肖能文, 劉勇波, 陳群英, 付夢(mèng)娣, 葉 瑤, 李俊生,*

1 中國(guó)環(huán)境科學(xué)研究院, 環(huán)境基準(zhǔn)與風(fēng)險(xiǎn)評(píng)估國(guó)家重點(diǎn)實(shí)驗(yàn)室, 北京 100012 2 中國(guó)科學(xué)院動(dòng)物研究所, 農(nóng)業(yè)蟲(chóng)鼠害綜合治理研究國(guó)家重點(diǎn)實(shí)驗(yàn)室, 北京 100080

轉(zhuǎn)基因油菜與野芥菜混作對(duì)土壤線蟲(chóng)群落的影響

肖能文1, 劉勇波1, 陳群英2, 付夢(mèng)娣1, 葉 瑤1, 李俊生1,*

1 中國(guó)環(huán)境科學(xué)研究院, 環(huán)境基準(zhǔn)與風(fēng)險(xiǎn)評(píng)估國(guó)家重點(diǎn)實(shí)驗(yàn)室, 北京 100012 2 中國(guó)科學(xué)院動(dòng)物研究所, 農(nóng)業(yè)蟲(chóng)鼠害綜合治理研究國(guó)家重點(diǎn)實(shí)驗(yàn)室, 北京 100080

隨著轉(zhuǎn)基因作物在全球的廣泛種植，轉(zhuǎn)基因作物對(duì)非靶標(biāo)生物的影響受到人們的廣泛關(guān)注。轉(zhuǎn)Bt基因抗蟲(chóng)作物與非轉(zhuǎn)基因作物種間混作是避免昆蟲(chóng)產(chǎn)生抗性的一種生態(tài)防御策略，但混作對(duì)非靶標(biāo)生物尤其是土壤線蟲(chóng)影響研究較少。設(shè)置不同比例的轉(zhuǎn)基因油菜(Brassicanapus)與野芥菜(B.juncea)混作5種處理，包括處理A、B、C、D、E分別為轉(zhuǎn)基因油菜和野芥菜比例0∶100；25∶75；50∶50；75∶25；100∶0，調(diào)查不同生育期土壤線蟲(chóng)種類與數(shù)量變化。結(jié)果表明，在油菜與野芥菜生育期內(nèi)，不同處理線蟲(chóng)優(yōu)勢(shì)類群均為擬麗突屬Acrobeloides和真滑刃屬Aphelenchus，各處理線蟲(chóng)屬數(shù)由多到少順序?yàn)樘幚鞡(30屬)>處理C(28屬)>處理A(26屬)>處理D和處理E(25屬)。線蟲(chóng)生活史策略(c-p值)組成一致，各處理間線蟲(chóng)生活史組成不存在明顯差異。不同處理的線蟲(chóng)總數(shù)以及各營(yíng)養(yǎng)類群均無(wú)顯著差異，線蟲(chóng)群落生態(tài)指數(shù)亦無(wú)顯著差異，但不同采樣時(shí)期間線蟲(chóng)Shannon-Wiener多樣性指數(shù)、Simpson優(yōu)勢(shì)度指數(shù)、均勻度指數(shù)、成熟度指數(shù)和線蟲(chóng)通路指數(shù)差異顯著。結(jié)果表明轉(zhuǎn)基因油菜與野芥菜單作和混作短期內(nèi)不影響土壤線蟲(chóng)群落結(jié)構(gòu)。

轉(zhuǎn)基因油菜; 野芥菜; 種間混作; 土壤線蟲(chóng); 群落結(jié)構(gòu)

隨著轉(zhuǎn)基因作物在全球的廣泛種植[1]，靶標(biāo)昆蟲(chóng)對(duì)抗蟲(chóng)基因的適應(yīng)以及次生害蟲(chóng)的爆發(fā)成為轉(zhuǎn)基因作物安全性評(píng)價(jià)的重要內(nèi)容[2]。通常用種內(nèi)混作或者建立避難所來(lái)延緩靶標(biāo)害蟲(chóng)抗性的產(chǎn)生[3-4]。轉(zhuǎn)基因作物種子與非轉(zhuǎn)基因種內(nèi)混作因?yàn)槿菀撞僮?，被推薦為昆蟲(chóng)減小或者避免抗性產(chǎn)生的一種策略[5]。不同品種作物間按不同行相間種植的“種間混作”模式可提高光能和土地的利用率，達(dá)到穩(wěn)產(chǎn)保收，同時(shí)能改變害蟲(chóng)與天敵群落結(jié)構(gòu)[6]，成為一種在中國(guó)常見(jiàn)的耕作模式[7]。

線蟲(chóng)是土壤動(dòng)物區(qū)系中最為豐富的無(wú)脊椎動(dòng)物，其營(yíng)養(yǎng)類群多樣，在土壤食物網(wǎng)中占有重要位置[8]。線蟲(chóng)具有培養(yǎng)分離鑒定相對(duì)簡(jiǎn)便、敏感性良好、以及對(duì)環(huán)境變化能做出迅速反應(yīng)等特點(diǎn)。線蟲(chóng)群落研究發(fā)展相對(duì)成熟的計(jì)算方法，如成熟度指數(shù)(MI)能直接地反映線蟲(chóng)群落的演替狀態(tài)，敏感地反映土壤環(huán)境的受脅迫程度[9]。線蟲(chóng)通路指數(shù)(NCR)能表示土壤有機(jī)質(zhì)的分解途徑[10]，因此常被廣泛地應(yīng)用于土壤質(zhì)量以及土壤污染的研究[11-12]。

自1986年Mathew等首先將新霉素磷酸轉(zhuǎn)移酶(neomycin phosphotransferase gene，NPT-Ⅱ)基因利用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)入芥菜型油菜以來(lái)，開(kāi)始對(duì)包括從細(xì)菌Bacillusthuringiensis[Berliner](Bt)獲得的轉(zhuǎn)基因油菜進(jìn)行種植與研究[13]。轉(zhuǎn)Bt油菜能有效控制小菜蛾P(guān)lutellaxylostellaL.和美洲棉鈴蟲(chóng)HelicoverpazeaBoddie[14]。但轉(zhuǎn)基因油菜也存在一定安全風(fēng)險(xiǎn)。如轉(zhuǎn)Bt油菜花粉逃逸能與野芥菜Brassicarapa雜交。Burgio等認(rèn)為轉(zhuǎn)基因油菜Bt蛋白能殘留在非靶標(biāo)昆蟲(chóng)桃蚜Myzuspersicae體內(nèi)[15]。但Pierre等認(rèn)為轉(zhuǎn)基因油菜對(duì)4種蜜蜂無(wú)顯著影響[16]。Howald等在油菜葉蜂Athaliarosae體內(nèi)檢測(cè)到Bt蛋白，但是對(duì)其生活史沒(méi)有影響[17]。

Bt毒素能通過(guò)花粉、作物殘?bào)w以及根分泌物進(jìn)入土壤環(huán)境[18-19]，可能對(duì)土壤環(huán)境以及土壤生物產(chǎn)生潛在風(fēng)險(xiǎn)，因此轉(zhuǎn)基因作物是否影響土壤生物多樣性及群落結(jié)構(gòu)受到社會(huì)廣泛關(guān)注[7, 20]。Zwahlen等用轉(zhuǎn)基因玉米喂食蚯蚓Lumbricusterrestris200 d后，蚯蚓體重顯著減少[19]。轉(zhuǎn)基因作物對(duì)線蟲(chóng)影響也有廣泛報(bào)道。某些Bt蛋白，如Cry5B、Cry6A、Cry14A和Cry21A發(fā)現(xiàn)對(duì)某些線蟲(chóng)有直接毒性[21]。但Yang等調(diào)查了連續(xù)種植轉(zhuǎn)基因棉田土壤線蟲(chóng)，并認(rèn)為無(wú)明顯影響[7]，H?ss等也認(rèn)為轉(zhuǎn)基因玉米對(duì)土壤自由生活線蟲(chóng)無(wú)顯著影響[20]。轉(zhuǎn)基因作物與常規(guī)物種“種間混作”常改變農(nóng)田害蟲(chóng)與天敵群落結(jié)構(gòu)，因此需要對(duì)轉(zhuǎn)基因作物生態(tài)安全進(jìn)行進(jìn)一步研究，但轉(zhuǎn)基因油菜與野芥菜之間混作是否影響土壤線蟲(chóng)群落結(jié)構(gòu)的研究未見(jiàn)報(bào)道。

本文選擇轉(zhuǎn)基因油菜與野芥菜為研究對(duì)象，按照不同比例移栽轉(zhuǎn)基因油菜與野芥菜幼苗，在不同時(shí)期對(duì)土壤進(jìn)行采樣，調(diào)查轉(zhuǎn)基因油菜與野芥菜種間混作對(duì)線蟲(chóng)群落結(jié)構(gòu)的影響，以期更全面地了解轉(zhuǎn)基因油菜與其他植物混作對(duì)非靶標(biāo)土壤動(dòng)物的影響，為轉(zhuǎn)基因油菜的潛在非靶標(biāo)生物生態(tài)風(fēng)險(xiǎn)評(píng)價(jià)提供科學(xué)依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)材料

野芥菜(Brassicajuncea, 2n= 36, AABB)為十字花科植物，是油菜(Brassicanapuscv “Westar”, 2n=38, AACC)的近緣種，可與油菜進(jìn)行雜交，種子由南京農(nóng)業(yè)大學(xué)提供，從當(dāng)?shù)靥镩g收集[22]。轉(zhuǎn)基因油菜為 pSAM 12質(zhì)粒轉(zhuǎn)化，包含CaMV 35S啟動(dòng)子控制編碼綠色熒光蛋白(Green fluorescent protein, GFP)和Bt(Bacillusthuringiensis) Cry1Ac蛋白基因油菜[23]。

1.2 實(shí)驗(yàn)處理

實(shí)驗(yàn)在北京順義實(shí)驗(yàn)基地進(jìn)行，按照隨機(jī)區(qū)組設(shè)計(jì)3個(gè)平行小區(qū)，每小區(qū)完全隨機(jī)設(shè)置轉(zhuǎn)基因油菜和野芥菜按照0∶100(處理A)；25∶75(處理B)；50∶50(處理C)；75∶25(處理D)；100∶0(處理E)不同比例5個(gè)處理。處理設(shè)置在2 m×2 m×2 m的50目尼龍網(wǎng)紗罩中，處理間距離6 m。轉(zhuǎn)基因油菜和野芥菜按比例相間排列，種植密度為6×6株(圖1)。轉(zhuǎn)基因油菜和野芥菜2012年4月16日溫室播種，5月20日移栽至試驗(yàn)地。每處理移栽灌溉以及雜草處理實(shí)行一致的管理方法，全生育期不施藥。

