Effects of editing DFR genes on flowers, leaves, and roots of tobacco.

BACKGROUND: DFR is a crucial structural gene in plant flavonoid and polyphenol metabolism, and DFR knockout (DFR-KO) plants may have increased biomass accumulation. It is uncertain whether DFR-KO has comparable effects in tobacco and what the molecular mechanism is. We employed the CRISPR/Cas9 method to generate a knockout homozygous construct and collected samples from various developmental phases for transcriptome and metabolome detection and analysis. RESULTS: DFR-KO turned tobacco blossoms white on homozygous tobacco (Nicotiana tabacum) plants with both NtDFR1 and NtDFR2 knockout. RNA-seq investigation of anthesis leaf (LF), anthesis flower (FF), mature leaf (LM), and mature root (RM) variations in wild-type (CK) and DFR-KO lines revealed 2898, 276, 311, and 101 differentially expressed genes (DEGs), respectively. DFR-KO primarily affected leaves during anthesis. According to KEGG and GSEA studies, DFR-KO lines upregulated photosynthetic pathway carbon fixation and downregulated photosystem I and II genes. DFR-KO may diminish tobacco anthesis leaf photosynthetic light reaction but boost dark reaction carbon fixation. DFR-KO lowered the expression of pathway-related genes in LF, such as oxidative phosphorylation and proteasome, while boosting those in the plant-pathogen interaction and MAPK signaling pathways, indicating that it may increase biological stress resistance. DFR-KO greatly boosted the expression of other structural genes involved in phenylpropanoid production in FF, which may account for metabolite accumulation. The metabolome showed that LF overexpressed 8 flavonoid metabolites and FF downregulated 24 flavone metabolites. In DFR-KO LF, proteasome-related genes downregulated 16 amino acid metabolites and reduced free amino acids. Furthermore, the DEG analysis on LM revealed that the impact of DFR-KO on tobacco growth may progressively diminish with time. CONCLUSION: The broad impact of DFR-KO on different phases and organs of tobacco development was thoroughly and methodically investigated in this research. DFR-KO decreased catabolism and photosynthetic light reactions in leaves during the flowering stage while increasing carbon fixation and disease resistance pathways. However, the impact of DFR-KO on tobacco growth steadily declined as it grew and matured, and transcriptional and metabolic modifications were consistent. This work offers a fresh insight and theoretical foundation for tobacco breeding and the development of gene-edited strains.

Correlation between the salivary microbiology and H2 S concentration of the oral cavity.

BACKGROUND: Hydrogen sulfide (H2 S) is the most important compound causing oral malodor, and its concentration is thought to be closely correlated with oral microorganism activity. Therefore, clarifying the correlation between oral microbes and metabolites is important. METHODS: This study tested with 16S rRNA gene amplicon and shotgun metagenomic sequencing of oral microorganisms and oral malodor tests. RESULTS: There were different of the microbial taxa between the low and high H2 S groups. And in the high H2 S group, most of the enriched taxa were genera which abundance was correlated with H2 S concentration. Fusobacterium periodonticum and Prevotella nanceiensis were significantly different in coverage breadth and depth and in LPS biosynthesis contributions between the two groups. The contribution of F. periodonticum to sulfur metabolism was significantly different between the two groups, and the relative F. periodonticum abundance was higher in the high H2 S group. CONCLUSIONS: The H2 S content is significantly associated with the oral cavity microorganism composition and abundance. Most microorganisms enriched in people with high H2 S levels are associated with oral diseases such as caries and periodontal diseases.

Proteome and physiological analyses reveal tobacco (Nicotiana tabacum) peroxidase 7 (POD 7) functions in responses to copper stress.

Copper is a micronutrient essential for plant growth and development. However, Cu is also a heavy metal element that has deleterious impacts on plants when excessively accumulated in the environment. To understand the molecular mechanism underlying tobacco in response to Cu stress, iTRAQ based technology was used to identify differentially expressed proteins (DEPs) and important metabolic pathways in tobacco plants treated with excessive CuSO4. The results showed that 180 DEPs were detected between the treatment and control, among which 78 were upregulated and 102 were downregulated. These DEPs can be functionally divided into 65 categories and are closely related to metabolic pathways, carbon metabolism, secondary metabolite biosynthesis, biosynthesis of antibiotics, glyoxylate and dicarboxylate metabolism, and glycolysis/gluconeogenesis. Peroxidase7 was significantly upregulated and was selected and overexpressed in tobacco. Then, positive transgenic lines and wild type plants were exposed to a Cu stress environment. The results showed that Peroxidase7 transgenic tobacco plants exhibited enhanced Cu stress resistance with decreased malondialdehyde and Cu contents, and increased shoot dry weight, root length, secondary root number, SOD, POD and CAT activity. The present study suggests that the ROS scavenging mechanism is essential for tobacco plants in response to Cu stress and that Peroxidase7 functions in tobacco plant resistance to excessive Cu environment.

