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Atractylodes macrocephala Koidz is a very common herbs in China, also famous for its high medicinal value (Lee et al., 2007). In summer of 2019, in Fuyang county of Zhejiang province, the main production area of China, 74 plants of A. macrocephala from a total of about 300 plants, showed black leaf spots . The incidence of the disease was 25% and increased under high temperature and humidity conditions. Initial leaf symptoms appeared as black or tan spots surrounded by brown margins and expanded irregularly. Finally, large blackish brown spots appeared on the leaves, elliptical or irregular, 1.0 to 1.5 cm in diameter, and then lesions turned necrotic. To isolate the pathogen, small pieces (5×5 mm) from the margin of symptomatic leaves were surface-sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite for 2 min, rinsed five times with sterile water, and incubated on potato dextrose agar (PDA) at 28°C in darkness. Purified colonies were white to pink with densely floccose to fluffy aerial mycelium and peach-orange pigmentation. Macroconidia, usually three-septate, were 26.7 to 43.3×3.1 to 5.3µm (n=50), thin-walled, slightly curved, with apical and basal cells curved when cultured in continuous darkness. Microconidia were mostly aseptate, ovate-oblong, straight to slightly curved, and measuring 5.9 to 14.3×2.3 to 3.9µm in size (n=50). Spherical chlamydospores were produced singly or in pairs from mycelium and spores. These characteristics were consistent with the description of Fusarium spp. (Leslie and Summerell, 2006). To identify the species, the translation elongation factor-1 alpha regions (TEF-1α) and the mitochondrial small subunit (mtSSU) were amplified using primers EF-3/ EF-22 (Palmore et al.,2010 and O'Donnell et al., 1998) and MS3F/ MS3R (Stenglein et al., 2010), respectively. Sequences were deposited in GenBank (MT263720, OM203177, OM203178, OM203179, OM203180, OM203181 and MN853662, MZ028170.1, MZ028171.1, MZ028172.1, MZ028173.1, MZ028174.1). These six isolates clustered in the Fusarium commune clade with 100% and 98% similarity, respectively. To test pathogenicity of every isolate, five 8-week-old potted A. macrocephalae plants were wound-inoculated and mycelial discs of 5-mm diameter were used to inoculate. As a control, five plants were inoculated with 5-mm PDA plugs. All plants were individually covered with a plastic bag and kept in a greenhouse at 25 ± 2°C with a 12-h photoperiod at 70 to 80% relative humidity. Typical symptoms similar to those of the field appeared only in inoculated plants after five days. In addition, a conidial suspension (1×105 spores/ml) was sprayed onto young leaves of three potted healthy plants. Three plants sprayed with sterile distilled water served as controls. After 7 days, typical symptoms were observed on all inoculated leaves. Experiments were replicated three times. F. commune was successfully re-isolated from diseased plants. Based on morphological and molecular identification, the pathogen was identified as F. commune. In China, Alternaria alternata (Zhuang, 2005), A. longipes (Tan et al., 2012), Phyllosticta commonsii (Sang et al., 2006) and Phoma exigua (Zhang et al., 2018) were reported as causal agents of the leaf spot disease of A. macrocephalae. To our knowledge, this is the first report of leaf spot disease on A. macrocephalae caused by F. commune in China. Effective control strategies need to be established to reduce the losses.

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Beet necrotic yellow vein virus (BNYVV) infections induce stunting and leaf curling, as well as root and floral developmental defects and leaf senescence in Nicotiana benthamiana. A microarray analysis with probes capable of detecting 1596 candidate microRNAs (miRNAs) was conducted to investigate differentially expressed miRNAs and their targets upon BNYVV infection of N. benthamiana plants. Eight species-specific miRNAs of N. benthamiana were identified. Comprehensive characterization of the N. benthamiana microRNA profile in response to the BNYVV infection revealed that 129 miRNAs were altered, including four species-specific miRNAs. The targets of the differentially expressed miRNAs were predicted accordingly. The expressions of miR164, 160, and 393 were up-regulated by BNYVV infection, and those of their target genes, NAC21/22, ARF17/18, and TIR, were down-regulated. GRF1, which is a target of miR396, was also down-regulated. Further genetic analysis of GRF1, by Tobacco rattle virus-induced gene silencing, assay confirmed the involvement of GRF1 in the symptom development during BNYVV infection. BNYVV infection also induced the up-regulation of miR168 and miR398. The miR398 was predicted to target umecyanin, and silencing of umecyanin could enhance plant resistance against viruses, suggesting the activation of primary defense response to BNYVV infection in N. benthamiana. These results provide a global profile of miRNA changes induced by BNYVV infection and enhance our understanding of the mechanisms underlying BNYVV pathogenesis.

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Plant microRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development, defense, and symptom development. Here, 547 known miRNAs representing 129 miRNA families, and 282 potential novel miRNAs were identified in Beta macrocarpa using small RNA deep sequencing. A phylogenetic analysis was performed, and 8 Beta lineage-specific miRNAs were identified. Through a differential expression analysis, miRNAs associated with Beet necrotic yellow vein virus (BNYVV) infection were identified and confirmed using a microarray analysis and stem-loop RT-qPCR. In total, 103 known miRNAs representing 38 miRNA families, and 45 potential novel miRNAs were differentially regulated, with at least a two-fold change, in BNYVV-infected plants compared with that of the mock-inoculated control. Targets of these differentially expressed miRNAs were also predicted by degradome sequencing. These differentially expressed miRNAs were involved in hormone biosynthesis and signal transduction pathways, and enhanced axillary bud development and plant defenses. This work is the first to describe miRNAs of the plant genus Beta and may offer a reference for miRNA research in other species in the genus. It provides valuable information on the pathogenicity mechanisms of BNYVV.

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BACKGROUND: Agarwood, is a resinous portion derived from Aquilaria sinensis, has been widely used in traditional medicine and incense. 2-(2-phenylethyl)chromones are principal components responsible for the quality of agarwood. However, the molecular basis of 2-(2-phenylethyl)chromones biosynthesis and regulation remains almost unknown. Our research indicated that salt stress induced production of several of 2-(2-phenylethyl)chromones in A. sinensis calli. Transcriptome analysis of A. sinensis calli treated with NaCl is required to further facilitate the multiple signal pathways in response to salt stress and to understand the mechanism of 2-(2-phenylethyl)chromones biosynthesis. RESULTS: Forty one 2-(2-phenylethyl)chromones were identified from NaCl-treated A. sinensis calli. 93 041 unigenes with an average length of 1562 nt were generated from the control and salt-treated calli by Illmunina sequencing after assembly, and the unigenes were annotated by comparing with the public databases including NR, Swiss-Prot, KEGG, COG, and GO database. In total, 18 069 differentially expressed transcripts were identified by the transcriptome comparisons on the control calli and calli induced by 24 h or 120 h salinity stress. Numerous genes involved in signal transduction pathways including the genes responsible for hormone signal transduction, receptor-like kinases, MAPK cascades, Ca(2+) signal transduction, and transcription factors showed clear differences between the control calli and NaCl-treated calli. Furthermore, our data suggested that the genes annotated as chalcone synthases and O-methyltransferases may contribute to the biosynthesis of 2-(2-phenylethyl)chromones. CONCLUSIONS: Salinity stress could induce the production of 41 2-(2-phenylethyl)chromones in A. sinensis calli. We conducted the first deep-sequencing transcriptome profiling of A. sinensis under salt stress and observed a large number of differentially expressed genes in response to salinity stress. Moreover, salt stress induced dynamic changes in transcript abundance for novel classes of responsive genes involved in signal transduction, including the genes responsible for hormone signal transduction, receptor-like kinases, MAPK cascades, Ca(2+) signal transduction, and transcription factors. This study will aid in selecting the target genes to genetically regulate A. sinensis salt-stress signal transduction and elucidating the biosynthesis of 2-(2-phenylethyl)chromones under salinity stress.

