Understanding the response of microbial communities and their potential functions is essential for sustainability of agroecosystems under long-term continuous cropping. However, limited research has focused on investigating the interaction between soil physicochemical factors and microbial community dynamics in agroecosystems under long-term continuous cropping. This study probed into the physicochemical properties, metabolites, and microbial diversity of tobacco rhizosphere soils cropped continuously for 0, 5, and 20 years. The relative abundance of bacterial genera associated with nutrient cycling (e.g., Sphingomonas) increased while potential plant pathogenic fungi and beneficial microorganisms showed synergistic increases with the duration of continuous cropping. Variations in soil pH, alkeline nitrogen (AN) content, and soil organic carbon (SOC) content drove the shifts in soil microbial composition. Metabolites such as palmitic acid, 3-hydroxypropionic acid, stearic acid, and hippuric acid may play a key role in soil acidification. Those results enhance our ability to predict shifts in soil microbial community structure associated with anthropogenic continuous cropping, which can have long-term implications for crop production.

The impact of various organic amendments on soil organic carbon (SOC) have rarely been reported. To address this, a laboratory experiment was designed to scrutinize the effects of different amendments on soil carbon fractions, microbial communities, and the underlying interactive mechanisms. The experiment encompassed a no-amendment control (CK), as well as treatments with corn straw (CS), tobacco stalks (TS), and peanut shell biochar (PB). Over a 70-day incubation, the SOC in plots amended with CS, TS, and PB displayed significant boosts of 13.9%, 17.5%, and 44.8%, respectively, compared to the CK. For soil carbon fractions, amendments with PB, TS, and CS led to a dramatic rise in particulate organic carbon (POC) of 27.4%, 20.2%, and 105.7%, respectively, in contrast to the CK plots. Mantel analysis and structural Equation Modeling uncovered strong interrelationships among the cbbL, cbbM, Bacteroidota, TOC, and POC. Organic amendments enhance soil carbon fractions, modulating the microbial community by increasing Bacteroidetes abundance and suppressing Acidobacteria richness, thereby influencing the abundance of key carbon cycle genes such as cbbL and cbbM. These results suggest that the addition of peanut shell biochar significantly boosted TOC and key carbon fractions, enhancing carbon content and soil fertility.

Tobacco (Nicotiana tabacum L.) is one of the most widely cultivated industrial crops worldwide. From April to July 2023, about 40% of tobacco seedlings in the greenhouse exhibited irregular taupe lesions in Zhengzhou, Henan Province, China. At an early stage of the lesion development, light grey spots with the diameter of 1-2 mm were observed, these spots gradually expanded and connected into large irregular lesions causing leaf wrinkling or withered. A total of 12 infected leaf tissues were sterilized with 75% ethanol for 45 s, rinsed three times in sterilized water and then plated on potato dextrose agar (PDA) medium for 10 days at 28°C in darkness. Seven fungal colonies that show the similar appearance were isolated and three of them (MB-1, MB-2 and MB-3) were used for subsequent identification. Colonies of these strains on PDA with loose mycelium and orange-red pigment on the underside, white aerial in the center and light yellow hyphae near the periphery, formed in the shape of a concentric ring pattern. Ascomata appeared from the 14th day, were black, spherical or ellipsoid with walls of textura angularis, and size was 53.8-101.1 μm × 50.3-104.3 μm (n=30). Terminal hairs were brown and straight, gradually tapering toward the tips. Asci clavate or fusiform, spore bearing part 16.2-29.2 × 7.3-11.4 μm (n=21), with 8 irregularly arranged ascospores, evanescent. Ascospores are brown at maturity, biapiculate, navicular or fusiform shapes with size of 8.7-12.8 μm × 4.8-6.9 μm (n=100), and more or less inaequilateral. Single spore strains derived from these strains exhibited the morphological features consistent with the original strains. The morphological characteristics of the fungus were consistent with the description of Arcopilus aureus (Chivers) X.W. Wang & Samson (= Chaetomium aureum Chivers) (Lee et al. 2019). Furthermore, the sequences of RPB2 region were amplified from these strains and the result sequences (GenBank accession no. OR513105-OR513108) all showed a 100.00% identity with A. aureus strain CBS 538.73 (GenBank accession no. KX976807.1). It was reported that the RPB2 gene was efficient in discriminating Arcopilus species (Tavares et al. 2022), thus a maximum likelihood (ML) phylogenetic tree based on the RPB2 gene sequences were constructed using MEGA 7.0 with 1000 replications of bootstrapping (Kumar et al. 2016), which revealed that these strains formed a well-supported clade with A. aureus strains of (CBS 153.52 and CBS538.73) (Wang et al. 2022). Pathogenicity analysis were performed on healthy flue-cured tobacco seedlings leaves (cv Y85) by using mycelial agar plugs (5 mm in diameter) and spore suspension (1×106 spores/mL), and the PDA plugs and sterile water were used for control group, respectively. Tobacco seedlings were incubated in a 25°C and 70% RH growth chamber. After seven days, the leaves showed obvious symptoms, with taupe lesions and yellow halos on the periphery, whereas no symptoms were found on the control leaves. The A. aureu was then reisolated from inoculated diseased leaves. Previously, A. aureus has been only reported to cause leaf black disease on Pseudostellaria heterophylla in China (Yuan et al. 2021). To our knowledge, this is the first reported of A. aureus causing tobacco leaf grey spot worldwide. Arcopilus aureus has been reported as a plant biocontrol fungus (Wang et al. 2013). However, due to the potential serious damage in tobacco seedlings caused by this fungus, the use of A. aureus as a plant biocontrol agent needs to be given more attention, and disease control measures of this pathogen should be developed.

