Transfer Of Spores Research Articles

SUBRECENT POLLEN ASSEMBLAGES IN ALLUVIAL DEPOSITS OF THE DON AND KUBAN RIVERS AND THEIR SIGNIFICANCE FOR PALAEOGEOGRAPHIC RECONSTRUCTIONS

The paper presents the results of studying modern alluvial deposits of the Don and Kuban rivers by the pollen analysis method. The study was carried out to clarify methodological issue, including the movement of pollen and spores by flowing waters. The obtained data show that pollen assemblages of river sediments formed in the process of pollen mixing by water and air characterize the composition of the regional vegetation cover rather than local nearriver plant groups. According to the results for the subrecent sediments of the Don River the transfer of pollen grains and spores by water was insignificant, which can be clearly seen at the border of the forest-steppe and steppe zones. Pollen assemblages of the forest-steppe zone contain 50% of tree species pollen, while in the steppe zone, including its northern part, tree species make up less than 30-35%. Sporadic forest elements such as Picea pollen, Lycopodium species spores and Sphagnum mosses in alluvial deposits of the steppe section of the Don River, as well as in the subrecent sediments of the Sea of Azov, could be neglected in the palaeoreconstructions. The composition of subrecent pollen assemblages of the Kuban River sediments is less variable from the middle course to the mouth with rather stable ratio of components. Pollen of the Caucasian flora elements (Fagus orientalis, Carpinus caucasica) is poorly dispersed by water from high and midmountain landscapes of the upper reaches of the river. The comparison of pollen assemblages from alluvial and marine (the Sea of Azov) sediments have shown that the distortion of fossil assemblages’ composition due to material input by water is minimal

The Hospital Environment as a Potential Source for Clostridioides difficile Transmission Based on Spore Detection Surveys Conducted at Paediatric Oncology and Gastroenterology Units.

Clostridioides difficile is an anaerobic, Gram-positive bacterium widely present in the hospital environment due to its ability to generate spores. The transfer of spores to patients through the hands of medical personnel is one of the most frequent paths of C. difficile transmission. In paediatric patients burdened with a serious primary illness requiring long-term hospitalisation and antibiotic therapy, C. difficile may be a significant risk factor for antibiotic-associated diarrhoea. The goal of the study was to assess the state of hospital environments as a potential source of C. difficile spores and to establish the share of hyperepidemic strains at the two paediatric units. The survey for C. difficile was conducted with a C. diff Banana BrothTM medium, used to detect spores and to recover vegetative forms of the bacteria. Environmental samples (n = 86) and swabs from the clothing of medical personnel (n = 14) were collected at two units of a paediatric hospital, where the cases of antibiotic-associated diarrhoea with a C. difficile aetiology constitute a significant clinical problem. In 17 samples, a change in the broth's colour was observed, indicating the presence of spores. Out of seven samples, C. difficile strains were cultured. The pathogenic isolates of C. difficile were obtained from swabs collected from elements of beds, a toilet, a door handle and a doctor's uniform. In our study, we indicated points of increased risk of pathogen transmission, which could constitute a source of infection. The clothing of medical personnel may be a dangerous carrier of pathogenic spores. Periodical surveys of hospital environments with the use of specialist microbiological mediums successfully indicate the direction of corrective actions to be undertaken by the medical facility in order to increase patient safety.

Influence of Structure of Detrital Food Webs on Fusarium Head Blight of Winter Wheat

