Exploring epiphytic and endophytic prokaryotice diversity in the phyllosphere of halophytes of subfamily Salicornioideae (Amaranthaceae)

Habitats with a high concentration of salt are commonly colonized by halophytes. Halophytes accumulate large amounts of salt, and they are susceptible to the colonization by halotolerant and halophilic microorganisms. Those microorganisms might be relevant in the development of the plant. However, there is a considerable lack of knowledge regarding the association halophyte-microorganism as well about the biogeographic patterns of these colonizers. This study has been initiated in order to understand the presence, abundance and diversity of halophilic microorganisms in halophytes specifically the association of endophytes and epiphytes to the phyllosphere of members of the subfamily Salicornioideae of five locations from Chile and Spain. Culture-dependent methods were used with five salinity concentrations to isolation the microorganisms. Colonies were screened by MALDI-TOF/MS and 16S rRNA gene sequencing for identification purposes. Additionally, RAPD fingerprinting was used to recognize clonality. To ensure the isolation of endophytes, a previous sterilization surface protocol was performed on the plant and the protocol efficiency was checked with SEM. Moreover, a method for isolation of endophytic DNA was developed and samples were sequenced by 454 pyrosequencing. Culturable abundances detected were until 106 CFUg-1 and 107 CFUg-1 for epiphytes and endophytes respectively. Significant differences between abundances in epiphytes vs endophytes were detected in each location. Pyrosequencing data exposed the efficiency of the endophytic DNA extraction method and organisms identified both in culture and independent-culture methods were principally affiliated to the bacterial Proteobacteria, Firmicutes and Actinobacteria. Results showed that community structures was highly related to the compartment where microorganisms thrive (plant surface or internal tissues), with no geographic pattern. However, the presence of specific microbial ecotypes in each location and compartment (i.e. specific endophytes or epiphytes) in a same plant, reflect a specific adaptation to conditions in each plant.