Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Seeding nets with neutral spores of the red alga Porphyra umbilicalis (L.) Kutzing for use in integrated multi-trophic aquaculture (IMTA)

Nets in traditional Porphyra mariculture are seeded with conchospores derived from the conchocelis phase, and spend a nursery period in culture tanks or calm coastal waters until they reach several centimeters in length. Some species of Porphyra can regenerate the foliose phase directly through asexual reproduction, which suggests that the time, infrastructure, and costs associated with conchocelis culture might be avoided by seeding nets with asexual spores. Here, we present work from a short-term mariculture study using nets seeded with asexual spores (neutral spores) of a native Maine species of Porphyra. Porphyra umbilicalis (L.) Kutzing was selected for this proof of concept research because of its reproductive biology, abundance across seasons in Maine, and evidence of its promise as a mariculture crop. We studied the maturation, release, and germination of the neutral spores to develop an appropriate seeding protocol for nets, followed by development of a nursery raceway to provide an easily manipulated environment for the seeded nets. Neutral spores were produced throughout the year on the central Maine coast,however, there was a temporal variability in the number and survival of released neutral spores, depending upon thallus position in the intertidal zone. Small thalli were strictly vegetative, but most thalli reproduced by neutral spores- sexual reproduction was absent. Neutral spores germinated quickly at 10 and 15 'C, but germination was delayed at 5 degrees C. Unlike some algal zygotes and spores, neutral spores of R umbilicalis required light to germinate; however, irradiances of 25 and 100 mu mol photons M-2 S-1 were equally sufficient for germination. Rafts of seeded nets were deployed in Cobscook Bay, Maine, at two distances from salmon aquaculture pens and at a control site on a nearby, fallow aquaculture site (no salmon). There was no difference in nitrogen content of harvested thalli; however, both the density and the surface area of harvested thalli were different among the sites. The possible causes of these differences are discussed in the context of potential use of P umbilicalis in IMTA. (C) 2007 Elsevier B.V. All rights reserved.

In vitro spore germination of Polystichum drepanum, a threatened fern from Madeira Island

Polystichum drepanum (Sw) C. Presl is a threatened fern endemic to a few forest areas in the north-west of Madeira Island. The aims of this work were to establish suitable culture conditions for in vitro germination of spores, and to evaluate short-term storage conditions for P drepanum spores. The highest frequency of germination was obtained in Murishage and Skoog (MS) liquid medium, without agitation. However, gametophytes maintained in MS liquid medium did not grow and, after 4 weeks, became anoxic and died. Thus, after germination in liquid medium, gametophytes were transferred to an MS double-phase culture system for further growth. The effects of storage period, temperature, and relative humidity during storage on in vitro spore germination were studied. Spore viability was assessed after 2, 4 and 6 months, and high viability (> 94%) was observed in all the assays. However, germination capability decreased with increased storage periods. The number of sporophytes obtained also decreased with prolonged storage periods. The results indicate that spores of R drepanum stored for 4 months at 21 degrees C maintain high viability and high germination frequency. ne sporophytes obtained were acclimatised in a mixture of peat and vermiculite [2:1 (v/v)] under high relative humidity (90-95%). Seventy-five sporophytes were successfully acclimatised to ex vitro conditions and showed active growth in the glasshouse.

Effect of small, acid-soluble proteins on spore resistance and germination under a combination of pressure and heat treatment

