SPORELING COALESCENCE AND INTRACLONAL VARIATION IN GRACILARIA CHILENSIS (GRACILARIALES,RHODOPHYTA)1

This study evaluates the hypothesis that spore coalescence may cause intraclonal variation. Spore coalescence might allow the occurrence of unitary thalli that in fact correspond to genetically different, coalesced individuals. Plant portions simultaneously derived from these chimeric individuals may exhibit dissimilar growth responses even when incubated under similar abiotic conditions. Testing of the hypothesis included various approaches. Transmission electron microscopy observations of early stages of sporeling coalescence indicated that polysporic plantlets were formed by groups of spores and their derivatives. Even though adjacent cells in two different groups may fuse, these groups maintained an independent capacity to grow and form uprights. Laboratory-grown plantlets showed a significant correlation between the initial number of spores and the total number of erect axes differentiated from the sporeling. Construction and growth of bicolor individuals indicated the chimeric nature of the coalesced individuals. Coalesced, bicolor holdfasts had green and red cells, which subsequently produced green and red uprights, respectively. Individuals fronds were also chimeric, as indicated by the production of green and red branchlets from single, red uprights. The existence of mixed tissues was further substantiated by random amplified polymorphic DNA analysis. The banding pattern produced by branchlets of a unisporic thallus was consistently monomorphic, whereas the patterns produced by the polysporic thallus were polymorphic. Growth rates of polysporic thalli had larger data dispersal and variation coefficients than oligosporic or monosporic thalli. Therefore, all results support the original hypothesis and suggest that coalescence might be ecologically more important than previously thought.     

Histidine acts as a co‐germinant with glycine and taurocholate for Clostridium difficile spores

Aims: It is well established that the bile salt sodium taurocholate acts as a germinant for Clostridium difficile spores and the amino acid glycine acts as a co‐germinant. The aim of this study was to determine whether any other amino acids act as co‐germinants. Methods and Results: Clostridium difficile spore suspensions were exposed to different germinant solutions comprising taurocholate, glycine and an additional amino acid for 1 h before heating shocking (to kill germinating cells) or chilling on ice. Samples were then re‐germinated and cultured to recover remaining viable cells. Only five amino acids out of the 19 common amino acids tested (valine, aspartic acid, arginine, histidine and serine) demonstrated co‐germination activity with taurocholate and glycine. Of these, only histidine produced high levels of germination (97·9–99·9%) consistently in four strains of Cl. difficile spores. Some variation in the level of germination produced was observed between different PCR ribotypes, and the optimum concentration of amino acids with taurocholate for the germination of Cl. difficile NCTC 11204 spores was 10–100 mmol l^?1. Conclusions: Histidine was found to be a co‐germinant for Cl. difficile spores when combined with glycine and taurocholate. Significance and Impact of the Study: The findings of this study enhance current knowledge regarding agents required for germination of Cl. difficile spores which may be utilized in the development of novel applications to prevent the spread of Cl. difficile infection.     

Development of a user-friendly delivery method for the fungus Metarhiziumanisopliae to control the ectoparasitic mite Varroa destructor in honey bee, Apis mellifera, colonies

A user-friendly method to deliver Metarhizium spores to honey bee colonies for control of Varroa mites was developed and tested. Patty blend formulations protected the fungal spores at brood nest temperatures and served as an improved delivery system of the fungus to bee hives. Field trials conducted in 2006 in Texas using freshly harvested spores indicated that patty blend formulations of 10 g of conidia per hive (applied twice) significantly reduced the numbers of mites per adult bee, mites in sealed brood cells, and residual mites at the end of the 47-day experimental period. Colony development in terms of adult bee populations and brood production also improved. Field trials conducted in 2007 in Florida using less virulent spores produced mixed results. Patty blends of 10 g of conidia per hive (applied twice) were less successful in significantly reducing the number of mites per adult bee. However, hive survivorship and colony strength were improved, and the numbers of residual mites were significantly reduced at the end of the 42-day experimental period. The overall results from 2003 to 2008 field trials indicated that it was critical to have fungal spores with good germination, pathogenicity and virulence. We determined that fungal spores (1 × 10¹⁰ viable spores per gram) with 98% germination and high pathogenicity (95% mite mortality at day 7) provided successful control of mite populations in established honey bee colonies at 10 g of conidia per hive (applied twice). Overall, microbial control of Varroa mite with M. anisopliae is feasible and could be a useful component of an integrated pest management program.     

