Honey-coloured,sessile Endogone spores: II. Changes in fine structure during spore development

Summary The fine structure of honey-coloured, sessile Endogone spores is described from initiation of the mother spore to dormancy of the resting spore. Three unusual organelles occur viz. pigment granules, large crystals and selfduplicating bacteria-like organisms. The first two are very numerous, and are specifically associated with spore formation. The pigment granules are involved in the deposition of the honey-coloured wall, and change into myelin-like figures when cytoplasm moves from the mother into the resting spore. The crystals, whose function is not known, are most conspicuous just before the resting spore reaches dormancy. The bacteria-like organisms, which may be actinomycete spores living symbiotically in the fungus, multiphy greatly as the spore enters dormancy. The dormant spore contains very little cytoplasm compressed into a fine network between very large polygonal oil globules and large round bodies thought to contain a storage polysaccharide.     

BIOCHEMISTRY OF FERN SPORE PROTEINS: GLOBULIN STORAGE PROTEINS IN ONOCLEA SENSIBILIS AND OSMUNDA CINNAMOMEA

Onoclea sensibilis was found to contain globulin storage proteins of 2.0S and 11.3S. These globulins, comparable in size and subunit composition to the spore storage proteins characterized in Matteuccia struthiopteris (Templeman, DeMaggio, and Stetler, 1987), declined during imbibition and the initial stages of spore germination. Osmunda cinnamomea, a fern that is only distantly related to Matteuccia, also contained globulin proteins, but these had S values of 5.5 and 11.3. SDS-PAGE analysis revealed extensive differences in banding patterns of the 11.3S protein between Onoclea and Osmunda. This work indicates that while globulin proteins are important storage materials in a variety of ferns they exhibit considerable molecular heterogeneity. The observed heterogeneity in the globulin proteins may be a useful tool to explore evolutionary relationships in the ferns.     

Effect of trehalose on the viability of sporangiospores of the mucorous fungus <Emphasis Type="Italic">Blakeslea trispora</Emphasis>

Sporangiospores of Blakeslea trispora are in a state of exogenous dormancy, and water is the key factor controlling their germination. A wide range of carbohydrates, ammonium salts, and yeast extract had a weak stimulating effect (less than 50%) on spore germination, whereas amino acids could significantly inhibit this process. Cultivation of B. trispora on sporogenous sucrose- and trehalose-containing media (S and T spores, respectively) resulted in significant changes in spore formation, as well as in the chemical composition of spores and their viability. In the presence of trehalose, the amount of spores increased twofold; spore viability during storage increased as well. All changes in the carbohydrate composition of the cytosol and in the composition of the spore membrane lipids indicated that the dormancy of T spores was deeper than that of S spores, which has a favorable effect on their viability.     

Evaluation of inoculum performance for enhancing gamma-linolenic acid production from Mucor rouxii

Aims: To facilitate a cost‐effective preparation of spore inoculum with good capacity for gamma‐linolenic acid (GLA) production from Mucor rouxii. Methods and Results: Sporangiospore production, mycelial growth ability and fatty acid composition of M. rouxii were determined. Compared with fungal cultivation on solid semi‐synthetic media, high spore production was achieved from M. rouxii grown on rice grains, particularly polished rice (30·7 g kg^?1 initial substrate). Variations in the fatty acid profiles were found in the spores grown on different types of solid media, whereas the spores obtained at different ages from cultivated polished rice showed a similar fatty acid profile. Using the inocula from different spore‐forming media and culture ages, and low temperature storage, not much change in the vegetative growth of submerged cultures or fatty acid composition of mycelia was observed. Conclusion: The spores generated on polished rice exhibited a high performance for GLA production. Age of spore and timing of spore storage at low temperature did not affect on fatty acid profile of the mycelial cultures. Significance and Impact of the Study: The simple, low cost method of inoculum preparation can be applied for large‐scale production of GLA‐rich oils, which reduce a time constraint and variation in fatty acid composition.     

