医学真菌菌株保藏方法概述

近年来,随着真菌感染的患者急剧增加,真菌病已经成为一个重要的公共卫生问题。对医学真菌流行病学、致病机理、耐药机理以及防治策略的研究尤为重要,而所有研究的基础都建立在对医学真菌的进行有效的保藏。医学真菌的保藏方法很多,包括传代法、蒸馏水法、冷冻干燥保藏法等,保藏方法的选择因实验室条件、菌种和研究要求不同而不同。本文对目前常用的几种医学真菌菌种保藏方法的优缺点及其应用等做了综述。     

嗜角蛋白真菌的界定、研究方法及其应用价值

嗜角蛋白真菌(keratinophilic fungi)是指能降解角蛋白并能利用其作为唯一营养源的真菌类群,大部分隶属于爪甲团囊菌目(Onygenales)、散囊菌目(Eurotiales)和肉座菌目(Hypocreales).由于该类群分布广、种类多、营养方式多样,其研究方法和技术手段也在不断进步,加深了其研究的深度...     

Methods for large-scale production of AM fungi: past, present, and future

Many different cultivation techniques and inoculum products of the plant-beneficial arbuscular mycorrhizal (AM) fungi have been developed in the last decades. Soil- and substrate-based production techniques as well as substrate-free culture techniques (hydroponics and aeroponics) and in vitro cultivation methods have all been attempted for the large-scale production of AM fungi. In this review, we describe the principal in vivo and in vitro production methods that have been developed so far. We present the parameters that are critical for optimal production, discuss the advantages and disadvantages of the methods, and highlight their most probable sectors of application.     

Quantification of predatory and endoparasitic nematophagous fungi in soil

Methods were developed to quantify predatory and endoparasitic fungi in soil. The methods were based on previously developed detection techniques and combined with a most probable number estimation. The methods were applied to an agricultural soil fertilized with farmyard manure. Large amounts of farmyard manure resulted in increased amounts of organic matter, numbers of propagules of predatory and endoparasitic fungi, and numbers of bacteria and nematodes.     

The comparison of isolation techniques for nematophagous fungi from soil

Using a single soil, a comparison was made of six well-known techniques for the isolation of nematophagous fungi. Each technique was tested for its ability to isolate the fungi present with regard to the number of species recovered, the number of plates needed to be 95% sure of isolating all the species possible and the amount of soil required. The advantages and disadvantages of each method are discussed. The results show that specific techniques are required for the isolation of endoparasites and predators respectively. The soil sprinkling technique and the Baermann funnel technique are shown to be the most efficient methods overall, with six and seven replicates required for 95% probability of isolating all the predator and endoparasite species respectively.     

Quantitative methods for the analysis of zoosporic fungi

Quantitative estimations of zoosporic fungi in the environment have historically received little attention, primarily due to methodological challenges and their complex life cycles. Conventional methods for quantitative analysis of zoosporic fungi to date have mainly relied on direct observation and baiting techniques, with subsequent fungal identification in the laboratory using morphological characteristics. Although these methods are still fundamentally useful, there has been an increasing preference for quantitative microscopic methods based on staining with fluorescent dyes, as well as the use of hybridization probes. More recently however PCR based methods for profiling and quantification (semi- and absolute) have proven to be rapid and accurate diagnostic tools for assessing zoosporic fungal assemblages in environmental samples. Further application of next generation sequencing technologies will however not only advance our quantitative understanding of zoosporic fungal ecology, but also their function through the analysis of their genomes and gene expression as resources and databases expand in the future. Nevertheless, it is still necessary to complement these molecular-based approaches with cultivation-based methods in order to gain a fuller quantitative understanding of the ecological and physiological roles of zoosporic fungi.     

Genetic manipulation in vesicular-arbuscular mycorrhizal fungi

The symbiosis between vesicular-arbuscular mycorrhizal (VAM) fungi and host plants develops after successful interactions between both partners. These interactions probably involve signal molecules produced by the host plant, by the fungi, or by both. So far the biotrophic status of VAM fungi has hampered the understanding of the processes regulating their physiology. However, among different methods for co-cultivating VAM fungi, root organ cultures (ROC) appear to be a useful technique for studying VAM development. This system has been useful in defining the nutritional requirements of VAM fungi in the precolonization stage and in obtaining axenic fungal material in various developmental stages. The work discussed here focuses on the application of Polymerase Chain Reaction (PCR) technology and the potential of promoting hyphal growth in the absence of the plant. These techniques are being used to study VAM fungi in two main areas. The first concerns the determination of the DNA sequences coding for the SSU ribosomal RNA of two VAM fungi. This approach has allowed the design of specific primers for the rapid identification and quantification of VAM fungi. The second area of research concerns the potential use of PCR technology to study selective expression of specific genes during fungal spore development in defined in vitro conditions. The achievement of this future prospect depends on the ability to prepare PCR-based cDNA libraries from small amounts of fungal material after stimulation of hyphal growth with CO₂ and plant flavonols.     

