A gene encoding alanine racemase is involved in spore germination in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Alanine racemase is a major component of the exosporium of Bacillus cereus spores. A gene homologous to that of alanine racemase (alrA) was cloned from Bacillus thuringiensis subsp. kurstaki, and RT-PCR showed that alrA was transcribed only in the sporulating cells. Disruption of alrA did not affect the growth and sporulation of B. thuringiensis, but promoted l-alanine-induced spore germination. When the spore germination rate was measured by monitoring DPA release, complementation of the alrA disruptant reduced the rate of l-alanine-induced spore germination below that of even wild-type spores. As previously reported for spores of other Bacillus species, d-alanine was an effective and competitive inhibitor of l-alanine-induced germination of B. thuringiensis spores. d-cycloserine alone stimulated inosine-induced germination of B. thuringiensis spores in addition to increasing l-alanine-induced germination by inhibiting alanine racemase. d-Alanine also increased the rate of inosine-induced germination of wild-type spores. However, d-alanine inhibited inosine-induced germination of the alrA disruptant spores. It is possible that AlrA converted d-alanine to l-alanine, and this in turn, stimulated spore germination in B. thuringiensis. These results suggest that alrA plays a crucial role in moderating the germination rate of B. thuringiensis spores.     

Investigation of factors influencing spore germination of Paenibacillus polymyxa ACCC10252 and SQR-21

Bioorganic fertilizer containing Paenibacillus polymyxa SQR-21 showed very good antagonistic activity against Fusarium oxysporum. To optimize the role of P. polymyxa SQR-21 in bioorganic fertilizer, we conducted a study of spore germination under various conditions. In this study, l-asparagine, glucose, fructose and K⁺ (AGFK), and sugars (glucose, fructose, sucrose, and lactose) plus l-alanine were evaluated to determine their ability to induce spore germination of two strains; P. polymyxa ACCC10252 and SQR-21. Spore germination was measured as a decrease in optical density at 600 nm. The effect of heat activation and germination temperature were important for germination of spores of both strains on AGFK in Tris–HCl. l-Alanine alone showed a slight increase in spore germination; however, fructose plus l-alanine significantly induced spore germination, and the maximum spore germination rate was observed with 10 mmol l⁻¹ l-alanine in the presence of 1 mmol l⁻¹ fructose in phosphate-buffered saline (PBS). In contrast, fructose plus l-alanine hardly induced spore germination in Tris–HCl; however, in addition of 10 mmol l⁻¹ NaCl into Tris–HCl, the percentages of OD₆₀₀ fall were increased by 19.6% and 24.3% for ACCC10252 and SQR-21, respectively. AGFK-induced spore germination was much more strict to germination temperature than that induced by fructose plus l-alanine. For both strains, fructose plus l-alanine-induced spore germination was not sensitive to pH. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination diversity, which will be beneficial for bioorganic fertilizer storage and transportation to improve the P. polymyxa efficacy as biological control agent.     

Streptomyces fragmentosporus,ein neuer thermophiler Actinomycet

Zusammenfassung Ein neuer thermophiler Actinomycet, Streptomyces fragmentosporus wird beschrieben. Kennzeichnend für die Art sind gabelig bis rechtwinklig verzweigte Sporophoren mit kurzen, geraden Ketten aus locker gestellten, stacheligen Fragmentationssporen und die Bildung von Fragmentationssporen im Substratmycel. Neben den Sporophoren werden lange Lufthyphen entwickelt, die teils Sporophoren tragen und gewöhnlich basipetal in Sporen zerfallen. Das Luftmycel ist weiß. Substrat-und Luftsporen sind polymorph. Die Art wächst nur auf wenigen komplexen Nährböden, Pigmente oder Melanin werden nicht gebildet. Streptomyces fragmentosporus ist am nächsten mit der mesophilen Art Streptomyces ramulosus verwandt. Die vergleichende morphologische Untersuchung beider Arten zeigte eine große Übereinstimmung in der Differenzierung des Substrat-und Luftmycels.

