First successful isolation of Entoloma clypeatum species complex from basidiospores

The Entoloma clypeatum species complex, known as “Harushimeji” in Japan, associates with Rosaceae and Ulmaceae plant species. In this study, we successfully isolated cultures of this fungal group via basidiospore isolation from tentative four Harushimeji species using modified Norkrans's C (MNC) medium and MNC medium containing n-butyric acid. Colony formations were observed on 22 of 25 basidioma samples; however, most exhibited slow and unsteady growth. The isolated mycelia contained dikaryotic hyphae and were identified through molecular phylogenetic analyses of the internal transcribed spacer region of fungal ribosomal RNA. Six isolates showing steady growth were deposited in the fungal culture collection of the Fungus/Mushroom Resource and Research Center, Faculty of Agriculture, Tottori University, Japan. This result indicates that basidiospore isolation is a useful method for obtaining Harushimeji strains.     

金针菇担孢子核相及遗传属性的研究

以3个不同的金针菇菌株为材料,研究了其担孢子的核相及遗传属性。荧光染色观察显示,担孢子核相以双核为主,双核孢子、单核孢子和无核孢子分别占80.2%、7.5%和12.3%。源于单孢分离物的菌丝为有隔膜、无锁状联合的多核菌丝。在交配试验中,源于不同菌株单孢分离物的菌丝原生质体的配对形成具锁状联合的菌落,而源于同一单孢分离物的菌丝原生质体的配对则形成无锁状联合的菌落,暗示担孢子中的两个核具有相同的交配型。RAPD分析显示,源于同一单孢分离物的菌丝原生质体为10个随机引物所扩增的图谱彼此完全相同,印证了担孢子中的双核是同质的。此外,观察表明,一个担子上着生有4个担孢子。因此,金针菇是一种具4个含同质双核担孢子的四极性蕈菌。     

Dikaryotic fruiting body development in a single dikaryon of <Emphasis Type="Italic">Agrocybe aegerita</Emphasis> and the spectrum of monokaryotic fruiting types in its monokaryotic progeny

Using monokaryotic offspring from several dikaryotic parental strains, the phenomenon of monokaryotic fruiting has been previously analysed in the commercially cultivated high-quality edible mushroom Agrocybe aegerita, revealing a variety of monokaryotic fruiting types. Here, we report a single dikaryotic A. aegerita strain, A. aegerita AAE-3, and 40 monokaryons derived from it, which exhibit a wide spectrum of monokaryotic fruiting types, including a rare, previously unknown type. Advantageously, the selected parental strain A. aegerita AAE-3 completes its life cycle within three weeks by the formation of dikaryotic fruiting bodies of typical agaric morphology on malt extract agar plates. In order to morphologically compare normal dikaryotic fruiting to monokaryotic fruiting, histology was performed from all dikaryotic fruiting body development stages and all fruiting types of monokaryotic origin. No clamp connections or dikaryotic hyphae were observed within the plectenchyma of monokaryotic fruiting stages. Among the monokaryotic fruiting types of the A. aegerita AAE-3-derived monokaryons, we also characterised the rare ‘stipe type’ here described as ‘elongated initials type’ as no differentiation into a future cap and stipe was seen. The two mating-compatible monokaryotic strains representing the extremes of the fruiting type spectrum observed, A. aegerita AAE-3-13 (‘mycelium type’) and A. aegerita AAE-3-32 (‘abortive?+?true homokaryotic fruiting fruiter type, AHF?+?THF fruiter type’), were also found to readily produce oidia (arthrospores). In order to obtain a set of mating-compatible monokaryons covering the whole observed spectrum of monokaryotic fruiting, the two monokaryons A. aegerita AAE-3-40 (‘initials type’) and A. aegerita AAE-3-37 (‘elongated initials type’) have been selected for their mating compatibility with A. aegerita AAE-3-32 and A. aegerita AAE-3-13, respectively. Together with the parental dikaryotic strain A. aegerita AAE-3, this set of standard monokaryons could prove useful for studies exploring the factors regulating monokaryotic fruiting in comparison to dikaryotic mushroom formation.     

