Tissue-Specific and Development-Dependent Accumulation of Phenylpropanoids in Larch Mycorrhizas

The tissue-specific and development-dependent accumulation of secondary products in roots and mycorrhizas of larch (Larix decidua Mill.; Pinaceae) was studied using high-performance liquid chromatography and histochemical methods. The compounds identified were soluble catechin, epicatechin, quercetin 3-O-[alpha]-rhamnoside, cyanidin- and peonidin 3-O-[beta]-glucoside, 4-O-[beta]-hydroxybenzoyl-O-[beta]-glucose, 4-hydroxybenzoate 4-O-[beta]-glucoside, maltol 3-O-[beta]-glucoside, and the wall-bound 4-hydroxybenzaldehyde, vanillin, and ferulate. In addition, we partially identified a tetrahydroxystilbene monoglycoside, a quercetin glycoside, and eight oligomeric proanthocyanidins. Comparison between the compounds accumulating in the apical tissue of fine roots, long roots, and in vitro grown mycorrhizas (L. decidua-Suillus tridentinus) showed elevated levels of the major compounds catechin and epicatechin as well as the minor compound 4-hydroxybenzoate 4-O-[beta]-glucoside specifically in the root apex of young mycorrhizas. The amounts of wall-bound 4-hydroxybenzaldehyde and vanillin were increased in all of the mycorrhizal sections examined. During the early stages of mycorrhization the concentrations of these compounds increased rapidly, perhaps induced by the mycorrhizal fungus. In addition, studies of L. decidua-Boletinus cavipes mycorrhizas from a natural stand showed that the central part of the subapical cortex tissue and the endodermis both accumulate massive concentrations of catechin, epicatechin, and wall-bound ferulate compared with the outer part of the cortex, where the Hartig net is being formed.     

Induction of resistance against Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial strain: ultrastructure and cytochemistry of the host response

The potential of Bacillus pumilus (PGPR strain SE 34), either alone or in combination with chitosan, for inducing defense reactions in tomato (Lycopersicon esculentum Mill.) plants inoculated with the vascular fungus, Fusarium oxysporum f. sp. radicis-lycopersici, was studied by light and transmission electron microscopy and further investigated by gold cytochemistry. The key importance of fungal challenge in the elaboration of defense mechanisms is discussed in relation to the possibility that an alarm signal provided by the pathogen itself is required for the expression of resistance in plants previously sensitized by biotic agents. Ultrastructural investigations of the infected root tissues from water-treated (control) plants showed a rapid colonization of all tissues including the vascular stele. In root tissues from bacterized tomato plants grown in the absence of chitosan, the limited fungal development coincided with marked changes in the host physiology. The main facets of the altered host metabolism concerned the induction of a structural response at sites of fungal entry and the abnormal accumulation of electron-dense substances in the colonized areas. A substantial increase in the extent and magnitude of the cellular changes induced by B. pumilus was observed when chitosan was supplied to bacterized tomato plants. These changes were characterized by a considerable enlargement of the callose-enriched wall appositions deposited onto the inner cell wall surface in the epidermis and the outer cortex. The use of the wheat germ agglutinin-ovomucoid-gold complex provided evidence that the wall-bound chitin component in Fusarium cells colonizing bacterized tomato roots was not substantially altered. One of the most-typical fungal cell reactions, observed only when bacterized tomato plants were grown in the presence of chitosan, was the formation of abnormal chitin-enriched deposits between the retracted plasma membrane and the cell wall. Results of the present study provide the first evidence that combination of biocontrol approaches is a promising step towards elaborating integrated pest management programmes. Received: 6 June 1997 / Accepted: 8 July 1997     

Primary attraction and kairomonal host discrimination in three species of Dendroctonus (Coleoptera: Scolytidae)

