微生物学报

Primers name	Primer sequences (5′→3′)
LsRPL18-F	CCAAGATGTTTACCTGCGAC
LsRPL18-R	CATTAAGCTGGCTACCTTCG
YLS-F	GCAGCGAAATGCGATAAGTAA
YLS-R	CCCTTAACCGGACGATGACCCAATA

Primers name	Primer sequences (5′→3′)
LsRPL18-F	CCAAGATGTTTACCTGCGAC
LsRPL18-R	CATTAAGCTGGCTACCTTCG
YLS-F	GCAGCGAAATGCGATAAGTAA
YLS-R	CCCTTAACCGGACGATGACCCAATA

Sample ID	Raw reads	Clean reads	Effective reads	Effective rate (%)
SL-1	38 810	38 810	38 751	99.85
SL-2	41 036	41 036	40 975	99.85
SL-3	39 910	39 910	39 803	99.73
SL-4	33 869	33 869	33 783	99.75
SL-5	35 322	35 322	35 276	99.87
SL-6	33 019	33 019	32 970	99.85
YC-1	42 260	42 260	41 499	98.20
YC-2	39 209	39 209	39 074	99.66
YC-3	39 992	39 992	39 905	99.78
YC-4	43 878	43 878	43 699	99.59
YC-5	38 380	38 380	38 313	99.83
YC-6	43 447	43 447	43 374	99.83
WF-1	34 601	34 601	34 233	98.94
WF-2	40 264	40 264	40 029	99.42
WF-3	37 843	37 843	37 395	98.82

Sample ID	Raw reads	Clean reads	Effective reads	Effective rate (%)
SL-1	38 810	38 810	38 751	99.85
SL-2	41 036	41 036	40 975	99.85
SL-3	39 910	39 910	39 803	99.73
SL-4	33 869	33 869	33 783	99.75
SL-5	35 322	35 322	35 276	99.87
SL-6	33 019	33 019	32 970	99.85
YC-1	42 260	42 260	41 499	98.20
YC-2	39 209	39 209	39 074	99.66
YC-3	39 992	39 992	39 905	99.78
YC-4	43 878	43 878	43 699	99.59
YC-5	38 380	38 380	38 313	99.83
YC-6	43 447	43 447	43 374	99.83
WF-1	34 601	34 601	34 233	98.94
WF-2	40 264	40 264	40 029	99.42
WF-3	37 843	37 843	37 395	98.82

Group	OTU	Phylum	Class	Order	Family	Genus
SL	35	1	3	3	3	4
YC	32	1	3	3	3	3
WF	15	1	1	1	1	2

Group	OTU	Phylum	Class	Order	Family	Genus
SL	35	1	3	3	3	4
YC	32	1	3	3	3	3
WF	15	1	1	1	1	2

Genus name	SL (%)	YC (%)	WF (%)
Symbiotaphrina	81.648 0	85.062 3	92.506 0
Fungi Incertae sedis	17.621 0	14.928 8	7.494 0
Aspergillus	0.641 3	0.006 5	0.000 0
Xeromyces	0.088 3	0.000 4	0.000 0
Leptobacillium	0.001 4	0.002 0	0.000 0

Genus name	SL (%)	YC (%)	WF (%)
Symbiotaphrina	81.648 0	85.062 3	92.506 0
Fungi Incertae sedis	17.621 0	14.928 8	7.494 0
Aspergillus	0.641 3	0.006 5	0.000 0
Xeromyces	0.088 3	0.000 4	0.000 0
Leptobacillium	0.001 4	0.002 0	0.000 0

Species name	SL (%)	YC (%)	WF (%)
Symbiotaphrina kochii	81.635 8	83.614 5	86.435 7
Fungi Incertae sedis	17.621 0	14.928 8	7.494 0
Symbiotaphrina buchneri	0.012 2	1.447 0	4.672 1
Symbiotaphrina microtheca	0.000 0	0.000 8	1.398 2
Aspergillus penicillioides	0.435 3	0.004 5	0.000 0
Aspergillus ruber	0.206 1	0.002 0	0.000 0
Xeromyces bisporus	0.088 3	0.000 4	0.000 0
Leptobacillium leptobactrum	0.001 4	0.002 0	0.000 0

Species name	SL (%)	YC (%)	WF (%)
Symbiotaphrina kochii	81.635 8	83.614 5	86.435 7
Fungi Incertae sedis	17.621 0	14.928 8	7.494 0
Symbiotaphrina buchneri	0.012 2	1.447 0	4.672 1
Symbiotaphrina microtheca	0.000 0	0.000 8	1.398 2
Aspergillus penicillioides	0.435 3	0.004 5	0.000 0
Aspergillus ruber	0.206 1	0.002 0	0.000 0
Xeromyces bisporus	0.088 3	0.000 4	0.000 0
Leptobacillium leptobactrum	0.001 4	0.002 0	0.000 0

Trophic mode	Guild	Relative abundance	Confidence ranking
Symbiotroph	Animal endosymbiont	81.640 4	85.048 2	92.504 2	Probable
Saprotroph	Undefined saprotroph	0.729 6	/	/	Possible

Trophic mode	Guild	Relative abundance	Confidence ranking
Symbiotroph	Animal endosymbiont	81.640 4	85.048 2	92.504 2	Probable
Saprotroph	Undefined saprotroph	0.729 6	/	/	Possible

Sample ID	Colony morphology	Spore morphology
SL-1	Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium	Spores are spherical or oval or irregularly shaped
SL-2	The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors	Conidiophores whorled on broomlike branches; peduncle erect, usually with transverse septum, hyaline; peduncle bearing multiple pedicels apically, pedicels almost equal in length, some pedicels inflated apically, conidiophores ellipsoidal or spindle-shaped
SL-3	Colonies are gray-green, powdery, yellow on the back, fading in color toward the edges	Conidiophores broomlike, in single whorls, vittae shorter, conidia globose
YC-1	Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium	Spores are spherical or oval or irregularly shaped
YC-2	The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors	Conidiophores in multiple whorls of broomlike branches; peduncle stems erect, usually with transverse septa, hyaline; peduncle stems bearing multiple columnar peduncles apically, peduncles almost equal in length, sometimes inflated apically, conidiophores globose
YC-3	The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors	Conidiophores in multiple whorls of broomlike branches; peduncle stems erect, usually with transverse septa, hyaline; peduncle stems bearing multiple columnar peduncles apically, peduncles almost equal in length, sometimes inflated apically, conidiophores globose
WF-1	Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium	Spores are spherical or oval or irregularly shaped
WF-2	Initial colony white filamentous, later colony surface color slowly become green powdery, the back of the colony slowly become yellow or orange, to the edge of the gradual lightening, the color becomes darker with time	Molecular spore pedicels broomlike, in single whorls, apical pedicels shorter, molecular spores globose, blue-green

