Location: Temperate Tree Fruit and Vegetable Research
2019 Annual Report
Objectives
Develop or identify new breeding lines, germplasm and named cultivars with superior quality, disease and pest resistance, and nutritional value. This will involve collaborative and independent work by our three-person team using our respective expertise in potato breeding, molecular physiology and plant pathology. The three objectives below undertake complimentary approaches to germplasm improvement. Objective 1 involves largely breeding for targeted traits. Objective 2 seeks to determine basic mechanisms that govern trait expression. Objective 3 will develop new or improved methods to evaluate breeding lines and germplasm. We will work closely with the TriState Breeding Program, as we have for over 20 years.
Objective 1: Evaluate, identify, breed, and release potato germplasm with improved traits of interest, especially improved disease and pest resistance, and increased amounts of phytonutrients.
Subobjective 1A. Develop breeding lines, cultivars or identify germplasm with enhanced amounts of phytonutrients and visual appeal.
Subobjective 1B. Develop breeding lines, cultivars or identify germplasm with superior disease resistance with a focus on soil-borne diseases.
Objective 2: Characterize genetic, environmental, molecular, physiological, and biochemical factors that control accumulation of potato phytonutrients and mechanisms that lead to plant disease resistance, and use this knowledge to produce new superior potato cultivars.
Subobjective 2A: Determine mechanisms that mediate tuber phytonutrient expression.
Subobjective 2B: Increase information and develop methods with potential to be used for control of Potato Cyst Nematode (PCN) and for improved disease resistance.
Objective 3: Develop improved pathogen diagnostic techniques and phenotyping approaches that can be used for potato germplasm evaluation, development of host-resistance, and identification of emerging potato diseases.
Subobjective 3A. Identify and characterize emerging and evolving pathogens and pests in the Pacific Northwest.
Subobjective 3B: Characterize Tobacco rattle virus (TRV)-potato interactions to develop better detection methods and determine the relationship between viral titer, cultivar, symptoms and resistance.
Approach
1A. Germplasm will be intercrossed and progeny evaluated in the field. Replicated plots will be grown in successive years across multiple locations. Lines will be analyzed for carotenoids, anthocyanins, antioxidants, total protein, potassium and iron. Molecular markers will be used to characterize high carotenoid lines. Liquid chromatography mass spectrometry (LC-MS) will be used to quantitate phytonutrients. If germplasm does not provide the desired traits, we will import additional germplasm.
1B. Resistance to nematodes, viruses and fungi will be developed using resistant lines to make crosses and evaluating progeny in field trials. Selected clones will be evaluated under high disease pressure and molecular markers used for Meloidogyne chitwoodi breeding. If progeny have lower selection rates than expected the size of the initial population will be increased.
2A. Expression of structural genes and transcription factors in potatoes or organs that have low or high amounts of phenolics, are cold-treated, or wounded will be analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and LCMS. We will use ribonucleic acid sequencing (RNA-seq) to generate transcriptomic data. Effect of environment on glycoalkaloids will be assessed by growing 13 genotypes in six locations and methanolic extracts from freeze-dried tubers analyzed by LCMS. If key genes are identified, resources will be redirected to apply this knowledge through precision breeding efforts.
2B. Potato cyst nematode (PCN) trap crop seed will be produced by sowing true seed directly into the soil at ¼ inch depth. Hatching factor purification will be tested on diverse High-performance liquid chromatography (HPLC) columns and fractions tested for activity. If a hatching factor is identified and quantitated by LCMS, increased resources will be directed.
3A. Samples from symptomatic plants will be collected. Grafting experiments will evaluate transmissibility. Established molecular tools will be used to detect any pathogens present. If targeting known pathogens does not identify a biological agent, primers that target unknown pathogens will be used. Psyllid involvement in beet-leafhopper transmitted virescence agent (BLTVA) will be tested using field and cage experiments. Development of improved diagnostic tools for BLTVA and Candidatus Liberibacter solanacearum (Lso) will be assessed using a single-tube nested PCR technique, RT-qPCR or Kompetitive allele-specific PCR. If unable to identify any known pathogen in a sample, next generation sequencing platforms will be used.
3B. Tobacco Rattle Virus (TRV) sampling methods will be evaluated for efficacy. Lines will be evaluated for resistance in field trials. PCR will be used to compare viral titer with symptom severity. Varieties will be exposed to TRV and differences in resistance/insensitivity and susceptibility compared. Daughter tuber symptoms and viral titer will be compared to mother tuber symptoms, viral titer, plant emergence, and daughter tuber yield. If TRV infection becomes sporadic, we will focus on the genotypes that were subject to sufficient disease pressure.
Progress Report
For Objective 1, researchers in Prosser, Washington, successfully conducted a potato crossing block in the spring of 2019 that generated thousands of recombinant seeds that will be evaluated in future field trials and initiated the process of discovering molecular markers linked to Tobacco Rattle Virus (TRV) resistance. Parental crossing material was selected with the goal of developing new potato varieties with improved nutritional quality, visual appeal, and disease resistance. Progress was achieved in generating new families by crossing individuals with elevated phytonutrient content, high tuber set, and favorable tuber shape within and between each major market class (russets, chipping varieties, fingerlings, and other specialty potatoes). Progress was also made by introgressing extreme resistance to viral diseases Potato Virus Y (PVY), TRV, Potato Mop Top Virus (PMTV) and nematode infection (Columbia Root Knot Nematode (CRKN), Pale Cyst Nematode (PCN)) into susceptible genetic backgrounds. In the winter of 2019, our team measured the incidence and severity of disease symptoms associated with TRV infection within a population known to be segregating for resistance to this virus and replanted this population in a TRV infested field (replicated in triplicate). We have genotyped a subset of the clones and will genotype the entire population this fall using genotyping by sequencing to assess the genetic architecture of TRV resistance using biparental linkage mapping. Potato germplasm and breeding lines were evaluated for phytonutrient content, including phenylpropanoids and carotenoids. Experiments were initiated that will further clarify how environment effects tuber nutritional content.
To address Objective 2, we have initiated a project focused on identifying physical and biological factors associated with soil health status. We have planted potato varieties that are susceptible (Russet Burbank) and resistant (Castle Russet) to TRV infection in a disease infested field and adjacent to a disease-free field. Each potato variety is replicated four times (four plots each, eight total) within each field and composite soil samples have been collected at the first time point from each plot. Nematodes have been extracted for each composite soil sample, the concentration of Stubby Root Nematodes (the vector species that transfers TRV between plants) has been determined, and samples have been submitted for chemical and physical soil testing. This sampling will be repeated at tuber bulking, after harvest, and total microbial composition will be assessed using next generation DNA sequencing. These data will establish how physical and biotic environmental factors interact with viruliferous Stubby Root Nematode infestation in fields planted with a TRV-resistant and TRV-susceptible potato variety.
We are also examining a line of reduced thorn Litchi Tomato for its efficacy against PCN in Idaho, working with Animal and Plant Health Inspection Service-Plant Protection and Quarantine (APHIS-PPQ). PCN is a quarantine pest and access to fields with PCN is restricted. This collaboration with APHIS will allow field testing of our Litchi Tomato line to examine its potential in reducing or eradicating the PCN population in Eastern Idaho. We also have preliminary evidence that Litchi Tomato may reduce the numbers of some other nematodes besides PCN and are working with researchers in Corvallis, Oregon, to gather more data on this potential effect.
For work on the nutritional quality of potatoes in Objective 2, we have discovered genes that regulate phenylpropanoids in potatoes, with a focus on health-promoting compounds called flavonols. This work also identified one of the first microRNAs ever found in potatoes, that we show regulates flavonol amounts.
Work is progressing on identifying factors that control glycoalkaloid potatoes and greening in potatoes, with major strides being made this past year showing some surprising environmental effects on glycoalkaloid and greening.
