Bovinus Siullus

Genotyping of mycorrhizas synthesized by spore inoculation of Suillus bovinus
Kikuchi Kensuke*1), Matsushita Norihisa 1) and Suzuki Kazuo2)
1) Laboratory of Forest Botany, Graduate School of Agricultural and Life Sciences, The University of Tokyo 2) Department of Forest Sciences and Resources, College of Bioresource Sciences, Nihon University

Introduction
Propagation of ectomycorrhizalfungi is primarily by spore dispersal and vegetative mycelial expansion. Recently, molecular ecological studies have revealed, mainly based on clonal sizes, relative importance of them depends on species. However, direct observations documenting the actual mechanism of mycelial clone establishment and early development by spore dispersal have been undescribed. Are the mycelia that constitutemycorrhizas formed by spore inoculation monokaryotic or dikaryotic?

Our previous experimental outcomes on Suillus bovinus spores
1. Mycorrhizas were formed by spore inoculation Mycorrhizas were observed about 4 weeks after inoculation of spores of Suillus bovinus to Pinus densiflora seedlings. 3. Monosporous strains had mycorrhizal ability
a) b)

2. Spore germination was induced by root exudates ofpine seedlings Spores of S. bovinus were germinated by combination of activated charcoal treatment of media and co-culture with seedlings of P. densiflora ( a) ).
Germination was observed 11 days after inoculation (b)). Some substances contained in root exudates other than abietic acid, which was reported to be specific activator of spore germination for Suillus species, seemed to inducegermination ( c)).

a)

b)

c)

Fig. Germinating spores of S. bovinus (R: root of a pine seedling)
Table. Effect of different treatments on germination rate (%) of spores of Suillus bovinus Charcoal treatment ＋ Additional treatment pine seedling abietic acid control pine seedling abietic acid control 1 week 0 0 0 0 0 0 2 weeks 5.4 0 0 0 0 0 3 weeks 7.5 0 0 0 0 0 4 weeks 7.7 0 0 0 0 0 5 weeks 8.20 0 0 0 0

Monosporous strains of S. bovinus had mycorrizal ability as reported for Laccaria bicolor, Hebeloma cylindrosporum and Pisolithus tinctorius.
Mycorrhizas were formed by inoculation of monokaryotic mycelia (a)). Presence of Hartig net was confirmed by light microscopy (b)).

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Materials and Methods
Seeds of Pinus densiflora were surfacesterilized by 30% H2O2 and germinated on ayeast-glucose agar medium. Each seedling of 4 weeks old was planted in a plastic box (75×225×15 mm) containing a sterilized substrate of Akadama or Kanuma soil and grown in a green house (25℃). Spores of S. bovinus were collected from 4 fruit bodies harvested in Ina, Nagano prefecture in September 2004 , which were heterozygous at the SSR locus Sb-CA1. Each spore collection was suspended insterilized distilled water to about 2×105　spores/ml and 2ml of the each suspension was inoculated to each seedling of 4 months old.

Molecular Analysis
　Among the 8 SSR (simple sequence repeat) loci isolated, the locus Sb-CA1 was confirmed to be polymorphic (a)), species-specific (b)) and co-dominant (c)). Therefore, in this study, the locus Sb-CA1 was used as a molecular marker for genotyping ofmycorrhizas.
b) a)

Fig. Observed polymorphism at the locus Sb-CA1.

c)

Fig. Evaluation of species-specificity of the SSR marker Sb-CA1.
Sb: Suillus bovinus ; 1: Suillus spectabilis ; 2: S. laricinus ; 3: S. grevillei ; 4: S. luteus ; 5: Gyroporus castaneus ; 6: Boletinus cavipes ; 7: Xerocomus nigromaculatus ; 8: X. sp. ; 9: Boletus griseus var. fuscus ; 10: B. ornatipes ; 11: B. speciosus; 12: B. pseudocalopus ; 13: Tylopilus neofelleus ; 14: T. castaneiceps ; 15: Leccinum extremiorientale ; 16: L. holopus ; 17: Boletellus chrysenteroides ; 18: B. russelli 19: Rhizopogon rubescens ; 20: Lyophyllum shimeji ; 21: Laccaria amethystea ; 22: L. laccata ; 23: Tricholoma flavovirens ; 24: T. auratum ; 25: T. robustum ; 26: T. fulvocastaneum ; 27: T. matsutake ; 28: T. bakamatsutake ;...