Project : USDA ARS

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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

2025 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 2025 progress for project 2072-22000-045-000D, "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 United States 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 manuscript was published in Molecular Plant-Microbe Interactions journal. To address Sub-objective 1.B, ARS researchers analyzed RNA-seq data and identified candidate genes of interest to host pathogen interactions. For Sub-objective 1.C, ARS researchers collected over 1,000 monoconidial isolates of Botrytis from red raspberry, blueberry, and grape tissues in Washington and Oregon and determined fungicide resistance profiles. Research focused on characterizing mutations in 250 isolates with resistance to succinate dehydrogenase inhibitor (SDHI) fungicides, which are effective for Botrytis management but at risk of resistance development. Alarmingly, 72 percent of the genetic mutations found confer resistance to multiple SDHI fungicides. This destabilizes disease management programs and emphasizes the importance of rotating fungicide chemistries for resistance management. A manuscript was accepted for publication in a peer-reviewed journal describing mutations conferring resistance to SDHI fungicides. Because the ascospore capture methods developed proved to be extremely inefficient, the contingency of monitoring field populations was pursued. All field results have indicated that the G143A mutation becomes undetected during the winter and is rarely found in the early spring but appear to be selected for during cool spring conditions. Growth chamber studies indicate that G143A isolates grow better at 15-19 degrees C than the wildtype for Sub-objecttive 1E. Thus, QoIs should not be used in the early spring, though they can be incorporated into a fungicide rotation when low temperatures are above 15 degrees C. Under Sub-objective 1.G, spores of the fungal pathogen (dry berry disease) Monilinia rubi were detected on impaction rods placed in red raspberry fields. DNA was extracted from several hundred more field-deployed impaction rods, but the samples have not been tested yet for the dry berry pathogen. The sexual reproductive phase of this berry mummification fungus, ascocarp formation, has not been observed in fields or on inoculated berries incubated under various environmental conditions. Isolates of M. rubi were found to be sensitive to fungicides used for crop protection. In support of Sub-objective 1.H, the blueberry stem gall pathogen was not detected in field soils by culturing and only isolated from gall tissues on blueberry stems. The lack of detection in soil was inconclusive. ARS researchers have selected pathogen isolates with spontaneous resistance to rifampicin, an antibiotic commonly used for microbial ecology studies, to allow tracking of bacteria through soils and plants. These rifampicin mutants should improve the ability to study the ecology of the bacterial pathogen in soils and in plants. For Sub-objective 1.I, ARS researchers in Corvallis, Oregon, sampled cranberry beds in Oregon and Washington in 2023 and 2024 and found low incidences (about 5 percent) of fruit rots in most beds. ARS researchers continued to isolate Neofabraea actinidiae, a new cranberry fruit rot pathogen that was previously described in a publication. ARS tested all of the 14 genera of cranberry fruit rot pathogens isolated in the Pacific Northwest (PNW) for sensitivity to site-specific fungicides used in cranberry production. As anticipated, some fungicides were ineffective against specific genera of pathogens. Researchers did observe a low to moderate frequency of tolerance to FRAC 11 fungicides in some of the genera that are managed with FRAC 11 materials. A manuscript was published on the cranberry fruit rot complex and incidence of field and storage rot in the PNW. Under Sub-objective 1.J, ARS researchers surveyed numerous commercial blackberry fields in three counties in Oregon for Gnomoniopsis idaeicola, a fungal pathogen associated with the newly described ‘blackberry collapse’ disease. They tested symptomatic tissues for the pathogen by culturing and used the new multiplex PCR assay that ARS developed for Gnomoniopsis idaeicola. The results were published in a peer-reviewed journal in 2024. In ARS's research blackberry plot, researchers did not observe the formation of spore producing structures (perithecia) on senescent blackberry leaves or stems over the last year, indicating that environmental conditions were not conducive for sporulation. Likewise, the incidence of detection of the pathogen in commercial fields was low. The low incidence could be due to a combination of unfavorable environmental conditions for sporulation, removal of diseased plants from fields, removal of spent floricanes from fields, and fungicides applied into the canopies with air-blast sprayers instead of boom sprayers. Emergence of resistance to various fungicides among isolates of G. idaeicola was not observed. The information gained and shared with growers about blackberry collapse provides targeted suggestions for sound management practices. To address Sub-objective 2.A, collaboration with commercial partners resulted in the design and construction of a double row Ultraviolet-C (UVC) unit that can deliver 1,200 watts (W) of germicidal UV energy. This device allows for almost three times the acreage covered compared to our prior approaches. Two years of testing have confirmed that effective control can be achieved with biweekly applications of 100J/m2. No direct impact on Botrytis bunch rot has been observed nor has there been any change ED50 among isolates. However, there remains a wide range of UV tolerance among isolates. In support of Sub-objective 2.B, ARS researchers in Corvallis, Oregon, 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 in PLoS Computational Biology. The approach was validated with a novel CRISPR-Cas assay using SHERLOCK technology to identify the sudden oak death pathogen Phytophthora ramorum and tested with bacteria, fungi, and oomycetes and found to be specific and sensitive. The krisp algorithm is expected to have wide use across many biological disciplines and accelerate development of traditional as well as CRISPR diagnostic assays. Assemblies of the mitochondrial genomes for P. ramorum samples were finished and developed a web-based implementation using nextstrain.

