Hae Keun Yun

Comparison of accumulation of stilbene compounds and stilbene related gene expression in two grape berries irradiated with different light sources

In this study, the accumulation of stilbene compounds and the expression of genes related to their syntheses in ‘Campbell Early’ and ‘Kyoho’ grapes (Vitis sp.) were investigated by irradiating the harvested berries with four different light sources (white fluorescent and purple, blue, and red LED lights) for 48 hours. The total concentrations of five stilbene derivatives at 24 hours after irradiation differed in response to different light sources and cultivars. The accumulation of stilbenic compounds in the skins of two grape cultivars and the expression of PAL and STS1 genes were mainly induced under red and blue LED lights. The expression of PAL, CHS, CHI, STS1, STS12, and ROMT was differently induced in response to irradiation with different light sources in both grape cultivars. The mRNA levels of PAL and STS1 were higher than those of CHS, CHI, STS12 and ROMT in the two grapes berries. Overall, the results of this study indicated that red and blue LED induced the accumulation of stilbene compounds and the expression of genes related to their syntheses in grape berries, suggesting that irradiation with LEDs can be used to induce the accumulation of phytochemicals that have positive impacts on human health via the induction of related genes.

Transcriptome analysis of grapevine shoots exposed to chilling temperature for four weeks

Low temperature is an important factor that can limit the growth of grapevine (Vitis spp.). In this study, we analyzed the transcriptome of grapevine shoots exposed to cold temperatures to identify genes expressed specifically at low temperature, determine their function based on GO-term analysis, and compare their differential expression. We analyzed two varieties that differed in cold tolerance, the more-tolerant ‘Campbell Early’ and the less-tolerant ‘Kyoho’ grapevine varieties. This was accomplished through annotation of data from sequencing short reads on the Solexa platform. We assembled more than 120 million high-quality trimmed reads using Velvet followed by Oases. Functional categorization of up-regulated transcripts revealed the conservation of genes involved in various biological processes including cellular processes, primary metabolic processes, and biological regulation. The major up-regulated genes in ‘Campbell Early’ included loci encoding response regulator 20, expansin-like B1, a leucine-rich repeat (LRR) family protein, and galactinol synthase 2. The major down-regulated genes in ‘Campbell Early’ included loci encoding fasciclinlike arabinogalactan 9, a GDSL-like lipase/acylhydrolase superfamily protein, early nodulin-like protein 14, and trichome birefringence-like 38. The differential expression observed by sequence analysis was confirmed by real-time PCR. Genes encoding a non-specific serine/threonine protein kinase, peroxidase, and ubiquitin-protein ligase showed reduced expression in response to low temperature in both grapevine varieties. Transcriptome analysis of shoots exposed to chilling could lead to new insights into the molecular basis of tolerance to low-temperature in grapevine.

Isolation of antibacterial compounds from hairy vetch (Vicia villosa) against grapevine crown gall pathogen

This study was conducted to investigate the efficacy of antibacterial compounds isolated from hairy vetch (Vicia villosa Roth) shoots against grapevine crown gall pathogen. The hairy vetch shoot powder was sequentially extracted and screened for antibacterial activity by the disc diffusion method. Ethyl acetate extract from the hairy vetch shoots was subjected to column chromatography on silica gel. The structures of the compounds were then elucidated by spectroscopic analysis and chromatographic assay. Three compounds were isolated and identified as bis (2-ethylhexyl) phthalate (compound 1), diethyl phthalate (compound 2), and p-hydroxybenzoic acid (compound 3). The compounds showed potential antibacterial effect as diameters of inhibition zones (7.3 ± 0.3 to 11.2 ± 0.4 mm) against Rhizobium vitis and Bacillus subtilis. The minimum inhibitory and minimum bactericidal concentrations of these compounds against the target bacteria were found to range from 62.5 to 125 and 125 to 250 μg·mL−1, respectively. The compounds also showed bactericidal activity based on the viable counts of the tested strain. These findings indicate that the isolated compounds could be used as possible candidates for control of crown gall disease in grapevines caused by R. vitis.

