Plant growth-promoting bacteria as affected by N availability as a suitable strategy to enhance the nutritional composition of lamb's lettuce affected by global warming.

Heat and nutritional stresses have a significantly effect on the accumulation of bioactive and other compounds harmful to human health, like nitrates, in green leafy vegetables like lamb's lettuce. Plant growth-promoting bacteria (PGPB) have shown to confer beneficial biochemical changes to various crops under different stresses. The hypothesis proposed here is that the combination of optimal N level (2.5 Mm, 12 mM or 20 mM of N) with the inoculation of PGPB in plants exposed to heat shock (43 °C) may be a good strategy to obtain healthier lamb's lettuce with a higher yield. Results showed that a dose of 20 mM N can be considered as overfertilization. Moreover, the inoculation of plants fed with fertilizers with reduced N and under heat stress, resulted in higher productivity and content of sugars (60 %), amino acids (94 %), nitrogen (21 %), and total phenolic compounds (30 %), and a reduced content of nitrates (27 %).

Persistence and survival of Cryptosporidium parvum oocysts on lamb's lettuce leaves during plant growth and in washing conditions of minimally-processed salads.

Cryptosporidium is the causative agent of cryptosporidiosis, which results, among others, in profuse diarrhoea. Transmission to humans occurs via the faecal-oral route directly by contact with infected hosts or indirectly by waterborne or foodborne routes. For the latter, parasite transmission is closely linked to the oocyst's ability to persist and survive in food matrices. In this study, we evaluated the persistence and survival of Cryptosporidium oocysts in lamb's lettuce: i) during plant growth and ii) in conditions mimicking the industrial washing process applied in minimally-processed vegetables (MPV). Results show that oocysts persisted during the growth of lamb's lettuce, i.e. two months from the 2-leaf stage until the 8-leaf harvest time (-0.89 Log10 of oocysts). However, their survival decreased from as early as one week (-0.61 Log10), and only 6 % of oocysts remained infective at the time of harvest. The washing process had a limited effect on parasite load (<0.5 Log10) and no effect on survival; chlorination of washing water did not improve the efficiency (removal and inactivation) of the process. The ability of C. parvum to persist and survive throughout the food chain may drive its transmission to humans through MPV products. Appropriate management measures should be implemented at each operational level to limit contamination and ensure food safety of fresh produce.

Calcium Phosphate Particles Coated with Humic Substances: A Potential Plant Biostimulant from Circular Economy.

Nowadays, the use of biostimulants to reduce agrochemical input is a major trend in agriculture. In this work, we report on calcium phosphate particles (CaP) recovered from the circular economy, combined with natural humic substances (HSs), to produce a plant biostimulant. CaPs were obtained by the thermal treatment of Salmo salar bones and were subsequently functionalized with HSs by soaking in a HS water solution. The obtained materials were characterized, showing that the functionalization with HS did not sort any effect on the bulk physicochemical properties of CaP, with the exception of the surface charge that was found to get more negative. Finally, the effect of the materials on nutrient uptake and translocation in the early stages of development (up to 20 days) of two model species of interest for horticulture, Valerianella locusta and Diplotaxis tenuifolia, was assessed. Both species exhibited a similar tendency to accumulate Ca and P in hypogeal tissues, but showed different reactions to the treatments in terms of translocation to the leaves. CaP and CaP-HS treatments lead to an increase of P accumulation in the leaves of D. tenuifolia, while the treatment with HS was found to increase only the concentration of Ca in V. locusta leaves. A low biostimulating effect on both plants' growth was observed, and was mainly scribed to the low concentration of HS in the tested materials. In the end, the obtained material showed promising results in virtue of its potential to elicit phosphorous uptake and foliar translocation by plants.

Factors Required for Adhesion of Salmonella enterica Serovar Typhimurium to Corn Salad (Valerianella locusta).

Salmonella enterica is a foodborne pathogen often leading to gastroenteritis and is commonly acquired by consumption of contaminated food of animal origin. However, frequency of outbreaks linked to the consumption of fresh or minimally processed food of nonanimal origin is increasing. New infection routes of S. enterica by vegetables, fruits, nuts, and herbs have to be considered. This leads to special interest in S. enterica interactions with leafy products, e.g., salads, that are mainly consumed in a minimally processed form. The attachment of S. enterica to salad is a crucial step in contamination, but little is known about the bacterial factors required and mechanisms of adhesion. S. enterica possesses a complex set of adhesive structures whose functions are only partly understood. Potentially, S. enterica may deploy multiple adhesive strategies for adhering to various salad species and other vegetables. In this study, we systematically analyzed the contributions of the complete adhesiome, of lipopolysaccharide (LPS), and of flagellum-mediated motility of S. enterica serovar Typhimurium (STM) in adhesion to Valerianella locusta (corn salad). We deployed a reductionist, synthetic approach to identify factors involved in the surface binding of STM to leaves of corn salad, with particular regard to the expression of all known adhesive structures, using the Tet-on system. This work reveals the contribution of Saf fimbriae, type 1 secretion system-secreted BapA, an intact LPS, and flagellum-mediated motility of STM in adhesion to corn salad leaves.IMPORTANCE Transmission of gastrointestinal pathogens by contaminated fresh produce is of increasing relevance to human health. However, the mechanisms of contamination of, persistence on, and transmission by fresh produce are poorly understood. We investigated the contributions of the various adhesive structures of STM to the initial event in transmission, i.e., binding to the plant surface. A reductionist system was used that allowed experimentally controlled surface expression of individual adhesive structures and analyses of the contribution to binding to leave surfaces of corn salad under laboratory conditions. The model system allowed the determination of the relative contributions of fimbrial and nonfimbrial adhesins, the type 3 secretion systems, the O antigen of lipopolysaccharide, the flagella, and chemotaxis of STM to binding to corn salad leaves. Based on these data, future work could reveal the mechanism of binding and the relevance of interaction under agricultural conditions.

Ready-to-Eat Salad Crops: A Plant Pathogen's Heaven.

The ready-to-eat salad sector, also called fresh-cut or bagged salads, is a fast-growing segment of the fresh-food industry. The dynamism and specialization of this sector, together with the lack of adequate crop rotation, the globalization of the seed market, and climate change, are the main causes of the development of many new diseases that cause severe production losses. Newly detected diseases of the most important crops grown (lettuce, wild and cultivated rocket, lamb's lettuce, chicory, endive, basil, spinach, and Swiss chard) are critically discussed. The management of these diseases represents a formidable challenge, since few fungicides are registered on these minor-use crops. An interesting feature of the ready-to-eat salad sector is that most crops are grown under protection, often in soilless systems, which provide an environment helpful to the implementation of innovative control methods. Current trends in disease management are discussed, with special focus on the most sustainable practices.

Adherence factors of enterohemorrhagic Escherichia coli O157:H7 strain Sakai influence its uptake into the roots of Valerianella locusta grown in soil.

Increasing numbers of outbreaks caused by enterohemorrhagic Escherichia coli (EHEC) are associated with the consumption of contaminated fresh produce. The contamination of the plants may occur directly on the field via irrigation water, surface water, manure or fecal contamination. Suggesting a low infectious dose of 10 to 102 cells, internalization of EHEC into plant tissue presents a serious public health threat. Therefore, the ability of EHEC O157:H7 strain Sakai to adhere to and internalize into root tissues of the lamb's lettuce Valerianella locusta was investigated under the environmental conditions of a greenhouse. Moreover, the influence of the two adherence and colonization associated genes hcpA and iha was surveyed regarding their role for attachment and invasion. Upon soil contamination, the number of root-internalized cells of EHEC O157:H7 strain Sakai exceeded 102 cfu/g roots. Deletion of one or both of the adherence factor genes did not alter the overall attachment of EHEC O157:H7 strain Sakai to the roots, but significantly reduced the numbers of internalized bacteria by a factor of between 10 and 30, indicating their importance for invasion of EHEC O157:H7 strain Sakai into plant roots. This study identified intrinsic bacterial factors that play a crucial role during the internalization of EHEC O157:H7 strain Sakai into the roots of Valerianella locusta grown under the growth conditions in a greenhouse.

Biological and Genetic Characterization of New and Known Necroviruses Causing an Emerging Systemic Necrosis Disease of Corn Salad (Valerianella locusta) in France.

An emerging systemic necrosis disease of corn salad was first observed in the Nantes region of France in the late 2000s. Classical virology and high-throughput sequencing approaches demonstrated that the disease is associated with four different necroviruses: tobacco necrosis virus A (TNVA), tobacco necrosis virus D (TNVD), olive mild mosaic virus (OMMV), and a novel recombinant Alphanecrovirus for which the name corn salad necrosis virus (CSNV) is proposed. Satellite tobacco necrosis virus was also frequently observed. Koch's postulates were completed for all four agents, each one alone being able to cause systemic necrosis of varying severity in corn salad. OMMV was the most frequently observed virus and causes the most severe symptoms. TNVA was the second, both in terms of prevalence and symptom severity while TNVD and CSNV were only rarely observed and caused the less severe symptoms. The emergence of this systemic disease may have been favored by the short and repeated cropping cycles used for corn salad, possibly allowing the selection of necrovirus isolates with an improved ability to systemically invade this specialty crop.

