[Identification and biological characterization of pathogen causing black spot of Pseudostellaria heterophylla in Fujian province].

In Zherong county, Fujian province, the black spot of Pseudostellaria heterophylla often breaks out in the rainy season from April to June every year. As one of the main leaf diseases of P. heterophylla, black spot seriously affects the yield and quality of the medicinal material. To identify and characterize the pathogens causing black spot, we isolated the pathogens, identified them as a species of Alternaria according to Koch's postulates, and then tested their pathogenicity and biological characteristics. The results showed that the pathogens causing P. heterophylla black spot were A. gaisen, as evidenced by the similar colony morphology, spore characteristics, sporulation phenotype, and the same clade with A. gaisen on the phylogenetic tree(the maximum likelihood support rate of 100% and the Bayesian posterior probability of 1.00) built based on the tandem sequences of ITS, tef1, gapdh, endoPG, Alta1, OPA10-2, and KOG1077. The optimum conditions for mycelial growth of the pathogen were 25 ℃, pH 5-8, and 24 h dark culture. The lethal conditions for mycelia and spores were both treatment at 50 ℃ for 10 min. We reported for the first time the A. gaisen-caused black spot of P. heterophylla. The results could provide a theoretical basis for the diagnosis and control of P. heterophylla leaf spot diseases.

[Identification and biological characteristics of violet root rot pathogen of Pseudostellaria heterophylla in Fujian province].

Violet root rot is one of the main root diseases in the production process of Pseudostellaria heterophylla. To clarify the pathogenic species that cause the violet root rot of P. heterophylla in Fujian province, the roots and the sclerotia with violet root rot symptoms were collected from the main producing areas of P. heterophylla(Fujian province) from 2017 to 2021, and the pathogens were isolated by tissue separation method and identified by morphology and multi-gene phylogenetic analysis. Additionally, the biological characteristics of the pathogens were studied and the fungicides were determined. The results showed that 78 strains of violet root rot were isolated from the collected root samples, which belonged to one type after preliminary morphological identification. Two represen-tative strains were selected from the pathogens for multi-gene phylogenetic analysis, and they were clustered with Helicobasidium mompa together. The suitable culture conditions for the mycelium were OA medium, 25 ℃, pH 6, and ammonium oxalate as the nitrogen source. The lethal temperature of the mycelium was 50 ℃ for 10 minutes. Moreover, 99.1% propiconazole and 98.7% azoxystrobin had the optimal bacteriostatic effect, and the concentrations with the 50% bacteriostatic rate were 16.85 and 12.24 µg·mL~(-1), respectively. On the basis of the above results, the pathogen causing violet root rot of P. heterophylla in Fujian province was H. mompa. The medium type, growth temperature, pH value, nitrogen source, etc. had significant effect on the growth of mycelium.

First Report of Corynespora cassiicola Causing Leaf spot of Strobilanthes cusia in China.

Strobilanthes cusia (Nees) Kuntze is a vital medicinal and industrial herb, planted extensively in southern China (Hu, et al. 2011.). In July and August of 2021, leaf spot incidence on >60% plants and reduced yields >20% for fresh leaves were observed in S. cusia cultivar 'Malan No.1' across the Shufeng whole Township, Xianyou County, Fujian province. Initial symptoms on leaves were observed as small, dark-brown, spots surrounded by a yellow halo, expanding irregularly or into semicircular spots. As symptoms developed, the spots became dark brown, thin and fragile, forming small holes. In severe cases plants were defoliated. The pathogen was isolated from the margin of 60 symptomatic leaf lesions， surfacesterilized with 75% ethanol for 45 s, rinsed three times with sterile water, air dried, and cultured on PDA at 25°C in the dark. Pure cultures were obtained by single-spore isolation after subculture. Ten representative single-spore isolates (MY-1 to MY-10) from 154 pathogens in 10 sampling points were selected for morphological characterization and identification. After 7 days, mycelial colonies were gray to dark gray with few aerial hyphae. Conidia (32.3 to 132.8 × 5.8 to 8.4 µm, average 81.4 × 6.3 µm, n=50) were pale to brown, erect or curved, solitary or in chains, with 0 to 15 pseudosepta. Based on morphological characteristics, the isolates were preliminarily identified as Corynespora cassiicola. Genomic DNA of isolate MY-2 (randomly selected from 10 isolates as representative) was extracted from mycelia using the Ezup DNA extraction kit (Sangon Biotech Co., Ltd. Shanghai, China). The ITS (internal transcribed spacer) region of rDNA, TEF1-α (translation elongation factor 1 alpha) and TUB2 (beta-tubulin) genes were amplified and sequenced with primers ITS4/ITS5, EF1-728F/EF-986R (Wang et al. 2021) and Bt2a/Bt2b (Glass et al. 1995), respectively. BLASTN sequence analyses of ITS (538 bp), TEF1-α (302 bp) and TUB2 (436 bp) of isolate MY-2 (GenBank accessions OK355515, OM339443, OM339442) showed 100%, 97.6%, 100% identity with C. cassiicola in GenBank (Accession numbers JX908713, MW961421, AB539228). A neighbor-joining phylogenetic analysis based on ITS and TEF1-α sequences using MEGA7 showed that MY-2 clustered in the same clade with C. cassiicola. For pathogenicity tests, five S. cusia plants were inoculated onto the adaxial surface of leaves with mycelial plugs from ten isolates of 8-day-oldcultures on PDA. Five leaves per plant were inoculated, covered with wet cotton, and kept in a controlled greenhouse (26~33 °C, RH 80% ~ 90%). Leaves inoculated with sterile PDA plugs served as a negative control. At 3-5 days post inoculation, all 25 inoculated leaves of each isolate showed leaf spot lesions similar to those observed in the field, and control leaves were symptomless. C. cassiicola was successfully reisolated from the diseased leaves. The pathogenicity tests were repeated three times under the same conditions and similar results were observed. In view of morphology, pathogenicity and sequence results, the isolates were identified as C. cassiicola, a pathogen reported from many important crops (Lu et al. 2021). This is the first report of C. cassiicola as a pathogen in China which poses a potential threat to leaf production and S. cusia processing. References: Glass, N. L., et al. 1995. Appl. Environ. Microb. 61:1323 Hu, J.Q., et al. 2011. Flora of China. Science Press, Beijing, China. Volume 19: 407 Li, Q.L., et al. 2013. Plant Dis. 97 (5): 690 Lu, P. et al. 2021. Plant Dis. 105:3753 Wang S. H., et al. 2021.Forest Pathology, 51(2):1 Keywords: fungal disease, Strobilanthes cusia, medicinal plants, etiology, leaf spot.

