李 藝,張海春,劉 媛,韋姣騰,王 聰,梁 映,劉可慧1,**,于方明*

泗頂?shù)V區(qū)剖層土固氮微生物群落結(jié)構(gòu)和豐度

李 藝1,2,張海春3,劉 媛2,韋姣騰2,王 聰2,梁 映2,劉可慧1,3**,于方明1,2*

(1.廣西師范大學(xué),珍稀瀕危動(dòng)植物生態(tài)與環(huán)境保護(hù)教育部重點(diǎn)實(shí)驗(yàn)室,廣西 桂林 541004；2.廣西師范大學(xué)環(huán)境與資源學(xué)院,廣西 桂林 541004；3.廣西師范大學(xué)生命科學(xué)學(xué)院,廣西 桂林 541004)

以廣西柳州泗頂?shù)V區(qū)的上游區(qū)、下游區(qū)和尾礦區(qū)12個(gè)剖層土(每個(gè)區(qū)域分為4層)為研究對(duì)象,采用高通量測(cè)序技術(shù)和熒光定量PCR技術(shù),分析了剖層土中固氮微生物的群落結(jié)構(gòu)、豐度和多樣性特征.結(jié)果表明,變形菌門在各區(qū)域剖層土中均為優(yōu)勢(shì)菌門,所占比例超過70%;α-變形菌綱在上游區(qū)和下游區(qū)剖層土中均為優(yōu)勢(shì)菌綱.上游區(qū)、下游區(qū)和尾礦區(qū)剖層土固氮微生物H基因豐度的范圍分別為3.02×106～1.17×107、2.55×106～7.78×106和8.19×105～3.14×106基因拷貝數(shù)/g (干土).主要影響H基因豐度的土壤環(huán)境因素是土壤氮(總氮、銨態(tài)氮和硝態(tài)氮)和磷(總磷和有效磷)的含量.礦區(qū)固氮微生物群落組成的差異性主要由土壤鉛、鋅和鎘含量的變化所引起,上游區(qū)剖層土的Shannon指數(shù)和ACE指數(shù)顯著高于下游區(qū)和尾礦區(qū),表明上游區(qū)剖層土固氮微生物群落的多樣性和豐富程度相對(duì)較高.另外,土壤鉀、鈣和鈉含量的變化對(duì)固氮微生物群落的ACE指數(shù)和NMDS1指數(shù)也產(chǎn)生了不同程度的影響.研究結(jié)果表明土壤環(huán)境因子變化影響了礦區(qū)剖層土固氮微生物的群落結(jié)構(gòu)、豐度和多樣性.研究為礦區(qū)的氮素調(diào)控、生態(tài)恢復(fù)和重建提供了理論依據(jù).

礦區(qū)；固氮微生物；剖層土；群落結(jié)構(gòu)；H基因豐度

采礦業(yè)在帶來巨大經(jīng)濟(jì)利益的同時(shí)也造成了礦區(qū)生態(tài)退化,產(chǎn)生了大量的礦業(yè)廢棄地,因此對(duì)礦區(qū)及其周邊廢棄地進(jìn)行生態(tài)恢復(fù)迫在眉睫.一般而言,礦區(qū)的生態(tài)恢復(fù)包括廢棄地的植被修復(fù)與復(fù)墾、土壤肥力修復(fù)以及植被演替[1].在礦區(qū)及周邊土壤肥力修復(fù)過程中,由于土壤理化性質(zhì)差,有機(jī)質(zhì)、水分、養(yǎng)分等缺乏,成為生態(tài)恢復(fù)與重建的主要限制因子[2-3].其中,土壤氮素的極端不足又是養(yǎng)分不足中的核心問題[4].

氮在所有生物體的生長(zhǎng)發(fā)育過程中具有不可替代的作用,是蛋白質(zhì)和核酸等關(guān)鍵細(xì)胞成分合成所必需的元素[5].因此,氮循環(huán)在重金屬污染嚴(yán)重的礦區(qū)生態(tài)恢復(fù)過程中具有重要作用.作為氮循環(huán)中重要的環(huán)節(jié)之一,生物固氮是陸地生態(tài)系統(tǒng)外部氮源輸入的最大自然來源;并且可以在缺乏人為氮源輸入時(shí)維持生態(tài)系統(tǒng)的可持續(xù)性[7-8].生物固氮由固氮酶催化,固氮酶由兩個(gè)多亞基的金屬蛋白酶組成,分別是D和K基因編碼的鉬鐵蛋白和H基因編碼的鐵蛋白組成[6];其中,H基因作為標(biāo)記基因,被廣泛用于檢測(cè)環(huán)境中固氮微生物的存在[5,9].固氮微生物廣泛存在于土壤環(huán)境中,如變形菌門(Proteobacteria),藍(lán)藻菌門(Cyanobacteria),擬桿菌門(Firmicutes)和疣微菌門(Verrucomicrobia)等[10-13].并且,固氮微生物的豐度、多樣性和群落組成會(huì)隨著土壤環(huán)境的變化而產(chǎn)生變化[14].

