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

Fusarium oxysporum f. sp. lactucae (FOLac) is a soil- and seedborne fungal pathogen that causes Fusarium wilt of lettuce, an important disease threatening global lettuce production. Based on pathogenicity 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, emerging race 4 known only in Europe. The development of race-specific diagnostic tools is hindered by insufficient genomic data to distinguish between the two races and FOLac from other F. oxysporum formae speciales and nonpathogenic isolates. Here, we describe a systematic approach for developing diagnostic markers for FOLac race 1 that utilized a comprehensive sequence database of F. oxysporum to identify 15 unique genomic sequences. Marker specificity was validated through an exhaustive screening process against genomic data from 797 F. oxysporum isolates representing 64 formae speciales and various plants and non-plant substrates. One of the unique sequences was used to develop a TaqMan quantitative polymerase chain reaction assay and a recombinase polymerase amplification assay, both exhibiting 100% sensitivity and specificity when tested against purified DNA from 171 F. oxysporum isolates and 69 lettuce samples. The relationship between qPCR C_t values and colony forming units (CFU)/g values was also determined. This study not only introduces a new marker for FOLac race 1 diagnostics and soil quantitation, but also underscores the value of an extensive genomic database and screening software pipeline for developing molecular diagnostics for F. oxysporum formae speciales and other fungal taxa.