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.

花椒的化感作用对凋落物分解的影响及机理研究

花椒（Zanthoxylum bungeanum）是川西干旱河谷地区重要的经济作物，化感作用是花椒连作障碍的重要原因之一。花椒凋落物分解是影响花椒林地土壤肥力及花椒产量的重要因素，因此系统研究花椒化感作用是否对花椒凋落物的分解产生影响可以为解决花椒连作障碍导致的产量下降等问题提供科学的理论依据。本文通过室内模拟实验研究了花椒凋落物的四个分解动态以及分解后凋落物浸提液对花椒林地土壤性质的影响；通过野外盆栽实验研究了花椒凋落物浸提液对花椒幼苗的生长、花椒凋落物的质量及土壤性质的影响。最终从生理生化角度揭示了花椒的化感作用对凋落物分解的影响机理，为深入解决花椒连作障碍问题、对花椒凋落物采取有效的人工措施提供了科学的理论依据。主要的研究结果如下： 1. 室内分解实验证明，花椒凋落物在分解的60 d 内分解速率呈现由大到小的变化趋势，并且凋落物分解呈现明显的毒性动态。凋落物在分解的10 d、30 d 时，分解速率较大，30 d 以后分解速率显著降低。凋落物分解的10 d 左右酚酸释放量最大，此时凋落物的毒性最大，凋落物分解到10 d 以后，酚酸释放量逐渐减少，凋落物的毒性也逐渐减小。 2. 四个分解动态的花椒凋落物浸提液对土壤化学性质产生了显著的影响。花椒凋落物在分解的60 d 内，其浸提液使土壤pH值均显著的增加。分解0 d 的凋落物浸提液显著的降低了土壤铵态氮的含量，抑制了纤维素分解菌的生长；分解60 d 的凋落物浸提液显著的降低了土壤酚酸含量，增加了土壤有效磷的含量；分解30 d 和60 d 的凋落物浸提液均显著的促进了好气性纤维素分解菌的生长。这说明花椒凋落物在分解过程中呈现出明显的毒性动态：凋落物分解的初期毒性作用较大，随着分解的继续进行特别是在分解的30 d 以后，其毒性作用慢慢降低。 3. 花椒凋落物浸提液对花椒幼苗表现出明显的化感作用。不同浓度的浸提液对花椒幼苗地上及地下生物量、叶面积均产生了显著的抑制作用，并且随着浸提液浓度的升高抑制作用加强。凋落物浸提液对叶片厚度的影响较小，只有Y1对叶片厚度的生长抑制作用显著。 4. 花椒的化感作用改变了凋落物的质量，并对凋落物分解产生了显著的影响。对花椒幼苗用不同浓度的凋落物浸提液进行处理，Y1使凋落物有机碳含量、木质素含量、C/N、木质素/氮显著降低，纤维素含量显著升高；Y3使凋落物有机碳含量、木质素含量、C/N、木质素/氮显著升高。花椒凋落物质量的改变显著的影响了凋落物的分解，凋落物的分解速率大小依次为：Y1（10.15 a-1）> Y2（8.71 a-1）> CK（6.41 a-1）> Y3（5.08 a-1）。 5. 花椒的化感作用改变了土壤性质，并对凋落物分解产生了显著的影响。对花椒幼苗用不同浓度的凋落物浸提液处理的同时，也改变了土壤性质。不同浓度的凋落物浸提液显著的升高了土壤pH值、有机碳含量。各种浓度的凋落物浸提液对土壤多酚氧化酶的活性均起到了显著的促进作用。凋落物浸提液Y1对土壤纤维素分解酶的活性、细菌和真菌的生长也具有显著的促进作用。土壤性质的改变显著的影响了凋落物的分解，凋落物的分解速率大小依次为：Y1（10.30 a-1）>Y2（9.60 a-1）>CK（6.41 a-1）>Y3（6.29 a-1）。 6. 不论是凋落物质量发生改变还是土壤性质发生改变，在凋落物分解的整个过程中，C元素始终处于单调净释放的状态，并且C释放量与分解速率成显著的正相关，即凋落物分解越快，凋落物C释放量越大。凋落物分解过程中，均出现了酚酸大量释放的情况，并与凋落物分解速率成显著正相关。凋落物分解后的木质素含量、木质素/氮均增加，并且随着浸提液浓度的升高，凋落物木质素含量、木质素/氮升高。 Zanthoxylum bungeanum is an important economic crop in dry valley of the Minjiang river (Sichuan, Southwest China), but allelopathy is one of the important reasons for its continuous cropping. Zanthoxylum bungeanum litter decomposition affects Zanthoxylum bungeanum soil fertility and its output. So systemically investigate if allelopathy affects litter decomposition could provide the scientific methods to solve the problem of output fall caused by the continuous cropping. In this paper, the releasing dynamics of phenolic acid during Zanthoxylum bungeanum litter decomposition (0, 10, 30 and 60 days) and the effects of its aqueous extract on soil chemical properties were investigated via the laboratory study. Effects of Zanthoxylum bungeanum litter aqueous extract on the growth of young Zanthoxylum bungeanum seedlings, litter qualities and the soil qualities were investigated via the field study. Finally, we open out the action manner of Zanthoxylum bungeanum allelopathic effect on the litter decomposition, and provide the theoretical basis to solve the Zanthoxylum bungeanum continuous cropping. The main results showed that: 1. The laboratory litter decomposition experiment showed a trend of decomposition rate from large to small and an occurrence of phytotoxicity with clear dynamic patterns during Zanthoxylum bungeanum litter decomposition. The litter decomposition rate was larger at the tenth and 30th day during 60-day litter decomposition and gradually decreased after 30 days of litter decompostion. The releasing quantity of the litter phenolic acid was the highest at the tenth day, and here, the litter toxicity was the biggest. The releasing quantity of the litter phenolic acid gradually decreased after 10 days of litter decomposition, so the phytotoxicity of litter was gradually decreased with the litter decomposition. 