Opportunities and challenges for microbiomics in ecosystem restoration.

Microbiomics is the science of characterizing microbial community structure, function, and dynamics. It has great potential to advance our understanding of plant-soil-microbe processes and interaction networks which can be applied to improve ecosystem restoration. However, microbiomics may be perceived as complex and the technology is not accessible to all. The opportunities of microbiomics in restoration ecology are considerable, but so are the practical challenges. Applying microbiomics in restoration must move beyond compositional assessments to incorporate tools to study the complexity of ecosystem recovery. Advances in metaomic tools provide unprecedented possibilities to aid restoration interventions. Moreover, complementary non-omic applications, such as microbial inoculants and biopriming, have the potential to improve restoration objectives by enhancing the establishment and health of vegetation communities.

Marine heatwave and reduced light scenarios cause species-specific metabolomic changes in seagrasses under ocean warming.

Climate change and extreme climatic events, such as marine heatwaves (MHWs), are threatening seagrass ecosystems. Metabolomics can be used to gain insight into early stress responses in seagrasses and help to develop targeted management and conservation measures. We used metabolomics to understand the temporal and mechanistic response of leaf metabolism in seagrasses to climate change. Two species, temperate Posidonia australis and tropical Halodule uninervis, were exposed to a combination of future warming, simulated MHW with subsequent recovery period, and light deprivation in a mesocosm experiment. The leaf metabolome of P. australis was altered under MHW exposure at ambient light while H. uninervis was unaffected. Light deprivation impacted both seagrasses, with combined effects of heat and low light causing greater alterations in leaf metabolism. There was no MHW recovery in P. australis. Conversely, the heat-resistant leaf metabolome of H. uninervis showed recovery of sugars and intermediates of the tricarboxylic acid cycle under combined heat and low light exposure, suggesting adaptive strategies to long-term light deprivation. Overall, this research highlights how metabolomics can be used to study the metabolic pathways of seagrasses, identifies early indicators of environmental stress and analyses the effects of environmental factors on plant metabolism and health.

Sulfide intrusion in a habitat forming seagrass can be predicted from relative abundance of sulfur cycling genes in sediments.

Sulfide intrusion from sediments is an increasingly recognized contributor to seagrass declines globally, yet the relationship between sediment microorganisms and sulfide intrusion has received little attention. Here, we use metagenomic sequencing and stable isotope (34S) analysis to examine this relationship in Cockburn Sound, Australia, a seagrass-dominated embayment with a gradient of sulfide stress and seagrass declines. There was a significant positive relationship between sulfide intrusion into seagrasses and sulfate reduction genes in sediment microbial communities, which was greatest at sites with long term seagrass declines. This is the first demonstration of a significant link between sulfur cycling genes present in seagrass sediments and sulfide intrusion in a habitat-forming seagrass that is experiencing long-term shoot density decline. Given that microorganisms respond rapidly to environmental change, the quantitative links established in this study can be used as a potential management tool to enable the prediction of sulfide stress on large habitat forming seagrasses; a global issue expected to worsen with climate change.

Natural attenuation of legacy hydrocarbon spills in pristine soils is feasible despite difficult environmental conditions in the monsoon tropics.

The Kimberley region of Western Australia is a National Heritage listed region that is internationally recognised for its environmental and cultural significance. However, petroleum spills have been reported at a number of sites across the region, representing an environmental concern. The region is also characterised as having low soil nutrients, high temperatures and monsoonal rain - all of which may limit the potential for natural biodegradation of petroleum. Therefore, this work evaluated the effect of legacy petroleum hydrocarbons on the indigenous soil microbial community (across the domains Archaea, Bacteria and Fungi) at three sites in the Kimberley region. At each site, soil cores were removed from contaminated and control areas and analysed for total petroleum hydrocarbons, soil nutrients, pH and microbial community profiling (using16S rRNA and ITS sequencing on the Illumina MiSeq Platform). The presence of petroleum hydrocarbons decreased microbial diversity across all kingdoms, altered the structure of microbial communities and increased the abundance of putative hydrocarbon degraders (e.g. Mycobacterium, Acremonium, Penicillium, Bjerkandera and Candida). Microbial community shifts from contaminated soils were also associated with an increase in soil nutrients (notably Colwell P and S). Our study highlights the long-term effect of legacy hydrocarbon spills on soil microbial communities and their diversity in remote, infertile monsoonal soils, but also highlights the potential for natural attenuation to occur in these environments.

Cable bacteria at oxygen-releasing roots of aquatic plants: a widespread and diverse plant-microbe association.

