取食洋白蜡和旱柳光肩星天牛幼虫肠道中的细菌多样性差异及关键纤维素降解细菌筛选

doi:10.11707/j.1001-7488.LYKX20210919

摘要/Abstract

摘要：

目的: 从肠道细菌多样性角度探究光肩星天牛幼虫对传统抗性树种洋白蜡的适应性原因。方法: 基于16S rDNA高通量测序技术，分别对用洋白蜡、旱柳饲喂的光肩星天牛幼虫肠道细菌进行了分子鉴定及多样性分析；然后通过富集培养，从未经饲喂的幼虫中筛选出了31株纤维素降解细菌，以透明水解圈直径（D）/菌落直径（d）为标准初步测定了各菌株纤维素降解能力；对降解能力最强的4个菌株进行了滤纸复筛试验。结果: 使用洋白蜡饲喂后，厚壁菌门、芽孢杆菌纲、乳杆菌目、链球菌科、乳球菌属为优势类群；使用旱柳饲喂后，厚壁菌门、芽孢杆菌纲、乳杆菌目、肠球菌科、肠球菌属为优势类群；变形菌门、γ-变形菌纲、肠杆菌目、肠杆菌科、拉乌尔菌属在两者中都为优势类群。两者肠道细菌群落最显著的差异在于，取食洋白蜡幼虫链球菌科、乳球菌属相对丰度显著高于取食旱柳的幼虫，而肠球菌科、肠球菌属相对丰度显著低于取食旱柳的幼虫。直接取食洋白蜡枝干的幼虫肠道纤维素降解细菌种类丰富，隶属于4门、5纲、5目、7科、7属，15种；优势属包括微杆菌属、纤维单胞菌属、假单胞菌属；其中纤维素降解能力最强的4个菌株分别为2株假单胞菌和2株产黄纤维单胞菌；二者在滤纸复筛试验中显著地分解了滤纸，进一步验证了其良好的纤维素降解能力。结论: 在取食洋白蜡光肩星天牛幼虫肠道中，链球菌科、乳球菌属为优势类群，可能发挥重要功能但不直接降解纤维素；放线菌门下的微杆菌属、纤维单胞菌属和变形菌门下的假单胞菌属非优势类群但纤维素降解能力强，可能发挥降解纤维素的功能；上述菌群共存于光肩星天牛肠道，参与其适应洋白蜡的过程。

关键词: 16S rDNA, 光肩星天牛, 洋白蜡, 旱柳, 肠道细菌, 纤维素降解细菌

Abstract:

Objective: This study aims to explore the adaptive reasons of Anoplophora glabripennis larvae to Fraxinus pennsylvanica, a traditional resistant species, in terms of intestinal bacteria diversity. Method: The A. glabripennis larvae were fed withF. pennsylvanica and Salix matsudana, respectively , and the high-throughput sequencing technology was used to sequence 16S rDNA of the larvae intestinal bacteria, with which the molecular identification and diversity analysis of intestinal bacteria were conducted. Subsequently, through accumulation culture, 31 strains of cellulose-degrading bacteria were screened out from unfed larvae. The cellulose-degrading ability of each strain was determined by the transparent circle diameter (D)/colony diameter (D) as the standard, and the filter paper re-screening test was conducted on the four strains with the strongest cellulose-degrading ability. Result: The results showed that Phylum Firmicutes, Class Bacilli, Order Lactobacillales, Family Streptococcaceae and the genus Lactococcus were predominant in the larvae intestinal tract after being fed with F. pennsylvanica. Whereas Phylum Firmicutes, Class Bacillus, Order Lactobacillales, Family Enterococcaceae and the genus Enterococcuswere predominant in the larvae intestinal tract after being fed with S. matsudana. Phylum Proteobacteria, Class Gammaproteobacteria, Order Enterobacteriales, Family Enterobacteriaceae and the genus Raoultella were predominant in the both feedings. The most significant difference between the two groups was that the relative abundance of Family Streptococcaceae and genus Lactococcusin the larvae feeding on F. pennsylvanica was significantly higher than that in the larvae feeding on S. matsudana , while the relative abundance of Family Enterococcaceae and genus Enterococcus was significantly lower than that in the larvae feeding onS. matsudana. In the intestinal bacterial community of larvae feeding directly on the branch of F. pennsylvanica, there were abundant species of cellulose-degrading bacteria, belonging to 4 phyla, 5 classes, 5 orders, 7 families, 7 genera and 15 species. At the genus level,Microbacterium, Cellulomonas and Pseudomonas were predominant. Among them, two strains ofPseudomonassp. and two strains of C. flavigena had the strongest cellulose-degrading ability. The filter paper was decomposed significantly in the re-screening test, which further verified that the strains had strong cellulose-degrading ability. Conclusion: Family Streptococcaceae and genus Lactococcus are the predominant bacteria in the intestinal tract of A. glabripennis larvae feeding onF. pennsylvanica, which may play an important role but do not degrade cellulose directly. Genera Microbacterium and Cellulomonas in Phylum Actinobacteria and genus Pseudomonas in Phylum Proteobacteria are not the predominant group of bacteria, but they have strong cellulose degradation ability and may play a role in cellulose degradation. These bacteria co-exist in the intestinal tract of A. glabripennis and are involved in its adaptation process to the F. pennsylvanica.