Giant kelp vegetative propagation: Adventitious holdfast elements rejuvenate senescent individuals of the Macrocystis pyrifera “integrifolia” ecomorph

Recent findings on holdfast development in the giant kelp highlighted its key importance for Macrocystis vegetative propagation. We report here for the first time the development of adventitious holdfasts from Macrocystis stipes. Swellings emerge spontaneously from different areas of the stipes, especially in senescent or creeping individuals. After being manually fastened to solid substrata, these swellings elongated into haptera, which became strongly attached after 1 month. Within 4 months, new thalli increased in size and vitality, and developed reproductive fronds. Our results suggest the usage of these structures for auxiliary attachment techniques. These could act as a backup, when primary holdfasts are weak, and thus improve the survival rate of the giant kelp in natural beds.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and micropropagation of <Emphasis Type="Italic">Givotia rottleriformis</Emphasis> Griff.

The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA₃). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts) supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively.     

Differential response of antioxidative enzymes in embryonic axes and cotyledons of germinating lupine seeds

Germination of lupine (Lupinus luteus L.) seeds was accompanied by an increase in concentration of free radicals with g ₁ and g ₂ values of 2.0056 ± 0.0003 and 2.0033 ± 0.0005, respectively. The highest intensity of free radical signal was observed in embryo axes immediately after radicle protruded through the seed coat. Hydrogen peroxide accumulated in embryonic axes and cotyledons during imbibition before the onset of germination in the seed population. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) rose progressively in embryo axes. In cotyledons SOD activity did not change significantly, while that of CAT increased during germination. The enhancement of Cu, Zn-SODs and Mn-SOD isoforms in embryonic axes was observed. A new isoform of catalase was synthesized, suggesting that it plays a relevant role during germination. SOD and CAT activities were detected in dry seeds. Free radical generation and response of antioxidative enzymes differed between embryo axes and cotyledons during the germination timecourse.     

SIZE INCREMENTS DUE TO INTERINDIVIDUAL FUSIONS: HOW MUCH AND FOR HOW LONG?1

Size increments following interindividual fusions appear as a general benefit for organisms, such as coalescing seaweeds and modular invertebrates, with the capacity to fuse with conspecifics. Using sporelings of the red algae Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira and Mazzaella laminarioides (Bory) Fredericq, we measured the growth patterns of sporelings built with different numbers of spores, and the magnitude and persistence of the size increments gained by fusions. Then we studied three morphological processes that could help explain the observed growth patterns. Results indicate that in these algae, coalescence is followed by immediate increase in total size of the coalesced individual and that the increment is proportional to the number of individuals fusing. However, the size increments in sporelings of both species do not last >60 d. Increasing reductions of marginal meristematic cells and increasing abundance of necrotic cells in sporelings built with increasing numbers of initial spores are partial explanations for the above growth patterns. Since sporelings formed by many spores differentiate erect axes earlier and in larger quantities than sporelings formed by one or only a few spores, differentiation, emergence, and growth of erect axes appear as a more likely explanation for the slow radial growth of the multisporic sporelings. Erect axis differentiation involves significant morphological and physiological changes and a shift from radial to axial growth. It is concluded that the growth pattern exhibited by these macroalgae after fusion differs from equivalent processes described for other organisms with the capacity to fuse, such as modular invertebrates.     

