Transcriptional analysis of pha genes in Pseudomonas mediterranea CFBP 5447 grown on glycerol

We analysed the draft genome sequence of Pseudomonas mediterranea CFBP 5447 in order to identify firstly the central metabolic pathways that convert fatty acids or carbohydrate intermediates into mcl-PHA and secondly the genes involved in glycerol metabolism (glpF, glpK, glpD, glpR). Absence of the glpF gene, which codifies for the “glycerol uptake facilitator protein”, was highlighted. In order to understand the expression of the pha gene cluster, we investigated the promoter activity of phaC1, phaC2, phaZ, phaD and phaI genes. When glycerol was present as the carbon source, PI was found to be the most active promoter. Expression analysis of the knock-out mutant of the phaD gene, which is a transcriptional regulator belonging to the TetR family, showed that PhaD acts as an activator of the phaI promoter which, in turn, triggers the transcription of the phaIF operon. The activation of PC1, which controls the phaC1ZC2D, by PhaD, was less efficient than PI

Pest categorisation of ‘Blight and blight-like’ diseases of citrus

The EFSA Panel on Plant Health performed a pest categorisation of ‘ Blight and blight-like ’ for the EU territory. Blight is a major disease of citrus. Similar ‘ blight-like ’ diseases are also known (e.g. declinio, declinamiento) and are addressed simultaneously with Blight in the present categorisation. The causal agent(s) remain(s) unknown and the potential role of a recently identi fi ed citrus endogenous pararetrovirus (Citrus Blight-associated pararetrovirus, CBaPRV) remains to be established. Transmissibility and ability to produce consistent (although poorly speci fi c) symptoms have been demonstrated and a combination of indirect approaches is used, with limits, for diagnosis. There are large uncertainties on the biology of the causal agent(s) and on the epidemiology of the disease, including the transmission mechanism(s) responsible for the observed fi eld spread. Blight has been reported from North, Central and South America, Africa and Oceania but is not known to occur in the EU. It is listed in Annex IIA of Directive 2000/29EC. It has the potential to enter, establish and spread in the EU territory. The main entry pathway (citrus plants for planting) is closed by existing legislation and entry is only possible on minor pathways (such as illegal import). Blight is a severe disease and a negative impact is expected should it be introduced in the EU, but the magnitude of this negative impact is very dif fi cult to estimate. ‘ Blight and blight like ’ satis fi es all criteria evaluated by EFSA to qualify as a Union quarantine pest. It does not meet the criterion of being present in the EU to qualify as a Union regulated non-quarantine pest (RNQP). Since the identity of the causal agent(s) of the Blight and blight-like disease(s) and the existence and ef fi ciency of natural spread mechanism(s) remain unknown, large uncertainties affect all aspects of the present pest categorisation

Emergence and phylodynamics of Citrus tristeza virus in Sicily, Italy

[EN] Citrus tristeza virus (CTV) outbreaks were detected in Sicily island, Italy for the first time in 2002. To gain insight into the evolutionary forces driving the emergence and phylogeography of these CTV populations, we determined and analyzed the nucleotide sequences of the p20 gene from 108 CTV isolates collected from 2002 to 2009. Bayesian phylogenetic analysis revealed that mild and severe CTV isolates belonging to five different clades (lineages) were introduced in Sicily in 2002. Phylogeographic analysis showed that four lineages co-circulated in the main citrus growing area located in Eastern Sicily. However, only one lineage (composed of mild isolates) spread to distant areas of Sicily and was detected after 2007. No correlation was found between genetic variation and citrus host, indicating that citrus cultivars did not exert differential selective pressures on the virus. The genetic variation of CTV was not structured according to geographical location or sampling time, likely due to the multiple introduction events and a complex migration pattern with intense co- and recirculation of different lineages in the same area. The phylogenetic structure, statistical tests of neutrality and comparison of synonymous and nonsynonymous substitution rates suggest that weak negative selection and genetic drift following a rapid expansion may be the main causes of the CTV variability observed today in Sicily. Nonetheless, three adjacent amino acids at the p20 N-terminal region were found to be under positive selection, likely resulting from adaptation events.A.W. and S.F.E. were supported by grant BFU2012-30805 from the Spanish Secretaria de Estado de Investigacion, Desarrollo e Innovacion and by a grant 22371 from the John Templeton Foundation. 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Exotic and Emergent Citrus Viruses Relevant to the Mediterranean Region

Citrus production in the Mediterranean area is of considerable importance, in both cultural and economic terms, and the viability of the industry greatly depends on proper phytosanitary management. In this review, we focus on exotic and emerging dangerous citrus viruses that have still not been reported in the countries of the Mediterranean area, that are not yet regulated or that are restricted to certain small areas. We also discuss the contribution that old and new technologies may offer for valuable surveys aimed at promoting the adoption and sharing of better control measures and the production of pathogen-tested citrus trees and rootstocks

A Simulation of the Use of High Throughput Sequencing as Pre-Screening Assay to Enhance the Surveillance of Citrus Viruses and Viroids in the EPPO Region

Citrus are affected by many viruses and viroids, some globally widespread and some restricted to particular countries or areas. In this study, we simulated the use of high throughput sequencing (HTS) and the bioinformatic analysis of small interfering RNAs (siRNA) as a pre-screening method to guide bioindexing and molecular detection to enhance the surveillance survey of some key or emerging citrus viruses, such as non-European citrus tristeza virus isolates (non-EU CTV), citrus tatter leaf virus, citrus leprosis virus, citrus yellow mosaic virus, and citrus bark cracking viroid, present in the EPPO lists, and the citrus yellow vein clearing virus. The HTS’s ability to detect other citrus viroids was also evaluated. The results demonstrate that HTS provides a comprehensive phytosanitary status of citrus samples either in single and multiple infections of viruses and viroids. It also provides effective information on citrus tristeza virus mixed infections despite not being able to identify the non-EU variants of the virus. Bioindexing checks each single virus infection but does not differentiate viroids on the Etrog citron indicator and is time-consuming. Molecular assays are valuable as confirmation tests of viruses and viroids but many pairs of primers are needed for a full screening and new or non-target pathogens remain undetected. In addition, the genomes of two isolates of the citrus yellow vein clearing virus and the citrus tatter leaf virus, detected in a sample from China, are described

High throughput sequencing of a stem pitting citrus tristeza virus isolate from Hunan Province P.R. China

A stem-pitting isolate of citrus tristeza virus (CTV), spreading in Hunan province of  China (HU-PSTS), was sequenced and indexed on indicator plants. Biological assays showed that HU-PSTS is a highly aggressive stem pitting isolate belonging to biogroup 5. Viral small RNAs (18-26 nt) of the isolate were deep sequenced by Illumina technology to gain genomic information on the CTV strain infecting the source plant. The reads mapping with 17 CTV reference genomes enabled us to re-assemble the genomes of VT, T68, T30 and T3 strains. Among the VT, the highest number of mapped reads (47-41%) was with SG29, T318A, CT11A, Nuaga and AT-1 genomes, whereas T68, T30 and T3 genomes were less represented (28-20%). Alignments with genomes belonging to T36 and RB strains revealed percentage of mapped reads ranging from 10 to 12%. This is the first sequenced genome of a CTV isolate from Hunan province. According to the results, further sequencing and bioindexing need to be developed to explore the potential of a local cross protective strategy

<i>F_st</i> values and <i>Ks</i>^*, <i>Z</i>^* and <i>S_nn</i> tests between pairs o CTV populations for the p20 gene.

*<p>Geographical regions: Argentina, California (USA), New Zealand, Pakistan, Spain, and Italy. Italy has been subdivided between the Sicily island and Apulia (peninsular Italy). The Italian sequences are: 110 from Sicily (108 determined in this work and two from GenBank), six from Apulia (Southeastern of Italian peninsula) and one from Calabria (Southwestern of Italian peninsula).</p>†<p><i>N</i> = number of CTV isolates.</p>‡<p><i>F_st</i> provides an estimate of the extent of gene flow between populations. A value of zero corresponds to genetically undifferentiate populations, whereas a value of one indicates genetically isolated populations.</p>§<p>No significant genetic differentiation (P>0.05) evaluated with the <i>Ks*</i>, <i>Z*</i> and <i>S_nn</i> tests.</p

Nucleotide diversity of p20 gene of CTV isolates from Italy corresponding to different introductions.

*<p>NA = non-applicable as there is only one CTV isolate.</p>†<p>Nucleotide diversities and standard errors of CTV isolates proceeding from a possible introduction (in italics) or between CTV isolates from different introductions.</p

Phylogenetic tree inferred for 108 CTV Sicilian isolates using the p20 gene.

<p>The ML tree was constructed using RAxML with the GTR+<i>Γ</i>₄ nucleotide substitution model, introducing three partitions (one for each codon position). Support >40% after 1000 bootstrap replicates is given.</p

Inferred migration graph for the different introductions of CTV in Sicily associated to clades B, C, D and E reconstructed from the MCC tree.

<p>The branches are colored according to the node height values to the colors between the specified maximal (red) and minimal boundary (black). Only introductions associated to clades B, C, D and E are shown since clade A has only a single Sicilian isolate.</p