The mechanism behind tenuazonic acid-mediated inhibition of plant plasma membrane H+-ATPase and plant growth.

The increasing prevalence of herbicide-resistant weeds has led to a search for new herbicides that target plant growth processes differing from those targeted by current herbicides. In recent years, some studies have explored the use of natural compounds from microorganisms as potential new herbicides. We previously demonstrated that tenuazonic acid (TeA) from the phytopathogenic fungus Stemphylium loti inhibits the plant plasma membrane (PM) H+-ATPase, representing a new target for herbicides. In this study, we further investigated the mechanism by which TeA inhibits PM H+-ATPase and the effect of the toxin on plant growth using Arabidopsis thaliana. We also studied the biochemical effects of TeA on the PM H+-ATPases from spinach (Spinacia oleracea) and A. thaliana (AHA2) by examining PM H+-ATPase activity under different conditions and in different mutants. Treatment with 200 µM TeA-induced cell necrosis in larger plants and treatment with 10 µM TeA almost completely inhibited cell elongation and root growth in seedlings. We show that the isoleucine backbone of TeA is essential for inhibiting the ATPase activity of the PM H+-ATPase. Additionally, this inhibition depends on the C-terminal domain of AHA2, and TeA binding to PM H+-ATPase requires the Regulatory Region I of the C-terminal domain in AHA2. TeA likely has a higher binding affinity toward PM H+-ATPase than the phytotoxin fusicoccin. Finally, our findings show that TeA retains the H+-ATPase in an inhibited state, suggesting that it could act as a lead compound for creating new herbicides targeting the PM H+-ATPase.

Effects of copper sulphate stress on the morphological and biochemical characteristics of Spinacia oleracea and Avena sativa.

Plants are subjected to various biotic and abiotic stresses that significantly impact their growth and productivity. To achieve balanced crop growth and yield, including for leafy vegetables, the continuous application of micronutrient is crucial. This study investigates the effects of different concentrations of copper sulphate (0, 75, 125, and 175 ppm) on the morphological and biochemical features of Spinacia oleracea and Avena sativa. Morphological parameters such as plant height, leaf area, root length, and fresh and dry weights were optimized at a concentration of 75 ppm copper sulfate. At this concentration, chlorophyll a & b levels increased significantly in Spinacia oleracea (462.9 and 249.8 𝜇𝑔/𝑔), and Avena sativa (404.7 and 437.63𝜇𝑔/𝑔). However, carotenoid content and sugar levels in Spinacia oleracea were negatively affected, while sugar content in Avena sativa increased at 125 ppm (941.6 µg/ml). Protein content increased in Spinacia oleracea (75 ppm, 180.3 µg/ml) but decreased in Avena sativa. Phenol content peaked in both plants at 75 ppm (362.2 and 244.5 µg/ml). Higher concentrations (175 ppm) of copper sulfate reduced plant productivity and health. Plants exposed to control and optimal concentrations (75 and 125 ppm) of copper sulpate exhibited the best health and growth compared to those subjected to higher concentrations. Maximum plant height, leaf area, root length, fresh and dry weights were observed at lower concentrations (75 and 125 ppm) of copper sulfate, while higher concentrations caused toxicity. Optimal copper sulfate levels enhanced chlorophyll a, chlorophyll b, total chlorophyll, protein, and phenol contents but inhibited sugar and carotenoid contents in both Spinacia oleracea and Avena sativa. Overall, increased copper sulfate treatment adversely affected the growth parameters and biochemical profiles of these plants.

Microwave seed priming and ascorbic acid assisted phytoextraction of heavy metals from surgical industry effluents through spinach.

