RESUMO
Burdock (Arctium lappa L., belongs to the family Asteraceae), is an edible plant and an oriental medicinal herb in Korea (Han and Koo, 1993). In July 2023, burdocks showing chlorotic ringspots and yellowing on the leaves were observed in nine of approximately 4,000 plants in a greenhouse in Daegu, South Korea. To determine the causal virus species, nine symptomatic leaves from each individual plant were collected and tested using commercially available immunostrips (Agdia, Elkhart, USA) for cucumber mosaic virus (CMV) and tomato spotted wilt virus (TSWV). Seven out of nine samples tested positive for TSWV only. TSWV in South Korea was first reported on sweet pepper from Yesan in 2004 (Kim et al., 2004) and has since spread to various crops. The first report of TSWV infecting burdock plants in the world was from Hawaii in 1995 (Bautista et al., 1995), but TSWV-infected burdock has not been reported in Korea. To further confirm the presence of TSWV, total RNA was extracted from TSWV-positive burdock leaves using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and used in reverse transcription-polymerase chain reaction (RT-PCR) assays with a specific primer set that amplifies 777 bp of nucleocapsid gene (N gene) of TSWV (Yoon et al., 2014). To obtain the complete genome sequence of this TSWV in the burdock plant, named TSWV-DG, fragments of L, M, and S segments were amplified and sequenced. The complete genome sequences of the L (8914 nt), M (4773 nt), and S (2946 nt) segments were obtained by overlapping RT-PCR amplicons. RT-PCR products were cloned into the pGEM-T Easy vector, and selected DNA clones were sequenced using Sanger method (Bioneer, Korea). The complete genome sequences were deposited to GenBank (LC790665, LC790666, and LC790667, respectively). BLASTn analysis showed that sequences of each TSWV-DG segment had maximum nucleotide identities of 99.5%, 99.5%, and 99.5% with TSWV-L, TSWV-M, and TSWV-S (OM154971, OM154970, and OM154969, respectively), which were isolated from water dropwort (Oenanthe crocata) in China (Qiu et al., 2023). To assess the biological activity of TSWV-DG, A. lappa and Nicotiana benthamiana were inoculated mechanically with sap from infected burdock leaves and maintained for visual inspection of virus symptoms at 25 â at 3 weeks. TSWV-DG produced symptoms on the systemic leaves of A. lappa, that included chlorotic spots and yellowing, and on the leaves of N. benthamiana, that included chlorotic spots and mosaic patterns from 14 days-post-inoculation. Meanwhile, mock-inoculated A.lappa and N.benthamiana remained symptomless. The presence of TSWV on the inoculated leaves was subsequently confirmed through Immunostrip and RT-PCR analyses. TSWV may pose a significant threat to the production of A. lappa, which is cultivated as both leafy greens and root vegetables in Korea. Furthermore, A. lappa may not only be at risk of damage from TSWV infection but also act as a potential source of TSWV infection, thereby posing a risk of transmission to other key crops in Korea, such as pepper or potato (Yoon et al., 2014). This is the first report TSWV infecting burdock in South Korea.