Abstract 1 Synthetic blends of bole and foliage volatiles of four sympatric species of conifers were released from pheromone‐baited multiple‐funnel traps to determine if three species of tree‐killing bark beetles (Coleoptera: Scolytidae): (i) exhibited primary attraction to volatiles of their hosts and (ii) discriminated among volatiles of four sympatric species of host and nonhost conifers. 2 Bole and foliage volatiles from Douglas‐fir, Pseudotsuga menziesii (Mirb.) Franco, increased the attraction of coastal and interior Douglas‐fir beetles, Dendroctonus pseudotsugae Hopkins, to pheromone‐baited traps. Primary attraction to bole volatiles was observed in interior D. pseudotsugae. Beetles were significantly less attracted to the pheromone bait when it was combined with volatiles of lodgepole pine, Pinus contorta var. latifolia Engelm. or interior fir, Abies lasiocarpa × bifolia. 3 The monoterpene myrcene synergized attraction of mountain pine beetles, Dendroctonus ponderosae Hopkins, to their aggregation pheromones, but there was no evidence of primary attraction to host volatiles or discrimination among volatiles from the four conifers. 4 There was significant primary attraction of the spruce beetle, Dendroctonus rufipennis Kirby, to bole and foliage volatiles of interior spruce, Picea engelmannii × glauca, but beetles did not discriminate among volatiles of four sympatric conifers when they were combined with pheromone baits. 5 Our results indicate that host volatiles act as kairomones to aid pioneer Douglas‐fir beetles and spruce beetles in host location by primary attraction, and that their role as synergists to aggregation pheromones is significant. For the mountain pine beetle, we conclude that random landing and close range acceptance or rejection of potential hosts would occur in the absence of aggregation pheromones emanating from a tree under attack.     

Differential selection of North American and Scandinavian conifer browse by northwestern moose (Alces alces andersoni) in winter

Scandinavian moose (Alces alces) eat Scots pine (Pinus sylvestris) in winter. Although North American moose are known to eat conifers such as true firs (Abies spp.) in winter, substantial consumption of pine by moose in North America has not been documented. Here, we document short-term winter preferences of human-habituated northwestern moose (Alces alces andersoni) for branches of mature North American and European conifer species as determined by a cafeteria-style feeding trial. Moose selected for species such as Douglas fir (Pseudotsuga menziesii; from which they took the smallest bite diameters) while avoiding species such as lodgepole pine (Pinus contorta; from which they took the largest bites) and hybrid white spruce (Picea glauca × engelmanii). The amount of species-specific biomass consumed by moose was negatively correlated with bite diameters taken from branches of those species and did not appear to be significantly influenced by differences in twig morphology between species. Our trial suggests that northwestern moose readily consume conifers in winter and, from the species we tested, prefer Douglas fir. While no clear preference existed between Scots pine and lodgepole pine, moose avoided lodgepole pine, but not Scots pine, relative to Douglas fir. Our trial suggests that northwestern moose are more likely to feed on the branches of Douglas fir than pine, which may be of interest to foresters managing conifers within the North American range of moose, particularly where Scots pine are being considered for planting.     

Coinoculation of aseptically grown Douglas fir with pairs of ectomycorrhizal fungi

