Escherichia coli, Pseudomonas aeruginosa, Salmonella paratyphoid B, and Shigella dysentery in live Bacillus licheniformis capsules, a polymerase chain reaction (PCR) method with high sensitivity and specificity was established. By combining bromide with propidium monoazide (PMA) -real-time quantitative PCR (qPCR) with microporous membrane filtration, excipients were removed, the filtrate was collected, and the bacteria were enriched using the centrifugal method. The optimal PMA working concentration, dark incubation time, and exposure time were determined. Specific E. coli, P. aeruginosa, S. paratyphoid B, and S. dysentery primers were selected to design different probes and a multiplex qPCR reaction system was established. The PMA-qPCR method was verified using different concentrations of dead and live bacteria. This method is efficient and accurate and can be widely applied to the detection of aforementioned pathogenic bacterial strains in live Bacillus licheniformis products.To eliminate the influences of excipients and interference of dead bacterial DNA on the detection of  Bacillus licheniformis capsule, play an important role in improving the intestinal microbiota imbalance and treating gastrointestinal diseases, such as acute and chronic enteritis and diarrhea , Shigella spp. (Shigella dysentery), Pseudomonas aeruginosa, and Escherichia coli. In accordance with relevant regulations and standards, the detection of the above pathogenic microorganisms or opportunistic pathogens requires preliminary enrichment culture, spread plate culture, and subsequent identification. However, probiotic products contain billions of live bacteria, which can grow in relevant culture media, greatly interfering with the detection of the above pathogenic bacteria. Furthermore, the traditional culture and identification methods are cumbersome and time-consuming, limiting the accurate and efficient detection of the above-mentioned pathogenic bacteria , the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), and the Chinese Pharmacopoeia (C. Ph) all require specific bacteria inspection in probiotic products, mainly including the detection of pathogenic microorganisms or opportunistic pathogens, such as bacteria .Salmonella spp., Shigella spp., P. aeruginosa, and E. coli in live Bacillus licheniformis capsule, wherein PMA-qPCR was combined with microporous membrane filtration to remove a large number of excipients in probiotic products and reduce their interference in subsequent bacterial detection. By optimizing the working concentration, dark incubation time, and exposure time of PMA, we established the optimal PMA treatment method. We designed specific primers and probes for the detection of aforementioned four species of bacteria. An efficient and accurate new method for the simultaneous detection of the aforementioned bacteria and live bacterial products was established.With the development of rapid live bacteria detection technology, the combination of PCR with nucleic acid dyes for the detection of pathogenic live bacteria has become a current research hotspot . PropidiThe following instruments were used in this study: PMA-LiteTMLED photolysis instrument , Viia7 qPCR instrument , Biodrop ultramicro protein-nucleic acid analyzer , ME2002E electronic balance , HFsafe-1200 biosafety cabinet , MLS-3781-PC high-pressure steam sterilizer , 5424 centrifuge , TW20 precision constant temperature water bath , and HTY-761 homogenizer .S. paratyphoid B, S. dysentery, P. aeruginosa, and E. coli , sterile sodium chloride solution , sterilized water for injection , aperture 1.2 and 5.0 μm polyether sulfone microporous filter membrane with a diameter of 50 mm , and Fc502 membrane filter .The following reagents and consumables were used: PMA (batch No. 19P0129), PMAxxTMdye , Takara bacterial genome DNA Extraction Kit (No. AI82587a), Probe qPCR Mix (with UNG) , primers and probes for For bacterial cell culture the following media were used: tryptose soy agar , trypticase soy broth , mannitol salt agar , and 7.5% sodium chloride broth . The applicability of the above media is in line with the provisions of C. Ph 2020 edition.Bacillus licheniformis capsule contained a live bacterial concentration of 1 × 109 CFU⋅g–1 .The live Salmonella paratyphoid B (CMCC [B] 50094), Shigella dysentery (CICC 23829), Proteus mirabilis (CICC 2516); Enterobacter sakazakii (CICC 21544), P. aeruginosa (CMCC [B] 10104), E. coli (CMCC [B] 44102), Stenotrophomonas maltophilia, Ralstonia Pichia, Citrobacter freundii (laboratory preserved strains), Burkholderia cepacia (ATCC 25416), Burkholderia cepacia (ATCC BAA-245), and Burkholderia cepacia (ATCC BAA-247).The following experimental strains were used: Salmonella spp., Shigella spp., P. aeruginosa, E. coli, P. mirabilis, E. sakazakii, S. maltophilia, R. Pichia, C. freundii, B. cepacia, and B. neocepacia, were inoculated into TSB medium. All strains were cultured at 33°C for 24 h. The number of live Salmonella spp., Shigella spp., P. aeruginosa, and E. coli bacteria were counted using TSA plates. The cell numbers of the live bacterial suspensions of Salmonella spp., Shigella spp., P. aeruginosa, and E. coli were enumerated as 7.0 × 108 CFU (colony forming units) mL–1, 5.0 × 108 CFU mL–1, 3.6 × 108 CFU mL–1, and 8.3 × 108 CFU⋅mL–1, respectively.The standard strains of Salmonella spp., Shigella spp., P. aeruginosa, and E. coli, prepared using the method described in section “Preparation of live bacterial suspensions,” were treated in a boiling water bath for different time periods , and then immediately placed on ice for cooling. The number of viable bacteria in the samples was obtained using the CFU counts. The best lethal heat treatment time of the strains was defined as the shortest boiling water bath treatment time required for sterile growth on the plate. At all selected time periods, 10, 20, 30, 40, 50, and 60 min, of boiling water bath treatment, no viable bacterial counts were obtained for Salmonella spp., Shigella spp., P. aeruginosa, and E. coli species, indicating that these bacterial strains could be killed after 10 min of boiling water bath treatment. Based on these results, 10 min of boiling water bath treatment was selected as the best lethal heat treatment time for further experiments.Live suspensions (10 mL) of Salmonella spp., Shigella spp., P. aeruginosa, and E. coli prepared using the method described in section “Preparation of live bacterial suspensions,” were treated in a boiling water bath for 10 min, centrifuged at 8,000 r.min–1 for 5 min, and the supernatant subsequently discarded. Then, the pellet containing the dead bacterial suspension of Salmonella spp., Shigella spp., P. aeruginosa, and E. coli (108 CFU⋅mL–1), was resuspended using 10 mL of sterile distilled water.Live bacterial suspensions (10 mL) of Bacillus licheniformis live capsule content was aseptically weighed, and added into 270 mL sterile sodium chloride solution dilution, homogenized at 3,500 r.min–1 for 30 s to obtain an 1:10 test solution (1 × 108 CFU⋅ mL–1). Then, 10 mL of the test solution was filtered using a sterile FC502 filter. The following microporous membranes: 0.45 μm mixed cellulose ester membrane, 1.2 μm, 5 μm polyethersulfone membrane, and 3, 5, and 8 μm nylon membranes were used for analysis and comparison. The results demonstrated no viable bacteria in the filtrate filtered using the mixed cellulose ester membrane. The rest of the membranes were weighed and the 5 μm polyethersulfone membrane excipients had the highest rejection rate.A total of 30 g of the –1 for 5 min. The supernatant was discarded, and the pellet was resuspended in sterile distilled water to a total volume of 500 μL. The resulting Bacillus licheniformis live capsule sample suspension (1 × 109 CFU⋅mL–1) was the pre-treatment sample.The 1:10 test solution was filtered using a 5 μm polyethersulfone filter membrane that matched the specifications of the FC502 sterile filter. The filter membrane was replaced for every 10 ml of the 1:10 test solution filtered, and the filtrate was collected by centrifugation at 8 000 r. minBacillus licheniformis capsule suspension prepared in section “Pre-treatment of live Bacillus licheniformis capsule” was added to each of the above centrifuge tubes and mixed. The mixtures were centrifuged at 8,000 r.min–1 for 5 min, the supernatant was discarded, and the pellet was resuspended in sterile distilled water to a final volume of 0.5 mL. PMA aqueous solution, at a concentration of 400 μg⋅mL–1, was added to each centrifuge tube to prepare solutions with PMA concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, and 110 μg⋅mL–1, respectively. The above mixtures were mixed, incubated in the dark at 20–25°C for 15 min, and vortexed to mix every 5 min. Then, they were placed in the centrifuge tube in the photolysis device for 20 min exposure and mixed every 5 min during exposure.The four live bacterial suspensions prepared in section “Preparation of live bacterial suspensions” were mixed in 1:1:1:1 ratio according to the cell counting results. The same procedure was followed to prepare a mixture of the dead bacterial suspensions prepared in section “Preparation of dead bacterial suspensions.” A total of 500 μL of each of the above live and dead bacterial mixtures was placed, respectively, in separate centrifuge tubes. Then, 500 μL of the live 2O. The primers and probes used in the reaction are shown in DNA from bacterial suspensions was isolated using the TaKaRa minibest bacterial genomic DNA extraction kit , following the manufacturer’s instructions. Then DNA was treated with PMA as described in section “Optimization of the propidium monoazide concentration.” Subsequently, qPCR detection was conducted to obtain the quantification cycle (Cq) values. The reaction mixture of quadruple qPCR (50 μL) contained: 25 μL of Probe qPCR Mix (with UNG), 1 μL of 0.2 μM upstream primers, 1 μL of 0.2 μM downstream primers, 2 μL of 0.4 μM probe, 5 μL of template DNA, and 4 μL of ddHBacillus licheniformis capsule prepared in section “Pre-treatment of live Bacillus licheniformis capsule” was added to each centrifuge tube and then mixed. The mixtures were centrifuged at 8,000 r.min–1 for 5 min, the supernatant was discarded, and the pellet was resuspended in sterile distilled water to a final volume of 0.5 mL. PMA aqueous solution, at a concentration of 400 μg⋅mL–1, was added to each centrifuge tube to a final PMA concentration of 40 μg⋅mL–1. The above mixtures were combined, incubated in the dark at 20–25°C, for 0, 5, 10, 15, 20, 25, and 30 min, vortexed to mix every 5 min, placed in the centrifuge tube in the photolysis device for 20 min exposure, and mixed every 5 min during exposure. Subsequently, DNA was extracted according to the method described in section “Quadruple quantitative polymerase chain reaction system” and then analyzed using qPCR.The bacterial