Physiological Responses of Microcystis aeruginosa PCC7806 to Nonanoic Acid Stress

A recent study has shown that nonanoic acid (NA) is one of the strongest allelochemicals to a cyanobacterium Microcystis aeruginosa, but the physiological responses of M. aeruginosa to NA stress remain unknown. In this study, physiological characters such as the growth rate, photosynthetic processes, phosphorus and nitrogen uptake kinetics, and the contents of intracellular microcystin of M. aeruginosa PCC7806 were studied under the NA stress. The results showed that the growth rates of M. aeruginosa PCC 7806 were significantly inhibited in all NA stress treatments during first 3 days after exposure, and the growth rate was recovered after 5-day exposure. After 2-day exposure, the contents of both phycocyanin and allophycocyanin per cell decreased at NA concentration of 4 mg L-1, and oxygen evolution was inhibited even at the concentration of 0.5 mg L-1, but carotenoid content per cell was slightly boosted in NA stress. Physiological recovery of M. aeruginosa PCC7806 was observed after 7-day exposure to NA. It was shown that NA stress had no effect on uptake of nitrogen, but could stimulate the uptake of phosphorus. The contents of intracellular microcystin have not been affected in all NA treatments in contrast with the control. (C) 2008 Wiley Periodicals, Inc. Environ Toxicol 24: 610-617, 2009.

Relationship between the allelopathic activity and molecular structure of hydroxyl derivatives of benzoic acid and their effects on cyanobacterium Microcystis aeruginosa

The antialgal activities of benzoic acid, 2-hydroxybenzoic acid (salicylic acid), 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 3,5-dihydroxybenzoic acid and 3,4,5-trihydroxybenzoic acid (gallic acid) were studied on the growth of two strains of Microcystis aeruginosa (toxic FACHB 942 and non-toxic 469). The results showed that the sequence of 50% growth inhibition concentration (ErC50) of 6- compounds for both strains of M. aeruginosa followed the same order: gallic acid > 3,5-dihydroxybenzoic acid > 4-hydroxybenzoic acid > salicylic acid > 3-hydroxybenzoic acid > benzoic acid. The position and the numbers of hydroxy groups between the hydroxy group and carboxyl influenced the antialgal effects of phenolic acids. We also investigated the joint effects of benzoic acid, 4-hydroxybenzoic acid and 3,4,5-trihydroxybenzoic acid on the growth of M. aeruginosa ( toxic FACHB 942). The mixture of phenolic allelochemicals showed the synergistic effects.

Allelopathic effects of the submerged macrophyte Potamogeton malaianus on Scenedesmus obliquus

Allelopathic effects of the submerged macrophyte Potamogeton malaianus on Scenedesmus obliquus were assessed using a twophase approach under controlled laboratory conditions. In the co- culture experiment ( phase I), the growth of S. obliquus at two different initial cell densities was significantly inhibited by P. malaianus. Moreover, the growth inhibition was dependent on the biomass density of P. malaianus. Antioxidant enzymes ( SOD, CAT and POD), MDA, APA, total soluble protein, protein electrophoretic pattern and morphology of S. obliquus were determined after the coculture experiment was terminated. The activities of SOD, CAT, POD and APA at the low initial cell density were stimulated, the contents of MDA and total soluble protein were increased, and some special protein bands disappeared in P. malaianus treatments. The macrophyte had no effect on the activities of SOD and APA at the high initial cell density, but significantly influenced other physiological parameters of S. obliquus with the increase of biomass density. The morphology of S. obliquus showed no difference in the macrophyte treatments and the controls, and the cultures were dominated by 4- celled coenobia. The results indicated P. malaianus had significant allelopathic effects on the growth and physiological processes of S. obliquus. Moreover, the allelopathic effects depended on initial algal cell density, biomass density of the macrophyte, and their interaction. In the experiment of P. malaianus culture filtrates ( phase II), filtrates from combined culture of plant and S. obliquus at the low initial cell density exhibited no apparent growth inhibitory effect on S. obliquus. The result showed that initial addition of growth- inhibiting plant filtrates had no allelopathic effect on S. obliquus. We concluded that the allelopathic effects on S. obliquus were found in the presence of P. malaianus, but not in P. malaianus filtrates. However, the absence of allelopathic effect on S. obliquus might be due to the very low concentrations of allelochemicals in the filtrates.

化感物质鉴定及其在土壤中的活性—以三裂叶豚草、水稻为例

化感物质鉴定及其活性研究是化感作用研究的核心问题。本研究以三裂叶豚草和化感水稻为例，在鉴定化感物质结构的基础上，深入探讨化感物质的生物活性，尤其是重点研究了化感物质与土壤生物因子的化学作用。 研究结果表明： (1) 三裂叶豚草能通过产生和释放挥发性化感物质到环境中抑制小麦等作物的萌发生长，而对稗草则显示出促进效应，并证实其挥发物可经土壤载体对其他植物及微生物显示化感效应。其挥发油对受试病原菌表现出不同程度的抑菌活性。顶空采样分析显示，三种单萜类物质berneol、alcanfor和 berneol acetate是三裂叶豚草周围空气中的主要化感物质。采用GC-MS结合滞留指数对三裂叶豚草挥发油进行了定性分析，在鉴定出的35个组分中，单萜类及其衍生物占总量的84.2%。 (2) 从三裂叶豚草发生地毒性土壤中分离鉴定出两种胡萝卜烷型倍半萜1α-angeloyloxycarotol和1α-(2-methylbutyroyloxy)-carotol，并检测出两种物质在毒性土壤中的浓度范围已超过其对小麦的抑制临界浓度(11.5µg/g和16.5µg/g)，进而推测两个萜类物质是三裂叶豚草干扰小麦生长的主要化感物质。 (3) 发现化感水稻组织含有尿囊素后，又从3个水稻化感品种和8个非化感品种的组织和培养基质中均检出尿囊素，证实尿囊素与水稻化感特性无相关性；水稻根分泌的尿囊素对稗草和稻田可培养微生物具有促生效应，而灭菌土壤中尿囊素半衰期(20.2 h, r2=0.95)超过非灭菌土壤(7.3 h, r2=0.92)3倍，表明土壤尿囊素中降解和转化速率较快，上述结果暗示尿囊素是参与水稻和其它生物间化学相互作用的一种促生物质。 (4) DGGE分析表明尿囊素能显著提高土壤细菌的丰富度和多样性指数；通过比较PLFA指纹图谱，一方面，尿囊素不但能显著增加PLFA总量，而且还能显著改变微生物的群落结构；主成分分析显示，在淹育和非淹育条件下，第一和第二主成分分别揭示了总方差的67.8%和55.3%。另一方面，添加黄酮甙和根分泌物均能显著改变土壤微生物的群落结构，第一和第二主成分累积方差变异之和占总变异的59.1%，并且化感品种根分泌物与黄酮甙处理的土壤样品呈正相关关系；而化感水稻根分泌物中化感物质含量与非化感水稻差异显著，暗示化感物质在影响微生物群落结构上的重要性。

