Comparative study on the antioxidant and biological activities of carvacrol, thymol, and eugenol derivatives

Four derivatives of thymol, carvacrol, and eugenol were synthesized: 4-(hydroxymethyl)-5-isopropyl-2-methylphenol, 4,4'-methylenebis(5-isopropyl-2-methyl)phenol, 4-allyl-6-(hydroxymethyl)-2-methoxyphenol, and 4-(hydroxymethyl)-2-isopropyl-5-methylphenol. The obtained derivatives showed remarkably better antioxidative properties according to 1,1-diphenyl-2-picrylhydrazyl assay (50% inhibitory concentrations = 4-156 microg/mL) and Rancimat assay (protection factors = 1.55-5.84) when compared with parent compounds and values similar to or better than those of butylated hydroxytoluene and vitamin C. At concentrations of 10 mM carvacrol derivatives had no toxic effect on viability of Escherichia coli K-12 (determined by minimum inhibitory concentrations). Other phenol derivatives showed reduced cytotoxic effect on E. coli K-12 at concentrations of 2-5 mM on the basis of 50% lethal dose measurements. In comparison with the parent compounds, phenol derivatives showed reduced cytotoxic effect for Saccharomyces cerevisiae cells (determined by yeast colony reduction). On the other hand, the majority of synthesized compounds had dose-dependent antiproliferative effects on human uterine carcinoma cells (HeLa), which makes them potentially interesting for the adjuvant experimental cancer treatments. The 4,4'-methylenebis(5-isopropyl-2-methyl)phenol derivative of carvacrol showed lower inhibiting capacity also for the HeLa cells, which makes this particular derivative attractive as an efficient antioxidant with negligible cytotoxic effects.     

Photosynthesis,chlorophyll fluorescence,and water relations in young sugar beet plants as affected by sulfur supply

The effect of different concentrations of sulfur (1 and 3 mM) and interruption of sulfur (S) supply for 25 days on the photosynthesis and leaf water relations in young sugar beet plants (Beta vulgaris L.) was studied in water culture, under greenhouse conditions. Interruption of S‐supply significantly reduced the content of sulfur, chlorophylls a+b and carotenoids, leaf area, density of stomatal and epidermal cells, transpiration rate and leaf water potential, while it increased the free proline content and stomatal diffusion resistance. An increase in S concentration in the nutrient medium from 1 to 3 mM did not significantly affect the tested parameters, except for an increase in leaf S content and a decrease of leaf water potential. Sulfur deficiency caused a pronounced decrease of the rate and quantum yield of photosynthetic oxygen evolution under non‐photorespiratory conditions. This was partly the result of the diminished photochemical efficiency of photosystem II reaction centers. Less efficient excitation of PSII reaction centers is most probably the consequence of higher thermal energy dissipation in the reaction centers of S‐starved plants. These data support that S nutrition is one of the factors regulating plant photosynthesis.     

Leukocyte subsets and specific antibodies in pigs vaccinated with a classical swine fever subunit (E2) vaccine and the attenuated ORF virus strain D1701

Total white blood cell (WBC) counts and percentages of CD4a+, CD8a+, CD5a+, CD45RA+, CD45RC+, wCD21+ and SWC3a+ cells in the peripheral blood of pigs were analysed in this study. Blood samples were collected before and on days 4, 10, 21 and 28 after vaccination. Group 1 pigs were vaccinated with a subunit E2 vaccine (gp E2 32 microg/dose), and Group 2 received a subunit vaccine combined with an attenuated ORF virus strain D1701 10(6.45) TCID50/dose. Control pigs received a placebo. The total WBC count and percentage of particular cell types were within the normal range in vaccinated and control pigs. Although the mechanism of attenuated ORF virus activity is not clear, changes were observed in CD4a+, CD5a+, CD8a+, CD45RA+ and CD45RC+ cells in pigs that received the combination of a subunit vaccine and ORF virus. However, the percentage of wCD21+ and SWC3a+ did not differ significantly from that recorded in pigs given only the subunit vaccine. At days 4 and 10 the number of pigs positive to E2 antibodies was higher in the group that received the subunit vaccine and ORF virus than in pigs vaccinated with the subunit vaccine only. A higher percentage of memory cells (CD45RC+) as well as Th and Tc lymphocytes in pigs that received the ORF virus and the subunit vaccine could be ascribed to a nonspecific influence of the ORF virus on the development (through cognate interactions between T and B cells) and the duration (presumed according to the finding of the clonal expression of memory cells) of humoral immunity (assessed by a higher number of seropositive pigs in this group). This seems likely since the proportion of these cells was found to be lower in the pigs that received E2 vaccine only.     