To find the range of pressure required for effective high-pressure inactivation of bacterial spores and to investigate the role of alpha/beta-type small, acid-soluble proteins (SASP) in spores under pressure treatment, mild heat was combined with pressure (room temperature to 65 degrees C and 100 to 500 MPa) and applied to wild-type and SASP-alpha(-/)beta(-) Bacillus subtilis spores. On the one hand, more than 4 log units of wild-type spores were reduced after pressurization at 100 to 500 MPa and 65 degrees C, On the other hand, the number of surviving mutant spores decreased by 2 log units at 100 MPa and by more than 5 log units at 500 MPa. At 500 MPa and 65 degrees C, both wild-type and mutant spore survivor counts were reduced by 5 log units. Interestingly, pressures of 100, 200, and 300 MPa at 65 degrees C inactivated wild-type SASP-alpha(+)/beta(+) spores more than mutant SASP-alpha(-)/beta(-) spores, and this was attributed to less pressure-induced germination in SASP-alpha(-)/beta(-) spores than in wild-type SASP-alpha(+)/beta(+) spores. However, there was no difference in the pressure resistance between SASP-alpha(+)/beta(+) and SASP-alpha(-)/beta(-) spores at 100 MPa and ambient temperature (approximately 22 degrees C) for 30 min. A combination of high pressure and high temperature is very effective for inducing spore germination, and then inactivation of the germinated spore occurs because of the heat treatment. This study showed that alpha/beta-type SASP play a role in spore inactivation by increasing spore germination under 100 to 300 MPa at high temperature.

Germination rate and sporeling development of Chondracanthus chamissoi (Rhodophyta, Gigartinales) varies along a latitudinal gradient on the coast of Chile

Chondracanthus chamissoi (C. Agardh) Kutzing is an economically important red seaweed with an extended latitudinal distribution along the south-east Pacific. Here we report on the seasonal in vitro germination of carpospores and tetraspores from four populations distributed from 27 to 41 degrees S on the Chilean coast. Our results show that both types of spores exhibited a different physiological behavior related to the geographic origin of the specimens. Germination occurred throughout the year for both spore types in the four populations. However, for the northern locations (Calderilla, La Herradura and Puerto Aldea) germination was higher in spring, while for the southern location (Lechagua), germination was higher in summer. The growth rate of carposporelings and tetrasporelings varied seasonally in ail locations studied, with higher growth in spring. Among all, carposporelings from Lechagua specimens reached the highest growth rates (9.3 +/- 0.2% d(-1)). However, spores from Herradura and P. Aldea had a good germination and SGR in all seasons and would be good candidates to start spores-based cultivation of this valuable resource in Chile. (C) 2009 Elsevier B.V. All rights reserved

Germination and mycelial growth of Bipolaris euphorbiae Muchovej & Carvalho as influenced by herbicides and surfactants

Bipolaris euphorbiae Muchovej & Carvalho é um forte candidato para o controle de Euphorbia heterophylla L. (amendoim bravo). Este fungo pode ser aplicado em combinação com herbicidas para controlar um maior espectro de espécies daninhas. Para tanto, experimentos laboratoriais foram realizados para verificar a possibilidade da utilização de mistura de tanque de esporos de B. euphorbiae e herbicidas ou surfatantes recomendados para a cultura da soja. Crescimento micelial e germinação de conídios foram avaliados em meio BDA acrescido dos herbicidas, nas concentrações recomendadas dos produtos comerciais, oxasulfuron (80 g/ha), glifosato (4 L/ha), bentazon (1.5 L/ha), fomesafen (1 L/ha), chlorimuron-ethyl (80 g/ha), lactofen (1 L/ha) e imazetaphyr (1 L/ha) e dos surfatantes Energic (2 ml/L), Aterbane (2,5 ml/L), Silwet L-77Ag (1 ml/L), Herbitensil (2 ml/L) e Natur L'óleo (10 ml/L). Diluições dos herbicidas de 50% e 25% foram avaliadas com um consumo de calda equivalente a 300 L/ha. Os surfatantes foram somente utilizados nas concentrações recomendadas. O crescimento micelial não foi afetado por bentazon e fomesafen e apenas levemente por oxasulfuron. Porém, glifosato, chlorimuron-ethyl, lactofen, Energic, Herbitensil, Silwet, e Aterbane o reduziram drasticamente. A redução observada com imazetaphyr foi intermediária e Natur L' óleo promoveu o crescimento micelial. Na presença dos surfatantes, observou-se que todos permitiram uma porcentagem de germinação equivalente àquela alcançada na presença de água. Energic e Herbitensil causaram um retardamento expressivo. Com Herbitensil, o processo germinativo iniciou somente aos 120 minutos. Com herbicidas, foi observado que somente na presença de glifosato e imazetaphyr a germinação dos conídios não seguiu a tendência observada com água, como ocorreu com os outros produtos testados.

