Location: Emerging Pests and Pathogens Research
2019 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. Two pathotypes of Globodera rostochiensis (golden nematode), Ro1 and Ro2, have been detected in New York state. Potato cultivars with resistance to Ro1 are available. Ro2 is a new virulent pathotype, for which resistant cultivars are very limited. Differentiation of the two pathotypes still relies on lengthy and laborious bioassays. Therefore, developing quick and reliable molecular diagnostic methods for nematode identification is urgently needed to help maintain the success of golden nematode quarantine in New York. ARS researchers have successfully completed the whole genome sequencing of a group of Ro1 and Ro2 single cyst lines. The analysis further identified 297 sequence variants that differentiate Ro1 from Ro2. These sequence variants will be validated and tested for molecular diagnostic marker development.
Sub-Objective 1.2: Monitor potato virus Y (PVY )strain diversity in the seed potato crop. Government shutdown precluded the collection of samples in January from the winter test plots. No further experiments were possible or planned.
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. In Ithaca, New York, completed studies investigating the effects of mode of transmission, either by aphid vector or vegetative propagation through tubers, on the evolution of PVY. Results confirmed that each transmission mode can shift the genetic makeup of the virus population. In collaboration with scientists from Spain, manuscripts are being prepared. A separate study investigated how movement of virus between different host plants can affect the genetic makeup of the virus and its ability to cause disease. Host switching can affect the host range of progeny virus. The changes in the virus genome due to host switching are being evaluated and manuscripts are being prepared.
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 (termed effectors) into plant root cells to promote successful infection. Understanding how nematode effectors function in the host cell is necessary for developing novel nematode control strategies. We continued to make progress on the functional characterization of a group of effector genes from the golden nematode. These functional studies have determined that a few effectors target different host cellular structures including nucleus and mitochondria to exert their role in host defense suppression. This knowledge may be utilized to develop durable nematode resistance in U.S. potato cultivars through a plant-mediated RNA interference approach. Researchers also established a proteomics approach for identifying host proteins that interact with nematode effectors and are in the process of characterizing a couple of the identified host proteins to discover the molecular details of the interaction. Researchers continued to make significant progress on understanding the function of the CLE effector family and studies indicated that a subgroup of nematode CLE effectors have a unique function in host plant cells and that multiple receptor proteins in the host are involved in perceiving nematode CLE effectors. Results of this aspect of research suggest targets in the host that may be manipulated for developing novel types of nematode resistance in U.S. potato cultivars.
Sub-Objective 2.2: Characterize infection and transmission competence of PVY strains and strain combinations in potato and aphid populations. The planned studies were completed in 2018. Continuing efforts have focused on how different strains of PVY compete in plants and how this competition affects their ability to be vegetatively propagated through tubers. Although the new recombinant strains will outcompete the older strain of the virus in the foliage of the plant. The older strain is efficiently transmitted along with the new strains to the tubers and will be present in the next year’s crop. Other studies have investigated if the virus transmission protein that acts to bind virus particles to the aphid vector mouthparts is specific only to itself or if it can facilitate the transmission of other virus strains. Findings indicate that the transmission protein from the old PVY strain will facilitate transmission of the new strains, but the reverse is not true. This may be helping to increase the prevalence of the new virus strains and suppress the incidence of the old strain.
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 proteins are involved in virus movement within and between hosts and how they affect disease severity. Studies this year focused on a conserved region of the virus protein involved in movement of the virus in plant hosts. A short 5 amino acid motif controlled how the virus moved long distance in the plant vasculature system. This movement was tied to the ability of the virus to associate with plant structures that allow virus to enter the plant “bloodstream”. Studies on another virus protein involved in suppression of plant defenses identified a single amino acid that regulated whether the virus could defeat plant defenses and proliferate severe disease or if the virus succumbed to plant defenses on only caused mild disease. Understanding how viruses move and cause disease allow for the development of strategies to disrupt the virus lifecycle and manage disease.
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 111 breeding clones with resistance to the golden nematode. These resistant clones will be retested multiple times and are in the pipeline for release as resistant cultivars. Joint efforts with Cornell has led to the release of the FIRST potato cultivar, named ‘Brodie’, which has resistance to both pathotypes (Ro1 and Ro2) of the golden nematode. This new cultivar provides the needed tool for combating Ro2, a more aggressive population, in the infested fields. Researchers have further screened 175 accessions of five wild potato species and identified 44 individual clones that showed resistance to Ro2. It is worth noting that a few clones from wild species Solanum brevicaule and S. boliviense exhibit resistance to multiple populations of potato cyst nematodes. These broad-spectrum resistant germplasm may be utilized by potato breeders to develop durable nematode resistance in U.S. potato cultivars.
Accomplishments
1. Release of potato cultivar with broad-spectrum resistance to the golden nematode. The potato cyst nematodes, including the golden nematode and the pale cyst nematode, are devastating pests for the U.S. potato production valued at approximately $4 billion. Deploying resistant cultivars is the most effective and sustainable means for combating nematode pests. ARS researchers at Ithaca, New York, in collaboration with scientists at Cornell University, have developed and released the first potato cultivar, named ‘Brodie’ that confers resistance to the two pathotypes (Ro1 and Ro2) of the golden nematode. This new resistant cultivar provides the needed tool for controlling Ro2, a more aggressive population, in the field, thereby helping maintain the success of golden nematode quarantine in the U.S.
Review Publications
Fletcher, J., Berenbaum, M., Gray, S.M., Groves, R., Scorza, R., Triplett, L., Trumble, J., Yang, B. 2018. A review of the Citrus Greening research and development efforts supported by the Citrus Research and Development Foundation: Fighting a Ravaging Disease. National Academy Press. 1-270. https://doi.org/10.17226/25026.
Gray, S.M., Power, A. 2018. Anthropogenic influences on emergence of vector-borne plant viruses: the persistent problem of Potato virus Y. Current Opinion in Virology. 33:177-183. https://doi.org/10.1016/j.coviro.2018.10.002.
Xu, Y., Dasilva, W., Qian, Y., Gray, S.M. 2018. An aromatic amino acid and associated helix in the C-terminus of the potato leafroll virus minor capsid protein regulate systemic infection and symptom expression. PLoS Pathogens. 14(11):e1007451. https://doi.org/10.1371/journal.ppat.1007451.
DeBlasio, S.L., Xu, Y., Johnson, R., Rebelo, A., MacCoss, M., Gray, S.M., Heck, M.L. 2018. The interaction dynamics of two potato leafroll virus movement proteins affects their localization to the outer membranes of mitochondria and plastids. Viruses. 10(11):585. https://doi.org/10.3390/v10110585.
Gal-On, A., Fuchs, M., Gray, S.M. 2017. Generation of novel resistance genes using mutation and targeted gene editing. Current Opinion in Virology. 26:98-103.
Gorny, A., Wang, X., Hay, F., Pethybridge, S. 2019. Development of a species-specific PCR for detection and quantification of Meloidogyne hapla in soil using the 16D10 Root-Knot Nematode Effector Gene. Plant Disease. https://doi.org/10.1094/PDIS-09-18-1539-RE.
