2.1. Sample Collection and Processing

A total of 41 F. rhynchophylla plant samples from various regions of Korea were collected in different time periods (Table 1). All samples were asymptomatic and collected: Jinju (n = 4 in March 2019; n = 8 in September 2019), Busan (n = 9 in October 2019; n = 6 in May 2020), Pocheon (n = 6 in September 2019), Jeonnam (n = 2 in September 2019), Yeongdong (n = 3 in September 2019), and Daegu (n = 2 in September 2019) (Figure 1). No insects were found or collected from any of these 41 plants. All samples were stored at −20 °C until processing. All leaf samples were sterilized by using 70% ethanol for 20–30 s and allowed to dry off from the ethanol with the air flow under the fume hood. Total DNA was extracted from leaf tissue samples using either a Viral Gene-Spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology) or a cetyl trimethylammonium bromide (CTAB)-based extraction protocol, following the manufacturer’s instructions [51]. Total DNA from each sample was used in RCA reaction with the TempliPhi™ kit (GE Healthcare, Chicago, IL, USA), as described by Shepherd et al. [52].

Ash tree (F. rhynchophylla) samples collection from various locations in Korea. (A) Targeted sites and the number of samples collected from each site. (B) Ash tree investigated in our study and the leaf samples collected from (C) Jinju, (D) Busan, (E) Pocheon, and (F) Yeongdong, respectively.

Tree ash samples collected from various locations in Korea.

* New Samples collected from Busan for the second time.