Detection and identification of selected cereal rust pathogens by TaqMan<sup>®</sup>real-time PCR
Bibliographic record
Abstract
AbstractThe rust species Puccinia graminis and P. striiformis sensu stricto are important cereal pathogens, most well-known for causing the diseases stem rust and stripe rust on wheat and generating significant yield losses. Early and accurate detection of the pathogens would facilitate effective control of the diseases. In the present study, we developed real-time PCR assays to detect the specific lineages that include the wheat pathogens for each species complex, as identified using multi-gene DNA sequence analyses. Four DNA loci, for a comprehensive set of target and closely related fungi collected from diverse hosts and geographic regions, were explored to search for suitable lineage-specific probes: β-tubulin (BT), cytochrome c oxidase subunit 1 (COI), rDNA internal transcribed spacer (ITS), and RNA polymerase II second largest subunit (RPB2). Four TaqMan® real-time PCR assays were designed based on either the BT or RPB2 genes: one targeting Puccinia Series Striiformis (PSBT), one targeting P. striiformis sensu stricto (PSstrRPB2), and two targeting the P. graminis lineages on wheat (Pg2+BT and Pg2RPB2). Sensitivities of the assays were determined to be 0.65 pg µL−1 (Pg2) and 5 pg µL−1 (PS). Specificity of each assay was confirmed using a broad diversity of rusts and other selected wheat-associated fungi. The ITS and COI loci were found to be unsuitable for diagnostic assay development but contributed phylogenetic signal to the multi-gene analyses.RésuméLes rouilles Puccinia graminis et P. striiformis sensu stricto sont d'importants agents pathogènes des céréales, connus particulièrement pour causer la rouille de la tige et la rouille jaune chez le blé ainsi que pour engendrer de lourdes pertes de rendement. La détection précoce et précise des agents pathogènes aiderait à lutter efficacement contre ces maladies. Au cours de cette étude, nous avons développé des analyses par PCR en temps réel pour détecter les lignées précises qui incluent les agents pathogènes du blé pour chaque complexe d'espèces, tel qu'ils ont été identifiés par analyses multigéniques de séquences d'ADN. Afin d'obtenir un ensemble complet de champignons cibles étroitement apparentés, collectés sur divers hôtes et provenant de régions géographiques différentes, quatre locus d'ADN ont été examinés afin de chercher des amorces appropriées, spécifiques des lignées: la β-tubuline (BT), la sous-unité 1 de la cytochrome c oxidase (COI), l'espaceur transcrit interne (ITS) de l'ADNr et la deuxième plus grosse sous-unité de l'ARN polymérase II (RPB2). Quatre analyses par PCR en temps réel avec sondes TaqManmd ont été conçues en fonction des gènes BT ou RPB2: une ciblant Puccinia Series Striiformis (PSBT), une ciblant P. striiformis sensu stricto (PSstrRPB2) et deux ciblant les lignées de P. graminis chez le blé (Pg2+BT et Pg2RPB2). Les sensibilités des analyses ont été de 0,65 pg µL−1 (Pg2) et 5 pg µL−1 (PS). La spécificité de chaque analyse a été confirmée grâce à l'utilisation d'une grande variété de rouilles et d'autres champignons sélectionnés, associés au blé. Les locus ITS et COI se sont avérés inappropriés à la mise au point de tests diagnostiques, mais ont fourni un signal phylogénétique pour les analyses multigéniques.Keywords: diagnosticsPuccinia graminisPuccinia Series StriiformisPuccinia striiformisstem ruststripe rustMots clés: diagnosticsPuccinia graminisPuccinia Series StriiformisPuccinia striiformisrouille de la tigerouille jaune AcknowledgementsWe are grateful to T. Fetch, S.-L. Jin and C.-J. Li and the following herbaria for generously providing specimens: B, BP, BPI, BR, BRNM, DAOM, HMAS, K, PRC, PRM, PUR, S. We thank the anonymous reviewers whose comments and suggestions led to an improved manuscript.Additional informationFundingThis study was part of a broader project (CRTI 04-0045RD) funded by Defence Research and Development Canada through the Canadian Safety and Security Program (CSSP; formerly CRTI).
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".