RESUMO
Passion fruit (Passiflora edulis) viral diseases caused by papaya leaf curl Guangdong virus, cucumber mosaic virus, East Asian Passiflora virus, and euphorbia leaf curl virus have been reported in South Korea (Joa et al. 2018; Kim et al. 2018). In June 2021, virus-like symptoms, e.g., mosaic pattern, curling, chlorosis, and deformation, were observed on leaves and fruits of greenhouse-grown P. edulis in Iksan, South Korea, with disease incidence greater than 2% (300 plants: 8 symptomatic plants and 292 asymptomatic plants). Total RNA was extracted from a pooled sample of symptomatic leaves of an individual P. edulis plant using the RNeasy Plant Mini Kit (Qiagen, Germany), and a transcriptome library was generated using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina, San Diego, CA). Next-Generation Sequencing (NGS) was performed using the Illumina NovaSeq 6000 system (Macrogen Inc., Korea). De novo assembly of the resulting 121,154,740 reads was performed using Trinity (Grabherr et al. 2011). A total of 70,895 contigs was assembled (>200 bp) and annotated against the NCBI viral genome database using BLASTn (ver. 2.12.0). One 827-nt contig was annotated as milk vetch dwarf virus (MVDV), a member of the genus Nanovirus in the family Nanoviridae (Bangladesh isolate, acc. no. LC094159, 96.0% nucleotide identity), and the other 3,639-nt contig corresponded to Passiflora latent virus (PLV), a member of the genus Carlavirus in the family Betaflexiviridae (Israel isolate, acc. no. DQ455582, 90.0% nucleotide identity). For further confirmation, total RNA was isolated from symptomatic leaves of the same P. edulis used for NGS analysis using a viral gene spin DNA/RNA extraction kit (iNtRON Biotechnology, Seongnam, Korea), and reverse transcription polymerase chain reaction (RT-PCR) was performed using specific primers: PLV-F/R (5'-GTGCCCACCGAACATGTTACCTC-3'/5'-CCATGCACTTGGAATGCTTACCC-3') targeting the coat protein region of PLV, MVDV-M-F/R (5'-CTAGTCAGCCATCCAATGGTG-3'/5'-GTGCAGGGTTTGATTGTCTGC-3') targeting the movement protein region, and MVDV-S-F/R (5'-GGATTTTAATACGCGTGGACGATC-3'/5'-AACGGCTATAAGTCACTCCGTAC-3') targeting the coat protein region of MVDV. An expected PCR product of 518 bp corresponding to PLV was amplified, while MVDV was not detected. The amplicon was directly sequenced, and its nucleotide sequence was deposited in GenBank (acc. no. OK274270). A BLASTn analysis showed that the nucleotide sequence of the PCR product shared 93.0% and 96.2% identity with PLV isolates from Israel (MH379331) and Germany (MT723990), respectively. In addition, six passion fruit leaves and two fruit samples with PLV-like symptoms were collected from a total of eight plants grown in the greenhouse in Iksan for RT-PCR analysis, and six samples tested positive for PLV. However, PLV was not detected in one leaf and one fruit among all samples. Mechanical sap inoculation was conducted using extracts of systemic leaves as inoculum on P. edulis and the indicator plants Chenopodium quinoa, Nicotiana benthamiana, N. glutinosa, and N. tabacum. In P. edulis, vein chlorosis and yellowing on systemic leaves were observed 20 days post inoculation (dpi). Necrotic local lesions were observed on inoculated leaves of N. benthamiana and N. glutinosa 15 dpi, and PLV infection was confirmed by RT-PCR assay in symptomatic leaf tissue. This study aimed to determine whether commercially grown passion fruit in the southern part of South Korea could be infected with and potentially spread PLV. Whereas PLV was asymptomatic in persimmon (Diospyros kaki) in South Korea, no pathogenicity testing in passion fruit was reported (Cho et al. 2021). Here, we have shown the natural infection of passion fruit with PLV in South Korea for the first time and associated infection with obvious symptoms. This suggests a need to evaluate potential losses in passion fruit and the selection of healthy propagation material.
RESUMO
Plant viral diseases accounts for major global economic losses in modern-day agriculture. Plant viral disease management is the primary challenge for both farmers and researchers. Detection and identification of plant viruses are of paramount importance for successful management of a viral disease. Recent advancements in molecular biology have contributed to significant progress in the development of new, sensitive, and effective diagnostic methods. However, most techniques are neither time/cost-effective nor user-friendly and require sophisticated labs. Hence, the past few decades of agricultural research have mainly focused on developing farmer-friendly, point-of-care diagnostic tools that provide high-sensitive rapid diagnosis. The current trend in plant virus diagnostic tools is cheaper, easy-to-use portable devices with no compromise on sensitivity and reproducibility.
