Can transcranial direct current stimulation combined with interactive computerized cognitive training boost cognition and gait performance in older adults with mild cognitive impairment? a randomized controlled trial.

BACKGROUND: Older adults with Mild Cognitive Impairment (MCI) are often subject to cognitive and gait deficits. Interactive Computerized Cognitive Training (ICCT) may improve cognitive function; however, the effect of such training on gait performance is limited. Transcranial Direct Current Stimulation (tDCS) improves cognition and gait performance. It remains unclear whether combining tDCS with ICCT produces an enhanced synergistic effect on cognition and complex gait performance relative to ICCT alone. This study aimed to compare the effects of tDCS combined with ICCT on cognition and gait performance in older adults with MCI. METHOD: Twenty-one older adults with MCI were randomly assigned to groups receiving either anodal tDCS and ICCT ( tDCS + ICCT ) or sham tDCS and ICCT ( sham + ICCT ). Participants played Nintendo Switch cognitive games for 40 min per session, simultaneously receiving either anodal or sham tDCS over the left dorsolateral prefrontal cortex for the first 20 min. Cognitive and gait assessments were performed before and after 15 training sessions. RESULTS: The global cognition, executive function, and working-memory scores improved in both groups, but there were no significant interaction effects on cognitive outcomes. Additionally, the group × time interactions indicated that tDCS + ICCT significantly enhanced dual-task gait performance in terms of gait speed (p = 0.045), variability (p = 0.016), and dual-task cost (p = 0.039) compared to sham + ICCT. CONCLUSION: The combined effect of tDCS and ICCT on cognition was not superior to that of ICCT alone; however, it had a significant impact on dual-task gait performance. Administering tDCS as an adjunct to ICCT may thus provide additional benefits for older adults with MCI. TRIAL REGISTRATION: This trial was registered at http://www. CLINICALTRIALS: in.th/ (TCTR 20,220,328,009).

Visualized detection of tobacco anthracnose by RPA-LFD.

Anthracnose caused by Colletotrichum spp. is a widespread fungal disease that is detrimental to tobacco growth and inflicts economic damage up to 100 million in tobacco-growing regions in China. An early diagnostic tool is vital for the accurate determination and management of anthracnose in the field. This study investigated the diversity of Colletotrichum spp. on tobacco leaves with anthracnose and developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) diagnostic method for the rapid and equipment-independent detection of the main Colletotrichum spp. causing tobacco anthracnose. This assay targeted the chitin synthase gene (chs1) and could be performed in a few minutes (6-10 min). All isolates of C. kastii, C. fructicola and C. gloeosporioides yielded positive results using the RPA-LFD assay, and no cross-reaction occurred with other fungal species from tobacco or other hosts. The detection threshold was 1 pg of genomic DNA under optimal reaction conditions. The entire RPA-LFD assay enabled the detection of pathogen visualization within 30 min without specialized equipment by combining a polyethylene glycol-KOH method for extracting DNA rapidly from tobacco leaves infected with C. kastii, C. fructicola and C. gloeosporioides. Based on these results, the RPA-LFD assay is easy to operate, rapid and equipment independent and is promising for development as a kit to diagnose tobacco anthracnose in resource-limited settings at point-of-care.

First Report of Tobacco Leaf Tip Blight Caused by Alternaria gossypina in China.

Tobacco (Nicotiana tabacum L.) belongs to the family Solanaceae, an economically significant crop (Zhou et al. 2023). Twelve samples with leaf spots were collected in Keti Village, Changshun County, Zunyi City, Guizhou province, China in 2022. Twenty-five percent of the samples had dry lesions near the leaf tip which resulted leaf tip blight after development. Fungi were isolated by a previous method (Wei et al. 2022). Six Alternaria strains were obtained and preserved in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. Among them, one strain YZU 221477 showed distinct cultural characteristics out of five A. alternata strains, which was again determined by growing on potato dextrose agar (PDA) at 25°C for 7 days in dark to evaluate. The colonies (60 mm in diameter) were white cottony in the center surrounded by vinaceous purple. To examine the morphology, mycelia were inoculated onto potato carrot agar (PCA) at 22°C, following an 8 h light/16 h dark photoperiod (Simmons 2007). Conidia were obclavate or ovoid, normally 3-5 conidial units per chain, 20-38 × 10-16.5 µm, 3 to 5 transverse septa, beakless or a short beak (4-30 µm). The observation results were consistent with those of A. gossypina (Zhang 2003). Total genomic DNA was extracted using the CTAB method and seven gene regions including internal transcribed spacer of rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1 alpha (TEF1), RNA polymerase second largest subunit (RPB2), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (EndoPG) and an anonymous gene region (OPA10-2) were amplified with ITS5/ITS4, gpd1/gpd2, EF1-728F/EF1-986R, RPB2-5F/RPB2-7cR, Alt-for/Alt-rev, PG3/PG2b and OPA10-2L/OPA10-2R primers, respectively. All sequences were deposited in GenBank (ITS: OR710806; GAPDH: PP057862; TEF1: PP158601; RPB2: PP057863; Alt a 1: PP057865; EndoPG: PP057861; OPA10-2: PP057864). Combining with relevant sequences retrieved from the NCBI database were used for the phylogenetic analysis. Maximum Likelihood (ML) tree was constructed with RAxML v.7.2.8 employing GTRCAT model using 1000 bootstrap (BS) replicates to assess statistical support. The results indicated that the present strain grouped with A. gossypina (type strain of CBS 104.32) supported with 73% bootstrap values, also having a support of 0.83 Bayesian posterior probabilities values. Based on morphology and molecular evidence, the strain YZU 221477 is identified as Alternaria gossypina. Pathogenicity was examined to fulfill Koch's postulates. Mycelial plugs (6 mm diameter) of the present strain and A. alternata cultivated on PDA were taken from the margin and inoculated onto viable tobacco leaves (Cultivar: Yunyan 87, n=3) growing forty days, while controls were inoculated with sterile PDA. The assay was conducted three times. The plants were maintained at 25°C with humidity levels over 85% in a greenhouse. Leaves were evaluated after 7 days, necrotic spots encircled by yellow halos were on both inoculums, except controls. Pathogen re-isolation confirmed that it was the same as inoculated fungus based on morphology. A. gossypina was firstly found on cotton (Hopkins 1931), late reported to induce disease on Minneola, Nopalea, Hibiscus, Citrus, Solanum and Ageratina. To our knowledge, this is the first report of A. gossypina causing tobacco leaf tip blight in China, and it also provides a basis for controlling of tobacco leaf tip blight.

