1 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77840, USA

Background and objectives
Among the fungal diseases that affect sorghum and maize crops worldwide, head smut (Sporisorium reilianum (Kuhn) Langdom and Fullerton) remains one of the most important. Over the past several decades, variations in the population dynamics of the sorghum head smut fungus (SHS) have resulted in losses of deployed resistance. Current sorghum breeding requires extensive field evaluation of resistance to at least three different pathotypes. In maize only one head smut (MHS) pathotype is known. The characterization of SHS populations requires the evaluation of uniform disease nurseries grown in areas with known high levels of smut infestation. However, most evaluation techniques are inaccurate or unreliable [1]. The objective of this study is to develop sensitive, selectively neutral genetic markers that could differentiate among isolates within each host, and use them for further studies on the population biology and epidemiology of S. ;reilianum.

Materials and methods
Ten SHS isolates from USA, Mexico, China, Mali and Uganda, as well as 10 MHS isolates from USA and Mexico were employed in this work. DNA from each isolate was digested with five different restriction enzymes. Two genomic libraries containing random S. ;reilianum DNA fragments were constructed using one SHS isolate. Two-hundred and ninety clones obtained from the libraries were used as potential probes for RFLP analysis. Sixty-four probes were screened using the five enzymes (320 probe-enzyme possible combinations within each host) and 82 probes were screened using two of the enzymes (164 probe enzyme combinations each for SHS and MHS isolates). The probe-enzyme combinations representing the most informative RFLP loci within each host were used to construct multilocus haplotypes for each individual. Each unique fragment or set of fragments at an RFLP locus was treated as an allele and assigned a unique number [2]. The alleles at different RFLP loci were combined in the same order for each isolate. The resulting haplotype was a nine-digit number per isolate that summarized which allele was present at each RFLP locus for each isolate.

Results and conclusions

When SHS isolates were compared with MHS isolates, a high level of variation was detected. Fifty-five per cent of the probe-enzyme combinations were polymorphic. Most of the differences given by the polymorphic probe-enzyme combinations between SHS and MHS hybridized to single or low copy sequences, where all the isolates within each host gave the same pattern. A lower genetic diversity was found within each host. In the case of SHS, 18 % of the probe-enzyme combinations tested were polymorphic among the 10 isolates. For MHS, 11% showed RFLPs; most of them had only one polymorphism among the 10 isolates evaluated. The high genetic diversity between SHS and MHS populations, and the limited genetic diversity found within each host suggests that these smuts have evolved independently from a source population, and strong selection for strict host specialization has maintained their distinct lineages. Nine of the probe-enzyme combinations which showed the greatest resolution were used to construct multilocus haplotypes for each individual. Each one of the nine selected probes displayed only two or three alleles within the isolates of each host screened. Among the 10 SHS isolates, six different multilocus haplotypes were present; among the 10 MHS isolates, only four. The probes selected in this study hybridized to one or few DNA fragments in suitable size ranges. The polymorphisms are easily differentiated, and displayed two or three alleles within the isolates of each host. The selected probes are currently being used to evaluate the genetic structure of nine SHS and four MHS populations collected from different locations of Mexico, USA and Niger.

1. Criag J, Frederiksen RA, 1992. Plant Disease 76, 314-18.
2. McDonald BA, Martinez JP, 1990. Phytopathology 80, 1368-73.