Progress 07/01/99 to 06/30/04
Outputs
Potato virus Y is one of the most common pathogens found in potato. It is found in all potato growing regions of the world and causes disease loss in the form of reduced yield or loss of quality. Recently, PVY infected potato samples were sent to the University of Idaho for analysis because they were unusual in their reaction to the serological assays commonly used to screen for potato virus certification. These assays were designed to detect strain O, but aberrant symptomological and serological results indicated the presence of a new PVY strain in U.S. certified potato seed lots. These virus isolates have been analyzed by host-range bioassay and by molecular techniques. There is considerable confusion in the scientific literature and GenBank accessions as to what isolates belong in what groups and in definitive assays for the delineation of these PVY strains. This report focuses on the correlation between biological observations of these new isolates and development
of molecular assays to identify and most effectively differentiate these PVY isolates. Serological and molecular analysis of the coat protein indicates that most of the isolates associated with veinal necrosis in tobacco belong to the necrotic strain of PVY that has not been previously observed in the potato growing regions of the North American continent. Of these, several isolates were also associated with tuber necrosis symptoms in potato (strain NTN). However, several isolates associated with veinal necrosis in tobacco and atypical tuber necrosis symptoms belonged to the O serotype. Because it is an O serotype, it will escape detection by any program that targets these new isolates using antibodies designed to detect N serotypes. This new type is similar to one recently reported to occur in Canada. Molecular analysis of the P1 region shows good correlation between banding patterns and observations of either veinal necrosis in tobacco or tuber necrosis in potato, with apparent
simultaneous infections with several distinct isolates observed. Sequence analysis of the P1 region shows that all new isolates associated with these symptoms belong to N, European NTN, or North American NTN PVY groups. So far, only Eu-NTN isolates produced tuber necrosis symptoms on cv. Ranger Russet in greenhouse tests. Molecular and biological characterization of these new isolates indicates the presence of natural recombinants between N serotype and O serotype in at least two instances. It is currently unknown if the recombination occurred before introduction or if N serotype strains are recombining with endemic O serotype strains. Only complete sequence analysis of the genome of the new isolates will provide definitive information on the origin of many of the new isolates. To accomplish this goal, we have generated full-length PCR products of the PVY genomes of the new isolates. Additional analysis of potato accessions used by U.S. potato breeders revealed a new strain of Potato
virus V that does not react to antisera prepared for the type virus. PVV has so far not been reported to occur in the U.S. and all known isolates of PVV have so far been confined to one serological strain.
Impacts
New viruses that have the potential to reduce yield and quality of potatoes are now present in the US. Results from this research have been directly used to assay for these new viruses in a sustained and coordinated effort to reduce and eradicate these pathogens from US seed potato stocks. In addition, this research has provided vital information on the prevalence and diversity of this virus threat to scientific and seed certification personnel within the state as well as nationally. Complete sequencing of several of these viruses has taken place and will provide definitive information on the identity and the source of these viruses. The discovery and characterization of a new strain of Potato virus V simultaneously provides information on a new virus and underscores the potential of these pathogens to circumvent the established detection system in place to prevent their entry into the US. Seed importation into the US requires strict screening for pathogens, often
relying on serological screening. Potato virus V is currently not present in the US and prevention of its importation may be hampered by the lack of serological reactivity of this newly described strain. The rapidly changing situation with both PVY and PVV indicate that there are still unknown sources of virus diversity present outside the US and that these sources can contribute to the pathogenicity and spread of these viruses.
Publications
- Shiel, P.J., Kopp, H.T., and Berger, P.H. 2003. Biological and molecular characterization of Potato Virus Y isolates from potato. Phytopathology 93:S78.
- Shiel, P.J., Miller, L., Slack, S.A., and Berger. P.H., 2003 Isolation and characterzatioon of a new isolate of Potato virus V with distinct biological and serological properties. Plant Disease (in press).
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Progress 01/01/02 to 12/31/02
Outputs
Work is proceeding to identify, characterize and determine evolutionary relationships of potyviruses. The Sunflower mosaic virus was characterized during the reporting period. Strains of Potato virus Y (PVY)that are either novel or were previously exotic to the US are in the process of being characterized. A reverse transcription-polymerase chain reaction-RFLP assay was modified from existing protocols to permit identification of these new isolates. Additional information on nucleotide sequence of these isolates was also obtained. The N strain (PVYN) was identified, as were isolates resembling European strains of NTN (PVYNTN). Isolates and strains identified as PVYNTN are, in fact, the result of recombination between PVYO (the common strain) and PVYN. PVYNTN is of particular concern because it is an international quarantine pest, and causes loss of quality of potatoes from infected plants as well as loss of yield. Furthermore, two additional isolates were identified.