ITS rDNA variation of the Coprinopsis phlyctidospora (syn.: Coprinus phlyctidosporus) complex in the Northern and Southern Hemispheres

 Coprinopsis phlyctidospora (syn: Coprinus phlyctidosporus) from the Netherlands, Japan, New Zealand, and Australia can be segregated into two groups, northern and southern, based on the nucleotide sequences of their ITS regions. The mating type of a C. phlyctidospora isolate was tetrapolar. Mating reactions were compatible between monokaryotic testers derived from basidiospores of a Japanese isolate and dikaryotic isolates obtained from a wide geographic area in Japan. In contrast, mating between the Japanese monokaryotic and dikaryotic isolates from Australia and New Zealand were incompatible. These results indicated that C. phlyctidospora was complex and individuals currently recognized as C. phlyctidospora in the Northern Hemisphere and those in the Southern Hemisphere are distinct taxa. The relationship between the clades and the biogeography of the C. phlyctidospora complex are also discussed. Received: January 22, 2002 / Accepted: March 15, 2002     

Ectomycorrhiza formation between Pseudotsuga menziesii seedling roots and monokaryotic and dikaryotic isolates of Laccaria bicolor

Seedling roots of Pseudotsuga menziesii were colonized with three monokaryotic isolates and one dikaryotic isolate of Laccaria bicolor to assess the effect of fungal genotype on ectomycorrhiza formation. Ectomycorrhizas resulting from colonization by the dikaryotic isolate had a multilayered mantle and a cortical Hartig net. One monokaryotic isolate (ss7) formed ectomycorrhizas comparable in anatomy to those induced by the dikaryotic isolate. Two other monokaryotic isolates (ss5, ss1) failed to form mantles or Hartig nets. Roots colonized by these isolates developed characteristics indicating an incompatible reaction.     

Monokariotic fruiting body and clamp cell formation in Mycoleptodonoides aitchisonii (Bunaharitake)

The ability to produce monokaryotic fruiting bodies and clamp cells in culture was examined in monokaryotic strain isolated from several dikaryotic parental strains of the edible mushroom, Mycoleptodonoides aitchisonii (Bunaharitake). We describe a single dikaryotic M. aitchisonii strain, TUFC50005, and 20 monokaryons derived from it, which exhibited a wide spectrum of monokaryotic fruiting types. Most strains formed primordia, or young fruiting body-like structures, but only one of the monokaryons, strain TUFC50005-4, formed a fruiting body, even though it had only one nucleus and produced only two spores after meiosis. We demonstrated that dikariotization was not required for clamp cell formation, fruiting body formation, or meiosis, in this mushroom.     

Nuclear selection in oidium formation from dikaryotic mycelia ofFlammulina velutipes

The effect of nuclear dominance in monokaryotic oidium formation from dikaryotic mycelia in a tetrapolar basidiomycete,Flammulina velutipes, was examined. A total of 46 monokaryotic stocks were used to produce 194 hybrid dikaryotic stocks by crossing. The proportion of homokaryons among the oidium isolates from dikaryotic mycelia was over 95%. The staining of nuclei of oidia with propidium iodide showed that over 90% of oidia were monokaryotic and suggested that these oidia had single haploid nuclei at the G1 stage. The monokaryotic oidium isolates from hybrid dikaryons were backrossed to parental monokaryotic stocks. Although most of the monokaryotic oidium isolates (except for those from 17 hybrid dikaryons from a total of 194 test stocks) showed nuclear types similar to only one of the parental stocks, the process seems to produce essentially the split nuclear type composition. Therefore, the monokaryotization in oidium formation from dikaryotic mycelia essentially involves the process of nuclear selection. The two separate results of hierarchies of relative dominance among two nuclei of the parental dikaryons in the monokaryotic oidium formation by grouping with incompatibility factor compositions were determined. Only a few discrepancies were found in the hierarchies between the two specific nuclear compositions of hybrid dikaryons.     

Variability of ligninolytic enzyme activities in basidiospore isolates of the fungusPleurotus ostreatus in comparison with that of protoplast-derived isolates

Variability of production of all tested ligninolytic enzymes (laccase, peroxidase and manganese-dependent peroxidase) was substantially higher in isolates derived from basidiospores of strain F6 of the white-rot basidiomycetePleurotus ostreatus than the relatively low variability of the dikaryotic mycelial colonies of this strain, and also higher than the variability of protoplast-derived isolates of the same strain. The difference was caused mainly but not completely by the monokaryotic nature of the isolates. To reach the best fructification of the fungus, two fructification media were chosen from twenty two tested and a modified technique of fructification was used.     

