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.

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. Liberibacter americanus' (CaLam) and 'Ca. Liberibacter africanus' (CaLaf), while 'Ca. Liberibacter solanacearum' (CaLsol) is associated with zebra chip disease in potatoes and vegetative disorders in apiaceous plants. Since these bacteria remain non-culturable and their symptoms are non-specific, their detection and identification are done by molecular methods, mainly based on PCR protocols. In this study, a new quantitative real-time PCR protocol based on TaqMan probe, which can also be performed in a conventional PCR version, has been developed to detect the four known phytopathogenic species of the genus Liberibacter. The new protocol has been validated according to European Plant Protection Organization (EPPO) guidelines and is able to detect CaLas, CaLam, CaLaf and CaLsol in both plants and vectors, not only using purified DNA but also using crude extracts of potato and citrus or psyllids. A comparative analysis with other previously described qPCR protocols revealed that this new one developed in this study is more specific and equally or more sensitive. Thus, other genus-specific qPCR protocols have important drawbacks regarding the lack of specificity, while with the new protocol there was no cross-reactions in 250 samples from 24 different plant and insect species from eight different geographical origins. Therefore, it can be used as a rapid and time-saving screening test, as it allows simultaneous detection of all plant pathogenic species of 'Ca. Liberibacter' in a one-step assay.

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.

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 Closteroviridae 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. This represents the first report of OLYaV in this country. A genomic sequence of 16,700 nt containing 11 open reading frames (ORFs) was recovered, representing the complete virus coding capacity. The knowledge of the nucleotide sequence of the genome including the gene that codes the coat protein will facilitate the development of diagnostic tests, which are limited so far to PCR-based methods targeting the HSP70h gene. Interestingly, a thaumatin-like protein (ORF2), previously reported in other unassigned viruses in the Closteroviridae family, persimmon virus B and actidinia virus 1, was identified in the OLYaV genome. Phylogenetic analysis of shared proteins (ORF1a, ORF1b, HSP70h, HSP90h and CP) with all members of the Closteroviridae family provides new insight into the taxonomic position of these three closteroviruses and suggests they could represent a new genus in the family.

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

Bacteria belonging to 'Candidatus Liberibacter spp.' are associated with various severe diseases in the five continents. The African citrus psyllid Trioza erytreae (Hemiptera: Triozidae) is an efficient vector of citrus huanglongbing-HLB disease, absent in the Mediterranean basin. This psyllid is currently present in the islands and mainland Portugal and Spain, where the prevalence of 'Ca. Liberibacter solanacearum' (CaLsol) associated to a carrot disease is high. Trioza erytreae normally feeds on citrus plants but has also been observed on other crops. It would be a great concern to the Mediterranean citrus industry if T. erytreae could transmit this bacterium from carrots to citrus and cause disease; therefore, the transmission of CaLsol from carrot plants to citrus plants was experimentally assessed. Although CaLsol was initially detected on receptor citrus plants in transmission assays by dodder and budding, the infection was not established. The feeding behavior by electrical penetration graphs and oviposition of T. erytreae on carrot plants versus citrus plants was evaluated. Trioza erytreae only reached the phloem in citrus plants. However, it was able to acquire CaLsol from infected carrots but unable to transmit it to citrus plants. CaLsol was detected in some carrot plants immediately after 7 and 14 days (inoculation access period), but it was not detected after one month. Trioza erytreae was unable to complete its life cycle on carrot plants. In conclusion, the efficient vector of bacteria associated to huanglongbing was unable to transmit CaLsol from carrot to citrus plants, but it acquired and transmitted the bacterium from carrot to carrot plants with low efficiency.

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. These validated user-friendly methods are recommended by IPPC-FAO standard for CTV diagnosis, detection, and identification. The methods are safe, not under current quarantine regulations because they do not involve any risk of introduction of exotic CTV isolates or vectors and are discrete and useful for epidemiological studies or screening for large-scale analyses. In this chapter, tissue-printing and squashing capture methods for direct sample preparation without extract preparation or nucleic acid extraction and purification were coupled with validated real-time RT-PCR detection protocols based on TaqMan chemistry for CTV detection.

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

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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. 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.

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.

