Location: Emerging Pests and Pathogens Research
2017 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. Rapid identification of different PCN (G. rostochiensis) populations is needed to maintain the success of PCN quarantine in New York State and to monitor the potential spread of PCN to new areas. We have generated multiple PCN lines and are in the process of propagating each population. These are critical materials for genome sequencing to identify sequence variations that can be used for diagnostic marker development.
Sub-Objective 1.2: Monitor potato virus Y (PVY) strain diversity in the seed potato crop. A total of 419 symptomatic leaf samples were collected from post harvest test potato plots grow by seed certification programs in nine 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. These data assist 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, many of which have been found to be genetic recombinants of two historic parental strains common in the U.S. during the 1900s. In a collaborative study with the Univ. of Idaho, we provided evidence that evolution of PVY proceeds through accumulation of mutations and more rapidly through recombination. The role of recombination in PVY evolution and the origin of common PVY recombinants were studied through whole genome analysis of 119 newly sequenced PVY isolates from U.S. potato, and an additional 166 whole PVY genomes from the GenBank database. Three new viruses were identified in the PVY complex. Sequence diversity in the parental sequences made it possible to trace the origins of all recombinant types of PVY, which also showed remarkable sequence diversity in most cases. The results indicate that recombination between different strains of PVY is relatively frequent and that the common recombinant PVY strains currently emerging in U.S. potatoes originated more than once, from different parental sequences.
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 facilitate successful infection. Nematodes utilize effector proteins to modulate various host cellular responses and cause infection. Understanding effector functions and their targets in host plant cells will uncover the molecular mechanisms of plant-nematode interactions. This knowledge may lead to novel strategies for the effective control of nematode pests. We have cloned a large group of effector genes from PCN (G. rostochiensis) and found that some of them belong to a multigene family. Two effector families, were found to have a major role in host defense suppression and each family has different targets in the host cell. One target was identified as a potato protein known to be involved in sugar metabolism and signaling. Two additional potato proteins were identified as receptors of effectors that are required for nematode infection The CRISPR/Cas9 genome editing technology was used to knockdown the expression of one of these receptors and the modified plants displayed enhanced resistance to nematode infection. This suggests that interference with receptors in the host may offer a novel tool for developing potatoes with broad-spectrum 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. Several emerging strains of potato virus Y (PVY) are more damaging to the U.S. potato industry because they can cause a tuber necrotic disease which reduces the quality of the potato disrupts trade and marketability. The rapid emergence and expansive distribution of the new strains is unprecedented and the factors driving the changes are unknown. We investigated whether the new strains of the virus are more efficiently transmitted by aphids when acquired from plants infected with one or more strains of PVY. Transmission of the new strains tended to be slightly more efficient if they were acquired from plants only infected by one strain. Interestingly, if aphids acquired virus from the rare plant infected with two or more strains, the old strain tended to be transmitted more efficiently although the differences were not always significant. Furthermore, transmission efficiency was significantly influenced by the potato cultivar and the isolate of the virus being used. If aphids visited multiple plants infected with different strains of PVY, the emerging strains were always transmitted with higher efficiently than the old strain regardless of the order in which aphids acquired both viruses. Most potato fields have plants infected with different strains of the virus and most aphids will visit multiple plants in the field. The ability of the new strains to outcompete the old strain during the transmission process likely contributes to the increase in the new strains that has been observed in recent years.
Sub-Objective 2.3: Define the mechanism of tissue tropism of poleroviruses in plants and aphid vectors. Phloem localization of plant viruses is advantageous for acquisition by sap sucking vectors but hampers host virus protein interaction studies. In collaborative studies with ARS researchers in Ithaca, New York, potato leafroll virus (PLRV) host protein complexes were isolated from systemically infected potato, a natural host of the virus. Comparing two different co-immunoprecipitation support matrices coupled to mass spectrometry, 44 potato proteins were identified that specifically associated with PLRV isolated from infected phloem tissue. An additional 142 proteins interacted in complexes involving virus and more than 80% of these proteins were previously found by us to interact with PLRV isolated from infected phloem tissue in the model host Nicotiana benthamiana. Bioinformatics identified these proteins are enriched for functions related to plasmodesmata, organelle membrane transport, translation, and mRNA processing. These results are proof that model system proteomics experiments are extremely valuable for understanding protein interactions regulating infection in phloem-limited viruses, and in identifying proteins that could be targeted in the host plant to disrupt the ability of viruses to cause infection or be transmitted by insect vectors.
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 > 430 breeding clones and four existing cultivars with resistance to PCN (G. rostochiensis). Excitingly, the four existing varieties were found to have broad-spectrum nematode resistance. These resistant clones and cultivars provide new resistant germplasm for potato breeding programs and options for growers to produce potatoes on PCN-regulated land. ‘Lamoka’ and ‘Waneta’, two recently-released resistant cultivars that were jointly developed with Cornell University, ranked 12th and 31th, respectively, in certified seed acres grown in 2016 in the U.S. In addition, each of the four resistant cultivars (Pike, Lehigh, Reba, and Keuka Gold) that were jointly developed with Cornell had a total seed acreage of more than 1000 acres and were grown in eight states in 2016. The rapid and widespread adoption of these resistant cultivars has provided an effective means of preventing a further spread of PCN within the U.S. We also initiated the screening of wild potato species and identified a few accessions in two wild potato species that have resistance to multiple populations or species of PCN. These studies identified new resistant germplasm that harbor new nematode resistance genes and are providing breeders with multiple sources of resistance to PCN to incorporate into U.S. potato cultivars.
