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The objective of this study was to comparative evaluate the protective efficacy of live attenuated and inactivated porcine reproductive and respiratory syndrome vaccines. Fifty-five healthy piglets free of PRRSV antigen and antibody were randomly assigned into four groups. Piglets of group 1 (n = 20) were inoculated with PRRS live vaccine TJM-F92 strain (Live vaccine TJM-F92). Piglets of group 2 (n = 20) were inoculated with commercial PRRS inactivated vaccine (Inactivated vaccine). Piglets of group 3 (n = 10) were inoculated with PBS (Negative control). Group 4 (n = 5) remained as a not-immunized and not-challenged control (Mock group). And half of piglets from groups 1 and 2 were randomly selected and challenged with HP-PRRSV TJ strain and PRRSV NADC30-like strain respectively at 49 days post immunization (dpi). Our data showed the level of antibodies arose from live vaccine was significantly higher and faster than that from inactivated vaccine after immunization. Neither high fever nor signs of clinical disease were observed in piglets immunized with live vaccine, whereas piglets in inactivated vaccine group exhibited serious clinical symptoms, pathological lesions, and viremia load after HP-PRRSV TJ strain challenge. No obvious clinical signs, gross pathology lesions and viremia load were found in live vaccine group, while severe clinical signs, pathology change of lungs and high viremia load were recorded in inactivated vaccine group after NADC30-like strain challenge. The results indicated that PRRS live vaccine (TJM-F92 strain) could effectively prevent the infection of HP-PRRSV TJ strain and virulent NADC30-like strain. Immunization with PRRS inactivated vaccine could not effectively arouse immune system and provide strong immune cross-protection. Therefore, it is necessary to select suitable, widely application and effective PRRS vaccine for PRRS control according to the situation of pig farms.

Context Previous studies indicate that compound Danshen Dripping Pill (CDDP) improves the adaptation to high-altitude exposure. However, its mechanism of action is not clear. Objective To explore the protective effect of CDDP on hypobaric hypoxia (HH) and its possible mechanism. Materials and methods A meta-analysis of 1051 human volunteers was performed to evaluate the effectiveness of CDDP at high altitudes. Male Sprague-Dawley rats were randomized into 5 groups (n = 6): control at normal pressure, model, CDDP-170 mg/kg, CDDP-340 mg/kg and acetazolamide groups. HH was simulated at an altitude of 5500 m for 24 h. Animal blood was collected for arterial blood-gas analysis and cytokines detection and their organs were harvested for pathological examination. Expression levels of AQP1, NF-κB and Nrf2 were determined by immunohistochemical staining. Results The meta-analysis data indicated that the ratio between the combined RR of the total effective rate and the 95% CI was 0.23 (0.06, 0.91), the SMD and 95% CI of SO2 was 0.37 (0.12, 0.62). Pre-treatment of CDDP protected rats from HH-induced pulmonary edoema and heart injury, left-shifted oxygen-dissociation curve and decreased P50 (30.25 ± 3.72 vs. 37.23 ± 4.30). Mechanistically, CDDP alleviated HH-reinforced ROS by improving SOD and GPX1 while inhibiting pro-inflammatory cytokines and NF-κB expression. CDDP also decreased HH-evoked D-dimer, erythrocyte aggregation and blood hemorheology, promoting AQP1 and Nrf2 expression. Discussion and conclusions Pre-treatment with CDDP could prevent HH-induced tissue damage, oxidative stress and inflammatory response. Suppressed NF-κB and up-regulated Nrf2 might play significant roles in the mechanism of CDDP.

Arbitrarily-oriented object detection is a challenging task. Since the object orientation in remote sensing images is arbitrary, using horizontal bounding boxes will lead to low detection accuracy. Existing regression-based rotation detectors can lead to the problem of boundary discontinuity. In this paper, we propose a remote sensing image object detection method based on angle classification that uses rotation detection bounding boxes with angle information to detect objects. Specifically, we incorporate the neural architecture search framework with feature pyramid network (NAS-FPN) module in a dense detector (RetinaNet) and use a binary encoding method in angle classification. This method reduces the background influence, so that there is almost no overlap between detection boxes. Based on the angles of the detection boxes, we can infer the information of the motion direction of the target and further determine the motion trajectory of the target. We conducted ablation experiments on a large publicly available for object detection in an aerial imagery (DOTA) to verify the effectiveness of each module in the method and compared the method with several other detection methods. The experimental results demonstrate the effectiveness of our method.