Conventional methods for Fusarium head blight (FHB) control are ineffective. A better understanding of the mechanisms linking the abundance of Fusarium species in soil before winter wheat flowering and mycotoxin content in mature grain may help to improve the effectiveness of methods for FHB control. In this study, we established a field experiment aimed to trace the impact of three types of organic mulch with different C:N ratios on the structure of detrital food webs and the manifestation of winter wheat FHB. T2-toxin content in grain was significantly higher in N-poor treatment (52.1 ± 0.2 µg g−1) compared to N-rich treatment (40.4 ± 1.6 µg g−1). The structure of detrital food webs in the studied treatments changed significantly after mulch addition; the abundance of soil saprophages and mycophages increased up to 50%. Based on the results of mixed-effects modeling, the abundance of herpetobionts and soil mesofauna were positively associated with an increase in Fusarium biomass in grain. The increase in the content of T2-toxin in the grain was associated with an increase in the abundance of earthworms and phytophagous macrofauna in the soil. Results suggest the existence of a previously undescribed mechanism for FHB infection by transfer of pathogenic spores by soil invertebrates, while the content of mycotoxins in grain can be triggered by the grazing activity of soil phytophagous invertebrates.

787. Evaluation of Clostridioides difficile Environmental Contamination Surrounding C. difficile Patients vs. non-C. difficile Patients in Outpatient Infusion Centers

Background Clostridioides difficile infection (CDI) is the most common cause of healthcare-associated infection. CDI and non-CDI patients (pts) are often treated at the same time in outpatient infusion centers (OICs). This proximity may allow horizontal transfer of spores. However, C. difficile (C. diff) spores are ubiquitous in nature and baseline contamination rates at OICs are unknown. The purpose of this pilot study was to determine toxigenic C. diff contamination in the OIC surrounding CDI pts receiving bezlotoxumab compared to non-CDI pts receiving another infusion in the same OIC before and after cleaning.MethodsOIC contamination rates were assessed at baseline, after infusion and after cleaning the environment of CDI pts receiving bezlotoxumab compared to non-CDI pts receiving other infusions. For each pt receiving an infusion, 11 areas were sampled at each time period; the infusion chair (n=4), medical and non-medical equipment (n=3), and the floor surrounding the infusion chair (n=4). Five high traffic control areas per sampling day were included. Swabs were cultured anaerobically and PCR was used to identify toxin genes. Proportion of toxigenic C. diff positive samples were compared between CDI and non-CDI pts for each time point. Cleaning was performed using a standard protocol of bleach (CDI pt) or non-bleach (non-CDI pt) products.ResultsSamples (n=709) were obtained from 10 pts in each group (329 CDI, 330 non-CDI, 50 high-traffic) from 7 OICs over 4 months. Overall, 55 patient area cultures (8%) were positive for C. diff. Positive sampling areas were highest for floors (13%) followed by infusion chairs (7%) and equipment (4%). Baseline contamination in high traffic areas was 6%. Contamination rates (Table 1) for CDI were 7% at baseline, higher after infusion (15%) and lower after cleaning (5%). For non-CDI pts, rates were similar at baseline (8%), after infusion (6%) and after cleaning (9%).Table 1. Proportion of toxigenic C. difficile-positive samples in the environment of CDI and non-CDI patients. ConclusionCompared to non-CDI pts, CDI pts had similar baseline but lower after cleaning contamination rates. These preliminary results suggest that with a proper cleaning protocol in place, the presence of CDI patients in an OIC does not increase the likelihood of C. diff transmission for other at-risk populations.Disclosures Kevin W. Garey, PharmD, MS, FASHP, Merck & Co. (Grant/Research Support, Scientific Research Study Investigator) Richard L. Hengel, MD, Merck & Co. (Other Financial or Material Support, Grant Steering Committee Member) Timothy E. Ritter, MD, Merck & Co. (Other Financial or Material Support, Grant Steering Committee Member) Ramesh V. Nathan, MD, FIDSA, Merck & Co. (Other Financial or Material Support, Grant Steering Committee Member) Engels N. Obi, PhD, Merck & Co. (Employee) Lucinda J. Van Anglen, PharmD, Merck & Co. (Grant/Research Support)

Sampling and inactivation of wet disseminated spores from flooring materials, using commercially available robotic vacuum cleaners.