Phytochemical Composition and Antitumor Activities of New Salad Greens: Rucola (Diplotaxis tenuifolia) and Corn Salad (Valerianella locusta).

D. tenuifolia and V. locusta, two greens, were analyzed for active compounds and antitumor actions on colorectal cancer cells. Phenolics were determined by UHPLC-Orbitrap-MS; carotenoids and glucosinolates by HPLC-MS; and sterols and fatty acids by gas-liquid chromatography (GLC). For antitumor effects, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) tests were run on HT-29 colorectal cancer cells, and in CCD-18 untransformed enterocyte cells. Six main carotenoids were identified in both vegetables, while total carotenoids accounted for 3520 and 2970 µg · g(-1) dry weight in D. tenuifolia and V. locusta, respectively. Six phenolics were detected in D. tenuifolia (68,600 µg · g(-1) dry weight) and five in V. locusta (139,000 µg · g(-1) dry weight). Three glucosinolates (GSL) were found in D. tenuifolia (1960 µg · g(-1) dry wt. total). Low-polarity extracts from V. locusta and D. tenuifolia showed IC50 ~ 150 and ~200 µg · mL(-1) on HT-29 cells, while both plants lacked actions on CCD-18 cells. V. locusta inhibited HT-29 cancer cells viability more efficiently than D. tenuiofolia, but induced less cytotoxicity. This work highlights the importance of functional foods for colorectal cancer prevention.

Uptake of azoles by lamb's lettuce (Valerianella locusta L.) grown in hydroponic conditions.

An uptake and translocation study of azole compounds was performed in lamb's lettuce (Valerianella locusta L.) grown in nutrient solution fortified with different azoles. Three azoles, (clotrimazole, fluconazole and propiconazole), which have different physico-chemical properties and are ubiquitous in the aquatic environment, were the compounds selected. An analytical method, based on matrix solid phase dispersion (MSPD) followed by LC-MS/MS determination, was developed to quantify these compounds in aqueous solution and in roots and leaves. The physicochemical properties of azoles are the main factors governing the uptake and plant accumulation. These azoles were detected in leaves indicating their transport within lamb's lettuce. Translocation from nutrient solution to the aerial part of lamb's lettuce was found to be highly dependent on the hydrophobicity of the azole. Clotrimazole accumulates in roots causing necrosis in roots and leaves, whereas fluconazole was the azole with the highest concentration in leaves without causing apparent phytotoxicity symptoms. The assessment of the levels of these azoles in leaves indicates that the risk for human health is negligible.

Monitoring of fresh-cut Valerianella locusta Laterr. shelf life by electronic nose and VIS-NIR spectroscopy.

The aim of this work was to investigate the applicability of non-destructive techniques in monitoring freshness decay of fresh-cut Valerianella locusta L. during storage at different temperature. The sampling was performed for 15 days for Valerianella samples preserved at 4 and 10 °C, and for 7 days for samples stored at 20 °C. The quality decay of samples was evaluated by quality parameters (pH, water content, total phenols, chlorophyll a fluorescence) and by non-destructive systems (electronic nose and visible-near infrared spectroscopy). Cluster Analysis (CA) was performed on quality indices and four clusters were identified, namely "fresh", "acceptable", "spoiled" and "very spoiled". Principal Component Analysis (PCA) was applied on the electronic nose data in order to evaluate the feasibility of this technique as a rapid and non-destructive approach for monitoring the freshness of fresh-cut Valerianella during storage. Linear Discriminant Analysis (LDA) and PLS-discriminant analysis (PLS-DA) models were developed to test the performance of electronic nose and VIS-NIR, respectively, to classify samples in the four classes of freshness. The average value of samples correctly classified using LDA was 95.5% and the cross validation error rate was equal to 8.7%. The results obtained from PLS-DA models, in validation, gave a positive predictive value (PPV) of classification between 74% and 96%. Finally, predictive models were performed using Partial Least Squares (PLS) regression analysis between quality indices and VIS-NIR data. RPD values <3 were obtained for water content and pH. Excellent results were obtained for total phenols with Rcv(2) and RPD equal to 0.89 and 3.19, and for chlorophyll a fluorescence with Rcv(2) and RPD equal to 0.92 and 3.22, respectively. Results demonstrated that electronic nose and VIS-NIR are complementary techniques able to support the conventional techniques in the shelf-life assessment of fresh-cut V. locusta L. providing information useful for a better management of the product along the distribution chain.

