pH值对Gigaspora margarita孢子萌发、菌丝生长与聚磷酸盐含量的影响

土壤pH值是影响AM真菌的生理与生态过程的重要因子之一,本试验在培养基上接种Gigasporamargarita的孢子,研究了pH值分别为5.2、6.0和6.8时孢子萌发率、菌丝生长和菌丝中聚磷酸盐(polyP)的含量。结果表明,不同pH条件下的孢子萌发率没有明显差异,培养12d后的萌发率为70%左右;随着pH的升高,菌丝的长度逐渐增加,表明低pH对菌丝的生长有一定的抑制效应;培养12d后,孢子中polyP含量低于菌丝中polyP含量,pH6.0和pH6.8的条件下菌丝中polyP含量明显高于pH5.2的含量,表明低pH也能降低菌丝中的聚磷酸盐含量。认为低pH对菌丝生长和polyP含量的抑制可能是其限制AM真菌功能发挥的重要机制之一。     

Intracellular pH in Arbuscular Mycorrhizal Fungi : A Symbiotic Physiological Marker

A method was developed to perform real-time analysis of cytosolic pH of arbuscular mycorrhizal fungi in culture using dye and ratiometric measurements (490/450 nm excitations). The study was mainly performed using photometric analysis, although some data were confirmed using image analysis. The use of nigericin allowed an in vivo calibration. Experimental parameters such as loading time and concentration of the dye were determined so that pH measurements could be made for a steady-state period on viable cells. A characteristic pH profile was observed along hyphae. For Gigaspora margarita, the pH of the tip (0–2 μm) was typically 6.7, increased sharply to 7.0 behind this region (9.5 μm), and decreased over the next 250 μm to a constant value of 6.6. A similar pattern was obtained for Glomus intraradices. The pH profile of G. margarita germ tubes was higher when cultured in the presence of carrot (Daucus carota) hairy roots (nonmycorrhizal). Similarly, extraradical hyphae of G. intraradices had a higher apical pH than the germ tubes. The use of a paper layer to prevent the mycorrhizal roots from being in direct contact with the medium selected hyphae with an even higher cytosolic pH. Results suggest that this method could be useful as a bioassay for studying signal perception and/or H⁺ cotransport of nutrients by arbuscular mycorrhizal hyphae.     

Heavy-Metal Stress and Developmental Patterns of Arbuscular Mycorrhizal Fungi

The rate of global deposition of Cd, Pb, and Zn has decreased over the past few decades, but heavy metals already in the soil may be mobilized by local and global changes in soil conditions and exert toxic effects on soil microorganisms. We examined in vitro effects of Cd, Pb, and Zn on critical life stages in metal-sensitive ecotypes of arbuscular mycorrhizal (AM) fungi, including spore germination, presymbiotic hyphal extension, presymbiotic sporulation, symbiotic extraradical mycelium expansion, and symbiotic sporulation. Despite long-term culturing under the same low-metal conditions, two species, Glomus etunicatum and Glomus intraradices, had different levels of sensitivity to metal stress. G. etunicatum was more sensitive to all three metals than was G. intraradices. A unique response of increased presymbiotic hyphal extension occurred in G. intraradices exposed to Cd and Pb. Presymbiotic hyphae of G. intraradices formed presymbiotic spores, whose initiation was more affected by heavy metals than was presymbiotic hyphal extension. In G. intraradices grown in compartmentalized habitats with only a portion of the extraradical mycelium exposed to metal stress, inhibitory effects of elevated metal concentrations on symbiotic mycelial expansion and symbiotic sporulation were limited to the metal-enriched compartment. Symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. Patterns exhibited by G. intraradices spore germination, presymbiotic hyphal extension, symbiotic extraradical mycelium expansion, and sporulation under elevated metal concentrations suggest that AM fungi may be able to survive in heavy metal-contaminated environments by using a metal avoidance strategy.     

Metabolic products of microorganisms

1. The total nitrogen content of spores of Penicillium notatum increased during swelling and reached a maximum before germ-tubes were formed. It subsequently decreased during germ-tube formation until a constant value in hyphae was reached.
2. An increase in the protein content of the spores was found during swelling, followed by a decrease when germ-tubes were formed. After germination, the protein content increased again to a constant level in hyphae.
3. The content of total lipids steadily decreased during swelling of spores. It reached a minimum value in germinating spores, followed by a net accumulation during hyphal growth. Similar changes occurred in free lipids, phospholipids and the acetone-soluble lipids but not in bound lipids.
4. After an initial decrease during swelling, no further change took place in the neutral carbohydrates content of the spores at the time of germ-tube formation. An accumulation of neutral carbohydrates occurred during late hyphal extension.
5. The nucleic acid content increased sharply during swelling, and reached the highest value in swollen spores just prior to the initiation of germ-tube formation. Afterwards, its content steadily decreased during germ-tube formation and hyphae elongation.

