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ARS Home » Pacific West Area » Wapato, Washington » Temperate Tree Fruit and Vegetable Research » Research » Research Project #425150

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

Location: Temperate Tree Fruit and Vegetable Research

2016 Annual Report


Objectives
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.


Approach
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
Given projections that agricultural yields must double by 2050 to meet future demand, food security is a national and global priority. As a staple food that provides a higher energetic yield per acre than any other major crop, potatoes have a central role in providing food security. Our research improves food security by identifying and developing potato germplasm with superior disease/pest resistance and nutritional qualities, along with identifying mechanisms that control these traits. For objective 1, we researched fundamental mechanisms that regulate potato disease resistance or nutritional value, because the lack of such knowledge slows down the development of new cultivars with superior resistance, flavor, appearance, and dietary value. We hypothesized that sugars play an important role in controlling phenylpropanoid content and our new data strongly supports a role for sugars in actual cropping conditions, not just in the lab or greenhouse experiments. Tuber sugars and phenylpropanoids were measured in over 100 different potato samples representing different genotypes, developmental stages, locations, and after Zebra chip infection or wounding. We used this data to develop a model for how genes and proteins in the sucrose and phenylpropanoids pathways interact to regulate each other. These findings create new opportunities to understand how sucrose influences tuber quality, and to develop potatoes with higher amounts of phenylpropanoids to enhance nutritional value, flavor, appearance, and stress and disease resistance. 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 and made potatoes with altered metabolic pathways that will be used to see if we can determine how hatching factors are made by potatoes. Plants use complicated, not fully understood, mechanisms to resist disease. We showed in increasing detail how multiple signals including salicylic acid, nitric oxide, fatty acids, and phenylpropanoids function in plants to regulate resistance. Salicylic acid is a key regulator of plant defenses that can be synthesized by two different pathways, only one of which was thought to be important for disease resistance. In a cooperative effort with University of Kentucky scientists, we showed that both pathways are essential for resistance to the pathogens Pseudomonas and Phytophthora. Signals that plants use to regulate defense responses must travel throughout the plant, and we determined azelaic acid and glycerol-3-phosphate are transported via the symplastic route and regulated by channels known as plasmodesmata. In contrast, salicylic acid moved via the apoplast. For objective 2, with collaborators in the TriState Breeding Program, new specialty potatoes were released with higher than average amounts of phytonutrients. Two new releases are Yukon Nugget, a yellow flesh potato with carotenoids and more antioxidants than Yukon Gold, and Smilin’ Eyes, a high-antioxidant yellow-flesh potato. As we work towards developing new cultivars with superior traits such as disease resistance, we screened numerous promising breeding lines to ensure the nutritional qualities of new breeding lines are not inadvertently lowered in the effort to improve other traits. This ensures that the phytonutrient content will be protected, if not increased, in new lines. To assist in the germplasm development, we tested various methods for their potential to allow a greater number of breeding lines to be screened for phytonutrients. Generally, phytonutrient screening is expensive and time-consuming, which limits the number of lines that can be screened. We tested higher throughput methods such as analyzing expressed juice from tubers in microplate assays. One of the most labor intensive parts of phytonutrient analysis is processing potatoes to get them ready for analysis. Thus, the more information one can get from a single sample, the more useful the analysis will be. Therefore, we are testing ways to use the same potato samples that are being analyzed for phytonutrients to concurrently test their resistance to browning/blackening/bruising and tuber soft rot. Results look promising and appeared to be providing useful information about discoloration and factors that contribute to browning/bruising. We found high