2. The Zanthoxylum bungeanum litter aqueous extract after four decomposition stages had significantly effect on the soil chemical qualities. The pH value in soil was significantly increased in litter aqueous extract of four decomposition stages. The NH+4-N concentration was significantly decreased in soil amended with litter aqueous extract of 10-day decomposition which inhibited the growth of Aerobic cellulose-decomposer. The growth of soil Aerobic cellulose-decomposer was promoted by the litter aqueous extract of 30-day decomposition. Available phosphorus concentration was significantly increased and phenolic acid content was significantly decreased in soil amended with litter aqueous extract of 60-day decomposition which promoted the growth of Aerobic cellulose-decomposer. The study results showed an occurrence of phytotoxicity with clear dynamic patterns during Zanthoxylum bungeanum litter decomposition. The phytotoxicity of litter was the largest at the initial stage, but the phytotoxicity gradually decreased with the litter decomposition, especially after 30 days of decomposition. 3. The field study indicated that the Zanthoxylum bungeanum litter aqueous extract had significant allelopathic effects on the growth of young seedlings.Different concentration aqueous extract had signinficant inhibiting effects on biomass and leaf area of young seedlings. The inhibiting effect on the biomass strengthened with the litter aqueous extract concentration augment. Litter aqueous extracts had less effect on the leaf thickness, and only Y1 had significant inhibiting effect on the leaf thickness. 4. The Zanthoxylum bungeanum allelopathy had significant effect on the litter qualities and the litter decomposition. Treating the young Zanthoxylum bungeanum seedlings with different concentration of litter aqueous extracts, the leaf litter organic C, lignin, C/N and lignin/N all decreased and the cellulose content increased under Y1 treatment. The leaf litter organic C, lignin, C/N and lignin/N all increased under Y3 treatment. So the litter decomposition was significant affectded by the litter qualities, and the litter decomposition rate was Y1(10.15 a-1)> Y2(8.71 a-1) > CK(6.41 a-1) > Y3(5.08 a-1). 5. The Zanthoxylum bungeanum allelopathy had significant effect on the soil qualities and the litter decomposition. Treating the young Zanthoxylum bungeanum seedlings with different concentration of litter aqueous extracts, also changed the soil qualities. Different concentration of litter aqueous extracts had significant effects on the soil pH and organic C content. Every concentration of litter aqueous extracts accelerated the soil Polyphenol Oxidase activity and Y1 accelerated the soil Cellulase activity, the number of soil bacteria and fungi. So the litter decomposition was significant affected by the soil qualities, and the litter decomposition rate was Y1(10.30 a-1) > Y2 (9.60 a-1) >CK(6.41 a-1)>Y3(6.29 a-1)。 6. Whether the litter or soil qualities changed, the litter C element at the state of release at all times during the litter decomposition, and the release quantity increased with the decomposition rate augment. Litter released plentiful total penolics content during decomposition, and the release quantity had the positive correlation with the litter decomposition rate. The litter lignin content and the lignin/N all increased with the litter aqueous extracts concentration augment after litter decomposition.

