Assembly and analysis of the genome of Notholithocarpus densiflorus.

Tanoak (Notholithocarpus densiflorus) is an evergreen tree in the Fagaceae family found in California and southern Oregon. Historically, tanoak acorns were an important food source for Native American tribes, and the bark was used extensively in the leather tanning process. Long considered a disjunct relictual element of the Asian stone oaks (Lithocarpus spp.), phylogenetic analysis has determined that the tanoak is an example of convergent evolution. Tanoaks are deeply divergent from oaks (Quercus) of the Pacific Northwest and comprise a new genus with a single species. These trees are highly susceptible to "sudden oak death" (SOD), a plant pathogen (Phytophthora ramorum) that has caused widespread deaths of tanoaks. In this study, we set out to assemble the genome and perform comparative studies among a number of individuals that demonstrated varying levels of susceptibility to SOD. First, we sequenced and de novo assembled a draft reference genome of N. densiflorus using cobarcoded library processing methods and an MGI DNBSEQ-G400 sequencer. To increase the contiguity of the final assembly, we also sequenced Oxford Nanopore long reads to 30× coverage. To our knowledge, the draft genome reported here is one of the more contiguous and complete genomes of a tree species published to date, with a contig N50 of ∼1.2 Mb, a scaffold N50 of ∼2.1 Mb, and a complete gene score of 95.5% through BUSCO analysis. In addition, we sequenced 11 genetically distinct individuals and mapped these onto the draft reference genome, enabling the discovery of almost 25 million single nucleotide polymorphisms and ∼4.4 million small insertions and deletions. Finally, using cobarcoded data, we were able to generate a complete haplotype coverage of all 11 genomes.

Rapid New Diagnostic LAMP (Loop-Mediated Isothermal Amplification) Assays to Distinguish Among the Four Lineages of Phytophthora ramorum.

Sudden oak death (SOD) is caused by Phytophthora ramorum, an invasive oomycete pathogen. This pathogen is of major regulatory concern for nurseries, horticulture, and forestry in the United States and around the world. Three of the 12 identified lineages of P. ramorum currently occur in the United States (NA1, NA2, and EU1) affecting wildland forests and nurseries. Rapid identification and lineage determination is essential to accelerate management decisions, detect introductions of new lineages, and control the spread of SOD. The objective of this study was to develop and validate diagnostic tools to rapidly identify P. ramorum and distinguish among the four common lineages of the pathogen and to accelarate management decision making. The loop-mediated isothermal amplification (LAMP) assays developed here are species specific with no cross reaction to common Phytophthora species found in Oregon, California, and Washington. The lineage-specific assays unambiguously distinguish among the four common clonal lineages. These assays are sensitive and able to detect P. ramorum DNA ranging in concentration from 30 to 0.03 ng/µl depending on the assay. These assays work effectively on a variety of sample types including plant tissue, cultures, and DNA. They have been integrated into the SOD diagnostic process in the forest pathology lab at Oregon State University. To date, 190 samples have been correctly identified from over 200 field samples tested for lineage determination. The development of these assays will help managers in forestry and horticulture identify and rapidly respond to new outbreaks of P. ramorum.

Comparative Efficacy of Chondrosterum purpureum and Chemical Herbicides for Control of Resprouts in Tanoak and Bay Laurel.

