DETECTION AND CHARACTERIZATION OF GEMINIVIRUSES AND MANAGEMENT OF GEMINIVIRUS-INDUCED DISEASES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0156874
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Plant Pathology

Non Technical Summary
Plant disease caused by geminiviruses, a unique group of single-stranded DNA viruses that are transmitted by leafhoppers or whiteflies, can cause devastating losses to crop production. The introduction of the exotic Tomato yellow leaf curl virus into southern California in 2007 poses a potential threat to the tomato industry of the state. New whitefly-transmitted geminiviruses are appearing worldwide due to the spread of the insect vector. In this project new highly sensitive molecular tools will be used to detect and characterize known and newly emerging geminiviruses. These tools also will be used to learn more about the biology of the virus and to develop improved management strategies. The results of this project will allow for more rapid detection of the appearance and spread of these viruses, allowing for implementation of management strategies before the viruses become an economic problem. The improve understanding of the biology of the viruses will allow for development of effective integrated pest management strategies that will minimize reliance on insecticide applications to control the insect vectors of these viruses. Finally, this information will be presented to growers, professional crop consultants and others through printed materials (flyers and publications in popular magazines and journals), grower talks and presentations and material made available on-line.

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
212	1410	1101	25%
212	1420	1101	25%
212	1460	1101	25%
212	1461	1101	25%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1460 - Tomato; 1410 - Beans (dry); 1461 - Peppers; 1420 - Melons;

Field Of Science
1101 - Virology;

Keywords

geminiviruses, begomoviruses, curotviruses, tomato yellow leaf curl

virus, curly top viruses, exotic pathogens, emerging diseases,

whiteflies, leafhoppers, insect-transmitted plant viruses, rapid

detection and diagnosis, polymerase chain reaction, plant disease

ecology and epidemiology, integrated pest management

Goals / Objectives
The overall goal of this project is to identify, understand and manage plant diseases caused by geminiviruses in California, the United States and worldwide. Geminiviruses are an emerging group of single-stranded DNA viruses that cause economically important diseases of vegetable crops including cotton, cucurbits, peppers and tomatoes. The importance of the diseases caused by these viruses is increasing worldwide and there is an urgent need to develop an improved understanding of these viruses as well as effective management strategies. This project involves the development and application of improved detection tools for geminiviruses, investigation of the biology and genetics of selected geminiviruses, and the development of disease management strategies. This research will allow for the rapid detection and identification of these important viruses, including known and new emerging viruses. Using molecular tools, many of which have been developed in our laboratory, studies of geminivirus biology and ecology will be conducted. In these studies, emphasis will be placed on the leafhopper-transmitted geminiviruses that cause curly top disease and the whitefly-transmitted viruses that cause tomato yellow leaf curl. In the latter case, TYLC is caused by the recently introduced Tomato yellow leaf curl virus (TYLCV). The results of these studies will be applied to developing management strategies for this group of viruses. The specific objectives of this project are: 1) Continue to develop and apply molecular techniques for the detection and characterization of geminiviruses infecting crop plants such as tomatoes, peppers and cucurbits; 2) generate full-length infectious clones of selected geminiviruses and develop and utilize efficient delivery systems to determine viral host range, screengerm plasm for disease resistance and/or to study molecular genetics of these viruses and 3) use molecular methods for detection and characterization of geminiviruses to gain better insight into the ecology and epidemiology of these viruses in California and/or other regions. Expected outputs of the project include an improved understanding of the diversity and nature of geminiviruses in California, the United States and other areas of the world. In particular, the spread and incidence of TYLCV in California will be followed and the potential impact of this exotic virus on the California tomato industry will be better understood. It is also expected that better and more sensitive detection technologies for these viruses also will be developed. An improved understanding of the biology of geminivirus diseases will be generated, including the interactions of the viruses with host and non-host plants, insect vectors, other viruses and the environment. Information generated from these studies will be used for the development of improved disease management strategies, including integrated pest management (IPM) approaches. Finally, the information generated from this study will be made available via publications, grower presentations, and on-line information.

Project Methods
In collaboration with our collaborators, we will collect samples of plants with virus-like disease symptoms and test for the presence of geminivirus infection. These samples will have symptoms such as stunted and distorted growth and leaf yellowing, crumpling, curling, distortion, and mottle/mosaic. For begomoviruses, we will focus on areas where the whitefly vector (B. tabaci) is prevalent (southern California), whereas for curotviruses, we will focus on counties that typically have curly problems (Fresno, Imperial, Kern, Kings and Merced). Particular emphasis will be placed on monitoring for TYLCV in Imperial and Riverside Counties, both in tomatoes and in weeds. The presence of geminivirus DNA in these samples will be determined with nucleic acid hybridization probes and/or PCR. These methods are now well developed in our laboratory. Nucleotide sequences of these PCR-amplified fragments will be determined, and compared with sequences of already characterized geminiviruses. In this way, we will be able to determine the precise identity of geminiviruses associated with these plant samples. In some cases, such as identification of a new virus or a variant of an existing virus, attempts may be made to clone the complete genome of the virus(es) involved. This will be accomplished using enriched double-stranded replicative form extracts or the more recently developed rolling circle amplification (RCA) method. In other cases, overlapping primers may be developed that will allow amplification of the viral DNA by PCR. Methods used to test infectivity of clones will include rub-inoculation of the cloned DNAs onto N. benthamiana plants and agroinoculation and particle bombardment. Particle bombardment and agroinoculation methods allow for inoculation of greater numbers of plants, such as in host range and germplasm screening experiments. We have already developed agroinoculation system for numerous geminiviruses including TYLCV and curly top viruses. The presence of viral DNA in inoculated plants will be determined by PCR. Establishment of the host range of these viruses will provide important information for disease management, and the germplasm screening will reveal useful sources of disease resistance that could be used in a breeding program and for eventual use in an IPM program. For selected geminivirus disease problems (i.e., those that pose a serious threat to agricultural production such as TYLCV), we will use molecular tools (e.g., DNA hybridization probes and PCR) to study the ecology/epidemiology of geminiviruses under field conditions, e.g., surveys of selected areas for alternate hosts or for monitoring the time and/or incidence of the appearance of geminivirus infections. The presence of the virus in insect vectors (leafhoppers or whiteflies) may also be monitored as a possible predictor of virus outbreaks and/or to provide insight into effects of cultural practices on the incidence of the virus. The results of these studies will provide new information about the importance and biology of geminiviruses and help in the development of disease management strategies.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:The target audience is tomato and other grower groups, people involved in the seed industry and virologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work was performed by a Project Scientist, Dr. Maria Rojas; two postdoctoral researchers, Dr. Monica Macedo and Dr. Tomas Melgarejo; and three graduate students, Mr. Minor Maliano, Ms. Juliana Souza and Mr. Yousef Mater. Being involved in this project, these young aspiring scientists gained valuable experience in molecular detection and characterization of gemoniviruses. Moreover, they learned how this information can be used for management of diseases caused by these viruses. These scientists also gained experience in writing their work for publication and contributing to the Annual Review of Phytopathology article entitled: World Management of Geminiviruses. In part due to this experience, Dr. Macedo recently was offered and accepted a faculty position in plant virology in Brazil. How have the results been disseminated to communities of interest?The results have been disseminated through the presentation of talks at grower and professional meetings as well as a number of scientific publications, including a prestigious Annual Review of Phytopathology article on World Management of Geminiviruses. The results also have been presented in reports to the California Tomato Reseach Institute and the California Department of Food and Agriculture (CDFA). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the five year period of this project, we have made progress in the following areas: 1) application of new detection technologies for geminiviruses, 2) monitoring for BCTV and TYLCV in California, 3) characterization of new geminiviruses in other parts of the world and 4) integrated pest management of geminiviruses. In terms of new detection technologies, we have extensively tested and demonstrated that the Whatman FTA card technology is very reliable for preparing, storing and sending plant samples from testing for geminivirus infection. In addition, rolling circle amplification (RCA) method has been successfully used to amplify and facilitate cloning of the complete genomes of geminiviruses, including begomoviruses (whitefly-transmitted geminiviruses) and curtoviruses (leafhopper-transmitted geminiviruses. Finally, we also initiated the use of high throughput sequencing (HTS) for geminivirus diagnostics, and this has helped us identify a novel geminivirus-like agent associated with Brazilian curly top disease. The FTA cards now allow for easy and efficient sample preparation, thereby obviating the need to send plant tissue samples. Although these detection methods do not replace the tried and true method of PCR and sequencing, they help to identify highly divergent geminivirus-like entities and to amplify small amounts of viral DNA for additional studies. During the duration of this project, we developed new tools for monitoring and predicting the severity of curly top disease in a given growing season. This involved the collection of leafhoppers and testing for the presence of BCTV by a PCR-based method. Then prediction of the severity of the disease the coming growing season is made based on the relative population of leafhoppers and the viral titer. In the years following the severe 2013 curly top outbreak in tomatoes in California, we have accurately predicted the severity of curly top disease in each of the subsequent growing seasons. We also have carefully monitored for BCTV infections in weeds and conducted studies to assess the capacity of leafhoppers to acquire BCTV from weeds with low virus titers. Together, we have learned much about the cause and nature of curly top disease and can now inform growers when the potential for curly top is high for a given year, thereby allowing for the implementation of management strategies. Following the introduction to TYLCV into Southern California in 2007, it was feared that the virus could move north and impact commercial processing tomato production in Kern County. Fortunately, surveys and tests continue to show that TYLCV has not moved north, most likely due to the early harvest and late appearance of whiteflies. However, every year, we get samples of tomato with yellow leaf curl symptoms from souther California and Arizona and confirm TYLCV infection in most of these samples. This demonstrates that TYLCV is persisting in these areas and that any commercial production of tomatoes in these areas will require management of this devastating virus. Of course, if warming climates allow for B. tabaci to overseason in northern locations in California. In the application of these methods for geminivirus detection and characterization, we identified a new strain of Beet curly top virus infecting tomatoes and peppers in California, BCTV-Spinach curly top (BCTV-SpCT). Previously, this strain had only been detected in Texas. Using RCA, the viral genome was amplified, cloned and sequenced. An agroinoculation system was generated and used to establish that this strain induces severe symptoms of curly top disease in tomatoes, peppers, and tobacco. Therefore, BCTV-SpCT has the potential to cause severe curly top symptoms in crops in California. Together, with previous studies, we have established that curly top disease of tomato in California is caused by at least six distinct strains of BCTV, with the predominant ones being BCTV-Colorado (BCTV-CO) and BCTV-LH71. In Brazil, we characterized a new begomovirus infecting tomatoes in Northwestern Brazil and demonstrated that it was a new species of New World monopartite begomovirus and it was named tomato leaf curl purple vein virus. A full-length clone of this viruse was generated and shown to induce disease symptoms following delivery by agroinoculation. Therefore, Koch's postulates were fulfilled for this new begomovirus species.This is yet another example of NW monopartite begomoviruses, which were first described in 2013 and represent a new direction in begomovirus evolution. In West Africa, we characterized a complex of monopartite begomoviruses and betasatellites that are associated with tomato leaf curl disease. The most effective management of geminiviruses involves the application of integrated pest management (IPM) approaches. The most effective IPM approaches are generated based upon proper identification of the viruses involved and a thorough understanding of the biology of the virus and options available for management. We have now brought all of this information together in an article on 'World Management of Geminiviruses', which was written in 2017 and published in 2018. In this collaborative article, the major geminivirus diseases and the viruses involved, their biology and available management strategies are presented. Then for each of these diseases an IPM approach is presented that included approaches that can be used before, during and after the growing season. We feel that this article will provide very useful information for growers, crop consultants, seed producer and academics and bring together efforts on management of these viruses.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Chen, L. F., Batuman, O., Aegerter, B. J., Willems, J., and Gilbertson, R. L. 2017. First report of curly top disease of pepper and tomato in California caused by the Spinach curly top strain of Beet curly top virus. Plant Dis. 101: 1334.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Macedo, M. A., Albuquerque, L. C., Maliano, M. R., Souza, J. O., Rojas, M. R., Inoue-Nagata, A. K., and Gilbertson, R. L. 2018. Characterization of tomato leaf curl purple vein virus, a new monopartite New World begomovirus infecting tomato in Northeast Brazil. Arch. Virol. 163:737-743.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Rojas, M.R., Macedo, M.A., Maliano, M.R., Soto-Aguilar, M., Souza, J. O. et al. 2018. World management of geminiviruses. Ann. Rev. Phytopathol. 56: 637-76.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Molecular characterization of begomoviruses and betasatellites associated with tomato leaf curl disease in Ghana reveals additional genetic diversity and betasatellite specificity

