三株散囊菌发酵茶叶的初步研究

散囊菌属真菌(Eurotium spp.)能赋予发酵茶独特的口感和香味。本研究利用前期从广西某六堡茶中筛选并鉴定的三株散囊菌属真菌Aspergillus chevalieri E2、Aspergillus chevalieri E3与Aspergillus cristatus E6,探讨在不同温度下以优化察氏液体培养基培养的生长状况,发酵前不同灭菌条件下的茶叶品质,以及所得茶汤中茶多酚含量、总抗氧化能力和DPPH·自由基清除能力。结果显示:三株真菌在优化的察氏液体培养基中31℃～34℃下都能良好生长。茶叶发酵温度为28℃,三株真菌在发酵初始含水量为20%以上生长良好,其中E3和混合发酵组的生长速度最快。E2在茶叶表面生长出大量金黄色子囊果以及大量浅绿色分生孢子梗; E3几乎只有浅绿色分生孢子梗; E6几乎只有金黄色子囊果。发酵茶叶制作的茶汤内茶多酚含量比未发酵低,抗氧化性指标也有所下降,说明本实验真菌发酵促进了茶内抗氧化物质的氧化。本研究对源于六堡茶不同散囊菌属真菌的茶叶发酵有重要参考价值。     

不同海水浓度和培养时间对海洋真菌抗菌活性的影响

用滤纸片琼脂扩散法研究不同海水浓度和培养时间对海洋真菌抗菌活性的影响,旨在为大规模培养海洋真菌和提高获得抗菌物质的几率提供理论依据。结果显示：用不同海水浓度培养基发酵海洋真菌,在供试的10株海洋真菌中,5株海洋真菌的抗菌活性和抗菌谱有明显差异;培养时间不同,6株海洋真菌的抗菌活性差别较大。实验结果表明,不同海水浓度和培养时间对海洋真菌抗菌活性有显著影响。     

海水真菌定量分析方法的初步研究

为定量分析海水真菌,分别采用不同的采样方法采样;并采用5种培养基,每种培养基分别用4种不同的处理方式,共20种不同处理组合,筛选出适合定量分析海水中真菌的平板计数方案.结果表明,不同的采样方法下,真菌在同一培养基上的生长无显著性差异.在同一海水水样中,马铃薯培养基和马铃薯葡萄糖琼脂培养基用海水配制时几乎没有真菌生长;麦芽汁培养基在不同的处理条件下真菌略有生长,且各种处理间无明显差别,其种类主要是霉菌;察氏培养基和酵母菌浸出粉胨葡萄糖琼脂培养基在不同处理条件下真菌长势差别较大,种类主要是酵母菌.通过对20种培养组合的比较,选择酵母浸出粉胨葡萄糖琼脂培养基,用海水来配制培养基并用海水稀释样品的处理,可以培养出最大数量的海洋真菌.     

地衣型真菌次级代谢产物抗菌活性初步研究

地衣是一种传统的民族药物,能产生多种具有活性的物质。该研究对地衣型真菌(Xanthoria elegans,Myelochroa indica,Ramalina peruviana,Cladonia macilenta,Nephromopsis pallescens,Cladonia coccifera)进行液体培养,2个月后,培养液用乙酸乙酯萃取后获得初提物。该研究采用抑菌圈法评价地衣型真菌初提物对7种致病细菌(Bacillus subtilis,Bacillus cereus,Vibrio parahaemolyticus,Straphylococcus haemolyticus,Pseudomonas aeruginosa,Staphylococcus aureus,Micrococcus luteus)的抗菌活性,并测定最低抑菌浓度(MIC)。结果表明:6种地衣型真菌的初提物均具有一定的抗菌活性,且不同培养基对地衣型真菌产生抗菌物质有显著影响。其中,R.peruviana在MY液体培养基中所产生的次级代谢产物对金黄色葡萄球菌、藤黄微球菌、溶血性葡萄球菌、铜尿假单胞菌具有抑制效果,但在YMG培养基中所得初提物对供试7种致病细菌不具有抑菌效果。X.elegans在YMG培养基中所得初提物对枯草芽孢杆菌具有明显抗菌活性,其抑菌圈直径可达17.77 mm。该研究证实不同地衣型真菌液体培养初提物具有抗菌活性,不同的培养基也直接影响地衣型真菌抗菌效果。该研究结果为地衣型真菌的进一步研究及民族药的开发利用奠定了基础。     

