Species diversity of freshwater hyphomycetes in some streams of Pakistan. Comparison of sampling techniques

Freshwater hyphomycete communities in four streams were sampled by filtration of water samples, trapping conidia in artificial foam, and examination of alder leaf pack baits and random sampling of naturally occurring submerged leaves. No two communities of freshwater hyphomycetes detected by different sampling techniques used singly in a stream showed 100% similarity. Based on relative frequency values, the same 10 top-ranking species in a stream were detected by artificial foam trap and on the baited leaves. These species differed in ranking, and the community in artificial foam was riche than on baited leaves. Of the various communities detected by different sampling techniques used singly or in combination, those detected by artificial foam trap or a combination of this technique with leaf baiting or with leaf baiting and randomly sampled leaves or filtration showed the highest similarity to the total community based on data generated using four sampling techniques simultaneously.This work was conducted under Grant No. PSF Project Environment 3 from the Pakistan Science Foundation.     

Ecological studies of spores of aquatic hyphomycetes in the cringle brook,lincs

The aquatic Hyphomycete spora of the Cringle Brook, Lincs, was examined by foam sampling and by the use of cellophane impaction traps between August, 1968 and January, 1970.The species most frequently found (Tetracladium marchalianum, Alatospora acuminata and Flagellospora curvula) were generally in agreement with those found by other workers in temperate areas. Impaction trap samples generally contained fewer species than foam samples but filiform spore types such as Flagellospora were more frequently found on traps than in foam suggesting that impaction is more selective towards the filiform spore type than is foam. Many species increased in frequency in autumn accompanying and following leaf fall, and the winter spora was dominated by Alatospora acuminata, Clavariopsis aquatica, Clavatospora stellata, Flagellospora curvula and Lemonniera aquatica. During the summer the spora was dominated by Tetracladium marchalianum.The role of foam and impaction in the balance of aquatic spore populations is discussed in relation to techniques available for their study.     

Simple Method for Preparation of Homogeneous Spore Suspensions Useful in Industrial Strain Selection

Homogeneous suspensions of streptomycete spores were desirable for plating after mutagenesis. Filtration through 8-mum membrane filters (Millipore Corp.) more effectively separated hyphae and spore clumps from single spores than did filtration through cotton wefts or paper.     

A modification of tooth germ cultivation in vitro and in ovo

Mandibular molar anlages excised from 17-day mouse foetuses were cultured in vitro or in ovo (on the chorioallantoic membrane). In both cases, the explants were underlain either with a Millipore filter or with a piece of fibrin foam. Tooth germs were harvested after 7 days of cultivation and processed histologically. Spatial arrangement was highly preserved in the tooth germs cultured in vitro on fibrin foam. In vitro cultures on Millipore filters revealed significant flattening of tooth germs, caused especially by the collapse of enamel organ and the pulp. The cytodifferentiation of tooth germs cultured in vitro on both substrates (Millipore filter, fibrin foam) was characterized by the presence of odontoblasts, polarizing ameloblasts and predentine. The cytodifferentiation of tooth germs cultured in ovo on Millipore filters placed on chorioallantoic membrane was characterized by the presence of odontoblasts, ameloblasts, predentine, dentine and enamel. However, the flattening of these explants was identical with the changes of the explants cultured on Millipore filters in vitro. In ovo cultivation on the fibrin foam failed to bring satisfactory results.     

Seasonal occurrence of water-borne fungi in Konaje stream (Mangalore), India

The Konaje stream near Mangalore University Campus has been surveyed during 1980–1981 for the occurrence of water-borne fungi by using three methods: leaf litter observations, foam analysis and water filtration. A total of twenty species of fungi has been recorded. Three species of fungi, Lunulospora curvula, Triscelophorus monosporus and Triscelophorus sp. were found to occur throughout the year. In all three methods Lunulospora curvula occurred in highest frequency. The occurrence of different species was found to correlate with the rainfall and leaf deposition in this region and not with water temperature.     