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience is tomato and other grower groups, people involved in the seed industry and virologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work was performed by a Project Scientist, Dr. Li-Fang Chen, nd this was part of her continued professional development in molecular virology and plant molecular biology. Indeed, during this time period, Dr. Chen obtained a job with Monsanto, in part due to the training she received in the laboratory. In addition, a number of undergraduate students assisted on this project and learned various techniques in molecular biology including DNA extraction and PCR. How have the results been disseminated to communities of interest?The results have been disseminated through the presentation of talks at grower and oprofessional meetings. The results also have been presented in reports to the California Tomato Reseach Institute and the California Department of Food and agriculture. Finally, a manuscript describing this finding has been written and will be submitted for publication. What do you plan to do during the next reporting period to accomplish the goals?We will continue to characterize geminiviruses associated with crops in the United States and world wide and develop improved methods for virus detection and screening for resistance to these viruses in crop germ plasm.

Impacts
What was accomplished under these goals? A major outbreak of curly top disease in 2013, caused by the beet leafhopper (Circulifer tenellus)-transmitted Beet curly top virus (BCTV, genus Curtovirus, family Geminiviridae), led to ~$100 million in losses to the processing tomato (Solanum lycopersicum) industry in California. In response to this outbreak, we have been characterizing strains of BCTV associated with the disease in tomato, other crops and weeds in the Central Valley of California to understand the nature of strain(s) involved. In May 2014, a tomato sample from the San Joaquin County with typical curly top symptoms, including stunting and crumpling, curling and vein purpling of leaves, was collected and tested for BCTV infection with a multiplex PCR and the general BCTV primers BGc396/BMCTVv2825/BSCTVv2688. A 0.7 kb DNA fragment, indicative of infection by the BCTV-Severe (Svr) and BCTV-Pepper curly top (PeCT) strains, was amplified from this sample. However, no DNA fragment was amplified in subsequent PCR tests with strain-specific primers for BCTV-Svr (Cfh-c197 5'-CTTGTGGGGACCACAATGATCTAC-3') and BCTV-PeCT (BV3-c1484 5'-GGATTGGGTACTGGAGCGTCAA-3') paired with BSCTVv2688. Sequence analysis of the 0.7 kb DNA fragment revealed 98% identity with the sequence of the spinach curly top strain of BCTV (BCTV-SpCT [US-Sp3-96], AY548948, previously named Spinach curly top virus), and <85% identities with sequences of other BCTV strains. The full-length genome of this BCTV isolate was generated with rolling-circle amplification and digestion with Xba I, and the resulting ~2.9 kb DNA fragment was cloned into pBluescript II (Stratagene) and sequenced. The complete nucleotide sequence (KT583749) was 2925 bp and 97% identical with the sequence of BCTV-SpCT [US-Sp3-96], which came from a spinach plant with curly top symptoms in Texas. This California isolate was named BCTV-SpCT [US-CA-14]. The infectivity of the cloned BCTV-SpCT [US-CA-14] genome was established by generating a 1.4-mer clone in the binary vector pCambia1300, and transforming into Agrobacterium tumefaciens EHA105. Tomato plants agroinoculated with this multimeric clone developed typical curly top symptoms by 12 days post inoculation. BCTV DNA in symptomatic plants was confirmed by PCR with the BGc396/BSCTVv2688 primer pair. In a partial host range test performed with agroinoculation and beet leafhopper transmission, BCTV-SpCT [US-CA-14] induced severe curly top symptoms in common bean, Nicotiana benthamiana (agroinoculation only), pepper, shepherd's purse (Capsella bursa-pastoris), spinach, sugar beet and tomato plants. These results also established the leafhopper transmissibility of BCTV-SpCT [US-CA-14]. To specifically detect BCTV-SpCT, a primer was designed (SpCT-c636, 5'-CCATACAGTCTGGAACTTGGGTG-3') that, when paired with BSCTVv2688, directs the amplification of a diagnostic ~0.9 kb DNA fragment. PCR tests with SpCT-c636/BSCTVv2688 and DNA extracts from 2013 and 2014 pepper and tomato curly top samples revealed BCTV-SpCT infection in 4 out of 316 tomato samples, including one each from Contra Costa and Solano counties, and two from San Joaquin Co.; and 3 out of 22 pepper samples from San Joaquin Co. This is the first report of BCTV-SpCT in California (and outside of Texas), and causing curly top of peppers and tomatoes in the field. These results also revealed additional genetic diversity in BCTV causing curly top disease in California.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Macedo, M. A., Costa, T. M., Barbosa, J. C., Pereira, J. C., Michereff-Filho, M., Gilbertson, R. L., Inoue-Nagata, A. K., and Bergamin Filho, A. 2016. Temporal and spatial dynamics of begomovirus disease in tomatoes in central Brazil. Doi: 10.1111/ppa.12632
• Type: Book Chapters Status: Published Year Published: 2016 Citation: Chen, L. F. and Gilbertson, R. L. Transmission of curtoviruses (Beet curly top virus) by the bee leafhopper (Circulifer tenellus). In: Vector-Mediated Transmission of Plant Pathogens. J.K Brown, ed. American Phytopathological Society., St. Paul, MN. p. 243-262.

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

Outputs
Target Audience:The target audience for this information is twofold. First, it includes virologists and other researchers who are interested in plant virus diversity and evolution as it pertains to geminiviruses, which are a group of single-stranded DNA viruses that are the largest (in terms of number of species) and most economically important. Such researchers are at academic and government institutions. Second, this information will be of interest to growers, professional crop consultants and breeders and pathologists in seed companies, particularly those active in tropical and sub-tropical locations, where whitefly-transmitted viruses can be a limiting factor. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two international graduate students worked on projects associated with this project during this reporting period: Mr. Yousef Mater from Kuwait (M.S. student) and Ms. Juliana Osse de Souza from Brazil (PhD student). Both students received extensive training in many areas of molecular biology and virology as well as scientific writing. Both participated in a journal club that helped them learn the scientific method. Mr. Mater completed his Master's thesis research during this period and was awarded his M.S. in Plant Pathology from the University of California-Davis. How have the results been disseminated to communities of interest?The results have been reported in informal discussions to growers and crop consultants, in presentations at scientific meetings as well as in a scientific publications. What do you plan to do during the next reporting period to accomplish the goals?We will continue to detect and characterize geminiviruses, understand the biology and genetics of these viruses and use this information to develop effective management strategies.

