張 偉,陳一峰

(1. 新疆師范大學(xué)生命科學(xué)學(xué)院，烏魯木齊 830046； 2. 新疆農(nóng)業(yè)科學(xué)院微生物應(yīng)用研究所， 烏魯木齊 830000)

棉花長期連作對新疆土壤細(xì)菌群落結(jié)構(gòu)的影響

張 偉1,2,*,陳一峰2

(1. 新疆師范大學(xué)生命科學(xué)學(xué)院，烏魯木齊 830046； 2. 新疆農(nóng)業(yè)科學(xué)院微生物應(yīng)用研究所， 烏魯木齊 830000)

新疆部分棉區(qū)發(fā)生連作障礙后與其他地區(qū)有所不同，能自發(fā)恢復(fù)并長期保持高產(chǎn)、穩(wěn)產(chǎn)，為了查明該類棉田土壤細(xì)菌群落結(jié)構(gòu)在連作障礙發(fā)生及自發(fā)恢復(fù)整個過程中的演替規(guī)律。以未開墾土地作為對照，利用16S rRNA-PCR-DGGE(polymerase chain reaction-density gradient gel electrophoresis)法對比研究了新疆阿克蘇棉區(qū)分別連作1、3、5、10、15和20a棉田1—30 cm深度土壤細(xì)菌群落結(jié)構(gòu)組成。結(jié)果表明未開墾土地細(xì)菌多樣性指數(shù)豐富度最高，多樣性和均勻度指數(shù)最低。隨著棉花連作年限的延長，土壤細(xì)菌群落豐富度指數(shù)不斷下降，而多樣性和均勻度指數(shù)逐漸增大。當(dāng)連作年限繼續(xù)延長至10a后各指數(shù)出現(xiàn)恢復(fù)或趨于達(dá)到一個新的相對穩(wěn)定狀態(tài)。聚類分析顯示7個樣品分別聚為3簇，其中連作3a的樣品差異最大，相似度僅有44%，而連作10a后的樣品和對照較為相似。主成分分析也有類似的結(jié)果。對比回收的部分序列顯示，序列間相似性在88%以上，分屬于Microbacterium、UnculturedChloroflexibacterium、TM7Phylumsp. Canine、Flavobacteria等4個不同菌屬。分析認(rèn)為棉花長期連作對該地區(qū)土壤細(xì)菌群落結(jié)構(gòu)組成影響很大，但隨著連作年限延長至5a以后，細(xì)菌群落結(jié)構(gòu)組成能自發(fā)趨于穩(wěn)定和回升。此外，對比棉田細(xì)菌群落結(jié)構(gòu)整體變化規(guī)律和棉花產(chǎn)量的增減及病蟲害發(fā)生規(guī)律發(fā)現(xiàn)，在棉花長期連作過程中兩者有很強的關(guān)聯(lián)性。

棉田土壤；細(xì)菌群落；16S rRNA-PCR-DGGE；聚類分析；主成分分析

新疆是我國最早種植棉花的地區(qū)之一，也是目前我國唯一的長絨棉種植基地。由于新疆地區(qū)具有日照充足，降水稀少，空氣干燥等多種適于種植棉花的自然環(huán)境條件，自20世紀(jì)80年代以來新疆棉花的種植面積不斷擴大，至2012年新疆棉花在種植面積、總產(chǎn)量、平均畝產(chǎn)等7項指標(biāo)上已連續(xù)21a位居全國首位[1- 2]。與此同時，由于新疆農(nóng)作物種類單一，長期以來棉花連作現(xiàn)象嚴(yán)重，尤其是主產(chǎn)區(qū)，棉花種植面積占到95%以上，休作、輪作幾乎是不可能的。連作障礙是植物和土壤兩個系統(tǒng)內(nèi)部諸多因素綜合作用的結(jié)果[3], 土壤微生物群落結(jié)構(gòu)的穩(wěn)定對維持土壤系統(tǒng)的健康和質(zhì)量非常關(guān)鍵[4], 對植物生長具有重要作用[5]。土壤微生物群落結(jié)構(gòu)的變化會直接影響土壤功能的發(fā)揮[6]。與其他地區(qū)作物連作一樣，新疆棉花連作也帶來了病蟲害持續(xù)加重[7]、土壤中農(nóng)藥、化肥、地膜污染長期積累等生態(tài)問題[8- 9]。但值得關(guān)注的是部分地區(qū)的棉花隨著連作年限的繼續(xù)延長，發(fā)生連作障礙后只需施用較少的農(nóng)藥、化肥，連作障礙現(xiàn)象會自發(fā)的緩解并長年保持高產(chǎn)、穩(wěn)產(chǎn)，因此連作年限達(dá)到30a以上, 這一現(xiàn)象引起人們的關(guān)注。

新疆棉花主產(chǎn)區(qū)存在著棉花連作時間跨度從0—30a的各類棉田，棉花長期的種植已經(jīng)形成了獨特的棉田土壤微生物群落[10- 11]。這些微生物參與土壤物質(zhì)轉(zhuǎn)化過程，在土壤形成、肥力演變、農(nóng)業(yè)污染物的降解和土壤結(jié)構(gòu)的形成與改良等方面起重要作用，但長期的棉花連作對于微生物群落結(jié)構(gòu)帶來的影響和微生物群落結(jié)構(gòu)的變化與棉花連作后產(chǎn)量的下降及病蟲害發(fā)生規(guī)律之間的關(guān)系還不清楚。PCR-DGGE技術(shù)近些年在微生物生態(tài)領(lǐng)域有廣泛的應(yīng)用[12- 16]，本文利用該技術(shù)著重研究隨著棉花連作年限的延長，土壤中細(xì)菌群落在組成、多樣性和演替等方面發(fā)生的變化，試圖從土壤微生物生態(tài)的角度解釋新疆棉花長期連作后連作障礙的發(fā)生及自發(fā)消除，以及該地區(qū)棉花種植過程中病蟲害的發(fā)生規(guī)律與土壤微生物群落結(jié)構(gòu)演替之間的聯(lián)系。

1 材料和方法

1.1 樣品的采集

采樣時間和地點： 2011年8月于新疆阿克蘇高產(chǎn)棉區(qū)，選擇棉花連作年限為0(尚未開墾的土地)、1、3、5、10、15和20a的土地分別收集土樣。方法：土鉆垂直打下30 cm(耕作層)深度取土，相同連作年限的棉田選擇不同的5處采集等量樣品混合后作為一個土樣，合計為7個土樣。采樣范圍在(E 80°16′77″—24′45″,N 41°07′47″—24′85″)之間。土樣采集后低溫保存帶回實驗室，-80 ℃儲存使用。

1.2 樣品總DNA的提取和純化

DNA的提取參照Zhou等的SDS based DNA extraction法[17- 18]，唯一改動之處是，在氯仿、異戊醇抽提前多加了一步等體積的酚、氯仿和異戊醇抽提。DNA的純化按Moreira的方法[19],每一DNA樣品做3個重復(fù)，等量混合后備用。

1.3 16s rRNA片段的PCR擴增

第一套PCR 用細(xì)菌的通用引物For/Dev擴增差不多全長的16s rRNA[20](表1)。25 μL反應(yīng)體系：2.5 mmol/L的dNTP 2.8 μL，2.5 μL 10x Buffer，1u Taq酶，引物各3 pmol。反應(yīng)程序：94 ℃，5 min；94 ℃，60 s；55 ℃，45 s；72 ℃，60 s，共3O個循環(huán)；72 ℃，5 min后穩(wěn)定在4 ℃。

第二套PCR 用F341GC/R534引物擴增細(xì)菌16 s rRNA v3區(qū)[21](表1)。25 μL反應(yīng)體系：稀釋100倍的第一套PCR產(chǎn)物1 μL，2.5 mmol/L的dNTP 2.8 μL，2.5 μL 10x Buffer，1 u Taq酶，引物各5 pmol。反應(yīng)程序：94 ℃，5 min；94 ℃，60 s；55 ℃，45 s；72 ℃，60 s；共30個循環(huán)：72℃，5 min后穩(wěn)定在4 ℃。

