New photoresponses of phycomyces

The influence of light on asexual fruiting and mycelial growth of Phycomyces blakesleeanus Burgeff was studied by means of fruiting body counts and size measurements in cultures on solid media under varied incubation conditions. Five types of photoresponses were shown by ATCC Strain 8743a: (a) photoinduction of giant sporangiophores; (b) interference by light with an endogenous system that otherwise induces fruiting when the mycelium approaches the rim of the Petri dish; (c) inhibition of mycelial growth rates by light; (d) inhibition of dwarf sporangiophore induction by light; and (e) postponement by light of death in clones maintained by serial transfer at low temperature. A second strain, designated G5, showed responses comparable to (a), (b), and (d). The magnitudes of the responses were greatly affected by temperature of incubation and available nitrogen (asparagine) supply. The photoinduction of giant sporangiophores could be demonstrated with light of wavelengths between 380 and 480 nanometers but not with 520 nanometers or above. At 480 nanometers, light doses as small as 40 ergs per square centimeter were effective in inducing giant sporangiophores in strain 8743a.     

The sensitive period for light and temperature regulation of sporangiophore development inPhycomyces

Light and temperature markedly influence sporangiophore development inPhycomyces blakesleeanus. Under normal conditions in the dark, low temperature drastically stimulates the production of dwarf sporangiophores (microphorogenesis) and inhibits that of giant sporangiophores (macrophorogenesis). These effects of low temperature could still be observed if applied only for a short period before sporangiophore initiation. Continuous white illumination strongly inhibits microphorogenesis and slightly stimulates macrophorogenesis. Short exposures to white light noticeably inhibit microphorogenesis and stimulate macrophorogenesis when given to mycelia grown for between 90 and 160 h at 14° C or 150 h or more at 10° C. These results indicate the existence in the mycelium of developmental stages for the regulation of sporangiophorogenesis by environmental signals.     

Vegetative regeneration on sexual organs inPhycomyces blakesleeanus

Phycomyces blakesleeanus produced an abundance of sexual organs when two mating types met on solid medium, but only about 14.7% of the sexual organs developed to the final stages. On the sexual organs showing arrested development, vegetative hyphae or dwarf sporangiophores (microphores) often regenerated. This vegetative regeneration was accelerated when the paired and looped progametangia were isolated from mycelia, when the counterparts of the progametangial cells constructing the loop were surgically incised, and whenPhycomyces was mated at high temperature (25–27°C). A leaky-carotenogenic mutant, whose sexual reaction was imperfect and arrested at an intermediate stage even when mated with the wild type, also regenerated hyphae with a high frequency on these arrested intermediate organs. The vegetative regeneration seems to result from interruption of a cell-to-cell recognition system between cells of different mating type, which is believed to be essential for the mating process of this fungus in addition to the pheromonal actions.     

EXPERIMENTAL CONTROL OF FRUIT-BODY FORMATION IN COPRINUS MACRORHIZUS

In order to establish a proper experimental system for studying the physiological and biochemical processes of fruit-body formation in a Basidiomycetes, Coprinus macrorhizus Rea f. microsporus Hongo, various culture conditions, especially light and temperature, were investigated. The use of homogenates of fruit-body primordia, instead of mycelia, as inocula was effective for maintaining cultures capable of active fruit-body formation during many successive cultivations. At least two successive intermittent illuminations every 12 hr during the 3rd and 4th days of incubation were required for the differentiation of pilei. The most effective wave lengths of incident light were found in the region from 440 to 485 mμ. Temperature effects on fruit-body formation were exhibited in two stages of morphogenesis; i.e., in the induction of primordia formation from vegetative hyphae (by 5°-treatment) and in the transition phase from primordia formation to fruit-body development (by 20°-treatment). Based on these observations, three experimental systems for physiological and biochemical studies on the fruit-body formation have been proposed.     

Formation of atypical fruiting structures inGanoderma lucidum isolates on a nutrient agar medium

Effects of light and ventilation on the formation of atypical fruiting structures (AFSs) and fruit body primordia (FBPs) ofGanoderma lucidum on nutrient agar media were investigated. Although the mycelial growth was inhibited by illumination and ventilation, brown AFSs appeared on the white mycelial colony, and basidia and basidiospores were produced on the AFSs. On the other hand, FBPs were induced by illumination alone, regardless of ventilation. However, the primordia could not develop to mature fruit bodies. In the dark, only vegetative growth of the fungus progressed. Twenty-three isolates ofG. lucidum collected from four countries were tested for the formation of AFSs and FBPs under light and ventilation. Thirteen isolates formed AFSs, and another five isolates produced FBPs. Of the remaining five isolates, one formed callus-like structures without elaborating basidiospores, and the other four did not induce AFSs or FBPs. Microscopical observation showed that the basidia were formed directly from generative hyphae on the surface of AFSs. Basidiospores formed on the basidia were brown and ellipsoid with an eccentric hilar appendix on the rounded spore base. They had a double wall and mostly contained one or two large vacuoles. The surface of basidiospores was smooth or wrinkled and had shallow holes. The spore size was (4.5–)6.4–9.6(–10.3) × (2.6–)3.2–5.1(–6.4), 7.3 × 4.2 µm on average.     