Decision-making impairment under ambiguity but not under risk may underlie medication overuse in patients with chronic migraine.

OBJECTIVE: To explore whether patients with chronic migraine and medication overuse headache (CM + MOH) present with decision-making deficit. BACKGROUND: Factors underlying MOH in patients with CM remain unclear. Whether the process of decision-making plays a role in MOH is still controversial. Decision-making varies in the degree of uncertainty: under ambiguity where the probability of outcome is unknown, and under risk where probabilities are known. METHODS: Decisions under ambiguity and risk were assessed with the Iowa Gambling Task and the Cambridge Gambling Task, respectively, whereas executive function was assessed by the Wisconsin Card Sorting Test. RESULTS: A total of 75 participants: 25 patients with CM + MOH, 25 with CM, and 25 age- and sex-similar healthy controls (HCs), completed this cross-sectional study. There was no significant difference in headache profiles except for more frequent analgesic use (mean ± SD: 23.5 ± 7.6 vs. 6.8 ± 3.4 days; p < 0.001) and higher Severity of Dependence Scores (median [25th-75th percentile]: 8 [5-11] vs. 1 [0-4]; p < 0.001) in patients with CM + MOH compared to CM. Total net score (mean ± SD) on the Iowa Gambling Task in patients with CM + MOH, CM, and HCs were - 8.1 ± 28.7, 10.9 ± 29.6, and 14.2 ± 28.8, respectively. There was a significant difference between the three groups (F(2, 72) = 4.28, p = 0.017), with patients with CM + MOH making significantly more disadvantageous decisions than patients with CM (p = 0.024) and HCs (p = 0.008), while the CM and HC groups did not differ (p = 0.690). By contrast, there was no significant difference between the groups in the Cambridge Gambling Task and the Wisconsin Card Sorting Test. Furthermore, performance on the Iowa Gambling Task was inversely correlated with analgesic consumption (r = -0.41, p = 0.003), suggesting that decision-making under ambiguity may be related to MOH. CONCLUSIONS: Our data suggest that patients with CM + MOH had impaired decisions under ambiguous, but not risky situations. This dissociation indicates disrupted emotional feedback processing rather than executive dysfunction, which may underlie the pathogenesis of MOH.

First Report of Target Spot Caused by Rhizoctonia solani AG-5 on Tobacco in China.

Tobacco is one of the vital economic crops in China. Nevertheless, tobacco diseases cause substantial economic losses each year. Tobacco target spot is a fungal disease which commonly found on the leaves. While both sexual and asexual reproduction can occur, asexual reproduction is much more common in tobacco. In June 2022, target spot was found on tobacco leaf samples from Yibin, Sichuan Province and Meitan, Guizhou Province, China. The typical symptoms were light brown tissue with concentric ring marks, and the necrotic part of the disease spot was fragile and forming perforation after falling off. The diseased tissue in the sample was cut off and sterilized in 75% ethanol for 1 min, and rinsed three times in sterilized distilled water. Finally, the tissues were placed on potato glucose agar (PDA) medium with kanamycin (0.1 mg/mL). After incubation at 28 °C in darkness for 3 days,the culture of the isolate grew in the form of radial mycelium on PDA dishes, the mycelium was white initially, turned brown generally at the later stage, and finally thickened and separated with the growth of the culture. Nine pathogenic strains were isolated, including four isolates from Yibin and five from Meitan. They were all used for pathogen identification. Genomic DNA of each isolate was extracted using the CATB method, and PCR analysis was performed with primers specifically designed to detect individual fusion groups or fusion subgroups of solani: AG-1 IA, IB, and IC; AG-3 PT; AG-4 HG-I, HG-II and HG-III; AGs-5-6 and P-21-22. Among the 11 specific primer pairs, only AG-5-specific primer amplified the fungal DNA, indicating that the nine isolates tested all belonged to the R. solani AG-5 fusion group. BLASTn search was performed on the gene sequences obtained from these strains and they deposited in GenBank under accession no. OP647851-OP647859. These gene sequences were aligned with the voucher specimen R. solani AG-5, with more than 99% similarity . The nine isolates were then tested for mycelial anastomosis reactions using the R. solani AG-5 standard strain following the method described by Ogoshi (1987). A decrease in the diameter of the mycelia at the anastomosis site and death of adjacent cells were observed, indicating their anastomosis response. Therefore, these nine strains were identified as R. solani AG-5 based on morphological and genetic analysis. Subsequently, one pathogenic strain from Meitan and another one from Yibin were selected for pathogenicity verification. Mycelial PDA blocks (6 mm in diameter) of the two isolates were inoculated on healthy tobacco plants, while leaves containing only PDA blocks were used as controls. A total of 6 replicates were conducted. After inoculation, they were incubated at 85% relative humidity and 15 to 25 °C. Koch's hypothesis was confirmed by reisolating pathogens from diseased leaves 5 days after inoculation. Typical symptoms were observed on tobacco plants inoculated with the pathogen strains but not on control tobacco plants. To the best of our knowledge, tobacco target spot has been reported caused by R. solani AG-3, AG-6 and AG-2.1 groups in the field in China and in Argentina. Up until now, this is the first report of R. solani AG-5 causing tobacco target spot on tobacco in the field in China. It was also found to be highly virulent to chickpea in Turkey. Due to serious damages caused by this disease in the last five years in China, more attention should be paid in disease control measures to avoid economic losses. In addition, it also provides some theoretical help for the damage caused by this pathogen on other hosts and helps people to better understand Rhizoctonia solani AG-5.

