Additional Evidence of Stable EMF-Induced Changes in Water Revealed by Fungal Spore Germination

Spore germination of some fungi was studied in EMF-tieated water samples. Water samples were exposed to 9.575 GHz microwave modulated with square waves of different pulse repetition frequencies. The study indicates varying inhibitory effects of different modulation frequencies on spore germination of Alternaria alternata, A. tenuissima, Fusarium udum, Helminthosorium oryzae, H. turcicumand Ustilago cynodontis. The pulse repetition frequency-dependent variation in spore germination caused by irradiated water illustrates that water may have the capability to store EMF frequency effects. EMF-treated water might have caused the effect by either changing the activities of and/or deactivating the spore enzymes.     

Effect of root exudates on the spore germiantion of rhizosphere and rhizoplane mycoflora of chilli (Capsicum annuum L.) cultivars

Summary From root exudates of three cultivars of chilli (Capsicum annuum L.) 12 amino acids and 7 sugars were detected. Methionine, d-1- phenylalanine, citrulline and d-xylose were detected only from the root exudates of resdistant cultivars. The root exudates of resistant variety inhibited spore germination of the pathogen (Fusarium oxysporum f. sp.capsici), but that of susceptible variety enhanced spore germiantion of the same. Spore germiantion of antagonistic fungi (Trichderma viride andAspergillus sydowi) was also influenced by the root exudates of resistant and susceptible varieties, but the influence was different.Spore germiantion of a number of rhizosphere fungi was studied and in general root exudate of susceptible cultivar enhanced spore germiantion of majority of fungi, but spore germination of antagonistic fungi against the pathogen was inhibited. However, root exudate of resistant cultivar stimulated spore germination of antagonistic fungi.     

缩球囊霉孢子萌发及细胞核在孢子和芽管中分布的研究*

本文研究了从本校分离的丛枝菌根真菌缩球囊霉Glomus constrictum的孢子萌发和萌发孢子的细胞核DAPI(46-diamidino-2-phenylindol)染色。结果显示,G. constrictum孢子直径为179.5-198.7μm ,顶生于产孢菌丝上。经表面消毒处理后,孢子在水琼脂平板上7天开始萌发。DAPI染色后,经稀释荧光计数,单个孢子细胞核数目约为5300,在孢子中无序分布, 细胞核直径约为9.9-11.2μm 。孢子萌发过程中,细胞核总数无明显变化,只是部分细胞核从孢子流向了萌发伸长的菌丝。     

INHIBITION OF FERN SPORE GERMINATION BY LIPOPHILIC SOLVENTS

Several alcohols and other solvents inhibit germination of spores of the fern, Onoclea sensibilis L. The inhibition is reversible when spores are transferred to solvent-free media. The effectiveness of different solvents, measured by the concentration needed to inhibit germination by 50%, increases with their lipid solubility. The activity of straight-chain alcohols from methanol through n-heptanol is highly correlated with lipid solubility, whereas the correlation is weaker for several other solvents. The results indicate that some lipophilic site in the spore is important in germination.     

Peculiarities of Exogenous Dormancy of Aspergillus nigerConidia

Aspergillus nigerconidia are characterized by exogenous dormancy: the first stage of their germination is accomplished in twice-distilled water. However, germ tube formation requires the availability of carbon and nitrogen sources. Exogenous dormancy in A. nigerconidia exhibits the following peculiar features: (i) nitrogen-containing substances are active stimulators of germination; (ii) temperature-dependent changes in the lipid bilayer and in the neutral lipid composition of conidia are virtually identical to those occurring in growing mycelium under temperature stress; and (iii) the spore viability threshold does not exceed 45°C; i.e., the spores are more heat-resistant than the mycelium, but they are less heat-resistant than the spores that are in the state of endogenous dormancy. According to the current classification of the types of cell metabolism arrest, the exogenous dormancy of A. nigerconidia resembles the pattern of metabolism characteristic of vegetative cells during the idiophase.     

Light dependent spore germination in Woodwardia radicans (L.) Sm

Abstract

The spores of Woodwardia radicans can germinate indifferently either in water or in culture media containing mineral salts at temperatures (15-24°C) falling within a range believed optimal for many other ferns (15-30 C).

The spores are photosensitive, will not germinate in the dark and the addition of gibberellic acid is ineffective in substituting a light requirement. Spore germination was induced by white and red light and phytochrome seems to be implicated in the control of germination since far-red light (and not the blue irradiation) can reverse the stimulating effect of the red light.