---

Streptomyces fragmentosporus, a new thermophilic actinomycete

Summary The new thermophilic species Streptomyces fragmentosporus is described. The organism is characterized by short, straight, often rectangularly branched aerial sporophores with loosely arranged fragmentation spores, and by the production of fragmentation spores in the substrate mycelium. Beside the sporophores long aerial hyphae are developed which may bear sporophores, and usually fragmentate into spores in basipetal direction. The spores are polymorph and bear small spines.Good growth and production of aerial mycelium was obtained only on manure- and hay-yeast-agar. No pigments or melanin were formed, and no antibiotic activity was observed. The optimum temperature was 50 to 60° C. Streptomyces fragmentosporus is most closely related to the mesophilic Streptomyces ramulosus. It differs from that species mainly by the colour of the aerial mycelium, the absence of pigments, and by the spiny spores which are connected in the chains by ± long parts of hyphal remnants.In a comparative morphological study Streptomyces ramulosus was found to produce abundantly short, ± rectangularly branched sporophores as reported by Shirling and Gottlieb (1968), and occasionally long aerial hyphae bearing sporophores as described by Ettlinger and coworkers (1958). In contrast to the latter authors we observed a production of spores also in these long hyphae like in Streptomyces fragmentosporus.

---

---

Die Untersuchung wurde durch eine Sachbeihilfe der Deutschen Forschungsgemeinschaft unterstützt.     

Germination of Basidiospores of Agaricus bisporus

The type of dormancy and conditions necessary for germination of Agaricus bisporus basidiospores were studied. Basidiospores failed to germinate on starvation agar and required the presence of carbon and nitrogen sources (asparagine and/or glucose) in the medium. Upon 3-week storage, basidiospores germinated after 4–5 days. Heat shock (20 min at 45°C) and decreased temperature facilitated activation of germination. Heterocyclic compounds stimulating germination of endogenously dormant spores, such as furfural, failed to activate germination. The data obtained suggested an endogenous dormancy of A. bisporus basidiospores differing from zygospores of Mucorales. Basidiospores contained 17–19% lipids with a composition of fatty acids differing from those of the pileus and stipe of the fruiting body. The soluble carbohydrates of the cytosol amounted to 12% dry spore weight and consisted of mannitol (74%) and trehalose (26%). Unlike basidiospores stored at 2°C, basidiospores stored for 5 months at 20°C lost their ability to germinate, which correlated with a decrease in the content of trehalose.     

Spore Liberation and Dispersal in Smut Fungi*

Teliospores are the most important diaspores of smut fungi, albeit not the only ones. The role of basidla, basidiospores, secondary spores, yeast cells, and infected parts of the host for dispersal has often been neglected. Many smut species have soral structures like galls, peridia, and elaters, which cause teliospores to be liberated over prolonged periods. This increases the chance that at least some spores are released under favourable wet climatic conditions and while host plants are susceptible. In this review, the diversity of dispersal units as well as vectors of smut fungi are presented. The importance of timing of diaspore liberation, flexibility in dispersal strategies, and the genetic and evolutionary implications of dispersal strategies of smut fungi are discussed. The general considerations are complemented by examples based on original field and laboratory observations: peridia of Farysia corniculata and certain species of Sporisorium expose the spore mass by hygroscopic movement under wet conditions (hygrochasy) favourable for teliospore germination and infection of a host plant. Basidia with firmly attached basidiospores liberated from spore balls of Doassansiopsis deformans, branched basidiospores of Rhamphospora nymphaeae, needle-shaped basidiospores of species of Entyloma, folded basidiospores of Mycosyrinx cissi, and stellate groups of yeast cells of Trichocintractia utriculicola show enlarged surfaces, which are advantageous for dispersal in water. Galls filled with spore balls of Doassansiopsis limnocharidis and witches' brooms formed by spikelets infected by Cintractia standleyana separate from the host and fall into water where they are dispersed.     

Seasonal and Diurnal Variation of Airborne Basidiomycete Spore Concentrations in Mexico City

Seasonal and diurnal changes in concentrations of airborne basidiomycete spores (basidiospores, rusts, smuts) were studied, using Burkard volumetric spore traps, in two areas of Mexico City with different degrees of urbanization and related to changes in climatic variables through 1991. Basidiomycete spores formed a large component of the total airborne fungal spore load in the atmosphere of Mexico City. They were the second most abundant spore type after Deuteromycotina (Hyphomycetes), forming 32% of the total fungal spores trapped in an urban-residential area and 28% in an urban-commercial area. The most abundant basidiomycete spores were basidiospores although smut-type spores were trapped on more days than basidiospores and rusts on fewer. Basidiospores occurred in concentrations up to 2,000 spores m-3 in the urban-residential area. Basidiospores showed a marked seasonal distribution, especially in the southern area, with their greatest abundance during the wet season. The correlation coefficients associated with regressions between basidiospore concentration and some environmental factors were increased when a lag period of 2 to 4 days was used between environmental measurements and the day of spore collection. Basidiospore concentrations exceeded the 75 percentile concentration (>400 spores m^-3) most often when rainfall was up to 6 mm and relative humidity was >70%. Basidiospores showed a diurnal periodicity with greatest concentrations in the early morning. The most common basidiospore type was Coprinus which formed 67% of basidiospores trapped in the southern area and 63% in the central area. Smut spores were trapped on 87% of days through the year while rust spores occurred in only 35%. Both rusts and smuts were present in only small concentrations.     

Die Beeinflussung der Farnsporen-Keimung [Osmunda cinnamomea (L.) undO. claytoniana (L.)] über das Phytochromsystem und die Photosynthese

Summary The control by light of the spore germination ofOsmunda cinnamomea L. andO. claytoniana L. has been investigated.This light control is complex, and in order to investigate this control quantitatively the process of germination had to be divided into at least two stages. The first stage of germination leads to the rupture of the exospore, the second step is characterized by the outgrowth of a rhizoid. In a very few percent of our spore population the first stage can occur in total darkness, the second step however does not ever occur in darkness.In the present paper it has been demonstrated, usingO. cinnamomea spores, that the first step of germination is phytochrome controlled, whereas the second step is under photosynthetic control. This was observed withO. claytoniana by a rough action spectrum and in experiments with a CO₂-less atmosphere.The germinating spores of these two species ofOsmunda show a very similar behaviour as the spores of the mossFunaria hygrometrica (L.) Sibth. (Bauer andMohr 1959). They behave very differently compared with spores of other ferns, e.g.Dryopteris filix-mas (L.) Schott, where the whole process of germination is exclusively phytochrome controlled (Mohr 1956).

---

---

Mit 10 Textabbildungen     

Correlation of spring spore concentrations and meteorological conditions in Tulsa, Oklahoma

Different spore types are abundant in the atmosphere depending on the weather conditions. Ascospores generally follow precipitation, while spore types such as Alternaria and Cladosporium are abundant in dry conditions. This project attempted to correlate fungal spore concentrations with meteorological data from Tulsa, Oklahoma during May 1998 and May 1999. Air samples were collected and analyzed by the 12-traverse method. The spore types included were Cladosporium, Alternaria, Epicoccum, Curvularia, Pithomyces, Drechslera, smut spores, ascospores, basidiospores, and other spores. Weather variables included precipitation levels, temperature, dew point, air pressure, wind speed, wind direction and wind gusts. There were over 242.57 mm of rainfall in May 1999 and only 64.01 mm in May 1998. The most abundant spore types during May 1998 and May 1999 were Cladosporium, ascospores, and basidiospores. Results showed that there were significant differences in the dry-air spora between May 1998 and May 1999. There were twice as many Cladosporium in May 1998 as in May 1999; both ascospores and basidiospores showed little change. Multiple regression analysis was used to determine which meteorological variables influenced spore concentrations. Results showed that there was no single model for all spore types. Different combinations of factors were predictors of concentration for the various fungi examined; however, temperature and dew point seemed to be the most important meteorological factors. Received: 5 July 2000 / Revised: 20 December 2000 / Accepted: 22 December 2000     