suspensions prepared in section “Preparation of dead bacterial suspensions” were mixed in 1:1:1:1 ratio according to the cell counting results. A total of 500 μL of each of the above live and dead bacterial mixtures were placed, respectively, in separate centrifuge tubes. Then, 500 μL of the live –1. The samples were mixed, incubated in the dark at 20–25°C for 10 min, vortexed to mix every 5 min, placed in the centrifuge tube in the photolysis device for 0, 5, 10, 15, 20, 25, and 30 min exposure, and mixed every 5 min during exposure. Subsequently, DNA was extracted according to the method described in section “Quadruple quantitative polymerase chain reaction system” and then analyzed using qPCR.The samples were prepared according to the method described in “Optimization of the propidium monoazide dark incubation time” with a PMA concentration of 40 μg⋅mLBacillus licheniformis capsule sample suspension was added according to the method described in section “Optimization of the propidium monoazide concentration” to a final volume of 0.5 mL. PMA aqueous solution, at a concentration of 400 μg⋅mL–1, was added to each centrifuge tube to a final PMA concentration of 40 μg⋅mL–1. The above mixtures were mixed, incubated in the dark at 20–25°C for 10 min, vortexed to mix every 5 min, and placed in the centrifuge tube in the photolysis device for 20 min exposure, and mixed every 5 min. Subsequently, DNA was extracted according to the method described in section “Quadruple quantitative polymerase chain reaction system” and then analyzed using qPCR. No PMA added was used as control.The four viable bacterial suspensions prepared in section “Preparation of live bacterial suspensions”, were mixed at a ratio of approximately 1:1:1:1 based on the counting results. Then, the same procedure was repeated to prepare a mixture of the dead bacterial suspensions as prepared in section “Preparation of dead bacterial suspensions.” The mixtures of the live and dead bacterial suspensions were mixed in proportion, and the proportions of live bacteria in the prepared mixed bacterial suspension were 0, 1, 10, 25, 50, and 100%. Then, 500 μL of the mixed bacterial suspension was placed into different 1.5 mL centrifuge tubes, and the Salmonella spp. live bacterial suspension at an initial concentration of 108 CFU⋅mL–1, was centrifuged and concentrated ten times. A concentration gradient was prepared by serially diluting the bacterial suspensions  by 10-fold. A total of 100 μL of each of the bacterial suspension concentrations were used in eight reaction systems, respectively. The E. coli, P. aeruginosa, and Shigella spp. bacterial suspensions prepared in “Preparation of live bacterial suspensions” were mixed, centrifuged, and concentrated ten times, and 100 μL of each suspension was added to the above reaction system. Next, 300 μL of the pre-treated live Bacillus licheniformis bacterial capsule sample was added to the reaction system. Subsequently, the samples were treated with PMA at a working concentration of 40 μg⋅mL–1, incubated in the dark for 10 min, and exposed for 15 min. DNA was extracted as described in section “Quadruple quantitative polymerase chain reaction system” and then analyzed using qPCR. The sensitivity test methods for E. coli, P. aeruginosa, and Shigella spp. were the same as detailed above.The Salmonella spp., Shigella spp., P. aeruginosa, E. coli, P. mirabilis, E. sakazakii, S. maltophilia, R. pickettii, C. freundii, B. cepacia, B. neocepacia, and B. polyphaga suspensions were added in separate 2 mL centrifuge tubes. Then, the Bacillus licheniformis capsule sample suspension was added following the method described in “Optimization of the propidium monoazide concentration” to a final volume of 0.5 mL. The optimal conditions of PMA treatment, extraction of genomic DNA, qPCR reaction, primer probes, and reaction conditions were used as the determined in “Optimization of the propidium monoazide treatment conditions and establishment of the quadruple quantitative polymerase chain reaction system.”A total of 1 mL of the P-values < 0.05.The obtained data are expressed as –1, the Cq values of E. coli, Salmonella, P. aeruginosa, and Shigella were 23.628, 22.037, 24.060, and 16.7602, respectively. With the increase in PMA concentration, the Cq value did not changed significantly (P > 0.05). As shown in As shown in –1 concentration, the binding of PMA to the dead Bacillus licheniformis DNA in the sample can be estimated, and the DNA amplification of dead E. coli, Salmonella, P. aeruginosa, and Shigella bacteria can be effectively inhibited. At the same time, PMA did not affect the DNA amplification of the above-mentioned live bacterial strains. Thus, 40 μg⋅mL–1 was selected as the optimal working concentration of PMA.Therefore, upon adding PMA at 40 μg⋅mLBacillus licheniformis capsule samples, 15 min was selected as the exposure time to ensure the effective combination of PMA and dead bacteria.As shown in As shown in Salmonella spp. sensitivity study are shown in Salmonella spp. in the live bacterial suspension (1.6 × 104∼1 × 109 CFU⋅mL–1) was obtained. With the standard curve equation: y = 3.1357x + 11.605 and R = 0.9537, the detection limit was estimated at 1.6 × 104 CFU⋅mL–1. The results of the Shigella spp. sensitivity study are shown in Shigella spp. in the live bacterial suspension (1.7 × 104∼1 × 109 CFU⋅mL–1) was obtained. With the standard curve equation: y = 2.3282x + 10.462 and R = 0.9929, the detection limit was estimated at 1.7 × 104 CFU⋅mL–1. The results of the P. aeruginosa sensitivity study are shown in P. aeruginosa in the live bacterial suspension (1.3 × 102∼1 × 109 CFU⋅mL–1) was obtained. With the standard curve equation: y = 1.7358x + 17.183 and R = 0.8838, the detection limit was estimated at 1.3 × 102 CFU⋅mL–1. The results of the E. coli sensitivity study are shown in E. coli live bacterial suspension (7.0 × 104∼7.0 × 109 CFU⋅mL–1) was obtained. With the standard curve equation: y = 2.613x + 13.833 and R = 0.9849, the detection limit was estimated at 7.0 × 104 CFU⋅mL–1.The results of the Salmonella spp. , Shigella spp. , P. aeruginosa , and E. coli  had an obvious exponentially higher fluorescence amplification signals. P. mirabilis, E. sakazakii, S. maltophilia, R. pickettii, C. freundii, B. cepacia, B. neocepacia, and B. polyphaga showed no obvious fluorescence amplification curve, indicating that the specificity of this method for the detection of Salmonella spp., Shigella spp., P. aeruginosa, and E. coli is higher compared to other tested bacteria.As shown in At present, the combination of PMA with qPCR is mainly used for the detection of common food and clinical pathogens . HoweverBacillus licheniformis capsules, we also emphasized on studying the effects of various other factors. We optimized the levels of four major factors: turbidity, dark incubation time and exposure-response, salt concentration, and percentage of dead cells in the samples. Firstly, when the turbidity of samples was too high, the light transmittance was reduced, causing incomplete photoreaction of PMA and weakening the effect of PMA on DNA modification easily (via specific penetration through the damaged cell membrane of dead cells, PMA intercalated into DNA in the dark. After that, under the irradiation of strong light, the azide group of the PMA molecule changed into the nitrene groups to form a covalent bond with DNA; free PMA that was not bound to DNA underwent photoreaction with water molecules and became inactive in solution (Bacillus licheniformis capsule sample contained a large number of dead bacteria. During the exposure time, the heat generated by the light would damage the cell membrane of the living bacteria and increase the risk of the cytotoxic effect of PMA. Therefore, 15 min was selected as the exposure time. Thirdly, studies have shown that the presence of salt ions such as sodium chloride could increase the osmotic pressure of the solution, making it easier for PMA to penetrate the cell membrane and reach the interior of the cell. However, Listeria monocytogenes found that when the number of dead/live bacteria exceeded 104 CFU⋅mL–1or the number of viable bacteria was less than 103 CFU⋅mL–1, the Cq value had no linear relationship with the log value of the number of viable bacteria in the samples. Moreover, 4 CFU⋅mL–1, the samples treated with PMA could effectively inhibit the amplification of DNA. The research object of this experiment was Bacillus licheniformis capsule products, in which the ratio of dead/live bacteria was not more than 10 generally. Under the PMA-qPCR detection conditions established in this study, a good linear relation was obtained, and the amplification DNA of dead bacteria was effectively suppressed.In the process of exploring the establishment of the PMA-qPCR method to detect the four strains of n easily . In thissolution . SufficiSalmonella spp. was estimated at 1.6 × 104 CFU⋅mL–1, of P. aeruginosa at 1.3 × 102 CFU⋅mL–1, of E. coli at 7.0 × 104 CFU⋅mL–1, of Shigella spp. at 1.7 × 104 CFU⋅mL–1. The detection limit of Salmonella spp. was consistent with the detection limit of Salmonella typhimurium and Salmonella Sonnei obtained in the single-plex SD-PMA-PCR and quadruple SD-PMA-PCR studied by P. aeruginosa was consistent with the research results obtained by E. coli and Eva There’s Gensberger’s research (2–103 CFU⋅mL–1, which may be due to the use of water as their research object. There were few matrix interferences in the samples, and less DNA loss in the extraction and purification process, thereby increasing the sensitivity for the detection of E. coli in this method. Shigella in milk, and attained the detection limit as low as 101 CFU⋅mL–1. The difference may be due to the use of liquid as the sample object, owing to much less interference from excipients. The interference of the sample matrix would affect the DNA extraction, PMA and DNA binding efficiency, thus sample preparation should get appropriate attention when optimizing the method.In the multiple qPCR method established in this study, the detection limit of research  result oOverall, the method proposed in this study is efficient, fast, real-time, and accurate. The whole process of screening and identification only takes 3 h, overcoming the problems of traditional methods, wherein the fast and accurate detection of the above-mentioned pathogenic bacteria in microecological live bacteria products remain an unresolved challenge. Thus, the method proposed in this study harbors a wide range of application prospects.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.XZ, JW, and YW conceived and designed the experiments. JL and QC contributed to reagents, materials, and analysis tools. XZ and YW performed the experiments. JW, XZ, YW, and WG wrote the manuscript. All authors analyzed the data, contributed to the article, and approved the submitted version.


