A convenient method to study fungal spores retention on a porous preformed support

A method was developed to determine engineering parameters of a preformed porous cellulose carrier, commercially known as Microcube. It demonstrated a high water retention (20 g g^–1 dry support) connected to a wet support density slightly higher than that of water (1.005 g ml^–1), making this material suitable for use in stirred vessels; a high porosity (96%) was also evidenced. Ungerminated spores of Aspergillus niger reversibly adsorbed on the carrier according to a Langmuir-type isotherm, while germinating conidia irreversibly attached to particles, allowing a further pellet-like development.     

Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/ microliters and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between C(T) value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.     

Differentiation of allergenic fungal spores by image analysis, with application to aerobiological counts

The ability of an image analysis routine to differentiate between spores of eleven allergenic fungal genera was tested using analysis based on seven basic and up to 17 more complex features, extracted from digitised images. Fungal spores of Alternaria, Cladosporium, Fusarium, Aspergillus, Penicillium, Botrytis, Epicoccum, Exserohilum, Ustilago, Coprinus and Psilocybe were examined in a series of experiments designed to differentiate between spores at the genus and species level. Linear and Quadratic Discriminant Analysis of feature measurements, recorded for 100 to 1600 spores per taxon, differentiated between genera and species with a high level of accuracy. Genus comparisons using only seven basic features resulted in 98% accuracy for the recognition of conidia belonging to Cladosporium, Fusarium and Epicoccum. Differentiation between conidia of Aspergillus and Penicillium was the least reliable, with 56% of Aspergillus conidia correctly identified and 41% misidentified as Penicillium. At the species level, conidia of Cladosporium macrocarpum, Fusarium moniliforme (microconidia), F. oxysporum (microconidia), F. solani (macroconidia), Alternaria helianthi and A. brassicae were consistently identified with 86--100% accuracy. Reduced levels of accuracy in the identification of spores by image analysis reflected similarities between species in their spore morphology. The application of image analysis to aerobiological counting methods is discussed in relation to the results obtained.     

泰山丛枝菌根真菌群落结构特征

2007年对泰山植被根围内丛枝菌根(arbuscular mycorrhiza,AM)真菌群落组成、数量、分布及其与植物多样性的关系进行了研究。从泰山傲徕峰、黑龙潭库区等样地共分离出4属16种AM真菌:球囊霉属Glomus 9种、无梗囊霉属Acaulospora 4种、巨孢囊霉属Gigaspora 2种和盾巨孢囊霉属Scutellospora1种。其中,球囊霉属Glomus及聚球囊霉Glomus fasciculatum的孢子密度、相对多度、分布频度和重要值均最高,分别为泰山植被区根围内AM真菌优势属和优势种。各样地之间Sorenson相似系数在0.60和0.85之间。植被数量与孢子密度(r=0.80,p0.01)、植物种的丰富度与AM真菌种的丰富度(r=0.77,p0.01)以及与孢子密度(r=0.59,p0.01)均呈极显著正相关关系。研究结果表明植物多样性对于提高AM真菌多样性发挥极为重要的作用。     

Viability testing and characterization of germination of fungal spores by automatic image analysis

Fungal spores are used in the laboratory for culture maintenance and at laboratory and other scales as inocula for fermentations. The spore swelling and germination processes constitute a major part of the lag phase, and the subsequent culture morphology and productivity can be greatly influenced by the initial concentration and condition of the spores. An image analysis method has been developed for assessing the viability and the germination characteristics of fungal spores in submerged cultures. Structural variations during germination, i.e., swelling, germ tube formation, and germ tube elongation, are measured in terms of distributions of spore volumes and of germ tube lengths and volumes. These measurements are fully automatic and give a very rapid assessment of spore viability. This image analysis method might be used as a tool in culture maintenance and for determining the quality of inocula for fungal fermentations. (c) 1993 John Wiley & Sons, Inc.     

An aerobiological study of pollen grains and fungal spores of Barcelona (Spain)

Summary A study of concentration of airborne pollen grains and fungal spores has been carried out in Barcelona (Spain) during 1989–90. The volumetric method of filtration, previously described for airborne pollen analysis (Suarez-Cervera and Seoane-Camba, 1983) has been used. In this case, the filters have also been cultivated in Czapecdox-agar, Sabouraud-agar and Sabouraud-agar with streptomycin for the identification of the fungal colonies. Analysis of the number of fungal spores growing on the filter shows that the maxima of colonies of spores developed in culture per m³ of air filtered, correspond to September–December. Pollen and spore concentrations start from November–December, reach a maximum in March–April and decline progressively until September–October. Therefore, in the city of Barcelona, the greatest concentration occurs in spring and the lowest in autumn.     