Sample ID	Colony morphology	Spore morphology
SL-1	Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium	Spores are spherical or oval or irregularly shaped
SL-2	The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors	Conidiophores whorled on broomlike branches; peduncle erect, usually with transverse septum, hyaline; peduncle bearing multiple pedicels apically, pedicels almost equal in length, some pedicels inflated apically, conidiophores ellipsoidal or spindle-shaped
SL-3	Colonies are gray-green, powdery, yellow on the back, fading in color toward the edges	Conidiophores broomlike, in single whorls, vittae shorter, conidia globose
YC-1	Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium	Spores are spherical or oval or irregularly shaped
YC-2	The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors	Conidiophores in multiple whorls of broomlike branches; peduncle stems erect, usually with transverse septa, hyaline; peduncle stems bearing multiple columnar peduncles apically, peduncles almost equal in length, sometimes inflated apically, conidiophores globose
YC-3	The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors	Conidiophores in multiple whorls of broomlike branches; peduncle stems erect, usually with transverse septa, hyaline; peduncle stems bearing multiple columnar peduncles apically, peduncles almost equal in length, sometimes inflated apically, conidiophores globose
WF-1	Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium	Spores are spherical or oval or irregularly shaped
WF-2	Initial colony white filamentous, later colony surface color slowly become green powdery, the back of the colony slowly become yellow or orange, to the edge of the gradual lightening, the color becomes darker with time	Molecular spore pedicels broomlike, in single whorls, apical pedicels shorter, molecular spores globose, blue-green

Strain number	Accession number	Identity (%)	Similar species
SL-1	PQ844595	99.46	Symbiotaphrina kochii
SL-2	PQ844596	100.00	Talaromyces pinophilus
SL-3	PQ844597	99.82	Penicillium chermesinum
YC-1	PQ844598	99.46	Symbiotaphrina kochii
YC-2	PQ844599	100.00	Talaromyces pinophilus
YC-3	PQ844600	100.00	Talaromyces pinophilus
WF-1	PQ844601	99.46	Symbiotaphrina kochii
WF-2	PQ844602	100.00	Penicillium mallochii

Strain number	Accession number	Identity (%)	Similar species
SL-1	PQ844595	99.46	Symbiotaphrina kochii
SL-2	PQ844596	100.00	Talaromyces pinophilus
SL-3	PQ844597	99.82	Penicillium chermesinum
YC-1	PQ844598	99.46	Symbiotaphrina kochii
YC-2	PQ844599	100.00	Talaromyces pinophilus
YC-3	PQ844600	100.00	Talaromyces pinophilus
WF-1	PQ844601	99.46	Symbiotaphrina kochii
WF-2	PQ844602	100.00	Penicillium mallochii

食料对烟草甲体内真菌群落结构及多样性的影响

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李艳青 ¹ , 万子凌 ¹ , 李昂 ¹ , 彭琛 ² , 梁增发 ² , 黄嘉杰 ² , 许小霞 ¹ , 金丰良 ¹

微生物学报 | 研究报告 2025,65(9): 3899-3920

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微生物学报 | 研究报告 2025, 65(9): 3899-3920

食料对烟草甲体内真菌群落结构及多样性的影响

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李艳青¹, 万子凌¹, 李昂¹, 彭琛², 梁增发², 黄嘉杰², 许小霞¹, 金丰良¹

作者信息

1 华南农业大学植物保护学院，绿色农药全国重点实验室，广东广州

2 广东中烟工业有限责任公司，广东广州

Diets influence the structure and diversity of the fungal community in the tobacco beetle Lasioderma serricorne

Yanqing LI¹, Ziling WAN¹, Ang LI¹, Chen PENG², Zengfa LIANG², Jiajie HUANG², Xiaoxia XU¹, Fengliang JIN¹

Affiliations

1 State Key Laboratory of Green Pesticide, College of Plant Protection, South China Agricultural University, Guangzhou, Guangdong, China

2 China Tobacco Guangdong Industrial Co. , Ltd. , Guangzhou, Guangdong, China

出版时间: 2025-09-04 doi: 10.13343/j.cnki.wsxb.20250066

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摘要

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【目的】 探究不同食料对烟草甲体内真菌群落结构和多样性的影响，为基于微生物调控的仓储害虫绿色防控策略提供理论依据。 【方法】 基于PacBio SMRT测序平台，采用内转录间隔区(internal transcribed spacer, ITS)全长扩增子测序技术对比分析人工饲料组(SL)、烟草驯化组(YC)和野生环境组(WF) 3组实验中烟草甲体内真菌的群落结构特征。运用传统培养方法分离可培养真菌，并通过荧光定量PCR技术定位核心共生菌Symbiotaphrina kochii的组织表达模式。 【结果】 群落结构特征分析显示，SL组、YC组和WF组分别鉴定出35、32和15个操作分类单元(operational taxonomic unit, OTU)，3组共有的核心OTUs占比分别为31.43%、34.38%、73.33%。多样性分析结果表明，SL组的Sobs指数(29.00±1.13)显著高于YC组(16.17±2.30)和WF组(12.33±1.33) (P<0.001)。Symbiotaphrina是3组共有核心功能菌群，其相对丰度均超过81.000 0%。曲霉属(Aspergillus)和耐干霉属(Xeromyces)是SL组和YC组的特征菌属，而Symbiotaphrina buchneri和Symbiotaphrina microtheca则形成了YC组和WF组特异性进化支。通过传统真菌分离技术，成功分离出8株子囊菌门真菌，其中包括Symbiotaphrina (3株)、Talaromyces (3株)和Penicillium (2株) 3个属。组织特异性表达证实，S. kochii在菌胞体中的表达量(10.42±1.03)显著高于脂肪体(0.74±0.08)和中肠(0.31±0.01)组织(P<0.001)，证实其胞内定殖特性。 【结论】 本研究揭示了食料类型通过“营养-菌群”互作网络调控烟草甲体内真菌群落的构建，证实了Symbiotaphrina属在宿主适应性进化中的关键作用，为构建基于微生物互作网络精准调控的害虫治理新技术提供了理论依据与关键靶标。

关键词

烟草甲 / 体内真菌 / 多样性 / 群落结构 / 食料 / 真菌分离

Abstract

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[Objective] This study investigates the effects of various diets on the structure and diversity of the fungal community in the tobacco beetle Lasioderma serricorne, aiming to provide a theoretical basis for developing green control strategies for stored-product pests through microbial regulation. [Methods] The PacBio SMRT platform was used for full-length internal transcribed spacer (ITS) amplicon sequencing, on the basis of which the community structure characteristics of fungi in L. serricorne were compared among three groups: artificial feed (SL), tobacco domestication (YC), and wild environment (WF). Additionally, culturable fungi were isolated via the culture method, and the tissue expression pattern of the core symbiotic fungus Symbiotaphrina kochii was localized by RT-qPCR. [Results] SL, YC, and WF groups contained 35, 32, and 15 operational taxonomic units (OTUs), respectively. The core OTUs shared by the three groups accounted for 31.43%, 34.38%, and 73.33% in SL, YC, and WF groups, respectively. The Sobs index of the SL group was 29.00±1.13, which was higher than those of the YC group (16.17±2.30) and WF group (12.33±1.33) (P<0.001). Symbiotaphrina was the core functional group shared by the three groups, and its relative abundance was more than 81.000 0% in all the three groups. Aspergillus and Xeromyces were the characteristic genera of the SL and YC groups, while Symbiotaphrinabuchneri and Symbiotaphrina microtheca formed the evolutionary clades specific to the YC and WF groups. Eight Ascomycota strains were isolated via the culture method, belonging to three genus: Symbiotaphrina (three strains), Talaromyces (three strains), and Penicillium (two strains). Tissue-specific expression analysis confirmed the higher expression level of S. kochii in mycetocytes (10.42±1.03) than in the fat body (0.74±0.08) and midgut (0.31±0.01) (P<0.001), validating its intracellular colonization. [Conclusion] This study for the first time reveals that diets regulate the fungal community assembly in L. serricorne through a “nutrient-microbiota” interaction network and demonstrates the pivotal role of Symbiotaphrina in adaptive evolution of the host. These findings establish a theoretical foundation and provide critical targets for developing precision pest control technologies based on targeted modulation of microbial interaction networks.

