Screening and characterization of biocontrol bacteria isolated from Ageratum conyzoides against Collectotrichum fructicola causing Chinese plum (Prunus salicina Lindl.) anthracnose.

Chinese plum (Prunus salicina Lindl.) is a nutritionally and economically important stone fruit widely grown around the world. Anthracnose, caused by Collectotrichum spp., is one of the primary biotic stress factors limiting plum production. Medicinal plants may harbor rhizospheric or endophytic microorganisms that produce bioactive metabolites that can be used as anthracnose biocontrol agents. Here, 27 bacterial isolates from the medicinal plant A. conyzoides with diverse antagonistic activities against C. fructicola were screened. Based on morphological, physiological, biochemical, and molecular characterization, 25 of these isolates belong to different species of genus Bacillus, one to Pseudomonas monsensis, and one more to Microbacterium phyllosphaerae. Eight representative strains showed high biocontrol efficacy against plum anthracnose in a pot experiment. In addition, several Bacillus isolates showed a broad spectrum of inhibitory activity against a variety of fungal phytopathogens. Analysis of the volatile organic compound profile of these eight representative strains revealed a total of 47 compounds, most of which were ketones, while the others included alkanes, alkenes, alcohols, pyrazines, and phenols. Overall, this study confirmed the potential value of eight bacterial isolates for development as anthracnose biocontrol agents.

Identification and Observation of Infection Processes of Colletotrichum Species Associated with Pearl Plum Anthracnose in Guangxi, China.

Pearl plum (Prunus salicina Lindl.) is mainly cultivated in Tian'e County in Guangxi Province, southern China. Anthracnose is a devastating disease on pearl plum, causing extensive leaf blight. Diseased leaves were sampled from 21 orchards in Tian'e County. Isolates were first screened for ones resembling Colletotrichum, and 21 representative isolates were selected for sequencing of portions of the ribosomal internal transcribed spacer (ITS), the intergenic region of apn2 and MAT1-2-1 genes (ApMAT), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), chitin synthase (CHS-1), and ß-tubulin 2 (TUB2). Based on colony, conidial, and appressorial morphology and sequence analyses, the Colletotrichum isolates associated with pearl plum anthracnose were identified as four species: Colletotrichum fructicola (16 isolates), C. gloeosporioides (3 isolates), C. cigarro (1 isolate), and C. siamense (1 isolate). The results of pathogenicity tests showed that isolates of all four species were pathogenic to wounded leaves of pearl plum seedlings. In this study, we microscopically observed the infection processes of isolates of these four species on attached pearl plum leaves. For C. cigarro and C. siamense, the entire infection processes took 120 h; for C. fructicola and C. gloeosporioides, it only took 72 h. This is the first report of C. fructicola and C. cigarro causing anthracnose on pearl plum worldwide, and also the first report of C. siamense causing anthracnose on pearl plum in China.

Banxia xiexin decoction affects drug sensitivity in gastric cancer cells by regulating MGMT expression via IL­6/JAK/STAT3­mediated PD­L1 activity.

Banxia xiexin decoction (BXXX) is a classic preparation used to treat gastrointestinal diseases, and also has certain therapeutic effects on gastrointestinal tumors. BXXX has been reported to regulate the expression of proteins associated with drug resistance and sensitivity in tumors, and thus, the aim of the present study was to investigate the mechanisms of BXXX drug sensitivity in gastric cancer (GC). The expression levels of programmed cell death 1 ligand 1 (PD­L1), 6­O­methylguanine­DNA methyltransferase (MGMT) and STAT3 were immunohistochemically detected in the cancer and adjacent non­cancer tissues of patients with GC, and in vitro experimentation was conducted using drug­resistant and ­sensitive GC cells. The expression levels of PD­L1, MGMT and STAT3 were determined using reverse transcription­quantitative PCR. Different concentrations of BXXX drug serum were used to treat the cells and the cellular inhibition rate was assessed using a Cell Counting Kit­8 assay. Flow cytometry was used to detect apoptosis, and western blot analysis was used to detect the expression levels of IL­6, IFN­Î³, JAK/STAT3 pathway proteins, PD­L1 and MGMT. The association between PD­L1 and MGMT protein expression levels was subsequently assessed via co­immunoprecipitation. Furthermore, in vivo studies were conducted following the establishment of a drug­resistant tumor­bearing mouse model, where GC tumor size was assessed under different treatment conditions, and western blot analysis was used to detect the expression of related pathway proteins. The expression levels of PD­L1, MGMT and STAT3 were significantly increased in GC tissues, GC cells and cisplatin­resistant cells. Furthermore, BXXX inhibited the proliferation of drug­resistant cells and promoted the inhibitory effects of chemotherapeutic drugs on drug­resistant cells. BXXX also inhibited the expression levels of IL­6, IFN­Î³ and JAK/STAT3 pathway proteins, as well as the expression levels of PD­L1 and MGMT. Colivelin, an activator of STAT3, reversed the effects of BXXX on drug­resistant GC cells, and significantly reversed the effect of BXXX on PD­L1 expression. In conclusion, BXXX was found to influence the drug sensitivity of GC cells by regulating the expression of MGMT. This process functions viaPD­L1, which was itself mediated by IL­6/JAK/STAT3 signaling.

