Spore Content Research Articles

Projections of the effects of climate change on allergic asthma: the contribution of aerobiology

Climate change is unequivocal and represents a possible threat for patients affected by allergic conditions. It has already had an impact on living organisms, including plants and fungi with current scenarios projecting further effects by the end of the century. Over the last three decades, studies have shown changes in production, dispersion and allergen content of pollen and spores, which may be region- and species-specific. In addition, these changes may have been influenced by urban air pollutants interacting directly with pollen. Data suggest an increasing effect of aeroallergens on allergic patients over this period, which may also imply a greater likelihood of the development of an allergic respiratory disease in sensitized subjects and exacerbation of symptomatic patients. There are a number of limitations that make predictions uncertain, and further and specifically designed studies are needed to clarify current effects and future scenarios. We recommend: More stress on pollen/spore exposure in the diagnosis and treatment guidelines of respiratory and allergic diseases; collection of aerobiological data in a structured way at the European level; creation, promotion and support of multidisciplinary research teams in this area; lobbying the European Union and other funders to finance this research.

Airborne fungal spore content in the atmosphere of the city of La Plata, Argentina

The aim of this paper is to present the first aeromycological study of the atmosphere of the city of La Plata. Air samples were taken using a Hirst-type volumetric spore trap sampler (Lanzoni, VPPS 2000) for a period of a year (July 2000–June 2001). Seventy-nine morphological types of spores belonging to the Phyla Myxomycota, Zygomycota, Oomycota, Ascomycota, Basidiomycota, as well as anamorphs of Higher Fungi were identified. A total of 171670.21 spores were recorded, with a daily mean of 540 spores/m³. The spores were present throughout the year the study was carried out. However, there was a wide daily fluctuation in the concentration values with a tendency toward an increase during the summer months. The fungal spores were classified in three categories: abundant, having five spore types; moderate having 12 types; and low, with 62 morphological types which represent 67.2, 24.2, and 8.6%, respectively. The most representative taxa were Cladosporium cladosporioides, Leptosphaeria, Cladosporium herbarum, Coprinus, and Agaricus, the first two taxa having a high frequency during the year of study. This scientific research reveals a great diversity of morphological types in this outdoor environment, showing a strong dominance of the Imperfect Fungi, whose components have seasonal rates. By this study, the first aeromycological profile of an urban center of Argentina has been portrayed, and new information on the field of aeromycology in the country has been provided.

In Planta Quantification of Plasmodiophora brassicae Using Signature Fatty Acids and Real-Time PCR.

Until now, molecular and biochemical methods have only been used to show whether or not Plasmodiophora brassicae is present in plant or soil samples but not to what extent. Here, in planta quantification of P. brassicae by whole-cell fatty acid (WCFA) measurements and real-time polymerase chain reaction (PCR) was evaluated. Arachidonic acid (ARA, 20:4) was the most abundant fatty acid in resting spores and was only found in infected roots, which indicates a potential of ARA as a biomarker for P. brassicae. A real-time PCR assay was developed using primers designed from the internal transcribed spacer region of the ribosomal DNA. Using these primers, it was possible to detect P. brassicae in infected roots 10 days after germination of plants sown in infested soil. A bioassay showed that the amounts of ARA found by WCFA analysis and the DNA found by real-time PCR in infected plants were well correlated. These measurements also correlated with the soil spore content and the assessed disease incidence and disease severity scores. Therefore, we conclude that WCFA analysis and real-time PCR are good tools for P. brassicae quantification that can be applied to basic studies of the pathogen and in resistance screens.

