Genetic characterization of the blood-sucking nematodes Libyostrongylus dentatus and Libyostrongylus douglassii supports their different evolutionary history.

Libyostrongylus sp. are nematodes that infect ostriches. Libyostrongylus douglassii was first described in ostriches from several countries in the world. Later Libyostrongylus dentatus was morphologically identified in ostriches in the USA and Brazil, and mixed infection is common in the latter country. The internal transcribed spacer (ITS) region of the ribosomal DNA gene is used for genetic variability assessment and phylogenetic reconstruction for many organisms. Through genetic analysis the status of different species morphologically defined was confirmed and a molecular method was developed to differentiate both species. ITS1, 5.8S, ITS2 regions of L. douglassii and L. dentatus were characterized. Regarding complete ITS region, the K2-p genetic distance between the species was 0.060 (SE 0.008) and the intra-specific distance was 0.002 (SE 0.001) for L. dentatus and 0.006 (SE 0.002) for L. douglassii. NJ and MP phylogenetic analysis of ITS1 and ITS2 regions indicated that both species belong to the Trichostrongylidae family, and are evolutionarily different, suported by high bootstrap value. Based on ITS DNA polymorphisms, a molecular approach was designed to detect both species. These results are the first molecular characterization of L. douglassii and L. dentatus, and provide new tools for the identification of these parasites of veterinary importance.

In situ probing of Xylella fastidiosa in honeydew of a xylem sap-feeding insect using 16S rRNA-targeted fluorescent oligonucleotides.

Xylella fastidiosa is a plant pathogen that threatens a US$ 4.6 billion worldwide wine and citrus industry. Monitoring its presence and distribution in plants and vectors is crucial for designing control strategies, as well as for understanding its ecological role and fate. We developed two fluorescent oligonucleotide probes complementary to different regions of the 16S rRNA gene of X. fastidiosa. The specificity of the newly designed probes S-S-X.fas-0067-a-A-18 and S-S-X.fas-1439-a-A-18 was demonstrated using fluorescence in situ hybridization (FISH) for 12 Xylella isolates, 15 closely related microorganisms and three plant endophytes. These probes were used to detect and quantify X. fastidiosa in plant sap (average value of 2.9 +/- 0.3 x 10(6) cells ml(-1)) from three different citrus orchards. In a second experiment, cells were quantified in honeydew (2.2 +/- 0.2 x 10(4) cells ml(-1)) collected from the insect vector Bucephalogonia xanthophis during the acquisition access period on an infected plant. The number of pathogen cells retained or digested by the insect is 10,000 times greater than the estimated minimum value to ensure an efficient transmission. Polymerase chain reaction (PCR) amplification using specific primers with plant sap and honeydew samples, followed by sequencing, confirmed the presence of the plant pathogen. This is the first demonstration of FISH being used for environmental samples, such as plant sap and insect honeydew, to estimate the abundance of a plant pathogen during infection.

Identification of citrus expressed sequence tags (ESTs) encoding pleiotropic drug resistance (PDR)-like proteins

Pleiotropic drug resistance (PDR) proteins, a subfamily of the ATP-binding cassette (ABC) transporters, have been recently shown to play a role in plant defense against biotic and abiotic stresses. However, nothing is known about their expression in citrus. To investigate the occurrence of PDR homologues in citrus species, we have surveyed EST sequences from different tissues and conditions of the Citrus Expressed Sequence Tags (CitEST) database, through sequence similarity search analyses and inspections for characteristic PDR domains. Multiple sequence alignments, prediction of transmembrane topology and phylogenetic analysis of PDR-like proteins were additionally performed. This study allowed the identification of nine putative proteins showing characteristic PDR features in citrus species under various conditions, which may indicate a potential correlation between PDRs and stress and metabolism of citrus plants. Moreover, a tissue-specific putative PDR-like protein was found in sweet orange fruits. To our knowledge, this is the first report regarding the identification of citrus ESTs encoding PDR-like proteins as well as the first to identify a putative full ABC transporter with specific expression in fruits.