铁皮石斛组培苗菌根真菌接种效应

本研究以铁皮石斛无菌组培苗为材料,通过接种菌株建立组培苗菌根共生培养体系,对接种共生的组培苗各生长阶段进行比较研究,以探讨接菌菌株对无菌组培苗生长的影响并筛选出最佳菌株,为铁皮石斛快速优质繁殖提供思考和借鉴。结果表明:D1菌株、D2菌株和D3菌株对铁皮石斛无菌组培苗地上部分生长和根系生长均有明显促进作用,组培苗D1、D2、D3处理的平均鲜重增长率分别比对照组(CK)高27.4%、9.8%、19.7%,与CK相比差异均达极显著水平(p0.01);组培苗D1、D2、D3、D5处理的根增长率分别比CK高23.1%、34.7%、49.1%、36.5%,与CK相比较差异达极显著水平(p0.01)。除了D4以外的大多数菌株可明显促进铁皮石斛组培苗叶片光合色素的累积,为植物生长提供充足的营养物质,进而促进组培苗的生长。     

微生物共同作用下的铁皮石斛组培苗生长效应研究

植物体和植物根际均是复杂的微生态系统,其内栖息着关系复杂的共生微生物,共同影响植物的生长发育.为探讨混合接种真菌与细菌对兰科植物生长的影响,筛选出真菌与细菌的优势促生组合,本研究选取经分离、筛选获得的4株促生内生真菌(铁皮石斛内生真菌C22、C35,美花石斛内生真菌L12、L28)和3株促生内生细菌(铁皮石斛菌内生细菌TX-7、TX-16、TX-19),以"真菌+细菌"的方式混合接种于铁皮石斛无菌组培苗中,共培养120 d.结果获得了3组优势组合:C22+TX-19、L28+TX-16和L28+TX-19,它们对铁皮石斛组培苗的生长均表现出正效应,其中C22+TX-19和L28+TX-19对促进组培苗生物量的增长具有协同效应,L28+TX-19对提高组培苗的根分枝率具有协同效应,3个组合对增加组培苗的分蘖数和根尖数均表现为累加效应.研究结果表明,内生真菌与内生细菌的共同作用可显著促进铁皮石斛的生长,混合接种有可能更大地发挥微生物的效能.     

春兰菌根菌的分离及共生培养体系的研究

采用根组织切片法自野生春兰菌根共分离到6株真菌菌株,接种春兰杂交组培苗,结果发现CL-3和CL-6菌株在菌-苗共生培养基Ⅰ中对幼苗生长有不同程度的促进作用,2 个月后处理苗的鲜重增长率分别达到47.4%和42.5%,与对照差异达显著水平(P＜0.05).在CL-3和CL-6接菌处理苗的营养根中均分离获得了原接种真菌,并观察到了菌根结构.结果表明,CL-3和CL-6菌株已与组培幼苗成功建立了共生培养体系.     

铁皮石斛组培苗与菌根真菌共培养过程中的相互作用

由于人为的滥采滥挖和野外生境的退化, 使得铁皮石斛 (Dendrobium officinale) 这种名贵的中药材一直处于极度濒危的状态。为了从菌根真菌的角度给人工保育铁皮石斛提供理论指导, 对铁皮石斛的组织培养苗人工接种‘GDB181’菌株 (Epulorhiza sp.) 。培养60d后, 接菌苗平均鲜重增长率比对照苗高出了84.8%。在营养元素含量方面, 接菌苗的B、Si、Fe、Cu和Mn元素含量的净增率分别为780%、533%、192%、191%和128%, 均在100%以上;其他元素含量也有不同程度的增加 (除Zn外), 结果证明两者有效地建立了共生关系。在显微和超微结构的观察中发现:真菌菌丝随机破坏铁皮石斛的根被入侵到外皮层, 并从外皮层细胞不断扩展延伸到皮层的大型细胞, 最后在大型细胞中被分解消化。在真菌侵染过程中, 被侵染的皮层细胞的细胞壁严重扭曲变形, 菌丝在皮层细胞形成菌丝结, 菌丝结常位于细胞核附近或包围细胞核。在皮层的大型细胞中, 菌丝细胞被植物的溶酶体包围, 部分或全部被消解, 出现脱壁或失去细胞质甚至成为空腔等变化, 最终形成衰败的菌丝残骸, 溶酶体也随之消失。溶酶体分布越多的部位, 菌丝细胞消解变形越严重。含有菌丝残骸的皮层细胞可被新侵染的菌丝重新定殖, 这一菌丝侵染被消化再侵染的过程在铁皮石斛生长发育过程中可不断重复发生。     

