Sexually active strains ofCandida albicans andCryptococcus albidus

Strains which are considered to be sexually active have been recovered from stock cultures ofCandida albicans andCryptococcus albidus. These sexually active strains are homothallic and are characterized by slow growth and the presence of small cells conjugating with their buds. Conjugation appears to result in the formation of monokaryotic zygotes.The zygotes may be converted directly into chlamydospores or may give rise to a large-celled, presumably diploid generation. The diploid generation produces large, dormant cells which become multinucleate and form sterigmata on which single and generally uninucleate and smaller conidia are formed. As the sexually active haplophase is recoverable from the diplophase, it is probable that the haplophase originates from these conidia.True asci are not formed, but endospore formation does occur apparently as a result of internal budding. The endospores could not be differentiated by the commonly employed ascospore stains.What are presumably the haplophase and diplophase can be cultivated separately provided that the necessary precautions are taken.     

A new aspect of the life cycle ofCandida tropicalis

The life cycle ofCandida tropicalis has been elucidated by nuclear staining. We were able to distinguish 5 stages: (1) the sexually active unstable haplophase which either can mutate directly to (2) the sexually inactive haplophase, or is converted to (3) the diplophase by “autoploidization”. The unstable diplophase changes into the sexually inactive haplophase, which is stable, by “budding meiosis’. Chlamydospores (4), which are possibly formed by sexually inactive haploid cells, are true resting spores, and during their germination only mitosis takes place. The buds formed during germination may develop into either the sexually active, or the inactive haplophase. During budding meiosis in the diplophase, multinucleate giant cells (5) may occur. The authors do not consider the latter to be metabasidia. Instead, they might in some way be homologus with asci, and their daughter nuclei might be comparable with the nuclei of ascospore initials. These new nuclei migrate successively into new buds which process results in a short chain of haploid cells. True ascospores have not been found. From these findings it is likely thatC. tropicalis is closely related to the ascomycetes rather than to the Hemibasidiomycetes.     

Saccharomyces Marxianus Hansen

III Conclusion and Summary Zygosaccharomyces Marxianus andSaccharomyces macedoniensis belong to the same species. This species is met with in the haplophase (Z. Marxianus) as well as in the diplophase (S. macedoniensis). It was possible to bring this yeast from the haplophase into the diplophase and vice versa. By keeping this yeast during long times on maltagar it showed a tendency to change from the haplophase into the diplophase, but not into the opposite direction.It seems quite possible thatHansen, who did not describe a conjugation in this yeast, had met with the diplophase.It has been once more emphasized — at whichWinge andLaustsen and alsoLindegren andLindegren have pointed —, that the genusZygosaccharomyces is no valid genus.The yeast studied here belongs to the genusSaccharomyces and must be designated with the original name given to it byHansen:Saccharomyces Marxianus.For the sake of completeness it is mentioned here that also an imperfect stage ofS. Marxianus has been describedviz., Candida macedoniensis (A. Castellani) Berkhout (I).Saccharomyces fragrans Beijerinck has to be considered as its synonym.     

Sporotrichum carthusio-viride rai & Mukerji,a new species from Indian soils

Summary A new species,Sporotrichum carthusio-viride Rai &Mukerji, isolated from a soil sample collected from Kukrail area, Lucknow, India is described. It differs from other species in its colony colour and conidial measurements. It also forms yeast-like colonies on Wort's agar which produce terminal and inter-calary chlamydospores and budding cells.     

Impact of nutritional conditions on yields,germination rate and shelf-life of Plectosporium alismatis conidia and chlamydospores as potential candidates for the development of a mycoherbicide of weeds in rice crops

The effect of nutritional conditions on spore qualities was investigated in order to select which propagules, conidia or chlamydospores, would be most suitable for mycoherbicide development. Plectosporium alismatis was grown in a liquid basal medium supplemented with glucose and a mineral nitrogen source (sodium nitrate) or an organic nitrogen source (casamino acids). Conidial and chlamydospore yields, germination rate and shelf-life were compared. Two growth models were developed: on one hand, sodium nitrate added as the sole nitrogen source was partially utilised (8%), resulting in poor growth (1.77±0.02 mg mL^?1; 6±1.7×10⁵ conidia mL^?1). Under these conditions, P. alismatis produced dense, melanised-like aggregates that contained chlamydospores (12.4±0.7×10⁴ chlamydospores mL^?1). Germination rates of chlamydospores and conidia produced under these conditions was high (80%). Twenty percent of chlamydospores were able to germinate after 4 months storage at 25°C, while survival of conidia declined rapidly (<2%). When casamino acids were added to the liquid medium as the sole nitrogen source, P. alismatis produced sparser pellets resulting in high dry weights (5.37±0.09 mg mL^?1 and high conidia numbers (9.6±1.5×10⁶ conidia mL^?1), while no chlamydospore were observed. The germination rate of conidia produced in casamino acids was low (33±13%) after 8 h incubation and microcycle conidiation occurred. Five percent of these conidia germinated after 4 months storage. These data indicate that chlamydospores may be suitable for mycoherbicide development, provided further optimisation of yields is achieved.     

