Aruncus dioicus var. kamtschaticus Hara (A. dioicus) and used it to generate basic data for the alternative meat market by comparing nutritional and microbiological components with commercially available vegetable and meat patties. Nutrient analysis, microbiological analysis, chromaticity, and texture analysis were performed on substitute meat patties (SMPs) with A. dioicus and commercially available vegetable and animal patties. Among sugars, the contents of fructose and maltose were respectively high in commercial meat patties (CMPs) and SMPs. SMPs were low in saturated and trans-fat, and high in ω-3 fatty acids. The contents (in descending order) of leucine > phenylalanine > threonine > isoleucine were high in SMPs and commercial vegetable patties (CVPs). Qualitative and quantitative findings of Escherichia coli (E. coli) and Staphylococcus aureus were all negative. Our SMPs had high lightness (L*), low redness (a*), and low yellowness (b*). The hardness, chewiness, and resilience of our SMPs were lower than those of other vegetable and animal patties. Considering our results, the method of manufacturing SMPs developed in the present study allows meat to be flavored without significant nutritional differences compared with commercially available CMPs. Our findings provide a base for studies on future meat alternatives.We developed a vegetable alternative to meat patties using  In the global context of COVID-19, contact rates between people have decreased, rates of staying home have increased, the amount of consumption has decreased, and the amount of physical activity has decreased. This has led to an increased prevalence of obesity, which is associated with an increasing incidence of colon cancer, breast cancer, cadiovascular disease, and high blood pressure ,3. Accor2 . By 2005Currently, plant-based meat substitutes, edible insects, and cultured meat are attracting attention as the most representative meat substitutes. Among them, plant-based alternatives to meat, representing the largest proportion, is a meat-like food manufactured using proteins extracted from plants. The main ingredients include wheat, pea protein, soybeans, peanuts, cottonseed, and rice ,7. The pAruncus dioicus var. kamtschaticus Hara is a perennial plant of the Rosaceae family that grows wild in alpine forests and propagates by seeds. The height is about 10–30 cm, the leaves are alternate, and the flowers are yellow-green [A. dioicus is rich in saponins, which is helpful for treating chronic degenerative diseases, and young shoots are rich in carbohydrates and minerals. The whole plant is used medicinally because it can detoxify, prevent tonsillitis, exert antioxidant and antibacterial effects, and enhance stamina. In addition, A. dioicus is used when boiling beef soup [A. dioicus [A. dioicus is cytotoxic and has antioxidant activities against prostate cancer [A. dioicus chloroplasts have been analyzed [A. dioicus inhibits the aging of UV-B-damaged skin [A. dioicus in food nutrition.ow-green . It is cow-green ,12,13,14eef soup . Lipid a dioicus ,16. A. de cancer ,18. It he cancer  and allee cancer . Aruncine cancer —and the analyzed . A. dioiged skin . HoweverA. dioicus, a natural vegetable herb that has a meaty taste and chewy texture. This study was conducted to provide basic data for research on substitute meat by comparing the developed meat patty with existing commercially available products.Therefore, in this study, an alternative meat patty was developed using w/w; distilled 6×) and then placed for 3 min in a W-110 dehydrator . The premix comprising salt 8%, sugar 32%, onion powder 7%, garlic powder 4.1%, mushroom powder 19%, ginger powder 1%, coriander powder 1.5%, white pepper 1%, and pomegranate was evenly mixed with 10.3% powder and 16.1% sahmyook seasoning. The gravy (based on 100% oil) of meat patties was reproduced by mixing 0.7% methylcellulose HV, 43.9% coconut oil, 21.9% cocoa butter, and 0.7% sahmyook seasoning in hot water (32.9%) for 5 min using an HG-15A homogenizer . The homogenate was stored at −18 °C. The color of meat patties was replicated (based on 100% bitcocoa water) by homogenizing 2.5% cocoa powder and 7.5% beet powder in 90% water for 30 s. Dehydrated TVP (45%), water (12.5%), water-soluble smoked flavor (0.3%), lemon juice (0.3%), and bitcocoa water (5.8%) were ground in an SMX-9400MD blender  for 1 min. The mixture and ground premix 5.1%; potato starch, 1.8%; mixed soy protein, 2.3%; methylcellulose HV, 1.2%; black pepper, 0.1%; xanthan gum, 0.2%; potassium chloride, 0.1%; smoke flavor powder, 0.5%; beef flavor powder, 0.3%; nutritional yeast flakes, 1.2%; oyster mushroom, 7%; and A. dioicus 3% were mixed and kneaded into a dough with canola oil (0.6%), barbecue flavor (0.7%), and fat-soluble smoked flavor (0.3%). The prepared oil (11.7%) was chopped and mixed, and then 80 ± 2 g of the dough was removed and molded into patties (content ratio determined in prior repeated experiments) (Textured vegetable protein was hydrated for 2 h with water (riments) . These wA. dioicus was added.The entire surface of the raw patties was photographed using an Ev-nxflzza2qkr digital camera , and the characteristics  of the patties were visually assessed. A. dioicus was determined using an FS-620 atmospheric pressure dryer , and crude protein was analyzed by the micro-Kjeldahl method using a KjeltecTM 2300 automated Nitrogen analyzer . This was also measured according to the ash content by direct painting using a KL-160 , and crude fat content was measured by the Soxhlet method using a SOX606 analyzer  [The moisture content of the , Korea) ,25. The g for 20 min. The supernatant was passed through a 0.45 μm filter and analyzed using an LC-20AD HPLC  with a carbohydrate column , with a mobile phase comprising acetonitrile in water  at a flow rate of 1.2 mL/min at 40 °C.Samples (1 g) were mixed with 40 mL of 80% ethanol at 100 rpm in a shaking incubator at 80 °C to extract free sugar as described in . The mixVitamin A: A mixture of ethanol (30 mL) and 10% pyrogallol in ethanol (1 mL) was saponified with 3 mL of potassium hydroxide (9→10), in a reflux condenser placed in a boiling water bath for 30 min. The solution was cooled to room temperature in a flask, and then 30 mL of water was added and the mixture was passed through a brown separatory funnel. The flask was washed with 10 mL of water, followed by 30 mL of petroleum ether . The wash was added to a separatory funnel, shaken well, and then left to separate into layers. The water layer was extracted twice with 30 mL of petroleum ether in a brown separatory funnel. All petroleum ether extracts were combined and washed once with 10 mL of water, followed by 50 mL each . Sodium sulfate was washed twice with 10 mL of petroleum ether, and the wash was added to the flask. All petroleum ether extracts were combined and evaporated to dryness under reduced pressure at 40–50 °C; the residue was dissolved in isopropanol  to 1.0 mL, serving as the test solution. The HPLC conditions were as follows: column; mobile phase, ethanol:water (95:5); flow rate, 0.5 mL/min; fluorescence detector with excitation and detection wavelengths of 340 and 460 nm, respectively.v/v; A) and 30% dichloromethane (B); flow rate, 1.0 mL/min; injection volume, 20 μL; run duration, 15 min; detector, 450 nm.β-carotene: Approximately 2 mg of β-carotene was dissolved in cyclohexane in 10 mL flasks, and then diluted with ethanol to prepare a standard solution. Portions (100–200 μg) of β-carotene were shaken with 10 mL of 3% pyrogallol in ethanol and 1 mL of 60% potassium hydroxide, and then saponified at 70 °C for 30 min. The saponified product was cooled with running water, and then shaken with 20 mL of 1% sodium chloride and 15 mL of hexane:ethyl acetate (9:1) for 10 min. After centrifugation at 2000 rpm for 5 min, the upper layer was concentrated, and 15 mL of hexane:ethyl acetate (9:1) was repeatedly added to the lower layer until the upper layer became colorless and the supernatants were combined. After concentration under reduced pressure, the solution was dissolved in 10 mL of ethanol to prepare test solutions. The LC-20AD HPLC conditions were as follows: Waters Atlantis dC18 column, C18 4.6 × 150 mm, 5 μm; mobile phase, 70% acetonitrile:methanol 85:15 (v/v), filtered with a 0.2 μm syringe filter, and then analyzed using the LC-20AD HPLC. The analytical conditions were as follows: column, LaChromUltra C18 ; fluorescence detector, excitation and emission at 298 and 320 nm, respectively; flow rate, 0.2 mL/min; column oven temperature, 40 °C; isocratic elution with mobile phase, acetonitrile:methanol 50:50 (v/v); sample injection volume, up to 20 μL.Vitamin E: Samples (~0.5 g) were saponified with 5 mL of 1 N KOH (in ethanol) containing a small amount of BHT and heated in capped 22 mL brown vials at 100 °C for 30 min. The vials were cooled at room temperature for ~15 min in the dark. Next, 5 mL of saturated saline and 10 mL of petroleum ether  were manually shaken for at least 1 min, and then left for 10 min to separate the layers. Supernatants (5 mL) were placed in 20 mL tubes, and petroleum ether was removed at 40 °C using a Turbo-Vap evaporator . This was dissolved in 2 mL of methanol:isopropanol 1:1 (2PO4:acetonitrile (60:40); flow rate, 1.0 mL/min; ultraviolet (UV) detector at 254 nm.S is the ascorbic acid concentration (μg/mL), a is the total amount (mL), and b is the dilution factor of test solutions.Vitamin C: Samples  were suspended in 10% methane phosphoric acid for 10 min, and then homogenized in 5% methane phosphoric acid. Homogenates were washed with a small amount of 5% methane phosphate, brought to 100 mL in volumetric flasks, and then centrifuged at 3000 rpm for 10–15 min. The supernatant was diluted with 5% methane phosphate to prepare test solutions that were analyzed by HPLC under the following conditions: column, YMC-Pack Polyamine II; mobile phase, 0.05 M KHSamples (~25 mg) without fat were mixed with 2 mL of 0.5 N methanolic sodium hydroxide in stoppered glass tubes, heated for ~10 min at 100 °C, and then cooled. Trifluoroborane methanol  was added; then, the mixture was heated at 100 °C for 10 min, cooled to 30–40 °C, and then vigorously shaken with 2 mL of isooctane for 30 s at this temperature. This mixture was shaken with 2 mL of saturated sodium chloride, cooled to room temperature, and then the isooctane layer was separated from the aqueous layer to serve as the test solution. The LC-20AD HPLC conditions were as follows: column, HP-FFAP ; oven temperature, 100 °C for 2 min, followed by 4 °C/min increments to 230 °C for 20 min; injector temperature, 230 °C; detector temperature, 250 °C; flow rate, 1.5 mL/min; and the instrument was set to GC-FID (Agilent 7890A) .Amino acid content analysis was performed by modifying the method of Grunau and Swiader . A 10 g 2PO4 in 500 mL of distilled water, adjusting the pH to 7.2 with 175 mL of 1 N NaOH, and then bringing the volume up to 1 L with distilled water. This stock buffer was sterilized by heating at 121 °C for 15 min, which was then stored at 4 °C. Immediately before use, 1 mL of the stock buffer was diluted in 800 mL of sterile phosphate buffered dilution water (Butterfield buffer). Samples were cut into small pieces using sterile scissors and tongs, and then 25 g of randomly selected pieces were homogenized in 225 mL of sterile butterfield buffer and used as test solutions. Plate count agar  was dissolved in 1 L of distilled water adjusted to pH 7.0 ± 0.2 and then sterilized at 121 °C for 15 min. Test solutions (1 mL) were aseptically mixed with 1 mL of 10-fold Butterfield buffer in at least two sterile Petri dishes, and then PCA (~15 mL) was aseptically dispensed into petri dishes at 43–45 °C. The dishes were rotated gently and tilted from side to side to solidify the medium, taking care to avoid attachment to the lid. The development of diffuse colonies