Medicago polymorpha L., a seasonal vegetable, is commonly grown in China. The increasing use of nanoparticles (NPs) such as ZnO and CuO NPs in agriculture has raised concerns about their potential risks for plant growth and for human consumption. There is a lack of research on the effects of ZnO and CuO NPs on agronomic performance of Medicago polymorpha L. and their potential risks for human health.−1) and CuO NPs  were used to determine their effects on the growth and nutrient absorption of Medicago polymorpha L., as well as their potential risk for human health.In this study, different treatment concentrations of ZnO NPs , potassium (K), magnesium (Mg), and calcium (Ca). The following micronutrients were also improved: iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), and manganese (Mn), with the exception of nitrogen (N) accumulation. Low treatment concentrations exhibited more efficient nutrient uptake than high treatment concentrations. A comprehensive analysis showed that the optimum concentrations were 25 mg kg−1 for ZnO NPs and 10 mg kg−1 for CuO NPs. The potential non-carcinogenic health risk of Medicago polymorpha L. treated with ZnO and CuO NPs was analyzed according to the estimated daily intake (EDI), the hazard quotient (HQ), and the cumulative hazard quotient (CHQ). Compared with the oral reference dose, the EDI under different ZnO and CuO NPs treatments was lower. The HQ and CHQ under different ZnO and CuO NPs treatments were far below 1. This indicated that Medicago polymorpha L. treated with ZnO and CuO NPs did not pose any non-carcinogenic health risk to the human body. Therefore, ZnO and CuO NPs were considered as a safe nano fertilizer for Medicago polymorpha L. production according to growth analysis and a human health risk assessment.The results showed that ZnO and CuO NPs increased the fresh weight of  Lolium perenne L., Medicago sativa L., etc.) and gramineous species  are common grass species in orchards in China , and trace elements (copper (Cu), manganese (Mn), iron (Fe), zinc (Zn), and magnesium (Mg)) ) . Additioum (Mg)) .−1 significantly decreased the root length and shoot length by 67–77% and 13–18%, respectively. ZnO NPs at 1,000 mg L−1 significantly decreased seed germination and the total dry weight. −1 significantly reduced the fresh shoot biomass and 1,000-grain weight of peanut. In addition, exposure to 50 and 500 mg kg−1 CuO NPs resulted in 36.2 and 21.1% decreases in the total amino acid content, respectively  (−1) significantly affected the potassium (K), phosphorus (P), Fe, Zn, Cu of bok choy, but had no significant effect on their boron (B), calcium (Ca), Mg, Mn, molybdenum (Mo), and sulfur (S) contents. −1) significantly reduced shoot elongation and relative chlorophyll content and decreased the macroelements and microelements B, Zn, Mn, Ca, P, and S in the shoots of cilantro. However, −1) increased root Ca, root Fe, bulb Ca, and the bulb Mg of green onion. The differences in results may be associated with the species of vegetables and the concentration of the treatments  . It shoueatments .Medicago polymorpha L. Medicago sativa L.). It should be noted that the effect of CuO NPs on plant growth and nutrient uptake was different from that of Cu NPs  provided guidelines on the estimated daily intake (EDI) of metal exposure by humans , and Nanjing Chemical Reagent Co., Ltd., . The properties of ZnO and CuO NPs are shown in ZnO and CuO NPs were purchased from Shanghai Macklin Biochemical Co., Ltd., Shanghai, China, and Sigma-Aldrich Chemistry , respectively. ZnSO4 at the concentrations of 25, 50, 100, 200 mg kg−1, ZnO NPs at the concentrations of 25, 50, 100, 200 mg kg−1, CuSO4 at the concentrations of 10, 25, 50, 100 mg kg−1, and CuO NPs at the concentrations of 10, 25, 50, 100 mg kg−1, respectively. These treatment concentrations were selected according to the soil environmental quality standard (−1) and Cu (∼20 mg kg−1) concentrations of the soil in the Experimental Station of Plant Science of Jiangsu Academy of Agricultural Science . The environmental quality standard value of Zn and Cu were 250 and 100 mg kg−1 in soils with the pH of 6.5−7.5, respectively . A total of 2 g of seeds were sown into a plastic cup filled with 110 g vegetable seedling substrate, and irrigated with 100 ml deionized water, with a subsequent covering with 5 g vegetable seedling substrate. The seedlings were thinned to 12 plants per cup ten days after emergence. The seedlings were cultured in a growth chamber . The maintained parameters during the plant growth period were set as follows: 22/20 °C day/night with a 16/8 h light/dark cycle with a light intensity of 42,000 lx and 50% relative humidity. The substrate was the “YouJia” vegetable seedling substrate, purchased from Zhongyuan Horticulture Development Co., Ltd. . The contents of nitrogen (N), P, K, and organic matter of the substrate were 12.8, 3.91, 19.2, and 406.9 mg g−1, respectively. The N, P, and K contents were measured with the methods described in our previous study  and 2.11 g compound fertilizer (N-P-K = 15%–15%-15%) were added to each cup after being dissolved in water. The fertilizers were applied every five days after the seedlings were thinned and the treatments were applied three times in total.There were seventeen treatments including the control, ZnSOstandard  and our ectively . The no us study . The orgus study . ZnSO4 aMedicago polymorpha L for each treatment.Five to six cups of plants were sampled to weigh the fresh shoot weight of The samples were manually ground into fine powders using a mortar. The powders were added to a digest tube containing 25 mL sulfuric acid (98%) and one mL hydrogen peroxide (30%). The concentrations of N, P, and K were measured with the methods used in our previous study . The conMedicago polymorpha L. treated with ZnO and CuO NPs. The EDI of the metal element was calculated according to the method of the US Environmental Protection Agency (The estimated daily intake (EDI), hazard quotient (HQ), and cumulative HQ (CHQ) were calculated to assess the potential non-carcinogenic health risk of n Agency . The HQ EDI (mg kg−1 d−1) was the index of estimated daily intake of metal element. CME (mg kg−1) was the metal element concentration of Medicago polymorpha L. DI (kg d−1) was the daily intake of Medicago polymorpha L. IF (d a−1) was the intake days per year. The values of DI and IF were obtained through investigation in southern Jiangsu. The values of DI were 0.10 kg d−1 for adult males, 0.08 kg d−1 for adult females, 0.02 kg d−1 for young children (<6 years old), respectively. The values of IF were 70 d a−1 for adults and 50 d a−1 for young children, respectively. EA (a) and AA (a) were the expected age and the average age for adults and young children, respectively. BW (kg) and AID (d) were average body weight and average total intake days. The values of EA, AA, BW, and AID were referred from the data reported by HQ represented the non-carcinogenic risk effect. RfD (mg kg−1d−1) was the oral reference dose of daily intake, and the values of RfD for Fe, nickel (Ni), Zn, Cu, Mn were 0.70, 0.02, 0.30, 0.037, 0.24 mg kg−1 d−1  test was used to determine the significance of differences between means at the 0.05 level. The p < 0.05 indicated that there were significant differences between different treatments. The p >  0.05 indicated that there were no significant differences between different treatmentsStatistical analysis was carried out using SPSS statistical software (SPSS 20.0). The experimental data were analyzed using analysis of variance (ANOVA) to evaluate the main effects of metal forms, metal treatment concentration and their interactions on the fresh weight per plant and the accumulation of macronutrients and micronutrients. The least significant difference  and CuO NPs (p = 0.39) on FWPP was not significantly different from those of Zn2+ and Cu2+ when averaged across treatment concentrations (2+ and Cu2+. Zn treatment concentrations (p = 0.002) and Cu treatment concentrations (p = 0.009) had a significant effect on FWPP  slightly increased the FWPP by 6.4 and 5.1, and 6.9 and 3.7%, respectively, but low concentrations of Cu2+ and CuO NPs (10 and 25 mg kg−1) significantly increased the FWPP by 10.0 and 11.2, and 7.0 and 8.1%, respectively, as compared with the control (trations . This in on FWPP . Zn2+ anectively . Cu2+ an control .Medicago polymorpha L. The form of Zn significantly affected the accumulation of N (p = 0.04), and N accumulation under Zn2+ was 21.6% higher than that of ZnO NPs  (2+ treatment concentrations had no significant effect on the accumulation of N (p >  0.05), although Zn2+ at concentrations of 100 and 200 mg kg−1 decreased the accumulation of N by 4.8 and 8.2%, respectively. There was no significant effect of ZnO NPs on the accumulation of N at concentrations of 25–100 mg kg−1 (p >  0.05). However, ZnO NPs at concentrations of 200 mg kg−1 significantly decreased the accumulation of N by16.2% (p < 0.05) . However, there was no significant difference between other treatments and the control ( ZnO NPs . The lar mg kg−1 . However = 0.90) . Zn2+ tr < 0.05) . For Cu  control .2+ when averaged across treatment concentrations (p = 0.39). Zn treatment concentrations significantly increased the P accumulation of plants (p < 0.001) when averaged across Zn forms . Cu treatment concentrations significantly increased the accumulation of P by 36.0–74.2% under Cu2+ treatments, and by 49.1–90.1% under CuO NPs treatment (p < 0.001), and the effect of CuO NPs on the accumulation of P was higher than that of Cu2+ treatments (p = 0.03). The Cu2+ at 25 mg kg−1 and CuO NPs at 10 mg kg−1 produced the highest P accumulation (ZnO NPs had no significant effect on the P accumulation compared with ZnZn forms . Compare mg kg−1 . The P amulation .2+ and ZnO NPs at 200 mg kg−1 when compared with the control (p <0.05). The highest K accumulation was obtained with Zn2+ and ZnO NPs at 25 mg kg−1 (p < 0.001) . Both Cu2+ at 10 and 100 kg−1 did not affect the K accumulation (p > 0.05). CuO NPs treatments significantly increased the K accumulation by 11.2–18.1% (p < 0.05), and K accumulation decreased with the increasing concentrations of CuO NPs (2+ and ZnO NPs (p = 0.14) when averaged across treatment concentrations (2+ (p = 0.002) (Zn treatment concentrations significantly increased the K accumulation of plants except for Zn mg kg−1 . Cu trea< 0.001) . The hig CuO NPs . No signtrations . However= 0.002) .p < 0.001 and p <0.001) and Ca (p = 0.004 and p = 0.02) when averaged across metal forms. ZnO and CuO NPs had no significant effect on the accumulation Mg and Ca compared with their ionic treatments (p > 0.05)  . Compareulations , and 2E.mulation  and 2J.2+ (p = 0.004). Zn treatment concentrations significantly affect the accumulation of Fe (p = 0.001) and Ni (p < 0.001)  and the interaction effect of Zn forms and Zn treatment concentrations (p = 0.02) on the accumulation of Cu were significant. However, the effect of Zn forms on the accumulation of Cu was not significant (p = 0.18) . Zn treatment concentrations increased the accumulation of Mn by 16.7–28.7% when averaged across the Zn forms. The highest Mn accumulation was obtained with the Zn2+ 50 mg kg−1 andZnO NPs at 25 mg kg−1 (< 0.001) . The hig ZnO NPs  and 3B. ncreased . The eff = 0.18) . Comparenificant . The Mn  mg kg−1 .p < 0.001) , while the high concentration of Cu treatments had no significant effect on the accumulation of Ni and Zn (p > 0.05) . The Mn accumulation of CuO NPs was 8.9% higher than that of Cu2+ when averaged across Cu treatment concentrations (p = 0.03). Compared with the control, Cu2+ at 10–50 mg kg−1 and CuO NPs at 10–100 mg kg−1 significantly increased the accumulation of Mn by 19.8–28.7%, and 20.7–50.9%, respectively (p < 0.05). The highest Mn accumulation was observed for Cu2+ at 25 mg kg−1 and CuO NPs at 10 mg kg−1 (Cu treatment concentrations significantly affected the accumulation of micronutrients (< 0.001) . Cu2+ an mg kg−1 . Low con > 0.05) . As expe mg kg−1 .2+ and Cu2+ treatments, the highest comprehensive score was observed at the concentration of 50 mg kg−1 and 25 mg kg−1. For ZnO NPs and CuO NPs treatments, the highest comprehensive score was observed at the concentration of 25 mg kg−1 and 10 mg kg−1  of metal elements by consuming the ZnO and CuO NPs treated vegetable was quite lower than the oral reference dose  on the growth of Medicago polymorpha L  in soil, CuO NPs increased the fresh weight of root, stem, and leaf of tomato by 198.5%, 106.2% and 77.5%, and dry weight of root, stem, and leaf by 462.4%, 154.5% and 242.8% compared to the control, respectively. At a relatively higher concentration, −1 did not affect the leaf, stem, and root biomass of kidney beans. 