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Summer Research

Summer 2019

Undescribed on Zostera marina and Z. japonica in the Puget Sound

Theresa Proctor University of Puget Sound

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Part of the Environmental Microbiology and Microbial Ecology Commons, Marine Commons, Commons, and the Terrestrial and Aquatic Ecology Commons

Recommended Citation Proctor, Theresa, "Undescribed oomycete pathogens on Zostera marina and Z. japonica in the Puget Sound" (2019). Summer Research. 353. https://soundideas.pugetsound.edu/summer_research/353

This Article is brought to you for free and open access by Sound Ideas. It has been accepted for inclusion in Summer Research by an authorized administrator of Sound Ideas. For more information, please contact [email protected]. Undescribed oomycete pathogens on Zostera marina and Z. japonica in the Puget Sound Theresa Proctor and Joel Elliott: Department of Biology, University of Puget Sound 1517 N. Union Ave, Tacoma, WA 98416 | Contact: [email protected]

Background Results

u : fungi-like , notorious terrestrial plant pathogens (a) (b) (c) (a) 1 u Previously undescribed in marine habitats but recent studies 0.9 0.8 introduced a number of novel marine 0.4 0.7 sample

Z. japonica Ratio u New species shown to reduce seagrass seed germination and Z. marina 0.6 Oomycete load 0.2 seedling development but little else known about pathogen/host 0.5 relationship or marine oomycete diversity 0.4 0.0 0.3 Z. japonica Z. marina u Oomycetes infect estimated 99% of seagrass populations worldwide, Seagrass species Species

Growth Rate (cm/day) 0.2 yet research into the rapid decline of seagrass populations seemingly RW Figure 3. (a) Host species affected mean (± 1 SE) pathogen load. The oomycete 0.1 overlooks oomycete infection as a contributor pathogen load was 94% lower in Z. japonica root tissue (n = 10) than Z. marina root PB 0 5 10 15 20 25 30 35 tissue (n = 10; p = 0.008); (b) Z. marina and (c) Z. japonica illustrations from The CP u Elliott lab isolated and cultured gemini as well as 5 Illustrated Flora of British Columbia (b) 1.8 Temperature (°C) WSU undescribed oomycetes from eelgrass (Z. marina) roots 1.6 P. gemini ² 2 spp. isolated from the Ruston Way sampling site, Pythium sp. 1.4

isolated from the Padilla Bay site, Halophytophthora sp. isolated from 1.2 Pythium the Cherry Point site, and Halophytophthora sp. first isolated by a WSU 1 lab (hereafter denoted as RW, RW2, PB, CP, and WSU, respectively) 0.8 Phytopythium

0.6 Growth Rate (cm/day)

0.4

Objectives Halophytophthora 0.2

0 0 5 10 15 20 25 30 35 40 45 1. Calculate the oomycete pathogen load of environmental samples Salinity (psu)

and compare Z. marina pathogen load to pathogen load of Z. Phytophthora Figure 4. (a) The effect of temperature on mean growth rate depended on species (2-way ANOVA, F = 6.69, df = 24, 77; p < 0.001). Maximum growth occurred at 20°C japonica, another species of seagrass in the Puget Sound for CP, PB, and RW while WSU and P. gemini experienced similar growth from 2. For each oomycete isolate, 10-30 °C (p < 0.05 for all); (b) The effect of salinity on mean growth rate also a. Characterize the physical range (temperature, salinity) Figure 5. Neighbor joining showing undescribed oomycetes and depended on species (2-way ANOVA, F = 7.43, df = 20, 60; p < 0.001). Maximum related terrestrial species based on COX1 and RPS10 sequences generated in growth occurred at 45 psu for RW and PB and 14-20 psu for CP (p < 0.001 for all). b. Sequence COX1 (traditional DNA barcode gene) and RPS10 Geneious 7.0 (related species’ sequences downloaded from GenBank and Salinity did not significantly affect mean daily radial growth for P. gemini (p = 0.157) (recently introduced barcode gene) and construct a phylogeny OomyceteDB). Bootstrap values are shown on the branches. or WSU (p = 0.057). Methods Conclusions Future Directions u Oomycetes preferentially infect Z. marina roots over u Characterize sporangia and production Amplify Ensure Purify and sequence Z. japonica roots to complete the life history description of each Isolate COX1/RPS10 target PCR product Molecular oomycetes genes amplification phylogeny u RW and PB are tolerant of high salinities and low isolate (e.g. Figure 7) QIAquick® PCR Purification from root PCR of hyphae Gel Kit, then GENEWIZ Sanger Geneious 7.0 temperatures; may prefer colder brine habitats u Sequence ITS and COX2 genes of all isolates for samples scrapings electrophoresis sequencing ² Slowest growth of all isolates – only become further phylogenetic analysis Embed competitive at 5-10 °C u Achieve higher resolution of eelgrass roots Temperature in selective u CP has highest growth of all isolates at salinities Growth curves Grow on 32 psu V8 agar at 5, 10, 15, 20, 25, 30, and oomycete detection via media (PARPH) and Measure radial growth rate (avg 35°C (3 replicates per isolate) above 6 psu, though is less successful at low salinities qPCR by designing - watch for cm/day) of each isolate’s hyphae and temperatures above 25 °C and below 20 °C; specific and isolate-specific hyphae by plating 4 mm plugs of hyphae Salinity growth indicates preference for highly saline habitats around on V8 agar Grow at 20°C on 0, 6, 14, 20, 32, and 45 psu agar primers plates (3 replicates per isolate) 20-25 °C u Correlate infection levels of u WSU and P. gemini have similar growth across all specific isolates with Figure 1. Flow chart illustrating the description process for each isolate, including generating molecular Figure 6. Typical life cycle of data and determining each isolate’s optimal range for temperature and salinity (latter measured in salinity levels and almost all temperatures (10-30 °C) Phytophthora spp. environmental data practical salinity units). ² May explain why WSU and P. gemini are easier Figure 2. Flow Quantify to isolate and culture from roots chart showing [oomycete DNA] Extract DNA u Phylogenetic analysis of COX1/RPS10 sequence data how oomycete from Z. SYBR Green® qPCR assay with Calculate Acknowledgments pathogen load in marina and Z. oomycete-specific 5.8s primers pathogen load indicates that the undescribed isolates are Z. marina and Z. japonica roots [oomycete DNA]/ significantly different from other known taxa and are Sincere thanks to the McCormick Foundation for funding and the UPS Biology japonica root Quantify [plant DNA] Department, Michal Morrison-Kerr and the biology storeroom staff, Amy samples was DNeasy® [plant DNA] in clade with other Halophytophthora, Pythium, and Replogle, Frank Martin and the Salinas USDA-ARS lab, and the WSU plant measured using PowerSoil® Kit SYBR Green® qPCR assay with Phytopythium species pathology lab for materials, protocols, and support. qPCR assays. plant-specific FMP primers