Ectomycorrhizal Fungal Assemblages of Nursery-Grown Scots Pine are Influenced by Age of the Seedlings

Scots pine (Pinus sylvestris L.) is the most widely distributed pine species in Europe and is relevant in terms of planted areas and harvest yields. Therefore, each year the demand for planting stock of Scots pine is exceedingly high, and large quantities of seedlings are produced annually throughout Europe to carry out reforestation and afforestation programs. Abundant and diverse ectomycorrhizal (ECM) symbiosis is critical for the success of seedlings once planted in the field. To improve our knowledge of ECM fungi that inhabit bare-root nursery stock of Scots pine and understand factors that influence their diversity, we studied the assemblages of ECM fungi present across 23 bare-root forest nurseries in Poland. Nursery stock samples were characterized by a high level of ECM colonization (nearly 100%), and a total of 29 ECM fungal taxa were found on 1- and 2-year-old seedlings. The diversity of the ECM community depended substantially on the nursery and age of the seedlings, and species richness varied from 3–10 taxa on 1-year-old seedlings and 6–13 taxa on 2-year-old seedlings. The ECM fungal communities that developed on the studied nursery stock were characterized by the prevalence of Ascomycota over Basidiomycota members on 1-year-old seedlings. All ecological indices (diversity, dominance, and evenness) were significantly affected by age of the seedlings, most likely because dominant ECM morphotypes on 1-year-old seedlings (Wilcoxina mikolae) were replaced by other dominant ones (e.g., Suillus luteus, Rhizopogon roseolus, Thelephora terrestris, Hebeloma crustuliniforme), mostly from Basidiomycota, on 2-year-old seedlings. Across all nurseries, negative correlations were identified for diversity metrics and soil N or C, indicating that mineral and organic fertilization contributes to the differences in the ECM fungal communities in nurseries. We discuss the ecological and practical implications of the composition and diversity of ECM fungi occurring on bare-root planting stock of Scots pine.

Molecular identification of ectomycorrhizas associated with ponderosa pine seedlings in Patagonian nurseries (Argentina)

Ponderosa pine ( Pinus ponderosa Dougl. ex P. & C. Laws.), an ectomycorrhiza (EM) dependent species, has been widely introduced in Patagonia, Argentina. This study used morphotyping, restriction analysis, and sequencing of EM root tips from ponderosa pine seedlings in two nurseries to assess the complete EM fungus (EMF) richness, to confirm doubtful identities of commonly reported morphotypes, and to evaluate the efficiency of morphotyping compared with molecular analysis. This interdisciplinary approach together with the fact that is the first study in which Patagonian nurseries EMF are genetically evaluated contributes to the general knowledge of this important group of fungi. Sequencing revealed the presence of 11 taxa. Basidiomycetes included Thelephoraceae ( Tomentella sp.), Atheliaceae ( Amphinema byssoides (Pers.) J. Erikss.), Hydnangiaceae ( Laccaria sp.), Rhizopogonaceae ( Rhizopogon roseolus (Corda) Th. Fr.), and Cortinariaceae ( Hebeloma mesophaeum (Pers.) Quel.). Ascomycetes included Pezizaceae ( Wilcoxina mikolae (Chin S. Yang & H.E. Wilcox) Chin S. Yang & Korf and Wilcoxina sp.) and Tuberaceae ( Tuber sp.). Morphotyping proved to be useful for certain EMF species (R. roseolus, H. mesophaeum, A. byssoides, and to a lesser extent Tuber sp.) in which some morphological features are conspicuous and unique. Our detection of W. mikolae and Wilcoxina sp. are new records for ponderosa pine in Patagonia. All of the EM taxa identified are common to pine plantations and nurseries around the world, and no indigenous EM associated with native Nothofagus spp. were found.

Host microtubules in the Hartig net region of ectomycorrhizas, ectendomycorrhizas, and monotropoid mycorrhizas

Various categories of mycorrhizas are recognized primarily by the structural changes that occur between fungi and roots. In all mycorrhiza categories, cytological modifications of root cells accompany the establishment of the functional symbiosis, and among these are alterations in the organization of the cytoskeleton. Using immuno labelling combined with confocal scanning laser microscopy, this study documents changes in microtubules (MTs) in root cells of ectendomycorrhizas and monotropoid mycorrhizas; in addition, ectomycorrhizas were reinvestigated to determine the effect of fungal colonization on host root cells. In Pinus banksiana L. – Laccaria bicolor (Maire) Orton ectomycorrhizas, MTs were present in epidermal and cortical cells adjacent to the Hartig net. The remaining cortical MTs had a different organization when compared with those of cortical cells of control roots. MTs were present in Hartig net hyphae. In ectendomycorrhizas formed when roots of P. banksiana were colonized by the ascomycete, Wilcoxina mikolae var. mikolae Yang & Korf, MTs were present adjacent to intracellular hyphae and host nuclei, but few cortical MTs were present. MTs were present within Hartig net and intracellular hyphae. In field-collected roots of Monotropa uniflora L., MTs were associated with fungal pegs, intracellular extensions of inner mantle hyphae within epidermal cells. The close association between MTs and fungal pegs may be related to the formation of the highly branched host-derived wall that envelops each fungal peg. The development of exchange interfaces in the three systems studied involve changes in the organization of microtubules.Key words: cytoskeleton, microtubules, Hartig net, mycorrhizas, immunolocalization, confocal microscopy.

Molecular systematics of E-strain mycorrhizal fungi: Wilcoxina and its relationship to Tricharina (Pezizales)

Nuclear-encoded ribosomal RNA gene sequences (rDNA) spanning 107 base pairs at the 3′ end of the 18S gene, the 5′ internal transcribed spacer region (ITS1), and across divergent domain D1 near the 5′ end of the 28S gene were analyzed to infer a phylogeny for taxa of the E-strain mycorrhizal fungal genus Wilcoxina and to determine their relationship to representatives of the genus Tricharina. The phylogeny suggests that Wilcoxina and Tricharina, although related, should be maintained as separate genera. Wilcoxina taxa formed a distinct group that exhibited interspecific variation of 37.6% in the ITS1 region. Wilcoxina alaskana was the most distant taxon, which is consistent with its growth on an unusual substrate (rotting wood). It remains to be confirmed that this taxon is mycorrhizal. A cryptic Wilcoxina species, known only from root isolates, was found. Sequence analysis of the ITS1 region distinguished two varieties of Wilcoxina mikolae: var. mikolae and var. tetraspora. Among the taxa of Tricharina examined there was 31.2% variation in the ITS1 region. The most divergent taxon in the Tricharina group was Tricharina praecox, although sequence analysis was unable to distinguish the varieties described within this species. The remaining taxa in Tricharina formed a tight group with only 10.2% interspecific divergence in the ITS1 region. There is sequence evidence that at least two taxa are included in Tricharina gilva as presently delineated. As the sole report of mycorrhiza formation in Tricharina is shown to be based upon a misidentification, it therefore appears that only Wilcoxina taxa are mycorrhizal. Keywords: E-strain, Wilcoxina, Tricharina, mycorrhiza, ribosomal DNA, phylogeny.

Structure of ectomycorrhizae formed by Wilcoxina mikolae var. mikolae with Picea mariana and Betula alleghaniensis

The structure of ectomycorrhizae synthesized between the E-strain fungus, Wilcoxina mikolae var. mikolae and two tree species, Picea mariana and Betula alleghaniensis, was characterized by light microscopy and scanning electron microscopy. For both mycorrhizal types, mantle formation was visible on lateral roots within 10 days of inoculation. Picea mariana ectomycorrhizae had a very thin mantle whereas B. alleghaniensis ectomycorrhizae had a mantle consisting of several layers. For both mycorrhizal types, the innermost mantle hyphae were embedded in a considerable amount of mucigel on the root surface. A well-developed Hartig net with labyrinthic growth occurred in both types of mycorrhizae. Betula alleghaniensis ectomycorrhizae had a paraepidermal Hartig net, and the root epidermal cells were radially elongate at an oblique angle. The Hartig net of P. mariana ectomycorrhizae penetrated the epidermis and all layers of the cortex. The cytoplasmic density of the intercellular hyphae was greatest towards the root apex. Ectomycorrhizal associations formed by E-strain fungi were similar to ectomycorrhizae formed by other fungi. Key words: E-strain, ectomycorrhizae, Wilcoxina, Picea, Betula, Hartig net.

Structure and development of Pinus banksiana – Wilcoxina ectendomycorrhizae

Seedlings of Pinus banksiana were grown in growth pouches and inoculated with Wilcoxina mikolae var. mikolae, Wilcoxina mikolae var. tetraspora, and Wilcoxina rehmii. Ectendomycorrhizae formed between P. banksiana and W. mikolae var. mikolae developed rapidly following inoculation. The mantle was of variable width, and a large amount of mucigel was evident on the root surface. Intracellular penetration of the cortical cells by hyphae occurred one to two cells distal to Hartig net formation. Both light and transmission electron microscopy revealed labyrinthic growth of Hartig net hyphae that were densely cytoplasmic during early penetration stages but became vacuolate as the association aged. Intracellular colonization of the cortex was extensive, with the hyphae highly branched and surrounded by an interfacial matrix and cortical cell plasma membrane. The external morphology and anatomy of ectendomycorrhizae formed between W. mikolae var. tetraspora and W. rehmii and P. banksiana were similar to those described for W. mikolae var. mikolae. Key words: ectendomycorrhizae, Wilcoxina, Pinus banksiana, intracellular, Hartig net, E-strain.