Evaluation of grapevine varietal resistance to anthracnose through treating culture filtrates from Elsinoe ampelina

The severe occurrence of anthracnose in grapevines has posed major difficulties for its control, thus, planting anthracnose resistant cultivars favored great advantages in grapevine production. Anthracnose varietal susceptibility in grapevines was evaluated through the use of culture filtrates produced from Elsinoe ampelina, which served as a substitute for pathogen inoculation or field screening. Bioassay of the grapevine leaves with culture filtrates exhibited active and host-selective phytotoxicity levels. Susceptible cultivars were found to be sensitive even at highly diluted culture filtrates. On the other hand, resistant cultivars were not affected even at original culture filtrates. Results of the varietal resistance evaluation by the culture filtrates treatments among the different grapevine cultivars, revealed that some cultivars, such as ‘Black Swan’, ‘Rizamat’, ‘Rosario Bianco’, and ‘Kaiji’ were found to be sensitive, while the other cultivars such as ‘Campbell Early’, ‘Niagara’, ‘Sheridan’, and ‘Izumo Queen’ were found to be anthracnose tolerant. Among the grapes tested, European grapes showed susceptibility and moderate susceptibility to anthracnose as compared to American varieties. These results were likewise consistent with those obtained from the pathogen inoculation screening.

Transcriptional profiles of Rhizobium vitis-inoculated and salicylic acid-treated `Tamnara` grapevines based on microarray analysis

The transcriptional profiles of `Tamnara` grapevine (Vitis labruscana L.) to Rhizobium vitis were determined using 12,000 gene oligonucleotide microarray chips constructed with 6,776 unigenes based on the EST sequencing. Among them, 95 clones were up-regulated more than three times and 90 were down-regulated more than 5-times in the R. vitis-inoculated grapevines relative to the control vines. Treatment of salicylic acid showed that 337 clones were upregulated and 52 clones were down regulated in grapevines. Microarray analysis, reverse transcription-polymer chain reaction, and slot blot hybridization analysis revealed that 5, 14, and 64 clones were up-regulated and 10, 12, and 61 clones were down-regulated in wounded, salicylic acid-treated, and R. vitis-inoculated `Tamnara` grapevine leaves, respectively. The expression patterns of -1,3-glucanase, proline-rich protein, and lipoxygenase genes of `Tamnara` moderately resistant to R. vitis were similar to those of resistant `Concord` and `Delaware` grapevines. However, chalcone synthase genes in `Tamnara` grapevines showed similar expression patterns to susceptible grapevines `Neomuscat` and `Rizamat`. Further expression studies with various clones for each gene should be conducted to elucidate their roles in resistant responses against pathogens or other stimuli in grapevines. These results could provide better resources for understanding the mechanism of defense responses against crown gall disease and clues for identifying new genes that may play a role in defense against R. vitis in grapevines.

Accumulation of stilbene compounds and induction of related gene expression by hairy vetch and ryegrass extracts in grape berries

The present study investigated the accumulation of stilbene compounds (trans- and cis-resveratrol, piceides, and piceatannol) and induction of gene expression related to their synthesis in the berries of ‘Campbell Early’ and ‘Kyoho’ grapes following treatment with water-based shoot extracts from hairy vetch and ryegrass. Trans-resveratrol was present in substantial amounts (4.1 ± 0.0 to 5.3 ± 0.07 μg/g fresh weight) in ‘Campbell Early’ and (7.6 ± 1.24 to 10.4 ± 0.35 07 μg/g fresh weight) in ‘Kyoho’, whereas trace amounts of cis-resveratrol were detected in treated grape berries. Moreover, the concentration of piceid and piceatannol in berries of both cultivars was markedly enhanced in response to treatment with plant extracts. Accumulation of stilbene compounds in berries increased at 24 h after treatment with the test extracts. Moreover, berries treated with extracts showed increased transcript levels of genes encoding enzymes involved in stilbene compound biosynthesis. Changes in the content of stilbene compounds were correlated with levels of stilbene synthase mRNA, indicating transcriptional control of stilbene synthase activity. Additionally, pre-treatment with the tested extracts resulted in reduced lesion size in berries inoculated with Botrytis cinerea. These results suggest that hairy vetch/ryegrass extracts can be used to elicit resistance responses against pathogen infection in grape berries.

