羊肚菌菌种生产车间气溶胶微生物群落研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0702

摘要/Abstract

摘要：

为研究羊肚菌菌种生产车间的气溶胶微生物群落动态变化，从菌种生产源头寻找影响羊肚菌安全生产的环境微生物因素。利用高通量扩增子测序对羊肚菌菌种大生产期间的接种室、原种室和栽培种室的气溶胶微生物群落进行监测。结果发现，随着菌种扩繁级别增加，扩繁车间空气中的真菌多样性显著升高，而细菌多样性无显著变化。车间的真菌群落以子囊菌门为主，细菌群落以变形菌门、厚壁菌门和放线菌门为主。假单胞菌属和乳杆菌属在3种扩繁车间的细菌群落均占主导地位，其比例随着菌种级别升高呈上升趋势；Phialemoniopsis sp.主导了3种扩繁车间的真菌群落，比例均超过96%。Phialemoniopsis sp.与羊肚菌不出菇的潜在风险因子——枝顶孢属具有极高的系统发育关系相关性，表明它可能影响羊肚菌生产的风险生态因子之一。研究为羊肚菌菌种安全生产的环境条件需求提供了理论补充。

关键词: 气溶胶微生物, 羊肚菌, 多样性, 假单胞菌, 菌种生产

Abstract:

This work is aimed to dissect the dynamic changes of aerosol microbial community in the production workshops of morel spawn and to find the environmental microbial factors affecting safe production of morel from the source of spawn production. High-throughput amplicon sequencing was used to monitor the aerosol microbial community in the inoculation room, original spawn cultivation room and cultivated spawn cultivation room during large-scale production of morel spawn. The results showed that the fungal diversity in the aerosol of the workshop increased significantly along with the increasing of amplification grade during morel-spawn production, while the bacterial diversity did not change significantly. The fungal community was dominated by Ascomycota, and the bacterial community was dominated by Proteobacteria, Firmicutes, and Actinomycetes. The bacterial communities of Pseudomonas and Lactobacillus were dominant in all types of workshops, and their proportions increased with the amplification level of morel strains. Phialemoniopsis sp. dominated the fungal communities in all types of workshops, with the proportions over 96%. There was a very high phylogenetic relationship between Phialemoniopsis sp. and Acremonium, a potential risk factor causing morel fructification failure, indicating that it may be one of the risk ecological factors affecting morel production. Our findings provide theoretical supplementation for environmental conditions required for safe production of morel spawn.

Key words: aerosol microorganism, morel, diversity, Pseudomonas, spawn production

余洋, 刘天海, 刘理旭, 唐杰, 彭卫红, 陈阳, 谭昊. 羊肚菌菌种生产车间气溶胶微生物群落研究[J]. 生物技术通报, 2023, 39(5): 267-275.

YU Yang, LIU Tian-hai, LIU Li-xu, TANG Jie, PENG Wei-hong, CHEN Yang, TAN Hao. Study on Aerosol Microbial Community in the Production Workshop of Morel Spawn[J]. Biotechnology Bulletin, 2023, 39(5): 267-275.

图/表 6

图1 3种扩繁车间气溶胶中真菌和细菌群落的α-多样性估计（A）和基于主坐标分析（PCoA）的真菌和细菌群落β多样性估计（B）

Fig. 1 α-diversity estimation (A) and β-diversity estimation (B) based on principal coordinate analysis（PCoA）of the fungal and bacterial communities in aerosol of three types of workshops

图2 气溶胶微生物群落组成

Fig. 2 Composition of aerosol microbial communities

图3 真菌和细菌属的热图（显示样本中前 20 个主要的属）

Fig. 3 Heatmap of fungal and bacterial genera (showing the top 20 prominent genera in the samples)

图4 3个车间的气溶胶微生物群落中主要细菌（A）和真菌属（B）的相对丰度柱子上相同的字母表示没有显著性差异（P>0.05）

Fig. 4 Relative abundance of the major bacterial (A)and fungal (B) genera in the aerosol microbial communities of 3 workshops The same letters on the column indicate no significant difference（P>0.05）

