Impact of chlorine dioxide disinfection of irrigation water on the epiphytic bacterial community of baby spinach and underlying soil

<div><p>The contamination of pathogenic bacteria through irrigation water is a recognized risk factor for fresh produce. Irrigation water disinfection is an intervention strategy that could be applied to reduce the probability of microbiological contamination of crops. Disinfection treatments should be applied ensuring minimum effective doses, which are efficient in inhibiting the microbial contamination while avoiding formation and accumulation of chemical residues. Among disinfection technologies available for growers, chlorine dioxide (ClO₂) represents, after sodium hypochlorite, an alternative disinfection treatment, which is commercially applied by growers in the USA and Spain. However, in most of the cases, the suitability of this treatment has been tested against pathogenic bacteria and low attention have been given to the impact of chemical residues on the bacterial community of the vegetable tissue. The aim of this study was to (i) to evaluate the continual application of chlorine dioxide (ClO₂) as a water disinfection treatment of irrigation water during baby spinach growth in commercial production open fields, and (ii) to determine the subsequent impact of these treatments on the bacterial communities in water, soil, and baby spinach. To gain insight into the changes in the bacterial community elicited by ClO₂, samples of treated and untreated irrigation water as well as the irrigated soil and baby spinach were analyzed using Miseq® Illumina sequencing platform. Next generation sequencing and multivariate statistical analysis revealed that ClO₂ treatment of irrigation water did not affect the diversity of the bacterial community of water, soil and crop, but significant differences were observed in the relative abundance of specific bacterial genera. This demonstrates the different susceptibility of the bacteria genera to the ClO₂ treatment. Based on the obtained results it can be concluded that the phyllosphere bacterial community of baby spinach was more influenced by the soil bacteria community rather than that of irrigation water. In the case of baby spinach, the use of low residual ClO₂ concentrations (approx. 0.25 mg/L) to treat irrigation water decreased the relative abundance of <i>Pseudomonaceae</i> (2.28-fold) and <i>Enterobacteriaceae</i> (2.5-fold) when comparing treated versus untreated baby spinach. Members of these two bacterial families are responsible for food spoilage and foodborne illnesses. Therefore, a reduction of these bacterial families might be beneficial for the crop and for food safety. In general it can be concluded that the constant application of ClO₂ as a disinfection treatment for irrigation water only caused changes in two bacterial families of the baby spinach and soil microbiota, without affecting the major phyla and classes. The significance of these changes in the bacterial community should be further evaluated.</p></div

Impact of chlorine dioxide disinfection of irrigation water on the epiphytic bacterial community of baby spinach and underlying soil - Fig 3

<p><b>Dendrogram generated by UPGMA clustering (A) and metric multidimensional scaling (MDS) (B) based on the Bray-curtis distance from the OTUs abundance matrix of irrigation water (IW), soil (Soil) and baby spinach (Crop).</b> Colors denote the cluster from samples.</p

Species richness (total species), diversity (Shannon, Fisher’s alpha, Simpson and Inverse Simpson indices), and evenness (Pielou’s index) of untreated and ClO₂ treated samples of irrigation water (IW), soil (Soil) and baby spinach (Crop).

<p>Species richness (total species), diversity (Shannon, Fisher’s alpha, Simpson and Inverse Simpson indices), and evenness (Pielou’s index) of untreated and ClO₂ treated samples of irrigation water (IW), soil (Soil) and baby spinach (Crop).</p

Dissimilarity analysis of bacterial community structured in irrigation water (IW), soil (Soil) and baby spinach (Crop).

<p>Dissimilarity analysis of bacterial community structured in irrigation water (IW), soil (Soil) and baby spinach (Crop).</p

Bacteria genera that showed significant differences (P < 0.05) in their relative abundances between untreated and ClO₂ treated samples of irrigation water (IW), soil (Soil) and baby spinach (Crop).

<p>Bacteria genera that showed significant differences (P < 0.05) in their relative abundances between untreated and ClO₂ treated samples of irrigation water (IW), soil (Soil) and baby spinach (Crop).</p

Composition of bacterial phyla and classes of untreated and ClO₂ treated samples.

<p>Percentage of relative abundance of bacterial phyla (A) and classes (B) of untreated and ClO₂ treated samples of irrigation water (IW), soil (Soil) and baby spinach (Crop). Bacterial community is the average of 5 individual samples. Data shown are phyla that comprised at least 1% of the sequences in at least one sample of a given agronomic habitat.</p

Metabolism of Oak Leaf Ellagitannins and Urolithin Production in Beef Cattle

Oak leaves have a high concentration of ellagitannins. These phytochemicals can be beneficial or poisonous to animals. Beef cattle are often intoxicated by oak leaf consumption, particularly after suffering feed restriction. The severity of the poisoning has recently been associated with the ruminal microbiota, as different bacterial populations were found in animals that tolerated oak leaves and in those that showed clinical and pathological signs of toxicity. Intoxication has previously been linked to the production of phenolic metabolites, particularly catechol, phloroglucinol, and resorcinol. This suggested that the microbial metabolism of ellagitannins could also be associated with its tolerance or intoxication in different animals. Therefore, it is essential to understand the metabolism of ellagitannins in cattle. Here we show that ellagitannins are metabolized in the cattle rumen to urolithins. Different urolithins were detected in ruminal fluid, feces, urine, and plasma. Oak leaf ellagitannins declined as they were converted to urolithins, mainly isourolithin A and urolithin B, by the ruminal and fecal microbiota. Urolithin aglycons were observed in rumen and feces, and glucuronide and sulfate derivatives were detected in plasma and urine. Sulfate derivatives were the main metabolites detected in plasma, while glucuronide derivatives were the main ones in urine. The main urolithins produced in cattle were isourolithin A and urolithin B. This is a relevant difference from the monogastric mammals studied previously in which urolithin A was the main metabolite produced. Low molecular weight phenolics of the benzoic, phenylacetic, and phenylpropionic groups and metabolites such as catechol, resorcinol, and related compounds were also detected. There was a large variability in the kinetics of production of these metabolites in individual animals, although they produced similar metabolites in all cases. This large variability could be associated with the large variability in the rumen and intestine microbiota that has previously been observed. Further studies are needed to demonstrate if the efficiency in the metabolism of ellagitannins by the microbiota could explain the differences observed in susceptibility to intoxication by the different animals