Use of Pseudomonas protegens to Control Root Rot Disease Caused by Boeremia exigua var. exigua in Industrial Chicory (Cichorium intybus var. sativum Bisch.).

Boeremia exigua var. exigua is a recurrent pathogen causing root rot in industrial chicory. Currently, there is no chemical or varietal control for this disease, and thus, management strategies need to be developed. This study determined the biocontrol effect of strains of Pseudomonas protegens bacteria with antimicrobial compounds on the fungus B. exigua var. exigua under in vitro, in vivo, and field conditions. In addition, root colonization by these bacteria was estimated by the phlD-specific PCR-based dilution end point assay. Eighteen isolates of Pseudomonas spp were evaluated, and the strains that showed the greatest in vitro inhibition of fungal mycelial growth (mm), Ca10A and ChB7, were selected. Inoculation with the strain ChB7 showed less severity (necrotic area) under in vivo conditions (root trials) compared with the control inoculated with the pathogen (p ≤ 0.05). The molecular analysis revealed that the root colonization of plants grown in pots was equal to or greater than 70%. Similar levels were observed in the field trials conducted at the Selva Negra and Canteras experimental stations (2015-2016 season), with values ranging from 85.7 to 70.5% and from 75.0 to 79.5%, respectively. Regarding yield (ton ha-1), values were higher in the treatments inoculated with strains Ca10A and ChB7 (p ≤ 0.05) at both experimental sites, while a lower incidence and severity of root rot were observed at Selva Negra. These results suggest that the Chilean strains of P. protegens are a promising tool for the control of root diseases in industrial chicory.

Indigenous Yeasts for the Biocontrol of Botrytis cinerea on Table Grapes in Chile.

One hundred twenty-five yeast strains isolated from table grapes and apples were evaluated for the control Botrytis cinerea of in vitro and in vivo. Ten strains were selected for their ability to inhibit mycelial growth of B. cinerea in vitro. In the in vivo assays, these yeasts were tested at 20 °C on 'Thompson Seedless' berries for 7 days; only three were selected (m11, me99 and ca80) because they significantly reduced the incidence of gray mold. These three yeast strains were then evaluated at different concentrations (1 × 107, 1 × 108 and 1 × 109 cells mL-1) on 'Thompson Seedless' grape berries at 20 °C. The strains m11, me99 and ca80 reduced the incidence of B. cinerea to 11.9, 26.1 and 32.1%, respectively, when the berries were submerged in a yeast suspension at a concentration of 1 × 109 cells mL-1 24 h before inoculation with B. cinerea. The most favorable pH for antifungal activity was 4.6 in the three isolates. The three yeast strains secreted the hydrolytic enzymes chitinase and ß-1-glucanase, and two strains (me99 and ca80) produced siderophores. The three yeast strains exhibited low oxidative stress tolerance and only strain m11 had the ability to produce biofilms. The strains were identified using 5.8S-ITS rDNA PCR-RFLP and correspond to the Meyerozyma guilliermondii (m11) and Aureobasidium pullulans (me99 and ca80) species.

Molecular and cultural characterization of Morchella spp. from disturbed environments of central-southern Chile.

In Northwestern Patagonia (Chile), three species of Morchella from undisturbed environments have been identified to date: Morchella tridentina, Morchella andinensis and Morchella aysenina, all belonging to the Elata clade and associated mainly with Nothofagus forests. In this study, the search for Morchella specimens was extended to disturbed environments in Central-Southern Chile, to further explore Morchella species diversity in the country, which is still very limited. The Morchella specimens were identified through multilocus sequences analysis, and the mycelial cultures were characterized, establishing comparisons with specimens from undisturbed environments. To the best of our knowledge, these results reveal for the first time in Chile the presence of the species Morchella eximia and Morchella importuna, and in the case of the last one also the first record in South America. These species were found associated almost exclusively with harvested or burned coniferous plantations. The in vitro mycelial characterization revealed certain inter- and intra-specific patterns of the morphology, such as pigmentation, mycelium type, and development and formation of sclerotia, which varied according to growth media and incubation temperature. The growth rates (mm/day) and mycelial biomass (mg) were significantly influenced by the temperature (p < 0.05), with maximum rates (>10 mm/day) and biomass (approx. 20 mg) between 20 and 24 °C, while a significant growth reduction (70-90%) was observed at 28 °C, mainly in the species from undisturbed environments. Potato-dextrose (PDA) medium stimulated the greatest mycelial density and sclerotia formation in most of the isolates, mainly in M. eximia (UDEC-LAF 236 isolate) which recorded the best mycelial growth performance. Among isolates, UDEC-LAF 236 also showed the best performance in sclerotia production (>350 sclerotia/dish) in 10 days of growth. This study contributes to the knowledge of the diversity of Morchella species in Chile by broadening the species range to those from disturbed environments. It also provides molecular and morphological characterization of the in vitro cultures of different Morchella species. The report on M. eximia and M. importuna, species known as cultivable, adapted to local climatic and edaphic conditions could represent the first step to developing artificial Morchella cultivation methods in Chile.

