Peculiarities of Exogenous Dormancy of Aspergillus nigerConidia

Aspergillus nigerconidia are characterized by exogenous dormancy: the first stage of their germination is accomplished in twice-distilled water. However, germ tube formation requires the availability of carbon and nitrogen sources. Exogenous dormancy in A. nigerconidia exhibits the following peculiar features: (i) nitrogen-containing substances are active stimulators of germination; (ii) temperature-dependent changes in the lipid bilayer and in the neutral lipid composition of conidia are virtually identical to those occurring in growing mycelium under temperature stress; and (iii) the spore viability threshold does not exceed 45°C; i.e., the spores are more heat-resistant than the mycelium, but they are less heat-resistant than the spores that are in the state of endogenous dormancy. According to the current classification of the types of cell metabolism arrest, the exogenous dormancy of A. nigerconidia resembles the pattern of metabolism characteristic of vegetative cells during the idiophase.     

泥炭地苔藓植物孢子生活力的快速检测

适量的烟气能够促进有性繁殖体萌发,但迄今尚无辅助烟气处理探究孢子生活力快速检测方法的研究报道。该文选择毛缘泥炭藓（Sphagnum fimbriatum）、中位泥炭藓（S.magellanicum）和粗叶泥炭藓（S.squarrosum）作为材料,分别使用亚甲基蓝染色法、四唑（TTC）染色法、碘-碘化钾（I₂-KI）染色法和红墨水染色法对泥炭藓孢子进行染色,并比照营养液、烟溶液+营养液培养的孢子萌发试验,对比研究泥炭地苔藓植物孢子生活力快速检测的最佳方法。结果表明：亚甲基蓝染色法的染色效果最为明显,TTC和I₂-KI均未能使泥炭藓孢子着色,孢子对红墨水虽有着色反应但不清晰;与营养液培养相比,添加烟溶液使毛缘泥炭藓、中位泥炭藓和粗叶泥炭藓孢子萌发率分别提高5%、5%和18%;使用亚甲基蓝染色的孢子染色率与经烟溶液处理过的孢子萌发率最为接近。综上认为,亚甲基蓝染色法能快速检测泥炭藓孢子的生活力。     

Resting Spore Dormancy and Infectivity Characteristics of the Potato Powdery Scab Pathogen Spongospora subterranea

The soil‐borne potato pathogen Spongospora subterranea persists in soil as sporosori, which are aggregates of resting spores. Resting spores may germinate in the presence of plant or environmental stimuli, but direct evidence for resting spore dormancy is limited. A soilless tomato bait plant bioassay and microscopic examination were used to examine features of S. subterranea resting spore dormancy and infectivity. Dried sporosori inocula prepared from tuber lesions and root galls were infective after both short‐ and long‐term storage (1 week to 5 years for tuber lesions and 1 week to 1 year for root galls) with both young and mature root galls inocula showing infectivity. This demonstrated that a proportion of all S. subterranea resting spores regardless of maturity exhibit characteristics of stimuli‐responsive dormancy, germinating under the stimulatory conditions of the bait host plant bioassay. However, evidence for constitutive dormancy within the resting spore population was also provided as incubation of sporosorus inoculum in a germination‐stimulating environment did not fully exhaust germination potential even after 2.4 years. We conclude that S. subterranea sporosori contain both exogenous (stimuli‐responsive) and constitutively dormant resting spores, which enables successful host infection by germination in response to plant stimuli and long‐term persistence in the soil.     

Characteristics of exogenous dormancy of Aspergillus niger conidia]

Aspergillus niger conidia are characterized by exogenous dormancy: the first stage of their germination is accomplished in twice distilled water. However, germ tube formation requires the availability of carbon and nitrogen sources. Exogenous dormancy in A. niger conidia exhibits the following peculiar features: (i) nitrogen-containing substances are active stimulators of germination; (ii) temperature-dependent changes in the lipid bilayer and in the neutral lipid composition of conidia are virtually identical to those occurring in growing mycelium under temperature stress; and (iii) the spore viability threshold does not exceed 45 degrees C; i.e., the spores are more heat-resistant than the mycelium, but they are less heat-resistant than the spores that are in the state of endogenous dormancy. According to the current classification of the types of cell metabolism arrest, the exogenous dormancy of A. niger conidia resembles the pattern of metabolism characteristic of vegetative cells during the idiophase.     