     

Effect of Light on Uredospore Germination and Germ Tube Growth of Soybean Rust (Phakopsora pachyrhizi Syd.)

The effect of light on uredospore germination and germ tube growth of Phakopsora pachyrhizi was studied. Frequency of uredospore germination was only partially reduced by high light intensity (> 1,9 * 10⁴ mW * m^?2). In uredospores unilaterally irradiated with polychromatic light germ tubes always emerged from the shadowed side. Already developed germ tubes showed a negative phototropic response. Both effects were inducible by low light intensities. Negative phototropism of germ tubes was a blue light effect. Light of 441 nm was more effective than that of 422 nm or 372 nm. Red light (> 600 nm) was ineffective, green light (513 nm) induced medium responses. In half-side illumination studies longitudinal halves of germ tube tips and spores were irradiated under a microscope. The tips of the germ tubes bent into the illuminating beam. In half-side illumination studies germ tubes always emerged from the illuminated spore halves. Under unilateral illumination liquid paraffin reversed this light “polarization” of spores and the negative phototropism of germ tubes. These results suggest that during unilateral illumination spores and germ tube tips act as a lens focussing the light on the wall farthest away from the light source., There, in uredospores emergence of germ tubes is stimulated and in germ tubes growth is inhibited. As a consequence, under unilateral illumination germ tubes emerge at the shadowed side of the spores and grow away from the light.     

Effect of host shoot clipping on carbon and nitrogen sources for arbuscular mycorrhizal fungi

The effect of clipping of the host-plant shoot on the sources of carbon and nitrogen for the arbuscular mycorrhizal (AM) fungus Gigaspora margarita was determined by measuring ¹³C in spores and hyphae in cocultures of C₃ and C₄ plants and by differential ¹⁵N labeling. C₃ and C₄ plants, which have different δ¹³C values, were grown in the same container separated by a series of hyphal compartments. The C₃ and C₄ plants were applied with ¹⁴N- and ¹⁵N-urea, respectively. After clipping of the C₃ shoots, spore δ ¹³C gradually approached that of the C₄ roots. Hyphal δ ¹³C paralleled that of spores. Spore % ¹⁵N was similar to that of mineral N in the C₄ plant compartment. Thus C in G. margarita coming from the clipped plants decreased with time. This demonstrates that C in AM fungi comes from living plants, whilst the N in spores comes mostly from the soil. Accepted: 28 November 2000     

Ultrastructure of Wall Swellings of Germinating Sporangia of Phytophthora palmivora (Butl.) Butl.

Germ tubes of directly germinating sporangia of P. palmivoraincubated in yeast extract solution at 30 ?C usually developedinto prominent swellings from which hyphae later emerged. Thegerm tubes arose as an extension of a new germination wall formedinternal to the sporangial wall prior to germination. The germtube swellings contained typical hyphal organelles. The germtube swelling possessed a thicker wall than both hyphae growingout of it and germ tubes that did not form swellings.     

Status of nuclear division in arbuscular mycorrhizal fungi during in vitro development

Summary The number of nuclei in spores and along hyphae of an arbuscular mycorrhizal fungiGigaspora margarita was measured in digital images of fluorescence arising from mithramycin stained cultures. Typical dormant spores (250 m diameter) contained 2000 nuclei. Eight hundred nuclei were mobilized during the first 3 days of germination. The number of nuclei in the spores nearly returned to the initial number after 22 days of hyphal growth. The average relative DNA content in the nuclei of dormant spores and in the nuclei of spores incubated for 22 days was comparable, as judged from fluorescence intensity. Hyphal elongation occurred with 460 nuclei per cm under a special set of in vitro conditions that promote extensive hyphal growth of arbuscular mycorrhizal fungi. We found an average total of 26000 hyphal nuclei per germinating spore after 22 days. The specific DNA polymerase inhibitor aphidicolin did not inhibit spore germination but it rapidly reduced the rate of hyphal growth and arrested growth after 4 days. No nuclei were produced de novo during this time. These results demonstrate thatG. margarita replicates nuclear DNA and undergoes nuclear division when grown in vitro even in the absence of a plant host.     