Composite Sampling Approaches for Bacillus anthracis Surrogate Extracted from Soil

Any release of anthrax spores in the U.S. would require action to decontaminate the site and restore its use and operations as rapidly as possible. The remediation activity would require environmental sampling, both initially to determine the extent of contamination (hazard mapping) and post-decon to determine that the site is free of contamination (clearance sampling). Whether the spore contamination is within a building or outdoors, collecting and analyzing what could be thousands of samples can become the factor that limits the pace of restoring operations. To address this sampling and analysis bottleneck and decrease the time needed to recover from an anthrax contamination event, this study investigates the use of composite sampling. Pooling or compositing of samples is an established technique to reduce the number of analyses required, and its use for anthrax spore sampling has recently been investigated. However, use of composite sampling in an anthrax spore remediation event will require well-documented and accepted methods. In particular, previous composite sampling studies have focused on sampling from hard surfaces; data on soil sampling are required to extend the procedure to outdoor use. Further, we must consider whether combining liquid samples, thus increasing the volume, lowers the sensitivity of detection and produces false negatives. In this study, methods to composite bacterial spore samples from soil are demonstrated. B. subtilis spore suspensions were used as a surrogate for anthrax spores. Two soils (Arizona Test Dust and sterilized potting soil) were contaminated and spore recovery with composites was shown to match individual sample performance. Results show that dilution can be overcome by concentrating bacterial spores using standard filtration methods. This study shows that composite sampling can be a viable method of pooling samples to reduce the number of analysis that must be performed during anthrax spore remediation.     

Use of a Foam Spatula for Sampling Surfaces after Bioaerosol Deposition

The present study had three goals: (i) to evaluate the relative quantities of aerosolized Bacillus atrophaeus spores deposited on the vertical, horizontal top, and horizontal bottom surfaces in a chamber; (ii) to assess the relative recoveries of the aerosolized spores from glass and stainless steel surfaces with a polyester swab and a macrofoam sponge wipe; and (iii) to estimate the relative recovery efficiencies of aerosolized B. atrophaeus spores and Pantoea agglomerans using a foam spatula at several different bacterial loads by aerosol distribution on glass surfaces. The majority of spores were collected from the bottom horizontal surface regardless of which swab type and extraction protocol were used. Swabbing with a macrofoam sponge wipe was more efficient in recovering spores from surfaces contaminated with high bioaerosol concentrations than swabbing with a polyester swab. B. atrophaeus spores and P. agglomerans culturable cells were detected on glass surfaces using foam spatulas when the theoretical surface bacterial loads were 2.88 × 10⁴ CFU and 8.09 × 10⁶ CFU per 100-cm² area, respectively. The median recovery efficiency from the surfaces using foam spatulas was equal to 9.9% for B. atrophaeus spores when the recovery was calculated relative to the theoretical surface spore load. Using a foam spatula permits reliable sampling of spores on the bioaerosol-exposed surfaces in a wide measuring range. The culturable P. agglomerans cells were recovered with a median efficiency of 0.001%, but staining the swab extracts with fluorescent dyes allowed us to observe that the viable cell numbers were higher by 1.83 log units than culturable organisms. However, additional work is needed to improve the analysis of the foam extracts in order to decrease the limit of detection of Bacillus spores and Gram-negative bacteria on contaminated surfaces.Surface sampling is performed on a frequent basis in all situations where clean environment monitoring is needed, e.g., in health care facilities and in the pharmaceutical industry and food industry. An anthrax bioterrorist event in the fall of 2001 has emphasized the importance of efficient sampling methods for detection of pathogenic microorganisms on surfaces within intentionally contaminated locations (22). Unfortunately, our knowledge on the most effective sampling methodology as well as the level of confidence we may have in the results obtained by wiping, swabbing, and other sample collection strategies is still limited (1). Moreover, in most of the studies performed so far, bacteria and/or spores were collected from test samples or coupons of various materials, inoculated with a suspension of microorganisms that had been placed and spread over the surface, and then dried (14, 15). This may not mimic the true situation of surface contamination by a pathogen that has been intentionally released. Edmonds et al. (12) recently reported lower swabbing efficiencies of different types of swab materials used for sampling glass, polycarbonate, and vinyl surfaces contaminated with dry aerosol-deposited Bacillus atrophaeus spores compared to the surfaces inoculated by spore suspensions. Solid surface contamination from exposure to aerosolized spores fits the real world better than the previous models.Therefore, in our study we decided to generate aerosols of various concentrations of B. atrophaeus spores as well as the vegetative cells of Pantoea agglomerans inside a chamber where the bioaerosol particles were allowed to gravitationally settle on solid surfaces. The aerosolization of P. agglomerans was performed to verify the recovery of Gram-negative bacteria according to the recommendations of Budowle et al. (5). The main goal of our study was to establish the range of detection when bioaerosol-contaminated surfaces were swabbed using a commercially available foam spatula.     

Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles

A method is described for preparing, by filtration on Millipore filters, very thin (about 10 µ) pellicles of packed particles. These pellicles can be embedded in Epon for electron microscopic examination. They are also suitable for cytochemical assays. The method was used with various particulate fractions from rat liver. Its main advantages over the usual centrifugal packing techniques are that it produces heterogeneity solely in the direction perpendicular to the surface of the pellicle and that sections covering the whole depth of the pellicle can be photographed in a single field. It thus answers the essential criterion of random sampling and can be used for accurate quantitative evaluations.     

Protostelids from German Beech forests

A survey for protostelids from old-growth beech forests of northeastern Germany resulted in 14 species found in both ground and aerial litter, constituting the first survey of these organisms from Central Europe. Additionally the myxomycete Echinostelium bisporum was recorded as new for Germany. A detailed investigation of randomly sampled dead Fagus leaves (16 leaves on ground, 16 aerial leaves, three replicate cultures per leaf) resulted in 7 species; a hyperbolic regression of the respective species accumulation curve gives the theoretical value of 8.3 species. Protostelids occurred especially at aerial leaves with a high frequency (0.94). A program simulating random spore hits was written to calculate the minimum spore fallout necessary to explain this frequency. The resulting average value of about 3 spores per leaf can well be matched by the potential spore productivity estimated to be about 1000 spores per leaf for the most common species, Protostelium mycophaga.     

中国产异蒴藓属（Lyellia）植物形态解剖比较观察

利用扫描电镜法、徒手切片法对中国产的2种异蒴藓进行观察分析。分别观察了孢子大小、颜色、叶片和茎的长度和宽度等。结果表明异蒴藓属植物叶片的一些形态学和解剖学性状表现出了金发藓科植物所共有的特征,异蒴藓属在金发藓科中气孔较明显属于进化性状,而孢壁纹饰则属于原始性状。     

Diversity and distribution of saprobic microfungi in leaf litter of an Australian tropical rainforest

The diversity and distribution of microfungal assemblages in leaf litter of a tropical Australian forest was assessed using two methods: (1) cultures were isolated using a particle filtration protocol (wet season 2001), and (2) fruit bodies were observed directly on leaf surfaces following incubation in humid chambers (wet and dry season of 2002). Four tree species were studied using both methods, namely Cryptocarya mackinnoniana (Lauraceae), Elaeocarpus angustifolius (Elaeocarpaceae), Ficus pleurocarpa (Moraceae), and Opisthiolepis heterophylla (Proteaceae). An additional two species, Darlingia ferruginea (Proteaceae) and Ficus destruens (Moraceae), were studied using direct observations. In total, fruiting bodies of 185 microfungal species were recorded on leaf surfaces (31–81 species per tree species), and 419 morphotypes were detected among isolates obtained by particle filtration (111–203 morphotypes per tree species). Although the observed microfungal diversity was higher with the particle filtration protocol, both methods concurred with respect to microfungal distributions. The overlap of microfungal species in pair wise comparisons of tree species was low (14–30 %), and only 2 and 3 % of microfungal species were observed in leaves of all tree species by particle filtration and by direct observations respectively. Multivariate analysis of data from direct observations confirmed the hypothesis that microfungal assemblages are strongly influenced by host phylogeny and are also affected by seasonal and site factors. The importance of host species in shaping microfungal distributions was also supported by the particle filtration data. Several taxa new to science, as well as some widespread saprotrophs, were detected on only one host. The underlying reasons for this affinity remain unclear, but we hypothesise that a number of factors may be involved such as fungal adaptation to plant secondary metabolites or the presence of a biotrophic phase in the fungus' life cycle.     

Disease prevalence and transmission of Microsporidium phytoseiuli infecting the predatory mite, Phytoseiulus persimilis (Acari: Phytoseiidae)

