First Report of Didymella uniseptata Causing Leaf Spot on Taraxacum mongolicum in China.

Dandelion (Taraxacum mongolicum), belonging to the Asteraceae family, is one of the main associated species in the alpine meadow, and is famous for its both feeding and medicinal values (Lin et al. 2022; Wang et al. 2022). In September 2021 (vigorous growth period), a newly emerging leaf spot disease of T. mongolicum were observed on natural grassland in Ruoergai County, Aba (Ngawa) Tibetan and Qiang Autonomous Prefect, China (33°59'51'' N, 102°44'57'' E, alt. 3414 m). Leaf disease incidence was ranged from 10% to 15%. The symptoms appeared as brown to dark brown, circular or irregular, sunken spots; eventually, the infected sites of leaves formed a hole in the middle position of lesions. For isolation, 21 tissue pieces (5mm × 5mm) from 7 symptomatic leaf samples of 4 different plants were surface sterilized with 70% ethanol for 30 s and rinsed three times with sterilized distilled water. Then, these tissues were placed on potato dextrose agar (PDA) at 25°C and incubated in the dark for 2 to 7 days. Finally, six pure Didymella strains with consistent colony characteristics were obtained from hyphal tips as described by Xue et al. (2023). Colonies on PDA were brown to black with concentric circles, and abundant black pycnidia were visible; reverse similar in color. Conidia were ellipsoidal, ovoid, hyaline, 0 to 1-septate, 4.07 to 8.67 × 2.74 to 5.35 µm (average 3.60 × 6.55 µm; n = 50). Seven-week-old healthy plants were obtained by growing T. mongolicum seeds in pots (two plants per pot). The six pure Didymella strains were subsequently used to inoculate healthy plants as follows: for each strain, eight pots were spray inoculated with a mycelial suspension of about 4 × 104 CFU/ml, referring to our previous method (Xue et al. 2023). In addition, eight pots considered as non-inoculated controls were sprayed with sterilized distilled water. All pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 23 to 29°C. After incubation for 10 days, the typical symptoms consisted of brown to dark brown, sunken spots, which were similar to those previously observed in nature grassland; however, symptoms were not observed on the non-inoculated plants (controls). The same fungus was reisolated from the lesions and confirmed by the morphological and molecular methods described in this note, thus fulfilling Koch's postulates. To further identify this fungal pathogen, ITS-rDNA, and two other protein-coding genes (rpb2 and tub2) of the representative strain REG28 were amplified with primers described by Chen et al. (2022). Sequences were deposited in GenBank (PP385777 for ITS, PP781948 for rpb2, and PP781947 for tub2). A maximum likelihood (RAxML) phylogenetic tree based on the combined ITS, rpb2, and tub2 alignments showed REG28, and ex-type CGMCC 3.20069 of D. uniseptata (Chen et al. 2022) formed a subclade with 100% bootstrap support (Fig. S1). The causal agent of this disease was confirmed as D. uniseptata by the morphological, molecular, and pathogenic features described above. Recently, D. macrophylla has been reported as the first record on T. officinale in Russia (Gomzhina et al. 2020). To our knowledge, this is the first report of D. uniseptata causing leaf spots on T. mongolicum worldwide. This information will be useful for the diagnosis, detection, pathogen identification and future control of this disease on T. mongolicum in natural grassland.

Identification, Pathogenicity, and Fungicide Sensitivity of Colletotrichum Species Associated with Anthracnose on Italian Ryegrass in Southwestern China.

Italian ryegrass (Lolium multiflorum L.) is widely cultivated as an important forage worldwide because of its high nutritional value and good palatability. Anthracnose caused by Colletotrichum species was a common and new emerging disease of Italian ryegrass. In this study, 88 Colletotrichum isolates were collected from diseased leaves of Italian ryegrass planting regions in Sichuan, Chongqing and Guizhou provinces of southwestern China between 2019 and 2022. By pure culture technique, 15 representative single-spore isolates were obtained for further study. Multi-locus phylogenetic analysis coupled with morphological features showed that these isolates were finally identified as six new record species: C. cereale of the C. graminicola species complex, C. fioriniae and C. nymphaeae of the C. acutatum species complex, C. boninense and C. citricola of the C. boninense species complex, and C. nageiae. Pathogenicity tests indicated that all species could induce anthracnose symptoms; of these, C. cereale was more invasive than other species, followed by C. fioriniae, C. nageiae, C. citricola and C. boninense; C. nymphaeae was weakest pathogenic to Italian ryegrass plants (P ≤ 0.05). Fungicide sensitivity assays showed that iprodione, propineb and oxime·tebuconazole had strong inhibitory effect on the mycelial growth of six Colletotrichum species; in addition, azoxystrobin and fludioxonil also significantly inhibited the mycelial growth of C. nymphaeae and C. fioriniae, respectively. These results provide the basis for the diagnosis and detection in the field, pathogen identification and management of anthracnose on Italian ryegrass.

