SspI (10 U/µL)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Fluorescently labeled products were digested with 10 U of restriction enzyme SspI or AvrII (Thermo Fisher Scientific, Italy) by following the manufacturer’s instructions.	Digested products were purified and analyzed on an ABI3730 capillary sequencer in genotyping mode with the size standard ROX-labeled GS500.

Protocol tips
Fluorescently labeled products were digested with 10 U of restriction enzyme SspI or AvrII (Thermo Fisher Scientific, Italy) by following the manufacturer’s instructions.
Downstream tips
Digested products were purified and analyzed on an ABI3730 capillary sequencer in genotyping mode with the size standard ROX-labeled GS500.

Publication protocol

To ensure that the Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis was not biased by biological sample, DNA extraction, and amplicon library preparation, two DNA pools, each consisting of equimolar DNA samples from biological replicates of each treatment, were prepared. Thus, the profile of the epiphytic bacterial community was derived analyzing 18 leaves from nine plants for each treatment. PCR amplification targeting the 16S rRNA gene was carried out using primer 8F (5′-AGAGTTTGATCCTGGCTCAG-3′), fluorescently labeled at the 5′ end with 6-FAM (6-carboxyfluorescein), and 1387R (5′-GGGCGGWGTGTACAAGGC-3′), with three replicates per DNA pool. PCR replicates were purified, using the Promega Wizard (Promega, Madison, WI, United States), quantified, using Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Italy), and pooled into a single tube to represent each amplicon library in equimolar amounts. Fluorescently labeled products were digested with 10 U of restriction enzyme SspI or AvrII (Thermo Fisher Scientific, Italy) by following the manufacturer’s instructions. Digested products were purified and analyzed on an ABI3730 capillary sequencer in genotyping mode with the size standard ROX-labeled GS500. Only peaks that achieved a prevalence of more than 1% have been considered. Replicate T-RF profiles of each DNA pool and T-RF profiles of distinct DNA pools from samples of each treatment gave reproducible fingerprints. Consensus profiles were created as suggested by Dunbar et al. (2001), using the average values for peak heights. Total richness (S), Shannon’s diversity index (H), and Simpson’s evenness index (E) were calculated using PRIMER (v7, PRIMER-E Ltd., Plymouth, United Kingdom)

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Manufacturer protocol

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