杉木连栽致害真菌拮抗菌Bacillus subtilis3728的研究

杉木是我国重要的速生丰产树种，分布在北纬21°41′到33°41′，东经102°到122°的广大地区，杉木人工林面积约占我国人工林总面积的1/4，随着连栽代数的增加，土壤中毒和生产力下降程度日趋严重。 本论文以分离自与红树林、珍珠贝、海兔子、海绵、软珊瑚等与海洋动、植物共栖或共生存的106株海洋微生物(54株放线菌，52株细菌)为资源，以杉木连栽致害真菌尖孢镰刀菌萎蔫专化型(Fusarium oxysporum f.sp.vasinfectum)菌株SF2为靶菌，通过平板对峙试验和土壤原位定殖试验，筛选到一株分离自红树林木榄(Bruguiera gymnorrhizo)根际土壤的海洋细菌3728菌株；该菌对SF2具有很强抑菌活性，能够高密度在杉木根际土壤中定殖，对杉木幼苗的生长有一定的促进作用。采用传统的细菌学和分子生物学的鉴定方法对其进行了菌种鉴定，为枯草芽孢杆菌(Bacillus subtilis)。 通过对抗菌谱的研究，发现海洋细菌3728除了对杉木连栽主要致害真菌尖孢镰刀菌萎蔫专化型菌株SF2有很强的抑菌活性外，对大豆连作致害菌 (Penicillium purpurogenum)，棉花枯萎病菌(Fusarium oxysporum)，棉花立枯病菌(Rhizoctonia solani)，大豆根腐病菌(Fusarium solani)以及小麦赤霉病菌(Fusarium graminerum)等也有较强的抑制作用。室内模拟试验还表明，在土壤中接种海洋细菌3728后，能够明显增加土壤中氨化细菌和氨化真菌的数量，能够增加土壤中功能性微生物——纤维素分解细菌和纤维素分解真菌的数量和种类，增强了纤维素分解能力。再添加C/N比较低的白三叶草凋落物，土壤中氨化细菌、氨化真菌的数量继续增加，土壤纤维素分解能力更显著提高。这为进一步开展对杉木连栽障碍的生物调控试验，提供了一定的科学依据。

中国竹材腐朽真菌多样性初步研究

生物多样性是人类赖以生存的物质基础，菌物多样性是生物多样性的重要组成。我国位于亚太竹区，是世界竹类的分布中心之一，有着极为丰富的竹种资源，并孕育着丰富的菌物资源。近年来，我国对木材腐朽菌，特别是多孔菌进行了广泛深入的研究，发表了许多新种和中国新记录种，但是对生于竹材上的腐朽菌却少见报道。因此，开展竹材腐朽真菌种类的研究对深入认识腐朽菌物种多样性，积极利用这些菌物资源，防治竹类病害以及竹材保藏都具有重要意义。 本论文按照多孔菌现代分类学方法对采集自我国10省市18个自然保护区、森林公园和林场的竹材腐朽真菌标本进行了初步的研究。对11种重要的竹材腐朽菌进行详细的描述和显微结构绘图，记载了每种的寄主、国内分布，并对每种与其相似种的联系和区别以及腐朽类型进行了讨论。 研究结果显示，我国范围内共记录及描述竹材腐朽真菌从5属增加到28属，新增属分别为：假芝属、薄孔菌属、小薄孔菌属、蜡孔菌属、拟蜡孔菌属、小集毛孔菌属、囊孔菌属、浅孔菌属、圆齿菌属、产丝齿菌属、耙齿菌属、容氏孔菌属、锐孔菌属、多年卧孔菌属、木层孔菌属、硬孔菌属、中国干腐菌属、干皮孔菌属、褶菌属、栓孔菌属、附毛孔菌属、孢孔菌属和中国记录属拟浅孔菌属 (Grammotheloposis)，其中广义多孔菌有20属，革菌2属，齿菌1属；有15属为白腐菌，1属褐腐。 新增竹材腐朽真菌19种，其中多孔菌有15种，革菌1种，齿菌1种，褶菌2属。相似干朽菌(Serpula similis)和棕榈浅孔菌（Grammothele fuligo）竹区分布较多，前者是腐朽病原菌，后者腐朽作用不强，但影响竹材美观。二者在中国南方竹区广泛分布，是对竹材危害较严重的种类。而长江以北分布极少。另外有药用菌4种，分别为浅黄囊孔菌（Flavodon flavus）、宽棱木层孔菌(Phellinus torulosus)、裂褶菌(Schizophyllum commune)和灵芝(Ganoderma lucidum)。待定种1种(Grammotheloposis sp.)。

模拟酸雨对马尾松菌根影响研究

本实验表明：外生菌根真菌彩色豆马勃、劣味乳菇、丝膜菌对PH的适应范围较广，最适生长BH呈酸性。模拟酸雨对马尾松幼苗菌根的外部形态和内部结构有明显影响。在温室栽培中，模拟酸雨（PH2.0）显著抑制菌根侵染率，在田间实验中，对菌根侵染率有一定的影响。菌根PH和土壤PH值随模拟酸雨PH下降而逐渐降低，接种菌根菌可略提高菌根PH和土壤PH值。菌根真菌过氧化氢酶对培养基中PH的变化不敏感，模拟酸雨对菌根过氧化氢酶活性影响也不明显。但沙培中，模拟酸雨（PH2.0）显著抑制菌根过氧化氢酶活性。模拟酸雨（PH2.0）显著刺激菌根过氧化物酶活性，接种菌根菌可以降低菌根过氧化物酶活性。不同PH的培养基对菌体硝酸还原酶活性有明显影响，而且菌体生长速度与硝酸还原酶活性呈正相关。模拟酸雨（PH2.0）显著抑制菌根硝酸还原酶活性，而接种菌根菌明显提高根系硝酸还原酶活性。菌体酸性磷酸酶活性对培养基中PH变化不敏感，同样菌根酸性磷酸酶活性对模拟酸雨的影响也不明显，但是接种菌根菌可明显提高根系酸性磷酸酶活性。模拟酸雨对马尾松幼苗茎的高生长影响不显著。但是对幼苗茎、根系的干重和侧根总长度有显著抑制作用。轻度酸雨（PH4.5-3.0）对马尾松幼苗生长有促进作用，接种菌可提高幼苗生长。从菌根形态结构和生理活性上看，接种菌根菌可减轻模拟酸雨对马尾松幼苗根系的危害，增强对模拟酸雨的抗性。4dThe result of experiment showed that ectomycorrhizal fungi Pisolithus tinctorins. Lactarius insulsus. Cortinarius russus can be growth in broad PH rang in pure culture, the optimum growth PH is acidity. The external morphology and internal structure of ectomycorrhiza of P. massoniana are affected with simulated acid rain. In greenhouse, simulated acid rain (PH2.0) treatment caused significant decrease in the percent infection, but it's not marked in field. The PH of mycorrhizal and soil are reduced with reducing rainfall PH. These PH are slight higher for inoculation with ectomycorrhizal fungi. Catalase activity of ectomycorrhizal fungus is not sensitive to medium with different PH. Mycorrhiza catalase activiyt is not affected significantly with simulated acid rain, but it's inhibited significantly with simulated acid rain (PH2.0) in the sand culture. Peroxidase atcivity of mycorrhiza is enhanced significantly with simulated acid rain (PH2.0), but it's universally lower for inoculation with ectomycorrhizal fungus. Ectomycorrhizal fungus nitrate reductase activity is affected significantly to medium with differdnt PH, the rates of these fungi growth and nitrate reductase activity is significant correlation. Nitrate reductase activity of mycorrhiza is inhibited significantly with simulated acid rain (PH 2.0), but it's increased significantly for inocnlation with mycorrhizal fungi. Ectomycorrhizal fungas acid phosphatase activity is not affected to medium with different PH, Mycorrhiza acid phosphatase activity is not affected with simulated acid rain too, the acid phosphatase activity of roots inoculated with mycorrhizal fungas is increased significantly. The highest acidity level simulated rain reduced signhficantly root system biomass and the dry weight of stem. Iower acidity level simulated rain can stimulated the growth of P. massoniana, the growth of seedling inocnlated with mycorrhizal fungus can be increased.

