First report of garlic common latent virus in elephant garlic (Allium ampeloprasum) in single and mixed infection in South Korea.

In South Korea, the cultivation area of elephant garlic (Allium ampeloprasum) is increasing as elephant garlic is milder and sweeter than garlic (A. sativum) (Kim et al., 2019; Lu et al., 2011). Viral diseases can decrease garlic productivity by up to 50% in South Korea (Nam et al., 2002). In 2022-2023, virus-like symptoms such as mosaic and yellow stripes were observed on leaves of elephant garlic in a 432㎡farm with disease incidence of approximately 40% in Yangpyeong-gun, Gyeonggi-do, South Korea. Seventy-two leaf samples were randomly collected from symptomatic plants in 2022 (n=46) and 2023 (n=26). Total RNAs were isolated from individual samples using the Total RNA Prep Kit (BioFact, Daejeon, Korea), and then two-steps RT-PCR was performed using the First Strand cDNA Synthesis kit (Thermo Fisher Scientific) and the TaKaRa TaqTM (TaKaRa Bio Inc.). These samples were tested for 13 viruses with virus-specific coat protein primers including garlic common latent virus (GarCLV) (supplementary Table 1). In 2022, GarCLV, garlic virus (GarV)-B, GarV-C, and GarV-D were detected with the expected amplicon sizes of their CP genes (960, 735, 780, and 753 bp, respectively) in four different plants. In 2023, the CP gene of GarCLV was detected in 26 samples and 4 of 26 samples were positive for GarV-B. The leaves infected with GarCLV and GarV-B in mixed infection showed synergistic effect with extended mosaic and yellow stripes than the leaves with single infection (supplementary Fig. 1). All amplicons were cloned into a pGEM-T Easy vector (Promega Co., USA), and sequenced at Bionics Co. Ltd., South Korea. The resulting nucleotide (nt) and amino acid (aa) sequences were analyzed using DNAMAN software version 5.1. Since all isolates were collected from a farm in Yangpyeong-gun, name of these isolates started with "YPG." The nt and aa sequences of the isolates were compared with those of other strains/isolates. All 27 GarCLV-YPG isolates sequences were deposited (Accessions: OP981636, and PP533185-PP533210). The GarCLV-YPG sequences shared 78.90%-94.40% nt and 92.10%-99.40% aa identities with other GarCLV strains and isolates, and they showed higher similarity (99.40% aa) to isolates produced from A. sativum in China and India (supplementary Table 2). GarV-C-YPG showed the highest similarity (99.20% aa) to isolate G81(GenBank MN059141) from A. sativum in China. GarV-D-YPG showed the highest similarity (99.20% aa) to isolates (G82, GenBank MN059388; BR, MT279193) from A. sativum in China and Brazil. Twenty-two quinoa plants (Chenopodium quinoa, local lesion host) were individually inoculated using the sap from 22 GarCLV infected plants. Chlorotic and necrotic spots appeared on inoculated leaves 12 days post-inoculation; no chlorotic and necrotic spots symptoms were observed on any other leaves except for the inoculated leaves. RT-PCR was performed and the targeted amplicon size for GarCLV was detected. In transmission electron microscope, filamentous particles of approximately 620-730 nm length and 12 nm diameter, similar to the particle description for members of the family Betaflexiviridae, were observed in the saps of symptomatic leaves of elephant garlic and quinoa plants infected with only GarCLV. To the best of our knowledge, this is the first report on GarCLV detection in elephant garlic in South Korea. We hypothesized that the presence of GarCLV in mixed infection with GarV-B might have increase the symptom severity in the elephant garlic. Further study is needed to proof the synergistic effect in mixed virus infection.

Metabolism of phenolic compounds catalyzed by Tomato CYP736A61.

