Evidence for 21-aminosteroid association with the hydrophobic domains of brain microvessel endothelial cells.

Studies were conducted to demonstrate 21-aminosteroid distribution into the hydrophobic or lipid domains of biological membranes, a presumed site at which these compounds inhibit lipid peroxidation. Bovine brain microvessel endothelial cells (BMECs) were labeled with diphenylhexatriene fluorophores and interactions with cell membranes characterized with fluorescence anisotropy and lifetimes. Two 21-aminosteroids (U-74500A and U74006F) were shown to preferentially alter the fluorescence anisotropy and lifetime parameters of the diphenylhexatriene probe distributing into membranes throughout the BMECs. Little or no effect of the compounds was observed on the fluorescence parameters of the probe localized on the surface of BMEC plasma membranes. By contrast, cholesterol used as a positive control substantially altered the fluorescence parameters of BMECs labeled with either diphenylhexatriene probe. Results suggest 21-aminosteroid-induced changes in the molecular packing order and drug: fluorescent probe interactions in membrane hydrophobic (or lipid) domains throughout the BMEC. Concentrations of 21-aminosteroids altering the fluorescence parameters of diphenylhexatriene labeled BMECs correspond to those concentrations of 21-aminosteroids effective in vitro in inhibition of lipid peroxidation.     

Metabolism and the triggering of germination of Bacillus megaterium. Concentrations of amino acids, organic acids, adenine nucleotides and nicotinamide nucleotides during germination.

A considerable amount of evidence suggests that metabolism of germinants or metabolism stimulated by them is involved in triggering bacterial-spore germination. On the assumption that such a metabolic trigger might lead to relatively small biochemical changes in the first few minutes of germination, sensitive analytical techniques were used to detect any changes in spore components during the L-alanine-triggered germination of Bacillus megaterium KM spores. These experiments showed that no changes in spore free amino acids or ATP occurred until 2-3 min after L-alanine addition. Spores contained almost no oxo acids (pyruvate, alpha-oxoglutarate, oxaloacetate), malate or reduced NAD. These compounds were again not detectable until 2-3 min after addition of germinants. It is suggested, therefore, that metabolism associated with these intermediates is not involved in the triggering of germination of this organism.     

Use of electron spin resonance to study Bacillus megaterium spore membranes.

Membranes from dormant and heat-activated spores were labelled with the fatty acid spin probe 5-doxyl stearate and analyzed using electron spin resonance spectroscopy. Membranes from dormant spores were slightly less fluid above 23° than membranes from heat-activated spores. Also L-proline caused a much larger increase in the upper transition temperature than did D-proline when added to membranes from heat-activated spores. Thus a compound known to trigger germination in this strain may interact stereospecifically to alter the biophysical properties of the spore membranes.     

Zinc release and the sequence of biochemical events during triggering of Bacillus megaterium KM spore germination.

Zinc release is the first quantitatively significant event detected during the triggering of Bacillus megaterium KM spore germination. Of the total spore Zn2+ pool 25% is released from non-heat-activated spores within 4 min of triggering germination. During this period only 10% of the spore population becomes irreversibly committed to germinate. The investigation of a putative role for Zn2+ in the germination trigger mechanism has established a relationship between the rate and extent of Zn2+ release and the stimulation of spore germination by heat activation. Furthermore, a correlation can be demonstrated between the extent of zinc release from spore populations and the time required to obtain 50% commitment of these populations to germinate over a wide temperature range. These findings have been used to expand a recently published model for the triggering of bacterial spore germination.     

Biochemistry of L-proline-triggered germination of Bacillus megaterium spores.

