陳 娜,劉 毅,黎 娟,袁 婧,葛體達,吳金水,孫志龍,徐華勤*
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長期施肥對稻田不同土層反硝化細菌豐度的影響
陳 娜1,2,劉 毅2,黎 娟1,袁 婧2,葛體達2,吳金水2,孫志龍3,徐華勤1*
(1.湖南農業(yè)大學農學院,湖南 長沙 410128;2.中國科學院亞熱帶農業(yè)生態(tài)研究所亞熱帶農業(yè)生態(tài)過程重點實驗室,湖南 長沙 410125;3.湖南省寧鄉(xiāng)市回龍鋪鎮(zhèn)農業(yè)綜合服務中心,湖南 寧鄉(xiāng) 410606)
為了探討長期施肥對稻田不同土層關鍵反硝化功能種群豐度的影響及核心驅動因子,以湖南寧鄉(xiāng)長期施肥定位試驗田為平臺,選取不施肥(CK)、全量化肥(NPK)和秸稈還田(ST)3個處理,結合實時熒光定量PCR(qPCR)技術,系統(tǒng)分析了稻田不同土層(0~10,10~20,20~30,30~40cm)關鍵反硝化功能基因(和)的豐度及其與土壤理化性質的內在聯(lián)系.結果表明,相比于不施肥處理(CK),施肥處理(NPK和ST)在0~40cm土層土壤SOC、TN、NO3--N、NH4+-N和Olsen-P分別顯著增加了2.2%~83.6%,3.5%~58.3%,70.8%~222.1%,0.9%~83.7%和16.5%~94.5%,pH值下降了0.31~0.67個單位;長期施用化肥和秸稈使和基因豐度分別增加0.75~7.18倍,1.57~3.02倍和0.53~3.81倍,其中秸稈還田對反硝化細菌數量的影響比單施化肥更顯著;稻田、和反硝化型細菌的豐度隨土層深度增加而逐漸降低,具有明顯的垂直分布特征;RDA分析結果顯示,土壤養(yǎng)分如SOC和TN是影響水稻土、和反硝化型細菌垂直分布的關鍵因子,而pH值是調控反硝化細菌在稻田底土分布的核心驅動因子.研究結果可為提升稻田土壤肥力和減少稻田氮素損失和溫室氣體排放提供理論依據.
水稻土;長期施肥;不同土層;反硝化細菌;種群豐度
農田土壤為N2O排放的主要人為源,年均排放量約為4.2Tg,占全球總釋放量的70%[1].我國是世界上最大的水稻生產國,稻田耕種面積占世界水稻生產面積的27%.因此,我國稻田N2O的排放日益受到關注[3].稻田土壤N2O的排放主要由土壤微生物參與的硝化和反硝化作用所驅動[2].由于稻田土壤長期處于淹水厭氧環(huán)境,因此反硝化作用被認為是稻田N2O產生的主要途徑[3].其中,基因編碼的硝酸還原酶是反硝化過程的第一步反應酶,和基因編碼的亞硝酸還原酶控制著關鍵的限速步驟,因此,、和常用作標記基因用以研究土壤反硝化細菌[4-5].大量研究結果表明[6-8],反硝化功能種群豐度的增加會導致反硝化作用的增強,進而引起N2O排放增多.因此,明確稻田土壤關鍵反硝化功能種群的豐度變化規(guī)律,對于減少稻田土壤氮素損失及N2O排放具有重要的意義.
近年來,由于稻田增值高產的需要,施肥成為保障糧食安全和產量的關鍵措施.但研究發(fā)現(xiàn),長期不同施肥會引起稻田土壤理化性質出現(xiàn)差異,進而影響反硝化微生物,從而影響稻田土壤反硝化作用及N2O的排放[9-10].因此,關于稻田土壤反硝化微生物對長期施肥響應的研究逐漸成為國內外學者關注的熱點.研究發(fā)現(xiàn)[11-12],施用化肥和有機肥均顯著提高反硝化細菌豐度,且施用有機肥處理與化肥處理間反硝化細菌豐度和群落結構差異顯著.這些研究表明不同施肥制度顯著影響反硝化微生物,然而到目前為止,關于長期施肥對稻田土壤反硝化微生物分布影響的研究主要集中在耕作層(0~20cm)土壤,而深層土壤反硝化微生物對長期不同施肥制度的響應的研究少有報道.因此,非常有必要針對長期施肥對稻田土壤剖面不同深度反硝化微生物的分布特征開展研究.
為了探究長期施肥對稻田不同土層關鍵反硝化功能種群豐度的影響,本研究依托湖南省寧鄉(xiāng)縣長期定位試驗田,選取無肥對照、全化肥、秸稈還田3種不同施肥處理,采集0~10,10~20,20~30,30~40cm土層土壤樣品,運用熒光定量PCR(qPCR)技術,系統(tǒng)分析了和等3種關鍵反硝化功能基因在不同施肥處理中沿土壤剖面的豐度變化特征及其核心驅動因子.研究結果可為提高稻田氮素利用效率和溫室氣體減排提供理論依據.
采樣地點位于湖南省寧鄉(xiāng)縣農業(yè)技術推廣中心,地理位置為113°00′20″E,北緯28°25′16″N,海拔36.1m,年平均氣溫為17.5℃,年均降雨量為1300mm,年均無霜期在274d左右,年日照為1663h.該地區(qū)為典型雙季稻產區(qū),于1986年設立田間試驗,為長期定位施肥實驗田.本研究采用其中3個施肥處理:①無肥對照(CK):不施加任何肥料;②全化肥處理(NPK):僅施氮磷鉀化肥,按每公頃施60kg氮((NH4)2SO4)、30kg磷(P2O5)和60kg鉀(K2O)的比例進行施肥;③秸稈還田處理(ST):施用上一季度收割的水稻秸稈為主(早季稻秸稈還田量為2775.0kg/hm2,晚季稻秸稈還田量為3600.0 kg/hm2),若與處理②比,總氮磷鉀量不足則用化肥補足.試驗小區(qū)面積為28.22m2,每個處理3次重復,隨機區(qū)組排列.
土壤樣品采集于2017年5月中旬,在每個小區(qū)采用五點法采集,土壤剖面0~40cm范圍內按10cm間距分段采樣.采樣時將鮮土分為2份:一份用錫箔紙包好立即用液氮速凍,存放于-80℃冰箱用于分子生物學實驗;另一部分室內剔除石塊和植物殘體后,存于4℃冰箱,進行理化指標的測定.土壤的基本理化性質見表1.
表1 供試土壤基本理化性質 Table 1 Characteristics of the soil in this study
土壤pH值按水土比2.5:1用Mettler- toledo320 pH計測定;土壤有機碳(SOC)和全氮(TN)采用碳氮元素分析儀(VARIO MAX C/N,德國)測定(干燒法);土壤速效磷(Olsen-P)含量采用Olsen法;硝態(tài)氮(NO3--N)和銨態(tài)氮(NH4+-N)用連續(xù)流動分析儀(Flastar 5000Analyzer)測定.
土壤DNA的提取方法參考陳哲[13]方法,并稍作修改:取0.5g經液氮速凍的土壤.DNA濃度測定使用核酸蛋白測定儀(Nanodrop ND-1000UV-Vis分光光度計),并取2μL用1%瓊脂糖膠進行電泳檢測,DNA樣品保存于-20℃冰箱.
實時熒光定量PCR所用儀器為ABI 7900 (Applied Biosystem),標準曲線的建立參照陳哲[13]的方法:、和PCR反應體系均為10μL,含有5μL SYBR GREEN Ⅰ (Takara),0.7μL ROX (Takara),0.3μL引物,1μL濃度為5ng的DNA模板,加水補至10μL.基因引物對為-571F-773R[13],引物序列為CCGATYCCGGCVAT- GTCSAT/GGNACGTTNGADCCCCA,反應條件為95℃預變性30s,95℃變性5s,60℃退火30s,共40個循環(huán);基因引物對為-1aCuF/K-3CuR[14],引物序列為ATCATGCTSCTGCCGCG/GCCTCGA- TCAGRTTGTGGTT,反應條件為95℃預變性30s, 95℃變性5s,56℃退火30s,共40個循環(huán);基因引物對為-cd3aF/-R3cd[15],引物序列為GTSAACGTSAAGGARACSGG/GASTTCGGRTGSGTCTTGA,反應條件為95℃預變性30s,95℃變性5s,56℃退火20s,72℃延伸30s,共40個循環(huán).溶解曲線均為95℃ 15s,60℃ 15s,90℃ 15s.
qPCR數據的導出采用SDS 2.3軟件,數據處理與統(tǒng)計分析采用Microsoft Excel 2016和SPSS 20.0,圖形繪制采用ORIGIN 9.0.不同處理顯著性用One-way ANOVA(單因素方差分析)進行檢驗,采用Duncan多重比較分析組間差異.冗余分析(RDA)用Canoco 5.0實現(xiàn).
