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.     

Gibberellin precursor is involved in spore germination in the moss Physcomitrella patens

Gibberellins are ent-kaurene derived phytohormones that are involved in seed germination, stem elongation, and flower induction in seed plants, as well as in antheridia formation and spore germination in ferns. Although ubiquitous in vascular plants, the occurrence and potential function(s) of gibberellins in bryophytes have not yet been resolved. To determine the potential role of gibberellin and/or gibberellin-like compounds in mosses, the effect of AMO-1618 on spores of Physcomitrella patens (Hedw.) B.S.G. was tested. AMO-1618, which inhibited ent-kaurene and gibberellin biosynthesis in angiosperms, also inhibited the bifunctional copalyl diphosphate synthase (E.C. 5.5.1.13)/ent-kaurene synthase (E.C. 4.2.3.19) of P. patens. AMO-1618 also caused a decrease in spore germination rates of P. patens, and this inhibitory effect was less pronounced in the presence of ent-kaurene. These results suggest that ent-kaurene biosynthesis is required by P. patens spores to germinate, implying the presence of gibberellin-like phytohormones in mosses.     

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.     

The effect of sporulation temperature on sporal characteristics ofBacillus subtilis A

Spores ofBacillus subtilis A were produced at different temperatures (23°–49°C) and examined for a number of sporal characteristics. Spore heat resistance increased with sporulation temperature to 45°C, with spores grown at 49°C showing a dramatic reduction in resistance. Spore crops showed biphasic thermal death curves whether enumerated on germination medium with or without calcium dipicolinate. This strain produces both rough and smooth variants. Of the spores produced at 23°C, 99% were rough, had a density of 1.305, and an average core/core + cortex volume ratio of 0.1838. At 49°C, 99% were smooth, had a density of 1.275, and an average volume ratio of 0.3098. Between these temperatures both spore types were produced. There appeared to be no direct correlation with sporulation temperature, heat resistance, and dipicolinate content. There was an increase in both the magnesium and calcium contents to 45°C with a dramatic reduction at 49°C. The 1.305 density spores had higher calcium and dipicolinate contents than the 1.275 spores, although both spore types showed biphasic thermal death curves. The mechanisms involved in determining which spore type (rough/smooth) is produced at a specific growth temperature is unknown.Florida Agricultural Experiment Station Journal Series Number R-00312.     

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.     

Resting spore formation ofLeptocylindrus danicus (Bacillariophyceae) during short time-scale upwelling and its significance as predicted by a simple model

Resting spore formation during short time-scale upwelling and its significance were investigated in the field and by a simple theoretical model. Field observations of spore formation ofLeptocylindrus danicus were made off Izu Peninsula, Japan. A rapid increase in ratio of resting spore to vegetative cell numbers indicated thatL. danicus formed resting spores quickly as a response to nutrient depletion in the upwelled water, although only a very low number of resting spores was found in the upwelling. A simple model was constructed to investigate the possible advantages of spore formation during short time-scale upwelling. This showed that there is a critical time-scale for resting spore formation to be advantageous. The nutrient depletion period of the upwelling off Izu was shorter than the critical time-scale determined by the model. Rapid-sinking of resting spores may increase further the critical time-scale, unless spores return with upwelling water. For short time-scale upwelling, the vegetative cell may be better suited than the resting spore for enduring a short period of nutrient depletion. Contribution from Shimoda Marine Research Center, University of Tsukuba, No. 475.     

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

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

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Spore ontogeny of the arbuscular mycorrhizal fungus<Emphasis Type="Italic"> Archaeospora trappei</Emphasis> (Ames &; Linderman) Morton &; Redecker (<Emphasis Type="Italic">Archaeosporaceae</Emphasis>)

Arbuscular mycorrhizal (AM) fungi in the genus Archaeospora (family Archaeosporaceae) contain both monomorphic and dimorphic species. The synanamorphism is often hard to discern without ontogenetic observations. Here, the spore ontogeny of Ar. trappei is reported from single species pot culture studies. The sporogenous hypha swelled up to a terminal sporiferous saccule and produced a lateral spore primordium on its neck. The saccule expanded fully before the spore primordium emerged. The saccule transferred its contents into the expanding spore and collapsed while wall differentiation continued inside the spore. The spore wall of Ar. trappei differentiated sequentially, in discrete steps, as in Acaulosporaceae members. In contrast, Ar. trappei produced a simplified spore wall in which the components differed in chemical and physical characteristics from those of the Acaulosporaceae members. Ontogenetic studies confirmed Ar. trappei to be monomorphic and producing acaulosporoid spores. The fungus is a new record to New Zealand.     

环境因子对中华双扇蕨孢子萌发的影响

为了解珍稀濒危植物中华双扇蕨(Dipteris chinensis)濒危原因,研究了温度和湿度对其孢子萌发的影响.结果表明,中华双扇蕨孢子萌发能力较强,萌发方式为Vittaria型,配子体萌发为Marattia型.中华双扇蕨孢子繁殖不受温度影响,而湿度显著影响孢子繁殖过程,湿润环境中孢子正常萌发,并形成心形配子体,顺利...     

New isolations of Endogone from Canadian soil

Summary Spores ofEndogone were found in additional soil samples taken from a variety of sites in Saskatchewan, Manitoba and Ontario, Canada. These spores were found in different soil environments some of which were cultivated, such as in wheat fields, and others uncultivated, as in densely wooded areas.Endogone spores varied in size, shape, thickness of wall and color. Many samples of soil contained more than one type of spore and, at present, these spore types are presumed to belong toEndogone. A large proportion of soil samples contained spores ofE. fasciculata in association with basidiospores of amelanogaster, previously identified asM. variegatus.     

THE “HYSTRICHOSPHAERID” RESTING SPORE OF THE DINOFLAGELLATE PYRODINIUM BAHAMENSE,PLATF, 19062

Germination experiments demonstrate that the “hystrichosphere” called Hemicystodinium zoharyi, which previously has been found only as a microfossil organism, is the resting spore stage in the life history of Pyrodinium bahamense, a modern bioluminescent, thecate dinoflagellate. The morphology of this spore, together with new details of the thecal structure and ontogeny of P. bahamense, is described, and it is concluded that Pyrodinium is closely related to Gonyaulax but worthy of retention as a discrete genus. The geological history of P. bahamense is traceable to the Eocene through fossil occurrences of its spore, and it is suggested that additional pyrodinioid dinoflagellates which now are extinct were represented in Lower Tertiary seas by another hystrichosphere genus, called Homotryblium. Selected aspects of the physiology and ecology of modern dinoflagellate resting spores are discussed briefly with special reference to Pyrodinium.     

Effect of spore concentration on germination and autotropism inTrichoderma hamatum

Summary The effect of spore concentration on spore germination and germtube growth ofTrichoderma hamatum on water agar and on potato dextrose agar (PDA) was studied. Increasing inoculum size up to 10⁹ spores/plate on PDA and up to 10⁷ spores/plate on water agar shortened the incubation period required for germtubes emergence and increased germination rate. However, on water agar germination was inhibited at 10⁸ and was completely arrested at 10⁹ spores/plate. Inhibition in germination of 10⁷ spores/plate was observed on water agar when the plates were preincubated with 10⁹ spores/plate for 5 h or more. Addition of glucose and ammonium nitrate to the water agar medium allowed only 25% of the spores to germinate at 10⁹ as compared to 78% at 10⁷ spores/plate after 8 h of incubation. Addition of polysaccharides to the C+N supplemented medium, significantly increased germination up to 84% as compared to 100% on PDA, after 8 h of incubation. Germlings ofTrichoderma hamatum phialospores exhibited positive autotropism and anastamosis on both media. The phenomenon was positively related to inoculum size, being most pronounced at 10⁷ spores/plate.     

Hydrolytic enzyme activities in germinating spores ofAdiantum capillus-veneris L.

Changes in hydrolytic enzyme activities were investigated during spore germination ofAdiantum capillus-veneris L. The spores were incubated for 3 days in the dark at 25 C for imbibition, and then germination of the spores was induced by continuous irradiation with red light. At day 2 after onset of the red light irradiation, rhizoids appeared out of spore coats and protonemal cells became visible on the following day. Lipase occurred in dry spores and its activity decreased during 3 days of dark incubation. The activity started to increase when the spore germination was induced by red light irradiation. On the other hand, amylolytic and aminopeptidase activities which were also detected in dry spores decreased continuously during the dark incubation and following the germination process. RNase activity also decreased during 3 days of dark incubation but the activity was retained thereafter at a constant level with or without red light irradiation. Developmental patterns of these hydrolytic enzymes were classified into two groups: One decreased during imbibition and dark incubation but increased after red light irradiation and the other continuously decreased during dark incubation and germination. These results are discussed in relation to compositional changes of cell constitutions such as lipid, sugars, proteins and amino acids during spore germination.     

Effect of volatile substances fromOriganum majorana andOcimum basilicum on spore respiration and germination of some soil fungi

Oxygen uptake by the spores ofFusarium moniliforme, F. oxysporum, F. semitectum, F. solani, Mucor racemosus andTrichoderma viride was increased in the presence of volatile substances extracted, fromOriganum majorana andOcimum basilicum. This increase was greater in the presence of volatile substances fromO. basilicum thanO. majorana, except in the case ofF. semitectum where the reverse was true. A drop in the RQ of all the germinating spores was observed in the presence of these substances. Volatile substances fromO. majorana reduced the spore germination ofM. racemosus whereas the spores ofT. viride were stimulated to germinate. Volatile substances fromO. basilicum stimulated the spore germination ofM. racemosus whereasT. viride spores were not affected.     