Key words

Lasioderma serricorne / internal fungi / diversity / community structure / diet / fungal isolation

引用本文

李艳青, 万子凌, 李昂, 彭琛, 梁增发, 黄嘉杰, 许小霞, 金丰良. 食料对烟草甲体内真菌群落结构及多样性的影响. 微生物学报, 2025 , 65 (9) : 3899 -3920 . DOI: 10.13343/j.cnki.wsxb.20250066

Yanqing LI, Ziling WAN, Ang LI, Chen PENG, Zengfa LIANG, Jiajie HUANG, Xiaoxia XU, Fengliang JIN. Diets influence the structure and diversity of the fungal community in the tobacco beetle Lasioderma serricorne[J]. Acta Microbiologica Sinica, 2025 , 65 (9) : 3899 -3920 . DOI: 10.13343/j.cnki.wsxb.20250066

正文

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烟草甲(Lasioderma serricorne)是一种分布广泛的鞘翅目昆虫，也是全球烟草储藏中的主要害虫之一。该昆虫食性广泛，其危害对象包括储藏中的烟草、禾谷类、豆类、动植物标本、书籍和胡椒等，其中烟叶及烟叶制品受到的影响尤为严重^[1]。烟草甲幼虫以烟叶为食，尤其偏好蛀食储存1-2年的正在醇化的烟叶。幼虫通常藏匿在烟叶的皱褶处，蛀食烟叶导致其穿孔和破碎。同时，取食过程中产生的虫粪、虫尸和虫蜕混杂在烟叶中，严重影响其品质^[2]。随着全球烟草产业的扩张，烟草的储存与销售规模日益扩大，烟草甲带来的损失也在不断增加^[3]。在我国由烟草甲造成的经济损失约占储烟害虫造成的总损失的98%以上^[4]。全球每年因烟草甲造成的烟叶经济损失超过3亿美元^[5]。目前，化学防治因其短期有效性而成为主要的防治策略，但由于化学药剂的不合理使用，加之烟草甲体型小、食性广、繁殖力强等特点，导致其对多种化学熏蒸剂产生较强的抗性，农药的效果逐渐减弱。在此背景下，基于昆虫共生微生物调控的生物防治技术因其环境友好性和作用靶向性优势正成为烟草甲综合治理体系中的研究热点。

昆虫体内的共生微生物在昆虫的生长和发育中发挥着重要作用^[6-7]，这些微生物不仅为宿主昆虫提供其自身无法合成的必需氨基酸^[8]，还参与宿主的物质代谢^[9]，并影响宿主的生长和繁殖^[10]、解毒^[11]、防御^[12]以及信息素合成^[13]等过程。昆虫体内的微生物主要包括细菌、古菌、原生动物、真菌和病毒等^[14]，其多样性显著受食物影响^[15]。研究表明食物会影响鞘翅目、膜翅目、鳞翅目、双翅目、直翅目和半翅目等昆虫的肠道微生物组成。例如，茶尺蠖^[16]、蚜虫^[17]、小菜蛾^[18]、家蚕^[19]和松墨天牛^[20]等昆虫在不同食料下的肠道菌群结构存在显著差异，其优势菌群也有所不同。此外，不同饮食习惯的甲虫(如肉食性、草食性、杂食性和腐食性)之间肠道真菌群落也表现出明显差异^[21]。研究表明昆虫共生菌对昆虫的抗药性具有显著影响。例如，点蜂缘蝽的内共生菌——伯克霍尔德菌氏菌属(Burkholderia)细菌能够降解有机磷农药杀螟松^[22]；小菜蛾肠道中的共生菌——蜡样芽孢杆菌(Bacillus cereus)能够降解茚虫威^[23]；褐飞虱体内的解脂假丝酵母共生菌对吡虫啉的抗药性与其体内羧酸酯酶和多功能氧化酶活性的增强密切相关^[24]；草地贪夜蛾的野生品系体内微生物多样性高于实验室杀虫剂敏感品系和抗杀虫剂品系^[25]。环丙沙星能够降低梨小食心虫体内微生物多样性，导致其幼虫发育周期延长、成虫寿命缩短，并降低繁殖力^[26]。因此微生物群落组成的多样性与害虫防治密切相关，其结构和功能的变化可能对害虫的抗药性和生存能力产生重要影响。

近年来，关于烟草甲体内微生物的研究逐渐深入，主要集中在烟碱降解菌的筛选与鉴定，以及体内细菌群落的多样性分析。研究表明烟草甲内生菌Mixta sp. J1具有显著的烟碱降解能力^[27]，这一特性为其在高烟碱含量的烟草环境中生存提供了关键支持。烟草甲体内还存在一种类酵母共生菌(yeast-like symbionts, YLS)——Symbiotaphrina kochii^[28]。该共生菌不仅能为宿主昆虫提供甾醇^[29]，还具有直接分解烟碱的能力^[30]。YLS可以产生水解酶代谢食物中的有毒物质^[31]。值得注意的是，外源化合物能够诱导YLS产生酯酶同工酶^[32]，这种酶对广谱性杀虫剂具有解毒作用，从而增强烟草甲对农药的抗性。

随着高通量测序技术的快速发展，研究者对仓储害虫体内微生物群落的多样性有了更深入的认识。通过对锈赤扁谷盗、长角扁谷盗、烟草甲、锯谷盗和印度谷螟5种仓储害虫体内微生物的16S rRNA基因V3-V4变异区进行测序分析，发现沃尔巴克氏体属(Wolbachia)是烟草甲和锯谷盗的优势菌属，物种的亲缘关系越近其体内微生物群落的组成也越相似^[33]，进一步研究揭示了烟草甲体内微生物群落的动态特性。研究表明当烟草甲在不同食物来源中连续饲养 6代(暴露阶段)后，其微生物群落的多样性和丰富度达到峰值；当烟草甲重新引入其原始食物来源并维持6代(回归阶段)时，其微生物群落的组成和结构逐渐恢复至初始状态^[34]。目前关于烟草甲体内真菌群落结构的研究仍相对有限，特别是不同食料对烟草甲体内真菌多样性和群落组成的影响尚未明确。本研究通过内转录间隔区(internal transcribed spacer, ITS)全长扩增子测序技术，分析不同食料条件下烟草甲体内真菌的多样性与群落结构，以期为构建基于微生物互作网络精准调控的害虫治理新技术提供理论依据与关键靶标。

1 材料与方法

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1.1 材料

1.1.1 供试虫源

野生环境组(wild environment, WF)的烟草甲采集自广东中烟公司烟叶贮藏仓库的烟叶。实验室早期已成功构建人工饲料组(artificial feed, SL)和烟草驯化组(tobacco domestication, YC)的烟草甲实验室种群。目前，人工饲料组和烟草驯化组的烟草甲均在华南农业大学绿色农药全国重点实验室进行饲养，并已连续培养超过15代。所有烟草甲均饲养于人工气候培养箱中，饲养条件设定为：温度(29±0.5) ℃、相对湿度(60±5)%、黑暗培养。

1.1.2 饲料

人工饲料组(SL)的饲料配方：全麦粉70%，麦麸20%和酵母粉10%；烟草驯化组(YC)的饲料配方：烟粉末80%和麦麸20%；野生环境组(WF)则直接取食烤烟烟叶。