Culture age impacts Plectosporium alismatis propagule yields and subsequent desiccation and UV-radiation tolerance

The effect of culture age on yields, desiccation tolerance and resistance to ultraviolet radiation of Plectosporium alismatis, a potential mycoherbistat of aquatic weeds in Australian rice fields, was studied. P. alismatis was grown in a liquid basal medium supplemented with malt extract and sodium nitrate and harvested after 7, 14 or 21 days incubation. Although chlamydospore yields harvested from 14-day-old liquid cultures were significantly higher (29.2×10⁵ chlamydospores mL^?1) than chlamydospore yields harvested from 7-day-old liquid cultures (1.07×10⁵ chlamydospores mL^?1) or from 21-day-old liquid cultures, the germination of freshly-harvested chlamydospores from 7-day-old cultures (72.7%) was significantly reduced when propagules were grown for 14 days (55.3%). When exposed to UV-radiation, conidia and chlamydospores harvested from 14-day-old cultures germinated at a lower rate (<20%) than conidia and chlamydospores harvested from 7-day-old cultures (>40%). When conidia and chlamydospores were dried and subsequently exposed to UV, less than 30% of propagules harvested from 7-day-old cultures germinated, whereas less than 10% of propagules harvested from 14-day-old cultures germinated. A three-way analysis of variance including culture age, UV exposure and type of propagules confirmed that the culture age had more impact on the germination of fresh or dry propagules (P=0.00001 and P=0.0004, respectively) than the type of propagules considered (P=0.5). These results demonstrate that the culture age impacts significantly propagule yields and germination of P. alismatis conidia and chlamydospores, particularly after stress caused by dehydration and/or exposure to UV-B radiation.     

The perfect and imperfect states ofSporobolomyces salmonicolor

A strain ofSporobolomyces salmonicolor from which both the haplophase and diplophase had been recovered was found to form teliospores which germinated usually by the formation of a non-septate promycelium bearing 2–4 sporidia. The strain has consequently been transferred to the new basidiomycetous yeast genusAessosporon, for which a diagnosis and type are given, and which has been assigned to the Tilletiaceae.     

Development of a submerged-liquid sporulation medium for the johnsongrass bioherbicide<Emphasis Type="Italic"> Gloeocercospora sorghi</Emphasis>

Submerged culture experiments were conducted in three phases to determine the optimal medium for rapidly producing conidia of the fungal bioherbicide Gloeocercospora sorghi. In phase I, 18 crude carbon sources were evaluated to determine which would support sporulation. Under the conditions tested, butter bean and lima bean brines (1.5–4.6 mS/cm) provided best conidiation. In phase II, a fractional-factorial design was utilized to screen 76 different medium adjuncts in combination with butter bean brine for improved sporulation. d-Mannitol and carboxymethylcellulose (CMC) were the only acceptable factors that resulted in a significant improvement. In phase III, a central composite design with response surface methodology was used to optimize concentrations of these critical factors. The model predicted optimal sporulation in a medium composed of 2.69 mS/cm butter bean brine +0.043 M d-mannitol +0.37% w/v CMC with an expected titer of 1.51×10⁷ conidia/ml. Actual mean titer attained with the model-derived medium was 1.91×10⁷ conidia/ml. Optimal sporulation occurred at 25.5°C in this medium and conidia remained viable up to 2.71 days when stored at 12°C. No significant difference was observed in virulence of conidia produced on agar vs washed conidia produced in the model-derived (liquid) medium.     

Anastomoses and nuclear condition in Cephalosporium coccorum petch

Nuclear condition and formation of anastomoses inC. coccorum Petch have been reported. Preliminary study on the nuclear condition shows that the vegetative mycelium and conidia are uninucleate. Anastomoses in mycelium and conidia are discernible. Hyphal fusion through anastomosing bridges and nuclear migration through them show the possibility of heterokaryosis in the organism. Nutrient free medium is suitable for the development of anastomoses both in mycelium as well as in conidia.     

