云南西双版纳几种热带兰菌根真菌的研究

:从云南西双版纳不同生境下的14 株热带兰菌根中分离到19 株真菌,分别鉴定为9 个属,其中优势 菌为镰孢霉属( Fusarium) 9 株占47. 37 %;组丝核菌属( Phacodium) 3 株占15. 79 %.

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。

虎杖对植物病原真菌的杀菌活性及有效成分研究

植物源杀菌剂的开发应用以及从植物中寻找杀菌活性物质作为先导化合物，是目前杀菌研究领域的热点之一。本文以蓼科植物虎杖（Polygonum cuspidatum Sieb.et Zucc.) 为材料，研究了虎杖提取物的杀菌活性和作用机理，确定了虎杖中的有效杀菌活性成分，并以此为先导化合物进行了衍生物合成与结构活性关系的研究。 不同溶剂提取物制备与杀菌活性测定结果表明，乙醇适合作为虎杖植物杀菌剂的提取溶剂，提取率高，对多种植物病原真菌具有广谱的杀菌和抑菌活性，除对黄瓜白粉病（Sphaerotheca fuliginea）表现出很好的防治效果外，对苹果腐烂病菌（Valsa mali）、玉米小斑病菌（Helminthosporium maydis）、葡萄炭疽病菌（Colletotrichum gloeosporioides）、小麦赤霉病菌（Fusarium graminearum）、油菜菌核病菌（Sclerotinia sclerotiorum）、水稻纹枯病菌（Rhizoconia solani）等也具有很好的抑制作用。虎杖回流提取物对黄瓜白粉病的杀菌作用以保护作用为主，兼具一定的治疗作用，并且具有一定的内吸活性，持效期约为4-7 d。温室试验结果表明，虎杖乙醇回流提取物10%可溶性液剂对黄瓜白粉病的EC90值为172.83 mg/L，田间小区试验表明该制剂在800-1600 mg/L的浓度下，对黄瓜白粉病的防效达到76.3-93.4%，具有较好的应用前景。 对苹果腐烂病菌的抑菌作用机理表明，虎杖乙醇提取物对该病原菌有明显的抑制作用，能够抑制蛋白质、葡萄糖等菌体细胞内物质的合成，从而使病菌代谢速度减慢，抑制其生长。虎杖提取物还能够使几丁质酶和β-1,3葡聚糖酶这两种细胞壁相关水解酶的活性升高，降解细胞壁而破坏菌体结构，使菌体自溶。 过测定虎杖乙醇回流提取物对黄瓜体内等一些防御酶和病程相关蛋白活性的影响，表明在40 mg/L和400 mg/L浓度下，虎杖乙醇提取物能够使黄瓜叶片内的过氧化物酶（POD）、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)、几丁质酶等不同程度的升高，从而在一定程度上提高植物对病原真菌的抗病能力。 通过生物活性跟踪测定以及pH梯度提取法确定了虎杖中的主要杀菌活性成分为蒽醌类化合物大黄素（emodin）和大黄素甲醚（physcion），结构通过了HPLC-MS和1H NMR确认，并且通过HPLC确定了虎杖乙醇回流提取物中二者的含量分别为3.28%和1.11%。 以虎杖中的有效成份之一的大黄素为原料，通过羟基的甲基化反应合成了包括已知物大黄素甲醚在内的11个大黄素衍生物，其中5个化合物为首次报道，并进行了初步结构活性关系研究。结果表明通过对大黄素3-OH位置以短直链烷基取代，其衍生物对黄瓜白粉病的活性大大提高，其中以甲基取代的衍生物大黄素甲醚的活性为母体大黄素的16.7倍，而以取代苄基修饰的衍生物的活性没有明显提高。一些目标化合物的活性明显优于三唑酮。研究中还意外发现大黄素的甲基化衍生物三甲氧基大黄素在4000 mg/L时能够明显抑制甜菜夜蛾幼虫的取食与生长发育，而大黄素和大黄素甲醚则无此作用。

