First report of pepper chlorosis-associated virus infecting tobacco (Nicotiana tabacum) plants in Sichuan Province in China.

Tobacco (Nicotiana tabacum) is an economically important crop in China, and more than 30 viruses have been reported to infect tobacco (Yin et al. 2022). In July 2022, we observed interveinal necrosis on tobacco leaves in fields in Sichuan Province (N 27.9172, E 105.6662) (Fig. 1). Total RNA was isolated from multiple leaves of one plant using an RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (TIANGEN, Beijing, China). Total RNAs were pooled, and a TruSeq Stranded Total RNA with RiboZero Gold Kit (Illumina, San Diego, CA, USA) was used to eliminate ribosomal RNA. An RNA-Seq library was constructed using VAHTS Universal V6 RNA-seq Library Prep (Nanjing Vazyme, China). High-throughput sequencing was performed on the Illumina DNBseq platform (BGI-ShenZhen, China), which yielded 20,102,087 reads with an average length of 150 nt (total size >6 Gb). Unaligned reads were assembled de novo using SPAdes (Bankevich et al. 2012). Contigs with length ≥200 nt were subjected to local BLASTn and BLASTx analyses against the GenBank nt and nr databases, respectively (Wang et al. 2022). A total of 23 contigs were identified through BLASTx (e-value cut-off = 10 -3), ranging from 631 to 1555 bp long, with 82% to 96% coverage to partial genomic sequences of pepper chlorosis-associated virus (PepCaV-Higashitsuno_2021; Accessions: LC719619 to LC719621) and one contig (6459 bp) with 99% similarity to tobacco mosaic virus (Accession: OP525281) isolate DSMZ PV-0109 from Germany. The complete genome sequence of PepCaV was obtained using primers based on the assembled contigs. The 5'- and 3'-terminal regions of the RNA genome were obtained by 5'- and 3'-rapid amplification of cDNA ends. These amplicons were cloned using the pEASY-Blunt Zero Cloning Kit (TRANSGEN, Nangjing, China) and sequenced by Sanger sequencing. Complete genome sequences of tripartite PepCaV from tobacco samples were 7697, 1808, and 1557 nucleotides long (Accession: OR451987 to OR451989) and showed genome organization typical of the genus Ophiovirus in the family Aspiviridae. The complete sequences of RNA1, RNA2 and RNA3 genome segments shared 92.36%, 90.43%, and 95.24%, nucleotide sequence identities, respectively, with the isolate PepCaV-Higashitsuno_2021 pepper isolate (Accession: LC719619 to LC719621) (Shimomoto et al. 2023), but PepCaV has not been reported to infect N. tabacum. In June 2023, 10 plants collected from each place of Macheng (N 27.9094, E 105.6740), Xiangyang (N 28.0936, E 105.6249) and Moni (N 27.8899, E 105.5936) showing interveinal necrosis symptoms were tested using RT-PCR using PepCaV-MP610-F (5'-TGTTCTCTGCTATGCGGTTG -3') and PepCaV-MP610-R (5'-AGCAATCTCGCACCTGAAGT-3') to product 610bp amplicon. Twenty-five tobacco plants were positive for PepCaV. Single sequence from each location was submitted to GenBank (Accession: PP728631 to PP728633). Sap extracts from the original field leaf samples collected from Sichuan Province were used to mechanically inoculate tobacco plants (10 plants) at the four-leaf stage. After 7 days, leaf samples were tested using RT-PCR assay specific to PepCaV and TMV while samples were positive only for TMV but failed to transmit PepCaV by mechanical inoculation. According to previous literature, ophioviruses may be transmitted though soilborne fungus (Jeong et al. 2014). Further research is needed to understand the transmission, epidemiology, and pathological properties of the PepCaV. To our knowledge, this is the first report documenting natural PepCaV infection of tobacco plants in China, providing a scientific basis for PepCaV infection control in tobacco plantations.

A Review of the Biological Activities of Heterocyclic Compounds Comprising Oxadiazole Moieties.

The oxadiazole core is considered a privileged moiety in many medicinal chemistry applications. The oxadiazole class includes 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, and 1,2,5-oxadiazole. Compounds bearing an oxadiazole ring show a wide range of biological activities, such as anticancer, antibacterial, anti-inflammatory, anti-malarial, and insecticidal properties. Among oxadiazoles, the 1,3,4-oxadiazole has been the most widely explored moiety in medicinal chemistry research. This review is primarily focused on the anticancer, antibacterial, and anti-inflammatory activities of compounds containing 1,2,4-oxadiazole, 1,3,4-oxadiazole and 1,2,5-oxadiazole reported in the last five years.

Development of Recombinase Polymerase Amplification Combined with Lateral Flow Detection Assay for Rapid and Visual Detection of Ralstonia solanacearum in Tobacco.

Bacterial wilt caused by Ralstonia solanacearum is a serious soilborne disease that results in severe losses to tobacco (Nicotiana tabacum) production in China. In this study, a novel RPA-LFD assay for the rapid visual detection of R. solanacearum was established using recombinase polymerase amplification (RPA) and lateral-flow dipstick (LFD). The RPA-LFD assay was performed at 37°C in 30 min without complex equipment. Targeting the sequence of the RipTALI-9 gene, we designed RPA primers (Rs-rpa-F/R) and an LF probe (Rs-LF-probe) that showed high specificity to R. solanacearum. The sensitivity of RPA-LFD assay to R. solanacearum was the same as that in conventional PCR at 1 pg genomic DNA, 103 CFU/g artificially inoculated tobacco stems, and 104 CFU/g artificially inoculated soil. The RPA-LFD assay could also detect R. solanacearum from plant and soil samples collected from naturally infested tobacco fields. These results suggest that the RPA-LFD assay developed in this study is a rapid, accurate molecular diagnostic tool with high sensitivity for the detection of R. solanacearum.

Characterisation of a novel alphaendornavirus isolated from balloon flower (Platycodon grandiflorus).

The complete genome sequence of a new virus, provisionally named "balloon flower endornavirus" (BfEV), was determined following virus isolation from leaves of a balloon flower (Platycodon grandiflorum) exhibiting severe leaf shrinkage disease in Anhui Province, China. The 15,180-nucleotide genome sequence of BfEV shares 40.52% amino acid (aa) sequence identity and 98% coverage with the polyprotein of Helianthus annuus endornavirus (HaEV). The open reading frame (ORF) encodes a deduced 4,887-aa polyprotein with RNA-dependent RNA polymerase (RdRp), viral helicase (Hel) and glycosyltransferase (GT) domains. Phylogenetic analysis of the amino acid sequences of RdRp places BfEV alongside members of the genus Alphaendornavirus in the family Endornaviridae. To our knowledge, this is the first report of a complete genome sequence of a novel alphaendornavirus identified in balloon flower.

Characterization of an RNA silencing suppressor encoded by maize yellow dwarf virus-RMV2.

Maize yellow dwarf virus-RMV2 (MYDV-RMV2) causes dwarfing and yellowing symptoms on leaves in field-grown maize plants in Anhui province in China. Herein, we evaluated the RNA silencing suppressor (RSS) activity of the P0 protein from MYDV-RMV2 by co-infiltration assays using wild-type and GFP-transgenic Nicotiana benthamiana (line 16C). The P0 of MYDV-RMV2 exhibited RSS activity and inhibited RNA silencing both locally and systemically. MYDV-RMV2 P0 acts as an F-box-like motif, and mutations to Ala at positions 67, 68, and 81 in the F-box-like motif (67LPxx81P) abolished the RSS activity of P0. However, MYDV-RMV2 P0 failed to interact with AGO1 from Arabidopsis thaliana. Expressing P0 induced developmental defects. P0 was targeted to both the nuclei and cytoplasm of plant cells. These findings expand our knowledge of the role of polerovirus P0 proteins in RNA silencing.

Modification, Biological Evaluation and SAR Studies of Novel 1H-Pyrazol Derivatives Containing N,N'-Disubstituted Urea Moiety as Potential Anti-melanoma Agents.

Malignant melanomas are amongst the most aggressive cancers. BRAF Inhibitors have exhibited therapeutic effects against BRAF-mutant melanoma. In continuation of our earlier studies on anti-melanoma agents based on 1H-pyrazole skeleton, two sets of novel compounds that include 1H-pyrazole-4-amines FA1 - FA13 and corresponding urea derivatives FN1 - FN13 have been synthesized and evaluated for their BRAFV600E inhibitory and antiproliferation activities. Compound FN10 displayed the most potent biological activity against BRAFV600E (IC50 = 0.066 µm) and the A375 human melanoma cell line (GI50 = 0.81 µm), which was comparable to the positive control vemurafenib, and more potent than our previously reported 1H-pyrazole-3-amines and their urea derivatives. The results of SAR studies and molecular docking can guide further optimization and may help to improve potency of these pyrazole-based anti-melanoma agents.

