High quality, phased genomes of Phytophthora ramorum clonal lineages NA1 and EU1

Phytophthora ramorum is the causal agent of sudden oak death in West Coast forests and currently two clonal lineages, NA1 and EU1, cause epidemics in Oregon forests. Here, we report on two high-quality genomes of individuals belonging to the NA1 and EU1 clonal lineages respectively, using PacBio long-read sequencing. The NA1 strain Pr102, originally isolated from coast live oak in California, is the current reference genome and was previously sequenced independently using either Sanger (P. ramorum v1) or PacBio (P. ramorum v2) technology. The EU1 strain PR-15-019 was obtained from tanoak in Oregon. These new genomes have a total size of 57.5 Mb, with a contig N50 length of ~3.5-3.6 Mb and encode ~15,300 predicted protein-coding genes. Genomes were assembled into 27 and 28 scaffolds with 95% BUSCO scores and are considerably improved relative to the current JGI reference genome with 2,575 or the PacBio genomes with 1,512 scaffolds. These high-quality genomes provide a valuable resource for studying the genetics, evolution, and adaptation of these two clonal lineages.

Innate Resistance and Phosphite Treatment Affect Both the Pathogen’s and Host’s Transcriptomes in the Tanoak-Phytophthora ramorum Pathosystem

Phosphites have been used to control Sudden Oak Death; however, their precise mode of action is not fully understood. To study the mechanism of action of phosphites, we conducted an inoculation experiment on two open-pollinated tanoak families, previously found to be partially resistant. Stems of treatment group individuals were sprayed with phosphite, and seven days later, distal leaves were inoculated with the Sudden Oak Death pathogen Phytophthora ramorum. Leaves from treated and untreated control plants were harvested before and seven days after inoculation, and transcriptomes of both host and pathogen were analyzed. We found that tanoak families differed in the presence of innate resistance (resistance displayed by untreated tanoak) and in the response to phosphite treatment. A set of expressed genes associated with innate resistance was found to overlap with an expressed gene set for phosphite-induced resistance. This observation may indicate that phosphite treatment increases the resistance of susceptible host plants. In addition, genes of the pathogen involved in detoxification were upregulated in phosphite-treated plants compared to phosphite-untreated plants. In summary, our RNA-Seq analysis supports a two-fold mode of action of phosphites, including a direct toxic effect on P. ramorum and an indirect enhancement of resistance in the tanoak host.

First reports of Phytophthora ramorum clonal lineages NA1 and EU1 causing Sudden Oak Death on tanoaks in Del Norte County, California

