Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Jan;62(1):87–93. doi: 10.1128/aem.62.1.87-93.1996

Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia indica, the Karnal bunt of wheat fungus.

M A Ferreira 1, P W Tooley 1, E Hatziloukas 1, C Castro 1, N W Schaad 1
PMCID: PMC167776  PMID: 8572716

Abstract

Mitochondrial DNA (mtDNA) from five isolates of Tilletia indica was isolated and digested with several restriction enzymes. A 2.3-kb EcoRI fragment was chosen, cloned, and shown to hybridize with total DNA restricted with EcoRI from T. indica and not from a morphologically similar smut fungus, Tilletia barclayana. The clone was partially sequenced, and primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2.3-kb fragment from total DNA of 17 T. indica isolates from India, Pakistan, and Mexico. DNA from 25 isolates of other smut fungi (T. barclayana, Tilletia foetida, Tilletia caries, Tilletia fusca, and Tilletia controversa) did not produce any bands, as detected by ethidium bromide-stained agarose gels and Southern hybridizations. The sensitivity of the assay was determined and increased by using a single nested primer in a second round of amplification, so that 1 pg of total mycelial DNA could be detected. The results indicated that the primers which originated from a cloned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contaminating wheat seeds.

Full Text

The Full Text of this article is available as a PDF (359.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amasino R. M. Acceleration of nucleic acid hybridization rate by polyethylene glycol. Anal Biochem. 1986 Feb 1;152(2):304–307. doi: 10.1016/0003-2697(86)90413-6. [DOI] [PubMed] [Google Scholar]
  2. Crowhurst R. N., Hawthorne B. T., Rikkerink E. H., Templeton M. D. Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplification of polymorphic DNA. Curr Genet. 1991 Nov;20(5):391–396. doi: 10.1007/BF00317067. [DOI] [PubMed] [Google Scholar]
  3. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ersek T., Schoelz J. E., English J. T. PCR amplification of species-specific DNA sequences can distinguish among Phytophthora species. Appl Environ Microbiol. 1994 Jul;60(7):2616–2621. doi: 10.1128/aem.60.7.2616-2621.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Garber R. C., Yoder O. C. Isolation of DNA from filamentous fungi and separation into nuclear, mitochondrial, ribosomal, and plasmid components. Anal Biochem. 1983 Dec;135(2):416–422. doi: 10.1016/0003-2697(83)90704-2. [DOI] [PubMed] [Google Scholar]
  6. Goodwin P. H., Annis S. L. Rapid identification of genetic variation and pathotype of Leptosphaeria maculans by random amplified polymorphic DNA assay. Appl Environ Microbiol. 1991 Sep;57(9):2482–2486. doi: 10.1128/aem.57.9.2482-2486.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. He Q., Marjamäki M., Soini H., Mertsola J., Viljanen M. K. Primers are decisive for sensitivity of PCR. Biotechniques. 1994 Jul;17(1):82, 84, 86-7. [PubMed] [Google Scholar]
  8. Kaji H., Xu Y., Takahashi Y., Abe H., Tamaki N., Chihara K. Human TRH receptor messenger ribonucleic acid levels in normal and adenomatous pituitary: analysis by the competitive reverse transcription polymerase chain reaction method. Clin Endocrinol (Oxf) 1995 Mar;42(3):243–248. doi: 10.1111/j.1365-2265.1995.tb01871.x. [DOI] [PubMed] [Google Scholar]
  9. Karlovsky P., de Cock A. W. Buoyant density of DNA-Hoechst 33258 (bisbenzimide) complexes in CsCl gradients: Hoechst 33258 binds to single AT base pairs. Anal Biochem. 1991 Apr;194(1):192–197. doi: 10.1016/0003-2697(91)90167-r. [DOI] [PubMed] [Google Scholar]
  10. Leite R. P., Jr, Minsavage G. V., Bonas U., Stall R. E. Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria. Appl Environ Microbiol. 1994 Apr;60(4):1068–1077. doi: 10.1128/aem.60.4.1068-1077.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Li K. N., Rouse D. I., German T. L. PCR primers that allow intergeneric differentiation of ascomycetes and their application to Verticillium spp. Appl Environ Microbiol. 1994 Dec;60(12):4324–4331. doi: 10.1128/aem.60.12.4324-4331.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Schesser K., Luder A., Henson J. M. Use of Polymerase Chain Reaction To Detect the Take-All Fungus, Gaeumannomyces graminis, in Infected Wheat Plants. Appl Environ Microbiol. 1991 Feb;57(2):553–556. doi: 10.1128/aem.57.2.553-556.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Simon L., Lalonde M., Bruns T. D. Specific amplification of 18S fungal ribosomal genes from vesicular-arbuscular endomycorrhizal fungi colonizing roots. Appl Environ Microbiol. 1992 Jan;58(1):291–295. doi: 10.1128/aem.58.1.291-295.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Steffan R. J., Atlas R. M. Polymerase chain reaction: applications in environmental microbiology. Annu Rev Microbiol. 1991;45:137–161. doi: 10.1146/annurev.mi.45.100191.001033. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES