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Research Project: Potato Germplasm Improvement for Disease Resistance and Superior Nutritional Content

Location: Temperate Tree Fruit and Vegetable Research

2017 Annual Report

Objective 1: Apply genetic analyses, metabolic engineering, and targeted metabolic profiling to elucidate genetic, molecular, and biochemical factors governing host disease resistance and accumulation of select phytonutrients and vitamins in potatoes. Sub-objective 1.A. Characterize molecular and biochemical factors that modulate phytonutrient content. Sub-objective 1.B. Characterize molecular and biochemical mechanisms involved in disease/pest resistance. Objective 2: Evaluate, breed, and release potato germplasm with increased amounts of phytonutrients, which are suitable for the processing and fresh potato market, as well as for niche markets. Objective 3: Identify and release germplasm or varieties with improved resistance to powdery scab, black dot, Columbia root-knot nematode, zebra chip, potato mop top virus, potato cyst nematode, and examine the role of micronutrients in host resistance to Verticillium wilt. Sub-objective 3.A: Nematodes: Focus on identifying and developing germplasm, including trap crops, that can provide superior control options for Columbia root-knot nematode or Potato Cyst Nematode. Sub-objective 3B: Soil borne pathogens: Develop superior germplasm or management options for soil borne pathogens including powdery scab, potato mop top virus, black dot and Verticillium wilt. Objective 4: Determine available host-plant resistance and epidemiological parameters, and develop diagnostic tests for emerging pests and pathogens of potato such as zebra chip.

Objective 1: We will utilize molecular physiology approaches, including measuring gene expression, enzyme activity and metabolite pools by hyphenated techniques. Structural genes and regulatory genes will be assessed using transient assays or stable transgenics. The phenylpropanoid pathway will be a focus. HQT expression will be reduced using RNAi. LCMS will be used to assess differences in phenylpropanoids between wild type and silenced lines and the expression of at least 10-20 phenylpropanoid genes measured by qPCR. Another gene targeted for silencing will be dihydroflavonol-4-reductase (DFR). LCMS and GCMS analysis will be used to examine how phenylpropanoid and primary metabolism is reprogrammed in plants with altered DFR metabolism. MYB transcription factors will be identified in silico based on phylogenetic and protein similarity with known transcription factors. Function will be assessed in transient and stable assays. Compounds that cause the hatching of potato cyst nematode eggs will be partially purified from root extracts using chromatographic methods. Objective 2: Tuberling populations will be assembled and grown two successive seasons in the Klamath Basin of Oregon in unreplicated plots. Promising material will be analyzed for carotenoids, anthocyanins, antioxidants, and a range of other metabolites to select clones with high phytonutrient content. Statistically the data will be analyzed as a mixed model with locations, clones and interaction as fixed effects and reps within locations as random effects. We will use molecular markers to characterize hybrids and assure that we intercross only duplex Zep1 hybrids. Objective 3: We will combine PVY extreme resistance and CRKN resistant germplasm. The genetic nature will be explored by determining segregation ratios in reciprocal crosses. Mitochondrial fingerprinting will be expulsed as a diagnostic genetic marker of the restored phenotype. Crosses will be made to select a less spiny version of Solanum sisymbriifolium for use as a PCN trap crop. A. rhizogenes will be used to attempt to make a version of the plant with greater root mass. Hatching assays will be used to screen for other plants that may be a superior PCN trap crop. Crosses will be made to generate potatoes with resistance to Black dot and Powdery scab and evaluated in field trials with a randomized complete block design with four replications and ten plants per replication. The crown and root will be scored for degree of galling and sclerotia. The effect of micronutrient supplements on Verticillium wilt resistance will be assessed in field and greenhouse trials. Macro and micronutrients will be applied in-furrow. Objective 4: Psyllids collected during the survey and additional insects collected in the Pacific Northwest will be subjected to high resolution melt (HRM) analysis of the cytochrome oxidase gene in order to differentiate genetic variants of the psyllid. Extracts will be tested by PCR methods reported in the literature at dilutions up to 1,000 to determine level of sensitivity and reliability of the various methods on different host plant tissues.