Canine parvovirus type 2 (CPV-2) is currently circulating in domestic and wild animals, but our knowledge about CPV-2 infections in raccoon dogs is limited. In this study, VP2 gene sequences of CPV-2 were amplified from rectal swabs of 14 diarrhetic raccoon dogs (Nyctereutes procyonoides) in Hebei province, China, in 2016 and 2017. Phylogenetic analysis of the VP2 gene sequences revealed that most of these sequences (11 of 14) belonged to the same subclade as raccoon dog strain CPV-2/Raccoon_Dog/China/DP-1/16 isolated from Shandong province in 2016. A comparison of deduced amino acid sequences revealed presence of the substitutions S297A and S27T in 11 of those 14 sequences. I418T was observed in a minority of the sequences (4 of 14). In addition, A300D and T301I, P13S and I219V, and N419K were found in three of the sequences. This study shows that CPV-2 strains with different substitutions in their VP2 amino acid sequences were spreading among raccoon dogs in Hebei during 2016 and 2017 and suggests that further studies are needed to monitor the distribution of these strains in China.

Getah virus (GETV) is a mosquito-borne alphavirus that is considered to be an emerging pathogen. To date, reverse transcription loop-mediated isothermal amplification (RT-LAMP) has not been used to detect GETV. Therefore, we describe a novel, fast, and sensitive LAMP method to detect GETV. Amplification of GETV RNA can be obtained within 50 min at 65°C. This RT-LAMP method was verified to be highly specific for GETV, with no cross detection of other viruses. The assay was 10

The molecular epidemiology and biological characteristics of Escherichia coli associated with hemorrhagic pneumonia (HP) mink from five Chinese Provinces were determined. From 2017 to 2019, 85 E. coli strains were identified from 115 lung samples of mink suffering from HP. These samples were subjected to serotyping, antimicrobial susceptibility, detection of virulence genes, phylogenetic grouping, whole-genome sequencing, drug resistant gene, multilocus sequence typing (MLST) and biofilm-forming assays. E. coli strains were divided into 18 serotypes. Thirty-nine E. coli strains belonged to the O11 serotype. Eighty-five E. coli strains were classified into seven phylogenetic groups: E (45.9%, 39/85), A (27.1%, 23/85), B1 (14.1%, 12/85), B2 (3.7%, 3/85), D (3.7%, 3/85), F (2.4%, 2/85) and clade I (1.2%, 1/85). MLST showed that the main sequence types (STs) were ST457 (27/66), All E. coli strains had ≥4 virulence genes. The prevalence of virulence was 98.8% for yijp and fimC, 96.5% for iucD, 95.3% for ompA, 91.8% for cnf-Ⅰ, 89.4% for mat, 82.3% for hlyF, and 81.2% for ibeB. The prevalence of virulence genes iss, cva/cvi, aatA, ibeA, vat, hlyF, and STa was 3.5–57.6%. All E. coli strains were sensitive to sulfamethoxazole, but high resistance was shown to tetracycline (76.5%), chloramphenicol (71.8%), ciprofloxacin (63.5%) and florfenicol (52.9%), resistance to other antibiotics was 35.3–16.5%. The types and ratios of drug-resistance genes were tet(A), strA, strB, sul2, oqxA, blaTEM-1B, floR, and catA1 had the highest frequency from 34%-65%, which were consistent with our drug resistance phenotype tetracycline, florfenicol, quinolones, chloramphenicol, the bla-NDM-I and mcr-I were presented in ST457 strains. Out of 85 E. coli strains, six (7.1%) possessed a strong ability, 12 (14.1%) possessed a moderate ability, and 64 (75.3%) showed a weak ability to form biofilm. Our data will aid understanding of the epidemiological background and provide a clinical basis for HP treatment in mink caused by E. coli.

Heart failure (HF) is the advanced heart disease with high morbidity and mortality. Compound DanShen Dripping Pill (CDDP) is a widely used Traditional Chinese Medicine for cardiovascular disease treatment. Herein, we investigated if CDDP can protect mice against doxorubicin (DOX) or isoprenaline (ISO)-induced HF. After 3 days feeding of normal chow containing CDDP, mice were started DOX or ISO treatment for 4 weeks or 18 days. At the end of treatment, mice were conducted electrocardiogram and echocardiographic test. Blood and heart samples were determined biochemical parameters, myocardial structure and expression of the related molecules. CDDP normalized DOX/ISO-induced heart weight changes, HF parameters and fibrogenesis. The DOX/ISO-impaired left ventricular ejection fraction and fractional shortening were restored by CDDP. Mechanistically, CDDP blocked DOX/ISO-inhibited expression of antioxidant enzymes and DOX/ISO-induced expression of pro-fibrotic molecules, inflammation and cell apoptosis. Additional DOX/ISO-impaired targets in cardiac function but protected by CDDP were identified by RNAseq, qRT-PCR and Western blot. In addition, CDDP protected cardiomyocytes against oxygen-glucose deprivation-induced injuries. Taken together, our study shows that CDDP can protect against myocardial injuries in different models, suggesting its potential application for HF treatment.

