Location: Emerging Pests and Pathogens Research
2018 Annual Report
Objectives
Our long-term objective is to develop improved management strategies for the range of pathotypes of the major invasive and emerging nematode and virus pathogens affecting the United States potato crop. While the potato industry is eager to improve cultural and genetic (i.e. resistance) management strategies that can be readily translated to the field, they are also interested in developing fundamental information on emerging pathogens to ensure appropriate and timely detection and the development of novel management strategies. Over the next 5 years we will focus on the following:
Objective 1: Define the genetic diversity and evolution of PCN and virus populations, and optimize associated diagnostic assays. [NP303, C1, PS1]
Sub-Objective 1.1: Compare genome sequences of G. rostochiensis pathotypes (Ro1 and Ro2) to identify sequence variations that may be used for developing molecular diagnostic markers.
Sub-Objective 1.2: Monitor PVY strain diversity in the seed potato crop.
Sub-Objective 1.3: Characterize PVY diversity and evolution.
Objective 2: Discover and characterize genes and proteins regulating virus-vector-host and nematode-host interactions. [NP303, C2, PS2A and PS2C]
Sub-Objective 2.1: Characterize candidate effector protein-encoding genes and their associated host proteins contributing to nematode parasitism and virulence.
Sub-Objective 2.2: Characterize infection and transmission competence of PVY strains and strain combinations in potato and aphid populations.
Sub-Objective 2.3: Define the mechanism of tissue tropism of poleroviruses in plants and aphid vectors.
Objective 3: Develop virus and nematode resistant potatoes that are acceptable to the potato industry and consumers. [NP303, C3, PS3A]
Sub-Objective 3.1: Determine the resistance of potato clones and wild potato species to G. rostochiensis pathotypes.
Sub-Objective 3.2: Collaborate with potato breeders to develop genetic markers for phenotypic traits useful in the development of durable virus resistance.
Approach
In general, nematode parasites and virus diseases of potatoes cause severe crop loss and effective control measures are lacking. Nematicides effective against the Potato cyst nematodes (PCN) are no longer available and alternative control strategies for emerging pathotypes/populations have not been developed. Similarly, virus disease control strategies are completely lacking. New plant biotechnologies will provide the basis for the development of novel methods of nematode and virus control, but the success of these methods will be dependent upon a more complete understanding of the fundamental mechanisms of host-nematode and host-virus-vector interactions. One approach is to define the genetic diversity and evolution of PCN and virus populations, and optimize associated diagnostic assays. Inbreed lines of different races of PCN will be sequenced. Candidate SNPs and other variations indicated to be unique for PCN pathotypes will be further analyzed and converted into PCR-based or other types of markers and finally validated by examining a range of PCN populations. Virus populations and strains will be monitored in the potato crop to identify new recombinants and facilitate diagnostic updates. Effects of vertical and horizontal transmission on virus populations will provide information on selection pressures most important in driving the emergence of new strains. A second approach is to discover and characterize genes and proteins regulating virus-vector-host and nematode-host interactions. Stylet-secreted PCN effector proteins that manipulate multiple host cellular processes to promote successful infection will be discovered and characterized by multiple technologies to better understand the function of these effectors in nematode parasitism and virulence. Virus work with focus on the infection and transmission competence of Potato virus Y (PVY) strains to identify factors that regulate virus acquisition and transmission by aphids and synergistic/antagonistic interactions of PVY strains in plants that limit the availability of virus to aphid vectors. We will continue to investigate how potato leafroll virus protein domains direct long distance virion movement while other domains direct virus cell to cell movement of RNA-protein complexes. The third approach is to develop virus and nematode resistant potatoes that are acceptable to the potato industry and consumers. Working with breeders we will continue to use bioassays and marker assisted selection to evaluate breeding clones and wild potato accessions for nematode resistance and develop genetic markers linked to phenotypic traits caused by PVY infection such as tuber necrosis and leaf necrosis. Conceptually novel information on the population genetics of nematode, aphid and virus pests of potato crops, and on host–pathogen-vector interactions will aid in the development of new effective biologically-based disease control strategies. Improved virus and nematode diagnostics as well as genetic markers for disease resistance and disease resistant cultivars developed through conventional breeding and genetic engineering can be transferred readily to customers.