1.4 PCR產(chǎn)物的DGGE電泳

用D-code System電泳儀(Bio-Rad公司)進行DGGE電泳分離。制備變性梯度凝膠，使聚丙烯酰胺凝膠(Polyacrylamide gel electrophoresis,PAGE)濃度為6%—8%，變性梯度30%—70% (7mol/L 尿素和4O%甲酰胺為100%變性)，電泳緩沖液為1x TAE，25 μL 第二套PCR產(chǎn)物在6O ℃，150 V條件下電泳4 h，取出后用硝酸銀法染色[22]。FR- 200紫外與可見分析裝置(復(fù)日科技)下進行拍照。

表1 用于16s rRNA擴增的引物

在5′端加入了一個富含GC序列的GC夾：CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G

1.5 數(shù)據(jù)處理

用Quantity One v4.62軟件對DGGE 圖譜進行數(shù)字化、標(biāo)準(zhǔn)化后, 得到一個記錄DGGE 膠中條帶遷移位置和亮度的數(shù)字化矩陣。系統(tǒng)自動依據(jù)條帶的有無按照非加權(quán)成對算術(shù)平均法(Unweighted Pair-Group Method with Arithmetic,UPGMA)對各泳道土壤樣品進行聚類和相似性比較。用Gel-Pro analyzer、SPSS16.0和Excel軟件直接讀取DGGE指紋圖譜條帶信息，以代表每一細(xì)菌種群的條帶在每一泳道中亮度峰值的百分含量為重要值對樣品進行主成分(Principal component analysis, PCA)和相關(guān)方差分析等工作。用Shannon-Wiener指數(shù)(H)、豐富度(S)和均勻度(EH)來評價土壤細(xì)菌群落的多樣性，其算式為：

EH=H/Hmax=H/lnS

式中，pi為某一條帶的強度與同泳道中所有條帶總強度的比值，S為每一泳道總的條帶數(shù)[23]。根據(jù)泳道的pi值進行PCA分析。

1.6 特異序列的回收及信息比對

選擇并回收DGGE凝膠上0a樣品中所占灰度比較大的條帶和連作樣品中出現(xiàn)的特異條帶?；厥蘸涂寺⒖糄aniela的方法[24]。對回收的序列上傳至GenBank,獲得的序列號是JN5725- 63。通過BLAST查詢及CLUSTAL X2.0、MEGA4.0 軟件處理構(gòu)建相關(guān)序列進化樹。

2 結(jié)果與討論

2.1 連作對棉田細(xì)菌多樣性的影響

細(xì)菌16s rRNA的PCR-DGGE圖譜顯示條帶數(shù)較多，可見各樣品中細(xì)菌多樣性都很豐富。其中不同連作年限土樣分離出有相同的帶也有不同的帶，但帶的多少及光密度都發(fā)生了較大變化(圖1)。通過軟件進一步數(shù)字化處理后得到表示土壤細(xì)菌多樣性的Shannon-Wiener指數(shù)(H)、豐富度(S)和均勻度(E)指數(shù)。由數(shù)據(jù)看出，香農(nóng)多樣性指數(shù)未開墾地最低為2.98，隨著連作年限的增加，細(xì)菌多樣性指數(shù)先增加后減少，其中連作5—15a的數(shù)值比較穩(wěn)定。豐富度未開墾土地最高為69，連作后都有所降低，最低為未開墾地為46。均勻度是未開墾地最低為0.668，連作后有所增加，其中10a的最高為0.887(表2)。由此可知棉花長期連作使土壤細(xì)菌群落結(jié)構(gòu)類型發(fā)生了很大改變[25- 28]。首先，未開墾土樣香農(nóng)多樣性和均勻度指數(shù)最低，但其豐富度最高，分析原因可能是原生態(tài)土壤中有機質(zhì)和水分等含量較少不利于細(xì)菌的大量繁殖，但細(xì)菌群落結(jié)構(gòu)組成較豐富[12]。其次，棉花的種植使土壤中細(xì)菌多樣性和均勻度增大，而豐富度下降，尤其是連作前5a各指數(shù)的變化較大，而隨著連作年限的繼續(xù)延長，各指數(shù)的變化趨緩。分析原因可能是棉花的種植給土壤輸入較多的有機質(zhì)等營養(yǎng)物質(zhì)導(dǎo)致細(xì)菌的大量繁殖，而某些種類的細(xì)菌由于環(huán)境的不利改變而消失[7]。