Spore morphology and spore germination pattern was studied using light and scanning electron microscopes.

It was concluded that the progressive disappearance of W. radicans from the Italian localities is not due to difficulties in spore germination but is related to problems that arise during the subsequent stages.     

Radiochemical investigation of the respiration of spores of Bacillus cereus

Summary The endogenous respiration of ¹⁴C-labelled spores of B. cereus was measured through the ¹⁴CO₂ produced, and the rate expressed as Q (l CO₂/hxmg). New upper limits for respiration in various conditions have been set.Dry spores had no measurable activity; Q<10^–4 at room temperature and <10^–3 at 35° C. For wet spores of different harvests, at 30°C, Q lay between 0.0013 to 0.067. Near 40° C, respiration showed a maximum. Thermal history has a great influence on Q. CO₂ production by heat-killed spores is attributed largely to infection.Water or 10^–3 m sodium phosphate buffer (pH=6.5) gave equal spore respiration, in strong NaCl it was less. Azide enhanced respiration dramatically. A temporary increase was also found with non-radioactive glucose. Exogenous respiration of spores in glucose exceeded endogenous respiration.Endogenous and exogenous respiration of vegetative forms were much larger than those of spores and were time-dependent. The ratio of minimum (endogenous, dry spores) and maximum (exogenous, wet vegetative cells) respiration was at least 3x10⁵.     

Endogenous respiration and regulation in a primitive marine fungus

The relationship between the respiration and the presence and utilization of endogenous and exogenous substrates was studied in the non-filamentous obligately marine fungus Thraustochytrium aureum. Using isotopic and manometric methods, it was shown that almost all exogenous glucose is assimilated, whilst almost all the oxygen consumption in the presence of exogenous glucose was due to oxidation of endogenous reserves. In contrast, exogenous glutamate, which cannot serve as the sole carbon source for growth, inhibits respiration of endogenous materials, and is itself rapidly oxidized. The uncoupler 2,4-dinitrophenol stimulates the oxidation of endogenous reserves without affecting the uptake and use of exogenous glucose. These data strongly support the idea of physiologic compartmentation in this organism.     

Dry mass of Fusarium roseum spores before and after germination

The total dry mass of Fusarium roseum spores and contained lipid bodies were determined before and after spores germinated using quantitative interference microscopy. The mean for spore dry mass before germination was about 57 pg. Lipid bodies accounted for about 61% of that. Areas of lipid bodies in spores before and after germination were about 23 % but the contents of the lipid bodies accounted for only 10% of the spore dry mass after germination. The total dry mass of the spore and germ tube(s) greatly exceeded that of the spore before germination. We infer that nutrients for germ tube growth are derived from within the germinating spore and from the medium which must contain nutrients leached from non-germinating spores.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

SEED DORMANCY AND GERMINATION IN MELAMPYRUM LINEARE

Immature seeds of Melampyrum lineare Desr. have very high germination percentages and dormancy is induced in a variable fraction of the seed crop during ripening. Correlated with this is the endogenous gibberellin-like activity which is found in considerable amounts in immature seeds, less in batches of ripe seeds, and is not detectable in batches containing only dormant seeds. For germination dormant seeds require activation followed by cold storage. In the laboratory activation is produced by allowing moist, dormant seeds to respire freely for several weeks at 20 C, or by treatment with exogenous GA₃. Dormancy appears to be most directly related to inability of the embryo to hydrolyze the thickened, mannan-containing endosperm cell walls. Embryos excised from dormant seed can be grown on agar enriched with whole macerated dormant seeds or with the ethanol-extractable materials from these (mostly sucrose and a glycoside). However, dormant seed material does not support growth when extracted to remove benzene- and ethanol-soluble materials.     

Observations on orchid seed mycorrhizae

Summary In the natural germination of orchid seeds, the fungus undoubtedly contributes sugars, auxins, and growth factors in stimulating the growth of the embryo. However, none of these factors alone are as successful in stimulating germination as they are when combined. No single factor can be identified as the specific germination agent in the mycorrhizal relationship. Seeds can germinate, although poorly and slowly, without added sugars or auxins. Rather, a combination of factors promoting vigorous aerobic respiration provides the cellular environment necessary for germination. Fungus infection in nature, or artificial media in the lab, can provide these conditions: either rapidly by providing exogenous substrates and growth factors for respiration; or slowly by stimulating the utilization of endogenous lipid seed reserves. In either case, cell division and synthesis of chlorophyll follow. A speculatory metabolic pathway is outlined.     

GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. V. ETHYLENE AND THE EARLY EVENTS OF SPORE GERMINATION

Early events during the germination of spores of the fern Onoclea sensibilis were studied to determine the time during germination when ethylene had its greatest inhibiting effect. Water imbibition by dry spores was rapid and did not appear to be inhibited by ethylene. During normal germination DNA synthesis occurred about four hours before the nucleus moved from a central position to the spore periphery. Following nuclear movement, mitosis and cell division occurred, partitioning the spore into a small rhizoid cell and a large protonemal cell. Cell division was complete approximately six hours after nuclear movement. Ethylene treatment of the spores blocked DNA synthesis, nuclear movement, and cell division. The earliest DNA replication in uninhibited spores was observed after 14 hours of germination, and the maximal rate of spore labeling with ³H-thymidine was between 16 and 20 hours. Spores were most sensitive to ethylene, however, during the stages of germination prior to DNA synthesis, and it was concluded that ethylene did not directly inhibit DNA replication but blocked germination at some earlier fundamental step. The effects of ethylene were reversible. since complete recovery from inhibition of germination was possible if ethylene was released and the spores were kept in light. Recovery was much slower in darkness. It was hypothesized that light acted photosynthetically to overcome the ethylene inhibition of germination. Consistent with this, it was shown that spores exhibit net photosynthesis after only two hours of germination.     

Characteristics of exogenous dormancy of Aspergillus niger conidia]

Aspergillus niger conidia are characterized by exogenous dormancy: the first stage of their germination is accomplished in twice distilled water. However, germ tube formation requires the availability of carbon and nitrogen sources. Exogenous dormancy in A. niger conidia exhibits the following peculiar features: (i) nitrogen-containing substances are active stimulators of germination; (ii) temperature-dependent changes in the lipid bilayer and in the neutral lipid composition of conidia are virtually identical to those occurring in growing mycelium under temperature stress; and (iii) the spore viability threshold does not exceed 45 degrees C; i.e., the spores are more heat-resistant than the mycelium, but they are less heat-resistant than the spores that are in the state of endogenous dormancy. According to the current classification of the types of cell metabolism arrest, the exogenous dormancy of A. niger conidia resembles the pattern of metabolism characteristic of vegetative cells during the idiophase.     

Utilization of trehalose during Dictyostelium discoideum spore germination

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.     

Membrane instability in respiring mitochondria: role of phosphate.

Metabolically-induced (spontaneous) high amplitude swelling of mitochondria has been shown to be due to a serial disruption of the mitochondrial membranes [D. Sambasivarao & V. Sitaramam (1985), Biochim Biophys Acta, 806, 195-209]. Phosphate- and arsenate-induced swelling was investigated in mitochondria to evaluate the role of phosphate transport in the instability created in the mitochondrial membranes. Phosphate-induced swelling in respiring mitochondria was similar to spontaneous swelling. Both represent essentially colloidal swelling due to the variable porosity induced in the inner membrane to polyols by respiration. Swelling of non-respiring mitochondria at high ammonium phosphate concentrations was, on the other hand, primarily due to high permeability to phosphate. This membrane instability created by phosphate transport in the surrounding lipid involves neither the endogenous nor the exogenous Ca2+.     

尖孢镰刀菌四个专化型细胞核的初步研究

本文对尖孢镰刀菌(Fusarium oxysporum)的4个专化型12株菌的孢子萌发、核分裂时间及核DNA含量进行了比较,结果表明:不同专化型菌株孢子萌发速度基本一致,而萌发过程中发生第一次核分裂的时间不同。黄瓜、西瓜、荸荠、大豆各专化型第一次核分裂时间分别为:5.67hr、5.45hr、7.35hr、7.82hr,其核DNA含量分别为:0.321pg、o.306pg、0.177pg 0.174pg。测定结果显示出不同专化型菌株的孢子核DNA含量可能存在倍数关系,并推测F.oxysporum的黄瓜专化型与西瓜专化型为同一类型,而荸荠专化型与大豆专化型为同一类型。     