The prevalence of inorganic pollutants in the environment, including heavy metals (HMs), necessitates a sustainable and cost-effective solution to mitigate their impacts on the environment and living organisms. The present research aimed to assess the phytoextraction capability of spinach (Spinach oleracea L.), under the combined effects of ascorbic acid (AA) and microwave (MW) irradiation amendments, cultivated using surgical processing wastewater. In a preliminary study, spinach seeds were exposed to MW radiations at 2.45 GHz for different durations (15, 30, 45, 60, and 90 seconds). Maximum germination was observed after the 30 seconds of radiation exposure. Healthy spinach seeds treated with MW radiations for 30 s were cultivated in the sand for two weeks, after which juvenile plants were transferred to a hydroponic system. Surgical industry wastewater in different concentrations (25 %, 50 %, 75 %, 100 %) and AA (10 mM) were provided to both MW-treated and untreated plants. The results revealed that MW-treatment significantly enhanced the plant growth, biomass, antioxidant enzyme activities and photosynthetic pigments, while untreated plants exhibited increased reactive oxygen species (ROS) and electrolyte leakage (EL) compared with their controls. The addition of AA to both MW-treated and untreated plants improved their antioxidative defense capacity under HMs-induced stress. MW-treated spinach plants, under AA application, demonstrated relatively higher concentrations and accumulation of HMs including lead (Pb), cadmium (Cd) and nickel (Ni). Specifically, MW-treated plants with AA amendment showed a significant increase in Pb concentration by 188 % in leaves, Cd by 98 %, and Ni by 102 % in roots. Additionally, the accumulation of Ni increased by 174 % in leaves, Cd by 168 % in roots, and Pb by 185 % in the stem of spinach plant tissues compared to MW-untreated plants. These findings suggested that combining AA with MW irradiation of seeds could be a beneficial strategy for increasing the phytoextraction of HMs from wastewater and improving overall plant health undergoing HMs stress.

Gene Regulatory Network Controlling Flower Development in Spinach (Spinacia oleracea L.).

Spinach (Spinacia oleracea L.) is a dioecious, diploid, wind-pollinated crop cultivated worldwide. Sex determination plays an important role in spinach breeding. Hence, this study aimed to understand the differences in sexual differentiation and floral organ development of dioecious flowers, as well as the differences in the regulatory mechanisms of floral organ development of dioecious and monoecious flowers. We compared transcriptional-level differences between different genders and identified differentially expressed genes (DEGs) related to spinach floral development, as well as sex-biased genes to investigate the flower development mechanisms in spinach. In this study, 9189 DEGs were identified among the different genders. DEG analysis showed the participation of four main transcription factor families, MIKC_MADS, MYB, NAC, and bHLH, in spinach flower development. In our key findings, abscisic acid (ABA) and gibberellic acid (GA) signal transduction pathways play major roles in male flower development, while auxin regulates both male and female flower development. By constructing a gene regulatory network (GRN) for floral organ development, core transcription factors (TFs) controlling organ initiation and growth were discovered. This analysis of the development of female, male, and monoecious flowers in spinach provides new insights into the molecular mechanisms of floral organ development and sexual differentiation in dioecious and monoecious plants in spinach.

High and low oxalate content in spinach: an investigation of accumulation patterns.

BACKGROUND: Oxalic acid is a common antinutrient in the human diet, found in large quantities in spinach. However, spinach is highly regarded by vegetable producers because of its nutritional content and economic value. One of the primary purposes of spinach-breeding programs is to improve the nutritional value of spinach by adjusting oxalate accumulation. Knowledge of the biosynthetic patterns of oxalic acid, and its different forms, is important for a better understanding of this process. RESULTS: We found three biosynthetic patterns of accumulation and concentration of oxalates. Two of them are related to the maximum type and one is related to the minimum type. We also developed a general model of variations in these compounds in the genotypes that were studied. CONCLUSION: This study introduced a unique type of spinach with high oxalate accumulation, which could be particularly suitable for consumption. This had the highest ratio of insoluble oxalate to soluble oxalate. It also accumulated more ascorbic acid (AA) than other types. Our findings in this study also indicate a small role for AA as a precursor to oxalate production in spinach, possibly confirming the significant role of glyoxylate as the most critical precursor in this plant. © 2021 Society of Chemical Industry.

Auxin regulated metabolic changes underlying sepal retention and development after pollination in spinach.

BACKGROUND: Pollination accelerate sepal development that enhances plant fitness by protecting seeds in female spinach. This response requires pollination signals that result in the remodeling within the sepal cells for retention and development, but the regulatory mechanism for this response is still unclear. To investigate the early pollination-induced metabolic changes in sepal, we utilize the high-throughput RNA-seq approach. RESULTS: Spinach variety 'Cornel 9' was used for differentially expressed gene analysis followed by experiments of auxin analog and auxin inhibitor treatments. We first compared the candidate transcripts expressed differentially at different time points (12H, 48H, and 96H) after pollination and detected significant difference in Trp-dependent auxin biosynthesis and auxin modulation and transduction process. Furthermore, several auxin regulatory pathways i.e. cell division, cell wall expansion, and biogenesis were activated from pollination to early developmental symptoms in sepals following pollination. To further confirm the role auxin genes play in the sepal development, auxin analog (2, 4-D; IAA) and auxin transport inhibitor (NPA) with different concentrations gradient were sprayed to the spinach unpollinated and pollinated flowers, respectively. NPA treatment resulted in auxin transport weakening that led to inhibition of sepal development at concentration 0.1 and 1 mM after pollination. 2, 4-D and IAA treatment to unpollinated flowers resulted in sepal development at lower concentration but wilting at higher concentration. CONCLUSION: We hypothesized that sepal retention and development might have associated with auxin homeostasis that regulates the sepal size by modulating associated pathways. These findings advanced the understanding of this unusual phenomenon of sepal growth instead of abscission after pollination in spinach.

Fine mapping and molecular marker development of the Fs gene controlling fruit spines in spinach (Spinacia oleracea L.).

KEY MESSAGE: The Fs gene, which controls spinach fruit spines, was fine mapped to a 0.27 Mb interval encompassing four genes on chromosome 3. There are two types of fruit of spinach (Spinacia oleracea L.), spiny and spineless, which are visually distinguishable by the spines of fruit coat. In spinach breeding, the fruit characteristic is an important agronomic trait that have impacts on "seed" treatment and mechanized sowing. However, the gene(s) controlling the fruit spiny trait have not been characterized and the genetic mechanism of this trait remained unclear. The objectives of the study were to fine map the gene controlling fruit spines and develop molecular markers for marker-assisted selection purpose. Genetic analysis of the spiny trait in segregating populations indicated that fruit spines were controlled by a single dominant gene, designated as Fs. Using a super-BSA method and recombinants analysis in a BC1 population, Fs was mapped to a 1.9-Mb interval on chromosome 3. The Fs gene was further mapped to a 0.27-Mb interval using a recombinant inbred line (RIL) population with 120 lines. From this 0.27 Mb region, four candidate genes were identified in the reference genome. The structure and expression of the four genes were compared between the spiny and spineless parents. A co-dominant marker YC-15 was found to be co-segregating with the fruit spines trait, which produced a 129-bp fragment specific to spiny trait and a 108-bp fragment for spineless fruit. This marker can predict spiny trait with a 94.8% accuracy rate when tested with 100 diverse germplasm, suggesting that this marker would be valuable for marker-assisted selection in spinach breeding.

Insight into morphological and molecular variations across Iranian spinach landraces.

Spinach is salt-tolerant leafy vegetable stemmed from central Asia. It has been well adapted to different climates of Iran. We aimed to study genetic diversity between several landraces as a prerequisite for crop improvement programs using molecular markers including microsatellites (SSRs) and morphological traits. Genetic diversity was studied among 22 spinach landraces using morphological and molecular tools. We developed 17 genic and genomic SSR markers based on the information acquired from NCBI resources. Morphological evaluation indicated high variability for economic traits including leaf color, leaf thickness, leaf wrinkle and seed type, across Iranian landraces. The molecular results exhibited that 12 out of 17 primer pairs successfully amplified genomic DNA with explicit bands. The results verified that genic markers were superior to genomic markers to detect polymorphism and genetic diversity. In this regard, PIC for genomic and genic SSRs was in the range of 0.4616-0.6621 and 0.5188-0.7394, respectively. Polymorphic genic SSRs were identified to be directly and indirectly involved in biotic/abiotic stresses. High degree of polymorphism, which was detected across the landraces by genic SSRs, could assist us to select 11 landraces for the second experiment. The second experiment was designed to evaluate the response of selected landraces to salinity stress. The results confirmed genetic variability among the landraces in terms of salinity tolerance. A highly diverse germplasm of Iranian spinach based on molecular and morphological characteristics along with the tolerance to oxidative stress provides an ample opportunity for plant breeders to select superior genotypes.

Prospective usage of magnesium potassium phosphate cement combined with Bougainvillea alba derived biochar to reduce Pb bioavailability in soil and its uptake by Spinacia oleracea L.

Combining biochar (BR) with other immobilizing amendments has additive effects on Pb immobilization and been recognized to be effective for the restoration of Pb polluted soils. However, the impacts of different proportions between BR and a highly efficient Pb immobilizing agent called "magnesium potassium phosphate cement (MC)" have never been earlier investigated. This work aimed to investigate the consequences of BR and MC alone and their mixtures of 25:75, 50:50, and 75:25 ratios on Pb bioavailability, Pb immobilization index (Pb-IMMi), and enzymatic activities in Pb polluted soil. Furthermore, amendments effects on Pb distribution in spinach, growth, antioxidant capacity, biochemical, and nutritional spectrum were also investigated. We found that MC alone performed well to immobilize Pb in soil and reducing its distribution in shoots, but was less efficient to improve soil enzymatic activities and plant attributes. Conversely, the application of BR alone stimulated soil enzymatic activities, plant growth, and quality but was less effective to immobilize Pb in soil and reducing shoot Pb concentrations. The combinations of BR and MC of various ratios showed variable results. Interestingly, the most promising outcomes were obtained with BR50%+MC50% treatment which resulted in enhanced Pb-IMMi (73%), activities of soil enzymes, plant growth and quality, and antioxidant capacity, compared to control. Likewise, significant reductions in Pb concentrations in shoots (85%), roots (78%), extractable Pb (73%) were also obtained with BR50%+MC50% treatment, compared to control. Such outcomes point towards a cost-effective approach for reducing Pb uptake by the plants via using MC and BR at a 50:50 ratio.

Effect of salt stress on the growth, mineral contents, and metabolite profiles of spinach.

BACKGROUND: Increased soil salt concentration decreases productivity and changes the physiological and chemical properties of plants. Various omics technologies have been used to understand the salt response in plants but overall changes in the metabolite profiles of spinach (Spinacia oleracea L.) under salt stress have not been studied. In this article, therefore, the changes in mineral and metabolite profiles of spinach plants cultivated with different NaCl concentrations of 0-200 mmol L-1 in the irrigation water were analyzed to investigate the effect of salt stress on nutritional quality. RESULTS: Increasing NaCl concentration decreased plant growth due to mineral imbalance. The amounts of minerals (K+ , Ca2+ , and Fe2+ ) were reduced with increasing NaCl concentration, resulting in altered ratios of Na+ :K+ and Na+ :Ca2+ . The change in the mineral ratios due to NaCl irrigation led to a decrease in the height and an increase in the weight of spinach. Moreover, the profiles of 32 metabolites, including flavonoids, amino acids, acidic compounds, sugars, and lipid-related compounds, were altered by NaCl irrigation; most of them showed decreased levels. In particular, at 200 mmol L-1 NaCl, the levels of sucrose, glutamic acid, hexose sugars, and acidic compounds significantly decreased upon NaCl irrigation. Based on these metabolites, a salt-stress-related spinach metabolomic pathway was proposed. CONCLUSION: Sodium chloride irrigation increased mineral imbalance, resulting in decreased plant growth, and the levels of most metabolites involved in energy production, sensory quality, and health benefits decreased with NaCl irrigation. The results suggest that NaCl irrigation negatively affects the nutritional quality of spinach. © 2020 Society of Chemical Industry.

