Fruiting Body Initiation Research Articles

In situ Localization of Specific RNAs in Whole Fruiting Colonies of Schizophyllum commune

cDNA clones representing eight specific mRNAs abundantly expressed in a dikaryon of Schizophyllum commune but not in the progenitor monokaryons, were used to localize these mRNAs in whole colonies by in situ hybridization. A genomic clone for 18S rRNA was used to probe for rRNA. The colonies were grown on hybridization membranes, fixed, and treated with RNAase-depleted wall-lytic enzymes from Trichoderma harzianum to facilitate permeation of the probes. RNAs in sectors of the colonies were then localized with [32P]DNA probes. Hybridization at different developmental stages showed that the specific mRNAs were only formed in parts of the colony where fruit-body initials arose. At later stages these mRNAs disappeared from abortive fruit bodies but remained high in fruit bodies continuing development.

A possible relation between cyclic-AMP levels and glycogen mobilization in Coprinus cinereus

Addition of cyclic AMP to specific sites in plate cultures of a mature dikaryotic colony of Coprinus cinereus caused a twofold increase in the amount of glycogen over the whole culture within 19 h. There was no evidence for non-random distribution of glycogen around the site of administration of the cAMP. In the normally developing fruit body levels of endogenous cAMP were considerably elevated in fruit body initials and remained unusually high in the fruit body until very late stages of development, when they declined to about the concentrations observed in mycelium. These cAMP accumulations were positively correlated with localized accumulations of glycogen within the fruit body. Published data from in vitro analyses suggest that cAMP may inhibit glycogen synthesis and activate glycogen breakdown in Coprinus. It is suggested that observations reported here would be consistent with this view if cAMP were specifically involved in the bulk translocation of glycogen, i.e. promoted glycogen breakdown in order to mobilize carbohydrate for transport, rather than immediate metabolism.

Karyogamy-dependent enzyme derepression in the basidiomycete [formula omitted

Observation of stained nuclei coupled with determination of activity of NADP-linked glutamate dehydrogenase (NADP-GDH) in the same tissue, showed that increase in enzyme activity was initiated as karyogamy became evident in normally-developing fruit bodies of the basidiomycete Coprinus cinereus . Enzyme activity stabilised for about 4 hours during meiosis, but resumed after meiosis II, and continued to increase until spore maturation. When the time of exposure to light was varied, karyogamy occurred only in tissues which received at least 24 h light exposure and, most significantly, derepression of NADP-GDH was apparent at the time of or very soon after karyogamy. It is concluded that expression of NADP-GDH in the fruit body cap of Coprinus cinereus is either a component part of the cellular programme involved in karyogamy, or is directly triggered by that programme. Parallel assays showed that protein content of fruit body cap tissue declined during development; the decline started before meiosis and also arrested during the division. A major flux in cyclic-AMP content occurred at a much earlier stage, a large accumulation in fruit body initials being rapidly reduced as these developed into primordia. Levels of cAMP similar to those recorded in vegetative cells were approached prior to meiosis, suggesting that this nucleotide has little involvement in development of the fruit body after formation of initials. Onset of large-scale utilization of accumulated glycogen proved to be a post-meiotic event.

Molecular cloning of RNAs differentially expressed in monokaryons and dikaryons of Schizophyllum commune in relation to fruiting

A complementary DNA library cloned in pBR327 was prepared from RNA of a fruiting dikaryon of the basidiomycete Schizophyllum commune. An improved colony-lysis method led to the isolation of complementary DNA clones hybridizing to nine different RNAs abundantly present in the fruiting dikaryon but present in very low or undetectable levels in the monokaryons. One of these RNAs, measuring 580 nucleotides, was exceptional in contributing more than 3% to the mRNA mass of the fruiting dikaryon. A role of the detected dikaryon-specific RNAs in fruiting was suggested by (i) their absence or low concentration in vegetatively growing monokaryons or the dikaryon, (ii) their steep increase in concentration during fruit-body formation in the dikaryon, (iii) a higher concentration of most of these RNAs in the fruit-body initials than in the supporting mycelium, and (iv) their prevalence in full-grown fruit bodies. The “fruiting-specific” RNAs were also present in a suspension-grown dikaryon, in the absence of fruiting, but in concentrations less than 10% of those in surface-grown fruiting dikaryon. The level of expression of these RNAs in the monokaryons was even lower or undetectable. Since the dikaryon and the monokaryons are otherwise coisogenic, it appears that the presence of two different incompatibility genes in the dikaryon allows for a low level of expression of the “fruiting genes” even in the absence of fruiting, whereas the genes are fully expressed under conditions permitting fruit-body formation.

