First Report of Leaf Spot on Stephania tetrandra Caused by Albifimbria verrucaria in China.

Stephania tetrandra S. Moore belongs to the family Menispermaceae and is a Chinese medicinal plant widely distributed in tropical and subtropical regions of Asia and Africa. The root can be used for a variety of treatments (Jiang et al. 2020). In August 2021, leaf spot symptoms were observed on S. tetrandra cultivated in Jiangxi (114.456E, 27.379N, southern China). The disease symptoms included a slight constriction of the leaves, with irregularly shaped brown to black spots with well-defined borders. Severely affected leaves were shed by the plant. In order to determine the cause, symptomatic leaves were surface-disinfested with 0.6% NaOCl for 2 min, and rinsed twice in sterile water, then incubated on moist paper towels at 26°C in the dark for 2 days. Cream-colored sporodochia were observed within the leaf spots, turning dark green to black within 16 hours. A slow-growing white fungus was isolated from 95% of the samples (n = 30) on PDA. Dark green sporodochia emerged after 7 to 10 days of incubation, and released tip-end oval, non-septate, hyaline conidia measuring 6.7 to 8.5 µm (mean 7.5 µm, n = 50) by 2.0 to 3.3 µm (mean 2.7 µm, n = 50). Concentric rings were interspersed with sporodochia on the continually incubated mycelium. The morphological characteristics of the isolates matched the description of Albifimbria (Lombard et al. 2016). Nucleotide sequences, amplified from isolate FJL5C using primers of the internal transcribed spacer (ITS) (White et al. 1990), calmodulin (cmdA; Carbone and Kohn 1999), and RNA polymerase II second largest subunit (rpb2; O'Donnell et al. 2007), were deposited in GenBank under accession numbers OM317911, OM386815, and OM386816. A BLASTn analysis of the sequences showed 100% identity with the type strain CBS 328.52 (Lombard et al. 2016) of Albifimbria verrucaria (syn. Myrothecium verrucaria) for ITS, and 99% for cmdA and rpb2 (KU845893, KU845875, and KU845931, respectively). A phylogenetic tree generated using the three sequences showed that the isolate from S. tetrandra grouped with the A. verrucaria isolates, but away from other species of Albifimbria. These results together with the lack of a pale luteus exudate produced by A. viridis (Lombard et al. 2016) implied that the isolate was A. verrucaria. The culture was deposited in Guangdong Microbial Culture Collection Center (GDMCC 3.716). To verify pathogenicity, conidial suspension (106 conidia/mL in 0.05% Tween 20 solution) was sprayed onto six healthy plants. Six other plants sprayed with the Tween 20 solution alone served as controls. All plants were incubated in the dark at 26°C and 95% humidity for 30 hours, then transferred to a greenhouse at 26°C and 12 hours of illumination per day for 2 to 3 days. Inoculated leaves developed similar symptoms to those described above, whereas control plants remained healthy. The same pathogen was isolated from the diseased leaves, with the same morphological and molecular traits as those from the field plants. This experiment fulfilled Koch's postulates and confirmed that A. verrucaria causes leaf spots on S. tetrandra. This pathogen has been reported to cause disease in a wide range of weeds, legumes, and crop plants (Herman et al. 2020). To our knowledge, this is the first report of A. verrucaria causing leaf spots on S. tetrandra in natural or controlled environments. The disease can seriously threaten S. tetrandra on growth and yield loss.

First Report of Sclerotium rolfsii Causing Southern Blight on Stephania tetrandra in China.