was suppressed by adding 3–5 mL of PCA and agitating the plates gently as described above. The petri dishes were inverted and incubated at 35 ± 1 °C for 48 ± 2 h. Colonies were counted in plates without diffuse colonies among 15–300 colonies per plate, and then the average number of colonies was multiplied by a dilution factor to calculate the number of bacteria (CFU/g).Phosphate buffer solution (PBS) was prepared by dissolving 34 g of KHColiforms: Samples were finely cut using sterile scissors and tongs, and then random pieces (25 g) were homogenized in 225 mL of Butterfield buffer. Next, 1.0 and 0.1 mL of test solutions ) in 1 L of distilled water were adjusted to a pH of 7.2 ± 0.1. Samples (10 mL) were dispensed into test tubes with fermentation tubes and sterilized at 121 °C for 15 min. Test solutions were incubated with BGLB medium at 35–37 °C for 48 ± 3 h, until gas was generated. Endo agar  was dissolved in 1 L of distilled water, adjusted to pH 7.4 ± 0.2, and sterilized at 121 °C for 15 min, or Eosin methylene blue agar  was dissolved in 1 L of distilled water, adjusted to pH 6.8 ± 0.2, and sterilized at 121 °C for 15 min. One typical or two or more atypical colonies in Endo agar or Eosin methylene blue agar were dissolved in nutrient agar  in 1 L of distilled water, adjusted to pH 6.8 ± 0.2, sterilized at 121 °C for 15 min, and incubated at 35–37 °C for 24 ± 2 h. Coliforms were identified in colonies cultured on nutrient agar using the VITEK 2 test Compact automated ID/AST instrument .Escherichia coli: Samples were cut finely using sterile scissors and tongs, and then randomly selected pieces (25 g) were homogenized in 225 mL of sterile Butterfield buffer. Test solutions (1 mL) according to preparation in 1 mL of distilled water with EC Broth  in 1 L of distilled water were adjusted to pH 6.9 ± 0.2. Next, 10 mL portions were dispensed into test tubes containing fermentation tubes, sterilized at 12 °C for 15 min, and then incubated at 44 ± 1 °C for 24 ± 2 h. Fermented material was inoculated onto EMB medium from the fermentations tubes and cultured at 35–37 °C for 24 ± 2 h, and then typical colonies were inoculated on ordinary agar medium and cultured at 35–37 °C for 24 ± 2 h. Colonies cultured on normal agar medium that were positive for E. coli were identified using VITEK 2 Compact.2O, 5 g; and agar 20 g) in 950 mL of distilled water, which was sterilized at 121 °C for 15 min, and then the pH was adjusted to 7.2. Glossy black colonies surrounded by transparent bands were placed on plain agar medium  and incubated at 35–37 °C for 18–24 h. Staphylococcus aureus was detected in cultured colonies using VITEK 2 Compact.Samples were into small pieces using sterile scissors and tongs, and then randomly selected pieces (25 g) in 225 mL of 10% NaCl were added to TSB medium  in 1 L of distilled water, adjusted to pH 7.3 ± 0.2, and sterilized at 121 °C for 15 min. Enrichment culture proceeded at 35–37 °C for 18–24 h on Baird–Parker agar  so that the total inoculum was 1 mL. The medium was completely dried to ensure that the inoculum was wholly absorbed into the medium and left for 10 min, before incubation at 35–37 °C for 48 ± 3 h; then, glossy black colonies surrounded by a transparent band were counted. Five or more typical colonies were selected from the counted plate, inoculated onto ordinary agar medium, and cultured at 35–37 °C for 18–24 h. Colonies positive for S. aureus were determined by multiplying the number of confirmed bacteria by the dilution factor.Chromaticity was analyzed using a CR-400 Konica colorimeter . The L*, a*, and b* values were measured three times and used to determine the average value. At this time, L* = 94.8, a* = 0.0, and b* = 2.9 of the white plate used .Analysis of the physical properties of the developed substitute meat patty product was measured using a texture analyzer  for patties cut to a size of 2 cm × 2 cm × 2 cm. Analysis conditions were as follows: pretest speed, 2.0 mm/s; test speed, 1.0 mm/s; and posttest speed, 2.0 mm/s; and measured using a cylindrical probe. Hardness, adhesiveness, springiness, cohesiveness, chewiness, and resilience were measured three times and the average value was used .p < 0.05).All experimental data are shown as the mean values with standard deviation and were statistically analyzed using SPSS version 18.0 . Mean values were compared using one-way analysis of variance, and the significance of differences between them was analyzed using Duncan multiple range tests (p < 0.05). Fat content was highest in the order of CMPs, CVPs, and SMPs . The crude content of CVPs was significantly higher among the three patties . The pH range was 5.59–6.12; that of CMPs was low, at 5.59 ± 0.10. The protein contents of substitute meat patties containing leghemoglobin are similar to those of animal patties, but have lower fat and carbohydrate contents [contents . The pH contents . The moicontents . In addicontents . Howevercontents ,35. Furtcontents . Therefocontents ,35, the p < 0.05). We found 0.8 ± 0.01 g of glucose/100 g for SMPs and 0.1 ± 0.01 g/100 g for CVPs and CMPs, as well as 1.7 ± 0.01 g of sucrose and 0.7 ± 0.01 g of maltose/100 g for SMPs and 0.6 ± 0.01 g of sucrose/100 g for CVPs (p < 0.05).p < 0.05). Vitamin C was undetectable in the three types of patties. We detected 2.3 ± 0.01, 4.8 ± 0.01, and 1.0 ± 0.01 g of vitamin E/100 g for SMPs, CVPs, and CMPs, respectively (p < 0.05).p < 0.05). The trans fat and cholesterol contents were 81.6 ± 0.01 g/100 g for CMPs, but were undetectable in SMPs and CVPs (p < 0.05). Levels of ω-3 fatty acids were 64.0 ± 0.01, 55.8 ± 0.01, and 37.5 ± 0.01 g/100 g for SMPs, CVPs, and CMPs. Levels of ω-6 fatty acids were significantly higher (4984.0 ± 0.01 g/100 g for CVPs) than those of any other fatty acids (p < 0.05). In this study, trans fat and cholesterol content were not observed in SMPs and CVPs, but were high in CMPs at 81.6 ± 0.01 g per 100 g. ω-3 fatty acid and ω-6 fatty acid showed the highest levels in SMPs at 64.0 ± 0.01 g and 635.9 ± 0.01 g per 100 g, respectively. As the amount of okara added increases, total saturated fatty acids and cholesterol decrease in pork patties, and unsaturated fatty acids increase [increase . Park etincrease  reportedp < 0.05). In particular, the contents of the essential amino acids, leucine > lysine > phenylalanine > threonine > isoleucine, were high in SMPs and CVPs, whereas those of lysine > threonine > valine > phenylalanine > isoleucine were high in CMPs. The non-essential glutamic acid contents were 2452.1 ± 0.01, 2427.8 ± 0.01, and 2780.7 ± 0.01 mg/100 g for SMPs, CVPs, and CMPs, respectively (p < 0.05). Among the non-essential amino acids, all three patties contained glutamic acid > aspartic acid > arginine, but urea, L-hydroxyproline, sarcosine, L-citrulline, DL-2-aminobutyric acid, DL-3-aminoisobutyric acid, DL-plus-allo-δ-hydroxylysine, L-1-methylhistidine, L-3-methylhistidine, and L-carnosine were all undetectable. However, the L-amino acids contents were as follows: L-alanine (41.9 ± 0.01 mg/100 g), L-lysine (34.8 ± 0.01 mg/100 g), and L-leucine (33.3 ± 0.01 mg/100 g) for vegetable SMPs; L-alanine (54.0 ± 0.01 mg/100 g), L-glutamic acid (51.7 ± 0.01 mg/100 g), L-cystine (27.0 ± 0.01 mg/100 g), L-carnosine (141.4 ± 0.01 mg/100 g) for CVPs; and L-alanine (30.6 ± 0.01 mg/100 g) for CMPs. The content of taurine was the highest at 22.0 ± 0.01 mg/100 g of patty (p < 0.05). In this study, we judged that there is no significant difference in the amino acid composition of SMPs compared to CVPs. Amino acids are used to predict palatability by heating them with nonvolatile compounds such as peptides, amines, nucleic acids, sugars, and organic acids [ic acids ,40. Aspaic acids , the mosE. coli was undetectable in all three types of patties in the qualitative evaluation. In addition, qualitative and quantitative findings of Staphylococcus aureus were also negative in all three patties. The total numbers of bacteria and numbers of coliforms significantly increase in pork patties with added parsley powder depending on the storage period, but these values do not significantly differ among groups with additives [dditives . The totdditives , and Readditives  found thp < 0.05). The L* values were 34.10 ± 1.45, 33.30 ± 0.50, and 27.90 ± 1.77, in SMPs, CVPs, and CMPs, respectively (p < 0.05). The a* of the three types of patties was significantly higher in CMPs (9.80 ± 0.17) than that in SMPs and CVPs . For b*, SMPs, CVPs, and CMPs were irradiated at 8.70 ± 0.30, 10.30 ± 0.45, and 10.40 ± 1.32, respectively (p < 0.05). The b value was the lowest in SMPs. Substitute meat patties developed by adding leghemoglobin have low lightness and yellowness but high redness [ redness . Adding p < 0.05). Values for physical properties such as chewiness and resilience were significantly higher for CVPs and CMPs than those for SMPs (p < 0.05). However, springiness and cohesiveness did not significantly differ among the three patties. The hardness, chewiness, and resilience of SMPs were lower than those of CVPs and CMPs. In SMPs developed by adding leghemoglobin, physical properties such as hardness, resilience, cohesiveness, and chewiness, excluding resilience, did not significantly differ among CVPs and animal patties. In particular, adding dietary fiber affected the physical properties of SMPs by increasing water retention [etention . The haretention , and Kasetention  reportedetention . Howeveretention .A. dioicus. Considering the results of this study, it is judged that there is no significant difference in amino acid composition in SMPs compared to CVPs. The results of the qualitative evaluation of E. coli and the qualitative and quantitative evaluation of Staphylococcus aureus were all negative. The chromaticity of the developed patties was high in L*, and low in a* and b*. The hardness, chewiness, and resilience of the developed substitute meat patties were observed to be lower than those of vegetable and animal patties. It is believed that the results of this study can provide good basic data for future research on meat alternatives.In this study, a substitute meat patty was developed using


