Preliminary findings on the development of a predictive model for BLCA based on disulfidptosis-associated IncRNAs signature.

BACKGROUND: Bladder urothelial carcinoma (BLCA) is the most common malignancy of the urinary tract, presenting with a wide range of clinical symptoms and prognosis. Disulfidptosis is a newly identified cell death method and closely associated with BLCA progression, prognosis, and treatment outcome. Currently, we need to construct a new prognostic model for disulfidptosis-related long noncoding RNAs (drlncRNAs) to improve the treatment strategy of BLCA. METHODS: The data for BLCA samples were obtained from The Cancer Genome Atlas (TCGA), and then 10 unique genes related to disulfidoptosis (DRGs) were identified from research papers. The differences between the two groups showed in this study were used to create the "disulfidptosis-related long noncoding RNAs score" (disulfidptosis-score) prognostic model. RESULTS: We identified two groups of drlncRNAs with high and low disulfidptosis scores in this study. Patients with low disulfidptosis scores had a better overall survival rate compared to those with high scores in bladder cancer, and the high disulfidptosis score subtype exhibited more active malignant pathways related to cancer than the low score subtype. We found that the low disulfidptosis-score subgroup had better prognosis than the high disulfidptosis-score subgroup. The expression of mutation burden was much higher in the low disulfidptosis-score group than in the high disulfidptosis-score group. The low disulfidptosis-score subgroup of patients exhibited significantly higher proportions of plasma cells, T cells CD8, and Tregs, while the high-risk subgroup had a greater abundance of Macrophages M0 and Macrophages M2. The disulfidptosis-score showed a strong correlation with the sensitivity of chemotherapeutic drugs, and patients in the low disulfidptosis-score group were more likely to exhibit an immune response and respond positively to immunotherapy. Additionally, we developed a nomogram to enhance the accuracy of the disulfidptosis-clinical score. CONCLUSION: Based on our investigation of disulfidptosis-score in BLCA, disulfidptosis-score may have an important role in TME, prognosis, and drug sensitivity. We also investigated the significance of the disulfidoptosis-score in relation to immunotherapy and immune response, providing a basis for improving prognosis and responding to immunotherapy among patients with BLCA.

First Report of Ilyonectria coprosmae Causing Root Rot of Bletilla striata in Yunnan province of China.

Bletilla striata is a valuable medicine in China, belonging to the Orchidaceae family, and is used for treating various ailments such as hemoptysis, pyocutaneous disease, and anal fissure by preventing blood flow, reducing swelling, and promoting granulation. In June 2022, a disease with symptoms similar to root rot was observed on B. striata in the pineland (the area was 0.4 hectare) of Lancang County (22°48'17" N, 99°46'58"22 E), Yunnan Province, China. The root rot incidence rate reached 16% (Table S1). The root rot incidence was calculated as follows: root rot incidence (%) = (number of root rot seedlings/total number of seedlings investigated) × 100. In May 2023, the similar symptoms were observed in the field, and the disease incidence was 17% (Table S1). Initially, there were no obvious symptoms on the leaves. Subsequently, the leaves wilted and brown spots appeared. Later, the entire leaf browned, withered and eventually died (Fig. S1A, B). The roots were brown and the browning spread from the root edge to the center, causing vascular bundle browning and dead lignified fibers in the cortex (Fig. S1C, D). To isolate the causal pathogen, 20 symptomatic root tissues were collected from 20 plants. Cutting the diseased tissues into small pieces (0.5 × 0.5 cm). After surface sterilization (30s with 75% ethanol and 3 min with 2% sodium hypochlorite, rinsed three times with sterile water), the disinfected root tissues were plated onto potato dextrose agar (PDA) and incubated at 25℃ for 4 to 6 days with 12 h light/dark photoperiod. A total of 10 single-spore isolates with similar morphology and conidial characteristics were obtained. one representative isolate BJG6 was selected for identification and further study. The fungal colony was reddish-brown or orange-white on PDA after 8 days of incubation at 25℃. The mycelium was like carpet or cotton, and the edge of colony was uniform (Fig. S1E). Large conidia were formed on simple conidial peduncles (Fig. S1F, G). The conidia with 1~3 septates and 1 mostly, with cylindrical shapes and narrow tops but sharp bases (Fig. S1H-J). Conidia with 1 septate measured as 5.5 (4.3-6.7) × 20.7 (16.0-25.4) µm (n=30), while those with 2 septates measured as 6.6 (5.8-7.4) × 26.5 (21.7-31.3) µm (n=30), and those with 3 septates was 6.9 (6.2-7.8) × 31.8 (29.3-34.3) µm (n=30). Ellipsoidal microconidia could be formed on conidiophore and measured as 2.4 (1.9-2.9) × 4.9 (5.9-3.9) µm to 2.7 (2.2-3.2) × 5.4 (4.3-6.5) µm (n=30). Spherical or subspherical chlamydospores were produced on low-nutrient agar, with an average size of 5.8(5.0-6.6) µm×5.3 (4.4-6.2) µm (n=30) (Fig. S1K, L). According to the morphology and conidial features, the pathogen was consistent with the description of Ilyonectria coprosmae (Cabral et al. 2012). The total genomic DNA was extracted, and primer pairs ITS4/ITS5 were used to amplify and sequence the rDNA-ITS region (ITS1-5.8 S rRNA-ITS2 gene regions) (White et al. 1990). The sequences were deposited in GenBank (SUB13905750 for ITS). BLAST searches revealed BJG6 showed 98% homology with corresponding sequences of Ilyonectria coprosmae in GenBank (JF735260). A phylogenetic tree (MEGA 7.0) was constructed using maximum-likelihood methods (Fig. S2). To identify pathogenicity, a cultured medium in a size of 6mm containing isolate BJG6 was inoculated onto ten healthy roots of B. striata, PDA plugs alone were used as the uninoculated controls. All samples were placed in a dark inoculation chamber at 25℃. The pathogenicity test was replicated three times. After two weeks, all inoculated roots appeared similar symptoms identical to those observed on field plants (Fig. S1M, N-P), while control plants remained healthy (Fig. S1Q, R). The same pathogenic fungus was reisolated from the symptomatic root rot, and the characteristics of colony and conidia were the same as the original isolates (Fig. S1S, T). These results confirmed I. coprosmae as the causal pathogen of root rot disease on B. striata in China by Koch's postulates tests for the first time. Further exploration should be conducted to understand the occurrence and migration of this disease, so as to develop specific and efficient disease management strategies in the future.