Stephania tetrandra S. Moore is a perennial liana and is widely cultivated in southern China for traditional Chinese medicine as a diuretic, anti-inflammatory, and antirheumatic treatment (Jiang et al. 2020). In August 2021, it was observed that a severe stem rot disease affected St. tetrandra cultivated in Anfu, Jiangxi province, China (114°27'26" E, 27°22'46" N). The disease symptoms included constriction and rot at the base of the stem, and covered with a layer of white mycelia. The plants above-ground finally wilted and dried with a disease incidence ranging from 8% to 16%. Lots of dried plants formed withered patches of field. Sections (1.0~2.0 cm) from browning stem tissues were surface-disinfected with 75% ethanol for 15 s, followed by 60 s in 4% NaClO, rinsed twice in sterile water, dried on sterilized filter paper, placed on potato dextrose agar (PDA), and incubated at 26°C in the dark for 3 days. A white rhizomorphic fungal mycelium, that is similar to the mycelium of strain FJSR0 on the surface of an infected plant in the field, was isolated from the cultured tissues with 67% frequency. When incubated on PDA, white and fluffy mycelia with even margins and a slight halo formed. Mycelia-produced clamp connections were observed. Colonies grew quickly and covered the dish (diameter: 9 cm) in 5 or 6 days. After that, sclerotia were initially white, then turned yellow, and chestnut brown at maturity. Spherical and subspherical sclerotia were observed after 8 days, with each plate containing 448 to 634 sclerotia (0.8 to 1.4 mm diameter; mean = 0.94 mm; n = 50). On the basis of morphology, the pathogen was similar to Sclerotium rolfsii Sacc. [teleomorph: Athelia rolfsii (Curzi) Tu & Kimbrough] (Sun et al. 2020; Ling et al. 2021). For molecular confirmation, the internal transcribed spacer (ITS) region with approximately 680 bp was amplified from strains FJRS0 and FJRS1 using primers ITS1/ITS4 (White et al. 1990). Two distinct types (different in one SNP and one 1-bp InDel) of ITS sequences were obtained from each isolate, and all isolates contain the two types (FJSR0: ON972516, ON972517; FJSR1: ON972520, ON972518). BLAST analysis of each type found that the hits, with identities >99%, are A. rolfsii except for two Sc. delphinii sequences (GU567775.1 and MK073010.1). Phylogenetic analysis placed strains FJSR0 and FJSR1 in the same clade as Sc. rolfsii but away from Sc. delphinii based on the previous method (Sun et al. 2021). Both morphological and molecular characteristics confirmed that the strains were Sc. rolfsii. For pathogenicity tests, PDA plugs (8 mm in diameter) covered with 5-day-old fungal mycelium were inoculated at the stem bases of three healthy St. tetrandra seedings and incubated at 26℃ and relative humidity of 80%. On the fifth day, inoculated plants were wilting. The infected stem bases turned brown to black and constricted as previously observed in the field. Some leaves, infected by the mycelium expanded from the PDA plugs, developed an orange and irregular spot. Sclerotia were observed 20 days post inoculation. In contrast, the leaves and stems of non-inoculated control plants remained symptomless. Pathogenicity tests were repeated three times. The fungus was reisolated consistently from each symptomatic tissue, thus completing Koch's postulates. Although Sc. rolfsii has been previously reported to cause a southern blight symptoms on vegetables, ornamentals, grass, and medicinal and leguminous crops (Sun et al. 2020; Ling et al. 2021), this is the first report of Sc. rolfsii causing similar symptoms of southern blight on St. tetrandra in China.

Effects and Mechanism of Berberine on the Dexamethasone-Induced Injury of Human Tendon Cells.

OBJECTIVE: To investigate the effects of berberine (Berb) on dexamethasone- (Dex-) induced injury of human tendon cells and its potential mechanism. METHODS: CCK-8 assay was used to explore the appropriate concentration of Dex-induced injury of tendon cells and the doses of Berb attenuates Dex cytotoxicity; cell wound healing assay was used to detect the effects (P < 0.05) of Berb and Dex on the migration ability of tendon cells; flow cytometry was used to measure cell apoptosis; DCF DA fluorescent probe was used to measure the ROS activity of cells. Western blotting was used to detect the expression of phenotype related factors including smooth muscle actin α (SMA-α), type I collagen (Col I), col III, apoptosis-related factors, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, and PI3K/AKT. RESULTS: CCK-8 assay showed that 1-100 µM Dex significantly inhibited the proliferation of tendon cells in a concentration-dependent manner (P < 0.05), where the inhibitory effect of 100 µM Dex was most significant (P < 0.005), and the pretreatment of 150, 200 µM Berb could reverse those inhibitions (all P < 0.05). Compared with the control group, Dex significantly inhibited cell migration (P < 0.05), while Berb pretreatment could enhance cell migration (P < 0.05). Flow cytometry and ROS assay showed that Dex could induce apoptosis and oxidative stress response of tendon cells (all P < 0.05), while Berb could reverse those responses (P < 0.05). Western blot showed that Dex could inhibit the expression of the col I and III as well as α-SMA (all P < 0.05) and enhance the expression of apoptosis-related factors including cleaved caspase-3 and cleaved caspase-9 (all P < 0.05). Besides, Dex could also inhibit the activation of the PI3K/AKT signaling pathway (all P < 0.05), thus affecting cell function, while Berb treatment significantly reversed the expression of those above proteins (all P < 0.05). CONCLUSION: Berb attenuated DEX induced reduction of proliferation and migration, oxidative stress, and apoptosis of tendon cells by activating the PI3K/AKT signaling pathway and regulated the expression of phenotype related biomarkers in tendon cells. However, further studies are still needed to clarify the protective effects of Berb in vivo.

Effect of modified Qing Long Bai Wei needling on the levels of IL-1β, IL-6 and INF-α in synovial fluid of knee osteoarthritis patients / 针灸推拿医学（英文版）

Objective:To compare the modified Qjng Long Bai Wei needling method and ordinary acupuncture method in the effects of improving the levels of interleukin (IL)-1β,IL-6 and tumor necrosis factor (TNF)-α in the treatment of knee osteoarthritis (KOA),and to determine the advantage of the modified Qing Long Bai Wei needling method for KOA.Methods:One hundred KOA patients were randomized into a treatment group and a control group by using the random number table,with 50 cases in each group.The treatment group was intervened by the modified Qing Long Bai Wei needling method,and the control group was given ordinary acupuncture.The two groups were observed before and after the treatment to determine the changes in the levels of IL-1β,IL-6 and TNF-α in synovial fluid,and the clinical efficacies were compared between the two groups.Results:The total effective rate and clinical recovery rate were 97.9％ and 52.1％ respectively in the treatment group,versus 85.1％ and 25.5％ in the control group,and the between-group differences were statistically significant (both P＜0.01).After the treatment,the levels of Ib1β,IL-6 and TNF-cα in synovial fluid changed significantly in both groups (all P＜0.01);there were significant differences in the levels of IL-1β,IL-6 and TNF-α in synovial fluid between the two groups (all P＜0.01).Conclusion:The modified Qjng Long Bai Wei needling is an effective method for KOA and it can significantly improve the levels of IL-1β,IL-6 and TNF-α in synovial fluid.

[Simultaneous determination of nine chemical markers of bletillae rhizoma by ultra performance liquid chromatography].

A UPLC method has been developed in the current investigation for simultaneous determination of nine chemical markers of Bletilla striata, 4-hydroxymethylphenyl beta-D-glucoside, blestroside, dactylorhin A, militarine, dihydrophenanthrene 5, gymnoside V, dihydrophenanthrene 1, benzylphenanthrene 3 and gymnosides IX. Separation was performed at 45 degrees C on an ACQUITY UPLC BEH C18 column (2.1 mm x 150 mm, 1.7 microm) with a gradient solvent system of acetonitrile-water as the mobile phase. The flow rate was 0.3 mL x min(-1), the detection wavelength was 280 nm. The results showed that the nine chemical markers could be well resolved and that in the selected linear range, all calibration curves of the nine chemical markers showed good linearity (r > or = 0.999 3). The recoveries (n = 6) were in the range of 98.15% - 102.2% and RSDs were between 2.1% - 3.6%. The data suggested that the developed UPLC-UV method had good reproducibility, robustness, and accuracy, which was suitable for the quality control of Bletilla striata. Applications of the method showed that the nine chemical markers had higher contents in the wild B. striata than in the cultivated ones.

