Comparative Genomics of a Polyvalent Escherichia-Salmonella Phage fp01 and In Silico Analysis of Its Receptor Binding Protein and Conserved Enterobacteriaceae Phage Receptor.

The polyvalent bacteriophage fp01, isolated from wastewater in Valparaiso, Chile, was described to have lytic activity across bacterial species, including Escherichia coli and Salmonella enterica serovars. Due to its polyvalent nature, the bacteriophage fp01 has potential applications in the biomedical, food and agricultural industries. Also, fundamental aspects of polyvalent bacteriophage biology are unknown. In this study, we sequenced and described the complete genome of the polyvalent phage fp01 (MH745368.2) using long- (MinION, Nanopore) and short-reads (MiSeq, Illumina) sequencing. The bacteriophage fp01 genome has 109,515 bp, double-stranded DNA with an average G+C content of 39%, and 158 coding sequences (CDSs). Phage fp01 has genes with high similarity to Escherichia coli, Salmonella enterica, and Shigella sp. phages. Phylogenetic analyses indicated that the phage fp01 is a new Tequintavirus fp01 specie. Receptor binding protein gp108 was identified as potentially responsible for fp01 polyvalent characteristics, which binds to conserved amino acid regions of the FhuA receptor of Enterobacteriaceae.

Characterization of Xanthomonas arboricola pv. juglandis Bacteriophages against Bacterial Walnut Blight and Field Evaluation.

Xanthomonas arboricola pv. juglandis (hereafter X. juglandis) is the etiological agent of walnut blight, the most important bacterial disease affecting walnut production worldwide. Currently, the disease is treated mainly with copper-derived compounds (e.g., CuSO4) despite the evidence of genetic resistance in these strains. Regarding the effectiveness and sustainability, the use of a bacteriophage appears to be a biocontrol alternative to reduce X. juglandis load and symptomatology of walnut blight. Here, the phages f20-Xaj, f29-Xaj, and f30-Xaj were characterized, and their effectiveness in walnut orchards against walnut blight was determined. These bacteriophages showed a specific lytic infection in X. juglandis strains isolated from Chile and France. Phylogenetic analysis of the complete genome of f20-Xaj and f30-Xaj indicates that these phages belong to the Pradovirus genus. In the field, the cocktail of these bacteriophages showed similar effectivity to CuSO4 in the reduction of incidence and severity in walnut tissue. Moreover, the bacterial load of X. juglandis was significantly reduced in the presence of bacteriophages in contrast to a CuSO4 treatment. These results show that the use of bacteriophages can be an alternative to combat the symptoms of walnut blight caused by X. juglandis.

Characterization of Bacteriophages against Pseudomonas Syringae pv. Actinidiae with Potential Use as Natural Antimicrobials in Kiwifruit Plants.

Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of a bacterial canker in kiwifruit plants and has caused economic losses worldwide. Currently, the primary strategies to control this pathogen include the use of copper-based compounds and even antibiotics. However, the emergence of isolates of Psa that are resistant to these agrochemicals has raised the need for new alternatives to control this pathogen. Bacteriophages have been proposed as an alternative to control bacterial infections in agriculture, including Psa. Here, we show the isolation and characterization of 13 phages with the potential to control Psa infections in kiwifruit plants. The phages were characterized according to their host range and restriction fragment length polymorphism (RFLP) pattern. Four phages were selected according to their lytic effect on the bacteria and their tolerance to different environmental conditions of pH (4-7), temperature (4-37 °C), and solar radiation exposure (30 and 60 min). The selected phages (CHF1, CHF7, CHF19, and CHF21) were sequenced, revealing a high identity with the podophage of Psa phiPSA2. In vitro assays with kiwifruit leaf samples demonstrated that the mixture of phages reduced the Psa bacterial load within three hours post-application and was able to reduce the damage index in 50% of cases. Similarly, assays with kiwifruit plants maintained in greenhouse conditions showed that these phages were able to reduce the Psa bacterial load in more than 50% of cases and produced a significant decrease in the damage index of treated plants after 30 days. Finally, none of the selected phages were able to infect the other bacteria present in the natural microbiota of kiwifruit plants. These results show that bacteriophages are an attractive alternative to control Psa infections in kiwifruit plants.

Encapsulation of specific Salmonella Enteritidis phage f3αSE on alginate-spheres as a method for protection and dosification

Background: Bacteriophages have been proposed as an alternative to control pathogenic bacteria resistant to antibiotics. However, they are not extensively used due to different factors such as vulnerability under environmental conditions and the lack of efficient administration methods. A potential solution is the encapsulation of bacteriophages in hydrogel polymers to increase their viability and as a controlled release method. This work describes the use of alginate-Ca+2 matrixes as mechanisms for protection and dosification of the phage f3αSE which has been successfully used to prevent infections produced by Salmonella Enteritidis. Results: The viability of the pure phage is reduced in near 100% after 1-h incubation at pH 2 or 3. However, the encapsulated phage remains active in 80, 6% at pH 3, while no differences were observed at pH 2, 4 or 7. Exposition of f3αSE to different T° showed that the viability of this phage decreased with increased T° to near 15% at 60°C, while the encapsulated phage remains with 50% viability at same temperature. Finally, the encapsulation of phages showed to extend their presence for 100 h in the medium compared to non-encapsulated phages in a water flow system, which simulate automatic birdbath used in poultry industry, maintaining the phage concentration between 102 and 104 PFU/mL during 250 h. Conclusions: Encapsulation in alginate-Ca+2 spheres can be a good alternative to extend viability of phages and can be used as a phage method dosification method in water flow systems.

Draft Genome Sequence of Xanthomonas arboricola pv. juglandis J303, Isolated from Infected Walnut Trees in Southern Chile.

Here, we report the draft genome sequence of Xanthomonas arboricola pv. juglandis J303, isolated from infected walnut trees in southern Chile. The size of the genome is 5,066,424 bp with a G+C content of 65.4%. X. arboricola pv. juglandis J303 has several genes related to virulence, antibiotic resistance, and copper resistance.

Relationship among color development, anthocyanin and pigment-related gene expression in 'Crimson Seedless' grapes treated with abscisic acid and sucrose.

'Crimson Seedless' is one of the most important table grape varieties in Chile, but under certain environmental conditions, the fruit exhibits inadequate red color development, causing economic losses due to lower product quality. The use of plant growth regulators, such as abscisic acid (ABA) and ethylene, during development increases the anthocyanin content of the skin, improving the color of the berry. Recently, sucrose has been identified as a signaling molecule capable of regulating the expression of genes of the anthocyanin biosynthesis pathway. The aim of this study was to analyze the effect of application of ABA and/or sucrose on color development and their relationship with anthocyanin metabolism. Applications of ABA (400 ppm or 200 ppm) and/or sucrose (90 mM) were performed close to the véraison stage. During development and at harvest, quality attributes such as berry firmness, total soluble solids and titratable acidity were not affected by these treatments. Increased red color development was observed in fruits treated with ABA and/or sucrose, due to accumulation of anthocyanins. Fruits subjected to sucrose treatment showed higher levels of anthocyanins than untreated fruits but lower levels than fruits treated with ABA. Increased expression of genes involved in anthocyanin biosynthesis was observed in ABA- and sucrose-treated fruits compared to untreated fruits. Based on these findings, we demonstrated that sucrose improved fruit color development by increasing synthesis and accumulation of anthocyanins, thus allowing earlier harvests and improving table grape quality.

Complete Genome Sequences of Lytic Bacteriophages of Xanthomonas arboricola pv. juglandis.

Three bacteriophages, f20-Xaj, f29-Xaj, and f30-Xaj, with lytic activity against Xanthomonas arboricola pv. juglandis were isolated from walnut trees (VIII Bío Bío Region, Chile). These lytic bacteriophages have double-stranded DNA (dsDNA) genomes of 43,851 bp, 41,865 bp, and 44,262 bp, respectively. These are the first described bacteriophages with lytic activity against X. arboricola pv. juglandis that can be utilized as biocontrol agents.

Stability inside hen eggs of a salmonella enterica serovar enteritidis bacteriophage

Background: Bacteriophages are viruses that infect bacteria and therefore are widespread in nature. Those that lyse the pathogen Salmonella enterica serovar Enteritidis (SE) should be expected in niches in which this bacterium thrives, among others the avian egg. Furthermore, bacteriophages could remain viable in this milieu. Results: Upon artificially infecting hen eggs with the SE bacteriophage f18 we found that the bacteriophage titer remains stable at least for up to 144 hrs post-infection , both in yolk and albumen at 25ºC. Conclusion: Bacteriophage f18 withstands the physico-chemical conditions of the egg inner milieu and could be considered for SE-controlling measures in the poultry industry.

Proteomic analysis of peach fruit mesocarp softening and chilling injury using difference gel electrophoresis (DIGE).

BACKGROUND: Peach fruit undergoes a rapid softening process that involves a number of metabolic changes. Storing fruit at low temperatures has been widely used to extend its postharvest life. However, this leads to undesired changes, such as mealiness and browning, which affect the quality of the fruit. In this study, a 2-D DIGE approach was designed to screen for differentially accumulated proteins in peach fruit during normal softening as well as under conditions that led to fruit chilling injury. RESULTS: The analysis allowed us to identify 43 spots -representing about 18% of the total number analyzed- that show statistically significant changes. Thirty-nine of the proteins could be identified by mass spectrometry. Some of the proteins that changed during postharvest had been related to peach fruit ripening and cold stress in the past. However, we identified other proteins that had not been linked to these processes. A graphical display of the relationship between the differentially accumulated proteins was obtained using pairwise average-linkage cluster analysis and principal component analysis. Proteins such as endopolygalacturonase, catalase, NADP-dependent isocitrate dehydrogenase, pectin methylesterase and dehydrins were found to be very important for distinguishing between healthy and chill injured fruit. A categorization of the differentially accumulated proteins was performed using Gene Ontology annotation. The results showed that the 'response to stress', 'cellular homeostasis', 'metabolism of carbohydrates' and 'amino acid metabolism' biological processes were affected the most during the postharvest. CONCLUSIONS: Using a comparative proteomic approach with 2-D DIGE allowed us to identify proteins that showed stage-specific changes in their accumulation pattern. Several proteins that are related to response to stress, cellular homeostasis, cellular component organization and carbohydrate metabolism were detected as being differentially accumulated. Finally, a significant proportion of the proteins identified had not been associated with softening, cold storage or chilling injury-altered fruit before; thus, comparative proteomics has proven to be a valuable tool for understanding fruit softening and postharvest.

Identification of woolliness response genes in peach fruit after post-harvest treatments.

Woolliness is a physiological disorder of peaches and nectarines that becomes apparent when fruit are ripened after prolonged periods of cold storage. This disorder is of commercial importance since shipping of peaches to distant markets and storage before selling require low temperature. However, knowledge about the molecular basis of peach woolliness is still incomplete. To address this issue, a nylon macroarray containing 847 non-redundant expressed sequence tags (ESTs) from a ripe peach fruit cDNA library was developed and used. Gene expression changes of peach fruit (Prunus persica cv. O'Henry) ripened for 7 d at 21 degrees C (juicy fruit) were compared with those of fruit stored for 15 d at 4 degrees C and then ripened for 7 d at 21 degrees C (woolly fruit). A total of 106 genes were found to be differentially expressed between juicy and woolly fruit. Data analysis indicated that the activity of most of these genes (>90%) was repressed in the woolly fruit. In cold-stored peaches (cv. O'Henry), the expression level of selected genes (cobra, endopolygalacturonase, cinnamoyl-CoA-reductase, and rab11) was lower than in the juicy fruit, and it remained low in woolly peaches after ripening, a pattern that was conserved in woolly fruit from two other commercial cultivars (cv. Flamekist and cv. Elegant Lady). In addition, the results of this study indicate that molecular changes during fruit woolliness involve changes in the expression of genes associated with cell wall metabolism and endomembrane trafficking. Overall, the results reported here provide an initial characterization of the transcriptome activity of peach fruit under different post-harvest treatments.