Yield advantage and cadmium decreasing of rice in intercropping with water spinach under moisture management.

Rice (Oryza sativa L.) intercropping with water spinach (Ipomoea aquatica Forsk) is an effective agricultural practice for safe crop production and for phytoremediation in cadmium-contaminated soil. A field and pot experiment were conducted to investigate the growth and cadmium absorption of rice intercropped with water spinach under different moisture management schemes (continuous flooding, interval flooding, and 75% field capacity). In the field experiment, the concentration of Cd in the grain of rice was significantly lower in the intercropping system than that permitted by the National Food Safety Standard of China (GB 2762-2017). Furthermore, the land equivalent ratio (1.42) was higher in the rice-water spinach intercropping system, indicating a significant advantage of the intercropping system in yield. At the same time, the bio-concentration amount (BCA) of Cd of rice and water spinach in intercropping system significantly increased by 17.99% and 31.98%, respectively (P＜0.05). However, the metal removal equivalent ratio (MRER) of Cd was 1.34, which showed the intercropping system of rice-water spinach had advantage in Cd removal. In the pot experiment, the total iron plaque concentration on the root surface of rice and the pH of the rhizosphere soil were higher under continuous flooding (TCF) than under the control conditions (75% field capacity, TCK), which could significantly decrease the available Cd in the rhizosphere soil and the accumulation of Cd in rice organs. So, this study demonstrated that iron plaque can obstruct and decrease the Cd absorbed by rice in a rice-water spinach intercropping system combined with water management. The intercropping rice with water spinach can achieve the goal of remediation while producing for farmland contaminated by Cd.

Biochar counteracts nitrification inhibitor DMPP-mediated negative effect on spinach (Spinacia oleracea L.) growth.

The use of nitrification inhibitors (NIs) such as 3,4-dimethylpyrazole phosphate (DMPP) has been suggested to diminish agricultural soil nitrate (NO3-) loss and increase nitrogen (N) use efficiency (NUE). However, the yield of ammonium (NH4+)-sensitive plants such as spinach (Spinacia oleracea L.) may be adversely affected by the application of NIs at high N levels and, on the other hand, the efficiency of the NIs may also be affected by soil amendments such as biochar. These two issues are still not adequately addressed. The aim of this study was to evaluate the effect of different N levels including DMPP or not in a calcareous soil with and without amendment of wheat straw biochar on spinach yield, NUE, nitrate concentration of spinach leaf, activity of enzymes nitrate reductase (NR) and nitrite reductase (NiR), and soil ammonium (NH4+) and NO3- concentration under greenhouse conditions. This experiment was carried out with different N rates factor at seven levels (un-fertilized, N0; fertilized with 50 mg N kg-1 soil, N50; fertilized with 75 mg N kg-1 soil, N75; fertilized with 100 mg N kg-1 soil, N100; fertilized with N50 + DMPP; fertilized with N75 + DMPP; and fertilized with N100 + DMPP) and biochar (BC) factor at two levels (0, 0%BC; and 2% (w/w), 2%BC) with six replications over a 56-day cultivation period of spinach. Results showed that the application of DMPP had no significant effect on the yield of spinach plant at low and medium levels of N (50 and 75 mg N kg-1 soil), but decreased the yield of this plant at the higher level of N (100 mg N kg-1 soil). However, application of BC decreased the negative effect of DMPP on spinach yield as the yield in spinach plants fertilized with N75 + DMPP and N100 + DMPP significantly increased. Both application of DMPP and addition of BC to soil decreased leaf NO3- concentration by 29.2% and 16.3% compared to control, respectively. Biochar compared to control decreased NR activity by 46.3%. With increasing N rate, NR and NiR activities increased, but DMPP decreased the activities of both enzymes. Biochar reduced the efficiency of DMPP as soil NH4+ concentration was higher in the treatments containing DMPP without BC at 56 days after planting. Biochar and DMPP could increase the quality of spinach plant through decreasing the leaf NO3- concentration. In general, wheat straw biochar counteracted DMPP-mediated negative effect on growth of spinach plant at high level of N by decreasing the efficiency of this inhibitor. These results provide the useful information for managing the application rate of N fertilizers including DMPP in biochar-amended soil.

Composts of poultry litter or dairy manure differentially affect survival of enteric bacteria in fields with spinach.

AIMS: The aim was to determine the survival and persistence of Escherichia coli in soil amended with compost from different manure sources. METHOD AND RESULTS: Complex interactions of abiotic and biotic factors on E. coli survival were characterized in field experiment plots receiving randomly assigned compost treatments: dairy windrow, dairy vermicompost, poultry windrow or no compost. Biomass, activity and function of indigenous microbial communities in the composts and soils were measured concurrently to determine whether mechanisms of compost were driven by biotic or abiotic properties. E. coli persisted in compost containing poultry amendments but not in composts containing dairy or no amendments. Poultry compost contained more NH4 -N and a distinct microbial community compared to dairy and no compost treatments. A laboratory experiment performed on compost extracts suggested that E. coli survived better in extracts devoid of indigenous microbes as long as bioavailable nutrients were plentiful. CONCLUSIONS: Dairy-based composts are less likely to support E. coli survival than poultry-based composts. SIGNIFICANCE AND IMPACT OF THE STUDY: Results aid in risk assessment of the use of different types of manure-based compost and soil amendments in fruit and vegetable production by elucidating the roles of nutrient and microbial community composition on survival of E. coli in amended field soils.

Metal(loid) induced toxicity and defense mechanisms in Spinacia oleracea L.: Ecological hazard and Prospects for phytoremediation.

A pot study was conducted to assess the phytoremediation potential of Spinach plants along with their physiological and biochemical response when grown in soil contaminated with heavy metal(loid)s (HMs). Plants were grown under different doses of Pb, Cd and As; and their metal(loid) accumulation efficiency was studied upon harvest; expressed in terms of bioabsorption coefficient (BAC), bioconcentration factor (BCF) and translocation factor (TF). Results showed significant (p ≤ 0.05) difference in physiological and biochemical mechanisms of plants as detected through decrease in concentration of cellular constituents (pigments, carbohydrates, total nitrogen content); and increase in antioxidants (both enzymatic and non-enzymatic). Despite of accumulating high amount of HMs in tissues, no visible signs of toxicity were seen; and hence the efficient survival and defense mechanism shown by spinach plants conclude that they are a viable option to be used for phytoremediation of sites contaminated with Cd and Pb. Since the content of Cd and Pb in edible part was higher than safe limits prescribed by USEPA, the present investigation also highlights the ecological hazards that may result upon cultivation of spinach in contaminated soil for agricultural purpose; or its accidental exposure to food chain when grown for phytoremediation.

Survival of Escherichia coli O157:H7 after application of lactic acid bacteria.

BACKGROUND: Establishing novel preharvest intervention strategies for leafy green growers is of critical need with the rise in foodborne outbreaks associated with these products. Recent studies have shown that lactic acid bacteria (LAB) are able to reduce the presence of Escherichia coli O157:H7 in various food matrices. Electrostatic application of organic acids has been shown to be effective as a postharvest safety intervention to reduce E.coli O157:H7 on leafy greens. The effect of LAB electrostatically applied and sprinkler irrigated once over a 4 week growth cycle was evaluated against E. coli O157:H7 on spinach. RESULTS: The results indicated that E. coli O157:H7 when applied once during the 4 week growth cycle will survive in the soil and spinach leaves at harvest. LAB applied electrostatically and by sprinkler irrigation water on the soil and/or leaf surface within the first 4 weeks of the growing cycle resulted in a significant reduction (almost a 3 log10 reduction) of E. coli O157:H7 both on the leaf and in the soil at harvest, regardless of the application time (P < 0.01). CONCLUSION: LAB surface treatments have the potential to improve the safety of leafy green plants as a preharvest food safety intervention when combined with good agricultural practices. © 2018 Society of Chemical Industry.

