Source: UNIV OF IDAHO submitted to NRP
PLANT VIRUS-HOST INTERACTIONS, EPIDEMIOLOGY, AND MANAGEMENT OF PLANT VIRUS DISEASES IN IDAHO CROPS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0226045
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jul 1, 2011
Project End Date
Jun 30, 2016
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
Plant Soil & Entomological Sci
Non Technical Summary
Idaho has a large and diverse crop production agriculture, with a significant impact on the state and national economies. The State of Idaho is the largest producer of potatoes in the U.S., with about one-third of the national production and more than 30% of the acreage in 2008. Other significant crops grown in Idaho include sugar beet, dry beans, peas and other cool season food legumes. In all these crops, virus diseases cause significant losses and require attention with respect to monitoring viruses and managing disease problems. Understanding the infection cycle, virus-host interactions, transmission, epidemiology, and strain composition of viruses affecting major Idaho crops facilitates development of more profitable and sustainable management practices for crop protection and production.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121310110170%
2121410110120%
2122010110110%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1310 - Potato; 2010 - Sugar beet; 1410 - Beans (dry);

Field Of Science
1101 - Virology;
Goals / Objectives
Idaho has large and diverse crop production agriculture, with a significant impact on the state and national economies. State of Idaho is the largest producer of potatoes in the U.S., with about one-third of the national production and more than 30% of the acreage in 2008; more than a quarter of U.S. potato processing capacity resides in Idaho. The Idaho potato industry has historically contributed over 5% of Idaho's gross state product. Other significant crops grown in Idaho include sugar beet, dry beans, peas and other cool season food legumes. In all these crops, virus diseases cause significant losses and require attention with respect to monitoring viruses and managing disease problems. Viruses, in particular Potato virus Y (PVY) and Potato leafroll virus (PLRV), are especially damaging in potato, a vegetatively propagated crop where special seed certification programs are employed to mitigate effects of degenerating virus diseases. Two viruses are important in beans in Idaho and throughout the entire western parts of North America, Bean common mosaic virus (BCMV) and Beet curly top virus (BCTV). The following specific objectives will be pursued. 1. To study plant virus-host interaction in the PVY-potato pathosystem, through screening the most common potato cultivars grown in Idaho for resistance to the wide-spread PVY strains in Idaho; 2. To study epidemiology and strain composition of PVY in Idaho, by subjecting our existing PVY isolate collection to whole-genome sequencing with subsequent correlations between sequences and specific biological, serological, and molecular features of the virus, to develop immunological and RT-PCR tools to identify isolates inducing PTNRD in susceptible potato cultivars; 3. To study virus genetic factors involved in aphid transmission through testing aphid transmission competence and efficiency for a series of mutants in the P4-gene of PLRV; 4. To facilitate breeding for virus resistance in beans through development of ELISA-based detection systems for viruses affecting common beans in Idaho. Understanding the infection cycle, virus-host interactions, transmission, epidemiology, and strain composition of viruses affecting major Idaho crops facilitates development of more profitable and sustainable management strategies for crop protection and production.
Project Methods
Objective 1. A large collection of potato germplasm (more than 50 named lines and more than 60 breeding lines) from the University of Idaho Tissue Culture Lab is available to the PI for screening. These represent all the potato cultivars now grown in Idaho and in the Pacific Northwest. Inoculation experiments will be done in an insect-proof greenhouse and growth chambers at the UI main campus. Virus-free plantlets of each cultivar will be mechanically inoculated by the PVY strain under testing, and PVY infection and strain type will be confirmed by ELISA and RT-PCR. Symptom scoring will take place 1-6 weeks post inoculation, and will be followed by an ELISA and RT-PCR based typing of the PVY isolate in each plant to confirm that no cross-contamination occurred. Objective 2. More than 4,000 field isolates of Potato virus Y have been collected through a combination of national and state surveys conducted between 2004 and 2010. We will subject all PVY isolates collected in Idaho to whole genome sequencing, and whole genome sequence analysis. The overall objective will be to correlate specific sequence signatures with defined biological, serological, and molecular properties, specifically with the ability of a given isolate to induce PTNRD in a susceptible potato cultivar. Objective 3. Through reverse genetics and site-directed mutagenesis, we have demonstrated that a non-structural protein of PLRV, p17, is involved in aphid transmission. We will continue construction of mutants in the P4 gene to localize domains involved in aphid transmission of PLRV, and also study physical nature and mechanisms of p17 involvement in transmission process. We will do it through a combination of reverse genetics approach, tissue immuno-printing, and analysis of PLRV presence in aphids. Objective 4. We have a small archive of several BCTV isolates in the Kimberly R&E Center, and in the adjacent USDA-ARS lab. For PCR-based detection, we will use several sets of primers designed for differentiation of viruses from the curly top virus complex. We will pursue studies of natural genetic diversity of BCTV in Idaho through systematic sequencing of field curly top virus isolates collected in the state. We also have a collection of BCMV and BCMNV strains at the Plant Virology Laboratory of the University of Idaho. We plan to propagate different pathotypes of BCMV in a susceptible bean cultivar Dubbele Witte with subsequent purification of the virus, and use this purified virus for antibody production in rabbits and guinea pigs for TAS-ELISA development. We will also clone the CP gene of BCMV for bacterial production of this antigen, with subsequent generation of antibodies against this bacterial BCMV antigen, similar to our BCTV project.

Progress 07/01/11 to 06/30/16

Outputs
Target Audience:Idaho commercial growers' groups involved in potato, sugar beet, bean production, plant pathologists, and researchers involved in managing virus diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Between 2011 and 2016, two Ph.D. and five M.S. students graduated from University of Idaho as a direct result of the project. Two additional M.S. students have been involved in various segments of the project, and their theses are in progress. Fifteen undergraduate students participated in various research activities related to this project. Five postdoctoral scientists were involved in the project between 2011 and 2016. How have the results been disseminated to communities of interest?Through presentations at the state commodity schools, at the professional meetings, and through peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Several distinct pathotypes of Potato virus Y (PVY) were identified based on their interactions with N genes present in potato cultivars, including PVYZ and PVYE, and characterized biologicallyand molecularly (Karasev et al., 2011; Kerlan et al., 2011; Galvino-Costa et al., 2012a,b). A previously postulated Nz gene was proved to be inherited as a dominant singleallele (Chikh-Ali et al., 2014). Multiple North American cultivars of potato were screened using five distinct strains of PVY to determine presence of the strain-specific resistance alleles in their genetic background, and both Ny and Nz genes were identified in these cultivars; one cultivar, Yukon Gem, was identified as carrying multiple resistance alleles, including four new, putative resistance alleles conferring resistance to recombinant strains of PVY, providing a new source of resistance to recombinants of the virus (Rowley et al., 2015). A new syndrome, tuber cracking was described in specialty potato cultivars expressed in PVY-infected plants (Benedict et al., 2015). Seven pathotypes of Bean common mosaic virus (BCMV) were biologically and molecularly characterized, and virus genetic determinants involved in interactions with three resistance alleles, I, bc-12, and bc-3, were tentatively identified (Feng et al., 2014a,b; Feng et al., 2015). A novel pathotype of BCMV overcoming both bc-2 and bc-3 resistance alleles was described and assigned to a new pathotype VIII (Feng et al., 2015). Objective 2. Strain composition of PVY was studied for several potato producing areas in the U.S. and outside of the country; a distinct shift from non-recombinant isolates of PVY to various recombinant types was documented in the U.S. (Benedict et al., 2015), Brazil (Galvino-Costa et al., 2012a,b), Japan (Chikh-Ali et al., 2013), Mexico (Quintero-Ferrer and Karasev, 2013; Quintero-Ferrer et al., 2014), Saudi Arabia (Chikh-Ali et al., 2016a), and Indonesia (Chikh-Ali et al., 2016b). Mappedlinear epitopes for three commercial N-specific monoclonal antibodies that are widely used for serotyping of the PVY isolates, these monoclonal antibodies are used in international potato trade toexclude tuber-necrotic strains of the virus (Nikolaeva et al., 2012). Defined a novel serotype of PVY, named AST-serotype, after an isolate of PVY from Brazil that first exhibited it (Galvino-Costa et al., 2012a,b).Developed and applied a new, simplifiedmethodology for PVY strain differentiation, based on immuno-capture RT-PCR, suitable for large-scale testing of field samples; this methodology can differentiate up to 14 recombinant structures of PVY(Chikh-Ali et al., 2013). Objective 3. Studied the roles of structural proteins in systemic movement, phloem retention,and transmission of Potato leafroll virus (PLRV), identified a large complex of cellular proteins interacting with PLRV virions, virus capsid protein, and the read-through protein of PLRV and facilitating these importantfunctions (DeBlasio et al., 2015a,b). Objective 4. A set ofpolyclonal antisera was produced in multiple animal species, specific to different strains of BCMV and Bean common mosaic necrosis virus (BCMNV), and an ELISA-based differentiation assay was developed to distinguish BCMV and BCMNV in a simple two-step ELISA format (Feng et al., 2014a). These BCMV and BCMNV antibodies were successfully used in a breeding project to screen for resistance to BCMV and BCMNV strains (Feng et al., 2015).

