Soil fungistasis: role of the microbial nutrient sink and of fungistatic substances in two soils.

Sensitivity of conidia of Cochliobolus victoriae to fungistasis decreased markedly following incubation on moist sand for at least 1 h. Germination was greater on Conover loam or on sand being leached with water than on an alkaline clay loam soil known to produce a volatile fungistatic substance. Evolution of 14CO2 began within 3 min after [14C]glucose was applied to the soils; the rate of 14CO2 evolution was faster with Conover loam. Germination of Thielaviopsis basicola conidia per unit of glucose remaining in agar discs initially containing 0-1% glucose, was lower for discs incubated on the clay loam soil than on Conover loam, and was greatest on a bed of sand undergoing aqueous leaching. Germination of ascospores of Neurospora tetrasperma and conidia of C. victoriae was suppressed on discs of washed, Purified Agar or polyacrylamide gel incubated on or over the clay loam soil, but no suppression resulted when discs were incubated on Conover loam. Extensive aeration of either soil did not remove its fungistatic effect. Fungistasis in Conover loam appears to be caused primarily by nutrient deprivation, whereas volatile fungistatic substances may play a major role in the clay loam soil.     

Soil bacteriostasis: inhibition of spore germination and microcolony development in agar discs incubated on nonsterile soils.

An agar-disc method showed that bacterial spores were subject to a bacteriostatic effect exerted by soil. Soil either inhibited spore germination or reduced microcolony development of the six Bacillus strains tested. Bacteriostasis was relieved by the addition of nutrients to soil-exposed discs after they had been removed from the soil.     

Studies on carbon material requirements for bacterial proliferation and spore germination under stress conditions: a new mechanism involving transmission of physical signals.

The growth of bacteria is often enhanced by addition of carbon materials such as graphite or activated charcoal to the growth medium. In this work, bacterial strains that strictly require such carbon materials under the ordinarily lethal stress caused by high concentrations of salt were isolated. The organisms were gram-positive, spore-forming, sugar-nonfermenting aerobic bacilli and were provisionally designated "Bacillus carbophilus" Kasumi after examination of their phenotypic traits. The growth- and germination-promoting effects of graphite and activated charcoal were demonstrated either quantitatively on agar plates containing fine crystals of the carbon materials mixed with a nonpermissive growth medium or qualitatively on agar plates on nonpermissive growth media half-covered with fine carbon particles. Further experiments demonstrated a novel feature of the phenomenon; i.e., the ability to induce colony formation on the nonpermissive plate was transmissible through the air, as well as through plastic or glass barriers. The mechanism probably involves transmission of physical signals regulating cell growth.     

Macrophage-mediated fungistasis in vitro: requirements for intracellular and extracellular cytotoxicity

Macrophage cytotoxicity for Cryptococcus neoformans was investigated by culturing mouse peritoneal macrophages with a thin-capsuled clone of cryptococcus under conditions permitting efficient phagocytosis. Yeast replication was quantitated by electronic particle counting after detergent lysis of macrophages, and viability was determined by quantitative plate counts. Under appropriate conditions, reproduction was completely inhibited; stasis began at 2 hr after addition of yeasts and lasted for 30 hr. During this time organisms in medium alone proliferated rapidly, doubling their number every 2.5 hr. After removal from macrophages, 60 to 100% of macrophage-inhibited cryptococci formed colonies, indicating that the cytotoxic effect was primarily fungistatic. When yeast cells were removed from macrophages, replication recommenced within 5 hr. Supernatant medium from fungistatic co-cultures was not inhibitory for fresh yeast cells. Conditions required for complete fungistasis were 1) peritoneal macrophages induced by peptone from BCG-infected mice, 2) endotoxin in nanogram per milliliter range added to serum-containing cell culture medium, 3) confluent macrophage monolayers, and 4) macrophage:cryptococci ratios of 20 to 100:1. Fungistasis occurred without phagocytosis but was more efficient when cryptococci were engulfed. For efficient fungistasis, macrophages must differentiate to and be maintained in the activated state. These results with yeast cells agree with the known requirements for macrophage effector function against neoplastic target cells.     

Influence of nitrogen and phosphorus on polyphosphate accumulation in Gigaspora margarita during spore germination

Polyphosphate (polyP) is the form in which phosphorus (P) is transferred from extraradical hyphae into arbuscles in the symbiotic stage of arbuscular mycorrhizal fungi. However, polyP dynamics in the presymbiotic stage are less understood. In this study, we aimed to investigate polyP accumulation in Gigaspora margarita as influenced by nitrogen (N) and/or P supply during germination. Spores of G. margarita were incubated on medium with or without P or N addition. PolyP content in the fungal tissue was monitored using a polyP kinase/luciferase system, and polyP synthetic activity was determined with ³²P labeling. The results showed that both N and P were necessary for polyP accumulation in germ tubes. Nitrate increased the polyP content in germ tubes, but ammonium did not. Along with germination, polyP content decreased in spores, but increased in germ tubes. ³²P labeling indicated that polyP synthetic activity increased in germ tubes along with germination, but was negligible in spores. Our results suggest that, in the presymbiotic stage of G. margarita, uptake of environmental N and P increases polyP content in germ tubes, and that polyP synthesis occurs mainly therein, leading to polyP accumulation. The possible mechanism of transfer of polyP from spores to hyphae remains to be elucidated.     

Effects of inorganic nitrogen sources on spore germination and gametophyte growth in Botrychium dissectum

Abstract. The effects of variations in nitrogen source upon spore germination and gametophyte growth of the eusporangiate fern Botrychium dissectum forma obliquum were investigated. Spore germination and early gametophyte growth were directly related to the oxidation level of the supplied nitrogen source. Nitrate and nitrite inhibited spore germination and at concentrations above 0.035 mol m^-3 prevented it entirely. Ammonium promoted germination well above the levels attained on media without nitrogen. Concentrations of ammonium greater than 0.035 mol m^-3 often resulted in germination above 90%. The growth of young gametophytes from spores was reduced on media without nitrogen or on media with high concentrations of nitrate. Ammonium stimulated the growth of both young and older gametophytes. However, older gametophytes were able to grow on media containing nitrate as the sole nitrogen source. We conclude that a reduced nitrogen source is necessary for spore germination and early growth of Botrychium gametophytes. This requirement has several ecological implications which may be related to the distribution of these ferns and the establishment of mycorrhizal associations.     

Initiation of Bacillus spore germination by hydrostatic pressure: effect of temperature.

Suspensions of Bacillus cereus T, B. subtilis, and B. pumilus spores in water or potassium phosphate buffer were germinated by hydrostatic pressures of between 325 and 975 atm. Kinetics of germination at temperatures within the range of 25 to 44 degrees C were determined, and thermodynamic parameters were calculated. The optimum temperature for germination was dependent on pressure, species, suspending medium, and storage time after heat activation. Germination rates increased significantly with small increments of pressure, as indicated by high negative deltaV values of -230 +/- 5 cm3/mol for buffered B. subtilis (500 to 700 atm) and B. pumilus (500 atm) spores and -254 +/- 18 cm3/mol for aqueous B. subtilis (400 to 550 atm) spores at 40 degrees C and -612 +/- 41 cm3/mol for B. cereus (500 to 700 atm) spores at 25 degrees C. The ranges of thermodynamic constants calculated at 40 degrees C for buffered B. pumilus and B. subtilis spores at 500 and 600 atm and for aqueous B. subtilis spores at 500 atm were: Ea = 181,000 to 267,000 J/mol; deltaH = 178,000 to 264,000 J/mol; deltaG = 94,000 to 98,300 J/mol; deltaS = 264 to 544 J/mol per degree K. These values are consistent with the concept that the transformation of a dormant to a germinating spore induced by hydrostatic pressure involves either hydration or a reduction in the visocosity of the spore core and a conformational change of an enzyme.     

