[Aprospective study of detection and clinical significance of bone marrow tumor cells in small cell lung cancer].

Objective: To investigate the detection of bone marrow tumor cells in small cell lung cancer (SCLC) patients and their relationship with clinical features, treatment response and prognosis. Methods: A total of 113patients with newly diagnosed SCLC from January 2018 to October 2022 at Beijing Chest Hospital were prospectively enrolled. Before treatment, bone marrow was aspirated and separately submitted for tumor cells detection by liquid-based cytology and disseminated tumor cells (DTCs) detection by the substrction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) platform. The correlation between the detection results of the two methods with patients' clinical features and treatment response was evaluated by Chi-square. Kaplan-Meier method was applied to create survival curves and the Cox regression model was used for multivariate analysis. Results: The positive rate of bone marrow liquid-based cytology in SCLC was 15.93% (18/113). The liver and bone metastases rates were significantly higher (55.56% vs 11.58% for liver metastasis, P＜0.001; 77.78% vs 16.84% for bone metastasis, P＜0.001) and thrombocytopenia was more common (16.67% vs 2.11%, P=0.033) in patients with tumor cells detected in liquid-based cytology than those without detected tumor cells. As for SE-iFISH, DTCs were detected in 92.92% of patients (105/113), the liver and bone metastasis rates were significantly higher (37.93% vs 11.90% for liver metastasis, P=0.002; 44.83% vs 20.23 % for bone metastasis, P=0.010), and the incidence of thrombocytopenia was significantly increased (13.79% vs 1.19%, P=0.020) in patients with DTCs≥111 per 3 ml than those with DTCs＜111 per 3 ml. The positive rates of bone marrow liquid-based cytology in the disease control group and the disease progression group were 12.00% (12/100) and 46.15% (6/13), respectively, and the difference was statistically significant (P=0.002). However, the result of SE-iFISH revealed the DTCs quantities of the above two groups were 29 (8,110) and 64 (15,257) per 3 ml, and there was no statistical difference between the two groups (P=0.329). Univariate analysis depicted that the median progression-free survival (PFS) and median overall survival (OS) of liquid-based cytology positive patients were significantly shorter than those of tumor cell negative patients (6.33 months vs 9.27 months for PFS, P=0.019; 8.03 months vs 19.50 months for OS, P=0.019, P=0.033). The median PFS and median OS in patients with DTCs≥111 per 3 ml decreased significantly than those with DTCs＜111 per 3 ml (6.83 months vs 9.50 months for PFS, P=0.004; 11.2 months vs 20.60 months for OS, P=0.019). Multivariate analysis showed that disease stage (HR=2.806, 95%CI:1.499-5.251, P=0.001) and DTCs quantity detected by SE-iFISH (HR=1.841, 95%CI:1.095-3.095, P=0.021) were independent factors of PFS, while disease stage was the independent factor of OS (HR=2.538, 95%CI:1.169-5.512, P=0.019). Conclusions: Both bone marrow liquid-based cytology and SE-iFISH are clinically feasible. The positive detection of liquid-based cytology or DTCs≥111 per 3 ml was correlated with distant metastasis, and DTCs≥111 per 3 ml was an independent prognostic factor of decreased PFS in SCLC.

Knowledge domain and emerging trends in sacubitril/valsartan study from 2008 to 2023 - a visualized conclusive analysis based on novel scientometric tools.

OBJECTIVE: This study aimed to determine the evolution of sacubitril-valsartan research and analyze the publications quantitatively and qualitatively. MATERIALS AND METHODS: We used the bibliometric method and a combination of CiteSpace_6.1.6 and VOSviewer_1.6.18 to identify top authors, countries, institutions, co-cited articles, co-cited journals, keywords, and trends. This study prioritized key aspects in the existing global research on Entresto (Sacubitril/Valsartan) to assess our depth of knowledge in this field and identify potential insights. The objective was to generate a reference for the utilization of the "angiotensin receptor-neprilysin inhibitor" (ARNI). RESULTS: From 2008 to 2022, citations of sacubitril-valsartan showed an upward trend. VOSviewer keyword analysis of 3,408 publications identified 624 keywords and divided them into seven different clusters. The clustered network was constructed based on 1,191 references cited by 3,408 publications that met the terms, where the clustered network of sacubitril-valsartan was presented. These publications can be regarded as fundamental to Entresto's research. Analysis of co-cited reference clusters showed that other than Entresto's novel application in other diseases, the new combination with other medication or mechanical assistance therapies against heart failure was Entresto's latest focus. Analysis of citation bursts showed that the rank of the top 25 keywords, according to the chronological sequence, marked Entresto's research entering a new era of exploring the extended application in other diseases and novel combinations with other diverse therapies. CONCLUSIONS: We found that emerging new mechanisms in sacubitril-valsartan therapy intended for more targets in the pathogenesis of specific diseases will be the focus of further studies.

[Basic competence for etiological diagnosis of lower respiratory tract infection after application of metagenomic next-generation sequencing].

In addition to clinical manifestations, medical history, and imaging, the diagnosis of low respiratory tract infection (LRTI) depends mainly on the ability of the clinical microbiology laboratory to detect the pathogens. However, conventional culture may be time-consuming, the sensitivity of microscopy is low, and nucleic acid-based targeted tests (e.g., PCR) could only cover limited range of pathogens. The use of mNGS technology has improved the diagnostic rate of LRTI, but the conventional microbiology detection has been neglected to some extent. This review addressed the appropriate use of these methods with the aim of strengthening the ability of traditional microbiology methods in LRTI diagnosis after mNGS application.

[Pulmonary mucormycosis after lung transplantation:3 cases report with literature review].

