Identifying the temporal contributors and their interactions during dynamic formation of black tea cream.

Tea cream formed in hot and strong tea infusion while cooling deteriorates quality and health benefits of tea. However, the interactions among temporal contributors during dynamic formation of tea cream are still elusive. Here, by deletional recombination experiments and molecular dynamics simulation, it was found that proteins, caffeine (CAF), and phenolics played a dominant role throughout the cream formation, and the contribution of amino acids was highlighted in the early stage. Furthermore, CAF was prominent due to its extensive binding capacity and the filling complex voids property, and caffeine-theaflavins (TFs) complexation may be the core skeleton of the growing particles in black tea infusion. In addition to TFs, the unidentified phenolic oxidation-derived products (PODP) were confirmed to contribute greatly to the cream formation.

Shedding light on cholecystokinin's role in hippocampal neuroplasticity and memory formation.

The hippocampus is a crucial brain region involved in the process of forming and consolidating memories. Memories are consolidated in the brain through synaptic plasticity, and a key mechanism underlying this process is called long-term potentiation (LTP). Recent research has shown that cholecystokinin (CCK) plays a role in facilitating the formation of LTP, as well as learning and memory consolidation. However, the specific mechanisms by which CCK is involved in hippocampal neuroplasticity and memory formation are complicated or poorly understood. This literature review aims to explore the role of LTP in memory formation, particularly in relation to hippocampal memory, and to discuss the implications of CCK and its receptors in the formation of hippocampal memories. Additionally, we will examine the circuitry of CCK in the hippocampus and propose potential CCK-dependent mechanisms of synaptic plasticity that contribute to memory formation.

Design and performance analysis of a new inferior vena cava filter.

This study proposes a novel inferior vena cava filter (IVCF) design, "Lotus," aiming to enhance release stability and endothelialization. A catheter-filter-vessel model was established for IVCF property analysis, validated by comparing numerical simulations and in vitro tests. Lotus's mechanical properties were analyzed, and optimization suggestions are provided. Compared to existing clinical filters, Lotus demonstrates improved release stability and thrombus capture ability. This work suggests Lotus as a potential technical reference for improved IVCF treatment.

Potentiated GABAergic neuronal activities in the basolateral amygdala alleviate stress-induced depressive behaviors.

AIMS: Major depressive disorder is a severe psychiatric disorder that afflicts ~17% of the world population. Neuroimaging investigations of depressed patients have consistently reported the dysfunction of the basolateral amygdala in the pathophysiology of depression. However, how the BLA and related circuits are implicated in the pathogenesis of depression is poorly understood. METHODS: Here, we combined fiber photometry, immediate early gene expression (c-fos), optogenetics, chemogenetics, behavioral analysis, and viral tracing techniques to provide multiple lines of evidence of how the BLA neurons mediate depressive-like behavior. RESULTS: We demonstrated that the aversive stimuli elevated the neuronal activity of the excitatory BLA neurons (BLACAMKII neurons). Optogenetic activation of CAMKII neurons facilitates the induction of depressive-like behavior while inhibition of these neurons alleviates the depressive-like behavior. Next, we found that the chemogenetic inhibition of GABAergic neurons in the BLA (BLAGABA ) increased the firing frequency of CAMKII neurons and mediates the depressive-like phenotypes. Finally, through fiber photometry recording and chemogenetic manipulation, we proved that the activation of BLAGABA neurons inhibits BLACAMKII neuronal activity and alleviates depressive-like behavior in the mice. CONCLUSION: Thus, through evaluating BLAGABA and BLACAMKII neurons by distinct interaction, the BLA regulates depressive-like behavior.

Development of Multifunctional Detection Robot for Roller Coaster Track.

Recent advances in roller coasters accelerate the creation of complex tracks to provide stimulation and excitement for humans. As the main load-bearing component, tracks are prone to damage such as loose connecting bolts, paint peeling, corroded sleeper welds, corroded butt welds, reduced track wall thickness and surface cracks under complex environments and long-term alternating loads. However, inspection of the roller coaster tracks, especially the high-altitude rolling tracks, is a crucial problem that traditional manual detection methods have difficulty solving. In addition, traditional inspection is labor-intensive, time-consuming, and provides only discrete information. Here, a concept of the multifunctional detection robot with a mechanical structure, electrical control system, camera, electromagnetic ultrasonic probes and an array of eddy current probes for detecting large roller coaster tracks is reported. By optimizing the design layout, integrating multiple systems and completing machine testing, the multifunctional roller coaster track detection robot exhibits outstanding performance in track appearance, thickness and crack detection. This study provides great potential for intelligent detection in amusement equipment, railcar, train and so on.