Ultrasonographic imaging findings of the shoulder in patients with Parkinson disease.

BACKGROUND: Shoulder disorders, including frozen shoulder, bursitis, and rotator cuff lesions, are common musculoskeletal problems in patients with Parkinson disease (PD). Because musculoskeletal ultrasound (US) can clearly image shoulder joints, we aimed to evaluate shoulder joints using US in patients with PD and healthy participants and correlation between US and PD severity. METHODS: This is a prospective case-control study. 50 patients with PD and 50 healthy subjects from the outpatient department were administered US for bilateral shoulders. For data analysis, we chose the more severely affected side in the PD group for matching with the corresponding shoulder in the control group according to age, sex, and body mass index. Pain and disability were measured using the Visual Analogue Scale (VAS) for pain, Shoulder Pain and Disability Index (SPADI), and the Shoulder Disability Questionnaire (SDQ). RESULTS: The PD group had higher VAS pain scores during activity (p = 0.003) and rest (p < 0.001), as well as the SPADI and SDQ scores (p < 0.001). In US findings, biceps long head tendon sheath effusion (p = 0.001), humeral head cortical irregularity (p = 0.012), and abnormality in the supraspinatus tendon (p = 0.003) were significantly greater in the PD group. Intra-group analysis in the PD group demonstrated a significant difference in passive flexion (p = 0.019) and supraspinatus tendinopathy (p = 0.033) on US examination during different disease stages. CONCLUSIONS: Patients with PD had more supraspinatus tendinopathy on US findings than control subjects. The lesion was significantly associated with disease severity. CLINICAL TRIAL NUMBER: NCT02702232.

Stem-end Rot Caused by Lasiodiplodia brasiliense on Tobacco in China.

Tobacco (Nicotiana tabacum L.), is a major cash crop grown worldwide for its leaves, which are dried and fermented before being put in tobacco products. Tobacco production is seriously threatened by numerous diseases (Qiu et al. 2021). In August 2021, plants with stem-end rot were observed in a tobacco field in Zunyi City, Guizhou Province, China. Surveys indicated a 22 to 35% disease incidence in five counties. Symptoms of black necrosis appeared at the base of stems, and leaves turned yellow. To isolate the pathogen, diseased stems were cut into small segments and placed on potato dextrose agar (PDA) at 25°C in darkness for 3 to 5 days. To obtain pure cultures, hyphal tips from colonies were transferred to fresh PDA plates. A representative strain, GZAX 110, was used for further identification. The fungal colonies were initially gray, later deepening to smoke-gray. Conidiogenous cells were fully divided, discrete, transparent, thin-walled, smooth and cylindrical. Conidia matured slowly, were ellipsoid to ovoid, containing granular content, with a rounded apex. The base was largely truncated, and conidia became dark brown with one central septum, 21.0-30.0 × 12.0-18.0 µm. On water-agar medium, teleomorph structures were not observed. These characteristics suggested the fungus was Lasiodiplodia sp. (Netto et al. 2014). For further confirmation, genomic DNA was extracted using the CTAB method (Watanabe et al. 2010); and the ITS (internal transcribed spacer region of rDNA) and EF-1α (translation-elongation factor) regions were amplified with primer pairs ITS1/ITS4 and EF1-688F/EF1-1251R (Cruywagen et al. 2017), respectively. The ITS and EF-1α sequences (OM534558 and OM632673) were analyzed by BLASTN searches. The ITS sequence showed 100% identity (490/490 bp) to L. brasiliense strain AGQMy0011 (MW274145) and the EF-1ɑ sequence showed 100% identity (551/551 bp) to L. brasiliense strain EX1 (MF580811). A multilocus phylogenetic tree was constructed via the Maximum-likelihood (RAxML v.7.2.8) and Bayesian Inference (MrBayes v.3.2.1) analyses (Elsie et al. 2017) using combined ITS and EF1-α reference sequences of Lasiodiplodia species. Phylogenetic analysis showed that GZAX 110 clustered monophyletically with strains of L. brasiliense. Thus, the isolate GZAX 110 was confirmed as L. brasiliense. Pathogenicity of GZAX 110 was tested on tobacco plants at the eight leaf stage by attaching mycelial plugs (5 mm in diameter) to stems and leaves according to Cruywagen et al. (2017). Inoculated plants were kept in a greenhouse (16 h light/8 h darkness, 22℃, relative humidity >85%). Control plants were inoculated with PDA plugs. The experiment was repeated three times with five plants. Seven days after inoculation, dark brown necrosis was observed at inoculation sites on stems and leaves, while the control plants remained healthy. The pathogen was re-isolated from the inoculated sites and further validated as the same fungus through morphological and phylogenetic analyses. Previously, this fungus has been reported on Mangifera indica (mango) in China (Zhang et al. 2018), and apple (Martins et al. 2018) and papaya (Netto et al. 2014) in Brazil. However, to our knowledge, this is the first worldwide report of L. brasiliense causing stem-end rot on tobacco. This report provides information for future diagnosis and management of the disease.

First Report of Target Spot Caused by Rhizoctonia solani AG-6 in Tobacco in China.

Tobacco is an annual and solanaceous crop, which is widely produced in China. In July 2020, tobacco target spot was observed on 50% of tobacco plants in a 5-ha commercial field of Bijie (27.32° N, 105.29° E), Guizhou province, China. Typical symptoms firstly appeared on the old leaves as round watery spots. Then the spots became a diameter of 2 to 20 cm, with concentric ring lines and dead spots. Fifteen small pieces (5 × 5 mm) of leaf tissue were cut from the edge of the lesions, surface sterilized and placed on potato dextrose agar (PDA) medium amended with kanamycin (0.1 mg/ml). Isolate J136, one of five isolates with similar morphology, was selected for pathogen identification. The culture of the isolate on PDA was brown and exhibited radial mycelial growth after incubation at 28 oC in darkness for 5 days. Hyphae of the fungus were white at the beginning, turned light brown to brown at the later stages, and finally became thick and separated. Sclerotia were brown and produced on PDA after 25 days of incubation in the dark. These characteristics were similar to the colony characteristics of R. solani. The genomic DNA of Isolate J136 was extracted using the CTAB method. PCR analyses were conducted using the following primers specifically designed for the detection of individual AGs or subgroups of R. solani: AG-1 IA, IB and IC (Kuninaga 2003), AG-2-1, AG-2-2, IIIB, IV and LP (Carling et al. 2002), AG-3 PT (Misawa 2015), AG-4 HG-I and HG-II (Kuninaga 2003), and AGs-5-6 (Arakawa and Inagaki 2014). Among the 12 specific primer pairs, only AG-6-specific primers amplified a fragment of ca. 230 bp product, indicating that the tested strain belonged to R. solani AG-6. The sequence was deposited in GenBank with accession no. MZ379468. Using BLASTN search, the sequence of the gene was aligned with the voucher specimen, R. solani AG-6. A phylogenetic tree was constructed based on these sequences. After wards, Isolate J136 was tested for hyphal anastomosis reaction using the R. solani AG-6 standard strain according to the method described by Ogoshi (1987). The hyphal diameter at the point of anastomosis was reduced, with obvious anastomosis point, and the death of adjacent cells, indicating their anastomosis reactions (Anderson 1982). Thus, based on the morphological and genetic analyses, the fungus was identified as R. solani AG-6. To verify its pathogenicity, six plants (cv. Yunyan87) at the 5-to-6 leaf stage were inoculated with mycelial PDA plugs (5 mm in diameter). Leaves inoculated with PDA-only plugs served as the controls. Treated tobacco plants were maintained at a temperature range of 15 to 25 oC in a greenhouse with 85% relative humidity. After 5 days inoculation, typical symptoms were observed on the inoculated leaves, whereas no symptoms were observed on the control leaves. Koch's postulates were fulfilled by re-isolation of the pathogen from the diseased leaves. R. solani AG-2-2 is the only previously reported group of R. solani, which causes tobacco target spot in the field (Gonzalez et al. 2011). Therefore, to our knowledge, this is the first report of R. solani AG-6 causing target spot of tobacco in the field in China. Since considerable losses caused by the disease have frequently happened in this region, addition of this new group pathogen in the disease pool can be more problematic. Proper disease control strategies are in need to be developed to prevent further losses.

First Report of Leaf spot Caused by Xylaria arbuscula on Flue-cured Tobacco in China.

Flue-cured tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves in China. Around 70% of tobacco production in China occurs in southwest China. In summer of 2019, leaf spot symptoms were observed on ten to twenty percent of tobacco plants in a 2 ha commercial field of Bijie (27.32° N, 105.29° E), Guizhou province, China. The leaf spots were white with dark-brown in edges, irregularly round and oval, and diseased tissue dropped out leaving the leaves ragged in appearance (Fig. 1A, 1B). One diseased leaf from each of five plants was sampled. From five leaves, a total of 15 small (5 mm × 5 mm) pieces of leaf tissue were cut from the edge of the lesions after surface sterilization and placed on potato dextrose agar (PDA) medium. Five fungal colonies that were similar in appearance were isolated and one was purified, BEZ22, was selected arbitrarily for identification. Mycelia of the pathogen was initally white and dense, and then black carbonized mycelia appeared from the center of the colony 7 days' after incubation. Mycelia was white, sparse and radiated when incubated on OA (oatmeal agar) (Fig. 1E, 1F, 1G, 1H). Genomic DNA of the isolate was extracted. The internal transcribed spacers (ITS) with primers ITS1/ITS4 (White et al. 1990), actin (ACT) gene with primers ACT-512F/ACT-738R (Hsieh et al. 2005), beta-tubulin (TUB2) with primers T1/T22 (O'Donnell & Cigelnik 1997) and RNA polymerase II second largest subunit gene (RPB2) with primers fRPB2-5F/ fRPB2-7cR (Liu et al. 1999) were amplified and sequenced, respectively. The generated sequences were deposited in GenBank with accession numbers MT804353 (ITS), MT809582 (ACT), MT799790 (TUB2) and MT799789 (RPB2). Using BLASTN searches, the sequences of each gene above were aligned with the voucher specimum, Xylaria arbuscula 89041211. The number of nucleotides that were similar for ITS (GU300090) was 550/551 (99%); for ACT (GQ421286), 266/266 bp (100%); for TUB2 (GQ478226), 1501/1501 bp (100%); and for RPB2 (GQ844805), 1135/1135 bp (100%), respectively (Fig. 2). A phylogenetic tree was constructed based on these four sequences with a final alignment of 3456 characters (ITS 551, ACT 266, TUB2 1501 and RPB2 1138). Thus, based on morphological and phylogenetic analyses, the isolate BEZ22 was identified as Xylaria arbuscula. To verify pathogenicity, six tobacco plants at seedling stage (5-6 leaves) without visible disease were inoculated using mycelial plugs (5 mm in diameter). Leaves inoculated with PDA only plugs served as controls. After inoculation, all tobacco plants were maintained in a greenhouse with 85% relative humidity at 25 oC under a 12/12 h light/dark cycle. Five days after inoculation, typical early symptoms were observed on the inoculated leaves, and not on the control leaves. Koch's postulates were fulfilled by re-isolation of the pathogen from diseased leaves. Xylaria arbuscula has also been reported as a pathogen of Macadamia in Hawaii (Wenhsiung et al. 2009) and sugarcane in Indonesia (Maryono et al. 2020). However, to our best knowledge, this is the first report of X. arbuscula causing leaf spot on tobacco in China. This leaf spot has the potential to cause serious damage to tobacco in this region that could result in reduced production, consequently disease management of this pathogen should be considered.

First Report of Leaf Spot Caused by Nigrospora aurantiaca in Tobacco in China.

Tobacco (Nicotiana tabacum L.) is one of the most important cash crops in China. In June 2019, tobacco (cv. Yunyan 87) samples with gray spots surrounded by yellowish ring were collected in Zhengan (107.43° N, 28.55° E), Guizhou province, China. Pieces of leaf tissue (3 mm × 3 mm) that were cut at the junction of diseased and healthy portion were surface sterilized and plated on potato dextrose agar (PDA). After incubation at 25°C in the dark for 7 days, an isolate (T22) was chosen and used for pathogen identification. The colonies had aerial hyphae, initially white and then turned grey, and produced a soluble red pigmen on PDA. The colonies were floccose aerial mycelia, dark grey, with pale brown hyphae, and produced conidia on oatmeal agar. Conidia were ovoid or ampulliform, black, smooth. Based on morphological characteristics, isolate T22 was identified as Nigrospora aurantiaca (Wang et al. 2017). For molecular identification, the large subunit (LSU) and internal transcribed spacer (ITS) of ribosomal RNA, ß-tubulin (TUB) and translation elongation factor 1-alpha (TEF1) genes of T22 were amplified by PCR with the primer sets LROR/LR5, ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF2 (Suwannarach et al.2019), then PCR products were sequenced. Their GenBank accession numbers were MT341787, MT328649, MT348395 and MT348394, respectively. Phylogenetic tree of combined LSU, ITS, TUB, and TEF sequences showed that isolate T22 was assigned to N. aurantiaca strain (CGMCC 3.18130 and LC 7034) with 100% bootstrap support. Based on morphological characteristics and multi-gene molecular analysis, isolate T22 was identified as N. aurantiaca. To fulfill Koch's postulates, PDA plugs grown with N. aurantiaca were placed on the leaves of four tobacco plants (cv. Yunyan 87) at the 10-leaf stage. Leaves inoculated with PDA only plugs served as the controls. Treated plants were maintained in a greenhouse with temperatures ranging from 18 to 28 °C. Five days after inoculation, typical symptoms were observed on inoculated leaves but not on the controls. N. aurantiaca was re-isolated from the diseased leaves but not from the controls. To our best of knowledge, this is the first report of N. aurantiaca causing leaf spot on tobacco in China. N. aurantiaca has been reported to cause leaf spot on Castanea mollissima in China (Luo et al. 2020). Due to potential serious damage caused by the disease in this region, proper disease management practices should be developed and implemented.

Leaf Spot Caused by Epicoccum latusicollum on Tobacco in China.

Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves in China. In continuing research on foliar diseases of tobacco in Guizhou province in August 2019, diseased leaves of tobacco that had sandy beige, elliptical or irregular shaped lesions, with brown in edge, and surrounded by yellow halos on 40% of leaves on 5% plants were obtained (cv. Yunyan 87) in Zhenan (28.55° N, 107.43° E), Guizhou, China (Fig. 1A, 1B). Diseased leaf segments were surface sterilized and plated on potato dextrose agar (PDA). Isolate (T41) was selected for identification. The colonies had white aerial hyphae, with orange-red on the underside when cultured on PDA (Fig. 1G, 1H). The colonies had woolly aerial hyphae, white to grey eventually, and produced pycnidia on oatmeal agar (OA) (Boerema et al. 2004) (Fig. 1I, 1J). Pycnidia were dark, spherical or flat spherical, and 69.2-178.0 µm in diameter. Conidia were oval mostly, aseptate, usually guttulate, and the size was 5.0 - 6.5 µm × 3.2 - 5.4 µm (Fig. 1K, 1L). The rDNA internal transcribed spacer region (ITS) with primers ITS1f/ITS4 (White et al. 1990; Gardes and Bruns 1993), 28S ribosomal RNA gene (LSU) with primers LROR/LR7 (Rehner and Samuels 1994), beta-tubulin gene (TUB2) with primers Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and RNA polymerase II second largest subunit gene (RPB2) with primers RPB2-5F2/fRPB2-7cR (Liu et al. 1999) of T41 were sequenced (GenBank accession numbers were MN704804, MN710367, MN718012 and MN718013, respectively). Maximum Likelihood (ML) analyses and Bayesian Inferences (BI) analyses based on concatenated these four sequences were conducted with RAxML v. 7.2.6 and MrBayes v. 3.2.1, respectively, which showed that T41 comprised a clade with Epicoccum latusicollum strains (CGMCC 3.18346 and LC 8153) (ML/BI = 100/1) (Fig. 2). Based on morphological and multi-gene molecular data, isolate T41 was identified as E. latusicollum described as a new taxon by Chen et al. (2017). To verify pathogenicity, tobacco plants at seedling stage (7-8 leaves) without visible disease were inoculated using conidial suspension (106 spores/ml), following Guo et al. (2020). All inoculated plants were maintained in a greenhouse with relative humidity ranging from 50% to 85% at 28 °C under a 12/12 h light/dark cycle. Seven days after incubation, typical symptoms were observed on inoculated leaves but not on control leaves (Fig. 1C, 1D, 1E, 1F). Koch's postulates were fulfilled by re-isolation of E. latusicollum from diseased leaves. E. latusicollum has been reported to cause black root on yam in China (Han et al. 2019). Meanwhile, there are many plants could be caused leaf spot by this genus, such as Lablab purpureus (Mahadevakumar et al. 2014) and Bletilla striata (Zhou et al. 2018). However, to the best of our knowledge, this is the first report of E. latusicollum causing leaf spot on tobacco in China. Because considerable loss occurred due to infection from E. latusicollum on tobacco leaves, this pathogen is worthy of further study and disease management practices need to be developed to prevent further losses.