Influence of growth medium on surface and wall lipid of fungal spores

Conidiospores of Alternaria tenuis, Botrytis fabae, Neurospora crassa and sporangiospores of Rhizopus stolonifer from cultures on various media were shown by microelectrophoresis to have lipid on their surface. Analyses of lipid fractions obtained by sequential solvent extraction demonstrated that surface lipid forms a small but discrete layer different in composition from that within the wall. Free fatty acids, alkanes, triglycerides and other acyl lipids were identified by GC-MS. Phospholipids and sterols were absent. The qualitative and quantitative composition of both the surface and wall lipid fractions was dependent upon the growth medium used.     

一种经济高效提取实蝇基因组DNA的方法

单头实蝇高质量基因组DNA的获得为实蝇分子生态学研究奠定了重要基础。本文提出一种经济高效的实蝇基因组DNA提取方法,该方法广泛适用于不同虫态、不同保存条件的实蝇标本,与传统的CTAB法和SDS法相比操作简单、耗时短,得率高。     

Give me a sample of air and I will tell which species are found from your region: Molecular identification of fungi from airborne spore samples

Fungi are a megadiverse group of organisms, they play major roles in ecosystem functioning and are important for human health, food production and nature conservation. Our knowledge on fungal diversity and fungal ecology is however still very limited, in part because surveying and identifying fungi is time demanding and requires expert knowledge. We present a method that allows anyone to generate a list of fungal species likely to occur in a region of interest, with minimal effort and without requiring taxonomical expertise. The method consists of using a cyclone sampler to acquire fungal spores directly from the air to an Eppendorf tube, and applying DNA barcoding with probabilistic species identification to generate a list of species from the sample. We tested the feasibility of the method by acquiring replicate air samples from different geographical regions within Finland. Our results show that air sampling is adequate for regional‐level surveys, with samples collected >100 km apart varying but samples collected <10 km apart not varying in their species composition. The data show marked phenology, and thus obtaining a representative species list requires aerial sampling that covers the entire fruiting season. In sum, aerial sampling combined with probabilistic molecular species identification offers a highly effective method for generating a species list of air‐dispersing fungi. The method presented here has the potential to revolutionize fungal surveys, as it provides a highly cost‐efficient way to include fungi as a part of large‐scale biodiversity assessments and monitoring programs.     

Dispersal of fungal spores from three types of air handling system duct material

Exposure to airborne microorganisms in indoor environments may result in infectious disease or elicit an allergic or irritant response. Air handling system components contaminated by fungi have been implicated in the dispersal of spores into the indoor environment, thereby serving as a route of exposure to occupants. This study was conducted to provide quantitative data on the dispersal of spores from fungal colonies growing on three types of duct material. Galvanized metal, rigid fibrous glass ductboard, and fiberglass duct liner were soiled and contaminated with a known concentration of Penicillium chrysogenum spores. The duct materials were incubated in humidity chambers to provide a matrix of growing, sporulating fungal colonies at a contamination level of 109 colony forming units (CFU) per duct section, consistent for all materials. For each experiment a contaminated duct section was inserted into the air handling system of an experimental room, and the air handling system was operated for three 5-minute cycles with an air flow of 4.2 m3 min–1. The duct air velocity was approximately 2.8 m sec–1. The airborne concentration of culturable P. chrysogenum spores (CFU m–3), total P. chrysogenum spores (spores m–3), and total P. chrysogenum-sized particles (particles m–3) were measured in the room using Andersen single-stage impactor samplers, Burkard slide impactor samplers, and an aerodynamic particle sizer, respectively. The highest airborne concentrations (104 CFU m–3; 105 spores m–3; 104 particles m–3) were measured during the first operating cycle of the air handling system for all duct materials with decreasing airborne concentrations measured during the second and third cycles. There was no significant difference in spore dispersal from the three contaminated duct materials. These data demonstrate the potential exposure for building occupants to high concentrations of spores dispersed from fungal colonies on air handling system duct materials during normal operation of the system.     

Airborne fungal spores in dust present in archives: pProposal for a detection method, new for archival materials

Libraries and archives are keepers of expressions of human thought, from the most ancient documents written on papyrus, parchement, etc., to the new photographic and electronic supports. These materials of vegetal, animal or synthetic origin are subject to deterioration by physical agents (light, heat, humidity), chemical agents (atmospheric pollution),and biological agents (bacteria, microfungi and insects). Micro-organisms, agents of biodeterioration, as it is known, can be carried by airborne particles which can eventually settle in the dust. This paper shows the results of a preliminary quantitative and qualitative study made on potentially deteriogenic airborne fungal spores present in the dust deposited on shelves and documents of an archive's repository. The hygrothermometric parameters and the water content of the materials were also measured. The dust samples were collected with Swab Millipore samplers, using electrostatic attraction. This kind of sampler, originally destined for the microbiological analysis of water and, in extension, used to isolate bacteria from the book-material, was successfully also used in this case of dust sampling. Compared with the data resulting from the bibliography on dust, the use of Swabs allowed the spores present in the dust on books and shelves to be isolated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Distribution of resting spores of the Lymantria dispar pathogen Entomophaga maimaiga in soil and on bark

