The Influence of Inoculated and Native Ectomycorrhizal Fungi on Morphology, Physiology and Survival of American Chestnut

The objective of this study was to evaluate the influence of five different species of ectomycorrhizal (ECM) fungi on root colonization of native fungi on putatively blight resistant chestnut hybrids (Castanea dentata x C. mollissima) in a reclaimed mine site in central Ohio. The five species were Hebeloma crustuliniforme, Laccaria bicolor, Scleroderma polyrhizum, Amanita rubescens, and Suillus luteus. We used a combination of DNA sequencing of the ITS region and phylogenetic analyses to indentify fungi found on roots after 12 and 18 months in the field. Non-metric multidimensional scaling (NMDS) ordinations were used to determine if ECM community composition was influenced by the fungal inoculum used. The results of this study demonstrated that the selected ECM species do not persist on chestnut after one year in the field. In addition, these selected ECM species did not impede natural root colonization of native fungi or influence ECM community composition after two growing seasons. Although these species did not persist in the field, the presence of ECM inoculum (with the exception of Amanita) greatly contributed to the survival of hybrid chestnut seedlings. Therefore, introduced inoculum that was present in the very early stages of outplanting had persisting effects with regard to seedling establishment in the field, even if the original inoculum did not persist. ECM fungi native to the area colonized chestnuts resulting in increased growth rates. These native assemblages may contain species better able to form functional mycorrhizas under these environmental extremes. Therefore, the conservation of these species may be necessary to facilitate long-term survival of deciduous tree species historically native to these lands

The Efficiency of Introduced Pisolithus tinctorius on Backcrossed Chestnut Germination and Survival.

American chestnut was eliminated as a canopy tree from the Appalachian region of North America with the introduction of chestnut blight in the early 1900s. Breeding programs initiated in the 1980s have produced seedling lines that display the pure American morphology with potential resistance to chestnut blight. More work is required to assess their field performance in field sites representative of their native range. This study used American (Castanea dentata) and backcrossed hybrid chestnuts (C. dentata × C. mollissima) on an abandoned coal mine in southeastern Ohio. Half of the seeds were planted with ectomycorrhizal fungus (ECM) Pisolithus tinctorius (Pt) as a granular inoculum. Germination, survival, percent ECM colonization, and ECM community were assessed. In addition, soil data such as pH, cation exchange capacity, nutrients, texture, temperature, moisture, and organic matter were evaluated to determine their influence on ECM. After the first and second growing season, germination and survival were not influenced by the Pt inoculum or chestnut genotypes. In addition, ECM root colonization, fungal community composition, and host response were similar between seedling types, regardless of the inoculum added. This indicated: 1) backcrossed breeding produces seedlings similar to pure American chestnuts with regard to growth, establishment, and fungal symbionts, and 2) the granular inoculum had no influence on ECM colonization in this field site. Introducing spore inoculum to a field site greatly deficient in nutrients and organic matter is not an efficient method to ensure ECM symbiosis. However, natural colonization by native ECM fungi, though limited, did result in larger chestnut seedlings. Cation exchange capacity significantly contributed to the percent of root colonization of ECM fungi on chestnut. Determination of planting protocols and factors that influence ECM root colonization will be useful for in future mine restoration projects using backcrossed American chestnut as a restoration tree

Facilitation of American Chestnut (Castanea dentata) Seedling Establishment by Pinus virginiana in Mine Restoration

This study evaluated the influence of planting sites on the establishment and ectomycorrhizal (ECM) colonization of American chestnut (Castanea denetata (Marsh.) Borkh.) on an abandoned coal mine in an Appalachian region of the United States. Root morphotyping and sequencing of the fungal internal transcribed spacer (ITS) region were used to identify the ECM species associated with the chestnut seedlings. Germination, survival, ECM root colonization, and growth were assessed in three habitats: forest edge, center (plots without vegetation), and pine plots (a 10-year-old planting of Pinus virginiana). Seedlings in pine plots had higher survival (38%) than the other plot types (center 9% and forest edge 5%; P = 0.007 ). Chestnuts found colonized by ECM within the pine plots were larger (P = 0.02), contributed by a larger root system (P = 0.03). Forest edge and pine plots had more ECM roots than seedlings in center plots (P = 0.04). ITS fungal sequences and morphotypes found among chestnut and pine matched Scleroderma, Thelephora, and Pisolithus suggesting these two plant species shared ECM symbionts. Results indicated that the presence of P. virginiana had a greater facilitative effect on growth and survival of chestnut seedlings