One of these isolates has sequences that are similar to PVYNTN , but these sequences are phylogenetically distinct from sequences of other strains and isolates. Another isolate, which may be novel, also has PVYNTN-related sequences, but recombination has resulted in a virus whose 3'-end has PVYO sequences. PVYO sequences in this virus include the coat protein region, which means that it will appear to PVYO on the basis of serological tests commonly used by state potato seed certification organizations. Since low levels of PVYO are permitted in seed, particularly in more advanced field generations, this recombinant virus will likely continue to be propagated in our seed systems. This isolate, as well as the other isolates new to the US represent a significant threat to the US potato industry. Work is underway to more thoroughly characterize these isolates of PVY biologically and molecularly, to obtain improved diagnostic reagents and procedures, and to obtain better information on the
distribution and prevalence of PVY in US potatoes.
Impacts
Strains of Potato virus Y (PVY) that were previously unknown in the US have been identified. Results from this work have provided improved diagnostic tools capable of identifying strains and isolates of PVY. These tools represent an intermediate step towards obtaining high-throughput tools that can be utilized by state potato seed certification organizations. Complete characterization of these strains and isolates will provide definitive information as to their identity and may permit identification as to the source of these viruses.
Publications
- Crosslin, J.M., Hamm, P.B., Eastwell, K.C., Thornton, R.E., Brown, C.R., Corsini, D., Shiel, P.J., and Berger, P.H. 2002. First report of PVYN potyvirus on potatoes in the northwestern United States. Plant Disease 86:1177.
- Gulya, T.J., Shiel, P.J., Freeman, T., Jordan, R.L., Isakeit, T., and Berger, P.H. 2002. Host range and characterization of Sunflower mosaic potyvirus. Phytopathology 92:694-702.
- Eigenbrode, S.D., Ding, H., Shiel, P., and Berger, P.H., 2002 Volatiles released from Potato leafroll virus-infected potatoes are attractants and arrestants for its vector, Myzus persicae. Proc. R. Soc. Lond. B 269:455-460.
- McCarthy,P.L., J.L. Hansen, R.S. Zemetra, and P.H. Berger. 2002. Rapid identification of transformed wheat using a half-seed PCR assay prior to germination. Biotechniques 32:560-564.
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Progress 01/01/01 to 12/31/01
Outputs
The purpose of this project is to study evolution of plant viruses, particularly viruses in family Potyviridae. Under the auspices of this project, a more general objective is characterization of viruses of importance to Idaho and US agriculture, as well as development of methods for their control, particularly using biotechnological approaches. There are several specific research projects intended to address the above objectives. 1) Characterization and detection of isolates of Potato virus Y (PVY) novel or exotic to the Pacific Northwest. At least two and possibly more isolates of PVY have been observed in the PNW. We are in the process of confirming the precise identity of these isolates and developing methods for their detection. Related to this project is work aimed at developing high throughput diagnostic methods for a range of potato viruses, with an aim towards eventually transferring this technology to state certification agencies. 2) Development of
transgenic wheat with virus resistance. This work emphasizes wheat cultivars grown in the PNW. We have created winter wheat with coat protein-mediated resistance to Barley yellow dwarf virus and to Wheat streak mosaic virus. Work underway includes field testing of potentially useful selections, and utilizing and/or developing methods to reduce or eliminate transcriptional gene silencing. Work is also progressing on studying the relationship between transgenic wheat and effects on virus-vector interactions. 3) Development of transgenic peas with virus resistance. This work emphasizes grain legumes typically grown in the PNW. We have extensive laboratory, greenhouse, and field data on peas with coat protein-mediated resistance to Pea enation mosaic virus (PEMV), and non-pathogen-derived resistance to both PEMV and pea streak virus. Work is in progress to provide detailed molecular genetic information on selected lines and to complete a third year of field testing. 4) Molecular evolution
of potyviruses. This work is described in the CRIS report for IDA09904-CG.
Impacts
The work on potato virus detection will have impact on the cost and effectiveness of potato seed certification. The work on PVY will also impact seed certification, and could also have an effect on export of US potato seed. Development of virus resistant wheat will decrease the cost of production as well as decreasing yield loss due to BYDV and WSMV. A similar benefit would be realized with resistance to virus diseases in peas.
Publications
- Gulya, T.J., Shiel, P.J., Freeman, T., Jordan, R.L., Isakeit, T., and Berger, P.H. 2002. Host range and characterization of Sunflower mosaic potyvirus. Phytopathology. (In Press).
- Eigenbrode, S.D., Ding, H., Shiel, P., and Berger, P.H. 2002. Volatiles released from Potato leafroll virus-infected potatoes are attractants and arrestants for its vector, Myzus persicae. Proc. Roy. Soc. London (in press)
- McCarthy, P.M., and Berger, P.H. 2002. Rapid identification of transformed wheat using a half-seed pcr assay prior to germination. Biotechniques (in press)
- Jimenez, E. S., N. A. Bosque-Perez, P. H. Berger, and R. S. Zemetra. 2001. Life history of the bird cherry-oat aphid, Rhopalosiphum padi., in untransformed and transgenic wheat challenged with barley yellow dwarf virus. Ann. Meeting Ent. Soc. America, San Diego, CA.
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Progress 01/01/00 to 12/31/00
Outputs
It was hypothesized and subsequently demonstrated that RNA viruses exist as a 'quasi-species', which has been the topic of a number of recent reviews and theoretical treatments. In essence, a quasi-species distribution describes a metastable equilibrium which will disintegrate and be replaced by another metastable equilibrium when a mutant appears that is more competitive. Thus, a quasi-species is defined as a mixture of viral sequences containing a predominant sequence and a variable mixture of related, but molecularly distinct, sequences. There is, however, little information on the plant viruses, particularly the potyviruses, yet this biological phenomenon raises a number of important questions. What is the relationship between a plant virus' quasi-species distribution of molecular sequences and its fitness? How do these characteristics change as specific environmental conditions change? How might they change if the virus infects a different host? Can we observe
evidence of adaptation at the nucleotide or amino acid sequence level? This proposal is aimed at obtaining information on sequence diversity in certain potyvirus populations, and determining the nucleotide substitution frequency in these populations. The effective date of this project was 1 July, 1999. We have focused our attention initially on bean common mosaic potyvirus (BCMV), and are now working with an infectious clone of soybean mosaic potyvirus (SMV). We have generated six populations of BCMV and four of SMV, by serial passage. We are also generating single passage populations of SMV derived directly from individual plants using the infectious DNA plasmid clone. Working with our initial passages of BCMV and concentrating on the region encompassing the amino terminus of the coat protein cistron, we have examined several hundred clones, of which only a few contain nucleotide substitutions. Thousands of clones remain to be screened, and we are utilizing single conformation
polymorphism assays to examine these. Furthermore, we are in the process of developing microarray-based methods that may allow us to screen very large numbers of clones for the presence of nucleotide substitutions. If this is successful, our throughput will be greatly increased.
Impacts
The goal of this research is to determine the frequency of mutations in potyvirus populations. This will provide necessary background information on population dynamics of these viruses and eventually information on the rate of mutation.
Publications
- Berger, P.H. 1999. The Bymoviruses. In: Encyclopedia of Plant Pathology. John Wiley & Sons.
- Berger, P.H., and German, T.L. 2000 Biotechnology and resistance to potato viruses. Chapt. 13 in: Identification and Control of Potato Viruses. Brunt, A., Berger, P.H., Lawson, R., and Loebenstein, G. (eds.). Kluwer Academic Publ., Berlin (in press)
- Shiel, P.J., and Berger, P.H. 2000. The nucleotide sequence of apple mosaic virus RNAs 1 and 2 and their relationship to other Ilarviruses and alfalfa mosaic virus. J. Gen. Virol. 81:273-278.
- Berger, P.H. and Parrish, J. 2000. Potyviruses. In: The Springer Index of Viruses. Springer-Verlag. Brunt, A., Berger, P.H., Lawson, R. and Loebenstein, G. (eds.) 2000. Identification and Control of Potato Viruses. Kluwer Academic Publ., Berlin (in press)
- Berger, P.H., Barnett, O.W., Brunt, A.A., Colinet, D., Edwardson, J.R., Hammond, J., Hill, J.H., Jordan, R.L., Kashiwazaki, S., Makkouk, K., Morales, F.J., Rybicki, E., Spence, N., Ohki, S.T., Uyeda, I., van Zaayen, A. and Vetten, H.J. 2000. The Potyviridae. in: Virus Taxonomy. 7th Report of the International Committee on Taxonomy of Viruses. Springer-Verlag, Berlin. pp. 703-724.
