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STRATEGIC RESEARCH PLAN
K. Hackett, E. Civerolo, R. Bennett
November 25, 2003
Agricultural Research Service (ARS) Researchers: D. Akey, E. Backus, K. Baumgartner, J. Blackmer, D. Boyd, J. Buckner, S. Castle, J. C. Chen, vice-Civerolo, A. Cohen, T. Coudron, P. Cousins, J. de Leon, G. Elzen, M. Glenn, R. Groves, J. Hagler, J. Hartung, T. Henneberry, W. Hunter, W. Jones, D. Kluepfel, J. Legaspi, C. Ledbetter, R. Leopold, H. Lin, G. Logarzo, R. Lynch, S. McKamey, M. McGuire, S. Naranjo, G. Puterka, D. Ramming, F. Ryan, N. Schaad, R. Scorza, J. Uyemoto, G. Yocum
ARS Research Associates: M. Francis, P. Joost, J. Habibi, W. Li, K. Tubajika, F. Zeng
Cooperation
Collaborators: P. Anderson [University of Florida (UF)-Quincy], D. Bartels [USDA-Animal and Plant Health Inspection Service (APHIS)-Mission], M. Blua [University of California (UC)-Riverside], D. Boucias (UF-Gainesville), G. Bruening (UC-Davis), F. Byrne (UC-Riverside), L. Cañas (University of Arizona-Tucson), C.-J. Chang (University of Georgia), A. Chaparro (Nacional Universidad, Colombia), M. Ciomperlik (APHIS), D. Cook (UC-Davis), H. Costa (UC-Riverside), K. Daane (UC-Berkeley), A. Dandekar (UC-Davis), M. Fatmi (Hassan II University, Agadir, Morocco), M. Gaiani (Facultad de Agricola, Universidad Central de Venezuela), C. Godoy (Instituto Nacional de Biodiversidad, Costa Rica), T. Gradziel (UC-Davis), B. Grafton-Cardwell (UC-Riverside), D. Gray (UF-Apopka), T. Freeman (Electron Microscope Center, North Dakota State University, Fargo), D. Gubler (UC-Davis), G. Gupta (Los Alamos National Laboratories, Los Alamos, NM), J. Hashim (UC-Cooperative Extension), A. Hicks (University of Colorado), R. Hix (UC-Riverside), M. Hoddle (UC-Riverside), M. Johnson (UC-Riverside), R. Jones (California Table Grape Commission), H. Kaya (UC-Davis), B. Kirkpatrick (UC-Davis), G. Lacy (Virginia Polytechnic Institute and State University, Blacksburg, VA), I. Lauziere (APHIS-Mission), E. Lopez (Clemson University-Charleston), J. Lu [Florida A&M; University (FAMU)-Tallahassee], D. Luvisi [Glassy-winged Sharpshooter (GWSS) Task Force of Kern and Tulare Counties, Project Coordinator], T. Miller (UC-Riverside), R. Mizell (UF-Quincy), D. Morgan [California Department of Food and Agriculture (CDFA)-Rubidoux], C. Pickett (CDFA-Bakersfield), A. Purcell (UC-Berkeley), R. Rakitov [Illinois Natural History Survey (INHS)], G. M. Raygoza (Universidad de Guadalajara, Mexico), G. Simmons (APHIS-Phoenix), D. Takiya (INHS), N. Toscano (UC-Riverside), S. Triapitsyn (UC-Riverside), M. Van Sluys (U. Sao Paolo, Brazil), E. Virla (PROIMI, Tucuman, Argentina), M. A. Walker (UC-Davis), G. Walker (UC-Riverside), L. Wendel (APHIS)
Partners/Cooperators: T. Batkin (Citrus Research Board), B. Drake (Drake Enterprises, Temecula, CA), C. Heintz (California Almond Board), P. Poole (Mt. Palomar Vineyards and Winery, Temecula, CA), A. Tariq (CDFA), C. Weaver (formerly of Calloway Vineyards and Winery, Temecula, CA), R. Wynn (CDFA)
Coordination: County Agricultural Commissioners, CDFA, National Invasive Species Council, UC-Cooperative Extension, California Pierce’s Disease (PD)-GWSS Board, UC-Department of Agriculture and Natural Resources (DANR) PD-GWSS Grant Program, USDA-APHIS
Executive Summary
The bacterium Xylella fastidiosa (Xf) causes serious diseases in many agronomic, horticultural, and landscape ornamental plants – including Pierce’s disease (PD) of grape, and leaf scorch diseases of almond, peach, plum, pear, and oleander in the U.S., and, citrus and coffee in South America. PD, alone, threatens a California wine, table, and raisin grape-dependent industry valued at $33 billion, and almond leaf scorch threatens the $730 million almond industry; oleander leaf scorch attacks the main roadside planting, oleander, in California. The Xf strain that causes citrus variegated chlorosis, if it were to be introduced into the U.S., would be particularly devastating to citrus production in California and Florida. The recent association of Xf with scorch disease-like symptoms in olive in southern California may also represent a potential risk to olive production.
While Xf strains that cause PD (and almond leaf scorch disease) have been vectored in California by several native sharpshooter (leafhopper) species, the introduction of the glassy-winged sharpshooter (GWSS) into southern California in the 1990’s has resulted in epidemics of disease in grape there (Temecula, in Riverside County) and in the lower San Joaquin Valley (Kern County). This is due to the GWSS’s large numbers, behavior of feeding at the base of canes (thus spreading the pathogen beyond prunable wood), and tendency to migrate deep into vineyards from areas where the insect overwinters in citrus and other plants.
ARS responded to the PD epidemic by organizing an Emergency PD/GWSS Research Response Team (PD/GWSS Team) that made site visits to areas of the PD epidemic in southern California in 2000, and developed a Strategic Plan of action. Since that time, the ARS effort on xylella diseases and GWSS has expanded greatly, through redirection of resources and personnel (there are currently 38 senior scientists engaged in full- or part-time PD/GWSS research at 16 locations), receipt of additional base-funding, and due to a greatly expanding network of Federal, state, and university collaborations. To coordinate the current research effort, ARS conducted a strategic planning process in 2003 that resulted in the document “ARS Xylella fastidiosa Diseases – Glassy-winged Sharpshooter, Strategic Plan,” submitted herein.
ARS has taken the approach of responding in the short-term to solutions that will suppress GWSS populations, as a means of interfering with transmission of Xf and reducing the incidence of xylella diseases. There has been considerable success in this effort (as acknowledged by a Secretary’s Honor Award to the PD/GWSS Team in 2003), brought about through a collaborative effort with our partners, achieved, in part, through our development of kaolin-clay based repellents, and evaluation and demonstration of the effectiveness of foliar and systemic insecticides. Having slowed the epidemic, ARS is now focusing on mid- and long-term research directed toward sustained control of xylella diseases and the sharpshooter. These research efforts include: i) comprehensive studies of the systematics, biologies, ecologies, epidemiologies, and genomics of Xf strains and sharpshooter vectors on a variety of crops (particularly, grape and almond) and reservoir hosts; ii) exploration of Xf-vector-plant interactions; and, using this baseline information, iii) evaluation of integrated pest management approaches for mitigating the impact of the Xf-caused diseases. An important component in this effort has been ARS’s support for sequencing, with Brazil, of Xf strains that cause scorch diseases in grape, almond, and oleander.
ARS has also supported grape genomics at the University of California, Davis, with the long-term goal of developing resistant varieties of grape, and has developed technology for transforming grape that can be used in elucidating gene function, identifying plant genes responsible for resistance and the disease process, and, primarily, facilitating breeding programs.
Introduction
Background of Problem
Recent invasions of exotic insect pests into California (e.g., pink hibiscus mealybug, vine mealybug, olive fruit fly, imported fire ant, and Africanized honey bee) highlight the threat of invasive species to agricultural production in the State.
