孫丕海,錢坤,李曉麗,楊志平,丁鑒鋒,孫雪瑩,馬悅欣

(1.大連海洋大學海珍品苗種培育基地,遼寧大連116023;2.大連金普新區(qū)海洋與漁業(yè)局,遼寧大連116100;3.大連海洋大學農業(yè)部北方海水增養(yǎng)殖重點實驗室,遼寧大連116023;4.大連匯新鈦設備開發(fā)有限公司,遼寧大連116039)

基于高通量測序分析海帶表面細菌群落結構

孫丕海1,錢坤2,李曉麗3,楊志平4,丁鑒鋒3,孫雪瑩3,馬悅欣3

(1.大連海洋大學海珍品苗種培育基地,遼寧大連116023;2.大連金普新區(qū)海洋與漁業(yè)局,遼寧大連116100;3.大連海洋大學農業(yè)部北方海水增養(yǎng)殖重點實驗室,遼寧大連116023;4.大連匯新鈦設備開發(fā)有限公司,遼寧大連116039)

為研究大連登沙河灣和正明寺海帶養(yǎng)殖區(qū)海帶Saccharina japonica表面及其周圍海域海水的細菌群落結構,分別于2015年1月、2月和4月,采用Illumina Miseq PE300高通量測序平臺對樣本基因組DNA 的16S rRNA基因V3～V4區(qū)PCR擴增片段進行測序分析。結果表明:從兩個海區(qū)健康海帶和海水中獲得優(yōu)化序列分別為130 158條和124 658條,海帶序列可歸為8個門和99個屬,海水序列可歸為14個門和135個屬;在門水平,海帶首要優(yōu)勢門為厚壁菌門 (＞69%),其次為變形菌門、藍細菌門、放線菌門和擬桿菌門 (＞1%),海水首要優(yōu)勢門為變形菌門 (＞46%),其次為厚壁菌門、擬桿菌門、藍細菌門和放線菌門 (＞1%);在屬水平,海帶主要優(yōu)勢屬為乳球菌屬 (＞40%)和芽孢桿菌屬 (＞10%),其次為 Solibacillus、假單胞菌屬和節(jié)桿菌屬 (＞3%),海水首要優(yōu)勢屬為弧菌屬 (21.58%)和乳球菌屬 (28.29%),其次為科爾韋爾氏菌屬、弓形桿菌屬和亞硫酸桿菌屬 (＞2%)。研究表明,兩個海區(qū)海帶細菌群落組成相似,海帶和海水細菌群落組成則明顯不同。

海帶;細菌群落;高通量測序

海帶 Saccharina japonica(原名 Laminaria japonica)[1]隸屬于褐藻門、褐子綱、海帶目、海帶科、海帶屬,具有較高的經濟價值,并廣泛應用于食品、醫(yī)藥、化工等方面,是大連沿海海域大規(guī)模栽培的主要藻類之一。研究人員曾使用傳統(tǒng)培養(yǎng)方法對海帶表面細菌進行了分離鑒定[2-4]。Balakirev 等[5]通過克隆文庫技術分析發(fā)現,太平洋西北地區(qū)廣泛分布的海帶典型種 (TYP)及其常見形態(tài)變種 (LON和SHA)的共生微生物群落存在顯著差異,認為宿主-共生體相互作用可導致高遺傳相似性形式 (TYP和LON)表型不同,細菌共生體可作為區(qū)分遺傳相似藻類形式的敏感標記。而假交替單胞菌屬Pseudoalteromonas、弧菌屬Vibrio和鹽單胞菌屬Halomonas可使海帶發(fā)生病害[6-9]。Bengtsson等[10]首次使用高通量測序技術,研究了Laminaria hyperborea表面細菌多樣性與次級生產力的關系。但有關海帶表面細菌群落的研究仍然較少。本試驗中采用高通量測序技術分析了大連登沙河灣和正明寺養(yǎng)殖區(qū)健康海帶表面和海水細菌群落結構,旨在為了解細菌與海帶間的相互作用提供參考。

1 材料與方法

1.1 材料

分別于2015年1月、2月和4月從大連登沙河灣和正明寺養(yǎng)殖區(qū)采集健康海帶孢子體和海水。

1.2 方法

1.2.1 樣品采集 剪取海帶葉片生長點附近葉片約10 g,經無菌海水沖洗3次,用無菌自封袋封裝。海水樣品經0.22 μm孔徑的濾膜抽濾,將濾膜及海帶樣品置于冰盒,郵寄美吉生物醫(yī)藥科技有限公司進行檢測,送樣24 h內,用1 L無菌生理鹽水反復沖洗樣品海帶,將濾液通過0.02 mm孔徑的濾膜過濾。

1.2.2 DNA提取及PCR擴增 依據說明書步驟用E.Z.N.A.?water DNA試劑盒 (Omega Bio-tek, Norcross,GA,USA)提取海帶及水體 (過膜后樣本)的總DNA,細菌16S rRNA基因V3～V4片段的擴增引物為338F(5′barcode-ACTCCTACGGGAGGCAGCA 3′)和806R(5′GGACTACHVGGG-TWTCTAAT 3′)[11-12]。PCR反應體系 (共20 μL): 5×FastPfu緩沖液4 μL,2.5 mmol/L dNTPs 2 μL,上、下游引物各0.8 μL(5 μmol/L),FastPfu聚合酶0.4 μL,模板DNA 10 ng,用ddH2O補足至20 μL。PCR反應程序為:95℃下預變性3 min;95℃下變性30 s,55℃下退火30 s,72℃下延伸45 s,共進行27個循環(huán),最后在72℃下再延伸10 min。每個PCR樣本設置3個平行,所得產物混合后經20 g/L瓊脂糖凝膠電泳檢測,使用AxyPrepDNA凝膠回收試劑盒 (AXYGEN公司)切膠回收PCR產物。

1.2.3 Illumina MiSeq測序及序列處理 將PCR產物用QuantiFluorTM-ST藍色熒光定量系統(tǒng) (Promega公司)進行DNA定量,之后按照每個樣本的測序量要求,進行相應比例的混合,按標準流程Illumina Miseq PE300測序平臺進行測序。

1.3 數據處理

使用 Trimmomatic和FLASH軟件對測序數據進行優(yōu)化,過濾序列尾部質量值20以下的堿基,拼接序列的重疊區(qū)錯配比率低于0.2,barcode錯配數為0,最大引物錯配數為2的優(yōu)化序列用于后續(xù)分析。將序列按照97%相似性進行OTUs(operational taxonomic units)聚類[13],在聚類過程中去除UCHIME嵌合體[14],得到 OTU的代表序列,與Silva數據庫 (http://www.arb-silva.de)[15]進行比對,采用RDP classifier貝葉斯算法對OTU代表序列進行分類學分析[16](http://sourceforge.net/ projects/rdp-classifier/),置信度閾值為0.7。使用Mothur 1.30.1軟件 (http://www.mothur.org/wiki/Schloss_SOP#Alpha_diversity)進行rarefaction分析和單樣本α多樣性指數分析,包括Coverage、Ace、Chao、Shannon和Simpson指數。使用Qiime軟件計算beta多樣性距離矩陣,進行層次聚類分析[17],使用非加權組平均法UPGMA(Unweighted pair group method with arithmetic mean)算法構建樹狀結構,比較樣本間細菌群落差異。利用R語言工具制作稀釋曲線圖、文氏圖、群落結構組分圖和多樣本相似性樹。

2 結果與分析

2.1 兩個海區(qū)海帶及海水細菌群落組成

將3個時間點測序數據合并分析,從大連登沙河和正明寺兩個海區(qū)海帶和海水中共獲得254 816條優(yōu)化序列。所有序列可歸為531個OTUs。海帶序列可歸為8個門、17個綱、37個目、63個科、99個屬;海水序列可歸為14個門、23個綱、44個目、78個科、135個屬。

海帶和海水門水平細菌群落組成如圖1所示。從圖1可見:海帶首要優(yōu)勢門為厚壁菌門Finnicutes,其在大連登沙河灣養(yǎng)殖區(qū)和正明寺養(yǎng)殖區(qū)所占比例分別為 69.25% (HBS)和 83.37% (HFS);次優(yōu)勢門為變形菌門Proteobacteria、藍細菌門Cyanobacteria、放線菌門Actinobacteria和擬桿菌門Bacteroidetes。海水第一優(yōu)勢門為變形菌門,其在兩個海區(qū)所占比例分別為67.88% (HWB) 和46.39%(HWF);厚壁菌門在HWF中為第二優(yōu)勢門,其比例為38.39%,而在HWB中比例為9.52%;其他次優(yōu)勢門為擬桿菌門、藍細菌門、放線菌門和疣微菌門Verrucomicrobia。