1.1.3 培养基

马铃薯葡萄糖琼脂培养基(PDA)的配制方法^[35](g/L)：去皮土豆200，葡萄糖20，琼脂20。

1.2 烟草甲样品收集

收集生长状态相对一致的烟草甲3龄幼虫，每个样品设置6个重复，每个重复包含15头幼虫(由于野生环境组样品较为珍贵，仅设置了3个重复)。使用毛笔清除虫体表面黏附的饲料碎屑，随后将其浸泡在75%的乙醇中30 s，用去离子水浸泡15 s，该过程重复2次。用厨房纸吸干水分，将样本迅速转移至1.5 mL离心管中，立即置于液氮中速冻，并存放于-80 ℃冰箱中备用。

1.3 样品建库测序

参照粪便/土壤基因组DNA提取试剂盒(南京诺唯赞生物科技股份有限公司)的说明书提取烟草甲内真菌基因组DNA的，采用引物F (中间为barcode) (5′-CTACACGACGCTCTTCCGAT CTTATCGGTGCACTTGGTCATTTAGAGGAAG TAA-3′)和引物R (中间为barcode) (5′-AAGCA GTGGTATCAACGCAGAGCTGCGTAACTCCGTGTTTCAAGACGGG-3′)进行PCR扩增^[36]。随后，对PCR产物进行纯化、定量和均一化处理后，以构建测序文库(SMRT Bell)。文库质检后使用PacBio平台进行测序。测序数据以BAM格式输出，通过SMRT Link分析软件导出CCS文件^[37]，根据barcode序列识别不同样品的数据，并转换为FASTQ格式。上述实验由康圣序源生物科技(武汉)有限公司完成。

1.4 数据分析

样品的全长ITS扩增测序后获得了HiFi数据。采用FASTP软件对原始序列(raw reads)进行质量过滤^[38]以获得高质量的测序数据(clean reads)。运用Cutadapt v4.4软件去除引物序列^[39]，并通过VSEARCH v2.22.1去除嵌合体序列^[40]。使用QIIME 2中的VSEARCH命令进行聚类分析^[41]。同时，进行了物种注释和丰度分析，以揭示样品的物种组成特征。对HiFi序列进行聚类与去噪，划分操作分类单元(operational taxonomic units, OTUs) (以下简称为Feature)，并根据Feature的序列组成进行了物种分类。基于特征分析结果，在门、纲、目、科、属、种水平上绘制了样品的群落结构图、系统发育树及分类树图。通过α多样性分析，研究单个样品内部的物种多样性，统计各样品的Sobs、Chao1、ACE、Shannon和Simpson指数，并绘制样品稀释曲线和丰度等级曲线。通过β多样性分析比较不同样品的物种多样性(群落组成及结构)差异。

1.5 烟草甲体内真菌的离体培养与鉴定

选取3种不同食料饲养的生长状态相对一致的烟草甲3龄幼虫，每组样品5头。重复1.2节步骤后，向离心管中加入适量PBS缓冲液进行匀浆处理，将匀浆液均匀涂布于PDA培养基表面进行培养。待真菌生长后进行纯化，直至获得单菌落。将纯化后的菌株接种至PDA培养基上于28 ℃进行培养，待菌丝充分生长后观察单个菌落的形态特征，包括菌落正反两面的颜色及菌丝特征。挑取少量菌丝置于载玻片上，在光学显微镜下观察分生孢子、产孢结构及菌丝等显微特征。对纯化后的真菌进行DNA提取，以真菌DNA为模板，采用真菌内转录间隔区(internal transcribed spacer, ITS)通用引物ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)和ITS4 (5′- TCCTCCGCTTATTGATATGC-3′)^[42]进行扩增。PCR反应体系(40 μL)：2×T8 High-Fidelity Master Mix 20 μL，DNA 2 μL，正、反向引物(10 μmol/L)各2 μL，ddH₂O 14 μL。PCR反应条件：98 ℃ 2 min；98 ℃ 10 s，58 ℃ 10 s，72 ℃ 20 s，72 ℃ 5 min，37个循环。PCR产物回收后进行Sanger测序。将测序结果在NCBI数据库中进行BLAST比对，筛选与菌株序列同源性和覆盖度较高的已知模式菌株，并下载其相关序列。使用MEGA软件构建maximum likelihood系统发育树^[43]。结合真菌的形态特征和分子鉴定结果，确定烟草甲体内的真菌物种。

1.6 优势菌YLS表达水平分析

在显微镜下解剖出烟草甲的表皮、脂肪体、菌胞体、肠道和马氏管5个组织，并参照粪便基因组提取试剂盒[天根生化科技(北京)有限公司]说明书进行DNA提取。以提取的DNA为模板，采用烟草甲LsRPL18作为内参基因^[44]，进行RT-qPCR。根据菌株序列，使用Primer 5.0设计特异性引物^[45]，引物序列见表1，并由广州擎科生物技术有限公司合成。使用ChamQ Blue Universal SYBR qPCR Master Mix试剂(南京诺唯赞生物科技股份有限公司)进行RT-qPCR扩增。RT-qPCR反应体系(20 μL)：2×ChamQ Blue Universal SYBR qPCR Master Mix 10 μL，DNA 1 μL，正、反向引物(10 μmol/L)各0.4 μL，ddH₂O 8.2 μL。RT-qPCR反应条件：95 ℃ 30 s；95 ℃ 10 s，60 ℃ 30 s，共40个循环。溶解曲线条件：95 ℃ 10 s，溶解曲线65.0-95.0 ℃，每次增加0.5 ℃，持续5 s；读板检测。采用2^-ΔΔ^C^t法^[46]分析比较同一目的基因在不同食料及不同组织的相对表达量。内参基因和目的基因均进行3个重复。

2 结果与分析

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2.1 物种丰度分析

在人工饲料组(SL)、烟草驯化组(YC)和野生环境组(WF)的15个样品中，真菌ITS区域的有效序列占原始序列的98.00%以上(表2)。当测序条数达到3 000后样品稀释曲线逐渐趋于平稳，表明测序深度已基本覆盖样品中的绝大多数微生物物种(图1A)。当测序条数达到3 000后Shannon指数曲线也趋于平稳，说明测序数据量充足，样本中的主要微生物物种已被充分捕获，进一步增加测序量不会显著提升分析效果(图1B)。在对不同食料条件下烟草甲体内真菌的等级丰度曲线分析中，发现人工饲料组(SL)的物种丰富度和均匀度均高于烟草驯化组(YC)和野生环境组(WF)(图1C)。这表明本次测序结果质量可靠，测序深度已充分覆盖样品中的绝大多数物种信息，能够真实反映样品的微生物群落组成特征。

2.2 OTU聚类及注释

在OTU水平上，烟草甲人工饲料组(SL)体内真菌的总物种数量为35种，高于烟草驯化组(YC)的32种和野生环境组(WF)的15种(表3)。3组共有的真菌物种数量为11种，分别占人工饲料组(SL)、烟草驯化组(YC)和野生环境组(WF)样品真菌总物种数的31.43%、34.38%和73.33%。韦恩图中重叠部分代表不同食料条件下烟草甲共有的特征数量，非重叠部分则表示特有的特征数量。通过韦恩图分析(图2)可见，人工饲料组(SL)和烟草驯化组(YC)烟草甲的OTU数量显著高于野生环境组(WF)，表明人工饲料组(SL)和烟草驯化组(YC)的烟草甲体内真菌多样性更高。

