呼倫貝爾沙區(qū)土壤細(xì)菌群落結(jié)構(gòu)與功能預(yù)測(cè)

杜宇佳,高廣磊*,陳麗華,丁國(guó)棟,張 英,曹紅雨

呼倫貝爾沙區(qū)土壤細(xì)菌群落結(jié)構(gòu)與功能預(yù)測(cè)

杜宇佳1,2,高廣磊1,2*,陳麗華1,丁國(guó)棟1,2,張 英1,曹紅雨1,2

(1.北京林業(yè)大學(xué)水土保持學(xué)院,水土保持國(guó)家林業(yè)與草原局重點(diǎn)實(shí)驗(yàn)室,北京 100083；2.寧夏鹽池毛烏素沙地生態(tài)系統(tǒng)國(guó)家定位觀測(cè)研究站,寧夏 鹽池 751500)

以呼倫貝爾沙區(qū)裸沙地、草地、沙地樟子松(var.)人工林和沙地樟子松天然林四種生境土壤為研究對(duì)象,采用野外調(diào)查、16S rRNA基因高通量測(cè)序和PICRUSt功能預(yù)測(cè)相結(jié)合的研究方法比較分析不同生境土壤細(xì)菌群落結(jié)構(gòu)和潛在功能組成特征.結(jié)果顯示:呼倫貝爾沙區(qū)沙地樟子松天然林土壤細(xì)菌多樣性最高,人工林土壤細(xì)菌多樣性最低,Shannon指數(shù)分別為(8.623±0.193)和(7.432±0.028),不同生境土壤細(xì)菌alpha和beta多樣性存在顯著差異.草地、沙地樟子松人工林和天然林土壤中變形菌門(Proteobacteria)相對(duì)豐度最高,均值分別為29.83%±1.14%、34.73%±1.99%、31.95%±0.21%,裸沙地土壤放線菌門(Actinobacteria)相對(duì)豐度最高,均值為26.13%±0.43%.不同生境土壤細(xì)菌主要優(yōu)勢(shì)屬為慢生根瘤菌屬()、RB41,其相對(duì)豐度在四種生境中的均值分別為5.29%±2.24%、4.22%±1.23%.PICRUSt功能預(yù)測(cè)共得到6個(gè)一級(jí)功能層,40個(gè)二級(jí)功能層,土壤細(xì)菌功能較為豐富,土壤細(xì)菌群落在環(huán)境信息處理、代謝、遺傳信息處理和有機(jī)系統(tǒng)方面功能活躍.沙地樟子松天然林核苷酸代謝、酶家族、氨基酸代謝、碳水化合物代謝功能基因較為豐富,保證了沙地樟子松天然林土壤細(xì)菌的存活,使其具有較高的多樣性.呼倫貝爾沙區(qū)不同生境土壤細(xì)菌功能基因豐度波動(dòng),反映了四種生境的土壤細(xì)菌群落組成及多樣性的變化,指示了不同生境功能基因?qū)ν寥兰?xì)菌群落的影響規(guī)律,可為預(yù)測(cè)和理解沙區(qū)土壤細(xì)菌代謝潛力和功能提供參考借鑒.

呼倫貝爾沙區(qū)；生境；細(xì)菌群落結(jié)構(gòu)；16S rRNA；功能預(yù)測(cè)

土壤細(xì)菌是土壤微生物中種類最多、數(shù)量最大、功能最豐富的類群[1],是陸地生態(tài)系統(tǒng)的重要組成部分.土壤細(xì)菌驅(qū)動(dòng)地球生物化學(xué)循環(huán),是生態(tài)系統(tǒng)物質(zhì)循環(huán)和能量流動(dòng)的關(guān)鍵環(huán)節(jié),對(duì)于穩(wěn)定、調(diào)節(jié)和修復(fù)陸地生態(tài)系統(tǒng)發(fā)揮著重要作用[2].土壤細(xì)菌的空間分布格局存在顯著的生境依賴性特征[3],受土壤性質(zhì)、植被、氣候等多種環(huán)境因素的綜合影響.在早期研究中,相關(guān)學(xué)者主要關(guān)注土壤細(xì)菌的群落結(jié)構(gòu)與多樣性[4-5],而后聚焦于土壤細(xì)菌群落結(jié)構(gòu)與分布的影響因素與驅(qū)動(dòng)過(guò)程[6].近年來(lái),隨著分子生物學(xué)技術(shù)與方法的不斷發(fā)展,土壤細(xì)菌相關(guān)研究開(kāi)始由結(jié)構(gòu)向功能轉(zhuǎn)變[7-8].各國(guó)學(xué)者共同致力于土壤細(xì)菌功能預(yù)測(cè)與分析,并試圖通過(guò)土壤細(xì)菌功能研究,揭示土壤細(xì)菌在陸地生態(tài)系統(tǒng)過(guò)程中發(fā)揮的重要作用[9-11].

