Chilling stress increases the amount of polyphenols, especially lignin, which protects tobacco (Nicotiana tabacum L. cv. k326) from chilling stress. To clarify the molecular biosynthesis mechanism of the key representative compounds, specifically lignin, RNA sequencing and ultra-high pressure liquid chromatography coupled to quadrupole-time of flight mass spectrometry technologies were used to construct transcriptomic and metabolomic libraries from the leaves of tobacco plants subjected to normal (25°C) and chilling (4°C) temperature treatments. Transcriptomic libraries from the different samples were sequenced, generating more than 40 million raw reads. Among nine samples, metabolomic analysis identified a total of 97 encoding enzymes that function in the key steps of pathways related to polyphenol biosynthesis, where 42 metabolites were also located. An integrated analysis of metabolic and transcriptomic data revealed that most of the intermediate metabolites and enzymes related to lignin biosynthesis were synthesised in the leaves under chilling stress, which suggests that the biosynthesis of lignin plays an important role in the response of tobacco leaves to cold temperatures. In addition, the cold insensitivity of chalcone synthase genes might be considered to be an important rate-limiting factor in the process of precursor substance flow to flavonoid biosynthesis under chilling stress. Furthermore, the upregulated expression of phenylalanine ammonia lyase (PAL), hydroxycinnamoyl transferase (HCT) and cinnamyl-alcohol dehydrogenase (CAD) under chilling stress is the key to an increase in lignin synthesis. This study provides a hypothetical basis for the screening of new active metabolites and the metabolic engineering of polyphenols in tobacco.

Consecutive monoculture practice facilitates enrichment of rhizosphere pathogenic microorganisms and eventually leads to the emergence of replant disease. However, little is known about the interaction relationship among pathogens enriched in rhizosphere soils, Nucleotide binding-leucine-rich repeats (NB-LRR) receptors that specifically recognize pathogens in effector-triggered immunity (ETI) and physiological indicators under replant disease stress in Rehmannia glutinosa. In this study, a controlled experiment was performed using different kinds of soils from sites never planted R. glutinosa (NP), replanted R. glutinosa (TP) and mixed by different ration of TP soils (1/3TP and 2/3TP), respectively. As a result, different levels of TP significantly promoted the proliferation of Fusarium oxysporum f.sp. R. glutinosa (FO). Simultaneously, a comparison between FO numbers and NB-LRR expressions indicated that NB-LRRs were not consecutively responsive to the FO proliferation at transcriptional levels. Further analysis found that NB-LRRs responded to FO invasion with a typical phenomenon of “promotion in low concentration and suppression in high concentration”, and 6 NB-LRRs were identified as candidates for responding R. glutinosa replant disease. Furthermore, four critical hormones of salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) had higher levels in 1/3TP, 2/3TP and TP than those in NP. Additionally, increasing extents of SA contents have significantly negative trends with FO changes, which implied that SA might be inhibited by FO in replanted R. glutinosa. Concomitantly, the physiological indexes reacted alters of cellular process regulated by NB-LRR were affected by complex replant disease stresses and exhibited strong fluctuations, leading to the death of R. glutinosa. These findings provide important insights and clues into further revealing the mechanism of R. glutinosa replant disease.

NB-ARC proteins are critical to effector-triggered immunity and play important roles in effector recognition and signal transduction in healthy plant growth. However, their primary protein traits, functions and roles remain incompletely understood in Rehmannia glutinosa. Here, we identified 45 NB-ARC protein sequences from the protein sequence sets and transcriptome of R. glutinosa. The CC type was the main one, accounting for 84.44% of these sequences. The most conserved motif was a 288 aa ADP-binding sequence. This motif belongs to the disease-resistance proteins. Differential expression of 36 expressed NB-ARC genes revealed that NB-ARC genes were rarely expressed 30 days after planting and were frequently expressed approximately 60 days after planting. To further understand the function of NB-ARC in replanted R. glutinosa, the genes encoding NB-ARC domains were profiled using qRT-PCR under the different stress states involved in the formation of consecutive monoculture problems. The results showed that NB-ARC genes might play a role in the formation of R. glutinosa consecutive monoculture problems. This study is the first to identify NB-ARC genes in R. glutinosa and to reveal their roles in consecutive monoculture problems in R. glutinosa. These findings provide insights into the mechanism of formation of consecutive monoculture problems.

Introduction: This study investigated the effects of a selective soil treatment agent on the microbial community structure of tobacco rhizosphere soil and the photosynthetic performance of tobacco plants at different temperatures (15 °C, 25 °C, 35 °C) through pot experiments. Methods: The control efficacy against black shank and root rot diseases was also evaluated. Results and discussion: The results indicated that 25 °C was the optimal temperature for the agent’s effectiveness. Under this condition, the agent significantly inhibited the enrichment of phytopathogenic fungi such as Fusarium spp. in the rhizosphere soil (the abundance of Fusarium in the T25SJ treatment decreased by 78.6% compared to the untreated group T25CK), while optimizing the microbial community structure, as evidenced by increased fungal α -diversity (Shannon index) and enhanced competitiveness of beneficial bacteria (e.g., Pseudomonas spp.). Notably, the bacterial community stability was maintained (no significant change in the Simpson index), confirming the agent’s selective regulatory effect rather than non-specific sterilization. Fluorescence imaging analysis revealed that the photosynthetic performance of tobacco plants treated with the agent at 25 °C was significantly higher than that of the untreated group, as showed by an increase in the maximum photochemical efficiency (Fv/Fm) (T25SJ vs. T25CK: 0.82 vs. 0.68), enhanced electron transport efficiency (ψ_Eo), and light energy conversion capacity (δ_Ro). In contrast, the untreated group exhibited severe damage to the photosynthetic system due to pathogen infection, leading to the inactivation of PSII reaction centers (increased ABS/RC and decreased RC/ABS values). At low (15 °C) and high (35 °C) temperatures, the agent still showed some inhibitory effects on certain pathogens (e.g., Thielaviopsis spp.), but the overall antibacterial efficiency and photosynthetic protection were significantly weaker than at 25 °C. Further analysis demonstrated that the agent alleviated oxidative stress caused by root damage and ensured the preferential allocation of light energy to photosynthesis by selectively suppressing dominant pathogens while preserving beneficial microbial populations. This study reveals that the antibacterial efficacy and photosynthetic protection of the soil treatment agent are significantly temperature-dependent, with 25 °C being the optimal application temperature. These findings provide a theoretical basis for the green control of soil-borne diseases in tobacco and the regulation of photosynthetic stress resistance, and offer a new paradigm for resolving the contradiction between “pathogen control” and “ecological protection” in soil-borne disease management.