Lake-mire deposition, earthquakes and wildfires along a basin margin fault; Rønne Graben, Middle Jurassic, Denmark

Regional uplift of structural highs took place in the greater North Sea region in early Middle Jurassic times, resulting in a change from marine shelf deposition in the Early Jurassic to widespread emergence, erosion and localized deposition in grabens. The Middle Jurassic Bagå Formation exposed on the island of Bornholm in the Baltic Sea was deposited in the Rønne Graben, which forms a dog-leg pull-apart basin linking the northern and southern branches of the Tornquist Zone. The studied succession is situated on the hanging wall immediately adjacent to the main eastern graben margin fault and the footwall consists of deeply weathered granite. It is the only locality along the northern branch of the Tornquist Zone where active tectonic influence on Middle Jurassic deposition can be demonstrated in outcrop. Small alluvial fans composed of poorly sorted washout sands and muddy debris flows with weathered granite boulders fringed the footwall scarp. Syn-depositional soft-sediment deformation and chaotic bedding structures interpreted as seismites caused by earthquake shocks are characteristic at several levels. On the hanging wall further away from the active fault the depositional environment was dominated by shallow freshwater lakes with deposition of clay. Lake deposition outpaced subsidence and open, oxygen-deficient freshwater mires were formed during periods of no or only incremental fault-movement. The mires were only occasionally isolated from clastic input due to doming and dense vegetation, resulting in accumulation of pure peat beds. The content of spores and pollen shows that the mires were covered by a vegetation of arborescent and herbaceous Filicopsida (ferns), Lycopsida (club mosses), Cycadales/Bennettitales, Ginkgoales, Caytoniales, Taxodiaceae or Cupressaceae, and Araucariaceae. Wildfires were common in the mires and also on the footwall probably followed by episodes of increased run-off and erosion of the upland. The presence of common washout sands, debrites, seismites and abundant lacustrine flooding surfaces indicating sudden drowning of the mires was caused by abrupt movements on the fault with associated earthquakes. The succession thus provides an example of temporal changes in deposition in response to changes in tectonic conditions. The study illustrates how regional uplift of structural highs caused a restriction of deposition to narrow grabens like the Rønne Graben. Facies patterns changed dramatically from laterally extensive uniform packages controlled by sea-level changes in the Early Jurassic to strongly localized deposition with marked facies changes controlled by faulting and earthquakes in the Middle Jurassic. The succession thus serves as an excellent example of tectonically controlled deposition under humid conditions in a continental graben.

Germination of phagocytosed E. cuniculi spores does not significantly contribute to parasitophorous vacuole formation in J774 cells

The obligate intracellular microsporidia have developed a unique invasion mechanism to infect their host cells. Spores explosively evert a tube-like structure and extrude the infectious spore content through this organelle into the host cell. Spores from species of the genus Encephalitozoon were also shown to be efficiently internalized by phagocytosis, which led to the hypothesis that spore germination from inside a phagosome might contribute to the infection process. Here, we challenge this hypothesis by quantifying Encephalitozoon cuniculi infection rates of J774 cells that were incubated with the phagocytosis inhibitor cytochalasin D. We demonstrate that the invasion rate in cytochalasin D-treated cells is identical to untreated controls, although phagocytic uptake of E. cuniculi spores was less than 10% of control samples. This study suggests that germination of phagocytosed spores is not a significant infection mode for E. cuniculi.

The use of artificial neural networks to forecast biological atmospheric allergens or pathogens only as Alternaria spores

The monitoring of atmospheric Alternaria spores is of major importance due to their adverse effects on crops and their role as human allergens. Most species act as plant pathogens, prompting considerable economic losses worldwide on important crops such as potato, tomato or wheat. Fungal spores can also have serious detrimental effects on human health, triggering respiratory diseases and allergenic processes. The aim of this study was not only to examine the relationship between the atmospheric Alternaria spore content and the prevailing meteorological parameters, but also to predict the atmospheric Alternaria spore content in the Northwest Spain using a novel data analysis technique, ANNs (Artificial Neural Networks). A Hirst-type LANZONI VPPS 2000 volumetric 7-day recording sampler was used to collect the airborne spores from 1997 to 2008. Neural networks provided us with a good tool for forecasting Alternaria airborne spore concentration, and thus could help the automation of the prediction system in the aerobiological information diffusion to the population suffering from allergic problems or the prevention of considerable economic worldwide losses on important crops. Our proposed model would be applied to different geographical areas; nevertheless, the adjustment of the model, by using the available and adequate variables, would be realised in each case.

