Bacterial richness and diversity decreased with N addition (N and NP) rather than only P input. Proteobacteria, Acidobacteria, and Actinobacteria were the major phylum across all treatments. Nitrogen addition increased the relative abundance of Proteobacteria and Actinobacteria by 42.0 and 10.5%, respectively, while it reduced that of Acidobacteria by 26.5%. Bacterial community structure in the CK and P treatments was different from that in the N and NP treatments upon principle coordinates analysis. Phosphorus addition did not significantly affect soil bacterial communities, and no interactions between N and P inputs on microbial traits were observed. Soil pH and mineral N availability appeared to have a cooperative effect on bacterial abundance and community structure, with soil pH being the key influencing factor by canonical correspondence analysis. These results indicate that inorganic N rather than P fertilization affected both bacterial abundance and community composition in subtropical forests.Nutrient inputs to forest ecosystems significantly influence aboveground plant community structure and ecosystem functioning. However, our knowledge of the influence of nitrogen (N) and/or phosphorus (P) inputs on belowground microbial communities in subtropical forests is still unclear. In this study, we used quantitative polymerase chain reaction and Illumina Miseq sequencing of the bacterial 16S rRNA gene to investigate bacterial abundance, diversity, and community composition in a Chinese fir plantation. The fertilization regimes were as follows: untreated control (CK), P amendment (P), N amendment (N), and N with P amendment (NP). Additions of N decreased soil pH and bacterial 16S rRNA gene abundance by 3.95 (from 4.69 to 3.95) and 3.95 × 10 Soil nutrient inputs such as nitrogen (N) and phosphorus (P) are critical to control plant growth in tropical forest plantations . BecauseSoil microorganisms are important drivers of energy flow and nutrient cycling, such as carbon (C), N, and P cycling in terrestrial ecosystems . TherefoCunninghamia Lanceolata), which covers over 9 million ha in China, is the most common coniferous timber species that has been extensively planted in southern China  assess the responses of bacterial abundance, diversity, and community composition to N and/or P addition; (2) identify which soil properties were correlated with bacterial community. We hypothesized that (1) the changes in soil pH and mineral N availability induced by N addition might affect bacterial community abundance, diversity, and composition and (2) the increased P content after P addition might not affect these variables of soil bacterial communities.2 of total land area in southern China. The site has a monsoon season and humid climate. The duration of the growing season is about 270 days, and the average annual precipitation and temperature are 1471.2 mm and 17.9°C, respectively . The soil in the forest, which is classified as Typical Hapludult Ultisols , developed from Quaternary Red Clay and covers over 60% of the 1.14 million kmectively . The ann−1 year−1), N addition (N) (100 kg N ha−1 year−1), and N and P addition (NP) (100 kg N ha−1 year−1 + 50 kg P ha−1 year−1). Nitrogen and phosphorus fertilizer were applied as NH4NO3 and NaH2PO4, respectively. Nitrogen and and/or phosphorus fertilizer were dissolved in 20 L of distilled water and, then, added into the corresponding plots. Twenty liters of distilled water was added into the control plots.In 2011, 16 experimental plots (20 m × 20 m each) were placed in a random manner with 10-m-wide buffer strip between plots. Four fertilization treatments (four replicates each) were set up in this fir plantation: without fertilizer (CK), P addition (P)  by shaking at 200 rpm for 1 h, and quantified by a Segmented Flow Analyzer . Dissolved organic carbon (DOC) was extracted by 0.5 M K2SO4 and determined using a total organic carbon analyzer  were collected using corers, after which they were pooled together to minimize within-plot variation. Subsequently, soil samples were mixed thoroughly and then passed through a 2-mm sieve and separated into two portions. One portion was used for soil chemical analysis and another that was frozen at −80°C for subsequent molecular analysis. The soil pH  was determined using a pH meter. Soil KCl-extractable NHTM DNA Isolation Kit . DNA concentration was checked using a NanoDrop ND-1000 spectrophotometry (United States), then DNA samples were stored at −80°C refrigerator for further analysis.The total DNA was isolated from 0.4 g of soil using MoBio PowersoilTM , 0.5 μL of each primer (10 mM), 1–10 ng of template DNA. The PCR condition was as follows: 3 min at 95°C, 35 cycles of 40 s at 95°C, 30 s at 54°C, 40 s at 72°C, and a plate read at 83°C for 10 s. The qPCR assay was performed in triplicate for each replicate. The amplification efficiency for all samples ranged between 91 and 100%.To estimate the bacterial abundance, quantitative polymerase chain reaction (qPCR) assays were performed using universal eubacterial 16S rRNA gene primers. The forward primer was 338F (5′-ACTCCTACGGGAGGCAGCA-3′), and the reverse primer was 806R  (Takara), and 9.5 μL of ddH2O. The thermocycling conditions consisted of initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 40 s, annealing at 54°C for 30 s, and elongation at 72°C for 6 min. PCR products were purified with a QIA quick Gel Extraction kit . The high-throughput sequencing was conducted on the Illumina MiSeq 2500 platform .The V3-V4 region of the bacterial 16S rRNA gene was amplified from DNA extracts using the same primers for qPCR. The adapter linked forward primer included a 5-bp barcode was used for sample identification. Each PCR mixture (25 μL) included 0.5 μL of each primer, 2 μL template DNA (1–10 ng), 12.5 μL of Premix TaqSRR6263277-SRR6263292. Observed OTU numbers, Pielou’s evenness, Chao1 richness, and ACE evenness, as well as Shannon’s diversity indices, were calculated using the Mothur software to estimate bacterial alpha diversity. Beta diversity was calculated based on Bray-Curtis distance matrices.Sequence data including raw data and clean data were merged and filtered using Mothur . BrieflyF-value was obtained (p < 0.05). On the effects of N, P, and their interactions on soil properties, bacterial 16S rRNA gene abundance, and alpha diversity indices were examined by two-way ANOVA. Spearman’s correlation coefficient was used to test the relationships among soil properties, bacterial 16S rRNA gene abundance, alpha diversity indices, and abundant phyla. A p < 0.05 was considered to indicate significance.One-way ANOVA was conducted to compare the effects of different fertilization treatments on bacterial 16S rRNA gene abundance and alpha diversity indices using SPSS version 19.0 . The mean values of four replicate plots were compared using Duncan’s test when a significant Principle coordinates analysis (PCoA) was conducted to assess the dissimilarities of the bacterial community composition between the treatments using R 3.4 software . Per-mut4+-N, NO3−-N, TN, and AP contents. These results indicated that pH remarkably decreased in response to N amendment, while was not significantly altered after P amendment (Table 1). The concentrations of NH4+-N, NO3−-N, and TN were significantly lower in both the CK and P treatments than the N addition treatments. However, the increases in NH4+-N, NO3−-N, and TN concentrations did not significantly differ between N and NP treatments (p > 0.05). Soil AP content was significantly elevated by P addition. Two-way ANOVA revealed no significant interactions between N and P additions on the soil properties (p > 0.05).The 6-year fertilization management significantly altered soil properties such as pH, NHFigure 1). Specifically, the abundance of bacterial 16S rRNA gene ranged from 3.95 × 109 to 9.27 × 109 copies g−1 dry soil. The bacterial abundance in the N and NP treatments was lower than in CK and P treatments, whereas no significant differences were observed in the CK and P treatments. 16S rRNA gene copies numbers were positively correlated with soil pH (p < 0.001), while negatively correlated with soil NH4+-N, NO3−-N, and TN . There were significant differences in bacterial alpha diversity for the observed OTU numbers, as well as Chao 1, ACE, and Shannon’s index values among treatments, but not for Pielou’s Evenness among the treatments. Pielou’s evenness was high among treatments, indicating that bacterial community structure was evenly distributed. The observed OTU numbers, Chao 1, ACE, and Shannon’s index values were much lower in the N and NP treatments than in the CK treatment. Moreover, there were no significant (p > 0.05) differences in these alpha diversity indexes between CK and P addition treatments.Bacterial community analysis of the 16 soil samples revealed a total of 493,859 high quality sequences with 25,504 to 41,155 sequences per sample , but were significantly and negatively correlated with NH4+-N, NO3−-N, and TN contents (p < 0.05).The correlations of bacterial alpha diversity indices with soil physicochemical parameters are shown in Figure 4A. The phyla Acidobacteria and Proteobacteria were most abundant in all treatments, comprising 67.1–74.3% of the bacterial sequences obtained from the soil, followed by Chloroflexi (8.9–9.8%), Actinobacteria (4.9–10.5%), Verrucomicrobia (2.1–2.9%), Planctomycetes (1.3–2.2%), and Bacteroidetes (1.7–2.1%). Different fertilization management strategies had various influences on bacterial composition at the phylum level. The relative abundance of the phyla Actinobacteria and Proteobacteria increased in N and NP treatments, while that of Acidobacteria decreased, relative to the CK and P treatments. There were no significant differences in the abundance of Acidobacteria, Actinobacteria, Proteobacteria, Chloroflexi, Actinobacteria, Planctomycetes, Bacteroidetes, and Verrucomicrobia between N and NP treatments. Similarly, the relative abundance of these phyla did not differ significantly between CK and P treatments (p > 0.05).All reads in each treatment were classified into eight phyla, and their relative abundances were shown in Figure 4B). There were significant differences between treatments for the four most abundant classes. Specifically, the relative abundance of class Acidobacteria was higher in the CK and P treatments than that in the N-related treatments. Furthermore, the relative abundance of Actinobacteria, Alphaproteobacteria, and Gammaproteobacteria significantly increased in the N and NP treatments.At class level, the 13 most abundant classes were obtained (>2%) among fertilizer treatments , which revealed that bacterial community composition separated clearly between N and CK treatments, while the bacterial community composition in the P treatment was similar to that in the CK treatment. The first two principal coordinates explained 31.8% (PC1) and 11.6% (PC2) of the variation in bacterial communities, respectively. PERMANOVA further revealed significant differences between N-free treatments (CK and P) and N-containing treatments (N and NP) , and the correlation coefficients followed the trend: pH > NH4+-N > NO3−-N > TN , which revealed that these soil variables explained 27.6% of the variation in the bacterial community by the first two constrained axes of CCA, with the first axis explaining 20.3% and the second 7.3%. Moreover, CCA clearly showed that soil pH and mineral N contents were the most important contributors to the variation in bacterial communities, and the direction of these arrows was closely correlated with the X axis.The Mantel