Background: Mirabilis himalaica, distributed in the high-altitude, arid, and semi-arid regions of Xizang, exhibits great tolerance to drought, which is rich in rotenoids and other secondary metabolites. It is still unknown, though, how drought stress influences rotenoid synthesis in M. himalaica. Methods: In this study, the calli of M. himalaica were subjected to 5% PEG6000 for 0, 20, and 40 h and divided into control group (CK), mild-drought-treated group (M), and high-drought-treated group (H), respectively. We then analyzed the relative content of three main rotenoids in M. himalaica using high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS/MS). Results: Our findings demonstrated that the content of rotenoids was significantly reduced under drought stress. Transcriptome analysis subsequently revealed 14,525 differentially expressed genes (DEGs) between the different treatments. Furthermore, these DEGs exhibited enrichment in pathways associated with isoflavone biosynthesis and hormone signaling pathways. Key genes with decreased expression patterns during drought stress were also found to be involved in rotenoid accumulation and drought-responsive phytohormone signaling, including abscisic acid (ABA), auxin (IAA), and jasmonic acid (JA). Conclusions: These findings elucidate the molecular processes of drought resistance in M. himalaica and shed light on the relationship between rotenoid production and drought stress in M. himalaica.

Dendrobium nobile, a rare orchid in Orchidaceae, is a traditional Chinese medicinal herb known for its diverse biological activities, including anti-tumor, anti-aging, and hypoglycemic effects (Fan et al. 2023). In February 2025, symptoms of leaf anthracnose were observed on D. nobile plants growing in the medicinal plant plantation of Zhejiang Chinese Medical University (30.08°N, 119.88°E), China. The affected area covered approximately 25 m2, with a disease incidence of around 55%. The disease symptoms began at the leaf edges, where light yellow spots first appeared. As the lesions expanded, they turned into irregular brown or black patches. The edges of the infected leaves dried out, forming a large necrotic area with dark brown spots, ultimately leading to defoliation. Six symptomatic leaf samples (5×5 mm) were surface sterilized with 75% ethanol for 30 s, followed by 2.5% NaClO for 1 minute. Afterair-drying, the tissues were placed on potato dextrose agar (PDA) medium and incubated at 28°C for five days. Two fungal isolates, DNLP07 and DNLP08, were obtained using the hyphal-tip isolation method. On PDA, the colonies appeared white and cottony on the surface, with a white reverse side, yellow-brown discoloration, and concentric rings visible on both sides. The conidia were single-celled, aseptate, cylindrical to oval, measuring 21.6-31.1 × 9.5-14.9 μm (average 25.9 × 11.7 μm; n=50). These morphological characteristics were consistent with those of Colletotrichum citricola (Fu et al. 2019). Six gene regions were amplified: internal transcribed spacer (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin 2 (TUB2). The following primer sets were used: ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF/GDR, and T1/Bt2b, respectively (Weir et al. 2012). The resulting sequences were submitted to GenBank with the following accession numbers: ITS: PV271806, PV271807; ACT: PV287640, PV287641; CAL: PV287638, PV287639; CHS: PV287642, PV287643; GAPDH: PV287644, PV287645; TUB2: PV287646, PV287647. BLASTn analysis showed that the isolate DNLP08 shared 99% to 100% sequence identity with the C. citricola type strain CBS 134228, including: ITS (KC293576; 468/468 bp), ACT (KC293616; 268/270 bp), CAL (KC293696; 424/425 bp), CHS (KC293792; 278/278 bp), GAPDH (KC293736; 239/239 bp), and TUB2 (KC293656; 487/488 bp). A maximum likelihood phylogenetic tree was constructed using the concatenated sequence dataset in MEGA 11, which showed that both isolates clustered with C. citricola. To confirm pathogenicity, a conidial suspension of isolate DNLP08 (1×106 spores/mL) was sprayed onto three healthy D. nobile plants. Three additional plants were treated with sterile distilled water as controls. All plants were maintained at 28°C with 85% relative humidity and a 12-hour photoperiod. After 12 days, the inoculated plants developed symptoms identical to those observed in the field, while the control plants remained asymptomatic. The pathogen was successfully re-isolated from symptomatic leaves and identified based on morphological features and sequence data from the six genes, fulfilling Koch's postulates. No pathogens were isolated from the control plants. This study first reports C. citricola causing leaf anthracnose on D. nobile in China. Accurate pathogen identification lays a crucial foundation for future effective prevention and control of the disease.

Mirabilis himalaica is a Tibetan medicinal plant whose tuberous root is its principal medicinal organ. To characterise molecular and metabolic differences associated with root enlargement, we integrated untargeted metabolomic and transcriptomic analyses of enlarged and non-enlarged roots. We identified 1465 metabolites, including 336 differentially accumulated metabolites, and 4058 differentially expressed genes. Flavonoids and phenolic acids were predominantly less abundant in enlarged roots, whereas several alkaloids showed higher relative abundance. Three gibberellin-related metabolites were lower in enlarged roots, while 1-Aminocyclopropanecarboxylic acid (ACC), L-tryptophan, tryptamine, and several cytokinin-related metabolites showed higher relative signals. Tryptophan metabolism was a shared enriched pathway in the integrated analysis. L-Tryptophan was positively correlated with Anthranilate synthase beta subunit 2 (ASB2) and negatively correlated with Tryptophan synthase alpha chain (TSA) and Probable indole-3-pyruvate monooxygenase (YUC) across the six samples; these exploratory correlations do not establish regulatory relationships. The observed patterns are consistent with coordinated changes in hormone-related metabolites, secondary metabolism, and gene expression during root enlargement. Because Indole-3-acetic acid (IAA) and lignin were not directly quantified, the study does not infer their concentrations or deposition. These findings provide a multi-omics resource for investigating tuberous-root development and for guiding future functional and targeted validation studies in this endangered medicinal species.