2+ at 75 150, 300, and 600 mg kg−1 did not cause significant changes in fresh weight, plant height, water content, and the chlorophyll content of green onion compared with the control. However, −1 increased the shoot biomass of lettuce by 16.3–19.1%. This indicated that different species have different sensitivity to metal oxide NPs, and the response of species to metal oxide NPs may be dependent on their concentrations. In addition, there was no significant difference in FWPP of Medicago polymorpha L. between ionic metal treatments and metal oxide nanoparticles treatments (p = 0.51) (2) NPs treatments was not different from that of ionic metal  treatments. This indicated that, at the tested concentrations, ZnO and CuO NPs application promoted the biomass production of Medicago polymorpha L., and the concentration tested were not toxic for biomass production of Medicago polymorpha L. compared with Zn2+ and Cu2+. Previous studies demonstrated that the toxicological effects of metal oxide NPs were dependent on the metal oxide particle size and treatment concentrations (−1) caused more physiological impairments compared with bulk CuO and Cu2+ in bok choy. However, in the present study, the treatment concentrations (10–100 mg kg−1) were lower than the treatment concentrations reported by 2+ at high treatment concentrations (25-100 mg kg−1). This may partially explain why there was no significant difference in biomass production between CuO NPs treatments and Cu2+ treatments.The beneficial effects of Zn and Cu treatments on FWPP was dependent on their concentrations. The metal ionic and metal oxide NPs exhibited higher efficiency for crop growth promotion at low concentrations than at relatively high concentrations . This wamorpha L . Du et a = 0.51) . Ebbs ettrations . For exa2+ and ZnO NPs did not affect N accumulation, but high concentrations of Zn2+ and ZnO NPs decreased N accumulation . In the present study, both the Zn2+ and ZnO NPs increased the aboveground dry weight of Medicago polymorpha L. plants (−1), ZnO NPs decreased the microbial biomass and diversity in the soil , and P accumulation showed decreasing trend with the increasing Zn treatment concentration   . Our res <0.001) . In addi <0.001) . On the  lettuce . Zuverzap < 0.001 and p = 0.002) significantly increased the K accumulation of plants except for the ZnO NPs treatment at 200 mg kg−1  had a negative effect on the K uptake compared with the lower concentration of CuO NPs. This was because the high concentration of CuO NPs induced a more complexation with K+, reducing their availability for plants uptake  and Cu treatments  (p < 0.001), and Cu treatments significantly increased the accumulation of Cu (p < 0.001). However, high concentrations of Zn (200 mg kg−1) decreased the uptake of Cu compared with the low treatment concentration (25 mg kg−1). Similarly, high concentrations of Cu (100 mg kg−1) decreased the uptake of Zn compared with the low treatment concentration (25 mg kg−1)  were the important nutrient element for plant growth. Zn and Cu treatments increased the accumulation of Fe . Howeverp <0.05) . Fe playp <0.05) . Mg is ep <0.05) . The incmg kg−1) . Zhao etntration . Our resnsporter . The uptnsporter . Zhao etMedicago polymorpha L. is essential for a better understanding of food safety and human health. Previous studies had assessed the potential non-carcinogenic health risk of metal NPs and metallic element  are essential elements for animals and human beings. However, excessive metal elements intake may cause toxic effects . Therefo element . In the  below 1 . Therefo below 1 . Zn play below 1 . Cu was  below 1 . In addi below 1 . The impMedicago polymorpha L. in a concentration-dependent fashion. Both ZnO and CuO NPs improved the absorption of macronutrients  and micronutrients absorption . Comprehensive analysis showed the optimum concentrations were 25 mg kg−1 for ZnO NPs and 10 mg kg−1 for CuO NPs. The EDI, HQ, and CHQ were further calculated to assess the non-carcinogenic health risk of consuming the Medicago polymorpha L. treated with ZnO and CuO NPs. Our results showed that the enhanced metal nutrients in Medicago polymorpha L. did not cause any non-carcinogenic health risk to human body. Therefore, our results indicated that the low concentration of ZnO and CuO NPs could enhance growth performance and improve the micronutrient absorption of Medicago polymorpha L. without posing any non-carcinogenic health risk to the human body. Taken together, according to growth analysis and a human health risk assessment, ZnO and CuO NPs were considered as a safe nano fertilizer for Medicago polymorpha L. production. However, the effect of ZnO and CuO NPs was impacted by many factors, including soil properties and biological effects  was referred from the studies of Click here for additional data file.


Neoseiulus californicus is a predatory mite with a wide global distribution that can effectively control a variety of pest mites. In this study, MaxEnt was used to analyse the potential distribution of N. californicus in China and the BCC-CSM2-MR model was used to predict changes in the suitable areas for the mite from 2021 to 2100 under the scenarios of SSP126, SSP245 and SSP585. The results showed that (1) the average of area under curve value of the model was over 0.95, which demonstrated an excellent model accuracy. (2) Annual mean temperature (Bio1), precipitation of coldest quarter (Bio19), and precipitation of driest quarter (Bio17) were the main climatic variables that affected and controlled the potential distribution of N. californicus, with suitable ranges of 6.97–23.27 °C, 71.36–3924.8 mm, and 41.94–585.08 mm, respectively. (3) The suitable areas for N. californicus were mainly distributed in the southern half of China, with a total suitable area of 226.22 × 104 km2 in current. Under the future climate scenario, compared with the current scenario, lowly and moderately suitable areas of N. californicus increased, while highly suitable areas decreased. Therefore, it may be necessary to cultivate high-temperature resistant strains of N. californicus to adapt to future environmental changes. Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) is a very effective predator of spider mites and was first described on lemons trees in California, USA, in 19541. N. californicus is widely distributed in Argentina, Chile, the United States, South Africa, Japan, Southern Europe, the Mediterranean coast and other subtropical and tropical areas, mainly inhabiting citrus, grapes, strawberries, avocados, maize, cassava, vegetables and other plants4. Controlling pest mites with N. californicus is a biological control method with many advantages. First, using N. californicus to control harmful mites does not cause environmental pollution, can effectively control the mites to a certain extent, and has many applications in orchard and vegetable fields6. Vidrih et al.7 reported that satisfactory results could be achieved in suppressing Tetranychus urticae Koch on a hop plantation by repeated use of the predatory mite N. californicus. Second, N. californicus can prey on a variety of pest mites, including Tetranychus cinnabarinus 8, Panonychus citri (McGregor)9, T. urticae2, Polyphagotarsonemus latus (Banks)10, and Panonychus ulmi (Koch)11, worldwide. Finally, N. californicus can be used in combination with agents to improve management and reduce the use of chemical pesticides. Sato et al.12 found that it was feasible to control T. urticae in strawberry fields using selective acaricides and N. californicus. Although abamectin, acephate, fenpropathrin, iprodione, mineral oil and tebuconazole were shown to be slightly harmful to N. californicus, a combination of the two can be used for integrated control13.16. Currently, the MaxEnt model17, GARP model18, Bioclim model19 and domain model are commonly used. MaxEnt models the geographical distribution of species according to the niche theory proposed by Jaynes22 and has been used widely since its launch in 2004 to model such distributions25. The salient features of MaxEnt are that it requires only presence data and can use both continuous and categorical data26. In addition, it is simple to use and can give reliable and stable output even with small datasets28. In addition, many studies have indicated that the MaxEnt model has a better simulation effect among various species distribution models. Elith et al.29 compared the simulation performance of various niche models, and the results showed that MaxEnt had the highest prediction accuracy among 16 models. Currently, MaxEnt can be used for the analysis of species habitat requirements30, impact of future climate change on species distribution31, species invasion monitoring34 and natural analysis of protected areas35.Species Distribution Models (SDMs) are designed to correlate the distribution data of species with the corresponding environmental variables  of their distribution sites, analyse the relationship between the geographical distribution of the species and the environmental variables, and build modelsN. californicus is a predatory mite that can control a variety of pest mites, the prediction and simulation of the potential distribution area of N. californicus is of great significance for its application in the future. In this paper, the distribution record points of N. californicus were determined by consulting the literature and the Global Biodiversity Information Facility (GBIF) and Centre Agriculture Bioscience International (CABI) websites. The future distribution of N. californicus in China was predicted with the MaxEnt model under the SSP126, SSP245 and SSP585 scenarios. This study had three objectives: (1) to evaluate the main environmental variables afecting the distribution of N. californicus, (2) to