The invasive Oomycete pathogen Phytophthora ramorum has killed millions of susceptible oak and tanoak trees in California and southern Oregon forests and is responsible for losses in revenue to the nursery industry through mitigation activities. In addition, infestation of forests in the United Kingdom by this organism has resulted in the destruction of many hectares of larch plantations. Resprouting stumps can be a reservoir for the inoculum of P. ramorum persisting on a site. In areas where the application of herbicides is not permitted, a biocontrol treatment would be an indispensable alternative. Treatment of stumps with the sap-rotting fungus Chondrostereum purpureum (Pers.) Pouzar has been shown to be an effective tool for the suppression of resprouting on several species, most notably red alder. In this project, the ability of C. purpureum to suppress resprouting was evaluated on stumps of two host species, tanoak (Notholithocarpus densiflorus) and California bay laurel (Umbellularia californica). Laboratory testing of three California isolates of C. purpureum indicated that the fungus can colonize bay laurel stems. Field trials were established near Brookings, Oregon, on tanoak and on bay laurel near Soquel, California. Early results of field testing showed that C. purpureum was able to colonize the stumps of tanoak following treatment and was found to occur naturally on tanoak logs and stumps. Formulations of C. purpureum appear to have some effect on reducing sprout survival in tanoak, but the most effective and rapid treatment for this host is the hack and squirt method of applying the herbicide imazapyr. Sprayed herbicide prevents sprouting on bay laurel, and there was evidence that resprouting was inhibited on stumps treated with C. purpureum. Over time, applications of C. purpureum may be a more permanent solution as the stumps begin to decay.

Mega-fire in redwood tanoak forest reduces bacterial and fungal richness and selects for pyrophilous taxa that are phylogenetically conserved.

Mega-fires of unprecedented size, intensity and socio-economic impacts have surged globally due to climate change, fire suppression and development. Soil microbiomes are critical for post-fire plant regeneration and nutrient cycling, yet how mega-fires impact the soil microbiome remains unclear. We had a serendipitous opportunity to obtain pre- and post-fire soils from the same sampling locations after the 2016 Soberanes mega-fire burned with high severity throughout several of our established redwood-tanoak plots. This makes our study the first to examine microbial fire response in redwood-tanoak forests. We re-sampled soils immediately post-fire from two burned plots and one unburned plot to elucidate the effect of mega-fire on soil microbiomes. We used Illumina MiSeq sequencing of 16S and ITS1 sequences to determine that bacterial and fungal richness were reduced by 38%-70% in burned plots, with richness unchanged in the unburned plot. Fire altered composition by 27% for bacteria and 24% for fungi, whereas the unburned plots experienced no change in fungal and negligible change in bacterial composition. Pyrophilous taxa that responded positively to fire were phylogenetically conserved, suggesting shared evolutionary traits. For bacteria, fire selected for increased Firmicutes and Actinobacteria. For fungi, fire selected for the Ascomycota classes Pezizomycetes and Eurotiomycetes and for a Basidiomycota class of heat-resistant Geminibasidiomycete yeasts. We build from Grime's competitor-stress tolerator-ruderal (C-S-R) framework and its recent microbial applications to show how our results might fit into a trait-based conceptual model to help predict generalizable microbial responses to fire.

Novel disturbance interactions between fire and an emerging disease impact survival and growth of resprouting trees.

Human-altered ecological disturbances may challenge system resilience and disrupt biological legacies maintaining ecosystem recovery. Yet, the extent to which novel regimes challenge these legacies varies. This may be partially explained by differences in the vulnerability of life history strategies to disturbance characteristics. In the fire-prone, resprouter-dominated coast redwood forests of California, the introduced disease sudden oak death (SOD) alters fuel profiles, fire behavior, and aboveground tree mortality; however, this system is dominated by resprouting trees that are well-adapted to aboveground damage, and belowground survival of individuals may represent the principal biological legacy connecting pre- and post-fire communities. Much of the research exploring altered disturbances and forest recovery has focused on legacies determined by seed dispersal and aboveground survival of adults. In this work, we use pre- and post-fire data from a long-term monitoring network to assess the impacts of novel disturbance interactions between wildfire and SOD on the belowground survival and vegetative reproduction of resprouters. We found that increasing accumulation of coarse woody surface fuels from SOD-killed hosts decreased the likelihood of belowground survival for resprouting tanoak trees, but not for redwoods. Tanoaks' belowground survival was negatively related to substrate burn severity, which increased with the volume of surface fuels from hosts, suggesting heat damage as a possible mechanism influencing altered patterns of resprouter mortality. These impacts increased with decreasing tree size. By contrast, redwood and tanoak trees that survived both disturbances resprouted more vigorously, regardless of post-fire infection by P. ramorum, and generated similar recruitment at the stand level. Our results demonstrate that disease-fire interactions can narrow recruitment filters for resprouters, which could impact long-term population and demographic structure; yet, compounded disturbance may also reduce stand density and disease pressure, allowing competitive release of survivors. Resprouters displayed vulnerabilities to altered disturbance, but our research suggests that legacies maintained by resprouting may be more resilient to certain compounded disturbances, compared to seed-obligate species, because of high rates of individual survival under increasingly severe events. These trends have important implications for conservation of declining tree species in SOD-impacted forests, as well as predictions of human impacts in other disturbance-prone systems where resprouters are present.