硝酸镧和兰科菌根真菌对铁皮石斛生理特性的影响

利用盆栽的方式研究了不同硝酸镧水平(0、1.0、3.0、5.0和7.0mg·L-1)下接种兰科菌根真菌对铁皮石斛生物量、多糖和蛋白质合成的影响,并分析了叶绿素含量、丙二醛(MDA)含量以及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性的变化,以探讨硝酸镧和兰科菌根真菌对铁皮石斛生理特性的影响。结果表明:(1)添加适量的硝酸镧有利于菌根真菌侵染和菌根发育,提高铁皮石斛幼苗生物量。(2)在接种菌根真菌的同时添加5.0mg·L-1的硝酸镧,铁皮石斛的根重、茎叶重和总生物量均达到最大,分别是未添加硝酸镧以及未接种对照组的4.26倍、4.98倍和4.87倍,其菌根侵染率也高达92.8%;而且可显著提高叶片中叶绿素含量,并显著降低细胞内的丙二醛含量。(3)在适量(5.0mg·L-1)的硝酸镧水平下接种菌根真菌能促进铁皮石斛幼苗多糖和蛋白质的合成,并显著提高细胞内SOD、CAT和POD活性。研究认为,菌根真菌与适宜浓度硝酸镧(5.0 mg·L-1)联合使用能显著促进铁皮石斛菌根的形成,增强植株的生理活性和适应能力,提高其生物量和多糖等活性成分的积累,有效改善铁皮石斛的药用品质。     

气调和乙烯对梅果叶绿素和内源激素含量的影响

本文研究了在 ( 2 5± 1 )℃下 ,气调包装和乙烯吸收剂处理对采后青梅果实叶绿素含量、内源激素IAA ,GA3,ABA含量和乙烯释放量的影响及它们之间的关系。结果表明 :气调包装果实叶绿素含量最高 ,其次是乙烯吸收剂处理的 ;各处理中气调包装果实的乙烯释放量始终很低 ,GA3含量较高 ,IAA和ABA含量则较低 ;对照果实的则相反 ,乙烯释放量很高 ,IAA和ABA含量较高 ,而GA3含量较低。乙烯吸收剂处理的处于二者之间。气调包装可以维持果实较高的GA3水平 ,降低ABA含量 ,保持较高的GA3/ABA值 ,抑制IAA和乙烯的生成 ,延缓梅果叶绿素的降解。     

气调和乙烯对梅果叶绿素和内源激素含量的影响

本文研究了在（25±1）℃下,气调包装和乙烯吸收剂处理对采后青梅果实叶绿素含量、内源激素IAA,GA3,ABA含量和乙烯释放量的影响及它们之间的关系。结果表明：气调包装果实叶绿素含量最高,其次是乙烯吸收剂处理的;各处理中气调包装果实的乙烯释放量始终很低,GA3含量较高,IAA和ABA含量则较低;对照果实的则相反,乙烯释放量很高,IAA和ABA含量较高,而GA3含量较低。乙烯吸收剂处理的处于二者之间。气调包装可以维持果实较高的GA3水平,降低ABA含量,保持较高的GA3／ABA值,抑制IAA和乙烯的生成,延缓梅果叶绿素的降解。     

不同种类柑橘的抗旱性及其与内源激素变化的关系

以红橘、朱橘、土橘、枳、枳橙、实生酸柚、实生甜橙、黄柑为材料,进行不同降水量的长期干旱胁迫后,测定生物量、抗旱系数及叶片中赤霉素(GA1+3)、玉米素核苷(ZR)、生长素(IAA)、脱落酸(ABA)含量,分析GA1+3、ZR、IAA、ABA的相对含量变化及ABA/GA1+3、ABA/ZR、ABA/IAA与8个种类柑橘抗旱性的关系.结果表明:在降水量低于1200 mm(对照)条件下,随降水量的减少不同种类柑橘的生物量明显降低,其抗旱系数及抗旱力大小顺序为朱橘红橘枳土橘枳橙实生酸柚实生甜橙黄柑.不同种类柑橘叶片中ABA含量随降水量的减少而增加,GA1+3、ZR和IAA含量随降水量的减少而降低.在抗旱性强的柑橘种类中,ABA/GA1+3和ABA/ZR的增加幅度大,ABA/IAA变化与种间抗旱力的关联度不大.ABA和GA1+3的相对含量与柑橘抗旱系数分别呈极显著正相关和极显著负相关,ZR的相对含量与抗旱性系数呈极显著或显著负相关,IAA的相对含量与抗旱性系数相关性不明显.对柑橘抗旱性的促进效果,以ABA最大,其次为GA1+3和ZR,IAA最小.     

春兰菌根真菌CL-3菌株的鉴定及共生体系的构建

采用分离自野生春兰(Cymbidium goeringii)根部的真菌CL-3菌株, 进行了春兰内生菌根真菌的人工接种、再分离及其共生培养研究。通过对CL-3菌株形态学观察和ITS序列同源性分析, 发现该菌株ITS序列与Acremonium strictum的亲缘关系最为接近, 序列同源性为100%。用CL-3菌株接种春兰组培苗, 接种后2个月可从组培苗中再分离获得该菌株, 且CL-3菌株处理苗的鲜重增长率达80.5%, 经方差分析, 与对照相比有显著差异。通过石蜡切片和染色, 在已接种的组培苗的根部组织中可观察到CL-3菌株存在。表明CL-3菌株能与组培幼苗成功建立共生培养体系。     

不同春石斛品种扦插苗茎尖激素含量与其成花品质关系

为探讨春石斛扦插苗生长发育过程中内源激素与成花品质的关系,本研究以成花品质不同的2个品种‘森禾H1’和‘森禾4001’为试材,对其一年生扦插苗生长发育过程中5个不同阶段茎尖内源激素含量进行了比较分析。结果显示:整个生长发育过程中,2个品种扦插苗茎尖中赤霉酸(GA3)、吲哚乙酸(IAA)、脱落酸(ABA)含量以及GA3/IAA、GA3/ABA、(GA3+IAA)/ABA变化趋势相似。成花品质较好的‘森禾H1’,其GA3、GA3/IAA、GA3/ABA、(GA3+IAA)/ABA,除了在休止叶后期外,其他时期均一直显著高于成花品质较差的‘森禾4001’,而其ABA含量在萌动期、展叶期、旺盛生长期显著低于‘森禾4001’,IAA含量没有显示出规律性。结果表明,春石斛扦插苗的一些内源激素含量及比例与其后期成花品质密切相关,可作为优良品种早期筛选的参考指标。     

产喜树碱喜树内生真菌的筛选及喜树内生真菌的SRAP分析

生活在宿主植物里的内生真菌是很重要的药用资源。喜树是中国的传统药用植物。从喜树植物中分离得到了大约50种菌株,其中一株产喜树碱的菌株通过形态学鉴定为青霉属,这是首次在喜树植物中发现产喜树碱的青霉属菌株。为研究简单序列重复相关序列扩增多态性(SRAP)方法在喜树内生真菌中应用的可行性,选择了十株喜树内生真菌进行SRAP多态性分析。SRAP引物共扩增出1 295条带,而这些菌株也被分为三大类。这些结果表明,SRAP研究喜树内生真菌具有高效性,是讨论喜树内生真菌的遗传多样性的有效方法。     

Cultivation of fungi from fecal specimens in cases of antibiotic associated diarrhea (AAD)

The aim of performed examinations was to isolate, identify and determine a drug susceptibility of fungi cultured from faecal specimens submitted for detection of Clostridium difficile in cases of antibiotic-associated diarrhoea (AAD). One hundred samples of diarrhoeic faeces were examined using routine bacteriological methods (isolation and identification of C. difficile), serological test (detection of C. difficile toxins A/B) and mycological methods (isolation, identification and drug susceptibility testing of fungi). Out of twenty seven specimens of diarrhoeic faeces fungal strains were isolated, in 20 samples C. difficile strain and/or C. difficile toxins A/B were detected, in 23 specimens fungal strains, C. difficile strains and/or toxins A/B of this species were present. The most active in vitro agent against cultured fungal strains was nystatin. In conclusion it can be stated, that fungal strains are responsible for some cases of antibiotic-associated diarrhoea. So, mycological diagnostics of faecal samples from patients with diarrhoea after antibiotic therapy is necessary. Cases of diarrhoea with mixed bacterial and fungal aetiology (C. difficile + yeast-like fungus) were observed.     

Effect of the dark septate endophytic fungus <Emphasis Type="Italic">Acrocalymma vagum</Emphasis> on heavy metal content in tobacco leaves

Dark septate endophytic fungi are symbiotes that play an important role in plant resistance to biotic and abiotic stress. In this study, 65 strains of endophytic fungi were obtained from tobacco roots using a tissue isolation method. Strains NYN8C05 and NYN8G01 were identified as Acrocalymma vagum based on ITS rDNA sequence analysis and morphological characteristics. Co-culture experiments showed that strains NYN8C05 and NYN8G01 can promote the growth of tobacco seedlings. Observations of roots after trypan blue staining showed that strains NYN8C05 and NYN8G01 colonized the roots and formed dark septate hyphae and microsclerotia. These results confirmed that strains NYN8C05 and NYN8G01 were dark septate endophytic fungi. Pot experiments showed that NYN8C05 and NYN8G01 significantly decreased the heavy metal content in leaves of tobacco plants. A. vagum strains NYN8C05 and NYN8G01 are dark septate endophytic fungi that have application potential for decreasing heavy metal content in tobacco leaves.     

临床酵母样真菌的感染特点及耐药性研究

目的了解临床酵母样真菌的感染类型、分布以及耐药情况,为临床诊断治疗提供合理的用药依据.方法采用常规方法进行真菌培养,用科玛嘉显色培养基联合法国生物梅里埃API 20C AUX鉴定条进行鉴定,药敏试验采用微量稀释法.结果864株酵母样真菌中,白色念珠菌619株（71.6%）,其次为热带念珠菌116株（13.4%）和克柔念珠菌47株（5.4%）,非白色念珠菌感染的比例逐年上升（21.5%）.其中,呼吸道标本酵母样真菌检出率最高,达79.7%,其次是消化道为8.8%,泌尿道为4.7%.科室分布依次为干部科、呼吸科、急诊内科、血液科等;白色念珠菌对两性霉素B、5-氟胞嘧啶高度敏感,达90%以上,对氟康唑和伊曲康唑的敏感性有所降低.结论酵母样真菌的检出率与患者基础疾病密切相关;对氟康唑等药物的敏感性有下降的趋势,未发现对4种药物同时耐药的菌株,提示在治疗中,药敏监测是非常必要的.     

医院内深部真菌感染分布及耐药性分析

目的探讨大连医科大学附属第一医院深部真菌感染的常见菌种,临床病区分布及其耐药性。方法对2008年1月1日至2008年12月31日749例住院患者深部真菌感染进行分析。结果常见菌种为白色假丝酵母菌,热带假丝酵母菌,近平滑假丝酵母菌,克柔假丝酵母菌,光滑球拟酵母菌。主要病区为ICU。从标本的来源看,痰标本中真菌检出率最高,占61.1%。两性霉素B抗菌作用最强,耐药率只有3.6%。结论医院内深部真菌感染分布广泛,非白色假丝酵母菌感染明显增多,耐药菌株增多,部分治疗需联合用药。     

Etiologic agents of fungemia in hospitalized patients]

The aim of performed examinations was the analysis of fungi as etiological agents of blood infections in patients hospitalized in surgical wards, internal medicine wards and intensive care units of the Medical Academy Central Clinical Hospital in Warsaw. Blood samples from patients hospitalized in 1997 were examined. Peripheral blood samples were incubated in BacT/Alert system (Organon Teknika, USA). Positive blood samples were inoculated on Sabouraud medium with chloramphenicol (bioMerieux, France or Oxoid, England). The time of cultivation was from 48 hours to 7 days at 30 degrees C. Fungal strains were identified by standard mycological procedures with the use of chromogenic medium BBL CHROMagar Candida (Becton Dickinson, USA) and biochemical test ID 32 C (bioMerieux, France). Susceptibility of strains to antifungal agents was determined by ATB FUNGUS method (bioMerieux, France). The total number of positive blood cultures in 1997 was 1380. Forty-two fungal strains were isolated from blood samples (3%). Strains belonged to the following species: C. albicans (17 isolates), C. parapsilosis (15), C. glabrata (3), melibiosica (2), C. pelliculosa (2), C. guilliermondii (1), C. tropicalis (1) and T. beigelii (1). Among fungi cultured from patients hospitalized in operative wards dominated C. parapsilosis (11) and C. albicans (10) strains, whereas from patients hospitalized in conservative wards most often C. albicans (6) strains were isolated. Candida strains were mostly susceptible to antifungal agents tested. It was interesting to culture Trichosporon beigelii (T. cutaneum) strain as an etiological agent of fungemia. This strain was multidrug-resistant.     