Sub-Objective 3.2: Collaborate with potato breeders to develop genetic markers for phenotypic traits useful in the development of durable virus resistance. Resistance to potato virus Y (PVY) has not been a priority of most North American potato breeding programs. This has contributed to a surge in virus incidence in recent decades and to the emergence of new virus strains that cause a tuber necrotic disease in some potato cultivars. In an attempt to link tuber necrosis to genetic markers, phenotypic and genotypic data were obtained for a population of 236 F1 progeny segregating for tuber necrosis and foliar symptom types. Genome analyses identified distinct regions on chromosomes 4 and 5 that have major effects on symptom expression. QTLs 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.
Accomplishments
1. Joint effort resulted in the first detection of the soybean cyst nematode in New York. The soybean cyst nematode (SCN) is the most damaging pathogen of soybean, causing > $1 billion in yield losses annually in the U.S. The SCN distribution map updated in 2014 showed that SCN was present in all major soybean-producing states in the U.S. except New York and West Virginia. Soybean shows great economic promise in New York State and its production area in the region has been expanding rapidly. ARS researchers in Ithaca, New York collaborated with researchers at Cornell University on a multi-year survey effort searching for the presence of SCN in New York soils. A recent soil sample from Cayuga County was identified to contain nematode cysts, which were further confirmed as the soybean cyst nematode by morphological, molecular, and bioassay analyses. This is the first detection of the soybean cyst nematode in New York, which signifies an urgent need for developing management strategies in order to protect New York soybean growers and the industry from nematode losses.
2. Strain-specific resistance to potato virus Y (PVY). Potato virus Y (PVY) threatens potato production due to effects on tuber quality and trade restrictions, in particular, due to emerging strains that cause potato tuber necrotic ringspot disease (PTNRD). These emerging recombinant strains have been spreading in the U.S. for the past decade, although the reasons for their rapid emergence were unclear. In collaboration with the University of Idaho and several state potato seed certification programs, the strain composition of PVY isolates circulating in the U.S. seed potato crop was determined from thousands of samples collected from all seed potato production states. The proportion of the ordinary strain of PVY, common in the U.S. potato crop prior to 2004, has dropped precipitously from nearly 70% 2004 to less than 10% in 2016. This drop in this strain was concomitant with the rise in proportion of the Wilga strain (from 25% in 2004 to 70% in 2016) and the tuber necrotic strain (from 5% in 2004 to nearly 25% in 2016). In collaboration with colleagues from several universities and ARS, controlled experiments revealed that a majority of the potato cultivars widely grown in the U.S. possess strain-specific resistance that reduces and localizes the systemic movement of the ordinary strain of PVY. This resistance reduces the transmission of the ordinary strain by aphids and through progeny tubers and thereby favors the spread of the emerging Wilga and tuber necrotic strains in the field and in the seed crop. The presence of strain-specific resistance genes inadvertently bred into potato cultivars likely represents the driving force behind the change in PVY strain composition observed in all U.S. potato production areas.
Review Publications
Green, K.J., Brown, C.J., Gray, S.M., Karasev, A.V. 2017. Phylogenetic study of recombinant strains of Potato virus Y. Virology. 507:40-52.
Wang, X., Bergstrom, G., Chen, S., Thurston, D., Cummings, J., Handoo, Z.A., Hult, M.N., Skantar, A.M. 2017. First report of the Soybean Cyst Nematode, Heterodera glycines, in New York. Plant Disease. https://doi.org/10.1094/PDIS-06-17-0803-PDN.
Carroll, J., Smith, D., Gray, S.M. 2016. Preferential acquisition and inoculation of PVYNTN over PVYO in potato by the green peach aphid Myzus persicae (Sulzer). Journal of General Virology. 97:797-802.
Deblasio, S.L., Johnson, R., Maccoss, M., Gray, S.M., Cilia, M. 2016. Model system-guided protein interaction mapping for virus isolated from phloem tissue. Journal of General Virology. 15:4601-4611.
Mondal, S., Lin, Y., Carroll, J.E., Wenninger, E.J., Perez, N., Whitworth, J.L., Gray, S.M. 2017. Potato virus Y transmission efficiency from different potato cultivars infected with single or multiple virus strains. Phytopathology. 107:491-498.
Funke, C., Nikolaeva, O.V., Green, K.J., Tran, L.T., Chikh-Ali, M., Quintero-Ferrer, A., Cating, R., Frost, K.E., Hamm, P.B., Olsen, N., Pavek, M.J., Gray, S.M., Crosslin, J.M., Karasev, A.V. 2017. Strain-specific resistance to Potato virus Y (PVY) in potato and its effect on the relative abundance of PVY strains in commercial potato fields. Plant Disease. 101:20-28.
Dejong, W.S., Halseth, D.E., Plaisted, R.L., Wang, X., Perry, K.L., Qu, X., Paddock, K.M., Falise, M., Christ, B.J., Porter, G.A. 2016. Lamoka, a variety with excellent chip color out of cold storage and resistance to the golden potato cyst nematode. American Journal of Potato Research. 94:148-152.
Tran, T., Chen, S., Wang, X. 2016. Root assays to study pattern-triggered immunity in plant-nematode interactions. European Journal of Plant Pathology. 147:955-961.
Pinheiro, P., Ghanim, M., Rebelo, A., Santos, R., Orsburn, B.C., Gray, S.M., Cilia, M. 2016. Host plants indirectly influence plant virus transmission by altering gut cysteine protease activity of aphid vectors. Molecular and Cellular Proteomics. DOI: 10.1074/mcp.M116.063495.