Objective: To report the risk factors, clinical characteristics and treatment courses of pulmonary mucormycosis after lung transplantation(LT). Methods: We included 3 cases with pulmonary mucormycosis after LT from March 2017 to July 2020 in the centre for lung transplantation of China-Japan Friendship Hospital. Twelve cases from Chinese and English literature from China National Knowledge Infrastructure (CNKI), China Biomedical Literature Service System and Pubmed Database from March 1980 to July 2020 were added. The risk factors, clinical characteristics and treatment courses of all cases were summarized and analyzed. Results: Pulmonary mucormycosis occurred in 1.06% (3/284) in our centre. A total of 15 cases with 12 cases from literature included 10 males and 5 females with a mean age of(47±20)years. Thirteen cases occurred after LT, and 2 cases occurred after heart-lung transplantation (HLT). Nine probable cases were diagnosed by positive isolation of the pathogen from bronchoalveolar lavage fluid or sputum. Three proven cases were diagnosed by transbronchial lung biopsy. Meanwhile, the other 3 proven cases diagnosed by CT-guided percutaneous lung biopsy, autopsy and surgical operation respectively. Ten cases (66.7%) were diagnosed with pulmonary mucormycosis within 90 days after lung transplantation. The mortality was as high as 46.67% (7/15), but if it occurred within 90 days, the mortality reached 70% (7/10). The average interval between transplantation and positive isolation of the pathogen was 112.3 (5-378) days. Conclusions: The clinical and radiographic features of pulmonary mucormycosis after LT were nonspecific. It had a high mortality, especially in those occurred within 90 days after LT. The combination of antifungal therapy and surgical resection may contribute to a better outcome of the disease.

[Risk factors and clinical prognosis analysis of carbapenem-resistant Enterobacterales bacteria nosocomial infection].

Objective: To explore the risk factors for carbapenem-resistant Enterobacterales (CRE) infection and death. Methods: A case-control analysis of 482 inpatients in 18 secondary or tertiary hospitals in Beijing in 2018 was conducted. Patients infected by CRE were selected as the case group (n=247), and infected by carbapenem susceptible Enterobacterales (CSE) as the control group (n=235). The risk factors and clinical prognosis of CRE infection were analyzed by single factor analysis and multivariate logistic regression analysis. Results: CRE were resistant to most antimicrobials, but were highly sensitive to colistin and tigecycline, with sensitivity of 94.0% and 99.5%, respectively. Multivariate analysis showed that prior 30-day tracheal intubation (OR=2.607, 95%CI: 1.655-4.108, P<0.001), empirical treatment using third or fourth generation cephalosporins (OR=2.339, 95%CI: 1.438-3.803, P=0.001), carbapenems (OR=2.468, 95%CI: 1.610-3.782, P<0.001) and quinolones (OR=2.042, 95%CI: 1.268-3.289, P=0.003) were independent risk factors for CRE infection. Mechanical ventilation (OR=3.390, 95%CI: 1.454-7.904, P=0.005), heart failure (OR=4.679, 95%CI: 1.975-11.083, P<0.001), moderate or severe liver disease (OR=3.057, 95%CI: 1.061-8.806, P=0.038), prior 30-day quinolones exposure (OR=2.882, 95%CI: 1.241-6.691, P=0.014) and septic shock (OR=7.772, 95%CI: 3.505-17.233, P<0.001) were independent risk factors for death after CRE infection. Conclusions: Reducing the use of antimicrobials and invasive procedures such as prior 30-day tracheal intubation may reduce the probability of CRE infection. Grading the severity of the underlying disease in patients with CRE infection, as well as predicting and preventing the occurrence of septic shock will help reduce the risk of death.

[Analysis of pathogen spectrum and antimicrobial resistance of pathogens associated with hospital-acquired infections collected from 11 teaching hospitals in 2018].

Objective: To investigate the spectrum and antimicrobial resistance of major pathogens causing nosocomial infections in China, 2018. Methods: Non-duplicated nosocomial cases as well as pathogens causing bloodstream infections (BSI), hospital-acquired pneumonia (HAP) and intra-abdominal infections (IAI) from 11 teaching hospitals across China were collected. The minimum inhibitory concentrations (MICs) of clinically significant strains were determined by agar dilution method or broth microdilution method. The Clinical and Laboratory Standards Institute (CLSI) M100-S29 criteria were used for interpretation, and the WHONET-5.6 software was used in data analysis. Results: A total of 1 590 cases were collected, including 831 cases from BSI, 450 cases from HAP and 309 cases from IAI. The most prevalent pathogens causing BSI were Escherichia coli (29.2%, 243/831), Klebsiella pneumoniae (16.2%, 135/831) and Staphylococcus aureus (10.1%, 84/831); the most prevalent pathogens causing IAI were E. coli (26.2%, 81/309), Enterococcus faecium (15.5%, 48/309) and K. pneumoniae (13.3%, 41/309); while Acinetobacter baumanii (24.7%, 111/450), Pseudomonas aeruginosa (20.7%, 93/450) and K. pneumoniae (16.2%, 73/450) were dominated in HAP. All S. aureus were susceptible to tigecycline, linezolid, daptomycin and glycopeptides; 77.8% (105/135) of S. aureus strains were susceptible to ceftaroline. Methicillin-resistant S. aureus (MRSA) accounted for 29.6% (40/135) of all the S. aureus, and was lower than the accounted rate of methicillin-resistant coagulase-negative Staphylococcus (MRCNS) (83.7%, 41/49). One E. faecium strain (1.1%, 1/95) resistant to vacomycin and teicoplanin and one E. faecalis strain (2.3%, 1/43) resistant to linezolid was found. The prevalence of extended-spectrum ß-lactamase (ESBL) was 56.1% (193/344) in E. coli and 22.1% (55/249) in K. pneumonia; the rate of carbapenem resistant E. coli and K. pneumonia was 4.1% (14/344) and 22.9% (57/249), respectively; the percentage of ceftazidime/avibactam resistant E. coli and K. pneumonia was 2.3% (8/344) and 2.0% (5/249), respectively; the percentage of colistin resistant E. coli and K. pneumonia was 1.5% (5/344) and 7.6% (19/249), respectively; no E. coli and K. pneumonia strains were found resistant to tigecycline. The rate of carbapenem resistant A. baumanii and P. aeruginosa were 78.9% (146/185) and 36.7% (66/180), respectively. A. baumanii showed low susceptibility to the antimicrobial agents except colistin (99.5%, 184/185) and tigecycline (91.4%, 169/185). Colistin, amikacin and ceftazidime/avibactam demonstrated high antibacterial activity against P. aeruginosa with susceptility rate of 100% (180/180), 93.3% (168/180) and 85.6% (154/180), respectively. Conclusions: Nosocomial Gram-negative pathogens show high susceptibilities to tigecycline, colistin and ceftazidime/avibactam in vitro. Antimicrobial resistance in A. baumannii is a serious problem. The prevalence of carbapenem-resistant Enterobacteriaceae has increased, which should be monitored continuously in China.

