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Background: Spinach downy mildew, caused by the obligate oomycete pathogen, Peronospora effusa remains a major concern for spinach production. Disease control is predominantly based on development of resistant spinach cultivars. However, new races and novel isolates of the pathogen continue to emerge and overcome cultivar resistance. Currently there are 20 known races of P. effusa. Here we characterized the transcriptomes of spinach, Spinacia oleracea, and P. effusa during disease progression using the spinach cultivar Viroflay, the near isogenic lines NIL1 and NIL3, and P. effusa races, R13 and R19, at 24 h post inoculation and 6 days post inoculation. A total of 54 samples were collected and subjected to sequencing and transcriptomic analysis., Results: Differentially expressed gene (DEG) analysis in resistant spinach interactions of R13-NIL1 and R19-NIL3 revealed spinach DEGs from protein kinase-like and P-loop containing families, which have roles in plant defense. The homologous plant defense genes included but were not limited to, receptor-like protein kinases (Spiol0281C06495, Spiol06Chr21559 and Spiol06Chr24027), a BAK1 homolog (Spiol0223C05961), genes with leucine rich repeat motifs (Spiol04Chr08771, Spiol04Chr01972, Spiol05Chr26812, Spiol04Chr11049, Spiol0084S08137, Spiol03Chr20299) and ABC-transporters (Spiol02Chr28975, Spiol06Chr22112, Spiol06Chr03998 and Spiol04Chr09723). Additionally, analysis of the expression of eight homologous to previously reported downy mildew resistance genes revealed that some are differentially expressed during resistant reactions but not during susceptible reactions. Examination of P. effusa gene expression during infection of susceptible cultivars identified expressed genes present in R19 or R13 including predicted RxLR and Crinkler effector genes that may be responsible for race-specific virulence on NIL1 or NIL3 spinach hosts, respectively., Conclusions: These findings deliver foundational insight to gene expression in both spinach and P. effusa during susceptible and resistant interactions and provide a library of candidate genes for further exploration and functional analysis. Such resources will be beneficial to spinach breeding efforts for disease resistance in addition to better understanding the virulence mechanisms of this obligate pathogen., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Spinach downy mildew, caused by the obligate oomycete pathogen Peronospora effusa , is a worldwide constraint on spinach production. The role of airborne sporangia in the disease cycle of P. effusa is well established, but the role of the sexual oospores in the epidemiology of P. effusa is less clear and has been a major challenge to examine experimentally. To evaluate seed transmission of spinach downy mildew via oospores in this study, isolated glass chambers were employed in two independent experiments to grow out oospore-infested spinach seed and noninfested seeds mixed with oospore-infested crop debris. Downy mildew diseased spinach plants were observed 37 and 34 days after planting in the two isolator experiments, respectively, in the chambers that contained one of two oospore-infested seed lots or seeds coated with oospore-infested leaves. Spinach plants in isolated glass chambers initiated from seeds without oospores did not show downy mildew symptoms. Similar findings were obtained using the same seed lot samples in a third experiment conducted in a growth chamber. In direct grow out tests to examine oospore infection on seedlings performed in a containment greenhouse with oospore-infested seed of two different cultivars, characteristic Peronospora sporangiophores were observed growing from a seedling of each cultivar. The frequency of seedlings developing symptoms from 82 of these oospore-infested seed indicated that approximately 2.4% of seedlings from infested seed developed symptoms, and 0.55% of seedlings from total seeds assayed developed symptoms. The results provide evidence that oospores can serve as a source of inoculum for downy mildew and provide further evidence of direct seed transmission of the downy mildew pathogen to seedlings in spinach via seedborne oospores., Competing Interests: The author(s) declare no conflict of interest.

Downy mildew of spinach, caused by Peronospora effusa , is a major economic threat to both organic and conventional spinach production. Symptomatic spinach leaves are unmarketable and spinach with latent infections are problematic because symptoms can develop postharvest. Therefore, early detection methods for P. effusa could help producers identify infection before visible symptoms appear. Recombinase polymerase amplification (RPA) provides sensitive and specific detection of pathogen DNA and is a rapid, field-applicable method that does not require advanced technical knowledge or equipment-heavy DNA extraction. Here, we used comparative genomics to identify a unique region of the P. effusa mitochondrial genome to develop an RPA assay for the early detection of P. effusa in spinach leaves. In tandem, we established a TaqMan quantitative PCR (qPCR) assay and used this assay to validate the P. effusa specificity of the locus across Peronospora spp. and to compare assay performance. Neither the TaqMan qPCR nor the RPA showed cross reactivity with the closely related beet downy mildew pathogen, P. schachtii . TaqMan qPCR and RPA have detection thresholds of 100 and 900 fg of DNA, respectively. Both assays could detect P. effusa in presymptomatic leaves, with RPA-based detection occurring as early as 5 days before the appearance of symptoms and TaqMan qPCR-based detection occurring after 24 h of plant exposure to airborne spores. Implementation of the RPA detection method could provide real-time information for point-of-care management strategies at field sites.