The Potential of Dark Septate Endophytes to Form Root Symbioses with Ectomycorrhizal and Ericoid Mycorrhizal Middle European Forest Plants

<div><p>The unresolved ecophysiological significance of Dark Septate Endophytes (DSE) may be in part due to existence of morphologically indistinguishable cryptic species in the most common <i>Phialocephala fortinii</i> s. l.—<i>Acephala applanata</i> species complex (PAC). We inoculated three middle European forest plants (European blueberry, Norway spruce and silver birch) with 16 strains of eight PAC cryptic species and other DSE and ectomycorrhizal/ericoid mycorrhizal fungi and focused on intraradical structures possibly representing interfaces for plant-fungus nutrient transfer and on host growth response. The PAC species <i>Acephala applanata</i> simultaneously formed structures resembling ericoid mycorrhiza (ErM) and DSE microsclerotia in blueberry. <i>A</i>. <i>macrosclerotiorum</i>, a close relative to PAC, formed ectomycorrhizae with spruce but not with birch, and structures resembling ErM in blueberry. <i>Phialocephala glacialis</i>, another close relative to PAC, formed structures resembling ErM in blueberry. In blueberry, six PAC strains significantly decreased dry shoot biomass compared to ErM control. In birch, one <i>A</i>. <i>macrosclerotiorum</i> strain increased root biomass and the other shoot biomass in comparison with non-inoculated control. The dual mycorrhizal ability of <i>A</i>. <i>macrosclerotiorum</i> suggested that it may form mycorrhizal links between Ericaceae and Pinaceae. However, we were unable to detect this species in Ericaceae roots growing in a forest with presence of <i>A</i>. <i>macrosclerotiorum</i> ectomycorrhizae. Nevertheless, the diversity of Ericaceae mycobionts was high (380 OTUs) with individual sites often dominated by hitherto unreported helotialean and chaetothyrialean/verrucarialean species; in contrast, typical ErM fungi were either absent or low in abundance. Some DSE apparently have a potential to form mycorrhizae with typical middle European forest plants. However, except <i>A</i>. <i>applanata</i>, the tested representatives of all hitherto described PAC cryptic species formed typical DSE colonization without specific structures necessary for mycorrhizal nutrient transport. <i>A</i>. <i>macrosclerotiorum</i> forms ectomycorrhiza with conifers but not with broadleaves and probably does not form common mycorrhizal networks between conifers with Ericaceae.</p></div

Relative abundances of the dominant fungal orders in Ericaceae roots detected by pyrosequencing.

<p>Tag-encoded pyrosequencing was performed with ten Ericaceae hair roots samples from three sites in NP České Švýcarsko (CS1—five samples, CS2—three samples, CS3—two samples) where the ectomycorrhizal morphotype <i>Pinirhiza sclerotina</i> formed by the DSE fungus related to PAC <i>Acephala macrosclerotiorum</i> was present. The obtained data were processed as described in Materials and Methods. OTUs with lower similarity and coverage than 88% were assigned as non-identified together with <i>incertae sedis</i> species. Orders less abundant than 0.1% were excluded from the figure.</p

Percentage fungal colonization of blueberry rhizodermal cells in Experiment 2.

<p>Blueberry seedlings were inoculated by 8 PAC species, 2 species related to PAC and <i>Rhizoscyphus ericae</i> as a positive control, two strains per each species. Blueberry seedlings were grown in a peat-based substrate for 3.5 months under <i>in vitro</i> conditions. The presented data are means of 6 replicates ± standard error of mean. Different letters above the columns indicate significant differences according to the non-parametric Kruskal-Wallis test followed by the multiple-comparison z-value test.</p

Principal component analysis of the relative abundance of OTUs.

<p>Only OTUs with component loadings for the first or second axis higher than 0.015 were visualized. For detailed information about the respective OTUs see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124752#pone.0124752.s004" target="_blank">S2 Table</a>.</p

The effect of inoculation on blueberry dry shoot weight in Experiment 2.

<p>Blueberry seedlings were inoculated by 8 PAC species, 2 species related to PAC and <i>Rhizoscyphus ericae</i> as a positive control, two strains per each species. Blueberry seedlings were grown in a peat-based substrate for 3.5 months under <i>in vitro</i> conditions. The presented data are means of 6 replicates ± standard error of mean. Different letters above the columns indicate significant differences according to the non-parametric Kruskal-Wallis test followed by the multiple-comparison z-value test.</p

The colonization patterns observed in European blueberry (<i>Vaccinium myrtillus</i>) roots in Experiment 2 and in silver birch in Experiment 3.

<p><b>2a)</b> Intracellular hyphal colonization resembling ericoid mycorrhiza formed by <i>Acephala applanta</i> AAP-1 in blueberry roots (asterisks). <b>2b)</b> An early stage of the development of an intracellular microsclerotium formed by <i>A</i>. <i>applanata</i> AAP-1 in a blueberry root (arrowhead). <b>2c)</b> An extraradical sclerotium formed on the surface of a blueberry root by <i>A</i>. <i>applanata</i> AAP-1 (arrow). <b>2d)</b> A blueberry hair root colonized in a manner resembling ericoid mycorrhiza by <i>Acephala macrosclerotiorum</i> AMA-11 (asterisks). <b>2e)</b> A detail of two blueberry rhizodermal cells intracellularly colonized by <i>A</i>. <i>macrosclerotiorum</i> AMA-11 in a manner resembling ericoid mycorrhiza (asterisks). <b>2f)</b> An extraradical sclerotium formed on the surface of a blueberry root by <i>A</i>. <i>macrosclerotiorum</i> AMA-11 (arrow). Note accompanying intracellular hyphal colonization (arrowheads). <b>2g)</b> A loose intracellular hyphal loop formed by <i>A</i>. <i>macrosclerotiorum</i> AMA-1 in a birch root (arrow). <b>2h)</b> A melanised intracellular microsclerotium formed by <i>Acephala applanata</i> AAP-1 in birch (arrowhead). All figures stained with trypan blue, observed with DIC, bars = 25 μm.</p

The effect of inoculation on blueberry fresh root weight in Experiment 2.

<p>Blueberry seedlings were inoculated by 8 PAC species, 2 species related to PAC and <i>Rhizoscyphus ericae</i> as a positive control, two strains per each species. Blueberry seedlings were grown in a peat-based substrate for 3.5 months under <i>in vitro</i> conditions. The presented data are means of 6 replicates ± standard error of mean. Different letters above the columns indicate significant differences according to the non-parametric Kruskal-Wallis test followed by the multiple-comparison z-value test.</p

Colonization, root fresh weight and shoot dry weight of birch seedlings inoculated by two strains of <i>A</i>. <i>macrosclerotiorum</i>, one strain of <i>A</i>. <i>applanata</i> and one strain of <i>P</i>. <i>involutus</i> as a positive EcM control fungus in Experiment 3.

<p>Plants were cultivated in a peat-agar medium in <i>in vitro</i> conditions for 6 months. Concentration of CO₂ in the microcosms was measured three days prior harvest. The presented data are means of 6 replicates ± SE. Different letters indicate significant differences according to the non-parametrical Kruscal-Wallis test followed by a multiple-comparison z-value test.</p><p>Colonization, root fresh weight and shoot dry weight of birch seedlings inoculated by two strains of <i>A</i>. <i>macrosclerotiorum</i>, one strain of <i>A</i>. <i>applanata</i> and one strain of <i>P</i>. <i>involutus</i> as a positive EcM control fungus in Experiment 3.</p

Some morphological characteristics of the novel basidiomycete (isolates JPK 87 and 90).

<p>2A) JPK 87 culture grown in a 9 cm diam. Petri dish on MMN for three weeks. 2B) JPK 90 grown under the same conditions. 2C) Capitate cystidium excreting a brownish substance around its apical part. Note abundant clamp connections (arrows) produced <i>in vitro</i> on the mycelium of JPK 87. DIC, bar  = 10 µm. 2D) Similar capitate cystidia were formed of sheathed ericoid mycorrhizal roots under natural conditions (arrows). <i>Vaccinium</i> sp. root, DIC, bar  = 50 µm.</p

Morphological and anatomical characteristics of sheathed ericoid mycorrhiza synthesized <i>in vitro</i> and observed under natural conditions.

<p>Sheathed ericoid mycorrhiza was synthesized <i>in vitro</i> between the basidiomycetous mycobiont (strains JPK 87 and JPK 90) and European blueberry (<i>Vaccinium myrtillus</i>) seedlings or observed in European blueberry hair roots collected in field. 3A) Terminal part of hair root is covered by a dense hyphal sheath (arrow). JPK 87, DIC, stained with trypan blue, bar  = 100 µm. 3B) Detail of a dense hyphal sheath (HS) covering terminal part of a hair root. Note the extensive extraradical hyphae (EH). JPK 90, DIC, stained with trypan blue, bar  = 50 µm. 3C) Dense intracellular hyphal coils (arrows) developing in the rhizodermal cells below a hyphal sheath. JPK 90, DIC, stained with trypan blue, bar  = 20 µm. 3D) Clamp connection (arrow) formed by the basidiomycete inside a rhizodermal cell. Roots from field, DIC, bar  = 10 µm. 3E) Terminally swollen capitate cystidium emerging from a sheathed ericoid mycorrhiza. Roots from field, DIC, bar  = 10 µm. 3F) Vigorous intracellular colonization (arrows) by the basidiomycete accompanied by numerous extraradical hyphae (EH). JPK 90, DIC, bar  = 50 µm.</p