Comparison of promidium monoazide and Reagent D for discrimination of viable Aphelenchoides besseyi using real-time PCR
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Comparison of promidium monoazide and Reagent D for discrimination of viable Aphelenchoides besseyi using real-time PCR Elvan Sert Çelik & Tevfik Özalp & Zübeyir Devran
Received: 25 March 2020 / Revised: 16 July 2020 / Accepted: 23 July 2020 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2020
Abstract Aphelenchoides besseyi Christie, 1942 is a seed-borne nematode and causes economic yield losses in rice. Information on the viability of A. besseyi in stored rice seeds is important to manage this nematode. In this study, propidium monoazide (PMA) and Reagent D, combined with qPCR, were used for discrimination of viable A. besseyi individuals. A. besseyi individuals that were live, stored for 1 year or heat-treated (40 or 60 °C, 15 min) were exposed to PMA and Reagent-D dyes. In addition, A. besseyi individuals not treated with any chemicals were used as a control. DNA was then isolated from all individuals and real-time PCR was carried out. Ct values for samples treated with Reagent-D and PMA were different from untreated samples. Results showed that live individuals of the rice white tipe nematode could be distinguished from nonviable individuals using Reagent-D or PMA combined with qPCR.
Keywords Aphelenchoides besseyi . Viability . Propidium monoazide . Reagent D . TaqMan probe
E. Sert Çelik : T. Özalp : Z. Devran (*) Department of Plant Protection, Faculty of Agriculture, University of Akdeniz, Antalya, Turkey e-mail: [email protected] E. Sert Çelik Argeto Company, Antalya, Turkey
Introduction Rice white tip nematode, Aphelenchoides besseyi Christie 1942, is one of the serious pathogens of rice. It is a seed-borne pathogen and, under dry conditions, hibernates beneath glumes for several years as adults and fourth-stage juveniles (Yoshii and Yamamoto 1950; Nandakumar et al. 1975). Thus, A. besseyi can spread rapidly in infested seed to rice-growing areas. In order to prevent dissemination, stored rice seeds should be treated to eliminate A. besseyi. One method of control is exposure of seed to hot-water; according to the International Rice Research Institute (IRRI), the rice seeds may be treated with hot water at 52–57ºC for 15 min, after presoaking for 3 h in cold water (Misra et al. 1994; Prot and Gergon 1994). However, old or damaged seeds, and varieties that have low heat tolerance are not suitable for seed treatments including chemicals and hot water (Misra et al. 1994). In addition, A. besseyi can sometimes survive hot-water treatment; therefore, it is important to distinguish between live and dead A. besseyi individuals in infected seeds. Propidium monoazide (PMA) and Reagent-D have been used as nucleic acid- intercalating dyes to prevent PCR amplification of DNA from dead or damaged cells (Nocker et al. 2007; Elizaquível et al. 2012). PMA has been widely used in such studies for a long time (Nocker et al. 2006; Christoforou et al. 2014), whereas ReagentD has been more recently released by Biotecon Diagnostics (Vesper et al. 2008; Martinon et al. 2012; Cattani et al. 2016). However, unt
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