Microbial introductions to apple leaves: Influences of altered immigration on fungal community dynamics

Fungal immigration to apple leaves in the field was altered by the introduction of populations ofChaetomium globosum orAureobasidium pullulans to surface-disinfested leaves either immediately following, or 6 days after, disinfestation. Total numbers of fungal individuals and numbers of filamentous fungal and yeast individuals were estimated and compared over time for 4–7 weeks on control leaves (leaves disinfested but no populations applied), onAureobasidium-treated, and onChaetomium-treated leaves. Fungal communities developing on leaves during three experiments in two different time frames (experiment 1: July 9–August 27; experiments 2 and 3: July 29–August 27), and thus under different immigration regimes, were also compared. Survival of introduced populations was not related to the presence of prior fungal immigrants. Rates of increase in total numbers of fungi and numbers of filamentous fungi and yeasts per leaf varied among experiments, apparently in relation to differences in immigration and environmental history. Differences among leaves in immigration had a short-term (days) influence on community size. However, no long-term effects of altered immigration on phylloplane fungal community size were evident.     

Fungal immigration dynamics and community development on apple leaves

Fungal immigration dynamics and community development were followed over time on sets of surface-disinfested apple leaves in the field. Immigration was defined as the arrival of viable propagules on the leaf surface. In three separate experiments (May, June, July), total numbers of fungal immigrants, numbers of filamentous fungal immigrants, and numbers of yeast immigrants per leaf were estimated for successive 12-hour immigration periods. Communities developing over 2–14 immigration periods (1–7 days) were compared with the corresponding estimates of cumulative immigration. There were significant differences among both experiments and immigration periods within each experiment in mean numbers of immigrants per leaf. Leaf area was often significantly correlated with numbers of immigrants. Developing communities supported progressively fewer individuals than the corresponding sums of immigrants, suggesting that losses due to emigration and/or death play a critical role in shaping these communities.     

Dichloran as an Inhibitor of Mold Spreading in Fungal Plating Media: Effects on Colony Diameter and Enumeration

Problems associated with overgrowth by spreading molds are not addressed by currently recommended fungal enumeration media. Twenty-two fungi, including 12 mold and 3 yeast genera, were evaluated for the effects of dichloran (2,6-dichloro-4-nitroaniline), previously identified as a mold-spreading inhibitor, on colony diameter and enumeration. On malt agar or antibiotic-potato-dextrose agar (APDA), colony diameters were effectively reduced when dichloran was added. Colony diameters decreased as the dichloran concentration increased. Counts obtained with mixed mold spore suspensions were lower on APDA supplemented with 25 μg of dichloran per ml than on APDA and were higher than APDA with the addition of 5 μg of dichloran per ml (APDA-D-5). Overall counts of mixed and individual mold spore and yeast suspensions were higher in APDA-D-5 than in APDA. The additional advantages of APDA-D-5 may be useful in routine enumeration of fungi.     

Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 × 10⁶ spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48°C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28°C.     

A ten year study of the incidence of spores of bracken, (Pteridium aquilinum (L.) Kuhn.) in an urban rooftop airstream in south east Scotland

Field studies of bracken during sporing haveindicated very high numbers of sporesreleased per cubic metre of air. Thecarcinogenic nature of bracken spores poses ahealth hazard to workers and visitors in areasof sporing strands.A retrospective study of bracken spores trappedon slides exposed during Augustand September over the ten year period, 1988–1997, on a rooftop site in Edinburgh,has added an urban dimension to the aerobiologyof bracken.Results have revealed, apart from 1989,consistently low counts. The spore samplerwas situated downwind approximately 1.6 km fromthe nearest sporing stand.A spore calendar, based on the ten years ofcumulative data, has been constructed.     

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10⁵ and 10⁷ spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 × 10⁷ to 2 × 10⁷ spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.     

