Analysis of the germination of individual Clostridium perfringens spores and its heterogeneity

Aims: To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens. Methods and Results: Germination of individual wild‐type Cl. perfringens spores was followed by monitoring Ca‐dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (T_lag) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔT_release) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex‐lytic enzyme (CLE) (sleC spores) exhibited the same features during GR‐dependent germination, but with longer average T_lag values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The T_lag of wild‐type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild‐type and sleC spores had identical average T_lag and ΔT_release values in dodecylamine germination that does not utilize GRs. Conclusions: Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE‐deficient Bacillus spores, indicating that germination of these species’ spores is not completely identical. Significance and Impact of the Study: This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.     

Analysis of the slow germination of multiple individual superdormant Bacillus subtilis spores using multifocus Raman microspectroscopy and differential interference contrast microscopy

Aim: To analyse the dynamic germination of hundreds of individual superdormant (SD) Bacillus subtilis spores. Methods and Results: Germination of hundreds of individual SD B. subtilis spores with various germinants and under different conditions was followed by multifocus Raman microspectroscopy and differential interference contrast microscopy for 12 h and with temporal resolutions of ≤30 s. SD spores germinated poorly with the nutrient germinant used to isolate them and with alternate germinants targeting the germinant receptor (GR) used originally. The mean times following mixing of spores and nutrient germinants to initiate and complete fast release of Ca‐dipicolinic acid (CaDPA) (T_lag and T_release times, respectively) of SD spores were much longer than those of dormant spores. However, the ΔT_release times (T_release?T_lag) of SD spores were essentially identical to those of dormant spores. SD spores germinated almost as well as dormant spores with nutrient germinants targeting GRs different from the one used to isolate the SD spores and with CaDPA that does not trigger spore germination via GRs. Conclusions: Since (i) ΔT_release times were essentially identical in GR‐dependent germination of SD and dormant spores; (ii) rates of GR‐independent germination of SD and dormant spores were identical; (iii) large increases in T_lag times were the major difference in the GR‐dependent germination of SD as compared with spores; and (iv) higher GR levels are correlated with shorter T_lag times, these results are consistent with the hypothesis that low levels of a GR are the major reason that some spores in a population are SD with germinants targeting this same GR. Significance and Impact of the Study: This study provides information on the dynamic germination of individual SD spores and improves the understanding of spore superdormancy.     

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.     

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。     

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.     

The Effect of Ultraviolet Radiation on the Germination of Nosema algerae Vávra and Undeen (Microsporida: Nosematidae) Spores1

Spores of Nosema algerae Vávra and Undeen were subjected to various dosages of 254 nm ultraviolet radiation (UV). Very high dosages of UV were required to block germination. Germination was normal immediately after UV dosages of 0.2 to 1.0 J/cm², followed by a delayed effect in which both percentage germination and the intrasporal concentration of trehalose decreased with time after UV exposure. Although a few spores were germinated, most of them were inactivated (rendered temporarily unable to germinate) by exposure to UV of 1.1 J/cm². Ultraviolet radiation between 1.1 and 3.4 J/cm² stimulated spores to germinate. However, spores were completely unable to germinate immediately after exposure to dosages above 3.8 J/cm². Ammonia had little effect on stimulation by UV but was inhibitory to germination after stimulation had occurred. These results demonstrate that UV behaves like a germination stimulus and are discussed in terms of the hypothesis that germination is initiated by the breakdown of barriers between trehalose and trehalase.     

Iron and temperature as sporangiospore germination factors of Pilobolus longipes

Sporangiospores of Pilobclus longipes germinated on a medium containing ascorbate and FeSO₄, but neither ascorbate nor FeSO₄ alone caused spores to germinate. The iron chelates (hemin, coprogen, and ferrichrome) that are known to promote mycelial growth of this and other species of Pilobolus had little or no effect on spore germination, suggesting that under these conditions dormant spores are unable to reduce iron III.Regardless of the medium used, maximum germination required treatment at two temperatures. The early stage of germination, spherical growth, was favored by treatment for several hours at about 38°C while optimum germ tube formation required incubation at lower temperatures (25°C). Under most conditions the requirement for a heat treatment was nearly absolute.When the iron-ascorbate and the heat treatments were separated it was found that they need not be applied simultaneously provided that iron and ascorbate are given first. Spores that were heated first and then given iron and ascorbate at lower temperatures did not germinate. Apparently dormancy of these spores is broken by available iron but a heat treatment is usually required to complete the germination process.     

