Genome-wide characterization of the bHLH gene family in Gynostemma pentaphyllum reveals its potential role in the regulation of gypenoside biosynthesis.

BACKGROUND: Gynostemma pentaphyllum, an ancient Chinese herbal medicine, serves as a natural source of gypenosides with significant medicinal properties. Basic helix-loop-helix (bHLH) transcription factors play pivotal roles in numerous biological processes, especially in the regulation of secondary metabolism in plants. However, the characteristics and functions of the bHLH genes in G. pentaphyllum remain unexplored, and their regulatory role in gypenoside biosynthesis remains poorly elucidated. RESULTS: This study identified a total of 111 bHLH members in G. pentaphyllum (GpbHLHs), categorizing them into 26 subgroups based on shared conserved motif compositions and gene structures. Collinearity analysis illustrated that segmental duplications predominately lead to the evolution of GpbHLHs, with most duplicated GpbHLH gene pairs undergoing purifying selection. Among the nine gypenoside-related GpbHLH genes, two GpbHLHs (GpbHLH15 and GpbHLH58) were selected for further investigation based on co-expression analysis and functional prediction. The expression of these two selected GpbHLHs was dramatically induced by methyl jasmonate, and their nuclear localization was confirmed. Furthermore, yeast one-hybrid and dual-luciferase assays demonstrated that GpbHLH15 and GpbHLH58 could bind to the promoters of the gypenoside biosynthesis pathway genes, such as GpFPS1, GpSS1, and GpOSC1, and activate their promoter activity to varying degrees. CONCLUSIONS: In conclusion, our findings provide a detailed analysis of the bHLH family and valuable insights into the potential use of GpbHLHs to enhance the accumulation of gypenosides in G. pentaphyllum.

First Report of Aspergillus westerdijkiae Causing Leaf Blight on Rehmannia glutinosa in China.

Rehmannia glutinosa (also known as Chinese foxglove) is a perennial dicotyledonous herb, which plays an important role in traditional Chinese medicine. Its active ingredients have a wide range of pharmacological effects on the blood system, endocrine system, immune system, cardiovascular system, and nervous system (Zhang et al. 2008). In May 2022, leaf blight was observed on 45-day-old R. glutinosa in a seedling nursery in Jiaozuo City (35°01'44.20″N, 113°05'30.63″E), Henan Province, China with an approximate disease incidence up to 54% (~1,300 plants). Irregular brown lesion initially appeared on the tips of basal leaves, then progressed to the entire leaf causing leaf drying out (Supple. Fig. 1-A, B, C). The same symptoms appeared successively in the leaves from the base to the top of the plant, which eventually caused the whole plant to die. To identify the pathogen, eight symptomatic leaves were randomly collected from eight individual plants, and cut into small pieces (5 × 5 mm) at the border of lesions. The pieces were surface disinfected in 75% ethanol for 15 s, followed by 1% NaClO for 1 min, rinsed in sterile water three times, and placed on potato dextrose agar (PDA) medium in the dark for 3 days at 25℃. Finally, 12 purified isolates (DHY1-DHY12) were obtained by using single spore method. Leaves of R. glutinosa seedlings were inoculated with conidial suspension (106 conidia/ml), three plants were inoculated per isolate. Controls were treated with sterilized water. All inoculated and control plants were incubated in a greenhouse at 25℃ under 80 ± 10% humidity and a 8-h/16-h dark/light cycle. This experiment was repeated three times. After 5 days, similar symptoms to those of diseased leaves in the seedling nursery appeared on leaves inoculated with DHY4-DHY10, while plants inoculated with DHY1-DHY3, DHY11-DHY12, and the controls remained asymptomatic (Supple. Fig.1-D, E). The same fungi were re-isolated from diseased leaves, fulfilling Koch's postulates. The causal agents DHY4 to DHY10, showed similar morphology, which were morphologically identified as Aspergillus sp. (Visagie et al. 2014). Isolate DHY5 was selected for further study. On PDA plates, the colonies were covered with white velutinous mycelia (Supple. Fig.1-F). Conidia were ochre yellow and outwards concentric circles. Vesicles were globose, and about 20.1-26.6 µm in diameter (Supple. Fig.1-G). Conidiophore stipes were smooth walled and hyaline, with conidial heads radiating. The conidia were light yellow to orange, exudate clear to orange droplets. The conidia were (2.53-3.25) µm × (2.58-3.47) µm in diameter (n=50) (Supple. Fig.1-H). For further molecular identification, the ITS and TUB gene sequences were amplified with primer pairs ITS1/ITS4 and BT2a/BT2b (Glass and Donaldson. 1995), respectively. BLASTn searches of the ITS (PP355445) and TUB (PP382788) sequences showed 100% and 98.42% similarity to those of A. westerdijkiae (OP237108 and OP700424), respectively. Phylogenetic analysis based on the concatenated sequences of ITS and TUB confirmed that the fungus was A. westerdijkiae, (Supple. Fig.2). A. westerdijkiae was mainly reported on its secondary metabolite ochratoxin A contamination of agricultural products, fruits, and various food products, such as coffee beans (Alvindia et al 2016), grapes (Díaz et al. 2009), oranges and fruit juice (Marino et al. 2009), etc. To our knowledge, this is the first report of A. westerdijkiae causing leaf blight on R. glutinosa in China.