BASIDIOSPOROGENESIS IN BOLETUS RUBINELLUS. I. STERIGMAL INITIATION AND EARLY SPORE DEVELOPMENT

Sterigmal initiation in Boletus rubinellus resembled hyphal tip growth. Four stages in early basidiospore development have been delineated based on gross morphology, and changes in wall layers and cytoplasm. Changes in wall layers and cytoplasm during spore development were stage-specific. During Stage 1 the spore wall consisted of two layers identical to those of the sterigmal wall with occasional pellicle remnants on the outer surface. The onset of wall differentiation began in Stage 2, and during Stage 3 wall layers characteristic of the mature spore developed. At Stage 4 there was a pronounced gradient in wall thickness from the apex to the base of the spore. Small vesicles (30–60 nm diam) were uniformly distributed in the cytoplasm of spherically enlarging spores (Stage 2), but during spore elongation (Stages 3 and 4) numerous larger vesicles as well as small vesicles aggregated at the spore apex. A variety of cytoplasmic organelles entered the spore during Stage 3; however, migration of storage materials and the nucleus to the spore did not occur until late basidiospore development. The hilar appendix body developed in the earliest spore primordium and persisted until Stage 3. Development of wall layers and their differential thickening, distribution of vesicles, and probable function of the hilar appendix body are discussed with reference to the control of spore shape. Systematic implications of the data are considered.     

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.     

SPORE GERMINATION AND DEVELOPMENT OF THE YOUNG GAMETOPHYTE OF THE OSTRICH FERN (MATTEUCCIA STRUTHIOPTERIS)

Light is required for the germination of spores of Matteuccia struthiopteris. Histochemical studies show that dormant spores contain no starch, but have an abundance of storage protein granules. Starch accumulates in the numerous chloroplasts of the spore on exposure to light and becomes gradually more extensive. Protein granules disappear as germination progresses. Following this, the centrally located nucleus migrates toward the proximal spore face. Concomitant with the nuclear migration, an increase of cytoplasmic RNA surrounding the nucleus occurs. An equal nuclear division and unequal cell division give rise to a 2-celled gametophyte consisting of a large prothallial cell and smaller rhizoidal cell. A new peripheral wall forms around the entire protoplast at the time of nuclear migration, while a transverse wall forms after nuclear division. The rhizoid emerges through the split raphe along the proximal spore face; it is rich in cytoplasmic RNA but contains very few chloroplasts and little starch. Electron microscopy of the 2-celled stage revealed a greater concentration of mitochondria, Golgi bodies, and a more extensive endoplasmic reticulum in the rhizoid than was found in the prothallial cell, which, however, was far richer in chloroplasts and lipid bodies. As the rhizoid elongates and becomes more vacuolated, cytoplasmic RNA decreases as cytoplasmic protein increases. The rhizoid undergoes no cell divisions, while the prothallial cell retains the potential for further cell division. The possible significance of the distribution of storage products, cell organelles, and other cell components were considered in relation to the non-equational cell division and differentiation of the 2 cells.     

Contamination levels of Clostridium estertheticum spores that result in gaseous spoilage of vacuum‐packaged chilled beef and lamb meat

Aims: To determine the contamination levels of Cl. estertheticum spores that result in gaseous spoilage of vacuum‐packaged chilled meats, beef and lamb, stored at two different temperatures, ?1·5 and 2°C. Methods and Results: The study consisted of two separate trials using the same processing parameters applied to beef and lamb at two different storage temperatures and six different inoculation concentrations of Cl. estertheticum. A threshold for pack blowing of c. 1 spore per vacuum pack was seen with both beef and lamb stored at ?1·5 and 2°C. These results highlight the detrimental effect that increasing Cl. estertheticum spore inoculum concentration has on the onset of blown pack spoilage for both meat species stored at ?1·5 and 2°C. Conclusions: This study demonstrates that storage temperature is an extremely important parameter influencing the onset of blown pack spoilage and that storing meat at ?1·5°C significantly delays the onset of blown pack spoilage in comparison with storage at 2°C. Significance and Impact of study: The results of this study indicate that 1 Cl. estertheticum spore may present a risk of spoilage, and thus hygienic carcass dressing is critical to keep contamination to a minimum and maximize storage life of the vacuum‐packed chilled product.     

Bacillus stearothermophilus spores on strips. Calibration of thermoresistance kinetics to prior biological validation

Filter-paper strips saturated with Bacillus stearothermophilus spores were prepared and then stored for up to 1470 days. The strips were used to measures spore activation time and death kinetics in two systems: a serum bottle/oil bath combination, and glassine packages in a gravity autoclave. The data collected were analyzed primarily by analysis of variance and the interaction of spore storage time, processing temperature, inertial time and exposure time was determined.     

