[Analysis of time for diagnosis of nontuberculous mycobacterial lung disease and its associated factors in a tuberculosis-designated hospital in Shanghai].

Objective: To investigate the pathogenic characteristics, bacteriological diagnosis time and its associated factors among patients with nontuberculous mycobacterial (NTM) lung disease in a large tuberculosis-designated hospital in Shanghai from 2020 to 2021, in order to improve diagnosis efficiency and formulate precision treatment. Methods: On the basis of the Tuberculosis Database in Shanghai Pulmonary Hospital, NTM patients diagnosed by the Department of Tuberculosis between January 2020 and December 2021 were screened. Demographic, clinical and bacterial information were retrospectively collected. Chi-square test, paired-sample nonparametric test and logistic regression model were used to analyze the factors associated with the diagnosis time of NTM lung disease. Results: A total of 294 patients with bacteriologically confirmed NTM lung disease were included in this study, 147 males and 147 females with a median age of 61(46, 69) years. Of them, 227 (77.2%) patients had comorbidity of bronchiectasis. Species identification results showed that Mycobacterium Avium-Intracellulare Complex was the main pathogen of NTM lung disease (56.1%), followed by Mycobacterium kansasii (19.0%) and Mycobacterium abscessus (15.3%). Species such as Mycobacterium xenopi and Mycobacterium malmoense were rarely identified, accounting for a total proportion of only 3.1%. Positive culture rates for sputum, bronchoalveolar lavage fluid and puncture fluid were 87.4%, 80.3% and 61.5%, respectively. Paired-sample analysis showed that the positive rate of sputum culture was significantly higher than that of smear microscopy (87.1% vs. 48.4%, P<0.01), while no statistical difference was observed between sputum and bronchoalveolar lavage fluid on positive culture rate (78.7% vs. 77.3%, P>0.05). Patients with cough or expectoration were observed with 4.04-fold (95%CI 1.80-9.05) or 2.95-fold (95%CI 1.34-6.52) higher probability of positive sputum culture, compared to those without. Regarding bronchoalveolar lavage fluid, female or patients with bronchiectasis had a 2.82-fold (95%CI 1.16-6.88) or 2.38-fold (95%CI 1.01-5.63) higher probability to achieve a positive culture. The median time to diagnosis of NTM lung disease was 32 (interquartile range: 26-42) days. The results of multivariable analysis showed that patients with symptom of expectoration (aOR=0.48, 95%CI 0.29-0.80) needed a shorter diagnosis time in comparison with patients without expectoration. With Mycobacterium Avium-Intracellulare Complex as a reference, lung disease caused by Mycobacterium abscessus needed shorter diagnosis time (aOR=0.43, 95%CI 0.21-0.88), whereas those caused by rare NTM species were observed to require a longer diagnosis time (aOR=8.31, 95%CI 1.01-68.6). Conclusion: The main pathogen causing NTM lung disease in Shanghai was Mycobacterium Avium-Intracellulare Complex. Sex, clinical symptoms and bronchiectasis had an impact on the positive rate of mycobacterial culture. The majority of patients in study hospital were timely diagnosed. Clinical symptoms and NTM species were associated with the bacteriological diagnosis time of NTM lung disease.

[The application of quantitative analysis of EBV DNA loads in plasma and peripheral blood mononuclear cells in Epstein-Barr Virus infection-related diseases in children].

Objective: To explore the correlation of EBV DNA load in two different types of plasma and peripheral blood mononuclear cells (PBMCs) in children with Epstein-Barr Virus (EBV) infection diseases. Methods: A retrospective evaluated was performed on EBV DNA quantification in plasma and PBMCs by qPCR between April, 2019 and December, 2020. The samples were collected from children of 456 cases with EBV infection and 2 306 healthy cases. In EBV infection group, boys were 253 and girls were 203, aged from 8 days to months to 16 years. In healthy group, boys were 1 267 and girls were 1 039, aged from 8 days to 16 years. Results: Infectious mononucleosis (IM) was the most common disease associated with EBV infection 73.68%(336/456). The detection rate of plasma and PBMCs in EBV infection group was 91.89% (419/456)and 99.34% (453/456)respectively, and was 100%(456/456) in plasma or PBMCs. The detection rate of plasma and PBMCs in healthy group was 1.13%(26/2 306) and 30.01%(715/2 306), respectively. Levels of EBV DNA in plasma and PBMCs in EBV infection group [IM, acute infections, pneumonia, post-transplantation lymphoproliferative disorder (PTLD), hemophagocytic lymphohistiocytosis, tonsillitis and lymphadenitis] was significantly higher than those in healthy group (In plasma, Z=-47.18,-34.41,-33.40,-31.71,-24.38,-20.86 and -20.59,respectively; In PBMCs, Z=-33.17,-16.45,-11.33,-9.45,-5.57,-5.16 and -5.45, respectively; P<0.05). In IM group, EBV DNA load in plasma and PBMCs in remission stage was significantly lower than those in infection stage (Z=-11.45, -8.53;P<0.05). In PTLD group, there was significant difference in EBV DNA load in plasma between infection and remission stage (Z=-4.13, P<0.05), while there was no significant difference in EBV DNA load in PBMCs (Z=-0.817, P>0.05). Conclusions: EBV infection was mainly caused by IM. Combined detection of plasma and PBMCs in EBV DNA is valuable for improving diagnosis ability of EBV infection-related diseases, and the load of EBV DNA could be used as a marker.

