鄧家軍,張仕祥,張富生,張艷玲,胡 鋒,李輝信,*

1 南京農(nóng)業(yè)大學(xué)資源與環(huán)境科學(xué)學(xué)院土壤生態(tài)實(shí)驗(yàn)室,南京 210095 2 江西省農(nóng)產(chǎn)品質(zhì)量安全檢測中心,南昌 330046 3 中國煙草總公司鄭州煙草研究院,鄭州 450001

煙草幼苗根系分泌自毒物質(zhì)種類及PAEs對(duì)根系抗氧化性能的影響

鄧家軍1,2,張仕祥3,張富生2,張艷玲3,胡 鋒1,李輝信1,*

1 南京農(nóng)業(yè)大學(xué)資源與環(huán)境科學(xué)學(xué)院土壤生態(tài)實(shí)驗(yàn)室,南京 210095 2 江西省農(nóng)產(chǎn)品質(zhì)量安全檢測中心,南昌 330046 3 中國煙草總公司鄭州煙草研究院,鄭州 450001

采用GC-MS技術(shù)鑒定水培煙草Burley及K326在幼苗期不同生長階段的根系分泌物；并用不同濃度鄰苯二甲酸二丁酯(DBP)、鄰苯二甲酸二異辛酯(DIOP)溶液澆灌盆栽煙草幼苗,研究其根系抗氧化性能變化。結(jié)果如下：(1)Burley根系分泌物主要有3類化合物,其中自毒物質(zhì)鄰苯二甲酸酯(PAEs)在二葉齡期、四葉齡期、六葉齡期的相對(duì)含量分別為7.6%、0.3%、未檢出；而K326根系分泌物主要有9類化合物,PAEs在二葉齡期、四葉齡期、六葉齡期的相對(duì)含量分別為35.6%、51.3%、2.2%。(2)濃度高于0.1 mmol/L的PAEs使根中超氧陰離子自由基產(chǎn)生的速率顯著(P<0.05)增加；隨著DIOP及DBP濃度的增加,超氧化物歧化酶、過氧化氫酶活性增加,在0.5 mmol/L時(shí)達(dá)到最大,然后隨著處理濃度的增加而下降。丙二醛的濃度隨著這兩種PAEs處理濃度的增加而增大。結(jié)果表明：煙草根系分泌的自毒物質(zhì)PAEs達(dá)到0.5 mmol/L時(shí),能降低根系的抗氧化性能,造成根尖細(xì)胞膜系統(tǒng)的氧化損傷,引起根吸收功能等一系列生理生化變化,并最終表現(xiàn)出自毒作用。

煙草；根系分泌物；自毒物質(zhì)；抗氧化酶活性

煙草(NicotianatabacumL.)屬于茄科(Solanaceae),煙草屬(Nicotiana)植物,是我國重要的經(jīng)濟(jì)作物之一。但隨著耕地面積減少,加之種植制度、經(jīng)濟(jì)效益等因素影響,我國煙草連作問題相當(dāng)突出。煙草長期連作,其根系分泌的某些物質(zhì)在土壤中累積到一定濃度時(shí),會(huì)對(duì)作物本身產(chǎn)生毒害[1-2],這就是煙草的自毒作用。自毒作用直接危害植株正常的生長發(fā)育,使煙葉產(chǎn)量及品質(zhì)下降[3-4],是煙草產(chǎn)生連作障礙的主要原因之一。

