Sporangiospore Viability and Oospore Production in the Spinach Downy Mildew Pathogen, Peronospora effusa

Downy mildew of spinach, caused by the obligate pathogen Peronospora effusa, remains the most important constraint in the major spinach production areas in the United States. This disease can potentially be initiated by asexual sporangiospores via “green bridges”, sexually derived oospores from seed or soil, or dormant mycelium. However, the relative importance of the various types of primary inoculum is not well known. The ability of P. effusa sporangiospores to withstand abiotic stress, such as desiccation, and remain viable during short- and long-distance dispersal and the ability of oospores to germinate and infect seedlings remain unclear. Thus, the primary objectives of this research were to evaluate the impact of desiccation on sporangiospore survival and infection efficiency and examine occurrence, production, and germination of oospores. Results indicate that desiccation significantly reduces sporangiospore viability as well as infection potential. Leaf wetness duration of 4 h was needed for disease establishment by spinach downy mildew sporangiospores. Oospores were observed in leaves of numerous commercial spinach cultivars grown in California in 2018 and Arizona in 2019. Frequency of occurrence varied between the two states-years. The presence of opposite mating types in spinach production areas in the United States was demonstrated by pairing isolates in controlled crosses and producing oospores on detached leaves as well as intact plants. Information from the study of variables that affect sporangiospore viability and oospore production will help in improving our understanding of the epidemiology of this important pathogen, which has implications for management of spinach downy mildew.

Genetic diversity of the spinach downy mildew pathogen based on hierarchical sampling

Downy mildew, caused by the obligate oomycete pathogen Peronospora effusa, is the most economically important disease of spinach. In the past 30 years, 14 new races and 13 strains with novel virulence have been identified. However, the mechanism(s) driving the rapid evolution of virulence remains unknown. To understand reproductive strategies potentially driving the emergence of new races in P. effusa, 67 composite isolates (a collection of symptomatic leaves from a single cultivar grown in a defined area) of P. effusa obtained from 13 states between 2010 and 2018 were used to analyze the population genetic diversity hierarchically. Genotypes at 33 SNP loci of 719 lesions from these 67 isolates were determined by targeted sequencing. Diversity was then evaluated among individual lesions within the composite isolates, between isolates, host cultivars, geographic locations, and years of isolates collected. A total of 380 genotypes were identified from 719 individual lesions. Of the 380 genotypes, 350 (92%) were unique while the most common genotype was identified in 110 lesions of 16 isolates collected from 13 cultivars from CA and AZ in 2016. Variation within composite isolates ranged from none (a single genotype among lesions from a composite isolate) to 38 unique genotypes recovered from 39 lesions of a composite isolate. An index of association analysis suggested asexual (clonal) and sexual reproduction play important roles in population structure. Based on discriminant analysis of principal components, four distinct subpopulations were identified. Host cultivar, origin, and time of collection had an effect on population differentiation, and genotypes specific to a certain location or collection period were identified. Some subpopulations were unique to certain areas, and were only detected after 2014-2016. The co-existence of sexual and asexual reproduction strategies may partially explain the rapid emergence and spread of new races and novel strains of P. effusa.

Spinach Growers Value Host Resistance and Synthetic Fungicides in the Fight against Downy Mildew

California spinach growers struggle to manage spinach downy mildew disease. The disease is especially difficult in the organic crop, which currently relies on resistant varieties to maintain disease-free crop. Alternative control measures are available, but it is not clear how growers perceive the efficacy of these methods. It is also not clear who growers contact to find out information on spinach downy mildew disease management. In this study, we conducted an online survey of people involved in spinach production, asking about their beliefs in the efficacy of different control methods and who they contact frequently to discuss spinach downy mildew control. We found that respondents were most positive about the efficacy of resistant varieties and synthetic pesticides, with much lower perceived efficacy for the practices of disking diseased fields, roguing diseased plants, and organic pesticides. Growers most frequently contacted pest control advisors (PCAs) about management strategies for spinach downy mildew. These results suggest that respondents are most confident about the efficacy of resistant varieties and synthetic pesticides and may be hesitant to adopt new control strategies like organic pesticides. The results also suggest that future extension efforts can be focused on PCAs to reach the most stakeholders with up to date research on downy mildew control.

Genome reconstruction of the non-culturable spinach downy mildew Peronospora effusa by metagenome filtering

Peronospora effusa (previously known as P. farinosa f. sp. spinaciae, and here referred to as Pfs) is an obligate biotrophic oomycete that causes downy mildew on spinach (Spinacia oleracea). To combat this destructive disease resistant cultivars are continually bred. However, new Pfs races rapidly break the employed resistance genes. To get insight into the gene repertoire of Pfs and identify infection-related genes, the genome of the first reference race, Pfs1, was sequenced, assembled, and annotated. Due to the obligate biotrophic nature of this pathogen, material for DNA isolation can only be collected from infected spinach leaves that, however, also contain many other microorganisms. The obtained sequences are, therefore, considered a metagenome. To filter and obtain Pfs sequences we utilized the CAT tool to taxonomically annotate ORFs residing on long sequences of a genome pre-assembly. This study is the first to show that CAT filtering performs well on eukaryotic contigs. Based on the taxonomy, determined on multiple ORFs, contaminating long sequences and corresponding reads were removed. Filtered reads were re-assembled to provide a clean and improved Pfs genome sequence of 32.40 Mbp consisting of 8,635 scaffolds. Transcript sequencing of a range of infection time points aided the prediction of a total of 13,277 gene models, including 99 RXLR(-like) effector, and 14 putative Crinkler genes. Comparative analysis identified common features in the secretomes of different obligate biotrophic oomycetes, regardless of their phylogenetic distance. Their secretomes are generally smaller, compared to hemibiotrophic and necrotrophic oomycete species. We observe a reduction in proteins involved cell wall degradation, in Nep1-like proteins (NLPs), proteins with PAN/apple domains, and host translocated effectors. The genome of Pfs1 will be instrumental in studying downy mildew virulence and for understanding the molecular adaptations by which new isolates break spinach resistance.

Research Advances in Adopting Drip Irrigation for California Organic Spinach: Preliminary Findings

The main objective of this study was to explore the viability of drip irrigation for organic spinach production and the management of spinach downy mildew disease in California. The experiment was conducted over two crop seasons at the University of California Desert Research and Extension Center located in the low desert of California. Various combinations of dripline spacings and installation depths were assessed and compared with sprinkler irrigation as control treatment. Comprehensive data collection was carried out to fully understand the differences between the irrigation treatments. Statistical analysis indicated very strong evidence for an overall effect of the irrigation system on spinach fresh yields, while the number of driplines in bed had a significant impact on the shoot biomass yield. The developed canopy crop curves revealed that the leaf density of drip irrigation treatments was slightly behind (1–4 days, depending on the irrigation treatment and crop season) that of the sprinkler irrigation treatment in time. The results also demonstrated an overall effect of irrigation treatment on downy mildew, in which downy mildew incidence was lower in plots irrigated by drips following emergence when compared to the sprinkler. The study concluded that drip irrigation has the potential to be used to produce organic spinach, conserve water, enhance the efficiency of water use, and manage downy mildew, but further work is required to optimize system design, irrigation, and nitrogen management practices, as well as strategies to maintain productivity and economic viability of utilizing drip irrigation for spinach.