Douglas fir seedlings grown under aseptic conditions in a peat-vermiculite substrate were inoculated with four pairs of ectomycorrhizal fungi to assess the relative inoculum dosages needed to establish two mycorrhizal fungi simultaneously in the same root system. The dual fungal combinations tested were: Pisolithus arhizus + Rhizopogon subareolatus, P. arhizus + R. roseolus, Laccaria bicolor + P. arhizus and L. bicolor + R. subareolatus. A total of 12 ml of inocula per plant was applied at the rates: 0+12, 3+9, 6+6, 9+3, 12+0, and 0+0 (v+v) for each combination. After 3 months growth, the number of mycorrhizas and uninfected short roots as well as the total plant biomass produced were recorded. Inoculations were successful with the fungal combinations P. arhizus + R. subareolatus and L. bicolor + P. arhizus. Plants developed P. arhizus and R. subareolatus mycorrhizas only at the rate 9Pa + 3Rs; at other rates tested, only monospecific mycorrhizas were formed. Plants developed L. bicolor and P. arhizus mycorrhizas at the three rates containing both fungi. L. bicolor behaved as an aggressive root colonizer and its level of root colonization remained constant at increasing rates of P. arhizus inoculum. L. bicolor displaced R. subareolatus at all inocula rates. P. arhizus displaced R. roseolus except at the rate 3Pa + 9Rr, with only a low number of mycorrhizas formed by either fungus. Total plant biomass was significantly increased by the presence of any fungal combination up to four times the values for uninoculated controls. P. arhizus and R. subareolatus were more effective in promoting plant growth and stimulating short root formation than either L. bicolor or R. roseolus.     

Structure and growth of infection threads in the legume symbiosis with Rhizobium leguminosarum

Specific antibodies and enzyme–gold probes were used to study the structure and development of infection threads in nodules induced by Rhizobium leguminosarum on the roots of Vicia, Pisum and Phaseolus. In Pisum nodules, the tubular infection thread wall contains polysaccharides antigenically similar to those of the cell wall, including cellulose, xyloglucan, methyl-esterified pectin and non-esterified pectin, but none of these wall components is present around the infection droplet structures from which bacteria are internalized by plant plasma membrane. As reported previously for pea nodules, the luminal matrix of infection threads and infection droplets contains a plant glycoprotein; this glycoprotein is also secreted by infected and uninfected cortical cells of a Vicia root at the earliest stages of nodule initiation. Synthesis of a transcellular infection thread apparently involves reorganized deposition of components normally targeted to the cell wall, and infection thread growth is orientated anticlinally through the outer cortex in the same plane observed for the deposition of new cell walls following mitosis. Both the development of infection threads in the outer cortex and the initiation of cell division in the inner cortex are preceded by a similar process of cell reactivation involving centralization of nuclei and the development of anticlinal transvacuolar strands. It is therefore suggested that the two Rhizobium-induced processes of infection thread growth and cortical cell division may both be consequences of a similar plant cell response in the inner and outer root cortex, respectively. Phaseolus nodules contained only short intracellular infection structures which terminated within individual cells and contained no luminal matrix material. The differences in infection thread structure between Pisum and Phaseolus nodules may reflect differences in ontogeny between “indeterminate” and “determinate” nodule meristems.     

Chemistry and occurrence of hydroxycinnamate oligomers

Hydroxycinnamates such as ferulic acid, sinapic acid and p-coumaric acid ester-linked to plant cell wall polymers may act as cross-links between polysaccharides to each other, but also to proteins and lignin. Although sinapates and p-coumarates also form cell wall cross-links by the formation of radically or photochemically formed dimers, ferulate derivatives are the quantitatively most important cross-links in the plant cell wall. While the first radically generated ferulate dimer was already identified almost 40 years ago, the spectrum of known ferulate dimers was considerably broadened within the last 15 years. Higher ferulate oligomers were generated in model systems, but also isolated from plant materials. Different model systems using either free hydroxycinnamic acids or their esters are reviewed, highlighting a discussion of the relevance of these models for the plant cell wall. The first ferulate trimer from plant material was discovered in 2003 and seven dehydrotrimers of ferulic acid were isolated from maize bran since. Some of these trimers were also identified in other plant materials such as wheat and rye grains, corn stover, sugar beet and asparagus. Formation mechanisms of ferulate trimers and implications for the plant cell wall are discussed. Ferulate tetramers are the highest oligomers isolated from plant materials so far. These compounds can theoretically cross-link up to four polysaccharide chains, assuming all cross-links are formed intermolecularly. Formation of intramolecular versus intermolecular polysaccharide cross-links is a key question to be answered in the future if we want to judge properly the importance of hydroxycinnamate cross-links in the plant cell wall.     

A morphological study of the mycorrhiza ofEntoloma clypeatum f.hybridum onRosa multiflora

Mycorrhizas ofEntoloma clypeatum f.hybridum onRosa multiflora in the field in Japan were studied by stereo, light and electron microscopy. In most mycorrhizas, the root cap, meristem, and apical region of the cortex disappeared, but in a few mycorrhizas, these tissues remained. Fungal hyphae of the mycorrhizas invaded root tissues and branched palmately. Hyphae in contact with cortical cells were larger than those far from the root cells and contained many mitochondria, cisternae of endoplasmic reticulum and transitional vesicles. Invading hyphae were undulate in the apical part of the mycorrhiza, and some of them lacked distinct organelles. Electron-dense granules accumulated in the root cells adjacent to the fungal hyphae. Both the remnants of the plant cells and the fungal hyphae were included in the amorphous materials on the tip of the stele. These observations suggest the destructive infection by fungal hyphae of the root cells and their collapse near the tip of the stele.     

Early colonization of red alder and Douglas fir by ectomycorrhizal fungi and Frankia in soils from the Oregon coast range

The potential for mycorrhizal formation and Frankia nodulation were studied in soils from six sites in the Pacific Northwest. The sites included young and old alder stands, a 1-year-old conifer clear-cut, a young conifer plantation, and rotation-aged and old-growth conifer stands. A bioassay procedure was used with both red alder and Douglas fir seedlings as hosts. After 6 weeks growth, seedlings of both hosts were harvested every 3 weeks for 21 weeks and numbers of nodules and ectomycorrhizal types estimated. Nodules formed on red alder and ectomycorrhizae formed on both alder and Douglas fir in soil from all sites. Nodulation potential was highest in soil from the alder stands and the conifer plantation. Seven morphologically distinct ectomycorrhizal types were recovered on Douglas fir and five on alder. Only Thelephora terrestris, a broad-host-range mycobiont, formed mycorrhizae on both hosts. New ectomycorrhizal types formed on both hosts throughout the bioassay. Ectomycorrhizal colonization of alder was greatest in the alder and clear-cut soils. Low ectomycorrhizal colonization on alder was found in soils from sites where conifers were actively growing. Ectomycorrhizal colonization of Douglas fir was highest in the young alder and conifer plantation soils and was low in the rotation-aged conifer soil. The highest diversity of ectomycorrhizal types was found on alder in the conifer clear-cut soil and on Douglas fir in the rotation-aged conifer soil. Effects of host specificity, nodulation and mycorrhiza-forming potential and nodule-mycorrhiza interactions on seedling establishment are discussed in relation to seral stage dynamics and attributes of pioneer ectomycorrhizal fungal species.     

Comparative study of mycorrhizal susceptibility and anatomy of four palm species

A morphological and anatomical study of the root systems of the palm species Brahea armata S. Watson, Chamaerops humilis L., Phoenix canariensis Chabaud and Phoenix dactylifera L. has been carried out to determine possible mycorrhizal colonization sites. Furthermore, the arbuscular mycorrhizal (AM) anatomical types formed by the four palm species in association with Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe have been examined. The presence of a continuous sclerenchymatic ring in the outer cortex and aerenchyma in the inner cortex that are anatomical indicators of mycorrhizal nonsusceptibility in all four palm species is observed. The root systems of B. armata and C. humilis present only one group of third-order roots, while the third-order roots of P. canariensis and P. dactylifera may be divided into five different groups: short thick roots, mycorrhizal thickened roots, fine short roots, fine long roots, and pneumatorhizas. Third-order and some second-order roots of B. armata and C. humilis are susceptible to colonization by AM fungi, while only the mycorrhizal thickened roots form mycorrhizas with arbuscules in the Phoenix species. The root system of the Phoenix species also presents AM colonization in fine roots with only intraradical hyphae and spores, but without arbuscules, and pseudomantles of spores anchored in the pneumatorings of the second-order roots, which are described for the first time. The mycorrhizas formed by the four palm species are of an intermediate type, between the Arum and the Paris types, and are characterized by intercalary arbusculate coils and not only by intracellular but also by intercellular fungal growth. Our study suggests that a different degree of adaptation may exist among palm mycorrhizas toward the slow growth of palms and low spore numbers in the soil where they grow.     

Compositional characterization and imaging of “wall-bound” acylesters of Populus trichocarpa reveal differential accumulation of acyl molecules in normal and reactive woods

Acylesterification is one of the common modifications of cell wall non-cellulosic polysaccharides and/or lignin primarily in monocot plants. We analyzed the cell-wall acylesters of black cottonwood (Populus trichocarpa Torr. & Gray) with liquid chromatography-mass spectrometry (LC-MS), Fourier transform-infrared (FT-IR) microspectroscopy, and synchrotron infrared (IR) imaging facility. The results revealed that the cell wall of dicotyledonous poplar, as the walls of many monocot grasses, contains a considerable amount of acylesters, primarily acetyl and p-hydroxycinnamoyl molecules. The "wall-bound" acetate and phenolics display a distinct tissue specific-, bending stress responsible- and developmental-accumulation pattern. The "wall-bound" p-coumarate predominantly accumulated in young leaves and decreased in mature leaves, whereas acetate and ferulate mostly amassed in the cell wall of stems. Along the development of stem, the level of the "wall-bound" ferulate gradually increased, while the basal level of p-coumarate further decreased. Induction of tension wood decreased the accumulation of the "wall-bound" phenolics while the level of acetate remained constant. Synchrotron IR-mediated chemical compositional imaging revealed a close spatial distribution of acylesters with cell wall polysaccharides in poplar stem. These results indicate that different "wall-bound" acylesters play distinct roles in poplar cell wall structural construction and/or metabolism of cell wall matrix components.     

THE ROOT APICAL MERISTEM OF EQUISETUM DIFFUSUM: STRUCTURE AND DEVELOPMENT

The root apical meristem of Equisetum diffusum Don has a prominent four-sided pyramidal apical cell with its base (distal face) in contact with the root cap. Derivatives (merophytes) that contribute to the main body of the root are produced from the three proximal faces of the apical cell. The first division of a proximal merophyte is periclinal to the root surface separating a small inner cell from a larger outer cell. The inner cell is the precursor of the vascular cylinder. The larger outer cell is the precursor of the epidermis, cortex, endodermis, and pericycle. Radial sectors, established early in the development of the cortex, alternate with sectors in the vascular cylinder. These developmental steps show quite clearly that early root development in Equisetum is markedly different from that of most ferns.     

Regulation of photoassimilate allocation in Pinus sylvestris seedlings by the nutritional status of the mycorrhizal fungus Suillus variegatus

Summary Seedlings of Pinus sylvestris L. were grown on defined nutrient solutions on carbon filters, either sterile or infected with the basidiomycete Suillus variegatus O. Kuntze. After mycorrhizas were established, the shoot of the seedling was subjected to ¹⁴CO₂ photosynthesis. ¹⁴C-labelled photoassimilates were translocated to both mycorrhizas and non-infected root tips. Microautoradiographs of mycorrhizas indicated that omission of external sugars did not affect the formation of mycorrhizas; ¹⁴C-photoassimilates were supplied to cortex, Hartig net and the mantle of hyphae surrounding the rootlet. Nutrient solution containing sugars (malt extract, glucose) enhanced the growth of the fungus. As a consequence, ¹⁴C-photoassimilates from the seedling were accumulated in the mantle, but defence mechanisms of the host cannot be excluded. When soluble nitrogen was omitted from the nutrient solution and replaced by chitin precipitated on the filter-bearing mycorrhizas, the fungus appeared strongly labelled in the mantle, where the fungal chitinase provided soluble nitrogen compounds, necessary for the growth of the seedling.     

