Fusarium wilt of spinach, caused by Fusarium oxysporum f. sp. spinaciae (Fos), is an important disease during warm conditions in production regions with acid soils, yet little is known about what confers pathogenicity to spinach in Fos genetically. To identify candidate fungal genes that contribute to spinach Fusarium wilt, each of 69 geographically diverse F. oxysporum isolates was tested for pathogenicity on each of three spinach inbreds. Thirty-nine isolates identified as Fos caused quantitative differences in disease severity among the inbreds that revealed two distinct pathogenicity groups of Fos. Putative effector gene profiles, predicted from whole-genome sequences generated for nine Fos isolates and five non-pathogenic, spinach-associated F. oxysporum (NPS) isolates, distinguished the Fos isolates from the NPS isolates, and separated the Fos isolates into two groups. Five of the putative effector genes appeared to be unique to Fos as they were not found in 222 other publicly available genome assemblies of F. oxysporum, implicating potential involvement of these genes in pathogenicity to spinach. In addition, two combinations of the 14 known Secreted in Xylem (SIX) genes that have been affiliated with host pathogenicity in other formae speciales of F. oxysporum were identified in genome assemblies of the nine Fos isolates: either SIX8 and SIX9, or SIX4, SIX8, and SIX14. Characterization of these putative effector genes should aid in understanding mechanisms of pathogenicity in Fos, developing molecular tools for rapid detection and quantification of Fos, and breeding for resistance to Fusarium wilt in spinach.

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Two pathogenicity groups of Fusarium oxysporum f. sp. spinaciae, the causal agent of Fusarium wilt of spinach (Spinacia oleracea L.), were described recently based on virulence of isolates on proprietary spinach inbreds. In this study, a wide range in severity of wilt was observed for 68 spinach cultivars inoculated with an isolate of each pathogenicity group, with 22 (32.4%) cultivars displaying differential responses to the isolates. In a second set of trials, seven spinach cultivars were inoculated with five isolates of each pathogenicity group. The cultivars had similar wilt responses to isolates within each group. In both sets of trials, the most severe wilt developed on cultivars inoculated with pathogenicity group 2 isolates when daylength was shorter and light intensity lower. To test whether light intensity exacerbates severity of Fusarium wilt, three spinach cultivars were inoculated with two isolates of each pathogenicity group and grown with or without shading. Shaded plants developed more severe wilt than non-shaded plants. This difference in wilt severity was greatest for plants inoculated with pathogenicity group 2 isolates. We propose naming isolates of pathogenicity groups 1 and 2 as races 1 and 2 of F. oxysporum f. sp. spinaciae, respectively, and recommend the cultivars Kiowa (susceptible to both races) and Magnetic (susceptible to race 2 and highly resistant to race 1) as differentials. Results of this study should help breeders screen spinach germplasm for resistance to both races of F. oxysporum f. sp. spinaciae.

Fusarium wilt of spinach, caused by Fusarium oxysporum f. sp. spinaciae, is a significant limitation for producers of vegetative spinach and spinach seed crops during warm temperatures and/or on acid soils. Identification of isolates of F. oxysporum f. sp. spinaciae, and distinction of isolates of the two known races entails time-intensive pathogenicity tests. In this study, two real-time PCR assays were developed: one for a candidate effector gene common to both races of F. oxysporum f. sp. spinaciae, and another for a candidate effector gene unique to isolates of race 2. The assays were specific to isolates of F. oxysporum f. sp. spinaciae (n = 44) and isolates of race 2 (n = 23), respectively. Neither assay amplified DNA from 10 avirulent isolates of F. oxysporum associated with spinach, 57 isolates of other formae speciales and Fusarium spp., or 7 isolates of other spinach pathogens. When the assays were used to detect DNA extracted from spinach plants infected with an isolate of race 1, race 2, or a 1:1 mixture of both races, the amount of target-DNA detected increased with increasing severity of wilt. Plants infected with one or both isolates could be distinguished based on the ratio in copy number for each target locus. The real-time PCR assays enable rapid diagnosis of Fusarium wilt of spinach and will facilitate research on the epidemiology and management of this disease, as well as surveys on the prevalence of this understudied pathogen in regions of spinach and/or spinach seed production.