polyphenol and high polyphenol oxidase (PPO) tubers that are resistant to discoloration, suggesting the mechanism of browning appears more complicated than traditionally assumed. We have a special interest in specialty potatoes because these potatoes are valued by consumers for their taste, appearance and nutritional value. By focusing on such traits, our program is a consumer-oriented project. Restauranteurs prefer fingerling type potatoes which are more elongate and narrow than those presently available in the market. With the help of the Potato Germplasm Collection at the ARS Potato Introduction Station (USDA/ARS, NRSP-6 Potato Genebank) in Sturgeon Bay Wisconsin, we grew out germplasm directly from collections made in South America that have a high frequency of highly elongate tuber shape. We identified fifty new selections meeting this criterion, from diploid, triploid, and tetraploid indigenous cultivars. Because these newly identified accessions are expected to lack adaptation to long days, they are being crossed with long day adapted specialty breeding lines with red skin and dark yellow flesh. For objectives 3 and 4, we worked closely in multi-state trials with other members of the Tri-State Potato Breeding Program, including university and industry personnel, to evaluate and develop potato varieties with superior disease resistance. Tobacco Rattle Virus (TRV) and Potato Mop-Top Virus (PMTV) are vectored by the stubby root nematode and the powdery scab fungus, respectively. After exposure to infested fields, potatoes showed a diverse range of internal necrosis that was highly variable between genotypes and location. In addition, significant numbers of tubers without visible symptoms were found to be TRV positive by PCR (Polymerase Chain Reaction). We found resistant potato genotypes that had few or no symptoms and importantly, were not latent carriers of these viruses. To assess potential changes in TRV titers that occur during storage, we assayed four varieties for TRV at harvest and after storage. The number of TRV positive tubers increased six-fold during storage in the samples exposed to infection in North Dakota plots. The same varieties (from the same seed source) exposed to virus in Washington State presented a much higher initial detection rate rising from sixty to ninety-five percent. This increase in TRV positive tubers was not correlated with poorer fry quality and did not appear to result in increased symptoms. However, it was clear that potatoes exposed to TRV harbored it in greater amounts after storage than at harvest. New TRV varieties will be the most useful if they also have no or very low occurrence of infection in the absence of symptoms. Castle Russet (POR06V12-3), a new line being released from a Prosser cross, has a very low incidence of symptomless infections. For additional screening, advanced breeding lines and varieties were planted in two fields, one with a history of PMTV and the other infested with TRV. The PMTV field resulted in a light infection, while the TRV field resulted in extremely high levels of internal defects. The cultivar Bintje and several breeding lines were among the highly resistant genotypes in the TRV test. A cross of one of these was made and several hundred seedlings were collected from a grow-out in Klamath Falls to determine the inheritance of resistance to TRV and to allow a genomics approach to identify resistance genes in collaboration with scientists at Oregon State University. One breeding line, POR06V12-3, displayed the highest resistance yet seen to PMTV, including a virtual lack of symptomless PMTV infection. TRV has been historically present in the Northwest, but recently the sudden appearance and long persistence of TRV, even after prolonged absence of potato cropping in fields, have led to questions of how it is introduced and maintained. Conversely, PMTV is a more recent problem resulting from the introduction and spread of its vector, Powdery Scab. We screened for both viruses in 350 seedlots intended for planting in Washington State, the first time a study for seed-borne TRV and PMTV has been carried out. We found that 0.5 % and 1.0 % of samples were positive for TRV and PMTV, respectively. This information suggests seed-borne infection has the potential to spread these diseasesand may support the need for establishment of testing protocols in the State-run potato seed certification agencies. This is a particularly important finding for PMTV, given the extreme difficulty in eradicating it once it is present in a field. Finally, after being short a scientist for two years since our virologist retired, our team gained a new plant pathologist, spring 2016.


