Sporangiospores of the fungus Mucor lusitanicus 12M: correlation between lipid composition, viability, and morphology of growth upon germination

We studied viability of sporangiospores from a surface culture of the fungus Mucor lusitanicus 12M grown on wheat bran. With culture ageing, the sporangiospores exhibited a tendency toward dimorphic growth upon germination and then lost the ability to germinate. This correlated with changes in the sporangiospore lipid composition, which involved a reduction in the total lipid pool and in the levels of reserve lipids and phospholipids in particular. We suggest that lipid catabolism in sporangiospores causes their defectiveness in the senescent culture.     

Dormant Cells in the Developmental Cycle of Blakeslea trispora: Distinct Patterns of the Lipid and Carbohydrate Composition

We revealed differences in lipid and carbohydrate composition between cells of mucorous fungi during endogenous and exogenous dormancy. Endogenous dormancy (zygospores) is characterized by high contents of phosphatidylcholine (about 70% of the total phospholipids) and triacylglycerol (over 90% of the total neutral lipids). By contrast, exogenous dormancy (sporangiospores) is accompanied by elevated amounts of sterols, sterol esters, and free fatty acids, which account for over 70% of the total neutral lipids. We established for the first time significant differences in the phospholipid composition between sporangiospores obtained from stylosporangia and sporangioles. Based on the data obtained, we regard the retardation of life-sustaining activities as a biochemical adaptation based on the dormancy state. We also discuss the taxonomical position of Blakeslea trispora.     

Sterols of the fungus Mucor hiemalis sporangiospores

We earlier reported that the viability and lipid composition of Mucor hiemalis F-1156 sporangiospores, as well as their capacity to develop yeast-like cells, depend on the age of the spore-forming culture [1]. With the increase in the time of the culture growth, sporangiospores exhibit a decrease in the levels of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, cardiolipin, and reserve lipids, as well as in the degree of fatty acid unsaturation, whereas the level of glycolipids increases. However, sterol composition was not been studied. Sterols are involved in morphogenetic processes [2, 3], and therefore we suggested that sporangiospores from senescent cultures, which, upon germination, develop mycelium and yeast-like cells, may exhibit a distinctive sterol pattern, along with the changed composition of fatty acids and polar and neutral lipids.     

Changes in the Lipid Composition of Mucor hiemalis Sporangiospores Related to the Age of the Spore-Forming Culture

Analysis of sporangiospore lipids of the fungus Mucor hiemalis F-1156 showed that alterations occur in the content of fatty acids and individual classes of lipids during long-term cultivation (for about 20 days). The changes in the lipid composition related to the age of the spore-forming mycelium suggest an important role of sporangiospore lipids in spore germination and in further development of the spherical cells formed in this processes. The M. hiemalis F-1156 sporangiospores with a lipid pool exhausted during long-term cultivation can give rise to both mycelial and yeastlike growth.     

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.     

Pinnaticoemansia, a new genus of Kickxellales, with a revised key to the genera of Kickxellales

Pinnaticoemansia coronantispora gen. sp. nov. (Kickxellaceae, Kickxellales) is described and illustrated. This species is characterized by imparipinnate sporocladia (sporocladia arranged in pairs), sporangiospores with a three-lobed corona, and the germination pattern of sporangiospores with downward hyphal growth and repeated dichotomy. The key to the genera of Kickxellales by Kurihara et al. (2001) is revised based on the observation of the germination pattern of this fungus in comparison with that of Asellariales.     

Impact of morphogenetic effectors on the growth pattern and the lipid composition of the mycelium and the yeastlike cells of the fungus Mucor hiemalis

We investigated the growth and the cell lipid composition of the mycelium and of the yeast-like form of Mucor hiemalis VKMF-1431 obtained under aerobic conditions by treatment with the morphogenetic agents itraconazole, exogenous triacylglycerols (TAGs), and trehalose. The sporangiospores of a 20-day culture were inoculated on the medium with glucose. Under these conditions, the fungus produced both mycelium and yeast-like cells. It was established that, upon the germination of old (20-day) sporangiospores, the fungus predominantly used the mycelium development strategy in the presence of trehalose and TAGs. It was characterized by a low ratio between the two bulk membrane lipids (PEA/PC) and increased levels of PC and polyunsaturated fatty acids (FA). Compared to the mycelium, the yeast cell morphotype obtained on the medium with glucose was distinguished by an elevated PEA/PC ratio, lowered TAG, free sterol (FS) and esterified sterol (ES) levels, a decreased ES/FS ratio that correlated with the reserve sterol pool size, and a lowered content of unsaturated fatty acids (the linoleic and the ??-linolenic acid). These peculiarities of the lipid composition of yeastlike cells correlated with the intensity of yeastlike growth. Light and electron microscopy revealed differences between the above cell morphotypes. With itraconazole, yeast-like cells were characterized by the destruction of the endoplasmic reticulum membranes and formation of a large number of vacuoles. The suggestion was confirmed that the state/age of inoculum sporangiospores exerts an influence on the capacity for dimorphism in mucorous fungi such as M. hiemalis. The data obtained testify to an involvement of lipids in the process of adaptation to environmental factors and to their regulatory role in morphogenetic processes associated with the formation of alternative morphotypes of the mucorous fungus.     

Dormant cells in the developmental cycle of Blakeslea trispora: distinct patterns of the lipid and carbohydrate composition

We revealed differences in the lipid and carbohydrate composition between cells of mucorous fungi during endogenous and exogenous dormancy. Endogenous dormancy (zygospores) is characterized by high contents of phosphatidylcholine (about 70% of total phospholipids) and triacylglycerol (over 90% of total neutral lipids). By contrast, exogenous dormancy (sporangiospores) is accompanied by elevated amounts of sterols, sterol esters, and free fatty acids, which account for over 70% of total neutral lipids. We established for the first time significant differences in the phospholipid composition between sporangiospores obtained from stylosporangia and sporangioles. Based on the data obtained, we regard the retardation of life-sustaining activities as a biochemical adaptation based on the dormancy state. We also discuss the taxonomical position of B. trispora.     

Effects of conditions for obtaining sporangiospores of the inoculum on the morphology and productivity of the fungus Mucor circinelloides var. lusitanicus 12 M, a producer of gamma-linolenic acid

Effects of lipid composition of sporangiospores of the fungus Mucor circinelloides var. lusitanicus 12 M, obtained within diverse time frames using distinct nutritive media, on the morphology of the fungus in submerged cultures, the yield of the biomass, and its content of gamma-linolenic acid have been studied. The levels of base phospholipids and individual fractions of neutral lipids in sporangiospores were correlated with the character of their germination. The spores characterized by a high rate of germination and giving rise to a well-developed mycelium contained more phosphatidylcholine and phosphatidylserine, but the level of diacylglycerols was low. An increase in diacylglycerols, free fatty acids, and sterols in lipids of sporangiospores of the inoculate was associated with deterioration of the mycelium development, dimorphism, and lowering of the yield of the biomass of the fungus.     

Lipid composition of the mycelium of the fungus Mucor hiemalis cultivated with trehalose, triacylglycerols, and itraconazole

We investigated the growth and cell lipid composition of the fungus Mucor hiemalis VKMF-1431 cultivated under aerobic conditions in the presence of the morphogenetic agents itraconazole, exogenous triacylglycerols, and trehalose. The sporangiospores of a 6-day culture were used as inocula. Under these conditions, the fungus produced mycelium; nevertheless, solitary yeastlike cells also developed on the glucose-containing medium and in the presence of itraconazole and sterilized triacylglycerols (sTAGs). No yeastlike growth occurred in the system with trehalose and with unsterilized (native) TAGs (nTAGs). With trehalose and nTAGs in the cultivation medium, the ratio between PEA and PC, the two main types of membrane lipids, was low. This testified to a relatively high PC percentage and, accordingly, a stable structure and a highly functional state of the membranes. Moreover, if the development of the fungus occurred exclusively as mycelium formation, the level of polyunsaturated fatty acids (??-linolenic and arachidonic acid) increased in the presence of trehalose and that of linoleic acid increased in the presence of nTAGs. These results may suggest that unsaturated fatty acids and membrane lipids are related to the cell wall formation and the implementation of morphogenetic programs in mucorous fungi.     