Effects of allyl isothiocyanate fumigation on medicinal plant root knot disease control, plant survival, and the soil bacterial community.

BACKGROUND: Allyl isothiocyanate (AITC) is a natural product with high volatility that is used as a biofumigant to alleviate soil-borne plant diseases, and problems such as root knot nematodes (RKNs) that necessitate continuous cropping. However, little research has assessed the effects of AITC fumigation on medicinal plants. RESULTS: AITC significantly reduced the population of RKNs in soil (p < 0.0001) and showed an excellent RKN disease control effect within 6 months after sowing Panax notoginseng (p < 0.0001). The seedling survival rate of 2-year-old P. notoginseng was approximately 1.7-fold higher after soil treatment with AITC (p = 0.1008). 16S rRNA sequencing indicated that the AITC treatment affected bacterial richness rather than diversity in consecutively cultivated (CC) soil. Furthermore, biomarkers with statistical differences between AITC-treated and untreated CC soil showed that Pirellulales (order), Pirellulaceae (family), Pseudomonadaceae (family), and Pseudomonas (genus) played important roles in the AITC-treated group. In addition, the microbiome functional phenotypes predicted using the BugBase tool suggested that AITC treatment is more conducive to improving CC soil through changes in the bacterial community structure. Crucially, our research also suggested that AITC soil treatment significantly increases soil organic matter (p = 0.0055), total nitrogen (p = 0.0054), and available potassium (p = 0.0373), which promotes the survival of a succeeding medicinal plant (Polygonatum kingianum). CONCLUSION: AITC is an ecologically friendly soil treatment that affects the top 10 bacterial richness but not diversity. It could also provide a basis for a useful agricultural soil management measure to alleviate soil sickness.

Insights into risk factors for urolithiasis: a mendelian randomization study.

BACKGROUND: Risk factors for urolithiasis have not been identified. Here, we aimed to identify potentially causal risk factors driving the risk of urolithiasis. METHODS: Two sets of instrumental variables were used for analysis, derived from publicly available databases. Summary-level statistical data for urolithiasis were obtained from the MRC-IEU Consortium and UK biobank (Neale Lab). Mendelian randomization (MR) was conducted to identify causal risk of urolithiasis. Finally, the results of the two databases were combined and a meta-analysis was performed. RESULTS: In the MRC-IEU consortium, the odds of urolithiasis increased per 1-SD increase of body mass index (BMI) (OR = 1.0016, 95% CI:1.0004-1.0029, p = 0.010), triglycerides (OR = 1.0016, 95% CI:1.0003-1.0029, p = 0.017), adiponectin (OR = 1.0027, 95% CI:1.0003-1.0050, p = 0.024), and body fat percentage (OR = 1.008, 95% CI:1.0001-1.0161, p = 0.047). In addition, alcohol intake also increased the incidence of urolithiasis (OR = 1.0030, 95% CI:1.0009-1.0051, p = 0.005). In the UK biobank, the odds of urolithiasis increased per 1-SD increase of waist circumference (OR = 1.0215, 95% CI:1.0061-1.0372, p = 0.008) and body fat percentage (OR = 1.0239, 95% CI:1.0043-1.0440, p = 0.020). Surprisingly, we found that the risk of urolithiasis decreased with increasing hip circumference (OR = 0.9954, 95% CI:0.9915-0.9992, p = 0.017). In a meta-analysis of MR results, higher BMI (OR = 1.0016, 95% CI:1.0004-1.0027, p = 0.009), waist circumference (OR = 1.0073, 95% CI:1.0020-1.0126, p = 0.007), adiponectin (OR = 1.0026, 95% CI:1.0008-1.0043, p = 0.004), triglycerides (OR = 1.0015, 95% CI:1.0004-1.0026, p = 0.008) and body fat percentage (OR = 1.0104, 95% CI:1.0030-1.0178, p = 0.006) increased the risk of urolithiasis. Furthermore, alcohol intake also increased the incidence of urolithiasis (OR = 1.0033, 95% CI:1.0012-1.0053, p = 0.002). CONCLUSIONS: Our MR study found that higher BMI, triglycerides, waist circumference, adiponectin, body fat percentage, and alcohol intake increased the risk of urolithiasis.