Impacts
What was accomplished under these goals? Until recently, all indigenous whitefly-transmitted geminiviruses (genus Begomovirus) from the New World had bipartite genomes, composed of two appox. 2.6 kb circular ssDNA components referred to as DNA-A and DNA-B. However, recently (2013-14) an indigenous NW monopartite begomovirus causing leaf curl disease in tomatoes was described from Peru, and it was named Tomato leaf deformation virus. In August 2014, tomatoes with symptoms typical of a monopartite begomovirus, i.e., stunting and upcurled leaves with purple, swollen veins, were observed in northeastern Brazil (Piaui State). These tomato plants also sustained high populations of whiteflies, Bemisia tabaci. Leaf samples from twenty plants with these symptoms were applied to FTA cards and then total genomic DNA was extracted. PCR analysis was performed with degenerate DNA-A and DNA-B primers and the expected ~1.2 kb DNA-A fragment was amplified, whereas no DNA-B fragment was amplified. Circular DNAs associated with these plants were amplified by rolling circle amplification (RCA) and digestion of these DNAs with the enzyme Msp I revealed DNA fragments that added up to ~2.6, consistent the presence of a single genomic DNA. DNA sequence analysis revealed that the PCR-amplified DNA-A fragment had the highest identity (>90%) with the DNA-A component of Tomato mottle leaf curl virus (ToMoLCV). To further characterize this genomic DNA, a full-length DNA of one isolate (C2) was generated by RCA, digested with Nde I and cloned. The C2 genomic DNA is 2630 nucleotides, shares 94% sequence identity with ToMoLCV and has a genome organization typical of the DNA-A components of NW bipartite begomoviruses. Phylogenetic analyses placed the C2 isolate in in a distinct clade with other isolates of ToMoLCV from Brazil. To confirm the monopartite nature of the virus and to fulfill Koch's postulates, a multimeric C2 clone was generated and transformed into Agrobacterium tumefaciens. Tomatoes agroinoculated with the C2 multimeric clone developed leaf curling and vein swelling and purpling similar to those observed in the field. Together, these results establish that ToMoLCV is the forst indigenous NW monopartite begomovirus from Brazil.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Inoue-Nagata, A. K., Lima, M. F., and Gilbertson, R. L. 2016. A review of geminivirus (begomovirus) diseases in vegetables and other crops in Brazil: current status and approaches for management. Horticultura Brasileira 34: 8-18.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Gilbertson, R. L., Batuman, O., Webster, C. G., and Adkins, S. Role of insect supervectors Bemisia tabaci and Frankliniella occidentalis in the emergence and global spread of plant viruses. Ann. Rev. Virology 2: 67-93.
• Type: Other Status: Published Year Published: 2015 Citation: Macedo, M., Gilbertson, R. L., and Bergamin Filho, B. 2015. Incidence of begomovirus and tospovirus diseases in processing tomato fields in three Midwest states of Brazil in 2013 and 2014. Phytopathology 105: S4.87
• Type: Other Status: Published Year Published: 2015 Citation: Mater, Y. J. Chen, L. F., Osei, M. K., and Gilbertson, R. L. 2015. Genetic analysis of begomoviruses and betasatellites associated with tomato leaf curl disease in Ghana. Phytopathology 105: S4.90.
• Type: Other Status: Published Year Published: 2015 Citation: Vu, S., de Souza, J., Melgarejo, T. A., Chen, L. F., Macedo, M., Nagata, A., and Gilbertson, R. L. 2015. Evidence that Tomato mottle leaf curl virus from Northeastern Brazil is an indigenous New World monopartite begomovirus. Phytopathology 105: S4.143.

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

Outputs
Target Audience: The target audience for this information is primarily virologists and other researchers who are interested in plant virus diversity and evolution. Such researchers are at academic and government institutions. To a less extent, this information will be of interest to growers and professional crop consultants in tropical and sub-tropical locations, where whitefly-transmitted viruses can be a limiting factor, as well as individuals that work for seed companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Mr. Tomas Melgarejo, a Ph.D student in the laboratory of Dr. Robert L. Gilbertson, performed most of the work on the Jatropha mosaic viruses. This provided Tomas with training in many areas of virology and molecular biology. In addition, he received valuable experience in scientific writing during the course of the preparation of the scientific paper on this topic. Finally, Mr. Melgarejo completed his Ph.D. research during this period and he was awarded his Ph.D. in Plant Pathology from the University of California-Davis. How have the results been disseminated to communities of interest? The results have been reported in informal discussions to growers and crop cunsultants, in presentations at scientific meetings as well as in a scientific publication in the journal Phytopathology. What do you plan to do during the next reporting period to accomplish the goals? We will continue to detect and characterize geminiviruses, understand the biology and genetics of these viruses and use this information to develop effective management strategies.

Impacts
What was accomplished under these goals? In tropical and subtropical countries in the Americas, weeds commonly show symptoms of infection by whitefly-transmitted geminiviruses (begomoviruses) including bright yellow mosaic and mottling. A long-standing questions is whether such weeds serve as inoculum sources of crop-infecting begomoviruses. Here, we characacterized the begomovirus(es) associated with yellow mosaic symptoms in the weed plant Jatropha spp. in the Dominican Republic. The complete nucleotide sequences of the DNA-A and DNA-B components of four viruses from three geographic regions were determined. Sequence comparisons revealed the highest identities (91-92%) with Jatropha mosaic virus (JMV), a begomovirus partially characterized from Jamaica, indicating that these viruses from the Dominican Republic were strains of JMV. When introduced into jatropha seedlings by particle bombardment, the cloned DNA-A and DNA-B components of the JMV strains from the Dominican Republic induced stunting and yellow mosaic, indistinguishable from symptoms observed in the field, thereby fulfilling Koch's Postulates for the disease. The JMV strains also induced disease symptoms in Nicotiana benthamiana, tobacco and several cultivars of common bean from the Andean gene pool, including several grown locally in the Dominican Republic. Thus, JMV in the DO is a complex of genetically distinct strains that have undergone local evolution and have the potential to cause disease in crop plants. However, the fact that JMV infections have been rarely observed in tobacco or bean crops in the Dominican Republic suggests that it is not economically important in these crops. This may be due to specialization of the whitefly vector on jatropha, which would minimize the spread of JMV to crop plants. Thus, jatropha infected with JMV does not serve as a source of crop-infecting begomoviruses, even though the JMV strains infecting this plant can infect crop plants under laboratory conditions. This work further illustrates the complexities of determining whether weeds are inoculum sources of crop infecting begomoviruses.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Melgarejo, T. A., Kon, T., and Gilbertson, R. L. 2014. Molecular and biological characterization of distinct strains of Jatropha mosaic virus from the Dominican Republic reveals local evolution and a potential to infect crop plants. Phytopathology 105: 141-153.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Kon, T., Melgarejo, T., and Gilbertson, R. L. 2014. Recent emergence of the mild strain of Tomato yellow leaf curl virus as a cause of tomato yellow leaf curl disease of processing tomatoes (Solanum lycopersicon) in the Dominican Republic. Plant Disease 98: 1592.

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

Outputs
Target Audience: The target audience for this information is a tomato growers, canners and professional crop consultants. In addition, basic information is of interest to virologists and researchers at academic and government institutions as well as at seed companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Mr. Tomas Melgarejo, a Ph.D student in the laboratory of Dr. Robert L. Gilbertson, performed most of the work on ToLDeV. This provided Tomas with training in many areas of virology and molecular biology. In addition, he and Dr. Gilbertson wrote the manuscript on this work, which provided valuable writing experience for Mr. Melgarejo. How have the results been disseminated to communities of interest? The results have been reported in presentations at scientific meetings as well as in a publication in the prestigious Journal of Virology. What do you plan to do during the next reporting period to accomplish the goals? We will continue to detect and characterize geminiviruses, understand the biology and genetics of these viruses and use this information to develop effective management strategies.

Impacts
What was accomplished under these goals? The etiology of tomato leaf curl disease in Peru was elucidated. We established that the disease is caused by a monopartite begomovirus that apparently evolved in the New World. This is the first example of a monopartite begomovirus in the New World, where most begomoviruses possess a bipartite genome (DNA-A and DNA-B components). This solved the mystery of why a DNA-B component could not be found associated with this disease. The key line of experimental evidence was the generation of an infectious clone of the DNA-A-like component associated with the disease and showing that, alone, this DNA induced the symptoms of tomato leaf curl disease. Thus, this completed Koch's Postulates for this disease. The virus was named Tomato leaf deformation virus (ToLDeV). Further evidence of the monopartite nature of this virus came from findings that it was not sap-transmitted, it was phloem-limited and the phenotypes of capsid protein and C4 gene mutants. We further demonstrated that tomato varieties with the Ty-1 resistance gene were resistant to ToLDeV. A number of ToLDeV variants were also characterized that differed in virulence and apparently evolved via recombination and mutation. Based upon these findings an integrated pest management program for ToLDeV in Peru is being developed.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Melgarejo, T. A., Kon, T., Rojas, M. R., Paz-Carrasco, L., Zerbini, F. M., and Gilbertson, R. L. 2013. Characterization of a new world monopartite begomovirus causing leaf curl disease of tomato in Ecuador and Peru reveals a new direction in geminivirus evolution. J. Virol. 87: 5397-5413.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Castro, R. M., Moreira, L., Rojas, M. R., Gilbertson, R. L., Hernandez, E., Mora, F., and Ramirez, P. 2013. Occurrence of Squash yellow mild mottle virus (SYMMoV) and Pepper golden mosaic virus (PepGMV) in potential new hosts in Costa Rica. Plant Pathol. J. 29: 285-293.

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

Outputs
OUTPUTS: One of the important questions in managing diseases of crops plants caused by whitefly-transmitted geminiviruses (genus Begomovirus) is the means by which the viruses persist in the absence of the crop plant host. The whitefly-transmitted begomovirus Tomato yellow leaf curl virus (TYLCV) was introduced into the Dominican Republic in the early 1990s and has spread into the southern United States where it poses a threat to tomato production. As part of an ongoing integrated pest management (IPM) program for TYLCV in the Dominican Republic, we are characterizing begomoviruses infecting weeds in and around tomato fields in various tomato-growing regions of the island. Here, we have characterized a begomovirus associated with yellow mosaic symptoms in Jatropha spp., a shrub-like plant that is also a source of biofuels. Leaves with yellow mosaic symptoms were collected from Jatropha plants in three tomato-growing regions in the Dominican Republic (Aguacatico, Clavellina and Azua). Using rolling circle amplification and restriction enzyme digestion, full-length DNA-A and DNA -B clones were obtained from a plant from each location. These clones were used to generate agroinoculation systems, and host range and component exchange (pseudorecombination) experiments were performed. Finally, the cloned DNAs were inoculated into Jatropha seedlings by particle bombardment. PARTICIPANTS: The work presented here was performed by Tomas Melgarejo a Ph.D. student in the laboratory of Dr. Robert L. Gilbertson. This work provided Tomas excellent training in many aspects of molecular virology and biology and many aspects of working with whitefly-transmitted geminiviruses. TARGET AUDIENCES: The target audience for this information is tomato growers, producers and professional crop consultants in areas where TYLCV is a problem. In addition, this information is important for the Minister of Agriculture in the Dominican Republic in their development and implementation of a mandatory host-free period imposed as part of an IPM program for TYLCV. This information also will also be of interest to geminivirologists. Eventually, the results of this research will be provided to the target audience through presentations and publications. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Jatropha plants with yellow mosaic symptoms are common observed in an around tomato fields in the Dominican Republic. Our results confirmed that these symptoms were associated with infection by a bipartite begomovirus, but not TYLCV. Thus, Jatropha does not appear to be a reservoir of TYLCV and efforts do not need to be taken to destroy symptomatic Jatropha plants for TYLCV control. Furthermore, sequence comparisons indicated that the begomoviruses from the Jatropha plants from the different geographical locations of the Dominican Republic were highly divergent strains of a new begomovirus species, for which the name Jatropha yellow mosaic virus (JYMV) is proposed. In agroinoculation tests, the full-length DNA-A and B clones of the isolates from Aguacatico (J-AG-A and -B), Azua (J-AZ-A and -B) and Clavellina (J-CL-A and -B) caused disease symptoms in various species of tobacco as well as common bean, but not in tomato, cucurbits or other plants. In particle bombardment tests, these clones induced distorted growth and yellow mosaic symptoms in Jatropha plants, thereby confirming that JYMV causes the yellow mosaic disease of Jatropha (i.e., Koch's postulated were performed). The finding that exchanging the DNA-A and DNA-B clones of the isolates from different locations (J-AG, J-AZ and J-CL) resulted in viruses that caused a range of symptom phenotypes (ranging from no symptoms to similar to the wild-type isolates) is consistent with these being distinct strains of JYMV that have undergone local evolution. The host range results indicated that JYMV does not infect tomato, further showing that the Jatropha plants do not pose a threat to tomato production. However, the capacity of JYMV to infect common bean and tobacco indicate the potential for JYMV to cause disease symptoms in these crops. Finally, the infectious clones of JYMV and the agroinoculation and biolistic delivery systems have the potential to be used as a gene vector that could be used for the genetic improvement of Jatropha grown for biofuels.