Progress Report
Sub-Objective 1.1: Compare genome sequences of potato cyst nematode (PCN) pathotypes (Ro1 and Ro2) to identify sequence variations that may be used in molecular diagnostics. Currently there are two pathotypes of Globodera rostochiensis (golden nematode or GN), Ro1 and Ro2, that have been detected in New York state. Ro1 is well controlled by resistant potato cultivars containing the H1 resistance gene. However, Ro2 can overcome H1-mediated resistance, posing a threat to the success of golden nematode quarantine in New York. Developing molecular tools for quick identification of GN pathotypes is urgently needed. Significant progress was made in generating and collecting multiple Ro1 and Ro2 single cyst lines, which are being prepared for genome sequencing at the Genomics Facility of Cornell University. Genome sequences of these lines will be analyzed and compared to discover sequence variations that are important for diagnostic marker development.
Sub-Objective 1.2: Monitor potato virus Y (PVY) strain diversity in the seed potato crop. Over 500 samples were tested from post-harvest test potato plots grow by seed certification programs in 9 states. These were tested for PVY infection and strain composition was determined. The Wilga strain remains the most prevalent (65%), with the tuber necrotic strain (24%), the N:O recombinant (4%) and the ordinary strain (3%) rounding out the field. This data assists seed certification programs and growers to monitor infection levels in the field and assist breeding and pathology programs in the development of appropriate resistance and diagnostics, respectively.
Sub-Objective 1.3: Characterize PVY diversity and evolution. Potato virus Y (PVY) exists as a complex of strains whose composition has shifted dramatically in recent years due to many biological and anthropomorphic factors. We studied how the mode of transmission, either by aphid vector or vegetative propagation through tubers can affect the evolution of PVY. Preliminary results indicate that each transmission mode can shift the genetic makeup of the virus population. In collaboration with scientists from Spain analysis of the data continues to determine the significance of these findings.
A separate study is investigating how movement of virus between different host plants can affect the genetic makeup of the virus and its ability to cause disease.
Sub-Objective 2.1: Characterize candidate effector protein-encoding genes and their associated host proteins contributing to nematode parasitism and virulence. Plant-parasitic nematodes secrete proteins, known as effectors, into plant root cells to promote infection. Understanding how nematode effectors function in the host cell may suggest novel methods for control of nematode pests. Significant progress was made on the functional characterization of three large effector families (GrSPRYSEC, Gr29D09 and GrCLE) from Globodera rostochiensis (golden nematode). Several new GrCLE effector genes were identified and cloned from various nematode populations. The receptors that these GrCLE proteins bind in potato were identified. RNA interference and genome editing technologies were used to alter or turn off these receptors in potato plants and those plants had enhanced resistance to nematode infection. The two other effector families (Gr29D09 and GrSPRYSEC) were demonstrated to have an important role in suppressing host defenses. Potato proteins that interact with Gr29D09 effectors were identified. Together, these results show that specific potato proteins interact with nematode effectors and that genetic manipulation of those host proteins is not detrimental to the potato plant, but can block their use by the nematode effectors and provide novel resistance to potato cyst nematodes.
Sub-Objective 2.2: Characterize infection and transmission competence of PVY strains and strain combinations in potato and aphid populations. Often multiple Potato virus Y (PVY) strains are found in the same potato field and occasionally within the same plant, but little is known about how strains of the virus interact or compete in the plant or in the aphid vector. The spatial and temporal dynamics of mixtures of PVY strains was examined in epidermal leaf cells of tobacco and potato leaves and in aphid mouthparts following virus acquisition. Two strains can systemically infect and co-localize in plant cells, but the rate of movement and their distribution in leaves changes over time so that both viruses would not always be available to aphids feeding on the infected plants. In general, the emerging recombinant strains of the virus tend to outcompete the ordinary strain of PVY that is becoming less common in potato fields. Two strains of PVY were also detected in the distal end of aphid mouthparts following virus acquisition from a plant infected with a strain mixture. Our data contrast the dogma of spatial separation of two closely related viruses and the antagonistic interaction between PVY strains we characterized could help explain the multitude of emerging recombinant PVY strains discovered in potato in recent years.
Sub-Objective 2.3: Define the mechanism of tissue tropism of poleroviruses in plants and aphid vectors. Potato leafroll virus (PLRV) and its relatives are responsible for economically significant disease in potato and other staple food crops. There is limited effective resistance in crops affected by these viruses. Management is limited to insecticidal control of vectors that is only partially effective. This study was designed to investigate how the virus regulates the expression of its proteins that control virus movement within and between hosts. Understanding these mechanisms identifies potential targets for controlling these viruses. We found protein expression is controlled by the interaction of two distant areas of the virus RNA genome, but the RNA folds into a complex 3-dimensional form, these two regions are in close proximity and bind together allowing the proteins to be produced. Small changes in either of the two regions can compromise their ability to interact and to produce the correct proteins. The result is a virus that does not move efficiently in its host and therefore it is not available to be transmitted by its vector to other susceptible hosts. All of the viruses related to PLRV have similar genome structures and function and therefore a single strategy to disrupt the interaction of these regions may provide a common management tool.