中国南海深海沉积物中的微生物资源

由于采样条件和培养条件的限制，深海微生物研究的较少，因此目前海洋微生物资源的研究主要集中在近海和浅海。而深海独特的生态环境，使微生物形成了独特的代谢体系，成为新颖化合物的重要来源，具有很好的开发应用前景。本文首次较为系统的研究了深度为1758-3600m南海海底沉积物中深海微生物资源的分离和活性菌株的筛选情况，旨在为探索我国南海深海微生物资源提供一定的科学依据。 采用多种分离培养基从6个不同深度（1758、2620、3200、3500、3587和3600m）的南海深海沉积物样品中，分离到225株深海微生物，包括40株放线菌和185株细菌。以分离得到的225株深海微生物为研究对象，从产蛋白酶、抗真菌、杀虫三个方面进行活性菌株的筛选，研究南海深海沉积物中的微生物资源状况： （1）.将分离到的225株深海微生物，同时在10℃、28℃、45℃三个温度下进行产低温蛋白酶、中温蛋白酶、高温蛋白酶的筛选。初筛结果表明：这225株深海微生物大都有大小不同的产蛋白酶能力。10℃有产蛋白酶的有109株，28 ℃产蛋白酶的有160株，45℃产蛋白酶的有117株。筛选到一株在45℃下有较强产蛋白酶能力的菌株B1394，其酶活可达873U/ml，有一定的应用前景。通过形态观察、生理生化测定、16SrDNA序列测定，将B1394定名为枯草芽孢杆菌（Bacillus subtilis）。其粗酶液性质研究发现：最适酶活温度60℃，最适pH 8.0， 40℃、50℃和60℃热稳定性较好， Mn2+ 、Mg2+ 、Ca2+对该蛋白酶有激活作用，Hg2+ 、Fe3+ 、Cu2+ 、Zn2+ 、 Fe2+对其有抑制作用，苯甲基磺酰氟（PMSF）几乎完全抑制其活性，推断为丝氨酸蛋白酶。 （2）.对其中100株深海细菌和40株深海放线菌，以立枯丝核菌（ Rhizoctonia solani）和白色念珠菌（Candida albicans）为靶菌进行了抗真菌活性的筛选。初筛结果表明：分别有37株和35株深海细菌对立枯丝核菌和白色念珠菌有抑菌活性，分别占被检测细菌数目的37%和35%；有18株深海放线菌对立枯丝核菌有抑制作用，占被检测放线菌总数的45%。筛选到一株有较强抗真菌活性的深海放线菌SHA6，其发酵液对包括尖孢镰刀菌（Fusarium oxysporum）在内的10种植物病原菌有明显的抑制作用。对其发酵液的理化性质进行初步研究发现SHA6发酵液具有良好的热稳定性和酸碱稳定性, 对SHA6进行分子生物学鉴定后将其初步定名为为橙色单胞菌（Aurantimonas altamirensis）。 （3）.以卤虫为初步筛选模型，对其中的20株深海放线菌进行了杀虫方面的筛选，结果发现有5个深海放线菌菌株表现有较强的杀虫活性。其中深海放线菌SHA4发酵液的乙酸乙脂提取物杀卤虫的活性最强，在浓度为100μg/ml时杀卤虫活性为83%,而在浓度400ppm时，48h可以杀死38%的甜菜夜娥。对SHA4进行分子生物学鉴定后将其初步定为拟诺卡氏菌属（Nocardiopsis sp）。 总之，本论文阐明了我国南海深海沉积物中微生物资源状况的初步研究结果，为进一步开发利用我国南海深海微生物资源奠定了基础。

