COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

Cognitive and neural mechanisms of voluntary versus forced language switching in Chinese-English bilinguals: an fMRI study.

The ecological validity of bilingual code-switching has garnered increasing attention in recent years. Contrary to traditional studies that have focused on forced language switching, emerging theories posit that voluntary switching may not incur such a cost. To test these claims and understand differences between forced and voluntary switching, the present study conducted a systematic comparison through both behavioral and neural perspectives. Utilizing fMRI alongside picture-naming tasks, our findings diverge from prior work. Voluntary language switching not only demonstrated switching costs at the behavioral level but also significantly activated brain regions associated with inhibitory control. Direct comparisons of voluntary and forced language switching revealed no significant behavioral differences in switching costs, and both shared several common brain regions that were activated. On the other hand, a nuanced difference between the two types of language switching was revealed by whole-brain analysis: voluntary switching engaged fewer language control regions than forced switching. These findings offer a comprehensive view of the neural and behavioral dynamics involved in bilingual language switching, challenging prior claims that voluntary switching imposes no behavioral or neural costs, and thus providing behavioral and neuroimaging evidence for the involvement of inhibitory control in voluntary language switching.

Developing Biarylhemiboronic Esters for Biaryl Atropisomer Synthesis via Dynamic Kinetic Atroposelective Suzuki-Miyaura Cross-Coupling.

We herein introduce biarylhemiboronic esters as a new type of bridged biaryl reagent for asymmetric synthesis of axially chiral biaryl structures, and the palladium-catalyzed asymmetric Suzuki-Miyaura cross-coupling of biarylhemiboronic esters is developed. This dynamic kinetic atroposelective coupling reaction exhibits high enantioselectivity, good functional group tolerance, and a broad substrate scope. The synthetic application of the current method was demonstrated by transformations of the product and a programmed synthesis of chiral polyarene. Preliminary mechanistic studies suggested that the reaction proceeded via an enantio-determining dynamic kinetic atroposelective transmetalation step.

Diffuse large B-cell lymphoma: the significance of CD8+ tumor-infiltrating lymphocytes exhaustion mediated by TIM3/Galectin-9 pathway.

BACKGROUND: Overexpression of T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is related to the exhaustion of CD8+ tumor-infiltrating lymphocytes (TILs) in diffuse large B-cell lymphoma (DLBCL). However, the mechanism of TIM3-mediated CD8+TILs exhaustion in DLBCL remains poorly understood. Therefore, we aimed to clarify the potential pathway involved in TIM3-mediated CD8+TILs exhaustion and its significance in DLBCL. METHODS: The expression of TIM3 and its correlation with CD8+TILs exhaustion, the key ligand of TIM3, and the potential pathway of TIM3-mediated CD8+TILs exhaustion in DLBCL were analyzed using single-cell RNA sequencing and validated by RNA sequencing. The biological significance of TIM3-related pathway in DLBCL was investigated based on RNA sequencing, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction data. Finally, the possible regulatory mechanism of TIM3-related pathway in DLBCL was explored using single-cell RNA sequencing and RNA sequencing. RESULTS: Our results demonstrated that CD8+TILs, especially the terminally exhausted state, were the major clusters that expressed TIM3 in DLBCL. Galectin-9, mainly expressed in M2 macrophages, is the key ligand of TIM3 and can induce the exhaustion of CD8+TILs through TIM3/Galectin-9 pathway. Meanwhile, high TIM3/Galectin-9 enrichment is related to immunosuppressive tumor microenvironment, severe clinical manifestations, inferior prognosis, and poor response to CHOP-based chemotherapy, and can predict the clinical efficacy of immune checkpoint blockade therapy in DLBCL. Furthermore, the TIM3/Galectin-9 enrichment in DLBCL may be regulated by the IFN-γ signaling pathway. CONCLUSIONS: Our study highlights that TIM3/Galectin-9 pathway plays a crucial role in CD8+TILs exhaustion and the immune escape of DLBCL, which facilitates further functional studies and could provide a theoretical basis for the development of novel immunotherapy in DLBCL.

