Performing Department
(N/A)
Non Technical Summary
Global trade and climate change accelerate and compound the emergence of plant pathogens and impacts on natural and managed ecosystems. Phytophthora pluvialis is a foliar pathogen of conifer trees that has repeatedly emerged in the United States (US) and invaded New Zealand (NZ) and more recently the United Kingdom (UK). Its emergence has caused severe epidemics on economically important conifer trees in both plantations and wild land forests. In its most recent emergence, P. pluvialis not only jumped to a new host but was associated with a devastating shift in disease etiology. Studying P. pluvialis is of direct relevance in support of goals to improve the long-term sustainability and competitiveness of U.S. agricultural systems. The change in disease etiology is of particular concern. Its potential effects on the forest industry in the Pacific Northwest of the US are significant. Western hemlock and Douglas-fir, the two main hosts of this pathogen, make up most of the crop trees planted on the 8.1 million acres of private industrial forest land. We aim to: 1) Characterize the variation in suscpetibility to P. pluvialis within and among populations of Douglas-fir and western hemlock; 2) use phylogeny guided phenotyping of pathogen populations; and 3) develop and deploy a multiplex CRISPR based SHERLOCK assay to detect major lineages of P. pluvialis in the field. These aims will allow us to evaluate and attenuate the risk of P. pluvialis to western hemlock and Douglas-fir forests in the USA.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Global trade and climate change accelerate and compound the emergence of plant pathogens and impacts on natural and managed ecosystems. Phytophthora pluvialis is a foliar pathogen of conifer trees that has repeatedly emerged in the United States (US) and invaded New Zealand (NZ) and more recently the United Kingdom (UK). Its emergence has caused severe epidemics on economically important conifer trees in both plantations and wildland forests. In its most recent emergence, P. pluvialis not only jumped to a new host but was associated with a devastating shift in disease etiology. In addition to infecting foliage, the pathogen infected the main stem of trees and caused cankers that led to tree mortality (Fig. 1). This change in disease etiology is of particular concern because of potential effects on the forest industry in the Pacific Northwest of the US. In this region western hemlock and Douglas-fir, the two main hosts of this pathogen, make up most of the crop trees planted on the 8.1 million acres of private industrial forest land. Testing the following specific hypotheses is essential for determining impacts of emergence by P. pluvialis on the US forest industrySpecific hypothesis 1: The invasion in the UK by P. pluvialis is the result of a genetic bottleneck of the host followed by accidental selection of susceptible western hemlock genotypes.Specific hypothesis 2: The shift in disease etiology is the result of emergence of a new clade of P. pluvialis able to infect hemlock and cause stem cankers.Specific hypothesis 3: Differences in environmental conditions among locations explain changes in the etiology of the disease and host shifts by P. pluvialis.The Objectives of this proposal are:Characterize the variation in susceptibility to P. pluvialis within and among populations of Douglas-fir and western hemlock.Genotype western hemlock and Douglas-fir populations from the US and the UK.Evaluate differences in susceptibility of leaves and stem tissue among populations of western hemlock and Douglas-fir.Evaluate the epidemiological risk to western hemlock and Douglas-fir tree farms in the USA.Evaluate growth and sporulation potential of representative P. pluvialis isolates across a range of temperatures and on inoculated seedlings.Contrast infection biology on Douglas-fir and western hemlock.Develop and deploy a multiplex Sherlock assay to detect major lineages of P. pluvialis.Validation of the SHERLOCK assay quantitation of sporulation.
Project Methods
Aim 1. Characterize the variation in susceptibility to P. pluvialis within and among populations of Douglas-fir and western hemlock. a. Genotype western hemlock and Douglas-fir populations from the US and UK. The range of both western hemlock and Douglas-fir in the western US is subdivided into provenances. These provenances were created to ensure that trees grown for forest regeneration purposes were adapted to the local climate. P. pluvialis is found within the coastal forests of Oregon, southern Washington, and northern California. This distribution spans approximately 17 seed zones. From each of the 17 seed zones, 100 randomly selected seeds for both Douglas-fir and western hemlock will be provided. The 1700 seeds/species will be germinated and grown under greenhouse conditions. Following germination and growth, seedlings will have needle tissue sampled, DNA extracted, and be genotyped using the Axiom array. To compare host population genetic diversity in the US to the UK, collaborators will provide lyophilized needle tissue from western hemlock and Douglas-fir trees collected in the two regions, where the P. pluvialis outbreak occurred. Needles will be collected from 100 individuals/species/region. Once seedlings have reached a sufficient size, ~100 mg of needle tissue will be collected, frozen, and lyophilized for DNA extraction with a QiagenPlant DNA mini kit. SNP data obtained from the Axiom array will be surveyed to determine population genetic structure and diversity within the two tree species. We will use ADMIXTURE and PCA plots to analyze SNP data to compare population genetic structure and gene flow between populations within the two tree taxa.b. Evaluate differences in susceptibility among populations of Douglas-fir and western hemlock. A total of 2,470 Douglas-fir (n = 1,235) and western hemlock (n = 1,235) will have been genotyped above (Table 1). We will use the