CONVERGENT BIOLOGICAL PROCESSES IN COALESCING RHODOPHYTA

Sporeling coalescence in Gracilaria chilensis Bird, McLachlan et Oliveira produces genetically polymorphic, chimeric individuals. If this is common in red algae, it may have significant biological consequences. In this study, we evaluate the hypotheses that coalescence is widespread among the Rhodophyta and that specific and convergent morphological and ecological responses characterize this as a unique growth style among marine algae. A literature survey on coalescence was undertaken to assess the distribution of this condition in the Florideophycidae. Sixty-two (54.9%) of 113 species considered germinated to form a disk. Subsequent development in 37 of these species showed crust formation and coalescence during development with other crusts in 31 species (84%). Coalescing red algae were members of the orders Ahnfeltiales, Corallinales, Gigartinales, Gracilariales, Halymeniales, Palmariales, and Rhodymeniales. Ultrastructural studies in species of Ahnfeltiopsis, Chondrus, Gracilaria, Mazzaella, and Sarcothalia suggested a common pattern of early development. Newly released, naked spores may fuse into a single cell, as they do in Chondrus canaliculatus, or they may develop individual cell walls that later are surrounded by a thickened common wall. Ultrastructural studies demonstrated two kinds of immediate development after the first mitotic division: direct development by symmetric divisions resulting in discoid sporelings or an indirect asymmetric arrangement of divisions before a diskoid sporeling was formed. Germination in coalescing species is a linear function of the initial spore density, whereas in noncoalescing species maximum germination occurs at intermediate densities. In the field, coalescing species may recruit either from solitary or aggregated spores. However, survival is significantly higher for plantlets grown from a larger number of coalescing spores. Total number of erect axes formed by the coalesced mass is a logarithmic function of the initial number of spores. Thus, germlings grown from a larger number of coalescing spores exhibited a larger photosynthetic canopy than do plantlets grown from a few spores. Juveniles and mature clumps grown from a coalescing mass may exhibit size inequalities among erect axes, with the larger axes located toward the center of the clump. These larger axes mature first or, in some cases, are the only to produce spores. The widespread occurrence of coalescence inroughly half the number of orders of the Florideophycidae, the similarity of the coalescence process, and the finding of various adaptive traits associated with coalescence characterizes this as a unique growth style, splitting the diversity of species now included in the Florideophycidae into two major groups: coalescing and noncoalescing Rhodophyta.     

Quantification of factors affecting the probability of development of pathogenic bacteria,in particularClostridium botulinum,in foods

Summary The success of a preservation method with respect toClostridium botulinum can be measured by its effect on the probability that a single spore would result in growth and formation of toxin. In canned, low-acid foods, the minimum heat-process is designed to reduce the probability of survival of a single, heat-resistant spore ofC. botulinum by a factor of 10¹². In some foods, safety depends on the combination of inactivation and inhibition ofC. botulinum. The degree of protection (Pr) can be expressed asPr=Ds+In, whereDs is the decimal destruction of spores ofC. botulinum andIn is the decimal inhibition. A similar approach can be used in the case of other bacteria.Pr=log 1/P, whereP=the probability that an individual spore or bacterium will survive and result in growth.P can be estimated as the number of spores or bacteria that survive and initiate growth in a culture medium or food divided by the number of viable spores or bacteria inoculated into the medium or food. The effects of combinations of preservative factors can be measured by their effect onP at a stated temperature for a stated time. In experiments to determine the effects of preservative factors on an anaerobic bacterium such asC. botulinum it is essential that oxygen should be eliminated, unless it is controlled as an inhibitory factor. Thus experiments in culture media should be done under strictly anaerobic conditions at a known, low redox potential. The results of experiments to determine the effects of preservative factors onP after a series of incubation times can be modeled by methods similar to those used to model the effect of preservative factors on rate of growth and on lag period. Experiments to determine the effect of preservative factors on the probability of growth from a single spore or bacterium ofC. botulinum are discussed. A few reports of similar experiments with other bacteria have been published and are described. This approach has the advantage that it takes account of the inoculum level of bacteria.Mention of brand of firm names does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.     

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l^–1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 10⁸ spores ml^–1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 10⁸ spores ml^–1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 10⁸ spores ml^–1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.     

Sporulation ofEnteromorpha spp. (Chlorophyta) and overwintering of spores in sediments of the Wadden Sea,Island Sylt,North Sea

For the last two decades dense mats of species of the filamentous green algaeEnteromorpha spp. have regulary occurred on tidal flats of Köningshafen Bay (island Sylt, North Sea, FRG). In calm areas overwintering of adult plants or plant fragments is a common process to guarantee the mass development during the next season. In contrast, the distribution ofEnteromorpha on exposed sandy tidal flats depends on recruitment by juvenile stages. In 1993Enteromorpha spore settlement was recorded regularly in the field. Polyethylene dishes were placed in the field and left for a period of seven days and lateron cultivated in the laboratory to checkEnteromorpha germling development. During summer 1993 — at a minimum distance of 200 m to the nearest adultEnteromorpha populations — a total of at least 82×10⁶ spores m^–2 settled. During winter the number of spores attached to the collecting dishes was close to zero and the adjacent sand flats were free of any visibleEnteromorpha plants. In further experiments it was shown that the development ofEnteromorpha juveniles in the next spring depended on the overwintering capacity of spores. More than 2×10⁶ spores m^–2 attached to large sand grains and other substrata (e.g. Hydrobia ulvae) survived the winter. In a laboratory experiment several species ofEnteromorpha were able to survive in total darkness for at least 10 months.     

144 Use of Fourier Transform Infrared (FT IR) Diffuse Reflectance Spectroscopy and Hamming Distances to Study the Phycocolloid Chemosystematics of the Red Algae (Rhodophta)

Coalescence in seaweeds is known to occur in the laboratory among young and older sporelings and in the field between neighboring conspecific clumps. However, because spores and germlings are difficult to study in the field, it is as yet unknown at which stage of population development coalescence is most important. Since many seaweeds disperse aggregated propagules, often with a sticky mucilagous envelope around the spores, aggregated recruitment and coalescence might be more important at early stages of population establishment than among fully grown, well established clumps. Using recruitment plates maintained during several experimental times in the field, we are evaluating the above idea with mid-intertidal populations of Mazzaella laminarioides. During high fertility seasons, close to 45% of the spores settling within or at close (<1 m) distances of the bed exhibited aggregated recruitment, forming groups of 2 to 150+spores. The probability of aggregated recruitments is a function of dispersal distance and spore density. The number of sporelings produced is a function of spore density and coalescence. Highest after-recruitment mortality (first 15 days) occurs among solitary recruits, followed by sporelings conformed by small number of spores (2–4). Approximately 50% of the spores recruited, isolated or in group, coalesce within these 15 days, gradually forming massive sporelings with increasingly larger basal areas. Thus, after recruitment, sporelings may disappear (die), survive or coalesce. These three alternatives are integrated in a new demographic model for coalescing seaweeds (supported by grant FONDECYT 1020855).     

Fusarium andDidymella-neglected spores in the air

Summary This paper reports about the occurence ofFusarium- andDidymella spores in the air of Essen/FRG. During the spore season 1990, the spore concentration was measured on several days with a volumetric pollen trap by hourly analysis. The calculated amount of spores per hour is compared to the data of a pluviometer and the values of the relative humidity during the same period.The occurence of both spore types in the air and high relative humidity (>80%) are correlated in a highly significant way (P<0.001). The dispersion of spores starts when rain begins or directly after the precipitation.Didymella reaches higher concentrations thanFusarium in the air (Maximum values:Didymella 30000 spores/m³,Fusarium only 800 spores/m³). During the emission of the spores the temperature varied between 10°C und 20°C degrees. Didymella andFusarium must be an important allergenic source in the outdoor area, because of their allergen-loaded biological aerosols. The question of providing well defined extracts ofDidymella exitialis is given to the pharmaceutical industry.     

Chloroplast activities of dark-imbibed and photoinduced spores of the fernOnoclea sensibilis

Summary Chloroplast activities of dark-imbibed (non-germinating) and photoinduced (germinating) spores of the sensitive fern,Onoclea sensibilis were compared to gain insight into the germination process. There were no changes in the number of chloroplasts or in the chlorophyll contents of the spore during dark-imbibition and during the early phase of germination. Levels of increase in the Chloroplast DNA content of dark-imbibed and photoinduced spores were nearly the same and were associated with autoradiographic incorporation of [³H]thymidine into the cytoplasm. However, incorporation of the label into the nucleus occurred only during photoinduction of spores. Analysis of Chloroplast and nuclear DNA contents by dot-blot hybridization with labeled gene-specific probes has confirmed that chloroplast DNA content of the spore increases during dark-imbibition and photoinduction, while increase in nuclear DNA occurs only in photoinduced spores. Chloroplasts isolated from dark-imbibed and photoinduced spores incorporated [³H]TTP into an acid-insoluble fraction identified as DNA. The results show that physiological activities of chloroplasts of dark-imbibed and photoinduced spores ofO. sensibilis are similar and support an exclusive role for nuclear DNA synthesis in spore germination.     

143 Coalescense During Early Recruitment of Mazzaella Laminarioides

Coalescence in seaweeds is known to occur in the laboratory among young and older sporelings and in the field between neighboring conspecific clumps. However, because spores and germlings are difficult to study in the field, it is as yet unknown at which stage of population development coalescence is most important. Since many seaweeds disperse aggregated propagules, often with a sticky mucilagous envelope around the spores, aggregated recruitment and coalescence might be more important at early stages of population establishment than among fully grown, well established clumps. Using recruitment plates maintained during several experimental times in the field, we are evaluating the above idea with mid‐intertidal populations of Mazzaella laminarioides. During high fertility seasons, close to 45% of the spores settling within or at close (<1 m) distances of the bed exhibited aggregated recruitment, forming groups of 2 to 150+spores. The probability of aggregated recruitments is a function of dispersal distance and spore density. The number of sporelings produced is a function of spore density and coalescence. Highest after‐recruitment mortality (first 15 days) occurs among solitary recruits, followed by sporelings conformed by small number of spores (2–4). Approximately 50% of the spores recruited, isolated or in group, coalesce within these 15 days, gradually forming massive sporelings with increasingly larger basal areas. Thus, after recruitment, sporelings may disappear (die), survive or coalesce. These three alternatives are integrated in a new demographic model for coalescing seaweeds (supported by grant FONDECYT 1020855).     

A simple method to preserve algal spores of <Emphasis Type="Italic">Ulva</Emphasis> spp. in cold storage with ampicillin

Preservation of algal spores of the green seaweed Ulva fasciata and U. pertusa was enhanced by the addition of ampicillin in f/2 medium at 4°C. The viability of preserved spores was determined by a spore germination assay at various time intervals. The germination rate of U. fasciata remained at 5% to 38% for the first five days, dropping to 1% to 6% on the 10th day of storage with various preservation treatments without ampicillin at 4°C during parameter-selecting experiments. In f/2 medium, 53% of U. fasciata spores were still viable on day 5 and 23% on day 10 at 4°C. By adding 100 μg mL⁻¹ ampicillin to f/2 medium, 90% of the spores were viable at day 40 and 61% after 100 days of storage at 4°C. Spores of U. pertusa had lower preservation rates, with viabilities of 70% at day 40 and 32% at day 100. Algal spore preservation was heavily dependent on the bacterial contamination and subsequent degradation in stock solutions. Handling editor: L. Naselli-Flores     

The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies

Within colony transmission of Paenibacillus larvae spores was studied by giving spore-contaminated honey comb or comb containing 100 larvae killed by American foulbrood to five experimental colonies respectively. We registered the impact of the two treatments on P. larvae spore loads in adult bees and honey and on larval mortality by culturing for spores in samples of adult bees and honey, respectively, and by measuring larval survival. The results demonstrate a direct effect of treatment on spore levels in adult bees and honey as well as on larval mortality. Colonies treated with dead larvae showed immediate high spore levels in adult bee samples, while the colonies treated with contaminated honey showed a comparable spore load but the effect was delayed until the bees started to utilize the honey at the end of the flight season. During the winter there was a build up of spores in the adult bees, which may increase the risk for infection in spring. The results confirm that contaminated honey can act as an environmental reservoir of P. larvae spores and suggest that less spores may be needed in honey, compared to in diseased brood, to produce clinically diseased colonies. The spore load in adult bee samples was significantly related to larval mortality but the spore load of honey samples was not.     

Spore production and mycorrhizal development in various tropical crop hosts infected with Glomus clarum

Plant growth, mycorrhizal development and vesicular arbuscular spore production were examined in five tropical crop host species inoculated with Glomus clarum and grown in a glasshouse. In one of the two experiments, sequential harvests of maize, sorghum and chickpea were made in order to study spore production in relation to plant growth and mycorrhizal development. Spore numbers in each of these hosts increased at a fairly constant rate until maximum plant dry weight, when spore production ceased. Sorghum and maize produced considerably more spores than chickpea, with spore numbers being closely correlated with mycorrhizal root length. In the second experiment, Glomus clarum was cultured on each of maize, millet, sorghum, groundnut and chickpea for three consecutive generations before cross-inoculation of the spores from each host onto all five hosts. Sporulation with respect to host size was generally greatest when the inoculum used to infect a host had been produced on that host. The growth-promoting effects of the fungus were not influenced by the source of the inoculum. More spores were produced on the cereals than the legumes. Differences in spore numbers amongst hosts and plant generations were apparently influenced mainly by infected root length and by the growth period.     

Evaluation of Lecanicillium longisporum, Vertalec for simultaneous suppression of cotton aphid, Aphis gossypii, and cucumber powdery mildew, Sphaerotheca fuliginea, on potted cucumbers

The commercial preparation of Lecanicillium longisporum, Vertalec^® was evaluated for simultaneous suppression of cotton aphid and cucumber powdery mildew on potted cucumber plants. Vertalec was applied onto cucumber plants that had been infested with either cotton aphid, spores of Sphaerotheca fuliginea or both. Irradiation-inactivated Vertalec (II Vertalec) was also applied to an identical series of cucumber plants as a control. The Vertalec was highly pathogenic against adult aphids with an LT₅₀ of 6.9 days. II Vertalec did not affect aphid survival. Application of either active or II Vertalec significantly suppressed spore production of S. fuliginea compared to the water control. For dual control assays, Vertalec applications were made one day after infestation of both aphid and S. fuliginea onto potted cucumbers. Fifteen days after the Vertalec treatments, the numbers of surviving aphids and the production of powdery mildew spores were significantly reduced compared with the water control. The presence of aphids also suppressed S. fuliginea spore production. Our results suggest the potential of a dual role for Vertalec as a microbial control agent of aphids and powdery mildew in cucumber.     

Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus

The production of biomass and lovastatin by spore-initiated submerged fermentations of Aspergillus terreus ATCC 20542 was shown to depend on the age of the spores used for inoculation. Cultures started from older spores produced significantly higher titers of lovastatin. For example, the lovastatin titer increased by 52% when the spore age at inoculation rose from 9 to 16 days. The lovastatin titer for a spore age of 16 days was 186.5±20.1 mg L⁻¹. The time to sporulation on surface cultures was sensitive to the light exposure history of the fungus and the spore inoculation concentration levels. A light exposure level of 140 μE m⁻² s⁻¹ and a spore concentration of 1,320 spore cm⁻² produced the greatest extent of sporulation within about 50 h of inoculation. Sporulation was slowed in the dark and with diluted inoculants. A rigorous analysis of the data of statistically designed experiments showed the above observations to be highly reproducible.     

The Beltsville method for soilless production of vesicular-arbuscular mycorrhizal fungi