海帶和海水綱水平細菌群落組成如圖2所示。從圖2可見:海帶首要優(yōu)勢綱為芽孢桿菌綱Bacilli,其在兩個海區(qū)所占比例分別為69.10%(HBS) 和83.03%(HFS);次優(yōu)勢綱包括藍細菌綱、γ-變形菌綱 γ-Proteobacteria、α-變形菌綱α-Proteobacteria、放線菌綱 Actinobacteria和黃桿菌綱Flavobacteriia。海水HWB中第一優(yōu)勢綱為γ-變形菌綱 (40.72%),γ-變形菌綱在HWF中為第二優(yōu)勢綱 (23.22%);HWF中第一優(yōu)勢綱為芽孢桿菌綱 (38.13%),在HWB中芽孢桿菌綱所占比例為9.51%;HWB和HWF的其余次優(yōu)勢綱包括α-變形菌綱、黃桿菌綱、藍細菌綱、ε-變形菌綱ε-Proteobacteria、δ-變形菌綱δ-Proteobacteria、β-變形菌綱β-Proteobacteria、放線菌綱、酸微菌綱Acidimicrobiia和噬纖維菌綱Cytophagia。

海帶和海水屬水平細菌群落組成如圖3所示。從圖3可見:HBS和HFS中主要優(yōu)勢屬為乳球菌屬Lactococcus(43.61%、53.46%)和芽孢桿菌屬Bacillus(11.19%、12.58%),次優(yōu)勢屬包括Solibacillus、假單胞菌屬 Pseudomonas、節(jié)桿菌屬 Arthrobacter、芽孢八疊球菌屬Sporosarcina、鏈球菌屬Streptococcus、Lysinibacillus、乳桿菌屬Lactobacillus、Loktanella和肉桿菌屬 Carnobacterium。HWB中首要優(yōu)勢屬為弧菌屬 Vibrio(21.58%),弧菌屬在HWF中的比例為6.22%,HWF中首要優(yōu)勢屬為乳球菌屬 (28.29%),所占比例為6.69%;HWB和HWF中次優(yōu)勢屬包括科爾韋爾氏菌屬Colwellia、弓形桿菌屬Arcobacter、亞硫酸桿菌屬Sulfitobacter、芽孢桿菌屬、Loktanella、假交替單胞菌屬Pseudo-alteromonas、Owenweeksia、Ulvibacter、馬賽菌屬Massilia、極地桿菌屬Polaribacter和Bacteriovorax。

圖1 兩個海區(qū)海帶及海水門水平細菌群落組成Fig.1 Bacterial communities in kelp and seawater in two sea areas at phylum level

2.2 海帶和海水細菌群落豐度和多樣性估計

海帶和海水優(yōu)化序列數量如表1所示,其中HBS序列66 812條,HFS序列63 346條,HWB序列62 807條,HWF序列61 851條。HBS和HFS的樣本OTU分別是220和198個,而HWB和HWF的樣本OTU數量高于海帶,分別為408和435個。在OTU水平上生成的4個樣品稀釋曲線(圖4)皆接近平坦,表明測序深度足夠反映樣本的多樣性。通過Chao和Ace指數可估計海帶和海水群落豐度,通過Shannon和Simpson指數可估計樣本群落多樣性。從表1可見,HBS和HFS群落豐度指數Chao和Ace為211～249,HWB和HWF的群落豐度指數Chao和Ace為415～451,表明海水(HWB和HWF)群落豐度高于海帶 (HBS和HFS)。同樣,Shannon和Simpson群落多樣性指數表明,海水群落多樣性也高于海帶。

圖3 兩個海區(qū)海帶及海水屬水平細菌群落組成Fig.3 Bacterial communities in kelp and seawater in two sea areas at genus level

圖4 兩個海區(qū)海帶及海水細菌群落稀釋曲線Fig.4 Dilution curve of bacterial communities in kelp and seawater in two sea areas

2.3 海帶和海水的細菌群落比較

圖5 兩個海區(qū)海帶及海水OTUs組成文氏圖Fig.5 Venn diagram of OTUs shared in kelp and seawater in two sea areas

海帶及海水樣本所共有和獨有的OTU數目見文氏圖 (圖5),HBS與HFS共有OTUs為153, Cyanobacteria_norank占其總 OTU的 57.74%, HWB與HWF共有OTUs為380,占其總OTU的82.07%。HBS與HWB共有OTUs為149,占其總OTU的31.11%,HFS與HWF共有OTUs為137,占其總OTU的27.62%,海帶 (HBS+HFS)與海水(HWB+HWF)共有OTUs為197,占總OTU (HBS+HFS+HWB+HWF)的37.10%。聚類分析結果顯示,HBS與HFS聚為一支,HWB與HWF聚為另一支 (圖6)。文氏圖和聚類分析結果均表明,兩個海區(qū)海帶之間細菌群落相似,海水之間細菌群落也相似;同一海區(qū)/兩個海區(qū)的海帶與海水之間細菌群落則存在明顯差異。

圖6 兩個海區(qū)海帶及海水OTU水平相似樹Fig.6 Similarity dendrogram of the OTU level betweenkelp and seawater in two sea areas

表1 兩個海區(qū)海帶及海水細菌群落豐富度及多樣性指數Tab.1 Richness and diversity indices of bacterial communities in kelp and seawater in two sea areas

3 討論

3.1 海帶細菌群落組成分析

高通量測序結果顯示,大連海區(qū)海帶細菌群落包括8個門,即厚壁菌門、變形菌門、藍細菌門、放線菌門、擬桿菌門、疣微菌門、浮霉菌門Planctomycetes和Armatimonadetes,研究人員通過培養(yǎng)方法和克隆文庫發(fā)現,海帶細菌群落由變形菌門、擬桿菌門 (CFB群)、放線菌門和厚壁菌門組成[2-5,18]。從海帶屬的其他種如 Laminaria hyperborea細菌群落可檢測出除Armatimonadetes之外的7個門 (通過培養(yǎng)方法、變性梯度凝膠電泳、克隆文庫、熒光原位雜交和高通量測序等方法檢測)[9,19-21],從細布海帶Laminaria religiosa檢測的細菌群落為變形菌門 (通過培養(yǎng)方法檢測)[22],從Laminaria longicruris細菌群落檢出變形菌門和擬桿菌門 (通過培養(yǎng)方法檢測)[23],Laminaria digitata附生細菌群落由變形菌門、放線菌門和擬桿菌門組成 (通過全細胞光譜方法檢測)[24],Laminaria saccharina細菌群落也可歸為變形菌門、擬桿菌門、放線菌門和厚壁菌門(通過培養(yǎng)方法和克隆文庫方法檢測)[25-26]。

在綱水平,從健康海帶檢測出17個綱,包括優(yōu)勢綱6個 (圖2)。其中,γ-變形菌綱、β-變形菌綱、放線菌綱、芽孢桿菌綱和黃桿菌綱為已報道海帶相關細菌群落成員[2-5,18,27]。從細布海帶可分離出 γ-變形菌綱和 β-變形菌綱細菌[22],從L.longicruris分離出 γ-變形菌綱和黃桿菌綱細菌[23],從L.digitata檢測出放線菌綱、γ-變形菌綱、α-變形菌綱和黃桿菌綱細菌[24],從L.saccharina檢測出α-變形菌綱、β-變形菌綱、γ-變形菌綱、ε-變形菌綱、放線菌綱、黃桿菌綱和芽孢桿菌綱細菌[25-26],從L.hyperborea檢測出α-變形菌綱、β-變形菌綱、γ-變形菌綱、δ-變形菌綱、藍細菌綱、芽孢桿菌綱和放線菌綱細菌[9,19-21]。

在屬水平,從健康海帶中共發(fā)現99個屬的細菌,包括優(yōu)勢屬12個 (圖5)。其中,Arenicella、芽孢桿菌屬、棒狀桿菌屬Corynebacterium、黃桿菌屬Flavobacterium、Granulosicoccus、假交替單胞菌屬、假單胞菌屬、嗜冷桿菌屬、嗜冷單胞菌屬Psychromonas、葡萄球菌屬Staphylococcus和弧菌屬為已報道海帶相關細菌群落成員[2-5,18,27]。 從L.longicruris分離出黃桿菌屬、假單胞菌屬和弧菌屬細菌[23]。從L.digitata檢測出節(jié)桿菌屬、副球菌屬Paracoccus、假交替單胞菌屬和假單胞菌屬細菌[24],從L.saccharina中發(fā)現節(jié)桿菌屬、芽孢桿菌屬、黃桿菌屬、假交替單胞菌屬、假單胞菌屬、鞘氨醇單胞菌屬 Sphingomonas、寡養(yǎng)單胞菌屬Stenotrophomonas、亞硫酸桿菌屬和弧菌屬細菌[26], 從L.hyperborea中分離出芽孢桿菌屬、Granulosicoccus和Loktanella細菌[21]。