[Advance and review: miniscrew-assisted rapid palatal expansion].

Rapid palatal expansion (RPE) is commonly used to correct transverse maxillary deficiencies and recognized as a reliable orthopedic procedure in children and adolescents. For adults, however, conventional RPE has been considered rarely successful and can produce undesirable dental effects. Along with the development of digital techniques and mini-implant anchorage, a novel method called miniscrew-assisted RPE (MARPE) has become available for the treatment of maxillary transverse deficiency in older patients recently. In this article, the biomechanical principles and indications of MARPE, the advances in device design, the clinical effects, the matters needing attention and limitations of this method, and the stability after expansion are discussed.

Presence of pldA and exoU in mucoid Pseudomonas aeruginosa is associated with high risk of exacerbations in non-cystic fibrosis bronchiectasis patients.

OBJECTIVES: To explore the association between the virulence genes exoU and pldA in isolated mucoid Pseudomonas aeruginosa and the clinical outcomes of patients with non-cystic fibrosis (CF) bronchiectasis. METHODS: A prospective observational cohort study was performed in the Shanghai Pulmonary Hospital from October 2012 to January 2015. We consecutively enrolled all non-CF bronchiectasis patients with mucoid P. aeruginosa isolates obtained from bronchoalveolar lavage fluid or sputum. The exposure variable was the presence of virulence gene, exoU or pldA, in the strains. The primary outcome was exacerbation of bronchiectasis. Logistic regression analysis was performed to evaluate the association between virulence genes and exacerbation. RESULTS: The final analysis included 147 patients (mean (SD) age, 57.86 (11.43) years, 101 female subjects) with median (interquartile range) follow-up of 18 (13-26) months. The following factors were relative to exacerbations: body mass index ≤18.5 kg/m2 (odds ratio (OR) = 5.05; 95% confidence interval (CI), 1.37-18.57), length of stay ≥8 days (OR = 2.65; 95% CI, 1.14-6.19) and positive for either virulence gene (OR = 6.80; 95% CI, 1.47-31.37). The gene-positive group had more exacerbations per year (mean 2.37, SD 2.10, n = 33 vs. mean 0.79, SD 0.83, n = 114) and a higher proportion of patients with exacerbation (31/33, 93.94% vs. 74/114, 64.91%). The proportion of patients being exoU or pldA positive increased as the exacerbation frequency of bronchiectasis increased. CONCLUSIONS: The virulence genes exoU and pldA in mucoid P. aeruginosa are significant risk factors for exacerbations in patients with non-CF bronchiectasis.

[Pathogen spectrum in enteroviral infections among children in Beijing from 2010 to 2016].

Objective: To understand the epidemiological and etiological characteristics of enterovirus (EV)-associated diseases among children in Beijing from 2010 to 2016. Methods: This was a repeated cross-sectional study. The throat swabs were collected from children with probable EV-associated diseases at the Children' s Hospital Affiliated to Capital Institute of Pediatrics from 2010 to 2016. The samples were sent for pan-EV, enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) detection by real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR) . The viral types of non-EV-A71 and non-CV-A16 EV-positive samples were identified using modified RT-PCR and sequencing with CV-A6, EV-A/B group and 5 'UTR universal primers. The constituent ratios of the prevalence of different EV types in different age and gender groups were compared. Results: Of the 2 703 throat swabs, 1 992 (73.7%) samples were positive for EV, including EV-A71 (19.1%, 516/2 703), CV-A16 (24.3%, 658/2 703), CV-A6 (22.2%, 600/2 703), CV-A10 (4.5%, 122/2 703) and other types of EV (3.5%, 95/2 703). There was 1 case of EV-A71 and CV-A16 co-infection. The positive detection rate of EV-A group (excluding EV-A71, CV-A16, CV-A6 and CV-A10) increased from 11.3% (7/62) to 95.2% (59/62) after using the modified VP1-specific primers and PCR amplification conditions. During the period between 2010 and 2012, CV-A16 and EV-A71 predominated in EV-positive samples. However, CV-A6 accounted for 60.7% (68/112) in 2013, much higher than CV-A16 (23.2%, 26/112) and EV-A71 (12.5%, 14/112). In 2014, EVs were mainly of CV-A16 and EV-A71, but CV-A6 was the predominant type in 2015 (68.2%, 232/340) and in 2016 (38.6%, 151/391). The epidemic season of EVs was mostly from April to August, but CV-A6 showed a small epidemic peak from October to November. The male-to-female ratio of EV-positive patients was 1.50∶1, and EV-associated diseases mostly occurred in children under 5 years of age. Younger children were more susceptible to CV-A6 than to EV-A71 and CV-A16. Conclusions: From 2010 to 2016, there was a significant change in the spectrum of EVs in children with EV-associated diseases in Beijing. Since 2013, non-EV-A71 and non-CV-A16 increased, and CV-A6 gradually became one of the major pathogens of EV-associated diseases. The modified PCR primers and amplification conditions can effectively improve the reliability of test results.

miR-483 is down-regulated in gastric cancer and suppresses cell proliferation, invasion and protein O-GlcNAcylation by targeting OGT.