Assuntos
Vírus de Plantas , Sistemas Automatizados de Assistência Junto ao Leito , Doenças das Plantas , Vírus de Plantas/genética , Plantas , Reprodutibilidade dos TestesRESUMO
Perilla mosaic virus (PerMV; the genus Emaravirus in the family Fimoviridae) has a multiple, negative-sense, single-stranded RNA genome (ICTV, 2018). PerMV has been reported in Japan, where it was transmitted by an eriophyid mite species (Acari: Eriophyidae) to Perilla frutescens (L.) Britton var. crispa (Kubota et al., 2020). In September 2021, typical symptoms of the virus including yellow flecks, mosaic symptoms, and malformation were observed in leaves of P. frutescens in a cultivated field in Iseo-myeon, Wanju, South Korea (Suppl. Fig. 1). Visual estimates indicated that symptom incidence reached 70%, and the top leaves of perilla plants exhibited more severe symptoms and leaf distortion. To identify the virus species accurately, total RNA was extracted from five symptomatic perilla leaves collected using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) then cDNAs were amplified by reverse-transcription polymerase chain reaction (RT-PCR) using two pairs of primers to PerMV specific primer set designed to amplify 412- and 491-bp cDNAs of the nucleocapsid protein gene RNA 3 and movement protein gene RNA 4, respectively (Suppl. Table). Single-infection of PerMV in symptomatic Korean perilla plants was confirmed by high-throughput sequence (HTS) analysis and de novo transcriptome assembly using the Illumina HiSeq 4000 platform (Macrogen Inc., Seoul, Korea). The assembled sequences were aligned with viral reference genomes through searches performed using the BLASTn tool. Seven contigs (597-7,213 bp) revealed 92.09-97.37% nucleotide homology with RNAs of the isolate PerMV_Kochi_Nankoku_2011 (accession numbers LC496090 to LC496099) in the GenBank database. Other viruses including turnip mosaic virus and cucumber green mottle mosaic virus were not identified by HTS analysis (Cho et al., 2021; Park et al., 2020; Song et al., 2022). Seven RNA genomes of PerMV were confirmed by RT-PCR using specific primer sets designed to amplify part of each genome (Suppl. Table 1 and Fig. 2). The complete nucleotide sequences of PerMV (named IS isolate) RNA 1-7 were determined to be 7,177, 2,089, 1,094, 1,302, 1,079, 1,098, and 995 bp in length, respectively; these were deposited in GenBank (LC721296-LC721303). Sap from a symptomatic leaf sample confirmed for single infection was inoculated mechanically onto the leaves of 10 healthy P. frutescens seedlings, which developed the same PerMV symptoms within 3 weeks. These results indicate that PerMV is the causal agent of viral disease in Korean perilla plants cultivated in South Korea. To our knowledge, this is the first report of a perilla mosaic emaravirus infecting to Korean perilla, P. frutescens in South Korea.
RESUMO
Cucurbita ficifolia Bouché, called fig-leaf gourd, is a cucurbitaceous climbing plant native to the Americas. During summer and autumn of 2020, almost all of fig-leaf gourds planted in an experimental plot in Jeonju (35°50'54â³N, 127°07'46â³E), Korea, were found to be infected by a powdery mildew with a 100% disease severity. Symptoms first appeared as white, small, irregular colonies, later coalesced into abundant hyphal growth on both sides of the leaves and young stems. Subsequently premature senescence and poor growth of affected leaves was observed. A voucher specimen was housed in the Korea University herbarium (KUS-F32173). For morphological characterization of the fungus, fresh materials were used for microscopy. Conidiophores (n = 30) were straight, 90 to 190 × 10 to 12 µm and produced three to six immature conidia in chains with a crenate outline. Conidia (n = 30) were ellipsoid-ovoid to barrel-shaped, measured 28 to 38 × 18 to 24 µm with a length/width ratio of 1.3 to 2.0, and contained distinct fibrosin bodies. No chasmothecia were found over the course of the season. The morphological feature of the fungus and the host genus were compatible with those of Podosphaera xanthii (Castagne) U. Braun & Shishkoff (Braun and Cook 2012), a well-known cucurbitaceous powdery mildew. For further confirmation, DNA was extracted from mycelium taken from above mentioned specimen. Internal transcribed spacers (ITS1 and ITS2) and large subunit (LSU) gene of the rDNA were amplified using primer pairs ITS1F/PM6 and PM3/TW14, respectively (Takamatsu and Kano 2001). Newly obtained sequences were registered to the GenBank under the accession numbers OL677355 for ITS and OL677356 for LSU. Our sequences shared 99.54~99.77% identity for ITS (MW559231 and MT250855) and 99.58% for LSU with sequences of P. xanthii (MK357445 and MK357438) in BLAST'n search. Pathogenicity tests were performed twice by pressing method of mycelial patches of the infected leaf onto the young leaves of five healthy fig-leaf gourd plants. Five non-inoculated plants were used as controls. Inoculated leaves started to develop powdery mildew signs after 5 days treatment, whereas the control plants remained symptomless. The fungus on the inoculated plants was morphologically identical to that originally observed on diseased plants, fulfilling Koch's postulates. To date three powdery mildew species have been reported on C. ficifolia (Farr and Rossman 2021). Among these reports, Golovinomyces orontii sensu lato was known from Chile, Mexico, United Kingdom, and Germany, Leveillula taurica from Korea and Podosphaera fuliginea (currently P. xanthii) was recorded Australia and India. To our knowledge, this is the first report of P. xanthii on C. ficifolia in Korea. Since this plant is used as grafting rootstock for preventing soil-borne gummy stem blight of cucumber caused by Didymella bryoniae in Korea (Choi et al. 2009), economic importance of the powdery mildew occurring on leaves of this plant is currently limited. Nevertheless, presence of a powdery mildew could be informative to breeding program of Cucurbita spp. and safe production of C. ficifolia fruits globally.
