微生物有機肥對櫻桃園土壤細(xì)菌群落的影響

張凱煜,谷 潔,王小娟,高 華

?

微生物有機肥對櫻桃園土壤細(xì)菌群落的影響

張凱煜,谷 潔*,王小娟,高 華

(西北農(nóng)林科技大學(xué)資源環(huán)境學(xué)院,陜西 楊凌 712100)

采用田間試驗,探究微生物有機肥對櫻桃園土壤細(xì)菌群落的影響.利用高通量測序和實時定量PCR技術(shù),研究不施肥(CK)、常規(guī)施肥(CN)和施微生物有機肥(CB)處理土壤細(xì)菌數(shù)量、多樣性和群落結(jié)構(gòu)的變化.結(jié)果表明,施微生物有機肥顯著提高了土壤有機質(zhì)、全氮、堿解氮和速效磷含量. 結(jié)合16S rRNA 基因拷貝數(shù)和α-多樣性指數(shù)結(jié)果,發(fā)現(xiàn)施微生物有機肥能提高細(xì)菌數(shù)量,且提高細(xì)菌多樣性和豐富度.不同施肥處理顯著改變了細(xì)菌群落結(jié)構(gòu).門水平上,變形菌門、酸桿菌門、厚壁菌門、芽單胞菌門、放線菌門為優(yōu)勢類群,共占細(xì)菌總量的74.3%~85.1%.目水平上, CB處理中Acidobacteria_Gp4和Gp6相對豐度顯著低于CK處理,而Acidobacteria_Gp7較CK處理增加了75.4%.冗余分析結(jié)果表明,環(huán)境因子解釋了細(xì)菌群落變化的92.3%,土壤有機質(zhì)、全氮含量和pH值是造成櫻桃園土壤細(xì)菌群落結(jié)構(gòu)差異的主要原因.因此,施用微生物有機肥能顯著提高土壤養(yǎng)分含量、土壤細(xì)菌數(shù)量及群落多樣性,對于培肥地力極為重要.

微生物有機肥；細(xì)菌群落；高通量測序；實時定量PCR

近年來,集約化農(nóng)業(yè)推動了農(nóng)業(yè)生產(chǎn)率的大幅提高,但長期大量施用化肥致使土壤肥力下降,影響土壤微生物活性,進而影響作物的產(chǎn)量和品質(zhì).土壤微生物不僅驅(qū)動著土壤物質(zhì)轉(zhuǎn)化和養(yǎng)分循環(huán),還可作為土壤有效養(yǎng)分的儲備庫[1].因此,土壤微生物在農(nóng)業(yè)土壤生態(tài)系統(tǒng)中的作用日益受到關(guān)注.施肥不僅能顯著增加土壤肥力,同時對土壤微生物的數(shù)量[1-2]、多樣性和活性[3-5]等也會產(chǎn)生顯著影響.微生物有機肥是指利用特定功能微生物與畜禽糞便、農(nóng)作物秸稈等為原料,經(jīng)腐熟混合處理而成的一類新型有機肥料,兼具微生物肥料和有機肥雙重效應(yīng),既有利于增產(chǎn)增收,又可以培肥土壤,減少化肥用量,同時還能實現(xiàn)農(nóng)業(yè)廢棄物資源化利用[6-7].

近年來的研究表明,微生物有機肥能夠調(diào)節(jié)土壤中微生物區(qū)系組成,使土壤向著健康方向發(fā)展[8-10].劉方春等[11]利用T-RFLP 研究了櫻桃根際土壤細(xì)菌群落,發(fā)現(xiàn)適當(dāng)?shù)母珊的芴岣吒H土壤細(xì)菌群落的多樣性.王偉華等[12]利用PLFA和MicroRespTM技術(shù)發(fā)現(xiàn)化肥配施有機肥顯著改善了土壤養(yǎng)分含量和土壤微生物量、微生物群落結(jié)構(gòu)和活性,對培肥地力和優(yōu)化土壤微生物群落極為重要.隨著測序技術(shù)的快速發(fā)展,新一代測序技術(shù)被廣泛應(yīng)用到環(huán)境微生物群落的研究中.高通量測序技術(shù)具有測序深度深和獲得數(shù)據(jù)量大等優(yōu)點,能更真實地揭示微生物群落的復(fù)雜性和多樣性,加快了環(huán)境中不可培養(yǎng)和痕量微生物的研究[13].目前,高通量測序已成為研究微生物群落變化的主要方法之一.前人利用高通量測序平臺探討了施肥和灌溉等農(nóng)業(yè)管理措施對土壤中微生物群落多樣性和結(jié)構(gòu)組成的影響[14-16].然而,化肥和微生物有機肥處理下,土壤細(xì)菌群落的響應(yīng)是否有差異,尤其是不同施肥影響了哪些關(guān)鍵菌群,進而調(diào)控了土壤養(yǎng)分循環(huán),亟需進行深入分析.