2.3 物种群落结构分析

分类堆积图(图3)中各颜色区域代表不同门类的真菌，其面积大小表示该类真菌在群落中的相对丰度。在门水平上，通过高通量测序共检测到2个真菌菌门。3种食料样品中共同存在的菌门包括子囊菌门(Ascomycota)和未鉴定真菌类群(Fungi Incertae sedis)。子囊菌门是3种食料中烟草甲的主要优势菌门，其占比分别为人工饲料组(SL) 82.38%、烟草驯化组(YC) 85.07%以及野生环境组(WF) 92.51%；未鉴定真菌类群的相对丰度在3种饲料中分别为17.62%、14.93%和7.49%，呈现出与子囊菌门相反的趋势(图3A)。在目水平上，Symbiotaphrinales是烟草甲体内的优势类群，其平均相对丰度高达86.41%；散囊菌目(Eurotiales)和肉座菌目(Hypocreales)仅在人工饲料组(SL)和烟草驯化组(YC)中检测到，其相对丰度较低，平均丰度分别为0.250%和0.001% (图3C)。在属水平上，共检测到4个真菌属。3种食料烟草甲样品中共有的是Symbiotaphrina，其在人工饲料组(SL)、烟草驯化组(YC)、野生环境组(WF)烟草甲体内的平均相对丰度分别为81.65%、85.06%和92.51%。此外，人工饲料组(SL)和烟草驯化组(YC)的烟草甲中还检测到其他真菌属，如曲霉属(Aspergillus)、耐干霉属(Xeromyces)和Leptobacillium，但其总平均相对丰度均低于1.00% (图3E)。综上所述，3种食料烟草甲体内的优势菌群是Symbiotaphrina真菌，优势菌群组成总体稳定，但其物种相对丰度存在动态变化。这说明饲料对烟草甲体内的优势真菌组成无明显影响，但对其相对丰度及真菌群落结构具有一定影响。

2.4 α多样性分析

人工饲料组(SL)的Sobs指数、Chao1指数和ACE指数显著高于烟草驯化组(YC)和野生环境组(WF)，表明人工饲料组(SL)烟草甲体内真菌群落的丰富度较高(图4A-4C)。其中，不同食料条件下烟草甲体内真菌OTU的Sobs指数存在显著差异(P<0.001)，而Chao1指数和ACE指数则未达到显著性水平。3组不同食料处理的烟草甲体内真菌的Shannon指数和Simpson指数均无显著差异(P>0.05)，说明其多样性和均匀度未受到显著影响(图4D、4E)。综合真菌α多样性指数，烟草驯化组(YC)和野生环境组(WF)烟草甲体内真菌群落的丰富度显著低于人工饲料组(SL)。这一现象表明，烟草驯化组(YC)和野生环境组(WF)样品中优势菌群的比例较高，但整体真菌种类较少。相比之下，人工饲料组(SL)烟草甲体内真菌群落呈现出较高的物种丰富度，提示食料可能影响了烟草甲体内真菌物种的存在和分布。该结果与物种丰度分布曲线一致，进一步证实了食料对烟草甲体内真菌群落的影响，同时表明3种不同食料处理下的真菌群落多样性和均匀度无显著差异。

2.5 β多样性分析

基于UPGMA聚类结果(图5A)，人工饲料组(SL)的烟草甲样品(SL-1、SL-2、SL-3、SL-4、SL-5、SL-6)聚为一组，野生环境组(WF)的样品(WF-1、WF-2、WF-3)则聚为另一组。烟草驯化组(YC)的样品(YC-1、YC-2、YC-3、YC-4)形成

第3组，且与野生环境组的距离较近，表明这2个组的物种同源性较高。针对烟草甲体内的真菌群落组成，人工饲料组(SL)、烟草驯化组(YC)和野生环境组(WF)样品可划分为3个不同的类群，反映这3组样品内的真菌群落存在显著差异(图5A)。基于Unweighted Unifrac距离的非限制性主坐标轴分析(PCoA)进一步验证了这一结果。PCoA分析结果显示，在不同食料条件下烟草甲的真菌群落显著分为3个类群，能够解释样本间96.44%的差异(R²=3.396 6，P=0.014)(图5B)。基于Unweighted Unifrac距离的非度量多维标定法(NMDS)分析也表明，不同食料条件下的烟草甲真菌群落存在显著差异。通常认为，当NMDS的stress值<0.2时，数据具有较高的可靠性。本研究中NMDS的stress值为0.027 4，表明分析结果可靠(图5C)。基于Unweighted Unifrac距离矩阵的箱型图显示，不同组间的差异显著大于组内差异，进一步证实了3种食料条件下β多样性的显著差异(图5D)。综上所述，研究表明食料是导致烟草甲体内真菌群落差异的重要影响因素之一。

2.6 物种组间差异分析

在属水平上，人工饲料组(SL)样品中Symbiotaphrina和曲霉属(Aspergillus)的相对丰度较高，分别为81.648 0%和0.641 3%；耐干霉属(Xeromyces)和Leptobacillium的相对丰度较低 (<0.100 0%)。在烟草驯化组(YC)样品中Symbiotaphrina的相对丰度高达85.062 3%；曲霉属(Aspergillus)、耐干霉属(Xeromyces)和Leptobacillium的相对丰度则较低(<0.010 0%)。在野生环境组(WF)样品中，仅检测到Symbiotaphrina，其相对丰度为92.506 0% (表4)。对比人工饲料组(SL)、烟草驯化组(YC)和野生环境组(WF)样品的结果表明，曲霉属(Aspergillus)和耐干霉属(Xeromyces)之间存在显著差异。同时，曲霉属(Aspergillus)和耐干霉属(Xeromyces)在人工饲料组(SL)烟草甲中的相对丰度显著高于烟草驯化组(YC)和野生环境组(WF)样品，而在野生环境组(WF)样品中均未检测到。

在种水平上，人工饲料组(SL)烟草甲中共检测到6个种(表5)，分别为Symbiotaphrina kochii、Symbiotaphrina buchneri、帚状曲霉(Aspergillus penicillioides)、赤曲霉(Aspergillus ruber)、双孢霉菌(Xeromyces bisporus)和Leptobacillium leptobactrum。烟草驯化组(YC)烟草甲中共检测到7个种，分别为S. kochii、S. buchneri、S. microtheca、帚状曲霉(A. penicillioides)、双孢霉菌(X. bisporus)和L. leptobactrum。在野生环境组(WF)烟草甲中仅检测到3个种，分别为S. kochii、S. buchneri和S. microtheca。在3种不同食料下，对烟草甲体内真菌的比较显示，人工饲料组(SL)中的帚状曲霉(A. penicillioides)和赤曲霉(A. ruber)的丰度显著高于烟草驯化组(YC)和野生环境组(WF)。然而，其S. kochii的丰度显著低于野生环境组(WF)，S. microtheca仅存在于烟草驯化组(YC)和野生环境组(WF)中，而双孢霉(X. bisporus)则仅存在于人工饲料组(SL)和烟草驯化组(YC)的烟草甲中。

LDA值分布柱状图显示，烟草甲中的真菌群落存在显著差异。柱状图的高度反映了不同真菌对组间差异的贡献程度，柱状图越高表明该物种对样品组间差异的贡献越大。在种水平上，人工饲料组(SL)中LDA值较高的物种包括耐干霉属的双孢霉(X. bisporus)、曲霉属的帚状曲霉(A. penicillioides)和赤曲霉(A. ruber)，表明这些物种是人工饲料组的特异性标志菌。相比之下，烟草驯化组(YC)和野生环境组(WF)的标志菌主要为S. buchneri (图6A-6C)。食料的差异显著影响了烟草甲体内真菌的种类和数量，部分真菌仅在某些特定的食料条件下存在。这一发现表明不同食料可以直接影响烟草甲及其体内真菌的群落结构。

