连作障碍与根际微生态研究 Ⅰ. 根系分泌物及其生态效应

作物、蔬菜、果树以及苗木长期连作后,皆出现生长衰退和产量降低。许多研究结果表明,连作条件下土壤生态环境对植物生长有很大的影响,尤以植物残体与病原微生物的分解产物,对植物有致毒作用,并影响植物根系分泌物正常代谢,以致于发生自毒作用。本文围绕根系分泌物与根际微生态的相互关系,系统地介绍连作障碍条件下,影响根系分泌物的环境因素(土壤空气、湿度、养分与微生物)、活性物质(自身毒素、残体分解物、微生物产生毒素)、土壤病原菌等的根际效应,为深入研究根系分泌物与连作障碍的相互作用机制提供启示。     

连作障碍与根际微生态研究Ⅰ.根系分泌物及其生态效应

作物、蔬菜、果树以及苗木长期连作后,皆出现生长衰退和产量降低．许多研究结果表明,连作条件下土壤生态环境对植物生长有很大的影响,尤以植物残体与病原微生物的分解产物,对植物有致毒作用,并影响植物根系分泌物正常代谢,以致于发生自毒作用．本文围绕根系分泌物与根际微生态的相互关系,系统地介绍连作障碍条件下,影响根系分泌物的环境因素（土壤空气、湿度、养分与微生物）、活性物质（自身毒素、残体分解物、微生物产生毒素）、土壤病原菌等的根际效应,为深入研究根系分泌物与连作障碍的相互作用机制提供启示．     

大量元素胁迫下南瓜化感作用研究

研究采用水培实验、生物测试及室内分析相结合的方法,研究在不同大量元素(N、P、K、Ca、S、Mg)营养胁迫下幼苗期南瓜根系分泌物及其提取物对南瓜幼苗生长的化感作用。结果表明：南瓜具有自毒作用,不同大量营养元素胁迫对其胚根和胚芽生长均产生了抑制作用。不同的营养条件对其根系分泌物的影响不同,营养元素的合理搭配可以减轻自身的自毒作用。南瓜根系分泌物经乙酸乙酯提取的活性有机物埘南瓜胚根牛长亦表现出抑制作用,且抑制作用程度较大。南瓜幼苗期根系十物质水浸液巾存在着抑制南瓜幼苗生长的化感物质：筛选出南瓜根系分泌物对自身幼苗生长产生抑制作用最弱的大量元素水平优化组合,为南瓜的营养胁迫机理提供依据。     

南瓜组培根根系分泌物的化感效应研究

使用组织培养技术、生物测试法及室内分析相结合的方法研究了南瓜组培根在不同生长时期与不同营养谐迫下（利用正交设计调节标准B₅培养基中的大量元素、微量元素、有机质和激素）根分泌物对南瓜、萝卜和小麦3种受体的种子萌发及幼苗生长的化感作用.结果表明,南瓜具有自毒作用,南瓜组培根根系分泌物对受体幼苗生长的抑制作用呈现出高抑低促的作用表型;南瓜组培根根系分泌物产生化感作用的活性与南瓜组培根生长速度有关,南瓜组培根根系分泌物的生长抑制活性以15～17 d即生长速度指数末期为最高,21 d生物量最大时的南瓜组培根根系分泌物的生长抑制活性最低;营养元素的改变明显影响了南瓜根系分泌物的产生,并通过筛选出能诱导南瓜组培根对受体具有强烈抑制作用的大量元素、微量元素、维生素和激素种类和含量的最优化组合,为研究南瓜化感作用营养胁迫机理提供依据.     