The responsivity, detectivity, and external quantum efficiency of the as-fabricated detector were 5.07 mA/W, 5.49 × 1011 Jones, and 24.8%, respectively. These findings demonstrate that the solution process employed in this study is suitable for the fabrication of mixed cation–halide perovskites which show immense potential for use as photodetectors.Photodetectors convert optical signals into electrical signals and demonstrate application potential in various fields, such as optical communication, image detection, environmental monitoring, and optoelectronics. In this study, a mixed cation–halide perovskite-based ultraviolet C photodetector was fabricated using a solution process. The higher the mobility of the perovskite carrier, which is one of the factors affecting the performance of electronic power devices, the better the carrier diffusion. The on/off ratio and responsivity indicate the sensitivity of the response, and together with the detectivity and external quantum efficiency, these parameters demonstrate the performance of the detector. The detector fabricated in this study exhibited a mobility of 202.2 cm A photodetector is a device that converts an optical signal into an electrical signal and exhibits a high detection rate, allowing the detection of weak optical signals ,2. PhotoWhen fabricating a UVC photodetector, it is necessary to consider whether a low-temperature and simple process is possible. This is because it is essential to develop sustainable electronic devices to reduce the impacts on the environment of energy consumption and waste ,16. Low Various characteristics of photodetectors, such as stability, light absorption, carrier mobility, band gap, reactivity, detectability, and on/off ratio, are dependent on the material composition. Accordingly, photodetectors comprising various materials have been investigated . PhotodeDL values) [Recently, perovskites have attracted attention in the field of optoelectronic devices, such as light-emitting diodes and solar cells ,20, owin values) . In addi values) ,23.3) has attracted attention as a material for developing hybrid organic–inorganic perovskites owing to its excellent photoelectric properties [3 perovskites exhibit phase instability at high temperatures and are more sensitive to moisture and light than FA-based perovskites, leading to the separation of halide components [3) have a higher thermal stability than MAPbX3 [3 exhibits low phase stability at room temperature and can be converted from the “black” α-phase to the “yellow” δ-phase [Methylammonium (MA) and formamidinium (FA) cations are commonly used in perovskite light absorption layers ,24, and operties . Howevermponents ,27. Formn MAPbX3 ; however δ-phase ,30.+ into FAPbI3-based perovskite via the crystal nucleation mechanism prevents the formation of δ-FAPbI3 [− improves phase stability [− with Br− maintains excellent light absorption properties and increases the carrier lifetime and mobility [DL. Therefore, mixed cation–halide perovskites have potential as materials for use in high performance UVC photodetectors with a low energy process and cost.Therefore, perovskite materials comprising only pure cation MA or FA are not optimal owing to their instability. However, the incorporation of MAδ-FAPbI3 , and thiδ-FAPbI3 . Additiotability ,34. The mobility . The mix3)1−x(MAPbBr3)x  photodetectors were fabricated by adding MAPbBr3 to FAPbI3, considering the low thermal stability of MABr at different mol% ratios. The fabricated mixed cation–halide perovskite-based photodetector exhibited improved stability, photocurrent, and responsiveness compared with a pure FAPbI3 photodetector. The performance of the photodetector was determined by investigating various characteristics, including responsivity, detectivity, carrier mobility, light absorption, dark environment, and current under 254 nm irradiation. The responsivity, detectivity, and external quantum efficiency (EQE) of the as-fabricated (FAPbI3)1−x(MAPbBr3)x-based photodetectors were 5.07 mA/W, 5.49 × 1011 Jones, and 24.8%, respectively, under 254 nm light irradiation with a −2 V bias and 0.6 mW/cm2 intensity. This value is more than six times larger than that of a pure FAPbI3 photodetector. These results show that the mixed cation–halide perovskite provides improved optical properties compared with the pure FAPbI3 perovskite, and therefore, is a promising candidate for UVC photodetector fabrication.In this study, , toluene (99.8%), dimethyl sulfoxide , FA iodide (FAI), MA iodide (MAI), and MA chloride (MACl).The materials and chemicals used were as follows: quartz indium tin oxide (ITO) patterned glass  with a thickness of 150 nm, lead (II) iodide , 3)1−x(MAPbBr3)x films. To remove organic matter, the ITO quartz substrate was ultrasonically cleaned using a neutral detergent, water, acetone, ethanol, and distilled water for 20 min each. After drying in an oven at 80 °C for 20 min, UV-ozone treatment was performed for 30 min to further remove moisture. A (FAPbI3)1−x(MAPbBr3)x solution was prepared by dissolving PbI2 and PbBr2 (1.4 M), FAI, and MABr (1.4 M) in DMF (1 mL), which was then mixed with 100 µL of DMSO. The perovskite precursor solution was then stirred for 1 h at 23 °C. MACl was then added, up to 40 mol% of FAI, and the solution was further stirred for 30 min. The solution was deposited on a quartz substrate via spin coating at 4000 rpm for 20 s, over which toluene was dropped for the last 10 s. After spin-coating, the thin film was annealed at 150 °C for 15 min.The crystal structure of the perovskite film was investigated using X-ray diffraction  with a scan rate of 3.00°/min. The morphology of the photodetector surface was observed using scanning electron microscopy . The light absorptivity of the device was measured using UV-visible (UV-vis) spectroscopy . The electrical response of the perovskite photodetector was measured with a combined source and measurement meter  and a UV lamp   under 254 nm irradiation.3)1−x(MAPbBr3)x films were fabricated with different ratios of x . Several characteristic perovskite peaks were observed in the XRD patterns. The peaks at ~11.7°, ~12.7°, and ~14.0° of the diffractogram were assigned to the δ-FAPbI3, PbI2, and α-FAPbI3 phases, respectively. Pure FAPbI3 (x = 0) exhibited the highest δ-FAPbI3 peak intensity and possessed low phase stability owing to the high concentration of δ-FAPbI3. As the MAPbBr3 content of the film increased, δ-FAPbI3 was converted to α-FAPbI3 [3. However, the reduction of the δ-FAPbI3 content did not improve the crystallinity of the all perovskite films. Although the α-FAPbI3 peak intensity of the (FAPbI3)1−x(MAPbBr3)x film increased up to x = 0.05, it started to decrease at x = 0.07 and exhibited the smallest peak intensity at x = 0.1. This is because the low thermal stability of MABr led to the degradation of MAPbBr3. As the “black” α-FAPbI3 phase is thermodynamically stable above 150 °C, FAPbI3 was annealed at 150 °C [+ has low thermal stability [3 thermally decomposes to form a PbI2 phase [2 peak intensity was observed when x = 0.1, as shown in α-FAPbI3 , which dt 150 °C . Howevertability , and theI2 phase ,35. The 3)x increased, the peak shifted to a high diffraction angle. This shows that the perovskite films were successfully formed, as lattice shrinkage is expected to occur when FA+ (2.79 Å) is replaced with MA+ (2.70 Å) [− (1.96 Å) replaces I− (2.2 Å), which shifts the corresponding peak to high diffraction angles [(2.70 Å) . Moreoven angles ,36.3 decreased compared to that of the pure FAPbI3, and the phase stability improved. In particular, the crystallinity of (FAPbI3)0.95(MAPbBr3)0.05 was the highest among the synthesized samples. However, when more than 7 mol% MAPbBr3 was added, a decrease in crystallinity, owing to the formation of a residue, was observed. This indicates that the overall crystallinity varies depending on the composition of the mixed cation perovskite.The results shown in 3)1−x(MAPbBr3)x perovskite films; it confirms that a change in the composition ratio x affects the film surface. The average diameter lengths of the grain of (FAPbI3)1−x(MAPbBr3)x films  were 1.629, 1.496, 1.465, 0.922, and 0.815 um. In + (2.70 Å) has a smaller particle size than FA+ (2.79 Å), and Br− (1.96 Å) has a smaller particle size than I− (2.2 Å) [+ and Br− replace FA+ and I− [ (2.2 Å) ,35; as s+ and I− . However3 was decomposed, at because of the high annealing temperature of 150 °C, forming PbI2 and PbBr2. This suggests that the perovskite was not well-formed at this composition ratio. A small grain size with many crystals observed on the surface leads to deterioration of performance. As residues are produced and replaced with smaller ions, resulting in a smaller grain size, the mobility of carrier transport decreases, which, in turn, increases carrier scattering, leading to poor performance [formance .3)1−x(MAPbBr3)x films . All (FAPbI3)1−x(MAPbBr3)x samples displayed a strong absorbance at 200–500 nm. This demonstrates that the (FAPbI3)1−x(MAPbBr3)x-based photodetector sufficiently detected light in the UVC region. The absorbance increased as the value of x increased but decreased when the value of x exceeded 0.07. This suggests that certain (FAPbI3)1−x(MAPbBr3)x compositions (x = 0.03 and 0.05) have better light absorption characteristics than pure FAPbI3.3)1−x(MAPbBr3)x  films. The sharp band energies indicate the presence of a direct bandgap. Semiconductors with wide bandgaps can absorb and emit high-energy photons, enabling the fabrication of high-performance photonic devices, including UV detectors [3 and (FAPbI3)1−x(MAPbBr3)x  was estimated to be 1.45 eV and 1.52 eV, respectively. The increase in the band gap was due to the structural distortion caused by the stress on the Pb–I bond resulting from the incorporation of Br [2 residues are likely to be present when x = 0.1 [etectors . Based oon of Br . Changeson of Br . The lar x = 0.1 .3)1−x(MAPbBr3)x thin films . The resistivity of (FAPbI3)1−x(MAPbBr3)x decreased as the x value increased, except for x = 0.1. In particular, (FAPbI3)0.95(MAPbBr3)0.05 displayed the lowest resistivity, i.e., 0.00157 Ω cm.3)1−x(MAPbBr3)x films had carrier concentration values higher than 1013 cm−3. The current depends on the mobility; the higher the mobility, the higher the current [3)1−x(MAPbBr3)x films were 120.3, 172.2, 202.2, 144, and 91.9 cm2/V.s for x values of 0, 0.03, 0.05, 0.07, and 0.1, respectively.Carrier concentration and mobility are significant parameters for semiconductor materials and electronic power devices . All  of the (FAPbI3)1−x(MAPbBr3)x films . D is related to the mobility, as shown in D is defined using the Einstein relation D = μKT/q, where μ is mobility, K is the Boltzmann constant, T is the temperature of the sample, and q is the amount of charge [D of the (FAPbI3)1−x(MAPbBr3)x films increased as the x value increased, except for x = 0.1. The charge carrier DL was calculated as DL = √Dτ, where τ is the lifetime [DL are proportional, and high mobility leads to an increase in the D and DL values. When I− is replaced by Br−, the lattice parameters shrink, leading to an increase in the photogenerated carrier lifetime and the charge carrier mobility [3)1−x(MAPbBr3)x film had the best electrical properties such as carrier concentration and mobility when x = 0.05.f charge . The D olifetime . The mobmobility ,45. Comb3)1−x(MAPbBr3)x  photodetectors, the current-voltage (I–V) characteristics were measured from −2 V to 2 V at a scan rate of 0.1 V. Measurements were performed in a dark environment and under a 254 nm light source with an output of 0.6 mW/cm2. In the perovskite photodetector, a Schottky barrier formed owing to the contact with the ITO electrode, and the current flowed through it.In addition, to analyze the performance of the (FAPbI3)1−x(MAPbBr3)x photodetectors were barely able to detect 254 nm light at 0.6 mW/cm2 in a dark environment. However, the (FAPbI3)0.95(MAPbBr3)0.05 photodetector generated significant photocurrents of −5.61 µA at −2 V and 4.2 µA at 2 V under 254 nm irradiation with an output of 0.6 mW/cm2, as shown in 2 were investigated. R) of the films. R is the main parameter that indicates the performance of the photodetector and the response of the device to the irradiated light. R is defined as R = (254I − darkI)/P × S mA/W [254I is the current when irradiated with 254 nm light, darkI is the dark environment current, P is the power of the incident light, and S is the irradiated area of the device [R gradually decreased as the bias decreased owing to the change in the photon-charge conversion efficiency of the photodetector [2, the R values of (FAPbI3)1−x(MAPbI3)x were 0.85, 2.77, 5.07, 0.68, and 0.26 mA/W at −2 V when x was 0, 0.03, 0.05, 0.07, and 0.1, respectively.To evaluate the performance of the photodetector, the significant parameters of the detector under a light of 254 nm with an output of 0.6 mW/cm× S mA/W , where Ie device . R gradudetector . Under 23)1−x(MAPbBr3)x photodetectors in the reverse bias region from −2 V to 0 V. A high on/off ratio is related to good performance in a photodetector [detector . The on/3)1−x(MAPbBr3)x photodetectors. An on/off switching test was performed to observe transient photoresponses under 254 nm radiation with an 0.6 mA/cm2 intensity at a 2 V bias. Response times, such as rise and decay times, could be inferred from the relative speed of response of the detector to changes in the optical signal. 