真菌的L型

随着广谱抗生素、免疫抑制剂的广泛应用及恶性肿瘤、AIDS 发病率的升高,真菌对人类的感染日益增多。尽管真菌细胞壁坚硬而复杂,但也因受到体内外某些因素的影响发生细胞壁缺损变为 L型。真菌 L 型的形成自发形成:真菌能自发形成 L 型,将念珠菌接种在营养缺乏的培养基上,使其处于饥饿状态,长出的菌30～50%缺乏细胞壁。若在培养基中加入人血,反而不利于 L 型的生长,若将此 L 型继续培养,菌落周边长出假菌丝,2周后又逐渐回复成典型的     

低氧对曲霉和假丝酵母体外两性霉素B、伊曲康唑和棘白素药敏试验的影响[英]／Warn PA…／／J Antimicrobial Chemother．

抗真菌药在体外和体内的效果有很大差异,可靠的体外药敏试验在对真菌病的用药是有帮助的。已知许多因素可影响真菌的最低抑菌浓度(MIC)测定,如培养基、菌悬液的浓度、试验方法的选择(多量稀释法或微量稀释法)和培养温度。作者研究了不同氧浓度对真菌体外药敏试验的影响。     

CHO细胞法检测百日咳毒素毒性的影响因素

目的考察不同培养基、不同牛血清、血清灭活与否及生产过程中添加的各外源物质对百日咳毒素(pertussis toxin, PT)在中华仓鼠卵巢细胞(chinese hamster ovary cell, CHO)簇集试验中的影响。方法分别使用3种培养基F-12K、DMEM/F12和1640培养CHO细胞,并进行CHO细胞簇集试验,观察细胞生长状态及PT引起细胞簇集的敏感性;分别选取2个厂家的牛血清(对2种血清进行灭活和不灭活处理)培养CHO细胞,观察4种牛血清对细胞生长及簇集的影响;选用生产过程中添加的物质进行CHO细胞簇集试验,观察细胞生长状态及是否出现簇集,确定不影响细胞生长的最高浓度,同时使用不影响细胞生长的各添加物质最高浓度进行小鼠组胺致敏试验,观察与CHO细胞簇集试验结果是否一致。结果 3种培养基对CHO细胞生长及CHO细胞簇集存在明显差异,F-12K培养基培养的细胞形态规则、典型,其他2种培养基培养的细胞生长缓慢,且对PT的敏感性均低于F-12K培养基;4种牛血清中胎牛血清培养的细胞生长最快且形态规则,簇集试验敏感性优于其他3组血清;添加的各外源物质均会导致细胞生长缓慢或死亡,在稀释至一定浓度后可以排除添加物质对CHO细胞簇集试验的影响,同时在小鼠组胺致敏试验中不会引起动物死亡。结论 F-12K培养基最适宜实验室CHO细胞生长,不同血清对细胞生长和簇集的敏感度有一定差异,添加的外源物质残留量应进行控制以保证试验结果的稳定可靠。     

油樟油对油樟内生真菌中活性化合物影响

YY26、YG42和YG71是从油樟中分离得到的三株内生真菌。本研究采用GC-FID、GC-MS技术,以油樟油作为诱导物,研究YY26、YG42、YG71菌株在不同培养基条件下对菌株中活性化合物1,8-桉叶油素、松油烯-4-醇、a-松油醇、r-松油烯产量的影响。结果表明:在PDB培养基中,一方面,油樟油可促进YY26、YG42菌株积累1,8-桉叶油素等物质,其中对1,8-桉叶油素的促进最强,分别提高1.41倍和1.30倍;另一方面,油樟油抑制YG71菌株中以上4种物质的积累。在察氏培养基中,油樟油对YY26、YG42及YG71菌株中4种物质的积累没有明显的促进作用。该研究为植物有用代谢产物的规模化生产提供了理论依据,有较强的实践意义。     