Next Generation Ingredients Based on Winemaking By-Products and an Approaching to Antiviral Properties.

Management of waste and use of winemaking by-products plays an important role in the development of new ingredients, especially with antiviral properties. Although the richness of bioactive compounds from wine waste is known, less is known about potential antiviral action. Bioactive compounds and health-enhancing effects of winery by-products make them potential candidates for use in antiviral ingredients. The design of new formulations by using nano-microencapsulation techniques will be necessary to successfully control diseases produced by viruses. Outcomes about the use of winery by-products, bioactive compounds found in winery wastes, green extraction techniques to concentrate these compounds, and development of formulations to obtain new ingredients were extracted from research around the world to be discussed and updated in this manuscript. The evidence collected in this review aims to encourage transfer of in vitro and in vivo knowledge to a new step for the development of antiviral and treatments.

Dietary Supplement of Grape Wastes Enhances Honeybee Immune System and Reduces Deformed Wing Virus (DWV) Load.

Ingredients rich in phenolic compounds and antioxidants of winemaking wastes, which play an important role in the prevention of various diseases and the control of viruses, are being explored. Currently, there is a concern about honeybee population loss, with deformed wing virus (DWV) being the most common virus infecting apiaries and one of the main causes of honeybee decline. Hence, the effect of grape pomace powder (GPP) as a dietary supplement to enhance the immune system of honeybees affected by DWV was evaluated. The characteristics of the ingredient GPP, obtained by spray-drying, revealed a high anthocyanin content (1102.45 mg 100 g-1), and it was applied at doses of 0.5, 1, 2.5 and 5% as a dietary supplement for bees infected by DWV. The results showed that the GPP treatments strengthened the immune response of honeybees against DWV. Moreover, the expression of the Relish gene was significantly higher in bees fed with GPP compared to the infected control. This study, which is framed in the search of food waste valorization for environmental sustainability, proves the feasibility of using grape wastes as dietary supplements for pollinators, and provides knowledge of the influence of polyphenols on the expression profiles of immune-related genes in honeybees.

Variant A of the Deformed Wings Virus Alters the Olfactory Sensitivity and the Expression of Odorant Binding Proteins on Antennas of Apis mellifera.

Insects have a highly sensitive sense of smell, allowing them to perform complex behaviors, such as foraging and peer recognition. Their sense of smell is based on the recognition of ligands and is mainly coordinated by odorant-binding proteins (OBPs). In Apis mellifera, behavior can be affected by different pathogens, including deformed wing virus (DWV) and its variants. In particular, it has been shown that variant A of DWV (DWV-A) is capable of altering the ultra-cellular structure associated with olfactory activity. In this study was evaluated olfactory sensitivity and the expression of OBP genes in honey bees inoculated with DWV-A. Electroantennographic analyses (EAG) were carried out to determine the olfactory sensitivity to the essential oils Eucalyptus globulus and Mentha piperita. The expression of nine antenna-specific OBP genes and DWV-A load in inoculated bees was also quantified by qPCR. We observed an inverse relationship between viral load and olfactory sensitivity and the expression of some OBP proteins. Thus, high viral loads reduced olfactory sensitivity to essential oils and the gene expression of the OBP2, OBP5, OBP11, and OBP12 proteins on the antennas of middle- and forager-age bees. These results suggest that DWV-A could have negative effects on the processes of aroma perception by worker bees, affecting their performance in tasks carried out in and outside the colony.