test indicated that bacterial community structure was closely correlated with soil chemical properties including pH, NHTable 4). Three dominant phyla  were significantly correlated with several soil chemical parameters (p < 0.05), while Bacteroidetes, Verrucomicrobia, Chloroflexi, and Planctomycetes were not obviously related to any of the soil chemical properties (p > 0.05). Only Acidobacteria was positively correlated with soil pH, whereas Actinobacteria and Proteobacteria were negatively correlated with this factor (p < 0.05). Moreover, Acidobacteria was negatively related to NH4+-N, NO3−-N, and TN, while Actinobacteria and Proteobacteria were positively correlated with these soil properties.We also evaluated the correlations between relative abundant phyla (>2%) and soil properties . Conversely, long-term P input did not affect bacterial abundance and α-diversity, which was supported by the result of recent studies (−2 year−1) and medium (5 g P m−2 year−1) P inputs. Additionally, 4+-N and NO3−-N rather than AP, further confirming that mineral N availability was a very important factor altering bacterial abundance and biodiversity  that had fast growth rates were more likely to increase in nutrient-rich conditions, while oligotrophic groups  that had slower growth rates would likely decline . Bacterial community composition in the N and NP treatments differed significantly from those not receiving N (CK and P), indicating that fertilizer addition had a stronger influence on it than P input. On the contrary, P input did not influence bacterial community structure in the present study, which was supported by previous reports , as revealed in previous studies  treatments were stronger than treatments that did not contain (P). This study provides valuable information that improves our understanding of the effects of N and/or P input on underground bacterial community and revealed the main factors influencing bacterial communities in subtropical forest ecosystems.QW and CW contributed to the design and implementation of the research, participated in drafting the manuscript. WY contributed to analyze the results. AT, DC, YH, JA, YJ, and ZH revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ralstonia solanacearum, a quarantine bacterium in many countries, and causative agent of bacterial wilt, one of the most important vascular plant diseases. Bacterial wilt management has been carried out with fluctuating effects, suggesting the need to find alternative treatments. In this work, three lytic phages were isolated from environmental water from geographically distant regions in Spain. They proved to specifically infect a collection of R. solanacearum strains, and some of the closely related pathogenic species Ralstonia pseudosolanacearum, without affecting non-target environmental bacteria, and were able to lyze the pathogen populations within a wide range of conditions comprising environmental values of water temperatures, pH, salinity, and lack of aeration found in storage tanks. The three bacteriophages displayed high efficiency in controlling R. solanacearum, with reductions of the bacterial populations of several orders of magnitude in just a few hours, and proved to be able to survive in freshwater for months at environmental temperatures keeping activity on R. solanacearum, pointing out their suitability for field application through irrigation. Concerning their biocontrol potential, they were effective in reducing high populations of the pathogen in environmental water, and bacterial wilt incidence in planta by watering with either one phage or their combinations in assays with more than 300 plants. This is the first report on effective R. solanacearum biocontrol by applying single or combined bacteriophages through irrigation water in conditions mimicking those of the natural settings. The three phages belong to the Podoviridae family and are members of the T7likevirus genus. They are the first isolated phages from river water with activity against R. solanacearum, showing the longest persistence in natural water reported until now for phages with biocontrol potential, and consistently being able to control the disease in the host plant under environmental conditions. Consequently, the use of these bacteriophages for the prevention and/or biocontrol of the bacterial wilt disease caused by R. solanacearum has been patented. Evidence provided reveals the suitability of these waterborne phages to be effectively considered as a valuable strategy within the frame of sustainable integrated management programs.Three new lytic bacteriophages were found to effectively control the pathogen  Ralstonia solanacearum bacterial species  isolated from potato (Solanum tuberosum) inR. solanacearum strains (R. pseudosolanacearum strains (R. solanacearum and R. pseudosolanacearum strains were grown on the non-selective Yeast Extract Peptone Glucose Agar (YPGA)   medium (Solanum lycopersicum) soil, rizhosphere and plant endophytes were grown either on YPGA, KB, Luria Bertani (LB)   for 48 hm B (KB)  for 48 h) medium  for 48 hani (LB) , or Wilbani (LB)  general R. solanacearum lytic bacteriophages was isolated according to European Directives ] in a modified Wilbrink broth (MWB)  . Bacterig method . A selecR. solanacearum species was determined for each of the bacteriophages tested by standard method in both, liquid and solid media, with 35 R. solanacearum and seven R. pseudosolanacearum strains from different hosts and geographical origins  and saline concentrations around 1.5–2.0 and 35%. Lytic activity was also determined in both, aeration by shaking at 200 rpm and static conditions, in MWB at 28°C. In all conditions, incubation was carried out until observation of lytic activity. All assays were performed at least in duplicate.Physiological characteristics of the bacteriophages tested were evaluated by lytic activity assays against Viral particles of selected bacteriophages were visualized by transmission electron microscopy (TEM) after negative staining with a solution of 1% phosphotungstic acid (PTA) in water, adjusted to pH 7 with 1 N NaOH solution. Drops of 0.22-μm-filtered lysates were placed on formvar and carbon covered grids. After 30–60 s drying, the excess liquid was blotted off, and drops of PTA solution were added on top of the lysate drops. After 1 min drying, the excess liquid was blotted off. Grids were air dried before TEM examination.R. solanacearum strain IVIA-1602.1 and each of the bacteriophages tested was initially performed in optimal conditions by coinoculation of the strain at about 109 CFU/mL and the phage tested at about 109 plaque-forming units (PFU)/mL, multiplicity of infection (MOI) = 1, both incubated in MWB at 28°C with shaking (200 rpm). The dynamics was subsequently performed between R. solanacearum and one selected bacteriophage by coinoculation of the strain at about 106 CFU/mL and the selected phage at about 104–103 PFU/mL, both incubated in MWB at 24 and 14°C with shaking (200 rpm). Additionally, the kinetics between the strain and the selected bacteriophage was monitored by using a Bioscreen C apparatus (Labsystems) with a discontinuous shaking regimen . Thus, strain IVIA-1602.1 and the phage were coinoculated at different proportions in MWB, with the strain at about 109 CFU/mL and the phage from 109 to 101 PFU/mL, and incubated at 26°C. The O.D.580 nm of the liquid cocultures were determined every 30 min and reported as the mean of five different measurements.The growth dynamics between R. solanacearum-free area, Southeastern (SE) region, with environmental water displaying temperature values from 11.5 to 22.0°C and mean pH value around 8.13, and the other region an originally R. solanacearum-contaminated area, Northwestern (NW) region, with environmental water displaying temperature values from 3.5 to 20.9°C and mean pH value around 7.36. With respect to the composition of the RW samples, SE-RW had values about 100 times higher for Mn, 10 times higher for Fe, between 5 and 10 times higher for chlorides and triple nitrates than NW-RW and mean pH value of 8.1, whereas NW-RW had values about four times higher for phosphate than SE-RW and mean pH value of 7.2. Both types of water were 0.22-μm-filtered and autoclaved previously to the survival experiments.River water from two regions in Spain was used to test survival of selected bacteriophages. One of the regions was a 7 to 105 PFU/mL. The microcosms were incubated at 4, 14, and 24 ± 1°C, and sampled for plaque counts at inoculation time (day 0), 1, 2, 3, 5 weeks postinoculation (wpi), 3, 5 months (mpi), and 3 years (ypi), performed from each microcosm according to a standard surface plating method after 48 h at 28°C. Survival experiments were performed in two separate assays at least in duplicate.For each of the SE-RW and NW-RW samples, three microcosms were prepared, each of them with a 300-mL volume of water, which were separately inoculated with the 0.22-μm-filtered lysates of the bacteriophages tested at a final concentration ranging from 10R. solanacearum strain IVIA-1602.1 at about 5 × 106 CFU/mL and each of the bacteriophages tested at about 104 PFU/mL (MOI = 0.01). Incubation was done with shaking (200 rpm) over 30 h at 24°C and 60 h at 14°C. Aliquots for bacterial and phage counts were regularly taken, serially 10-fold diluted in PBS and plated onto YPGA as abovementioned. Assays were done at least in duplicate, in separate experiments.Volumes of 100 mL of 0.22-μm-filtered and autoclaved RW samples were inoculated with R. solanacearum lytic phages that could be naturally present in the water samples. As the assays did not detect any initial lytic activity, the presence of R. solanacearum indigenous phages was discarded, and the water samples were then inoculated with a selected bacteriophage at about 104 PFU/mL. Volumes of 100 mL were processed in three different ways as follows: (i) 0.22-μm-filtration and autoclaving, with no alive microbiota (AW), (ii) 0.22-μm-filtration, only containing the indigenous phages without activity against R. solanacearum and the inoculated phage (FW), or (iii) without treatment, with the whole native microbiota and the inoculated phage (W). All of them were subsequently inoculated with R. solanacearum strain IVIA-1602.1 at about 5 × 106 CFU/mL (MOI = 0.01). Incubation was done in static conditions at 24 and 14°C for a month. Aliquots for R. solanacearum counts were taken at inoculation time (day 0), at 30 h at 24°C and 60 h at 14°C, and then weekly for a month, serially 10-fold diluted in PBS and plated onto YPGA as abovementioned. Assays were done at least in duplicate, in separate experiments.River water samples with the whole microbiota  were processed by enrichment assays for detection of in planta was tested in a susceptible host. Sets of 10 tomato plants cv. Roma aged 3 weeks were coinoculated in duplicate in two independent experiments (Experiment 1 and Experiment 2) by watering once with 20 mL of R. solanacearum strain IVIA-1602.1 at concentrations of 104 or 105 CFU/mL, and a selected bacteriophage at concentrations of 109 or 106 PFU/mL and their 10-fold dilutions. Another experiment (Experiment 3) was performed in triplicate by watering once with 20 mL of R. solanacearum strain IVIA-1602.1 at 105 CFU/mL and the same selected bacteriophage at 108 PFU/mL. Tomato plants inoculated either with the bacterial strain or non-sterile water were also included as controls. Plants were maintained in a climatic chamber of adequate dimensions, in cycles of 16 h light 8 h darkness at 26°C and 70% humidity in optimal conditions for disease development, under