Key words: 16S rDNA, Anoplophora glabripennis, Fraxinus pennsylvanica, Salix matsudana, intestinal bacteria, cellulose-degrading bacteria

中图分类号:

S763.38

贾若峰,谷奇,孙一鸣,陆鹏飞,俱世博,乔海莉. 取食洋白蜡和旱柳光肩星天牛幼虫肠道中的细菌多样性差异及关键纤维素降解细菌筛选[J]. 林业科学, 2023, 59(4): 117-131.

Ruofeng Jia,Qi Gu,Yiming Sun,Pengfei Lu,Shibo Ju,Haili Qiao. Differences in Bacterial Diversity and Key Cellulose-Degrading Bacteria in the Intestinal Tract of Anoplophora glabripennis (Coleoptera: Cerambycidae)Larvae Feeding on Fraxinus pennsylvanica and Salix matsudana[J]. Scientia Silvae Sinicae, 2023, 59(4): 117-131.

图/表 9

表1

图1

图2

图3

表2

取食洋白蜡光肩星天牛幼虫肠道匀浆液代表纤维素降解细菌菌株NCBI比对结果"

编号 No.	门 Phylum	纲 Class	目 Order	科 Family	属 Genus	种 Species	NCBI登陆号 NCBI accession No.	相似度 Similarity(%)
BLY-30	放线菌门 Actinobacteria	放线菌纲 Actinobacteria	微球菌目 Micrococcales	微杆菌科 Microbacteriaceae	微杆菌属 Microbacterium	叶片微杆菌 Microbacterium foliorum	CP041040.1	100.00
BLY-35						氧化微杆菌 Microbacterium oxydans	HQ202812.1	99.65
BLY-34						叶球形微杆菌 Microbacterium phyllosphaerae	EU370409.1	98.80
BLY-10						氧化烃微杆菌 Microbacterium hydrocarbonoxydans	MN685257.1	99.72
BLY-19						氧化烃微杆菌 Microbacterium hydrocarbonoxydans	JF700471.1	99.79
BLY-18						微杆菌 Microbacterium sp.	KM253118.1	99.78
BLY-36						微杆菌 Microbacterium sp.	KF454846.1	99.72
BLY-1				纤维单胞菌科 Cellulomonadaceae	纤维素单胞菌属 Cellulomonas	产黄纤维单胞菌 Cellulomonas flavigena	KF040991.1	99.93
BLY-28						纤维单胞菌 Cellulomonas sp.	JQ660025.1	99.50
BLY-26	厚壁菌门 Firmicutes	芽孢杆菌纲 Bacilli	芽孢杆菌目 Bacillales	芽孢杆菌科 Bacillaceae	芽孢杆菌属 Bacillus	芽孢杆菌 Bacillus sp.	MG371992.1	99.93
BLY-25				类芽孢杆菌科 Paenibacillaceae	类芽孢杆菌属 Paenibacillus	类芽孢杆菌 Paenibacillus sp.	JF768724.1	100.00
BLY-3	变形菌门 Proteobacteria	γ-变形菌纲 Gammaproteobacteria	假单胞杆菌目 Pseudomonadales	假单胞菌科 Pseudomonadaceae	假单胞菌属 Pseudomonas	类芽孢杆菌 Pseudomonas sp.	MH815093.1	99.86
BLY-11						类芽孢杆菌 Pseudomonas sp.	DQ991143.2	99.72
BLY-23						类芽孢杆菌 Pseudomonas sp.	KY852302.1	100.00
BLY-21	拟杆菌门 Bacteroidetes	噬几丁质杆菌纲 Chitinophagia	噬几丁质菌目 Chitinophagales	噬几丁质菌科 Chitinophagaceae	Niastella	Niabella hibiscisoli	NR_156824.1	98.23
BLY-31		拟杆菌纲 Bacteroidia	鞘脂杆菌目 Sphingobacteriales	鞘脂杆菌科 Sphingobacteriaceae	鞘氨醇杆菌属 Sphingobacterium	鞘氨醇杆菌 Sphingobacterium sp.	KC252768.2	97.49

表2

图4

表3

取食洋白蜡光肩星天牛幼虫肠道匀浆液纤维素降解细菌菌株的纤维素降解能力①"

菌株编号 Strain No.	细菌类群 Species of bacteria	NCBI登陆号 NCBI accession No.	D/d
BLY-30	叶片微杆菌 Microbacterium foliorum	CP041040.1	2.31
BLY-35	氧化微杆菌 Microbacterium oxydans	HQ202812.1	2.081
BLY-34	叶球形微杆菌 Microbacterium phyllosphaerae	EU370409.1	3.216
BLY-5	氧化烃微杆菌 Microbacterium hydrocarbonoxydans	MN685257.1	2.54
BLY-10	氧化烃微杆菌Microbacterium hydrocarbonoxydans	MN685257.1	2.86
BLY-19	氧化烃微杆菌 Microbacterium hydrocarbonoxydans	JF700471.1	2.826
BLY-20	氧化烃微杆菌 Microbacterium hydrocarbonoxydans	JF700471.1	2.549
BLY-18	微杆菌 Microbacterium sp.	KM253118.1	2.5
BLY-36	微杆菌 Microbacterium sp.	KF454846.1	4.37
BLY-1	产黄纤维单胞菌 Cellulomonas flavigena ¹	KF040991.1	5.594
BLY-2	产黄纤维单胞菌 Cellulomonas flavigena ¹	KF040991.1	5.444
BLY-4	产黄纤维单胞菌 Cellulomonas flavigena	KF040991.1	3.333
BLY-7	产黄纤维单胞菌 Cellulomonas flavigena	KF040991.1	3.36
BLY-8	产黄纤维单胞菌 Cellulomonas flavigena	KF040991.1	3.965
BLY-9	产黄纤维单胞菌 Cellulomonas flavigena	KF040991.1	3.811
BLY-14	产黄纤维单胞菌 Cellulomonas flavigena	KF040991.1	5.2
BLY-15	产黄纤维单胞菌 Cellulomonas flavigena	KF040991.1	3.882
BLY-24	产黄纤维单胞菌 Cellulomonas flavigena	KF040991.1	4.515
BLY-28	纤维单胞菌 Cellulomonas sp.	JQ660025.1	3.106
BLY-29	纤维单胞菌 Cellulomonas sp.	JQ660025.1	2.865
BLY-26	芽孢杆菌 Bacillus sp.	MG371992.1	2.087
BLY-25	类芽孢杆菌 Paenibacillus sp.	JF768724.1	5.233
BLY-3	假单胞菌 Pseudomonas sp. ¹	MH815093.1	6.645
BLY-12	假单胞菌 Pseudomonas sp. ¹	MH815093.1	9.836
BLY-11	假单胞菌 Pseudomonas sp.	DQ991143.2	1.362
BLY-13	假单胞菌 Pseudomonas sp.	KY852302.1	2.029
BLY-23	假单胞菌 Pseudomonas sp.	KY852302.1	2.171
BLY-21	Niabella hibiscisoli	NR_156824.1	2.649
BLY-22	Niabella hibiscisoli	NR_156824.1	2.462
BLY-31	鞘氨醇杆菌 Sphingobacterium sp.	KC252768.2	1.288
BLY-33	鞘氨醇杆菌 Sphingobacterium sp.	KC252768.2	1.438

表3

图5

表1

BL、HL的分类系统及各属丰度值"