廣西被譽(yù)為“有色金屬之鄉(xiāng)”,擁有各類礦山6800多座[15],生物固氮在礦區(qū)及周邊生態(tài)系統(tǒng)的恢復(fù)和重建中發(fā)揮著重要作用.研究表明,土壤微生物在礦區(qū)碳、氮循環(huán)和能量流動(dòng)過程中起著關(guān)鍵作用[16].而土壤pH值、養(yǎng)分含量、金屬濃度、含水率和覆土深度等被認(rèn)為是礦區(qū)及周邊固氮微生物群落多樣性和豐度的主要限制因子[17-20].因此,提高固氮微生物的多樣性和群落組成,促進(jìn)礦區(qū)氮素的累積,已成為礦區(qū)生態(tài)恢復(fù)的研究焦點(diǎn).

因此,本研究以廣西柳州泗頂鉛鋅礦區(qū)為研究對(duì)象,運(yùn)用高通量測(cè)序和熒光定量PCR技術(shù),對(duì)泗頂?shù)V區(qū)上游、下游和尾礦區(qū)剖面各層土壤中固氮微生物群落結(jié)構(gòu)、豐度和多樣性進(jìn)行了研究,探討礦區(qū)不同區(qū)域剖層土土壤固氮微生物群落結(jié)構(gòu)、豐度和多樣性的分布特征及其與土壤環(huán)境因子之間的關(guān)系,以期深入認(rèn)識(shí)礦區(qū)不同區(qū)域剖層土壤中固氮微生物的群落結(jié)構(gòu)特征,為礦區(qū)的氮素調(diào)控、生態(tài)恢復(fù)和重建提供理論依據(jù).

1 材料與方法

1.1 研究區(qū)概況

土壤樣品采自廣西柳州泗頂?shù)V區(qū)(北緯24°46′～25°34′,東經(jīng)109°13′～109°47′).區(qū)域地貌為外圍環(huán)形突起、中間洼地的峰林地,環(huán)形隆起高程400m以上,盆地高程為320m左右.該地區(qū)屬中亞熱帶季風(fēng)氣候,常年溫暖潮濕,年均氣溫17.8℃,年降水量189.9mm.

礦區(qū)面積約為13.64km2,已于2006年開采完畢;冶煉廠附近有一條河流,冶煉污水通過排污口排入河流.其中,上游區(qū)位于河流上游,土壤屬于黏質(zhì)黃土,植被類型主要有蜈蚣草、五節(jié)芒、蘆葦、馬唐等,植物生長(zhǎng)茂盛;下游區(qū)位于河流下游,土壤屬于砂質(zhì)棕土,植被類型主要有五節(jié)芒、蜈蚣草、辣蓼等;尾礦區(qū)位于礦山尾礦庫,土壤類型主要是選礦后篩出的泥土與礦石廢渣混合物,屬于砂質(zhì)黑土,植被類型主要有五節(jié)芒、蘆葦、節(jié)節(jié)草、狗牙根等.因此,本文選擇上述3個(gè)區(qū)域作為代表區(qū)域進(jìn)行研究.

1.2 土壤采集與理化性質(zhì)分析

2020年7月上旬,在泗頂?shù)V區(qū)的上游區(qū)(北緯25°02′53′′～25°02′58′′,東經(jīng)109°41′60′′～109°41′ 63′′)、尾礦區(qū)(北緯25°06′60′′～25°06′61′′,東經(jīng)109°41′66′′～109°41′67′′)、下游區(qū)(北緯25°08′73′′～25°08′75′′,東經(jīng)109°41′56′′～109°41′57′′)隨機(jī)均勻設(shè)置3個(gè)采樣點(diǎn).每個(gè)采樣點(diǎn)自上而下垂直分為4層,每層15cm,每層隨機(jī)采集3個(gè)土壤樣品.其中上游區(qū)土壤簡(jiǎn)稱為S,按照自上而下分別對(duì)應(yīng)S-1(0～15cm), S-2(15～30cm), S-3(30～45cm)和S-4(45～60cm);同理,尾礦區(qū)土壤簡(jiǎn)稱為W,下游區(qū)土壤簡(jiǎn)稱為X.采集的同一類型土壤,在去除植物根系和碎石等雜物后充分混勻,采用四分法選取適當(dāng)樣品置于無菌袋中,冷藏保存運(yùn)回實(shí)驗(yàn)室.每個(gè)土樣分為2個(gè)部分,一部分鮮土樣品通過2mm篩后置于-80℃冰箱保存以提取土壤微生物DNA;另一部分土壤自然風(fēng)干后過0.149mm篩,用于分析土壤理化性質(zhì).土壤重金屬含量及其他理化性質(zhì)采用《土壤農(nóng)業(yè)化學(xué)分析法》進(jìn)行測(cè)定[21],結(jié)果見表1.