High Diversity of Epicoccum Associated with Leaf Spot on Italian Ryegrass in Southwestern China: Three New Species and Six New Records.

Italian ryegrass is widely cultivated for the productions of forage, hay and silage, due to its high nutritional value and good palatability. Leaf spots caused by fungi pose a serious threat to forage crops. In order to expand knowledge of fungi causing leaf spot in ryegrass (Lolium multiforum) in Sichuan, Yunnan, Chongqing and Guizhou of southwestern China, a comprehensive survey was undertaken from 2015 to 2022. Survey discovered that Epicoccum leaf spot (ELS) was a common and widespread disease, more serious at the late stage of growth (after late May); symptomatic leaf samples collected from the four different provinces were analyzed, and a total of 202 Epicoccum isolates were obtained; based on both multilocus phylogeny (ITS, LSU, TUB2, and RPB2) and morphology, 10 Epicoccum species were finally identified, including three novel species (E. endololii sp. nov., E. lolii sp. nov. and E. loliicola sp. nov.), six new host records (E. draconis, E. endophyticum, E. oryzae, E. plurivorum, E. thailandicum and E. tobaicum), and an unknown species (Epicoccum sp.1). Pathogenicity tests showed that E. endophyticum, E. endololii and Epicoccum sp.1 were non-pathogenic to Italian ryegrass, which were confirmed as endophytes in this study; other six species could infect Italian ryegrass and cause leaf lesions to different degrees, of which E. draconis was more aggressive (P ≤ 0.05). Coupling with the isolation rates and geographical distributions of these species, E. plurivorum was the predominant pathogen in Yunnan while E. oryzae and E. tobaicum in other three provinces. This work provides an initial understanding of the taxonomies, virulence and distributions of Epicoccum species associated with ELS of southwestern China, and lays a solid foundation for the diagnosis in the field, and scientific control of ELS on Italian ryegrass.

The impact of the gut microbiome on tumor immunotherapy: from mechanism to application strategies.

Immunotherapy is one of the fastest developing areas in the field of oncology. Many immunological treatment strategies for refractory tumors have been approved and marketed. Nevertheless, much clinical and preclinical experimental evidence has shown that the efficacy of immunotherapy in tumor treatment varies markedly among individuals. The commensal microbiome mainly colonizes the intestinal lumen in humans, is affected by a variety of factors and exhibits individual variation. Moreover, the gut is considered the largest immune organ of the body due to its influence on the immune system. In the last few decades, with the development of next-generation sequencing (NGS) techniques and in-depth research, the view that the gut microbiota intervenes in antitumor immunotherapy through the immune system has been gradually confirmed. Here, we review important studies published in recent years focusing on the influences of microbiota on immune system and the progression of malignancy. Furthermore, we discuss the mechanism by which microbiota affect tumor immunotherapy, including immune checkpoint blockade (ICB) and adoptive T-cell therapy (ACT), and strategies for modulating the microbial composition to facilitate the antitumor immune response. Finally, opportunity and some challenges are mentioned to enable a more systematic understanding of tumor treatment in the future and promote basic research and clinical application in related fields.

First Report of Pseudopithomyces palmicola Causing Leaf Spot on Italian Ryegrass in China.

Italian ryegrass (Lolium multiflorum) is widely cultivated for the production of forage, hay and silage worldwide, due to its high nutritional value and palatability (Feng et al. 2021). It has been infected by a number of foliar fungal diseases caused by different fungal pathogens (Xue et al. 2017, 2020; Victoria Arellano et al. 2021; Liu et al. 2023). In August 2021, three Pseudopithomyces isolates with similar colony characteristics were isolated from fresh samples of leaf spots on Italian ryegrass that was collected from the Forage Germplasm Nursery, Maming town of Qujing city, Yunnan province, China (25.53833°N, 103.60278°E). For specific isolation, tissue pieces (about 0.5 cm × 1 cm) from symptomatic leaves were surface-disinfested in 75% ethanol solution for 40s, rinsed three times in sterilized distilled water and air dried, plated on potato dextrose agar (PDA), and incubated at 25°C for 3 to 7 days in the dark. Following initial isolation, a representative isolate, KM42, was selected for further study. Colonies growing on PDA were cottony, white to grey, 53.8 to 56.9 mm diameter after 6 days in the dark at 25°C, with white and regular edge; in reverse, colonies were brown to dark brown in the center, and pale yellow toward the periphery with concentric rings. Under near-UV light at room temperature (20 ± 3°C), colonies were incubated on PDA for ten days to produce conidia. Conidia were globose or ellipsoid to amygdaloid, 1- to 3-transversely septate, 0- to 2-vertical septate, light brown to brown, measuring 11.6 to 24.4 × 7.7 to 16.8 µm (avg. 17.3 × 10.9 µm). The internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA(ITS), large subunit nrRNA (LSU), and partial DNA-directed RNA polymerase II second largest subunit (RPB2) genes were amplified with primers described by Chen et al. (2017). Sequences were deposited in GenBank (OQ875842 for ITS, OQ875844 for LSU, and OQ883943 for RPB2). BLAST analysis of all these three segments showed 100% (ITS: MF804527), 100% (LSU: KU554630), and 99.4% (RPB2: MH249030) identity with sequences of reported isolate CBS 143931 (= UC22) of Pseudopithomyces palmicola (Lorenzini et al. 2016; Liu et al. 2018). To fulfill Koch's postulates, four 12-week-old healthy Italian ryegrass plants were spray-inoculated separately with a mycelial suspension of about 5.4 × 102 CFU/ml of an isolate of P. palmicola. In addition, four control plants were sprayed with sterilized distilled water. All plants were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 18 to 22°C. Small brown to dark brown spots developed on leaves 10 days after inoculation; symptoms were not observed on control plants. Pathogenicity tests were conducted three times by the same method. The same fungus was re-isolated from the lesions, and confirmed by morphological and molecular methods as described above. To the best of our knowledge, this is the first report of P. palmicola causing leaf spot on Italian ryegrass in China or worldwide. This information will be useful to forage grass managers and plant pathologists in recognizing the disease and developing effective control measures.

Metabolomics-based discovery of XHP as a CYP3A4 inhibitor against pancreatic cancer.

Background: Xihuang Wan (XHW), a purgative and detoxifying agent, is commonly utilized in modern medicine as a treatment and adjuvant therapy for various malignancies, including breast cancer, liver cancer, and lung cancer. A clinical study demonstrated the potential usefulness of the combination of XHW and gemcitabine as a therapy for pancreatic cancer (PC), indicating that XHW's broad-spectrum antitumor herbal combination could be beneficial in the treatment of PC. However, the precise therapeutic efficacy of XHW in treating pancreatic cancer remains uncertain. Aim: This study assessed the biological activity of XHW by optimizing the therapeutic concentration of XHW (Xihuang pills, XHP). We performed cell culture and developed an animal test model to determine whether XHP can inhibit pancreatic cancer (PC). We also applied the well-known widely targeted metabolomics analysis and conducted specific experiments to assess the feasibility of our method in PC therapy. Materials and Methods: We used UPLC/Q-TOF-MS to test XHP values to set up therapeutic concentrations for the in vivo test model. SW1990 pancreatic cancer cells were cultured to check the effect the anti-cancer effects of XHP by general in vitro cell analyses including CCK-8, Hoechst 33258, and flow cytometry. To develop the animal model, a solid tumor was subcutaneously formed on a mouse model of PC and assessed by immunohistochemistry and TUNEL apoptosis assay. We also applied the widely targeted metabolomics method following Western blot and RT-PCR to evaluate multiple metabolites to check the therapeutic effect of XHP in our cancer test model. Results: Quantified analysis from UPLC/Q-TOF-MS showed the presence of the following components of XHP: 11-carbonyl-ß-acetyl-boswellic acid (AKBA), 11-carbonyl-ß-boswellic acid (KBA), 4-methylene-2,8,8-trimethyl-2-vinyl-bicyclo [5.2.0]nonane, and (1S-endo)-2-methyl-3-methylene-2-(4-methyl-3-3-pentenyl)-bicyclo [2.2.1heptane]. The results of the cell culture experiments demonstrated that XHP suppressed the growth of SW1990 PC cells by enhancing apoptosis. The results of the animal model tests also indicated the suppression effect of XHP on tumor growth. Furthermore, the result of the widely targeted metabolomics analysis showed that the steroid hormone biosynthesis metabolic pathway was a critical factor in the anti-PC effect of XHP in the animal model. Moreover, Western blot and RT-PCR analyses revealed XHP downregulated CYP3A4 expression as an applicable targeted therapeutic approach. Conclusion: The results of this study demonstrated the potential of XHP in therapeutic applications in PC. Moreover, the widely targeted metabolomics method revealed CYP3A4 is a potential therapeutic target of XHP in PC control. These findings provide a high level of confidence that XHP significantly acts as a CYP3A4 inhibitor in anti-cancer therapeutic applications.

