Jasmonic acid (JA) is known to be an important phytohormone that orchestrates plant defence mechanisms against a range of herbivores and pathogens. Studies have suggested allene oxide synthase (AOS; E.C 4.2.1.92), the first committed step in JA biosynthesis, is essential for JA biosynthesis, yet clear evidence of its role as a biosynthetic regulatory point is lacking, in the main due to conflicting results derived from transgenic studies. However other studies lend support to a biosynthetic regulatory role for AOS. These studies have suggested that certain amino acid substitutions can increase the biosynthetic capacity of the enzyme and consequently improve pathogen tolerance in plants. To explore the role of AOS in Grapevine we isolated and functionally characterised this enzyme for the first time from Vitis vinifera L. Sauvignon blanc. The cloned AOS consisted of a single 1563 bp open reading frame. Comparative sequence analysis showed that the cloned gene (VvAOS) was highly conserved compared to those from other species. Complementation of an Arabidopsis AOS null mutant (aos) with VvAOS recovered the male sterile mutant phenotype and confirmed its function. Transcript analysis showed that VvAOS was wound responsive in leaves and was detectable in most tissues, with the highest levels of transcript in the mesocarp (pulp) of mature berries. Sub-cellular localisation of the VvAOS protein indicated that VvAOS is associated with the chloroplast membrane. Unexpectedly high levels of VvAOS transcript in complemented aos lines did not lead to predicted increases in JA. We have functionally characterised the sole AOS from Grapevine. Patterns of transcript accumulation in grapevine suggest roles in growth, development as well as an important role for JA in fruit ripening. Expression of VvAOS in Arabidopsis suggest complex epigenetic interactions between transgenic and endogenous AOS alleles, providing a possible explanation for why transgenic studies of AOS have delivered conflicting data pointing to a questionable role of AOS as a key regulatory point in JA biosynthesis.

In Korea, most of the grafted watermelons are a fusion of bottle gourd (Lagenaria siceraria) as a rootstock and watermelon (Citrullus lanatus) as a scionstock (Lee et al., 2010). Currently, we have collected several samples from grafted watermelon displaying symptoms of yellowing, withered and wilting leaves. When the symptomatic stem was excised, browning vascular tissues were observed due to the colonization of fungal pathogen. From the samples obtained, 25 fungal isolates were identified as species of Fusarium. Among 25 isolates, 18 were identified as Fusarium oxysporum, four as Fusarium solani, and three as Fusarium equiseti (F. equiseti) . Initial assessment showed that one of the F. equiseti isolates (NIHHS 16-126) was highly virulent to rootstock. Interestingly, this is the first time F. equiseti has been identified pathogenic to grafted watermelon. NIHHS 16-126 isolate was collected from watermelon cultivation field around Buyeo-gun (36.25951°N, 126.92044°E) county. Disease incident was estimated to infect approximately 10% of the watermelon plants cultivated in this area. NIHHS 16-126 isolate was examined to confirm its identity. On potato dextrose agar, colonies appeared yellowish-brown while the aerial mycelium was whitish to peach in color. Macroconidia were relatively long (20.21 – 51.13 × 2.30 – 4.5 μm, n=50), comprise of 3-6 septa, curvature shape and its conidiophores were with monophialides. However, microconidia formation was not observed. These morphological characteristics resemble F. equiseti characters as described by Hyun (2019). For molecular identification, an internal transcribed spacer of ribosomal DNA (ITS-rDNA), elongation factor-1α (EF-1α), and beta-tubulin (β-tub) genes were sequenced using primer pairs of ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Glass and Donaldson 1995), and Bt2a/Bt2b (Carbone and Kohn 1999). BLASTN analysis revealed that ITS-rDNA (LC648248), EF-1α (LC648250), and β-tub (LC648249) sequences were 99-100% identical to F. equiseti reference sequences (KF515650, KF747331, and KF747330) infected Avicennia marina in China (Lu 2014). Phylogenetic analysis of concatenated ITS-rDNA, EF-1α and β-tub sequences showed that this isolate clustered in the same clade as F. equiseti, confirming its identity as F. equiseti. For the inoculation, roots of 12-days-old seedlings (watermelon and bottle gourd, n=10 each) were dipped in the conidia suspension (1x106 conidia/µL) for 30 min. Inoculated seedlings were planted in the soil before being transferred to the greenhouse (temperature; 30°C, daylight; 14 hours). Control plants were inoculated with sterile water. Results showed that after 21 days post-inoculation, all inoculated bottle gourd seedlings (n=10) wilted and eventually died. In contrast, none of the inoculated watermelons or control seedlings were affected. Re-isolation of three fungal isolates (infected root) showed that their morphology and gene markers sequence were identical to the original isolates thus fulfilled Koch’s postulates. Bottle gourd is the most preferred rootstock for grafted watermelons among Korean farmers due to its ability to resist Fusarium spp. infection. Therefore, the identification of F. equiseti as a fungal that is pathogenic to rootstock is crucial information to manage fusarium wilt disease among grafted watermelon. To our knowledge, this is the first report confirming F. equiseti infection in grafted watermelon plants in Korea.

