Identification and Differentiation of Tilletia indica and T. walkeri Using the Polymerase Chain Reaction

Abstract

Karnal bunt of wheat, caused by Tilletia indica, was found in regions of the southwestern United States in 1996. Yield losses due to Karnal bunt are slight, and the greatest threat of Karnal bunt to the U.S. wheat industry is the loss of its export market. Many countries either prohibit or restrict wheat imports from countries with Karnal bunt. In 1997, teliospores morphologically resembling T. indica were isolated from bunted ryegrass seeds and wheat seed washes. Previously developed PCR assays failed to differentiate T. indica from the recently discovered ryegrass pathogen, T. walkeri. The nucleotide sequence of a 2.3 kb region of mitochondrial DNA, previously amplified by PCR only from T. indica, was determined for three isolates of T. indica and three isolates of T. walkeri. There was greater than 99% identity within either the T. indica group or the T. walkeri group of isolates, whereas there was =3% divergence between isolates of these two Tilletia species. Five sets of PCR primers were made specific to T. indica, and three sets were designed specifically for T. walkeri based upon nucleotide differences within the mitochondrial DNA region. In addition, a 212 bp amplicon was developed as a target sequence in a fluorogenic 5' nuclease PCR assay using the TaqMan system for the detection and discrimination of T. indica and T. walkeri.

Real-Time PCR Assays for Detection and Quantification of Sweetpotato Viruses

“…In plant pathology, this technology increasingly is used for studying various causal agents of plant diseases (1,9,19,23). This is the first report of the use of real-time PCR technology and TaqMan fluorogenic chemistry for the detection and quantification of sweetpotato viruses.…”

Rapid and specific detection of Tilletia indica using loop-mediated isothermal DNA amplification

“…Several strategies have been designed to distinguish T. indica from other Tilletia species when using PCR-based technique. An early strategy capitalized on the unique property of Taq polymerase which required a perfect match between the primers and the template at the 3′ terminal ends for amplification (Frederick et al 2000), or the special features of the genomic DNA that allowed species differentiation by repetitive-sequence-based polymerase chain reaction (rep-PCR) fingerprinting (McDonald et al 2000) or random amplified polymorphic DNA (RAPD) analysis (Mishra et al 2002). Another strategy involved using PCR in conjunction with anther technique, such as polymorphism of restriction fragment lengths in the PCR products (Levy et al 2001) or fluorescence resonance energy transfer (FRET) analysis (Tan and Murray 2006).…”

Diagnostics of Seed-Borne Plant Pathogens for Safe Introduction and Healthy Conservation of Plant Genetic Resources

“…Plasmopara halstedii , causing sunfl ower downy mildew in sunfl ower seeds (Says-Lesage et al 2001 ), could be detected by PCR assay. Tilletia indica , causing Karnal bunt disease in wheat seeds (Frederick et al 2000 ), Rhynchosporium secalis , causing scald disease in barley seeds, Alternaria alternata , A. radicina , and A. dauci in carrot seeds (Konstantinova et al 2002 ), A. brassicae , A. brassicola , and A. japonica , causal agents of black spot in crucifers (Iacomi-Vasilescu et al 2002 ) could be effectively detected by PCR assays. Furthermore, a number of Fusarium species, such as F. culmorum , F. graminearum , F. poae , F. crookwellense , F. sporotrichoides , F. sambucinum , F. avenaceum , F. trinctum and F. nivale , causing fusarium head blight/scab disease in cereals have been differentiated by PCR assay using trichodiene synthase gene (Tri5) (Edwards et al 2001 ).…”