First report of basal stem rot caused by three Fusarium species on Chinese yam in China.

The cultivated variety of Chinese yam (Dioscorea polystachya Turcz. cv. Tiegun) is an economically important plant, capable of producing tubers that are used as food and traditional Chinese medicine. The basal stem rot was found on approximately 65% of yam (tuber expansion stage) in a total of 10 ha field in Wuzhi, Wen, and Hua counties, Henan, China (Sep 2021). Dark brown fusiform lesions initially occurred at the stems basal, irregularly extending to join together and leading to loop-stem necrotic indentation. Three diseased samples from Wuzhi county were collected, cut into 5 × 5 mm pieces, surface sterilized in 75% ethanol (30 s) and 1% NaClO (1 min), washed in sterile water 3 times, and placed on PDA in the dark for 3 days at 28℃. A total of 44 isolates forming three groups of Fusarium colonies were obtained using monosporic isolation, of which 19, 8, and 17 isolates were identified as F. oxysporum, F. solani, and F. proliferatum based on colony morphology, respectively. Typical isolates SYJJ6, 9, and 10 for each group were further studied. The SYJJ6 colonies showed gray white abundant fluffy aerial mycelium with rough edges, formation of ellipsoid, unicellular microconidia without septa, 5.6 to 13.4 × 2.4 to 4.7 µm (n = 50), and sickle-shaped, slightly curved macroconidia with 2 to 4 septa, 14.0 to 23.9 × 3.4 to 5.1 µm (n = 50). Isolate SYJJ9 produced flocculent white colonies, grew in a circular pattern with a sharp edge, forming oval or oblong microconidia with zero or one septum, 11.2 to 18.8 × 3.4 to 6.2 µm (n = 50), and slightly curved macroconidia with 2 to 3 septa, 27.6 to 44.0 × 3.9 to 7.4 µm (n = 50). SYJJ10 produced whitish or pinkish white colonies with fluffy aerial mycelium and a red pigmentation, produced renal or oval microconidia with no septa, 5.1 to 11.8 × 1.8 to 4.2 µm (n = 50), and falcate, slightly curved macroconidia with 3 to 4 septa, 16.1 to 30.2 × 3.1 to 5.9 µm (n = 50). Additionally, TUB, EF-1α, and RPB2 genes were amplified with primers BT2a/BT2b, EF1/EF2, and 5f2/-7cr, respectively (Glass and Donaldson 1995; O'Donnell et al. 1998, 2010). BLASTn analysis on SYJJ6 (OR047663, OR047666, OR047669), SYJJ9 (OR047665, OR047667, OR047670), and SYJJ10 (OR047664, OR047668, OR047671) gene sequences were over 99% identical to those of F. oxysporum (100%, MK432917; 100%, MN417196; 99.61%, MN457531), F. solani (100%, MF662662; 100%, MN223440; 99.80%, CP104055), and F. proliferatum (100%, ON557521; 100%, ON458137; 99.90%, LT841266), respectively. Pathogenicity tests of three isolates were separately performed on 60-day-old yam seedlings. The basal stems were wounded using needle, and the wounds were wrapped with cotton balls soaked with conidial suspension (1 mL, 3×106 conidia/mL) or water (control). Each isolate treated three plants and repeated three times. All plants were grown at 28℃ under a 16/8-h light/dark cycle. Typical symptoms emerged on basal stems at 16, 13, and 17 days after inoculation with the conidia of isolates SYJJ6, 9, and 10, while the control basal stems appeared healthy. The re-isolated fungi were identical to the original three isolates. Fusarium species (F. oxysporum, F. commune, F. humuli, etc.)were previously reported to cause wilt or stem rot on different D. polystachya cultivars (Fang et al. 2020; Li et al. 2023; Zhao et al. 2013), or basal stem rot on Panax ginseng (Ma et al. 2020). This is the first report of Chinese yam basal stem rot caused by Fusarium species, which threatens the production of Chinese yam 'Tiegun' and should be further studied.

First Report of Cercospora Leaf Spot Caused by Cercospora cf. flagellaris on Lonicera japonica Thunb. in Henan, China.

Honeysuckle flower (Lonicera japonica Thunb.) is a traditional Chinese medicinal plant. It is perennial and widely cultivated in China, Japan and Korea. From late August to October in 2021 and 2022, leaf spots symptoms were observed on L. japonica in different planting fields in Yuzhou, Yuanyang and Fenqiu districts, Henan province, China. The disease incidence was above 85% which reduce photosynthesis. Early disease symptoms appeared as small, circular to elliptical, brown spots on the leaves and later the lesions (1 to 5 mm × 1 to 4 mm) slowly developed yellow haloes. The different brown lesions seldom merge and form larger irregular lesions. Small fragments (3 to 5 mm) of leave tissue were excised from the lesion margins and surface-sterilized in 3% NaClO for 3 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. A total number of 8 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The colonies on PDA were whitish to gray, with cottony aerial mycelium. Conidiophores were fasciculate, olivaceous brown, straight or geniculate, uniform in width, multiseptate, and ranged from 290 to 700 µm (560 µm on average, n = 20). Conidia were hyaline, slightly curved or straight, needle shaped, truncate at the base, and terminal at the tip, 3 to 17-septate, and measuring 150 to 240 µm (180 µm on average, n = 20). The morphological features were consistent with Cercospora cf. flagellaris Ellis & G. Martin (Groenewald et al. 2013). The genomic DNA was extracted using CTAB method. The nuclear ribosomal internal transcribed spacer region (ITS), portions of the actin (ACT), histone H3 (HIS3), and translation elongation factor 1-α (TEF1) genes were amplified using primers ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone and Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006), and EF1-728F/EF1-986R (Carbone and Kohn 1999). The resulting 537-bp ITS, 226-bp ACT, 410-bp HIS3, and 306-bp TEF1 sequences of isolate JDJ002 were deposited in GenBank (accession nos. OR492367, OR548247, OR548248 and OR548248, respectively). Sequence analysis revealed that ITS, ACT, HIS3 and TEF1α sequences exhibited ≥99% of identity with the ITS (KP896013), ACT(KP895965), HIS3(MK991295) and TEF1 (MN180408) sequences of C. cf. flagellaris, respectively. A pathogenicity test was conducted on healthy of L. japonica leaves. The healthy leaves pricked from L. japonica plants, rinsed in autoclaved distilled water three times and dried with distilled filter paper. Then twelve healthy leave were inoculated with a mycelial plug (0.4 cm diameter) harvested from the periphery of two week-old colony. As negative control, leaves inoculated with PDA medium plugs. Inoculated leaves were covered with plastic bags to maintain high relative humidity and incubated at 25°C in growth chamber. After 7 days, the inoculated leaves showed symptoms identical to those observed in the field under natural conditions, whereas negative control remained symptom-free. Re-isolation of the fungus from lesions on inoculated leaves confirmed that the causal agent was C. cf. flagellaris. Pathogenicity tests were repeated three times by the same methods with the same results. To our knowledge, this is the first report of C. cf. flagellaris except Cercospora rhamni Fack., Alternaria alternata, Corynespora cassiicola or Phomopsis sp. causing leave spots on L. japonica in China.

The FomYjeF Protein Influences the Sporulation and Virulence of Fusarium oxysporum f. sp. momordicae.

Fusarium oxysporum causes vascular wilt in more than 100 plant species, resulting in massive economic losses. A deep understanding of the mechanisms of pathogenicity and symptom induction by this fungus is necessary to control crop wilt. The YjeF protein has been proven to function in cellular metabolism damage-repair in Escherichia coli and to play an important role in Edc3 (enhancer of the mRNA decapping 3) function in Candida albicans, but no studies have been reported on related functions in plant pathogenic fungi. In this work, we report how the FomYjeF gene in F. oxysporum f. sp. momordicae contributes to conidia production and virulence. The deletion of the FomYjeF gene displayed a highly improved capacity for macroconidia production, and it was shown to be involved in carbendazim's associated stress pathway. Meanwhile, this gene caused a significant increase in virulence in bitter gourd plants with a higher disease severity index and enhanced the accumulation of glutathione peroxidase and the ability to degrade hydrogen peroxide in F. oxysporum. These findings reveal that FomYjeF affects virulence by influencing the amount of spore formation and the ROS (reactive oxygen species) pathway of F. oxysporum f. sp. momordicae. Taken together, our study shows that the FomYjeF gene affects sporulation, mycelial growth, pathogenicity, and ROS accumulation in F. oxysporum. The results of this study provide a novel insight into the function of FomYjeF participation in the pathogenicity of F. oxysporum f. sp. momordicae.

[Identification of terpene synthase gene family in Gynostemma pentaphyllum and expression pattern analysis under abiotic stresses].

The present study aimed to investigate the composition of the terpene synthase(TPS) gene family in Gynostemma pentaphyllum and its role in abiotic stresses. The G. pentaphyllum TPS gene family was identified and analyzed at the genome-wide level using bioinformatics analysis, and the expression patterns of these family members were analyzed in different tissues of G. pentaphyllum as well as under various abiotic stresses. The results showed that there were 24 TPS gene family members in G. pentaphyllum with protein lengths ranging from 294 to 842 aa. All of them were localized in the cytoplasm or chloroplasts and unevenly distributed on the 11 chromosomes of G. pentaphyllum. The results of the phylogenetic tree showed that the G. pentaphyllum TPS gene family members could be divided into five subfamilies. As revealed by the analysis of promoter cis-acting elements, TPS gene family members in G. pentaphyllum were predicted to respond to a variety of abiotic stresses such as salt, low temperature, and dark stress. The analysis of gene expression patterns in different tissues of G. pentaphyllum revealed that nine TPS genes were tissue-specific in expression. The qPCR results showed that GpTPS16, GpTPS17, and GpTPS21 responded to a variety of abiotic stresses. This study is expected to provide references in guiding the further exploration of the biological functions of G. pentaphyllum TPS genes under abiotic stresses.