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BACKGROUND: Rhizomania is one of the most devastating diseases of sugar beet. It is caused by Beet necrotic yellow vein virus (BNYVV) transmitted by the obligate root-infecting parasite Polymyxa betae. Beta macrocarpa, a wild beet species widely used as a systemic host in the laboratory, can be rub-inoculated with BNYVV to avoid variation associated with the presence of the vector P. betae. To better understand disease and resistance between beets and BNYVV, we characterized the transcriptome of B. macrocarpa and analyzed global gene expression of B. macrocarpa in response to BNYVV infection using the Illumina sequencing platform. RESULTS: The overall de novo assembly of cDNA sequence data generated 75,917 unigenes, with an average length of 1054 bp. Based on a BLASTX search (E-value ≤ 10-5) against the non-redundant (NR, NCBI) protein, Swiss-Prot, the Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, there were 39,372 unigenes annotated. In addition, 4,834 simple sequence repeats (SSRs) were also predicted, which could serve as a foundation for various applications in beet breeding. Furthermore, comparative analysis of the two transcriptomes revealed that 261 genes were differentially expressed in infected compared to control plants, including 128 up- and 133 down-regulated genes. GO analysis showed that the changes in the differently expressed genes were mainly enrichment in response to biotic stimulus and primary metabolic process. CONCLUSION: Our results not only provide a rich genomic resource for beets, but also benefit research into the molecular mechanisms of beet- BNYV Vinteraction.

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BACKGROUND: Beet necrotic yellow vein virus (BNYVV) is the infectious agent of sugar beet rhizomania, which consists of four or five plus-sense RNAs. RNA4 of BNYVV is not essential for virus propagation in Nicotiana benthamiana but has a major effect on symptom expression. Early reports showed that RNA4-encoded P31 was associated with severe symptoms, such as curling and dwarfing, in N. benthamiana. RESULTS: We discovered that the pathogenesis-related protein 10 (PR-10) gene can be up-regulated in BNYVV-infected N. benthamiana in the presence of RNA4 and that it had a close link with symptom development. Our frame-shift, deletion and substitution analysis showed that only the entire P31 could induce PR-10 up-regulation during BNYVV infection and that all the tryptophans and six cysteines (C174, C183, C186, C190, C197 and C199) in the cysteine-rich P31 had significant effects on PR-10 expression. However, P31 could not interact directly with PR-10 in yeast. CONCLUSIONS: Our data demonstrated that only integrated P31 specifically induced PR-10 transcription, which coincided closely with the appearance of severe symptoms in BNYVV-infected N. benthamiana, although they could not interact directly with each other in yeast.

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BACKGROUND: Beet necrotic yellow vein virus (BNYVV), encodes either four or five plus-sense single stranded RNAs and is the causal agent of sugar beet rhizomania disease, which is widely distributed in most regions of the world. BNYVV can also infect Nicotiana benthamiana systemically, and causes severe curling and stunting symptoms in the presence of RNA4 or mild symptoms in the absence of RNA4. RESULTS: Confocal laser scanning microscopy (CLSM) analyses showed that the RNA4-encoded p31 protein fused to the red fluorescent protein (RFP) accumulated mainly in the nuclei of N. benthamiana epidermal cells. This suggested that severe RNA4-induced symptoms might result from p31-dependent modifications of the transcriptome. Therefore, we used next-generation sequencing technologies to analyze the transcriptome profile of N. benthamiana in response to infection with different isolates of BNYVV. Comparisons of the transcriptomes of mock, BN3 (RNAs 1+2+3), and BN34 (RNAs 1+2+3+4) infected plants identified 3,016 differentially expressed transcripts, which provided a list of candidate genes that potentially are elicited in response to virus infection. Our data indicate that modifications in the expression of genes involved in RNA silencing, ubiquitin-proteasome pathway, cellulose synthesis, and metabolism of the plant hormone gibberellin may contribute to the severe symptoms induced by RNA4 from BNYVV. CONCLUSIONS: These results expand our understanding of the genetic architecture of N. benthamiana as well as provide valuable clues to identify genes potentially involved in resistance to BNYVV infection. Our global survey of gene expression changes in infected plants reveals new insights into the complicated molecular mechanisms underlying symptom development, and aids research into new strategies to protect crops against viruses.