CO2浓度升高对红桦幼苗生理与生长的影响

大气CO2浓度的增加已经成为不可争议的事实。预计本世纪末大气CO2浓度将增加到约700µmol mol-1。森林年光合产量约占陆地生态系统年光合产量的70%。森林树木是一个巨大的生物碳库，约占全球陆地生物碳库的85%。森林树木对CO2的固定潜力是缓解由大气CO2浓度升高引起的未来全球气候变化问题的决定性因子之一。红桦（Betula albosinensis Burk.）是川西亚高山采伐迹地自然或人工恢复的重要树种。本研究以1a红桦幼苗为模式植物，采用人工模拟的方法，研究CO2浓度升高对不同种内竞争强度（种群水平）下红桦幼苗的生理特征、生长、干物质积累及其分配的影响，探讨在种内竞争生长条件下红桦幼苗的“光合适应机理”与生长特征，为西南亚高山森林生产力对未来全球变化的预测提供重要参考。 本研究的主要结果如下： 1）在种内竞争生长条件下红桦幼苗经过CO2浓度升高熏蒸4个月后，叶片出现“光合适应”现象。与对照相比，低种植密度（28株m-2）和高种植密度（84株m-2）条件下的红桦幼苗净光合速率（A）、气孔导度（gs）、蒸腾速率（E）、表观量子产量（AQY）和羧化速率（CE）显著降低，而水分利用效率（WUE）则显著提高。CO2浓度升高处理的红桦幼苗叶片Rubisco活性、单位叶面积N浓度、叶绿素a、叶绿素b和类胡萝卜素浓度都显著降低。但CO2浓度对红桦幼苗的叶绿素a与叶绿素b的比值没有显著影响。CO2浓度升高显著增加红桦幼苗单位叶面积的非结构性碳水化合物（TNC）浓度，结果是红桦幼苗的比叶面积（SLA，cm2 g-1）显著降低。 2）与对照相比，CO2浓度升高处理的红桦幼苗高、基径、单叶面积和侧枝的相对生长速率（R GR）显著提高，尤其在试验处理的早期。CO2浓度升高既增加单株红桦幼苗总叶片数量又增加单叶面积，结果是单株红桦幼苗的总叶面积比对照显著增加。 3）CO2浓度升高处理显著增加红桦幼苗干物质积累（尤其是细根生物量），改变了红桦幼苗生物量的分配格局。与对照相比，CO2浓度升高处理的红桦幼苗叶重比（LWR）、叶面积比（LAR）、叶根重比（Wl/Wr）和源汇重比（leaf weight to non-leaf weight ratio, Wsource/Wsink）显著下降（高种植密度的LWR除外），而根冠比（R/S）则显著增加。在两种种植密度条件下，CO2浓度升高显著增加红桦幼苗根生物量的分配比率，显著降低叶片的生物量分配比率，对主茎、侧枝以及地上生物量的分配比率不变或约有下降。 总之，长期生长在CO2浓度升高条件下的红桦幼苗光合能力下降，并伴随Rubisco活性、叶N浓度、光合色素浓度的显著降低以及TNC浓度的显著增加。支持树木光合速率下降与Rubisco活性、叶N浓度下降以及TNC浓度增加紧密相关的假设。CO2浓度升高处理红桦幼苗的早期相对生长速率大大高于对照，而后期迅速下降，说明红桦幼苗生物量的显著增加主要归功于CO2浓度升高的早期促进作用和叶面积的显著增加。CO2浓度升高显著增加红桦幼苗根系生物量和根冠比，表明红桦幼苗“额外”固定的C向根系转移。 The steady increae of atmospheric CO2 concentration（[CO2]）has been inevitable fact. Models predict that the atmospheric [CO2] will increase to about 700µmol mol-1 at the end of the twenty-first century. As trees constitute a majoor carbon reservoir–85% of total plant carbon is found in forest, and their ability to sequester carbon is a key determinant of future global change problems caused by increases in atmospheric CO2. In addition to the role of forests in the global carbon cycle, inceased growth could be of economic benefit, for example, offsetting deleterious effects of climatic changes. Betula albosinensis (Burk.) usually emerges as the pioneer species in initial stage and as constructive species in later stages of forest community succession of mountain forest area, and also is one of important tree species for afforestation in logged area, in southwesten China. In this experinment, Betula albosinensis seedling (one-year-old) was used as the model plant. B. albosinensis seedlings were grown under two all-day [CO2], ambient (about 350 µmol·mol-1) and elevated [CO2] (about 700 µmol·mol-1), and two planting densities of 28 plants per m2 and 84 plants per m2. The objectives were to characterize birch mature leaf photosynthesis, growth, mass accumulation and allocation responses to long-tern elevated growth [CO2] under the influences of neighbouring plants, and to assess whether elevated [CO2] regulated birch mature leaf photosynthetic capacity, in terms of leaf nitrogen concentration (leaf [N]), activity of ribulose bisphosphate carboxygenase (Rubisco), Rubisco photosynthetic efficiency, and total nonstructural carbohydrates (TNC) concentration, and also to provide a strong reference to predict the productivity of subalpine forests under the future global changes. The results