Activation ofStreptomyces viridochromogenes spores by detergents

Constitutively dormant spores ofStreptomyces viridochromogenes germinate rapidly following treatment with 1.0% of the detergents Tween 80, sodium dodecyl sulfate (SDS), or sodium heptadodecyl sulfate. Six other detergents did not activate the spores. Activation by SDS was studied further. The spores were not activated following treatment with 0.09% or less of SDS for 60 min at 37°C. Activation was complete within 1 to 2 min of treatment with 1.0% SDS. the SDS-activated spores became deactivated during incubation in buffer. Deactivation was slow at 4°C and complete after incubation for 12 h at 25°C or 6 h at 37°C. The endogenous respiratory rate of the spores was increased 3-fold by SDS activation.     

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.     

Effects of temperature on the spores of thermophilic actinomycetes

The effects of temperature on the germination properties of spores of thermophilic actinomycetes were examined. Temperatures above and below the growth temperature of 55° C were found to produce marked changes in the germination properties of spores. High temperatures caused reductions in the germinative activities of spores. However, heated spore populations regained original germinative activities after maintaining them for suitable periods of time at 25°C. Recovery from the effects of heat on spore germination was also observed at 4°C, but at a much slower rate compared with 25°C. Spores of two strains of thermophilic actinomycetes, grown and prepared at 55°C, failed to germinate. Storage of dormant (nonactivated) spore populations at different temperatures demonstrated a low temperature requirement for the activation of these spores; while little or no activation occurred at 55°C, rapid activation took place at 25°C. Heating the spores at 80°C for 30 min slightly delayed the activation (rates) of spores at 25°C. The requirement of low temperature for spore activation was strain dependent and was influenced by the composition of the germination medium.     

Germination and water dispersal of seeds from a threatened plant species Penthorum chinense

To conserve a threatened plant species (Penthorum chinense Pursh) in Japan, seed germination responses to pretreatment (imbibition and/or chilled), temperature and light, and seed dispersal by water were examined. The seeds collected from abandoned paddy fields in a warm temperate region, central Japan, germinated in light (14 h photoperiod; light 22°C, dark 21°C) after a moist-chilled treatment. After this pretreatment, the seeds germinated well at 10–25°C (optimum temperature 15°C), but did not germinate in darkness even at the optimum temperature. Most of the seeds floated on distilled water, but 20–60% of the seeds that were collected from several populations sank in distilled water, indicating dimorphism in seed dispersal by water. The floating and sunken seeds did not show significant differences in weight and germination rate within a population. The addition of a surface-active agent in distilled water submerged the seeds, indicating that the buoyancy of the seeds is attributable to an oil coating on the seed surface that enhances the interfacial tension on the seeds. Three times the number of seeds sank in river water collected from a rural area than in distilled water. A greater number of seeds also sank in water that had increasing concentrations of linear alkylbenzenesulfonate, which is a major component of synthetic detergents. This suggests that the water dispersal of this species is suppressed by surface-active agents, including detergents, in river water.     

Inducing sporulation in Alternaria solani I. Effect of water treatment

The sporulation was induced when fully grown cultures were given dip or spray treatment with distilled water (cold or hot) and thereafter, kept partially covered at different temperatures. Cultures dipped in cold water (4° C) for 4 minutes or sprayed with cold water (4° C) or hot water (58° C) and thereafter incubated at room temperature (13–26° C) in diffused sunlight, produced maximum number of spores within 60 hours. Incubating water treated cultures in diffused sunlight or complete darkness and age and scraping of the cultures had a considerable effect upon intensity of sporulation. The cultures yield a number of subsequent crops of spores when scraped and given dip treatment with cold or hot water, after obtaining each crop of spores.     

Influence of salinity and temperature on the germination of Kochia scoparia

Kochia scoparia is one of the most common annual halophytes foundin the Great Basin. Seeds were collected from a population growing in asalt playa at Faust, Utah and were germinated at 5 temperature regimes(12 h night/12 h day, 5–15 ^°C, 10–20 ^°C, 15–25 ^°C,20–30 ^°C and 25–35 ^°C) and 6 salinities (0, 200, 400,600, 800 and 1000 mM NaCl) to determine optimal conditions forgermination and recovery of germination from saline conditions after beingtransferred to distilled water. Maximum germination occurred in distilledwater, and an increase in NaCl concentration progressively inhibited seedgermination. Few seeds germinated at 1000 mM NaCl. A temperatureregime of 25 ^°C night and 35 ^°C day yielded maximumgermination. Cooler temperature 5–15 ^°C significantly inhibited seedgermination. Rate of germination decreased with increase in salinity.Germination rate was highest at 25–35 ^°C and lowest at5–15 ^°C. Seeds were transferred from salt solutions to distilled waterafter 20 days and those from high salinities recovered quickly at warmertemperature regimes. Final recovery germination percentages in high salttreatments were high, indicating that exposure to high concentration ofNaCl did not inhibit germination permanently.     

Biological control of postharvest blue mold of oranges by <Emphasis Type="Italic">Cryptococcus laurentii </Emphasis>(Kufferath) Skinner

Cryptococcus laurentii (Kufferath) Skinner was evaluated for its activity in reducing postharvest blue mold decay of oranges caused by Penicillium italicum in vitro and in vivo. The results showed that washed cell suspensions of yeast provided control of blue mold decay better than yeast in culture broth. Autoclaved cell culture and cell-free culture filtrate failed to provide protection against the pathogen. The concentrations of antagonist had significant effects on biocontrol effectiveness. When the washed yeast cell suspension reached the concentration of 1 × 10⁹ CFU/ml, challenged with pathogen spore suspension at 1 × 10⁴ spores/ml, the blue mold decay was completely inhibited during 5 days of incubation at 20 °C. No complete control was obtained when oranges were stored at 4 °C for 30 days, but the decay was distinctly prevented. Efficacy of C. laurentii was maintained when applied simultaneously or prior to inoculation with P. italicum. Efficacy was reduced when C. laurentii was applied after inoculation. In drop-inoculated wounds of oranges, the populations of C. laurentii increased by approximately 50-fold during the first 24 h at 20 °C. The maximum yeast populations, approximately 250-fold over the initial populations, were reached 15 days after inoculation at 4 °C.     

Spore germination in Dictyostelium discoideum

Mutant spores of Dictyostelium discoideum, strain SG-10, differ from wild type spores in their ability to spontaneously germinate, to be activated with 5% dimethyl Sulfoxide (DMSO), and to be deactivated with 0.2 M sucrose. Both heat-activated wild type and mutant spores began to swell after a lag of 60–75 min at ambient temperature. Suspension of heat activated spores in 5% DMSO resulted in blockage of spore swelling and a concomitant severe inhibition of respiration; removal of 5% DMSO allowed resumption of respiration and the spores began to swell after a lag of only 15 min. It was concluded that 5% DMSO allowed the early reactions (M) to proceed but blocked the later reactions (R) of post-activation lag.Treatment of one day old spores with 20% DMSO solution for 30–120 min quantitatively activated the population. The post-activation lag time was directly dependent on the time of 20% DMSO treatment. Spores activated with 20% DMSO treatment could be deactivated by incubation at 0°C; the spores most quickly deactivated at 0°C were those within 10 min of swelling. Mitochondrial transport inhibitors such as azide and cyanide caused deactivation in an analogous manner. It is hypothesized that spores proceed to the second portion of the lag phase called (R) before the environment determines if dormancy is reimposed or if germination will proceed. The sensitive strain (SG-10) showed a greater degree of damage than the wild type after supraoptimal treatment with 40% DMSO. The spores became more resistant with age to the damaging action of 40% DMSO. All the observed effects of DMSO treatment were compatible with our multistate model of activation which suggests that the early portion of the lag phase (M) may involve a relative uncoupling of oxidative phosphorylation while the later portion (R) may require tight coupling.     