Alterations in the carbohydrate composition of the cytosol of fungal spores caused by temperature variations and the storage process

Differences in the carbohydrate composition were revealed among spores of fungi belonging toZygomycetes, Ascomycota, Basidiomycota, andOomycota, part of the novel kingdomChromista. It was shown for the first time thatPhytophthora infestons contains arabitol in addition to glucose and trehalose. Sucrose was detected inPleurotus ostreatus basidiospores. It was established thatBlakeslea trispora stylospores contain inositol. The dependence of the spore carbohydrate composition on the temperature of the habitat of the corresponding species is discussed. It was shown that the cytosol of the conidia is dominated by trehalose and inositol under hypothermic conditions and by mannitol and glucose under hyperthermic conditions.Neomycota andEomycota were shown to differ in their responses to stress (starvation), which correlated with the differences in the carbohydrate composition of the spore cytosols. Assuming that cytosol carbohydrates perform a protective function, we explain the higher viability of conidia compared to stylo- and sporangiospores.     

Dry/wet cycling reduces spore germination and viability in six peatland bryophytes

• Dry/wet cycling driven by water level fluctuation in wetlands may strongly influence the destiny of seeds. However, how dry/wet cycling affects spore survival and germinability in peatland bryophytes is poorly understood.
• Six peatland bryophytes, three hummock- and three hollow-dwelling Sphagnum species, were chosen as study species. We tested the effects of dry (60% air RH)/wet (waterlogging) cycle frequency (once per 12, 8 or 4 days for low, medium or high, respectively) and ratio (3:1, 1:1 or 1:3 dry:wet time per cycle) on spore germinability, viability, dormancy percentage and protonema development.
• Dry/wet cycling significantly reduced spore germination percentage and viability and slowed protonema development in all Sphagnum species, being more pronounced with higher dry/wet cycling frequencies. The hummock species S. capillifolium and S. fuscum had higher spore germination percentage after the continuous dry treatment, while the hollow species S. angustifolium, S. squarrosum and S. subsecundum showed the opposite response, compared to the continuously wet treatment. Except for S. squarrosum, spore viability was higher after the dry than after the wet treatment. Spore viability and dormancy percentage were higher after a dry/wet ratio of 1:3 than after ratios of 3:1 and 1:1.
• Our study shows that both germinability and viability of bryophyte spores are reduced by dry/wet cycling (especially when frequent) in peatlands. This emphasizes the need to ensure constant water levels and low frequencies of water level fluctuation, which are relevant in connection with wetland restoration, to promote Sphagnum spore survival and establishment in peatlands after disturbances.

     

“INTERNAL THECAE” OF EUNOTIA SOLEIROLII (BACILLARIOPHYCEAE): DEVELOPMENT,STRUCTURE AND FUNCTION AS RESTING SPORES1

The “double thecae” or “internal septa” of Eunotia soleirolii (Kütz.) Rabenh, are shown to represent the thecae of resting spores, as characterized by their physiology, as well as morphology. They differ from all resting spores of centric diatoms by the formation of both their valves as a result of unequal cell divisions; and, from the majority of centric spores by the presence of several girdle bands in both their thecae. Spore formation can be induced by high or low pH, high temperature (24 C), and iron, silica, phosphate or nitrate deficiencies, whereas low temperatures defer it. Spores do not germinate directly, but dormancy can be removed by dark treatments (–2 to 15 C) for a minimum of 4–5 wk. Longer dark treatments result in higher germination rates. At 15 C, a minimum of 2 mo is required and 4 mo is better. Heat treatments (27–42 C) are ineffective, but may shorten the dormancy-breaking subsequent cold period. Instances of secondary dormancy, as well as relative dormancy, were observed. Germination usually occurs in the light between 2 and 21 C. An equal division of the spore is followed by unequal divisions of both new cells with only the two resulting large cells being viable. The experiences in the laboratory aided the discovery of stages of spore germination in nature.     

Influence of Temperature and Relative Humidity on Germinability of Mucor piriformis Spores

Studies were conducted to determine the influence of temperature and relative humidity (RH) on germinability and viability of Mucor piriformis spores. Spores did not survive when stored at 35 °C and their survival rate decreased rapidly at 30 °C; however, spores remained viable for more than 1 year at 0 °C. RH also significantly affected spore viability. Spores held at 26 °C and 100% RH no longer germinated after 35 days, while those held at 75 or 90% RH germinated for 65 days. At 20 °C, RH had little effect on spore germinability. The effect of temperature and RH on percentage spore germination also varied. At all temperatures studied, spore viability decreased more rapidly with time at 100% RH than at 75 or 90% RH. The least favorable, temperature-humidity combination, 30 °C and 100% RH, decreased spore germination from 100% to less than 1% in 14 days.     

Effect of inhibitors of trypsin-like proteolytic enzymes Bacillus cereus T spore germination.