杉木人工林土壤中毒性化感物质的鉴定与来源

针对杉木化感物质种类的争论，我们以生测活性做导向，对杉木人工林土壤中毒性化感物质进行了分离，并通过质谱、核磁共振等仪器对化感物质的化学结构进行了鉴定，然后研究了其活性和来源，同时从土壤养分和化感作用两个方面对杉木火力楠混交林解决杉木连栽障碍的作用机制进行了研究。结果表明： (1) 虽然不同连栽代数土壤养分与杉木幼苗苗高存在正相关关系，即随着杉木连栽代数的增加，土壤养分含量逐渐降低，土壤处理的杉木幼苗苗高也逐渐降低，但是同一林分中，距杉木根系不同距离的土壤养分含量与杉木幼苗根长存在负相关关系。在一代和二代杉木林中，与杉木林地土相比，杉木根际土能够显著地抑制杉木幼苗根生长。单用土壤养分匮缺来解释连栽杉木生产力下降是不充分的，杉木化感作用也是连栽障碍的重要原因之一。 (2) 从杉木人工林土壤中检测到8种酚酸类物质：对羟基苯甲酸、香草酸、阿魏酸、苯甲酸、肉桂酸、香草醛、异香草醛和香豆素。自然浓度的酚类物质没有抑制杉木幼苗的生长，反而促进了杉木幼苗的生长。从连栽杉木林土壤分离到一种新环二肽(6-hydroxy-1,3-dimethyl-8-nonadecyl-[1,4]-diazocane-2,5-diketone)，它在自然浓度条件下能够显著地抑制杉木幼苗的生长。而且当浓度达到40nmol•ml-1时，环二肽对杉木幼苗根生长的抑制率接近50%，显示其具有较高的化感活性。因此，引起连栽杉木生产力下降的化感物质不是对羟基苯甲酸等酚酸类物质，而是从杉木林土壤中分离得到的环二肽。 (3) 杉木各组织中环二肽含量最多的为叶凋落物，而且用杉木叶凋落物处理的土壤中环二肽含量显著增加，这说明杉木叶凋落物是环二肽的重要来源之一。杉木根系分泌物中含有环二肽，而且随着连栽代数的增加，杉木根系分泌环二肽的速率也增加，这说明杉木根系分泌物也是环二肽的重要来源之一。杉木成熟林每年每公顷凋落的叶凋落物中含有约0.173~0.418 mol的环二肽，而每年每公顷杉木根系分泌的环二肽可达1.289~1.362 mol，显著高于前者。因此，杉木根系分泌物是杉木化感物质环二肽最重要的来源。 (4) 杉木火力楠混交林能够提高土壤有机质、全氮等养分含量，增加土壤微生物数量及土壤蔗糖酶、脲酶和磷酸酶等土壤酶活性，从而提高了混交林土壤熟化程度，加速了土壤有机质的转化速率，增强了土壤养分的供应能力，改善了土壤养分匮缺的状况。与二代杉木林相比，杉木火力楠混交林中杉木根系分泌环二肽的速率降低了33.2%，杉木火力楠混交林能减少环二肽的释放量，从而降低土壤中化感物质的含量，减弱林地土壤对杉木幼苗生长的抑制作用，缓解了连栽杉木自毒作用。因此，杉木火力楠混交林是一种高生产力和生态协调的造林模式。

稻田化感物质对土壤微生物和酶的作用及其迁移行为

本论文以化感水稻品种PI312777和非化感水稻品种辽粳9为试验材料，研究田间全生育时期内两种水稻根际土壤微生物及酶活性的变化；室内添加两种水稻苗期根系分泌物、水稻及稗草化感物质，探讨其对土壤微生物及酶活性的影响。测定指标有：土壤微生物生物量碳、微生物数量(细菌、放线菌、真菌、氨化细菌和好气性自生固氮菌)、土壤酶(脲酶、转化酶、脱氢酶和多酚氧化酶)活性和磷酸脂肪酸谱图分析。同时利用土壤薄层层析结合生物测定的方法研究化感物质在不同类型土壤中的迁移性能，结果表明： 1.种植水稻明显提高了土壤微生物生物量碳、微生物数量和酶活性。在生长的初始阶段(苗期到分蘖期)，化感水稻PI312777根际土壤微生物各项指标及酶活性均高于非化感水稻辽粳9。 2.两种水稻苗期根系分泌物均刺激土壤微生物并间接提高土壤酶活性，改变土壤微生物群落组成，处理间的效应因培养时间和水分条件而不同。 3.稗草化感物质——对羟基扁桃酸对水稻生长及土壤微生物的影响因浓度而呈现不同的效应。水稻化感物质对土壤微生物有不同的影响。苯甲酸和阿魏酸刺激土壤微生物及酶活性；黄酮对土壤微生物生物量碳和真菌数量有抑制作用。水分条件的变化导致微生物群落结构的改变，淹水条件下环丙基脂肪酸(cy17:0和cy19:0)及真菌脂肪酸含量减少。 4.不同化感物质在同一土壤中的迁移性能不同，而同一化感物质在不同土壤中的移动性也有差异；比移值Rf与土壤有机质含量、粘粒含量、阳离子交换量呈负相关，与土壤pH呈正相关。

