Examination of spores and young mycelia of Rhizopogon roseolus by scanning electron microscopy

Abstract

Rhizopogon Fr. is one oi' the most important without development ofyoung mycelium. After 60 genera of ectomycorrhizal fungi. Although basi­ days sorne spores produced spherical germ vesicles, diospore inocula have been used in nurseries for without protrusion of a germ tube or development sorne years, few reports of attempts to germinate of young mycelium. One or several germ-hyphae basidiospores oi' Rhizopogon are found in the grew out from the vesicle. In many cases, the literature. Bulmer (1964) appears to be the first hyphal apices ceased elongation and started investigator to report germination experiments swelling up forming one or more vesicles. Deacon with species of this genus. (1988) reported that the process of interruption­ Fries (1941l developed a method to induce spore swelling-ramification is frequently observed germination in several species of gasteromycetes during apical growth in a great number offungi. and ectomycorrhizal hymenomycetes. The first To obtain more information, we studied the spore fruitful experiment was the result of a happy morphology under the scanning electron micro­ coincidence: on a malt extract agar plate with scope (SEM), using both dried material and agar spores of Lycoperdon umbrinum Pers.: Pers. sorne cultures from the same fruitbody. The principal contamination appeared, and close to one uniden­ problem, to select the best method to prepare tified yeast colony a few germinating Lycoperdon samples for SEM, is discussed in this paper. spores were detected. This was the first step to developing a method based on the introduction ofa Methods germination-inducing colony ofyeast, for instance Dried fragments ofthe fruitbody used in germina­ Rhodotorula glutinis (Fr.i Harrison, aIl10ng the tion experiments were prepared following three spores plated on to agar media (Fries, 1943). The different protocols (A-C): (A) Mounted directly on same author improved Laccaria laccata (Scop.: Fr.) standard SEM stubs using double-sided adhesive Berk. germination 10 to 20% by adding activated tape and coated with gold (Martin et al., 1993). (B) charcoal to the agar media (Fries, 1977). Fixed with 70% ethanol. Rehydrated 15 min in Following the methodology of Fries one of us distilled water, 15 min in ammonia and 15 min in (MPM) had the opportunity of observing spore distilled water. Dehydrated in a graded ethanol germination and different patterns ofdevelopment series (50%,60% 70%, 80%, 90%, 95%, 95%, 100%, of young mycelia, from spore cultures of Rhizopo­100%) in 15 min exchanges. Passed through four gon roseolus (Corda) Th. MFr., collected in Teruel, exchanges (15 min) of a graded isoamyl acetate Spain (herbarium number BCC-MPM 1859). series and dehydrated with critical point drying Under the light microscope, spores of R. roseo­(CPD), using carbon dioxide as the drying fluid, lus are cylindrico-ellipsoid to fusoid (6.5-8 x 2.5­ mounted on standard SEM stubs using alu­ 3.5 !-lm), smooth and with one or two strongly minium tape and coated with gold (Martin & light-refractive droplets. One sign ofthe germina­ Rocabruna, 1988). (C) Hydrated in saturated tion process is the increment of the spore length, chloral hydrate for 24 h, directly fixed and accompanied by the coalescence of its droplets. dehydrated in 95% ethanol for 15 min, exchanged Twenty three days afer plating, direct germina­ to 100% ethanol for 15 mino Passed through four tion occurred by an apical germ tube, with or exchanges (15 min) of a graded isoamyl acetate