Sequencing and phylogenetic analysis of tobacco virus 2, a polerovirus from Nicotiana tabacum.

The complete genome sequence of a new virus, provisionally named tobacco virus 2 (TV2), was determined and identified from leaves of tobacco (Nicotiana tabacum) exhibiting leaf mosaic, yellowing, and deformity, in Anhui Province, China. The genome sequence of TV2 comprises 5,979 nucleotides, with 87% nucleotide sequence identity to potato leafroll virus (PLRV). Its genome organization is similar to that of PLRV, containing six open reading frames (ORFs) that potentially encode proteins with putative functions in cell-to-cell movement and suppression of RNA silencing. Phylogenetic analysis of the nucleotide sequence placed TV2 alongside members of the genus Polerovirus in the family Luteoviridae. To the best our knowledge, this study is the first report of a complete genome sequence of a new polerovirus identified in tobacco.

Ecogenomic survey of plant viruses infecting Tobacco by Next generation sequencing.

BACKGROUND: The invasion of plant by viruses cause major damage to plants and reduces crop yield and integrity. Devastating plant virus infection has been experienced at different times all over the world, which are attributed to different events of mutation, re-assortment and recombination occurring in the viruses. Strategies for proper virus management has been mostly limited to eradicating the vectors that spreads the plant viruses. However, development of prompt and effective diagnostic methods are required to monitor emerging and re-emerging diseases that may be symptomatic or asymptomatic in the plant as well as the genetic variation and evolution in the plant viruses. A survey of plant viruses infecting field-grown Tobacco crop was conducted in Anhui Province of China by the deep sequencing of sRNAs. METHODS: Survey of plant viruses infecting Tobacco was carried based on 104 samples collected across the province. Nine different sRNA libraries was prepared and custom-made bioinformatics pipeline coupled with molecular techniques was developed to sequence, assemble and analyze the siRNAs for plant virus discovery. We also carried out phylogenetic and recombination analysis of the identified viruses. RESULTS: Twenty two isolates from eight different virus species including Cucumber mosaic virus, Potato virus Y, Tobacco mosaic virus, Tobacco vein banding Mosaic virus, Pepper mottle virus, Brassica yellow virus, Chilli venial mottle virus, Broad bean wilt virus 2 were identified in tobacco across the survey area. The near-complete genome sequence of the 22 new isolates were determined and analyzed. The isolates were grouped together with known strains in the phylogenetic tree. Molecular variation in the isolates indicated the conserved coding regions have majorly a nucleotide sequence identity of 80-94 % with previously identified isolates. Various events of recombination were discovered among some of the isolates indicating that two or more viruses or different isolates of one virus infect the same host cell. CONCLUSION: This study describes the discovery of a consortium of plant viruses infecting Tobacco that are broadly distributed in Anhui province of China. It also demonstrates the effectiveness of NGS in identifying plant viruses without a prior knowledge of the virus and the genetic diversity that enhanced mixed infection.

Discovery of N-(benzyloxy)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives as potential antiproliferative agents by inhibiting MEK.

Mitogen activated protein kinase (MAPK) signal transduction pathway has been proved to play an important role in tumorigenesis and cancer development. MEK inhibitor has been demonstrated significant clinical benefit for blocking MAPK pathway activation and possibly could block reactivation of the MAPK pathway at the time of BRAF inhibitor resistance. Twenty N-(benzyloxy)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives have been designed and synthesized as MEK inhibitors, and their biological activities were evaluated. Among these compounds, compound 7b showed the most potent inhibitory activity with IC50 of 91nM for MEK1 and GI50 value of 0.26µM for A549 cells. The SAR analysis and docking simulation were performed to provide crucial pharmacophore clues that could be used in further structure optimization.

Aphicidal Activity of Illicium verum Fruit Extracts and Their Effects on the Acetylcholinesterase and Glutathione S-transferases Activities in Myzus persicae (Hemiptera: Aphididae).

