Development of SCAR Markers for the Identification ofPhytophthora katsuraeCausing Chestnut Ink Disease in Korea

Abstract

Sequence characterized amplified region (SCAR) markers are one of the most effective and accurate tools for microbial identification. In this study, we applied SCAR markers for the rapid and accurate detection of Phytophthora katsurae, the casual agent of chestnut ink disease in Korea. In this study, we developed seven SCAR markers specific to P. katsurae using random amplified polymorphic DNA (RAPD), and assessed the potential of the SCAR markers to serve as tools for identifying P. katsurae. Seven primer pairs (SOPC 1F/SOPC 1R, SOPC 1-1F/SOPC 1-1R, SOPC 3F/SOPC 3R, SOPC 4F/SOPC 4R, SOPC 4F/SOPC 4-1R, SOPD 9F/SOPD 9R, and SOPD 10F/SOPD 10R) from a sequence derived from RAPD fragments were designed for the analysis of the SCAR markers. To evaluate the specificity and sensitivity of the SCAR markers, the genomic DNA of P. katsurae was serially diluted 10-fold to final concentrations from 1 mg/mL to 1 pg/mL. The limit of detection using the SCAR markers ranged from 100 µg/mL to 100 ng/mL. To identify the limit for detecting P. katsurae zoospores, each suspension of zoospores was serially diluted 10-fold to final concentrations from 10 × 105 to 10 × 101 zoospores/mL, and then extracted. The limit of detection by SCAR markers was approximately 10 × 101 zoospores/mL. PCR detection with SCAR markers was specific for P. katsurae, and did not produce any P. katsurae-specific PCR amplicons from 16 other Phytophthora species used as controls. This study shows that SCAR markers are a useful tool for the rapid and effective detection of P. katsurae.

Development of two novel specific SCAR markers by cloning improved RAPD fragments from the medicinal mushroom Ganoderma lucidium (Leysser: Fr) Karst

“…In a previous study, Su et al (2007) developed a 'strain-specific' G. lucidum ISSR-based SCAR marker, which could identify certain strains of G. lucidum, but it was not a species-specific marker. RAPD-SCAR markers have been successfully developed for the identification of some other organisms, including plants, animals, and microbes (Jiang et al, 2009;Dutta et al, 2012;Lee et al, 2013;Yang et al, 2013, Fu et al, 2015Zhang et al, 2015;Mei et al, 2015).…”

Effects of Water Stress on the Endophytic Fungal Communities of Pinus koraiensis Needles Infected by Cenangium ferruginosum

“…The size of the amplified PCR product was 477 bp, and the nucleotide sequence was a 100% match with that of CF . In fact, species-specific markers have been produced by various methods and utilized to detect tree pathogens [ 25 , 26 ].…”

SCAR marker forPhytophthora nicotianaeand a multiplex PCR assay for simultaneous detection ofP. nicotianaeandCandidatusLiberibacter asiaticus in citrus

“…The SCAR DNA fragment represents a single locus in the genome and is therefore specific for a given organism and has proven to be more reproducible, robust and reliable for the authentication and identification of genotypes (Babaei et al 2014;Bhagyawant 2016). Due to the advantages of low workload, rapidity and high efficiency compared with traditional identification methods, RAPD-based SCAR markers were extensively used in designing of species-specific primers as well as for the in planta detection and identification of P. cactorum (Causin et al 2005), Phytophthora alni (Ioos et al 2005), Phytophthora katsurae (Lee et al 2013), Phytophthora infestans (Hussain et al 2017) and Phytophthora sojae (Xiong et al 2019). Among all the 20 RAPD primers tested in this study, OPA4 has given the single distinguished amplicon (380 bp size) in all the P. nicotianae isolates.…”