Evaluation of phenolic compounds in virgin olive oil by direct injection in high-performance liquid chromatography with fluorometric detection

Hydrophilic phenols are the most abundant natural antioxidants of virgin olive oil (VOO), in which tocopherols and carotenes are also present. The prevalent classes of hydrophilic phenols found in VOO are phenyl alcohols, phenolic acids, secoiridoids such as the dialdehydic form of decarboxymethyl elenolic acid linked to (3,4-dihydroxyphenyl)ethanol or (p-hydroxypheny1)ethanol (3,4-DHPEA-EDA or p-HPEA-EDA) and an isomer of the oleuropein aglycon (3,4-DHPEA-EA), lignans such as (+)-1-acetoxypinoresinol and (+)-pinoresinol, and flavonoids. A new method for the analysis of VOO hydrophilic phenols by direct injection in high-performance liquid chromatography (HPLC) with the use of a fluorescence detector (FLD) has been proposed and compared with the traditional liquid-liquid extraction technique followed by the HPLC analysis utilizing a diode array detector (DAD) and a FLD. Results show that the most important classes of phenolic compounds occurring in VOO can be evaluated using HPLC direct injection. The efficiency of the new method, as compared to the liquid-liquid extraction, was higher to quantify phenyl alcohols, lignans, and 3,4-DHPEA-EA and lower for the evaluation of 3,4-DHPEA-EDA and p-HPEA-EDA.     

Treatment of cereal products with a tailored preparation of trichoderma enzymes increases the amount of soluble dietary fiber

Nutritionists recommend increasing the intake of soluble dietary fiber (SDF), which is very low in most cereal-based products. Conversion of insoluble DF (IDF) into SDF can be achieved by chemical treatments, but this affects the sensorial properties of the products. In this study, the possibility of getting a substantial increase of SDF from cereal products using a tailored preparation of Trichoderma enzymes is reported. Enzymes were produced cultivating Trichoderma using durum wheat fiber (DWF) and barley spent grain (BSG) as unique carbon sources. Many Trichoderma strains were screened, and the hydrolysis conditions able to increase by enzymatic treatment the amount of SDF in DWF and BSG were determined. Results demonstrate in both products that it is possible to triple the amount of SDF without a marked decrease of total DF. The enzymatic treatment also causes the release of hydroxycinnamic acids, mainly ferulic acid, that are linked to the polysaccharides chains. This increases the free phenolic concentration, the water-soluble antioxidant activity, and, in turn, the phenol compounds bioavailability.     

Tuta absoluta,a South American pest of tomato now in the EPPO region: biology,distribution and damage

Tuta absoluta (Meyrick) is an invasive pest of tomato native to South America, where it is responsible for extensive damage. It rapidly spread into several European countries, becoming a key pest. Knowledge of its main biological traits can be used to develop effective plant protection management strategies. It is a multivoltine species with a homodynamous behaviour, and the length of its life cycle depends on environmental conditions, particularly temperature. The larvae feed and develop inside tomato leaves, stems and fruits throughout the entire growing cycle. The adults have crepuscular habits, and in Mediterranean conditions they can be easily detected throughout the year. Damage is directly related to the reduction of plants' photosynthetic capacity and of production levels in both protected and open‐field tomato crops; indirect damage can be also caused by secondary infections, with pathogens developing on the infested plant and fruit tissues.     

Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides

BACKGROUND: QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is caused by mutations (most frequently G143A) in the mitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary) Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. RESULTS: B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild‐type strains, or from naturally infected plants on a medium amended with 1–3 mg L^?1 trifloxystrobin and 2 mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. CONCLUSIONS: The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. In laboratory mutants the heteroplasmic state of mutated mitochondria could likely hamper the G143A detection, otherwise other gene(s) underlying different mechanisms of resistance could be involved. Copyright © 2012 Society of Chemical Industry     

Towards a rapid molecular identification of the common phlebotomine sand flies in the Mediterranean region

