Optimizing the green synthesis of antibacterial TiO2 - anatase phase nanoparticles derived from spinach leaf extract.

Titanium dioxide nanoparticles, renowned for their abundance, non-toxicity, and stability, have emerged as indispensable components in various fields such as air purification, healthcare, and industrial processes. Their applications as photocatalysts and antibacterial agents are particularly prominent. The synthesis methods significantly influence the properties and subsequent applications of these nanoparticles. While several techniques exist, the biological approach using plant extracts offers advantages such as simplicity, biocompatibility, and cost-effectiveness. This study focused on the green synthesis of titanium dioxide nanoparticles utilizing spinach leaf extract. Within the scope of this investigation, the green synthesis of titanium dioxide nanoparticles through spinach leaf extract were synthesized and optimized, followed by a comprehensive examination of their morphological, structural, and chemical attributes with UV-visible spectroscopy, FTIR spectroscopy, XRD, FESEM, and EDX. The minimum inhibitory concentration (MIC) against E. coli and S. aureus was determined to evaluate their antibacterial potential. Optimal synthesis conditions were identified at 50 °C, using a 1/30 concentration and 20 ml of spinach leaf extract. Spherical anatase nanoparticles, ranging from 10 to 40 nm, were produced under these conditions. The change in the color of the extract, absorption at 247 nm, change and increase of the peak at 800 - 400 wavelengths, and the maximum intensity of X-ray diffraction at the angle of 25.367 with the crystal plane 101 were indications of the synthesis of these nanoparticles. Notably, the synthesized nanoparticles exhibited antibacterial activity with MIC values of 0.5 mg/ml against E. coli and 2 mg/ml against S. aureus. This research presents a novel, eco-friendly approach to synthesizing titanium dioxide nanoparticles with promising antibacterial properties.

First report of Cotton leaf curl Multan virus of Spinach in China.

Cotton leaf curl Multan virus(CLCuMuV; Begomovirus gossypimultanese, family Geminiviridea) is a single-stranded circular DNA virus, with a genome size of about 2.7 kb, CLCuMuV, which is commonly associated with its satellite DNA, Cotton leaf curl Multan betasatellite (CLCuMuB) (Mansoor et al., 2003), is a serious threat in cotton production causing cotton leaf curl disease (CLCuD) (Briddon et al., 2000). The spread of CLCuMuV is closely linked to its insect vector, whitefly (Bemisia tabaci), which is the exclusive vector species for CLCuMuV transmission (Pan et al., 2018). In May 2019, two spinach (Spinacia oleracea L) samples (XJBC01, XJBC02) showing upward curling of the leaf margins, vein thickening, and enation, symptoms were collected in Shihezi City, Xinjiang, China (Fig. 1B). A 570 bp fragment was amplified from the two symptomatic spinach samples using Begomovirus universal primer pair AV494 (5'-GCCYATRTAYAGRAAGCCMAG-3') and COPR (5'-GANGSATGHTRCADGCCAT ATA-3'), Sequences generated from these amplicons shared 99% nucleotide sequence identities with CLCuMuV DNA-A sequences, suggesting CLCuMuV infection in spinach. To our knowledge CLCuMuV has not been reported in spinach previously. The complete sequences of CLCuMuV and CLCuMuB were then sequenced using CLCuMuV-specific primers GD37-F (5'-GGATCCATTGTTAAACGAATTTCC-3') and GD37-R (5'-GGATCCCACATGTTTGAATTTGA-3') (Gu et al., 2015), as well as betasatellite universal primers ß01 (5'-GGTACCACTACGCTACGCAGCAGCC-3') and ß02 (5'-GGTACCTACCCTCCCAGGGGTACAC-3') (Zhou et al.,2003). The full length CLCuMuV DNA-A in spinach spans 2737 nt (GenBank accession number: MW561346), while CLCuMuB in spinach covers 1343 nt (GenBank accession number: MW561347). The 2737 nt full length CLCuMuV DNA-A and the associated 1343 nt CLCuMuB genome sequences generated from spinach samples were deposited in the GenBank with accession numbers MW561346 and MW561347. The MW561346 shared 99.5% sequence identity with CLCuMV GD37 from Hibiscus rosasinensis. Whereas the MW561347 shared 98.4% sequence identity with CLCuMuB GD37ß. Therefore, we used infectious clones of CLCuMuV (GD37) and CLCuMuB (GD37ß), provided by Xueping Zhou (Gu et al., 2015), to inoculate healthy spinach via Agrobacterium. Infected plants showed typical symptoms 14 days post-inoculation, including leaf edge curling, shrinkage, and vein enlargement, which is consistent with symptoms observed in infected spinach plants in the field (Fig. 1C). The expected 570 bp fragments were amplified in the uninoculated upper leaves of spinach showing symptoms, while not detected in the control spinach, indicating that the symptoms on spinach plants were caused by CLCuMuV associated with CLCuMuB. The transmission efficiency of CLCuMuV to spinach was assessed using two whitefly species, MEAM1 and MED, which were fed on h. rosasinensis infected with CLCuMuV. To compare the transmission efficiency between the two species, 14 spinach plants were inoculated with MEAM1, and 11 spinach plants were inoculated with MED. Each spinach plant was inoculated by releasing 10 whiteflies. After 30 days, MEAM1 transmitted CLCuMuV to spinach inducing typical symptoms (Fig. 1D), with a 78.57% (11/14) transmission efficiency. Similarly, MED also transmitted CLCuMuV to spinach but with a lower efficiency of 54.54% (6/11). These results suggested both MEAM1 and MED could transmit CLCuMuV to spinach, with MEAM1 demonstrating higher efficiency than MED. To the best of our knowledge, this study marks the first report of CLCuMuV infecting spinach, indicating an expanded host range for the virus.

