Synergic interactions between 2-deoxy-D-glucose and Candida saitoana enhances citrus green mould control.

The activity of 2-deoxy-D-gLucose (2-DG) alone or in combination with a biocontrol yeast (Candida saitoana, strain 8C) was evaluated in vitro and in vivo against citrus green mould (Penicillium digitatum Sacc.). The in vitro assays were performed on amended potato dextrose agar (PDA) containing 0, 1.5, 3.0, 6.0, 15.0, 30.0 or 60.0 mM of 2-DG. P. digitatum conidia were sown on the amended media and growth inhibition occurred starting from 6.0 mM. A nearly total inhibition of the growth and spore germination occurred with 60.0 mM of 2-DG. The antagonist was not affected by any of the 2-DG concentrations employed and the amended plates resulted well colonized within 2 d post-treatment. In vivo assays were carried out with 'Hamlin' oranges, inoculated with P. digitatum 24 h before treating with: the antagonist; the above reported concentrations of 2-DG, or by combining the two treatments. Seven days post-treatment the inhibition activity exerted by 3.0, 6.0, 15.0, 30.0 and 60.0 mM of 2-DG combined with the yeast was 15, 37, 42, 63 and 84%, respectively. While that exerted by the antagonist was 22% and that by the different concentrations of 2-DG were 7, 11, 27, 42 and 57%, respectively. Compared to single treatments, the co-application significantly and in a synergic mode improved the control of decay. Alterations to the hyphae were observed by SEM when the pathogen was cultured on amended media and into the wounds of inoculated oranges.

Predictive models for carcinogenicity and mutagenicity: frameworks, state-of-the-art, and perspectives.

Mutagenicity and carcinogenicity are endpoints of major environmental and regulatory concern. These endpoints are also important targets for development of alternative methods for screening and prediction due to the large number of chemicals of potential concern and the tremendous cost (in time, money, animals) of rodent carcinogenicity bioassays. Both mutagenicity and carcinogenicity involve complex, cellular processes that are only partially understood. Advances in technologies and generation of new data will permit a much deeper understanding. In silico methods for predicting mutagenicity and rodent carcinogenicity based on chemical structural features, along with current mutagenicity and carcinogenicity data sets, have performed well for local prediction (i.e., within specific chemical classes), but are less successful for global prediction (i.e., for a broad range of chemicals). The predictivity of in silico methods can be improved by improving the quality of the data base and endpoints used for modelling. In particular, in vitro assays for clastogenicity need to be improved to reduce false positives (relative to rodent carcinogenicity) and to detect compounds that do not interact directly with DNA or have epigenetic activities. New assays emerging to complement or replace some of the standard assays include Vitotox, GreenScreenGC, and RadarScreen. The needs of industry and regulators to assess thousands of compounds necessitate the development of high-throughput assays combined with innovative data-mining and in silico methods. Various initiatives in this regard have begun, including CAESAR, OSIRIS, CHEMOMENTUM, CHEMPREDICT, OpenTox, EPAA, and ToxCast. In silico methods can be used for priority setting, mechanistic studies, and to estimate potency. Ultimately, such efforts should lead to improvements in application of in silico methods for predicting carcinogenicity to assist industry and regulators and to enhance protection of public health.

Host-pathogen-biocontrol agent interaction as affected by sequential application of Na2CO3 and CaCl2.

Among the alternatives to synthetic postharvest fungicides encouraging results have been reported with biocontrol agents, and on Citrus fruits, their efficacy was improved when co-applied with GRAS compounds or with physical means. Still, the reason for this increased efficacy has not been explained and therefore a study was performed using orange fruit (Citrus sinensis Osbec. cv 'Washington navel') as host, P. digitatum as the pathogen, a yeast (Pichia guiliermondii, isolate 5A) as the biocontrol agent, white 2% Na2CO3 (SC) and 1% CaCl2 were employed as GRAS compounds. When treatments were combined salts were applied sequentially, and SC preceded CaCl2 followed by the yeast. As a result of large scale trait with inoculated and un-inoculated fruit a clear beneficial interaction occurred when treatments were combined. SC exerted a direct fungistatic activity and an indirect one by inducing scoparone in host tissue. Also the isolate A5 induced the phytoalexin accumulation and when combined with SC a greater accumulation occurred within the first 7 days post-treatment. The application of CaCl2 alone had no effect on pathogenesis, while when combined with SC or with the yeast, decay was towered. The yeast growth on an amended medium was negatively affected by the addition of SC; while in vivo this effect was missing. The antagonist growth in vivo was enhanced when applied together with 1% CaCl2 also when applied with SC. The results reported improve our knowledge on the complex interactions among host, pathogen and the antagonist as affected by SC and CaCl2.