Effect of liming on spore germination,germ tube growth and root colonization by vesicular-arbuscular mycorrhizal fungi

Summary The effect of soil acidity on spore germination, germ tube growth and root colonization of vesicular-arbuscular mycorrhizal (VAM) fungi was examined using a Florida Ultisol. Soil samples were treated with 0, 4, 8 and 12 meq Ca/MgCO₃/100 g soil and each lime level received 0, 240, and 720 ppm P as superphosphate. Corn (Zea mays L.) was planted in the soil treatments, inoculated with eitherGlomus mosseae orGigaspora margarita spores and grown for 31 days. Acid soil inhibits mycorrhizal formation byG. mosseae through its strong fungistatic effect against the spores. The dolomitic lime increased mycorrhizal formation by both fungal species.G. margarita is much less sensitive to acidic conditions thanG. mosseae. Al ions are a very important component of the fungistatic property against the VAM symbiosis. VAM fungus adaptation may be important for plants growing on infertile acid soils if soil inoculation with these fungi is to contribute significantly to low-input technology for tropical agricultural systems.     

Biochemical aspects on the germination of conidiospores of Aspergillus niger

Summary Biochemical events occurring in synchronously germinating spores of Aspergillus niger strain 1617 were investigated. The spores were found to require l-proline (or l-alanine), glucose and phosphate for the complete germination. The germination process in the above synthetic medium could be divided into three phases: endogenous swelling, exogenous swelling and sprouting. The first swelling phase was not influenced by the severe environmental factors so far tested, while the second phase was found to be affected by them, especially the CO₂ concentration. Rates of increase in cellular substances and in consumption of environmental substances changed markedly after germ tubes sprouted. The first cellular synthesis thus far detected was nucleic acid synthesis in the exogenous swelling phase. At the end of this phase accumulation of free amino acids, mainly glutamic acid and alanine, was observed. Protein synthesis then followed. A conspicuous increase in O₂-uptake commenced in parallel with the active synthesis of protein, when germ tubes began to sprout.During the course of germination a shift of metabolic pattern from that of the spore to the mycelium was indicated by the ratios of total nitrogen/dry weight, RNA/DNA, oxygen consumed/glucose consumed, and oxygen consumed/total nitrogen taken at various time intervals.Rosalie B. Hite Post-doctoral Fellow of the University of Texas.     

Traits related to differences in function among three arbuscular mycorrhizal fungi

Diversity in phosphorus (P) acquisition strategies was assessed among three species of arbuscular mycorrhizal fungi (AMF) isolated from a single field in Switzerland. Medicago truncatula was used as a test plant. It was grown in a compartmented system with root and root-free zones separated by a fine mesh. Dual radioisotope labeling (³²P and ³³P) was employed in the root-free zone as follows: ³³P labeling determined hyphal P uptake from different distances from roots over the entire growth period, whereas ³²P labeling investigated hyphal P uptake close to the roots over the 48 hours immediately prior to harvest. Glomus intraradices, Glomus claroideum and Gigaspora margarita were able to take up and deliver P to the plants from maximal distances of 10, 6 and 1 cm from the roots, respectively. Glomus intraradices most rapidly colonized the available substrate and transported significant amounts of P towards the roots, but provided the same growth benefit as compared to Glomus claroideum, whose mycelium was less efficient in soil exploration and in P uptake and delivery to the roots. These differences are probably related to different carbon requirements by these different Glomus species. Gigaspora margarita provided low P benefits to the plants and formed dense mycelium networks close to the roots where P was probably transiently immobilized. Numerical modeling identified possible mechanisms underlying the observed differences in patterns of mycelium growth. High external hyphal production at the root-fungus interface together with rapid hyphal turnover were pointed out as important factors governing hyphal network development by Gigaspora, whereas nonlinearity in apical branching and hyphal anastomoses were key features for G. intraradices and G. claroideum, respectively.     

Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

Factors affecting spore germination in Aspergillus niger

Temperature markedly affected germination and germ tube length of A. niger. More than 90% of the spores were germinated in the range 30°–34 °C and formed maximum length of germ tubes. At temperatures from 38° to 43 °C, the proportion of the spores that germinated as well as the germ tube length were both gradually decreased. However, at 47°C germ tube formation was completely inhibited up to 15 hrs. after inoculation.High relative humidity was found necessary for the spore germination of A. niger. Germination failed to occur at 76% relative humidity. At 78 and 81% relative humidity germination was detected 15 hrs. after inoculation while at the higher humidities germination was started after 6 hrs. only.Conidiospores of A. niger were very sensitive to changes in the hydrogen ion concentration, pH. Complete inhibition of germination was found at pH less than 3.5. The germination and the length of the formed germ tubes increased with pH to reach their maximum rates at pH 4.5.     

The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.     

