长白山哈泥泥炭地丘间生境泥炭藓孢子的寿命

长寿有性繁殖体对于植物种群的长存具有重要意义,迄今,泥炭地苔藓植物孢子长寿性研究还很少。在长白山哈泥泥炭地钻取丘间表层泥炭样品,测定泥炭腐殖化度和烧失量,逐层提取和培养泥炭藓孢子,研究埋藏时间对孢子萌发的影响。结果表明,丘间泥炭藓孢子埋藏环境中,随着埋深的增加即埋藏年限的增加,泥炭腐殖化度和烧失量总体上分别呈现增加和递减的趋势,而地层泥炭藓孢子萌发率呈现直线递减的规律,但在埋藏近150余年后孢子萌发率仍可达40%。研究进一步证明泥炭藓具有长期持久孢子库,根据推算,泥炭地丘间埋藏环境中,泥炭藓孢子最大寿命可超过400a。     

Effect of the osmotic conditions during sporulation on the subsequent resistance of bacterial spores

The causes of Bacillus spore resistance remain unclear. Many structures including a highly compact envelope, low hydration of the protoplast, high concentrations of Ca-chelated dipicolinic acid, and the presence of small acid-soluble spore proteins seem to contribute to resistance. To evaluate the role of internal protoplast composition and hydration, spores of Bacillus subtilis were produced at different osmotic pressures corresponding to water activities of 0.993 (standard), 0.970, and 0.950, using the two depressors (glycerol or NaCl). Sporulation of Bacillus subtilis was slower and reduced in quantity when the water activity was low, taking 4, 10, and 17 days for 0.993, 0.970, and 0.950 water activity, respectively. The spores produced at lower water activity were smaller and could germinate on agar medium at lower water activity than on standard spores. They were also more sensitive to heat (97 degrees C for 5-60 min) than the standard spores but their resistance to high hydrostatic pressure (350 MPa at 40 degrees C for 20 min to 4 h) was not altered. Our results showed that the water activity of the sporulation medium significantly affects spore properties including size, germination capacity, and resistance to heat but has no role in bacterial spore resistance to high hydrostatic pressure.     

Trehalose breakdown in germinating spores of Mucor rouxii

Germinating spores of Mucor rouxii rapidly broke down their large (23% of the dry weight) trehalose reserve. More than 50% of this trehalose was broken down to ethanol. About one-third of the trehalose was converted to glycerol, which started to leak out of the spores after some 20 min germination. The synthesis of glycerol was not associated with any major change in glycerol 3-phosphatase activity in the spores. Since its rate of leaking was much smaller and the internal concentration reached was much higher in spores subjected to osmotic stress, glycerol might play a role in the initial water uptake and swelling of the germinating spores.     

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.     

Expression of proteolytic enzymes during Dictyostelium discoideum spore germination

The specific activity of cathepsin B-like, cathepsin D-like, and leucine aminopeptidase enzymes was measured in dormant, aging, and germinating spores of wild-type and mutant Dictyostelium discoideum.The activity of leucine aminopeptidase was relatively constant during spore aging and spore germination. The level of cathepsin D-like activity was highest in young dormant spores but decreased during germination or aging.The level of cathepsin B-like activity remained constant in wild-type spores which were aged for 13 days. The dormant spores of spontaneous germination mutants initially contained low levels of cathepsin B-like activity which increased during aging. Thus, there was no correlation between the level of endogenous cathepsin B activity and the ability to be autoactivated or heat-activated. The level of cathepsin B-like activity does not have a role in the generation of energy for the swelling stage of germination. Finally, the combined level of endogenous and exogenous cathepsin B activity increased more than 20-fold during the emergence of myxamoebae suggesting that the enzyme(s) may play a role at this development stage of germination.     

Lipids stimulate spore germination in the entomopathogenic ascomycete <Emphasis Type="Italic">Ascosphaera aggregata</Emphasis>

The alfalfa leafcutting bee (Megachile rotundata) is solitary and managed on a large scale for pollination of alfalfa seed crops. The bees nest in holes drilled in wood or polystyrene blocks, and their larvae are highly prone to a fungal disease called chalkbrood. The most prevalent form of chalkbrood is caused by Ascosphaera aggregata, but this ascomycete is difficult to culture. Hyphae will grow on standard fungal media, but spore germination is difficult to achieve and highly variable. We found that germination can be enhanced with oils. Lipids derived from plants and bee larvae increased germination from 50% (without oil) to 75–85% (with oil). Percent germination was significantly greater in the presence of lipids but germination was not significantly different when different oils, including mineral oil, were used. A. aggregata spores oriented along the oil--aqueous interface in the broth in a polar fashion, with swelling and germ tube formation always occurring into the aqueous portion of the broth. The other half of the spore tended to attach to a lipid droplet, where it remained, without swelling, during germ tube formation. The physical attachment of spores to the oil--aqueous interface is what most probably stimulates spore germination, as opposed to some nutritional stimulation. However, further research is needed to determine if and where the spores encounter such an interface when germinating in the host gut, where germination normally occurs.     

Chloroplast activities of dark-imbibed and photoinduced spores of the fernOnoclea sensibilis

Summary Chloroplast activities of dark-imbibed (non-germinating) and photoinduced (germinating) spores of the sensitive fern,Onoclea sensibilis were compared to gain insight into the germination process. There were no changes in the number of chloroplasts or in the chlorophyll contents of the spore during dark-imbibition and during the early phase of germination. Levels of increase in the Chloroplast DNA content of dark-imbibed and photoinduced spores were nearly the same and were associated with autoradiographic incorporation of [³H]thymidine into the cytoplasm. However, incorporation of the label into the nucleus occurred only during photoinduction of spores. Analysis of Chloroplast and nuclear DNA contents by dot-blot hybridization with labeled gene-specific probes has confirmed that chloroplast DNA content of the spore increases during dark-imbibition and photoinduction, while increase in nuclear DNA occurs only in photoinduced spores. Chloroplasts isolated from dark-imbibed and photoinduced spores incorporated [³H]TTP into an acid-insoluble fraction identified as DNA. The results show that physiological activities of chloroplasts of dark-imbibed and photoinduced spores ofO. sensibilis are similar and support an exclusive role for nuclear DNA synthesis in spore germination.     

Soybean and sunflower oils increase the infectivity of Colletotrichum gloeosporioides f. sp. aeschynomene to Northern Jointvetch

Soybean and sunflower oils increased the level of infection of northern jointvetch, Aeschynomene virginica, plants by Colletotrichum gloeosporioides f. sp. aeschynomene. Inoculation of seedlings with spore suspensions containing 10% (v:v) soybean oil or 10% sunflower oil resulted in more disease than when inoculated with suspensions of spores in water alone. The lengths of the dew periods required to establish equivalent levels of disease by spore suspensions containing 10% soybean or 10% sunflower oil were approximately 4–8 h less compared to aqueous suspensions. Incubation of spores in 10% soybean oil followed by removal and resuspension in water did not affect the infectivity of spores when compared to spores incubated in aqueous suspensions. Spore germination and appressoria formation were unaffected by either of the oils tested in in vitro assays; however, in in vivo assays, 10% soybean oil and 10% sunflower oil increased spore germination in comparison to spores that were suspended in water.     

Activation and killing of Dictyostelium discoideum spores with urea.

The optimal conditions for activation of Dictyostellium discoideum spores are an 8 M urea treatment for 30 min. The lag between activation and swelling is 45 min. Lower concentrations of urea do not activate entire spore populations. Incubating spores in 8 M urea for 60 min or treatment with 10 M urea for 30 min results in a lengthening of the post-activation lag and a decrease in the final percentage of germination. Urea-activated spores can be deactivated by azide, cyanide, osmotic pressure, and low-temperature incubation. Activated spores do not germinate if incubated in 1 M urea for 24 h but will complete germination upon resuspension in urea-free buffer. Shocking spores at 45 degrees C in 8 M urea or incubating spores in 4-8 M urea for 10 h at 23.5 degrees C causes inactivation. When suspended in urea-free buffer, a larger percentage of these dead spores release spheroplasts through a longitudinal split in the spore case. Sequential enzyme treatment of spheroplasts with cellulase and pronase causes them to release lysable protoplasts. The data of these experiments suggest that shedding of the outer and middle wall layers during physiological spore swelling may be a physical process rather than an enzymatic one.     