Gut microbiome-derived ammonia modulates stress vulnerability in the host.

Ammonia has been long recognized as a metabolic waste product with well-known neurotoxic effects. However, little is known about the beneficial function of endogenous ammonia. Here, we show that gut ammonia links microbe nitrogen metabolism to host stress vulnerability by maintaining brain glutamine availability in male mice. Chronic stress decreases blood ammonia levels by altering gut urease-positive microbiota. A representative urease-producing strain, Streptococcus thermophilus, can reverse depression-like behaviours induced by gut microbiota that was altered by stress, whereas pharmacological inhibition of gut ammonia production increases stress vulnerability. Notably, abnormally low blood ammonia levels limit the brain's availability of glutamine, a key metabolite produced by astrocytes that is required for presynaptic γ-aminobutyric acid (GABA) replenishment and confers stress vulnerability through cortical GABAergic dysfunction. Of therapeutic interest, ammonium chloride (NH4Cl), a commonly used expectorant in the clinic, can rescue behavioural abnormalities and GABAergic deficits in mouse models of depression. In sum, ammonia produced by the gut microbiome can help buffer stress in the host, providing a gut-brain signalling basis for emotional behaviour.

Cholecystokinin B receptor antagonists for the treatment of depression via blocking long-term potentiation in the basolateral amygdala.

Depression is a common and severe mental disorder. Evidence suggested a substantial causal relationship between stressful life events and the onset of episodes of major depression. However, the stress-induced pathogenesis of depression and the related neural circuitry is poorly understood. Here, we investigated how cholecystokinin (CCK) and CCKBR in the basolateral amygdala (BLA) are implicated in stress-mediated depressive-like behavior. The BLA mediates emotional memories, and long-term potentiation (LTP) is widely considered a trace of memory. We identified that the cholecystokinin knockout (CCK-KO) mice impaired LTP in the BLA, while the application of CCK4 induced LTP after low-frequency stimulation (LFS). The entorhinal cortex (EC) CCK neurons project to the BLA and optogenetic activation of EC CCK afferents to BLA-promoted stress susceptibility through the release of CCK. We demonstrated that EC CCK neurons innervate CCKBR cells in the BLA and CCK-B receptor knockout (CCKBR-KO) mice impaired LTP in the BLA. Moreover, the CCKBR antagonists also blocked high-frequency stimulation (HFS) induced LTP formation in the BLA. Notably, CCKBR antagonists infusion into the BLA displayed an antidepressant-like effect in the chronic social defeat stress model. Together, these results indicate that CCKBR could be a potential target to treat depression.

Structural insight into polyphenol oxidation during black tea fermentation.

Polyphenol oxidation during fermentation plays a critical role in the formation of flavor and function of black tea. However, how the specific structures of tea polyphenols affect their oxidation kinetics during black tea fermentation is still unknown. Here, we found that the oxidations of tea polyphenols, including 7 catechins, 4 phenolic acids and 11 flavonoid glycosides followed pseudo-first-order kinetics during fermentation. Molecular structure and oxygen concentration collaboratively regulated the oxidation rate of different polyphenols. Pyrogallol structure was more easily to be oxidized than catechol and monophenol structure in B-ring, the gallic group in C-ring could inhibit oxidation of catechins, while the role of sugar moiety of flavonoid glycosides was differentiating. In addition, oxygen was found to be the key factor limiting the oxidation rate of polyphenols in regular black tea fermentation, and the oxidation rate constants of tea polyphenols were linearly and positively correlated with oxygen concentration.

Zein nanospheres assisting inorganic and organic drug combination to overcome stent implantation-induced thrombosis and infection.