[Clinical features and prognostic factors of parainfluenza virus infections in adult patients].

Objective: To explore the clinical features and prognostic factors of parainfluenza viral lower respiratory tract infections in adults. Methods: A total of 70 patients withpositive nucleic acid of parainfluenza virus (PIV) admitted to China-Japan Friendship Hospital between August 2016 and November 2019 were enrolled. Multiplex real-time polymerase chain reaction (RT-PCR) assays for viral detection were implemented to specimens (nasopharynx swab, sputum or bronchoalveolar lavage) obtained from all the patients, which was consistent with the diagnosis of lower respiratory tract infection. Patients were divided into two groups depending on the status of immune function (immunocompromised group, n=26; immunocompetent group, n=44), and patients were divided into PIV infection group (n=43) and mixed infection group (n=27) according to whether there was mixed infection. Characteristics including age, gender, underlying diseases, symptoms, disease severity, imaging manifestations, etiology, respiratory failure, mechanical ventilation, vasoactive drug, antimicrobial drug and 30-day mortality between the groups were compared, and the prognostic factors of PIV infections were investigated using Cox regression. Results: The peak incidence of PIV infection time was in May, August, September and December, accounting for 58.6% of all cases. The enrolled 70 cases included 43 cases with pneumonia, 16 cases with interstitial lung disease and infection, 7 cases with bronchiectasis and infection, and 4 cases with acute exacerbation of chronic obstructive pulmonary disease. Eight patients (11.4%) had no underlying diseases, 21 patients (30.0%) had respiratory failure, 18 patients (25.7%) were treated with mechanical ventilation, 15 patients (21.4%) died within 30 days after admission. The pneumonia severity index score, percentage of patients with ground-glass opacity according CT scan, with honeycomb or reticular pattern, with mechanical ventilation, with respiratory failure, with ICU admission, and 30-day mortality in immunocompromised group were higher than those of immunocompetent group [(91.5 vs 84.0), (60.0% vs 34.1%), (44.0% vs 11.4%), (42.3% vs 15.9%), (50.0% vs 18.2%), (38.5% vs 22.7%), (34.6% vs 13.6%)] (all P<0.05). There were 27 cases (38.6%) with mixed infection, including 17 viruses (24.3%), 19 bacteria (27.1%), 14 (20.0%) fungi (PCP, aspergillus) and 1 (1.4%) Mycobacterium intracellulare. Sixteen patients (59.3%) in the mixed infection group were immunocompromised patients and 21 patients (77.8%) had chronic lung disease. Cox regression analysis showed that mechanical ventilation and interstitial lung disease were independent predictors of prognosis in all patients, and mechanical ventilation was an independent predictor of prognosis in PIV infection group. Conclusions: Most of the patients with PIV lower respiratory tract infection in adults are complicated with underlying diseases and mixed infection, with a high 30-day mortality. Interstitial lung disease and mechanical ventilation indicate poor prognosis in these patients.

High levels of phosphatase and tensin homolog expression predict favorable prognosis in patients with non-small cell lung cancer.

OBJECTIVE: The prognostic role of phosphatase and tensin homolog (PTEN) in non-small cell lung cancer (NSCLC) has been controversial. PATIENTS AND METHODS: In this study, levels of PTEN expression were investigated in NSCLC patients and their prognostic value in NSCLC was assessed. PTEN expression in tumor tissues from 68 NSCLC patients was analyzed using immunohistochemistry and confocal microscopy. Survival analysis was performed using the log-rank test and Cox proportional hazards regression analysis. RESULTS: NSCLC patients classified as expressers of high levels of PTEN (n = 46) had better prognoses than those classified as expressers of low levels (mean survival 17.1 versus 12.9 months, log-rank p = 0.038). In patients with adenocarcinoma (AC), high PTEN expression (n = 9) was associated with significantly longer survival than low PTEN expression (mean survival 23.50 versus 15.54 months, log-rank p = 0.043). High levels of PTEN expression resulted in 43% reduction in risk for all NSCLC patients (HR = 0.57, 95% CI: 0.33-0.98, p = 0.041). PTEN expression and clinical stage remained significantly associated with survival after adjustment for age, sex and tumor type (HR = 0.56, 95% CI: 0.32-0.99; p = 0.048; HR = 0.54, 95% CI: 0.36-0.97; p = 0.045). No significant difference in continuous PTEN expression levels was observed among groups with different clinical or pathological characteristics (p > 0.17). When levels of PTEN expression were binarized using the optimal cutpoint, higher levels of PTEN expression were observed in patients with T1/T2 than in those with T3/T4 (80% and 58% respectively, p = 0.049) and in patients with AC than in those with squamous-cell carcinoma (SCC) (78% and 58% respectively, p = 0.08). No significant difference in binarized PTEN expression levels was found among groups with any other clinical/pathologic characteristic (p > 0.28). CONCLUSIONS: Our results suggest that high levels of PTEN expression may be favorable prognostic marker in NSCLC patients.

First Report of Botrytis Leaf Blight on Eleutherococcus senticosus Caused by Botrytis cinerea in China.