植物的自毒作用通常發(fā)生在幼苗階段,因?yàn)橛酌绾艽嗳?容易受到自毒物質(zhì)的影響[5]。目前,直接評(píng)價(jià)植物自毒作用的文獻(xiàn)較少：Jia等[6]研究了連作煙草產(chǎn)生的自毒物質(zhì)對(duì)種子活力、幼苗根系活力、根系的長度及數(shù)量、幼苗高度及生物量的影響。Sun等[7]研究了番茄的自毒物質(zhì)對(duì)其生長、抗氧化酶活力及光合作用的影響。劉蘋等[8]研究結(jié)果顯示,當(dāng)土壤中自毒物質(zhì)長鏈脂肪酸含量達(dá)到一定濃度時(shí),對(duì)花生植株的生長和土壤酶活性能產(chǎn)生顯著地抑制作用。郭亞利等[9]研究表明,煙草根系分泌物能顯著降低其幼苗根系活力及對(duì)營養(yǎng)元素的吸收,且分泌物的不同組分的抑制效應(yīng)存在顯著差異,推測煙草根系分泌物中含有多種自毒物質(zhì)。Yeasmin等[10]在連續(xù)移栽系統(tǒng)中以不同輪作方式培養(yǎng)兩種蘆筍(AsparagusofficinalisL.),發(fā)現(xiàn)所產(chǎn)生的自毒物質(zhì)(草酸、琥珀酸和酒石酸等)對(duì)蘆筍根和芽的生長及營養(yǎng)物質(zhì)(P、N、K、Ca、Mg)的吸收產(chǎn)生不同程度的抑制作用。而環(huán)境中的pH值降低更易使根細(xì)胞膜脂受到傷害[11],Mazzoleni等[12]認(rèn)為植物殘?bào)w對(duì)同物種的抑制作用有兩種：一種是由殘?bào)w腐解出的不穩(wěn)定的自毒物質(zhì)所產(chǎn)生的非特異性抑制,另一種是由殘?bào)w的DNA發(fā)起的特異性抑制。但煙草幼苗期各生長階段自毒物質(zhì)的分泌特征及自毒機(jī)制尚不明確。本研究擬對(duì)兩個(gè)煙草品種Burley及K326幼苗期不同生長階段的根系分泌物進(jìn)行分離鑒定,并研究主要自毒物質(zhì)對(duì)土培煙草根系中超氧陰離子產(chǎn)生的速率、抗氧化酶活性、脂質(zhì)過氧化等的影響,從生理生化方面闡明煙草自毒作用可能的生理機(jī)制。此研究將對(duì)深入研究煙草根系分泌物中自毒物質(zhì)的作用機(jī)理、減弱乃至消除煙草的自毒作用等方面具有重要意義。

1 材料與方法

1.1 供試植物

煙草Burley和K326的種子,由中國煙草總公司鄭州煙草研究院提供。

1.2 幼苗期煙草根系分泌物的鑒定

1.2.1 收集根系分泌物

預(yù)培養(yǎng)煙草Burley及K326的種子,出芽后,分別放入裝有適量無瓊脂MS培養(yǎng)基的玻璃培養(yǎng)皿中。無瓊脂MS 培養(yǎng)基成分為：

(1)大量元素 1.65 g NH4NO3,1.9 g KNO3,0.37 mg MgSO4·7H2O,0.17 g KH2PO4,0.44 g CaCl2·2H2O；

(2)微量元素 22.3 mg MnSO4·4H2O,0.83 mg KI,0.025 mg CuSO4·5H2O,6.25 mg H3BO5,0.025 mg CoCl·6H2O,8.65 mg ZnSO4·7H2O,0.25 mg Na2MoO4·2H2O；

鐵鹽 27.8 mg FeSO4·7H2O,37.3 mg Na2EDTA；

蔗糖30 g于1 L水中,用Tris調(diào)節(jié)pH至5.70,高壓滅菌20 min[13]。

每個(gè)培養(yǎng)皿放入20顆,每種煙草設(shè)3個(gè)平行。然后將培養(yǎng)皿放入22—26 ℃、16 h光照/8 h黑暗的光照培養(yǎng)箱中,每天更換1次培養(yǎng)基。當(dāng)幼苗生長至2葉齡期、4葉齡期及6葉齡期時(shí),分別將3個(gè)時(shí)期的煙苗從培養(yǎng)皿中的培養(yǎng)基中取出,沖洗干凈后,放入去離子水中。分泌24 h后再將煙苗放回培養(yǎng)基中,收集含有根系分泌物的去離子水。

1.2.2 pH值測定

采用pH值測定儀(BPH-200D, 上海益?zhèn)惌h(huán)境科技有限公司),測定收集煙草幼苗各葉齡期根系分泌物時(shí)的培養(yǎng)基pH值。