MicroRNAs (miRNAs) are involved in the gastric carcinogenesis and progression. Here, we confirmed that miR-483 was frequently decreased in gastric cancer patients. The expression levels of miR-483 were negatively correlated with tumor stage, node metastasis and stromal invasion. Log-rank tests demonstrated that low expression of miR-483 was strongly correlated with poor overall survival in patients with gastric cancer. Moreover, ectopic expression of miR-483 remarkably suppressed gastric cancer cell proliferation by enhancing cell apoptosis and significantly inhibited the invasion of gastric cancer cells, while low expression of miR-483 exhibited the opposite effect. Bioinformatics analysis revealed that OGT was a potential target of miR-483, and miR-483 inhibited the expression level of OGT mRNA by direct binding to its 3'-untranslated region (3'UTR). Expression of miR-483 was negatively correlated with OGT in gastric cancer tissues. In addition, modulation of miR-483 expression could affect the global cellular protein O-GlcNAcylation in gastric cancer cells. Furthermore, silencing of OGT counteracted the effects of miR-483 repression, while its overexpression reversed tumor inhibitory effects of miR-483. In conclusion, our study revealed that miR-483 functions as a tumor suppressor by inhibiting proliferation, invasion and protein O-GlcNAcylation of gastric cancer via targeting OGT, and that miR-483 may serve as prognostic or therapeutic target for gastric cancer.

[Effect of occupational exposure on job burnout in nurses].

Objective: To investigate the effect of occupational exposure on job burnout in nurses, and to analyze the mediating effect of negative emotion between occupational exposure and job burnout and the regulatory effect of supervisor support on occupational exposure and negative emotion. Methods: From September to December, 2015, simple random sampling was used to select 543 nurses from six tertiary hospitals in Zhejiang Province, China. The questionnaires consisted of occupational exposure risk questionnaire, negative emotion questionnaire, supervisor support questionnaire, and job burnout questionnaire. Results: The total score of occupational exposure risk in nurses was 11.43±7.19; the score of emotional exhaustion was 3.19±1.24, the score of low sense of personal accomplishment was 3.02±1.21, and the score of sense of working indifference was 2.24±1.06. There were significant differences in occupational exposure score between nurses with different sexes (t=2.61, P<0.01) and working years (F=4.49, P<0.01) . There were significant differences in the scores of emotional exhaustion and low sense of personal accomplishment in nurses with different sexes (t=5.25, P<0.001) and working years (t=-3.48, P<0.01) . Occupational exposure had positive effects on negative emotion (ß=0.41, P<0.05) , emotional exhaustion (ß=0.47, P<0.05) , sense of working indifference (ß=0.42, P<0.05) , and low sense of personal accomplishment (ß=0.17, P<0.05) . Negative emotion had a partial mediating effect between occupational exposure and emotional exhaustion (total effect size 30.5%, P<0.05) and between occupational exposure and sense of working indifference (total effect size 37.1%, P<0.05) . Negative emotion had a complete mediating effect between occupational exposure and low sense of personal accomplishment (ß=0.08, P>0.05) . Supervisor support negatively regulate the effects of occupational exposure and negative emotion (F=21.73, P<0.001) . Conclusion: In nurses, occupational exposure has a direct positive effect on job burnout and indirectly influences job burnout via negative emotion. Supervisor support can reduce the negative impact of occupational exposure on negative emotion.

[Application of mind mapping in the teaching of oral and maxillofacial development].

Oral and maxillofacial development is the course that the oral students first learn, and their mastery directly influences the study of other courses that follow. The application of mind mapping in the teaching of the development of oral maxillofacial region can make the temporal and spatial features of oral and maxillofacial development in the form of visual presentation, help students build development mode of thinking, stimulate students' interest in learning, improve the quality of teaching of oral histopathology, and promote the new teaching concept in oral pathology teaching.

Identification of amino acid residues involved in hemin binding in Porphyromonas gingivalis hemagglutinin 2.

Porphyromonas gingivalis (P. gingivalis) is a major etiological agent in the development and progression of chronic periodontitis. It produces cysteine proteases (gingipains), including a lysine-specific gingipain and two arginine-specific gingipains. Heme binding and uptake are fundamental to the growth and virulence of P. gingivalis. The recombinant hemagglutinin 2 domain (rHA2) of gingipain binds hemin with high affinity. The aim of the present work was to identify the key residues involved in its hemin-binding activity. A functional rHA2 was expressed and bound to hemin-agarose, and then digested with endopeptidases. The peptides bound to hemin-agarose were identified by mass spectrometry and the amino acids were assessed by mutation and peptide binding inhibition analysis. The DHYAVMISK sequence was identified in peptides derived from both Asp-N and Lys-C endopeptidase digestions of rHA2. A monoclonal antibody, mAb QB, was produced and its epitope was associated with the DGFPGDHYAVMISK peptide within the HA2 domain. Hemin was shown to competitively inhibit the immunoreactivity of rHA2 or the peptide to mAb QB. The peptide DHYAVMISK inhibited hemin-binding activity; although, this inhibition was not seen when the peptide contained the H1001E mutation (DEYAVMISK). Based on these results, we propose that residue His1001 is involved in the hemin-binding mechanism of the P. gingivalis rHA2 and the peptide containing this residue, DHYAVMISK, may be an inhibitor of hemin binding.

A systematic review and meta-analysis on the efficacy of low-level laser therapy in the management of complication after mandibular third molar surgery.

The successful reduction of postoperative discomfort is of great significance. This review aims to evaluate the efficacy of low-level laser therapy (LLLT) for the reduction of complication caused by impacted mandibular third molars extraction. An extensive literature search up to October 2013 for randomized controlled trials (RCTs) was performed through CENTRAL, PubMed, Embase, Medline, and CNKI. Six RCTs in which involves 193 participants are included in the meta-analysis. Among them, three RCTs exhibit a moderate risk of bias, while the other three show a high bias risk. Compared with placebo laser/control group, pain is significantly reduced with LLLT on the first day (mean difference [MD] = -2.63, 95% confidence interval [CI] -4.46 to -0.79, P = 0.005). The superiority of LLLT in pain control persists on the second day (MD = -2.34, 95% CI -4.61 to -0.06, P = 0.04) and the third day (MD = -3.40, 95% CI -4.12 to -2.68, P < 0.00001). Moreover, LLLT reduces an average of 4.94 mm (MD = 4.94, 95 % CI 1.53 to 8.34, P = 0.004) of trismus compared with placebo laser irradiation in the first 3 days. On the seventh day, the superiority of LLLT also persists (MD = 3.24, 95% CI 0.37 to 6.12, P = 0.03). In the first 3 days after surgery, extraoral irradiation (MD = -0.69, 95% CI -1.30 to -0.08, P = 0.03) and intraoral combined with extraoral irradiation (MD = -0.65, 95% CI -1.15 to -0.15, P = 0.01) reduced facial swelling significantly. On the seventh day, the intraoral combined with extraoral irradiation group (MD = -0.32, 95% CI -0.59 to -0.06, P = 0.02) still showed benefit in relieving facial swelling. However, because of the heterogeneity of intervention and outcomes assessment and risk of bias of included trials, the efficacy is proved with limited evidence. In the future, well-designed RCTs with larger sample size will be required to provide clearer recommendations.

Fibroblasts regulate variable aggressiveness of syndromic keratocystic and non-syndromic odontogenic tumors.

UNLABELLED: Keratocystic odontogenic tumors (KCOTs) are jaw lesions that can be either sporadic or associated with nevoid basal cell carcinoma syndrome, which typically occurs as multiple, aggressive lesions that can lead to large areas of bone destruction and resorption and cause major impairment and even jaw fracture. To clarify the role of fibroblasts in the aggressivness of syndromic (S-) as compared with non-syndromic (NS-) KCOTs, we assessed fibroblasts derived from 16 S- and NS-KCOTs for differences in cell proliferation, multilineage differentiation potential, alkaline phosphatase activity, and osteoclastogenic potential. S-KCOT fibroblasts had proliferative and osteoclastogenic capacity higher than those from NS-KCOTs, as evidenced by higher numbers of tartrate-resistant acid-phosphatase-positive multinuclear cells, expression of cyclooxygenase 2, and ratio of receptor activator of nuclear factor-kappa B ligand to osteoprotegerin. The osteogenic potential was higher for S- than for NS-KCOT fibroblasts and was associated with lower mRNA expression of runt-related transcription factor 2, collagen type I α1, osteocalcin, and osteopontin as well as reduced alkaline phosphatase activity. These results suggest that the distinct characteristics of fibroblasts in KCOTs are responsible for the greater aggressiveness observed in the syndromic subtype. ABBREVIATIONS: AP, alkaline phosphatase; CK, cytokeratin; COL1A1, collagen type I α1; COX-2, cyclooxygenase-2; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-1α, interleukin 1α; KCOT, keratocystic odontogenic tumor; NBCCS, nevoid basal cell carcinoma syndrome; NS-KCOT, non-syndrome-associated KCOT; OCN, osteocalcin; OPG, osteoprotegerin; OPN, osteopontin; RANKL, receptor activator of nuclear factor-kappa B ligand; Runx2, runt-related transcription factor 2; S-KCOT, syndrome-associated KCOT; TAF, tumor-associated fibroblast; and TRAP, tartrate-resistant acid phosphatase.