Eleutherococcus senticosus (Acanthopanax senticosus, manyprickle acathopanax) is a perennial herb belonging to the family Araliaceae and is mainly distributed in northeastern China, Siberia, Korea, and Japan. It is used for the treatment of rheumatism and neurasthenia. With the development of its cultivation, many diseases began to occur (2) and a previously unknown leaf blight on manyprickle was first observed in July of 2010 in Linjiang City, Jilin Province. The same symptoms were detected in other areas of Jilin Province, such as Baishan and Hunchun cities. The disease has resulted in serious loss of production of manyprickle acanthopanax, with 5 to 10% of leaves infected. The infection initially manifested as irregular lesions on the tips or margins of the leaves, which gradually developed into a V-shaped blight with concentric rings that was grayish brown in the center and dark brown at the margins. The blight eventually spread to cover one third of the entire leaf. Severely infected leaves were rolled or distorted, eventually desiccated and became brittle. Under continuously humid conditions, scattered gray mycelium and conidia appeared on the surface of affected leaf tissue. To isolate the causal agent, tissues were excised from diseased leaves, immersed in 0.1% mercuric chloride, suspended in sterile water, and plated on potato dextrose agar (PDA). Conidiophores arose singly or in groups, straight or flexuous, septate, with an inflated basal cell and dendriform near the apex, brown to light brown, and measured 5.0 to 10.0 × 100.0 to 150.0 µm (n = 50). Conidia were single-celled, globoid or oval-shaped, colorless, measuring 6.0 to 10.0 × 7.0 to 13.0 µm (n = 50). In culture, dark, irregular sclerotia were produced. The morphological descriptions and measurements of the fungi were similar to Botrytis cinerea (4). The ITS region of rDNA was amplified and sequenced. BLAST analysis of the 567-bp segment (JX840481) showed 100% identity with the sequence of Botryotinia fuckeliana (perfect stage of B. cinerea). To further identify the species of B. cinerea, three nuclear protein-coding genes (G3PDH, HSP60, and RPB2) (3) were sequenced and the sequences (KJ018759, KJ018757, and KJ018755) all showed 100% identity with those of B. fuckeliana. Pathogenicity tests were carried out on potted, healthy, 1-year-old plants (n = 10). A conidial suspension of 105 conidia/ml was sprayed with each strain (five strains total) on five leaves still on plants, and five plants were sprayed with water as controls. Plants were covered with polyethylene bags and incubated for 3 days at 25°C in a greenhouse. Symptoms appeared 7 days after inoculation, and were similar to those originally observed on plants under natural conditions, whereas control plants remained healthy. The pathogen was successfully re-isolated from inoculated leaves and was identified as B. cinerea on the basis of its morphological characteristics and related gene sequences. B. cinerea has been previously reported on E. senticosus in Korea (1). However, to our knowledge, this is the first report of Botrytis leaf blight of E. senticosus caused by B. cinerea in China. These results lay the foundation for the disease control. References: (1) K. J. Choi et al. Korean J. Med. Crop Sci. 15:199, 2007. (2) J. Gao et al. Plant Dis. 95:493, 2011. (3) M. Staats et al. Mol. Biol. Evol. 22:333, 2005. (4) Z. Y. Zhang. Flora Fungorum Sinicorum. 26. Botrytis, Ramularia. Science Press, Beijing, 2006.

First Report of Bacterial Soft Rot of Horn Lian (Typhonium giganteum) Caused by Pectobacterium carotovorum subsp. carotovorum in Jilin Province of China.

Horn lian (Typhonium giganteum) is a perennial herb of the family Aracea and is commonly used for expelling phlegm and as an antispasmodic treatment. In August 2012, horn lian grown in Changchun, Jilin Province of China, exhibited soft rot disease with ~60% incidence and experienced great losses. Water-soaked and dark green lesions on leaves expanded along main veins. Semitransparent, water-soaked, and sunken lesions on stems expanded rapidly and caused the whole plant to collapse with a foul smell. Nine representative strains were isolated from infected leaves and stems on nutrient agar (NA) medium after 36 h incubation at 28°C (1). Colonies were round, shiny, grayish white, and convex on NA medium. All strains were gram-negative, non-fluorescent on King's B medium (KB), facultatively anaerobic, motile with three to six peritrichous flagella (observed by electron transmission microscope), positive for catalase and pectolytic activity test on potato slices, but negative for oxidase, urease, and lecithinase. Strains grew at 37°C and in yeast salts broth medium containing 5% NaCl. They also liquefied gelatin and reduced nitrate, but did not reduce sucrose. Strains were also negative for starch hydrolysis, malonate utilization, gas production from glucose and indole. Results were variable for the Voges-Proskauer test. The strains utilized sucrose, arabinose, fructose, D-galactose, D-glucose, inositol, lactose, D-mannose, D-mannitol, melibiose, rhamnose, salicin, trehalose, maltose, raffinose, glycerol, D-xylose, and cellobiose as carbon sources, but not melezitose, α-CH3-D-gluconate, sorbitol, or dulcitol. Species identity was confirmed by molecular characterization of one of the nine strains, DJL1-2. DNA GC content indicated by high performance liquid chromatography (HPLC) was 51.7%. The 16S rDNA sequence (KC07897) of DJL1-2 showed 99% identity to that of a Pectobacterium carotovorum subsp. carotovorum (Pcc) strain (CP001657) and the sequence of the 16S-23S rDNA spacer region (KJ623257) was 93% similar to that of another known strain of Pcc (CP003776). As a result, the strains were identified as Pcc (2). Pathogenicity of the nine strains was evaluated by spraying 1 ml of bacterial cell suspension (108 CFU/ml) onto healthy leaves and injecting 0.1 ml of cell suspension into stems of 3-year-old horn lian plants with a sterile pipette tip. Three seedlings were used for each strain and sterilized water served as negative controls. Pcc SMG-2 reference strain (from milk thistle) was also inoculated into horn lian leaves and stems. Inoculated plants were covered with plastic bags for 24 h in a greenhouse at 28 to 30°C. After 72 h, water-soaked lesions similar to the naturally infected plants were observed on leaves and stems inoculated by the nine isolated strains and Pcc SMG-2, while negative control plants remained symptomless. Biochemical tests and 16S rDNA sequence analysis confirmed that the re-isolated bacteria were Pcc. To our knowledge, this is the first report of Pcc causing bacterial soft rot of horn lian in Changchun, Jilin Province, China. References: (1) Z. D. Fang. Research Method of Phytopathology. China Agricultural Press, 1998. (2) N. W. Schaad, et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. American Phytopathological Society, St. Paul, MN, 2001.

First Report of Bacterial Root Rot of Ginseng Caused by Pseudomonas aeruginosa in China.

Ginseng (Panax ginseng) is an economically valuable medicinal herb mainly planted in Jilin Province, China. In September 2013, during harvest, suspected bacterial rots were observed on ginseng roots with about 10% incidence in Fusong County, Jilin Province, China (127°29.48' N, 42°11.12' E). Rotted roots completely lost their economic value. Symptoms on roots began as water-soaked lesions, and developed rapidly into a soft, watery, decayed mass within 3 to 5 days. Three diseased root tissues were surface-sterilized in 70% ethanol for 30 s, rinsed 3 times in sterilized water and cut into small pieces (2 to 3 mm). Tissues were then macerated for 5 min in sterilized water, streaked onto nutrient agar (NA) medium, and incubated at 28°C for 2 days. Representative colonies were selected from each plate and further purified by sub-culturing onto NA medium. Five strains of the bacteria were gram-negative, short straight rods, 0.5 to 1.0 × 1.5 to 3.0 µm with a single, polar flagellum. Colonies were round, smooth, translucent, and yellowish green on NA medium. The bacteria were identified based on physiological and biochemical tests as follows (3): They were levan and potato rot negative, oxidase, aerobic, and arginine dihydrolase positive, converted nitrate to N2, hydrolyzed gelatin, produced nitrites from nitrates, produced pyocyanin, and grew at 41°C. Bacterial identity was further confirmed by amplifying the 16S rRNA (1,461 bp), gyrB (1,134 bp), and 16S-23S ITS genes (523 bp) with 27F/1492R, UP1/UP2, and L1/L2 primer sets, respectively. The 16S rRNA gene sequence (NCBI Accession No. KJ156527), gyrB gene sequence (KJ748373), and 16S-23S ITS gene sequence (KJ748374) had 99% identity to that of Pseudomons aeruginosa strain BS01 (JQ229778), ATCC25011 (FJ652721), and ATCC15522 (AB547908), respectively. The strains were also identified by using BD Phoenix-100 Automated Microbiology System (BD Ltd., New Jersey) as P. aeruginosa with 99% confidence. A pathogenicity test was conducted by spraying a suspension of five strains individually (108 CFU/ml) onto 4-year-old ginseng roots (cv. Damaya) wounded with a sterilized needle. Five ginseng roots were inoculated with each strain and five ginseng roots were inoculated with sterilized water as controls. All inoculated plants were maintained at 28°C with 80 to 85% relative humidity. Soft, watery tissue rot symptoms developed 3 to 5 days after inoculation, and were similar to those observed on the diseased plants under natural conditions. In contrast, no symptoms developed on control plants. The bacteria were readily re-isolated from inoculated plants and identified as P. aeruginosa using bacterial colony morphology, physiological and biochemical tests, as well as sequence analysis of the 16S rRNA gene, fulfiling Koch's postulates. The bacterium was not isolated from control plants. P. aeruginosa has been reported to cause diseases in a variety of plants including onion (1,2), arabidopsis, and sweet basil (4). To our knowledge, this is the first report of P. aeruginosa causing ginseng root rot in China. References: (1) E. J. Cother et al. Phytopathology 66:828, 1976. (2) X. J. Hao, and G. L. Xie. J. Plant Pathol. 88:340, 2006. (3) N. W. Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria, 3rd edition. APS Press, St. Paul, MN, 2001. (4) T. S. Walker et al. Plant Physiol. 134:320, 2004.

First Report of Botrytis cinerea Causing Stem Blight of Scutellaria baicalensis in Jilin Province, China.

Chinese skullcap (Scutellaria baicalensis) is a perennial herb in the Lamiaceae, and is grown in Jilin and 10 other provinces in China. Skullcap root extracts are used for treating allergies and respiratory conditions (5), and have inhibitory effects on some plant pathogens (2). In 2012, stem blight on 3-year-old Chinese skullcap plants were observed from mid-June to late-September in a herb garden at Jilin Agricultural University, Jilin Province, China. More than 90% of the 15 ha of skullcap plants were infected, and almost 60% of the infected plants died. A similar situation occurred in other regions of Jilin in 2012 and 2013. Oblong to irregular stem lesions, each 3 to 5 cm long, appeared on the basal stem. Lesions were initially light brown, and turned dark brown because of velvety, thick, gray fungal sporulation that formed over the lesions. The lesions eventually encircled the stems, preventing water and nutrient uptake. The infected plants often turned chlorotic and wilted. Symptomatic stem tissues were collected from three gardens in Jilin Province. Small pieces of diseased stems were surface-disinfested in 3% NaOCl for 2 min, rinsed twice in sterilized distilled water, plated on potato dextrose agar (PDA) medium, and incubated at 22°C for 5 days. Ten isolates were recovered, and all produced gray hyphae and dark sclerotia on PDA. The conidia formed on tree-like, branched conidiophores and were colorless, single, lemon-shaped, smooth-walled, and 9.0 to 16.9 × 5.7 to 9.7 µm. Sclerotia formed when cultures on PDA plates were incubated for 20 days at 22°C. The sclerotia were dark, irregular, and 2.2 to 3.8 × 1.1 to 2.6 mm. To confirm pathogenicity of each of the 10 isolates, five 3-year-old Chinese skullcap plants were each inoculated on the basal stem with an 8-mm-diameter colonized PDA plug of the appropriate isolate. Five plants were inoculated similarly with non-colonized PDA plugs as a control treatment. All plants were maintained at 22°C in a greenhouse. The first lesions appeared on stems 5 days after inoculation, whereas control plants remained healthy. The same fungus was consistently recovered from inoculated lesions as the original isolates, and no fungus was re-isolated from control plants. DNA was extracted from isolate RSL-1 and amplified using the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region primers ITS5/ITS4 (1), glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH) primers G-F/G-R, heat-shock protein 60 gene (HSP 60) primers H-F/H-R, and DNA-dependent RNA polymerase subunit II gene (RPB2) primers R-F/R-R (3). The ITS, G3PDH, HSP 60, and RPB2 sequences (GenBank Accession Nos. JX840480, KJ018760, KJ018758, and KJ018756, respectively) of isolate RSL-1 showed 100% similarity to the ITS sequence of strain WM6 of Botryotinia fuckeliana (anamorph Botrytis cinerea) (JN164269) (1), 100% identity to the G3PDH sequence of isolate Ice-2 of B. fuckeliana (AB546620) (3), 100% identity to the HSP60 sequence of isolate MUCL1152 of B. fuckeliana (AJ716090) (4), and 99.8% identity to the RPB2 sequence of isolate WM6 of B. fuckeliana (JN164272) (1). Based on these characteristics, the fungus was identified as B. cinerea. To the best of our knowledge, this is the first report of B. cinerea causing stem blight on S. baicalensis in Jilin Province, China. This disease may potentially cause great losses under favorable conditions. References: (1) X. Li. Plant Dis. 95:1592, 2011. (2) J. Y. Liu et al. Hubei Agric. Sci. 50:1809, 2011. (3) K. Maeda et al. J. Gen. Plant Pathol. 76:303, 2010. (4) M. Staats et al. Mol. Biol. Evol. 22:333, 2005. (5) K. Zandi et al. BMC Compl. Alternat. Medicine 13:1472, 2013.

