Jiahong Dong

Tsaoko stripe mosaic virus (TkSMV) has been identified as the predominant viral pathogen of Amomum tsaoko in Yunnan Province of China. This virus induced various foliar symptoms, including chlorosis, stripe mosaic, and necrosis, while some infected A. tsaoko plants remain asymptomatic. Comprehensive investigation of the population genetic structure and molecular detection protocols for TkSMV is crucial for elucidating its evolutionary dynamics, epidemiology, and disease control strategies. Phylogenetic analysis based on 15 full-length genomic sequences and 29 coat protein (CP) gene fragments revealed three distinct evolutionary lineages (S1, S2, and S3) within the TkSMV population. Neutrality test analysis indicates that the genetic polymorphism of the TkSMV population is consistent with a neutral evolution model (Tajima’s D > 0, not significant). The comparative evolutionary analysis demonstrated significant selective pressures across viral proteins: codon usage bias analysis revealed strong purifying selection (dN/dS < 1) in nine structural/functional proteins, eliminating deleterious mutations. The 9K gene exhibited the highest nucleotide diversity (π = 0.13567), suggesting its role in adaptive evolution. NIa-Pro protease showed the lowest diversity (π = 0.09497), indicating functional constraint. Remarkably, the CP gene displayed polarized evolutionary patterns. The N-terminal region (amino acids 1 to 100) acted as a hypervariable hotspot, while the C-terminal region (amino acids 151 to 240) maintained evolutionary conservation. Furthermore, multiplex RT-PCR assays for accurate discrimination of TkSMV-S1/S2/S3 strains in mixed infections were developed. These advancements provide a technical foundation for epidemiological surveillance through strain-specific monitoring and seed certification programs for A. tsaoko cultivation.

Stephania epigaea Lo is an herb of significant medicinal and ornamental value in China. In May 2024, leaf spot disease was observed on S. epigaea in the medicinal plant nursery base of Yunnan University of Chinese Medicine, Kunming, Yunnan Province, China (24°33'59.21"N, 99°55'43.71"E), with an incidence rate of 75%. The symptomatic leaves exhibited irregular brown lesions along the margins, surrounded by a yellow halo. To determine the causal agents, symptomatic leaf tissues (5 × 5 mm) were excised, surface-sterilized in 75% ethanol for 30 s, followed by immersion in 1% sodium hypochlorite (NaClO) for 180 s, and rinsed three times with sterile distilled water. The sterilized tissues were transferred onto potato dextrose agar (PDA) and incubated at 28°C in the dark for 3 d. Pure cultures were obtained using the single-spore purification method. A total of five single-spore isolates were obtained. The isolates exhibited white, cottony aerial mycelia with undulated colony margins. Conidia were fusiform and four-septate, with three versicolor median cells and hyaline apical and basal cells (17.3-26.8 µm long × 3.7-6.2 µm wide; n = 30). The apical cells bore two to three appendages (19.6-28. 6 μm long; n = 30), while the basal cells bore a single unbranched appendage. To further identify the fungus, isolate SeF01 was randomly selected for sequencing. The nuclear ribosomal internal transcribed spacers (ITS), large subunit ribosomal RNA gene (LSU), β-tubulin gene (TUB2), and translation elongation factor-1α (TEF-1α) regions were amplified and sequenced using primer pairs ITS1/ITS4 (White et al., 1990), LR0R/LR5 (Schoch et al., 2012), T1/T2 (O'Donnell and Cigelnik, 1997), and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively. BLASTn homology analysis revealed that the ITS (GenBank accession no. PV363391), LSU (PV363539), TUB2 (PV384116), and TEF-1α (PV384117) sequences of isolate SeF01 exhibited 99.44-100% identity with Neopestalotiopsis rosae strain CBS 101057 (KM199359, KM116245, KM199429, and KM199523). A phylogenetic tree of Neopestalotiopsis species was built based on concatenated nucleotide sequences of ITS, LSU, TUB2, and TEF-1α using the maximum likelihood method. Isolate SeF01 clustered with N. rosae on the same branch. Morphological and molecular analyses confirmed that SeF01 was identical to N. rosae. To confirm pathogenicity of the isolate, conidial suspension with 1 × 106 conidia/mL was sprayed onto S. epigaea plants until runoff, while the control plants were sprayed with sterile distilled water. Two replicates (6 plants/replicates) were performed. All plants were incubated at 28℃ and 80% relative humidity with a 12 h light/12 h dark photoperiod. After 15 d, inoculated plants developed leaf spot symptoms, whereas control plants remained symptom-free. Fungal strains re-isolated from symptomatic leaves displayed morphological characteristics identical to SeF01. N. rosae has been reported to cause leaf spot on Fragaria × ananassa, Punica granatum, and Lonicera caerulea. To our knowledge, this is the first report of leaf spot caused by N. rosae on S. epigaea in China. The disease significantly reduces the value of S. epigaea, and pathogen-specific control measures are recommended.