A low-cost, low-maintenance system for soilless production of vesicular-arbuscular mycorrhizal (VAM) fungus spores and inoculum was developed and adapted for production of acidophilic and basophilic isolates. Corn (Zea mays) plants were grown with Glomus etunicatum, G. mosseae or Gigaspora margarita in sand automatically irrigated with modified Hoagland's solution. Sand particle size, irrigation frequency, P concentration, and buffer constituents were adjusted to maximize spore production. Modified half-strength Hoagland's solution buffered with 4-morpholine ethane-sulfonic acid (MES) automatically applied 5 times/day resulted in production of 235 G. etunicatum spores/g dry wt. of medium (341000 spores/pot) and 44 G. margarita spores/g dry wt. of medium (64800 spores/pot). For six basophilic isolates of G. mosseae, CaCO₃ was incorporated into the sand and pots were supplied with the same nutrient solution as for acidophilic isolates. The increased pH from 6.1±0.2 to 7.2±0.2 resulted in spore production ranging from 70 to 145 spores/g dry wt. (102000–210000 spores/pot). Spore production by all isolates grown in the soilless sand system at Beltsville has exceeded that of traditional soil mixtures by 32–362% in 8–12 weeks.     

长白山哈泥泥炭地丘间生境泥炭藓孢子的寿命

长寿有性繁殖体对于植物种群的长存具有重要意义,迄今,泥炭地苔藓植物孢子长寿性研究还很少。在长白山哈泥泥炭地钻取丘间表层泥炭样品,测定泥炭腐殖化度和烧失量,逐层提取和培养泥炭藓孢子,研究埋藏时间对孢子萌发的影响。结果表明,丘间泥炭藓孢子埋藏环境中,随着埋深的增加即埋藏年限的增加,泥炭腐殖化度和烧失量总体上分别呈现增加和递减的趋势,而地层泥炭藓孢子萌发率呈现直线递减的规律,但在埋藏近150余年后孢子萌发率仍可达40%。研究进一步证明泥炭藓具有长期持久孢子库,根据推算,泥炭地丘间埋藏环境中,泥炭藓孢子最大寿命可超过400a。     

A new point of view on the taxonomy ofPottia starckeana agg. (Musci,Pottiaceae)

A new taxonomic treatment is proposed for thePottia starckeana species complex. The peristome development is not considered to be a useful feature to separate the taxa. On the basis of spore morphology only two species are accepted:P. starckeana, with spores wavy in outline, andP. davalliana, with variously-shaped and developed processes on the spores.Pottia starckeana var.brachyoda is reduced to synonymy withP. starckeana; P. conica andP. commutata are treated as synonyms ofP. davalliana. The speciesP. mutica, P. affinis, P. salina, P. microphylla, P. texana, andP. arizonica (included var.mucronulata) are considered taxa of doubtful affinity, as they have spore features intermediate between the two spore types established for the group. The identity ofP. appertii andP. recurvifolia has not been elucidated because the type material has been destroyed.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

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.     

Seasonal variation in the spore bank of ferns in grasslands on dolomite rock

Composition and seasonal patterns of the fern spore bank were compared to the surface vegetation of grasslands on dolomite rock in Hungary. Viability and potential dormancy of spores were tested through storage experiments. Although Asplenium ruta-muraria L. was the only species found at the study sites, five others, probably originating from air-borne spores from nearby areas, emerged from the soil samples. Considerable seasonal variability was detected in the number of prothallia emerging from soil samples from different sampling dates, with a peak after spore dispersal. The increased number of emerging prothallia after 1 year of storage suggests that a part of the spores stored in the soil samples were presumably dormant. Investigations on the dormancy of fern spores might be of great interest, especially in species adapted to seasonally unfavourable habitats.     

Application of response surface methodology in medium optimization for spore production of <Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in solid-state fermentation

Summary Spore production of Coniothyrium minitans was optimized by using response surface methodology (RSM), which is a powerful mathematical approach widely applied in the optimization of fermentation process. In the first step of optimization, with Plackett–Burman design, soluble starch, urea and KH²PO⁴ were found to be the important factors affecting C. minitans spore production significantly. In the second step, a 2³ full factorial central composite design and RSM were applied to determine the optimal concentration of each significant variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. The optimum values for the critical components for the maximum were obtained as follows: soluble starch 0.643 (36.43 g. l⁻¹), urea −0.544 (3.91 g l⁻¹) and KH²PO⁴ 0.049 (1.02 g l⁻¹) with a predicted value of maximum spore production of 9.94 × 10⁹ spores/g IDM. Under the optimal conditions, the practical spore production was 1.04 × 10¹⁰ spores/g IDM. The determination coefficient (R²) was 0.923, which ensure an adequate credibility of the model.