Publications

• Zhou, Y., Rojas, M. R., Park, M.-R., Seo, Y.-S., Lucas, W. J., and Gilbertson, R. L. 2011. Histone H3 interacts and co-localizes with the nuclear shuttle protein and movement protein of a geminivirus. J. Virol. 85: 11821-11832.
• Kon, T., and Gilbertson, R. L. 2012. Two genetically related begomoviruses causing tomato leaf curl disease in Togo and Nigeria differ in virulence and host range but do not require a betasatellite for induction of disease symptoms. Arch. Virol. 157: 107-120.
• Muniyappan, R., Watson, G. W., Vaughan, L., Gilbertson, R. L. and Noussourou, M. 2012. New records of mealybugs, scale insects, and whiteflies (Hemiptera: Sternorrhyncha)from Mali and Senegal. J. Agricult. and Urban Hort. 28: 1-7.

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

Outputs
OUTPUTS: One of the most effective and desirable methods for managing diseases caused by geminiviruses is by the use of host resistance. However, for most crops, the identification and utilization of host resistance for diseases caused by whitefly-transmitted geminiviruses (genus Begomovirus) has been difficult, due to lack of known sources of resistance and difficulties in screening large number of diverse germplasm. Here, we have attempted to address both of these limitations through screening germplasm representing the genetic diversity of common bean with two bean-infecting begomoviruses (Bean dwarf mosaic virus [BDMV] and Bean golden yellow mosaic virus [BGYMV]) using the agroinoculation method. The germplasm screened represented the two major common bean gene pools (Andean and Middle American) and the races within these gene pools. In control experiments with the susceptible cultivar Topcrop, all plants developed severe symptoms that were typical and expected for each virus. A range of responses were observed among germplasm, and the responses differed among the two viruses. Furthermore, for many materials, individual plants showed a range of responses. Materials in which the majority of plants showed resistance to both viruses included A-429 and Sanilac. For BGYMV, additional materials that showed resistance included Cardinal (95% of plants), DOR 390 (70% of plants), 91-1 and Yolano (65% of plants). Materials that showed susceptibility (including <50% resistant plants) to both viruses included Etna, L-10-2, T 39, and TC-75. . PARTICIPANTS: We worked closely with Dr. Shree Singh, a bean breeder from the University of Idaho. In the course of this project, training was provided to Ms. Maria Hernandez, a worker in the laboratory. TARGET AUDIENCES: The target audience that much of this information is common bean breeders and pathologists. Eventually, the results of this research will be provided to the target audience through presentations and publications. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Begomoviruses, including those that infect common bean continue to be major constraints on crop production worldwide as well as in the United States (e.g., Florida). Here, we have evaluated the agroinoculation technique for BDMV and BGYMV for screening a diversity of common bean germplasm for resistance to these two viruses. This approach revealed differential responses in the germplasm to the two viruses, including some that had resistance to both viruses and others that had resistance to only BGYMV. Another interesting finding was the fact that for many of the materials, individual plants showed a range of responses, including susceptible and resistant plants. This suggested that a number of these materials were not genetically pure. The results of this study provide preliminary information that can be used in breeding programs for bean-infecting begomoviruses.

Publications

• Stewart, C. S., Kon, T., Gilbertson, R. L., and Roye, M. E. 2011. First report of the complete sequence of Sida golden yellow vein virus from Jamaica. Arch. Virol. 156: 1481-1484.
• Gilbertson, R. L., Natwick, E. T., and Rojas, M. R. 2011. Development of integrated pest management (IPM) strategies for whitefly (Bemisia tabaci)-transmissible geminiviruses. Pages 323-356. In: W. Thompson (ed.), The whitefly, Bemisia tabaci (Homoptera: Aleyrodidae) Interaction with Geminivirus-Infected Host Plants. Springer, Dordrecht, The Netherlands.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Curly top disease is caused by various species in the genus Curtovirus (family Geminiviridae) and affects many vegetable crops in California, including tomatoes and peppers. Research conducted to date has indicated that curly top of tomato is caused by two curtovirus species: Beet mild curly top virus (BMCTV), and Beet severe curly top virus (BSCTV) and that a PCR-based test can be used to detect these viruses in the beet leafhopper vector. Curly top virus monitoring in pepper and tomato in 2010 revealed high rates of viruliferous leafhoppers early in the season (January-March) and that the predominant curtoviruses in leafhoppers and tomatoes and peppers were BMCTV and BSCTV. However, A putative new curtovirus (BV3) was associated with curly top disease in tomato. A full-length clone of this isolate was infectious, and induced typical curly top symptoms in Nicotiana benthamiana plants. The BV3 clone was 2931 nucleotides and had a typical curtovirus genome organization. Total genome sequence comparisons revealed that it is most similar (~96%) to a putative new curtovirus species, Pepper curly top virus (PeCTV), previously identified from pepper in New Mexico. Sequence comparisons of BV3 with other curtoviruses revealed high levels of identity (95-100%) with the C1 and C4 open reading frames and right intergenic region of BSCTV. Host range experiments involving agroinoculation and beet leafhopper transmission revealed that BV3 has a similar host range to BSCTV, including a severe symptom phenotype in sugar beet. A second recombinant curtovirus isolate was identified from beet leafhoppers collected from the Central Valley of California in 2010. The genome of this isolate was composed of BMCTV (major parent) and BV3 (minor parent). PARTICIPANTS: We worked closely with colleagues from the California Department of Food and Agriculture (CDFA), and they provided leafhopper samples and some plant samples. We also worked closely with a number of UCCE Farm Advisors. Training and professional development was received by Dr. Li-Fang Chen, a postdoctoral researcher who did most of this research. TARGET AUDIENCES: The target audience that much of this information is for farmers and professional crop consultants. The results of this research was presented at a number of growers meetings and commodity board meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Curly top disease can cause devastating losses in vegetable crops such as common beans and tomatoes. It is critical to understand the key curtovirus(es) involved in the disease in order to develop diagnostic tools and for selection of appropriate isolates for disease resistance screening. We continued to apply our PCR-based diagnostics for curtoviruses to determine the curtoviruses involved in curly top of tomato in California. Our results showed high levels of viruliferous leafhoppers early in the season, consistent with the concept that adult leafhoppers migrating down from the foothills were already carrying the virus. Pepper and tomato samples were infected with either BMCTV or BSCTV, consistent with these being the major curtoviruses associated with tomato curly top. However, a new curtovirus species, PeCTV, was associated with curly top of tomato in California. Moreover, sequence analyses revealed that BSCTV has a recombinant genome, including sequences of PeCTV. This is the first evidence that BSCTV may be a recombinant and raises questions about its' origin. The finding of a second recombinant isolate from a leafhopper sample, composed of BMCTV and PeCTV, revealed additional evidence for the role of recombination in the emergence of new curtovirus species. Indeed, this isolate may represent another new curtovirus species. Together, these results suggest that a third curtovirus (PeCTV) may play a role in curly top disease in California, and a more important role for recombination in evolution and emergence of new curtoviruses than previously recognized.

Publications

• Chen, L.-F., Vivoda, E. and Gilbertson, R. L. Genetic diversity in curtoviruses: A highly divergent strain of Beet mild curly top virus associated with an outbreak of curly top disease in pepper in Mexico. Archives of Virology. Online First, 30 December 2010

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Curly top is reported in the literature to be an important disease of cucurbits. However, anecdotal reports indicate that the disease is rarely observed in cucurbits in California. We investigated this conundrum by conducting surveys of cucurbit fields in California for curly top, and testing samples of cucurbits and other crops from Idaho for curly top infection. The relative susceptibility of cucurbits (cantaloupe, pumpkin and watermelon) to infection by three curly top viruses (curtoviruses), (Beet curly top virus [BCTV], Beet mild curly top virus [BMCTV], and Beet severe curly top virus [BSCTV]), was assessed via agroinoculation and leafhopper transmission; and compared with that of known susceptible hosts (tomato, Nicotiana benthamiana and shepherd's purse). In addition, the role of the viral acquisition host in influencing transmission of curtoviruses to different hosts was also assessed. PARTICIPANTS: We worked closely with colleagues from the California Department of Food and Agriculture (CDFA), and they provided leafhopper samples and some plant samples. We also worked closely with a number of UCCE Farm Advisors. Training and professional development was received by Dr. Li-Fang Chen, a postdoctoral researcher who did most of this research. TARGET AUDIENCES: The target audience that much of this information is for farmers and professional crop consultants. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Curly top disease can cause devastating losses in vegetable crops such as common beans and tomatoes, and it is also supposed to present a threat to cucurbit crops. Thus, the CDFA Curly top Control Program includes melons in their assessment. However, anecdotal reports indicate that cucurbits rarely, if ever, get curly top disease in California. Surveys of melon fields conducted in Central California did not reveal curly top symptoms, although the disease was observed in nearby tomato fields. However, cucurbit samples from Idaho with curly top-like symptoms were confirmed to be infected with BMCTV. Irrespective of the curtovirus species or inoculation method, low rates of infection and mild or symptomless disease phenotypes were observed in inoculated cucurbits; whereas all inoculated tomato, N. benthamiana and shepherd's purse plants were infected and developed severe symptoms. In addition, it was found that the host from which the virus was acquired impacted transmission of the virus to cucurbits, but not to the more susceptible hosts. Thus, leafhopper transmission of curtoviruses to cucurbits occurred only when the virus was acquired from a high titer host like shepherd's purse, whereas transmission to susceptible hosts occurred when the virus was acquired from hosts with high or low titers. Together, our results indicate that although cucurbits can develop curly top disease experimentally and in the field (e.g., in Idaho), they are relatively poor hosts for these curtoviruses. It is not clear why cucurbits get curly top in Idaho but not in California; this may reflect higher disease pressure in Idaho or a benefit of the curly top control program.

Publications

• Chen, L.F., and Gilbertson, R. L. 2009. Curtovirus-cucurbit interaction: Acquisition host plays a role in leafhopper transmission in a host-dependent manner. Phytopathology 99:101-108.
• Miklas, P. N., Seo, Y.-S., and Gilbertson, R. L. 2009. Quantitative resistance to Bean dwarf mosaic virus in common bean is associated with the Bct gene for resistance to Beet curly top virus. Plant Dis. 93:645-648.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: We utilized a method developed in our laboratory to monitor for the viruses causing curly top disease in tomato (Beet curly top virus [BCTV], Beet mild curly top virus [BMCTV], and Beet severe curly top virus [BSCTV]) in California in the leafhopper vector. Leafhopper samples were received at monthly intervals and test for the presence of the viruses over the course of the growing season. Tomato fields were monitored for curly top development and samples were also received from growers, PCAs and the California Department of Food and Agriculture. These samples were tested for the presence of curly top viruses using a PCR test with general and virus-specific primers. This information has been disseminated through growers meetings, a Farm Advisor's conference and meetings with the California Department of Food and Agriculture. PARTICIPANTS: We worked closely with colleagues from the California Department of Food and Agriculture (CDFA), and they provided leafhopper samples and some plant samples. We also worked closely with a number of UCCE Farm Advisors. Training and professional development was received by Dr. Li-Fang Chen, a postdoctoral researcher who did most of this research. TARGET AUDIENCES: The target audience that much of this information is for is tomato farmers and PCAs. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Enter narrative in this block. The fact that the CDFA was not allowed to conduct their annual spray program for the beet leafhopper vector of these viruses in the foothills raised concerned about whether curly top disease was going to be a problem in 2008. Indeed, leafhoppers collected in March-May had relatively high levels of curly top virus. Consistent with this finding, our surveys revealed curly top appearing in tomato field relatively early in the season, though the overall severity of curly top was moderate in 2008. This finding helped support the idea that level of curly top virus in leafhoppers early in the season may be a predictor of disease development later in the season. This information could improve the implementation of the spray program for leafhoppers conducted by CDFA. We also determined that the initial disease symptoms induced by curly top viruses and the thrips-transmitted Tomato spotted wilt virus (TSWV) both involve upcurling of levels, purpling of the veins and a pale green coloration. This leads to the possibility of mis-diagnosis, especially early in the season. Indeed, a number of putative curly top samples that were received in 2008 were negative for curly top virus, but were positive for TSWV. We also found no evidence of mixed infections of curly top viruses and TSWV. This shows the problem with visual identification of these diseases and the need to use diagnostic methods (e.g., PCR tests for curly top virus and antibody tests for TSWV). This is very important because it impacts the nature of the disease management efforts, as different approaches are used for these two different virus diseases. This information should improve the capacity to correctly identify these diseases in the field which, in turn, will lead to better disease management.

Publications

• Chen, L.-F., and Gilbertson, R. L. 2008. Beet mild curly top virus. Pages 195-215 in: Characterization, Diagnosis & Management of Plant Viruses. G. P. Rao, P. Khurana, and S. M. Lenardon, eds. Studium Press LLC, Texas.
• Hagen, C., Rojas, M. R., Sudarshana, M. R., Xoconostle-Cazares, B., Natwick, E. T., Turini, T. A., and Gilbertson, R. L. 2008. Biology and molecular characterization of Cucurbit leaf crumple virus, an emergent cucurbit-infecting begomovirus in the Imperial Valley of California. Plant Dis. 92:781-793.
• Hagen, C., Rojas, M. R., Kon, T., and Gilbertson, R. L. 2008. Recovery from Cucurbit leaf crumple virus (Family Geminiviridae, Genus Begomovirus) infection is an adaptive antiviral response associated with changes in viral small RNAs. Phytopathology 98: 1029-1037.

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

Outputs
We have continued to conduct surveys for whitefly-transmitted viruses in the Imperial Valley and other southern parts of California. In March 2007, tomato plants growing in a greenhouse in Brawley, California showed symptoms similar to those caused by Tomato yellow leaf curl virus (TYLCV), including stunted upright growth and small upcurled leaves with crumpling and strong interveinal chlorosis. These plants also sustained high populations of whiteflies. Samples of diseased tomato plants and whiteflies were collected from these plants and DNA extracted for testing in the polymerase chain reaction test with primers specific for TYLCV. Amplified DNA fragments were cloned and sequenced. Sequences were compared to previously characterized viruses. This information has been disseminated through growers meetings, a press conference at the Annual Meeting of the American Phytopathological Society and meetings with the California Department of Food and Agriculture. A flyer has been prepared on TYLCV that provides photographs and information on the biology of the virus. This flyer has been handed out at meetings and is also available through the University of California IPM website.

Impacts
The results of the analyses of the tomato disease samples revealed that the plants were infected by TYLCV. The complete nucleotide sequence of the virus was determined and found to be greater than 95% identical to TYLCV from other locations. The virus also was detected in the bodies of whiteflies collected from the plants in the greenhouse and from weeds surrounding the greenhouse. This is the first report of this exotic virus in California. This finding led the CDFA to quarantine this greenhouse and destroy all the tomato plants and close down the house for plant growth. Surveys have been conducted in Imperial County for tomatoes and a number of these plants were positive for TYLCV infection and these were destroyed. Surveys have been initiated to determine whether the virus has persisted in the Imperial Valley.

Publications

• Rojas, M.R., Kon, T., Natwick, E.T., Polston, J.E., Akad, F., and Gilbertson, R.L. 2007. First report of Tomato yellow leaf curl virus associated with tomato yellow leaf curl disease in California. Plant Dis. 91:1056.
• Gilbertson, R.L., Rojas, M.R., Kon, K., and Jaquez, J. 2007. Introduction of Tomato yellow leaf curl virus into the Dominican Republic: the development of a successful integrated pest management strategy. Pages 279-303. In H. Czosnek (ed.). Tomato Yellow Leaf Curl virus disease. Springer, Dordrecht, The Netherlands.

Progress 01/01/06 to 12/31/06

Outputs
We have continued our molecular genetic analysis of geminivirus gene function, with emphasis on understanding the differential susceptibility of common bean to the bean-infecting whitefly-transmitted geminiviruses Bean dwarf mosaic virus (BDMV) and Bean golden yellow mosaic virus (BGYMV). We had previously identified the BV1 gene, which encodes the nuclear shuttle protein (NSP), as the determinant of avirulence of BDMV in cultivars Othello and UI 114. To further identify the region of this protein responsible for recognition, a series of recombinant viruses with hybrid BDMV/BGYMV BV1 genes was generated and inoculated into common bean genotypes representing the two major gene pools. The recombinant with an NSP with the N-terminal 42 amino acids of BGYMV was able to break the resistance of UI 114, whereas the other recombinant viruses were not. This established that the avirulence determinant was in the N-terminus, a variable and surface-exposed region of the protein. Another recombinant, with amino acids 43-92 of BGYMV, was not infectious, revealing a virus-specific domain in the NSP. When the BDMV BV1 was inserted into the BGYMV genome, in place of the capsid protein (CP) gene, it functioned as a virulence factor in the Andean cv. Topcrop and an avirulence factor in the Middle American cultivars, but only in the absence of the BGYMV BV1. Finally, the CP played a gene pool-specific role in viral infectivity, it was dispensable for infectivity in the Andean cv. Topcrop, but was required for infectivity in the Middle American cultivars. Redundancy of the CP and NSP, which are nuclear proteins directly or indirectly involved in movement, appears to provide a masking effect that may allow the virus to avoid host defense responses.

Impacts
The identification of the specific region of a viral protein that is recognized by the product of a host resistance gene may provide information that could be useful for engineering disease-resistant plants. This information also demonstrates the genetic complexity and specificity that underlies the begomovirus-common bean interaction and will complicate breeding for resistance to multiple viruses.

Publications

• Seo, Y.-S., Zhou, Y.-C., Turini, T.A., Cook, C.G., Gilbertson, R.L., and Natwick, E.T. 2006. Evaluation of cotton germplasm for resistance to the whitefly (Bemisia tabaci) and cotton leaf crumple (CLCr) disease and etiology of CLCr in the Imperial Valley of California. Plant Dis. 90:877-884.

Progress 01/01/05 to 12/31/05

Outputs
We continued our investigation of the importance of curly top disease in melon. Our approach has involved 1) surveying melon fields for curly top symptoms (stunted growth and upcurling, crumpling and yellowing) and 2) inoculating various cucurbits, including cantaloupe and honeydew melon, with the three curly top viruses known to occur in California (Beet curly top virus, BCTV; Beet severe curly top virus, BSCTV; and Beet mild curly top virus, BMCTV) and determining their susceptibility compared with tomato and tobacco. In 2005, we also expanded our survey efforts to get samples from other states. Surveys were conducted in California failed to reveal obvious symptoms of curly top were observed in any fields, and curly top virus was not detected in any samples from any California melon fields. However, in late August/early September we received samples of melons (cantaloupe, honeydew and charentais) from Idaho that had curly top-like symptoms (leaf curling, crumpling and yellowing). Using the PCR technique we confirmed that most of these were infected with BMCTV. Thus, this appears to be a real case of curly top of melon, and it correlated with one of the worst curly top years in Idaho history. Results of CTV inoculation experiments using two inoculation methods indicated that melon can get infected by curly top viruses and even develop symptoms, but that melon is a poor host compared with tomato or tobacco. By agroinoculation, BCTV and BSCTV had very low rates of infection in melon; whereas 100% of inoculated tomato and tobacco plants were infected and showed severe symptoms. A similar situation was observed with leafhopper transmission of BMCTV; however, it was discovered that the host from which the leafhopper acquires the virus influences the ability of the insects to infect cucurbits. Thus, when leafhoppers acquired the virus from the highly susceptible host, Shepherds purse, cucurbits get infected with BMCTV, and a number of pumpkin (8/17; 47%) and cantaloupe (2/20; 10%) plants even developed curly top symptoms (leaf curling, crumpling and yellowing). In contrast, no infections were detected when insects acquired the virus from sugar beet, a less susceptible host. Considered together with the finding of curly top in melons in Idaho, our results indicate that under certain conditions curly top can develop in melons, which is in agreement with the early reports of curly top in melon. A threshold model is proposed to explain these results, in which high CTV pressure is needed for curly top to develop in less susceptible hosts (melons) and little pressure is needed to infect highly susceptible hosts (tomato and tobacco).

