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Enzyme-Linked Immunosorbent Assay for Detection of Thielaviopsis basicola. Brent A. Holtz, Former graduate student researcher, Department of Plant Pathology, University of California, Berkeley 94720, Current address: Department of Plant Pathology, University of California, Davis, Kearney Agricultural Center, 9240 S. Riverbend Ave., Parlier 93648; Alexander E. Karu, and Albert R. Weinhold. director of the College of Natural Resources Hybridoma Facility, and professor, respectively, Department of Plant Pathology, University of California, Berkeley 94720. Phytopathology 84:977-983. Accepted for publication 7 June 1994. Copyright 1994 The American Phytopathological Society. DOI: 10.1094/Phyto-84-977.

Field isolates of Thielaviopsis basicola, the causal agent of black root rot of cotton (Gossypium hirsutum), were grown in Czapek-Dox broth amended with dialyzed carrot extract. Soluble protein extracts of chlamydospores and mycelium were used to raise polyclonal mouse ascites antibodies. The immunoglobulin G antibody fraction was purified and biotin-labeled to devise a fungal capture sandwich enzyme-linked immunosorbent assay (ELISA). ELISA detected both brown and gray cultural types of T. basicola and had negligible cross-reactivity with other soilborne fungi commonly found in the San Joaquin Valley of California cotton field soils. The minimum detection limit of ELISA was between 1 and 20 ng of T. basicola protein depending on the assay. T. basicola could be detected in cotton roots 2 days after inoculation. At this time, initial symptoms were apparent. The antibody also was used to observe T. basicola on cotton roots with immunofluorescence microscopy.