图5 扩繁车间主要的真菌和细菌属的相对丰度（A，B）和标志性类群（C，D）

Fig. 5 Relative abundances of fungal and bacterial (A, B) phyla and biomarker taxa (C, D) in the strain production workshop

图6 基于ITS序列的系统发育分析

Fig. 6 Phylogenetic analysis based on ITS sequence

参考文献 35

[1]	彭卫红, 唐杰, 何晓兰, 等. 四川羊肚菌人工栽培的现状分析[J]. 食药用菌, 2016, 24(3): 145-150.
	Peng WH, Tang J, He XL, et al. Status analysis of morel artificial cultivation in Sichuan[J]. Edible Med Mushrooms, 2016, 24(3): 145-150.
[2]	谭昊, 刘天海, 闫世杰, 等. 羊肚菌栽培对沙漠砂基质中微生物群落及基质理化性质的影响[J]. 生物技术通报, 2021, 37(11): 166-177. doi: 10.13560/j.cnki.biotech.bull.1985.2021-1110
	Tan H, Liu TH, Yan SJ, et al. Impacts of morel cultivation on the microbial community and physiochemical characteristics in a substratum of desert sand[J]. Biotechnol Bull, 2021, 37(11): 166-177. doi: 10.13560/j.cnki.biotech.bull.1985.2021-1110
[3]	唐杰, 王勇, 许瀛引, 等. 羊肚菌产业发展关键问题研究进展[J]. 菌物研究, 2021, 19(4): 217-231.
	Tang J, Wang Y, Xu YY, et al. Research progress on key issues of morel industry development[J]. J Fungal Res, 2021, 19(4): 217-231.
[4]	刘天海, 羊淑琴, 刘付彭, 等. 麦秸鸡粪发酵有机肥对六妹羊肚菌连作的影响[J]. 生物技术通报, 2022, 38(12): 263-273. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0123
	Liu TH, Yang SQ, Liu FP, et al. Effect of organic fertilizers fermented with wheat straw and chicken manure on continuous cultivation of Morchella sextelata[J]. Biotechnol Bull, 2022, 38(12): 263-273.
[5]	谭昊, 苗人云, 刘天海, 等. 成都平原地区梯棱羊肚菌人工栽培生态环境参数范围及变化规律[J]. 西南农业学报, 2021, 34(1): 27-39.
	Tan H, Miao RY, Liu TH, et al. Ranges and dynamics of environmental factors for Morchella importuna cultivation in Chengdu plain area[J]. Southwest China J Agric Sci, 2021, 34(1): 27-39.
[6]	Du XH, Yang ZL. Mating systems in true morels(Morchella)[J]. Microbiol Mol Biol Rev, 2021, 85(3): e0022020. doi: 10.1128/MMBR.00220-20 URL
[7]	贺新生, 王茂辉, 王珏, 等. 羊肚菌菌种规模化生产技术[J]. 食药用菌, 2020, 28(5): 346-350.
	He XS, Wang MH, Wang J, et al. The large-scale production technology of Morel spawn[J]. Edible Med Mushrooms, 2020, 28(5): 346-350.
[8]	Liu QZ, Ma HS, Zhang Y, et al. Artificial cultivation of true morels: current state, issues and perspectives[J]. Crit Rev Biotechnol, 2018, 38(2): 259-271. doi: 10.1080/07388551.2017.1333082 pmid: 28585444
[9]	顾红政, 曹卫平. 牙科手机高速旋转形成的气溶胶与医院感染关系研究[J]. 口腔生物医学, 2011, 2(3): 145-147.
	Gu HZ, Cao WP. Levels of bacterial aerosols and their relationship with hospital infection by dental handpieces[J]. Oral Biomed, 2011, 2(3): 145-147.
[10]	He XL, Peng WH, Miao RY, et al. White mold on cultivated morels caused by Paecilomyces penicillatus[J]. FEMS Microbiol Lett, 2017, 364(5): 2017 Mar 1;364(5).
[11]	刘天海, 周洁, 王迪, 等. 一种六妹羊肚菌的新型柄腐病害[J]. 菌物学报, 2021, 40(9): 2229-2243.
	Liu TH, Zhou J, Wang D, et al. A new stipe rot disease of the cultivated Morchella sextelata[J]. Mycosystema, 2021, 40(9): 2229-2243.