目前,生物信息技術(shù)發(fā)展迅速.PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), Tax4Fun和FAPROTAX (Functional Annotation of Prokaryotic Taxa)等方法基于系統(tǒng)發(fā)育與功能充分聯(lián)系的假設(shè),根據(jù)16S rRNA測(cè)序基因與參考基因組數(shù)據(jù)庫(kù)的相似性,進(jìn)而判斷和預(yù)測(cè)標(biāo)記基因的功能組成,為我們深入理解微生物功能提供了新途徑[12-13].其中,FAPROTAX依據(jù)物種名進(jìn)行功能預(yù)測(cè),但僅涉及元素循環(huán)相關(guān)功能,無(wú)法對(duì)整體功能進(jìn)行預(yù)測(cè),算法存在明顯的局限性;Tax4Fun基于SILVA數(shù)據(jù)庫(kù)進(jìn)行基因功能預(yù)測(cè),雖然該數(shù)據(jù)庫(kù)擁有細(xì)菌、真菌、古菌rRNA基因序列,但數(shù)據(jù)庫(kù)剛剛建立,尚不完善,應(yīng)用仍處于起步階段;PICRUSt的比對(duì)數(shù)據(jù)庫(kù)為Greengenes,該數(shù)據(jù)庫(kù)涵蓋細(xì)菌、古菌基因序列,數(shù)據(jù)庫(kù)相對(duì)完善,具有預(yù)測(cè)準(zhǔn)確,功能先進(jìn),方便快捷,成本低廉的鮮明特點(diǎn).目前,PICRUSt算法已在土壤、海洋和湖泊細(xì)菌功能分析得到應(yīng)用[14-15],為土壤微生物群落結(jié)構(gòu)與功能研究提供了新途徑[16-17].

呼倫貝爾沙區(qū)是我國(guó)防沙治沙的重點(diǎn)地區(qū)之一,也是構(gòu)建“兩屏三帶”國(guó)家生態(tài)格局“北方防沙帶”的核心地區(qū).相關(guān)學(xué)者已對(duì)呼倫貝爾沙區(qū)土壤微生物數(shù)量、多樣性、群落結(jié)構(gòu)進(jìn)行了大量的研究探索,取得了豐碩的研究成果[18-19].隨著研究的不斷深入,相關(guān)研究重點(diǎn)已由土壤微生物群落結(jié)構(gòu)向功能轉(zhuǎn)移.鑒于此,本研究以呼倫貝爾沙區(qū)裸沙地、草地、樟子松天然林、樟子松人工林四種典型生境為研究對(duì)象,采用PICRUSt方法分析土壤細(xì)菌群落結(jié)構(gòu)與功能特征,以期研究揭示不同生境土壤微生物群落結(jié)構(gòu)與功能的相互關(guān)系,為預(yù)測(cè)和理解沙區(qū)土壤細(xì)菌代謝潛力和功能提供參考借鑒.

1 材料與方法

1.1 研究區(qū)概況

呼倫貝爾沙區(qū)位于內(nèi)蒙古自治區(qū)東北部(115°31′~126°04′E,47°05′~53°20′N),屬半濕潤(rùn)大陸性季風(fēng)氣候區(qū).年均氣溫-0.3℃,年均降水量280~ 355mm,年均蒸發(fā)量1400~1900mm,無(wú)霜期121d.地帶性土壤主要由黑鈣土和栗鈣土組成,風(fēng)沙土則多分布于沙帶沙丘及沙質(zhì)草原沙區(qū),境內(nèi)包括海拉爾河流域和伊敏河流域.研究區(qū)喬木植被主要有沙地樟子松(var)、白樺()等,灌木植被主要有小葉錦雞兒()、黃柳()和沙蒿()等,草本植被主要有羊草()、針茅()和沙蓬()等.

1.2 土壤樣品采集

2017年7月,在裸沙地、草地、沙地樟子松人工林和沙地樟子松天然林4種典型生境地勢(shì)平坦地段分別布設(shè)3個(gè)20×20m的樣地(表1),開(kāi)展土壤樣品采集工作,樣品采集深度為0~10和10~20cm.其中,沙地樟子松天然林和人工林樣地內(nèi)分別選取3株長(zhǎng)勢(shì)相當(dāng)?shù)臉?biāo)準(zhǔn)木,在樹(shù)冠投影處采集土壤樣品,草地和裸沙地則在樣地內(nèi)隨機(jī)選取3個(gè)采樣點(diǎn)采集土壤樣品.新鮮土壤樣品去除石子、根系和碎草等雜質(zhì)后裝入無(wú)菌袋,置于4 ℃便攜式保溫箱中冷凍保存,運(yùn)回實(shí)驗(yàn)室后采用-80 ℃的冰箱保存.

表1 樣地基本概況

注:HLB:bare sandy land,裸沙地;HLG:grass land,草地;HLN:natural forest,樟子松天然林;HLP:plantation,樟子松人工林.

1.3 土壤樣品處理及分析

1.3.1 土壤樣品處理及理化性質(zhì)分析 將土壤樣品風(fēng)干后,過(guò)2mm篩后進(jìn)行樣品理化性質(zhì)的分析.土壤含水量采用烘干測(cè)定法;土壤pH值采用電位法使用便攜式pH計(jì)測(cè)定;土壤有機(jī)質(zhì)采用稀釋熱法測(cè)定;土壤全氮含量、速效氮含量采用靛酚藍(lán)比色法測(cè)定;土壤全磷含量、速效磷含量采用鉬銻抗比色法測(cè)定[20].

1.3.2 土壤細(xì)菌的分離鑒定 取5g采集的土壤樣品于加入液氮的研缽中研磨,使用Power Soil DNA Kit(MoBio,USA)試劑盒提取土壤微生物基因組DNA,置-20 ℃的冰箱中保存.對(duì)16S rRNA基因的V3-V4區(qū)域進(jìn)行擴(kuò)增,引物序列為338F(ACTCCT- ACGGGAGGCAGCAG)、806R(GGACTACHVGG- GTWTCTAAT).PCR采用25mL反應(yīng)體系:1mL× Forward Primer(5mmol/L),1mL×Reverse Primer (5mmol/L),3mL BSA(2ng/mL),12.5mL 2×Taq PCR MasterMix,30ng DNA模板,最后用7.5mL ddH2O補(bǔ)足至總體積25mL.PCR擴(kuò)增程序?yàn)?95℃預(yù)變性300s; 95℃變性45s,55℃退火50s,72℃延伸45s,持續(xù)28個(gè)循環(huán)周期;最后72℃延伸10min.將樣本PCR產(chǎn)物混合后用2%瓊脂糖凝膠電泳檢測(cè),使用AxyPrepDNA凝膠回收試劑盒(AXYGEN公司)切膠回收PCR產(chǎn)物,Tris-HCl洗脫;2%瓊脂糖電泳檢測(cè).合格的PCR之后進(jìn)行MiSeq文庫(kù)構(gòu)建并測(cè)序.本研究涉及的所有測(cè)序數(shù)據(jù)可在NCBI Sequence Read Archive下載(PRJNA551926).

1.3.3 測(cè)序數(shù)據(jù)處理 Miseq測(cè)序得到的序列利用Trimmomatic對(duì)雙端序列數(shù)據(jù)進(jìn)行過(guò)濾處理,過(guò)濾read尾部質(zhì)量值20以下的堿基,設(shè)置50bp的窗口,如果窗口內(nèi)的平均質(zhì)量值低于20,從窗口開(kāi)始截去后端堿基,過(guò)濾質(zhì)控后50bp以下的read;利用Pear對(duì)含有barcode的數(shù)據(jù)進(jìn)行質(zhì)控過(guò)濾(根據(jù)-value和-value校正,-value值0.0001);然后根據(jù)PE測(cè)序的overlap關(guān)系,利用FLASH將成對(duì)的序列拼接(merge)成一條序列,拼接序列的overlap區(qū)允許的最大錯(cuò)配比率為0.1.根據(jù)序列首尾兩端的barcode和引物區(qū)分樣品,并調(diào)整序列方向,barcode允許的錯(cuò)配數(shù)為0;利用usearch軟件去除嵌合體.下機(jī)數(shù)據(jù)在去除barcode和primer并拼接后得到raw tags,raw tags經(jīng)進(jìn)一步去除嵌合體、短序列后得到優(yōu)質(zhì)序列clean tags,共得到優(yōu)質(zhì)序列1472261條.其中,裸沙地、草地、沙地樟子松天然林和人工林優(yōu)質(zhì)序列分別有303383、353675、422576、392627條.為最小化樣本變化對(duì)測(cè)序效率的影響[21],根據(jù)優(yōu)質(zhì)序列進(jìn)一步去除singlton序列,得樣本最低序列數(shù)99173條,按此樣本進(jìn)行隨機(jī)抽平.利用QIIME v.1.8軟件對(duì)優(yōu)質(zhì)序列進(jìn)行質(zhì)控,通常對(duì)97%的相似水平的物種分類單元(OTU)進(jìn)行信息統(tǒng)計(jì)分析[22].

1.4 數(shù)據(jù)分析

將QIIME軟件得到的biom文件上傳到Galaxy網(wǎng)站進(jìn)行PICRUSt功能基因預(yù)測(cè)分析,得到土壤細(xì)菌功能基因組成.采用mothur軟件計(jì)算細(xì)菌群落Chao1豐富度指數(shù)、Shannon-Wiener多樣性指數(shù)、Simpson優(yōu)勢(shì)度指數(shù).采用SPSS 24.0進(jìn)行單因素方差分析(ANOVA)、最小顯著差異法(LSD)比較、Pearson相關(guān)性分析.weighted Nearest Sequenced Taxon Index(weighted NSTI)是將樣本中的每個(gè)OTU與已知數(shù)據(jù)庫(kù)中參考細(xì)菌基因組分開(kāi)的平均分支長(zhǎng)度,通過(guò)樣本中OTU的豐度加權(quán)得到,使用predict_metagenomes.py命令-a選項(xiàng)計(jì)算NSTI.采用Excel 2010繪制相對(duì)豐度圖.采用Origin 2018繪制土壤細(xì)菌群落組成與功能熱圖.采用Canoco for Windows進(jìn)行土壤細(xì)菌PCA與RDA分析.

2 結(jié)果與分析

2.1 不同生境土壤細(xì)菌多樣性特征

圖1 不同生境土壤細(xì)菌群落多樣性指數(shù)

不同大小寫字母分別表示0~10cm、10~20cm土層不同生境土壤細(xì)菌多樣性差異顯著(<0.05).橫坐標(biāo)中a為0~10cm土層深度,b為10~20cm 土層深度,下同

呼倫貝爾沙區(qū)不同生境土壤細(xì)菌的Chao1豐富度指數(shù)、Shannon多樣性指數(shù)、Simpson優(yōu)勢(shì)度指數(shù)組內(nèi)和組間差異顯著(<0.05)(圖1).草地和沙地樟子松天然林的土壤細(xì)菌多樣性指數(shù)較高,分別為8.60±0.06和8.62±0.19,其次是裸沙地為8.52±0.26,沙地樟子松人工林土壤細(xì)菌多樣性指數(shù)最低為7.43±0.03.草地和沙地樟子松天然林土壤細(xì)菌群落豐富度指數(shù)無(wú)顯著性差異(0.05),且明顯高于裸沙地與沙地樟子松人工林.裸沙地、草地、沙地樟子松天然林土壤細(xì)菌多樣性指數(shù)和優(yōu)勢(shì)度指數(shù)均無(wú)顯著性差異(0.05),均高于沙地樟子松人工林.由四種生境土壤細(xì)菌分布的垂直空間分析,裸沙地、草地、沙地樟子松人工林,土壤細(xì)菌群落豐富度指數(shù)和多樣性指數(shù)均隨著土層深度的增加而增加,而沙地樟子松天然林變化規(guī)律與之相反.

2.2 不同生境土壤細(xì)菌群落結(jié)構(gòu)特征

不同生境土壤細(xì)菌類群主要優(yōu)勢(shì)細(xì)菌門(圖2)是變形菌門(Proteobacteria)、放線菌門(Actinobacteria)、酸桿菌門(Acidobacteria),其在四種生境中的均值分別為29.76%±4.43%、22.11%±4.46%、18.71%±4.62%,主要優(yōu)勢(shì)細(xì)菌屬是慢生根瘤菌屬()、RB41,其相對(duì)豐度的均值分別為5.29%±2.24%、4.22%±1.23%.草地、沙地樟子松人工林、沙地樟子松天然林中變形菌門的相對(duì)豐度最高,其均值分別為29.83%±1.14%、34.73%±1.99%和31.95%±0.21%,裸沙地中放線菌門的相對(duì)豐度最高,為26.13%±0.43%.酸桿菌門在沙地樟子松天然林中相對(duì)豐度最高,為25.80%±4.33%.變形菌門的相對(duì)豐度在不同土層分布無(wú)差異,放線菌門和酸桿菌門在不同土層深度其相對(duì)豐度表現(xiàn)出波動(dòng).