Prediction of Sweet Corn Seed Germination Based on Hyperspectral Image Technology and Multivariate Data Regression.

Vigor identification in sweet corn seeds is important for seed germination, crop yield, and quality. In this study, hyperspectral image (HSI) technology integrated with germination tests was applied for feature association analysis and germination performance prediction of sweet corn seeds. In this study, 89 sweet corn seeds (73 for training and the other 16 for testing) were studied and hyperspectral imaging at the spectral range of 400-1000 nm was applied as a nondestructive and accurate technique to identify seed vigor. The root length and seedling length which represent the seed vigor were measured, and principal component regression (PCR), partial least squares (PLS), and kernel principal component regression (KPCR) were used to establish the regression relationship between the hyperspectral feature of seeds and the germination results. Specifically, the relevant characteristic band associated with seed vigor based on the highest correlation coefficient (HCC) was constructed for optimal wavelength selection. The hyperspectral data features were selected by genetic algorithm (GA), successive projections algorithm (SPA), and HCC. The results indicated that the hyperspectral data features obtained based on the HCC method have better prediction results on the seedling length and root length than SPA and GA. By comparing the regression results of KPCR, PCR, and PLS, it can be concluded that the hyperspectral method can predict the root length with a correlation coefficient of 0.7805. The prediction results of different feature selection and regression algorithms for the seedling length were up to 0.6074. The results indicated that, based on hyperspectral technology, the prediction of seedling root length was better than that of seed length.

An enhanced drought-tolerant method using SA-loaded PAMPS polymer materials applied on tobacco pelleted seeds.

Drought is one of the most important stress factors limiting the seed industry and crop production. Present study was undertaken to create novel drought-resistant pelleted seeds using the combined materials with superabsorbent polymer, poly(2-acrylamide-2-methyl propane sulfonic acid) (PAMPS) hydrogel, and drought resistance agent, salicylic acid (SA). The optimized PAMPS hydrogel was obtained as the molar ratio of 2-acrylamido-2-methyl-propanesulfonic acid (AMPS) to potassium peroxydisulfate (KPS) and N, N'-methylene-bis-acrylamide (MBA) was 1 : 0.00046 : 0.00134. The hydrogel weight after swelling in deionized water for 24 h reached 4306 times its own dry weight. The water retention ratio (RR) of PAMPS was significantly higher as compared with the control. It could keep as high as 85.3% of original weight after 30 min at 110 °C; even at 25 °C for 40 d, the PAMPS still kept RR at 33.67%. PAMPS disintegration ratio increased gradually and reached around 30% after embedding in soil or activated sludge for 60 d. In addition, there were better seed germination performance and seedling growth in the pelleted treatments with SA-loaded PAMPS hydrogel under drought stress than control. It suggested that SA-loaded PAMPS hydrogel, a nontoxic superabsorbent polymer, could be used as an effective drought resistance material applied to tobacco pelleted seeds.