are as follows: 1) B.albosinensis seedlings exposed to elevated [CO2] for 120 days, photosynthetic acclimation phenomena occurred. At two planting densities, leaves of birch seedlings grown under elevated [CO2] had lower net photosynthetic rate (A), stomatal conductance (gs), transpiration (E), apparent quantum yield (AQY) and carboxylated efficiency (CE) and higher water use efficiency (WUE), compared to those of B.albosinensis seedlings grown under ambient [CO2]. Based on the leaf area, leaf [N], Rubisco activity and photosynthetic pigments concentrations of B. albosinensis seedlings grown under elevated [CO2] were significantly lower than those grown under ambient [CO2]. The ratio of chlorophyll a to chlorophyll b concentration was not affected by elevated [CO2]. Under elevated [CO2], the TNC concentration per unit leaf area significantly increased, resulting in significant decrease in specific leaf area. Thus leaf photosynthetic capacity of B. albosinensis seedlings would perform worse under rising atmospheric [CO2] and the influences of neighbouring plants. 2) Under elevated [CO2], the relative growth rate (RGR) of B. albosinensis seedlings height, basal diameter, a leaf area and branch length significantly increased, especially at the initial stage of exposure to elevated [CO2], and a leaf area and leaf numbers per B. albosinensis seedling also significantly increased. Thus the total leaf area per B. albosinensis seedling was significantly increased under elevated [CO2]. 3) As the increase of RGR and total leaf area, biomass of B. albosinensis seedling grown elevated [CO2] was higher, compared to that of B.albosinensis seedlings grown at ambient [CO2]. Elevated [CO2] changed the biomass allocation pattern of B. albosinensis seedling. At two planting densities, B. albosinensis seedlings grown elevated [CO2] had lower leaf weight to total weight ratio (LWR), leaf area to total weight ratio (LAR) and leaf weight to non-leaf weight ratio (Wsource/Wsink), but higher root weight to shoot weight ratio (R/S), compared to those of B.albosinensis seedlings grown at ambient [CO2]. Under elevated [CO2], roots biomass to total biomass ratio was signigicantly increased, leaves biomass to total biomass ratio was significantly decreased. The main stem and branch biomass to total biomass ratio were not affected by elevated [CO2]. In conclusion, our results supported the hypothesis that the decline in photosynthetic capacity of C3 plants will appear after long-term exposure to elevated [CO2], accompanying with the significant decrease in Rubisco activity, leaf N concentration, photosynthetic pigments concentration, and significant increase in total non-structural carbohydrates concentration. Our results also have shown that the increase of biomass of B. albosinensis seedlings should be attributed to initial stimulation on RGR and total leaf area resulted from elevated [CO2]. Under elevated [CO2], the extra carbon sequestered by B.albosinensis seedlings transferred into under-ground part because of increase in root biomass and R/S.

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.