Three new diphenyl ether derivatives from the fermentation products of an endophytic fungus Phomopsis fukushii.

Three new diphenyl ethers (1-3), together with four known isopentylated diphenyl ethers derivatives (4-7), were isolated from the fermentation products of an endophytic fungus Phomopsis fukushii. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR techniques. Compounds 1-3 were evaluated for their anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity. The results revealed that compounds 1 and 2 showed strong inhibitions with inhibition zone diameters (IZD) of 20.2 ± 2.5 mm and 17.9 ± 2.2 mm, respectively. Compound 3 also showed good inhibition with IZD 15.2 ± 1.8 mm. The IZD data of compound 1 is close to that of positive control with IZD 21.9 ± 2.1 mm.

[A new isobenzofuranone derivative from Chaenomeles sinensis and its antibacterial activity].

A new isobenzofuranone derivative was isolated from Chaenomeles sinensis by using various chromatographic techniques,including silica gel,Sephadex LH-20,MCI-gel resin and RP-HPLC. This compound was determined as 2,2-dimethyl-5-( 2-oxopropyl)-2 H-furo[3,4-h]chromen-7( 9 H)-one( 1) by NMR,MS,IR and UV spectra,and was also evaluated for its antibacterial activity. The results showed that it showed prominent antibacterial activity with MIC90 value of( 53. 7±4. 5) mg·L-1 for methicillin resistant Staphylococcus aureus( MRSA) strain. This value is close to that of levofloxacin [with MIC90 value( 50. 2± 4. 2) mg·L-1].

Sphingomonas tabacisoli sp. nov., a member of the genus Sphingomonas, isolated from rhizosphere soil of Nicotiana tabacum L.

A Gram-staining-negative, aerobic, motile and rod-shaped bacterium, designated strain X1-8T, was isolated from rhizosphere soil of Nicotiana tabacum L. collected from the tobacco produce base located in Kunming, south-west PR China. Cells showed oxidase-negative and catalase-positive reactions and were motile by means of peritrichous flagella. Growth occurred at 25-40 °C and pH 6.0-8.0 with optimal growth at 30-35 °C, pH 7.0. The major respiratory lipoquinone was Q-10. C16 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) were identified as major cellular fatty acids. The profile of polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, sphingoglycolipid, phosphatidylcholine and one unidentified glycolipid. The major polyamine was sym-homospermidine. The genomic DNA G+C content was 66.5 mol%. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that X1-8T should be affiliated to the genus Sphingomonasand formed a clade with most closely related species Sphingomonas changbaiensisNBRC 104936T. The results of 16S rRNA gene sequences similarity analysis indicated that X1-8T had the highest similarity with S. changbaiensisNBRC 104936T (98.4 %) and lower than 96.0 % with other species of the genus Sphingomonas. DNA-DNA hybridization data indicated that X1-8T represented a novel genomic species of the genus Sphingomonas. The characteristics determined in the polyphasic taxonomic study indicated that X1-8T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas tabacisoli sp. nov. (type strain X1-8T=KCTC 62032T=CGMCC 1.16275T) is proposed.

The Efficacy of a Chewing Gum Containing Phyllanthus emblica Fruit Extract in Improving Oral Health.

Phyllanthus emblica: (PE) fruit extract has pharmacological activity and exert anti-bacterial, anti-oxidative, anti-inflammatory and anti-cancer effects, but few study exist for evaluating its improved effects on the imbalance of oral ecology, which may contribute to series of oral diseases. In this study, an examiner-blinded, randomized, and gum-base-controlled crossover manner was conducted to evaluate the efficacy of a sugar-free chewing gum containing PE fruit extract in changing the oral microbiome. Twenty healthy young adults were randomly instructed to chew either PE gum or placebo gum. Saliva samples were collected at baseline and from 0 to 2, 2 to 5, 5 to 10, 10 to 15, and 75 to 80 min after each intervention. The following outcomes were measured: (i) salivary flow rate and pH value; (ii) total bacteria, Streptococcus mutans (S. mutans) and Porphyromonas gingivalis (P. gingivalis) counts; and (iii) volatile sulfur compound (VSC) concentrations. The results showed similar data between groups at baseline and significantly higher salivary flow rates and pH levels in the PE fruit gum group after 0-2, 2-5, and 5-10 min of chewing. Assessment of total bacteria, S. mutans, P. gingivalis, and VSC levels revealed significant differences between the PE and control gum groups at 75-80 min. No adverse effects were registered. The present finding indicated chewing gum containing PE fruit extract stimulated salivary flow and significantly reduced clinical test indexes in the short term. Chewing PE gum might be a safe means of improving oral hygiene.

