Research Project: Knowledge Based Tools for Exotic and Emerging Diseases of Small Fruit and Nursery Crops

Location: Horticultural Crops Disease and Pest Management Research Unit

2023 Annual Report

Objectives
Objective 1: Describe the pathogen biology and disease epidemiology of exotic and emerging plant pathogens affecting perennial fruit and nursery crops. Sub-objective 1.A: Conduct comparative genomic analyses of Phytophthora ramorum. Sub-objective 1.B: Investigate genes differentially expressed in the EU1 and NA1 clonal lineages causing sudden oak death in Oregon forests. Sub-objective 1.C: Characterize fungicide resistance of Botrytis populations from small fruit and grape. Sub-objective 1.D: Assess fitness of sensitive and resistant QoI and DMI E. necator isolates. Sub-objective 1.E: Conduct mating studies of sensitive and resistant QoI and DMI E. necator isolates. Sub-objective 1.F: Determine relationship between mutation frequency and fungicide tolerance. Sub-objective 1.G: Describe the pathogen and epidemiology of the re-emerging disease, dry berry of Rubus. Sub-objective 1.H: Insight into the ecology and pathobiology of the aerial gall pathogen of blueberry. Sub-objective 1.I: Identify cranberry fruit rot pathogens, their fungicide sensitivity, and estimate yield loss to rots in Oregon and Washington production beds. Sub-objective 1.J: Elucidate the disease cycle of Gnomoniopsis idaeicola, an emerging pathogen of blackberry. Objective 2: Apply knowledge of biology, ecology, and epidemiology to the development of improved integrated disease management approaches. Sub-objective 2.A: Examine the utility of UVC for management of grape diseases. Sub-objective 2.B: Develop and improve plant pathogen diagnostics, detection, and identification.

Approach
The long-term goal of this project is to develop the knowledge and tools needed to respond to plant disease epidemics using approaches that are economically and environmentally sustainable, with emphasis on increasing our ability to respond to exotic, emerging, and re-emerging pathogens. This will be accomplished through trans-disciplinary approaches that: (1) improve methods for pathogen monitoring and conduct pathogen surveys to ascertain changes in diversity and specific genetic traits in critical pathogen populations; (2) increase knowledge of pathogen biology and life cycles; (3) integrate this knowledge into decision aids to enhance the economic and environmental sustainability of horticultural crops while improving disease management. The globalization of agricultural markets, increased human, plant, and animal intercontinental travel, and climate change will continue to enhance pathogen spread and introduction of exotic pathogens that threaten natural and agronomic ecosystems. Comparative genomics and transcriptomics will be used to identify new variants and tract population diversity and alleles associated with differences in pathogenesis and fungicide resistance. The consequences of pathogen movement will depend on the speed with which we can detect and track their introduction and adjust management practices in response. We will use the latest advances in CRISPR technology to develop inexpensive diagnostic assays suitable for tracking pathogen variants. The fitness costs associated with genetic variation associated with fungicide resistance will be examined using traditional phenotype characterization and by examining allele segregation and prevalence in laboratory experiments and natural environments. Crop production is also threatened by pathogens currently considered manageable or insignificant but may emerge or re-emerge due to pesticide resistance development, overcoming available host resistance, and/or removal of pesticides from the commercial market. These threats can result in direct and indirect economic impacts, such as reduced yield or quality, loss of foreign or domestic markets, and non-crop impacts. The epidemiology of numerous emerging fungal diseases will be examined using a series of in-field and laboratory studies that elucidate the causal agents, the environmental conditions suitable for disease development, and methods to reduce disease progression. The utility and constraints of germicidal ultraviolet radiation treatments for disease management will be examined in laboratory and field settings. Greater knowledge of the factors influencing establishment and spread of pathogens, and subsequent disease development is needed to develop economically and environmentally sustainable management strategies. For these reasons, this project focuses on a multitude of pathosystems that cause major impacts on horticulture crops, including sudden oak death, botrytis blight and grape powdery mildew.

Progress Report
This report documents progress for project 2072-22000-045-000D, ‘Knowledge Based Tools for Exotic and Emerging Diseases of Small Fruit and Nursery Crops’. In support of Sub-objective 1A, we continue to conduct a comparative genomic analysis of long-read sequenced and fully assembled genomes of major U.S. and Asian variants of the sudden oak death pathogen (Phytophthora ramorum). We conducted an experiment using tanoak inoculated with P. ramorum EU1 and NA1 strains. The transcriptome was sequenced and is currently being analyzed. To address Sub-objectives 1B and 2B, we used resequenced P. ramorum genomes and after variant calling to identify genomic regions diagnostic for differentiating species and clonal lineages. These regions are being used in Sub-objective 2A for a diagnostic assay. For Sub-objective 1D, phenotyping of eight isolates of Erysiphe necator for effects of temperature on germination and infection was conducted. Results demonstrated that there is a fitness advantage conferred by the A143 resistant mutation under cool growing conditions (15-19 C) with some reduction in growth at temperatures greater than 33 C. These data agree with field monitoring where the A143 population starts to grow at low frequency and rapidly becomes the dominant population with or without selecting Qol (quinone outside inhibitor) fungicides being used. The mating studies described in Sub-objective 1E have proven to be too inefficient to obtain the numbers of F1 progeny needed to draw accurate conclusions; thus, the contingency of monitoring field populations will be pursued. Field data indicates that A143 (resistant genotype) does not survive the winter as well as the G143 (wildtype) genotype. Towards Sub-objective 1F, data from 10 E. necator isolates indicated that there is a positive correlation of CYP51 copy number in relation to demethylation inhibitor (DMI) tolerance level among isolates. The data appears to indicate that increased copy number alone is sufficient to confer tolerance to some DMI fungicides. In support of Sub-objective 1G, the genomes of the type strain of the fungal pathogen Monilinia rubi (1959) and a recent isolate were sequenced with Illumina Hi-Seq and Pac-Bio. Assembly of the genomes is underway and will be examined for conserved genetic regions and unique sequences for development of diagnostic tools. For Sub-objective 1H, Illumina Hi-Seq was used to generate whole genome sequences of eight blueberry bacterial stem gall isolates from two fields. The genomes were nearly identical. We will sequence isolates from additional fields to examine diversity of the blueberry stem gall bacterium. Between 3 and 30 percent (%) of Washington and Oregon cranberry beds had cranberry fruit rot in 2022. In support of Sub-objective 1I, we isolated 11 of the known cranberry fruit rot fungal pathogens. We frequently isolated two types of fungi not known to cause cranberry fruit rot. The ability of these fungi to cause cranberry fruit rot is being tested. For Sub-objective 1J, we sampled 13 commercial blackberry fields and isolated Gnomoniopsis idaeicola, the fungal pathogen associated with ‘blackberry collapse’, from 12 fields located in three counties in Oregon. To address Sub-objective 2A, a delivery unit was designed and built that can deliver 1200 watts of Ultraviolet-C radiation. A collection of 15 isolates had ED50 (effective dose that inhibits 50% of growth) ranging from 83 TO 380 joules/square meter. A small plot field trail was initiated that examines four doses of UVC in conjunction with various fungicide application regimes. Results indicated that 120 joules/square meter applied twice per week significantly reduced grape powdery mildew but did not impact Botrytis bunch rot. To address Sub-objective 2.B, we assembled the mitochondrial genomes of currently available P. ramorum isolates and developed a web-based, implementation using nextstrain. This implementation still requires further work and is not yet final.

Accomplishments

Review Publications
Bocardo, F.S., Weisberg, A.J., Riutta, E., Kilday, K., Bonkoswki, J.C., Cresswell, T., Daughtrey, M.L., Rane, K., Grunwald, N.J., Chang, J.H., Putnam, M.L. 2022. Whole genome sequencing-based tracing of a 2022 introduction and outbreak of Xanthomonas hortorum pv. pelargonii. Phytopathology. https://doi.org/10.1094/PHYTO-09-22-0321-R.
Cox, M.P., Guo, Y., Winter, D.J., Sen, D., Cauldron, N.C., Shiller, J., Bradley, E.L., Ganley, A.R., Gerth, M.L., Lacey, R.F., McDougal, R.L., Panda, P., Williams, N.M., Grunwald, N.J., Mesarich, C.H., Bradshaw, R.E. 2022. Chromosome-level assembly of the Phytophthora agathidicida genome reveals adaptation in effector gene families. Frontiers in Microbiology. 13. Article 1038444. https://doi.org/10.3389/fmicb.2022.1038444.
Mullett, M., Van Poucke, K., Haegeman, A., Focquet, F., Cauldron, N., Knaus, B., Horta Jung, M., Kageyama, K., Hieno, A., Masuja, H., Uematsu, S., Webber, J., Brasier, C.M., Bakonyi, J., Heungens, K., Grunwald, N.J., Jung, T. 2023. Phylogeography and population structure of the global, wide host-range hybrid pathogen Phytophthora x cambivora. IMA Fungus. 14. Article 4. https://doi.org/10.1186/s43008-023-00109-6.
Mahaffee, W.F., Margairaz, F., Ulmer, L., Bailey, B.N., Stoll, R. 2023. Catching spores: Linking epidemiology, pathogen biology, and physics to ground-based airborne inoculum monitoring. Plant Disease. 107(1):13-33. https://doi.org/10.1094/PDIS-11-21-2570-FE.
Baumgartner, K., Mahaffee, W.F. 2022. Esca and young vine decline. In: Moyer, M.M., O'Neal, S.D., editors. Field Guide for Integrated Pest Management in Pacific Northwest Vineyards. 2nd edition. Pacific Northwest Extension Publication #PNW644. Pullman, WA: Washington State University Press. p. 89-91.
Perelet, A.O., Ward, H.C., Stoll, R., Mahaffee, W.F., Pardyjak, E. 2022. Quantifying turbulence heterogeneity in a vineyard using eddy-covariance and scintillometer measurements. Boundary Layer Meteorology. 184:479-504. https://doi.org/10.1007/s10546-022-00714-9.
Moody, M.J., Bailey, B.N., Pardyjak, E., Mahaffee, W.F., Stoll, R. 2021. Adaption and validation of a voxel based energy transport model for conifer species. Urban Climate. 39. Article 100967. https://doi.org/10.1016/j.uclim.2021.100967.
Ulmer, L., Margairaz, F., Bailey, B., Mahaffee, W.F., Pardyjak, E., Stoll, R. 2022. A fast-response, wind angle-sensitive model for predicting mean winds in row-organized canopies. Agricultural and Forest Meteorology. 329. Article 109273. https://doi.org/10.1016/j.agrformet.2022.109273.
Zaccaron, A.Z., Neill, T.M., Corcoran, J., Mahaffee, W.F., Stergiopoulos, I. 2023. A chromosome-scale genome assembly of the grape powdery mildew pathogen Erysiphe necator reveals its genomic architecture and previously unknown features of its biology. mBio. Article e00645-23. https://doi.org/10.1128/mbio.00645-23.
Lowder, S.R., Neill, T.M., Peetz, A., Miles, T.M., Moyer, M.M., Oliver, C., Stergiopoulos, I., Ding, S., Mahaffee, W.F. 2023. A rapid glove-based inoculum sampling technique to monitor Erysiphe necator in commercial vineyards. Plant Disease. https://doi.org/10.1094/PDIS-02-23-0216-RE.
Stergiopoulos, I., Aoun, N., Huynh, Q., Neill, T.M., Lowder, S., Newbold, C., Cooper, M.L., Ding, S., Moyer, M., Miles, T.D., Oliver, C.L., Urbez-Torres, J., Mahaffee, W.F. 2022. Identification of putative SDHI target site mutations in the SDHB, SDHC, and SDHD subunits of the grape powdery mildew pathogen Erysiphe necator. Molecular Plant Pathology. 106(9):2310-2320. https://doi.org/10.1094/PDIS-09-21-1993-RE.
Grunwald, N.J., Brown, C., Ip, H., Chang, J. 2022. Genetic processes facilitating pathogen emergence. In: Cardwell, K.F., Bailey, K.L., editors. Tactical Sciences for Biosecurity in Animal and Plant Systems. Hershey, PA: IGI Global. p. 32-53. https://doi.org/10.4018/978-1-7998-7935-0.ch002.
Carleson, N., Press, C.M., Grunwald, N.J. 2022. High-quality, phased genomes of Phytophthora ramorum clonal lineages NA1 and EU1. Molecular Plant-Microbe Interactions. 35(4):360-363. https://doi.org/10.1094/MPMI-11-21-0264-A.
Weisberg, A.J., Miller, M., Ream, W., Grunwald, N.J., Chang, J.H. 2021. Diversification of plasmids in a genus of pathogenic and nitrogen-fixing bacteria. Proceedings of the Philosophical Transactions of The Royal Society B: Biological Sciences. 377(1842). Article 20200466. https://doi.org/10.1098/rstb.2020.0466.
Sparks, A.H., Del Ponte, E., Alves, K., Foster, Z.S., Grunwald, N.J. 2023. Openness and computational reproducibility in plant pathology: Where we stand and a way forward. Phytopathology. https://doi.org/10.1094/PHYTO-10-21-0430-PER.
LeBoldus, J.M., Navarro, S.M., Kline, N., Ritokova, G., Grunwald, N.J. 2022. Repeated emergence of sudden oak death in Oregon: Chronology, impact, and management. Plant Disease. 106(12):3013-3021. https://doi.org/10.1094/PDIS-02-22-0294-FE.