Four commercially available robotic vacuum cleaners were assessed for sampling efficiency of wet disseminated Bacillus atrophaeus spores on carpet, polyvinyl chloride (PVC) and laminate flooring. Furthermore, their operability was evaluated and decontamination efficiency of one robot was assessed, using a sodium hypochlorite solution. In an environmental chamber, robots self-navigated around 4m2 of flooring containing a single contaminated 0·25m2 tile (c.104 spores per cm2 ). Contamination levels at predetermined locations were assessed by macrofoam swabs (PVC and laminate) or water soluble tape (carpet), before and after sampling. Robots were dismantled postsampling and spore recoveries assessed. Aerosol contamination was also measured during sampling. Robot sampling efficiencies were variable, however, robots recovered most spores from laminate (up to 17·1%), then PVC and lastly the carpet. All robots spread contamination from the 'hotspot' (all robots spread <0·6% of the contamination to other areas) and became surface contaminated. Spores were detected at low levels during air sampling (<5·6 spores per litre). Liquid decontamination inactivated 99·1% of spores from PVC. Robotic vacuum cleaners show promise for both sampling and initial decontamination of indoor flooring. In the event of a bioterror incident, e.g. deliberate release of Bacillus anthracis spores, areas require sampling to determine the magnitude and extent of contamination, and to establish decontamination efficacy. In this study, we investigate robotic sampling methods against high concentrations of bacterial spores applied by wet deposition to different floorings, contamination spread to other areas, potential transfer of spores to the operators and assessment of a wet vacuum robot for spore inactivation. The robots' usability was evaluated and how they can be employed in real life scenarios. This will help to reduce the economic cost of sampling and the risk to sampling/decontamination teams.

Transfer and Reaerosolization of Biological Contaminant following Field Technician Servicing of an Aerosol Sampler

Over the last several years, aerosol samplers have been fielded in many locations to collect biological agents in the air, providing a sample that, once analyzed, will alert safety and public health officials of potential bioterrorism events. If a biological agent was present at the sampling location, the collector and surrounding area may be contaminated due to bioaerosol deposition, possibly posing a hazard to the technician maintaining the aerosol sampler. The technician may, in turn, serve as a source for cross-contamination to clean areas subsequently visited, potentially producing a hazard to others if transferred to indoor settings, such as a job site or analysis laboratory. To investigate our hypothesis about these potential exposure sources and cross-contamination, a study was performed to: (1) examine biological material transfer from a contaminated site to an individual; and (2) determine aerosol resuspension levels due to typical personnel activity at a contaminated, paved bioaerosol sampling site. Analysis of air samples indicated reaerosolization of spore-containing particles upon disturbance of a contaminated site by a field technician, and analysis of swatches taken from the technician’s clothing indicated substantial transfer of spores. These results provide insight into sources of cross-contamination and potential steps to mitigate consequences of infectious contaminant transfer, and also demonstrate potential exposure hazards for technicians servicing fielded bioaerosol collectors.

Asexual reproduction induces a rapid and permanent loss of sexual reproduction capacity in the rice fungal pathogen Magnaporthe oryzae: results of in vitroexperimental evolution assays

BackgroundSexual reproduction is common in eukaryotic microorganisms, with few species reproducing exclusively asexually. However, in some organisms, such as fungi, asexual reproduction alternates with episodic sexual reproduction events. Fungi are thus appropriate organisms for studies of the reasons for the selection of sexuality or clonality and of the mechanisms underlying this selection. Magnaporthe oryzae, an Ascomycete causing blast disease on rice, reproduces mostly asexually in natura. Sexual reproduction is possible in vitro and requires (i) two strains of opposite mating types including (ii) at least one female-fertile strain (i.e. a strain able to produce perithecia, the female organs in which meiosis occurs). Female-fertile strains are found only in limited areas of Asia, in which evidence for contemporary recombination has recently been obtained. We induced the forced evolution of four Chinese female-fertile strains in vitro by the weekly transfer of asexual spores (conidia) between Petri dishes. We aimed to determine whether female fertility was rapidly lost in the absence of sexual reproduction and whether this loss was controlled genetically or epigenetically.ResultsAll the strains became female-sterile after 10 to 19 rounds of selection under asexual conditions. As no single-spore isolation was carried out, the observed decrease in the production of perithecia reflected the emergence and the invasion of female-sterile mutants. The female-sterile phenotype segregated in the offspring of crosses between female-sterile evolved strains and female-fertile wild-type strains. This segregation was maintained in the second generation in backcrosses. Female-sterile evolved strains were subjected to several stresses, but none induced the restoration of female fertility. This loss of fertility was therefore probably due to genetic rather than epigenetic mechanisms. In competition experiments, female-sterile mutants produced similar numbers of viable conidia to wild-type strains, but released them more efficiently. This advantage may account for the invasion of our populations by female-sterile mutants.ConclusionsWe show for the first time that, in the absence of sexual reproduction, female-sterile mutants of M. oryzae rice strains can arise and increase in abundance in asexual generations. This change in phenotype was frequent and probably caused by mutation. These results suggest that female fertility may have been lost rapidly during the dispersion of the fungus from Asia to the rest of the world.

Viability of Bacillus licheniformis and Bacillus thuringiensis Spores as a Model for Predicting the Fate of Bacillus anthracis Spores during Composting of Dead Livestock

Safe disposal of dead livestock and contaminated manure is essential for the effective control of infectious disease outbreaks. Composting has been shown to be an effective method of disposal, but no information exists on its ability to contain diseases caused by spore-forming bacteria, such as Bacillus anthracis. Duplicate composters (east and west), each containing 16 dead cattle, were constructed (final capacity, 85,000 kg). Spores (10(7) CFU/g manure) of Bacillus licheniformis and Bacillus thuringiensis were mixed with autoclaved feedlot manure and placed in either sterile vials or porous nylon bags. Compost temperatures in the west composter were slightly higher than in the east composter. Viable B. thuringiensis spores were reduced to ≤10(2) CFU in all samples after 112 days but were isolated from bags (west composter) at ≤10(2) and at 10(5) CFU (east composter) after 230 days. In contrast, B. licheniformis was at ≤10(2) CFU in vials (west composter) after 112 days but remained at 10(6) CFU after 230 days (east composter). Similarly, B. licheniformis in bags was not detected after 230 days in the west composter but remained at 10(7) CFU in the east composter. Our study suggests that spore viability was reduced in the west composter by exposure to compost and elevated temperatures over time. Different temperature profiles may explain why spores remained viable in the east structure but were largely rendered nonviable in the west structure. Under practical conditions, variation in composting microclimates may preclude the complete inactivation of Bacillus spores, including those of B. anthracis, during composting. However, composting may still have merit as a method of biocontainment, reducing and diluting the transfer of infectious spores into the environment.

Strain-specific colonization pattern of Rhizoctonia antagonists in the root system of sugar beet

To develop effective biocontrol strategies, basic knowledge of plant growth promotion (PGP) and root colonization by antagonists is essential. The survival and colonization patterns of five different biocontrol agents against Rhizoctonia solani AG2-2IIIB in the rhizosphere of greenhouse-grown sugar beet plants were analysed in single and combined treatments. The study included bacteria (Pseudomonas fluorescens L13-6-12, Pseudomonas trivialis RE(*) 1-1-14, Serratia plymuthica 3Re4-18) as well as fungi (Trichoderma gamsii AT1-2-4, Trichoderma velutinum G1/8). Microscopic analysis by confocal laser scanning microscopy revealed different colonization patterns for each DsRed2/green fluorescent protein-labelled strain. Bacteria and T. velutinum G1/8 colonized the root surface and the endorhiza in single and co-culture, while for T. gamsii AT1-2-4, only the transfer of spores was observed. Whereas Pseudomonas strains formed large microcolonies consisting of hundreds of cells, S. plymuthica was arranged in small endophytic clusters or clouds around the entire root system. In co-culture, each strain showed its typical pattern and occupied specific niches on the root, without clear evidence of morphological interactions. PGP was only observed for four strains with rhizosphere competence and not for T. gamsii AT1-2-4. The results provide useful information on which combination of strains to test in larger biocontrol experiments directed to applications.

