Distribution of polyphosphates in cell-compartments of Chlorella fusca as measured by 31P-NMR-spectroscopy

In suspensions of the green alga Chlorella fusca the influence of high pH and high ethylene-diamine-tetraacetic acid concentrations in the external medium, of French-press and perchloric acid extraction of the cells and of alkalization of the intracellular pH on the polyphosphate signal in ³¹P-nuclear magnetic resonance (³¹P NMR) spectra was investigated.The results show that part of the polyphosphates of asynchronous Chlorella cells are located outside the cytoplasmic membrane and complexed with divalent metal-ions. These polyphosphates are tightly bound to the cell wall and/or the cytoplasmic membrane and are not susceptible to hydrolyzation by strong acid at room temperature, in contrast to the intracytoplasmic polyphosphates.Upon alkalization of the internal pH of Chlorella cells, polyphosphates, previously not visible in the spectra become detectable by ³¹P-NMR-spectroscopy. ³¹P-NMR spectroscopic monitoring of polyphosphates during gradual alkalization of the extra-and intracellular space is proposed as a quick method for the estimation of the cellular polyphosphate content and distribution.Abbreviations CCCP Carbonylcyanide-m-chlorophenyl-hydrazone - NTP/NDP Nucleotide triphosphate/-diphosphate - PCA Perchloric acid - ³¹P-NMR ³¹P-nuclear magnetic resonance - PolyP polyphosphates - PP₁, PP₂, PP₃ terminal, second and third phosphate residue of polyphosphates, respectively - PP₄ core phosphate residues of polyphosphates     

<Superscript>31</Superscript>P NMR study of polyphosphate levels during different growth phases of <Emphasis Type="Italic">Phycomyces blakesleeanus</Emphasis>

The changes in relative polyphosphate content, estimated as the intensity ratio of core polyphosphate signal and intracellular inorganic phosphate signal from ³¹P NMR spectra, during the growth of Phycomyces blakesleeanus are reported. The ratio increases from 16 h to 28 h of growth, the minimum occurs at 32 h, followed by sharp increase up to 36 h, and a steady decrease afterwards. The changes in the biomass during mycelium growth showed steady increases, with a stagnation period between 32 h and 36 h during which a pronounced increase in the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal occurred. The reduction of growth temperature from 22°C to 18°C significantly decreased the rate and intensity of growth, but the pattern of polyphosphate changes remained unchanged. The changes of the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal are linked to characteristic stages of sporangiophore development. Analysis of core polyphosphates, intracellular inorganic phosphate and β-ATP signal intensities suggest the role of polyphosphates as an energy and/or a phosphate reserves during Phycomyces development.     

Ammonia fungi of iriomote Island in the southern Ryukyus,Japan and a new ammonia fungus,Hebeloma luchuense

Species composition and fruiting season of ammonia fungi were investigated in Iriomote Island of the southern Ryukyus, Okinawa Prefecture, Japan.Castanopsis andPinus forests were surveyed and 10 species of ammonia fungi were collected, including one new agaric species,Hebeloma luchuense sp. nov. This new fungus is characterized by having a rooting, squamulose-scaly stipe and cortinate veil and forms ectomycorrhizae withCastanopsis cuspidata var.sieboldii. Although the general mushroom season in theCastanopsis forest in Iriomote island was very short and restricted to summer, ammonia fungi were observed to fruit throughout the year in urea-treated plots.     

Influence of aluminum on in vitro formation of Pinus caribaea mycorrhizae

The ability of Pinus caribaea var. hondurensis to form mycorrhizae was determined in vitro with seven species of ectomycorrhizal fungi in the presence of six levels of Al (added as AlCl₃) in a nutrient solution. The time required for mycorrhizal formation, the number of mycorrhizal root tips and the percent mycorrhizae were measured after 15, 30, 60, 90 and 120 days. Cenococcum graniforme was susceptible to Al toxicity at all Al concentrations. Pisolithus tinctorius and Suillus sp. were depressed at lower but stimulated at higher Al concentrations. The inverse was shown for Rhizopogon reaii and Hebeloma cylindrosporum. Tolerance to Al was verified for R. nigrescens and H. crustuliniforme. Pisolithus tinctorius had the largest mycorrhizal capacity, defined as the sum of the values for time, percent and number of mycorrhizae. Ectomycorrhizal fungi appeared to ameliorate Al damage to plant roots even in treatments where no mycorrhizae formed. Inoculation of pine seedlings with Al-tolerant mycorrhizal fungi is likely to improve reforestation efforts in highly-weathered tropical soils.     

B-group vitamins production by mycorrhizal fungi in response to pH (in vitro studies)

Studies were carried out on B-group vitamins production by mycorrhizal fungi grown in vitro at different pH values. It was found that not all the fungi investigated produced all the B-group vitamins studied. Production of the vitamins varied between species and was influenced by the pH of the medium. Out of seven fungal species studied three did not produce biotin. Suillus bovinus synthesized this vitamin both in the acidic and neutral medium. Thiamin was produced by the fungi in minute amounts mainly in the acidic medium. The greatest amounts of nicotinic acid were produced by Hebeloma crustuliniforme (No 5392). Pantothenic acid was not detected only in the culture of Cenococcum graniforme.     

Occurrence ofHebeloma vinosophyllum on the forest ground after decomposition of crow carcass

Hebeloma vinosophyllum (Basidiomycota, Agaricales), a member of the ammonia fungi, occurred on the ground in the close vicinity of the decomposed carcass of a jungle crow,Corvus macrorhynchos, in a forest dominated byQuercus spp. in Urawa, Saitama Pref., central Japan. This is the first report of an ammonia fungus occurring at the site of a decomposed wild bird carcass.     

Inorganic Polyphosphates and Exopolyphosphatases in Cell Compartments of the Yeast Saccharomyces cerevisiae Under Inactivation of PPX1 and PPN1 Genes

Purified fractions of cytosol, vacuoles, nuclei, and mitochondria of Saccharomyces cerevisiae possessed inorganic polyphosphates with chain lengths characteristic of each individual compartment. The most part (80–90%) of the total polyphosphate level was found in the cytosol fractions. Inactivation of a PPX1 gene encoding ~40-kDa exopolyphosphatase substantially decreased exopolyphosphatase activities only in the cytosol and soluble mitochondrial fraction, the compartments where PPX1 activity was localized. This inactivation slightly increased the levels of polyphosphates in the cytosol and vacuoles and had no effect on polyphosphate chain lengths in all compartments. Exopolyphosphatase activities in all yeast compartments under study critically depended on the PPN1 gene encoding an endopolyphosphatase. In the single PPN1 mutant, a considerable decrease of exopolyphosphatase activity was observed in all the compartments under study. Inactivation of PPN1 decreased the polyphosphate level in the cytosol 1.4-fold and increased it 2- and 2.5-fold in mitochondria and vacuoles, respectively. This inactivation was accompanied by polyphosphate chain elongation. In nuclei, this mutation had no effect on polyphosphate level and chain length as compared with the parent strain CRY. In the double mutant of PPX1 and PPN1, no exopolyphosphatase activity was detected in the cytosol, nuclei, and mitochondria and further elongation of polyphosphates was observed in all compartments.     

Comparative structural study ofQuercus serrata andQ. acutissima formed byPisolithus tinctorius andHebeloma cylindrosporum

Ectomycorrhizas were synthesized in pots and growth pouches betweenQuercus serrata, Q. acutissima, and two ectomycorrhizal fungi,Pisolithus tinctorius andHebeloma cylindrosporum. Root morphology and the structure of the mantle and Hartig net were compared using light, fluorescence, scanning and transmission electron microscopy.P. tinctorius initially colonized root cap cells, and eventually produced a highly branched lateral root system with a complete mantle, whereasH. cylindrosporum promoted root elongation with few hyphae on the root apex surface indicating that interaction between roots differs with fungal species. Hartig net structure and hyphal inclusions varied between all the combinations tested. There were structural differences between mycorrhizas ofH. cylindrosporum/Q. acutissima grown in soil and growth pouches, which indicate that the growth pouch environment can induce artefacts in roots. Fruit bodies ofH. cylindrosporum developed in pots withQ. acutissima. AlthoughP. tinctorius has been used to inoculate oak seedlings in the nursery, results of this study indicate thatH. cylindrosporum may also be an effective ectomycorrhizal fungus forQ. serrata andQ. acutissima.     

The fungus-specificity of mycorrhization helper bacteria (MHBs) used as an alternative to soil fumigation for ectomycorrhizal inoculation of bare-root Douglas-fir planting stocks with Laccaria laccata

Mycorrhization helper bacteria (MHBs) isolated and selected from the Douglas fir-Laccaria laccata symbiotic system have previously been shown to be fungus-specific: they promote ectomycorrhizal establishment of Laccaria laccata but inhibit mycorrhiza formation by other fungi. In this paper, two experiments in a nursery producing two years-old bare-root Douglas-fir planting stocks confirm the specificity of MHBs under field conditions. They also show that, by selectively helping the introduced L. laccata against the resident symbionts, MHBs are an interesting alternative (safer and easier) to soil fumigation for the success of routine controlled mycorrhization of planting stocks in forest nurseries.     

