Detection of Ralstonia solanacearum in ginger rhizomes by real-time PCR

Abstract

Bacterial wilt of ginger (Zingiber officinale), caused by Ralstonia solanacearum (Rs), has emerged as an important disease of ginger production in Thailand and throughout Asia. Real-time PCR assays were developed for detection of Rs in ginger rhizomes. A unique 329-bp DNA fragment from Rs biovar 4 from ginger was identified using amplified fragment length polymorphisms, and the nucleotide sequence was determined. PCR primer and probe sequences were designed for real-time PCR assays and screened against 86 strains of Rs. The primers RSAF1 and RSAR1 and probe RSP1 were shown to react with all strains of Rs race 1 biovars 3 and 4 but not biovars 1 and 2. Real-time TaqMan^® PCR protocols were developed for two real-time PCR platforms, the ABI 7700 sequence detection system (Applied Biosystems, Foster City, Calif.) and the portable Smart Cycler (Cepheid, Sunnyvale, Calif.). A comparison between classical real-time PCR and real-time BIO-PCR protocols, using 15 asymptomatic ginger rhizomes collected from different fields and markets, showed that 13 and 9 were positive by standard PCR and BIO-PCR, respectively. This is the first description of a real-time PCR assay capable of detecting Rs in asymptomatic ginger rhizomes and the first report of Rs in asymptomatic ginger rhizomes being sold in markets in Thailand.

Keywords:

• BIO-PCR
• Ralstonia solanacearum
• bacterial wilt
• ginger
• Zingiber officinale