Bacillus endozanthoxylicus sp. nov., an endophytic bacterium isolated from Zanthoxylum bungeanum Maxim leaves.

A Gram-stain-positive, rod-shaped, motile bacterium, designated as 1404T, was isolated from leaves of Chinese red pepper (Huajiao) (Zanthoxylum bungeanum Maxim) collected from Gansu, north-west China. Spores were not observed under a range of conditions. Strain 1404T was observed to grow at 15-45 °C and pH 6.0-10.0 and in presence of 0-5 % (w/v) NaCl concentration. The cell wall of strain 1404T was found to contain meso-diaminopimelic acid, and the predominant respiratory quinone was identified as MK-7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid as well as three unidentified polar lipids. The major fatty acids profile of strain 1404T consisted of iso-C15 : 0 (25.6 %), anteiso-C15 : 0 (18.4 %) and iso-C14 : 0 (12.1 %). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 1404T was affiliated to the genus Bacillus and was closely related to Bacillusoryzisoli 1DS3-10T, Bacillusbenzoevorans DSM 5391T and Bacilluscirculans DSM 11T with sequence similarity of 98.3, 98.2 and 96.9 %, respectively. The G+C content of the genomic DNA was determined to be 39.4 mol%. DNA-DNA hybridization values indicated that relatedness between strain 1404T and the type strains of closely related species of the genus Bacillus was below 41 %. Therefore, on the basis of the data from the polyphasic taxonomic study presented, strain 1404T represents a novel species of the genus Bacillus, for which the name proposed is Bacillus endozanthoxylicus sp. nov. The type strain is 1404T (=CCTCC AB 2017021T=KCTC 33827T).

[A new naphthalene derivative from Aloe vera and its antibacterial activity].

A new naphthalene derivative has been isolated from Aloe vera by using various chromatographic techniques, including silica gel, sephadex, MCI-gel resin, and RP-HPLC. The new compound was determined as 3-hydroxy-1-(1,7-dihydroxy-3,6-dimethoxynaphthalen-2-yl)propan-1-one (1). In the biological activity assay, compound 1 disglayed prominent antibacterial activity with a MIC90 value of (48±4) mg•L⁻¹ for methicillin resistant Staphylococcus aureus (MRSA) strain which was stronger than that of the positive control levofloxacin with a MIC90 value (58±5) mg•L⁻¹.

The 14-3-3 protein nt GF14e interacts with CIPK2 and increases low potassium stress in tobacco.

Potassium (K+) plays a role in enzyme activation, membrane transport, and osmotic regulation processes. An increase in potassium content can significantly improve the elasticity and combustibility of tobacco and reduce the content of harmful substances. Here, we report that the expression analysis of Nt GF14e, a 14-3-3 gene, increased markedly after low-potassium treatment (LK). Then, chlorophyll content, POD activity and potassium content, were significantly increased in overexpression of Nt GF14e transgenic tobacco lines compared with those in the wild type plants. The net K+ efflux rates were severely lower in the transgenic plants than in the wild type under LK stress. Furthermore, transcriptome analysis identified 5708 upregulated genes and 2787 downregulated genes between Nt GF14e overexpressing transgenic tobacco plants. The expression levels of some potassium-related genes were increased, such as CBL-interacting protein kinase 2 (CIPK2), Nt CIPK23, Nt CIPK25, H+-ATPase isoform 2 a (AHA2a), Nt AHA4a, Stelar K+ outward rectifier 1(SKOR1), and high affinity K+ transporter 5 (HAK5). The result of yeast two-hybrid and luciferase complementation imaging experiments suggested Nt GF14e could interact with CIPK2. Overall, these findings indicate that NtGF14e plays a vital roles in improving tobacco LK tolerance and enhancing potassium nutrition signaling pathways in tobacco plants.

NtERF283 positively regulates water deficit tolerance in tobacco (Nicotianatabacum L.) by enhancing antioxidant capacity.

