Pseudomonas lurida (P. lurida) and its thermostable alkaline proteases can seriously damage raw milk quality.The psychrophilic bacterium P. lurida’s gyrB and aprX genes, and a real-time loop-mediated isothermal amplification  rapid detection method was developed for the early monitoring of P. lurida and its proteases in raw milk. A phylogenetic tree of the gyrB and aprX genes of P. lurida was constructed to analyze the homology of the design sequence of the RealAmp primer. The DNA of 2 strains of P. lurida and 44 strains of non-P. lurida were detected via RealAmp to analyze the specificity of the primer.In this study, specific primers were designed for aprX-positive proteases were produced by P. lurida-positive strains only when Pseudomonas fluorescens was negative. The dissociation temperatures of gyrB and aprX in the RealAmp-amplified products were approximately 85.0°C and 90.0°C, respectively. Moreover, DNA was detected through a 10-fold dilution of P. lurida in a pure bacterial solution and artificially contaminated skimmed milk. The limit of detection of P. lurida DNA copy number in the pure bacterial solution was 8.6 copies/μL and that in the 10% skimmed milk was 5.5 copies/μL. Further, 144 raw milk samples throughout the year from three farms in Hebei province were analyzed using RealAmp. The highest detection rate of P. lurida was 56% in the first and third quarters, and that of proteases was 36% in the second quarter. The detection rates of P. lurida and its proteases were the highest in samples collected from pasture 2 , and the ability of P. lurida to produce proteases reached 88%.It was found that P. lurida and its proteases in raw milk samples, allowing the identification and control of contamination sources in a timely manner to ensure the quality of milk and dairy products.In conclusion, RealAmp established an early and rapid method for the detection of  Pseudomonas lurida and Pseudomonas strains were cultured in a medium of Brain Heart Infusion broth for 18 h at 30°C. The pure cultures of nine non-Pseudomonas strains were incubated in nutrient broth at their optimum temperatures for 18 h.Two strains of 2.2.P. fluorescens-produced thermostable alkaline proteases (Deoxyribonucleic acid (DNA) was extracted using a bacterial DNA extraction kit and boiling water bath. The DNA extraction kit purchased from TIANGEN Biotech C., Ltd.  and was used according to the manufacturer’s protocol. The boiling water bath method was previously used in real-time LAMP identification of roteases .2.3.gyrB gene of P. lurida (GenBank: MT941328.1) were designed using Primerexplorer V5, an online primer-designing software.RealAmp primers for the The outer primer of forward (Q-F3) was 5′-TGGGAACAGACCTACGT-3′ and backward (Q-B3) was 5′-GGAAGGACAGTTCACG-3′. The inner primer of forward (Q-FIP) was 5′-GCGTACCGGTGCCACGGTGTTCCACAGGA-3′ and backward (Q-BIP) was 5′-CATTTCAAGCCATCGGCTGATACGCTTGGCCAGGATGT-3′. The loop primer of forward (Q-LF) was 5′-GTCGCCGACGATTTTCATC-3′ and backward (Q-LB) was 5′-CCTTCAAGAATATCCACT-3′.aprX gene of P. lurida were obtained from the study of The primer sequences of the 2.4.P. lurida RealAmp reaction system based on the gyrB gene was as follows: 2 mM of MgSO4, 0.4 mM of dNTP, 1.2 μM each of Q-FIP and Q-BIP, 0.2 μM each of Q-F3 and Q-B3, 0.4 μM each of Q-LF and Q-LB, 0.2 M of betaine , 2.5 μL of 10× ThermoPol Buffer, 8 U of Bst polymerase , 1/300 dilution of 0.3 μL of 10,000× SYBR green I, 1 μL of DNA template, and added sterilized distilled water up to a final volume of 25 μL.The P. lurida RealAmp reaction system based on the aprX gene was as follows: 2 mM of MgSO4, 0.4 mM of dNTP, 1.4 μM each of L-FIP and L-BIP, 0.28 μM each of L-F3 and L-B3, 0.28 μM each of L-LF and L-LB, 0.2 M of betaine (Sigma-Aldrich), 2.5 μL of 10× ThermoPol Buffer, 8 U of Bst polymerase (New England Biolabs Inc.), 1/300 of dilution of 0.3 μL of 10,000× SYBR green I, 1 μL of DNA template, and added sterilized distilled water up to a final volume of 25 μL.The gyrB and aprX genes, reaction tubes were held at 61°C and 62°C, respectively, for 40 min in the fluorescent quantitative PCR instrument . After the RealAmp reaction was complete, the detection result was determined based on the peak time.Further, 20 μL of mineral oil was used to cover the reaction system to prevent contamination. For the 2.5.gyrB and aprX genes of P. lurida and the sequences with more than 95 and 94% identity with non-P. lurida strains in the GenBank, respectively. By separately aligning the gyrB and aprX sequences of the genus Pseudomonas and their related species from the GenBank using the Clustalx1.81 software, 38 sequences of each were determined. After these sequences were aligned using the DAMBE software, the similar sequences were removed. Finally, 19 unique sequences of gyrB and 30 unique sequences of aprX were retained, which were used to construct their respective phylogenetic trees.Two phylogenetic trees were constructed separately targeting the amplified regions of the 2.6.gyrB and aprX genes of P. lurida, the DNA of 2 P. lurida and 44 related strains was extracted and used as the template for RealAmp amplification. The specificity of the RealAmp primers of P. lurida gyrB and aprX genes was verified by observing the amplification curve and the corresponding peak of dissociation temperatures. The blank control DNA was replaced with sterilized distilled water, and all experiments were repeated five times.To determine the specificity of the RealAmp assay for 2.7.P. lurida was quantified, which was found to be 5.6 × 108 colony-forming units (CFU)/mL. DNA was extracted from 1 ml of Brain Heart Infusion broth with P. lurida cultured after 18 h at 30°C. The concentration of the extracted DNA was approximately 5.75 fg/μL–57.5 ng/μL after a 10× gradient dilution. The content of P. lurida was 5.6 × 101–5.6 × 108 CFU/ml. The RealAmp reaction was performed using P. lurida DNA copy number with a 10× gradient variation of approximately 8.6 × 100–8.6 × 106 copies/μL as the template, and the detection limit of P. lurida in pure bacterial solution via RealAmp method was verified according to the peak time. The formula for calculating copies/μL was as follows:The bacterial suspension of P. lurida in the formula was approximately 6.1 × 106 bp. Under the same conditions, 10 measurements were performed, and their mean and standard deviation (SD) were calculated. The repeatability of the method was evaluated by obtaining the variation coefficient (CV) of the peaking time (Ct) according to the following formula:DNA length of 2.8.P. lurida contamination before adding P. lurida. The bacterial suspension of P. lurida (4 ml) was centrifuged at 13,523× g for 2 min. The collected bacteria were resuspended in sterile water (1 ml) as the original bacterial solution and added to 10% sterile skimmed milk (9 ml), after which plate counting was performed. The content of P. lurida in artificially contaminated 10% skimmed milk was 2.4 × 108 CFU/ml. Further, 1 ml of the artificially contaminated 10% skimmed milk was used to extract the bacterial DNA. After 10× gradient dilution of the extracted DNA, the DNA concentration was approximately found to be 3.70 fg/μL–37.0 ng/μL. The corresponding P. lurida content was 2.4 × 101–2.4 × 108 CFU/ml. The RealAmp reaction was performed using different DNA copy number with a 10× gradient variation of approximately 5.5 × 100–5.5 × 106 copies/μL as the template to determine the limit of detection for P. lurida in artificially contaminated milk samples. Under the same conditions, 10 measurements were performed, and their mean and SD were calculated. The CV of the Ct was calculated using the above-mentioned formula.Sterile skimmed milk (10%) was inoculated on KB plates and cultured at 30°C for 48 h. As no subsequent colony growth was observed, the skimmed milk was confirmed to be free of 2.9.g for 10 min. The upper fat and supernatant were discarded, 50 μL Tris-EDTA buffer solution (TE) was added to the precipitate, and the mixture was shaken and then boiled at 100°C for 10 min. Subsequently, it was resuspended and centrifuged at 13,523× g for 5 min, following which the supernatant (DNA) was collected and stored at −20°C. RealAmp detection was performed using the DNA extracted from 144 raw milk samples. The primers designed for the aprX gene were used to detect proteases  contained 463 cows; the second (east longitude 114°39′57.93″ and north latitude 38°15′57.82″) contained 388 cows; and the third (east longitude 115°43′11.27″ and north latitude 38°14′6.90″) contained 342 cows. Raw milk was collected once a week from the mixed milk samples (about 4–6 tons per pasture) obtained from all cows in each pasture in 1 day. All cows in each pasture were milked three times a day: in the morning, middle of the day, and evening, and the fresh milk from each collection was temporarily stored in a storage tank at 4°C. All samples collected in 1 day were combined into a mixed milk sample, which was then sent to the milk and dairy processing plant. Each sample was divided into three parts, two of which were used for the separate detection of both genes, and one was retained at −80°C for storage. Each raw milk sample (1 ml) was collected into a centrifuge tube (2.0 ml), to which anhydrous ethanol (0.2 ml), ammonia (0.2 ml), and petroleum ether (0.2 ml) were added, mixed, and centrifuged at 13,523× roteases .2.10.SPSS 26.0 was used for statistical analysis. The Kruskal–Wallis test for nonparametric data was used for the statistical analysis of data that did not conform to normal distribution.3.3.1.Pseudomonas targeting the gyrB gene, was constructed using the neighbor-joining method  of P. lurida amplified by RealAmp had high intraspecific homology with P. lurida species and low interspecific homology with other Pseudomonas species.A phylogenetic tree of the genus g method . This trPseudomonas targeting the aprX gene, was constructed using the neighbor-joining method  of P. lurida amplified by RealAmp had high homology with P. lurida and P. fluorescens. The phylogenetic tree showed that P. lurida and P. fluorescens could not be distinguished between themselves, but they could be distinguished from other Pseudomonas species.A phylogenetic tree of the genus g method . This tr3.2.P. lurida strains and 44 related strains were analyzed. Based on the primers designed for the gyrB gene, the fluorescence intensity of 2 P. lurida strains showed an S-type amplification curve with the extension of amplification time, and the results were positive. In contrast, the fluorescence intensity (delta Rn) of non-P. lurida strains and the blank control were fixed at approximately zero; no amplification reaction occurred, and the results were negative  and P. fluorescens  were 90.36°C, 90.18°C, 90.07°C, 90.36°C, 90.18°C, and 90.49°C, respectively. There were no dissociation temperatures for non-P. lurida and blank control samples  in pure culture ; however, there was no significant difference among the other quarters (p > 0.05). Moreover, there was no significant difference in the detection ratio of aprX among the different quarters (p > 0.05). The detection rate of the thermostable alkaline proteases of P. lurida in all quarters was lower than that of P. lurida itself, and the highest ability of P. lurida to produce thermostable alkaline proteases was observed in the second quarter (81%). The highest detection rate of P. lurida and thermostable alkaline proteases appeared in different seasons.The average detection rate of the analyzed ; the detP. lurida-positive in raw milk obtained from three pastures