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Chikh-Ali, M., Naidu, R.A., and Karasev, A.V. (2016) First report of Potato virus Y (PVY) strain PVYC associated with a tomato disease in Kenya. Plant Disease 100: 864.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Idaho commercial growers' groups involved in potato, sugar beet, bean production, plant pathologists, and researchers involved in managing virus diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Five graduate students,4 M.S. and1 Ph.D., were involved in various virus research projects during this year. Four undergraduate students were involved in research projects during this year. How have the results been disseminated to communities of interest?The results have been published as peer-reviewed papers, presented at professional meetings, and at the growers conferences and trade shows. What do you plan to do during the next reporting period to accomplish the goals?Continue research on virus-host interactions in pathosystems important for Idaho agriculture.

Impacts
What was accomplished under these goals? 1. Potato virus Y (PVY) strains have been defined based on genetic reactions in potato indicators expressing hypersensitive reaction (HR) response due to the presence of three different N genes, and also based on genomic information. Nine strains are known currently, with five PVY strains defined biologically, PVYO, PVYC, PVYZ, PVYN, and PVYE. The genetic background of the majority of North American potato cultivars has so far been poorly characterized for the presence of N genes inducing HR towards different PVY strains. The HR response was studied in eight potato cultivars, elicited by five strains of PVY circulating in North America. These PVY isolates included representative isolates of PVYN-Wi, PVYNA-N, PVYO, PVYZ, and PVYN strains. Potato cultivars tested included Russet Burbank, Russet Norkotah, Shepody, Ranger Russet, Western Russet, Alturas, Rio Grande Russet, and Yukon Gem, grown in the U.S., and standard indicators Desiree and Maris Bard with the known genetic background. Three additional strains, PVYN:O, PVY-NE11, and PVYE, were tested on Yukon Gem. Virus-free potato plants were mechanically inoculated with PVY inoculum, and local and systemic foliar symptoms were observed for eight weeks post-inoculation under different climate-controlled conditions. Virus status of the inoculated plants was tested starting at three weeks post-inoculation, by serotype-specific ELISA and RT-PCR, in order to monitor successful infections and confirm the identity of the inoculated PVY isolate. This systematic approach allowed us to identify Nytbr and Nztbr genes present in several North American cultivars. Two more new, putative N genes were postulated to be expressed in the cultivar Yukon Gem, and one additional putative N gene was postulated to be expressed in two cultivars, Yukon Gem and Rio Grande Russet. These N genes may represent valuable sources of resistance against multiple strains of PVY. 2. Identification of host proteins interacting with the aphid-borne, Potato leafroll virus (PLRV) from the genus Polerovirus, family Luteoviridae, is a critical step towards understanding how PLRV and related viruses infect plants. A polyclonal antibody raised against purified PLRV virions was used to immunoprecipitate virus-host protein complexes from Nicotiana benthamiana tissue agro-inoculated with PLRV. Isolated protein complexes were characterized using high-resolution mass spectrometry and were comprised of host proteins complexed directly and indirectly with virions as well as the non-incorporated readthrough protein (RTP) including three phosphorylated positional isomers of the RTP. A bioinformatics analysis using ClueGo and STRING show plant proteins in the PLRV protein interaction network regulate key biochemical processes including, carbon fixation, amino acid biosynthesis, ion transport, protein folding and trafficking. 3. Bean common mosaic virus (BCMV) exists as a complex of strains that can be distinguished biologically, on a set of bean differentials, into pathogenicity groups (PG) numbered I to VII. In order to establish molecular signatures characteristic of different pathogenicity groups of BCMV, we performed a complete biological and partial molecular study of 12 field BCMV isolates collected in Oregon, and also biological and molecular characterization of two control isolates from PG-I (US1) and PG-V (NY15P). All but one field isolates were found to have B-serotype, with four of them belonging to PG-I, five to PG-III, two to PG-IV, and one to a new PG-VIII. One isolate had A-serotype and was classified as Bean common mosaic necrosis virus. Partial sequences of the HC-Pro region were determined for eight out of 12 field isolates of BCMV, and no correlation was found between the PG assignment of a BCMV isolate and the HC-Pro sequence similarities. The complete genomes of US1 and NY15P isolates were sequenced and found closest to the BCMV strain NL1 (PG-I, 94% identity to US1 and 97% identity to NY15P). Sequence analysis revealed that strain US1 might be a product of recombination between strains US10 and NY15P. The data obtained suggest that the comparative genomics of BCMV isolates may be useful to identify genetic determinants of BCMV pathogenicity in common bean.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Feng, X., Poplawsky, A.R., and Karasev, A.V. (2014) A recombinant of Bean common mosaic virus induces temperature-insensitive necrosis in an I gene-bearing line of common bean. Phytopathology 104: 1251-1257.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Chikh-Ali, M., Rowley, J.S., Kuhl, J.C., Gray, S.M., and Karasev, A.V. (2014) Evidence of a monogenic nature of the Nz gene conferring resistance against Potato virus Y strain Z (PVYZ) in potato. American Journal of Potato Research 91: 649-654.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Rowley, J.S., Gray, S.M., and Karasev, A.V. (2015) Screening potato cultivars for new sources of resistance to Potato virus Y. American Journal of Potato Research 92: 38-48.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: DeBlasio, S.L., Johnson, R., Mahoney, J., Karasev, A.V., Gray, S.M., MacCoss, M.J., and Cilia, M. (2015) Insights into the polerovirus-plant interactome revealed by co-immunoprecipitation and mass spectrometry. Molecular Plant-Microbe Interactions 28: 467-481.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: DeBlasio, S.L., Johnson, R., Sweeney, M.M., Karasev, A.V., Gray, S.M., MacCoss, M.J., and Cilia, M. (2015) The Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection. Proteomics 15: 2098-2112.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Idaho commercial growers' groups involved in potato, sugar beet, bean production, plant pathologists, and researchers involved in managing virus diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Five graduate students,3 M.S. and 2 Ph.D., were involved in various virus research projects during this year. One student, K. Evans, submitted and defended her M.S. thesis in 2014. One student, X. Feng, submitted and defended her Ph.D. thesis in 2014. How have the results been disseminated to communities of interest? The results have been published as peer-reviewed papers, presented at professional meetings, and at the growers conferences and trade shows. What do you plan to do during the next reporting period to accomplish the goals? Continue research on virus-host interactions in pathosystems important for Idaho agriculture.

Impacts
What was accomplished under these goals? The I gene is a single, dominant gene conferring temperature-sensitive resistance to all known strains of Bean common mosaic virus (BCMV) in common bean (Phaseolus vulgaris L.). However, the closely related Bean common mosaic necrosis virus (BCMNV) induces whole plant necrosis in I-bearing genotypes of common bean, and the presence of additional, recessive genes is required to prevent this severe whole plant necrotic reaction caused by BCMNV. Almost all known BCMNV isolates have so far been classified as having pathotype VI based on their interactions with the five BCMV resistance genes, and all have a distinct serotype A. We described a new isolate of BCMV, RU1M, capable of inducing whole plant necrosis in the presence of the I gene, that appears to belong to pathotype VII and exhibits B-serotype. Unlike other isolates of BCMV, RU1M was able to induce severe whole plant necrosis below 30oC in bean cultivar 'Jubila' that carries the I gene and a protective recessive gene bc-1. The whole genome of RU1M was cloned and sequenced and determined to be 9,953-nt long excluding poly(A), coding for a single polyprotein of 3,186 aa. Most of the genome was found almost identical (>98%) to the BCMV isolate RU1-OR (also pathotype VII) that did not induce necrotic symptoms in 'Jubila'. Inspection of the nucleotide sequences for BCMV isolates RU1-OR, RU1M and US10 (all pathotype VII), and three closely related sequences of BCMV isolates RU1P, RU1D, and RU1W (all pathotype VI) revealed that RU1M is a product of recombination between RU1-OR and a yet unknown potyvirus. A 0.8-kb fragment of an unknown origin in the RU1M genome may have led to its ability to induce necrosis regardless of temperature in beans carrying the I gene. This is the first report of a BCMV isolate inducing temperature-insensitive necrosis in an I gene containing bean genotype. Hypersensitive resistance (HR) to Potato virus Y (PVY) in potato (Solanum tuberosum) is conferred by strain-specific N genes. Two such genes have been identified in potato so far, Nytbr conferring HR to PVYO, and Nctbr conferring HR to PVYC. A third, putative gene Nztbr was proposed to confer HR against a distinct strain PVYZ. However, due to the scarcity of the PVYZ isolates of PVY, no formal proof of the monogenic nature of this new gene, Nztbr, was available until now. We reported on a genetic study of the Nztbr inheritance in three crosses between cultivars Maris Bard (Ny:Nz) and King Edward (ny:nz), and Maris Bard (Ny:Nz) and Russet Norkotah (ny:nz). A fully-sequenced PVYZ isolate, L26, was used to screen the parents and progeny for a virus-induced HR phenotype in foliage. Based on the phenotypic analysis of 203 progeny, segregation of HR phenotype in the PVYZ-infected plants was found to be 1:1, indicating a monogenic, dominant nature of the Nztbr gene. Since the PVYZ strain includes PVYNTN isolates associated with tuber necrotic ringspot disease (PTNRD) in susceptible potato cultivars, the Nztbr gene represents a valuable source of HR against PTNRD-inducing PVY isolates. This is the first demonstration that Nztbr is a single, dominant N gene in potato conferring resistance to the PVYZ-NTN strain.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Feng, X., Poplawsky, A.R., Nikolaeva, O.V., Myers, J.R., and Karasev, A.V. (2014) Recombinants of Bean common mosaic virus (BCMV) and genetic determinants of BCMV involved in overcoming resistance in common bean. Phytopathology 104: 786-793.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Quintero-Ferrer, A., Robles-Hernandez, L., Gonzalez-Franco A.C., Kerlan, C., and Karasev, A.V. (2014) Molecular and biological characterization of a recombinant isolate of Potato virus Y from Mexico. Archives of Virology 159: 1781-1785.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Swisher, K.D., Sengoda, V.G., Dixon, J., Munyaneza, J.E., Murphy, A.F., Rondon, S.I., Thompson, B., Karasev, A.V., Wenninger, E.J., Olsen, N., and Crosslin, J.M. (2014) Assessing potato psyllid haplotypes in potato crops in the Pacific Northwestern United States. American Journal of Potato Research 91: 485-491.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Idaho commercial growers' groups involved in potato, sugar beet, bean production, plant pathologists, and researchers involved in managing virus diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Four graduate students,2 MS and2 PhD, were involved in various virus research projects during this year. One student, J. Rowley, submitted and defended her MS thesis in 2013. Two visiting MS students from University of Chihuahua (Mexico) received 6-mo training in molecular genetcs methods of plant virus characterization, using Potato virus Y and Beet curly top virus as models. How have the results been disseminated to communities of interest? The results have been publishedas peer-reviewed papers, presented at professional meetings, and at the growers conferences and trade shows. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In order to understand the genetic determinants of pathogenicity for Bean common mosaic virus (BCMV), the whole genome was cloned and sequenced for the BCMV strains US10 and RU1-OR (pathotype VII), and also for the strain RU1p (pathotype VI). Inspection of the nucleotide sequences for BCMV RU1-OR and US10, and closely related sequences BCMV RU1p, RU1d, and RU1-W (all pathotype VI) revealed that BCMV RU1 isolates originated through a series of recombination events between US10 and a yet unknown parental genome, resulting in changes in virus pathology. The data obtained suggest that a fragment of the US10 genome in the P1-HC-Pro region may be involved in its ability to overcome the BCMV resistance in beans conferred by the bc-22 gene. Beet severe curly top virus (BSCTV) is one of three curtoviruses affecting sugar beet in dry desert climate of western U.S. BSCTV is a member of the genus Curtovirus, family Geminiviridae. It is a phloem-limited virus, transmitted by leafhoppers, with a ca. 3.0-kb genome represented by a single-stranded, circular DNA. In order to create a tool for convenient screening of the curly top-resistant sugar beet germplasm, we generated two full-length, infectious clones of BSCTV (1.2 and 1.8 genomes) in a binary construct suitable for delivery into plants via agroinoculation. The infectivity for both clones was confirmed for three varieties of sugar beet, two varieties of tomato, and for Nicotiana benthamiana. The time-course of BSCTV symptom development was compared to symptoms caused by another curtovirus, Beet curly top virus (BCTV, strain Logan). Infectivity of BSCTV and BCTV infectious clones was monitored through a combination of symptom observations, ELISA, and PCR tests. A multiplex RT-PCR assay was previously developed to identify a group of PVY isolates with unusual recombinant structures, e.g. PVYNTN-NW and SYR-III, and to differentiate them from other PVY strains. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. The results obtained using well-characterized reference isolates revealed for the first time that this multiplex RT-PCR assay is an accurate and robust method to identify and differentiate nine PVY strains reported so far, including PVYO (both PVYO and PVYO-O5), PVYN, PVYNA-N, PVYZ, PVYE, PVY-NE11, PVYN-Wi and PVYN:O which is not possible by any of the previously reported RT-PCR. This would make this RT-PCR a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Karasev, A.V. and Gray, S.M. (2013) Genetic diversity of Potato virus Y complex. American Journal of Potato Research 90: 7-13. *Quintero-Ferrer, A. and Karasev, A.V. (2013) First report of Potato virus Y in potato in Jalisco, Mexico. Plant Disease 97: 430-430. Karasev, A.V. and Gray, S.M. (2013) Continuous and emerging challenges of Potato virus Y in potato. Annual Review of Phytopathology 51: 571-586. Chikh Ali, M., Karasev, A.V., Furutani, N., Taniguchi, M., Kano, Y., Sato, M., Natsuaki, T., and Maoka, T. (2013) Occurrence of Potato virus Y strain PVYNTN in foundation seed potatoes in Japan, and screening for symptoms in Japanese potato cultivars. Plant Pathology 62: 1157-1165. Chikh Ali, M., Gray, S.M., and Karasev, A.V. (2013) An improved multiplex IC-RT-PCR assay distinguishes nine strains of Potato virus Y. Plant Disease 97: 1370-1374. Feng, X. and Karasev, A.V. (2013) A novel recombinant of Bean common mosaic virus. Phytopathology 103: S2.42. Eid, S.G., Poplawsky, A.R., and Karasev, A.V. (2013) Mutations in the Potato leafroll virus non-structural protein p17 impair aphid transmission but do not affect virion assembly. Phytopathology 103: S.2.38. Feng, X., Poplawsky, A.R., and Karasev, A.V. (2013) A recombinant of Bean common mosaic virus induces necrosis in an I gene bearing line of common beans. Phytopathology 103: S3.13. Poplawsky, A.R., Eid, S.G., and Karasev, A.V. (2013) A preliminary host-range study of two infectious full-length clones of Beet severe curly top virus. Phytopathology 103: S3.15.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Curly top disease of sugar beet in Idaho is caused by a complex of at least three curtoviruses which are transmitted by the beet leafhopper. In the field, some of these viruses are found preferentially in sugar beet and others in beans. To address the mechanisms of these host preferences, an infectious, dimeric clone was constructed for Beet curly top virus (BCTV, Logan), and maintained in a binary vector suitable for agroinoculation. Infectivity of this BCTV clone was tested on a range of host plants following agroinoculation with subsequent ELISA, tissue printing, and PCR to confirm successful infection. Nicotiana benthamiana, two cultivars of sugar beet, and six cultivars of tomatoes supported replication of this BCTV infectious clone, and the typical symptoms of stunting, leaf curl and chlorosis at the growing point started appearing at 3 weeks post-inoculation in most cases. These symptoms correlated with the laboratory tests for the presence of BCTV. Both the infectivity rate and the