于2012年移栽前(5月20日)，以及移栽后按照生育期花期(7月4日)和收獲期(8月14日)分別采樣。在樣方內(nèi)選擇3個(gè)點(diǎn)取土，用土鉆采集深度為0—10 cm的表土，將其均勻混合后制成約500 mL混合土樣帶回實(shí)驗(yàn)室分離線蟲(chóng)和進(jìn)行理化分析。土壤pH值為7.3±0.5，有機(jī)碳含量為(6.8±0.4)mg/g。

1.3 線蟲(chóng)分離、鑒定

每個(gè)土樣取土100 cm3，3 d內(nèi)用改進(jìn)的Baermann漏斗法分離線蟲(chóng)48 h[24]，收集線蟲(chóng)懸浮液并濃縮至2 mL，用4%福爾馬林溶液固定。光學(xué)顯微鏡下參照Goodey的分類系統(tǒng)[25]和《中國(guó)土壤動(dòng)物檢索圖鑒》[26]以及《植物線蟲(chóng)志》[27]，將線蟲(chóng)鑒定到屬，并統(tǒng)計(jì)各屬線蟲(chóng)數(shù)量。

圖1 轉(zhuǎn)基因油菜與野芥菜單作以及混作實(shí)驗(yàn)設(shè)計(jì)布局圖Fig.1 Experimental design layout for sole cropping or mixed intercropping between transgenic oilseed rape and wild mustard

1.4 線蟲(chóng)群落結(jié)構(gòu)分析

土壤線蟲(chóng)依據(jù)Yeates等分為4個(gè)營(yíng)養(yǎng)類型[10]，分別為食細(xì)菌類(Ba)、食真菌類(Fu)、植物寄生類(PP)和雜食捕食類(OP)。根據(jù)線蟲(chóng)不同的生活史策略，將線蟲(chóng)劃分為5個(gè)類群，即不同的colonizer persister (c-p)類群[28]。

研究采用生態(tài)學(xué)評(píng)價(jià)指數(shù):Shannon-Wiener多樣性指數(shù)H′，H′=-∑PilnPi，Pi=ni/N，式中Pi為樣品中屬于第i種的個(gè)體的比例；ni為第i類群的個(gè)體數(shù)；N為所有類群的個(gè)體總數(shù)[29]。Pielou均勻度指數(shù)J′，J′=H′/lnS，式中S為類群數(shù)[30]。Simpson優(yōu)勢(shì)度指數(shù)λ，λ=∑(ni/N)2，λ=∑H′2[31]。線蟲(chóng)成熟指數(shù)，MI=∑v(i)f(i)，式中v(i)是第i種線蟲(chóng)的c-p值；f(i)第i種線蟲(chóng)的個(gè)體數(shù)占自由生活線蟲(chóng)數(shù)量的比例[9]。線蟲(chóng)通路比值(NCR)，NCR=NBa/(NBa+NFu)，式中NBa為食細(xì)菌線蟲(chóng)數(shù)量；NFu為食真菌線蟲(chóng)數(shù)量[10]。

1.5 數(shù)據(jù)分析

數(shù)據(jù)分析采用SPSS 軟件(16.0版, SPSS Inc.)。采樣雙因子方差分析(Two-way ANOVA)不同處理與采樣時(shí)期間線蟲(chóng)數(shù)量以及多樣性指數(shù)差異，處理組間差異顯著性采用Duncan檢驗(yàn)(P<0.05)。

2 結(jié)果與分析

2.1 線蟲(chóng)科屬及營(yíng)養(yǎng)類群

在所采集的45個(gè)土壤樣品中，土壤線蟲(chóng)分屬21科33屬 (表1)，其中食細(xì)菌類15屬、食真菌類5屬、植物寄生類8屬和雜食捕食類5屬。各處理線蟲(chóng)屬數(shù)：處理B(30屬)>處理C(28屬)>處理A(26屬)>處理D和處理E(25屬)。在所有處理中，優(yōu)勢(shì)類群為擬麗突屬Acrobeloides和真滑刃屬Aphelenchus，分別占總數(shù)的12.3%和37.4% (表1)。常見(jiàn)類群有13屬，占總數(shù)的47.9%，而稀有類群19屬，占總數(shù)的2.39%。潛根屬Hirschmanniella僅出現(xiàn)在處理C中。墊刃屬Tylenchus和胞囊屬Heterodera僅在處理A和處理B中出現(xiàn)。按照線蟲(chóng)功能類群，食真菌線蟲(chóng)所占比例最大，為47.5%，其次為食細(xì)菌線蟲(chóng)，所占比例為33.7%，植食性線蟲(chóng)，占14.0%和捕食雜食性線蟲(chóng)，占線蟲(chóng)總數(shù)4.8%。