Biosecurity Level 3 (BSL3). After inoculation, plants were monitored periodically for symptom development by visual inspection during 6–8 weeks. Wilting symptoms in less than 25% of leaves were recorded as first symptoms to be confirmed in subsequent evaluations. Progressive increase in wilting incidence to 50–75% of the leaves and branches was recorded as positive wilting in the plant. More than 75% of leaves and branches affected were recorded as near collapse or collapse of the wilted plant. Bacterial reisolation from wilted plants was performed according to European Directives  in all the experimental conditions. Tomato plants inoculated either with the bacterial strain or non-sterile water were also included as controls. Plants (more than 300 per assay) were incubated under BSL3 conditions, monitored for a period of 1.5–2 months, and then sampled and processed as abovementioned.The abilities of selected RW phages for bacterial wilt biocontrol versus no wilting were compared using the Chi-square test. A p-value below 0.05 was considered significant. Data were analyzed with SPSS 20.0 software (IBM SPSS Statistics).For each biocontrol assay in host plants, the relation among different treatments  and wilting Molecular biology techniques for DNA isolation and sequencing were performed . Genomicde novo genome assembly. For each bacteriophage genome, a single large contig was generated by reads assembly, and genomes were defined as complete if the single contig assembled without error. These contigs were used to identify closely related homologs with the NCBI BlastN tool by comparison with the sequences published in the GenBank. Preliminary functional annotations of the assembled genomes were also performed by sequence comparison with the NCBI public database, and the best aligning results were selected to annotate the unigenes.Then, massive-scale sequencing was carried out by Illumina SBS Technology. Paired-end libraries with approximate average insert lengths of 200 base pairs were synthesized from the DNA using the NexteraXT (Illumina) protocol, and library concentrations and sizes were assayed on a 2100 Bioanalyzer. Libraries were sequenced using an Illumina sequencer HiSeq 2500/1500 (2 × 150 bp). The obtained sequences were analyzed and preliminary annotated after cleaning of the sequences of the FASTQ file. Low-quality sequences  were removed, and only the high-quality reads were assembled to construct unique consensus sequences by analysis against a reference sequence and R. solanacearum contaminated areas in Spain. They were purified, and eight of them selected according to their plaque size and different geographical origins. These were further characterized in a range of environmental conditions, and then three of them selected on the basis of their lytic characteristics, mainly specificity against R. solanacearum, kinetics of their lytic activity, and survival in environmental water. They were isolated in different years from RW from geographically distant Spanish regions that do not share any tributary with each other. Bacteriophages were initially named F2 , M2 , and R2 . They produced transparent plaques on a lawn of R. solanacearum after 36–48 h on YPGA. An initial collection of lytic bacteriophages was isolated from former R. solanacearum (former phylotype II) from different sources, hosts and years of isolation, all of them of biovar 2 (R. pseudosolanacearum (former phylotypes I and III) of biovars 1, 3, 4, and 5 , and negative at 9 and between 32 and 39°C , in MWB at pH 7. In water from river, lake, and irrigation sources, phage activity was positive at pH values ranging between 6.5 and 9.0 at 14, 20, and 29°C. Lytic activity was also positive in freshwater from different sources with salinity contents up until approximately 2.1% , and negative in saline water at 35% pH 8 from seaport and beach. With respect to aeration, lytic activity was similarly observed after incubation in MWB at 28°C with aeration by shaking at 200 rpm as well as in static conditions, being faster with aeration.Lytic activity of the three phages was positive for 35 strains of biovar 2 . Among t4, and 5 . Lytic a4, and 5  and 46 uPodoviridae family. Bacteriophages F2, M2, and R2 were then renamed vRsoP-WF2, vRsoP-WM2, and vRsoP-WR2, respectively, where “v” stands for “virus,” “Rso” for R. solanacearum, “P” for “podovirus,” and “W” for “water.”Viral particles adsorbed to the host cell and viral shapes were observed in detail by TEM of the bacteriophage lysates. 9 CFU/mL and each of the three phages at 109 PFU/mL in MWB at 28°C, since initial turbid suspensions became transparent in 5–6 h, corresponding to bacterial concentrations of 104–103 CFU/mL, for each of the bacteriophages tested. Reductions were also observed and quantified in cocultures of strain IVIA-1602.1 at 106 CFU/mL and the phage vRsoP-WF2 at 104–103 PFU/mL in MWB at 24 and 14°C, since the inoculated bacterial populations decreased 5–6 log units in 9 h postinoculation (hpi) at 24°C (9 CFU/mL and the phage vRsoP-WF2 at 109 PFU/mL and their 10-fold dilutions to 101 PFU/mL in MWB at 26°C (9 CFU/mL): vRsoP-WF2 (109 PFU/mL) being the most effective, since the reduction achieved in R. solanacearum populations was the highest in less than 24 hpi.Reductions in the inoculated bacterial populations caused by the lytic action of the phages were observed when monitoring cocultures of strain IVIA-1602.1 at 10 at 24°C , and 4–5 at 24°C , so bein at 26°C . Reducti4–105 PFU/mL at 4 and 14°C, and 103–104 PFU/mL at 24°C until 5 mpi  within 10 hpi, with one additional log unit decrease by 24 hpi (3 CFU/mL (3 log units) within 23 hpi, keeping these levels in the following 24 hpi   . No resiter (FW) . These dR. solanacearum populations at 104 CFU/mL (2 × 105 CFU/pot) yielded no disease symptoms in all condition after 6–8 wpi, with or without phage. Biocontrol activity could be demonstrated in assays by watering the host with 20 mL of bacterial populations of about 105 CFU/mL (2 × 106 CFU/pot) mixed with the phage vRsoP-WF2 at 109 or 106 PFU/mL or their 10-fold dilutions (from 2 × 1010 to 2 × 107 PFU/pot) in Experiments 1 and 2 (5 CFU/mL (2 × 106 CFU/pot) mixed with the phage vRsoP-WF2 at 108 PFU/mL in Experiment 3 (p < 0.001 or p < 0.05) in most of the phage treatments in Experiments 1 and 2 (p < 0.001) in Experiment 3. R. solanacearum-like colonies were reisolated from wilted plants and PCR identified.Bacterial wilt biocontrol by the phage vRsoP-WF2 against the strain IVIA-1602.1 was observed in susceptible tomato plants cv. Roma after being watered once with a mixture of the phage and the pathogen in non-sterile environmental water. Assays performed by watering with 20 mL of  1 and 2 , and by riment 3 . A repre 1 and 2  and (p <6 CFU/mL (2 × 107 CFU/pot) and total initial concentration of phages of about 107 PFU/mL (2 × 108 PFU/pot). Bacterial wilt percentages obtained with positive controls were about 80% and values obtained after irrigation with single phages ranged between 20–6%, depending on the phage (p < 0.001) in all the applied phage treatments (5 CFU/mL (2 × 106 CFU/pot) and total initial concentration of bacteriophages of about 107 PFU/mL (2 × 108 PFU/pot) in all experimental conditions. Percentages of bacterial wilt affected plants in the positive controls were about 50% and values obtained after irrigation with single phages ranged between 45–5%, depending on the phage (p < 0.001) in every mixture of two phages and the three, and also for phage vRsoP-WR2 when applied separately , 40,861 bp for SEQ ID N°2 (vRsoP-WM2), and 40,408 bp for SEQ ID N°3 (vRsoP-WR2). Initial bioinformatic analysis and annotation pointed out that each of the major sequences in SEQ ID N°1 (vRsoP-WF2), SEQ ID N°2 (vRsoP-WM2), and SEQ ID N°3 (vRsoP-WR2) were easily identifiable as complete genomes of bacteriophages belonging to the genus of T7-like viruses, whose type species is the Enterobacteria phage T7, which belongs to the Podoviridae family.Genomic DNA was obtained from each of the three bacteriophages, with ARalstonia phage RsoP1EGY , Ralstonia phages P-PSG-11 and P-PSG-11-1 (GenBank accession no. MN270889.1 and MN270890.1), and Ralstonia phage RPSC1 (GenBank accession no. MF893341.1), respectively. Regions with high identity correspond to 5–23% of the complete genome of vRsoP-WF2, vRsoP-WM2, and vRsoP-WR2, and belong to highly conserved regions, mainly related to replication and encapsidation. Consequently, except in these conserved zones within the T7-like bacteriophages, the genomes of vRsoP-WF2, vRsoP-WM2, and vRsoP-WR2 seem to contain a nucleotide sequence divergent from that of the other bacteriophages deposited in GenBank. The three nucleotide sequences SEQ ID N°1 (vRsoP-WF2), SEQ ID N°2 (vRsoP-WM2), and SEQ ID N°3 (vRsoP-WR2) have been deposited in GenBank with accession numbers MN685189, MN685190, and MN685191, respectively.Comparison of the genomes of the three bacteriophages showed >99% identity among them. Thus, between SEQ ID N°1 (vRsoP-WF2) and SEQ ID N°2 (vRsoP-WM2) as reference sequence, coverage and identity values were of 99 and 99.82%, respectively; between SEQ ID N°1 (vRsoP-WF2) and SEQ ID N°3 (vRsoP-WR2) as reference sequence, coverage and identity values were of 100 and 99.87%, respectively; and between SEQ ID N°2 (vRsoP-WM2) and SEQ ID N°3 (vRsoP-WR2) as reference sequence, coverage and identity values were of 98 and 99.77%, respectively. However, the analysis of these sequences revealed the presence of different small mutations, insertions and deletions distributed in their genomes. These differences were higher between SEQ ID N°2 (vRsoP-WM2) and the other two, SEQ ID N°1 (vRsoP-WF2) and SEQ ID N°3 (vRsoP-WR2). Thus, SEQ ID N°2 contains an insertion of 468 nucleotides in comparison to SEQ ID N°1 and SEQ ID N°3. The small differences found in the nucleotide sequences point out that vRsoP-WF2, vRsoP-WM2, and vRsoP-WR2 may be distinct bacteriophages of the same viral species. Bioinformatic analysis also indicated that they may be new phages, closely related to T7 bacteriophages. Genomes of vRsoP-WF2, vRsoP-WM2, and vRsoP-WR2 exhibited coverage and identity values of 97–98 and >99.7%, respectively, with R. solanacearum-contaminated environmental water samples from geographically distant origins in Spain, confirming the choice of environmental reservoirs as the better sources for antibacterial phages (R. solanacearum (former phylotype II) strains, known to be a genetically homogeneous group  the three new phages being the first isolated from environmental water with activity against R. solanacearum, (ii) displaying the longest persistence in environmental water reported until now for phages with biocontrol potential, and (iii) consistently proving to be able to control the disease in the host plant under environmental conditions. It has been the subject of a recent patent in order to be effectively used with other biocontrol strategies in an integrated management program with less impact than agrochemicals and closer approach to sustainability of agricultural systems.Effective and environmentally friendly biocontrol of All datasets generated for this study are included in the article/supplementary material.BÁ, ML, and EB planned and designed the experiments and wrote the manuscript. BÁ performed the laboratory assays. BÁ and EB carried out the assays with host plants under BSL3 conditions.The University of Valencia (UV) and the Valencian Institute for Agricultural Research (IVIA) have the following interests: a granted Spanish patent ES2592352 B2 and the patents filed in EU and EEUU–P16799402.9 and US15/576,798.