in the Hebei province was analyzed (aprX gene in pasture 2 was significantly higher than that in pastures 1 and 3 (p < 0.05). Moreover, the detection rate of the thermostable alkaline proteases of P. lurida in the 3 pastures was lower than that of P. lurida itself, and the highest ability of P. lurida to produce thermostable alkaline proteases was observed in pasture 2 (88%). The highest detection rate of P. lurida and thermostable alkaline proteases both appeared in the same pasture.In addition, the detection rate of analyzed . The det4.P. lurida, and its potential harm to food is easy to ignore, particularly the damage it causes to raw milk stored at low temperatures. In addition, the development of rapid detection methods for this bacterium is still limited. In the present study, we developed a fluorescent RealAmp method with high sensitivity and specificity to detect P. lurida and its thermostable alkaline proteases. This method enables the early detection and control of contamination to ensure the quality of milk and dairy products and reduce economic losses of enterprises.At present, there are few studies on P. lurida-positive detection ratio was observed in the first and third (56%), followed by the second (44%) and fourth (25%) quarters of the year. These data show that the growth of P. lurida was suitable for nearly 3/4 seasons in the Hebei province. Like P. fluorescens, P. lurida has become the main psychrophilic bacterium affecting the quality of raw milk in Hebei province of China. This observation is consistent with the growth characteristics of P. lurida as a psychrophilic bacterium, growing at a wide temperature range of 4–30°C and having an optimal growth temperature of 28°C  and third (60%) quarters was significantly higher than that of the first (40%) and fourth (44%) quarters, showing a strong correlation with quarters. Additionally, the range of temperatures at which proteases were produced was narrower than the range of the growth temperature of P. lurida. The optimal temperature for protease production was also lower than that of P. lurida growth, and considerably low temperatures were not conducive to the production of P. lurida thermostable alkaline proteases. These results are consistent with previous research , which may be attributed to the pasture environment. Pseudomonas lurida is present in the surrounding environment, such as the water, soil, and cushion materials . This ability is related to the characteristics of the P. lurida strain; some P. lurida strains have the aprX gene, which can produce proteases, while other strains lack this gene and, therefore, do not produce proteases  were analyzed. The detection rate of aterials ; thereforoteases . In a re regions . In our aprX gene, which are the total numbers of thermostable alkaline proteases-producing samples of P. lurida and P. fluorescens , which is equivalent to detecting the DNA of the same sample in one reaction tube (three sets of primers/tube).In a previous study, positive numbers of thermostable alkaline proteases were detected using RealAmp with primers designed for the orescens . However of 100% . Therefo5.P. lurida and its thermostable alkaline proteases. The detection limits in 10% skimmed milk within 40 min were 5.5 copies/μL. This method rapidly detected and analyzed the contamination with P. lurida and its thermostable alkaline proteases in raw milk samples collected from three pastures throughout the year. This rapid method provides a scientific basis for enterprises to identify and control contamination sources in a timely manner to ensure the quality of milk and dairy products.The present study established a rapid RealAmp detection method for The data presented in this study are deposited in the NCBI GenBank repository, accession number MT941328.1.All the authors contributed to the conception and design of the study. LH and YX conceived the study. SZ and LH performed the experiments. SZ and DZ analyzed the data. YZ and SW developed the methodology. SZ and LH wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Key R&D Program of China (grant 2018YFC1604305), the Program of Hebei Provincial Department of Science and Technology (19222805D), the Natural Science Foundation of Hebei Province (C2022106010), the Key Research and Development Projects of Shijiazhuang (211170212A), and the Doctoral Research Startup Fund Project of Shijiazhuang University (grant number 20BS007).Author(s) YX, DZ, YZ, and SW were employed by Junlebao Dairy Group Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dendropanax morbifera (DM), a medicinal plant, is rich in polyphenols and commonly used to treat cancer, inflammation, and thrombosis. However, to date, no study has been conducted on DM regarding the enormous drift of secondary metabolites of plants in different regions of the Republic of Korea and their effects on antiobesity, to explore compounds that play an important role in two major obesity-related pathways. Here, we present an in-depth study on DM samples collected from three regions of the Republic of Korea . We used high-performance liquid chromatography (HPLC) and multivariate component analyses to analyze polyphenol contents , followed by discrimination of the samples in DMJG using single nucleotide polymorphism and chemometric analysis. In silico and in vitro evaluation of major compounds found in the plant extract on two major anti-obesity pathways (adipogenesis and thermogenesis) was carried out. Furthermore, two extraction methods (Soxhlet and ultrasound-assisted extraction) were used to understand which method is better and why. Upon quantifying plant samples in three regions with the polyphenols, DMJG had the highest content of polyphenols. The internal transcribed region (ITS) revealed a specific gel-based band for the authentication of DMJG. PCA and PLS-DA revealed the polyphenol’s discriminative power of the region DMJG. The anti-obesity effects of plant extracts from the three regions were related to their polyphenol contents, with DMJG showing the highest effect followed by DMJ and DMB. Ultrasound-assisted extraction yielded a high number of polyphenols compared to that of the Soxhlet method, which was supported by scanning electron microscopy. The present work encourages studies on plants rich in secondary metabolites to efficiently use them for dietary and therapeutic purposes. DM is golden in color and medium-sized, approximately 15 meters in height, with leaves with a duck-feet composition. DM produces black fruits between September and November and grows in the Republic of Korea. The genus Dendropanax has the maximum species diversity, consisting of 91–95 species distributed across China, the Republic of Korea, Malaysia, Vietnam, Japan, Thailand, Taiwan, Laos, Mexico, Central America, Colombia, Peru, Bolivia, Venezuela, and Brazil (in vitro and in vivo as it is rich in plant secondary metabolites (PSMs). However, little attention has been given to PSMs found in DM and their variability across different regions in the Republic of Korea. PSMs are chemical compounds that are abundantly produced by plant cells through metabolic pathways and do not directly influence the growth and normal function of plants  and modern [ultrasound-assisted extraction (UAE)] methods. Furthermore, solvent ratio, quantity of plant material, time, extraction temperature, and other factors are considered. A prominent method for this purpose is response surface methodology (RSM). RSM maximizes extraction yield in a low number of trials by determining the quadruple effect of a factor or interaction between several factors to acquire a high-precision prediction of an optimum value , and compared the anti-obesity efficacy of DM from three regions using the 3T3-L1 cell lines for the first time.Here we provide detailed information on the distribution of PSMs  in three regions of the Republic of Korea. Firstly, we do this by comparing Soxhlet and UAE extraction methods, which process yields high bioactive components, before debating and discussing the possible mechanisms. Furthermore, we used single nucleotide polymorphism (SNP) and chemotactic analysis for the discrimination of plants and simultaneous identification of CGA, NCGA, CCGA, and rutin. We performed 2.2.1.2.1.1.A total of 10 batches of 12-years-old DM from DMJG (34°45′36.6”N 126°53′55.5″E), DMJ (33°22′34.3”N 126°49′51.3″E), and DMB (34°08′58.9”N 126°32′35.7″E) in the Republic of Korea were obtained in July 2021. The samples were washed with tap water to remove any residue and then dried at 35°C for 20 h in a L’EQUIP dehydrator.2.1.2.Four reference compounds, NCGA, CGA, CCGA, and rutin, were purchased from ChemFaces . HPLC-grade water, acetonitrile, and analytical-grade phosphoric acid were acquired from Honeywell (Republic of Korea).2.1.3.Dried samples were ground for approximately 5 min using a Chuhen grinder  to obtain fine powder. For extraction of PSMs, percentage of ethanol, solvent-to-sample ratio, and time were applied according to RSM software (Design Expert 12). For both Soxhlet and UAE methods, we applied the same conditions that were generated using RSM. We underlined the lower, middle, and upper levels of the three factors. The solvent/plant ratio, time, and extraction temperature were 20, 30, and 40 mL/g; 100, 140, and 180 min; and 40, 60, and 80°C, respectively, which were considered based on the values obtained in preliminary experiments. The total number of experimental runs was 17. Out of the 17 tests, we selected only one condition that resulted in the highest amounts of extracted polyphenols. Detailed information on RSM conditions is provided in 2.1.4.After extraction, the samples were dried and analyzed using scanning electron microscopy . For good conduction, the samples were coated with platinum using Quorum (Q150R S).2.1.5.The HPLC system consisted of the Agilent 1260 infinity Quaternary Pump (G1311B), Agilent 1260 infinity Standard Auto Sampler (G1329B), Agilent 1260 Infinity Column Thermostat Compartment (G1316A), and Agilent 1260 Infinity Variable Wavelength Detector (G1314F) as instrumental system, and ZORBAX Eclipse Plus C18 column   was used as the stationary phase. For simultaneous detection of the four compounds, gradient elution composition was as follows: . Channel A contained 0.4% phosphoric acid in HPLC-grade water, and channel B contained acetonitrile. The wavelength was 327 nm, with a column temperature of 35°C and an injection volume of 5 μL. The four analytical standards and DM plant extract under observation HPLC generated peaks are merged and provided in 2.1.6.For calibration curves, working standard solutions of different concentrations were analyzed by plotting the peak areas of each analyte with respect to its concentration. The limit of detection (LOD) and limit of quantification (LOQ) for each compound were defined as the concentration that produced peaks with signal-to-noise values of 3 and 10, respectively.We developed an intra-day precision method by repetitively analyzing the sample six times within the same day with a gap of 2 h. For inter-day precision, the sample was examined in triplicates for the following 3 days. In the stability test, the sample was stored at −80°C for a day, brought to room temperature, and analyzed at 0, 2, 4, 6, 8, 10, 12, 24, and 48 h. For confirmation of repeatability, the same sample was extracted in sextuplicate and analyzed independently. Precision, stability, and repeatability were evaluated by relative standard deviations (RSD) of the established method.2.2.2.2.1.PetD and trnL-trnF) and one from rDNA (ITS), were analyzed to find SNP or insertion and deletion (INDEL) regions. Upon analyzing the sequences using the web tooltrnL-trnF was considered unsuitable for designing a specific primer, and PetD had no SNPs or INDEL regions. Therefore, ITS was chosen to discriminate the plant samples.Genomic DNA was isolated from freshly collected samples using a Plant DNA Extraction kit  according to the manufacturer’s instructions. The quality and quantity of DNA were determined by measuring the absorbances at 260 and 280 nm using BioTek Synergy HT . For barcoding, three genes, two from chloroplast  with a pair of primers was performed to analyze primer efficiency on gel-based results. PCR was carried out in a 20 μL mixture, which consisted of 10 μL 2× Taq PCR premix (Solgent), 10 ng template DNA, 10 μM each of forward and reverse primers, and 10 μM and 0.6 μM specific primer. The conditions for optimized multiplex PCR involve pre-denaturation for 2 min at 95°C, followed by 36 cycles of 30 s at 95°C for denaturation, and 2 min at 72°C for extension, followed by 6 min at 72°C for final extension. PCR products were analyzed on 1% agarose gel stained with Biofact dye and visualized using ultraviolet light (DNR Bio-Imaging System Mini BIS Pro). For sequencing, the PCR products were purified using a PCR extraction kit  and sequenced at Genotech Corp. .2.3.2.3.1.