Effect of ozone on spore germination,spore production and biomass production in two <Emphasis Type="Italic">Aspergillus</Emphasis> species

The ability of ozone gas to reduce food spoilage is relatively well documented, but the developmental effects of the gas on food spoilage fungi are not well known. In this study two model aspergilli, Aspergillus nidulans and Aspergillus ochraceus were used to study the effects of ozone on spore germination, sporulation and biomass production. These effects were examined under three levels of ozone; two high level ozone exposures (200 and 300 μmol mol⁻¹) and one low level exposure (0.2 μmol mol⁻¹). The two species behaved noticeably different to each other. Ozone was more effective in reducing growth from spore inocula than mycelia. No spore production could be detected in A. nidulans exposed to continuous low level O₃, whereas the same treatment reduced spores produced in A. ochraceus by 94%. Overall the work suggests that ozone exposure is an effective method to prevent spread of fungal spores in a food storage situation.     

Interactions between<Emphasis Type="Italic"> Trichoderma pseudokoningii</Emphasis> strains and the arbuscular mycorrhizal fungi<Emphasis Type="Italic"> Glomus mosseae</Emphasis> and<Emphasis Type="Italic"> Gigaspora rosea</Emphasis>

The interaction between Trichoderma pseudokoningii (Rifai) 511, 2212, 741A, 741B and 453 and the arbuscular mycorrhizal fungi Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe BEG12 and Gigaspora rosea Nicolson & Schenck BEG9 were studied in vitro and in greenhouse experiments. All T. pseudokoningii strains inhibited the germination of G. mosseae and Gi. rosea except the strain 453, which did not affect the germination of Gi. rosea. Soluble exudates and volatile substances produced by all T. pseudokoningii strains inhibited the spore germination of G. mosseae. The germination of Gi. rosea spores was inhibited by the soluble exudates produced by T. pseudokoningii 2212 and 511, whereas T. pseudokoningii 714A and 714B inhibited the germination of Gi. rosea spores by the production of volatile substances. The strains of T. pseudokoningii did not affect dry matter and percentage of root length colonization of soybean inoculated with G. mosseae, except T. pseudokoningii 2212, which inhibited both parameters. However, all T. pseudokoningii strains decreased the shoot dry matter and the percentage of AM root length colonization of soybean inoculated with Gi. rosea. The saprotrophic fungi tested seem to affect AM colonization of root by effects on the presymbiotic phase of the AM fungi. No influence of AM fungi on the number of CFUs of T. pseudokoningii was found. The effect of saprotrophic fungi on AM fungal development and function varied with the strain of the saprotrophic species tested.     

Germination responses of <Emphasis Type="Italic">Erica andevalensis</Emphasis> to different chemical and physical treatments

Erica andevalensis Cabezudo & Rivera is a threatened edaphic endemic species of Andalusia (SW Spain). Under natural conditions, the plants produce a very large number of small seeds (0.3–0.4 mm) but very few seedlings survive. Different treatments (high temperature, cold pre-treatment, nitrogen salts, and gibberellic acid applications) were tested to assess germination patterns in different populations and to determinate the most favorable conditions for germination. Gibberellic acid was provided in five different concentrations from 0 to 400 ppm GA₃, while nitrogen was applied as 10 mM of either KNO₃ or NH₄NO₃. The effect of pH on germination was also tested. The species always showed a low germination rate (6.50–22%) that was not stimulated either by 1 or 4 months in dry cold pre-treatment, nitrogen application, acid pH medium, or by high temperature (80°C for 10 min); although gibberellic acid application (100–400 ppm) significantly enhanced germination. The highest percentage of germination (41.6%) was achieved with a mean germination time to start germination (t ₀) of 7.6 ± 0.54 days when the seeds were subjected to 400 ppm gibberellic acid treatment. The population origin did not have a significant effect on germination percentage.     

