Caruaru Soil Replicate 2
Roots, leaves and rizospheric soil from ten samples of A. nummularia were collected from each city between the 15th, 16th and 17th of September 2020. Samples were collected from different points of the field. Soils, roots and leaves were collected, separated in duplicates and stored in sterile plastic bags and kept inside a thermic box with ice until transfer to a freezer in the lab. Soil was collected near the roots of each saltbush and sieved (2mm). Small fragments of roots were collected, preferably thinner ones, where microbial activity is supposedly higher. To collect leaves, branches from different parts of the plants were cut. Leaves and roots samples were thawed and five grams of the material was submitted to superficial disinfection, to remove microbial contaminants from the outside of the tissues. The protocol consisted of: washing under running water; immersion in 70% alcohol for 1 minute; immersion in 2% sodium hypochlorite for 2 minutes; immersion in 70% alcohol for 1 minutes; 5 washings in sterile distilled water. Washed roots and leaves were transferred to erlenmeyers with 45 ml of PBS (1:10, organic material to PBS), and grinded and stirred with T 18 digital ULTRA-TURRAX at about 20000 rpm, until all the material was triturated, and avoiding overheating of the material. Ten ml of the remaining liquid was transferred to falcons and liofilized. Around 200 mg of the liofilized material was used to DNA extraction with Pure Link Microbiome DNA Purification Kit (Thermo Fisher Scientific, USA), with an adaptation on the 7th step of the manufacturers manual (we perfomed 2 runs in a FAST PREP-24 5G, with 6.0 m/sec for 40 seconds). About 200 mg of soil was also used for extraction with the same kit, but without the disinfection procedures. DNA was assessed using a NanoDrop 1000 device (Thermo Fisher Scientific, USA) and concentration with a Qubit 4 fluorometer (Thermo Fisher Scientific, USA). Extracted DNA was submitted to the sequencing of the V4 region of the gene that encodes the 16S rRNA with primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT). The runs were paired-end 250 bp in a Illumina NovaSeq 6000 (Illumina, USA) with a sequencing depth of 100K reads.