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Fusarium oxysporum f.sp. lactucae (Fol) causes a vascular disease in lettuce that results in significant yield losses. Race-specific and sensitive real-time PCR assays were developed for Fol races 1 and 4, which are prevalent in Europe. Using genotyping-by-sequencing, unique DNA loci specific to each race were identified and subsequently used for the design of primers and hydrolysis probes. Two assays per race were developed to ensure specificity. The two assays of each race could be run in duplex format, while still giving a sensitivity of 100 fg genomic DNA for all assays. Sample preparation methods were developed for plant tissue, soil, and surfaces, with an extra enrichment step when additional sensitivity was required. By controlling the incubation conditions during the enrichment step, the real-time PCR signal could be matched to the number of spore equivalents in the original sample. When enriching naturally infested soil, down to six conidiospore equivalents L-1 soil could be detected. As enrichment ensures sensitive detection and focuses on living Fol propagules, it facilitates the evaluation of control measures. The developed detection methods for soil and surfaces were applied to samples from commercial lettuce farms and confirmed the prevalence of Fol race 4 in Belgium. Monitoring of soil disinfestation events revealed that despite a dramatic decrease in quantity, the pathogen could still be detected either immediately after sheet steaming or after harvesting the first new crop. The detection method for plant tissue was successfully used to quantify Fol in lettuce inoculated with race 1, race 4 or a combination of both. Under the temperature conditions used, race 4 was more aggressive than race 1, as reflected in larger amounts of DNA of race 4 detected in the roots. These newly developed assays are a promising tool for epidemiological research as well as for the evaluation of control measures. [ABSTRACT FROM AUTHOR]

Fusarium oxysporum f. sp. lactucae race 4 causes vascular necrosis and wilting of lettuce. First observed in Belgium in 2015, the lack of disease resistance in commercial cultivars allowed this pathogen to spread to nearly the entire Belgian production area within 4 years. Different levels of disease development were observed in different commercial greenhouses. To help explain this variation, we collected 78 Fusarium isolates and characterized them both physiologically and genetically. Molecular race identification indicated that 91% of the isolates belonged to race 4, while 6% of the isolates belonged to race 1, which was not previously reported in Belgium. Pathogenicity assays using differential cultivars confirmed the molecular race assignment of selected isolates. Cultivar Patriot was identified as a suitable new differential cultivar to race 4. Race 4 isolates were more aggressive than race 1 isolates at 24°C, but only when using chlamydospore inoculum instead of a root dip assay containing microconidia. Variation in pathogenicity and aggressiveness of the races may explain differences in disease development in commercial greenhouses. Based on genotyping‐by‐sequencing (GBS), race 1 and race 4 isolates were highly similar to reference isolates. Fusarium curvatum, F. oxysporum f. sp. tulipae and F. oxysporum f. sp. rhois were phylogenetically separated from F. oxysporum f. sp. lactucae races 1 and 4 based on the GBS data, but not when using multilocus sequence data. Within F. oxysporum f. sp. lactucae race 4, the GBS data differentiated two rather homogeneous groups, suggesting at least two introductions. However, the two groups did not differ in aggressiveness. [ABSTRACT FROM AUTHOR]