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

2024 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 FY 2024 progress for project 20-000D, “Knowledge Based Tools for Exotic and Emerging Diseases of Small Fruit and Nursery Crops”, which began in May 2022. In support of Sub-objective 1.A, ARS researchers in Corvallis, Oregon, finished 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. These genomes revealed a loss in some effectors relative to the most recent common ancestor of the clade. A large transcriptomics experiment using tanoak infected with P. ramorum with EU1 and NA1 strains has been analyzed, and a publication is being drafted. ARS researchers discovered large stretches of loss of heterozygosity in one clonal lineage relative to others that is being studied further. To address Sub-objectives 1.B and 2.B, ARS researchers identified regions and designed a novel clustered regularly interspaced short palindromic repeats (CRISPR)-Cas assay to differentiate Phytophthora ramorum from other Phytophthora species. For Sub-objective 1.C, ARS researchers obtained over 300 isolates from vineyards in Oregon and Washington and screened them for resistance to seven fungicide classes. Over 50% of the isolates had tolerance to more than one fungicide and there was tolerance to all of the tested fungicide classes. These results indicate that growers should only use a fungicide class once per season and rotate chemistries across seasons. The mating studies described in Sub-objective 1.E are too inefficient to obtain the numbers of F1 progeny needed to draw accurate conclusions; thus, the contingency of monitoring field populations has been pursued. Field data continues to indicate that A143 (resistant genotype) does not survive the winter as well as the G143 (wildtype) genotype. Studies to ascertain the decline and potential causes have been initiated. Preliminary indications are that there is a temperature dependent response. In support of Sub-objective 1.G, spores of the fungal pathogen Monilinia rubi were detected on impaction rods placed in red raspberry fields. However, it is unclear if a sexual reproductive phase is important in the disease cycle because ascocarps were not seen in fields nor formed on inoculated berries incubated under various environmental conditions. Isolates of M. rubi were sensitive to fungicides used for crop protection. For Sub-objective 1.H, the blueberry stem gall pathogen was not detected in field soils by culturing and only isolated from gall tissues on plants. Given the tools at hand, the lack of detection was not conclusive. ARS researchers are proceeding with their contingency plan to introduce a plasmid carrying two markers, kanamycin resistance and the green fluorescent protein. These markers will improve their ability to study the ecology of the bacterial pathogen in soils and in planta. Under Sub-objective 1.I, ARS researchers observed low incidences (~6%) of cranberry fruit rots in Oregon and Washington in 2023. They isolated a new cranberry fruit rot pathogen (Neofabraea actinidiae) and showed that it causes rot in fields and during storage. The researchers tested several genera of fruit rot pathogens for sensitivity to site-specific fungicides used in cranberry production. Most isolates were sensitive, but in some cases, researchers observed a low to moderate frequency of tolerance to FRAC 11 fungicides. For Sub-objective 1.J, a dozen commercial blackberry fields in three counties in Oregon were surveyed for Gnomoniopsis idaeicola, a fungal pathogen associated with ‘blackberry collapse’. Emergence of resistance to various fungicides among isolates of G. idaeicola was not observed. The incidence of detection of the pathogen was low, as growers have been removing diseased plants from fields. ARS researchers developed a diagnostic tool based on multiplex PCR to detect G. idaeicola in planta and to identify it in culture. Identification or detection of G. idaeicola can be completed in a day with multiplex PCR, much quicker than the traditional culture-based methods that took several weeks. To address Sub-objective 2.A, a delivery unit was designed and built that can deliver 1200 watts of Ultraviolet-C (UVC) radiation. A second year of monitoring isolates indicates that a change in the ultraviolet tolerance is not observed. A small plot field trial was repeated to examine four doses of UVC in conjunction with various fungicide application regimes. Results confirmed that approximately 100 joules/square meter applied twice per week is needed to significantly reduce grape powdery mildew, and there does not appear to be a direct effect on Botrytis bunch rot. A second double row UVC field unit is being designed and constructed that can deliver approximately 200 J/m^2 to two rows simultaneously. In support of Sub-objective 2.B, ARS researchers developed the krisp python package that can scan whole genome sequences to identify regions diagnostic for distinguishing a target from a non-target group that is now published. The researchers validated a novel CRISPR-Cas assay using SHERLOCK technology to identify the sudden oak death pathogen, Phytophthora ramorum, and tested it with bacteria, fungi, and oomycetes and found it to be specific and sensitive. They finished assemblies of the mitochondrial genomes for P. ramorum samples and developed a web-based, implementation using nextstrain.

Accomplishments
1. Development of the krisp python package for development of CRISPR Cas diagnostic assays using whole genome sequences. Pathogens continue to emerge at accelerated rates affecting animals, plants, and ecosystems. Rapid development of novel diagnostic tools is needed to monitor novel pathogen variants or groups. ARS researchers in Corvallis, Oregon, developed the computational tool krisp to identify genetic regions suitable for development of clustered regularly interspaced short palindromic repeats (CRISPR) diagnostics. Krisp scans whole genome sequence data for target and non-target groups to identify diagnostic regions based on DNA or RNA sequences. This computational tool has been validated using genome data for the sudden oak death pathogen. Krisp is a released open source under a permissive license with all the documentation needed to quickly design CRISPR-Cas diagnostic assays. This technology provides a much needed, novel tool for biosurveillance of emerging and invasive pathogens.