The present study reports two simple molecular approaches allowing a rapid identification of the most prevalent species of phlebotomine sand flies in the Mediterranean region. A PCR protocol for the amplification of ITS2 ribosomal region and a PCR-RFLP on a mitochondrial DNA fragment (cytb-nd1) were settled in order to identify and discriminate among Phlebotomus perniciosus, Phlebotomus neglectus, Phlebotomus perfiliewi, Phlebotomus papatasi and Sergentomyia minuta. The ITS2 regions showed a certain degree of interspecific variability, which led to PCR amplicons of different sizes, i.e., 450, 490, 460, 480 and 530 bp for P. perniciosus, P. neglectus, P. perfiliewi, P. papatasi, and S. minuta, respectively. Analogously, the digestion of a mitochondrial DNA amplicon with Ase I enzyme showed five different restriction profiles, which allowed the unequivocal differentiation of the sand fly species examined. These methods might represent useful tools for a molecular large scale screening of phlebotomine sand fly species caught in areas where leishmaniasis is endemic, in order to plan appropriate epidemiological surveillance programs for both Leishmania spp. and their vectors.     

A duplex real-time polymerase chain reaction assay for the detection of and differentiation between Dirofilaria immitis and Dirofilaria repens in dogs and mosquitoes

This study describes a duplex real-time polymerase chain reaction (PCR) assay for the detection and differentiation between Dirofilaria immitis and Dirofilaria repens in dog blood and mosquitoes. Regions of a cytochrome oxidase 1 (cox1) mitochondrial DNA fragment and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA were amplified from microfilariae and adult worm samples, using a sensitive SsoFast? EvaGreen(?) based real-time PCR method coupled with melting-curve analysis. The limit of the real-time PCR in detecting microfilaria and adult worm DNA was also tested both in dog blood and in artificially infected microfilarial. Two peaks at different melting temperatures (T(m)) for D. immitis (mean ± SD=75.7 ± 0.3°C) and D. repens (mean ± SD=70 ± 0.7°C), respectively, were obtained for microfilarial and adult positive controls of both species when examined separately and together. The real-time PCR protocol was also efficient in detecting microfilarial and adult DNA of both species when tested in samples spiked with DNA from Aedes albopictus, in Aedes aegypti experimentally infected by D. repens and in Culex pipiens naturally infected by D. repens and D. immitis. The high sensitivity of real-time PCR confirmed its reliability in detecting small amounts of genomic DNA either in dog blood or mosquitoes (2.5 pg/μl and 3 × 10(-1)pg/μl for D. immitis and D. repens, respectively). This assay is proposed as a tool for the epidemiological surveillance of the two most important Dirofilaria species in areas where they are endemic and sympatric.     

Potential for using Datura alba leaf extracts against two major stored grain pests, the khapra beetle Trogoderma granarium and the rice weevil Sitophillus oryzae

Various plants are well known for their insecticidal activity and their use was maintained for millennia throughout all the agricultural regions of the world. In a current context, the use of Botanical insecticides represents one of the best alternatives to chemicals for the development of environmental-friendly strategies for stored grain pest control. Datura alba Nees is a plant found extensively in the warmer regions of the world and it is used as a medicinal plant. This study aims to assess the contact toxicity and the trans-generational effect of D.?alba leaf extract (DLE) against two important insect pests on stored rice, Trogoderma granarium and Sitophilus oryzae, under laboratory conditions. Filter papers were soaked in three DLE concentration solutions and in two control treatments: water and acetone. The survivor specimens (F₀) were transferred to a new untreated feeding substrate and the population build up of the two following generations (F₁ and F₂) were counted after 30 and 60?days, respectively. The highest DLE concentration (2.5?%) induced the significantly highest mortality with 33.5 and 45?% mortality in T.?granarium and S.?oryzae after 7?days of exposure, respectively. The DLE long-term effect toward both tested species was also proved by the high demographic decrease in the F₂ generations, when compared to control groups. This study is the first step toward establishing a scientific basis for the effective application of D.?alba plant materials as biorational tools to control stored grain pests.     