Benefiting from the development of network pharmacology, Traditional Chinese Medicine (TCM) shows great potential in modern drug discovery. Recently, more and more TCM-related databases have been established for both academic and industry research, but they are still insufficient in data standardization, integrity, and precision. To better accelerate the TCM research and overcome these shortcomings, we construct a web-based TCM platform, LTM-TCM, which is currently the most comprehensive TCM database that includes the following advantages: (1) High-quality data integration from fourteen TCM authoritative databases, especially with additional manual collected 41,025 clinical treatment records and 213 ancient Chinese medical books. (2) Accurate correction of multi-source TCM interactions (between symptoms, prescriptions, herbs, ingredients and targets) through in-house Biomedical Natural Language Processing (BioNLP) approaches in more than 30 million articles. (3) Diverse cross-field pipelines (e.g., bioactive ingredients screening, targets prediction, and mechanism prediction, etc.) help integrating traditional medicine with modern science in common aspects at both the molecular and phenotypic levels. In summary, LTM-TCM contains 1928 symptoms, 48,126 prescriptions, 9122 plants, 34,967 ingredients, 13,109 targets and 1170,133 interactions among all TCM related components. LTM-TCM has both Chinese and English interfaces, and it is accessible at http://cloud.tasly.com/#/tcm/home.

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.以国内商品化水貂犬瘟热病毒疫苗所用毒株CDV-3为模板，构建犬瘟热病毒感染性cDNA克隆，为犬瘟热病毒新型疫苗研制、致病机理研究提供理论基础。设计13对引物对其全基因组序列测定，分析单一酶切位点，将CDV-3的全长分5个片段进行RT-PCR扩增。经酶切拼接，将5个片段顺次插入到酶切位点改造后的真核载体pcDNA3.2的多克隆酶切位点处，同时在F1首端和F5末端分别加入锤头状核酶和丁型肝炎核酶序列，获得CDV-3株的全长cDNA质粒 (pcDNA3.2-CDV-3)。构建表达CDV-3 N、P、L蛋白的3个辅助质粒。利用转染试剂Lipofectamine™ 2000将全长质粒和3个辅助质粒共转染293T细胞，3 d后，将上清接种到Vero细胞。观察犬瘟热病毒典型合胞体病变，对重组病毒进行免疫荧光鉴定和标签鉴定。最后，比较wtCDV-3和rCDV-3的生长特性。全长质粒和辅助质粒的酶切鉴定和序列测序均正确。拯救的重组病毒能在Vero细胞上形成典型的合胞体病变，经RT-PCR、间接免疫荧光和电镜观察鉴定，证明成功拯救出重组病毒rCDV-3株。rCDV-3的病毒滴度最高达到10⁷·⁶⁶⁷ TCID₅₀/mL，比wtCDV-3的滴度10⁶·⁶⁶⁷ TCID₅₀/mL高出约10倍。rCDV-3感染Vero细胞后，迅速大量增殖，于感染后36 h达到最高病毒滴度。而wtCDV-3增殖平缓，感染后72 h时病毒含量达到最大。文中建立的高效CDV-3株反向遗传操作平台，为犬瘟热病毒新型疫苗研制和致病机理研究奠定基础。.

Canine adenovirus type 1 (CAdV-1) is the etiologic agent of fox encephalitis, and a virus strain from fox encephalitis is isolated and related researches are conducted. In this experiment, the results showed that the F1301 strain was confirmed to be the CAdV-1. The whole genome of the CAdV-1 F1301 strain isolated from fox was 30535 bp and had higher homology to the other reported CAdV-1 strains. After 0 h, 12 h, and 36 h of CAdV-1 infection, the difference gene of the 592 LncRNA and 11215 mRNA were involved in cell responses to CAdV-1 infection through the PI3K-AKT, Wnt, Herpes simplex, Hepatitis C, and Epstein-Barr virus infection pathway in MDCK. The results indicate that the biological characterization of the CAdV-1 and the MDCK cell-CAdV-1 interaction are clarified. This article is protected by copyright. All rights reserved.