Control of &lt;i&gt;Tribolium confusum&lt;/i&gt; J. du val by diatomaceous earth (Protect It™) on stored groundnut (&lt;i&gt;Arachis hypogaea&lt;/i&gt;) and aspergillus flavus link spore dispersal

Environmental and human health problems associated with the use of synthetic pesticides have prompted the demand for non-polluting, biologically specific insecticides. The current study assessed the losses caused by Tribolium confusum and its control by diatomaceous earth and the effect on Aspergillus flavus spore dispersal during storage of groundnuts. When losses due to Tribolium confusum were assessed over a period of 60 days, it was found that an increase in insect population in stored groundnuts resulted in increased weight loss of stored groundnuts. The true weight loss due to insect feeding was 0.60 g per 400 g of stored groundnuts. When diatomaceous earth (DE) was applied to groundnuts followed by the introduction of insects in a compartment (A), increased mortality of insects with increased diatomaceous earth concentration was observed. For a concentration range of 0-2.5 g DE/kg groundnut, 2.5 g/kg treatment was the most effective (only 5 surviving T. confusum adults out of 50 were recovered in samples treated with 2.5 g/kg compared to 38 adults in the control samples). Larval emergence from groundnuts treated with DE also declined with increased diatomaceous earth concentration. When groundnuts were inoculated with A. flavus spores, followed by DE application and T. confusum introduction into compartment A, the transfer of spores between inoculated groundnut samples in compartment A and uninoculated samples in compartment B was reduced. The mean A. flavus spore concentration recovered from initially sterile compartment B was1.08x103; while it was 45 in the control and in samples treated with 2.5 g/kg dosage respectively. There was a significant difference in the mean numbers of spores recovered from groundnuts in different compartments (A, B) (H = 13.99, df = 4 and P = 0.007). Thus, from this study, losses due to T. confusum on groundnuts and fungal spore transfer in storage by this insect can be minimized by the application of diatomaceous earth (Protect-ItO) to stored groundnuts. Key words: Groundnut, Tribolium confusum , Diatomaceous earth

Competition with filamentous fungi and its implication for a gregarious lifestyle in insects living on ephemeral resources

Abstract. 1. Recent studies have demonstrated the existence of positive density dependence in the survival and development ofDrosophila(the so‐called Allee effect); however the underlying mechanisms of such Allee effects have remained elusive. Competition with filamentous fungi have often been suggested to be involved in causing high mortality at low larval density, but it has not yet been explicitly tested if the well known spatial aggregation of insect eggs yields a fitness benefit for the developing larvae in the presence of noxious moulds.2. UsingDrosophila melanogaster, the present study tested whether larval survival is greater in aggregations when confronted with various combinations of three representative mould species (Aspergillus,Alternaria, andPenicillium) and a head start for fungal development.3. High rates of fungal‐dependent mortality and significant positive density‐dependent larval survival (i.e. Allee effects) were observed when larvae were confronted with food resources containing established colonies ofAspergillusorAlternaria. Neither the simultaneous transfer ofAspergillusorAlternariaspores with larvae to food patches nor food infections withPenicilliumaffected insect larval development.4. Significant correlations between mould growth and larval survival could be identified, although the patterns that emerged were shown to be inconsistent when the effects were compared between fungal species and fungal priority. Because mould growth only partly explained larval survival, the influence of other fungal‐borne factors, e.g. mycotoxins, needs to be elucidated in order to understand the mechanistic basis of insect–mould interactions.5. These results are the first to argue convincingly for moulds being involved in mediating Allee effects for insects on ephemeral resources; however they also demonstrate an unexpected diversity in insect–mould interactions. Considering this diversity may be important in understanding insect spatial ecology.

Indicators to assess biological diversity: Weyerhaeuser's coastal British Columbia forest project

Adaptive management is a key component of a forest project being implemented across all of Weyerhaeuser's coastal forest tenures. This project uses two main tools to accomplish the British Columbia (BC) Coastal Group's ecological and socio-economic goals: variable retention (VR) harvesting and broad zoning of the land base. The adaptive management program was designed to examine the effectiveness of retention systems and zoning in maintaining those forest attributes necessary to sustain biological richness and essential ecosystem functions, such as nutrient transfer, energy flow, decomposition, and dispersal of seeds, spores, and animals. The program is grounded on three biological indicators evaluated in both operational and experimental contexts: 1) representation of habitat types in a relatively unmanaged state to ensure that little-known species are retained; 2) structure of stands and landscapes to ensure that key elements are present through time; and 3) indicator organisms to track whether retaining structures and patterns, while addressing representation, will maintain species and populations whose life needs are well understood. Representation of ecosystems in unmanaged conditions has been examined. Habitat structure is being assessed in VR blocks and in unmanaged blocks. Studies on several organisms (breeding birds, owls, gastropods, amphibians, bryophytes, lichen, squirrels, mycorrhizae, and carabid beetles) have been underway for various lengths of time. These studies collected baseline information to begin comparisons of the effectiveness of the various types of VR for maintaining biological richness. The current focus in the adaptive management program is refining the lists of specific elements to monitor and beginning to create tools to help extrapolate relationships and findings over large areas and long time frames, with the expectation of operational implementation in 2003. The first two years of pilot work are also being used to examine how the results will link to management practices to strengthen areas that most need improvement. This paper describes three indicators used in Weyerhaeuser's adaptive management program. Key words: adaptive management, variable retention harvesting, indicators for monitoring forests

Susceptibility of Vespula vulgaris (Hymenoptera: Vespidae) to Generalist Entomopathogenic Fungi and Their Potential for Wasp Control

The pathogenicity of Vespula vulgaris wasp workers and larvae to a range of fungi was determined. All fungi were isolated in New Zealand and included isolates from Vespula, known generalist insect pathogens, and isolates generally nonpathogenic to insects. Workers and larvae were highly susceptible to pathogenic isolates at high spore concentrations (>1.75 × 105 cfu/individual). Eight isolates, two of Metarhizium anisopliae, five of Beauveria bassiana, and one of Aspergillus flavus were pathogenic while a single isolate of M. flavouiride var. novazealandicum, Cladosporium sp., and Paecilomyces sp. were not. The transfer of spores between workers, and between workers and larvae, was also investigated using several different application methods. Transfer of spores occurred between treated and untreated individuals, and for some of the application methods sufficient spores were transferred to cause mortality of the nontreated individuals. These findings are related to the potential of fungi for the control of wasps.

The track structures of ionizing particles and their application to radiation biophysics. I. A new analytical method for investigating two biophysical models.

A new approach to the interpretation of the effects of radiation on cells is described, in which sample particle tracks are constructed using a Monte Carlo computer program and the exposure of cellular targets to these tracks is simulated using a second program known as BIOPHYS. Data on the shapes and DNA contents of the cell nuclei are obtained from the literature. It is assumed that the sensitive material is DNA, and that the target is divided into cubes of approximately 2 nm (the diameter of the DNA helix) per side; the numbers of these cubes containing different numbers of ionizations are derived. Two different methods of analysing the output of BIOPHYS are described. In the first, it is assumed that lethality is caused by the occurrence of a number of ionizations equal to or greater than a certain threshold in one cube; in the second method, it is assumed that only two ionizations are required, in different parts of the cube, but that only some fraction of the cube is sensitive. These models have been applied to the interpretation of the variation of radiosensitivity with a linear energy transfer (LET) of spores of Bacillus subtilis exposed wet and dry, and good fits to the published experimental data were obtained using both models. Fits to experimental data for a range of other cell lines will be presented in a second paper.