Life history and reproductive potential of the agarophyte Gelidium robustum in California

The reproductive potential of the tetrasporangial phase of Gelidium robustum was studied for 16 months at two sites off Santa Barbara, California. In all samples tetrasporangial thalli were always more abundant than gametangial ones. Tetratrasporangial sori were present throughout the duration of the study but relative fecundity was highest [300–400 sori g^–1 (w. wt)] in spring/summer samples of consecutive years, as a result of increasing numbers both of tetrasporangial branchlets per plant and of sori per branchlet. On the other hand, laboratory experiments showed that tetraspore release per sorus was highest (150–250 spores sorus^–1 d^–1) in winter. Inferring from these field and laboratory data plants released up to ± 34 000 tetraspores g^–1 (w. wt) d^–1 in the spring/summer of the second study year. Tetraspore germination, under defined culture conditions, also showed a marked seasonality increasing sharply from less than 10% in winter up to almost 60% in spring/summer, thus coinciding with the period of maximal spore output per plant. These results suggest that although relatively high numbers of tetraspores may be released by G. robustum plants all year round these might not always have the potential to germinate and recruit.     

Effects of long-term NP-fertilization on abundance and diversity of arbuscular mycorrhizal fungi under a maize cropping system

Diversity of arbuscular mycorrhizal fungi (AMF) in 27-year long-term NP-fertilization plots under a maize cropping system in Thailand was studied through spore morphological characterization. The plots received 0–0, 60–60, 120–120 and 180–180 kg N-P₂O₅ ha^–1 year^–1 as ammonium sulfate and triple superphosphate. The plots were sampled monthly for one year, the AMF spores were counted and morphotyped, and taxa were identified after morphotyping and monospecific pot culture. Spore number g^–1 soil, relative spore abundance and Shannon-Wiener indexes were calculated. Sixteen putative taxa were recorded from the field of which nine sporulated on maize roots in pot culture. The long-term fertilization caused decreases in AMF total spore numbers and variation in species diversity depended on sampling time. Effects of fertilization on spore number and also relative spore abundance varied with species and sampling time. Among the nine species sporulating under maize, only Acaulospora sp.1 showed no change (P > 0.003 after Bonferroni correction) in spore number with fertilization in the field; and was therefore classified as an AMF species insensitive to fertilization. Spores of Entrophospora schenckii, Glomus mosseae, Glomus sp.1, Glomus geosporum-like and Scutellospora fulgida, though they decreased in absolute numbers in response to fertilization, showed no change (P > 0.003 after Bonferroni correction) in relative abundance; these species were classified as AMF species slightly sensitive to fertilization. Three unidentified species of Glomus, though they decreased in absolute numbers in response to fertilization, showed decreases (P < 0.003 after Bonferroni correction) in relative abundance; these species were classified as AMF species highly sensitive to fertilization.     

An improved method for rearing Halotydeus destructor (Acari: Penthaleidae) in the laboratory

We developed an improved method for rearing Halotydeus destructor in the laboratory. Mite numbers increased rapidly over the summer of 1992–1993 when fed on vetch, Vicia sativa cv Blanchefleur; in contrast mite numbers were low and fell over the second half of the summer of 1990–91 when fed on subterranean clover, Trifolium subterraneum cv Junee. On V. sativa at a fluctuating temperature from 11 to 18°C 200 mites produced a mean of 1308 and 1455 progeny per transfer in 2 years, a 6–7-fold rate of increase per transfer. The mean transfer time was 35 days and using the interval between transfers as a measure of generation time, the mites completed six to seven generations with no evidence for development of diapause eggs from August until April. There was considerable variability in the numbers of mites produced per pot, associated in part with the presence of Verticillium sp. fungus in 35% of the pots during transfers. Improvement in H. destructor rearing resulted from the use of V. sativa cv Blanchefleur as a food source, maintenance of high humidity and adequate ventilation within cages, and the transfer of mites to fresh food sources at early nymphal stages, which reduced the spread of fungal infections.     

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

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

Marine bacterioplankton at the Weddell Sea ice edge,distribution of psychrophilic and psychrotrophic populations

Summary In the eastern Weddell Sea on several transects from ice-covered, through ice melt, to open-ocean stations, total and heterotrophic bacteria were estimated to document an enhanced bacteriological biomass expected near the ice edge. The highest numbers of bacteria were found in melted ice cores, with 4.2·10³ CFUml^–1 and 1.1·10⁷ Cells ml^–1. Although brine from pore water samples average more than one order of magnitude less cells per ml, the highest bacterial production, 2.2·10⁷ cells l^–1 day^–1, was recorded in brine samples. All quantitatively studied bacterial parameters were lower under the ice than in the ice samples but there were no clear vertical gradients in the water column. In the studied spring situation, sea ice occurrence seems to play only a minor role in the general distribution of the seawater bacterioplankton. The bacterial community structure was investigated by carrying out 29 morphological and biochemical tests on 118 isolated strains. The bacterial communities inhabiting Antarctic pack ice differ from those found in underlying seawater. Although non fermentative Gram-negative rods were always dominant in seawater, Vibrio sp. represented more than 25% of the strains isolated from some ice samples. The results clearly indicated that a large majority of the bacteria isolated from seawater must be considered psychrotrophic but that truly psychrophilic strains occurred in melted ice and brine samples.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

A habitat colonisation model for spore-dispersed organisms—Does it work with eumycetozoans?

Spore productivities and establishment probabilities of eumycetozoans were estimated and compared with quantitative data obtained from field surveys, using series of cultures of a given substrate. Spore numbers per spore case were found to increase from one to four in protostelids to up to 10⁵–10⁶ in myxomycetes, whereas average spore size decreased slightly from 14.8 μm for protostelids to 10.3 μm in myxomycetes. Spore numbers of fructifications calculated from dimensions of spores and fruit bodies were in good agreement with direct counts carried out for six species of myxomycetes. A colonisation model is presented that estimates frequencies (as a percent of successfully colonized habitat islands), which is independent of a given density of spore rain and the sexual system of the species being considered. Whereas asexual species need a minimum spore rain of ca 0.7 spores per habitat island to reach a frequency of 50 %, this figure is at least 2.4-fold higher for sexual species, depending from the incompatibility system assumed. Data from cultures indicate that the maximum potential spore rain is usually three orders of magnitude higher than the minimum figure required to create the observed frequencies. Eumycetozoans seem to follow the evolutionary trends predicted by the model. Species with sexual reproductive systems produce often more spores than asexual ones; many morphospecies have sexual and asexual strains; and back-conversion from sexual to asexual reproduction occurs occasionally.     

Production of Aseptic Spores of Vesicular-Arbuscular Endophytes and Their Viability After Chemical and Physical Stress

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30°C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30°C for Glomus caledonius or at 35°C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 μg of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 μg/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40°C for 80 min, 55°C for 10 min, and 60°C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 × 10⁸ ergs cm⁻² from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.     

Polycell-gel assay of water for spores of Saprolegniaceae (fungi), especially those of the Saprolegnia pathogen of fish

Large aliquot water samples (30 ml) were enriched with nutrients, then gelled in the cold with Polycell (wallpapering paste) and incubated to derive Saprolegniaceae colonies and hence deduced spore counts, from water collections of about 1 litre. The Saprolegnia pathogen of fish was a component of the total recoveries; it was recognised on the basis of its secondary zoospore cyst ornamentation. Windermere water entering The Ferry House fish hatchery gave spore assays of 2 to 28 1^–1 for the pathogen, a significant component of total Saprolegnia, which was 6–73 spores per litre.     

