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肠道为昆虫的一个重要器官,由于肠道的特殊生态环境,其中栖息着大量的微生物。昆虫肠道微生物与宿主昆虫的生命活动密切相关,参与摄食、消化、排泄及繁殖等一系列过程,同时还与有毒物质的降解、信息素的合成以及宿主免疫反应等有关[1-3]。植食性昆虫肠道微生物群落的结构与多样性受到所食植物的影响。在对棉铃虫(Helicoverpa armigera)[4]、菜粉蝶(Pieris rapae)[5]、小菜蛾(Plutella xylostella)[6]、松异舟蛾(Thaumetopoea pityocampa)[7]等昆虫的研究中证实,肠道细菌群落的组成与昆虫所食植物叶际细菌群落的组成相似,表明食物是植食性昆虫肠道微生物的重要来源。植食性昆虫啃食植物的同时会给植物造成大量伤口,这些伤口使植物更易受到病原微生物的侵入[8-9]。同时,肠道是病原微生物定殖的重要场所,部分病原微生物可在昆虫肠道内存活,昆虫则成为了病原微生物的越冬场所或来年病害的初侵染来源[10]。
油桐尺蛾(Buzura suppressaria)是为害桉树的重要灾害性食叶害虫,近年来的为害率和为害面积均呈快速上升趋势[11]。在以往的林间调查过程中,我们发现油桐尺蛾虫害与叶部病害经常在同一片桉树林中发生,但究竟是油桐尺蛾先为害然后引起病害侵入?还是病害先侵染桉树,引起植株产生生理生化改变,继而吸引油桐尺蛾为害?或者上述现象仅仅只是一个巧合?由于目前尚未有相关方面的报道,因此有必要对上述问题开展研究。目前对油桐尺蛾的研究主要集中在生物学、生理学、生态学、化学或生物防治等方面[12-14]。鉴于肠道微生物对油桐尺蛾的生命活动有重要影响,因此分析油桐尺蛾肠道微生物群落的结构和多样性,寻找与桉树叶部微生物的关联,对于研究油桐尺蛾与桉树间的互作机理、明确为害规律、寻找新的防治方法均具有重要意义。
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结果显示(表1),5个BS样品的细菌16s rDNA文库测序共获得3 271条有效序列,基于97%的序列相似性进行聚类分析,得到199个OTUs,共注释到6门、11纲、19目、38科、43属;真菌ITS rDNA文库测序共获得280 392条有效序列,得到4 279个OTUs,共注释到8门、29纲、67目、159科、257属。5个BCN样品细菌16s rDNA文库测序共获得11 413条有效序列,得到379个OTUs,共注释到10门、19纲、26目、54科、74属;真菌ITS rDNA文库为287 330条有效序列,获得4 236个OTUs,共注释到8门,31纲,79目,171科,291属。从属水平上看,BCN样品的细菌和真菌数目均多于BS样品。
表 1 BCN和BS样品OTU聚类及RDP分类水平统计
Table 1. OTU clustering and RDP classification level statistics of BCN and BS samples
样品
Sample检测项目
Test items有效reads数目/条
Reads numOTU数目/个
OUT numRDP分类
Classification of RDP门
Phylum纲
Class目
Order科
Family属
GenusBS 细菌 Bacteria 3 271 199 6 11 19 38 43 真菌 Fungus 280 392 4 279 8 29 67 159 257 BCN 细菌 Bacteria 11 413 379 10 19 26 54 74 真菌 Fungus 287 330 4 236 8 31 79 171 291 -
细菌和真菌群落的α多样性检测结果显示(表2),BCN和BS样品物种覆盖率均达97%以上,表明样品所构建的细菌和真菌文库均能有效反应其物种的多样性,且各组样品多样性指数组内偏差较小,数据可靠。BCN样品的细菌ACE指数和Chao l指数均明显高于BS样品,说明病害桉树叶内生细菌的群落丰富度较高,而上述两组样品的真菌ACE指数和Chao l指数则无显著差异,即两者的真菌群落丰富度差异较小。此外,BCN细菌和真菌的Shannon指数、覆盖率、Simpson指数较BS样品均无统计学差异,表明两组样品在细菌、真菌群落多样性、优势菌种的集中程度方面无明显差异。
表 2 BCN和BS样品α多样性指数统计
Table 2. Alpha diversity index statistics of BCN and BS samples
样品
Sample检测项目
Test itemsOTU数量
OUT numSimpson指数
Simpson indexShannon指数
Shannon indexACE指数
ACE indexChao1指数
Chao1 index覆盖率
coverageBS 细菌 Bacteria 39.80 ± 14.82 0.30 ± 0.10 1.90 ± 0.43 61.37 ± 24.07 53.55 ± 14.68 0.97 ± 0.01 真菌 Fungus 855.80 ± 168.88 0.10 ± 0.05 3.60 ± 0.47 1 098.59 ± 164.90 1 058.26 ± 156.59 1.0 ± 0.00 BCN 细菌 Bacteria 75.80 ± 32.71 0.21 ± 0.07 2.33 ± 0.57 109.82 ± 33.31a 104.12 ± 38.89a 0.98 ± 0.01 真菌 Fungus 847.20 ± 142.69 0.13 ± 0.13 3.50 ± 0.71 1 167.82 ± 103.06 1 094.02 ± 147.21 1.0 ± 0.00 注:a为与BS样品细菌检测组相比,P < 0.05
Note: P is compared with the BS samples in the bacterial detection group, P < 0.05 -