稗草根系分泌物化感物质的分离鉴定及其功能

稗草是水稻田常见伴生杂草。近年来对稗草化感物质分离鉴定及其在诱导水稻化感作用方面的研究已经成为热点。本文对稗草通过根系释放的化感物质进行了分离鉴定和植物活性测定；研究了稗草及其根系分泌物对3叶期的两个水稻品种化感活性的诱导作用；以及诱导过程中水稻内源水杨酸和茉莉酸的响应关系。 研究结果显示： (1)稗草根系分泌物化感活性较强的极性部分鉴定2,4-二叔丁基酚这一具有强化感活性的物质，2,4-二叔丁基酚对供试的莴苣和两个水稻品种的生长具有明显的抑制作用。 (2)稗草在伴生和诱导条件下可以显著提高水稻化感物质含量，增强水稻的化感活性。用稗草生长过的培养液来培养水稻，使水稻化感物质含量增加，表明稗草诱导水稻化感物质含量的增加是由其根系分泌物引起的，水稻化感活性对稗草根系分泌物的响应比对稗草的响应要快速。稗草根系分泌物中不同极性部分的诱导作用表明，低极性部分的乙酸乙酯相对水稻化感物质诱导作用更强。从稗草根系分泌物低极性部分鉴定出的硬脂酸、2,4-二叔丁基酚和邻苯二甲酸二乙酯对水稻化感物质具有显著的诱导作用，显示出稗草根系分泌物中多个组分对水稻化感物质具有诱导作用，而硬脂酸具有更强的诱导效应。 (3)稗草及其根系分泌物诱导水稻化感活性增强过程中，受试水稻品种的内源水杨酸含量变化都较小，而在化感水稻品种PI312777和非化感水稻品种辽粳9之间含量差异较大，Pearson’s相关分析表明水杨酸基础含量与水稻品种化感潜力特性显著相关。 稗草及其根系分泌物诱导后，在含量高的水稻根部茉莉酸含量成倍增加，表明茉莉酸在水稻识别稗草后的信号传导起着重要作用。暗示水稻是通过内源茉莉酸的含量变化来启动防御机制，产生和释放更多的化感物质来抑制稗草。

花椒主效化感物质分离鉴定及化感作用研究

近年来，随着对作物重茬障碍原因的深入研究，植物的化感作用越来越受到国内外众多学者的重视。花椒（Zanthoxy piperitum.）为芸香科植物，是一种收益早、用途广、价值高的经济树种，是川西干旱河谷地区的重要经济作物，其连作障碍也倍受关注，系统研究花椒化感作用将有助于理解和最终解决花椒连作障碍问题。本文首先通过萃取、层析等方法分离花椒主效化感成分；通过外加不同浓度的花椒叶水浸液研究了对土壤氮素养分循环的影响；研究了花椒叶水浸液对苜蓿生理生化、光合作用、氮素养分吸收的影响，并对外施氮肥对这种化感影响的缓解作用做了研究；研究了花椒化感潜力对全球变化——UV-B增强辐射的响应。主要研究结果如下： 1．用不同极性的有机溶剂对花椒叶水浸液浓缩浸膏萃取、柱层析，结合生物活性检测，分离得到主效化感作用组分的一种化感物质——对甲氧基苯酚。采用该物质纯品进行生物活性检测，证明其具有化感作用。 2．花椒叶水浸液处理土壤30天后，土壤硝态氮、铵态氮、无机氮（硝态氮＋铵态氮）与对照相比，随着花椒叶水浸液浓度的增加呈现降低的趋势，其中土壤铵态氮含量显著降低，而硝态氮含量的变化则不显著，无机氮含量也显著降低。土壤脲酶和蛋白酶的活性与无机氮含量的变化趋势相同。随着花椒叶水浸液浓度的增加，氨化细菌数量显著降低，固氮菌的数量变化不显著，硝化细菌和反硝化细菌数量有减少的趋势。60天后，硝态氮含量、铵态氮含量、无机氮随水浸液浓度增加的变化趋势与30天时相似；随着花椒叶水浸液浓度的增加，氨化细菌、固氮菌的数量显著减少，硝化细菌数量、反硝化细菌数量仍呈减少趋势；土壤脲酶、蛋白酶活性与第30天的变化趋势相同。第60天与第30天的结果相比，相同水浸液浓度处理的硝态氮、铵态氮、无机氮均有下降的趋势，但除了25g.L－1水浸液处理的外，其它相同浓度的处理间差异均不显著；除了12.5 g.L的处理外土壤脲酶活性均呈增强的趋势；蛋白酶活性都有不同程度的增加；花椒叶水浸液处理的土壤硝化细菌和反硝化细菌数量呈增加趋势。 3．随着花椒叶水浸液浓度的增加，显著抑制了苜蓿根长、地上地下生物量、叶绿素含量、叶片中可溶性蛋白的含量，净光合速率。苜蓿体内四种抗氧化酶（POD、SOD、CAT、APX） 活性随着水浸液浓度的增加而降低，而丙二醛含量则增加。苜蓿氮初级同化相关酶硝酸还原酶（NR）、谷氨酰合成酶(GS)、谷氨酸脱氢酶（GDH）的活性随着水浸液浓度的增加受到不同程度的影响。总的来说，苜蓿硝酸还原酶、谷氨酰合成酶的活性受到抑制，而谷氨酸脱氢酶活性的变化则比较复杂，根呈先降低后增加的趋势，叶片则无显著变化。外施两种不同浓度的硝酸铵氮肥后，对12.5、25 g.L－1花椒叶水浸液处理的苜蓿化感作用有显著的缓解作用，表现在株高、生物量、光合作用等方面，大多达到与对照（0 g.L－1）未施氮肥无显著差异的水平，而对50 g.L－1水浸液处理的苜蓿幼苗，虽有一定的缓解作用，但这种作用均未达到与对照（0 g.L－1）未施氮肥时无显著差异的水平。 4． UV-B增强辐射处理花椒后，花椒的化感潜力显著增强。花椒叶片内UV-B吸收物质的含量和总酚含量均显著增加。 In recent years, with profound research on the reasons of continuous cropping obstacles, allelopathy received increasing attention to many scholars at home and abroad. Zanthoxy bungeanum as a Rutaceae plant is a high economic value species which gains early and uses widely. Zanthoxylum is an important economic crop in the arid valley of western Sichuan region, and its not even has received much concern for the continuous cropping obstacles. The systematic study of allelopathy of Zanthoxylum will contribute to the understanding and final settlement of this issue. The major allelopathic composition was separated through the extraction, chromatography combined with other methods. The impact on soil nutrient cycling was also studied through the addition of different concentrations of water extracts of Zanthoxylum. Furthermore, the effects of water extracts of Zanthoxylum leaves on alfalfa leaf physiological and biochemical indexes, photosynthesis, soil enzymes and nutrient uptake of nitrogen and the mitigation of allelopathy through using external fertilizer were studied to put forward scientific resolvent for Zanthoxylum continuous cropping obstacles .The response of allelopathic potential of Zanthoxylum to global change - UV-B enhanced radiation was studied . The main findings are as follows: 1. Through extraction with different polar organic solvents on concentrated water extract of Zanthoxylum leaf and then using column chromatography combined with detection of biological activity, one of the main allelopathic components- methoxy-phenol was isolated. The biological activity testing of the pure material of methoxy-phenol proved that it does have allelopathic potential. 2. Thirty days after treating soil with water extract of Zanthoxylum leaf, as compared with the control, the contents of soil nitrate, ammonium, nitrate plus ammonium nitrogen showed a trend of decrease with the increase of the concentration of water extract whereas the content of ammonium nitrogen showed a significant reduction, and the content of nitrate did not change significantly, the content of nitrate plus ammonium nitrogen also showed a significant (P <0.05) redction. The activity of soil urease and protease showed the same trend as the content of nitrate nitrogen plus ammonium nitrogen. With the increase in the concentration of water extract, the number of ammonification bacteria significantly reduced but nitrogen-fixing bacteria did not change significantly and there was a decreasing trend in the number of nitrifying bacteria and denitrifying bacteria. Sixty days after the treatment, with the increase in solution concentration of water extract of Zanthoxylum leaf, the content of nitrate、 ammonium nitrogen, nitrate plus ammonium nitrogen showed a similar change trend to 30 days’; the number of ammonification bacteria, nitrogen-fixing bacteria significantly reduced ; the number of nitrifying bacteria, denitrifying bacteria was still an downward trend; the activity of soil urease and protease showed the same trend as the 30th days’. Compared to the results of the 30th days’, the content of nitrate, ammonium, nitrate plus ammonium nitrogen showed a decrease trend between the treatment of same concentration, but there was no significant difference except the treatment of 25g.L-1 between the same concentration; the activity of soil urease showed enhanced trend except the treatment of 12.5 g.L-1; the activity of protease increased to varying degrees; the number of ammonification bacteria、 nitrifying bacteria and denitrifying bacteria were growing while nitrogen-fixing bacteria reduced.. 3. With the increase of the concentration of water extract of Zanthoxylum leaf, the water extract significantly inhibited the root length, aboveground biomass, content of chlorophyll and soluble protein in leaf and net photosynthetic rate. The activity of four antioxidant enzymes (POD, SOD, CAT, APX) reduced with the increase in concentration of the water extract but the content of MDA increased. The activity of enzymes related to primary nitrogen assimilation such nitrate reductase (NR), glutamyl synthetase (GS), glutamate dehydrogenase (GDH) were subject to different degrees with an increase in the concentration of water extracts. In general, the activity of nitrate reductase, glutamyl synthetase were inhibited, while change in the activity of glutamate dehydrogenase was more complex. The activity of glutamate dehydrogenase in leaf was first reduced and then increase,but did not change significantly in root. After using two external different concentrations of nitrogen fertilizer, there was a significant mitigation in inhibiton in plant height, biomass, photosynthesis, etc. in the treatment of 12.5,25 gL-1 of water extract of Zanthoxylum leaf, and most of these indexes showed no significant difference with the control (0 g.L-1, no external fertilizer was added) .Although there showed a certain degree of ease in the treatment of 50 g.L-1 , there was still a significant difference compared with the control (0 gL-1) in which no external fertilizer was used. 4.The allelopathic potential of Zanthoxylum positively responded to enhanced UV-B significantly. The content of UV-B absorbing compounds and the total phenol also significant increased.