explore the distribution of N. californicus under current and future climatic scenarios, and to provide a theoretical basis for how to use N. californicus to control pests in different periods and places in the future, (3) determine the changes in the suitable distribution area under the future scenario model, and provide biological protection recommendations on how to respond to climate change.Since The accuracy test of the MaxEnt model is shown in Table N. californicus was compared by the Jackknife method, and the results are shown in Fig. N. californicus were Bio1 (29.4%), Bio19 (15%), and Bio17 (16.4%).The importance of the six main variables that had a great influence on the distribution of N. californicus. The distribution probability of N. californicus increased with the increase in environmental variables within a certain range and decreased with the increase in variables after reaching a certain peak value. The results showed that the appropriate range ranges of Bio1, Bio19, Bio17, Bio4, Bio12, and Bio2 were 6.97–23.27 °C, 71.36–3924.8 mm, 41.94–585.08 mm, 65.63–958.57 °C, 488.74–4975.51 mm and -0.96–22.57 °C, respectively  and Jiangxi (15.01 × 104 km2) had the largest distribution area. The moderately suitable areas were mainly distributed in Guangxi, Guizhou, Guangdong, Hubei, Sichuan, Fujian, Anhui, Jiangsu and other provinces, with a total area of 77.09 × 104 km2, accounting for 8.04% of China's land area. Among these provinces, the largest distribution areas were Guangxi (13.23 × 104 km2), Guizhou (12.67 × 104 km2) and Guangdong (12.03 × 104 km2). The unsuitable area was located in northern Sichuan, Shaanxi, Henan and Shandong, with a total area of 733.03 × 104 km2, accounting for 76.42% of China's land area.As shown in Fig. N. californicus in China in the 2030s, 2050s, 2070s and 2090s under the SSP126 scenario. As shown in Fig. 4 km2 to 75.41 × 104 km2 (2030s), 93.11 × 104 km2 (2050s), 69.14 × 104 km2 (2070s) and 67.45 × 104 km2 (2090s) in the future. The moderately suitable area was projected to increase from a current area of 77.09 × 104 km2 to 101.17 × 104 km2 (2030s), 94.85 × 104 km2 (2050s), 103.93 × 104 km2 (2070s) and 107.05 × 104 km2 (2090s). The highly suitable area was projected to decrease from a current area of 90.92 × 104 km2 to 29.79 × 104 km2 (2030s), 19.76 × 104 km2 (2050s), 35.98 × 104 km2 (2070s) and 35.98 × 104 km2 (2090s).Figure N. californicus in China in the 2030s, 2050s, 2070s and 2090s under the SSP245 scenario. As shown in Fig. 4 km2 to 65.58 × 104 km2 (2030s), 66.62 × 104 km2 (2050s), 105.01 × 104 km2 (2070s) and 108.05 × 104 km2 (2090s) in the future. The moderately suitable area was projected to increase from a current area of 77.09 × 104 km2 to 88.60 × 104 km2 (2030s), 110.18 × 104 km2 (2050s), 79.03 × 104 km2 (2070s) and 88.73 × 104 km2 (2090s). The highly suitable area was projected to decrease from a current area of 90.92 × 104 km2 to 50.13 × 104 km2 (2030s), 29.43 × 104 km2 (2050s), 16.18 × 104 km2 (2070s) and 12.49 × 104 km2 (2090s).Figure N. californicus in China in the 2030s, 2050s, 2070s and 2090s under the SSP585 scenario. As shown in Fig. 4 km2 to 62.78 × 104 km2 (2030s), 98.20 × 104 km2 (2050s), 103.28 × 104 km2 (2070s) and 148.50 × 104 km2 (2090s) in the future. The moderately suitable area was projected to increase from a current area of 77.09 × 104 km2 to 92.49 × 104 km2 (2030s), 92.62 × 104 km2 (2050s), 89.03 × 104 km2 (2070s) and reduced to 36.69 × 104 km2 (2090s). The highly suitable area was projected to decrease from a current area of 90.92 × 104 km2 to 55.67 × 104 km2 (2030s), 20.52 × 104 km2 (2050s), 14.90 × 104 km2 (2070s) and 5.57 × 104 km2 (2090s).Figure In the SSP126 scenario, the species centroids from the current position were 15.64 km (2030s) in the northeast, 0 km (2050s), 8.74 km (2070s) in the west, and 13.47 km (2090s) in the southwest. In the SSP245 scenario, the species centroids from the current position were 23.26 km (2030s) in the northeast, 10.25 km (2050s) in the north, 22.28 km (2070s) in the southwest, and 18.09 km (2090s) in the northwest. In the SSP585 scenario, the species centroids from the current position were 10.71 km (2030s) in the northeast, 34.39 km (2050s) in the northeast, 28.30 km (2070s) in the southwest, and 30.75 km (2090s) in the southeast , precipitation in June (prec6), precipitation in October (prec10), and precipitation in December (prec12) had important effects on the distribution of N. californicus. Because mites are small in size, they are affected by factors such as wind and rain in addition to temperature, and rainfall can negatively affect them by drowning them or knocking them into the soil47. The scouring of heavy rain and torrential rain has been reported to have a significant inhibitory effect on T. cinnabarinus48.Species distribution is highly susceptible to the influence of the environment, and the environment directly or indirectly affects the physiological and ecological functions of species, thereby limiting their distributionN. californicus was 226.22 × 104 km2. Of this area, the proportion of lowly suitable area accounted for 25.73%, moderately suitable area accounted for 34.08%, and highly suitable area accounted for 40.19%. In general, the boundaries of suitable and unsuitable areas for N. californicus were generally consistent with the findings of Wang et al.31 In addition, there were also some differences in the locations and areas of different suitable areas. The reason for these differences may be that we obtained 118 points for our distribution prediction after removing redundant points, while Wang et al. obtained 65 points. We performed a model optimization for the parameters in R before running the MaxEnt fitness simulations31.Our results indicated that the total suitable area for 49. The latest CMIP6 model shows that the world will be significantly warmed in the future. In the future SSP126, SSP245, and SSP585 scenarios, the temperature is predicted to increase by 1.3–2.9 °C, 2.1–4.3 °C, and 3.8–7.4 °C, respectively50. Therefore, based on the current distribution of N. californicus, we predicted the potential redistribution of N. californicus in response to climate change under these three climate scenarios in the twenty-first century. Our results showed that the size and distribution of the areas of N. californicus were different under the three future climate scenarios, but all showed basically the same trends. Compared with the current distribution, the distribution areas of the lowly and moderately of N. californicus showed an upwards trend (except in the 2090s under the SSP585 scenario), and the highly suitable area showed a downwards trend. Combined with existing research reports, in the temperature range of 30–35 °C, the mortality of female adult mites significantly increased, indicating that high temperatures above 30 °C have an adverse effect on the development, survival rate and reproduction of N. californicus46. At 37.5 °C, the females of N. californicus can lay eggs, but the eggs cannot hatch; at 40 °C, the females of N. californicus cannot lay eggs51. This may be one reason why the area of highly suitable area for N. californicus was projected to decrease in the future due to climate change. In addition, due to climate change, the overall migration trend of N. californicus was to the west (SSP126), northwest (SSP245), and northeast (SSP585).As the global climate warms, the structure and function of terrestrial ecosystems may be significantly altered, resulting in significant changes in the extent and distribution of biological habitats52 found that high-temperature exposure had significant effects on the egg hatching rate, survival rate and development duration of N. californicus, but had little effect on the pre-oviposition and survival rate of the adults.Thus, to adapt to the future climate and continue to effectively and continuously control pests and mites in a high-temperature environment, high-temperature resistant strains of N. californicus may need to be cultivated in the future as Zhang et al.53 selected the high-temperature resistant strain of Neoseiulus barkeri.Climate change and the frequent occurrence of extreme weather will restrict the continuous control of phytoseiid mites in agricultural ecosystems and high-temperature will often interfere with the biological control of tetranychus mites using phytoseius mites. Yuan et al.54 was downloaded from the Museum of Natural History website (https://biodiversityinformatics.amnh.org/open_source/maxent/); Java software was downloaded from its official website (https://www.oracle.com/java/); R (version 4.1.2)55 and RStudio software were downloaded from their official website ; ArcGIS software (version 10.8.1) was downloaded from the ESRI website (https://support.esri.com/en/Products/Desktop/arcgis-desktop/arcmap); and the base map was provided by the National Meteorological Information Centre of China.MaxEnt software (version 3.4.1)N. californicus were downloaded from the GBIF (https://www.gbif.org/) and CABI (https://www.plantwise.org/knowledgebank/) websites and combined with relevant literature on the occurrence of N. californicus66. The latitude and longitude recorded in the literature for N. californicus were determined using Google Earth. Through the above procedure, a total of 118 distribution data points was obtained  and included 19 bioclimatic variables (2.5′ resolution)   Fig. .Table 5LAfter completing the above steps, six environmental variables were finally retained and used to build the final model, including annual mean temperature (Bio1), mean diurnal range (Bio2), temperature seasonality (Bio4), annual precipitation (Bio12), precipitation of driest quarter (Bio17), and precipitation of coldest quarter (Bio19).68 in R was used to test the Akaike information criterion correction (AICc), which generally provides priority to parameters with small AICc values for simulation and is considered to be a standard measure of the goodness of fit of a model. Finally, we selected the model with a delta AICc equal to 0 according to the result of the ENMeval procedure.The ENMeval packageN. californicus were input into MaxEnt, and then ‘Create Response Curve’, ‘Do Jackknife to Measure Variable Importance’, and output format as ‘Logistic’ were selected. In the settings, the ‘Random test percentage’ was set to 25 , and the ‘Regularization Multiplier’ was set to 1, ‘Replicates’ was set to 10 times; the model was set to ‘Hinge Features’ and the other parameters were set to the default software parameters. The receiver operating characteristic (ROC) curve output by MaxEnt was used to evaluate the accuracy of the model. Model performance was classified as failing (0.5–0.6), poor (0.6–0.7), fair (0.7–0.8), good (0.8–0.9), and excellent (0.9-) according to the AUC value69. Moreover, the True Skill Statistic (TSS) also have performed to test the performance of MaxEnt model70. For future potential distribution analysis of different scenarios, the corresponding future environment data will be placed in the ‘Projection Layers Directory/file’ in MaxEnt and run under the same settings.The occurrence data and seven environmental variables of N. californicus in China were extracted.We imported the ASC II file obtained after the MaxEnt operation into ArcGIS 10.8.1, converted it into ‘raster’ format by using ‘ArcToolbox’, used the ‘Reclass’ function of the ‘Spatial Analyst tool’ to reclassify layers and divided regions according to their suitability level. Jenks' natural breaks were used to reclassify the suitability and classify the suitability into four categories: unsuitable area (P < 0.078008), lowly suitable area (0.078008 ≤ P < 0.269478), moderately suitable area (0.269478 ≤ P < 0.47513) and highly suitable area. Finally, based on a map of China, the potential distribution areas of


