Location: Emerging Pests and Pathogens Research
2020 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. The potato cyst nematode, Globodera rostochiensis (golden nematode), is a quarantine pest that poses serious threat to the U.S. potato industry. Although the golden nematode has been confined to New York State for several decades, a new virulent pathotype, Ro2, has emerged, for which control measures are very limited. Differentiation of Ro2 from the common pathotype Ro1, which can be controlled by resistant potato cultivars, still relies on laborious and lengthy bioassay methods. Developing reliable and rapid molecular diagnostic tools for identifying different pathotypes of the golden nematode is necessary to help ensure the continued success of golden nematode quarantine in New York. Researchers in Ithaca, New York previously completed the whole genome sequencing of a collection of Ro1 and Ro2 single cyst lines and identified 297 sequence variations that may be targeted for diagnostic marker development. The Researchers initially analyzed approximately 50 sequence variants using a method called cleaved amplified polymorphic sequence (CAPS) analysis and found that one sequence variant is potentially unique for the Ro2 pathotype. Researchers plan to validate the utility of this unique sequence variant as a diagnostic marker by testing more Ro1 and Ro2 populations from different geographic regions, and analyze the rest of the sequence variants for molecular marker development.
Sub-Objective 1.2: Monitor potato virus Y (PVY) strain diversity in the seed potato crop. This sub-objective has been completed.
Sub-Objective 1.3: Characterize PVY diversity and evolution. This sub-objective has been completed.
Sub-Objective 2.1: Characterize candidate effector protein-encoding genes and their associated host proteins contributing to nematode parasitism and virulence. Plant-parasitic nematodes, including the golden nematode (Globodera rostochiensis) and the pale cyst nematode (G. pallida), are among the most devastating plant pathogens causing significant crop yield losses annually. These nematode pests utilize their secreted proteins (called effectors) to manipulate various host cellular processes for their own parasitic advantage. Identifying host proteins targeted by nematode effectors may uncover the molecular function of nematode effectors in the host plant cell. The obtained knowledge may be utilized to develop novel nematode control strategies. Studies this year focused on a group of effectors that were previously confirmed to have a role in host defense suppression. By using a technique to discover protein-protein interactions and a proteomics approach, several candidate host proteins that may interact with nematode effectors were identified. Further functional characterization of these effectors led to some interesting discoveries. For example, one of the effectors was found to induce a hypersensitive response (which often relates to effector recognition by a resistance gene in the plant) when transiently expressed in the leaves of a wild potato species that was confirmed to have broad- spectrum resistance to potato cyst nematodes (G. rostochiensis and G. pallida). The results suggest that this effector may be recognized by an endogenous resistance gene in the wild potato species. A further study of this important effector may accelerate the cloning of the resistance gene in the potato plant.
Researchers in Ithaca, New York, continued to make progress on the study of nematode secreted CLAVATA3/ESR-like (CLE) effectors, previously demonstrated to function as mimics of plant CLE peptides that regulate many aspects of plant growth and development. Transcriptome analysis of potato lines overexpressing a CLE effector gene (GrCLE1-1 from G. rostochiensis) provided a list of genes encoding receptor proteins and transcription factors that may be regulated by nematode CLE effectors. The researchers are in the process of evaluating a role of the host receptor proteins and downstream components in CLE signaling pathways in nematode parasitism. Studies using microscopic examinations of cross sections of roots of CLE overexpression lines revealed that the CLE effector could cause cellular alterations in root tissues. The results support our previous hypothesis that nematode CLE effectors can activate multiple developmental pathways in host roots by interacting with different receptor proteins. A paper describing these results is under preparation. A further understanding of how CLE effectors function in the host plant cell may allow the development of novel resistance in potato and other crop species. Plant CLE peptides are a group of peptide hormones essential for plant growth and development. Nematode-secreted CLE peptides are unique as they function as a versatile peptide that can simultaneously trigger different developmental pathways in plant cells. This type of CLE peptides has not been identified in any plant species. Thus, a further study of this unique group of nematode CLE peptide may add new knowledge of CLE peptide signaling in plant development.
Sub-Objective 2.2: Characterize infection and transmission competence of PVY strains and strain combinations in potato and aphid populations. This sub-objective has been completed.
Sub-Objective 2.3: Define the mechanism of tissue tropism of poleroviruses in plants and aphid vectors. This sub- objective has been completed.
Sub-Objective 3.1: Determine the resistance of potato clones and wild potato species to PCN pathotypes. Researchers in Ithaca, New York, continue the collaboration with potato breeders at Cornell University and other major U.S. potato breeding programs to screen potato breeding clones for resistance against the golden nematode. A total of one-hundred-and-six (106) potato clones were screened using marker test and pot bioassay and thirty-two (32) of them showed resistance to the golden nematode. These clones will be retested for nematode resistance multiple times and further evaluated in fields by University collaborators until they may be released as a resistant cultivar. Efforts continued to screen wild potato accessions that were requested from the U.S. Potato Genebank for nematode resistance. A list of clones was obtained that showed strong resistance to Ro2, an emerging and more aggressive pathotype of the golden nematode. Tissue culture plantlets of the resistant clones were obtained and their responses evaluated to Ro2 infection. The infection assay revealed that nematodes can penetrate into roots of the resistant clones but they fail to develop after root penetration. The results suggest that these resistant clones may contain a nematode resistance (R) gene(s). More excitingly, some of these clones were found to have resistance to Globodera pallida, a second species of the potato cyst nematode that has been detected in Idaho. These resistant clones are valuable material for downstream identification and cloning of the novel R genes and for use in potato breeding programs to develop durable nematode resistance in U.S. potato cultivars.
Subobjective 3.2: Collaborate with potato breeders to develop genetic markers for phenotypic traits useful in the development of durable virus resistance. This sub-objective has been completed.
Accomplishments
1. Wild potato species have resistance to potato cyst nematodes. The potato cyst nematodes, including the golden nematode and the pale cyst nematode, are quarantine pests important to agriculture. Deploying resistant cultivars is the most effective and sustainable means for combating potato cyst nematodes. ARS scientists in Ithaca, New York, have identified a list of clones that showed broad and robust resistance against multiple kinds of potato cyst nematodes. Potato breeders are using these wild clones to generate new nematode-resistant potato cultivars in the U.S.
Review Publications
Butler, K.J., Chen, S., Smith, J.M., Wang, X., Bent, A.F. 2019. Soybean resistance locus Rhg1 confers resistance to multiple cyst nematodes in diverse plant species. Phytopathology. https://doi.org/10.1094/PHYTO-07-19-0225-R.
Park, J., Hackett, C.A., Dandurand, L., Wang, X., Dejong, W.S. 2019. QTL for resistance to Globodera rostochiensis pathotype Ro2 and G. pallida pathotype Pa2/3 in autotetraploid potato. American Journal of Potato Research. https://doi.org/10.1007/s12230-019-09745-4.
Manohar, M., Tenjo-Castano, F., Chen, S., Zhang, Y.K., Kuman, A., Williamson, V.M., Wang, X., Klessig, D.F., Shroeder, F.C. 2020. Plant metabolism of nematode pheromones mediates plant-nematode interactions. Nature Communications. 11(208). https://doi.org/10.1038/s41467-019-14104-2.
Xu, Y., Gray, S.M. 2020. Aphids and their transmitted potato viruses: Continuing challenges in potato crops. Journal of Integrative Agriculture. 19(2):367-375. https://doi.org/10.1016/S2095-3119(19)62842-X.
