Comparison of the performance of the main real-time and conventional PCR detection tests for ‘Candidatus Liberibacter’ spp., plant pathogenic bacteria causing the Huanglongbing disease in Citrus spp

23 Pág.Huanglongbing disease affects the Rutaceae family and is associated with three phloem-limited bacterium species: Candidatus Liberibacter asiaticus, africanus and americanus. These species are considered quarantine pathogens in the world, and pose major risks for citrus production and industry. Due to the low titer and the uneven distribution of the bacteria within its host plant, conventional PCR detection protocols can lead to false negative results, especially for early detection. Herein, three real-time PCR diagnostic methods recommended by the EPPO and FAO for asiaticus and africanus species detection were evaluated for their performance and compared with a conventional duplex PCR. Assessments were done as part of an international cooperative project under the EUPHRESCO guidance. Intra-laboratory assessment of the analytical specificity and analytical sensitivity was performed on 33 target or non-target DNA samples and seven target DNA samples were used to determine the sensitivity. Thereafter, repeatability, reproducibility, and concordance odds ratio were assessed on 20 target or non-target DNA samples through a collaborative test performance study organized among eight international laboratories. Results showed that the Li protocol proved to be the best method for asiaticus and africanus species detection, along with the conventional duplex PCR; whereas the Morgan protocol showed high performance only for asiaticus species. Interlaboratory reproducibility was high, suggesting that these real-time PCR methods can be readily transferred to diagnostic laboratories.This research was funded by Anses - Plant Health Laboratory (LSV).Peer reviewe

Hybrid genome assembly and evidence-Based annotation of the egg parasitoid and biological control agent trichogramma brassicae

Trichogramma brassicae (Bezdenko) are egg parasitoids that are used throughout the world as biological control agents and in laboratories as model species. Despite this ubiquity, few genetic resources exist beyond COI, ITS2, and RAPD markers. Aided by a Wolbachia infection, a wild-caught strain from Germany was reared for low heterozygosity and sequenced in a hybrid de novo strategy, after which several assembling strategies were evaluated. The best assembly, derived from a DBG2OLC-based pipeline, yielded a genome of 235 Mbp made up of 1,572 contigs with an N50 of 556,663 bp. Following a rigorous ab initio-, homology-, and evidence-based annotation, 16,905 genes were annotated and functionally described. As an example of the utility of the genome, a simple ortholog cluster analysis was performed with sister species T. pretiosum, revealing over 6000 shared clusters and under 400 clusters unique to each species. The genome and transcriptome presented here provides an essential resource for comparative genomics of the commercially relevant genus Trichogramma, but also for research into molecular evolution, ecology, and breeding of T. brassicae.</p

A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World.

The Old World bollworm, Helicoverpa armigera (Hübner), and the corn earworm, H. zea (Boddie), are two of the most important agricultural pests in the world. Diagnosing these two species is difficult-adults can only be separated with a complex dissection, and larvae cannot be identified to species using morphology, necessitating the use of geographic origin for identification in most instances. With the discovery of H. armigera in the New World, identification of immature Helicoverpa based on origin is no longer possible because H. zea also occurs in all of the geographic regions where H. armigera has been discovered. DNA barcoding and restriction fragment length polymorphism (RFLP) analyses have been reported in publications to distinguish these species, but these methods both require post-PCR processing (i.e., DNA sequencing or restriction digestion) to complete. We report the first real-time PCR assay to distinguish these pests based on two hydrolysis probes that bind to a segment of the internal transcribed spacer region 2 (ITS2) amplified using a single primer pair. One probe targets H. armigera, the second probe targets H. zea, and a third probe that targets a conserved segment of 18S rDNA is used as a control of DNA quality. The assay can be completed in 50 minutes when using isolated DNA and is successfully tested on larvae intercepted at ports of entry and adults captured during domestic surveys. We demonstrate that the assay can be run in triplex with no negative effects on sensitivity, can be run using alternative real-time PCR reagents and instruments, and does not cross react with other New World Heliothinae

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids.

Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the PospiSense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests

Cq values for each probe resulting from testing different real-time PCR master mixes with the triplex assay.

<p>ANOVA statistics for comparisons of each data set are provided in the last row of the table.</p

Alignment of ITS2 for <i>H</i>. <i>armigera</i> and <i>H</i>. <i>zea</i> showing sequence variation along with primer and probe locations.

<p>Alignment of ITS2 for <i>H</i>. <i>armigera</i> and <i>H</i>. <i>zea</i> showing sequence variation along with primer and probe locations.</p

Taxa sampled for study with country of origin, life stage, and number of specimens.

<p>Taxa sampled for study with country of origin, life stage, and number of specimens.</p

Standard curve of Cq values for serial dilutions of <i>H</i>. <i>armigera</i> and <i>H</i>. <i>zea</i> DNA run with the real-time PCR assay in triplex, duplex, and simplex for the ITS2 probe (FAM or HEX) and in triplex and duplex for the control probe (Quasar 670).

<p>Standard curve of Cq values for serial dilutions of <i>H</i>. <i>armigera</i> and <i>H</i>. <i>zea</i> DNA run with the real-time PCR assay in triplex, duplex, and simplex for the ITS2 probe (FAM or HEX) and in triplex and duplex for the control probe (Quasar 670).</p