The microbiome associated to the marine fanerogames, studies and applications

Seagrasses are benthic ecosystems that provide important services in the coastal zones, yet are declining worldwide at an alarming rate. Seagrasses provide habitat to commercially important fish and crustaceans, and contribute to coastal protection through the stabilization of sediment run-off. They play a critical role in the maintenance of biogeochemical cycles, burying approximately 12% of the global atmospheric carbon production. Rising anthrophogenic and natural threats, including human-induced climate change may negatively affect seagrasses, resulting in permanent habitat loss and reduction of biodiversity. With most of seagrass environmental monitoring based on long term responses to environmental pressures, there is a growing interest to develop alternative diagnostic tools that more effectively identify changes in seagrasses conservation status at an early stage. Diversified microbial communities are known to be associated with seagrasses above (leaves) and belowground (rhizomes and roots). These microbial communities are tightly associated with plants and have the capacity to adapt rapidly to changes in environmental conditions, helping secure meadow health and conservation. This research activity has been devoted to study the microbial communities of seagrass, to describe their structure and composition even in different environmental conditions. These studies give the necessary basic knowledge to understand the relationship microbiome/seagrass plants and their possible use as a putative marker of environmental change.

Next Generation Sequencing techniques has been applied (in the beginning, 454-pyrosequencing, then Illumina Platform) to the microbial metagenome associated with plants of Halophila stipulacea from Red Sea, Thalassia hemprichi from Faafu atoll (Maldives) and Posidonia oceanica from Cyprus and Crete. These analyses showed how different the microbial communities are, in both different plant parts and environmental conditions, varying along environmental gradients and according to the eco-physiological state of the plant. This depends on the ability of microbial communities to rapidly respond to environmental changes, and implies that the host plant/associated microbiome interaction plays a crucial role in the plant's fitness, but also that the host and all associated microorganisms represent a complex functional unit, called holobiont, which significantly changes the concept of living organism. Therefore, microbial communities are able to provide indications on the conservation status of plants and, consequently, could represent a sensitive putative monitoring tool and ecological indicator, to detect signs of stress in marine plants, before an irreversible decline occurs.

 

This work was made possible thanks to the collaboration with: Gidon Winters from the Dead Sea & Arava Science Center, Hazeva (Israel); Rodrigo Costa of the University of Lisbon (Portugal); Pedro Beca-Carretero from the School of Natural Sciences of the National University of Ireland, Galway (Ireland), Paolo Galli and Davide Seveso from the University of Milano Bicocca and Marlen Vasquez from the Cyprus Technological University, Limassol (Cyprus). Field studies were made possible thanks to the numerous grants won on the ASSEMBLE, ASSEMBLE plus and COST projects, funded by the European Community, but also thanks to the MaRHE prize of the University of Milano Bicocca, awarded to Astrid Mejia, during the XXIV Congress of the Italian Society of Ecology in Ferrara (2014).

 

Conte C., Rotini A., Manfra L., D’Andrea M.M., Winters G., Migliore L. (2021). THE SEAGRASS HOLOBIONT: WHAT WE KNOW AND WHAT WE STILL NEED TO DISCLOSE FOR ITS POSSIBLE USE AS AN ECOLOGICAL INDICATOR. Water, 13(4): 406. DOI: 10.3390/w13040406

 Microbes and seagrass establish symbiotic relationships constituting a functional unit called the holobiont that reacts as a whole to environmental changes. Recent studies have shown that the seagrass microbial associated community varies according to host species, environmental conditions and the host’s health status, suggesting that the microbial communities respond rapidly to environmental disturbances and changes. These changes, dynamics of which are still far from being clear, could represent a sensitive monitoring tool and ecological indicator to detect early stages of seagrass stress. In this review, the state of art on seagrass holobiont is discussed in this perspective, with the aim of disentangling the influence of different factors in shaping it. As an example, we expand on the widely studied Halophila stipulacea’s associated microbial community, highlighting the changing and the constant components of the associated microbes, in different environmental conditions. These studies represent a pivotal contribution to understanding the holobiont’s dynamics and variability pattern, and to the potential development of ecological/ecotoxicological indices. The influences of the host’s physiological and environmental status in changing the seagrass holobiont, alongside the bioinformatic tools for data analysis, are key topics that need to be deepened, in order to use the seagrass-microbial interactions as a source of ecological information.