本研究在試驗區(qū)設(shè)置了不同施肥處理試驗,采用實時定量PCR 和高通量測序方法,探究土壤細(xì)菌群落結(jié)構(gòu)和多樣性對施用不同肥料的響應(yīng)差異,并結(jié)合土壤理化性狀揭示其驅(qū)動因素,以期闡明不同施肥類型影響土壤肥力的生物學(xué)機制,為指導(dǎo)櫻桃合理施肥提供依據(jù).

1 材料與方法

1.1 試驗設(shè)計

試驗于2016年3月在陜西省興平市西吳鎮(zhèn)進行(108o49′N,34o32′E),供試櫻桃品種為艷陽,樹齡6年,栽培密度2m×4m.本試驗所在地是陜西優(yōu)質(zhì)櫻桃生產(chǎn)區(qū)域之一.目前櫻桃園管理粗放,農(nóng)民以施化肥為主,施肥的盲目性很大,不僅增加了生產(chǎn)成本且效果不佳.供試土壤基本性質(zhì)為:有機質(zhì)21.3g/kg、全氮0.89g/kg、全磷1.29g/kg、全鉀17.8g/kg、堿解氮70.5mg/kg、速效磷18.4mg/kg、速效鉀496mg/kg、pH=7.9.供試微生物有機肥為西北農(nóng)林科技大學(xué)資源環(huán)境學(xué)院制作的微生物有機肥,其有益微生物(主要為解磷菌及解鉀菌)數(shù)量為2.8′108cfu/g,有機質(zhì)含量28.3%,氮(N) 3.8%,磷(P2O5)2.6%,鉀(K2O) 3.1%.試驗設(shè)3個處理: CK,不施肥; CN,常規(guī)施肥,施肥量為復(fù)合肥1350kg/hm2(復(fù)合肥含N 15%、P2O515%、K2O 15%); CB,施微生物有機肥,施肥量為5325kg /hm2(以化肥養(yǎng)分氮含量折算施用等養(yǎng)分微生物有機肥,即N 202.5kg/hm2、P2O5138kg/hm2、K2O 165kg/hm2).小區(qū)面積60m2,3次重復(fù),隨機區(qū)組排列.肥料于2016年3 月15日一次施入,方法為將肥料均勻地撒在樹冠內(nèi)外的地面上,樹冠內(nèi)2/3,樹冠外1/3,施肥后深翻入土壤.

1.2 樣品采集

土樣采集于2017年9月20日進行,采用"S形"五點采樣法取0~20cm土壤樣品.將新鮮土樣裝入無菌自封袋,用冰盒迅速帶回實驗室,去除雜物后分為2部分.一部分樣品經(jīng)冷凍干燥后,用于土壤微生物分析.另一部分土樣風(fēng)干后過篩1mm和0.25mm,用于土壤理化性質(zhì)測定.

1.3 測定項目與方法

1.3.1 土壤DNA提取和PCR擴增 土壤樣品DNA利用FastDNA SPIN Kit for Soil(MP Biomedicals,美國)由0.100g樣品中提取,具體步驟根據(jù)使用手冊進行.利用Nanodrop Spectrophotometer ND-1000(Thermo Fisher Scientific,美國)檢測DNA濃度和純度,保存于-80℃冰箱.

1.3.2 細(xì)菌16s rRNA基因定量檢測 16S rRNA基因豐度定量檢測:16S rRNA基因采用細(xì)菌特異性引物341F(CCTACGGGAGGCAGCAG),518R(ATTA- CCGCGGCTGCTGG)[17].首先將樣品DNA進行普通PCR (Bio-Rad ALD1244,美國)將PCR產(chǎn)物進行1%的瓊脂糖電泳檢測,切割清晰單一的電泳條帶進行回收DNA.其次,將回收的DNA進行克隆,本文采用pGEM-T Easy載體將目的基因片段轉(zhuǎn)化到大腸桿菌(DH5α)中,并將大腸桿菌菌液涂布于含氨芐的LB培養(yǎng)基上,加IPTG和X-gal做藍白斑篩選.將陽性克隆子在LB液體培養(yǎng)基中過夜培養(yǎng),抽提質(zhì)粒.檢測提取的質(zhì)粒DNA濃度,并送往生工(上海,中國)進行測序分析,確定目的基因是否存在.利用公式(1)換算成拷貝數(shù)(copies).其中目的基因長度(bp)由表示,所用pGEM-T Easy載體長度為3015.標(biāo)準(zhǔn)曲線由質(zhì)粒DNA進行10倍稀釋梯度制成.

在Bio-Rad CFX ConnectTM(Bio-Rad,美國)中進行定量分析.采用20μL反應(yīng)體系,包括模板DNA 1μL,正向和反向引物各0.2μL(20pmol/L),熒光定量PCR預(yù)混液10μL(北京康為,中國),滅菌超純水8.6μL,退火溫度為55℃.擴增效率在90%~110%則符合反應(yīng)要求.將土壤DNA原液稀釋10倍后,作為反應(yīng)體系中所用的模板DNA,可消除樣品DNA中抑制物對整個反應(yīng)的影響.通過溶解性曲線判斷反應(yīng)過程中的非特異性擴增情況.

1.3.3 高通量測序 在北京諾禾致源科技股份有限公司利用Illumina HiSeq PE250平臺進行16S rRNA 基因V4可變區(qū)高通量測序.根據(jù)每個測序樣品特異的barcode,將原始測序結(jié)果準(zhǔn)確分配至各測序樣品.利用FLASH將原DNA片段中測定的雙端序列進行合并,并采用QIIME質(zhì)量過濾器對合并的序列進行過濾,隨后使用UCHIME剔除PCR嵌體,獲得高質(zhì)量的序列[18].通過UPARSE將序列進行聚類(97%相似度),獲得可操作分類單元(OTUs).利用QIIME軟件對樣品中OTUs進行抽平分析,然后將每個樣品中抽平的OTUs通過RDP分類器進行分類,得到OTUs詳細(xì)的注釋結(jié)果,進行后續(xù)分析.

1.3.4 土壤理化性質(zhì)的測定 參考鮑士旦的相關(guān)方法[19].土壤pH值采用電位法;有機質(zhì)采用重鉻酸鉀-外加熱法;全氮采用凱氏定氮法;土壤全磷采用HCLO3-H2SO4.消煮-鉬銻抗比色法;土壤全鉀采用NaOH熔融-火焰光度計法;堿解氮用堿解擴散法;速效磷采用NaHCO3浸提-鉬銻抗比色法;速效鉀用NH4OAc浸提-火焰光度計法測定.

1.4 數(shù)據(jù)處理與分析

數(shù)據(jù)采用SPSS v19.0進行統(tǒng)計分析和方差分析(LSD,<0.05),用Microsoft Excel 2013進行繪圖.利用CANOCO 4.5軟件進行細(xì)菌群落和環(huán)境因子間冗余分析(RDA).采用R軟件繪制細(xì)菌目水平相對豐度Heatmap圖,并進行基于OTUs的相對豐度主坐標(biāo)分析(PCoA)[20].

2 結(jié)果與分析

2.1 不同施肥處理對土壤理化性質(zhì)的影響

不同施肥處理顯著影響了土壤理化性質(zhì)(表1).不同處理間土壤pH值無顯著差異.與CK處理相比,施用微生物有機肥CB處理中土壤有機質(zhì)、全氮、堿解氮和速效磷分別增加了28.5%、61.4%、67.8%和185.7%. CN處理與CK處理相比土壤有機質(zhì)、堿解氮和速效磷分別增加了11.9%、52.7%、77.8%,全氮、全磷、全鉀和速效鉀含量與CK處理差異不顯著(<0.05).由此可見,施肥可以顯著提高土壤養(yǎng)分,改善土壤理化性質(zhì),但常規(guī)施肥效果不如微生物有機肥.

表1 不同施肥處理土壤理化指標(biāo) (1)Table 1 Soil properties in different fertilization treatments

注:平均值±標(biāo)準(zhǔn)誤差,=3; 同1行數(shù)據(jù)中不同字母表示處理間差異顯著(<0.05).

2.2 不同施肥處理對細(xì)菌16s rRNA豐度和多樣性指數(shù)的影響

利用qPCR對土壤細(xì)菌16s rRNA基因定量檢測(圖1),不同施肥處理中16s rRNA基因豐度變化為1.48×109~8.26×1010copies/g.其中CB處理較CK和CN處理提高了1個數(shù)量級,差異顯著.利用 Illumina HiSeq平臺對細(xì)菌16S rRNA基因V4區(qū)測序,各樣品的測序覆蓋度為0.9657~0.9698.樣品OTU稀釋性曲線均趨于平坦,表明測序深度包括了樣品中的絕大多數(shù)細(xì)菌類型,測序數(shù)據(jù)量合理

香農(nóng)指數(shù)(Shannon)、辛普森指數(shù)(Simpson)和Chao1指數(shù)是表征微生物α-多樣性的指標(biāo),其值越高表示微生物多樣性越高,物種豐富度越高[15].CB處理較CN和CK處理的香農(nóng)指數(shù)分別提高了6.92%和6.13%, Chao1指數(shù)分別提高了15.26%和28.65%,并表現(xiàn)出顯著差異(<0.05).結(jié)果表明微生物有機肥能提高細(xì)菌數(shù)量,且提高細(xì)菌多樣性和豐富度.

2.3 不同施肥處理對土壤細(xì)菌群落結(jié)構(gòu)的影響

圖2 不同施肥處理土壤細(xì)菌群落結(jié)構(gòu)主坐標(biāo)分析 (1) Fig.2 Principal coordinate analysis of the soil bacterial community structure under different fertilizer treatments

基于OTU水平對細(xì)菌群落結(jié)構(gòu)進行主坐標(biāo)分析(PCoA).由圖2可知,PC1和PC2共解釋總變量的92.0%.CK,CN,CB處理分別位于第3象限,第2象限和第4象限,說明不同施肥顯著改變了細(xì)菌群落結(jié)構(gòu).

