Search

Erwinia pyrifoliae is a gram-negative bacterial pathogen that commonly causes black shoot blight in pear and apple tree. Although the pathogenicity of this bacterial species is very similar to E. amylovora, there is no specific explanation of its pathogenic genes and mechanisms. In this study, our investigation into E. pyrifoliae pathogenicity involved generating seven YKB12327 mutant strains using Tn5 transposon mutagenesis. Observations revealed weakened growth rate and loss of pathogenicity in these mutants. Whole-genome sequencing and alignment analysis identified transposon insertions within the coding sequences of five strains and in the intergenic region of two strains. Annotation analysis elucidated genes directly or indirectly associated with pathogenicity. Notably, mutant strain MT16 displayed a transposon insertion mutation in the cyclic-di-GMP phosphodiesterase (pdeF) gene, a key player in bacterial signaling, governing microbial behavior and adaptation to environmental changes. Our findings provide insights into the genetic regulation of E. pyrifoliae pathogenicity, suggesting potential avenues for further research aimed at understanding and controlling this bacterial pathogen by targeting pdeF to mitigate apple black shoot blight disease.

Abstract Background Viruses have notable effects on agroecosystems, wherein they can adversely affect plant health and cause problems (e.g., increased biosecurity risks and economic losses). However, our knowledge of their diversity and interactions with specific host plants in ecosystems remains limited. To enhance our understanding of the roles that viruses play in agroecosystems, comprehensive analyses of the viromes of a wide range of plants are essential. High-throughput sequencing (HTS) techniques are useful for conducting impartial and unbiased investigations of plant viromes, ultimately forming a basis for generating further biological and ecological insights. This study was conducted to thoroughly characterize the viral community dynamics in individual plants. Results An HTS-based virome analysis in conjunction with proximity sampling and a tripartite network analysis were performed to investigate the viral diversity in chunkung (Cnidium officinale) plants. We identified 61 distinct chunkung plant-associated viruses (27 DNA and 34 RNA viruses) from 21 known genera and 6 unclassified genera in 14 known viral families. Notably, 12 persistent viruses (7 DNA and 5 RNA viruses) were exclusive to dwarfed chunkung plants. The detection of viruses from the families Partitiviridae, Picobirnaviridae, and Spinareoviridae only in the dwarfed plants suggested that they may contribute to the observed dwarfism. The co-infection of chunkung by multiple viruses is indicative of a dynamic and interactive viral ecosystem with significant sequence variability and evidence of recombination. Conclusions We revealed the viral community involved in chunkung. Our findings suggest that chunkung serves as a significant reservoir for a variety of plant viruses. Moreover, the co-infection rate of individual plants was unexpectedly high. Future research will need to elucidate the mechanisms enabling several dozen viruses to co-exist in chunkung. Nevertheless, the important insights into the chunkung virome generated in this study may be relevant to developing effective plant viral disease management and control strategies.

This work investigated the viral infection in jujube plants in Korea. A total of 61 samples with the symptoms of putative viral infection were collected from experimental fields and orchards. Thereafter, the samples were subjected to metatranscriptome analysis, Reverse transcription polymerase chain reaction analysis, and nucleotide sequence analysis. These analyses identified the presence of two DNA viruses, jujube-associated badnavirus (JuBV), jujube mosaic-associated virus (JuMaV), and one RNA virus, jujube yellow mottle-associ-ated virus (JYMaV). All samples collected were confirmed to be infected by at least one of the three viruses, with most showed multiple infections. The detection rates of JuBV, JYMaV, and JuMaV were 100%, 90.2%, and 8.2%, respectively. Only three combinations of viral infections were found: 9.8% of samples showed single infection of JuBV, 82.0% showed double infection of JuBV+JYMaV, and 8.2% showed triple infection of JuBV+JYMaV+JuMaV. Sequence analysis of the three viruses showed very high homology with respective virus isolates reported in China. This study is predicted to provide fundamental data to produce virus-free jujube seedlings and represents the first report of JuBV and JuMaV infection in Korea.

Abstract The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations.

Asian pear (Pyrus pyrifolia) is a widely cultivated and commercially important fruit crop, which is occasionally subject to severe economic losses due to latent viral infections. Thus, the aim of the present study was to examine and provide a comprehensive overview of virus populations infecting a major pear cultivar (‘Singo’) in Korea. From June 2017 to October 2019, leaf samples (n = 110) of pear trees from 35 orchards in five major pear-producing regions were collected and subjected to RNA sequencing. Most virus-associated contigs matched the sequences of known viruses, including apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV). However, some contigs matched the sequences of apple green crinkle-associated virus and cucumber mosaic virus. In addition, three complete or nearly complete genomes were constructed based on transcriptome data and subjected to phylogenetic analyses. Based on the number of virus-associated reads, ASGV and ASPV were identified as the dominant viruses of ‘Singo.’ The present study describes the virome of a major pear cultivar in Korea, and looks into the diversity of viral communities in this cultivar. This study can provide valuable information on the complexity of genetic variability of viruses infecting pear trees.

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), and Apple scar skin viroid (ASSVd) are economically important viruses that infect pear tree species worldwide. To evaluate the prevalence of these viruses in Korea, we investigated infection degree of three viruses and one viroid for the commercial nursery trees of the pear cultivars, Niitaka, Chuwhang, Wonwhang, and Whasan in 2017 and 2018. The results showed that the infection ratio of ACLSV, ASPV, ASGV, and ASSVd for the scion of pear cultivar Niitaka was 10%, 45%, 77%, and 50%, respectively. From the scion of pear cultivar Chuwhang, infection ratios of ASPV, ASGV, and ASSVd were found to be 70%, 50%, and 60%, respectively. From the scion of pear cultivar Whasan, infection ratios of ACLSV, ASPV, ASGV and ASSVd were found to be 40%, 60%, 93%, and 20%, respectively. From the root stock of pear cultivar Wonwhang, infection ratios of ACLSV, ASPV, ASGV, and ASSVd showed 28%, 57%, 100%, and 14%, respectively. ASGV had the highest recorded infection rate, and ACLSV was characterized by the lowest infection rate. The mixed infection ratio of Niitaka, Chuwhang, Whasan, and Wonwhang was 45%, 60%, 70%, and 85%, respectively.

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as 4.7 ng/μl of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of 42°C. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.

In 2010, foliar symptoms were observed in the paprika leaves in Jinju city, Korea. Beet western yellows virus (BWYV) was identified in paprika by using the large-scale oligonucleotide chip assay. To investigate the occurrence of BWYV, a survey was performed on various crops, including paprika, from 2011 to 2014. Further, the presence of BWYV was consistently verified through literature survey from 2015 to 2017. BWYV infection has been identified in Solanaceae crops (bell pepper, hot pepper, and paprika), various weeds, and green peach aphids and it occurs on a nationwide scale. Cultivation using organic methods involved natural enemies and showed a high BWYV infection rate, which was more than that for conventional cultivation methods in greenhouse. The complete genome sequence of BWYV isolated from paprika was determined for the first time. The genome of the BWYV-Korea isolate consists of 5750 nucleotides and has six open reading frames. Sequence identity results showed maximum similarity between the BWYV-Korea isolate and the BWYV LS isolate (identity ＞ 90%). This study is the first report of BWYV infecting paprika in Korea. The survey revealed that BWYV is naturalized in the domestic ecology of Korea.

To isolate Bacillus velezensis mutants with improved antifungal activity for use in the biological control of phytopathogenic fungi, wild-type Bacillus velezensis KRF-001 producing iturin, surfactin, and fengycin was irradiated by ultraviolet (UV) rays. The in vitro and in vivo antifungal activities of UV mutants and characterization of the cyclic lipopeptides produced by a selected mutant were examined. A mutant strain yielding high levels of iturin showed over 2-fold higher antifungal activity than the wild-type against Fusarium oxysporum. A potent suppressive effect of the mutant was also observed on spore germination of Botrytis cinerea, the causative agent of cucumber gray mold, at different butanol extract concentrations. Further analysis of the mutant by real-time PCR and high-performance liquid chromatography revealed increased expression of iturin and surfactin biosynthesis genes as well as enhanced production of iturin and surfactin metabolites. However, the amounts of fengycin obtained from the mutant strain BSM54 were significantly lesser than those of iturin and surfactin. Particularly, iturin A production by the mutant was 3.5-fold higher than that of the wild-type, suggesting that the higher antifungal activity of the mutant against F. oxysporum resulted from the increased expression of biosynthesis genes associated with iturin production. The commercial greenhouse experiment using soil naturally infested with Sclerotinia sclerotiorum (sclerotinia rot) and F. oxysporum (fusarium wilt) showed that the mutant strain reduced sclerotinia rot and fusarium wilt diseases (P = 0.05) more effectively than the wild-type and commercially available product Cillus® in Korea. These results suggest that the mutant with high iturin yield is a potential candidate for the development of a biological control agent in agriculture.

The full-length sequence of a new isolate of Apple chlorotic leaf spot virus (ACLSV) from Korea was divergent, but most closely related to the Japanese isolate A4, at 84% nucleotide identity. The full-length cDNA of the Korean isolate of ACLSV was cloned into a binary vector downstream of the bacteriophage T7 RNA promoter and the Cauliflower mosaic virus 35S promoter. Chenopodium quinoa was successfully infected using in vitro transcripts synthesized using the T7 promoter, detected at 20 days post inoculation (dpi), but did not produce obvious symptoms. Nicotiana occidentalis and C. quinoa were inoculated through agroinfiltration. At 32 dpi the infection rate was evaluated; no C. quinoa plants were infected by agroinfiltration, but infection of N. occidentalis was obtained.

Deep sequencing has generated 52 contigs derived from five viruses; Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple green crinkle associated virus (AGCaV), and Apricot latent virus (ApLV) were identified from eight apple samples showing small leaves and/or growth retardation. Nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. Sequences of ASPV and ASGV were the most abundantly represented by the 52 contigs assembled. The presence of the five viruses in the samples was confirmed by RT-PCR using specific primers based on the sequences of each assembled contig. All five viruses were detected in three of the samples, whereas all samples had mixed infections with at least two viruses. The most frequently detected virus was ASPV, followed by ASGV, ApLV, ACLSV, and AGCaV which were withal found in mixed infections in the tested samples. AGCaV was identified in assembled contigs ID 1012480 and 93549, which showed 82% and 78% nt sequence identity with ORF1 of AGCaV isolate Aurora-1. ApLV was identified in three assembled contigs, ID 65587, 1802365, and 116777, which showed 77%, 78%, and 76% nt sequence identity respectively with ORF1 of ApLV isolate LA2. Deep sequencing assay was shown to be a valuable and powerful tool for detection and identification of known and unknown virome in infected apple trees, here identifying ApLV and AGCaV in commercial orchards in Korea for the first time.

Virus-induced gene silencing (VIGS) is an effective tool for the study of soybean gene function. Successful VIGS depends on the interaction between virus spread and plant growth, which can be influenced by environmental conditions. Recently, we developed a new VIGS system derived from the Soybean yellow common mosaic virus (SYCMV). Here, we investigated several environmental and developmental factors to improve the efficiency of a SYCMV-based VIGS system to optimize the functional analysis of the soybean. Following SYCMV: Glycine max-phytoene desaturase (GmPDS) infiltration, we investigated the effect of photoperiod, inoculation time, concentration of Agrobacterium inoculm, and growth temperature on VIGS efficiency. In addition, the relative expression of GmPDS between non-silenced and silenced plants was measured by qRT-PCR. We found that gene silencing efficiency was highest at a photoperiod of 16/8 h (light/dark) at a growth temperature of approximately 27°C following syringe infiltration to unrolled unifoliolate leaves in cotyledon stage with a final SYCMV:GmPDS optimal density (OD)₆₀₀ of 2.0. Using this optimized protocol, we achieved high efficiency of GmPDS-silencing in various soybean germplasms including cultivated and wild soybeans. We also confirmed that VIGS occurred in the entire plant, including the root, stem, leaves, and flowers, and could transmit GmPDS to other soybean germplasms via mechanical inoculation. This optimized protocol using a SYCMV-based VIGS system in the soybean should provide a fast and effective method to elucidate gene functions and for use in large-scale screening experiments.

Gibberellic acid (GA) is one of the factors that promotes flowering in radish (Raphanus Sativus L.), although the mechanism mediating GA activation of flowering has not been determined. To identify this mechanism in radish, we compared the effects of GA treatment on late-flowering (NH-JS1) and early-flowering (NH-JS2) radish lines. GA treatment promoted flowering in both lines, but not without vernalization. NH-JS2 plants displayed greater bolting and flowering pathway responses to GA treatment than NH-JS1. This variation was not due to differences in GA sensitivity in the two lines. We performed RNA-seq analysis to investigate GA-mediated changes in gene expression profiles in the two radish lines. We identified 313 upregulated, differentially expressed genes (DEGs) and 207 downregulated DEGs in NH-JS2 relative to NH-JS1 in response to GA. Of these, 21 and 8 genes were identified as flowering time and GA-responsive genes, respectively. The results of RNA-seq and quantitative PCR (qPCR) analyses indicated that RsFT and RsSOC1-1 expression levels increased after GA treatment in NH-JS2 plants but not in NH-JS1. These results identified the molecular mechanism underlying differences in the flowering-time genes of NH-JS1 and NH-JS2 after GA treatment under insufficient vernalization conditions.

Garlic generally becomes coinfected with several types of viruses belonging to the Potyvirus, Carlavirus, and Allexivirus genera. These viruses produce characteristically similar symptoms, they cannot be easily identified by electron microscopy (EM) or immunological detection methods, and they are currently widespread around the world, thereby affecting crop yields and crop quality adversely. For the early and reliable detection of garlic viruses, virus-specific sets of primers, including species-specific and genus-specific primers were designed. To effectively detect the twelve different types of garlic viruses, primer mixtures were tested and divided into two independent sets for multiplex polymerase chain reaction (PCR). The multiplex PCR assays were able to detect specific targets up to the similar dilution series with monoplex reverse transcription (RT)-PCR. Seventy-two field samples collected by the Gyeongbuk Agricultural Technology Administration were analyzed by multiplex RT-PCR. All seventy two samples were infected with at least one virus, and the coinfection rate was 78%. We conclude that the simultaneous detection system developed in this study can effectively detect and differentiate mixed viral infections in garlic.

In May 2013, an angel’s trumpet leaves showing mosaic and malformation symptoms were collected from Suwon city, Gyeonggi-do. An analysis of the collected sample by transmission electron microscopy observation showed filamentous rod particles of 720-800 nm in length. On the basis of the these observations, we performed PCR against three reported Potyviruses (Brugmansia mosaic virus, Colombian datura virus and Brugmansia suaveolens mottle virus), and the sample was positive for BruMV. Pathogenicity and host range test of BruMV was determined by mechanical inoculation. Solanaceae (tobacco, tomato and eggplant) and Amaranthaceae (ground cherry) appeared typical virus symptoms. To determine coat protein of this virus, we designed specific primer pairs, and performed PCR amplification, cloning, and sequencing. Phylogenetic analysis showed that BruMV-SW was most closely related to BruMV isolate SK. Comparison of the BruMV-SW coat protein nucleotide sequences showed 92% to 99% identities to the other BruMV isolates.

Bacillus subtilis subsp. krictiensis ATCC55079 produces the cyclic lipopeptide antibiotics iturin A-F as well as several surfactins. Here, we analyzed and characterized the biosynthetic genes associated with iturin and surfactin production in this strain. We aligned the sequences of each iturin and surfactin synthetase ORF obtained from a genomic library screen and next generation sequencing. The resulting 37,249-bp and 37,645-bp sequences associated with iturin and surfactin production, respectively, contained several ORFs that are predicted to encode proteins involved in iturin and surfactin biosynthesis. These ORFs showed higher sequence homologies with the respective iturin and surfactin synthetase genes of B. methylotrophicus CAU B946 than with those of B. subtilis RB14 and B. subtilis ATCC6633. Moreover, comparative analysis of the secondary metabolites produced by the wild-type and surfactin-less mutant (with a spectinomycin resistance cassette inserted into the srfAB gene within the putative surfactin gene region) strains demonstrated that the mutant strain showed significantly higher antifungal activity against Fusarium oxysporum than the wild-type strain. In addition, the wild-type strain-specific surfactin high performance liquid chromatography (HPLC) peaks were not observed in the surfactin-less mutant strain. In contrast, the iturin A peak detected by HPLC and liquid chromatography-mass spectrometry (LC/MS) in the surfactin-less mutant strain was 30% greater than that in the wild-type strain. These results suggested that the gene cluster we identified is involved in surfactin biosynthesis, and the biosynthetic pathways for iturin and surfactin in Bacillus strains producing both iturin and surfactin may utilize a common pathway.

Lettuce mosaic virus (LMV) causes disease of plants in the family Asteraceae, especially lettuce crops. LMV isolates have previously been clustered in three main groups, LMV-Yar, LMV-Greek and LMVRoW. The first two groups, LMV-Yar and LMV-Greek, have similar characteristics such as no seed-borne transmission and non-resistance-breaking. The latter one, LMV-RoW, comprising a large percentage of the LMV isolates contains two large subgroups, LMV-Common and LMV-Most. To date, however, no Korean LMV isolate has been classified and characterized. In this study, LMV-Muju, the Korean LMV isolate, was isolated from lettuce showing pale green and mottle symptoms, and its complete genome sequence was determined. Classification method of LMV isolates based on nucleotide sequence divergence of the NIb-CP junction showed that LMV-Muju was categorized as LMV-Common. LMV-Muju was more similar to LMV-O (LMV-Common subgroup) than to LMV-E (LMV-RoW group but not LMV-Common subgroup) even in the amino acid domains of HC-Pro associated with pathogenicity, and in the CI and VPg regions related to ability to overcome resistance. Taken together, LMV-Muju belongs to the LMV-Common subgroup, and is expected to be a seed-borne, non-resistance-breaking isolate. According to our analysis, all other LMV isolates not previously assigned to a subgroup were also included in the LMV-RoW group.

A large-scale oligonucleotide (LSON) chip was developed for the detection of the plant viruses with known genetic information. The LSON chip contains two sets of 3,978 probes for 538 species of targets including plant viruses, satellite RNAs and viroids. A hundred forty thousand probes, consisting of isolate-, species- and genus-specific probes respectively, are designed from 20,000 of independent nucleotide sequence of plant viruses. Based on the economic importance, the amount of genome information, and the number of strains and/or isolates, one to fifty-one probes for each target virus are selected and spotted on the chip. The standard and field samples for the analysis of the LSON chip have been prepared and tested by RT-PCR. The probe’s specific and/or nonspecific reaction patterns by LSON chip allow us to diagnose the unidentified viruses. Thus, the LSON chip in this study could be highly useful for the detection of unexpected plant viruses, the monitoring of emerging viruses and the fluctuation of the population of major viruses in each plant.

