Progress 06/01/24 to 05/31/25

Outputs
Target Audience:Scientists and researchers who work on Phytophthora diseases in forest ecosystems. Natural resource managers, forest pathologists, and stakeholders responsible for managing forest land in the western United States. Foresters and landowners esponsible for managing forest land in the western United States. USFS scientists. Industrial partners and landowners. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two post docotoral scholars and one undergraduate have been trained using the funds from the project to date. The postdocs will have the opportunity to present the results from their research in year 2 and year 3 of the project at scientific meetings. This project has also given the opportunity for the post-docs to help mentor an undergraduate student interested in research. They have trained the student in both laborotory techniques and exposed the student to computation biology, statistics, and bioinformatics. How have the results been disseminated to communities of interest?The results will be disseminated in presentations at scientific meetings and filed tours with natural resource managers. What do you plan to do during the next reporting period to accomplish the goals?1.Characterize the variation in susceptibility to P. pluvialis within and among populations of Douglas-fir and western hemlock. 1. Genotype western hemlock and Douglas-fir populations from the US and the UK. The1,721 DF and 561 WH will have a needles collected, frozen, and lyophilized. DNA will be extracted using an Opentrons OT-2 lab robot with a QiagenPlant DNA mini kit. DNA from each sample will then be adjusted to a minimum concentration of 20 ng/uL. DNA samples will be processed using the standard Affymetrix Axiom array protocols. 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. ADMIXTURE models the probability of genotypes for different ancestor groups similar to the software STRUCTURE. The degree of genetic connectivity among populations will be evaluated using a minimum spanning network (MSN) constructed in the R package poppr 2.1.5 . Population differentiation will be evaluated at the different hierarchies with Nei's Gst, an Fst analog estimated in the mmod 1.13 package . Population heterozygosity will be estimated and statistically contrasted for each population, using the Hs.test function with 499 replicates using the same package. The results from this analysis wil be used to guide the selection of WH and DF for inoculation in goal 2 below. 2. Evaluate differences in susceptibility of leaves and stem tissue among populations of western hemlock and Douglas-fir. Inoculations will be conducted as follows. Briefly, 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. The significance of the fixed effects of provenance, geographic origin, and clade and the random effects of block and seedling will be determined using a likelihood ratio chi-squared test. The order of testing will be determined using the default z- test. All statistical analyses will be conducted in R 4.2.1 (R Core Team 2022). Difference among isolates(clades) , and geographic origins will be compared using the emmeans package in R. Significance will be evaluated at an α = 0.05. 2. Evaluate the epidemiological risk to western hemlock and Douglas-fir tree farms in the USA. 1. Evaluate growth and sporulation potential of representative P. pluvialis isolates across a range of temperatures and on inoculated seedlings. Seedling sporulation assays. Seedlings will be inoculated as described above. Approximately 20 ml of inoculum, with a concentration of 200 to 300 sporangia/ml, will be applied, using an airbrush sprayer, to each seedling. Racks of inoculated trees will be enclosed in polyethylene bags for 48 h. Following incubation, trees will be returned to growth chambers set to 15, 20, 25°C with 12-h photoperiod. Following 14 days of incubation, infected trees will be misted with water and individually bagged to induce sporulation. Sporangia will be collected and counted. Briefly, sporulation will be determined at the end of each experiment by washing sporangia from leaves and enumerating concentration using a hemocytometer. Sporulation will be expressed as the number of sporangia produced per cm2 of leaf area. Needle infection efficiency. Following washing, a subset of symptomatic needles (n =10) will be selected for the needle colonization assay. The 10 needles will be ground in liquid nitrogen and 125 mg (wet weight) will be placed in individual tubes. A Nucleospin Plant II kit, in a 96-well plate format, will be used to extract genomic DNA from frozen needles samples. 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 as described below. 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 4.2.1 (R Core Team 2022). A likelihood ratio chi-squared test will be used to evaluate the significance of variables in each model. Significance will be evaluated at an α = 0.05. 2. Contrast infection biology on Douglas-fir and western hemlock. Samples from the large scale inocualtion experiment, seedling sporulation assay, and needle infection assay will be collected. These samples will be stored and processsed in year 3 fo the project to compare the infection biology on WH and DF. 3. Develop and deploy a multiplex Sherlock assay to detect major lineages of P. pluvialis. 1. Validation of the SHERLOCK assay quantitation of sporulation. In the next reporting period we will ue the genomes we have assembled and annotated to selct target sites for teh SHERLOCK assay. We will test these sites to determine which we will use for the diagnostic assay and to quantify sporulation. lab and field testing will be conducted to valiadate the assay.

Impacts
What was accomplished under these goals? 1.Characterize the variation in susceptibility to P. pluvialis within and among populations of Douglas-fir and western hemlock. 1. Genotype western hemlock and Douglas-fir populations from the US and the UK. We gathered seeds of Douglas-fir (DF) and western hemlock (wh) from their native range, including Oregon (OR) and Washington (WH) in the United States, and British Columbia (BC) in Canada. We also received seeds from the United Kingdom (UK) where DF and WH were introduced from North America. In total, we collected seeds from 28 populations of DF (BC=9, WH=3, OR=12, UK=4), and 11 populations of WH (BC=3, WA=3, OR=1, UK=4). The seeds of both tree species were fully submerged in cool tap water for 24 hours, drained and surface dried at room temperature and cold stratified at 4 C? for 21 days for DF and 28 days for wh. After the cold stratification process, the seeds were spread out on a filter paper placed on top of on water- saturated kimpack (a highly absorbent 100% cellulose sterile germination paper) in a transparent tightly closed box (1 box per provenance). The boxes were then placed in growth chambers set to 30 C day, 20 C night 16/8 photo period for DF, and 20 C day, 20 C night 16/8 photo period for wh. The seedlings of both tree species were sown right upon germination in a green house maintained at 20-22 C . In total, we obtained 2,282 trees from 39 populations, 1,721 DF and 561 WH. These seedlings are currently growing in the greenhouse and and needles will be collected during the summer of 2025 for extraction and genotyping. 2. Evaluate differences in susceptibility of leaves and stem tissue among populations of western hemlock and Douglas-fir. Preliminary work has begun on this to develop a consistent and reliable assay. We will begin inoculations once the genotyping is complete and a genetically diverse panel of DF and WH host genotyopes from the US and UK can be selected for screening. 2. Evaluate the epidemiological risk to western hemlock and Douglas-fir tree farms in the USA. 1. Evaluate growth and sporulation potential of representative P. pluvialis isolates across a range of temperatures and on inoculated seedlings. We randomly selected total of 31 P. pluvialis isolates from the United States (n=10), New Zealand (n=10), United Kingdom (n=10), and Belgium (n=1). The isolates were grown on 20 ml clarified V8 media poured into 90 mm Petri dishes at 20 C? for 10-14 days. The plugs were taken from the actively growing edge of the mycelia and placed in a center of a Petri dish (V8; 20ml), on the intersection of two lines, previously drawn on each plate, in three replicates for five different temperatures, and were grown at 20 C for four days to initiate the growth. After four days, the growth was recorded by taking a photograph, and the isolates and their replicates were distributed to different temperatures (5°C, 10°C, 15°C, 20°C, 25°C). The growth of each replicate for each temperature was then recorded at days seven, ten, and fourteen. Radial growth was measured using ImageJ software and data analysis was performed in RStudio. 2. Contrast infection biology on Douglas-fir and western hemlock. This aim of the project has not yet been initiatited. Once the large scale inocualtions experiments are conducted this spring we will sample a subset of of DF and WH genotypes to conduct the comparative infection biology studies. 3. Develop and deploy a multiplex Sherlock assay to detect major lineages of P. pluvialis. 1. Validation of the SHERLOCK assay quantitation of sporulation. In order to improve the the genome assemble and annotation of P. pluvialis to facilitate the identification of variant sites for the SHERLOCK assay we conducted oxford nanopore seqeuncing of 6 representative isolates. We have used the short read sequences formt he preliminary data to polish these genome and are currently phasing and annotating the genomes. These genomes will be used to conduct assemblies of the remaining short read genome and the genetic variation captured in thepopulation will be used to selct and test sites for the CRISPR assay.

Publications