A microarray to measure repair of damaged plasmids by cell lysates.

DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.

Induction of phytoalexins biosynthesis in orange fruit by the biocontrol yeast Rhodotorula glutinis.

The biocontrol yeast Rhodotorula glutinis, isolate 21A, obtained from tomato fruit was used to control Penicillium digitatum, P. italicum and Botrytis cinerea on artificially wounded citrus fruit. Orange and satsuma mandarin fruit were treated with the biocontrol yeast, inoculated with the pathogens and stored for 7 days at 23 degrees C. On orange fruit the antagonist compared to the control reduced decay by 92.2, 88.4 and 96.2% for P. digitatum, P. italicum and B. cinerea, respectively. On satsuma mandarin fruit the same pathogens were inhibited by 96.2, 91.2 and 90.0%, respectively. Scanning electron microscope observations, focusing on the antagonist-pathogen interactions, revealed a fast colonization of the growing fungal mycelia by the yeast cells, but no sign of lytic activity on hyphae was observed. Moreover, the fruit accumulated the phytoalexins scoparone and scopoletin into artificial wounds previously treated by the yeast and either inoculated or uninoculated with the pathogen. The concentration of scoparone, which showed higher accumulation in fruit tissues, varied significantly in relation to the time lag between the application of the antagonist and the inoculation with the pathogen. In particular, the concentration of scoparone 4 days after application of the sole yeast was 69.0 microg x g(-1) fresh weight (FW), 6.3 times higher than in the uninoculated wounded tissues (11.0 microg x g(-1) FW). The phytoalexin accumulation was low (13.0 microg x g(-1)FW) applying the yeast jointly with P. digitatum into wounds, while it increased consistently (74.0 microg x g(-1)FW) when the antagonist was applied 24 h before the pathogen.

Inhibitory activity of 2-deoxy-D-glucose and Candida saitoana against Penicillium digitatum.

The toxic activity of 2-deoxy-D-glucose (2-DG) alone or combined with the biocontrol yeast Candida saitoana strain 8C was evaluated in vitro and in vivo against the postharvest fungal pathogen Penicillium digitatum. In order to assess the effect of the 2-DG on both the biocontrol yeast and fungal pathogen, in vitro tests were performed in Petri dishes containing potato dextrose agar amended with different concentrations (1.5, 3.0, 6.0, 15.0, 30.0, 60.0 mM) of the sugar. The plates were then seeded with 25 microl of a P. digitatum conidial suspension at 10(5) conidia/mL. Result of the assays showed an enhanced inhibitory activity as concentration increased from 15.0 to 60.0 mM. Corroborated by SEM observations showing a reduced growth and the appearance of damaged hyphae were found. At 60 mM of 2-DG, a total inhibition occurred while concentrations from 1.5 to 6.0 mM resulted ineffective. The same tests evidenced no adverse effects on the yeast 8C at all tested concentrations. In vivo assays were carried out on orange fruit cv 'Biondo comune', wounded in 5 sites around the calyx. Each wound (2.5 wide and 3.4 mm depth) was first filled with 25 microl of a 0, 3.0, 6.0, 15.0, 30.0 or 60.0 mM 2-DG-water solution alone or combined with the yeast 8C at 10(8) cells/mL and then a 25 microl of the P. digitatum conidial suspension was added. Each treatment consisted of 3 replicates of 8 fruit (5 wounds/fruit) for a total 120 wounds per treatment. Oranges were maintained at 20 degrees C and high RH (95-98%) for up to 5 days, during which infection was monitored and the inhibitory activity calculated. The tests in vitro evedenced a significant slowing of the pathogen growth with the highest concentrations of 2-DG (15.0, 30.0 and 60.0 mM) with respect to the control; while at lower concentrations (1.5, 3.0, 6.0 mM) the development of the fungi was not significantly reduced. C. saitoana was resistant to all the doses employed to the abovementioned compound. In vivo the yeast alone was more effective compared to the sugar alone up to 6.0 mM while, at higher concentrations an additive effect was founded.

