Study of the nuclear cycle of four species of strictly anaerobic rumen fungi by fluorescence microscopy

The nuclear cycles of four species of strictly anaerobic rumen fungi (Neocallimastix frontalis, Piromonas communis, Neocallimastix joyonii, andSphaeromonas communis) were compared by fluorescence microscopy. The existence of two types of sporangium development and thalli was confirmed in this study. The first type involved monocentric species fitted with an endogenously developing single sporangium and with rhizoids, such asN. frontalis, P. communis, andS. communis, characterized by the presence of rhizoid-like vesicles. The second type concerned polycentric species with an exogenous sporangium development such asN. joyonii. This species is characterized by a rhizomycelium and gamma particle-like bodies within the zoospore.     

BIOCHEMICAL AND MICROCHEMICAL STUDY OF THE CELL WALLS OF RHIZIDIOMYCES SP.

Fuller , Melvin S. (Brown U., Providence, R. I.) Biochemical and microchemical study of the cell walls of Rhizidiomyces sp. Amer. Jour. Bot. 47(10): 838–842. lllus. 1960.—The presence of chitin in the cell walls of the fungus Rhizidiomyces is demonstrated by qualitative analysis of enzymatic and hydrochloric-acid hydrolysates of partially cleaned cell walls. Qualitative examination of the enzymatic and acid hydrolysates did not, however, serve for the detection of cellulose present in the cell walls of Rhizidiomyces. With microchemical tests, both chitin and cellulose can be detected. These microchemical tests served to indicate the localization of the chitin and cellulose in the cell walls of mature plants before and during zoospore discharge.     

The influence of the developing bacterial spore on the mother cell

Mutants unable to develop a completely engulfed forespore do not lose their viability, i.e., their ability to resume cell division, for at least 10 hr after the end of exponential growth. In contrast, mutants, which are blocked at later stages in development and which are able to produce completely engulfed forespores, lose their ability to divide. The time course of this decrease in viability coincides with the time course for the appearance of completely enclosed forespores. Experiments with the sporulating standard strains of Bacillus subtilis and B. megaterium suggest that the mother cells also lose their viability at about the time of forespore enclosure. These results indicate that the forespore, as soon as it is completely engulfed and thus committed to continue differentiation, somehow prevents the mother cell (sporangium) from resumption of growth.     

Regulation of hyphal morphogenesis by Ras and Rho small GTPases

The fungal kingdom is extremely diverse – comprised of over 1.5 million species including yeasts, molds and mushrooms. Essentially, all fungi have cell walls that contain chitin and the cells of most fungi grow as tube-like filaments called hyphae. These filamentous fungi, such as the mold Neurospora crassa, develop branched radial networks of hyphae referred to as mycelium. In contrast, non-filamentous fungi do not form radial mycelia, but grow as single cells, which reproduce by either budding or fission such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, respectively. Finally, there are fungi that are capable of switching between single cell, yeast form growth and filamentous growth such as Candida albicans. The switch from yeast to filamentous growth in these so-called dimorphic fungi is a virulence trait in many human and plant pathogens. Highly conserved master regulators of all three fungal growth modes – filamentous, non-filamentous and dimorphic – are the Ras and Rho small GTPases, which spatially and temporally control cell polarity establishment and maintenance. This review summarizes the key roles of the Ras and Rho GTPases during hyphal morphogenesis in a range of fungi.     

Flagellar Development During the Cell Cycle in Chlamydomonas reinhardtii

Flagellar development during the asexual synchronous cell cycle of Chlamydomonas reinhardtii (11.32 aM) was studied by light microscopy. Cell walls of sporangia of different developmental status were dissolved using gamete lysin (g-lysin) enabling direct observation of flagellar development. Flagellar growth in progeny cells exhibits a linear kinetic with a growth rate of 28 nm/min at 30°C leading to a flagellar length of 7–7.5 μm in 4–4.5 h. After this time the flagellar growth rate drops to 2.8 nm/min (as in interphase). Both flagella of a single cell and all flagella within a sporangium grow out at the same time and with the same rate. Cycloheximide (10 μg/ml) completely blocks flagellar development. If cycloheximide is removed flagellar growth resumes at the normal rate with no lag-phase. Flagellar development during the cell cycle in C. reinhardtii differs considerably from the well-studied model system of flagellar regeneration following amputation in the same species.     

