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.     

Secondary leaf fall of Hevea brasiliensis: factors affecting the production, germination and viability of spores of Colletotrichum gloeosporioides

The optimum temperature for growth and sporulation of Colletotrichum gloeosporioides from Hevea brasiliensis was between 26 and 32 ^oC, whereas spore germination exceeded 90% between 21.5 and 30.5 ^oC. Germination decreased in culture after 3 days, and on exposure of spores to sunlight or oven heat (46 ^oC) for 10 min. Spore viability and germination were sensitive to atmospheric humidity; at 99% r.h. germination was half that at 100% r.h. and was negligible below 97% r.h. Germination decreased by up to 30% after 3 h storage at 80% r.h. Continuous light favoured spore production in vitro, but spores produced in the dark had a higher percentage germination. No differences were detected between the numbers of spores germinating on leaves of different ages, although there were slightly more on susceptible cultivars and in the presence of extracts of uninfected susceptible leaves. Extracts from, infected leaves depressed spore germination, as did concentrations above 5 times 10⁵ spores/ml. The highest % germination was observed when naturally infected leaves were dry-stored for up to 20 days and then incubated for 2 days in a moist chamber.     

Production of Aseptic Spores of Vesicular-Arbuscular Endophytes and Their Viability After Chemical and Physical Stress

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30°C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30°C for Glomus caledonius or at 35°C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 μg of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 μg/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40°C for 80 min, 55°C for 10 min, and 60°C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 × 10⁸ ergs cm⁻² from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.     

Spore pool glutamic acid as a metabolite in germination

Spore glutamic acid pools were examined in dormant and germinating spores using colorimetric and (14)C analytical procedures. Germination of spores of Bacillus megaterium (parent strain), initiated by d-glucose, was accompanied by a rapid drop in the level of spore pool glutamate, from 12.0 mug/mg of dry spores to 7.7 mug/mg of dry spores after 30 sec of germination. Similar decreases in extractable spore pool glutamate were observed with l-alanine-initiated germination of B. licheniformis spores. On the other hand, glutamate pools of mutant spores of B. megaterium, with a requirement of gamma-aminobutyric acid for spore germination, remained unchanged for 9 min of germination, at which time more than 50% of the spore population had germinated. Evidence for conversion of spore pool glutamate to gamma-aminobutyric acid during germination of spores of B. megaterium (parent strain) was obtained.     

Heat Activation and Heat-induced Dormancy of Bacillus stearothermophilus Spores

Heat-induced dormancy was observed when spores of two strains of Bacillus stearothermophilus were heated in distilled water at 80, 90, and 100 C. At temperatures above 100 C, true activation occurred; however, maximal activation was not achieved until temperatures of 110 to 115 C were employed. A heat treatment of 115 C for 3 min was required to induce maximal activation in one suspension of strain 1518 spores, whereas a heat treatment of 110 C for 7 to 10 min was adequate for the other suspension of strain 1518 spores. Spores from both strain M suspensions required heat treatments of 110 C for 9 to 15 min for maximal activation. The degree to which the spores could be activated was strain dependent and variable among spore suspensions of the same strain.     

Temporal control of autoactivator synthesis and secretion during spontaneous spore germination in Dictyostelium discoideum

SG mutant and aged wild type spores of the cellular slime mold Dictyostelium discoideum germinate in the absence of an externally applied activation treatment. This type of germination is referred to as autoactivation. During the swelling stage of autoactivation, spores release a factor, the autoactivator, capable of stimulating germination in subsequent spore populations. The autoactivator was not present in the dormant spore, but it or a precursor was produced internally during the first hour of autoactivation. This production was sensitive to moderately high temperatures (+31° C) and was completely destroyed by heat activation (45° C for 30 min). Internal production of the autoactivator was not sensitive to protein synthesis inhibitors. However, the release of the activator from the spore appeared to be regulated by protein synthesis. Internal autoactivator was also produced in the aged wild type strain during the postautoactivation lag phase. The activator could not be directly isolated from within the germinating spore. Its activity on the rest of the spore population was dependent upon its release from the germinating spore. A model is presented integrating the effects of heat, cycloheximide, autoinhibitor and autoactivator on spores of D. discoideum.     