Theobroxide Treatment Inhibits Wild Fire Disease Occurrence in Nicotiana benthamiana by the Overexpression of Defense-related Genes

Theobroxide, a novel compound isolated from a fungus Lasiodiplodia theobromae, stimulates potato tuber formation and induces flowering of morning glory by initiating the jasmonic acid synthesis pathway. To elucidate the effect of theobroxide on pathogen resistance in plants, Nicotiana benthamiana plants treated with theobroxide were immediately infiltrated with Pseudomonas syringae pv. tabaci. Exogenous application of theobroxide inhibited development of lesion symptoms, and growth of the bacterial cells was significantly retarded. Semi-quantitative RT-PCRs using the primers of 18 defense-related genes were performed to investigate the molecular mechanisms of resistance. Among the genes, the theobroxide treatment increased the expression of pathogenesis-related protein 1a (PR1a), pathogenesis-related protein 1b (PR1b), glutathione S-transferase (GST), allen oxide cyclase (AOC), and lipoxyganase (LOX). All these data strongly indicate that theobroxide treatment inhibits disease development by faster induction of defense responses, which can be possible by the induction of defense-related genes including PR1a, PR1b, and GST triggered by the elevated jasmonic acid.

In vitro Test of Mycelial Growth Inhibition of 5 Fungi Pathogenic to Strawberries by Ultraviolet-C (UV-C) Irradiation

In strawberry production, among others, the high incidence of diseases by pathogenic fungi resulting in the reduction of fruit yield and quality requires the development of eco-friendly management systems rather than chemical sprays to control them. The diameter of colonies grown in media at 25 o C for 5 days was measured to evaluate the in vitro inhibition of mycelial growth of 5 pathogenic fungi by irradiation with ultraviolet (UV-C, 264 nm). The mycelial growth of 5 pathogenic fungi was inhibited in potato dextrose agar (PDA) by the irradiation of UV-C for 1 hour a day, and was dramatically inhibited by the irradiation of UV-C for 9-12 h a day. The irradiation of UV-C for 9-12 h a day inhibited completely the growth of the late blight pathogen, Phytophthora cactorum. The irradiation distance of 40 to 50 cm was effective for the inhibition of mycelial growth of fungi. The mycelial growth of fungi without pre-incubation was inhibited strongly by UV-C irradiation compared to fungi pre-incubated for 2 days without light. The mycelia growth of Colletotrichum gloeosprioides and Fusarium oxysporum was inhibited strongly by UV-C irradiation in vegetable 8 juice agar compared to PDA.

In vitro evaluation system for varietal resistance against ripe rot caused by Colletotrichum acutatum in grapevines

Ripe rot caused by Colletotrichum spp. occurs severely in grapevines grown in warm and humid climatic regions such as Korea. Research was initiated to develop an efficient screening system for grapevine resistance against ripe rot through the inoculation of C. acutatum spore suspensions grown in potato dextrose agar. Resistant grapevine cultivars against ripe rot were easily distinguished from susceptible ones through lesion scorings of the detached leaves and immature fruits inoculated with mycelial blocks or spore suspension of C. acutatum at 25 °C Resistant grape cultivars identified through pathogen inoculation include: ‘Zabalkanski’, ‘Benianyo’, and ‘Beniyamabico’. The evaluation of grapevines varietal resistance against ripe rot revealed some specificity in the culture filtrates for the pathogens. Hence, the application of culture filtrates treatment of C. acutatum can be an alternative approach for screening the ripe rot varietal resistance in grapevines.

Evaluation of resistance to Pierce’s disease among grapevine cultivars by using the culture filtrates produced from Xylella fastidiosa

포도나무 피어스병에 대한 품종간 저항성을 검정하는 데에 이용되는 병원균 접종법이나 포장저항성 검정법을 대체할 수 있는 방법으로 병원균(Xylella fastidiosa)이 생산하는 배양여액을 이용할 수 있는 검정법을 개발하고자 하였다. 상처를 가한 포도나무 잎에 병원균의 배양여액을 처리한 결과 독성이 발현되었으며 품종간에 차이가 나타났다. 병원균 배양여액의 에틸아세테이트 추출물은 공시한 포도나무에서 배양여액 처리와 동일한 반응을 유도하였다. 병원균 배양여액의 독성과 감수성은 에틸아세테이트 추출물과 동일하였다. 감수성 품종은 높은 비율로 희석된 배양여액에도 감수성을 나타내었으며, 저항성품종은 원액에서도 반응하지 않았다. 감수성 품종은 희석된 배양여액보다 원액에서 감수성이었으나 저항성 품종은 원액에도 저항성을 나타내었다. 본 연구에서 유럽종 포도 품종이 중도 감수성이었으나, 배양여액을 이용한 피어스병해 저항성 검정은 병해에 저항성인 유전자원 선발과, 교배조합의 저항성 실생의 조기 에 선발에 중요한 기술로서, 향후 병해에 저항성인 포도품종 육성에 크게 기여할 것으로 여겨진다.