Isolated colonies of the predatory mite, Phytoseiulus persimilis, were used to gain information regarding prevalence and transmission of Microsporidium phytoseiuli. Two colonies of P. persimilis were reared on spider mite (Tetranychus urticae)-infested bean plants in isolated cages. Disease prevalence of predators from Colony 1 remained relatively low (between 0 and 15%) over 57 weeks of observation whereas disease prevalence of predators from Colony 2 increased over 3 months (from 12 to 100%). Disease prevalence among predators from Colony 1 had increased to 100% 2 months after weekly sampling had ceased for this colony and periodic sampling confirmed that disease prevalence among individuals of both colonies remained at 100%. Microsporidian spores were not detected in randomly chosen samples of T. urticae prey mites that were removed and examined biweekly during this period. Although numerous microsporidian spores were observed in smear preparations of fecal pellets examined by light microscopy, spores were not observed on leaf surfaces or predator feces when examined by SEM. The latter appeared as intact aggregates composed of numerous dumbbell-shaped crystals and it is unlikely that spores are liberated from intact fecal pellets onto leaf surfaces. Vertical transmission of M. phytoseiuli was 100%; horizontal transmission was low (14.3%) and occurred only when immature P. persimilis were permitted to develop in contact with infected immature and adult predators. The mean number of eggs produced per mated pair was highest when uninfected females were mated with uninfected males (63.2 eggs per mated pair). Although mean egg production decreased when one or both parents were infected, not all differences were significant. Male predatory mites did not contribute to infection of their progeny. Results suggest that routine examination of P. persimilis for microsporidian spores is essential for the management of M. phytoseiuli within P. persimilis colonies. Low disease prevalence and lack of obvious disease signs or symptoms, as in the case of M. phytoseiuli, increase the probability that these pathogens will escape notice unless individuals are routinely examined for pathogens.     

Subgroups in Group III of RNA Phages

In our previous studies, RNA phage strains were separated into 3 major groups on the basis of filtration efficiency through Millipore filters. In the present study, the strains of group III were shown to be further divided into 3 subgroups: (a) Qβ, NH, SG; (b) VK, SO; (c) ST. This subgrouping was found to be compatible with the serological grouping and pronase sensitivity with the exception of strain NM. Strain NM was classified in subgroup (a) by the Millipore filtration and in subgroup (b) by the other two methods.     

Community structure of arbuscular mycorrhizal fungi in a primary successional volcanic desert on the southeast slope of Mount Fuji

Community structure of arbuscular mycorrhizal fungi (AMF), evaluated as spore samples and mycorrhizal roots of four herbaceous plant species, was investigated at different altitudes in a primary successional volcanic desert on Mount Fuji using molecular methods (fragment and sequence analysis of the large ribosomal subunit RNA gene). In total, 17 different AMF clades were identified, and most were members of the Glomaceae, Acaulosporaceae, and Gigasporaceae. The AMF community structures detected by spore sampling were inconsistent with those from plant roots. Of all AMF clades, six (35.3%) were detected only on the basis of spores, six (35.3%) only in roots, and five corresponded to both spores and roots (29.4%). Although an Acaulospora species was the most dominant among spores (67.1%), it accounted for only 6.8% in root samples. A species analysis of AMF communities at different altitudes demonstrated that AMF species diversity increased as altitude decreased and that the species enrichment at lower altitudes resulted from the addition of new species rather than species replacement. The inconsistencies in the species composition of spore communities with those in roots and the change in species diversity with altitude are discussed.     

Spore productivity in Cladosporium

The high incidence ofCladosporia in the airspora indicates a prolific production of spores. Six species ofCladosporium were sampled over a period of 9 weeks, using dry and wet (mist-laden) air, and over a period of 4 weeks using humid air. Many more spores were released in wet air than in dry air: numbers released in humid air were generally intermediate between those of wet and dry samples. None of the cultures was exhausted of spores at the end of the sampling periods although samples generally decreased in size from the fifth or sixth week onwards. Removal of spores would seem to be conducive to further sporulation provided the substrate is not exhausted. Maximum productivities recorded for the six species (all in mist-laden air) ranged from 730 to 26 100 spores per mg dry weight of mycelium. Differences in the levels of spore production in culture by the six species do not correlate with their individual frequencies in the airspora, indicating that the latter are more dependent on the distribution and substrate relationships of each species.     

Phlorizin increases the permeability of intestinal mucosal membrane to sodium

We reported previously that when jejunal transmural glucose transport was inhibited by phlorizin the ratio of Na:glucose transport increased from 2.0:1 (in controls) to 3.3:1. To elucidate the mechanism of this increased ratio of Na:glucose transport, in the present study we have investigated the effect of phlorizin on Na uptake by brush border membrane vesicles and by everted sacs of hamster jejunum. In experiments on membrane vesicles the following observations were made. The time course of Na uptake showed that the control vesicles were in complete equilibrium with a Na-containing (100 mM) medium between 30 and 90 min incubation. In these periods of incubation, the vesicles incubated with phlorizin presumably also equilibrated with the medium, but lost their intravesicular Na during Millipore filtration and washing, and consequently the residual Na content was lower than that of controls. This effect of phlorizin was concentration dependent, and appeared to be unrelated to Na-coupled glucose transport, because it was also observed in the absence of glucose. This loss of Na during Millipore filtration and washing was also observed (i) when vesicles were equilibrated in a Na-containing solution in the absence of phlorizin and then exposed to a similar solution containing phlorizin, or (ii) when vesicles were equilibrated in a Na-containing solution in the presence of phlorizin and then washed repeatedly following Millipore filtration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative study of methods for the quantification of fungal spores at the leaf surfaces ofPopulus xeuramericana

Summary The removal of fungal spores, urediniospores ofMelampsora medusae and conidia ofPestalozzia sp., from the leaf surfaces ofPopulus xeuramericana (Dode) Guinier cv. I-488 was assessed using three cultural techniques conventionally employed in phylloplane studies. The method of removal and the original density of spore deposition, but not the interaction of these factors, were significant determinants of variability in spore removal. Irrespective of the original density of deposition, the leaf print method was the most, and the leaf washing technique the least, efficient means of spore removal from the leaf surface. Factors which could contribute to this difference in efficiency are discussed.     

Cladosporium airborne spore incidence in the environmental quality of the Iberian Peninsula

Cladosporium spores are ubiquitous in the air and are included in the fungal contaminants that define environmental quality. This paper provides an updated review of their concentration in the Iberian Peninsula, based on data from 12 sampling stations collected using a common non-viable volumetric sampling method and the same subsequent data-treatment techniques. The database comprised 84 annual periods, covering the period 1993–2009. Mean annual total spore concentrations for each station, calculated as the sum of daily concentrations, ranged from 54 459 in Cartagena to 933 485 in Sevilla; other sampling stations recorded annual total spores between 93 052 (Porto) and 579 953 (Mérida). Analysis of annual spore-distribution patterns revealed either one or two peaks (spring and fall) depending on the location and prevailing climate of the area where the sampling stations have been placed. For all stations, average temperature was the meteorological parameter displaying the strongest positive correlation with airborne spore concentrations.     

Comparison of pressure chamber and psychrometer estimates of leaf water potential in rice

Summary The need to compare pressure-chamber estimates of leaf water potential with a psychrometric method has been established for several crop species. We investigated this relationship for rice (Oryza sativa L.) as well as the need to protect leaves from water loss during sampling and measuring period in the pressure chamber. Two rice cultivars grown in containers on a clay-loam soil were stressed to varying degrees by withholding water. Fully expanded leaves were sampled for estimation of leaf water potential by the dew point hygrometer and pressure-chamber techniques. The same leaf was used in both methods allowing direct comparison. Additionally, two alternative methods of leaf handling for measurement by the pressure chamber technique were compared. Protection of leaf samples against water loss during excision, transport and handling was found to be more important at higher leaf water potentials (>−1.0 MPa). The two cultivars used appeared to differ in their response to protection of the leaf sample. These results serve to further caution pressure chamber users on extrapolating comparisons between the two measurement methods and between tissue handling techniques even within a crop species.     

Comparison of gauze swabs and membrane filters for isolation of Campylobacter spp. from surface water

The epidemiology of Campylobacter jejuni indicates that waterborne transmission is important; the organism has been isolated from seawater, fresh water, and estuarine sites. Membrane filtration, with and without use of an enrichment broth, has been the most common method for isolating C. jejuni from water. We evaluated two methods for isolating C. jejuni from water: membrane filtration and gauze filtration. The membrane filters evaluated included 0.22- and 0.45-micron-pore Millipore filters (Millipore Corp., Bedford, Mass.), 0.2- and 0.4-micron-pore Nuclepore filters (Nucleopore Corp., Pleasanton, Calif.), and a 0.45-micron-pore Zetapor filters (AMF Cuno, Meridian, Conn.). The gauze filters included both Moore and Spira swabs. Of the membrane filters evaluated, the 0.45-micron-pore Millipore and Zetapor filters were the most sensitive for recovery of C. jejuni from seeded waters. The 0.45-micron-pore Millipore filter placed in Oosterom broth was better for recovery of C. jejuni from seeded stationary surface waters than either the Spira or Moore swab. However, the 0.45-micron-pore Millipore filter placed on a plate or in enrichment broth was equivalent to the Spira gauze swab when used to examine water from Atlanta area streams. C. jejuni organisms were isolated from 9 of 24 surface water samples representing 5 of 12 streams.