Development of a prognostic model to predict BLCA based on anoikis-related gene signature: preliminary findings.

BACKGROUND: The prevalence of bladder urothelial carcinoma (BLCA) is significant on a global scale. Anoikis is a type of procedural cell death that has an important role in tumor invasion and metastasis. The advent of single-cell RNA sequencing (scRNA-seq) approaches has revolutionized the genomics field by providing unprecedented opportunities for elucidating cellular heterogeneity. Understanding the mechanisms associated with anoikis in BLCA is essential to improve its survival rate. METHODS: Data on BLCA and clinical information were acquired from the databases of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). ARGs were obtained from Genecards and Harmonizome databases. According to univariate Cox regression analysis, the least absolute shrinkage and selection operator (LASSO) algorithm was utilized to select the ARGs associated with the overall rate (OS). A multivariate Cox regression analysis was carried out to identify eight prognostic ARGs, leading to the establishment of a risk model. The OS rate of BLCA patients was evaluated using Kaplan-Meier survival analysis. To explore the molecular mechanism in low- and high-risk groups, we employed Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSVA). Immune infiltration landscape estimation was performed using ESTIMATE, CIBERSOT, and single sample gene set enrichment analysis (ssGSEA) algorithms. Patients were categorized into different subgroups through consensus clustering analysis. We employed biological functional enrichment analysis and conducted immune infiltration analysis to examine the disparities in potential biological functions, infiltration of immune cells, immune activities, and responses to immunotherapy. RESULTS: We identified 647 ARGs and 37 survival-related genes. We further developed a risk scoring model to quantitatively assess the predictive capacity of ARGs. The high-risk score group exhibited an unfavorable prognosis, whereas the low-risk score group demonstrated a converse effect. We also found that the two groups of patients might respond differently to immune targets and anti-tumor drugs. CONCLUSION: The nomogram with 8 ARGs may help guide treatment of BLCA. The systematic assessment of risk scores can help to design more individualized and precise treatment strategies for BLCA patients.

Comprehensive analysis of cuproptosis-related lncRNAs signature to predict prognosis in bladder urothelial carcinoma.

BACKGROUND: Cuproptosis-related genes (CRGs) have been recently discovered to regulate the occurrence and development of various tumors by controlling cuproptosis, a novel type of copper ion-dependent cell death. Although cuproptosis is mediated by lipoylated tricarboxylic acid cycle proteins, the relationship between cuproptosis-related long noncoding RNAs (crlncRNAs) in bladder urothelial carcinoma (BLCA) and clinical outcomes, tumor microenvironment (TME) modification, and immunotherapy remains unknown. In this paper, we tried to discover the importance of lncRNAs for BLCA. METHODS: The BLCA-related lncRNAs and clinical data were first obtained from The Cancer Genome Atlas (TCGA). CRGs were obtained through Coexpression, Cox regression and Lasso regression. Besides, a prognosis model was established for verification. Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, gene ontology (GO) analysis, principal component analysis (PCA), half-maximal inhibitory concentration prediction (IC50), immune status and drug susceptibility analysis were carried out. RESULTS: We identified 277 crlncRNAs and 16 survival-related lncRNAs. According to the 8-crlncRNA risk model, patients could be divided into high-risk group and low-risk group. Progression-Free-Survival (PFS), independent prognostic analysis, concordance index (C-index), receiver operating characteristic (ROC) curve and nomogram all confirmed the excellent predictive capability of the 8-lncRNA risk model for BLCA. During gene mutation burden survival analysis, noticeable differences were observed in high- and low-risk patients. We also found that the two groups of patients might respond differently to immune targets and anti-tumor drugs. CONCLUSION: The nomogram with 8-lncRNA may help guide treatment of BLCA. More clinical studies are necessary to verify the nomogram.

First report of Leptosphaeria biglobosa 'brassicae' causing leaf spots in Eutrema japonicum in Yunnan, China.

Wasabi (Eutrema japonicum) is a root vegetable that is cultivated at large scales in southwestern China. In November 2021, approximately 40% of plants in a forested plantation in Dadishui, Yunnan Province, China (25.47°N, 103.22°E), showed leaf spot symptoms. The early symptoms were small black spots that gradually expanded into irregular brown to black lesions (0.5-1.5 cm), which were restricted by leaf veins. Yellow halos were observed at the outer edges of necrotic lesions. To identify the causal agent, we collected 20 diseased leaves and obtained fungal isolates from symptomatic leaf tissues. Following surface sterilization with 75% ethanol for 30 s, the tissues were cultured on potato dextrose agar (PDA) plates and incubated at 25°C under a 12 h light/12 h dark light cycle. After 7 days of incubation, a total of 12 isolates were obtained through single-spore culture. All isolates had similar colony morphology, and produced fluffy white mycelia and yellow pigment after 1 week of PDA culture at 25°C, and blackish- brown mycelium, tan pigment, and conidia after 2 weeks. The conidia were hyaline and cylindrical, with an average size of 4.6 µm × 2.2 µm. These morphological characteristics similar to the description of Leptosphaeria biglobosa (Shoemaker et. al, 2001) and Leptosphaeria maculans (Vincenot et al. 2008). Genomic DNA was extracted from mycelium of isolate SK-1, which was harvested from 10-day-old PDA culture using a FAST plant genomic DNA Extraction Kit (Biomed, China), following the manufacturer's instructions. The species-specific primers LbigF, LmacF, and LmacR (Liu et al. 2006) were used for identification via polymerase chain reaction (PCR). A 444-bp fragment characteristic of L. biglobosa 'brassicae' (Lbb), and a 330-bp of L. maculans 'brassicae' (Lmb) were amplified, respectively. Internal transcribed spacer (ITS) sequences (592 bp), part of the 5' end of beta-tubulin (968 bp), and actin (899 bp) were also amplified using the primers ITS1/ITS4, BT1/BT2, and ACTF/ACTR (Vincenot et al. 2008), respectively. PCR was performed in a volume of 25 µL containing 12.5 µL 2 × T5 Super PCR Mix (Tsingke Biotech, Beijing, China), 1 µL 10 µM primer (Tsingke Biotech), 1 µL DNA template, and an aliquot of sterile water to attain the total volume. The thermal cycler settings were 5 min at 98°C; 35 cycles of 10 s at 98°C, 10 s at 58°C, and 30 s at 72°C; and extension for 2 min at 72°C. The ITS sequence of isolate SK-1 (GenBank accession no. OQ216838), the partial ß-tubulin gene sequence (OQ241183), and the actin gene sequence (OQ241184) indicated 100% query cover and 100% identity with L. biglobosa (DQ458906), Lbb strain B3.6 (AY748995), and Lbb strain 2379-4 (AY748949), respectively. Phylogenetic analysis (King et al. 2022) also identified of isolate SK-1 as Lbb. To determinate its pathogenicity, isolate SK-1 was grown on PDA incubated at 28°C for 2 weeks, and conidial suspensions were prepared at a concentration of 106 conidia/mL. Then, 15 leaves of 4-month-old E. japonicum seedlings were needle-wounded on the front and inoculated by syringe injection of 10 µL of the appropriate conidial suspension. We used 10 µL of the sterilized distilled water as the control under forest growth conditions. All inoculation sites were covered with cotton strips and moistened with 1.0 mL sterile water to maintain humidity. After 12 days of incubation, the leaves developed symptoms similar to those observed in the field, and the fungus was reisolated from diseased leaves, whereas the controls remained healthy. Based on these results, we identified L. biglobosa 'brassicae' as the causal agent of leaf spot on E. japonicum in China. This fungus has been reported to cause blackleg in many Brassica crops in China such as Brassica napus (Fitt et al. 2006), Brassica oleracea (Zhou et al. 2019), B. juncea var. tumida (Deng et al. 2020), Brassica rapa subsp. pekinensis (Yu et al. 2021). To the best of our knowledge, this is the first report of L. biglobosa causing leaf spots in E. japonicum in China. Our data provide a basis for disease management in E. japonicum production in China.

Characterization of a Root-Knot Nematode Infecting Aconitum carmichaelii in Southwest China.

Growth of the Chinese herbal medicine industry has resulted in several new pests and diseases. China is one of the world largest producers of monkshood (Aconitum carmichaelii Debx.), but an unidentified root-knot nematode has become a significant pest in the southwestern provinces of Yunnan and Sichuan. Morphological characteristics and the ribosomal DNA-internal transcribed spacer and D2-D3 region of the 28S ribosomal RNA gene sequences were used to identify the nematode as Meloidogyne hapla. Through investigation, this is the first report of M. hapla infecting monkshood in Yunnan and Sichuan Provinces.

A new Panax notoginseng Leaf Spot Caused by Boeremia linicola in Yunnan, China.