Study of the presence of the spores of Clostridium botulinum in honey in Brazil

The isolation of Clostridium botulinum from honey samples is described. Botulism is characterized as an intoxication provoked by ingestion of contaminated foods with this toxin. Infant botulism happens by the ingestion of spores of C. botulinum together with food that in special conditions of the intestinal tract, such as those present in babies of less than 1 year old, will allow the germination and colonization of the intestine with production and absorption of botulinic toxin. The samples were subjected to dilution and to a thermal shock and cultivated in modified CMM (Difco). Cultures were subjected to Gram smears and toxicity tests in mice. The toxic cultures were purified in RFCA (Oxoid) plates and incubated in anaerobic jars. Positive samples were typed using the mouse assay neutralization test. From the 85 honey samples analyzed, six were positive for C. botulinum (7.06%), and identified as producers of type A, B, and D toxins.

Influence of light, plant density and disinfection in spore germination of fern blechnum brasiliense desv. blechnaceae

The present assay had as objective evaluating spore germination of Blechnum brasiliense in relation to light, plant density and disinfection. The assay was carried out at Jaboticabal, Sào Paulo State, Brazil, from February, 22 to June, 30, 1996. The experimental design was randomized blocks on a factorial scheme (3x2x2), consisting of 12 treatments, three environments (shade-house, dark-house and germination camera), 2 densities (0.005 grs and 0.010 grs of spores/treatment) and presence or absence of disinfection. The leaf coverage area (130 days) and the number of days necessary to germinate were evaluated. The germination camera data were not analysed because they were insignificant; consequently, the remining data were analysed on a 2×2×2 scheme. The shade-house provided larger green covering area and a faster germination. The density of 0.0 lOg of spore/treatments presented the largest green covering area. The supply of partial light was necessary for good germination. The interaction between the environment and the density had significant effect on the green covering area.

Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance

Bacterial endospores derive much of their longevity and resistance properties from the relative dehydration of their protoplasts. The spore cortex, a peptidoglycan structure surrounding the protoplasm, maintains, and is postulated to have a role in attaining, protoplast dehydration. A structural modification unique to the spore cortex is the removal of all or part of the peptide side chains from the majority of the muramic acid residues and the conversion of 50% of the muramic acid to muramic lactam. A mutation in the cwlD gene of Bacillus subtilis, predicted to encode a muramoyl-l-alanine amidase, results in the production of spores containing no muramic lactam. These spores have normally dehydrated protoplasts but are unable to complete the germination/outgrowth process to produce viable cells. Addition of germinants resulted in the triggering of germination with loss of spore refractility and the release of dipicolinic acid but no degradation of cortex peptidoglycan. Germination in the presence of lysozyme allowed the cwlD spores to produce viable cells and showed that they have normal heat resistance properties. These results (i) suggest that a mechanical activity of the cortex peptidoglycan is not required for the generation of protoplast dehydration but rather that it simply serves as a static structure to maintain dehydration, (ii) demonstrate that degradation of cortex peptidoglycan is not required for spore solute release or partial spore core rehydration during germination, (iii) indicate that muramic lactam is a major specificity determinant of germination lytic enzymes, and (iv) suggest the mechanism by which the spore cortex is degraded during germination while the germ cell wall is left intact.

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.

Water-, pH- and temperature relations of germination for the extreme xerophiles Xeromyces bisporus (FRR 0025), Aspergillus penicillioides (JH06THJ), and Eurtotium halophilicum (FRR 2471)