2.7 真菌群落功能预测分析

根据FUNGuild分析可获得样本中真菌的功能分类及其在不同样本中的丰度信息。在相对丰度大于0.01%的条件下，不同食料烟草甲真菌的群落功能组成与丰度可分为2种营养模式和2个生态功能群(表6)。对于不同食料烟草甲真菌样品，烟草甲人工饲料组(SL)的35个OTUs中有25个OTUs被划分为2个真菌功能类群，约占总OTUs数量的71.43%。其中，共生营养型为主要营养类型，主要功能类群为动物共生菌，主要涉及Symbiotaphrina。曲霉属(Aspergillus)和耐干霉属(Xeromyces)的营养类型为腐生营养型，功能群为未定义腐生真菌。在烟草甲烟草驯化组(YC)的32个OTUs中有7个OTUs被划分为1个真菌功能类群，约占总OTUs数量的21.89%。在烟草甲的野生环境组(WF)中，15个OTUs中有9个OTUs被划分为一个真菌功能类群，约占总OTUs数量的60.00%。这2个组的营养类型均为共生营养型，功能类群均为动物共生菌。此外，烟草甲烟草驯化组(YC)的7个OTUs和野生环境组(WF)的 9个OTUs均只涉及1个门[子囊菌门(Ascomycota)]和1个属(Symbiotaphrina)。

2.8 烟草甲体内真菌的离体培养和鉴定

对不同食料的烟草甲3龄幼虫体内的可培养真菌进行了分离和鉴定。在PDA培养基上，人工饲料组(SL)分离得到3株真菌，分别命名为SL-1、SL-2和SL-3；烟草驯化组(YC)分离得到3株真菌，分别命名为YC-1、YC-2和YC-3；野生环境组(WF)分离得到2株真菌，分别命名为WF-1和WF-2。所有真菌的菌落形态、孢子形态及光学显微特征如表7、图7、图8所示。

为进一步鉴定这些分离的真菌，进行了分子生物学分析，结果如表8、图9所示。通过NCBI数据库进行BLAST比对，选取ITS序列相似的片段构建系统发育树。结合形态学特征，初步鉴定以下菌株：SL-1、YC-1、WF-1为Symbiotaphrina kochii；SL-2、YC-2、YC-3为嗜松蓝状菌(Talaromyces pinophilus)；SL-3为鲜红青霉(Penicillium chermesinum)；WF-2为马洛青霉(Penicillium mallochii)。SL-1、YC-1、WF-1的序列一致性为100.00%，SL-2、YC-2、YC-3的序列一致性为99.64%。

2.9 YLS在不同食料以及不同组织的表达情况

RT-qPCR结果显示，YLS在野生环境组(WF)中的表达量显著高于人工饲料组(SL)和烟草驯化组(YC) (图10A)，这与高通量测序结果一致(图3)。此外，研究发现烟草甲YLS在菌胞体中的表达水平最高，同时在脂肪体和肠道中也有表达，且表达水平存在显著差异，约为脂肪体的14倍和肠道的33倍，而在表皮和马氏管中表达量很低或不表达(图10B)。

3 讨论与结论

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本研究采用PacBio三代全长扩增子测序技术对不同食料烟草甲体内的真菌多样性进行了分析。结果显示，烟草甲体内的优势真菌为子囊菌门，但其整体真菌丰富度相对较低(表3、图3)。与传统的第二代测序技术相比，第三代测序技术的长读长优势能够更准确地鉴定真菌物种，特别是基因组中含有复杂重复序列的物种^[47]。研究表明烟草中含有茄尼醇、绿原酸、芸香苷及生物碱等具有抑菌作用的活性物质^[48-49]。在3种食料来源的烟草甲中，人工饲料组(SL)的真菌群落丰富度显著高于烟草驯化组(YC)和野生环境组(WF)，这可能与仓库环境的单一性以及烟草中的抑菌活性物质有关。

类酵母共生菌(YLS)是生活在昆虫体内、形态和功能与酵母相似的微生物，它们通常寄生在昆虫的特定组织或器官，如脂肪体、菌胞体、马氏管或中肠，并与宿主形成互利共生关系。类酵母共生菌通过出芽生殖的方式繁殖，并经由卵母细胞垂直传播，通常附着在卵壳表面，幼虫取食卵壳时被摄入^[50]。此类共生菌广泛存在于半翅目^[51-53](如灰飞虱、白背飞虱、蚜虫、紫胶虫)和鞘翅目^[54](如烟草甲、药材甲)昆虫中。研究表明烟草甲体内的类酵母共生菌S. kochii在其营养需求和外源物质的解毒中具有重要作用。该共生菌不仅能为烟草甲提供甾醇^[29]，还能够分解烟碱并产生多种解毒酶^[31]，例如水解酶和乙酰胆碱酯酶，从而帮助宿主代谢有毒物质。进一步研究发现，食物种类显著影响烟草甲体内的类酵母共生菌数量和活性。这些共生菌的数量与烟草甲的生长发育呈负相关关系，表明共生菌的数量可能直接影响烟草甲的生长发育及其对农药的敏感性^[30]。研究发现同为鞘翅目窃蠹科的仓储害虫药材甲的类酵母共生菌YLS为Symbiotaphrina buchneri^[25]，但其功能尚未见报道。本研究表明食料不会改变烟草甲的优势菌，但其相对丰度存在动态变化(表5)。YLS主要存在于烟草甲前肠与中肠交接处的菌胞体中，其在菌胞体的表达量显著高于其他组织，约为脂肪体的14倍，肠道的33倍(图10)。这一结果与前人关于食料对烟草甲体内YLS数量影响以及YLS在烟草甲体内分布的研究结果一致。在烟草驯化组(YC)和野生环境组(WF)中检测到了S. buchneri，推测其存在可能与烟草有关，可能参与烟草甲有毒物质的降解和代谢。

单宁是一类难以降解且对生物具有毒害作用的次生代谢产物^[55]，能够与蛋白质、淀粉、纤维素和矿物质等物质形成稳定的复合物。这种复合物会减少动物的摄食量，降低蛋白质的消化率，并抑制消化酶的活性^[56]。经过单宁酶处理的饲料中单宁含量显著降低，营养成分得到改善，饲料的适口性和消化吸收率也有所提高^[57]。赤曲霉(A. ruber)在固态发酵条件下能够产生单宁酶^[58]，麦麸曾作为微生物产单宁酶的底物^[59]。本研究中人工饲料组(SL)的烟草甲体内赤曲霉(A. ruber)的相对丰度(0.206 1%)显著高于烟草驯化组(YC) (0.002 0%)，而在野生环境组(WF)的烟草甲体内未检测到赤曲霉(A. ruber)(表5)。由于人工饲料组(SL)和烟草驯化组(YC)的饲料中均含有麦麸，并且人工饲料组(SL)的营养成分在3种食料中最为丰富，因此推测赤曲霉(A. ruber)的相对丰度可能与食料的营养成分有关。