花椒水浸液及酚酸类化感物质对作物种子萌发和幼苗生长的影响效应

花椒（Zanthoxylum piperitum）是川西干旱河谷地区的重要经济作物，化感作用是花椒连作障碍的原因之一。系统研究花椒化感作用有助于深入理解并最终解决花椒连作障碍。本文通过研究花椒叶、林下土壤浸提液及单一纯化感物质对花椒幼苗生长、苜蓿种子萌发及幼苗生长的影响作用，从生理生化角度揭示浸提液及纯化感物质的作用方式。通过室外和室内模拟实验，对浸提液及纯化感物质的化感效应进行比较，为花椒连作障碍的解决和化感作用机制的深入理解提供依据。主要结果如下： 1.花椒叶及林下土壤浸提液对地下生物量影响作用强与对地上生物量的化感效应，两种浸提液的化感效应强度不同，叶浸提液作用表现更显著。其中在Y6、Y8 、T6和T8处理时，花椒幼苗地下生物量分别降低了31.2%、32.1%、31.6%和31.7%。 2.两种浸提液均能显著影响花椒幼苗体内的保护性酶活性，总体说来，在高浓度下抑制各种抗氧化物酶活性，幼苗体内丙二醛含量增加，幼苗受害严重；在较低浓度下，各种保护性酶活性有所增加，丙二醛含量减少，幼苗伤害减轻。同时，不同月份里，各种酶的活性高低显著不同，9月份的活性显著低于7月份的酶活性。对于养分含量的影响，Y8、T8的影响强度最大，分别使碳元素含量降低了27.8%和30.8%，使钾元素含量降低了34.7%和25.6%。 3.花椒叶及林下土壤浸提液对苜蓿种子萌发及幼苗生长有化感作用，表现在最终萌发率、不同物质代谢及保护性酶活性的差异上。两种浸提液对苜蓿种子萌发过程中蛋白质的含量均无显著性影响，对淀粉和可溶性糖的影响作用类似，高浓度处理无明显化感效应，较低浓度处理显著降低二者在萌发苜蓿种子中的含量。Y2、Y4与T4处理分别使可溶性糖含量减少了32.3%、29.1%和18.8%，Y2与T2处理分别使淀粉含量降低了29.3%和26.8%。 4.苜蓿种子在4种单一化感物质最高浓度即10-3 mol•L-1处理下，萌发率显著降低，半数萌发时间推迟，随着处理浓度降低，抑制作用逐渐减弱，当降低到10-6 mol•L-1时，又能够表现出对苜蓿种子萌发的促进作用。 5.纯化感物质在10-6 mol•L-1时使苜蓿幼苗叶片的保护性酶活性显著升高，丙二醛含量显著降低；在10-3 mol•L-1时使苜蓿叶片中保护性酶活性显著降低，丙二醛含量增加，膜脂过氧化程度加重。 Zanthoxylum piperitum is one of the most important cash crops and has been extensively cultivated in Eastern Tibetan Plateau, especially in the fragile dry valley areas. Allelopathic effects could be a reason for Z. piperitum’s continuous cropping impediment. Systemmatically research of the effect of Z. piperitum allelopathy could help to comprehend the continuous cropping impediment. The allelopathic effects on seedlings growth and seed germination of aqueous extracts of Zanthoxylum piperitum and phenolic allelochemicals were studied, and the action mechanism of the two substances was also discussed from physiology. Indoor and outdoor experiments were set to compare the difference between aqueous extracts and pure allelochemicals. The main results showed that: 1. The aqueous extracts of leaf and soil had significant allelopathic effects on aboveground and underground biomass, but the effect on underground biomass was stronger than the effect on underground evidently. Treated with Y6、Y8 、T6 and T8, the underground biomass was reduced 31.2%、32.1%、31.6% and 31.7% respectively. 2. The activity of activities of superoxide dismutase, catalase, peroxidase and ascorbate peroxidase were significantly reduced, while the content of MDA was increased and the seedlings were suffered stronger, when treated by the high concentration; but at the low concentration, these were reversed. And then, at the different month, the activities of antioxidant enzyme were significantly distinct. As for the contents of nutrient element, Y8、T8 had the more intensive effects than other treatments. 3. The results showed that the two types of aqueous extracts had significant allelopathic effects on seed germination, substances metabolize and the activity of antioxidant enzyme. But the aqueous extracts had no effects on the content of protein, while had the similar effects on the content of starch and soluble sugar. At Y2、Y4 and T4, the content of soluble sugar decreased 32.3%、29.1% and 18.8% respectively. 4. Treated with 10-3 mol•L-1 of the four allelochemicals, the seed germination of alfalfa was significantly inhibited. Ferulic acid, coumarin and vanillic acid at 10-3 mol•L-1 significantly reduced the activities of antioxidant enzyme, while the content of MDA in alfalfa seedling was significantly increased. The restrain effects became weakened with the treat concentration falled. However, ferulic acid, coumarin and vanillic acid could increase the activities of antioxidant enzyme at 10-6mol•L-1.