圖1 棉花連作0、1、3、5、10、15、20a的土壤細(xì)菌16s rRNA DGGE圖譜及聚類分析Fig.1 DGGE patterns and cluster analysis of 16s rRNA from 0, 1, 3, 5, 10, 15 and 20 years cotton continuous cropping field, respectively

Table 2 Shannon-Weaver diversity (H), eveness index (E) and abundance index (S) of the cotton soils bacterial community with difference continuous cropping years

樣品Number樣地Sample香農(nóng)-威納指數(shù)Shannon-Wienerdiversity豐富度指數(shù)Abundanceindex均勻度指Evenessindex1連作0a2.98±0.13b*69±4.5a0.668±0.06a2連作1a3.12±0.14b46±0.6a0.841±0.03b3連作3a3.41±0.17a67±0.5a0.811±0.02b4連作5a3.28±0.13b56±3.5b0.815±0.01b5連作10a3.44±0.11a48±4.0b0.887±0.07a6連作15a3.65±0.10a67±0.5a0.868±0.02b7連作20a3.06±0.09a48±0.5b0.790±0.04a

*a, b表示在P<0.05水平下有顯著差異

2.2 連作對棉田細(xì)菌群落結(jié)構(gòu)組成的影響

聚類分析結(jié)果表明：土壤中細(xì)菌群落結(jié)構(gòu)組成發(fā)生了較大的變化。其中，連作1、5和15a的聚為一小類，相似度達(dá)55%以上。連作0、10和20a的聚為另一小類，相似度達(dá)50%以上。而連作3a的最后才聚到一起，相似度只有44%(圖1)?？梢娺B作3a的土樣細(xì)菌群落結(jié)構(gòu)變化最大，而隨著棉花連作年限的延長，細(xì)菌群落結(jié)構(gòu)呈現(xiàn)反復(fù)波動并有恢復(fù)的趨勢。對DGGE圖譜各泳道每個條帶的Pi值進行主成分分析,結(jié)果表明，第一主成分方差貢獻率達(dá)55.37%，第二主成分方差貢獻率達(dá)15.5%，積累方差達(dá)70.87%。在這兩個主成分為坐標(biāo)軸構(gòu)建的二維坐標(biāo)系中，不同連作年限樣品與對照相比都發(fā)生了明顯的位置變化，但又明顯被分為兩組，其中連作5、10、和20a樣品與對照較為一致，而連作1、3和15a樣品變化趨勢較一致，其中棉花連作3a對土壤細(xì)菌群落組成影響最大，這點和聚類分析結(jié)果一致(圖2)?？傮w分析原因可能在外界環(huán)境發(fā)生長期一致的改變后，土壤細(xì)菌群落為應(yīng)對外界環(huán)境的變化自發(fā)的調(diào)整結(jié)構(gòu)類型，表現(xiàn)在反復(fù)波動中逐漸恢復(fù)并穩(wěn)定下來，同時顯示出一定的穩(wěn)定性[25]。由此可見棉花的長期種植不僅改變了原土壤細(xì)菌群落的微環(huán)境，并影響了土壤細(xì)菌群落結(jié)構(gòu)組成。

圖2 樣品細(xì)菌群落結(jié)構(gòu)主成分分析Fig.2 Principal component analysis of the monoculture cotton soil bacterial with different years

2.3 序列同源性分析

條帶1—8是0a樣品強度較大的，條帶a—k是棉花連作不同年限后新出現(xiàn)的強度較大的(圖1)。這19個條帶之間同源性在88%—100%之間。部分序列與已知的Microbacteriuminsulae、Devrieseaagamarum、Atopobiumrimae等微生物同源性達(dá)到100%，并隸屬于多個屬。查閱已知的與DGGE凝膠上回收條帶序列同源性最近的微生物信息，都屬于土壤中常見的類型(進化樹結(jié)果)(圖3)。由于土壤細(xì)菌數(shù)量和種類繁多，DGGE凝膠上出現(xiàn)的條帶很多，而每一條帶代表的是一類細(xì)菌[26]，雖然回收了部分特異的條帶，但測序后并沒有發(fā)現(xiàn)有導(dǎo)致棉花病蟲害的微生物信息，至于哪些菌群發(fā)生的改變會影響棉花產(chǎn)量和病蟲害的發(fā)生還有待深入研究[26]。

