The character of endogenous growth regulators in the course of development in the fungus Lentinus tigrinus

The character of endogenous growth regulators in different stages of growth and development ofLentinus tigrinus was followed by means of bioassays. The methanolic extract of differently old mycelium and fruiting body was chromatographically separated and tested for auxins by means of Avena coleoptile segments, for gibberellins by using lettuce seedlings and for cytokinins by measuring the content of chlorophyl in leaf segments of barley. Auxins were found only in young vegetative mycelium and in the growing young stalk. The level of gibberellins is more significant and it reaches the highest values in the vegetative mycelium, in the growing young stalk and in the differentiated cap. The content of auxin and gibberellin substances did not increase even in the medium after cultivation of mycelium. Bioassays of cytokinins show a slight stimulation in the vegetative mycelium and in the primordia of caps. Significant inhibition was ascertained in the fully developed stalk. The high quantities of cytokinins were found in caps when basidiospores were formed. The results show a close connection of endogenous growth substances with the growth and the development of the fungus and indicate their participation in differentiation processes.     

Successful large-scale production of fruiting bodies of Laetiporus sulphureus (Bull.: Fr.) Murrill on an artificial substrate

Laetiporus sulphureus is an edible wood-rotting basidiomycete fungus whose fruiting bodies contain substances with verified therapeutic evidences and large amounts of α-(1 → 3)-glucan which is used as an effective inducer of microbial α-(1 → 3)-glucanases. However, production of mature fruiting bodies of this species under artificially controlled conditions has not been reported until now. Here, we provide the first report of successful initiation and development of L. sulphureus fruiting bodies in large-scale experiments. Twelve Laetiporus strains were isolated from a natural habitat. A synthetic log production system with a substrate composed of a mixture of sawdust enriched with organic and inorganic additives was developed. It was found that shocking the fungus mycelium with cold water or low temperature was the only suitable method for forced fruiting of L. sulphureus strains. Primordia of two strains were initiated already after 5–6 days from induction, and after another 2 days, they began to develop into fruiting bodies. Carpophores appeared fastest on substrates with high organic supplementation (40–45 %) and a low moisture content (40 %). The resulting mature fruiting bodies reached a weight of 200–300 g. The method of cultivation presented in this paper opens the way to commercial production of this valuable basidiomycete.     

Exogenous regulators in the mycelium ofPleurotus ostreatus after exogenous application

The mycelium of the fungusPleurotus ostreatus was cultured on a synthetic medium with growth regulators. Best growth was observed on media with 100 ppm IAA, 200 ppm GA₃ and 200 ppm of kinetin. Growth of terminal hyphae and the appearance of colonies differed characteristically. Auxin bio-assays showed that the content of substances in younger mycelium slowly increased during cultivation without conspicuous changes in the medium. After exogenous application of IAA the content of auxins in younger mycelium increased and the content of IAA decreased gradually in the culture medium. Gibberellin bio-assays showed their content in the mycelium fell during cultivation and while it increased in the medium. Application of GA₃ to the medium leads to a significant increase in these substances in the young mycelium and in the medium their level remains the same. Cytokinin bio-assays showed their content to vary during cultivation. An addition of kinetin to the nutrient medium results in an increase of cytokinins in the mycelium while in the medium the level of cytokinins is maintained. Addition of one substance thus influences the levels of other endogenous regulators.     

Stimulation of the formation of fruiting bodies of the fungusLentinus tigrinus (Bull.) Fr. by growth regulators

Growth regulators, indole-3-acetic acid (IAA), gibberellic acid (GA₃), and kinetin (KIN), were used in different concentrations to stimulate the initiation and further development of the fruiting bodies of the fungusLentinus tigrinus. Vegetative mycelium of the fungus was cultivated on cellulose cylinders soaked with a synthetic nutrient solution or with a 3% malt extract. When the mycelium covered the surface of the cylinders, further cultivation was carried out in graduated concentrations of the growth regulators mentioned above. The number of developed fruiting bodies showed that the optimum IAA and GA₃ concentrations were in both media 300 p.p.m. The optimum concentration of kinetin was 400 p.p.m. The individual growth regulators influenced characteristically also the shape of the fruiting bodies and changed in them the natural level of endogenous growth regulators. The addition of IAA into the medium raised the level of endogenous auxins in the cap. The presence of gibberellic acid and of kinetic in the medium resulted in an increase in the level of these regulators in the fruiting bodies.     