采用PCA法对BCN和BS样品的细菌和真菌群落进行主成分分析(图1A、1B)。从总体上看,BCN与BS样品大致分布在不同区域,表明BCN与BS样品间细菌、真菌的组成和结构存在较大差异。其中,细菌群落分析结果中有1组重复、真菌群落分析结果中有2组重复与其各自所属组别不在同一区域,可能是因为上述3组重复存在的偏差较大,从而导致PCA分析时出现误差,但还需做进一步验证。
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在门水平上(图2A),BCN样品和BS样品的细菌均主要由变形菌门(Proteobacteria)组成,分别占(92.66%~99.65%)和(91.16%~97.56%),即变形菌门为两组样品的优势菌门。
图 2 BCN和BS样品细菌在门(A)和属(B)水平的组成
Figure 2. Composition of bacteria in BCN and BS samples at the level of phylum (A) and genus (B)
在属水平上(图2B),BCN样品排名前五位的优势菌属分别为假单胞菌属(Pseudomonas)(12.24%~59.14%)、不动杆菌属(Acinetobacter)(6.37%~39.86%)、甲基杆菌属(Methylobacterium)(8.76%~39.11%)、拜叶林克氏菌属(Beijerinckia)(0.85%~18.53%)、鞘氨醇单胞菌属(Sphingomonas)(1.22%~12.09%);BS样品的优势菌属为假单胞菌属(52.10%~86.97%)和甲基杆菌属(4.42%~27.97%),而不动杆菌属(0.41%~1.66%)、拜叶林克氏菌属(0.2%~0.87%)和鞘氨醇单胞菌属(0.2%~0.62%)的占比较少。
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在真菌门分类水平上(图3A),BCN和BS样品的优势菌门均为担子菌门(Ascomycota),分别占78.68%~93.44%和64.42%~91.55%。
图 3 BCN和BS样品真菌在门(A)和属(B)水平的组成
Figure 3. Composition of BCN and BS samples at the phylum (A) and genus (B) levels
在属水平上(图3),BCN样品中排名前三位的优势菌属分别为平脐疣孢属(Zasmidium)(16.18%~60.82%)、假尾孢属(Pseudocercospora)(5.53%~16.58%)、球腔菌属(Mycosphaerella)(0.84%~10.47%),均为球腔菌科真菌;BS样品中排名前三位的优势菌属分别为平脐疣孢属(12.88%~40.68%)、假尾孢属(10.63%~28.59%)和叶点霉属(Phyllosticta)(4.06%~34.50%),球腔菌属(2.45%~12.33%)则排名第四位。
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BCN和BS样品细菌菌群丰度的T-Test分析结果表明(图4),BCN样品中硝化杆菌属(Nitrobacter)和苯基杆菌属(Phenylobacterium)的丰度明显低于BS样品(P < 0.05),而Terriglobus菌属和鞘脂菌属(Sphingobium)的丰度显著高于BS样品(P < 0.05)。真菌菌群丰度的T-Test分析结果显示(图5),BCN样品中叉丝单囊壳属(Podosphaera)、Pseudoteratosphaeria属、Zymoseptoria属的丰度均明显高于BS样品(P < 0.05);而炭疽菌属(Colletotrichum)、节担菌属(Wallemia)、曲霉菌属(Aspergillus)、Graphiola属、黑孢属(Nigrospora)、青霉菌属(Penicillium)、梗孢酵母属(Sterigmatomyces)的丰度均明显低于BS样品(P < 0.05)。
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采用T-Test分析法分别对BCN和BS样品中桉树主要病害病原菌所属菌属的丰度进行比较。结果显示(表3),BS样品中引起桉树炭疽病的炭疽菌属丰度明显高于BCN样品(P < 0.05)。而BCN和BS样品中引起青枯病的假单胞菌属(Pseudomonas)、引起叶斑病类的叶点霉属和假尾孢属(Pseudocercospora)、引起花斑病的短梗霉属(Aureobasidium)、引起紫斑病的壳针孢属(Septoria)、引起枝枯病的毛色二孢属(Lasiodiplodia)、引起梢枯病的新壳梭孢属(Neofusicoccum)的菌群丰度则无显著差异。
表 3 BCN和BS样品中桉树病害病原菌菌群丰度差异T-Test比较(属级水平)
Table 3. Comparison of abundance difference of Eucalyptus disease pathogenic bacteria in BCN and BS samples by T-Test (genus level)
菌属 Genus Freq1 Freq2 P值
P valueq值
q value效应量
Effect size95.0% 置信区间 95.0% CI 下限
Lower上限
UpperPseudomonas 40.534 71.146 0.071 1.000 −30.612 −64.790 3.566 Phyllosticta 0.081 14.517 0.059 1.000 −14.436 −29.774 0.903 Colletotrichum 0.005 0.020 0.046 1.000 −0.015 −0.029 0.000 Aureobasidium 0.000 0.009 0.115 1.000 −0.009 −0.021 0.003 Septoria 0.023 0.001 0.192 1.000 0.022 −0.017 0.062 Pseudocercospora 10.120 15.362 0.225 1.000 −5.242 −14.630 4.146 Lasiodiplodia 0.000 0.007 0.264 1.000 −0.007 −0.023 0.008 Neofusicoccum 0.083 0.033 0.267 0.990 0.050 −0.056 0.156 注:Freq1:桉树叶内菌属丰度;Freq2:油桐尺蛾中肠菌属丰度;q值:P值的校验值。
Note:Freq1: Abundance of bacteria or fungus in Eucalyptus leaves (genus level); Freq2: Abundance of bacteria or fungus inmidgut of Buzura suppressaria larvae (genera level); q value: The check value of the P value.
油桐尺蛾肠道菌群与桉树叶片内生菌差异分析
Comparison and Analysis of Diversified Intestinal Flora of Buzura suppressaria Larvae and Endophytes in Eucalyptus Leaves
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摘要:
目的 植食性昆虫肠道微生物的多样性与其食物密切相关,本研究通过探讨油桐尺蛾(Buzura suppressaria)幼虫肠道菌群及其取食的桉树叶内生菌群的多样性,分析两者之间的关系。 方法 利用Illumina Miseq技术对油桐尺蛾5龄幼虫肠道和桉树叶部内生的细菌16s rDNA和真菌ITS rDNA进行测序,分析油桐尺蛾肠道和桉树叶部内生的细菌和真菌的多样性和丰度。 结果 油桐尺蛾肠道细菌共注释到6门、11纲、19目、38科、43属,真菌共注释到8门、29纲、67目、159科、257属;桉树叶部内生细菌共注释到10门、19纲、26目、54科、74属,真菌共注释到8门,31纲,79目,171科,291属。桉树叶内和油桐尺蛾肠道中均发现桉树主要病害病原菌所属菌属,分别为炭疽菌属(Colletotrichum)、假单胞菌属(Pseudomonas)、假尾孢属(Pseudocercospora)、短梗霉属(Aureobasidium)、壳针孢属(Septoria)、毛色二孢属(Lasiodiplodia)、新壳梭孢属(Neofusicoccum)。 结论 油桐尺蛾肠道的细菌、真菌的多样性和丰度与桉树叶之间存在差异,桉树叶对油桐尺蛾肠道微生物的群落结构有重要影响,而携带某些病原菌的油桐尺蛾啃食或其排泄物接触可能是导致该病原菌侵染健康桉树叶的原因。 Abstract:Objective This study is to investigate the diversified intestinal flora of Buzura suppressaria larvae and Eucalyptus leaves which are the major food of B. suppressaria, and to analyze the inner relationship between them. Method Illumina Miseq technology was applied to sequence the bacteria 16S rDNA and fungi ITS rDNA in the intestinal of the 5th instar larvae and Eucalyptus leaves. Both the diversity and abundance of the bacteria and fungi in the intestinal and Eucalyptus leaves were analyzed. Result The results of bioinformatics analysis showed that the bacteria populations in the intestinal tract of B. Suppressaria larvae were annotated into 6 phyla, 11 classes, 19 orders, 38 families and 43 genus, whereas the fungi populations were categorized into 8 phyla, 29 classes, 67 orders, 159 families and 257 genus. The Endophytic bacteria found in Eucalyptus leaves included 10 phyla, 19 classes, 26 orders, 54 families and 74 genus, and the fungi included 8 phyla, 31 classes, 79 orders, 171 families and 291 genus. The main pathogenic bacteria of Eucalyptus were found in both of the Eucalyptus leaves and the larvae intestinal tract, including the orders of Colletotrichum, Pseudomonas, Pseudocercospora, Aureobasidium, Septoria, Lasiodiplodia and Neofusicoccum. Conclusion The diversity and abundance of bacteria and fungi populations in the intestinal tract of B. suppressaria larvae were different from those of the Eucalyptus leaves, which had an important influence on the intestinal microbial community structure. The gnawing or fecal contact of B. suppressaria larvae carrying certain pathogens may be the cause of the pathogen infecting healthy Eucalyptus leaves. -