Microtubules,organelle movement,and cross-wall formation at the sporangial-rhizoidal interface in the fungus,Chytridium confervae

Summary Chytridium confervae is a eucarpic, monocentric chytrid. We have used light and electron microscopy to study the relationship between the nutrient absorbing rhizoids and the asexually reproductive sporangium during growth. We have also examined the induction of zoosporogenesis by starvation, and subsequent differentiation until zoospore release. During growth the cytoplasm of the rhizoids and the developing sporangium was continuous and similar. At the start of starvation a bundle of fibers that were visible with light microscopy appeared at the junction between the rhizoids and the sporangium. Two hours after initiation of starvation a wall, that was also visible with light microscopy, formed to separate the rhizoids from the sporangium. Electron microscopy revealed a large, ordered array of microtubules in the thallus at the same time that the fibers appeared, and a sharp difference in the density of ribosomes in the cytoplasm of the sporangium and that of the rhizoids that was apparent immediately after starvation. This cytoplasmic difference was preserved by the formation of a cross-wall that was penetrated by plasmodesmata. After the wall was formed the cytoplasm of the rhizoids senesced. Comparison ofC. confervae with other organisms that use arrays of microtubules to move organelles is made and speculation on the role of the microtubules in organelle movement and wall formation inC. confervae is offered.     

Germination and parasitation of the resting sporangia ofSynchytrium endobioticum

Summary The cytoplasmic organization of the long-lived, thick walled resting stage of the sporangium ofSynchytrium endobioticum (Schilb.) Perc. is described. The cytoplasm of the resting sporangium contains a large number of closely packed lipid bodies and irregular electron dense bodies, which are interspaced with fine channels of cytoplasm. These ultrastructural observations are discussed in relation to the hypothesis ofBally (1912) andCurtis (1921) that zoospore primordia are already present during the resting stage. It is shown that the zoospore primordium is actually a lipid body and an osmiophilic body and the strands postulated to connect the individual zoospore primordia are actually the fine channels of cytoplasm.A new inner wall layer is laid down prior to the start of the germination. It is this wall layer which will protrude to form the vesicle in which sporogenesis takes place. The germination process observed, protrusion of a vesicle through a crack in the sporangial wall, the migration of the sporangial content into the vesicle, and the formation of a single, membrane-bound sporangium within this vesicle, is in full agreement with the recent light microscopic studies ofSharma andCammack (1976). These observations support the transfer ofS. endobioticum from the subgenusMesochytrium to the subgenusMicrosynchytrium (bothsensu Karling 1964).A major objective of the study, to obtain ultrastructural evidence for the location of the meiotic divisions in the life cycle, was not fulfilled.Three different fungi were observed to parasitize the resting sporangium ofS. endobioticum. These infections are discussed in relation to other mycoparasites of plant pathogenic fungi. The possibility of using a mycoparasite for the biological control of potato wart disease is considered to be without practical relevance.     

The influence of phosphorus limitation of the diatomAsterionella Formosa on the zoospore production of its fungal parasiteRhizophydium Planktonicum

The influence of phosphorus limitedAsterionella on the zoospore production of its fungal parasiteRhizophydium planktonicum was measured, using laboratory cultures of host and parasite. At saturated phosphorus concentrations the host reached a specific growth rate of 0.95.d^–1. Growing on these host cells, the mean parasite zoospore production was 26 spores per sporangium, and the mean development time of a sporangium was 45 hours. Growing on phosphorus limited hosts, the zoospore production decreased to less then 9 spores per sporangium, and the development time decreased to 40 hours. On phosphorus limited hosts, zoospores were produced at a slower rate. The algal growth rate was reduced to a greater extent than the fungal growth rate. Therefore, it could be concluded that phosphorus limitation ofAsterionella will facilitate the development of an epidemic of its parasiteRhizophidium, at least at high diatom densities, when possible differences in infectability of the algae play a minor role.     