PC1軸的可信度是27.50%,PC2軸的可信度為17.40%(圖3).同一生境樣點(diǎn)相對(duì)聚集,不同生境樣點(diǎn)相對(duì)分開(kāi),表明四種生境細(xì)菌群落組成存在明顯差異.草地、沙地樟子松人工林和天然林3種生境內(nèi)樣點(diǎn)相對(duì)聚集,表明這3種生境不同土層細(xì)菌群落組成較為接近.裸沙地內(nèi)樣點(diǎn)距離其他3種生境最遠(yuǎn),由此推斷,裸沙地中土壤細(xì)菌群落結(jié)構(gòu)與其他3種生境相比存在較大差異.裸沙地0~10和10~20cm兩個(gè)土層中,各有一個(gè)點(diǎn)與樣地內(nèi)其他樣點(diǎn)距離較遠(yuǎn),表明裸沙地0~10和10~20cm土層細(xì)菌群落組成存在變異性

圖2 不同生境土壤細(xì)菌群落組成(門水平)

圖中僅列出相對(duì)豐度大于1%的細(xì)菌門,小于此值則計(jì)為other

圖3 不同生境土壤細(xì)菌群落組成主成分分析

2.3 不同生境土壤理化性質(zhì)與細(xì)菌群落的RDA分析及與細(xì)菌多樣性指數(shù)的相關(guān)性

土壤理化性質(zhì)見(jiàn)表2.由圖4可見(jiàn),第一排序軸解釋了不同生境土壤理化性質(zhì)與細(xì)菌群落關(guān)系的63.7%,第二排序軸解釋了18.6%的變異,2個(gè)排序軸累計(jì)貢獻(xiàn)率為82.3%.不同生境樣本點(diǎn)分布相對(duì)離散且距離原點(diǎn)較遠(yuǎn),說(shuō)明不同生境細(xì)菌群落結(jié)構(gòu)特征存在差異且受土壤理化性質(zhì)影響較大.裸沙地土壤細(xì)菌群落結(jié)構(gòu)與土壤含水量有相關(guān)性(<0.05);沙地樟子松人工林土壤細(xì)菌群落結(jié)構(gòu)與土壤有機(jī)質(zhì)含量、土壤速效磷含量有顯著的相關(guān)性(<0.05);沙地樟子松天然林土壤細(xì)菌群落結(jié)構(gòu)主要受到土壤pH值的影響.土壤理化性質(zhì)與土壤細(xì)菌群落多樣性指數(shù)有相關(guān)關(guān)系(表3),其中,土壤細(xì)菌群落豐富度指數(shù)與土壤速效氮含量達(dá)到顯著相關(guān)(<0.05),與土壤速效磷含量達(dá)到極顯著相關(guān)(<0.01),土壤細(xì)菌群落Shannon指數(shù)與土壤速效磷含量達(dá)到顯著負(fù)相關(guān)(<0.05),表現(xiàn)為沙地樟子松人工林土壤細(xì)菌多樣性在4種生境中最低且土壤速效磷含量最高.

表2 不同生境土壤理化性質(zhì)

圖4 不同生境土壤細(xì)菌群落結(jié)構(gòu)與環(huán)境因子的冗余分析(RDA)

SWC:土壤含水量,pH:土壤pH值,SOM:土壤有機(jī)質(zhì)含量,TN:土壤全氮含量,TP:土壤全磷含量,AN:土壤速效氮含量,AP:土壤速效磷含量

表3 土壤理化性質(zhì)與土壤細(xì)菌多樣性指數(shù)之間的相關(guān)關(guān)系

注:**:相關(guān)性顯著(<0.01),*:相關(guān)性顯著(<0.05).

2.4 不同生境土壤細(xì)菌PICRUSt基因預(yù)測(cè)

2.4.1 功能基因家族組成 Weighted NSTI是用來(lái)評(píng)估給定樣品中微生物與測(cè)序基因組相關(guān)的程度.裸沙地、草地、沙地樟子松天然林和人工林NSTI指數(shù)分別為0.202~0.223、0.184~0.187、0.172~0.182、0.171~0.172(表4).NSTI指數(shù)的數(shù)值越小,說(shuō)明樣品功能預(yù)測(cè)與已知數(shù)據(jù)庫(kù)的匹配度越高.

