Improved PCR detection of potyviruses in Allium species

Abstract

Protocols for producing virus-free Allium plants require an indexing system that is more sensitive than DAS-ELISA and can detect low virus concentrations in infected plants. In the present work, degenerate primers were designed and a one-step IC-RT-PCR protocol was developed to differentiate between Leek yellow stripe virus (LYSV) and Onion yellow dwarf virus (OYDV) in single and mixed infections in several Allium spp. A 566-bp band was observed for LYSV, a 489-bp band for OYDV in single infections, and two bands of the same sizes in mixed infections in different species of Alliaceae. A 508-bp band of Shallot yellow stripe virus and a 594-bp band of Turnip mosaic virus were also amplified with the same primers. RTnested-PCR was also conducted directly in microtitre plate wells after negative or questionable reactions were produced in an ELISA experiment. The detection limit of the DAS-ELISA for LYSV was 16.5-27.3 ng ml )1 . The RT-nested-PCR done after DAS-ELISA was 10 2 times more sensitive than the DAS-ELISA alone. In parallel, an IC-RT-nested-PCR in microcentrifuge tubes was 10 4 times more sensitive than the DAS-ELISA. The DAS-ELISA-RT-nested-PCR enables the initial screening of samples by DAS-ELISA to eliminate a high percentage of virus-positive plants, considerably reducing the number of plants to analyze further by RT-PCR.

Detection of Garlic Viruses Using SYBR Green Real-time Reverse Transcription-Polymerase Chain Reaction

“…The problem with the use of PCR methods for virus diagnosis is that the gene for coat protein (CP) used for detection has a high level of sequence variability among isolates of the same virus species (Tsuneyoshi et al. 1997, 1998; Lunello et al. 2005).…”

“…TaqMan real-time RT-PCR assay was developed for the detection of OYDV and LYSV (Lunello et al 2004). The problem with the use of PCR methods for virus diagnosis is that the gene for coat protein (CP) used for detection has a high level of sequence variability among isolates of the same virus species (Tsuneyoshi et al 1997(Tsuneyoshi et al , 1998Lunello et al 2005).…”

Molecular variability and genetic structure of the population of Onion yellow dwarf virus infecting garlic in Iran

“…OYDV also has been considered the most pathogenic potyvirus to Allium plants and is recognized as the major viral pathogen in garlic [3,11,21]. In garlic, OYDV produces different types of mosaic symptoms on leaves depending on virus isolate and plant cultivars [11] and decreases garlic bulb weight in a range from 24 to 60% [4]. The CP gene is most often used for OYDV identification and classification [11,22].…”

“…Viruses infecting garlic are widespread globally and affect a great number of other members of Alliaceae [4]. These viruses are categorized in three main genera including Potyvirus, Carlavirus, and various members of the recently established genus Allexivirus [5].…”

“…These viruses are categorized in three main genera including Potyvirus, Carlavirus, and various members of the recently established genus Allexivirus [5]. Among them, Onion yellow dwarf virus (OYDV), which is a member of Potyvirus, is one of the most important viral pathogens of garlic crops worldwide [3,4].…”