Impacts
The curly top disease is known to be an important disease of crops such as tomato, sugar beet and common bean in California, and a statewide spray program is conducted to control the beet leafhopper vector. However, it has been a conundrum trying to reconcile old reports of curly top disease in melons and the more recent inability to find the disease in melon, even in areas having curly top in tomato. Our findings that curly top can develop in melons, but only under high virus pressure, may help explain this conundrum. Thus, when leafhoppers are fed on a host having a high titer of curly top virus, they deliver sufficient virus into melon for some plants to develop curly top symptoms. Consistent with this finding was the identification of curly top in melon in Idaho in 2005, when disease pressure was extremely high. It is therefore possible that the curly top spray program could benefit melons by keeping levels of the virus below the threshold needed for melons to develop the disease.

Publications

• Rojas, M. R., Hagen, C., Lucas, W. J., and Gilbertson, R. L. 2005. Exploiting chinks in the plants armor: Evolution and emergence of geminiviruses. Ann. Rev. Phytopathol. 43:361-394.
• Soto, M. J., Chen, L. F., Seo, Y.-S., and Gilbertson, R. L. 2005. Identification of regions of the Beet mild curly top virus (family Geminiviridae) capsid protein involved in systemic infection, virion formation, and leafhopper transmission. Virology 341: 257-270.

Progress 01/01/04 to 12/31/04

Outputs
Surveys were conducted to assess the incidence and severity of curly top disease in melons in California. No obvious symptoms of curly top infection were observed in 11 melon fields in two surveys conducted on July 14 and 28, 2004. A number of these fields were considered high risk, i.e., they were near tomato fields that had plants showing curly top symptoms or they were near the foothills, where curly top virus carrying leafhoppers originate. A total of 52 samples showing the closest approximation to virus symptoms were collected from these fields and tested for curly top infection by PCR. Three samples were positive and found to be infected with the Beet severe curly top virus (BSCTV). A series of inoculations were next performed in which curly top viruses were introduced into melons and other hosts by agroinoculation (BSCTV and Beet curly top virus, BCTV) or leafhopper inoculation (Beet mild curly top virus, BMCTV). All three viruses were highly infectious and caused curly top symptoms in tomato. In contrast, none of the inoculated honeydew melon or watermelon plants developed symptoms. In cantaloupe, BCTV and BMCTV did not induce symptoms, whereas BSCTV induced leaf curling and crumpling in a small proportion of plants (6%). In pumpkin, symptoms developed in a small proportion of plants inoculated with BCTV and BSCTV, whereas no plants inoculated with BMCTV developed symptoms. Together, these results suggest that cucurbits are poor hosts of the curly top viruses, and that curly top may not be an important disease of melons in California.

Impacts
The curly top disease is known to be an important disease of crops such as tomato, sugar beet and common bean in California and a statewide spray program is conducted to control the beet leafhopper vector. However, there is conflicting opinion about whether this disease is a threat to melon production in California. The results of this study support anecdotal reports that curly top is not an important disease of melon. This will have important implications in curly top disease management efforts in California.

Publications

• Gilbertson, R. L. 2004. Annual report to the California Melon Research Board.

Progress 01/01/03 to 12/31/03

Outputs
Surveys were conducted to assess the incidence and severity of a newly described whitefly-transmitted begomovirus, Cucurbit leaf crumple virus (CuLCrV), in melons in the Imperial Valley of California. No symptoms of CuLCrV infection were observed in spring melons (March-May 2003). However, whitefly populations increased in the summer and CuLCrV symptoms were observed in fall melon production (September-October 2003), with some fields having a high incidence of infection. PCR tests with CuLCrV-specific primers confirmed CuLCrV infection in representative symptomatic plants. Interestingly, infected cantaloupe plants underwent recovery from severe symptoms and no major losses were reported. A whitefly-independent CuLCrV agroinoculation system was developed and used to screen melons and other cucurbits for susceptibility to CuLCrV. Squash was highly susceptible, watermelon was moderately susceptible, cantaloupe was moderately resistant and honeydew melon was highly resistant. Cantaloupe plants that developed symptoms after agroinoculation underwent recovery from symptoms. Together, these results suggest that CuLCrV does not pose a major threat to California melon production and that there are potential sources of virus resistance in melon germplasm.

Impacts
Our results indicate that CuLCrV does not reach high levels in California melons until late in the season and that melon plants have the ability to recover from viral symptoms. A whitefly-independent inoculation system was developed that allows for efficient screening of melons for resistance to CuLCrV and indicated a high level of resistance to CuLCrV in cantaloupe and honeydew melon. Thus, melon production should not be greatly impacted by this virus, which is good news for melon growers in the Imperial Valley of California.

Publications

• Gilbertson, R. L. 2003. Annual report to the California Melon Research Board.

Progress 01/01/02 to 12/31/02

Outputs
Cotton (Gossypium hirsutum L.) cultivars and breeding lines were evaluated in the field for resistance to cotton leaf crumple disease (CLCrD) caused by whitefly-transmitted begomovirus, Cotton leaf crumple virus (CLCrV) transmitted by the silverleaf whitefly (Bemisia argentifolii). Cultivars and breeding lines were evaluated in field plots in 2001 in the Imperial Valley, CA. The cultivars were Texas 121, AP 4103, AP 6101, Stoneville 474, and breeding lines NK2387C, NK2108SS, NKX2207,NKX2907, NK2165C and NKXC429-93-2ct. Cotton plants were surveyed weekly for the incidence of whitefly adults and nymphs. CLCr disease severity was assessed in each plot in three times each in August and September. CLCrV incidence and levels were determined in leaves of selected plants using dot blot hybridization analysis with a CLCrV DNA probe. Differences in whitefly infestation levels and CLCr virus symptoms were detected among cultivars. The highest levels of whiteflies were found on NKXC429-93-2ct (30.7/per leaf) followed by Stoneville 474 (16.6); the lowest level of whiteflies were detected on NK2387C (8.7). The cultivar AP4103 had the highest CLCrD rating (3.5) and the second highest dot blot rating, followed by AP6101 and NKXC429-93-2ct. The breeding line, NK2387C, with Cedix parentage, had the lowest CLCrD rating and the lowest dot blot rating, suggesting a high level of resistance to CLCrV.

Impacts
Cotton can be infected by the whitefly-transmitted begomovirus, CLCrV, resulting in yield losses. In Pakistan and India, another cotton begomovirus, Cotton leaf curl virus (CLCuV) causes devastating losses. Thus, the identification of a cotton breeding line, NK2387C, that is highly resistant to CLCrD should allow for the development of CLCrV-resistant cultivars. It will be important to determine if this breeding line is also resistant to infection by CLCuV.

Publications

• Natwick, E. T., Cook, C. G., Gilbertson, R. L., and Seo, Y.-S. 2000. Resistance in upland cotton to the silverleaf whitefly-transmitted cotton leaf crumple disease. In Abstracts Beltwide Cotton Prod. Res. Conf. National Cotton Council, Memphis, TN. 1: 164-176.

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

Outputs
Cotton (Gossypium hirsutum L.) cultivars and breeding lines were evaluated in the field for resistance to cotton leaf crumple disease (CLCrD) caused by whitefly-transmitted begomovirus, Cotton leaf crumple virus (CLCrV) transmitted by the silverleaf whitefly (Bemisia argentifolii). Cultivars and breeding lines were evaluated in field plots in 2001 in the Imperial Valley, CA. The cultivars were Texas 121, AP 4103, AP 6101, Stoneville 474, and breeding lines NK2387C, NK2108SS, NKX2207,NKX2907, NK2165C and NKXC429-93-2ct. Cotton plants were surveyed weekly for the incidence of whitefly adults and nymphs. CLCr disease severity was assessed in each plot in three times each in August and September. CLCrV incidence and levels were determined in leaves of selected plants using dot blot hybridization analysis with a CLCrV DNA probe. Differences in whitefly infestation levels and CLCr virus symptoms were detected among cultivars. The highest levels of whiteflies were found on NKXC429-93-2ct (30.7/per leaf) followed by Stoneville 474 (16.6); the lowest level of whiteflies were detected on NK2387C (8.7). The cultivar AP4103 had the highest CLCrD rating (3.5) and the second highest dot blot rating, followed by AP6101 and NKXC429-93-2ct. The breeding line, NK2387C, with Cedix parentage, had the lowest CLCrD rating and the lowest dot blot rating, suggesting a high level of resistance to CLCrV.

Impacts
Cotton can be infected by the whitefly-transmitted begomovirus, CLCrV, resulting in yield losses. In Pakistan and India, another cotton begomovirus, Cotton leaf curl virus (CLCuV) causes devastating losses. Thus, the identification of a cotton breeding line, NK2387C, that is highly resistant to CLCrD should allow for the development of CLCrV-resistant cultivars. It will be important to determine if this breeding line is also resistant to infection by CLCuV.

Publications

• Natwick, E. T., Cook, C. G., Gilbertson, R. L. and Seo, Y.-S. 2000. Resistance in upland cotton to the silverleaf whitefly-transmitted cotton leaf crumple disease. In Abstracts Beltwide Cotton Prod. Res. Conf. National Cotton Council, Memphis, TN. 1:164-176.

Progress 01/01/00 to 12/31/00

Outputs
A new whitefly-transmitted bipartite begomovirus (genus Begomovirus, Family Geminiviridae) infecting cucurbits in the Imperial Valley of California has been identified. This virus has been named Cucurbit leaf crumple virus (CuLCrV). CuLCrV was originally found in watermelon volunteers showing leaf crumpling and distortion symptoms, but in the 2000 melon-growing season it was detected in commercial watermelon and cantaloupe fields in Southern California and Arizona. Thus, this virus has become established in Southern California and Arizona. To gain further insight into the nature of CuLCrV, full-length CuLCrV clones were generated using PCR and overlapping primers. The complete nucleotides sequences of these clones were determined and sequences of genes and the common region (a noncoding region shared between the two components of a given geminivirus species) were compared with those of other geminiviruses. The results of these analyses confirmed that CuLCrV is a distinct geminivirus species (i.e., nucleotide sequences of CuLCrV genes were no more than 87 percent identical to other begomoviruses). Phylogenetic analyses revealed that the closest relatives of CuLCrV are Squash leaf curl virus (SLCV) and Bean calico mosaic virus (BCMoV), which are found in Southern California and Northern Mexico, respectively. Thus, CuLCrV is a new member of the SLCV cluster of begomoviruses.

Impacts
A new whitefly-transmitted begomovirus, infecting cucurbits in Southern California and Arizona has been identified and characterized. This virus has been named Cucurbit leaf crumple virus (CuLCrV). The complete nucleotide of full-length clones has been determined and can be used to generate tools for the rapid detection of the virus, which should be useful if this virus becomes a major problem in the near future.

Publications

• Guzman, P., Sudarshana, M. R., Seo, Y.-S., Rojas, M. R., Natwick, E., Turini, T., Mayberry, K., and Gilbertson, R. L. 2000. A new bipartite geminivirus (begomovirus) causing leaf curl and crumpling in cucurbits in the Imperial Valley of California. Plant Disease. 84:488.
• Hernandez, N. A., Sudarshana, M. R., Guzman, P., and Gilbertson, R. L. 2000. Generation and characterization of infectious clones of Cucurbit leaf crumple virus, a new bipartite geminivirus from the Imperial Valley of California. Phytopathology 90:S35.

Progress 01/01/99 to 12/31/99

Outputs
Transgenic tomato plants expressing wild-type or mutated BV1 or BC1 movement proteins from bean dwarf mosaic geminivirus (BDMV) were generated and examined for phenotypic effects and resistance to tomato mottle geminivirus (ToMoV). Fewer transgenic plants were recovered with the wild-type or mutated BC1 genes compared with the wild-type or mutated BV1 genes. Transgenic tomato plants expressing the wild-type or mutated BV1 proteins appeared normal. Interestingly, although BDMV induces only a symptomless infection in tomato (i.e., BDMV is not well adapted to tomato), transgenic tomato plants expressing the BDMV BC1 protein showed a viral disease-like phenotype (i.e., stunted growth and leaf mottling, curling, and distortion). This suggests that the symptomless phenotype of BDMV in tomato is not due to a host-specific defect in the BC1 protein. One transgenic line expressing the BC1 gene did not show the viral disease-like phenotype. This was associated with a deletion in the 3 prime region of the gene, which resulted in expression of a truncated BC1 protein Several R0 plants, expressing either wild-type or mutated BV1 or BC1 proteins, showed a significant delay in ToMoV infection compared with non-transformed plants. R1 progeny plants also showed a significant delay in ToMoV infection, but this delay was less than that in the R0 parents. These results also demonstrate that expression of geminiviral movement proteins, in transgenic plants, can have deleterious effects on various aspects of plant development.

Impacts
A strategy to engineer tomato plants to be resistant to geminivirus infection was tested in which bean dwarf mosaic geminivirus (BDMV) movement proteins (MPs) were expressed. The transgenic tomatoes showed a delay in tomato mottle virus infection, but were not highly resistant. Expression of certain BDMV MPs in tomatoes caused disease symptoms, indicating possible problems with this strategy.

Publications

• Gilbertson, R. L., Garrido-Ramirez, E., Sharp, L. P., Salati, R. S., Rojas, M. R., and Sudarshana, M. R. 1999. Geminiviruses: A scourge to vegetable production or promising tools for plant biology. InVitro 35:19A.
• Sharp, L. P., Hou, Y.-M., Garrido-Ramirez, E. R., Guzman, P. and Gilbertson, R. L. 1999. A synergistic interaction between geminivirus DNA components results in increased symptom severity and viral DNA levels in infected plants. Phytopathology 89:S71.

Progress 01/01/98 to 12/01/98

Outputs
We have been investigating the etiology of the chino del tomate disease of tomato. Though known to be caused by whitefly-transmitted geminivirus(es) the etiology of the disease remains unclear. A tomato sample with chino del tomate disease was received from Sinaloa, Mexico and three geminivirus DNA components were cloned from this sample. Two of these components were the DNA-A and DNA-B of pepper huasteco geminivirus (PHV), whereas the other component was a DNA-A of unknown identity. This unknown DNA-A component had the capacity to replicate autonomously. To assess the role of this unknown DNA-A in chino del tomate disease, N. BENTHAMIANA, tomato, and pepper plants were inoculated with this unknown DNA-A alone or in combination with the infectious cloned DNA components of PHV or another begomovirus associated with chino del tomate disease, tomato leaf crumple (TLCrV). Alone, the new DNA-A was not infectious. In combination with PHV, this new DNA-A component systemically infected all hosts. Moreover, resulting symptoms were more severe than those induced by PHV alone, and were similar to chino del tomate symptoms. In contrast, the new DNA-A component only infected N. BENTHAMIANA in the presence of TLCrV, and the resulting symptoms were not more severe than those in plants infected with TLCrV alone. Together, these results reveal a novel synergistic interaction between this new DNA-A component and PHV, and provide evidence that chino del tomate may be caused by a complex of geminivirus components.

Impacts
(N/A)

Publications

• GARRIDO-RAMIREZ, E. R. and GILBERTSON, R.L. 1998. First report of tomato mottle geminivirus infecting tomatoes in Yucatan, Mexico.
• RIBEIRO, S.G., DE AVILA, A. C., BEZERRA, I. C., FERNANDES, J.J., FARIA, J.C., LIMA, M.F., GILBERTSON, R.L., MACIEL-ZAMBOLIM, E., and ZERBINI, F. M. 1998. Widespread occurrence of tomato geminiviruses in Brazil associated with the new bio.
• SHARP, L. P., HOU, Y.M., GARRIDO-RAMIREZ, E. R., GUZMAN, P., and GILBERTSON, R.L. 1998. Characterization of a new geminivirus DNA-A component from tomatoes with chino del tomate disease. Phytopathology

Progress 01/01/97 to 12/01/97

Outputs
Whitefly-transmitted geminiviruses continue to cause significant losses in vegetable production in tropical and sub-tropical areas of the world. We have continued our studies on the genetic diversity of geminiviruses infecting tomatoes. Tomato samples showing symptoms of geminivirus infection were received from Venezuela. The presence of geminivirus nucleic acids in these samples was demonstrated using DNA probes and the polymerase chain reaction and degenerate primers for whitefly-transmitted geminiviruses. To further characterize the geminiviruse(es) infecting these samples, PCR-amplified DNA-A and DNA-B fragments were cloned and sequenced. DNA sequence analysis indicated that there were two distinct geminiviruses infecting these samples. One of these was a strain of potato yellow mosaic geminivirus (PYMV), a previously described geminivirus from Venezuela, and the other is a new bipartite geminivirus. Thus, at least two distinct bipartite geminiviruses are involved with recent geminiviral disease outbreaks in tomatoes in Venezuela. In order to develop more efficient methods for screening plants for resistance to geminiviruses, we have developed agroinoculation systems for two bipartite geminiviruses: pepperhuasteco (PHV) and bean golden mosaic (BGMV). For PHV, we have successfully infected tomato, pepper, and Nicotiana benthamiana plants using agroinoculation, whereas for BGMV, we have successfully infected beans. We are now screening various bean genotypes for BGMV resistance using agroinoculation.

Impacts
(N/A)

Publications

• GUZMAN, P., ARREDONDO, C.R., EMMATTY, D., PORTILLO, R.J., and GILBERTSON, R.L. 1997. Partial characterization of two whitefly-transmitted geminiviruses infecting tomatoes in Venezuela.
• STOUT, J.T., LIU, H.T., POLSTON, J.E., GILBERTSON, R.L., NAKHLA, M.K., HANSON, S.F., and MAXWELL, D.P. 1997. Engineered rep gene-mediated resistance to tomato mottle geminivirus in tomato.

Progress 01/01/96 to 12/30/96

Outputs
Whitefly-transmitted geminiviruses are significant constraints on vegetable production in Central America, the Caribbean Basin, and the southern United States. Basic studies on geminivirus evolution have established that intermolecular recombination can occur between the DNA components of a pseudorecombinant geminivirus, leading to increased pathogenicity. This may be an important mechanism by which new geminivirus evolve. Full-length clones of a geminivirus causing chlorosis and yellow vein symptoms of tomato in Sinaloa, Mexico were generated. The infectivity of these clones was established in N. BENTHAMIANA, pepper and tomato plants by particle bombardment. Nucleotide sequence comparisons of the common region and AC1, BC1, and BV1 open reading frames revealed that the Sinoloa tomato geminivirus is similar but not identical to pepper huasteco geminivirus (PHV), indicating that the Sinaloa tomato geminivirus is a strain of PHV (PHV-Sin). A geminivirus associated with bean golden mosaic disease in Chiapas, Mexico was characterized. Sequence analysis of cloned PCR fragments representing DNA-A and DNA-B components revealed a high degree of similarity with bean golden mosaic geminivirus (BGMV) from Guatemala and the Dominican Republic (93 and 91%, respectively). These results indicate that the Mexican geminivirus is a type II BGMV isolate. Full-length infectious clones of BGMV-MX were obtained and shown to be infectious by particle bombardment.