Beneficial effects of silicon on hydroponically grown corn salad (Valerianella locusta (L.) Laterr) plants.

Soil-less cultivation of horticultural crops represents a fairly recent innovation to traditional agriculture which has several advantages including higher water-use efficiency. When plants are grown with this system, their roots come in contact with nutrients solely via the hydroponic solution. Although its beneficial effects have been widely demonstrated, silicon (Si) is mostly omitted from the composition of nutrient solutions. Therefore, the objective of this study was to assess the beneficial effect of Si addition to hydroponic solution on quali-quantitative aspects of edible production of two cultivars of corn salad (Valerianella locusta (L.) Laterr.) grown in soil-less floating system. Impacts on shelf life of this food were also studied. Results show that the supply of Si increased the edible yield and the quality level reducing the nitrate concentration in edible tissues. This result might be attributed to changes either in the metabolism (such as the nitrate assimilation process) or to the functionality of root mechanisms involved in the nutrient acquisition from the outer medium. In fact, our results show for the first time the ability of Si to modulate the root activity of nitrate and Fe uptake through, at least in part, a regulation of gene expression levels of the proteins involved in this phenomenon. In addition, the presence of Si decreased the levels of polyphenoloxidase gene expression at harvest and, in post-harvest, slowed down the chlorophyll degradation delaying leaf senescence and thus prolonging the shelf life of these edible tissues. In conclusion, data showed that the addition of Si to the nutrient solution can be a useful tool for improving quali-quantitatively the yield of baby leaf vegetable corn salad as well as its shelf life. Since the amelioration due to the Si has been achieved only with one cultivar, the recommendation of its inclusion in the nutrient solution does not exclude the identification of cultivars suitable for this cultivation system and the comprehension of agronomical and environmental factors which could limit the Si benefits.

Detection of Acidovorax valerianellae, the causing agent of bacterial leaf spots in corn salad [Valerianella locusta (L.) Laterr.], in corn salad seeds.

AIM: The black leaf spot disease on corn salad caused by the bacterium Acidovorax valerianellae has been observed in Europe for several years and causes economic losses in corn salad cropping. Contaminated seeds or infested soil are considered as the major infection sources. The use of healthy seed material is the only way to prevent disease outbreaks. Therefore, a sensitive diagnostic method for seed testing should be developed. METHODS AND RESULTS: Using a triple antibody sandwich ELISA with a high-specific monoclonal antibody, a quick and reliable detection method for contamination of seed lots with the pathogen was developed. This method allowed to detect contaminated seed lots as well as contamination with A. valerianellae in single seeds. Furthermore, the occurrence and distribution of the pathogen could be shown in symptomatic corn salad leaves and in naturally infested seeds by transmission electron microscopy and immunogold labelling for the first time. CONCLUSION: Our results confirm the seed transmission of this corn salad disease. Pathogen load and distribution vary between positively tested seed lots. SIGNIFICANCE AND IMPACT OF THE STUDY: With this method, not only routine testing of seed material to eliminate contaminated seed lots from production is possible but also the control of sanitation procedures to reduce contamination.

Influence of hydroponic and soil cultivation on quality and shelf life of ready-to-eat lamb's lettuce (Valerianella locusta L. Laterr).

BACKGROUND: Nowadays, there is an increasing interest in the hydroponic floating system to cultivate leafy vegetables for ready-to-eat salads. It is reasonable that different growing systems could affect the quality and shelf life of these salads. RESULTS: The quality and shelf life of ready-to-eat lamb's lettuce grown in protected environment in soil plot or in soil-less system over hydroponic solution with or without the addition of 30 µmol L⁻¹ silicon were evaluated. Minimum effects were observed on colour, firmness and microbial counts. Hydroponic cultivation largely affected plant tissue hydration, leading to weight loss and structural modifications during refrigerated storage. The shelf life of lamb's lettuce was limited by the development of visually detectable unpleasant sensory properties. Shelf life, calculated by survival analysis of consumer acceptability data, resulted about 7 days for soil-cultivated salad and 2 days for the hydroponically grown ones. The addition of silicon to the hydroponic solution resulted in an interesting strategy to increase plant tissue yield and reduce nitrate accumulation. CONCLUSIONS: Although hydroponic cultivation may have critical consequences on product quality and shelf life, these disadvantages could be largely counterbalance by increased yield and a reduction of nitrate accumulation when cultivation is performed on nutritive solutions with supplemental addition of silicon.