seedlings from the US provenances for the inoculation experiments (Douglas-fir = 1,105 and western hemlock = 1,105). Isolates for the inoculation experiments will be selected from our re-sequenced population representing major clades within P. pluvialis pandemic population [US (n = 9), UK (n = 2) and NZ (n = 2)]. One isolate from each clade will be selected to inoculate 10 trees from each provenance (13 P. pluvialis clades X 17 Provenances X 10 reps X = 2,210 individuals). The experimental design will be a randomized complete block design.Sporangia produced from 13 isolates will be used to inoculate 2-year-old Douglas-fir and western hemlock seedlings. Approximately 20 ml of inoculum, with a concentration of 200 to 300 sporangia/ml, will be applied to each seedling using an airbrush sprayer. Racks of inoculated trees will be enclosed in polyethylene bags for 48 h with supplemental mist. Following incubation, trees will be placed in growth chambers at 16 to 18°C with 12-h photoperiod. Phenotyping of inoculated trees will be conducted 14 days post-inoculation. Phenotypes will include: a count of the number of necrotic lesions, visual assessment of defoliation, and color spectrum of infected needles. Phenotyping data will be analyzed using mixed models.Aim 2. Evaluate the epidemiological risk to western hemlock and Douglas-fir tree farms in the USA. a. Evaluate growth and sporulation potential of representative P. pluvialis isolates across a range of temperatures in vitro and in planta.Radial growth assays. Isolates will be grown in Petri dish experiments and over a range of temperatures to determine radial growth rate and temperature optimums. Radial growth will be measured for all isolates in six replicates at 15, 20, 25°C on V8 agar medium daily.Seedling sporulation assays. Seedlings will be inoculated as described above and placed in growth chambers set to 15, 20, 25°C with 12-h photoperiod. Two weeks later infected trees will be misted with water and individually bagged to induce sporulation for counting.Needle infection efficiency. A subset of symptomatic needles (n =10) will be selected for the needle colonization assay. The 10 needles will be sampled. We will use the ratio of pathogen to host DNA as a means of quantifying host infection efficiency. Comparisons between clades and clades nested within the USA, UK, and NZ populations will be made.Statistical analysis. Analysis of variance, with linear and non-linear regression modeling, will be used to detect significant differences among treatments. Models will be developed describing dependence of disease variables (radial growth, sporulation, needle colonization) on independent variables (temperature, isolate, and isolate nested within USA, UK, or NZ populations). All statistical analyses will be conducted in R 4.2.b. Contrast infection biology of P. pluvialis on Douglas-fir and western hemlock. One susceptible provenance of Douglas-fir (n = 60 seedlings) and western hemlock (n = 60 seedlings) will be used. These plants will be inoculated, as described above, using the most virulent variant from the USA and UK. Needles and stem tissue from inoculated trees will be sampled at five different time points: 24h post-inoculation, 48h post-inoculation, 96h post inoculation, 1-week post-inoculation, and 2 weeks post inoculation. Imaging will be performed using a scanning electron microscope.We will study infection and colonization of susceptible trees simultaneously.Aim 3. Develop CRISPR-Cas diagnostic assays differentiating variants belonging to select clades. a. Validation of the SHERLOCK assay quantification of sporulation in the lab and the field. SHERLOCK. All assays will be validated using extracted DNA as well as plant samples infected with the pathogen genotype or variant of interest in live plant leaves or stems. Because validation is done in a diagnostic setting, there is opportunity for us to compare findings from conventional approaches versus those from the CRISPR-dx methods. Infections will be done under containment following USDA APHIS protocols where required. All assays will include positive controls (target lineage/species/plasmid of interest) as well as negative controls (non-target species/lineages/plasmids, etc.; water-only controls).Specificity. Specificity for all assays will be determined in triplicate using extracted DNA, sporangial (Phytophthora) suspensions, and infected plant material or pure culture DNA. Specificity will be determined with a panel of non-target relatives including a selection of Phytophthora species (P. nemorosa, P. pseudosyringae, P. psychrophila and P. ilicis). Negative controls will include non-target oomycete, fungal, and bacterial species.Sensitivity. Sensitivity will be defined as the limit of detection (LOD) that successfully detects 95% or greater of samples at the lowest concentration of the target organism. We will serially dilute either a known number of sporangia/cells (starting with 10,000 sporangia/cells per ul over a log range from 0.1-10,000 sporangia/cells per µl) or a known DNA concentration. A regression analysis of Ct values versus the log of DNA concentration will be conducted. Water will be included as a negative control. LOD will be evaluated for each concentration with 6 replications in three independent runs performed on different days.Field validation. Select field samples from Oregon forests will be used to validate the assay. A total of 30 symptomatic needles will be collected from field sites with symptomatic trees. We will use qPCR, SHERLOCK, and conventional plating on selective media to compare the specificity and sensitivity of SHERLOCK to traditional diagnostic tools.Data analysis. Student t-tests will be used to determine significance of differences (P < 0.05) of background-subtracted fluorescence between targets and controls. LFA test line intensity will be measured based on grayscale pixel values using ImageJ software. All analyses will be done in the R computer and statistical language.