ATTACHMENT NO. 3: Critical Review of NC-501. Initiated October 1, 1997.

Objective 1. Gain an improved understanding of Claviceps africana biology in the Central Great Plains.
a) Overwintering of ergot in Kansas and Nebraska.
Although conditions were not conducive for the development of ergot in Kansas and Nebraska m 1998, researchers at Kansas State University and the University of Nebraska were able to conduct experiments to evaluate the over-wintering capacity of ergot in the Central Great Plains. Ergot infected heads collected during the fall of 1997 were stored under natural field conditions as surface and sub-surface residue. Researchers in Kansas were able to show that ergot conidia collected from these heads were viable and able to germinate in vitro as late as June of 1998. Researchers in Nebraska conducted similar experiments and were able to induce ergot infection in greenhouse grown plants using inoculum collected from field locations as late as May of 1998. Similar studies are planned for 1999.

b) Alternate ergot hosts.
To determine the source of C. africana inoculum for infection of florets during the initial flowering period in each growing region, the role of potential collateral hosts to ergot is being assessed. Researchers in Kansas and Nebraska are screening an array of range grasses as well as other sorghum species for susceptibility to ergot. Research to date has indicated that all Sorghum spp. are susceptible to ergot including Sorghum almum. S. bicolor, S. dmrnmondii, S aethiopicum S. vertilliflorum. S. virgatum, S. arundinaceum, and S. halepence. No susceptible hosts have been identified among common range grasses that have been evaluated to date including Big and Little Bluestem, Osage Indian Grass, Switch Grass, Canada Wild Rye, or Gama Grass. Studies of additional sorghum and rangeland grass species are underway.

c) Taxonomic, phylogenetic, and biochemical characterization of Claviceps africana.
A library of C. africana isolates collected from ergot infected fields in the U S Mexico, the Caribbean and South American is being assembled by researchers at the University of Nebraska. Mitochondrial and chromosoma1 ITS fragments wi11 be isolated from these samples by PCR and will be sequenced determine genetic differences that can be used to separate Claviceps sp.

Objective 2. Develop rapid screening techniques for factors contributing host-plant resistance to ergot.
Cold temperature induced male sterility predisposes sorghum to ergot infection Researchers m Kansas and Nebraska are conducting experiments to systematically characterize the response of sorghum to cold temperature stress. The first objective was to develop a sorghum pollen germination assay to quantify pollen viability. A pollen germination assay was developed and optimized for use in sorghum. Pollen germination was used to quantify chants m sorghum pollen viability following anthesis and in response different temperature treatments. The results of this study have been prepared for publication in the Australian Journal of Plant Physiology.

Studies are currently being conducted to determine the mode of action of cold temperature induced male sterility in sorghum. Studies to date indicate that cold temperature stress delays anther dehiscence but does not appear to significantly reduce pollen viability as measured by pollen germination and seed set. Large differences in cold tolerance were detected among elite parent lines. Studies are planned to evaluate the inheritance of cold tolerance among these elite parent lines.