New Meroterpenoids and α-Pyrone Derivatives Isolated from the Mangrove Endophytic Fungal Strain Aspergillus sp. GXNU-Y85.

Two new meroterpenoids, aspergienynes O and P (1 and 2), one new natural compound, aspergienyne Q (3), and a new α-pyrone derivative named 3-(4-methoxy-2-oxo-2H-pyran-6-yl)butanoic acid (4) were isolated from the mangrove endophytic fungal strain Aspergillus sp. GXNU-Y85, along with five known compounds (5-9). The absolute configurations of those new isolates were confirmed through extensive analysis using spectroscopic data (HRESIMS, NMR, and ECD). The pharmacological study of the anti-proliferation activity indicated that isolates 5 and 9 displayed moderate inhibitory effects against HeLa and A549 cells, with the IC50 values ranging from 16.6 to 45.4 µM.

Diisoprenyl Cyclohexene-Type Meroterpenoids with Cytotoxic Activity from a Mangrove Endophytic Fungus Aspergillus sp. GXNU-Y85.

Five new diisoprenyl cyclohexene-type meroterpenoids, aspergienynes J-N (1-5), along with three known analogues (6-8), were obtained from the mangrove endophytic fungal strain Aspergillus sp. GXNU-Y85. The chemical structures, including their absolute configurations, were established via spectroscopic data and comparison of experimental and calculated ECD spectra. Cytotoxicity assay results indicated that compound 8 had strong cytotoxicity against HeLa cancer cells, and its IC50 value was 11.8 µM. In addition, flow cytometry analysis revealed that the cytotoxicity of 8 was due to the induction of G1 cell cycle arrest and apoptosis in HeLa cells.

Polysiloxane-Based Molecular Logic Gate for Dual-Channel Visualizing Mitochondrial pH and Sulphite Changes during Cuproptosis.

Intracellular Cu-induced regulated cell death, characterized by the aggregation of lipidizing mitochondrial enzymes, is called cuproptosis. Mitochondria play a vital role in the metabolic regulation of cell injury and stressful immune responses. The pH levels and sulfur dioxide (SO2) content in mitochondria have important indicative roles in the regulation of cuproptosis. However, fluorescent probes that simultaneously detect changes in pH and SO2 in mitochondria during cuprotosis have not been reported. To fill this blank, in this study, we dexterously used functional polysiloxane as a fluorescent platform to propose a molecular logic gate probe P0-pH-SO2 for detecting changes in intramitochondrial pH and SO2 content through a dual-channel mode. In addition, we defined a new function to reflect the cellular state of the elesclomol-induced cuproptosis process based on the input and output of the relevant logic relationship. This new fluorescent molecular logic gate probe P0-pH-SO2 can be rapidly activated by mitochondrial sulfites to induce green fluorescence, while the red fluorescence is quenched with the proton in the mitochondria. Overall, this study developed a novel logic-gated molecular probe that provided a versatile strategy for monitoring the role played by intramitochondrial sulfites and H+ in cuproptosis. This work will open the way to broaden the applications of molecular logic gates and fluorescent polysiloxanes.

Versatile Switchable Targeted Polysiloxanes for High-Resolution Visualization of Mitochondrial and Lysosomal Interactions during Ferroptosis.

Ferroptosis is an iron-dependent process that regulates cell death and is essential for maintaining normal cell and tissue survival. The explosion of reactive oxygen species characterizes ferroptosis in a significant way. Peroxynitrite (ONOO-) is one of the endogenous reactive oxygen species. Abnormal ONOO- concentrations cause damage to subcellular organelles and further interfere with organelle interactions. However, the proper conduct of organelle interactions is critical for cellular signaling and the maintenance of cellular homeostasis. Therefore, investigating the effect of ONOO- on organelle interactions during ferroptosis is a highly attractive topic. To date, it has been challenging to visualize the full range of ONOO- fluctuations in mitochondria and lysosomes during ferroptosis. In this paper, we constructed a switchable targeting polysiloxane platform. During the selective modification of NH2 groups located in the side chain, the polysiloxane platform successfully constructed fluorescent probes targeting lysosomes and mitochondria (Si-Lyso-ONOO, Si-Mito-ONOO), respectively. Real-time detection of ONOO- in lysosomes and mitochondria during ferroptosis was successfully achieved. Remarkably, the occurrence of autophagy during late ferroptosis and the interaction between mitochondria and lysosomes was observed via the differentiated responsive strategy. We expect that this switchable targeting polysiloxane functional platform will broaden the application of polymeric materials in bioimaging and provide a powerful tool for further deeper understanding of the ferroptosis process.

