Application of Doehlert design to determine the combined effects of temperature, water activity and pH on conidial germination of Penicillium chrysogenum

AIMS: The influence of temperature, water activity and pH on the time necessary for germination of 90% of Penicillium chrysogenum conidia inoculated (T90) was determined. METHODS AND RESULTS: A new experimental device was developed for easy monitoring of the germination process. Experiments were carried out according to a Doehlert matrix at 11-31 degrees C, 0.86-0.98 water activity (a(w)) and pH 3.5-6.5. In these conditions, a second order polynomial relationship between T90 and the environmental factors was established for the different humectants used throughout this study (e.g. glycerol and sorbitol) with regression coefficients close to 0.97. CONCLUSIONS: For both humectants, the major effect of temperature and water activity on T90 was highlighted, whereas the effect of pH on T90 in these experimental conditions was not significant. The combined effect of temperature and water activity on T90 was also demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Both the experimental set-up and the Doehlert matrix were well suited to determine the influence of environmental factors on mould germination.     

芹菜斑枯病菌生物学特性研究

以沈阳地区芹菜斑枯病菌的纯培养菌株为试材,通过对菌丝生长、孢子萌发的营养及环境条件的多因子试验,明确了芹菜斑枯病菌菌丝生长以在CDAMS—VI培养基上最好,其次是CSA、CCSA和PDA,菌丝生长及产孢对碳源、氮源种类有明显的选择性。菌丝生长的最适pH值为4～5,最适温度为21℃,致死温度为39℃（处理40min）,光照明显抑制菌丝生长。病菌分生孢子在水和芹菜汁中不能萌发,在水琼脂上萌发最好;孢子萌发的适宜pH值为4．6～6．6,适温为17—25℃,致死温度为42℃（处理20min）,RH100％最适宜孢子萌发。光照和变温处理可促进分生孢子器的形成,病菌在PDA上从孢子萌发到分生孢子器形成并释放出分生孢子需要16～18d。     

Bacillus megaterium spore germination is influenced by inoculum size

AIMS: The effect of spore density on the germination (time-to-germination, percent germination) of Bacillus megaterium spores on tryptic soy agar was determined using direct microscopic observation. METHODS AND RESULTS: Inoculum size varied from approximately 10(3) to 10(8) cfu ml(-1) in a medium where pH=7 and the sodium chloride concentration was 0.5% w/v. Inoculum size was measured by global inoculum size (the concentration of spores on a microscope slide) and local inoculum size (the number of spores observed in a given microscope field of observation). Both global and local inoculum sizes had a significant effect on time-to-germination (TTG), but only the global inoculum size influenced the percentage germination of the observed spores. CONCLUSIONS: These results show that higher concentrations of Bacillus megaterium spores encourage more rapid germination and more spores to germinate, indicating that low spore populations do not behave similarly to high spore populations. SIGNIFICANCE AND IMPACT OF THE STUDY: A likely explanation for the inoculum size-dependency of germination would be chemical signalling or quorum sensing between Bacillus spores.     

The effects of temperature,water activity and pH on the growth of Aeromonas hydrophila and on its subsequent survival in microcosm water

AIMS: The influence of temperature, water activity and pH on the growth of Aeromonas hydrophila, and on its survival after transfer in nutrient-poor water were assessed. METHODS AND RESULTS: Experiments were carried out according to a Box-Behnken matrix at 10-30 degrees C, 0.95-0.99 water activity (aw) and pH 5-9. The effect of each factor on the kinetic parameters of growth (i.e. the maximal specific growth rate, mumax, and the lag time, lambda) and on the decline of the bacteria in microcosm water (time to obtain a reduction of 5 log, T5 log) were studied by applying central composite design. CONCLUSIONS: The major effect of temperature and water activity on the growth of A. hydrophila was highlighted, whereas the effect of pH in these experimental conditions was not significant. Models describing the effect of environmental parameters on the growth of A. hydrophila were proposed. The effect of the growth environment, and particularly the incubation temperature, have an influence on the survival ability of the bacteria in nutrient-poor water. SIGNIFICANCE AND IMPACT OF THE STUDY: The Box-Behnken design was well suited to determine the influence of environmental factors on the growth of A. hydrophila and to investigate the effect of previous growth conditions on its survival in microcosm water.     

No evidence for a Ganoderma spore dispersal mutualism in an obligate spore-feeding beetle Zearagytodes maculifer

The role of spore dispersal mutualism remains equivocal in many fungus-insect assemblages. We tested experimentally whether an obligate spore-feeding beetle Zearagytodes maculifer has a mutualistic relationship with its host bracket fungus Ganoderma cf. applanatum via spore dispersal. We asked three specific questions: (1) whether or not Ganoderma spore germination rate is increased via beetle digestive activity and (2) is dependent on temperature and sporocarp identity. Spore germination rates were examined in 2×3×2 factorial experiments (spores consumed by beetles or not×temperature 20, 25, and 30°C×two independent pairs of sporocarp-beetle populations) replicated five times in an array of 60 experimental cultures. Analysis showed that consumption by beetles significantly reduced germination rate of Ganoderma spores. The effect of temperature was modulated by the effect of individual sporocarp, and was overridden by beetle feeding. Microscopic analysis revealed that spores from beetle faecal pellets exhibited extensive damage to their thin outer walls (pellicles) and thick inner walls, as well as significant loss of cytoplasm, while control spores were intact. The overall evidence argued against our spore dispersal mutualism hypothesis, suggesting that Z. maculifer can potentially exert a negative, if vanishingly small, fitness effect on its host fungus G. cf. applanatum.     

Control of Aspergillus growth and aflatoxin production using antioxidants at different conditions of water activity and pH

AIMS: The effect of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trihydroxybutyrophenone (THB) and propyl paraben (PP) (at concentrations of 1, 10 and 20 mmol l(-1)) on germination, growth and aflatoxin B1 production by Aspergillus section Flavi was evaluated. METHODS AND RESULTS: Studies on the percentage of spore germination, elongation rate, growth rate and aflatoxin B1 production were carried out in vitro in relation to water activity (aw) at 0.982, 0.937, 0.809 and 0.747 values. At 0.809 and 0.747aw values none of the isolates was able to germinate. Overall, PP and BHA were the antioxidants most effective at inhibiting germination of both species. In the presence of the lowest concentration of BHA and PP (1 mmol l(-1)) the conidial germination percentage ranged from 2 to 19% after 15 h of incubation at the highest water activity tested. BHA and PP at 10-20 mmol l(-1) completely inhibited conidial germination. The antioxidants more efficient in controlling Aspergillus elongation rate were PP, BHT and BHA. All strains were much more sensitive to all antioxidants tested on the percentage of spore germination and growth rate at 0.937aw. The antioxidants PP and BHA completely inhibited aflatoxin B1 production by all strains when added at 1 mmol l(-1). Decreased aflatoxin B1 levels in comparison with the control, were observed with BHT at 1, 10 and 20 mmol(-1) with the strain T20 at 0.982aw. In contrast, stimulation was observed with the antioxidant THB at 10 and 20 mmol l(-1) at 0.937aw with the strains T20 and T23. The effect of BHA and PP at 1 mmol l(-1) on lag phase and growth rate was maintained in the pH range between 6 and 8. At all pH values the inhibitory effect of BHA was higher than PP. No aflatoxin B1 was detected at all pH values. CONCLUSIONS: The data show that BHA and PP could be considered as effective fungitoxicants for A. flavus and A. parasiticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The information obtained show promise for controlling growth and aflatoxin B1 in stored maize. Futher studies should be carried out to examine the potential for antioxidants, such as BHA and PP to effectively control both growth and aflatoxin production.     

乌蕨孢子萌发研究

用随机区组试验和方差分析方法, 研究了培养温度、贮藏温度、GA 处理和光照强度对乌蕨( Sphenomeris chinensis) 孢子萌发的影响。与室温(20～25℃ ) 相比, 培养温度在28℃时, 孢子最大萌发率相近而萌发速率明显较高。贮藏温度(A) 极显著( P < 0 . 01 ) 影响孢子萌发率, - 20℃ 贮藏降低萌发率; GA (B) 对孢子萌发率无显著影响。光照强度(C) 极显著( P < 0.01) 影响孢子萌发率, 充足光照和弱光照无显著差异, 黑暗处理降低萌发率。A×B, A×C, B×C 及A×B×C 交互效应不显著。     

Nutritional and physiological factors affecting germination of heterothallic Saccharomyces cerevisiae ascospores.

Saccharomyces cerevisiae strain AP-3 was examined with respect to those nutritional requirements and physiological conditions which influence its germination rate. It was found that glucose as a carbon source supported the most rapid rate of germination for this heterothallic strain. In contrast, strain AP-3 spore germination was supported the least by the carbon sources potassium acetate and lactose. Of the nitrogen sources tested in culture medium containing glucose, the complex nitrogen sources peptone and casein hydrolysate appeared to be capable of stimulating germination better than a control culture containing ammonium sulphate. None of the amino acids screened were found to stimulate strain AP-3 germination compared with ammonium sulphate. The optimal culture medium pH for ascospore germination was 4.5 although spore germination could still be initiated by glucose between pH 3.0 and pH 7.5. Germination initiation by glucose was observed over a temperature range from 25 degrees C to 50 degrees C, but the optimal temperature appeared to be 40 degrees C.     

Effects of temperature, pH, water activity and CO2 concentration on growth of Rhizopus oligosporus NRRL 2710

AIMS: To investigate the effects of temperature, pH, water activity (aw) and CO2 concentration on the growth of Rhizopus oligosporus NRRL 2710. METHODS AND RESULTS: Hyphal extension rates from mycelial and spore inocula were measured on media with different aw (approximately 1.0, 0.98 and 0.96) and pH (3.5, 5.5 and 7.5) incubated at 30, 37 or 42 degrees C in atmospheres containing 0.03, 12.5 or 25% (v/v) CO2. The effects of environmental conditions on hyphal extension rate were modelled using surface response methodology. The rate of hyphal extension was very sensitive to pH, exhibiting a pronounced optimum at pH 5.5-5.8. The hyphal extension rate was less sensitive to temperature, aw or CO2, exhibiting maximum rates at 42 degrees C, a(w) approximately 1.0 and 0.03% (v/v) CO2. CONCLUSIONS: The fastest hyphal extension rate (1.7 mm h(-1)) was predicted to occur at 42 degrees C, pH 5.85, a(w) approximately 1.0 and 0.03% CO2. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work is the first to model the simultaneous effects of temperature, pH, aw and CO2 concentration on mould growth. The information relates to tempe fermentation and to possible control of the microflora in Tanzanian cassava heap fermentations.     

Spore fitness components do not differ between diploid and allotetraploid species of Dryopteris (Dryopteridaceae)

BACKGROUND AND AIMS: Although allopolyploidy is a prevalent speciation mechanism in plants, its adaptive consequences are poorly understood. In addition, the effects of allopolyploidy per se (i.e. hybridization and chromosome doubling) can be confounded with those of subsequent evolutionary divergence between allopolyploids and related diploids. This report assesses whether fern species with the same ploidy level or the same altitudinal distribution have similar germination responses to temperature. The effects of polyploidy on spore abortion and spore size are also investigated, since both traits may have adaptive consequences. METHODS: Three allotetraploid (Dryopteris corleyi, D. filix-mas and D. guanchica) and three related diploid taxa (D. aemula, D. affinis ssp. affinis and D. oreades) were studied. Spores were collected from 24 populations in northern Spain. Four spore traits were determined: abortion percentage, size, germination time and germination percentage. Six incubation temperatures were tested: 8, 15, 20, 25 and 32 degrees C, and alternating 8/15 degrees C. KEY RESULTS: Allotetraploids had bigger spores than diploid progenitors, whereas spore abortion percentages were generally similar. Germination times decreased with increasing temperatures in a wide range of temperatures (8-25 degrees C), although final germination percentages were similar among species irrespective of their ploidy level. Only at low temperature (8 degrees C) did two allotetraploid species reach higher germination percentages than diploid parents. Allotetraploids showed faster germination rates, which would probably give them a competitive advantage over diploid parents. Germination behaviour was not correlated with altitudinal distribution of species. CONCLUSIONS: The results of this study suggest that (i) relative fitness of allopolyploids at sporogenesis does not differ from that of diploid parents and (ii) neither does allopolyploidization involve a change in the success of spore germination.     

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.     

Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions

AIMS: Limited information is available on the germination triggers for spores of non-proteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. METHODS AND RESULTS: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate (50 mmol l(-1) of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5.5-8.0, germination occurred at 1-40 degrees C, but not at 50 degrees C, and was optimal at 20-25 degrees C. CONCLUSIONS: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum. SIGNIFICANCE AND IMPACT OF THE STUDY: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.     

马铃薯干腐病菌硫色镰孢的生物学特性

从碳源、氮源、酸碱度及生长温度等方面对引起马铃薯干腐病的硫色镰孢Fusarium sulphureum的生物学特性进行了研究。结果表明,该病原菌在不同发育阶段对营养和环境条件的要求存在差异。在固体培养基上,菌落生长最佳碳源为葡萄糖、麦芽糖,最佳氮源为蛋白胨,pH8?为最佳;在液体培养基中,菌丝体生长以麦芽糖为最佳碳源,以硝酸钠为最佳氮源,pH6?为最佳;在分生孢子萌发阶段,在以羧甲基纤维素钠、蛋白胨和谷氨酸为碳、氮源的营养液中,分生孢子萌发率最高,最适pH 6–8。该病菌最适生长温度为25℃,分生孢子致死     

A rapid staining technique for the detection of the initiation of germination of bacterial spores

AIMS: We propose to apply the Wirtz-Conklin staining technique to evaluate spore germination. METHODS AND RESULTS: Spores at different stages of germination were stained with modified spore stain (Wirtz-Conklin) and evaluated for staining properties. Bacillus spores suspended in deionized water, which does not support germination, stained greenish-blue. Spores suspended in germination enhancers that did not form bacilli stained pink, indicating the initiation of germination. Spores suspended in culture media, which promotes bacterial outgrowth, formed bacilli and were also stained pink. CONCLUSIONS: Modified spore stain (Wirtz-Conklin) was found to be useful to detect the initiation of spore germination as early as 30 min following incubation in a germination environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple staining procedure is useful in detecting the initiation of germination of bacterial spores.     

Factors Affecting Spore Germination of Volvariella volvacea

Environmental factors affecting basidiospore germination were studied with Volvariella volvacea, the edible straw mushroom which is common in South East Asia. A relatively mild heal shock is necessary for spore germination. The spores give best germination at 40°C although early hyphal growth is better at 35°C the germination of spores is affected by temperature. pH. a presoaking treatment and spore density. Higher pH supports more germination but seems unfavourable for early mycelial growth. Presoaking treatment in phosphate buffer solution or distilled water also stimulates germination markedly.     

温度、相对湿度和pH对蜜蜂球囊菌孢子萌发的影响

研究了温度、相对湿度和pH对蜜蜂球囊菌(Ascosphaeraapis)孢子萌发3个阶段(活化、膨大、产生萌发管)的影响．结果表明,孢子活化和膨大在15～40和(25～40)±0．5℃范围内受温度的影响不明显(P＞0．05);萌发管仅发生在25～37±0．5℃,最适温度位于(31～35)±0．5℃．相对湿度越大,越有利于孢子萌发,而相对湿度低于80％对孢子萌发极为不利．孢子萌发的3个阶段在pH为5～7．8时几乎不受pH变化的影响,而在pH值较低影响很大．可见,A．apis是一种高度专一的蜜蜂幼虫病原体．     

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.     

Relationship of nuclear segregation and macroconidial germination in Microsporum gypseum

Microsporum gypseum macroconidia germinated at 37 C possessed from one to eight nuclei per germinated spore compartment. The distribution of nuclei per spore compartment was the result of a random packaging of nuclei from the available nuclear population. Partial inhibition of germination by incubation at 25 C or at 37 C in the presence of 10(-4)m phenyl methyl sulfonyl-fluoride resulted in an enrichment of germinated spores containing high numbers of nuclei per compartment. The selection for higher nuclear numbers was statistically significant. Compartments possessing high numbers of nuclei appeared to be precommitted to spore germination since they were not sensitive to germination inhibition. The effect of incubation temperature variation on spore germination is discussed with respect to the organism's natural environment.     

Levels of H+ and other monovalent cations in dormant and germinating spores of Bacillus megaterium.

Previous investigators using the extent of uptake of the weak base methylamine to measure internal pH have shown that the pH in the core region of dormant spores of Bacillus megaterium is 6.3 to 6.5. Elevation of the internal pH of spores by 1.6 U had no significant effect on their degree of dormancy or their heat or ultraviolet light resistance. Surprisingly, the rate of methylamine uptake into dormant spores was slow (time for half-maximal uptake, 2.5 h at 24 degrees C). Most of the methylamine taken up by dormant spores was rapidly (time for half-maximal uptake, less than 3 min) released during spore germination as the internal pH of spores rose to approximately 7.5. This rise in internal spore pH took place before dipicolinic acid release, was not abolished by inhibition of energy metabolism, and during germination at pH 8.0 was accompanied by a decrease in the pH of the germination medium. Also accompanying the rise in internal spore pH during germination was the release of greater than 80% of the spores K+ and Na+. The K+ was subsequently reabsorbed in an energy-dependent process. These data indicate (i) that between pH 6.2 and 7.8 internal spore pH has little effect on dormant spore properties, (ii) that there is a strong permeability barrier in dormant spores to movement of charged molecules and small uncharged molecules, and (iii) that extremely early in spore germination this permeability barrier is breached, allowing rapid release of internal monovalent cations (H+, Na+, and K+).     

Nutritional conditions for the germination of streptomyces galbus 5ME-13 spores

The nutritional conditions for the germination of spores of Streptomyces galbus 5ME-13 were determined under laboratory conditions. The germination of the spores was intiated by the emergence of 1–2 germ tubes after the second hour of incubation and attained its maximum at the sixth hour. This was accompanied by a steady rise in the optical density of the germinating spore suspension. A malt-extract yeast-extract medium was found to be the best medium for the germination of the spores. Glycerol as the sole source of carbon was the best supporter for spore germination while, as N source, L-alanine was preferred. The optimum pH and temperature for spore germination were 7.2 and 30°C, respectively.