Towards assessing the sustainability of European logging operations

The forest-based sector has been at the forefront in operationally implementing the sustainability concept, its associated principles and indicators for sustainable forest management. Several methods have been developed to study environmental impacts of forestry activities, but none of the existing tools address all the dimensions of sustainability along the whole forest wood chain (FWC) in a balanced way. Consequently, the decision was made to develop a tool for sustainability impact assessment (ToSIA), the modelling framework for sustainability impact assessment of FWCs. The objective of the EU Project Eforwood was to develop ToSIA, a decision support tool. Within ToSIA, a FWC is modelled as a number of interconnected processes. For each process, a range of economic, environmental and social indicators and their respective values are calculated, thus representing the three pillars of sustainability. By this method, the multifunctionality of forests can be assessed and supply chains can be compared with respect to sustainability. Sensitivity analysis and scenario techniques can be applied to learn about the effect of expected changes to the structure of the chain, the material flows and the indicator values. In order to provide the tool with information about forest and logging operations, data were collected at two fundamental levels: (1) a regional level with case studies in Scandinavia, Iberia and Baden-Württemberg and (2) a European level with a case study that reflects conditions in the 27 countries of the European Union. This paper describes and details the harvesting and logging processes for the European countries. The results are displayed for each of the three regional case studies as well as aggregated to five principal areas in Europe: Eastern, Northern, Western, Central and Southwest Europe.     

Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot blot hybridisation

Detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by polymerase chain reaction (PCR) is often hampered by the lack of efficient methods for sample treatment. We report a protocol for analysis of faecal samples based on buoyant density centrifugation in Percoll and IS900 sequence capture PCR combined with a dot blot assay for detection of low-grade infection of M. paratuberculosis. Serial dilutions of M. paratuberculosis genomic DNA and M. paratuberculosis bacteria were used to assess the sensitivity of the method. The final evaluation was performed with spiked faecal samples, which also were analysed by culture. The presence of PCR inhibitory substances in processed faecal samples was evaluated by including a PCR internal control. By using buoyant density centrifugation, sequence capture PCR, and dot blot hybridisation, we achieved a sensitivity of 10(3)CFU (colony forming units)/g of faeces. The detection limit by culture was assessed to 10(2)CFU/g of faeces. We conclude that the described protocol is a fast and sensitive alternative to bacterial culture of faecal samples.     

Genetic analysis and phenotypic associations for drought tolerance in Hordeum spontaneum introgression lines using SSR and SNP markers

Associations between markers and drought related traits were investigated on a set of 57 advanced barley breeding lines, carrying various levels of introgression from Hordeum spontaneum lines 41-1 and 41-5, the best sources of drought tolerance in the ICARDA barley breeding program, using 74 simple sequences repeats (SSR) and 20 single nucleotide polymorphism markers. The 57 lines were evaluated for grain yield and drought related traits for three years (2003/04, 2004/05, 2005/06) in nine Mediterranean low rainfall environments. A high level of polymorphism was found with SSR markers, and the mean polymorphism information content and gene diversity values were 0.67 and 0.71, respectively. The number of alleles per locus varied from 2 to 11, with an average of 5.8 alleles per marker. Considering all the 57 lines, the linkage disequilibrium (LD) analysis was significant at a comparison-wise P?<?0.01 level in nearly 9 % of the SSR marker pairs used and a decay of LD was observed to a value of r ²?<?0.2 at a genetic distance of 40?cM. The association analysis revealed a total of 147 significant marker?Ctrait associations for grain yield and drought related traits. A total of 72 (49 %) marker?Ctrait associations showed favorable effects of the exotic germplasm where the H. spontaneum lines contributed to an improvement of the trait under drought stress conditions. The number of significant marker?Ctrait associations per trait were: 12 for growth habit; 2 for growth vigor; 11 for peduncle extrusion; 5 for number of grains per spike; 20 for peduncle length; 16 for days to heading; 20 for plant height; 8 for spike length; 17 for thousand kernel weight; 30 for grain yield; 4 for harvest index and 2 for biological yield. The phenotypic variation explained by individual marker?Ctrait associations ranged from 7.6 % to 36.2 %. The identification of genomic regions associated with grain yield and drought related traits is useful for the genetic improvement of cultivars better adapted to drought stress environments. Thus, the present study is encouraging in identifying significant marker?Ctrait associations through LD based association mapping analysis, which could complement and augment previous quantitative trait loci information for the potential use of Marker Assisted Selection for drought.