Panax notoginseng-also known as Tianqi and Sanqi-is one of the most highly valued medicinal perennial herbs in the world (Wang et al. 2016). In August 2021, leaf spot was observed on P. notoginseng leaves in Lincang sanqi base (23º43´10˝N, 100º7´32˝E, 13.33 hm2). Symptoms expanded from water soaked areas on the leaves to form irregular round or oval leaf spots with transparent or grayish-brown centers containing black granular matter, with an incidence of 10 to 20%. To identify the causal agent, ten symptomatic leaves were randomly selected from ten P. notoginseng plants. Symptomatic leaves were cut into small pieces (5 mm2) with asymptomatic tissue margins, disinfected in 75% ethanol for 30s and in 2% sodium hypochlorite for 3 min, and rinsed three times with sterile distilled water. The tissue portions were placed on potato dextrose agar (PDA) plates incubated at 20℃ with a 12 h light/dark photoperiod. Seven pure isolates were obtained with similar colony morphology, dark gray (top view) or taupe (back view) coloration, with flat and villous surfaces. Pycnidia were globose to subglobose, glabrous or with few mycelial outgrowths, dark brown to black, 22.46 to 155.94 (av. 69.57) µm × 18.20 to 130.5 (av. 57.65) µm (n=50) in size. Conidia were ellipsoidal to cylindrical, thinwalled, smooth, hyaline, aseptate, and measured 1.47 to 6.81 (av. 4.29) µm long and 1.01 to 2.97 (av. 1.98) µm thick (n=100). The isolated strains were preliminarily identified as Boeremia sp. based on the morphological characteristics of colonies and conidia. (Aveskamp et al. 2010; Schaffrath et al. 2021). To confirm pathogen identity, the total genomic DNA of two isolates (LYB-2 and LYB-3) was extracted using the T5 Direct PCR kit. The internal transcribed spacer (ITS), 28S large subunit nrRNA gene (LSU), and ß-tubulin (TUB2) gene regions were PCR-amplified using primers ITS1/ITS4, LR0Rf/LR5r, and BT2F/BT4R (Chen et al. 2015), respectively. Sequences have been deposited in GenBank (ON908942-ON908943 for ITS, ON908944-ON908945 for LSU, ON929285-ON929286 for TUB2). BLASTn searches of generated DNA sequences from 2 purified isolates (LYB-2 and LYB-3) against GenBank showed high similarity (>99%) with the sequences of Boeremia linicola. Moreover, a phylogenetic tree was constructed based on the neighbor-joining method in MEGA-X (Kumar et al. 2018) and revealed that the 2 isolates were closest to B. linicola (CBS 116.76). Pathogenicity tests were conducted with the 2 isolates (LYB-2 and LYB-3) as described by Cai et al. (2009) with slight modifications. Each isolate was inoculated with three healthy annual P. notoginseng plants, and each leaf was inoculated with three drops of conidia suspension (106 spores/mL). Three P. notoginseng plants inoculated with sterile water were used as controls. All plants were covered with plastic bags incubated in a greenhouse (20℃, 90%RH, 12 h light/dark photoperiod). Fifteen days post-inoculation, all inoculated leaves showed similar lesions, and the symptoms were identical to those in the field. The pathogen was reisolated from symptomatic leaf spots, and the colony characteristics were identical to the original isolates. Control plants remained healthy, and no fungus was re-isolated. Morphological characteristics, sequence alignment and pathogenicity tests confirmed that B. linicola was the cause of P. notoginseng leaf spot disease. This is the first report of B. linicola causing leaf spot on P. notoginseng in Yunnan, China. The identification of B. linicola as the causal agent of the observed leaf spot on P. notoginseng is critical to the prevention and control of this disease in the future.

Effects of different soil moisture on the growth, quality, and root rot disease of organic Panax notoginseng cultivated under pine forests.

The under-forest economy in the agroforestry system can improve land use efficiency, protect ecological environment, and promote arable land sustainable development. However, the effects of soil moisture in the forest and irrigation strategies on the healthy growth of intercropping crops are still incomplete. Here, considering the organic Panax notoginseng cultivated under pine forests (PPF) as the research object, we explored the effects of different soil moisture on the physiological state, yield, quality and disease occurrence of PPF. Our results suggested that 80-85% and 95-100% field capacity (FC) treatments were more conducive to increased photosynthetic rate and biomass accumulation of PPF, but 50-55% and 65-70% FC treatments were more conducive to the accumulation of saponins in PPF leaves. Notably, the root rot index of PPF was highest under 95-100% FC (19.51) treatment, significantly higher than that under 65-70% FC (8.44) and 80-85% FC (10.21) treatments. Further, the rhizosphere microorganisms of PPF under different soil moisture treatments were sequenced, and the sequencing data analysis revealed that high soil moisture (95-100% FC) could destroy the microbial diversity balance and cause the accumulation of pathogens (Fusarium oxysporum and Ilyonectria radicicola), leading to a high incidence of root rot. The incidence of PPF root rot was negatively correlated with rhizosphere microbial diversity. Overall, our results highlight that the quantitative irrigation (80-85% FC) is conducive to maintaining the balance between yield, saponin content and disease occurrence of PPF, providing a practical basis for PPF irrigation strategy and promoting the sustainable development of PPF agroforestry system.

Association of triglyceride levels and prostate cancer: a Mendelian randomization study.

BACKGROUND: The association between triglyceride and prostate cancer (PCa) has been reported in observational studies. However, the causality from triglyceride on PCa remained unknown. METHOD: Two-sample Mendelian randomization (MR) was performed with triglyceride genome-wide association study (GWAS) data from 177,861 individuals and GWAS summary statistics of PCa from 463,010 individuals. Then, 48 single nucleotide polymorphisms (SNPs) of triglyceride were used as instrumental variables (IVs) to conduct MR analysis on PCa. Inverse-variance weighted (IVW), Weighted median, MR-Egger regression, Simple mode and Weighted mode were used for MR analysis. To verify the sensitivity of the data, heterogeneity test, pleiotropy test and leave-one-out sensitivity test were performed. RESULTS: Association for an effect of triglyceride on PCa risk was found in IVW (odds ratio [OR]: 1.002, 95% confidence interval (CI): 1.000-1.004, p = 0.016). However, opposing results were observed using the weighted median (OR: 1.001, 95% CI: 0.999-1.003, p = 0.499) and MR-Egger (OR: 0.999, 95% CI: 0.995-1.002, p = 0.401) approach. After MRPRESSO, the same result was obtained by using IVW method (OR: 1.002, 95% CI: 1.001-1.004, p = 0.004). CONCLUSIONS: The large MR analysis indicated that the potential causal effect of triglyceride on PCa. The odds of PCa would increase with high levels of triglyceride.

First Report of Leaf Spot on Panax notoginseng Caused by Caryophylloseptoria pseudolychnidis in Yunnan, China.

Panax notoginseng is a unique traditional medicinal plant in China, which has the effects of improving myocardial ischemia, protecting liver and preventing cardiovascular diseases (Jiang, 2020). In July 2021, gray-brown round spots were found on the leaves of P. notoginseng in the plantations of Lincang City (23º43´10˝N, 100º7´32˝E). By September, the symptoms were observed on more P. notoginseng plants, with incidence reaching 31%. Initial symptoms on leaves were small, brown spots that expanded, with black granular bulges on the lesions, often surrounded with yellow halo. As the disease progressed, multiple lesions merged, leaves became yellow, and abscission occurred. To isolate the causal pathogen, twelve symptomatic leaves were randomly obtained from twelve P. notoginseng plants. Small pieces of infected leaf tissues (about 5 mm2) were disinfected with 75% ethanol for 30 s, soaked in 2% sodium hypochlorite for 3 min, and then rinsed 3 times with sterile water and blotted dry. Sample tissues were plated on potato dextrose agar (PDA) plates incubated at 25℃ for 5 days with 12 h light/dark photoperiod. Hyphal-tips from the growing edge of colonies were transferred to fresh PDA to obtain pure cultures. Eight isolates were obtained with similar colony morphology, gray (top view) or black (back view) coloration, with a villous surface, and slow-growing on PDA. Conidia were hyaline, slender and obtuse to subobtuse at both ends, 10.3 to 52.62 (av. 25.2) µm × 1.4 to 4.0 (av. 2.4) µm (n=200) in size. Characteristics of the colonies and conidia were consistent with Caryophylloseptoria pseudolychnidis as described by Quaedvlieg et al. (2013) and Verkley et al. (2013). Genomic DNA of three representative isolates (LINC-4 to LINC-6) was extracted, and the rDNA-ITS region, ACT, and LSU gene regions were amplified and sequenced using the primer pairs ITS4/ITS5, 512F/783R, and LSU1Fd/LR5, respectively. Sequences have been deposited in GenBank (OK614104-OK614106 for ITS, OK614109-OK614111 for LSU, OK628350-OK628352 for ACT). BLAST search showed that all sequences were 98% to 100% homology with the corresponding sequences of C. pseudolychnidis. ITS sequences of the three isolates (LINC-4 to LINC-6) showed 99.21% identity (500/504 bp) to C. pseudolychnidis strain CBS 128630 (GenBank accession no. NR156266). LSU sequences of the three isolates showed 99.76% identity (823/825 bp) to C. pseudolychnidis strain CBS 128630 (MH876481). For ACT sequences, LINC-4 and LINC-5 showed 98.53% identity (201/204 bp) to C. pseudolychnidis strain 128614 (KF253599); LINC-6 showed 99.02% identity (202/204 bp) to C. pseudolychnidis strain 128614 (KF253599). Further, the neighbor-joining and maximum-likelihood method were used for multilocus phylogenetic analysis of the obtained sequences using MEGA-X (Kumar et al. 2018). The three isolates were clustered in the same clade with two C. pesudolychidis from database. Three isolates (LINC-4 to LINC-6) were tested for pathogenicity to confirm Koch's postulates. Annual potted P. notoginseng was inoculated with spore suspension (105 spores.mL-1). Each isolate was inoculated onto two leaves each of five P. notoginseng plants. The controls were similarly mock-inoculated with sterile water. To maintain high humidity (>90% RH), all plants were placed in transparent plastic boxes in a greenhouse at 25℃ with a 12 h light/dark photoperiod. Fifteen days post-inoculation, inoculated leaves showed similar symptoms to those observed in the field, and control plants remained healthy. The pathogen were reisolated from symptomatic leaf spots, and the colony characteristics were the same as those of the original isolates. Morphological characteristics, molecular data, and Koch's postulates tests confirmed C. pseudolychnidis as the cause of P. notoginseng leaf spot disease. To our knowledge, this is the first report of C. pseudolychnidis causing leaf spot on P. notoginseng in Yunnan, China. The spread of this disease might pose a serious threat to the production of P. notoginseng. The occurrence and spread of this pathogen should be further studied in order to formulate reasonable control measures.

Multi-Element Analysis and Origin Discrimination of Panax notoginseng Based on Inductively Coupled Plasma Tandem Mass Spectrometry (ICP-MS/MS).

Panax notoginseng is an important functional health product, and has been used worldwide because of a wide range of pharmacological activities, of which the taproot is the main edible or medicinal part. However, the technologies for origin discrimination still need to be further studied. In this study, an ICP-MS/MS method for the accurate determination of 49 elements was established, whereby the instrumental detection limits (LODs) were between 0.0003 and 7.716 mg/kg, whereas the quantification limits (LOQs) were between 0.0011 and 25.7202 mg/kg, recovery of the method was in the range of 85.82% to 104.98%, and the relative standard deviations (RSDs) were lower than 10%. Based on the content of multi-element in P. notoginseng (total of 89 mixed samples), the discriminant models of origins and cultivation models were accurately determined by the neural networks (prediction accuracy was 0.9259 and area under ROC curve was 0.9750) and the support vector machine algorithm (both 1.0000), respectively. The discriminant models established in this study could be used to support transparency and traceability of supply chains of P. notoginseng and thus avoid the fraud of geographic identification.