First Report of Pindo Palm Heart Rot Caused by Ceratocystis paradoxa in China.

On January 12th, 2012, a novel disease with an incidence of 50% was discovered in Pindo palm Butia capitata (Mart.) Becc from the Coconut Grant View Garden (19°33.137' N, 110°47.482' E) located in Wenchang, Hainan Province. Diseased leaflets at the base of the rotted heart leaves had reddish brown lesions; when the infection progressed, the leaves turned yellow and became blighted from the inner to the outer part of the crown. Once the growing point was destroyed, the entire tree ultimately died. Tissues from the edges of lesions from diseased leaflet samples were placed onto potato dextrose agar (PDA) and incubated at 25°C for 3 days. The color of colonies of five isolates obtained turned from white to black in 48 h. The optimum temperature for mycelium growth was from 20 to 30°C, and no growth occurred at temperatures higher than 40°C or lower than 5°C (n = 5). The cylindrical colorless to pale brown conidia were 7.5 to 17.5 µm long × 5.0 to 7.5 µm wide (n = 100); oval black chlamydospores were 12.5 to 22.5 × 7.5 to 15.0 µm (n = 100). The sequence (497 bp) of the internal transcribed spacer (ITS) region of the representative isolate BX3 (China Center for Type Culture Collection No. CCTCC AF2014002) was amplified using primer pair ITS1/ITS4 (GenBank Accession No. KF939052) and shared 99% sequence identity with Ceratocystis paradoxa strain xie331-4 (JQ039332). Based upon these biological characteristics and ITS sequence, this pathogen was identified as C. paradoxa (Dade) C. Moreau (anamorph Thielaviopsis paradoxa (de Seynes) Höhn.) (3). Pathogenicity tests were conducted on 8-cm-long sections of young leaflets excised from a 12-year-old pindo palm tree. One side of the midrib of 10 sections was wounded with a sterilized scalpel at the center and the other side was non-wounded, then a PDA plug (4 to 6 × 4 to 6 mm) from the edge of an actively growing colony of BX3 incubated for 3 days were inoculated onto each wounded or non-wounded site. As controls, plain PDA plugs were placed on wounded and non-wounded spots of another 10 sections following the above procedure. Pathogenicity was tested twice. Each inoculated section was then put into a 9-cm petri dish in which two filter papers (Φ = 9 cm) were placed and 8 ml of sterile water were added to maintain high humidity, and then all dishes were placed in a dark incubator at 25°C. After 5 days, typical symptoms developed only on the wounded points inoculated with mycelium plugs. C. paradoxa was re-isolated from the margins of the expanding lesions. C. paradoxa causing fruit rot of B. capitata was reported in Uruguay (2), but to our knowledge, there are no previous reports of this species in China or infecting leaves of B. capitata worldwide (1). We report here a new Ceratocystis disease on B. capitata, and it was named as pindo palm heart rot based on its symptoms. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , Feb 21, 2014. (2) V. Gepp et al. New Dis. Rep. 27:12, 2013. (3) F. Y. Yu et al. Plant Dis. 96:290, 2012.

First Report of Leaf Spot Disease in Coconut Seedling Caused by Bipolaris setariae in China.

Coconut (Cocos nucifera L.), an important oilseed as well as a multipurpose perennial plantation crop, is distributed and planted in humid tropical areas. In October 2012, a new leaf spot disease was observed on 3-year-old coconut seedlings in Wenchang, Hainan Province, China. The symptom first appeared as spindly or elliptical and brown flecks with water-soaked lesions that became yellow with the progress of the disease. In the later stage of the disease, the lesions merged together, gradually expanding to the leaf apex. In recent years, the disease has been prevalent in all the nursery gardens surveyed. Once young leaves got infected and nearly all the leaves of the tree showed diseased symptoms, the coconut eventually became defoliated. The pathogen was isolated from the lesion margin, surface sterilized with 75% ethanol and 0.1% mercury bichloride, washed by sterile distilled water, and then placed excising pieces of leaves from the leision margin onto potato dextrose agar (PDA). Plates were incubated at 25°C for 4 days. After 7 days, the colony was grayish black and produced black pigment in the medium. Aerial mycelium was fluffy, septate, and branched, the conidiophores were slightly flexuous or straight, 5 to 11 µm thick, and produced curved, spindle-shaped, or fusiform, septate conidia with 4 to 10 septa, measuring 39 to 86 × 9 to 16 µm, with a slightly protuberant hilum, truncated. Based on the symptoms and mycelial and conidial characters above, the fungus was identified as Bipolaris setariae (1). The pathogenicity was established and repeated for six times by following Koch's postulates. Two 1-year-old coconut seedlings were washed with sterilized water and six leaves were wounded with a sterile needle and then inoculated by spraying them with a suspension of conidia of the isolate. The seedlings were kept in two incubators at 25°C for 12 days. Inoculated leaves showed typical symptoms similar to those described above. The pathogen was re-isolated from inoculated leaves. Morphological characteristics were identical to the original isolated fungus. In contrast, the control leaves did not show any symptoms. The genomic DNA of this fungus was extracted, amplification of the internal transcribed spacer (ITS) region was performed with primer ITS1 and ITS4, and the purified PCR product was sequenced (GenBank Accession No. KJ605157). BLASTn analysis revealed 99% sequence similarity with four B. setariae isolates (HE792936.1, JX462256, GU073108.1, and FJ606786.1). Morphologic characters and sequence analysis of the ITS rDNA confirmed that the pathogen was B. setariae. Bipolaris incurvata has been reported causing disease on coconut (2), but B. setariae was not previously reported on coconut. So far, this is the first report of B. setariae caused coconut seedling leaf spot disease in Hainan, China. References: (1) K. C. da Cunha et al. J. Clin. Microbiol. 50:4061, 2012. (2) A. Kamalakannan et al. New Dis. Rep. 12:18, 2005.

First Report of Leaf Spot of Tea Oil Camellia (Camellia oleifera) Caused by Lasiodiplodia theobromae in China.

Tea oil camellia (Camellia oleifera Abel.), one of the most famous woody oil plants, is distributed and cultivated widely in central and southern China for its strong adaptability. In September 2013, tea oil camellia plants with severe leaf spots were observed in commercial production fields located in Wenchang, Hainan Province. Spots were initially chlorotic, became necrotic and black with a chlorotic halo, developing to cover the entire width of the leaves, and leading to leaf death. Isolations were performed by excising pieces of symptomatic leaves from the lesion margin, surface sterilized with 90% ethanol and 0.6% sodium hypochlorite, and then placed them on potato dextrose agar (PDA). Plates were incubated in a sterile chamber at 26 ± 2°C for 2 days. A fungus was consistently isolated on PDA from all 23 diseased leaf samples. Pure cultures were obtained by monosporic culture technique. After 2 to 3 days of incubation at 26 ± 2°C with a 12-h photoperiod, the fungus initially produced white colonies with dense aerial mycelia, which later turned black (6 to 7 days). The mycelium was fast spreading, branched, and septate. Pycnidia were black, globose, ostiolate, and produced in stroma on the medium surface after 28 days at the same culture conditions as above. Conidia were initially unicellular, subovoid, hyaline, thick-walled with granular content, and 19.8 to 28.9 × 11.5 to 15.7 µm (avg. 25.1 × 13.5 µm). Mature conidia were one-septate and dark brown with longitudinal striations. These observed morphological features suggested that the fungus possessed the same characteristics as previously described for Lasiodiplodia theobromae (Pat.) Griffon & Maubl (syn = Botryodiplodia theobromae) (2). For molecular identification, the ITS1-5.8S-ITS2 region and fragments of the ß-tubulin and elongation factor 1-alpha (EF1-α) genes were sequenced and BLASTn searches done in GenBank. Accession numbers of gene sequences submitted to GenBank were KF811055 for ITS region; KJ639047 for ß-tubulin; and KJ639048 for EF1-α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS164.96 of L. theobromae reported in GenBank (NR_111174, EU673110, and AY640258). Hence, both morphological and molecular characteristics confirmed the fungus as L. theobromae. To confirm fungal pathogenicity, ten 1-year-old healthy plants of C. oleifera were inoculated with the fungus. Mycelial plugs (5 mm) taken from a 7-day-old colony growing on PDA were deposited on wounds with a sterilized knife on leaves and covered with moist cotton. Ten additional control plants were treated similarly but with sterile PDA plugs. Plants were maintained in a moist chamber at 26 ± 2°C for 3 days and then in a greenhouse at 25°C and 40% relative humidity. All the inoculated plants produced typical leaf spot symptoms 3 weeks after inoculation. The fungus was consistently re-isolated from all inoculated plants. Control plants did not show any symptoms. L. theobromae has been reported to cause cankers and dieback in a wide range of hosts and is common in tropical and subtropical regions of the world (1,2), but not previously reported causing disease on C. oleifera. To our knowledge, this is the first report worldwide of leaf spot of C. oleifera caused by L. theobromae. References: (1) S. Mohali et al. For. Pathol. 35:385, 2005. (2) E. Punithalingam. Page 519 in: CMI Descriptions of Pathogenic Fungi and Bacteria. Commonwealth Mycological Institute, Kew, Surrey, UK, 1976.

First Report of Burkholderia andropogonis Causing Bacterial Leaf Spot of Betel Palm in Hainan Province, China.