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum

The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.     

Tricholoma matsutake– an assessment of in situ and in vitro infection by observing cleared and stained whole roots

 Structures present within field-collected Tricholoma matsutake/Pinus densiflora ectomycorrhizas and in vitro infections of P. densiflora roots by T. matsutake were observed by clearing, bleaching and staining whole lateral roots and mycorrhizas. Field mycorrhizas were characterized by a lack of root hairs, by the presence of a sparse discontinuous mantle composed of irregularly darkly staining hyphae over the root surface, primarily behind the root cap, and by the presence of Hartig net mycelium within the root cortex. Hartig net 'palmettis' were classified into three basic structures, each with distinctive morphologies. Aerial hyphae, bearing terminal swellings, were observed emanating from the mantle. Cleared, bleached and stained in vitro-infected roots possessed multibranched hyphal structures within the host root cortex and aerial hyphae bearing terminal swellings were observed arising from the mycelium colonizing the root surface. T. matsutake on P. densiflora conforms to the accepted morphology of an ectomycorrhiza. This staining protocol is particularly suited to the study of Matsutake mycorrhizal roots and gives rapid, clear, high-contrast images using standard light microscopy while conserving spatial relationships between hyphal elements and host tissues. Accepted: 26 August 1999     

Fungal adhesion pad formation and penetration of root cuticle in early stage mycorrhizas ofPicea abies andLaccaria amethystea

Summary Primary events during the establishment of the fungus-root symbiosis in ectomycorrhizas are still little understood. No attention has been paid so far to the adhesion of hyphae to the root cuticle and penetration of this barrier, although the importance of the cuticle has been shown for pathogen-plant interactions. Early developmental stages of in vitro mycorrhization ofLaccaria amethystea onPicea abies after short periods of incubation in growth chambers under elevated CO₂ concentrations were studied by light and transmission electron microscopy. No structural changes in mycorrhization related to elevated CO₂ were found, but fine roots and mycorrhizas developed faster. Adhesion pad formation was observed at hyphal tips in contact with the root cuticle. The adhesion pad was connected to the outer cell wall layer of the hypha and reacted positively to the Swift reaction for cysteine rich proteins. Although the reaction cannot be considered as totally specific, findings are discussed in respect to hydrophobins, which have recently been found to be expressed during early steps in ectomycorrhizal development. The root cuticle was dissolved and penetrated by fungal tips of the fingerlike branching mycelium attached to the root surface. The findings are compared with well documented pathogenic fungus-plant interactions at the cuticle. The possibility of restriction of hyphal attack to that part of the cuticle covering cell junctions is discussed.     

Structure and permeability of the fungal sheath in thePisonia mycorrhiza

Summary The tracer Cellufluor has been used to test the apoplastic permeability of the fungal sheath inPisonia grandis R. Br. mycorrhizas. In the tip region in the immediate vicinity of the root cap, where the sheath is not yet fully differentiated, Celluflor penetrates as far as the root epidermal cells. Behind this (i.e. just proximal to it) in differentiated regions, where the ultrastructure of both the root and fungal cells indicates that the mycorrhiza is likely to be functionally active, the sheath is impermeable to Cellufluor. During the development and differentiation of the sheath, the interhyphal spaces become filled with extracellular material. In the outer and middle regions this becomes electron opaque after fixation and staining. It is proposed that the dramatic decrease in apoplastic permeability over a short distance back from the root apex as the fungal sheath differentiates results from secretion of extracellular material by the fungus and its modification by deposition of phenolic substances. The symplastic pathway within the fungus may be very important for radial transfer of materials across the sheath. Blockage of the sheath apoplast could provide a sealed apoplastic compartment at the fungus-root interface, with resulting increase in efficiency of transfer between partners. The implications of these observations are discussed in relation to radial transfer across the sheath and transfer between partners in sheathing mycorrhizas in general.     