咖啡根系分泌物对嫁接后植株生长及叶片保护酶活性的影响

咖啡根系分泌物引起的自毒作用是造成咖啡连作障碍的主要原因,而种间嫁接是抑制咖啡自毒的有效方式。为探明嫁接抑制自毒机制,采用盆栽试验研究咖啡主栽品种根系分泌物对嫁接植株的化感效应。结果表明:根系分泌物抑制了咖啡自根苗生长,且随着根系分泌物浓度升高,抑制作用增强;低浓度根系分泌物能促进咖啡嫁接苗生长,经根系分泌物处理后的嫁接苗株高、根长、地上部干重和根干重分别较未处理的对照嫁接苗平均增加了22.15%、6.52%、44.80%和20.67%,根系活力、叶片保护酶活性也明显增加;高浓度根系分泌物虽未能促进嫁接苗生长,但嫁接苗较自根苗生长优势显著,经根系分泌物处理后嫁接苗POD、CAT和SOD活性分别较自根苗增加了60.61%、27.27%和18.24%,MDA含量显著减少,根系活力显著增加。因此,在根系分泌物处理下,嫁接苗能通过提高自身保护酶活性等方式,促进植株生长或使植株生长所受抑制减少。     

凤丹(Paeonia ostii T.)自毒物质的检测及其作用机制

药用牡丹的种植中存在明显的连作障碍现象,为探讨其机制,利用已萌发长根的凤丹(Paeonia ostii)种子对凤丹根际土壤和根的水浸提液进行了自毒化感检测;采用高效液相色谱法(HPLC)对种植4a的凤丹根际土壤及其根皮中的化感自毒物质进行了定性和定量分析;在此基础上,利用外源法研究了盆栽凤丹幼苗对5种检出物质(阿魏酸、肉桂酸、香草醛、香豆素和丹皮酚)及其混合物在3种不同浓度下对植株生长的影响和生理响应.结果表明:(1)连续种植4a的凤丹根际土壤和根浸提液对已萌发种子根的生长有显著的抑制作用(p＜0.05),显示凤丹根分泌物具有自毒化感的潜势;(2)HPLC分析表明在凤丹根际土壤中存在5种以上的酚酸类物质 (阿魏酸、肉桂酸、香草醛、香豆素和丹皮酚),其中阿魏酸、肉桂酸、香草醛、香豆素为前人已报道的化感物质;(3)在实验室条件下,所设浓度范围内,5种物质及其混合物对凤丹幼苗的高度、根长及地下和地上生物量均有明显影响,尤其对根长和地下生物量的影响最为明显(p＜0.05),此结果与连作移栽幼苗时,根部首先发黑死亡的现象相一致;(4)凤丹幼苗的根系活力和叶绿素含量在各处理中表现出相似性,即低浓度时根系活力和叶绿素含量高,高浓度时根系活力和叶绿素含量低,低浓度的阿魏酸和香草醛对幼苗根系活力和叶绿素合成有促进作用.此结果显示自毒物质可能是通过影响根系酶活性和叶绿素合成来影响群体中其它个体的生长.本实验的结果显示栽培4a的凤丹土壤水提液具有抑制自身种子根系生长的活性,此提示栽培凤丹连作障碍可能主要与自身分泌到土壤中的酚酸类物质的自毒作用有关.     

花生植株和土壤水浸液自毒作用研究及土壤中自毒物质检测

通过盆栽试验及生物测试研究花生植株、土壤水浸液对其种子萌发和幼苗生长的自毒作用,利用单因子方差分析结合多重比较分析各处理组生物活性差异;采用液相色谱法(HPLC)对正茬、连作2、4 a和6 a的花生根际土壤中酚酸类物质进行定性和定量分析。结果表明:花生植株不同部位及根际土壤水浸液对花生种子萌发和幼苗生长均具有不同程度的抑制作用,且抑制作用具有浓度梯度效应,浓度越高抑制作用越强,16 g/10 mL水浸液的抑制作用最大。花生生长受自毒物质的影响,此影响作用强度因自毒物质的来源部位不同而存在差异。种子萌发以茎水浸液的自毒作用最为明显。但针对幼苗生长的不同测定指标其部位差异不一致,其中以茎水浸液对幼苗株高和叶面积的自毒作用最为明显,根际土壤水浸液对幼苗主根长、单株鲜重和单株干重的自毒作用最为明显。在根际土壤水浸液中鉴定到4种酚酸物质,分别为对羟基苯甲酸、香草酸、香豆酸和香豆素,其中香草酸和香豆素含量较高且变化规律性明显,在土壤中的含量随连作年限的增加而上升,连作6 a后土壤中的含量达到0.289μg/g和0.025μg/g干土。     

