Mitosis in the basidiomycete fungusTulasnella araneosa

Summary This is a report of a light and electron microscopic study of mitosis in the basidiomycetous fungusTulasnella araneosa. The study employs serial section analyses of nuclei preselected with fluorescence microscopy. It is the first such study of nuclear division in theTulasnellaceae and the first of conjugately dividing nuclei in basidiomycetous hyphal segments lacking clamp connections. Mitosis inT. araneosa is unusual in that the spindle pole body (SPB) develops asymmetrically; the SPB middle piece is large and transversely curved; and the nuclear envelopes of adjacent late anaphase nuclei fuse. Analyses of mitotic characteristics used for phylogenetic purposes indicate that, of the many characters available, only SPB characteristics are presently valuable. Available evidence indicates that the SPB ofT. araneosa is more different from that ofUredinales than it is from representatives of the other four orders ofBasidiomycotina that have been thoroughly studied.     

Hyperinduction of nitrilases in filamentous fungi

2-Cyanopyridine proved to act as a powerful nitrilase inducer in Aspergillus niger K10, Fusarium solani O1, Fusarium oxysporum CCF 1414, Fusarium oxysporum CCF 483 and Penicillium multicolor CCF 2244. Valeronitrile also enhanced the nitrilase activity in most of the strains. The highest nitrilase activities were produced by fungi cultivated in a Czapek-Dox medium with both 2-cyanopyridine and valeronitrile. The specific nitrilase activities of these cultures were two to three orders of magnitude higher than those of cultures grown on other nitriles such as 3-cyanopyridine or 4-cyanopyridine.     

Meiosis,basidiospore development and post-meiotic mitosis in the basidiomycete,Phallus impudicus

The meiotic spindles of Phallus impudicus had a similar ultrastructure to those seen in other basidiomycetes. After meiosis the basidal apex differentiated to form a number of broadly based protuberances which subsequently developed into basidiospores (up to 9 per basidium). The nuclei then underwent a post-meiotic mitosis within the cytoplasm of each basidiospore. After this mitosis one nucleus remained in the basidiospore and a complete septum developed at the base. The significance of this arrangement of meiosis, spore production and post-meiotic mitosis is discussed with respect to the taxonomy and life cycle of this unusual basidiomycete.     

Community interactions between the filamentous alga Cladophora glomerata (L.) Kuetzing,its epiphytes,and epiphyte grazers

Summary Interactions between epiphytes, epiphyte grazers and the filamentous green alga Cladophora glomerata (L.) Kuetzing were explored with smaples from rivers in Montana. Extracts of C. glomerata lowered photosynthetic rates of Nitzschia fonticola Grunow (an epiphytic diatom). Nutrient enrichment showed that C. glomerata from the Madison River was N deficient and its epiphytes were P deficient on 2 dates and N deficient on one date, while no nutrient deficiencies were detected in samples from 3 other rivers; this implies there was little nutrient competition between the epiphytes and C. glomerata. Epiphytes lowered drag on C. glomerata tufts and current velocity inside the tufts, apparently by decreasing the effective surface area. Lower drag may decrease detachment, but lowering current velocity from 8 to 0 cm s^-1 resulted in a 100 % decrease in photosynthesis. Light absorption by epiphyte pigments may lower photosynthetic rate of C. glomerata when irradiance is below 200–500 E m^-2 s^-1, and protect against photoinhibition above this irradiance range. Invertebrate grazers (predominantly Baetis tricaudatus Dodds, Trycorythodes minutus Traver and Brachycentrus occidentalis Banks) at high densities removed 75% of epiphytes and B. occidentalis grazed on C. glomerata. Invertebrates regenerated a mean of 0.16 mol NH _inf4 ^sup+ individual^-1 d-1 which could have enhanced growth of downstream C. glomerata. Competition and grazing were not the only interactions in the C. glomerata community, positive (mutualistic) interactions were also important.     

Expression of the autofluorescent protein, DsRed2, in the recombinants of the ectomycorrhizal basidiomycete, Suillus grevillei, generated by Agrobacterium-mediated transformation

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5′ gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.     

Agrobacterium-mediated transformation of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces commercially valuable fruit bodies, matsutake

Using agroinfection with a T-DNA vector carrying a hygromycin resistance marker, the recombinants were generated for the first time from the ectomycorrhizal basidiomycete Tricholoma matsutake, which produces commercially valuable fruit bodies, matsutake, during association with Pinus sp. plants. The transformation system may be useful in the genetic analysis of T. matsutake.     

Flow cytometric analysis of nuclear genome of the Ethiopian cereal tef [Eragrostis tef (Zucc.) Trotter]

Flow cytometric analysis of nuclear DNA content was performed by using nuclei isolated from young leaf tissue of tef (Eragrostis tef). The method was very useful for rapid screening of ploidy levels in cultivars and lines of tef representing the phenotypic variability of this species in Ethiopia. The results of the analysis showed that all cultivars were tetraploid. Flow cytometry was also used to determine nuclear DNA content in absolute units (genome size) in four tef cultivars. Nuclei isolated from tomato (Lycopersicon esculentum, 2C=1.96 pg) were used as an internal reference standard. The 2C DNA content of individual tef cultivars ranged from 1.48 to 1.52 pg (1C genome size: 714 Mbp-733 Mbp), the differences among them being statistically nonsignificant. The fact that the nuclear genome of tef is only about 50% larger than that of rice should make it amenable for analysis and mapping at the molecular level.     

Application of fluorescent probes to study structural changes in Aspergillus fumigatus exposed to amphotericin B, itraconazole, and voriconazole

The broad objective of this study was to document patterns of structural changes following antifungal treatment, and to determine any relationship with minimum inhibitory concentration (MIC) of an antifungal. Three clinical isolates of Aspergillus fumigatus, with high, intermediate, and low amphotericin B (AB), itraconazole (IZ), and voriconazole (VZ) MICs were studied in 24-well plates with cover slips. The fluorescent probes used were Calcofluor White (cell wall), propidium iodide (nucleus), and MitoTracker Green FM (mitochondria). Fluorescent microscopy as early as 3-h after exposure revealed that AB treated hyphae had intact cell wall with deformed mitochondria and nuclei while IZ and VZ treated hyphae revealed no intact cell wall, and deformation of mitochondria and nuclei. At 48 h, AB treated cells revealed rupture of hyphae and disintegration of mitochondria, and nuclei, IZ treated hyphae were swollen with disintegration of mitochondria, and nuclei while VZ treated hyphae showed rupture and disintegration of mitochondria and nuclei. The structural changes for the three strains studied were similar in fluorescent microscopy as long as the incubation time and their respective MICs were used. Thus, AB, IZ, and VZ induced gross organelle defects in A. fumigatus nuclei, mitochondria, and cell wall, which were consistent with respective MICs of antifungals used.     

Chimeric pheromone receptors in the basidiomycete Schizophyllum commune

The pheromone receptor system of the basidiomycete Schizophyllum commune is capable of ligand discrimination to confer mating specificity. The pheromone receptors of the B alpha locus were investigated for ligand discrimination in a strategy of domain swapping experiments. Several altered phenotypes of chimeric receptors have been found. These include constitutive pheromone receptors which need no ligand for activation of the downstream cascade of events. In addition, receptors still dependent on ligand were identified that had altered pheromone activation profiles, including promiscuous receptors that are activated by pheromones of all nine specificities, including the former self. In addition, highly discriminative receptors were created which are activated by only two of the eight non-self-specificities. The chimeric receptors identify the last third of the receptor as the determinant for B alpha 1 specificity, whereas B alpha 2 specificity resides in noncontiguous domains covering the first and middle parts of the receptor molecule.     

Bipolar incompatibility system of an ectomycorrhizal basidiomycete, Rhizopogon rubescens

The mating systems of most ectomycorrhizal fungi have not been elucidated because of two reasons. One is the difficulty of obtaining homokaryotic isolates for mating tests caused by the low germination rate of basidiospores, and another is the difficulty of checking dikaryotization caused by the absence or inconsistent production of clamp connections on heterokaryotic mycelia under laboratory conditions. Basidiospore germination of a few ectomycorrhizal fungi has been induced by living roots of their host plants. Based on this information, we examined methods to obtain homokaryotic isolates of Rhizopogon rubescens using its host plant, Pinus thunbergii. The basidiospores of R. rubescens appeared to germinate well on an agar plate, on which axenic pine seedlings were grown in advance to induce germination, even when the seedlings were removed from the plate at the time of spore inoculation. To enhance the production rate of clamp connections on the heterokaryotic mycelia of R. rubescens, the culture medium composition was modified. The pH of the medium was critical for the production of clamp connections, and the optimal pH was higher for the production of clamp connections than for mycelial growth. These findings made it possible to conduct mating tests, and we found that the mating system of R. rubescens is bipolar with a multiallelic mating type factor.     

Parasexual analysis of common-A crosses in the basidiomycete Agrocybe aegerita

Summary Crosses were performed between homokaryons of Agrocybe aegerita having the same allele at the A incompatibility gene but different B alleles. Heterokaryotic mycelia originating from crosses between two complementary auxotrophs were characterized by their instability on complete medium and extensive anastomosis between hyphae. Diploid mycelia were selected by plating oidia recovered from these heterokaryons onto minimal medium. These mycelia were characterized by the production of larger oidia than those of homokaryons, the release of a brown pigment when growing on complete medium and extensive hyphal anastomoses. Diploids retained the two B incompatibility functions of their homokaryotic parents and gave rise to a diploid/haploid dikaryon when crossed with a compatible homokaryon. Nearly 1% of the oidia recovered from heterokaryons were diploid. These nuclear fusion frequencies as well as the production of brown pigments enabled the identification of diploid strains on complete medium. In this way, crosses between wild prototrophic strains were successfully performed. Somatic recombination was induced following the treatment of diploid mycelia with haploidizing compounds. Selection based on the inability of mycelia to produce the brown pigments on complete medium led to selection of strains homoallelic at the B locus.     

Population structure and responses to disturbance of the basidiomycete Resinicium bicolor

Summary Resinicium bicolor (Alb. & Schw. ex Fr.) Parm. [=Odontia bicolor (Alb. & Schw. ex Fr.) Bres.] is an outcrossing resupinate basidiomycete associated with root and butt rots of trees, but is itself only very weakly pathogenic. The distribution of genets among every spruce stump in a 70-year-old 1250 m² spruce stand was analysed using somatic incompatibility testing. R. bicolor was present on 40% of 8-to 10-year-old stumps. Nineteen genets were found occupying 32 stumps; yielding probabilities of colonisation following establishment by basidiospores of 0.20–0.24 and by mycelial extension or dispersal of 0.16–0.20. The probability of colonisation decreased with increasing distance from a point of establishment. R. bicolor responded to both enrichment and destructive disturbances by the formation of an extensive cord system which enabled it to colonise discontinuously distributed resources and to overgrow fungi adjacent to it in a single resource unit, including Heterobasidion annosum.     

Cell wall metabolism during fruiting of the basidiomycete Schizophyllum commune

During the development of fruit bodies of the basidiomycete Schizophyllum commune, the alkali-insoluble (R glucan) and alkali-soluble (S glucan) cell wall fractions are synthesized during the entire course of morphogenesis. The water soluble glucan (WSG) is not synthesized after an early stage. There is also a relative increase in the proportion of S glucan during development which appears related to a change in the proportion of the components synthesized. Data are also presented to show that several fruiting mutants also have specific cell wall differences, and that there is a significant contribution to cell wall structure by genes which do not cause a macroscopically observable change in phenotype.     

Ultrastructure of the motile cells of the prostrate filamentous green algaeProtoderma sarcinoidea andChamaetrichon capsulatum

The biflagellate zoospores ofProtoderma sarcinoidea and the quadriflagellate zoospores ofChamaetrichon capsulatum are each covered by an amorphous, mucous material and a single layer of square scales, and the pyrenoid matrix is traversed by one or more thylakoid membranes. In the flagellar apparatus the basal bodies ofP. sarcinoidea and the upper basal bodies ofC. capsulatum are displaced in the counterclockwise absolute orientation, while the lower basal bodies ofC. capsulatum are directly opposed. Other components of the flagellar apparatus observed in each alga include: cruciately arranged d and s rootlets, each associated with an electron-dense component; simple terminal caps comprised of large and small subunits; a terminal electron-dense mass located near the proximal end of each basal body inP. sarcinoidea and near the upper basal bodies inC. capsulatum; and two rhizoplasts. Components specific to one or the other species include a single accessory basal body inP. sarcinoidea and a fibrous, electron-opaque band that links the upper and the lower basal bodies inC. capsulatum. The flagellar apparatus architecture ofP. sarcinoidea resemblesGayralia oxysperma, while that ofC. capsulatum is similar toTrichosarcina polymorphum andUlothrix species, all of which are included in theUlothrix-group,Ulotrichales, Ulvophyceae.     

Closed spindle nuclear division in the plasmodial phase of the acellular slime moldEchinostelium minutum

Summary Mitosis in the plasmodium ofEchinostelium minutum is intranuclear (closed spindle) and centrioles are not present at the spindle poles. The nuclear envelope remains essentially intact throughout mitosis with polar fenestrae appearing in anaphase and persisting through telophase. During anaphase there is a shortening in the distance of the chromosomes to the poles followed by a further separation of the poles. Zippering of microtubules may be the basis for these two anaphasic movements. During telophase the polar MTOCs are extruded into the cytoplasm through the polar fenestrae prior to reconstitution of the nuclear envelope. It is proposed that during sporulation such MTOCs are responsible for the differentiation of the centrioles which subsequently persist in the myxamoebal phase of this species.Based on the doctoral dissertation of the first author presented to the Department of Botany, University of Washington, Seattle, WA 98195, U.S.A.     

Predicting the onset of filamentous bulking in biological wastewater treatment systems by exploiting image analysis information

The performance of the activated sludge process is limited by the ability of the sedimentation tank (1) to separate the activated sludge from the treated effluent and (2) to concentrate it. Apart from bad operating strategies or poorly designed clarifiers, settling failures can mainly be attributed to filamentous bulking. Image analysis is a promising technique that can be used for early detection of filamentous bulking. The aim of this paper is therefore twofold. Foremost, correlations are sought between image analysis information (i.e., the total filament length per image, the mean form factor, the mean equivalent floc diameter, the mean floc roundness and the mean floc reduced radius of gyration) and classical measurements (i.e., the Sludge Volume Index (SVI)). Secondly, this information is both explored and exploited in order to identify dynamic ARX and state space-type models. Their performance is compared based on two criteria.     

Sporulation in the filamentous cyanobacterium Anabaena cylindrica

Sporulation in the filamentous cyanobacterium Anabaena cylindrica involves the transformation of a vegetative cell into a thick-walled resistant structure. Because this process occurs at predictable loci in each filament and involves a significant increase in cell size, the course of sporulation in a culture can be quantitatively determined. Sporulation occurs during the late logarithmic phase of a culture, a time of slow but unbalanced growth. Under the conditions imployed here, sporulation is not a synchronous event either between or within filaments. The information in this paper provides an estimate of the rate of spore differentiation and supports the previous notion that in the formation of strings of more than one spore, a gradient of spore maturation exists.     

Molecular diversity of soil basidiomycete communities in northern-central New South Wales, Australia

Basidiomycete communities were profiled using terminal RFLP (TRFLP) and amplified ribosomal DNA restriction analysis (ARDRA) approaches at seven field sites under differing land use in northern-central New South Wales (NSW), Australia. TRFLP data indicated greater basidiomycete species richness at sites with natural vegetation. Sixty-seven basidiomycete ARDRA-types were detected. Various putatively ectomycorrhizal fungi were detected at all sites with native vegetation. Most ectomycorrhizal taxa had affinities to the genus Tomentella, while two Pisolithus taxa and putatively ectomycorrhizal Cantharellales taxa were also detected. Although soils under woodland or grassland communities supported a range of putatively saprotrophic taxa, only members of the Ceratobasidiales were detected in soils under agricultural land use. This study is the first investigation of fungal communities in soils of northern-central NSW, Australia.     

Lack of relationship between gliding cyanobacteria and filamentous gliding heterotrophic eubacteria: comparison of 16S rRNA catalogues of Spirulina,Saprospira, Vitreoscilla,Leucothrix, and Herpetosiphon

Spirulina platensis and Spirulina maxima were characterized by 16S ribosomal RNA oligonucleotide cataloguing. Both species are highly related. They fall into the main group of the cyanobacteria, showing a remote relationship to Nostoc, Fischerella, Aphanocapsa, and also to Prochloron. Low similarity coefficients were found between the Spirulina species and certain organotrophic and sulfide oxidizing bacteria, viz. Saprospira grandis, Vitreoscilla stercoraria, Leucothrix mucor, Herpetosiphon aurantiacus, and Beggiatoa leptomitiformis, respectively. This result does not support the classical hypothesis that certain filamentous gliding bacteria are apochlorotic cyanobacteria.     

Suppression of the growth of the basidiomycete yeast, Rhodotorula minuta, by cinnamic acid

Rhodotorula minuta, a basidiomycete fungus, prefers neutral pH for growth and its growth inhibition by food preservatives such as benzoic acid and cinnamic acid has not been reported. Cinnamic acid at 1 g l^–1 arrested the growth and decreased the respiration of Rhodotorula but did not kill the yeast. The inhibitory effect was stronger in a mutant strain, 5-286, deficient in the -ketoadipate pathway than in the wild, suggesting that -ketoadipate pathway functions to detoxify this acid by restoring the decreased respiration.