Cytochrome P450s (CYPs) regulate plant growth and stress responses by producing diverse primary and secondary metabolites. However, the function of many plant CYPs remains unknown because, despite their structural similarity, predicting the enzymatic activity of CYPs is difficult. In this study, one member of the CYP736A subfamily (CYP736A61) from tomatoes was isolated and characterized its enzymatic functions. CYP736A61 was successfully expressed in Escherichia coli through co-expression with molecular chaperones. The purified CYP736A61 showed hydroxylation activity toward 7-ethoxycoumarin, producing 7-hydroxycoumarin or 3-hydroxy 7-ethoxycoumarin. Further substrate screening revealed that dihydrochalcone and stilbene derivates (resveratrol and polydatin) are the substrates of CYP736A61. CYP736A61 also mediated the hydroxylation of resveratrol and polydatin, albeit with low activity. Importantly, CYP736A61 mediated the cleavage of resveratrol and polydatin as well as pinostilbene and pterostilbene. Interestingly, CY736A61 also converted phloretin to naringenin chalcone. These results suggest that CYP736A61 is a novel CYP enzyme with stilbene cleavage activity.

A multiplex PCR assay for the simultaneous detection of five potexviruses infecting cactus plants using dual-priming oligonucleotides (DPOs) primers.

Five potexviruses, namely, cactus virus X (CVX), opuntia virus X, pitaya virus X (PiVX), schlumbergera virus X (SchVX) and zygocactus virus X (ZyVX), have been reported in cactus plants. In this report, a multiplex RT-PCR assay, based on specific dual-priming oligonucleotide (DPO) primers, was developed to detect these five viruses simultaneously in field samples. Using 18 field plants comprising 16 cactus species, these viruses were detected among nine of the 18 plants, including the simultaneous detection of CVX, PiVX, SchVX and ZyVX co-infecting an Aporocactus flagelliformis and a Notocactus leninghausii f. cristatus plant. The multiplex PCR assay was thus applied successfully in the field plants and it would be useful in the diagnosis of viral infections in cactus plants.

Genetic differences between Korean and American isolates of Petunia vein clearing virus.

Petunia plants are used for urban landscaping in many parts of the world, including South Korea. In this study, we aimed to investigate the occurrence of petunia vein clearing virus (PVCV) infection in petunia plants in Seoul, South Korea. PVCV was detected from 23 of 79 petunia samples collected from Seoul. We obtained the complete genome sequences of the Korean isolates in this study (called PVCV-Kr, Kr2, and Kr3), which were compared with the genome sequence of the USA isolate of the virus (PVCV-USA). The genomic DNA of the three PVCV isolates was found to comprise 7210-7267 nucleotides (nts), which is 4-15 nts longer than the PVCV-USA genome. The genomes of the Kr and Kr2 isolates encode a large polyprotein of 252 kDa (2180 amino acids (aa)). The genome of the Kr3 isolate encodes a large polyprotein of 255 kDa (2203 aa). The polyprotein has six protein domains: a movement protein (MP; 72 aa), a coiled-coil domain (CC; 33 aa), an RNA-binding domain (RB; 18 aa), a protease (PR; 21 aa), a reverse transcriptase (RT; 196 aa), and an RNase H (RH; 121 aa). The large polyprotein and six domains of the three isolates showed 93.9-100.0% sequence homology with those of PVCV-USA. Furthermore, the polymerase polyprotein gene (PR, RT, and RH) of the four PVCV isolates containing the USA isolate grouped with those of Rice tungro bacilliform virus and Soybean chlorotic mottle virus, which belong to the same family (Caulimoviridae). Our findings suggested that the Korean isolates represent a new isolate of PVCV. To our knowledge, this is the first report of PVCV detection in South Korea.

Detection of Co-Infection of Notocactus leninghausii f. cristatus with Six Virus Species in South Korea.

Co-infection with two virus species was previously reported in some cactus plants. Here, we showed that Notocactus leninghausii f. cristatus can be co-infected with six different viruses: cactus mild mottle virus (CMMoV)-Nl, cactus virus X (CVX)-Nl, pitaya virus X (PiVX)-Nl, rattail cactus necrosis-associated virus (RCNaV)-Nl, schlumbergera virus X (SchVX)-Nl, and zygocactus virus X (ZyVX)-Nl. The coat protein sequences of these viruses were compared with those of previously reported viruses. CMMoV-Nl, CVX-Nl, PiVX-Nl, RCNaV-Nl, SchVX-Nl, and ZyVX-Nl showed the greatest nucleotide sequence homology to CMMoV-Kr (99.8% identity, GenBank accession NC_011803), CVX-Jeju (77.5% identity, GenBank accession LC12841), PiVX-P37 (98.4% identity, GenBank accession NC_024458), RCNaV (99.4% identity, GenBank accession NC_016442), SchVX-K11 (95.7% identity, GenBank accession NC_011659), and ZyVX-B1 (97.9% identity, GenBank accession NC_006059), respectively. This study is the first report of co-infection with six virus species in N. leninghausii f. cristatus in South Korea.

A pepper mottle virus-based vector enables systemic expression of endoglucanase D in non-transgenic plants.

Plant-virus-based expression vectors have been used as an alternative to the creation of transgenic plants. Using a virus-based vector, we investigated the feasibility of producing the endoglucanase D (EngD) from Clostridium cellulovorans in Nicotiana benthamiana. This protein has endoglucanase, xylanase, and exoglucanase activities and may be of value for cellulose digestion in the generation of biofuels from plant biomass. The EngD gene was cloned between the nuclear inclusion b (NIb)- and coat protein (CP)-encoding sequences of pSP6PepMoV-Vb1. In vitro transcripts derived from the clone (pSP6PepMoV-Vb1/EngD) were infectious in N. benthamiana but caused milder symptoms than wild-type PepMoV-Vb1. RT-PCR amplification of total RNA from non-inoculated upper leaves infected with PepMoV-Vb1/EngD produced the target band for the CP, partial NIb and EngD-CP regions of PepMoV-V1/EngD, in addition to nonspecific bands. Western blot analysis showed the CP target bands of PepMoV-Vb1/EngD as well as non-target bands. EngD enzymatic activity in infected plants was detected using a glucose assay. The plant leaves showed increased senescence compared with healthy and PepMoV-Vb1-infected plants. Our study suggests the feasibility of using a viral vector for systemic infection of plants for expression of heterologous engD for the purpose of digesting a cellulose substrate in plant cells for biomass production.

Antiviral Activities of Trichothecenes Isolated from Trichoderma albolutescens against Pepper Mottle Virus.

A bioassay-guided isolation using a green fluorescence protein (GFP)-tagged pepper mottle virus (PepMoV-GFP) based leaf-disk method to obtain new antiviral agents led to the isolation of trichodermin, 1, and a new compound trichoderminol, 2, from EtOAc extract of Trichoderma albolutescens culture medium. The structures of compounds 1 and 2 were determined by MS and NMR experiments, and the absolute configurations of the compounds were established by experimental and calculated vibrational circular dichroism spectra. Compounds 1 and 2 were evaluated for their anti-PepMoV potential in systemic host plants, such as tobacco and pepper, by PepMoV-GFP based systemic host method. All compounds exhibited inactivation effects against PepMoV. Furthermore, compound 1 showed protective effects against PepMoV.

Quassinoids isolated from Brucea javanica inhibit pepper mottle virus in pepper.

A green fluorescent protein (GFP)-tagged pepper mottle virus (PepMoV) based leaf-disc method and systemic host method were developed to identify antiviral agents. Preliminary experiments using a PepMoV-GFP based leaf-disc method led to the isolation of five quassinoids, including brusatol (1), bruceantin (2), brucein A (3), bruceantinol (4), and brucein B (5), from the CH3OH extract of Brucea javanica. All isolated compounds exhibited inactivation effects in systemic host plants, and compounds 3 and 4 were potent, with a minimum inhibitory concentration of 10µM. Furthermore, compound 3 was found to have a protective effect at the tested concentration of 40µM.

Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/µl of viral RNA under monoplex conditions and 10 pg/µl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed.