Multi-omics and chemical profiling approaches to understand the material foundation and pharmacological mechanism of sophorae tonkinensis radix et rhizome-induced liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sophorae tonkinensis Radix et Rhizoma (STR) is an extensively applied traditional Chinese medicine (TCM) in southwest China. However, its clinical application is relatively limited due to its hepatotoxicity effects. AIM OF THE STUDY: To understand the material foundation and liver injury mechanism of STR. MATERIALS AND METHODS: Chemical compositions in STR and its prototypes in mice were profiled by ultra-performance liquid chromatography coupled quadrupole-time of flight mass spectrometry (UPLC-Q/TOF MS). STR-induced liver injury (SILI) was comprehensively evaluated by STR-treated mice mode. The histopathologic and biochemical analyses were performed to evaluate liver injury levels. Subsequently, network pharmacology and multi-omics were used to analyze the potential mechanism of SILI in vivo. And the target genes were further verified by Western blot. RESULTS: A total of 152 compounds were identified or tentatively characterized in STR, including 29 alkaloids, 21 organic acids, 75 flavonoids, 1 quinone, and 26 other types. Among them, 19 components were presented in STR-medicated serum. The histopathologic and biochemical analysis revealed that hepatic injury occurred after 4 weeks of intragastric administration of STR. Network pharmacology analysis revealed that IL6, TNF, STAT3, etc. were the main core targets, and the bile secretion might play a key role in SILI. The metabolic pathways such as taurine and hypotaurine metabolism, purine metabolism, and vitamin B6 metabolism were identified in the STR exposed groups. Among them, taurine, hypotaurine, hypoxanthine, pyridoxal, and 4-pyridoxate were selected based on their high impact value and potential biological function in the process of liver injury post STR treatment. CONCLUSIONS: The mechanism and material foundation of SILI were revealed and profiled by a multi-omics strategy combined with network pharmacology and chemical profiling. Meanwhile, new insights were taken into understand the pathological mechanism of SILI.

Leaf Spot Caused by Didymella glomerata on Chaihu (Bupleurum falcatum L.) in China.

Bupleurum falcatum is a Apiaceae family herbal medicinal plant, which has the functions of soothing liver, relieving depression, relieving fever, dispelling stagnation, and regulating menstruation. B. falcatum roots have been used in Chinese herbal formbulary for at least 2000 years (Ahmadimoghaddam et al. 2021). In June 2021, infected leaves of B. falcatum that had dark brown, circular, elliptical or irregular shaped lesions or severely withered were obtained in Yichang (30.75 ° N，111.24 ° E), Hubei, China. Disease incidence was approximately 40% in the 20 hm2 B. falcatum plantation base. Fifteen small pieces (3 mm) were cut from the junction between disease and health of surface sterilized (with 75% alcohol) leaves and then plated on potato dextrose agar (PDA). After 3 days incubation, eight isolates with the same colony morphology were sub-cultured and purified by hyphal tip isolation. Isolate CHYB1 cultured on potato dextrose agar (PDA) was selected for identification. The colony was initially white and later producing gray and brown. Pycnidia were dark, spherical or flat spherical, and 78.3 to 137.4 µm in diameter. Conidia were oval mostly, smooth, aseptate, and 18 the size was 3.7 to 5.1 × 1.6 to 2.5 µm. Following DNA extraction, PCR was performed using the TSINGKE 2×T5 Direct PCR Mix kit. Target areas of amplification were the internal transcribed spacer (ITS) and beta-tubulin gene (TUB2) using ITS1/4 (White et al. 1990) Btu-F-F01/Btu-F-R01 primers (Wang et al. 2014), respectively. BLAST analysis of the ITS sequence (MZ818334.1) had 99% similarity to a 498 bp portion of D. glomerata sequence in GenBank (KR709012.1) and TUB2 sequence (OL439060) had 100% similarity to a 323 bp portion of D. glomerata sequence in GenBank (LT592974.1). All isolates (CHYB1-8) were taken for a pathogenicity test in laboratory on surface-disinfested leaves of B. falcatum. Mycelial plugs (5 mm) were excised from the margin of colony cultured for 5 days, and placed on surface-disinfested leaves of potted B. falcatum which involved creating small wounds. The potted plants were placed in a closed bucket to keep 80% relative humidity. Controls were inoculated with non-colonized PDA plugs (5 mm). All treatments had three replicates. On the inoculated B. falcatum, the leaves of B. falcatum appeared brown spot and been covered with off-white hyphae 7 DPI. By comparision, the control leaves had no symptoms. The pathogen was reisolated from the inoculated leaves and exhibited same morphological characteristics and ITS sequence as those of D. glomerata. D. glomerata was reported to cause round leaf spot on Sophora tonkinensis Gagnep and black spot disease of Actinidia chinensis in China (Pan et al. 2018; Song et al. 2020). To our knowledge, this is the first report of leaf spot caused by D. glomerata on B. falcatum in China.

Coptischinensis Franch root rot infection disrupts microecological balance of rhizosphere soil and endophytic microbiomes.