帚状曲霉(A. penicillioides)具有嗜旱、嗜渗透和嗜盐的特性^[60-61]，而双孢霉菌(X. bisporus)则是一种耐糖嗜旱菌^[62]。这2种真菌主要生活在低水活度、高糖和高盐的环境中。本研究发现，在人工饲料组(SL)的烟草甲中帚状曲霉 (A. penicillioides)和双孢霉菌(X. bisporus)的相对丰度显著高于烟草驯化组(YC)和野生环境组(WF) (表5)。这种现象可能与食物的物理性质密切相关。人工饲料组(SL)中的全麦粉和酵母粉吸水性较差，导致环境相对干燥，且全麦粉中含有淀粉、纤维素等糖类物质。因此推测帚状曲霉(A. penicillioides)与双孢霉菌X. bisporus的存在可能是烟草甲为了适应人工饲料的干燥环境而采取的一种生存策略。

相较于昆虫体内细菌的研究，目前国内外关于昆虫体内真菌的研究报道相对较少。薛宝燕等^[28]从烟草甲菌胞体中分离出了S. kochii。何欢^[63]采用研磨法从不同食料的大蜡螟中分离出 6株青霉属真菌和1株蓝状菌属真菌，并发现不同饲养条件下真菌种类存在差异。张珊等^[64]运用传统微生物分离纯培养法对思茅松毛虫4龄幼虫的肠道内生真菌进行分离，获得8株青霉属真菌。刘柳^[65]通过传统微生物分离方法从秦岭鳞翅目昆虫中分离出24株青霉属真菌。房晗^[66]从茶尺蠖肠道中分离出青霉属真菌，并对其抗菌活性进行了研究。刘丽君^[67]从蜻蜓肠道中分离到6株蓝状菌属真菌，发现T. pinophilus和Talaromyces sp.能够抑制球孢白僵菌。Makulana等^[42]从金龟子科昆虫Pachylomerus femoralis、Anachalcos convexus、Euoniticellus intermedius幼虫中筛选和分离木聚糖酶丝状真菌，共分离出28株青霉属真菌以及7株蓝状菌属真菌。本研究通过体外分离获得了8株真菌，均属于子囊菌门。其中，3株属于Symbiotaphrina，3株属于蓝状菌属(Talaromyces)，2株属于青霉属(Penicillium)(图7、图8)。具体而言，SL-1、YC-1和WF-1为S. kochii，属于Symbiotaphrina属；SL-2、YC-2和YC-3为嗜松蓝状菌(T. pinophilus)，属于蓝状菌属(Talaromyces)；SL-3为鲜红青霉(P. chermesinum)，WF-2则为P. mallochii，二者均属于青霉属(Penicillium) (图9)。在3种不同食料条件下成功分离出了S. kochii，进一步证实了Symbiotaphrina是烟草甲体内的优势菌群，这一结果与高通量测序结果一致。本研究分离到的S. kochii与薛宝燕^[30]的研究结果相符，但其研究仅限于YLS。此外，还分离到了其他属的真菌，如青霉属(Penicillium)，这一结果与张珊等^[64]和刘柳^[65]的研究结果一致。这表明在不同食料条件下烟草甲体内的真菌组成存在差异。由于传统分离培养技术的局限性，无法全面反映烟草甲体内真菌的多样性。因此进一步对3种食料进行了ITS全长扩增子测序，以排除食料对烟草甲体内真菌的潜在污染(结果未展示)。在相对丰度排名前10的菌群中，Symbiotaphrina作为烟草甲优势菌群未在人工饲料(YCFD)中检测到，但在烟草驯化组饲料(YCFD)和野生环境组饲料(WFFD)中均被发现。值得注意的是，未检测到青霉属(Penicillium)和蓝状菌属(Talaromyces)真菌，表明烟草甲离体培养获得的真菌并非来源于饲料。然而在饲料中发现了Leptobacillium和曲霉属(Aspergillus)真菌，推测其可能来源于饲料。目前关于青霉属(Penicillium)和蓝状菌属(Talaromyces)真菌是否为烟草甲的共生菌仍存在诸多不确定性。尽管通过一系列实验对青霉属等真菌与烟草甲之间的关系进行了初步探究，但由于共生关系的复杂性，短期内难以确凿验证二者之间是否存在真正的共生关系。

本研究利用ITS全长三代扩增子测序技术分析和鉴定了3种不同食料对烟草甲体内真菌群落结构和多样性的影响。研究发现饲料类型显著重塑烟草甲肠道真菌群落结构，但其核心菌群组成保持相对稳定。通过传统微生物分离培养技术成功获得8株子囊菌门菌株，并运用RT-qPCR技术揭示了优势共生菌Symbiotaphrina kochii在宿主体内的特异性组织表达模式。本研究阐明了食料通过“营养-菌群”互作网络调控烟草甲肠道真菌群落构建的生态机制，从进化生物学角度证实Symbiotaphrina属真菌在宿主适应性进化中的关键作用，为研发基于微生物互作网络精准调控的害虫生态防控技术提供了理论依据与关键靶标。

基金

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广东省自然科学基金(2023A1515010305)
广东中烟工业有限责任公司项目(粤烟工[2021]科字第031号)

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2025年第65卷第9期

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doi: 10.13343/j.cnki.wsxb.20250066

接收时间：2025-01-22
首发时间：2026-02-07
出版时间：2025-09-04

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收稿日期：2025-01-22
录用日期：2025-05-17

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Natural Science Foundation of Guangdong Province(2023A1515010305)

广东省自然科学基金(2023A1515010305)

Project of Guangdong China Tobacco Industry Co., Ltd. (GDTPI [2021] Science & Technology 031)

广东中烟工业有限责任公司项目(粤烟工[2021]科字第031号)

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1 华南农业大学植物保护学院，绿色农药全国重点实验室，广东广州

2 广东中烟工业有限责任公司，广东广州

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Primers name	Primer sequences (5′→3′)
LsRPL18-F	CCAAGATGTTTACCTGCGAC
LsRPL18-R	CATTAAGCTGGCTACCTTCG
YLS-F	GCAGCGAAATGCGATAAGTAA
YLS-R	CCCTTAACCGGACGATGACCCAATA

Primers name

Primer sequences (5′→3′)

LsRPL18-F

CCAAGATGTTTACCTGCGAC

LsRPL18-R

CATTAAGCTGGCTACCTTCG

YLS-F

GCAGCGAAATGCGATAAGTAA

YLS-R

CCCTTAACCGGACGATGACCCAATA

Sample ID	Raw reads	Clean reads	Effective reads	Effective rate (%)
SL-1	38 810	38 810	38 751	99.85
SL-2	41 036	41 036	40 975	99.85
SL-3	39 910	39 910	39 803	99.73
SL-4	33 869	33 869	33 783	99.75
SL-5	35 322	35 322	35 276	99.87
SL-6	33 019	33 019	32 970	99.85
YC-1	42 260	42 260	41 499	98.20
YC-2	39 209	39 209	39 074	99.66
YC-3	39 992	39 992	39 905	99.78
YC-4	43 878	43 878	43 699	99.59
YC-5	38 380	38 380	38 313	99.83
YC-6	43 447	43 447	43 374	99.83
WF-1	34 601	34 601	34 233	98.94
WF-2	40 264	40 264	40 029	99.42
WF-3	37 843	37 843	37 395	98.82

Sample ID

Raw reads

Clean reads

Effective reads

Effective rate (%)