Wood biodeterioration control potential of Acalypha hispida leaf phenolic extract in combination with Trichoderma viride culture filtrate

The phenolic extract of Acalypha leaves inhibited growth of Gloeophyllum sepiarium and Pleurotus sp. (test wood-rot fungi) in potato dextrose agar, starch agar, starch glucose agar, carboxyl methyl cellulose agar and carboxyl methyl cellulose glucose agar. Fungicidal or fungistatic concentration of the extract (10–14 mg/ml) depended on the medium. However a lower concentration of the extract (8–10 mg/ml) in combination with Trichoderma viride culture filtrate caused a similar inhibitory pattern. Degradation of obeche (Triplochiton scleroxylon), mahogany (Khaya ivorensis) and walnut (Lovoa trichilioides) by the test fungi was limited or prevented by extract treatment of 8–10 mg/g wood. A similar inhibitory effect again occurred when a combination of T. viride filtrate and lower extract concentration (6–8 mg extract per gram of wood) was used. On-going wood decay was limited or halted by a combined treatment involving 8–12 mg extract per gram of wood depending on the fungal residence period. Treated stakes exposed to 6 months of tropical wet season retained resistance to fungal attack including soft rot. The phenolic extract of A. hispida may prove useful in an integrated chemical and biological approach to wood treatment.     

Susceptibility of the cattle tick Boophilus microplus (Acari: Ixodidae) to isolates of the fungus Beauveria bassiana

The susceptibility of the tick Boophilus microplus to Beauveria bassiana was evaluated by inoculating eggs, larvae and engorged females of the tick with five fungal isolates at concentrations of 10⁶, 10⁷ and 10⁸ conidia/ml. Tick eggs (0.25 g) were immersed in 1 ml of a suspension of the different conidial concentrations for 1 min. Similar exposure was performed by immersion of 2000 larvae and homogeneous groups of nine engorged females in 2 and 20 ml of conidial suspension, respectively. Treated eggs, larvae and adults were placed in an incubator at 27 ± 1 °C and relative humidity above 80% for evaluation of the fungal action. All fungal isolates applied at all conidial concentrations reduced the hatching rate of larvae from treated eggs by 1.36–65.58% and increased the mortality rate of inoculated larvae by 0.8–70.49%. In the bioassay with engorged females, oviposition period was reduced by 9.69–47.80%, egg mass weight by 4.71–53.87%, estimated reproduction by 8.3–60.62%, egg production index by 5.03–54.20%, percent larval hatching by 0.27–13.96%, and the mortality rate of treated females was increased by 96.60–100%. The reduction of the estimated reproduction obtained for the treated groups ranged from 8.37 to 64.52%. The sporulation of the pathogen on dead females ranged from 3.70 to 88.88% depending on the isolate and concentration used. Isolates AM 09, CB 7 and JAB 07 were the most effective and effectiveness increased with increasing concentrations of conidia in the suspensions.     

Initial Colonization, Nutrient Supply, and Fungal Activity on Leaves Decaying in Streams

Aquatic hyphomycetes dominate leaf decomposition in streams, and their biomass is an important component in the diet of leaf-eating invertebrates. After 2 weeks of exposure in a first-order stream, maple leaf disks had low levels of fungal biomass and species diversity. Spore production by aquatic hyphomycetes also was low. Subsets of these disks were left in the stream for another 3 weeks or incubated in defined mineral solutions with one of three levels of nitrate and phosphate. Stream disks lost mass, increased ergosterol levels and spore production, and were colonized by additional fungal species. External N and P significantly stimulated mass loss, ergosterol accumulation, and spore production of laboratory disks. On disks incubated without added N and P, ergosterol levels declined while conidium production continued, suggesting conversion of existing hyphal biomass to propagules. In all other treatments, approximately equal amounts of newly synthesized biomass were invested in hyphae and conidia. Net yield (fungal biomass per leaf mass lost) varied between 1% (in the laboratory, without added N or P) and 31% (decay in stream). In most treatments, the three aquatic hyphomycete species that dominated spore production during the first 2 weeks in the stream also produced the largest numbers of conidia in the following 3 weeks. Principal-component analysis suggested two divergent trends from the initial fungal community established after 2 weeks in the stream. One culminated in the community of the second phase of stream exposure, and the other culminated in the laboratory treatment with the highest levels of N and P. The results suggest that fungal production in streams, and, by extension, production of invertebrates and higher tropic levels, is stimulated by inorganic N and P.     

Marine bacterial community structure resilience to changes in protist predation under phytoplankton bloom conditions

To test whether protist grazing selectively affects the composition of aquatic bacterial communities, we combined high-throughput sequencing to determine bacterial community composition with analyses of grazing rates, protist and bacterial abundances and bacterial cell sizes and physiological states in a mesocosm experiment in which nutrients were added to stimulate a phytoplankton bloom. A large variability was observed in the abundances of bacteria (from 0.7 to 2.4 × 10⁶ cells per ml), heterotrophic nanoflagellates (from 0.063 to 2.7 × 10⁴ cells per ml) and ciliates (from 100 to 3000 cells per l) during the experiment (∼3-, 45- and 30-fold, respectively), as well as in bulk grazing rates (from 1 to 13 × 10⁶ bacteria per ml per day) and bacterial production (from 3 to 379 μg per C l per day) (1 and 2 orders of magnitude, respectively). However, these strong changes in predation pressure did not induce comparable responses in bacterial community composition, indicating that bacterial community structure was resilient to changes in protist predation pressure. Overall, our results indicate that peaks in protist predation (at least those associated with phytoplankton blooms) do not necessarily trigger substantial changes in the composition of coastal marine bacterioplankton communities.     

The long-term trends and seasonal variation of the aeroallergen Alternaria in Derby, UK

Very little is known in the UK about long term trends of theAlternaria spore although it is known to trigger asthma. It hasrecently become apparent that Alternaria spore levels areincreasing in Derby and a detailed study of Alternaria wasundertaken to investigate the increase in numbers, seasonal variationand diurnal periodicity. The seasonal (June—October)Alternaria spore concentrations show a distinct upward trendand there is evidence of an earlier seasonal start and an increase inthe seasonal duration. There has been a dramatic rise in the number ofdays with an Alternaria spore count above 50 spores per cubicmetre, with the peak daily count usually occurring in August butoccasionally in late July or early September. August generally has thehighest monthly total and for 1991–1998 there was a positivecorrelation with monthly rainfall and average temperature. Day to dayspore levels show a positive correlation between Alternariaspore concentrations and maximum temperature but a slight negativecorrelation with daily rainfall. The peak time for spore capture is14.00–22.00, and more than half the daily Alternariacatch is caught between 18.00 and 24.00 hours. The upward trend inAlternaria spore concentrations may be responsible forincreasing levels of respiratory disease, especially during harvesttime.     

Validation of an analytical method for a potent antitumor agent, TZT-1027, in plasma using liquid chromatography–mass spectrometry

A sensitive and specific analytical method for a potent antitumor agent, TZT-1027, in plasma has been developed using liquid chromatography–mass spectrometry (LC–MS) with [²H₄]TZT-1027 as an internal standard (I.S.). A plasma sample was purified by solid-phase extraction on a C₁₈ cartridge, followed by solvent extraction with diethyl ether. The extract was then injected into the LC–MS system. Chromatography was carried out on a C₁₈ reversed-phase column using acetonitrile–0.05% trifluoroacetic acid (TFA) (55:45) as a mobile phase. Mass spectrometric analysis was performed in atmospheric pressure chemical ionization (APCI) mode with positive ion detection, and the protonated molecular ions ([M+H]⁺) of TZT-1027 and I.S. were monitored to allow quantitation. The method was applied to the determination of TZT-1027 in human, monkey, dog, rat and mouse plasma. As far as the sample preparation was concerned, good recoveries (73.5–99.1%) were obtained. The calibration curves were linear over the range of 0.25–100 ng per 1 ml of human, dog and rat plasma, per 0.5 ml of monkey plasma, and per 0.1 ml of mouse plasma. From the intra- and inter-day accuracy and precision, the present method satisfies the accepted criteria for bioanalytical method validation. TZT-1027 was stable when stored below −15°C for 6 months in human plasma and for 3 weeks in plasma from other species. TZT-1027 was also stable in plasma through at least three freeze–thaw cycles.