表4 不同生境土壤細(xì)菌NSTI指數(shù)

呼倫貝爾沙區(qū)不同生境中獲得的一級(jí)生物代謝功能通路分析相對(duì)豐度大于1%的功能基因包括4種(圖5):環(huán)境信息處理(Environmental Information Processing)、代謝(Metabolism)、遺傳信息處理(Genetic Information Processing)、有機(jī)系統(tǒng)(Organismal Systems).每種功能基因在不同生境中相對(duì)豐度基本一致,除沙地樟子松天然林功能基因相對(duì)豐度隨土層深度的變化而改變,其他3種生境在不同土層功能基因的相對(duì)豐度沒(méi)有改變.且呼倫貝爾沙區(qū)不同生境中樟子松天然林0~10cm土層功能基因與其他3種生境0~10cm土層功能基因存在顯著差異(<0.05).沙地樟子松天然林中代謝、遺傳信息處理、有機(jī)系統(tǒng)功能基因的相對(duì)豐度顯著高于其他生境,環(huán)境信息處理功能基因的相對(duì)豐度顯著低于其他生境.

圖5 不同生境土壤細(xì)菌功能預(yù)測(cè)(一級(jí)功能層)

以代謝通路相對(duì)豐度大于1%作圖

2.4.2 功能基因家族差異分析 6個(gè)一級(jí)功能層預(yù)測(cè)得到40個(gè)二級(jí)功能層中,其中相對(duì)豐度在0.1%以上的二級(jí)功能層有13個(gè)(圖6).4種生境的功能基因在二級(jí)功能層存在差異.其中,0~10cm土層中沙地樟子松天然林膜運(yùn)輸(Membrane Transport)的功能基因豐度顯著低于其他生境,核苷酸代謝(Nucleotide Metabolism)、酶家族(Enzyme Families)、氨基酸代謝(Amino Acid Metabolism)、碳水化合物代謝(Carbohydrate Metabolism)的功能基因豐度顯著高于其他生境.10~20cm土層中裸沙地中核苷酸代謝與酶家族的功能基因豐度顯著高于其他生境,草地和沙地樟子松天然林在該土層中氨基酸代謝的功能基因豐度偏低.

PC1軸的可信度是72.1%,PC2軸的可信度是19.7%(圖7).草地與沙地樟子松人工林兩種生境樣點(diǎn)相對(duì)聚集,表明兩種生境內(nèi)土壤細(xì)菌功能組成情況較為相似.裸沙地與沙地樟子松天然林內(nèi)樣點(diǎn)與另兩種生境距離較遠(yuǎn),由此推斷,這兩種生境與草地和沙地樟子松人工林土壤細(xì)菌功能組成存在較大差異.沙地樟子松天然林10~20cm土層中,有一個(gè)點(diǎn)與樣地內(nèi)其他樣點(diǎn)距離較遠(yuǎn),表明該生境10~20cm土層細(xì)菌功能組成情況存在變異性.同一生境距離之間的歐幾里得距離較同一土層之間的歐幾里得距離較近,因此,不同生境對(duì)細(xì)菌功能組成情況影響大于不同土層深度對(duì)細(xì)菌功能組成情況的影響.

圖6 不同生境土壤細(xì)菌功能預(yù)測(cè)熱圖(二級(jí)功能層)

僅展示代謝通路相對(duì)豐度大于0.1%功能

圖7 不同生境土壤細(xì)菌功能組成主成分分析

3 討論

3.1 生境對(duì)土壤細(xì)菌多樣性影響

呼倫貝爾沙區(qū)不同生境土壤細(xì)菌多樣性存在差異,這與前人研究結(jié)果一致.主要是因?yàn)椴煌惩寥乐兴牡蚵湮飻?shù)量、有機(jī)質(zhì)含量、根系分泌物均存在差異,這使得每種生境有其特定的微環(huán)境,從而影響細(xì)菌多樣性[23-24],土壤細(xì)菌多樣性與土壤理化性質(zhì)有關(guān),沙地樟子松人工林土壤含水量最低,pH值酸性最強(qiáng),全磷、速效磷含量顯著降低,從而使得該生境土壤細(xì)菌多樣性最低[25].不同生境土壤細(xì)菌群落多樣性隨土層深度的變化而改變,這與前人研究結(jié)果一致,裸沙地、草地、沙地樟子松人工林3種生境中10~20cm土層土壤含水量高于0~10cm土層土壤含水量,該生境土壤細(xì)菌多樣性隨土層深度增加而增加.但在沙地樟子松天然林中卻出現(xiàn)了相反的趨勢(shì),由于天然林表層土壤有良好的營(yíng)養(yǎng)和通氣條件,土壤結(jié)構(gòu)疏松,細(xì)菌群落結(jié)構(gòu)與土壤孔隙顯著相關(guān),天然林土壤的孔隙度不同于其他3種生境,細(xì)菌在表層土壤生長(zhǎng)迅速,從而提高了0~10cm土層的細(xì)菌多樣性[26-27].磷元素是植物生長(zhǎng)過(guò)程中所需要的最重要元素之一,植物對(duì)土壤中相對(duì)較高含量的磷元素有相對(duì)較低的吸收率[28],土壤速效磷含量與變形菌門、酸桿菌門存在相關(guān)性[29-30],土壤中存在的這些微生物能夠幫助植物吸收磷元素[31].

3.2 生境對(duì)土壤細(xì)菌群落結(jié)構(gòu)的影響

呼倫貝爾沙區(qū)土壤細(xì)菌優(yōu)勢(shì)門與陜北沙化區(qū)、黃土高原區(qū)、內(nèi)蒙荒漠草原的細(xì)菌優(yōu)勢(shì)門類別一致[32-34].不同生境中土壤優(yōu)勢(shì)細(xì)菌門的相對(duì)豐度不同是由各生境環(huán)境狀況的不同引起的,不同生境會(huì)影響土壤氮、磷含量,導(dǎo)致細(xì)菌群落結(jié)構(gòu)發(fā)生變化[35].由于變形菌門是營(yíng)養(yǎng)性的,對(duì)碳有很高的利用率[36],草地、沙地樟子松人工林和天然林中相對(duì)較高的土壤有機(jī)質(zhì)含量保證了沙地地區(qū)細(xì)菌的生長(zhǎng).由于放線菌門除對(duì)干旱有很強(qiáng)的耐受性外,還可以產(chǎn)生刺激促進(jìn)植物生長(zhǎng)的物質(zhì)[37].酸桿菌門在沙地樟子松天然林中相對(duì)豐度最高,這與前人研究不一致[38-39].沙地樟子松天然林與土壤pH值有顯著相關(guān)性,且該生境土壤pH值在4種生境中酸性最強(qiáng),多數(shù)研究認(rèn)為酸桿菌是一種土壤寡營(yíng)養(yǎng)菌,其生長(zhǎng)與土壤酸堿性密切相關(guān)[40-41].沙地樟子松天然林中土壤細(xì)菌群落豐富度和多樣性指數(shù)最高,且表層土有較高的營(yíng)養(yǎng)性,酸桿菌門起到降解植物纖維素的作用[42].不同生境土壤理化性質(zhì)與土壤細(xì)菌群落結(jié)構(gòu)有顯著的相關(guān)性[43-44],其中,裸沙地細(xì)菌群落結(jié)構(gòu)與土壤含水量有極強(qiáng)的相關(guān)性,土壤含水量影響土壤微生物呼吸,細(xì)菌群落結(jié)構(gòu)對(duì)土壤含水量變化敏感[45-46].

3.3 生境對(duì)土壤細(xì)菌功能基因的影響

在呼倫貝爾沙區(qū)四種典型生境的一級(jí)功能層中,因?yàn)榧?xì)菌群落的功能特征會(huì)影響細(xì)菌群落結(jié)構(gòu)與多樣性,沙地樟子松天然林中較高豐度的的代謝、遺傳信息處理、有機(jī)系統(tǒng)3類功能基因使土壤細(xì)菌代謝旺盛,生長(zhǎng)力好,從而提高了細(xì)菌群落結(jié)構(gòu)的多樣性[47].二級(jí)功能層的功能基因存在差異性,表現(xiàn)為樟子松天然林0~10cm土層中核苷酸代謝、酶家族、氨基酸代謝、膜運(yùn)輸?shù)墓δ芑蜇S度顯著高于其他樣本.功能基因代謝主要作用是從土壤中吸收營(yíng)養(yǎng)物質(zhì),通過(guò)攝取氨基酸、能量、碳水化合物等來(lái)保證細(xì)菌的存活,膜運(yùn)輸?shù)墓δ芑蚩梢匀芙忤F和小分子,保證細(xì)菌快速成活[48].樟子松天然林通過(guò)這些功能基因從土壤中攝取更多的核苷酸、氨基酸,提高了細(xì)菌多樣性.功能基因的豐度通過(guò)影響微生物過(guò)程進(jìn)而影響生態(tài)過(guò)程的轉(zhuǎn)化[49].

