Metabolic enzyme activity and microbial composition of the air-curing and fermentation processes determine the quality of cigar tobacco leaves (CTLs). In this study, we reveal the evolution of the dominant microorganisms and microbial community structure at different stages of the air-curing and fermentation processes of CTLs. The results showed that the changes in metabolic enzymes occurred mainly during the air-curing phase, with polyphenol oxidase (PPO) being the most active at the browning phase. Pseudomonas, Bacteroides, Vibrio, Monographella, Bipolaris, and Aspergillus were the key microorganisms in the air-curing and fermentation processes. Principal coordinate analysis revealed significant separation of microbial communities between the air-curing and fermentation phases. Redundancy analysis showed that bacteria such as Proteobacteria, Firmicutes, Bacteroidota, and Acidobacteriota and fungi such as Ascomycota and Basidiomycota were correlated with enzyme activity and temperature and humidity. Bacteria mainly act in sugar metabolism, lipid metabolism, and amino acid metabolism, while fungi mainly degrade lignin, cellulose, and pectin through saprophytic action. Spearman correlation network analysis showed that Firmicutes, Proteobacteria, and Actinobacteria were the key bacterial taxa, while Dothideomycetes, Sordariomycetes, and Eurotiomycetes were the key fungal taxa. This research provides the basis for improving the quality of cigars by improving the air-curing and fermentation processes. Key points: • Changes in POD and PPO activity control the color change of CTLs at the air-curing stage. • Monographella, Aspergillus, Pseudomonas, and Vibrio play an important role in air-curing and fermentation. • Environmental temperature and humidity mainly affect the fermentation process, whereas bacteria such as Proteobacteria, Firmicutes, Bacteroidota, and Acidobacteriota and fungi such as Ascomycota and Basidiomycota are associated with enzyme activity and temperature and humidity. Graphical

The surface color of cigar tobacco leaves (CTLs) is largely determined by the browning response and is one of the most important quality traits affecting consumer preferences. The physiological changes and metabolic network of browning in CTLs after harvest have not been reported. We investigated the molecular mechanism of browning in CTLs by comparing the physiological characteristics and metabolites of CTLs browning at five postharvest periods. Phenotypic traits and browning-related enzyme activity data indicated that the browning reaction was most intense at approximately 12 d when polyphenol oxidase and peroxidase activities reached their maximum. Postharvest CTLs polyphenols, polyacids, and carbon and nitrogen compounds were rapidly degraded during the yellowing phase, and the biosynthesis rate of polyphenols and carotenoids was greater than the degradation rate during the browning phase. Metabolomic analysis discovered 2027 metabolites that were annotated mainly to lipids and lipid-like molecules, organic acids and their derivatives, and organic heterocyclic components. Hierarchical cluster analysis discovered glutamate, serine, threonine, ornithine, and arginine as the major amino acids involved in the browning reaction. KEGG pathway enrichment analysis indicated that the metabolic pathways with important effects on enzymatic browning were phenylpropanoid biosynthesis, flavonoid biosynthesis, isoquinoline alkaloid biosynthesis, phenylalanine metabolism, and linoleic acid metabolism. The metabolic pathway network was mined, and the main polyphenols involved in the browning reaction of CTLs were found to be cyanidin, rutin, caffeoylquinic acid, kaempferol, naringin, and neohesperidin. This study provides a reference for the browning physiology and metabolism network of postharvest CTLs. Graphical Abstract

Paris polyphylla var. yunnanensis is an important medicinal plant with abundant saponins that are widely used in the pharmaceuticals industry. It is unclear why the levels of active ingredients increase as these plants age. We speculated that the concentrations of those components in the rhizomes are mediated by fungal endophytes. To test this hypothesis, we took both culture-dependent and -independent (metagenomics) approaches to analyze the communities of endophytic fungi that inhabit those rhizomes in plants of different age classes (four, six, and eight years old). In all, 147 isolates representing 18 fungal taxa were obtained from 270 segments (90 per age class). Based on morphological and genetic characteristics, Fusarium oxysporum (46.55% frequency of occurrence) was the predominant endophyte, followed by Leptodontidium sp. (8.66%) and Trichoderma viride (6.81%). Colonization of endophytic fungi was maximized in the eight-year-old rhizomes (33.33%) when compared with four-year-old (21.21%) and six-year-old (15.15%) rhizomes. Certain fungal species were present only at particular ages. For example, Alternaria sp., Cylindrocarpon sp., Chaetomium sp., Paraphaeosphaeria sporulosa, Pyrenochaeta sp., Penicillium swiecickii, T. viride, and Truncatella angustata were found only in the oldest plants. Analysis of (metagenomics) community DNA extracted from different-aged samples revealed that, at the class level, the majority of fungi had the highest sequence similarity to members of Sordariomycetes, followed by Eurotiomycetes and Saccharomycetes. These results were mostly in accord with those we obtained using culture methods. Fungal diversity and richness also changed over time. Our investigation is the first to show that the diversity of fungi in rhizomes of P. polyphylla var. yunnanensis is altered as plants age, and our findings provide a foundation for future examinations of useful compounds.