[12]	陈诚, 李强, 王剑, 等. 羊肚菌烂柄病发生对土壤真菌群落结构的影响[J]. 微生物学杂志, 2018, 38(5): 39-45.
	Chen C, Li Q, Wang J, et al. Effects of morel(Morchella sp.) stipe rot disease occurrence on soil fungal community structure[J]. J Microbiol, 2018, 38(5): 39-45.
[13]	Tan H, Liu TH, Yu Y, et al. Morel production related to soil microbial diversity and evenness[J]. Microbiol Spectr, 2021, 9(2): e0022921. doi: 10.1128/Spectrum.00229-21 URL
[14]	Yu Y, Liu TH, Liu LX, et al. Application of the mushroom volatile 1-octen-3-ol to suppress a morel disease caused by Paecilomyces penicillatus[J]. Appl Microbiol Biotechnol, 2022, 106(12): 4787-4799. doi: 10.1007/s00253-022-12038-2
[15]	Noble R, Dobrovin-Pennington A, Hobbs PJ, et al. Volatile C8 compounds and pseudomonads influence primordium formation of Agaricus bisporus[J]. Mycologia, 2009, 101(5): 583-591. doi: 10.3852/07-194 URL
[16]	Pion M, Spangenberg JE, Simon A, et al. Bacterial farming by the fungus Morchella crassipes[J]. Proc Biol Sci, 2013, 280(1773): 20132242.
[17]	蔡婧, 常堃, 边银丙, 等. 通过动态监测的方法分析金针菇工厂洁净区空气微生物状况[J]. 食用菌学报, 2020, 27(3): 52-60.
	Cai J, Chang K, Bian YB, et al. Dynamic monitoring of airborne microorganisms in clean area of Flammulina filiformis factory[J]. Acta Edulis Fungi, 2020, 27(3): 52-60.
[18]	施维属, 潘腾飞, 钟凤林, 等. 柑橘基因组DNA快速提取及ISSR-PCR扩增体系优化[J]. 生物技术通报, 2009(10): 109-113.
	Shi WS, Pan TF, Zhong FL, et al. Improvement of genomic DNA extraction method and optimization of ISSR-PCR amplification for Citrus[J]. Biotechnol Bull, 2009(10): 109-113.
[19]	尹国丽, 李亚娟, 张振粉, 等. 不同草田轮作模式土壤养分及细菌群落组成特征[J]. 生态学报, 2020, 40(5): 1542-1550.
	Yin GL, Li YJ, Zhang ZF, et al. Characteristics of soil nutrients and bacterial community composition under different rotation patterns in grassland[J]. Acta Ecol Sin, 2020, 40(5): 1542-1550.
[20]	赵庆庆, 解金昆, 高永超, 等. 不同水文条件下黄河口滨海湿地土壤真菌群落的分布特征[J]. 环境科学学报, 2022, 42(1): 95-103.
	Zhao QQ, Xie JK, Gao YC, et al. The distribution pattern of soil fungal community in coastal wetlands with different hydrologic conditions in the Yellow River Estuary[J]. Acta Sci Circumstantiae, 2022, 42(1): 95-103.
[21]	Tan H, Yu Y, Tang J, et al. Build your own mushroom soil: microbiota succession and nutritional accumulation in semi-synthetic substratum drive the fructification of a soil-saprotrophic morel[J]. Front Microbiol, 2021, 12: 656656. doi: 10.3389/fmicb.2021.656656 URL
[22]	Caporaso JG, Kuczynski J, Stombaugh J, et al. QIIME allows analysis of high-throughput community sequencing data[J]. Nat Methods, 2010, 7(5): 335-336. doi: 10.1038/nmeth.f.303 pmid: 20383131
[23]	Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads[J]. Nat Methods, 2013, 10(10): 996-998. doi: 10.1038/nmeth.2604 pmid: 23955772