γ in a complex with rosiglitazone (PDB ID: 7AWC)  and uncoupling protein 1 (UCP1) with the phytochemicals present in DM. The chemical structures of the phytochemicals and control drugs were obtained from the PubChem  databaseD: 7AWC)  was attaD: 7AWC) . The obtrmations , were cormations  to predirmations  to dock rmations  webserve2.4.2.4.1.3T3-L1, a preadipocyte cell line obtained from mouse embryo, was purchased from the American Type Culture Collection. High glucose Dulbecco’s Modified Eagle Medium (DMEM) and bovine calf serum (BCS) were obtained from Welgene . Penicillin–streptomycin was purchased from GeneDEPOT. Cocktails for cell differentiation, human recombinant insulin, 3-isobutyl-1-methylxanthine, and dexamethasone were purchased from Wako .2.4.2.2. For differentiation of preadipocytes, cells were treated with an adipogenesis-inducing medium (MDI) 2 days after post-confluence (day 0). MDI consists of 3-isobutyl-1-methylxanthine, dexamethasone, and insulin. After 72 h (on day 2), cells were stimulated with a maturation medium, which consists of 10 μg/mL insulin with or without the addition of DM extracts. Media was changed every 2 days, followed by incubation for 5–8 additional days. On day 8, fully differentiated adipocytes were viewed using a microscope to observe lipid droplets. Cells cultured in a complete medium only were considered the control.3T3-L1 cells were cultured in DMEM supplemented with 10% BCS and 1% penicillin–streptomycin and maintained in a humidified incubator at 37°C under 5% CO2.4.3.4 cells/well and kept to attach overnight at 37°C under 5% CO2 within an incubator. Cells were treated with DM extracts of different concentrations after discarding old medium, and cells without any treatment were used as the control. After incubation for 24 h, 20 μL 3--2,5 diphenyl tetrazolium bromide was added to each well and incubated for 3 h. Finally, 100 μL dimethyl sulfoxide was added to each well. Absorbance was measured at 570 nm using a microplate reader .Preadipocytes were seeded in a 96-well plate at a density of 1 × 102.4.4.In matured cells lipid droplets were visualized using oil red O to confirm adipogenesis in 3T3-L1 cells following a previously described method . Briefly2.4.5.Total RNA was isolated from mature adipocytes treated with DM extracts using TriZol LS reagent following manufacturer’s instructions . Total RNA (1 μg) was used to synthesize cDNA using a cDNA synthesis kit  following manufacturer’s instructions. The conditions for cDNA synthesis were 42°C for 1 h followed by 72°C for 5 min. cDNA was used to amplify the targeted genes.PPARγ, C/EBPα, and perilipin were amplified using the following conditions: 94°C for 30 s for denaturation; annealing at 58°C; and extension at 72°C for 5 min. PCR amplicons were visualized with 1% agarose gel.RT-PCR was performed using gene-specific primers listed in 2.5.2.5.1.To measure reactive oxygen species (ROS) levels, cells were washed with PBS at room temperature and exposed to 10 μM 2′,7′-dichlorofluorescein diacetate , a common cell-permeable fluorogenic substance. Cells were then incubated for 30 min in the dark at 37°C. Finally, fluorescence was measured between 485 and 495 nm using a Spectra Fluor multi-well fluorescence reader .2.5.2.Free radical scavenging activity of the plant extracts was assessed using DPPH as previously described by Akter et al. . The rea2.5.3.Reducing potential was evaluated according to Akter et al. . The rea2.5.4.The assay was performed using a commercial kit following manufacturer’s instructions. The sample (10 μL) was mixed with 190 μL ABTS reagent and incubated at room temperature for 6 min. Absorbance was measured at 414 nm using BioTek Synergy HT.2.6.2CO3 was added and incubated in the dark for 1 h. The mixture was poured in a 96-well plate, and absorbance was measured at 715 nm using BioTek Synergy HT. Total phenolic content was assessed from the standard curve using gallic acid as the standard. Results were expressed as μmol gallic acid equivalent per gram dry weight (μmol GAE/g DW).Total phenolic contents of the samples were determined using the Folin–Ciocalteu reagent . The pla2.7.t-test and two-way analysis of variance were used to determine total variations between treated and untreated (control) groups. The difference was considered significant at *p < 0.05, **p < 0.01, and ***p < 0.001.PCA, PLS-DA, and hierarchical clustering analysis (HCA) were carried out using SIMCA v.14.1 software . The extraction conditions used for comparison between UAE and Soxhlet were based on Design Expert 12. All data are expressed as mean ± standard error (SE) of at least three independent experiments. GraphPad Prism  was used for statistical analysis. Student’s 3.3.1.3.1.1.Extraction of PSMs from plants is the first crucial step for obtaining the desired secondary metabolites. For the extraction of polyphenols, two of the most frequent methods used are Soxhlet  and UAE . To maximize the efficiency of extraction, techniques are often compared to search for the best method to achieve high yield from extraction. For instance, Palmieri et al.  comparedWe used RSM only to correlate the values of independent factors, as randomly connecting independent values often leads to a mix-up of the values, thereby overlapping the independent factors. For an unbiased comparison between Soxhlet and UAE techniques, we applied the same values of parameters. The final values selected for subsequent experiments were 20 mL/g, 140 min, and 40°C.3.1.2.Mitragyna speciosa, and Peng et al. . The results are shown in g et al.  isolatedA possible explanation for obtaining high extraction yields may be efficient disruption of plant tissues in UAE, which was supported by the SEM pictures . SEM was3.2.3.2.1.The developed method was used to simultaneously determine four polyphenols  in three regions  of the Republic of Korea . The low3.2.2.HCA is a clustering method that provides information on large amounts of data by organizing them into groups and subgroups depicting a hierarchy. In our experiment, HCA was carried out using Ward’s method as the cluster method. DM samples from the three regions were successfully classified into three groups, i.e., DMJG, DMJ, and DMB, according to the ratio of compounds. This indicates that the samples from different regions are undoubtedly different in terms of their polyphenol contents .PCA and PLS-DA are mathematical approaches that can be useful to biological or chemical data to identify a pattern and classify them. Both methods are frequently used to discriminate and compare herbal medicinal plants . To provR2Y = 0.89 and Q2 = 0.882 . The LOD and LOQ of CCGA were 0.004 and 0.012 mg/mL, respectively, which were lowest. The relative standard deviations for inter-day and intraday precision were <1.937% and 1.636%, respectively. The stability was presented as RSD and was <1.827%. Furthermore, the repeatability of the four compounds was in the range between 0.584%–1.99%. The details of the method validation are provided in The developed method was validated using standard validation methods including linearity, LOD, LOQ, stability, precision, repeatability, and recovery. The acceptable linear correlation was set as  that is has the least evolution rate and one (ITS2) from ribosomal region (rDNA) that is considered one of the best regions for DNA barcoding . In DM, 3.3.2.After analyzing the two genes in cpDNA, we switched to rDNA. The ITS2 region was sequenced, and a stable SNP was detected upon multi-alignment in DMJG, where a nucleotide G was substituted with T. The SNP region is illustrated in To analyze the efficiency and reproducibility of specific primers, DM plant samples were purchased online to perform a blind test. The results revealed the accuracy and reproducibility to be 100%. The test was carried out using eight samples, of which five belonged to the DMJG region, which was confirmed by the specific band of 581 bp and a common gel-based band of 1,400 bp . Herein,3.4.3.4.1.Molecular docking is a widely used method in computer-aided drug design to identify potential drugs for various diseases. DM has been previously reported for its efficacy on various metabolic disorders , 33. In in silico studies, the compounds present in DM showed promising results. Therefore, DM could potentially be used to manage obesity and metabolic disorders by synergizing the activities of PPARγ and UCP1.Rutin, CCGA, NCGA, and CGA had significant binding energy of −8.4, −7.1, −7.2, and −6.8 kcal/mol for UCP1, respectively. Rutin showed the highest binding affinity among the compounds and drugs used in this study . Further3.5.3.5.1.Adipose tissue stores excess energy in the form of triglycerides derived from food. Mouse 3T3-L1 preadipocyte cells were used as a cellular model because they present all adipocytes . We inve3.5.2.in vitro results in hyperplasia (an increased number of adipocytes) and hypertrophy (an increased size of adipocytes)  . Our respocytes) .in vitro. However, when DM extract was added into the MDI medium, triglyceride content significantly reduced. DMJG (100 μg/mL) demonstrated a significant lipid-reducing effect via suppressing lipid accumulation by approximately 45%, and DMB (100 μg/mL) suppressed lipid accumulation by up to 20% . PPARγ, CEPBα, and perilipin are major regulators that are closely related to lipid contents in adipocytes .The ITS sequences of DM can be found in the NCBI, DMJG (OR263193), DMJ (OR263482), and DMB (OR263483). The sequences for in silico analysis. JA helped with the HPLC analysis. JL helped with plant samples. Y-JK, RM, G-YK, MR, and GL edited the manuscript. MA, DY, Y-JK, and S-KJ contributed to the conception and design of the manuscript. All authors contributed to the article and approved the submitted version.MA contributed to the data collection and plant material experiment and wrote the first draft of the manuscript. RA performed experiments on cell work. VB carried out This work was supported by Southwest Coast Hwangchil Cooperative.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To evaluate changes in objective optical quality following intense pulsed light (IPL) treatment combined with meibomian gland (MG) expression (MGX) in patients with MG dysfunction (MGD).This retrospective cross-sectional study included MGD-related dry eye disease (DED) patients who received IPL treatment between March and December 2021 at Kim’s Eye Hospital, Seoul, Republic of Korea. Each patient underwent four sessions of IPL treatment using Lumenis M22  and MGX at three-week intervals.This study included 90 eyes from 45 patients with MGD. The mean age was 52.3 ± 16.1 years , and 53.3% (24/45) of patients were female. Compared with the baseline, all clinical symptoms and signs significantly improved after IPL treatment combined with MGX. All optical quality parameters obtained with an optical quality analysis system  have improved significantly over the baseline (p < 0.001).In patients with MGD, IPL treatment combined with MGX improved the objective optical quality and clinical signs and symptoms of DED.The online version contains supplementary material available at 10.1186/s12886-023-02939-9. Dry eye disease (DED) caused by meibomian gland (MG) dysfunction (MGD) is a common disorder . The preWell-maintained meibum lipids in tear film prevent evaporation and provide a smooth optical surface . In patiIn recent years, intense pulsed light (IPL) treatment has emerged as a useful treatment option for DED with MGD –15. NumeThis study was approved by the Institutional Review Board of Kim’s Eye Hospital, Seoul, Republic of Korea (2021-09-006) and adhered to the tenets of the Declaration of Helsinki. We conducted a retrospective cross-sectional study on MGD-related DED patients who received IPL treatment between July and December 2021 at Kim’s Eye Hospital, Seoul, Republic of Korea. Considering the retrospective nature of the study and the use of de-identified patient data, the requirement for written informed consent was waived by the Institutional Review Board of Kim’s Eye Hospital, Seoul, Republic of Korea. The inclusion criteria were adults aged 20–80 years who met all three of the following criteria: OSDI score ≥ 13 points ; TBUT < The primary endpoint was the change in the objective optical quality, which was analyzed using an optical quality analysis system . The secondary endpoints were as follows.Changes in the best-corrected distance VA (BCVA), OSDI score, lipid layer thickness (LLT), partial blinking rate (PBR), TBUT, CFS score, Schirmer I test value, and MGE. To avoid affecting test results, the tests were conducted in the less invasive to more invasive order. Consequently, BCVA, OSDI, OQAS, LLT, PBR, TBUT, CFS, Schirmer I test, and MGE were tested in this order. All of the above tests were conducted by two ophthalmologists (JK and KK) prior to treatment and three weeks after four IPL treatment sessions were completed.BCVA was assessed according to a standardized protocol following manifest refraction assessment in both eyes. The Early Treatment of Diabetic Retinopathy Study chart was placed 4 m from the patient in a standard light box . BCVA reTo estimate the magnitude of patients’ discomfort, participants were requested to complete the OSDI questionnaire containing 12 questions before and after receiving IPL treatment , 29. OSDOQAS measurements were performed by an ophthalmologist (JK) on both eyes of each patient under low light conditions with a pupil diameter of 4.0 mm, prior to instillation of eye drops, as recommended by the manufacturer . OQAS usThe MTF indicates a quantified visual acuity value, and the MTF limit is the spatial rate at which the MTF declines to zero. MTF is a widely agreed upon and validated parameter for quantifying image quality of intraocular lenses (IOLs) –38. The LLT and PBR measurements were performed using the LipiView® II ocular surface interferometer  that was applied to the lower palpebral conjunctiva. Participants were asked to blink several times for few seconds to ensure adequate mixing of the dye on the cornea. Then, the eye was checked using a slit lamp  with maximum cobalt blue light. The participants were asked to open their eyes wide and look straight, and a single ophthalmologist (KK) measured the time taken for a black spot or line to appear on the cornea. Interval between last blink to the first sign of tear film rupture was recorded as TBUT , 46. WhiThe Schirmer I test was conducted without anesthesia using sterile Schirmer strips. Strips were placed in the center of the lower fornix for 5 min . During MGE was measured from the five central glands of the upper and lower eyelids with compression forceps  . Only onThe induced meibomian secretion (meibum) assessed from 0 to 3 points as follows: 0, clear meibum; 1, cloudy meibum on mild compression; 2, cloudy meibum on moderate compression; and 3, no meibum or toothpaste-like meibum expressed through intense compression , 50. A h2), as described in earlier studies [A single ophthalmologist (JY) managed Lumenis M22  throughout the study. All patients received four IPL treatment sessions at 3-week intervals. Both eyes were treated on the same day, with the right eye being treated before the left eye. The therapeutic process was based on a previously published technique . Topical studies –56. MGX Data were analyzed using the IBM Statistical Package for the Social Sciences (SPSS) version 22 , and statistical significance was set at p < 0.05. Continuous variables are presented as the mean ± standard deviation. Paired analysis was used to compare the pre- and post-treatment data. Normality of the data was assessed using the Kolmogorov–Smirnov test. All paired analyses showed nonparametric distributions and were performed using the Wilcoxon signed-rank test.This retrospective study included 90 eyes from 45 patients with MGD. Each eye underwent four IPL treatment and MGX sessions at 3-week intervals. The mean age was 52.3 ± 16.1 years , and 53.3% (24/45) of the patients were female. The objective optical quality and dry eye parameters improved significantly after the IPL treatment. Table 1 summarizes the clinical signs and symptoms before and after IPL treatment combined with MGX. No systemic or skin-related side effects or ocular complications were noted.All optical quality parameters improved significantly after treatment . The baseline OSI, MTF cutoff, and SR improved from 3.37 ± 3.05, 21.06 ± 12.30, and 0.12 ± 0.06, to 1.45 ± 0.92, 31. 62 ± 9.58, and 0.18 ± 0.04, respectively. Following treatment, BCVA showed a significant improvement from logMAR 0.04 ± 0.07 to logMAR 0.03 ± 0.05 (p = 0.08). Mean and maximum LLT showed significant improvement after treatment. The baseline mean, maximum, and minimum LLT improved from 75.30 ± 25.03, 89.48 ± 18.70, and 61.90 ± 28.73 to 82.73 ± 20.51, 95.00 ± 12.37, and 67.64 ± 25.92. A significant decline in PBR from 0.51 ± 0.42 to 0.39 ± 0.40 was observed after treatment (p = 0.037). The baseline MGE, CFS, Schirmer I test (mm), TBUT (s), and OSDI score improved from 2.06 ± 0.72, 1.18 ± 0.82, 8.41 ± 1.69, 3.18 ± 1.46, and 31.46 ± 6.29 to 0.92 ± 0.64, 0.61 ± 0.55, 9.64 ± 1.72, 6.60 ± 1.96, and 23.26 ± 5.96, respectively. All these parameters improved significantly after treatment .MGD is a common ocular surface disease and is one of the most common diseases encountered in ophthalmology . ObstrucVisual complaints due to DED is often clinically overlooked due to its sometimes diurnal variation , fluctuaThe irregularity of the tear film may cause an uneven corneal surface and, consequently, subject the retina to larger optical aberrations . Tear fiThe strength of our study is that it is the first study to show the improvement in objective optical quality after combined IPL treatment and MGX for MGD. In this study, BCVA and objective optical quality improved significantly from baseline after four sessions of IPL treatment combined with MGX.There are limitations to the interpretation of our findings. First, this study was limited by its uncontrolled design and small number of participants. Second, because this study used a before- and after-treatment observational design, no controls were enrolled. This may have introduced bias into the efficacy findings. It would make the study more credible if the test results were included after each IPL treatment rather than only the baseline and final treatment. As the IPL treatment sessions are relatively long, 3 weeks apart, changes in the indicators after each IPL treatment would have been very informative. Third, in addition to IPL treatment, patients in this study used artificial tears. It could not be inferred from this study how the ocular surface parameters and optical quality would change with artificial tears alone. Fourth, There was no assessment of eyelid margins or MG dropout, which would provide additional information on MG changes. Fifth, the last follow-up was completed three weeks after the completion of all treatment sessions. Thus, the observation period was insufficient to determine the long-term effects of IPL treatment combined with MGX. Because the subjective results have been reported in many other articles, our main purpose was to focus on changing the objective optical quality. To validate the current findings, a prospective case–control study comparing patients with and without IPL treatment  with a larger number of participants and longer follow-up is necessary.In summary, IPL treatment combined with MGX improved the objective optical quality and DED parameters in patients with MGD. Based on our findings, we hope that the indications and extent of IPL use can be expanded.Below is the link to the electronic supplementary material.Supplementary Material 1


Ralstonia solanacearum is a widespread, severe plant disease. Tomato (Solanum lycopersicum), one of the most important vegetable crops worldwide, is particularly susceptible to this disease. Biological control offers numerous advantages, making it a highly favorable approach for managing bacterial wilt. In this study, the results demonstrate that treatment with the biological control strain Bacillus subtilis R31 significantly reduced the incidence of tomato bacterial wilt. In addition, R31 directly inhibits the growth of R. solanacearum, and lipopeptides play an important role in this effect. The results also show that R31 can stably colonize the rhizosphere soil and root tissues of tomato plants for a long time, reduce the R. solanacearum population in the rhizosphere soil, and alter the microbial community that interacts with R. solanacearum. This study provides an important theoretical basis for elucidating the mechanism of B. subtilis as a biological control agent against bacterial wilt and lays the foundation for the optimization and promotion of other agents such as R31.Bacterial wilt disease caused by  Ralstonia solanacearum is a widespread, severe plant disease, affecting more than 200 plant species of more than 53 botanical families. This disease is especially prevalent in tropical, subtropical, and warm-temperature regions, where it significantly reduces the yields of various crops such as tomato (Solanum lycopersicum), pepper (Capsicum annuum), potato (Solanum tuberosum), peanut (Arachis hypogaea), and banana . The seedlings were watered with 10-fold diluted Hoagland nutrient solution (Qingdao Haibo Biotechnology). When the seedlings had developed four true leaves, they were transplanted into planting bags (16 × 14 cm) filled with a mixture of yellow clay soil and vegetable planting soil (3:1 weight ratio). The plants were fertilized once a week with 10-fold diluted Hoagland nutrient solution. The strain inoculation experiment was conducted when the plants had reached the six-leaf stage.The tomato plant variety used in this study was R. solanacearum, the disease severity index of the plants was observed at 7 days post inoculation, with scores of 0 , 1 (0%–25% wilting leaves), 2 (25%–50% wilting leaves), 3 (50%–75% wilting leaves), and 4 (75%–100% wilting leaves). The disease incidence, disease severity, and biocontrol efficiency were calculated based on the disease severity index. The data obtained from the calculations were analyzed using one-way analysis of variance (ANOVA) and multiple comparisons (Duncan’s test) using SPSS 21.0.Following inoculation with Bacillus subtilis strains R31 and 168  agar plate  using the root immersion method (7 colony-forming units (CFUs)/mL R. solanacearum was inoculated at the wound site. An equal volume of sterile water was used as the control treatment.For inoculation of the R31 strain, each root of a tomato seedling with six leaves was inoculated with 50 mL of n method . For inon method . One sid8 CFU/mL R. solanacearum was mixed with 20 mL of cooled CPG agar medium that had not yet solidified. After incubating the plates at 30°C for 0, 12, or 14 h, sterile filter paper disks were placed on the medium and 10 μL of 1 × 108 CFU/mL B. subtilis R31 fermentation broth was dropped onto the disks. The plates were incubated at 30°C for 3 days.For the strain confrontation experiment using agar medium mixed with bacteria, 0.5 mL fermentation broth containing 1 × 108 CFU/mL R. solanacearum was spread evenly on the surface of CPG agar medium. A 10-μL aliquot of 1 × 108 CFU/mL B. subtilis R31 fermentation broth was dropped onto the plate. Alternatively, 10 μL of 1 × 108 CFU/mL R31 bacterial suspension that was prepared by washing the bacterial cells with sterile water twice and resuspending in sterile water was dropped onto the plate. The plates were incubated at 30°C for 3 to 4 days.For the strain confrontation experiment using agar medium spread with bacteria, 100 μL fermentation broth containing 1 × 10B. subtilis R31 were predicted using the sequenced whole-genome data of strain R31 (B. subtilis R31 using the acid precipitation method (The gene clusters responsible for the biosynthesis of the secondary metabolites of rain R31  and antirain R31 . Crude ln method . The ferm/z.Liquid chromatography–mass spectrometry (LC-MS) was used to identify crude lipopeptides. The high-performance liquid chromatography conditions were as follows: flow rate of 1.0 mL/min and detection wavelength of 200–400 nm. The MS conditions were as follows: an electrospray ionization source was used, the capillary voltage was 4.5 KV, desolvation gas flow rate was 5.0 L/min, atomizer pressure was 0.8 Bar, and scanning range was 100–2,000 R. solanacearum, 100 μL of 106 CFU/mL R. solanacearum suspension was evenly spread onto CPG agar medium. Sterile Oxford cups were placed on the plate, and 100 μL crude lipopeptide extract was added to each cup. An equal volume of PBS was added as the control group. The culture was incubated at 30°C for 4 days.When using agar culture medium to detect the antibacterial effect of crude lipopeptide extracts on R. solanacearum biofilm, 1,980 μL of liquid CPG medium was added to a 24-well culture plate and mixed with 10 μL of 108 CFU/mL R. solanacearum suspension. The experimental group was combined with 10 μL crude lipopeptide extract, and the control group was combined with phosphate buffer. After 48 and 72 h of culture, the amount of R. solanacearum biofilm formation was quantitatively measured by crystal violet staining. Briefly, the bacterial suspension was removed , 1% (w/v) crystal violet was added through the hole, and the sample was allowed to be stained for 20 min. Each hole was washed with deionized water until the hole was colorless, and the crystal violet fixed in the biofilm was eluted using 33% (v/v) glacial acetic acid. Finally, the absorbance at 570 nm of each eluant was measured.To detect the effect of crude lipopeptide extracts on To accurately isolate and detect the quantity of R31 in the samples, strain R31 carrying a rifampicin resistance cassette was used for soil and plant inoculation. Strain cultivation and inoculation were performed as described above. When collecting rhizosphere soil samples, 5 g of tomato roots (keeping the soil close to the root surface) was collected, placed in 200 mL of PBS, and incubated at 30°C with shaking at 180 rpm for 30 min. After removing the roots, the soil suspension was centrifuged at 8,000 rcf for 10 min at 30°C. The supernatant was removed, and 1 g of soil pellet was transferred to 9 mL of sterile water. The sample was vortexed at maximum speed for 10 min. Each sample was diluted six times in a 10-fold concentration gradient. A 100-μL aliquot of each concentration of sample was placed on a plate containing 300 μg/mL rifampicin. The plates were incubated at 37°C for 24 h before counting the colonies.To collect root tissue samples, 1 g of tomato root tissues was collected, incubated in 75% (v/v) ethanol for 30 s followed by 2.5% (w/v) sodium hypochlorite for 60 s for surface sterilization, and washed four times with sterile water. Each sample was placed in a sterilized mortar, and sterile quartz sand was added for grinding. After grinding thoroughly, 9 mL of sterile water was added, and the sample was transferred to a 15-mL centrifuge tube. The sample was vortexed at maximum speed for 1 min and diluted six times in a 10-fold concentration gradient. A 100-μL aliquot of each concentration of sample was placed on a plate containing 300 μg/mL rifampicin. The plates were incubated at 37°C for 24 h before counting. To collect plant stem samples, a 1-cm stem sample was cut from the plant at 3 cm above the ground. The surface of the sample was disinfected by immersion in 75% (v/v) ethanol for 60 s, followed by immersion in 2.5% (w/v) sodium hypochlorite for 90 s. The other steps were the same as described for root sample collection and treatment.RipAK  was used to clone the gene in a plasmid for the production of quantitative standard curves. Using the above method to obtain soil samples, DNA was extracted from 0.3 g soil samples according to the instructions of the HiPure Soil DNA Mini Kit (D3142-03). Following qPCR amplification, the titer of R. solanacearum in the soil was calculated based on the Ct value.To accurately detect the titer of GMI1000 in the samples, a fluorescence quantitative PCR system was used. The GMI1000 gene RipAK  was used2O2 contents in roots, root samples were collected from tomato plants at specific time points and placed in a 2 mL grinding tube. After quickly added 0.1 M PBS , the roots were ground in a homogenizer. The ratio of root tissue to PBS was 1:9 . A 2-mL aliquot of homogenate was centrifuged at 4,000 rcf at 4°C for 10 min. The supernatant was transferred to a clean centrifuge tube and stored at 4°C for later use. The NO and H2O2 contents in the root tissue were determined using an H2O2 detection kit (S0038) and an NO detection kit (S0021S) according to the manufacturer’s instructions .To detect NO and H16S rDNA V3-V4 region  was subjected to PCR amplification. The amplification products were sent to GeneDenovo Biotechnology Co., Ltd. for high-throughput sequencing. The 16S sequences of R. solanacearum strains R31 and GMI1000 were compared to the OTU  representative sequences obtained from microbial group analysis using BLAST. Based on the results, the R31 strain was determined to be OTU000251 in the microbial group, while the R. solanacearum GMI1000 strain was determined to be OTU001444.All rhizosphere samples were collected as described above and used to extract soil DNA. The p < 0.05. Additionally, the VEGAN package (version 2.5-7) in R was used to visualize the differences in taxonomic diversity among samples. Principal coordinate analysis (PCoA) was performed utilizing a Bray-Curtis distance matrix of taxon relative abundances  was employed to cluster effective tags across all samples . Defaultundances . To idenon 3.8.2 .p ≤ 0.05 was used. Duncan’s test, ANOVA, and t-tests were used to determine statistically significant differences between data sets.Unless stated otherwise, each experiment was conducted with a minimum of three biological replicates, and representative results are presented. Group similarity was assessed using Levene’s test and tested for normality using the Shapiro–Wilk test. Statistical analysis was carried out using Microsoft Excel (Office 365) and GraphPad Prism (version 8.02). A significance level of The data supporting the findings of this study are included in the paper and Due to the good results achieved in the prevention and control of banana wilt disease , the useR. solanacearum. We then inoculated the plants with 107 CFU/mL R. solanacearum cell suspension using the root injury method and scored disease incidence, disease severity, and biological efficiency 7 days after infection. Tomato plants showed varying degrees of disease occurrence in different experimental groups, with reduced disease severity observed in plants inoculated with R31  alone, the other treatments had little effect on the richness of the microbial community , pointing to an apparent negative correlation between the relative abundances of these two strains in soil; however, this correlation was not significant. Therefore, we reasoned that R31 might also play a role in controlling bacterial wilt by affecting other bacterial communities that coexist with R. solanacearum. We thus analyzed the potential interacting microbiota with R31 in the absence and presence of R. solanacearum. We selected bacterial genera for each group based on the thresholds “average relative abundance > 10−5” and “detected in ≥50% of samples.” We performed Spearman correlation analysis of the selected bacterial genera based on their relative abundance information. We identified significant correlations based on the thresholds “correlation coefficient R absolute value > 0.8 (extremely strong correlation)” and “p < 0.01.” In soil lacking R. solanacearum, R31 was positively correlated with Sphingobacterium, Brevundimonas, Algoriphagus, and Bacteriovorax and negatively correlated with bacteria such as Dyella and Paenibacillus  with R31 in soil containing R. solanacearum; these interactions are consistent with those observed in soil in the absence of R. solanacearum. R31 positively regulates Acidovorax abundance via Glutamicibacter, and Acidovorax and R. solanacearum are mutually exclusive  can be found in the article/YS: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Visualization, Writing – original draft, Writing – review & editing. YS: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Writing – original draft. ZM: Conceptualization, Data curation, Investigation, Methodology, Software, Writing – original draft. JZ: Formal analysis, Software, Visualization, Writing – review & editing. LZ: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. SM: Data curation, Formal analysis, Software, Writing – original draft. DQ: Data curation, Investigation, Methodology, Writing – review & editing. YR: Formal analysis, Methodology, Software, Writing – original draft. YW: Data curation, Formal analysis, Writing – original draft, Writing – review & editing. LX: Conceptualization, Data curation, Formal analysis, Software, Writing – original draft. XY: Data curation, Formal analysis, Methodology, Writing – review & editing. ZD: Formal analysis, Methodology, Resources, Writing – review & editing. PC: Data curation, Validation, Visualization, Writing – review & editing. MS: Data curation, Formal analysis, Supervision, Writing – review & editing. GY: Resources, Supervision, Visualization, Writing – review & editing.