Impact of Mixed Infections of Gut Parasites Lotmaria passim and Nosema ceranae on the Lifespan and Immune-related Biomarkers in Apis mellifera.

Lotmaria passim currently appears to be the predominant trypanosome in honey bees worldwide. Although, the specific effects of L. passim by single or mixed with other gut parasites such as Nosema ceranae on honey bees' health is still unclear. We consequently measured bees' survival, parasite loads, the expression of antimicrobial peptides (AMPs) and vitellogenin gene. Thus, (1) bees naturally infected with L. passim, (2) healthy bees inoculated with Nosema ceranae, (3) bees naturally infected with L. passim and inoculated with N. ceranae and (4) healthy bees (control) were maintained under controlled conditions. Honey bees infected with N. ceranae or with mixed infections of L. passim and N. ceranae had significantly lower survival rates than the control group at 20 days post-inoculation (dpi). A competitive suppression was also detected, provided that the L. passim load was significantly affected by the presence of N. ceranae at 15 dpi. Expressions of the AMPs defensin and hymenoptaecin rapidly (two hours post-inoculation) increased in bees infected with N. ceranae and mixed infections. However, this effect was not continuous. In fact, expressions of abaecin, defensin, hymenoptaecin and vitellogenin decreased drastically at 15 dpi in bees with both single and mixed infections. The decrease in the expression of AMPs and vitellogenin throughout this period was consistent with the reduced survivals observed in this study, indicating that mixed infections of L. passim and N. ceranae, and even into a scenario of competition between them, may have a synergic effect on the survival and immune-related gene expressions (biomarkers) of worker bees.

PCR-specific detection of recently described Lotmaria passim (Trypanosomatidae) in Chilean apiaries.

The recently described trypanosome Lotmaria passim is currently considered the most predominant trypanosomatid in honey bees worldwide and could be a factor in honey bee declines. For a specific and quick detection of this pathogen, we developed primers based on the SSU rRNA and gGAPDH genes for the detection of L. passim in Chilean honey beehives. PCR products amplified and sequenced for these primers shared 99-100% identity with other sequences of L. passim. The designed primers were specific and we were able to detect a high prevalence (40-90%) of L. passim in bee hives distributed throughout Chile. Our described PCR-based method offers a feasible and specific detection of L. passim in any honey bee samples.

Efficient double-stranded RNA production methods for utilization in plant virus control.

Double-stranded RNA (dsRNA) is an inducer molecule of the RNA silencing (RNA interference, RNAi) pathway that is present in all higher eukaryotes and controls gene expression at the posttranscriptional level. This mechanism allows the cell to recognize aberrant genetic material in a highly sequence specific manner. This ultimately leads to degradation of the homologous target sequence, rendering the plant cell resistant to subcellular pathogens. Consequently, dsRNA-mediated resistance has been exploited in transgenic plants to convey resistance against viruses. In addition, it has been shown that enzymatically synthesized specific dsRNA molecules can be applied directly onto plant tissue to induce resistance against the cognate virus. This strongly implies that dsRNA molecules are applicable as efficacious agents in crop protection, which will fuel the demand for cost-effective dsRNA production methods. In this chapter, the different methods for dsRNA production-both in vitro and in vivo-are described in detail.

Razones de consumo de frutas y vegetales en escolares costarricenses de una zona urbana / Reasons for fruit and vegetable consumption in Costa Rican students of an urban zone