Introduction: The ecological balance of the plant microbiome, as a barrier against pathogens, is very important for host health. Coptis chinensis is one of the important medicinal plants in China. In recent years, Illumina Miseq high-throughput sequencing technology was frequently used to analyze root rot pathogens and the effects of root rot on rhizosphere microorganisms of C. chinensis. But the effects of root rot infection on rhizosphere microecological balance of C. chinensis have received little attention. Methods: In this study, Illumina Miseq high-throughput sequencing technology was applied to analyze the impact on microbial composition and diversity of C. chinensis by root rot. Results: The results showed that root rot infection had significant impact on bacterial α-diversity in rhizome samples, but had no significant effect on that in leaf samples and rhizosphere soil samples, while root rot infection exhibited significant impact on the fungal α-diversity in leaf samples and rhizosphere soil samples, and no significant impact on that in rhizome samples. PCoA analysis showed that the root rot infection had a greater impact on the fungal community structure in the rhizosphere soil, rhizome, and leaf samples of C. chinensis than on the bacterial community structure. Root rot infection destroyed the microecological balance of the original microbiomes in the rhizosphere soil, rhizome, and leaf samples of C. chinensis, which may also be one of the reasons for the serious root rot of C. chinensis. Discussion: In conclusion, our findings suggested that root rot infection with C. chinensis disrupts microecological balance of rhizosphere soil and endophytic microbiomes. The results of this study can provide theoretical basis for the prevention and control of C. chinensis root rot by microecological regulation.

Development of EST-SSR primers and genetic diversity analysis of the southern blight pathogen Sclerotium rolfsii using transcriptome data.

Introduction: Sclerotium rolfsii Sacc. is a globally dispersed pathogenic fungus that causes southern blight disease in many crops and Chinese herbal medicine. The high degree of variation and diversity in the fungi altered population genetic structure. Therefore, the important factors of variation within the pathogen population should be considered during the development of management strategies for the disease. Methods: In this study, S. rolfsii isolates from 13 hosts in 7 provinces of China were collected and analyzed to identify their morphological features and perform molecular characterization. To develop EST-SSR primers, transcriptome sequencing was performed on isolated CB1, and its SSR loci were comprehensively analyzed. In addition, we analyzed the polymorphisms among different populations based on screened EST-SSR primers. Results: The results showed that all of these clean reads with total 36,165,475 assembled bases were clustered into 28,158 unigenes, ranged from 201 bp to 16,402 bp on the length, of which the average length was 1,284 bp. Of these, the SSR sequence appeared at an average interval of 15.43 kB, and the frequency of SSR was 0.0648 SSR/kB. Polymorphism of 9 primers was observed among 22 populations, and was verified by the Shannon's index (average = 1.414) and polymorphic information index (> 0.50). The genetic diversity analysis revealed diversity in all host populations and geographical populations. Further, molecular variance analysis (AMOVA) showed that the differences between groups were mainly related to geographical location. Based on cluster analysis, the 7 populations were roughly divided into 3 groups, and the results were highly consistent with those based on the geographical location, ultimately aligning with the results of STRUCTURE analysis. Discussion: The findings build on current knowledge of the distribution of S. rolfsii in the southwest area of China, adding value to current knowledge base on the population structure and genetic diversity of S. rolfsii, specifically in the context of Chinese herbal medicine cultivation in China. Overall, our findings may provide valuable information for breeding of crops with enhanced resistance toward S. rolfsii.

Spectrum-Effect Relationship Analysis of Bioactive Compounds in Zanthoxylum nitidum (Roxb.) DC. by Ultra-High Performance Liquid Chromatography Mass Spectrometry Coupled With Comprehensive Filtering Approaches.

Zanthoxylum nitidum (Roxb.) DC. (ZN), with strong effects of anti-inflammation and antioxidant activities is treated as a core herb in traditional Chinese medicine (TCM) preparation for treating stomachache, toothache, and rheumatoid arthritis. However, the active ingredients of ZN are not fully clarified due to its chemical complexity. In the present study, a double spectrum-effect analysis strategy was developed and applied to explore the bioactive components in herbs, and ZN was used as an example. Here, the chemical components in ZN were rapidly and comprehensively profiled based on the mass defect filtering-based structure classification (MDFSC) and diagnostic fragment-ion-based extension approaches. Furthermore, the fingerprints of 20 batches of ZN samples were analyzed by high-performance liquid chromatography, and the anti-inflammatory and antioxidant activities of the 20 batches of ZN samples were studied. Finally, the partial least squares regression (PLSR), gray relational analysis models, and Spearman's rank correlation coefficient (SRCC) were applied to discover the bioactive compounds in ZN. As a result, a total of 48 compounds were identified or tentatively characterized in ZN, including 35 alkaloids, seven coumarins, three phenolic acids, two flavonoids, and one lignan. The results achieved by three prediction models indicated that peaks 4, 12, and 17 were the potential anti-inflammatory compounds in ZN, whereas peaks 3, 5, 7, 12, and 13 were involved in the antioxidant activity. Among them, peaks 4, 5, 7, and 12 were identified as nitidine, chelerythrine, hesperidin, and oxynitidine by comparison with the standards and other references. The data in the current study achieved by double spectrum-effect analysis strategy had great importance to improve the quality standardization of ZN, and the method might be an efficiency tool for the discovery of active components in a complex system, such as TCMs.

[Identification, biological characteristics, and control of pathogen causing Pinellia ternata soft rot in Hubei province].

This study was designed to identify the pathogen causing soft rot of Pinellia ternata in Qianjiang of Hubei province and screen out the effective bactericides, so as to provide a theoretical basis for the control of soft rot of P. ternata. In this study, the pathogen was identified based on molecular biology and physiological biochemistry, followed by the detection of pathogenicity and pathogenicity spectrum via plant tissue inoculation in vitro and the indoor toxicity determination using the inhibition zone method to screen out bactericide with good antibacterial effects. The control effect of the bactericide against P. ternata soft rot was verified by the leave and tuber inoculation in vitro. The phylogenetic tree was constructed based on the 16 S rDNA, dnaX gene, and recA gene sequences, respectively, and the result showed that the pathogen belonged to the same branch as the type strain Dickeya fangzhongdai JS5. The physiological and biochemical tests showed that the pathogen was identical to D. fangzhongdai, which proved that the pathogen was D. fangzhongdai. The pathogenicity test indicated that the pathogen could obviously infect leaves at 24 h and tubers in 3 d. As revealed by the indoor toxicity test, 0.3% tetramycin, 5% allicin, and 80% ethylicin had good antibacterial activities, with EC_(50) values all less than 50 mg·L~(-1). Tests in tissues in vitro showed that 5% allicin exhibited the best control effect, followed by 0.3% tetramycin and 10% zhongshengmycin oligosaccharide, and their preventive effects were better than curative effects. Therefore, 5% allicin can be used as the preferred agent for the control of P. ternata soft rot, and 0.3% tetramycin and 10% zhongshengmycin oligosaccharide as the alternatives. This study has provided a certain theoretical basis for the control of P. ternata soft rot.

Efficacy and Safety of Traditional Chinese Medicine in Coronavirus Disease 2019 (COVID-19): A Systematic Review and Meta-Analysis.

Introduction: Until now, there is no clinically approved specific medicine to treat COVID-19. Prior systematic reviews (SRs) have shown that traditional Chinese medicine (TCM) reduces the number of patients with severe disease and time to fever clearance, promotes clinical effectiveness, and improves chest images and the negativity rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid test. Few SRs arrived at a definitive conclusion, and more randomized controlled trials (RCTs) were published. We conducted this study to summarize the latest evidence of TCM in COVID-19. Methods: Eight online databases were searched from December 2019 to July 2020, updated to March 2021. Only RCTs evaluating the clinical efficacy and safety of TCM in the treatment of COVID-19 were included. Primary outcomes were clinical cure and the negativity of the SARS-CoV-2 nucleic acid test. Secondary outcomes included clinical deterioration, ARDS, mechanical ventilation, death, time to fever clearance, duration of hospitalization, and chest imaging improvement. Safety outcomes included adverse events and serious adverse events during treatment. Two reviewers selected the included articles, assessed the risk of bias, and extracted data independently and in duplicate. Results: A total of 25 RCTs involving 2222 participants were selected in the systematic review, and seven RCTs were included in the meta-analysis. The results showed that TCM plus routine treatment was significantly better than routine treatment alone in clinical cure (risk ratio [RR] = 1.20, 95% confidence interval (CI) [1.04, 1.38], P = 0.01) and chest image improvement (RR = 1.22, 95% CI [1.07, 1.39], P = 0.01) and could reduce clinical deterioration (RR = 0.39, 95% CI [0.18, 0.86], P = 0.02), ARDS (RR = 0.28, 95% CI [0.11, 0.69], P = 0.01), mechanical ventilation (RR = 0.30, 95% CI [0.12, 0.77], P = 0.01), or death rate (RR = 0.28, 95% CI [0.09, 0.84], P = 0.02). No significant difference between TCM and routine treatment in the negativity of SARS-CoV-2 nucleic acid test (RR = 1.08, 95% CI [0.94, 1.23], P = 0.29) was observed. Finally, there was no overall significant difference in the incidence of adverse events between the two groups. The summary of evidence showed moderate confidence of a benefit of 11.8% in clinical cure and 14.0% in chest image improvement and a reduction of 5.9% in clinical deterioration, 25.4% in ARDS, 18.3% in mechanical ventilation, and 4.5% in death with TCM plus routine treatment compared to routine treatment alone in patients with COVID-19. A low confidence of a benefit of 5.4% in the negativity of SARS-CoV-2 nucleic acid test was also observed. Conclusions: Synethized evidence of 21 outcomes in 8 RCTs showed moderate certainty that TCM treatment plus routine treatment may promote a clinical cure and chest image improvement compared to routine treatment alone while reducing clinical deterioration, development of ARDS, use of mechanical ventilation, and death in patients with COVID-19. TCM treatment plus routine treatment may not promote the negativity of the SARS-CoV-2 nucleic acid test compared to routine treatment alone. TCM treatment was found to be safe for patients with COVID-19.

Duohua huangjing (Polygonatum cyrtonema Hua) seedling basal rot caused by Fusarium redolens in China.

Duohua huangjing (Polygonatum cyrtonema Hua) seedling basal stem rot caused by Fusarium redolens in China Tao Tang1, Fanfan Wang1, Jie Guo1, Xiaoliang Guo1, Yuanyuan Duan1,Jingmao You1* 1 Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences, Enshi, 445000, China. Duohua huangjing (Polygonatum cyrtonema Hua), a herbal medicine, that is mostly planted in several provinces in China. In April 2020, severe diseases with about 40% seedling losse was found in the Huangjing seedling base in Shiyan city, Hubei province. The symptoms included softening and decay of the roots and stem bases, a progressive yellowing and wilting of leaves, and finally being completely rotted. Small pieces of symptomatic stems (0.5 cm in length) and leaves (0.5 × 0.5 cm in size) were surface sterilized with 75% ethanol for 30 s, followed by 0.1% HgCl2 for 1 min, rinsed three times with sterile water, and then dried with sterilized absorbent paper. The sections were placed on potato dextrose agar (PDA) medium containing 10 µg/ml of ampicillin and incubated at 25°C in the dark. After 3 days incubation, eight isolates with the same colony morphology were sub-cultured and purified by hyphal tip isolation. Macroconidia were sickle-shaped, 15.8 - 32.3 × 3.1 - 5.6 µm (n = 25), and three to five septate. Microconidia were oval or kidney-shaped, 5.2 - 11.4 × 2.0 - 3.2 µm (n = 25), and zero to one septate. To confirm the identity of the pathogen, molecular identification was performed with strain HJCD1. Following DNA extraction, PCR was performed using the TSINGKE 2×T5 Direct PCR Mix kit. Target areas of amplification were the internal transcribed spacer (ITS) and translation elongation factor 1α (TEF-1α) using ITS1/4 (White et al. 1990) , EF1/EF2 (Taylor et al. 2016), respectively. Following BLAST searches and phylogenetic reconstruction, the ITS region (GenBank MW485770.1) showed 99% identity with those of Fusarium redolens in GenBank (KU350713.1) and the TEF-1α (GenBank MW503930.1) showed 100% identity with F. redolens GenBank (MK922537.1). Pathogenicity tests were performed to fulfill Koch's postulates. Huangjing seedlings were rinsed with sterile water, wiped clean with sterile absorbent paper, and transferred to a tray covered with wet filter paper to maintain high humidity. The mycelial piugs of F. redolens HJCD1 were inoculated onto the surface of leaves and basal stems. Controls were inoculated with sterile PDA plugs. The inoculated seedlings were sealed with plastic wrap, and then cultivated in a 25 ℃ growth chamber with 16 h of light per day. The pathogen-inoculated plants exhibited etiolation and typical wilt symptoms after 4 days, whereas no symptoms were observed in the control plants. F. redolens was reisolated from the infected tissues, and colony morphology and ITS sequence of re-isolates were same as that of HJCD1. The pathogen has been reported previously in american ginseng in China (Fan et al. 2021), lentil in Pakistan (Rafique et al. 2020), and wild rocket in United Kingdom (Taylor et al. 2019). However, to the best of our knowledge, this is the first report of F. redolens causing seelding basal rot on Duohua huangjing in China. References: White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Taylor, A., et al. 2016. Mol. Plant Pathol. 17:1032. https://doi.org/10.1111/mpp.12346 Fan, S. H., et al. 2021. Plant Dis. https://doi.org/10.1094/PDIS-11-19-2519-PDN Rafique, K., et al. 2020. Plant Dis. 9:104. https://doi.org/10.1094/PDIS-11-19-2519-PDN Taylor, A., et al. 2019. Plant Dis.6:103. https://doi.org/10.1094/PDIS-12-18-2143-PDN Funding: Science Funds for Young Scholar of Hubei Academy of Agricultural Science (grant no. 2020NKYJJ20), National Modern Agricultural Industrial Technology System (grant no. CARS-21), Technology R&D Program of Enshi (grant no. D20190015), Science Funds for Young Scholar of Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences (grant no. 2019ZYCJJ03), Key Laboratory of Integrated Management of Crops of Central China, Ministry of Agriculture, P. R. China / Hubei Key Laboratory of Crop Disease, Insect Pests and Weeds Control (grant no.2020ZTSJJ6).

Occurrence of Dickeya fangzhongdai Causing Soft Rot of Banxia (Pinellia ternata) in China.