活性炭纤维(ACF)吸附法处理河流突发污染事故模拟研究

环境突发污染事故给人民生活、经济发展和生态环境造成重大影响，研究污染物泄漏造成河流突发污染事故的应急处理方法十分必要。本论文选取苯酚、苯胺和亚甲基蓝等典型污染物为实验对象，采用吸附容量大、密度与水接近的活性炭纤维(ACF)为吸附剂。在自制的河流模型中，研究了ACF以苯酚、苯胺和亚甲基蓝为典型污染物的吸附过程，考察了吸附剂投加量、污染物浓度、吸附剂比表面积、吸附剂投加方式、水流速度与水质等对吸附速率与吸附效果的影响。实验结果表明，ACF能以较快的速率吸附苯酚、苯胺和亚甲基蓝，吸附率都在95％以上； ACF投加量是影响吸附速率最重要的因素，当一次性投加ACF质量之比为 1:2:4时，吸附速率常数之比近似为1:2:4；污染物浓度对吸附速率的影响显著，浓度较低时吸附速率较高。苯酚初始浓度为7mg·L-1时，经过86分钟的吸附，处理后的浓度可以达到地表水Ⅴ类水中挥发酚的限值要求(0.1mg·L-1)；在吸附11分钟左右追加适量的ACF，能够明显提高吸附速率；河水流速和河流中的天然有机物、浊度、河水硬度对ACF吸附都不产生显著影响，这说明ACF作为河流突发污染事故应急处理的吸附剂，有广泛的适应性。在实际河水中，ACF对苯酚的吸附过程与在模拟河水中相似，吸附效果显著。实验结果还表明，ACF对苯酚的吸附是放热反应，符合Freundlich模型和Langmuir模型。事故应急处理后，应该及时将吸附了污染物的ACF打捞上来，有利于进行后续处理。 Emergency environmental pollution accidents pose significant impacts on our living, economic development and ecological environment. The study on the approach of emergency control for the contingency caused by leakage of pollutants in rivers is very necessary. In the experiment, phenol, aniline and methylene blue were selected as representative pollutant and activated carbon fiber (ACF) was selected as adsorbent, which has strong adsorption capacity and similar density to water. In the self-made river model, the effects of ACF dosage, pollutant concentration, ACF surface area, ACF adding ways, water flow rate and water quality on adsorption courses were investigated. The experimental results showed that ACF could adsorb pollutant quickly and effectively. The ACF dosage was the most important factor that affected adsorption rate .When the ACF dosage rate was 1:2:4, the constants of adsorption rate was approximately 1:2:4. The effect of pollutant concentrations on the adsorption rate was notable. Faster adsorption rates were achieved at low pollutant concentrations. Phenol concentration reached the limits of volatile phenol in Category Ⅴ surface water (0.1 mg·L-1) after 86 minutes of adsorption with initial phenol concentration of 7 mg·L-1. After 11 minutes of adsorption, certain amount of ACF was added and the adsorption rate was improved significantly. River flow rate and water quality have little effect on the adsorption rate. The adsorption results obtained in actual river water were comparable with that in simulating river water. The results also showed that, ACF on the absorption of phenol is exothermic reaction, witch matched with the Freundlich model and the Langmuir model. After emergency treatment, the ACF absorbed pollutants should be promptly salvaged for follow-up treatment.

菌剂强化处理炼油废水及苯酚冲击下功能菌响应机制研究

近年来，我国炼油行业发展迅速，炼油能力全世界第二，炼油行业已成为污染大户。本研究针对炼油废水生物处理中存在的稳定达标难、抗冲击负荷能力差、建设投资与运行成本高等问题，就菌剂强化处理炼油废水中试与工程应用展开了研究，以期为菌剂的工程应用与推广提供理论参考与技术支持；并以炼油废水中的主要特征污染物苯酚为研究对象，考察了不同浓度苯酚冲击下功能菌的响应机制，并以此为指导研制功能菌激活促进剂，考察其对功能菌生物学指标的调控效果，以期为废水生物处理有毒污染物冲击调控提供理论依据与技术支持。 中试研究表明，菌剂强化处理炼油废水，出水COD、NH4+-N 平均值为86.7、7.6 mg/L，其平均去除率较常规生物处理系统分别提高了35.47%、59.28%，其耐受COD、NH4+-N 容积负荷分别高达2.42、0.139kg/(m3·d)，具有良好的耐冲击能力。工程应用研究表明，菌剂强化处理炼油废水，出水COD、NH4+-N 平均值分别为85.05、8.4mg/L，其去除率较常规生物处理系统提高了25.1%、28.7%，出水水质各项指标均达到了国家《污水综合排放标准GB 8978-1996》一级排放标准。技术经济分析表明，菌剂强化处理炼油废水在建设成本、运行成本上分别降低38%、49%，具有良好的技术经济优势。 苯酚冲击下功能菌响应机制研究表明：不同浓度苯酚冲击下，生物学指标生物量、脱氢酶酶活、1,2-双加氧酶酶活对冲击都有不同程度的响应，其响应敏感程度为脱氢酶酶活＞生物量＞1,2-双加氧酶酶活。1,2-双加氧酶酶活与COD 降解率相关性良好，可表征苯酚降解过程，确认为调控重点。以此为指导研制出苯酚降解功能菌抗冲击激活促进剂，可有效调控功能菌对有毒污染物苯酚的降解效果，1000mg/L 苯酚冲击下，经调控，其COD 去除率较对照提高20%，降解时间缩短16%以上。其对生物学指标的调控效果为1,2-双加氧酶酶活＞生物量＞脱氢酶酶活，验证了功能菌在苯酚冲击下的响应机制。研究表明菌剂强化处理炼油废水切实可行，具有良好的技术经济优势。有毒污染物冲击下废水生物处理系统响应机制研究为抗冲击调控提供了新的研究思路。 