Hourly predictive artificial neural network and multivariate regression tree models of Alternaria and Cladosporium spore concentrations in Szczecin (Poland)

A study was made of the link between time of day, weather variables and the hourly content of certain fungal spores in the atmosphere of the city of Szczecin, Poland, in 2004-2007. Sampling was carried out with a Lanzoni 7-day-recording spore trap. The spores analysed belonged to the taxa Alternaria and Cladosporium. These spores were selected both for their allergenic capacity and for their high level presence in the atmosphere, particularly during summer. Spearman correlation coefficients between spore concentrations, meteorological parameters and time of day showed different indices depending on the taxon being analysed. Relative humidity (RH), air temperature, air pressure and clouds most strongly and significantly influenced the concentration of Alternaria spores. Cladosporium spores correlated less strongly and significantly than Alternaria. Multivariate regression tree analysis revealed that, at air pressures lower than 1,011 hPa the concentration of Alternaria spores was low. Under higher air pressure spore concentrations were higher, particularly when RH was lower than 36.5%. In the case of Cladosporium, under higher air pressure (>1,008 hPa), the spores analysed were more abundant, particularly after 0330 hours. In artificial neural networks, RH, air pressure and air temperature were the most important variables in the model for Alternaria spore concentration. For Cladosporium, clouds, time of day, air pressure, wind speed and dew point temperature were highly significant factors influencing spore concentration. The maximum abundance of Cladosporium spores in air fell between 1200 and 1700 hours.

Mycological complexes in Holocene and Late Pleistocene paleohorizons and in fragments of paleosols

Fragments of buried Late Pleistocene (30000-year-old) and Early Holocene (10000-year-old) paleosols contained viable complexes of microscopic fungi. The mycobiota of these paleosols represents a pool of fungal spores that is lower in number and species diversity as compared to that in the recent humus horizons and higher than that in the inclosing layers. The central part of the paleosol profiles is greatly enriched in microscopic fungi. In the intact humus horizons of the Late Holocene (1000–1200 years) paleosols, actively functioning fungal complexes are present. These horizons are characterized by their higher level of CO2 emission. The buried horizons, as compared to the recent mineral ones, contain a greater fungal biomass (by several times) and have a higher species diversity of microscopic fungi (including fungi that are not isolated from the recent horizons). Nonsporulating forms are also present there as sterile mycelium. The seasonal dynamics of the species composition and biomass of the fungal complexes were more prominent and differed from those inherent to the surface soil horizons. In the buried humus horizons, the dynamics of the fungal biomass were mainly due to the changes in the content of spores. The data on the composition of the fungal complexes in the buried soils confirm (due to the presence of stenotopic species) the results of paleobotanic analyses of the past phytocenoses or do not contradict them.

Comparison of Two Analytical Methods for Detecting (1-3)-β-D-Glucan in Pure Fungal Cultures and in Home Dust Samples

There are two methods available for the analysis of (1-3)-β-D-glucan: the Limulus Amebocyte Lysate assay (LAL) and the inhibition Enzyme Immunoassay (EIA). The aim of this study was to compare the accuracy and specificity of these two methods in detecting eight alpha and beta-glucan standards, and their sensitivity for the analysis of (1-3)-β- D-glucan content of common indoor fungal species and indoor dust samples. The results show that the LAL assay is more accurate, specific, and sensitive in measuring linear and branched β-D-glucans than the EIA. The greatest LAL-analyzed (1-3)-β-D-glucan content per spore (241 pg/spore) was found with E. nigrum, which also had the largest spore size (28 μm). The biomass-normalized (1-3)-β-D-glucan content of fungal spores from pure cultures was within similar range with the two assays but no correlation was found between the results from the two assays. In contrast, there was a significant correlation between the EIA and LAL-measured (1-3)-β-D-glucan concentrations (μg/m2 of floor area) in field dust samples.

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45 degrees C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45 degrees C by wet heat, 2 M HCl, 2 M NaOH and 2 M H(2)O(2) was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45 degrees C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45 degrees C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45 degrees C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.

MICROSPORIDIAN PARASITES OF MOSQUITOES

The Microsporidia are a large diverse group of obligate, intracellular parasites. They are singlecelled eukaryotic microorganisms that have small genomes in the size range of prokaryotic cells and are now thought to be highly evolved fungi (Keeling and Fast 2002). They are exclusive parasites of other eukaryotes and possess a unique and highly specialized mechanism for invading host cells via infectious spores. Spores are the only stage that can exist outside a living host cell and they are the primary vehicles for horizontal transmission between hosts (peroral) and vertical transmission (transovarial) within the host. Spores are diagnostic, especially at the ultrastructural level (Fig. 1), and are distinguished by their small size (2–20 mm), thick walls (consisting of an endospore and exospore), and presence of a unique set of organelles that function as an extrusion apparatus. These include a tightly coiled polar filament (tube) that is attached to an anchoring disc at the anterior pole of the spore, a membranous polaroplast, and a posterior vacuole which collectively function to explosively inoculate the spore content or ‘‘sporoplasm’’ through the polar filament into a host cell to initiate infection. Microsporidia are ubiquitous in nature and exhibit a very broad host range within the animal kingdom. They have been described as parasites in all classes of vertebrates, including humans, and most invertebrates, but are particularly common to arthropods and fish. Microsporidia represent one of the largest and most diverse groups of parasitic organisms associated with mosquito populations in nature. They have been described from 14 different genera worldwide, and it is quite likely that all mosquitoes serve as hosts for one or more of these parasites. The group currently includes a heterogeneous assemblage of over 150 described species from 23 recognized genera, 15 of which are monotypic (only one species is known) (Table 1). Members of these genera exhibit extensive variation in their development and life cycles but generally fall into 2 broad categories. The first includes the monomorphic forms such as Anncaliia (formerly Nosema and then Brachiola) (Franzen et al. 2006) and Vavraia. These microsporidia have comparatively simple life cycles involving only one sporogonic sequence (Fig. 2). They develop asexually (merogony or schizogony) and produce a single spore type that is orally infectious to mosquito larvae. Vertical transmission may additionally occur via oral ingestion of spores on contaminated eggs (transovum), but there is no separate developmental sequence leading to ovarian infection in female hosts. These microsporidia have a very broad host range and are mildly pathogenic to mosquitoes, generally producing low larval mortality. Other genera from which only one developmental sequence has been described include Aedispora, Crepidulospora, Polydispyrenia, Senoma, Trichotosporea, and Tricornia. However, the complete life cycles and modes of transmission of members of these genera have yet to be resolved and they may be polymorphic. The second group includes the true polymorphic forms. These parasites are more common in nature and exhibit some of the most complex life cycles yet described for any microsporidia (Fig. 2). These include elements of asexual (schizogony, merogony, and sporogony) and sexual (karyogamy, gametogenesis, and plasmogamy) reproduction; the formation of multiple spore types in various stages of the host; host sexand tissue-dependent development; and separate developmental sequences leading to vertical (transovarial) and horizontal transmission. Many species, such as Edhazardia aedis, require two successive host generations to complete their life cycle, and at least 4 genera, Amblyospora, Duboscqia, Hyalinocysta and Parathelohania require obligatory development in an intermediate copepod host. These microsporidia generally exhibit higher levels of host specificity and although they do not cause any acute mortality or detectable morbidity in adult female hosts that go on to transmit infections transovarially, they have at least one phase of development that typically kills larval hosts during the last stadium. Mortality in larvae results from destruction of various host tissues and subsequent depletion of essential energy reserves necessary for pupation. The production of entomopathogenic toxins has never been documented. Copyright E 2007 by The American Mosquito Control Association, Inc.