A review: recent advances and future prospects of taxol-producing endophytic fungi

In the urgent search for more effective ways to treat cancer, new extraction methods of taxol from endophytic fungus have demonstrated high potential in increasing the efficiency of taxol extraction for more efficient and sustainable production of taxol and cancer treatment products. This paper summarizes recent advances in taxol-producing endophytic fungi, both in China and abroad, in the following areas: isolation and identification of endophytic fungi types, extraction and detection methods of endophytic taxol in plants, and improved efficiency of the extraction process. With the advancement of science and technology, new techniques in biotechnology, such as fungal strain improvement and recombining technique and microbial fermentation engineering, have increased the extraction yield from taxol-producing fungi, thereby improved the overall efficiency of taxol production.     

Extracellular enzymes and the pathogenesis of nematophagous fungi

Nematophagous fungi are an important group of soil microorganisms that can suppress the populations of plant-parasitic nematodes. The pathogenic mechanisms of nematophagous fungi are diverse: They can be parasitical–mechanical through producing specialized capturing devices, or toxin-dependent. During infections, a variety of virulence factors may be involved against nematodes by nematophagous fungi. In this review, we present up-to-date information on the modes of infection by nematophagous fungi. The roles of extracellular hydrolytic enzymes and other virulence factors involved in infection against nematodes were summarized. The biochemical properties and peptide sequences of a special group of enzymes, the serine proteases, were compared, and their implications in infections were discussed. We also discussed the impact of emerging new techniques on our understanding of this unique group of fungi.     

Elaboration and validation of methods for quantitative analysis of phenolic compounds and flavonoids in the extracts of higher fungi

Simple express methods for the quantitative evaluation of phenolic compounds and flavonoids in the extracts of basidiomycetes and plants were developed using ethanol extracts of the Fomes fomentarius xylotrophic fungus as a model system. The analysis of phenolic compounds was based on the photocolorimetric Folin–Ciocalteu method. The spectrophotometric method based on the reaction of complex formation with aluminum chloride was chosen for the analysis of flavonoids. The composition of the reactants and their concentrations in the reaction mixtures were optimized for both techniques. The advantages of the developed techniques were the following: the simplicity, speed, the use of commonly used laboratory equipment, and the small volumes of the test samples and reagents. The proposed methods allow for serial analyses of up to four dozen samples. It was found that the decrease of the volume of the studied sample by factors of 5–10 did not reduce the sensitivity of the techniques in comparison with the classic versions of the methods. The proposed methods were examined for linearity, accuracy, repeatability, and convergence. The validation showed that the methods meet the eligibility criteria. The proposed methods for the quantitative analysis of phenolic compounds and flavonoids can be recommended for the characterization of fungi and plant extracts of both natural and biotechnological origin.     

Genetic transformation of filamentous fungi

Many filamentous fungi of all taxa can now be subject to DNA-mediated transformation. Many dominant selectable markers are available and the range available is increasing as new genes are cloned. Transformation is especially valuable in cloning genes defined by mutations with selectable phenotypes and is allowing investigation of many problems in fungi with good genetic systems. Increasingly sophisticated techniques for inactivating genes, targetingin vitro generated mutations to specific loci, and altering gene expression and its regulation are being developed. These approaches are being used to investigate the wealth of basic and applied biological problems available in filamentous fungi.     

A comprehensive catalogue of polyketide synthase gene clusters in lichenizing fungi

Lichens are fungi that form symbiotic partnerships with algae. Although lichens produce diverse polyketides, difficulties in establishing and maintaining lichen cultures have prohibited detailed studies of their biosynthetic pathways. Creative, albeit non-definitive, methods have been developed to assign function to biosynthetic gene clusters in lieu of techniques such as gene knockout and heterologous expressions that are commonly applied to easily cultivatable organisms. We review a total of 81 completely sequenced polyketide synthase (PKS) genes from lichenizing fungi, comprising to our best efforts all complete and reported PKS genes in lichenizing fungi to date. This review provides an overview of the approaches used to locate and sequence PKS genes in lichen genomes, current approaches to assign function to lichen PKS gene clusters, and what polyketides are proposed to be biosynthesized by these PKS. We conclude with remarks on prospects for genomics-based natural products discovery in lichens. We hope that this review will serve as a guide to ongoing research efforts on polyketide biosynthesis in lichenizing fungi.     