如圖1所示,經長期施肥后,各處理間0~40cm土層土壤SOC、TN、NO3--N、NH4+-N、Olsen-P含量和pH值差異顯著.同時多重比較(表2)結果發(fā)現(xiàn),施肥和土壤深度及其交互作用均對土壤SOC、TN、NO3--N、NH4+-N和Olsen-P含量及pH值有極顯著影響(<0.01).相比于CK處理,施肥處理(NPK和ST)在0~40cm土層土壤SOC、TN、NO3-- N、NH4+-N和Olsen-P分別增加了2.2%~83.6%, 3.5%~58.3%,70.8%~222.1%,0.9%~83.7%和16.5%~ 94.5%, pH值下降了0.31~0.67個單位;相比于全化肥處理,秸稈還田使0~30cm土層土壤SOC、TN、NO3--N和NH4+-N含量分別增加了20.3%~26.6%、18.1%~25.0%、3.2%~85.3%和22.8%~78.4%,pH值下降0~0.16個單位,使20~40cm土層土壤Olsen-P含量增加6.0%~34.4%.長期施肥增加土壤養(yǎng)分,降低pH值,這一結論在施肥試驗中具有普遍性,如Zhao等[16]在稻麥種植制度的施肥試驗中發(fā)現(xiàn)所有施肥處理包括化肥及化肥配施秸稈處理土壤有機質、有效磷和全氮含量均增加;孟紅旗等[17]和于冰等[18]的研究表明施用化肥及秸稈配施化肥處理較不施肥處理pH值降低,秸稈配施化肥處理的有機質、TN、NH4+-N、NO3--N和有效磷含量高于其他處理.
表2 施肥處理和土壤深度對土壤理化性質的多重比較Table 2 Effects of fertilization and soil depth on soil properties
注:數值及*號為值和顯著水平.**為極顯著(<0.01).
由于耕作層土壤養(yǎng)分的向下遷移,使得土壤養(yǎng)分具有垂直分布特征.圖1中土壤C、N、P含量均在0~10cm土層中最高,與土層深度呈顯著負相關.杜林森等[19]通過38a的長期培肥試驗發(fā)現(xiàn),土壤碳氮含量隨土壤深度增加而逐漸降低,且施用秸稈和化肥處理是不施肥處理的1.1~13.7倍,單艷紅等[20]研究發(fā)現(xiàn)0~30cm土層土壤Olsen-P含量與土層深度呈負相關,這與本研究結果一致.長期施肥稻田土壤養(yǎng)分垂直分布在水稻生產上具有雙重意義[21]:一方面,養(yǎng)分適度下移,可以豐富稻田底土養(yǎng)分含量,對于培育稻田土壤肥力極為有利;另一方面,當養(yǎng)分下移超過水稻根系所能吸收的范圍,將造成養(yǎng)分的淋失,進而造成地下水的污染.由此可見,長期施化肥或秸稈秸稈對提高稻田土壤肥力和促進稻田可持續(xù)性具有顯著的效果,同時需對施肥可能導致的環(huán)境問題引起注意.
