Characterization of Laccases and Peroxidases from Wood-Rotting Fungi (Family Coprinaceae)

Panaeolus sphinctrinus, Panaeolus papilionaceus, and Coprinus friesii are described as producers of ligninolytic enzymes. P. papilionaceus and P. sphinctrinus both produced a laccase. In addition, P. sphinctrinus produced a manganese peroxidase. C. friesii secreted a laccase and two peroxidases similar to the peroxidase of Coprinus cinereus. The purified laccases and peroxidases were characterized by broad substrate specificities, significant enzyme activities at alkaline pH values, and remarkably high pH optima. The two peroxidases of C. friesii remained active at pH 7.0 and 60°C for up to 60 min of incubation. The peroxidases were inhibited by sodium azide and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), whereas the laccases were inhibited by sodium azide and N,N-diethyldithiocarbamic acid. As determined by native polyacrylamide gel electrophoresis and isoelectric focusing, all three fungi produced laccase isoenzymes.     

Detection and Identification of Fungi Intimately Associated with the Brown Seaweed Fucus serratus

The filamentous fungi associated with healthy and decaying Fucus serratus thalli were studied over a 1-year period using isolation methods and molecular techniques such as 28S rRNA gene PCR-denaturing gradient gel electrophoresis (DGGE) and phylogenetic and real-time PCR analyses. The predominant DGGE bands obtained from healthy algal thalli belonged to the Lindra, Lulworthia, Engyodontium, Sigmoidea/Corollospora complex, and Emericellopsis/Acremonium-like ribotypes. In the culture-based analysis the incidence of recovery was highest for Sigmoidea marina isolates. In general, the environmental sequences retrieved could be matched unambiguously to isolates recovered from the seaweed except for the Emericellopsis/Acremonium-like ribotype, which showed 99% homology with the sequences of four different isolates, including that of Acremonium fuci. To estimate the extent of colonization of A. fuci, we used a TaqMan real-time quantitative PCR assay for intron 3 of the beta-tubulin gene, the probe for which proved to be species specific even when it was used in amplifications with high background concentrations of other eukaryotic DNAs. The A. fuci sequence was detected with both healthy and decaying thalli, but the signal was stronger for the latter. Additional sequence types, representing members from the Dothideomycetes, were recovered from the decaying thallus DNA, which suggested that a change in fungal community structure had occurred. Phylogenetic analysis of these environmental sequences and the sequences of isolates and type species indicated that the environmental sequences were novel in the Dothideomycetes.     

Detection and Identification of Bacterial Endosymbionts in Arbuscular Mycorrhizal Fungi Belonging to the Family Gigasporaceae

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.     

条盖盔孢伞子实体及菌丝体中鹅膏毒素检测

条盖盔孢伞(Galerina sulciceps)是一种有毒蘑菇,近年来在中国南方发生了多起因误食该菌引起的中毒事件。在分离获得纯培养物的基础上,采用高效液相色谱(HPLC)法检测分析了条盖盔孢伞子实体和菌丝体中的鹅膏毒素。结果显示野生条盖盔孢伞子实体(干质量)含有α-amanitin和β-amanitin分别为868.32μg/g和406.56μg/g,液体发酵培养的菌丝体中未检测到α-amanitin和β-amanitin。     

Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay

Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, background-free ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5′ and 3′ ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 10⁴. A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.Clinical diagnostics and disease management strategies increasingly require fast and accurate methods for the detection and identification of multiple pathogenic microorganisms from complex samples. Conventional techniques used to detect and identify pathogenic microorganisms have typically relied upon culture-based morphological approaches. Unfortunately, these methods are often time-consuming, laborious, and restricted to those microorganisms that can be cultured routinely.Several recently developed molecular techniques, such as conventional and real-time PCRs, circumvent some of these drawbacks. PCR-based detection mechanisms are sensitive, accurate, and relatively fast and allow the detection of difficult-to-culture microorganisms. This last aspect is of considerable importance, given the fact that the majority of the microorganisms present in environmental samples still elude conventional cultivation efforts (1, 21). Although PCR-based methods for microbial identification and detection offer several advantages over conventional microbiological approaches, they still often have serious limitations. The attainable level of multiplexing is relatively low and is typically restricted to the detection of only a few target pathogens per assay (15, 40). Adding multiple specific primer pairs to a single reaction mixture can result in undesired amplification products (16), and for TaqMan PCR, the attainable level of multiplexing is low due to the limited number of fluorescent probes (8, 34, 41). Reliable detection and identification of several pathogens in a single sample, therefore, requires separate reaction mixtures, making large-scale screening of samples more laborious, time-consuming, and expensive. To increase efficiency and reduce expenses, it is desirable to develop simple and rapid multiplex assays that can specifically detect and identify several pathogens simultaneously.DNA microarray- and macroarray-based technologies offer the possibility of adding a highly multiplexed aspect to PCR-based pathogen detection and identification (9, 37, 54, 58). Array-based pathogen detection strategies typically involve PCR amplification of universal phylogenetic target genes (e.g., 16S, 18S, and 23S rRNA genes) or a number of microorganism-specific genetic markers (10, 19, 58) or random amplification of genomic DNA (gDNA) fragments (54). The combination of nucleic acid amplification strategies with array-based detection has resulted in the development of sensitive, high-throughput microbial diagnostic microarrays (MDMs) (4, 38, 50, 60). Although several array-based detection technologies have been realized to detect pathogens, only a minority of these methods can discriminate target pathogens from closely related nontarget organisms, which may differ from the target organisms by only a single nucleotide in the probe-binding region (36). Designing sufficiently discriminating oligonucleotide detectors for arrays, however, is relatively complicated, requiring extensive hybridization specificity testing. Moreover, the oligonucleotide detectors spotted onto the microarray are target organism specific, making it necessary to redesign microarrays if accommodation of additional probes is required for the detection of new targets.DNA ligase requires a double-stranded match to allow ligation, facilitating the development of ligation-based systems to discriminate point mutations (29). This feature of ligation detection (LD) has led to the development of several strategies for genotyping single-nucleotide polymorphisms (SNPs) and detecting pathogens (6, 7, 11, 46). However, current LD assays require two adjacent detection oligonucleotide probes with the same melting temperature (T_m) for each target sequence, although the use of intramolecular ligation, as in padlock probe (PLP) technologies, has been demonstrated to hold clear advantages (44, 45, 51). PLPs are long oligonucleotides, ∼100 bases long, containing target complementary arms at both termini of the probe. In the assay developed in this study, the target complementary arms are connected via a compound linker sequence containing spacer sequences, a thymine-linked desthiobiotin moiety for specific capture and release (25, 53), deoxyuracil nucleotides for probe cleavage, and a unique sequence identifier, the so-called ZipCode, for standardized microarray hybridization (18) (Fig. ​(Fig.1A).1A). The unimolecular nature of the PLP allows asymmetric target complementary arm design, whereby a long 5′ arm serves as an anchor sequence and the short 3′ arm, with a low T_m, facilitates extremely specific target detection (17, 51, 53). Microarray-based PLP technology was previously shown to provide reliable detection of multiple pathogenic microorganisms, but PCR amplification of residual, unligated PLPs resulted in significant background signals, thereby complicating data analysis and decreasing the overall dynamic range of reliable detection (3, 51).Open in a separate windowFIG. 1.Schematic overview of the novel single-molecule LD system. (A) PLP design. T1a and T1b are asymmetric target complementary regions. Each PLP contains a unique ZipCode sequence for universal array hybridization, two spacer sequences (S1 and S2), a desthiobiotin moiety (dBio) for probe capture, a polyoligo(dT) linker sequence, and a polydeoxyuracil sequence for probe cleavage. (B) Multiple target-specific PLPs are ligated to PCR-preamplified DNA samples. T1a and T1b bind to adjacent sequences of the target, and in the case of a perfect match, the probe is circularized by enzymatic ligation. The PLPs are reversibly captured and washed via the desthiobiotin moiety with magnetic streptavidin-coated beads. Next, the washed probes are cleaved at the polydeoxyuracil sequences with UNG and endonuclease enzymes. The sample containing the cleaved PLPs is hybridized on a universal microarray. Finally, only the hybridized PLPs that were originally ligated can be labeled and visualized with streptavidin R-PE by using the desthiobiotin moiety.Here, we describe the development, testing, and implementation of a novel, background-free, LD-dependent strategy in which multiple PLPs are ligated on fragmented, PCR-preamplified DNA sequences. The target complementary regions recognize adjacent sequences on the target DNA, and ligation occurs only if the end nucleotides perfectly match their target, resulting in a circular molecule (Fig. ​(Fig.1B).1B). Next, the probes are captured with streptavidin-coupled magnetic beads, allowing separation from the rest of the sample. Subsequently, the washed probes are eluted from the beads and cut at the internal polydeoxyuracil probe region by enzymatic cleavage. Thus, the desthiobiotin moiety needed for fluorescent labeling of unligated PLPs is removed, while the ligated probes are linearized (Fig. ​(Fig.1B).1B). Finally, the sample is hybridized on a universal complementary ZipCode (cZipCode) microarray (18) and visualized via fluorescent labeling of the desthiobiotin moiety (Fig. ​(Fig.1B1B).In this paper, we report the development and application of cleavable PLPs combined with LD for the simultaneous, background-free detection and identification of multiple plant pathogens in environmental samples. The specificity, sensitivity, and dynamic range of detection of the developed assay were determined by using nine target-specific PLPs, and the robustness of the assay was evaluated by using samples collected from hydroponic horticultural water recirculation systems.     