Spores of microorganisms

Calcium-dipicolinate (Ca-DPA)-rich and Ca-DPA-deficientBacillus cereus spores were incubated in a synthetic medium with germination stimulants and in bactopeptone medium with a fairly high calcium ion concentration. In the complex medium the germination of Ca-DPA-rich spores was completely blocked at a concentration of 0.5m CaCl₂, whereas the complete blockage of germination in the synthetic medium required higher concentrations (0.6–0.8m) of calcium chloride. Ca-DPA-deficient spores germinated more slowly and less completely in the synthetic medium than in the bactopeptone medium. The germination of these spores took place, however, even at higher calcium ion concentrations (0.6–0.8m). On the contrary, lower calcium chloride concentrations (0.1–0.4m) accelerated the germination of these spores in the synthetic medium and the final percentage of phase-dark and stainable spores was higher. “H-forms” of the Ca-DPA-rich and Ca-DPA-deficient spores prepared by acid titration germinated in both media. The germination of the latter spores being slower and proceeding less completely. “H-forms” germinated completely or partially in media with a high concentration of calcium chloride. The percentage of germinated spores, however, was strongly influenced by the concentration of this cation, especially the “H-forms” of Ca-DPA-deficient spores. Moreover, the germination of Ca-DPA-deficient spores in this medium was affected by the length of previous storage and, in the case of “H-forms” by the pH at which they were titrated. It was assumed that the increased permeability of calcium into the calciumundersaturated spore periphery in Ca-DPA-deficient and in “H-forms” of spores of both types co-determines (in the presence of germinants) the germinability of bacterial spores.     

Licht- und elektronenmikroskopische Untersuchungen von Entwicklungsstadien der FlechteEndocarpon pusillum unter Kulturbedingungen

The lichenEndocarpon pusillum Hedw. was cultivated under laboratory conditions on agar, silica gel and soil substrate. Selected developmental stages of the life cycle (germination, contact between the symbionts, cortex, squamule and perithecia development) were studied by light and scanning electron microscope.—It could be shown that the spores had no rigid spore walls with germination colpies and the spore cells which are in contact with the substrate were formed directly into germination tubes.— Further studies showed that the initial contact between the components was thigmotropic and both the form and the gelatinous matrix around the algal cells play an important role in this process. — The development of the cortex occurs under reduced moisture conditions resulting in a reduced algal reproduction. The thickness of the cortex was dependent on light intensity during cultivation. The cortex originated from hyphae, which developed beyond the algal layer and were combined to a tight network.—Fruiting bodies with spores and hymenial algae were only formed in cultures on soil substrate.

Frau Prof. Dr.Elisabeth Tschermak-Woess zu ihrem 70. Geburtstag gewidmet.     

The Role,Interaction and Regulation of the Velvet Regulator VelB in Aspergillus nidulans

The multifunctional regulator VelB physically interacts with other velvet regulators and the resulting complexes govern development and secondary metabolism in the filamentous fungus Aspergillus nidulans. Here, we further characterize VelB’s role in governing asexual development and conidiogenesis in A. nidulans. In asexual spore formation, velB deletion strains show reduced number of conidia, and decreased and delayed mRNA accumulation of the key asexual regulatory genes brlA, abaA, and vosA. Overexpression of velB induces a two-fold increase of asexual spore production compared to wild type. Furthermore, the velB deletion mutant exhibits increased conidial germination rates in the presence of glucose, and rapid germination of conidia in the absence of external carbon sources. In vivo immuno-pull-down analyses reveal that VelB primarily interacts with VosA in both asexual and sexual spores, and VelB and VosA play an inter-dependent role in spore viability, focal trehalose biogenesis and control of conidial germination. Genetic and in vitro studies reveal that AbaA positively regulates velB and vosA mRNA expression during sporogenesis, and directly binds to the promoters of velB and vosA. In summary, VelB acts as a positive regulator of asexual development and regulates spore maturation, focal trehalose biogenesis and germination by interacting with VosA in A. nidulans.     