Based on HPLC and HS-GC-IMS Techniques, the Changes in the Internal Chemical Components of Schisandra chinensis (Turcz.) Baill. Fruit at Different Harvesting Periods Were Analyzed.

Schisandra chinensis, as a traditional Chinese herbal medicine, has clear pharmacological effects such as treating asthma, protecting nerves and blood vessels, and having anti-inflammatory properties. Although the Schisandra chinensis fruit contain multiple active components, the lignans have been widely studied as the primary pharmacologically active compound. The volatile chemical components of Schisandra chinensis include a large amount of terpenes, which have been proven to have broad pharmacological activities. However, when to harvest to ensure the highest accumulation of pharmacologically active components in Schisandra chinensis fruits is a critical issue. The Schisandra chinensis fruit trees in the resource nursery were all planted in 2019 and began bearing fruit in 2021. Their nutritional status and tree vigor remain consistently good. The content of lignans and organic acids in the fruits of Schisandra chinensis over seven different harvest periods was tested, and the results of high-performance liquid chromatography (HPLC) indicated that the lignan content was higher, at 35 mg/g, in late July, and the organic acid content was higher, at 72.34 mg/g, in early September. If lignans and organic acids are to be selected as raw materials for pharmacological development, the harvest can be carried out at this stage. Using HS-GC-IMS technology, a total of 67 volatile flavor substances were detected, and the fingerprint of the volatile flavor substances in the different picking periods was established. It was shown by the results that the content of volatile flavor substances was the highest in early August, and 16 flavor substances were selected by odor activity value (OAV). The variable importance in projection (VIP) values of 16 substances were further screened, and terpinolene was identified as the key volatile flavor substance that caused the aroma characteristics of Schisandra chinensis fruit at different harvesting periods. If the aroma component content of Schisandra chinensis fruit is planned to be used as raw material for development and utilization, then early August, when the aroma component content is higher, should be chosen as the time for harvest. This study provides a theoretical basis for the suitable harvesting time of Schisandra chinensis for different uses, and promotes the high-quality development of the Schisandra chinensis industry.

A Fully Amorphous, Dynamic Cross-Linked Polymer Electrolyte for Lithium-Sulfur Batteries Operating at Subzero-Temperatures.

Solid-state lithium-sulfur batteries have shown prospects as safe, high-energy electrochemical storage technology for powering regional electrified transportation. Owing to limited ion mobility in crystalline polymer electrolytes, the battery is incapable of operating at subzero temperature. Addition of liquid plasticizer into the polymer electrolyte improves the Li-ion conductivity yet sacrifices the mechanical strength and interfacial stability with both electrodes. In this work, we showed that by introducing a spherical hyperbranched solid polymer plasticizer into a Li+ -conductive linear polymer matrix, an integrated dynamic cross-linked polymer network was built to maintain fully amorphous in a wide temperature range down to subzero. A quasi-solid polymer electrolyte with a solid mass content >90 % was prepared from the cross-linked polymer network, and demonstrated fast Li+ conduction at a low temperature, high mechanical strength, and stable interfacial chemistry. As a result, solid-state lithium-sulfur batteries employing the new electrolyte delivered high reversible capacity and long cycle life at 25 °C, 0 °C and -10 °C to serve energy storage at complex environmental conditions.

Anti-inflammatory effects of steroidal alkaloids of Solanum lyratum Thunb.

Solanum lyratum Thunb., a traditional Chinese herbal medicine, has a promising background. However, the anti-inflammatory effects of its component steroid alkaloid have not been explored. In this study, animal and cell experiments were performed to investigate the anti-inflammatory effects and mechanism of action of Solanum lyratum Thunb steroid alkaloid (SLTSA), in order to provide evidence for its potential utilization. SLTSA effectively inhibited ear swelling and acute abdominal inflammation of mice. We observed concentration-dependent inhibition of pro-inflammatory cytokines by SLTSA, as confirmed by the ELISA and RT-qPCR results. Flow cytometry, immunofluorescence and RT-qPCR analyses revealed that SLTSA suppressed TLR4 expression. Western blot results indicated that SLTSA inhibited the activation of the TLR4/MyD88/NF-κB signaling pathway. Our study demonstrated that SLTSA possesses anti-inflammatory properties.