This study aims to explore the aphicidal activity and underlying mechanism of Illicium verum Hook. f. that is used as both food and medicine. The contact toxicity of the extracts from I. verum fruit with methyl alcohol (MA), ethyl acetate (EA), and petroleum ether (PE) against Myzus persicae (Sulzer), and the activities of acetylcholinesterase (AChE) and glutathione S-transferases (GSTs) of M. persicae after contact treatment were tested. The results showed that MA, EA, and PE extracts of 1.000 mg/l caused, respectively, M. persicae mortalities of 68.93%, 89.95% and 74.46%, and the LC50 of MA, EA, and PE extracts were 0.31, 0.14 and 0.27 mg/l at 72 h after treatment, respectively; the activities of AChE and GSTs in M. persicae were obviously inhibited by the three extracts, as compared with the control, with strong dose and time-dependent effects, the inhibition rates on the whole reached more than 50.00% at the concentration of 1.000 mg/l at 72 h after treatment. The inhibition of the extracts on AChE and GSTs activities (EA extract > PE extract > MA extract) were correlated with theirs contact toxic effects, so it is inferred that the decline of the metabolic enzymes activities may be one of important reasons of M. persicae death. The study results suggested that I. verum extracts have potential as a eco-friendly biopesticide in integrated pest management against M. persicae.

Complete genome sequence of tobacco virus 1, a closterovirus from Nicotiana tabacum.

The complete genome sequence of a novel virus, provisionally named tobacco virus 1 (TV1), was determined, and this virus was identified in leaves of tobacco (Nicotiana tabacum) exhibiting leaf mosaic and yellowing symptoms in Anhui Province, China. The genome sequence of TV1 consists of 15,395 nucleotides with 61.6 % nucleotide sequence identity to mint virus 1 (MV1). Its genome organization is similar to that of MV1, containing nine open reading frames (ORFs) that potentially encode proteins with putative functions in virion assembly, cell-to-cell movement and suppression of RNA silencing. Phylogenetic analysis of the heat shock protein 70 homolog (HSP70h) placed TV1 alongside members of the genus Closterovirus in the family Closteroviridae. To our knowledge, this study is the first report of the complete genome sequence of a closterovirus identified in tobacco.

Effects of Phospholipase C on Fusarium graminearum Growth and Development.

Phospholipase C (PLC) plays important roles in regulating various biological processes in eukaryotes. Currently, little is known about the function of PLC in filamentous fungi, especially the plant pathogenic fungi. Fusarium graminearum is the causal agent of Fusarium head blight in many cereal crops. BLAST search revealed that Fusarium genome contains six FgPLC genes. Using quantitative RT-PCR, different FgPLC gene expressions in mycelia were analyzed. To investigate the role of FgPLC in F. graminearum biology, a pharmacological study using a known inhibitor of PLC (U73122) was conducted. Results showed that inhibition of FgPLC resulted in significant alterations of mycelial growth, conidiation, conidial germination, perithecium formation, and expressions of Tri5 and Tri6 genes. As expected, the treatment of F. graminearum with U73343, an inactive analog of U73122, showed no effect on F. graminearum biology. Our results suggested strongly that FgPLC plays important roles in F. graminearum growth and development.

Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells.

Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H2O2) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 µM FA and then analyzed for H2O2 accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H2O2 production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H2O2 accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells.

Fusaric acid induction of programmed cell death modulated through nitric oxide signalling in tobacco suspension cells.

Fusaric acid (FA) is a nonhost-selective toxin mainly produced by Fusarium oxysporum, the causal agent of plant wilt diseases. We demonstrate that FA can induce programmed cell death (PCD) in tobacco suspension cells and the FA-induced PCD is modulated by nitric oxide (NO) signalling. Cells undergoing cell death induced by FA treatment exhibited typical characteristics of PCD including cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane plasmolysis, and formation of small cytoplasmic vacuoles. In addition, caspase-3-like activity was activated upon the FA treatment. The process of FA-induced PCD was accompanied by a rapid accumulation of NO in a FA dose-dependent manner. Pre-treatment of cells with NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or NO synthase inhibitor N(G)-monomethyl-arginine monoacetate (L-NMMA) significantly reduced the rate of FA-induced cell death. Furthermore, the caspase-3-like activity and the expression of PAL and Hsr203J genes were alleviated by application of cPTIO or L-NMMA to FA-treated tobacco cells. This indicates that NO is an important factor involved in the FA-induced PCD. Our results also show that pre-treatment of tobacco cells with a caspase-3-specific inhibitor, Ac-DEVD-CHO, can reduce the rate of FA-induced cell death. These results demonstrate that the FA-induced cell death is a PCD and is modulated by NO signalling through caspase-3-like activation.