Survival of inoculated Laccaria bicolor in competition with native ectomycorrhizal fungi and effects on the growth of outplanted Douglasfir seedlings

The survival, development and mycorrhizal efficiency of a selected strain of Laccaria bicolor along with naturally occurring ectomycorrhizal fungi in a young plantation of Douglas fir was examined. Symbionts were identified and their respective colonization abilities were determined. Eight species of symbiotic fungi, which may have originated in adjacent coniferous forests, were observed on the root systems. Mycorrhizal diversity differed between inoculated (5 taxa) and control (8 taxa) seedlings. Ectomycorrhizal fungi which occurred naturally in the nursery on control seedlings (Thelephora terrestris and Suillus sp.) did not survive after outplanting. Both inoculated and naturally occurring Laccaria species, as well as Cenococcum geophilum, survived on the old roots and colonized the newly formed roots, limiting the colonization by other naturally occurring fungi. Other fungi, such as Paxillus involutus, Scleroderma citrinum and Hebeloma sp. preferentially colonized the old roots near the seedling's collar. Russulaceae were found mainly in the middle section of the root system. Mycorrhizal colonization by Laccaria species on inoculated seedlings (54%) was significantly greater than on controls (13%) which were consequently dominated by the native fungi. Significant differences (up to 239%) were found in the growth of inoculated seedlings, especially in root and shoot weight, which developed mainly during the second year after outplanting. Seedling growth varied with the species of mycorrhizae and with the degree of root colonization. Competitiveness and effectiveness of the introduced strain on improving growth performances of seedlings are discussed.     

HORMONAL CONTROL OF PEROXIDASE ACTIVITY IN CULTURED PELARGONIUM PITH

Freshly excised Pelargonium pith tissue lacks peroxidase activity toward guaiacol or benzidine, but it develops such activity within 24–36 hr in aseptic culture. All the activity is manifested as a single enzyme moving toward the cathode during electrophoresis on starch gel at pH 9.0. This development of peroxidase activity is at first (up to ca. 50 hr in culture) inhibited and later (ca. 100–150 hr in culture) promoted by IAA. This dual effect of IAA resembles that previously reported for specific isoperoxidases in tobacco pith cells. Kinetin alone also inhibits peroxidase formation, but in the presence of IAA those concentrations which enhance growth enhance peroxidase formation as well.     

Development of the resting sporangia ofSynchytrium endobioticum,the causal agent of potato wart disease

Summary An ultrastructural study of the development of the resting sporangium ofSynchytrium endobioticum (Schilb.) Perc. infecting potato cells is presented. The resting sporangium is found to have a single large, centrally placed nucleus with a prominent nucleolus through its entirein situ development. The cytoplasmic organization of the resting sporangium is further characterized by numerous membrane-bound lipid bodies and osmiophilic bodies. The latter have a characteristic sieve-like appearance, probably because certain storage components have been extracted during preparation for electron microscopy. Because of the similar location and appearance of these osmiophilic bodies it is suggested that they are identical to what has earlier (based on light microscopy) been described as chromatin granules; and the ultrastructural studies presented here show that nucleolar discharge which was described from light microscopic observations as leading to chromatin granules in the cytoplasm, and finally forming the nuclei of the zoospores (bally 1912,curtis 1921,percival 1910) simply does not occur.The appearance of dense fibrillar-like structures on the sporangial surface at an early stage of resting sporangium development ultrastructurally distinguishes the resting sporangium from the zoosporangium. The development of the layered portion of the thick sporangial wall is shown to be due to the fusion of vacuoles containing pre-made wall fibrils with the cell membrane. It is suggested that the inner compact wall layer which is essentially substructureless is formed by the membrane itself.The characteristic wings of the matureS. endobioticum resting sporangium originate from the potato host cell wall. Remnants of host cell organelles in the outermost layer of the resting sporangium wall show that degradation of the host cell cytoplasm contributes to wall formation of the parasite.     

FUNGI FROM THE LOWER DEVONIAN RHYNIE CHERT: CHYTRIDIOMYCETES

Several different chytridiomycetes are described from the Lower Devonian (Siegenian) Rhynie chert. Included are both eucarpic and apparently holocarpic forms that occur in Palaeonitella, Aglaophyton, Lyonophyton, Horneophyton, and clusters of algal cells, as well as in the surrounding chert matrix. Holocarpic types consist of endobiotic sporangia, each characterized by one discharge tube. Sporangia can be traced from the thallus stage to the discharge of zoospores. Monocentric and polycentric eucarpic chytrids are associated with the miospores of Aglaophyton and various thick-walled fungal spores. In these forms the sporangia are variable in size and shape ranging up to 30 μm. Most appear to be inoperculate and there is evidence that the sporangium ruptured on the distal surface. Some contain zoospores with flagella. One operculate eucarpic form had parasitized the cellular gametophyte emerging from the proximal surface of an Aglaophyton spore. Several of the Rhynie chert chytrids are comparable with a number of extant forms (e.g., Olpidiaceae and Spizellomycetaceae), while others possess features that encompass several groups. These fossil fungi are discussed in the context of their interactions with other organisms in this Lower Devonian freshwater paleoecosystem.     

Analysis of growth and rotational behavior of sporangiophores in Pilobolus crystallinus (Wiggers) Tode

The growth and rotation of the sporangiophore of Pilobolus crystallinus, which are important factors in its phototropic behavior, were analyzed throughout its development. The sporangiophore initial emerged from the trophocyst and elongated at the extreme tip without rotating. The elongation rate of the sporangiophore apex then gradually decreased and the apex expanded radially to produce the sporangium, but no rotation occurred. A transient cessation of elongation after sporangium development was followed by resumption of both elongation and radial expansion in the region beneath the sporangium developing the subsporangial vesicle. Rotation was not obvious at this stage. Radial expansion of the subsporangial vesicle continued at a decreasing rate until full size was reached. Elongation then recommenced in the newly established growth zone in the upper region of the sporangiophore just beneath the subsporangial vesicle. During this period of growth, the sporangiophore rotated in a clockwise direction as viewed from above. All growth and rotation ceased about 1 h before ejection of the sporangium into the air. Based on these results, a modified classification of the developmental stages has been proposed.This work was carried out under the Joint Research Program of the Institute of Genetic Ecology, Tohoku University, Japan (892006). The authors please to thank Kaori Koga and Hiroko Kikuchi for their helpful assistance.     

Germination of the resting sporangia ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The structural and developmental characteristics of the resting sporangium in uniflagellate phycomycetes, together with the type of zoospore, are of high taxonomic value. Among these fungi, however, only a few electron microscopic investigations have been published on this topic, mainly due to technical problems. In the present study ofPhysoderma maydis (Blastocladiales) these problems were overcome as the resting sporangia in this species are formed synchronously, in large numbers, the germination is readily induced and the impermeability of the resting sporangium wall can be circumvented by shaking the prefixed sporangia with glass beads.The germination of the resting sporangia ofP. maydis is described by correlative light and electron microscopic studies and discussed in relation to related investigations on sporogenesis: The germination process starts by a breakdown of large electron-dense accretions found in the resting stage. Simultaneously, the peripheral location of the lipid bodies is lost. The large operculum is pushed open by a protrusion of the inner sporangial wall; an additional wall layer is formed during this process. Synaptonemal complexes are found in the nuclei at this stage, as are nuclear division figures which suggests anEuallomyces type of life cycle for this fungus. Cleavage vesicles, formed from dictyosomes or endoplasmic reticulum, ultimately separate the sporangial content into meiospores. The sequential assembly of organelles into the side body complex is described. Sequestering of the ribosomes into a nuclear cap is interpreted as taking place immediately prior to zoospore discharge.     

TWO NEW MEMBERS OF THE BLASTOCLADIACEAE. II. MORPHOGENETIC RESPONSES TO O2 AND CO2

The development of Microallomyces dendroideus and Allomyces reticulatus was studied under various mixtures of O₂, CO₂ and N₂. All thalli grown in broth or agar media in air produce abundant resistant sporangia but rarely produce any zoosporangia. In both fungi, the proportion of zoosporangia borne by the thalli increases as the oxygen level decreases. In addition, increased CO₂ strongly stimulates resistant sporangium germination in M. dendroideus. Neither fungus grows in the absence of oxygen; therefore, they seem to be adapted for rapid multiplication in a microaerophilic niche.     

