Real-Time PCR (qtPCR) to Discover the Fate of Plant Growth-Promoting Rhizobacteria (PGPR) in Agricultural Soils.

To optimize the application of plant growth-promoting rhizobacteria (PGPR) in field trials, tracking methods are needed to assess their shelf life and to determine the elements affecting their effectiveness and their interactions with plants and native soil microbiota. This work developed a real-time PCR (qtPCR) method which traces and quantifies bacteria when added as microbial consortia, including five PGPR species: Burkholderia ambifaria, Bacillus amyloliquefaciens, Azotobacter chroococcum, Pseudomonas fluorescens, and Rahnella aquatilis. Through a literature search and in silico sequence analyses, a set of primer pairs which selectively tag three bacterial species (B. ambifaria, B. amyloliquefaciens and R. aquatilis) was retrieved. The primers were used to trace these microbial species in a field trial in which the consortium was tested as a biostimulant on two wheat varieties, in combination with biochar and the mycorrhizal fungus Rhizophagus intraradices. The qtPCR assay demonstrated that the targeted bacteria had colonized and grown into the soil, reaching a maximum of growth between 15 and 20 days after inoculum. The results also showed biochar had a positive effect on PGPR growth. In conclusion, qtPCR was once more an effective method to trace the fate of supplied bacterial species in the consortium when used as a cargo system for their delivery.

Overexpression of ZNT1 and NRAMP4 from the Ni Hyperaccumulator Noccaea caerulescens Population Monte Prinzera in Arabidopsis thaliana Perturbs Fe, Mn, and Ni Accumulation.

Metalliferous soils are characterized by a high content of metal compounds that can hamper plant growth. The pseudometallophyte Noccaea caerulescens is able to grow on metalliferous substrates by implementing both tolerance and accumulation of usually toxic metal ions. Expression of particular transmembrane transporter proteins (e.g., members of the ZIP and NRAMP families) leads to metal tolerance and accumulation, and its comparison between hyperaccumulator N. caerulescens with non-accumulator relatives Arabidopsis thaliana and Thlaspi arvense has deepened our knowledge on mechanisms adopted by plants to survive in metalliferous soils. In this work, two transporters, ZNT1 and NRAMP4, expressed in a serpentinic population of N. caerulescens identified on the Monte Prinzera (Italy) are considered, and their expression has been induced in yeast and in A. thaliana. In the latter, single transgenic lines were crossed to test the effect of the combined over-expression of the two transporters. An enhanced iron and manganese translocation towards the shoot was induced by overexpression of NcZNT1. The combined overexpression of NcZNT1 and NcNRAMP4 did perturb the metal accumulation in plants.

Smart agriculture for food quality: facing climate change in the 21st century.

Climate change, with increasing temperatures and atmospheric carbon dioxide levels, constitutes a severe threat to the environment and all living organisms. In particular, numerous studies suggest severe consequences for the health of crop plants, affecting both the productivity and quality of raw material destined to the food industry. Of particular concern is the reduction of proteins and essential micronutrients as iron and zinc in crops. Fighting this alarming trends is the challenge of Climate-Smart Agriculture with the double goal of reducing environmental impacts (use of pesticides, nitrogen and phosphorus leaching, soil erosion, water depletion and contamination) and improving raw material and consequently food quality. Organic farming, biofertilizers and to a lesser extent nano-carriers, improve the antioxidant properties of fruits, but the data about proteins and micronutrients are rather contradictory. On the other hand, advanced devices and Precision Agriculture allow the cultivations to be more profitable, efficient, contributing more and more to reduce pest diseases and to increase the quality of agricultural products and food safety. Thus, nowadays adoption of technologies applied to sustainable farming systems is a challenging and dynamic issue for facing negative trends due to environmental impacts and climate changes.

Antimicrobial activity of cellulosic pads amended with emulsions of essential oils of oregano, thyme and cinnamon against microorganisms in minced beef meat.

Cellulosic pads, amended with emulsions containing essential oils of thyme and oregano, exhibited antimicrobial activity against the psychrophilic microbiota of minced beef. In addition, the pads were active against specific meat bacterial species (Pseudomonas putida, Pseudomonas fragi, Pseudomonas fluorescens, Enterococcus faecalis and Lactococcus lactis) and some common foodborne pathogens (Salmonella enterica, Campylobacter jejuni and Staphylococcus aureus). Three emulsions, IT131017, Mediterranean and Etnic, containing different percentages of carvacrol, thymol, linalool, and ɑ and ß-pinene, significantly reduced the growth of S. enterica and P. putida. Pads derived from emulsions Mediterranean and Etnic induced slight (0.3-0.8 Log10 CFUs/g) but reproducible reduction of the psychrophilic microbiota in minced meat and hamburger stored for 12 and 15 days at 4 °C.

Application of real-time PCR (qPCR) for characterization of microbial populations and type of milk in dairy food products.

Dairy foods represent an important sector of the food market for their nutritional qualities and their organoleptic characteristics, which are often linked to tradition and to region. These products are typically protected by labels such as PDO (Protected Designation of Origin) and PGI (Protected Geographical Indication). Real-time PCR (qPCR) is a fundamental tool in "Food Genomics;" a discipline concerned with the residual DNA in food, which, alongside traditional physical and chemical methods, is frequently used to determine product safety, quality and authenticity. Compared to conventional or "end-point" PCR, qPCR incorporates continuous monitoring of reaction progress, thereby enabling quantification of target DNA. This review describes qPCR applications to the analysis of microbiota, and to the identification of the animal species source of milk from which dairy products have been made. These are important aspects for ensuring safety and authenticity. The various applications of qPCR are discussed, as well as advantages and disadvantages in comparison with other analytical methods.

A novel ß-propeller phytase from the dioxin-degrading bacterium Sphingomonas wittichii RW-1.

ß-propeller phytase-like sequences (BPP-like sequences) are widespread in the microbial world and have been found in the sequenced genomes of aquatic, soil, and plant bacteria. Exploring NCBI microbial genome database for putative genes encoding phytase, a BPP-like sequence from Sphingomonas wittichii RW-1 (Sequence ID: CP000699.1), known for its capacity of degrading polychlorinated dibenzo-p-dioxins and dibenzofurans, was recognized. The putative phytase gene (phySw) was amplified with specific primers, cloned, and overexpressed in Escherichia coli and the catalytic properties of the recombinant PhySw protein were analyzed. The results show that phySw encodes an enzyme with the properties of ß-propeller phytases: it requires the presence of Ca2+ ions, it is optimally active at 55 °C, and it has a pH optimum of 6.0 with good activity in the range 6.0-8.0. Furthermore, the enzyme exhibits a good thermostability, recovering 68% of its original activity after treatment at 80 °C for 10 min, and shows a good substrate specificity for phytic acid. These properties render this enzyme a candidate as an animal feed additive (e.g., for aquaculture industry). The isolation of phytases from a hydrocarbon-utilizing microorganism also opens new scenarios for their possible application in combating oil pollution.

Nucleo-mitochondrial interaction of yeast in response to cadmium sulfide quantum dot exposure.

Cell sensitivity to quantum dots (QDs) has been attributed to a cascade triggered by oxidative stress leading to apoptosis. The role and function of mitochondria in animal cells are well understood but little information is available on the complex genetic networks that regulate nucleo-mitochondrial interaction. The effect of CdS QD exposure in yeast Saccharomyces cerevisiae was assessed under conditions of limited lethality (<10%), using cell physiological and morphological endpoints. Whole-genomic array analysis and the screening of a deletion mutant library were also carried out. The results showed that QDs: increased the level of reactive oxygen species (ROS) and decreased the level of reduced vs oxidized glutathione (GSH/GSSG); reduced oxygen consumption and the abundance of respiratory cytochromes; disrupted mitochondrial membrane potentials and affected mitochondrial morphology. Exposure affected the capacity of cells to grow on galactose, which requires nucleo-mitochondrial involvement. However, QDs exposure did not materially induce respiratory deficient (RD) mutants but only RD phenocopies. All of these cellular changes were correlated with several key nuclear genes, including TOM5 and FKS1, involved in the maintenance of mitochondrial organization and function. The consequences of these cellular effects are discussed in terms of dysregulation of cell function in response to these "pathological mitochondria".