Atta Fabricius, 1804 are consumed as a protein-rich food source and sometimes used for medical purposes. Little is known, however, about the species identity of collected ants and the accuracy of identification when ants are sold, ambiguities that may impact the conservation status of Atta species as well as the nutritional value that they provide to consumers. Here, 21 samples of fried ants bought in San Gil, Colombia, were identified to species level using Cytochrome Oxidase I (COI) barcoding sequences. DNA was extracted from these fried samples using standard Chelex extraction methods, followed by phylogenetic analyses with an additional 52 new sequences from wild ant colonies collected in Panama and 251 publicly available sequences. Most analysed samples corresponded to Atta laevigata , even though one sample was identified as Atta colombica Guérin-Méneville, 1844 and another one formed a distinct branch on its own, more closely related to Atta texana  and Atta mexicana . Analyses further confirm paraphyly within Atta sexdens  and A. laevigata clades. Further research is needed to assess the nutritional value of the different species.Leaf-cutting ants are often considered agricultural pests, but they can also benefit local people and serve important roles in ecosystems. Throughout their distribution, winged reproductive queens of leaf-cutting ants in the genus  Atta Fabricius, 1804 and Acromyrmex Mayr, 1865—both included in the tribe Attini Smith, 1858 (Hymenoptera: Formicidae [Leucoagaricus gongylophorus (Möller) Singer (1986), originating approximately 18 MYA [Atta in was the subject of several taxonomical revisions using morphology characters  and Atta sexdens  were found to be paraphyletic, and it was suggested that wider sampling would be needed to resolve these relationships.Relatively few taxa are still sustainably harvested from the wild for human consumption . Of thes Singer 186, origiy 18 MYA ,11,12, ay 18 MYA . They ary 18 MYA ,15. Thro.g., see ,17,18), ) Singer 86, origiAtta species found they contain on average 42–52 g protein per 100 g dry weight (corresponding mainly to the massive thoracic flight muscles), a protein to body mass ratio similar to that of game meat [Atta spp. per year per family [Leaf-cutting ants can also provide nutritional value and have positive economic impacts. In several Latin-American countries, winged female leaf-cutting alates  are also collected for human consumption as they disperse from their colony of birth for mating flights ,21. Throame meat . Today, ame meat . With rer family . In Color family  to $68 UAtta ants listed above, a flag must be raised with regard to the detrimental effects that unlimited collecting of local populations might pose. Firstly, over-collecting of certain species could lead to a population collapse , given .g., see ,29. Seco.g., see . Moreove.g., see , thus reA. laevigata  were sampled from a packet bought at a local store in San Gil and used for DNA extraction and sequencing of the mitochondrial COI gene. In addition, fifty individuals belonging to various Atta species were gathered from either field or laboratory colonies previously collected near Gamboa —one Atta cephalotes , 47 Atta colombica Guérin-Méneville, 1844, and two A. sexdens. In addition, 251 COI sequences of different Atta species , were also sequenced and used as an outgroup—Acromyrmex echinatior , Acromyrmex octospinosus , and Acromyrmex insinuator Schultz, Bekkevold & Boomsma, 1998.Twenty-one individuals of fried rated in ,33) wereAtta and Acromyrmex genomes [2, 7.8 µL ddH2O, and 1 µL of template DNA, and the program used included 5 min denaturing at 95 °C, followed by 25 cycles of 30 s denaturing at 95 °C, 45 s annealing at 60–55 °C with a touchdown of −0.2 °C per cycle, and 1 min extension at 72 °C followed by 20 cycles of 30 s denaturing at 95 °C, 45 s annealing at 55 °C and 1 min extension at 72 °C, and a final 10 min extension at 72 °C; (2) for LCO1490-PK and HCO2198-PK primers, reactions contained 10 µL VWR Red Taq DNA polymerase Master Mix , 0.6 µL for each of the 10 µM primers, 0.4 µL of 25 mM MgCl2, 7.4 µL ddH2O, and 1 µL template DNA, and used a program of 2 min initial denaturing at 95 °C, followed by 40 cycles of 30 s denaturing at 95 °C, 45 s annealing at 55 °C, and 1 min extension at 72 °C, with a final 5 min extension at 72 °C. PCR products were purified by adding 0.4 volumes of a master mix containing 3.33% Exonuclease I  and 6.67% Alkaline Phosphatase  and ddH2O, followed by 15 min incubation at 37 °C and 15 min incubation at 85 °C. Purified products were sequenced bidirectionally using the PCR primers. Sequencing reactions were prepared in a 5 µL final volume containing 1 µL BigDye sequencing buffer , 1.3 µL ddH2O, 0.3 µL MgCl2 (25 mM), 0.2 µL primer (10 µM), 0.2 µL BigDye3.1 (ABI), and 2 µL purified PCR product and used a program of 2 min initial denaturing at 95 °C, followed by 60 cycles of 10 s denaturing at 95 °C, 10 s annealing at 50 °C, and 2 min extension at 60 °C. Sequenced products were precipitated using ethanol precipitation as described by the manufacturer [2O before they were loaded on an ABI PRISM 3730 automated DNA sequencer .Genomic DNA was extracted using standard protocols by boiling the thorax or several legs in 150 µL of 10% Chelex X-100  at 99 °C for 60 min following the method from Walsh et al. . A regio genomes ,38,39: LSequence fragments were subjected to BLAST queries for an initial verification of their identities. They were then assembled with Geneious v8.1.7 . Using UApis mellifera Linnaeus, 1758  are removed in the frying process. Single origin polytomies of A. colombica and A. cephalotes, visible in Before running MrBayes, the AIC in jModelTest 2.0 ,52 was uAtta taxonomy. The majority-rule consensus tree resulting from the Bayesian analysis is shown in The final alignment included 18 species (incl. 3 outgroup species), 190 sequences and a total of 1098 positions of which 329 were informative, thus enabling us to confidently place the fried queen samples in the existing Atta phylogeny including multiple genes [A. sexdens is also paraphyletic, and includes the monophyletic clade containing Atta robusta Borgmeier, 1939, and possibly several other species currently registered as subspecies, although more data are needed to confirm this result. The main difference compared to the previously published phylogeny is the polyphyly of A. laevigata reported here for the first time, with two main monophyletic groups: (1) A. laevigata group 1, recovered in the section Epiatta and nested within a clade including the species Atta bisphaerica Forel, 1908, Atta capiguara Gonçalves, 1944, Atta opaciceps Borgmeier, 1939, Atta saltensis Forel, 1913 and Atta vollenweideri Forel, 1893 and (2) A. laevigata group 2, placed amongst the paraphyletic taxa of the section Archeatta with an unresolved position. Two additional sequences were reconstructed elsewhere, potentially due to sequence errors or misidentifications: One as sister to the species A. capiguara (EU848069) and another one recovered within the A. sexdens clade (EU848090). These results were consistent among all three tree reconstructions.Our COI phylogeny was mostly concordant with an earlier le genes , and we A. laevigata, as currently defined, might include more than one species, presenting a case of cryptic diversity in this lineage. Regarding the ‘hormigas culonas’ sampled from the bag bought in Colombia, we confirmed that the majority belonged to A. laevigata group 1. Since A. laevigata group 1 clade includes most A. laevigata samples, it is likely that it represents this species in a strict sense. If this is the case, then our results confirm that the identification given by the shop owners as well as a newspaper-clipping present at the shop was correct  as A. laevigata, which suggests the potential of a species in the section Archeatta with similar morphology as the true A. laevigata. Additional data will be needed to disentangle the current status of this species. Although we were able to reconstruct most of the known Atta topology and species clades using a single marker (COI), support in deep branches of the tree was low, and several conflicts arose in deep internodes, due mostly to lack of characters. Recently developed high-throughput sequencing tools, such as Restriction site Associated DNA (RAD) markers [Our results also corroborate those of Bacci et al. , that A.onçalves  and Borgonçalves ,18 had porgmeier  in an ea markers  and Ultr markers ,65, haveAtta spp. are regularly used in traditional medicine by indigenous tribes in the Americas. The heads, and in particular the mandibles, of the soldiers have traditionally been used as stitches to close wounds [Regarding the three genetically distinct species found in a single bag, it is plausible that misidentifications could vary throughout the collecting season with different species available throughout the year due to different times of mating flights . The cone wounds , and it e wounds . Given te wounds , it is tAtta queens before they reach the age of 5 years old—the age needed for an Atta colony to start reproducing—is approximately of 1% [The survival rate of ly of 1% ,70. Suchly of 1%  and remoAtta should be investigated to test for potential effects on nutritional quality. We also confirm the paraphyly of A. sexdens and A. laevigata and suggest further research is needed to fully understand Atta diversity. We suggest that the diversity of Atta needs further exploration through morphological and genetic analyses on existing and new collections.In summary, our results show that occasional misidentifications occur by ant collectors, which potentially affects the nutritional value of the food items. We suggest that nutritional values for the different species of


