葱地种蝇幼虫伴生细菌多样性及两种培养基分离效果比较

刘梅; 张新建; 周方园

doi:10.19303/j.issn.1008-0384.2022.01.012

葱地种蝇幼虫伴生细菌多样性及两种培养基分离效果比较

doi: 10.19303/j.issn.1008-0384.2022.01.012

刘梅^{1, 2,},
张新建¹,
周方园^1, ,

1.
齐鲁工业大学（山东省科学院）生态研究所/山东省应用微生物重点实验室，山东济南　250013
2.
湖北大学生命科学学院，湖北武汉　430062

基金项目: 山东省重点研发计划项目（2019GSF109056）；山东省重点研发计划（重大科技创新工程）项目（2021TZXD002）；国家自然科学基金（31700426）；山东省自然科学基金（ZR2017BC050）

详细信息

作者简介:
刘梅（1995−），女，博士研究生，主要从事昆虫伴生菌相关研究（E-mail：liumeigrace@163.com）

通讯作者:
周方园（1990-），男，副研究员，主要从事昆虫生态、虫菌互作研究（E-mail: fangyuan_zhou@outlook.com）

中图分类号: S 436
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- 被引次数: 0
出版历程
- 收稿日期: 2021-09-11
- 修回日期: 2021-11-22
- 网络出版日期: 2022-01-21
- 刊出日期: 2022-01-28

Diversity and Isolation of Symbiotic Bacteria on Delia antiqua Larvae

LIU Mei^{1, 2
,},
ZHANG Xinjian¹,
ZHOU Fangyuan^{1
, ,}

1.
Shandong Provincial Key Laboratory of Applied Microbiology/Ecology Institute, Qilu University of Technology (Shandong Academy of Sciences), Ji’nan, Shandong　250013, China
2.
School of Life Sciences, Hubei University, Wuhan, Hubei　430062, China

摘要

摘要: 目的探索蒜蛆幼虫伴生细菌的多样性，并比较两种常用培养基对其伴生细菌的分离效果，为开展蒜蛆伴生细菌研究提供技术指导。方法采用高通量扩增子测序方法检测蒜蛆幼虫体表和肠道的细菌多样性；使用LB和TSB从蒜蛆幼虫的体表和肠道分离细菌，并比较两种培养基分离结果、测序结果和两种培养基分离效果的差异。结果高通量测序从蒜蛆幼虫体表和肠道共检测到4个门、66个科、115个属的细菌，其中绝大部分不能够鉴定到种。使用LB培养基从蒜蛆幼虫体表和肠道共分离得到463株细菌，鉴定后共得到29种细菌，分别属于11个科18个属。使用TSB培养基从蒜蛆幼虫体表和肠道分离得到391株细菌，经测序后共鉴定到44种细菌，分别属于19个科28个属。总体来说，TSB分离到的细菌种类更多，部分细菌可同时用LB与TSB分离到；此外，与高通量测序结果相比，两种培养基均只能分离到很小一部分种类的细菌。结论在蒜蛆伴生细菌的分离试验中，最好同时使用两种培养基对细菌进行分离，以获得更多种类的细菌。
- 蒜蛆 /
- 肠道菌 /
- 分离鉴定 /
- 微生物多样性
Abstract: Objective Diversity of the symbiotic bacteria on Delia antiqua larvae was studied, and effectiveness of two commonly used culture media in isolating them compared. Methods Diversity of the bacterial population on the surface and in the guts of D. antiqua larvae were determined using the high-throughput amplicon sequencing technique. The effectiveness of the LB and TSB media to culture and isolate the bacteria were compared based on the sequencing results. Results The inhabitation bacteria on the larvae were from 115 genera, 66 families, and 4 phyla according to the sequencing. Most of them could not be annotated to species level. On LB medium, 463 strains were isolated that belonged to 29 species, 18 genera, and 11 families, while on TSB, 391 strains from 44 species, 28 genera, and 19 families. TSB isolated more species than LB, and some of the species could be isolated by either method. However, the high-throughput amplicon sequencing revealed that these culture methods could provide only a minute portion of the bacteria population that coinhabited on the host. Conclusion It was necessary to use both TSB and LB media in order to maximize the isolation of all symbiotic bacteria on D. antiqua larvae.
- Onion maggot /
- gut microbe /
- bacterial isolation and identifications /
- microbial diversity

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图 1 蒜蛆幼虫体表伴生细菌测序样本中物种属水平（a）和种水平（b）相对丰度

注：图中“u_”开头的分类阶元为未鉴定到属或种，下同。

Figure 1. Relative abundance of bacteria on D. aniqua larval surface at genus (a) and species (b) levels

Note: Taxonomic categories started with “u_” represent sequencing reads not assigned to species or genus. Same for below.

