Jenise M. Bauman

Soil preparation methods promoting ectomycorrhizal colonization and American chestnut Castanea dentata establishment in coal mine restoration

Summary 1. The objective of this research was to evaluate soil subsurface methods that may aid in seedling establishment and encourage root colonization from a diverse group of ectomycorrhizal (ECM) fungi during restoration projects. 2. American chestnut Castanea dentata Marsh. Borkh. and backcrossed chestnuts seedlings were planted on a reclaimed coal mine site in central Ohio, USA. Roots from chestnut seedlings planted in the plots that were cross-ripped, plowed and disked, or a combination of treatments were sampled for ECM fungi and compared with control plots. The presence and identification of native ECM were determined by fungal DNA sequencing of the internaltranscribed (ITS) region. 3. After two growing seasons, mechanical soil treatments resulted in seedlings with significantly more ECM species when compared to seedlings grown in the control plots (P <0 0001). A nonmetric multidimensional scaling ordination followed by a permutational MANOVA confirmed significant dissimilarities in community composition between the control and mechanically treated plots (F =0 24, P =0 015). 4. Ectomycorrhizal root colonization was significantly higher on the chestnut seedlings sampled from the mechanically treated plots when compared to the control plots (F =1 063, P <0 0001). Differences did not exist among the three mechanical treatments. There was also a significant increase in above-ground seedling growth in the plots that were treated with a surface soil method (F =1 572, P <0 0001). It is not clear whether ECM activity was the driver of plant growth; regardless, both are strong indicators of healthy tree establishment. 5. Synthesis and applications. This study illustrates that the use of soil subsurface methods increased ectomycorrhizal (ECM) activity and seedling growth. Employing methods that encourage the root colonization by beneficial ECM and promote healthy seedling establishment may aid the long-term survival of chestnuts in restoration projects. This can be applied to other hardwood seedlings used in reforestation in soils compacted after anthropogenic disturbances.

Vegetation Communities of a Coal Reclamation Site in Southeastern Ohio

Abstract Laws regulating mine reclamation following coal extraction mandate the establishment of vegetative cover, which often includes the introduction of non-native plant species. We evaluated the vegetative community composition of a recovering, reclaimed surface mine at The Wilds, a conservation center in southeastern Ohio. In 2007 and 2009, we identified a total of 109 species within a 1885-ha grassland area. After >30 years postreclamation, invasive species were the predominant plants at the site, with no evidence of succession towards a mixed mesophytic forest typical of the region. Our study illustrates how non-native plantings followed by passive management can result in the development and stability of non-native communities even decades after reclamation. Strategic and longterm management efforts, such as careful preparation of the rooting zone for trees, or the establishment of deep-rooted native plants, along with frequent monitoring, are needed to recover native vegetation and associated wildlife.

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.

Growth performance and chestnut blight incidence (Cryphonectria parasitica) of backcrossed chestnut seedlings in surface mine restoration

Anthropogenic disturbances such as mining for coal have caused significant disturbance to the Appalachian forests of North America. Recovery of these disturbances is highly dependent on restoration methods that encourage natural succession. Unfortunately, current reclamation protocols have resulted in soil compaction and aggressive herbaceous groundcovers that impede the recovery of native trees. To overcome this, methods such as deep ripping and plow and disking were applied to a reclaimed mine land in Ohio, USA Plantings of pure American chestnut (Castanea dentata) and two seedling types (BC1F3 and BC2F3) bred for resistance to chestnut blight fungus (Cryphonectria parasitica) were assessed among different soil preparation treatments over five field seasons. Seedling mortality due to natural infection by chestnut blight was recorded and related to the disease resistance potential of the BC1F3 and BC2F3 seedlings. The growth and survival of chestnuts in plots that employed either ripping, plow disking, or the combination of the two methods were significantly greater than the control plots. After five seasons, differences existed among the soil treatments; plots that applied deep ripping had the highest survival and growth. When chestnut types were compared, pure American chestnut was the tallest. However, BC2F3 chestnut seedlings had the highest survival and lower disease incidence. Results suggest that employing deep ripping with backcrossed chestnut seedlings provides a method for establishing hardwood seedlings in soils impacted by surface mining. Planting methods that promote vigorous growth can be applied more broadly to other regions where anthropogenic disturbances create soil conditions that hinder seedling establishment.