Penicillium roqueforti conidia induced by L-amino acids can germinate without detectable swelling

Penicillium roqueforti is used for the production of blue-veined cheeses but is a spoilage fungus as well. It reproduces asexually by forming conidia. Germination of these spores can start the spoilage process of food. Germination is typically characterized by the processes of activation, swelling and germ tube formation. Here, we studied nutrient requirements for germination of P. roqueforti conidia. To this end,?>?300 conidia per condition were monitored in time using an oCelloScope imager and an asymmetric model was used to describe the germination process. Spores were incubated for 72 h in NaNO₃, Na₂HPO₄/NaH₂PO₄, MgSO₄ and KCl with 10 mM glucose or 10 mM of 1 out of the 20 proteogenic amino acids. In the case of glucose, the maximum number of spores (P_max) that had formed germ tubes was 12.7%, while time needed to reach 0.5 P_max (τ) was about 14 h. Arginine and alanine were the most inducing amino acids with a P_max of germ tube formation of 21% and 13%, respectively, and a τ of up to 33.5 h. Contrary to the typical stages of germination of fungal conidia, data show that P. roqueforti conidia can start forming germ tubes without a detectable swelling stage.

     

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.     

Dry mass changes in germinating spores of Diplodia maydis

Summary The dry mass of two-celled Diplodia maydis spores was measured both before and after germination by quantitative interference microscopy. The dry mass of spores declined approximately 50% during germination. However, the dry mass of germinating spores plus the dry mass of their germ tubes was greater than the dry mass of spores before germination. We conclude that the germinating spores absorbed nutrients released from non-germinating spores.The dry mass of fungal spores can be estimated by weighing large numbers of spores and determining the mean from sample spore counts. Mumford and Pappelis(4) determined the total dry mass of individual spores of Fusarium roseum and the contained lipid bodies before and after spores germinated using quantitative interference microscopy. The mean spore dry mass before germination was 57 pg. Lipid bodies accounted for about 61% of that mass and decreased as spores germinated. The total dry mass of the spore and germ tube 24 hr later greatly exceeded that of the spore before germination. Quantitative interference microscopy has been used to measure the dry mass of various types of cells. Kulfinski and Pappelis (3) recently reviewed how this technique has been applied to plant cells. Technical aspects of interference microscopy have been described by Ross (6).The purpose of this study was to examine the dry mass changes in Diplodia maydis (Berk.) Sacc. with and without germ tubes through the use of interference microscopy.     

The polarisome component SPA-2 localizes at the apex of Neurospora crassa and partially colocalizes with the Spitzenkörper

In fungal hyphae multiple protein complexes assemble at sites of apical growth to maintain cell polarity and promote nucleation of actin. Polarity allows the directional traffic of vesicles to the Spitzenkörper (Spk) prior to fusing with the plasma membrane to provide precursors and enzymes required for cell extension and nutrition. One of these complexes is the polarisome, which in Saccharomyces cerevisiae contains Spa2p, Pea2p, Bud6p/Aip3p and Bni1p. To investigate the localization and role of the polarisome during Spk establishment in Neurospora crassa we tagged SPA-2 with the green fluorescent protein (GFP) and examined growing cells by laser scanning confocal microscopy in elongating germ tubes and mature hyphae. SPA-2-GFP accumulated gradually at the apex of germ tubes, when a FM4-64 stained Spk was not still detectable. When the germlings reached about 40 μm in length, a FM4-64 stained Spk started to be apparent and from this point on SPA-2-GFP was observed in the apical region of both germ tubes and mature hyphae, as a hand fan shape with a brighter spot at the base. Fusion of the N. crassa SPA-2-GFP strain with a N. crassa strain expressing chitin synthase 1 (CHS-1) labeled with mCherryFP indicated only partial colocalization of the polarisome and the Spk core. N. crassa SPA-2-GFP was also found at the apex of forming branches but not in septa, suggesting that it participates only in areas of tip growth. A Δspa-2 strain displayed hyphae with uneven constrictions, apices with an unstable Spk, reduced growth rate and higher number of branches than the wild type strain, indicating that SPA-2 is required for the stability, behavior and morphology of the Spk and maintenance of regular apical growth in hyphae of N. crassa, although not for polarity or Spk establishment.     

Effect of undecanoic acid on germination of microconidia of wild and undecanoic acid resistant mutant of trichophyton rubrum

Effects of undecanoic acid (UDA) on germination of microconidia and elongation of germ tubes in UDA sensitive (uda _s) wild type Trichophyton rubrum and UDA resistant (uda _r) mutant derived from it, were studied. UDA inhibited conidial germination of uda _s and uda _r strains at 30 g/ml and 120 g/ml respectively which were minimum inhibitory concentrations of UDA for these two strains. When spores from both uda _s and uda _r were germinated in presence of subinhibitory concentration of UDA, germ tube growth was short. The elongation of germ tubes of spores pregerminated in absence of UDA was also inhibited by dose of UDA not sufficient to inhibit germination.