relative level of virus replication were reduced for cultivars with partial resistance to curly top. Grapevine fleck virus (GFkV) belongs to the genus Maculavirus, family Tymoviridae comprising positive-sense, single-stranded RNA viruses with ca. 7.6-kb genome, it is one of five non-mechanically transmitted viruses associated with the fleck disease complex and has been previously documented to occur in the neighboring state of Washington. Main sources of wine grape nursery material imported to Idaho reside in Washington or in California, and it is important to monitor virus status of the planting material brought to the state. However, no information was available on the occurrence and prevalence of GFkV in wine grapes in Idaho. During three growing seasons in 2009-2011, random grapevine samples were collected in 14 vineyards in Canyon, Elmore, Ada, and Nez Perce counties. A total of 434 samples were tested by one step RT-PCR using GFkV-specific primers. Twenty-four samples were found positive by RT-PCR producing the expected bandDNA fragment of expected size, 179-bp; these came from five vineyards from across all sampled counties, and were found in seven wine grape cultivars, Pinot Noir, Cabernet Sauvignon, Syrah, Lemberger, Riesling, Chardonnay, Pinot Gris, and one unknown table grape cultivar. Twelve amplicons PCR products were cloned into the plasmid vector, sequenced, and confirmed to represent fragments of the GFkV CP gene between positions 6,453 and 6,631 in the genome of GFkV isolate MT48 (GenBank accession number AJ309022.1). In the absence of symptoms expressed in wine grape cultivars infected with GFkV, laboratory methods remain the only tool to detect the virus. This is the first report of GFkV found in wine grapes in Idaho demonstrating its substantial presence in production areas. PARTICIPANTS: Alexander Karasev, Associate Professor; Allan Poplawsky, Research Associate, Olga Nikolaeva, Research Associate; Sahar Eid, Postdoctoral Scientist; Mohamad Chikh Ali, Postdoctoral Scientist, Xue Feng, Graduate Student, Eunice Kanuya, Graduate student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In 2011, two isolates of Bean common mosaic virus (BCMV) were collected in the Willamette Valley of Oregon from bean plants exhibiting mild mosaic and stunting symptoms. Both isolates appeared to be seedborne in the line L192, a brown-seeded dry bean with determinant growth habit. Based on a biological characterization on twelve bean differential lines, these two BCMV isolates were classified as belonging to the non-necrotic pathotype VII, similar to a control isolate US10. Both isolates had B-serotype but exhibited serology distinct from the isolate US10 (pathotype VII). Both isolates were subjected to cDNA cloning and sequencing and were found to be 98% identical to the BCMV isolate RU1 originally intercepted in the mid-1980s in a dry bean entry imported from Russia. However, the original RU1 isolate was classified as a non-necrotic strain belonging to pathotype VI. It is possible that the weakly expressed, attenuated symptoms are responsible for the spread of these BCMV isolates in beans. This is the first report of a BCMV RU1-like strain isolated from field plants in the U.S.

Publications

  • Feng, X., Poplawsky, A.R., Nikolaeva, O.V., Myers, J.R., and Karasev, A.V. (2012) Genome sequences of two field isolates of Bean common mosaic virus. Phytopathology 102: S6.9.
  • Poplawsky, A.R., Nikolaeva, O.V., Feng, X., Myers, J.R., and Karasev, A.V. (2012) Characterization of two field isolates of Bean common mosaic virus. Phytopathology 102: S4.93.
  • Poplawsky, A.R., Eid, S., and Karasev, A.V. (2012) Host range studies of a Beet curly top virus (Logan) infectious clone. Phytopathology 102: S4.93.
  • Chikh Ali, M., Maoka, T., Natsuaki, T., and Karasev, A.V. (2012) A new recombinant Potato virus Y isolate classified as belonging to PVYZ strain group may help to define viral determinant responsible for tuber necrosis in potato. Phytopathology 102: S.4.23.
  • Chikh Ali, M., Karasev, A.V., Furutani, N., Taniguchi, M., Kano, Y., Sato, M., Natsuaki, T., and Maoka, T. (2012) An outbreak of the Potato virus YNTN (PVYNTN) strain in foundation seed potatoes in Japan. Phytopathology 102: S4.23.
  • Kanuya, E., Clayton, L.A., Naidu, R.A., and Karasev, A.V. (2012) First report of Grapevine fleck virus in Idaho grapevines. Plant Disease 96: 1705-1705.