Atopic dermatitis (AD) is one of the most common, relapsing, chronic inflammatory skin disease, being regarded as a global health issue. Recent studies have shown that Th2 cell-mediated type 2 immunity plays a central role in AD. The type 2 inflammatory cytokines such as IL-4, IL-13, IL-22, IL-31, IL-17 and IL-5 mediate the pathogenesis of AD. A variety of antibody drugs targeting these cytokines have been developed to treat AD in clinics. Notably, several antibody drugs have exhibited high efficacy in treating atopic dermatitis in previous studies, demonstrating that they could be therapeutic methods for AD patients. Herein, we reviewed the clinical trials of antibody drugs in the treatment of AD, which provides a useful guideline for clinicians to treat patients with AD in clinics. Atopic dermatitis (AD), also known as atopic eczema, is a chronic relapsing inflammatory skin disorder , 2. ClinBased on the mechanism of atopic dermatitis, various treatments have been developed for patients at different stages . Currentp < 0.001)]; and pruritus scores decreased by 55.7% in the dupilumab group versus 15.1% in the placebo group (p < 0.001). In addition, Zhao et al. evaluated the efficacy and safety of Dupilumab in 165 adult Chinese patients with moderate-to-sever AD in a phase III study (p < 0.001) respectively (p < 0.0001)  . Noticea 0.0001)  Ocular s 0.0001) , are typ 0.0001) . Others  0.0001) . Moreove 0.0001) . Surpris 0.0001) , was see 0.0001) . Similar 0.0001) , 51. Thup = 0·002) and 22·2% vs. 10·9% in ECZTRA 2  and 20 weeks . Meanwhile, the improvements in body surface area were considerably higher compared to placebo-treated patients , as well as the decline in IGA versus placebo groups    . Brunner 10−19]) . The pre 10−19]) .p < 0.01 for all comparisons)]. Changes on the EASI were − 23.0, −42.3, and − 40.9%, respectively, in the nemolizumab groups, vs. −26.6% in the placebo group, whereas the respective changes in body-surface area affected by atopic dermatitis were − 7.5, −20.0, and − 19.4% with nemolizumab, versus −15.7% with placebo. In 2018, Nemolizumab reached to second phase, Silverberg et al., in 2021, conducted a randomized phase 2B trial in order to compare its efficacy with multiple dimensions (p ≤ 0.001) at week 16, whereas the sleep disturbance was improved significantly. Furthermore, the least square mean EASI score at the end of the trial was reduced by 68.6% in drug-treated group as compared with 42.6% in placebo group (p = 0.002). Later, two long-term phase III studies carried by Kabashima et al. showed clinically meaningful improvements in nemolizumab patients . EASI-75 and EASI-90 also show a similar trend  is highly expressed in human cutaneous epithelial cells in AD patients, however, this overexpression triggers robust itch-evoked scratching, which induces AD skin phenotype . Thereforend see . Furtherpost hoc analysis of Asian patients was also conducted by them as a higher TH17 activation was demonstrated in these patients, however, a similarly no significant changes were found. Adverse events occurred in this trial were orbital cellulitis, upper respiratory infection and streptococcal pharyngitis, both data were the same for secukinumab and placebo group (4% vs. 0%).Interleukin-17 (IL-17) is an essential proinflammatory cytokine, which is mainly secreted by the CD4+ helper T cells and is associated with the pathogenesis of inflammatory diseases, including atopic dermatitis. The IL-17 axis thereby is an important pathway for targeted therapy for AD . SecukinMepolizumab is a humanized immunoglobulin monoclonal antibody that binds to IL-5 . A multiPreclinical studies of skin inflammation and asthma models have supported that OX40-OX40L signaling interactions are pivotal to the efficiency of the responses that are regulated by memory Th2 cells ; Figure . TherefoIn a phase 2a study in 2019, GBR830, which is another humanized mAb against OX40, was investigated. Patients were divided into 2 groups, 62 in ITT and 40 in BAS population, respectively. The results indicated that GBR830 was well tolerated, with a greater proportion of drug-treated patients achieving 50 and 75% reduction in EASI. A higher reduction in SCORAD score in day 71 was also observed in GBR830 group than in placebo group (−45.4 ± 26.9 vs. -31.0 ± 16.9 for ITT and − 44.0 ± 27.6 vs. -31.4 ± 18.2 for BAS). Moreover, the mean change from baseline in pruritus NRS score in week 10 showed the similar pattern, which is −2.7 ± 2.5 vs. -1.5 ± 1.6 and − 3.0 ± 2.6 vs. -1.3 ± 1.6 for each population. Lesional skin, epidermal hyperplasia and proliferation were reduced significantly after treating with GBR830. However, 63% of participants in GBR830 group reported TEAEs, which was numerically identical to that in placebo group, including headache, nasopharyngitis, upper respirator tract infection, fatigue, etc. .Amlitelimab inhibits OX40-OX40L pathway by binding OX40L and blocking interaction with OX40. Weidinger et al. evaluated its tolerability and safety by conducting a phase 2a, randomized, placebo-controlled, double-blind multicentre trial. According to this investigation, while a higher proportion of clinically meaningful improvements in EASI were seen in amlitelimab low dose and high dose groups than placebo group (−80.12% and −69.97% vs. −49.37%), more patients in drug-treated group achieved vIGA 0/1 at week 16 (44% for low dose and 37% for high dose vs. 8%), and this clinical response sustained to week 36 . This work was supported by the National Natural Science Foundation of China (81603119) and the Natural Science Foundation of Beijing Municipality (7174316).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Few studies have reported the differential outcomes of Small Incision Lenticule Extraction (SMILE) on myopic astigmatism. Given this, we examined the effectiveness of SMILE for up to one year, comparing with-the-rule (WTR), against-the-rule (ATR), and oblique astigmatism, conducting a retrospective review of patients who underwent correction of myopic astigmatism using the 500-kHz VisuMax femtosecond laser (Carl Zeiss Meditec) at two refractive clinics in Poland between 2016–2017. Patients were aged ≥21 with stable refractive errors between −0.5 and −10.0 diopter (D) with astigmatism up to 5D. The mean age of the 209 patients (355 eyes) available was 32 years; 58.4% were female. Of these, 247 had WTR, 62 oblique, and 46 ATR astigmatism. The mean pre-operative spherical equivalent (SE) was −5.4 ± 2.57D and the cylinder −1.7 ± 1.0D. The mean SE for WTR reduced from −5.60 ± 2.37D to −0.31 ± 0.67D at 2 months and −0.38 ± 0.70D at 12 months; the mean cylinder improved from −1.90 ± 1.10D to −0.31 ± 0.39D and −0.36 ± 0.43D, respectively. Eyes with oblique astigmatism also improved from a mean SE of −5.8 ± 3.4 D to −0.82 ± 1.50D and −0.69 ± 1.15D and a cylinder of −1.4 ± 0.73D to −0.17 ± 0.33D at 2 months and −0.1 ± 0.32D at 12. For ATR, the mean SE improved from −4.0 ± 1.8D to −0.08 ± 0.22D and −0.04 ± 0.12D; and the mean cylinder from −1.25 ± 0.53 to −0.02 ± 0.09D −0.08 ± 0.21D at 2 and 12 months, respectively. There were statistically significant improvements in SE, manifest sphere and cylinder refraction, and UDVA and CDVA scores for each cylinder type at 2 months with ATR cylinders having better outcomes. Although missing data limited interpretation at one year, differences were maintained. The magnitude of error calculations suggests that WTR was more prone to under-correction, particularly for high astigmatism (>1.5D). SMILE for myopic astigmatism reliably corrects SE, irrespective of the subtype of astigmatism. The choice of using Small Incision Lenticule Extraction (SMILE) for the surgical correction of refractive errors has become increasingly popular since its introduction in 2011 ,4,5.Although multiple studies have reported refractive outcomes after SMILE ,4,6, fewGiven that few studies have investigated the outcomes of SMILE on astigmatism, further investigation is required to confirm the results of Ivarsen et al.  and Pére® Eye Health Care Clinics), between January 2016 and December 2017, were reviewed. All eyes with an attempted cylinder correction of 0.75D or higher were identified from surgical logbooks. In this retrospective observational cohort study (case series design), the charts of consecutive patients who underwent correction of myopic astigmatism using SMILE at two laser refractive centres in Poland  with astigmatism up to 5D were eligible for surgery and included. Only those with a baseline central corneal thickness of 500 microns were included. Medical charts were reviewed and patients were divided into three groups according to their type of astigmatism: WTR, ATR, and oblique. WTR was defined as a positive cylinder between 90° ± 29° (61° and 119°), ATR from 180° to 151° or 0° to 29°, and oblique between 120° and 150° and 30° and 60°. Each group was then divided according to the amount of astigmatism based on pre-operative subjective analysis of the cylinder axis into low astigmatism with an attempted correction from 0.75 to 2.25D and high astigmatism with a cylinder of 2.50D and greater. Demographic data including age and sex were collected. Data from pre-operative assessments including Uncorrected Distance Visual Acuity (UDVA), manifest, and Best Corrected Distance Visual Acuity (BCDVA) were also recorded. Distance visual acuity (VA) was measured in bright light at 6 meters (reported as 20 feet) and then recorded using the Snellen chart. Ocular residual astigmatism (ORA), an important metric to describe the discrepancy between anterior corneal astigmatism and refractive astigmatism, calculated to the corneal plane was used to quantify the astigmatism from the crystalline lens, posterior corneal surface, and retina. A trained optometrist performed the clinical refraction. All patients underwent comprehensive eye examination by the eye surgeon conducting the procedure, which included anterior segment examination, assessment of the tear film, measurement of intraocular pressure with Goldmann tonometry, and dilated fundoscopy. Corneal high-resolution Scheimpflug tomography  was performed pre-operatively. All patients were fully informed of the details and possible risks of the procedure. Written informed consent to perform the surgical procedure was obtained from all patients pre-operatively. The study was conducted in accordance with the tenets of the Declaration of Helsinki. As this was a quality control study, under Polish law, no ethical approval was required. All surgeries were performed by three experienced refractive surgeons . Pre-operatively, all eyes were anesthetized with Proxymetacaini hydrochloridum and the peripheral cornea was marked in the 0°, 90°, and 180° axis using a marker pen . Under the slit lamp in the patient’s upright position, SMILE was performed using a 500-kHz Visumax femtosecond laser . Laser settings used were as follows: cut energy of 140 nJ with a spot separation of 4.5 μm, 6.4–7.0 mm optical zone with a 0.1mm transition zone, the diameter of the cap ranged from 7.6 to 7.9 mm, and the cap thickness varied from 110 μm to 135 μm. The treatment was centered on the visual axis and the patient was instructed to look at the green flashing fixating light. Once the proper centration was achieved, the suction was applied. If any cyclotorsion (incyclotorsion or excyclotorsion) was present on the screen, a gentle rotation of the contact lens was performed to manually compensate cyclotorsion and align horizontal marks of the eye to the 0° and 180° axis of the reticule. Once