In May 2009, a severe bacterial disease of arecanut (Areca catechu L.) with an incidence of 100% was observed in a plantation of about 8,400 plants in Wenchang City, Hainan Province, China (19°47.171' N, 110°54.335' E). Symptoms consisted of small circular to elongated brown lesions, ranging from 1 to 105 mm in length and 1 to 21 mm in width, surrounded by yellow halos. White colonies, without fluorescent or diffusible pigments, were consistently recovered on King's B Medium plates from lesions surface-sterilized in 70% ethyl alcohol for 1 min. All isolates were gram-negative and each had a single, polar, sheathed flagellum. Isolates were identified as a Burkholderia sp. based on physiological and biochemical tests: oxidase and catalase positive, negative for arginine dihydrolase, gelatin hydrolysis and starch hydrolysis, and negative for acid production from levan (1,3). Sequences (approx. 1,400 bp each) of the 16S rRNA gene amplified from four isolates using primer pair 27F/1492R (2) (GenBank Accession Nos. JX415481, JX415479, JX415482, and JX415483) shared 99% sequence identity with that of Burkholderia andropogonis strain 6369 (DQ786951). Representative isolates Y11 (China General Microbiological Culture Collection Center No. CGMCC 1.12337), Y30 (CGMCC 1.12338), W15, and W20 were compared with B. andropogonis strain NCPPB No. 1012 and all caused a hypersensitive reaction on leaves of Nicotiana benthamiana. Isolate pathogenicity was tested twice with a total of three replications per isolate. Two young leaves each of 2-year-old arecanut plants were infiltrated with a bacterial suspension of 108 CFU/ml, then covered individually with plastic bags for 48 h, and incubated at 100% relative humidity with 16 h of daylight at 25°C by day and 8 h of darkness at 20°C by night. After 7 days, small water-soaked spots with yellow halos were observed and 60 days after inoculation, lesions developed similar to those caused by B. andropogonis in the field. Koch's postulates were fulfilled by reisolating bacteria from typical lesions on inoculated plants. These bacteria were identical to inoculated strains in colony morphology and sequences of the 16S ribosomal RNA gene. To our knowledge, this is the first report of B. andropogonis infection on betel in Hainan Province, mainland China. This disease was first reported in Taiwan, a province of China. Conditions of high humidity and high temperature support disease outbreaks and infection can result in severe economic losses. In 2012, this disease also appeared on a number of plantations located in other counties. As betel is, economically, the second most important crop in Hainan Province, measures should be required to control this disease, especially in typhoon seasons. References: (1) S. H. Hseu et al. Plant Pathol. Bull. 16:131, 2007. (2) D. J. Lane. In: E. Stackebrandt, et al. Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Chichester, United Kingdom, pp. 115-175, 1991. (3) X. Li and S. H. De Boer. Plant Dis. 89:1132. 2005.

First Report of Stem Bleeding in Coconut Caused by Ceratocystis paradoxa in Hainan, China.

Stem bleeding of coconut was discovered in 2009 in Hainan, China. Affected trunk areas exhibited dark discoloration and a reddish brown or rust-colored liquid bleeding from different points. Stem tissues under the lesions rotted and became brownish yellow to black. Affected plants died within 3 to 4 months after stem symptoms first appeared. Stem bleeding of coconut is known to occur in production areas worldwide. The disease was first reported in Sri Lanka (1), caused severe damage to PB-121 hybrids in Indonesia (2), and is now known to occur in many other coconut-producing countries. However, to our knowledge, this is the first report of the disease in China. A fungus was isolated from lesion margins of diseased coconut trees. Colonies on potato dextrose agar (PDA) were white, became black 1 to 2 days later, and emitted a strong, fruity aroma. The fungus produced conidia, which were cylindrical, colorless to pale brown, and 6.9 to 14.9 × 3.1 to 6.0 µm, and oval, black chlamydospores that were 7.9 to 19.4 × 4.6 to 11.0 µm. The optimum temperature for mycelial growth ranged from 25 to 35°C and it did not grow at temperatures lower than 5°C or higher than 40°C. On the basis of these characteristics, the fungus was identified as Ceratocystis paradoxa (Dade) C. Moreau (anamorph Thielaviopsis paradoxa (de Seynes) Höhn). The internal transcribed spacer (ITS) region was amplified from genomic DNA with primers ITS1 and ITS4 and the PCR products were sequenced (GenBank Accession No. JQ039332). BLAST analysis showed 99% sequence similarity with C. paradoxa (GenBank Accession No. HQ248205.1). Pathogenicity of the fungus was tested by inoculating 10, 3-year-old coconut trees of the cv. green tall at the 12-leaf stage in the field. Agar plugs (5 mm in diameter) from the periphery of 7-day-old C. paradoxa colonies grown on PDA were placed on healthy trunks, rachis, and leaves, which were either wounded or unwounded. Wounds were made with a sterilized cork borer. Sites of the inoculations were wrapped with plastic tape to prevent desiccation; the experiment was repeated three times. Controls received plain PDA discs. Two weeks after inoculation, characteristic rusty brown lesions appeared only on wounded plants that were inoculated with the fungus. A brownish liquid oozed from the points of inoculation. Controls did not show signs of disease development. C. paradoxa was reisolated from the diseased tissues. Infection occurred on wounded sites only, suggesting that wounds may be required for infection. To prevent stem bleeding of coconut trees by C. paradoxa, vigilant cultural practices must be maintained to avoid causing wounds on the trees. References: (1) S. A. Alfieri. Plant Pathol. Circular No. 53. Florida Department of Agriculture Division of Plant Industry, 1967. (2) D. R. N. Warwick and E. E. M. Passos. Trop. Plant Pathol. 34:175, 2009.