Ethylene responsive factor (ERF) is a plant-specific transcription factor that plays a pivotal regulatory role in various stress responses. Although the genome of tobacco harbors 375 ER F genes, the functional roles of the majority of these genes remain unknown. Expression pattern analysis revealed that NtERF283 was induced by water deficit and salt stresses and mainly expressed in the roots and leaves. Subcellular localization and transcriptional activity assays confirmed that NtERF283 was localized in the nucleus and exhibited transcriptional activity. In comparison to the wild-type (WT), the NtERF283-overexpressing transgenic plants (OE) exhibited enhanced water deficit tolerance, whereas the knockout mutant erf283 displayed contrasting phenotypes. Transcriptional analysis demonstrated that several oxidative stress response genes were significantly altered in OE plants under water deficit conditions. 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining showed that erf283 accumulated a higher level of reactive oxygen species (ROS) compared to the WT under water deficit conditions. Conversely, OE plants displayed the least amount of ROS accumulation. Furthermore, the activities of POD and SOD were higher in OE plants and lower in erf283, suggesting that NtERF283 enhanced the capacity to effectively eliminate ROS, consequently enhancing water deficit tolerance in tobacco. These findings strongly indicate the significance of NtERF283 in promoting tobacco water deficit tolerance through the activation of the antioxidant system.

Overexpression of NtLHT1 affects the development of leaf morphology and abiotic tolerance in tobacco.

LYSINE HISTIDINE TRANSPORTER1 (LHT1) is a crucial broad-specificity and high-affinity amino acid transporter affecting the uptake of nitrogen and probably the tolerance to abiotic stress in plants. However, little is known about the phenotypic functions of LHT1 in plant growth and development and abiotic stress tolerance. In this study, we identified the NtLHT1 gene from the tobacco variety Honghuadajinyuan (HD) and determined its important roles in leaf morphological development and plant resistance to abiotic stress. Comprehensive functional analyses using knockout and overexpression transgenic lines (ntlht1 and OE) revealed overexpression of NtLHT1 accelerated leave senescence and increased plant height, leaf number and plant tolerance under cold, salt and drought stresses. In addition, NtLHT1 overexpression significantly decreased the leaf elongation of HD, causing the leaves to change from a long-elliptical shape to an elliptical shape. However silencing NtLHT1 decreased the seed germination rate under NaCl and PEG stresses. Moreover, NtLHT1 significantly affected the contents of various amino acids, such as the neutral, acidic, non-polar and aromatic amino acids, ethylene precursor (ACC), GA3 and IAA in tobacco. These results suggested that the amino acid and ethylene precursor ACC transport activities of NtLHT1 provide fine regulatory function for plant growth and development and plant tolerance to abiotic stress.

Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into nematode-trap formation.

Nematode-trapping fungi are "carnivorous" and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.

In vitro micronucleus assay for the analysis of total particulate matter in cigarette smoke: comparison of flow cytometry and laser scanning cytometry with microscopy.

The possible genotoxicity of the total particulate matter (TPM) in cigarette smoke has typically been evaluated using the in vitro micronucleus assay. In recent years, automated scoring techniques have been developed to replace the manual counting process in this assay. However, these automated scoring techniques have not been applied in routine genotoxicity assays for the analysis of TPM to improve the assay efficiency. Chinese hamster ovary (CHO) cells were treated with TPM produced from 14 types of cigarettes at five concentrations (25-200µg/ml) without exogenous metabolic activation. The three following methods were used to score the micronucleus (MN) frequency: (a) flow cytometry with SYTOX and EMA dyes, which differentially stain micronuclei and apoptotic/necrotic chromatin to enhance assay reliability; (b) laser scanning cytometry with FITC and PI dyes, which is a system that combines the analytical capabilities of flow and image cytometry; and (c) visual microcopy with Giemsa dye. The test results obtained using the three methods were compared using correlation analysis. The key findings for this set of compounds include the following: (a) both flow cytometry- and laser scanning cytometry-based methods were effective for MN identification, (b) the three scoring methods could detect dose-dependent micronucleus formation for the 14 types of TPM, and (c) the MN frequencies that were measured in the same samples by flow cytometry, laser scanning cytometry, and visual microscopy were highly correlated, and there were no significant differences (p>0.05). In conclusion, both flow cytometry and laser scanning cytometry can be used to evaluate the MN frequency induced by TPM without exogenous metabolic activation. The simpler and faster processing and the high correlation of the results make these two automatic methods appropriate tools for use in in vitro micronucleus assays for the analysis of TPM using CHO cells.