In vitro activation and behavior of the ameboid sperm of Ascaris suum (Nematoda)

Summary A system is described for the study of activation and motility of Ascaris spermatozoa in vitro. Activation was accomplished by addition of the sperm-activating substances (SAS), extracted from the male accessory gland, to cells incubated in phosphate-buffered saline (pH 7.4) at 37–39° C under anaerobic conditions (95% N₂, 5% CO₂). Activation is characterized by a change from spherical to ameboid shape with coalescence of the refringent granules. The normal ameboid spermatozoa bear several stubby and needle-like filopodia at the lamellipodial margin. Within the lamellipodium are bundles of microfilament-like structures extending toward the pseudopodial membrane and concentrating within the needle-like filopodia. These filopodia exhibit a pendulous, sweeping motion with subsequent retraction and disappearence within the main lamellipodium. Membranes of the ameboid cells interact at the pseudopodial regions with partial fusion, as suggested by apparent membrane breakdown between interdigitating portions of the pseudopodia. Activation is complete in 5–15 min, is totally inhibited at 4° C and/or by an atmospheric environment, but can be reinitiated by transfer to anaerobic conditions at 22–9° C. Activation also requires favorable pH (6.8–8.7) and continual exposure to sufficiently high sodium concentrations (134–154mM), i.e., lowering of sodium concentration to 10 mM causes irreversible inactivation. Sodium may be replaced by potassium or lithium but not by Tris or sucrose. Proteinases (10 g/ml) can act as activators even though SAS lack detectable proteolytic activity against azoalbumin, azocasein, TAME and BTEE and SAS activation was not inhibited by TLCK or soybean trypsin inhibitor.Adult Ascaris suum were provided through the generosity of Wilson and Company, Cedar Rapids, Iowa, U.SA. This study was supported by grant number 5T01 HD00152 and postdoctoral fellowship 1F 32AI05646 from the National Institute of Health, U.S.A.     

Agars from Gelidium rex (Gelidiales,Rhodophyta)

Gelidium rex grows in the rocky intertidal of central Chile. Extraction of vegetative G. rex with water at 95 °C yielded 17.9% of agar with a gel strength value of 590 g cm^–2. The gel strength increased up to 1272 g cm^–2 when the alga was treated with alkali prior to extraction. Cystocarpic and tetrasporic thalli of G. rex were extracted with distilled water at 95 °C, affording soluble polysaccharides in 36.0% and 15.7% yield respectively. Polysaccharides of both life history phases were fractionated by chromatography on DEAE Sephadex. Elution with distilled water gave fractions devoid of sulfate; the fraction from cystocarpic plants contained 45.5% of 3,6-anhydrogalactose whereas the neutral fraction from tetrasporic plants contained 40.0% of 3,6-anhydrogalactose. Further elution with 0.1, 0.4, 0.8 and 1.5 molar aqueous solutions of KCl afforded four fractions for each polysaccharide. Chemical analysis of these fractions showed that the agars from cystocarpic and tetrasporic Gelidium rex are mixtures of related polysaccharides that range from neutral polymers to highly sulfated galactans.     

The effect of gamma radiation on breaking of dormancy in Phycomyces spores

Dormant sporangiospores of Phycomyces blakesleeanus were activated by Cs-137 gamma rays. After a dose of about 300 krad, between 80 and 90% of the spores germinated normally. However, further development of the mycelium was inhibited. Even with dry spores a partial activation was obtained. Activation by gamma rays was not accompanied by an increase in trehalase activity.     

Experiments inducing prospective polar body nuclei to participate in embryogenesis of the sawfly Athalia rosae (Hymenoptera)

Summary Mature eggs dissected from ovaries of unmated females of Athalia rosae (Hymenoptera: Tenthredinidae), if placed on a filter-paper soaked with distilled water, are activated and develop to haploid males. Occasionally, however, diploid females develop from these artificially activated eggs. Treatment of mature unfertilized eggs dissected from diploid females with ice-cold temperatures immediately before activation and with a high temperature (36° C) upon and immediately after activation resulted in the production of diploid males, diploid females, triploid females and gynandromorphs at high frequency. The same treatment of mature unfertilized eggs dissected from triploid females resulted in the production of only triploid survivors. These results, together with the results on the segregation of a marker mutation, yellow fatbody (yfb), appear to indicate that meiotic divisions were complete in the treated eggs, and that all four nuclei became potentially capable of participating in development with or without automictic fusion.Studies on the sawfly, Athalia rosae (Insecta, Hymenoptera, Tenthredinidae), part V     

