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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., Competing Interests: C-yH was employed by Guizhou Provincial Tobacco Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hu, Li, Wang, Sun, Huang and Wang.)

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., Competing Interests: Author Z-NG was employed by MGI Tech Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from the China National Tobacco Corporation and Guizhou Tobacco Company. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication., (Copyright © 2023 Li, Hu, Li, Wang, Guo, Cheng, Cai and Shi.)

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, β-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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Shi, Huang, Wang, Li and Cai.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xiang, Wang, Cai, Guo, Luo, Hsiang and Yu.)

Phyllospheric microbial composition of tobacco ( Nicotiana tabacum L.) is contingent upon certain factors, such as the growth stage of the plant, leaf position, and cultivar and its geographical location, which influence, either directly or indirectly, the growth, overall health, and production of the tobacco plant. To better understand the spatiotemporal variation of the community and the divergence of phyllospheric microflora, procured from healthy and diseased tobacco leaves infected by Alternaria alternata , the current study employed microbe culturing, high-throughput technique, and BIOLOG ECO. Microbe culturing resulted in the isolation of 153 culturable fungal isolates belonging to 33 genera and 99 bacterial isolates belonging to 15 genera. High-throughput sequencing revealed that the phyllosphere of tobacco was dominantly colonized by Ascomycota and Proteobacteria, whereas, the most abundant fungal and bacterial genera were Alternaria and Pseudomonas . The relative abundance of Alternaria increased in the upper and middle healthy groups from the first collection time to the third, whereas, the relative abundance of Pseudomonas, Sphingomonas , and Methylobacterium from the same positions increased during gradual leaf aging. Non-metric multi-dimensional scaling (NMDs) showed clustering of fungal communities in healthy samples, while bacterial communities of all diseased and healthy groups were found scattered. FUNGuild analysis, from the first collection stage to the third one in both groups, indicated an increase in the relative abundance of Pathotroph-Saprotroph, Pathotroph-Saprotroph-Symbiotroph, and Pathotroph-Symbiotroph. Inclusive of all samples, as per the PICRUSt analysis, the predominant pathway was metabolism function accounting for 50.03%. The average values of omnilog units (OUs) showed relatively higher utilization rates of carbon sources by the microbial flora of healthy leaves. According to the analysis of genus abundances, leaf growth and leaf position were the important drivers of change in structuring the microbial communities. The current findings revealed the complex ecological dynamics that occur in the phyllospheric microbial communities over the course of a spatiotemporal varying environment with the development of tobacco brown spots, highlighting the importance of community succession., Competing Interests: Y-fD was employed by Bijie Tobacco Company. J-xL was employed by Guizhou Tobacco Company of CNTC, China National Tobacco Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dai, Wu, Wang, Li, Cai, Li, Wang, Sehar and Shamsi.)

In the tobacco phyllosphere, some of the microbes may have detrimental effects on plant health, while many may be neutral or even beneficial. Some cannot be cultivated, so culture-independent methods are needed to explore microbial diversity. In this study, both metagenetic analysis and traditional culture-dependent methods were used on asymptomatic healthy leaves and symptomatic diseased leaves of tobacco plants. In the culture-independent analysis, asymptomatic leaves had higher microbial diversity and richness than symptomatic leaves. Both asymptomatic and symptomatic leaves contained several potentially pathogenic bacterial and fungal genera. The putative bacterial pathogens, such as species of Pseudomonas , Pantoea , or Ralstonia , and putative fungal pathogens, such as species of Phoma , Cladosporium , Alternaria , Fusarium , Corynespora , and Epicoccum , had a higher relative abundance in symptomatic leaves than asymptomatic leaves. FUNGuild analysis indicated that the foliar fungal community also included endophytes, saprotrophs, epiphytes, parasites, and endosymbionts. PICRUSt analysis showed that the dominant functions of the bacterial community in a symptomatic leaf were cellular processes and environmental information processing. In the other five foliar samples, the dominant functions of the bacterial community were genetic information processing, metabolism, and organismal systems. In the traditional culture-dependent method, 47 fungal strains were isolated from 60 symptomatic tobacco leaf fragments bearing leaf spots. Among them, 21 strains of Colletotrichum (29%), Xylariaceae (14%), Corynespora (14%), Pestalotiopsis (10%), Alternaria (10%), Epicoccum (10%), Byssosphaeria (5%), Phoma (5%), and Diaporthe (5%) all fulfilled Koch's postulates and were found to cause disease on detached tobacco leaves in artificial inoculation tests. Symptoms on detached leaves caused by three strains of Corynespora cassiicola in artificial inoculation tests were similar to the original disease symptoms in the tobacco field. This study showed that the combined application of culture-dependent and independent methods could give comprehensive insights into microbial composition that each method alone did not reveal., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xiang, Wang, Wang, Cai, Li, Hsiang and Yu.)

Introduction: Engaging in a secondary task while walking increases motor-cognitive interference and exacerbates fall risk in older adults with mild cognitive impairment (MCI). Previous studies have demonstrated that Tai Chi (TC) may improve cognitive function and dual-task gait performance. Intriguingly, with emerging studies also indicating the potential of transcranial direct current stimulation (tDCS) in enhancing such motor-cognitive performance, whether combining tDCS with TC might be superior to TC alone is still unclear. The purpose of this study was to investigate the effects of combining tDCS with TC on dual-task gait in patients with MCI. Materials and Methods: Twenty patients with MCI were randomly assigned to receive either anodal or sham tDCS, both combined with TC, for 36 sessions over 12 weeks. Subjects received 40 min of TC training in each session. During the first 20 min, they simultaneously received either anodal or sham tDCS over the left dorsolateral prefrontal cortex. Outcome measures included dual-task gait performance and other cognitive functions. Results: There were significant interaction effects between groups on the cognitive dual task walking. Compared to sham, the anodal tDCS group demonstrated a greater improvement on cadence and dual task cost of speed. Conclusion: Combining tDCS with TC may offer additional benefits over TC alone in enhancing dual-task gait performance in patients with MCI. Clinical Trial Registration: [www.ClinicalTrials.gov], identifier [TCTR20201201007]., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liao, Liu, Wang, Walsh and Lau.)