Banxia [Pinellia ternata (Thunb.) Breit., Araceae] is a perennial herbaceous plant, where the tuber is commonly used in traditional Chinese herbal medicine. In the summer of 2020, an outbreak of soft rot of Banxia was observed in Zhugentan Town (30°50'N, 112°91'E), Qianjiang City, Hubei Province, with about 56% percentage of infected plants. Symptomatic plants initially appeared with small water-soaked spots on leaves that progressed into extensive translucent spots when facing a light source. The bacteria further spread to the stems and tubers. Infected tubers appeared normal, but inner macerated inclusions exuded when touched. The whole plant was macerated and collapsed within a few days. Ten leaves with typical symptoms were obtained from a diseased field, by surface sterilizing in 75% ethanol for 30 s and 0.3% NaClO for 5 min, washing the tissue sections three times in sterile water. Small pieces of tissue (5 × 5 mm) were removed from lesion borders, plated on nutrient ager medium, and cultivated at 37 ℃ for 48 h. Five representative isolates were selected for further identification. Colonies were all smooth and transparent. In addition, these strains were Gram-negative, and had the ability to reduce D-arabinose, melibiose, galactose, raffinose, rhamnose, inositol, and mannitol, but not reduce 5-keto-D-gluconate, L-xylose, amygdalin, and sorbitol. Genomic DNA was extracted from isolate stain ZG5. The 16S rDNA gene, recombinase A (recA) gene, and DNA polymerase III subunits gamma and tau (dnaX) were amplified by PCR with the primers 27f/1492r (Weisburg et al. 1991), recF/recR (Waleron et al. 2002), and dnaXf/dnaXr (Slawiak et al. 2009), respectively. The PCR products were sequenced, then submitted to GenBank (GenBank MW332472, MW349833, MW349834, respectively). BLAST search showed that the sequences of 16S rDNA, recA, and dnaX respectively matched ≥99% with D. fangzhongdai strains DSM 101947 (CP025003), QZH3 (CP031507), and PA1 (CP020872). Pathogenicity tests were performed on 10 healthy, 3-month-old P. ternate plants. Five plants were injected with 20 µl of bacterial suspension (108 CFU/ml) of isolate ZG5, and other plants were injected with sterile water as a negative control. All tested plants were incubated at 28 ℃ and individually covered with a plastic bag. After 24 h, soft rot symptoms all appeared on the pathogen-inoculated leaves, whereas no symptoms on the control leaves. The pathogenicity test was repeated three times and obtained same results. Koch's postulates were fulfilled by reisolating D. fangzhongdai from inoculated plants. Meanwhile, PCR were performed on the reisolated bacteria as above described, and the pathogen was identified and confirmed as D. fangzhongdai. Here we report that D. fangzhongdai causes soft rot of P. ternata in China. The disease progressed very rapidly, and reduced the yield and quality of tubers. Thus, more research is needed to implement effective strategies to manage this disease.

Occurrence of Stem Blight Caused by Pseudomonas extremorientalis on Pinellia ternata in China.

Pinellia ternata is a perennial herbaceous plant, which tubers can be used for anti-inflammatory and has a significant position in Traditional Chinese Medicine (Marki et al. 1987). In April 2020, bacterial stem blight first occurred on P. ternata in Jingmen City (30°32'N, 111°51'E), Hubei Province, China. In the follow-up investigation, the disease also appeared in plantations of P. ternata in Qianjiang City, Tianmen City. Initial symptoms showed orange-red streak on the stem, then progressed into chlorotic and water-soaked lesions, which caused roots to be necrotic and leaves to stunting, fading, and wilting. In the end, the leaves withered, the stems rotted completely, and the incidence of plant collapse reached 20~30%. To isolate the plant pathogenic bacteria, twenty P. ternata plant samples with distinct chlorotic stem symptoms were obtained from two fields in Jingmen City. Symptomatic samples were cut to 1-cm-long pieces by sterile scalpel, then were streaked onto nutrient agar medium and grow at 28℃ for 48 h. Four pure typical aerobic, gram-negative bacteria were isolated by characterized with transparent, smooth, round, convex surfaces. The isolated colonies did not produce fluorescent pigments on King's B medium. In addition, the isolates were positive for nitrate reduction, arabinose, mannitol, D-ribose, sucrose, D-sorbitol, and were negative for gelatin liquefaction, rhamnose, D-glucose, D-melibiose. These characteristics were identified as Pseudomonas extremorientalis (Ivanova et al. 2002). One representative colony ZJH1 was selected randomly for further verification. The 16s rRNA, gyrB, and rpoD regions were obtained with primers 27F/1492R (Weisburg et al. 1991), gyrB-Fps/ gyrB-Rps, and rpoD-Fps/ rpoD-Rps, respectively (Sarkar and Guttman. 2004). These sequences were deposited in GenBank as accession nos. MT459234.1, MT469887.1 and MT469886.1, which revealed 99% homology with P. extremorientalis strain BS2774 (accession nos. LT629708.1). The pathogenicity of P. extremorientalis strain ZJH1 was confirmed by using 3-month-old, healthy, greenhouse-grown P. ternata plants. The stems were stabbed and inoculated 10 µL of the bacterial suspension (108 CFU / ml), inoculating the same amount of sterile water as a control, repeated 5 times for each treatment. The plants were cultivated in a greenhouse at 28 °C and a humidity of 80%. Three days later, the stems showed necrosis, followed by the withered leaves and died plants, whereas the control had no symptoms. P. extremorientalis were reisolated and verified again from symptomatic plants, which was consistent with Koch's postulates. This experiment was repeated thrice to get the same result. To our knowledge, this is the first report of bacterial stem blight caused by P. extremorientalis on P. ternata in China. Stem blight caused by P. extremorientalis poses a significant threat to yield and marketability of P. ternata. Further research on selecting resistant variety and effective chemical control is needed. References: Ivanova, E. P., et al. 2002. Int J Syst Evol Micr. 2113:2120. https://doi.org/10.1099/00207713-52-6-2113 Marki, T., et al. 1987. Planta Med. 53:412. Sarkar, S. F., Guttman, D. S. 2004. Appl. Environ. Microbiol. 70:1999. https://doi.org/10.1128/AEM.70.4.1999-2012.2004 Weisburg, W. G., et al. 1991. J. Bacteriol. 173:697. https://doi.org/10.1128/jb.173.2.697-703.1991 F. F. Wang and Y. J. You contributed equally to this work. The author(s) declare no conflict of interest. Funding: National Modern Agricultural Industrial Technology System (grant no. CARS-21), Technology R&D Program of Enshi Tujia and Miao Autonomous Prefecture (grant no. D20190015), Science Funds for Young Scholar of Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences (grant no. 2019ZYCJJ01), Key R&D Program of Hubei Province (grant no. 2020BCA059), Key Technology R&D Projects of Hubei Agricultural Science and Technology Innovation Center (grant no. 2020-620-000-002-04).