2.2 土壤線蟲(chóng)群落c-p值變化

在不同c-p中，以c-p 2所占比例最高，為77.7%，其次是c-p 3，占11.2%，c-p 1占5.88%，c-p 5和c-p 4類群較少，分別占4.86%和0.35%。De Goede等認(rèn)為線蟲(chóng)主要為c-p 2—4的類群，而c-p1和c-p5相對(duì)較少，可以用對(duì)c-p 2, c-p 3和c-p 4類群按比例做成三角形圖[33]。調(diào)查結(jié)果表明c-p 1, c-p 2和c-p 3類群較多，而c-p 4和c-p 5類群較少，c-p 1, c-p 2和c-p 3類群所占比例作圖(圖2)，結(jié)果表明，幾個(gè)處理線蟲(chóng)c-p值比例差異不大，基本生活史類型組成相似，說(shuō)明不同處理類型在生活史組成沒(méi)有差異。

2.3 不同處理線蟲(chóng)總量以及各營(yíng)養(yǎng)類型線蟲(chóng)數(shù)量比較

方差分析結(jié)果表明，不同處理間土壤線蟲(chóng)總量差異不顯著，但不同取樣時(shí)間線蟲(chóng)總數(shù)差異顯著(表2)，實(shí)驗(yàn)開(kāi)始時(shí)數(shù)量最多，隨后在花期和收獲期線蟲(chóng)總數(shù)減少(圖3)。取樣時(shí)間與處理間不存在相互作用(表2)。不同營(yíng)養(yǎng)類型的線蟲(chóng)數(shù)量在不同采樣時(shí)期間有顯著差異，但處理與時(shí)間之間的交互作用不顯著(表2)。

表1 不同處理線蟲(chóng)科屬豐富度與功能類群

試驗(yàn)開(kāi)始時(shí)，處理D線蟲(chóng)總數(shù)為(756±131)條/100 cm3，而處理C線蟲(chóng)數(shù)量最少，為(493±147)條/100 cm3。但到花期7月4日，各處理線蟲(chóng)總數(shù)有所下降，但仍以處理D線蟲(chóng)數(shù)量最多，但處理間差異縮小。到成熟期，線蟲(chóng)總數(shù)進(jìn)一步減少，處理C線蟲(chóng)總數(shù)最多，為(339±123)條/100 cm3，處理E線蟲(chóng)最少，僅(141±205)條/100 cm3。多重比較表明，處理間均無(wú)顯著差異(圖3)。

對(duì)不同營(yíng)養(yǎng)類型的線蟲(chóng)數(shù)量進(jìn)行了進(jìn)一步的比較，植食性線蟲(chóng)在5月20日差異顯著(P<0.05，圖4 I)，處理B數(shù)量最高，達(dá)(87±37)條/100 cm3，而處理D植食性線蟲(chóng)數(shù)量最少，僅(20±14)條/100 cm3。其他時(shí)間段不同處理間差異均不顯著(P>0.05，圖4)。

圖2 不同處理線蟲(chóng)c-p值相對(duì)豐富度 Fig.2 Relative abundances of nematode taxa classified as c-p 1, c-p 2 and c-p 3—5數(shù)據(jù)點(diǎn)代表5個(gè)不同處理(A、B、C、D、E)平均值

圖3 不同處理線蟲(chóng)總量 Fig.3 The total number of nematodes at different treatments圖中數(shù)據(jù)為平均值±標(biāo)準(zhǔn)誤，小寫(xiě)字母相同表示組間無(wú)顯著性差異，字母不同表示有顯著性差異，α=0.05

方差分析結(jié)果表明，食細(xì)菌線蟲(chóng)、食真菌線蟲(chóng)與捕食雜食性線蟲(chóng)在不同處理間均無(wú)顯著差異(P>0.05)(圖4)。

圖4 不同處理線蟲(chóng)各營(yíng)養(yǎng)類型數(shù)量比較Fig.4 The number of four feeding types of nematode against different treatments at three sampling times字母相同表示組間無(wú)顯著性差異，字母不同表示有顯著性差異，α=0.05

2.4 不同處理線蟲(chóng)群落結(jié)構(gòu)比較

方差分析結(jié)果表明，各處理間多樣性、優(yōu)勢(shì)度、均勻度、成熟度和線蟲(chóng)通路比值(NCR)差異不顯著，不同采樣時(shí)期間差異顯著，但處理與時(shí)間不存在交互作用(表2)。

表2 不同處理線蟲(chóng)數(shù)量組成及群落結(jié)構(gòu)方差分析結(jié)果

對(duì)不同處理線蟲(chóng)數(shù)量群落多樣性分析(圖5)，線蟲(chóng)群落Shannon-Wiener多樣性指數(shù)(H′)、Simpson優(yōu)勢(shì)度指數(shù)(λ)和均勻度指數(shù)(J′)在5月20日處理間無(wú)顯著差異(圖4)。到7月4日，指數(shù)間均出現(xiàn)差異，處理D有更高的多樣性指數(shù)、優(yōu)勢(shì)度和均勻度，而處理A多樣性、優(yōu)勢(shì)度和均勻度均最低，且與處理D存在顯著差異(P<0.05)(圖5)。到油菜成熟期，處理間多樣性差異不顯著。但是各處理間優(yōu)勢(shì)度與均勻度仍然存在顯著差異，處理D優(yōu)勢(shì)度與均勻度最高，處理C的優(yōu)勢(shì)度與均勻度最低。

各處理線蟲(chóng)成熟度指數(shù)(MI)在2.15—3.63之間，NCR(圖5)在0.28—0.57之間，在3個(gè)取樣時(shí)間成熟度指數(shù)和NCR差異均不顯著(P>0.05)。但方差分析結(jié)果表明，不同取樣時(shí)間差異極顯著(P<0.01)，不同時(shí)間與處理間對(duì)線蟲(chóng)MI和NCR指數(shù)均不存在相互作用(P>0.05)。

圖5 不同處理土壤線蟲(chóng)群落多樣性分析Fig.5 The diversity of soil nematode communities at different treatments

3 討論

抗蟲(chóng)轉(zhuǎn)Bt基因作物種植能控制靶標(biāo)害蟲(chóng)并減少殺蟲(chóng)劑的使用[34-35]，減少殺蟲(chóng)劑進(jìn)入土壤環(huán)境，從而減少對(duì)土壤生物的環(huán)境壓力。轉(zhuǎn)基因植物的毒蛋白能通過(guò)植物殘?bào)w和根基分泌物等土壤環(huán)境[36-37]。混作常用于提高作物產(chǎn)量，也是常見(jiàn)的控制害蟲(chóng)的耕作模式，混作可以提高作物光能和土地的利用率，增加田間生物多樣性。

利用轉(zhuǎn)基因油菜與野芥菜混作，鑒定出土壤線蟲(chóng)33屬，線蟲(chóng)總數(shù)范圍為在141.5—756.0條/100 cm3。線蟲(chóng)總數(shù)以及不同營(yíng)養(yǎng)類型線蟲(chóng)數(shù)量均無(wú)顯著差異。雖然在5月20日處理B(轉(zhuǎn)基因油菜∶野芥菜25∶75)植食性線蟲(chóng)數(shù)量顯著高于處理D(75∶25)。但本次采樣為作物移栽時(shí)土壤本底的差異，而本次采樣其他處理間不存在顯著差異。在隨后的2次采樣中，不同處理間不存在差異。本文結(jié)果說(shuō)明轉(zhuǎn)基因油菜、野芥菜以及兩種作物混作，土壤線蟲(chóng)數(shù)量不存在顯著差異，說(shuō)明混作以及單作不影響土壤線蟲(chóng)的數(shù)量。其結(jié)果與Li和Liu等結(jié)論一致，認(rèn)為長(zhǎng)期種植轉(zhuǎn)基因棉花對(duì)土壤線蟲(chóng)總數(shù)影響很小[38]。H?ss等也認(rèn)為表達(dá)Cry1Ab和Cry3Bb1蛋白的Bt玉米對(duì)土壤線蟲(chóng)多樣性無(wú)顯著影響[20]。Griffiths等調(diào)查歐洲3個(gè)研究區(qū)表達(dá)CryIAb蛋白的轉(zhuǎn)基因玉米(ZeamaysL.)土壤線蟲(chóng)，認(rèn)為對(duì)線蟲(chóng)的動(dòng)態(tài)變化在正常農(nóng)業(yè)變化范圍內(nèi)[39]。Al-Deeb等也證明轉(zhuǎn)Bt基因玉米與非轉(zhuǎn)基因玉米土壤中的線蟲(chóng)數(shù)量相當(dāng)[40]。這些結(jié)果均表明，大田種植的不同轉(zhuǎn)Bt作物，對(duì)土壤線蟲(chóng)數(shù)量影響均不顯著。

研究表明轉(zhuǎn)基因油菜單作以及與野芥菜混作不改變線蟲(chóng)生活史組成和群落組成。不同比例轉(zhuǎn)基因油菜與野芥菜混作以及單作處理，土壤線蟲(chóng)中優(yōu)勢(shì)類群均為擬麗突屬Acrobeloides和真滑刃屬Aphelenchus，土壤線蟲(chóng)群落組成變化較??；且5個(gè)處理線蟲(chóng)c-p值組成一致(圖1)，聚成一類，轉(zhuǎn)基因油菜單作以及與野芥菜混作后，各處理間線蟲(chóng)群落組成不存在明顯差異。結(jié)果與Yang等結(jié)論一致[12]，認(rèn)為轉(zhuǎn)基因棉花的種植不影響土壤線蟲(chóng)群落組成。但不同作物農(nóng)田土壤線蟲(chóng)優(yōu)勢(shì)類群不一樣，Yang等棉田中優(yōu)勢(shì)類群為擬麗突屬Acrobeloides、真頭葉屬Eucephalobus, 和真滑刃屬Aphelenchus。但Li和Liu調(diào)查多年轉(zhuǎn)基因棉種植田間主要類群為螺旋屬Helicotylenchus、絲尾墊刃屬Filenchus和擬麗突屬Acrobeloides，不同作物種植類型和不同種植年限，可能導(dǎo)致線蟲(chóng)優(yōu)勢(shì)類群不同。