胡颓子属三株根瘤内生菌培养条件的比较研究

从胡颓子属植物翅果油树、沙枣和牛奶子的根瘤中,分离获得Frankia sp. Eml108、EaI827和EuI620三株内生菌。对它们的培养条件作了比较研究。结果表明,三株菌对培养基的pH和培养温度的要求基本相似。EmI108和EuI620菌株的最适宜培养基为Qmod/Tween80,而EaI827菌株则为Qmod培养基。在以NH+4作氮源的基础培养基中,EmI108和EuI620菌株生长最适宜的碳源为Tween80,而EaI827菌株则为琥珀酸。EaI827和EuI620菌株能较好地利用无机氮源NH4Cl,但是对于三株菌来说,最适宜的还是有机氮源聚胨和酪蛋白水解物。     

光照和培养基对内生真菌棘豆链格孢Alternaria oxytropis苦马豆素含量的影响

以小花棘豆内生真菌棘豆链格孢Alternaria oxytropis野生株OW7.8及其酵母氨酸还原酶基因缺失突变株M1为材料,研究它们在不同培养基上和不同光照下菌落生长速率与苦马豆素含量。结果表明:OW7.8在胡萝卜葡萄糖琼脂培养基上暗培养时生长速度最快,为(2.57±0.17)mm/d,而M1则在马铃薯葡萄糖琼脂培养基上暗培养时生长速度最快,为(4.93±0.10mm)/d。不同培养基和光照条件下,相同菌株苦马豆素的含量差异不显著(P0.05);不同菌株在相同培养条件下其苦马豆素含量差异显著(P0.05)。     

Studies on fungi cultivated by ants

A collection of 36 fungi cultivated by leaf-cutting ants has been established at The New York Botanical Garden. These fungi grow on a variety of natural media and on a synthetic medium with mineral salts, dextrose, casein hydrolysate, purine and pyrimidine bases and vitamins. Tests of the fungi for antibacterial activity were all negative againstStaphylococcus aureus andEscherichia coli. Only four isolates of ant fungi, each cultivated by a different species of ant, produced basidiocarps on oatmeal agar. Taxonomic studies indicate that these belong to the same species of fungus (Lepiota sp.). Eighteen isolates produced bromatia characteristic of the form species,Attamyces bromatificus Kreisel, one produced a mycelium with clamp connections, and thirteen produced sterile mycelia without clamped hyphae and without bromatia.     

Effect of aposymbiotic conditions on colony growth and secondary metabolite production in the lichen-forming fungus Ramalina dilacerata

The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is thought to be influenced by environmental conditions. The effects of the environment may be studied by culturing fungi under defined growing parameters to provide a better understanding of the role of the large number of polyketide synthase (PKS) gene paralogs detected in the genomes of many fungi. The objectives of this study were to examine the effects of culture conditions (media composition and pH level) on the colony growth, the numbers of secondary products, and the expression of two PKS genes by the lichen-forming fungus Ramalina dilacerata. Four types of growth media at four different pH levels were prepared to culture spore isolates of R. dilacerata. Colony diameter and texture were recorded. The number of secondary compounds were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Expression of two PKS genes (non-reducing (NR) and 6-MSAS-type PKS) were compared with expression of an internal control mitochondrial small subunit gene (mtSSU). The results showed that media containing yeast extracts produced the largest colony diameters and the fewest number of secondary metabolites. Colony growth rates also varied with different media conditions, and a significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of the NR PKS gene was significantly higher at pH 6.5 on the glucose malt agar than any other media, and expression of the 6-MSAS-type (partially-reducing) PKS gene was significantly higher at pH 8.5 on (malt agar) malt agar than on the other types of agar. Gene expression was correlated with the pH level and media conditions that induced the production of the larger number of secondary substances. This is the first study to examine secondary metabolite production in R. dilacerata by comparing the number of polyketides detected with quantitative polymerase chain reaction (qPCR) of two PKS genes under different culture conditions.     

Effects of various media and supplements on laccase production by some white rot fungi

White rot fungi produce three main extracellular enzymes involved in ligninolysis; laccase, lignin peroxidase and manganese peroxidase. Though all white rot fungi do not produce all three enzymes, laccase occupies an important place in ligninolysis. The present paper reports its production by some white rot fungi; Daedalea flavida, Phlebia brevispora, Phlebia radiata and Polyporus sanguineus under different nutritional conditions. Of the various basal media tested, mineral salts malt extract broth proved to be the best medium for laccase production. Sugarcane bagasse proved to be the best laccase inducer among the various supplements added to different media.     