在門水平上(圖3a),變形菌門(Proteobacteria)、酸桿菌門(Acidobacteria)、厚壁菌門(Firmicutes)、芽單胞菌門(Gemmatimonadetes)、放線菌門(Actinobacteria)為優(yōu)勢類群,共占細(xì)菌總量的74.3%~85.1%.在目水平上(圖3b),梭菌目(Clostridiales), Acidobacteria-GP6,芽單胞菌目(Gemmatimonadales)為主要優(yōu)勢類群. Acidobacteria_Gp4、Gp6、Gp7為酸桿菌門的主要類群,它們對不同施肥處理響應(yīng)不同.CB和CN處理中Acidobacteria_Gp4相對豐度在較CK處理分別降低了40.4%和24.6%, Acidobacteria_Gp6較CK處理分別降低了41.3%和34.3%.而Acidobacteria_Gp7變化趨勢相反,在CN和CB處理分別較CK處理增加了91.6%和75.4%.

進一步分析環(huán)境因子對細(xì)菌群落的影響,在門水平上與土壤pH值、有機質(zhì)、全氮、全磷、全鉀、堿解氮、速效磷、速效鉀等理化指標(biāo)進行冗余分析(圖4).結(jié)果顯示,所選取的環(huán)境因子共解釋細(xì)菌群落變化的92.3%,其中土壤有機質(zhì)、全氮含量和pH值均顯著影響了細(xì)菌群落結(jié)構(gòu).

圖4 不同施肥處理環(huán)境因子對細(xì)菌群落結(jié)構(gòu)的冗余分析 Fig.4 Redundancy analysis based on the soil properties and the bacterial community structure

3 討論

土壤微生物是土壤生態(tài)系統(tǒng)中的重要組成部分,在營 (1)養(yǎng)物質(zhì)循環(huán)過程中起著重要作用, 對保持土壤肥力及土壤可持續(xù)利用至關(guān)重要[21-22].作為土壤中重要的生命有機體,微生物生物學(xué)特性和群落結(jié)構(gòu)與土壤質(zhì)量有密切關(guān)系,它們對生存環(huán)境的改變極其敏感,微生物數(shù)量、多樣性和群落結(jié)構(gòu)等均能作為判斷土壤健康狀況的重要指標(biāo).向土壤中人為大量輸入外源物質(zhì),尤其肥料,被認(rèn)為是導(dǎo)致生態(tài)系統(tǒng)穩(wěn)定性及土壤肥力發(fā)生變化的關(guān)鍵因素.施肥和肥料類型對土壤微生物數(shù)量、組成以及結(jié)構(gòu)多樣性差異緊密相關(guān)[23].

施用微生物有機肥較不施肥處理土壤理化性質(zhì)得到明顯改善.微生物有機肥的施入,使土壤有機質(zhì)、全氮含量均顯著提高(<0.05).雖然化肥也可使土壤養(yǎng)分得到一定提高,但提高效果不如微生物有機肥.土壤速效養(yǎng)分也表現(xiàn)出類似趨勢,尤其是土壤速效磷含量,CB處理較CK處理增加了184.8%.這可能主要是因為微生物有機肥中含有解磷菌,可作用于土壤中難溶或不溶的磷并使土壤釋放出大量速效磷.而土壤速效鉀含量變化不大(無顯著差異,<0.05),可能是因為土壤本身速效鉀含量較高,解鉀菌所釋放的速效鉀不足以使微生物有機肥處理速效鉀含量高于其他處理.微生物有機肥能在作物生長前期快速提供一定的速效養(yǎng)分,還可在作物生長過程中,通過微生物的生命活動分解有機質(zhì)和礦物質(zhì)釋放養(yǎng)分,或固定空氣中的游離氮,不斷地供作物生長需要.這種速效和長效兼有的作用能為微生物提供更穩(wěn)定的棲息繁殖環(huán)境[24-25].本研究利用qPCR和高通量測序技術(shù)分析了不同施肥處理土壤細(xì)菌16s rRNA 基因豐度和土壤細(xì)菌群落結(jié)構(gòu)及組成的特征.結(jié)果表明,施用微生物有機肥顯著增加了土壤細(xì)菌數(shù)量,較不施肥和常規(guī)施肥處理分別提高了16.9和3.4倍.這與張奇春等[26]研究一致,他發(fā)現(xiàn)使用有機肥可以迅速提高土壤環(huán)境細(xì)菌種類和數(shù)量,從而改善土壤的生態(tài)環(huán)境,可能是因為施肥處理較高的有機質(zhì)和養(yǎng)分,能為更多的微生物提供生長和繁殖的條件.與不施肥相比,傳統(tǒng)施肥盡管顯著提高了細(xì)菌數(shù)量,但卻顯著降低了細(xì)菌的多樣性.而施用微生物有機肥不但增加了細(xì)菌數(shù)量,還增加了細(xì)菌群落多樣性,其香農(nóng)指數(shù)、辛普森指數(shù)和Chao1指數(shù)均為最高. Liu等[27]利用Biolog方法研究了土壤微生物群落多樣性,結(jié)果表明施用有機肥處理香農(nóng)指數(shù)顯著高于化肥處理.綜上表明施微生物有機肥能將特定功能微生物帶入土壤,從而提高細(xì)菌數(shù)量和群落多樣性,這些功能菌的生命活動是微生物有機肥優(yōu)于普通有機肥的關(guān)鍵因素,這與前人研究結(jié)果相似[28-30].

