双孢蘑菇发酵培养料细菌多样性分析

袁滨; 柯丽娜; 连燕萍; 赵光辉; 冯伟林; 蔡为明

doi:10.19303/j.issn.1008-0384.2022.009.018

双孢蘑菇发酵培养料细菌多样性分析

doi: 10.19303/j.issn.1008-0384.2022.009.018

1.
漳州市农业科学研究所，福建　漳州　363005
2.
福州市农业科学研究所，福建　福州　350014
3.
浙江省农业科学院园艺研究所，浙江　杭州　310021

基金项目: 国家现代农业产业技术体系建设专项（CARS-20）；福建省科技计划农业引导性（重点）项目（2019N0052）

详细信息

作者简介:
袁滨（1984−），男，硕士，副研究员，研究方向：食药用菌育种和栽培（E-mail：yuanbin07031@163.com）

通讯作者:
蔡为明（1967−），男，博士，研究员，研究方向：食药用菌育种、生理生态及栽培技术（E-mail：caiwm527@126.com）

中图分类号: S 646
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出版历程
- 收稿日期: 2022-04-02
- 修回日期: 2022-06-20
- 网络出版日期: 2022-10-05

Changes of Microbial Community in Agaricus bisporus Culture Substrate during Fermentation

1.
Zhangzhou Agricultural Scientific Research Institute, Zhangzhou, Fujian　363005, China
2.
Fuzhou Institute of Agricultural Sciences, Fuzhou, Fujian　350014, China
3.
Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang　310021, China

摘要

摘要: 目的了解双孢蘑菇培养料发酵过程中微生物的群落动态变化趋势及其发挥的作用。方法以优化的复合菌渣（金针菇和杏鲍菇菌渣）作为双孢蘑菇培养料的主要成分，采用PacBio平台、高通量 16S rDNA全长测序方法，分析双孢蘑菇培养料由建堆、第一次发酵、第二次发酵过程中的7个阶段（Ag1~Ag7）的细菌群落特征。结果在7个阶段的发酵培养料中共获得的OUT数量分别为328、340、294、377、364、166、174个，共计715个，其中有161个 OTU 存在于发酵的7个阶段，涵盖了21 门 299 属399种的细菌。Fimicutes（厚壁菌门）、Proteobacteria（变形菌门）、Bacteroidetes（拟杆菌门），Gemmatimonadetes（芽单胞菌门）在7个阶段中丰度较高。在建堆和第一次发酵过程中Ureibacillus（解脲芽孢杆菌属）为主要优势类群，在第二次发酵过程中Limnochordaceae、S0134 terrestrial group、Thermobacillus（嗜热杆菌属）、Ruminiclostridium（瘤胃梭菌属）的相对丰度更高。在种分类水平，Ureibacillus thermophilus和Ureibacillus terrenus是建堆和第一次发酵过程中的优势菌种，Limnochordaceae属的菌种在第二次发酵中相对丰度最高。上述研究结果表明：在第二次发酵之前细菌种类和丰度随着发酵过程不断升高，第一次发酵和第二次发酵样本间细菌群落结构差异较大，并在二次发酵后显著降低，而且这些优势菌群主要参与物质降解，从而提高了双孢蘑菇培养料质量。结论通过全长测序的方法能更好地在种水平对不同发酵阶段的优势菌种进行鉴定，同时还发现了很多未分类的细菌物种，为优化发酵培养料和提高双孢蘑菇产量提供了理论依据。
- 双孢蘑菇 /
- 培养料 /
- 发酵 /
- 16S全长扩增子测序 /
- 细菌群落
Abstract: Objective Changes of the microbial community in fermentation of substrates for cultivating Agaricus bisporus were investigated. Method The microbial community characteristics of a compound A. bisporus culture substrates containing spent Flammulina velutiper and Pleurotus eryngii materials were monitored during the 7 stages, Ag1 to Ag7 from pile building to 1^st and 2^nd fermentation, using the high throughput 16S rDNA full-length sequencing of Pacbio platform. Result A total of 715 OTUs were obtained in the fermentation process (i.e., 328, 340, 294, 377, 364, 166, and 174 for each stage) with 161 OTUs commonly found in all stage. The microbes included 21 phyla, 299 genera, and 399 species. At phylum level, Fimicutes, Proteobacteria, Bacteroidetes, and Gemmatimonadetes had higher abundance in all 7 stages. The dominant genus in the substrate during the pile building and 1^st fermentation stages was Ureibacillus, while Limnochordaceae, S0134_terrestrial_group, Thermobacillu, and Ruminiclostridium stood out in the 2^nd fermentation stage. At species level, Ureibacillus thermophilus and Ureibacillus terrenus were dominant in the pile building and 1^st fermentation stages. In the 2^nd fermentation stage, the species in the genus of Limnochordaceae were the greatest in relative abundance. It appeared that the microbial abundance and diversity increased gradually before the onset of 2^nd fermentation. The community structure of the substrate varied significantly between the 1^st and the 2^nd fermentation, but the deviation diminished significantly after the 2^nd fermentation leaving mostly the dominant species that basically functioned to degrade the waste mushroom material enriching the fertilization effect. Conclusion The full-length sequencing technology clearly identified the dominant species unveiling many previously unclassified microorganisms. The results helped to better optimize the substrate fermentation process for an improved cultivation of A. bisporus.
- Agaricus bisporus /
- culture substrates /
- fermentation /
- 16s full-length sequencing /
- bacterial community

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图 1 各样品 OTUs 稀释曲线

Figure 1. Rarefaction curves on OTUs of individual samples

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图 2 Alpha多样性指数组间差异柱状图

A−B：不同样品的ACE和Chao1指数，纵坐标表示样品中的OUT数目；C−E: 不同样品的PD_whole_tree，Shannon和Simpson指数，纵坐标表示样品中物种多样性。*. 差异显著 (P＜0.05)； **. 差异极显著 (P＜0.01)。

Figure 2. Histogram of alpha diversity index

A-B: ACE and Chao1 indices of sample, Y-axis for number of OUT; C-E: PD_whole_tree and Shannon and Simpson indices of sample, respectively, Y-axis for species diversity; *: Significant difference at P＜0.05; **: Extremely significant difference at P＜0.01.

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图 3 Beta 多样性的主坐标分析(PCoA)

Figure 3. Principal coordinate analysis plot of beta diversity scores

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图 4 在门水平细菌群落的相对丰度

Figure 4. Relative abundance of microbial communities at phylum level

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图 5 在属水平细菌群落的相对丰度

Figure 5. Relative abundance of microbial communities at genus level

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图 6 在种水平细菌群落的相对丰度

Figure 6. Relative abundance of microbial communities at species level

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表 1 培养料理化指标

Table 1. Physiochemical indicators of substrate

编号 Sample ID	取样时期 Sampling period	干重 Dry weight/g	水分 Moisture	pH
Ag1	建堆	47.07	73.92%	5.16
Ag2	一次翻堆	50.00	71.85%	5.35
Ag3	二次翻堆	50.48	72.26%	5.66
Ag4	三次翻堆	34.8	71.58%	5.55
Ag5	四次翻堆	52.24	71.56%	6.71
Ag6	二次发酵巴氏消毒结束	28.42	66.50%	8.36
Ag7	二次发酵全过程结束	43.95	62.08%	7.64

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