化感物质香豆素对苜蓿种子萌发及幼苗初级氮同化的影响

香豆素类物质是苯丙酸内酯（环酯）类化合物，绝大部分高等植物通过次生代谢途径都能合成。研究表明，香豆素类物质是花椒体内最重要的化感物质，系统研究香豆素类物质的作用机理有助于理解和最终解决花椒连作障碍。本文通过研究香豆素对几种植物种子特别是苜蓿种子萌发、苜蓿幼苗初级氮同化的影响，从生理生化角度揭示香豆素的作用方式，为花椒连作障碍的解决和化感作用机制的深入理解提供依据。主要研究结果如下：1. 研究了香豆素对6 种常见作物种子萌发的影响，并对一组数据采用4个不同的指标进行评价，对生物测定化感作用中存在的问题进行了讨论。结果发现1.0mM的香豆素对采用的6 种作物的种子萌发均表现出一定的化感作用，4 个指标的敏感程度依次为S (发芽速度)＞AS(累积发芽速度)＞CRG(发芽指数)＞GT(最终发芽率)。种子萌发实验是化感作用研究中最重要、应用最广泛的生物测定方法之一，应根据不同的研究目的合理采用指标和实验方法。2. 采用培养皿试纸法进行种子萌发试验，研究了香豆素水溶液在苜蓿种子萌发过程中对其吸水、电导率及抗氧化保护酶活性的影响。结果表明，影响苜蓿种子发芽的香豆素浓度阀值为0.3mM。香豆素在1.0mM 的浓度下降低了苜蓿种子吸水阶段Ⅱ的吸水速度，使其外渗物质增多，电导率增大，并显著抑制了超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)的活性，同时种子体内丙二醛（MDA）的含量显著增大。高浓度香豆素破坏了膜的结构、影响了抗氧化保护酶的活性是香豆素降低苜蓿发芽率的原因之一，也可能是影响花椒-苜蓿间作的关键因素之一。3. 不同浓度（0、25 μM、50 μM、0.1 mM、1.0 mM）化感活性物质香豆素对10 日龄苜蓿幼苗初级氮同化的影响的结果表明25 µM～50 µM 的香豆素加快了苜蓿幼苗对硝态氮的吸收。高浓度的香豆素导致苜蓿根系和叶片内可溶性蛋白含量降低、鲜重减小、地下鲜重/地上鲜重（R/S）的比值升高，根系中初级氮同化的关键酶硝酸还原酶（NR）、谷氨酸胺合成酶（GS）、谷氨酸脱氢酶（GDH）的活性降低，叶片中NR、GS 的活性减低、叶绿素含量减少，而GDH 的活性升高。香豆素影响苜蓿幼苗氮代谢和氨同化的关键酶，导致体内养分的缺失是香豆素抑制苜蓿幼苗生长的机理之一。Coumarins are lactones of o-hydroxycinnamic acid, and are allelopathiccompounds that originate in the phenylpropanoid pathway. They are synthesized byalmost all higher plants. According to previous studies, coumarins were mostimportant allelochemicals in Chinese prickly ash. Systematically research of theeffect of coumarin could help to comprehend the continuous cropping impediment.The effects of coumarin on seed germination and primary nitrogen assimilation ofalfalfa were studied. The main results showed that:1. We compared four common germination indices (S, AS, CRG, GT)preciously calculated with the same date. The results showed that, at theconcentration of 1.0 mM, coumarin inhibited seeds germination. Among all indices,the S index was most sensitive, followed by the AS and CRG indices. Andsuggestions on the expression of bioassay results were also provided.2. At concentrations above 0.3 mM, coumarin inhibited seed germination in aconcentration-dependent manner. During seed imbibitionⅡ, coumarin at 1.0 mMsignificantly reduced the activities of superoxide dismutase (SOD), catalase (CAT),peroxidase (POD), while the content of malonyldialdehyde (MDA) in alfalfa seedssignificantly increased. The higher concentration coumarin destroyed structure ofmembrane and influenced activities of antioxidant enzymes, which might be one ofthe reasons that coumarin decreased germination rate of alfalfa, and one of the keyfactors influencing Chinese prickly ash-alfalfa intercropping.3. Alfalfa plants were exposed to different concentration of coumarin (0、25μM、50 μM、0.1 mM、1.0 mM) grown for 10 days on control medium. Coumarin, in the range of 25 μM～50 μM, significantly stimulated the net nitrate uptake.Increasing coumarin concentration led to a decrease of protein contents in theleaves and roots. The root to shoot (R/S) FW ratio was increased by increasingcoumarin concentration. Under high coumarin concentration, the activities of nitratereductase (NR) and glutamine synthetase (GS) were repressed in the roots andleaves. Glutamate dehydrogenase (GDH) was inhibited in the roots, while enhancedin the leaves. Chlorophyll contents in the leaves were also decreased under highcoumain concentration. Coumarin decreased alfalfa growth by (i) nutritionaldeficiencies shown by the decrease of nitrate, (ii) lowered N compound synthesisvia inhibition of nitrate reduction and ammonium assimilation.