[Effects of methyl cantharidimide tablets on urinary protein and enzymes in Beagle dogs].

OBJECTIVE: To investigate the nephrotoxic effects of methyl cantharidimide tablets on urinary protein and enzymes in Beagle dogs. METHOD: Beagle dogs were randomly divided into negative control group(blank tablet), methyl cantharidimide tablets group (6.11,12.21, 24.42 mg x kg(-1)), continuously 30 days of oral adminiStration, once a day. The drug and control group were collected and determined fresh urine in 1, 2, 3 and 4 weeks of the administration; Serum urea nitrogen (BUN), creatinine (Crea), total protein (TP) and albumin (ALB) as well as sodium, potassium, chloride electrolyte were determined on 15 and 30 days of the administration; Urine albumin (mAlb), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin( NGAL), N-acetyl-beta-D-glucosaminidase (NAG), clusterin, beta2-microglobulin (beta2-MG), alpha1-microglobulin (alpha1-MG), alanine aminopeptidase( AAP) and im- munoglobulins IgG were tested on 15 and 30 days of the administration. RESULT: Compared with the control group, urine protein and white blood cells was significantly increased in each dose group. On 15 days of the administration, mAlb were higher in each dose group, KIM-1, NGAL, clusterin, NAG and AAP were significantly higher in high-dose group, while the middle and low dose group had no significant difference, as well as blood SCr and BUN no obvious abnormalities. On 30 days, mAlb, KIM-1, clusterin, NAG, AAP were increased in each dose group, appearing dose-effect relationship, beta2-MG and NGAL levels were significantly increased in high-dose group. Contents above indicators were increased with significant dose and time relationship, and serum BUN, Scr were correlated, suggesting that urine mAlb, KIM-1, clusterin, NAG and AAP indicators that can sensitively respond the changes of proteins and enzymes in urine. CONCLUSION: Methyl cantharidimide tablets has a renal toxicity, urine mAlb, KIM-1, clusterin, NAG and AAP can be used as the early nephrotoxic biomarkers of methyl cantharidimide tablets.

[Experiment study of total anthraquinone in cassiae semen on lipid peroxidation and PPAR-gamma expression in liver tissues of rats with alcoholic fatty liver].

OBJECTIVE: To investigate the effect of total anthraquinone in Cassiae Semen on lipid peroxidation and peroxisome proliferator activated receptors gamma (PPAR-gamma) expression in liver tissues of rats with alcoholic fatty liver. METHOD: Referring to literature, it was established animal models of fatty liver feeding with alcohol. Rats were randomized into 6 groups, except the normal group, the other 5 groups of rats had been administered alcohol two times a day for 3 months. Rats were killed at the end of this experiment. It were respectively measured that the contents of ALT, AST, AKP, TG, TC, HDL-C, LDL-C, MDA, SOD, FFA in the serum and TG, TC, MDA, SOD, HL, LPL, FFA in the liver. The left leaf of liver was observed by histopathological staining, the immunohistochemical staining and RT-PCR were used to observe the effects on the expressions of PPAR-gamma mRNA. RESULT: Compared with the model group, total anthraquinone in Cassiae Semen could remarkably decrease the content of ALT, AST, TC, TG, MDA and increase the content of SOD in the serum of the experimental fatty liver induced by alcohol; remarkably decrease the content of TC, TG, FFA and increase the content of HL, LPL, SOD in the liver of the experimental fatty liver with induced by alcohol. Total anthraquinone in Cassiae Semen group was the similar the model group, but remarkably lighten inflammatory cell intiltration and fibrosis increasing. The RT-PCR and immunohistochemical staining results showed that: compared with the normal group,the model group could remarkably decrease the expression of PPAR-gamma mRNA in the liver (P < 0.01). Compared with the model group, total anthraquinone in Cassiae Semen could remarkably increase the expression of PPAR-gamma mRNA in the liver of the experimental fatty liver (P < 0.01). CONCLUSION: The results showed that total anthraquinone in Cassiae Semen has good effects on the treatment of hepatic fat induced by alcohol diet in rats. the possible action mechanism of total anthraquinone in Cassiae Semen possess obvious effect of regulating the disorder of lipid metabolism, ameliorating hepatic function, as well as anti-lipidperoxidation, increasing the expression of PPAR-gamma in hepatic cells of rats.