Color, dye and DOC removal, and acid generation during fenton oxidation of dyes.

The removal of color, dye and dissolved organic carbon by Fenton discoloration was investigated using the synthetic dye wastewaters containing various dyes (reactive blue 19, Eriochrome Black T or Fast Green FCF). The results indicated that discoloration of dyes was very rapid but mineralization of dyes was insignificant based on the removal of dissolved organic carbon. The rates of color, dye and dissolved organic carbon removal were in the order of reactive blue 19 > Fast Green FCF > Eriochrome Black T. The generation of SO(2-)4, and N(O-)3, increased with the progress of the Fenton reaction. The concentrations of SO(-2)4 and N(O-)3, generated are in the order of reactive blue 19 > Fast Green FCF> Eriochrome Black T. A mathematic model was proposed to formulate the formation of SO(2-)4 and N(O-)3 during dye degradation. Results indicated that one S-containing and two N-containing functional groups are involved in the oxidation reaction, and that S-containing groups are involved in the oxidation reaction earlier than N-containing functional groups.

Development of a polyclonal antibody-based sensitive enzyme-linked immunosorbent assay for fumonisin B(4).

Polyclonal antibodies (PAb) against fumonisin B(4) (FmB(4)), which have good cross-reactivity with four major fumonisins, were produced by immunizing a rabbit with FmB(4)-keyhole limpet hemocyanin conjugate. A sensitive competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for fumonisins was developed. Because of the limited supply of FmB(4), both FmB(1)-horseradish peroxidase conjugate (HRP) and FmB(3)-HRP were tested as the toxin-enzyme markers in the CD-ELISA. In the FmB(1)-HRP-based CD-ELISA, the concentrations of FmB(1), FmB(2), FmB(3), and FmB(4) causing 50% inhibition of binding of enzyme marker (IC(50)) were 9.0, 2.1, 9.0, and 6.5 ng/mL (or the relative cross-reactivities toward FmB(1), FmB(2), FmB(3), and FmB(4) were 58.5, 309.5, 58.5, and 100%), respectively. In the FmB(3)-HRP-based CD-ELISA, the IC(50) values for FmB(1), FmB(2), FmB(3), and FmB(4) were 7.1, 1.9, 7.6, and 5.3 ng/mL (or the relative cross-reactivities toward FmB(1), FmB(2), FmB(3), and FmB(4) were 74, 280, 70, and 100%), respectively. The FmB(3)-HRP-based CD-ELISA was then used in a series of analytical recovery experiments using Fusarium moniliforme corn culture material spiked with FmB(1) and with clean corn spiked with a FmB(3)/FmB(4)-containing extract. The overall recovery of FmB(1) from culture material in the range of 10-100 ppm was 65%. The detection limit for FmB(1) with clean corn as matrix was between 100 and 500 ppb. F. moniliforme cultures were analyzed with the developed CD-ELISA and a well-established FmB(1) antibody-based ELISA, which is not sensitive to FmB(4). Differences in the fumonisin levels found by the two assays were used as an indication of the presence of FmB(4) in the culture material and, therefore, as a method to identify FmB(4)-producing strains. Using ELISA in combination with HPLC individual B-series fumonisins were quantified. The ELISA developed in the present study would be a useful supplement to FmB(1) antibody-based ELISA for screening of Fusarium strains for the production of major fumonisins.

Monitoring of microcystin-protein phosphatase adduct formation with immunochemical methods.

Using anti-microcystin-LR monoclonal antibodies, an immunoblotting procedure was developed to monitor the formation of microcystin-protein phosphatase adducts in vitro and in vivo. The detection limits for the covalent binding of MCYST-LR with the recombinant protein phosphatase 1 (PP1) and rabbit liver cytosol proteins were found to be 0.1 ng and 0.3 ng per assay, respectively. MCYST-PP1 adducts were detected 30 s after the addition of MCYST-LR into the reaction mixture. Reduction of the methyldehydroalanine (Mdha) residue of MCYST-LR with ethanethiol totally abolished the covalent binding of the toxin to PP1, but retained its inhibitory toxicity on PP1. Immunoblotting analyses and enzyme-linked immunosorbant assay showed that between 5 min to 16 h after i.p. injection of single dose (35 microg/kg) of MCYST-LR into mice, approximately 0-27% of the injected toxin was found covalently bound while 0.2-9.2% existed as free form in liver cytosol.