Key words:
- Buzura suppressaria
- / Eucalyptus
- / intestinal flora
- / endophyte
- / microbial diversity
- / high-throughput sequencing
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表 1 BCN和BS样品OTU聚类及RDP分类水平统计
Table 1. OTU clustering and RDP classification level statistics of BCN and BS samples
样品
Sample检测项目
Test items有效reads数目/条
Reads numOTU数目/个
OUT numRDP分类
Classification of RDP门
Phylum纲
Class目
Order科
Family属
GenusBS 细菌 Bacteria 3 271 199 6 11 19 38 43 真菌 Fungus 280 392 4 279 8 29 67 159 257 BCN 细菌 Bacteria 11 413 379 10 19 26 54 74 真菌 Fungus 287 330 4 236 8 31 79 171 291 表 2 BCN和BS样品α多样性指数统计
Table 2. Alpha diversity index statistics of BCN and BS samples
样品
Sample检测项目
Test itemsOTU数量
OUT numSimpson指数
Simpson indexShannon指数
Shannon indexACE指数
ACE indexChao1指数
Chao1 index覆盖率
coverageBS 细菌 Bacteria 39.80 ± 14.82 0.30 ± 0.10 1.90 ± 0.43 61.37 ± 24.07 53.55 ± 14.68 0.97 ± 0.01 真菌 Fungus 855.80 ± 168.88 0.10 ± 0.05 3.60 ± 0.47 1 098.59 ± 164.90 1 058.26 ± 156.59 1.0 ± 0.00 BCN 细菌 Bacteria 75.80 ± 32.71 0.21 ± 0.07 2.33 ± 0.57 109.82 ± 33.31a 104.12 ± 38.89a 0.98 ± 0.01 真菌 Fungus 847.20 ± 142.69 0.13 ± 0.13 3.50 ± 0.71 1 167.82 ± 103.06 1 094.02 ± 147.21 1.0 ± 0.00 注:a为与BS样品细菌检测组相比,P < 0.05
Note: P is compared with the BS samples in the bacterial detection group, P < 0.05表 3 BCN和BS样品中桉树病害病原菌菌群丰度差异T-Test比较(属级水平)
Table 3. Comparison of abundance difference of Eucalyptus disease pathogenic bacteria in BCN and BS samples by T-Test (genus level)
菌属 Genus Freq1 Freq2 P值
P valueq值
q value效应量
Effect size95.0% 置信区间 95.0% CI 下限
Lower上限
UpperPseudomonas 40.534 71.146 0.071 1.000 −30.612 −64.790 3.566 Phyllosticta 0.081 14.517 0.059 1.000 −14.436 −29.774 0.903 Colletotrichum 0.005 0.020 0.046 1.000 −0.015 −0.029 0.000 Aureobasidium 0.000 0.009 0.115 1.000 −0.009 −0.021 0.003 Septoria 0.023 0.001 0.192 1.000 0.022 −0.017 0.062 Pseudocercospora 10.120 15.362 0.225 1.000 −5.242 −14.630 4.146 Lasiodiplodia 0.000 0.007 0.264 1.000 −0.007 −0.023 0.008 Neofusicoccum 0.083 0.033 0.267 0.990 0.050 −0.056 0.156 注:Freq1:桉树叶内菌属丰度;Freq2:油桐尺蛾中肠菌属丰度;q值:P值的校验值。
Note:Freq1: Abundance of bacteria or fungus in Eucalyptus leaves (genus level); Freq2: Abundance of bacteria or fungus inmidgut of Buzura suppressaria larvae (genera level); q value: The check value of the P value. -
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