Microcycle conidiation and the conidial properties in the entomopathogenic fungus Metarhizium acridum on agar medium

Conidiation of the entomopathogenic fungus Metarhizium acridum on agar media was investigated. M. acridum CQMa102 exhibits two different conidiation patterns on agar media: normal conidiation in which conidia are formed on extended hyphae and microcycle conidiation in which conidiation occurs directly after conidia germination. Microcycle conidiation resulted in a mass of conidia produced via budding by accelerated development at the inoculation site. The mean total conidial yield (conidiation at day 10) was 4–5-fold greater after microcycle conidiation than during normal conidiation. Insect pathology assays indicated that microcycle conidia produced on SYA agar were as effective as normal aerial conidia against the locust. Ultraviolet (UV)-resistance tests showed no significant differences between the two types of cell propagules. However, microcycle conidia were more heat resistant than normal aerial conidia, and accumulated higher levels of trehalose in response to heat induction compared to normal aerial conidia.     

New species ofAcremonium, Cylindrocarpon andVerticillium from soil in the Bonin (Ogasawara) Islands, Japan

Three new soil fungi from the Bonin (Ogasawara) Islands, Japan are described:Acremonium macroclavatum, characterized by large clavate guttulate conidia;Cylindrocarpon boninense, characterized by 3–7-septate clavate macroconidia, terminal or intercalary chlamydospores, rarely produced unicellular clavate microconidia; andVerticillium hahajimaense, characterized by conidial heads bearing cylindrical conidia, and catenulate chlamydospores.     

Beauveria bassiana submerged conidia production in a defined medium containing chitin,two hexosamines or glucose

Summary Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH₄Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 × 10⁵/ ml after two days in GlcNAc medium as compared to 8.9 × 10⁵/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 10⁶/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.     

Two morphological groups derived fromClonostachys cylindrospora and their relationship totrans-4-hydroxy-(l)-proline productivity

Thetrans-4-hydroxy-(l)-proline productivity and morphological characteristics of strains isolated from two different conidium-producing structures inClonostachys cylindrospora were compared.trans-4-Hydroxy-(l)-proline was found to be produced only by strains originating from conidia on penicillate conidiophores, not by strains originating from conidia on verticillate conidiophores. Strains from each conidium-producing structure were also segregated by morphological properties; i.e., the amount of aerial hypha produced on modified Weitzman's agar, production of water-soluble yellow pigment in potato-dextrose agar, the primarily formed conidium-producing structures, and sclerotium formation. These phenomena indicate that morphological characteristics and metabolite productivity are correlated and destined at the stage of conidial production inC. cylindrospora.     

The hyphomycete Sorosporetta — Syngliocladium from mole cricket,Scapteriscus vicinus

The hyphomycete Sorosporella sp. was isolated from the mole cricket, Scapteriscus vicinus collected in Alachua Co., Florida. Scanning (SEM) and/or transmission (TEM) electron microscopy were used to study the chlamydospores characteristic of this genus and the conidial production in the alternate state, Syngliocladium. Brick-red chlamydospores, which occur in clusters in insect cadavers, have a fibrous cell wall as revealed by TEM. Pores often occur between walls of adjacent cells. Lipids, including a large central lipid droplet and smaller droplets along the periphery of the cell, are abundant in the cytoplasm. Most organelles were difficult to distinguish due to the density of the cytoplasmic material. Chlamydospores germinate on water or media. On Sabouraud maltose agar, germinating chlamydospores produce a white mycelial mat; synnematous-type growth was sometimes observed. Mycelia may bear conidiophores and ellipsoid conidia.Florida Agricultural Experiment Station Journal Series No. 7789.     

Comparative media studies in the isolation ofCandida albicans from pregnant women

Summary Rice agar and corn meal agar, with and without Tween 80, were evaluated clinically as directly inoculable selective and differential media for the isolation ofC. albicans from vulvovaginal specimens taken from pregnant women. Chlamydospore formation on these media was investigated as a criterion for the identification ofC. albicans.Of 301 patients cultured, 118 (39.2 %) gave positive cultures for yeast-like fungi of the genusCandida. Of 118 strains for which fermentation patterns were determined, 69 (58.5 %) gave the pattern forC. albicans. Of these, 56 (81.1 %) formed chlamydospores.Tween 80 was found to exert a very stimulating influence on chlamydospore production. Rice agar with Tween 80 appeared to be the most efficient medium for eliciting chlamydospores. However, since strains of 4 out of 6 species ofCandida isolated were found to sporulate it was concluded that chlamydospore formation is not a reliable criterion for the speciation ofC. albicans.Each of the 4 media served satisfactorily as a directly inoculable selective medium for the isolation of yeast-like fungi of the genusCandida from vulvovaginal specimens. None of the media appeared to preferentially stimulate chlamydospore production inC. albicans.Dr.Smith is Associate Professor in the Department of Microbiology; Dr.Taubert is a Fellow in the Department of Obstetrics and Gynecology; Mr.Towns is Laboratory Assistant in the Department of Microbiology.Supported in part by a grant from the Lederle Medical Faculty Awards Committee and in part by United States Public Health Service Grant E-3068.     