Transcriptome-guided selection of stable reference genes for expression analysis in spinach.

Accurate measurement of gene expression levels is vital for advancing plant biology research. This study explores the identification and validation of stable reference genes (RGs) for gene expression analysis in Spinacia oleracea. Leveraging transcriptome data from various developmental stages, we employed rigorous statistical analyses to identify potential RGs. A total of 1196 candidate genes were initially screened based on expression variability, with subsequent refinement using criteria such as low variance and stability. Among 12 commonly used candidate RGs, EF1α and H3 emerged as the most stable across diverse experimental conditions, while GRP and PPR exhibited lower stability. These findings were further validated through qRT-PCR assays and comprehensive statistical analyses, including geNorm, NormFinder, BestKeeper, and RefFinder. Our study underscores the importance of systematic RG selection to ensure accurate normalization in gene expression studies, particularly in the context of S. oleracea developmental stages and physiological processes like flowering. These validated RGs provide a robust foundation for future gene expression analysis in S. oleracea and contribute to the advancement of molecular research in plant biology.

Endophytic Colletotrichum fructicola KL19 and Its Derived SeNPs Mitigate Cd-Stress-Associated Damages in Spinacia oleracea L.

The application of nanotechnology in agriculture has received much attention in order to improve crop yield, quality and food safety. In the present study, a Cd-tolerant endophytic fungus Colletotrichum fructicola KL19 was first ever reported to produce SeNPs, and the production conditions were optimized using the Box-Behnken design in the Response Surface Methodology (RSM-BBD), achieving a peak yield of 1.06 mM under optimal conditions of 2.62 g/20 mL biomass, 4.56 mM Na2SeO3, and pH 6.25. Following this, the properties of the biogenic SeNPs were elucidated by using TEM, DLS, and FTIR, in which the 144.8 nm spherical-shaped SeNPs were stabilized by different functional groups with a negative zeta potential of -18.3 mV. Furthermore, strain KL19 and SeNPs (0, 5, 10, 20 and 50 mg/L) were inoculated in the root zone of small-leaf spinach (Spinacia oleracea L.) seedlings grown in the soil with 33.74 mg/kg Cd under controlled conditions for seven weeks. Impressively, compared with Cd stress alone, the strain KL19 and 5 mg/L SeNPs treatments significantly (p < 0.05) exhibited a reduction in Cd contents (0.62 and 0.50 folds) within the aboveground parts of spinach plants and promoted plants' growth by improving the leaf count (0.92 and 1.36 folds), fresh weight (2.94 and 3.46 folds), root dry weight (4.00 and 5.60 folds) and root length (0.14 and 0.51 folds), boosting total chlorophyll synthesis (0.38 and 0.45 folds), enhancing antioxidant enzymes (SOD, POD) activities, and reducing the contents of reactive oxygen species (MDA, H2O2) in small-leaf spinach under Cd stress. Overall, this study revealed that utilizing endophytic fungus C. fructicola or its derived SeNPs could mitigate reactive oxygen species generation by enhancing antioxidant enzyme activity as well as diminish the absorption and accumulation of Cd in small-leaf spinach, promoting plant growth under Cd stress.