1.3 土壤DNA提取與高通量測(cè)序

稱取0.5g土壤樣品,采用Fast?DNA SPIN Kit(MP Biomedical, Solon, OH, USA)對(duì)土壤DNA進(jìn)行提取.引物為:HF(5′-AAAGGYGGWATCG GYAARTCCACCAC-3′)和HR(5′-TTGTTSGC SGCRTACATSGCCATCAT-3′)[22].PCR擴(kuò)增體系(20μL)為:引物各0.8μL(5μmol/L), 2.0μL DNA, 4.0μL 5×FastPfu緩沖液, 2.0μL dNTPs (2.5 μmol/L), 0.4μL TransStart?FastPfu DNA聚合酶, 補(bǔ)無菌ddH2O至20μL. PCR的擴(kuò)增條件為:95℃預(yù)變性3min, 95℃變性30s, 55℃退火30s, 72℃延伸45s,循環(huán)35次,最后72℃延伸10min.使用EZNA Gel Extraction Kit (Omega, USA)回收PCR混合產(chǎn)物進(jìn)行純化.土壤樣品完成DNA提取后,送至上海美吉生物科技有限公司運(yùn)用Illumina Miseq測(cè)序平臺(tái)進(jìn)行高通量測(cè)序.

表1 泗頂?shù)V區(qū)剖層土土壤金屬含量及其理化性質(zhì)

1.4 nifH基因豐度測(cè)定

利用熒光定量PCR(Real-time PCR)技術(shù)分析剖面土H基因數(shù).反應(yīng)引物同1.3所示,擴(kuò)增片段長(zhǎng)度為461bp.反應(yīng)體系(20μL)為: 2X Taq Plus Master Mix 10μL,引物各0.8μL(5μmol/L), 1.0μL DNA,補(bǔ)無菌ddH2O至20μL.熒光定量PCR反應(yīng)條件為:95℃預(yù)變性5min, 95℃變性30s, 55℃退火30s, 72℃延伸1min,循環(huán)35次.根據(jù)標(biāo)準(zhǔn)曲線計(jì)算基因豐度,H基因豐度最終被計(jì)算為每克干土的拷貝數(shù)(基因拷貝數(shù)/g(干土)).

1.5 數(shù)據(jù)處理與分析

數(shù)據(jù)處理采用Microsoft Excel 2019軟件完成,繪圖采用Origin 2019b軟件完成.利用Uparse軟件對(duì)高通量測(cè)序樣本進(jìn)行聚類,默認(rèn)以97%的一致性將序列聚類成為OTUs(Operational Taxonomic Units)[23].采用QIIME(Version 1.9.1)軟件對(duì)樣本序列進(jìn)行抽平,α多樣性指數(shù)(Shannon、Simpson和ACE指數(shù))和β多樣性指數(shù)(NMDS1指數(shù))的計(jì)算利用QIIME平臺(tái)完成.H基因拷貝數(shù)和多樣性指數(shù)的單因素方差分析(One-way ANOVA,=3)、環(huán)境因子對(duì)Shannon、Simpson、ACE和NMDS1指數(shù)影響的多元線性回歸分析、環(huán)境因子對(duì)固氮微生物H基因數(shù)屬水平相對(duì)豐度影響的Spearman等級(jí)相關(guān)系數(shù)分析均采用SPSS 19.0軟件完成.其中,礦區(qū)不同區(qū)域間H基因數(shù)和α-多樣性的差異性通過T檢驗(yàn)(Student’s-test)進(jìn)行分析,采用SPSS 19.0軟件完成.環(huán)境因子對(duì)固氮微生物H基因數(shù)和屬水平相對(duì)豐度影響的熱圖采用Origin 2019b軟件完成.固氮微生物群落與環(huán)境因子關(guān)系的冗余度分析及繪圖采用Canoco 5.0軟件完成.

2 結(jié)果與分析

2.1 土壤固氮微生物群落豐度和多樣性分析

利用Illumina Miseq測(cè)序平臺(tái)對(duì)泗頂?shù)V區(qū)上游、下游和尾礦區(qū)12個(gè)剖層土固氮微生物H基因進(jìn)行測(cè)序分析,獲得質(zhì)控后的序列數(shù)分別為:上游區(qū)(S-1～S-4)18569、15973、13758和15566條;下游區(qū)(X-1～X-4)15485、17134、16177和13933條;尾礦區(qū)(W-1～W-4)19238、12175、14873和17003條.把相似度水平397%的序列聚為OTUs,置信度閾值設(shè)為0.8.