地黄自毒物质提取及其生物指标测定

连作障碍在药用植物地黄(Rehmannia glutinosa L.)栽培中尤其明显,同一地块上种植一茬地黄后,须经8-10a后方可再种。从怀地黄主产区河南省焦作地区采集种植1a地黄的茬后土壤,采用不同溶剂提取地黄土壤自毒物质并利用生物测试确定抑制率最强的土壤提取液作温室栽培实验以确定对植物生理指标的影响。利用高效液相色谱法(HPLC)对土壤提取液和根系分泌物进行比较,并利用高效液相色谱、傅立叶转换红外光谱(FTIR)和电喷雾离子阱质谱(ESI-MS)技术鉴定地黄连作自毒物质的化学成分。生物测试结果表明水和甲醇提取的地黄茬后土壤自毒物质对地黄生长具有最强的抑制作用;在培养器中加入浓度0.5g/mL的水和甲醇提取液对地黄胚根的抑制率分别达17%和26%,当浓度增至5.0g/mL时,抑制率增加到70%以上。盆栽结果显示甲醇提取的自毒物质导致地黄地上部叶绿素含量减少,根系活力下降,明显抑制了地黄体内超氧化物歧化酶、过氧化物歧化酶、过氧化物酶的活性,加剧了地黄膜质过氧化,以及引起地黄植体内生长素类激素含量下降。高效液相色谱法显示土壤提取液和根系分泌物具有相似图谱。电喷雾离子阱质谱分析共检测到6个特征物质:香草酸、D-甘露醇、二十六烷酸苯羟基乙酯、毛蕊花糖苷、β-谷甾醇和胡萝卜苷。     

自毒胁迫对甜瓜种子萌发与幼苗保护酶活性和转录组的影响

甜瓜自毒作用是导致其栽培实践中连作障碍严重的关键原因之一。该实验以甜瓜植株水浸提液处理模拟甜瓜自毒胁迫,通过测定自毒条件下甜瓜种子萌发、幼苗根系保护酶活性和MDA含量的变化及转录组分析,以探讨甜瓜自毒作用机理。结果显示:(1)甜瓜自毒胁迫总体上抑制了甜瓜种子萌发和后续生长,胁迫处理的阈值为0.03 g/mL。(2)甜瓜植株水浸提液处理后其幼苗根系保护酶活性和MDA含量变化非常剧烈,SOD活性表现为降-升-降趋势,POD活性先降后升,CAT活性先升后降,MDA含量持续增加。(3)转录组分析结果共鉴定出2 599个差异表达基因(differentially expressed genes, DEGs),胁迫2 d后共产生2 251个DEGs,显著多于胁迫4 d后的329个DEGs;且2 d与0 d对比有923个DEGs上调,1 328个DEGs下调,表明甜瓜幼苗在基因水平对自毒胁迫产生了积极响应。(4)相关生物信息学分析表明,自毒胁迫导致的DEGs主要与苯丙烷代谢、活性氧代谢、光合作用和植物激素信号转导有关,同时也涉及到渗透调节、膜和蛋白保护等过程;且这些DEGs主要富集于2 d与0 d,说明自毒胁迫条件下幼苗光合作用发生了改变,且这种变化主要发生在胁迫早期。(5)对6个甜瓜自毒胁迫密切相关DEGs(AP2-2、bZIP1、bZIP2、AP2-1、bHLH和HIS)的qRT-PCR分析显示,AP2-1、bHLH和HIS等3个基因在自毒胁迫2 d时表达量出现峰值,对照组AP2-2基因在2 d时表达量达到峰值,结果与转录组测序分析结果一致。研究表明,甜瓜自毒胁迫引起了其植物细胞的异常,进而会对生长和器官结构产生不良影响并诱发了大量与刺激或胁迫相关的基因差异表达,且甜瓜自毒胁迫下短期响应的基因或转录因子数量明显多于长期响应的数量,暗示甜瓜幼苗可以对自毒胁迫做出快速响应。     