吸水链霉菌、放线菌Sp.253和金色毛壳菌的次生代谢产物研究

链霉菌是十分重要的一类放线菌，绝大多数的抗生素都由该类细菌产生。毛壳属真菌是一类重要的丝状真菌，从中也发现有很多结构新颖、活性独特的活性物质。因此本论文对两株链霉菌的活性成分及一株金毛壳菌的次生代谢产物进行了研究。 1．从吸水链霉菌（Streptomyces hygroscopicus 1.358）液态发酵产物（乙酸乙酯提取物）中分离得到3个化合物，通过波谱方法鉴定为RK955A (1)、Nigericin（2）、Elaiophylin（3）。以青霉素耐药-金黄色葡萄球菌作为指示菌的抗菌活性测定表明，三者均具有较强抗菌活性。 2．通过抗肿瘤体外活性筛选模型筛选得到得到一株链霉属土壤放线菌，从中分离得到六个化合物：苯乙酰胺（4）、苯丙酰胺（5）、肉桂酰胺（6）、3-（N-（甲酰胺基）乙酰基）吲哚（7）、鸟苷磷酸（8）、鸟苷（9）。 3．从金色毛壳菌（Chaetomium aureus）的固态培养物中分离得到13个化合物，利用波谱方法将其鉴定为：金毛壳菌素A（10）、金毛壳菌素B（11）、Eugenetin（12）、Eugenitol（13）、Chaetoquadrin A（14）、Chaetoquadrin B（15）、Chaetoquadrin G（16）、Chaetoquadrin H（17）、Chaetochromin A（18）、Sterigmatocystin（19）、O-methylsterigmatocystin（20）、3β-羟基-麦角甾-5，7，22-三烯（21）和过氧麦角甾醇（22）。 4．综述了聚醚类抗生素的结构、生物合成、生物活性及作用机理。 The genus Streptomyces (Actinomycetes) is an important group of microbe. Most antibiotics known nowdays are discovered from species of Streptomyces. The fungi of the genus Chaetomium have attracted much attention because various kinds of secondary metabolites with diverse bioactivities have been found from them. Thus, the bioactive compounds from two strains of Streptomyces and the secondary metabolites of Chaetomium aureus were investigated. 1. Three compounds were isolated from the ethyl acetate extract of the fermentation broth of Streptomyces hygroscopicus. They are identified to be elaiophylin (1), nigericin (2), and antibiotic RK955A (3) on the basis of their spectroscopic data. Compounds 1-3 possess antibacterial activities against Staphyloccocus aureus. 2. It was found that the extract of the fermented broth of a strain of Actinomycetes could inhibit some tumor cel lines. Separation of the bioactive fraction led to the isolation of six compounds. They were characterized to be phenylacetamide (4), phenylpropylamide (5), trans-cinnamamide (6), 3- (N- (formylmethyl) acetamide) indole (7), guanylicacid (8), and guanosine (9). 3. From the fermented broth of Chaetomium aureus, 13 compounds were isolated for the first time. They were determined to be chaetomiumycin A (10), chaetomiumycin B (11), eugenetin (12), eugenitol (13), chaetoquadrin A (14), chaetoquadrin B (15), chaetoquadrin G (16), chaetoquadrin H (17), chaetochromin A (18), sterigmatocystin (19), O-methylsterigmatocystin (20), 3β-hydroxyergosta-5, 7, 22-triene (21) and peroxy-ergosterol (22). Compounds 10 and 11 are new ones. 4. Structure, biosynthesis, biological activity, and mechanisms of polyether antibiotics were reviewed.

五种毛壳霉属和曲霉属真菌代谢产物及细胞毒活性

毛壳霉属(Chaetomium)和曲霉属(Aspergillus)真菌产生多种具有生物活性的化合物。为系统阐明两属微生物的次生代谢物，对三种毛壳霉、两种曲霉真菌分别进行固态发酵，以色谱和波谱技术研究发酵物中的成分，分离鉴定了51个化合物，其中23个为新化合物，测试了部分化合物对肿瘤细胞的活性。 1、从螺卷毛壳霉（C. cochloides）固态发酵物中分离鉴定了11个化合物，3个新化合物为螺卷毛壳霉素A~C（1~3）。化合物1、3及dethio-tetra (methylthio) chetomin（4）对Bre-04、Lu-04和N-04细胞株生长抑制的GI50值为0.05~7.0 μg/mL。 2、从印度毛壳霉（C. indicum）固态发酵物中鉴定的三个异喹啉生物碱印度毛壳霉素A~C（12~14）代表两类骨架新颖的异喹啉生物碱。 3、从巴西毛壳霉（C. brasiliense）固态发酵物中鉴定了11个化合物，其中Mollicellins I~J（15~16）、2-Hydroxymethyl-6-methylmethyleugenin（19）为新化合物。化合物16和Mollicellin H（18）对Bre-04、Lu-04、N-04细胞株生长抑制的GI50值在2.5~8.6 μg/mL。 4、从土曲霉（A. terreus）固态发酵物中鉴定了18个化合物。5个为新化合物为Terretonin A~D（24~27）和Asterrelenin（28），24~27为二倍半萜化合物，28为吲哚生物碱。 5、从杂色曲霉(A. versicolor）固态发酵物中鉴定了16个化合物。9个新的化合物Brevianamides K~N (40~43)、Averins A~C (44~46)和Glyanphenines A~B (47~48)代表三种类型的生物碱。 6、综述了1997－2007年间新的二倍半萜的研究进展。 The fungi of the genera Chaetomium and Aspergillus produce various secondary metabolites with biological activities. In order to systematically study the secondary metabolites, the solid-state fermented rice culture of three species of Chaetomium and two of Aspergillus were chemically studied. By the means of chromatograhy and spectroscopy, 55 compounds were isolated and identified, among of them 23 were new ones. The biological activities of some compounds were investigated. 1. From the fungus C. cochliodes, three new epipolythiodioxopiperazines, chaetocochins A-C (1-3) were isolated, together with 8 known ones (4-11). Compounds 1, 3 and 4 showed growth inhibitory effects against cancer cell lines Bre-04, Lu-04 and N-04 with GI50 values from 0.05 to 7.0 μg/mL. 2. Three novel isoquinolines Chaetoindicins A-C (12-14) were isolated and identified from the fungus C. indicum. Chaetoindicin A, Chaetoindicins B-C represented two classes of novel carbon skeletons. 3. Three new compounds, Mollicellins I-J (15-16), and 2-hydroxymethyl-6-methylmethyleugenin (19), were isolated from C. brasiliense. Compound 16 and Mollicellin H (18) showed growth inhibitory effects against cancer cell lines Bre-04, Lu-04 and N-04 with GI50 values from 2.5 to 8.6 μg/mL. 4. Eighteen compounds were isolated from the fungus A. terreus. Terretonin A-D（24 - 27）and Asterrelenin（28） are new compounds belonging to sesterterpoids and indole-ralated alkaloid, respectively. 5. From the fungus A. versicolor, sixteen secondary metabolites, including nine new ones, Brevianamides K-N (40-43), Averins A-C (44-46), and Glyanphenines A-B (47-48), were isolated and identified. Brevianamides K-N (40-43), Averins A-C (44-46), and Glyanphenines A-B (47-48) represented three classes of alkaloids. 6. New sesterterpenes and their bioactivities reported from 1997 to 2007 were summarized.

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.

氮肥对黄瓜根际土壤线虫群落组成、结构及其多样性的影响

土壤线虫是土壤动物的主要功能类群之一，在土壤养分分解和循环中起到重要的作用。本研究通过施用两种形态氮肥，硝态氮(NO-3–N)和铵态氮(NH+4–N)，对黄瓜整个生长期内根际土壤线虫的群落组成、结构及其多样性等的影响进行了比较研究。为增进土壤健康，提高土壤质量以及合理施用氮肥提供科学的理论依据。研究结果如下： 1. 氮肥处理后，不同浓度NO-3–N处理提高了根际土壤线虫数量，而NH+4–N处理（202.5 kg N/ hm2）抑制了线虫数量的增加。线虫群落结构相对较稳定，营养类群变化不大。且少量优势科/属会对土壤线虫群落特征起着至关重要的作用。 2. 在整个生长季节内，非寄生线虫的群体动态变化与寄生线虫的群体的动态变化具有相反的变化趋势。其中，NO-3–N处理和NH+4–N处理后植物寄生线虫出现频率的变化趋势相近，都是由高到低；非植物寄生线虫出现频率的变化趋势则都是由低到高。这说明非植物寄生线虫数量增长和空间占位对植物寄生线虫群体有一定的抑制作用。另外，也反映了适量氮肥在一定程度上能够减轻植物寄生线虫对黄瓜的危害。 3. 由多样性指数变化可知，NO-3–N和NH+4–N在低肥（67.5 kg N/hm2）和高肥（202.5 kg N/hm2）处理较中肥（135.0 kg N/hm2）处理，更不利于提高土壤线虫多样性地提高和线虫群落的稳定性。中肥不同形态氮肥处理与对照相比，H´指数在初花期和结果期显著增加了土壤线虫的H´指数，说明施入两种形态的氮肥能够提高线虫生物多样性程度。NH+4–N处理在初花期和结果期显著降低了土壤线虫种类的丰富性。土壤线虫生物多样性变化中，H´和SR指数在一定程度上能够反映施用无机氮肥对土壤线虫的多样性的影响，而J指数和λ指数效果不明显。NO-3–N和NH+4–N处理相比较，NO-3–N处理对黄瓜土壤线虫的多样性指数影响更大，促进了土壤线虫群体的多样化和种类的丰富度，更有利于提高土壤线虫的多样性，增加其稳定性。这些结果表明适量无机氮肥特别是NO-3–N的施用对黄瓜土壤线虫的生物多样性有一定的维护和提高作用。 4. 线虫数量与土壤质量指标的相关分析表明：线虫数量与有关土壤理化生指标，如土壤NO-3–N、NH+4–N、有机质含量等的正相关程度高，与总酚含量等显著负相关；与根际土壤微生物，细菌、真菌、放线菌数量等呈显著正相关。另外，线虫数量与土壤含水量未表现出显著相关关系。 Nematodes play a major role in decomposition and nutrient cycling in soil. Nematode community analyses are useful in assessing soil ecosystem status and function. The effects of two forms of mineral nitrogenous fertilizers (NO-3–N and NH+4–N) on nematode community composition, structure and diversity in rhizosphere of cucumber were investigated during different growing seasons of cucumber. Systematically research of effects of nitrogenous fertilizers could help to obtain better undstanding of a healthy soil and using nitrogenous fertilizers in reason. The main results are as follows: 1. The total numbers of nematode were more abundant in NH+4–N treatments than other teatments. However, NH+4–N teatment（202.5 kg N/hm2）dramatically inhibited it. All the tropic groups in the soil nematode communities were stable, and the dominant family or genus had an important function in the nematode community structure. 2. There was similar trend of the frequency of plant parasitic nematodes between NO-3–N and NH+4–N treatment, the similar trend of the frequency of non-plant parasitic nematodes was also found. But the frequency of plant parasitic nematodes exhibited a contrary trend to that of plant parasitic nematodes after different nitrogenous fertilizer treatments. The results showed that the increasing trend of the frequency and the niche of non-plant parasitic nematodes inhibited the plant parasitic nematodes, and indicated that right chemical fertilizers dosage could abate plant parasitic nematodes harm to cucumber. 3. The changes of the biodiversity index showed that the nitrogen treatment（135.0 kg N/hm2）promoted the stabilization of soil nematode diversity than other nitrogen treatments(67.5 kg N/hm2 and 202.5 kg N/hm2). In the treatment（135.0 kg N/hm2）,The changes in nematode diversity between the control plots and treated plots were compared by the biodiversity index (H´, J, SR, λ). Among these tested index, H´ and SR were effective in reflecting the effects of different nitrogenous fertilizers on the diversity of soil nematodes. In comparison with the NH+4–N treatment, the NO-3–N treatment promoted the stabilization of soil nematode diversity. 4. Correlation coefficients between nematode abundance and soil quality indices indicated that the total numbers of nematode were affected positively by NO-3–N, NH+4–N and the organic matter, and negatively by total phenolic acids; the total num- bers of nematode had positive correlation with bacteria, fungi and actinomycetes nu- mbers. Soil water contents had only a weak negative influence on it.