The complication of stent implantation is the biggest obstacle to the success of its clinical application. In this study, we developed a combination way of 3D printing and the coating technique for preparation of functional polyurethane stents against stent implantation-induced thrombosis and postoperative infection. SEM, XPS, static water contact angle, and XRD demonstrated that the functional polyurethane stent had a 37 µm-thickness membrane composed of zein nanospheres (250-350 nm). Meanwhile, ZnO nanoparticles were encapsulated in zein nanospheres while heparin was adsorbed on the surface, causing 97.1 ± 6.4 % release of heparin in 120 min (first-order kinetic model) and 62.7 ± 5.6 % release of Zn2+ in 9 days (Korsmeyer-Peppas model). The mechanical analysis revealed that the functional polyurethane stents had about 8.61 MPa and 2.5 MPa tensile strength and bending strength, respectively. The in vitro biological analysis showed that the functional polyurethane stents had good EA.hy926 cells compatibility (97.9 ± 3.8 %), anti-coagulation response (comparable plasma protein, platelet adhesion and suppressed clotting) and sustained antibacterial activities by comparison with the bare polyurethane stent. The preliminary evaluation by rabbit ex vivo carotid artery intervention experiment demonstrated that the functional polyurethane stents could maintain blood circulation under the continuous stresses of blood flow. Meanwhile, the detailed data from the simulated implant infection experiment in vivo showed the functional polyurethane stents could effectively reduce microbial infection by 3-6 times lower and improve fibrosis and macrophage infiltration.

Artemisinin exerts a protective effect in the MPTP mouse model of Parkinson's disease by inhibiting microglial activation via the TLR4/Myd88/NF-KB pathway.

AIMS: We performed cell and animal experiments to explore the therapeutic effect of artemisinin on Parkinson's disease (PD) and the TLR4/Myd88 signaling pathway. METHODS: C57 mice were randomly divided into the blank, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced and artemisinin-treated groups. Clinical symptoms, the number of dopaminergic (DAergic) neurons in the substantia nigra, and microglial cell activation were compared among the three groups. Subsequently, BV-2 cell activation and TLR4/Myd88 pathway component expression were compared among the blank, MPP+ -treated, artemisinin-treated, and TLR4 activator-treated groups. RESULTS: Behavioral symptoms were improved, the number of DAergic neurons in the substantia nigra of the midbrain was increased, and microglial cell activation was decreased in artemisinin-treated MPTP-induced PD model mice compared with control-treated MPTP-induced PD model mice (p < 0.05). The cell experiments revealed that artemisinin treatment reduced MPP+ -induced BV-2 cell activation and inhibited the TLR4/Myd88 signaling pathway. Moreover, the effect of artemisinin on the BV-2 cell model was inhibited by the TLR4 activator LPS (p < 0.05). CONCLUSION: Artemisinin may reduce damage to DAergic neurons in a PD mouse model by decreasing microglial activation through the TLR4-mediated MyD88-dependent signaling pathway. However, this finding cannot explain the relationship between microglia and DAergic neurons.

Altered neurotransmission in stress-induced depressive disorders: The underlying role of the amygdala in depression.

Depression is the second leading cause of disability in the world population, for which currently available pharmacological therapies either have poor efficacy or have some adverse effects. Accumulating evidence from clinical and preclinical studies demonstrates that the amygdala is critically implicated in depressive disorders, though the underlying pathogenesis mechanism needs further investigation. In this literature review, we overviewed depression and the key role of Gamma-aminobutyric acid (GABA) and Glutamate neurotransmission in depression. Notably, we discussed a new cholecystokinin-dependent plastic changes mechanism under stress and a possible antidepressant response of cholecystokinin B receptor (CCKBR) antagonist. Moreover, we discussed the fundamental role of the amygdala in depression, to discuss and understand the pathophysiology of depression and the inclusive role of the amygdala in this devastating disorder.

Microglia-dependent excessive synaptic pruning leads to cortical underconnectivity and behavioral abnormality following chronic social defeat stress in mice.

Synapse loss in medial prefrontal cortex (mPFC) has been implicated in stress-related mood disorders, such as depression. However, the exact effect of synapse elimination in the depression and how it is triggered are largely unknown. Through repeated longitudinal imaging of mPFC in the living brain, we found both presynaptic and postsynaptic components were declined, together with the impairment of synapse remodeling and cross-synaptic signal transmission in the mPFC during chronic stress. Meanwhile, chronic stress also induced excessive microglia phagocytosis, leading to engulfment of excitatory synapses. Further investigation revealed that the elevated complement C3 during the stress acted as the tag of synapses to be eliminated by microglia. Besides, chronic stress induced a reduction of the connectivity between the mPFC and neighbor regions. C3 knockout mice displayed significant reduction of synaptic pruning and alleviation of disrupted functional connectivity in mPFC, resulting in more resilience to chronic stress. These results indicate that complement-mediated excessive microglia phagocytosis in adulthood induces synaptic dysfunction and cortical hypo-connectivity, leading to stress-related behavioral abnormality.

Water-responsive 4D printing based on self-assembly of hydrophobic protein "Zein" for the control of degradation rate and drug release.