Genome- and transcriptome-wide identification of trehalose-6-phosphate phosphatases (TPP) gene family and their expression patterns under abiotic stress and exogenous trehalose in soybean.

BACKGROUND: Trehalose-6-phosphate phosphatase (TPP) is an essential enzyme catalyzing trehalose synthesis, an important regulatory factor for plant development and stress response in higher plants. However, the TPP gene family in soybean has not been reported. RESULTS: A comprehensive analysis of the TPP gene family identified 18 GmTPPs classified into eight groups based on the phylogenetic relationships and the conservation of protein in six monocot and eudicot plants. The closely linked subfamilies had similar motifs and intron/exon numbers. Segmental duplication was the main driving force of soybean GmTPPs expansion. In addition, analysis of the cis-regulatory elements and promoter regions of GmTPPs revealed that GmTPPs regulated the response to several abiotic stresses. Moreover, RNA-seq and qRT-PCR analysis of the tissue-specific GmTPPs under different abiotic stresses revealed that most GmTPPs were associated with response to different stresses, including cold, drought, saline-alkali, and exogenous trehalose. Notably, exogenous trehalose treatment up-regulated the expression of most TPP genes under saline-alkali conditions while increasing the carbohydrate and trehalose levels and reducing reactive oxygen species (ROS) accumulation in soybean sprouts, especially in the saline-alkali tolerant genotype. Furthermore, the interaction network and miRNA target prediction revealed that GmTPPs interacted with abiotic stress response-related transcription factors. CONCLUSIONS: The findings in this study lay a foundation for further functional studies on TPP-based breeding to improve soybean development and stress tolerance.

Cumulus provides cloud-based data analysis for large-scale single-cell and single-nucleus RNA-seq.

Massively parallel single-cell and single-nucleus RNA sequencing has opened the way to systematic tissue atlases in health and disease, but as the scale of data generation is growing, so is the need for computational pipelines for scaled analysis. Here we developed Cumulus-a cloud-based framework for analyzing large-scale single-cell and single-nucleus RNA sequencing datasets. Cumulus combines the power of cloud computing with improvements in algorithm and implementation to achieve high scalability, low cost, user-friendliness and integrated support for a comprehensive set of features. We benchmark Cumulus on the Human Cell Atlas Census of Immune Cells dataset of bone marrow cells and show that it substantially improves efficiency over conventional frameworks, while maintaining or improving the quality of results, enabling large-scale studies.

Automatic synchrotron tomographic alignment schemes based on genetic algorithms and human-in-the-loop software.

Tomography imaging methods at synchrotron light sources keep evolving, pushing multi-modal characterization capabilities at high spatial and temporal resolutions. To achieve this goal, small probe size and multi-dimensional scanning schemes are utilized more often in the beamlines, leading to rising complexities and challenges in the experimental setup process. To avoid spending a significant amount of human effort and beam time on aligning the X-ray probe, sample and detector for data acquisition, most attention has been drawn to realigning the systems at the data processing stages. However, post-processing cannot correct everything, and is not time efficient. Here we present automatic alignment schemes of the rotational axis and sample pre- and during the data acquisition process using a software approach which combines the advantages of genetic algorithms and human intelligence. Our approach shows excellent sub-pixel alignment efficiency for both tasks in a short time, and therefore holds great potential for application in the data acquisition systems of future scanning tomography experiments.

The Relationship between the Serum NLRP1 Level and Coronary Lesions in Patients with Coronary Artery Disease.

Background: The pathogenesis of coronary artery disease is complex, and inflammation is one of the regulatory factors. The nucleotide-binding oligomerization domain (NOD)-like receptor protein 1 (NLRP1) plays an important role in the cellular inflammatory response, cell apoptosis, cell death, and autoimmune diseases. Whether the level of NLRP1 is related to the severity of coronary artery stenosis in patients with coronary artery disease (CAD) has not been reported. Objective: To test the serum level of NLRP1 in unstable angina (UA) patients and investigate the effect of NLRP1 on coronary stenosis severity of the coronary artery disease (CAD). Methods: 307 patients hospitalized in the Department of Cardiology of the Affiliated Hospital of Xuzhou Medical University for coronary angiography from January 1, 2021, to December 31, 2022 were included. We detect the level of NLRP1 in the serum of the included patients. Patients were divided into UA group and control group according to coronary angiography results and other clinical data. We use logistic regression to screen the influencing factors of UA. Then, subgroups were divided according to the Gensini score and the number of coronary artery lesions, and the difference of serum NLRP1 level between the groups was compared. Spearman correlation analysis was used to explore the correlation between the serum NLRP1 level and Gensini score. We analyze the diagnostic value of NLRP1 for UA by drawing ROC curve. Results: The median level of serum NLRP1 in patients with UA (n = 257) was 49.71 pg/ml, IQR 30.15, 80.21, and that in patients without UA (n = 50) was 24.75 pg/ml, IQR 13.49, 41.95. Serum NLRP1 levels were significantly different among different subgroups. The patient's Gensini score was correlated with the patient's serum NLRP1 level. Conclusion: The serum NLRP1 level is increased in patients with UA, which is increased with the increasing severity of coronary lesions.

Synaptotagmin-7 is a key factor for bipolar-like behavioral abnormalities in mice.

The pathogenesis of bipolar disorder (BD) has remained enigmatic, largely because genetic animal models based on identified susceptible genes have often failed to show core symptoms of spontaneous mood cycling. However, pedigree and induced pluripotent stem cell (iPSC)-based analyses have implicated that dysfunction in some key signaling cascades might be crucial for the disease pathogenesis in a subpopulation of BD patients. We hypothesized that the behavioral abnormalities of patients and the comorbid metabolic abnormalities might share some identical molecular mechanism. Hence, we investigated the expression of insulin/synapse dually functioning genes in neurons derived from the iPSCs of BD patients and the behavioral phenotype of mice with these genes silenced in the hippocampus. By these means, we identified synaptotagmin-7 (Syt7) as a candidate risk factor for behavioral abnormalities. We then investigated Syt7 knockout (KO) mice and observed nocturnal manic-like and diurnal depressive-like behavioral fluctuations in a majority of these animals, analogous to the mood cycling symptoms of BD. We treated the Syt7 KO mice with clinical BD drugs including olanzapine and lithium, and found that the drug treatments could efficiently regulate the behavioral abnormalities of the Syt7 KO mice. To further verify whether Syt7 deficits existed in BD patients, we investigated the plasma samples of 20 BD patients and found that the Syt7 mRNA level was significantly attenuated in the patient plasma compared to the healthy controls. We therefore concluded that Syt7 is likely a key factor for the bipolar-like behavioral abnormalities.

Five glycosylation-related gene signatures predict the prognostic risks of lung adenocarcinoma.

This study aimed to identify glycosylation-related genes associated with lung adenocarcinoma (LUAD) prognosis through comprehensive bioinformatic analysis. Glycosylation-related genes were identified from the Human Gene Nomenclature Committee, and LUAD prognostic genes were screened from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO)-GSE68465 datasets. Glycosylation risk score (GLRS) was calculated to predict LUAD prognostic risk. Samples were grouped into GLRS-high and GLRS-low and compared. The Tumor Immune Dysfunction and Exclusion (TIDE) score was computed to assess the antitumor immune escape possibility after immunotherapy. From 213 glycosylation-related genes, five gene signatures served as prognostic LUAD predictors using univariate and stepwise Cox regression analyses. GLRS-based models were constructed using TCGA and GSE68465 samples; their sensitivity and specificity in predicting LUAD prognosis were confirmed. GLRS was an independent LUAD prognostic factor and contributed to the nomogram to predict patient survival. High GLRS was associated with advanced tumor stage and higher mutation frequencies, estimate scores, and TIDE scores. GLRS-high and GLRS-low patients differed in immune cell infiltration and epithelial-mesenchymal transition (EMT)-related gene expression. Thus, we propose five glycosylation-related gene signatures to predict overall survival and prognostic risks of LUAD. Their regulatory roles may be related to immune invasion, immunotherapy response, mutation, and EMT.

Analysis of Volatile Components in Dried Fruits and Branch Exudates of Schisandra chinensis with Different Fruit Colors Using GC-IMS Technology.

To investigate the volatile components of Schisandra chinensis (Turcz.) Bail (commonly known as northern Schisandra) of different colors and to explore their similarities and differences, to identify the main flavor substances in the volatile components of the branch exudates of northern schisandra, and finally to establish a fingerprint map of the volatile components of the dried fruits and branch exudates of northern Schisandra of different colors, we used GC-IMS technology to analyze the volatile components of the dried fruits and branch exudates of three different colors of northern Schisandra and established a fingerprint spectra. The results showed that a total of 60 different volatile chemical components were identified in the branch exudates and dried fruits of Schisandra. The components of germplasm resources with different fruit colors were significantly different. The ion mobility spectrum and OPLS-DA results showed that white and yellow fruits were more similar compared to red fruits. The volatile components in dried fruits were significantly higher than those in branch exudates. After VIP (variable importance in projection) screening, 41 key volatile substances in dried fruits and 30 key volatile substances in branch exudates were obtained. After screening by odor activity value (OAV), there were 24 volatile components greater than 1 in both dried fruits and branch exudates. The most important contributing volatile substance was 3-methyl-butanal, and the most important contributing volatile substance in white fruit was (E)-2-hexenal.

MicroRNA-590-3p inhibits T helper 17 cells and ameliorates inflammation in lupus mice.

T helper 17 (Th17) cells have a pathogenic effect in many autoimmune diseases. Inhibition of Th17 cells can alleviate the inflammatory damage in autoimmune diseases. Our previous study found that microRNA-590-3p (miR-590-3p) was involved in the differentiation of Th17 cells in systemic lupus erythematosus (SLE). Here, we demonstrated that an increase in Th17 cells was correlated with low expression of miR-590-3p in patients with SLE and in lupus mice. Upregulation of miR-590-3p reduced the differentiation and promoted apoptosis of Th17 cells. Subsequent experiments demonstrated that miR-590-3p promoted apoptosis in Th17 cells by inhibiting autophagy. Autophagy-related 7 (Atg7) was the direct target of miR-590-3p that blocked the autophagy pathway. Finally, treatment of MRL/lpr mice with miR-590-3p agomir ameliorated lupus nephritis and skin lesions. Our work revealed that miR-590-3p inhibited Th17 cells by suppressing autophagy and that increased miR-590-3p expression was able to ameliorate the clinical symptoms of lupus. Therefore, miR-590-3p may be a promising therapeutic target for SLE and other Th17 cell-dependent autoimmune diseases.

Robust Heterostructures in Two-Dimensional Perovskites by Threshold-Dominating Anion Exchange.

Heterostructures play an irreplaceable role in high-performance optoelectronic devices. However, the preparation of robust perovskite heterostructures is challenging due to spontaneous interdiffusion of halogen anions. Herein, a vapor-phase anion exchange method universally suitable for the preparation of robust 2D Ruddlesden-Popper perovskite (RPP) heterostructures is developed. A variety of heterostructures are fabricated based on exfoliated RPP microplates (MPs). Depending on the specific organic cations, the heterostructures can be either sharp and uniform, or broad and gradient, suggesting a new anion diffusion behavior different from that in 3D perovskites. Further experimental studies reveal that the lateral transport of anions follows a threshold-dominating mechanism, while the vertical transport can be partially or completely suppressed by organic cations. Subsequently, quantitative investigation of anion diffusion in 2D perovskites is conducted. The lateral diffusion coefficient of halogen anions is calculated to be 6 to 7 orders of magnitude larger than the vertical coefficient, consistent with the observed highly anisotropic anion diffusion. In addition, it is shown that the anion exchange threshold can also enhance the thermodynamic stability of the heterostructures at elevated temperature. These results provide a general method to fabricate robust lateral RPP heterostructures, and offer important insights into anion behavior in low-dimensional perovskites.

In situ determination of the extreme damage resistance behavior in stomatopod dactyl club.

The structure and mechanical properties of the stomatopod dactyl club have been studied extensively for its extreme impact tolerance, but a systematic in situ investigation on the multiscale mechanical responses under high-speed impact has not been reported. Here the full dynamic deformation and crack evolution process within projectile-impacted dactyl using combined fast 2D X-ray imaging and high-resolution ex situ tomography are revealed. The results show that hydration states can lead to significantly different toughening mechanisms inside dactyl under dynamic loading. A previously unreported 3D interlocking structural design in the impact surface and impact region is reported using nano X-ray tomography. Experimental results and dynamic finite-element modeling suggest this unique structure plays an important role in resisting catastrophic structural damage and hindering crack propagation. This work is a contribution to understanding the key toughening strategies of biological materials and provides valuable information for biomimetic manufacturing of impact-resistant materials in general.

Dually Responsive Poly(N-vinylcaprolactam)-b-poly(dimethylsiloxane)-b-poly(N-vinylcaprolactam) Polymersomes for Controlled Delivery.

Limited tissue selectivity and targeting of anticancer therapeutics in systemic administration can produce harmful side effects in the body. Various polymer nano-vehicles have been developed to encapsulate therapeutics and prevent premature drug release. Dually responsive polymeric vesicles (polymersomes) assembled from temperature-/pH-sensitive block copolymers are particularly interesting for the delivery of encapsulated therapeutics to targeted tumors and inflamed tissues. We have previously demonstrated that temperature-responsive poly(N-vinylcaprolactam) (PVCL)-b-poly(dimethylsiloxane) (PDMS)-b-PVCL polymersomes exhibit high loading efficiency of anticancer therapeutics in physiological conditions. However, the in-vivo toxicity of these polymersomes as biocompatible materials has not yet been explored. Nevertheless, developing an advanced therapeutic nanocarrier must provide the knowledge of possible risks from the material's toxicity to support its future clinical research in humans. Herein, we studied pH-induced degradation of PVCL10-b-PDMS65-b-PVCL10 vesicles in-situ and their dually (pH- and temperature-) responsive release of the anticancer drug, doxorubicin, using NMR, DLS, TEM, and absorbance spectroscopy. The toxic potential of the polymersomes was evaluated in-vivo by intravenous injection (40 mg kg-1 single dose) of PVCL10-PDMS65-PVCL10 vesicles to mice. The sub-acute toxicity study (14 days) included gravimetric, histological, and hematological analyses and provided evidence for good biocompatibility and non-toxicity of the biomaterial. These results show the potential of these vesicles to be used in clinical research.

Assessing the Ratios of Formaldehyde and Glyoxal to NO2 as Indicators of O3-NOx-VOC Sensitivity.

Ozone (O3) pollution has a negative effect on the public health and crop yields. Accurate diagnosis of O3 production sensitivity and targeted reduction of O3 precursors [i.e., nitrogen oxides (NOx) or volatile organic compounds (VOCs)] are effective for mitigating O3 pollution. This study assesses the indicative roles of the surface formaldehyde-to-NO2 ratio (FNR) and glyoxal-to-NO2 ratio (GNR) on surface O3-NOx-VOC sensitivity based on a meta-analysis consisting of multiple field observations and model simulations. Thresholds of the FNR and GNR are determined using the relationship between the relative change of the O3 production rate and the two indicators, which are 0.55 ± 0.16 and 1.0 ± 0.3 for the FNR and 0.009 ± 0.003 and 0.024 ± 0.007 for the GNR. The sensitivity analysis indicated that the surface FNR is likely to be affected by formaldehyde primary sources under certain conditions, whereas the GNR might not be. As glyoxal measurements are becoming increasingly available, using the FNR and GNR together as O3 sensitivity indicators has broad potential applications.