2.4 連作棉花發(fā)病規(guī)律、產(chǎn)量變化與土壤細(xì)菌群落結(jié)構(gòu)變化的關(guān)系

耕作土中細(xì)菌群落的結(jié)構(gòu)組成和分布特征受土壤理化性質(zhì)、當(dāng)?shù)厮臍夂虻鹊挠绊慬27]，此外耕作制度對土壤細(xì)菌結(jié)構(gòu)組成的影響也是非常重要的[28- 29]。目前關(guān)于農(nóng)作物連作對土壤細(xì)菌群落組成的研究比較多，但還是不能完全清楚土壤微生物與作物之間的復(fù)雜相互關(guān)系。新疆作為我國長絨棉種植基地，與其他地區(qū)作物連作一樣，起初棉花連作也帶來了病蟲害加重、產(chǎn)量下降等負(fù)面影響[30]，但隨著連作年限的繼續(xù)延長，連作障礙的現(xiàn)象能自發(fā)緩解[7]。針對這一現(xiàn)象本文研究了0—20a棉花連作過程中土壤細(xì)菌群落結(jié)構(gòu)組成的變化規(guī)律。在參考當(dāng)?shù)剞r(nóng)技部門記載的數(shù)據(jù)和走訪棉花種植戶后發(fā)現(xiàn)：棉花連作障礙的發(fā)生與土壤細(xì)菌群落結(jié)構(gòu)的變化規(guī)律在時間上比較一致[31- 32]。比如，連作3a是產(chǎn)量較低、病蟲害較重的階段，而土壤細(xì)菌群落結(jié)構(gòu)變化也最大。隨著連作年限延長至5a之后各種負(fù)面影響趨于減緩，10a之后甚至出現(xiàn)恢復(fù)的跡象。研究后發(fā)現(xiàn)土壤細(xì)菌群落組成也在這個階段出現(xiàn)穩(wěn)定及恢復(fù)的趨勢。但由于土壤細(xì)菌組成和功能的復(fù)雜性，對于土壤細(xì)菌群落組成與農(nóng)作物之間的互作關(guān)系了解較少，因此還需要做更深入的研究才能更好的給當(dāng)?shù)孛藁ㄟB作造成的土傳病蟲害防治及耕作管理制度的改善提供指導(dǎo)[33]。

3 結(jié)論

首先：與利用其他方法的相關(guān)研究報道有相似之處[33- 34]，都發(fā)現(xiàn)棉花單一作物長期連作對本地區(qū)土壤細(xì)菌群落結(jié)構(gòu)組成及功能有重大影響，表現(xiàn)在隨著連作年限的增加，土壤細(xì)菌結(jié)構(gòu)組成發(fā)生反復(fù)波動。當(dāng)連作年限超過5a后，土壤細(xì)菌群落組成逐漸趨于穩(wěn)定，并與原生態(tài)土壤細(xì)菌結(jié)構(gòu)組成有一定的相似性。其次，長期一致的外界脅迫可以使土壤微生物群落結(jié)構(gòu)組成發(fā)生適應(yīng)性改變，但在某些特殊的環(huán)境下也有可能形成新的、穩(wěn)定的、健康的、適合作物長期種植的結(jié)構(gòu)類型。此外，在長期的連作過程中，土壤會發(fā)生“細(xì)菌型”向“真菌型”的轉(zhuǎn)變[35]，因此也有必要研究土壤真菌的結(jié)構(gòu)組成及多樣性隨著連作年限延長發(fā)生的變化。

圖3 對回收的序列和相似性序列繪制系統(tǒng)樹Fig.3 The closest sequence match of known phylogenetic affiliation to band sequences extracted from DGGE gel