Alternaria brassicicola Causes a Leaf Spot on Isatis indigotica in China.

Chinese woad (Isatis indigotica) is a biennial herb in the Brassicaceae that is widely cultivated in China. Extracts from the roots and leaves have potential pharmaceutical use for treatment of flu, encephalitis, measles, hepatitis, and mumps (2). In June 2012, a leaf spot was observed on 1-year-old plants of I. indigotica in the medicinal garden of Jilin Agricultural University, Changchun, Jilin Province, China. More than 50% of the leaves and 100% of the plants in the garden were symptomatic. In the initial stage of infection, irregular to circular, dark gray spots, each surrounded by a chlorotic halo, appeared on leaves. The spots ranged from pinpoint to 5 mm in diameter. Some spots enlarged and coalesced, forming concentric rings. Black, sunken, fusiform lesions were observed on the petioles. Lesions gradually dried and exhibited a shot-hole appearance, and entire infected leaves desiccated. Small pieces of infected leaves and petioles were surface-disinfested in 75% ethanol for 60 s, rinsed thrice in sterilized distilled water, dried, and plated on potato dextrose agar. Olive-green mycelium developed after 2 days of incubation at 25°C, turned dark green, and covered the petri dish 10 days later. The periphery of each colony was gray and velvety. On potato carrot agar medium, conidia formed on branched chains. Conidiophores arose singly or in clusters, were straight or flexuous, separated, and measured 6.8 to 26.7 × 3.1 to 11.9 µm Conidia on host plant tissues were olivaceous, cylindrical or inverted clavate, and 25.8 to 65.2 × 10.9 to 18.3 µm Larger conidia were cylindrical or obclavate, and smaller conidia were oval. Transverse and longitudinal septa of conidia ranged from 3 to 10 and from 0 to 7 µm, respectively. A very small conidial beak or no beak was observed on each conidium. On the basis of these morphological characteristics,the fungus was identified as Alternaria brassicicola (3). A PCR assay with the ITS4 and ITS5 primers was used to amplify DNA extracted from each of four isolates (1). The sequence (567 bp) of isolate Sl-8 was submitted to GenBank (Accession No. KF531832), and showed 100% similarity to that of an A. brassicicola isolate (AF392985.1), confirming the species identification. Pathogenicity assays with 10 single-conidium isolates were done by spraying a conidial suspension (1 × 106 conidia/ml) of each isolate, or sterilized water for the control treatment, onto healthy leaves and petioles of five 3-month-old plants of I. indigotica. Inoculated and control plants were enclosed in plastic bags for 48 h. After 7 days, symptoms on inoculated plants were similar to those on the original diseased plants, while control plants remained symptomless. Re-isolation from inoculated plants produced mycelial colonies with morphological characteristics of A. brassicicola, fulfilling Koch's postulates. No fungus was isolated from control plants. A. napiformis and A. brassicae have been reported as causal agents of Alternaria leaf spot on I. indigotica in China (3). To our knowledge, however, this is the first report of A. brassicicola as a pathogen on I. indigotica in China. References: (1) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (2) A. J. Li et al. Flora Reipublicae Popularis Sinicae Tomus 33, 1998. (3) T. Y. Zhang. Alternaria. Pages 99-100 in: Flora Fungorum Sinicorum, 2003.

First Report of Root Rot Caused by Fusarium redolens on Ginseng (Panax ginseng) in Jilin Province of China.

