Colletotrichum horii and Colletotrichum fioriniae Causing Anthracnose on Star Anise (Illicium verum) in China.

Star anise (Illicium verum) is an important economic and medical plant widely cultivated in Guangxi province, China. Its fruit can be used as spice and medicine (Wang et al. 2011). In recent years, anthracnose led to a serious decline in the production of star anise in Guangxi. In 2021, a survey conducted in CenwangLaoshan Reserve of Guangxi (24°21'N; 106°27'E) showed that the 2500 ha planting area had disease incidence greater than 80%. The leaf symptoms initially appeared as small spots, then expanded to round spots, finally becoming withered with grayish-white centers, surrounded by dark brown margins. Sometimes, small black acervuli were observed in the later stage. To explore the pathogen, infected leaves were collected and cut into small pieces (about 5 mm2) from the edge of the lesion, disinfected with 75% ethanol for 10 s, 1% NaClO for 1 min, washed with sterilized water and incubated on potato dextrose agar (PDA) plates at 28 °C in the dark. Ten single-spore isolates were obtained from the cultures. After 7 days on PDA at 28 °C, the colonies of 7 isolates were white with abundant aerial hyphae, gray-black with white-gray margins, and the other 3 isolates were light gray on the upper surface, and pink or orange on the underside. Representative isolates BS3-4 and BS3-1 were selected from 3 isolates and 7 isolates, respectively. Conidia of BS3-4 and BS3-1 were both hyaline, cylindrical, aseptate, smooth, apex obtuse, base truncate, and no significant differences (P > 0.05) in size between BS3-1 (13.22 to 5.38 × 3.89 to 1.99 µm) (n = 50) and BS3-4 (12.04 to 4.34 × 3.48 to 1.64 µm) (n = 50). These morphological characteristics were consistent with the Colletotrichum ssp. (Damm et al. 2012). The species identification of BS3-4 and BS3-1 was performed based on DNA sequence analysis. Genomic DNA was extracted as a template. Partial sequences of the rDNA internal transcribed spacer (ITS), actin gene (ACT), ß-tubulin2 (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified and sequenced (Weir et al. 2012). The sequences were deposited in GenBank (ITS:OQ062642-43; ACT:OQ067614-15; GAPDH:OQ067616-17;TUB2：OQ067618-19). Based on the concatenated sequences of the 4 genes (ITS-ACT- GAPDH -TUB2) of BS3-4 and BS3-1 as well as sequences of other Colletotrichum spp. obtained from GenBank, the Maximum likelihood (ML) tree which produced with IQ-TREE (Minh et al. 2020) revealed that the isolate BS3-1 was Colletotrichum horii, and BS3-4 was Colletotrichum fioriniae. Pathogenicity was confirmed on healthy leaves of 1-year-old star anise seedlings (cultivar Dahong), and the leaves were wounded by sterilized toothpicks, and were inoculated with 10 µl of conidial suspensions of BS3-1 and BS3-4 (106 conidia/ml). Control seedlings were inoculated with sterilized distilled water. Five leaves per plant and 3 plants per treatment were selected. All inoculated seedlings were maintained in the greenhouse (12/12h light/dark, 25 ± 2℃, 90% relative humidity). Wound sites inoculated with BS3-1 and BS3-4 both turned greenish-brown after 2 days and then turned light brown with water-soaked spots. Black (BS3-1) or orange (BS3-4) dots of acervuli developed after 6 days. The lesion diameter of BS3-1 (14.4 mm) was larger than that of BS3-4 (8.1 mm). No symptoms were observed on controls. BS3-1 and BS3-4 were re-isolated from inoculated leaves, fulfilling Koch's postulates. Anthracnose of star anise caused by C.horii has been reported in China (Liao et al. 2017). However, to our knowledge, this is the first report of C.fioriniae infecting star anise in China. Accurate pathogen identification in this study could provide a reference for the control of anthracnose on star anise.

[Expression of IL-34 in chronic periapical lesions and its clinical significance].

PURPOSE: To observe the expression of IL-34 mRNA in chronic periapical lesions and healthy periodontal ligaments and discuss the role of IL-34 in the etiology of chronic apical periodontitis. METHODS: A total of 25 periapical tissues from chronic apical periodontitis and 22 normal periodontal ligament tissue from extracted healthy teeth for orthodontic reason were selected. The expression of IL-34mRNA was detected by real-time PCR; the expression of IL-34 protein was detected by immunohistochemical analysis. Statistical analysis was performed using SPSS 13.0 software package. RESULTS: The level of IL-34 mRNA expression in periapical lesions (3.53±3.07) was significantly higher than that of the normal control (1.07±0.76); IL-34 was positively expressed in lymphocytes, plasma cells and macrophages. Image analysis software indicated that the level of IL-34 protein was significantly higher in periapical lesions than that in normal control (P<0.01). CONCLUSIONS: IL-34 may be closely related to inflammation of chronic apical periodontitis.

[Effect of cone angle on fracture resistance force of root and post-core system].

OBJECTIVE: To study the effect of cone angle on fracture resistance force of root and post-core system. METHODS: Forty-two simulated tooth roots made of polymethacrylate (PMMA) were divided into six groups according to the root canal cone angles of 0 degrees, 3.93 degrees, 5.71 degrees, 7.48 degrees, 11.31 degrees and 14.71 degrees. Cast post and cores were manufactured and cemented with zinc polycarboxylate cement (PC). Casting the post and core to restore every simulated tooth root. Each specimen was embedded in acrylic resin and then fixed in a special jig on the universal load-testing machine. A compressive load was applied at a 90-degree angle to the long axis of the core until fracture, at a crosshead speed of 1 mm/min. The maximum of load was recorded. RESULTS: The mean values of load in root and post-core system were 0.1225 kN, 0.1498 kN, 0.1600 kN, 0.1893 kN, 0.1078 kN and 0.0945 kN. There were significant differences in the fracture resistance among groups. CONCLUSION: The fracture resistance force of cast post and core increased with the enlargement of post cone angles under certain conditions.

[Expression of Wnt5a in chronic apical periodontitis and its clinical significance].

PURPOSE: To detect the expression of Wnt5a in lesions of chronic apical periodontitis and determine the relationship between expression of Wnt5a and inflammation degree. METHODS: Ten patients with chronic apical periodontitis and 5 healthy controls were enrolled in this study. According to the inflammatory cell infiltration, the specimens were divided into 2 groups: severe inflammation group and mild inflammation group. The expression of Wnt5a was measured by real-time PCR (RT-PCR) and immunohistochemical analysis in the lesions of chronic apical periodontitis. The amount of Wnt5 expression was assayed and compared in different inflammation levels. Statistical analysis was performed using SPSS 13.0 software package. RESULTS: Wnt5a was detected in both groups. Expression of Wnt5a mRNA in patients were significantly higher than the controls (P<0.05). According to inflammation level, the positive expression rate of Wnt5a in severe inflammation group was significantly higher than the controls (P<0.01), and Wnt5a positive expression in mild inflammation group was also significantly higher than the controls (P<0.05). The expression of Wnt5a was significantly different between severe inflammation group and mild inflammation group (P<0.05). CONCLUSIONS: The expression of Wnt5a increases as the severity of tissue inflammation increases, which indicates that Wnt5a plays an important role in the development of chronic apical periodontitis.

[Effect of Ca(OH)2 on the cytotoxicity of lipopolysaccharide extracted from Porphyromonas endodontalis in vitro].

PURPOSE: To detect the degradation of Ca(OH)2 on lipopolysaccharide (LPS) extracted from Porphyromonas endodontalis (P.e) in vitro and estimate the influence of P.e LPS pretreated with Ca(OH)2 on the proliferation of MC3T3-E1 cells. METHODS: The effect of Ca(OH)2 on MC3T3-E1 cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay. Then P.e LPS was treated with Ca(OH)2 for 30 mins or 60 mins at 37 degrees centigrade in vitro and the activity of P.e LPS was evaluated by Chromogenic End-point Tachypleus Amebocyte Lysate (CE TAL) test. Finally, MC3T3-E1 cells were exposed to P.e LPS pretreated with 15% Ca(OH)2 for 1, 3 and 5 d, and the cell proliferation was measured using the MTT assay comparing with the P.e LPS control group. SPSS 13.0 software package was used for statistical analysis. RESULTS: Compared with the negative control, exposing cells to 5%, 10% and 15% Ca(OH)2 had greatly promoted MC3T3-E1 cell proliferation. P.e LPS treated with 10% and 15% Ca(OH)2 both presented the best results by CE TAL and significant difference compared with P.e LPS control group. When 10 µg/mL P.e LPS was pretreated with 15% Ca(OH)2, no inhibition of MC3T3-E1 cell proliferation was noted. CONCLUSIONS: Ca(OH)2 detoxifies P.e LPS in vitro, mitigates the impact of P.e LPS on MC3T3-E1 cell proliferation. Supported by Science and Technology Projects of Liaoning Province (2011225020).