Mutants ofPhycomyceswith Decreased Gallic Acid Content

Weinkove, D., Poyatos, J. A., Greiner, H., Oltra, E., Avalos, J., Fukshansky, L., Barrero, A. F., and Cerdá-Olmedo, E. 1998. Mutants ofPhycomyceswith decreased gallic acid content.Fungal Genetics and Biology25, 196–203. Most plants and some fungi accumulate phenols. Two hydroxybenzoic acids, gallic and protocatechuic acids, are abundant in the giant sporangiophores of the zygomycetePhycomyces blakesleeanus,much more so than in the basal mycelium or the culture medium. The actual concentrations vary with illumination, age of the culture, and composition of the medium. We devised a simple screening procedure to isolatehbamutants whose sporangiophores contained less gallic acid than the wild type. The most useful mutant had very low concentrations of hydroxybenzoic acids in the sporangiophores, but about the same as the wild type in the basal mycelium and the medium. The mutant was only slightly different from the wild type in growth and morphology. Mutant and wild-type sporangiophores grew away from ultraviolet C sources (260 nm) equally well. Contrary to previous conjectures, ultraviolet tropism does not depend on the ultraviolet absorption of gallic acid or other free hydroxybenzoic acids in the sporangiophore. Against expectations, phenols did not impair DNA extraction: sporangiophores produced better DNA preparations than basal mycelia and thehbamutant only slightly better than the wild type.     

Nuclear fusion,meiosis and the origin of dikaryotic hyphae in Armillariella mellea

The behaviour of nuclei during the growth and differentiation of basidiocarp primordia of Armillariella mellea (Vahl) Karst. is described. The primordial initials which arose from monokaryotic rhizomorphs were also monokaryotic. In older primordia, at the site of initiation of gill folds, multinucleate cells formed at the tips of monokaryotic hyphae and gave rise to the dikaryotic hyphae bearing clamp connections. These formed the gills of the older primordia. Cytological studies suggested that the nuclei in monokaryotic cells were diploid. In young basidial primordia haploidization occurred in the cells which were to become multinucleate prior to giving rise to dikaryotic hyphae of the gills. In mature basidia after nuclear fusion and meiosis had occurred, each of the four haploid daughter nucleic migrated into a basidiospore and then divided mitotically. One of the mitotic daughter nucleic migrated from each spore back into the basidium so that mature spores were uninucleate.Abbreviations M.T.O.C. microtubule organizing centre     

Crystalloids of phycomyces sporangiophores: nature and photosensitive accumulation

Crystalloids accumulate in the vacuoles of the giant sporangiophores of Phycomyces blakesleeanus Burgeff during growth. On the basis of solubility in alkaline solutions, cytochemical staining reactions, trypsin sensitivity, optical absorption and response in the Lowry protein test, the crystalloids have been judged to consist principally of an acidic protein. In assays by Lowry test and by reference to optical absorption at 280 mμ, dark-grown sporangiophores were consistently found to contain from 2 to 4 times as much crystalloid material as light-grown counterparts. Concurrent assays of soluble phenolic materials revealed no significant effect of culture illumination, while carotene content of sporangiophores and mycelium was found to be raised from 2 to 4-fold by illumination during growth.     

Microcycle speculation in theClaviceps purpurea 244

The mutant strainClaviceps purpurea 244 forming hyphae composed mainly from sclerotiumlike cells was found to sporulate both in liquid and solid media, particularly in the form of terminal chlamydospores (4.0 × 6.5 μm). Chlamydospores produced during submerged cultivation germinated, new chlamydospores being formed directly from germ tubes, or, occasionally, conidia (the so-called microcycle) or new vegetative mycelium were formed. The ultrastructures of the chlamydospores and vegetative cells was identical. The cytoplasm was filled with ribosomes and contained lipid inclusions and vacuoles with membrane invaginations. Strain 244 cultivated under submerged conditions produced 150 μg/ml clavins, with elymoclavin predominating (82 %). The parent strainClaviceps purpurea 129 only produced chlamydospores on the vegetative mycelium, whereas no microcycle was detected; under submerged conditions it produced mainly agroclavin (85 %) at a concentration of 4 mg/ml.     