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图 2 蒜蛆幼虫肠道细菌测序样本中属水平（a）和种水平（b）物种丰度

Figure 2. Relative abundance of bacteria in D. aniqua guts at genus (a) and species (b) levels

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图 3 两种培养基分离蒜蛆幼虫细菌的16S rDNA最大似然树

注：图中L、M开头的菌株为LB、TSB培养基分离得到的；其中标为橙色、绿色的菌株为从蒜蛆幼虫体表和肠道分离得到的，下同。

Figure 3. Maximum likelihood trees for 16S rDNA sequences of strains on D. antiqua larva isolated on two media

Note: Strains numbered L and M are isolations from LB and TSB media, respectively. Orange and green indicate strains from surface and guts of D. antiqua larvae, respectively. Same for below.

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图 4 变形菌门（Proteobacteria，a）、放线菌门（Actinobacteria，b）和拟杆菌门（Bacteroidetes，c）的16S rDNA最大似然树

Figure 4. Maximum likelihood trees for 16S rDNA sequences of Proteobacteria (a), Actinobacteria (b), and Bacteroidetes (c)

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图 5 LB培养基上分离蒜蛆幼虫体表（a）和肠道（b）细菌分离频率

Figure 5. Frequency of isolating bacteria on D. antiqua surface (a) and in guts (b) by using LB medium

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图 6 TSB培养基上分离蒜蛆幼虫体表（a）和肠道（b）细菌分离频率

Figure 6. Frequency of isolating bacteria on D. antiqua surface (a) and in guts (b) by using TSB medium

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图 7 蒜蛆幼虫使用LB、TSB培养基以及测序分离细菌种类比较

注：图中a、b为种水平细菌种类比较；c-h为属水平细菌种类比较；a、c、e、g为蒜蛆幼虫体表细菌种类比较；b、d、f、h为蒜蛆幼虫肠道细菌种类比较。

Figure 7. Bacteria from D. antiqua larva as identified by LB, TSB, and high throughput sequencing methods

Note: Figs. a and b represent bacteria on species level; Figs. c-h, on genus level; Figs. a, c, e, and g, from D. antiqua larval surface; and Figs. b, d, f, and h, from D. antiqua larval guts.

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表 1 使用两种培养基分离得到的蒜蛆幼虫伴生细菌菌株的GenBank号以及种属鉴定信息

Table 1. GenBank codes and species identification information on bacteria associated with D. antiqua larvae isolated on two media