Tolerance of Penicillium expansum to postharvest fungicide treatments in apple packingshouses in Lerida (Spain)

Thiabendazole tolerant isolates of Penicillium expansum were recovered from sampling of natural spore populations in storage rooms and partially decayed apples collected from packinghouses in Lerida (Spain). The parasitic fitness of the resistant population of Penicillium expansum was studied.     

Optimization of the spray drying of a Paenibacillus polymyxa-based biopesticide on pilot plant and production scales

Spore survival and moisture content are two important properties of biopesticides, and both are related to field biocontrol efficacy and storage shelf life. In this study, Paenibacillus polymyxa (HY96-2) was spray-dried on both pilot plant and production scales, and the effects of inlet and outlet temperatures on spore survival and moisture content were investigated. The results showed that inlet temperatures ranging from 170 to 230 °C (at an outlet temperature of 80 °C) had no obvious effect on the two properties during pilot scale processing, although an inlet temperature of 230 °C resulted in higher feed speed. When the outlet temperature on the pilot scale was reduced from 100 to 80 °C, no obvious variations in spore survival and moisture content were found, while a further reduction from 80 to 65 °C resulted in a decline in spore survival from 81.0 to 67.0% and an increase in moisture content from 2.3 to 31.7%. These results indicate that both outlet temperature and moisture content have an effect on spore survival. Optimum inlet and outlet temperatures for P. polymyxa processing were 230 °C and 85–90 °C on a production scale. Under these conditions, spore survival and moisture content were 83.5–86.6% and 2.73––4.12%, respectively.     

Bacillus anthracis spore suspensions: determination of stability and comparison of enumeration techniques

Aim: To determine the stability and variability in concentration of spore suspensions of Bacillus anthracis (BA) spore suspensions by comparing different methods of enumeration and to detect changes, if any, under different storage conditions. Methods and Results: Plate and microscope counts were compared to measuring the genomic equivalents based on DNA content BA spore suspensions. We developed chemical methods to extract spore DNA and extra-spore (ES) DNA. DNA mass was determined by gel electrophoresis and QPCR assays were developed using the markers on the chromosome (rpoB) and the pXO1 plasmid (pag). The plate counts and microscope counts were very stable (for up to 900 days). The effect of freezing and the presence of additives in samples were tested for up to 300 days, and the results indicated that the additives tested and freezing did not decrease the viability or microscope counts. Conclusions: Bacillus anthracis spore suspensions can be stored for long periods of time without significant loss of viability or clumping. The content of ES DNA was variable and changed with time. Significant and Impact of the Study: The study shows that BA spore suspensions can be developed for reference materials providing a uniform basis for comparing detection equipment and results from different laboratories.     

GIBBERELLIC ACID-INDUCED GERMINATION OF SPORES OF ANEMIA PHYLLITIDIS: NUCLEIC ACID AND PROTEIN SYNTHESIS DURING GERMINATION

The distribution and synthesis of nucleic acids and proteins during gibberellic acid-induced germination of spores of Anemia phyllitidis were studied in order to relate biochemical activity with morphogenetic aspects of germination. Germination is accompanied by the hydrolysis of storage protein granules and the localized appearance of cytoplasmic RNA, protein, and insoluble carbohydrates in a small area adjoining the spore wall and surrounding the nucleus. The protoplast of the spore enlarges in this region, the spore wall breaks and a protonemal cell is formed which contains many chloroplasts. A second division in the spore at right angles to the first yields a rhizoid cell. Autoradiography of ³H-thymidine incorporation has shown that DNA is synthesized both in the nucleus and in the immediately surrounding cytoplasm of the germinating spore until some time after the first division, although a strictly nuclear DNA synthesis is observed later. Synthesis of RNA and proteins is limited to the presumptive regions of the germinating spore which become the protonema and rhizoid, shifting to specific sites in these cells as germination proceeds. The nucleus of the spore continues to be biosynthetically active long after it ceases to divide.     

GENETIC VARIABILITY WITHIN A POPULATION AND BETWEEN DIPLOID/HAPLOID TISSUE OF MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Multi-locus DNA fingerprints using an M13 probe were obtained for eight individuals of giant kelp Macrocystis pyrifera (L.) C. Ag. collected from Monterey Bay, California. For each individual, DNA was extracted from a diploid blade and from ca. 10⁹ haploid spores that were released from four to Jive sporophylls. Viable or swimming spores from one individual were pooled and referred to as a spore group. A total of 34 bands (4–19 kb) was detected in DNA fingerprints from the eight blades and eight spore groups, with individual blade or spore groups exhibiting 7–18 bands (mean = 12.6). One band (4.5 kb) was present in all 16 samples. Eight bands were detected in 11–14 of the 16 samples. Similarity indices were calculated for all pairwise comparisons of fingerprint bands among all possible combinations of blades and spore groups. Mean similarity indices for the eight blades (0.51, SE = 0.032) and spore groups (0.56, SE = 0.031) were significantly lower than for the eight comparisons of the blade and spore groups from a single individual (0.86, SE = 0.052). The data indicate that DNA fingerprints can be used to measure genetic variation within populations of M. pyrifera because variation of DNA fingerprints associated with meiotic products (spores) of a given individual is small relative to variation observed among individuals within the population. Additionally, fingerprint variation between diploid vegetative tissue and haploid meiotic products may be a measure of genetic change due to recombination or DNA turnover mechanisms.     

Aerobiological monitoring of Aspergillus/Penicillium spores during the potato storage

Aerobiological studies are widely used to determine the fungal spectrum in the air. These studies have revealed that Aspergillus/Penicillium spores are the most abundant spores in both outdoor and indoor environments. In this study, we have presented the variations in the concentration of these spores in an indoor environment (a potato store). Aerobiological sampling was conducted during five storage period (from 2002 to 2008 year) using a Hirst-type spore trap. The maximum spore concentrations were counted during the second fortnight of January and in the months of February and March, with values higher than 6,000 spores/m³ per day. A correlation analysis between the Aspergillus/Penicillium spores and the main environmental parameters was performed; significant coefficients were obtained for spores present in the store previous days and mean temperature of the same day and previous days (P < 0.001). Moreover, a regression model was established and predicted 53% variability of the data included in the analysis. The best obtained model took into account the Aspergillus/Penicillium spore type levels of 1 previous day and the mean temperature in the preceding 2 days.     

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.     

23种顶蒴藓类孢子形态的观察

报道了中国２３种顶蒴藓类孢子的形态特征。观察结果显示：孢子形状有两类,球形和卵球形。孢子直径可分为４组：１０μm以下、１１－２０μm、２１－３０μm和３１μm以上。萌发孔有３种类型：近极薄壁区、单裂缝和无萌发孔。外壁纹饰有４种：棒状、瘤状、疣状和鼓槌状。孢子颜色有６种：黄褐色、黑褐色、金黄色、草绿色和紫红色。藓类孢子体积不同物种间有差异,但球形和卵球形是基本类型。苔藓孢子的萌发孔有３种类型：三裂缝为类     

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.     

A respiratory nitrate reductase active exclusively in resting spores of the obligate aerobe Streptomyces coelicolor A3(2)

The Gram‐positive aerobe Streptomyces coelicolor undergoes a complex life cycle including growth as vegetative hyphae and the production of aerial hyphae and spores. Little is known about how spores retain viability in the presence of oxygen; however, nothing is known about this process during anaerobiosis. Here, we demonstrate that one of the three respiratory nitrate reductases, Nar‐1, synthesized by S. coelicolor is functional exclusively in spores. A tight coupling between nitrite production and the activity of the cytoplasmically oriented Nar‐1 enzyme was demonstrated. No exogenous electron donor was required to drive nitrate reduction, which indicates that spore storage compounds are used as electron donors. Oxygen reversibly inhibited nitrate reduction by spores but not by spore extracts, suggesting that nitrate transport might be the target of oxygen inhibition. Nar‐1 activity required no de novo protein synthesis indicating that Nar‐1 is synthesized during sporulation and remains in a latently active state throughout the lifetime of the spore. Remarkably, the rates of oxygen and of nitrate reduction by wetted spores were comparable. Together, these findings suggest that S. coelicolor spores have the potential to maintain a membrane potential using nitrate as an alternative electron acceptor.