Possible involvement of Ca<Superscript>2+</Superscript>-dependent protein kinases in spore germination of the fern<Emphasis Type="Italic">Osmunda Japonica</Emphasis>

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca²⁺ for the germination. Furthermore, we showed that Sr²⁺ and Mn²⁺, which could substitute for Ca²⁺ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.     

Light germination ofAnthoceros miyabeanus spores

The effects of light on the spore germination of a hornwort species,Anthoceros miyabeanus Steph., were investigated. Spores of this species were photoblastic, but their sensitivities to light quality were different. Under either continuous white, red or diffused daylight, more than 80% of the spores germinated, but under blue light none or a few of them germinated. Under continuous far-red light or in total darkness, the spores did not germinate at all.Anthoceros spores required red light irradiation for a very long duration, i.e., over 12–24 hr of red light for saturated germination. However, the spore germination showed clear photo-reversibility by repeated irradiation of red and far-red light. The germination pattern clearly varied with the light quality. There were two fundamental patterns; (1) cell mass type in white or blue light: spores divide before germination, and the sporelings divide frequently and form 1–2 rhizoids soon after germination, and (2) germ tube type in red light: spores germinate without cell division, and the single-cell sporelings elongate without cell division and rhizoid formation.     

The preparation,germination properties and stability of superdormant spores of Bacillus cereus

Aims: To determine yields, germination and stability of superdormant Bacillus cereus spores. Methods and Results: Superdormant B. cereus spores were isolated by germination with high concentrations of inosine or l ‐alanine in 2–5% yield and did not germinate with high concentrations of either of these germinants, but germinated like starting spores with Ca‐DPA, dodecylamine, l ‐alanine plus inosine or concentrated complete medium. Yields of superdormant spores from germinations with low inosine concentrations were higher, and these spores germinated poorly with low inosine, but relatively normally with high inosine. Yields of superdormant spores were also higher when nonheat‐activated spores were germinated. Superdormant spores stored at 4°C slowly recovered some germination capacity, but recovery was slowed significantly at ?20°C and ?80°C. Conclusions: Factors that influence levels of superdormant B. cereus spores and the properties of such spores are similar to those in B. megaterium and B. subtilis, suggesting there are common mechanisms involved in superdormancy of Bacillus spores. Significance: Superdormant spores are a major concern in the food industry, because the presence of such spores precludes decontamination strategies based on triggering spore germination followed by mild killing treatments. Studies of the properties of superdormant spores may suggest ways to eliminate them.     

A New Regulatory Function of A-Factor: Stimulation of the Germination of Streptomycete Spores

Spore germination in streptomycetes was shown to be stimulated by exogenously added A-factor. Agar medium either containing or not containing A-factor was inoculated with spore suspensions of three strains differing in their ability to produce regulators of the A-factor group: Streptomyces griseus 773, which produces A-factor and two its lower homologs; S. coelicolor A3(2), which forms six Acl-factors (A-factor analogues); and S. avermitilis JCM5070, which fails to form regulators of this group. A count of the grown colonies showed that exogenous A-factor stimulated spore germination in strains that were themselves able to synthesize regulators of the A-factor group. In S. griseus 773, the number of germinated spores increased by 67% on average after the addition of A-factor to the medium in an amount of 10 g/ml. In strain S. coelicolor A3 (2), the number of germinated spores increased by 75% after the addition of 1 g/ml of A-factor. During germination of the S. avermitilis JCM5070 spores, no changes in the CFU number was observed after the addition of A-factor.     