半夏内生真菌的初步研究

从健康无病害的半夏(Pinellia ternate)根、茎、叶和花组织中分离获得内生真菌共计61株,对其进行形态学鉴定后,针对不产孢的菌株,测定了ITSrDNA序列以进行分子鉴定。分离到的内生真菌分别来自11个属,以半知菌为主要群落,其中Hyphomycetes,Zygomycetes和Coelomycetes的比例分别为62.2%,18.1%和8.2%;镰刀菌属(Fusarium spp.)是半夏植物中分离率最高(6%)的属,其余主要优势属为Alternaria,Mucor,Epicoccum,Mortierella和Plectosphaerella。研究表明:从半夏球茎组织中分离到的内生真菌数量多于其他组织。     

Identification of C3 acceptors responsible for complement activation in Crithidia fasciculata

Crithidia fasciculata, an insect trypanosomatid is readily lysed by normal human serum at concentrations as low as 3%. Lysis occurs in the presence of Mg+2-EGTA and is antibody independent, indicating that the alternative pathway of complement activation is involved. Analysis of [131I]C3 deposition on C. fasciculata cells using C8-deficient serum, revealed that about 4 x 10(5) C3 molecules bound to each cell. Most of the C3 was bound to cells as C3b, part of it forming high molecular weight complexes, which could be dissociated by methylamine treatment at alkaline pH. To characterize the C3 acceptors on C. fasciculata, surface-iodinated cells were incubated with C8D or heat-inactivated serum, extracted and immunoprecipitated with anti-C3 or anti-arabinogalactan antisera. Analysis of the immunoprecipitated material on SDS gels showed high-molecular weight components, which disappeared after methylamine treatment, giving rise to a component of 200 kDa molecular size. This 200-kDa component corresponded to a purified arabinogalactan complex, which was immunoprecipitated from labeled cell extracts, without incubation with C8D, using anti-arabinogalactan antibodies. These results suggest that the arabinogalactan glycoconjugate is a C3 acceptor in C. fasciculata during complement activation. Purified arabinogalactan complexes were able to inactivate C3 in vitro. Solubilization in KOH to cleave the peptide moiety rendered it unable to inactivate C3. Apparently, the aggregated state of the purified arabinogalactan component at the cell surface is important for C3 deposition and activation.     