Banxia Xiexin Decoction Inhibits the Expression of PD-L1 Through Multi-Target and Multi-Pathway Regulation of Major Oncogenes in Gastric Cancer.

PURPOSE: Banxia xiexin decoction (BXXX) is a classical Chinese herbal compound for the treatment of gastrointestinal diseases. Its ingredients are also considered helpful for cancer rehabilitation. Here, we will explore the regulatory mechanism of BXXX acting on PD-L1 in gastric cancer (GC). METHODS: GC samples and the general baseline data of the patients were collated. Immunohistochemical (IHC) detected the expression of programmed cell death-ligand 1(PD-L1), hypoxia-inducible factor-1 (HIF-1), epidermal growth factor receptor (EGFR), interferon-γ receptor (IFNGR) and Toll-like receptor 4 (TLR4). ELISA detected the expressions of EGF, IFNG and IL-6 in serum samples. Network tools were used to analyze the potential molecules of BXXX. In the cell experiment, CCK-8 detected the cell proliferation. Tunel detected the apoptosis. Western blot detected the expression of related proteins. In animal experiments, the tumor volume of GC-bearing mice was observed. Expression of EGF, IFNG and IL-6 in the serum of tumor-bearing GC mice were detected by ELISA. Western blot detected the expression of related proteins. RESULTS: The expressions of PD-L1, HIF-1, EGFR, IFNGR and TLR4 in the tissues of GC patients were significantly increased, and the expressions of EGF, IFNG and IL-6 in serum were increased. The molecular results of the network tools showed that BXXX and its main components have a targeting effect on the key molecules of each pathway in the PD-L1 regulatory network. Cell experiments showed that BXXX can inhibit the expression of PD-L1, HIF-1, EGFR and TLR4, but has no significant effect on the expression of IFNGR, thus inhibiting the proliferation and promoting the apoptosis of GC cells. The results were consistent with the animal experiments on tumor-bearing gastric cancer mice. CONCLUSION: BXXX inhibited the expression of PD-L1 through multi-target and multi-pathway regulation of major oncogenes in GC, thus effect cell proliferation and apoptosis.

First Report of Colletotrichum siamense Causing Leaf Spot on Alocasia macrorrhiza in China.