Effect of nicotine on cholesterol gallstone formation in C57BL/6J mice fed on a lithogenic diet.

Gallstones are diseases of the biliary system caused by cholesterol supersaturation and/or deficiency in bile salts in bile. Early studies have shown that symptomatic gallstones are primarily a disease of non-smokers, raising the possibility that nicotine can prevent gallstone formation. The present study investigated the effect of nicotine on the formation of cholesterol gallstone in C57BL/6J mice. C57BL/6J mice (eight-weeks-old) were fed a normal or lithogenic diet (basic feed 82.45%, fat 15.8%, cholesterol 1.25% and sodium cholate 0.5%) and divided into five groups: normal diet (ND); ND + high dose nicotine (H); lithogenic diet (LD); LD + low dose nicotine (L) and LD + nicotine (H). They were treated with or without nicotine injection for 10 weeks. Nicotine treatment did not change the rate of cholesterol gallstone formation. There was no difference in TNFα, IL-1ß and IL-6 among the five groups. The LD group showed the highest cholesterol levels and there was significant suppression of the total cholesterol, low-density lipoprotein-cholesterol and total bile acid levels in the serum of the nicotine-treated mice. Quantitative PCR showed nicotine altered few bile acid metabolism-related genes expression in liver tissue and significantly altered cholesterol-metabolism genes in gallbladder tissue. Hematoxylin and eosin staining and western blotting showed that protein levels of farnesoid X receptor (FXR) and megalin in the gallbladder increased in the lithogenic-diet mice, which was significantly suppressed in the nicotine-treated mice. In vitro studies using gallbladder epithelial cells showed that chenodeoxycholic acids increased megalin expression, which could be attenuated by nicotine. Nicotine could regulate bile acid metabolism via the FXR-megalin/cubilin pathways, which potentially contribute to cholesterol nucleation and subsequent gallstone formation.

Isochromenes from the Nicotiana tabacum-derived endophytic fungus Aspergillus versicolor and their anti-tobacco mosaic virus activities.

Three new isochromenes, (5-methoxy-7-prenyl-1H-isochromen-3-yl)methanol (1), 3-(3-(hydroxymethyl)-5-methoxy-1H-isochromen-7-yl)propan-1-ol (2), and (5-methoxy-7-methyl-1H-isochromen-3-yl)methanol (3), along with three known analogues (4-6) were isolated from the fermentation products of a Nicotiana tabacum-derived endophytic fungus Aspergillus versicolor. Their structures were elucidated by spectroscopic methods, including extensive 1 D and 2 D NMR techniques. Compounds 1-3 and 6 were evaluated for their anti-tobacco mosaic virus (anti-TMV) activities. The results showed that compound 2 exhibited high anti-TMV activity with inhibition rate of 46.4%, and this rate is higher than that of positive control. Compounds 1, 3, and 6 also showed potential anti-TMV activity with inhibition rates of 28.6, 30.5, and 26.2%, respectively. The IC50 of compounds 1-3 and 6 were also tested, and showed IC50 values of 49.3, 22.4, 42.2, and 54.1 µM, respectively.

Highly efficient transgene-free genome editing in tobacco using an optimized CRISPR/Cas9 system, pOREU3TR.

CRISPR/Cas9 genome-editing technology has revolutionized plant science and holds enormous promise for crop improvement. The exploration of this system received much attention regarding plant genome editing. Here, by editing the NtPDS gene in tobacco, we first verified that incorporating an OsU3-tRNA promoter combination into the CRISPR/Cas9 system contributed to the highest editing efficiency, as the sgRNA expression level was greater than that resulting from the AtU6-tRNA and AtU6 promoters. Then, we optimized the existing tobacco CRISPR/Cas9 system, pORE-Cas9, by using the OsU3-tRNA promoter combination instead of AtU6 and by fusing an AtUb10-Ros1 expression cassette to the T-DNA to monitor the transgene events. The new system was named pOREU3TR. As expected, 49 transgene-free and homozygous gene-edited green plants were effectively screened in the T1 generation as a result of editing the NtLHT1 gene in tobacco, and the plant height and the contents of most free amino acids in the leaves of the T2 mutant plants were significantly different from those in the leaves of WT plants, demonstrating the high efficiency of the new editing system. This OsU3-tRNA-sgRNA/AtUb10-Ros1 system provides essential improvements for increasing the efficiency of plant genome editing.