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. Therefore, the main aim of this work was to determine the sensitivity, specificity, and likelihood ratios of three commonly used methods, including one serological test (double-antibody sandwich enzyme-linked immunosorbent assay [DAS-ELISA]) and two nucleic acid-based techniques (spot and conventional real-time reverse transcription-polymerase chain reaction [RT-PCR]). Latent class models using a Bayesian approach have been applied to determine diagnostic test parameters and to facilitate decision-making regarding diagnostic test selection. Statistical analysis has been based on the results of a total of 281 samples, which were collected during the dormant period from three different populations. The best-fit model out of the 49 implemented models revealed that DAS-ELISA was the most specific method (value = 0.99) and provided the highest degree of confidence in positive results. Conversely, conventional real-time RT-PCR was the most sensitive method (value = 0.98) and produced the highest degree of confidence in negative results. Furthermore, the estimation of likelihood ratios showed that in populations with low GLRaV-3 prevalence the most appropriate method could be DAS-ELISA, while conventional real-time RT-PCR could be the most appropriate method in medium or high prevalence populations. Combining both techniques significantly increases detection accuracy. The flexibility and power of Bayesian latent class models open new possibilities for the evaluation of diagnostic tests for plant viruses.

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. Moreover, these two species share phenotypic and molecular characteristics. Two specific and sensitive conventional and real-time PCR protocols were developed to identify and detect E. piriflorinigrans and to differentiate it from E. amylovora and other species of this genus. These protocols were based on sequences from plasmid pEPIR37, which is present in all strains of E. piriflorinigrans analyzed. After the stability of the plasmid was demonstrated, the specificities of the protocols were confirmed by the amplification of all E. piriflorinigrans strains tested, whereas 304 closely related pathogenic and nonpathogenic Erwinia strains and microbiota from pear trees were not amplified. In sensitivity assays, 10(3) cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 10(2) cells/ml were detected in DNA extracted from spiked plant material by real-time PCR. The protocols developed here succeeded in detecting E. piriflorinigrans in 102 out of 564 symptomatic and asymptomatic naturally infected pear samples (flowers, cortex stem tissue, leaves, shoots, and fruitlets), in necrotic Pyracantha sp. blossoms, and in necrotic pear and apple tissues infected with both E. amylovora and E. piriflorinigrans. Therefore, these new tools can be used in epidemiological studies that will enhance our understanding of the life cycle of E. piriflorinigrans in different hosts and plant tissues and its interaction with E. amylovora.

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. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect 'Ca. L. solanacearum'. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of 'Ca. Liberibacter' were observed using electron microscopy in celery plant tissues. A fifth haplotype of 'Ca. L. solanacearum', named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.

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. In addition, 158 grapevine plants collected in a survey of the Protected Designation of Origin in Alicante, Spain were compared using the three methods. The results of the molecular methods were very similar, with only four discordant results, and both were able to detect many more infected plants than the ELISA. The high prevalence of Grapevine fleck virus, Grapevine leafroll-associated virus 3 and Grapevine fanleaf virus suggests that the main pathways of viral introduction are infected plant material that has escaped controls and/or uncontrolled traffic of propagating plant material. Real-time multiplex RT-PCR could be used to facilitate a better control of grapevine viruses.

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 largescale analyses.

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 largescale analyses (AU)

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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. Nucleic acid extraction is still necessary in many cases and in practice inhibition problems are decreasing the theoretical sensitivity of molecular detection. For these reasons, integrated protocols that include the use of molecular techniques as screening methods, followed by confirmation by other techniques supported by different biological principles are advisable. Overall, molecular techniques based on different types of PCR amplification and very especially on real-time PCR are leading to high throughput, faster and more accurate detection methods for the most severe plant pathogens, with important benefits for agriculture. Other technologies, such as isothermal amplification, microarrays, etc. have great potential, but their practical development in plant pathology is still underway. Despite these advances, there are some unsolved problems concerning the detection of many plant pathogens due to their low titre in the plants, their uneven distribution, the existence of latent infections and the lack of validated sampling protocols. Research based on genomic advances and innovative detection methods as well as better knowledge of the pathogens' lifecycle, will facilitate their early and accurate detection, thus improving the sanitary status of cultivated plants in the near future.

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.

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 (AU)

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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. These samples were tested using methods recommended by the European and Mediterranean Plant Protection Organization to detect PPV (DASI-ELISA, RT-PCR and Co-PCR) and by NASBA-FH. Winter PPV diagnostic results by ELISA and NASBA-FH coincided in 90.8%, while ELISA and PCR-based methods coincided in 91.6% and PCR-based methods with NASBA-FH agreed in 95.4%. In spring, diagnostic results were similar with all the molecular techniques, which agreed with ELISA results for 98.8% of the trees. NASBA-FH was able to detect more positive infections in winter, which were later confirmed in spring. These results indicate that NASBA-FH is a suitable molecular method for routine PPV detection in the winter and spring. This user-friendly isothermal RNA amplification coupled with a very fast flow-through hybridisation (15 min) opens up new possibilities for rapid and reliable diagnosis of a variety of pathogens.