Four-dimensional (4D) printing is a promising technology that provides solutions for compelling needs in various fields. Most of the reported 4D printed systems are based on the temporal shape transformation of printed subjects. Induction of temporal heterogenicity in functions in addition to shape may extend the scope of 4D printing. Herein, we report a 4D printing approach using plant protein (zein) gel inspired by the amyloid fibrils formation mechanism. The printing of zein gel in a specialized layered-Carbopol supporting bath with different water concentrations in an ethanol-water mixture modulates hydrophobic and hydrogen bonding that causes temporal changes in functions. The part of the construct printed in a supporting bath with higher water content exhibits higher drug loading, faster drug release and degradation than those printed in the supporting bath with lower water content. Tri-segment conduit and butterfly-shaped construct with two asymmetrical wings are printed using this system to evaluate biomedical function as nerve conduit and drug delivery system. 4D printed conduits are also effective as a drug-eluting urethral stent in the porcine model. Overall, this study extends the concept of 4D printing beyond shape transformation and presents an approach of fabricating specialized baths for 4D printing that can also be extended to other materials to obtain 4D printed medical devices with translational potential.

Injectable zein gel with in situ self-assembly as hemostatic material.

Zein is a biocompatible and biodegradable corn protein with promising properties for biomedical applications. It is hydrophobic with the ability to self-assemble in an aqueous medium. It can also form a gel in hydroalcoholic solvents at higher concentrations. Few studies have investigated the biomedical significance of zein gels. Herein, we exploited the injectability and water-responsive increase in stiffness of zein gel to achieve hemostasis by physical blockage of the wound and clot formation. The release of components from the gel further aided blood clotting and gave a higher clot strength than a natural clot, which can prevent rebleeding. Rabbit aortic injury and swine femoral artery injury models were used to evaluate the hemostatic efficacy of the zein gel. Zein gel was effective in both hemostatic models without applying external compression due to an in situ increase in stiffness, while the control (Celox™ Gauze) required external compression at the wound site. The zein gel was easily removed after hemostasis due to hydrophobic self-assembly. Overall, zein gel is proposed as an effective hemostatic product for any wound shape owing to its good shape adaptability and rapid in situ blood-responsive stiffness increase.

Analysis of volatile metabolite variations in strip green tea during processing and effect of rubbing degree using untargeted and targeted metabolomics.

Strip green tea (SGT) is widely distributed in China owing to its unique appearance and aroma but the evolution and formation mechanisms of volatile metabolites (VMs) during SGT processing, and especially in the unique process of rubbing, remain unclear. In this study, based on untargeted metabolomics, 217 VMs (8 categories) were identified, and fixation and rubbing processes were found to be key for SGT aroma formation. Moreover, targeted metabolomics was applied to obtain 38 differential VMs and their related substances, of which fatty acid-derived volatiles (14 VMs) and glycoside-derived volatiles (8 VMs) showed significant contributions to SGT aroma, and their derivation laws during SGT manufacturing were clarified. Furthermore, the effect of rubbing degree on volatile metabolite formation was explored, and 11 key differential VMs were screened by variable importance in projection, and odor activity value analyses. Appropriate rubbing promoted the loss of grassy VMs (such as 1-octanol and 2-pentyl-furan) and enrichment of floral/fruity VMs (such as trans-ß-ionone, nonanal, geraniol, citral, (Z)-3,7-dimethyl-2,6-octadien-1-ol, and (Z)-hexanoic acid, 3-hexenyl ester). Our study not only enriches the chemical theory of green tea processing but also provides technical support for the precision directional processing of high-quality SGT.

Long noncoding RNA Gm2694 drives depressive-like behaviors in male mice by interacting with GRP78 to disrupt endoplasmic reticulum homeostasis.

Long noncoding RNAs (lncRNAs) are involved in various biological processes and implicated in the regulation of neuronal activity, but the potential role of lncRNAs in depression remains largely unknown. Here, we identified that lncRNA Gm2694 was increased in the medial prefrontal cortex (mPFC) of male mice subjected to chronic social defeat stress (CSDS). The down-regulation of Gm2694 in the mPFC alleviated CSDS-induced depressive-like behaviors through enhanced excitatory synaptic transmission. Furthermore, we found that Gm2694 preferentially interacted with the carboxyl-terminal domain of 78-kilodalton glucose-regulated protein (GRP78), which abrogated GRP78 function and disrupted endoplasmic reticulum homeostasis, resulting in a reduction of the surface expression of AMPA receptors (AMPARs). Overexpression of GRP78 in the mPFC promoted the surface expression of AMPARs and attenuated the CSDS-induced depressive-like behaviors of mice. Together, our results unraveled a previously unknown role of Gm2694 in regulating endoplasmic reticulum homeostasis and excitatory synaptic transmission in depression.

First Report of Stem Canker on Dangshen (Codonopsis pilosula) Caused by Binucleate Rhizoctonia AG-K in China.

Dangshen (Codonopsis pilosula) is a well-known medicinal and food homologous plant in China, which is widely used as a tonic agent and has good immunomodulatory effects (Bai et al. 2020; Luan et al. 2021). To retain the best medicinal properties, growers imitated the original ecological planting method for cultivating C. pilosula in hillside fields in Wutai county, Shanxi province, China. In July and August 2021 and 2022, stem canker disease was observed in C. pilosula. The basal part of the stems showed slightly sunken brown lesions, and the disease incidence was up to 20% in the investigated fields (6.67 ha). To identify the causal agents of stem canker, 12 small pieces (approximately 5 mm long) from 12 diseased samples (one piece per sample) were cut from the border of the lesions, surface-sterilized (70% ethanol for 30 s, 0.5% NaClO for 3 min), washed three times with sterile water, and then incubated on water agar (WA) at 25 °C for 24 h. Isolates with right-angle branching, a septum near the branch, and a slight constriction at the branch base were selected, and their hyphal tips were transferred onto potato dextrose agar (PDA) plates. After incubation at 25 °C, 12 Rhizoctonia-like isolates (Dcp-19 to Dcp-30) with white colonies were obtained. White monilioid cells in aerial mycelia formed as they aged but did not produce sclerotia. Based on nuclear fluorescence staining with 1 µg·mL-1 4'-6-diamidino-2-phenylindole as described by Ahvenniemi et al. (2009), there were two nuclei per hyphal cell for all the 12 isolates. Moreover, the sequences of internal transcribed spacer region of ribosomal DNA (rDNA-ITS) of all the 12 isolates were amplified using the primers ITS1/ITS4 (White et al. 1990). For identical sequences, only the rDNA-ITS sequence (674 bp) of Dcp-19 has been deposited in GenBank (accession no. ON004932) and BLASTn analyses showed 100% homology with Rhizoctonia AG-K (MF070696). Maximum likelihood phylogenetic analysis further confirmed the identification. Healthy C. pilosula plants grown for two years in hillside fields were transplanted into sterile soil for pathogenicity testing. And the 12 isolates were all done in this test. Sterilized wheat seeds were placed on a 2-day-old colony of the isolate and incubated for 7 days. One fungus-infested seed was placed at the base of the stem and covered with sterilized soil. Control plants were inoculated with sterilized wheat seeds. Tests were performed on three plants for each isolate. The experiment was repeated twice. All the plants were cultivated at 22 °C and 50% relative humidity. After three weeks, the basal stems of the control plants were still healthy and did not have lesions, but the treated plants exhibited sunken brown canker lesions. The mean disease incidence of all the 12 isolates was 58.33%. The AG-K isolates re-isolated from the lesions of treated plants were confirmed by the morphological and molecular characteristics mentioned above, fulfilling Koch's postulates. To our knowledge, this is the first report of stem canker on C. pilosula caused by binucleate Rhizoctonia AG-K in China.

Analysis of non-volatile and volatile metabolites reveals the influence of second-drying heat transfer methods on green tea quality.

Second-drying is a key process of green tea manufacturing, however, hitherto the effect of second-drying methods on green tea quality has not been assessed. In this study, we compared the effect of three heat transfer drying methods (heat radiation, heat convection, and heat conduction) on green tea quality. Gas chromatography-tandem dual mass spectrometry was used to detect volatile compounds, while absolute quantitative methods were used to detect the non-volatile ones. We identified 45 non-volatile metabolites, 101 volatile metabolites, and 15 objective flavor indicators. Seventeen differential non-volatiles and 8 differential volatiles were screened. Microwave second-drying in heat radiation was the optimal method for green tea flavor, as it can promote the retention of chlorophyll, the degradation of flavonoid glycosides, and the enrichment of amino acids, soluble sugars, nonanal, trans-ß-ionone, linalool, and jasmone. The results provide a theoretical basis and technical guidance for the precise and directional processing of high-quality green tea.

Assessment of Variations in Round Green Tea Volatile Metabolites During Manufacturing and Effect of Second-Drying Temperature via Nontargeted Metabolomic Analysis.

Round green tea (RGT) is widely distributed and presents a high yield in China. The quality of RGT can be determined by its aroma; however, the transformation and formation of volatile metabolites during RGT processing remain unclear. In this study, 173 volatile compounds (nine categories) were identified totally from RGT via gas chromatography-mass spectrometry with infrared-assisted headspace-solid phase microextraction. These substances exhibited different changing trends during various procedures, with the most intense transformation occurring during fixation, followed by pan-frying and second drying; moreover, 51 substances were screened, mainly containing fatty acid-derived volatiles (i.e., (E)-2-hexen-1-ol, Hexanal, pentanal, hexanal) and glycoside-derived volatiles (i.e., linalool, geraniol, benzyl alcohol, benzaldehyde), and their evolution during processing was clarified. Furthermore, the effect of the second-drying temperature on volatile compound metabolism was clarified, and 90°C was the best temperature for RGT aroma. This research lays a foundation for in-depth quality control and the aroma formation mechanism of RGT.

First Report of Alternaria alternata Inciting Leaf Spot of Dangshen (Codonopsis pilosula) in China.

As a commonly traditional Chinese medicine, the perennial herb Dangshen (Codonopsis pilosula) has superior curative effects including regulating immunity, strengthening the spleen, and tonifying lungs (Bai et al. 2020). To imitate natural ecological conditions, plants were grown on hillside fields with stems prostrate on the ground, tangle-up with each other. In August 2020, leaf spots were observed on C. pilosula in Wutai county, Shanxi province, China, and indicated a high disease incidence (70%-80%) in investigated fields (6.67 ha). Small brown necrotic spots, occasionally enclosed by chlorotic halos, were observed on leaves, stems, and sepals. For identification of the pathogen, 15 small pieces (5×5 mm) of symptomatic tissues from 5 randomly-collected diseased plants were surface sterilized, placed on potato dextrose agar plates, and incubated for 4 d in darkness at 25 °C to obtain the colonies. Cultures were purified by single spore isolation from these colonies. A total of 15 isolates named as Dcp-3, and Dcp-5~Dcp-18 were recovered. They produced ovoid or obclavate spores with 15.9-57.5×9.1-20.1 µm in size, 1-6 transverse septa, and 0-4 longitudinal septa. The conidial chains with 4 to 6 spores had numerous secondary and occasionally tertiary chains on potato carrot agar plates. Because all isolates had identical morphological traits, five genes from the representative isolate Dcp-3, actin (ACT), Alternaria major allergen (Alt a1), plasma membrane ATPase (ATP), histone 3 (H3), and rDNA ITS, were amplified with primer pairs ACTDF1/ACTDR1, Alt-for/Alt-rev, ATPDF1/ATPDR1, H3-1a/H3-1b, and ITS1/ITS4, respectively (Hong et al. 2005; Lawrence et al. 2013; Ma et al. 2020). BLASTn searches indicated species of Dcp-3 could not be accurately confirmed by rDNA ITS, ATP, ACT, and Alt a1 (GenBank accession nos. OM334894, OM362504, OM326344, OM362500). Phylogenetic analysis showed it was most closely related to Alternaria alternata, A. arborescens, and A. tenuissima based on concatenated sequences of above four genes. The H3 sequence (OM362508) shared 100% homology with that of A. alternata (MN481948). The phylogenetic tree using H3 also confirmed Dcp-3 as A. alternata. Heathly, two-year-old C. pilosula were transplanted to a greenhouse. A surface-sterilized leaf was sprayed with 50 µL spore suspension (106 conidia/mL) of Dcp-3. A leaf sprayed with isometric sterile water was used as controls. Each treatment used six plants (five leaves per plant). Plants were covered with sterilized plastic bags and incubated at 22 ℃. The test was repeated twice. A week later, control leaves were healthy, but brown necrotic spots similar to field symptoms emerged on treated leaves. The A. alternata isolates were re-isolated from the border of lesions, and confirmed by morphological and molecular characteristics mentioned above, fulfilling Koch's postulates. Leaf spot of C. pilosula caused by Septoria codonopsidis has been reported in China (Wang et al. 2011). However, to our knowledge, this is the first report of A. alternata inciting leaf spot of C. pilosula in China. Our report would promote growers to enhance the field management and consider associated strategies on controlling Alternaria leaf spot of C. pilosula.