大量研究證實土壤全氮含量和有機質(zhì)含量顯著影響細(xì)菌群落結(jié)構(gòu)[23,31-32].冗余分析結(jié)果表明,驅(qū)動土壤細(xì)菌群落結(jié)構(gòu)變化的主要因素有土壤有機質(zhì)、全氮含量和pH值.各處理優(yōu)勢門類群為變形菌門、放線菌門、和酸桿菌門,與前人在不同類型農(nóng)田土壤中得到的細(xì)菌優(yōu)勢類群相似,但不同研究中各優(yōu)勢類群的相對豐度差異很大.這些結(jié)果表明農(nóng)田土壤中細(xì)菌的優(yōu)勢類群相似,但優(yōu)勢類群的相對豐度受土壤類型或質(zhì)地和種植作物品種的影響[33-34].變形菌門是豐度最高的細(xì)菌,不同處理豐度不同(CK

綜上,傳統(tǒng)施肥和微生物有機肥均可提高土壤養(yǎng)分,然而傳統(tǒng)施肥顯著降低了土壤細(xì)菌多樣性.因此,微生物有機肥具有較好的應(yīng)用效果.但微生物有機肥中有益菌存活時間短,有益菌在不同土壤中存活率差異大,限制了微生物有機肥的應(yīng)用[42].此外,微生物有機肥發(fā)酵耗時長,占地較多,電力、熱源消耗高等因素極大地增加了微生物有機肥的生產(chǎn)及使用成本[43].在后續(xù)研究中應(yīng)該進一步完善微生物有機肥生產(chǎn)技術(shù),降低成本,提高肥料活性.同時,結(jié)合投入及產(chǎn)出,探索經(jīng)濟最佳施肥量,或者微生物有機肥與化肥配施從而達到降低成本的目的.

4 結(jié)論

4.1 施微生物有機肥使土壤有機質(zhì)、全氮、堿解氮和速效磷含量分別增加了28.5%、61.4%、67.8%和185.7%,且效果優(yōu)于常規(guī)施肥.

4.2 施微生物有機肥能顯著提高土壤細(xì)菌數(shù)量和多樣性,并改變細(xì)菌群落結(jié)構(gòu)組成.

4.3 冗余分析結(jié)果表明, 環(huán)境因子共解釋了細(xì)菌群落變化的92.3%,土壤有機質(zhì)、全氮含量和pH值是影響細(xì)菌群落變化的主要因子.

[1] 譚周進,周衛(wèi)軍,張楊珠,等.不同施肥制度對稻田土壤微生物的影響研究[J]. 植物營養(yǎng)與肥料學(xué)報, 2007,13(3):430-435.Tan Z J, Zhou W J, Zhang Y Z, et al. Effect of fertilization systems on microbes in the paddy soil [J]. Plant Nutrition and Fertilizer Science. 2007,13(3):430-435.

[2] 袁紅朝,秦紅靈,劉守龍,等.長期施肥對紅壤性水稻土細(xì)菌群落結(jié)構(gòu)和數(shù)量的影響[J]. 中國農(nóng)業(yè)科學(xué), 2011,44(22):4610-4617.Yuan H C, Qin H L, Liu S L, et al. Response of Abundance and Composition of the Bacterial Community to Long-term Fertilization in Paddy Soils [J]. Scientia Agriculture Sinica, 2011,44(22):4610- 4617.

[3] Berthrong S T, Buckley D H, Drinkwater L E. Agricultural management and labile carbon additions affect soil microbial community structure and interact with carbon and nitrogen cycling [J]. Microbial Ecology. 2013,66(1):158-170.

[4] 喬 潔,畢利東,張衛(wèi)建,等.長期施用化肥對紅壤性水稻土中微生物生物量、活性及群落結(jié)構(gòu)的影響[J]. 土壤, 2007,39(5):772-776.Qiao J, Bi L D, Zhang W J, et al. Effects of Long-Term Chemical Fertilization on Soil Microbial Biomass, Activity and Community in Paddy Soil in Red Soil Region of China [J]. Soils, 2007,39(5):772- 776.

[5] Dan W, Qian Y, Zhang J Z, et al. Bacterial community structure and diversity in a black soil as affected by long-term fertilization [J]. Pedosphere. 2008,18(5):582-592.

[6] 楊興明,徐陽春,黃啟為,等.有機(類)肥料與農(nóng)業(yè)可持續(xù)發(fā)展和生態(tài)環(huán)境保護[J]. 土壤學(xué)報, 2008,45(5):925-932.Yang X M, Xu Y C, Huang Q W, et al. Organic fertilizers and agricultural sustainable development and ecological and environmental protection [J] Acta Pedologica Sinica. 2008,45(5):925- 932.

[7] Liu L, Li T, Wei X, et al. Effects of a nutrient additive on the density of functional bacteria and the microbial community structure of bioorganic fertilizer [J]. Bioresource Technology. 2014,172(172): 328-334.

[8] 陳曉芬,李忠佩,劉 明,等.不同施肥處理對紅壤水稻土團聚體有機碳、氮分布和微生物生物量的影響[J]. 中國農(nóng)業(yè)科學(xué), 2013,46(5): 950-960. Chen X F , Li Z P , Liu M , et al. Effect of Different Fertilizations on Organic Carbon and Nitrogen Contents in Water-Stable Aggregates and Microbial Biomass Content in Paddy Soil of Subtrobial China [J]. Scientia Agricultura Sinica, 2013,46(5):950-960.

[9] 王麗麗,石俊雄,袁賽飛,等.微生物有機肥結(jié)合土壤改良劑防治煙草青枯病[J]. 土壤學(xué)報, 2013,50(1):150-156.Wang L L, Shi J X, Yuan S F, et al. Control of tobacco bacterial wilt with bio manure plus soil amendments [J]. Acta Pedologica Sinica, 2013,50(1):150-156.

[10] 袁英英,李敏清,胡 偉,等.生物有機肥對番茄青枯病的防效及對土壤微生物的影響[J]. 農(nóng)業(yè)環(huán)境科學(xué)學(xué)報, 2011,30(7):1344-1350.Yuan Y Y, Li M Q, Hu W, et al. Effect of Biological Organic Fertilizer on Tomato Bacterial Wilt and Soil Microorganism [J]. Journal of Agro-Environment Science, 2011,30(7):1344-1350.

[11] 劉方春,邢尚軍,馬海林,等.持續(xù)干旱對櫻桃根際土壤細(xì)菌數(shù)量及結(jié)構(gòu)多樣性影響[J]. 生態(tài)學(xué)報, 2014,34(3):642-649.Liu F C, Xing S J, Ma H L, et al. Effects of continuous drought on soil bacteria populations and community diversity in sweet cherry rhizosphere [J]. Acta Ecologica Sinica, 2014,34(3):642-649.

[12] 王偉華,劉 毅,唐海明,等.長期施肥對稻田土壤微生物量、群落結(jié)構(gòu)和活性的影響[J]. 環(huán)境科學(xué), 2018,39(1):430-437.Wang W H, Liu Y, Tang H M, et al. Effects of Long-term Fertilization regimes on Microbial Biomass, Community Structure and Activity in a Paddy Soil [J]. Environment Science. 2018,39(1):430- 437.

[13] Shokralla S, Spall J L, Gibson J F, et al. Next-generation sequencing technologies for environmental DNA research [J]. Molecular Ecology. 2012,21(8):1794-1805.

[14] 楊亞東,王志敏,曾昭海.長期施肥和灌溉對土壤細(xì)菌數(shù)量、多樣性和群落結(jié)構(gòu)的影響[J]. 中國農(nóng)業(yè)科學(xué), 2018,51(2):290-301.Yang Y D, Wang Z M, Ceng Z H. Effects of Long-Term Different Fertilization and Irrigation Managements on Soil Bacterial Abundance, Diversity and Composition [J]. Scientia Agricultura Sinica, 2018, 51(2):290-301.

[15] 朱金山,張 慧,馬連杰,等.不同沼灌年限稻田土壤微生物群落分析[J]. 環(huán)境科學(xué), 2018,(5):2400-2411.ZHU J S, ZHANG H, MA L J, et al. Diversity of the Microbial Community in Rice Paddy Soil with Biogas Slurry Irrigation Analyzed by Illumina Sequencing Technology [J]. Environmental Science, 2018,(5).

[16] Mchugh T A, Schwartz E. Changes in plant community composition and reduced precipitation have limited effects on the structure of soil bacterial and fungal communities present in a semiarid grassland [J]. Plant and Soil, 2015,388(1/2):175-186.

[17] Aminov R I, Chee-Sanford J C, Garrigues N, et al. Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria [J]. Appl Environ Microbiol., 2002,68(4):1786-1793.

[18] Caporaso J G, Kuczynski J, Stombaugh J, et al. QIIME allows analysis of high-throughput community sequencing data [J]. 2010,7(5):335- 340.

[19] 鮑士旦.土壤農(nóng)化分析 [M]. 北京:中國農(nóng)業(yè)出版社, 2000:25-108.Bao S D. Soil agrochemical analysis [M]. Beijing: China Agriculture Press, 2000:25-108.

[20] 鄭 涵,田昕竹,王學(xué)東,等.鋅脅迫對土壤中微生物群落變化的影響[J]. 中國環(huán)境科學(xué), 2017,37(4):1458-1465.Zheng H, Tian X Z, Wang X D, Effects of Zn pollution on soil microbial community in field soils and its main influence factors [J]. China Environmental Science, 2017,37(4):1458-1465.

[21] Denef K, Roobroeck D, Wadu M C W M, et al. Microbial community composition and rhizodeposit-carbon assimilation in differently managed temperate grassland soils [J]. Soil Biology & Biochemistry, 2009,41(1):144-153.

[22] Sturz A V, Christie B R. Beneficial microbial allelopathies in the root zone: the management of soil quality and plant disease with rhizobacteria [J]. Soil Tillage Research, 2003,72(2):107-123.

[23] 王慧穎,徐明崗,周寶庫,等.黑土細(xì)菌及真菌群落對長期施肥響應(yīng)的差異及其驅(qū)動因素[J]. 中國農(nóng)業(yè)科學(xué), 2018,51(5):914-925.Wang H Y, Xu M G, Zhou B K, et al. Response and Driving Factors of Bacterial and Fungal Community to Long-Term Fertilization in Black Soil [J]. Scientia Agricultura Sinica, 2018,51(5): 914-925.

[24] Xun W, Zhao J, Xue C, et al. Significant alteration of soil bacterial communities and organic carbon decomposition by different long-erm fertilization management conditions of extremely low-productivity arable soil in South China [J]. Environmental Microbiology, 2015, 18(6):1907-1917.

[25] 魏 巍,許艷麗,朱 琳,等.長期施肥對黑土農(nóng)田土壤微生物群落的影響[J]. 土壤學(xué)報, 2013,50(2):372-380.Wei W, Xu Y L, Zhu L et al. Effects of long-term fertilization on soil microbial community in black soil farmlands [J]. Acta Pedologica Sinica, 2013,50(2):372-380.

[26] 張奇春,王雪芹,時亞南,等.不同施肥處理對長期不施肥區(qū)稻田土壤微生物生態(tài)特性的影響[J]. 植物營養(yǎng)與肥料學(xué)報, 2010,16(1): 118-123.Zhang Q C, Wang X Q, Shi Y N, et al.Effects of different fertilization treatments on soil microbial ecological characteristics of paddy fields in long-term no-fertilization areas [J]. Plant Nutrition and Fertilizer Science, 2010,16(1):118-123.

[27] Liu B, Cong T, Hu S, et al. Effect of organic, sustainable, and conventional management strategies in grower fields on soil physical, chemical, and biological factors and the incidence of Southern blight [J]. Applied Soil Ecology, 2007,37(3):202-214.

[28] Ming G X, Tang H J, Yang X Y, et al. Best soil managements from long-term field experiments for sustainable agriculture [J]. Journal of Integrative Agriculture, 2015,14(12):2401-2404.

[29] Chen S, Jie G, Hua G, et al. Effect of microbial fertilizer on microbial activity and microbial community diversity in the rhizosphere of wheat growing on the Loess Plateau [J]. African Journal of Microbiology Research, 2011,5(2):137-143.

[30] 張風(fēng)革,霍云倩,孫 藝,等.連續(xù)施用生物有機肥對草地生物量及土壤微生物區(qū)系的影響[J]. 南京農(nóng)業(yè)大學(xué)學(xué)報, 2018,41(2):382-388.Zhang F G, Huo Y Q, Sun Y, et al. Effect of consecutive biofertilizer application on aboveground biomass and management of soil microflora in grassland [J]. Journal of Nanjing Agricultural University, 2018,41(2):382-388.

[31] 徐永剛,宇萬太,馬 強,等.長期不同施肥制度對潮棕壤微生物生物量碳、氮及細(xì)菌群落結(jié)構(gòu)的影響[J]. 應(yīng)用生態(tài)學(xué)報, 2010,21(8): 2078-2085. Xu Y G, Yu W T, Ma Q, et al. Effects of long-term fertilizations on microbial biomass C and N and bacterial community structure in an aquic brown soil [J]. Chinese Journal of Applied Ecology, 2010,21(8): 2078-2085.

[32] Zhou J, Guan D, Zhou B, et al. Influence of 34-years of fertilization on bacterial communities in an intensively cultivated black soil in northeast China [J]. Soil Biology Biochemistry. 2015,90:42-51.

[33] Liu J, Sui Y, Yu Z, et al. High throughput sequencing analysis of biogeographical distribution of bacterial communities in the black soils of northeast China [J]. Soil Biology & Biochemistry, 2014,70(2): 113-122.