Streptomyces strains. Starting from this scaffold new xanthine derivatives have been synthetized and evaluated for their antibacterial and antifungal effects. The antibacterial action was investigated against Gram positive  and Gram negative (Escherichia coli ATCC 25922) bacterial strains. The antifungal potential was investigated against Candida spp. . In order to improve the antimicrobial activity, the most active xanthine derivatives with thiazolidine-4-one scaffold  were included in a chitosan based polymeric matrix (CS). The developed polymeric systems (CS-XTDs) were characterized in terms of morphological , physic-chemical properties (swelling degree), antibacterial and antifungal activities, toxicity, and biological functions . The presence of xanthine-thiazolidine-4-one derivatives into the chitosan matrix was confirmed using Fourier transform infrared (FT-IR) analysis. The size of developed polymeric systems, CS-XTDs, ranged between 614 µm and 855 µm, in a dry state. The XTDs were encapsulated into the chitosan matrix with very good loading efficiency, the highest entrapment efficiency being recorded for CS-6k, which ranged between 87.86 ± 1.25% and 93.91 ± 1.41%, depending of the concentration of 6k. The CS-XTDs systems showed an improved antimicrobial effect with respect to the corresponding XTDs. Good results were obtained for CS-6f, for which the effects on Staphylococcus aureus ATCC 25923 (21.2 ± 0.43 mm) and Sarcina lutea ATCC 9341 (25.1 ± 0.28 mm) were comparable with those of ciprofloxacin (25.1 ± 0.08 mm/25.0 ± 0.1 mm), which were used as the control. The CS-6f showed a notable antifungal effect, especially on Candida parapsilosis ATCC 22019 (18.4 ± 0.42 mm), the effect being comparable to those of nystatin (20.1 ± 0.09 mm), used as the control. Based on the obtained results these polymeric systems, consisting of thiazolidine-4-one derivatives loaded with chitosan microparticles, could have important applications in the food field as multifunctional  packaging materials.In the past many research studies have focused on the thiazolidine-4-one scaffold, due to the important biological effects associated with its heterocycle. This scaffold is present in the structure of many synthetic compounds, which showed significant biological effects such as antimicrobial, antifungal, antioxidant, anti-inflammatory, analgesic, antidiabetic effects. It was also identified in natural compounds, such as actithiazic acid, isolated from  In the last time the xanthine scaffold was widely investigated based on its high biological potential. Some of the most important derivatives are caffeine, theophyline, theobromine, and paraxanthine . These cStreptomyces spp., which showed high and specific action against Mycobacterium tuberculosis [In the medicinal chemistry the thiazolidine heterocycle is also known as an important scaffold that is used to modulate the classical structures in order to improve their biological effects and to induce other new ones. For the first time this structure was identified as an actithiazic acid, a natural compound isolated from rculosis . Later, rculosis ,11,12. Irculosis .Gram-positive bacterial strains are more susceptible to chitosan than Gram-negative ones [In a previous study the researchers focused their studies on micro and nanotechnology as new strategies for increasing solubility, improving the biological properties, and decreasing the toxicity of the drugs. Some of the most important applications are in the regenerative medicine or tissue engineering area, as biosensors or as controlled drug release systems. The polymeric systems such as nano and microparticels are largely used as drug delivery systems due to their small particle size and large surface area . Lately,ive ones .Referring to the food industry, it is known that the packaging components have an important role for preservation of food as well as for environment protection, in a previous study the researchers focused on the use of renewable material and on the development of active packaging in order to increase the quality and safety of food . The embIn this study we report the preparation, physic-chemical characterization, and biological evaluation of new polymeric systems based on chitosan, which have been loaded with new xanthine derivatives with a thiazolidine-4-one scaffold. Based on the unique properties of chitosan  and of the biological effects of xanthine derivatives with a thiazolidine-4-one scaffold, the developed polymeric systems could have important applications in the food industry as active packaging materials and also in the medical field. Staphyloccoccus aureus ATCC 25923, Sarcina lutea ATCC 9341, Escherichia coli ATCC 25922) and yeast strains  were obtained from the Department of Microbiology, “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania. Ampicillin (30 µg/disc), ciprofloxacin (25 µg/disc) and nystatin (100 µg/disc) were used as positive controls for antibacterial and antifungal activity respectively. Swiss mice were purchased from the Biobase of “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania.Chitosan , pentasodium tripolyphosphate (TPP), disodium hydrogen phosphate, sodium dihydrogen phosphate, hydrochloric acid 37%, acetic acid , dimethyl sulfoxide , Mueller–Hinton agar medium (Oxoid), Sabouraud agar medium were purchased from Sigma Aldrich. Xanthine derivatives with thiazolidine-4-one scaffold  have been synthesized by our research group and were reported in a previous paper . Bacteriv/v) and the mixture was left under stirring overnight. 3 mL of chitosan solution were dropped, using a syringe needle , into 20 mL of TPP (2%–3%), under light stirring and the mixture was left at room temperature, under stirring for 4–8 h in order to achieve an efficient reticulation. After 24 h the formed chitosan beads were separated from the TPP solution, washed three times with distillated water, and air-dried at room temperature.Chitosan microparticles were prepared by the ionic gelation method. Briefly, chitosan  was dispersed in 100 mL of acetic acid 1% (v/v) in order to obtain three different concentrations  for each derivative . The resulted mixtures were dropped, using a syringe needle , into 20 mL of TPP (2%–3%), under light stirring and the mixture was left at room temperature, under stirring for 4–8 h to achieve an efficient reticulation. After 24 h the formed chitosan loaded beads (CS-XTDs) were separated fromTPP solution, washed with distillated water three times, and air-dried at room temperature.The xanthine derivatives with thiazolidine-4-one scaffold  were loaded into chitosan microparticles by the ionic gelation method . BrieflyThe CS-XTDs microparticle size (in wet and dry state) was measured using a Leica DM750 microscope  equipped with a video model ICC50 W0366. The values were recorded with 10× objective by LAS EZ program. The morphology of the microparticles was studied using a scanning electronic microscope (SEM), Vega II SBH model, produced by the Tescan Company . All measurements were performed in triplicate and the results are expressed as mean size ± standard deviation.1). At the end of the study the microparticles were dried again and weighted (W2). All tests were carried out in triplicate and the results are expressed as mean SD ± standard deviation. The analysis of Variance (ANOVA) was used to analyze the experimental data. Statistical significance was set to p value ≤ 0.05.The swelling studies were carried out using distilled water and simulated gastric fluid  at 37 °C (in a thermostated water bath) according to the literature references ,39. Brie1—the weight of the swollen CS-XTDs microparticles; W2—the weight of the dried CS-XTDs microparticles.The swelling degree (SD) at different times was calculated using the following formula:2 = 1 for 6c, y = 27.606x − 0.0151; R2 = 1 for 6e, y = 28.89x + 0.0219; R2 = 0.9996 for 6f, y = 20.914x + 0.0406; R2 = 1 for 6k) was used. The loading efficiency of XTDs  into the chitosan matrix was analyzed using a GBC Cintra 2010 UV-VIS spectrophotometer  equipped with Cintral Software, according to the literature references  with sli1 = the amount of CS-XTD microparticles, after drying (mg); W3 = the amount of XTD loaded into the chitosan matrix (mg).The drug loading (DL) was calculated using the following formula :(2)DL(%)2 = the mount of XTD used in study (mg); W3 = the amount of XTD loaded into chitosan matrix(mg).The entrapment efficiency (EE) was calculated using the following formula : (3)EE was performed using simulated gastric fluid  and simulated intestinal fluid , according to the literature references, with slight modifications ,43,44. T1 = concentration of the XTD (mg/mL) in the release media at different times; C0 = concentration of the XTD (mg/mL) in the CS-XTD microparticles.The Drug release (DR) was calculated using the following formula :(4)DR(%)All tests were performed in triplicate and the results are expressed as mean DR ± standard deviation.−1, after 16 scans, in the 4000–650 cm−1 range and processed with the Horizon MBTM FT-IR Software . FT-IR spectra of chitosan, XTDs  and of CS-XTDs  were recorded using ABB-MB3000 FT-IR MIRacleTM Single Bounce ATR  at a resolution of 4 cm50). The Swiss mice (22–35 g) were housed in polyethylene cages, at constant conditions: temperature of 24 ± 2 °C, humidity of 40–70%, and cycle of 12 h light and 12 h dark. The acclimatization of the animals to laboratory conditions was performed 7 days before the experiment, receiving standard food and water ad libitum. The animals were kept fasting for 24 h before starting the experiment. Each group (six mice/group) received XTDs, orally (p.o.), as suspensions in Tween 80 in doses ranging between 1500–6500 mg/kg b.w. The survival rate was determined at 1 day, 2 days, 3 days, 7 days, and 14 days after administration.The acute toxicity of the XTDs  was evaluated by determining the lethal dose of 50 (LD50) was determined by the Karber method using the following formula [a = the difference between two successive doses of the XTDs; b = the arithmetic average of the mice died from two successive series; n = the number of animals from each group; LD100 = the 100% lethal dose.The lethal dose 50 LDThis study was performed according to the ethics guidelines on laboratory animal studies  and with agreement (no 17826/2016) of the Ethics Committee for Animal Research of “Grigore T. Popa” University of Medicine and Pharmacy Iasi.3 (5 mg/mL) were evaluated using the agar disc diffusion and broth micro-dilution methods.The antibacterial and antifungal activity of XTDs and CS-XTDs6 CFU/mL (McFarland standard no 5). The suspensions were diluted 1:10 in Mueller Hinton agar (bacteria) and Sabouraud agar (yeasts) and then spread on sterile Petri plates (25 mL/Petri plate). Sterile stainless steel cylinders  were placed on the agar surface in Petri dishes. In each cylinder 200 µL of XTDs, solutions in DMSO (20 mg/mL) and an equivalent concentration of CS-XTDs were added. Commercial discs containing ampicillin (25 µg/disc), ciprofloxacin (30 µg/disc), and nystatin (100 µg/disc) were used as a positive control. DMSO was used as a negative control. The plates were incubated for 24 h at 37 °C (bacteria) and for 48 h at 24 °C (yeasts). After incubation the diameters of inhibition