1.2.3 測試樣品的制備

取50 mL含有煙草幼苗根系分泌物的水樣及100 mL 正己烷,倒入250 mL分液漏斗中,振蕩30 min,靜置。分層后,將有機(jī)相通過裝有無水硫酸鈉的玻璃漏斗,接入旋轉(zhuǎn)蒸發(fā)瓶中。水相再萃取1次,有機(jī)相轉(zhuǎn)入旋轉(zhuǎn)蒸發(fā)瓶,然后于45 ℃水浴中旋轉(zhuǎn)蒸發(fā)至干,正己烷定容至2 mL,過0.22 μm濾膜,經(jīng)硅烷化處理后于 -20 ℃下保存待測。

1.2.4 GC-MS檢測

檢測儀器：氣質(zhì)聯(lián)用儀450GC-320MS(Bruker Daltonics Inc., USA)。

色譜條件：DB-5MS 色譜柱(Agilent 19091S-433, HP-5MS, 5% Phenyl Methyl Siloxane,30 m×0.25 mm×0.25 μm,美國安捷倫公司)；進(jìn)樣口溫度250 ℃；程序升溫：柱溫 70 ℃保持4 min,以5 ℃/min升溫至130 ℃,保持5 min,再以10 ℃/min升至250 ℃,保持15 min；質(zhì)譜接口溫度為270 ℃；載氣為He,流速為1.0 mL/min。

質(zhì)譜條件：EI源,-70 eV；掃描范圍為m/z 50—500 amu；掃描時(shí)間為0.5 s；離子源溫度為200 ℃；四級(jí)桿溫度為150 ℃；傳輸線溫度220 ℃。

進(jìn)樣方式：不分流進(jìn)樣,進(jìn)樣量為1 μL。

與標(biāo)準(zhǔn)質(zhì)譜譜庫Wiley 7n.L 及NIST 05.L比對(duì),確定各種化合物,采用面積歸一化法(JY/T003—1996)計(jì)算其相對(duì)含量。

1.3 鄰苯二甲酸酯(PAEs)對(duì)煙草根系抗氧化酶活性的影響

將6片真葉的K326幼苗移栽至直徑11 cm,1.5 L的瓦盆中,內(nèi)裝有取自河南省鄭州市郊土壤(表1)。每盆中移栽1株,隨機(jī)置于溫室中：每天光照16 h,有效輻射為320 μmol m-2s-1,溫度范圍為35 ℃/25 ℃(晝/夜),相對(duì)濕度為60%[14]。在幼苗正常生長后,每星期澆100 mL用鄰苯二甲酸二丁酯(DBP)、鄰苯二甲酸二異辛酯(DIOP) (優(yōu)級(jí)純,Aladdin試劑有限公司,中國上海) 分別配制0、0.01、0.1、0.5、1.0、5.0 mmol/L及10.0 mmol/L 6個(gè)濃度梯度溶液,濃度參照煙草連作土壤中檢測的濃度設(shè)置的[15],每個(gè)梯度有3個(gè)重復(fù),連續(xù)澆5周。然后采集植株根系,洗凈、晾干后備用。

表1 供試土壤的基本理化性質(zhì)

1.4 數(shù)據(jù)分析

采用SPSS 16.0 (SPSS Inc., Chicago, USA) 的One-way ANOVA分析不同處理之間的差異性(P<0.05)。同一梯度3個(gè)重復(fù),用于計(jì)算平均值和標(biāo)準(zhǔn)偏差。