High Bioavailability of Spinach Folate Evaluated by Functional Biomarkers in a Folate Depletion-Repletion Mouse Model.

The bioavailability of natural folates is 50% lower than that of synthetic folic acid (FA); however, it remains unclear whether this value is universally applicable to all foods. Therefore, the present study investigated the bioavailability of folate from spinach using multiple biomarkers in a folate depletion-repletion mouse model. Mice were fed a folate-deficient diet for 4 wk and subsequently divided into three groups: folate-deficient, FA, and spinach folate. The folate repletion group received either FA or spinach folate at 2 mg/kg diet for 9 d. On the 7th day of repletion, half of each group underwent low-dose total body X-ray irradiation to induce chromosomal damage in bone marrow. Folate bioavailability biomarkers included measurements of folate levels in plasma, liver, and bone marrow along with an analysis of plasma homocysteine levels and chromosome damage, both of which are functional biomarkers of body folate. The consumption of a folate-deficient diet led to decreased tissue folate levels, increased plasma homocysteine levels, and chromosomal damage. Repletion with spinach folate restored folate levels in plasma, liver, and bone marrow to 69, 13, and 68%, respectively, of FA levels. Additionally, spinach folate repletion reduced plasma homocysteine levels and chromosome damage to 83% and 93-117%, respectively, of FA levels. Collectively, the present results demonstrated that the bioavailability of spinach folate exceeded 83% of FA, particularly when assessed using functional biomarkers.

Corrigendum: Reduction in residual cyantraniliprole levels in spinach after various washing and blanching methods.

[This corrects the article DOI: 10.3389/fnut.2022.948671.].

Developing a plant microbial fuel cell by planting water spinach in a hanging-submerged plant pot system.

To plant crops (especially dry crops such as water spinach) with concomitant electricity recovery, a hanging-submerged-plant-pot system (HSPP) is developed. The HSPP consists of a soil pot (anodic) partially submerged under the water surface of a cathode tank. The microbial communities changed with conditions were also investigated. It was found that with chemical fertilizers the closed-circuit voltage (CCV, with 1 kΩ) was stable (approximately 250 mV) within 28 d; however, without fertilizer, the water spinach could adjust to the environment to obtain a better power output (approximately 3 mW m-2) at day 28. The microbial-community analyses revealed that the Pseudomonas sp. was the only exoeletrogens found in the anode pots. Using a secondary design of HSPP, for a better water-level adjustment, the maximum power output of each plant was found to be approximately 27.1 mW m-2. During operation, high temperature resulted in low oxygen solubility, and low CCV as well. At this time, it is yet to be concluded whether the submerged water level significantly affects electricity generation.

Application of EFSA EU menu database and R computing language to calculate the green chlorophyll intake in the European population.

The growing evidence of the health benefits of chlorophyll pigments and the claims that could arise from industry and academia require data on their common dietary intakes. This study presents data on the chronic intake of green chlorophyll in 23 European countries using standardised methodologies to manage food consumption data within the EU Menu methodology. A mean intake of 207.12 mg of green chlorophylls/(d × person) for the adult population was calculated, considering significant covariates. The hierarchical cluster and partial least squares discriminant analysis (PLS-DA) techniques were applied to analyse intake disparities by region and age groups, identifying common food sources of green chlorophylls, such as olive oil, kale, and spinach. This paper presents a modern mathematical approach for obtaining novel information from existing databases of food composition data. Future challenges include building a comprehensive chlorophyll composition database for foods and extending the estimation to non-green chlorophyll pigments and metallo-chlorophyll food colourants.

Characterization of Stemphylium species associated with Stemphylium leaf spot of spinach (Spinacia oleracea).

Stemphylium leaf spot can result in significant losses to spinach seed, processing, and fresh market crops. Stemphylium isolates (n = 1,775) collected from 2000 to 2022 from spinach seed, leaves, and seed crop stem residues were used to assess the diversity of species associated with spinach. Eleven Stemphylium species were identified based on cmdA sequences: S. vesicarium (63.6% of isolates), S. beticola (48.9%), S. amaranthi (5.1%), S. eturmiunum (4.5%), S. astragali (4.0%), S. simmonsii (3.4%), and S. lucomagnoense, S. drummondii, S. gracilariae, S. lycopersici, and S. chrysanthemicola (each 0.6 to 1.7%). Only isolates of S. beticola, S. drummondii, and S. vesicarium were pathogenic to spinach. The incidence of spinach seed on which Stemphylium was observed ranged from 2.5 to 73.5% per seed lot, with S. vesicarium and S. beticola predominant. However, only 60.7 and 62.3% of isolates tested for these two species were pathogenic to spinach, respectively. Therefore, the incidence of Stemphylium species on spinach seed may not reflect accurately the risk of a seed lot carrying pathogenic isolates. Fused MAT1-1 and MAT1-2 genes were detected in isolates of S. vesicarium, but only MAT1-1 was detected in S. beticola isolates, which corroborates previous studies that have proposed the two species to be self-fertile. The duration of ascospore dispersal of S. beticola and S. vesicarium from spinach seed crop stem residues in western Washington, the primary region of spinach seed production in the USA, occurred from mid-winter to late spring or early fall, potentially serving as inoculum for the next season's spinach seed crops. Growers should incorporate residues into the soil after harvest to reduce inoculum production of these pathogens on spinach seed crop residues.

Isolation, characterization, and application of a lytic bacteriophage SSP49 to control Staphylococcus aureus contamination on baby spinach leaves.

Staphylococcus aureus, a major foodborne pathogen, is frequently detected in fresh produce. It often causes food poisoning accompanied by abdominal pain, diarrhea, and vomiting. Additionally, the abuse of antibiotics to control S. aureus has resulted in the emergence of antibiotics-resistant bacteria, such as methicillin resistant S. aureus. Therefore, bacteriophage, a natural antimicrobial agent, has been suggested as an alternative to antibiotics. In this study, a lytic phage SSP49 that specifically infects S. aureus was isolated from a sewage sample, and its morphological, biological, and genetic characteristics were determined. We found that phage SSP49 belongs to the Straboviridae family (Caudoviricetes class) and maintained host growth inhibition for 30 h in vitro. In addition, it showed high host specificity and a broad host range against various S. aureus strains. Receptor analysis revealed that phage SSP49 utilized cell wall teichoic acid as a host receptor. Whole genome sequencing revealed that the genome size of SSP49 was 137,283 bp and it contained 191 open reading frames. The genome of phage SSP49 did not contain genes related to lysogen formation, bacterial toxicity, and antibiotic resistance, suggesting its safety in food application. The activity of phage SSP49 was considerably stable under various high temperature and pH conditions. Furthermore, phage SSP49 effectively inhibited S. aureus growth on baby spinach leaves both at 4 °C and 25 °C while maintaining the numbers of active phage during treatments (reductions of 1.2 and 2.1 log CFU/cm2, respectively). Thus, this study demonstrated the potential of phage SSP49 as an alternative natural biocontrol agent against S. aureus contamination in fresh produce.

Genome-wide analysis of MADS-box genes and their expression patterns in unisexual flower development in dioecious spinach.

Evolution of unisexual flowers involves extreme changes in floral development. Spinach is one of the species to discern the formation and evolution of dioecy. MADS-box gene family is involved in regulation of floral organ identity and development and in many other plant developmental processes. However, there is no systematic analysis of MADS-box family genes in spinach. A comprehensive genome-wide analysis and transcriptome profiling of MADS-box genes were undertaken to understand their involvement in unisexual flower development at different stages in spinach. In total, 54 MADS-box genes found to be unevenly located across 6 chromosomes and can be divided into type I and type II genes. Twenty type I MADS-box genes are subdivided into Mα, Mß and Mγ subgroups. While thirty-four type II SoMADSs consist of 3 MIKC*, and 31 MIKCC -type genes including sixteen floral homeotic MADS-box genes that are orthologous to the proposed Arabidopsis ABCDE model of floral organ identity determination, were identified in spinach. Gene structure, motif distribution, physiochemical properties, gene duplication and collinearity analyses for these genes are performed in detail. Promoters of both types of SoMADS genes contain mainly MeJA and ABA response elements. Expression profiling indicated that MIKCc genes exhibited more dynamic and intricate expression patterns compared to M-type genes and the majority of type-II genes AP1, SVP, and SOC1 sub-groups showed female flower-biased expression profiles, suggesting their role in carpel development, while PI showed male-biased expression throughout flower developmental stages, suggesting their role in stamen development. These results provide genomic resources and insights into spinach dioecious flower development and expedite spinach improvement.

Assessing global-warming induced soil organic matter and iron oxides depletion: Empirical insights into sorption and uptake of atrazine by plants.

Recent pesticide use is alarmingly high and unregulated in several parts of the world. Pesticide fate in soil is controlled by sorption processes which affect the subsequent transport and chemical reactivity in the environment, as well as uptake by plants. Sorption processes are dependent on soil composition and properties, but these are beginning to be affected by global warming-linked factors leading to soil depletion. Thus, it is vital to decipher soils' response, especially in the sub-Sahara (SS), to the depletion of some inherent components in the presence of pesticides. This was ascertained by monitoring a model pesticide (atrazine) sorption and desorption on whole SS soil (WS), and the same soil whose organic matter (OMR) and iron oxides (IOR) were substantially depleted, as well as studying atrazine uptake from these soils by fast-growing vegetables. Organic matter depletion enhanced equilibrium in OMR. Sorption was enhanced at lower ambient pH, higher initial atrazine concentration, and higher temperature. Hysteresis was low resulting in high desorption. Overall, atrazine desorption of ≥65 % was observed; it was higher in OMR (≥95 %) since SOM enhanced hysteresis. Though sub-Saharan soils are rich in iron oxides, SOM played a significantly higher role in sorption than iron oxides in this soil. This result suggests a high potential for atrazine to leach into the aquifer in the sub-Saharan. Atrazine uptake experiment by waterleaf and spinach showed that it could be detected in soil after 63 d, and its presence significantly affected the growth of both vegetables especially in soils with depleted SOM and iron oxides, and at high (100 µg/kg) atrazine spiking. Spinach may be a higher atrazine accumulator than waterleaf. It may be concluded that waterleaf and spinach grown on atrazine-contaminated soils, especially on SOM/iron oxide-depleted soils, are likely to accumulate atrazine.

A longitudinal study on the bacterial quality of baby spinach cultivated in Arizona and California.

In the U.S., baby spinach is mostly produced in Arizona (AZ) and California (CA). Characterizing the impact of growing region on the bacterial quality of baby spinach can inform quality management practices in industry. Between December 2021 and December 2022, baby spinach was sampled after harvest and packaging for microbiological testing, including shelf-life testing of packaged samples that were stored at 4°C. Samples were tested to (i) determine bacterial concentration, and (ii) obtain and identify bacterial isolates. Packaged samples from the Salinas, CA, area (n = 13), compared to those from the Yuma, AZ, area (n = 9), had a significantly higher bacterial concentration, on average, by 0.78 log10 CFU/g (P < 0.01, based on aerobic, mesophilic plate count data) or 0.67 log10 CFU/g (P < 0.01, based on psychrotolerant plate count data); the bacterial concentrations of harvest samples from the Yuma and Salinas areas were not significantly different. Our data also support that an increase in preharvest temperature is significantly associated with an increase in the bacterial concentration on harvested and packaged spinach. A Fisher's exact test and linear discriminant analysis (effect size), respectively, demonstrated that (i) the genera of 2,186 bacterial isolates were associated (P < 0.01) with growing region and (ii) Pseudomonas spp. and Exiguobacterium spp. were enriched in spinach from the Yuma and Salinas areas, respectively. Our findings provide preliminary evidence that growing region and preharvest temperature may impact the bacterial quality of spinach and thus could inform more targeted strategies to manage produce quality. IMPORTANCE: In the U.S., most spinach is produced in Arizona (AZ) and California (CA) seasonally; typically, spinach is cultivated in the Yuma, AZ, area during the winter and in the Salinas, CA, area during the summer. As the bacterial quality of baby spinach can influence consumer acceptance of the product, it is important to assess whether the bacterial quality of baby spinach can vary between spinach-growing regions. The findings of this study provide insights that could be used to support region-specific quality management strategies for baby spinach. Our results also highlight the value of further evaluating the impact of growing region and preharvest temperature on the bacterial quality of different produce commodities.

A high-efficiency transient expression system mediated by Agrobacterium tumefaciens in Spinacia oleracea leaves.

BACKGROUND: Optimization of a highly efficient transient expression system is critical for the study of gene function, particularly in those plants in which stable transformation methods are not widely available. Agrobacterium tumefaciens­mediated transient transformation is a simple and low-cost method that has been developed and applied to a wide variety of plant species. However, the transient expression in spinach (Spinacia oleracea L.) is still not reported. RESULTS: We developed a transient expression system in spinach leaves of the Sp75 and Sp73 varieties. Several factors influencing the transformation efficiency were optimized such as Agrobacterium strain, spinach seedling stage, leaf position, and the expression time after injection. Agrobacterium strain GV3101 (pSoup-p19) was more efficient than AGL1 in expressing recombinant protein in spinach leaves. In general, Sp75 leaves were more suitable than Sp73 leaves, regardless of grow stage. At four-leaf stage, higher intensity and efficiency of transient expression were observed in group 1 (G1) of Sp75 at 53 h after injection (HAI) and in G1 of Sp73 at 64 HAI. At six-leaf stage of Sp75, group 3 (G3) at 72 HAI were the most effective condition for transient expression. Using the optimized expression system, we detected the subcellular localization of a transcriptional co-activator SoMBF1c and a NADPH oxidase SoRbohF. We also detected the interaction of the protein kinase SoCRK10 and the NADPH oxidase SoRbohB. CONCLUSION: This study established a method of highly efficient transient expression mediated by Agrobacterium in spinach leaves. The transient expression system will facilitate the analysis of gene function and lay a solid foundation for molecular design breeding of spinach.

Diaminonaphthalene functionalized LUS-1 as a fluorescence probe for simultaneous detection of Hg2+ and Fe3+ in Vetiver grass and Spinach.

One of the important problems in the environment is heavy metal pollution, and fluorescence is one of the best methods for their detection due to its sensitivity, selectivity, and relatively rapid and easy operation. In this study, 1,8-diaminonaphthalene functionalized super-stable mesoporous silica (DAN-LUS-1) was synthesized and used as a fluorescence probe to identify Hg2+ and Fe3+ in food samples. The TGA and FT-IR spectra illustrated that 1,8-diaminonaphthalene was grafted into LUS-1. XRD patterns verified that the LUS-1 and functionalized mesoporous silica have a hexagonal symmetrical array of nano-channels. SEM images showed that the rod-like morphology of LUS-1 was preserved in DAN-LUS-1. Also, surface area and pore diameter decreased from 824 m2 g⁻1 and 3.61 nm for the pure LUS-1 to 748 m2 g⁻1 and 3.43 nm for the DAN-LUS-1, as determined by N2 adsorption-desorption isotherms. This reduction demonstrated that 1,8-diaminonaphthalene immobilized into the pore of LUS-1. The DAN-LUS-1 fluorescence properties as a chemical sensor were studied with a 340/407 nm excitation/emission wavelength that was quenched by Hg2+ and Fe3+ ions. Hg2+ and Fe3+ were quantified using the fluorescence response in the working range 8.25-13.79 × 10-6 and 3.84-10.71 × 10-6 mol/L, with detection limits of 8.5 × 10-8 M and 1.3 × 10-7 M, respectively. Hg2+ and Fe3+ were measured in vetiver grass and spinach. Since the Fe3+ quenching can move in the opposite direction with sodium hexametaphosphate (SHMP) as a hiding compound for Fe3+, consequently, the circuit logic system was established with Fe3+, Hg2+, and SHMP as inputs and the fluorescent quench as the output.

Impact of nitrogen fertilizer type and application rate on growth, nitrate accumulation, and postharvest quality of spinach.