Efficacy of formulation and storage on rice straw waste on the activation of bioagents against root-rot diseases of bean plants

Trichoderma harzianum and Trichoderma viride, which were grown on modified media of rice straw waste, were used as suitable natural media for determining the population of the bioagents and stimulating the production of antimicrobial substances, i.e. toxins, enzymes at different periods (1, 2, 3, 4, 5 and 6 weeks). Also, the evaluation of synergistic effect between biocontrol fungi and bacteria may play a key role in the natural process of biocontrol. Synergism can occur when different agents are applied together and cell wall degrading enzymes produced by fungi can increase the efficacy of bacteria. The evaluation of the bioagents formulated on rice straw waste has influenced the percentage of infection of root rot on bean plants under green house and field conditions. The formulation of the Trichoderma spp. to reduce the incidence of the diseases caused by soil-borne fungi in the field has great importance in the biocontrol of the diseases. This work was aimed to determine and improve the efficacy of application and formulation of Trichoderma spp. on the rice straw waste against root-rot disease of bean under green house and field conditions.     

Fungal culture of musculoskeletal tissue: what’s the point?

There have not been any studies that review the prevalence of fungal isolates using selective media from samples of banked musculoskeletal tissue retrieved from living and cadaveric donors. A total of 2,036 swab and 2,621 biopsy samples of musculoskeletal tissue from tissue banks were received from the 1st August 2008 till 31st December 2010. Routine culture for fungi using selective media with a prolonged incubation period failed to demonstrate a greater prevalence of fungal isolates than by using non-selective culture media alone. Using selective culture fungi were recovered from only two Sabouraud agar plates (0.1%) but not from non-selective media. During the same period fungi were isolated from three graft samples cultured in non-selective broth media only (0.1%). There was no correlation of fungal isolates from selective or non-selective media inoculated at the same time nor from multiple graft samples collected from the same donor supporting the possibility of an exogenous source for fungal isolates rather than an endogenous source.     

Simultaneous specific in planta visualization of root-colonizing fungi using fluorescence in situ hybridization (FISH)

In planta detection of mutualistic, endophytic, and pathogenic fungi commonly colonizing roots and other plant organs is not a routine task. We aimed to use fluorescence in situ hybridization (FISH) for simultaneous specific detection of different fungi colonizing the same tissue. We have adapted ribosomal RNA (rRNA) FISH for visualization of common mycorrhizal (arbuscular- and ectomycorrhiza) and endophytic fungi within roots of different plant species. Beside general probes, we designed and used specific ones hybridizing to the large subunit of rRNA with fluorescent dyes chosen to avoid or reduce the interference with the autofluorescence of plant tissues. We report here an optimized efficient protocol of rRNA FISH and the use of both epifluorescence and confocal laser scanning microscopy for simultaneous specific differential detection of those fungi colonizing the same root. The method could be applied for the characterization of other plant–fungal interactions, too. In planta FISH with specific probes labeled with appropriate fluorescent dyes could be used not only in basic research but to detect plant colonizing pathogenic fungi in their latent life-period.     

Acquired thermotolerance,membrane lipids and osmolytes profiles of xerohalophilic fungus Aspergillus penicillioides under heat shock

Xerophilic fungi accumulate a large amount of glycerol in the cytosol to counterbalance the external osmotic pressure. But during heat shock (HS) majority of fungi accumulate a thermoprotective osmolyte trehalose. Since glycerol and trehalose are synthesized in the cell from the same precursor (glucose), we hypothesised that, under heat shock conditions, xerophiles growing in media with high concentrations of glycerol may acquire greater thermotolerance than those grown in media with high concentrations of NaCl. Therefore, the composition of membrane lipids and osmolytes of the fungus Aspergillus penicillioides, growing in 2 different media under HS conditions was studied and the acquired thermotolerance was assessed. It was found that in the salt-containing medium an increase in the proportion of phosphatidic acids against a decrease in the proportion of phosphatidylethanolamines is observed in the composition of membrane lipids, and the level of glycerol in the cytosol decreases 6-fold, while in the medium with glycerol, changes in the composition of membrane lipids are insignificant and the level of glycerol is reduced by no more than 30%. In the mycelium trehalose level have increased in both media, but did not exceed 1% of dry weight. However, after exposure to HS the fungus acquires greater thermotolerance in the medium with glycerol than in the medium with salt. The data obtained indicate the interrelation between changes in the composition of osmolytes and membrane lipids in the adaptive response to HS, as well as the synergistic effect of glycerol and trehalose.     