Synchronous Initiation and Sporulation of Fruit Bodies by Coprinus cinereus on a Defined Medium

As in many Basidiomycetes, dikaryotic mycelia of Coprinus cinereus neither initiates nor produces normal fruit bodies in the absence of light. However, once light-dependent fruit body initiation takes place, alternating periods of light and dark are required for normal fruit body development. Cultures grown on a defined medium designated as Coprinus Fruiting Medium (CFM) at 25 C and exposed to continuous light or alternating 12-h periods of light and dark, invariably produced fruit body primordia at the point of inoculation. Cultures kept in the light following fruit body initiation gave rise to abortive fruit bodies. Cultures grown in alternating light and dark periods produced fruit body initials after 140 to 144 hours of incubation. If these incubation conditions were continued, 4–5 fruit bodies per culture sporulated synchronously during the 5th 12-h dark period following fruit body initiation. Cultures incubated in the dark did not produce fruit body initials. The presence of both DL-alanine and L-asparagine in CFM was essential for both fruit body initiation and fruit body maturation given the appropriate light and dark regimes. The synchronous nature of both fruit body initiation and fruit body maturation exhibited by C. cinereus grown on CFM under the conditions described here make this procedure useful for a variety of developmental studies.

Synchronous Initiation and Sporulation of Fruit Bodies byCoprinus Cinereuson a Defined Medium

As in many Basidiomycetes, dikaryotic mycelia of Coprinus cinereus neither initiates nor produces normal fruit bodies in the absence of light. However, once light-dependent fruit body initiation takes place, alternating periods of light and dark are required for normal fruit body development. Cultures grown on a defined medium designated as Coprinus Fruiting Medium (CFM) at 25 C and exposed to continuous light or alternating 12-h periods of light and dark, invariably produced fruit body primordia at the point of inoculation. Cultures kept in the light following fruit body initiation gave rise to abortive fruit bodies. Cultures grown in alternating light and dark periods produced fruit body initials after 140 to 144 hours of incubation. If these incubation conditions were continued, 4-5 fruit bodies per culture sporulated synchronously during the 5th 12-h dark period following fruit body initiation. Cultures incubated in the dark did not produce fruit body initials. The presence of both DL-alanine and L-asparagine in CFM was essential for both fruit body initiation and fruit body maturation given the appropriate light and dark regimes. The synchronous nature of both fruit body initiation and fruit body maturation exhibited by C. cinereus grown on CFM under the conditions described here make this procedure useful for a variety of developmental studies.

Sequence analysis of a split gene involved in fruiting from the fungus Schizophyllum commune

The sequence of a gene and its mRNA, which is abundantly expressed during fruiting body initiation in the Basidiomycete Schizophyllum commune, is described. This gene (1G2), the first to be analyzed in this group of fungi, contains an open reading frame coding for a polypeptide of 94 amino acids and a mol. wt. of 9842. A possible signal peptide of 20 residues and one glycosylation site were found. The sequence analysis was hampered by a sequence rearrangement in one of the cDNA clones, probably due to base pairing between short complementary sequences present at the 5' and 3' ends of the mRNA. The 5' untranslated leader sequence is 57 bp long and harbors a possible ribosome binding site close to the AUG start codon. A TATA box is found at position -31 upstream of transcription initiation. The 3' untranslated sequence is 200 bp long and contains the sequence -TATATAAT-, which most likely represents the polyadenylation signal. Some heterogeneity as to the site of addition of the poly(A) tail was observed. The coding region of the gene is interrupted by three very small introns of 53, 49 and 49 bp, respectively. The 5' and 3' splice junctions are conserved: GTGAGT- and -AG-, respectively. Each intron contains a sequence complementary to the 5' end of the intron. These sequences are compared with internal conserved sequences in yeast and filamentous fungi with regard to their possible role in splicing.

Molecular cloning of a gene abundantly expressed during fruiting body initiation in Schizophyllum commune.

Complementary DNA was synthesized on polyadenylated RNA from a dikaryotic mycelium of the basidiomycete Schizophyllum commune bearing fruiting body initials. The complementary DNA was cloned into the PstI site of pBR327 by the deoxyguanidylate-deoxycytidylate tailing approach. After transformation into Escherichia coli cells, a differential screening was performed by colony hybridization with complementary [32P]DNA made on the RNAs of the monokaryon and dikaryon strains. Two clones were selected for further analysis by Northern blotting and hybrid release translation. Clone 1D10 hybridized with an mRNA of 775 nucleotides, coding for a polypeptide with an Mr of 15,000. Although this RNA was present in both monokaryotic and dikaryotic mycelia, its concentration appeared to change considerably over time and with different cultivation conditions. This mRNA is probably the most abundantly expressed sequence in S. commune. Clone 1G2 and its homologs hybridized with an mRNA of 650 nucleotides, coding for a polypeptide with an Mr of 13,000. This gene was exclusively expressed in the dikaryon strain. In liquid-grown cultures, the concentration of this mRNA was low but increased ca. 20-fold during the establishment of fruiting body primordia. A chromosomal fragment of 9 kilobase pairs which contained the 1G2 gene was cloned into pBR327 and used as a probe in Northern blot hybridization. It was found that surrounding sequences were not expressed at the same time or to the same extent as the 1G2 gene.

Response of the sporophores of the cultivated mushroom ( Agaricus bisporus) to volatile substances

Propylene had no observable effect on the morphology, growth and yield of the mushroom. Acetone, ethanol, ethyl acetate, acetaldehyde and acetic acid, when vaporised into the air, had little or no effect on fruiting and yield, but some encouraged the development of brown spots and stained areas on the caps of the sporophores. Apparently associated with the discolouration resulting from exposure to vapour was a mould, or moulds, growing over the surface of the casing, sometimes profusely. From one sample of discoloured sporophores, a Penicillium sp. was isolated from the stained areas. Gaseous formaldehyde tended to reduce the number of sporophores harvested and 1.25% formaldehyde solution watered onto the casing delayed fruiting, reduced yield and caused severe cracking of the sporophore caps. Toluene and xylene vapour reduced the number and weight of sporophores harvested. Diesel-oil vapour delayed and reduced sporophore production and also gave rise to distorted gill development (rosecomb) only when the sporophores, 2 – 10 mm in diameter, were exposed to diesel fumes. Mixing diesel oil into the casing at 5 ml kg −1 (5 l tonne −1) did not delay fruitbody initiation, but all the sporophores died before reaching 10 mm in diameter. Subsequent attempts to produce gill distortion with hydrocarbons known to be constituents of diesel oil, such as hexane, decane, tetradecane and hexadecane, were unsuccessful. The mushroom is sensitive to certain chemicals, and the response is progressive depending on the severity and duration of exposure.

Changes in complex RNA during fruit-body initiation in the fungus Schizophyllum commune

Total RNA was isolated from 4-day surface cultures of two monokaryotic strains (vegetative mycelium) and a resultant dikaryotic strain bearing fruit-body initials (fruiting mycelium) of the basidiomycete Schizophyllum commune. Single-copy DNA (scDNA) and complementary DNA (cDNA) hybridizations with these RNAs showed that both mycelial types expressed 10, 000 to 13, 000 different RNA sequences of average size. scDNA combination-hybridization experiments did not reveal differences between the RNA populations. However, cDNA cross-hybridization experiments indicated that about 5% of the complex RNA mass present in fruiting mycelium was absent in vegetative mycelium. Homologous and heterologous hybridization of total RNA with cDNA probes corresponding to more and less frequently occurring RNA sequences demonstrated that the fruiting mycelium contained about 35 specific abundant RNAs. Comparison of cell-free translation products of total RNA showed a similar difference between the abundant RNA species in vegetative and fruiting mycelium. Of about 400 polypeptides detected after two-dimensional gel electrophoresis, 18 were encoded exclusively by RNA from the fruiting dikaryon.

Genetic studies of the basisdiomycete Agrocybe aegerita

In the edible white rot fungus Agrocybe aegerita the threshold from mycelial growth to fruit body formation is under control of a single gene in both monokaryons and dikaryons.The allele su opens the pathway for fruiting and allows the subsequent expression of the fruiter genes fi(+) (fruit body initials) and fb (+) (fruit bodies). Its allele, su (+), suppresses monokaryotic fruiting completely and restricts dikaryotic fruiting drastically.The detection of this threshold gene su (+)/su and its action and interactions has practical implication in that an opportunity for concerted breeding is created.First results indicate that the fruiter genes are involved in two essential parameters of productivity. Both time of fruiting and biomass production depend on the two fruiter genes fi (+) and fb (+).Comparable results obtained with two other basidiomycetes suggest that the genetic control of fruiting in Agrocybe aegerita is a general mechanism which may be made use of in breeding work with other basidiomycetes of economic value.

Gamma-ray Induced Delay of Fruiting Body Initiation in a Basidiomycete, Hebeloma vinosophyllum

The effect of gamma-radiation on fruiting body initiation in a basidiomycete, Hebeloma vinosophyllum, was investigated. Fruiting of this fungus is induced by visible light, but irradiation of the mycelium before or after light treatment delayed fruiting body initiation. The time required for fruiting body initiation increased with the radiation dose. The induction of fruiting bodies had two gamma-radiation sensitive stages, one immediately before fruiting body initiation and the other 15 to 20 h after the start of photoinduction.

Genetics of fruit body production in higher basidiomycetes II. Monokaryotic and dikaryotic fruiting in schizophyllum commune

In the wood destroying fungus Schizophyllum commune, a well known subject for genetic studies, fruit bodies are produced not only in the course of the sexual cycle but also asexually. Sexual fruiting requires the establishment of a dikaryon which is under the control of the incompatibility factors A and B. Asexual fruiting, however, starts directly from a monokaryon. The initiation of monokaryotic fruiting requires the presence of a single gene leading to the differentiation of fruit body initials. The action of at least two more genes is required for the further morphogenetic steps resulting eventually in the production of fruit bodies which differ in shape from dikaryotic fruit bodies. As a consequence of a mitosis their basidia produce two spores only. The three genes responsible for monokaryotic fruiting are pleiotropic and determine synergistically the timing of dikaryotic fruiting within a range between 6 to 20 d or longer. A fourth gene was found which codes epistatically for the formation of dome-like masses of stromatic tissue, thus directing morphogenesis into a side track.

A developmental variant of Agaricus bisporus

Studies on sporophore initiation in the cultivated mushroom, Agaricus bisporus (Lange) Imbach are hampered by the difficulty of inducing strains to fruit in axenic culture. Two isolates from a single basidium have been found which readily produce fruit-body primordia in axenic culture on both complex and defined media. The chitin content, level of intracellular laccase, and response to carbon dioxide of these isolates indicated that the structures formed are directly comparable to the initial stages of normal sporophore development. The fruit-body initials from these two strains are produced at high frequency, synchronously and during active growth. This is in marked contrast to the behaviour of other mushroom strains in axenic culture.The origin of these isolates is described and their genetic basis discussed.

A common genetic control of dikaryotic and monokaryotic fruiting in the basidiomycete Agrocybe aegerita

In the edible mushroom Agrocybe aegerita (Agaricales) fruit bodies may be formed in both the sexual and asexual cycle. The major difference between the two types of fruit bodies is that the latter are smaller and contain only two spores on each basidium. Sexual fruiting requires the establishment of a dikaryon which is under the control of the well known incompatibility factors A and B. Asexual fruiting starts directly from a monokaryon. In both dikaryotic and monokaryotic fruiting the same two genes (fi+, fb+)are responsible for the initiation and differentiation of fruit bodies respectively. This shows that the morphogenetic procedures leading to fruit body formation in higher basidiomycetes are not necessarily correlated with the sexual cycle. These findings are significant for basic and applied research.

Phenoloxidase activity in relation to substrate and development stage in the mushroom, Agaricus bisporus

The activities of laccase and tyrosinase were assayed during the development of crops of Agaricus bisporus (Lange) Sing., the cultivated mushroom. Laccase activity characterizes the vegetative stage and tyrosinase activity develops at the time of initiation and development of fruit bodies. The lack of tyrosinase activity in the compost may be due in part to inhibition by products of laccase activity.

Cultivation of the Paddy Straw Mushroom, Volvariella volvacea (Bull, ex Fr.) Sing.1

Abstract The paddy straw mushroom was grown on the type of compost commonly used to produce the cultivated mushroom, Agaricus bisporus (Lange) Sing. An appreciable quantity of light (15 min full sunlight) is required for initiation of fruit bodies. The average yield of paddy straw mushroom was 84 g mushrooms fresh wt/kg dry wt of compost at spawning time. Such production is in the range reported for straw mushroom production on rice straw in Asia but only 20% of the yield of the cultivated mushroom or champignon, A. bisporus.

Environmental Control of Fruitbody Development in Boletus rubinellus in Axenic Culture

SUMMARYThe influence of temperature, humidity, aeration and gas composition, light and nutrition on fruitbody development in Boletus rubinellus was studied. The fruitbody index was used to quantify the degree of fruitbody maturation. Fruitbody development required 4–6 weeks depending on the conditions used. Fruitbody initiation occurred at 17–29 C, and maturation from 22–25 C. In chambers supplied with continuously changing, humidified air, stipe development and pileus formation occurred only in the light. Primordia were initiated in the light or in the dark in aerated or unaerated chambers, but they developed further only in aerated chambers in the light. Increased carbon dioxide concentration prevented stipe elongation and appeared to be the cause of stipe inhibition in unaerated cultures. Fruitbodies were produced on a defined medium solidified with purified agar. In culture, regeneration, the dependence of fruitbody induction on the presence of carpophore tissue, does not explain the ability of B. rubinellus to form fruitbodies, but the small size of the carpophore may have a bearing on its fruiting ability.