To address Objective 3, researchers at Prosser, Washington, sought to identify a potentially new or changing pathogens causing mysterious disease-like symptoms in the Columbia Basin of Washington and Oregon. During the 2018 growing season in this region, potato plants were identified for the second year with mysterious disease-like symptoms of leaf purpling, leaf distortion and stem blistering and necrotic lesions. Commercial fields of five different cultivars were observed, and varying symptoms and symptom severity was observed across the cultivars. Umatilla Russet was consistently the most severely impacted cultivar, while Russet Burbank was only mildly impacted. Tissue from roughly 50 field-collected symptomatic plants was grafted to healthy greenhouse-grown potato plants during each year of the two-year study. No symptoms associated with the mysterious disease were transmitted to the healthy plants. Molecular detection of known viral and bacterial pathogens were assayed from all field-collected tissue in the laboratory. Despite the presence of PVY in several plant samples, no pathogen was detected that correlated to the disease symptoms. Tubers from commercial and research fields showing the mystery disease-like symptoms in the Columbia Basin were grown out in the greenhouse to assess the health of emerged plants. No tubers produced plants with any of the disease-like symptoms of leaf purpling, leaf distortion or stem lesions. These combined results indicate that the cause of the mysterious disease-like symptoms across the Columbia Basin in not pathogenic in nature.
Also, under Objective 3, researchers at Prosser, Washington, conducted experiments to identify the role that potato psyllid might have in the epidemiology of the beet leafhopper transmitted virescence agent (BLTVA) phytoplasma in potato. Previous data from our laboratory has indicated that wild-caught potato psyllids in central Mexico can test positive for the phytoplasma pathogen. We subsequently tested potato psyllids collected in Washington State for the presence of the BLTVA phytoplasma. Psyllids were tested in groups (up to 30 psyllids) and individually, and 0.8 to 18 percent tested positive for BLTVA. With the finding of psyllids in Washington State with BLTVA, we assessed the acquisition of the pathogen by the psyllid in a controlled environment. Here we conducted three different greenhouse experiments each of which forced potato psyllids to feed on BLTVA-infected potato plants. Potato psyllids were subsequently recovered from the plants and tested for the presence of the pathogen by molecular diagnostics. In these controlled experiments, psyllids acquired BLTVA at very low levels despite being caged on infected plants.
Researchers at Prosser, Washington, began an investigation to identify the vector of the novel ‘Candidatus Liberibacter solanacearum’ (Lso) haplotype F bacterium that was identified in their laboratory in a potato tuber with zebra chip disease symptoms. In collaboration with university and USDA scientists, psyllids were collected from the Klamath Basin in Oregon on sticky cards placed in or near commercial potato fields. Psyllids were generally identified as belonging to the Aphalara genus through visual identification technique and molecular analysis. Over 1000 individual psyllids were removed from the sticky cards and tested for the presence of Lso. Twenty-five psyllids tested positive for Lso by molecular analysis. Work is ongoing to identify the haplotype of Lso in these samples in order to determine if Aphalara psyllids are responsible for vectoring Lso haplotype F to potato in the Klamath Basin.
Progress was made on Objective 3 to determine if sampling method, variety, and tuber symptoms have a role in TRV detection. Here tubers were sampled using five different sampling methods to identify the most reliable and efficient method for sampling TRV-infected tubers for viral detection. Additionally, samples were collected from over 10 different potato cultivars with varying internal tuber necrotic symptoms (asymptomatic, mild, moderate, and severe) for assaying viral titer. Work to optimize a reverse transcription quantitative-polymerase chain reaction method for assessing viral titer has begun in the laboratory, as well as nucleic acid extraction of all samples. Further progress for Objective 3 involves applying proximal and remote sensing technology to assess plant health and environmental variables associated with disease symptoms. We have contracted with UpAngle Drone Services out of Richland, Washington, to collect aerial orthomosaic images of all our experimental plots at four different time points throughout the growing season (seedling emergence, row closure, flowering, and harvest). We have completed data collection of our first time point and we are currently constructing computer pipelines to extract information from this and future datasets. In two of these fields we have deployed six soil moisture sensors (one-foot depth) to estimate the availability and fluctuation of water across the field and throughout the growth season. Integration of these sensors, climate data from AgWeatherNet, with drone, and satellite imagery, will help us better understand how these data relate to pathogen infection and disease symptoms of potato. In addition, we have set up a controlled environment experiment to evaluate the use of hyperspectral sensors to discriminate between plant genotypes, plant growth medium, and TRV infection status.