Development of nitrogen-assimilating enzymes in sunflower cotyledons during germination as affected by the exogenous nitrogen source

Activities of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.3) were measured in cotyledons of sunflower (Helianthus annuus L. cv Peredovic) seedlings during germination and early growth under various external nitrogen sources. The presence of NO ₃ ^- in the medium promoted a gradual increase in the levels of NR and NiR activities during the first 7 d of germination. Neither NR nor NiR activities were increased in a nitrogen-free medium or in media with either NH ₄ ⁺ or urea as nitrogen sources. Moreover, the presence of NH ₄ ⁺ did not abolish the NO ₃ ^- -dependent appearance of NR and NiR activities. The increase of NR activity was impaired both by cycloheximide and chloramphenicol, which indicates that both cytoplasmic 80S and plastidic 70S ribosomes are involved in the synthesis of the NR molecule. By contrast, the appearance of NiR activity was only inhibited by cycloheximide, indicating that NiR seems to be exclusively synthesized on the cytoplasmic 80S ribosomes. Glutamine-synthetase activity was also strongly increased by external NO ₃ ^- but not by NH ₄ ⁺ or urea. The appearance of GS activity was more efficiently suppressed by cycloheximide than chloramphenicol. This indicates that GS is mostly synthesized in the cytoplasm. The cotyledons of the dry seed contain high levels of GDH activity which decline during germination independently of the presence or absence of a nitrogen source. Cycloheximide, but not chloramphenicol, greatly prevented the decrease of GDH activity.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase     

Effect of carbon source and pH of the growth medium on spore germination inPhycomyces blakesleeanus

Phycomyces blakesleeanus sporangiospores responded differently to activation by physical and chemical stimuli. Spores that were physically (heat shock) activated or chemically (ammonium acetate) activated germinated and grew at pH 4.5 with the hexoses glucose, fructose, galactose, andN-acetylglucosamine, and with glycerol and amino acids. Under these conditions, physically activated spores showed a lower, although significant growth with the hexoses fructose, galactose,N-acetylglucosamine and with glycerol. On the other hand, physically activated spores incubated at alkaline pH (pH 7.3) required glucose to germinate; a requirement not observed with chemically activated spores, which showed significant growth in the other hexoses tested. Both physically and chemically activated spores incubated at pH 7.3 were unable to germinate and grow with amino acids and glycerol. These results suggest that there are different targets for activation of the spores by physical and chemical treatments. The levels of the fermentative enzymes alcohol dehydrogenase and lactate dehydrogenase and of the oxidative enzyme NAD⁺-isocitrate dehydrogenase were higher in cells grown at pH 4.5 in medium containing glucose; however, alcohol dehydrogenase and lactate dehydrogenase appear not to be affected by a change in the pH of the growth medium.     

A unique method for studying the initiation of Bacillus megaterium spore germination.

The sequence of events that characterize initiation of $\text{Bacillus megaterium}$ QM B1551 spore germination can be interrupted by the presence of 1 mM HgCl₂. Whereas these was a complete loss in spore dipicolinic acid, only partial losses in absorbance, refractility, density and resistance to staining occurred. This simple technique allows one to determine the necessity of certain reactions for initiation of germination.     

Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore.

The GerAA, -AB, and -AC proteins of the Bacillus subtilis spore are required for the germination response to L-alanine as the sole germinant. They are likely to encode the components of the germination apparatus that respond directly to this germinant, mediating the spore's response; multiple homologues of the gerA genes are found in every spore former so far examined. The gerA operon is expressed in the forespore, and the level of expression of the operon appears to be low. The GerA proteins are predicted to be membrane associated. In an attempt to localize GerA proteins, spores of B. subtilis were broken and fractionated to give integument, membrane, and soluble fractions. Using antibodies that detect Ger proteins specifically, as confirmed by the analysis of strains lacking GerA and the related GerB proteins, the GerAA protein and the GerAC+GerBC protein homologues were localized to the membrane fraction of fragmented spores. The spore-specific penicillin-binding protein PBP5*, a marker for the outer forespore membrane, was absent from this fraction. Extraction of spores to remove coat layers did not release the GerAC or AA protein from the spores. Both experimental approaches suggest that GerAA and GerAC proteins are located in the inner spore membrane, which forms a boundary around the cellular compartment of the spore. The results provide support for a model of germination in which, in order to initiate germination, germinant has to permeate the coat and cortex of the spore and bind to a germination receptor located in the inner membrane.