2 結(jié)果

2.1 根系分泌物的鑒定

2.1.1 Burley幼苗期根系分泌物

Burley幼苗期根系分泌物主要有3類(表2)。其中2葉齡期主要分泌2類物質(zhì)：

(1)烷烴；(2) 鄰苯二甲酸酯: 鄰苯二甲酸二異丁酯(DIBP)、鄰苯二甲酸3-己基異丁酯。

Burley 4葉齡期主要分泌3類物質(zhì)：(1)烷烴；(2) 鄰苯二甲酸酯: 鄰苯二甲酸異丁基壬酯；(3) 2-甲氧基苯酚。

Burley 6葉齡期主要分泌2類物質(zhì)：(1) 烷烴；(2)鄰苯二甲酸酯: 鄰苯二甲酸4-庚基異丁酯。

表2 Burley幼苗不同階段的根系分泌物

ND: not detected

2.1.2 K326幼苗期根系分泌物

K326幼苗期根系分泌物主要有9類(見表3)。其中,2葉齡期根系分泌物中主要含有 7類物質(zhì)：(1) 烷烴；(2) 鄰苯二甲酸酯：DIOP、鄰苯二甲酸二正辛酯(DOP)、鄰苯二甲酸丁基環(huán)己酯 (BCHP)；(3) 二十三烷酸甲酯；(4) 十二烷基-三苯基溴化磷；(5) 1,4-二醇, 2, 3-二甲基-5-三氟甲基苯；(6) 十一丁基亞硫酸酯；(7) 反油酸-芐基二甲硅基脂。

表3 K326幼苗不同階段的根系分泌物

ND: not detected

K326 4葉齡期根系分泌物中主要含有3類物質(zhì)：(1) 烷烴；(2) 鄰苯二甲酸酯： DIBP、鄰苯二甲酸丁基環(huán)己酯、鄰苯二甲酸丁基庚酯； (3) 檸檬酸三乙酯。

K326 6葉齡期主要含有4類物質(zhì)：(1) 烷烴；(2) 鄰苯二甲酸酯：DBP、鄰苯二甲酸丁基己基酯；(3) 2-十九烷酮-2,4-二硝基苯肼；(4)十一丁基亞硫酸酯。

常見的自毒物質(zhì)主要有水溶性有機(jī)酸、直鏈醇、脂肪族醛與酮、簡單不飽和內(nèi)酯、長鏈脂肪酸與多炔、蒽醌與復(fù)合醌、簡單酚、苯甲酸及其衍生物、肉桂酸及其衍生物、香豆素類、類黃酮、單寧、類萜及甾類化合物、氨基酸及多肽、生物堿與氰醇、硫化物與芥子油苷、嘌呤及核苷等[20-22]。在Burley幼苗期根系分泌物中具有自毒作用的是鄰苯二甲酸酯及二甲氧基苯酚。鄰苯二甲酸酯在二葉齡期及四葉齡期所占的比率分別為7.6%及0.3%,而在六葉齡期未檢出鄰苯二甲酸酯。在K326幼苗期根系分泌物中具有自毒作用的是鄰苯二甲酸酯,分別在二葉齡期、四葉齡期、六葉齡期所占的比率為35.6%、51.3%、2.2% 。具有潛在自毒作用的是烷酸酯、檸檬酸三乙酯等。

2.2 煙草幼苗不同葉齡期培養(yǎng)基pH值

煙草幼苗不同葉齡期培養(yǎng)基pH值如表4。本試驗(yàn)用于培養(yǎng)煙草幼苗的MS無瓊脂培養(yǎng)基pH為5.70。Burley與K326兩種煙草幼苗各個(gè)葉齡期培養(yǎng)基的pH值都比培養(yǎng)前低。兩種煙草幼苗隨著葉齡期的增加,培養(yǎng)基的pH值升高,但都比培養(yǎng)前低。

表4 Burley與K326幼苗不同葉齡期培養(yǎng)基的pH值

表中不同字母表示煙草幼苗不同葉齡期培養(yǎng)基pH值在P<0.05水平上差異顯著,數(shù)據(jù)為平均值±標(biāo)準(zhǔn)差(n=3)

2.3 兩種肽酸酯對(duì)煙草根系抗氧化活性的影響

2.3.2 兩種肽酸酯對(duì)SOD活性的影響

煙草根系SOD活性隨著DBP濃度的增加而升高。而SOD活性隨著DIOP濃度的增加分別在0.5 mmol/L時(shí)達(dá)到最大,然后隨著DIOP濃度的增大而降低(圖2)。兩種試劑處理對(duì)SOD活性影響有顯著性差異(P<0.05)。