Effects of foliar selenium application on growth and rhizospheric soil micro-ecological environment of Atractylodes macrocephala Koidz.

Atractylodes macrocephala (A. macrocephala), a famous medicinal herb in China, is widely cultivated and consumed in China with various beneficial effects. Numerous studies have shown that selenium (Se) plays an important role in promoting plant growth, although Se has not been considered an essential element for higher plants. The objectives of this research were to determine the effects of foliar Se application (0, 2.5, 5.0, 10.0 and 20.0 mg m-2 Se in sodium selenite, sprayed monthly from May to August) on the growth and rhizospheric soil micro-ecological environment of A. macrocephala, and explore the possible mechanisms underlying plant response to foliar Se application through a field experiment. The results were: The foliar application of 5.0 mg m-2 Se significantly increased the survival rate of A. macrocephala compared to the control. The yield of A. macrocephala was increased when the Se level maintained belowed 10.0 mg m-2 but decreased when Se level reached 20.0 mg m-2. The Se content in the rhizome of A. macrocephala showed a significant positive correlation with the Se level, while the insect attack rate was significantly negatively correlated with the Se level. However, foliar Se application hardly affected the concentration of bioactive compound atractylenolide in the rhizome of A. macrocephala. Notably, the application of foliar Se changed the content of partial soil nutrients, microbial diversity and composition in the rhizosphere soil of A. macrocephala. Bacterial diversity was positively correlated with A. macrocephala growth whereas fungal diversity was negatively correlated, suggesting that microbial diversity in the rhizosphere soils is closely related to plant growth. Moreover, correlation analysis showed that available potassium, Burkholderia and Cupriavidus in rhizospheric soil might be critical factors for promoting the growth of A. macrocephala. Overall, the foliar application of Se at moderate concentration was beneficial for the growth of A. macrocephala, and 5.0-10.0 mg m-2 Se level was the optimum. Our findings revealed novel insights into the response of A. macrocephala to foliar Se application from plant growth, rhizospheric soil nutrient and microbial community composition .

Traditional Chinese exercise for COVID-19: A protocol for systematic review and meta-analysis.

BACKGROUND: A new type of coronavirus (COVID-19), is spreading all over the world. Under the background of the comprehensive medical treatment and strict prevention and control in China, the number of discharged patients increased substantially. By the end of July, more than 80,000 patients had been cured and discharged from hospital in China. In order to effectively promote the full recovery of the patient's physical and mental functions and quality of life, gradually shift the emphasis of clinical work to convalescence therapy is very important, thus Chinese experts draw up Expert Consensus on Rehabilitation of Chinese Medicine for COVID-19. This systematic review and meta-analysis will assess studies of the effects of traditional Chinese exercise (TCE) for COVID-19 patients. METHODS AND ANALYSIS: We will search 6 English and 4 Chinese databases by 01, December 2020. After a series of screening, Randomized Clinic Trials (RCTs) will be included related to TCE for COVID-19. Two assessors will use the Cochrane bias risk assessment tool to assess the RCTs. Finally, the evidence grade of the results will be evaluated. RESULTS: This study will provide a reliable evidence for the selection of TCE therapies for COVID-19. CONCLUSION: The results of this study will provide references for the selection of TCE treatment for COVID-19, and provide decision making references for clinical research. PROSPERO REGISTRATION NUMBER: CRD42020179095.

