Development of TaqMan Real-Time PCR Protocols for Simultaneous Detection and Quantification of the Bacterial Pathogen and Their Specific Lytic Bacteriophages.

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 bacteriophages was recently patented for bacterial wilt biocontrol in environmental water and in plants.

A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus 'Candidatus Liberibacter' in plants and insects.

Four pathogenic bacterial species of the genus 'Candidatus Liberibacter', transmitted by psyllid vectors, have been associated with serious diseases affecting economically important crops of Rutaceae, Apiaceae and Solanaceae families. The most severe disease of citrus plants, huanglongbing (HLB), is associated with 'Ca. Liberibacter asiaticus' (CaLas), 'Ca.

Genomic Analysis of the First European Bacteriophages with Depolymerase Activity and Biocontrol Efficacy against the Phytopathogen .

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 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.

Molecular Characterization of the Complete Coding Sequence of Olive Leaf Yellowing-Associated Virus.

Genome organization and phylogenetic relationships of olive leaf yellowing-associated virus (OLYaV) with other members of the family were determined. The complete coding sequence of OLYaV was obtained by high throughput sequencing of total RNA from a 35-year-old olive tree (cv. Zarzaleña) from Brazil, showing olive leaf yellowing disease and deformations in the wood.

' Liberibacter Solanacearum' Is Unlikely to Be Transmitted Spontaneously from Infected Carrot Plants to Citrus Plants by .

Bacteria belonging to ' Liberibacter spp.' are associated with various severe diseases in the five continents. The African citrus psyllid (Hemiptera: Triozidae) is an efficient vector of citrus huanglongbing-HLB disease, absent in the Mediterranean basin.

Tissue-Print and Squash Capture Real-Time RT-PCR Method for Direct Detection of Citrus tristeza virus (CTV) in Plant or Vector Tissues.

Direct systems to process samples allow high-throughput testing or identification of Citrus tristeza virus (CTV) by the sensitive real-time reverse transcription coupled to polymerase chain reaction (RT-PCR) neither with extract preparation nor nucleic acid purification. Immobilized CTV targets are amplified from fresh sections of plant tissues or squashes of fresh or already caught vectors onto paper, nitrocellulose, or positively charged nylon membranes. The printed or squashed support can be stored or mailed at room temperature.

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.

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

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.

Modeling the Accuracy of Three Detection Methods of Grapevine leafroll-associated virus 3 During the Dormant Period Using a Bayesian Approach.

Grapevine leafroll-associated virus 3 (GLRaV-3) has a worldwide distribution and is the most economically important virus that causes grapevine leafroll disease. Reliable, sensitive, and specific methods are required for the detection of the pathogen in order to assure the production of healthy plant material and control of the disease. Although different serological and nucleic acid-based methods have been developed for the detection of GLRaV-3, diagnostic parameters have not been established, and there is no gold standard method.

Conventional and real-time PCRs for detection of Erwinia piriflorinigrans allow its distinction from the fire blight pathogen, Erwinia amylovora.

Erwinia piriflorinigrans is a new pathogenic species of the bacterial genus Erwinia that has been described recently in Spain. Accurate detection and identification of E. piriflorinigrans are challenging because its symptoms on pear blossoms are similar to those caused by Erwinia amylovora, the causal agent of fire blight.

Association of 'Candidatus Liberibacter solanacearum' with a vegetative disorder of celery in Spain and development of a real-time PCR method for its detection.

A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with 'Candidatus Liberibacter solanacearum' and not with phytoplasmas.

Virus-viroid interactions: Citrus Tristeza Virus enhances the accumulation of Citrus Dwarfing Viroid in Mexican lime via virus-encoded silencing suppressors.

An assay to identify interactions between Citrus Dwarfing Viroid (CDVd) and Citrus Tristeza Virus (CTV) showed that viroid titer was enhanced by the coinfecting CTV in Mexican lime but not in etrog citron. Since CTV encodes three RNA silencing suppressors (RSSs), p23, p20 and p25, an assay using transgenic Mexican limes expressing each RSS revealed that p23 and, to a lesser extent, p25 recapitulated the effect observed with coinfections of CTV and CDVd.

Real-time multiplex RT-PCR for the simultaneous detection of the five main grapevine viruses.

A real-time multiplex RT-PCR has been developed for the simultaneous detection and identification of the major RNA viruses that infect grapevines (Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 3 and Grapevine fleck virus). Serial dilutions of infected plant extracts were tested using the new method, and the results were compared with those obtained using a commercially available ELISA and real-time singleplex RT-PCR. The two real-time RT-PCR versions detected up to the same level of dilution and were at least 10,000 times more sensitive than the ELISA.

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.

Multiplex Nested Reverse Transcription-Polymerase Chain Reaction in a Single Tube for Sensitive and Simultaneous Detection of Four RNA Viruses and Pseudomonas savastanoi pv. savastanoi in Olive Trees.

ABSTRACT A multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) in a single closed tube was developed for the simultaneous detection of four RNA viruses: Cucumber mosaic virus, Cherry leaf roll virus, Strawberry latent ringspot virus, and Arabis mosaic virus, and the bacterium Pseudomonas savastanoi pv. savastanoi. The method enabled, for the first time, the sensitive and simultaneous detection of RNA and DNA targets from plant viruses and a bacterium, saving time, decreasing risks of contamination, and reducing costs compared with conventional monospecific nested amplifications.

Are molecular tools solving the challenges posed by detection of plant pathogenic bacteria and viruses?

Plant pathogenic bacteria, phytoplasmas, viruses and viroids are difficult to control, and preventive measures are essential to minimize the losses they cause each year in different crops. In this context, rapid and accurate methods for detection and diagnosis of these plant pathogens are required to apply treatments, undertake agronomic measures or proceed with eradication practices, particularly for quarantine pathogens. In recent years, there has been an exponential increase in the number of protocols based on nucleic-acid tools being those based on PCR or RT-PCR now routinely applied worldwide.

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.

Isothermal amplification coupled with rapid flow-through hybridisation for sensitive diagnosis of Plum pox virus.

A nucleic acid sequence-based amplification method coupled with rapid flow-through hybridisation (NASBA-FH) was developed for diagnosis of Plum pox virus (PPV). The sensitivity level achieved by NASBA-FH was 10 times higher than that obtained by Co-PCR and 1000 times higher than the sensitivity afforded by RT-PCR. In addition, samples from 262 stone-fruit trees collected during winter and spring seasons were analysed.

Real-time PCR for simultaneous and quantitative detection of quarantine phytoplasmas from apple proliferation (16 SrX) group.

A real time PCR assay conjugated with the fluorescent SYBR Green I dye has been developed for rapid, sensitive and quantitative detection of 'Ca. Phytoplasma pyri', 'Ca. P.

Real-time assay for quantitative detection of non-persistently transmitted Plum pox virus RNA targets in single aphids.

A TaqMan real-time RT-PCR was developed to detect and quantify RNA-targets from the non-circulative, non-persistently transmitted Plum pox virus (PPV) in individual fresh or aphids captured previously and squashed on paper. Reliable quantitation ranged from 40 up to 4 x 10(8) copies of control transcripts. This technique was applied successfully to plant material and to individual PPV vector (Myzus persicae) and non-vector of PPV (Aphis nerii) aphid species demonstrating acquisition of viral targets by both vector and non-vector aphids.

Seasonal variation of Ralstonia solanacearum biovar 2 populations in a Spanish river: recovery of stressed cells at low temperatures.

The presence of Ralstonia solanacearum biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14 degrees C or higher, but its isolation was usually unsuccessful at temperatures below 9 degrees C.

Estimation of the number of aphids carrying Citrus tristeza virus that visit adult citrus trees.

Aphid species were counted on citrus trees in orchards in Valencia, Spain, in the spring and autumn of 1997, 1998 and 1999. Moericke yellow water traps, the 'sticky shoot' method and counts of established colonies were used in extensive surveys in which 29,502 aphids were recorded and identified. Aphis spiraecola and Aphis gossypii were the most abundant aphid species.

A new and sensitive Co-operational polymerase chain reaction for rapid detection of Ralstonia solanacearum in water.

Three primers from 16S rRNA were successfully assayed simultaneously in one reaction for sensitive detection of Ralstonia solanacearum in watercourses. The protocol is a modification of the Co-operational polymerase chain reaction (Co-PCR), which allows the simultaneous and co-operational action of the primers. It specifically amplified R.

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.