Location: Emerging Pests and Pathogens Research
2022 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
This project terminated on February 25, 2022, and this is the final report summarizing the project.
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 or GN), is a quarantine pest that poses a serious threat to the U.S. potato industry. Two GN pathotypes, Ro1 and Ro2, currently exist in infested potato fields in New York. Ro1 is effectively controlled by resistant potato cultivars containing the H1 resistance gene. However, Ro2 can reproduce on H1-
containing potato cultivars, and control measures for this emerging pathotype are very limited. Determining the presence of Ro2 from the endemic pathotype Ro1 still relies on laborious bioassays that take almost twelve months to achieve a reliable determination. Thus, developing reliable and rapid molecular diagnostic tools for
identifying different pathotypes of GN is urgently needed to help ensure the continued success of GN quarantine in New York. We successfully completed the whole genome sequencing of a collection of Ro1 and Ro2 single cyst lines and identified about 300 sequence variations that may be targeted for diagnostic marker developed. A paper describing the results of the work was published. The researchers further analyzed most of the sequence variants using dCAPS (derived cleaved amplified polymorphic sequences) and PCR assays and identified four sequence variants indicated to be good candidates for diagnostic marker development. Subsequent analysis of the four variants by testing a group of GN populations originated from New York, Scotland, France, Germany, Netherlands, Hungary, Portugal, and Lebanon, determined that two of the sequence variants can be used as diagnostic markers that differentiate the two GN pathotypes. These molecular diagnostic tools may be introduced into the quarantine program to assist the integrity of GN quarantine in New York.
Sub-Objectives 1.2 & 1.3: Monitor PVY strain diversity in the seed potato crop and 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. Studies were performed to investigate the effects of mode of transmission, either by aphid vector or vegetative propagation through tubers, on the evolution of PVY. Papers describing the results of the work were published.
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 potato cyst nematodes (PCN; Globodera rostochiensis and G. pallida), are among the most devastating plant pathogens that pose serious threats to food security globally. These nematode pests secrete an array of proteins called effectors into host root cells to induce disease. PCNs encode a large group of effector proteins; however, the exact function of these effectors and the nature of the host cellular program that they may target to promote infection are mostly unknown. Understanding how nematode effectors function in host root cells is necessary for developing effective and sustainable means for nematode control. During the past five years, we concentrated on the cloning and functional characterization of several key effector families, including the SPRYSEC (secreted SPRY domain-containing proteins) effector family, the 29D09 effector family, and the CLE (CLAVATA3/EMBRYO SURROUNDING REGION) effector family, as well as a few pioneer effectors such as NMAS1 (nematode manipulator of autophagy system 1), from PCN. Studies revealed some mechanistic details of the molecular function of the SPRYSEC and 29D09 effector families in plant defense suppression. Studies on the CLE effector family resulted in the identification of a novel class of CLE effectors from cyst nematodes. Several receptors in the host plant that perceive nematode-secreted CLE peptides and components of nematode CLE-mediated signaling pathways were identified. Work further discovered that nematode CLE effectors contain a unique trafficking sequence that allows CLE effectors to function in the apoplast of plant cells. Collectively, the studies have uncovered some underlying mechanisms of CLE effector-mediated signaling in nematode parasitism and suggested methods for developing engineered resistance in crop plants against nematode infection. Lastly, we identified and characterized a novel effector named NMAS1 from PCN and other cyst nematode species. These studies revealed for the first time that plant autophagy plays an important role in plant-nematode interactions and that cyst nematodes including PCN have evolved NMAS1 effectors to target host autophagy system to promote successful infection. Papers describing results generated under this sub-objective either have been published or are being prepared.
Sub-Objective 2.2: Characterize infection and transmission competence of PVY strains and strain combinations in potato and aphid populations. The researchers 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 indicated that the transmission protein from the old PVY strain facilitates 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. Studies were performed to investigate how the virus proteins are involved in virus movement within and between hosts and how they affect disease severity. Papers describing the results of the work were published.
Sub-Objective 3.1: Determine the resistance of potato clones and wild potato species to PCN pathotypes. This research program in Ithaca is one of the only two programs in the U.S. that are authorized to conduct research on potato cyst nematodes (PCN), including the golden nematode (GN) and the pale cyst nematode (Globodera pallida) that is only detected in Idaho. One of the key factors contributing to the success of GN quarantine in New York is the availability of potato cultivars resistant to GN. Researchers in Ithaca, New York, have been collaborating with potato breeders at Cornell University and other institutions to develop and release nematode resistant potato cultivars.