Chromium toxicity mediated by application of chloride and sulfate ions in Vertisol of Central India.

Chromium (Cr) is one of the toxic metals adversely affecting organisms including humans in the ecosystems, and it is present in considerable concentration in the tannery industrial effluent. Toxicity expression of Cr is suspected to be influenced considerably by other accompanying ions present in the effluent used for irrigation. In a screen house experiment, interactive effects of chloride and sulfate ions in a Vertisol on uptake of Cr by spinach crop were investigated and treatments imposed were three levels each of Cr (0, 50, 100 mg kg-1), chloride (Cl-) (0, 25, 50 mM kg-1), and sulfur (S) (0, 4, 8 mM kg-1) in possible combinations. Plant growth parameters and leaf Cr concentrations were recorded to find out the effect of anions on Cr dynamics in the plant. Increasing the concentration of Cl- ions in soil reduced the Cr concentration in both root and shoot. Similarly, increasing the concentration of S from 4 to 8 mM kg-1 also reduced the concentration and uptake of Cr. Application of sulfate ions augmented the plant growth and counters the negative effect of Cl- ions and Cr. Thus, the study revealed that the addition of S fertilizers could minimize the Cr toxicity in high Cr contaminated soils.

Quantitative microbial exposure modelling as a tool to evaluate the impact of contamination level of surface irrigation water and seasonality on fecal hygiene indicator E. coli in leafy green production.

The use of Quantitative Microbial Exposure Assessment (QMEA) modelling of faecal hygiene indicator microorganisms (e.g. E. coli), is proposed as an alternative approach to the use of Quantitative Microbiological Risk Assessment (QMRA) models of enteric pathogenic microorganisms in the fresh produce chain. As more field data and quantitative microbial models become available, the potential use of QMEA models as a tool to assess the impact of different risk mitigation strategies increases helping growers to make the right decisions. This paper focuses on the pros and cons of previously published QMRAs as well as on the proposal of an alternative approach based on the use of a quantitative microbial contamination modelling to investigate how the selection of the irrigation water sources affect the E. coli loads in leafy greens at harvest. The modified model describes the final E. coli levels of baby spinach when different water sources with different contamination levels are applied and the impact of seasonality. Substantial differences were observed between the irrigation water sources while seasonality only had small effects on the simulated levels of E. coli in the harvested baby spinach. Based on the results, the produce grown using irrigation water from drainage ditches show E. coli levels above threshold levels (2 log CFU/g) while less than 1% of baby spinach irrigated with water obtained from water reservoirs where above this limit. The use of this QMEA model will help growers in the decision-making process to reduce microbial contamination of leafy greens.

Effect of harvest time on the levels of phytochemicals, free radical-scavenging activity, α-amylase inhibition and bile acid-binding capacity of spinach (Spinacia oleracea).

BACKGROUND: Spinach is a green leafy vegetable that is rich in health-promoting compounds. The present study analyzed the levels of phytochemicals and health-promoting properties of spinach harvested at 20, 30, 40, 50 and 60 days after planting. RESULTS: The time of harvest had a significant effect on nitrate levels, which increased from 1909 ± 70.6 µg g-1 (20 days) to 3668 ± 101.3 µg g-1 (40 days) and then decreased to 974 ± 164 µg g-1 (60 days). Lutein and chlorophylls a and b were found to be maximum at 60 days, whereas ß-carotene was higher at 50 days. Liquid chromatography/high-resolution quadrupole time-of-flight tandem mass spectrometry (LC/HR-QTOF-MS) was used to identify 12 flavonoids, and their tentative fragmentation pathways have been proposed. Spinach harvested at 30 and 60 days exhibited significantly higher 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical-scavenging activities and inhibition of amylase. The levels of total phenolics ranged from 885 ± 35.1 to 1162 ± 112.4 µg g-1 in the samples. In vitro bile acid-binding capacity showed that glycochenodeoxycholate and glycodeoxycholate were bound to maximum levels in all spinach samples. CONCLUSION: The harvest time has a major effect on the levels of phytochemicals and health-beneficial properties, which indicates that consumption of both baby and mature spinach will provide maximum health benefits. © 2017 Society of Chemical Industry.