Water activity, temperature and pH are determinants for biotic activity of cellular systems, biosphere function and, indeed, for all life processes. This study was carried out at high concentrations of glycerol, which concurrently reduces water activity and acts as a stress protectant, to characterize the biophysical capabilities of the most extremely xerophilic organisms known. These were the fungal xerophiles: Xeromyces bisporus (FRR 0025), Aspergillus penicillioides (JH06THJ) and Eurotium halophilicum (FRR 2471). High-glycerol spores were produced and germination was determined using 38 media in the 0.995–0.637 water activity range, 33 media in the 2.80–9.80 pH range and 10 incubation temperatures, from 2 to 50°C. Water activity was modified by supplementing media with glycerol+sucrose, glycerol+NaCl and glycerol+NaCl+sucrose which are known to be biologically permissive for X. bisporus, A. penicillioides and E. halophilicum respectively. The windows and rates for spore germination were quantified for water activity, pH and temperature; symmetry/asymmetry of the germination profiles were then determined in relation to supra- and sub-optimal conditions; and pH- and temperature optima for extreme xerophilicity were quantified. The windows for spore germination were ~1 to 0.637 water activity, pH 2.80–9.80 and > 10 and < 44°C, depending on strain. Germination profiles in relation to water activity and temperature were asymmetrical because conditions known to entropically disorder cellular macromolecules, i.e. supra-optimal water activity and high temperatures, were severely inhibitory. Implications of these processes were considered in relation to the in-situ ecology of extreme conditions and environments; the study also raises a number of unanswered questions which suggest the need for new lines of experimentation.

Real time detectionof airborne fungal spores and investigations into their dynamics in indoor air

Concern regarding the health effects of indoor air quality has grown in recent years, due to the increased prevalence of many diseases, as well as the fact that many people now spend most of their time indoors. While numerous studies have reported on the dynamics of aerosols indoors, the dynamics of bioaerosols in indoor environments are still poorly understood and very few studies have focused on fungal spore dynamics in indoor environments. Consequently, this work investigated the dynamics of fungal spores in indoor air, including fungal spore release and deposition, as well as investigating the mechanisms involved in the fungal spore fragmentation process. In relation to the investigation of fungal spore dynamics, it was found that the deposition rates of the bioaerosols (fungal propagules) were in the same range as the deposition rates of nonbiological particles and that they were a function of their aerodynamic diameters. It was also found that fungal particle deposition rates increased with increasing ventilation rates. These results (which are reported for the first time) are important for developing an understanding of the dynamics of fungal spores in the air. In relation to the process of fungal spore fragmentation, important information was generated concerning the airborne dynamics of the spores, as well as the part/s of the fungi which undergo fragmentation. The results obtained from these investigations into the dynamics of fungal propagules in indoor air significantly advance knowledge about the fate of fungal propagules in indoor air, as well as their deposition in the respiratory tract. The need to develop an advanced, real-time method for monitoring bioaerosols has become increasingly important in recent years, particularly as a result of the increased threat from biological weapons and bioterrorism. However, to date, the Ultraviolet Aerodynamic Particle Sizer (UVAPS, Model 3312, TSI, St Paul, MN) is the only commercially available instrument capable of monitoring and measuring viable airborne micro-organisms in real-time. Therefore (for the first time), this work also investigated the ability of the UVAPS to measure and characterise fungal spores in indoor air. The UVAPS was found to be sufficiently sensitive for detecting and measuring fungal propagules. Based on fungal spore size distributions, together with fluorescent percentages and intensities, it was also found to be capable of discriminating between two fungal spore species, under controlled laboratory conditions. In the field, however, it would not be possible to use the UVAPS to differentiate between different fungal spore species because the different micro-organisms present in the air may not only vary in age, but may have also been subjected to different environmental conditions. In addition, while the real-time UVAPS was found to be a good tool for the investigation of fungal particles under controlled conditions, it was not found to be selective for bioaerosols only (as per design specifications). In conclusion, the UVAPS is not recommended for use in the direct measurement of airborne viable bioaerosols in the field, including fungal particles, and further investigations into the nature of the micro-organisms, the UVAPS itself and/or its use in conjunction with other conventional biosamplers, are necessary in order to obtain more realistic results. Overall, the results obtained from this work on airborne fungal particle dynamics will contribute towards improving the detection capabilities of the UVAPS, so that it is capable of selectively monitoring and measuring bioaerosols, for which it was originally designed. This work will assist in finding and/or improving other technologies capable of the real-time monitoring of bioaerosols. The knowledge obtained from this work will also be of benefit in various other bioaerosol applications, such as understanding the transport of bioaerosols indoors.