土壤微生物和酶对大气CO2浓度与温度升高的响应

土壤微生物(Soil microbes)是生态系统的重要组成部分，它参与土壤中复杂有机物质的分解和再合成，也参与C、N、S、P等的循环。土壤酶(Soil enzyme)是土壤中具有生物活性的蛋白质，它与微生物一起推动着土壤的生物化学过程，并在树木营养物质的转化中起着重要的作用。鉴于土壤微生物和土壤酶对环境变化的敏感性，它们在CO2浓度和温度升高时的反应将在很大程度上影响森林生态系统的结构和功能。因此，要全面评价大气CO2浓度和温度升高对整个生态系统的影响，有必要对CO2浓度和温度升高条件下的土壤微生物的反应进行深入的研究与探讨。本文应用自控、封闭、独立的生长室系统，研究了川西亚高山岷江冷杉（Abies faxoniana）根际、非根际土壤微生物数量，红桦（Betula albosinensis）根际微生物数量以及根际、非根际土壤酶活性对大气CO2浓度(环境CO2浓度+350±25μmol·mol-1，EC)和温度(环境温度+2.0±0.5℃，ET)升高及两者同时升高(ECT)的响应。结果表明： 1) EC和ET显著增加岷江冷杉根际微生物数量，但不同微生物种类对EC和ET的反应有所差异。6、8和10月，岷江冷杉根际微生物数量与对照(CK)相比，EC处理的根际细菌数量分别增加了35%、164%和312%，ET处理增加了30%、115%和209%；EC和ET处理对根际放线菌和根际真菌数量影响不显著。ECT处理的根际放线菌数量分别增加了49%、50%和96%，根际真菌数量增加了151%、57%和48%；而ECT对根际细菌数量影响不显著。EC、ET和ECT处理对岷江冷杉土壤微生物总数的根际效应明显，其R/S值分别为1.93、1.37和1.46(CK的R/S值为0.81)。 2) 红桦根际微生物数量对EC、ET和ECT的响应不同。生长季节(5~10月)，高密度的红桦根际细菌数量与CK 相比，EC的根际细菌数量分别增加28%、33%、423%、65%、43%和79%，而低密度的红桦根际细菌数量增加不显著。ET能显著增加根际细菌数量(7~10月)，其中高密度的根际细菌数量分别增加了377%、107%、35%、22%，而低密度的根际细菌数量分别增加了27%、27%、64%、48%；ECT对两个密度水平下根际细菌数量均未产生有显著的影响。高、低密度的红桦根际放线菌和根际真菌数量与 CK 相比，EC显著增加了低密度的红桦根际放线菌数量，而对高密度的根际放线菌数量无显著影响；ET和ECT对高低密度的红桦根际放线菌数量均未产生显著影响。EC和ET对高低密度的根际真菌数量也无显著影响，而ECT却显著增加了高低密度的根际真菌数量。 3) EC、ET和ECT处理的低密度红桦根际微生物(细菌、放线菌和真菌)数量没有显著高于或低于高密度根际微生物数量，表明短期内密度对红桦根际微生物数量不产生影响。 4) 不同种类的氧化还原酶对EC、ET和ECT的响应不同。5~10月，EC的红桦根际过氧化氢酶活性是CK 的1.44、1.06、1.11、1.10、1.12和1.24倍，差异显著(6月除外)；ET和ECT处理根际过氧化氢酶活性无显著增加。EC的红桦根际多酚氧化酶活性比CK显著增加；ET的根际多酚氧化酶活性显著高于CK(8月除外)。ECT的根际多酚氧化酶活性高于CK，差异不显著。EC的根际脱氢酶活性分别增加了46%、40%、133%、48%、17%和26%，差异显著。5~7月，ET和ECT的根际脱氢酶活性高于CK的脱氢酶活性，而8~9月则相反，差异性均不显著。 5) EC、ET和ECT对不同种类的水解酶的影响不同。EC能显著增加红桦根际脲酶活性，5~10月分别增加了29%、42%，、70%、67%、59%和57%。ET和ECT 对根际脲酶活性未产生显著影响。EC显著提高根际转化酶活性，5、6和9月EC的根际转化酶活性分别比CK高51%、42%和40%。5和10月，ET的根际转化酶活性低于CK，而其余月份却高于CK，但均具有显著性差异。ECT的根际转化酶活性与CK的根际转化酶活性有显著性差异(9月除外)，5、6和7月的根际转化酶活性分别提高了94%、198%和67%。 6) 与CK相比，EC、ET和ECT的非根际土壤微生物数量以及非根际土壤酶活性均无显著提高。EC、ET和ECT的过氧化氢酶、脲酶的根际效应明显，而多酚氧化酶和脱氢酶根际效应不明显。EC和ECT的转化酶根际效应明显，而ET的转化酶根际效应不明显。 It is well known that atmospheric CO2 concentration and temperature are increasing as a consequence of human activities. In past decades, considerable efforts had been put into investigating the effects of climate change on processes of forest ecological system. In general, studies had been mainly focused on the effects of elevated atmospheric CO2 on plant physiology and development, litter quality, and soil microorganisms. Studies showed that there was variation in the responses of root development and below-ground processes to climate between different plant communities. Since the concentration of CO2 in soil was much higher (10~50 times) than in the atmosphere, increasing levels of atmospheric CO2 may not directly in fluence below ground processes. Betula albosinensis and Abies faxoniana, as the dominated tree species of subalpine dark coniferous forest in the western Sichuan province, which play an important role in the structure and function of this kind of forest ecosystem. In our study, effects of elevated atmospheric CO2 concentration (350±25μmol·mol-1), increased temperature (2.0±0.5℃) and both of the two on the number of rhizospheric microbe and rhizospheric enzyme activity were studied by the independent and enclosed-top chamber’ system under high-frigid conditions. Responses of rhizospheric bacteria, actinomycetes and fungi number of Betula albosinensis and Abies faxoniana under different densities(high density with 84 stems·m-2, low density with 28 stems·m-2 ), and rhizospheric enzyme activity of Betula albo-sinensis to elevated CO2 concentration and increased temperature were analyzed and discussed. The results are as the following, 1) In comparion with the control, the numbers of rhizospheric bacteria of Abies faxoniana were increased by 35%, 164% and 312% significantly in June, August and October respectively of EC, and were increased by 30%, 115% and 209% respectively of ET.However the effect of EC and ET on rhizospheric actinomycetes and fungi was not significant. The number of rhizospheric actinomycetes of ECT were increased significantly by 49%, 50% and 96% respectively, and the increment of rhizospheric fungi were 151%, 57% and 48% respectively .The effect of ECT on rhizospheric bacteria was not significant. Rhizospheric effect of soil microbe for all treatments was significant, with the R/S of 1.93, 1.27 and 1.46 for EC, ET and ECT, respectively. 2) Treatment EC improved the number of rhizospheric bacteria of Betula albosinensis under high density significantly in comparison with the control, over the growing season, the greatest increment of rhizospheric bacteria was from July. However, EC had no effect on the number of rhizospheric bacteria under low density. Except May and June, treatment ET improved the number of rhizospheric signifcantly. The effect of treatment ECT on the number of rhizospheric bacteria under different densities was not significant. Of treatment EC, the number of rhizospheric actinomycetes of Betula albosinensis under low density were increased significantly, however, treatment EC did not stimulate the number of rhizospheric actinomycetes under high density. Simultaneously, treatment ET and ECT did not stimulate the number of rhizospheric actinomycetes. Finally, in treatment ECT, the number of rhizospheric fungi under high density were increased significantly, however treatment EC and ET did not stimulate the number of rhizospheric fungi under different densities. 3) Of treatment EC, ET and ECT, the number of rhizospheric microbe of Betula albosinensis under low density were not more or fewer than that of microbe under hign density along the growing season, which showed that plant density had no effect on the nmber of microbe. 4) From May to October, 2004，rhizospheric catalase activity of Betula albosinensis of treatment EC was 1.44, 1.06, 1.11, 1.10, 1.12 and 1.24 times as treatment CK respectively, and the difference was statistically significant(except June). Treatment ET and ECT did not increase rhizospheric catalase activity significantly. In treatment EC, the rhizospheric pohyphenol oxidase activity was higher than treatment CK significantly. The rhizospheric pohyphenol oxidase activity of treatment ET was higher than CK significantly (except August). The rhizospheric pohyphenol oxidase activity of treatment ECT was higher than CK, but the difference was not statistically significant. Over the growing period, the rhizospheric dehydrogenase activity were increased 46%, 40%, 133%, 48%, 17% and 26% respectively by treatment EC, and the difference was statistically significant. From May to July, the rhizospheric dehydrogenase activity in treatment ET and ECT was higher than CK, but from August to October, the rhizospheric dehydrogenase activity was lower than CK, the difference was not significant. 5) Treatment EC increased rhizospheric urease activity significantly, from May to October, rhizospheric urease activity were increased 29%, 42%, 70%, 67%, 59% and 57% respectively by EC. Treatment ET and ECT had no effect on rhizospheric urease activity. Treatment EC improved rhizospheric invertase activity significantly, in May, June and September, the rhizospheric invertase activity of treatment EC were increased 51%, 42% and 40% in comparison with the control. Except May and October, the rhizospheric invertase activity of treatment ET was markly higher than CK. The rhizospheric invertase activity of treatment ECT was significantly different from CK (except September), in May, June and July treatment ECT increased rhizospheric invertase activity by 94%, 198% and 67% respectively. 6) In comparison with the control, treatment EC, ET, and ECT had no effect on the number of non-rhizospheric microbe and non-rhizospheric enzyme activity. Rhizospheric effect of catalase and urease for all treatments was significant, but rhizospheric effect of pohyphenol oxidase and dehydrogenase was not significant. Rhizospheric effect of invertase of EC and ECT was significant, but rhizospheric effect of invertase of ET was not significant.