Identification of C3 Acceptors Responsible for Complement Activation in Crithidia fasciculata1

Crithidia fasciculata, an insect trypanosomatid is readily lysed by normal human serum at concentrations as low as 3%. Lysis occurs in the presence of Mg⁺²-EGTA and is antibody independent, indicating that the alternative pathway of complement activation is involved. Analysis of [¹³¹I]C3 deposition on C. fasciculata cells using C8-deficient serum, revealed that about 4 times 10⁵ C3 molecules bound to each cell. Most of the C3 was bound to cells as C3b, part of it forming high molecular weight complexes, which could be dissociated by methylamine treatment at alkaline pH. To characterize the C3 acceptors on C. fasciculata, surface-iodinated cells were incubated with C8D or heat-inactivated serum, extracted and immunoprecipitated with anti-C3 or anti-arabinogalactan antisera. Analysis of the immunoprecipitated material on SDS gels showed high-molecular weight components, which disappeared after methylamine treatment, giving rise to a component of 200 kDa molecular size. This 200-kDa component corresponded to a purified arabinogalactan complex, which was immunoprecipitated from labeled cell extracts, without incubation with C8D, using anti-arabinogalactan antibodies. These results suggest that the arabinogalactan glycoconjugate is a C3 acceptor in C. fasciculata during complement activation. Purified arabinogalactan complexes were able to inactivate C3 in vitro. Solubilization in KOH to cleave the peptide moiety rendered it unable to inactivate C3. Apparently, the aggregated state of the purified arabinogalactan component at the cell surface is important for C3 deposition and activation.     

Galerina Earle: A polyphyletic genus in the consortium of dark-spored agarics

The basidiomycete genus Galerina Earle accommodates more than 300 small brown-spored agarics worldwide, predominantly described from the Northern hemisphere. The delimitation of species and infrageneric units hitherto has been based on morphological and, to some extent, ecological characters. In this study we have analyzed nuclear ribosomal LSU and ITS sequences to reveal infrageneric phylogeny and the phylogenetic placement of Galerina among the dark-spored agarics. Sequences from 36 northern hemisphere Galerina species and 19 other dark-spored taxa were analyzed, some of them obtained from EMBL/GenBank. Our results, received from Bayesian and distance methods, strongly suggest that Galerina is a polyphyletic genus. The LSU analysis shows that Galerina is composed of three or four separate monophyletic main groups. In addition, a few species cluster together with other dark-spored agarics. The same groups are recognized in the ITS tree and they correspond roughly to previously recognized subgenera or sections in Galerina. With high support our LSU analysis suggests that Gymnopilus is a monophyletic genus and that Gymnopilus and one of the Galerina lineages ("mycenopsis") are sister groups. The analyses further indicate that the Galerina lineages, as well as the genus Gymnopilus, could be referred to a strongly emendated family Strophariaceae, which corresponds largely to the family as circumscribed by Kühner (1980). Our results affirm that morphological characters often are highly homoplastic in the agarics. At the present stage formal taxonomic consequences or nomenclatural changes are not proposed.     

中国盔孢伞属的分类

根据采自全国20个省、自治区的盔孢伞属Galerina真菌标本整理、鉴定,确认出36种,其中包含28个已知种和8个中国新记录种。对中国新记录种褐柄盔孢伞G. badipes、帆孢盔孢伞G. calyptrata、迦佩盔孢伞G. jaapii、假拟提灯藓盔孢伞G. pseudomniophila、沼泽盔孢伞G. paludosa、泡孢盔孢伞G. physospora、萨列里盔孢伞G. sahleri和胫囊盔孢伞G. tibiicystis进行了详尽的形态学描述,提供了原生态照片和显微特征线条图以及分亚属、分种检索表。通过相关研究材料进行DNA提取,所得21条ITS序列与73条下载序列利用贝叶斯法及最大似然法构建系统发育树,验证鉴定结果的准确性。     

Detection and Identification of Decay Fungi in Spruce Wood by Restriction Fragment Length Polymorphism Analysis of Amplified Genes Encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.     