Biosynthesis and beneficial effects of microbial gibberellins on crops for sustainable agriculture

Soil microbes promote plant growth through several mechanisms such as secretion of chemical compounds including plant growth hormones. Among the phytohormones, auxins, ethylene, cytokinins, abscisic acid and gibberellins are the best understood compounds. Gibberellins were first isolated in 1935 from the fungus Gibberella fujikuroi and are synthesized by several soil microbes. The effect of gibberellins on plant growth and development has been studied, as has the biosynthesis pathways, enzymes, genes and their regulation. This review revisits the history of gibberellin research highlighting microbial gibberellins and their effects on plant health with an emphasis on the early discoveries and current advances that can find vital applications in agricultural practices.     

Diffusible and Extractable Gibberellins in Bean Cotyledons in Relation to Dwarfisni

Dwarfism in bean plants (Phaseolus vulgaris L.) is investigated in relation to the diffusible and extractable gibberel-lins in cotyledonary tissues. These gibberellins were partitioned into four parts prior to thin layer chromatograplty: non-acidic and acidic ethyl acetate fractions, and non-acidic and acidic butanol fractions. Cotyledonary segments from a tall plant (cv. Kentucky Wonder) seem to diffuse, preferentially acropetally, more gibberellins in each fraction than those from a dwarf plant (cv. Masterpiece). The diffusion increases with the length of the segments and decreases with period after sowing. From experiments on extraetable gibberellins, however, it is concluded that these phenomena actually result from differences in the gibbereliin contents of the tall and the dwarf plants, from differences in the gibberellin contents of the distal and proximal parts of cotyledons, and from the gibberellin contents before and after imbibition. Ten kinds of gibberellin-Iike substances are detectable in the diffusates, exudates and extracts from the cotyledons of both the plants; two in the non-acidic ethyl acetate, three in the acidic ethyl acetate (GA₁, GA₆ and another), two in the non-acidic butanol, and three in the acidic butanol frac—tion. They are almost identical in quality in the dwarf and tall plants, but in the latter they are more abundant in the cotyledons, particularly in their distal part. With respect to the change in content during the period after sowing, the gibberellin-Iike substances are classified in three groups; unchanging, decreasing (GA₁, GA₆, others) and increasing (butanol soluble glucosyl esters and glucosides of gibberellins)-. The increase of glucose-bound gibberellins and the decrease of free gibberellins during the sowing period suggest the occurrence of conversion. This is obvious only in the tall plants. The gibberellin content in cotyledons is higher in the tall plant than in the dwarf plant. Thus, the marked hypocotyl growth in the tall plants may be dependent on the higher content of gibberellins in their cotyledons and on the higner rate of conversion from free to bound forms.     

Alterations in the composition of membrane glycero-and sphingolipids in the course of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> surface culture development

Qualitative and quantitative characteristics of the alterations in the lipid composition of the membrane of the basidial fungus Flammulina velutipes in the course of surface culture development were investigated. Modifications of the lipid composition were shown to be timed to specific ontogeny stages, such as changes in the growth rate of the colonies, the appearance of differentiated vegetative cells, and the formation of generative structures. A slowdown of growth correlated with an alteration in the ratio of major classes of phospholipids, namely, with a decrease of phosphatidylcholine relative content and an increase in phosphatidylethanolamines. The differentiation of vegetative cells of the mycelium proceeded along with modifications of molecular composition of glycoceramides. In the course of the first week of growth, the surface culture of F. velutipes produced monohexosylceramides with epoxidized methyl sphingadienine as a sphingoid base. Later on, along with culture growth and specialization of mycelium cells, molecular species with methyl sphingadienine, common for basidiomycetes, start to prevail among the fungal glycoceramides. The formation of fruit bodies is accompanied by enrichment of molecules of phospholipids, mainly, the phosphatidylcholines, with unsaturated fatty acids.     

Phytohormonal changes in intact shoots of wheat and oilseed rape treated with the acylcyclohexanedione growth retardant prohexadione calcium

The effects of the acylcyclohexanedione-type growth retardant prohexadione calcium on seedling growth and endogenous levels of immunoreactive phytohormone-like substances in shoots of wheat ( Triticum aestivum L. cv. Kanzler) and oilseed rape ( Brassica napus L. ssp. napus cv. Lirajet) were studied. After treatment of seedlings with increasing retardant concentrations in hydroponics, plant height and fresh weight of shoots were reduced by up to 40%. Concomitantly, the amount of immunoreactive gibberellins decreased, on a fresh weight basis, when compared with levels in the shoots of control plants. In contrast, the levels of abscisic acid and dihydrozeatin riboside and isopentenyladenosine-type cytokinins were considerably elevated by the growth retardant. The content of 3-indoleacetic acid decreased slightly. These results suggest that, in addition to its effect on gibberellin content, prohexadione calcium also influences the levels of endogenous abscisic acid and cytokinins.     