Background: A balanced supply of nitrogen is essential for spinach, supporting both optimal growth and appropriate nitrate (NO3 -) levels for improved storage quality. Thus, choosing the correct nitrogen fertilizer type and application rate is key for successful spinach cultivation. This study investigated the effects of different nitrogen (N) fertilizer type and application rates on the growth, nitrate content, and storage quality of spinach plants. Methods: Four fertilizer types were applied at five N doses (25, 50, 200, and 400 mg N kg-1) to plants grown in plastic pots at a greenhouse. The fertilizer types used in the experiment were ammonium sulphate (AS), slow-release ammonium sulphate (SRAS), calcium nitrate (CN), and yeast residue (YR). Spinach parameters like Soil Plant Analysis Development (SPAD) values (chlorophyll content), plant height, and fresh weight were measured. Nitrate content in leaves was analyzed after storage periods simulating post-harvest handling (0, 5, and 10 days). Results: The application of nitrogen fertilizer significantly influenced spinach growth parameters and nitrate content. The YRx400 treatment yielded the largest leaves (10.3 ± 0.5 cm long, 5.3 ± 0.2 cm wide). SPAD values increased with higher N doses for AS, SRAS, and CN fertilizers, with AS×400 (58.1 ± 0.8) and SRAS×400 (62.0 ± 5.8) reaching the highest values. YR treatments showed a moderate SPAD increase. Fresh weight response depended on fertilizer type, N dose, and storage period. While fresh weight increased in all fertilizers till 200 mg kg-1 dose, a decrease was observed at the highest dose for AS and CN. SRAS exhibited a more gradual increase in fresh weight with increasing nitrogen dose, without the negative impact seen at the highest dose in AS and CN. Nitrate content in spinach leaves varied by fertilizer type, dose, and storage day. CNx400 resulted in the highest NO3 - content (4,395 mg kg-1) at harvest (Day 0), exceeding the European Union's safety limit. This level decreased over 10 days of storage but remained above the limit for CN on Days 0 and 5. SRAS and YR fertilizers generally had lower NO3 - concentrations throughout the experiment. Storage at +4 °C significantly affected NO3 - content. While levels remained relatively stable during the first 5 days, a substantial decrease was observed by Day 10 for all fertilizers and doses, providing insights into the spinach's nitrate content over a 10-day storage period. Conclusion: For rapid early growth and potentially higher yields, AS may be suitable at moderate doses (200 mg kg-1). SRAS offers a more balanced approach, promoting sustained growth while potentially reducing NO3 - accumulation compared to AS. Yeast residue, with its slow nitrogen release and consistently low NO3 - levels, could be a viable option for organic spinach production.

Nitrogen Mineralization of Selected Organic Materials and Their Combined Effects with Nitrogen Fertilizer on Spinach Yield.

A 2-month incubation study was carried out using two soil types to determine the nitrogen mineralization of different inorganic-organic amendments. The following seven treatments (Ts) were established: T1 = control (no amendment), T2 = 5 g of dry algae per kg of soil (100%DA), T3 = 136 g of agri-mat per kg of soil (100%GAM), T4 = 61 g of ground grass per kg of soil (100%GG), T5 = 0.6 g of N using lime-ammonium nitrate (LAN) + 2.5 g of dry algae (50%DA50NF), T6 = 50%GAM50NF, and T7 = 50%GG50NF. Three samples per treatment were obtained at 0, 3, 7, 15, 30, 45, and 60 days for N mineral determination. A 2-month glasshouse experiment was established afterward with the following five treatments: T1 = control, T2 = 50%DA, T3 = 50%GAM, T4 = 50%GG, and T5 = 100 NF. The results indicate that nitrogen mineralization was significantly higher in organic-inorganic amendments compared with singular organic amendments. The percentage differences ranged from 157% to 195%. The 50%DA treatment increased the spinach yield by 20.6% in sandy loam and 36.5% in loam soil. It is difficult to fully recommend the 50%DA treatment without field-scale evaluation, but it is a promising option to be considered.

Development and evaluation of a vacuum impregnation system for enhancement of biochemical properties of food materials.

