We describe our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous ascomycetes, Rosellinia necatrix, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei. Hyphal anastomosis between incompatible strains, which were field and single ascospore isolates, were observed with cell death showing fused hyphae, and nuclei debris which were intensified by staining with ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide but were intensified by staining with acridine orange. A strain was found which did not form a barrier reaction, but which could be shown to undergo cell death and therefore showed a positive vegetative incompatibility reaction. We also examined the vegetative incompatibility among five single ascospore isolates and the putative parent strain from the same perithecium; all strains were incompatible. These results strongly suggest that vegetative incompatibility in R. necatrix is regulated by many loci.

Our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous basidiomycete, Helicobasidium monpa, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei is described. Hyphal anastomoses between strains belonging to different mycelium compatibility groups (MCG) were observed with cell death in fused hyphae, whose nuclei were intensified by ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide, but were intensified by staining with acridine orange. These results indicate that in H. monpa, ethidium bromide staining is a useful method for detecting dead cells. We also examined the relationships between the alternation of ploidy and hyphal anastomosis formation using the newly developed method on filamentous fungi. The tetraploid monokaryon strain derived from the original dikaryon strain by continuous subculture could not be fused to any wild type strains, but the original dikaryon strain could be fused without cell death to only the same MCG strain. In contrast, the haploid dikaryon strain derived from the original monokaryon strain fuses to several strains belonging to different MCGs without cell death. These results suggested that the cellular ploidy of this fungus is closely related to its mating system and, H. monpa may be a self-fertilizing fungus.

The telomere associated DNA sequence pTel46, which was isolated from Coprinus cinereus, was hybridized with Rosellinia necatrix genomic DNA. The DNA fragments hybridized with pTel46 were more sensitive to Bal31 nuclease. This result suggests that the DNA fragments hybridized with pTel46 were located at the end of chromosomes in R. necatrix. Telomere-linked restriction fragment length polymorphism (RFLP) was found in strains of R. necatrix isolated from various field and single ascospores. Thus, this marker appears to be an excellent tool to show the great polymorphism of R. necatrix. However, RFLP could not be found among several field isolated strains belonging to the same mycelial compatibility group (MCG) isolated in the same field. Therefore the strains belonging to the same MCG might be the same strain that could be anastomosed with each other without cell death except for strain W718 carrying a double-stranded RNA (dsRNA) virus. Therefore the RFLP corresponded to a MCG group, and none of the strains belonging to the same MCG group showed different RFLP in R. necatrix. Moreover, the presence of a kind of dsRNA virus might imply anastomosis between compatible strains.

Variation in the number of nuclei and cellular ploidy were observed in eight strains of Helicobasidium 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 of H. mompa in nature, and the alternation of ploidy may be due to a feature of the mating system of this fungus.Key Words--cellular ploidy; cytology; fluorescent microscopy; haploidaization; Helicobasidium.Mating between compatible monokaryons of the tetrapolar filamentous basidiomycete Coprinus cinereus (Schaeffer ex Fries) S. F. Gray involves dikaryotization and clamp-cell formation, followed by development of fruiting bodies, meiosis and basidiospore formation. The number of nuclei and their ploidy are generally stable during mycelial growth, except during development of fruiting bodies and meiosis (Wong and Gruen, 1977; Casselton and Olesnicky; 1998; KQes, 2000), though subculturing the bipolar filamentous basidiomycete Pholiota nameko (T. Ito) S. Ito et Imai frequently induces dedikaryotization of the dikaryotic mycelium (Masuda et al., 1995; Kumata et al., 1995). Moreover, strains containing both diploid and haploid nuclei have been reported for Cryptococcus neoformans (Sanfelice) Vuillemin, a bipolar basidiomycete yeast, and shifts in cellular ploidy occur widely (White and Tacobson., 1985;Tanaka et al 1999; Hata et al., 2000). These phenomena suggest that the number of nuclei of dikariotic mycelium in bipolar basidiomycetes is less stable than in tetrapolar basidiomycetes, regardless of whether the mating type locus is a multiple allele. This may be because certain mating-type genes associated with nuclear migration, present in tetrapolar basidiomycetes, are lacking in bipolar basidiomycetes (Casselton and Olesnicky 1998; KLies, 2000).The filamentous basidiomycete Helicobasidium mompa Tanaka is a commercially important, soil-borne, root pathogen affecting a wide range of plant species, tt is the causal agent of purple root rot disease, and host plants infected by the fungus quickly wither and die. In Japan, this fungal disease, which spreads rapidly and is ~Corresponding author. very difficult to prevent, has done great damage to commercially grown grape vines and apple and pear trees. Unfortunately, despite its importance, little is known about the cytology and genetics of this fungus.Investigation of the cellular ploidy of H. mompa is very important for understanding its pathogenesis and mating system. Pulsed-field electrophoresis is a good tool for differentiating chromosomes among strains, especially with respect to molecular size (Zolan and Pukkila, 1995), but it r...

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