Cadavers of late instar Lymantria dispar (gypsy moth) larvae killed by the fungal pathogen Entomophaga maimaiga predominantly contain resting spores (azygospores). These cadavers frequently remain attached to tree trunks for several weeks before they detach and fall to the ground. Density gradient centrifugation was used to quantify resting spores in the soil and on tree bark. Titers of resting spores were extremely high at 0–10 cm from the base of the tree and the number decreased with distance from the trunk of the tree. Titers were also highest in the organic layer of the soil with numbers decreasing precipitously with increasing depth in the soil. While resting spores were obtained from tree bark, densities per unit area were much lower than those found in the organic soil layer at the base of the tree. Field bioassays were conducted with caged L. dispar larvae to compare infection levels with distance from the tree trunk as well as on the trunk. Highest infection levels were found at 50cm from the tree base with lowest infection on the tree trunk at 0.5 m height, although we expected the highest infection levels among larvae caged at the bases of trees, where highest spore titers occurred. Laboratory experiments demonstrated that L. dispar larvae exposed to resting spore- bearing soil at the soil surface became infected while larvae exposed to soil with resting spores buried at least 1 cm below the surface did not become infected.     

Evaluation of rayon swab surface sample collection method for Bacillus spores from nonporous surfaces

AIM: To evaluate US Centers for Disease Control and Prevention recommended swab surface sample collection method for recovery efficiency and limit of detection for powdered Bacillus spores from nonporous surfaces. METHODS AND RESULTS: Stainless steel and painted wallboard surface coupons were seeded with dry aerosolized Bacillus atrophaeus spores and surface concentrations determined. The observed mean rayon swab recovery efficiency from stainless steel was 0.41 with a standard deviation (SD) of +/-0.17 and for painted wallboard was 0.41 with an SD of +/-0.23. Evaluation of a sonication extraction method for the rayon swabs produced a mean extraction efficiency of 0.76 with an SD of +/-0.12. Swab recovery quantitative limits of detection were estimated at 25 colony forming units (CFU) per sample area for both stainless steel and painted wallboard. CONCLUSIONS: The swab sample collection method may be appropriate for small area sampling (10 -25 cm2) with a high agent concentration, but has limited value for large surface areas with a low agent concentration. The results of this study provide information necessary for the interpretation of swab environmental sample collection data, that is, positive swab samples are indicative of high surface concentrations and may imply a potential for exposure, whereas negative swab samples do not assure that organisms are absent from the surfaces sampled and may not assure the absence of the potential for exposure. SIGNIFICANCE AND IMPACT OF THE STUDY: It is critical from a public health perspective that the information obtained is accurate and reproducible. The consequence of an inappropriate public health response founded on information gathered using an ineffective or unreliable sample collection method has the potential for undesired social and economic impact.     

An unusual microfungus in a fungal spore from the Lower Devonian Rhynie chert

Abstract: A new fossil microfungus, Kryphiomyces catenulatus gen. et sp. nov., occurs as an endobiotic mycelial thallus in a large spore of a glomeromycotan fungus from the Lower Devonian Rhynie chert. The thallus consists of branched (?pseudo‐)septate hyphae with numerous catenulate swellings. Some hyphal tips produce spherical reproductive structures or propagules. Hyphal morphology in K. catenulatus is reminiscent of that in certain extant Hyphochytridiomycota, Chytridiomycota, and even Ascomycota, but specific diagnostic features that allow assignment of the fossil to modern groups are absent. The discovery of this interfungal association broadens our knowledge about the diversity of microfungi and their intricate associations in early continental ecosystems.     

从动物组织提取高质量总RNA方法的改进

RNA提取技术是分子生物学研究中的重要实验技术。介绍一种高纯度、高产量的从动物组织中提取总RNA的改进的方法,该法实用性强、重复性好。提取的RNA无DNA等污染物,其产量、纯度完全能满足分子克隆和基因表达研究的需要。利用改进后的方法提取牛组织的总RNA,研究了NRDR基因在牛组织中的表达分布。     

Efficiency of different methods of extracting the rice root nematode Hirschmanniella oryzae from rice and wheat soil and root samples

The nematode extraction method of centrifugal-floatation proved to be more efficient and significant (p ≤ 0.01) in extracting the rice root nematode, Hirschmanniella oryzae adults and larvae from soil or roots of rice and wheat crops than those extracted by sieving and tissue paper filtration technique. The extracted nematode from rice roots using incubation method is time-dependent and the peak of nematodes occurred four days after incubation. The number of extracted nematode varied according to crop, nematode mobility in soil particles, the number of nematodes present and tissue paper permeability.     

一种快速高效提取病原真菌DNA作为PCR模板的方法

真菌rDNA-ITS序列分析适合于较高等级水平的生物群体间的系统分析。真菌DNA的提取采用传统的方法,步骤繁琐,需要较长时间。采用Chelex-100法提取真菌DNA,使用PCR扩增rDNA-ITS序列评价提取核酸的质量。结果显示,该方法具有经济、简便、快速、高效的特点,是一种比较理想的提取真菌基因组DNA作为PCR模板的方法。