We developed a reliable, inexpensive, and simple method for staining arbuscular-mycorrhizal fungal colonizations in root tissues. Apart from applications in research, this nontoxic, high-quality staining method also could be of great utility in teaching exercises. After adequate clearing with KOH, an ink-vinegar solution successfully stained all fungal structures, rendering them clearly visible.

The study of arbuscular mycorrhizal (AM) fungi and the AM symbiosis formed with host plant roots is complicated by the biotrophic and hypogeous nature of the mycobionts involved. To overcome this, several attempts have been made during the last three decades to obtain this symbiosis in vitro. The use of root-organ cultures has proved particularly successful. In this review, we describe the method by which root-organ cultures (transformed and nontransformed) have been obtained, together with the choice of host species, inoculation techniques, and culture media. We also outline the potential use of continuous cultures and cryopreservation of in vitro produced spores for long-term germ plasm storage. Furthermore, this review highlights the considerable impact that in vitro root-organ cultures have had on studies of AM fungal morphology, taxonomy, and phylogeny and how they have improved our understanding of the processes leading to root colonization and development of the extraradical mycelium. This is supported by a summary of some of the most important findings, regarding this symbiosis, that have been made at the physiological, biochemical, and molecular levels. We also summarize results from studies between AM fungi and certain pathogenic and nonpathogenic soil microorganisms. We describe some of the limitations of this in vitro system and propose diverse avenues of AM research that can now be undertaken, including the potential use of a similar system for ectomycorrhizal research.Key words: arbuscular mycorrhiza, root-organ cultures, Glomales, in vitro, root symbioses, source of inoculum, cryopreservation, intraradical and extraradical mycelium, mycorrhizosphere.

Acer saccharum Marsh. (sugar maple) is one of only few arbuscular mycorrhizal trees to form extensive stands in northern temperate biomes. Recent maple decline could result from altered intensity and quality of root colonization by associated mycobionts or possible shifts in symbiotic fungal community composition following environmental stresses. In this study the effects on arbuscular mycorrhizal fungi of soil acidification, one of several proposed causal stresses underlying forest decline, and remedial liming were investigated under glasshouse conditions. Acer saccharum seedlings were grown in unsterilized, pH altered, forest soils from healthy and declining maple stands. Over a range of treatment pHs normally tolerated by A. saccharum, fungal populations and responses to pH changes differed between the two soils. The declining site with more acidic soil had an initially larger spore population but lower taxonomic diversity than the healthy site. However, liming stimulated sporulation of several taxa initially apparently absent from the declining site spore population. The quantity of colonization generally increased with pH for both sites. Five Glomus taxa and Scutellospora calospora (Nicol. & Gerd.) Walker & Sanders are added to the list of fungi known to form arbuscular mycorrhizas with A. saccharum, and the known range of Acaulospora cavernata Blaszkowski is extended from Poland to eastern North America.Résumé : Acer saccharum Marsh. (érable à sucre) est l'un des rares arbres endomycorhiziens à former des peuplements étendus dans les biomes tempérés nordiques. Suite au dépérissement des érablières, l'acidification des sols a été suggérée comme stress causal. Bien qu' A. saccharum puisse tolérer l'acidité des sols, les effets de cette acidification sur les symbioses endomycorhiziennes de l'érable ont été jusqu'à maintenant largement ignorés. L'impact du pH du sol sur le taux et la qualité de la colonisation endomycorhizienne ainsi que sur la diversité des Glomales a été étudié en serres sur des semis d'A. saccharum dans des sols provenant respectivement d'érablières en santé et en dépérissement, ajustés à différents pH. Initialement, le site en dépérissement au sol plus acide, avait un nombre plus élevé de spores malgré une diversité taxonomique réduite. Le chaulage subséquent de ce sol a stimulé la sporulation de plusieurs taxons apparemment absents au début de l'expérience. Cette étude a aussi démontré une corrélation positive du pH avec la quantité et la qualité de la colonisation intra-racinaire.

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To elucidate the effect of cold storage on spore dormancy in the arbuscular mycorrhizal (AM) fungus Glomus intraradices, spores were cold stratified at 4 degrees C, for either 0, 3, 7, 14, 90 or 120 days, prior to germination tests at 25 degrees C. The results showed that cold stratification longer than 14 days significantly increased spore germination. Moreover, the longer cold storage periods clearly reduced spore mortality from 90% to 50% and considerably altered the hyphal growth pattern. Long polarized hyphae were only observed after cold stratification periods longer than 14 days, involving consequences for root infectivity. The results clearly show that environmental factors, e.g., coldness, can affect the physiology of AM fungal spores.