门 Phylum	纲 Class	目 Order	科 Family	属 Genus	丰度值 Bacterial abundance
门 Phylum	纲 Class	目 Order	科 Family	属 Genus	BL	HL
变形菌门 Proteobacteria	α-变形菌纲 Alphaproteobacteria	根瘤菌目 Rhizobiales	德沃斯氏菌科 Devosiaceae	德沃斯菌属 Devosia	5	1
			Kaistiaceae	Kaistia	2	0
			根瘤菌科 Rhizobiaceae	Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium	2	6
				苍白杆菌属 Ochrobactrum	11	1
		红细菌目 Rhodobacterales	红细菌科 Rhodobacteraceae	大不里士菌属Tabrizicola	16	0
				副球菌属Paracoccus	6	0
	γ-变形菌纲 Gammaproteobacteria	肠杆菌目 Enterobacteriales	肠杆菌科 Enterobacteriaceae	肠杆菌属 Enterobacter	228	1383
				Gibbsiella	97	214
				拉乌尔菌属 Raoultella	12739	14029
				未知属 Unclassified	331	6204
		假单胞菌目 Pseudomonadales	莫拉氏菌科 Moraxellaceae	不动杆菌属 Acinetobacter	0	104
			假单胞菌科 Pseudomonadaceae	假单胞菌属 Pseudomonas	4	3738
		黄单胞菌目 Xanthomonadales	黄单胞菌科 Xanthomonadaceae	藤黄单胞菌属Luteimonas	11	0
		Betaproteobacteriales	伯克氏菌科 Burkholderiaceae	青枯菌属 Ralstonia	0	1
				嗜异生质菌属 Xenophilus	8	0
			嗜甲基菌科 Methylophilaceae	Methylopila	1	0
		未知目 Unclassified	未知科 Unclassified	未知属 Unclassified	3	624
厚壁菌门 Firmicutes	芽孢杆菌纲 Bacilli	乳杆菌目 Lactobacillales	链球菌科 Streptococcaceae	乳球菌属 Lactococcus	20581	0
			肠球菌科 Enterococcaceae	肠球菌属 Enterococcus	1603	17484
			乳酸菌科 Lactobacillaceae	乳杆菌属 Lactobacillus	8	1
放线菌门 Actinobacteria	放线菌纲 Actinobacteria	微球菌目 Micrococcales	微杆菌科 Microbacteriaceae	微杆菌属 Microbacterium	26	8
				亮杆菌属 Leucobacter	3	1
			纤维单胞菌科 Cellulomonadaceae	纤维素单胞菌属 Cellulomonas	0	989
			微球菌科 Micrococcaceae	Glutamicibacter	0	31
			表皮菌科 Dermabacteraceae	短状杆菌属 Brachybacterium	0	1
			未知科 Unclassified	未知属 Unclassified	13	0
			未知科 Unclassified	未知属 Unclassified	2	0
		丙酸杆菌目 Propionibacteriales	丙酸杆菌科 Propionibacteriaceae	Propionicicella	6	0
		棒状菌目 Corynebacteriales	诺卡氏菌科 Nocardiaceae	红球菌属Rhodococcus	0	3
				戈登氏菌属Gordonia	5	9
			棒状杆菌科 Corynebacteriaceae	棒状杆菌属Corynebacterium_1	0	6
		土壤红杆菌目 Solirubrobacterales	土壤红杆菌科 Solirubrobacteraceae	Patulibacter	1	0
		放线菌目 Actinomycetales	放线菌科 Actinomycetaceae	放线菌属 Actinomyces	21	0
		双歧杆菌目 Bifidobacteriales	双歧杆菌科 Bifidobacteriaceae	双歧杆菌属 Bifidobacterium	13	0
		弗兰克氏菌目 Frankiales	中村氏菌科 Nakamurellaceae	中村菌属 Nakamurella	6	0
拟杆菌门 Bacteroidetes	拟杆菌纲 Bacteroidia	黄杆菌目 Flavobacteriales	藏红花黄色线菌科 Crocinitomicaceae	Fluviicola	2	0
		鞘脂杆菌目 Sphingobacteriales	鞘脂杆菌科 Sphingobacteriaceae	鞘氨醇杆菌属 Sphingobacterium	22	0
蓝细菌门 Cyanobacteria	产氧光细菌纲 Oxyphotobacteria	叶绿体 Chloroplast	缺少信息 Norank	缺少信息 Norank	1	0
			缺少信息 Norank	缺少信息 Norank	0	9
未知门 Unclassified	未知纲 Unclassified	未知目 Unclassified	未知科 Unclassified	未知属 Unclassified	4	0

合计 Total					35781	44847

表1

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