Excitation of mirror symmetry higher-order rational soliton in modulation stability regimes on continuous wave background.

We study the relationship between the structures of the nonlinear localized waves and the distribution characteristics of the modulation stability regime in a nonlinear fiber with both third-order and fourth-order effects. On the background frequency and background amplitude plane, the modulation stability region consists of two symmetric curves on the left and right and a point on the symmetry axis. We find that the higher-order excitation characteristics are obviously different at different positions in the modulation stability region. Their excitation characteristics are closely related to the modulation instability distribution characteristics of the system. It is shown that asymmetric high-order rational solitons are excited at the left and right stable curves, and the symmetric one is excited at the stable points. Interestingly, the asymmetric higher-order rational solitons on the left and right sides are mirror-symmetrical to each other, which coincides with the symmetry of the modulation instability distribution. These results can deepen our understanding of the relationship between nonlinear excitation and modulation instability and enrich our knowledge about higher-order nonlinear excitations.

First Report of Tobacco Mild Green Mosaic Virus Infecting Rehmannia glutinosa in China.

Rehmannia glutinosa (family Scrophulariaceae) is an important traditional medicinal plant, whose root is used to treat anemia, hemoptysis, and gynecological diseases in China (Matsumoto et al. 1989). This plant is native to China and cultivated in China, Korea, Japan, and northern Vietnam (Kwak et al. 2020). Viral diseases caused remarkable loss in the yield and quality of R. glutinosa (Ling et al. 2009). To date, ten viruses have been identified globally to infect R. glutinosa and seven of these viruses reported in China (Liu et al. 2018; Zhang et al. 2021). Most plants of R. glutinosa are infected with one or more of these viruses (Kwak et al. 2018; Zhang et al. 2004). In July 2020, a survey of the viral disease infecting R. glutinosa was conducted in commercial plantations of Wenxian, Wuzhi, Mengzhou, and Yuzhou counties in Henan Province, China. The disease symptoms included mosaic, chlorosis, leaf distortion, and the percentage of symptomatic plants was over 70% in the surveyed fields (n=9). Sixty leaf samples of symptomatic R. glutinosa plants were collected from nine cultivation fields in Wenxian, Wuzhi, Mengzhou, and Yuzhou counties (five to seven plants for each field). Total RNA was extracted from one pooled sample containing a portion of all above-mentioned leaf samples using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China) and analyzed by high-throughput sequencing (HTS) to identify viral pathogens. A transcriptome library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), and sequenced on an Illumina NovaSeq6000 sequencing system at Berry Genomics Corporation (Beijing, China). A total of 27,664,949 high-quality clean reads were obtained after trimming and used for contig assembly. The assembled contigs (n=109,180) were searched using Basic Local Alignment Search Tool (BLAST) at GenBank. BLASTn analysis showed that the R. glutinosa plants were infected with known viruses, including broad bean wilt virus, rehmannia mosaic virus, youcai mosaic virus, and cucurbit chlorotic yellows virus. In addition, one contig (6,418 nt in length) had a nucleotide sequence identity of 99.64% with the TN29 isolate of tobacco mild green mosaic virus (TMGMV, GenBank accession no. MF139550). To confirm the presence of this virus, sixty above-mentioned samples were screened by reverse transcription-polymerase chain reaction (RT-PCR) using the specific primer pairs (Supplementary Table1) TMGMG-CPF/TMGMG-CPR targeting a 545-nt fragment within the CP gene. Amplicons with expected sizes were detected from 47 of 60 samples but not from the negative control (virus-free healthy plant through the tip meristem culture). Seventeen amplicons (11#, 13#, 14#, 21#, 22#, 23#, 25#, 26#, 27#, 31#, 32#, 33#, 37#, 52#, 57#, 59#, and 60#) of TMGMV-CP were selected, and purified. The PCR products were cloned into the pMD19-T vector (TAKARA Biotech, Dalian, China) and sequenced. The sequences were deposited into the GenBank (accession nos. MZ395944 to MZ395960). The near-full-length genomic sequence of TMGMV-Rg14 isolate was obtained from one positive sample (sample no. 14) by RT-PCR amplification of two overlapping fragments using the following primer pairs: TMGMV-40F/TMGMV-3570R and TMGMV-3220F/TMGMV-6400R. The near-full-length genomic sequence of the TMGMV-Rg14 isolate was 6 304 nucleotides (nt) in length and deposited into GenBank (accession no. MZ395975). BLASTn analysis demonstrated that the TMGMV-Rg14 isolate shared a sequence identity ranging from 96.89% (AB078435) to 99.60% (MF139550) with the other TMGMV isolates. Furthermore, the virus-free healthy R. glutinosa plants were inoculated with sap from the positive sample (14#) to confirm the infection of TMGMV. Mosaic symptoms were induced on the systemically infected leaves of the inoculated plants 14 days post inoculation. The systemically infected leaves of inoculated plants were assayed by RT-PCR using the primer pairs TMGMV-CPF/CPR. Amplicons of expected size were detected from the inoculated plants but not from non-inoculated plants. To our knowledge, this is the first report of TMGMV infection on R. glutinosa. Further studies are necessary to select a suitable indicator plant for this TMGMV, its host range, and the symptoms it induces in single infection. Since R. glutinosa is cultivated by vegetative propagation, production of virus-free healthy plants is necessary. This study will help to generate virus-free healthy plants and prevent viral disease on R. glutinosa. Further study is needed to determine its pathological implications and economic impact on R. glutinosa in China.