2. Demonstrated that low doses of germicidal ultraviolet (UV) are effective for management of grape powdery mildew. Grape powdery mildew can result in complete crop loss and require up to 17 fungicide applications to manage. This has resulted in the emergence of fungicide resistance and field-level control failures. Alternatives to fungicide applications are needed. ARS researchers in Corvallis, Oregon, demonstrated that low doses of germicidal UV are effective in reducing both leaf and cluster disease. Results indicate, that under low disease pressure, Ultraviolet-C (UVC) applications twice a week could be effective in managing powdery mildew at a commercial scale.

3. Development of a multiplex polymerase chain reaction (PCR) assay to detect the emerging fungal pathogen Gnomoniopsis idaeicola. Oregon is the top producing state of blackberries in the United States. An emerging disease called blackberry collapse is associated with the fungus Gnomoniopsis idaeicola. As the name implies, affected plants show reduced growth, wilting, and eventual death in two to three years. Symptomatic plants are removed to reduce disease spread in the field. Early symptoms of blackberry collapse are difficult to distinguish from other non-lethal cane diseases. ARS researchers in Corvallis, Oregon, developed a PCR assay to detect and identify G. idaeicola. The assay provides rapid diagnosis of blackberry collapse, which may allow growers to deploy methods to reduce the establishment of the disease and minimize long-term losses.

Review Publications
Sharma, N., Neill, T.M., Yang, H., Oliver, C., Mahaffee, W.F., Naegele, R.P., Moyer, M., Miles, T. 2023. Development of a PNA-LNA-LAMP assay to detect an SNP associated with QoI resistance in Erysiphe necator. Plant Disease. 107(1):3238-3247. https://doi.org/10.1094/PDIS-09-22-2027-RE.
Sudermann, M., Foster, Z.S., Chang, J., Grunwald, N.J. 2023. Metabarcoding for plant pathologists. Canadian Journal of Plant Pathology. 46(2):142-160. https://doi.org/10.1080/07060661.2023.2290041.
Oliver, C., Cooper, M., Lewis Ivey, M., Brannen, P., Miles, T., Lowder, S., Mahaffee, W.F., Moyer, M. 2024. Fungicide use patterns in select United States wine grape production regions. Plant Disease. 108(1):104-112. https://doi.org/10.1094/PDIS-04-23-0798-RE.
Ulmer, L., Margairaz, F., Mahaffee, W.F., Stoll, R. 2024. A fast-response model of turbulence and passive scalar transport in row-organized canopies. Agriculture and Forest Meterology. 349. Article 109919. https://doi.org/10.1016/j.agrformet.2024.109919.
Kozanitas, M., Knaus, B.J., Tabima, J.F., Grunwald, N.J., Garbelotto, M. 2024. Climatic variability, spatial heterogeneity and the presence of multiple hosts drive the population structure of the pathogen Phytophthora ramorum and the epidemiology of Sudden Oak Death. Ecography. Article e07012. https://doi.org/10.1111/ecog.07012.
Stockwell, V.O., Shaffer, B.T. 2024. Multiplex PCR assay for detection and identification of Gnomoniopsis idaeicola, the causal agent of blackberry collapse. Plant Health Progress. 25(2):214-217. https://doi.org/10.1094/PHP-09-23-0080-BR.
Check, J., Harkness, R., Heger, L., Sakalidis, M.L., Chilvers, M.I., Mahaffee, W.F., Miles, T. 2024. It’s a trap! Part I: Exploring the applications of rotating-arm impaction samplers in plant pathology. Plant Disease. 108(7):1910-1922. https://doi.org/10.1094/PDIS-10-23-2096-FE .
Check, J., Harkness, R., Heger, L., Chilvers, M.I., Mahaffee, W.F., Sakalidis, M.L., Miles, T.D. 2024. It’s a trap! Part II: An approachable guide to constructing and using rotating-arm air samplers. Plant Disease. 108(7):1923-1936. https://doi.org/10.1094/PDIS-01-24-0131-SR.
Belisle, W.H., Rooney-Latham, S., Soriano, M.C., Grunwald, N.J., Blomquist, C.L. 2024. First report of Phytophthora ramorum causing leaf spots on Cornus capitata (evergreen dogwood) in the United States. Plant Disease. 108(4):1119. https://doi.org/10.1094/PDIS-12-23-2638-PDN.
Grunwald, N.J., Bock, C.H., Chang, J.H., Alves De Souza, A., Del Ponte, E.M., du Toit, L.J., Dorrance, A.E., Dung, J., Gent, D.H., Goss, E.M., Lowe-Power, T., Madden, L.V., Martin, F.N., McDowell, J., Naegele, R.P., Potnis, N., Quesada-Ocampo, L.M., Sundin, G.W., Thiessen, L., Vinatzer, B.A., Zeng, Q. 2024. Open access and reproducibility in plant pathology research: Guidelines and best practices. Phytopathology. 114(5):910-916. https://doi.org/10.1094/PHYTO-12-23-0483-IA.