Research Project: Disease Management in Small Fruit and Nursery Crops Based on Knowledge of Pathogen Diversity, Biology, and Environmental Effects

Location: Horticultural Crops Disease and Pest Management Research Unit

2023 Annual Report

Objectives
Objective 1: Improve and expand knowledge of the prevalence of genotypic and phenotypic diversity in existing and emerging plant pathogens of small fruits and nursery crops. Sub-objective 1.A: Evaluate boxwood blight in Oregon nurseries. Sub-objective 1.B: Develop molecular diagnostics and tools to facilitate research on a nematode/virus disease complex. Sub-objective 1.C : Identify factors that cause blueberry shock virus recurrence in commercial fields and blueberry breeding lines. Objective 2: Understand how pathogen biology and diversity interacts with environmental factors to cause disease of small fruits and nursery crops. Sub-objective 2.A: Influence of plant spacing and irrigation frequency on the spread of boxwood blight from infected plants to healthy plants. Sub-objective 2.B: Cellular level response of Meloidogyne spp. to nematicides. Objective 3: Develop chemical and host resistance disease management strategies for nursery crops and small fruits. Sub-objective 3.A: Evaluate newer fungicide chemistries for control of Phytophthora root rot of rhododendron by sensitivity assays. Sub-objective 3.B: Novel nematicide discovery: Solanum sisymbriifolium as a source of nematicidal compounds. Sub-objective 3.C: Screen selected Rubus idaeus (red raspberry) accessions for resistance to raspberry bushy dwarf virus Objective 4: Develop robust and reliable diagnostic assays for plant virus detection in small fruits. Sub-objective 4.A: Compare graft indexing, RT-PCR, and high-throughput sequencing methods for virus detection in strawberry. Sub-objective 4.B: Compare graft indexing, RT-PCR, and high-throughput sequencing methods for virus detection in Rubus.

Approach
The long-term goal of this project is to develop sustainable disease management strategies that are based on a knowledge of the identity and biology of the causal agent(s) and on knowledge of pathogen co-infections and interactions with the environment. This will be accomplished by: (1) determining the prevalence of key pathogens and nematodes constraining production of nursery and small fruit crops; (2) understanding how environment and management practices influence disease; (3) identifying new pesticides and disease resistant crop genotypes for the management of nursery and small fruit diseases; and (4) developing pathogen detection protocols for nursery certification and quarantine plant material. Knowledge about the prevalence of fungal pathogens, nematodes, and viruses in agricultural systems is key for establishing effective disease control methods. Surveys will be conducted to assess the incidence of fungal pathogens in nurseries, and viruses and nematodes in small fruit research and production fields. Molecular diagnostic tools will be developed to evaluate the ability of nematodes to vector plant viruses and to assess for virus coinfections in small fruit crops. Pathogen and nematode prevalence is influenced by their response to multiple environmental factors. Therefore, studies will be established to assess the influence of irrigation and plant spacing on the spread of fungal plant pathogens in outdoor container trials, and on the effect of nematicides on nematode fitness in laboratory trials. New pesticide chemistries are needed because multiple oomycete pathogens have developed resistance to fungicides and many traditional nematicides have been phased out because of harmful environmental and human health effects. New pesticide chemistries will be evaluated for their efficacy against oomycete plant pathogens and nematodes in laboratory, greenhouse and field experiments. In addition, host resistance plays a crucial role in successful disease management and diagnostic assays are needed to allow growers, regulatory agencies, and diagnosticians to quickly and accurately identify the pathogens causing disease. Small fruit genotypes from grower fields, breeding programs, and national germplasm collections will be screened for resistance or tolerance to key viruses and both traditional bioassays and modern DNA- or RNA-based technologies will be compared for their ability to detect a wide range of viral pathogens for the small fruit industry. Together, results from this research will identify chemical and nonchemical practices to reduce plant disease, and that can be deployed in horticultural systems in the future.

Progress Report
This report documents progress for project 2072-22000-046-000D, “Disease Management in Small Fruit and Nursery Crops based on Knowledge of Pathogen Diversity, Biology, and Environmental Effects”. Progress towards Sub-objective 1A has been made by sampling for boxwood blight at three additional boxwood nurseries in Oregon. Single-spore cultures of the boxwood blight pathogen have been prepared to share with collaborators. For Sub-objective 1B, locations with both virus and vector nematodes were identified in the Pacific Northwest (PNW). Plant material was collected and high throughput sequencing was used to generate genomes of tomato ringspot virus (ToRSV). The genomes were then used to develop inclusive primers that will amplify diverse isolates of ToRSV. A system is under development to allow nematodes go feed on ToRSV-infected plants in order to have viruliferous nematodes to work with to continue to develop a quantitative polymerase chain reaction diagnostic assay. For Sub-objective 1C, we surveyed and collected about 1,800 blueberry samples from Washington and Oregon. We have collected phenotypic data on all samples. All samples were processed for RNA extraction. All samples were tested for blueberry shock virus and for the new luteovirus (recently named blueberry virus L). For Sub-objective 2A, a potted plant experiment was installed to test the influence of irrigation and plant spacing on disease progress. Plants in each plot are irrigated once, twice, or three times a day and spaced so that the pots are touching or are six inches apart. Preliminary evidence shows disease spreads fastest on plants that are irrigated three times a day and are spaced closer together compared to the other treatments. For Sub-objective 2B, preliminary experiments were conducted to develop a system to expose nematodes to nematicides. Once a method was in place, M. incognita second-stage juveniles were exposed to four nematicides and a water control for two hours. After exposure, nematodes were collected and processed immediately for RNA extraction. The samples have been sent for sequencing. For Sub-objective 3A, tests to evaluate Phytophthora isolates against the first fungicide, mefenoxam, have been completed. Tests to determine sensitivity to the second fungicide, phosphorous acid, are in progress. For Sub-objective 3B, experiments were conducted to evaluate the host status of S. sisymbrifolium to plant-parasitic nematodes. Plants were inoculated with M. incognita, M. hapla, M. chitwoodi, and P. penetrans in a greenhouse assay; appropriate positive controls were included. At the end of the experiment nematode reproduction was determined as either egg production (Meloidogyne) or recovered mixed-stage nematode (Pratylenchus). Results showed that S. sisymbrifolium is a poor host for the plant-parasitic nematode considered in the study. For Sub-objective 3C, 50 red raspberry plants have been collected from the USDA ARS National Clonal Germplasm Repository and we have propagated sufficient quantity for downstream applications. For Sub-objective 4A, we have propagated 600 plants and concluded graft experiment 1. We extracted RNA from strawberry donor plants. RNA was used for high throughput sequencing (HTS). HTS is now complete. For Sub-objective 4B, we propagated 225 plants and have concluded graft experiment 1. We have extracted RNA from Rubus donor plants. RNA was used for HTS, which is now complete.

Accomplishments
1. Genomic characterization and distribution of newly described virus infecting blueberries. An ARS scientist in Corvallis, Oregon, collaborated with the University of Arkansas to complete the genomic sequence and characterization of a new blueberry virus, Blueberry Virus L, a luteovirus in the family Tombusviridae. The genetic information will be applied to develop and optimize virus detection in blueberries. A survey was conducted, and it was found that this virus is widespread throughout the United States, including the Pacific Northwest. Incidence of the virus ranges from around 5 to 80 percent, depending on plant age, variety, and location.

Review Publications
Silva, J., Melo, F., Elena, S., Candresse, T., Sabanadzovic, S., Tzanetakis, I., Blouin, A., Villamor, D., Mollov, D.S., Constable, F., Cao, M., Saldarelli, P., Cho, W., Nagata, T. 2022. Virus classification based on in-depth sequence analyses and development of demarcation criteria using the Betaflexiviridae as a case study. Journal of General Virology. 103(11). Article 001806. https://doi.org/10.1099/jgv.0.001806.
Weiland, G.E., Scagel, C.F., Grunwald, N.J., Davis, E.A., Beck, B.R., Mitchell, J.N. 2022. Irrigation frequency and volume has little influence on Phytophthora root rot in container-grown rhododendron. Journal of Environmental Horticulture. 40(2):67-78. https://doi.org/10.24266/2573-5586-40.2.67.
Ohkura, M., Scagel, C.F., Weiland, G.E. 2023. Rapid and scalable DNA extraction and real-time PCR assay from boxwood tissue for the detection of Calonectria pseudonaviculata, causal agent of boxwood blight. Plant Disease. 107(5):1279-1283. https://doi.org/10.1094/PDIS-06-22-1453-SR.
Scagel, C.F., Weiland, G.E., Beck, B.R., Mitchell, J.N. 2023. Temperature and fungicide sensitivity in three prevalent Phytophthora species causing Phytophthora root rot in rhododendron. Plant Disease. https://doi.org/10.1094/pdis-11-22-2670-re.
Anderson, O.P., Wram, C.L., Zasada, I.A. 2022. What is the optimal way to assess Meloidogyne spp. reproduction in greenhouse pot experiments? Journal of Nematology. 54(1). Article 3922. https://doi.org/10.2478/jofnem-2022-0012.
Harrington, S., Knox, J., Burns, A., Choo, K., Au, A., Kitner, M.L., Haeberli, C., Pyche, J., D'Amata, C., Kim, Y., Volpatti, J., Guiliani, M., Snider, J., Wong, V., Palmeira, B., Redman, E., Vaidya, A., Gilleard, J., Stagljar, I., Cutler, S., Kulke, D., Dowling, J., Yip, C., Keiser, J., Zasada, I.A., Lautens, M., Roy, P. 2022. Egg-laying and locomotory screens with C. elegans yield a nematode-selective small molecule stimulator of neurotransmitter release. Communications Biology. 5. Article 865. https://doi.org/10.1038/s42003-022-03819-6.
Zasada, I.A., Nunez-Rodriguez, L., Rivedal, H.M., Peetz, A.B., Ocamb, C. 2023. First report of the root-lesion nematode, Pratylenchus penetrans, parasitizing hemp (Cannabis sativa) in Washington. Plant Health Progress. https://doi.org/10.1094/PHP-12-22-0122-BR.
Stainton, D., Villamor, D., Sierra Mejia, A., Srivastava, A., Mollov, D.S., Martin, R., Tzanetakis, I. 2023. Genomic analyses of a widespread blueberry virus in the United States. Virus Research. 333. Article 199143. https://doi.org/10.1016/j.virusres.2023.199143.
Mestas, A., Weiland, G.E., Scagel, C.F., Davis, E.A., Mitchell, J.N., Beck, B.R. 2023. Greater rate of nitrogen fertilizer application increases root rot caused by Phytophthora cinnamomi and P. plurivora in container-grown rhododendron. Plant Pathology. https://doi.org/10.1111/ppa.13776.