Effects of the nematode Litylenchus crenatae subsp. mccannii and beech leaf disease on leaf fungal and bacterial communities on Fagus grandifolia (American beech).

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.

Interannual variation in spring weather conditions as a driver of spring wildflower coverage: a 15-year perspective from an old-growth temperate forest.

Spring ephemerals are wildflowers found in temperate deciduous forests that typically display aboveground shoots for a period of 2 months or less. Early spring, before the canopy leaves out, marks the beginning of the aboveground growth period where ephemerals acquire nutrients and resources via aboveground tissues. Several studies have shown that spring ephemeral reproduction is affected by spring temperature, but few have looked at how weather conditions of the current and previous seasons, including precipitation and temperature, influence aboveground growth. Here, we examine the response of a spring ephemeral community in a temperate hardwood forest to weather conditions during their current and previous growing seasons. For 15 years we estimated percent cover of each species within our community. We highlighted five dominant spring ephemerals within this community: wild leek (Allium tricoccum), cutleaf toothwort (Cardamine concatenata), spring beauty (Claytonia virginica), squirrel corn (Dicentra canadensis) and trout lily (Erythronium americanum). We compared changes in cover on both a community and species level from 1 year to the next with average precipitation and temperature of the year of measurement as well as the year prior. We found precipitation and temperature influence a change in cover at the community and species level, but the strength of that influence varies by species. There were few significant correlations between plant cover in the current year and temperature and precipitation in the 30 days preceding measurement. However, we found significant correlations between plant cover and precipitation and temperature during the previous spring; precipitation and cover change were positively correlated, whereas temperature and cover change were negatively correlated. Overall, cooler, wetter springs lead to an increase in aboveground cover the next year. Learning how individual species within a forest plant community respond to weather conditions is a crucial part of understanding how plant communities will respond to climate change.

Development of Primers Specific for Detection of Litylenchus crenatae, the Causal Agent of Beech Leaf Disease, in Plant Tissue.

Beech leaf disease (BLD), an emerging threat to American beech (Fagus grandifolia) in the northern United States and Canada, was recently confirmed to be caused by the nematode Litylenchus crenatae subsp. mccannii (hereafter L. crenatae). Consequently, there is a need for a rapid, sensitive, and accurate method for detecting L. crenatae for both diagnostic as well as control purposes. This research developed a new set of DNA primers that specifically amplify L. crenatae and allow for accurate detection of the nematode in plant tissue. These primers have also been used in quantitative PCR (qPCR) to determine relative differences in gene copy number between samples. This primer set provides an improved, effective tool for monitoring and detecting L. crenatae in temperate tree leaf tissue which is necessary to understand the spread of this emerging forest pest and to develop management strategies.

Beech Leaf Disease Severity Affects Ectomycorrhizal Colonization and Fungal Taxa Composition.

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.

The effects of increased hay-to-grain ratio on behavior, metabolic health measures, and fecal bacterial communities in four Masai giraffe (Giraffa camelopardalis tippelskirchi) at Cleveland Metroparks Zoo.

We evaluated whether increasing the hay-to-grain ratio offered to Masai giraffe (Giraffa camelopardalis tippelskirchi) at Cleveland Metroparks Zoo would reduce oral stereotypies and alter feeding behaviors, maintain or increase serum calcium-to-phosphorus ratio, decrease serum insulin-to-glucose ratio and salivary insulin, and alter fecal bacterial community structure. Giraffe transitioned to a ∼90:10 hay-to-grain ratio in even increments over 8 weeks. A ration balancer was added during the seventh week of transition to ensure proper mineral and nutrient balance. We collected (1) behavioral data collected approximately daily using instantaneous focal sampling; (2) serum collections every other week for insulin-to-glucose and calcium-to-phosphorus ratio analysis and saliva weekly for insulin analysis; and (3) weekly fecal sample collections to examine changes in bacterial community structure during the 8 weeks preceding and following the diet change. After the diet change, giraffe spent significantly more time feeding and less time performing tongue and mouth stereotypies, people-directed and alert behaviors. Salivary and serum insulin and serum insulin-to-glucose ratio decreased, and fecal bacterial community structure changed significantly. However, serum calcium-to-phosphorus ratio remained >1:1 throughout the study. While further studies are needed to elucidate the nature and implications of the change in fecal bacterial community structure and metabolic hormones, the results of this study show promise for incremental improvements in health and welfare from feeding a higher proportion of forage in the diet.

Mycorrhizal fungal communities respond to experimental elevation of soil pH and P availability in temperate hardwood forests.

Many forests are affected by chronic acid deposition, which can lower soil pH and limit the availability of nutrients such as phosphorus (P), but the response of mycorrhizal fungi to changes in soil pH and P availability and how this affects tree acquisition of nutrients is not well understood. Here, we describe an ecosystem-level manipulation in 72 plots, which increased pH and/or P availability across six forests in Ohio, USA. Two years after treatment initiation, mycorrhizal fungi on roots were examined with molecular techniques, including 454-pyrosequencing. Elevating pH significantly increased arbuscular mycorrhizal (AM) fungal colonization and total fungal biomass, and affected community structure of AM and ectomycorrhizal (EcM) fungi, suggesting that raising soil pH altered both mycorrhizal fungal communities and fungal growth. AM fungal taxa were generally negatively correlated with recalcitrant P pools and soil enzyme activity, whereas EcM fungal taxa displayed variable responses, suggesting that these groups respond differently to P availability. Additionally, the production of extracellular phosphatase enzymes in soil decreased under elevated pH, suggesting a shift in functional activity of soil microbes with pH alteration. Thus, our findings suggest that elevating pH increased soil P availability, which may partly underlie the mycorrhizal fungal responses we observed.

