Identification and Comparison of Arbuscular Mycorrhizal Fungi and Soil Microbiomes Between American Chestnuts and Surrounding Hardwoods

Background/Questions/Methods The introduction of the Chinese chestnut blight in 1904 decimated native American chestnut (Castanea dentata (Marshall) Borkh.) populations. In this study we aim to: 1) document the location of individual chestnuts in our vicinity; 2) document healthy versus blight infected individuals; 3) characterize putative differences in the soil microbiome between infected and non-infected individuals, as well as between chestnuts and hardwood neighbors. With this information we hope to provide new insights into mechanisms that may enhance blight and fungal resistance in American chestnuts through the understanding of the interactions between the trees and their surrounding soil microbiome. Soil samples were collected 1 m north of the base of each of six of the chestnut trees located and 1m from the base of one hardwood tree within 5 m of each chestnut and aligned to one cardinal direction. Also, 2-3 root samples were collected approximately 5 m from the base of each individual chestnut. DNA was extracted from soil and root samples using extraction kits, replicated, confirmed using nanodrop dsDNA analysis and prepared for outsource sequencing. Results/Conclusions Thirty-two individual chestnut trees have been identified, with a subset of six mature trees used for this study. Only one is currently known to be infected with Chinese chestnut blight. We are in the process of DNA extraction from soil and root samples, after which samples will be sent off for analysis to examine the metagenomics of taxa present in the soil microbiome around the mature American chestnut trees. Thus far, nine samples from three different trees have successfully undergone extraction and have been analyzed for dsDNA content using a Nanodrop

ABOVE AND BELOW GROUND COMMUNITY STRUCTURES IN A LONGLEAF PINE RESTORATION SITE: DOMINANT HERBACEOUS SPECIES AND THEIR SOIL BACTERIAL MICROBIOMES

The longleaf pine (Pinus palustris Mill.) ecosystem is one of the most diverse and endangered temperate ecosystems in North America. The native herbaceous species serve as fuel for the low-intensity, recurring fires the system depends on and thus play a major role in the maintenance of ecosystem structure. Soil microorganisms, such as bacteria and fungi, are known to aid greatly in many plant processes, including nutrient and water acquisition, resistance to infection, decomposition, and nutrient cycling. However, little is known about the relationship between dominant herbaceous species and soil bacterial compositions within montane longleaf pine ecosystems. In this preliminary study, we completed a multiyear, seasonal analysis of the soil bacterial microbiome composition upon removal of the two most dominant herbaceous species in a longleaf pine restoration site. The objective was to identify the major soil bacterial taxa and establish if changes occurred in their community structures following above-ground community manipulation. We also wanted to investigate relationships between bacterial community structure and environmental factors. Six randomized blocks (2.4m2) were established at Sheffield Wildlife Management Area in Paulding County, Georgia. Each block contained four subplots (50cm2), each with one of the following treatments: 1) control; 2) most dominant herbaceous species removed; 3) second most dominant herbaceous species removed; 4) soil disturbance: no species removed. DNA was extracted from soil samples from subplots each season and PCR amplified for bacterial gene sequences. Preliminary results indicated that the most abundant bacteria were Proteobacteria, Acidobacteria, and Actinobacteria, and soil bacterial relative abundances changed with plant removal and seasons. This information will serve as baseline data for future studies focused on the intricate relationships between ecologically important plants and their equally important microbial symbionts. Understanding these relationships will help inform the restoration process

Soil Bacterial Community Shifts in a Montane Longleaf Pine Restoration Site: Effects of Dominant Herbaceous Species Removal, Prescribed Burn, and Changes in Season

Longleaf pine (Pinus palustris Mill.) ecosystems are one of the most endangered and ecologically diverse systems in North America. The native herbaceous species serve as fuel for low-intensity, recurring fires this system relies on. The herbaceous species play major roles in maintaining ecosystem structure and diversity. Soil microorganisms like bacteria are known to aid greatly in many different plant and ecosystem processes. However, little is known about the dominant herbaceous species and soil bacterial compositions within montane longleaf pine ecosystems, especially before and after a prescribed fire. We completed a multiyear, seasonal analysis of the soil bacterial community in a longleaf pine restoration site in the Piedmont region of Georgia, USA to identify major soil bacterial taxa and analyze for changes in their community structures following plant removal, seasonal environmental changes, and use of prescribed burn. Six randomized blocks (2.4 m2) in Sheffield Wildlife Management Area contained four subplots (50 cm2), each with one of the following treatments: 1) control; 2) Pityposis nervosa (Willd.) Dress removed; 3) Andropogon virginicus L. removed; or 4) soil disturbance. DNA was extracted from soil samples and PCR amplified for bacterial rRNA gene sequences. The most abundant bacteria were common bulk and rhizosphere-dwelling phyla such as Planctomycetota, Proteobacteria, and Acidobacteria, with most unclassified at the family level. Plant removal did not significantly change soil bacterial community structures, but microbiomes did shift with changes in seasons. This serves as baseline data for future studies on interactions between ecologically important plants and their microbial communities, which may help inform the restoration process of native ecosystems

Above and Below Ground Community Structures in a Longleaf Pine Restoration Site: Native Herbaceous Species and Their Soil Microbiomes

The longleaf pine (Pinus palustris Mill.) ecosystem is one of the most diverse ecosystems on the planet and once covered close to 92 million acres of land across the American Southeast. This ecosystem was historically maintained by fires, but due to fire suppression and agricultural and logging techniques, only 3% of it remains. Restoration of this ecosystem has been of interest because of its economic importance and high ecological diversity. The native herbaceous species within this ecosystem have major effects on the maintenance of community structure by serving as fuel for the low-intensity, recurring fires the system depends on. Soil microorganisms, such as bacteria and fungi, are known to aid greatly in many plant processes, primarily nutrient and water acquisition, resistance to infection by pathogens and diseases, and decomposition and nutrient cycling. However, little is known about the relationship between the dominant herbaceous species and soil microbiome compositions within montane longleaf pine ecosystems. In this study, we completed a multiyear, seasonal analysis of the soil bacterial and fungal microbiome compositions upon removal of the two most dominant herbaceous species in a longleaf pine restoration site. Six randomized blocks (2.4m2) were established at Sheffield Wildlife Management Area in Paulding County, GA. Each block contained four subplots (50cm2), each with one of the following treatments: 1) control; 2) most dominant herbaceous species removed; 3) second most dominant herbaceous species removed; 4) soil disturbance: no species removed. DNA was extracted from soil samples from subplots each season and PCR amplified for both fungal and bacterial gene sequences. Results from this study will provide us with baseline data on the microbiome composition within the endangered longleaf pine ecosystem that will be used to better inform restoration practices as more knowledge is gained on the intricate relationships between plants and their associated soil microbiomes

Setting Up a Study to Investigate the Impact of Removal of Understory Species on the Soil Bacterial Microbiome of a Longleaf Pine Restoration Site

The longleaf pine (Pinus palustris) ecosystem is one of the most diverse ecosystems on the planet. For thousands of years, this ecosystem was maintained by fires, and its historic land cover spread from the Carolinas all the way to eastern Texas and down through Florida. Today, only 3% of the ecosystem remains, and only 0.01% of that is untouched land. This is largely due to fire suppression and heavy harvesting of these trees. Restoration of the longleaf ecosystem has been of interest because of its economic importance and high ecological diversity. In this study, we propose to investigate the effect of the removal of two grass species on the soil bacterial microbiome of the longleaf pine in a restoration site located in the Piedmont region of Georgia. These two grasses are thought to have major effects on the overall maintenance of the ecosystem because they contribute to the spread of forest fires. This helps to clear the understory and makes it possible for longleaf pines to dominate the savannah. We have surveyed the vegetation and set up six randomized blocks. Within each block, we will have four subplots with the following treatments: 1) control; 2) dominant grass removal; 3) second most dominant grass removal; 4) soil disturbance with no grass species removal. We will present our methodology to study the effect of the removal of the two grass species on the bacterial community of the area under restoration. We expect that results from this project will provide baseline data to help inform and improve restoration practices for the longleaf pine