Effect of CO2 on the germination of conidiospores of Aspergillus niger

Summary The effect of different concentrations of CO₂ on the germination of conidiospores of Aspergillus niger A 5 has been studied using Pardee's buffer mixtures which maintain constant CO₂ tensions. The beneficial effect of CO₂ on germination is maximum at 0.5% CO₂ concentration, when 70–90% of the spores germinate within 6 hours, whereas in controls with air containing 0.03% CO₂ there is only 15–20% germination at 6 hours. At higher CO₂ concentrations this beneficial effect of CO₂ on germination diminishes and at 3% there is a complete inhibition of spore germination.The spore density and the ph of the medium have a noticeable effect on germination rates in presence of 0.5% CO₂. The germination rates decrease at spore densities higher than 5 · 10⁵/ml and at a ph of 6.8.Communication No. 431.     

Secondary leaf fall of Hevea brasiliensis: factors affecting the production, germination and viability of spores of Colletotrichum gloeosporioides

The optimum temperature for growth and sporulation of Colletotrichum gloeosporioides from Hevea brasiliensis was between 26 and 32 ^oC, whereas spore germination exceeded 90% between 21.5 and 30.5 ^oC. Germination decreased in culture after 3 days, and on exposure of spores to sunlight or oven heat (46 ^oC) for 10 min. Spore viability and germination were sensitive to atmospheric humidity; at 99% r.h. germination was half that at 100% r.h. and was negligible below 97% r.h. Germination decreased by up to 30% after 3 h storage at 80% r.h. Continuous light favoured spore production in vitro, but spores produced in the dark had a higher percentage germination. No differences were detected between the numbers of spores germinating on leaves of different ages, although there were slightly more on susceptible cultivars and in the presence of extracts of uninfected susceptible leaves. Extracts from, infected leaves depressed spore germination, as did concentrations above 5 times 10⁵ spores/ml. The highest % germination was observed when naturally infected leaves were dry-stored for up to 20 days and then incubated for 2 days in a moist chamber.     

The response to a specific germinant by <Emphasis Type="Italic">Bacillus anthracis</Emphasis> spores in primary mouse macrophages is modulated by a protein encoded on the pXO1 plasmid

A Bacillus anthracis Sterne pXO1 plasmid-encoded protein designated Cot43 was found in coat extracts of purified spores. Cot43 is a tetratricopeptide repeat domain protein related to those which function as phosphatases in the sporulation phosphorelay and as regulators of competence and pathogenic factors. The synthesis of Cot43 began in the late exponential phase downstream from a sigmaA promoter (as mapped by RACE) and it was present at least until the formation of phase white endospores. There was specificity in the association of Cot43 with B. anthracis spores since Bacillus cereus producing Cot43 from a cloned gene had very little of this protein in spore coat extracts. In addition, Cot43 was synthesized by B. anthracis cells to the same extent in glucose-yeast extract and nutrient sporulation media, but was essentially absent from spores formed in the former. l-histidine is an important germinant for B. anthracis spores in macrophages, Spores produced by a mutant with a disruption of cot43 germinated in response to l-histidine both in vitro and within primary mouse macrophages earlier and more extensively than Sterne strain spores. The germination delay due to the presence of Cot43 would enhance spore survival and thus increase the chances for a successful infection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Study of airborne fungal spores in Madrid, Spain

The concentration of fungal spores in the atmosphere of Madrid was recorded and analyzed for the year 2003. Airborne spores were sampled continuously with a Hirst-type spore trap located on the roof of a building of the School of Pharmacy, at about 8 m above ground level. Correlation between the mean daily spore concentrations and meteorological variables were explored by means of Spearman’s correlation analyses. Seventy spore types were identified, of which the most numerous were Cladosporium, Aspergillaceae (conidia), Coprinus, Agaricales (basidiospores), Ustilago (teliospores) and Pleospora (ascospores). These six types of spores represented more than 70% of the total. Cladosporium represented 41% of the total fungal spores, while Ustilago spores, the concentrations of which in May and June exceeded 47% of the monthly total spore count, constituted the second most important group. Spores reached their highest concentrations in the spring months, and in the autumn, mainly in October. A␣positive significant correlation was found between airborne spore counts and temperature and relative humidity. The results provide a picture of the spectrum of airborne fungal spores present in the atmosphere of Madrid and of the `peak' periods of their presence. Future studies will provide more detailed information on the seasonal dynamics of the spores most frequently found in the air as well as on the extent to which atmospheric conditions influence their release, dispersion and sedimentation processes.     