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. Additional Keywords: root colonization of fungi, chestnut restoration.

Sustainable Landscapes: Evaluating Strategies for Controlling Autumn Olive (Elaeagnus umbellata) on Reclaimed Surface Mineland at The Wilds Conservation Center in Southeastern Ohio

Autumn olive (Elaeagnus umbellata) was planted during the reclamation process to reduce erosion and improve nitrogen content of the soil. However, since its establishment, E. umbellata has spread prolifically and control measures are difficult. The primary objective of this case study was to evaluate the effectiveness of various control methods on eradication of E. umbellata in varying degrees of infestation. A two- phase case study was conducted at The Wilds conservation center in Cumberland, OH. Phase 1 began in 2007-2008 to evaluate three treatments in areas with moderate cover (15-30%) of E. umbellata: mechanical removal, foliar herbicide, and dormant stem herbicide. Nine 200m 2 study plots were established with three replications of each treatment. Effectiveness of each treatment was evaluated in 2009 through tracking 225 individual shrubs. The foliar herbicide controlled 98% of E. umbellata; dormant stem herbicide achieved 71 % and the mechanical treatment controlled only 15 %. Statistical comparisons indicated the foliar and dormant stem herbicides were more effective (P = 0.0008) than mechanical removal. This suggests that foliar applications can be a reliable tool for control of E. umbellata in areas with a 15-30 % density level. Based on these findings, phase 2 of this study was initiated in 2010 to evaluate removal techniques in dense shrub infestations (95-100 %). Treatments included a combination of mechanical clearing then a chemical treatment of stumps to reduce re-sprouts. The fracture treatment was most effective during the second phase (63 %), when compared to the cut-stump (46 %) mechanical treatment (P = 0.004). Results demonstrate that a combined mechanical- chemical approach is efficient in dense infestations. Mechanical land clearing through fracture and re-sprout treatment appeared to be most effective in E. umbellata control and the most cost effective in dense cover; however replicated studies are needed to provide conclusive information about the fracture re-sprout treatment.

AILANTHUS ALTISSIMA INTERFERS WITH BENEFICIAL SYMBIONTS AND NEGETIVELY IMPACTS OAK REGENERATION 1

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 plants 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.

REPRODUCTION POTENTIAL AND NATURAL CANKER INFECTION ON BACKCROSSED CHESTNUT TREES 1

American chestnut (Castanea dentata Marsh. Borkh.) is a once- dominant hardwood species with the potential to be a valuable restoration tree for use on surface mined lands in the Appalachian region. Coupling soil ripping and plowing and disking with plantings of American and backcrossed chestnuts has resulted in high seedling survival on a reclaimed surface mine site in southeast Ohio. The objective of this study was to evaluate flowering, chestnut bur production (seed), and natural cankers caused by chestnut blight fungus (Cryphonectria parasitica) on three chestnut breeding lines, in three soil tillage treatments after seven field seasons. Pure American (Castanea dentata), and two types of C. dentata × C. mollissima hybrids (BC2F1 and BC3F1) were documented. Reproduction potential was measured via flower production in June. Canker incidence was recorded as necrotic bark lesions with the presence of orange spore bearing structures. When reproduction potential was compared among seed types, there were no differences; all chestnuts trees were flowering and producing chestnut burs after 7 seasons. Soil treatment had no impact on flowering incidence; however, hybrid breeding lines had a mild effect. Canker incidence and presence of flowers were not related statistically. When natural canker incidence was compared, pure American chestnut exhibited the most infection (P < 0.0001). There were also notable treatment effects, plots that applied the deep ripping had greater disease incidence on pure American chestnuts (P < 0.0001). Long-term survival and stand stability will depend on chestnuts tolerance to the blight at an age of fruiting and flowering. Results after seven years suggest that hybrids were exhibiting a decrease in blight incidence and were flowering and producing burs. Employing deep ripping methods to backcrossed American chestnut plantings provide a viable method for hardwood seedling establishment in soils impacted by surface mining.

