Chronic acid deposition affects many temperate hardwood forests of the northeastern United States, reduces soil pH and phosphorus (P) availability, and can alter the structure and function of soil microbial communities. The strategies that microorganisms possess for survival in acidic, low P soil come at a carbon (C) cost. Thus, how microbial communities respond to soil acidification in forests may be influenced by plant phenological stage as C allocation belowground varies; however, this remains largely unexplored. In this study, we examined microbial communities in an ecosystem level manipulative experiment where pH and/or P availability were elevated in three separate forests in Northeastern Ohio. Tag-encoded pyrosequencing was used to examine bacterial and fungal community structure at five time points across one year corresponding to plant phenological stages. We found significant effects of pH treatment and time on fungal and bacterial communities in soil. However, we found no interaction between pH treatment and time of sampling for fungal communities and only a weak interaction between pH elevation and time for bacterial communities, suggesting that microbial community responses to soil pH are largely independent of plant phenological stage. In addition, fungal communities were structured largely by site, suggesting that fungi were responding to differences between the forests, such as plant community differences.

Beech leaf disease (BLD) is an emerging forest infestation affecting beech trees (Fagus spp.) in the midwestern and northeastern United States and southeastern Canada. BLD is attributed to the newly recognized nematode Litylenchus crenatae subsp. mccannii. First described in Lake County, Ohio, BLD leads to the disfigurement of leaves, canopy loss, and eventual tree mortality. Canopy loss limits photosynthetic capacity, likely impacting tree allocation to belowground carbon storage. Ectomycorrhizal fungi are root symbionts, which rely on the photosynthesis of autotrophs for nutrition and growth. Because BLD limits tree photosynthetic capacity, ECM fungi may receive less carbohydrates when associating with severely affected trees compared with trees without BLD symptoms. We sampled root fragments from cultivated F. grandifolia sourced from two provenances (Michigan and Maine) at two timepoints (fall 2020 and spring 2021) to test whether BLD symptom severity alters colonization by ectomycorrhizal fungi and fungal community composition. The studied trees are part of a long-term beech bark disease resistance plantation at the Holden Arboretum. We sampled from replicates across three levels of BLD symptom severity and compared fungal colonization via visual scoring of ectomycorrhizal root tip abundance. Effects of BLD on fungal communities were determined through high-throughput sequencing. We found that ectomycorrhizal root tip abundance was significantly reduced on the roots of individuals of the poor canopy condition resulting from BLD, but only in the fall 2020 collection. We found significantly more ectomycorrhizal root tips from root fragments collected in fall 2020 than in spring 2021, suggesting a seasonal effect. Community composition of ectomycorrhizal fungi was not impacted by tree condition but did vary between provenances. We found significant species level responses of ectomycorrhizal fungi between levels of both provenance and tree condition. Of the taxa analyzed, two zOTUs had significantly lower abundance in high-symptomatology trees compared with low-symptomatology trees. These results provide the first indication of a belowground effect of BLD on ectomycorrhizal fungi and contribute further evidence to the role of these root symbionts in studies of tree disease and forest pathology.

Beech leaf disease (BLD) is a newly emerging disease in North America that affects American beech ( Fagus grandifolia ). It is increasingly recognized that BLD is caused by a subspecies of the anguinid nematode Litylenchus crenatae subsp. mccannii (hereafter L. crenatae ) , which is likely native to East Asia. How nematode infestation of leaves affects the leaf microbiome and whether changes in the microbiome could contribute to BLD symptoms remain uncertain. In this study, we examined bacterial and fungal communities associated with the leaves of F. grandifolia across nine sites in Ohio and Pennsylvania that were either symptomatic or asymptomatic for BLD and used qPCR to measure relative nematode infestation levels. We found significantly higher levels of infestation at sites visibly symptomatic for BLD. Low levels of nematode infestation were also observed at asymptomatic sites, which suggests that nematodes can be present without visible symptoms evident. Bacterial and fungal communities were significantly affected by sampling site and symptomology, but only fungal communities were affected by nematode presence alone. We found many significant indicators of both bacteria and fungi related to symptoms of BLD, with taxa generally occurring in both asymptomatic and symptomatic leaves, suggesting that microbes are not responsible for BLD but could act as opportunistic pathogens. Of particular interest was the fungal genus Erysiphe, which is common in the Fagaceae and is reported to overwinter in buds—a strategy consistent with L. crenatae . The specific role microbes play in opportunistic infection of leaves affected by L. crenatae will require additional study. IMPORTANCE: Beech leaf disease (BLD) is an emerging threat to American beech ( Fagus grandifolia ) and has spread quickly throughout the northeastern United States and into southern Canada. This disease leads to disfigurement of leaves and is marked by characteristic dark, interveinal banding, followed by leaf curling and drop in more advanced stages. BLD tends to especially affect understory leaves, which can lead to substantial thinning of the forest understory where F. grandifolia is a dominant tree species. Understanding the cause of BLD is necessary to employ management strategies that protect F. grandifolia and the forests where it is a foundation tree species. Current research has confirmed that the foliar nematode Litylenchus crenatae subsp. mccannii is required for BLD, but whether other organisms are involved is currently unknown. Here, we present a study that investigated leaf-associated fungi and bacteria of F. grandifolia to understand more about how microorganisms may contribute to BLD.

Forest ecosystems with altered nutrient limitations are a common legacy of acidic deposition in North America. Continued acidic deposition has lowered soil pH and revealed phosphorus (P) limitations in many temperate forest ecosystems. Previous studies exploring P limitations or co‐limitations are often short term, and thus may potentially show a response to limitation that is not sustained over time. To better understand how a forest's response to P limitation and acidic deposition can change over time, we added P, limestone to raise pH, and a cross‐treatment where both P and limestone were added to 3 different northeastern Ohio forest stands over a 12‐year period. We tracked diameter at breast height of the trees annually, conducted foliar nutrient analyses, and collected tree roots to assess treatment impacts on mycorrhizal colonization. We analyzed our dataset in three sections: the first 6 years after manipulation, the latter 6 years, and the entire 12‐year period. These sections allowed us to compare differences between early responses to manipulation and later responses. Here, we found that P additions increased basal area growth across multiple species and throughout the entire study, confirming that our forest trees are P‐limited. Cross‐treatments similarly increased basal area growth, but not as much as P additions alone. Some species saw waning effects of treatment in the second half of the study. This could be due to changes in weather patterns, an adjustment of the study system's equilibrium, or the emergence of beech leaf disease in 2014, which has led to the decline of Fagus grandifolia. Early successional species such as Acer rubrum began to benefit from treatments after beech leaf disease killed canopy F. grandifolia trees, perhaps first being light‐limited, but later able to take advantage of the nutrient additions and pH alteration of their soils. Our results suggest that in forests subject to acidic deposition, soil P may co‐limit tree growth, but responses are species dependent.