Currently, China’s oil refining industry is developing rapidly and has become the second largest all over the world. The oil refining industry is one of the major pollution industries in our country. The pilot scale study and engineering application research were conducted aiming at the problems in refining wastewater such as poor treatment stability and water quality, poor anti-shock capacity and expensive running cost, etc., so as to provide theoretical references and technological supports for the engineering application and popularization of microbial preparation in wastewater treatment. Also, the response mechanism of functional microbe under shock of different phenol concentrations, which is the main pollutants in refinery wastewater, was studied. Based on this result, functional microbe activation accelerator was developed, and the regulation effect of functional microbe biological index under phenol shocking were studied, in order to provide theoretical basis and technological support for regulation of toxic shocking of wastewater biological treatment. The result of pilot scale research indicated: for treatment of refinery wastewater in bioaugmention treatment system of microbial preparation, the COD and NH4+-N average value of effluent was 86.7 and 7.6 mg/L, Comparing with normal biological treatment system, the average removal rates of COD, NH4+-N increased 35.47%,59.28% separately by bioaugmention treatment system, which showed better anti-shocking capacity, the volumetric load r of COD and NH4+-N reached 2.42 kg/(m3·d) and 0.139 kg/(m3·d), respectively. The research on engineering application of refinery wastewater bioaugmentation treatment by microbial preparation indicated:the average concentrations of effluent COD and NH4+-N in the bioaugmentation treatment system were 85.05 and 8.4mg/L, which increased by 25.1% and 28.7% comparing with normal biological treatment system of refinery wastewater, And the effluent quality meets the first grade of discharging standard of National Integrated Wastewater Discharge Standard GB 8978-1996. The economic analysis of technology indicated: the demonstration project of bioaugmentation treatment of refinery wastewater by microbial preparation decreased by 38% in construction cost and 49% in running cost. This technology has economic benefits. The response mechanism of functional microbe under phenol shock indicated: biological index such as the biomass concentration, dehydrogenase and 1,2-dioxygenase had different responses under phenol shocking of different concentrations. The response sensitivity of different biological index under phenol shocking of different concentrations is: dehydogenase activity ＞ biomass ＞1,2-dioxygenase activity, and high correlation of 1,2-dioxygenase and COD degradation percentage is achieved, thus 1,2-dioxygenase could be used to reflect the degradation situation of pollutants. So, 1,2-dioxygenase is the keypoint of regulation. The anti-shock activation accelerator of phenol degradation functional microbe was primarily developed. The results indicated: the activation accelerator could regulate the degradation effect of toxic substance-phenol by functional microbe effectively. For the functional microbe treatment system under phenol shocking of 1000mg/L, the COD degradation rate increased by 20% and the degradation time reduced by more than 16% under regulation of activation accelerator. The regulation effects of biological index are: 1,2-dioxygenase ＞ biomass ＞ dehydrogenase. In this way, the response mechanism of functional microbe under toxic shocking is verified. The result indicated: the augmented microbial preparation treatment of refinery wastewater is applicable. It has many technical and economical advantages. The research results of responses mechanism of wastewater treatment system on toxic pollutants would offer a new idea for regulation of anti-shock.