Ethylene requirement during germination of Botrytis cinerea spores

Application of 2,5‐norbornadiene, a competitive inhibitor of ethylene, effectively inhibited the germination of Botrytis cinerea Pers. ex Fr. spores. The transfer of spores from 2,5‐norbornadiene to air relieved inhibition by norbornadiene, indicating that its effects are non‐toxic and reversible. Ethephon (2‐chloroethylophosphonic acid), which stimulates spore germination of B. cinerea, does not affect germination in the presence of norbornadiene. However, ethephon appeared to be effective in relieving inhibition, when norbornadiene was removed from the atmosphere surrounding spores. The addition of ethylene to an atmosphere enriched with norbornadiene, counteracted the inhibition of spore germination. The inhibition of spore germination by 2,5‐norbornadiene and the reversal of this effect by ethephon or ethylene, indicate that the action of ethylene is indispensable for germination of B. cinerea spores.

Transfer of spores, bacteria and yeast into toluene containing phospholipids and low amounts of water: Preservation of the bacterial respiratory chain

A method that allows the transfer of spores, bacteria and yeasts into a ternary system composed of toluene, phospholipids and low amounts of water is described. Initially an emulsion is formed by sonication of cells suspended in water in presence of toluene and phospholipids. The emulsion formed was subsequently clarified by blowing N2 on its surface and transparency was achieved when the water content of the system was reduced to 1-3 microliter/ml of organic solvent. The cells in ternary systems exhibit a reduction of cell volume, but the general structure is preserved. About 1/10,000 bacilli or yeast were viable, but spores were viable after 30 days in the ternary system. Yeast cells transferred to the ternary system, and back to an all water media failed to show oxygen uptake. In contrast bacilli that remained in the ternary system for 2 days respired to 80% of their maximal capacity when returned to an aqueous media.

Comparison of three media used for the enumeration of heat-injured Botrytis cinerea

The efficacy of three media (antibiotic-supplemented plate count agar (3 days, 21 °C); antibiotic–pyruvate–supplemented plate count agar (3 days, 21 °C); and trypticase soy agar (16 h, followed by 2.5 days on antibiotic-supplemented plate count agar, 21 °C)) for recovery of heat-injured Botrytis cinerea spores was compared using hydrophobic grid membrane filters. The filters restrict spreading of fungal colonies and facilitate the transfer of fungal spores from pre-enrichment trypticase soy agar to antibiotic-supplemented plate count agar. Counts obtained from pre-enrichment on trypticase soy agar were significantly larger than counts obtained from the other media (α = 0.05). Addition of sodium pyruvate to antibiotic-supplemented plate count agar did not increase spore recovery (α = 0.05). The use of trypticase soy agar as a pre-enrichment medium should be considered for use when antibiotic-supplemented plate count agar is employed in the enumeration of heat-stressed fungi.