Angiotensin-converting enzyme inhibitors and bacterial pneumonia in patients with Parkinson disease.

BACKGROUND: To evaluate the association of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers with pneumonia development in patients with Parkinson's disease (PD). METHODS: The study cohort consisted of patients aged 50 years or older who were initially diagnosed with PD and had hypertension. We assessed the patients' exposure statuses and accumulated doses of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers. We then evaluated the risk of pneumonia development in the patients who were exposed to these drugs and those who were not. RESULTS: We examined 2,310 patients. During the observation period, 608 patients developed pneumonia. Angiotensin-converting enzyme inhibitors were associated with a lower risk of pneumonia. This association was dose-dependent. CONCLUSION: Angiotensin-converting enzyme inhibitor use was associated with a dose-dependent reduction in the risk of pneumonia in patients with PD and hypertension.

Comparison of activities of daily living impairments in Parkinson's disease patients as defined by the Pill Questionnaire and assessments by neurologists.

OBJECTIVES: To compare the clinical judgment of experienced neurologists after interviewing Parkinson's disease (PD) patients and their caregivers with the use of the Pill Questionnaire to determine the presence of impairments on activities of daily living (ADL). BACKGROUND: ADL impairment is a criterion for the diagnosis of dementia associated with PD. The Pill Questionnaire has been recommended as a screening tool to assess ADL impairment in PD patients, but its usefulness and validity have not been fully investigated. METHODS: We recruited idiopathic PD patients from 12 hospitals in Taiwan, and the patients underwent clinical assessments, a neuropsychological test battery and the Unified Parkinson Disease Rating Scale evaluation. The Pill Questionnaire was administered by study assistants. Patient and caregiver interviews were performed by experienced neurologists who were blinded to the Pill Questionnaire results. RESULTS: In total, 284 PD patients (mean age 71.8±9 years, mean education 8.7±5.3 years, mean disease duration 5.4±5.3 years) were recruited. 63 patients showed ADL impairment by the Pill Questionnaire, and 108 patients showed ADL impairment by neurologists' clinical interviews. κ Statistics showed moderate agreement between the two methods (κ=0.521, p<0.001). Of the 108 patients who were diagnosed with ADL impairment by neurologists, only 56 patients (51.9%) showed impairment according to the Pill Questionnaire. Most of the missed patients had milder cognitive impairment and lower motor disability. CONCLUSIONS: A comprehensive interview is essential to determine the presence of ADL impairment in PD patients, especially in patients with early PD.

Dominance of Tau Burden in Cortical Over Subcortical Regions Mediates Glymphatic Activity and Clinical Severity in PSP.

BACKGROUND: Progressive supranuclear palsy (PSP) is a tauopathy that involves subcortical regions but also extends to cortical areas. The clinical impact of different tau protein sites and their influence on glymphatic dysfunction have not been investigated. PATIENTS AND METHODS: Participants (n = 55; 65.6 ± 7.1 years; 29 women) with PSP (n = 32) and age-matched normal controls (NCs; n = 23) underwent 18 F-Florzolotau tau PET, MRI, PSP Rating Scale (PSPRS), and Mini-Mental State Examination. Cerebellar gray matter (GM) and parametric estimation of reference signal intensity were used as references for tau burden measured by SUV ratios. Glymphatic activity was measured by diffusion tensor image analysis along the perivascular space (DTI-ALPS). RESULTS: Parametric estimation of reference signal intensity is a better reference than cerebellar GM to distinguish tau burden between PSP and NCs. PSP patients showed higher cortical and subcortical tau SUV ratios than NCs ( P < 0.001 and <0.001). Cortical and subcortical tau deposition correlated with PSPRS, UPDRS, and Mini-Mental State Examination scores (all P 's < 0.05). Cortical tau deposition was further associated with the DTI-ALPS index and frontal-temporal-parietal GM atrophy. The DTI-ALPS indexes showed a significantly negative correlation with the PSPRS total scores ( P < 0.01). Finally, parietal and occipital lobe tau depositions showed mediating effects between the DTI-ALPS index and PSPRS score. CONCLUSIONS: Cortical tau deposition is associated with glymphatic dysfunction and plays a role in mediating glymphatic dysfunction and clinical severity. Our results provide a possible explanation for the worsening of clinical severity in patients with PSP.