Vacuum impregnation is a novel methodology for adding various substances to porous foods. This study aimed to develop a cost effective automate system for vacuum impregnation of food materials to enhance their nutritional, functional and sensory properties depending on the functionality of the impregnation solution. The developed vacuum impregnation system includes a vacuum chamber, vacuum pump and an automation setup for creating and maintaining vacuum conditions, feeding impregnated solutions to the samples and releasing vacuum. Fresh-cut spinach leaves were impregnated with ascorbic acid (AsA) and calcium chloride (Cacl2) (10% concentration) in the setup in order to test the effect of the process on some biochemical properties. Statistical analysis revealed significant effect of vacuum impregnation on the biochemical properties (total soluble solids, total phenolic content, flavonoid content and free radical scavenging activity) and color of spinach leaves during storage up to 4 days. Impregnation process showed significant increase in the total phenolic and flavonoid content of the spinach leaves. Increment up to 78% in antioxidant activity was seen for the uncoated impregnated leaves as compared to 59% activity in untreated samples. Thus, products with desired parameters can be produced with this process with minimal impact on their properties at a lower cost and in a shorter time period.

Enhancing zinc biofortification and mitigating cadmium toxicity in soil-earthworm-spinach systems using different zinc sources.

Cadmium (Cd) pollution poses significant threats to soil organisms and human health by contaminating the food chain. This study aimed to assess the impact of various concentrations (50, 250, and 500 mg·kg-1) of zinc oxide nanoparticles (ZnO NPs), bulk ZnO, and ZnSO4 on morphological changes and toxic effects of Cd in the presence of earthworms and spinach. The results showed that Zn application markedly improved spinach growth parameters (such as fresh weight, plant height, root length, and root-specific surface area) and root morphology while significantly reducing Cd concentration and Cd bioconcentration factors (BCF-Cd) in spinach and earthworms, with ZnO NPs exhibiting the most pronounced effects. Earthworm, spinach root, and shoot Cd concentration decreased by 82.3 %, 77.0 %, and 75.6 %, respectively, compared to CK. Sequential-step extraction (BCR) analysis revealed a shift in soil Cd from stable to available forms, consistent with the available Cd (DTPA-Cd) results. All Zn treatments significantly reduced Cd accumulation, alleviated Cd-induced stress, and promoted spinach growth, with ZnO NPs demonstrating the highest Cd reduction and Zn bioaugmentation efficiencies compared to bulk ZnO and ZnSO4 at equivalent concentrations. Therefore, ZnO NPs offer a safer and more effective option for agricultural production and soil heavy metal pollution management than other Zn fertilizers.

Investigating the carcinogenic and non-carcinogenic health hazards of heavy metal ions in Spinacia oleracea grown in agricultural soil treated with biochar and humic acid.

This study addressed the bioaccumulation and human health risk among the consumption of Spinacia oleracea grown in agricultural soil treated with humic acid (189-2310 ppm) and biochars (0.00-5.10%.wt). The biochars came from two local feedstocks of rice-husk (RH) and sugar-beet-pulp (SBP) pyrolyzed at temperatures 300 and 600 °C. Total concentrations of Cu, Cd, and Ni found in both the soil and biomass/biochar exceeded global safety thresholds. The bioaccumulation levels of HMs in spinach leaves varied, with Fe reaching the highest concentration at 765.27 mg kg-1 and Cd having the lowest concentration at 3.31 mg kg-1. Overall, the concentrations of Zn, Cd, Pb, and Ni in spinach leaves exceeded the safety threshold limits, so that its consumption is not recommended. The assessment of hazard quotient (HI) for the HMs indicated potential health hazards for humans (HI > 1) from consuming the edible parts of spinach. The biochar application rates of 4.35%wt and 0.00%.wt resulted in the highest (3.69) and lowest (3.15) HI values, respectively. The cumulative carcinogenic risk (TCR) ranged from 0.0085 to 0.0119, exceeding the cancer risk threshold. Introducing 5.10%wt biomass/biochar resulted in a 36% rise in TCR compared to the control. The utilization of humic acid alongside HMs-polluted biochars results in elevated levels of HMs bioaccumulation exceeding the allowable thresholds in crops (with a maximum increase of 49% at 2000 ppm humic acid in comparison to 189 ppm). Consequently, this raised the HI by 46% and the TCR by 22%. This study demonstrated that the utilization of HMs-polluted biochars could potentially pose supplementary health hazards. Moreover, it is evident that the utilization of HMs-polluted biochars in treating metal-contaminated soil does not effectively stabilize or reduce pollution.