[24]	Gdanetz K, Benucci GMN, Vande Pol N, et al. CONSTAX: a tool for improved taxonomic resolution of environmental fungal ITS sequences[J]. BMC Bioinformatics, 2017, 18(1): 538. doi: 10.1186/s12859-017-1952-x pmid: 29212440
[25]	Segata N, Izard J, Waldron L, et al. Metagenomic biomarker discovery and explanation[J]. Genome Biol, 2011, 12(6): R60. doi: 10.1186/gb-2011-12-6-r60 URL
[26]	张静, 徐武敏, 张兰珍, 等. 血液透析室空气微生物气溶胶分布与医院感染的相关性及健康风险评价[J]. 中华医院感染学杂志, 2022, 32(8): 1261-1265.
	Zhang J, Xu WM, Zhang LZ, et al. Distribution characteristics of microbial aerosol in air of hemodialysis room and its correlation with nosocomial infection and health risk assessment[J]. Chin J Nosocomiology, 2022, 32(8): 1261-1265.
[27]	陈露安, 杨传钧, 谷雨倩, 等. 超声波加湿室内微生物气溶胶浓度与优化方法[J]. 中国环境科学, 2022, 42(4): 1594-1600.
	Chen L, Yang CJ, Gu YQ, et al. Impacts of ultrasonic humification on size-distribution and concentration variations of indoor bioaerosol and its optimization strategy[J]. China Environ Sci, 2022, 42(4): 1594-1600.
[28]	陈锷, 褚可成, 罗涛, 等. 兰州市不同季节大气微生物气溶胶粒径及分布特征研究[J]. 中国环境监测, 2022, 38(2): 85-95.
	Chen E, Chu KC, Luo T, et al. Study on the particle size and distribution characteristics of atmospheric microbial aerosol in different seasons in Lanzhou[J]. Environ Monit China, 2022, 38(2): 85-95.
[29]	吴翔, 黄忠乾, 苗人云, 等. 毛木耳发菌棚内外空气真菌的检测[J]. 西南农业学报, 2015, 28(2): 723-727.
	Wu X, Huang ZQ, Miao RY, et al. Detection of airy fungi in and out of Auricularia polytricha's culture room[J]. Southwest China J Agric Sci, 2015, 28(2): 723-727.
[30]	Carrasco J, Preston GM. Growing edible mushrooms: a conversation between bacteria and fungi[J]. Environ Microbiol, 2020, 22(3): 858-872. doi: 10.1111/1462-2920.14765 pmid: 31361932
[31]	Berendsen RL, Kalkhove SIC, Lugones LG, et al. Effects of the mushroom-volatile 1-octen-3-ol on dry bubble disease[J]. Appl Microbiol Biotechnol, 2013, 97(12): 5535-5543. doi: 10.1007/s00253-013-4793-1 pmid: 23467828
[32]	Tietel Z, Masaphy S. Aroma-volatile profile of black morel(Morchella importuna)grown in Israel[J]. J Sci Food Agric, 2018, 98(1): 346-353. doi: 10.1002/jsfa.8477 URL
[33]	Tan H, Kohler A, Miao RY, et al. Multi-omic analyses of exogenous nutrient bag decomposition by the black morel Morchella importuna reveal sustained carbon acquisition and transferring[J]. Environ Microbiol, 2019, 21(10): 3909-3926. doi: 10.1111/emi.v21.10 URL
[34]	Eastwood DC, Herman B, Noble R, et al. Environmental regulation of reproductive phase change in Agaricus bisporus by 1-octen-3-ol, temperature and CO₂[J]. Fungal Genet Biol, 2013, 55: 54-66. doi: 10.1016/j.fgb.2013.01.001 pmid: 23354075
[35]	陈诚, 李强, 黄文丽, 等. 羊肚菌白霉病发生对土壤真菌群落结构的影响[J]. 微生物学通报, 2017, 44(11): 2652-2659.
	Chen C, Li Q, Huang WL, et al. Effects of Morchella white mold disease on soil fungal community structure[J]. Microbiol China, 2017, 44(11): 2652-2659.