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Analysis of the bacterial communities in continuous cotton fields of Xinjiang Province

ZHANG Wei1,2,*,CHEN Yifeng2

1CollegeofLifeScience,XinJiangNormalUniversity,Urumqi830046,China2InstituteofMicrobiology,XinjiangAcademyofAgriculturalSciencesUrumqi830000,China

Long-term continuous cropping of cotton has caused dramatic soil-borne diseases in many places, leading to substantial agricultural losses. However, in some areas of Xingjang Province, the obstacles caused by continuous cotton cropping can spontaneously restore and maintain high yields of cotton for many years. To analyze the variable spectrum of soil bacterial communities and changes in the community structure in these spontaneously restored fields during cropping, soils at depths from 1 to 30 cm were sampled from cotton fields with a history of 0, 1, 3, 5, 10, 15 or 20 years of cotton cropping in the Akesu region of Xinjiang Province. The bacterial communities in these samples were studied using 16S rRNA-based polymerase chain reaction-density gradient gel electrophoresis (PCR-DGGE) with samples from uncultivated land as a control. Bacterial community diversity indices including the Shannon-Wiener diversity (H), Abundance index (S) and Evenness index (EH) were compared among these samples. Samples from uncultivated land had relatively high levels of the richness indices but both the Diversity and Evenness Indices were at lower levels. With increasing years of cotton cropping, both the bacterial Diversity and Evenness Indices increased, whereas the richness indices showed a general decrease. However, after 10 years of continuous cropping all these indices were restored to their original values or reached a relative stable level. Cluster analysis of DGGE fragments indicated that the seven samples were clustered into three branches: fragments from samples under successional cropping for 0, 10 and 20 years formed one small branch with a similarity of approximately 50%; fragments from successional cropping for 1, 5 and 15 years formed another branch with a similarity of 53%; and the last branch comprised fragments from successional cropping for 3 years with a similarity of 44%. Principal component analysis (PCA) showed that all of the samples were statistically correlated with the major component and fluctuated on the right of the major component between the positive and negative axes of the second principal component. Both cluster analysis and PCA results suggested that, compared to those from original uncropped fields, the bacterial community structure showed the most variation in samples from the field of 3-year cropping, whereas similar patterns of bacterial community structure were found between samples from fields of 10 years of cotton cropping and those from uncropped fields. Nineteen clones were sequenced from each band and among them one sequence was selected and submitted to GenBank (accessory no. JN572545-JN572563). By aligning with the GenBank database, all sequences from DGGE were classified into four groups:Microbacterium, UnculturedChloroflexibacterium, TM7Phylumsp. canine, andFlavobacteria. Further analysis demonstrated that the isolated V3 sequences showed a homology of 88%—100% to known sequences in GenBank and 47% of the sequences belonged to bacteria which were not cultured. No microbial data were correlated with soil-borne plant diseases of cotton. The study demonstrated that the age of cotton fields had significant effects on soil bacterial diversity. Continuous cotton cropping exerted significant influences on the community structure of soil bacteria in Xinjiang Province, with an initial suppression effect on bacterial diversity. However, the bacterial community reached a stabilized or even increased level compared with its original state after 5 years of continuous cropping. In addition, correlations between variations in the bacterial community structure at a depth of 1—30 cm and the yield of cotton and pest disease attacks were also found in this study.

cotton field soils; bacterial communities; 16S rRNA-PCR-DGGE; cluster analysis; principal component analysis

國家自然科學(xué)基金項目(30860016);新疆師范大學(xué)科研處(XJNUBS1004); 微生物重點學(xué)科資助項目

2012- 12- 18; 網(wǎng)絡(luò)出版日期:2014- 03- 04

10.5846/stxb201212181817

*通訊作者Corresponding author.E-mail: zw0991@sohu.com

張偉,陳一峰.棉花長期連作對新疆土壤細(xì)菌群落結(jié)構(gòu)的影響.生態(tài)學(xué)報,2014,34(16):4682- 4689.

Zhang W, Chen Y F.Analysis of the bacterial communities in continuous cotton fields of Xinjiang Province.Acta Ecologica Sinica,2014,34(16):4682- 4689.