Corn salad (Valerianella locusta (L.) Laterr.) growth in a water-saving floating system as affected by iron and sulfate availability.

BACKGROUND: Unbalanced nutrient availability causes disequilibrated plant growth, which can result in a worsening of harvested product quality, such as high nitrate content in edible tissues. To cope with this problem, improved knowledge of the mechanisms involved in nutrient acquisition and regulation is necessary. For this purpose the responses of acquisition mechanisms of N, Fe and S were studied as a function of Fe and S availability using two corn salad cultivars grown hydroponically, considering also aspects related to N metabolism. RESULTS: The results showed that an increase in Fe or S availability enhanced nitrate uptake and assimilation, which in turn increased biomass production of leaves with lower nitrate content. In particular, high S availability exerted a positive effect (gene expression and functionality) both on the uptake and metabolism of N and on Fe acquisition mechanisms. CONCLUSION: The data presented here show close interactions between N, S and Fe, highlighting that relevant improvements in yield and quality from soilless culture might also be obtained through appropriate adjustments of nutrient availability. In this respect, concerning the role of S in the acquisition mechanisms of N and Fe and in N metabolism, its level of availability should be taken into high consideration for equilibrated plant growth.

Phylogenetic placement and biogeography of the North American species of Valerianella (Valerianaceae: Dipsacales) based on chloroplast and nuclear DNA.

Although Valerianaceae is a relatively small group of angiosperms (ca. 350 species), sample sizes of previous phylogenetic studies have been limited and taxonomic sampling has been usually geographically biased to species from Europe and/or South America. One group that has never been included in any analyses to date is the North American representatives of Valerianella. In this study I sampled 17 additional accessions from Valerianaceae, including six of the 12 described North American Valerianella species and four additional samples of European Valerianella species. Phylogenies based on parsimony and Bayesian methods show strong support for placing the North American Valerianella nested within the European species. These analyses also found Fedia to be nested within Valerianella, making Valerianella paraphyletic, a result consistent with several previously published molecular phylogenies. Divergence times estimated from the molecular data using recently proposed Bayesian 'relaxed' clock methods, suggest Valerianella was in North America by the middle Miocene (ca. 16-14 million years ago). These dates would suggest the North American species of Valerianella arrived in the New World just prior to the North Atlantic land bridge (NALB) no longer being a viable conduit for migration. Given the large amount of uncertainty in the divergence time estimates, dispersal across the North Atlantic may not be the only viable alternative to explain the current distribution of Valerianella. However, biogeographic analyses using an explicit model based approach, strongly favor the NALB as the route of dispersal for Valerianella.

Acidovorax valerianellae sp. nov., a novel pathogen of lamb's lettuce [Valerianella locusta (L.) Laterr].

Bacterial spot disease of lamb's lettuce [Valerianella locusta (L.) Laterr.] was first observed in fields in 1991. This new bacterial disease is localized in western France in high-technology field production of lamb's lettuce for the preparation of ready-to-use salad. Nineteen strains isolated in 1992 and 1993 from typical black leaf spots of naturally infected lamb's lettuce were characterized and compared with reference strains of Acidovorax and Delftia. The pathogenicity of the 19 strains was confirmed by artificial inoculation. Biochemical and physiological tests, fatty acid profiles, DNA-DNA hybridization and other nucleic acid-based tests were performed. A numerical taxonomic analysis of the 19 lamb's lettuce strains showed a single homogeneous phenon closely related to previously described phytopathogenic taxa of the genus Acidovorax. DNA-DNA hybridization studies showed that the lamb's lettuce strains were 91-100% related to a representative strain, strain CFBP 4730(T), and constituted a discrete DNA hybridization group, indicating that they belong to the same novel species. Results from DNA-rRNA hybridization, 16S rRNA sequence analysis and fatty acid analysis studies confirmed that this novel species belongs to the beta-subclass of the Proteobacteria and, more specifically, to the family Comamonadaceae and the genus Acidovorax. The name Acidovorax valerianellae sp. nov. is proposed for this novel taxon of phytopathogenic bacteria. The type strain is strain CFBP 4730(T) (= NCPPB 4283(T)).