Biological and physical approaches to improve induced resistance against green mold of stored citrus fruit.

Health and environmental concerns have point out the need to improve or change several manufacturing steps in the food chain. In this context particular attention should be given to the technologies involved in fruits and vegetables production. Nearly all fresh fruit and vegetables are subjected to different periods of storage and/or shelf-life before of their consumption. This implies the need to protect the commodities from microbial spoilage. Some Citrus species (e.g. lemon and grapefruit) may be stored for several months before consumption and then post-harvest treatments are essential to contain green (Penicillium digitatum) and blue (P. italicum) moulds. Alternative approaches to chemicals usually have a lower efficacy in containing rots but fulfill the consumer's expectation. Among the alternative strategies, the improvement of host natural resistance is promising. In this regard, we report some results concerning the use of biotic (yeast) and abiotic agents as inducers of phytoalexin (i.e. scoparone and/or scopoletin) accumulation in Citrus rind and its importance in the control of fungal decay. In all experiments the inducers were applied on fruits before or 24 h after inoculation with P. digitatum and the rot severity was monitored 7 days later. The accumulation of phytoalexins was monitored according to a standard methodology by HPLC. In all experiments a positive correlation was found between increase of the phytoalexin scoparone in host tissue and reduction of decay.

Fungitoxic activity of 12 essential oils against four postharvest citrus pathogens: chemical analysis of thymus capitatus oil and its effect in subatmospheric pressure conditions.

The fungitoxic activity against Penicillium digitatum, Penicillium italicum, Botrytis cinerea, and Alternaria citri of 12 essential oils (EOs) distilled from medicinal plants is reported. The results of the in vitro trials show strong fungitoxic activity of Thymus capitatus (L.) Hofmgg EOs, which inhibited the growth of the four fungi at a concentration of 250 ppm (vol/vol). The other 11 essences reduced the development of the fungi from 95 to 9% at 250 ppm (vol/vol). The fungitoxic activity of T. capitatus EOs (75, 150, and 250 ppm) on healthy orange fruits, inoculated with P. digitatum (10(8) conidia ml(-1)) by spraying and placed in 10-liter desiccators, was weak at atmospheric pressure (3 to 10% inhibition at all three concentrations), while in vacuum conditions (0.5 bar), conidial mortality on the exocarp was high (90 to 97% at all three concentrations). These data proved not to be statistically different from treatments with thiabendazole-TBZ (2,000 ppm). Scanning electron microscope observations showed that T. capitatus EO vapors altered the morphology of P. digitatum hyphae and conidia. The gas-chromatographic analyses of thyme EO indicated that carvacrol was present at 81 to 83%, p-cymene at 4.5 to 5%, gamma-terpinene at 2.6 to 3.3%, caryophyllene at 1.5 to 1.6%, beta-myrcene at 1.6%, and linalool at 1.1 to 1.2%. Carvacrol proved to be the most important fungitoxic compound among the thyme EO constituents, but, unlike thyme EO, it caused alterations to the fruit at the concentration of 75 ppm.

Nutritional status and body composition of adolescent female gymnasts.

Nutritional status and body composition of 26 young female gymnasts (average age 12 years) were studied. The body fat percentage (15% of total body weight) is low, still consistent with an excellent health. The caloric intake is lower (1552 kcal/day) than that recommended for their age group, however it is still within the standard by body weight (43 kcal/kg). They show and insufficient assumption of carbohydrates (47.7%), mineral salts and vitamins, so that a more detailed alimentary information is required in order to avoid undesired consequences on sport performance and growth rate.

Prolactin release induced by physical exercise is independent from peripheral vasoactive intestinal polypeptide secretion.

The plasma concentrations of vasoactive intestinal polypeptide and prolactin were measured before and after an exhaustive and a submaximal exercise test in 7 male marathon runners. A significant increase of vasoactive intestinal polypeptide was recorded after both tests, whereas the prolactin increase was observed only after the exhaustive exercise test. No significant correlation was found between the plasma vasoactive intestinal polypeptide and the plasma prolactin values recorded during the two exercise tests. Data suggest that the exercise-induced prolactin release occurs independently from variations of the vasoactive intestinal polypeptide levels in peripheral circulation.