Materials and Reagents

Gloves
1.5 ml sterile microcentrifuge tubes
10, 100, 1,000 micropipette tips
Sterile craft paper
Ziploc bags (ethanol rinsed)
Sterile swabs (preferably sponge top)
Plant leaves (Espeletia sp.) ~10 g (Maybe more depending on the downstream application). Leaves were collected 1-2 days prior to processing, transported on dry ice and stored in sterile plastic bags at 4 °C
Sterile dH₂O
100% ethanol (Sigma-Aldrich, catalog number: E7023 )
5.25% sodium hypochlorite (Quidiscol Ltda, Bogotá, Colombia)
Power Soil DNA Isolation Kit (MO BIO Laboratories, catalog number: 12888-50 )
Sodium phosphate dibasic, Na₂HPO₄ (Sigma-Aldrich, catalog number: S3264 )
Malt extract solid medium (OXOID, catalog number: CM0059 )
Tris base (Promega, catalog number: H5133 )
EDTA (Sigma-Aldrich, catalog number: E9884 )
HCl (Mol Labs, catalog number: V4653 )
0.5 M NaH₂PO₄ (see Recipes)
NAP buffer (see Recipes)
Malt extract solid medium (see Recipes)
TE buffer (see Recipes)

Note: All reagents used have a specific manufacturer and catalog number. Nonetheless, the user can use any molecular-grade reagent for the protocol.

Equipment

Sterile 500-1,000 ml beakers, autoclaved
1-25 µl micropipette
10-100 µl micropipette
100-1,000 µl micropipette
Sterile razors
Vortex
Sterile tweezers
25 °C incubator
Sterile glass beads, autoclaved
Laminar flow hood, preferably a class II BSC to avoid contamination
Mini-Bead beater-96 (BioSpec Products)
Qubit^® 2.0 fluorometer (Thermo Fisher Scientific, USA)
Autoclave

Procedure

Leaves taken from Espeletia sp. plants (50 to 100 g) are transported to the laboratory inside sterile craft paper and Ziploc bags on dry ice. One set of leaves from each plant is then used for endophyte DNA extraction.
Whole, intact leaves are first washed with sterile dH₂O to remove dirt; this wash is performed by first submerging the leaves in sterile dH₂O, sufficient to cover the entire leaf (about 500 ml in a 1 L beaker), and then rinsing with clean dH₂O using a 100-1,000 µl micropipette.
The intact leaves are placed in NAP buffer (500 ml) in a 1 L sterile beaker and vortexed for 1 min to dislodge epiphytic microbes residing on the plant surface. The fine short hairs, or surface pubescence, present on Espeletia plant leaves are removed by shaving using sterile razors since this facilitates the subsequent sterilization process.
After shaving, leaves are rinsed by pipetting three times with sterile H₂O (for large leaves this is approximately 100 ml) and then submerged in:

500 ml of 90% ethanol for 60 sec in a 1,000 ml beaker.
Note: This depends on the size of the leaves; the beaker and the volume of ethanol can be smaller.
5.25% sodium hypochlorite solution in a similar beaker for 6 min.
70% ethanol for 30 sec.
During this sterilization process it is important to completely submerge the leaf samples in each solution, solutions should be sterile and everything should be conducted preferably in a hood to avoid possible contamination from air. Leaf samples are transferred from one solution to another using sterile tweezers.

The intact leaf samples are finally rinsed with sterile distilled H₂O using the 1,000 µl micropipette.
The sterilization is checked by making an imprint of each leaf on a plate of solid malt extract medium using sterile tweezers.
Plates are incubated at 25 °C for up to three weeks, and checked daily for growth. Incubation is done at 25 °C because environmental isolates tend to prefer temperatures below 37 °C. The sterilization process can also be done with liquid medium.
The leaf is cut into small pieces using the sterile razor and one gram is weighed out. These 1 g leaf fragments are further cut into 0.1 to 0.5 mm sections using sterile razors and placed in a 1.5 ml Eppendorf tube containing 1 g of sterile 0.1 mm diameter glass beads and 1 ml TE buffer. Alternatively, leaves can also be cut first into 0.1 to 0.5 mm sections, mixed and then weighed to obtain one gram of sample material. The plant material is processed immediately for extraction of DNA, but only those samples that proved to be sterile upon prolonged incubation on solid or in liquid media (step 7) are used for analysis of microbial communities.
The plant material is then homogenized at room temperature (20-25 °C) in a Mini-Bead Beater for 5 min at 36 oscillations/sec.
The DNA is extracted using the Power Soil DNA Isolation Kit, according to the manufacturer’s instructions, adding the recommended volume of the solution for a single extraction.
Note: In some cases more than one individual extraction can be performed for a single sample.
Extracted DNA is quantified using a Qubit^® 2.0 fluorometer, following the manufacturer’s instructions. It is also possible to visualize the quality of the DNA by gel electrophoresis, but this depends on the amount of DNA obtained, which is usually low.

Notes

During the sterilization, the leaves are processed intact and the amount of leaf material and water or NAP buffer depends on the size of the leaves.
The sterilization of leave samples was checked using solid media, but liquid media could also be used. In this case, sterilized leaves can be submerged briefly in liquid medium (1-2 min), rinsed, and then the medium incubated for several days or weeks, checking for growth daily. Growth in any of the media indicates incomplete sterilization and therefore the respective samples must be discarded or re-sterilized.
Depending on the amount of DNA required for downstream applications, more plant material can be used. If you have access to a bead beater that can accommodate larger volumes, you could add more leaf tissue and probably increase the DNA yields at the end.

Recipes

0.5 M NaH₂PO₄ (500 ml)
34.50 g NaH₂PO₄ (F.W. 137.99)
~450 ml dH₂O
Dissolve, then bring up to volume with dH₂O
Sterilize by autoclaving at 121 °C for 15 min
Note: A more concentrated solution (0.5 M) is prepared so it is easier to dilute to the desired concentration (124 mM).
NAP buffer
124 mM Na₂HPO₄
Malt extract solid medium
50 g MEA powder
1 L of dH₂O
Sterilize by autoclaving at 115 °C for 10 min
TE buffer
10 mM Tris
10 mM EDTA
Adjust pH to 8.0

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

Araujo, W. L., Marcon, J., Maccheroni, W., Jr., Van Elsas, J. D., Van Vuurde, J. W. and Azevedo, J. L. (2002). Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl Environ Microbiol 68(10): 4906-4914.
Miles, L. A., Lopera, C. A., González, S., de García, M. C., Franco, A. E. and Restrepo, S. (2012). Exploring the biocontrol potential of fungal endophytes from an Andean Colombian Paramo ecosystem. BioControl 57(5): 697-710.
Yan, Q., Bi, Y., Deng, Y., He, Z., Wu, L., Van Nostrand, J. D., Shi, Z., Li, J., Wang, X., Hu, Z., Yu, Y. and Zhou, J. (2015). Impacts of the Three Gorges Dam on microbial structure and potential function. Sci Rep 5: 8605.