轉(zhuǎn)基因油菜與野芥菜混作不改變土壤線蟲(chóng)多樣性。雖然在7月4日，線蟲(chóng)多樣性指數(shù)、優(yōu)勢(shì)度指數(shù)和均勻度指數(shù)在100%野芥菜處理A和轉(zhuǎn)基因油菜與野芥菜75∶25處理D間存在顯著差異，8月14日，處理D的優(yōu)勢(shì)度與均勻度明顯高于處理C，但在整體方差分析結(jié)果表明各指數(shù)在處理間無(wú)線蟲(chóng)差異(表3)。在成熟度MI指數(shù)與線蟲(chóng)通路指數(shù)NCR指數(shù)也有著相似的變化規(guī)律。其結(jié)果與轉(zhuǎn)基因棉田土壤線蟲(chóng)一致[12, 38]。轉(zhuǎn)基因油菜與野芥菜混作不影響土壤線蟲(chóng)群落結(jié)構(gòu)。時(shí)培建等認(rèn)為作物物種豐富度顯著性影響害蟲(chóng)物種豐富度，混栽田中節(jié)肢動(dòng)物群落穩(wěn)定性高于單一種植田中節(jié)肢動(dòng)物群落穩(wěn)定性[41]。但本實(shí)驗(yàn)表明單作以及混作處理后，植食性土壤線蟲(chóng)數(shù)量沒(méi)有發(fā)生顯著變化，不同營(yíng)養(yǎng)類型線蟲(chóng)數(shù)量不存在顯著差異，轉(zhuǎn)基因油菜與野芥菜混作與單作相比并沒(méi)有增加土壤線蟲(chóng)多樣性。

本文實(shí)驗(yàn)結(jié)果表明，轉(zhuǎn)基因油菜與野芥菜按不同比例混作，土壤線蟲(chóng)優(yōu)勢(shì)類群相同，線蟲(chóng)總數(shù)與不同營(yíng)養(yǎng)類型線蟲(chóng)數(shù)量不存在顯著差異，線蟲(chóng)生活史策略(c-p值)組成相似。線蟲(chóng)群落參數(shù)亦不存在顯著差異，轉(zhuǎn)基因油菜與野芥菜混作短期內(nèi)沒(méi)有使土壤線蟲(chóng)群落結(jié)構(gòu)發(fā)生明顯改變。

[1] James C. Global Status of Commercialized Biotech/GM Crops: 2012. The International Service for the Acquisition of Agri-biotech Applications Briefs No. 44. Ithaca, NY, 2012: 1-18.

[2] Lu Y H, Wu K M, Jiang Y Y, Xia B, Li P, Feng H Q, Wyckhuys K A G, Guo Y Y. Mirid bug outbreaks in multiple crops correlated with wide-scale adoption of Bt cotton in China. Science, 2010, 328(5982): 1151-1154.

[3] Ramachandran S, Buntin G D, All J N, Raymer P L, Stewart C N. Intraspecific competition of an insect-resistant transgenic canola in seed mixtures. Agronomy Journal, 2000, 92(2): 368-374.

[4] Wu K, Feng H, Guo Y. Evaluation of maize as a refuge for management of resistance to Bt cotton byHelicoverpaarmigera(Hübner) in the Yellow River cotton-farming region of China. Crop Protection, 2004, 23(6): 523-530.

[5] Hokkanen H M T, Wearing C H. Assessing the risk of pest resistance evolution toBacillusthuringiensisengineered into crop plants: a case study of oilseed rape. Field Crops Research, 1996, 45(1/3): 171-179.

[6] Yang B, Ge F, Ouyang F, Parajulee M. Intra-species mixture alters pest and disease severity in cotton. Environmental Entomology, 2012, 41(4): 1029-1036.

[7] Yang B, Parajulee M, Ouyang F, Wu G, Ge F. Intraspecies mixture exerted contrasting effects on nontarget arthropods ofBacillusthuringiensiscotton in northern China. Agricultural and Forest Entomology, 2014, 16(1): 24-32.

[8] Yeates G W, Bongers T, De Goede R G M, Freckman D M, Georgieva S S. Feeding habits in soil nematode families and genera -an outline for soil ecologists. Journal of Nematology, 1993, 25(3): 315-331.

[9] Bongers T. The maturity index: an ecological measure of environmental disturbance based on nematode species composition. Oecologia, 1990, 83(1): 14-19.

[10] Yeates G W. Nematodes as soil indicators: functional and biodiversity aspects. Biology and Fertility of Soils, 2003, 37(4): 199-210.

[11] 肖能文, 謝德燕, 王學(xué)霞, 閆春紅, 胡理樂(lè), 李俊生. 大慶油田石油開(kāi)采對(duì)土壤線蟲(chóng)群落的影響. 生態(tài)學(xué)報(bào), 2011, 31(13): 3736-3744.

[12] Yang B, Chen H, Liu X H, Ge F, Chen Q Y. Bt cotton planting does not affect the community characteristics of rhizosphere soil nematodes. Applied Soil Ecology, 2014, 73: 156-164.

[13] Stewart Jr C N, Adang M J, All J N, Raymer P L, Ramachandran S, Parrott W A. Insect control and dosage effects in transgenic canola containing a syntheticBacillusthuringiensiscryIAc gene. Plant Physiology, 1996, 112(1): 115-120.

[14] Stewart C N, All J N, Raymer P L, Ramachandran S. Increased fitness of transgenic insecticidal rapeseed under insect selection pressure. Molecular Ecology, 1997, 6(8): 773-779.

