Abstract
Alternaria burnsii is the causal agent of cumin blight, a seed-borne disease of economic concern for all cumin growing areas. Current detection and identification methods for the pathogen are based on visual examination of morphological features, which are time-consuming and laborious. The present study describes conventional and real-time PCR assays for rapid and accurate detection of A. burnsii in cumin seeds. Based on sequence differences in Alternaria allergen a1 (Alt a1) gene, two primer pairs, Ab35/326 and AB177/403, were designed for conventional and real-time PCR assays, respectively. Both primer pairs amplified the expected target PCR fragment from A. burnsii genomic DNA. The sensitivity of conventional PCR with primer pairs Ab35/326 was 1 pg of genomic DNA and allowed the detection of pathogen in cumin seeds samples with 0.2% infestation rate. Real-time PCR assay was highly sensitive and allowed the quantification of 0.1 pg pathogen DNA. Also, this assay confirmed the presence of pathogen in cumin seeds up to 0.1% infestation level. The standard curve (r2 = 0.99) showed a good correlation between fungal DNA quantities and Cq values. The specificity of primer pairs was confirmed by the absence of amplified product with DNA of related fungi species and healthy plant tissue. The assays developed in this study provide a rapid and sensitive tool for the detection and quantification of Alternaria burnsii in cumin seed.
Zusammenfassung
Alternaria burnsii ist der Erreger der Kreuzkümmelfäule, einer durch Samen übertragenen Krankheit, die für alle Anbaugebiete von Kreuzkümmel von wirtschaftlicher Bedeutung ist. Die aktuellen Nachweis- und Identifikationsmethoden für den Erreger basieren auf der visuellen Untersuchung morphologischer Merkmale, die zeitaufwändig und mühsam ist. Die vorliegende Studie beschreibt konventionelle und Real-Time-PCR-Assays für den schnellen und genauen Nachweis von A. burnsii in Kreuzkümmelsamen. Basierend auf Sequenzunterschieden im Alternaria-Allergen a1 (Alt a1) Gen wurden zwei Primerpaare, Ab35/326 und AB177/403, für konventionelle bzw. Real-Time-PCR-Assays entwickelt. Beide Primerpaare amplifizierten das erwartete Ziel-PCR-Fragment aus der genomischen DNA von A. burnsii. Die Sensitivität der konventionellen PCR mit den Primerpaaren Ab35/326 betrug 1 pg genomische DNA und ermöglichte den Nachweis von Krankheitserregern in Kreuzkümmelproben mit einer Befallsrate von 0,2 %. Der Real-Time-PCR-Assay war hochsensitiv und erlaubte die Quantifizierung von 0,1 pg pathogener DNA. Außerdem bestätigte dieser Test das Vorhandensein von Krankheitserregern in Kreuzkümmelsamen bis zu einer Befallsrate von 0,1 %. Die Standardkurve (r2 = 0,99) zeigte eine gute Korrelation zwischen DNA-Mengen und Cq-Werten. Bei der Verwendung von DNA verwandter Pilzarten und gesundem Pflanzengewebe wurde kein amplifiziertes Produkt gebildet, wodurch die Spezifität der Primerpaare bestätigt werden konnte. Die in dieser Studie entwickelten Assays stellen ein schnelles und sensitives Verfahren für den Nachweis und die Quantifizierung von Alternaria burnsii in Kreuzkümmelsamen dar.
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This study was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK), grant TOVAG 116O036.
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G. Özer and H. Bayraktar declare that they have no competing interests.
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Özer, G., Bayraktar, H. Development of Conventional and Real-Time PCR Assays to Detect Alternaria burnsii in Cumin Seed. Gesunde Pflanzen 71, 205–212 (2019). https://doi.org/10.1007/s10343-019-00466-6
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DOI: https://doi.org/10.1007/s10343-019-00466-6