乙草胺、甲胺磷及其复合对黑土真菌的生态效应

农药对土壤微生物区系结构和功能的影响以及潜在的生态风险成为人们关注的热点之一。本文以中科院海伦生态站农田黑土作为实验土壤，采用室内模拟的方法，利用传统的（CFU和ergosterol）及分子微生物生态学技术（DGGE，real-time PcR，clolle library）研究了乙草胺、甲胺磷及其复合对黑土真菌的生态效应，并得出以下结果：经8周处理，中、高浓度乙草胺（150和250mgkg-1）对土壤真菌数量、生物量和可培养真菌种群多样性具有长期抑制效应。乙草胺处理8周后可培养真菌和土壤固氮微生物n州基因的种群结构不能得到恢复，而总的真菌结构可基本恢复。甲胺磷对土壤可培养真菌数量和生物量具有促进作用，以高浓度（250mgkg~(-1)）尤为显著。高浓度甲胺磷（25omgkg~(-1)）对nifH基因多样性有长期抑制效应。甲胺磷处理8周后可培养真菌种群结构不可恢复，而总的真菌和n积基因种群结构可部分恢复。两者复合后对真菌数量，生物量，多样性及n担基因多样性的影响无论是促进还是抑制其作用强度都大于单因子。处理8周后可培养真菌、总的真菌和n州基因三者的种群结构均不可恢复。克隆测序分析发现乙草胺、甲胺磷及其复合可明显促进植物致病真菌（colletolrichum；truncatum，Rhizoctonia zeae，Fusarium oxysporum）的生长，同时使土壤中常见的青霉菌数量减少，使农药处理后具有潜在的植物病害爆发的风险。本试验结果表明，乙草胺、甲胺磷及其复合对土壤真菌数量、结构、多样性和功能基因nifH的多样性及其种群组成有不同程度的影响，甚至产生某些不可逆的长期生态效应。复合处理对土壤真菌的影响要大于两个单因子作用，表现了明显的复合生态效应。一般来说受到午扰的真菌种群结构不容易自然恢复，因此建议在施用这两种农药过程中要避免大量、频繁的单独或复合施用。

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.