SL-1

38 810

38 751

99.85

SL-2

41 036

40 975

99.85

SL-3

39 910

39 803

99.73

SL-4

33 869

33 783

99.75

SL-5

35 322

35 276

99.87

SL-6

33 019

32 970

99.85

YC-1

42 260

41 499

98.20

YC-2

39 209

39 074

99.66

YC-3

39 992

39 905

99.78

YC-4

43 878

43 699

99.59

YC-5

38 380

38 313

99.83

YC-6

43 447

43 374

99.83

WF-1

34 601

34 233

98.94

WF-2

40 264

40 029

99.42

WF-3

37 843

37 395

98.82

Group	OTU	Phylum	Class	Order	Family	Genus
SL	35	1	3	3	3	4
YC	32	1	3	3	3	3
WF	15	1	1	1	1	2

Group

OTU

Phylum

Class

Order

Family

Genus

Genus name	SL (%)	YC (%)	WF (%)
Symbiotaphrina	81.648 0	85.062 3	92.506 0
Fungi Incertae sedis	17.621 0	14.928 8	7.494 0
Aspergillus	0.641 3	0.006 5	0.000 0
Xeromyces	0.088 3	0.000 4	0.000 0
Leptobacillium	0.001 4	0.002 0	0.000 0

Genus name

SL (%)

YC (%)

WF (%)

Symbiotaphrina

81.648 0

85.062 3

92.506 0

Fungi Incertae sedis

17.621 0

14.928 8

7.494 0

Aspergillus

0.641 3

0.006 5

0.000 0

Xeromyces

0.088 3

0.000 4

0.000 0

Leptobacillium

0.001 4

0.002 0

0.000 0

Species name	SL (%)	YC (%)	WF (%)
Symbiotaphrina kochii	81.635 8	83.614 5	86.435 7
Fungi Incertae sedis	17.621 0	14.928 8	7.494 0
Symbiotaphrina buchneri	0.012 2	1.447 0	4.672 1
Symbiotaphrina microtheca	0.000 0	0.000 8	1.398 2
Aspergillus penicillioides	0.435 3	0.004 5	0.000 0
Aspergillus ruber	0.206 1	0.002 0	0.000 0
Xeromyces bisporus	0.088 3	0.000 4	0.000 0
Leptobacillium leptobactrum	0.001 4	0.002 0	0.000 0

Species name

SL (%)

YC (%)

WF (%)

Symbiotaphrina kochii

81.635 8

83.614 5

86.435 7

Fungi Incertae sedis

17.621 0

14.928 8

7.494 0

Symbiotaphrina buchneri

0.012 2

1.447 0

4.672 1

Symbiotaphrina microtheca

0.000 0

0.000 8

1.398 2

Aspergillus penicillioides

0.435 3

0.004 5

0.000 0

Aspergillus ruber

0.206 1

0.002 0

0.000 0

Xeromyces bisporus

0.088 3

0.000 4

0.000 0

Leptobacillium leptobactrum

0.001 4

0.002 0

0.000 0

Trophic mode	Guild	Relative abundance	Confidence ranking
Symbiotroph	Animal endosymbiont	81.640 4	85.048 2	92.504 2	Probable
Saprotroph	Undefined saprotroph	0.729 6	/	/	Possible

Trophic mode

Guild

Relative abundance

Confidence ranking

SL (%)

YC (%)

WF (%)

Symbiotroph

Animal endosymbiont

81.640 4

85.048 2

92.504 2

Probable

Saprotroph

Undefined saprotroph

0.729 6

Possible

Sample ID	Colony morphology	Spore morphology
SL-1	Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium	Spores are spherical or oval or irregularly shaped
SL-2	The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors	Conidiophores whorled on broomlike branches; peduncle erect, usually with transverse septum, hyaline; peduncle bearing multiple pedicels apically, pedicels almost equal in length, some pedicels inflated apically, conidiophores ellipsoidal or spindle-shaped
SL-3	Colonies are gray-green, powdery, yellow on the back, fading in color toward the edges	Conidiophores broomlike, in single whorls, vittae shorter, conidia globose
YC-1	Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium	Spores are spherical or oval or irregularly shaped
YC-2	The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors	Conidiophores in multiple whorls of broomlike branches; peduncle stems erect, usually with transverse septa, hyaline; peduncle stems bearing multiple columnar peduncles apically, peduncles almost equal in length, sometimes inflated apically, conidiophores globose
YC-3	The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors	Conidiophores in multiple whorls of broomlike branches; peduncle stems erect, usually with transverse septa, hyaline; peduncle stems bearing multiple columnar peduncles apically, peduncles almost equal in length, sometimes inflated apically, conidiophores globose
WF-1	Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium	Spores are spherical or oval or irregularly shaped
WF-2	Initial colony white filamentous, later colony surface color slowly become green powdery, the back of the colony slowly become yellow or orange, to the edge of the gradual lightening, the color becomes darker with time	Molecular spore pedicels broomlike, in single whorls, apical pedicels shorter, molecular spores globose, blue-green

Sample ID

Colony morphology

Spore morphology

SL-1

Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium

Spores are spherical or oval or irregularly shaped

SL-2

The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors

Conidiophores whorled on broomlike branches; peduncle erect, usually with transverse septum, hyaline; peduncle bearing multiple pedicels apically, pedicels almost equal in length, some pedicels inflated apically, conidiophores ellipsoidal or spindle-shaped

SL-3

Colonies are gray-green, powdery, yellow on the back, fading in color toward the edges

Conidiophores broomlike, in single whorls, vittae shorter, conidia globose

YC-1

Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium

Spores are spherical or oval or irregularly shaped

YC-2

The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors

Conidiophores in multiple whorls of broomlike branches; peduncle stems erect, usually with transverse septa, hyaline; peduncle stems bearing multiple columnar peduncles apically, peduncles almost equal in length, sometimes inflated apically, conidiophores globose

YC-3

The mycelium is white and fluffy at the beginning of the colony, turning green at a later stage, and the back of the colony shows concentric whorls of yellowish or pinkish colors

WF-1

Colonies are round, slightly elevated in the center, smooth and shiny surface, creamy white, without mycelium

Spores are spherical or oval or irregularly shaped

WF-2

Initial colony white filamentous, later colony surface color slowly become green powdery, the back of the colony slowly become yellow or orange, to the edge of the gradual lightening, the color becomes darker with time

Molecular spore pedicels broomlike, in single whorls, apical pedicels shorter, molecular spores globose, blue-green

Strain number	Accession number	Identity (%)	Similar species
SL-1	PQ844595	99.46	Symbiotaphrina kochii
SL-2	PQ844596	100.00	Talaromyces pinophilus
SL-3	PQ844597	99.82	Penicillium chermesinum
YC-1	PQ844598	99.46	Symbiotaphrina kochii
YC-2	PQ844599	100.00	Talaromyces pinophilus
YC-3	PQ844600	100.00	Talaromyces pinophilus
WF-1	PQ844601	99.46	Symbiotaphrina kochii
WF-2	PQ844602	100.00	Penicillium mallochii

Strain number

Accession number

Identity (%)

Similar species

SL-1

PQ844595

99.46

Symbiotaphrina kochii

SL-2

PQ844596

100.00

Talaromyces pinophilus

SL-3

PQ844597

99.82

Penicillium chermesinum

YC-1

PQ844598

99.46

Symbiotaphrina kochii

YC-2

PQ844599

100.00

Talaromyces pinophilus

YC-3

PQ844600

100.00

Talaromyces pinophilus

WF-1

PQ844601

99.46

Symbiotaphrina kochii

WF-2

PQ844602

100.00

Penicillium mallochii