R. L. Peterson

Modifications to clearing methods used in combination with vital staining of roots colonized with vesicular-arbuscular mycorrhizal fungi

Leek, maize, and pigmented soybean roots colonized by vesicular-arbuscular mycorrhizal (VAM) fungi were assessed for succinate dehydrogenase (SDH) activity using the nitro blue tetrazolium chloride (NBT)-succinate method. NBT-succinate-reacted roots, cleared in a 55° C drying oven in 5% (w/v) KOH for 24 h or longer and observed as whole mounts, revealed signs of intraradical VAM fungus colonization more clearly than roots cleared by the standard 20% (w/v) boiling chloral hydrate method. Combined clearing of NBT-succinate-reacted roots using boiling chloral hydrate followed by clearing in 5% KOH at 55° C for prolonged periods also improved the visualization of intraradical fungal structures. Bleaching of NBT-succinate-reacted roots using the standard NH3-H2O2 method removed pigmentation from roots and did not alter the viability indicator, formazan. Pigmented, field-collected soybean roots were successfully cleared and bleached to reveal signs of viable and nonviable intraradical fungal structures. Counterstaining of NBT-succinate-reacted roots with acid fuchsin clearly revealed both viable and nonviable intraradical fungal structures. The NBT-succinate solution infiltrated all intraradical fungal structures after 24 h; formazan products were observed at similar concentrations in viable structures after 24, 36, and 48 h.

Anatomical aspects of field ectomycorrhizas on Polygonum viviparum (Polygonaceae) and Kobresia bellardii (Cyperaceae)

Abstract Root systems of the herbaceous species Polygonum viviparum and Kobresia bellardii were excavated from an alpine site in the Rocky Mountains, Colorado, and processed for microscopic examination. Several ectomycorrhizal morphotypes were present on root systems of both species;K. bellardii often had complex clusters of mycorrhizal roots present. A mantle and Hartig net were present on all mycorrhizal root tips processed. The Hartig net was confined to the epidermis, and the parenchyma cells of this layer were radially elongated, vacuolated and contained densely staining inclusions. Intracellular hyphae and structures typical for vesicular-arbuscular mycorrhizas were never observed. Both herbaceous species, therefore, had ectomycorrhizal associations comparable to those described for woody angiosperm species.

Survival of the external mycelium of a VAM fungus in frozen soil over winter

The present investigation examines (1) whether the external VAM mycelium survives winter freezing to act as a source of inoculum in the spring, and (2) whether soil disturbance reduces the infectivity of the external VAM mycelium following freezing of the soil. Sealed pouches of fine nylon mesh were placed in pots containing soil inoculated with a Glomus species. The mesh was impervious to roots but not to hyphae. Following two 3-week growth cycles of maize in the pots, the pouches were transplanted to the field. Pouches were removed from the field once during the 4 months when the soil was frozen, and once after spring thaw. Measurements were made of VAM spore density, hyphal length and viability in the pouches. Bioassays for infectivity were conducted on all pouches. Some VAM hyphae survived freezing and remained infective following winter freezing, in the absence of plant roots. Soil disturbance did not reduce the infectivity of hyphae following exposure to freezing temperatures. We observed a change in the distribution of viable cytoplasm within hyphae over winter, which we hypothesize represents an adaptation allowing hyphae to survive freezing temperatures. We suggest that the effect of disturbance on hyphal infectivity may be related to this seasonal change in the distribution of hyphal viability.

Suppression of common root pathogens by helper bacteria and ectomycorrhizal fungi in vitro

Abstract Root pathogens cause considerable loss of tree seedlings in nurseries and are generally difficult to control using conventional methods. Inoculation with ectomycorrhizal fungi may provide some suppression of pathogens. Bacteria (so-called mycorrhization helper bacteria) have been isolated that stimulate mycorrhiza formation on seedling roots and enhance seedling growth; however, their role in pathogen inhibition has not been explored. Four strains of helper bacteria were inoculated together with the ectomycorrhizal fungal species Laccaria bicolor, L. proxima and Suillusgranulatus on culture plates to determine inhibition of the pathogens Fusariumoxysporum and Cylindrocarpon sp. Buffered medium was used to rule out acidification of the medium as a mechanism of inhibition. None of the ectomycorrhizal fungal species alone inhibited the growth of Fusarium but all showed slight inhibition of Cylindrocarpon growth. Helper bacterium strain MB3 (Bacillussubtilis) was effective in inhibiting both pathogens and, when inoculated with either L. proxima or S. granulatus, inhibition of Fusarium growth was enhanced over MB3 alone. With Cylindrocarpon, however, only S. granulatus inoculated along with MB3 showed enhanced inhibition over MB3 alone. The other three bacterial strains had little effect on the growth of Fusarium or Cylindrocarpon. More research is necessary to determine if these inhibitory effects are reproducible in situ.