The germination of Bacillus cereus T spore suspensions is partially prevented by several inhibitors of trypsin-like enzymes. Leupeptin, antipain, and tosyl-lysine-chloromethyl ketone are effective inhibitors, whereas chymostatin, elastatinal, and pepstatin are inactive. A synthetic substrate of trypsin, tosyl-arginine-methyl ester, also inhibits germination. Its inhibitory effect decreases as a function of incubation time in the presence of spores and is abolished by previous hydrolysis with trypsin. Germinating, but not dormant, spore suspensions hydrolyze tosyl-arginine-methyl ester; its hydrolysis is insensitive to chloramphenicol, sulfhydryl reagents, and EDTA. A crude extract of germinated B. cereus spores contains a trypsin-like enzyme whose activity, as measured by hydrolysis of benzoyl-arginine p-nitroanilide, is sensitive to germination-inhibitory compounds such as leupeptin, tosyl-arginine-methyl ester, and tosyl-lysine-chloromethyl ketone. Spore suspensions exposed to the above inhibitors under germination conditions lose only part of their heat resistance and some 10 to 30% of their dipicolinic acid content. Part of the germinating spore population becomes "phase grey" under phase optics. Based on a study of the inhibition of germination by protease inhibitors and the activity of a protease in germination spores and spore extracts, it is suggested that the activity of a trypsin-like enzyme may be involved in the mechanism of the breaking of dormancy in spores of B. cereus T.     

Germination of spores of mycelial fungi in relation to exogenous dormancy

Comparative analysis of germination of asexual sporulation spores (conidia and sporangiospores) and of specific features of dormancy release was carried out for ascomycete mycelial fungi Aspergillus tamarii VKM F-64 and A. sydowii VKM F-441, as well as for zygomycete fungi Cunninghamella echinulata VKM F-663 and Umbelopsis ramanniana VKM F-582. The spores of these strains were shown to be in a state of exogenous dormancy and differed in lag phase duration and germination rate, which depended on the presence of nutrients in the medium. Only the strain C. echinulata VKM F-663 exhibited 100% spore germination, with the germination rate and lag phase duration not depending on the composition of the medium. While in A. tamarii strain VKM F-64, the total number of spores germinating on rich and poor media was also almost the same, in the absence of nutrients lag phase duration increased and the germination rate decreased. For strains U. ramanniana VKM F-582 and A. sydowii VKM F-441, the degree of spore germination in the absence of nutrients in the medium was considerably lower than on the rich medium, while the lag phase was longer. These data indicate that the spores of C. echinulata VKM F-663 are in the state of exogenous dormancy, which does not require for release any compounds except water. The spores of U. ramanniana strain VKM F-582 and of the Aspergillus strains exhibited another variant of exogenous dormancy, which required for release, apart from water, also the sources of carbon and nitrogen. Thus, the character of dormancy release may differ even within a single genus (Aspergillus).     

Fungal spores: dormancy, germination, chemical composition, and role in biotechnology (review)

This review is focused on one of the stages of ontogenesis distinctive by its particular tolerance to the action of unfavorable factors and ability to retain the genomic material for a long period of time, i.e., fungal spores. The major part is devoted to the characterization of the specific stage typical for spores, which is called dormancy. Data are presented characterizing the carbohydrate and lipid composition of spores, with special attention being paid to the role of carbohydrate protectors, in particular, trehalose and mannite, as well as to the role of rafts in the process of sporogenesis. The role of special compounds called autoinhibitors and autostimulators in the process of exit from dormancy is discussed. The final section deals with the role of spore seeding material in biotechnological processes. Data on the correlation between the chemical composition of spores, their ability to remain dormant, and the germination process are considered. Special biotechnological approaches are presented for the first; they allow for the preservation of the germinating ability of spores, intensification of sporogenesis, changes in the ratio of final fermentation products, and an increase in their yield.     

Fungal spores: Dormancy, germination, chemical composition, and role in biotechnology (review)

This review is focused on one of the stages of ontogenesis distinctive by its particular tolerance to the action of unfavorable factors and ability to retain the genomic material for a long period of time, i.e., fungal spores. The major part is devoted to the characterization of the specific stage typical for spores, which is called dormancy. Data are presented characterizing the carbohydrate and lipid composition of spores, with special attention being paid to the role of carbohydrate protectors, in particular, trehalose and mannite, as well as to the role of rafts in the process of sporogenesis. The role of special compounds called autoinhibitors and autostimulators in the process of exit from dormancy is discussed. The final section deals with the role of spore seeding material in biotechnological processes. Data on the correlation between the chemical composition of spores, their ability to remain dormant, and the germination process are considered. Special biotechnological approaches are presented for the first; they allow for the preservation of the germinating ability of spores, intensification of sporogenesis, changes in the ratio of final fermentation products, and an increase in their yield.     

Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore properties

Aims: To determine effects of inner membrane lipid composition on Bacillus subtilis sporulation and spore properties. Methods and Results: The absence of genes encoding lipid biosynthetic enzymes had no effect on B. subtilis sporulation, although the expected lipids were absent from spores’ inner membrane. The rate of spore germination with nutrients was decreased c. 50% with mutants that lacked the major cardiolipin (CL) synthase and another enzyme for synthesis of a major phospholipid. Spores lacking the minor CL synthase or an enzyme essential for glycolipid synthesis exhibited 50–150% increases in rates of dodecylamine germination, while spores lacking enzymes for phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysylphosphatidylglycerol (l‐PG) synthesis exhibited a 30–50% decrease. Spore sensitivity to H₂O₂ and tert‐butylhydroperoxide was increased 30–60% in the absence of the major CL synthase, but these spores’ sensitivity to NaOCl or Oxone^? was unaffected. Spores of lipid synthesis mutants were less resistant to wet heat, with spores lacking enzymes for PE, PS or l‐PG synthesis exhibiting a two to threefold decrease and spores of other strains exhibiting a four to 10‐fold decrease. The decrease in spore wet heat resistance correlated with an increase in core water content. Conclusions: Changing the lipid composition of the B. subtilis inner membrane did not affect sporulation, although modest effects on spore germination and wet heat and oxidizing agent sensitivity were observed, especially when multiple lipids were absent. The increases in rates of dodecylamine germination were likely due to increased ability of this compound to interact with the spore’s inner membrane in the absence of some CL and glycolipids. The effects on spore wet heat sensitivity are likely indirect, because they were correlated with changes in core water content. Significance and Impact of the Study: The results of this study provide insight into roles of inner membrane lipids in spore properties.     

Oxidative Damage Involves in the Inhibitory Effect of Nitric Oxide on Spore Germination of <Emphasis Type="Italic">Penicillium expansum</Emphasis>

The effects of nitric oxide (NO) on spore germination of Penicillium expansum were investigated and a possible mechanism was evaluated. The results indicated that NO released by sodium nitroprusside (SNP) significantly suppressed fungal growth. With the use of an oxidant sensitive probe and Western blot analysis, an increased level of intracellular reactive oxygen species (ROS) and enhanced carbonylation damage were detected in spores of P. expansum under NO stress. Exogenous superoxide dismutase (SOD) and ascorbic acid (Vc) could increase the resistance of the spore to the inhibitory effect of NO. The activities of SOD and catalase (CAT), as well as ATP content in spores under NO stress were also lower than those in the control. We suggest that NO in high concentration induces the generation of ROS which subsequently causes severe oxidative damage to proteins crucial to the process of spore germination of P. expansum.     

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.     

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。     

The effect of sunlight on allergen release from spores of the fungus Alternaria

Airborne fungal spores are known carriers of allergen. Correlations between spore counts and allergen concentrations are poor. It is known that germination increases allergen release, implicating spore viability as a determinant of allergen release. During aerial dispersal, spores can be exposed to prolonged periods of ultraviolet (UV) light which can reduce viability of spores. We examined the relation between spore viability and allergen release in two experiments: firstly spores from culture were treated with a UV wavelength of 254?nm (not present in sunlight reaching the earth's surface) or autoclaved, and secondly, spores were exposed to simulated sunlight over three days. In both studies viability was measured (by germination on agar and by metabolic activity with nitro-blue tetrazolium vital stain) and allergen release by the Halogen immunoassay. The UV light reduced the proportion of spores able to germinate but did not affect metabolic activity or allergen release. Autoclaving reduced the proportion of spores releasing allergen by half (p<0.0001). Three days' exposure to simulated sunlight correlated negatively with spore germination and metabolic activity (p<0.0001), but did not affect allergen release (p=0.799). In conclusion, simulated sunlight reduced the metabolic activity and germinability of spores however the proportion releasing allergen remained unaffected. These findings suggest that spore counts may reflect allergen concentrations in the air if spores are dead or dormant. The contribution of viable spores to concentrations of airborne allergen, as well as the role of germination in allergen delivery to the respiratory tract, remains to be resolved.     

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.     

Effect of the composition of the medium and magnesium and calcium ions on the germination of spores of Thermoactinomyces vulgaris]

The germination of the spores of Thermoactinomyces vulgaris formed on a complex medium is stimulated by suspending them in solutions containing Mg2+ and Ca2+ ions. The stimulation is not the result of the initiation of the spores in the presence of the ions since the experiments were carried out at a temperature of 20 degrees C at which the initiation did not virtually take place. The ions of Na+ and K+ have almost no effect on the germination of the spores. The fraction of the resting spores of Thermoactinomyces vulgaris depends on the composition of the growth medium, especially on its amino acid composition. The addition of Mg2+ and Ca2+ ions to a minimal synthetic growth medium stimulates the growth of the cultures and decreases the dormancy of the spores. The spores formed on the synthetic medium are less thermostable than the spores formed on the complex medium. Thermostability of the spores increases upon the addition of Mg2+ to the synthetic medium. Spore suspensions obtained on the synthetic medium with Mg2+ or Ca2+ are initiated more completely than spore suspensions obtained on the complex medium.