长根小奥德蘑双孢菌株与四孢菌株核相变化的比较

用双苯并咪唑（Hoechst 33258）染色法分别对长根小奥德蘑Oudemansiella radicata双孢菌株和四孢菌株的菌丝、子实体、担孢子进行染色观察,结果表明：双孢长根小奥德蘑菌丝细胞多为单核,无锁状联合;原担子中单核进行一次有丝分裂形成两个横向或纵向排列的子核,这2个子核分别进入2个担孢子中,留下无核的空担子;成熟担孢子具有一个核。四孢长根小奥德蘑菌丝细胞大多数为双核,具有锁状联合;进入原担子中的两个单倍性细胞核先发生核配,形成一个二倍性的核,再经过减数分裂形成四个染色体减半的单倍性子核,     

Nuclear selection in monokaryotic oidium formation from dikaryotic mycelia in a basidiomycete,Pholiota nameko

The effect of nuclear dominance in monokaryotic oidium formation from dikaryotic mycelia inPholiota nameko was examined. Over 90% of oidium isolates from dikaryotic mycelia were monokaryotic. Although only one parental nuclear type was recovered from an average of about 80% in these isolates, the nuclear selection process in oidium formation seems essentially to produce split nuclear type composition in oidium products. The hierarchy of relative dominance among the nuclear types of the parental dikaryons in monokaryotic oidium formation was determined. The two hierarchies in nuclear selection between monokaryotic oidium formation and monokaryotic mycelium formation coincided at a level of at least 75%.     

Alternation of nuclear phase in the filamentous basidiomycete,Helicobasidium mompa

Variation in the number of nuclei and cellular ploidy were observed in eight strains ofHelicobasidium mompa. The basidiospores, single-spore isolates and field-isolated strains were all dikaryons. The cellular ploidy, which was assessed by analyzing the fluorescence emitted by DAPI-stained nuclei, was unstable: monokaryotic strains derived from the original dikaryotic strains by successive subcultures were mainly tetraploid, although the original dikaryon was in most cases diploid. On the other hand, a dikaryotic strain derived by treatment with benomyl was haploid. These results suggest that diploid dikaryon is a normal nuclear phase ofH. mompa in nature, and the alternation of ploidy may be due to a feature of the mating system of this fungus.     

Characterization of protoplasts prepared from the edible fungus, Stropharia rugoso-annulata

Protoplast preparation and regeneration conditions of the edible fungus, Stropharia rugoso-annulata Farlow apud Murrill were studied, and the regenerated progenies were characterized in this study. The optimal condition for protoplast preparation was incubation of young mycelia with gentle shaking in 1.5%(w/v) Lywallzyme at 30 °C for 3 h. PGPM (potato/glucose/peptone/mannitol) was the most suitable regeneration medium. Served as osmotic stabilizer, sugars (mannitol and sucrose) were better than inorganic salts (MgSO₄) for clone development and growth. Pre-incubation of protoplasts in liquid regeneration medium resulted in a significantly decreased regeneration rate. Both dikaryotic isolates and monokaryotic isolates could be identified from protoplast-regenerated progenies, with a much higher frequency of monokaryotic isolates identified from the early-developed and fast-growing regenerated clones. Two parental mating types were also identified from protoplasted monokaryotic isolates, but not segregated by 1:1. The mycelial growth rate of protoplasted monokaryotic isolates showed a mating type-dependent model when cultured at different incubation temperatures and pH values, with A2B2 mating type monokaryotic isolates growing faster than those of A1B1 mating type monokaryotic isolates.     

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     

Physiological variation among Tricholoma matsutake isolates generated from basidiospores obtained from one basidioma

Matsutake (Tricholoma matsutake) is a commercially valuable edible ectomycorrhizal mushroom. The physiological traits of T. matsutake have been previously assessed using mycelial isolates isolated from basidiomata; however, few studies have focused on basidiospores. Here, we report that sibling T. matsutake isolates generated from basidiospores on a single basidioma show distinct physiological variation. We first established 145 isolates of T. matsutake on modified Norkrans' C (MNC) agar medium and found that their radial growth varied significantly. The mycelial biomasses of nine isolates with different growth rates were reduced on low-carbon and low-nitrogen MNC media. However, the colony diam of one isolate was significantly elevated on low-carbon medium, and the colony diam of two isolates were significantly elevated on low-nitrogen medium. In co-cultures of two or three isolates, commensal and amensal interactions were observed. The physiological variation induced by low carbon and nitrogen levels and the mycelial interactions between sibling isolates imply mechanisms for the genetic and functional characteristics of mycelia of T. matsutake.     