Tree‐ring stable isotopes record the impact of a foliar fungal pathogen on CO2 assimilation and growth in Douglas‐fir

Swiss needle cast (SNC) is a fungal disease of Douglas‐fir (Pseudotsuga menziesii) that has recently become prevalent in coastal areas of the Pacific Northwest. We used growth measurements and stable isotopes of carbon and oxygen in tree‐rings of Douglas‐fir and a non‐susceptible reference species (western hemlock, Tsuga heterophylla) to evaluate their use as proxies for variation in past SNC infection, particularly in relation to potential explanatory climate factors. We sampled trees from an Oregon site where a fungicide trial took place from 1996 to 2000, which enabled the comparison of stable isotope values between trees with and without disease. Carbon stable isotope discrimination (Δ¹³C) of treated Douglas‐fir tree‐rings was greater than that of untreated Douglas‐fir tree‐rings during the fungicide treatment period. Both annual growth and tree‐ring Δ¹³C increased with treatment such that treated Douglas‐fir had values similar to co‐occurring western hemlock during the treatment period. There was no difference in the tree‐ring oxygen stable isotope ratio between treated and untreated Douglas‐fir. Tree‐ring Δ¹³C of diseased Douglas‐fir was negatively correlated with relative humidity during the two previous summers, consistent with increased leaf colonization by SNC under high humidity conditions that leads to greater disease severity in following years.     

Mycorrhizal associations in Pinus massoniana Lamb. and Pinus elliottii Engel. inoculated with Pisolithus tinctorius

Dichotomous mycorrhizas were induced in Pinus massoniana and Pinus elliottii seedlings inoculated with Pisolithus tinctorius growing under non-axenic conditions. Six months after inoculation, Pinus massoniana seedlings exhibited a higher degree of infection, bore more mycorrhizas and had developed more abundant extramatrical mycelium than seedlings of Pinus elliottii. Nevertheless, seedlings of Pinus massoniana were stunted and exhibited chorosis of the needles, indicating a possible nutrient deficiency. Histological examination of these pine mycorrhizas showed an ectomycorrhizal association typical of gymnosperms with an intercellular Harting net penetrating between several layers of cortical cells close to the endodermis. However, strong polyphenolic reactions, intracellular hyphae and wall modifications were occasionally observed, indicating that both host-tissue incompatibility and ectendomycorrhizal association can occur in pine species under stressed conditions.     

Structural characteristics of root–fungus associations in two mycoheterotrophic species, Allotropa virgata and Pleuricospora fimbriolata (Monotropoideae), from southwest Oregon, USA

All members of the Monotropoideae (Ericaceae), including the species, Allotropa virgata and Pleuricospora fimbriolata, are mycoheterotrophs dependent on associated symbiotic fungi and autotrophic plants for their carbon needs. Although the fungal symbionts have been identified for A. virgata and P. fimbriolata, structural details of the fungal–root interactions are lacking. The objective of this study was, therefore, to determine the structural features of these plant root–fungus associations. Root systems of these two species did not develop dense clusters of mycorrhizal roots typical of some monotropoid species, but rather, the underground system was composed of elongated rhizomes with first- and second-order mycorrhizal adventitious roots. Both species developed mantle features typical of monotropoid mycorrhizas, although for A. virgata, mantle development was intermittent along the length of each root. Hartig net hyphae were restricted to the host epidermal cell layer, and fungal pegs formed either along the tangential walls (P. fimbriolata) or radial walls (A. virgata) of epidermal cells. Plant-derived wall ingrowths were associated with each fungal peg, and these resembled transfer cells found in other systems. Although the diffuse nature of the roots of these two plants differs from some members in the Monotropoideae, the structural features place them along with other members of the Monotropoideae in the “monotropoid” category of mycorrhizas.