20.500.12556/RUL-145002 Highly specific qPCR and amplicon sequencing method for detection of quarantine citrus pathogen Phyllosticta citricarpa applicable for air samples The fungus Phyllosticta citricarpa is a quarantine pathogen in the EU and is of high economic importance in many parts of the world where favourable climate conditions drive the development of citrus black spot (CBS) disease. Disease symptoms include necrotic lesions on leaves and fruits. Low disease pressure can reduce crop market-ability, while higher disease pressure can cause premature fruit drop, significantly increasing crop losses. The wind-dispersed spores of P. citricarpa are especially prob-lematic for rapid pathogen dispersal, but also provide an opportunity for early detec-tion of the disease spreading into a new area. In this study we have developed and validated a quantitative PCR (qPCR) assay based on the TEF1-α sequence. Specificity testing demonstrated that it is currently the only qPCR assay that does not cross- react with closely related Phyllosticta species. The assay is sensitive and can detect a single copy of the TEF1 gene in a reaction, it is highly repeatable and reproducible and can be used for testing of the sticky tapes from spore traps as well as citrus fruit sam-ples. High-throughput sequencing (HTS) of the DNA barcodes ITS1 and TEF1 was also explored for the detection and discrimination of P. citricarpa. The limit of detection of the HTS was 1000 spores on a daily spore trap tape. This study makes an important improvement to the diagnostics of the CBS and the methods developed can also be applied to improve the surveillance and early detection of the pathogen when linked to spore samplers in the field. detection fungal spore sampling internal transcribed region ITS translation elongation factor 1-α TEF1 Phyllosticta citricarpa detekcija citrusi zrak true false true Angleški jezik Slovenski jezik Članek v reviji 2023-03-29 15:04:44 2023-03-29 15:04:54 2023-04-03 11:23:23 0000-00-00 00:00:00 2023 0 0 Str. 548-563 iss. 3 Vol. 72 Apr. 2023 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 632 1365-3059 10.1111/ppa.13679 136443139 Plant_Pathology_-_2022_-_Zajc_-_Highly_specific_qPCR_and_amplicon_sequencing_method_for_detection_of_quarantine_citrus.pdf Plant_Pathology_-_2022_-_Zajc_-_Highly_specific_qPCR_and_amplicon_sequencing_method_for_detection_of_quarantine_citrus.pdf 1 395843FC39960EFF623DACFAF0DFD590 39ecc12ffc90e80c8e63d93922ba6ffa2427e3c2f0bf05d8164664e6af637d98 803a1486-ce32-11ed-a314-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=167620 https://bsppjournals.onlinelibrary.wiley.com/doi/10.1111/ppa.13679 0 5550ef19-ce32-11ed-a314-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=167619 Biotehniška fakulteta 0 0 0