Mating type of isolates derived from the spermogonial state ofPuccinia coronata var.coronata

Hyphal confrontation between two haploid cultures originating from single basidiospores was used to determine the mating type ofPuccinia coronata var.coronata. Pairs of 15 single-basidiospore cultures were placed approximately 1 mm apart on the medium in all possible combinations. Hyphae of the pairs of colonies came into contact with each other in all combinations approximately two weeks after confrontations. When the nuclear number of hyphal cells in a contact zone was investigated one month after confrontation, monokaryotic hyphae were observed in selfing combination. On the other hand, dikaryotic hyphae were observed in 90.5% of crossing combinations between different cultures. Two isolates are considered compatible if dikaryotic hyphae are present in the contact zone but incompatible if they are absent. The mating type of the fungus was found to be characterized by multiple-allelomorphic tetrapolar incompatibility controlled by the “A” and “B” incompatible factors. Contribution No. 116, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

CYTOLOGICAL STUDIES IN LACCARIA (AGARICALES). I. MEIOSIS AND POSTMEIOTIC MITOSIS

In the genus Laccaria, basidial formation, dikaryotic basidia, karyogamy, and meiosis were generally similar to structures and phenomena reported for other Agaricales. The haploid nuclei of dikaryotic basidia resided side by side in the basidium prior to karyogamy. Following karyogamy a single nucleolus was observed in L. montana (four-spored species); several nucleoli remained in the nucleus of L. tortilis (two-spored species). The haploid number of chromosomes for L. montana appeared to be n = 9. Postmeiotic mitosis typically occurred in the basidiospores resulting in binucleate basidiospores for four-spored species and quadrinucleate basidiospores for two-spored species. Postmeiotic mitosis sometimes occurred in the sterigmata and basidia proper. In instances where postmeiotic mitosis occurred in basidia, mature basidiospores were not formed and the basidia were collapsed, and contained up to eight nuclei.     

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.     

Comparative study of cultures of four species of the genusOudemansiella

Thirty stationary and submerged cultures of species of the genusOudemansiella Speg.,viz. two narrow-region species (O. brunneomarginata, O. canarii) and two primarily wideregion species (O.mucida, O. radicata) were compared. Stationary cultures have identical macroscopic and microscopic features (they produce crust, teleomorph, large thick-walled basidiospores with an apiculum and an uneven surface, dikaryotic hyphae have clamp connections). “Coils” were detected in the mycelium of two species by SEM. Submerged cultures produce antibacterial and particularly antifungal antibiotics, although with various intensity.O. brunneomarginata was first studied in pure culture and its microstructures were first studied by SEM. It could be demonstrated that this species is related with other representatives of the genusOudemansiella. In the present taxonomy of basidiomycetes it is advisable to extend and deepen data concerning a single genusOudemansiella with a series of interspecies taxons, without impairing “borders” of the genus.     

Nuclear selection in monokaryotization of dikaryotic mycelia ofPholiota nameko as described by leading and following nuclei

To examine monokaryotization of dikaryotic mycelia ofPholiota nameko, 18 monokaryotic stocks were used to produce a total of 130 dikaryotic stocks by reciprocal crossing. Monokaryotized mycelium was raised from dikaryotic mycelium in the peripheral zone of the growing colony. The stocks mated with a particular group of monokaryons produced wide-range monokaryotization at higher rates than the other combinations of hybridization. The growth rates of the monokaryotized mycelia exceeded from those of the corresponding parental dikaryons. The monokaryotized mycelium was isolated and back-crossed to parental monokaryotic stocks. Most of the isolates had nuclear types similar to only one of the parental stocks, while the replicates of isolates from two dikaryotic hybrids showed split nuclear type compositions. It is suggested that a relative dominance is active in the selection of one of the two nuclei of the dikaryotic cells in monokaryotization. The hierarchy of relative dominance among nuclei of 18 parental monokaryotic stocks in the monokaryotization of their reciprocal crossing products was estimated. We propose the involvement of a cascade process in dikaryotic cell division, in which the first dividing nucleus (to be found in the monokaryotized cell) may act as the leading nucleus and the other one as the following nucleus.     

Genetic analysis of nuclear ribosomal DNA of Lentinula edodes

Genetic analysis of nuclear ribosomal DNA (rDNA) of Lentinula edodes was carried out using rDNA restriction fragment length polymorphisms (RFLPs) as genetic markers. Two compatible monokaryotic strains that differed in the endonuclease digestion patterns of their rDNA were used. The dikaryotic strain established by crossing them produced mixed RFLP patterns. Single-spore isolates derived from the dikaryotic strain showed three types of rDNA RFLP patterns: either one of the two parental types or a mixed type. From the frequency of the mixed type, the recombination value of rDNA tandem repeats was calculated to be 31.4%. Linkage analysis between rDNA and two incompatibility factors (A and B) revealed that rDNA was not linked to either factor. The rDNA genotypes did not affect mycelial growth among the single-spore isolates.