A quadruplex PCR (qxPCR) assay for adulteration in dairy products.

This study describes the development of a quadruplex quantitative Real Time PCR (qxPCR) based on SYBR®GreenER chemistry, for rapid identification of DNA of cow, goat, sheep and buffalo in dairy products, and for quantification of cow DNA in these products. The platform was applied to: (i) mixes of milks at fixed percentages; (ii) cheeses prepared with the same mixes; (iii) commercial dairy products. The methodology enabled the detection of DNA from cow in mixes of milk and cheeses with a limit of detection (LOD) of 0.1%. When applied to commercial dairy products the qxPCR gave results comparable with each single-plex Real Time PCR. A good correlation (R(2)>0.9) between peaks' area of derivative of melting curves of amplicons and percentages of cow milk in milk mixes and cheeses, allows for an estimation of cow DNA in a dynamic range varying from 0.1-5% to 1-25%.

Detection of plant oil DNA using high resolution melting (HRM) post PCR analysis: a tool for disclosure of olive oil adulteration.

Extra virgin olive oil is frequently subjected to adulterations with addition of oils obtained from plants other than olive. DNA analysis is a fast and economic tool to identify plant components in oils. Extraction and amplification of DNA by PCR was tested in olives, in milled seeds and in oils, to investigate its use in olive oil traceability. DNA was extracted from different oils made of hazelnut, maize, sunflower, peanut, sesame, soybean, rice and pumpkin. Comparing the DNA melting profiles in reference plant materials and in the oils, it was possible to identify any plant components in oils and mixtures of oils. Real-Time PCR (RT-PCR) platform has been added of the new methodology of high resolution melting (HRM), both were used to analyse olive oils mixed with different percentage of other oils. Results showed HRM a cost effective method for efficient detection of adulterations in olive oils.

Development of a multiplex real time PCR to detect thermophilic lactic acid bacteria in natural whey starters.

A multiplex real time PCR (mRealT-PCR) useful to rapidly screen microbial composition of thermophilic starter cultures for hard cooked cheeses and to compare samples with potentially different technological properties was developed. Novel primers directed toward pheS gene were designed and optimized for multiple detection of Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus thermophilus and Lactobacillus fermentum. The assay was based on SYBR Green chemistry followed by melting curves analysis. The method was then evaluated for applications in the specific detection of the 4 lactic acid bacteria (LAB) in 29 different natural whey starters for Parmigiano Reggiano cheese production. The results obtained by mRealT-PCR were also compared with those obtained on the same samples by Fluorescence in Situ Hybridization (FISH) and Length-Heterogeneity PCR (LH-PCR). The mRealT-PCR developed in this study, was found to be effective for analyzing species present in the samples with an average sensitivity down to less than 600 copies of DNA and therefore sensitive enough to detect even minor LAB community members of thermophilic starter cultures. The assay was able to describe the microbial population of all the different natural whey starter samples analyzed, despite their natural variability. A higher number of whey starter samples with S. thermophilus and L. fermentum present in their microbial community were revealed, suggesting that these species could be more frequent in Parmigiano Reggiano natural whey starter samples than previously shown. The method was more effective than LH-PCR and FISH and, considering that these two techniques have to be used in combination to detect the less abundant species, the mRealT-PCR was also faster. Providing a single step sensitive detection of L. helveticus, L. delbrueckii, S. thermophilus and L. fermentum, the developed mRealT-PCR could be used for screening thermophilic starter cultures and to follow the presence of those species during ripening of derived dairy products. A major increase in understanding the starter culture contribution to cheese ecosystem could be harnessed to control cheese ripening and flavor formation.