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Progress 01/01/99 to 12/31/99
Outputs
It was hypothesized and subsequently demonstrated that RNA viruses exist as a 'quasi-species', which has been the topic of a number of recent reviews and theoretical treatments. In essence, a quasi-species distribution describes a metastable equilibrium, which will disintegrate and be replaced by another metastable equilibrium when a mutant appears that is more competitive. Thus, a quasi-species is defined as a mixture of viral sequences containing a predominant sequence and a variable mixture of related, but molecularly distinct, sequences. There is, however, little information on the plant viruses, particularly the potyviruses, yet this biological phenomenon raises a number of important questions. What is the relationship between a plant virus' quasi-species distribution of molecular sequences and its fitness? How do these characteristics change as specific environmental conditions change? How might they change if the virus infects a different host? Can we observe
evidence of adaptation at the nucleotide or amino acid sequence level? This proposal is aimed at obtaining information on sequence diversity in certain potyvirus populations, and determining the nucleotide substitution frequency in these populations. We have focused our attention initially on bean common mosaic virus (BCMV), since it has well described strains with known pathotypes. Initially, we are examining the amino terminal region of the coat protein, since this is known to be a highly variably region of potyviruses. By reverse transcriptase-polymerase chain reaction, we obtained several hundred clones encompassing this region from a virus population. So far, we have seen only a single nucleotide substitution in 70 clones. We will continue to collect data from this population, but in the meantime we have passaged the virus twice. In the second passage we observed two clones with single nucleotide changes. One introduces a silent mutation while the other introduces a stop codon
which is presumably a fatal mutation. Several hundred clones remain to be screened. We have examined seven clones from the last passage, and observed no mutations. We plan to repeatedly passage this population, and also introduce it to another host determine if the frequency of mutations is affected. As this project progresses, we will also work with an infectious plasmid clone of the related soybean mosaic virus (SMV), which should serve to reduce heterogeneity in the starting population.
Impacts
This research provides necessary background information on population dynamics of these viruses and eventually information on the rate of mutation.
Publications
- Shukla, D.D., Ward, C.W., Brunt, A.A., and Berger, P.H. 1999. Potyviridae. AAB Descriptions of Plant Viruses. No.366 (No. 245 revised)
- Berger, P.H. 1999 Potyviridae. In: Encyclopedia of Life Sciences. Macmillan Reference Limited, London, UK.
- Berger, P.H. 1999. The Bymoviruses. In: Encyclopedia of Plant Pathology. John Wiley & Sons. (in press)
- Berger, P.H., Barnett, O.W., Brunt, A.A., Colinet, D., Edwardson, J.R., Hammond, J., Hill, J.H., Jordan, R.L., Kashiwazaki, S., Makkouk, K., Morales, F.J., Rybicki, E., Spence, N., Ohki, S.T., Uyeda, I., van Zaayen, A, Vetten, H.J. 2000. The Potyviridae. in: Virus Taxonomy. 7th Report of the International Committee on Taxonomy of Viruses. Springer-Verlag, Berlin. (in press)
- Shiel, P.J., and Berger, P.H. 2000. The nucleotide sequence of apple mosaic virus RNAs 1 and 2 and their relationship to other Ilarviruses and alfalfa mosaic virus. J. Gen. Virol. (in press)
- Eberlein, C.V., Guttieri, M.J., Berger, P.H., Fellman, J.K., Mallory-Smith, C.A., Thill, D.C., Baerg, R.J., and Belknap, W.R. 1999. Physiological effects of mutation for resistance in a near isonuclear ALS-inhibitor resistant Lactuca sativa line. Weed Science 47:383-392.
- Mink, G. I., Vetten, H. J., Wyatt, S. D., Berger, P. H., and Silbernagel, M. J. 1999. Epitopes located on the N terminal portion of the capsid protein can be used to subdivide serotype B viruses within the bean common mosaic virus subgroup. Arch. Virol. 144:1173-1189.
- Berger, P.H., and Cavileer, T.D. 1999. The frequency of nucleotide substitutions in populations of bean common mosaic potyvirus in the amino terminal region of the coat protein cistron. XIth Int. Congr. Virology, 9-13 August, Sydney, Aust.
- Berger, P.H., and Cavileer, T.D. 1999. Nucleotide substitution frequencies in populations of bean common mosaic potyvirus in the amino terminal region of the coat protein cistron. XVth Int. Working Group on Legume Viruses, 15-17 August, Freemantle, Aust.
- Berger, P.H., Hammond, J., Stenger, D.L., 1999. Taxonomy of the family Potyviridae. XIth Int. Congr. Virology, 9-13 August, Sydney, Aust.
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