Gibberellin-induced delay of leaf senescence of Alstroemeria cut flowering stems is not caused by an increase in the endogenous cytokinin content

The effects of various chemically pure gibberellins and cytokinins on leaf yellowing of Alstroemeria were described. The loss of chlorophyll was measured both in leaves of cut flowering stems and in a model system consisting of detached leaf tips. It was demonstrated that plant growth substances affected chlorophyll loss in both systems to the same extent. Leaf senescence was delayed by various gibberellins and cytokinins. The results demonstrated that some of the gibberellins (GA₄ and GA₇) are far more effective in delaying chlorophyll loss than GA₃, which is commonly used as a postharvest treatment for Alstroemeria cut flowering stems. Immunoassays were used to demonstrate that the effect of gibberellins on leaf yellowing does not involve an increase in the endogenous cytokinin concentrations in the leaves as an intermediate step.Abbreviations GA gibberellin A - HPLC high performance liquid chromatography - GA_3Mc GA₃-methyl ester - ZR zeatin riboside - IPAR isopentenyl adenine riboside.     

Growth and mycelial strand production of Rigidoporus lignosus with various nitrogen and carbon sources

Growth and differentiation of mycelial strands in Rigidoporus lignosus have been shown to depend on suitable combinations of the pH of the media and the nature of the nitrogen and carbon sources. Amino acids as sole nitrogen sources gave rise to vegetative mycelium. At pH 4.5, growth and mycelial strand differentiation required asparagine, as the fungus failed to grow in the absence of this amino acid. However, at pH 6, differentiation of strands occurred appreciably in asparagine-deficient media, suggesting a close balance between pH and amino acid requirements. Ammonium was required for strand differentiation, while nitrate, as a sole nitrogen source, maintained the fungus undifferentiated. Of the carbohydrates tested, only glucose, fructose and mannose supported strand differentiation. Starch was found to be particularly effective in promoting growth of vegetative mycelium. Strand differentiation required more specific conditions than growth of the vegetative mycelium.     

Flowering in tobacco needs gibberellins but is not promoted by the levels of active GA1 and GA4 in the apical shoot

Flowering of Nicotiana tabacum cv Xhanti depends on gibberellins because gibberellin-deficient plants, due to overexpression of a gibberellin 2-oxidase gene (35S:NoGA2ox3) or to treatment with the gibberellin biosynthesis inhibitor paclobutrazol, flowered later than wild type. These plants also showed inhibition of the expression of molecular markers related to floral transition (NtMADS-4 and NtMADS-11). To investigate further the role of gibberellin in flowering, we quantified its content in tobacco plants during development. We found a progressive reduction in the levels of GA1 and GA4 in the apical shoot during vegetative growth, reaching very low levels at floral transition and beyond. This excludes these two gibberellins as flowering-promoting factors in the apex. The evolution of active gibberellin content in apical shoots agrees with the expression patterns of gibberellin metabolism genes: two encoding gibberellin 20-oxidases (NtGA20ox1 = Ntc12, NtGA20ox2 = Ntc16), one encoding a gibberellin 3-oxidase (NtGA3ox1 = Nty) and one encoding a gibberellin 2-oxidase (NtGA2ox1), suggesting that active gibberellins are locally synthesized. In young apical leaves, GA1 and GA4 content and the expression of gibberellin metabolism genes were rather constant. Our results support that floral transition in tobacco, in contrast to that in Arabidopsis, is not regulated by the levels of GA1 and GA4 in apical shoots, although reaching a threshold in gibberellin levels may be necessary to allow meristem competence for flowering.     

Cytokinins and gibberellins in sap exudate of the oil palm

Exudates were collected from stumps of pre-anthesis inflorescences of oil palm and analysed for cytokinin and gibberellin content using combined HPLC-ELISA techniques. Three antisera, for zeatin-type, dihydrozeatin-type and isopentenyladenine-type cytokinins, were used in ELISAs to identify members of these three groups of cytokinins. Ribotides, 9-glucosides, free bases and ribosides were detected for each of the groups with zeatin riboside the most abundant cytokinin identified in the exudate. Isopentenyladenine-type and dihydrozeatin-type cytokinins were also identified but at lower levels. In addition, two monoclonal antibodies were used in the development of novel ELISAs for members of the 13-hydroxylated and non-13-hydroxylated families of gibberellins. The new ELISAs allow the determination of gibberellins in smaller amounts of tissue than are required for GC-MS. The most abundant gibberellins identified in exudates were GA19 and GA44, as well as other members of the early 13-hydroxylation pathway. Gibberellins were confirmed by GC-MS. The presence of these types of growth regulators in exudate supplying immature inflorescences suggest they have a role in growth and development of these structures.     