were measured. All tests were performed in triplicate and the results are expressed as mean diameter ± standard deviation.Antibacterial and antifungal activity of XTDs and CS-XTDs, expressed as the diameter of inhibition area, were evaluated using the standard disk diffusion assay according to literature reference  with sliCandida strains. After the incubation, the culture media were diluted in order to obtain a final concentration of 106 CFU/mL. In a 96-well microplate, different dilutions were prepared from the stock solution of the XTDs (20 mg/mL), in order to obtain a volume of 100 μL in each well with final concentrations in the range of 10 mg/mL, 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL, 0.312 mg/mL, 0.156 mg/mL, 0.078 mg/mL, 0.039 mg/mL, 0.0195 mg/mL, 0.009 mg/mL, and 0.0048 mg/mL. A volume of 100 μL strain suspension was inoculated onto the wells and the microplates were incubated for 24 h at 37 °C  and at 24 °C for . The MIC value was established as the lowest concentration, which determined the visual inhibition of the strain’s growth. For MBC/MFC determination, 1 µl from each visually complete inhibition was transferred onto a plate with solid media and incubated for 24 h at 37 °C  and at 24 °C for . The MBC/MFC values were considered the lowest concentration, which killed 99.9% of the tested strains. All tests were performed in triplicate.The minimum inhibitory concentration (MIC) and the minimum bactericidal/fungicidal concentration (MBC/MFC) of XTD were determined using the standard two-fold dilution method standardized by , with slThe synthesis of new xanthine derivatives with thiazolidine-4-one scaffold (6a–k)  were preThe chitosan microparticles could be prepared using chemical and physical methods. The chemical method is based on the covalent bond formation between the functional groups of chitosan while the physical process involves electrostatic hydrophobic interactions or hydrogen bonds formation in the polymer matrix ,49. It iFour XTDs  were selected based on their biological effects and were inglobated into a polymeric matrix based on chitosan using the ionic gelation method. In order to optimize the preparation procedure of chitosan microparticles loaded with xanthine derivatives with thiazolidine-4-one scaffold (CS-XTDs) different parameters were applied: chitosan concentration (1.2%–1.7%), TPP concentration (2%–3%), and reticulation time (4–8 h). The most stable CS-XTDs systems were obtained using the following parameters: CS concentration of 1.7%, TPP concentration of 2%, and reticulation for 8 h. Using three concentrations  for each XTD, twelve CS-XTD systems were developed and characterized. The mass ration between CS, TPP, and XTD, based on the concentration of used XTD  was as follows:5.7:44:1, 4.25:33:1 and 3.4:27:1.The size of the CS-XTDs ranged between 858–953 μm (wet state) and 611–855 μm (dry state) and it was higher than CS , thus confirming the loading process . The dat3 (5 mg/mL), for which the spherical shape is lost and pronounced flattening was recorded (The SEM showed that chitosan microparticles (CS) have a spherical and regular shape, regular outline and a slightly rough surface. Upon loading of XTD, a deformation of microparticles was observed, which is intensified by the increasing concentration described in other studies . The mosrecorded .The results showed that a higher concentration of the cross-linking agent was associated with decreased stability of microparticles during the gelation process. Also, it was highlighted that the deformation of CS-XTDs microparticles during the air-drying process was directly proportional to the concentration of the TPP solution, which resulted in strongly flattened microparticles being obtained for a value of 3% TPP. The reticulation time is also an important parameter for the stability of microparticles; increasing the time was associated with the increased stability of the developed polymeric systems ,55.The swelling degree is an important parameter in the release of the drug from the polymer matrix, having an important influence on the drug’s bioavailability. The results for CS-XTDs systems recorded in distilled water and simulated gastric fluid  are presented in 3) and 206 ± 2.9 (CS-6k1). In similar conditions the recorded value for CS was 209 ± 3.5  ; the res1) and 93.91% (CS-6k3) and is directly proportional to the concentration. The highest entrapment efficiency, at all three concentrations , was recorded for CS-6k, the values being: 87.86 ± 1.25% (3 mg/mL), 91.58 ± 0.55% (4 mg/mL) and 93.91 ± 1.41% (5 mg/mL). These results could be explained based on the structure of the XTD (6k), which has hydrophobic methyl group at para position of the aromatic ring. The XTD with hydrophilic methoxy group attached at the ortho (6e) and para (6f) position on the aromatic ring have showed lower values of entrapment efficiency.The results obtained for drug loading (DL) and entrapment efficiency (EE) parameters are presented in 1) and 22.02 ± 0.74% (CS-6k3). As a previous parameter, the highest values were obtained for derivative 6k, which has a methyl group at the para position of the aromatic ring as follows: 14.32 ± 1.12% (CS-6k1), 18.90 ± 0.67% (CS-6k2), and 22.02 ± 0.74% (CS-6k3). A good correlation was also observed between DL and EE values, which proves the accuracy of the study. Thus, it has been demonstrated that the optimal CS-XTD formulation was obtained for 5 mg/mL of XTD.Concerning the DL of XTDs into the polymeric matrix, it was noted that this parameter increases with the concentration. The obtained values ranged between 10.04 ± 0.49% , while the highest values were recorded for CS-6c . In the SGF, the drug release from CS-XTD was ranging between 51.34% and 98.42%, while the drug release in SFI was lower, being around 5%, excepting the CS-6k for which the drug release in SIF was 17% . The lowThe confirmation of the loading process was performed by highlighting in FTIR spectra the CS-XTDs of the specific functional groups of the components of the polymeric matrix: CS, XTDs, and TPP. The FTIR spectra of CS-XTDs  at different concentrations in reference with the FTIR spectra of CS and XTDs  are presented in In acetic acid solution, chitosan has a pKa of 6.3 and a polycationic structure with positively charged amino groups. In water, TPP dissociates to form both hydroxyl and phosphoric ions. In the crosslinking process the cationic amino groups of chitosan react with negatively charged TPP to form ionic complexes by electrostatic bonds. A secondary mechanism based on hydrogen-hydrogen bonds between the hydroxyl groups of CS and of TPP is also possible . The XTD−1 , 2981–2872 cm−1, 1431–1414 cm−1 and 1377–1375 cm−1 (–CH2–CH3), 1661–1647 cm−1 (C=O), 1267–1250 cm−1 (C–N), 1034–1026 cm−1 (C–O–C). The presence of XTDs  into the polymer matrix was confirmed by identification of the specific absorption bands: NH bond (3362–3285 cm−1), thiazolidine-4-one ring: 2981–2872 cm−1 (–CH2–), 1724–1711 cm−1 (C=O) and 671–660 cm−1 (C–S), aromatic ring: 3362–3285 cm−1, 810–746 cm−1 (=C–H), 1558–1549 cm−1 and 1494–1452 cm−1 (C=C), amide group, which are overlapping with valence vibrations of the same group in the chitosan structure, and not the substituents on the aromatic ring at 1063–1059 cm−1 (C–O from –OCH3 in case of 6e and 6f), 822–818 cm−1 (–Cl in case of 6c) and 1377–1375 cm−1 . The reticulation agent, TPP, was identified by specific absorption bands in the 1151–1149 cm−1 region (P=O) and the 895–893 cm−1 region (P–O–P).The specific vibrations of functional groups of CS have been identified in the following regions: 3362–3285 cm50 values: 2125 mg/kg bw (6c), 1687.5 mg/kg bw (6e), 1937.5 mg/kg bw (6f) and 1312.5 mg/kg bw (6k). These compounds proved to be less toxic than theophylline, which is used as a starting reagent in their synthesis (LD50 = 332 mg/kg bw) [The toxicological screening highlighted that all tested XTDs  have a low toxicity, with the following LDg/kg bw)  and supp3  are active on both bacterial and fungal strains, the diameter of the inhibition area being in correlation with structure of the XTD and also with bacterial and yeasts strains, respectively. The data presented in Staphylococcus aureus ATCC 25923 was 6d, for which the diameter of the inhibition area was 17.1 ± 0.24 mm. The compounds 6a, 6f and 6g showed improved antibacterial effects on Sarcina lutea ATCC 9341, their effect  being higher than the effect of CS and comparable with ciprofloxacin (25.0 ± 0.1 mm). The effect of XTDs on Gram negative bacterial strain Escherichia coli ATCC 25922 was reduced in reference to Gram positive bacterial strains , the diameter of the inhibition area ranging between 10.4 mm and 12.2 mm.Referring to the XTDs it was noted the most active compound on Candida albicans ATCC 10231 (15.2 ± 0.21 mm and 15.2 ± 0.26 mm respectively), for 6h and 6g on Candida glabrata ATCC MYA 2950 (19.1 ± 0.17 mm and 16.1 ± 0.28 mm respectively) and for 6f and 6g on Candida parapsilosis ATCC 22019 (15.2 ± 0.11 mm and 15.4 ± 0.31 mm respectively). Appreciable antifungal effects were noted for compounds 6c and 6h on As we expected, the loading of the XTDs into the polymer matrix based on chitosan was associated with increasing the antimicrobial effects, all CS-XTD systems  show improved antibacterial and antifungal effects in reference to corresponding XTD  .The antibacterial effect of 6d was confirmed by the broth micro-dilution assay, for which the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 0.3125 mg/mL and 10 mg/mL, respectively . The simIn this study new polymeric systems based on chitosan (CS) loaded with new xanthine derivatives with thiazolidine-4-one scaffold (XTDs) were developed and characterized. The success of the loading process was proved by highlighting in FTIR spectra of the CS-XTDs of the specific functional groups of XTD. The optimized polymeric systems were evaluated in terms of particle size, morphology, swelling degree, drug loading and entrapment efficiency. The results demonstrated a good swelling degree for CS-XTDs and the entrapment efficiency of the XTD into polymer matrix was between 63% and 94%. In the simulated biological fluids , an increased cumulative release, between 54.52% and 97.44% was observed. The data supports also improved antimicrobial effects for CS-XTDs in reference with XTDs. Based on the obtained results the developed polymeric systems could have important applications in the food field as active multifunctional  packaging materials and in medical and pharmaceutical fields.