The anti-TMV potency of the tobacco-derived fungus Aspergillus versicolor and its active alkaloids, as anti-TMV activity inhibitors.

Nicotiana tabacum (tobacco) has attracted interest as one of the most economically important industrial crops widely cultivated in China, whose dried leaves are popularly consumed medicinally and recreationally by human societies. In this study, five undescribed alkaloids derivatives, isoaspergillines A-E, together with eight known alkaloids, notoamide D, (1R,4S)-4-benzyl-1-isopropyl-2,4-dihydro-1H-pyrazino-[2,1-b]quinazoline-3,6-dione, protuboxepin K, notoamide C, notoamide M, deoxybrevianamide E, cyclo (D-Pro-L-Trp), and versicolamide B, were obtained from the culture of the Nicotiana tabacum-derived fungus Aspergillus versicolor. Their structures were mainly elucidated through comprehensive analyses of spectroscopic data. Bioactivity evaluation of all isolated compounds revealed that isoaspergilline A and notoamide M exhibited anti-TMV activities with IC50 values of 20.0 and 22.8 µM, respectively. Molecular docking suggested that isoaspergilline A and notoamide M were well located into the active site of anti-TMV by interacting with SER138, SER143, and ASN73 residues. This study enlightens the therapeutic potential of the endophytic fungus A. versicolor and it is helpful to find undescribed anti-TMV activity inhibitors, as well as searching for new anti-TMV candidates from natural sources.

Autotoxins in continuous tobacco cropping soils and their management.

Tobacco belongs to the family Solanaceae, which easily forms continuous cropping obstacles. Continuous cropping exacerbates the accumulation of autotoxins in tobacco rhizospheric soil, affects the normal metabolism and growth of plants, changes soil microecology, and severely reduces the yield and quality of tobacco. In this study, the types and composition of tobacco autotoxins under continuous cropping systems are summarized, and a model is proposed, suggesting that autotoxins can cause toxicity to tobacco plants at the cell level, plant-growth level, and physiological process level, negatively affecting soil microbial life activities, population number, and community structure and disrupting soil microecology. A combined strategy for managing tobacco autotoxicity is proposed based on the breeding of superior varieties, and this approach can be combined with adjustments to cropping systems, the induction of plant immunity, and the optimization of cultivation and biological control measures. Additionally, future research directions are suggested and challenges associated with autotoxicity are provided. This study aims to serve as a reference and provide inspirations needed to develop green and sustainable strategies and alleviate the continuous cropping obstacles of tobacco. It also acts as a reference for resolving continuous cropping challenges in other crops.

Influence of cigarette smoking on oral microbiota in patients with recurrent aphthous stomatitis.

Recurrent aphthous stomatitis (RAS) is a common recurrent ulcerative disease of the oral mucosa which is closely related to oral microbial composition. However, the specific effect and the mechanism of smoking in RAS are unclear. In this study, 16S rRNA sequencing technology was used to compare the differences in saliva microbial community between 28 non-smoking healthy controls (NSctrl), 31 non-smoking RAS patients (NSras), and 19 smoking RAS patients (Sras). The results showed that the bacterial community diversity in patients with RAS (NSras and Sras) was lower than that of NSctrl. The microbial community in smoking-associated RAS is less diverse and distinct from that of non-smokers. The RAS groups have higher abundance of Veillonella, Rothia, and Sneathia and lower abundance of Bacteroidales, Bacteroides, Wolinella, Moryella, Pyramidobacter, and Christensenellaceae at the genera level. A significantly different abundance of Anaerovorax, Candidatus Endomicrobium, Lactococcus, Sneathia, Veillonella, and Cloacibacterium was observed between the Sras and the NSras group. Notably, there was a significant difference in many species from the genus Prevotella and Treponema between the NSras and the Sras group. Further, the relative abundance of several taxa is correlated with smoking age or frequency, including Megasphaera, Haemophilus, Leptotrichia, and Rothia at the genera level, and Prevotella melaninogenica, Prevotella salivae, Megasphaera micronuciformis, Haemophilus parainfluenzae, Alloprevotella tannerae, Actinomyces naeslundii, Lautropia mirabilis, and Capnocytophaga sputigena at the species level. Among patients with RAS, smoking aggravated the pathways of respiration and human pathogens. Our results suggest that smoking is closely related to changes in the oral microbiota, which may contribute an opposite effect to the pathogenesis of RAS. This study provides new insight and theoretical basis for the cause and pathogenesis of RAS and better prevention and treatment.