Does the dopamine hypothesis explain schizophrenia?

The dopamine hypothesis has been the cornerstone in the research and clinical practice of schizophrenia. With the initial emphasis on the role of excessive dopamine, the hypothesis has evolved to a concept of combining prefrontal hypodopaminergia and striatal hyperdopaminergia, and subsequently to the present aberrant salience hypothesis. This article provides a brief overview of the development and evidence of the dopamine hypothesis. It will argue that the current model of aberrant salience explains psychosis in schizophrenia and provides a plausible linkage between the pharmacological and cognitive aspects of the disease. Despite the privileged role of dopamine hypothesis in psychosis, its pathophysiological rather than etiological basis, its limitations in defining symptoms other than psychosis, as well as the evidence of other neurotransmitters such as glutamate and adenosine, prompt us to a wider perspective of the disease. Finally, dopamine does explain the pathophysiology of schizophrenia, but not necessarily the cause per se. Rather, dopamine acts as the common final pathway of a wide variety of predisposing factors, either environmental, genetic, or both, that lead to the disease. Other neurotransmitters, such as glutamate and adenosine, may also collaborate with dopamine to give rise to the entire picture of schizophrenia.

Characteristics of Epicoccum latusicollum as revealed by genomic and metabolic phenomic analysis, the causal agent of tobacco Epicoccus leaf spot.

Epicoccum latusicollum is a fungus that causes a severe foliar disease on flue-cured tobacco in southwest China, resulting in significant losses in tobacco yield and quality. To better understand the organism, researchers investigated its optimal growth conditions and metabolic versatility using a combination of traditional methods and the Biolog Phenotype MicroArray technique. The study found that E. latusicollum exhibited impressive metabolic versatility, being able to metabolize a majority of carbon, nitrogen, sulfur, and phosphorus sources tested, as well as adapt to different environmental conditions, including broad pH ranges and various osmolytes. The optimal medium for mycelial growth was alkyl ester agar medium, while oatmeal agar medium was optimal for sporulation, and the optimum temperature for mycelial growth was 25°C. The lethal temperature was 40°C. The study also identified arbutin and amygdalin as optimal carbon sources and Ala-Asp and Ala-Glu as optimal nitrogen sources for E. latusicollum. Furthermore, the genome of E. latusicollum strain T41 was sequenced using Illumina HiSeq and Pacific Biosciences technologies, with 10,821 genes predicted using Nonredundant, Gene Ontology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes, and SWISS-PROT databases. Analysis of the metabolic functions of phyllosphere microorganisms on diseased tobacco leaves affected by E. latusicollum using the Biolog Eco microplate revealed an inability to efficiently metabolize a total of 29 carbon sources, with only tween 40 showing some metabolizing ability. The study provides new insights into the structure and function of phyllosphere microbiota and highlights important challenges for future research, as well as a theoretical basis for the integrated control and breeding for disease resistance of tobacco Epicoccus leaf spot. This information can be useful in developing new strategies for disease control and management, as well as enhancing crop productivity and quality.

Analysis of growth dynamics in five different media and metabolic phenotypic characteristics of Piriformospora indica.

Piriformospora indica is an important endophytic fungus with broad potential for alleviating biotic and abiotic stress on host plants. This study monitored the growth dynamics of P. indica on five commonly used artificial media for microorganisms and analyzed its metabolic characteristics using Biolog Phenotype Microarray (PM) technology. The results showed that P. indica grew fastest on Potato Dextrose Agar (PDA), followed by Kidney Bean Agar (KBA), Alkyl Ester Agar (AEA), Oatmeal Agar (OA), and Luria-Bertani Agar (LB), and the most suitable medium for spore production was OA. Using Biolog PM1-10, 950 metabolic phenotypes of P. indica were obtained. P. indica could metabolize 87.89% of the tested carbon sources, 87.63% of the tested nitrogen sources, 96.61% of the tested phosphorus sources, and 100% of the tested sulfur sources. P. indica displayed 92 kinds of tested biosynthetic pathways, and it could grow under 92 kinds of tested osmotic pressures and 88 kinds of tested pH conditions. PM plates 1-2 revealed 43 efficient carbon sources, including M-Hydroxyphenyl acid, N-Acetyl-D-Glucosamine, Tyramine, Maltotrios, α-D-Glucosine, I-Erythritol, L-Valine, D-Melezitose, D-Tagatose, and Turanose. PM plates 3,6-8 indicated 170 efficient nitrogen sources, including Adenosine, Inosine Allantoin, D, L-Lactamide, Arg-Met, lle-Trp, Ala-Arg, Thr-Arg, Trp-Tyr, Val-Asn, Gly-Gly-D-Leu, Gly-Gly-Phe, and Leu-Leu-Leu. This study demonstrates that P. indica can metabolize a variety of substrates, such as carbon and nitrogen sources, and has a wide range of environmental adaptability. The growth dynamics on artificial culture media and metabolic phenotypes of P. indica can be used to investigate its biological characteristics, screen for more suitable growth and sporulation conditions, and elucidate the physiological mechanisms that enhance the stress resistance of host plants. This study provides a theoretical basis for its better application in agriculture.

Phenotypic analysis and genome sequence of Rhizopus oryzae strain Y5, the causal agent of tobacco pole rot.