圖1 兩種肽酸酯對(duì)煙草根系產(chǎn)生速率的影響Fig.1 Effects of two phthalate esters on rates of generation in tobacco roots圖中不同字母表示相同處理不同PAE濃度在P<0.05水平上差異顯著,數(shù)據(jù)為平均值±標(biāo)準(zhǔn)差(n=3)；DBP：鄰苯二甲酸二丁酯dibutyl phthalate；DIOP：鄰苯二甲酸二異辛酯 diisooctyl phthalate

圖2 兩種肽酸酯對(duì)煙草根系中SOD活性的影響Fig.2 Effect of two phthalate esters on SOD activity in tobacco roots圖中不同字母表示相同處理不同PAE濃度在P<0.05水平上差異顯著,數(shù)據(jù)為平均值±標(biāo)準(zhǔn)差(n=3)；DBP：鄰苯二甲酸二丁酯 dibutyl phthalate；DIOP：鄰苯二甲酸二異辛酯 diisooctyl phthalate

2.3.3 兩種肽酸酯對(duì)CAT活性的影響

煙草根系CAT活性隨著DIOP濃度的增加而急劇升高。可是,隨著DBP濃度的增加,CAT的活性在0.5 mmol/L時(shí)達(dá)到最大,然后下降至與對(duì)照相似的水平(圖3)。兩種試劑處理對(duì)CAT活性影響有顯著性差異(P<0.05)。

2.3.4 兩種肽酸酯對(duì)煙草根系MDA含量的影響

圖3 兩種肽酸酯對(duì)煙草根系中CAT活性的影響Fig.3 Effects of two phthalate esters on CAT activity in tobacco roots圖中不同字母表示相同處理不同PAE濃度在P<0.05水平上差異顯著,數(shù)據(jù)為平均值±標(biāo)準(zhǔn)差(n=3)；DBP (dibutyl phthalate)：鄰苯二甲酸二丁酯；DIOP (diisooctyl phthalate)：鄰苯二甲酸二異辛酯

圖4 兩種肽酸酯對(duì)煙草根系中MDA 濃度的影響Fig.4 Effects of two phthalate esters on the concentration of MDA in tobacco roots圖中不同字母表示相同處理不同PAE濃度在P<0.05水平上差異顯著,數(shù)據(jù)為平均值±標(biāo)準(zhǔn)差(n=3)；DBP：鄰苯二甲酸二丁酯 dibutyl phthalate：；DIOP：鄰苯二甲酸二異辛酯 diisooctyl phthalate

3 討論

自毒作用是個(gè)體間為了利用有限的資源而進(jìn)行的一種特殊形式的種內(nèi)競爭,結(jié)果會(huì)導(dǎo)致競爭個(gè)體適合度的下降,通常表現(xiàn)為密度制約或自疏現(xiàn)象[23]。這將會(huì)調(diào)解種群數(shù)量,使種群由較健康、有活力、更適合周圍環(huán)境、避免種內(nèi)競爭的較大個(gè)體組成,這樣的種群有利于自然選擇[24]。

4 結(jié)論

煙草Burley及K326在幼苗期的不同葉齡期根系分泌物的種類及相對(duì)含量是不同的：Burley幼苗期根系分泌物主要有3種,K326幼苗期根系分泌物主要有9種。兩種煙草不同葉齡期根系分泌物的種類和相對(duì)含量有顯著性差別。而且這兩種煙草都能產(chǎn)生自毒物質(zhì)PAEs,及潛在自毒物質(zhì)烷酸酯等。在煙草幼苗在生長過程中,所產(chǎn)生的PAEs的相對(duì)含量逐漸減少。

[1] Walker T S, Bais H P, Grotewold E, Vivanco J M. Root exudation and rhizosphere biology. Plant Physiology, 2003, 132(1): 44-51.

[2] Xie X N, Kusumoto D, Takeuchi Y, Yoneyama K, Yamada Y, Yoneyama K. 2′-epi-orobanchol and solanacol, two unique strigolactones, germination stimulants for root parasitic weeds, produced by tobacco. Journal of Agricultural and Food Chemistry, 2007, 55(20): 8067-8072.

[3] 晉艷, 楊宇虹, 段玉琪, 龍玉華, 葉成碧. 烤煙連作對(duì)煙葉產(chǎn)量和質(zhì)量的影響研究初報(bào). 煙草科技, 2002, (1): 41-45.