Native mycorrhizal fungi replace introduced fungal species on Virginia pine and American chestnut planted on reclaimed mine sites of Ohio

Plant-microbe community dynamics influence the natural succession of plant species where pioneer vegetation facilitates the establishment of a distantly related, later successional plant species. This has been observed in the case of restoration of the American chestnut (Castanea dentata) on abandoned mine land where Virginia pine (Pinus virginiana) facilitated the establishment of chestnut seedlings. This was apparently due to the natural mycorrhizal networks of pine, which aided the survival and growth of chestnut seedlings. In this study, we assessed the survival and propensity of introduced mycorrhizal fungi on Virginia pine to colonize pure American and backcrossed American chestnut. Seedlings were planted in Perry State Forest located in southeastern Ohio. This area was mined for coal in the 1950s and had very little reclamation done aside from experimental tree plantings. The selected site, with little topsoil or organic matter, was characterized by high concentrations of Al, high soil temperatures, and a pH of 3.6. Virginia pine seedlings were inoculated using ectomycorrhizal (ECM) cultures of Amanita rubescens, Laccaria laccata, and Pisolithus tinctorius via liquid media. After three months, roots were tested for the presence of mycorrhizae. They were then transplanted and grown for two years in the greenhouse. After verifying mycorrhizal colonization, 600 pines were out planted in May of 2005. Chestnut seedlings (100 one-year-old seedlings) inoculated with P. tinctorius by the Ohio state tree nursery had been planted by other researchers at the same time. After eight growing seasons, pines and chestnuts were measured and sampled for ECM colonization. Growth measurements showed that pines and hybrid chestnuts had significantly more aboveground biomass compared to pure American chestnut (P = 0.01). Eleven fungal species were detected using DNA sequencing. With the exception of Amanita, the inoculum that were out planted with both chestnut and Virginia pine were replaced after 8 field seasons by fungi native to the site. More fungal species were sampled from the Virginia pines than from chestnut roots, which contributed to the significant differences in ECM fungal community composition between the two species (P = 0.005).

CASE STUDY: Restoring Remnant Hardwood Forest Impacted by Invasive Tree-of-Heaven (Ailanthus altissima)

Tree-of-heaven (Ailanthus altissima) is a fast growing tree native to China. Introduced as an ornamental plant, A. altissima has spread throughout North American landscapes, imposing a threat to the biodiversity of native ecosystems. Recommended control methods include basal bark treatments using herbicide with an oil-based carrier around the base of Ailanthus stems. Land managers value application methods that maximize efficiency while also reducing environmental impacts when applied over landscape scales. The focus of this study was to assess the efficiency of herbicide concentrations and carriers on the mortality of A. altissima. This study was conducted in a 105 ha hardwood forest at the Wilds Conservation Center in Cumberland, OH. The forest is directly adjacent to areas mined for coal and reclaimed in the 1980s. Twenty-five plots were established consisting of 732 target trees. Two carriers (AX-IT basal oil and diesel fuel) mixed with Garlon 4 Ultra herbicide were tested at two different concentrations: 1) 10% Garlon in 90% diesel fuel carrier, 2) 20% Garlon with 80% diesel carrier, 3) 10% Garlon with 90% AX-IT carrier, and 4) 20% Garlon with 80% AX-IT carrier. Basal bark treatments were applied using a backpack sprayer. After one year, treatments were similar (89-100% mortality) with one exception, the 10% Garlon in 90% diesel treatment was least effective (69% mortality; P< 0.0001). This was more apparent as the diameter at breast height (DBH) increased (P < 0.0001). When canopy dieback was compared across treatments, AX-IT basal oil remained more effective regardless of the DBH or concentration. Cost comparisons show 10% Garlon solution in AX-IT oil base can be the most economically and ecologically beneficial treatment when applied on a large scale. Long-term monitoring will determine the occurrence of re-sprouts (via seed and root sprouting) and the impact each treatment has on the plant communities within this forest system. Additional