The electrochemical properties of the prepared nanohybrids with various composition ratios were examined by cyclic voltammetry (CV), and the trace additives in food and/or beverage was detected by using differential pulse voltammetry (DPV). The experimental results indicated that the prepared nanohybrids for fabrication of electrochemical modified electrodes possess active electroresponse, marked redox current, and good electrochemical reversibility, which could be mediated by changing the system formulations. The nanohybrid modified electrode sensors had a good peak current linear dependence on the analyte concentration with a wide detection range and a limit of detection as low as about 1.35 × 10−9 mol L−1, and the amount of aspartame was measured to be 35.36 and 40.20 µM in Coke zero, and Sprite zero, respectively. Therefore, the developed nanohybrids can potentially be used to fabricate novel electrochemical sensors for applications in the detection of beverage and food safety.An electrochemical sensor for detection of the content of aspartame was developed by modifying a glassy carbon electrode (GCE) with multi-walled carbon nanotubes decorated with zinc oxide nanoparticles and in-situ wrapped with poly(2-methacryloyloxyethyl ferrocenecarboxylate) (MWCNTs@ZnO/PMAEFc). MWCNTs@ZnO/PMAEFc nanohybrids were prepared through reaction of zinc acetate dihydrate with LiOH·H Most extensively adopted detection methods for aspartame have capillary electrophoreses [Aspartame is a dipeptide artificial sweetener, and extensively used in the manufacturing of many sugar-free, low calorie, and dietary products ,2. It haphoreses ,8 and hiphoreses . These tphoreses . Electrophoreses ,12. NeveUp to now, the electrode modification remains an effective method to improve the sensitivity. Since carbon nanotubes (CNTs) exhibit the activity of edge-plane-like graphite sites at the CNTs ends, they can promote the electron transfer in electroanalytical applications , and theInspired by the above description, our aim is to prepare MWCNTs scattered with ZnO (MWCNTs@ZnO) nanocomposites, and then to wrap poly(2-methacryloyloxyethyl ferrocenecarboxylate) (PMAEFc) around MWCNTs@ZnO to achieve a novel MWCNTs@ZnO/PMAEFc organic–inorganic nanohybrid for modification of amperometric sensing electrodes. By investigating the influence of the system composition on the electrochemical performances, the fabricated modified electrode sensors are anticipated to be capable of improving the electrochemical signals and lowering the LOD, thus providing a simple, rapid, highly-sensitive, and accurate electrochemical approach for detection of aspartame in real samples.3COO)2·2H2O, and lithium hydroxide monohydrate  were obtained from J & K Reagent Company, Beijing, China, and used as received. 2-Hydroxyethyl methacrylate  was offered by Zahn Chemical Technology Co., Ltd., Shanghai, China and used dirrectly. 4-Dimethylaminopyridine  and dicyclohexylcarbodiimide  were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., Shanghai, China. Azobisisobutyronitrile  and 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid  as chain transfer agent (CTA) were supplied by Aladdin  Reagent Company, China. Ethanol (98%), ether (98%), and N,N-dimethylformamide  were afforded by Sinopharm Chemical Reagent Co., Ltd., Shanghai, China, and used as received. Aspartame (98%) and perfluorinated ion-exchange resin  mixture) were furnished by Aladdin (Shanghai) Reagent Company, Shanghai, China. The samples to be detected—including Coke Zero, Sprite Zero, Mango juice, kiwi fruit juice, strawberry juice, and Lipton lipya tea sachet—were acquired from a local supermarket .The original pristine solid powdered MWCNTs, with particle diameter of 10–30 nm, and length of 10–30 μm, were provided by Chengdu Institute of Organic Chemistry, CAS, and dried in an oven at 120 °C for 24 h before use. Ferrocenecarboxylic acid , zinc acetate dihydrate  in 50 mL of ethanol was dropwise added to the flask under vigorous magnetic stirring. The reaction solution was stirred for 3 h to obtain a fine ZnO sol containing MWCNTs. The resulting sol dispersoid was filtered to achieve a solid product mainly containing MWCNTs and ZnO NPs. The solid samples were sintered at 85 °C in vacuum for 2 h to afford MWCNTs@ZnO powder [Nanostructured ZnO was prepared using chemical route through reaction of zinc acetate dihydrate with LiOH·Heviously ,35,36,37O powder ,35,36. T2Cl2. Afterwards, DCC  dissolved in 30 mL desiccative CH2Cl2 was dropwise added to the above mixed solution under the condition of N2 atmosphere at 0 °C at a speed of 3–4 drop s−1. The reaction mixture was stirred at 0 °C for 2 h, and then at room temperature overnight. The resulting solution was filtered to get rid of the sediment 1,3-dicyclohexylurea (DCU). The filtrate was extracted twice by using saturated sodium bicarbonate solution and deionized water to remove DMAP and unreacted FCA until the supernatant is colorless. After concentrated, the extract was purified by column chromatography using a mixture of n-hexane and ethyl acetate (v/v = 9/1) as an eluent. The collected AEFc solution was evaporated by a rotary evaporator to remove solvents and then dried in vacuum at 30 °C for 24 h, giving an orange solid product with a mean yield of 72%. 1H NMR , δ (ppm): 4.18 , 4.40 , 4.81 , 4.48 OCH2-CH2OC(O)-), 5.63 OCH2-), 6.18 OCH2-), and 1.98 .To prepare MWCNTs@ZnO/PMAEFc organic–inorganic nanohybrids, 2-methacryloyloxyethyl ferrocenecarboxylate (MAEFc) was synthesized via esterification reaction of FCA with HEMA using DMAP as a catalyst and DCC as a dehydration agent in a molar radio of FCA:HEMA:DMAP:DCC of 1:1.4:1:1.2. In a sealed 500 mL three-neck flask, FCA , HEMA , and DMAP  were dissolved in 50 mL dried CH2, and the flask was immerged in an oil bath of 75 °C to start the polymerization. After 24 h, the polymerization was terminated by putting the flask into liquid N2. The reaction mixture solution was vacuum filtered, and the solid was flushed with DMF thrice to remove the unreacted MAEFc. The crude product was precipitated with ether thrice, then filtered, and the solid sample was dried in a vacuum oven at 60 °C overnight, giving a MWCNTs@ZnO/PMAEFc nanohybrid with a PMAEFc mass content of 25% (mean yield: 90.1%), named C1. Similarly, the MWCNTs@ZnO/PMAEFc nanohybrids with a PMAEFc mass percentage of 50% and 75% was prepared in the same method as the above operation, giving a mean yield of 91.8% and 93.4%, respectively, denominated C2 and C3.Then, MWCNTs@ZnO/PMAEFc organic–inorganic nanohybrids were prepared by an in situ RAFT polymerization of MAEFc at a molar ratio of monomer:initiator:CDSP of 100:1:0.25. The mass percentage of the resulting PMAEFc theoretically retained 25%, 50%, and 75% through changing the amount of MWCNTs@ZnO. In a typical procedure,0.17 g dried MWCNTs@ZnO was dispersed in 5 mL DMF under ultrasonication for 2 h in a 25 mL dried Schlenk flask, and then 0.51 g (1.49 mmol) MAEFc, 6.00 mg (0.0149 mmol) CDSP, and 0.61 mg (0.0037 mmol) AIBN were added into the above flask in succession. The reaction system was degassed by freezing, vacuumizing, and purging with NThe above C1, C2, or C3 nanohybrids of 0.01 g were dispersed in 1 mL DMF by means of an ultrasonic technique for 2 h. Uniformly dispersed conducting nanohybrid dispersions (6 µL) were evenly deposited on the surface of the clean pre-treated glass carbon electrodes (GCEs) using a micro-syringe, and then the modified electrodes were dried with an infrared lamp to obtain a finished GCEs coated with the nanohybrids. To ensure good adherence of the nanohybrids on the surface of the electrodes, 5 µL of 1 wt % Nafion solution was cast on the modified layers and dried in air before use. The MWCNTs@ZnO/PMAEFc modified electrode sensors with MAEFc contents of 25%, 50%, and 75% severally denominated as C1, C2, and C3.−1. Raman spectra were analyzed by an AlMEGA dispersive Raman spectrometer  employing Ar+ laser with excitation wavelength of 532 nm. Thermal gravimetric analysis (TGA) was performed on a thermoanalyzer system  at a heating rate of 10 °C min−1 under N2 atmosphere (flow rate: 40 mL min−1). X-ray photoelectron spectroscopy (XPS) spectra were recorded on an AXIS ULTRA spectrometer  at a voltage of 15 kV and room temperature. Fourier transformation infrared (FI-IR) spectra were determined by EQUINX55 Fourier transform infrared spectrophotometer  using KBr pellets. X-ray diffraction (XRD) analyses were conducted on a D/Max-2550 VB+/PC X-ray diffractometer  using Cu radiation at a voltage of 40 kV, a current of 30 mA and a scanning rate of 10° minAtomic force microscope  was employed to observe surface topography and distribution of MWCNTs@PMAEFc at a scan rate of 0.977 Hz, and to estimate the layer thickness of MWCNTs@PMAEFc. The sample morphologies were recorded on a transmission electron microscope  at an accelerating potential of 200 kV. Before observation, the samples to be determined were ground into powders, and fully dispersed in ethanol through an ultrasonic clearer for about 20 min. After that, a drop of the dispersion solution was dripped on the copper grid with carbon films, and dried for 6 h. Scanning electron microscopy (SEM) images were acquired via a SU-8020 field emission electron microscope  at an operating voltage of 15 kV. All measurements were carried out at room temperature. Electrochemical analyses including cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were conducted on a CHI 660E electrochemical workstation  consisting of a conventional three-electrode cell, which contains a bare or modified glassy carbon electrode with a diameter of 3.0 mm as a working electrode, a platinum electrode as a counter electrode, and a saturated calomel electrode ) as a reference electrode. For detection of aspartame, DPV analyses were conducted in PBS of pH = 2, with a modulation amplitude of 50 mV, a pulse width of 50 ms and a step potential of 5 mV.