Endospores of Sporosarcina halophila: characteristics and ultrastructure

Sporosarcina halophila forms endospores. Electron micrographs revealed ultrastructural similarity to spores of S. ureae. Spore germination indicated by loss of refractility, darkening, swelling and formation of new vegetative cells was followed by phase contrast light microscopy. To induce spore germination, the endospores needed to be heat avtivated. After activation, they were inoculated into nutrient broth medium supplemented with sea-water. Double concentrated sea-water was found to be optimal for germination. Similar to other bacterial endospores, the spores were found to be resistant to heat and ethanol. An ultraviolet absorbing substance was isolated from suspensions of free spores; it was identified to be pyridine-2,6-dicarboxylic acid (DPA) usually present in bacterial spores. DPA was detected in amounts ranging from 5–7% of the spore dry weight; it was not detected in extracts of vegetative cells.Abbreviation DPA 2,6-pyridine-dicarboxylic acid     

Nutritional conditions for the germination of streptomyces galbus 5ME-13 spores

The nutritional conditions for the germination of spores of Streptomyces galbus 5ME-13 were determined under laboratory conditions. The germination of the spores was intiated by the emergence of 1–2 germ tubes after the second hour of incubation and attained its maximum at the sixth hour. This was accompanied by a steady rise in the optical density of the germinating spore suspension. A malt-extract yeast-extract medium was found to be the best medium for the germination of the spores. Glycerol as the sole source of carbon was the best supporter for spore germination while, as N source, L-alanine was preferred. The optimum pH and temperature for spore germination were 7.2 and 30°C, respectively.     

The Effect of Water Activity and the aw Controlling Solute on Spore Germination of Bacillus stearothermophilus

The germination of spores of Bacillus stearothermophilus was studied in nutrient broth in relation to the water activity ( a _w) of the medium, the nature of the a _w controlling solutes glycerol, sucrose, KCl, and NaCl, and temperature. Quantitation of germination was based on the change of the phase-bright spore to phase-dark. Activation of spores was by exposure to 100°C/10 min in a medium of the same composition as that used for germination.
Of the four solutes used, sucrose proved most inhibitory to germination, especially in the upper part of the temperature range 38-75°C, glycerol was the most favourable whereas KCl and NaCl, whose effect was almost identical, occupied an intermediate place. The glycerol effect became more pronounced as the a _w of the medium decreased towards 0.960, becoming inhibitory thereafter.
The solute effect on spore germination followed a pattern that related to the class of solute, i.e. electrolyte or non-electrolyte, and its cell penetration characteristics.
Solute penetration during heat activation and germination was considered as the major germination factor and was associated with the osmoregulation mechanism within the spore proposed recently as the basis of spore dormancy and resistance.     

Utilization of trehalose during Dictyostelium discoideum spore germination

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.     

Reactive products formed by peroxidase catalyzed oxidation of p-phenetidine

The drug 4-nitroquinoline 1-oxide (4NQO) is a potent inhibitor of Dictyostelium discoideum spore germination. This inexpensive, water soluble drug is active at a concentration of 5 micrograms/ml (26 microM) and permeates the spore at all stages in germination. Spores subjected to 4NQO treatment exhibit an irreversible blockage of myxamoebae emergence, but spore activation, post-activation lag, and swelling are not affected. Swollen 4NQO-treated spores lose the outer two spore walls but lack the ability to degrade the innermost wall. The drug does not affect oxygen uptake during post-activation lag or swelling, and only a stage specific depression in O2 uptake is observed when control spores begin to release myxamoebae. When added early in germination, 4NQO blocks the incorporation of [3H] uracil into a cold trichloroacetic acid (TCA) insoluble fraction by 98%. However, when the drug is added midway through germination and followed by a pulse labelling period of 1 h, only 65% inhibition of RNA synthesis is observed. This lack of complete inhibition may occur because the drug requires metabolic activation; thus, new rounds of RNA synthesis may have initiated before the drug became fully activated. 4NQO also blocks the de novo expression of beta-glucosidase activity when added early in germination. Additionally, we observe that vegetative cellular slime mold cells are 100 times more resistant than spores to 4NQO-induced damage. Taken together, our results support the observation that RNA synthesis is only required for the emergence stage of germination and that dormant D. discoideum spores may lack efficient excision repair mechanisms.     

The identification of a self-inhibitor from Syncephalastrum racemosum and its effect upon sporangiospore germination

High concentrations of Syncephalastrum racemosum spores germinated less readily than low concentrations. Extensive washing of spores alleviated this inhibition of germination. Analysis of the spore washings revealed the main constituent to be nonanoic acid. Exogenously added nonanoic acid was found to mimic the self-inhibition, in that it delayed the time of germ tube emergence and increased the lag before spore swelling commenced.     

The physiological effects of restrictive environmental conditions on Dictyostelium discoideum spore germination.