番茄（Lycopersicon）的化感作用研究

采用室内生测、室外盆栽和水培相结合的研究方法,以多种受体品种来探讨番茄 的化感作用．结果表明,番茄植物不仅具有自毒作用,番茄植株的水提液对黄瓜、萝卜、生 菜、白菜、包心菜的幼苗生长均有显著的抑制作用;番茄植株的挥发物对黄瓜的生长具有 明显的抑制作用,但对绿豆、白菜、生菜及番茄自身的幼苗生长则无明显的影响;番茄移苗 后４０ ｄ之内,其根分泌物对黄瓜生长有明显抑制作用,但对生菜作用不明显认为自毒作 用是造成番茄连作障碍的原因之一,指出番茄种植应采用轮作方式,水培或大棚种植番茄 时,应避免与黄瓜间种．     

Autotoxic Ginsenosides in the Rhizosphere Contribute to the Replant Failure of Panax notoginseng

MethodsThe autotoxicities were measured using seedling emergence bioassays and root cell vigor staining. The ginsenosides in the roots, soils, and root exudates were identified with HPLC-MS.ResultsThe seedling emergence and survival rate decreased significantly with the continuous number of planting years from one to three years. The root exudates, root extracts, and extracts from consecutively cultivated soils also showed significant autotoxicity against seedling emergence and growth. Ginsenosides, including R₁, Rg₁, Re, Rb₁, Rb₃, Rg₂, and Rd, were identified in the roots and consecutively cultivated soil. The ginsenosides, Rg₁, Re, Rg₂, and Rd, were identified in the root exudates. Furthermore, the ginsenosides, R₁, Rg₁, Re, Rg₂, and Rd, caused autotoxicity against seedling emergence and growth and root cell vigor at a concentration of 1.0 µg/mL.ConclusionOur results demonstrated that autotoxicity results in replant failure of Sanqi ginseng. While Sanqi ginseng consecutively cultivated, some ginsenosides can accumulate in rhizosphere soils through root exudates or root decomposition, which impedes seedling emergence and growth.     

Autotoxicity of wheat (Triticum aestivum L.) as determined by laboratory bioassays

Wheat varietal autotoxicity and varietal allelopathy were assessed based on plant extract and root exudate bioassays under laboratory conditions. Aqueous extract of wheat differed in varietal autotoxicity and varietal allelopathy, inhibiting wheat germination by 2–21%, radicle growth by 15–30%, and coleoptile growth by 5–20%, depending on the combination of the receiver and donor. Extracts of cv Triller or cv Currawong were more allelopathic to other wheat varieties than cv Batavia and cv Federation. Triller extract was more autotoxic than Federation. Assessment of root exudates by the equal-compartment-agar-method further identified the significant differences in varietal autotoxicity and varietal allelopathy of root exudates between wheat varieties, with root exudates of Triller or Batavia showing stronger autotoxic or allelopathic effects than Currawong or Federation. The varietal autotoxicity and allelopathy of root exudates also showed a characteristic radial inhibitory pattern in the agar growth medium. These results suggest that careful selection of suitable wheat varieties is necessary in a continuous cropping system in order to minimize the negative impacts of varietal allelopathy and varietal autotoxicity. Factors affecting autotoxicity in the field and strategies in autotoxicity management are discussed. Resposible Editor: Philippe Hinsinger     