四种嗜热真菌的分离与鉴定

目的:在对嗜热真菌的资源调查中,分离到嗜热真菌20株。方法:通过形态学比较研究并结合分子分析方法。结果:鉴定出嗜热真菌4种,即杜邦青霉Penicillium dupontii、疏绵状嗜热丝孢菌Thermomyceslanuginosus、嗜热子囊菌Thermoascus aurantiacus、嗜热革节孢Scytalidium thermophilum。此外,还分离到耐热真菌1种,鉴定为不规则头梗霉Cephaliophora irregularis,为中国新记录种。结论:这些研究结果新增了嗜热真菌在中国的分布记载,丰富了我国西南地区嗜热真菌的菌种资源库,另外对分离获得的嗜热真菌进行木聚糖酶活性测试,发现嗜热子囊菌为高产木聚糖酶活力的菌株。     

千层塔内生真菌的分离与鉴定

目的：为从千层塔中分离具有药用价值的内生真菌奠定基础。方法：新鲜千层塔茎段,经酒精和升汞消毒后,接种于PDA平板培养基上进行内生真菌的分离、纯化;根据菌落形态和孢子等形态特征,结合核糖体基因居间序列（ITS序列）进行菌株鉴定。结果：从千层塔的茎中分离出4株内生真菌。内生真菌I菌落形态和孢子特征与枝状枝孢霉属的特征相符合,ITS序列与GenBank中多条属于枝状枝孢霉的ITS序列相似,鉴定该菌株属于枝状枝孢霉;内生真菌II菌落形态和孢子特征与黄青霉的特征相符合,鉴定该菌株属于黄青霉;内生真菌III菌落形态和孢子特征与尖孢镰刀菌的特征相符合,ITS序列与GenBank中多条属于尖孢镰刀菌的ITS序列相似程度高,鉴定该菌株属于尖孢镰刀菌;内生真菌Ⅳ菌落形态与盾壳霉相似,ITS序列与GenBank中6条属于盾壳霉的ITS序列具有较高的相似性,鉴定该菌株属于盾壳霉。结论：从千层塔中分离和鉴定出4株内生真菌,分别属于枝状枝孢霉、黄青霉、尖孢镰刀茵和盾壳霉。     

Polysaccharides of Crithidia fasciculata: Identification and partial characterization of a cell surface constituent

A carbohydrate-containing fraction was extracted from the trypanosomatid Crithidia fasciculata by a phenol-water procedure. Ion-exchange chromatography separated this fraction into three components: a polysaccharide which was not retained on the column; RNA which eluted upon addition of salt; and, another polysaccharide which eluted upon addition of detergent. The unretained fraction was shown to be composed solely of d-mannose. The mannan, which was heterodisperse on Sephadex G-100, had an average molecular weight of approx. 14 000 as based on analysis of reducing groups. The detergent-eluted material yielded arabinose and galactose upon acid hydrolysis. The arabinogalactan was excluded from Sephadex G-100 and Sephacryl S-200 molecular sieve columns, suggesting a molecular weight ≥ 200 000. Cell fractionation studies showed the bulk of extractable polysaccharide was associated with a particulate fraction. Further determination of the cellular localization of the polysaccharide was accomplished by employing a specific antiserum prepared from rabbits immunized with the polysaccharide extract. The cell surface localization of the arabinogalactan was demonstrated by cell agglutination studies as well as immunocytochemical techniques using fluorescein and ferritin conjugated antibodies.