Morphological and cytological aspects of oidium formation in a basidiomycete,Pholiota nameko

Pholiota nameko produced abundant oidia on aerial hyphae from monokaryotic and dikaryotic test stocks, but oidia were rare on submerged hyphae. The oidia from the former stocks had a layer of hydrophobic protein between the cell wall and the inner cell membrane which was absent in the oidia from the latter. The only remarkable differences in the morphological features of the oidia from monokaryotic and dikaryotic mycelia was the slightly larger size of the latter. Observation of various test stocks on slide cultures revealed that about 80% of oidia were produced from the secondary branched hypha, and about 20% from the terminal hyphal, cell of the main hypha. In the former, the secondary hyphae were segmented to form several oidium cells; in the latter, a single or several oidia were formed at the terminal end of the main hypha. Most oidia from monokaryons and dikaryons had only one haploid nucleus, while the remainders were multinucleate. Among the stocks tested, most oidia had a DNA content with a haploid amount at the G1 phase of the cell cycle, but a few contained twice that amount corresponding to the G2 phase     

In vitro evidence of mycoparasitism of the ectomycorrhizal fungus <Emphasis Type="Italic">Laccaria laccata</Emphasis> against <Emphasis Type="Italic">Mucor hiemalis</Emphasis> in the rhizosphere of <Emphasis Type="Italic">Pinus sylvestris</Emphasis>

Interactions between the ectomycorrhizal fungus Laccaria laccata and the soil fungus Mucor hiemalis f. hiemalis in co-culture, and in the rhizosphere of in vitro-grown Pinus sylvestris seedlings were investigated by light- and scanning electron-microscopy. In co-culture, mycelial growth away from the L. laccata colony reduced the number of aerial hyphae at the contact zone and increased the density and compactness of the mycelium-characterized gross morphology of the saprobic fungus. Although the growth of M. hiemalis was suppressed, no penetration of M. hiemalis hyphae after the colony was entered by L. laccata was observed. Instead, dense coiling of L. laccata hyphae around sporangiophores, overpowering them and causing them to disappear, was quite common. On nonmycorrhizal roots, sporangiospores germinated heavily and formed long hyphae for 2 days post inoculation, whereas their germination was totally inhibited on mycorrhizal roots. At 3 days after inoculation, only sporangia were seen with mycelial mats firmly attached to the roots by the mantle hyphae, whereas some remnants of sporangiophores, ruptured sporangial walls and degraded hyphae of M. hiemalis were overgrown by the mantle hyphae. During the next 3 days, the mantle-hyphae-invading sporangia formed short, thin branches that grew directly towards individual spores, tapering off upon contact.     

MORPHOLOGY AND CYTOLOGY OF TILLETIA CARIES AND T. CONTROVERSA IN AXENIC CULTURE

On a wheat-based medium, the pathogenic phase of the common and dwarf bunt fungi grew slowly at 15–18 C and continued to produce massive quantities of teliospores in all subcultures for over 2 years. At warmer temperatures or on a chemically defined medium, the teliosporogenic colonies reverted to haploid mononucleate colonies. The hyphae of the teliosporogenic colonies were stained with a modified Giemsa technique and found to be thick, contorted, highly branched and short celled with usually one or two nuclei per cell. In contrast, the haploid mononucleate hyphae were thinner, straighter, and longer and never contained more than one nucleus per cell. The vegetative hyphae of T. caries and T. controversa were indistinguishable. Teliospores, formed at the terminal end of binucleate hyphae, were initially binucleate but became mononucleate before the mature cell wall formed.     

DIFFERENTIATION OF THE GREEN ALGA CODIUM FRAGILE

Clonal cultures of Codium fragile were established from both swimming cells and vegetative filaments. In the laboratory axis primordia differentiate from heterotrichous juveniles only when cultures are agitated on a reciprocating shaker. The shear forces created by mechanical agitation are essential both for initiation and maintenance of primordia. Contact guidance of growing coenocytic filaments indicates mutual adhesion of filaments as the basis for the differentiation process.     

Growth of fruit-bodies inFavolus arcularius

The fruiting ofFavolus arcularius in culture is described. When the cultures, which have been pre-incubated in darkness to allow the inoculum mycelia to become thick and white wooly in texture, are exposed to light, fruit-body primordia, 1 mm in height, are formed about 4 days after the start of illumination. The primordium develops into a cylindrical stipe, the growth of which mainly occurs in the final 1 mm of the terminal region. Hyphal elongation in the region within 3 mm of the apex is predominant in the growth of the pileate stipe. With maturation of the stipe, changes in hyphal orientation occur on the periphery of the subapical region, and then the pileus-primordium is formed. The differentiation into the inner layer and the outer layer (pre-hymenial layer) in the pileus tissue is completed at this stage. The early growth of the pileus may be due to rapid elongation of the hyphae on the margin in addition to gradual expansion of the hyphae in the preformed pseudo-tissue. When the pileus has grown to about 3 mm in diameter, the subsequent three to four fold increase in size may be due to parallel expansion of the hyphae constituting the young pileus tissue.     