Soybean yellow mottle mosaic virus (SYMMV) and Soybean yellow common mosaic virus (SYCMV) were recently isolated in Korea, and it has not been reported how two viruses were dispersed in Korea. In 2012, we performed nationwide survey in subsistence soybean farming. Suspicious virus-infected infected leave were collected from the field and a total of 682 soybean tissue samples were assayed by RT-PCR using triplex primers detecting SYMMV, SYCMV, and Soybean mosaic virus (SMV). On hundred two samples showed SMV positive, and SYMMV and SYCMV were detected in 116 and 17 tissue samples, respectively. No sample showed double infection of SYMMV and SYCMV, but there were double infection tissues indicating two viruses positive of SMV plus SYMMV (5 tissue samples) and SMV plus SYCMV (1 tissue sample). Through this first subsistence soybean farming field survey, we assumed soybean viruses were originated from home seed production managed by farmer. Thus, in order to prevent possible seed transmission and further damage caused by virus transmission, virus-free commercial soybean seeds are recommended to be planted.

Barley stripe mosaic virus (BSMV) induces massive actin filament thickening at the infection front of infected Nicotiana benthamiana leaves. To determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the BSMV triple gene block (TGB) proteins were coexpressed in cells with the actin marker DsRed: Talin. TGB ectopic expression experiments revealed that TGB3 is a major elicitor of filament thickening, that TGB2 resulted in formation of intermediate DsRed:Talin filaments, and that TGB1 alone had no obvious effects on actin filament structure. Latrunculin B (LatB) treatments retarded BSMV cell-to-cell movement, disrupted actin filament organization, and dramatically decreased the proportion of paired TGB3 foci appearing at the cell wall (CW). BSMV infection of transgenic plants tagged with GFP-KDEL exhibited membrane proliferation and vesicle formation that were especially evident around the nucleus. Similar membrane proliferation occurred in plants expressing TGB2 and/or TGB3, and DsRed: Talin fluorescence in these plants colocalized with the ER vesicles. TGB3 also associated with the Golgi apparatus and overlapped with cortical vesicles appearing at the cell periphery. Brefeldin A treatments disrupted Golgi and also altered vesicles at the CW, but failed to interfere with TGB CW localization. Our results indicate that actin cytoskeleton interactions are important in BSMV cell-to-cell movement and for CW localization of TGB3.

In 2005, a survey was conducted to identify virus diseases on victory onion, Allium victorialis var. platyphyllum grown in Ulleung island located in the East Sea. A total of 61 samples were collected from victory onion in the neighborhood of Seonginbong. The identification of viruses from the samples were carried out by electron microscopy and RT-PCR using primers species specific to GCLV, LYSV, SLV, OYDV and genus specific to Allexivirus, respectively. From sixty-one samples, filamentous rod particles (600-900 nm) were detected from four victory onion samples in EM, three samples containing SLV and one sample containing both SLV and Allexivirus in RT-PCR analysis, respectively. Victory onions naturally infected by the viruses were asymptomatic apparently. The viruses detected by RT-PCR were further characterized by the nucleotide sequence analysis of the coat protein region. Three isolates of SLV showed approximately 99% identities in the nucleotide and amino acid sequences, suggesting that they were likely to be the same strain. On the other hand, they showed approximately 75.7~83.7% identities in the nucleotide and 89.2~97.0% in amino acid sequences compared with the previously reported SLV isolates in Allium. The CP gene of the Allexivirus showed approximately 99.2% nucleotide identities and 98.8% amino acid identities with Garlic virus A. However, there was relatively low homology ranging from 60.6% to 81.5% compared with other Allexiviruses (GarV-C, GarV-E, GarV-X, GMbMV, and Shal-X). These data suggested that two viruses, SLV and GarV-A identified from victory onion, are named SLV-Ulleungdo and GarV-A-Ulleungdo, respectively. This is the first report of viruses infecting victory onion.