组合白腐真菌预处理木质纤维素原料的研究

木质纤维素原料种类多、分布广、数量巨大，通过燃料乙醇生产技术、厌氧沼气发酵技术将其转化成乙醇、沼气等二次能源，一定程度上可以缓解化石能源的不断消耗所带来的能源危机，也解决了农林废弃物引起的环境污染问题。其中以木质纤维素原料生产燃料乙醇，还可以避免以淀粉类和糖类原料生产燃料乙醇时带来的“与人争粮”等一系列问题。因此具有重要的经济效益、环境效益和社会效益。 然而，木质纤维素原料结构致密，木质素包裹在纤维素、半纤维素外围，导致其很难被降解利用，必须进行适当的预处理，去除木质素，打破原有的致密结构，利于原料的后续利用。因此，预处理成为木质纤维素原料能源化利用的关键。而目前预处理环节的费用过于昂贵，于是寻找一种高效、低成本的预处理方法是当今研究的热点。 本论文采用组合白腐真菌对木质纤维素原料进行生物预处理研究，与其他物理化学法相比，该法有着专一性较强、反应温和、不造成环境污染、成本低等优势。白腐真菌主要通过分泌木质素降解酶对木质素进行降解，从而破坏原料的致密结构，提高后续利用效率。所以木质素降解酶酶活的高低是影响原料预处理效果的一个关键因素。于是本论文首先通过将白腐真菌进行组合的方式提高木质素降解酶（漆酶，Lac）酶活；接着对组合菌的菌株相互作用机理进行研究，阐明组合菌Lac 酶活提高的原因，为菌株组合提高Lac 酶活这种方法的应用提供理论依据，同时也为后续组合白腐真菌预处理木质纤维素原料提供指导；进一步采用固态发酵和木质素降解酶两种方式对木质纤维素原料进行预处理研究，最大化去除木质素成分，破坏原料的致密结构；最终对预处理后原料的酶解糖化进行初步研究，为原料后续的能源化应用奠定基础。具体研究结果如下： （1） 以实验室保存的三株主要分泌Lac 的白腐真菌为出发菌株，筛选得到一组Lac 酶活明显提高的组合菌55+m-6，其中菌株55 为Trametes trogii sp.，m-6 为Trametes versicolor sp.，组合后Lac 酶活较单菌株分别提高24.13倍和4.07 倍。组合菌的最适产酶条件为pH 6.5、C/N 16:1、Tween 80 添加量为0.01%，在该条件下组合菌的Lac 酶活峰值比未优化时提高4.11倍。 （2） 对组合菌55+m-6 菌株间相互作用机理进行研究，发现菌株之间不存在抑制作用；平板培养时，菌丝交界处Lac 酶活最高并分泌棕色色素；液体培养时，菌株m-6 对组合后Lac 酶活的提高起着更为重要的作用：菌株m-6的菌块、过滤灭菌胞外物以及高温灭菌胞外物均能明显刺激菌株55 的Lac产生；菌株55、m-6 进行组合后，同工酶种类未发生增减，但有三种Lac同工酶浓度有所提高；对菌株胞外物进行薄层层析和质谱分析，结果表明组合前后菌株胞外物中各物质在浓度上存在较大的变化。推测组合菌Lac酶活的明显提高，主要是由于菌株m-6 胞外物中的一些物质能刺激菌株55 分泌大量Lac 进行代谢，且这些刺激物质并非菌株m-6 特有，菌株55自身也可以代谢生成，但是适当的浓度才能刺激Lac 的大量分泌。 （3） 将组合菌55+m-6 用于固态发酵预处理木质纤维素原料，发现其对玉米秆的降解程度最大，在粉碎度40 目、含水率65%的最优处理条件下，处理至第15d，秸秆失重率为41.24%，其中木质素、纤维素、半纤维素均有降解，且Lac 和纤维素酶（CMC）酶活以及还原糖量均达到峰值。 （4） 对玉米秆进行木质素降解酶预处理，发现Lac/1-羟基苯并三唑（HBT）系统对玉米秆木质素的降解效果最好，在最优处理条件时，即HBT 用量0.2%、处理时间1d、Lac 用量50U/g，木质素降解率可达12.60%。预处理后玉米秆的致密结构被破坏，比表面积增大，利于后续酶与纤维素、半纤维素成分的结合。 （5） 对预处理后的玉米秆进行酶解糖化，其中组合菌固态发酵预处理后玉米秆的糖化率比对照高4.33 倍；Lac/HBT 系统预处理后玉米秆的糖化率比对照高2.99％，糖化液中主要含有木糖、葡萄糖两种单糖。 There are many kinds and large quantities of lignocellulosic biomass widely distributed on the earth. They can be converted into secondary energy such as fuel ethanol, biogas, et al., which can relieve the energy crisis caused by consumption of fossil energy resources and solve the problem of environmental pollution caused by agriculture and forestry waste. Meanwhile, the production of fuel ethanol from lignocellulosic biomass can ensure food supply to human kind instead of starch- and sugar-containing raw materials. So the energy conversion of lignocellulosic biomass contributes considerable economic, environment and social benefits. However, lignocellulosic biomass has the compact structure, in which lignin surrounds cellulose and hemicellulose, so it must be pretreated before energy usage and pretreatment is one of the most critical steps in the energy conversion of lignocellulosic biomass. At present, the cost of pretreatment is too expensive, so looking for an efficient and low-cost pre-treatment method is one of recent research hot spots. In this research, combined white rot fungi pretreatment method was used, which had some advantages in low cost, high specificity, mild reacting conditions and friendly environmental effects compared with the other physical and chemical methods. White rot fungi secrete lignin degrading enzymes to degrade the content of lignin and damage the contact structure of lignocellulosic biomass, so the activity of the lignin degrading enzymes is the key factor to the degradation effect of raw materials. Firstly, the combined fungi with high laccase activity were screened; secondly, the interaction mechanism between strains was studied, and the cause of higher laccase activity after strains combination