all of the corneal marks were aligned, laser treatment was started to create an intra-stromal lenticule. A blunt spatula was used  to break tissue bridges after the laser treatment and the lenticule was removed with a pair of forceps .Post-operative treatment included topical antibiotics (0.3% of ofloxacin) four times per day for a week, and preservative-free eye drops  three times a day initially and then tapered down over the next four weeks along with preservative-free artificial tears for two to three months after surgery. There were no intraoperative complications noted such as suction loss, black spots, or incomplete lenticule extraction. Post-operative follow-up examinations were scheduled for day one, one week, and two months. Surgeons who performed the surgery conducted all examinations. All follow-up visits included assessment of UDVA, manifest refraction, BCDVA, and tomography. All complications or adverse events were recorded.t-test was used to compare normal data and the Wilcoxon Rank Sum Test for non-normal data; distribution frequencies were compared using Chi-Square Tests. One-way analysis-of-variance (ANOVA) was used to compare two or more independent variables. Distance VA was converted to the decimal equivalent. A modified version of the Journal of Refractive Surgery Standard for Reporting Astigmatism Outcomes of Refractive Surgery was used to report refractive outcomes [Data were analysed using SPSS for Mac . The Shapiro–Wilk test was used to test normality. The paired Student’s outcomes  using thoutcomes .In all, 355 eyes from 209 patients with astigmatism of 0.75D or more were identified and included. Most, 185/355 (52%), were right eyes. Of these, 122 were female (58.4%) and 87 were male (41.6%). The median age of the sample was 32 years  with a mean age of 32.9 and standard deviation (SD) of 8.1 years. At the time of the data collection, follow-up data were available for 250 eyes at 2 months and 131 at 12 months. Pre-operative (baseline), one week and two, eight and twelve-month post-operative parameters including spherical equivalent (SE), manifest sphere and cylinder, UDVA, and BCDVA are presented in p < 0.001), manifest sphere (p = 0.004), manifest cylinder refraction (p < 0.001), and BCDVA (p = 0.035) scores between all three groups but no difference in the mean UDVA scores (p = 0.96). The WTR group had a greater degree of pre-operative astigmatism (−1.9), compared to the oblique (−1.4) and ATR groups (−1.25).Eyes were categorized into three groups according to the type of astigmatism: WTR 90° ± 29° (61° and 119°), ATR from 180° to 151° or 0° to 29°, and oblique between 120° and 150° and 30° and 60°. Of those included, 247 (70%) eyes had WTR astigmatism, 62 (17.5%) eyes were oblique, and 46 (13%) eyes had ATR astigmatism. Pre-operative parameters by cylinder classification are presented in Target refraction was plano for the majority of eyes, 322/355 (91%); the target for the remainder ranged from −0.5D to −6.5D. The mean post-operative SE for the total sample at 2 months and 12 months was −0.36 ± 0.85D and −0.38 ± 0.75D, respectively; the mean cylinder scores were −0.18 ± 0.35D and −0.27 ± 0.40D, respectively. The mean UDVA for those included, irrespective of cylinder, was 0.87 ± 0.35D at month 2 and 0.86 ± 0.19D by month 12. At all available time points, the majority of the eyes achieved a post-operative refraction that was within 0.5D of the intended target; at 12 months 86% of eyes were within 0.50D and 95% were within 1.00D A. There was no change in BCDVA at 12 months for most patients (82% of eyes), while only 0.8% lost two or more lines of BCVA B. ExaminPost-operative parameters for all three types of cylinders over time, where available, compared with baseline, are presented in p < 0.001), manifest sphere (p = 0.007), manifest cylinder (p < 0.001), UDVA (p < 0.001), and BCDVA (p < 0.001) scores at 2 months with better outcomes for patients with ATR astigmatism. These differences remained significant at 12 months for SE (p = 0.02), manifest sphere (p = 0.017), manifest cylinder refraction (p = 0.002), and UDVA (p = 0.006) but not BCDVA (p = 0.08) scores. The mean pre-operative ORA for all eyes was low (0.76 ± 0.39D) and there were no statistically significant differences between the three subtypes of astigmatism (p = 0.279). Comparing refractive outcomes by cylinder type, there were statistically significant differences in SE , showed that optimal correction of astigmatism was achieved for the ATR group. Of these, 100% of eyes were within ≤0.50D of target astigmatism by 2 months, while 95% remained within ≤0.50D of the target, where follow-up was available, at 12 months. Fewer (90%) of those with oblique astigmatism remained within ≤0.50D of the target at 2 and 12 months. This compared to 84% at 2 months and only 71% of the WTR astigmatism group, albeit 94% were within 1.00D at 12 months. These results are presented in The ME at 2 and 12 months for WTR was low for WTR astigmatism, irrespective of stratification by magnitude and axis of the preoperative cylinder (close to zero for those with lower manifest cylinder scores at baseline). Mean postoperative ORA remained low (<1.0 D) for all subtypes across follow-up. Vector analyses at 2 and 12 months are compared in This paper presents our experience of myopic astigmatism correction with SMILE in a large sample of patients (355 eyes) and in different types of astigmatism among those attending two laser refractive clinics in Poland. The results showed that SMILE provides safe and reliable correction of refractive error with mean SE and cylinder values improving for all three groups at 2 months . The results also suggest that SMILE likely provides long-term, at least up to one year, reliable correction of SE, irrespective of the subtype of astigmatism examined. This study adds to the existing, as yet limited literature, reaffirming data on astigmatism correction with SMILE ,9. It alIn a sample of Danish patients (n = 829) in 2018, researchers showed that approximately one-quarter of the variation in outcomes with SMILE for myopic astigmatism could be accounted for by the attempted cylinder correction and the type of astigmatism (ATR versus WTR), albeit they did not provide data for oblique astigmatism . HoweverVector analysis in our study found that the magnitude of error was significantly different for all three groups at both 2 and 12 months. In particular, the WTR group had more negative values, also suggesting that patients with WTR astigmatism may be more prone to under-correction than those with either ATR or oblique cylinders. This was especially true for those with high astigmatism. A small to moderate correlation was seen between TIA and SIA for all groups, although it was stronger for the WTR group i.e., the target TIA and SIA showed that WTE had greater correlation than ATR or oblique cylinders. This conflicts with the refractive outcomes , which showed worse outcomes for WTR, which may be due to the small sample size of the latter and because we reported the keratometric SIA, which measures corneal astigmatism.p = 0.279) at baseline. ORA for each subtype was <1.0D at both 2-month and 12-month follow-ups, supporting the efficiency of astigmatism correction with SMILE for patients in this study. In our study, SE refraction stability was high with only 13% changing >0.5D by 12 months, compared to the study from the US, which found that 19% changed . There wSeveral studies have examined the differential effects of either Photorefractive Keratectomy (PRK) or Laser-assisted in situ keratomileusis (LASIK) versus SMILE on astigmatism. Studies examining the effectiveness of astigmatism correction with PRK and LASIK suggest that high pre-operative astigmatism is associated with lower predictability and higher risk for residual astigmatism after both LASIK and PRK ,20. Of tth LASIK ,24. Seveth LASIK ,26. Induth LASIK ,28. Studth LASIK . No diffth LASIK . In addith LASIK . In anotth LASIK . To our The strengths of this study include its large sample size of “real-life” patients, with different types of myopic astigmatism followed for up to 12 months after SMILE in two centres in Poland, where relatively few studies have examined outcomes of SMILE. While other studies have presented outcome data, few have followed patients with astigmatism, and none, to our knowledge, with three types for up to one-year follow-up. The study has a number of limitations. There was a high degree of loss to follow-up as not all patients had yet completed 12 months of follow-up or declared they would not be attending for prolonged follow-up, which may be a source of bias including a bias towards a successful outcome. This is a common issue in cross-sectional studies and may also reflect high levels of satisfaction and, given the location of these clinics near Western European countries, a degree of “healthcare tourism”, rather than poor outcomes. Attempts to update and expand the analysis were delayed due to the COVID-19 pandemic and staff have now moved on, limiting the ability to add to the analysis. There is a wide range of targets of refraction (−0.50 to −6.50D) in this study, which may have introduced bias. This was because certain patients could only have a partial reduction of their myopia and for this reason, the target in these cases ranged widely. In all cases, the entire amount of astigmatism was treated. Here we selected 2.50D of astigmatism to categorize the low and high astigmatism groups, possibly resulting in bias. As this study was a retrospective, observational study of existing patients who were treated in our two refractive surgery clinics, the design inherently limited our sample size, the characteristics of included patients, and selection criteria. However, there is no accepted cut-off and other studies have selected either identical ,32. HencMost patients (70%) had WTR astigmatism, again potentially creating bias and limiting the generalisability of findings. By examining the effectiveness of SMILE for all three groups separately, we were able to compare differential outcomes and limit the potential for this. Nevertheless, at baseline, the larger WTR group had a cylinder indicating a greater degree of pre-operative astigmatism, potentially predisposing these patients to under-correction. Indeed, there were significant differences in the pre-operative astigmatism between the different subtypes examined in this paper, which could have introduced bias. Evidence suggests that a higher amount of pre-operative cylinder is associated with a greater degree of under-correction of astigmatism post-operatively . Hence, In summary, this study affirms the growing body of evidence showing that SMILE for myopic astigmatism is an effective procedure with long-term refractive stability in a relatively large sample of patients in two refractive laser clinics in Eastern Europe (Poland), followed for up to one year. It also adds to data suggesting that the type of astigmatism as well as the cylinder are important and should be considered in pre-operative planning to provide more consistent results in high cylinder corrections. While the sample mainly included those having WTR astigmatism, almost one-third had oblique and against-the-rule cylinders, suggesting that irrespective of type, SMILE is useful for the surgical correction of myopic astigmatism. Long-term follow-up to demonstrate safety and satisfaction of SMILE for low versus high astigmatism according to subtype over a longer period of follow-up, beyond one year, is now required.


