2.3. Sampling of S. takesimense

RP and RS of host plants from each site were sampled; full-grown adult S. takesimense species were sampled as targets species. In the Dokdo Islands, soil loss because of steeply inclined planes is often observed [21]. Host plant sites were selected such that they bore no marks of soil loss. Over 15 individual S. takesimense were sampled, and the sampled soil depth was above 30 cm. Specific permission was required for one location (Dokdo) no endangered or protected species live in the geographical regions disturbed. In each site, three soil samples (1 kg each) were collected in the vertices of 1 m side equilateral triangle and mixed in a unique representative analytical sample [22]. Soil samples from each site were subdivided in two representative sub-samples: the first one was air-dried, 2 mm sieved, then chemically and physically analyzed, whereas the second one was stored at −80 °C and later processed for the pyrosequencing analysis.

RS represents soil areas affected by the plant physiological activity and physiologically interacting microorganisms [23]. An RS soil core (depth: 30 cm, diameter: 2 mm) was sampled from each site. The RP region represents the region in contact with the soil of plant root surfaces containing fungal mycelia or microbial community [24]. Three RP and RS samples from the three geographical regions were pooled per site. In each geographically isolated region, the RP and RS soil were obtained from three well-developed halophytic colonies and combined in one last sample for sequencing. In poor condition plant objects that make up, colonies were excluded in the sampling and pretreatment stages. For representative samples, the pooling was conducted by region, and each of the three S. takesimense colonies in a particular region was within the permitted population in an administrative manner, in the natural resources and natural cultural heritage protection zones. Soils strongly associated with plant roots were separated by vigorous vortex with 50 ml of sterile phosphate-buffered saline (PBS) solution. The roots were stirred vigorously with sterile forceps to remove all soil from the root surface. Suspensions were centrifuged (Sigma-Aldrich, St. Louis, MO, USA) at 5000 rpm for 10 min at 4 °C, and the sediment was considered RS soil. After removing all the soils, the roots were put into a tube with 50 ml sterile PBS and then sonicated for 10 s (three times) at 50 Hz in a sealed state (ultrasonic cleaner, Branson Ultrasonics, Danbury, CT, USA). The roots were then removed using sterile forceps, and the solution was centrifuged at 5000 rpm for 10 min at 4 °C. The resulting pellets were considered as RP soil.