Objetivo: Determinar cuáles son las razones del consumo y no consumo de frutas y vegetales por parte de los escolares de una zona urbana. Método: Estudio de tipo descriptivo, transversal y cualitativo. Participaron 72 escolares entre 10 y 12 años de cuarto, quinto y sexto grado de tres escuelas de los cantones de Montes de Oca y Curridabat, de la provincia de San José, Costa Rica, en el año 2004. Se realizaron 6 grupos focales, de los cuales 3 fueron con niñas y 3 con niños. La información obtenida fue categorizada a partir del análisis de contenido y de matrices en las que se consideraron las opiniones más comunes dadas por los escolares, así como los conceptos derivados de las dimensiones establecidas en la literatura y las opiniones de expertos en educación y nutrición. Resultados: Los escolares refieren que la principal razón de consumo de frutas y vegetales, son las características físicas, para el caso de los vegetales estas características son también motivo de rechazo. Entre las razones de no consumo están la poca disponibilidad tanto en el hogar como en las sodas y comedores escolares, los inadecuados hábitos alimentarios familiares, el desconocimiento de la importancia del consumo de estos alimentos y un poder de compra restringido. Los escolares reconocen que las frutas y los vegetales son alimentos que contribuyen a la buena salud porque aportan de manera general vitaminas y minerales, no obstante, no los consumen diariamente porque prefieren la "comida chatarra". Discusión: El consumo de frutas y vegetales está asociado con una serie de factores intrínsecos y extrínsecos que potencian o limitan el consumo de estos alimentos.

Objective: To determine the reasons for consumption and non-consumption of fruits and vegetables in school students of an urban zone. Methods: The present study is descriptive, cross-sectional and qualitative in nature. During 2004, seventy two schoolchildren between ten and twelve years of age, coming from the fifth and sixth grades of three schools in the municipalities of Montes de Oca and Curridabat, province of San José, participated in the study. The purpose of the work was to determine the reasons for fruit and vegetable intake. Six focal groups were organized, three with girls and three with boys. The information was classified on the basis of content and matrices analysis, in which the most common opinions given by the children were considered, in addition to the concepts derived from dimensions established in the literature, and expert opinion on education and nutrition. Results: The schoolchildren mentioned that the main reasons for consumption of both food groups are their physical characteristics. For vegetables, the physical characteristics were also a motive of rejection. Other reasons for non-consumption are reduced availability at home, restaurants and school cafeterias, inadequate family nutritional habits, ignorance of the importance of intake of these foods, and a reduced buying ability. The schoolchildren acknowledge that fruits and vegetables are foods that contribute to good health, because in general they provide vitamins and minerals. However, they do not eat them on a daily basis because they prefer "junk foods". Discussion: Fruit and vegetable consumption is associated with several intrinsic and external factors that can increase or limit their intake.

Transient expression of homologous hairpin RNA interferes with PVY transmission by aphids.

Hairpin RNAs have been used to confer resistance to viruses in plants through RNA silencing. However, it has not been demonstrated that RNA silencing was effective against inoculation by aphids of non-persistently transmitted viruses, the major route of plant virus spread in nature. As a proof-of-principle strategy, we made use of Agrobacterium tumefaciens to transiently express a hairpin RNA homologous to Potato virus Y (PVY) in plant tissues. A complete and specific interference with aphid transmission of PVY was achieved by inducers of RNA silencing, as demonstrated by specific siRNAs accumulation in agroinfiltrated tissues. To our knowledge, this is the first report of successful interference with non-persistent transmission of a plant virus using RNA interference.

Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections.

BACKGROUND: Double-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals. We have previously shown and patented that mechanical inoculation of in vitro-transcribed dsRNA derived from viral sequences specifically prevents virus infection in plants. The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour. RESULTS: We have developed an in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria, with a view to providing a practical control of virus diseases in plants. Partially purified bacterial dsRNAs promoted specific interference with the infection in plants by two viruses belonging to the tobamovirus and potyvirus groups. Furthermore, we have demonstrated that easy to obtain, crude extracts of bacterially expressed dsRNAs are equally effective protecting plants against virus infections when sprayed onto plant surfaces by a simple procedure. Virus infectivity was significantly abolished when plants were sprayed with French Press lysates several days before virus inoculation. CONCLUSION: Our approach provides an alternative to genetic transformation of plant species with dsRNA-expressing constructs capable to interfere with plant viruses. The main advantage of this mode of dsRNA production is its simplicity and its extremely low cost compared with the requirements for regenerating transgenic plants. This approach provides a reliable and potential tool, not only for plant protection against virus diseases, but also for the study of gene silencing mechanisms in plant virus infections.