Due to their susceptibility to several human viruses, the mink has been proposed as potential animal models for the study of human viral infections. However, there are no specific monoclonal antibody (mAbs) currently available for the detection of mink-specific interferon-gamma (miIFN-γ). The BALB/c mice were immunized intraperitoneally with purified recombinant miIFN-γ protein. The splenocytes were obtained and fused with murine myeloma cells. Five of 24 hybridoma clones were obtained to produce mAbs steadily with the strongest affinity to recombinant miIFN-γ protein. The isotype of the 31A, 31B and 31G were lgG 2b. The isotype of 44 and 46 were lgG 2a and 1. All five mAbs were κ light chains. Western blotting and indirect ELISA method showed that 5 mAbs were positive to miIFN-γ. Immunofluorescence showed that 2 mAbs (44 and 46) had a positive reaction to miIFN-γ. The hybridoma clone 46 had the highest sensitivity for the detection of miIFN-γ. Most importantly, our primary sandwich ELISA system (mAbs 46 and polyclonal antiserum) detected endogenous IFN-γ in mink lymphocytes infected with canine distemper virus (CDV). We have thus developed a novel mAbs could recognize miIFN-γ, and have demonstrated the first ELISA-based measurement of IFN-γ in lymphocyte of the mink.

Acute kidney injury (AKI) is often accompanied by inflammation. Echinacea polysaccharide (EP) is an active ingredient that has been demonstrated to possess anti‑oxidative, anti‑inflammatory, antimicrobial and immunomodulatory functions. However, the role of EP in AKI has not been examined. The present study investigated the effects of EP on lipopolysaccharide (LPS)‑induced AKI. Western blotting, immunohistochemistry and immunofluorescence analyses were performed to detect protein expression levels. Administration of EP significantly attenuated LPS‑induced renal tissue injury, along with a decrease in blood urea nitrogen and creatinine levels. EP decreased the levels of inducible nitric oxide synthase and cyclo‑oxygenase‑2 in LPS‑treated mice. Furthermore, LPS‑induced inflammation was inhibited by EP in renal tissues and HBZY‑1 cells, as demonstrated by the downregulation of tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6, nitric oxide and prostaglandin E2 levels. Similarly, EP administration decreased oxidative stress (OS) via decreasing reactive oxygen species, malondialdehyde and oxidized glutathione levels, and increasing superoxide dismutase, catalase, glutathione reductase and reduced glutathione activity. Notably, EP induced a marked decrease in the expression levels of phospho‑extracellular signal‑regulated protein kinase (p‑ERK), phospho‑c‑Jun N‑terminal kinase (p‑JNK) and p‑p38 in vivo and in vitro. In addition, in LPS‑treated HBZY‑1 cells, EP enhanced cell viability and inhibited nuclear translocation of p‑ERK, p‑JNK and p‑p38. Overall, the present findings demonstrated that EP alleviated LPS‑induced AKI via the suppression of inflammation, OS and the mitogen‑activated protein kinase signaling pathway, providing insight into potential avenues for the treatment of AKI.

The aim of this study was to establish a multiplex PCR (mPCR) method that can simultaneously detect canine parvovirus (CPV-2), canine coronavirus (CCoV) and canine adenovirus (CAV), thereby eliminating the need to detect these pathogens individually. Based on conserved regions in the genomes of these three viruses, the VP2 gene of CPV-2, the endoribonuclease nsp15 gene of CCoV, and the 52K gene of CAV were selected for primer design. The specificity of the mPCR results showed no amplification of canine distemper virus (CDV), canine parainfluenza virus (CPIV), or pseudorabies virus (PRV), indicating that the method had good specificity. A sensitivity test showed that the detection limit of the mPCR method was 1 × 104 viral copies. A total of 63 rectal swabs from dogs with diarrheal symptoms were evaluated using mPCR and routine PCR. The ratio of positive samples to total samples for CPV-2, CCoV, and CAV was 55.6% (35/63) for mPCR and 55.6% (35/63) for routine PCR. Thirty-five positive samples were detected by both methods, for a coincidence ratio of 100%. This mPCR method can simultaneously detect CCoV (CCoV-II), CAV (CAV-1, CAV-2) and CPV-2 (CPV-2a, CPV-2b, CPV-2c), which are associated with viral enteritis, thereby providing an efficient, inexpensive, specific, and accurate new tool for clinical diagnosis and laboratory epidemiological investigations.