毛壳菌产抗真菌活性物质菌株筛选及种间原生质体融合研究

毛壳菌属很多种类具有重要生防价值，其生防机理包括对植物病原真菌的重寄生作用、诱导植物产生抗病性、产生抗真菌活性的次生代谢产物等。迄今，学界对毛壳菌的研究主要集中在毛壳菌的生防机理，毛壳菌活性次生代谢产物的分离等方面。本研究致力于产抗生素的毛壳菌的种间原生质体融合，从产抗生素毛壳菌菌株的筛选开始，进而对产抗生素的角毛壳菌进行诱变选育，最终用产不同抗生素的角毛壳菌与球毛壳菌进行种间原生质体融合。主要有以下五方面研究结果。 1、毛壳菌抗真菌活性物质产生菌株的筛选：不同毛壳菌菌株发酵液采用琼脂扩散法对植物病原真菌进行抑菌活性试验，结果显示，菌株CH08和CH23的发酵液对芒果炭疽、苹果炭疽和马铃薯晚疫菌具有抑制作用。菌株CH16和CH17的发酵液对芒果炭疽菌、苹果炭疽菌有抑制作用。菌株CH21发酵液对辣椒炭疽菌和西瓜枯萎菌有抑制作用。经形态学研究，菌株CH08、CH16、CH17和CH23鉴定为球毛壳菌，菌株CH21鉴定为角毛壳菌。对角毛壳菌与球毛壳菌菌株发酵液抑菌谱比较，发现角毛壳菌与球毛壳菌发酵液具有明显不同的抑菌谱，表明角毛壳菌与球毛壳菌产生不同的抗真菌活性物质。 2、角毛壳菌（CH21）和球毛壳菌（CH08）原生质体制备和再生条件研究：考察了菌龄、酶浓度、稳渗剂及其浓度、酶解温度、酶解时间及再生培养基对原生质体制备和再生的影响。用菌龄为生长54 h的角毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1.5 h，原生质体释放量2.02×107个/g；以PDA为再生培养基，0.7 mol/L的蔗糖再生稳渗剂，再生率可达51.45%。用菌龄为生长48 h的球毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1 h，原生质体释放量达1.57×108个/g；以PDA为再生培养基，0.7 mol/L的蔗糖为再生稳渗剂，再生率可达41.48%。 3、角毛壳菌（CH21）原生质体紫外诱变选育：以CH21为出发菌株，制备原生质体进行紫外诱变，诱变条件为：15 w紫外灯，距离30 cm，照射90 s，致死率80%~85%。建立了诱变菌株初筛的双层平板筛选模型。经平板初筛和摇瓶复筛，获得一株突变菌株CH21-I-402，其发酵液抑菌活性较出发菌株提高18.3%。 4、抗性标记菌株的获得：菌株CH21-I-402和CH08抗生素药敏试验表明， CH21-I-402菌株对潮霉素有抗性、对G418（Geneticin）敏感，菌株CH08对潮霉素和G418都敏感。根癌农杆菌EHA105介导的新霉素磷酸转移酶基因转化球毛壳菌，经PCR检测，新霉素磷酸转移酶基因成功转化进菌株CH08-GR70，CH08-GR120。转化子对G418抗性提高3～4倍，对潮霉素仍然比较敏感。 5、以G418和潮霉素抗性为筛选标记的原生质体融合与融合菌株AFLP分析：制备角毛壳菌CH21-I-402和球毛壳菌CH08-GR70原生质体，以35％的PEG6000为助融剂进行原生质体融合，以65 μg/ml的潮霉素和60 μg/ml G418为抗性筛选标记，获得46个再生菌株。再生菌株连续传代5代后，再生菌株表现出多种形态类型。利用AFLP技术对再生菌株及亲本菌株基因组DNA分析表明，再生菌株PF1、PF26为融合菌株。抑菌活性测试表明，融合菌株PF26发酵液对芒果炭疽菌和苹果轮纹菌有强的抑制作用，且抑菌活性比亲本球毛壳菌明显提高。 Chaetomium spp. have great potentials as biocontrol agents against a range of plant pathogens on the basis of its mycoparasitism, induced plant disease resistance, production of antifungal metabolites, and so on. Previous researches on C. spp. mostly focused on the mechanisms of its biocontrol and the isolation of secondary metabolites. In this study, screening antifungal C. spp., mutation breeding of C. cupreum and interspecies protoplast fusion between C. cupreum and C. globosum were carried out, respectively. The corresponding results are as follows: Firstly, among more than 40 C. spp., the strains produced anti-fungal antibiotics were screened by agar diffusion experiments. Results showed that both CH08 and CH23 had inhibition against Colletotrichum gloeosporioides, Cladosporium fulvum, and Phytophthora infestans. Both CH16 and CH17 had inhibition against Colletotrichum gloeosporioides and Cladosporium fulvum. In addition, CH21 exhibited anti-fungal activity against Fusarium oxysporum f. sp niveum and Colletotrichum capsici. Furthermore, CH08, CH16, CH17 and CH23 were identified as C. globosum, CH21 was proved to be C. cupreum based on morphology. The comparison of the anti-fungal spectrum between C. cupreum and C. globosum, showed they could produce different antibiotics. Secondly, specified protocols for preparing and regenerating protoplasts from mycelia of C. cupreum CH21 and C. globosum CH08 were studied. The effects of the age mycelia, the concentration of enzyme, digestion temperature and time, kinds of osmotic stabilizer and regeneration medium on protoplasts preparation and regeneration were all optimized, respectively. In one protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1.5 h at 30 ºC, 2.02×107 protoplasts from each gram mycelia were obtained from cultures of C. cupreum CH21 grown in potato dextrose broth (PDB) medium for 54 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA (potato dextrose agar with osmotic stabilize), the regeneration efficiency of protoplasts was 51.45%. In another protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1 h at 30 ºC, 1.57×108 protoplasts from each gram mycelia were obtained from cultures of C. globosum CH08 grown in PDB for 48 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA, the regeneration efficiency of protoplasts was 41.48%. Thirdly, the mutagenesis conditions and secondary screening model of C. cupreum CH21 were explored. An 80% to 85% death rate could be achieved when the protoplasts of C. cupreum CH21 were irradiated by 15 w UV lamp from 30 cm distance for 90 s. In addition, the doublelayer plate’s method for the primary screening of high-producing antibiotics strains was established. A high yielding antibiotic mutant CH21-I-402 was obtained through the primary screening on plate and the secondary selection in Erlenmeyer flask, compared to the original CH21 strain, the antifungal activity of the mutant CH21-I-402 was increased by 18.3%. Fourth, the sensitivity to antibiotics of both C. cupreum CH21-I-402 and C. globusm CH08 was detected. Results showed C. cupreum CH21-I-402 was sensitive to G418 (Geneticin) (Gs) and resistant to Hygromycin B(Hr), and C. globusm CH08 was sensitive to both G418 (Geneticin) (Gs) and Hygromycin B(Hs). At the same time, neomycin phosphotransferase II (npt II) gene was transformed into C. globusm CH08(Gs, Hs) mediated by Agrobacterium tumefaciens EHA105, and the npt II gene was verified by polymerase chain reaction in resistance to G418 strains CH08-GR70 and CH08-GR120. The transformants still showed sensitive to Hygromycin B(Hs). Finally, a selection system for hybrids was set up by interspecies protoplast fusion between C. cupreum and C. globusm using dominant selective drug resistance markers. At first, protoplasts of C. cupreum CH21-I-402 (Hr, Gs) and C. globusm CH08-GR70 (Hs, Gr) were prepared, then the protoplasts were fused in the presence of 35% polyethylene glycol 6000 and regenerated on OPDA medium with 65 μg/ml Hygromycin B and 60μg/ml G418, at last 46 colonies with Hr and Gr were obtained. Even after 5 generations’ subculture, most of the colonies displayed significant difference in taxonomic characteristics with their parental strains. Regenerated strains PF1 and PF26 were confirmed as fusants by amplified fragment length polymorphisms analysis with the genomic DNA as the model. PF26 showed higher inhibitory activity against Colletotrichum gloeosporioides and Macrophoma kuwatsukai than that of the parental strain C. globusm.