Nematicidal activity of (E,E)-2,4-decadienal and (E)-2-decenal from Ailanthus altissima against Meloidogyne javanica

Methanol extracts of various plant parts of Ailanthus altissima were tested against the root knot nematode Meloidogyne javanica . Extracts of bark (ABE), wood (AWE), roots (ARE), and leaves (ALE) from A. altissima were investigated against freshly hatched second-stage juveniles (J(2)). AWE was the most active extract, with EC(50/3d) of 58.9 mg/L, while ALE, ARE, and ABE did not show nematicidal activity. The chemical composition of the extracts of A. altissima was determined by gas chromatography-mass spectrometry, and (E,E)-2,4-decadienal, (E)-2-undecenal, (E)-2-decenal, hexanal, nonanal, and furfural were the most prominent constituents. (E,E)-2,4-Decadienal, (E)-2-decenal, and furfural showed the highest nematicidal activity against M. javanica , with EC(50/1d) = 11.7, 20.43, and 21.79 mg/L, respectively, while the other compounds were inactive at the concentrations tested. The results obtained showed that AWE and its constituents (E,E)-2,4-decadienal and (E)-2-decenal could be considered as potent botanical nematicidal agents.     

Insect Pests

The fall armyworm, Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), is a major pest of maize and frequently demands control measures. The timing of insecticide application is a key factor in determining its efficiency, so an experiment was designed to investigate this. Application of insecticide was based on three criteria: (i) the number of trap-caught moths in a Delta-type trap with a commercial sex pheromone lure placed in the center of the target area, soon after plant emergence; (ii) the percentage of plants exhibiting pinhole-type damage (10% or 20%) and (iii) the percentage of plants exhibiting shot hole-type damage (10% or 20%) compared to a check plot without any control measures. We found that the number of trap-caught moths was, compared to the other methods, the best means of deciding on insecticide application in maize to control the fall armyworm. Using pheromone traps, we obtained the best performance of the insecticide Spinosad, causing >90% larval mortality. Without insecticide application, maize yield reduction due to fall armyworm larva damage was 39%.     

Susceptibility to flavescence dorée of different Vitis vinifera genotypes from north-western Italy

The aim of this work was to evaluate the susceptibility to flavescence dorée (FD) of 12 Vitis vinifera cultivars grown in Piedmont, and representative of the wine-making tradition of this area. The experiments were conducted under controlled conditions to ensure constant infection pressure. Test plants were ex vitro potted vines, singly inoculated with four Scaphoideus titanus infected by FD-C phytoplasma (FDp), under greenhouse conditions. Vines were tested for FDp at 5 and 8 weeks postinoculation (wpi) and the phytoplasma load was measured in leaves and roots at 8 wpi. Within the 14 V. vinifera accessions (belonging to 12 cultivars), three susceptibility clusters were identified. Cultivars within the low susceptibility group showed low phytoplasma loads and low percentages of infected plants, suggesting a tolerant behaviour. To confirm these results, four Vitis cultivars, representing extremes of FD susceptibility from low to high, were grafted onto Kober 5BB rootstocks and inoculated with laboratory-infected S. titanus, under semi-field conditions. The transmission experiments onto grafted cuttings confirmed that susceptibility to the disease depends on the scion genotype. The data indicated that none of the tested V. vinifera genotypes are resistant to FD, although some cultivars with low susceptibility are available, and can be explored for identifying genetic traits involved in disease tolerance/resistance. Moreover, ranking Vitis genotypes for their susceptibility to FD is in itself a valuable tool to support vine growers in their decision management, by helping them to choose the most appropriate varieties according to their specific FD epidemiological contexts.     