The mechanism by which L-proline triggers germination in Bacillus megaterium QM B1551 spores was investigated. First, brief exposure of spores to L-proline, followed by dilution, was sufficient to trigger germination. Once germination was triggered, the spores continued initiation of germination and did not require high concentrations of L-proline. Triggering of germination was pH and temperature dependent. Second, enzymes for L-proline catabolism were absent in spores, and several non-metabolizable analogs of L-proline were effective trigger compounds. Third, triggering of germination occurred in the presence of inhibitors of proton motive force production, oxygen uptake, and metabolism. Fourth, uptake of L-proline occurred after the triggering of germination. These results argue that neither uptake nor metabolism of L-proline was necessary to trigger germination. Instead, L-proline probably causes a biophysical alteration in the spores that triggers the biochemical changes in germination.     

Lethal germination of spores ofBacillus megaterium 9885

Washed spore suspensions germinated promptly without prior heat shock in a basal germination solution containingl-leucine.Germination was inhibited by dipicolinic acid. The inhibition was reversed by eitherl-leucine or phosphate.Phosphate accelerated the rate and increased the extent of germination, which was accompanied by an uncommonly large fall in the optical density of the suspension, but phosphate also caused a massive lysis after germination. This was accompanied by a sudden shedding of the spore coats. The suspensions consisted of shrivelled, cellular walls and membranes attached to the empty spore coats.Lysis of the germinated cells was prevented by fairly high concentrations of Ca or Mg.During germination, exogenous Ca we used Ca⁴⁵ was absorbed by the cells. Both cells and sonically disrupted cellular particles firmly retained the calcium, and evidence suggested that much of the Ca was bound in the cytoplasmic membranes.The cations contained in plain agar enabled spores which germinated on tryptone soya agar plates to develop into colonies; in the corresponding broth medium these spores lysed upon germination.Hypertonic sucrose delayed but did not prevent lysis.     

L-Proline site for triggering Bacillus megaterium spore germination.

Germination of $\text{Bacillus megaterium}$ QM B1551 spores can be triggered by L-proline chloromethyl ketone at ～ 10 fold lower concentrations than L-proline. [³H] L-proline chloromethyl ketone bound to several protein fractions, one of which was decreased in a mutant (JV137) that cannot be triggered by L-proline. Treatment of spores with [³H] acetic anhydride specifically inhibited L-proline triggered germination, and also covalently modified the same protein fraction which appears to be bound to the spore membrane. These results indicate that it is possible to identify a protein fraction in spores that may play a key role in triggering spore germination.     

Germination of Bacillus megaterium spores after various extraction procedures

The initiation of germination of Bacillus megaterium QM B1551 spores, grown in supplemented nutrient broth, has been studied. The initiation properties depend on buffer concentrations and the particular batch of spores. Initiation in l-alanine, KBr, calcium dipicolinate, or in buffer alone increases as a function of the spore age; whereas initiation in glucose, l-leucine, or l-proline remains relatively constant. Extraction of spores with alkali, sodium dodecyl sulfate-dithiothreitol, or lithium diiodosalicylate removes variable amounts of dipicolinic acid, hexosamine, and protein. These extracted spores are still capable of initiation and, in some cases, initiation is stimulated. However, extraction of spores with 8 M urea-10% mercaptoethanol inhibits subsequent initiation.     

Cooperativity between different nutrient receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by alterations in the GerB receptor

The GerA nutrient receptor alone triggers germination of Bacillus subtilis spores with L-alanine or L-valine, and these germinations were stimulated by glucose and K+ plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K+ (GFK) (termed cogerminants) plus GerK for triggering of germination with a number of L-amino acids. GerB and GerA also triggered spore germination cooperatively with l-asparagine, fructose, and K+ and either L-alanine or L-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to D-alanine do not respond to D-alanine but respond to the same L-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these L-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to l-alanine germination via GerA, spore germination via L-alanine and GerB or GerB* was not inhibited by D-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K+ somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to L-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to L-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to L-amino acids, and GerA interacts only with L-valine, L-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to L-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore 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.     

Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore properties

Aims: To determine effects of inner membrane lipid composition on Bacillus subtilis sporulation and spore properties. Methods and Results: The absence of genes encoding lipid biosynthetic enzymes had no effect on B. subtilis sporulation, although the expected lipids were absent from spores’ inner membrane. The rate of spore germination with nutrients was decreased c. 50% with mutants that lacked the major cardiolipin (CL) synthase and another enzyme for synthesis of a major phospholipid. Spores lacking the minor CL synthase or an enzyme essential for glycolipid synthesis exhibited 50–150% increases in rates of dodecylamine germination, while spores lacking enzymes for phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysylphosphatidylglycerol (l‐PG) synthesis exhibited a 30–50% decrease. Spore sensitivity to H₂O₂ and tert‐butylhydroperoxide was increased 30–60% in the absence of the major CL synthase, but these spores’ sensitivity to NaOCl or Oxone^? was unaffected. Spores of lipid synthesis mutants were less resistant to wet heat, with spores lacking enzymes for PE, PS or l‐PG synthesis exhibiting a two to threefold decrease and spores of other strains exhibiting a four to 10‐fold decrease. The decrease in spore wet heat resistance correlated with an increase in core water content. Conclusions: Changing the lipid composition of the B. subtilis inner membrane did not affect sporulation, although modest effects on spore germination and wet heat and oxidizing agent sensitivity were observed, especially when multiple lipids were absent. The increases in rates of dodecylamine germination were likely due to increased ability of this compound to interact with the spore’s inner membrane in the absence of some CL and glycolipids. The effects on spore wet heat sensitivity are likely indirect, because they were correlated with changes in core water content. Significance and Impact of the Study: The results of this study provide insight into roles of inner membrane lipids in spore properties.     

Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis

The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K(+) (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.     

Chloroplast activities of dark-imbibed and photoinduced spores of the fernOnoclea sensibilis

Summary Chloroplast activities of dark-imbibed (non-germinating) and photoinduced (germinating) spores of the sensitive fern,Onoclea sensibilis were compared to gain insight into the germination process. There were no changes in the number of chloroplasts or in the chlorophyll contents of the spore during dark-imbibition and during the early phase of germination. Levels of increase in the Chloroplast DNA content of dark-imbibed and photoinduced spores were nearly the same and were associated with autoradiographic incorporation of [³H]thymidine into the cytoplasm. However, incorporation of the label into the nucleus occurred only during photoinduction of spores. Analysis of Chloroplast and nuclear DNA contents by dot-blot hybridization with labeled gene-specific probes has confirmed that chloroplast DNA content of the spore increases during dark-imbibition and photoinduction, while increase in nuclear DNA occurs only in photoinduced spores. Chloroplasts isolated from dark-imbibed and photoinduced spores incorporated [³H]TTP into an acid-insoluble fraction identified as DNA. The results show that physiological activities of chloroplasts of dark-imbibed and photoinduced spores ofO. sensibilis are similar and support an exclusive role for nuclear DNA synthesis in spore germination.     

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.     

Electrostatic interactions at charged lipid membranes Kinetics of the electrostatically triggered phase transition

Charged lipid membranes of dimyristoylmethylphosphatidic acid were mixed rapidly in a stopped-flow cell with protons or Ca²⁺ to compensate the charges and thereby trigger the ordered-fluid phase transition. The kinetics of the transition was studied by following the time development of the fluorescence anisotropy of diphenylhexatriene. A relaxation process was observed with a characteristic time in the range 10–200 ms. By comparison with existing theories of non-equilibrium relaxation it was concluded that the relaxation process is governed by a nucleation step.     

Germination of Nosema algerae (Microspora) Spores: Conditional Inhibition by D2O, Ethanol and Hg2+ Suggests Dependence of Water Influx upon Membrane Hydration and Specific Transmembrane Pathways

ABSTRACT. The germination of microsporidian spores under conditions expected to affect water flow across the plasma membrane-wall complex was studied by assessing their responses to in vitro stimulation with Na⁺ or K⁺. Partial or full substitution of common water with D₂O, which more effectively coats ions and electrostatically-charged cell surfaces with relatively stable hydration layers, delayed and inhibited spore germination in a concentration-dependent manner; yet, preincubation in 100% D₂O did not change the normal response to standard stimulation. Water structure-breaking conditions, such as an increase in temperature (within the 15° C to 40° C range) or in ionic strength (2- to 10-fold normal), opposed the inhibition by D₂O and allowed significant stimulation by Li⁺, the monovalent cation with the largest hydration diameter and a usually weak stimulant action on the spores. Ethanol, known to reduce water permeation across cell membranes and phospholipid bilayers, also caused a powerful and dose-dependent (1% to 4% v/v) inhibition of spore germination, but pretreatment with ethanol did not affect the normal response. HgCl₂, an inhibitor of specific water channels, blocked spore germination at just 250 μM in the normal stimulation solution irrespective of the temperature, and permitted only a delayed response in high salt stimulation solutions. However, the inhibition by Hg²⁺ was abolished by the simultaneous presence of 2-mercaptoethanol in the medium. These results suggest (1) that spore germination is keenly dependent upon the hydration states of both the plasma membrane-wall complex and the stimulant ions, and (2) that osmotic water flows into the spores through specific transmembrane pathways with critical sulfhydryl groups, i.e. analogous to the water channels that facilitate water movements across the plasma membranes of highly permeable cells.     

Isolation and properties of membranes from Bacillus megaterium spores.

Membranes from dormant and heat-activated spores of Bacillus megaterium QM B1551 were isolated and purified by gentle lysis procedures followed by differential and sucrose density gradient centrifugations. The purified membranes were enriched for inner membranes and were characterized by their density and content of proteins, phospholipids, enzymes, cytochromes, and carotenoids. These purified spore membranes could be used to investigate their role in the triggering of germination.     

Germination of spores of Bacillus subtilis with dodecylamine

AIMS: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination. METHODS AND RESULTS: Spores of B. subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while spores prepared on solid medium germinated more poorly with this agent. Dodecylamine germination of spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the spores. The dodecylamine-germinated spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did spores germinated by nutrients. As measured by DPA release, dodecylamine readily induced germination of B. subtilis spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes. The DNA in dodecylamine-germinated wild-type spores was readily stained, while the DNA in dodecylamine-germinated spores of strains that were incapable of spore cortex degradation was not. These latter germinated spores also did not release their pool of free adenine nucleotides. CONCLUSIONS: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general. New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated spores are much more sensitive to processing treatments than are dormant spores.     

Possible involvement of Ca<Superscript>2+</Superscript>-dependent protein kinases in spore germination of the fern<Emphasis Type="Italic">Osmunda Japonica</Emphasis>

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca²⁺ for the germination. Furthermore, we showed that Sr²⁺ and Mn²⁺, which could substitute for Ca²⁺ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.     

Soybean and sunflower oils increase the infectivity of Colletotrichum gloeosporioides f. sp. aeschynomene to Northern Jointvetch

Soybean and sunflower oils increased the level of infection of northern jointvetch, Aeschynomene virginica, plants by Colletotrichum gloeosporioides f. sp. aeschynomene. Inoculation of seedlings with spore suspensions containing 10% (v:v) soybean oil or 10% sunflower oil resulted in more disease than when inoculated with suspensions of spores in water alone. The lengths of the dew periods required to establish equivalent levels of disease by spore suspensions containing 10% soybean or 10% sunflower oil were approximately 4–8 h less compared to aqueous suspensions. Incubation of spores in 10% soybean oil followed by removal and resuspension in water did not affect the infectivity of spores when compared to spores incubated in aqueous suspensions. Spore germination and appressoria formation were unaffected by either of the oils tested in in vitro assays; however, in in vivo assays, 10% soybean oil and 10% sunflower oil increased spore germination in comparison to spores that were suspended in water.