Progress Report
Some estimate that global agricultural yields must double by the year 2050 in order to meet projected demand. Our research enhances food security by identifying and developing potato germplasm with superior disease and pest resistance and nutritional qualities, along with identifying mechanisms that control these traits. Objective 1 addresses the lack of knowledge about fundamental mechanisms that regulate potato disease resistance or nutritional value. This deficiency slows down the development of new cultivars with superior disease resistance, flavor, appearance, and dietary value. Sugars play an important role in controlling phenylpropanoid content. New data strongly supports a role for sugars in actual cropping conditions, not just in the lab or greenhouse experiments. High phenylpropanoid tubers would be expected to have superior nutritional value, disease resistance, and potentially better flavor and appearance. A potential negative for these potatoes is the possibility of more browning and/or bruising, which might be expected in a high phenylpropanoid potato. Phenylpropanoid content and other metabolites have been characterized in different potato genotypes to more accurately determine the factors that regulate browning and bruising in tubers. Preliminary results suggest a lack of surprising correlation between phenylpropanoids, browning enzymes, and discoloration. This suggests that discoloration may be more complex than typically thought, and that other components besides the usual suspects have a sizeable role. Breeders can produce many more breeding lines that can be phenotyped for traits like nutritional value. We have been optimizing methods designed to improve the speed of analysis and increase the amount of trait information that can be gained from each sample. Together, these result in a higher efficiency of analysis. This approach has been used to more completely phenotype advanced breeding lines. Results showed a considerable range of phytonutrient content among potato germplasm and helps establish what reasonable phytonutrient targets are for breeding programs. Potato Cyst Nematode (PCN) is a quarantine pest that can persist in the soil for 30 years and is extremely difficult to eliminate from farmland. Potatoes produce compounds called hatching factors that stimulate PCN eggs in the soil to hatch. If these hatching factors were better understood, they could be used to eradicate PCN by causing a “suicide hatch”, but nothing is known about the biochemical pathway that potatoes use to synthesize hatching factors. To identify the metabolic pathway involved, we cloned several candidate genes, which were then used to attempt to increase or decrease the target pathway. Over seven types of transgenic potatoes were generated to allow us to evaluate the effect on the pathway. Preliminary results, such as differences in the phenotyes, show the potatoes were successfully transformed and that the pathway was altered. Further testing will confirm that the pathway flux was successfully reprogrammed, and will determine the effect of altering the pathway on the overall potato phenotype and hatching factor production. Plant diseases threaten food security by reducing yield and quality, and reduce grower profitability. Plants use complicated, not fully understood, mechanisms to resist disease. Working with scientists at the University of Kentucky, a group of plant proteins were identified that bind ribonucleic acid (RNA) and have key roles in how plants resist bacterial and viral diseases. These proteins are a part of a complex plant response to disease that ultimately can result in the plant resisting disease. As mechanisms like these are discovered, it will become increasingly possible to produce plants with superior disease resistance and preserve plant yields, reduce pesticide inputs and protect profitability. For objective 2, with collaborators in the TriState Breeding Program, new potato crosses were made and evaluated in field trials. One new release is Castle Russet, an attractive russet potato with good resistance to Tobacco rattle virus (TRV), potato virus Y (PVY) and Potato mop top virus (PMTV). Advanced breeding lines were harvested from fields in Oregon, Washington and Idaho, then processed and screened for phytonutrients including phenolics, antioxidants, protein, chlorogenic acid, vitamin C, vitamin B6 and B9, potassium, zinc, iron and magnesium, as well as disease resistance to TRV and PMTV. For objective 3 and 4, new advanced germplasm with insensitivity to TRV was identified. We collaborated with the University of Idaho in the exposure of four standard oblong russets to TRV and monitored the development of internal symptoms and increase in detectability of TRV in tubers that were symptomless at the beginning of cold storage. Approximately 20 percent of a progeny of the cross POR06V12-3 x POR08BD1-3 showed extreme insensitivity in a field exposure to TRV. POR06V12-3 is an advanced breeding line with extreme insensitivity to TRV. TRV infection, not accompanied by internal symptoms, is a common phenomenon which increases over a period of nine months of storage. We detected three percent symptomless infection by PMTV in four hundred seed samples from seed growers. In collaboration with ARS scientists in Corvallis, Oregon, the presence of Paratrichodorus minor, a potential new vector of TRV, was detected in the state of Oregon. An initial attempt to transfer TRV to tobacco via P. minor failed to transmit virus. The identity of P. minor was confirmed using polymerase chain reaction amplification patterns of an internal transcribed spacer region. These findings show that both TRV and PMTV insensitivities should be combined with resistance to virus infection as a breeding goal to enhance resistance and avoid future varieties running into regulatory barriers in international trade. In order to assess virus transmission under natural conditions, work to assess the role of virus titer in Tobacco rattle virus transmission through seed potato in a large field trial was begun. Ten potato lines representing those resistant (tolerant) and those susceptible to TRV with varying disease symptoms in the tuber were planted, and emergence dates are currently being observed and recorded. Leaf tissue from each emerged plant has been collected to detect any virus in the foliage, and at the end of the growing season, the daughter tubers will be harvested, scored for disease symptoms, and viral titer will be assessed for each treatment. Testing is currently underway to assess the role of the potato psyllid insect in transmission of the beet leafhopper transmitted virescence agent phytoplasma, a bacteria-like pathogen of potatoes. A source of phytoplasma-positive potato plants has been acquired, and potato psyllids maintained in a laboratory colony have been released on these plants. Preliminary studies indicate that the potato psyllid can acquire the pathogen, albeit at low levels. This is the first step in understanding the role of the insect pest in phytoplasma disease transmission, and will provide beneficial information to growers trying to prevent the spread of phytoplasma in their fields. In addition to enhancing food security, this work will protect grower profitability by improving disease and pest resistance, and provide consumers and growers additional options for new types of potatoes that can satisfy evolving consumer preferences.