The Nature of Systemic Invasion of Stem and Leaves of Sunflowers by Plasmopara helianthi Novot. var. helianthi Novot. with Mechanism of Sporulation and Zoospore Release

During the systemic development of Plasmopara helianthi Novot. var. helianthi Novot. the hyphae in the stem advance especially through the intercellular spaces of loose parenchyma forming haustoria in adjacent cells. The hyphae which reach leaf blades through petioles continue their growth along the main veins intercellularly in non-vascular tissues enclosing the vascular bundles. At the same time, the hyphae spread to intercellular spaces of spongy parenchyma where the growth is limited by the veinlets resulting in angular chlorotic lesions. Under humid conditions the sporangiophores arising from the aggregated hyphae in a substomatal cavity emerge through the stomatal pore on the lower side of the leaf and zoosporangia are borne terminally on sporangiophores. Soon after biflagellated zoospores are liberated into distilled water from the zoosporangia, they retract their flagellae and then lyse.     

Fixation technique to preserve the vacuolation ofPenicillium chrysogenum hyphae

Summary It has been shown that a paraformaldehyde/glutaraldehyde/phosphate buffer saline fixative can be used to preserve vacuolation ofPenicillium chrysogenum hyphae from submerged cultures for later image analysis characterisation. After fixing, the proportion of the hyphase as vacuoles and empty cells (in several samples from different fermentations with different initial percentage vacuolations) did not change significantly over about 70h, and there were only minor changes towards smaller vacuoles in the vacuolation size distributions over the same period. This fixation method will permit the storage of vacuolated hyphae, for later analysis either for physiological studies or for dynamic studies of the links between hyphal vacuolation and fragmentation.     

ROOT GLOCHIDS AND ROOT SPURS OF OPUNTIA ARENARIA (CACTACEAE)

Root glochids of Opuntia arenaria Engelm. are produced by adventitious buds (areoles) that arise endogenously on fleshy roots. If they remain active, root areoles become dwarf shoots that initiate only glochids and trichomes unless the roots are uncovered or the shoots bearing the roots are removed. Then they will expand, producing aerial shoot joints. Root spurs are formed in great numbers on the profusely branched system of small roots. They consist of clusters of rootlets about one mm long that are initiated in sympodial sequence, each new rootlet arising endogenously near the base of the preceding one. On older spurs this results in the formation of a short axial peg. Rootlet primordia lack a root cap from the beginning and appear to mature quickly, losing all their meristematic characteristics and becoming completely covered with root hairs, even over the tip. They may represent a mechanism for the rapid production of root hairs during the infrequent periods when moisture is available. In any case, they seem to be transitory structures because cork soon forms beneath the older ones on a spur.     

Cytology of Thamnidium elegans link

The resting nuclei in hyphae, sporangiophores and sporangiospores of sporangia and sporangiola of Thamnidium elegans consist of a large centrals nucleolus and a shell of chromatin surrounding the nucleolus. Division of the nucleus in hyphae and sporangiospores is achieved by elongation and constriction.     

Differentiation and anaerobiosis in standing liquid cultures of Streptomyces coelicolor

Streptomyces coelicolor differentiates on solid agar media by forming aerial hyphae that septate into spores. We here show that differentiation also occurs in standing liquid minimal media. After a period of submerged growth, hyphae migrate to the air interface, where they become fixed by a rigid reflecting film. Colonies that result from these hyphae form sporulating aerial hyphae. In addition, submerged hyphae in the liquid minimal medium may attach to the surface. Liquid standing cultures easily become anoxic only 1 to 2 mm below the surface. Yet, biomass increases, implying the existence of metabolic pathways supporting anaerobic growth.     

cAMP promotes hyphal branching in <Emphasis Type="Italic">Mucor globosus</Emphasis>

The growth pattern of Mucor globosus cultured on a medium with or without cyclic adenosine monophosphate (cAMP) was examined. Branching remarkably increased in a mycelium grown on an agar medium containing cAMP. In submerged culture containing cAMP, some sporangiospores grew spherically and formed yeast-like cells, and others showed hyphal growth. These hyphae showed septation and swelling and formed spore-like structures. When these hyphae were transferred to cAMP-free medium, a germ tube emerged from each compartment. These results show that cAMP has two different effects on the development of hyphae: one is the promotion of branching, and the other is the suppression of polarized growth.