Seasonal variation in mycorrhizal fungi colonizing roots of Allium tricoccum (wild leek) in a mature mixed hardwood forest.

The community of arbuscular mycorrhizal (AM) fungi colonizing roots of the forest herb Allium tricoccum Ait. (wild leek) was examined to assess whether colonization varied seasonally and spatially within the forest. Whole plants were collected to coincide with observed phenological stages, and the perennial tissue (i.e., the bulb) was used to analyze total C, N, and P over the growing season. AM fungal community composition, structure, and abundance were assessed in roots by terminal restriction fragment length polymorphism analysis and quantitative PCR. It was found that A. tricoccum rDNA co-amplified using the general AM primers NS31/AM1, and a new primer for qPCR was designed that discriminated against plant DNA to quantify AM colonization. Community structure of AM fungi did not vary seasonally, but did change spatially within the forest, and AM fungal communities were correlated with the presence of overstory tree species. Fungal colonization of roots, however, did change seasonally with a maximum observed in late winter and early spring following leaf emergence. Maximum AM fungal colonization was associated with declines in bulb N and P, suggesting that leaf emergence and growth were responsible for both declines in stored nutrients and increases in AM fungal colonization. Plant N and P contents increased between late summer and early spring while C contents remained unchanged. The observed increase in nutrient content during a time when A. tricoccum lacks leaves indicates that the roots or AM fungi are metabolically active and acquire nutrients during this time, despite an absence of photosynthesis and thus a direct supply of C from A. tricoccum.

Shotgun metagenomic analysis of metabolic diversity and microbial community structure in experimental vernal pools subjected to nitrate pulse.

BACKGROUND: Human activities have greatly increased nitrogen (N) levels in natural habitats through atmospheric N deposition and nutrient leaching, which can have large effects on N cycling and other ecosystem processes. Because of the significant role microorganisms play in N cycling, high inputs of nitrogenous compounds, such as nitrate (NO3-), into natural ecosystems could have cascading effects on microbial community structure and the metabolic processes that microbes perform. To investigate the multiple effects of NO3- pollution on microbial communities, we created two shotgun metagenomes from vernal pool microcosms that were either enriched with a solution of 10 mg NO3--N (+NO3-) or received distilled water as a control (-N). RESULTS: After only 20 hours of exposure to NO3-, the initial microbial community had shifted toward one containing a higher proportional abundance of stress tolerance and fermentation environmental gene tags (EGTs). Surprisingly, we found no changes to N metabolism EGTs, even though large shifts in denitrification rates were seen between the +NO3- and -N microcosms. Thus, in the absence of NO3- addition, it is plausible that the microbes used other respiratory pathways for energy. Respiratory pathways involving iron may have been particularly important in our -N microcosms, since iron acquisition EGTs were proportionally higher in the -N metagenome. Additionally, we noted a proportional increase in Acidobacteria and Alphaproteobacteria EGTs in response to NO3- addition. These community shifts in were not evident with TRFLP, suggesting that metagenomic analyses may detect fine-scale changes not possible with community profiling techniques. CONCLUSIONS: Our results suggest that the vernal pool microbial communities profiled here may rely on their metabolic plasticity for growth and survival when certain resources are limiting. The creation of these metagenomes also highlights how little is known about the effects of NO3- pollution on microbial communities, and the relationship between community stability and function in response to disturbance.

Mycorrhizal response to experimental pH and P manipulation in acidic hardwood forests.

Many temperate forests of the Northeastern United States and Europe have received significant anthropogenic acid and nitrogen (N) deposition over the last century. Although temperate hardwood forests are generally thought to be N-limited, anthropogenic deposition increases the possibility of phosphorus (P) limiting productivity in these forest ecosystems. Moreover, inorganic P availability is largely controlled by soil pH and biogeochemical theory suggests that forests with acidic soils (i.e.,

The effects of pH change and NO3- pulse on microbial community structure and function: a vernal pool microcosm study.

Forest vernal pools experience strong environmental fluctuations, such as changes in water chemistry, which are often correlated with changes in microbial community structure. However, very little is known about the extent to which these community changes influence ecosystem processes in vernal pools. This study utilized experimental vernal pool microcosms to simulate persistent pH alteration and a pulse input of nitrate (NO3 -), which are common perturbations to temperate vernal pool ecosystems. pH was manipulated at the onset and microbial respiration was monitored throughout the study (122 days). On day 29, NO3 - was added and denitrification rate was measured and bacterial, fungal, and denitrifier communities were profiled on day 30 and day 31. Microbial respiration and both bacterial and fungal community structure were altered by the pH treatment, demonstrating both structural and functional microbial responses. The NO3 - pulse increased denitrification rate without associated changes in community structure, suggesting that microbial communities responded functionally without structural shifts. The functioning of natural vernal pools, which experience both persistent and short-term environmental change, may thus depend on the type and duration of the change or disturbance.

Temporal and spatial patterns of eukaryotic and bacterial communities found in vernal pools.

In this study, we examined the effects of physicochemical variability on the microbial communities of vernal pools. Denaturing gradient gel electrophoresis revealed temporal changes to be more pronounced than spatial changes in eukaryotic and bacterial communities. Sequencing revealed high degrees of richness in decomposers, which supports the notion that vernal pools are heterotrophic habitats.