Change of CMTM7 expression, a potential tumor suppressor, is associated with poor clinical outcome in human non-small cell lung cancer.

BACKGROUND: CKLF-like MARVEL transmembrane domain-containing 7 (CMTM7) located at 3p22.3, is a frequent deletion site and a tumor suppressor gene (TSG) locus in many cancer, which suggests CMTM7 may be a potential TSG. The aim of this study was to investigate the correlations of CMTM7 expression and survival rate in patients with non-smallcell lung cancer (NSCLC). METHODS: Surgical specimens of 180 cases with pathologically confirmed NSCLC were grouped into 18 tissue microarray slides. CMTM7 expression in these specimens were detected by immunohistochemistry staining and representative cases were confirmed by Western blotting. Univariate and multivariate analyses were performed to identify the association of CMTM7 expression with pathological features and survival of patients with NSCLC. RESULTS: A total of 78.9% of the 180 patients had variations of CMTM7 protein expression, either up-regulated or down-regulated. Univariate analysis showed that the patients' survival rate after surgery was highly correlated with CMTM7 expression (P = 0.0091). In addition, prognostic factors were examined by multivariate Cox regression analysis, and results suggested that CMTM7 expression was a unique prognostic factor in NSCLC survival. CONCLUSIONS: The CMTM7 expression may be related to survival of patients with NSCLC and a unique prognostic factor. CMTM7 may play an important role in NSCLC development.

[Preparation and characterization of monoclonal antibody against HCCR protein].

AIM: Preparation of monoclonal antibody (mAb) to HCCR, which is a candidate biomarker for human hepatocellular carcinoma (HCC). METHODS: The recombinant protein HCCR-1(167-360); was expressed and was used as immunogen to immunize mouse for generation of mAb against HCCR. The protein Ep-HCCR, which displayed a epitope of HCCR, was also expressed and purified to use to detect serum antibody titer and to screen the positive clones of hybridmas. The properties of HCCR antibody were analyzed by ELISA, Western blot, immunofluorescence and immunohistochemistry. RESULTS: A hybridmas clone, which secreted anti-HCCR mAb, was obtained. The affinity constant (Kaff) of the mAb is 5.4×10(6); L/mol analyzed by ELISA; Western blot showed that the mAb could specifically recognize HCCR-1 and HCCR-2 expressed in HepG2 cells; The mAb was also used to detect the expression of HCCR proteins in hepatoma cells and HCC tissues. The results of immunofluorescence indicated that HCCR proteins mainly localized on the plasma membrane and cytoplasm of HepG2 cells. In addition, HCCR was found high-expressed in HCC tissues but not in normal liver tissue detected by Immunohistochemistry. CONCLUSION: A specific mAb against HCCR was successfully generated, which laid the foundation for establishing HCC detection method based on HCCR.

[Generation of monoclonal antibody to GP73 based on antigen epitope].

AIM: Preparation of monoclonal antibody (mAb) against GP73 protein. METHODS: The N-terminal peptide (AAAERGAVELK) of GP73 protein was displayed on T7 phage, the recombinant phage was amplified and used as the immunogen to immunize mouse to produce antibody. The titer of the antiserum and the positive hybridoma clones which secreted the mAb against GP73 protein were detected by ELISA. The mAb specificity was assayed by ELISA and Western blot. RESULTS: The high specificity mAb against GP73 protein was selected from the mouse immunized with the recombinant T7 phages displaying the epitope of GP73 by cell fusion and screening. CONCLUSION: The appropriate protein epitope displayed on T7 phage could be used as alternative antigen to immunize animals to make specific antibody against the corresponding native protein.

[Production and identification of a monoclonal antibody against human HPPCn].

AIM: To make monoclonal antibody(mAb) against human HPPCn for the use in research on HPPCn's function and its relationship with liver diseases. METHODS: The female BALB/c mice were immunized with the recombinant HPPCn proteins. Splenocytes and Sp2/0 cells were fused with PEG-1500. The positive clone was identified through indirect ELISA and then subcloned by limited dilution. Indirect ELISA, Western blot and Ig sub-class identification kit were used to identify the mAb's properties. By immunofluorescence experiments, we studied the cellular localization of HPPCn. The mAb epitope was also analyzed using peptide phage display technology. RESULTS: An anti-HPPCn mAb, named W2-D5, was obtained. It belongs to IgG1 subclass. It could specially bind to human HPPCn. Furthermore, by immunofluorescence results, wo confirmed HPPCn located in the nucleus and our mAb could combined with the natural protein. With the mAb, the minimal detectable concentration was 0.1 µg/L for HPPCn; The peptide sequence of HPPCn7₋13;(IHLELRN)was identified as the epitope of the mAb. CONCLUSION: An anti-HPPCn mAb with high specificity and high affinity was successfully obtained.