Survey for the Presence of Phytophthora cinnamomi on Reclaimed Mined Lands in Ohio Chosen for Restoration of the American Chestnut

We have been planting blight resistant American chestnut seedlings on reclaimed coal mined areas in Southeastern Ohio, which was once within the natural range of the American chestnut. Towards the goal of restoring the American chestnut, we are testing suitable sites that can aid survival, growth and establishment of planted seedlings pre-inoculated with ectomycorrhizal fungi. Prior to the arrival of the chestnut blight fungus, pathogens of the genus Phytophthora were introduced in the USA that were responsible for the “ink disease” or “root-rot” resulting in wide-spread death of chestnut trees in southern states. Although these pathogens were not observed elsewhere, recent reports indicate their presence in some northern states, including Ohio. We have been testing each location targeted for chestnut plantings for the presence of Phytophthora, specifically P. cinnamomi. The work reported here shows results obtained from seven different sites in southeastern Ohio where reclamation was done 3-20 years ago. Soil was collected at a depth of 4-5” at several locations within each site. A positive control containing ~4 cfu/10 g soil was used in the analysis. We used two different techniques for identifying the pathogen: 1. Direct isolation of the pathogen from the soil using selective media; 2. Using chestnut leaves as a baiting technique followed by selection on plates. In both cases, final identification was done by DNA isolation and sequencing using Phytophthora-specific primers. Our results showed that, at least in the locations we tested, P. cinnamomi was not detected. Because most of these lands were only recently reclaimed, it is possible that the pathogen may not have established there yet. However, samples from locations that were reclaimed more than 2 decades ago also showed absence of this fungus. Results suggest that this pathogen is either not as wide-spread in Ohio as in southern states or mined sites are not favorable for its existence and spread

Ailanthus altissima (Tree-of-Heaven) Interferes with Beneficial Symbionts with Negative Impacts on Oak Regeneration

The invasion of Tree-of-Heaven (Ailanthus altissima) has been documented in disturbed landscapes leading to biodiversity loss and degradation of ecosystem function. Ailanthus interferes with the restoration of native species by its aggressive growth habit, alteration of nutrient cycles, and allelopathic chemical production. Recent studies suggest that allelopathy has a negative effect on the growth of red oak (Quercus rubra), possibly by interfering with the symbiosis of beneficial ectomycorrhizal fungi (ECM). This fungal symbiont is essential for healthy tree growth and the unavailability of these fungi may impede the success of seedling regeneration. This study investigated the effects of Ailanthus on biomass production and ectomycorrhizal fungal (ECM) colonization of red oak (Q. rubra) seedlings on a reclaimed coal mine site in eastern Ohio. Six plots were designated in an existing riparian buffer zone in a wetland at The Wilds Conservation Center in Muskingum County. Three of the plots were in an area where mature Ailanthus was present. The other three plots were located in the same riparian zone that was without Ailanthus. Naturally regenerating two- year-old red oak seedlings were selected for study (10 seedlings per plot, 60 seedlings total). The oak seedlings were sampled for biomass (g) and ECM root colonization. Two-year-old oak seedlings growing among mature Tree-of- Heaven produced significantly less biomass, specifically in root production, than the oaks growing without the invasive tree (P = 0.02). There was a decrease in ECM colonization (P = 0.001) and a shift in ECM community composition in plots where the Tree-of-Heaven was present (P = 0.0004). The increase in root biomass and ECM colonization may aid in the plant’s competitive ability for belowground resources, important for reestablishment. These data suggest that areas impacted by the invasion of Tree-of-Heaven may require restoration with plant species less reliant on ECM colonization when planting in soils immediately following invasive species removal