花椒叶和土壤浸提液对土壤微生物、土壤酶及土壤化学性质的影响

花椒（Zanthoxylum bungeanum Maxim.）是川西地区重要的经济植物，化感作用是花椒连作障碍的原因之一，而花椒凋落物和根系分泌物对土壤质量的影响是花椒化感作用的一个重要方面。系统研究花椒如何影响土壤有助于深入理解和解决花椒连作障碍。本文主要以大红袍（10a生）花椒叶和种植过花椒的土壤的浸提液浇灌花椒幼苗进行试验，分析叶浸提液与土壤浸提液对非花椒生长土壤中土壤微生物、土壤酶及土壤化学性质的影响。主要结果如下： 1.花椒叶浸提液和土壤浸提液减少了土壤中微生物的种类、组成和数量。本试验中未施加浸提液的土样中根际微生物明显高于非根际区，在经过花椒叶浸提液处理后，根际细菌、真菌和放线菌数量以及微生物总数都有所减少，这样将会导致土壤中的有效养分的供给减少，进而可能影响植物的生长。 2．施加花椒叶浸提液和土壤浸提液，以及花椒幼苗的栽种，对不同土样中的土壤酶各有促进和抑制作用。在浸提液处理下，水解酶之间及氧化还原酶之间各存在相互促进作用。 3.施加花椒叶浸提液和土壤浸提液均抑制了根际土中全氮和有机质含量，叶浸提液还抑制了无苗土中全磷含量，土壤浸提液还抑制了无苗土中全氮含量与根际土全磷、有机质含量。但两种浸提液均促进了根际土中有效磷和水解性氮含量、根外土中全磷含量，叶浸提液促进了根际土中全磷含量，土壤浸提液促进了根外土中有效磷含量。全氮和有机质含量的下降可能对植物生长发育不利。 4．土壤化学性质与土壤酶活性在不同土样中有不同的相关性。全氮含量在施加叶浸提液的土样中与蛋白酶活性呈正相关。水解性氮含量在施加叶浸提液的土样中与蛋白酶活性、蔗糖酶活性呈正相关。全磷含量在施加叶浸提液的土样中与多酚氧化酶活性呈正相关；在施加土壤浸提液的土样中与蛋白酶活性、蔗糖酶活性呈正相关，与多酚氧化酶活性呈负相关。有效磷含量在施加叶浸提液的土样中与多酚氧化酶活性呈正相关，与蛋白酶活性呈负相关；在施加土壤浸提液的土样中与蛋白酶活性、过氧化氢酶活性呈正相关。有机质含量在施加叶浸提液的土样中与蛋白酶活性、蔗糖酶活性呈正相关。 Zanthoxylum bungeanum is one of the most important cash crops in Eastern Tibetan Plateau. Allelopathic effects could be one of reasons for Z. bungeanum’s continuous cropping impediment. The effects of secretion of leaf and root of Z. bungeanum on soil quality is a important way of Z. bungeanum’s allelopathic effects. However, allelopathic effect of Z.bungeanum on soil microbes, enzyme activities and chemical property were seldom studied. In this study, leaf and soil extracts of Da Hongpao(DHP), the most common varieties of Z.bungeanum in this area, were used to assess allelopathic effect of Z. bungeanum on soil biology and biochemistry by pot experiments . The main results showed that: 1. The irrigation of two kinds of extracts reduced the species, component and quantity of soil microbes. In rhizosphere soil which irrigated by distilled water, the quantity of soil microbes is significantly different from exoroot soil. In rhizosphere soil which irrigated by leaf extracts, the quantity of bacterial, fungi, actionmycete and gross of microbes were decreased, it may resulted in reduce of Available nutrient in soil, and influenced the growth of plants. 2．The irrigation of two kind of extracts reduced or enhanced the enzyme activities in different soils. Interaction between hydrolytic ferments and redoxases were promoted each other. 3. The irrigation of two kinds of extracts reduced the total N and organic matter in rhizosphere soil. Leaf extracts also reduced the total P in soil without seedling. Soil extracts reduced total N in soil without seedling and total P, organic matter in rhizosphere soil. But both extracts also enhanced available P and hydrolysable N in rhizosphere soil, total P in exoroot soil. Leaf extracts enhanced total P in rhizosphere soil. Soil extracts enhanced available P in exoroot soil. The reduction of total N and organic matter may influence growth of plants. 4．Positive correlations between total N and prolease, hydrolysable N and prolease, hydrolysable N and saccharase, total P and polyphenol oxidase, available P and polyphenoloxidase, organic matter and prolease, organic matter and saccharase, were studied in soil irrigated by leaf extracts. In soil irrigated by soil extracts, there are positive correlations between total P and prolease, total P and saccharase, available P and prolease, available P and catalase, while negative correlation between total P and polyphenoloxidase, available P and prolease, available P and catalase was found.

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.

白腐菌选择性降解木质素的研究

对15株白腐真菌进行了以玉米秸秆为基质的初步筛选，从中获得一株选择性系数较高的菌株Y10，并对其降解玉米秸秆的情况进行了研究。结果表明，在30天的培养过程中菌株Y10对玉米秸秆降解的选择性系数都大于1，第15天选择性系数最高为3.88。对未经降解和降解过的玉米秸秆分别作了紫外光谱和红外光谱分析，结果表明，经该菌降解后玉米秸秆的化学成分发生了很大变化，且木质素的降解程度要大于纤维素的降解程度。对菌株Y10进行了ITS-5.8S rDNA序列鉴定，初步判定其为Cerrena sp.。 为了考查不同的外源添加物对菌株Y10降解玉米秸秆的影响，在以玉米秸秆为基质的固态发酵培养基中分别添加了7种金属离子、8种碳源、6种氮源。结果显示，这7种金属离子均能促进木质素的降解，并且一定浓度的某些离子明显抑制纤维素的降解；其中添加0.036%的MnSO4·H2O和0.36%的MgSO4·7H2O对纤维素降解的抑制作用比较强，降解率分别为0.96%和1.31%，木质素的选择性系数分别达到了34.40和20.17。8种碳源中除麦芽糖外都能促进木质素的降解，除微晶纤维素外都明显促进纤维素的降解。6种氮源中酒石酸铵、硫酸铵、草酸铵和氯化铵的添加都会使该菌生长变慢，而且氮源浓度越高菌丝生长越慢。外加碳源和金属离子对半纤维素降解和选择性系数的影响不大。 同时对菌株Y10在液态培养下产木质素降解酶的条件和培养基做了优化。结果表明，在初始产酶培养基中，菌株Y10的漆酶酶活在第10d达到最高，锰过氧化物酶酶活在第11d达到最高，基本上检测不到木质素过氧化物酶。菌株Y10产漆酶的最适温度为32℃，最适PH为6.0；产锰过氧化物酶的最适温度为32℃，最适PH为6.5。菌株Y10产漆酶的最佳碳源为甘露糖，最佳氮源为酒石酸铵，最适诱导剂VA浓度为3 mmol/L，最适表面活性剂TW-80浓度为1%。 利用响应面法对其产漆酶的培养基进行优化，优化后的培养基配方为葡萄糖10.00 g/L，酒石酸铵0.50 g/L，大量元素296.50 ml/L，微量元素100.00 ml/L，NTA 1.40 g/L，VA 5.00 mmol/L，吐温-80加入量为0.10%。进行了菌株Y10产漆酶的验证实验，实测酶活为5282.56 U/L，与预测酶活5162.73 U/L接近。在优化后培养基中，菌株Y10在第14 d达到生长的最高峰，第20 d时，漆酶酶活最高，为11325.00 U/L；第16 d时，锰过氧化物酶酶活最高，为30.77 U/L。 对菌株Y10的漆酶酶学性质做了初步的研究，结果显示，酶反应的最适温度为40℃-65℃，最适PH为3.0。在40℃，PH=3.0时，漆酶催化ABTS反应的米氏方程为 。 Fifteen white-rot fungi based on corn stalk were screened. One white-rot fungus Y10 with high selectivity value was obtained. The degradation of corn stalk was initially studied. The results indicated that the selectivity value was above 1 during the 30 day-cultivation and the highest was 3.88 after 15 days. The composition of untreated and treated stalk was analyzed through ultraviolet spectroscopy and infrared spectroscopy. It was found that the composition of treated stalk was greatly altered and the degree of the degradation of lignin is greater than the cellulose. Y10 was identified as Cerrena sp. by ITS -5.8S rDNA sequence analysis. The influence of metal ions, carbon sources and nitrogen sources on corn stalk degradation by white-rot fungus was studied. While all seven metal ions could promote lignin degradation, the cellulose degradation was best inhibited at certain ion concentrations. Notably, when 0.036% MnSO4·H2O and 0.36% MgSO4·7H2O were added into the medium, the cellulose degradation was restrained to the extents that the coefficients of lignin selectivity rose to 34.40 and 20.17 respectively. It was also found that all carbon sources except maltose can promote lignin degradation. The addition of carbon sources other than microcrystalline cellulose significantly promoted cellulose degradation. The addition of the nitrogen sources, ammonium tartrate, ammonium sulfate, oxalate, ammonium chloride, resulted in remarkable inhibition to mycelium growth; the larger the concentrations of nitrogen sources are, the slower the mycelium grew. The addition of carbon sources and metal ions had less impact on the degradation of hemicellulose and selectivity value. Meanwhile, we optimized the conditions and culture medium of the lignin-degrading enzyme production of strain Y10. The results showed that in the initial culture medium, the Lac activity was highest at the 10th day, the MnP activity was highest at the 11th day and the LiP could not be detected. The optimum condition of Lac was at temperature 32 and PH =6.0 and the optimum condition of MnP was at temperature 32 and PH =6.5. The optimum carbon source for Lac was seminose, the optimum nitrogen source was ammonium tartrate, the optimum content of VA was 3 mmol/L, the optimum content of TW-80 was 1%. PB and RSM were used to optimize the culture medium of laccase by white-rot fungus Y10. The optimum culture medium was consist of glucose 10.00 g/L, ammonium tartrate 0.50 g/L, macro elements 296.50 ml/L, trace elements 100.00 ml/L, NTA 1.40 g/L, VA 5.00 mmol/L, TW-80 0.10%. Under the optimal conditions, the activity of laccase was 5282.56 U/L and the experimental value agreed with the predicted value 5162.73 U/L. The biomass was highest at the 14th day, the Lac activity was highest at the 20th day, the MnP activity was highest at the 16th day. The results of the studies on the characteristics of Lac showed that the optimum temperature for Lac activity is 40℃-65℃ ; the optimum PH for Lac activity is 3.0 and under 40℃，PH=3.0, the Michaelis-menten equation of Lac catalized ABTS oxidation was .

捷安肽素对采后柑桔青绿霉菌抑制效果及作用机理研究

捷安肽素是一种由枯草芽孢杆菌（Bacillus subtilis）ZK 产生的抗真菌多肽。本文以柑桔青霉菌（Penicillium italicum）和绿霉菌（Penicillium digitaum）为供试真菌，研究了捷安肽素的抑菌性能及作用机理，为捷安肽素开发为有效的生物杀菌剂提供理论依据。全文共分两部分：第一部分：捷安肽素对柑桔青霉菌和绿霉菌抑制效果研究。采用琼脂扩散法测定捷安肽素对柑桔青霉菌和绿霉菌的抑菌活性。53.9 µg/mL 捷安肽素对绿霉菌和青霉菌的抑菌圈直径分别为26.7mm 和24.1mm。结果表明捷安肽素能够抑制柑桔青绿霉菌的生长，柑桔绿霉菌比青霉菌对捷安肽素敏感。在柑桔果实上，研究了不同浓度、不同接入时间的捷安肽素对柑桔青霉病和绿霉病的防治效果，并与常用化学杀菌剂抑霉唑、咪鲜胺、甲基硫菌灵和多菌灵作比较。53.9 µg/mL捷安肽素处理柑桔果实，柑桔青霉病和绿霉病发病率分别为5.0 %和5.3 %,比对照低95.0 %和94.7 %；柑桔青霉病和绿霉病的病情指数分别为1.87 和2.18，比对照低73.73 和97.82。结果表明，捷安肽素能够有效地防治柑桔青绿霉病。与对照相比，捷安肽素先于或后于柑桔青绿霉菌接入时，对柑桔青绿霉菌均有抑制作用，但抑制效果随接入间隔时间的增长而降低。第二部分：捷安肽素对绿霉菌作用机理研究。首先在光学显微镜和透射电镜下观察捷安肽素处理后绿霉菌菌丝表面形态结构与菌丝体内超微结构的变化。形态观察发现，捷安肽素处理24h以内，绿霉菌菌丝结构无变化。捷安肽素作用36h后，绿霉菌菌丝不规则缢缩和膨大。48h后，在绿霉菌菌丝顶端、中部、末端的多处细胞均可发生畸形的球状结构，这种畸变结构随处理的延长而增加，致使细胞成为捻珠状。处理72 h后，畸变球形细胞开始断裂离解。处理96h后，镜下几乎无完整菌丝，成单个的球状细胞，部分细胞出现破裂。而对照菌丝表面光滑，结构完整。通过透射电镜观察发现，与对照相比，捷安肽素处理后，绿霉菌细胞壁、细胞膜轮廓模糊不清，细胞质外泄。推测捷安肽素能够使绿霉菌细胞膜通透性发生改变。进一步实验利用紫外-可见分光光度计检测捷安肽素作用后绿霉菌胞外液紫外吸光度的变化，表明捷安肽素作用于绿霉菌菌丝后，细胞内蛋白质、核酸缓慢泄漏。通过Atomscan Advantage单道扫描等离子体发射光谱仪(ICP)测定捷安肽素作用后菌丝体内K+浓度的改变，结果表明捷安肽素作用于柑桔绿霉菌1h内，菌丝体内K+含量迅速下降，为对照绿霉菌K+含量的37.53 %，1 h后菌丝体内K+含量变化趋于平缓。K+的迅速泄漏，以及蛋白质、核酸的泄漏表明捷安肽素通过迅速改变绿霉菌细胞膜通透性，使绿霉菌菌丝生长受到抑制。Jiean-peptide produced by Bacillus subtilis ZK has broad-spectrumresistance to plant pathogens. In this study, we investigated the antifungal propertyand the possible antifungal mechanism of jiean-peptide against two commonphytopathogenic fungi of citrus fruits: blue molds (P. italicum) and green molds (P.digitatum).The paper involved two parts:Part 1 is the study of the antifungal property of jiean-peptide against blue moldsand green molds of citrus fruits. The in vitro inhibition effect of jiean-peptide againstblue molds and green molds was detected by agar diffusion method. The diameters ofinhibition zones of green molds and blue molds are 26.7mm and 24.1mm respectivelyby treating with 53.9 µg/mL jiean-peptide. It shows that jiean-peptide effectivelyinhibits the both phytopathogenic fungi, and it is more effective for inhibiting greenmolds than blue molds. The effectiveness of jiean-peptde to inhibit green molds andblue molds in vivo was investigated compared with four conventional fungicides thatare imazalil, prochloraz, carbendazin and methylthiophanate. The result is that the incidences of the blue mold disease and green mold disease are 5.0 % and 5.3 %, thedisease severities are 1.87 and 2.18 respectively when citrus are inoculated with 53.9µg/ml jiean-peptide. The decay incidences and disease severities were significantlyreduced by treating with jiean-peptide compared with the control. The results indicateJiean-peptide is effective for controlling blue molds and green molds on citrus. Theoptimized inoculation time was also investigated. When inoculated with jiean-peptideat 0 h, 6 h, 12 h, 24 h and 48 h before or after pathogens’ inoculation, Jiean-peptidecan suppress the occurrence of blue molds and green molds compared with the control, but the effect of later inoculation decreases compared with the inoculation at the sametime.In Part 2, we investigated the possible antifungal mechanism against greenmolds of citrus. At first, we observed the exterior morphological changes andultrastructural changes of blue molds under light microscopy (LM) and transmissionelectron microscopy (TEM). Compared with untreated control cells which aregenerally uniform in shape, the appearances of treated hyphae change obviously. Itshows that some cells of hyphae irregularly shrink or enlarge when cultured for 36h.When the treating time of jiean-peptide increases, the aberrance of the hyphaebecomes more obvious, and hyphae exhibit the moniliform appearances. Finally, thereis no intact hypha leaved except only single cells, and some of which appear fractured.By transmission electron microscopy (TEM) observation, we find that the outline ofthe cell wall and the cell membrane of hyphae are blurry, and the cytoplasma oozesout. The observation result under LM and TEM suggests that jiean-peptide mightchange the permeability of the cell membrane. So we conducted further experiment todetect the change of permeability when the cells of blue molds were treated withjiean-peptide. And the effect of jiean-peptide on non-growing cells of blue molds wastested. By the spectrophotometer measurement, we found that compounds with lightabsorption at 260 nm and 280 nm were released and amounts increased within 12 hcompared with the control. Moreover, by the ICP measurement, the leakage of K+occurred immediately in the presence of jiean-peptide within 1 h, but with nearly nofurther change after 1 h. All these results indicate that jiean-peptide could change themembrane permeability of blue molds immediately and result in leaking nucleotides,proteins and K+ from cells.

齐墩果酸的微生物转化

齐墩果酸（OA）是一个分布广泛、含量丰富的天然三萜化合物，常以皂苷元的形式广泛存在于植物中,具有多种重要生物活性。但是OA许多活性较弱，且生物利用度低，限制了其在临床上的应用。一是OA水溶性差;二是抗癌活性仍与临床应用的抗癌药物相差比较大。 真菌在微生物转化中具有种类多、培养条件比较简单等特点，为了寻找到具有转化OA能力的菌株，采取一步发酵的方法，在18株实验室保藏真菌菌株中筛选到5株目的菌株,TLC分析显示有转化效果。 随后采用二步发酵的方法作为复筛，验证5株菌株转化能力，波谱分析结果表明5株菌株对OA确实有转化作用。 选择5株菌种中代号1F-2 2菌株作为放大实验菌株，分离转化产物，得到OA衍生物108（相对分子量414m/z）和1010（相对分子量340 m/z），分离出的产物用于活性检测。寻找到产物108的RP-HPLC分离条件，质谱得出二者相对分子质量。 为验证OA转化产物抗肿瘤活性，首次研究了OA对卵巢癌细胞株IGROV1和人乳腺癌细胞株MDA-MB-231作用，通过细胞增殖抑制实验、用MTT法检测细胞活性，结果表明齐墩果酸可降低卵巢癌细胞株IGROV1和乳腺癌细胞MDA-MB-231细胞增殖能力并呈剂量依赖性，对肿瘤细胞株的半数有效抑制浓度化IC50 分别为36.58μg/mL和38.8μg/mL (P<0．01)。OA能抑制肿瘤细胞活性，并且OA对卵巢癌细胞株IGROV1抑制活性高于乳腺癌细胞MDA-MB-231。 在此基础上，转化产物108和1010对卵巢癌细胞株IGROV1和人乳腺癌细胞株MDA-MB-231的抑制作用也进行研究，MTT实验结果表明，转化产物对两株癌细胞也有抑制活性（P<0．01）。 总之，本文工作为进一步开展齐墩果酸类化合物结构改造和抗肿瘤活性的研究奠定了基础。 Oleanolic acid (OA) is a triterpenoid widely distributed in the nature which possesses various important bioactivities. OA also serves as aglycon of many natural saponins. However, the relatively weak activities and poor bioavailability hinder its clinical use. Firstly, poor water-solubility results in worse bioavailability. Secondly, compared with clinical antitumor drug, the antitumor effect of OA has a great difference, it is worse. Many fungi have ability to transform nature products into a variety of derivatives, and transformation conditions of fungi are simple. Attempt to obtain fungi strains able to biotransform OA, we carried out the following experiments: To investigate the biotransformation 0f OA by strains supplied firstly, we used one-step fermentation method to screen the aimed strains from 18 fungus strains stored in our laboratory. On the basis of the initial screening experiments, we found 5 aimed strains. The TLC results showed that the 5 fungi strains could transform OA into other components derivatives. Then we used two-step fermentation method as secondly screening. We repeated the five strains to do the experiments, analytical data of the results proved the transformation indeed. In the followed experiments work, we chose 1F-2 2 strain as large-scale transformation fungus from the aimed fungi. We got two biotransformation products of OA by 1F-2 2, and named those derivatives 108 and 1010. We found RP-HPLC separation conditions of product 108. The two products were characterized by ESI-MS. To verify the anti-tumor activity of biotransformation products of OA, we studied the inhibition effect of oleanolic acid on the ovarian carcinomas IGROV1 and breast cancer cell line MDA-MB-231 firstly. With an assay based on a tetrazolium dye (MTT), the effects of various concentrations of oleanolic acid on ovarian carcinomas IGROV1 and breast cancer cell line MDA-MB-231 were studied. MTT method was used to measure the tumor cells viability. Compared with the control group, oleanolic acid can significantly inhibit the viability of the ovarian carcinoma cells IGROV1 and MDA-MB-231 breast cancer cell line (P<0．01), IC50 values were 36.58μg/mL or 38.8μg/mL. Oleanolic acid can inhibit the malignant tumor cells viability, and inhibitory activity of OA to ovarian carcinomas IGROV1 was higher than to breast cancer cell line MDA-MB-231. On this basis, we studied the anti-tumor activity of the two derivatives of OA [called 108 (414 m/z) and 1010(340 m/z)]. It came to the conclusion that the two derivatives also showed potent inhibitory effect on the growth of these tumor cells（P<0．01）. Therefore, the results of studies will benefit the further investigating on the relationships of structures and antitumor activities of OA.

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.

泸州老窖古酿酒作坊空气微生物分布特征研究

本文主要研究了泸州老窖古酿酒作坊内外环境空气真菌和空气细菌的群落结构和分布特征。结果如下： 作坊内外环境空气微生物浓度差别显著，并随季节变换而变化，春、夏季微生物浓度较高，秋、冬季较低，空气真菌在夏季达到最高，细菌在春季最高。 古作坊内外环境检测到的真菌均为16 属，但优势菌属不同，作坊外的优势菌属为青霉属(Penicillium)、曲霉属(Aspergillus)、无孢菌(non-sporing)、枝孢霉属(Cladosporium)和链格孢属(Alternaria)；而作坊内优势菌属为曲霉属、青霉属、酵母菌(Yeast)、无孢菌，作坊内还含有较高浓度的根霉属（Rhizopus）、毛霉属（Mucor）、短梗霉属（Aureobasidiu），枝孢霉属和链格孢属等，曲霉属、酵母菌、根霉属、毛霉属为古酿酒作坊重要的酿酒真菌，青霉属、链格孢属为酿酒不利菌群。对古作坊内曲霉属进行了初步鉴定，主要是小冠曲霉（A.cristatellus）、米曲霉（A.oryzae）、黑曲霉（A.niger）和白曲霉（A.cadidus）。 空气细菌10 属21 种，作坊内外环境的优势菌属均为芽孢杆菌属（Bacillus）、微球菌属(Micrococcus)、葡萄球菌属(Staphylococcus)、假单胞菌属(Pseudomonad)，其中芽孢杆菌属在作坊内占有绝对的优势，浓度比在40℅以上，是古酿酒作坊重要的酿酒细菌，另外还检测到较高浓度的乳酸杆菌（lactobucillus），这类菌容易使酒味发涩发苦，为酿酒不利菌。 作坊内外环境空气微生物表现出明显的交流现象。作坊内，青霉属、枝孢霉属、链格孢属、葡萄球菌属等杂菌占有一定比例；而在作坊外,芽孢杆菌属、曲霉属、根霉属(Rhizopus)、酵母菌等处于相对较高水平，绿化环境较好的营沟头作坊内的短梗霉属，枝孢霉属和链格孢属等杂菌含量低于什字头和新街子作坊。 The community structure and distribution characteristic of airborne microbes was investigated in ancient brewage workshops of luzhoulaojiao. The results are as follows: The concentration of airborne microbes was different in interior and exterior environment of ancient workshops, and also varied by seasons. microbial concentration was higher in spring and summer, and lower in fall and winner. The highest levels of airborne bacteria was in spring, but the fungal’s in summer. The identified genus of fungi were 16 in interior and exterior environment of the ancient workshops. But the dominant genus were different , The advantage genus in the interior were Aspergillus, Yeasts, Penicillum and Nonsporing and in the exterior were Penicillum, Nonsporing, Cladosporium, Aspergillus and Aureobasidiu. Rhizopus ,mucor, Aureobasidiu, Cladosporium, Alternaria and all also were at a higher level. Among these, Aspergillus, Yeasts, Rhizopus ,mucor are important vintage flora . Penicillum, Alternaria do harm to vintage. Aspergillus of ancient workshops was identified , the preponderant aspergillus species were A.cristatellus, A.oryzae, A.niger and A.cadidus in ancient brewage workshops. 10 genus 21 species bacteria were identified, the advantage genuses among the interior and exterior of the three workshops were bacillus, microccus, Staphylococcus Pseudomonas. Bacillus, which account for beyond 40℅ of the total bacteria concentration in all sampling pots, was the most dominant genus. Lactobacillus was identified at a high level in ancient workshops, it makes spirit taste bitter and astringent. So it is not a kind of good bacterium for vintage. The fungus in the interior and exterior atmosphere characterized intercommunion phenomenon. Obviously, the concentration of profitless fungus such as Penicillum, Cladosporium, Alternaria appeared in the interior, and the fungus such as Bacillus, Aspergillus, Rhizopus and Yeasts in the exterior were at a relatively high level. the harmfull fungus in yinggoutou workshops such as Aureobasidiu, Cladosporium, Alternaria and all were lower than shenzitou and xinjiezi workshops.