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.     

Studies on the prolonged storage of Metarhizium anisopliae conidia: effect of growth substrate on conidial survival and virulence against mosquitoes

Metarhizium anisopliae was grown on six complex mycological media and on three types of rice at three moisture levels to determine the effect of growth substrate on conidial yield, viability, and virulence against mosquitoes immediately after spore maturation and after the storage of conidia at four different temperature-relative humidity (RH) combinations over a 1-year period. Conidial yields varied with the mycological media, but the viability and virulence of conidia against mosquitoes produced on all substrates were similar when spores were stored under the same conditions. The storage conditions were more critical to spore survival and virulence than the substrate upon which conidia were produced. The comparison of rice types for conidial production indicated that conidial yield, viability, and virulence to mosquitoes were more dependent upon the moisture level during growth and on the storage conditions that upon the rice used. The best storage conditions among those tested for the retention of both spore viability and virulence against mosquitoes were 19°C–97% RH and 4°C–0% RH.     

Germination and inactivation of Bacillus subtilis spores induced by moderate hydrostatic pressure

In this study, we investigated the mechanisms of spore inactivation by high pressure at moderate temperatures to optimize the sterilization efficiency of high‐pressure treatments. Bacillus subtilis spores were first subjected to different pressure treatments ranging from 90 to 550 MPa at 40°C, with holding times from 10 min to 4 h. These treatments alone caused slight inactivation, which was related to the pressure‐induced germination of the spores. After these pressures treatments, the sensitivity of these processed spores to heat (80°C/10 min) or to high pressure (350 MPa/40°C/10 min) was tested to determine the pressure‐induced germination rate and the advancement of the spores in the germination process. The subsequent heat or pressure treatments were applied immediately after decompression from the first pressure treatment or after a holding time at atmospheric pressure. As already known, the spore germination is more efficient at low pressure level than at high pressure level. Our results show that this low germination efficiency at high pressure seemed not to be related either to a lower induction or a difference in the induction mechanisms but rather to an inhibition of enzyme activities which are involved in germination process. In fact, high pressure was necessary and very efficient in inducing spore germination. However, it seemed to slow the enzymatic digestion of the cortex, which is required for germinated spores to be inactivated by pressure. Although these results indicate that high‐pressure treatments are more efficient when the two treatments are combined, a small spore population still remained dormant and was not inactivated with any holding time or pressure level. Biotechnol. Bioeng. 2010;107: 876–883. © 2010 Wiley Periodicals, Inc.     

Influence of Temperature and Relative Humidity on Germinability of Mucor piriformis Spores

Studies were conducted to determine the influence of temperature and relative humidity (RH) on germinability and viability of Mucor piriformis spores. Spores did not survive when stored at 35 °C and their survival rate decreased rapidly at 30 °C; however, spores remained viable for more than 1 year at 0 °C. RH also significantly affected spore viability. Spores held at 26 °C and 100% RH no longer germinated after 35 days, while those held at 75 or 90% RH germinated for 65 days. At 20 °C, RH had little effect on spore germinability. The effect of temperature and RH on percentage spore germination also varied. At all temperatures studied, spore viability decreased more rapidly with time at 100% RH than at 75 or 90% RH. The least favorable, temperature-humidity combination, 30 °C and 100% RH, decreased spore germination from 100% to less than 1% in 14 days.     

Analysis of the action of compounds that inhibit the germination of spores of Bacillus species

AIMS: To determine the mechanism of action of inhibitors of the germination of spores of Bacillus species, and where these inhibitors act in the germination process. METHODS AND RESULTS: Spores of various Bacillus species are significant agents of food spoilage and food-borne disease, and inhibition of spore germination is a potential means of reducing such problems. Germination of the following spores was studied: (i) wild-type B. subtilis spores; (ii) B. subtilis spores with a nutrient receptor variant allowing recognition of a novel germinant; (iii) B. subtilis spores with elevated levels of either the variant nutrient receptor or its wild-type allele; (iv) B. subtilis spores lacking all nutrient receptors and (v) wild-type B. megaterium spores. Spores were germinated with a variety of nutrient germinants, Ca2+-dipicolinic acid (DPA) and dodecylamine for B. subtilis spores, and KBr for B. megaterium spores. Compounds tested as inhibitors of germination included alkyl alcohols, a phenol derivative, a fatty acid, ion channel blockers, enzyme inhibitors and several other compounds. Assays used to assess rates of spore germination monitored: (i) the fall in optical density at 600 nm of spore suspensions; (ii) the release of the dormant spore's large depot of DPA; (iii) hydrolysis of the dormant spore's peptidoglycan cortex and (iv) generation of CFU from spores that lacked all nutrient receptors. The results with B. subtilis spores allowed the assignment of inhibitory compounds into two general groups: (i) those that inhibited the action of, or response to, one nutrient receptor and (ii) those that blocked the action of, or response to, several or all of the nutrient receptors. Some of the compounds in groups 1 and 2 also blocked action of at least one cortex lytic enzyme, however, this does not appear to be the primary site of their action in inhibiting spore germination. The inhibitors had rather different effects on germination of B. subtilis spores with nutrients or non-nutrients, consistent with previous work indicating that germination of B. subtilis spores by non-nutrients does not involve the spore's nutrient receptors. In particular, none of the compounds tested inhibited spore germination with dodecylamine, and only three compounds inhibited Ca2+-DPA germination. In contrast, all compounds had very similar effects on the germination of B. megaterium spores with either glucose or KBr. The effects of the inhibitors tested on spores of both Bacillus species were largely reversible. CONCLUSIONS: This work indicates that inhibitors of B. subtilis spore germination fall into two classes: (i) compounds (most alkyl alcohols, N-ethylmaleimide, nifedipine, phenols, potassium sorbate) that inhibit the action of, or response to, primarily one nutrient receptor and (ii) compounds [amiloride, HgCl2, octanoic acid, octanol, phenylmethylsulphonylfluoride (PMSF), quinine, tetracaine, tosyl-l-arginine methyl ester, trifluoperazine] that inhibit the action of, or response to, several nutrient receptors. Action of these inhibitors, is reversible. The similar effects of inhibitors on B. megaterium spore germination by glucose or KBr indicate that inorganic salts likely trigger germination by activating one or more nutrient receptors. The lack of effect of all inhibitors on dodecylamine germination suggests that this compound stimulates germination by creating channels in the spore's inner membrane allowing DPA release. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the steps in spore germination that are inhibited by various chemicals, and the mechanism of action of these inhibitors. The work also provides new insights into the process of spore germination itself.     

Preservation, mass production and storage ofStagonospora convolvuli, a bioherbicidecandidate for field bindweed (Convolvulusarvensis)

Different solid substrates were investigated as spore production methods for Stagonospora convolvulistrain LA39, a potential bioherbicide for field bindweed (Convolvulusarvensis L.). Up to 4 × 10⁸ spores/g of substratewere yielded on cous-cous (cracked hard wheat). Thespores were as pathogenic as those grown on artificial medium (V-8-juice agar). The air-drying on kaolin and storage at 3 °C kept spores viable and pathogenic for 180 days. Spore germination exceeded70% for the first 140 days and then declined to 50%after 175 days. Less than 5% of spores were still viable after 17 months. The preservation of stock cultures in 10% glycerine at −80 ° C and in liquid nitrogen did not affect viability orpathogenicity of the spores. This revised version was published online in July 2006 with corrections to the Cover Date.     

Viability testing and characterization of germination of fungal spores by automatic image analysis

Fungal spores are used in the laboratory for culture maintenance and at laboratory and other scales as inocula for fermentations. The spore swelling and germination processes constitute a major part of the lag phase, and the subsequent culture morphology and productivity can be greatly influenced by the initial concentration and condition of the spores. An image analysis method has been developed for assessing the viability and the germination characteristics of fungal spores in submerged cultures. Structural variations during germination, i.e., swelling, germ tube formation, and germ tube elongation, are measured in terms of distributions of spore volumes and of germ tube lengths and volumes. These measurements are fully automatic and give a very rapid assessment of spore viability. This image analysis method might be used as a tool in culture maintenance and for determining the quality of inocula for fungal fermentations. (c) 1993 John Wiley & Sons, Inc.     

Effect of temperature and dark pretreatment on the germination of three species of Jamesonia (Pteridaceae,Polypodiopsida)

To study the influence of temperature on the germination ability of three species of Jamesonia (Jamesonia imbricata, Jamesonia scammaniae and Jamesonia rotundifolia), spores were cultured at 10°C, 15°C and 20°C. A temperature of 15°C was selected as representative of the natural annual average temperature of the paramo environment that Jamesonia species inhabit. In addition, a dark pretreatment of 2 days was tested to verify if germination was enhanced. The results indicated that germination of Jamesonia, considering the three species as a whole, is affected by temperature, but is independent of the dark treatment. All species showed higher and faster germination at 20°C, and exhibited a threshold minimum temperature around 10°C, below which germination is avoided or extremely low and delayed. This could suggest that spore germination in Jamesonia is adapted to establish gametophyte populations during frost‐free periods.     

Effect of mechanical abrasion on the viability, disruption and germination of spores of Bacillus subtilis

AIMS: To elucidate the factors influencing the sensitivity of Bacillus subtilis spores in killing and disrupting by mechanical abrasion, and the mechanism of stimulation of spore germination by abrasion. METHODS AND RESULTS: Spores of B. subtilis strains were abraded by shaking with glass beads in liquid or the dry state, and spore killing, disruption and germination were determined. Dormant spores were more resistant to killing and disruption by abrasion than were growing cells or germinated spores. However, dormant spores of the wild-type strain with or without most coat proteins removed, spores of strains with mutations causing spore coat defects, spores lacking their large depot of dipicolinic acid (DPA) and spores with defects in the germination process exhibited essentially identical rates of killing and disruption by abrasion. When spores lacking all nutrient germinant receptors were enumerated by plating directly on nutrient medium, abrasion increased the plating efficiency of these spores before killing them. Spores lacking all nutrient receptors and either of the two redundant cortex-lytic enzymes behaved similarly in this regard, but the plating efficiency of spores lacking both cortex-lytic enzymes was not stimulated by abrasion. CONCLUSIONS: Dormant spores are more resistant to killing and disruption by abrasion than are growing cells or germinated spores, and neither the complete coats nor DPA are important in spore resistance to such treatments. Germination is not essential for spore killing by abrasion, although abrasion can trigger spore germination by activation of either of the spore's cortex-lytic enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanisms of the killing, disruption and germination of spores by abrasion and makes the surprising finding that at least much of the spore coat is not important in spore resistance to abrasion.     

Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure

AIMS: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination. METHODS AND RESULTS: Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50 degrees C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60 degrees C, in contrast to an optimum temperature of 40 degrees C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23 degrees C to 44 degrees C, and decreased twofold when 1 mol l(-1) salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg(2+), but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment. CONCLUSIONS: VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear. SIGNIFICANCE AND IMPACT OF THE STUDY: There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.     

Characterization of the germination of Bacillus megaterium spores lacking enzymes that degrade the spore cortex

Aims:  To determine roles of cortex lytic enzymes (CLEs) in Bacillus megaterium spore germination.
Methods and Results:  Genes for B. megaterium CLEs CwlJ and SleB were inactivated and effects of loss of one or both on germination were assessed. Loss of CwlJ or SleB did not prevent completion of germination with agents that activate the spore's germinant receptors, but loss of CwlJ slowed the release of dipicolinic acid (DPA). Loss of both CLEs also did not prevent release of DPA and glutamate during germination with KBr. However, cwlJ sleB spores had decreased viability, and could not complete germination. Loss of CwlJ eliminated spore germination with Ca²⁺ chelated to DPA (Ca-DPA), but loss of CwlJ and SleB did not affect DPA release in dodecylamine germination.
Conclusions:  CwlJ and SleB play redundant roles in cortex degradation during B. megaterium spore germination, and CwlJ accelerates DPA release and is essential for Ca-DPA germination. The roles of these CLEs are similar in germination of B. megaterium and Bacillus subtilis spores.
Significance and Impact of the Study:  These results indicate that redundant roles of CwlJ and SleB in cortex degradation during germination are similar in spores of Bacillus species; consequently, inhibition of these enzymes will prevent germination of Bacillus spores.     

Preservation of Phakopsora pachyrhizi Uredospores

This study compared different temperatures and dormancy‐reversion procedures for preservation of Phakopsora pachyrhizi uredospores. The storage temperatures tested were room temperature, 5°C, ?20°C and ?80°C. Dehydrated and non‐dehydrated uredospores were used, and evaluations for germination (%) and infectivity (no. of lesions/cm²) were made with fresh harvested spores and after 15, 29, 76, 154 and 231 days of storage. The dormancy‐reversion procedures evaluated were thermal shock (40°C/5 min) followed or not by hydration (moist chamber/24 h). Uredospores stored at room temperature were viable only up to a month of storage, regardless of their hydration condition. Survival of uredospores increased with storage at lower temperatures. Dehydration of uredospores prior to storage increased their viability, mainly for uredospores stored at 5°C, ?20°C and ?80°C. At 5°C and ?20°C, dehydrated uredospores showed increases in viability of at least 47 and 127 days, respectively, compared to non‐dehydrated spores. Uredospore germination and infectivity after storage for 231 days (7.7 months), could only be observed at ?80°C, for both hydration conditions. At this storage temperature, dehydrated and non‐dehydrated uredospores exhibited 56 and 28% of germination at the end of the experiment, respectively. Storage at ?80°C also maintained uredospore infectivity, based upon levels of infection frequency, for both hydration conditions. Among the dormancy‐reversion treatments applied to spores stored at ?80°C, those involving hydration allowed recoveries of 85 to 92% of the initial germination.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

Summer meeting 2013 – when the sleepers wake: the germination of spores of Bacillus species

Spores of Bacillus species can remain dormant and resistant for years, but can rapidly ‘come back to life’ in germination triggered by agents, such as specific nutrients, and non‐nutrients, such as CaDPA, dodecylamine and hydrostatic pressure. Major events in germination include release of spore core monovalent cations and CaDPA, hydrolysis of the spore cortex peptidoglycan (PG) and expansion of the spore core. This leads to a well‐hydrated spore protoplast in which metabolism and macromolecular synthesis begin. Proteins essential for germination include the GerP proteins that facilitate germinant access to spores' inner layers, germinant receptors (GRs) that recognize and respond to nutrient germinants, GerD important in rapid GR‐dependent germination, SpoVA proteins important in CaDPA release and cortex‐lytic enzymes that degrade cortex PG. Rates of germination of individuals in spore populations are heterogeneous, and methods have been developed recently to simultaneously analyse the germination of multiple individual spores. Spore germination heterogeneity is due primarily to large variations in GR levels among individual spores, with spores that germinate extremely slowly and termed superdormant having very low GR levels. These and other aspects of spore germination will be discussed in this review, and major unanswered questions will also be discussed.     

The Role of Osmotic Pressure in the Germination of Nosema algerae Spores1

ABSTRACT. Both the lag period and the time required for the filament and sporoplasm to emerge from Nosema algerae spores were prolonged when germination occurred under hyperosmotic conditions. Polyethylene glycol (PEG) and sucrose inhibited germination, first by preventing eversion of the filament, and then at higher concentrations by preventing stimulation. The size of the spore cases decreased by about 21% following germination, indicating an elastic spore wall and turgor pressure in the dormant spores. Increased pressure during germination was indicated by less osmotically-induced shrinkage in stimulated than in dormant spores and by higher concentration of solutes in the homogenates of germinated than ungerminated spores. These results are consistent with the hypothesis of a pressure increase during germination that is caused by an endogenous increase in solute concentration.     

Differences between Brachiola (Nosema) algerae isolates of human and insect origin when tested using an in vitro spore germination assay and a cultured cell infection assay

Brachiola (Nosema) algerae is a microsporidian species generally believed to be an intracellular parasite of insects, especially mosquitoes. However, both mosquito and human isolates have been shown to infect mammalian cells. The present study was undertaken to determine if spores of two insect and two human isolates of B. algerae cultured at 30 degrees C and 37 degrees C differed in their ability to germinate and infect cultured green monkey kidney cells at these two temperatures. Spores from all four isolates exhibited an optimum pH of 9.5 for germination. Mercury (Hg2+) inhibited germination of all isolates equally. Germination of spores from all four isolates was significantly greater when the parasite was cultured at 30 degrees C than when cultured at 37 degrees C. However, spores from the insect isolates cultivated at 30 degrees C or 37 degrees C infected significantly fewer mammalian cells at 37 degrees C than did spores from the human isolates under the same conditions. Thus, there is no correlation between the effects of temperature on the germination and the infectivity of an isolate. In addition, while exposure of B. algerae to 37 degrees C has been reported to cause spore dysmorphism, we failed to observe any consistent ultrastructural changes that explained the greater infectivity of the human isolates at 37 degrees C.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spore germination of Frankia strains isolated from Colletia hystrix and Retanilla ephedra (Rhamnaceae)

Two Frankia strains, ChI1 from Colletia hystrix and ReI6 from Retanilla ephedra, were assayed to determine the germinability of their spores and the stages of the life cycle in different media. In BAP medium, both strains showed approximately 18% spore germination. However, in BAP medium lacking micronutrients and FeEDTA, strain ChI1 spores exhibited approximately 53% germination and strain ReI6 spores about 42%. The survey also showed that the spores were not heat-resistant, the ReI6 spores being more sensitive than ChI1 spores. Both strains exhibited a small increase in their percentage of germination with a 15-pulse ultrasonic treatment.     

A correlated response of a parasite’s virulence and life cycle to selection on its host’s life history

We demonstrate a correlated response of the virulence and the mode of transmission of the microsporidian parasite Edhazardia aedis to selection on the age at pupation of its host, the mosquito Aedes aegypti. We selected three lines of mosquitoes each for early or late pupation and exposed the larvae after zero, two and four generations of selection to a low and a high concentration of the parasite’s spores. Before selection the parasites induced a similar level of mortality in the six lines; after four generations of selection mortality was higher in the mosquitoes selected for late pupation than in those selected for early pupation. Overall, parasite-induced mortality was positively correlated with the mean age at pupation of the matching uninfected line. When they died, mosquitoes selected for early pupation harboured mostly binucleate spores, which are responsible for vertical transmission. Mosquitoes selected for late pupation were more likely to harbour uninucleate spores, which are responsible for horizontal transmission. The parasite enhanced this tendency for horizontal transmission by prolonging the larval period in the lines selected for late pupation, but not in the ones selected for early pupation. These results suggest that the genetic basis of the mosquito’s age at pupation helps to determine the parasite’s mode of transmission: parasites in rapidly developing mosquitoes are benign and transmit vertically, while parasites in slowly developing mosquitoes are virulent and transmit horizontally. Thus, as the host’s life history evolves, the parasite’s performance changes, because the host’s evolution changes the environment in which the parasite develops.     

光照和温度对扇蕨孢子萌发的影响

用组织培养法和光学显微镜技术初步研究光照和温度影响扇蕨孢子萌发的结果表明,扇蕨孢子发芽是需光型,具有明显的光休眠现象。光照是孢子萌发的主要影响因子,25℃下,孢子萌发率达（85．1±5．1）％。相同温度下孢子即使在黑暗中培养50d也不能萌发,转入光照下后萌发率可达（82．4±6．6）％。孢子在光下的最适发芽温度为21．6-26．5℃,7d开始萌发,6-7周完全萌发,温度升高或下降均降低孢子萌发率。