Expression of early growth response factor-1 in rats with cerulein-induced acute pancreatitis and its significance.

AIM: To observe the expressions of early growth response factor-1 (Egr-1) and tissue factor (TF) in rats with cerulein-induced acute pancreatitis and to explore its significance. METHODS: A large dose of cerulein was used to create the experimental acute pancreatitis model in rats. The changes of Egr-1 mRNA and protein in rats were observed during 30 min to 4 h after the treatment and immunohistochemical method was used to observe the localized expression of Egr-1 in tissues. In addition to the mRNA expression of Egr-1 target gene, TF was also observed. A blank control group, and a bombesin-administered group were used for comparison. RESULTS: After the stimulation of a large dose of cerulein, the rats showed typical inflammatory changes of acute pancreatitis. Thirty minutes after the stimulation, the mRNA expression of Egr-1 in the pancreatic tissue reached its peak and then declined, while the expression of Egr-1 protein reached its peak 2 h after the stimulation. Histologically, 2 h after the stimulation, almost all pancreatic acinar cells had the expression of Egr-1 protein, which was focused in the nuclei. The mRNA expression of TF occurred 1 h after the stimulation and gradually increased within 4 h. However, a large dose of bombesin only stimulated the pancreatic tissue to produce a little mRNA expression of Egr-1 and no mRNA expression of Egr-1 protein and TF. CONCLUSION: Egr-1 as a pro-inflammatory transcription factor may play an important role in the pathogenesis of acute pancreatitis by modulating the expression of TF.

[Expressions of early growth response 1 and tissue factor in caerulein-induced acute pancreatitis tissues in rats].

OBJECTIVE: To observe the expressions of early growth response 1 (Egr-1) and tissue factor (TF) in rat tissues of acute pancreatitis induced by caerulein and to explore their significance. METHODS: Pancreatitis was induced in rats by high-dose intraabdominal caerulein injection. The changes of Egr-1 mRNA and protein in pancreas were measured by quantitative PCR and Western blotting, and the localization of Egr-1 protein in acinar cells was visualized by immunohistochemistry. TF mRNA levels were also measured by quantitative PCR. High-dose bombesin-stimulated rats served as the negative control. RESULTS: Egr-1 mRNA was rapidly increased in the pancreas of rats stimulated by high-dose cearulein, and reached the peak level 30 min after the stimulation, whereas band for peak Egr-1 protein level was visualized by Western blotting till 2 h after stimulation. Immunohistochemistry showed that almost every acinar cell in the pancreas was Egr-1-positive, especially in the nucleus. In line with Egr-1 activation, TF mRNA was detected 1 h after the stimulation and increased steadily within the initial 4 h. Only a small quantity of Egr-1 mRNA expression was observed in bombesin-stimulated rats, in which no Egr-1 protein or TF mRNA were detected. CONCLUSION: Egr-1 mRNA and protein were up-regulated in the early stage of pancreatitis. Egr-1, as a pro-inflammatory transcriptional factor, probably plays an important role in the initiation of acute pancreatitis, and its action might be partially mediated through the up-regulation of TF expression.