Interrelationship betweenCoprinus lagopus andFusarium udum in soil in relation to competitive saprophytic ability and microbial antagonism

Summary The interrelationship between the growth ofCoprinus lagopus andFusarium udum on pigeon pea substrates in soil was studied at 22-2°C and 30±2°C in relation to their competitive saprophytic ability and antagonism.C. lagopus was observed to be a potent and frequent colonizer of pigeon pea substrates in soil along withF. udum. Saprophytic colonization of pigeon pea substrate byF. udum precolonized byC. lagopus was inhibited in all inoculum soil mixtures. Saprophytic colonization of the substrate byF. udum was better at 22°C than at 30°C and that byC. lagopus better at 30°C than at 22°C. The colonization of substrate by each fungus was suppressed by the other fungus depending upon the temperature used. Hyphal parasitism and colony interactions between these fungi and also with a number of other saprophytic fungi were investigated. No antagonistic reaction was observed betweenF. udum andC. lagopus. However, these fungi were observed to be potent antagonists against other microfungi tested. The formation of fruiting bodies ofC. lagopus was also observed in the laboratory on nutrient media but more frequently on pigeon pea substrates.     

Heat Activation and Heat-induced Dormancy of Bacillus stearothermophilus Spores

Heat-induced dormancy was observed when spores of two strains of Bacillus stearothermophilus were heated in distilled water at 80, 90, and 100 C. At temperatures above 100 C, true activation occurred; however, maximal activation was not achieved until temperatures of 110 to 115 C were employed. A heat treatment of 115 C for 3 min was required to induce maximal activation in one suspension of strain 1518 spores, whereas a heat treatment of 110 C for 7 to 10 min was adequate for the other suspension of strain 1518 spores. Spores from both strain M suspensions required heat treatments of 110 C for 9 to 15 min for maximal activation. The degree to which the spores could be activated was strain dependent and variable among spore suspensions of the same strain.     

Effect of nitrite,storage temperature and time onClostridium botulinum type A toxin formation in liver sausage

Summary The occurrence ofClostridium botulinum-type A toxin in inoculated, commercially processed Finnish liver sausages that were kept under different storage conditions was studied. Two levels of sodium nitrite addition, three different storage times and three temperatures were used. An addition of 100 mg of sodium nitrite per kg of sausage emulsion prevented toxin formation during a 2-week period. In an inoculated sausage without nitrite the toxin was not produced when stored at 15°C, but was formed at 20 and 25°C.     

Water vapor, aqueous ethyl alcohol, and heat activation of Bacillus megaterium spore germination

Dormant spores of Bacillus megaterium were activated for germination on glucose by heating them in aqueous suspension (but not if heated dry), by treating them with aqueous ethyl alcohol at 30 C, or by exposing them to water vapor at room temperature. The degree of water vapor activation depended upon the relative humidity, the time, and the temperature of exposure. Activation increased the extent and rate of glucose-induced germination and decreased the average microlag. Extended water vapor treatment also activated spores for germination induced by KI and by l-alanine. Spores activated by any of the three treatments were deactivated by treatment at 66 C, either for 18 hr in 100% ethyl alcohol or for 40 hr over P(2)O(5). Deactivated spores were reactivated by heat, by 5 m ethyl alcohol, or by water vapor. It is postulated that heating and ethyl alcohol may change the structure of liquid water, so that it is more like water vapor and can more readily penetrate to and hydrate a critical (enzymatic?) spore site, leading to activation.     