First Report of Youcai Mosaic Virus Infecting Yam in China.

Yam (Dioscorea opposita Thunb.) is cultivated mainly as a functional food and for nutritional and medicinal purposes in China (1). It is propagated through tubers and this facilitates the spread and accumulation of viruses in the crop, eventually leading to yield losses (2). At present, different virus species belonging to the genera Aureusvirus, Badnavirus, Carlavirus, Comovirus, Cucumovirus, Fabavirus, Macluravirus, Potexvirus and Potyvirus have been reported in yams (3) and fifteen viruses in these genera have been detected in China. In July 2020, a survey of viral diseases on yam was conducted in plantations of Wenxian and Mengzhou counties in Henan Province, China. Fifty-four leaf samples of Dioscorea opposite showing mosaic and leaf discoloration (Supplementary Fig1) were collected from eight fields (five to ten plants per field). These leaf samples were ground in liquid nitrogen and total RNA was extracted from a portion of the mixed powder using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China). A cDNA library was constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) after ribosomal RNA depletion using Ribo-off rRNA Depletion Kit (Vazyme Biotech, Nanjing, China), and sequenced on the Illumina NovaSeq 6000 system at the Berry Genomics Corporation (Beijing, China). A total of 87,075 contigs (>200 bp) were generated from de novo assembly (CLC Genomic Workbench 10.0) from a total of 34,656,172 paired-end reads. After BLASTn analysis, three contigs with the length of 1009, 1340 and 1859 nucleotides shared 96.33%, 96.72% and 96.29% nt identity respectively with youcai mosaic virus SX isolate, a tobamovirus (YoMV GenBank accession no. JX422022). In addition to YoMV, broad bean wild virus 2 and yam latent virus were also identified, which had previously been reported in yams in China. To confirm the NGS result, total RNAs were extracted from fifty-four above-mentioned samples and RT-PCR was carried out to amplify a 528 bp fragment of the coat protein (CP) of YoMV by using a pair of specific primers CP gene. PCR products with expected size were obtained from 26 out of 54 samples, and seventeen amplicons of YoMV-CP were sequenced (accession nos. ON052726 to ON052742). The nt sequence identities of CP gene among these seventeen isolates were 99.6%-100%. Furthermore, the near-full-length genomic sequence of YoMV-Do41 isolate was obtained from sample 41 by RT-PCR amplification of four overlapping fragments using the following primer pairs: YoMV-15F/YoMV-1910R, YoMV-1770F/YoMV-3750R, YoMV-3645F/YoMV-5404R and YoMV-4921F/YoMV-6280R (Supplementary Table1). The YoMV-Do41 isolate was 6, 274 nt in length (accession no. ON149803) and shared 89.65% and 97.31% nt identities to As1-2 isolate (GenBank accession no. MW307290) and to SX isolate (accession no. JX422022), respectively.To the best of our knowledge, this is the first report of YoMV infecting yam in China. YoMV has a wide host range including genera Impatiens, Rehmannia, Brassica, Chelidonium, Trifolium, Crossandro, Alstroemeria, Stellaria. This study will serve as an important reference for the host range of YoMV. According to the detection rate infections with YoMV in yam are common in these producing regions. Further studies will be required to determine the infection rate in other producing regions and the potential threat posed by YoMV on yam production should be considered.

First Report of Dasheen Mosaic Virus Infecting Typhonium giganteum Engl. (Baifuzi) in Henan Province of China.

