Abstract
The plant phyllosphere, which represents all plant parts that are above the ground, is considered one of the most extensive ecosystems to be colonized by microorganisms, both at the surface as epiphytes or as endophytes within the plant. These plant-associated microbial communities are reservoirs of microbial diversity and they can be important for plant health. The characterization of microbial communities in diverse plants, such as Espeletia plants that are endemic to the Paramo ecosystem in the Andes Mountains, can shed light regarding possible interactions among microorganisms and microbial functional properties. Obtaining DNA from plant endophytic microbial communities involves various steps to ensure that samples are free of contamination from microorganisms present on the plant surface (epiphytes). Plant leaves are first surface sterilized, cut into pieces, homogenized using glass beads, and then used for DNA extraction using a commercially available kit. DNA samples are then quantified and analyzed using Qubit® 2.0 for use in PCR amplification of 16S rRNA genes.
Keywords: Endophyte, Phyllosphere, Metagenomics, Epiphyte, 16S rRNA
Background
Extraction of endophytic DNA from plant samples has been done by several research groups and usually involves steps to minimize contamination from surface microbes. However, protocols must also be adapted to the plant material being studied and as such can incorporate different steps. Extraction of epiphyte DNA must also ensure that there is no contamination from endophytic microorganisms. The protocol described integrates elements from previous reports (Miles et al., 2012; Araujo et al., 2002), but was not identical given the characteristics of the plant material used. In this work we used leaves from Espeletia hartwegiana, a plant that is endemic to the Paramo ecosystem present in the Colombian Andean mountains. These leaves are characterized to be large and succulent with the presence of short hairs on the surface (pubescence) that require removal prior to leaf surface sterilization and DNA isolation. Here, although the pubescence is removed, the microorganisms associated with it are dislodged first to ensure a complete picture of the epiphyte community. In this case, sufficient DNA of good quality was recovered for PCR amplification and 16S rRNA gene analysis and for functional analysis using the GeoChip (Yan et al., 2015). Nonetheless, other downstream applications could require more DNA and hence more plant material.
Materials and Reagents
Note: All reagents used have a specific manufacturer and catalog number. Nonetheless, the user can use any molecular-grade reagent for the protocol.
Equipment
Procedure
Notes
Recipes
Acknowledgments
This work was financed by Colciencias (contract Nos. 573-2012 and 649-2013) and was performed under MADS contract No. 76-2013 for access to genetic resources and UAESPNN Research permit No. PIDB DTAO 021-10. The protocol was adapted from previously published work (Miles et al., 2012; Araujo et al., 2002). The authors declare that they have no conflict of interest that could impact on the design of implementation of this protocol.
References
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Hi, at the top of the online page (DOI: 10.21769/BioProtoc.2142), there is a link to download the pdf.