Investigating the Impact of the Removal of Andropogon Virginicus on the Soil Bacterial Microbiome of the Longleaf Pine

The longleaf pine (Pinus palustris), native to the Southeastern United States, is part of an endangered, pyrophytic ecosystem. The longleaf pine plays a significant role in the environment and is an important economic resource. It is currently considered the third most endangered ecosystem in the United States. Little is known about the microbial community within this ecosystem. The aim of this study is to investigate the effect of the removal of two dominant herbaceous species (Pityopsis nervosa and Andropogon sp.) on the bacterial soil microbiome of an area under restoration for the longleaf pine in the Piedmont region of Georgia. We focused on the effect of removing Andropogon sp., the second-most dominant grass species in the longleaf pine ecosystem. This species is notable for its ability to contribute to forest fires, which are necessary to the regeneration of the longleaf pine. Two longleaf pine restoration sites were chosen for this study. At each, six blocks were randomly set up containing four randomized subplots with one of the following treatments: control, removal of Andropogon sp., removal of Pityopsis nervosa, and soil disturbance. Soil samples were taken from each subplot in each block and DNA was extracted from soil using a commercially available kit. Further genetic analysis was performed to identify the bacteria that inhabit the soil microbiome in each treatment. Preliminary results include the presence of Proteobacteria and Planctomycetes. Planctomycetes are a phylum of terrestrial bacteria that inhabit soil microbial communities and are most notable for their ability to go through anammox in the nitrogen cycle. Proteobacteria are well known for their role in carbon, nitrogen, and sulfur cycling in soil. These results indicate key microbial groups and their functions to better understand the relationship between microbes and the herbaceous species the longleaf pine relies on

Effect of removing Pityopsis Nervosa on the Soil Bacterial Microbiome of a Longleaf Pine Ecosystem

The longleaf pine (Pinus palustris) is an integral part of a diverse, endangered ecosystem in the Southeastern United States. Longleaf pines are better equipped to survive strong winds and droughts than other native species and are notable for their dependence on fire for successful regeneration and establishment. The herbaceous vegetation within this ecosystem provides suitable conditions for the longleaf pine by supporting a positive feedback loop with fire. The herbaceous community’s importance is well known, but the microbial community associated with these key players is currently understudied. The aim of this study is to investigate the association between soil microorganisms and two important herbaceous species in the longleaf pine ecosystem, Pityopsis nervosa and Andropogon sp. Pityopsis nervosa is the most dominant herbaceous species in the montane longleaf pine restoration sites studied; this forb plays a major role in ecosystem maintenance by serving as fuel for low-intensity fires. Two longleaf pine restoration sites in North Georgia were chosen. Six blocks were randomly set up at each site, and each block contained four randomized subplots with one of the following treatments: control, removal of Andropogon sp., removal of Pityopsis nervosa, and soil disturbance. DNA was extracted using a commercial extraction kit, measured for concentration, and analyzed to identify bacteria inhabiting the soil samples from each sublot. Preliminary results of these analyses indicated that Acidobacteria and Actinobacteria were among the most abundant phyla of bacteria present in each treatment group. Acidobacteria is a keystone taxon known to regulate biochemical cycles and promote plant growth. Actinobacteria are widely distributed in soil and secrete enzymes involved in degrading chitin and chitosan, play roles in carbon cycling, and degrade plant residues. Additional findings will add to the knowledge of these key microbial groups inhabiting the ecosystem and interacting with dominant herbaceous species such as Pityopsis nervosa