A Myriad of biotic and abiotic factors inevitably affects the growth and production of tobacco ( Nicotiana tabacum L.), which is a model crop and sought-after worldwide for its foliage. Among the various impacts the level of disease severity poses on plants, the influence on the dynamics of phyllospheric microbial diversity is of utmost importance. In China, recurring reports of a phyto-pathogen, Didymella segeticola , a causal agent of tobacco leaf spot, accentuate the need for its in-depth investigation. Here, a high-throughput sequencing technique, IonS5 TM XL was employed to analyze tobacco leaves infected by D. segeticola at different disease severity levels, ranging from T1G (least disease index) to T4G (highest disease index), in an attempt to explore the composition and diversity of phyllospheric microbiota. In all healthy and diseased tobacco leaves, the most dominant fungal phylum was Ascomycota with a high prevalence of genus Didymella , followed by Boeremia , Meyerozyma and Alternaria , whereas in the case of bacterial phyla, Proteobacteria was prominent with Pseudomonas being a predominant genus, followed by Pantoea . The relative abundance of fungi, i.e., Didymella and Boeremia (Ascomycota) and bacteria, i.e., Pseudomonas and Pantoea (Proteobacteria) were higher in diseased groups compared to healthy groups. Healthy tissues exhibited relatively rich and diverse fungal communities in contrast with diseased groups. The infection of D. segeticola had a complex and significant effect on fungal as well as bacterial alpha diversity. FUNGuild analysis indicated that the relative abundance of pathotrophs and saprotrophs in diseased tissues proportionally increased with disease severity. PICRUSt analysis of diseased tissues indicated that the relative abundance of bacterial cell motility and membrane transport-related gene sequences elevated with an increase in disease severity from T1G to T3G and then tended to decrease at T4G. Conclusively, the current study shows the typical characteristics of the tobacco leaf microbiome and provides insights into the distinct microbiome shifts on tobacco leaves infected by D . segeticola., Competing Interests: The authors declare that this study received funding from Guizhou Tobacco Company Project. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication., (Copyright © 2021 Huang, Wang, Cai, Li, Pan, Xiang, Su, Li, Adil and Shamsi.)

Rhizopus oryzae causes tobacco pole rot in China during tobacco flue-curing. Flue-curing is a post-harvest process done to prepare tobacco leaves and involves three different stages: the yellowing stage has the lowest temperatures and highest humidity, then the color-fixing stage has higher temperatures and medium humidity, and finally the stem-drying stage has the highest temperatures and lowest humidity. In this study, fungal culturing and IonS5XL high-throughput sequencing techniques were used to reveal the fungal community of the petioles and lamina of tobacco leaves infected with pole rot during flue-curing. A total of 108 fungal isolates belonging to 6 genera were isolated on media. The most common fungal species isolated was the pathogen, R. oryzae , that was most often found equally on petioles and laminas in the color-fixing stage, followed by saprotrophs, mostly Aspergillus spp. High-throughput sequencing revealed saprotrophs with Alternaria being the most abundant genus, followed by Phoma , Cercospora , and Aspergillus , whereas Rhizopus was the tenth most abundant genus, which was mostly found on petioles at the yellowing stage. Both culturable fungal diversity and fungal sequence diversity was higher at stem-drying stage than the yellowing and color-fixing stages, and diversity was higher with leaf lamina than petioles revealing that the changes in fungal composition and diversity during the curing process were similar with both methods. This study demonstrates that the curing process affects the leaf microbiome of tobacco during the curing process, and future work could examine if any of these saprotrophic fungi detected during the curing of tobacco leaves may be potential biocontrol agents for with pole rot in curing chambers., (Copyright © 2020 Chen, Cai, Wang, Cai, Goodwin, Ma, Wang and Li.)

Botrytis cinerea , which causes gray mold, is an important pathogen in four important economic crops, tomato, tobacco, cucumber and strawberry, in China and worldwide. Metabolic phenomics data on B. cinerea isolates from these four crops were characterized and compared for 950 phenotypes with a BIOLOG Phenotype MicroArray (PM). The results showed that the metabolic fingerprints of the four B. cinerea isolates were similar to each other with minimal differences. B. cinerea isolates all metabolized more than 17% of the tested carbon sources, 63% of the amino acid nitrogen substrates, 80% of the peptide nitrogen substrates, 93% of the phosphorus substrates, and 97% of the sulfur substrates. Carbon substrates of organic acids and carbohydrates, and nitrogen substrates of amino acids and peptides were the significant utilization patterns for B. cinerea . Each B. cinerea isolate contained 94 biosynthetic pathways. These isolates showed a large range of adaptabilities and were still able to metabolize substrates in the presence of the osmolytes, including up to 6% potassium chloride, 10% sodium chloride, 5% sodium sulfate, 6% sodium formate, 20% ethylene glycol, and 3% urea. These isolates all showed active metabolism in environments with pH values from 3.5 to 8.5 and exhibited decarboxylase activities. These characterizations provide a theoretical basis for the study of B. cinerea in biochemistry and metabolic phenomics and provide valuable clues to finding potential new ways to manage gray mold.