Cable bacteria are sulfide-oxidising, filamentous bacteria that reduce toxic sulfide levels, suppress methane emissions and drive nutrient and carbon cycling in sediments. Recently, cable bacteria have been found associated with roots of aquatic plants and rice (Oryza sativa). However, the extent to which cable bacteria are associated with aquatic plants in nature remains unexplored. Using newly generated and public 16S rRNA gene sequence datasets combined with fluorescence in situ hybridisation, we investigated the distribution of cable bacteria around the roots of aquatic plants, encompassing seagrass (including seagrass seedlings), rice, freshwater and saltmarsh plants. Diverse cable bacteria were found associated with roots of 16 out of 28 plant species and at 36 out of 55 investigated sites, across four continents. Plant-associated cable bacteria were confirmed across a variety of ecosystems, including marine coastal environments, estuaries, freshwater streams, isolated pristine lakes and intensive agricultural systems. This pattern indicates that this plant-microbe relationship is globally widespread and neither obligate nor species specific. The occurrence of cable bacteria in plant rhizospheres may be of general importance to vegetation vitality, primary productivity, coastal restoration practices and greenhouse gas balance of rice fields and wetlands.

Cutting out the middle clam: lucinid endosymbiotic bacteria are also associated with seagrass roots worldwide.

Seagrasses and lucinid bivalves inhabit highly reduced sediments with elevated sulphide concentrations. Lucinids house symbiotic bacteria (Ca. Thiodiazotropha) capable of oxidising sediment sulphide, and their presence in sediments has been proposed to promote seagrass growth by decreasing otherwise phytotoxic sulphide levels. However, vast and productive seagrass meadows are present in ecosystems where lucinids do not occur. Hence, we hypothesised that seagrasses themselves host these sulphur-oxidising Ca. Thiodiazotropha that could aid their survival when lucinids are absent. We analysed newly generated and publicly available 16S rRNA gene sequences from seagrass roots and sediments across 14 seagrass species and 10 countries and found that persistent and colonising seagrasses across the world harbour sulphur-oxidising Ca. Thiodiazotropha, regardless of the presence of lucinids. We used fluorescence in situ hybridisation to visually confirm the presence of Ca. Thiodiazotropha on roots of Halophila ovalis, a colonising seagrass species with wide geographical, water depth range, and sedimentary sulphide concentrations. We provide the first evidence that Ca. Thiodiazotropha are commonly present on seagrass roots, providing another mechanism for seagrasses to alleviate sulphide stress globally.

Soil microbes of an urban remnant riparian zone have greater potential for N removal than a degraded riparian zone.

Soils in the riparian zone, the interface between terrestrial and aquatic ecosystems, may decrease anthropogenic nitrogen (N) loads to streams through microbial transformations (e.g., denitrification). However, the ecological functioning of riparian zones is often compromised due to degraded conditions (e.g., vegetation clearing). Here we compare the efficacy of an urban remnant and a cleared riparian zone for supporting a putative denitrifying microbial community using 16S rRNA sequencing and quantitative polymerase chain reaction of archaeal and bacterial nitrogen cycling genes. Although we had no direct measure of denitrification rates, we found clear patterns in the microbial communities between the sites. Greater abundance of N-cycling genes was predicted by greater soil ammonium (N-NH4 ), organic phosphorus, and C:N. At the remnant site, we found positive correlations between microbial community composition, which was dominated by putative N oxidisers (Nitrosomonadaceae, Nitrospiraceae and Nitrosotaleaceae), and abundance of ammonia-oxidizing archaea (AOA), nirS, nirK and nosZ, whereas the cleared site had lower abundance of N-oxidisers and N cycling genes. These results were especially profound for the remnant riparian fringe, which suggests that this region maintains suitable soil conditions (via diverse vegetation structure and periodic saturation) to support putative N cyclers, which could amount to higher potential for N removal.

Root microbiomes as indicators of seagrass health.

The development of early warning indicators that identify ecosystem stress is a priority for improving ecosystem management. As microbial communities respond rapidly to environmental disturbance, monitoring their composition could prove one such early indicator of environmental stress. We combined 16S rRNA gene sequencing of the seagrass root microbiome of Halophila ovalis with seagrass health metrics (biomass, productivity and Fsulphide) to develop microbial indicators for seagrass condition across the Swan-Canning Estuary and the Leschenault Estuary (south-west Western Australia); the former had experienced an unseasonal rainfall event leading to declines in seagrass health. Microbial indicators detected sites of potential stress that other seagrass health metrics failed to detect. Genera that were more abundant in 'healthy' seagrasses included putative methylotrophic bacteria (e.g. Methylotenera and Methylophaga), iron cycling bacteria (e.g. Deferrisoma and Geothermobacter) and N2 fixing bacteria (e.g. Rhizobium). Conversely, genera that were more abundant in 'stressed' seagrasses were dominated by putative sulphur-cycling bacteria, both sulphide-oxidising (e.g. Candidatus Thiodiazotropha and Candidatus Electrothrix) and sulphate-reducing (e.g. SEEP-SRB1, Desulfomonile and Desulfonema). The sensitivity of the microbial indicators developed here highlights their potential to be further developed for use in adaptive seagrass management, and emphasises their capacity to be effective early warning indicators of stress.

Oxygen loss from seagrass roots coincides with colonisation of sulphide-oxidising cable bacteria and reduces sulphide stress.

Seagrasses thrive in anoxic sediments where sulphide can accumulate to phytotoxic levels. So how do seagrasses persist in this environment? Here, we propose that radial oxygen loss (ROL) from actively growing root tips protects seagrasses from sulphide intrusion not only by abiotically oxidising sulphides in the rhizosphere of young roots, but also by influencing the abundance and spatial distribution of sulphate-reducing and sulphide-oxidising bacteria. We used a novel multifaceted approach combining imaging techniques (confocal fluorescence in situ hybridisation, oxygen planar optodes, and sulphide diffusive gradients in thin films) with microbial community profiling to build a complete picture of the microenvironment of growing roots of the seagrasses Halophila ovalis and Zostera muelleri. ROL was restricted to young root tips, indicating that seagrasses will have limited ability to influence sulphide oxidation in bulk sediments. On the microscale, however, ROL corresponded with decreased abundance of potential sulphate-reducing bacteria and decreased sulphide concentrations in the rhizosphere surrounding young roots. Furthermore, roots leaking oxygen had a higher abundance of sulphide-oxidising cable bacteria; which is the first direct observation of these bacteria on seagrass roots. Thus, ROL may enhance both abiotic and bacterial sulphide oxidation and restrict bacterial sulphide production around vulnerable roots, thereby helping seagrasses to colonise sulphide-rich anoxic sediments.

Low Light Availability Alters Root Exudation and Reduces Putative Beneficial Microorganisms in Seagrass Roots.

Seagrass roots host a diverse microbiome that is critical for plant growth and health. Composition of microbial communities can be regulated in part by root exudates, but the specifics of these interactions in seagrass rhizospheres are still largely unknown. As light availability controls primary productivity, reduced light may impact root exudation and consequently the composition of the root microbiome. Hence, we analyzed the influence of light availability on root exudation and community structure of the root microbiome of three co-occurring seagrass species, Halophila ovalis, Halodule uninervis and Cymodocea serrulata. Plants were grown under four light treatments in mesocosms for 2 weeks; control (100% surface irradiance (SI), medium (40% SI), low (20% SI) and fluctuating light (10 days 20% and 4 days 100%). 16S rDNA amplicon sequencing revealed that microbial diversity, composition and predicted function were strongly influenced by the presence of seagrass roots, such that root microbiomes were unique to each seagrass species. Reduced light availability altered seagrass root exudation, as characterized using fluorescence spectroscopy, and altered the composition of seagrass root microbiomes with a reduction in abundance of potentially beneficial microorganisms. Overall, this study highlights the potential for above-ground light reduction to invoke a cascade of changes from alterations in root exudation to a reduction in putative beneficial microorganisms and, ultimately, confirms the importance of the seagrass root environment - a critical, but often overlooked space.

The role of root exuded low molecular weight organic anions in facilitating petroleum hydrocarbon degradation: current knowledge and future directions.

Rhizoremediation is a bioremediation technique whereby enhanced microbial degradation of organic contaminants occurs within the plant root zone (rhizosphere). It is considered an effective and affordable 'green technology' for remediating soils contaminated with petroleum hydrocarbons (PHCs). This paper critically reviews the potential role of root exuded compounds in rhizoremediation, with emphasis on commonly exuded low molecular weight aliphatic organic acid anions (carboxylates). The extent to which remediation is achieved shows wide disparity among plant species. Therefore, plant selection is crucial for the advancement and widespread adoption of this technology. Root exudation is speculated to be one of the predominant factors leading to microbial changes in the rhizosphere and thus the potential driver behind enhanced petroleum biodegradation. Carboxylates can form a significant component of the root exudate mixture and are hypothesised to enhance petroleum biodegradation by: i) providing an easily degradable energy source; ii) increasing phosphorus supply; and/or iii) enhancing the contaminant bioavailability. These differing hypotheses, which are not mutually exclusive, require further investigation to progress our understanding of plant-microbe interactions with the aim to improve plant species selection and the efficacy of rhizoremediation.