−3 mol L−1 aspartame in PBS (pH = 2) solution was prepared, and then the solution of 5 mL was mixed with 100 mL Coke Zero and 100 mL Sprite Zero to get a 14 mg l−1 (4.76 × 10−5 mol L−1) aspartame solution. As for fruit juice, 0.3 mg of each sample was weighed and diluted using 20 mL PBS (pH = 2), whilst Lipton lipya tea sachet (2 g) was fetched out and dissolved in deionized water, and then transferred in a 1 L volumetric flask. For reducing the error, all solutions were filtered via a 0.45 µm membrane before measurements. The aspartame concentration was determined by the electrochemical modified electrodes with the method of DPV. The percentage of recovery was determined for five different concentrations, and relative standard deviations (RSDs) were measured for 20 times detection for the same concentration.The two carbonate beverages (Coke Zero and Sprite Zero), three fruit juices  and Lipton lipya tea sachet were selected as real sample for residual aspartame detection. For carbonate beverages, their aqueous samples were ultrasonicated for 30 min and diluted with deionized water. Subsequently, the Coke Zero and Sprite Zero were directly spiked with appropriate amounts of aspartame standard solution. Specifically, a 50 mL 1 × 10θ of about 25.9°, 43.0°, and 44.2° that are assigned to the diffraction signals of the (002) hexagonal graphite structure, the (100) and (101) diffraction planes, respectively [θ of 31.6°, 34.3°, 36.1°, 47.4°, and 56.5°, which originate from (100), (002), (101), (110), and (102) crystal planes of hexagonal ZnO that matches with the JCPDS card (no. 36-1451) [kλ/βcosθ, where k is constant (about 0.9), λ is the wavelength of X-ray (0.15406 nm), β is the full width of half-maxima (FWHM) of the diffraction line corresponding to (101) having high intensity and θ is the Bragg’s angle. This value is in agreement with that estimated by the following SEM and TEM observations. However, the three peaks reflecting the diffraction features of MWCNTs are clearly weakened due to the incorporation of ZnO NPs; but the combination of ZnO NPs with MWCNTs does not destroy the MWCNTs structures. These results show the formation of MWCNTs@ZnO nanocomposites. After PMAEFc is grafted onto the surface of the MWCNTs@ZnO nanocomposites, as shown in The phase structure of MWCNTs@ZnO/PMAEFc nanohybrid and its precursors was examined by XRD, as shown in ectively . In XRD 36-1451) . The ave−1 is assigned to the stretch band of the -OH groups due to the moisture absorbed from the air and the residues of carboxylic groups during preparation, and the vibration peak at 1638 cm−1 is attributed to the C=C stretch vibration band of the benzenoid (νC=C) structure in the MWCNTs skeleton, signifying the graphite structure of MWCNTs [−1, which is attributable to the Zn-O characteristic peak [−1 are ascribed to the characteristic C=C mode, as stated. The increased peak intensity at 3445 cm−1 is due to the stretching vibration band of water molecules on the surface of ZnO or MWCNTs/ZnO and MWCNTs [−1 is assigned to the =C-H stretch mode of the ferrocenyl rings (Cp); the bands at 2960–2986 cm−1 are attributable to the C-H stretch mode; the peak at 1719 cm−1 is attributed to the C=O ester carbonyl stretch characteristic; the peak at 1589 cm−1 are assigned to the C=C stretch modes of the Cp rings; the peak at 1138–1276 cm−1 is correlated with the C-O stretch mode; and the bands at 772, 828, and 968 cm−1 reflect the =C-H stretching modes in the Cp rings and Fe-C or Cp-Fe stretch modes, respectively [−1 are attributed to the Fe-C/Cp-Fe and Zn-O stretching modes.FTIR spectroscopy was adopted to qualitatively confirm the chemical structure of MWCNTs@ZnO/PMAEFc nanohybrids, as depicted in f MWCNTs ,41. Aftetic peak ,43. The −1, which is known as typical D band (disorder induced band) and G band (graphite band), respectively [G/ID, as a rough measure of specimen quality, can be used to indicate the integrity of crystalline graphitic structure of the grown MWCNTs. In comparison with the high IG:ID ratio (1.94) of the original pristine MWCNTs, the MWCNTs decorated with ZnO NPs gives a decreased IG/ID value of 1.46, signifying an increased interaction between the MWCNTs and ZnO NPs presumably because of the growth of Zn NPs on the defect dots of MWCNTs [G/ID values further decrease to 1.18, 1.20, and 1.32; this correspondto the MWCNTs@ZnO/PMAEFc nanohybrids with the PMAEFc mass percentage of 25%, 50%, and 75%, respectively. However, the increased IG/ID values with increasing the PMAEFc mass percentage are maybe due to more π–π conjugation interactions between MWCNTs and Cp, which have a contribution to good crystalline graphitic structure of the MWCNTs. These findings indicate that the ZnO NPs and PMAEFc homopolymer are decorated or covered at the defect dots or/and on the surface of MWCNTs successively.Raman spectroscopy was further adopted to confirm the preparation of the prepared nanohybrids, as shown in ectively . The peaf MWCNTs . After tThermogravimetric (TGA) was implemented to describe the thermal behavior of the products in the process from 0 to 800 °C in TEM observations were further used to characterize the morphologies of MWCNTs@ZnO and MWCNTs@ZnO/PMAEFc, as presented in CV was adopted to examine the electrochemical behavior of various materials, as displayed in E  lower than about 50 mV and the ratio of the anodic to cathodic peak currents  almost close to unity 1, which is due to the reversible Fe(III)/Fe(II) redox electrochemical process [XPS and AFM were further used to interpret the electrochemical response, as illustrated in  process . Consequ process . These rIp) dependence on the scan rates was examined in a potential range from 10 to 120 mV s−1 at a voltage step of 10 mV in PBS solution of pH = 2.0. −1, showing a good linear correlation between the scan rates and peak currents, and offering a well-defined linear equation Ip,a(10−4 A) = 0.0206ν (mV s−1) + 0.0016 (R2 = 0.9925) for anodes and Ip,c(10−4 A) = −0.0223ν (mV s−1) − 0.0021 (R2 = 0.9962) for cathodes. These findings suggest that the redox processes are a diffusion-controlled electrode reaction process for the CNTs@ZnONPs/PMAEFc modified electrode sensors and the electrode processes are quasi-reversible [Given that the scan rate may have effect on the electroactivity of the nanohybrids, viz., the conducting rate of the electrons, the peak currents  by changing the aspartame content. The experimental results were shown in −8 to 1 × 10−12 mol L−1, and the increment in peak currents (I-I0) is proportional to the logarithm of aspartame concentrations, giving a linear calibration equation Ip,C1(1 × 10−5 A) = −0.0354(−log C) − 0.3303  for C1 and Ip,C2(1 × 10−5 A) = −0.0960(−log C) − 0.1080  for C2, Ip,C3(1 × 10−5 A) = −0.0483(−log C) − 0.2232  for C3, where Ip and C represent the peak current and the concentration of aspartame, respectively. The limit of detection (LOD) is calculated as per the following equation using three times noise, viz., signal/noise = 3 (S/N = 3) [−10 A for C1, 4.320 × 10−11 A for C2 and 1.547 × 10−9 A for C3; K is a coefficient based on a certain confidence level factor (K = 3); and r is the sensitivity of the detection method, viz., the slope of the calibration curve . The LOD values are estimated to approximately be 3.12 × 10−8, 1.35 × 10−9 and 9.61 × 10−8 mol L−1 corresponding to the C1, C2, and C3 modified electrode sensor, respectively. These LOD values signify that an effective combination of the three constituents can significantly lower the LOD value of the modified electrodes, and thus detect an extremely trace amount of additives in food or beverage. These values are superior to those obtained from any other technique such as flow injection analysis (FIA), surface-bound molecular imprinting technology (SMIT), capillary zone electrophoresis (CZE), square-wave voltammetry (SWV), FTIR, DPV, hydrophilic interaction liquid chromatography (HILIC), and UV visible simultaneous analysis (CZE-UV) [To practically measure trace aspartame in food or beverages, calibration curves of the nanohybrid modified electrode sensors should be prepared by detecting the change of peak currents with the concentrations of aspartame using DPV methods. The detection was conducted at room temperature in a traditional electrochemical cell having 10 mL 0.2 mol LS/N = 3) LOD = KS(CZE-UV) ,66,67,68(CZE-UV) , as summ(CZE-UV) ,30. In cTo evaluate the applicability of the MWCNTs@ZnO/PMAEFc/GCE sensors, the C2 sensor was selected to determine the aspartame concentrations in the real samples. For this purpose, the Coke Zero, Sprite Zero, mango juice, kiwi fruit juice, strawberry juice, and Lipton lipya tea sachet sample solutions of 50 mL, which contain aspartame, were stirred for 30 min and served as stock solutions with no dilution. Then, the solution was added to the sample cells and the alteration in current density was detected by DPV. The electrochemical signals of each sample were recorded five times and averaged, with the RSD as low as 0.67%, 0.17%, 2.6%, 1.8%, 3.3%, and 4.1% for Coke Zero, Sprite Zero, mango juice, kiwi fruit juice, strawberry juice, and Lipton lipya tea sachet samples, respectively. The concentrations of aspartame in Coke Zero, Sprite Zero, mango juice, kiwi fruit juice, strawberry juice and Lipton lipya tea sachet were calculated using the calibration graph to be approximately 35.36 µM, 40.20 µM, 0.96 mM, 1.34 mM, 0.88 mM, and 1.53 mM, respectively in this way. Compared with any other method presented in the literatures  28,60,6,66,67,68−9 mol L−1 can be applied to detect trace aspartame in a μM concentration level in food or beverage, particularly for sample C2. Compared with previously-reported techniques, the developed electrochemical sensors have the advantages of good electrochemical response, repeatedly stability with low RSD, low LOD, and wide detection range. Therefore, the prepared MWCNTs@ZnO/PMAEFc nanohybrid modified electrode electrochemical sensors can potentially applied in the monitoring of beverage and food safety.In summary, we have shown an effective means to prepare novel organic–inorganic nanohybrids constructed by carbon nanotube hybridized with ZnO NPs and wrapped with PMAEFc homopolymer, as confirmed by FTIR, Raman, XRD, TGA, XPS, AFM, TEM, and SEM measurements. The MWCNTs@ZnO/PMAEFc nanohybrid modified electrodes exhibit a quasi-reversible electrochemical redox response, reproducibility, stability, and a good linear correlation between the oxidation peak current and the aspartame concentration over a wide concentration range, hinging on the effective combination of the three components, and/or their composition ratios. A significantly low LOD of 1.35 × 10