The perennial herbaceous vine Stephania epigaea Lo, known as Di Burong in Chinese, is generally used as an anti-inflammatory and analgesic drug, as well as an ornamental plant (Dong et al., 2018). In May 2024, symptoms resembling anthracnose were observed on S. epigaea plants in an herbal nursery (33 m2, approximately 400 plants) in Kunming, Yunnan Province (24°33' 59.21" N, 99°55' 43.71" E), China, with an incidence rate of 75%. The disease initially occurred as small, round brown spots on the leaf margins, which later expanded into irregular lesions. The centers of these lesions were dark brown with yellow edges. To isolate and identify the pathogen, 0.5 × 0.5 mm sections of diseased leaf tissue were sterilized by dipping them in 75% ethanol for 30 s, then in 1% NaCIO for 180 s, followed by three rinses in sterile water. The samples were then cultured on potato dextrose agar (PDA) in the dark at 28 °C for 3 days. The fungal isolates were purified using the single-spore purification method, and 10 isolates with similar morphological characteristics were obtained. The colonies on PDA were initially white, changing to a grayish-white color over time. The ascomata were dark brown, spherical, and either on the surface or partially immersed in the PDA. The conidiophores were smooth, septate, branched, and hyaline to light brown. The conidia were cylindrical, aseptate, hyaline, and smooth, with dimensions of 10.0 to 16.7 × 5.2 to 6.9 μm (average 13.5 × 6.0 μm, n = 30). A 24-hour slide culture (Cai et al., 2009) of the conidia suspension revealed solitary appressoria that were light to dark brown, nearly spherical to oval, and smooth-walled to undulate, with dimensions of 6.8 to 10.5 × 4.7 to 10.0 μm (average 8.7 × 6.7 μm, n = 30). To further characterize the pathogen, isolate SeF03 was randomly selected for further testing. Total genomic DNA was extracted using the cetyl trimethylammonium bromide method, and the nuclear ribosomal internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 3 (HIS3), actin (ACT), and calmodulin (CAL) genes were amplified using the primer pairs ITS1/ITS4 (White et al., 1990), gpd1/gpd2 (Berbee et al., 2019), CYLH3F/CYLH3R (Crous et al., 2006), ACT-512F/ACT-783R (Carbone and Kohn, 1999), and CL1C/CL2C (Weir et al., 2012), respectively. BLASTn homology search showed that the ITS (PQ963009), GAPDH (PV021394), HIS3 (PV021395), ACT (PV021396), and CAL (PV021397) sequences of SeF03 were 100, 98.81, 100, 98.15, and 100% identical to Colletotrichum karstii strain CBS 128500 (JQ005202, JQ005289, JQ005463, JQ005550, and JQ005723), respectively. A phylogenetic tree of Colletotrichum species was built based on concatenated nucleotide sequences of ITS, GAPDH, HIS3, ACT, and CAL using the maximum likelihood method with the Tamura-Nei model. SeF03 and C. karstii clustered on the same branch. Morphological and molecular characteristics also confirmed that SeF03 was identical to C. karstii. Pathogenicity was evaluated through foliar spray inoculation with a conidial suspension (1 × 10⁶ conidia/mL) applied until runoff, using sterile water-treated plants as controls. All sprayed plants were placed in an incubator at a constant temperature of 28 ℃ with alternating light and dark periods (L/D = 12 h/12 h, 80% humidity). Each treatment consisted of 3 plants, with 2 replicates. After 15 days, obvious brown spots appeared on leaves treated with the SeF03 spore solution, while the control plants sprayed with sterile water remained asymptomatic. The fungal strains isolated from the symptomatic leaves shared the same morphological characteristics as SeF03. C. karstii has been reported to cause anthracnose on Fatsia japonica, Dalbergia odorifera, and Piper nigrum. To our knowledge, this is the first report of anthracnose caused by C. karstii on S. epigaea in China. The disease significantly threatens S. epigaea's ornamental value, requiring urgent and targeted control measures.

A novel macluravirus, tentatively named "tsaoko stripe mosaic virus" (TkSMV), was identified in Amomum tsaoko through high-throughput sequencing. The complete genome sequence of TkSMV was determined using RT-PCR and RACE. The genome sequence consists of 8218 nucleotides, excluding the poly(A) tail, and contains a large open reading frame encoding a polyprotein of 2625 amino acids with a molecular weight of approximately 297.13 kDa. TkSMV is most closely related to Alpinia oxyphylla mosaic virus, sharing 71.5% nucleotide and 75.9% amino acid sequence identity. These values are below the species demarcation threshold for the family Potyviridae. These results suggest that TkSMV should be considered a distinct member of the genus Macluravirus.