Influence of arbuscular mycorrhizal fungi on mercury accumulation in rice (Oryza sativa L.): From enriched isotope tracing perspective.

The microorganisms that co-exist between soil and rice systems in heavy metal-contaminated soil environments play important roles in the heavy metal pollution states of rice, as well as in the growth of the rice itself. In this study, in order to further examine the effects of soil microorganisms on the mercury (Hg) uptake of rice plants and determine potential soil phytoremediation agents, an enriched 199Hg isotope was spiked in a series of pot experiments to trace the absorption and migration of Hg and rice growth in the presence of arbuscular mycorrhizal fungi (AMF). It was observed that the AMF inoculations significantly reduced the Hg concentration in the rice. The Hg concentration in rice in the AMF inoculation group was between 52.82% and 96.42% lower than that in the AMF non-inoculation group. It was also interesting to note that the presence of AMF tended to cause Hg (especially methyl-Hg (Me199Hg)) to migrate and accumulate in the non-edible parts of the rice, such as the stems and leaves. Under the experimental conditions selected in this study, the proportion of Me199Hg in rice grains decreased from 9.91% to 27.88%. For example, when the exogenous Hg concentration was 0.1 mg/kg, the accumulated methyl-Hg content in the grains of the rice in the AMF inoculation group accounted for only 20.19% of the Me199Hg content in the rice plants, which was significantly lower than that observed in the AMF non-inoculated group (48.07%). AMF also inhibited the absorption of Hg by rice plants, and the decrease in the Hg concentration levels in rice resulted in significant improvements in growth indices, including biomass and micro-indexes, such as antioxidant enzyme activities. The improvements occurred mainly because the AMF formed symbiotic structures with the roots of rice plants, which fixed Hg in the soil. AMF also reduce the bioavailability of Hg by secreting a series of substances and changing the physicochemical properties of the rhizosphere soil. These findings suggest the possibility of using typical co-existing microorganisms for the remediation of soil heavy metal contamination and provide valuable insights into reducing human Hg exposure through rice consumption.

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.

Corydalis saxicola Bunting: A Review of Its Traditional Uses, Phytochemistry, Pharmacology, and Clinical Applications.

Corydalis saxicola Bunting (CSB), whose common name in Chinese is Yanhuanglian, is a herb in the family Papaveraceae. When applied in traditional Chinese medicine, it is used to treat various diseases including hepatitis, abdominal pain, and bleeding haemorrhoids. In addition, Corydalis saxicola Bunting injection (CSBI) is widely used against acute and chronic hepatitis. This review aims to provide up-to-date information on the botanical distribution, description, traditional uses, phytochemistry, pharmacology, and clinical applications of CSB. A comprehensive review was implemented on studies about CSB from several scientific databases, such as SciFinder, Elsevier, Springer, ACS Publications, Baidu Scholar, CNKI, and Wanfang Data. Phytochemical studies showed that 81 chemical constituents have been isolated and identified from CSB, most of which are alkaloids. This situation indicates that these alkaloids would be the main bioactive substances and that they have antitumour, liver protective, antiviral, and antibacterial pharmacological activities. CSBI can not only treat hepatitis and liver cancer but can also be used in combination with other drugs. However, the relationships between the traditional uses and modern pharmacological actions, the action mechanisms, quality standards, and the material basis need to be implemented in the future. Moreover, the pharmacokinetics of CSBI in vivo and the toxicology should be further investigated.

Prediction of in-hospital mortality in patients with exertional heatstroke: a 13-year retrospective study.

Hyperactivity of coagulation is common in exertional heatstroke (EHS). Disseminated intravascular coagulation (DIC) is the most severe form of coagulation dysfunction and associated with poor outcome. DIC, temperature and Glasgow coma scale score were identified as independent risk factors for in-hospital mortality by multivariate logistic regression analysis, and we developed a nomogram for predicting in-hospital mortality in a 13-year EHS patient cohort. The nomogram was assessed by calibration curves and bootstrap with 1,000 resamples. The receiver operating characteristic curve was constructed, and the area under the curve (AUC) was compared. Two hundred and ten patients were included. The in-hospital mortality was 9.0%, and the incidence of DIC was 17.6%. The AUC of the nomogram was 0.897 (95% CI 0.848-0.935, p < .0001) and was non-inferior to SOFA and APACHE II scores but superior to SIRS score, which were widely-used score systems of disease severity. The nomogram contributed to the adverse outcome prediction of EHS.

(±)-Corysaxicolaine A: a pair of antitumor enantiomeric alkaloid dimers from Corydalis saxicola.

(±)-Corysaxicolaine A (1), isolated from the aerial parts of Corydalis saxicola for the first time, is a pair of novel dimeric alkaloids, each of which is directly coupled by the rare 6, 12' C-C σ-bond between benzophenanthridine and protoberberine. The enantiomeric separation was achieved using chiral chromatography. Their structures, including stereochemistry, were clarified by carrying out extensive spectroscopic techniques and an electronic circular dichroism (ECD) calculation. (-)-Corysaxicolaine A was observed to exhibit an apparent cytotoxic effect against T24 cells with an IC50 value of 9.45 µM.

Pseudomonas cichorii causing leaf spot disease on Banxia (Pinellia ternata) in China.

Banxia (Pinellia ternata) is an important Chinese medicinal material in the family Araceae and is a widely grown herb in China. In September 2021, a leaf spot disease was observed on Banxia field, with an incidence rate of 35 to 40 % in a 4-ha field, in Zhongxiang City, Hubei Province of China. Symptoms were observed as yellow-white centers, water-soaked edges, irregular lesions, and gradually developed into a yellowish-brown center and a dark-brown edge. Necrotic spots gradually increased, leading to leaf chlorosis and plant death. Margins of leaf lesions were excised form diseased tissue and were plated on nutrient agar (NA) using serial dilution. Growth on NA was predominantly cream-colored circular bacterial colonies with undulated margins. Characterization of three randomly chosen bacterial isolates (JYB1, JYB7 and JYB8) suggests they are Gram-negative, levan negative, arginine dihydrolase negative, oxidase positive, potato soft rot positive, and tobacco hypersensitive positive. Isolates were identified as Pseudomonas cichorii based on the LOPAT scheme (Cottyn et al. 2009). Taxonomic positioning was confirmed genetically by PCR analysis using primers set: 16S rRNA gene universal primers 27F/1492R (Weisburg et al. 1991) and hrcRST gene specific primers Hcr1/Hcr2 (Cottyn et al. 2011). Homology search of 16S rRNA gene sequences (GenBank accessions: JYB1, MZ749668; JYB7, MZ823822; and JYB8, MZ823823) indicated 99.93 % (1396 bp) identity with P. cichorii strains (GenBank accessions: MK356431, JX913785, MZ723344). Similarly, comparison of the hrcRST locus (GenBank accessions: JYB1, MZ977010; JYB7, MZ977011; and JYB8, MZ977012) shared 99.38% (812 bp) with P. cichorii strains (GenBank accessions: CP007039, CP074349, GU324131). Koch's postulate was performed on healthy 30-day-old Banxia plants to confirm pathogenicity of the isolated strains. Leaves were injected with 50 µL bacterial suspensions (1x108 cfu/ml) by sterile syringe. The negative control was inoculated with sterile water. The inoculated Banxia plants were incubated at 28 °C, 70 to 80 % relative humidity, and exhibited water-soaked lesions on the leaf surface within two days around the inoculation sites. Within seven days, all leaves withered and plants died. In contrast, control plants remained healthy and symptomless. The pathogen was consistently reisolated from diseased plants and morphologically and molecularly identified as P. cichorii, while no bacterial colonies were isolated from the control plants, fulfilling Koch's postulates. To our knowledge, this is the first report of bacterial leaf spot caused by P. cichorii on Banxia in China. As one of the main producing areas of Banxia in China, Jianghan Plain of Hubei Province has a planting area of nearly 20 square kilometers. The occurrence of this bacterial disease has the potential threat to the Banxia industry, more research is needed for breeding disease resistance and for developing chemical control.

Effectiveness and safety of percutaneous kyphoplasty combined with zoledronic acid in treatment of osteoporotic vertebral compression fractures: a meta-analysis.