 

 Rotini A., Conte C., Seveso D., Montano S., Galli P., Vai M., Migliore L., Mejia A. (2020). DAILY VARIATION OF THE ASSOCIATED MICROBIAL COMMUNITY AND THE HSP60 EXPRESSION IN THE MALDIVIAN SEAGRASS THALASSIA HEMPRICHIIJournal of Sea Research, 156: 101835. DOI: 10.1016/j.seares.2019.101835

 

The microbial communities associated with plant’s compartments and the expression of the chloroplast chaperonin Hsp60 have been simultaneously analyzed during the diel cycle in the marine seagrass Thalassia hemprichii from the lagoon of Magoodhoo island (Maldives), characterized by remarkable daily shifts in temperature and light. Plants showed a significant up-regulation of Hsp60 expression from 8.00 a.m to 2.00 p.m., in correspondence with the increase in temperature and light, confirming their role as defence mechanism against photoinhibition and oxidative damage. However, a further significant increase of the Hsp60 level was also observed when irradiance and temperature dropped, suggesting that the cellular stress was still in progress. The plant-associated microbial communities showed differences by plant compartment and sampling time, with the aboveground compartment (leaves) being much more dynamic than the belowground one (roots/rhizomes). In the phyllosphere, a progressive shift during the day from the absolute dominance of the Gammaproteobacteria class, mainly constituted of Enterobacteraceae family, to the increase of the biodiversity due to the rise of Alphaproteobacteria was observed. Belowground, the microbial diversity was much lower than aboveground, being Gammaproteobacteria the most represented class throughout all sampling times. Vibrionaceae family was the most abundant at 8:00 a.m. and 6.00 p.m. decreasing slightly at 2.00 p.m., partially replaced by Halomonadaceae. The combined biochemical and microbial markers allows to assess the plant stress response and to deepen the knowledge on the seagrasses adaptation to harsh and changing environmental conditions, resulting useful to detect early signs of change in an organism's physiological state. Furthermore, the variation of Hsp60 expression and associated bacterial communities in response to light and temperature fluctuations support the ‘holobiont’ theory, which considers the plant/microorganisms association as a functional unit, as suggested for corals.

 

Rotini A., Mejia A.Y., Costa R., Migliore L., Winters G. (2017) - THE SEAGRASS HOLOBIONT HALOPHILA STIPULACEA: ECOPHYSIOLOGICAL PLASTICITY AND BACTERIOME STRUCTURE ALONG A DEPTH GRADIENT IN THE NORTHERN RED SEA. Frontiers in Plant Science, 7: 2015