A Method to Use Lichen Spores in Quantitative Studies on Germination

Lichen spores are discharged onto a parafilm sheet, thoroughly mixed in water, dried, stored, and returned at will into an aqueous suspension to be distributed homogeneously on a culture surface. The randomness of distribution has been verified, allowing the calculation of a confidence interval for the percentage of germinated spores according to the law of binomial distribution. Spores of Xanthoria parietina and Physcia pulverulenta stored on parafilm retain their germinative ability at least one month. The spores of lichen fungi have rarely been used in quantitative experiments on germination because it is difficult to obtain homogeneous samples of such spores. The classical method of collecting them aseptically on an agar nutrient medium has been described by Werner (1927): An agar plate is placed above moistened apothecia a short distance from the epithecium and the spores that are ejected upwards usually adhere to the agar. For monospore cultures, one spore can then be removed with a glass needle and transferred to another agar plate. But for statistical investigations, such as those concerning the average rate of germination, individual transfer of spores is not practical. Germination must be observed directly on the original plate and the drawbacks are the following: a) The spores discharged by one ascus usually remain packed together so that the beginning of germination is often difficult to detect. Later on, especially for spores giving rise to more than one filament, it is even more difficult to see to which spores the hyphae belong. b) Some areas are densely crowded with spores (above the apothecia) while others are nearly devoid of them. Up to now, group effects, commonly described for other kinds of spores, have not been noticed in lichens but they may exist in some cases and it is better to avoid them if possible. c) Spores ejected from different apothecia, and most of all from different thalli (even if these have been collected near to each other), rarely have the same germinative capacity. Therefore, such a distribution of spores is far from random and it becomes necessary to deal with large numbers of spores and to make many replicate experiments to obtain significant results. An improvement of the method is thus required. Scattering spores by gently brushing the surface of the medium with a thin brush should overcome the first difficulties (a and b). However, this procedure is not entirely efficient because it does not suppress the heterogeneity (c) between populations from different culture dishes. The best solution is to mix the spores thoroughly before sowing them and then to scatter them as well as possible. Contrary to other kinds of spores (mosses, ferns), lichen spores are extremely I thank the Institute of Applied Mathematics of Grenoble for providing probability tables of the binomial distribution. I am also grateful to Dr. F. LeBlanc for revising the English text. 2 Laboratoire de Physiologie V~g6tale, Universit6 de Grenoble, Grenoble, France. This content downloaded from 207.46.13.163 on Fri, 08 Jul 2016 05:38:40 UTC All use subject to http://about.jstor.org/terms 1970] KOFLER: LICHEN SPORE GERMINATION 603 hydrophilic. They may adhere to glass surfaces, especially if they have been allowed to dry out. Water itself remains partly on such polar surfaces when drops are rolled across or when it is pipetted. Consequently a smooth hydrophobic surface was looked for, one onto which spores could be ejected and from which these could be easily collected in a suitable liquid. Parafilm, which is commonly used to cap culture vials, was found entirely suitable for this purpose.

THE ECOLOGY AND PHYSIOLOGY OF SOIL FUNGI INVOLVED IN THE DEGRADATION OF LIGNIN AND RELATED AROMATIC COMPOUNDS

SummaryA study has been made of the colonization by soil fungi of kaolin pellets in which was incorporated either a lignin preparation extracted from Phragmites or the lignin‐related substances, vanillic acid or α‐conidendrol. Fungi isolated from the lignin pellets by the dilution method were grown in liquid culture containing the Phragmites lignin. Subsequent analysis by UV absorption spectrometry indicated up to 40 per cent loss of this material. IR analysis of the residual lignin showed that it was little altered.Fungi from the pellets containing vanillic acid and α‐conidendrol were isolated by direct transfer of spores and mycelium from the pellets to agar plates. The range of soil fungi already known to metabolize vanillic acid in pure culture was extended by this method and included Stilbum spp., Humicola sp., and two unidentified species. Dry‐weight determinations of mycelia combined with UV absorption spectrometry and paper chromatography of culture filtrates indicated that, with the exception of Acremoniella sp., all isolates metabolized vanillic acid when it was supplied as sole C source. Growth‐promoting substances present in yeast extract were required by Volutella spp. before the vanillic acid was metabolized.The pellet technique offers many possibilities for studying the microbiological degradation of soil substances and in addition enables the soil micro‐habitats to be explored in greater detail than has previously been possible.

The Viability of Cultures of Rhizopus nigricans

From time to time, various investigators have given data, either directly or indirectly, which provide some information concerning a possible time period during which cultures or spores of fungi may retain their viability. Povah (2) has shown that cultures of fungi which fail to grow when transferred to fresh agar slants may do so when given a certain hot agar treatment. This proves that the longevity of a culture is not necessarily correlated with the success or failure to obtain a subculture by means of a simple transfer of spores or mycelium, and therefore the latter condition should not be used as a criterion of the lack