Polyamines stimulate hyphal branching and infection in the early stage of <Emphasis Type="Italic">Glomus etunicatum</Emphasis> colonization

Polyamines are known to strongly stimulate hyphal growth in arbuscular mycorrhizal fungi. The effect of the polyamines putrescine, spermidine and spermine on spore germination, hyphal elongation and branching by the AM fungus Glomus etunicatum was investigated in this study. The effect of spermine on infection and the development of the host and of daughter spores was further investigated using the dual monoaxenic culture system comprised of Gl. etunicatum fungal cultures in Ri T-DNA transformed carrot hairy roots. Spermidine and spermine showed positive effects on germination and all three polyamines significantly promoted hyphal growth. Hyphal branching was also strongly stimulated by treatment with polyamines, such as an increase in the number of branches. Infection during the early stages of the in vitro co-culture life cycle was enhanced in the presence of spermine, and daughter spores appeared at earlier timepoints compared to the control. Our results demonstrate that polyamines stimulate germination and hyphal branching in the early stage of AM fungal colonization. Moreover, results from the investigations conducted in the fungus-root co-culture suggest that polyamines may be involved in establishing the symbiotic relationship between root and fungus.     

Penicillium roqueforti conidia induced by L-amino acids can germinate without detectable swelling

Penicillium roqueforti is used for the production of blue-veined cheeses but is a spoilage fungus as well. It reproduces asexually by forming conidia. Germination of these spores can start the spoilage process of food. Germination is typically characterized by the processes of activation, swelling and germ tube formation. Here, we studied nutrient requirements for germination of P. roqueforti conidia. To this end,?>?300 conidia per condition were monitored in time using an oCelloScope imager and an asymmetric model was used to describe the germination process. Spores were incubated for 72 h in NaNO₃, Na₂HPO₄/NaH₂PO₄, MgSO₄ and KCl with 10 mM glucose or 10 mM of 1 out of the 20 proteogenic amino acids. In the case of glucose, the maximum number of spores (P_max) that had formed germ tubes was 12.7%, while time needed to reach 0.5 P_max (τ) was about 14 h. Arginine and alanine were the most inducing amino acids with a P_max of germ tube formation of 21% and 13%, respectively, and a τ of up to 33.5 h. Contrary to the typical stages of germination of fungal conidia, data show that P. roqueforti conidia can start forming germ tubes without a detectable swelling stage.

     

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.     

Seed germination ecology of the threatened endemic Iberian <Emphasis Type="Italic">Delphinium fissum</Emphasis> subsp. <Emphasis Type="Italic">sordidum</Emphasis> (Ranunculaceae)

Seeds of Delphinium fissum subsp. sordidum are physiologically dormant at maturity, with underdeveloped embryos; thus they have morphophysiological dormancy (MPD). The aims of this study were to determine the requirements for embryo growth, dormancy break and germination, to characterise the type of seed dormancy and to evaluate the effects of light, seed age, pollination mechanism, and inter-annual and inter-population variability on germinative ability. After 3 months of incubation at 5°C (cold stratification) in darkness conditions, the mean embryo length increased from 5.6 to 2.07 mm, with 76% of seeds germinating. Conversely, embryos of seeds incubated during 3 months at 20/7 or 28/14°C hardly grew and no germination was recorded. Since cold stratification was the only requirement for the loss of MPD, and both dry storage in laboratory conditions and warm stratification prior to cold stratification shortened the cold stratification period required for germination, it could be concluded that D. fissum subsp. sordidum seeds have intermediate complex MPD. Cold stratification and incubation in darkness conditions promoted higher germination percentages than those in light. In addition, germinative ability increased with seed age up to 8 months (reaching 96% at 5°C in darkness), showed a pronounced inter-annual and inter-population variability, as well as a significant decrease in seeds coming from pollination by geitonogamy. High temperatures (25/10 or 28/14°C) induced seeds to secondary dormancy, so seedling emergence in the greenhouse was restricted to February–March. The requirements for dormancy break and germination reflect an adaptation to trigger germination in late winter. This study is the first one to document a gradual increase in germination percentage with seed age for plant species with intermediate complex MPD.     

Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

Background  

During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.     