4 結(jié)論

4.1 呼倫貝爾沙區(qū)不同生境土壤細(xì)菌多樣性指數(shù)存在顯著性差異,且沙地樟子松天然林土壤細(xì)菌多樣性最高,沙地樟子松人工林中土壤細(xì)菌多樣性最低.其Shannon指數(shù)分別為8.623±0.193、7.432±0.028.

4.2 呼倫貝爾沙區(qū)不同生境土壤細(xì)菌群落組成及優(yōu)勢(shì)細(xì)菌門的豐度存在差異.草地、沙地樟子松人工林、沙地樟子松天然林3種生境中變形菌門相對(duì)豐度最高,其均值分別為29.83%±1.14%、34.73%±1.99%、31.95%±0.21%,裸沙地的主要優(yōu)勢(shì)細(xì)菌門為放線菌門,其相對(duì)豐度的均值為26.13%±0.43%.

4.3 呼倫貝爾沙區(qū)不同生境土壤細(xì)菌功能較為豐富.四種生境中土壤細(xì)菌群落在環(huán)境信息處理、代謝、遺傳信息處理、有機(jī)系統(tǒng)方面表現(xiàn)活躍.

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Soil bacteria community structure and function prediction in the Hulun Buir Sandy Area.

DU Yu-jia1,2,GAO Guang-lei1,2*, CHEN Li-hua1, DING Guo-dong1,2, ZHANG Ying1, CAO Hong-yu1,2

(1.Key Laboratory of State Forestry and Grassland Administration on Soil and Water Conservation, School of Soil and Water Conservation, Beijing Forestry University, Beijing 100083, China；2.Yanchi Ecology Research Station of the Mu Us Desert, Yanchi 751500, China)., 2019,39(11)：4840~4848

Soil samples were collected from four habitats in the Hulun Buir sandy area including bare sandland, grassland,varplantation andnatural forest. Subsequently, 16S rRNA genes high-throughput sequencing and PICRUSt-based functional prediction were performed to detect soil bacterial community structure and potential functional component. The results indicated that in the Hulun Buir sandy area, the natural forest and plantation had the highest and lowest soil bacterial Shannon Index (8.623±0.193 and 7.432±0.028), respectively. There were significant differences in alpha and beta diversity of soil bacteria in different habitats. The relative abundance of Proteobacteria was the highest in the grassland (29.83%±1.14%),plantation (34.73%±1.99%) and natural forest (31.95%±0.21%).The relative abundance of Actinobacteriawas the highest in the bare sand (26.13%±0.43%). The dominant soil bacteria genera with highest relative abundance were, RB41with the mean values of 5.29%±2.24% and 4.22%±1.23%, respectively. Soil bacterial functions were classified into 6 and 40 functional categories at hierarchy level 1and 2,which implied the abundant soil bacteria functions. Soil bacteria was active in environmental information processing, metabolism, genetic information processing and organic systems. Further, the functional genes of soil bacterial from the natural forest was abundant in nucleotide metabolism, enzyme family, amino acid metabolism and carbohydrate metabolism, which ensured soil bacteria survival with higher diversity. Conclusively, the functional genes fluctuation of soil bacteria associated with different habitats in the Hulun Buir sandy area reflected the changes of soil bacterial community structure and diversity, and indicated the effects of functional genes on soil bacterial community. Our study will provide a firm basis for better prediction and understanding of soil bacteirial metabolic potential and functions in sandy area.

Hulun Buir sandy area；habitat；community structure；16S rRNA；functional prediction

X172

A

1000-6923(2019)11-4840-09

杜宇佳(1996-),女,山西忻州人,北京林業(yè)大學(xué)碩士研究生,主要從事荒漠生態(tài)學(xué)研究.

2019-04-25

國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2018YFC0507101);國(guó)家自然科學(xué)基金項(xiàng)目(31600583);中央高?；究蒲袠I(yè)務(wù)費(fèi)專項(xiàng)資金項(xiàng)目(2017PT03, 2015ZCQ-SB-02)

* 責(zé)任作者, 副教授, gaoguanglei@bjfu.edu.cn