Molecular identification of arbuscular mycorrhizal fungi in roots: Perspectives and problems

Molecular identification methods are about to revolutionize studies on ecology of arbuscular mycorrhiza. These techniques offer the unique opportunity to investigate communities of arbuscular mycorrhizal fungi (AMF) within roots. Recent technical advances are reviewed, discussing their drawbacks and advantages. An experimental approach to analyze AMF communities within roots using a molecular identification method is presented. Sample results from the analysis of trap cultures from a current project are shown.     

丛枝菌根观察与侵染率测定方法的比较

菌根生长状况观察与侵染率测定是菌根学研究中一项重要的基础性工作。综述了丛枝菌根（AM）染色观察与侵染率测定方法研究概况,并对其进行比较和评价。认为采用醋酸墨水染色观察AM生长状况与采用根段侵染率加权法和放大交叉法测定AM真菌侵染率是目前较为科学、准确、易行的方法。根据不同需要也可选择其他适宜的方法,如要了解丛枝发育状况,可采用放大交叉法;如要了解泡囊和侵入点数量,可采用直接计数法,从而使其研究结果具有可比性。有必要建立基于分子生物学技术和脂肪酸定量分析技术测定一种或数种AM真菌侵染状况,这将有力推动AM真菌生理、生态功能研究的发展。     

三种杓兰根相关真菌多样性和生态功能

【背景】除了菌根真菌(Orchid mycorrhizal fungi,OrMF)外,兰科植物根中还有其它内生真菌,称为根相关真菌(Root-associated fungi,RAF)。【目的】采用分离培养的方法获得同一栖息地针叶林和灌木林两种不同生境西藏杓兰、黄花杓兰和无苞杓兰的RAF菌株,研究其真菌谱系、多样性和生态功能结构。【方法】从杓兰根碎屑中分离RAF,通过总DNA提取、PCR扩增及测序得到ITS(Internaltranscribedspacer)序列;进行系统发育和多样性分析,并通过NCBI数据库比对得到相似性最高序列的注释信息来分析RAF生态学特性。【结果】共分离得到278株RAF,25种OTU类型,包括23个子囊菌门OTU,2个毛霉菌门OTU。RAF物种丰富度分析发现西藏杓兰的较黄花杓兰高,不同生境没有显著差异;不同杓兰物种较不同生境的RAF群落分化程度高。生态功能分析显示25个OTU包括共生型、腐生型和致病型3种营养型,以及外生菌根菌群、植物病原菌群、内生真菌群、动物病原菌群、真菌寄生菌群、杜鹃花类菌根群、未定义的腐生菌群和不确定型8种共位群。【结论】阐明不同生境采集的不同杓兰中RAF的分布特点和生态功能,为未来研究RAF与杓兰属植物的共生关系奠定基础。     

Modern morphological engineering techniques for improving productivity of filamentous fungi in submerged cultures

Morphological engineering techniques have recently gained popularity as they are used for increasing the productivity of a variety of metabolites and enzymes in fungi growing in submerged cultures. Their action is mainly associated with the changes they evoke in fungal morphology. Traditional morphological engineering approaches include manipulation of spore concentration, pH-shifting and mechanical stress exerted by stirring and aeration. As the traditional methods proved to be insufficient, modern techniques such as changes of medium osmolality or addition of mineral microparticles to the media (microparticle-enhanced cultivation, MPEC) were proposed. Despite the fact that this area of knowledge is still being developed, there are a fair amount of scientific articles concerning the cultivations of filamentous fungi with the use of these techniques. It was described that in Ascomycetes fungi both MPEC or change of osmolality successfully led to the change of mycelial morphology, which appeared to be favorable for increased productivity of secondary metabolites and enzymes. There are also limited but very promising reports involving the successful application of MPEC with Basidiomycetes species. Despite the fact that the mineral microparticles behave differently for various microorganisms, being strain and particle specific, the low cost of its application is a great benefit. This paper reviews the application of the modern morphology engineering techniques. The authors critically assess the advantages, shortcomings, and future prospects of their application in the cultivation of fungi.     