固定化微生物降解土壤中菲和芘的研究

Immobilized with PVA,sodium alginate and activated carbon,both Zoogloea sp. and Fusarium sp.strains could degrade phenanthrene and pyrene efficiently.The optimal carrier was made of 100ρ·g -1 L PVA,5 sodium alginateρ·g -1 L and 50 activated carbon ρ·g -1 L.The degradation rates of phenanthrene and pyrene in 10 days were 87.48% and 75.34% by the immobilized bacterium,37.04% and 20.85% higher than those by the free bacterium,and the rates in 15 days were 84.36% and 74.87% by the immobilized fungus,5.35% and 11.23% higher than those by the free fungus.

海洋细菌3512A对黄瓜枯萎病的防治及促进植株生长的效应

自南海柳珊瑚(Pacifigorgia sp.)中分离得到一株对黄瓜枯萎病菌尖孢镰刀菌黄瓜专化型(Fusarium oxysporum f.sp.cucumer-inum)有较强抑制作用的海洋枯草芽孢杆菌(Bacillus subtilis)3512A。经室内模拟试验表明,该菌株能够在灭菌土和有菌土中高密度定殖,促进黄瓜幼苗的生长。为探讨该菌株在室外对黄瓜枯萎病的生防作用及对黄瓜生长和产量的影响,进行了盆栽试验,结果表明,海洋细菌3512A能够有效地防治黄瓜枯萎病,促进黄瓜的生长,提高叶绿素含量,增加黄瓜的产量。

江苏省云台山7种野生中草药植物根区土壤真菌多样性研究

通过对江苏省云台山地区7种不同野生中草药植物烟台百里香(T.quinquecostatusCelak.)、掌叶半夏(P.pedatisectaSchott.)、何首乌(P.multiflorumThunb.)、海洲香薷(E.splendensNakai ex F.)、野葛[P.lobata(Willd)Ohwi]、紫金牛(A.japonicaBl.)和菝葜(S.sieboldiiMiq.)根区土壤真菌进行分离鉴定,共分离出真菌16属126种。多样性分析结果表明,7种野生中草药植物根区土壤真菌种群多样性丰富,其中青霉属(Penicillium)、曲霉属(Asper-gillus)和木霉属(Trichoderma)是中草药植物根区土壤中的优势种群,镰孢菌属(Fusarium)、交链孢属(Alternaria)、腐霉属(Pythium)、毛霉属(Mucor)真菌分布丰度也较高。结果也表明根区土壤真菌群落在一定程度上受到中草药植物的影响,大部分野生中草药植物根区土壤的真菌群落的均匀度指数低于裸地非根区土壤,而丰富度指数却高于裸地非根区土壤。不同野生中草药植物根区土壤真菌区系的结构和组成存在一定的差异性,紫金牛根区土壤中真菌种类最多,达到14属,而野葛根区土壤中真菌种类最少,只有8属。