Periplaneta americana, which is one of the most common insects in the nature, can be extracted to treat UC. However, the active components in Periplaneta americana extract (PAE) can be degraded in the stomach due to its extreme acidic environment and enzyme. In this study, we developed a pH-dependent drug delivery method using polymer cellulose acetate (Eudragit S100) as a carrier to deliver high concentration PAE to inflamed colon. Both in vitro and in vivo results showed the PAE-Eudragit-S100 could treat UC through delivering active drug components to colon without degradation.Ulcerative colitis (UC) is a chronic disease, which can result the inflammation of the rectum, mucosa of the colon, and submucosa. The active component such as polypeptide in  Eudragit S100 polymer solution was mixed with PAE ethanol solution by sonicating. Then mixture was then mixed with lecithin and glycerol monooleate in a blender . The emulsion was stirred at room temperature with a magnetic stir bar at 420 rpm. Them the microparticles were freeze-dried in a freeze-dryer. Finally, When mice were treated with this drugs. the drugs were not decomposed in stomach and small intestine but damaged in the colon, which had a good curative effeet on colitis. Tianjin Bodi Chemical Co. Ltd  supplied Tween 80, paraffin, acetic acid, ninhydrin, ascorbic acid. Hydrochloric acid, phosphate buffer solution, acetic acid, sodium sulfate, dinitrochlorobenzene, normal saline, and formalin were purchased from Shanghai Yuanye Biotechnology Co. Ltd. . Coomassie bright blue R250 and SDS-PAGE gel preparation kit were purchased from Biyuntian Biotechnology Co. Ltd . Protein standard molecular weight marker 26610 was obtained from Beijing Solaibao Technology Co. Ltd . ELISA kits were acquired from Shanghai Fanke Biotechnology Co. Ltd . Deionized water was filtered by 0.22 μm PES filter  to remove any contaminants.Inosine, aminopropionic acid, bovine serum albumin, and hypoxanthine were supplied from Shanghai McLean Biochemical Technology Co. Ltd . Soybean lecithin was purchased from Beijing Biotechnology Co. Ltd . Glyceryl monooleate was obtained from Shanghai Jiafashi Trading Co. Ltd . Methanol, ethanol, phenol reagent, ethyl ether, glacial acetic acid, NaC2.2.Animal Experiment Center in Shenyang Pharmaceutical University supplied SD mice . All animals were kept in the housing system under 24 ± 1 °C, relative humidity (40-70%), and 12-12 hours cycles of light-dark with free water and fodder access. Before the experiment, the animals were deprived of food for 12 hours but with free access to water. The protocols were performed strictly according to guidelines issued by Animal Experiment Center in Shenyang Pharmaceutical University.2.3.Periplaneta americana body was blended to fine powder and was soaked in 90 v/v % ethanol solution for 12 hours  was used to purify the PAE. The PAE was loaded into the packed column and eluted by 0.22 μm filtered deionized (DI) water at 100 mL/hour for 2 hours at room temperature. 95 v/v % ethanol was used for desorption at 100 mL/hour for 1.5 hours and the elution was collected. Finally, the PAE mixture was freeze-dried in a freeze-dryer  at −100 Pa, −40 °C for 24 hours.2.4.The protein content in PAE was detected by high performance liquid chromatooraphy (Hitachi Scientific Instruments (Beijing) Co. LTD). The mobile phase was 0.1 M Na2SO4, 0.05 M Na2HPO4 and 0.05 M NaH2PO4. The flow rate was 0.8 ml/min at 25 °C. The chromatographic column was TSKgel G3000SWxl  for 30 minutes to form fine emulsion. The mixture was then mixed with 0.33 g/L lecithin and 30.3 g/L glycerol monooleate in a blender  for 2 minutes to form 110 mL emulsion  at 3000 rpm for 15 minutes, and supernatant was collected. The supernatant was used to calculate the concentration of polypeptides using method 2.3.1. The encapsulation efficiency (EE) of polypeptide microparticles was calculated according to  at 37 °C and 140 rpm for 20 hours, and 1 mL solution was taken out at 0.3, 0.6, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 20 hours, respectively  of PAE at different time;1, K2, and K3 denoted the release-rate constants for zero-order, first-order, and Korsmeyer-Peppas;n was the release exponent.K2.9.The equipment was filled with sealing glue ) , separating glue  and concentrating glue. . 4 mg/mL PAE microparticles were prepared in simulated gastric solution, simulated small intestine juice and simulated colonic juice, respectively. They were agitated in an incubator 37 °C and 140 rpm for 24 hours, and centrifugated at 12000 rpm for 15 minutes to obtain supernatant. 35 μL supernatant was mixed with 15 μL loading buffer  in 1.5 mL tube. Then the liquid was boiled for 5 minutes and centrifuged at 12000 rpm for 5 minutes. 30 μL protein samples and protein marker were loaded into the gel. When bromophenol blue line was closed to sealing glue, the gel was collected and stained by Coomash bright blue R-250 for 12 hours. Then the gel was de colorized by eluent, and the images of gel were recorded , and its morphology was observed under the microscope Olympus BX 60  with eyepiece 40 and objective 10  for 2 hours. Only two clear bands were observed at about 25 kDa and 35 ∼ 45 kDa. Since the microparticles were still intact , one of the most important members of the peroxidase family, mainly exists in neutrophils and mononuclear macrophages (Podrez et al., 5.In this study, we used Eudragit S100 as a drug carrier to deliver PAE in colon. PAE-S100 microparticles could research the colon without degradation in the digestion process and release PAE in colon to treat UC. Both DAI score and MPO were significantly reduced using the PAE-S100 drug delivery system. In addition, this system could be potentially used for other oral protein formulation administration.All authors contributed to this paper, with Meng Li taking the supervision and writing the original draft; Hao Wu reviewing and checking the content and editing the final version of the manuscript; Jing Han, Yang Han designing the framework and research, conceptualization;Hao Wu, Meng Li, Shuang Wang, Shengshun Wu conducting the experimental operation; All authors contributed extensively to this work. All authors have read and agreed to the published version of the manuscript.Click here for additional data file.Click here for additional data file.


Cd) associated with injury to kidney tubular cells, a defective tubular reabsorption of filtered proteins, and a reduction in the estimated GFR (eGFR). Data were from 289 males and 445 females, mean age of 48.1 years of which 42.8% were smokers, while 31.7% had hypertension, and 9% had chronic kidney disease (CKD). The BMDL value of ECd associated with kidney tubular cell injury was 0.67 ng/L of filtrate in both men and women. Therefore, an environmental Cd exposure producing ECd of 0.67 ng/L filtrate could be considered as Cd accumulation levels below which renal effects are likely to be negligible. A reduction in eGFR and CKD may follow when ECd rises from 0.67 to 1 ng/L of filtrate. These adverse health effects occur at the body burdens lower than those associated with ECd of 5.24 µg/g creatinine, thereby arguing that current health-guiding values do not provide a sufficient health protection.Cadmium (Cd) is a toxic metal pollutant that accumulates, especially in the proximal tubular epithelial cells of kidneys, where it causes tubular cell injury, cell death and a reduction in glomerular filtration rate (GFR). Diet is the main Cd exposure source in non-occupationally exposed and non-smoking populations. The present study aimed to evaluate the reliability of a tolerable Cd intake of 0.83 μg/kg body weight/day, and its corresponding toxicity threshold level of 5.24 μg/g creatinine. The PROAST software was used to calculate the lower 95% confidence bound of the benchmark dose (BMDL) values of Cd excretion (E Cadmium (Cd) is an environmental toxicant of worldwide public health significance because numerous population-based studies suggest that exposure to Cd even at low levels adversely impacts the functions of most organs of the body . VolcaniFoods that are frequently consumed in large quantities such as rice, potatoes, wheat, leafy salad vegetables, and other cereal crops form the most significant dietary sources of Cd ,14,15. SCd accumulates most extensively in the proximal tubular epithelial cells of the kidneys, and the kidney burden of Cd as µg/ g tissue weight increases with age ,19. Appr2-microglobulin  above 300 μg/g creatinine to be the “critical” toxicity endpoint, the tolerable intake level of Cd was set at 0.83 µg/kg body weight/day, and a urinary Cd excretion of 5.24 µg/g creatinine was considered to be the toxicity threshold level u and [cr]p) was based on the Jaffe reaction. The urinary NAG assay was based on colorimetry . The urinary β2M assay was based on the latex immunoagglutination method .Simultaneous blood and urine sampling is required to normalize Eu was determined by inductively-coupled plasma mass spectrometry  because it had the high sensitivity required to measure very low Cd concentrations. Multi-element standards  were used to calibrate the Cd analyses. The accuracy and precision of those analyses were ascertained with reference urine . The low limit of detection (LOD) of urine Cd, calculated as 3 times the standard deviation of blank measurements was 0.05 µg/L. The Cd concentration assigned to samples with Cd below the detection limit was the detection limit divided by the square root of 2 oot of 2 .Cd data reported for Mae Sot residents that could be reliably quantified by atomic absorption spectrophotometry (AAS), the [Cd]u in samples from Mae Sot group was determined by AAS . Urine standard reference material No. 2670  was used for quality assurance and control purposes. The LOD of Cd quantitation, calculated as 3 times the standard deviation of blank measurements was 0.06 µg/L. None of the urine samples from this group was found to have a [Cd]u below the detection limit.Based on EThe GFR is the product of nephron number and mean single nephron GFR, and in theory the GFR is indicative of nephron function ,33,34. Iy × 0.993age, where y = −0.411 if serum creatinine ≤ 0.9 mg/dL or −1.209 if serum creatinine > 0.9 mg/dL.Male eGFR = 141 × [serum creatinine/0.9]y × 0.993age, where y = −0.329 if serum creatinine ≤ 0.7 mg/dL or −1.209 if serum creatinine > 0.7 mg/dL.Female eGFR = 144 × [serum creatinine/0.7]These CKD-EPI equations have been validated with inulin clearance, and they are considered to be the most accurate eGFR calculating equations .x was normalized to Ecr as [x]u/[cr]u, where x = Cd, β2M or NAG; [x]u = urine concentration of x (mass/volume); and [cr]u = urine creatinine concentration (mg/dL). The ratio [x]u/[cr]u was expressed in μg/g of creatinine.Ex was normalized to Ccr as Ex/Ccr = [x]u[cr]p/[cr]u, where x = Cd, β2M or NAG; [x]u = urine concentration of x (mass/volume); [cr]p = plasma creatinine concentration (mg/dL); and [cr]u = urine creatinine concentration (mg/dL). Ex/Ccr was expressed as the excretion of x per volume of filtrate p and mean [cr]u in females were lower than males (p < 0.001 for both parameters) as were mean [Cd]u (p = 0.020) and mean ECd/Ccr (p = 0.021). In contrast, mean ENAG/Ecr in females was higher than that of males (p < 0.001).For the Bangkok group, mean [cr]2, respectively p, mean [cr]u and mean [Cd]u all were the highest in the eGFR level 3, middle in eGFR level 2, and the lowest in eGFR level 3 as were the means for Ecr- and Ccr-normalized ECd, Eβ2MG, ENAG.The percentages of females and those with hypertension in each eGFR group did not differ, while % of diabetes (6.1%) and smoking (63.6%) were the highest in the lowest eGFR group. None of subjects from a low exposure area had low eGFR. Subjects of the low eGFR group was the oldest (mean age 63.2), who also had the lowest mean BMI [. These findings were consistent with data from a conventional dose–response analysis, where an inverse relationship between ECd and eGFR became statistically significant only when ECd was normalized to Ccr [The BMDL values of Eectively . The reseatinine . In contd to Ccr .cr-normalization, the effect of Cd on eGFR was not realized. In a systematic review and meta-analysis of pooled data from 28 studies, the risk of proteinuria was increased by 1.35-fold when comparing the highest vs. lowest category of Cd dose metrics, but an increase in the risk of low eGFR was statistically insignificant (p = 0.10) [Because of high variance in datasets introduced by E = 0.10) . An erro = 0.10) .Cd/Ecr for tubular injury (ENAG) was 1.67 μg/g creatinine [Cd/Ecr BMD/BMDL figures based on Eβ2M and ENAG were 1.24/0.62 and 0.85/0.49 μg/g creatinine in men, respectively [Cd/Ecr BMD/BMDL figures for Eβ2MG were higher than those for ENAG. This was seen in a Swedish study [In a meta-analysis of pooled data from 30 publications, the BMD of Eeatinine . In anotectively . The corsh study  and our Cd/Ecr BMD figures for Eβ2M endpoint were 0.6–1.2 and 0.6–2.3 µg g creatinine in men and women, respectively [In a study of data from 410 men and 418 women, aged 40–59 years, who were residents of non-pollution areas of Japan, the Eectively .β2M/Ecr ≥ 300 μg/g creatinine, the BMDL value ECd/Ecr values were 0.469 and 0.733 µg/g creatinine in men and women, respectively . Eβ2M is a function of nephron numbers and tubular reabsorption activity, a substantial increase in Eβ2M can be expected when nephrons are lost [β2M as a “critical” effect of Cd accumulation in kidney tubular cells is inappropriate.Consistent with the above notions are data in are lost . AccordiCd/Ccr associated with a 5% increase of kidney tubular cell injury are 0.67/3.94 and 0.67/3.99 ng/L filtrate in men and women, respectively. The respective BMDL/BMDU values of ECd/Ccr associated with a 5% eGFR decline in men and women are 0.80/7.14 and 0.98/8.12 ng/L filtrate. A Cd accumulation level resulting in Cd excretion at 0.67 ng/L filtrate in men and women could be viewed as NOAEL equivalent and is a maximally permissible Cd accumulation level. A reduction in eGFR and CKD may follow when Cd excretion levels exceed this maximal permissible accumulation level of 0.67 µg/L of filtrate. Health guidance values for Cd derived from these NOAEL equivalents of Cd excretion levels are more likely to be low enough to carry a negligible adverse effect on kidneys.The BMDL/BMDU values of E


Meanwhile, we determined rhizosphere soil physicochemical properties, phenolic acids content, and bacterial community diversity by high-throughput sequencing. The results show that FOFs improved the tobacco plant agronomic traits, leaf yield (by 2.9–42.8%), value (by 1.2–47.4%), and chemical quality when compared with CF. More content of NH4+-N, available P, and available K were discovered in the rhizosphere soil in VOF, HOF, and WOF. The rhizosphere sinapic acid and total phenolic acids content declined in the FOF treatments  versus those in the CF treatment . Moreover, the rhizosphere bacterial community structure changed under FOF functions: the beneficial microbes Actinobacteria, Firmicutes, Streptomyces, and Bacillus increased, and the harmful microbes Acidobacteria and Gemmatimonadota decreased in abundance. There was a positive correlation between the tobacco leaf yield and soil NH4+-N, TC content, and the relative abundance of Proteobacteria and Actinobacteriota. In summary, the application of VOF and WOF is a modest, practical, and environmentally friendly strategy to alleviate tobacco CCO from the standpoint of recycling solid waste.Continuous cropping obstacle (CCO) in tobacco is a prevalent and intractable issue and has not yet been effectively solved. Many researchers have favored exploring environmentally friendly and sustainable solutions to CCO  organic fertilizers). Therefore, to study the effects of functional organic fertilizers (FOFs) on tobacco CCO, we applied five types of fertilizers in a tobacco continuous cropping field with red soil . The tobacco plant agronomic traits, leaf yield, economic value, and chemical quality (nicotine, total sugar, K Capsicum annuum L.) (Lycopersicon esculentum Miller) (Nicotiana tabacum L.) (Panax quinquefolius L.) (Arachis hypogaea L.) (Cucumis sativus L.)   in the same field is a serious and widespread phenomenon in China, and even throughout the world . Crop CCnuum L.) , tomato  Miller) , tobaccoacum L.) , Americalius L.) , peanut gaea L.) , cucumbeivus L.) , apple (a Mill.) , and so a Mill.) . Plantina Mill.) .There are three main well-known causes for crop CCO: deterioration of the soil’s physicochemical properties, an accumulation of crop allelopathic substances (primarily phenolic acids-PA), and a change of soil microbial community structure, which can be collectively referred to as an imbalance of soil micro-ecology environment . Based oRapeseed cakes and mushroom residues are two by-products of agricultural activities that are available in large quantities and need to be properly utilized. Due to their high N/P/K contents and other micro-elements, they can be the main raw resources of organic fertilizers. Vermicompost is an organic compost product that is derived from organic matter decomposition and degradation processes by the action of earthworm digestion systems and the action of related microbes . It has The use of a combination of rapeseed cakes and mushroom residues as the primary resources of N/P/K with soil functional amendments  has not recently been reported or applied in tobacco production. Indeed, how FOFs perform in soil and alleviate crop CCO needs to be further deciphered and validated. Returning quantities of biomass residues to the soil is expected to not only achieve the cycle utilization of biomass but also improve soil condition, crop yield, and quality. The objectives of this study are: 1) to study the effects of FOFs on tobacco CCO ; 2) to explore the possibility of using FOFs to replace tobacco-special chemical fertilizers as base fertilizers; and 3) to elucidate the mechanisms of FOFs’ function by paying attention to the changes of rhizosphere soil physicochemical properties, phenolic acids content, and bacterial community structure under field conditions. We assume that FOFs can alleviate tobacco CCO, including the improvement of tobacco yield and chemical quality, by ameliorating the soil’s physicochemical properties and bacterial community structure when compared to CF. Knowledge of FOFs’ efficacy and action mechanisms will provide great potential for the sustainable plantation of tobacco and other crops, and will help to improve soil quality in an environmentally friendly way.Vicia faba L.) in the same year. The tobacco variety and soil type are K326 and red soil, respectively. The basic properties of the soil are as follows: pH 7.3, NH4+-N 7.3 mg kg−1, NO3−-N 8.3 mg kg−1, available phosphorous (AP) 37.4 mg kg−1, cation exchange capacity (CEC) 20.2 cmol+ kg−1, organic matter (OM) 26.1 g kg−1, total carbon (TC) 26.6 g kg−1, total nitrogen (TN) 2.1 g kg−1, total phosphorous (TP) 0.9 g kg−1, and total potassium (TK) 7.5 g kg−1. Tobacco growers usually apply tobacco-special compound chemical fertilizers  as base fertilizers. We used four types of functional organic fertilizers (FOFs) to displace chemical fertilizers. Rapeseed cakes and mushroom residues are the primary components in the four FOFs, which are added with respective functional substances . Therefore, five fertilizer application programs were included in the study, as follows: CF , VOF (vermicompost-based FOF), HOF (humic acid-based FOF), WOF (wood biochar-based FOF), and COF . KNO3, K2SO4, (NH4)2HPO4, and CH4N2O were used to balance the N/P/K content in the FOFs compared to CF. There were four repetitions in each treatment as a randomized block design. Each block owned about 66 m2 and was surrounded by guard rows. The CF and FOFs were applied in the planting hole as base fertilizer in April 2020, before transplanting tobacco seedlings at a rate of 225 and 1,000 kg ha−1, respectively. Tobacco seedlings were planted with 1.20 × 0.55 m and cultivated according to the local optimal production technology. There were a total of five fertilizer applications during the tobacco planting period—one application of base fertilizer and four applications of topdressing.The field experiment was conducted from April to September 2020 and was located on Longjie Street, Chengjiang County, Yuxi City, Yunnan Province in China . The climate of the study location is as follows: temperature 16–24°C, annual sunshine duration 2, 100–2,300 h; average annual precipitation 837 mm; annual frost-free period 244–365 days; and altitude 1745 m. Tobacco had been continuously cultivated for 2 years in the experimental field before 2020. The tillage regime is the rotation of tobacco and broad bean  was removed by shaking heavily and the rhizosphere soil (0–5 mm away from the root) of these four tobacco plants was gently collected to form one sample. Immediately, about 50 g of rhizosphere soil per sample was put into an incubator with ice bags, taken back to the laboratory, and put into a –80°C refrigerator for the determination of PA content and bacterial community diversity. The remaining rhizosphere soil was subjected to analysis of the physicochemical properties. Meanwhile, 10 representative tobacco plants in each block were chosen and labeled to determine the agronomic characteristics, including the number of productive leaves, plant height, stem thickness, largest leaf width, and length. The labeled tobacco leaves in each block were separately harvested and flue-cured. After a total of five harvests, we classified the flue-cured tobacco leaves in each block according to the local protocol, weighed each classification, and then calculated economic parameters. The cured leave samples belonging to the C3F classification  were used to measure chemical components  based on the continuous flow method  .3−-N, NH4+-N, AP, and AK contents and through a 0.15 mm sieve to determine the CEC, OM, total organic matter (TOC), TC, and TN contents. Soil pH was analyzed by a pH parameter    . Soil NO, China) . Soil AK, China) . Soil ex, China) . The TOC, China) .g for 10 min 10 ml of supernate was acidified with 12 M HCl to pH 2.5 and was then put for 2 h for humic acid precipitation. After that, the suspension was centrifuged at 8,000 × g for 10 min and the supernate was passed through a 0.22 μm organic filter subjected to UPLC . The UPLC analytical conditions for PA were as follows: chromatographic column, C18 ; column temperature, 40°C; detector wavelength, 280 nm; flow velocity, 1 ml min−1; and injection volume, 10 μL. The mobile phase consisted of 0.1% phosphoric acid solution (A-phase) and acetonitrile (B-phase). In total, 17 types of standard PA samples  were measured for the retention time and peak size under a certain concentration. The PA kinds and concentrations in rhizosphere soil were identified by comparing retention time and peak size with respective standards.The method to determine the PA content was drawn from ® Spin Kit for Soil . The purity and concentration of DNA were determined based on the 260/280 and 260/230 nm ratios through a micro-spectrophotometer . DNA integrity was determined by 1.0% agarose gel electrophoresis and visualized. The hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified using a barcode sequences primer pair 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR reactions were performed in 20 uL reaction mixtures: 5 × TransStart FastPfu buffer 4 μL, 2.5 mM dNTPs 2 μL, 5 uM forward primer 0.8 μL, 5 uM reverse primer 0.8 μL, TransStart FastPfu DNA Polymerase 0.4 μL, BSA 0.2 μL, template DNA 10 ng, and finally ddH2O up to 20 uL. The PCR thermal cycling conditions were performed as the regime: initial denaturation (95°C for 3 min), followed by 27 cycles of denaturation (95°C for 30 s), annealing (55°C for 30 s), extension (72°C for 45 s), and a final extension (72°C for 10 min). Each sample was amplified in triplicate. PCR product was extracted from 2% agarose gels, purified using the AxyPrep DNA Gel Extraction Kit  according to manufacturer’s instructions, and quantified using Quantus™ Fluorometer .According to the manufacturer’s protocol, the microbial community genomic DNA of rhizosphere soil was extracted with the instructions of Fast DNAThe purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform  by Majorbio Bio-Pharm Technology Co. Ltd. .https://www.arb-silva.de/) with a classification confidence of 0.7.The raw 16S rRNA gene sequencing reads were demultiplexed and quality-filtered by fastp v0.19.6 . Pair-enp < 0.05). OTUs counts were normalized based on the minimum in all samples before bioinformatic analysis. All of the sequences were processed using QIIME v1.9.1  and WOF  increased apparently (p < 0.05) than CF  (p ≥ 0.05) among HOF , COF , and CF. The tobacco yield and value increased by 40.6% and 35.6% in VOF, and by 41.5% and 46.1% in WOF than those in CF, respectively. Simultaneously, the tobacco leaf mean price, high class leaf rate, class index, and production index in FOFs were all higher than those in CF, with VOF and WOF presenting extraordinary enhancements  . Howeverncements . The tob3−-N and TN content did not show any obvious variance under the five treatments (p ≥ 0.05). Although the OM, TOC, and TC contents increased in VOF and HOF, they did not present a significant difference (p ≥ 0.05). Meanwhile, the FOFs had a greater effect on the NH4+-N, AP, AK contents, and CEC, particularly on the AP content. The NH4+-N, AK contents, and CEC showed an increasing trend under FOFs, except for COF, when compared with CF. The AP content increased by 31.6–81.0% in the four FOFs treatments when compared to CF.Rhizosphere Soil pH showed an acidification trend after planting tobacco, which changed from 7.34 to 6.73–6.94 . This acp-hydroxybenzoic acid, phthalic acid, vanillic acid, syringic acid, vanillin, ferulic acid, and sinapic acid) could be detected in the rhizosphere soil (p-hydroxybenzoic acid average content was the highest (1.52–1.64 mg kg−1 dry soil), followed by the sinapic acid (1.23–2.01 mg kg−1 dry soil), vanillin (1.28–1.38 mg kg−1 dry soil), ferulic acid (1.11–1.42 mg kg−1 dry soil), vanillic acid (1.18–1.29 mg kg−1 dry soil), and phthalic acid contents (1.14–1.26 mg kg−1 dry soil). The syringic acid content was the lowest (0.60–0.62 mg kg−1 dry soil). The total PA content ranged from 7.95 to 10.10 mg kg−1 dry soil in all treatments. Although the sinapic acid and total PA contents decreased apparently under FOFs when compared to CF (p < 0.05), they did not show an obvious variance among the four FOF treatments. There was no significant difference (p ≥ 0.05) for the other PAs among the five treatments.Although we prepared 17 varieties of the PA’s standard reference samples, only seven types , Chloroflexi (20.6–25.0%), Proteobacteria (13.5–16.4%), Acidobacteria (1.8–7.7%), and Firmicutes (3.0–9.1%), with an average total relative abundance of 90.0% , Arthrobacter (4.1–5.7%), norank_f__Roseiflexaceae (3.7–5.5%), norank_f__JG30-KF-CM45 (3.6–5.6%), and Gaiella (3.4–4.5%), with the average total relative abundance of 22.5%  different from the other four treatments, and there was no significant difference (p ≥ 0.05) among CF, VOF, HOF, and WOF. Based on PCoA and PerMANOVA analyses on the OTUs level, the five treatments were divided from each other, particularly at the first primary component, with the first two primary components explaining 41.87% and 14.05% variance, respectively  (p = 0.001). CF was prominently distinct from COF, and VOF, HOF, and WOF clustered relatively closer, presenting a more pronounced difference in the bacterial community’s β-diversity between CF and COF than that among VOF, HOF, and WOF.For the α-diversity of the bacterial community on the OTUs level, COF had the minimum of the Ace, Chao, and Shannon indexes, and the maximum of the Simpson index. This indicates that the α-diversity in COF was the lowest in all treatments . Moreove= 0.001) . This ph= 0.001) , which sAcidobacteriota in CF, family-Microbacteriaceae in HOF, genus-norank_f__Gemmatimonadaceae in WOF, and phylum-Firmicutes in COF. From the significance analysis for the bacterial community at the phylum level among groups based on the Kruskal–Wallis H test, there was a significant increase in the relative abundance of Firmicutes, and a significant decrease of Acidobacteriota, Gemmatimonadota, Elusimicrobiota, WS2 and Sumerlaeota in the four FOF treatments compared to CF, particularly in COF  with more genera  was improved in the four FOF treatments. Similarly, no apparent difference in tobacco leaf quality was discovered among the four FOF treatments. The enhancement of organic fertilizers on plant yield and quality has been demonstrated in many studies  in the four FOF treatments, and no significant difference (p ≥ 0.05) was observed for the other PAs, which are a class of small organic substances and secondary metabolites that are released from plants and have strong allelopathy (Microvirga and Nocardioides (p ≤ 0.05) in the rhizosphere soil based on the Spearman correlation analysis. Therefore, PA has changed the bacterial structure and diversity to some extent.Different plants release their unique varieties of PA. Meanwhile, elopathy . PAs canelopathy . After aelopathy . Meanwhielopathy . This alOur results show that the bacterial structure and diversity in rhizosphere soil were changed under the function of the FOFs. Different fertilizer management programs can significantly change the soil’s microbial community, biomass, and diversity, and functional activities can be enhanced under balanced fertilization . There wActinobacteria, Chloroflexi, Proteobacteria, Acidobacteria, Firmicutes, and Gemmatimonadota were the most abundant bacterial phyla in the rhizosphere soil in our study, which is generally consistent with previous studies  and was found in more abundance in organic fertilizer treatment. Firmicutes increased in peanut-planting soil with the application of humic acid fertilizer, which has been reported as one type of plant-growth beneficial bacteria. From the LEfSe analysis, every group owned their biomarkers except VOF. CF and COF owned more biomarkers than other groups, which further verified a significant difference in bacterial community diversity among CF, COF, and the other three groups. Based on the significance analysis for the bacterial community at the phylum and genus level, some beneficial bacteria  increased in the four FOF treatments, which was in line with the results of Streptomyces can control tomato diseases by producing antibiotics . It is reported that Streptomyces can stimulate plant growth by fixing atmospheric N into soil NH4+ and Bacillus can improve the soil AK content by effectively dissolving K minerals in soil , and soil NH4+-N content indirectly contributed to cucumber yield with a positive effect on bacterial numbers.From the Spearman correlation analysis and RDA between the bacterial community and soil environmental factors, soil TC, TN, CEC, pH, and AP were the most important factors influencing bacterial composition. Simultaneously, the correlation pattern between soil TC and microbiomes was similar to that between soil TN and microbiomes. Soil C, N, and P provide soil microbiomes with principal nutrients and energy sources, which are the common environmental factors that are related to microbial composition . The dem in soil . Althoug content . Wang et4+-N, AP, AK, TOC, and OM content), which could enable tobacco to absorb more nutrients, and enable the soil to be used more progressively and sustainably. Second, FOFs decreased the accumulation of rhizospheric PA, primarily sinapic acids, which weakened the PA’s allelopathy function. Third, FOFs changed the rhizospheric bacterial community structures, with an increase in beneficial bacteria and a decrease in harmful bacteria, which encouraged the normal growth of tobacco. This study has discussed the effects of different types of FOFs on continuous cropping tobacco yield and quality, focusing on the variance of soil physicochemical properties, the accumulation of PA, and the bacterial community structure. The application of FOFs can be extended to other broad crops at different experimental sites with a larger time scale in a further study. The interactions among FOF applications, the shift of soil environmental factors, soil microbial communities , and crop yield, quality, and disease-suppressing ability need to be considered and revealed using various technologies. An antagonistic effect in compound FOF is observed in the present study, and the corresponding mechanisms may emerge from carefully designed experiments.In summary, the mechanisms of FOFs on tobacco yield and quality improvement can be linked to the direct function of the plant growth-promoting substances in FOFs, and the indirect function through altering soil nutrient conditions and bacterial communities. First, FOFs changed the soil nutrient conditions , and inducing a healthier rhizospheric bacterial community structure. Additionally, soil AP, TC, TN, CEC, and pH were the important environmental factors that influenced the bacterial community structure. Tobacco yield showed an active correlation with soil TC, NH