3)1−x(MAPbBr3)x (x = 0.03 and 0.05) photodetectors exhibited a higher photo-response than pure FAPbI3. In addition, 3)1−x(MAPbBr3)x photodetectors had the smallest rise and decay times, i.e., 0.048 and 0.1 s, respectively. Therefore, the (FAPbI3)0.95(MAPbBr3)0.05 photodetector had the fastest photo-response among all the photodetectors, and is highly sensitive.3)0.95(MAPbBr3)0.05 photodetector. The on/off switch was irradiated for 1000 s using a 254 nm light source with an output of 0.6 mW/cm2. In the first on/off iteration, the photocurrent was 523 nA. The light was irradiated for 5 s, and the photodetector was cycled on/off 100 times. After 100 cycles, the photocurrent decreased slightly to 468 nA, but remained constant from the 16th cycle to the 100th cycle. Although a slight decrease in photocurrent was observed, the maintenance of the photocurrent over a relatively long time proves its reproducibility.EQE, which is the number of electrons produced per incident photon, is another important parameter that indicates the detector performance. EQE is defined as Rhc/eλ, where R is the responsivity, h is the Planck constant, c is the speed of light, e is the electron charge, and λ is the wavelength of the light [EQE from −2 V to 2 V bias as a box plot under 254 nm light irradiation with 0.6 mA/cm2 intensity. EQE increases up to x = 0.05 and decreases at x = 0.07. These results exhibit the same pattern as the other measurements  and demonstrate that among the (FAPbI3)1−x(MAPbBr3)x  photodetectors, the (FAPbI3)0.95(MAPbBr3)0.05 composition exhibited the best optical properties.he light . Figure D* is an important photodetector parameter, defined as D* = R/(2darkqJ)1/2, where R is the responsivity, q is the electron charge, and darkJ is the current density of the dark current [R, D, and EQE, of UVC photodetectors fabricated using various materials are listed. The R and EQE values of the (FAPbI3)0.95(MAPbBr3)0.05 photodetector fabricated in this study were superior to those of photodetectors reported in other studies. This suggests that the perovskite-based UVC photodetectors fabricated in this study are promising candidates in the field of UVC sensing, as the perovskites can be fabricated using a simple process at lower temperatures than those of previously reported photodetectors. In addition, the R and EQE values were superior to those of perovskite-based photodetectors reported in other studies, while the D* values were similar. These results indicate that (FAPbI3)0.95(MAPbBr3)0.05 is a promising material for developing perovskite-based photodetectors.Detectivity  current ,38. In T3)1−x(MAPbBr3)x -based photodetectors were investigated. (FAPbI3)1−x(MAPbBr3)x films exhibited the most improved stability and light absorption when the MAPbBr3 ratio was 5 mol%. The (FAPbI3)0.95(MAPbBr3)0.05 UVC photodetector exhibited an EQE of 24.8%, a mobility of 202.2 cm2/Vs, a response of 5.07 mA/W, and a detection rate of 5.49 × 1011 Jones. These are excellent parameter values compared with previously reported values for perovskite-based photodetectors fabricated using the solution process. By applying the process described in this study, it is possible to fabricate a photodetector with improved optical properties through a low-energy process. This suggests that mixed cation–halide perovskite-based photodetectors have significant potential for use in UVC photodetectors.Mixed cation–halide perovskite (FAPbI


Our previous studies revealed that certain endogenous low molecular weight lipids have potent anti-diabetic actions. Of all, arachidonic acid (AA) and its anti-inflammatory and inflammation resolving metabolite lipoxin A4 (LXA4) are the most potent anti-diabetic molecules. Similar anti-diabetic action is also shown by resolvins. In our efforts to identify other similar lipid based anti-diabetic molecules, we investigated potential anti-diabetic action of protectin DX that also has anti-inflammatory and inducer of inflammation resolution action(s) like LXA4. Protectin DX {10(S),17(S)-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid, also called as 10(S),17(S)-DiHDoHE)} prevented the development of streptozotocin-induced type 1 and type 2 diabetes mellitus in Swiss male albino mice. Protectin DX showed potent anti-inflammatory, antioxidant and anti-apoptotic actions that could explain its anti-diabetic action. In view of these beneficial actions, efforts need to be developed to exploit PDX and other similar compounds as potential anti-diabetic molecule in humans. Diabetes mellitus (DM) is common across the globe. Of the two major types of DM, type 1 and type 2, the latter is more common. Both type 1 and type 2 DM are associated with inappropriate inflammation, immune dysfunction and gut dysbiosis.Type 1 DM is an autoimmune disease in which pancreatic β cells are infiltrated by T cells, macrophages and NK cells that secrete excess pro-inflammatory cytokines IL-6, TNF-α, MIF (macrophage migration inhibitory factor), and IFN-γ which induce the production of excess of reactive oxygen species (ROS). Both cytokines and ROS induce apoptosis of β cells and consequently lead to the development of insulin deficient type 1 DM . It is sin vitro and animals with STZ-induced type 2 DM have low plasma levels of LXA4 and BDNF , alpha-linolenic acid (ALA), gamma-linolenic acid (GLA), dihomo-GLA (DGLA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have potent anti-diabetic actions especially against chemical -induced type 1 DM –26. Of aand BDNF . These rand BDNF , 31–33. and BDNF –35. Thisand BDNF , 37. TheAll reagents and chemicals were purchased from Sigma Aldrich Chemical Pvt Ltd (USA). 10(S), 17(S) DiHDHA  was obtained from Cayman Chemical Company (USA). ELISA kits were procured from BioLegend (USA).0C room temperature with 12-hr dark and 12-hr light cycle. The study was approved by the Institutional Animal Ethics Committee.6-8 weeks old male Swiss albino mice procured from National Institute of Nutrition  were used in this study. Animals were acclimatised for a week before the start of the study. The animals were housed at 25 ± 2th, 14th, 21st and 28th day of the study. Experiment was terminated on 30th day of the study after OGTT estimation. Blood was collected by cardiac puncture. Plasma and pancreas organ obtained and stored at -80°C for further analysis.Animals weighing ~30 g were divided into 4 groups with 6 animals in each group: control, diabetic (Type 1DM group), PDX and PDX + Type 1DM. Control group received PBS as vehicle. T1DM was induced by a single dose of streptozotocin  on day 0. PDX group was treated initially with PDX  for 5 consecutive days (Day 0 to Day 4) and subsequently once in a week for the next 4 weeks. PDX + Type 1DM group received STZ and PDX simultaneously on day 0 and subsequently once in a week for the next 4 weeks , PDX and PDX + Type 2DM. Control group received PBS as vehicle. Type 2 DM was induced by administration of STZ  on day 0 and day 2. Nicotinamide  was injected 15 min prior to STZ administration on day 0. PDX group received PDX  for 5 consecutive days (Day 0 to Day 4) and subsequently once in week for 4 weeks. PDX + Type 2DM group received simultaneously NAD, STZ and PDX , the animals were given an oral glucose load (2 g/kg) by gavage. Further blood samples were collected at the end of 30, 60, 90 and 120 min from the retro orbital plexus. ISI was computed by HOMA IR, HOMA B and QUICKI (Quantitative insulin check index) methods in Type 1 DM and Type 2 DM studies.Plasma and pancreatic lysates were used for the estimation of antioxidant enzymes: catalase, superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione peroxidase (GPX) as described previously –28. LipiMice plasma samples were analysed for the concentrations of insulin , TNF-α , IL-6  and IL-10  as per manufacturer instructions.Total RNA was isolated from mice pancreatic tissue by using Trizol method; cDNAs were synthesized by reverse transcriptase from 1 µg of total RNA using SuperScript First-Strand Synthesis for qRT–PCR (Invitrogen) kit by following manufacturer instructions. Semiquantitative Polymerase Chain Reaction (PCR) was performed to study the expression of Bcl2, Bax, NFκB, IκB, iNOS and β-actin as follows: 94°C for 2 min initial denaturation, 94°C for 30 s denaturation, 30 s annealing, followed by 72°C for 30 s extension and 72°C for 5 min final extension, and overall, 35 cycles were performed. PCR products were observed and analysed by electrophoresis on 1.5% (w/v) agarose gel in 1X TAE buffer at 100V. Quantification was performed by taking the ratio of gene of interest and β-actin and calculated as percentage comparing with the respective control. The details of primer genes, respective annealing temperatures and product size are mentioned in Experiments were performed by taking six animals in each group. Analysis was done twice in triplicate to ensure the repeatability of the results obtained. All results are expressed as mean ± SEM. The obtained values were analysed by paired t-test with equal variance using Microsoft Excel statistical analysis tool. Additionally, the results were also analysed using ANOVA test wherever necessary which showed that the statistical significance was like the Student’s T test.th day of the study. In contrast, treatment with PDX improvement bodyweight by 9% in Type 1 DM animals  fasting blood glucose levels to near normal levels in both Type 1  in both glucose intolerance and insulin resistance tests Table 2 vs STZ: 14.10 ± 2.07 vs PDX + STZ: 6.11 ± 0.89) in type 1 DM animals. In a similar fashion, PDX treatment reduced significantly levels of plasma NO in PDX + STZ group (control: 20.65 ± 1.43 vs PDX + STZ: 15.22 ± 1.92); plasma LPO (control: 1.13 ± 0.01 vs 0.81 ± 0.12) and enhanced plasma SOD (control: 2.66 ± 0.25 vs PDX + STZ: 5.42 ± 0.10) and GST (control: 1.47 ± 0.11 vs PDX + STZ: 2.22 ± 0.22) activities and reduced pancreatic LPO levels (control: 3.79 ± 0.10 vs PDX + STZ: 3.04 ± 0.29); and reduced SOD (control 23.12 ± 1.51 vs PDX + STZ: 15.79 ± 1.68); GST (control: 6.10 ± 0.22 vs PDX + STZ: 4.58 ± 0.44) and GPX activities (control: 5122.24 ± 337.69 vs PDX + STZ: 3822.27 ± 232.91) significantly in type 2 DM animals  which suppressed gluconeogenic program -induced cytotoxicity but also prevented insulin resistance in C2C12 myotubes by decreasing protein kinase C (PKC)-θ activation and restoring cellular acylcarnitine profile, insulin-dependent AKT phosphorylation and glucose uptake. It was reported that DHA suppressed the production of IL-6 and TNF-α that is responsible for its beneficial action. In experimental animals that were fed high-cholesterol–high-sucrose diet, supplementation with DHA enhanced the insulin-dependent AKT phosphorylation and reduced the PKC-θ activation in skeletal muscle. These results imply that DHA prevents lipotoxicity and inflammation . It was ammation . These isistance . These rsistance . In a ex program . In addisitivity . These rsitivity  and the sitivity –47 depensitivity .C. Elegans gene, fat-1, encoding an n-3 fatty acid desaturase catalysing the conversion of n-6 to n-3 PUFAs  formed from AA, presumably by displacing AA from the cell membrane lipid pool that, in turn, gets converted to LXA4. In studies that reported the beneficial action of EPA or DHA in DM  to M2 (anti-inflammatory) type, instruct suppressive immune cells and adaptive immune responses to deal with subsequent encounters, able to induce tissue repair and restore homeostasis . In the Since LXA4, resolvins , 36, 37 The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author/s. The datasets presented in this study are included in the article.The animal study was reviewed and approved by Ethics committee of GVP Medical College. Written informed consent was obtained from the owners for the participation of their animals in this study.UD generated the idea, planned the studies, provided the facilities, supervised the studies, interpreted the data, wrote the manuscript. RP and SP performed the studies, wrote the manuscript. All authors contributed to the article and approved the submitted version.


Vibrio sp. Ni1. The ORF of the algB gene has 1824 bp, encoding 607 amino acids. Homology analysis shows that AlgB belongs to the PL7 family. There are two catalytic domains with the typical region of QIH found in AlgB. The purified recombinant enzyme of AlgB shows highest activity at 35 °C, pH 8.0, and 50 mmol/L Tris-HCl without any metal ions. Only K+ slightly enhances the activity, while Fe2+ and Cu2+ strongly inhibit the activity. The AlgB preferred polyM as substrate. The end products of enzymatic mixture are DP2 and DP3, without any metal ion to assist them. This enzyme has good industrial application prospects.The applications of alginate lyase are diverse, but efficient commercial enzymes are still unavailable. In this study, a novel alginate lyase with high activity was obtained from the marine bacteria  Alginatcing end ,4. In a Pseudomonas aeruginosa [Original research on alginate lyase was from ruginosa ,6,7. Curruginosa ,11,12,13ruginosa ,14,15.The products produced by alginate lyases, alginate oligosaccharides, have diverse bioactivities, such as anti-oxidant , anti-inVibrio sp. Ni1 and over-expressed in Escherichia coli. The purified recombinant enzyme has high activity toward alginate, poly M and poly G at high temperature and pH, independent of ions. The properties of the recombinant enzyme and its products were analyzed.In the present study, a novel alginate gene was cloned from Vibrio sp. Ni1 and their alginate-degraded circles were observed and measured 2SO4 precipitation. The majority of the extracellular proteins were exhibited in the range of 20–75 kDa in SDS-PAGE gel . The amino acid sequence displays high similarity of 93.87% identity with three alginate lyases . These three alginate lyases have not been characterized. Other alginate lyases have about 70–80% identities, such as AIY22661.1 from Vibrio sp. W13, 81.13%; and WP_118120558.1 from Vibrio sp. Dhg, 77.86% .Based on the sequencing result of the partial lgB gene . It cons, 77.86% a. The alAlgB is classified in the polysaccharide lyase family 7 (PL7). Conserved domain analysis revealed two structural domains in AlgB b. Each salgB gene was cloned into the expression vector pET32a (+) and transformed to E. coli BL21. The fusion proteins of AlgB were soluble when expressed in host cells and purified by nickel affinity chromatography. Since the mass of the fusion tag protein (TrxA) is approximately 20 kDa, the recombinant AlgB is about 88 kDa. The SDS-PAGE analysis is consistent with the prediction . E. coli BL21(DE3) was used as an expression host. The sodium alginate donated by Haizhilin Biotechnology Development Co. Ltd.  was used as the substrate to test alginate lyase activity. The viscosity of this sodium alginate sample was 1250 mPa.s. The sodium alginate used to prepare medium was from Solarbio .The strain of Vibrio sp. Ni1 grown in liquid Luria-Bertani (LB) medium, was transferred to an alginate plate containing 1% (w/v) sodium alginate, 0.2% (w/v) glucose, 0.3% (w/v) NaCl, 0.2% (w/v) NaNO3, 0.05% (w/v) MgSO4·7H2O, 0.1% (w/v) K2HPO4, 0.01% (w/v) CaCl2, and 1% (w/v) agar, pH 7.5. The plate was incubated at 28 °C for 48 h. The plate was stained with 1 mL Lugol’s reagent (5% (w/v) I2, 10% (w/v) KI) for 1 min [A 5 μL aliquot from a culture of or 1 min . The plaVibrio sp. Ni1 were cultured in the liquid fermentation medium containing 1% (w/v) sodium alginate, 3% (w/v) NaCl, 0.2% (w/v) NH4Cl, 0.05% (w/v) MgSO4·7H2O, 0.1% (w/v) K2HPO4, and 0.01% (w/v) CaCl2, pH 7.5, at 28 °C for 24 h. The supernatant was harvested by centrifugation at 4 °C, 8000× g, for 10 min. Solid (NH4)2SO4 was gradually added to the supernatant to a final concentration of 70% (w/v), then stirred continuously at 4 °C for 12 h. The protein precipitant was collected by centrifugation  then dissolved in 0.05 mol/L Tris-HCl (pH 7.5). The suspended solution was dialyzing in 0.05 mol/L Tris-HCl (pH 7.5) 12–15 h. The crude protein solution was obtained after centrifugation  to get rid of insoluble impurity inside the dialysate, then stored at 4 °C. The cells of 4HCO3. The gel slice was dried in a vacuum centrifuge. The proteins were reduced in-gel with dithiothreitol (10 mmol/L DTT and 100 mmol/L NH4HCO3) for 30 min at 56 °C, then alkylated with iodoacetamide (20 mmol/L IAA and 100 mmol/L NH4HCO3) in the dark at 25 °C for 30 min. The gel slice was briefly rinsed with 100 mmol/L NH4HCO3 and acetonitrile, followed by digestion with 12.5 ng/μL trypsin in 25 mmol/L NH4HCO3 overnight. The digested peptides were extracted three times with 60% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid. The extracts were pooled and dried completely in a vacuum centrifuge. The peptide mass and sequence were determined by Liquid Chromatography (LC)-Electrospray Ionization (ESI) Tandem mass spectrometry (MS/MS) in a Q Extractive mass spectrometer which was coupled to Easy nLC . The MS data were analyzed using Max Quant (Version 1.6.4.0) by searching the data against the amino acid sequence of alginate lyases from Vibrio, and the intensity of sequenced peptide in the target protein was calculated.The crude alginate lyase was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 10% acrylamide gel was stained with Coomassie blue and detained with 30% acetonitrile and 0.1 mol/L NHalgB gene. According to the sequencing result of the partial algB gene, a series of primers were designed to apply the SiteFinding-PCR method to extend the target gene [algB-QF/R was designed to amplify the full-length gene. The expression recombinant plasmid was constructed with pET32a on sites of BamH I and Xhol I and transformed into E. coli BL21(DE3). Based on the LC-MS/MS results, a pair of degenerate primers  was designed to amplify fragments of the get gene ,35. The E. coli cells harboring the recombinant plasmid pET32a-algB were cultured at 37 °C in liquid LB medium with 100 μg/mL ampicillin until OD600 reached about 0.6. Gene expression was induced with the addition of isopropyl-β-d-thiogalactoside (IPTG) to a final concentration of 0.8 mmol/L and then grown at 20 °C for 24 h. The cells were harvested by centrifugation 8000 r/min, 5 min, at 4 °C, then suspended in 0.05 mol/L Tris-HCl buffer (pH 7.4) to concentrate 1/10 volume. The cells were disrupted by ultrasonication. The lysate was harvested by centrifugation 10,000× g, 30 min, at 4 °C. The supernatant was filtered with 0.22 micrometer cellulose acetate membrane before passage through a Ni-NTA Sepharose column . The column was washed with washing buffer (0.05 mol/L Tris-HCl buffer (pH 7.5), 0.5 mol/L NaCl, 20 mmol/L imidazole), and the recombinant alginate lyase AlgB was eluted with elution buffer (0.05 mol/L Tris-HCl buffer (pH 7.5), 0.5 mol/L NaCl, 0.3 mol/L imidazole). The eluate was dialyzed in 0.05 mol/L Tris-HCl buffer (pH 7.5) overnight and analyzed by 12% SDS-PAGE. The protein concentration was measured by the Bradford method.The w/v) sodium alginate dissolved in 0.05 mol/L Tris-HCl buffer (pH 8.0) (0.9 mL) and enzyme solution (0.1 mL) at 40 °C for 30 min. Enzyme activity was calculated by constructing a standard curve with uronic acid under the same reaction conditions with DNS solution [The activity of the alginate lyase AlgB was measured in a mixture solution containing 1% (solution . One unisolution . The activity of AlgB at a specific pH range (3–10) and temperature range (4–90 °C) was determined by the enzyme assay described above using 1% alginate as substrate. The thermostability was evaluated by measuring the residual activity after different treatment time at different temperatures. Kinetic parameters of the enzyme towards alginate were calculated by the substrate at different concentrations (1–5 mg/mL). The enzyme activity was measured as described previously. 4, ZnSO4, FeSO4, CuSO4, MnSO4, CaCl2, CoCl2 and NiCl2) to a final concentration of 10 mmol/L. The parallel reaction without metal ions served as the control. The activity on different concentrations of NaCl (1–5 mol/L) was also measured.The effect of metal ions on the activity of AlgB was investigated. The reaction mixtures were added with different metal ions  were 6–8 kDa. 2O (3:2:3) and visualized by heating 80 °C for 15 min after spraying a diphenylamine/aniline/phosphate (1:1:5) reagent. To further investigate the enzymatic products, ESI-QqQ-MS/MS was conducted. Five microliter samples were injected into a triple quadrupole tandem mass spectrometry (QqQ-MS/MS)  after filtration with 0.22 µm cellulose acetate membrane. The products were detected in a negative ion mode using the following settings: pressure of the atomizer (N2), 50 psi; flow rate of the dryer (N2), 10 L/min; temperature of the dryer (N2), 350 °C; capillary voltage, 4000 V; scanning the mass range, 100–1000 m/z [The mixture of enzymatic products was purified by centrifugation  after react 12 h at optimal conditions. The degraded products of AlgB were analyzed by thin-layer chromatography (TLC)  . The enzVibrio sp. Ni1. The purified recombinant enzyme of AlgB showed highest activity at 35 °C, pH 8.0, 50 mmol/L Tris-HCl without metal ions. The AlgB preferred polyM as substrate. The predominant end products of the enzymatic reaction are DP2 and DP3. In this study, a novel alginate lyase of AlgB with high activity was obtained from Agarivorans sp. JAM and Serratia marcescens, respectively. The optima pH and temperature of AlyPL6 [Some alginate lyases can catalyze the degradation in extreme environments. The optimal pH of A1m  and AlgNf AlyPL6  and AlgHf AlyPL6  is 45 °Cf AlyPL6  and A1-If AlyPL6 . As repoVibrio splendidus OU02 [Vibrio splendidus 12B01 [Sphingomonas sp. A1 [Flavobacterium sp. UMI-01 [Aplysia Kurodai [Vibrio sp. Ni. Work to identify additional alginate lyases from this strain are in progress.Many organisms are reported to encode more than one alginate lyase gene, such as dus OU02 ,37, Vibrus 12B01 , Sphingos sp. A1 ,43, Flav. UMI-01 , and Apl Kurodai . As indi