Diffusion in immobilized-cell agar layers: influence of microbial burden and cell morphology on the diffusion coefficients ofl-malic acid and glucose

Summary The diffusivities ofl-malic acid and glucose in an agar membrane entrapping small amounts ofEscherichia coli orRhodospirillum rubrum whole cells were measured using time lag (TL) and steady state (SS) methods. Diffusivities were overestimated by the SS method. For concentrations of immobilizedR. rubrum cells ranging between 10⁴ and 10⁹ organisms cm^–3 agar (20 ng-2 mg dry weight cm^–3 agar), the diffusion coefficient ofl-malic acid, determined by both methods, was related to the logarithm of the membrane cell content by a decreasing linear relationship. The diffusion coefficient of glucose obtained by TL analysis was not significantly affected by the presence in the membrane of 3 ng-0.3 mg dry wt.E. coli cm^–3 agar. However, values arising from the SS method decreased linearly as a function of the amount of immobilized organisms. Membranes containingR. rubrum cells offered higher diffusional resistance tol-malic acid and glucose than those loaded with the same amount ofE. coli cells.     

A new nematode-destroying Harposporium

A mucedinaceous parasite, observed destroying eelworms in a maize-meal-agar plate culture prepared by the addition of some forest duff from western Maryland, is newly described as Harposporium cycloides. Its conidia differ from the crescentic or semicircular conidia of the familiar H. anguillulae in that their curvature extends to an angular magnitude of approximately a full circle. Its chlamydospores, though sometimes found united in pairs, are much more often produced singly than are the chlamydospores of H. anguillulae.     

Morphogenesis and Ultrastructure of Claviceps purpurea During Submerged Alkaloid Formation

Criteria for morphogenetic and ultrastructural distinction between conidia and chlamydospores of a submerged culture of Claviceps purpurea (Fr.) Tul. are described. Both the hyphae of the sphacelia (asexual) stage and the conidia contained granular cytoplasm. Cytoplasmic invaginations in vacuoles were transformed to electron-opaque bodies and disintegrated prior to germination. The budding of conidia had basipetal succession. The chlamydospores were formed by rounding up the terminal cells of filamentous hyphae. Homogeneous nonvacuolized cytoplasm with lipid droplets and lipid-forming bodies was characteristic of young chlamydospores. Cristate mitochondria did not appear in the chlamydospores before the alkaloid production phase. Simultaneously a specific organelle in the chlamydospores, a dense body, appeared to absorb intracellular lipids and form large deposits of phospholipid material. No germination of chlamydospores was observed. The ultrastructural pattern described for chlamydospores was also observed in hyphae with reduced proliferation during the alkaloid production phase.     

Isolation of halophilic and halotolerant bacteria from a Japanese salt field and comparison of the partial 16S rRNA gene sequence of an extremely halophilic isolate with those of other extreme halophiles

Halophilic and halotolerant bacteria were isolated from soil samples of a Japanese salt field, an environment where salt concentrations vary annually. From 1 g of each of the five samples collected, over 1×10³ bacterial colonies (colony forming units (cfu)g^-1) grew on agar medium containing 2M Na⁺. In contrast, 0–4 bacterial colonies (cfu g^-1) were observed on agar medium containing 4M Na⁺. Two of the five samples contained numerous bacteria (10²–10³ cfu g^-1) capable of growth on a 2M Na⁺ alkaline (pH=9.5) medium, while few bacterial colonies were observed from the other three samples. Only one of the five samples was shown to contain bacteria capable of growth on a 4M Na⁺ alkaline medium. Most of the bacteria isolated on 4M Na⁺ agar were eubacteria, but one extreme halophile (TR-1, already described as Haloarcula japonica JCM7785) was also isolated. The 16S rRNA sequence of TR-1 was determined and shows high homology (94.4–98.5%) to Ha. marismortui and Ha. sinaiiensis. These results suggested that: 1) environments with seasonally varying salinity can harbour halotolerants as well as halophiles and, 2) closely related halophiles can be isolated from geographically distant habitats.     

Isozyme Polymorphism in Natural Populations of a Liverwort, PLAGIOCHILA ASPLENIOIDES

Assays of five Polish populations of Plagiochila asplenioides (Hepaticae, Plagiochilaceae) for peroxidase isozymes revealed three bands differing in electrophoretic mobility. The phenotypes observed appear to be governed by three loci; each locus has two alleles, one of which produces a band and the other no band, i.e., the second allele is silent, or null. Marked variability in enzyme phenotypes was found both within and between populations. The evolutionary implications of this variability are discussed in terms of the persistent haplophase and ephemeral diplophase of this organism.