1. A new type of Tobacco rattle virus (TRV) host-resistance. TRV is vectored by the stubby root nematode and can cause severe internal discoloration of tubers. ARS researchers in Prosser, Washington, identified a breeding line with an unusual type of host resistance that shows no internal necrosis, does not have asymptomatic infection, and prevents the nematode from maintaining its infectiousness by converting the nematode to an aviruliferous state. Analysis of progeny from a cross of this breeding line found that approximately twenty percent of the progeny displayed this high level of resistance. The heritability of this type of resistance will permit the breeding of future varieties with strong TRV resistance. It will also reduce the TRV levels in the nematode vector and mitigate the effect on yields, quality and marketability.

2. More efficient analysis of potato breeding lines. Bottlenecks for potato cultivar development include the need for faster methods of phenotyping the large number of lines generated by breeding programs. ARS scientists in Prosser, Washington, optimized ways to more quickly assess a greater number of traits in advanced breeding lines; including minerals, phytonutrients, protein, browning, bruising, glycoalkaloids and disease resistance. Using the same set of potatoes, these methods were used to show over a 2 to 5 fold difference occurs in most minerals and phytonutrients among the different breeding lines. Concurrently, these methods were used to determine whether multiple tuber components predispose a line to browning or bruising. This work will facilitate the development of new potato cultivars with superior quality, phytonutrient content, and disease resistance, protect grower profitability and provide increased choices for consumers.

3. Asymptomatic occurrence of potato mop-top virus (PMTV) in seed potatoes. PMTV causes serious internal tuber discoloration, is impacting exports, and is becoming a disease of growing concern for the North American potato industry. By screening numerous cultivars in certified seedlot trials planted by Washington State University scientists in 2016 and 2017, ARS researchers in Prosser, Washington, discovered over 2 percent of tested seedlots were positive for PMTV, despite showing no PMTV symptoms. These results demonstrate a means by which previously clean fields can become infected with PMTV, and show that selecting for the absence of PMTV symptoms in breeding lines is not a reliable identifier of resistant lines. These findings establish a need to breed varieties that are truly resistant, not just asymptomatic, and show that the current methods of producing certified seed can fail to detect PMTV.