REVERSIBLE ACTIVATION FOR GERMINATION AND SUBSEQUENT CHANGES IN BACTERIAL SPORES

Lee, W. H. (University of Illinois, Urbana) and Z. John Ordal. Reversible activation for germination and subsequent changes in bacterial spores. J. Bacteriol. 85:207-217. 1963.-It was possible to isolate refractile spores of Bacillus megaterium, from a calcium dipicolinate germination solution, that were activated and would germinate spontaneously in distilled water. Some of the characteristics of the initial phases of bacterial spore germination were determined by studying these unstable activated spores. Activated spores of B. megaterium were resistant to stains and possessed a heat resistance intermediate between that of dormant and of germinated spores. The spontaneous germination of activated spores was inhibited by copper, iron, silver, or mercury salts, saturated o-phenanthroline, or solutions having a low pH value, but not by many common inhibitors. These inhibitions could be partially or completely reversed by the addition of sodium dipicolinate. The activated spores could be deactivated and made similar to dormant spores by treatment with acid. Analyses of the exudates from the variously treated spore suspensions revealed that whatever inhibited the germination of activated spores also inhibited the release of spore material. The composition of the germination exudates was different than that of extracts of dormant spores. Although heavy suspensions of activated spores gradually became swollen and dark when suspended in solutions of o-phenanthroline or at pH 4, the materials released resembled those found in extracts of dormant spores rather than those of normal germination exudates.     

Sperm cryopreservation of the critically endangered olive barb (Sarpunti) Puntiussarana (Hamilton, 1822)

The present study focused on development of a sperm cryopreservation protocol for the critically endangered olive barb Puntiussarana (Hamilton, 1822) collected from two stocks within Bangladesh and reared in the Fisheries Field Laboratory, Bangladesh Agricultural University (BAU). The sperm were collected in Alsever’s solution prepared at 296 mOsmol kg⁻¹. Sperm were activated with distilled water (24 mOsmol kg⁻¹) to characterize motility. Maximum motility (90%) was observed within 15 s after activation, and sperm remained motile for 35 s. Sperm activation was evaluated in different osmolalities and motility was completely inhibited when osmolality of the extender was ?287 mOsmol kg⁻¹. To evaluate cryoprotectant toxicity, sperm were equilibrated with 5%, 10% and 15% each of dimethyl sulfoxide (DMSO) and methanol. Sperm motility was noticeably reduced within 10 min, when sperm were equilibrated with 15% DMSO, indicating acute toxicity to spermatozoa and therefore this concentration was excluded in further trials. Sperm were cryopreserved using DMSO at concentrations of 5% and 10% and methanol at 5%, 10% and 15%. The one-step freezing protocol (from 5 °C to −80 °C at 10 °C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia) and 0.25-ml straws containing spermatozoa were stored in liquid nitrogen for 7–15 days at −196 °C. The highest motility in thawed sperm 61 ± 8% (mean ± SD) was obtained with 10% DMSO. The fertilization and hatching rates were 70% and 37% for cryopreserved sperm, and 72% and 62% for fresh sperm. The protocol reported here can be useful for hatchery-scale production of olive barb. The use of cryopreserved sperm can facilitate hatchery operations, and can provide for long-term conservation of genetic resources to contribute in the recovery of critically endangered fish such as the olive barb.     

Germination of Tagetes minuta L. II. Role of gibberellins

The effect of the application of gibberellins to Tagetes minuta L. achenes (seeds) was determined at both 25°C, the optimal germination temperature, and 35°C, at which temperature the achenes are thermoinhibited. Both GA₃ and GA₄₊₇ accelerated germination at 25°C. Seed germination at 25°C was inhibited by paclobutrazol, but on subsequent application of GA₄₊₇ rapid germination was induced. Following application of GA₃ or GA₄₊₇ to thermoinhibited seeds, a significantly higher final germination percentage was observed than in the distilled water control. However, endogenous gibberellin levels in germinating and thermoinhibited seeds did not differ significantly.     

Amperometric determination of sodium nitrite by a microbial sensor

Summary A microbial sensor was prepared to determine sodium nitrite. This microbial sensor consisted of immobilized Nitrobacter sp. and an oxygen electrode. When a sample solution containing sodium nitrite was tested, nitrite was changed to NO₂ gas in the buffer (pH 2.0) and the current of the electrode decreased with time until a steady state was reached. The steady state current was attained within 10 min and the maximum decrease in current was obtained at 30°C and pH 2.0. A linear relationship was observed between the current decrease and the sodium nitrite concentration below 0.59 mM, the minimum sodium nitrite concentration that could be determined was 0.01 mM. The current decrease was reproducible (5% relative error). The current output of the sensor was almost constant for more than 21 days and 400 assays.