Typhonium giganteum Engl. (Baifuzi ) is a perennial plant of the family Araceae. In China, its root is commonly used as an antispasmodic for stroke and cancer treatment (Chi et al. 2010; Gao et al. 2014; Khalivulla et al. 2019). Yuzhou city in Henan Province is the main producing area of T. giganteum Engl., and in July 2020, a survey of viral disease infecting T. giganteum Engl. was conducted in the city. In the surveyed fields (n =5), over 60% of plants displayed varying levels of virus-like symptoms, including mosaic, chlorotic and leaf distortion (Supplementary Figure S1) . To identify possible viral pathogens associated with the disease symptoms afflicting T. giganteum Engl., one leaf each from 25 symptomatic plants was collected and analyzed by high-throughput sequencing (HTS) as well as PCR. For HTS analysis, total RNA was extracted from one pooled sample containing a portion of all abovementioned leaves using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China). After removing ribosomal RNA with Ribo-off rRNA depletion kit (Vazyme Biotech, Nanjing, China), a sequencing library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) and sequenced on an Illumina Novaseq6000 sequencing system at Berry Genomics Corporation (Beijing, China). A total of 6,899,143 high-quality clean reads were obtained after trimming and used for contig assembly. BLASTn and BLASTx analyses on the contigs (n = 128,400) showed that one contig (9,245 bp in length) exhibited a sequence identity of 84.0% with the reference sequence of dasheen mosaic virus (DsMV, NCBI reference seq. NC_003537, genus Potyvirus, family Potyviridae) , suggesting infection of the plants by DsMV. No other viral sequences were detected in the sample. To confirm these results, a near full-length genomic sequence of DsMV was obtained from one sample (sample no. 39) by reverse transcription polymerase chain reaction (RT-PCR) of three overlapping fragments with the following primer pairs: DsMV-1F (5'-AAATTAAAACATCTCAACAAAACCTACA-3') /DsMV-4130R (5'-TTCATGGTCCTCGTGGAGTATA-3'), DsMV-3870F (5'-GAGGACGTGAGAATTCAAAGTCT-3')/DsMV-8250R (5'-GTCCAACCTTGCTTGATGCATGC-3'), DsMV-7690F (5'-GGAGCGACTCCTCTTCCAAAGTTGTG-3')/DsMV-10100R (5'-TGAACACCGTGCACGAAGCATCTC-3'). The PCR products were cloned into pMD19-T vector (TAKARA Biotech, Dalian, China) and sequenced. The near full-length genomic sequence of the isolate (DsMV-BF39) was 9,737 nt in length and deposited into GenBank under the accession no. MZ043618. BLASTn analysis of this sequence demonstrated that it shared an identity ranging from 78.6% (MG602234) to 85.6% (MG602227) with various DsMV isolates. To determine whether DsMV was closely associated with the symptoms observed in T. giganteum Engl., leaf tissues from 30 symptomatic plants and 22 asymptomatic plants were analyzed by RT-PCR using primer pairs DsMV-CPF (5'-TGTTCTGTGAACATGATGAAGTTG-3', sense) and DsMV-CPR (5'-GTAACTGTGGCCTGTTTACCAG-3', antisense) targeting a 916 bp fragment of the CP gene of DsMV. Amplicons with the expected size were detected from the 30 symptomatic plants but not from the 22 asymptomatic plants, suggesting a close association between DsMV infection and the observed symptoms. To our knowledge, this is the first report of DsMV infecting T. giganteum Engl.. Further study is needed to identify the specific symptoms induced by this virus in T. giganteum Engl. and to understand the biological characteristics, epidemiology, prevalence of this virus in China.

[Analysis of gene variant in a Chinese pedigree with preaxial polydactyly].

OBJECTIVE: To analyze the pathogenic variant of preaxial polydactyly in a Chinese Han pedigree and identify the cause of polydactyly. METHODS: The peripheral blood DNA of the proband and her parents was extracted. The polydactyly-related genes were detected by trio whole exome sequencing, and the suspected pathogenic gene was screened out. Sanger sequencing was applied to other members of the pedigree. RESULTS: The results of gene sequencing showed that the LMBR1 gene had a heterozygous variant of c.423+4909(IVS5)C>T in 6 patients of the pedigree. The same variant was not detected in family members with normal phenotype. Based on the ACMG guidelines, c.423+4909(IVS5)C>T of the LMBR1 gene was predicted to be pathogenic (PM1+PM2+PP1-S(PS)+PP4+PP5). CONCLUSION: The heterozygous C>T variant at position 4909 of intron 5 of the LMBR1 gene probably underlies the disease in this pedigree.

Characteristics and complete genome sequence of the virulent Vibrio alginolyticus phage VAP7, isolated in Hainan, China.

A novel Vibrio alginolyticus phage, VAP7, was isolated from seawater collected from Sanya, Hainan province, China. Whole-genome sequencing analysis revealed that phage VAP7 has a linear, double-stranded DNA genome of 144,685 bp with an average G+C content of 41.9% and a high degree of sequence similarity to Vibrio phage VP-1. Annotation results identified 193 open reading frames and one transfer RNA-encoding gene in the phage genome. The morphology and the results of phylogenetic analysis suggest that VAP7 should be classified as a new member of the family Ackermannviridae. Moreover, phage VAP7 grew over a wide pH (5.0-10.0) and temperature (4-40 °C) range. Host-range experiments revealed that VAP7 could infect 31 Vibrio alginolyticus strains. Thus, VAP7 infecting Vibrio alginolyticus strains represents a potential new candidate for use in phage therapy.

