Understanding the Ways of the Water

Coastal ecosystems are biodiverse and under threat from human activities and climate change. While microorganisms are an important component of biodiversity in many ecosystems, little is known about microbial communities in coastal environments of the Gulf of Mexico, and few studies have investigated microbial communities in coastal Mississippi. Beaches are especially important along the Mississippi coast as they account for approximately a third of the coastline. We characterized the bacterial communities in sand and seawater at 50 beach sites spanning much of the Mississippi Gulf coast. DNA was extracted from seawater and sand collected from each site, and a portion of the 16S rRNA gene was sequenced via Illumina NextSeq. Sequences were separated into amplicon sequence variants (ASVs), each representing a different 16S rRNA gene sequence type. Samples averaged an observed richness of approximately 1,900 ASVs per sample, with bacterial communities in sand typically richer than those in seawater. Community composition differed between sand and seawater, but both communities were dominated by members of the Bacteroidetes, Gammaproteobacteria, and Alphaproteobacteria. Seawater tended to have greater proportions of Betaproteobacteria, and Verrucomicrobia, while sand tended to contain greater proportions of Planctomycetes and Acidobacteria. Ongoing work is relating these bacterial communities to spatial variation and physicochemical parameters collected at the time of sampling.

Ralstonia solanacearum is a bacterial pathogen that causes the devastating bacterial wilt disease in major crops such as tomato, potato, banana, pepper, etc. Recently, this bacterium was discovered in association with floating pennywort (Hydrocotyle ranunculoides) and Pennsylvania smartweed (Polygonum pennysylvanicum), two previously undescribed aquatic hosts. Duckweed is an aquatic plant with a high level of resilience and short doubling time. Common duckweed (Lemna minor) has previously demonstrated its capability as a versatile infection model for human pathogens. Given this newly found association of R. solanacearum with aquatic hosts and the fact that duckweed is a common pond weed previously identified in several ponds located nearby infected crops, the goal of this project is to evaluate its capability as a host for the spread of R. solanacearum. In order to establish both common duckweed and giant duckweed (Spirodela polyrhiza) as a potential host for this pathogen, we developed an infection assay in which the pondweeds were placed into a water-based solution containing the bacterium. Following inoculation, the plants were surface sterilized, homogenized, and dilution plated in order to quantify bacterial presence inside the plant material. The colonies isolated from duckweed were then used to inoculate 3-week-old Bonny Best tomato plants. Plant wilting symptom was recorded over a course of two weeks. The results of this study indicate that both common and giant duckweed can serve as a host to R. solanacearum and S.polyrhiza is capable of serving as an agent for the spread of Ralstonia.

Understanding the spatial and temporal dynamics of marine bacterial communities is crucial for assessing ecosystem health and the impacts of environmental changes along the Gulf Coast. Bacterioplankton communities in seawater were sampled monthly at five shoreline sites (Pass Christian Central, Long Beach, East Courthouse, Biloxi West, and Biloxi East beaches) along the Mississippi coast and characterized by next generation sequencing of the 16S rRNA gene. The structure of these bacterial communities varied between sampling months and location along the coast, with significant interactions between sampling month and site. Positive correlations were observed between both temporal and spatial distance and bacterial community composition, with temporal differences being more pronounced. Salinity and pH significantly influenced the structure of seston bacterial communities. Bacterial species richness varied by month and ranged from 1,145-1,599, while bacterial species diversity (Inverse Simpson's Index) ranged from 88.5-186.2. Bacterial species diversity in seawater at Pass Christian Central beach was significantly lower than that at East Courthouse, Biloxi West, and Biloxi East beaches. Seawater at Pass Christian Central and Biloxi East beaches had significantly lower bacterial species richness compared to water at Long Beach and East Courthouse. All samples were dominated by members of the Bacteroidetes, Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, and Cyanobacteria, although the relative abundances of these phyla varied spatially and temporally. Proportions of Cyanobacteria were consistent across sites but increased significantly from January to April, correlating with increasing water temperatures. These findings highlight the dynamic nature of marine bacterial communities along the Mississippi Gulf Coast and provide valuable insights for managing and preserving the health of coastal ecosystems.

The Grand Bay National Estuarine Research Reserve (GNDNERR) has chronically elevated fecal coliform counts; however, the source of the contamination is unknown. Due to the poor understanding of the local fecal pollution sources, this area has not been opened to shellfish harvesting since 2007. According to past sanitary surveys, there has been a history of malfunctioning residential septic systems and inadequate wastewater treatment in the upper watershed. Feral hogs and birds are also abundant in the estuary and could be a potential contributor to elevated fecal coliform levels in this area. The GNDNERR is located on the northern Gulf of Mexico coastline which is known to receive some of the highest annual precipitation totals in the United States. The intense rainfall can lead to a large fecal load from upland sources into the estuary. To better understand the temporal and spatial variation of fecal coliforms in the GNDNERR and inform oyster reef management, monthly sampling took place over the course of a year at six sites within the main watershed of the GNDNERR and two sites upstream in the bayous. Having the two sites upstream allows for a better understanding of how rainfall affects fecal contamination throughout the estuary. Quantitative polymerase chain reactions (qPCR) are being used to identify levels of potential sources from humans, feral hogs, and birds within the estuary as well as the concentrations of Enterococcus. Preliminary data analysis has shown the presence of temporal and spatial patterns for the feral hog and bird markers.

Coastal waters can experience elevated levels of fecal contamination as a result of human populations, unsupported infrastructure, and terrestrial watershed drainage. Pathogens can be conveyed to waterways from human and non-human (e.g., wildlife, livestock, domesticated animals) sources, posing health risks through contaminated fisheries and recreational waters. Microbial source tracking seeks to identify fecal contamination, and different approaches vary in capability and effort. There is a need for reliable and efficient methods to improve understanding of fecal sources for land use and waterway management. Molecular methods (e.g., environmental DNA, quantitative PCR) can identify DNA-based viral and bacterial communities as well as quantify Bacteroides (a dominant group of fecal bacteria that enables source identification due to high host specificity). This study aims to identify fecal sources to the Mississippi-Alabama coast using a novel multi-species approach. Water samples were collected monthly for one year from 13 sites that represent potential fecal inputs to Alabama's coast. Total DNA was extracted and metagenomes sequenced on an Illumina NovaSeq platform with 6X coverage to capture the rare biosphere. An aliquot was reserved for qPCR that targeted multiple species (fowl, pig and feral hog, human, cow, dog, general Bacteroides). Bacteria associated with feces were prevalent in Mobile Bay, Alabama year-round, with human-associated as the predominant source, followed episodically by pig-associated feces. Overall, greater concentrations of Bacteroides were found in the summer months, and north Mobile Bay had higher average concentrations than the south bay. Coupled with an eDNA metabarcoding approach, species and trophic relationships not captured by qPCR methods can be identified (such as free-living or algal-associated Bacteroides) as well as potential pathogenicity. To enhance monitoring and management efforts, we are developing an eDNA Toolkit training workshop to provide partners guidance on implementing advanced source tracking methods.