Manejo de suinos

Trabalho de Conclusão de Curso - Universidade Federal de Santa Catarina. Curso de Agronomia

Development of TaqMan Real-Time PCR protocols for simultaneous detection and quantification of the bacterial pathogen ralstonia solanacearum and their specific lytic bacteriophages

Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most destructive diseases of solanaceous plants, affecting staple crops worldwide. The bacterium survives in water, soil, and other reservoirs, and is difficult to control. In this sense, the use of three specific lytic R. solanacearum bacteriophages was recently patented for bacterial wilt biocontrol in environmental water and in plants. To optimize their applications, the phages and the bacterium need to be accurately monitored and quantified, which is laborious and time-consuming with biological methods. In this work, primers and TaqMan probes were designed, and duplex and multiplex real-time quantitative PCR (qPCR) protocols were developed and optimized for the simultaneous quantification of R. solanacearum and their phages. The quantification range was established from 108 to 10 PFU/mL for the phages and from 108 to 102 CFU/mL for R. solanacearum. Additionally, the multiplex qPCR protocol was validated for the detection and quantification of the phages with a limit ranging from 102 targets/mL in water and plant extracts to 103 targets/g in soil, and the target bacterium with a limit ranging from 103 targets/mL in water and plant extracts to 104 targets/g in soil, using direct methods of sample preparation

Recovery of Pseudomonas savastanoi pv. savastanoi from symptomless shoots of naturally infected olive trees

Seasonal dynamics of Pseudomonas savastanoi pv. savastanoi (Psv) on stems and leaves from symptomless shoots of naturally infected olive trees was monitored in Spanish olive orchards. Data inferred from the comparison between washing of leaves and dilution-plating versus leaf printing of individual leaves suggested that Psv population sizes varied by over several orders of magnitude, among leaves sampled concurrently from the same shoot. We did not find significant differences between leaves and stems, in respect to the number of samples where Psv was isolated or detected by PCR, showing that Psv colonizes both leaves and stems. The frequencies of Psv isolation and average populations were highly variable among field plots. No correlation between Psv populations and those of non-Psv bacteria in any plant material or field plot was observed. However, where both Psv and yellow Pantoea agglomerans colonies were isolated a positive correlation was found. In a selected field plot, dynamics of Psv over three years showed significant differences between summer and the rest of seasons. The highest Psv population occurred in warm, rainy months, while low numbers were generally found in hot and dry months. [Int Microbiol 2007; 10(2):77-84

Interference between variants of peach latent mosaic viroid reveals novel features of its fitness landscape: implications for detection

[EN] Natural populations of peach latent mosaic viroid (PLMVd) are complex mixtures of variants. During routine testing, TaqMan rtRT-PCR and RNA gel-blot hybridization produced discordant results with some PLMVd isolates. Analysis of the corresponding populations showed that they were exclusively composed of variants (of class II) with a structural domain different from that of the reference and many other variants (of class I) targeted by the TaqMan rtRT-PCR probe. Bioassays in peach revealed that a representative PLMVd variant of class II replicated without symptoms, generated a progeny with low nucleotide diversity, and, intriguingly, outcompeted a representative symptomatic variant of class I when co-inoculated in equimolecular amounts. A number of informative positions associated with the higher fitness of variants of class II have been identified, and novel sets of primers and probes for universal or specific TaqMan rtRT-PCR detection of PLMVd variants have been designed and tested.We thank A. Ahuir for excellent technical assistance and Dr. Francesco Di Serio for suggestions. This work was supported by grant BFU2014-56812-P (to R.F.) from Ministerio de Economia y Competitividad (MINECO) of Spain. P.S. was the recipient of a postdoctoral contract from MINECO and E. Bertolini of an INIA-CCA2011-2016 contract also from MINECO.Serra Alfonso, P.; Bertolini, E.; Martinez, MC.; Cambra, M.; Flores Pedauye, R. (2017). Interference between variants of peach latent mosaic viroid reveals novel features of its fitness landscape: implications for detection. Scientific Reports. 7. https://doi.org/10.1038/srep42825S7Diener, T. O. Discovering viroids - a personal perspective. Nat. Rev. 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Transmission of ‘Candidatus Liberibacter solanacearum’ by Bactericera trigonica Hodkinson to vegetable hosts

The bacterium ‘Candidatus Liberibacter solanacearum’ is a recent plant pathogen of several crops in Solanaceae and Apiaceae and is associated with economically important diseases. The bacterium is a carrot seed borne pathogen that can also be transmitted from potato mother tubers and by psyllid vectors. The psyllid Bactericera trigonica Hodkinson was described carrying CaLso associated with vegetative disorders in carrot and celery crops in Spain and its competence to transmit this phloem-limited bacterium among vegetables is currently being investigated. Here electrical penetration graphs showed that B. trigonica fed in the phloem of carrot and celery and probed the phloem in potato, but not in tomato plants. The bacterium was efficiently transmitted to carrot and celery plants when either single B. trigonica or groups of ten fed on these species. An inoculation access period of 24 hours was sufficient for a single B. trigonica to transmit the bacterium to carrot (67.8%), celery (21.1%) and eventually to potato and tomato (6.0%). Higher transmission rates were obtained with 10 individuals on celery (100%), carrot (80%), potato (10%) and tomato (10%). Bactericera trigonica laid eggs, and the hatched nymphs develop into adult on carrot and celery, but not on potato and tomato. CaLso was detected in 20% of the eggs laid by females carrying the bacterium. The results confirmed that B. trigonica is a vector of the bacterium to carrot and celery, and it is discussed the potential role of this psyllid in the transmission of the pathogen to potato and tomato plants

Genomic analysis of the first european bacteriophages with depolymerase activity and biocontrol efficacy against the phytopathogen Ralstonia solanacearum

Ralstonia solanacearum is the causative agent of bacterial wilt, one of the most destructive plant diseases. While chemical control has an environmental impact, biological control strategies can allow sustainable agrosystems. Three lytic bacteriophages (phages) of R. solanacearum with biocontrol capacity in environmental water and plants were isolated from river water in Europe but not fully analysed, their genomic characterization being fundamental to understand their biology. In this work, the phage genomes were sequenced and subjected to bioinformatic analysis. The morphology was also observed by electron microscopy. Phylogenetic analyses were performed with a selection of phages able to infect R. solanacearum and the closely related phytopathogenic species R. pseudosolanacearum. The results indicated that the genomes of vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 range from 40,688 to 41,158 bp with almost 59% GC-contents, 52 ORFs in vRsoP-WF2 and vRsoP-WM2, and 53 in vRsoP-WR2 but, with only 22 or 23 predicted proteins with functional homologs in databases. Among them, two lysins and one exopolysaccharide (EPS) depolymerase, this type of depolymerase being identified in R. solanacearum phages for the first time. These three European phages belong to the same novel species within the Gyeongsanvirus, Autographiviridae family (formerly Podoviridae). These genomic data will contribute to a better understanding of the abilities of these phages to damage host cells and, consequently, to an improvement in the biological control of R. solanacearum

Innovative tools for detection of plant pathogenic viruses and bacteria

Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative- PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is microarray technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms

Genetic diversity reflects geographical origin of Ralstonia solanacearum strains isolated from plant and water sources in Spain

The characterization and intraspecific diversity of a collection of 45 Ralstonia solanacearum strains isolated in Spain from different sources and geographical origins is reported. To test the influence of the site and the host on strain diversity, phenotypic and genotypic analysis were performed by a polyphasic approach. Biochemical and metabolic profiles were compared. Serological relationship was evaluated by Indirect-ELISA using polyclonal and monoclonal antibodies. For genotypic analysis, hrpB and egl DNA sequence analysis, repetitive sequences (rep-PCR), amplified fragment length polymorphism (AFLP) profiles and macrorestriction with XbaI followed by pulsed field gel electrophoresis (PFGE) were performed.The biochemical and metabolic characterization, serological tests, rep-PCR typing and phylogenetic analysis showed that all analysed strains belonged to phylotype II sequevar 1 and shared homogeneous profiles. However, interesting differences among strains were found by AFLP and macrorestriction with XbaI followed by PFGE techniques, some profiles being related to the geographical origin of the strains. Diversity results obtained offer new insights into the biogeography of this quarantine organism and its possible sources and reservoirs in Spain and Mediterranean countries.Keywords: Bacterial wilt · potato · soil · PFGE · AFL

Direct sample preparation methods for the detection of Plum pox virus by real-time RT-PCR

Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 +/- 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for large-scale analyses. [Int Microbiol 2009; 12(1): 1-6