由圖1(a)可知,礦區(qū)不同區(qū)域剖層土固氮微生物H基因豐度差異顯著(<0.05).上游區(qū)、下游區(qū)和尾礦區(qū)的剖面各層土壤H基因豐度范圍分別為3.02×106～1.17×107、2.55×106～7.78×106和8.19× 105～3.14×106基因拷貝數(shù)/g(干土).各區(qū)域表層土(0～15cm)的H基因豐度相對(duì)于其他層最大;上游區(qū)和下游區(qū)的H基因豐度隨著剖面深度的增加而顯著減小(<0.05).

由圖1(b)～圖1(d)可知,礦區(qū)不同區(qū)域剖層土固氮微生物群落的α-多樣性指數(shù)(Shannon、Simpson和ACE指數(shù))差異顯著(<0.05).上游區(qū)剖層土的Shannon指數(shù)和ACE指數(shù)范圍分別為4.31～4.39和595.12～795.89,顯著高于下游區(qū)和尾礦區(qū)(圖1(b)和圖1(d));表明上游區(qū)剖層土固氮微生物群落的多樣性和豐富程度相對(duì)較高.同時(shí),下游區(qū)和尾礦區(qū)表層土(0～15cm)的Shannon指數(shù)和ACE指數(shù)相對(duì)于其他層最大.另外,上游區(qū)剖層土的Simpson指數(shù)范圍為0.03～0.04,顯著低于下游區(qū)和尾礦區(qū)(圖1(c));表明上游區(qū)剖層土固氮微生物的均勻性相對(duì)較低.

*<0.05, **<0.01, ***<0.001.其中: 右側(cè)箱型圖分別對(duì)應(yīng)表示礦區(qū)不同區(qū)域間H基因數(shù)和α-多樣性指數(shù)的差異性

2.2 土壤固氮微生物群落組成分析

泗頂?shù)V區(qū)上游區(qū)、下游區(qū)和尾礦區(qū)12個(gè)剖層土的固氮微生物群落相對(duì)豐度如圖2所示,相對(duì)豐度<0.03%的門種類合并為其他(Others).其中,變形菌門在礦區(qū)各區(qū)域剖層土中均為優(yōu)勢(shì)菌門;在上游區(qū)、下游區(qū)和尾礦區(qū)的剖層土中所占比例范圍分別為82.1%～87.1%、92.8%～96.6%和71.4%-95.9%.其中,上游區(qū)和下游區(qū)剖層土的優(yōu)勢(shì)菌綱為α-變形菌綱(Alphaproteobacteria),所占比例范圍分別為26.2%～32.8%和60.6%～87.7%.另外,上游區(qū)剖層土β-變形菌綱(Betaproteobacteria)的豐度范圍為1.6%～3.1%.而在尾礦區(qū)剖層土中,除了α-變形菌綱,在W-1層中優(yōu)勢(shì)菌綱還有δ-變形菌綱(Deltaproteobacteria) (27.8%),在W-3層中優(yōu)勢(shì)菌綱還有γ-變形菌綱(Gammaproteobacteria) (30.4%).在上游區(qū)、下游區(qū)和尾礦區(qū)剖層土中,α-變形菌綱下的根瘤菌目(Rhizobiales)相對(duì)豐度較高,所占比例范圍分別為8.4%～14.6%、3.7%～67.4%和8.2%～32.7%.其中,在上游區(qū)剖層土中,根瘤菌目下的慢生根瘤菌屬()相對(duì)豐度較高,所占比例范圍為3.6%～11.9%.

除了變形菌門固氮微生物,在礦區(qū)上游區(qū)、下游區(qū)和尾礦區(qū)的剖層土中還存在藍(lán)藻菌門、擬桿菌門和放線菌門(Actinobacteria)固氮微生物.其中,尾礦區(qū)W-1層的藍(lán)藻菌門的相對(duì)豐度最高,為6.9%,主要為念珠藻科(Nostocaceae).尾礦區(qū)W-3層中擬桿菌門的相對(duì)豐度最高,為4.1%,主要為沼小桿菌屬().上游區(qū)各層剖層土、下游區(qū)X-4層和尾礦區(qū)W-1層中還存在放線菌門固氮微生物,主要為弗蘭克氏菌屬().

圖2 泗頂?shù)V區(qū)剖層土土壤固氮微生物屬水平群落組成

2.3 土壤環(huán)境變化對(duì)固氮微生物群落結(jié)構(gòu)多樣性的影響

表2 多元線性回歸分析環(huán)境因子多樣性指數(shù)的影響

續(xù)表2

注: *<0.05, **<0.01, ***<0.001.