GA3浸种处理对五种晚花杜鹃种子萌发的影响

晚花杜鹃(late flower Rhododendron)是一类花期较晚的杜鹃品种,具有较高的观赏价值,被广泛应用于庭院种植和园林绿化。随着旅游以及经济发展的需要,开发和利用晚花杜鹃资源显得非常迫切。自然条件下晚花杜鹃萌发率相对较低,因此为提高晚花杜鹃萌发率,探究不同浓度赤霉素(GA_3)处理对五种杜鹃种子萌发的影响,该文以五种晚花杜鹃(小白杜鹃、大白杜鹃、桃叶杜鹃、长蕊杜鹃和九龙山杜鹃)为实验材料,通过不同浓度(0、300、400、500、600、700 mg·L~(-1))的GA_3对五种晚花杜鹃种子进行24 h浸种处理,测定其发芽指数、发芽势、发芽率以及成苗率等指标,分别确定五种杜鹃种子萌发的最适GA_3浓度,并对相同处理下五种杜鹃种子的萌发率和成苗率进行比较。结果表明:(1) GA_3浸种对五种晚花杜鹃种子萌发具有促进作用,在适当GA_3浓度下五种杜鹃种子的发芽指数、发芽势和发芽率均显著高于对照组,萌发时滞、萌发高峰期和持续萌发时间均较对照组浓度相对缩短。(2)大白杜鹃、桃叶杜鹃和九龙山杜鹃种子在GA_3浓度为600 mg·L~(-1)处理时各项萌发指标相对较好;长蕊杜鹃以GA_3浓度为700 mg·L~(-1)浸种处理萌发效果相对较好;小白杜鹃以GA_3浓度为400和700 mg·L~(-1)处理最好。因此,在杜鹃的栽培中,可以采用赤霉素GA_3处理法提高种子发芽率,缩短萌发时间。     

The effect of <Emphasis Type="Italic">Baccharis glutinosa</Emphasis> extract on the growth of mycotoxigenic fungi and fumonisin B<Subscript>1</Subscript> and aflatoxin B<Subscript>1</Subscript> production

The aim of this study was to evaluate the effect of Baccharis glutinosa isolated extract on the growth of Aspergillus flavus and Aspergillus parasiticus, and their aflatoxin B₁ production; and growth of Fusarium verticillioides, and their fumonisin B₁ production. The three fungi were exposed to an antifungal fraction, designated as fraction F6-1, isolated from B. glutinosa by methanolic extraction followed by silica gel chromatography. The growth of the fungi was evaluated in kinetics of radial extension growth, kinetics of spores germination, length and diameter of hyphae, spores diameter, as well as in aflatoxin B₁ and fumonisin B₁ production. Fraction F6-1 caused radial growth inhibition of the three fungi mainly F. verticillioides. Spores germination of A. flavus and A. parasiticus was delayed in the early stage of the incubation time, although they completely germinated at 27 h. In contrast, spore germination of F. verticillioides was inhibited 87.7% up to 96 h. The lengths and diameters of hyphae, and spore diameters of the three fungi, were significantly smaller in comparison with those of the controls, and several morphological alterations were observed. Concerning aflatoxin B₁ and fumonisin B₁, fraction F6-1 did not show any inhibition effect at the concentration used. Fraction F6-1 was able to significantly inhibit the development of the three fungi, mainly F. verticillioides. The strong inhibitory effect of F6-1 on hyphae and spores suggests that it interacted with the fungi cell walls, which caused severe deformities. Nevertheless, this fraction was unable in inhibiting mycotoxin production from the three fungi at the concentration tested.     