Effects of temperature on the spores of thermophilic actinomycetes

The effects of temperature on the germination properties of spores of thermophilic actinomycetes were examined. Temperatures above and below the growth temperature of 55° C were found to produce marked changes in the germination properties of spores. High temperatures caused reductions in the germinative activities of spores. However, heated spore populations regained original germinative activities after maintaining them for suitable periods of time at 25°C. Recovery from the effects of heat on spore germination was also observed at 4°C, but at a much slower rate compared with 25°C. Spores of two strains of thermophilic actinomycetes, grown and prepared at 55°C, failed to germinate. Storage of dormant (nonactivated) spore populations at different temperatures demonstrated a low temperature requirement for the activation of these spores; while little or no activation occurred at 55°C, rapid activation took place at 25°C. Heating the spores at 80°C for 30 min slightly delayed the activation (rates) of spores at 25°C. The requirement of low temperature for spore activation was strain dependent and was influenced by the composition of the germination medium.     

Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis

Phase intensity changes of individual germinating spores of Bacillus subtilis were determined by phase-contrast light microscopy and image analysis. Two germination phases were investigated. The length of the time period before a change in phase brightness was evident and the duration of the phase intensity change until a constant greylevel was maintained. The incubation temperature (37 and 20 °C) and heat activation (10 min at 65 °C) had a distinct effect on both phases. At 37 °C, spores of B. subtilis 604 started to show a decrease in brightness in l -alanine buffer after 3–39 min and needed 10–39 min to complete the phase change. At 20 °C, lag times of 10–100 min were observed and the spores needed 30–100 min to reach a constant greylevel. Heat activation and subsequently exposure to l -alanine buffer at 20 °C reduced the lag phase to 6–90 min and the phase change was finished after 30–60 min. Our results indicate enzymatic involvement before and during the phase intensity change of germinating spores.     

Novel strains of Moorella thermoacetica form unusually heat-resistant spores

Two strains of Moorella thermoacetica, JW/B-2 and JW/DB-4, isolated as contaminants from autoclaved media for chemolithoautotrophic growth containing 0.1% (wt/vol) yeast extract, formed unusually heat-resistant spores. Spores of the two strains required heat activation at 100 degrees C of more than 2 min and up to 90 min for maximal percentage of germination. Kinetic analysis indicated the presence of two distinct subpopulations of heat-resistant spores. The decimal reduction time (D10-time=time of exposure to reduce viable spore counts by 90%) at 121 degrees C was determined for each strain using spores obtained under different conditions. For strains JW/DB-2 and JW/ DB-4, respectively, spores obtained at approximately 25 degrees C from cells grown chemolithoautotrophically had D10-times of 43 min and 23 min; spores obtained at 60 degrees C from cells grown chemoorganoheterotrophically had D10-times of 44 min and 38 min; spores obtained at 60 degrees C from cells grown chemolithoautotrophically had D10-times of 83 min and 111 min. The thickness of the cortex varied between 0.10 and 0.29 microm and the radius of the cytoplasm from 0.14 to 0.46 microm. These spores are amongst the most heat-resistant noted to date. Electron microscopy revealed structures within the exosporia of spores prior to full maturity that were assumed to be layers of the outer spore coat.     

粗茎鳞毛蕨孢子萌发研究

以经过3年低温储藏的粗茎鳞毛蕨孢子为实验材料,从孢子离心、孢子消毒、培养基种类、光质等4方面对孢子萌发进行研究,结果表明:在离心转数≤14 000 r.min-1、离心时间≤30 min条件下,离心处理对孢子萌发基本无影响;对孢子进行1%NaClO水溶液浸泡处理20～30 min为最佳消毒条件;改良Knop’s培养基为最佳孢子萌发培养基;黑暗条件下孢子不能萌发,但是黑暗处理能够明显提高孢子萌发整齐性;红光比白光能促进孢子提早萌发1 d左右,但对提高萌发率效果不显著。     

Effects of temperatures above the maximum for germination on the endospore of Bacillus cereus

Knaysi, Georges (Cornell University, Ithaca, N.Y.). Effects of temperatures above the maximum for germination on the endospore of Bacillus cereus. J. Bacteriol. 87:1129-1136. 1964.-This is chiefly a study of heat activation and of the instability observed in spores of Bacillus cereus exposed to high temperatures. Temperatures up to 80 C for periods up to 6 hr were used. The spores were in colonies grown on collodion films, and were observed with the phase microscope, in dark contrast, for any evidence of change in their optical properties, the structure of their spodograms, and any other evidence of germination or growth. Exposure of the endospores to 80 C for 4 hr or more, whether in distilled water or in glucose broth, entirely eliminated stage II (i.e. the germcell stage, when the germinating spore begins to grow), and also tended to render the spores unstable and to produce uniformly dark spodograms. This instability involves some destructive processes and is not equivalent to normal entry into stage I (the initial stage). True heat-activation seemed to take place at a stage prior to the one which yielded uniformly dark spodograms. It was shown to consist of two factors: accumulation of germination-promoting components of the medium and activation of some spore components involved in germination. Activation of the medium components by preheating did not stimulate the spores of the strain employed. Unstable or activated spores can not be instantaneously stabilized, deactivated, or their changes arrested by exposure to acid buffers. It is concluded that the optimal temperature for activation, without danger of injury to the spore, in a given strain, is just above the maximum for stage I in that strain.     

Response of Micromonospora echinospora (NCIMB 12744) spores to heat treatment with evidence of a heat activation phenomenon

The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 degrees C treatment of spores in phosphate buffer for 10 min led to an approximately five-fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy-yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 degrees C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 degrees C and above with D-values of 21.43, 2.67, 0.45 and 0.09 min being recorded at 70, 80, 90 and 100 degrees C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells.     

Properties and attempted culture of Pasteuria penetrans, a bacterial parasite of root-knot nematode (Meloidogyne javanica)

When they were subjected to a range of physical and chemical treatments, spores of Pasteuria penetrans showed properties similar to those of other endospore-forming bacteria. The spores did not take up some stains, were resistant to desiccation and sonication and showed extrusion of spore contents ('spore popping') on prolonged exposure to 0.1% KMnO₄ in 0.3 n HNO₃. Calcium and dipicolinic acid (DPA) were present at concentrations of 0.28% and 0.96% of the spore dry weight respectively, giving a Ca: DPA molar ratio of 1.2. The infectivity of P. penetrans spores was reduced to a low level after heating at 100°C for 5 min, but spore attachment was not markedly affected by heating at 100°C for 15 min. Evidence for the presence of catalase in P. penetrans spores was equivocal because the low levels of catalase activity observed in spore suspensions may have been due to contamination from catalase-positive nematode tissue. When P. penetrans spores were exposed to a range of substances known to act as germinants for spores of Bacillus spp., germination or loss of refractility was not observed by phase microscopy. In vitro culture of P. penetrans was attempted by inoculating either spores or vegetative mycelial bodies onto a diverse range of simple and complex media and incubating them in aerobic, reduced oxygen, anaerobic and increased CO₂ environments. Signs of spore germination or growth of vegetative stages were never observed.     

Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species.

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.     

The Effect of Water Activity and the aw Controlling Solute on Spore Germination of Bacillus stearothermophilus

The germination of spores of Bacillus stearothermophilus was studied in nutrient broth in relation to the water activity ( a _w) of the medium, the nature of the a _w controlling solutes glycerol, sucrose, KCl, and NaCl, and temperature. Quantitation of germination was based on the change of the phase-bright spore to phase-dark. Activation of spores was by exposure to 100°C/10 min in a medium of the same composition as that used for germination.
Of the four solutes used, sucrose proved most inhibitory to germination, especially in the upper part of the temperature range 38-75°C, glycerol was the most favourable whereas KCl and NaCl, whose effect was almost identical, occupied an intermediate place. The glycerol effect became more pronounced as the a _w of the medium decreased towards 0.960, becoming inhibitory thereafter.
The solute effect on spore germination followed a pattern that related to the class of solute, i.e. electrolyte or non-electrolyte, and its cell penetration characteristics.
Solute penetration during heat activation and germination was considered as the major germination factor and was associated with the osmoregulation mechanism within the spore proposed recently as the basis of spore dormancy and resistance.     

Activation and killing of Dictyostelium discoideum spores with urea.