Mycorrhiza and root-associated fungi in Spitsbergen

Summary Roots of 76 plant species collected in West Spitsbergen (Svalbard), in the middle-northern Arctic zone, were examined for mycorrhiza and root-associated fungi. Dryas octopetala, Pedicularis dasyantha and Salix polaris were ectomycorrhizal and Cassiope tetragona and Empetrum hermaphroditum ericoid mycorrhizal. Pedicularis dasyantha was only slightly infected. Structures resembling vesicular-arbuscular mycorrhizal (VAM) fungi were not found in the roots, and soil samples screened for VAM fungi contained only one spore. Root endophytic fungi commonly occurred in Spits-bergen, but only Olpidium brassicae, Pleospora herbarum, Papulaspora, Microdochium bolleyi and Rhizoctonia solani were identified with reasonable certainty. A sterile endophytic dark-septate fungus (DSF) was in 39.5% of the flowering-plant species examined, especially in the Brassicaceae, Caryophyllaceae, Saxifragaceae and Poaceae. DSF were categorized into four slightly overlapping groups. Sterile endophytic hyaline septate fungi were rare. In the literature it is suggested that at least some of the DSF species or the hyaline septate fungi are functionally mutualistic rather than saprophytic or pathogenic. The literature on ectomycorrhizal fungi and plants in Spitsbergen is reviewed, including about 50 species, mainly of the genera Cortinarius, Hebeloma, Inocybe, Laccaria, Lactarius and Russula. These are symbiotic with the above-mentioned ectomycorrhizal plants. Four further ectomycorrhizal plants (Betula nana, Salix spp.) are very rare in the area.     

Biodiversity and distribution of fungi associated with decomposing Nypa fruticans

Fungi associated with the decomposition of Nypa fruticans in Malaysia are under investigation. Forty-one fungi have been identified including 35 ascomycetes, four mitosporic fungi and two basidiomycetes. The distribution of intertidal fungi on palm structures including leaves, leaf veins, rachides, petiole bases, and inflorescences, and fungi on terrestrial parts have also been examined. No fungi were found on the leaf material, although several fungi were found on the leaf midribs, and possible reasons for this are given. Very few taxa developed on the inflorescences, but those that were present were abundant. The greatest density of fruiting structures occurred on the rachides, and the greatest diversity of fungal species occurred on the petiole bases. The terrestrial fungi differed from the intertidal fungi, although Linocarpon nipae occurred in both habitats. Reasons for the differences in fungal numbers and diversity on the various palm parts are discussed. The diversity of fungi at Morib mangrove was low when compared to previous studies on fungi on Nypa palm at Kampong Api Api in Brunei and in this study at Kuala Selangor mangrove in Malaysia.     

Glomalean mycorrhizal fungi from tropical Australia

 A comparison of different methods for isolation of vesicular-arbuscular mycorrhizal (VAM) fungi into open-pot cultures was undertaken as part of a study of the diversity of these fungi. Four different isolation techniques using spores separated from soil, soil trap cultures, root samples, or transplanted seedlings grown in intact soil cores were used to obtain as many fungi as possible from each site. Isolation methods were compared using paired samples from the same locations within natural (savanna, rocky hill, wetland, rainforest) and disturbed (minesite) habitats in a seasonally dry tropical region in the Northern Territory of Australia. There were large differences in (i) the efficiency (rate of increase in mycorrhizal colonisation), (ii) the proportion of successful cultures, (iii) fungal diversity (number of fungal species in each culture) and (iv) specificity (identity of species isolated) between these four procedures. However, the less-efficient procedures generally resulted in a higher proportion of cultures of one fungus, which could be used without further isolation steps. Most species of Scutellospora, Acaulospora and Gigaspora were obtained primarily from field-collected spores, but only 50% of these culture attempts were successful. Spores from these initial cultures produced mycorrhizas much more rapidly and successfully when used to start second-generation cultures. Several species of fungi, rarely recovered as living spores from field soils, were dominant in many trap cultures started from soil or roots. Most of these fungi were Glomus species, that were first distinguished by colonisation patterns in roots and eventually identified after sporulation in second- or third-generation trap cultures. These experiments demonstrated that glomalean fungi in the habitats sampled belonged to two functional categories, based on whether or not spores were important propagules. The "non-sporulating" fungi were dominant in many trap cultures, which suggests that these fungi had higher total inoculum levels in soils than other fungi. Pot-culturing methods provided additional information on fungal diversity which complemented spore occurrence data obtained using the same soil samples and provided valuable new information about the biology of these fungi. Accepted: 26 December 1998     

病原真菌生态学研究现状及方法

通过形态学、生理生化学及分子生物学等方法鉴定由自然界分离的真菌菌种,构建系统发生树,了解菌种间亲缘关系,评估种间和种内的进化规律,了解真菌与其所处环境的相互关系,是真菌生态学的主要研究目的之一。本文就真菌与自然界和动物宿主的相互关系,环境中真菌的分离和鉴定方法及系统发生学研究及现状进行综述,以期了解致病真菌的生存环境、种类发生、感染途径等,达到尽早控制感染源、探讨致病性及防治疾病传播的目的。