Bacillus cereus Spores During Housing of Dairy Cows: Factors Affecting Contamination of Raw Milk

The contamination of raw milk with Bacillus cereus spores was studied during the indoor confinement of dairy cattle. The occurrence of spores in fresh and used bedding material, air samples, feed, feces, and the rinse water from milking equipment was compared with the spore level in bulk tank milk on 2 farms, one of which had 2 different housing systems. A less extensive study was carried out on an additional 5 farms. High spore concentrations of >100 spores/L in the raw milk were found on 4 of the farms. The number of spores found in the feed, feces, and air was too small to be of importance for milk contamination. Elevated spore contents in the rinse water from the milking equipment (up to 322 spores/L) were observed and large numbers of spores were found in the used bedding material, especially in free stalls with >5cm deep sawdust beds. At most, 87,000 spores/g were found in used sawdust bedding. A positive correlation was found between the spore content in used bedding material and milk (r=0.72). Comparison of the genetic fingerprints obtained by the random amplified polymorphic DNA PCR of isolates of B. cereus from the different sources indicated that used bedding material was the major source of contamination. A separate feeding experiment in which cows were experimentally fed B. cereus spores showed a positive relationship between the number of spores in the feed and feces and in the feces and milk (r=0.78). The results showed that contaminated feed could be a significant source of spore contamination of raw milk if the number of spores excreted in the feces exceeded 100,000/g.

Spore detection in aerobic granules by different dipicolinic acid releasing methods

This work aimed at developing a procedure for spore quantification. Spore content was determined by analyzing dipicolinic acid (dpa) extracted from aerobic granules by 13 methods. Concentrated HCl was able to release dpa completely. Results showed that dpa constituted 33.7 mg per g SS, meaning that about 337 mg per g SS were spores, not the normal vegetative cells.

Effect of Different Premilking Manual Teat-Cleaning Methods on Bacterial Spores in Milk

Different teat-cleaning methods were evaluated to determine their effect on the presence of spores from anaerobic bacterial spore-formers in the milk. Artificial contamination was used to achieve uniform contamination of teats to reduce the number of cows and samples needed in the experiments and still obtain adequate power to detect differences among tested methods. Teats were contaminated experimentally with a large amount of Clostridium tyrobutyricum spores in a manure–water slurry. Various types of dry and moistened towels and different combinations of methods using soap or 2 types of towels, together with cleaning times of 10 or 20s, were compared in 2 Latin square-designed experiments with 7 cows, 7 treatments, and 4 replications in each experiment. In comparison with control (no cleaning and no forestripping), cleaning teats with dry paper towels for 10s reduced concentration of spores in milk by 45 to 50%. A 50 to 74% reduction was achieved using different types of moist towels for 10s. Methods using 2 towels, soap, or a longer cleaning time reduced bacterial contamination by 85 to 91%. The most effective methods in reducing milk spore content (96% reduction) were use of a moist washable towel with or without soap followed by drying with a dry paper towel, for a total time of 20s per cow. One of the best cleaning methods was studied in an additional experiment to determine the effect of different teat contamination mixtures. The Latin square-designed experiment with 8 cows, 8 treatments, and 2 replications showed that cleaning was independent of the tested contamination matrix (manure, soil, or sawdust), type of spores (Cl. tyrobutyricum and Bacillus cereus), or degree of contamination (manure or extra manure).

Spore production in solid-state fermentation of rice by Clonostachys rosea, a biopesticide for gray mold of strawberries

Gray mold caused by Botrytis cinerea is an important disease of strawberry. Clonostachys rosea is a mycoparasite of B. cinerea that reduces fruit losses when used as a biocontrol agent. Since spore production by C. rosea has not been optimized, we investigated factors affecting sporulation under aseptic conditions on white rice grains. The greatest spore production in glass flasks, 3.4 × 10 9 spores/g-dry-matter (gDM), occurred with an initial moisture content of 46% (w/w wet basis), inoculated with 1 × 10 6 spores/gDM and hand shaken every 15 days. However, a lower inoculum density (9 × 10 3 spores/gDM) and no shaking also gave acceptable sporulation. In plastic bags 1.1 × 10 8 spores/gDM were produced in 15 days, suggesting that larger scale production may be feasible: with this spore content, 24 m 2 of incubator space would produce sufficient spores for the continued treatment of 1 ha of strawberry plants.