A year of forest health surveys has led to the first detection of Phytophthora ramorum in Del Norte County followed by the first wildland detection of the EU1 clonal lineage (Grunwald et al. 2009) of this pathogen in California. In July 2019, leaves were sampled from two tanoaks (Notholithocarpus densiflorus) and 16 California bay laurels (Umbellularia californica) in Jedediah Smith State Park in Del Norte County, the northernmost coastal County of California. Leaves displayed lesions normally associated with Sudden Oak Death (SOD) caused by P. ramorum and were discovered during the citizen science-based survey known as SOD Blitz (Meentemeyer et al. 2015). Samples were surface sterilized using 75% Ethanol and plated on PARPH-V8 agar (Jeffers and Martin 1986). After plating, DNA was extracted and amplified using two P. ramorum-specific assays (Hayden et al. 2006, Kroon et al. 2004). Leaves from two tanoaks exhibiting twig die-back had typical SOD lesions along the midvein, gave positive PCR results and yielded cultures with colony morphology, sporangia and chlamydospores typical of the NA1 lineage of P. ramorum originally isolated in California from tanoaks and coast live oaks (Quercus agrifolia) (Rizzo et al. 2002). The ITS locus and a portion of the Cox-1 locus were sequenced from DNA extracts of each culture using primers DC6-ITS4 (Bonants et al. 2004) and COXF4N-COXR4N (Kroon et al. 2004), respectively. ITS sequences (GB MN540639-40) were typical of P. ramorum and Cox-1 sequences (GB MN540142-3) perfectly matched the Cox-1 sequence of the NA1 lineage (GB DQ832718) (Kroon et al. 2004). Microsatellite alleles were generated as described in Croucher et al. (2013) for the two Del Norte cultures and for eight P. ramorum cultures, representative of the four main multilocus genotypes (MLGs) present in California, namely c1 (Santa Cruz/Commercial Nurseries), c3 (San Francisco Bay Area), c2 (Monterey County), and c4 (Humboldt County) (Croucher et al. 2013). The two Del Norte MLGs were identical to one another and most similar to MLG c1, with a single repeat difference at a single locus. SSR results suggest the inoculum source may not be from Humboldt County, neighboring to the South, but from a yet unidentified outbreak, possibly associated with ornamental plants. Jedediah Smith State Park was surveyed for 12 months following the initial detection, however the pathogen has yet to be re-isolated in that location. In July 2020, SOD symptomatic leaves from two tanoak trees exhibiting twig cankers were collected 8 Km north of Jedediah Smith State Park, where three additional tanoak trees displayed rapidly browned dead canopies consistent with late stage SOD. Leaves were processed as above. Colonies from these samples produced chlamydospores and sporangia typical of P. ramorum on PARPH-V8 agar, but displayed a growth rate faster than that of NA1 genotypes and were characterized by aerial hyphae, overall resembling the morphology of EU1 lineage colonies (Brasier 2003). The EU1 lineage was confirmed by the perfect match of the sequence of a portion of the Cox-1 gene (GB MW349116-7) with the Cox-1 sequence of EU1 genotypes (GB EU124926). The EU1 clonal lineage has been previously isolated from tanoaks in Oregon forests, approximately 55 Km to the North (Grünwald et al. 2016), but this is the first report for California wildlands and will require containment and government regulations. It is unknown whether the EU1 strains in Del Norte County originated from Oregon forests or elsewhere.

Estimating the Potential for Forest Degradation in the Eastern United States Woodlands from an Introduction of Sudden Oak Death

Research Highlights: This research identified areas of the eastern United States forests that may be at particular risk from Sudden Oak Death (SOD) if it becomes established in the region. The highest suitability for SOD occurs where there are both species at risk of serious infection and species that could spread SOD to new hosts. Background and Objectives: SOD, caused by Phytophthora ramorum, can cause serious damage to forests. Although SOD is not believed to exist in the wild in the eastern United States, the presence of potential P. ramorum hosts in the area has led to questions about the potential for its establishment there. The present project expanded on previous efforts by examining both the possibility and potential consequences of P. ramorum establishment based on the presence of suitable hosts. Materials and Methods: We developed a map of the potential severity of tree loss that could result from a P. ramorum establishment in the eastern United States as well as a habitat suitability index based on the density of P. ramorum susceptible and spreader host species. We also implemented a connectivity analysis method for determining patches important to the spread of P. ramorum in the Great Smoky Mountains National Park. Results: The Ozarks and Appalachians both had a high potential severity of infection due to their high percentage of susceptible species, but the Appalachians had a higher overall level of risk due to the presence of more spreader tree species in the area. Connectivity analysis in the Great Smoky Mountains National Park identified patches likely to be particularly important to P. ramorum spread. Conclusions: Analysis of the presence of potential P. ramorum hosts in the eastern United States identified suitable conditions for SOD establishment there, particularly in the Appalachians. Techniques developed in this research can identify critical areas for SOD spread at both landscape and regional scales.

Citizen Science Uncovers Phytophthora ramorum as a Threat to Several Rare or Endangered California Manzanita Species

The Sudden Oak Death (SOD) Blitzes consist of yearly surveys led by citizen scientists designed to map the distribution of Phytophthora ramorum, cause of the forest disease called SOD, across northern California. During the 2017 Santa Cruz County SOD Blitz, six rare or endangered Arctostaphylos (manzanita) species were found to be possibly symptomatic for the first time. Symptoms included branch cankers and associated canopy mortality, and affected multiple individuals per species. Isolates of P. ramorum were obtained from each of the six species and, through a 30-day-long inoculation experiment on live plants, Koch’s postulates were completed for each one of them, conclusively determining that they all are hosts of this pathogen. Two additional manzanita species were later found to be apparently symptomatic in Marin County. Inoculations on detached branches using an isolate of P. ramorum obtained from one of the six rare species from Santa Cruz County were successful, suggesting that these two species may also be hosts of P. ramorum. Detached leaves of all eight species were also successfully inoculated at the University of California-Berkeley in fall 2018 and then again in spring 2019. In these cases, the same isolate was used for all inoculations, in order to obtain information on the comparative susceptibility of the eight species in question. Both branch and leaf inoculations identified significant interspecific differences in susceptibility. The production of sporangia was low on all species but it was not zero, suggesting that sporulation may cause within-plant and limited across-plant contagion, especially in rainy years.