4. Insect-vectored pathogens in solanaceous crops. Psyllid and leafhopper insects are known to transmit pathogens to many crops, including solanaceous plants like potatoes, tomatoes, and peppers that impact production globally. ARS researchers in Prosser and Wapato, Washington, in collaboration with scientists at the Institudo Nacional de Investigaciones Forestales, Agricolas y Pecuaries (INIFAP) or the National Institute of Forestry, Agriculture, and Livestock Research, in Mexico, assessed symptomatic pepper plants collected from central Mexico for the presence of several common pathogens transmitted by psyllids and leafhoppers. Several pathogens were detected within each field, and surprisingly, several pathogens were detected in individual plants within a field. Similarly, the psyllid and leafhopper insect vectors also contained mixed-infections. These results suggest that mixed-infections are more common than previously thought. Growers in Central and North America will need to effectively monitor and control the psyllids and leafhoppers to prevent the decline of their crops and any economic losses that may result from these pathogens.

5. Detection of Beet Leafhopper-Transmitted Virescence Agent (BLTVA) in solanaceous crops. Plant pathogens of pepper and other solanaceous crops like potato and tomato are a serious threat to production in the U.S. and around the world. ARS researchers in Prosser and Wapato, Washington, in collaboration with scientists at Institudo Nacional de Investigaciones Forestales, Agricolas y Pecuaries (INIFAP) or the National Institute of Forestry, Agriculture, and Livestock Ressearch, in Mexico, assessed whether pathogens were associated with diseased pepper plants found in commercial fields in the states of Durango and Zacatecas, Mexico. A specific strain of group 16SrVI-A phytoplasma, known as BLTVA, was identified in the symptomatic pepper plants. BLTVA is transmitted to pepper by the beet leafhopper insect, and samples of this insect collected in and around pepper fields in Zacatecas, Mexico, were also positive for BLTVA. This pathogen is one of economic importance in solanaceous crops in North America, and its presence in peppers and beet leafhoppers suggests that growers will need to effectively monitor and control the insect vector to decrease overall disease pressure and the economic losses that result.

Review Publications
Bali, S., Sathuvalli, V., Brown, C.R., Novy, R.G., Ewing, L., Debons, J., Douches, D., Coombs, J., Navarre, D.A., Whitworth, J.L., Charlton, B., Yilma, S., Shock, C., Stark, J., Pavek, M., Knowles, R. 2017. Genetic fingerprinting of potato varieties from the Northwest Potato Variety Development Program. American Journal of Potato Research. 94:54-63.
Brown, C.R., Haynes, K.G., Moore, M., Pavek, M., Hane, D., Love, S., Novy, R.G., Miller Jr., C. 2014. Stability and broad-sense heritaibility of mineral content in potato: copper and sulfur. American Journal of Potato Research. 91:618-624.
Davies, L., Brown, C.R., Elling, A. 2015. Calcium is involved in the RMc1(blb)-mediated hypersensitive response against Meloidogyne chitwoodi in potato. Plant Cell Reports. 34:167-177.
Swisher, K.D., Munyaneza, J.E., Velasquez-Valle, R., Mena-Covarrubias, J. 2017. First report of BLTVA phytoplasma in Capsicum annuum and Circulifer tenellus in Mexico. Plant Disease. 101(6):1032. doi:10.1094/PDIS-12-16-1723-PDN.
Cress, B.F., Leitz, Q.D., Kim, D.C., Amore, T.D., Suzuki, J.Y., Linhardt, R.J., Koffas, M.A. 2017. CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production. Microbial Cell Factories. 16:10. doi:10.1186/s12934-016-0623-3.
Dinh, P., Zhang, L., Mojtahedi, H., Brown, C.R., Elling, A. 2015. RNA interference of effector gene 16D10 leads to broad meloidogyne resistance in potato. Journal of Nematology. 47:71-78.
Ramesh, S., Raikhy, G., Brown, C.R., Pappu, H. 2014. Complete genomic characterization of potato mop-top virus isolate from the United States. Archives of Virology. 159(12):3427-3433.
Singh, R.K., Navarre, D.A., Brown, C.R. 2016. Relationship between sugars and phenylpropanoids in tubers from diverse genotypes. American Journal of Potato Research. 93:581-589.
Boydston, R.A., Navarre, D.A., Collins, H.P., Chaves-Cordoba, B. 2017. The effect of nitrogen rate on vine kill, tuber skinning injury, tuber yield and size distribution, and tuber nutrients and phytonutrients in two potato cultivars grown for early potato production. American Journal of Potato Research. 94:425-436. doi:10.1007/s12230-017-9579-z.