Studies on the Metabolism of Aspergillii

Endogenous respiration of Aspergillus sojae K.S. was studied in terms of biochemical analysis. It was found that the different kind of substrates was utilized for the endogenous respiratoin according to C:N ratio of the agar medium on which the mold was grown. In the mycelial mats grown on the medium of low C:N value, pool amino acids, protein, and nucleic acids were mainly utilized from the beginning while carbohydrate or lipid displayed a minor role. The corresponding amount of ammonia was formed. On the other hand, in the mycelial mats grown on the medium of rather high C:N value, carbohydrate or lipid was the major substrate of endogenous respiration in the early stages of incubation. The utilization of the nitrogenous materials and the accompanying formation of ammonia got to start only after the lapse of several hours of incubation.     

ANALYSIS OF STARCH ACCUMULATION AND GERMINATION IN ONOCLEA SPORES

Previous studies have shown that low fluences of light accelerate starch accumulation and enhance germination of Onoclea spores. Fluence response curves for induction of starch accumulation were compared with fluence response curves for enhancement of germination in order to determine if the two photoresponses in Onoclea spores have a common photoreceptor. Fluence response curves indicate that both responses were proportional to the log of the fluence and that the relative fluence efficiencies of the four wavelength regions tested were similar for induction of both germination and starch accumulation. Red (600–720 nm) irradiation was the most efficient, while green (500–600 nm), blue (400–520 nm), and far-red (720–900 nm) irradiations showed a decreasing order of efficiency for induction of the responses. A correlation coefficient between the amount of starch accumulated as a result of red irradiation and the final percent germination was calculated to be 0.964. These results support the hypothesis that a common photoreceptor mediates both photoinduced germination and starch accumulation. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) inhibits photosynthesis by blocking the flow of electrons from Photosystem II to Photosystem I. At 0.1 mM DCMU failed to inhibit both photoinduced starch acumulation and germination. This result and the greater efficiency of red than blue light, the low fluence functional for induction, and the fluence dependency argue against the participation of photosynthesis in photoinduced starch accumulation. A similar conclusion has been previously drawn for photoenhancement of Onoclea spore germination. Additionally, the effects 0.01–1.0 mm cycloheximide and 100 μl/l ethylene on photoinduced starch accumulation were investigated. Neither agent inhibited starch accumulation, whereas both substances inhibited germination 70–90% when applied at a time coincidental with the period of rapid starch accumulation. These results indicate that the photoinduction of starch accumulation does not have an ethylene sensitive stage nor does it require protein synthesis as does photoenhancement of germination of Onoclea spores.     

STUDIES IN THE PHYSIOLOGY OF FUSARIA. THE RESPIRATORY AND FERMENTATIVE MECHANISMS

1.Fusarium tricothecoides was selected for a study of the respiratory and fermentative activities of Fusaria. "Resting cell" suspensions were investigated by the Barcroft manometric technique. 2. The results of the investigation indicate clearly that the mechanism of endogenous metabolism (respiration) is distinct from the exogenous mechanism (fermentation). Anaerobically no significant CO₂ production is apparent without added substrate. In the presence of glucose the anaerobic CO₂ evolution is practically equal to the added CO₂ evolved aerobically in the presence of added glucose. Low concentrations of iodoacetate or fluoride selectively poison the exogenous mechanism but do not affect the endogenous mechanism. Alcohol is not produced in the course of endogenous metabolism, but is produced in the presence of added glucose. 3. A study of the metabolism of the organism throughout its entire growth phase from 1 to 7 days has been made. 4. The ability of suspensions of Fusarium sp. H., obtained by growth on a variety of common substrates, to attack a large number of carbon sources with the production of exogenous CO₂ was determined. It is found that organisms grown on glucose will attack only glucose, mannose, and fructose, but none of the common intermediary metabolites except pyruvic acid. Organisms grown on galactose attack galactose, as well as the other hexoses, indicating an adaptive mechanism. 5. An identical mechanism for the dissimilation of glucose, mannose, and galactose is indicated since no additive effects with these substrates were observed. Growths on non-hexose carbon sources attack glucose slightly under the experimental conditions with the evolution of CO₂, but do not attack any other substrate. This would indicate a residual glucose-dissimilating mechanism in all growths investigated. 6. Striking similarities between the general metabolism of resting suspensions of Fusarium sp. H. and resting suspensions of yeast cells are apparent.