Ultrastructure of chitin in hyphae ofCandida albicans and other dimorphic and mycelial fungi

Summary Chitin microfibrils exposed by chemical extraction of hyphal walls ofCandida albicans, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidiodes brasiliensis, Coprinus cinereus andMucor mucedo were of variable morphology but gave identical infrared spectra and behaved as pure chitin in chromatographic analyses. The microfibrils of the four dimorphic fungi studied were shorter than those in the mouldsC. cinereus andM. mucedo but were similar to those reported for the yeastSaccharomyces cerevisiae. InC. albicans the microfibrils in the septal plates of hyphae were predominantly tangentially orientated and were longer than those in the lateral walls. Microfibrils produced by chitin synthasein vitro were very much longer than any observed from hyphal preparations.     

Inorganic polyphosphates and exopolyphosphatases in different cell compartments of Saccharomyces cerevisiae

The cytosol, nuclei, vacuoles, and mitochondria of the yeast Saccharomyces cerevisiae possess inorganic polyphosphates (polyPs). PolyP levels, spectra of polyP chain lengths, and their dependence on the growth phase are distinguished in the mentioned compartments. Inactivation of the PPX1 gene has no effect on the polyP metabolism under cultivation of the yeast in medium with glucose and 5–7 mM P_i. Inactivation of the PPN1 gene results in elimination of the high-molecular-mass exopolyphosphatases (∼120 to 830 kD) of the cytosol, nuclei, vacuoles, and mitochondria of S. cerevisiae suggesting that it is just PPN1 that encodes these enzymes. Expression of the low-molecular-mass exopolyphosphatase of ∼45 kD encoded by the PPX1 gene decreases under PPN1 inactivation as well. While PPN1 inactivation has negligible effect on polyP levels, it results in increase in the long-chain polyPs in all the compartments under study. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 11, pp. 1445–1450.     

Comparison of the Influence of Carbon Substrates on the Fibrolytic Activities of Anaerobic Rumen Fungi

Cellulolytic and xylanolytic activities among genera of anaerobic fungi when grown on glucose, xylan and the cellulosic substrates, filter paper and Avicel were compared. All the fungi had basal extracellular fibrolytic activities that could be enhanced by growth on xylan or the cellulosic substrates. However,Piromyces communisstrain 22 andNeocallimastix patriciarumstrain 27 had substantially greater levels of fibrolytic activity thanOrpinomyces joyoniistrain 19-2 orNeocallimastix frontalisstrain RE1. Zymogram analysis suggested both structural and regulatory differences amongst the enzyme systems of the fungi. Numerous and varied enzyme bands were evidenced for all the fungi, with substantial substrate influences seen in the xylanase activities. Most commonly the smaller molecular weight bands, found exclusively extracellularly, appeared under the greatest regulatory control. Endoglucanase activities ofP. communisandO. joyoniidemonstrated similar regulatory control, while those of the twoNeocallimastixstrains did not appear to exert such control. These results suggest that while the enzymatic activities are functionally similar, there are likely significant variations in the enzyme systems of the anaerobic fungi.     

Comparative studies on ectomycorrhizae synthesized with various in vitro techniques using Picea abies and two Hebeloma species

Summary Various in vitro synthesis techniques with Picea abies and two Hebeloma species showed that structures of the mantle and Hartig net of synthesized ectomycorrhizae within the given two fungus species are stable. However, thickness of mantle, and penetration depth and number of hyphal cell rows between cortical cells of the Hartig net are dependent on techniques and substrates. Porous glass balls as substrate in the Erlenmeyer technique seem to suppress or delay mantle and Hartig net formation. With the other techniques (growth pouch, open cuvette, Erlenmeyer with a vermiculite-peat moss mixture) development of the mantle is simultaneous with or shortly in advance of Hartig net formation. The ectomycorrhizae of the two tested Hebeloma species are similar and cannot be morphologically differentiated by the in vitro techniques used.     