Hormonal Profile in Vegetative and Floral Buds of Azalea: Levels of Polyamines, Gibberellins, and Cytokinins

The floral transition includes a complex system of factors that interact and involve various biochemical signals, including plant growth regulators. The physiological signals involved in the control of the floral transition have been sparsely studied and mainly in plant species whose genetics are poorly known. In this work, the role of polyamines, gibberellins, and cytokinins was investigated by analyzing their endogenous content in vegetative and floral buds of azalea. The results showed that there is a clear distinction between floral and vegetative buds with respect to the levels of these plant hormones, with floral buds containing higher amounts of conjugated polyamines, gibberellins (GAs) from the non-13-hydroxylation pathway (GA₉, GA₇, and GA₄), and cytokinins (particularly isopentenyl-type species), and vegetative buds containing higher amounts of free polyamines and gibberellins from the early 13-hydroxylation pathway and fewer cytokinins. In conclusion, there is a specific pattern of endogenous hormone profiles in both vegetative and floral bud development in azalea, which may be relevant for future research on the control of flowering by exogenous hormone applications.     

The Location of Cytokinins and Gibberellins in Wheat Seeds

Wheat seeds (Triticum aestivum L. cvs. Yorkstar and Sirius) were cut transversely into embryoless and embryo-containing (embryonated) halves and the content of endogenous cytokinins and gibberellins in both halves determined before and after the seeds imbibed water for 12–15 h at 22°C in the light. Dry seeds contained little ethyl acetate-extractable gibberellin activity as measured by bioassay but n-butanol-soluble cytokinins were detected mainly in the embryoless halves. Dry, embryonated half-seeds contained water-soluble gibberellins which could be extracted into acidic ethyl acetate after treatment of the aqueous extract with either alkaline phosphatase, β-glucosidase or a crude pectolytic enzyme preparation. When half-seeds were allowed to imbibe water for 12 h and then extracted, cytokinin activity was largely lost from the embryoless halves and completely from the embryonated halves and water-soluble gibberellins were also lost from the embryonated halves. However, ethyl acetate-soluble gibberellins were present in the latter suggesting that “bound’ gibberellins were released during imbibition. The hormones present in normal and naturally embryoless dry grains of cv. Yorkstar were also determined. Both gibberellin and cytokinin activity was higher in normal grains suggesting that the presence of an embryo is essential for synthesis or accumulation of these hormones in the grain during development.     

Heterogeneity of the content of endogenous gibberellins in the leaves of winter wheat in relation to their insertion and ontogeny

Free and bound gibberellins were estimated in individual leaves of winter wheat using TLC a lettuce hypocotyl bioaasay. Bound gibberellins were extracted from the hydrolyzate of the water remnant after the removal of free gibberellins. Each leaf exhibits its own dynamics of free and bound gibberellins during its individual ontogeny, the gradual changes tending to be identical in every leaf. In young developing leaves the level of free gibberellins is high and that of bound is low. Further, inhibitors are sometimes present. The free gibberellin level rises with the activisation of growth and vital activity of a given leaf. Towards the end of the life cycle the content of free gibberellins is reduced to its minimum and inhibitory substances appear. The dynamics of bound gibberellins is opposite to that of free ones during ontogeny.     

Chemical Composition of the Edible Mushroom Pleurotus ostreatus Produced by Fermentation

Pleurotus ostreatus `Florida' was grown in submerged liquid culture. The biomass yield of the fungus, grown for 3 days in 2-liter fermentors, where the mycelial pellets measuring 5 mm in diameter were formed, was 11.7 g (dry weight)/liter. Comparing the chemical constituents of fruiting bodies produced on cotton straw and mycelial pellets revealed several similarities in total nitrogen, protein, glycogen, fatty acids, RNA, and ash content. Differences were observed in the contents of six amino acids. Although the total fatty acid content was similar, there were more saturated fatty acids in the mycelium. Cell wall composition, typical for basidiomycetes, was observed in both mycelium and fruiting bodies, with laminarin as the main polymer.     

Bioluminescence characteristics of a tropical terrestrial fungus (Basidiomycetes).

Freshly collected samples of luminous mycelium of a terrestrial fungus from Panama were investigated for their bioluminescence characteristics. Taxonomic identification of fungal species could not be determined because of the lack of fruiting bodies. Fluorescence excited by 380 nm illumination had an emission spectrum with a main peak at 480 nm and additional chlorophyll peaks related to the wood substrate. Bioluminescence appeared as a continuous glow that did not show any diel variation. The light production was sensitive to temperature and decreased with temperatures higher or lower than ambient. Bioluminescence intensity was sensitive to hydration, increasing by a factor of 400 immediately after exposure to water and increasing by a factor of 1 million after several hours. This increase may have occurred through dilution of superoxide dismutase, which is a suppressive factor of bioluminescence in fungus tissue. The mycelium typically transports nutritive substances back to the fruiting body. The function of luminescent mycelium may be to increase the intensity of light from the fungus and more effectively attract nocturnal insects and other animals that serve as disseminating vectors for fungal spores.