[4] 鄧陽春, 黃建國. 長期連作對(duì)烤煙產(chǎn)量和土壤養(yǎng)分的影響. 植物營養(yǎng)與肥料學(xué)報(bào), 2010, 16(4): 840-845.

[5] Weir T L, Park S W, Vivanco J M. Biochemical and physiological mechanisms mediated by allelochemicals. Current Opinion in Plant Biology, 2004, 7(4): 472-479.

[6] Jia Z H, Yi J H, Su Y R, Shen H. Autotoxic substances in the root exudates from continuous tobacco cropping. Allelopathy Journal, 2011, 27(1): 87-96.

[7] Sun Y Y, Jiang G Y, Wei X C, Liu J G. Autotoxicity effects of soils continuously cropped with tomato. Allelopathy Journal, 2011, 28(2): 135-144.

[8] 劉蘋, 趙海軍, 仲子文, 孫明, 龐亞群, 馬征, 萬書波. 三種根系分泌脂肪酸對(duì)花生生長和土壤酶活性的影響. 生態(tài)學(xué)報(bào), 2013, 33(11): 3332-3339.

[9] 郭亞利, 李明海, 吳洪田, 袁玲, 黃建國. 烤煙根系分泌物對(duì)烤煙幼苗生長和養(yǎng)分吸收的影響. 植物營養(yǎng)與肥料學(xué)報(bào), 2007, 13(3): 458-463.

[10] Yeasmin R, Nakamatsu K, Matsumoto H, Motoki S, Nishihara E, Yamamoto S. Inference of allelopathy and autotoxicity to varietal resistance of asparagus (AsparagusofficinalisL.), Australian Journal of Crop Science, 2014, 8(2): 251-256.

[11] Liu P, Wan S B, Jiang L H, Wang C B, Liu Z H, Zhao H J, Yu S F, Yang L. Autotoxic potential of root exudates of peanut (ArachishypogaeaL.). Allelopathy Journal, 2010, 26(2):197-205.

[12] Mazzoleni S, Bonanomi G, Incerti G, Chiusano M L, Termolino P, Mingo A, Senatore M, Giannino F, Cartenì F, Rietkerk M, Lanzotti V. Inhibitory and toxic effects of extracellular self-DNA in litter: a mechanism for negative plant-soil feedbacks?. New Phytologist, 2015, 205(3): 1195-1210.

[13] Murashige T, Skoog F. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiologia Plantarum, 1962, 15(3): 473-497.

[14] Walch-Liu P, Neumann G, Bangerth F, Engels C. Rapid eects of nitrogen form on leaf morphogenesis in tobacco. Journal of Experimental Botany, 2000, 51(343): 227-237.

[15] Yi J H, Jia Z H, Lin Q, Lv H Z, Shen H. Allelopathic effects of decaying tobacco leaves on tobacco seedlings. Allelopathy Journal, 2012, 29(1): 51-61.

[16] Dhindsa R S, Matowe W. Drought tolerance in two mosses: correlated with enzymatic defence against lipid peroxidation. Journal of Experimental Botany, 1981, 32(1): 79-91.

[17] Beers R F Jr, Sizer I W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. The Journal of Biological Chemistry, 1952, 195(1): 133-140.

[18] Heath R L, Packer L. Photoperoxidation in isolated chloroplast. I. Kinetics and stochiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics, 1968, 125(1): 189-198.

[19] Elstner E F, Heupel A. Formation of hydrogen peroxide by isolated cell walls from horseradish (ArmoracialapathifoliaGilib.). Planta, 1976, 130(2): 175-180.

[20] Tang C S, Takenaka T. Quantitation of a bioactive metabolite in undisturbed rhizosphere-benzyl isothiocyanate FromCaricaPapayaL. Journal of Chemical Ecology, 1983, 9 (8):1247-1253.

[21] Rice E L. Allelopathy. 2nd ed. Orlando, Florida: Academic Press, 1984.

[22] Inderjit. Plant phenolics in allelopathy. The Botanical Review, 1996, 62(2):186-202.

