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.

真菌单宁酶发酵五倍子和有机溶剂中酶法合成没食子酸丙酯的研究

本文报告了丝状真菌单宁酶发酵五倍子及有机溶剂中酶法合成没食子酸丙酯的研究。利用单宁和/或五倍子诱导丝状真菌产生单宁 酶的原理，借助二级发酵程序，对从天然源得到的75株菌进行了生物转化实验研究。选择出既能水解单宁或五倍子成没食子酸，又 能把没食子酸和丙醇合成没食子酸丙酯，而且生物催化活性都较高的1株菌，这株菌经初步鉴定为黑曲霉(Aspergillus niger No.17)。随后对它开展了产酶条件和参数优化实验，得出了最佳培养条件。立足于参数优化实验方案的基础上，经由液体培养发酵 制备单宁酶制剂，并把该酶通过化学手段共价结合到一种新型载体—聚乙烯醇和戊二醛反应生成的缩醛上，制备得到固定化单宁酶 。这种固定化生物催化剂在两种有机介质体系中都具有逆向催化合成没食子酸丙酯的能力。最后建立起来一条有效可行的微生物酶 法制备没食子酸的技术途径，没食子酸产率达到70%。对这种物质进行元素 分析：含C,49.45%;含H,3.63%。它的熔点为237℃～243 ℃，三种溶剂系统的TLC均只给出一个斑点。这些数据都与标准品一致。有机溶剂中酶法合成没食子酸丙酯的技术途径已经建立。 水溶性单宁酶在潜溶剂体系中也能催化上述酯化反应，反应混合物中的PG浓度为16.4mmol/L，制备薄层被用于分离反应混合物所含 的PG，这种产物被红外、质谱及三种溶剂系统的TLC等方法鉴定，确证为目标产物。在这一学位论文的实验研究过程中，还包括一 些生化分析方法的建立和应用，这些方法用于鉴定底物和产物及测定它们的浓度，其内容主要包括TLC定性/半定量分析、元素分析 、质谱、红外等手段的综合运用。本工作为开发我国特有的天然产物资源—五倍子的生物化工加工技术及非水相生物催化技术的开 发，提供了有用的基础数据资料，具有应用基础研究工作的重要性。In this thesis, the studies on the fermentation of Chinese gallotannin by filamentous fungi with tannase activity and enzymatic synthesis of propyl gallate(PG) in organic solvents were described through these biocatalysts. Based on the principles of induction enzyme, the tannase produced from filamentous fungi by adding tannic acid(TA) and/or Chinese gallotannin into media was investigated, and the screening experiments of bioconversion were done with 75 strains by means of a two-stage fermentation procedure. These strains were isolated with the enrichment culture technique from natural sources. Hence we selected one strain (Aspergillus niger No.17) that can not only catalyze the hydrolyses of TA and/or Chinese gallotannin into gallic acid(GA) in the liquid cultures, but also be used to synthesize PG from propanol and GA in the non-aqueous media. At the same time both of its biocatalytical activities were higher. This strain was calssified to be Aspergillus niger by the primary identification. Then optimum conditions for production of the tannase and its parameters were examined. In this way, one set of optimum culture conditions was selected. Making use of the optimum proposal, the tanase was prepared through a liquid fermentation procedure. The enzyme was convalently coupled to a new type of carrier which was made chemically from polyvinyl alcohol(PVA)and glutaraldehyde. The immobilized enzymes were able to synthesize PG reversely in two organic media. Finally, an effective enzymatic technique for production of GA was developed. The yield of GA products was up to 70%。Element analysis for this substance: calce: C, 49.42%; H, 3.56%; found: C, 49.45%, H, 3.63%. Its melting point was 237℃～ 243℃ and TLCs on three solvent systems gave only one spot respectively. These data were identical with theauthentic GA. The enzymatic synthesis of PG in organic solvents was extablished with reverse route of tannase catalytical hydrolysis. Aqueous enzyme perparation also catalyzed above esterification in a buffer system. The PG concentration in the reaction mixture was 16.4mmol/L. The reparative-scale TLC was used to isolate PG from the reaction mixture. This product separated was identified by IR, MS and TLC on three solvent systems. In this study of thesis, some biochemical analytical mehtods were developed and used to identify substrates and products, and to determinate their concentration. These methods, including TLC qualitative/half quantitative analysis, element analysis, MS, IR and so on, were useful, available and performable. This work provided basic data and information for developing the biochemical engineering and bio-processing of Chinese gallotannin-a special natural resource in China and the non-aqueous phase biocatalysis. Thus, this study possesses importance in the applied and basic research work.