Soil fungal isolates produce different organic acid patterns involved in phosphate salts solubilization

Phosphorus availability is a major limiting factor for yield of most crop species. The objective of this study was to compare the solubilization of three sources of phosphorus (P) by different fungal isolates and to determine the possible mechanisms involved in the process. Talaromyces flavus (S73), T. flavus var flavus (TM), Talaromyces helicus (L7b) and T. helicus (N24), Penicillium janthinellum (PJ), and Penicillium purpurogenum (POP), fungal strains isolated from the rhizosphere of crops, are known to be biocontrol agents against pathogenic fungi. The P solubilization efficiency of these fungal strains in liquid media supplemented either with tricalcium phosphate (Ca₃(PO₄)₂; PC), aluminum phosphate (AlPO₄; AP), or phosphorite (PP) depended on the source of P and the fungal species. The type and concentration of organic acids produced by each species varied according to the source of available P. In the medium supplemented with PC, the highest proportion was that of gluconic acid, whereas in the media supplemented with the other P sources, the highest proportion was that of citric and valeric acids. This suggests that the release of these organic compounds in the rhizosphere by these microorganisms may be important in the solubilization of various inorganic P compounds. Results also support the hypothesis that the simultaneous production of different organic acids by fungi may enhance their potential for solubilizing insoluble phosphate.     

Immune complex-based vaccine for pig protection against parvovirus

The insoluble immune complexes (ICs) were prepared under the conditions of double immunodiffusion in gel, using the suspension of the ultrasound treated PK-15 cell-line infected with porcine parvovirus (PPV) containing both viral particles and viral proteins, as well as pig or rabbit anti-PPV polyclonal immune sera. The immunodiffusion performed in an agarose gel allows only viral subunits with a molecular mass equal to or less than 1000 kDa, rather than the viral particles, to diffuse through the gel and reach the point where the immunoprecipitate is to be formed. The immunoprecipitation under the conditions of the diffusion ensures the optimal, i.e. equimolar ratio of both immunoprecipitating components, antibody/antigen in the IC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blot analyses showed the ICs were composed of two proteins, a protein in which molecular mass corresponded to the VP2 of the PPV and a protein with a molecular mass of the IgG. This suggests that the ICs are mainly composed of the VP2 antigen and IgG class antibodies. The potency of the IC-vaccines prepared in the form of a water-in-oil-in-water emulsion was compared with that of a commercially available, inactivated oil vaccine. The vaccination of gilts, 6 weeks before mating, with the IC containing allogeneic pig antibodies, resulted in the development of high and long-lasting anti-PPV antibody titres, similar to those generated by the licenced vaccine (P > 0.01). The content of the virus material administered by the IC was twice lower than that in the licenced vaccine. Neither systemic nor local reactions were observed in the gilts during the period of the trial with the IC vaccine. The number of viable piglets per litter varied between 9 and 12 and no signs of the PPV infection were detected. Rabbits were used as one of the alternative laboratory animal models accepted for the testing of the vaccine against the PPV. The rabbit humoral immune response generated by the IC containing the allogeneic antibodies were higher than that generated by the ICs containing the xenogeneic pig antibodies. It was similar to that generated by two-times higher content of the virus material administered by a commercially available vaccine. The IC-based vaccines belong to non-replicating, subunit vaccines, which are both ecologically convenient and the safest vaccines of all.     

Characterization of Clavibacter michiganensis subsp. michiganensis strains from recent outbreaks of bacterial wilt and canker in Serbia

Sixty-eight Clavibacter michiganensis subsp. michiganensis (Cmm) strains from recent outbreaks of bacterial wilt and canker in Serbia were collected from several tomato growing regions during a three-year period. The pathogen was identified based on bacteriological characteristics and pathogenicity tests and the identity of strains was confirmed by DAS ELISA and PCR amplification using primers CMM5/6 and PSA4/R. The strains showed homogeneity in biochemical and physiological properties. However, pathogenicity tests revealed differences in virulence that are presumably due to a loss of the pat-1 gene. Further strain characterization using DNA-based methods revealed a high diversity of the Serbian Cmm strains. Based on multi-locus sequence typing (MLST) analyses of five genes, Cmm strains were divided into seven groups. The pulsed-field gel electrophoresis (PFGE) pattern of a selection of strains supported the groupings based on trees of the kdpA/sdhA sequences. On the other hand, groupings made according to PFGE and MLST were not correlated to plasmid content in all cases. This study suggested that high genetic variability of the Serbian Cmm strains was detected both in MLST and PFGE analyses, and could have resulted either from new Cmm strains being introduced by seeds from different origins or as a consequence of an intraspecific hybridization process. In addition, this study proposed MLST as an efficient tool in epidemiological studies, population biology investigations and tracking the routes of transmission of pathogens. Four of the five house-keeping genes (kdpA, sdhA, ligA and gyrB) selected to characterize Cmm strains proved to be suitable for the MLST analysis. This is the first study carried out on the characterization of Cmm using MLST.