Can rodents enhance germination rates in rainforest seeds?

The decline of large coevolved frugivorous species within fragmented habitats can have an effect on ecological processes, for example, seed dispersal and germination. It is therefore necessary for more resilient species to ensure essential processes are maintained within the system. This study investigates the influence of two rodent species, Melomys cervinipes (Fawn-footed Melomys) and Rattus fuscipes (Bush Rat), on the germination process of rainforest fruits. Both species are endemic to north Queensland rainforest and commonly found in fragmented habitats in high densities. We found in 85% of fruit species tested, rodent feeding increased seed germination rate by a factor of 3.5. Our results suggest that rodents can play a significant role in enhancing germination rates of fruits in the tropical rainforest of far north Queensland.

Seed germination may explain differences in invasiveness and prevalence : a case study using cat's claw creeper (Dolichandra unguis-cati)

High germination rates and rapid germination behavior in response to different environmental cues are traits that may be associated with invasiveness. Cat’s claw creeper (Dolichandra unguis-cati (L.) Lohmann (syn. Macfadyena unguis-cati (L.) Gentry), a Weed of National Significance has two forms, a long-pod (LP) form and a short-pod (SP) from. The LP form occurs in only a few localities in southeast Queensland while the SP form is widely distributed in Queensland and New South Wales. The aims of this investigation were: to evaluate whether there are significant differences in germination traits between the two forms of cat’s claw creeper; and if there are any significant differences, to find out whether the differences in germination can be related to prevalence and invasiveness levels for the two forms. Long pod and short pod seeds collected in 2009, 2010, 2011, 2012 and 2013 from various localities in Qld were germinated in growth chambers in early 2013. The growth chambers were set to 10/20 ºC, 15/25 ºC and 20/30 ºC temperature cycles. Seeds from 2009-2012 of either form did not germinate, while for the fresh seeds (2013), SP exhibited significantly higher total germination percentage and rates than LP. Assuming that the two forms were introduced in Australia at around the same period, these results could explain why SP is widely distributed (and therefore more invasive) in Qld and NSW while LP is only confined to a few localities in southeast Qld.

Germination and hardseededness in desmanthus

The mechanisms and control of hardseededness in the 3 Australian cultivars of the genus Desmanthus were investigated in a series of experiments in which the effects of various seedsoftening treatments, particularly boiling water, were measured. Desmanthus seed is predominantly hard, only defective seeds being normally otherwise. As it has only very brief, early embryo dormancy, hardseededness is the only serious barrier to germination. Seed is most readily softened through rupture of the palisade at the lens (strophiole). The lens is of a typically mimosaceous type which is readily ruptured by immersion in boiling water or less readily by application of pressure to adjacent parts of the testa. Ruptures may consist only of separation of the palisade from underlying tissue, which alone does not confer permeability; mostly they also result in fractures to the palisade that then render seeds irreversibly permeable. The palisade becomes reflective as it separates, which allows the event to be witnessed at the moment of separation if suitable pressure is applied to the testa of an individual seed while it is viewed under magnification. Brief (4–10 seconds) immersion of highquality seed in boiling water consistently softened a high proportion of seeds without causing serious damage. Extending the duration of immersion led to a progressive increase in the proportion of seed deaths. Neither previous boiling water treatment nor scarification damage to the testa materially affected results of treatment, but immature and small seeds behaved differently, being more vulnerable to damage than mature seed, and less likely to undergo lens rupture. Adaptation of boiling water treatment to farm-scale seed handling was simple and reliable. Commercial treatment of seed by an alternative method suitable for greater bulks and consisting of passage through a rice-whitener was checked and found to be successful through a combination of gentle scarification and lens rupture, both attributable to the numerous minor impacts of the process. Percentage emergence of seedlings from soil in the greenhouse closely followed percentage laboratory germination, except when inferior seed grades were included in the comparison, when emergence was poor. Very little seed softened in soil. Already-permeable seed either germinated rapidly or died, while buried hard seed mostly remained hard and viable even more than a year after sowing.