Accomplishments
1. New methods to increase phytonutrients in potatoes. Potatoes with increased phytonutrient concentrations are needed to ensure producers have products that appeal to changing markets and consumer preferences that prioritize nutritional value. A limitation of breeding even more nutritious potatoes is the lack of knowledge of the mechanisms that control amounts of phytonutrients like phenylpropanoids, which are highly desirable in the diet, but low in most white potatoes. ARS researchers in Prosser, Washington, and scientists at Washington State University, identified genes and a microRNA that control the amount of health-promoting phenylpropanoids in potatoes. These discoveries reveal new opportunities to breed potatoes with increased phytonutrients that not only appeal to health-conscious consumers, but improve plant stress resistance, appearance, and flavor.
2. Impact of ‘Candidatus Liberibacter solanacearum’ haplotypes A and B on plant emergence from infected potato. Zebra chip is an economically devastating disease of potato in the U.S., where two Lso haplotypes (A and B) cause vascular darkening and necrotic flecking that render the potatoes unmarketable. ARS researchers in Prosser and Wapato, Washington, identified differences in plant emergence and daughter tuber yield produced from seed potato tubers infected with either Lso A or B and healthy control seed. Plant emergence from Lso A and B infected seed potato were slower to emerge, had lower overall rates of emergence, and produced reduced tuber numbers and yield compared to the healthy seed. Seed potato infected with Lso B generally had lower plant emergence and daughter tuber production compared to seed potato infected with Lso A, confirming earlier analyses that identified Lso B as a more severe haplotype. The results from this study indicate that potato tuber seed infected with either Lso A or B should not be a significant concern to growers, as poor emergence allows plants emerging from healthy seed to outgrow and outproduce plants emerging from infected seed of either Lso haplotype.
3. Cause of mysterious disease-like symptoms in Columbia Basin potatoes is not caused by a pathogen. Some potatoes grown in the Columbia Basin in the last few years have shown unusual symptoms of unknown origin, raising alarm in the industry that this may be due to a new, emerging disease. ARS researchers in Prosser, Washington, collected potato foliar tissue from commercial and research plots throughout the Columbia Basin of Washington and Oregon that showed leaf purpling, leaf deformity and necrotic stem lesions. Molecular diagnostic analyses to detect known pathogens and greenhouse grafting analyses to determine if infected tissue could transmit symptoms to the healthy plants was performed. Two years of molecular diagnostics did not identify a known pathogen associated with all symptomatic plants, and grafting analyses did not show transmission of the disease symptoms to healthy plants. These results suggest that the cause of the mysterious disease-like symptoms in the Columbia Basin is not pathogenic in nature, suggesting that an insect pest or environmental stimuli is likely responsible, providing critical answers to potato growers worried about a spreading disease.
Review Publications
Shock, C., Brown, C., Sathuvalli, V., Charlton, B., Yilma, S., Hane, D., Quick, R.A., Rykbost, K., James, S., Mosley, A., Feibert, E., Whitworth, J.L., Novy, R.G., Stark, J., Pavek, M., Knowles, R., Navarre, D.A., Miller, J., Holm, D., Jayanty, S., Debons, J., Vales, I., Wang, X., Hamlin, L. 2018. TerraRossa: A mid-season specialty potato with red flesh and skin and resistance to common scab and golden cyst nematode. American Journal of Potato Research. 95(5):597-605. https://doi.org/10.1007/s12230-018-9667-8.
Navarre, D.A., Sathuvalli, V., Brown, C. 2019. Potato vitamins, minerals and phytonutrients from a plant biology perspective. American Journal of Potato Research. 96(2):111-126. https://doi.org/10.1007/s12230-018-09703-6.
Bali, S., Patel, G., Novy, R.G., Vining, K., Thompson, A., Brown, C., Holm, D., Porter, G., Endleman, J., Sathuvalli, V. 2018. Evaluation of genetic diversity among Russet potato clones and varieties from breeding programs across the United States. PLoS One. 13(8): e0201415. https://doi.org/10.1371/journal.pone.0201415.
Si, M., Navarre, D.A. 2018. Optimization of hairy root induction in solanum tuberosum. American Journal of Potato Research. 95(6):650-658. https://doi.org/10.1007/s12230-018-9671-z.
Sun, X., Du, M., Navarre, D.A., Zhu, M. 2018. Purple potato extract promotes intestinal epithelial differentiation and barrier function by activating AMP-activated protein kinase. Molecular Nutrition and Food Research. 62(4):1700536. https://doi.org/10.1002/mnfr.201700536.