[15] Burgio G, Lanzoni A, Accinelli G, Dinelli G, Bonetti A, Marotti I, Ramilli F. Evaluation of Bt-toxin uptake by the non-target herbivore,Myzuspersicae(Hemiptera: Aphididae), feeding on transgenic oilseed rape. Bulletin of Entomological Research, 2007, 97(2): 211-215.

[16] Pierre J, Marsault D, Genecque E, Renard M, Champolivier J, Pham-Delègue M H. Effects of herbicide-tolerant transgenic oilseed rape genotypes on honey bees and other pollinating insects under field condtions. Entomologia Experimentalis et Applicata, 2003, 108(3): 159-168.

[17] Howald R, Zwahlen C, Nentwig W. Evaluation of Bt oilseed rape on the non-target herbivoreAthaliarosae. Entomologia Experimentalis et Applicata, 2003, 106(2): 87-93.

[18] Saxena D, Stotzky G.Bacillusthuringiensis(Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil. Soil Biology and Biochemistry, 2001, 33(9): 1225-1230.

[19] Zwahlen C, Hilbeck A, Howald R, Nentwig W. Effects of transgenic Bt corn litter on the earthwormLumbricusterrestris. Molecular Ecology, 2003, 12(4): 1077-1086.

[20] H?ss S, Nguyen H T, Menzel R, Pagel-Wieder S, Miethling-Graf R, Tebbe C C, Jehle J A, Traunspurger W. Assessing the risk posed to free-living soil nematodes by a genetically modified maize expressing the insecticidal Cry3Bb1 protein. Science of the Total Environment, 2011, 409(13): 2674-2684.

[21] Wei J Z, Hale K, Carta L, Platzer E, Wong C, Fang S C, Aroian R V.Bacillusthuringiensiscrystal proteins that target nematodes. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(5): 2760-2765.

[22] Liu Y B, Tang Z X, Darmency H, Stewart C N Jr, Di K, Wei W, Ma K P. The effects of seed size on hybrids formed between oilseed rape (BrassicaNapus) and wild brown mustard (B.Juncea). PLoS ONE, 2012, 7(6): e39705.

[23] Halfhill M D, Richards H A, Mabon S A, Stewart C N. Expression of GFP and Bt transgenes inBrassicanapusand hybridization withBrassicarapa. Theoretical and Applied Genetics, 2001, 103(5): 659-667.

[24] Ingham R E. Nematodes. Methods of Soil Analysis. Part 2. Microbiological and Biochemical Properties//Weaver R W, Angle S, Bottomley P, Bezdicek D, Smith S, Tabatabai A, Wollum A, eds. Society of America Book Series: Madison, WI.1994, 459-490.

[25] Goodey T. Soil and Freshwater Nematodes. New York: John Wiley & Sons Inc., 1963.

[26] 尹文英. 中國(guó)土壤動(dòng)物檢索圖鑒.北京: 科學(xué)出版社, 1998.

[27] 劉維志. 植物線蟲(chóng)志.北京: 中國(guó)農(nóng)業(yè)出版社, 2004.

[28] Neher D A. Role of nematodes in soil health and their use as indicators. Journal of Nematology, 2001, 33(4): 161-168.

[29] Shannon C E. A mathematical theory of communication. Bell System Technical Journal, 1948, 27(3):379-423.

[30] Pielou E C. Mathematical Ecology. New York: John Wiley & Sons Inc., 1977.

[31] Simpson E H. Measurement of diversity. Nature, 1949, 163(4148): 688-688.

[32] Neher D A, Peck S L, Rawlings J O, Campbell C L. Measures of nematode community structure and sources of variability among and within agricultural fields. Plant and Soil, 1995, 170(1): 167-181.

[33] De Goede R G M, Bongers T, Ettema C H. Graphical presentation and interpretation of nematode community structure: c-p triangles. Medical Faculty Landbouww University of Gent, 1993, 58(2b): 743-750.

[34] Lu Y H, Wu K G, Jiang Y Y, Guo Y, Desneux N. Widespread adoption of Bt cotton and insecticide decrease promotes biocontrol services. Nature, 2012, 487(7407): 362-365.

[35] Cattaneo M G, Yafuso C, Schmidt C, Huang C Y, Rahman M, Olson C, Ellers-Kirk C, Orr B J, Marsh S E, Antilla L, Dutilleul P, Carrière Y. Farm-scale evaluation of the impacts of transgenic cotton on biodiversity, pesticide use, and yield. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(20): 7571-7576.

[36] Wang Y M, Hu H W, Huang J C, Li J H, Liu B, Zhang G. Determination of the movement and persistence of Cry1Ab/1Ac protein released from Bt transgenic rice under field and hydroponic conditions. Soil Biology and Biochemistry, 2013, 58: 107-114.

[37] Saxena D, Stotzky G. Insecticidal toxin fromBacillusthuringiensisis released from roots of transgenic Bt corn in vitro and in situ. FEMS Microbiology Ecology, 2000, 33(1): 35-39.

[38] Li X G, Liu B. A 2-year field study shows little evidence that the long-term planting of transgenic insect-resistant cotton affects the community structure of soil nematodes. PLoS ONE, 2013, 8(4): e61670.