莲藕状固定化真菌(镰刀菌)对土壤中菲、芘的降解

采用莲藕状聚乙烯醇复合载体对镰刀菌(Fusarium sp.)固定化包埋,分别进行了不同接种量的固定化真菌对菲、芘的降解,固定化真菌对不同系列浓度菲、芘的降解试验,以及对固定化真菌在自然土壤中对菲、芘降解的各项参数作了测定,结果表明,固定化真菌具有较好的降解效果,同时用电镜观察研究了镰刀菌在固定化载体中的分布形态.自然土壤中固定化真菌在360h时,对菲、芘的降解效率分别为76.96%和20.69%,而土著菌仅达到33.37%和15.28%.

牛蒡根际土壤致害菌Fusariumsolani分离鉴定

目的:探索牛蒡根际镰刀菌与牛蒡之间的相互关系。方法:从全国30个地区采集牛蒡根际土壤样品,进行了根际土壤真菌数量和群落的生态学研究,测定镰刀菌发酵液对牛蒡幼苗和牛蒡子萌发的影响,并对其中毒性最强的两株镰刀菌F130和F131进行了形态学和分子生物学的鉴定。结果:镰刀菌为牛蒡根际的最优势类群,贡献率为34.297%,其次为木霉,贡献率为22.519%;绝大多数镰刀菌对牛蒡有明显的毒性作用,其中F130和F131被鉴定为Fusarium solani。结论:Fusarium solani是牛蒡根际土壤中的致害菌。

杉木连栽根际土壤致害Fusariumoxysporum拮抗菌的筛选

尖孢镰刀菌萎蔫专化型(Fusarium oxysporumf.sp.vasinfectum,SF2)及其分泌的毒素是造成杉木连栽土壤中毒的重要因素之一,基于此,笔者从106株海洋微生物中筛选到23株具有拮抗SF2特性的菌株,在湖南会同杉木中心产区进行的室外盆栽试验结果表明,其中海洋细菌3728菌株能够在杉木根际土壤中定殖,抑制杉木致害菌的生长繁殖,促进了杉木幼苗生长,经鉴定为枯草芽孢杆菌(Bacillus subtilis)。该研究对杉木人工林生产具有重要的理论与实践意义。

真菌对石油污染土壤的降解研究

利用微生物对石油污染土壤进行生物降解，具有操作简单，费用低廉，场地适用性强等特点。挑选了2种菌株，进行了室内油降解实验，在摇床实验油降解率：微生物真菌（Faserium．LK）（土著）和真菌（Phanerochaete．Chrysosprium），在20d分别为41.2％和28．1％，真菌（Fusarium．LK）高于真菌（Phane－rochate．Chrysosprium）的降解率，而在培养箱石油污染土壤中，真菌（Fusarium．LK）（土著）和真菌（Phane－rochaete.Chrysosprium），在50d分别为61.8％和66．1％，（Fusarium.LK）低于（Phanerochaete.Chrysosri-um）。

Biosynthesis of gold nanoparticles assisted by Escherichia coli DH5 alpha and its application on direct electrochemistry of hemoglobin

In this communication, biosynthesis of gold nanoparticles assisted by Escherichia coli DH5 alpha and its application on direct electrochemistry of hemoglobin are reported. The gold nanoparticles formed on the bacteria surface are mostly spherical. The direct electrochemistry of hemoglobin can be achieved by incorporated into the bio-nanocomposite films on a glassy carbon electrode.