First Report of Brown Rot of fruit on Vitis amurensis Caused by Monilinia polystroma in China.

Vitis amurensis Rupr. (Amur grape) is a wild grape genetic resource widely distributed in Heilongjiang, Jilin, Liaoning, and Inner Mongolia, among other places in China (Song et al. 2009) and the Russian Far East and Korean Peninsula. In September 2018, brown rot symptoms were observed at ripening stage on the fruits of a 5-year-old Amur grapevine germplasm resources nursery of the cultivar 'Beibinghong' and a few Russian resources in Zuojia Town, Jilin City, Jilin Province, China. The diseased fruit surface became brown with soft rot and produced buff to brownish-grey sporodochia with conidia. Around 180 plants of 'Beibinghong' were examined which had 8 % incidence. Forty five samples were collected from symptomatic fruits of 15 randomly sampled 'Beibinghong' grape clusters, cut into 5-mm2 pieces of diseased tissue, surface sterilized with 1% NaOCl for 2 min, rinsed three times with sterile water, dried on sterilized filter paper, and plated on potato dextrose agar (PDA). Thirteen monosporic isolates were obtained using the single-spore isolation method with incubation at 25°C and a 12-h light/12-h dark cycle. The average colony diameter was 46-49 mm after 4 days of culture on PDA. Colonies were white to grayish with even margins. Irregular black stromatal crusts were observed on the reverse side of dishes 10 days after inoculation. Conidial spores were produced when cultured on cherry agar at 25°C under near-ultravolet light. Spores were single-celled and hyaline, limoniform or ellipsoid, and were produced in branched monilioid chains, 12-22 × 8-13 µm (mean: 15.4 ± 1.03 × 9.01 ± 0.72 µm, n = 50). When conidia were cultured on water agar at 25°C for 18 h, the germ tubes were straight, 700-1,000 µm long, and often with two germ tubes per conidium. Morphological characteristics were consistent with those of Monilinia polystroma (van Leeuwen et al. 2002). To confirm the species identification, two DNA regions of the selected isolate 'VAMPWYZSH8' were amplified by polymerase chain reaction (PCR) and sequenced: the internal transcribed spacer region (ITS) was generated using primers ITS1/ ITS4 (Munda 2015) and ß-tubulin (TUB2) was amplified using primers Bt2a/Bt2b (Zhu et al. 2016). A BLAST analysis of the nucleotide sequence of the PCR products revealed 100% identity with two M. polystroma sequences in the NCBI GenBank (KJ814976 for ITS, KR778970 for TUB2). Our sequences were deposited in GenBank with accession nos. MT038413 for ITS and MT038414 for TUB2. On the basis of these results, the isolate was identified as M. polystroma. To confirm pathogenicity, 78 fresh and healthy bunches of 'Beibinghong' grapes at ripening were collected, surface disinfected by immersion in 1% NaOCl for 1 min, rinsed three times with sterile water, then allowed to air dry. Under dry aseptic conditions, the fruits were inoculated using the pin prick method. Each wound was inoculated with 10 µl conidial suspension (106 spore ml-1) and incubated at 25°C with about 90% relative humidity and natural light. Inoculation with water was used as control and the experiment was repeated three times. After a 10-day incubation, typical symptoms of brown rot developed on inoculated fruits, while control fruits were symptomless. The fungus was consistently re-isolated only from diseased fruits and showed the same morphological characteristics as the original isolates, thus fulfilling Koch's postulates. This is the first report of M. polystroma on V. amurensis in China. The resulting disease decreases fruit quality and yield, necessitating the development of effective control measures.