Enhancement in Particulate Organic Nitrogen and Light Absorption of Humic-Like Substances over Tibetan Plateau Due to Long-Range Transported Biomass Burning Emissions.

To elucidate the influence of long-range transported biomass burning organic aerosols (BBOA) on the Tibetan Plateau, the molecular compositions and light absorption of HUmic-Like Substances (HULIS), major fractions of brown carbon, were characterized during the premonsoon season. Under the significant influence of biomass burning, HULIS concentrations increased to as high as 26 times of the background levels, accounting for 54% of water-soluble organic carbon (WSOC) and 50% of organic carbon (OC). The light absorption of HULIS also enhanced up to 42 times of the background levels, contributing 61% of the WSOC absorption and 50% of OC absorption. Meanwhile, elevated nitrogen-containing compounds (NOCs) among HULIS were observed. The NOCs from fresh and aged BBOA were unambiguously identified on the molecular level, through comparing with the molecular compositions of NOCs from lab-controlled and field burning experiments. N-Heterocyclic bases represent major fractions in the reduced nitrogen compounds from fresh BBOA, and nitroaromatic compounds are important groups among the oxidized nitrogen compounds from aged BBOA. The nitrogen-containing compounds, including nitroaromatics and N-heterocyclic compounds, were also important chromophores, which contributed to the enhanced light absorption of extracted HULIS during biomass burning-influenced periods.

Gender Differences in Vitamin D Status in China.

BACKGROUND Vitamin D insufficiency is widespread in China. Various factors influence vitamin D level in the body. The present study investigated vitamin D status of residents in Jinzhong city, China, and analyzed the influence of gender on vitamin D status. MATERIAL AND METHODS In this cross-sectional study, 302 participants (176 men and 126 women) were recruited. Anthropometric data (body circumferences and height, weight) were collected, and serum vitamin D concentration was tested. RESULTS Inadequate levels of vitamin D were found in 69% of men and 75% of women. Women's 25(OH)D level (38.40±12.37 nmol/l) was substantially lower than that of the men (43.49±14.78 nmol/l) (p<0.01). The young women group had the lowest vitamin D level, which was even significantly below that of the elderly women group. Multiple linear regression analysis showed gender was significantly associated with vitamin D status (p<0.01). CONCLUSIONS Vitamin D deficiency is common in residents of Jinzhong during the winter. Compared to men, women are more prone to have inadequate vitamin D levels.

A Ratiometric and Colorimetric Hemicyanine Fluorescent Probe for Detection of SO2 Derivatives and Its Applications in Bioimaging.

Based upon the intramolecular charge transfer (ICT) mechanism, a novel ratiometric fluorescent probe EB was developed to detect SO32-/HSO3-. The probe displayed both colorimetric and ratiometric responses toward SO32-/HSO3-. It displayed a quick response (within 60 s), good selectivity and high sensitivity (a detection limit of 28 nM) towards SO32-/HSO3-. The SO32-/HSO3- sensing mechanism was confirmed as the Michael addition reaction by ESI-MS. Moreover, the probe could be applied to measure the level of sulfite in real samples, like sugar and chrysanthemum, and it could also be used to detect SO32-/HSO3- in HepG2 cells through confocal fluorescence microscopy, which proved its practical application in clinical diagnosis.

Several localized waves induced by linear interference between a nonlinear plane wave and bright solitons.

We investigate linear interference effects between a nonlinear plane wave and bright solitons, which are admitted by a pair-transition coupled two-component Bose-Einstein condensate. We demonstrate that the interference effects can induce several localized waves possessing distinctive wave structures, mainly including anti-dark solitons, W-shaped solitons, multi-peak solitons, Kuznetsov-Ma like breathers, and multi-peak breathers. Specifically, the explicit conditions for them are clarified by a phase diagram based on the linear interference properties. Furthermore, the interactions between these localized waves are discussed. The detailed analysis indicates that the soliton-soliton interaction induced phase shift brings the collision between these localized waves which can be inelastic for solitons involving collision and can be elastic for breathers. These characters come from the fact that the profile of solitons depends on the relative phase between bright solitons and a plane wave, and the profile of breathers does not depend on the relative phase. These results would motivate more discussions on linear interference between other nonlinear waves. Specifically, the solitons or breathers obtained here are not related to modulational instability. The underlying reasons are discussed in detail. In addition, possibilities to observe these localized waves are discussed in a two species Bose-Einstein condensate.

Potential of secondary aerosol formation from Chinese gasoline engine exhaust.