297例浅部真菌病临床及病原菌菌种分析

目的 探讨皮肤浅部真菌病致病真菌菌种的构成.方法 对297例真菌涂片阳性和培养阳性的浅部真菌病患者,取标本进行分离培养及菌种鉴定,培养阳性标本在形态学上不能准确鉴定的,进行梅里埃API 20C AUX酵母菌鉴定试剂盒或核糖体DNA （rDNA） ITS区序列测定,确切鉴定菌种.使用SPSS 17.0统计软件对于结果进行统计分析.结果 共分离培养出致病菌13种,其中红色毛癣菌86株（29.0％）,须癣毛癣菌68株（22.9％）,念珠菌属59株（19.9％）,暗色真菌属13株（4.4％）,曲霉菌属13株（4.4％）,红酵母菌12株（4.0％）,青霉菌属9株（3.0％）,毛霉菌9株（3.0％）,犬小孢子菌5株（1.7％）,浅白隐球菌3株（1.0％）,毛孢子菌属2株（0.7％）,絮状表皮癣菌1株（0.3％）,混合感染17株（5.7％）.结论 本地区浅部真菌病以甲癣为主,主要致病真菌是红色毛癣菌,但其他种类真菌感染尤其是念珠菌属有明显上升趋势.     

Yeast-like fungi as etiologic agents of blood infections in patients hospitalized in 1998-1999

The aim of the study was the analysis of frequency of yeast-like fungi as etiological agents of fungemias in patients hospitalized in operative and conservative wards of Medical Academy Central Clinical Hospital in Warsaw in 1998-1999. Peripheral blood samples and collected from vascular catheters were incubated in BacT/Alert system(Organon Teknika, USA). Positive blood samples were inoculated on Sabouraud medium with chloramphenicol (bioMerieux, France) (the time of cultivation from 48 h to 7 days at 30 C) and on chromogenic medium BBL CHROMagar Candida (Becton Dickinson, USA). Fungal strains were identified by standard mycological procedures using ID 32 C strips (ATB system, bioMerieux, France) and tests of Sanofi Diagnostics Pasteur (France). The total number of positive blood cultures was 1724. Fifty eight fungal strains were isolated from blood samples (3.36%). Strains belonged to 4 genera: Candida (55), Trichosporon (1), Saccharomyces (1) and Pichia (1). Thirty eight fungal strains were isolated from peripheral blood samples. Forty seven fungal strains were cultured from patients hospitalized in operative wards. Among fungi isolated from peripheral blood samples C. albicans (10), C. glabrata (9) and C. parapsilosis (5) strains dominated. From blood samples collected from vascular catheters most often C. albicans (7), C. glabrata (4) and C. parapsilosis (3) were isolated.     

Analysis of fatty acid composition of anaerobic rumen fungi

The fatty acid (FA) composition of fresh mycelia of anaerobic rumen fungi was determined. The fatty acids methyl esters (FAME) of six strains belonging to four genera (Neocallimastix, Caecomyces, Orpinomyces, Anaeromyces) and one unknown strain were analyzed by gas chromatography. All studied fungi possess the same FAs but differences were found in their relative concentrations. The FA profile of anaerobic fungi comprises carbon chains of length ranging from 12 to 24; the most common fatty acids were stearic (C(18:0)), arachidic (C(20:0)), heneicosanoic (C(21:0)), behenic (C(22:0)), tricosanoic (C(23:0)) and lignoceric (C(24:0)) with relative amount representing >4% of total FA. Significant differences were determined for heptadecanoic, oleic, behenic and tricosanoic acids. Rumen anaerobic fungi can contain very long chain fatty acids; we found unsaturated fatty acids including cis-11-eicosenoic (C(20:1)), cis-11,14-eicosadienoic (C(20:2)), erucic (C(22:1n9)), cis-13,16-docosadienoic (C(22:2)) and nervonic (C(24:1)) acids in very small amounts but their presence seems to be unique for anaerobic fungi.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.