We isolated Getah virus from infected foxes in Shandong Province, eastern China. We sequenced the complete Getah virus genome, and phylogenetic analysis revealed a close relationship with a highly pathogenic swine epidemic strain in China. Epidemiologic investigation showed that pigs might play a pivotal role in disease transmission to foxes.

The root of Panax notoginseng is a famous traditional Chinese medicine used to stop bleeding, and to decrease inflammation and heart disease, in which saponins are the major active components. The current methods for saponins quantification are unsatisfactory to separate multi-components in short time. An improved, simple, and reliable HPLC method with potential for wide use was developed to simultaneously quantify five major saponins extracted from P. notoginseng. Five saponins, including ginsenoside Rg1, Re, Rb1, Rd and notoginsenoside R1 were well separated within 54 min. The best program for saponins extraction was that 0.2 g samples was soaked by 8 ml 70% methanol with overnight immersion, and extracted by ultrasonic bath for 1 h. P. notoginseng had been cultivated hundreds years as an industrial crop in China and the accumulative characteristics of five main saponins were first reported in this study. During three growing seasons, root was always the main organ to accumulate saponins when compared with aboveground part. During three growing seasons, the total content of five saponins was above 5% in the root of P. notoginseng cultivating more than one year. Among the accumulation of five saponins, ginsenoside Rg1 and Rb1 in root had higher content, and occupied larger proportion of total. September in the third year was the best harvest time due to the highest content of five major saponins in roots at that time. Besides, removing the flower bud in the third year would significantly increase the content of saponins in root of P. notoginseng.

Mink enteritis virus (MEV) is a major pathogen inducing acute hemorrhagic enteritis in mink. This study aims to determine the pathogenicity of the isolated MEV strain (SMPV-11) compared with the attenuated MEV strain (MEV-F61) in the mink. The two MEV strains were inoculated in the two mink groups, respectively. Then the clinical symptom, hematological, serological, and histopathological change were evaluated. Our findings showed that there were differences in the clinical features and pathological changes of the SMPV-11 and MEV-F61 in the mink. It indicates that SMPV-11 is a virulent strain, and it can be the potential MEV vaccine strain in the mink.

An outbreak of severe pseudorabies virus (PRV) infection in farmed mink occurred in northern China in late 2014, causing significant economic losses in the local fur industry. Here, we report the first case of a PRV outbreak in mink in northeastern China, caused by feeding farmed mink with raw pork or organs contaminated by PRV. Mink infected with virulent PRV exhibited diarrhea, neurologic signs, and higher mortality, which can be misdiagnosed as highly pathogenic mink enteritis virus (MEV), canine distemper virus (CDV), and food poisoning. However, these were excluded as causative agents by PCR or bacteria isolation. The duration of disease was 3-7 days, and the mortality rate was 80-90%. PRV was characterized using indirect immunofluorescence assays (IFA) and electron microscopy (EM). Phylogenetic analysis based on full-length genome sequences and those of individual genes of this novel virus strain showed that it clustered in an independent branch with several other PRV isolates from China.

Among Panax genus, only three endangered species Panax notoginseng, P. vietnamensis, and P. stipuleanatus that have a similar morphology are mainly distributed in Southeast Asia. These three plants are usually misidentified or adulterated. To identify them well, their chemical chromatographic fingerprints were established by an effective high-performance liquid chromatography method. By comparing the chromatograms, the three Panax species could be distinguished easily using the 22 characteristic peaks. Besides, the data of the chromatographic fingerprints aided by chemometric approaches were applied for the identification and investigation the relationship of different samples and species. Using similarity analysis, the chemical components revealed higher similarity between P. vietnamensis and P. stipuleanatus. The results of hierarchical clustering analysis indicated that samples belonging to the same species could be clustered together. The result of principal component analysis was similar with hierarchical clustering analysis and the three principal components accounted for >80.5% of total variability.

Salvia miltiorrhiza, a traditional Chinese medicinal herb, contains two groups of bioactive components: lipid-soluble tanshinones and water-soluble phenolic acids. Many researchers have recently focused on elicitation of secondary metabolites to improve their production. In this paper, folic acid was used as an elicitor to stimulate phenolics production in S. miltiorrhiza hairy root cultures. The results showed that folic acid use was effective for eliciting rosmarinic acid (RA), salvianolic acid B (SAB), caffeic acid (CA), and total phenolics from S. miltiorrhiza hairy roots and that 50 μM is the most effective folic acid concentration for elicitation. Roots treated with 50 μM folic acid showed 97.6, 76.7, and 47 % increases in RA, SAB, and CA contents, respectively, relative to the control. The expression of three key genes involved in SAB biosynthesis (i.e., PAL, TAT, and RAS) were upregulated by folic acid. In conclusion, we report for the first time that folic acid is an effective elicitor of phenolics production in S. miltiorrhiza hairy roots. Our results suggest that folic acid is a potential elicitor that may improve phenolic acid production in medicinal plants.

In order to evaluate whether the two wild species, Panax vietnamensis (from Vietnam) and Panax stipuleanatus (from primeval forest, Yunan Province) could markedly increase the genetic diversity of cultivated Panax notoginseng (Wenshan, Yunnan Province), both start codon targeted (SCoT) markers and internal transcribed spacer (ITS) DNA barcode were firstly employed in this genus. A total of 173 amplification bands were generated by 16 selected SCoT primers, in which 153 (89.5%) were polymorphic. Nei's gene-diversity indicated that the genetic diversity of three species (h = 0.16 and I = 0.27) was obviously higher than that of P. notoginseng (h = 0.09). Similarly, 38 different ITS sites out of 639 (5.9%) were detected among three species, but only one was different within 22 samples of P. notoginseng. Analysis of molecular variance (AMOVA) showed a greater proportion of genetic diversity existed within (61.3%) rather than among (38.7%) groups at genus level. In addition, P. vietnamensis had a closer relationship with P. notoginseng than P. stipuleanatus. These results would be significant for increasing the genetic diversity of P. notoginseng population by hybridization with P. vietnamensis and P. stipuleanatus, thus obtaining more varieties for future cultivar breeding and germplasm resources management.

Background Blind and excessive application of fertilizers was found during the cultivation of Panax notoginseng in fields, as well as increase in root rot disease incidence. Methods Both "3414" application and orthogonal test designs were performed at Shilin county, Yunnan province, China, for NPK (nitrogen, phosphorus, and potassium) and mineral fertilizers, respectively. The data were used to construct the one-, two-, and three-factor quadratic regression models. The effect of fertilizer deficiency on root yield loss was also analyzed to confirm the result predicted by these models. A pot culture experiment was performed to observe the incidence rate of root rot disease and to obtain the best range in which the highest yield of root and saponins could be realized. Results The best application strategy for NPK fertilizer was 0 kg/667 m 2 , 17.01 kg/667 m 2 , and 56.87 kg/667 m 2 , respectively, which can produce the highest root yield of 1,861.90 g (dried root of 100 plants). For mineral fertilizers, calcium and magnesium fertilizers had a significant and positive effect on root yield and the content of four active saponins, respectively. The severity of root rot disease increased with the increase in soil moisture. The best range of soil moisture varied from 0.56 FC (field capacity of water) to 0.59 FC, when the highest yield of root and saponins could be realized as well as the lower incidence rate of root disease. Conclusion These results indicate that the amount of nitrogen fertilizer used in these fields is excessive and that of potassium fertilizer is deficient. Higher soil moisture is an important factor that increases the severity of the root rot disease.

Four Panax genus species (Panax notoginseng (PN), Panax stipuleanatus (PS), Panax vietnamensis (PV) and Panax japonicus (PJ)) are all distributed in Southeast Asia. Besides PN, the other three species attracted little attention because of their limited distribution area, less wide application or relatively low quantity. PN had been cultivated as an industrial crop for hundreds years in China while the other three species as substitutes for PN in minority ethnic groups had no large-scale cultivation until now. The saponins of the four medical plants had been investigated in previous study while there has been virtually no reports describing their other compounds. In this study, the gas chromatography-mass spectrometry (GC–MS) was used to compare the volatile oils compounds of the four species. A total of 146 compounds were identified among all the species and every species contained compounds with similar quantity (all about 70). The falcarinol (28.86%) and andrographolide (38.35%) were the main compositions for PN. Furthermore, falcarinol was the dominant composition for PV (70.65%) and PS (64.61%), while pentadecanoic acid and ledene oxide-(II) were the high content compounds for PJ, accounting 9.31% and 8.39%, respectively. In addition, hierarchical clustering analysis was conducted to evaluate the relationship between the four Panax species based on contents (%) of chemical constituents in volatile oils. The results indicated that the PV and PS exhibited high similarity of volatile oils compounds. The PN, PV and PS could be used as the raw materials of falcarinol presenting well biological activities.