[34] Chu H, Fierer N, Lauber C L, et al. Soil bacterial diversity in the Arctic is not fundamentally different from that found in other biomes [J]. Environmental Microbiology, 2010,12(11):2998-3006.

[35] Fierer N, Bradford M A, Jackson R B. Toward an ecological classification of soil bacteria [J]. Ecology, 2007,88(6):1354-1364.

[36] Michaels S, Johannes R. Considering fungal: bacterial dominance in soils Methods, controls, and ecosystem implications [J]. Soil Biology Biochemistry. 2010,42(9):1385-1395.

[37] 王伏偉,王曉波,李金才,等.施肥及秸稈還田對砂姜黑土細(xì)菌群落的影響[J]. 中國生態(tài)農(nóng)業(yè)學(xué)報, 2015,23(10):1302-1311.Wang F W, Wang X B, Li J C, et al. Effects of Fertilization and Straw Incorporation on Bacterial Communities in Lime Concretion Black Soil [J]. Chinese Journal of Eco-Agriculture, 2015,23(10):1302- 1311.

[38] 劉彩霞,董玉紅,焦如珍.森林土壤中酸桿菌門多樣性研究進展[J]. 世界林業(yè)研究, 2016,29(6):17-22.Liu C X, Dong Y H, Jiao R Z. Research Progress in Acidobacteria Diversity in Forest Soil [J]. World Forestry Research, 2016,29(6): 17-22.

[39] Janssen P H. Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes [J]. Applied Environmental Microbiology. 2006,72(3):1719-1728.

[40] M?nnist? M K, Tiirola M, H?ggblom M M. Bacterial communities in Arctic fjelds of Finnish Lapland are stable but highly pH-dependent [J]. Fems Microbiology Ecology, 2007,59(2):452-465.

[41] Ling N, Zhu C, Xue C, et al. Insight into how organic amendments can shape the soil microbiome in long-term field experiments as revealed by network analysis [J]. Soil Biology Biochemistry, 2016,99:137-149.

[42] 張余莽,周海軍,張景野,等.生物有機肥的研究進展 [J]. 吉林農(nóng)業(yè)科學(xué), 2010,35(3):37-40.Zhang Y M, Zhou H J, Zhang J Y, et al. Progress of Studies of Bio- organic Fertilizer [J]. Journal of Jilin Agricultural Sciences, 2010, 35(3):37-40.

[43] 何蔚娟.生物有機肥料生產(chǎn)問題研究 [J]. 陜西農(nóng)業(yè)科學(xué), 2018, 64(6):90-92.H W J.Study on the production of bio-organic fertilizer [J]. Shannxi Journal of Agricultural Sciences, 2018,64(6):90-92

Effects of bio-organic fertilizer on the soil bacterial community in a cherry orchard.

ZHANG Kai-yu, GU Jie*, WANG Xiao-juan, GAO Hua

(College of Natural Resources and Environment, Northwest A&F University, Yangling 712100, China)., 2019,39(3)：1245~1252

Field experiment was conducted to investigate the effects of bio-organic fertilizer on the diversity of the bacterial community in a cherry orchard. High-throughput sequencing and quantitative real-time PCR were used to determine the bacterial abundance, diversity, and composition under different no fertilizer (CK), conventional fertilizer (CN), bio-organic fertilizer (CB). The results showed that CB significantly increased the soil organic matter, total nitrogen, alkali nitrogen, and available phosphorus. The bacterial 16S rRNA gene copy numbers andadiversity indexes showed that CB increased the number, diversity, and richness of bacteria. Principal coordinate analysis showed that the different fertilizer treatments significantly changed the bacterial community structure. At the phylum level, Proteobacteria, Acidobacteria, Firmicutes, Gemmatimonadetes, and Actinobacteria were the predominant phyla, accounting for 77.22%~86.28% of the total reads. CB significantly decreased the abundances of Acidobacteria_Gp4and Gp6compared with CK, whereas Acidobacteria_Gp7exhibited the opposite trend with an increase of 75.4% compared with CK. Redundancy analysis showed that environmental factors explained 92.3% of bacterial community changes. Soil organic matter, total nitrogen content, and pH were the main factors related to the variations in the bacterial community in cherry orchards. Therefore, bio-organic fertilizer could significantly increase soil nutrient content, quantity of soil bacteria, and bacterial community diversity, which was important for improving soil fertility.

bio-organic fertilizer；bacterial community；high-throughput sequencing；qPCR

X712, S144

A

1000-6923(2019)03-1245-08

張凱煜(1990-),女,陜西榆林人,西北農(nóng)林科技大學(xué)博士研究生,主要研究方向為環(huán)境微生物及固體廢資源化利用.發(fā)表論文4篇.

2018-08-31

陜西省科技統(tǒng)籌創(chuàng)新工程項目(2016KTCL02-29);國家自然科學(xué)基金資助項目(41671474)

* 責(zé)任作者, 教授, gujie205@sina.com