JUICE: a data management system that facilitates the analysis of large volumes of information in an EST project workflow.

BACKGROUND: Expressed sequence tag (EST) analyses provide a rapid and economical means to identify candidate genes that may be involved in a particular biological process. These ESTs are useful in many Functional Genomics studies. However, the large quantity and complexity of the data generated during an EST sequencing project can make the analysis of this information a daunting task. RESULTS: In an attempt to make this task friendlier, we have developed JUICE, an open source data management system (Apache + PHP + MySQL on Linux), which enables the user to easily upload, organize, visualize and search the different types of data generated in an EST project pipeline. In contrast to other systems, the JUICE data management system allows a branched pipeline to be established, modified and expanded, during the course of an EST project. The web interfaces and tools in JUICE enable the users to visualize the information in a graphical, user-friendly manner. The user may browse or search for sequences and/or sequence information within all the branches of the pipeline. The user can search using terms associated with the sequence name, annotation or other characteristics stored in JUICE and associated with sequences or sequence groups. Groups of sequences can be created by the user, stored in a clipboard and/or downloaded for further analyses. Different user profiles restrict the access of each user depending upon their role in the project. The user may have access exclusively to visualize sequence information, access to annotate sequences and sequence information, or administrative access. CONCLUSION: JUICE is an open source data management system that has been developed to aid users in organizing and analyzing the large amount of data generated in an EST Project workflow. JUICE has been used in one of the first functional genomics projects in Chile, entitled "Functional Genomics in nectarines: Platform to potentiate the competitiveness of Chile in fruit exportation". However, due to its ability to organize and visualize data from external pipelines, JUICE is a flexible data management system that should be useful for other EST/Genome projects. The JUICE data management system is released under the Open Source GNU Lesser General Public License (LGPL). JUICE may be downloaded from http://genoma.unab.cl/juice_system/ or http://www.genomavegetal.cl/juice_system/.

A rapid and efficient method for purifying high quality total RNA from peaches (Prunus persica) for functional genomics analyses.

Prunus persica has been proposed as a genomic model for deciduous trees and the Rosaceae family. Optimized protocols for RNA isolation are necessary to further advance studies in this model species such that functional genomics analyses may be performed. Here we present an optimized protocol to rapidly and efficiently purify high quality total RNA from peach fruits (Prunus persica). Isolating high-quality RNA from fruit tissue is often difficult due to large quantities of polysaccharides and polyphenolic compounds that accumulate in this tissue and co-purify with the RNA. Here we demonstrate that a modified version of the method used to isolate RNA from pine trees and the woody plant Cinnamomun tenuipilum is ideal for isolating high quality RNA from the fruits of Prunus persica. This RNA may be used for many functional genomic based experiments such as RT-PCR and the construction of large-insert cDNA libraries.

A Rapid and Efficient Method for Purifying High Quality Total RNA from Peaches (Prunus persica) for Functional Genomics Analyses

Prunus persica has been proposed as a genomic model for deciduous trees and the Rosaceae family. Optimized protocols for RNA isolation are necessary to further advance studies in this model species such that functional genomics analyses may be performed. Here we present an optimized protocol to rapidly and efficiently purify high quality total RNA from peach fruits (Prunus persica). Isolating high-quality RNA from fruit tissue is often difficult due to large quantities of polysaccharides and polyphenolic compounds that accumulate in this tissue and co-purify with the RNA. Here we demonstrate that a modified version of the method used to isolate RNA from pine trees and the woody plant Cinnamomun tenuipilum is ideal for isolating high quality RNA from the fruits of Prunus persica. This RNA may be used for many functional genomic based experiments such as RT-PCR and the construction of large-insert cDNA libraries.