was also preliminary clarified; under the guidance of the mechanism, lignocellulosic biomass was pretreated by the combined fungi; lastly, the enzymatic hydrolysis of pretreated lignocellulosic biomass was also preliminary studied; all of the researches could lay the foundation for the energy application of lignocellulosic biomass. The specific research results were as follows: (1) The combined fungi 55+m-6 with significant higher laccase activity were screened from the three white rot fungi stored in our lab which mainly secreted laccase. Strain 55 and strain m-6 were Trametes trogii sp. and Trametes versicolor sp., respectively. The laccase activity of combined fungi was 24.13 and 4.07-fold than strain 55 and strain m-6, respectively. The optimized condition for laccase production of the combined fungi in liquid medium was pH 6.5, C/N 16:1 and Tween 80 0.01%. In this optimized condition, the laccase activity of combined fungi was 4.11-fold higher comparing with which in non-optimized medium. (2) The interaction mechanism between strain 55 and strain m-6 was further studied, and no inhibition effect was observed. Brown pigment was secreted on the junction of the two strains on the plate, where the highest laccase activity was detected. Strain m-6 was much important to boost laccase activity of combined fungi in liquid medium, and strain 55 was stimulated by fungal plug, filter sterilized extracellular substances and high temperature sterilized extracellular substances of strain m-6 to produce laccase. The types of laccase isozymes did not change after combining strain 55 and strain m-6, but the concentrations of three types increased. Mass Spectrometry and TLC analysis of extracellular substances of each strain showed that concentration of some substances considerably changed after strains were combined. It was supposed that the cause of higher laccase activity of combined fungi was mainly due to some extracellular substances of strain m-6 with the appropriate concentration which stimulated laccase secretion of strain 55 and generated not only by strain m-6 but also by strain 55. (3) Combined fungi 55+m-6 were used to lignocellulosic biomass pretreatment with the type of solid-state fermentation. The highest degree of degradation of corn straw was obtained, including the rate of weight loss was 41.24% and the lignin, cellulose and hemicellulose were degraded partially under the optimized condition of 40 mesh, 65% water content on 15th day. Laccase, CMCase activities and content of reducing sugar reached the maximum value on that day. (4) Lignin degrading enzymes from combined fungi 55+m-6 were used for corn straw pretreatment. The most remarkable degradation of lignin in corn straw with Lac/1-hydroxybenzotriazole (HBT) system was observed, and the 12.60% lignin degradation was obtained under the optimized condition of 0.2% HBT, 50 U/g laccase for 1 d. After pretreated by Lac/HBT, the tight structure of corn straw was demolished and specific surface area increased, which had advantages for accessible of enzyme to cellulose and hemicellulose. (5) The corn straws pretreated by combined fungi 55+m-6 with the type of solid-state fermentation and Lac/HBT were used for enzymatic hydrolysis, and the saccharification rates of each pretreatment type were 4.33 times and 2.99% higher than CK, respectively. The enzymatic hydrolysis liquid of corn straw pretreated by Lac/HBT mainly contained xylose and glucose.