Demonstration tests of irrigation water disinfection with chlorine dioxide in open field cultivation of baby spinach.

BACKGROUND: Treatments for the disinfection of irrigation water have to be evaluated by demonstration tests carried out under commercial settings taking into account not only their antimicrobial activity but also the potential phytotoxic effects on the crop. The consequences of the treatment of irrigation water with chlorine dioxide (ClO2 ) used for sprinkler irrigation of baby spinach in two commercial agricultural fields was assessed. RESULTS: Residual ClO2 levels at the sprinklers in the treated field were always below 1 mg L-1 . ClO2 treatment provoked limited but statistically significant reductions in culturable Escherichia coli counts (0.2-0.3 log reductions), but not in the viable E. coli counts in water, suggesting the presence of viable but non-culturable cells (VBNC). Although disinfected irrigation water did not have an impact on the microbial loads of Enterobacteriaceae nor on the quality characteristics of baby spinach, it caused the accumulation of chlorates (up to 0.99 mg kg-1 in plants) and the reduction of the photosynthetic efficiency of baby spinach. CONCLUSION: Low concentrations of ClO2 are effective in reducing the culturable E. coli present in irrigation water but it might induce the VBNC state. Presence of disinfection by-products and their accumulation in the crop must be considered to adjust doses in order to avoid crop damage and chemical safety risks. © 2017 Society of Chemical Industry.

Genetic diversity and association mapping of mineral element concentrations in spinach leaves.

BACKGROUND: Spinach is a useful source of dietary vitamins and mineral elements. Breeding new spinach cultivars with high nutritional value is one of the main goals in spinach breeding programs worldwide, and identification of single nucleotide polymorphism (SNP) markers for mineral element concentrations is necessary to support spinach molecular breeding. The purpose of this study was to conduct a genome-wide association study (GWAS) and to identify SNP markers associated with mineral elements in the USDA-GRIN spinach germplasm collection. RESULTS: A total of 14 mineral elements: boron (B), calcium (Ca), cobalt (Co), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), molybdenum (Mo), sodium (Na), nickel (Ni), phosphorus (P), sulfur (S), and zinc (Zn) were evaluated in 292 spinach accessions originally collected from 29 countries. Significant genetic variations were found among the tested genotypes as evidenced by the 2 to 42 times difference in mineral concentrations. A total of 2402 SNPs identified from genotyping by sequencing (GBS) approach were used for genetic diversity and GWAS. Six statistical methods were used for association analysis. Forty-five SNP markers were identified to be strongly associated with the concentrations of 13 mineral elements. Only two weakly associated SNP markers were associated with K concentration. Co-localized SNPs for different elemental concentrations were discovered in this research. Three SNP markers, AYZV02017731_40, AYZV02094133_57, and AYZV02281036_185 were identified to be associated with concentrations of four mineral components, Co, Mn, S, and Zn. There is a high validating correlation coefficient with r > 0.7 among concentrations of the four elements. Thirty-one spinach accessions, which rank in the top three highest concentrations in each of the 14 mineral elements, were identified as potential parents for spinach breeding programs in the future. CONCLUSIONS: The 45 SNP markers strongly associated with the concentrations of the 13 mineral elements: B, Ca, Co, Cu, Fe, Mg, Mn, Mo, Na, Ni, P, S, and Zn could be used in breeding programs to improve the nutritional quality of spinach through marker-assisted selection (MAS). The 31 spinach accessions with high concentrations of one to several mineral elements can be used as potential parents for spinach breeding programs.