[23] Weller D E. The interspecific size-density relationship among crowded plant stands and its implications for the -3/2 power rule of self-thinning. The American Naturalist. 1989, 133(1): 20-41.

[24] Singh H P, Batish D R, Kohli R K. Autotoxicity: concept, organisms, and ecological significance. Critical Reviews in Plant Sciences, 1999, 18(6): 757-772.

[25] Inderjit, Callaway R M, Vivanco J M. Can plant biochemistry contribute to understanding of invasion ecology? Trends in Plant Science, 2006, 11(12): 574-580.

[26] Kato-Noguchi H. Allelopathic substance in rice root exudates: Rediscovery of momilactone B as an allelochemical. Journal of Plant Physiology, 2004, 161(3): 271-276.

[27] El-Halmouch Y, Benharrat H, Thalouarn P. Effect of root exudates from different tomato genotypes on broomrape (O.aegyptiaca) seed germination and tubercle development. Crop Protection, 2006, 25(5): 501-507.

[28] Neumann G, Massonneau A, Martinoia E, R?mheld V. Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin. Planta, 1999, 208(3): 373-382.

[29] Tyler G, Str?m L. Differing organic acid exudation pattern explains calcifuge and acidifuge behaviour of plants. Annals of Botany, 1995, 75(1): 75-78.

[30] Schumacher W J, Thill D C, Lee G A. Allelopathic potential of wild oat (Avenafatua) on spring wheat (Triticumaestivum) growth. Journal of Chemical Ecology, 1983, 9(8): 1235-1245.

[32] 屠振力, 鐘儒杰. 環(huán)境激素鄰苯二甲酸丁基芐酯對(duì)家蠶生殖的影響. 生態(tài)學(xué)報(bào), 2014, 34(19): 5470-5476.

[33] Alías J C, Sosa T, Escudero J C, Chaves N. Autotoxicity against germination and seedling emergence inCistusladaniferL. Plant and Soil, 2006, 282(1/2): 327-332.

[34] Ma Y Q, Jia J N, An Y, Wang Z, Mao J C. Potential of some hybrid maize lines to induce germination of Sunflower Broomrape. Crop Science, 2013, 53(1): 260-270.

[35] Foyer C H, Descourvières P, Kunert K J. Protection against oxygen radicals: an important defence mechanism studied in transgenic plants. Plant, Cell & Environment, 1994, 17(5): 507-523.

[36] Halliwell B, Gutteridge J M C. Free radicals in biology and medicine. 4th ed. Oxford: Glarendon Press, 1985.

[37] Papadakis A K, Roubelakis-Angelakis K A. The generation of active oxygen species differs in tobacco and grapevine mesophyll protoplasts. Plant Physiology, 1999, 121(1): 197-206.

[38] 鄧家軍, 胡繼偉, 李繼新, 蘇賢坤, 黃先飛, 劉峰. 重金屬離子對(duì)烤煙葉片中銅鋅超氧化物歧化酶活性的影響. 中國煙草學(xué)報(bào), 2010, 16(3): 1-6.

[39] Mobin M, Khan N A. Photosynthetic activity, pigment composition and antioxidative response of two mustard (Brassicajuncea) cultivars differing in photosynthetic capacity subjected to cadmium stress. Journal of Plant Physiology, 2007, 164(5): 601-610.

[40] Van Camp W, INZé D, Van Montagu M. The regulation and function of tobacco superoxide dismutases. Free Radical Biology and Medicine, 1997, 23(3): 515-520.

[41] Willekens H, Chamnongpol S, Davey M, Schraudner M, Langebartels C, Van Montagu M, INZé D, Van Camp W. Catalase is a sink for H2O2and is indispensable for stress defense in C3plants. the EMBO Journal, 1997, 16(16): 4806-4816.

[42] Bais H P, Vepachedu R, Gilroy S, Callaway R M, Vivanco J M. Allelopathy and exotic plant invasion: from molecules and genes tospecies interactions. Science, 2003, 301(5638): 1377-1380.

[43] 李志萍, 張文輝, 崔豫川. NaCl和Na2CO3脅迫對(duì)栓皮櫟種子萌發(fā)及幼苗生長的影響. 生態(tài)學(xué)報(bào), 2015, 35(3): 742-751.

[44] Foyer C H, Lopez-Delgado H, Dat J F, Scott I M. Hydrogen peroxide-and glutathione-associated mechanisms of acclimatory stress tolerance and signalling. Physiologia Plantarum, 1997, 100(2): 241-254.

[45] Baziramakenga R, Leroux G D, Simard R R. Effects of benzoic and cinnamic acids on membrane permeability of soybean roots. Journal of Chemical Ecology, 1995, 21(9): 1271-1285.

[46] Ye S F, Zhou Y H, Sun Y, Zou L Y, Yu J Q. Cinnamic acid causes oxidative stress in cucumber roots, and promotes incidence ofFusariumwilt. Environmental and Experimental Botany, 2006, 56(3): 255-262.

[47] Yu J Q, Matsui Y. Effects of root exudates of cucumber (Cucumissativus) and allelochemicals on ion uptake by cucumber seedlings. Journal of Chemical Ecology, 1997, 23(3): 817-827.

Autotoxins exuded from roots and the effects of PAEs on antioxidant capacity in roots of tobacco seedlings

DENG Jiajun1,2, ZHANG Shixiang3, ZHANG Fusheng2, ZHANG Yanling3, HU Feng1, LI Huixin1,*

1SoilEcologyLaboratory,CollegeofResourcesandEnvironmentalSciences,NanjingAgriculturalUniversity,Nanjing210095,China2TestingCenterofAgro-ProductQualityandSafetyofJiangxiProvince,Nanchang330046,China3ZhengzhouTobaccoResearchInstitute,ChinaNationalTobaccoCorporation,Zhengzhou450001,China

Root exudates of two varieties (Burley and K326) of tobacco seedlings cultured in a hydroponic medium were collected, and identified using gas chromatography-mass spectrometry (GC-MS). Further, the effects of different concentrations of dibutyl phthalate (DBP) and diisooctyl phthalate (DIOP) on root antioxidant capacity were assessed using pot culture experiments. The results revealed three main compounds in Burley root exudates. The relative content of autotoxin phthalate esters (PAEs) at the two-, four-, and six-leaf stages were 7.6%, 0.3%, and not detected, respectively. However, there were nine main compounds in K326 root exudates. The relative PAE content at the two-, four-, and six-leaf stages were 35.6%, 51.3%, and 2.2%, respectively. PAEs such as DIOP and DBP were identified as the major autotoxins in root exudates of both tobacco seedlings. The rate of superoxide anion radical generation in roots significantly increased at concentrations greater than 0.1 mmol/L DIOP and DBP (P< 0.05). Both superoxide dismutase and catalase activities increased with increasing DIOP concentrations, with a peak at 0.5 mmol/L, and subsequently decreased at higher concentrations. Accumulated malondialdehyde concentrations increased with increasing DIOP and DBP concentrations, and the magnitude of malondialdehyde content was DIOP > DBP, which indicated the order of their toxic effect. Finally, our findings also revealed that when PAEs reached 0.5 mmol/L, they decreased the antioxidant capacity of the root system, initiated oxidative damage of the root cell membrane system, and caused a further decrease in root absorption and mineral metabolism, and thereby led to autotoxicity in the tobacco plants.

tobacco; root exudates; autotoxins; antioxidant enzyme activity

中國煙草總公司鄭州煙草研究院科學(xué)技術(shù)合作項(xiàng)目(YKJSS201101)

2015-08-02;

日期:2016-06-13

10.5846/stxb201508021630

* 通訊作者Corresponding author.E-mail: huixinli@njau.edu.cn

鄧家軍,張仕祥,張富生,張艷玲,胡鋒,李輝信.煙草幼苗根系分泌自毒物質(zhì)種類及PAEs對(duì)根系抗氧化性能的影響.生態(tài)學(xué)報(bào),2017,37(2):495-504.

Deng J J, Zhang S X, Zhang F S, Zhang Y L, Hu F, Li H X.Autotoxins exuded from roots and the effects of PAEs on antioxidant capacity in roots of tobacco seedlings.Acta Ecologica Sinica,2017,37(2):495-504.