During the past five years, the program evaluated approximately 820 breeding clones and identified 452 clones to have GN resistance, which represent candidate clones that could be eventually released as commercial cultivars after further evaluation. Importantly, the joint effort has led to the release of three GN resistant potato cultivars, including ‘Algonquin’, ‘Upstate Abundance’, and ‘Brodie’, since 2017. ‘Brodie’ and ‘Upstate Abundance’ are the first two cultivars ever developed that are resistant to both pathotypes (Ro1 and Ro2) of GN. These two cultivars provide the first realistic option for affected New York farmers who still want to grow potatoes. Many Cornell developed resistant cultivars and breeding lines have been used as parents in other breeding programs, not because of their nematode resistance, but because of their exceptionally good processing quality. As a result, although < 0.1% U.S. potato land is infested with GN, > 7% of U.S. potato acreage is currently planted with cultivars resistant to GN Ro1, which has greatly helped prevent GN to appear elsewhere in the country. Additionally, the researchers have mapped a gene in potato that confers resistance to Ro2 and partial resistance to G. pallida. A PCR marker linked to this broad-spectrum resistance gene has been developed and is being evaluated for its reliability to predict resistance in a wide range of potato germplasm. The researchers have pioneered the study on exploring wild potato species of the U.S. Potato Genebank collection for novel resistance against the two PCN species, and this effort has resulted in the identification of a group of wild potato clones indicated to have strong and broad spectrum PCN resistance, and the generation of mapping populations for subsequent cloning of novel resistance genes. The identified new resistant potato germplasm can be used to accelerate breeding potatoes for novel and durable nematode resistance, which are critically needed to help support the nematode quarantine programs and to help sustain the profitability of the U.S. potato industry. Papers describing results generated under this subobjective have been published or are being prepared.
Accomplishments
1. New potato germplasm identified to have novel and broad-spectrum resistance to potato cyst nematodes. The potato cyst nematodes (PCN), including the golden nematode and the pale cyst nematode, are quarantine pests that pose serious threats to the multi-billion-dollar U.S. potato industry. Deploying resistant potato cultivars is the most effective and sustainable means for PCN control. ARS scientists in Ithaca have screened many accessions of wild potato species of the collection at the U.S. Potato Genebank and identified several new clones that showed strong and broad-spectrum resistance against multiple populations or species of PCN. These new resistant clones are valuable for discovering novel resistance genes against potato cyst nematodes and are being introduced into U.S. potato breeding programs to accelerate breeding potatoes for durable resistance against potato cyst nematodes.
2. New knowledge on the mechanistic details of nematode infection of plants. Plant-parasitic nematodes including the two species of potato cyst nematodes (golden nematode and the pale cyst nematode) utilize secreted effector proteins to induce disease. Identification of nematode effectors and understanding how they function in host roots is necessary for the development of strategies to disrupt nematode infection and manage disease. ARS scientists in Ithaca, New York, have functionally characterized several key nematode effectors and identified host components or host plant cellular programs that are targeted by these effectors. Their studies have added new knowledge on the mechanistic details of nematode infection of plants and suggested methods for generating engineered resistance in crop plants.
Review Publications
Chen, S., Cui, L., Wang, X. 2021. A plant cell wall-associated kinase encoding gene is dramatically downregulated during nematode infection of potato. Plant Signaling and Behavior. 17(1):e2004026. https://doi.org/10.1080%2F15592324.2021.2004026.
Shock, C., Brown, C., Sathuvalli, V., Charlton, B., Yilma, S., Hane, D., Quick, R.A., Rykbost, K., James, S., Mosley, A., Feibert, E., Whitworth, J.L., Novy, R.G., Stark, J., Pavek, M., Knowles, R., Navarre, D.A., Miller, J., Holm, D., Jayanty, S., Debons, J., Vales, I., Wang, X., Hamlin, L. 2018. TerraRossa: A mid-season specialty potato with red flesh and skin and resistance to common scab and golden cyst nematode. American Journal of Potato Research. 95(5):597-605. https://doi.org/10.1007/s12230-018-9667-8.
Ngatat, S., Hanna, R., Kumar, P.L., Gray, S.M., Cilia, M., Ghogomu, R.T., Fontem, D.A. 2017. Relative susceptibility of Musa genotypes to banana bunchy top disease in Cameroon and implication for disease management. Crop Protection. 101:116-122.
Fulladolsa, A., Charkowski, A., Cai, X., Whitworth, J.L., Gray, S.M., Jansky, S.H. 2019. Germplasm with resistance to Potato Virus Y derived from Solanum chacoense: Clones M19 (39-7) and M20 (XD3). American Journal of Potato Research. 96(4):390-395. https://doi.org/10.1007/s12230-019-09719-6.
Xu, Y., Ju, H., DeBlasio, S.L., Carino, E.J., Johnson, R., MacCoss, M., Heck, M.L., Miller, W., Gray, S.M. 2018. A stem-loop structure in potato leafroll virus ORF5 that is essential for readthrough translation of the coat protein ORF stop codon 700 bases upstream. Journal of Virology. https://doi.org/10.1128/JVI.01544-17.
Pinheiro, P.V., Wilson, J.R., Xu, Y., Zheng, Y., Rebelo, A., Fattah-Houseini, S., Kruse, A., Santos Dos Silva, R., Xu, Y., Kramer, M.H., Giovannoni, J.J., Fei, Z., Gray, S.M., Heck, M.L. 2019. Plant viruses transmitted in two different modes produce differing effects on small rna-mediated processes in their aphid vector. Phytobiomes Journal. 3(1):71-81. https://doi.org/10.1094/PBIOMES-10-18-0045-R.