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Garlic (Allium sativum L. cv.namdo) is one of the most popular vegetables grown in Korea due to its high demand from the food industry. However, garlic is susceptible to a wide range of pest infestations and diseases that cause a significant decrease in garlic production, locally and globally (Schwartz and Mohan 2008). In early 2019, the occurrence of leaf blight disease was found spreading in garlic cultivation areas around Jeonnam (34.9671107, 126.4531825) province, Korea. Disease occurrence was estimated to affect 20% of the garlic plants and resulted in up to a 3-5% decrease in its total production. At the early stage of infection, disease symptoms were manifested as small, white-greyish spots with the occurrence of apical necrosis on garlic leaves. This necrosis was observed to enlarge, producing a water-soaked lesion before turning into a black-violet due to the formation of conidia. As the disease progressed, the infected leaves wilted, and the whole garlic plants eventually died. To identify the causal agent, symptomatic tissues (brown dried water-soak lesion) were excised, surface sterilized with 1% NaOCl and placed on the Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 5 days. Among ten fungal isolates obtained, four were selected for further analyses. On PDA, fungal colonies were initially greyish white in colour but gradually turned to yellowish-brown after 15 days due to the formation of yellow pigments. Conidia were muriform, brown in colour, oblong (almost round) with an average size of 18 – 22 × 16 – 20 μm (n = 50) and possessed 6 - 8 transverse septa. Fungal mycelia were branched, septate, and with smooth-walled hyphae. Morphological characteristics described above were consistent with the morphology of Stemphylium eturmiunum as reported by Simmons (Simmons, 2001). For molecular identification, molecular markers i.e. internal transcribed spacer (ITS) and calmodulin (cmdA) genes from the selected isolates were amplified and sequenced (White et al., 1990; Carbone and Kohn 1999). Alignment analysis shows that ITS and cmdA genes sequence is 100% identical among the four selected isolates. Therefore, representative isolate i.e. NIHHS 19-142 (KCTC56750) was selected for further analysis. BLASTN analysis showed that ITS (MW800165) and cmdA (LC601938) sequences of the representative isolates were 100% identical (523/523 bp and 410/410 bp) to the reference genes in Stemphylium eturmiunum isolated from Allium sativum in India (KU850545, KU850835) respectively (Woudenberg et al. 2017). Phylogenetic analysis of the concatenated sequence of ITS and cmdA genes confirmed NIHHS 19-142 isolates is Stemphylium eturmiunum. Pathogenicity test was performed using fungal isolate representative, NIHHS 19-142. Conidia suspension (1 × 106 conidia/µL) of the fungal isolate was inoculated on intact garlic leaves (two leaves from ten different individual plants were inoculated) and bulbs (ten bulbs were used) respectively. Inoculation on intact leaves was performed at NIHHS trial farm whereas inoculated bulbs were kept in the closed container to maintain humidity above 90% and incubated in the incubator chamber at 25°C. Result show that the formation of water-soaked symptoms at the inoculated site was observed at 14 dpi on intact leaves whereas 11 dpi on bulbs. As a control, conidia suspension was replaced with sterile water and the result shows no symptoms were observed on the control leaves and bulbs respectively. Re-identification of fungal colonies from symptomatic leaf and bulb was attempted. Result showed that the morphological characteristics and molecular marker sequences of the three colonies selected were identical to the original isolates thus fulfilled Koch’s postulates. Early identification of Stemphylium eturmiunum as a causal agent to leaf spot disease is crucial information to employ effective disease management strategies or agrochemical applications to control disease outbreaks in the field. Although Stemphylium eturmiunum has been reported to cause leaf spot of garlic disease in China, France and India (Woudenberg et al. 2017), to our knowledge, this is the first report of causing leaf spot disease on garlic in 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.