伊贝母根系分泌物自毒作用研究

采用生物测定的方法,研究了伊贝母(Fritillaria pallidifloraSchvek)根系分泌物及其主要成分1,3,5-三烯丙基-1,3,5-三嗪-2,4,6(1H,3H,5H)三酮、苯酚和二者的混合液对伊贝母生长的影响。结果表明,伊贝母根系分泌物对其种子萌发及胚根胚轴的生长有明显抑制作用,各浓度处理液对种子萌发及发芽势的影响多表现为抑制作用,随浓度升高而增强,但当超过一定浓度以后抑制作用有所下降。各浓度处理液对胚根胚轴生长的抑制作用表现为随浓度升高而增强,在较低浓度时对胚轴的生长表现为一定的促进作用。     

草莓重茬病菌的分离及其生物防治

选取河北省主要草莓产区的8个连作地块草莓病株,对草莓根部重茬病菌进行了分离、鉴定,结果表明草莓重茬病主要是由于镰刀菌属、丝核菌属和轮枝菌属的真菌单独或复合侵染造成的。室内平板实验结果表明,T42木霉发酵液能够抑制草莓重茬病菌菌落生长,其中T42木霉发酵液10倍稀释液对镰刀菌、立枯丝核菌菌落生长的抑制率分别为87．8％、85．3％。以该木霉发酵产物作为生物制剂施入田间能够明显控制草莓重茬病害的发生,使草莓重茬死苗率由52．9％降低到14．5％,使草莓产量增加77．8％。     

Rice seedlings release momilactone B into the environment

Since the growth inhibitor momilactone B was found recently in root exudates of rice (Oryza sativa L.), 3-day-old rice seedlings were transferred to hydroponic culture and the level of momilactone B released into the environment from the seedlings was measured. At day 15 after transfer, the level of momilactone B in the culture solution was 1.8 nmol per seedling compared with endogenous levels of 0.32 and 0.63 nmol per root and shoot, respectively, suggesting that rice seedlings actively releases momilactone B into the culture solution. This release must occur from the roots because only rice roots were immersed in the culture solution. Momilactone B inhibited the growth of ten cress (Lepidium sativum L.) seedlings at concentrations greater than 3 microM. Ten rice seedlings were incubated with ten cress seeds in a Petri dish containing 1 ml of medium, the medium contained 18 nmol of momilactone B, which came to 18 microM. This level of momilactone B was enough to reveal growth inhibition of the cress seedlings. Release level of momilactone B and its effectiveness as a growth inhibitor suggest that it may play an important role in rice allelopathy.     

Potential role of allelopathy in the soil and the decomposing root of Chinese-fir replant woodland

The role of allelopathy in the Chinese-fir (Cunninghamia lanceolata) replant problem was studied. The failure of Chinese-fir seedlings to grow normally in Chinese-fir replant woodland was not only caused by the depletion of nutrients and the deterioration of the structure of replant woodland soil, but also by biotic factors and allelopathy. Extracts of soils collected from replant woodlands significantly reduced the growth of Chinese-fir seedlings. Extracts and decomposing root residues also significantly inhibited the growth of Chinese-fir seedlings. Extracts of the replant soil and of the decomposing roots from Chinese-fir replant woodland were both toxic to other plants. The combination of the decomposing root residues and the pathogenic fungi reduced the growth of Chinese-fir seedlings the most when compared to the decomposing root residue alone and the control. It appears that allelopathy is at least partly involved in the Chinese-fir replant problem. ei]Section editor: R Rodriguez-Kabana     