First report of Sclerotinia blight caused by Sclerotinia sclerotiorum on Pinellia ternata in China.

Pinellia ternata is a perennial herb that belongs to the Araceae Family. The tuber of the P. ternata plant contains active ingredients such as alkaloids and pinellia starch, and can be used as a Chinese medicine for antineoplastic and anti-inflammatory disorders in humans (Marki et al. 1987). In April 2019, lesions with rotting were observed during the flowering phase on spathes of P. ternata in a field in Qianjiang City (30°50'N, 112°92'E), Hubei Province, which is one of the main production areas of P. ternata in China. Approximately 10% of P. ternata plants were symptomatic. The initial symptoms of infection were reddish-brown lesions, followed by the appearance of white cottony mycelia. Subsequently, lesions became gray-brown, and rotted with white mycelium that eventually formed on the lesions after 2 to 3 days. In the later phases, spathes were completely rotted and mycelia began to spread to the stems, until the plant wilted and died. Ten spathes at the initial stage of infection were collected in Zhugentan Town, Qianjiang City disinfested with 0.5% sodium hypochlorite for 1 min and 75% alcohol for 20 sec, then washed with sterile distilled water three times, dried, and placed on Petri plates with potato dextrose agar (PDA) and incubated at 22℃ for two days. Six fungal isolates were obtained and purified by hyphal tip isolation in fresh culture, respectively. Culture media was covered with white hyphae after 3 to 4 days of incubation, and dark-gray, rough, irregular sclerotia (1.5 to 5.5 mm in length × 1.0 to 3.5 mm in width) formed on the margins of the media, followed by the melanization as the culture aged. Eventually, black sclerotia were formed and wrapped by white hyphae. All isolates were preliminarily identified as Sclerotinia sclerotiorum (Lazarovits et al. 2000). To further identify the pathogens, molecular identification was performed with one of the six isolates (BXH1). Polymerase chain reaction was performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990) and primers SSasprF/SSasprR for the aspartyl protease gene (Abd-Elmagid et al. 2013). BLAST search analysis revealed that the 456-bp ITS sequence (GenBank MT436756.1) was ≥99% similar to S. sclerotiorum (MT177216.1, MN105884.1, MG931017.1, etc.), and the 173-bp aspartyl protease gene sequence (GenBank MT584031.1) was too (MK028159.1, MK028161.1, AF271387.1, etc.). Pathogenicity tests were carried out by inoculating disease-free, surface-disinfested spathes of thirty 30-day-old P. ternata plants in plastic pots with a sterilized mixture of peat moss and vermiculite (3:1). Five mycelial plugs (6 mm) were excised from the margin of a colony cultured for 5 days. The plugs were placed on five spathes covered with wet sterile cotton at 22±1℃, and 80% relative humidity, with a 12-h photoperiod. Five control plants were inoculated with noncolonized PDA plugs. Lesions formed on the second day, then rot and white hyphae began to appear on the third day, while the controls had no symptoms. Similar results were obtained in three repeated experiments with S. sclerotiorum being re-isolated from all diseased plants, in accordance with Koch's postulates. This disease is an emerging problem in P. ternata fields in Qianjiang, leading to extensive yield reduction and significant economic losses. To our knowledge, this is the first report of Sclerotinia blight on P. ternata in China. References: Abd-Elmagid, A., et al. 2013. J. Microbiol. Methods 92:293. https://doi.org/10.1016/j.mimet.2012.12.020 Marki, T., et al. 1987. Planta Med. 53:412. Lazarovits, G., et al. 2000. Pestic Biochem Phys. 54:62. https://doi.org/10.1006/pest.2000.2474 White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 Funding: National Modern Agricultural Industrial Technology System (grant no. CARS-21), Technology R&D Program of Enshi (grant no. D20190015), Science Funds for Young Scholar of Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences (grant no. 2019ZYCJJ01), Key Laboratory of Integrated Management of Crops of Central China, Ministry of Agriculture, P. R.China / Hubei Key Laboratory of Crop Disease, Insect Pests and Weeds Control (grant no.2019ZTSJJ6).

[Effects of biocontrol strain BZJN1 and streptoprofen on physicochemical properties and bacteria structure of rhizosphere soil of Atractylodes macrocephala].

Soil microorganisms are one of the important biological indictors of soil quality and can reflct the comprehensive ecological environment characteristics of the soil. The research of soil microbial diversity is the key to know the ecological functions and balance with soil. In this paper, high-throughput sequencing on PCR-amplified 16 S rRNA gene V3-V4 fragments was used to determine the bacterial diversity in rhizosphere soil of A. macrocephala under the treatment with BZJN1 or streptoprofen. The results showed that there were no significant differences of the bacteria in A. macrocephala rhizosphere soil of the streptoprofen treatment group and the biocontrol BZJN1 treatment group. All the soil bacteria was classified into 25 categories,67 classes, 108 orders, 167 families and 271 generas, except some unidentified bacteria. Proteobacteria(30.7%-34.8%) was the dominant phylum, of which Alphaproteobacteria(16.8%-18.5%) was the dominant subgroup. Compared with the control group, the relative abundance of multiple phylums bacteria in the rhizosphere soil of A. macrocephala was significantly changed in the streptoprofen treatment group and the biocontrol BZJN1 treatment group. In addition, RDA analysis showed that there was connection with different environmental factors and microbial communities. The abundance of the three genera in the rhizosphere soil of A. macrocephala was significantly positively correlated with Invertase, Urease and AP. PICRUSt function prediction results showed that BZNJ1 could enhance some bacterial functions and promote the plant growth. Biocontrol is a new type of green and safety control pest method. BZNJ1 significantly enhances some bacterial functions on the basis of effectively preventing root rot of A. macrocephala and promoting plant growth, and has no significant effect on the soil bacterial community structure. All the results can provide theoretical support for popularization of BZNJ1.

[Isolation and identification of pathogen of Dendrobium officinale gray mold and its prevention and control].

Through investigation,it was found that the main disease of leaves was grey mold on Dendrobium officinale in Hubei province,which has a great impact on the yield and quality of D. officinale. The identification of morphological and molecular biological was used to prove that the pathogen was Botrytis cinerea. Through test the effect of 5 plant source fungicides and 4 antibiotic fungicides on mycelial growth of strain HS1,which proved 0. 3% eugenol had the best inhibitory effect,EC50 was 0. 29 mg·L-1,the second was1% osthol and EC50 was 1. 12 mg·L-1,the EC50 of 0. 5% matrine was 9. 16 mg·L-1,the EC50 of the other six fungicides was higher than 10 mg·L-1. The field control effect test proved that 0. 3% eugenol had the best control effect,reaching 89. 44%,secondly for 1%osthole,which was 77. 17%,0. 5% matrine was in the third place with 62. 37% of effective rate. However,the control effect of the other fungicides was less than 60%. The three plant-derived fungicides were safe for the produce of D. officinale and showed no phytotoxicity. The effect of these fungicides on the growth of D. candidum was tested,and proved that all the fungicides were safe and harmless to D. candidum. This study provides a research basis for the safe and effective prevention and control gray mold of D. officinale.

[Identification of endophytic bacteria BZJN1 and research on biological control of root rot of Atractylodes macrocephala].

In this study， an endophytic bacteria strain BZJN1 was isolated from Atractylodes macrocephala， and identified as Bacillus subtilis by physiological and biochemical tests and molecular identification. Strain BZJN1 could inhibit the growth of mycelia of Ceratobasidium sp. significantly， and the inhibition rate was more than 70%. The mycelium growth deformity with bulge as spherical and partially exhaustible in apex or central with microscopic observation. The inhibitory rates under 3% and 6% concentrations of the cell free fermentation were 22.7% and 38.7% expectively. The field test proved that the control efficacy of treatment of 1×108 cfu·mL⁻¹ is 75.27% and 72.37% after 10 and 20 days. All the treatments of strain BZJN1 was able to promote the growth of A. macrocephala， the treatment of 1×108 cfu·mL⁻¹ could able to increase the yield to 14.1%.

Effect of ceramic infrastructure on the failure behavior and stress distribution of fixed partial dentures.

OBJECTIVES: The effect of the ceramic infrastructure (IS) on the failure behavior and stress distribution of fixed partial dentures (FPDs) was evaluated. METHODS: Twenty FPDs with a connector cross-section of 16 mm(2) were produced for each IS and veneered with porcelain: (YZ) Vita In-Ceram YZ/Vita VM9 porcelain; (IZ) Vita In-Ceram Zirconia/Vita VM7 porcelain; (AL) Vita In-Ceram AL/Vita VM7 porcelain. Two experimental conditions were evaluated (n = 10). For control specimens, load was applied in the center of the pontic at 0.5 mm/min until failure, using a universal testing machine, in 37°C deionized water. For mechanical cycling (MC) specimens, FPDs were subjected to MC (2 Hz, 140 N, 10(6) cycles) and subsequently tested as described for the control group. For YZ, an extra group of 10 FPDs were built with a connector cross-section of 9 mm(2) and tested until failure. Fractography and FEA were performed. Data were analyzed by ANOVA and Tukey's test (α = 0.05). RESULTS: YZ16 showed the greatest fracture load mean value, followed by YZ16-MC. Specimens from groups YZ9, IZ16, IZ16-MC, AL16 and AL16-MC showed no significant difference for the fracture load. SIGNIFICANCE: The failure behavior and stress distribution of FPDs was influenced by the type of IS. AL and IZ FPDs showed similar fracture load values but different failure modes and stress distribution. YZ showed the best mechanical behavior and may be considered the material of choice to produce posterior FPDs as it was possible to obtain a good mechanical performance even with a smaller connector dimension (9 mm(2)).