Aspergillus on tree nuts: incidence and associations

California exports tree nuts to countries where they face stringent standards for aflatoxin contamination. Trade concerns have stimulated efforts to eliminate aflatoxins and Aspergillus flavus from almonds, pistachios and walnuts. Incidence of fungi on tree nuts and associations among fungi on tree nuts were studied. Eleven hundred pistachios, almonds, walnuts and brazil nuts without visible insect damage were plated on salt agar and observed for growth of fungi. Samples came both from California nut orchards and from supermarkets. To distinguish internal fungal colonization of nuts from superficial colonization, half the nuts were surface-sterilized before plating. The most common genera found were Aspergillus , Rhizopus and Penicillium . Each species of nut had a distinct mycoflora. Populations of most fungi were reduced by surface sterilization in all except brazil nuts, suggesting that they were present as superficial inoculum on (rather than in) the nuts. In general, strongly positive associations were observed among species of Aspergillus ; nuts infected by one species were likely to be colonized by other species as well. Presence of Penicillium was negatively associated with A. niger and Rhizopus in some cases. Results suggest that harvest or postharvest handling has a major influence on nut mycoflora, and that nuts with fungi are usually colonized by several fungi rather than by single species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Lipofuscins and sclerotial differentiation in phytopathogenic fungi

Lipofuscins of lipidic and proteinaceous origin were identified by their excitation and emission spectra in phytopathogenic fungal representatives of different sclerotial differentiation types. Lipofuscin pigments in Sclerotium rolfsii, Rhizoctonia solani, Sclerotinia minor and Sclerotinia sclerotiorum showed similar excitation and emission maxima (ex-em 330–450, 330–450, 330–470 and 3307–470 nm, respectively). Sclerotial differentiation of these fungi was proceeded by a 4.2, 2.5, 2.7, 2.5 and 6, 2.9, 3.8, 3.1 fold increase of lipofuscin accumulation (per lipid and protein content), per respective fungus, as compared to their undifferentiated stage. Lipofuscin levels were higher in older than in younger mycelia and this phenomenon was more profound in S. rolfsii. Since lipofuscins are considered as indicators of oxidative stress, these data are in accordance with the hypothesis that suggests oxidative stress to be a common underlying factor in sclerotial differentiation of sclerotia-forming filamentous phytopathogenic fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structural and kinetic characterization of native laccases from Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii

A comparative study has been performed on five native laccases purified from the three basidiomycete fungi Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii to relate their different catalytic capacities to their structural properties. Spectroscopic absorption features and EPR spectra at various pH values of the five enzymes are very similar and typical of the blue oxidases. The analysis of the dependence of kinetic parameters on pH suggested that a histidine residue is involved in the binding of nonphenolic substrates, whereas both a histidine and an acidic residue may be involved in the binding of phenolic compounds. His and an Asp residue are indeed found at the bottom of a cavity which may be regarded as a suitable substrate channel for approaching to type 1 copper in the 3D homology models of the two laccases from Pleuorotus ostreatus (POXC and POXA1b) whose sequences are known.     

Five onygenalean fungi from Japan

Soils from various areas in Japan were screened for isolation of cleistothecial ascomycetes. From the recent collections, two new species of onygenalean fungi,Gymnascella japonica andGymnostellatospora frigida, are described and illustrated. Three interesting fungi,Aphanoascus saturnoideus, Arachnotheca glomerata andLeucothecium emdenii, are also described as the first records from Japan.     

广陈皮外源真菌的组成及产毒真菌分析

[背景]广陈皮为药食同源中药材,在高温、高湿且贮存不当的条件下容易发霉,从而产生毒素,严重威胁陈皮的质量安全.[目的]分析广陈皮表面外源真菌的组成及其产生毒素的真菌.[方法]采用平板稀释法分离广陈皮表面外源真菌,利用分生孢子形态特征及DNA序列分析进行真菌鉴定,采用高效液相色谱-三重串联四极杆质谱联用技术对青霉属和曲霉...     

<Emphasis Type="Italic">Penicillium frequentans</Emphasis> isolated from<Emphasis Type="Italic"> Picea glehnii</Emphasis> seedling roots as a possible biological control agent against damping-off

Picea glehnii seedlings are affected by damping-off fungi in nurseries. The aims of this study were (1) to isolate fungi grown in the seedling rhizosphere in forest soil of P. glehnii, (2) to select fungi that produce antifungal compounds against Pythium vexans, and (3) to examine whether or not selected fungi can protect seedlings from P. vexans. Penicillium frequentans from Picea glehnii seedling roots produced antibiotic penicillic acid. Penicillic acid did not cause significant phytotoxicity to the seedlings. Penicillium frequentans increased the average percentage of surviving seedlings when inoculated together with Pythium vexans, but the increase was not significant. Vigorous mycelial growth of P. frequentans around seedling roots seems to be one of the mechanisms for protection, but the amount of penicillic acid was too low to show antifungal activity in the seedling rhizosphere.