Identification of Tilletia indica, Cause of Karnal Bunt of Wheat

Introduction

Accurate identification of Tilletia indica is essential for making appropriate plant quarantine decisions. Tilletia indica can be identified accurately using morphological characters if the teliospores are found on infected wheat seeds. Identification to species can be problematic when the host is not known, especially if few teliospores are found. In general if sufficient teliospores are present with diameters mainly in the upper half of the size range for T. indica, identification is possible based on spore size and morphology, even if the host is not certain. If the host is not known and few teliospores are present or if their diameter is in the lower half of the size range for T. indica, then confirmation requires the use of a molecular technique such as that developed by Smith et al. (1996).

Mature teliospore and several sterile cells.

Identification

Tilletia indica is characterized by the production of dark brown, globose to subglobose teliospores measuring 22-47 um diameter with an average that ranges from 35 - 39 um. Young teliospores have spines that are pointed, straight or sometimes curved, becoming truncate with age. These teliospore characteristics distinguish T. indica from the other species of Tilletia that infect wheat. The latter produce smooth or minutely punctate or reticulate teliospores and are smaller in size.

A partially mature teliospore showing ornamentation in median view.	A partially mature teliospore showing ornamentation in surface view.

When the host is not certain the situation is more difficult because there are other described species of Tilletia that produce teliospores similar in morphology to T. indica. The species that can be confused with T. indica include Tilletia horrida, Tilletia barclayana, T. pulcherrima, T. pennisetina, and others. These have been considered one species, distinct from T. indica, under the name Tilletia barclayana based on work by Tullis and Johnson (1952). However, more recent evidence suggests that, in fact, T. horrida, the rice kernel smut fungus, may be a distinct species (Duran and Gray, 1989; Whitney, 1989). The other species of Tilletia that have teliospores morphologically similar to T. indica are from grass hosts such as Panicum and Digitaria. These latter species have not been studied adequately to draw conclusions about their taxonomic and biological relationships. Data on when their grass hosts produce seed and teliospores, if infected, in relation to when wheat is harvested are necessary to determine whether teliospores similar to T. indica might be present as contaminants of wheat seed samples.

Working with a statistician, G. Peterson (unpubl.) developed a morphometric test based on a logistics index to differentiate teliospores of T. indica from those of "T. barclayana." Because the T. barclayana teliospores were from rice, those measurement data are for T. horrida in the strict sense. The test is being evaluated further and modified as necessary based on data from measurements of teliospores of the other Tilletia species listed above, in order to account for the possibile presence of other T. indica-like spores.

Smith et al. (1996) developed a PCR-based method for identifying T. indica. Primers were developed that were specific for T. indica and could reliably differentiate that species from T. barclayana as well as other species of Tilletia on wheat. Ferreira et al. (1996) isolated a species-specific mitochondrial DNA sequence that might be useful in identifying Tilletia indica.

Mature teliospores

Summary

At present, all possible steps are being taken to ensure accurate identification of T. indica. For consistency among samples and sites, all measurements must be made using a 100X objective and teliospores mounted in Shear's mounting medium (Graham, 1959). For future reference voucher specimens of teliospores and bunted kernels or both are deposited in the U.S. National Fungus Collection, Beltsville, Maryland. Additional methods are being evaluated to facilitate accurate identification of T. indica in the U.S. national survey and for certification of U.S.-grown wheat for export. Morphological and molecular means for rapid identification of teliospores of T. indica are being explored. Morphological and molecular work also is underway to enable accurate identification of species similar to T. indica, particularly to avoid falsely identifying them as T. indica in the absence of host material. Methods for rapid observation of bunted kernels in a sample are under consideration. Finally, no matter which approach is used for identification, basic knowledge of the biosystematics of Tilletia indica and similar species is essential, using morphological, molecular, and biological characteristics.

References Cited

Ferreira, M. A. S. V., P. W. Tooley, E. Hatziloukas, C. Castro, and N. W. Schaad. 1996. Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia indica, the Karnal bunt of wheat fungus.

Appl. Environ. Microbiol. 1996 (Jan.):87-93. Graham, S. 1959. The effects of various reagents, mounting media, and dyes on the teliospore walls of Tilletia controversa. Mycologia 51:477-491.

Smith, O., G. L. Peterson, R. J. Beck, N. W. Schaad, and M. R. Bonde. 1996. Development of a PCR-based methods for identification of Tilletia indica, causal agent of Karnal bunt of wheat. Phytopathology 86:115-122.

Tullis, E., and A. Johnson. 1952. Synonymy of Tilletia horrida and Neovossia barclayana. Mycologia 44:773-788.