、和型反硝化細菌基因拷貝數如圖2所示,、和基因豐度分別在1.20′107~5.02′109,5.10′106~3.36′109,4.71′106~2.75′109拷貝數/g干土,其在不同土壤深度土層的分布對不同施肥的響應趨勢較相似.長期施肥提高了土壤、和型反硝化細菌基因豐度,相比較于不施肥處理,施用秸稈和化肥使基因豐度增加0.75~7.18倍,基因豐度增加1.57~3.02倍,基因豐度增加0.53~3.81倍.這與之前的研究結論基本一致,如解開治等[22]指出無機肥和無機有機肥配施均能顯著提高稻田土壤和基因豐度;靳振江等[26]也發(fā)現(xiàn)施用化肥和化肥配施秸稈處理的基因豐度較無肥處理提高了0.51倍和0.80倍.其原因為,反硝化微生物主要為異養(yǎng)厭氧型微生物,在稻田土壤長期的淹水厭氧環(huán)境下,土壤養(yǎng)分的增加能極大地滿足反硝化微生物生長繁殖對養(yǎng)分的需求,從而刺激反硝化微生物的大量繁殖.同時,從圖中可得,秸稈還田處理中、和基因豐度較僅施用化肥高,表明長期施用秸稈對稻田反硝化微生物數量的促進作用更顯著.這與很多國內外研究結果相似[23-24],如尹昌等[25]研究表明施用有機肥顯著促進型反硝化細菌的生長,而施用化肥需配施有機肥才促進反硝化微生物的生長;Chen[30]等發(fā)現(xiàn)有機肥處理、和反硝化細菌數量顯著高于無機肥處理.其原因為,首先,秸稈有機肥的長期輸入為反硝化細菌和其他微生物提供了大量生物有效性碳源,促進微生物的大量繁殖,微生物活性的提高反過來又促進有機物的降解,從而增加土壤中速效養(yǎng)分,為微生物的生長繁殖提供適宜的環(huán)境[26];其次,NO3--N是反硝化作用的底物,NO3--N顯著增加(圖1)為其提供了豐富的反應底物[27];最后,有機質增加刺激了土壤微生物活性,呼吸作用強,加速了厭氧環(huán)境的形成[28].化肥雖然也能為反硝化微生物直接快速地提供NO3--N底物,但并不能直接增加土壤碳養(yǎng)分,而是通過促進植株根系生長和產生根系分泌物來保障土壤反硝化微生物對碳源的需求[29].
圖2 不同施肥處理下不同土壤深度反硝化細菌基因豐度 Fig.2 Gene Abundance of denitrifying bacteria in soil profile with different fertilizer application
之前有報道指出,土壤養(yǎng)分的垂直分布也會引起土壤微生物具有垂直分布特征[30].如Taylor等[31]發(fā)現(xiàn)農田土壤表層細菌數量遠高于底層;Fierer等[32]指出草地土壤微生物數量隨土壤深度的增加而減少.圖2中隨著土壤深度的增加,不同施肥處理、和基因豐度均顯著降低,具有明顯的垂直分布特征, 類似結果在森林土壤中也有發(fā)現(xiàn)[33].造成這種變化趨勢的主要原因可能為土壤表層受耕作等影響較大,肥料和凋落物等直接施入表土,土壤表層養(yǎng)分如有機碳、硝態(tài)氮和速效磷等含量較高(圖1),因而土壤表層微生物更易于獲取足夠的養(yǎng)分和底物;隨著深度增加,土壤養(yǎng)分含量逐漸減少(圖1),微生物生長受限,微生物數量減少.另外,本研究表明,相對于施用化肥處理,秸稈還田處理中不同土層反硝化微生物的豐度都要更高(圖2),類似試驗結果在長期定位施肥的旱地(菜地)土壤中也有所發(fā)現(xiàn)[34].水稻田不同于旱地土,其土壤養(yǎng)分向下遷移速率和滲漏損失量相對較小,大多沉積于底底土中,因此不同施肥處理所引起的稻田耕作層養(yǎng)分含量的差異,其類似特征也能反映在底土上.正是因為不同施肥處理間土壤養(yǎng)分垂直分布的差異,導致了、和反硝化型細菌在不同施肥處理中垂直分布存在差異.
RDA排序結果(圖3)表明,第一和第二排序軸占總特征值的72.76%,表明這6項環(huán)境因子能解釋大部分反硝化細菌基因豐度的變化.土壤SOC、TN、NH4+-N、NO3--N、Olsen-P和pH值均極顯著影響反硝化功能種群豐度(蒙特卡羅檢驗值分別為=0.002,0.002,0.002,0.002,0.006,0.002),其中土壤SOC和TN在第一排序軸上的投影長度較長, NH4+-N、NO3--N和pH值次之,Olsen-P最短,表明在6項理化因子中SOC和TN對土壤反硝化基因豐度的影響較大.因此,土壤養(yǎng)分如SOC和TN是影響稻田土壤、和反硝化型細菌垂直分布的關鍵因子,尤其對稻田耕作層的影響更為顯著(圖3).類似的結果在其他生態(tài)系統(tǒng)也有發(fā)現(xiàn),如Fierer等[32]認為土壤微生物數量隨土壤深度的增加而減少主要是由于土壤碳的有效性降低;Liu等[35]認為在森林土壤中,土壤DOC、DON、NH4+和NO3-垂直分布是、和型反硝化細菌呈垂直分布特征的關鍵驅動因子.同時RDA結果顯示,pH值與20~30cm和30~40cm土層反硝化細菌的豐度緊密相關,暗示pH值可能是驅動、和型反硝化細菌在稻田底土分布的核心驅動因子. pH值對反硝化細菌具有選擇效應,因而在不同的土壤環(huán)境中反硝化細菌對pH值的響應不同[36]. Dandie等[37]發(fā)現(xiàn),農田土壤中pH值是限制型反硝化細菌群落結構的唯一影響因子;而王亞男等[43]在設施菜地中的研究和Enwall[38]在稻田的研究發(fā)現(xiàn)pH值與反硝化細菌豐度并沒有相關性.本研究pH值顯著影響反硝化基因豐度,與前者研究結果相似.0~20cm施肥處理、10~ 20cm、20~30cm和30~40cm土層處理基因豐度在二維排序圖中彼此分離,表明土壤深度影響反硝化基因豐度.在0~ 30cm土層中,全化肥處理、秸稈還田處理與不施肥處理彼此分離,表明施用化肥及秸稈還田顯著影響土壤反硝化細菌基因豐度.
圖3 影響反硝化細菌基因豐度因素的RDA分析 Fig.3 RDA analysis of influencing factors to gene abundance of denitrifying bacteria
3.1 長期施肥顯著提升水稻土不同土層碳、氮、磷等含量,降低土壤pH值.
3.2 稻田、和反硝化型細菌的豐度隨土層深度增加而逐漸降低.長期施肥顯著增加水稻土不同土層、和反硝化型細菌的豐度,其中秸稈還田對反硝化細菌數量的影響比施用化肥更顯著.
3.3 土壤養(yǎng)分如SOC和TN是影響水稻土、和反硝化型細菌垂直分布的關鍵因子,而pH值是調控反硝化細菌在稻田底土分布的核心驅動因子.
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Effects of long-term fertilization on the abundance of the key denitrifiers in profile of paddy soil profiles.
CHEN Na1,2, LIU Yi2, LI Juan1, YUAN Jing2, GE Ti-da2, WU Jin-shui2, SUN Zhi-long3, XU Hua-qin1*
(1.College of Agronomy, Hunan Agriculture University, Changsha 410128, China;2.Key Laboratory of Subtropical Agriculture Ecology, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China;3.Integrated Service for Agriculture Ningxiang County Huilongpu Town, Ningxiang 410606, China)., 2019,39(5):2154~2160
The aims of this study were to explore the effect of long-term fertilization on the abundance of the key denitrifiers in paddy soil profiles (0~40cm), and the core factors driving denitrifiers. Soils with non-fertilization (CK), inorganic fertilizer (NPK) and organic fertilizer (ST) were collected in Ning xiang County, Hunan Province, and real-time fluorescent quantitative PCR technology was used to analyze the abundance of-,- and-containing communities in paddy soil profile (0~10cm, 10~20cm, 20~30cm, 30~40cm) and their relationship with soil properties. The results showed that compared with CK, SOC、TN、NO3--N、NH4+-N and Olsen-P in soil profile under NPK and ST increased by 2.2%~83.6%、3.5%~58.3%、70.8%~222.1%、0.9%~ 83.7% and 16.5%~94.5% respectively, and pH decreased by 0.31~0.67 units. Long-term application of inorganic fertilizer and organic fertilizer increased,, andgene abundance by 0.75~7.18 times, 1.57~3.02 times, and 0.53~3.81 times, respectively. And the effect of organic fertilizer on the abundance of denitrifiers was more significant than that of inorganic fertilizer application; The abundance of-,- and-containing communities decreased gradually with soil depth increasing, which presented an obvious vertical distribution; RDA analysis showed that soil nutrients such as SOC and TN were the core factors affecting the vertical distribution of-,- and-containing populations in paddy soil, especially in the cultivated horizon, while pH was the core driving factor regulating the distribution of denitrifying bacteria in paddy field subsoil. The results can provide theoretical basis for improving soil fertility and reducing nitrogen loss and greenhouse gas emission in paddy soils.
paddy soil;long-term fertilization;soil profile;denitrifying bacteria;abundance
X172
A
1000-6923(2019)05-2154-07
陳 娜(1994-),女,湖南婁底人,湖南農業(yè)大學碩士研究生,主要從事環(huán)境生態(tài)修復.發(fā)表論文1篇.
2018-10-14
國家自然基金資助項目(41771300,41301274)
*責任作者, 副教授, xu7541@163.com