本研究中首次發(fā)現海帶細菌群落成員:Armatimonadetes、藍細菌門、浮霉菌門 Planctomycetes和疣微菌門;酸微菌綱、α-變形菌綱、擬桿菌綱Bacteroidia、梭菌綱Clostridia、藍細菌綱、噬纖維菌綱、δ-變形菌綱、ε-變形菌綱、Negativicutes、Phycisphaerae、Sphingobacteriia和疣微菌綱 Verru-comicrobiae;優(yōu)勢屬乳球菌屬、Solibacillus、節(jié)桿菌屬、芽孢八疊球菌屬、鏈球菌屬、Lysinibacillus、乳桿菌屬、Loktanella和肉桿菌屬。本研究結果擴展了人們對海帶表面微生物群落的認識。

海帶表面相關細菌組成有多方面的功能,有的是潛在的抗菌資源,如一種芽孢桿菌、一種假單胞菌和一種黃桿菌Flavobacterium sp.具有抗微生物活力[28];有的可促進植物生長,如 Pseudoalteromonas porphyrae MMM18菌株可提高蘿卜、小麥、大豆、燕麥、豌豆和白菜種子的發(fā)芽率和芽長[18];但有些細菌對宿主是有害的,如Pseudoalteromonas bacteriolytica[7]和P.elyakovii[8]分別是海帶紅點病和葉斑點傷病害的病原菌。7株假交替單胞菌屬和4株弧菌屬細菌可能是海帶孔爛病的潛在病原[9]。本研究從海帶中發(fā)現芽孢桿菌屬、假單胞菌屬、黃桿菌屬、假交替單胞菌屬和弧菌屬,其生態(tài)作用及其與宿主的相互作用有待于進一步研究。

3.2 海帶和海水細菌群落比較

本試驗中海帶養(yǎng)殖區(qū)海水中的細菌群落主要由變形菌門、厚壁菌門、擬桿菌門、藍細菌門和放線菌門組成 (圖1)。這些門也分布在其他海藻生長海域水體中,如L.saccharina生長海域水體中的細菌屬于變形菌門和擬桿菌門[25]。澳洲石莼生長海域水體中優(yōu)勢門為變形菌門、擬桿菌門和放線菌門,而藍細菌門和厚壁菌門豐度較低。在屬和OTU水平上海帶細菌群落與其周圍海水的細菌群落明顯不同 (圖3～圖5)。這一結果與其他學者的研究結果類似[20,25,29]。L.saccharina和海水細菌群落的DGGE條帶圖譜明顯不同,相似性僅為20%[14]。L.hyperborea表面生物膜與其周圍海水細菌群落重疊較少[20]。在門水平澳洲石莼附生細菌群落與其周圍海水細菌群落類似,而種水平兩細菌群落則明顯不同[29]。其原因可能是兩種環(huán)境的物理差異引起群落組成有差異。海帶上細菌的可能來源之一是其周圍的海水,因此,海帶表面的細菌可能是通過選擇過程從海水較少的細菌種群中吸收或經表面直接接觸細菌所致[20]。

[1] Lane C E,Mayes C,Druehl L D,et al.A multi-gene molecular investigation of the kelp(Laminariales,Phaeophyceae)supports substantial taxonomic re-organization[J].Journal of Phycology, 2006,42(2):493-512.

[2] Duan D,Xu L,Fei X,et al.Marine organisms attached to seaweed surfaces in Jiaozhou Bay,China[J].World Journal of Microbiology and Biotechnology,1995,11(3):351-352.

[3] Wang Zifeng,Xiao Tian,Pang Shaojun,et al.Isolation and identification of bacteria associated with the surfaces of several algal species[J].Chinese Journal of Oceanology and Limnology,2009,27 (3):487-492.