First report of Meloidogyne arenaria infecting Maidong (Ophiopogon japonicus) in China.

Maidong (Ophiopogon japonicus) is a perennial evergreen plant of the Asparagaceae, occurring mainly in China, Japan, Vietnam, and India. It grows in the damp place on the hillside below 2000 meters above sea level, under the forest or beside the stream;It has been widely cultivated in the Sichuan ofhina for medicinal uses; and it is included in the Chinese Pharmacopoeia. During April 2019, Maidong plants exhibiting symptoms of stunting, leaf wilting, and multiple galls in the roots associated with root-knot nematode (Meloidogyne sp.) were detected in a commercial field in near the city of Mianyang (N105°42', E30°93'), Sichuan, China. The second-stage juveniles (J2) were collected from the soil in the root zone, and adult females were dissected from roots. Population densities of J2 ranged from 190 to 255 per 100 cm3. Subsequently, individual females (n=20) were extracted from root samples and submitted to Meloidogyne species identification by perineal pattern morphological analysis (n=20), and morphometric measurements of second stage juveniles (J2) (n = 20). The J2 showed the following morphometric characters：body length = 475.5 ± 24.2 µm, tail length = 55.2 ± 6.43µm, stylet length = 12.4 ± 1.56 µm and distance from dorsal esophageal gland opening to the stylet knot (DGO) = 2.97 ± 0.44 µm; perineal patterns of females showed a low dorsal arch, with lateral field marked by forked and broken striae, no punctate markings between anus and tail terminus were observed. These morphological characteristics are consistent with Meloidogyne arenaria (Neves et al. 2016). In addition, to confirm species identification, DNA was extracted from females (Blok, et al. 1997) and D2/D3 fragments of the 28S rRNA was amplified using the universal primers D2A/D3B. The DNA fragment obtained showed a 754 bp length (GenBank accession no. MW965614) that was sequenced and analyzed, sequences were 99.8% identical to the MH359158, KX151138 and EU364889 M. arenaria sequences. Furthermore, species-specific SCAR primers Far/Rar were used as described by Zijlstra et al. 2000. The PCR produced approximately 420 bp sequences, which was identical to that previously reported for M. arenaria (Zijlstra et al. 2000). Morphological and molecular characterization supports the identification of the isolate found on Ophiopogon japonicus as M. arenaria. To verify the nematode pathogenicity on Maidong plants, Maidong seed were planted in 20-cm diameter, 10-cm deep plastic pots containing 1000 cm3 sterilized soil and infested with 2000 M. arenaria J2 per seedling, using a sterilized micropipette. Plants were maintained at 20-25°C in a greenhouse. Control plants received sterile water, and the pathogenicity test was repeated three times. After 60 days, all inoculated plants showed reduced growth compared with control. The symptoms were similar to those observed in the field, a large number of galls (38.5 ± 2.4) and egg masses (18.5 ± 0.2) were found on each root system. Maidong was considered a good host for M. arenaria in Mianyang. M. arenaria is one of the most important plant parasitic nematode with a wide geographic distribution and causes great losses in many crops around the world (Perry et al. 2009). Through investigation, this is the first report worldwide of M. arenaria infecting Ophiopogon japonicus.

Pathogen identification of Gentiana macrophylla root-knot nematode disease in Yulong, China.

In September 2020, samples of galled roots with rhizosphere soil were collected from declining Gentiana macrophylla in Yulong County, China. The pathogenic nematodes were identified by observing morphological characteristics of females, second-stage juveniles and perineal pattern, sequence alignments, and specific amplification of sequence characterized amplified region (SCAR). The results showed that the perineal pattern of this nematode was round or oval, the dorsal arch was moderately high or low, one side or both of the lateral field extended to form a wing shape, the tail region had punctations, and the morphological characteristics and morphometric values of second-stage juveniles and females were similar to those of Meloidogyne hapla. The ITS region fragment of this nematode were highly similar to those of M. hapla in NCBI database, with a similarity of over 99.35%. Using the SCAR specific primers, a specific band with an expected size of approximately 440 bp was amplified from this nematode. Morphological and molecular identification supports the nematode species found on Gentiana macrophylla as M. hapla. This is the first report of this regulated root-knot nematode on Gentiana macrophylla in China.

Inhibitory mechanism of 6-Pentyl-2H-pyran-2-one secreted by Trichoderma atroviride T2 against Cylindrocarpon destructans.

Root rot caused by Cylindrocarpon destructans is one of the most devastating diseases of Panax notoginseng, and Trichoderma species are potential agents for the biocontrol of fungal diseases. Thus, we screened a total of 10 Trichoderma isolates against C. destructans and selected Trichoderma atroviride T2 as an antagonistic strain for further research. 6-Pentyl-2H-pyran-2-one (6PP) was identified as an important active metabolite in the fermentation broth of the strain and exhibited antifungal activity against C. destructans. Transcriptome and metabolome analyses showed that 6PP significantly disturbed the metabolic homeostasis of C. destructans, particularly the metabolism of amino acids. By constructing a gene coexpression network, ECHS1 was identified as the hub gene correlated with 6PP stress. 6PP significantly downregulated the expression of ECHS1 at the transcriptional level and combined with the ECHS1 protein. Autophagy occurred in C. destructans cells under 6PP stress. In conclusion, 6PP may induce autophagy in C. destructans by downregulating ECHS1 at the transcriptional level and inhibiting ECHS1 protein activity. 6PP is a potential candidate for the development of new fungicides against C. destructans.