[39] Griffiths B S, Caul S, Thompson J, Birch A N E, Scrimgeour C, Andersen M N, Cortet J, Messéan A, Sausse C, Lacroix B, Krogh P H. A comparison of soil microbial community structure, protozoa and nematodes in field plots of conventional and genetically modified maize expressing theBacillusthuringiensis CryIAb toxin. Plant and Soil, 2005, 275(1/2): 135-146.

[40] Al-Deeb M A, Wilde G E, Blair J M, Todd T C. Effect of Bt corn for corn rootworm control on nontarget soil microarthropods and nematodes. Environmental Entomology, 2003, 32(4): 859-865.

[41] 時(shí)培建, 惠蒼, 門(mén)興元, 趙紫華, 歐陽(yáng)芳, 戈峰, 金顯仕, 曹海鋒, Larry L B. 作物多樣性對(duì)害蟲(chóng)及其天敵多樣性的級(jí)聯(lián)效應(yīng). 中國(guó)科學(xué): 生命科學(xué), 2014, 44(1): 75-84.

Effect of interspecific mixed cropping between transgenic oilseed rape and wild mustard on soil nematode communities

XIAO Nengwen1, LIU Yongbo1, CHEN Qunying2, FU Mengdi1, YE Yao1, LI Junsheng1,*

1StateKeyLaboratoryofEnvironmentalCriteriaandRiskAssessment，ChineseResearchAcademyofEnvironmentalSciences,Beijing100012,China2StateKeyLaboratoryofIntegratedManagementofPestInsectsandRodents,InstituteofZoology,ChineseAcademyofSciences,Beijing100080,China

As genetically modified (GM) crops are cultivated worldwide, the effects of GM crops on non-target organisms are of concern. Interspecific mixed cropping between transgenic and non-transgenic crops is generally regarded as a strategy against insects to minimize the development of resistance to otherwise insect-resistant transgenic crops. The toxin fromBacillusthuringiensis(Bt) is introduced into the soil primarily through root exudates and by the incorporation of plant residues after harvest, with probable help from pollen. Such incorporation of the toxin poses potential risks to soil organisms, including microbes, nematodes, collembolans, and other invertebrates. However, its effects on non-target soil organisms have rarely been assessed. We evaluated the effect on soil nematodes of mixed cropping with transgenic canolaBrassicanapusL. expressing Bt and wild brown mustardB.juncea. The abundance and genera composition of soil nematodes in the flowering and fruiting period of canola were investigated in five mixed proportions of transgenic canola and wild brown mustard: 0∶100 (A), 25∶75 (B), 50∶50 (C), 75∶25 (D), and 100∶0 (E). The results showed the following order of genera composition with each treatment: B (30 genera) > C (28 genera) > A (26 genera) > D and E (25 genera). The dominant nematode genera wereAcrobeloidesandAphelenchus, accounting for 37.4% and 12.3% of total abundance, respectively. The common and rare groups belonging to 13 and 19 genera accounted for 47.9% and 2.39% of the total, respectively.Hirschmanniellaappeared only in treatment C.TylenchusandHeteroderaappeared only in treatments A and B. Depending on the trophic structure based on the functional group, fungivorous nematodes formed the largest proportion at 47.5%, followed by bacterivorous, herbivorous, and omnivorous-predatory nematodes at 33.7%, 14%, and 4.8% of the total, respectively. The colonizer-persister (c-p) values of nematodes had the same composition among the five treatments. Further, similar life histories were noted following the treatments. The total number of nematodes was in the range of 141.5—756.0/100 cm3. The total abundance and number of four feeding types of nematodes were not significantly different among treatments. The generic composition and community parameters of nematodes did not differ significantly among the five treatments. The Shannon-Wiener diversity index (H′), Simpson index (λ), and evenness index (J′) of soil nematode communities showed no significant differences among treatments on May 20. However, treatment D showed a high diversity index, dominance, and evenness index on July 4, and the highest Simpson index and evenness index on August 22. Nematode maturity index (MI) was in the range of 2.15—3.63; nematode channel ratio (NCR) was 0.28—0.57 for the three sampling times in each treatment. Thus, theH′,λ,J′,MI, and NCR of the nematodes varied with time. These results suggest that sole cropping or mixed cropping of transgenic canola with wild brown mustard had no short-term impact on the soil nematode community.

transgenic canola; wild brown mustard; interspecific mixed cropping; soil nematodes; community structure

中央級(jí)公益性科研院所基本科研業(yè)務(wù)專項(xiàng)(2013-YSKY-16); 國(guó)家自然科學(xué)基金青年項(xiàng)目(31200288)

2014-01-21;

日期:2014-11-19

10.5846/stxb201401210157

*通訊作者Corresponding author.E-mail: lijsh@craes.org.cn

肖能文, 劉勇波, 陳群英, 付夢(mèng)娣, 葉瑤, 李俊生.轉(zhuǎn)基因油菜與野芥菜混作對(duì)土壤線蟲(chóng)群落的影響.生態(tài)學(xué)報(bào),2015,35(18):6189-6198.

Xiao N W, Liu Y B, Chen Q Y, Fu M D, Ye Y, Li J S.Effect of interspecific mixed cropping between transgenic oilseed rape and wild mustard on soil nematode communities.Acta Ecologica Sinica,2015,35(18):6189-6198.