[4] 徐滌,秦松,龐國興,等.青島和大連海區(qū)海帶(Laminaria japonica)外生細菌的16S rRNA基因序列分析[J].高技術通訊, 2004(2):81-86.

[5] Balakirev E S,Krupnova T N,Ayala F J.Symbiotic associations in the phenotypically-diverse brown alga Saccharina japonica[J]. PLoS One,2012,7(6):e39587.

[6] Yumoto I,Yamaguchi K,Yamada K,et al.Relationship between bacterial flora and occurrence of the Alteromonas sp.,the causative agent of red-spots on the culture bed of makonbu Laminaria japonica,in the coastal area of Funka Bay[J].Nippon Suisan Gakkaishi,1989,55(11):1907-1914.

[7] Sawabe T,Makino H,Tatsumi M,et al.Pseudoalteromonas bacteriolytica sp.nov.,a marine bacterium that is the causative agent of red spot disease of Laminaria japonica[J].International Journal of Systematic Bacteriology,1998,48(3):769-774.

[8] Sawabe T,Tanaka R,Iqbal M M,et al.Assignment of Alteromonas elyakovii KMM 162T and five strains isolated from spot wounded fronds of Laminaria japonica to Pseudoalteromonas elyakovii comb. nov.and the extended description of the species[J].International Journal of Systematic and Evolutionary Microbiology,2000,50(1): 265-271.

[9] Wang Gaoge,Shuai Li,Li Yun,et al.Phylogenetic analysis of epiphytic marine bacteria on Hole-Rotten diseased sporophytes of Laminaria japonica[J].Journal of Applied Phycology,2008,20 (4):403-409.

[10] Bengtsson M M,Sj?tun K,Lanzén A,et al.Bacterial diversity in relation to secondary production and succession on surfaces of the kelp Laminaria hyperborea[J].The ISME Journal,2012,6(12): 2188-2198.

[11] Stackebrandt E,Goodfellow M D.Nucleic Acid Techniques in Bacterial Systematics[M].Chichester:John Wiley and Sons Press,1991:115-175.

[12] McBain A J,Bartolo R G,Catrenich C E,et al.Microbial characterization of biofilms in domestic drains and the establishment of stable biofilm microcosms[J].Applied and Environmental Microbiology,2003,69(1):177-185.

[13] Edgar R C,Haas B J,Clemente J C,et al.UCHIME improves sensitivity and speed of chimera detection[J].Bioinformatics,2011, 27(16):2194-2200.

[14] Edgar R C.UPARSE:highly accurate OTU sequences from microbial amplicon reads[J].Nature Methods,2013,10(10):996-998.

[15] Quast C,Pruesse E,Yilmaz P,et al.The SILVA ribosomal RNA gene database project:improved data processing and web-based tools[J].Nucleic Acids Research,2013,41(D1):D590-D596.

[16] Wang Qiong,Garrity G M,Tiedje J M,et al.Na?ve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J].Applied and Environmental Microbiology,2007,73(16):5261-5267.

[17] Jiang Xiaotao,Peng Xin,Deng Guanhua,et al.Illumina Sequencing of 16S rRNA tag revealed spatial variations of bacterial communities in a mangrove wetland[J].Microbial Ecology,2013,66 (1):96-104.

[18] Dimitrieva G Y,Crawford R L,Yüksel G ü.The nature of plant growth-promoting effects of a pseudoalteromonad associated with the marine algae Laminaria japonica and linked to catalase excretion[J].Journal of Applied Microbiology,2006,100(5):1159-1169.

[19] Bengtsson M M,?vre?s L.Planctomycetes dominate biofilms on surfaces of the kelp Laminaria hyperborea[J].BMC Microbiology,2010(10):261.

[20] Bengtsson M M,Sj?tun K,?vre?s L,et al.Seasonal dynamics of bacterial biofilms on the kelp Laminaria hyperborea[J].Aquatic Microbial Ecology,2010,60(1):71-83.

[21] Bengtsson M M,Sj?tun K,Storesund J E,et al.Utilization of kelp-derived carbon sources by kelp surface-associated bacteria [J].Aquatic Microbial Ecology,2011,62(2):191-199.

[22] Vairappan C S,Suzuki M,Motomura T,et al.Pathogenic bacteria associated with lesions and thallus bleaching symptoms in the Japanese kelp Laminaria religiosa Miyabe(Laminariales,Phaeophyceae)[J].Hydrobiologia,2001,445(1-3):183-191.

[23] Laycock R A.The detrital food chain based on seaweeds:I.Bacteria associated with the surface of Laminaria fronds[J].Marine Biology,1974,25(3):223-231.

[24] Salaün S,Kervarecd N,Potin P,et al.Whole-cell spectroscopy is a convenient tool to assist molecular identification of cultivatable marine bacteria and to investigate their adaptive metabolism[J]. Talanta,2010,80(5):1758-1770.

[25] Staufenberger T,Thiel V,Wiese J,et al.Phylogenetic analysis of bacteria associated with Laminaria saccharina[J].FEMS Microbiology Ecology,2008,64(1):65-77.

[26] Wiese J,Thiel V,Nagel K,et al.Diversity of antibiotic-active bacteria associated with the brown alga Laminaria saccharina from the Baltic Sea[J].Marine Biotechnology,2009,11(2):287-300.

[27] Hollants J,Leliaert F,De Clerck O,et al.What we can learn from sushi:a review on seaweed-bacterial associations[J].FEMS Microbiology Ecology,2013,83(1):1-16.

[28] Zheng Li,Han Xiaotian,Chen H M,et al.Marine bacteria associated with marine macroorganisms:the potential antimicrobial resources[J].Annals of Microbiology,2005,55(2):119-124.

[29] Burke C,Thomas T,Lewis M,et al.Composition,uniqueness and variability of the epiphytic bacterial community of the green alga Ulva australis[J].The ISME Journal,2011,5(4):590-600.

Characterization of bacterial community structure on surface of kelp Saccharina japonica by high-throughput sequencing

SUN Pi-hai1,QIAN Kun2,LI Xiao-li3,YANG Zhi-ping4,DING Jian-feng3,SUN Xue-ying3,MA Yue-xin3
(1.Seafood Seedling Breeding Base,Dalian Ocean University,Dalian 116023,China;2.Dalian Jinzhou District Oceanic and Fishery Administration, Dalian 116100,China;3.Key Laboratory of Mariculture and Stock Enhancement in North China's Sea,Ministry of Agriculture,Dalian Ocean University,Dalian 116023,China;4.Dalian Huixin Titanium Equipment Development Company Limited,Dalian 116039,China)

The genomic DNA was extracted from surface bacteria sampled from healthy kelp Saccharina japonica and surrounding seawater in the sea areas of Dengshahe bay and Zhengmingsi in Dalian in January,February and March of 2015,and a gene segment from the V3-V4 portion of the 16S rRNA gene was sequenced using Illumina Miseq PE300 high-throughput sequencing platform to examine the bacterial community on the surface of the kelp in two sea areas in coastal Dalian.A total of 130 158 and 124 658 optimized sequences were obtained from the kelp and seawater,respectively.Sequences from kelp were classified into 8 phyla and 99 genera,and sequences from seawater were identified as 14 phyla and 135 genera using 16S rRNA gene database.In the kelp community,phylum Firmicutes(＞69%)was predominantly observed,followed by Proteobacteria,Cyanobacteria,Actinobacteria and Bacteroidetes(＞1%).In seawater community,Proteobacteria(＞46%)was most abundant,followed by Firmicutes,Bacteroidetes,Cyanobacteria and Actinobacteria.At genus level,Lactococcus(＞40%)andBacillus(＞10%)were dominant kelp members,followed by Solibacillus,Pseudomonas and Arthrobacter(＞3%),while Vibrio(21.58%)and Lactococcus(28.29%)were the dominant members in seawater from Dengshahe bay and Zhengmingsi,respectively,followed by Colwellia,Arcobacter and Sulfitobacter(＞2%).Dendrogram of hierarchical cluster analysis revealed that there were similar bacterial communities in the two sea areas,and different bacterial communities between the seawater and kelp surface.

Saccharina japonica;bacterial community;high-throughput sequencing

Q939.1

A

10.16535/j.cnki.dlhyxb.2017.01.002

2095-1388(2017)01-0007-06

2016-09-22

遼寧省自然科學基金資助項目 (2015020800);大連市金州新區(qū)海洋與漁業(yè)局項目 (2014115,2014118)

孫丕海 (1959—),男,高級工程師。E-mail:dlsph@aliyun.com

馬悅欣 (1963—),女,博士,教授。E-mail:mayuexin@dlou.edu.cn