The antifungal protein AFP from Aspergillus giganteus prevents secondary growth of different Fusarium species on barley

Secondary growth is a common post-harvest problem when pre-infected crops are attacked by filamentous fungi during storage or processing. Several antifungal approaches are thus pursued based on chemical, physical, or bio-control treatments; however, many of these methods are inefficient, affect product quality, or cause severe side effects on the environment. A protein that can potentially overcome these limitations is the antifungal protein AFP, an abundantly secreted peptide of the filamentous fungus Aspergillus giganteus. This protein specifically and at low concentrations disturbs the integrity of fungal cell walls and plasma membranes but does not interfere with the viability of other pro- and eukaryotic systems. We thus studied in this work the applicability of AFP to efficiently prevent secondary growth of filamentous fungi on food stuff and chose, as a case study, the malting process where naturally infested raw barley is often to be used as starting material. Malting was performed under lab scale conditions as well as in a pilot plant, and AFP was applied at different steps during the process. AFP appeared to be very efficient against the main fungal contaminants, mainly belonging to the genus Fusarium. Fungal growth was completely blocked after the addition of AFP, a result that was not observed for traditional disinfectants such as ozone, hydrogen peroxide, and chlorine dioxide. We furthermore detected reduced levels of the mycotoxin deoxynivalenol after AFP treatment, further supporting the fungicidal activity of the protein. As AFP treatments did not compromise any properties and qualities of the final products malt and wort, we consider the protein as an excellent biological alternative to combat secondary growth of filamentous fungi on food stuff.     

The genus Blastomycoides as a mycological entity

Summary Studies were made on some environmental factors promoting yeast-phase growth ofBlastomycoides in chemically defined media. With inclusion of different tryptophane desaminase inhibitors, pure yeast phase of growth was obtained. The same factors facilitate the development of perfect sexual forms and the observation of the cytological characteristics. Therefore,Blastomycoides, represented byBlastomyces dermatitidis, Bl. brasiliensis, Bl. cerolytica andBl. immitis (formerlyCoccidioides immitis), form a special genus which is very near to the Protomyces and represents a transitory phase between the Endomycetales and the Taphrinales. Although the vegetative mycelium of the Blastomycoides resembles the Aleurismaceae, they cannot be classified as such, especially because they are dimorphic fungi (filamentous-yeast) and their sexual forms are characteristic asci on a very primitive level.Blastomycoides not only represent a group of fungi causing grave visceral mycoses (blastomycoses) but they also are an indiscutable mycological entity, as yet represented in the genusBlastomycoides     

Comparison of plant and fungal gravitropic responses using imitational modelling

The mechanisms of gravity perception are still hypothetical, but there are sufficient data from experiments with plants to enable mathematical modelling to imitate the behaviour of gravitropic response systems. We have a much less complete picture of gravitropic kinetics in agaric mushrooms. However, some existing mathematical models which imitate plant responses are in principle universal because their conceptual components are not limited to any specific cellular entities. In this work we have used such models to compare plant and fungal gravitropism, using recently acquired kinetic data from the agarics Coprinus cinereius and Flammulina velutipes. The results show striking similarities between plants and fungi. First, it is evident that the basic assumptions of the plant models are logically applicable to fungi. Secondly, the mechanism of bending is the same (differential growth of opposite flanks of the growing organ). Thirdly, the distribution of growth seems very similar: in both plants and fungi growth of the organ is most intensive just behind the apex and is almost absent at the apex and at the base. Fourthly, in both fungi and plants the gravitropic response exhibits a substantial time delay suggesting that many time-consuming processes are involved in reception, transduction and realization of gravitropic stimuli. Important differences in plant and fungal gravitropism kinetics were: (i) the agaric stem apex always returned to the vertical, whereas some plant organs show stable plagiogravitropic growth; (ii) inflections were usually seen in C. cinereus stem gravitropism time courses suggesting that a curvature compensation process delayed bending for a time; (iii) C. cinercus stems very rarely overshot or oscillated around the vertical although many plant subjects oscillate and the (limited) data for F. velutipes showed a single, exaggerated overshoot and oscillation. In this latter case, experimental modelling with parameters characteristic of a low level of perception improved the fit to the F. velutipes data, indicating that the two fungi may differ in this factor. Application of the plant models focused future research attention on the urgent need for data bearing on angle-response and acceleration–response relationships in fungi, and their detection–level thresholds for gravitational acceleration. Since the modelling also highlighted some fundamental kinetic differences between the only two fungi for which sufficient data are available at the moment, it is also clear that detailed observations need to be made of gravitropism kinetics in a larger number and wider range of fungi.