UV‐A/BLUE LIGHT–INDUCED REACTIVATION OF SPORE GERMINATION IN UV‐B IRRADIATED ULVA PERTUSA (CHLOROPHYTA)1

Recent reduction in the ozone shield due to manufactured chlorofluorocarbons raised considerable interest in the ecological and physiological consequences of UV‐B radiation (λ=280–315 nm) in macroalgae. However, early life stages of macroalgae have received little attention in regard to their UV‐B sensitivity and UV‐B defensive mechanisms. Germination of UV‐B irradiated spores of the intertidal green alga Ulva pertusa Kjellman was significantly lower than in unexposed controls, and the degree of reduction correlated with the UV doses. After exposure to moderate levels of UV‐B irradiation, subsequent exposure to visible light caused differential germination in an irradiance‐ and wavelength‐dependent manner. Significantly higher germination was found at higher photon irradiances and in blue light compared with white and red light. The action spectrum for photoreactivation of germination in UV‐B irradiated U. pertusa spores shows a major peak at 435 nm with a smaller but significant peak at 385 nm. When exposed to December sunlight, the germination percentage of U. pertusa spores exposed to 1 h of solar radiation reached 100% regardless of the irradiation treatment conditions. After a 2‐h exposure to sunlight, however, there was complete inhibition of germination in PAR+UV‐A+UV‐B in contrast to 100% germination in PAR or PAR+UV‐A. In addition to mat‐forming characteristics that would act as a selective UV‐B filter for settled spores under the parental canopy, light‐driven repair of germination after UV‐B exposure could explain successful continuation of U. pertusa spore germination in intertidal settings possibly affected by intense solar UV‐B radiation.     

Kinetic study of the toxicity of zinc and lead ions to the heavy metals accumulating fungus Paecilomyces marquandii

Studies have been carried out to determine the toxicity of zinc and lead ions to germinating spores and hyphal growth of heavy metal accumulating fungus Paecilomyces marquandii (former Verticillium marquandii). Inhibitive concentration (IC₅₀) of zinc and lead ions was assayed by three different methods: image analysis, nephelometric on-line measurement and microcalorimetry. A kinetic model of spore germination and germ tube elongation was formulated and used as an auxiliary tool to determine IC₅₀ values upon image analysis data. The inhibitive effect of Zn²⁺ and Pb²⁺ to P. marquandii spores was mathematically described by the Edwards equation. Comparing the obtained IC₅₀ values, lead ions occurred to be more toxic to the germinating spores of P. marquandii than zinc ions (2.80 and 5.20 mM, respectively), although zinc ions induced a more significant delay in the development of the hyphae (13.84 h for 5 mM of Zn²⁺ and 9.30 h for 5 mM of Pb²⁺), which was demonstrated by the lengthened lag-phase (spore-swelling phase).     

Phytotoxic effects of fumonisin B1 on maize seedling growth

Fumonisin B₁ toxin is produced by the fungusFusarium moniliforme Sheldon, which is systemic to maize (Zea mays L.) and maize seeds. The effects of zero to 100 parts per million fumonisin B₁ on the germination process of maize seeds was determined. The presence of fumonisin had no effect on percent seed germination, but fumonisin inhibited radicle elongation by up to 75% after 48 hours of imbibition. An analysis of amylase secretion in the maize endosperm indicated that fumonisins inhibited amylase production in the germinating seed. Isoelectric focusing of endosperm extracts indicated that secretion of the low pI class of amylases was affected more that other amylase isozymes. The results suggested that the presence of high levels of fumonisin in maize seed may have deleterious effects on seedling emergence.     

Isolation and characterization of a spore germination inhibitor from Streptomyces sp. CB-1-1, a phytopathogen causing root tumor of melon

The germination rate and activation conditions of spores were examined for four strains of Streptomyces sp., a phytopathogen causing root tumor of melon. An inhibitor was isolated from the agar-cultured material of strain CB-1-1 and then characterized. The inhibitor selectively acted on spore germination and did not affect hyphal growth, and inhibition was abolished by washing the spores in water. The inhibitor was produced by an agar culture, and most of the inhibitor existed in the spores. The IC(50) value for the inhibitor was approximately 0.25 microg/ml.     

Germination of Bacillus thuringiensis var. israelensis spores in the gut of Aedes larvae (Diptera: Culicidae)

In laboratory experiments, germination and growth of Bacillus thuringiensis israelensis in the gut of Aedes aegypti and A. vexans larvae (Culicidae: Diptera) was observed. The number of spores and vegetative cells in the gut of living larvae and in cadavers was estimated by plaing homogenized larvae on selective agar plates. The number of spores per gut increased in the first 40–140 min of exposure to a maximum, and decreased in the subsequent time, demonstrating spore germination in living larvae, moribunds, and in cadavers. Twenty-four hours after the death of the larvae, a minimal amount of spores, but an increased number of vegetative cells, was found in cadavers. In A. aegypti larvae, germination and growth of B. thuringiensis israelensis in the larval gut was photographically documented.