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.

一株丝状真菌对半纤维素利用的初步研究

本文筛选出一株能利用木糖产乙醇的丝状真菌Z7，对其利用木糖和半纤维素水解产物产乙醇的发酵条件进行了研究，并对Z7 利用玉米芯产木聚糖酶的条件进行了优化。全文分为三部分： 第一部分：目标微生物筛选、纯化及系统发育分析。以木糖为唯一碳源，采用梯度稀释和平板化线法从高温、中温酒曲中分离到16 株能利用木糖良好生长的丝状真菌；通过发酵试验复筛，获得一株能产乙醇的丝状真菌Z7；综合形态学和ITS 序列分析，初步鉴定为Aspergillus flavus。 第二部分：Z7 的乙醇发酵条件研究。以木糖为碳源，通过单因素试验确定最佳氮源和发酵温度；通过正交试验及SPSS 软件分析得到了不同N、P、K 成分对乙醇、残糖和菌体干重的影响。获得最佳的发酵条件为：（g/L）木糖50，尿素1, NH4NO3 1, K2HPO4 2 , KCl 0.5 , MgSO4.7H2O 0.5 , NaNO3 1 , pH 自然，培养温度33 ℃。以玉米芯半纤维素稀酸水解液为底物进行乙醇发酵，根据稀酸水解的单糖释放量和乙醇产量，确定115 ℃，1 h 为最佳玉米芯预处理条件；结合最佳发酵条件，添加1 g/L 的吐温20 能获得最大的乙醇浓度8.31 g/L。因此，Aspergillus flavus Z7 能利用半纤维素水解产物产乙醇，其中木糖的利用率80%以上。 第三部分：Z7 利用玉米芯产木聚糖酶条件优化。Aspergillus flavus Z7 在具有产乙醇能力的同时还具有产木聚糖酶的能力。本文通过单因素和正交试验得到最佳产酶培养基组分为：(g/L)玉米芯20，尿素2, 酵母膏2.5, K2HPO4 5，NaNO31, MgSO4.7H2O 1。单因素试验表明，用纱布代替塑料布密封摇瓶封口能显著提高产酶量；Z7 在碱性条件下具有更强的产酶性能。在最优条件下发酵，能产生最大木聚糖酶活122.23IU/mL。通过薄层分析，验证了Z7 产生的木聚糖酶具有水解木聚糖生成木糖及木寡糖的能力。 A strain of filamentous fungus which can produce ethanol by using the xylose was isolated in this research. The ethanol fermention conditions from xylose and dilute-acid hydrolyzate of the corn core were studied. The conditions of xylanase production by Z7 were also optimized. The paper involved three parts. Part1: Isolation, purification and phylogenetic analysis of the microbe. By using xylose as the single carbon source and the pla te streaking method, several filamentous fungi were isolated from the wine starter; through the fermentation test, a filamentous fungus Z7 which can produce ethanol was further recognized; furthermore, according to the morphologic observation and ITS seque nces analysis, Z7 was identified as Aspergillus flavus at the first step. Part2: Research on the condition of ethanol fermentation by Z7. By single factor experiment, the optional nitrogen resource and temperature of the fermentation were fixed; meanwhile, through the orthogonal array tests and the analysis of statistic software SPSS, the optional component of the culture medium and the fermentation condition were organized as follows: (g/L) xylose 50, urea 1, NH4NO3 1, K2HPO4 2, KCl 0.5 , MgSO4.7H2O 0.5, NaNO31, pH nature, temperature 33℃. Based on these optimal parameters, the fermentation of dilute-acid hydrolyzate of the corn core was carried on by Z7. According to the quantities of released sugar monomers and content of the ethanol, 115℃ in 1h is the best pretreatment condition; the maximal ethanol content can be obtained when 1g/L Tween 20 was added to. Therefore, the filamentous fungus Aspergillus flavus can use the hydrolysate of hemicellulose to produce ethanol, and the rate of xylose utilization was over 80%. Part3: Optimization of Z7’s xylanase producing condition from corn core. Aspergillus flavus Z7, which can utilize xylose or the hydrolysate of hemicellulose to produce ethanol, also had the ability of xylanase production. The optional component of the culture medium were fixed by the single factor experiment and the orthogonal array tests, and they were organized as follows: (g/L) corn core 20, Urea 2, Yeast extract 2.5, K2HPO4 5, NaNO31, MgSO4.7H2O 1; it was testified by the single factor experiment that sealing the shaking flasks with pledget other than plastic paper can obviously increase the xylanase activity; moreover, Z7 showed better xylanase production ability when in the alkali environment. Under the optional fermentation condition, the maximal xylanase activity 122.23IU/mL was proved. Through the analysis of thin- layer chromatography (TLC), the ability of xylanase from Z7, which can hydrolyze xylan to xylose monomer and oligomer, was vividly displayed.