Alocasia macrorrhiza (L.) Schott, known as Alocasia is found in the Araceae, and is widely planted in southern China for its ornamental and medicinal value. This plant has a wide range of pharmacological effects, and has potential anti-tumor activity (Lei et al. 2013). In July of 2019, leaf spots were observed on A. macrorrhiza in the Xixiangtang Area, Nanning, Guangxi, China. Disease symptoms began with water-soaked yellow-green spots and progressed to form brown, round or oval lesions with yellow halos. Under severe conditions, spots merged into larger irregular lesions. More than 60% of the plants in a 0.5 ha field showed disease symptoms. Symptomatic leaves were collected and cut into small pieces (3×3 mm). Leaf pieces from the margin of the necrotic tissue were surface sterilized in 75% alcohol for 10 s, followed by 2% sodium hypochlorite solution for 2 min, then rinsed three times in sterile distilled water. Tissues were plated on potato dextrose agar (PDA) and incubated at 28°C for 5 days in the dark. Among over 30 isolates, most shared a similar morphology, the isolation rate of these was 86.7% and three of these (GY1-1A, GY1-1B, and GY1-1C) were chosen for single-spore purification and used for fungal morphological characterization and identification. White feathery aerial mycelia with olivaceous gray mycelia below were observed in 7-day cultures. After 14 days, orange conidia were observed. Conidia were hyaline, guttulate, smooth, one-celled, and cylindrical, averaged 13.79 µm × 5.26 µm, 13.89 µm × 5.33 µm and 13.92 µm × 5.42 µm for GY1-1A, GY1-1B and GY1-1C, respectively. Appressoria were mostly irregular in outline, deeply lobed or lightly lobed, gray brown to dark brown, conidial appressoria were 7.93 to 8.74 µm × 5.26 to 5.42 µm, mycelial appressoria were 7.15 to 10.11 µm × 5.60 to 7.44 µm. These morphological characteristics were similar to the C. siamense as previously described (Weir et al. 2012). The partial internal transcribed spacer (ITS) regions, actin (ACT), chitin synthase (CHS-1), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), ß-tubulin (TUB2), and the intergenic region of apn2 and MAT1-2-1 (ApMAT) were amplified from genomic DNA for the three isolates using primers ITS4/ITS1 (White et al. 1990), ACT-512F/ACT-783R, CHS-79F/CHS-354R, GDF1/GDR1, CL1C/CL2C, Bt2a/Bt2b (Weir et al. 2012), and AM-F/AM-R (Silva et al. 2012) and sequenced. All sequences showed over 99% identity with C. siamense and were deposited in GenBank (ITS, MW040179-MW040181; ACT, MW049220-MW049222; CHS-1, MW049229-MW049231; GAPDH, MW049232-MW049234; CAL, MW049226-MW049228; TUB, MW049235-MW049237; ApMAT, MW049223-MW049225). Maximum Likelihood (ML) phylogenetic tree was constructed with MEGA 5 using the concatenation of multiple sequences (ACT, CHS-1, GAPDH, ITS, TUB2, CAL). According to the phylogenetic tree, all three isolates were found with C. siamense with 95% bootstrap support. To confirm pathogenicity, three sets (three plants per set) of healthy leaves were slightly scratched with autoclaved toothpicks at each of eight locations. Each inoculation location was a cross (2 mm length) and inoculation location was at least 3 cm apart. Ten µl of conidial suspension (106 conidia /ml in 0.1% sterile Tween 20) was applied to the inoculation areas. A control group was mock inoculated with 0.1% sterile Tween 20. Plants were covered with plastic bags to maintain a high humidity environment and placed in a 28°C growth chamber with constant light for 7 days. Inoculated leaves showed yellowish brown spots (0.4 × 0.65 cm), but no symptoms were observed in the control group. The fungus was reisolated from inoculated leaves, and these isolates matched the molecular and morphological characteristics of the original isolates confirming Koch's postulates. Reported hosts of this pathogen include Coffea arabica, Carica papaya, Melilotus indicus and Litchi chinensis (Weir et al. 2012; Qin et al. 2017; Ling et al. 2019) and so on. To our knowledge, this is the first report of C. siamense causing leaf spot on A. macrorrhiza in China. The identification of this pathogen provides a foundation for the management of leaf spot on this medicinal plant.

Efficacy and safety of Xiangsha Liujunzi granules for functional dyspepsia: A multi-center randomized double-blind placebo-controlled clinical study.

AIM: To assess the efficacy and safety of a Chinese herbal medicine (CHM), Xiangsha Liujunzi granules, in the treatment of patients with functional dyspepsia (FD). METHODS: We performed a randomized, double-blind, placebo-controlled trial with patients from three centers. Two hundred and sixteen subjects diagnosed with FD according to ROME III criteria and confirmed by upper gastrointestinal endoscopy and spleen-deficiency and Qi-stagnation syndrome were selected to receive Xiangsha Liujunzi granules or placebo for 4 wk in a 2:1 ratio by blocked randomization. The subjects also received follow-up after the 4-wk intervention. Herbal or placebo granules were dissolved in 300 mL of water. Participants in both groups were administered 130 mL (45 °C) three times a day. Participants were evaluated prior to and following 4 wk of the intervention in terms of changes in the postprandial discomfort severity scale (PDSS) score, clinical global impression (CGI) scale score, hospital anxiety and depression scale (HADS) score, traditional Chinese medicine symptoms score (SS), scores of various domains of the 36-item short form health survey (SF-36), gastric emptying (GE) and any observed adverse effects. RESULTS: Compared with the placebo group, patients in the CHM group showed significant improvements in the scores of PDSS, HADS, SS, SF-36 and CGI scale (P < 0.05 or P < 0.01). They also showed the amelioration in the GE rates of the proximal stomach and distal stomach (P < 0.05 or P < 0.01). CONCLUSION: Xiangsha Liujunzi granules offered significant symptomatic improvement in patients with FD.

[Analysis of Animal Modeling Methods for Functional Dyspepsia].

Functional dyspepsia (FD) is a common disease in clinics. It is necessary to establish suitable animal models for clarifying the pathogenesis of FD. FD belongs to "Piman" (abdominal disten- sion) , "Weiwantong" (epigastric pain) , "Caoza" (epigastric upset) in Chinese medicine (CM). It is inor- ganic disease but functional disease. There is no unified standard for FD animal models. Pi deficiency syndrome model is often used as FD animal model now, but they are not completely the same thing. Au- thors summarized and analyzed common methods for FD modeling.

Efficacy of Gastrosis No.1 compound on functional dyspepsia of spleen and stomach deficiency-cold syndrome: a multi-center, double-blind, placebo-controlled clinical trial.

OBJECTIVE: To assess the efficacy and safety of Gastrosis No.1 compound in the treatment of functional dyspepsia with Spleen (Pi) and Stomach (Wei) deficiency-cold syndrome. METHODS: A randomized, double-blind, placebo-controlled trial was performed in 5 centers. Patients with functional dyspepsia (FD) of Spleen-deficiency and qi-stagnation syndrome (162 cases) were randomly assigned to groups given Chinese herbal medicine (CHM) Gastrosis No.1 compound or placebo in a 2:1 ratio. This trial included a 4-week treatment period and a 4-week follow-up period. The outcomes were the dyspepsia symptom scores (measured by total dyspepsia symptom scale and single dyspepsia symptom scale) and syndromes of traditional Chinese medicine score (measured by traditional Chinese medicine syndrome scale). The outcomes were noted at weeks 0, 4 and 8. RESULTS: Compared with patients in the placebo group, patients in the CHM group showed significant improvement in the dyspepsia symptom scores as rated by patients and investigators (P <0.01), and also showed improvement in syndromes of traditional Chinese medicine score (P <0.01). No serious adverse event was reported. Safety tests obtained after 4 weeks of treatment showed no abnormal values. CONCLUSION: CHM Gastrosis No.1 compound was effective and safe in the treatment of functional dyspepsia with Spleen and Stomach deficiency-cold syndrome.

Efficacy of modified ban xia xie xin decoction on functional dyspepsia of cold and heat in complexity syndrome: a randomized controlled trial.

Background. Chinese herbal medicine (CHM) has been used in China and elsewhere to treat patients with functional dyspepsia (FD). However, controlled studies supporting the efficacy of such treatment are lacking. Objective. To assess the efficacy and safety of modified Ban xia xie xin decoction in patients with FD of cold and heat in complexity syndrome. Methods. We performed a randomized, double-blind, placebo-controlled trial involving patients from five centers. Patients with FD of cold and heat in complexity syndrome (n = 101) were randomly assigned to groups given either CHM modified Ban Xia Xie Xin decoction or placebo in a 2 : 1 ratio. Herbal or placebo granules were dissolved in 300 mL of boiled water cooled to 70°C. Patients in both groups were administered 150 mL (50°C) twice daily. The trial included a 4-week treatment period and a 4-week followup period. The primary outcomes were dyspepsia symptom scores, measured by the total dyspepsia symptom scale and the single dyspepsia symptom scale at weeks 0, 1, 2, 3, 4, and 8. Results. Compared with patients in the placebo group, patients in the CHM group showed significant improvements according to the total and single dyspepsia symptom scores obtained from patients (P < 0.01) and investigators (P < 0.01). Conclusions. CHM modified Ban Xia Xie Xin decoction appears to offer symptomatic improvement in patients with FD of cold and heat in complexity syndrome. Trial Registration. Chinese Clinical Trial Registry (ChiCTR): ChiCTR-TRC-10001074.