Applicability of SSR markers to the traceability of monovarietal olive oils.

BACKGROUND: To protect the features and authenticity of food products, the European Commission enforces two certification labels: Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI). EEC Regulation No. 510/2006 imposes criteria for labelling, production and commercialisation of olive oil. Since plant genotype is a major determinant in establishing the PDO and PGI labels, methods to ascertain the varieties present in a batch of olive oil are essential in validating product conformity. The traceability of olive oil can be assessed through simple sequence repeat (SSR) co-dominant markers targeted to specific regions of DNA from olive cultivars. RESULTS: Twenty-one monovarietal olive oils were analysed with nine nuclear and two shortened SSRs. For each marker the correspondence of allelic profile with the reference cultivar, the reproducibility of profiles in different DNA extractions and the polymorphism information content were determined. CONCLUSION: The results showed that using a panel of SSR markers such as those described in this paper allows one to make a reliable attribution of an olive oil to a specific cultivar.

Applicability of SCAR markers to food genomics: olive oil traceability.

DNA analysis with molecular markers has opened a shortcut toward a genomic comprehension of complex organisms. The availability of micro-DNA extraction methods, coupled with selective amplification of the smallest extracted fragments with molecular markers, could equally bring a breakthrough in food genomics: the identification of original components in food. Amplified fragment length polymorphisms (AFLPs) have been instrumental in plant genomics because they may allow rapid and reliable analysis of multiple and potentially polymorphic sites. Nevertheless, their direct application to the analysis of DNA extracted from food matrixes is complicated by the low quality of DNA extracted: its high degradation and the presence of inhibitors of enzymatic reactions. The conversion of an AFLP fragment to a robust and specific single-locus PCR-based marker, therefore, could extend the use of molecular markers to large-scale analysis of complex agro-food matrixes. In the present study is reported the development of sequence characterized amplified regions (SCARs) starting from AFLP profiles of monovarietal olive oils analyzed on agarose gel; one of these was used to identify differences among 56 olive cultivars. All the developed markers were purposefully amplified in olive oils to apply them to olive oil traceability.

Control of the synthesis and subcellular targeting of the two GDH genes products in leaves and stems of Nicotiana plumbaginifolia and Arabidopsis thaliana.

Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an alpha- or a beta-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the alpha- and beta-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the alpha-subunit in the stems when the expression of the beta-subunit is impaired in the leaves, and of the beta-subunit in the leaves when the expression of the alpha-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.

Traceability of plant contribution in olive oil by amplified fragment length polymorphisms.

Application of DNA molecular markers to traceability of foods is thought to bring new benefit to consumer's protection. Even in a complex matrix such as olive oil, DNA could be traced with PCR markers such as the amplified fragment length polymorphisms (AFLPs). In this work, fluorescent AFLPs were optimized for the characterization of olive oil DNA, to obtain highly reproducible, high-quality fingerprints, testing different parameters: the concentrations of dNTPs and labeled primer, the kind of Taq DNA polymerase and thermal cycler, and the quantity of DNA employed. It was found that correspondence of fingerprinting by comparing results in oils and in plants was close to 70% and that the DNA extraction from olive oil was the limiting step for the reliability of AFLP profiles, due to the complex matrix analyzed.

Chemical and biological diversity of Bergamot (Citrus bergamia) in relation to environmental factors.

Oil of bergamot is receiving renewed popularity in aromatherapy. The biovariability of Citrus bergamia grown wild in Calabria (Italy) was investigated as far as chemical markers (linalool, linalyl acetate and bergapten) content and antioxidant and antifungal activities of the methanolic extracts. The average content in the markers presents slight variations with the altitude and more evident changes with the latitude of the areas of plant collection.