Current research on tobacco aroma predominantly focuses on single-omics approaches. In this study, we conducted a comprehensive investigation of the relationships between tobacco metabolite profiles, microbial communities, and aroma characteristics. Untargeted metabolomics and metagenomic analyses were performed on flue-cured upper tobacco leaves to compare light aromatic tobacco (LAT) and strong aromatic tobacco (SAT). The results showed that sugar metabolite levels in LAT were significantly higher than those in SAT, whereas levels of specific acids and amino acid metabolites in SAT exceeded those in LAT. Redundancy analysis (RDA) and metabolomic correlation analyses indicated that the genera Methylorubrum and Pseudomonas may promote sugar metabolite accumulation, while Pseudokineococcus potentially regulates both sugar and acid metabolites. In contrast, Methylobacterium and Sphingomonas were associated with acid and amino acid metabolism, with Methylobacterium additionally exhibiting inhibitory effects on sugar metabolism. Metagenomic analysis revealed that Methylorubrum, Pseudomonas, and Pseudokineococcus were abundant in LAT, whereas Methylobacterium and Sphingomonas dominated in SAT. Notably, the bidirectional regulation of aromatic metabolites by microbial genera such as Pseudokineococcus highlights the universality of plant-microbe interactions in shaping metabolic networks—a mechanism potentially applicable to other crop systems. These findings reveal conserved microbial functional traits (e.g., metabolic pathway modulation) that may drive plant phenotypic differentiation beyond tobacco, offering insights into microbiome-mediated crop quality improvement. The results provide theoretical guidance for tobacco aging and aroma regulation and underscore the broader significance of microbial community engineering in agriculture for manipulating plant metabolic outputs.

As is well known, continuous cropping can lead to a decrease in crop yield and quality. Despite this, continuous cropping remains prevalent in practical agricultural production, particularly in the case of tobacco cultivation, owing to its high economic value. The samples for this study were collected from a flue-cured tobacco planting base located in Huili County, Liangshan Yi Autonomous Prefecture, Sichuan Province, China. After years of continuous planting, the yield of tobacco in this base has significantly decreased. In order to explain the microecological causes of this phenomenon, we collected non-continuous cropping, continuous cropping for 5 years, and continuous cropping for 10 years of tobacco rhizosphere soil, and analyzed the effects of long-term continuous cropping on nutrients, enzyme activities, microbial community structure, and function of tobacco rhizosphere soil. The results showed that with the continuous cropping, the majority nutrients (except for phosphorus and manganese) in rhizosphere soil decreased significantly, and the rhizosphere microbial community structure changed significantly. Correlation network analysis results showed that changes in the rhizosphere microbial community of tobacco were closely related to soil urease, active organic carbon, and available iron content. The results of functional analysis based on microorganisms and genes showed that the rhizosphere microbiota may change the content of soil nutrients through iron_respiration, sulfur_respiration, and Carbon fixation in prokaryotes pathways. The results of the correlation network analysis and the functional analysis mutually confirmed each other, both emphasizing the important role of soil carbon and iron in shaping the structure of the tobacco rhizosphere microbial community. Based on the results of this study, we propose to improve the microbial community structure of tobacco rhizosphere soil by increasing the levels of readily oxidizable organic carbon, available iron, and soil urease activity in the future, so as to alleviate the negative impact of continuous cropping on crop yield. The results of this study provide theoretical support for modifying the rhizosphere microbial environment through nutrient regulation, thereby enhancing plant growth in the context of continuous tobacco cropping.