Facilitation of American Chestnut ( Castanea dentata

This study evaluated the influence of planting sites on the establishment and ectomycorrhizal (ECM) colonization of American chestnut (Castanea denetata (Marsh.) Borkh.) on an abandoned coal mine in an Appalachian region of the United States. Root morphotyping and sequencing of the fungal internal transcribed spacer (ITS) region were used to identify the ECM species associated with the chestnut seedlings. Germination, survival, ECM root colonization, and growth were assessed in three habitats: forest edge, center (plots without vegetation), and pine plots (a 10-year-old planting of Pinus virginiana). Seedlings in pine plots had higher survival (38%) than the other plot types (center 9% and forest edge 5%; P=0.007). Chestnuts found colonized by ECM within the pine plots were larger (P=0.02), contributed by a larger root system (P=0.03). Forest edge and pine plots had more ECM roots than seedlings in center plots (P=0.04). ITS fungal sequences and morphotypes found among chestnut and pine matched Scleroderma, Thelephora, and Pisolithus suggesting these two plant species shared ECM symbionts. Results indicated that the presence of P. virginiana had a greater facilitative effect on growth and survival of chestnut seedlings

MOLECULAR MARKER GENES FOR ECTOMYCORRHIZAL SYMBIOSIS

Mycorrhizal symbiosis is a mutually beneficial association very commonly found among most vascular plants. Formation of mycorrhiza happens only between compatible partners and predicting this is often accomplished through a trial and error process. We investigated the possibility of using expression of symbiosis specific genes as markers to predict the formation of ectomycorrhiza. We used antibodies and cDNA probes corresponding to PF6.2 and Lbras genes of Laccaria bicolor to detect presence and expression of similar genes in other ectomycorrhiza fungi during their association with the red pine. Analyses using transmission electron microscopy and northern hybridization showed that such genes were expressed in several other fungi forming mycorrhiza with red pine. The results suggest that these genes may have a role in many ectomycorrhizal symbiosis systems and may serve as molecular markers for monitoring symbiosis development

The impact of utilizing different optical coherence tomography devices for clinical purposes and in multiple sclerosis trials.

Optical coherence tomography (OCT) derived retinal measures, particularly peri-papillary retinal nerve fiber layer (RNFL) thickness, have been proposed as outcome measures in remyelinating and neuroprotective trials in multiple sclerosis (MS). With increasing utilization of multiple centers to improve power, elucidation of the impact of different OCT technologies is crucial to the design and interpretation of such studies. In this study, we assessed relation and agreement between RNFL thickness and total macular volume (in MS and healthy controls) derived from three commonly used OCT devices: Stratus time-domain OCT, and Cirrus HD-OCT and Spectralis, two spectral-domain (SD) OCT devices. OCT was performed on both Cirrus HD-OCT and Stratus in 229 participants and on both Cirrus HD-OCT and Spectralis in a separate cohort of 102 participants. Pearson correlation and Bland-Altman analyses were used to assess correlation and agreement between devices. All OCT retinal measures correlated highly between devices. The mean RNFL thickness was 7.4 µm lower on Cirrus HD-OCT than Stratus, indicating overall poor agreement for this measurement between these machines. Further, the limits of agreement (LOA) between Cirrus HD-OCT and Stratus were wide (-4.1 to 18.9 µm), indicating poor agreement at an individual subject level. The mean RNFL thickness was 1.94 µm (LOA: -5.74 to 9.62 µm) higher on Spectralis compared to Cirrus HD-OCT, indicating excellent agreement for this measurement across this cohort. Although these data indicate that these three devices agree poorly at an individual subject level (evidenced by wide LOA in both study cohorts) precluding their co-utilization in everyday practice, the small difference for mean measurements between Cirrus HD-OCT and Spectralis indicate pooled results from these two SD-devices could be used as outcome measures in clinical trials, provided patients are scanned on the same machine throughout the trial, similar to the utilization of multiple different MRI platforms in MS clinical trials

Boxplots of interscanner agreement for measures of RNFL thickness and TMV between Stratus OCT and Cirrus HD-OCT and Spectralis OCT and Cirrus HD-OCT.

<p>Boxplots illustrating the inter-quartile range for interscanner agreement indices show stronger agreement between Cirrus HD-OCT and Spectralis OCT measurements than between Cirrus HD-OCT and Stratus OCT. The lower and upper boundaries of each rectangle represent the 25^th and 75^th percentiles respectively, while the horizontal lines in between these represent the 50^th percentile.</p