生姜化感作用机理及其栽培模式研究

近年来，随着对作物重茬（连年种植）障碍原因的深入研究，植物的化感作用越来越受到国内外众多学者的重视。而作为重要调料和药用植物的生姜，其连作障碍也备受关注，系统地研究生姜化感作用将有助于理解和最终解决生姜连作障碍问题。本文通过研究生姜不同部位、不同浓度的水浸液对与其间作的两个物种（大豆和四季葱）种子的萌发及幼苗生长的影响，从而证明生姜化感作用的存在；并通过温室盆栽实验研究了生姜的自毒作用（即研究生姜不同部位、不同浓度的水浸液对其幼苗的形态、生理生化、光合作用、土壤酶、土壤微生物多样性及土壤养分的影响），从而揭示生姜退化和衰老的机制，并为生姜筛选出合适的间作物种提供科学依据，对生姜连作障碍提出科学的解决方法。主要研究结果如下： 1. 与对照相比，生姜所有部位（根茎、茎、叶）、所有浓度（10、20、40、 80 g l-1）的水浸液均抑制了大豆种子和葱籽的萌发率、幼苗生长、水分吸收和脂肪酶活性，并且其抑制程度随着水浸液浓度的增加而增强，其生姜各部位水浸液抑制效应的强弱顺序为茎＞叶＞根茎。这一结果表明生姜根茎、茎、叶含有能够抑制大豆种子和葱籽种子萌发和幼苗生长的水溶性化感物质。根茎是生姜的主要收获部位，而生姜的残株（主要是茎和叶）应该从大田中处理掉以减轻其抑制效应。生姜水浸液中主要化感成分包括：根茎水浸液中主要是丁香酸和伞花内脂；茎水浸液中主要是阿魏酸，且其含量最高为73.4 ug/g；叶水浸液中除了阿魏酸，其他六种物质均检测出来，但含量较高的主要有丁香酸、伞花内脂和香豆酸。 2. 生姜茎和叶不同浓度的水浸液均显著抑制了生姜幼苗的株高、每株叶片数和叶面积，其抑制程度随着水浸液浓度的增加而有所增强，而生姜幼苗每株分枝数差异不显著；同时生姜水浸液也极大程度地影响了生姜幼苗的生物量（包括地下生物量、地上生物量和总生物量，均为鲜重）。在同一浓度下，茎水浸液对生姜幼苗形态指标及生物量指标均显示出最强的抑制作用，叶水浸液次之，根茎水浸液最弱。与对照相比，低浓度的生姜根茎水浸液提高了生姜幼苗叶片内四种抗氧化酶（SOD、POD、CAT、APX）活性，高浓度的根茎水浸液抑制了四种抗氧化酶活性，而茎和叶水浸液均随着浓度的增加而抑制了四种抗氧化酶活性，三种水浸液均随着浓度的增加降低了生姜幼苗叶片内叶绿素的含量，而增加了生姜幼苗叶片的相对电导率和丙二醛含量。同时，三种水浸液均随着浓度的增加降低了生姜幼苗的光合参数（包括胞间CO2浓度、气孔导度、蒸腾速率及净光合速率）。 3. 三种生姜水浸液对所测六种土壤酶活性均产生了不同程度的影响，其中影响最大的是酸性磷酸酶和蔗糖酶，在10 g l-1 时就达到了显著水平，并且所有酶均有随着水浸液浓度增加而增大的趋势；相同部位的水浸液随着浓度的增加，细菌和真菌的数量呈增加趋势，而放线菌的数量呈减少趋势；三种生姜水浸液均随着浓度的增加降低了土壤中有机质的含量，加剧了土壤中硝态氮含量的积累，根茎水浸液对土壤有效磷、速效钾和铵态氮均显示出低浓度提高其含量而高浓度降低其含量的趋势，而茎和叶水浸液则随着浓度的增加均降低了其含量。 4. 与生姜单作相比，所有间作系统均在旺盛生长期和收获期不同程度地提高了土壤酶活性，同时也增加了土壤细菌数量及土壤微生物总数但不显著；所有间作系统在旺盛生长期和收获期均不同程度地影响了土壤真菌及放线菌数量（增加或减少），所有间作系统间的多样性指数差异不显著，除了旺盛生长期四种作物（生姜-大豆-四季葱-大蒜）的间作模式显著降低了多样性指数，其值仅为生姜单作的33.18%；生姜与大豆间作不仅提高了19.6%的生姜产量而且获得了较好的经济效益，并且，所有间作系统均显著抑制了生姜姜瘟病的发生。 5. 不同栽培模式不同程度地影响了收获期生姜的株高、分枝数、根茎产量及内在品质。其中处理2显著地促进了生姜的分枝（10.5%），同时处理2、3和4也促进了生姜的生长（株高分别增加了15.0%、11.4%和14.0%），并且这三个处理提高了生姜的产量；处理2和3能有效提高生姜块茎中维生素C（分别较单作生姜显著提高了3.29%和4.05%）、处理3显著提高了可溶性糖（8.2%）、姜辣素（4.6%）和蛋白质等有益物质的含量，降低硝酸盐有害物质的含量（处理2显著降低了14.0%），改善了姜块的外观和内在品质。并且，生姜与大豆间作具有最高的纯收入和产投比，分别较生姜单作提高了24.80%和8.8%。Recently, allelopathy has been more and more paid attentions by national and foreign scholars with profound research on reasons of crop replanted (continuous planted) obstacle. Ginger rhizome is valuable all over the world either as a spice or herbal medicine and ginger replanted obstacle is also paid attentions. Systematic research on ginger allelopathy will contribute to understanding and ultimate solving problem of ginger replanted obstacle. The effects of ginger aqueous extracts with different parts and concentrations on seed germination and early seedling growth of soybean and chive were studied in this article to testify that ginger existed allelopathy. Furthermore, ginger autotoxicity was also studied by pot experiment in greenhouse (namely research on effects of ginger aqueous extracts with different parts and concentrations on morphological indexes, physiological and biochemical indexes, photosynthesis, soil enzymes, soil microbial diversity and soil nutrients) to reveal mechanism of ginger degeneration and senescence, provide scientific basis for selecting appropriate intercropping species and put forward scientific resolvent for ginger replanted obstacle. The main results were as follows: 1. All aqueous extracts at all concentrations inhibited seed germination, seedling growth, water uptake and lipase activity of soybean and chive compared with the control, and the degree of inhibition increased with the incremental extracts concentration. The degree of toxicity of different ginger plant parts can be classified in order of decreasing inhibition as stem＞leaf＞rhizome. The results of this study suggested that rhizome, stem and leaf of ginger contained water soluble allelochemicals which could inhibit seed germination and seedling growth of soybean and chive. The rhizome is the main harvested part of ginger. The residue (mainly stems and leaves) of the ginger plant should be removed from the field so as to diminish its inhibitory effect. The main allelopathic components of three kind of aqueous extracts were as follows: Rhizome extract chiefly contained syringic acid and vmbelliferone and stem extract mainly contained frulic acid whose content was the highest (73.4 ug/g). The other six substances were detected except of frulic acid, but only contents of syringic acid, vmbelliferone and p-coumaric acid were higher. 2. Stem and leaf aqueous extracts of ginger with different concentrations significantly inhibited plant height, leaf numbers per plant and leaf area, and the degree of inhibition increased with the incremental extracts concentration. However, tiller number per plant of ginger seedling showed no significant difference. At the same time, ginger aqueous extracts also influenced biomass including under-ground biomass, above-ground biomass and total biomass (fresh weight) to a large extent. Under the same concentration, stem aqueous extract showed the mostly inhibitory effect on morphological indexes and biomass indexes of ginger seedling. Rhizome aqueous extract showed the leastly inhibitory effect and leaf aqueous extract was intervenient. Enhanced concentration of ginger aqueous extracts significantly reduced total chlorophyll content, accompanying with increases in memberane permeability (REL) and lipid peroxidation (MDA). Compared with the control, rhizome ginger aqueous extract of lower concentration (10 g l-1) increased the activities of major antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POD; catalase, CAT; ascorbate peroxidase, APX) of ginger leaf tissue and higher concentration inhibited the activities of four antioxidant enzymes. However, stem and leaf aqueous extract inhibited the activities of four antioxidant enzymes with increase in concentration. Meanwhile, enhanced concentration of ginger aqueous extracts significantly reduced photo-parameters of ginger seedling (including CO2 concentration, stoma conductivity, net photosynthesis rate and transpiration rate). 3. Rhizome, stem and leaf ginger aqueous extract showed different effect on six soil enzyme activities, and acid phosphatase and invertase showed significant effect when aqueous extract concentration got 10 g l-1. Furthermore, six soil enzyme activities increased with increase in aqueous extract concentration. Bcterial and fungi number tended to increase while antinomyces tented to decrease with the increase in aqueous extract concentration of identical part. Ginger aqueous extracts reduced soil organic matter content with increased concentration, accompanying with NO3-—N accumulation in soil. Rhizome aqueous extract showed the same tendency for available P, available K and NH4+—N, namely lower concentration increased their contents in soil and higher concentration reduced their contents. While stem and leaf aqueous extracts reduced their contents with the increamental concentration. 4. All intercropping systems increased soil enzyme activities to different extent both at VGS and at HS compared to solo ginger. All intercropping systems increased the colony numbers of soil bacteria and total of soil microbe but not significantly either at VGS or at HS. All intercropping systems increased the colony numbers of soil fungi and actinomytes to a different extent (increase or decrease) both at VGS and at HS. For DI, difference between all cultivation patterns and S-G was not significant either at VGS or at HS except that G-S-C-G whose value was only 33.18% of S-G at VGS significantly decreased. G-S not only increased ginger yield by 19.6% but also obtained better economic benefit. Furthermore, all intercropping systems significantly inhibited occurrence of bacterial wilt of ginger. 5. Different cultivated pattern influenced plant height, tiller numbers, rhizome yields and intrinsic quality of ginger. Treatment 2 significantly facilitated tiller occurring (10.5%). Treatment 2, 3 and 4 promoted ginger growth (plant height respectively increased 15.0%、11.4% and 14.0%) and enhanced rhizome yields. Treatment 2 and 3 effectively increased vitamin C content (significantly increased 3.29% and 4.05% compared to solo ginger). Treatment 3 significantly increased contents of beneficial substances such as soluble sugar (8.2%), gingerols (4.6%) and protein. Treatment 2 significantly decreased contents of deleterious substance namely nitrate (14.0%) and improved appearance and intrinsic quality of ginger rhizome. Furthermore, treatment 2 (ginger/soybean intercropping) could obtain better economic benefit and showed the highest net income and ratio of benefit and cost whose values respectively increased by 24.80% and 8.8% compared to solo ginger.

Allelopathic interactions between the macroalga Ulva pertusa and eight microalgal species

HETEROSIGMA-AKASHIWO RAPHIDOPHYCEAE; CENTRAL VENICE LAGOON; ALEXANDRIUM-TAMARENSE; RED-TIDE; COASTAL LAGOONS; PHYTOPLANKTON; GROWTH; BAY; DINOFLAGELLATE; COMPETITION

Interactions between the bloom-forming dinoflagellates Prorocentrum donghaiense and Alexandrium tamarense in laboratory cultures

Interactions between Prorocentrum donghaiense and Alexandrium tamarens, two bloom-forming dinoflagellates, were investigated using bi-algal cultures. All R donghaiense died, but A. tamarense was hardly affected by the end of the experiment when the initial cell density was set at 1.0 X 10(4) cells mL(-1) for P. donghaiense and 0.28 x 10(4) cells mL(-1) for A. tamarense. However, significant growth suppression occurred in either species when the initial cell density of P donghaiense increased to I. 0 X 105 Cells mL(-1) in the bi-algal culture, but no out-competement was observed. The simultaneous assay on the culture filtrates showed that P donghaiense filtrate prepared at a lower initial density (1.0 X 10(4) cells mL(-1)) stimulated growth of the co-cultured A. tanzarense (0.28 x 10(4) cells mL(-1)), but filtrate at a higher initial density (1.0 x 10(5) cells mL(-1)) depressed its growth. The filtrate of A. tamarense at a density of 0.28 x 10(4) cells mL(-1) killed all R donghaiense at a lower density (1.0 x 10(4) cells mL(-1)), but only exhibited an inhibitory effect on it at a higher density (1.0 x 10(5) cells mL(-1)). It is likely that these two species of microalgae interfere with each other mainly by releasing allelochemical substance(s) into the culture medium, and a direct cell-to-cell contact was not necessary for their mutual interaction. The allelopathic test further proved that A. tamarense could affect the growth of co-cultured P. donghaiense by producing allelochemical(s); moreover, A. tamarense culture filtrate at the stationary growth phase (SP) had a strongly inhibitory effect on P donghaiense compared to that at the exponential phase (EP). Results also demonstrated a dose-dependent relationship between the microalgal initial cell density and the degree of the allelopathic effect. The growth of R donghaiense and A. tamarense in the bi-algal cultures was simulated using a mathematical model to quantify the interaction. The estimated parameters from the model showed that the inhibition exerted by A. tamarense on P. donghaiense was about 17 and 8 times stronger than the inhibition P. donghaiense exerted on A. tamarense, when the initial cell density was set at 1.0 X 10(4) and 1.0 X 10(5) cells mL(-1) for P donghaiense, respectively. and 0.28 x 10(4) cells mL(-1) for A. tamarense in the bi-algal cultures. A. tamarense seems to have a survival strategy that is superior to that of P. donghaiense in bi-algal cultures under controlled laboratory conditions. (c) 2006 Elsevier B.V. All rights reserved.