First report of Passiflora latent virus(Passiflora latent virus;PLV) infecting persimmon in Korea.

Passiflora latent virus (PLV), a member of the genus Carlavirus in the family Betaflexiviridae has been reported in Passiflora species in Australia, Germany, Israel, the United States, and New Zealand (Tang et al., 2008). In September 2019, leaves showing a virus-like disease with mosaic, curling and necrosis were collected from ten persimmon (Diospyros kaki Thunb.) orchards in Gyeongsang province, Korea. Total RNA from a pooled sample of leaves from 21 trees was extracted using RNeasy Plant Mini Kit (Qiagen, Germany) and subjected to high throughput sequencing. After pre-processing and Ribo-Zero rRNA Removal, a cDNA library was prepared using an Illumina TruSeq Stranded Total RNA Kit and sequenced on an Illumina NovaSeq 6000 system (Macrogen Inc. Korea). De novo assembly of the 74,862,810 reads was performed using Trinity software (r20140717); the initially assembled 213,476 contigs were screened against the NCBI viral genome database using BLASTN. By these means, 12 contigs derived from PLV were identified. Contigs with lengths of 209 to 802 nt shared nt identities of 90.70 to 94.82% with PLV isolates, covering a total of 5,169 nt (~61.6% of the full PLV genome). Two additional viruses were also detected from the pooled sample: persimmon cryptic virus (PeCV) and persimmon virus A (PeVA). To confirm PLV infection, reverse transcription-polymerase chain reaction (RT-PCR) was performed using virus-specific primers, PLV-F (5'-ACACAAAACTGCGTGTTGGA-3') and PLV-R (5'-CAAGACCCACCTACCTCAGTGTG-3'), designed based on a 633 nt contig sequence in the polymerase gene. RT-PCR products of the expected 571 bp were obtained from two of 21 individual original samples; no asymptomatic plants were tested. Amplicons were cloned into the pGEM-T Easy Vector, and two clones per sample Sanger sequenced bidirectionally (BIONEER, Korea). The identical Sequence (GenBank LC556232) showed 99.65% nt identity to the contig, and 93.87% identity with the corresponding polymerase sequence of PLV-Rehovot isolate from passion fruit in Israel (MH379331). The two PLV positive samples showing leaf necrosis were also co-infected with PeVA, identified by RT-PCR using previously reported primers PeVAfor/ PeVArev (Morell et al., 2014), but not with PeCV (mixed with PeVA in only 1/21 plants; PeVA was found in 19/21 plants). None of the tested viruses were detected in two trees, displaying mosaic, and leaf curling, respectively. The foliar symptoms of PLV infection on passionfruit have been reported to vary throughout the year (Spiegel et al., 2007). No such observations in persimmon was possible, as the infected persimmon trees were removed and destroyed because they might pose a threat to the cultivation of passion fruits in Korea. To our knowledge, this is the first report of persimmon as a host of PLV anywhere in the world, and the first report of PLV in Korea in any host. A further survey is needed to determine possible presence of PLV on persimmon and Passiflora species.

First report of bacterial shot-hole disease caused by Xanthomonas arboricola pv. pruni on plumcot in South Korea.

Plumcot is an interspecific hybrid product between Japanese plums (Prunus salicina) and apricots (Prunus armeniaca) obtained by the NIHHS, Korea in 1999 [1]. At the early of 2017, black spots-like symptoms were observed on plumcot fruits and leaves at cultivation areas in Naju (34.965595, 126.665853) province. Further investigation shows that approximately 60% of the plumcot leaves in the affected orchard were infected, which caused 40% total production loss. At the early stage of infection, disease symptoms appear as small, angular and water-soaked spots and develop into circular brown spots at the later stages of infection. As the disease progresses, the leaf tissues around the spots became yellow and the lesions enlarged. When the adjacent lesions merged and the necrotic tissues fall off, shot-hole symptoms appear on the leaves. To identify the causal agent of this disease, infected leaf tissues were excised and surface-sterilized with 1% NaOCl for 30 secs prior to rinsing with sterile water, thrice . Tissue samples were then placed in sterile water (0.5 mL) for 5 min before its aliquots were streaked onto Luria-Bertani (LB) agar. Plates then were incubated at 28°C. To obtain pure colonies, bacteria were re-streak into a new LB agar and colonies showing typical Xanthomonas spp. morphology (i.e. convex, smooth, yellow, and mucoid) were subjected to Gram staining assay. For molecular identification, 16S ribosomal DNA (16S-rDNA) and gyrase B (gyrB) genes were amplified using a 9F/1512r and UP-1/UP-2Sr primers [2,3] respectively from 5 gram-negative isolates. PCR products were sequenced and analysed using BLASTN. Result shows that 16S-rDNA and gyrB genes are 99-100% identical to a similar genomic region of Xanthomonas arboricola pv. pruni (Xap) isolated in almond (MK156163), peach (MG049922) and apricot (KX950802) respectively [4,5,6]. 16S-rDNA and gyrB gene sequences were deposited in the GenBank (LC485472 and LC576824), whereas pathogen isolate was deposited into Korean Agricultural Culture Collection (KACC19949). Pathogenicity test was performed using Xap bacterial suspension (108 cfu/mL) inoculated on the abaxial and adaxial surface of plumcot detached leaves. For inoculation, 10 healthy young leaves were used whereas, 5 young leaves mock-inoculated with sterile LB broth were used as a control. Both leaf samples were kept in a closed container to maintain 100% humidity before being incubated at 25°C. The water-soaked symptoms were observed visually on the inoculated leaves 2 to 3 days post-inoculation. No water-soaked symptoms were observed on the control leaves. Morphology and sequences of molecular markers used showed that the 3 bacterial colonies re-isolated from the inoculated leaves were identical to the original isolate, fulfilling Koch's postulate. Pathogenicity tests were repeated twice and the results obtained were consistent with the first experiment. As a new variety of stone fruit cultivated in Korea, information about pathogens and registered agrochemicals to control disease outbreak in plumcot are still limited. Therefore, the identification of Xap as a causal agent to the black spot disease is critical for the development of disease management strategies and to identify appropriate agrochemicals to control the occurrence of this disease in the field. To our knowledge, this is the first report of Xap as a causal agent to the shot-hole disease on the plumcot in Korea.