Brachiaria humidicola, a tropical grass, could release root exudates with biological nitrification inhibition (BNI) capacity and reduce soil nitrous oxide (N2O) emissions from grasslands. However, evidence of the reduction effect in situ in tropical grasslands in China is lacking.B. humidicola on soil N2O emissions, a 2-year (2015–2017) field experiment was established in a Latosol and included eight treatments, consisting of two pastures, non-native B. humidicola and a native grass, Eremochloa ophiuroide, with four nitrogen (N) application rates. The annual urea application rates were 0, 150, 300, and 450 kg N ha−1.To evaluate the potential effects of E. ophiuroides biomass with and without N fertilization were 9.07–11.45 and 7.34 t ha−1, respectively, and corresponding values for B. humidicola increased to 31.97–39.07 and 29.54 t ha−1, respectively. The N-use efficiencies under E. ophiuroide and B. humidicola cultivation were 9.3–12.0 and 35.5–39.4%, respectively. Annual N2O emissions in the E. ophiuroides and B. humidicola fields were 1.37 and 2.83 kg N2O-N ha−1, respectively, under no N fertilization, and 1.54–3.46 and 4.30–7.19 kg N2O-N ha−1, respectively, under N fertilization.The average 2-year B. humidicola cultivation increased soil N2O emissions, especially under N fertilization. This is because B. humidicola exhibited the more effective stimulation effect on N2O production via denitrification primarily due to increased soil organic carbon and exudates than the inhibition effect on N2O production via autotrophic nitrification. Annual yield-scaled N2O emissions in the B. humidicola treatment were 93.02–183.12 mg N2O-N kg−1 biomass, which were significantly lower than those in the E. ophiuroides treatment. Overall, our results suggest that cultivation of the non-native grass, B. humidicola with BNI capacity, increased soil N2O emissions, while decreasing yield-scaled N2O emissions, when compared with native grass cultivation.According to the results,  The glo3) to nitrite and further to nitrate (NO3−) and producing N2O as a byproduct , but are less effective in Andosol during the 60-day incubation.Natural compounds with biological nitrification inhibition (BNI) have been found from litters, root exudates, tissue extracts, and rhizosphere of plants such as grasses, trees, and crops , includila grass . Some rotivities , while 1tivities . Up to de tested . For exail-assay . GopalakB. humidicola, reportedly exhibits the strongest BNI function among tropical grasses reduces the NH3 oxidation rate and N2O emissions significantly during a 3-year field experiment, when compared with soybean-planted or plant-free soils  capacity exhibit approximately 2-fold greater productivity than those lacking such capacity in nutrient-limited ecosystems, based on an estimate of a newly developed model . The B. ee soils . During mendment . In conttal site .B. humidicola and a native grass species, Eremochloa ophiuroides. We hypothesized that in situ N2O emissions from grasslands under cultivation with Brachiaria with higher BNI capacity are lower than in those cultivated with Eremochloa. The objectives of the present study were: (1) to determine whether the N2O emissions from a B. humidicola field are lower than those from an E. ophiuroides field in tropical Hainan Province, China; and (2) to evaluate the mitigation effects of B. humidicola on yield-scaled N2O emissions under the different N application rates. We also established an incubation with soils from the field experiment using a 15N tracing technique to evaluate the influence of B. humidicola on the N transformation process rates and N2O production rates via nitrification and denitrification . The region is characterized by a tropical monsoon climate, with a rainy season from May to October, and a dry season from November to April. The mean annual temperature and precipitation are 23.1°C and 1,823 mm, respectively. The soil is derived from granite and classified as a Latosol, with a sandy loam texture. Latosol is a most widely distributed soil in Hainan Province. The properties of surface soil (0–20 cm) prior to the field experiment are shown in 2.2.Brachiaria humidicola CIAT679 and Eremochloa ophiuroides, with four N application rates. The annual urea application rates were 0, 150, 300, and 450 kg N ha−1, which were designated as BCK, BN1, BN2, and BN3, respectively, for B. humidicola, and ECK, EN1, EN2, and EN3, respectively, for E. ophiuroides. The plots measured 3 m × 4 m. The treatments, which had three replicates, were set up based on a randomized complete block design. During the first season from August 2015 to August 2016, 60 and 40% of urea was applied as basal fertilizer and top-dressing fertilizer, respectively, in the fertilized treatments. In the second season from August 2016 to August 2017, urea was added with three splits: 40% as basal fertilizer, and 30% as top-dressing fertilizer on 13 March and 9 June 2017, respectively. Calcium superphosphate (150 kg P2O5 ha−1) and potassium chloride (105 kg K2O ha−1) were applied as basal fertilizer. All the fertilizers were dissolved in water and uniformly spread into the soil. Harvested plant samples were oven-dried at 60°C to a constant weight, and then ground to less than 0.2 mm for analysis. Field management practices were similar to local practices and standardized at all plots. Specific dates are listed in A field experiment was established in August 2015 and included eight treatments, consisting of two pastures, 2.3.2O concentrations were analyzed using a gas chromatograph  equipped with a 63Ni electron capture detector and a thermal conductivity detector. The N2O fluxes were calculated using the following equation  and a 5-cm groove around the upper edge was permanently fit 10 cm into the soil. During gas sampling, a stainless chamber (50 cm × 50 cm × 50 cm) was inserted into the groove, which was filled with water to ensure airtightness. The chamber was covered with reflective film and foam to minimize air temperature change inside the chamber. A rubber plug with a mercury thermometer was fit tightly into the hole on the top of the chamber for use in measuring the chamber temperature while gas sampling. Two vents welded with stainless tubes were punched on top of the chamber, one connected to a rubber tube with a three-way stopcock for gas collection, and another one for ensuring air pressure equilibrium inside and outside the chamber. Gas samples were obtained once every other day during 1 week after each fertilization and once a week during the other period. Sampling was conducted between 7:00 am and 12:00 pm to minimize diurnal variation. Four gas samples were extracted from the chamber at 0, 10, 20, and 30 min after chamber closure using airtight plastic syringes and instantly injected into 20-ml pre-evacuated vials fitted with butyl rubber stoppers. The Nequation :F is the flux in N2O (μg N2O-N m−2 h−1); ρ is the density of N2O at 0°C and 760 mm Hg (kg m−3); V is the effective volume of the chamber (m3); S is the soil area covered by the chamber (m2); ΔC/Δt is the rate of N2O concentration increase in the chamber (ppbv N2O-N h−1); and, T is mean air temperature inside the chamber during sampling (°C).where 2.4.Soil temperature was measured at 5 cm depth using a geothermometer. Soil water content was measured at three different positions in each plot with time domain reflectometry (TDR) probes and expressed as water-filled pore space  as follows :−3).where total soil porosity = 1 - (soil bulk density/2.65), 2.65 being the soil particle density (g cm4+-N) and nitrate-N (NO3−-N) were extracted with 2 M KCl (soil/KCl solution ratio of 1:5) by agitating for 1 h on a rotary shaker, and the concentrations were measured using a colorimetric method on a segmented flow analyzer . Subsequently, the DOC was analyzed using the combustion oxidation nondispersive infrared absorption method on a TOC analyzer .Surface (0–20 cm) soil samples were collected at five different positions in each plot fortnightly using a stainless steel soil sampler and thoroughly mixed to form a composite sample. The samples were taken to the laboratory immediately and stored at −20°C before analysis. Soil exchangeable ammonium-N  using a pH meter . Soil organic C (SOC) was measured using the wet oxidation-redox titration method . Total Nc method . Availabc method .2.5.2O emissions  were calculated using the following equation ; i is the ith measurement; (ti + 1 –ti) is the interval between the i + 1)th measurement time (d); n is the total number of measurements; and 24 × 10−5 was used for unit conversion.where 2O emission factor of applied fertilizer N  was calculated using the following equation:The NEfertilizer and Econtrol are the cumulative N2O emissions from the fertilized and control treatments, respectively; and Napplied is the amount of fertilizer N applied to the corresponding treatment.where 2O emission (mg N2O-N kg−1 biomass) was computed using the following equation ; and yield is the amount of grass biomass harvested annually (kg ha−1).where Fertilizer N-use efficiency  was calculated as follows:Nfertilizer and Ncontrol are the amount of N uptake in aboveground biomass (kg N ha−1) in the fertilized and control treatments, respectively; and Napplied is the amount of the N applied to the corresponding treatment (kg N ha−1).where p < 0.05. Spearman’s correlation analysis was used to determine the relationships between N2O flux and soil WFPS, soil inorganic N, soil dissolved organic C, and air temperature. All statistical analyses were performed using IBM SPSS Statistics 26 for Windows .Significant differences among treatments were evaluated using one-way ANOVA followed by the Duncan test at 3.3.1.B. humidicola cultivation and only by 5.8–15.1% under E. ophiuroides, when compared with the pre-treatment soil. Cultivation of both pastures promoted soil N accumulation significantly (p < 0.05); however, there were no significant differences in soil N accumulation among treatments under different N application rates  but did not increase the biomass yield of E. ophiuroides, during the first season. During the 2016–2017 season, however, the biomass of both grasses was enhanced with N fertilization, and increased with an increase in the N application rate (p < 0.05).The biomass of  seasons . The N aB. humidicola under no N fertilization were 220.08 and 188.74 kg N ha−1 during the 2015–2016 and 2016–2017 seasons, respectively, and decreased to 44.22 and 69.82 kg N ha−1 for E. ophiuroide, respectively . Mean rainfall was 2,341 and 2,373 mm during the 2015–2016 and 2016–2017 seasons, respectively. Precipitation mainly occurred in the rainy season, from May to October, accounting for 87% of the total annual precipitation. Soil moisture fluctuated from 5.1 to 58.3% WFPS, and the mean WFPS in all the treatments was 33.5–38.3%, with no significant differences among treatments.Air temperature (AT) ranged from 5.4 to 32.6°C, with an average of 24.8°C over the 2-year measurement period, and there was no apparent difference between two growth seasons . Soil te4+-N concentration peaks occurred approximately 1 week after each fertilization, and decreased to a constant level 40 days later .Soil NHys later . The meaectively . Under N3.4.2O-N m−2 h−1 in the BN3 treatment on 31 August 2016, which was 3-fold that in the EN3 treatment (2O-N m−2 h−1) was also observed on 20 June 2017, which, however, was only 1.2-fold greater than that in the EN3 treatment. The N2O fluxes were significantly (p < 0.01) correlated with soil moisture, NH4+-N, NO3−-N, and air temperature  were 1.38 and 1.35 kg N2O-N ha−1 during the 2015–2016 and 2016–2017 seasons, respectively, and increased to 1.43–3.28 and 1.65–3.64 kg N2O-N ha−1 under N fertilization, respectively. The annual N2O emissions increased linearly with an increase in the N application rate in the B. humidicola fields ; conversely, they exhibited exponential correlations with the N application rate in the E. ophiuroides fields .Annual N2O emission factor (EF) of the N applied ranged from 0.74 to 0.98% for B. humidicola, and decreased to 0.11–0.47% for E. ophiuroides over the 2 years  ha−1 day−1 BNI activity in the South American savannas, which is equivalent to the application of 6.2–18 kg ha−1 nitrapyrin based on 1 ATU being equal to 0.6 μg of nitrapyrin. B. humidicola, like dicyandiamide, significantly suppresses soil nitrification potential. In a previous study, using the 15N tracing incubation with soils collected from the B. humidicola and E. ophiuroides plots at the field experiment end, we found that B. humidicola decreased the autotrophic nitrification rate and N2O production rate via nitrification by 27.3 and 14.7%, respectively when compared with E. ophiuroides (B. humidicola efficiently inhibited nitrification and N2O production via nitrification.Previous study suggested that ee soils . Byrnes periment . The actmidicola . Brachiamidicola . The inhmidicola . Subbaraiuroides . This inB. humidicola reduced the abundance of both ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a Vertisol with pH 7.40 when compared with soil cultivated with soybean. 2O production under high NH4+-N concentrations, the contribution of AOB was greater in a soil with pH 6.50. In the test acid soil with pH 5.42, it is likely that both AOA and AOB participated in NH3 oxidation and N2O production. Further investigations are required to determine the relative importance of AOA and AOB in N2O production, and the suppression effects of B. humidicola on AOA and AOB activity , we found that the N2O production rate during denitrification in the B. humid cola soil increased by 7.7-fold when compared with the E. ophiuroides soil and the contribution of denitrification to N2O emissions sharply enhanced from 9.7% in the E. ophiuroides soil to 47.1% . In addition, we observed interannual shifts in yield-scaled N2O emissions in both grasslands, which was primarily driven by changes in annual N2O emission in B. humidicola fields, whereas they were driven by changes in biomass yield in E. ophiuroides fields. The lower yield-scaled N2O emissions under B. humidicola cultivation compared with under E. ophiuroides indicated that although B. humidicola increased annual N2O emissions, it was more environmentally friendly based on its higher forage productivity and NUE.Comparing yield-scaled N methods . In the 5.B. humidicola exhibited higher yields and NUE, and in contrast and unexpectedly, induced higher soil N2O emissions when compared with E. ophiuroides. Although cultivation of B. humidicola with high BNI capacity reduced N2O production rate via nitrification, however, it more efficiently enhanced N2O production rate than E. ophiuroides via denitrification due to increased SOC and exudate concentrations, thereby increasing N2O emissions , the International Partnership Program of Chinese Academy of Sciences (151432KYSB20200001), and International Atomic Energy Agency coordinated research project (D15020).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.