INTRODUCTION: To investigate the clinical effectiveness of combination treatment of percutaneous kyphoplasty (PKP) and zoledronic acid (ZOL) in the treatment of osteoporotic vertebral compression fracture (OVCF). MATERIALS AND METHODS: We searched studies investigating the PKP combined with ZOL in the treatment of OVCF. We used a fixed-effects or random-effects model to analyze the bone mineral density (BMD), visual analogue scale (VAS), Oswestry disability index (ODI), bone markers (N-MID, ß-CTX, and P1NP) and adverse events, expressed as weight mean difference (WMD) and risk ratio (RR) with 95% confidence interval (95% CI). RESULTS: We identified 5 cohort studies with a total of 440 patients. Compared with PKP alone, the combination treatment of PKP and ZOL significantly reduced the VAS score at 6 months (WMD = - 0.78, 95% CI - 1.42, - 0.14; P = 0.018), and 12 months (WMD = - 0.98, 95% CI - 1.46, - 0.51; P < 0.001). Moreover, the combination treatment also improved the BMD at 6 (WMD = 0.06, 95% CI 0.01, 0.11, P = 0.016) and 12 months (WMD = 0.20, 95% CI 0.03, 0.36, P = 0.018) after treatment. The ODI score in the combination group was significantly lower than in PKP group at 6, 12 and 24 months after treatment (at 6 months: WMD = - 9.25, 95% CI - 13.62, - 4.87 P < 0.001; at 12 months: WMD = - 9.21, 95% CI - 11.91, - 6.50, P < 0.001; at 24 months: WMD = - 7.26, 95% CI - 11.39, - 3.14, P = 0.001). The N-MID and P1NP values were found to be significantly lower in the combination group than the PKP group, but the ß-CTX value was similar between the two groups. There was no significant difference in incidence of adverse events between the two groups, but more adjacent vertebral fractures and bone cement leakage occurred in PKP alone group. CONCLUSION: In patients with OVCF, combination treatment of PKP and ZOL showed more effective than PKP alone in improving BMD and bone marker levels, relieving pain, as well as reducing the risk of new fractures. More large-scale RCTs are needed to verify our findings.

[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.

Artemisinin inhibits TLR4 signaling by targeting co-receptor MD2 in microglial BV-2 cells and prevents lipopolysaccharide-induced blood-brain barrier leakage in mice.

Artemisinin and its derivatives have been the frontline drugs for treating malaria. In addition to the antiparasitic effect, accumulating evidence shows that artemisinins can alleviate neuroinflammatory responses in the central nervous system (CNS). However, the precise mechanisms underlying their anti-neuroinflammatory effects are unclear. Herein we attempted to delineate the molecule target of artemisinin in microglia. In vitro protein intrinsic fluorescence titrations and saturation transfer difference (STD)-NMR showed the direct binding of artemisinin to Toll-like receptor TLR4 co-receptor MD2. Cellular thermal shift assay (CETSA) showed that artemisinin binding increased MD2 stability, which implies that artemisinin directly binds to MD2 in the cellular context. Artemisinin bound MD2 showed much less collapse during the molecular dynamic simulations, which supports the increased stability of MD2 upon artemisinin binding. Flow cytometry analysis showed artemisinin inhibited LPS-induced TLR4 dimerization and endocytosis in microglial BV-2 cells. Therefore, artemisinin was found to inhibit the TLR4-JNK signaling axis and block LPS-induced pro-inflammatory factors nitric oxide, IL-1ß and TNF-α in BV-2 cells. Furthermore, artemisinin restored LPS-induced decrease of junction proteins ZO-1, Occludin and Claudin-5 in primary brain microvessel endothelial cells, and attenuated LPS-induced blood-brain barrier disruption in mice as assessed by Evans blue. In all, this study unambiguously adds MD2 as a direct binding target of artemisinin in its anti-neuroinflammatory function. The results also suggest that artemisinin could be repurposed as a potential therapeutic intervention for inflammatory CNS diseases.

Cannabidiol-dihydroartemisinin conjugates for ameliorating neuroinflammation with reduced cytotoxicity.

Cannabidiol (CBD) and dihydroartemisinin (DHA) can alleviate neuroinflammatory responses. However, they show cytotoxicity, which severely limits their therapeutic windows. Therefore, there is a great need to develop neuroprotective agents with improved safety. Drug-drug conjugate is an emerging approach for enhancing therapeutic index. Herein, the development, synthesis, and the pharmacological characterization of CBD-DHA conjugates were performed. Meanwhile, the combination of CBD and DHA as separate entities was also quantitatively analyzed for direct comparison with CBD-DHA conjugates. In this study, BV-2 microglial cell line was used to mimic primary microglia and the effects of CBD, DHA, the combination of CBD and DHA, as well as CBD-DHA conjugates on LPS-activated signaling molecules and pro-inflammatory factors were assessed. The interaction of CBD and DHA in inhibiting LPS-induced nitric oxide (NO) production was found to be additive. In contrast, DHA was found to synergize with CBD in inhibiting BV-2 cellular viability which implies that the combination of CBD and DHA amplifies their cytotoxicity. CBD-DHA conjugate C3D eliminated the cytotoxicity associated with single CBD/DHA use without significantly compromising the anti-neuroinflammation activity. C3D was more potent than C2D and C4D in inhibiting LPS-induced NO and mRNAs of iNOS and IL-1ß, which implies that the linker length is critical for CBD-DHA conjugates' anti-inflammatory activities. Further signaling characterizations showed that C3D inhibited LPS-induced NF-κB but not MAPKs activation in BV-2 cells, therefore blocking LPS-induced neuroinflammation. This work provides a good example that conjugated drug-drug approach may improve the therapeutic index by increasing the maximum tolerated concentration/dose compared to traditional combination strategy.

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 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).

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.

Baseline Sensitivity and Control Efficacy of Strobilurin Fungicide Pyraclostrobin Against Sclerotium rolfsii.

Sclerotium rolfsii is a fungi pathogen of southern blight with broad host range. The quinone outside inhibitor fungicide pyraclostrobin was officially approved for controlling many diseases in 2015. In this study, baseline sensitivity of S. rolfsii to pyraclostrobin was established by measuring the 50% effective concentration (EC50) values of 155 isolates of S. rolfsii. The EC50 values ranged from 0.0291 to 1.0871 µg/ml, with a mean EC50 of 0.4469 ± 0.2490 µg/ml (mean ± standard deviation). In a preventive fungicide in vitro experiment and a field experiment, pyraclostrobin preventive efficacy reached 90% and 80%, respectively, which were much higher than that of the control agent carbendazim. Curative efficacy of pyraclostrobin was significantly lower than its preventive efficacy. Pyraclostrobin at 0.1, 0.5, and 2 µg/ml significantly reduced the number of sclerotia produced on potato dextrose agar medium, but had no significant influence on their total weight. Pyraclostrobin had no significant influence on mycelial cell membrane permeability, but it significantly reduced oxalate secretion and protein synthesis of S. rolfsii. Our findings are of great significance for resistance monitoring of S. rolfsii and also provide new insight into the action mechanism of pyraclostrobin against S. rolfsii.

Specific biotests to assess eco-toxicity of biodegradable polymer materials in soil.

Eco-toxicity investigation of polymer materials was considered extremely necessary for their potential menace, which was widely use as mulching materials in agricultural. In this study, polyethylene (PE), polystyrene (PS) and synthetic biomaterials-Ecoflex and cellulose were applying into soil cultivated with two potential indicator plants species: oat (Avena sativa) and red radish (Raphanus sativum). Variety of chemical, biochemical parameters and enzyme activity in soil were proved as effective approach to evaluate polymers phytotoxicity in plant-soil mesocosm. The F-value of biomass, pH, heavy metal and electoral conductivity of Raphanus behaved significant different from T0. Significant analysis results indicated biodegradation was fast in PE than PS, besides, heavy metals were dramatically decrease in the end implied the plant absorption may help decrease heavy metal toxicity. The increase value at T2 of Dehydrogenase activity (0.84 higher than average value for Avena & 0.91 higher for Raphanus), Metabolic Index (3.12 higher than average value for Avena & 3.81 higher for Raphanus) means during soil enzyme activity was promoted by biodegradation for its heterotrophic organisms' energy transportation was stimulated. Statistics analysis was carried on Biplot PC1 (24.2% of the total variance), PC2 (23.2% of the total variance), versus PC3 (22.8% of the total variance), which indicated phosphatase activity and metabolic index was significant correlated, and high correlation of ammonium and protease activity. Furthermore, the effects were more evident in Raphanus treatments than in Avena, suggesting the higher sensitivity of Raphanus to polymers treatment, which indicate biodegradation of polymers in Raphanus treatment has produced intermediate phytotoxic compounds.

Upconversion nanoparticle and gold nanocage satellite assemblies for sensitive ctDNA detection in serum.

A rapid molecular diagnostic technique targeting circulating tumor DNA (ctDNA) has become one of the most clinically significant liquid biopsy methods for non-invasive and timely diagnosis of cancer. Herein, a sensitive detection system of ctDNA based on a fluorescence resonance energy transfer (FRET) system using upconversion nanoparticles (UCNPs) and gold nanocages (AuNCs) was constructed. Through the doping of Yb and Tm ions, the excitation and emission wavelengths of UCNPs were adjusted to 980 nm and 806 nm, respectively. Subsequently, UCNPs and AuNCs with the corresponding wavelength absorption were linked by complementary pairing of surface-modified DNA to form near-infrared fluorescent nanoprobes (NIR probes). Targeting DNA mutation recognition and signal transduction were realized by using NIR probes through the toehold-mediated strand displacement reaction. This method could detect a single point mutation of the KRAS gene with a wide detection range from 5 pM to 1000 pM and the limit of detection reached 6.30 pM. More importantly, the stable and highly specific NIR probes could be directly used in the serum environment without complicated pretreatment and amplification processes in advance. It could be envisioned that this specific and sensitive ctDNA detection strategy has great potential in clinical diagnosis and monitoring of diverse malignant tumors.