Impacts
(N/A)

Publications

• ZERBINI, F. M., ZAMBOLIM, E. M., CARRIJO, I. V., and GILBERTSON, R. L. 1996. A new bipartite geminivirus infecting tomatoes in Minas Gerais State, Brazil. Phytopathology 86: S1.
• HOU, Y.-M., and GILBERTSON, R. L. 1996. Increased pathogenicity in a pseudorecombinant bipartite geminivirus correlates with intermolecular recombination. J. Virol. 70:5430-5436.
• HOU, Y.-M., E. R. GARRIDO-RAMIREZ, S. STINGLEY, P. GUZMAN, and GILBERTSON, R. L.1996. Characterization of the infectious clones of the Sinloa strain of pepper hausteco virus. Phytopathology 86:S43.
• GARRIDO-RAMIREZ, E. R., and GILBERTSON, R. L. 1996. Molecular detection and characterization of bean golden mosaic geminivirus (BGMV) from Chiapas, Mexico. Phytopathology 86: S17.
• SANCHEZ, V., FRIAS, G., AGUIRRE, L., BRAVO, L., CORTEZ, E., QUEZADA, J., RUIZ, R., HERNANDEZ, M., RANGEL, U., GARZON, J., RIVERA, R., GILBERTSON, R., and ROJAS, M. 1996. Agroecology of a whitefly-transmitted geminivirus.

Progress 01/01/95 to 12/30/95

Outputs
Whitefly-transmitted geminiviruses continue to be one of the most severe pests of beans, tomatoes, and other crops produced in Central America, the Caribbean basin and the southern United States. There is considerable concern that these viruses may enter California. We continue to develop improved and more rapid methods for detecting and characterizing these viruses. We routinely screen samples from California for the presence of whitefly-transmitted geminiviruses and, in the 1995 season, we did not detect WFT geminiviruses in California tomatoes. A screening method for resistance to one tomato geminivirus (tomato mottle geminivirus, ToMoV) was developed and resistance to this geminivirus was identified in wild tomato spp. (e.g. accessions of LYCOPERSICON CHILENSE and L. HIRSUTUM). Because numerous WFT geminiviruses are found in Mexico and these viruses could be introduced into California, we have been characterizing a geminivirus associated with one such disease, which has become prevalent in recent growing seasons in the Sinaloa state of northwestern Mexico. This disease is characterized by vein yellowing and chlorosis on newly infected leaves followed by striking stunting and distortion of leaves emerging later. It was confirmed that the disease was caused by a geminivirus and genetic analysis revealed that this geminivirus is a distinct strain of pepper hausteco geminivirus (PHV), which had been previously reported from eastern Mexico.

Impacts
(N/A)

Publications

• GILBERTSON, R. L., HOU, Y.-M., GUZMAN, P., and CARVALHO, M. G. 1995. A new geminivirus associated with chlorosis of tomatoes in northwestern Mexico. Phytopathology 85: 1138.

Progress 01/01/94 to 12/30/94

Outputs
Whitefly-transmitted geminiviruses continue to be severe threats to crop production in Central America, the Caribbean basin and in the southern United States. There is considerable concern that these viruses may enter California. In 1993-1994, a severe virus outbreak occurred in tomatoes in the Dominican Republic. Plants were severely stunted, and leaves were curled, small and often had yellow margins. These symptoms were very similar to those induced by tomato yellow leaf curl (TYLCV), which occurs in the Middle East and Europe. When hybridized with a general DNA probe for whitefly-transmitted geminiviruses, virus infected tomatoes hybridized weakly. However, when hybridized with a specific probe for TYLCV, the infected tomatoes hybridized strongly. A 2.8 kb DNA fragment was amplified from the infected tomato tissue by the polymerase chain reaction (PCR) and overlapping TYLCV-specific PCR primers. The fragment was cloned and partial nucleotide sequences were determined. A partial nucleotide sequence of the intergenic region was 97, 66, and 55% identical to homologous regions of TYLCV from Israel (TYLCV-ISR), TYLCV from Sardinia (TYLCV-SAR), and tomato mottle geminivirus (ToMoV). Similarly, a partial sequence of the coat protein open reading frame was 98, 79, and 69% identical to homologous sequences from TYLCV-ISR, TYLCV-SAR, ToMoV.

Impacts
(N/A)

Publications

• NAKHLA, M. K., MAXWELL, D. P., MARTINEZ, R. T., CARVALHO, M. G., and GILBERTSON, R. L. 1994. Occurrence of the eastern Mediterranean strain of tomato yellow leaf curl geminivirus in the Dominican Republic. Phytopathology 84: 1072.
• NAKHLA, M. K., MAXWELL, D. P., MARTINEZ, R. T., CARVALHO, M. G., and GILBERTSON, R. L. 1994. Widespread occurrence of the Eastern Mediterranean strain of tomato yellow leaf curl geminivirus in the Dominican Republic. Plant Disease 78:926.
• ISAKEIT, T., ROBERTSON, N. L., BROWN, J. K., and GILBERTSON, R. L. 1994. First report of squash leaf curl virus on watermelon in Texas. Plant Disease 78:1010.
• PAPLOMATAS, E. J., PATEL, V. P., HOU, Y.-M., NOUEIRY, A. O., and GILBERTSON, R. L. 1994. Molecular characterization of a new sap-transmissible bipartite genome geminivirus infecting tomatoes in Mexico. Phytopathology 84: 1215-1224.

Progress 01/01/93 to 12/30/93

Outputs
Whitefly-transmitted geminiviruses continue to be severe threats to crop production in Central America, the Caribbean basin and in the southern United States. There is considerable concern that these viruses may enter California. Now we have used the molecular methods that we have developed to detect and differentiate these viruses to identify outbreaks of squash leaf curl geminivirus (SqLCV) in Texas and tomato yellow leaf curl geminivirus in the Dominican Republic. This latter find is the first report of this devastating virus from the Middle East being present in the Western Hemisphere. We have partially characterized a different tomato-infecting geminivirus from Mexico and named it tomato leaf crumple geminivirus (TLCrV). It is one of a complex of geminiviruses affecting tomatoes in Mexico and we are trying to identify the other viruses involved. To screen tomato cultivars for resistance to geminiviruses we have generated an agroinoculation system for the Florida tomato mottle geminivirus and TLCrV. Plants can be efficiently infected without the use of whiteflies and this technique is being evaluated for screening tomato cultivars for resistance to geminiviruses.

Impacts
(N/A)

Publications

• PATEL, V. P., ROJAS, M. R., PAPLOMATAS, E. J., and GILBERTSON, R. L. 1993. Cloning of biologically active geminivirus DNA using PCR and overlapping primers. Nucleic Acids Research 21:1325-1326.
• ROJAS, M. R., GILBERTSON, R. L., RUSSELL, D. R., and MAXWELL, D. P. 1993. Use of degenerate primers in the polymerase chain reaction to detect whitefly-transmitted geminiviruses. Plant Disease 77: 340-347.
• HIDAYAT, S. H., GILBERTSON, R. L., RUSSELL, D. R., HANSON, S. F., MORALES, F. J., AHLQUIST, P. G., and MAXWELL, D. P. 1993. The nucleotide sequence of the infectious DNA components of bean dwarf mosaic geminivirus. Phytopathology 83: 181-.
• GILBERTSON, R. L., FARIA, J. C., LEONG, S. A., AHLQUIST, P. G., and MAXWELL, D. P. 1993. Genetic diversity in geminiviruses causing bean golden mosaic disease: the nucleotide sequence of the infectious cloned DNA components of a Brazilian.
• GILBERTSON, R. L., PAPLOMATAS, E. J., GRIECO, P. D., PATEL, V. P., and MAXWELL, D. P. 1993. Agroinfection of bean, tobacco, and tomato with two bipartite geminiviruses from tomato. Phytopathology 83:1350.
• FARIA, J. C., GILBERTSON, R. L., HANSON, S. F., MORALES, F. J., AHLQUIST, P., LONIELLO, A. O., and MAXWELL, D. P. 1994. Bean golden mosaic geminivirus type II isolates from the Dominican Republic and Guatemala: Nucleotide sequences, infect.

Progress 01/01/92 to 12/30/92

Outputs
The tremendous outbreaks of sweetpotato whitefly (BEMISIA TABACI) populations inthe southern Untied States, Mexico, and Central America has also brought an increased incidence of plant diseases caused by geminiviruses. These single-stranded DNA viruses cause very severe plant diseases, and apparently new geminiviruses infecting tomatoes and/or peppers have appeared in Florida, Texas, and Mexico. We have cloned the complete DNA genome of two gemini-viruses infecting tomatoes in Florida and Mexico, respectively. We have demonstrated that these viruses are very different from each other, and that the cloned viral DNA will cause disease symptoms in plants similar to symptoms seen in the field. The Florida geminivirus has been named tomato mottle geminivirus (ToMoV) and it is different from previously characterized geminiviruses. The closest relative to ToMoV is abutilon mosaic geminivirus, which we have found in California ornamental abutilon plants. We have also shown that the cloned DNA or ToMoV induces very different disease symptoms in different tomato cultivars, which further complicates field identification. We have developed and are using DNA probes and a polymerase chain reaction technique to detect and differentiate these viruses.

Impacts
(N/A)

Publications

• GILBERTSON, R.L., HIDAYAT, S.H., ROJAS, M.R., and MAXWELL, D.P. 1991. Infectious DNA clones of a new geminivirus associated with tomatoes in Florida. Phytopathology 81:1184.
• PAPLOMATAS, E.J., HIDAYAT, S.H., MAXWELL, D.P., and GILBERTSON, R.L. 1991. Molecular cloning and characterization of a geminivirus infecting tomatoes in Mexico. Phytopathology 81:1220.
• GILBERTSON, R.L., PAPLOMATAS, E.J., ROJAS, M.R., and MAXWELL, D.P. 1992. Advances in the molecular detection and characterization of geminiviruses infecting leguminous plants. Fitopatologia Brasileira 17:1441-142.
• PAPLOMATAS, E.J., GRIECO, P.D., ROJAS, M.R., MAXWELL, D.P., and GILBERTSON, R.L. 1992. Geminivirus complexes associated with tomato and pepper diseases in Mexico. Phytopathology 82:1070.
• HOU, Y.-M., MAXWELL, D.P., and GILBERTSON, R.L. 1992. Pseudo-recombination between two distinct geminivirus: Host range determinants reside on the DNA-B component. Phytopathology 82:1070.
• GILBERTSON, R.L., HIDAYAT, S.H., PAPLOMATAS, E.J., ROJAS, M.R., and MAXWELL, D.P. 1993. Pseudorecombination between the cloned infectious DNA components of tomato mottle and bean dwarf mosaic geminiviruses. Journal of General Virology.