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An Er3+/Yb3+ co-doped bismuth germanate glass is synthesized and analyzed. The radiative characteristics and mid-infrared spectroscopic properties are investigated under excitation of a conventional 980 nm laser diode. The prepared glass possesses higher spontaneous transition probability (68.82 s(-1)) and larger calculated emission cross-section (7.73×10(-21) cm2) corresponding to the 4I11/2→4I13/2 transition. The calculated energy migration coefficient (CDD) among Yb3+ ions is larger than the energy transfer coefficient (CDA) from Yb3+ to Er3+, indicating the energy transfer process assisted with energy migration. The excellent spectroscopic properties along with the outstanding thermal stability suggest that this glass may become an attractive host for developing solid state lasers operating in the mid-infrared range.

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Beet black scorch virus (BBSV) encodes three movement proteins (P7a, P7b and P5') that facilitate its cell-to-cell movement. An arginine-rich motif of P7a N-terminus was found to determine nuclear and nucleolar localization. Amino acids substitution or deletion of the R-rich motif interfered with P7a nuclear and nucleolar localization. Bimolecular fluorescence complementation (BiFC) assays revealed that P7a protein interacted with Nicotiana benthamiana nuclear import factor importin α, suggesting that P7a is translocated into the nucleus by the classical importin α/ß-dependent pathway. Moreover, P7a also interacted with the nucleolar protein fibrillarin. Mutations in the R-rich motif of P7a diminished P7a interactions with importin α and fibrillarin, influenced viral replication in Nicotiana benthamiana protoplasts and altered the symptom phenotype and viral RNA accumulation in Chenopodium amaranticolor plants. These results demonstrate that the R-rich motif of P7a is correlated with nuclear and nucleolar localization, viral replication and virus infection.

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BACKGROUND: Beet Necrotic Yellow Vein virus (BNYVV) is a member of the genus Benyvirus causing a worldwide sugar beet disease rhizomania. BNYVV contains four or five plus-sense single stranded RNAs. In altered selective conditions, multipartite RNA viruses of plant are prone to undergoing internal deletions, thus turning into Defective RNAs (D RNAs). Although several D RNAs have been reported in BNYVV infection, the spontaneous internal deletion mutants responsible for severe symptom in systemic host Nicotiana benthamiana (N. benthamiana) are not described so far. RESULTS: Systemic host N. benthamiana was inoculated by Chinese BNYVV isolates. RT-PCR and Northern blot showed that the D RNAs forms of BNYVV RNA3 were present in the systemic infection of the N. benthamiana. Three distinct D-RNA3s, named as D-RNA 3α, D-RNA 3ß and D-RNA 3γ, were made into infectious clones. When inoculated on the N. benthamiana, the in vitro transcripts of D forms exhibited more stable than that of wild-type RNA3 in systemic movement. Among the detected mutant, the p25 protein frame-shift mutant (D-RNA3α) induced obvious necrotic lesions on Tetragonia.expansa (T. expansa) and pronounced systemic symptom on the N. benthamiana. The D-RNA3α was further mutated artificially to pre-terminate the downstream N protein, leading to the abolishment of the pathogenicity, indicating the N protein was responsible for the necrotic symptom. CONCLUSION: Our studies demonstrated the internal deletion mutants of BNYVV-RNA3 were spontaneously generated in the systemic infection on N. benthamiana. The internal deletions didn't affect the efficient replication of D-RNA3s, instead by improving the stability and pathogenicity of RNA3 in the systemic host N. benthamiana. Besides, our results also suggested the downstream N protein of RNA3, but not the upstream p25 protein, may play an important role in the systemic infection on N. benthamiana.

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