RESUMO
Tomato spotted wilt virus (TSWV) is economically important in Korea as it causes significant losses to a wide range of important ornamental and vegetable crops. Therefore, a rapid detection method is imperative for TSWV diagnosis. Specific primers and probes were designed based on the conserved sequences of the TSWV coat protein gene. In this study, an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) assay, combined with lateral flow strips (LFS), was established for rapid detection of TSWV in pepper infected leaves. The RT-RPA reaction was performed at an optimal condition of 38 °C for 10 min and an LFS incubation time of approximately 5 min. There was no cross-reactivity with other viruses infecting pepper such as cucumber mosaic virus, pepper mottle virus, pepper mild mottle virus, and broad bean wilt virus 2, thus confirming the specificity of RT-RPA-LFS. The sensitivity of the RT-RPA assay was similar to that of RT-PCR, and RT-RPA-LFS was successfully applied to detect TSWV in the pepper samples collected from the field. Thus, RT-RPA-LFS assay might be a promising candidate for quick diagnosis of TSWV-infected pepper plants.
Assuntos
Tospovirus , Primers do DNA , Folhas de Planta , Recombinases/genética , Transcrição Reversa , Tospovirus/genéticaRESUMO
Peach latent mosaic viroid (PLMVd) represents a continuing threat to peach tree production worldwide. In this study, a sensitive and accurate quantification of PLMVd in peach leaves was established using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The quantitative linearity, accuracy, and sensitivity of RT-ddPCR for the detection of PLMVd were comparatively assessed to those of reverse-transcription real-time quantitative polymerase chain reaction (RT-qPCR) assay. The specificity assay shows no amplification in major peach viruses, apple chlorotic leaf spot virus and prunus necrotic ring spot virus and negative control. Furthermore, the levels of PLMVd transcripts determined using RT-ddPCR and RT-qPCR showed a high degree of linearity and quantitative correlation. Our results also indicated that the RT-ddPCR assay is at least two-fold more sensitive than qPCR and could therefore, be used to detect PLMVd in field samples. Moreover, optimization of RT-ddPCR was found to enhance the sensitivity of PLMVd detection in the peach leaf samples with low viral loads. In summary, the established RT-ddPCR assay represents a promising alternative method for the precise quantitative detection of PLMVd; it would be particularly applicable for diagnosing PLMVd infections in plant quarantine inspection and PLMVd-free certification program.
