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.

[1]  K. Livak,et al.  Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. , 1995, PCR methods and applications.

[2]  L. M. Carris,et al.  Genetic variability among isolates of Tilletia barclayana, T. indica and allied species , 1998 .

[3]  M. Mitra.  A New Bunt on Wheat in India , 1931 .

[4]  K. Livak,et al.  Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes , 1995, Applied and environmental microbiology.

[5]  C. Schoen,et al.  Detection of potato leafroll virus in dormant potato tubers by immunocapture and a fluorogenic 5' nuclease RT-PCR assay , 1996 .

[6]  T. White.  Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics , 1990 .

[7]  J M Henson,et al.  The polymerase chain reaction and plant disease diagnosis. , 1993, Annual review of phytopathology.

[8]  K. Mullis,et al.  Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. , 1987, Methods in enzymology.

[9]  Y. Berthier-Schaad,et al.  Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by BIO-PCR and an Automated Real-Time Fluorescence Detection System. , 1999, Plant disease.

[10]  G. Peterson,et al.  Development of a PCR-based method for identification of Tilletia indica, causal agent of Karnal bunt of wheat , 1996 .

[11]  J. Devereux,et al.  A comprehensive set of sequence analysis programs for the VAX , 1984, Nucleic Acids Res..

[12]  J. Phillips,et al.  Comparison of the virulence of isolates of Tilletia indica, causal agent of Karnal bunt of wheat, from India, Pakistan, and Mexico , 1996 .

[13]  N. Schaad,et al.  Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia indica, the Karnal bunt of wheat fungus , 1996, Applied and environmental microbiology.

[14]  J. Smilanick,et al.  Karnal Bunt of Wheat. , 1997, Plant disease.

[15]  L. Castlebury,et al.  Tilletia walkeri, a new species on Lolium multiflorum and L. perenne , 1999 .

[16]  M. Palm.  Mycology and world trade: a view from the front line , 1999 .

[17]  L. Mayer,et al.  Utility of Random Amplified Polymorphic DNA PCR and TaqMan Automated Detection in Molecular Identification ofAspergillus fumigatus , 1998, Journal of Clinical Microbiology.

[18]  R. Durán.  Aspects of teliospore germination in North American smut fungi. II. , 1972 .

[19]  G. Peterson,et al.  Separation of Tilletia indica chromosomes using CHEF gel electrophoresis , 1995 .

[20]  G. Peterson,et al.  First report of karnal bunt of wheat in the United States. , 1996 .

[21]  F. Nolte,et al.  Rapid Identification of up to ThreeCandida Species in a Single Reaction Tube by a 5′ Exonuclease Assay Using Fluorescent DNA Probes , 1999, Journal of Clinical Microbiology.

[22]  K. Mullis,et al.  9 – Specific Synthesis of DNA in Vitro via a Polymerase-Catalyzed Chain Reaction , 1989 .