Rhizopus oryzae is a destructive pathogen that frequently causes tobacco pole rot in curing chambers. Phenotypic characterization of the pathogen was conducted to provide basic biological and pathological information using Biolog Phenotype MicroArray (PM). In addition, the Y5 strain of R. oryzae was sequenced using Illumina HiSeq and Pacific Biosciences (PacBio) technologies. Using PM plates 1-8, 758 growth conditions were tested. Results indicated that R. oryzae could metabolize 54.21% of tested carbon sources, 86.84% of nitrogen sources, 100% of sulfur sources, and 98.31% of phosphorus sources. About 37 carbon compounds, including D-xylose, N-acetyl-D-glucosamine, D-sorbitol, ß-methyl-D-glucoside, D-galactose, L-arabinose, and D-cellobiose, significantly supported the growth of the pathogen. PM 3 indicated the active nitrogen sources, including Gly-Asn, Ala-Asp., Ala-Gln, and uric acid. PM 6-8 showed 285 different nitrogen pathways, indicating that different combinations of different amino acids support the growth of the pathogen. Genome sequencing results showed that the R. oryzae Y5 strain had raw data assembled into 2,271 Mbp with an N50 value of 10,563 bp. A genome sequence of 50.3 Mb was polished and assembled into 53 contigs with an N50 length of 1,785,794 bp, maximum contig length of 3,223,184 bp, and a sum of contig lengths of 51,182,778 bp. A total of 12,680 protein-coding genes were predicted using the Nonredundant, Gene Ontology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes, and SWISS-PROT databases. The genome sequence and annotation resources of R. oryzae provided a reference for studying its biological characteristics, trait-specific genes, pathogen-host interaction, pathogen evolution, and population genetic diversity. The phenomics and genome of R. oryzae will provide insights into microfungal biology, pathogen evolution, and the genetic diversity of epidemics.

Molecular characterization of six heat shock protein 70 genes from Arma chinensis and their expression patterns in response to temperature stress.

Arma chinensis is an important predatory enemy of many agricultural and forest pests. Heat shock protein 70 (Hsp70) plays an essential role in insect adaptation to various stress factors. To explore the functions of Hsp70s in relation to thermal tolerance of A. chinensis, full-length cDNAs of six Hsp70 genes (AcHsp70Ba, AcHsp70-4, AcHsp68a, AcHsp68b, AcHsp70-2, and AcHsc70-4) were cloned. Their open reading frames (ORFs) were 1902, 2454, 1884, 1905, 1872, and 1947 bp, respectively. Developmental expression profiles showed that AcHsp70Ba, AcHsp70-4, and AcHsc70-4 were extremely highly expressed in adult stages. AcHsp68a and AcHsp70-2 showed the highest level of expression in nymph stages, and AcHsp68b was mainly expressed in male adults. Tissue distribution analysis demonstrated that the AcHsp70s were ubiquitously expressed but showing gene-specific and sex-driven patterns of expression. High temperature induced the expression of the six AcHsp70s. Among them, AcHsp70Ba, AcHsp70-4, AcHsp68a, and AcHsc70-4 were significantly induced at 38 °C for 6 h, while all six AcHsp70s were significantly induced at 38 °C for 24 h. There were differences in responses of the six AcHsp70s to low-temperature stress. The expressions of AcHsp70-4, AcHsp68a, and AcHsp68b in male adults were significantly repressed at 4 °C for 6 h, whereas AcHsp70Ba and AcHsp70-2 were significantly induced. The levels of AcHsp70Ba, AcHsp68b, and AcHsp70-2 in female adults were significantly repressed at 4 °C for 24 h, whereas AcHsc70-4 was significantly induced. These results suggested that AcHsp70s play important roles in various developmental stages and tissue function, and contribute to the tolerance of A. chinensis to extreme temperatures.

Variations in leaf phyllosphere microbial communities and development of tobacco brown spot before and after fungicide application.

In recent years, STROBY (50% Kresoxim-methyl) has been widely used to control tobacco brown spot in Guizhou Province, China. As a broad-spectrum fungicide, STROBY targets not only phytopathogens, but also affects many other microorganisms including those pathogenic, beneficial, or neutral to the plant hosts. To understand the effects of STROBY on the phyllosphere microbial communities of tobacco leaves during the development of tobacco brown spot, the fungal and bacterial communities of symptomatic and asymptomatic leaves at four time points, before spraying (August 29) and after spraying (September 3, 8, and 13), were investigated using the Illumina high-throughput sequencing. The results showed that STROBY had significant effects on the phyllosphere microbial communities of tobacco leaves. Microbial communities in asymptomatic leaves were more greatly affected than their counterparts in symptomatic leaves, and fungal communities were more sensitive than bacterial communities. Throughout the experiment, the most common genera in symptomatic leaves were Alternaria, Pseudomonas, Pantoea, and Sphingomonas, and in asymptomatic leaves, these were Golubevia and Pantoea. After spraying, the alpha diversity of fungal communities increased in symptomatic leaves and decreased in asymptomatic leaves, while the alpha diversity of bacteria increased in both types of leaves. Beta diversity showed that in asymptomatic leaves, the fungal communities in the first stage was significantly different from the remaining three stages. In contrast, the fungal communities in symptomatic leaves and the bacterial communities in all leaves did not fluctuate significantly during the four stages. Before spraying (August 29), the dominant functions of the fungal community were animal pathogen, endophyte, plant pathogen, and wood saprotroph. Whereas after spraying (September 3, 8, and 13), the proportion of the above fungal functions decreased and the unassigned functions increased, especially in asymptomatic leaves. This study describes the effects of STROBY application and tobacco brown spot presence in shaping the leaf phyllosphere microbial communities, and provides insights into the microbial community effects on tobacco leaves of a strobilurin fungicide.