Temporal control of autoactivator synthesis and secretion during spontaneous spore germination in Dictyostelium discoideum

SG mutant and aged wild type spores of the cellular slime mold Dictyostelium discoideum germinate in the absence of an externally applied activation treatment. This type of germination is referred to as autoactivation. During the swelling stage of autoactivation, spores release a factor, the autoactivator, capable of stimulating germination in subsequent spore populations. The autoactivator was not present in the dormant spore, but it or a precursor was produced internally during the first hour of autoactivation. This production was sensitive to moderately high temperatures (+31° C) and was completely destroyed by heat activation (45° C for 30 min). Internal production of the autoactivator was not sensitive to protein synthesis inhibitors. However, the release of the activator from the spore appeared to be regulated by protein synthesis. Internal autoactivator was also produced in the aged wild type strain during the postautoactivation lag phase. The activator could not be directly isolated from within the germinating spore. Its activity on the rest of the spore population was dependent upon its release from the germinating spore. A model is presented integrating the effects of heat, cycloheximide, autoinhibitor and autoactivator on spores of D. discoideum.     

Lethal germination of spores ofBacillus megaterium 9885

Washed spore suspensions germinated promptly without prior heat shock in a basal germination solution containingl-leucine.Germination was inhibited by dipicolinic acid. The inhibition was reversed by eitherl-leucine or phosphate.Phosphate accelerated the rate and increased the extent of germination, which was accompanied by an uncommonly large fall in the optical density of the suspension, but phosphate also caused a massive lysis after germination. This was accompanied by a sudden shedding of the spore coats. The suspensions consisted of shrivelled, cellular walls and membranes attached to the empty spore coats.Lysis of the germinated cells was prevented by fairly high concentrations of Ca or Mg.During germination, exogenous Ca we used Ca⁴⁵ was absorbed by the cells. Both cells and sonically disrupted cellular particles firmly retained the calcium, and evidence suggested that much of the Ca was bound in the cytoplasmic membranes.The cations contained in plain agar enabled spores which germinated on tryptone soya agar plates to develop into colonies; in the corresponding broth medium these spores lysed upon germination.Hypertonic sucrose delayed but did not prevent lysis.     

Forecasting spore concentrations: A time series approach

Fungal basidiospores andCladosporium spores are the two most numerous spore types in the air of Dublin and its surroundings. They are known to have allergenic components, and the aim of the study described here is to develop a predictive model for these spores. A very simple model, which combines an estimated diurnal rhythm with a simple, one-parameter time series model, provided golld short-term forecasts. The one-step prediction error variance was reduced by 88% forCladosporium spores and by 98% for basidiospores.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

Airborne basidiospores in the atmosphere of Seville (South Spain)

Basidiomycetes produce large quantities of spores that are dispersed by the wind over long distances. Some species can release billions of spores in a day, many of which are important aeroallergens associated with asthma and allergic rhinitis. The results of a survey carried out in the city of Seville during two consecutive years, using a Hirst-type spore trap, showed that airborne basidiospores are present throughout the year, although there is a certain seasonal distribution, with the highest average concentration occurring in November (23,586 basidiospores). Eighteen different types of basidiospores were identified, with Coprinus and Ustilago being the most frequent, followed in descending order of abundance by Agaricus, Phylacteria, Boletaceae, Ganoderma, Cortinarius, Calvatia, Agrocybe, Bovista and Puccinia; the remaining spore types comprised less than 1% of the total basidiospore count.