Ginseng (Panax ginseng C.A. Meyer) is a treasure of traditional Chinese medicine resources and has been used in food, health care products, and medicines in China for over 4,000 years. Jilin Province is the largest producing region in the world. Ginseng root rot disease caused by Fusarium spp. is the chief disease of ginseng with more than 25% incidence. Infected ginseng roots could not be used, which brought economic losses, and the species of the pathogens were not clear. The above-ground leaves of infected ginseng plants became red in the early stages and later wilted or died. All parts of root including rhizome, taproot, and fibrous root can be infected. Brown lesions were mainly formed on the surface and expanded gradually. Finally, multiple lesions converged and caused root rot. Diseased roots collected in June 2012 from 4-year-old ginseng (cv. Damaya) from Jingyu County and Jiaohe City, Jilin Province. Six isolates were obtained by cutting the roots into 5 mm3 pieces, disinfecting in 3% NaOCl for 3 min, and isolating on half-strength acidified potato dextrose agar (PDA). All isolates were purified from single spore. They produced white and cottony aerial mycelium cultured on PDA, microconidia were in the majority, oval or reniform, and length ranged from 5.97 to 12.01 µm. Macroconidia were in the minority, fusiform to conical, usually 3 to 5 septa, length ranged from 13.99 to 26.50 µm, thin-walled, with a hooked apical cell and a foot-shaped basal cell. A variety of structures of chlamydospores were produced, in chains or in pairs, basidixed or in the middle of the hyphae. Preliminary conclusions were made according to the morphological characteristics, which were consistent with the descriptions of Fusarium redolens (2). All the isolates of Fusarium spp. were accurately identified based on molecular biological methods of rDNA internal transcribed spacer (ITS) and the elongation factor (EF1-α) gene sequences analysis. ITS sequences analysis (GenBank Accession No. KF499035) resulted in a 100% match for one accession of F. redolens (X94169) by BLAST in the NCBI nucleotide database, and EF1-α gene (KF499036) had 100% similarity with 12 accessions of F. redolens (e.g., AF324319) by BLAST in the FUSARIUM-ID database (1). All isolates of F. redolens were tested for pathogenicity on ginseng root in vitro and healthy plants in a greenhouse, respectively. The surface of healthy ginseng roots were washed and disinfested in 70% alcohol for 3 min and plant in flower pots with sterile sand and sorghum grain infested with 14-day-old isolates and no inoculation as the control. Each experiment was repeated three times. Root rots were assessed 21 days after planting and all the isolates consistently caused root rot on inoculated plants, taproot and fibrous root exhibited brown to black root rot and plant wilting, whereas no root rot was observed in the controls and the pathogen was successfully re-isolated. The results were confirmed by the in vitro and healthy plant inoculation of ginseng root. F. redolens was identified previously as F. oxysporum, but recent molecular studies have shown that they are distant species in phylogenetic analysis (3). To our knowledge, this is the first report of F. redolens causing root rot of ginseng in China. References: (1) M. D. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) K. O'Donnell et al. Mycologia 90:465,1998.

First Report of Bacterial Soft Rot of Milk Thistle (Silybum marianum) Caused by Pectobacterium carotovorum subsp. carotovorum in Jilin Province of China.

Milk thistle (Silybum marianum) is an annual or biannual plant of the Asteraceae family that produces the hepaprotectant silymarin. In 2012, almost all milk thistle grown in the medicinal herbal garden of Jilin Agricultural University (Changchun, Jilin Province, China) exhibited symptoms of a previously undetected soft rot disease. Initial symptoms on stems appeared as tan, semitransparent, and water-soaked, then became sunken. The rotted lesions expanded rapidly and inner stem tissues were rotten with a foul smell. Eventually, the whole plant became black, then collapsed and died. Economic losses were significant as the seed crop was almost completely lost. Nine bacterial strains were isolated from tissues on nutrient agar (NA) medium after 36 h incubation at 28°C (1). Colonies of the nine strains were round, shiny, grayish white, and convex on NA medium. All strains were gram-negative, non-fluorescent, facultatively anaerobic, motile with two to four peritrichous flagella (observed by electron transmission microscope), positive for catalase and potato rot, but negative for oxidase and lecithinase. Strains grew at 37°C and in yeast salts broth medium containing 5% NaCl. They also liquefied gelatin. Strains were also negative for starch hydrolysis, malonate utilization, gas production from glucose, and indole. Results were variable for the Voges-Proskauer test and production of H2S from cysteine. The strains utilized esculin, fructose, D-galactose, D-glucose, inositol, lactose, D-mannose, D-mannitol, melibiose, rhamnose, salicin, trehalose, D-xylose, and cellobiose as carbon sources, but not melezitose, α-CH3-D-gluconate, sorbitol, or starch. Glycerol and maltose were only weakly utilized. Species identity was confirmed by molecular analysis of one of the strains, SMG-2. HPLC indicated a DNA GC content of 50.55%. The 16S rDNA sequence (KC207898) of SMG-2 showed 99% sequence identity to that of a Pectobacterium carotovorum subsp. carotovorum strain (DQ333384) and the sequence of the 16S-23S rDNA spacer region (KJ415377) was 95% similar to that of another known strain of P. carotovorum subsp. carotovorum (AF232684). Based on biochemical and physiological characteristics (2), as well as 16S rDNA gene analysis, the strains were identified as P. carotovorum subsp. carotovorum. Pathogenicity of the nine strains was evaluated by depositing a bacterial suspension (108 CFU/ml) on wounded stems (made with a disinfected razor blade) of 3-month-old milk thistle plants. Three plants were inoculated with each strain and three plants were treated with sterilized water as negative controls. Inoculated plants were covered with plastic bags for 24 h in a greenhouse at 28 to 30°C. After 48 h, the plants inoculated with bacteria showed similar symptoms as the naturally infected plants, while control plants remained symptomless. The symptoms observed on inoculated stems were rotten and sunken tissues. Bacteria were re-isolated from the inoculated plants and confirmed to be identical to the original strains based on 16S rDNA sequence analysis. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum causing bacterial soft rot of milk thistle in Changchun, Jilin Province, China. References: (1) Z. D. Fang. Research Method of Phytopathology. China Agricultural Press (In Chinese), 1998. (2) N. W. Schaad et al., eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. American Phytopathological Society, St. Paul, MN, 2001.

First Report of Phoma rhei as a Pathogen of Rheum rhabarbarum in China.