Progress 07/01/11 to 12/31/11

Outputs
OUTPUTS: Potato virus Y (PVY) strains were originally defined by interactions with different resistance genes in standard potato cultivars. Five distinct strain groups are defined that cause local and/or systemic hypersensitive responses in genetic background with a corresponding N gene, these are PVYO, PVYN, PVYC, PVYZ and PVYE. The nucleotide sequences of multiple isolates of PVYO and PVYN differ from each other by about 8% along their genomes. Additionally, complete genome sequences of multiple recombinant isolates are composed of segments of parental PVYO and PVYN sequences. Here, we report that recombinant isolate PVY-L26 induces a hypersensitive response (HR) in potato cv. Maris Bard carrying the putative Nz gene, and is not recognized by two other resistance genes, Nc and Nytbr. These genetic responses in potato, combined with the inability of PVY-L26 to induce vein necrosis in tobacco, clearly define it as an isolate from the PVYZ strain group and provide the first information on genome structure and sequence of PVYZ. The genome of PVY-L26 displays typical features of PVYEU-NTN isolates, i.e. European NTN type with three recombinant junctions, and hence the PVY-L26 is named PVYZ-NTN. Three typical PVYNTN isolates and two PVYN isolates all inducing vein necrosis in tobacco were compared with PVY-L26. One PVYNTN isolate elicited HR reactions in cv. Maris Bard, similar to PVY-L26, while two induced a severe systemic HR-like reaction quite different from the quasi-symptomless reaction induced by two PVYN isolates. Cv. Yukon Gold from North America produced HR against several PVYNTN isolates, including PVY-L26, but only late and limited systemic necrosis against one PVYN isolate. Consequently, according to symptoms in potato indicators, both PVYZ and PVYNTN isolates appeared biologically very close and clearly distinct from PVYO and PVYN strain groups. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Curly top is a serious problem in many irrigated crops in the semi-arid areas in the western U.S. The disease is caused by a complex of leafhopper-transmitted curtoviruses, one of which, Beet mild curly top virus (BMCTV), was previously found in chili pepper in Zacatecas and Aguascalientes, Mexico (3). In the past few years, sporadic symptoms similar to curly top disease were observed in jalapeno pepper in the South Central area of Chihuahua State. Symptomatic plants were scattered in otherwise healthy looking pepper stands, and displayed stunting and yellowing. Affected leaves were brittle, showed upward curling, and distinct green vein pattern with interveinal yellowing. In June and August of 2010, field surveys were conducted in Cordillera-Escuadra, Meoqui-Estacion Consuelo, Meoqui-Lomas del Consuelo and Delicias-Presa Francisco I Madero. Ninety-four leaf samples were collected from symptomatic jalapeno pepper plants and subjected to ELISA and PCR testing for curly top. Of the 94 samples, 11 were found positive by TAS-ELISA with polyclonal antibodies against curly top (2). To confirm the identification of curly top and type the specific curtovirus identified, four ELISA-positive samples were subjected to a PCR analysis using virus-specific primer set for curtovirus typing designed by Chen et al. (1). All four samples tested produced a single 720-bp band with primers BSCTVv2688 and BGc396 (1) characteristic of the Beet severe curly top virus (BSCTV). These curly top-specific PCR amplicons were sequenced and found 99% similar to the BSCTV nucleotide sequence in the C1 gene region (e.g. accession number X97203); corresponding sequences were deposited in GenBank under accession numbers JF437870-JF437873. This is the first report of the curly top virus in the State of Chihuahua, demonstrating that curly top is established and common in jalapeno pepper here, and will need surveillance in other vegetable crops under irrigation.

Publications

  • Robles-Hernandez, L., Gonzalez-Franco, A.C., Gill-Langarica, E.M., Sago, C., Nikolaeva, O.V., and Karasev, A.V. (2011) First report of Beet severe curly top virus in jalapeno pepper in Chihuahua, Mexico. Plant Disease 95: 778-778.
  • Karasev, A.V., Hu, X., Brown, C.J., Kerlan, C., Nikolaeva, O.V., Crosslin, J.M., and Gray, S.M. (2011) Genetic diversity of the ordinary strain of Potato virus Y (PVY) and origin of recombinant PVY strains. Phytopathology 101: 778-785.
  • Kerlan, C., Nikolaeva, O.V., Hu, X., Meacham, T., Gray, S.M.,and Karasev, A.V. (2011) Identification of the molecular make-up of the Potato virus Y strain PVYZ: Genetic typing of PVYZ-NTN. Phytopathology 101: 1052-1060.