Novel introductions and epidemic dynamics of the sudden oak death pathogen Phytophthora ramorum in Oregon forests

Sudden oak death caused by Phytophthora ramorum has been actively managed in Oregon since the early 2000’s. To date, this epidemic has been driven mostly by the NA1 clonal lineage of P. ramorum, but an outbreak of the EU1 lineage has recently emerged. Here we contrast the population dynamics of the NA1 outbreak first reported in 2001 to the outbreak of the EU1 lineage first detected in 2015. We tested if any of the lineages were introduced more than once. Infested regions of the forest were sampled between 2013-2018 (n = 903) and strains were genotyped at 15 microsatellite loci. Most genotypes observed were transient, with 272 of 358 unique genotypes emerging one year and disappearing the next. Diversity of EU1 was very low and isolates were spatially clustered (< 8 km apart), suggesting a single EU1 introduction. Some forest isolates are genetically similar to isolates collected from a local nursery in 2012, suggesting introduction of EU1 from this nursery or simultaneous introduction to both the nursery and latently into the forest. In contrast, the older NA1 populations were more polymorphic and spread over 30 km2. Principal component analysis supported two to four independent NA1 introductions. The NA1 and EU1 epidemics infest the same area but show disparate demographics owing to initial introductions of the lineages spaced 10 years apart. Comparing these epidemics provides novel insights into patterns of emergence of clonal pathogens in forest ecosystems.

Sudden Oak Death Taking a Toll on U.S. West Coast

Researchers have been modeling effects of the plant pathogen Phytophthora ramorum on coastal forests in California and Oregon since it arrived on the West Coast 3 decades ago.

Applying optimal control theory to a spatial simulation model of sudden oak death: ongoing surveillance protects tanoak while conserving biodiversity

Sudden oak death has devastated tree populations across California. However, management might still slow disease spread at local scales. We demonstrate how to unambiguously characterize effective, local management strategies using a detailed, spatially explicit simulation model of spread in a single forest stand. This pre-existing, parameterized simulation is approximated here by a carefully calibrated, non-spatial model, explicitly constructed to be sufficiently simple to allow optimal control theory (OCT) to be applied. By lifting management strategies from the approximate model to the detailed simulation, effective time-dependent controls can be identified. These protect tanoak—a culturally and ecologically important species—while conserving forest biodiversity within a limited budget. We also consider model predictive control, in which both the approximating model and optimal control are repeatedly updated as the epidemic progresses. This allows management which is robust to both parameter uncertainty and systematic differences between simulation and approximate models. Including the costs of disease surveillance then introduces an optimal intensity of surveillance. Our study demonstrates that successful control of sudden oak death is likely to rely on adaptive strategies updated via ongoing surveillance. More broadly, it illustrates how OCT can inform effective real-world management, even when underpinning disease spread models are highly complex.

Phytophthora pathogens threaten rare habitats and conservation plantings

Phytophthora pathogens are damaging native wildland vegetation including plants in restoration areas and botanic gardens. The infestations threaten some plants already designated as endangered and degrade high-value habitats. Pathogens are being introduced primarily via container plant nursery stock and, once established, they can spread to adjacent areas where plant species not previously exposed to pathogens may become infected. We review epidemics in California – caused by the sudden oak death pathogen Phytophthoraramorum Werres, De Cock & Man in ‘t Veld and the first USA detections of P. tentaculata Kröber & Marwitz, which occurred in native plant nurseries and restoration areas – as examples to illustrate these threats to conservation plantings.