Efficacy of modified LiuJunZi decoction on functional dyspepsia of spleen-deficiency and qi-stagnation syndrome: a randomized controlled trial.

BACKGROUND: Chinese herbal medicine (CHM) has been used in China and some other countries for the treatment of patients with functional dyspepsia (FD). However, controlled studies supporting the efficacy of such treatments in patients with FD are lacking. In this trial, we aimed to assess the efficacy and safety of modified LiuJunZi decoction in patients with FD of spleen-deficiency and qi-stagnation syndrome. METHODS: We performed a randomized, double-blind, placebo-controlled trial with patients from five centers. Patients with FD of spleen-deficiency and qi-stagnation syndrome (n = 160) were randomly assigned to groups given CHM modified LiuJunZi decoction or placebo in a 2:1 ratio. Herbal or placebo granules were dissolved in 300 ml of boiled water cooled to 70°C. Patients in both groups were administered 150 ml (50°C) twice daily. The trial included a 4-week treatment period and a 4-week follow-up period. The primary outcomes were dyspepsia symptom scores, measured by the total dyspepsia symptom scale and the single dyspepsia symptom scale at weeks 0, 1, 2, 3, 4 and 8. The secondary outcome was the change of radiopaque barium markers emptied from the stomach between week 0 and week 4 of treatment. RESULTS: Compared with patients in the placebo group, patients in the CHM group showed significant improvements according to the scores of total dyspepsia symptoms and single dyspepsia symptoms obtained from patients (P < 0.01) and investigators (P < 0.01). They also showed an improvement in the number of radiopaque barium markers emptied from the stomach (P < 0.05). CONCLUSIONS: CHM modified LiuJunZi decoction appears to offer symptomatic improvement in patients with FD of spleen-deficiency and qi-stagnation syndrome. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR): http://ChiCTR-TRC-10001074.

Bioinformatics research on chronic superficial gastritis of Pi-deficiency syndrome by gene arrays.

OBJECTIVE: To determine the bioinformatical characteristics of differential gene expression in patients with chronic superficial gastritis (CSG) with the Pi-deficiency syndrome (PDS) and those of the non-Pi-deficiency syndrome (non-PDS), i.e. patients of CSG with Pi-Wei dampnese-heat syndrome and healthy persons. METHODS: With the BRB-Array Tools software package, original data collection and bioinformatic: analysis of gene arrays were conducted in 6 CSG patients of PDS (CSG-PDS), 6 CSG patients of non-PDS (CSG-nPDS), and 6 healthy volunteers (Normal). RESULTS: Compared with non-PDS, the gene expressions: in PDS with regards to protein synthesis, energy metabolism, immune reaction and ionic transport tended to be down-regulated, while those concerning secretion, cytoskeleton and ubiquitinization were up-regulated dominantly. CONCLUSIONS: The two kinds of samples, CSG-PDS/Normal and CSG-PDS/CSG-nPDS, have their respective gene expression profiles with different characteristics. Gene expression profile has certain referential significance in syndrome classification.

[Primary exploration on immune associated genome of patients with Pi-Qi deficiency syndrome].

OBJECTIVE: To investigate the abnormal change of immune function in patients with Pi-Qi deficiency Syndrome, and to explore the genomic mechanism of its genesis by cDNA chip techniques. METHODS: The cross probe was made by extracting and microamplifying the total RNA and mRNA of peripheral white blood cells (WBC) in healthy subjects and patients with chronic gastritis and ulcerative colitis, which were labeled by Cy3 and Cy5 respectively. Then equal quantity of the two labeled probes were mixed and hybridized with cDNA chip, fluorescent signal of the chips were scanned with scanner. Data obtained were analyzed for comparing the difference of the expressive levels of immune associated genome in peripheral WBC in healthy subjects with those in patients. RESULTS: Expressions of CD9, CD164, PF4 and RARB gene in WBC of patients, both gastritis and colitis, were down-regulated while those of IGKC, DEFA1 and GNLY were up-regulated. CONCLUSION: The genesis of Pi-Qi deficiency syndrome has its immune associated genomic basis, and the immune functions are disordered in patients with that syndrome.