Lipid and elemental composition as indicators of the physiological state of sporangiospores in Mucor hiemalis cultures of different ages

The number of sporangiospores used as inocula may significantly affect the development of the morphogenetic programs in mucoraceous fungi, including manifestations of dimorphism. In order to assess the physiological state of sporangiospores differing in their viability and germination patterns, lipid composition of sporangiospores from 6-, 14-, and 20-day cultures of Mucor hiemalis F-1431 was determined and the element ratios in these spores (Ca/K and P/S) were measured using X-ray microanalysis. While the lipid composition of the “old” (20 days) and “young” (6 days) spores was generally similar, older spores contained no cerebrosides, had half the level of polar lipids and γ-linolenic acid, and also contained 6 times more monoacylglycerols and 2.5 times more phosphatidic acid. The ratio of esterified to free sterols (ES/FS) characterizing the sterol storage pool was lowest in the spores from 20-day cultures. X-ray microanalysis revealed the highest P/S ratio in 6-day spores and lowest ratio in 14-day ones. Mature 14-day spores had the highest Ca/K ratio, ten times exceeding that for the 20-day spores. Higher values of P/S and Ca/K ratios in young (ripening) spores than in old 20-day spores indicate their higher metabolic activity and correlate with their higher viability and mycelial type of germination. Together with the lipid characteristics, the Ca/K and P/S ratios are the parameters which may be used to develop the criteria for assessment of the physiological state of the cells, including viable fungal sporangiospores. This complex may be also used to assess the capacity of mucoraceous fungi for dimorphism, including the species (M. hiemalis) for which this capacity has not been demonstrated previously.     

Influence of growth medium on surface and wall lipid of fungal spores

Conidiospores of Alternaria tenuis, Botrytis fabae, Neurospora crassa and sporangiospores of Rhizopus stolonifer from cultures on various media were shown by microelectrophoresis to have lipid on their surface. Analyses of lipid fractions obtained by sequential solvent extraction demonstrated that surface lipid forms a small but discrete layer different in composition from that within the wall. Free fatty acids, alkanes, triglycerides and other acyl lipids were identified by GC-MS. Phospholipids and sterols were absent. The qualitative and quantitative composition of both the surface and wall lipid fractions was dependent upon the growth medium used.     

Effects of conditions for obtaining sporangiospores of the inoculum on the morphology and productivity of the fungus Mucor circinelloides var. lusitanicus 12 M, a producer of γ-linolenic acid

Effects of the lipid composition of sporangiospores of the fungus Mucor circinelloides var. lusitanicus 12 M, obtained within diverse time frames using distinct nutrient media, on the morphology of the fungus in submerged cultures, the yield of the biomass, and its content of g-linolenic acid have been studied. The levels of base phospholipids and individual fractions of neutral lipids in sporangiospores correlated with the character of their germination. The spores that were characterized by a high rate of germination and gave rise to a well-developed mycelium contained more phosphatidylcholine and phosphatidylserine, but the level of diacylglycerols was low. The increase in diacylglycerols, free fatty acids, and sterols in lipids of sporangiospores of the inoculate was associated with deterioration in mycelium development, dimorphism, and a decreasing yield of the biomass of the fungus.     

CULTURE AND NUTRITION OF AMOEBIDIUM PARASITICUM

Whisler , Howard C. (McGill U., Montreal, Canada.) Culture and nutrition of Amoebidium parasiticum. Amer. Jour. Bot. 49(3): 193–199. Illus. 1962.—The little known Trichomycete, Amoebidium parasiticum, has been isolated into pure culture Maximum growth was obtained on a thiamine-enriched Tryptone-glucose medium. Growth also occurred on a defined medium consisting of thiamine, glucose, methionine, ammonium and inorganic salts. Methionine could not be replaced by sulfate or l -cystine. Both mannose and fructose could satisfy the carbon requirement. At present, reproduction in pure culture is always by sporangiospores, but the amoeboid phase can be induced by the addition of crushed host material.     

