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ARS Home » Pacific West Area » Salinas, California » Crop Improvement and Protection Research » Research » Publications at this Location » Publication #414677
Research Project: Disease Management and Improved Detection Systems for Control of Pathogens of Vegetables and Strawberries

Location: Crop Improvement and Protection Research

Title: A systematic approach for identifying unique genomic sequences for Fusarium oxysporum f. sp. lactucae race 1 and development of molecular diagnostic tools

Author
item LI, NINGXIAO - Pennsylvania State University
item GEISER, DAVID - Pennsylvania State University
item STEENWYK, JACOB - University Of California
item TSUCHIDA, CAYLA - Trical Inc
item KOIKE, STEVE - Trical Inc
item SLINSKI, STEPHANIE - University Of Arizona
item Martin, Frank

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/21/2024
Publication Date: 10/22/2024
Citation: Li, N., Geiser, D.M., Steenwyk, J.L., Tsuchida, C., Koike, S., Slinski, S., Martin, F.N. 2024. A systematic approach for identifying unique genomic sequences for Fusarium oxysporum f. sp. lactucae race 1 and development of molecular diagnostic tools. Phytopathology. 115(2):204-217. https://doi.org/10.1094/PHYTO-04-24-0142-R.
DOI: https://doi.org/10.1094/PHYTO-04-24-0142-R

Interpretive Summary: This submission describes the development of a software pipeline that provides a systematic approach for designing diagnostic markers for detection of different lineages of Fusarium oxysporum. Diagnostic assays developed with this software pipeline for detection of race 1 of the lettuce pathogen Fusarium oxysporum f. sp. lactucae are described. One assay is laboratory based and uses real time PCR to detect and quantify the pathogen. A second isothermal assay using recombinant polymerase amplification is much quicker and can be run directly in the field in as little as 20 minutes.

Technical Abstract: Fusarium oxysporum f. sp. lactucae (FOL) is a soil- and seedborne fungal pathogen that causes Fusarium wilt of lettuce, an important disease threatening global lettuce production. Based on the ability to cause disease on differential lettuce cultivars, four races (1-4) have been identified, with race 1 the only race detected in the United States and the closely related race 4 a more recently emerged race found only in Europe. However, the lack of genomic data greatly impedes our progress in developing diagnostic tools that can accurately distinguish race 1 from other races and other formae speciales. In this study, we identified 15 unique genomic sequences for FOL race 1 by generating a comprehensive sequence database of F. oxysporum and underwent a stringent screening process to ensure specificity using genomic data from 797 F. oxysporum isolates representing 60 formae speciales and ecologically diverse isolates associated with various hosts and soils. One of the unique sequences was used to develop both a TaqMan quantitative polymerase chain reaction assay and a recombinase polymerase amplification assay for identification and diagnostics of Fusarium wilt from fungal cultures, diseased lettuce plants, and potentially for soil quantification. Both assays had 100% sensitivity and specificity by screening purified DNA from 171 F. oxysporum isolates representing diverse taxa. Both assays were capable of multiplexing with internal controls to eliminate false negatives and were validated with 69 lettuce samples. Together, this work demonstrates the value of using a systematic approach to identify sequences for pathogen identification and provides valuable tools to monitor for FOL race 1.