烟草幼苗根系分泌自毒物质种类及PAEs对根系抗氧化性能的影响

采用GC-MS技术鉴定水培烟草Burley及K326在幼苗期不同生长阶段的根系分泌物;并用不同浓度邻苯二甲酸二丁酯(DBP)、邻苯二甲酸二异辛酯(DIOP)溶液浇灌盆栽烟草幼苗,研究其根系抗氧化性能变化。结果如下:(1)Burley根系分泌物主要有3类化合物,其中自毒物质邻苯二甲酸酯(PAEs)在二叶龄期、四叶龄期、六叶龄期的相对含量分别为7.6%、0.3%、未检出;而K326根系分泌物主要有9类化合物,PAEs在二叶龄期、四叶龄期、六叶龄期的相对含量分别为35.6%、51.3%、2.2%。(2)浓度高于0.1 mmol/L的PAEs使根中超氧阴离子自由基产生的速率显著(P0.05)增加;随着DIOP及DBP浓度的增加,超氧化物歧化酶、过氧化氢酶活性增加,在0.5 mmol/L时达到最大,然后随着处理浓度的增加而下降。丙二醛的浓度随着这两种PAEs处理浓度的增加而增大。结果表明:烟草根系分泌的自毒物质PAEs达到0.5 mmol/L时,能降低根系的抗氧化性能,造成根尖细胞膜系统的氧化损伤,引起根吸收功能等一系列生理生化变化,并最终表现出自毒作用。     

甜瓜自毒相关基因CmGST的克隆及其对自毒胁迫的响应

植物谷胱甘肽硫转移酶基因(GST)在清除生物和非生物胁迫产生的氧化损伤中扮演着重要的角色,为探究GST在根系分泌物介导的甜瓜自毒胁迫中的响应机制,该实验在转录组测序基础上,以甜瓜叶片cDNA为模板,采用RT PCR技术获得了一个根系分泌物介导的甜瓜自毒作用密切相关的GST基因,命名为CmGST(GenBank 登录号：AYU66762.1);对CmGST基因进行了相关生物信息学分析,同时对自毒胁迫过程中,CmGST的差异表达情况、谷胱甘肽硫转移酶(GST)活性和谷胱甘肽(GSH)含量进行了相关测定。结果显示：(1)CmGST基因含有完整开放阅读框(ORF)654 bp,编码217个氨基酸;CmGST蛋白为酸性亲水蛋白,且较为稳定,具有GST家族Tau亚家族的2个典型的保守结构域;CmGST蛋白与黄瓜的亲缘关系较近。(2)亚细胞定位结果表明CmGST定位于细胞质中。(3)qRT PCR分析结果表明,CmGST在甜瓜根系和幼苗中均有表达,在正常生长的甜瓜幼苗中表达量相对稳定;自毒胁迫后在叶中的表达先降后升,后期迅速增加;在根系中变化趋势类似,但程度较为缓和。(4)GST活性和GSH含量分析表明,叶片中GST活性变化趋势与CmGST基因表达变化基本吻合;在根系中,GST活性呈现先升再降最后又升高的趋势;随胁迫时间增加,GSH含量在根系和叶片中均都有不同程度的增加,但根部含量低于叶片。研究认为,在自毒胁迫过程中,CmGST基因在甜瓜植株根和叶片均有相应表达水平的变化,参与了对根系分泌物介导的自毒胁迫响应。     

Fine-root system development and susceptibility to pathogen colonization

Root development may exert control on plant–pathogen interactions with soil-borne pathogens by shaping the spatial and temporal availability of susceptible tissues and in turn the impact of pathogen colonization on root function. To evaluate the relationship between root development and resistance to apple replant disease (ARD) pathogens, pathogen abundance was compared across root branching orders in a bioassay with two rootstock genotypes, M.26 (highly susceptible) and CG.210 (less susceptible). Root growth, anatomical development and secondary metabolite production were evaluated as tissue resistance mechanisms. ARD pathogens primarily colonized first and second order roots, which corresponded with cortical tissue senescence and loss in second and third order roots. Defense compounds were differentially allocated across root branching orders, while defense induction or stress response was only detected in first order and pioneer roots. Our results suggest disease development is based largely on fine-root tip attrition. In accordance, the less susceptible rootstock supported lower ARD pathogen abundance and altered defense compound production in first order and pioneer roots and maintained higher rates of root growth in both the ARD soil and pasteurized control compared to the more susceptible. Thus, this rootstock’s ability to maintain shoot growth in replant soil may be attributable to relative replant pathogen resistance in distal root branches as well as tolerance of infection based on rates of root growth.