Handbook Part 2 - International Mycological Association

PS4-406-0257
A homothallic mutant induced by UV irradiation in Lentinula edodes
Norihiro Shimomura 1, Shigeyuki Murakami 2, Teruyuki Matsumoto 1, Nitaro Maekawa1, Kozaburo Hasebe 2
1 Faculty of Agriculture, Tottori University, Koyama-Minami, Tottori, Japan, 2 The Tottori Mycological Institute, Kokoge,
Tottori, Japan
Shiitake, Lentinula edodes (Berk.) Pegler, is the major edible mushroom in Asia. The mating system of this fungus,
leading to the formation of a dikaryon that can produce fruit bodies, is known to be bifactorial heterothallism
(tetrapolality) controlled by two unlinked multialletic incompatibility factors, A and B. Mutants of A and B factors
(Amut and Bmut) are useful for elucidating the structure and function of mating type genes. Particularly, an Amut
Bmut double mutant is expected to have helpful properties that facilitate the recovery and analysis of developmental
mutants. In previous studies, B mutants of this fungus had been obtained. However, no Amut Bmut double mutant has
been isolated so far. To recover an Amut Bmut double mutant, basidiospores of the common Bmut dikaryon (A1B1mut
x A2B1mut) were treated with UV irradiation. Of a total of 5000 monosporous isolates, a single basidiospore isolate was
found to produce the hyphae bearing clamp connections without mating. The mutant could form fruit bodies on a
sawdust medium, and all of the single basidiospore isolates from the mutant grew into colonies which were dikaryotic
in appearance. These homothallic behaviors of the mutant were stably expressed for four generations. Microscopical
observations of basidiospores treated with DAPI revealed that the mutant produced DAPI-negative, uninucleate or
binucleate basidiospores. When basidiospores of the mutant were incubated on a thin film of malt extract agar
medium, about half of basidiospores showed no sign of germination; the other half were able to germinate. During
the germination and following hyphal elongation, a clamp connection was not observed at the first septum in two
cells hypha, but clearly detected in a subsequent multicellular one which contained two nuclei in each cell. The
clamp connections of the mutant were morphologically variable, viz., pseudo-, abnormal- and true-clamps. The
germilings of the mutant were characterized by abnormal swelling and branching. To examine genetic relatedness
among basidiospore isolates of the mutant, amplified fragment length polymorphism (AFLP) analysis was conducted.
AFLP profiles among the basidiospore isolates were identical, indicating that the mutant produced isogenic
basidiospores. These results suggest that the mutant recovered in this study is mutated in both A and B incompatibility
factors.
PS4-407-0274
Growth parameters, morphological and genetic variability of the medicinal mushroom Flammulina
velutipes (Curt. : Fr.) Sing.
S.M. Badalyan 1, K.W. Hughes 2, C.Z. Sakeyan 1, E. Helmbrecht 2
1 Yerevan State University,Laboratory of Fungal Biology and Biotechnology, Yerevan, Armenia, 2 University of
Tennessee, Department of Ecology and Evolutionary Biology, Knoxville, United States
Recent advances in biotechnology have increased the interest in fungal organisms for developing new biotechproducts
and functional food additives. The F. velutipes contains different groups of bioactive compounds and
enzymes with medicinal properties.
Mycelial micro-, macromorphological and genetic variability, as well as growth parameters, laccase activity test,
pigmentation and telemorph formation of 31 collections of Flammulina velutipes have been investigated on maltextract
and potato-glucose agar media. The strains were isolated from fruiting bodies collected in different
geographical regions and wood substrates.
Two morphotypes (A, B) and 3 subtypes (A1, A2, A-B) of mycelial colonies with different growth parameters were
described in F. velutipes collections. Two forms with different intensity of agar pigmentation correlated with described
morphotypes were observed. All strains of F. velutipes, except one, formed normal fruiting bodies on tested media.
Correlations between morphotypes and growth characteristics with substrate nature and geographical origination of
strains have been revealed. However, in order to evaluate species-specific media responses for described
morphotypes further research with more strains is required. Mycelial macromorphology and growth parameters were
more variable than described microstructures (form, shape of clamp-cells, crystals, oidia). Mycelia of tested F.
velutipes strains were laccase positive. Significant correlation between the mycelial morphotypes and genetic
variability of F. velutipes strains has not been revealed. Genes for morphological and cultural variability may be
unlinked to genes for the nuclear ribosomal repeat.
Collections of putative F. velutipes were confirmed by nuclear rDNA-ITS sequencing to be F. velutipes with the
exception of four collections identified as an unknown biotype, F. elastica and F. rossica. High genetic diversity was
observed in Armenian collections of F. velutipes when compared to collections from Eurasia. At least 16 haplotypes
were recovered in Armenian collections. This sequence diversity may be a consequence of the survival of an ancient
genetic variation in the Caucasus and Armenia during glaciation period, while in Europe, the genetic variation was
extirpated. A subset of Armenian genetic diversity of F. velutipes is found in Europe.
Revealed macro-, micromorphological and growth characteristics of mycelium will assist in further biotechnological
cultivation of F. velutipes. High genetic variability of Armenian collections may be used for improvement of strains to
obtain novel biotech-products and health-enhancing functional food additives from this valuable medicinal
mushroom.
Research was supported in part by NATO (#980764), DAAD (#548.104401.174) and NSF PEET (DEB-9521526) grants.
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