First report of Alternaria alternata causing leaf spot on Polygonatum kingianum in China.

Polygonatum kingianum is a Chinese herbal medicine that belongs to the genus Polygonatum of the family Liliaceae. In June 2023, Polygonatum kingianum Coll. et Hemsl. in nurseries in Qujing, Yunnan Province, China, showed irregular brown spots on the leaves, whole leaf necrosis, and plant death in serious cases, with an incidence of 10-20% (Fig. S1). To identify the pathogens of P. kingianum, six diseased samples were collected from nurseries with 0.6 acre. These diseased sample leaves were soaked in 0.1% HgCl2 for 1 min and 75% ethanol for 2 min and then rinsed thrice with sterile water. Treated leaves were cut into small pieces (5×5 mm) and cultured on potato dextrose agar (PDA) for five days at 28°C. Total thirteen fungal strains were isolated from PDA medium. The nuclear ribosomal internal transcribed spacer of ribosomal DNA (ITS rDNA) region of these 13 strains was amplified by polymerase chain reaction (PCR) using universal primers ITSI/ITS4 (White et al. 1990). Sequencing and BLAST of the ITS region on NCBI showed that 11 out of 13 fungal strains belonged to the genus Alternaria, with an identity ≥99%. We selected one of the Alternaria strains, HJ-A1, for further study. The HJ-A1 colony appeared grayish brown white-to-gray with a flocculent texture on the front side and a dark gray underside on the PDA medium (Fig. S1). The conidiophores appeared brown, either single or branched, and produced numerous short conidial chains. The conidia were obclavate to obpyriform or ellipsoid in shape and contained 1-4 transverse septa and 0-2 oblique septa. The conidial diameter was 27.30µm in length and 12.27µm in width. (Fig. S1). To further determine the species of HJA1, the genomic DNA of HJ-A1 was extracted using the Lysis Buffer for PCR (AG, Hunan, China). Four Alternaria genomic DNA regions including the ITS, translation elongation factor 1-α gene (TEF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Alternaria major allergen gene (Alt a1) were amplified by PCR using the primers as previously reported (Woudenberg et al. 2013, Hong et al. 2005). Sequence analysis revealed that the ITS (484bp) of HJ-A1 (NCBI No. PP082633), TEF1-α (267bp) of HJ-A1 (NCBI No. PP419893), GAPDH (582bp) of HJ-A1 (NCBI No. PP419892), and Alt a1 (522bp) of HJ-A1 (NCBI No. PP228046) shared the highest identity with A. alternata respectively (99≥%). A maximum likelihood phylogenetic tree was constructed with the combined sequence data sets of ITS, GAPDH, TEF, and Alt a1 using MEGA 7. The results showed that HJ-A1 strain clustered with A. alternate (Fig. S2). The pathogenicity of HJ-A1 was tested according to Koch's postulates by inoculating HJ-A1 conidia suspension (2×105 conidia/mL) into leaves of 1-year-old P. kingianum, with sterile water as a control. Each treatment group included 3 plants with 3 replicates. The tested plants were planted in a phytotron at 28℃ and 90% humidity. Three days after inoculation, symptoms similar to those under natural conditions were observed in the HJ-A1-inoculated plants, whereas no symptoms were observed in the control plants (Fig. S1). The same fungal strains were re-isolated from inoculated leaves and identified by morphologically and sequence of ITS. Previous studies showed that Alternaria alternata funji cause many plant diseases, such as fig fruit rot (Latinovic N et al. 2014)，daylily leaf spot (Huang D et al. 2022), fruit blight on sesame (Cheng H et al. 2021)，leaf spot of Cynanchum atratum Bunge (Sun H et al. 2021) and so on. To our knowledge, this is the first report of A. alternata causing P. kingianum leaf spot in China. The discovery of this pathogen will help to guide the protection and control of P. kingianum disease.

[3-Deazaadenosine (3-DAA) accelerates Japanese encephalitis virus replication and down-regulates levels of inflammatory factors in mouse and hamster cells].

Objective To investigate the effect of 3-deazaadenosine (3-DAA), an N6-methyladenosine (m6A) methylation modification inhibitor, on the replication of the Japanese encephalitis virus (JEV). Methods Neuro2a mouse neuroblastoma cells, N9 mouse microglial cells, and BHK baby hamster kidney cells were exposed to JEV and then treated with 3-DAA. JEV was also injected into the footpad of adult C57BL/6 mice, which were then administered 3-DAA intraperitoneally. Real-time quantitative PCR was utilized to measure mRNA expression levels of JEV, interleukin 1ß (IL-1ß), IL-6, tumor necrosis factor α (TNF-α), monocyte chemoattractant protein 1 (MCP-1), inducible nitric oxide synthase (iNOS), arginase 1 (Arg1), interferon (IFN)-α, IFN-ß, IFN-γ, and C-X-C motif chemokine ligand 10 (CXCL10) in the cells and mouse brain tissues. Western blot analysis was used to detect JEV protein expression in the cells and mouse brain tissues. Furthermore, the survival of the mice was monitored and pathological changes in mouse brains were observed via hematoxylin and eosin (HE) staining. Results 3-DAA had a dose-dependent effect on the replication of RNA and protein expression of JEV in both BHK, N9, Neuro 2α cells and mouse brain tissues, which resulted in rapid progression of JEV infection in mice and a decrease in their survival rate. Furthermore, 3-DAA suppressed the expression of inflammatory factors such as IL-6, TNF-α, CXCL10, IL-1ß and iNOS, thus weakening the immune response. Conclusion 3-DAA promotes JEV infection and hastens death of infected cells and mice, indicating that m6A modification may negatively regulate JEV replication.

N6-methyladenosine modification positively regulate Japanese encephalitis virus replication.

N6-methyladenosine (m6A) is present in diverse viral RNA and plays important regulatory roles in virus replication and host antiviral innate immunity. However, the role of m6A in regulating JEV replication has not been investigated. Here, we show that the JEV genome contains m6A modification upon infection of mouse neuroblast cells (neuro2a). JEV infection results in a decrease in the expression of m6A writer METTL3 in mouse brain tissue. METTL3 knockdown by siRNA leads to a substantial decrease in JEV replication and the production of progeny viruses at 48 hpi. Mechanically, JEV triggered a considerable increase in the innate immune response of METTL3 knockdown neuro2a cells compared to the control cells. Our study has revealed the distinctive m6A signatures of both the virus and host in neuro2a cells infected with JEV, illustrating the positive role of m6A modification in JEV infection. Our study further enhances understanding of the role of m6A modification in Flaviviridae viruses.

[Analysis for 171 cases of esophageal foreign bodies impacted in upper esophagus with endoscopic treatment].

OBJECTIVE: To assess the effectiveness and complications of rigid endoscopy (RE) and flexible endoscopy (FE) for the extraction of esophageal foreign bodies (FBs) in adults. METHODS: A retrospective analysis was conducted on the medical records of 171 adult patients with the upper esophageal FB impaction treated at Peking University Third Hospital, Beijing, China, between January 2008 and December 2012. RESULTS: In the study, 126 patients with the upper esophageal foreign body impaction were treated with RE, while 45 patients received FE. (1)The size of FBs in FE group was the same as RE group(P = 0.495, P = 0.125). (2)The period impacted in the esophagus of RE group (25.8 ± 28.6) h was longer than that of FE group (13.9 ± 14.5) h (P = 0.009). (3)71.4% of the patients in RE and 88.9% in FE group went to hospital for treatment within 24 hours from being impacted, while 15.1% in RE group and 8.9% in FE group were between 24 and 48 hours.13.5% in RE and 2.2% in FE group went to hospital beyond 48 hours.(4)The proportion of FBs puncturing into one or two esophageal walls in RE group (67.5%) was higher than that in FE group (35.6%).(5) The positive rates with the upper gastrointestinal barium contrast and chest X-ray or abdominal plain film were 98.3%,23.6% and 100%,14.3% for diagnosing esophageal FBs in RE and FE groups.(6)The successful rate, complication rate and perforation rate were 100%,38.1% and 6.3%and 95.6%,48.9%,and 2.2% in RE and FE groups, respectively with no statistical difference (P > 0.05). CONCLUSION: Both RE and FE were effective in the extraction of upper esophageal FBs with no difference in the complication and perforation rates. But FE was cheaper and no need for general anesthesia.

[Complication related factors and efficacy with flexible endoscopy of 101 esophageal foreign bodies].

OBJECTIVE: To analyze the related factors of complications and treatment efficacy with flexible endoscopy for esophageal foreign body (FB). METHODS: In a retrospective study with consecutive data, 101 adults including 52 males and 49 females with esophageal FB impaction between January 2005 and December 2012 admitted into Department of Gastroenterology's Endoscopic Unit at Peking University Third Hospital were included, aged (49 ± 21) years. RESULTS: (1) FB impaction in upper and middle esophagus accounted for 87.1% (n = 88) of all esophageal FBs. No significant difference existed in interval time from impaction to removal of FB impacted between upper, middle and lower esophagus (P > 0.05) . (2) Patients with esophageal FB seeking hospital treatment accounted for 82.2% (n = 83) within 24 h and 99.0% (n = 100) within 48 h. Food lump, fish bone, chicken bone and fruit seeds accounted for 76.2% (n = 77). (3) Positive rates were 91.3% (21/23) and 24.1% (7/29) with upper gastrointestinal barium contrast and chest or abdominal plain film. The success rate was 94.1% (n = 95) with flexible endoscopy for removal of FB. (4) Denture was the most difficult FB for removal. Four patients in all 11 patients with denture impacted were not removed successfully with flexible endoscopy. (5) The complication (except for mild scratch) rate was 48.5% (n = 49) and the perforation rate 3.0% (n = 3) . Whether complications took place or not was independent of age, location of impaction, time from impaction to removal and size of FB (all P > 0.05) , but dependent on piercing into esophageal wall, concomitant with esophageal stricture and types of FB (all P < 0.01) . Whether perforation or not was independent of any above factor. CONCLUSION: Esophageal FB should be removed as soon as possible within 24 h especially for those with sharp edges and piercing into esophageal wall.