Endophytic prokaryotic diversity associated with sea grass beds of Thalassia testudinum from Cabo Rojo, Lajas,and Vieques, Puerto Rico

Sea grass beds are considered a vital component of marine ecosystems, which serve as a nutrient source for many marine organisms, while also providing a critical habitat for many endangered species. In Puerto Rico sea grass bed communities are beneficial to different marine ecosystems, though this habitat has been threatened by developmental projects, pollution and global warming which consequently are causing its decay. The scope of this research project was to determine the prokaryotic endophytic diversity of T. testudinum beds and to establish differences in community structure of four geographical areas in Puerto Rico that have been exposed to different anthropogenic impacts. The objectives were addressed by culture dependent and culture independent techniques. A total of 1,279 strains were recovered from surface sterilized leaves. Phylogenetic analysis revealed the prevalence of gamma-Proteobacteria (Enterobacter (5%), Pseudomonas(1%) and Cobetia(4%), Actinobacteria (Cellulosimicrobium, Nesterenkonia and Micrococcus (1%)), and the Firmicutes (Bacillus (55%), Geobacillus (<1%), Staphylococcus (18%) and Halobacillus (11%). Furthermore the complexity and diversity patterns of the prokaryote community associated with total DNA extracted was evaluated based on Terminal Fragment Length Polymorphism (TRFLP) profiles and the analysis of clone libraries of PCR amplified 16S rRNA genes. TRFLP patterns revealed shared similarities in community structure as well as unique patterns for each sampling site, while the analysis of 16S rRNA clone libraries revealed the dominance of alpha-Proteobacteria, gamma-Proteobacteria, epsilon-Proteobacteria and Firmicutes. Staphylococcus isolates predominated in Buye Beach (39%) in contrast to the reduced community at the other sites (<7%). In addition, Bacillus isolates were predominant in all sites, excluding Buye which confirms the unfavorable influence of higher impact to this site, since members of Bacillus are recognized for establishing beneficial associations with terrestrial plants. Culture independent techniques revealed low diversity of TRF's and shifts of predominant OTU's for Buye clone libraries. Statistical analysis confirmed that this site is significantly different from the others, suggesting that Buye higher levels of impact are causing alterations to the basal endophytic communities. By knowing the assemblages of these endophytes is setting bases for the development of new strategies for restoration programs to address the decay of sea grasses.