Light-duty gasoline vehicles have drawn public attention in China due to their significant primary emissions of particulate matter and volatile organic compounds (VOCs). However, little information on secondary aerosol formation from exhaust for Chinese vehicles and fuel conditions is available. In this study, chamber experiments were conducted to quantify the potential of secondary aerosol formation from the exhaust of a port fuel injection gasoline engine. The engine and fuel used are common in the Chinese market, and the fuel satisfies the China V gasoline fuel standard. Substantial secondary aerosol formation was observed during a 4-5hr simulation, which was estimated to represent more than 10days of equivalent atmospheric photo-oxidation in Beijing. As a consequence, the extreme case secondary organic aerosol (SOA) production was 426±85mg/kg-fuel, with high levels of precursors and OH exposure. The low hygroscopicity of the aerosols formed inside the chamber suggests that SOA was the dominant chemical composition. Fourteen percent of SOA measured in the chamber experiments could be explained through the oxidation of speciated single-ring aromatics. Unspeciated precursors, such as intermediate-volatility organic compounds and semi-volatile organic compounds, might be significant for SOA formation from gasoline VOCs. We concluded that reductions of emissions of aerosol precursor gases from vehicles are essential to mediate pollution in China.

The effect of nursing participation in the design of a critical care information system: a case study in a Chinese hospital.

BACKGROUND: Intensive care information systems (ICIS) are continuously evolving to meet the ever changing information needs of intensive care units (ICUs), providing the backbone for a safe, intelligent and efficient patient care environment. Although beneficial for the international advancement in building smart environments to transform ICU services, knowledge about the contemporary development of ICIS worldwide, their usage and impacts is limited. This study aimed to fill this knowledge gap by researching the development and implementation of an ICIS in a Chinese hospital, nurses' use of the system, and the impact of system use on critical care nursing processes and outcomes. METHODS: This descriptive case study was conducted in a 14-bed Respiratory ICU in a tertiary hospital in Beijing. Participative design was the method used for ICU nurses, hospital IT department and a software company to collaboratively research and develop the ICIS. Focus group discussions were conducted to understand the subjective perceptions of the nurses toward the ICIS. Nursing documentation time and quality were compared before and after system implementation. ICU nursing performance was extracted from the annual nursing performance data collected by the hospital. RESULTS: A participative design process was followed by the nurses in the ICU, the hospital IT staff and the software engineers in the company to develop and implement a highly useful ICIS. Nursing documentation was fully digitized and was significantly improved in quality and efficiency. The wrong data, missing data items and calculation errors were significantly reduced. Nurses spent more time on direct patient care after the introduction of the ICIS. The accuracy and efficiency of medication administration was also improved. The outcome was improvement in ward nursing performance as measured by ward management, routine nursing practices, disinfection and isolation, infection rate and mortality rate. CONCLUSIONS: Nurses in this ICU unit in China actively participated in the ICIS development and fully used the system to document care. Introduction of the ICIS led to significant improvement in quality and efficiency in nursing documentation, medication order transcription and administration. It allowed nurses to spend more time with patients to improve quality of care. These led to improvement in overall nursing performance. Further study should investigate how the ICIS system contributes to the improvement in decision making of ICU nurses and intensivists.

Diversity of Sweepoviruses Infecting Sweet Potato in China.

Sweepoviruses (a group of begomoviruses that infect plants in the family Convolvulaceae) have monopartite genomes that consist of a circular, single-stranded DNA molecule. Seventy-three complete genomic sequences of sweepoviruses were characterized from the sweet potato samples collected in China. Eight sweepovirus species, including two novel species with proposed names of Sweet potato leaf curl China virus 2 and Sweet potato leaf curl Sichuan virus 2, were identified among these samples. One species, Sweet potato leaf curl Canary virus, was first identified in China. Among the 13 identified strains of Chinese sweepoviruses, 4 were newly discovered. Sweet potato leaf curl virus had the highest frequency (53.4%) of occurrence in the sweet potato samples from China. The similarities among the 73 sweepovirus genomic sequences were between 77.6 and 100.0%. Multiple recombination events were identified, and 16 recombinant sequences were determined. Recombination was observed between different species and between different strains of the same species. Recombination breakpoints were mainly localized on the intergenic region and in three open reading frames (AC1, AV1, and AV2). This study is the first comprehensive report on the genetic diversity of sweepoviruses in China.

Complete genome sequence of a novel monopartite begomovirus infecting sweet potato in China.

The complete genome sequence of a novel monopartite begomovirus, isolate G-YU-12-10, was obtained from sweet potato samples exhibiting severe leaf curl symptoms in Xinxiang, Henan Province, China. The genome sequence consisted of 2766 nucleotides and encoded two open reading frames (ORFs) (AV1 and AV2) in the viral-sense strand and four ORFs (AC1-AC4) in the complementary-sense strand. The genome of isolate G-YU-12-10 was closely related to other sweet-potato-infecting begomoviruses (sweepoviruses) and shared the highest nucleotide sequence identity (89.0 %) with sweet potato leaf curl China Sichuan virus (SPLCCSV, KC488316). Thus, the G-YU-12-10 isolate represents a novel species according to the demarcation criteria of species in the genus Begomovirus, for which the name Sweet potato leaf curl Henan virus (SPLCHnV) is proposed. Interspecific recombination analysis supported the recombination hypothesis, indicating that recombination with other begomoviruses had taken place within AC2 and AC3 ORFs of SPLCHnV and also in the non-coding intergenic region (IR).