以土壤重金屬含量(鉛、鋅、鎘、銅和錳含量)、土壤pH值、含水率、有機(jī)質(zhì)、總氮、銨態(tài)氮、硝態(tài)氮、總磷和有效磷含量以及土壤中鉀、鈣、鈉和鎂含量作為環(huán)境變量,采用多元線性回歸分析環(huán)境因子變化對(duì)土壤固氮微生物群落的α-多樣性指數(shù)(Shannon、Simpson和ACE指數(shù))和β-多樣性指數(shù)(NMDS1指數(shù))的影響(表2).結(jié)果表明,土壤鉛、鋅和鎘含量的變化顯著影響了Shannon指數(shù)、ACE指數(shù)和NMDS1(<0.05),其中,鋅含量變化對(duì)NMDS1指數(shù)的影響尤其顯著(=33.89,<0.001).土壤鉀、鈣和鈉含量變化顯著影響了ACE指數(shù)(<0.05),其中,鈉含量變化對(duì)ACE指數(shù)的影響尤其顯著(=46.91,<0.001).同時(shí),土壤鉀和鈉含量變化顯著影響了NMDS1指數(shù)(<0.05).另外,土壤含水率、有機(jī)質(zhì)、總氮和硝態(tài)氮含量變化顯著影響了Shannon指數(shù)(<0.05).土壤含水率和總氮含量變化顯著影響了Simpson指數(shù)(<0.05).土壤總磷和銨態(tài)氮含量變化顯著影響了ACE指數(shù)(<0.05).土壤有機(jī)質(zhì)和總磷含量變化顯著影響了NMDS1指數(shù)(<0.05).

2.4 土壤環(huán)境變化對(duì)固氮微生物nifH基因豐度和群落組成的影響

圖(a) 固氮微生物與重金屬、pH值和含水率的冗余分析; 圖(b) 固氮微生物與有機(jī)質(zhì)、總氮、銨態(tài)氮、硝態(tài)氮、總磷、有效磷和H基因數(shù)的冗余分析.其中:紅色實(shí)線箭頭代表屬水平上的土壤固氮微生物OUT,綠色虛線箭頭代表環(huán)境因子,紫色實(shí)線箭頭代表H基因數(shù)

土壤環(huán)境變化對(duì)固氮微生物H基因豐度和群落組成的冗余分析如圖3和表3所示.圖3(a)的RDA結(jié)果表明,第一軸(Axis 1)和第二軸(Axis 2)分別解釋了52.57%和47.92%的變異;同時(shí),微生物群落數(shù)據(jù)變化的累計(jì)解釋量為98.94%;微生物組分變化-土壤環(huán)境因子累計(jì)解釋量為97.13%.表明土壤重金屬含量、土壤pH值和含水率對(duì)固氮微生物群落組成有顯著影響.圖3(b)的RDA結(jié)果表明,第一軸(Axis 1)和第二軸(Axis 2)分別解釋了53.11%和86.06%的變異;同時(shí),微生物群落數(shù)據(jù)變化的累計(jì)解釋量為98.26%;微生物組分變化-土壤環(huán)境因子累計(jì)解釋量為98.83%.表明土壤有機(jī)質(zhì)、氮(總氮、銨態(tài)氮和硝態(tài)氮)和磷(總磷和有效磷)對(duì)固氮微生物群落組成有顯著影響.

表3 土壤固氮微生物群落結(jié)構(gòu)差異的解釋變量冗余分析

基于Spearman等級(jí)相關(guān)系數(shù)分析環(huán)境因子對(duì)固氮微生物H基因數(shù)和屬水平相對(duì)豐度影響的熱圖如圖4所示.可以看出,α-變形菌綱和根瘤菌目的豐度與鉛、鋅、鎘和有機(jī)質(zhì)含量呈正相關(guān)(<0.05或<0.01).其中,根瘤菌目的豐度還與土壤pH值呈正相關(guān)(<0.05).慢生根瘤菌屬、珠藻科和弗蘭克氏菌屬的豐度與土壤中鉀和鈉含量呈正相關(guān)(<0.05或<0.01),與土壤鉛和鋅的豐度呈負(fù)相關(guān)(<0.05或<0.01).并且,慢生根瘤菌屬的豐度還與土壤中銨態(tài)氮、總磷和有效磷含量呈正相關(guān)(<0.05).另外,慢生根瘤菌屬、地桿菌屬()、珠藻科、除硫單胞菌目(Desulfuromonadales)、弗蘭克氏菌屬的豐度與土壤中鈣含量呈負(fù)相關(guān)(<0.05).同時(shí),固氮微生物H基因豐度與土壤中銅、錳、鈉、總氮、銨態(tài)氮、硝態(tài)氮、總磷、有效磷含量及含水率呈正相關(guān)(<0.05或<0.01);與土壤中鈣和鎂的含量呈負(fù)相關(guān)(<0.01).表明土壤環(huán)境因子變化在不同程度上影響了固氮微生物H基因豐度和群落組成.

圖4 環(huán)境因子對(duì)固氮微生物nifH基因數(shù)和屬水平相對(duì)豐度影響的熱圖

*<0.05, **<0.01

3 討論

微生物在生態(tài)系統(tǒng)的功能保持和維護(hù)過程中起著至關(guān)重要的作用,尤其是在氮循環(huán)過程中,固氮微生物直接影響土壤的固氮效率,對(duì)緩解土壤生態(tài)系統(tǒng)的氮素極端不足和實(shí)現(xiàn)氮循環(huán)的正常運(yùn)轉(zhuǎn)至關(guān)重要[24].礦區(qū)及周邊土壤重金屬污染嚴(yán)重,各區(qū)域土壤中碳、氮和磷等含量差異較大,這會(huì)對(duì)土壤固氮微生物的豐度、多樣性和群落組成產(chǎn)生影響[9,25].

變形菌門在礦區(qū)各區(qū)域剖層土中均為優(yōu)勢(shì)菌門(圖2).研究表明,變形菌門在土壤養(yǎng)分循環(huán)中發(fā)揮著重要的作用,廣泛的存在于各種環(huán)境中,并且呈現(xiàn)出高度的多樣化[8,24].變形菌門是加拿大安大略省北部礦區(qū)(主要污染物:鎳和銅)土壤中的優(yōu)勢(shì)菌門[26],也是斯洛伐克西南部礦區(qū)(主要污染物:鎳、鈷和鋅)土壤中的優(yōu)勢(shì)菌門[27].在本研究中,變形菌門固氮微生物主要由α-變形菌綱組成.α-變形菌綱下的根瘤菌目、慢生根瘤菌科、慢生根瘤菌屬和厭氧粘細(xì)菌等可以與豆科植物形成根瘤并進(jìn)行共生固氮[28].其中,這些固氮微生物物種如根瘤菌目、慢生根瘤菌科和慢生根瘤菌屬被認(rèn)為是可以定殖于礦區(qū)的先鋒微生物物種[29].礦區(qū)各區(qū)域剖層土中根瘤菌目相對(duì)豐度較高,并且在上游區(qū)剖層土中,慢生根瘤菌屬相對(duì)豐度較高;這些固氮微生物的高比例存在,表明這些區(qū)域存在共生固氮的可能.另外,在礦區(qū)各區(qū)域剖層土中還存在藍(lán)藻菌門固氮微生物.研究表明,藍(lán)藻菌門微生物是自養(yǎng)型微生物,廣泛存在于植物的根瘤中[11,30].這表明這些區(qū)域的剖層土壤中存在一定豐度的自養(yǎng)型固氮微生物.在尾礦區(qū)W-3層中還檢測(cè)到擬桿菌門固氮微生物,研究表明,擬桿菌門微生物也是常見的固氮微生物[31].Li等[32]指出擬桿菌門微生物對(duì)重金屬的耐受性較好,可以在重金屬污染嚴(yán)重的環(huán)境中生存.

礦區(qū)上游剖層土固氮微生物H基因豐度較高,并且各區(qū)域表層土(0～15cm)中的H基因豐度相對(duì)于同區(qū)域的其他層最大;上游區(qū)和下游區(qū)的H基因豐度隨著土壤剖層深度的增加而顯著減小(<0.05)(圖1(a)).Zhao等[33]的研究表明,重金屬污染可能對(duì)土壤微生物產(chǎn)生兩個(gè)明顯的影響,一是不適應(yīng)高濃度或重金屬毒性的微生物種群數(shù)量減少;二是對(duì)高污染環(huán)境適應(yīng)性較強(qiáng)的微生物種群數(shù)量增加.在本研究中,土壤銅和錳的含量在一定程度上影響了固氮微生物H基因豐度(圖4);然而,相對(duì)于尾礦區(qū),下游區(qū)的鉛、鋅和鎘含量更大(表1),H基因豐度更高.因此,本文認(rèn)為土壤重金屬含量對(duì)泗頂?shù)V區(qū)各區(qū)域剖層土固氮微生物H基因豐度產(chǎn)生了一定的影響,但主要影響剖層土固氮微生物H基因豐度的因素是土壤氮(總氮、銨態(tài)氮和硝態(tài)氮)和磷(總磷和有效磷)的含量(圖4).研究表明,與氮相關(guān)的參數(shù)如總氮、銨態(tài)氮和硝態(tài)氮水平的變化是影響土壤固氮微生物豐度和群落結(jié)構(gòu)的主要因素[25,34].磷作為生物生長(zhǎng)所必須的營(yíng)養(yǎng)元素之一,參與微生物的細(xì)胞生理過程,包括能力存儲(chǔ)、代謝和細(xì)胞分裂[35],也會(huì)對(duì)土壤固氮微生物豐度和群落結(jié)構(gòu)產(chǎn)生影響[36-37].并且,由于礦區(qū)各區(qū)域剖層土中存在一定豐度的根瘤菌目和慢生根瘤菌屬固氮微生物,表明礦區(qū)土壤存在共生固氮的可能;Israel指出[38],土壤磷含量的增加可以有效促進(jìn)共生固氮進(jìn)程.因此,在氮和磷含量更為豐富的上游區(qū)和下游區(qū),固氮微生物H基因豐度相對(duì)更高.

同時(shí),土壤氮和磷的含量變化,對(duì)固氮微生物群落的α-多樣性和β-多樣性也產(chǎn)生了不同程度的影響(表2).在總氮含量較高的上游區(qū),固氮微生物群落的Shannon指數(shù)較高而Simpson指數(shù)較低,表明土壤總氮含量對(duì)固氮微生物群落的多樣性和均勻性產(chǎn)生了影響;在銨態(tài)氮含量較高的上游區(qū),ACE指數(shù)相對(duì)較高,表明銨態(tài)氮含量對(duì)固氮微生物群落豐富程度產(chǎn)生了影響.說明在泗頂?shù)V區(qū),土壤氮含量的變化主要影響了土壤固氮微生物群落的多樣性、豐富程度和均勻性.這與Li等[39]和Wang等[25]的研究一致,表明土壤氮含量變化是影響土壤固氮微生物群落結(jié)構(gòu)和多樣性的最主要因素之一.在總磷含量較高的上游區(qū),ACE指數(shù)相對(duì)較高,表明總磷含量也對(duì)固氮微生物群落豐富程度產(chǎn)生了影響;并且總磷含量同時(shí)影響了NMDS1指數(shù),說明在泗頂?shù)V區(qū),土壤總磷含量變化主要影響了土壤固氮微生物群落的均勻性和固氮微生物在各區(qū)域間的物種差異性.研究表明,銨態(tài)氮、總磷和有效磷的含量變化顯著影響慢生根瘤菌屬()的豐度(<0.05)(圖4).這與Wang等[40]的研究一致,表明土壤磷含量和銨態(tài)氮含量是影響土壤固氮微生物群落組成和多樣性的重要因素之一.

研究表明,土壤重金屬含量變化對(duì)固氮微生物群落的多樣性和組成均產(chǎn)生了不同程度的影響.其中土壤鉛、鋅和鎘含量變化對(duì)固氮微生物群落的Shannon指數(shù)和ACE指數(shù)產(chǎn)生了顯著的影響(< 0.05)(表2).在土壤鉛、鋅和鎘含量較低的上游區(qū), Shannon指數(shù)和ACE指數(shù)相對(duì)更高.表明土壤重金屬含量較高,會(huì)降低群落的Shannon指數(shù)和ACE指數(shù),影響微生物群落的多樣性和均勻性,這與Chodak等[41]的研究結(jié)果相似.并且,土壤重金屬含量過高,會(huì)對(duì)土壤的生物固氮過程產(chǎn)生抑制作用[42].同時(shí),礦區(qū)土壤鉛、鋅和鎘含量的變化對(duì)固氮微生物群落的NMDS1指數(shù)也產(chǎn)生了顯著的影響(<0.01)(表2);其中鋅含量變化的影響最大(=33.89,<0.001).表明礦區(qū)各區(qū)域固氮微生物群落組成的差異性主要由土壤鉛、鋅和鎘含量的變化所引起,這也與Wang等[43]的研究結(jié)果相似.在土壤鉛、鋅和鎘含量相對(duì)更高的下游區(qū),α-變形菌綱和根瘤菌目的豐度更大.另外,土壤鉀、鈣和鈉含量的變化對(duì)固氮微生物群落的ACE指數(shù)和NMDS1指數(shù)也產(chǎn)生了不同程度的影響(表2);其中,土壤鈣(=15.63,<0.01)和鈉(= 46.91,<0.001)含量的變化對(duì)ACE指數(shù)的影響最大.表明礦區(qū)各區(qū)域微生物群落組成的豐富度變化主要由土壤鈣和鈉含量的變化所引起.并且,土壤鈉含量變化對(duì)固氮微生物群落的NMDS1指數(shù)也產(chǎn)生了顯著的影響(<0.01).Quesada等[44]的研究表明,在稻田土壤中,鈉含量與微生物固氮酶活性呈正相關(guān)關(guān)系.本研究的結(jié)果也表明鈉含量與土壤固氮微生物H基因豐度呈顯著的正相關(guān)關(guān)系(<0.01) (圖4);然而,在重金屬污染的礦區(qū)土壤中,鈉含量變化如何影響固氮微生物群落結(jié)構(gòu)和多樣性組成還有待進(jìn)一步研究.除此之外,土壤有機(jī)質(zhì)含量變化對(duì)固氮微生物群落的Shannon指數(shù)和NMDS1指數(shù)產(chǎn)生了顯著的影響(<0.05) (表2).Eo等[45]指出,土壤有機(jī)質(zhì)的來源主要是植物根系碎片和滲出物,其與微生物的活性呈正相關(guān)關(guān)系,是影響土壤微生物群落結(jié)構(gòu)的主要因素之一[46].本研究中,在有機(jī)質(zhì)含量較高的上游區(qū),Shannon指數(shù)相對(duì)更高;同時(shí)有機(jī)質(zhì)含量變化也會(huì)引起礦區(qū)各區(qū)域微生物群落組成的差異性.

4 結(jié)論

4.1 變形菌門在上游區(qū)、下游區(qū)和尾礦區(qū)剖層土中均為優(yōu)勢(shì)菌門,占比超過70%;α-變形菌綱在上游區(qū)和下游區(qū)剖層土中均為優(yōu)勢(shì)菌綱.

4.2 上游區(qū)、下游區(qū)和尾礦區(qū)剖層土固氮微生物H基因豐度的范圍分別為3.02×106～1.17×107、2.55×106～7.78×106和8.19×105～3.14×106基因拷貝數(shù)/g(干土).各區(qū)域表層土(0～15cm)的H基因豐度相對(duì)于其他層最大.主要影響H基因豐度的土壤環(huán)境因素是土壤氮和磷的含量;在氮和磷含量更為豐富的上游區(qū)和下游區(qū),H基因豐度相對(duì)更高.

4.3 上游區(qū)剖層土的Shannon指數(shù)和ACE指數(shù)顯著高于下游區(qū)和尾礦區(qū),表明上游區(qū)剖層土固氮微生物群落的多樣性和豐富程度相對(duì)較高.礦區(qū)各區(qū)域固氮微生物群落組成的差異性主要由土壤鉛、鋅和鎘含量的變化所引起.

4.4 土壤鈣和鈉含量的變化對(duì)ACE指數(shù)的影響最大.表明礦區(qū)各區(qū)域微生物群落組成的豐富度變化主要由土壤鈣和鈉含量的變化所引起.

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Characteristics on the community structure and abundance of diazotrophsfrom the soil profile in the Siding mine area.

LI Yi1,2, ZHANG Hai-chun3, LIU Yuan2, WEI Jiao-teng2, WANG Cong2, LIANG Ying2, LIU Ke-hui1,3**, YU Fang-ming1,2*

(1.Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Ministry of Education, Guangxi Normal University, Guilin 541004, China；2.College of Environment and Resources, Guangxi Normal University, Guilin 541004, China；3.College of Life Science, Guangxi Normal University, Guilin 541004, China)., 2022,42(4)：1819～1828

In the present study, twelve soil profile samples (4 soil layers in each area) were collected from upstream, downstream and mine tailing areas in the Siding mine located in Liuzhou, Guangxi Province. The community composition, abundance and diversity of diazotrophs from the soil profile were examined using Illumina MiSeq high-throughput sequencing technology and fluorogenic quantitative real-time PCR technology. The results indicated that the phylum Proteobacteria was the most dominant taxon, with an abundance higher than 70%. Alphaproteobacteria was the dominant class in the soil profile from the upstream and downstream areas. TheH gene abundance in the soil profile ranged from 3.02×106～1.17×107, 2.55×106～7.78×106and 8.19×105～3.14×106gene copies/g (DW) in upstream, downstream and mine tailing areas, respectively. Nitrogen-related soil properties (including total nitrogen, ammonia and nitrate) and phosphorus-related soil properties (including total phosphorus and available phosphorus) were the main factors influencingH gene abundance. Soil lead, zinc and cadmium concentrations were found to mainly influence diazotrophic community composition. The Shannon index and ACE index of the diazotrophic community in upstream area were higher than those in the downstream and mine tailing areas, which indicated that the diversity and richness of the diazotrophic community in the soil profile were relatively higher in the upstream area. In addition, the soil potassium, calcium and sodium contents contributed to the ACE index and NMDS1index of the diazotrophic community to different degrees. Hence, the results indicated that variation in soil environmental factors had an impact on the community composition, abundance and diversity of diazotrophs from the soil profile. Our research will help to provide a scientific basis for nitrogen regulation, ecological restoration and reconstruction in mining areas.

mine area；diazotrophs；soil profile；community structure；H gene abundance

X172

A

1000-6923(2022)04-1819-10

李 藝(1986-),女,遼寧蓋州人,副教授,博士,主要從事環(huán)境污染生物修復(fù)研究.發(fā)表論文30余篇.

2021-09-16

國(guó)家自然科學(xué)基金資助項(xiàng)目(41967019,41907096)

*責(zé)任作者, 教授, fmyu1215@163.com; **教授, coffeeleave@126.com