Accomplishments
1. Developing high-phytonutrient potatoes. Health professionals and consumers are placing increased importance on the nutritional value of foods, and nutrition is also a key component of food security. ARS scientists in Prosser, Washington, developed improved methods to screen germplasm for phytonutrients, identified regulatory mechanisms that control tuber phytonutrient content, developed new advanced breeding lines with higher amounts of phytonutrients, and with collaborators in the TriState Breeding Program released new high-phytonutrient cultivars. Lab and field research provided additional evidence that sucrose regulates tuber phytonutrient content and allowed development of a model for how genes and proteins in the sucrose and phenylpropanoid pathways interact to regulate each other and consequently effect nutritional value, flavor, appearance, stress and disease resistance. This research helps growers and industry produce potatoes that meet evolving consumer food preferences, thereby helping to protect grower profitability and tens of thousands of potato related jobs.

2. New breeding line resistant to Tobacco Rattle Virus (TRV). TRV, a soil-borne disease of increasing importance, is vectored by a nematode ubiquitous in the Pacific Northwest, the stubby root nematode. ARS scientists in Prosser, Washington, developed a new breeding line, POR06V12-3 (V12-3) with extreme resistance to TRV. In collaboration with scientists from AgNema, a Washington company, V12-3 was tested in pot experiments and found to greatly reduce TRV occurrence in susceptible potato cultivars planted subsequent to V12-3 planting. This is the first example of a resistant potato breeding line counteracting the pathogen so that a following planting with a susceptible potato cultivar was free of TRV. The extreme resistance of V12-3 to TRV can reduce the amount of potatoes culled for quality defects, reduce use of nematicides and soil fumigants, and potentially lower the TRV pressure in fields allowing use of common cultivars that are susceptible to TRV.

3. Detection of two potato viruses in seed. Because potatoes are planted from vegetatively propagated seed tubers, they are susceptible to pathogens transmitted through seed, a liability minimized by State administered certified seed programs that ensure that seedstock is free of pathogens. ARS scientists at Prosser, Washington, and collaborators at Washington State University, examined over 350 seedlots for the presence of Tobacco Rattle Virus (TRV) and Potato Mop Top Virus (PMTV) and found that 0.5 % and 1.0 % of seed pieces tested harbored TRV and PMTV, respectively. This is the first time that the presence of these two viruses has been confirmed in potato seed, and is particularly important for PMTV, which is vectored by powdery scab, a pathogen that is extremely difficult to eradicate once introduced into a field. The detection of these two viruses in potato seed provides information that State certification agencies can use to evaluate whether screening for these viruses should be added to the list of pathogens currently being screened in seed.


ARS scientists in Wapato, Washington, mentored research projects by two undergraduate students for the Research for Undergraduates (REU) program at Heritage University, a predominantly Hispanic-serving liberal arts college located on the Yakama Indian Reservation. The projects consisted of 1) isolating and characterizing the salivary proteins of pear psylla, and 2) assessing the effects of Phytoplasma infection on pear psylla behavior.


Review Publications
Bamberg, J., del Rio, A., Navarre, D.A. 2016. Intuitive visual impressions (cogs) for identifying clusters of diversity within potato species. American Journal of Potato Research. 93(4):350-359. doi: 10.1007/s12230-016-9508-6.
Moehninsi, M., Navarre, D.A., Brown, C.R. 2015. Phytonutrient analysis of Solanum sisymbriifolium Lam. berries. Journal of Food Composition and Analysis. 44:73-79.
Lim, G., Shine, M.B., De Lorenzo, L., Yu, K., Weier, C., Navarre, D.A., Hunt, A.G., Lee, J., Kachroo, A., Kachroo, P. 2016. Plasmodesmata localizing proteins regulate transport and signaling during systemic acquired immunity in plants. Cell Host and Microbe. 19:541-549.
Horton, D.R., Cooper, W.R., Munyaneza, J.E., Swisher, K.D., Echegaray, E., Murphy, A., Rondon, S., Wohleb, C., Waters, T., Jensen, A. 2015. A new problem and old questions: potato psyllid in the Pacific Northwest. American Entomologist. 61(4):234-244.
Swisher, K.D., Velasquez-Valle, R., Mena-Covarrubias, J., Munyaneza, J.E. 2016. Occurrence and molecular detection of Spiroplasma citri in carrots and Circulifer tenellus in Mexico. Journal of Plant Pathology. 98:355-360.