空气后的微生物多样性

Microbial Diversity of Whole-Plant Maize during Ensilage and after Air Exposure Analyzed by Metagenomics Technology

本试验旨在分析全株玉米青贮发酵期间和暴露空气后的发酵品质及微生物多样性,监测其微生物群落组成动态变化。试验分别在3个发酵时期取样,即青贮第5天(F5组)、青贮第40天(F40组)和青贮40 d开袋暴露空气后第3天(A3组),每次取3袋作为3个重复。采用宏基因组学技术,测定全株玉米青贮发酵期间和暴露空气后微生物的16S r DNA V3~V4区序列,比较3个发酵时期样品微生物群落的组成和丰度信息,通过Alpha多样性和主成分分析,考察和比较全株玉米青贮发酵期间和暴露空气后的微生物多样性。结果表明:全株玉米经过40 d的青贮发酵,可使p H及中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)含量显著下降(P<0.05),乳酸含量显著升高(P<0.05),具有良好的发酵品质和营养价值,且发酵40 d后短时间开袋暴露空气对其发酵品质和营养价值无显著影响(P>0.05)。3组样本通过Illumina Miseq测序平台共获得122 371条高质量有效序列,聚类为239个操作分类单元,经分类学鉴定分属16个门,163个属。在门水平上,厚壁菌门(Firmicutes)始终占优势地位,其丰度在F5、F40和A3组中分别为57.57%、74.65%、78.82%,呈增长趋势。发酵前期(F5组)、发酵后期(F40组)和开袋期(A3组)优势菌属均为乳杆菌属(Lactobacillus),其丰度分别为49.78%、64.46%和45.34%,但在开袋期芽孢乳杆菌属(Sporolactobacillus)的比例上升明显,至28.46%。综上,全株玉米青贮自然发酵升高了产乳酸乳杆菌属的丰度,有利于提高全株玉米青贮的发酵品质,但开袋暴露空气3 d对全株玉米青贮的微生物多样性产生了影响,而通过Miseq高通量测序技术,能够全面了解全株玉米青贮期间和暴露空气后微生物群落组成及丰度上的变化。

The objective of this experiment was to analyze the fermentation quality and microbial diversity o f whole-plant maize during ensilage and after air exposure, and to monitor the dynamic changes o f microbial community composition. There were 3 sampling time points in this experiment including fermentation at the 5th day （F5 group） , fermentation at the 40th day （F40 group） and exposed air at the 3rd day after ensilage for 40 days （A 3 group） , and each sampling time point took 3 bags as 3 replicates. Microbial 16S rD N A V 3 to V 4 re-gion of whole-plant maize during ensilage and after air exposure were sequenced by metagenomics technology, and the microbial community composition and abundance o f 3 groups were compared. Alpha diversity and prin-cipal component analysis were carried out to investigate the microbial diversity o f whole-plant maize during en-silage and after air exposure. The results showed that the contents o f p H , neutral detergent fiber （N D F ） and acid detergent fiber （ADF ） of the whole-plant maize after 40 days o f silage fermentation were significantly de-creased （P〈0.05） , and the lactic acid content was significantly increased （P〈0.05） , which had a good fer-mentation quality and nutritional value, and short-time exposed air after 40 days for silage had no significantly effects on fermentation quality and nutritional value （P〉0.05）. A total o f 122 371 high-quality effective se-quences and 239 operational taxonomic units （OT U ） were obtained by Illumina Miseq sequencing p latform, and totally 16 phyla, 163 genuses o f bacteria were identified in samples o f 3 groups. A t the level o f phylum , Firmicutes was dominant from beginning to end, and its abundance in F5, F40 and A 3 groups was 57.57% , 74.65% and 78.82% , respectively, and showed a growth trend. Dominant species o f the early stage for fer-mentation （F5 group） , the late stage for fermentation （F4 0 ） and the aerobic period （A 3 group） were Lacto-bacillus ,which abundance was 49.78% , 64.46% and 45.34% , respectively, but the proportion o f Sporolacto- bacillus in aerobic period was obvious increased by 28.46%. In conclusion, the natural fermentation o f whole- plant maize silage significantly increases the abundance o f Lactobacillus, which is beneficial to improve the fer-mentation quality of whole-plant maize silage, but exposed air at the 3rd day after ensilage has effect on the mi-crobial diversity of whole-plant maize silage. By Miseq high-throughput sequencing technology, we can fu l ly understand the changes of microbial community composition and abundance o f whole-plant maize during ensi-lage and after air exposure.