Structure and histochemistry of mycorrhizae synthesized between Arbutus menziesii (Ericaceae) and two basidiomycetes, Pisolithus tinctorius (Pisolithaceae) and Piloderma bicolor (Corticiaceae)

Arbutoid mycorrhizae were synthesized in growth pouches between Arbutus menziesii Pursch. (Pacific madrone) and two broad host range basidiomycete fungi, Pisolithus tinctorius (Pers.) Coker and Couch and Piloderma bicolor (Peck) Jülich. P. tinctorius induced the formation of dense, pinnate mycorrhizal root clusters enveloped by a thick fungal mantle. P. bicolor mycorrhizae were usually unbranched, and had a thin or non-existent mantle. Both associations had the well-developed para-epidermal Hartig nets and intracellular penetration of host epidermal cells by hyphae typical of arbutoid interactions. A. menziesii roots developed a suberized exodermis which acted as a barrier to cortical cell penetration by the fungi. Ultrastructurally, the suberin appeared non-lamellar, but this may have been due to the imbedding resin. Histochemical analyses indicated that phenolic substances present in epidermal cells may be an important factor in mycorrhiza establishment. Analyses with X-ray energy dispersive spectroscopy showed that some of the granular inclusions present in fungal hyphae of the mantle and Hartig net were polyphosphate. Other inclusions were either protein or polysaccharides.

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular “fungal pegs” within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.

Comparative structural study ofQuercus serrata andQ. acutissima formed byPisolithus tinctorius andHebeloma cylindrosporum

Ectomycorrhizas were synthesized in pots and growth pouches betweenQuercus serrata, Q. acutissima, and two ectomycorrhizal fungi,Pisolithus tinctorius andHebeloma cylindrosporum. Root morphology and the structure of the mantle and Hartig net were compared using light, fluorescence, scanning and transmission electron microscopy.P. tinctorius initially colonized root cap cells, and eventually produced a highly branched lateral root system with a complete mantle, whereasH. cylindrosporum promoted root elongation with few hyphae on the root apex surface indicating that interaction between roots differs with fungal species. Hartig net structure and hyphal inclusions varied between all the combinations tested. There were structural differences between mycorrhizas ofH. cylindrosporum/Q. acutissima grown in soil and growth pouches, which indicate that the growth pouch environment can induce artefacts in roots. Fruit bodies ofH. cylindrosporum developed in pots withQ. acutissima. AlthoughP. tinctorius has been used to inoculate oak seedlings in the nursery, results of this study indicate thatH. cylindrosporum may also be an effective ectomycorrhizal fungus forQ. serrata andQ. acutissima.

Comparative studies of ectomycorrhiza formation in Alnus glutinosa and Pinus resinosa with Paxillus involutus

Abstract Mycorrhiza ontogeny and details of Hartig net and mantle structure were compared in ectomycorrhizas synthesized in growth pouches between the broad host range fungus Paxillus involutus and the tree species European black alder (Alnus glutinosa) and red pine (Pinus resinosa). In Alnus glutinosa, a paraepidermal Hartig net was restricted to the proximal (basal) portion of first-order laterals; the hypodermal layer appeared to be a barrier to fungal penetration. Phi-thickenings were present in some cortical cells but these were not related to lack of fungal ingress into the cortex. The mantle was often present close to the root apex but in many roots it was loosely organized and patchy. In several instances, the mantle formed around the root apex was only temporary; renewed root growth occurred without the formation of a mantle. In Pinus resinosa, the Hartig net developed between cortical cell layers of monopodial and dichotomously branched first–order laterals. Fungal hyphae in the Hartig net exhibited a complex labyrinthine mode of growth. The mantle had a pseudoparenchymatous structure and covered the root, including apices of dichotomously branched roots. The Paxillus–Pinus resinosa interaction had all the characteristics of a compatible ectomycorrhizal association. The Paxillus–Alnus glutinosa interaction, however, showed only aspects of superficial ectomycorrhizas, including the presence of a minimal (sometimes absent) and mostly proximal Hartig net and variable mantle development. Sclerotia were produced in the extraradical mycelium of Paxillus involutus when associated with either Alnus glutinosa or Pinus resinosa.

Ectomycorrhiza formation between Pseudotsuga menziesii seedling roots and monokaryotic and dikaryotic isolates of Laccaria bicolor

Seedling roots of Pseudotsuga menziesii were colonized with three monokaryotic isolates and one dikaryotic isolate of Laccaria bicolor to assess the effect of fungal genotype on ectomycorrhiza formation. Ectomycorrhizas resulting from colonization by the dikaryotic isolate had a multilayered mantle and a cortical Hartig net. One monokaryotic isolate (ss7) formed ectomycorrhizas comparable in anatomy to those induced by the dikaryotic isolate. Two other monokaryotic isolates (ss5, ss1) failed to form mantles or Hartig nets. Roots colonized by these isolates developed characteristics indicating an incompatible reaction.