Fusarium species are among the most significant pathogens causing root rot in Panax notoginseng. In this study, a strain of Trichoderma hamatum was isolated from the rhizosphere soil of P. notoginseng and subjected to whole-genome sequencing. Plate confrontation experiments were conducted to investigate the antagonistic effects of T. hamatum against Fusarium oxysporum, Fusarium solani, and Fusarium acutatum, the primary Fusarium species causing root rot. Whole-genome sequencing revealed 10,774 predicted genes in T. hamatum, of which 454 were associated with carbohydrate-active enzymes (CAZymes) involved in fungal cell wall degradation. Additionally, 11 biosynthetic gene clusters (BGCs) associated with antimicrobial production were identified, highlighting the biocontrol potential of T. hamatum. In plate confrontation experiments, T. hamatum showed substantial inhibition rates of 68.07%, 70.63%, and 66.12% against F. oxysporum, F. solani, and F. acutatum, respectively. Scanning electron microscopy suggested the hyperparasitism of T. hamatum against F. solani, which was characterized by spore production that adhered to the pathogen, thereby inhibiting its growth. These findings provide a theoretical foundation to enhance understanding of the biological control mechanisms of T. hamatum, supporting its potential applications in sustainable agriculture.

The B115 strain, isolated from the inter-root soil of healthy plants in a continuous cropping site of Panax notoginseng, was identified as Bacillus velezensis B115 by 16S rDNA sequence comparison and comparative genomic analysis. B115 is a strain of beneficial microorganisms present in the inter-root zone of plants, with favorable plant growth-promoting properties and antagonistic effects against the plant pathogen Fusarium oxysporum. However, the whole genome of B115 remains unclear, thus restricting its potential applications. To address this gap, the whole genome of B115 has been sequenced and annotated to elucidate the molecular mechanisms underlying its plant growth-promoting and antimicrobial activities. The genome analysis revealed that B115 comprises a single circular chromosome of 4,200,774 bp and a plasmid region 16,878 bp long, possessing a GC content of 45.95%. Moreover, 4349 protein-coding genes were predicted. Notably, the B115 genome contains a substantial number of genes (103) involved in the biosynthesis, transport, and catabolism of secondary metabolites. Through genome mining, 13 BGCs and 540 genes encoding secondary metabolites with predicted roles were identified, including members of the surfactin and fengycin families. Utilizing LC–MS/MS technologies, 2318 metabolites were detected in the fermentation broth of B. velezensis B115, encompassing compounds such as Corynebactin, Gamabufotalin, Pracinostat, Indoleacetic acid, (8)-Gingerol, Luteolin, Liquiritigenin, and other metabolites with antimicrobial, growth-promoting, antioxidant, and antitumor properties. By exploring secondary metabolite-related genes and predicting potential secondary metabolites from the B115 genome based on the whole-genome sequence results, we further elucidate the genomic basis for its ability to promote plant growth and inhibit pathogen activity.

Paris polyphylla var. yunnanensis, one of the important medicinal plant resources in Yunnan, China, usually takes 6–8 years to be harvested. Therefore, it is urgent to find a method that can not only shorten its growth years, but also improve its quality. In this study, we examined the effects of a combination treatment of arbuscular mycorrhizal fungi (AMF) and plant growth-promoting endophytes (PGPE) and drought stress on the accumulation of saponins in it. P. polyphylla var. yunnanensis was infected with a mixture of AMF and PGPE under drought stress. The content of saponins, as well as morphological, physiological, and biochemical indicators, were all measured. The UGTs gene related to saponin synthesis was obtained from transcriptome data by homologous comparison, which were used for RT-PCR and phylogenetic analysis. Regardless of water, AMF treatment could infect the roots of P. polyphylla var. yunnanensis, however double inoculation with AMF and PGPE (AMF + PGPE) would reduce the infection rate of AMF. Plant height, aboveground and underground fresh weight did not differ significantly between the single inoculation AMF and the double inoculation treatment under different water conditions, but the inoculation treatment significantly increased the plant height of P. polyphylla var. yunnanensis compared to the non-inoculation treatment. Single inoculation with AMF considerably increased the net photosynthetic rate, stomatal conductance, and transpiration rate of P. polyphylla var. yunnanensis leaves under various water conditions, but double inoculation with AMF + PGPE greatly increased the intercellular CO₂ concentration and chlorophyll fluorescence parameter (Fv/Fm). Under diverse water treatments, single inoculation AMF had the highest proline content, whereas double inoculation AMF + PGPE may greatly improve the amount of abscisic acid (ABA) and indoleacetic acid (IAA) compared to normal water under moderate drought. Double inoculation AMF + PGPE treatment improved the proportion of N, P, and K in the rhizome of P. polyphylla var. yunnanensis under various water conditions. Under moderate drought stress, AMF + PGPE significantly enhanced the contents of P. polyphylla var. yunnanensis saponins I, II, VII, and total saponins as compared to normal water circumstances. Farnesyl diphosphate synthase (FPPS), Geranyl pyrophosphate synthase (GPPS), Cycloartenol synthase (CAS), and Squalene epoxidase (SE1) were the genes that were significantly up-regulated at the same time. The amount of saponins was favorably linked with the expression of CAS, GPPS, and SE1. Saponin VI content and glycosyl transferase (UGT) 010922 gene expression were found to be substantially associated, as was saponin II content and UGT010935 gene expression. Under moderate drought, AMF + PGPE was more conducive to the increase of hormone content, nutrient absorption, and total saponin content in P. polyphylla var. yunnanensis, and AMF + PGPE could up regulate the expression of key genes and UGTs genes in one or more steroidal saponin synthesis pathways to varying degrees, thereby stimulating the synthesis and accumulation of steroidal saponins in the rhizome of P. polyphylla var. yunnanensis. The combination of AMF and PGPE inoculation, as well as adequate soil drought, reduced the buildup of saponins in P. polyphylla var. yunnanensis and increased its quality.