Rheum rhabarbarum L., rhubarb, is a perennial herb planted mainly in Hebei, Hubei, Shanxi, Heilongjiang, and Jilin provinces as well as Inner Mongolia, China. The plant grows about 1,000 meters above sea level (4), and is used widely in China to treat constipation and gout. From June to September 2012, a leaf spot was observed on R. rhabarbarum in the medicinal garden of Jilin Agricultural University, Changchun, Jilin Province, causing significant effects on the leaves of all infected plants. In the early stage of disease development, small red lesions were visible on infected leaves, which subsequently developed into irregularly shaped or circular necrotic spots, each with a light colored center, pink-red alternating concentric rings, and surrounded by a chlorotic halo. Some lesions became perforated in the center. Lesions ranged from 1 to 15 mm in diameter. Extensive spotting resulted in general browning and yellowing of entire leaves. As lesions enlarged and coalesced, some leaves died from the margin inwards. Lesions on the stem were fusiform and sunken. Small pieces of diseased leaves and stems were surface-disinfested in 75% ethanol for 60 s, rinsed twice in distilled water, dried, and plated on potato dextrose agar (PDA). A Phoma species was isolated that produced a gray or dark gray colony after 5 to 7 days. The isolate was transferred to oatmeal agar (OA) (3). Pycnidia were dark brown to black, globose to subglobose, and 121 to 354 × 100 to 262 µm. Conidia were ellipsoidal or reniform, colorless, unicellular, and 3.8 to 6.5 × 1.7 to 4.1 µm. On the basis of these characteristics, the fungus was identified as Phoma rhei (1). A PCR assay with the ITS4 and ITS5 primers was used to amplify the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) (2). The amplified product (567 bp) was sequenced and the sequence submitted to GenBank (Accession No. KF531831). The ITS sequence exhibited 99% identity to that of a P. rhei isolate in GenBank (GU237743.1), confirming the morphological identification. Pathogenicity of eight isolates on rhubarb was confirmed by spraying a spore suspension (1 × 106 spores/ml) produced on PDA on the leaves of each 6-year-old R. rhabarbarum (cv. Boyedahuang) plant. Each isolate was inoculated onto five plants, and five plants were sprayed similarly with sterilized water as a control treatment. The plants were then covered with plastic bags for 48 h, and kept in a greenhouse (20 to 30°C with a 12-h photoperiod/day). Initial symptoms on inoculated leaves were observed 3 to 4 days after inoculation, while the control plants remained healthy. Re-isolations from lesions on the inoculated leaves, using the same protocol as the original isolations, produced fungal colonies with the same morphological characteristics as the original isolates of P. rhei, but no fungi were re-isolated from the control plants. This fungus has been found on R. rhaponticum in New Zealand (1), but to our knowledge this is the first report of P. rhei on R. rhabarbarum in China. References: (1) G. H. Boerema et al. Phoma Identification Manual. Diffferentiation of Specific and Infra-Specific Taxa in Culture. CABI Publishing. Wallingford, UK, 2004. (2) D. E. L. Cooke et al. Mycol. Res. 101:667, 1997. (3) Z. D. Fang. Research Method of Phytopathology. China Agricultural Press (In Chinese), 1998. (4) A. J. Li et al. Flora Reipublicae Popularis Sinicae. Tomus 25:171, 1998.

First Report of Aphelenchoides besseyi Causing White Tip Disease of Rice in Jilin Province, China.

White tip disease of rice caused by the plant-parasitic nematode Aphelenchoides besseyi is an important domestic quarantine issue causing heavy yield losses of rice. During a survey for rice diseases in 2013, suspect white tip disease of rice was observed in the cities of Changchun and Gongzhuling, Jinlin Province, China. Leaf tips of susceptible rice varieties were white or yellow, becoming brown or black as necrosis set in. Tips of developing leaves were twisted and wrinkled. The flag leaf became twisted above the panicle until development was inhibited. General stunting of the plant accompanied leaf injury. Panicles were severely reduced and produced small deformed kernels while spikelets were reduced in number. Maturity of panicles was delayed, and secondary panicles arising from the lower nodes of the panicle were sterile. Nematodes were isolated from rice grains of diseased panicles. Key morphological features were determined for females and males. Measurements of females (n = 15) were: body length 612.50 to 735.00 µm (mean 673.75 µm), body width 17.37 to 22.21 µm (mean 19.79 µm), esophagus to gland 64.44 to 68.07 µm (mean 66.26 µm), and tail 34.41 to 41.29 µm (mean 37.85 µm). Females had a relatively short ovary with oocytes arranged in several lines; posterior uterine branch two to four times as long as body width; and tail tapering, conoid, with terminus bearing a mucro with four processes arranged in a shape somewhat that of a star. Measurements of males (n = 22) were: body length 483.39 to 580.00 µm (mean 531.70 µm), body width 14.46 to 17.12 µm (mean 15.79 µm), esophagus to gland 63.97 to 66.42 µm (mean 65.20 µm), and tail 30.38 to 36.45 µm (mean 33.41 µm). Males had a curved tail about 180° when relaxed, three pairs of ventrosubmedian papillae with the first one adanal, spicula curved with a slight basal process, and terminus bearing four mucrones arranged variably. Both males and females had lateral field occupying one fourth of the body width, marked by four incisures; delicate cephalic framework; small spear with moderate-size basal knobs; excretory pore was anterior to nerve ring; and intestine joined to esophagus immediately behind median bulb (3). All morphological data and characters were consistent with A. besseyi. Molecular diagnosis as A. besseyi was confirmed after DNA was extracted from nematodes (n = 16) and the templates were used in PCR analysis. The internal transcribed spacer region (ITS) of rDNA was amplified with primers TW81 and AB28 (2). The size of PCR product was 842 bp, and this sequence (KJ009342) was submitted to GenBank and was 98% similar with that of A. besseyi isolates from India (JF826519, JF93390, JF826517, JF826518) and Russia (EU186069). Molecular identification was further confirmed by amplifying part of the ITS region and part of the 5.8 gene of rDNA using the A. besseyi-specific primers BSF and BSR (1). The amplification yielded a 312-bp product specific to A. besseyi. Morphological and molecular data confirmed that the pathogen responsible for white tip disease in Changchun and Gongzhuling was A. besseyi. While this nematode has been reported from many rice-producing areas in China, this is the first detection of A. besseyi in Jilin Province, China. References: (1) R. Q. Cui et al. Plant Quarantine (Chinese) 24:10-12, 2010. (2) S. A. Subbotin et al. Nematology 2:153, 2000. (3) G. Thorne. Principles of Nematology. McGraw-Hill, New York, 1972.

[Assessment of present nutrition status with weight for height method in children of Guangxi].

According to the method of weight for height recommended by WHO in 1979. The present nutritional status of 9459 7-10 year old children in 5 prefectures and 3 cities of Guangxi was assessed. The results showed that the deficient nutrition rate was 19.94% and rate of obese children was 1.85%. The latter was lower than that reported by other provinces and foreign countries. The deficient nutrition rate (26.43%) of city children was higher than that of rural children (15.90%), but no statistically significant difference was found between male and female (P greater than 0.05).