Growth induction and stabilization of population composition in <Emphasis Type="Italic">Saccharopolyspora erythraea</Emphasis> by catecholamine compounds

Dopamine and epinephrine, hormone compounds belonging to the catecholamine group, produced a polymodal effect on the culture of the erythromycin-producing actinobacterium Saccharopolyspora erythraea. They stimulated spore germination and stabilized the composition of its population. Plating of monospore suspensions of strains RIA 1387 and RIA 120 in the presence of catecholamines increased the colony number by 2- and 2.3-fold, respectively (with epinephrine), and 3- and 4-fold, respectively (with dopamine). The optimum effect of both catecholamines was attained at a concentration of 10⁻⁵ M. The influence of exogenous catecholamines resulted in a significant shift in the population structure of the tested strains with an increased share of the colonies of the dominant morphotype, which is the most efficient antibiotic producer. Considerable differences in the biological activity of the catecholamines were revealed after exposure to ultralow temperatures both during medium-term storage (for three months) and after short-term freezing (for 10 min) in liquid nitrogen. An appreciable effect was produced by exogenous dopamine introduced at the resuscitation stage, resulting in a two- to three-fold increase in the viability of actinobacterial spores. Almost 100% of the resulting populations were composed of colonies of the dominant morphotype. In contrast to dopamine, epinephrine failed to produce a growth-stimulating effect when added to spore suspensions exposed to ultralow temperatures.     

2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

Coordinated action of β‐galactosidases in the cell wall of embryonic axes during chickpea germination and seedling growth

The plant cell wall is a dynamic structure whose constant modification is necessary for plant cells to grow and divide. In the cell walls of chickpea (Cicer arietinum) there are at least four β‐galactosidases, whose presence and location in embryonic axes during the first 48 h of seed imbibition are discussed in this paper. We examined their roles as cell wall‐modifying enzymes in germinative and/or post‐germinative events. At the start of germination, only βV‐Gal, and to a lesser extent βIV‐Gal, appear in the axes before rupture of the testa, suggesting they are related to germination sensu stricto. Once the testa has broken, the four β‐galactosidases are involved in growth and differentiation of the axes. Immunolocation of the different proteins in axes, which in part confirms previous results in seedlings and plants, allows assignment of post‐germinative roles to βI‐Gal and βIII‐Gal as cell wall modifiers in vascular tissue elements. βIV‐Gal and βV‐Gal participate in the initial events of germination in which cell walls are involved: βV‐Gal in cell proliferation, detachment of root cap cells and initial vascular tissue differentiation; both of them in xylem maturation; and βIV‐Gal in thickening of the primary cell wall. Together with other cell wall‐modifying enzymes, such as expansins and XTH, chickpea galactosidases might function in a sequential order in turnover of the primary cell wall, allowing the elongation of embryonic axes during seed germination.     

The effects of smoke derivatives on in vitro seed germination and development of the leopard orchid Ansellia africana

Plant‐derived smoke and smoke‐isolated compounds stimulate germination in seeds from over 80 genera. It has also been reported that smoke affects overall plant vigour and has a stimulatory effect on pollen growth. The effect of smoke on orchid seeds, however, has not been assessed. In South Africa, orchid seeds from several genera may be exposed to smoke when they are released from their seedpods. It is therefore possible that smoke may affect their germination and growth. Therefore, the effects of smoke [applied as smoke‐water (SW)] and two smoke‐derived compounds, karrikinolide (KAR₁) and trimethylbutenolide (TMB), were investigated on the germination and growth of orchid seeds in vitro. The effect of SW, KAR₁ and TMB were investigated on the endangered epiphytic orchid, Ansellia africana, which is indigenous to tropical areas of Africa. Smoke‐water, KAR₁ and TMB were infused in half‐strength MS medium. The number of germinated seeds and number of seeds and protocorm bodies to reach predetermined developmental stages were recorded on a weekly basis using a dissecting microscope for a 13‐week period. Infusing SW 1:250 (v:v) into half‐strength MS medium significantly increased the germination rate index (GRI) and the development rate index (DRI) of the A. africana seeds. All the SW treatments significantly increased the number of large protocorm bodies at the final stage of development. Infusing KAR₁ into the growing medium had no significant effect on germination or development of the seeds. The TMB treatment, however, significantly reduced the GRI and DRI of A. africana seeds.