Eucalyptus plantations have expanded toward the warm and humid regions of northern and northeastern Brazil, where Calonectria leaf blight (CLB) has become the primary fungal leaf disease of this crop. CLB can be caused by different Calonectria species, and previous studies have indicated that Calonectria might have high species diversity in Brazil. During a disease survey conducted in three commercial plantations of Eucalyptus in northeastern Brazil, diseased leaves from Eucalyptus trees with typical symptoms of CLB were collected, and Calonectria fungi were isolated. Based on phylogenetic analyses of six gene regions  and morphological characteristics, two new species of Calonectria were identified. Five isolates were named as C.paragominensissp. nov. and four were named as C.imperatasp. nov. The pathogenicity to Eucalyptus of both species was confirmed by fulfilling the Koch’s postulates.In recent decades, commercial  Calonectria species are widely distributed around the world and cause diseases in more than 335 plant species, distributed among nearly 100 plant families, including forestry, agricultural and horticultural crops , damping-off, cutting rot and root rot in commercial plantations and nurseries of Eucalyptus , and the Sphaero-Naviculate Group (C.kyotensis and C.naviculata species complexes)  .Eucalyptus, ten species isolated from soil samples of Eucalyptus plantations, seven species isolated from different plant species, six species isolated from soil samples of tropical rainforests, and one mycoparasite species . In 2020, the total area of Eucalyptus plantations was 7.47 million hectares, with an average productivity of 36.8 m3/ha per year . However, in recent decades, commercial Eucalyptus plantations have expanded toward the warm and humid regions of northern and northeastern Brazil, where CLB has become the primary fungal leaf disease of this crop , were observed and collected for fungal isolation and species characterization. On average, 50 diseased leaves were sampled from each Eucalyptus genotype, one leaf per tree, depending on the planted areas. The sampled Eucalyptus genotypes corresponded to E.urophylla, localized in the municipalities of Cidelândia  and Itinga do Maranhão , in the state of Maranhão, and to the E.grandis × E.brassiana hybrid genotype, in the microregion of Paragominas , in the state of Pará, Brazil.In February 2020, during a disease survey conducted in three commercial plantations of 2 from the transition section between healthy and diseased tissue were cut and the surface was disinfected by washing with 1% sodium hypochlorite for 1 min, with 70% ethanol for 30 s and with sterilized water three times before culture on 2% malt extract agar  plates at 25 °C. After 48 h of incubation, Calonectria-like mycelial plugs, 5 mm in diameter, were transferred to a fresh MEA plate and incubated at 25 °C until the fungus covered the plate completely. Induction of sporulation on MEA plates and single spore cultures was obtained following the procedures described by UFLA (CCDCA) at Universidade Federal de Lavras (UFLA), Minas Gerais, Brazil. Ex-paratypes were deposited as pure cultures in the Laboratory of Forest Pathology (PFC) at UFLA.Samples were stored in paper bags and transported to the Laboratory of Forest Pathology at the Universidade Federal de Lavras. From each leaf, small segments of 1 cm-1, yeast extract 2 g·L-1, sucrose 5 g·L-1) for ten days at 25 °C in the dark. The protocol described by Total genomic DNA was extracted from fresh mycelia of single spore cultures grown on malt extract broth , calmodulin (cmdA), histone H3 (his3), RNA polymerase II (rpb2), translation elongation factor 1-alpha (tef1), and β-tubulin (tub2) genes were used as DNA barcodes due to provide a stable and reliable resolution to distinguish all Calonectria species. The primers ACT-512F and ACT-783R  mix , 1 U GoTaq Flexi DNA Polymerase , 0.2 mM each primer, and 30 ng DNA template. DNA amplifications were conducted in a thermal cycler . The PCR conditions for the act, cmdA, his3, tef1, and tub2 gene regions were as follows: an initial denaturation step at 95 °C for 5 min; then 35 amplification cycles at , and a final extension step at 72 °C for 5 min. For the rpb2 gene region, a touchdown PCR protocol was used: an initial denaturation step at 95 °C for 5 min, then  × 10 cycles,  × 30 cycles, and a final extension step at 72 °C for 10 min.The PCRs were carried out in a 25 μL final volume containing molecular biology-grade water  1X PCR buffer , 2.5 mM MgClhttp://www.ncbi.nlm.nih.gov).PCR products were separated by electrophoresis at 120 V for 1 h in a 1.2% agarose gel, stained with Diamond Nucleic Acid Dye , and visualized using an ultraviolet light transilluminator. Successful PCR products were purified and sequenced in both directions using the same primer pairs used for amplification by Macrogen Inc. . Raw sequences from each gene region were edited, consensus sequences were generated using SeqAssem software ver. 07/2008 , and theCalonectria spp. obtained from GenBank  with the alignment strategy FFT-NS-i . Alignments were manually corrected using MEGA7  described by MP), maximum likelihood (ML), and bayesian inference (BI) were conducted on the individual gene regions and their concatenated dataset, depending on the sequence availability.The partition homogeneity test (TBR) branch swapping. Gaps were treated as missing data, and all characters were unordered and weighted equally. The measures calculated for parsimony included the tree length (TL), consistency index (CI), homoplasy index (HI), retention index (RI), and rescaled consistency index (RC). Statistical support for branch nodes was assessed with 1,000 bootstrap replicates.Maximum parsimony analysis was performed using PAUP 4.0b10 , with phAIC) using MODELTEST v. 3.4 , TVM + G (cmdA), TrN + G (his3), SYM + G (rpb2), TVM + I + G (tef1) and HKY + I (tub2). Statistical support for branch nodes was assessed with 1,000 bootstrap replicates. A partitioned ML analysis was performed using IQ-TREE . The BI models used were K80 + G (act), GTR + G (cmdA and his3), SYM + G (rpb2), GTR + I + G (tef1) and HKY + I (tub2). A Markov Chain Monte Carlo (MCMC) algorithm was employed, and two independent runs of four MCMC chains (three hot and one cold) were run in parallel simultaneously starting from random trees for 107 generations  and 307 generations (concatenated dataset), sampling trees every 1,000 generations. The distribution of log-likelihood scores was examined with TRACER v.1.5 (ESS), the potential scale reduction factor (PSRF), and the average standard deviation of split frequencies (ASDSF) (PP) were computed using the remaining trees. Trees were visualized in FIGTREE v. 1.4.4 (https://inkscape.org).Individual and partitioned v.3.2.7a  on XSEDEER v.1.5  to deter (ASDSF) . The firv. 1.4.4  and editGCPSR) model (as described by https://uni-tuebingen.de)   to deterngen.de) . BootstrCalonectria species isolate was determined through PCR by using the primer pairs Cal_MAT111_F/Cal_MAT111_R and Cal_MAT121_F/Cal_MAT121_R, which amplify the MAT1-1-1 and MAT1-2-1 genes using the protocol described by MSA; The mating-type idiomorph of each Calonectria species identified by phylogenetic analyses was performed as described by Morphological characterization of representative isolates of each Calonectria species was selected for inoculation. Healthy leaves of three short cut branches from an approximately eleven-month-old Eucalyptus plants were inoculated with suspensions of 1 × 104 conidia·mL-1 obtained from single spore cultures. The conidia suspensions for each isolate were prepared using the method described by Calonectriaparagominensis was inoculated on E.grandis × E.brassiana hybrid genotype and C.imperata on E.urophylla genotype. The inoculation consisted of spraying the conidia suspension until the suspension run off the leaves. Sterile water was sprayed onto healthy leaves as the negative control. The branches with inoculated leaves were covered with plastic bags to maintain high humidity and kept at 25 °C under a photoperiod of 12 h for 72 h. After that time, the plastic bags were removed, and necrotic symptoms were observed.One representative isolate of each Calonectria species were obtained from infected leaves of the Eucalyptus genotypes sampled. Based on preliminary phylogenetic analyses of the tef1 and tub2 gene regions (data not shown), nine isolates were selected for further studies , and four resided in the Calonectriacandelabrum species complex (CCSC). Both Calonectria complexes belong to the Prolate Group, whose species are characterized by their clavate to pyriform to ellipsoidal vesicles , cmdA , his3 , rpb2 , tef1 , tub2  and concatenated . The PHT generated a p value of 0.01 for the concatenated dataset, suggesting some incongruence in the datasets for the six regions and the accuracy of the combined data could have suffered relative to the individual partitions .Tree topologies derived from the CSSC were clustered in an independent clade  and high values of posterior probability (1.0)  and high values of posterior probability (1.0)  was performed to determine the recombination level among C.paragominensis and its phylogenetically closely related species, C.densa, C.humicola, C.spathiphylli and C.pseudospathiphylli. A value of Φw = 0.2879 revealed no significant genetic recombination events, and this relationship was supported with a high bootstrap value (100%) in the phylogenetic network analysis, indicating that they are different species  was performed to determine the recombination level among C.imperata and its phylogenetically closely related species, C.brassiana, C.glabeicola, C.piauiensis, and C.venezuelana. A value of Φw = 0.1587 revealed no significant genetic recombination events, and this relationship was supported with a high bootstrap value (94%) in the phylogenetic network analysis, indicating that they are different species 135–207(–281) × (2–)2.6–3.5(–4) μm; stipe extension septate, straight to flexuous, (123–)147–220(–295) μm long, (1.5–)1.9–2.4(–3) μm wide at the apical septum, terminating in a globose to sphaeropedunculate vesicle, (8–)8.5–10.5(–12) μm diam; lateral stipe extensions (90° to the axis) also present. Conidiogenous apparatus was (40–)56–88(–113) μm long, (45–)67–107(–129) μm wide; primary branches aseptate or 1-septate, (15.7–)18.4–25.9(–30.6) × (3.3–)4–6(–6.5) μm; secondary branches aseptate, (12.7–)14.3–19.6(–22.1) × (3–)3.5–5(–6) μm; tertiary branches aseptate, (9.9–)11.6–15.3(–17.9) × (2.8–)3.6–5.3(–6.4) μm; additional branches (–4), aseptate, (10.3–)11–13.2(–14) × (3–)3.2–4.4(–5) μm; each terminal branch produced 2–4 phialides; phialides doliiform to reniform, hyaline, aseptate, (8–)9.1–11.8(–14) × (2–)2.7–4.1(–6) μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia were cylindrical, rounded at both ends, straight, (47–)56–66(–71) × (4–)4.8–5.9(–7) μm (av. = 61 × 5 μm), (1–3) septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colorless slime. Megaconidia and microconidia were not observed.Colonies formed abundant white aerial mycelium on MEA at 25 °C after seven days, with irregular margins and moderate sporulation. The surface had white to buff outer margins, and sienna to amber in reverse with abundant chlamydospores throughout the medium, forming microsclerotia. The optimal growth temperature was 23.8 °C, with no growth at 5 °C; after seven days, colonies at 10 °C, 15 °C, 20 °C, 25 °C, and 30 °C reached 7 mm, 23 mm, 38.3 mm, 36.1 mm, and 31.8 mm, respectively.E.grandis × E.brassiana.Leaves of Northeast Brazil.E.grandis × E.brassiana; 20 Feb. 2020; M.A. Ferreira; cultures PFC2, PFC3, PFC4, PFC5.Brazil,• Pará state, Paragominas microregion; From infected leaves of C.paragominensis is a new species in the C.spathiphylli species complex  are longer than those of C.densa (av. 54 × 6 μm), C.humicola (av. 51 × 5 μm) and C.pseudospathiphylli (av. 52 × 4 μm) but smaller than those of C.spathiphylli (av. 70 × 6 μm). complex . MorpholTaxon classificationFungiHypocrealesNectriaceaeE.I.Sanchez, T.P.F.Soares & M.A.Ferreirasp. nov.4843A76F-0B34-56D4-B28F-1E0EEFAC60E7843461imperata” is in honor of the city of Imperatriz, Brazil, which was close to the place where the fungus was collected.The term “Calonectriaimperata differs from the phylogenetically closely related species C.brassiana, C.glaebicola, C.piauiensis and C.venezuelana with respect to the number of unique alleles and stipe dimensions.Brazil,• Maranhão state, Cidelândia municipality; 5°09'24"S, 47°46'26"W; From infected leaves of E.urophylla; 20 Feb. 2020; M.A. Ferreira; holotype: UB24350, ex-type: CCDCA 11649 = PFC6. GenBank: act = ON009351; cmdA = OM974330; his3 = OM974339; rpb2 = OM974348; tef1 = OM974357; tub2 = OM974366.Sexual morph unknown. Macroconidiophores consisted of a stipe, a suite of penicillate arrangements of fertile branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, (135–)151–198(–227) × (2–)2.6–3.4(–4) μm; stipe extension septate, straight to flexuous, (151–)169–220(–254) μm long, (1.5–)1.9–2.7(–3) μm wide at the apical septum, terminating in an ellipsoidal to narrowly obpyriform vesicle (3–)3.1–4.6(–6) μm diam. Conidiogenous apparatus was (50–)66–100(–127) μm long, (41–)62–89(–110) μm wide; primary branches aseptate, (14.6–)19–24.8(–28.5) × (2.5–)3.2–4(–4.5) μm; secondary branches aseptate, (12.1–)13.5–18.2(–24.2) × (2.3–)2.8–3.7(–4) μm; tertiary branches aseptate, (10.1–)11–15(–18.1) × (1.9–)2.3–3.2(–4.1) μm; each terminal branch producing 2–4 phialides; phialides doliiform to reniform, hyaline, aseptate, (8–)9.1–13(–15) × (2–)2.7–3.3(–4) μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia were cylindrical, rounded at both ends, straight, (38–)43–49(–52) × (2–)2.7–3.2(–4) μm (av. = 46 × 3 μm), (–1) septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colorless slime. Megaconidia and microconidia were not observed.Colonies formed moderate aerial mycelium on MEA at 25 °C after seven days, with moderate sporulation. The surface had white to buff outer margins, and sepia to umber in reverse with abundant chlamydospores throughout the medium, forming microsclerotia. The optimal growth temperature was 25 °C, with no growth at 5 °C; after seven days, colonies at 10 °C, 15 °C, 20 °C, 25 °C, and 30 °C reached 10.1 mm, 25.5 mm, 29.1 mm, 44.5 mm, and 40.6 mm, respectively.E.urophylla.Leaves of Northeast Brazil.5°09'24"S, 47°46'26"W; From infected leaves of E.urophylla; 20 Feb. 2020; M.A. Ferreira; cultures PFC7, PFC8, PFC9. Brazil• Maranhão state, Itinga do Maranhão; 4°34'43"S, 47°29'48"W; from infected leaves of E.urophylla; 20 Feb. 2020; M.A. Ferreira; culture PFC9.Brazil,• Maranhão state, Cidelândia municipality; C.imperata is a new species in the C.candelabrum species complex  is larger than those of C.piauiensis (50–110 × 4–6 μm), C.glaebicola (50–130 × 5–7 μm), and C.venezuelana (35–100 × 4–8 μm) but narrower than those of C.brassiana (55–155 × 5–8 μm). Additionally, C.imperata lacks lateral stipe extensions, which are present in C.piauiensis. complex . MorpholC.paragominensis and C.imperata produced lesion symptoms on leaves (Fig. The conidia suspensions of the representative isolates of ves Fig. , but no ves Fig. . The patCalonectria isolated from diseased Eucalyptus leaves were identified based on phylogenetic analyses of six gene regions and on morphological comparisons. These two species were named C.paragominensis and C.imperata.Two new species of Calonectriaparagominensis is a new species in the C.spathiphylli complex. The five species identified and described in C.spathiphylli complex are C.densa, C.humicola, C.spathiphylli, C.pseudospathiphylli, and C.paragominensis, where C.paragominensis can be differentiated morphologically with respect to the macroconidia dimensions (Calonectriaspathiphylli is described as heterothallic (C.densa as putatively heterothallic (C.pseudospatiphilly as homothallic (C.humicola mating type has not been indicated (C.paragominensis is described as putatively homothallic based on PCR amplification of the mating-type genes. Regarding pathogenicity, C.paragominensis is pathogenic to Eucalyptus sp., C.spathiphylli is pathogenic to Sapthiphyllum sp. Heliconia sp. Ludwigia sp. Strelitzia sp. and Eugenia sp. (Calonectriadensa, C.humicola, and C.pesudospathiphylli were isolated from soil, and their pathogenicity has not been indicated (C.paragominensis, only C.spathiphylli has been indicated to be present in Brazil (mensions . These smensions . Calonecothallic , C.densaothallic  and C.psothallic . The C.hndicated . Here, Cenia sp. . Calonecndicated . In addin Brazil .Calonectriaimperata is a new species in the C.candelabrum complex. Species in this complex are characterized by presenting ellipsoidal to obpyriform terminal vesicles, in both heterothallic and homothallic species, and occur in Africa, Asia, Europe, North and South America, and Oceania (C.candelabrum complex (Calonectriaimperata is phylogenetically closely related to C.brassiana, C.glaebicola, C.piauiensis and C.venezuelana, which can be differentiated with respect to the number of unique alleles and stipe dimensions (Calonectriabrassiana, C.glaebicola, and C.piauiensis were found in Brazil, isolated from soil samples of Eucalyptus plantations, but only C.glaebicola has been confirmed to be pathogenic to Eucalyptus sp. (Calonectriavenezuelana was reported in Venezuela, similarly, isolated from soil samples, but its pathogenicity has not been indicated (C.brassiana, C.glaebicola, C.piauiensis and C.venezuelana has not been determined ( Oceania . Of the  complex , 17 have complex . Calonecmensions . Calonecptus sp. . Calonecndicated . The mattermined .C.paragominensis and C.imperata are pathogenic to E.grandis × E.brassiana hybrid genotype and E.urophylla genotype, respectively. Although the death of Eucalyptus trees due to CLB is not common, it affects Eucalyptus plants most severely from six months to 2–3 years after planting (CLB has not been quantified directly, according to artificial pruning studies conducted by E.grandis seedlings of one year of age, the volumetric productivity has decreased by 45% by the time they reach seven years old. Therefore, it has been inferred that in susceptible clones, the pathogen can cause economic losses, since under favorable conditions, infection by Calonectria can result in severe defoliation (CLB, and by using the estimates of growth reduction from Eucalyptus wood as R$ 38.70 per m3 (3·ha-1 in the first 7-year rotation. Therefore, accurate diagnoses of plant diseases and identification of their casual agents are fundamental in promoting the development of effective disease management strategies (Pathogenicity tests showed that planting . Althougoliation . Additio0 per m3  and a prrategies .Calonectria species, both isolated from diseased Eucalyptus leaves from commercial plantations localized in a tropical zone. These results suggest that there are still more Calonectria species to be discovered in Brazil, and that they require careful monitoring, since this knowledge could facilitate the development of resistant Eucalyptus clones.In this study, we described two new


Solanum nigrum L. is a Cd hyperaccumulator. In the present experiment, after applying different types of N fertilizers 2SO4, CH4N2O), root and shoot biomasses and Cd phytoextraction efficiency of S. nigrum effectively improved (p < 0.05). Shoot biomasses of S. nigrum harvested at the first florescence stage plus the amounts at the second florescence stage were higher than those harvested at the maturation stage, which indicates that S. nigrum Cd phytoaccumulation efficiency was higher in the former compared to the latter as there was no clear change in Cd concentration (p < 0.05). The pH value and extractable Cd contents showed no changes, regardless of whether N fertilizer was added or not at different growth stages. In addition, after N fertilizer was applied, H2O2 and malondialdehyde (MDA) contents in S. nigrum in vivo were lower compared to those that had not received N addition (CK); similarly, the concentration of proline was decreased as well (p < 0.05). The activity of the antioxidant enzyme catalase (CAT), harvested at different growth periods after four types of N fertilizer applications, obviously decreased in S. nigrum shoots, while peroxidase (POD) and superoxide dismutase) (SOD) activities increased (p < 0.05). Our study demonstrated that (NH4)2SO4 treatment exerted the most positive effect and CH4N2O the second most positive effect on S. nigrum Cd phytoremediation efficiency in double harvests at florescence stages, and the growth conditions were better than others.It is very important to increase phytoremediation efficiency in practice in suitable climatic conditions for plant growth through multiple harvests.  