WHOLE-CELL FATTY ACID PROFILES - A TOOL FOR SPECIES AND SUBSPECIES CLASSIFICATION IN THE PUCCINIA RECONDITA COMPLEX

The fatty acid profiles of 145 telial isolates of Puccinia recondita sensu lato (Uredinales: Pucciniaceae), from different host species and different geographical origins, were determined by gas chromatography (GC). The isolates clustered into three different GC groups, according to consistent quantitative differences in their fatty acid compositions. Isolates collected from bread, durum and wild emmer wheats, with Thalictrum speciosissimum (Ranunculaceae) as aecial host, regardless of their geographical origin, formed one group. The second group consists of isolates collected on Aegilops spp. and some from durum wheat, all alternating on Boraginaceous species. The third group contains isolates collected on rye, alternating on Boraginaceae. The grouping of P. recondita isolates by fatty acid profiles coincides with a similar one, suggested by several studies involving pathogenicity on alternate hosts, cross ability, teliospore dimensions and relative DNA content of haploid spores. It is concluded that the three groups represent two species and one subspecies: Puccinia triticina (on wheats, group 1), P. recondita subsp. recondita (on rye, group 3) and P. recondita subsp. aegilopis (on Aegilops spp. and durum wheat, group 2).

Rapid differentiation and enumeration of the total, viable vegetative cell and spore content of thermophilic bacilli in milk powders with reference to Anoxybacillus flavithermus

The development of a rapid method for the selective detection and enumeration of the total and viable vegetative cell and spore content of thermophilic bacilli in milk powder by PCR. Quantitative PCR and microscopy indicate the presence of up to 2.9 log units more cells in milk powder than accounted for by plate counting due to the majority of cells being killed during milk processing. Two approaches for viable and dead cell differentiation of thermophilic bacilli by quantitative PCR were evaluated, these being the nucleic binding dye ethidium monoazide (EMA) and DNase I digestion. The former agent exposed to a viable culture of Anoxybacillus flavithermus caused considerable cell inactivation. In contrast, DNase I treatment had no effect on cell viability and was utilized to develop DNA extraction methods for the differential enumeration of total, viable vegetative cells and spores in milk powder. Moreover, the methods were further applied and evaluated to 41 factory powder samples taken throughout eight process runs to assess changes in numbers of vegetative cells and spores with time. DNase I treatment reduced vegetative cell numbers enumerated with PCR by up to 2.6 log units. The quantification of spores in the factory milk powders investigated indicates on average the presence of 1.2 log units more spores than determined by plate counting. The method presented in this study provides the ability to selectively enumerate the total and viable cell and spore content of reconstituted milk. The current study provides a tool to monitor the extent of thermophilic contamination during milk powder manufacturing 60-90 min after sampling.

Contribution of ExsFA and ExsFB Proteins to the Localization of BclA on the Spore Surface and to the Stability of the Bacillus anthracis Exosporium

Spores of Bacillus anthracis, the etiological agent of anthrax, and the closely related species Bacillus cereus and Bacillus thuringiensis, possess an exosporium, which is the outermost structure surrounding the mature spore. It consists of a paracrystalline basal layer and a hair-like outer layer. To date, the structural contribution of only one exosporium component, the collagen-like glycoprotein BclA, has been described. It is the structural component of the hair-like filaments. Here, we describe two other proteins, ExsFA and ExsFB, which are probably organized in multimeric complexes with other exosporium components, including BclA. Single and double exsF deletion mutants were constructed and analyzed. We found that inactivation of exsF genes affects the BclA content of spores. BclA is produced by all mutants. However, it is partially and totally released after mother cell lysis of the DeltaexsFA and DeltaexsFA DeltaexsFB mutant strains, respectively. Electron microscopy revealed that the exsF mutant spores have defective exosporia. The DeltaexsFA and DeltaexsFA DeltaexsFB spore surfaces are partially and totally devoid of filaments, respectively. Moreover, for all mutants, the crystalline basal layer appeared unstable. This instability revealed the presence of two distinct crystalline arrays that are sloughed off from the spore surface. These results indicate that ExsF proteins are required for the proper localization of BclA on the spore surface and for the stability of the exosporium crystalline layers.