Sub-Objective 3.1: Determine the resistance of potato clones and wild potato species to PCN pathotypes. Collaborations with Cornell University and other major U.S. potato breeding programs identified 142 breeding clones with resistance to G. rostochiensis (golden nematode). These resistant clones will be retested in following years and are in the pipeline for release as resistant cultivars. Our joint effort with the University has led to the release of two new golden nematode resistant cultivars, ‘Algonquin’ and ‘Upstate Abundance‘, both of which are early season table-stock cultivars. Golden nematode resistant cultivars, most of which were jointly developed with Cornell University, now account for more than 7% of U.S. potato acreage. This widespread adoption of these resistant cultivars has offered an effective method that prevents a further spread of the golden nematode within the U.S. We also screened around 60 wild potato accessions and identified a few accessions in three wild potato species that exhibit resistance to multiple populations of potato cyst nematodes. This new resistant germplasm may be introduced into potato breeding programs for developing desirable potato cultivars with durable resistance to potato cyst nematodes.
Sub-Objective 3.2: Collaborate with potato breeders to develop genetic markers for phenotypic traits useful in the development of durable virus resistance. Genome analyses identified distinct regions on chromosomes 4 and 5 that have major effects on symptom expression. Quantitative trait loci for tuber necrosis and foliar mosaic were found near each other on chromosome 4, suggesting that markers diagnostic for specific haplotypes of this region may prove useful for breeders who want to select genes that confer resistance to infection and/or determine susceptibility to tuber necrosis. This work has been published and no further experiments are planned.
Accomplishments
1. Joint effort resulted in the release of two new resistant potato cultivars. Potato cyst nematodes (PCN) are serious pests for the U.S. potato production valued at $4 billion. Host resistance is the most effective and environmentally-sound method for PCN control. ARS researchers at Ithaca, New York, in collaboration with scientists at Cornell University have developed and released two new golden nematode resistant potato cultivars, ‘Algonquin’ and ‘Upstate Abundance’, both of which are early season table-stock cultivars. These cultivars can serve as an effective tool for PCN control and eradication in the U.S. and can be utilized by potato breeders as parent material in crosses to develop more resistant cultivars.
Review Publications
Gorny, A., Hay, F., Wang, X., Pethybridge, S. 2018. Isolation of nematode DNA from 100 grams of soil using Fe3O4 super paramagnetic nanoparticles. Nematology. 20:271-283.
Park, J., Yang, H., Dejong, W.S., Wang, X. 2017. An evaluation of two H1-linked markers and their suitability for selecting Globodera rostochiensis resistant potatoes in the New York breeding program. American Journal of Potato Research. 95:170-177.
Guo, X., Wang, J., Gardner, M., Fukuda, H., Kondo, Y., Etchells, P., Wang, X., Mitchum, M.G. 2017. Identification of cyst nematode B-type CLE peptides and modulation of the vascular stem cell pathway for feeding cell formation. PLoS Pathogens. 13(2):e1006142.
Mondal, S., Gray, S.M. 2017. Sequential acquisition of Potato virus Y strains by Myzus persicae favors the transmission of the emerging recombinant strains. Virus Research. 241:116-124. https://doi.org/10.1016/j.virusres.2017.06.023.
DeBlasio, S.L., Rebelo, A., Parks, K., Gray, S.M., Heck, M.L. 2018. Disruption of chloroplast function through downregulation of phytoene desaturase enhances the systemic accumulation of an aphid-borne, phloem-restricted virus. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-03-18-0057-R.
Da Silva, W., Ingram, J.T., Hackett, C., Coombs, J., Douches, D., Bryan, G., Dejong, W., Gray, S.M. 2017. Mapping loci that control tuber and foliar symptoms caused by PVY in Autotetraploid Potato (Solanum tuberosum L.). G3, Genes/Genomes/Genetics. doi: 10.1534/g3.117.300264.
Couture, J.J., Singh, A., Charkowski, A., Groves, R., Gray, S.M., Bethke, P.C., Townsend, P. 2018. Integrating spectroscopy with potato disease management. Plant Disease. https://doi.org/10.1094/PDIS-01-18-0054-RE.