Accomplishments
1. A comparative genomic analysis of pathogen variants provides insights into sudden oak death pathogen adaptation. Sudden oak death caused by Phytophthora ramorum is causing large epidemics in West Coast forests and large losses in U.S. nurseries. Multiple pathogen variants exist with differences in how they attack hosts. Understanding their underlying genetics could lead to a better understanding of these observed differences and may facilitate control efforts. Recent discovery of P. ramorum in Asia provides a new opportunity for a deeper investigation of the genetic history of the species. ARS researchers in Corvallis, Oregon, sequenced and analyzed the genomes of P. ramorum samples, including three from Asia and three representing variants causing epidemics in western US forests. This information provides novel tools for understanding pathogen evolution and inferring which genetic changes are important for pathogenicity.

2. Identification of emerging fungicide resistance in the gray mold pathogen. Washington and Oregon lead the nation in production of blueberries and red raspberries with a total crop value of $482 million. The disease 'gray mold', caused by the fungus Botrytis, is a significant threat to these crops in the field and in storage. Succinate dehydrogenase inhibitor (SDHI) fungicides are very effective for gray mold control, but there is a risk of Botrytis becoming resistant. ARS scientists in Corvallis, Oregon, in collaboration with Washington State University scientists, surveyed blueberry and red raspberry fields over two years and consistently recovered Botrytis isolates that were resistant to one or more of SDHI fungicides recently registered for disease control. Sequence analysis of the Botrytis isolates revealed genetic mutations that confer cross-resistance to several SDHI fungicides. Growers are using the documented emerging multi-SDHI fungicide resistance from these studies to refine their pest management programs for resistance management as well as sustainable gray mold control.

3. Deciphering the array of fungi causing cranberry fruit rots in the Pacific Northwest. Oregon and Washington rank in the top five cranberry producing states with a crop value >$40 million. Growers had limited knowledge of which pathogens are present and causing crop losses. ARS scientists in Corvallis, Oregon, in collaboration with Oregon State University Extension scientists, surveyed cranberry beds over four years and documented crop losses of up to 40% to fruit rots at harvest and additional losses up to 69% of cranberries during cold storage. A new cranberry fruit rot pathogen was identified and 15 different fungal pathogens were documented to be present in Pacific Northwest cranberry production beds. Growers now have a better understanding of the pathogen diversity present which will result in more targeted pest management practices for cranberry.

4. New tools for monitoring pathogen dispersion. Movement of airborne plant debris, particularly during harvest, and other particles have the potential of dispersing associated pathogens. However, the particle size associated with successful dispersion is difficult to assess but critical for predicting disease risk since there is an exponential decrease in distance traveled with increase in size. A modular, 3D-printed device was designed by ARS researchers in Corvallis, Oregon, to collect and size-segregate particles deposited under realistic atmospheric conditions. This device will allow researchers to accurately assess particle size and assess the same particles for viable pathogen propagules. This device gives researchers a low-cost tool that will enable the development of experiments that were previously too expensive or not practical and thereby improve our understanding of airborne pathogen spore dispersion.

U.S. DEPARTMENT OF AGRICULTURE

Horticultural Crops Disease and Pest Management Research Unit: Corvallis, OR