Assuntos
Prunus , Transcrição Reversa , Doenças das Plantas , Vírus de Plantas , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Verbena bonariensis L., named as purple-top vervain or Argentinian vervain, is native to tropical South America. It is cultivated worldwide as an ornamental plant. During summer and autumn of 2020, over 50% of the leaves of V. bonariensis were found infected with powdery mildew in a flower garden in Seoul (37°35'19"N 127°01'07"E), Korea. White, superficial mycelia developed initially on the leaves and subsequently covered surfaces of leaves and stems, are resulting in leaf discoloration, early defoliation, and shoots distortion. Heavily infected plants lost ornamental value. A representative voucher specimen was deposited in the Korea University herbarium (KUS-F32168). Morphological characterization and measurements of conidiophores and conidia were carried out using fresh samples. Microscopic observation showed that aAppressoria on the superficial hypha were nipple-shaped, but rarely found or nearly absent. Conidiophores (n = 30) were cylindrical, 110 to 220 × 10 to 12 µm, and produced 2 to 5 immature conidia in chains with a sinuate outline, followed by 2 to 3 short cells. Foot-cells of conidiophores were straight, cylindrical, and 46 to 90 µm long. Conidia (n = 30) were hyaline, ellipsoid to doliiform, 28 to 40 × 18 to 24 µm with a length/width ratio of 1.3 to 2.0, and contained small be like oil-like drops, but without distinct fibrosin bodies. Primary conidia were apically rounded and sub-truncate at the base. Germ tubes were produced at perihilar position of the conidia. Chasmothecia were not observed. These morphological characteristics were typical of the conidial stage of the genus Golovinomyces (Braun and Cook 2012, Qiu et al. 2020). To identify the fungus, rDNA was extracted from the voucher sample. PCR products were amplified using the primer pair ITS1F/PM6 for internal transcribed spacer (ITS), and PM3/TW14 for the large subunit (LSU) of the rDNA (Takamatsu and Kano 2001). The resulting sequences were registered to GenBank (MW599742 for ITS, and MW599743 for LSU). Using Blast'n search of GenBank, sequences showed 100% identity for ITS and LSU with G. ambrosiae (MT355557, KX987303, MH078047 for ITS, and AB769427, AB769426 for LSU), respectively. Thus, based on morphology and molecular analysis, the isolate on V. bonariensis in Korea was identified as G. ambrosiae (Schwein.) U. Braun & R.T.A. Cook. Pathogenicity tests were carried out by touching an infected leaf onto healthy leaves of disease-free pot-grown plants using a replication of five plants, with five non-inoculated plants used as controls. After 7 days, typical powdery mildew colonies started to appear on the inoculated leaves. The fungus on inoculated leaves was morphologically identical to that originally observed in the field. All non-inoculated control leaves remained symptomless. On different global Verbena species, tThere have been many reports of Golovinomyces powdery mildews including G. cichoracearum s.lat., G. longipes, G. monardae, G. orontii s.lat., and G. verbenae (Farr and Rossman 2021). In China, G. verbenae was recorded on V.erbena phlogiflora (Liu et al. 2006). Golovinomyces powdery mildew has not been reported on Verbena spp. in Korea. Powdery mildew has been reported on V. bonariensis in California, but identity of the causal agent had not been reported. To our knowledge, this is the first report on the identity of the powdery mildew caused by G. ambrosiae on V. bonariensis in Korea. Since heavily infected plants lost ornamental value, appropriate control measures should be developed.
RESUMO
Benincasa hispida (Thunb.) Cogn. (syn. B. cerifera Savi, Cucurbita hispida Thunb.), called wax gourd or ash gourd, is a cucurbitaceous vine grown for medicinal purposes and commercial values of its large fruits in Southeast Asia (Al-Snafi 2013). During the summer and autumn of 2020, leaves of wax gourd were observed to be affected by powdery mildew with 100% disease incidence in an experimental plot of Jeonbuk National University (35°50'55â³N, 127°07'48â³E), Korea. Fungal colonies were initially circular to irregular, forming white patches on both sides of the leaves and young stems, finally covering entire leaves and causing premature senescence of the leaves and poor growth. A representative voucher specimen was deposited in the Korea University herbarium (KUS-F32171). At least 30 measurements were taken for each asexual diagnostic features. Conidiophores arising from superficial hyphae were straight, 100 to 210 µm long, and produced 3 to 7 immature conidia in chains with a crenate outline. Foot-cells were cylindrical, 46 to 74 ×10 to 12 µm, followed by 1 to 2 shorter cells. Conidia were ellipsoid-ovoid to barrel-shaped, 30 to 40 × 18 to 23 µm with a length/width ratio of 1.4 to 2.0 and contained conspicuous fibrosin bodies. Germ tubes were produced from a lateral position on conidia. Sexual stage was not observed during the growing season. The morphological characteristics of the fungus were compatible with those of Podosphaera xanthii (Castagne) U. Braun & Shishkoff (Braun and Cook 2012), a well-known cucurbitaceous powdery mildew. DNA was extracted from mycelium, and primer sets ITS1F/PM6 and PM3/TW14 were used for amplification of ITS1-5.8S-ITS2 regions and 5´-end of 28S rDNA gene, respectively (Takamatsu and Kano 2001). Sequences determined in this study were deposited to the GenBank under the accession numbers MW559231 and MW559420, respectively. The sequences for ITS regions and 28S rDNA gene showed 99.78% and 99.07% similarity respectively with those of P. xanthii (MH465242, MH465243, MT250855 for ITS, and MK357436, MT826247 for LSU). Pathogenicity was confirmed twice by pressing a diseased leaf onto young leaves of five wax gourd plants. Five non-inoculated plants were used as controls. Inoculated leaves developed symptoms after 5 days, whereas the control plants remained symptomless. The fungus present on the inoculated plants was identical morphologically to that originally observed on diseased plants. Sphaerotheca fuliginea (syn. P. xanthii) on B. hispida has been listed in Hungary, India, Japan, Singapore, and Taiwan so far (Farr and Rossman 2021). Recently, the identity of P. xanthii on B. hispida in Taiwan was confirmed with morphological examination and molecular analysis by Wu and Kirschner (2017). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on B. hispida in Korea. Since wax gourd production is only recently started on a commercial scale in the southern part of Korea, powdery mildew infections pose a serious threat to the safe production of the fruits, especially in organic farming where chemical control options are limited.
RESUMO
Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein.IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed.
Assuntos
Proteínas do Capsídeo/biossíntese , Códon de Terminação/genética , Sequências Repetidas Invertidas/genética , Luteoviridae/genética , Conformação de Ácido Nucleico , RNA Viral/metabolismo , Sequência de Aminoácidos/genética , Sequência de Bases , Proteínas do Capsídeo/genética , Fases de Leitura Aberta/genética , Doenças das Plantas/virologia , Biossíntese de Proteínas/genética , Deleção de Sequência/genética , Solanum/virologia , Nicotiana/virologiaRESUMO
We report the complete genome sequence of a novel nucleorhabdovirus, apple rootstock virus A (ApRVA), isolated from Malus spp. in South Korea. ApRVA has a 14,043-nt single-stranded negative-sense RNA genome. In the antigenome sense, it contains seven open reading frames, encoding the putative nucleocapsid protein, phosphoprotein, cell-to-cell movement protein, matrix protein, glycoprotein, RNA-dependent RNA polymerase, and an additional hypothetical protein, the gene for which is located between the genes for the matrix protein and glycoprotein. The complete genome sequence of ApRVA showed 47.45% nucleotide sequence identity to that of black currant-associated rhabdovirus 1. The genome organization, phylogenetic relationships, and sequence similarities to other nucleorhabdoviruses suggest that ApRVA is a new member of the genus Nucleorhabdovirus.
Assuntos
Genoma Viral , Malus/virologia , Raízes de Plantas/virologia , Rhabdoviridae/classificação , Rhabdoviridae/genética , Análise de Sequência de DNA , Ordem dos Genes , Fases de Leitura Aberta , Filogenia , RNA Viral/genética , República da Coreia , Rhabdoviridae/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Proteínas Virais/genéticaRESUMO
Polymyxa graminis, a root endoparasite of several cereal species, is considered to be non-pathogenic but serves as a vector of various plant viruses belonging to the genera Bymovirus, Furovirus, and Pecluvirus. Specifically, it reduces barley productivity by transmitting the Barley Yellow Mosaic Virus (BaYMV). To date, due to its obligate biotrophic property, no artificial culturing of P. graminis was reported and its quantification was also technically challenging. Here, we developed a novel and simple method to infect P. graminis within sterile barley roots in contamination free by preparing nearly pure zoospore inoculum. Such artificial maintenance of P. graminis was verified based on the presence of various developmental stages in infected barley roots under microscope. In addition, the population of resting spores in host tissue was determined by establishing standard curve between manually counted number of spores and Ct values of 18S rDNA amplification using quantitative real-time PCR. Furthermore, it was validated that standard curve generated was also applicable to estimate the abundance of P. graminis in soil environments. In conclusion, the present study would help to generate a system to investigate the etiological causes as well as management of plant diseases caused by P. graminis and BaYMV in tissue and soil.
RESUMO
Soybean (Glycine max L.) is one of the most widely planted and used legumes in the world, being used for food, animal feed products, and industrial production. The soybean mosaic virus (SMV) is the most prevalent virus infecting soybean plants. This study developed a diagnostic method for the rapid and sensitive detection of SMV using a reverse transcription-recombinase polymerase amplification (RT-RPA) technique combined with a lateral flow strip (LFS). The RT-RPA and RT-RPA-LFS conditions to detect the SMV were optimized using the selected primer set that amplified part of the VPg protein gene. The optimized reaction temperature for the RT-RPA primer and RT-RPA-LFS primer used in this study was 38â for both, and the minimum reaction time was 10 min and 5 min, respectively. The RT-RPA-LFS was as sensitive as RT-PCR to detect SMV with 10 pg/µl of total RNA. The reliability of the developed RT-RPA-LFS assay was evaluated using leaves collected from soybean fields. The RT-RPA-LFS diagnostic method developed in this study will be useful as a diagnostic method that can quickly and precisely detect SMV in the epidemiological investigation of SMV, in the selection process of SMV-resistant varieties, on local farms with limited resources.
RESUMO
The Colletotrichum gloeosporioides species complex includes many phytopathogenic species, causing anthracnose disease on a wide range of host plants and appearing to be globally distributed. Seventy-one Colletotrichum isolates in the complex from different plants and geographic regions in Korea were preserved in the Korean Agricultural Culture Collection (KACC). Most of them had been identified based on hosts and morphological features, this could lead to inaccurate species names. Therefore, the KACC isolates were re-identified using DNA sequence analyses of six loci, comprising internal transcribed spacer, gapdh, chs-1, his3, act, and tub2 in this study. Based on the combined phylogenetic analysis, KACC strains were assigned to 12 known species and three new species candidates. The detected species are C. siamense (n = 20), C. fructicola (n = 19), C. gloeosporioides (n = 9), C. aenigma (n = 5), C. camelliae (n = 3), C. temperatum (n = 3), C. musae (n = 2), C. theobromicola (n = 2), C. viniferum (n = 2), C. alatae (n = 1), C. jiangxiense (n = 1), and C. yulongense (n = 1). Of these, C. jiangxiense, C. temperatum, C. theobromicola and C. yulongense are unrecorded species in Korea. Host plant comparisons showed that 27 fungus-host associations are newly reported in the country. However, plant-fungus interactions need to be investigated by pathogenicity tests.
RESUMO
Legumes are primarily grown agriculturally for human consumption, livestock forage, silage, and as green manure. However, production has declined primarily due to fungal pathogens. Among them, this study focused on Fusarium spp. that cause Fusarium wilt in minor legumes in Korea. Diseased legume plants were collected from 2020 to 2021, and diverse fungal genera were isolated from the internal tissues of the plant roots and stems. Fusarium spp. were the most dominant, accounting for 71% of the isolates. They were identified via morphological characteristics and molecular identification. In the pathogenicity test, Fusarium oxysporum and Fusarium fujikuroi generally exhibited high virulence. The host range investigation revealed that the NC20-738, NC20-739, and NC21-950 isolates infected all nine crops, demonstrating the widest host range. In previous studies, the focus was solely on Fusarium wilt disease in soybeans. Therefore, in this study, we aimed to investigate Fusarium wilt occurred in minor legumes, which are consumed as extensively as soybeans, due to the scarcity of data on the diversity and characteristics of Fusarium spp. existing in Korea. The diverse information obtained in this study will serve as a foundation for implementing effective management strategies against Fusarium-induced plant diseases.
Assuntos
Fabaceae , Fusarium , Humanos , Fabaceae/microbiologia , Glycine max , Verduras , República da CoreiaRESUMO
Protein interactions are critical determinants of insect transmission for viruses in the family Luteoviridae. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the Potato leafroll virus (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector-virus and plant-virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus.
Assuntos
Afídeos/virologia , Interações Hospedeiro-Patógeno , Insetos Vetores/virologia , Luteoviridae/patogenicidade , Mapeamento de Interação de Proteínas/métodos , Solanum tuberosum/virologia , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Sequência de Aminoácidos , Animais , Afídeos/metabolismo , Sítios de Ligação , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Comportamento Alimentar , Luteoviridae/isolamento & purificação , Luteoviridae/fisiologia , Espectrometria de Massas , Dados de Sequência Molecular , Doenças das Plantas/virologia , Plasmídeos/genética , Plasmídeos/metabolismo , Nicotiana/virologia , Montagem de VírusRESUMO
Emerging evidence supports the notion that selenium (Se) plays a beneficial role in plant development for modern crop production and is considered an essential micronutrient and the predominant source of plants. However, the essential role of selenium in plant metabolism remains unclear. When used in moderate concentrations, selenium promotes plant physiological processes such as enhancing plant growth, increasing antioxidant capacity, reducing reactive oxygen species and lipid peroxidation and offering stress resistance by preventing ferroptosis cell death. Ferroptosis, a recently discovered mechanism of regulated cell death (RCD) with unique features such as iron-dependant accumulation of lipid peroxides, is distinctly different from other known forms of cell death. Glutathione peroxidase (GPX) activity plays a significant role in scavenging the toxic by-products of lipid peroxidation in plants. A low level of GPX activity in plants causes high oxidative stress, which leads to ferroptosis. An integrated view of ferroptosis and selenium in plants and the selenium-mediated nanofertilizers (SeNPs) have been discussed in more recent studies. For instance, selenium supplementation enhanced GPX4 expression and increased TFH cell (Follicular helper T) numbers and the gene transcriptional program, which prevent lipid peroxidase and protect cells from ferroptosis. However, though ferroptosis in plants is similar to that in animals, only few studies have focused on plant-specific ferroptosis; the research on ferroptosis in plants is still in its infancy. Understanding the implication of selenium with relevance to ferroptosis is indispensable for plant bioresource technology. In this review, we hypothesize that blocking ferroptosis cell death improves plant immunity and protects plants from abiotic and biotic stresses. We also examine how SeNPs can be the basis for emerging unconventional and advanced technologies for algae/bamboo biomass production. For instance, algae treated with SeNPs accumulate high lipid profile in algal cells that could thence be used for biodiesel production. We also suggest that further studies in the field of SeNPs are essential for the successful application of this technology for the large-scale production of plant biomass.
Assuntos
Ferroptose , Selênio , Animais , Antioxidantes/farmacologia , Biomassa , Peroxidação de Lipídeos , Lipídeos , Selênio/farmacologiaRESUMO
An integrative taxonomic analysis of Hierodula patellifera (Audinet-Serville) is presented based on morphological and molecular characters (COI, 28S rDNA). During repeated trips to the Korean peninsula, we collected unusual specimens from Wanju-gun. They were similar to H. patellifera, but can be distinguished by a larger body size, the number and shape of spines on foreleg, and the shape of male genitalia. To examine the phenotypes and delimit H. patellifera from fourteen sampled populations, we used forecoxal spines and male genitalia as key morphological characters, as well as molecular data including gene tree monophyly and genetic divergence data. The molecular analyses (p-distance, neighbor-joining, and parsimony analyses) did not separate the specimens as two distinct species. The diagnostic characters of H. patellifera are illustrated with habitus images.
Assuntos
Mantódeos , Animais , Deriva Genética , Masculino , Mantódeos/anatomia & histologia , Mantódeos/classificação , Mantódeos/genética , FilogeniaRESUMO
Leaf spot on lemon balm is frequently observed in Korea, causing considerable damage to crops. In 2014 and 2015, the occurrence of leaf spot was observed in several production greenhouses at Suwon, Gongju, and Namwon in Korea. Symptoms on lower leaves initially developed as small, distinct, discolored lesions, which enlarged progressively turning into dark brown, angular spots surrounded by purplish-brown margins. Based on the morphological characteristics and sequence analysis of actin (ACT), translation elongation factor 1-alpha (EF-1α), internal transcribed spacer (ITS), 28S nrDNA (LSU), and RNA polymerase II second largest subunit (RPB2), the fungus associated with the lemon balm leaf spot was determined as Septoria melissae. To the best of our knowledge, this is the first report of lemon balm leaf spot caused by S. melissae in Asia as well as in Korea.