The optimal conditions for activation of Dictyostellium discoideum spores are an 8 M urea treatment for 30 min. The lag between activation and swelling is 45 min. Lower concentrations of urea do not activate entire spore populations. Incubating spores in 8 M urea for 60 min or treatment with 10 M urea for 30 min results in a lengthening of the post-activation lag and a decrease in the final percentage of germination. Urea-activated spores can be deactivated by azide, cyanide, osmotic pressure, and low-temperature incubation. Activated spores do not germinate if incubated in 1 M urea for 24 h but will complete germination upon resuspension in urea-free buffer. Shocking spores at 45 degrees C in 8 M urea or incubating spores in 4-8 M urea for 10 h at 23.5 degrees C causes inactivation. When suspended in urea-free buffer, a larger percentage of these dead spores release spheroplasts through a longitudinal split in the spore case. Sequential enzyme treatment of spheroplasts with cellulase and pronase causes them to release lysable protoplasts. The data of these experiments suggest that shedding of the outer and middle wall layers during physiological spore swelling may be a physical process rather than an enzymatic one.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

Activate to Eradicate: Inhibition of Clostridium difficile Spore Outgrowth by the Synergistic Effects of Osmotic Activation and Nisin

Background

Germination is the irreversible loss of spore-specific properties prior to outgrowth. Because germinating spores become more susceptible to killing by stressors, induction of germination has been proposed as a spore control strategy. However, this strategy is limited by superdormant spores that remain unaffected by germinants. Harsh chemicals and heat activation are effective for stimulating germination of superdormant spores but are impractical for use in a hospital setting, where Clostridium difficile spores present a challenge. Here, we tested whether osmotic activation solutes will provide a mild alternative for stimulation of superdormant C. difficile spores in the presence of germinants as previously demonstrated in several species of Bacillus. In addition, we tested the hypothesis that the limitations of superdormancy can be circumvented with a combined approach using nisin, a FDA-approved safe bacteriocin, to inhibit outgrowth of germinated spores and osmotic activation solutes to enhance outgrowth inhibition by stimulating superdormant spores.

Principal Findings

Exposure to germination solution triggered ∼1 log₁₀ colony forming units (CFU) of spores to germinate, and heat activation increased the spores that germinated to >2.5 log₁₀CFU. Germinating spores, in contrast to dormant spores, became susceptible to inhibition by nisin. The presence of osmotic activation solutes did not stimulate germination of superdormant C. difficile spores exposed to germination solution. But, in the absence of germination solution, osmotic activation solutes enhanced nisin inhibition of superdormant spores to >3.5 log₁₀CFU. The synergistic effects of osmotic activation solutes and nisin were associated with loss of membrane integrity.

Conclusions

These findings suggest that the synergistic effects of osmotic activation and nisin bypass the limitations of germination as a spore control strategy, and might be a novel method to safely and effectively reduce the burden of C.difficile spores on skin and environmental surfaces.     

Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

Germination of Bacillus subtilis spores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores'' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the “germinosome,” and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with ≥75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased ∼50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ∼2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds.     

Kinetics of germination of wet-heat-treated individual spores of Bacillus species, monitored by Raman spectroscopy and differential interference contrast microscopy

Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the kinetics of nutrient and nonnutrient germination of multiple individual untreated and wet-heat-treated spores of Bacillus cereus and Bacillus megaterium, as well as of several isogenic Bacillus subtilis strains. Major conclusions from this work were as follows. (i) More than 90% of these spores were nonculturable but retained their 1:1 chelate of Ca2+ and dipicolinic acid (CaDPA) when incubated in water at 80 to 95°C for 5 to 30 min. (ii) Wet-heat treatment significantly increased the time, T(lag), at which spores began release of the great majority of their CaDPA during the germination of B. subtilis spores with different nutrient germinants and also increased the variability of T(lag) values. (iii) The time period, ΔT(release), between T(lag) and the time, T(release), at which a spore germinating with nutrients completed the release of the great majority of its CaDPA, was also increased in wet-heat-treated spores. (iv) Wet-heat-treated spores germinating with nutrients had higher values of I(release), the intensity of a spore's DIC image at T(release), than did untreated spores and had much longer time periods, ΔT(lys), for the reduction in I(release) intensities to the basal value due to hydrolysis of the spore's peptidoglycan cortex, probably due at least in part to damage to the cortex-lytic enzyme CwlJ. (v) Increases in T(lag) and ΔT(release) were also observed when wet-heat-treated B. subtilis spores were germinated with the nonnutrient dodecylamine, while the change in I(release) was less significant. (vi) The effects of wet-heat treatment on nutrient germination of B. cereus and B. megaterium spores were generally similar to those on B. subtilis spores. These results indicate that (i) some proteins important in spore germination are damaged by wet-heat treatment, (ii) the cortex-lytic enzyme CwlJ is one germination protein damaged by wet heat, and (iii) the CaDPA release process itself seems likely to be the target of wet-heat damage which has the greatest effect on spore germination.