Energetic aspects of spore germination in filamentous fungi

The antifungal activity of substances interfering with the function and biogenesis of mitochondria was studied. Strict anaerobiosis, cyanide, azide, oligomycin, bongkrekic acid and ethidium bromide were found to prevent spore germination ofAspergillus niger andPenicillium italicum in liquid germination medium. The effect of azide, oligomycin and ethidium bromide was fungicidal. Cyanide and azide completely inhibited the incorporation of¹⁴C-leucine and¹⁴C-uracil into germinating conidia ofA. niger. Oligomycin and ethidium bromide reduced the extent of incorporation of both precursors in the first few hours of conidial germination and at later stages stopped it completely. The inhibition of both spore germination and macromolecules synthesis during the germination ofA. niger conidia were in relation to the specific inhibitory effect of the agents on respiratory activity of dormant conidia and mycelial cells. The results indicate that both the function of mitochondrial genetic and protein synthesizing systems and the function of oxidative phosphorylation are essential for normal spore germination and fungal growth.     

Sequential release of cellulose enzymes during germination ofTrichoderma reesei spores

The pattern of release of extracellular cellulase during the germination ofTrichoderma reesei spores has been examined. The four enzymes namely, filter paper degrading enzyme, Β-1,4 endoglucanase, Β-glucosidase and xylanase appear sequentially in the culture broth during germination of the spores. The order of enzyme appearance is not altered by the type of carbon source in the germination medium. Measureable quantities of filter paper degrading enzyme is detected only after the outgrowth has occurred. A possible mechanism of spore germination and induction of the enzymes by insoluble cellulose is suggested. An erratum to this article is available at .     

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

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

Enhanced spore production ofBacillus thuringiensis by fed-batch culture

Summary Fed-batch culture was carried out to increase cell mass followed by batch culture for spore production ofbacillus thuringiensis. High cell mass obtained by increasing the feeding glucose concentration in constant fed-batch culture which supported fast cell growth resulted in good sporulation during subsequent batch culture, and the maximum cell mass of 72.6 g/L and spore concentration of 1.25×10¹⁰ spores/mL could be obtained.     

Gas discharge plasmas are effective in inactivating <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Clostridium</Emphasis> spores

Bacterial spores are the most resistant form of life and have been a major threat to public health and food safety. Nonthermal atmospheric gas discharge plasma is a novel sterilization method that leaves no chemical residue. In our study, a helium radio-frequency cold plasma jet was used to examine its sporicidal effect on selected strains of Bacillus and Clostridium. The species tested included Bacillus subtilis, Bacillus stearothermophilus, Clostridium sporogenes, Clostridium perfringens, Clostridium difficile, and Clostridium botulinum type A and type E. The plasmas were effective in inactivating selected Bacillus and Clostridia spores with D values (decimal reduction time) ranging from 2 to 8 min. Among all spores tested, C. botulinum type A and C. sporogenes were significantly more resistant to plasma inactivation than other species. Observations by phase contrast microscopy showed that B. subtilis spores were severely damaged by plasmas and the majority of the treated spores were unable to initiate the germination process. There was no detectable fragmentation of the DNA when the spores were treated for up to 20 min. The release of dipicolinic acid was observed almost immediately after the plasma treatment, indicating the spore envelope damage could occur quickly resulting in dipicolinic acid release and the reduction of spore resistance.     

Arbuscular mycorrhizal fungi on adjacent semi-natural grasslands with different vegetation in Japan

Arbuscular mycorrhizal (AM) fungi on Japanese semi-natural grasslands were investigated at three adjacent sites with different vegetation. The predominant grasses at the three sites were 1)Pleioblastus chino, 2)Miscanthus sinensis andArundinella hirta (M. sinensis/A. hirta), and 3)Zoysia japonica, respectively. The degree of colonization was higher inM. sinensis/A. hirta than inP. chino andZ. japonica. AM fungi were recovered by spore extraction and by pot cultures started from soil inoculum or from transplanting of field plants. Total spore number obtained by the spore extraction method was highest in the rhizosphere ofM. sinensis/A. hirta and lowest in that ofP. chino. AGlomus sp. resemblingG. geosporum predominated in association withM. sinensis/A. hirta andP. chino. FromZ. japonica, three species,Acaulospora gerdemannii, Glomus leptotichum, and a species resemblingG. clarum, were isolated by pot culture from soil and two species,A. longula andScutellospora cerradensis, by pot culture from transplanting ofZ. japonica. FromM. sinensis/A. hirta, one species,A. longula, was found by pot culture from soil. FromP. chino, no AM fungus was detected by either method. Single-spore culture confirmed thatG. leptotichum andA. gerdemannii are conspecific.