Effect of volatiles from bacteria and yeast on the growth and pigmentation of sapstain fungi

Sapstain fungi affect the appearance of wood due to colonisation by pigmented hyphae but without producing significant strength losses. This is due to the production of melanin in the fungal cell walls of the staining fungi. Any biological control strategy targeted against this type of deterioration would therefore be considered successful if it inhibited either fungal growth or pigment production. Previous work has established that specific bacterial and yeast isolates selected on the basis of agar screening studies could significantly reduce levels of staining in wood block tests. This paper presents the results of a study to examine the role of volatile organic compounds (VOCs) produced by four bacterial and three yeast isolates on the growth and pigment production by a range of five sapstain fungi on three media types. VOCs from three of the four bacterial strains tested completely inhibited growth of the five target sapstain fungi but only when the antagonists were grown on tryptone soya media. When antagonists were grown on either malt agar or a low nutrient medium levels of inhibition were either significantly reduced or non-existent. Yeast antagonists generally produced lower levels of growth inhibition than the bacteria but a Williopsis mrakii isolate gave 100% inhibition of three of the five sapstain fungi. Production of inhibitory VOCs was highly dependent on the specific antagonist as well as its growth substrate and all five sapstain fungi showed varying sensitivities to the VOCs produced. Not all fungi were inhibited, growth of O. piliferum and A. pullulans being stimulated by the VOCs from antagonists but only when grown under low nutrient conditions. In some instances, where growth was only slightly reduced, the level of pigmentation of the sapstain colony was significantly reduced compared with corresponding controls. The implications of this work for the biological control of sapstain fungi are discussed.     

两个苏丹草品系成熟种子的组织培养和植株再生研究

该研究以苏丹草品系S722和Sa的成熟种子为外植体、MS培养基为基础培养基,2,4-D和NAA各3个浓度共6个处理对这两个苏丹草品系成熟种子进行愈伤诱导,探讨不同品系在不同植物生长物质浓度及植物生长物质组合中诱导愈伤组织和继代培养以及分化的能力。结果表明：苏丹草S722和Sa成熟种子的愈伤诱导率差异不显著,平均诱导率为17.19％。诱导培养基中2,4-D浓度为0.5或1 mg?L-1时,诱导效果最佳,而添加NAA不能提高愈伤诱导率。在继代培养中,设定2,4-D和6-BA各两个浓度共4个处理组合,处理1(2,4-D 1 mg?L-1＋6-BA 0 mg?L-1)的继代培养效果最佳。为了解不同植物生长物质对愈伤分化的影响,设定6-BA、NAA 各两个不同浓度、KT 3个不同浓度共5个处理组合对继代培养的愈伤进行分化培养。在5个处理中,处理1(6-BA 2 mg?L-1＋NAA 0 mg?L-1＋KT 0 mg?L-1)对 S722成熟种子诱导的愈伤分化率最高,达33.3％。在这两个苏丹草品系中,S722更容易分化培养。综上结果表明,2,4-D浓度为1 mg?L-1时诱导愈伤和继代培养效果较好,6-BA浓度为2 mg?L-1时分化效果较好。另外,针对不同苏丹草品系进行组织培养和植株再生时,适当调整植物生长物质浓度能提高植株再生的成功率。     

Production of auxins and gibberellin-like substances by mycorrhizal fungi,bacteria and actinomycetes isolated from soil and the mycorrhizosphere of pine (Pinus silvestris L.)

Summary Production of auxins and gibberellin-like substances by mycorrhizal fungi, bacteria and actinomycetes isolated from the mycorrhizosphere of Scots pine was studied. Chromatography and biossays were used.Most of the organisms required tryptophan for auxins production. The highest biological activity exhibited substances located at R_f 0.2–0.4.The organisms produced minute amounts of gibberellin-like substances which appeared at different R_f values. It was stated that auxins production is much more common among the root zone organisms of pine than the production of gibberellin-like substances.This research was carried out under problem MR.II.16 coordinated by the Institute of Dendrology, Polish Academy of Sciences.