Some hyulescens , Solanumigrum L.  and Violhanensis , Cr hypea Swartz , Zn hypeii Hance ,10, Ni hm murale  and As h vittata ,13. Howe vittata , and thu vittata , as wells supply  and intes supply , could i2−), hydrogen peroxide (H2O2) and hydroxyl radicals (OH), and maintains the balance of the generation and elimination of ROSs so as to withstand the secondary oxidative stress [AsA-GSH recycle system composed of ascorbic acid peroxidase (APX), glutathione reductase (GR), dehydroascorbic acid reductase (DHAR) and monodehydroascorbic acid reductase (MDAR) to alleviate Cd toxicity in plants, thereby protecting the plant itself [Nitrogen (N) is an essential major element that improves soil fertility and biochemical properties and increases crop biomass yields  induced in vivo by biotic and abiotic stresses, such as superoxide radicals  [The used soil sample was collected from a local agricultural field with meadow-brown soil, and the soil sample was taken from neutral soils. Seeds of y Check) .2∙2.5H2O, and the Cd concentration in all treatment groups was 2 mg∙kg−1. Compared with the Soil Environmental Quality Standard in China (GB-15618-2018), the pollution level was moderate [4HCO3, NH4Cl, (NH4)2SO4 and CH4N2O) were purchased at the local market and added to Cd. The detailed experimental design is shown in Distilled water was spiked with Cd with no Cd detected in the form of CdClmoderate . The purS. nigrum seedlings with four leaves and of a uniform height were transplanted from the seedling tray into each pot, and each treatment was repeated 3 times. Plants were irrigated with tap water twice per day to maintain approximately 80% soil moisture content. Single-cropped plants were harvested at maturity, while double-cropped plants were harvested at the first florescence stage (S. nigrum seedlings were transplanted until harvest at the florescence stage (the second florescence stage). The maturation stage (118 days) was roughly equal to the time it took for seedlings to evolve from the first florescence stage (59 days) to the second florescence stage (59 days) in one year.Six ce stage , i.e., t2. Cd concentrations in plant and soil samples were digested using concentrated HNO3 and HClO4, and determined by AAS . The certified standard reference material  was used for quality control. Likewise, organic matter, nitrogen and extractable P contents were measured using a method described by Lu (2000) [v/w) [Plant biomass and Cd concentration were measured according to Yang et al. (2019) . In brieu (2000) . The pH 0) [v/w) .w/v) (TCA) with 0.25% (w/v) 2-thiobarbituric acid (TBA). The mixture was heated at 95 °C for 30 min, and then quickly cooled in an ice bath and centrifuged at 10,000 rpm for 10 min. The data of the supernatant were measured at 532 nm. H2O2 content was measured using the previous method [4 added to 20% H2SO4 (v/v) were obtained at 410 nm after 5 min at room temperature.Malondialdehyde (MDA) content, which is was used as a parameter for the evaluation of lipid peroxidation, was measured as the degree of damage to the plants under abiotic or biotic stress. In this experiment, MDA concentration was determined as described by Zhu et al. (2019) . In bries method . BrieflyThe proline content was determined according to Bates et al. (1973) . Fresh t2O2, which is a modified method described by Hasanuzzaman et al. (2011) [−1 phosphate buffer, pH 7.8, containing 1.0 mmol L−1 EDTA and 2% (w/v) polyvinylpyrollidone (PVP). Then, the homogenate was centrifuged at 5000× g for 10 min at 4 °C, and the supernatant was used for the enzymatic assays. Superoxide dismutase (SOD) and peroxidase (POD) activities were determined according to the provided references [2O2 consumption at 470 nm absorbance. All treatments were performed with three replicates.CAT activity was measured by a decrease revealed at 240 nm absorbance for 3 min in presence of H. (2011) . A totalferences . One unip < 0.05 based on the assumption of normal distribution and homogeneity of variance [Data processing and summary statistics were performed using Microsoft Excel 2000 . Means of different treatments were compared using one-way ANOVA and two-way ANOVA with DPS software . For post hoc test, LSD multiple comparison was performed, and the significance level was variance .S. nigrum, was no significant change in Cd concentration in roots and aboveground parts (shoots) of S. nigrum after applying different fertilizers 2SO4 and CH4N2O) compared to CK with no fertilizer addition (p < 0.05).As seen in −1) in the shoots after application of four types of nitrogen fertilizers 2SO4 and CH4N2O) significantly increased compared to CK (p < 0.05). When S. nigrum was harvested at the maturation stage, N additions significantly increased Cd phytoremediation efficiency compared to the CK (17.66 μg plant−1): NH4HCO3 increased by 1.20-fold (F3), NH4Cl increased by 1.14-fold (F6), (NH4)2SO4 increased by 1.88-fold (F9), and CH4N2O increased by 1.84-fold (F12), respectively.Nevertheless, the analysis of the single- and double-harvest experiments showed that Cd phytoremediation efficiency  in the shoots was basically the same , and their averages were 24.16 μg plant−1, 25.61 μg plant−1, 34.01 μg plant−1 and 33.03 μg plant−1, i.e., significantly increased by 36.8%, 45.0%, 92.6% and 87.0%, respectively, compared to the CK.When −1) in the shoots at the first florescence stages plus the second florescence stages (double harvest) were 48.32 μg plant−1 (F1+F2), 51.31 μg plant−1 (F4+F5), 68.02 μg plant−1 (F7+F8) and 66.60 μg plant−1 (F10+F11), i.e., significantly increased by 1.74-fold (NH4HCO3), 1.90-fold (NH4Cl), 2.85-fold ((NH4)2SO4) and 2.77-fold (CH4N2O) compared to the CK, respectively. Obviously, the Cd phytoremediation efficiency (μg plant−1) in the shoots of double-harvested plants increased by 24.3% (F1+F2 vs. F3), 35.7% (F4+F5 vs. F6), 33.8% (F7+F8 vs. F9) and 32.3% (F10+F11 vs. F12), respectively.In one year, the Cd phytoremediation efficiency (μg plantS. nigrum markedly increased (p < 0.05) in all treatment groups after applying the same amount of the four types of N fertilizers 2SO4 and urea (CH4N2O)); dry weights of roots and aboveground parts were: 0.41 g plant−1, 1.84 g plant−1, 0.42 g plant−1, 1.86 g plant−1, 0.69 g plant−1, 2.41 g plant−1 and 0.70 g plant−1 and 2.41 g plant−1, respectively, and in comparison to CK, they were increased by 1.64-, 1.15-, 1.66-, 1.18-, 3.43-, 1.81- and 3.47- and 1.81-fold, respectively (p < 0.05). Using four types of N fertilizer 2SO4 and urea (CH4N2O)) treatments, our study confirmed that root and shoot biomasses harvested at the mature stage were higher than those harvested at the first and second florescence stages. In addition, it was calculated that total dry biomasses (g plant−1) of the aboveground S. nigrum parts harvested at the first and second florescence stages with the double-cropping method over the course of one year were much larger than those harvested at the maturation stage with the single-cropping method over one year; of all treatments, the (NH4)2SO4 and urea (CH4N2O) treatment groups obtained the highest shoot biomasses, and urea (CH4N2O) treatment showed the second highest (−1)  .2O2 (mg g−1) and MDA (μmol g−1) contents in S. nigrum shoots with four types of N fertilizer 2SO4 and urea (CH4N2O)) markedly decreased (p < 0.05); at the maturation stage, H2O2 contents (mg g−1) decreased by 16.55%, 17.27%, 20.14% and 17.99%, respectively (p < 0.05). Similarly, MDA content (μmol g−1) decreased by 4.20%, 3.94%, 3.67% and 4.20%, respectively, compared to CK (p < 0.05).2O2 and MDA contents in S. nigrum shoots harvested at the first and second florescence stages were the lowest.In addition, H4HCO3, NH4Cl, (NH4)2SO4 and urea (CH4N2O)) at different growth stages resulted in a significant decrease in antioxidant proline contents (mg g−1) and catalase (CAT) activities (U g−1 min−1) in S. nigrum shoots in comparison to CK with no fertilizer addition (p < 0.05); at the maturation stage, the proline contents (mg g−1) decreased by 13.63%, 14.56%, 15.10% and 15.34%, respectively, and CAT activities (U g−1 min−1) decreased by 6.64%, 6.17%, 5.38% and 7.54%, respectively, compared to CK (p < 0.05). Additionally, proline contents and CAT activities were the lowest at the first and second florescence stages (e stages .−1 min−1) and SOD activities (U g−1) were markedly increased in comparison to CK with no fertilizer addition (p < 0.05); at the maturation stage, POD activities (U g−1 min−1) increased by 12.20%, 11.35%, 11.80% and 11.39%, respectively, and SOD activities (U g−1) increased by 29.55%, 27.95%, 28.62% and 29.95%, respectively  and superoxide dismutase (SOD), the results show that after the application of different fertilizers, POD 2SO4 and urea (CH4N2O)) did not cause any significant difference in extractable Cd concentration in soil planted with S. nigrum harvested at different growing periods when compared to CK, and treatments were in the range of 1.11–1.15 mg∙kg−1 (p < 0.05).As shown in S. nigrum, the results show that the pH value in the rhizosphere soil used for S. nigrum cultivation under all N fertilizer treatment groups 2SO4 and urea (CH4N2O) did not differ significantly compared to the control with no fertilizer supply and ranged from 6.60 to 6.70 (p < 0.05) (Comparing the one-stage and two-stage phytoremediation Cd experiments involving  < 0.05) .S. nigrum could accumulate Cd from slightly-to-moderately Cd-contaminated soil by double harvesting during the growing season, thereby showing an enormous potential for improving Cd accumulation efficiency in practice [−1) of S. nigrum under all treatments due to the significantly increased root and shoot biomasses (g plant−1) and maintenance of the Cd concentration in S. nigrum (4NO3 and Ca(H2PO4)2 application did not increase biomass, while Cd concentrations significantly decreased compared to unfertilized plants [4)2SO4 had no obvious effect on Cd accumulation by maize [In a previous experiment, Wei et al. (2006) showed that the Cd hyperaccumulator practice , which i. nigrum  and lead. nigrum . In contd plants . These aby maize .Tagetes patula grown in Cd-contaminated soil [T. caerulescens [Carpobrotus rossii [However, other pot culture experiments showed that N fertilizer significantly improved the biomass and Cd phytoremediation efficiency of ted soil . Nitrogeulescens , while us rossii , which fs rossii ,41.Brassica juncea grown in contaminated sediments; the research showed that N fertilizer not only increases boron phytoextraction, but also alleviates B stress [2O2 and MDA in wheat [2O2 and MDA are the major oxidation products of lipid peroxidation and can directly reflect the degree of oxidative stress. Accordingly, in our pot culture experiment, it was discovered that after applying nitrogen fertilizer, the contents of H2O2 and MDA in S. nigrum shoots at the maturation stage effectively decreased compared to CK (p < 0.05)  , which r2O2 generation under stress conditions [2O2 and proline concentration were considerably decreased after N treatment in S. nigrum shoots in different growth stages  was the highest during flowering stages [In addition, POD and SOD activities in e stages c,d, whicg stages .p < 0.05) (Our study showed that different types of N fertilizers did not significantly increase extractable Cd concentration compared to CK ( < 0.05) . General4+ ion exchange and assimilation [p < 0.05)  . NeverthS. nigrum improved with the addition of N fertilizers 2SO4, CH4N2O) (p < 0.05); moreover, the Cd phytoaccumulation capacity of S. nigrum was higher during double harvests compared to single harvests due to the increase in S. nigrum shoot biomass and no obvious changes in Cd concentration. The pH value and extractable Cd content did not show a significant variation trend. In addition, by analyzing the content and activity of non- and enzymatic antioxidants, it was found that compared to the single-cropping system, S. nigrum grew better after fertilization, and showed the highest efficiency in terms of Cd phytoremediation when harvested using a double-cropping system. The differences were associated with the harvesting mode, rather than fertilizer styles.Cd phytoremediation efficiency of