菌株号 Strain No.	Genbank登录号 GenBank accession No.	最相似菌株 Closest type strains	相似率 Similarity/%
M6595	OK642282	谷氨酸棒状杆菌 Corynebacterium glutamicum DQ173747.1	99.79
M4615	OL304276	红杆菌 Rhodobacter sp. MT036106.1	98.53
M4614	OK642283	蜡样芽孢杆菌 Bacillus cereus KY495215.1	99.86
M4613	OL304277	红杆菌 Rhodobacter sp. JF710650.1	96.67
M4110	OK642284	假苍白杆菌 Pseudochrobactrum sp. KF735806.1	99.92
M4061	OK642285	微小芽胞杆菌 Brevundimonas diminuta LC420058.1	99.78
M4025	OK642286	Paenochrobactrum glaciei NR112750.1	99.00
M2014	OK642287	拟黄杆菌 O. pseudogrignonensis MK517588.1	99.93
M0685	OK642288	食酸菌 Acidovorax sp. AB646315.1	99.79
M0684	OK642289	肺炎克雷氏伯杆菌 Acinetobacter guillouiae KF873017.1	98.63
M0681	OK642290	红斑丹毒丝菌 Erysipelothrix rhusiopathiae MN933918.1	97.61
M0678	OK642291	摩根氏菌 Morganella morganii HQ407316.1	99.18
M0662	OK642292	亮杆菌某种 Leucobacter sp. GQ344412.1	99.42
M0596	OK642293	腐殖质类芽胞杆菌 Paenibacillus humicus AM411529.2	99.73
M0564	OK642294	不动杆菌某种 Acinetobacter sp. KT261003.1	99.71
M0545	OK642295	溶血假单胞菌 P. asaccharolyticum KU597492.1	99.79
M0512	OK642296	产酸克雷伯菌 Klebsiella oxytoca LC049195.1	99.65
M0497	OK642297	微杆菌某种 Microbacterium sp. KR906366.1	99.78
M0483	OK642298	节杆菌某种 Arthrobacter sp. JX566636.1	99.72
M0432	OK642299	琥珀色葡萄球菌 Staphylococcus succinus GU084442.1	100.00
M0419	OK642300	人苍白杆菌 Ochrobactrum anthropi MG897139.1	99.85
M0417	OK642301	微杆菌某种 Microbacterium sp. LT009507.1	99.93
M0351	OK642302	俊片菌 Lampropedia sp. MK733277.1	99.28
M0332	OK642303	氧化硫副球菌 Paracoccus sulfuroxidans JQ291588.1	99.63
M0321	OK642304	雷氏普罗威登斯菌 Providencia rettgeri MN006177.1	99.73
M0291	OK642305	苍黄假棍状杆菌 Pseudoclavibacter helvolus MG694511.1	99.86
M0281	OK642306	马红球菌 Rhodococcus equi LC020105.1	99.86
M0251	OK642307	木糖葡萄球菌 Staphylococcus xylosus KT260686.1	99.59
M0211	OK642308	节杆菌某种 Arthrobacter sp. MK087736.1	99.78
M0207	OK642309	干丘亮杆菌 Leucobacter aridicollis NR 042288.1	99.65
M0201	OK642310	铬还原白杆菌 Leucobacter chromiireducens NR042287.1	99.58
M0181	OK642311	鞘氨醇杆菌 Sphingobacterium sp. NR159136.1	100.00
M0144	OK642312	酯香微杆菌 M. esteraromaticum NR026468.1	99.44
M0126	OK642313	亮杆菌某种 Leucobacter sp. KP340979.1	100.00
M0064	OK642314	粪产碱菌 Alcaligenes faecalis MK968769.1	99.86
M0055	OK642315	血杆菌某种 Sanguibacter sp. JX997905.1	99.79
M0054	OK642316	微杆菌某种 Microbacterium sp. EU841532.1	99.09
M0051	OK642317	贝莱斯芽孢杆菌 Bacillus velezensis MK780002.1	100.00
M0049	OK642318	短稳杆菌 Empedobacter brevis MN596020.1	99.93
M0047	OK642319	气味类香菌 Myroides odoratus JQ407801.1	99.85
M0021	OK642320	亮杆菌某种 Leucobacter sp. MN099406.1	97.56
L5111	OK642321	雷氏普罗威登斯菌 Providencia rettgeri MN006177.1	99.66
L0506	OK642322	摩根氏菌 Morganella morganii HQ407316.1	99.81
L0505	OK642323	土白蚁丛毛单胞菌 Comamonas odontotermitis NR 043859.1	99.72
L0502	OK605023	氧化微杆菌 Microbacterium oxydans MF459692.1	99.79
L0501	OK642324	铬还原白杆菌 Leucobacter chromiireducens NR 042287.1	99.93
L0491	OK642325	微杆菌某种 Microbacterium sp. LC133735.2	99.86
L0481	OK642326	芽孢杆菌某种 Bacillus sp. MN590524.1	99.80
L0401	OK642327	芽孢杆菌某种 Bacillus sp. MN582995.1	99.80
L0391	OK642328	海泥芽胞杆菌 Bacillus oceanisediminis MN606212.1	100.00
L0361	OK642329	稳杆菌 Empedobacter sp. MK224650.1	99.79
L0351	OK642330	芽孢杆菌某种 Bacillus sp. AB211009.1	99.80
L0331	OK642331	芽孢杆菌某种 Bacillus sp. AM913937.1	99.39
L0301	OK642332	类芽胞杆菌某种 Paenibacillus sp. KM521215.1	99.53
L0294	OK642333	铬还原白杆菌 Leucobacter chromiireducens NR 042287.1	99.93
L0281	OK642334	金肠球菌 Enterococcus gilvus NR 043793.1	99.93
L0271	OK642335	稳杆菌 Empedobacter sp. MK224650.1	99.79
L0267	OK642336	雷氏普罗威登斯菌 Providencia rettgeri MN006177.1	99.66
L0221	OK642337	谷氨酰菌 Glutamicibacter arilaitensis MG788347.1	99.93
L0202	OK642338	枯草芽孢杆菌 Bacillus subtilis KX901787.1	100.00
L0192	OK642339	谷氨酰菌 Glutamicibacter arilaitensis MG788347.1	99.93
L0182	OK642340	土白蚁丛毛单胞菌 Comamonas odontotermitis NR 043859.1	99.72
L0166	OK642341	摩根氏菌 Morganella morganii HQ407316.1	99.18
L0145	OK642342	类芽胞杆菌某种 Paenibacillus sp. KR055030.1	99.59
L0138	OK642343	拟黄杆菌 O. pseudogrignonensis KC342237.1	98.64
L0119	OK642344	欧文氏菌 Erwinia sp. KP836251.1	99.38
L0092	OK642345	巨大芽孢杆菌 Bacillus megaterium MH144290.1	99.86
L0081	OK642346	Paenochrobactrum sp. MG596953.1	98.81
L0079	OK642347	类芽胞杆菌某种 Paenibacillus sp. KF465974.1	99.59
L0078	OK642348	类芽胞杆菌某种 Paenibacillus sp. AM162345.1	99.52
L0062	OK642349	谷氨酸杆菌 Glutamicibacter sp. MH725567.1	99.93
L0051	OK642350	枯草芽孢杆菌 Bacillus subtilis KX901787.1	100.00
L0042	OK642351	滋养节杆菌 Arthrobacter pascens GQ360068.1	98.96
L0031	OK642352	欧文氏菌 Erwinia sp. KP836251.1	99.38
L0021	OK642353	皮特不动杆菌 Acinetobacter pittii MN490071.1	99.86
L0013	OK642354	溶血假单胞菌 P. asaccharolyticum MF285796.1	98.08
L0011	OK605542	苍黄假棍状杆菌 Pseudoclavibacter helvolus NR 029264.1	99.93

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