Palaeoecological constraints of some Lepidostrobus cones and their parent plants from the Late Palaeozoic continental basins of the Czech Republic

About 60 strobilar specimens of six Lepidostrobus species ( Lepidostrobus sternbergii, Lepidostrobus nemejcii sp. nov., Lepidostrobus thomasii sp. nov., Lepidostrobus obovatus, Lepidostrobus ronnaensis sp. nov. and Lepidostrobus stephanicus) and their microspores from the Bohemian Late Palaeozoic continental basins were studied. The stratigraphical range of cones is from the Langsettian to the Stephanian B. Microspores isolated from six species of Lepidostrobus cones belong to six dispersed spore species of the genus Lycospora. Lepidostrobus crassus was synonymised with L. sternbergii based on identical spore content and closely similar morphology of the type specimens of both species. Lepidostrobus nemejcii, L. thomasii and L. ronnaensis have been erected as new species according to the cone morphology, spore content and parent plant. Ecological constraints of all the species are discussed. Two groups of species preferring different habitats were distinguished. The first group consists of species that preferred clastic to mixed peat/clastic substrates and high water table. Their parent plants grew either in clastic swamps along the lake margins and shallows or in planar mires frequently disturbed by clastic input during floodings. This group involves most of the studied species and their parent plants. Differences between habitats of the species of this group are indicated but their precise specifications needs further investigation. The second group involves only L. ronnaensis and its parent plant Lepidodendron ophiurus ( sensu Němejc). It preferred peat substrate of planar eutrophic mires with only minor clastic disturbances. The palynological comparison with in situ lycospores from compressed and petrified Lepidostrobus cones is made.

Isolation of Bacillus pumilus from in vitro grapes as a long-term alcohol-surviving and rhizogenesis inducing covert endophyte.

To isolate and characterize the alcohol-surviving covert bacterium associated with grape tissue culture. Single colony was isolated by plating the spent rectified spirit used during grape culturing and the organism was identified as Bacillus pumilus using partial 16S rDNA sequence data. Spotting tests (1 microl) using 3-day-old broth culture having a spore content of 20-30% showed similar bacterial survival in 25-90% (v/v) aqueous ethanol for 14 days. Survival in 90% ethanol and 90% rectified spirit appeared affected thereafter with no colony growth from 1 microl samples after 4 months. Plating the samples at this stage gave similar CFU ml(-1) for 25, 50 and 70% ethanol, a significant reduction in 80% ethanol and very few colonies in 90% ethanol and rectified spirit. B. pumilus-inoculated grape microcuttings showed substantial endophytic colonization of original cuttings (7.4 x 10(6) CFU g(-1)) followed by the sprout (5.9 x 10(5)) and roots (2.0 x 10(4)). The bacterium although a poor root colonizer, induced early rooting and more roots in vitro. Inoculation at ex vitro planting resulted in significantly more roots, root weight and shoot growth. Bacillus pumilus could remain as a covert endophyte in grape tissue cultures and survive in aqueous ethanol for extended periods. 90% ethanol was the most effective bactericidal concentration. The bacterium showed endophytic colonization and root and shoot growth promotion. The revelation that general recommendation of 70-80% ethanol may not be the most effective bactericidal concentration for all bacteria, elucidation of the possibility of covert bacterial survival in vitro plant cultures and isolation of a potential plant growth promoting endophyte in grape.