Colonization of endophyte Acremonium sp. D212 in Panax notoginseng and rice mediated by auxin and jasmonic acid.

Endophytic fungi can be beneficial to plant growth. However, the molecular mechanisms underlying colonization of Acremonium spp. remain unclear. In this study, a novel endophytic Acremonium strain was isolated from the buds of Panax notoginseng and named Acremonium sp. D212. The Acremonium sp. D212 could colonize the roots of P. notoginseng, enhance the resistance of P. notoginseng to root rot disease, and promote root growth and saponin biosynthesis in P. notoginseng. Acremonium sp. D212 could secrete indole-3-acetic acid (IAA) and jasmonic acid (JA), and inoculation with the fungus increased the endogenous levels of IAA and JA in P. notoginseng. Colonization of the Acremonium sp. D212 in the roots of the rice line Nipponbare was dependent on the concentration of methyl jasmonate (MeJA) (2-15 µmol/L) and 1-naphthalenacetic acid (NAA) (10-20 µmol/L). Moreover, the roots of the JA signaling-defective coi1-18 mutant were colonized by Acremonium sp. D212 to a lesser degree than those of the wild-type Nipponbare and miR393b-overexpressing lines, and the colonization was rescued by MeJA but not by NAA. It suggests that the cross-talk between JA signaling and the auxin biosynthetic pathway plays a crucial role in the colonization of Acremonium sp. D212 in host plants.

Molecular Mechanisms Underlying Microbial Disease Control in Intercropping.

Many reports indicate that intercropping, which usually consists of growing two species next to each other, reduces the incidence of microbial diseases. Besides mechanisms operating at the field level, like inoculum dilution, there is recent evidence that plant-centered mechanisms with identified plant molecules and pathways are also involved. First, plants may trigger the induction of resistance in neighboring plants by the well-known mechanism of induced resistance. Second, molecules produced by one plant, either above- or belowground, can directly inhibit pathogens or indirectly trigger resistance through the induction of the plant immune system in neighboring plants. Third, competition for resources such as light or nutrients may indirectly modify the expression of the plant immune system. The conceptual frameworks of nonkin/stranger recognition and competition may be useful to further investigate the molecular mechanisms underlying crop protection in interspecific plant mixtures.

Transcriptome analysis of 1- and 3-year-old Panax notoginseng roots and functional characterization of saponin biosynthetic genes DS and CYP716A47-like.

MAIN CONCLUSION: Transcriptome analysis revealed high expression of saponin biosynthetic genes may account for highly accumulated saponins in 3-year-old Panax notoginseng roots and DS and CYP716A47 - like were functionally verified by transgenic tobacco. Panax notoginseng is a well-known traditional medical herb that contains bioactive compounds known as saponins. Three major dammarene-type triterpene saponins including R1, Rb1, and Rg1 were found to be highly accumulated in the roots of 3-year-old plants when compared to those of 1-year-old plants. However, the underlying cellular mechanism is poorly understood. In this study, transcriptome analysis revealed that most genes involved in saponin biosynthesis in P. notoginseng roots augmented during their growth periods. The analysis of the KEGG pathway indicated that the primary metabolism, cell growth, and differentiation were less active in the roots of 3-year-old plant; however, secondary metabolisms were enhanced, thus providing molecular evidence for the harvesting of P. notoginseng roots in the 3rd year of growth. Furthermore, the functional role of DS and CYP716A47-like, two of the candidate genes involved in saponin biosynthesis isolated from P. notoginseng, were verified via overexpression in cultivated tobacco. Approximately, 0.325 µg g-1 of dammarenediol-II and 0.320 µg g-1 of protopanaxadiol were recorded in the dry leaves of transgenic tobacco overexpressed with DS and both DS and CYP716A47-like, respectively. This study provides insights into the molecular mechanisms for saponin accumulation in P. notoginseng roots during its growth period and paves a promising way to produce dammarenediol-II and protopanaxadiol via transgenic techniques.

Benzothiazole inhibits the growth of Phytophthora capsici through inducing apoptosis and suppressing stress responses and metabolic detoxification.

Benzothiazole (BZO) is an antimicrobial secondary metabolite volatilized by many plants and microbes. However, the mechanism of BZO against phytopathogens is still unclear. Here, we found that BZO has antimicrobial activity against the oomycete pathogen Phytophthora capsici. Transcriptome and proteome analyses demonstrated that BZO significantly suppressed the expression of genes and proteins involved in morphology, abiotic stress defense and detoxification, but induced the activity of apoptosis. Annexin V-FITC/PI staining confirmed that the process of apoptosis was significantly induced by BZO at concentration of 150 mg L-1. FITC-phalloidin actin-cytoskeleton staining combined with hyphal cell wall staining and hyphal ultrastructure studies further confirmed that BZO disrupted the cell membrane and hyphal morphology through disrupting the cytoskeleton, eventually inhibiting the growth of hyphae. These data demonstrated that BZO has multiple modes of action and may act as potential leading compound for the development of new oomycete fungicides. These results also showed that the combination of transcriptomic and proteomic approaches was a useful method for exploring the novel antifungal mechanisms of natural compounds.