Nonanoic Acid, a Fungal Self-Inhibitor, Prevents Germination of Rhizopus oligosporus Sporangiospores by Dissipation of the pH Gradient

Germination of Rhizopus oligosporus sporangiospores is characterized by swelling of the spores and subsequent emergence of germ tubes. Changes in spore morphology and alterations in intracellular pH (pH(infin)) of the sporangiospores were assessed during the germination process by flow cytometry. Sporangiospores were stained with carboxyfluorescein by incubation with carboxyfluorescein diacetate. The nonfluorescent carboxyfluorescein diacetate is passively transported into intact cells and subsequently cleaved by esterases, which results in intracellular accumulation of the fluorescent carboxyfluorescein. Given that the fluorescence of carboxyfluorescein is pH dependent, the pH(infin) of the individual spores could be assessed simultaneously with spore size. For R. oligosporus, swelling of the sporangiospores was accompanied by an increase of pH(infin). In the presence of nonanoic acid, a self-inhibitor produced by various fungi, increase of the pH(infin) was prevented and swelling was inhibited at concentrations of less than 1 mM. Octanoic acid and decanoic acid were equally effective. Acetic acid also inhibited germination but at much higher concentrations (>8 mM). The mechanism of action of these fatty acids is most likely dissipation of the pH gradient. A model is proposed in which the pH(infin) plays a crucial role in the germination of R. oligosporus sporangiospores.     

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.     

Transformation of steroids by fungal spores

Summary Treatment of Cunninghamella elegans sporangiospores with dilute KOH, EDTA or Helix pomatia digestive enzymes (HpE) followed by cortexolone transformation resulted in stimulation of cortisol and epicortisol formation. The increased ability to hydroxylate steroids was accompanied by swelling of spores and increased permeability of their envelopes to exogenous citrate.Activated spores, unlike the untreated controls, exhibited enhanced degradation of intracellular amino acids, especially alanine and glutamic acid-the main constituents of the amino acid pools. We also observed a higher NADPH: (NADP⁺+NADPH) ratio probably due to the operation of more effective NADPH-generating system(s) in HpE, KOH or EDTA pre-treated spores.     

Bioprospecting for novel oxylipins in fungi: The presence of 3-hydroxy oxylipins in Pilobolus

As previously found in various members of the Mucorales, 3-hydroxy oxylipins in Mucor genevensis are associated with the sporangia, i.e. mainly the columella structure and between aggregating sporangiospores. To determine if this phenomenon is also true in distantly related members, the mucoralean fungus Pilobolus was examined. This fungus is characterized by relatively large sub sporangial-columella structures which actively eject sporangia in a sticky liquid for attachment onto herbage surrounding its growth medium – in this case horse dung. Strikingly, this fungus produced a novel oxylipin i.e. a 3-hydroxy monounsaturated fatty acid, possibly a nonenoic acid, which is mainly associated with the sub sporangial-columella structure and aggregating sporangiospores. The specificity of the antibody against 3-hydroxy oxylipins used in immunofluorescence mapping of the mucoralean fungi, was further confirmed in the yeast, Saccharomycopsis malanga which produces 3-hydroxy palmitate in crystal form. These crystals occur between aggregating yeast cells. On the basis of the available data, we hypothesize that 3-hydroxy oxylipins probably function as adhesives, attaching fungal cells to each other or to other surfaces through entropic based hydrophobic forces and/or hydrogen bonds.     

The effect of gamma radiation on breaking of dormancy in Phycomyces spores

Dormant sporangiospores of Phycomyces blakesleeanus were activated by Cs-137 gamma rays. After a dose of about 300 krad, between 80 and 90% of the spores germinated normally. However, further development of the mycelium was inhibited. Even with dry spores a partial activation was obtained. Activation by gamma rays was not accompanied by an increase in trehalase activity.     

Changes in the organisation of surface rodlets during germination ofSyncephalastrum racemosum sporangiospores

Summary Dormant sporangiospores ofSyncephalastrum racemosum have a regular pattern of rodlets on their surface. During the spherical growth phase of spore germination these rodlets become displaced and areas free from rodlets appear. At the time of germ tube emergence the rodlets remain on the spherical portion of the spore whilst the germ tube has a smooth surface.