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.

Study on bioactivity of extracts from marine sponges in Chinese Sea

The bioactivity screening of fractions from two inter-tidal sponges collected from the north of China Yellow Sea and one sponge collected from the South Chinese Sea was reported in this study. In sponge Hymeniacidon perleve there were 9 fractions out of 15 from CHCl3 extract with anti Staphylococcus aureus activity, 9 fractions out of 19 from BuOH extract with anti Escherichia coli activity, and three fractions from CHCl3 extract which had moderate to strong activity in inhibiting Bacillus subtilis, Candida albicans, and Aspergilus niger. The fractions of Reniochalina sp. showed bioactivity against bacteria and fungi. The fractions of Acanthella acuta Schmidt showed bioactivity against S. aureus and fungi. One compound from H. perleve obtained by the bioactively directing isolation was tested for bioactivity against the human hepatoma cell line Qgy7701 (IC50 10.1 mug/ml), Burkitt's lymphoma cell line Raji (IC50 9.76 mug/ml) and chronic myelogenous leukemia K562 (IC50 1.90 mug/ml). (C) 2003 Elsevier B.V. All rights reserved.

产于海南的中国革菌一新记录属—乳孔菌属Theleporus(英文)

<正>INTRODUCTION One resupinate, poroid specimen of wood-inhabiting fungi was collected on angiosperm stump from

长白山森林生态系统中的稀有和濒危多孔菌

In last 10 years,extensive field inventories were carried out to investigate Polypore species, the major wood decaying fungi in the Changbaishan Nature Reserve of Northeastern China. The following 27 species were treated as rare or threathened species: Amylocystis lapponica (Romell) Singer, Anomoporia albolutescens (Romell) Pouzar, Anomoporia bombycina (Fr.) Pouzar, Anomoporia vesiculosa Y.C. Dai & Niemel, Antrodia carbonica (Overh.) Ryvarden & Gilb., Antrodia crassa (P. Karst.) Ryvarden, Antrodiella citrinella Niemel & Ryvarden, Diplomitoporus flavescens (Bres.) Dománski, Donkioporia expansa (Desm.) Kotl. & Pouzar, Gloeophyllum carbonarium (Berk. & M.A. Curtis) Ryvarden, Haploporus odorus (Sommerf.) Bondartsev & Singer, Inonotopsis subiculosa (Peck) Parmasto, Nigroporus ussuriensis (Bondartsev & Ljub.) Y.C. Dai & Niemela, Oxyporus sinensis X.L. Zeng, Parmastomyces taxi (Bondartsev) Y.C. Dai & Niemela, Phellinidium sulphurascens (Pilat) Y.C. Dai, Phellinus vaninii Ljub., Polyporus vassilievae Thorn, Pycnoporellus fulgens (Fr.) Donk, Skeletocutis brevispora Niemela, Skeletocutis ochroalba Niemela, Skeletocutis perennis Ryvarden, Trechispora candidissima (Schwein.) Bondartsev & Singer, Wolfiporia dilatohypha Ryvarden & Gilb., Wolfiporia curvispora Y.C. Dai, Wrightoporia avellanea (Bres.) Pouzar and Wrightoporia lenta (Oveh. & J. Lowe) Pouzar. Polypores are richer in East Asia than in Europe and North America, not only because of destructive galciations and fewer hosts in the latters, but also because of the geography. NE Asia is a link between Europe and North America. Changbaishan Nature Reserve is very rich in polypores, and over 260 species were recorded in the reserve. Some rare species in North America and Europe, for instance, Anomoporia albolutescens, Antrodia crassa, Diplomitoporus flavescens, Inonotopsis subiculosa and Skeletocutis ochroalba etc. were found in Changbaishan Nature Reserve as well, and these species are in fact rare in the earth. Most of the 27 species occurred on fallen trunks or rotten wood in the reserve, but some of them grew on living trees. 18 species occured on substrate of gymnosperms, and 9 species grew on wood of angiosperm.Among the 27 species, 7 species caused a brown rot,and 20 species produced a white rot. The morphology, substrate and ecology of each species were briefly discussed. The most important tool for polypore conservation is the conservation of their habitats, and it is necessary to study the ecology of the rare and threathened species of polypores in the Changbaishan Nature Reserve. Because most of polypores live on the substrate of fallen trunks and rotten wood, it is very important to keep such substrate in the ecosystem.

Soil microbial community analysis using terminal restriction fragment length polymorphisms

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR-fingerprinting methods. Sizing of terminal restriction fragments (T-RFs) can now be done using capillary sequencing technology allowing samples contained in 96- or 384-well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T-RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T-RFLP data. Due to biases inherent in the method, linking the size of T-RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T-RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T-RFLP method is a user-friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.

Introduction to Molecular Analysis of Ectomycorrhizal Communities

A number of methods are available for those researchers considering the addition of molecular analyses of ectomycorrhizal (EcM) fungi to their research projects and weighing the various approaches they might take. Analyzing natural EcM fungal communities has traditionally been a highly skilled, time-consuming process relying heavily on exacting morphological characterization of EcM root tips. Increasingly powerful molecular methods for analyzing EcM communities make this area of research available to a much wider range of researchers. Ecologists can gain from the body of work characterizing EcM while avoiding the requirement for exceptional expertise by carefully combining elements of traditional methods with the more recent molecular approaches. A cursory morphological analysis can yield a traditional quantification of EcM fungi based on tip numbers, a unit with functional and historical significance. Ectomycorrhizal root DNA extracts may then be analyzed with molecular methods widely used for characterizing microbiota. These range from methods applicable only to the simple mixes resulting from careful morphotyping, to community-oriented methods that identify many types in mixed samples as well as provide an estimate of their relative abundances. Extramatrical hyphae in bulk soil can also be more effectively studied, extending characterization of EcM fungal communities beyond the rhizoplane. The trend toward techniques permitting larger sample sets without prohibitive labor and time requirements will also permit us to more frequently address the issues of spatial and temporal variability and better characterize the roles of EcM fungi at multiple scales.

石油污染土壤的生物降解研究

Biodeyradation has the characteristics of simple operation,low cost,and easy application.In this research,two fungus were chosen to degrade the oil in polluted soil in laboratory and fields.In the laboratory culture test with temperature controlled,the rate of oil degradation by native fungi (F) and foreign fungi (P) are 61.8% and 66.1% respectively after 50 days.In the field test the rate of oil degradation by native fungi (F) and foreign fungi (P) are 59.8% and 70.1% respectively after 150 days.

石油污染土壤生物修复过程中微生物生态研究

The analysis on microbiological ecology for four types of oil contaminates soils showed that the bacteria utilizing the oil as carbon sources increase,wheras the fugi become less .Zooloea and Bacillu are the dominant bacteria ; Mocor and Cunninghamella ,and Fursarium are the dominant fungi streptomyces take the superiority among the actinomyces.The anaiysis on esterase activity showed that the microbes above mentioned have abilies of degrading esters. The biodeg radationrates are 55.45%,56.74%,38.37% and 45.19%respectively,after 53 days,the biodegradation rate can be increased by 12.6% when the dominant microbes are added.