Halophila stipulacea is a small tropical seagrass species. It is the dominant seagrass species in the Gulf of Aqaba (GoA; northern Red Sea), where it grows in both shallow and deep environments (1–50 m depth). Native to the Red Sea, Persian Gulf, and Indian Ocean, this species has invaded the Mediterranean and has recently established itself in the Caribbean Sea. Due to its invasive nature, there is growing interest to understand this species’ capacity to adapt to new conditions, which might be attributed to its ability to thrive in a broad range of ecological niches. In this study, a multidisciplinary approach was used to depict variations in morphology, biochemistry (pigment and phenol content) and epiphytic bacterial communities along a depth gradient (4–28 m) in the GoA. Along this gradient, H. stipulacea increased leaf area and pigment contents (Chlorophyll a and b, total Carotenoids), while total phenol contents were mostly uniform. H. stipulacea displayed a well conserved core bacteriome, as assessed by 454-pyrosequencing of 16S rRNA gene reads amplified from metagenomic DNA. The core bacteriome aboveground (leaves) and belowground (roots and rhizomes), was composed of more than 100 Operational Taxonomic Units (OTUs) representing 63 and 52% of the total community in each plant compartment, respectively, with a high incidence of the classes AlphaproteobacteriaGammaproteobacteria, and Deltaproteobacteria across all depths. Above and belowground communities were different and showed higher within-depth variability at the intermediate depths (9 and 18 m) than at the edges. Plant parts showed a clear influence in shaping the communities while depth showed a greater influence on the belowground communities. Overall, results highlighted a different ecological status of H. stipulacea at the edges of the gradient (4–28 m), where plants showed not only marked differences in morphology and biochemistry, but also the most distinct associated bacterial consortium. We demonstrated the pivotal role of morphology, biochemistry (pigment and phenol content), and epiphytic bacterial communities in helping plants to cope with environmental and ecological variations. The plant/holobiont capability to persist and adapt to environmental changes probably has an important role in its ecological resilience and invasiveness.

microbioma-2

Mejia A., Rotini A., Lacasella F., Bookman R., Thaller M.C., Winters G., Migliore L. (2016) - MORPHOLOGY, BIOCHEMICAL DESCRIPTORS AND MICROBIAL COMMUNITY ANALYSIS: A PROMISING APPROACH FOR ASSESSING THE ECOLOGICAL STATUS OF SEAGRASSES. A CASE STUDY, HALOPHILA STIPULACEA FROM THE RED SEA. Ecological Indicators, 60: 1150-1163.

Seagrasses are one of the most valuable marine ecosystems on earth, yet they are declining worldwide at alarming rates. With most of seagrass monitoring based on long term responses to environmental pressures, there is growing interest in developing alternative diagnostic tools that more effectively identify changes in seagrass ecological status at an early stage. Besides morphological indicators, functional and biochemical descriptors may provide a good understanding of plant's responses to environmental changes. Moreover, the epiphytic microbial communities of seagrasses may also shift in response to changes in environmental conditions, although these have been seldom used as a descriptor of environmental change. In this study three Halophila stipulacea (Forsk.) Aschers meadows, found in the Gulf of Aqaba (northern Red Sea), were characterized using an integrated approach to highlight possible differences in the meadows ecological status. Plant descriptors, including leaves morphometrics (leaf size, leaf number/plant, leaves with lost apex), photosynthetic pigments (Chlorophylls, Carotenoids) and total phenols contents, were investigated and coupled with the plants’ epiphytic microbial community structure and composition, studied using pyrosequencing. The entire suite of descriptors highlighted differences among the meadows ecological status based on changes in plants’ morphology and biochemistry, and their associated microbial communities, in response to the different environmental conditions (water column turbidity, seawater and sediment nutrients) and the geomorphological features (bottom slope, granulometry) of the stations. Leaf morphology and photosynthetic pigment content were modulated in H. stipulacea in response to light availability and hydrodynamics in the Gulf of Aqaba. The highest leaf surface area and photosynthetic pigment contents were observed at the lowest irradiance and hydrodynamics/granulometry among stations. Total phenol content showed differences among stations with increasing concentrations from north to south. The microbial communities showed differences among stations and plant compartments, with high incidence of Gammaproteobacteria and Bacteroidetes in light limiting conditions, while Cyanobacteria and Rhodobacteraceae thrived in conditions of high light availability and hydrodynamics. The mutual response of the seagrass plants and the microbial communities provided evidence of their functional relationship, which undoubtedly needs further investigation. To the best of our knowledge, this is the first time that such descriptors have been used in an integrated approach. We provide evidence of their effectiveness in discriminating seagrass ecological status, even at small spatial scales. This work constitutes a new approach to the assessment of seagrasses and a stepping stone in the application of microbial communities as a putative marker in a changing environment.

microbioma-1