Ecology and pathology of Phytophthora ITS clade 3 species in forests in western Oregon, USA.

Phytophthora species are widespread and diverse in forest ecosystems, but little is known about their ecology. We explore ecological attributes of the closely related clade 3 species that occur sympatrically in western North American forests. We address the population structure, pathology, and epidemiology of P. ilicis, P. nemorosa, P. pluvialis, P. pseudosyringae, and P. psychrophila. Phytophthora species were isolated from plant tissues, rainwater falling through the forest canopy, streams, and soils in forests in western Oregon. Species identifications were based on morphology in culture with molecular confirmation using COX spacer and internal transcribed spacer (ITS) sequences. All five clade 3 Phytophthora species are present in western Oregon forests, although P. ilicis (only 1 forest isolate) and P. psychrophila (only 12 isolates) are apparently rare. P. ilicis is known only from holly in horticultural situations and once from a naturalized seedling in an urban forest. The known distribution of P. nemorosa in forest settings coincides with the ranges of its principle hosts, tanoak and myrtlewood, in Oregon and California. Although it is regularly identified from streams within the tanoak range, it has not been recovered from streams beyond that range. P. pluvialis is primarily associated with Douglas-fir canopies. It was identified from scattered locations throughout western Oregon in rain traps beneath Douglas-fir plantations and from diseased needles. P. pseudosyringae is also isolated from tanoak and myrtlewood in southwest Oregon and California, but its distribution, in streams at least, extends throughout much of western Oregon. P. psychrophila in Oregon is known only from rain traps beneath tanoak trees. Little intraspecific variation was detected in the nuclear rDNA ITS of clade 3 species. Variation in the mitochondrial COX spacer region was more frequent, with 2 to 10 haplotypes identified in the clade 3 species, for which we had multiple isolates.

Wildfire and forest disease interaction lead to greater loss of soil nutrients and carbon.

Fire and forest disease have significant ecological impacts, but the interactions of these two disturbances are rarely studied. We measured soil C, N, Ca, P, and pH in forests of the Big Sur region of California impacted by the exotic pathogen Phytophthora ramorum, cause of sudden oak death, and the 2008 Basin wildfire complex. In Big Sur, overstory tree mortality following P. ramorum invasion has been extensive in redwood and mixed evergreen forests, where the pathogen kills true oaks and tanoak (Notholithocarpus densiflorus). Sampling was conducted across a full-factorial combination of disease/no disease and burned/unburned conditions in both forest types. Forest floor organic matter and associated nutrients were greater in unburned redwood compared to unburned mixed evergreen forests. Post-fire element pools were similar between forest types, but lower in burned-invaded compared to burned-uninvaded plots. We found evidence disease-generated fuels led to increased loss of forest floor C, N, Ca, and P. The same effects were associated with lower %C and higher PO4-P in the mineral soil. Fire-disease interactions were linear functions of pre-fire host mortality which was similar between the forest types. Our analysis suggests that these effects increased forest floor C loss by as much as 24.4 and 21.3 % in redwood and mixed evergreen forests, respectively, with similar maximum losses for the other forest floor elements. Accumulation of sudden oak death generated fuels has potential to increase fire-related loss of soil nutrients at the region-scale of this disease and similar patterns are likely in other forests, where fire and disease overlap.