Spores may be reversibly activated by the application of heat, dimethyl sulfoxide, urea, or ethylene glucol. Severe changes in four environmental variables (high osmotic pressure, low oxygen tension, low or high pH, and low or high temperature) interfere with the germination process. Spores at the end of the postactivation lag phase of germination were usually deactivated if exposed to severe environmental conditions and thus did not swell; spores in the swelling and oxygen uptake which began during spore activation was primarily attributable to a cyanide-sensitive pathway and secondarily to a salicylhydroxamic acid (SHAM) sensitive pathway. Inhibition of the SHAM-sensitive pathway did not cause spore deactivation while the addition of cyanide resulted in rapid spore deactivation. Treatment of activated spores with azide or environmental shifts also resulted in inhibition of oxygen uptake and spore deactivation. Deactivating spores did not demonstrate the amino acid incorporation, uridine incorporation, and expression of trehalase activity which is found in the later stages of germinating control spores. Protein synthesis inhibitors did not cause spore deactivation or a decrease in oxygen uptake but they inhibited amino acid incorporation and the expression trehalase activity in swollen spores. It is concluded that control of respiratory activity is involved in regulation of reversible activation.     

Morphological characterization and germination of aerial and submerged spores of the entomopathogenic fungus Verticillium lecanii

Under solid-state and liquid-state cultivations, the entomopathogenic fungus Verticillium lecanii F091 produced different types of spores. The aerial spores (AS) on cooked rice formed clusters on the tips of conidiophores, while the submerged spores (SS) were dispersed in the medium. The aerial spore appeared relatively uniform in size, which was 6.1 ± 0.9 m long, and 2.2 ± 0.3 m wide. The submerged spore varied in shape and size, with a mean length of 5.0 ± 1.0 m and width of 1.9 ± 0.5 m. Under scanning electron microscopy, the AS had a tendency to have rough, brittle surface characteristics; however, the SS appeared smooth on the surface. These spores were compared in two different germination media. On SMAY (Sabouraud maltose, agar, yeast extract, and neopeptone) coated coverslips, the AS did not show germ tubes until 8 h of incubation; while the SS showed many germ tubes. However, over 90% spore germination ratio was reached for both types of spores at 18-h of incubation. In the liquid medium, the SS germinated rapidly and many spores even produced spores on the spores; while the AS germinated, grew, and branched in the submerged culture gradually, and some sporulated on the tips of the short branches, or on the mycelia until 18 h of incubation. Evidently, the germination, growth patterns of aerial or submerged spores differed greatly under the different culture conditions. The virulence of the pathogen in relation to the type of spore of V. lecanii is discussed.     

Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination

Germination of mutant spores of Bacillus subtilis unable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.     

Water availability affects the growth,accumulation of compatible solutes and the viability of the biocontrol agent Epicoccum nigrum

Growth of the biocontrol fungus Epicoccum nigrum was more sensitive to ionic solute water stress (NaCl) than non-ionic (glycerol) on potato dextrose-based media at –0.5, –3.0 and –5.5 MPa water potentials. Subsequent physiological manipulation of growth of E. nigrum in glycerol-modified media to –3.0 MPa water potential resulted in a significant increase in the accumulation of compatible solutes in both mycelial liquid cultures and spores, but no enhanced accumulation of the desiccation protectant trehalose, when compared to unmodified media (–0.5MPa). The main solute accumulated was glycerol, followed by arabitol. In temporal studies over 20 days maximum accumulation of glycerol occurred in 5-d old cultures with water stressed cultures having 250× greater amounts than those from unmodified medium. The arabitol content was also higher in mycelium and spores produced under water stress. The difference was maximum after 15 days growth. Glucose content decreased over time in mycelial colonies but increased in spores. The germination of conidia from the two treatments was similar, regardless of compatible solute content, even at –9.25 MPa water potential stress. However, germ tube extension was significantly increased at this water potential level. The production of E. nigrum spores at –3.0 MPa water potential resulted in improved survival when stored fresh at 4 and 25 °C. However, freeze-drying severely affected the viability of spores produced on both media (–0.5 or 3.0 MPa). Accumulation of compatible solutes may assist the fungus in better ecological competence and establishment in the phyllosphere, where water availability is often limited.This revised version was published online in October 2005 with corrections to the Cover Date.     

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

A major event in the nutrient germination of spores of Bacillus species is release of the spores'' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores'' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.