One recent introduction, the glassy-winged sharpshooter (GWSS), Homalodisca coagulata (Say), is an effective vector of Xylella fastidiosa (Xf), a bacterial pathogen that causes devastating diseases in a wide variety of agronomic and horticultural crops, as well as landscape ornamentals and shade trees (Hopkins & Purcell, 2002; Purcell 1979; Purcell & Hopkins, 1996; Purcell et al., 1979, Purcell et al., 1999). Some of these diseases are emerging as serious, destructive diseases of important crops (e.g., almond, citrus, coffee, grapevines, peaches, plum) in North and/or South America. In addition, a number of other non-crop plant species are hosts for Xf (Purcell et al., 1999). The threat to wine, table, and raisin grapes (>750,000 acres; total economic value >$33B) and almonds (>525,000 acres; value >$730M), alone, if realized, would be devastating to the California economy.
Currently, there are no effective treatments for xylella diseases, including Pierce’s disease (PD) of grapevines, almond leaf scorch disease (ALSD), plum leaf scald disease (PLSD) and phony peach disease (PPD), each of which can result in serious economic losses during crop production. Control of xylella diseases is currently directed toward reduction of vector numbers.
Although Xf strains can multiply to some degree within a large number of plants that are inoculated with the bacterium, relatively few plants support moderate to high bacterial populations and fewer still allow systemic movement of Xf (Purcell et al., 1999). Further, high bacterial titer, in itself, is not always closely correlated with disease (D. Cook, pers. comm.).
Considerable genetic and pathogenic variability occurs among Xf strains (Chen et al., 1995, 2000; da Costa et al., 2000; Hendson et al., 2001; Hopkins & Purcell, 2002; Mehta & Rosato, 2001; Pooler & Hartung, 1995; Purcell & Hopkins, 1996). Additionally, phylogenetic studies divide X. fastidiosa into three subspecies, X. fastidiosa subsp. piercei (grape, alfalfa, almond and maple), X. fastidiosa subsp. multiplex (peach, plum, sycamore, elm, and almond), and X. fastidiosa subsp. pauca (citrus) (Schaad et al., 2004, in press).
PD occurs in some California vineyards every year. Serious losses have traditionally occurred in the Napa Valley, but, with the introduction of GWSS into southern Califormia, losses are now extensive in parts of the San Joaquin Valley (e.g., Kern County) and the Temecula Viticulture Area. This has resulted in the need for extensive grape replanting. In Florida and other southeastern states (usually with native GWSS populations present), PD has precluded commercial production of European varieties. Some muscadine grapes and American wild grape x European grape hybrids (Vitis vinifera) are tolerant or resistant to PD (Hopkins & Purcell, 1996; Purcell & Hopkins, 2002; Varela et al., 2001).
Xf is transmitted by xylem-feeding insects. The primary insect vectors of Xf are leafhoppers (family Cicadellidae), particularly sharpshooters (subfamily Cicadellinae); however, spittlebugs (family Cercopidae) are also Xf vectors (Hopkins & Purcell, 2002; Purcell & Hopkins, 1996). Transmission of Xf, especially early in the season, by native sharpshooter species in California occurs primarily by adult insects that migrate into vineyards and other crop plantings from outside hosts (Varela et al., 2001). In California, at least 20 species of xylem-feeding insects are capable of transmitting Xf (Hill & Purcell, 1997); however, only four species are considered epidemiologically significant for PD (Varela et al., 2001).
GWSS was first detected in California in 1989 (Sorenson and Gill, 1996), following its introduction from the southeastern U.S. Since then, it has become invasive, spreading and becoming established throughout southern California and into the southern San Joaquin Valley (Blua et al., 1999, 2001). The GWSS feeds on a wide variety of horticultural crop and ornamental plants, and transmits Xf prolifically by virtue of its large populations, and tendencies to migrate far into vineyards, and to feed at the base of the cane, beyond the prunable wood; consequently, the PD epidemic has exploded in areas in which this vector is present. The epidemiologies of other xylella diseases [e.g., ALSD, oleander leaf scorch disease (OLSD), and scorch diseases of other landscape plants] are also likely to change in areas in which the GWSS is present. The presence of the GWSS also poses a potential threat to other landscape and ornamental plants, as well as cultivated horticultural crops. This threat will be amplified if other exotic plant pathogens are introduced into California [e.g., Xf strains that cause citrus variegated chlorosis (CVC), PPD, PLSD, and pear leaf scorch disease (PeLSD)], and if other Xf strains or pathotypes emerge (e.g., olive scorch).
Thus, xylella diseases are caused by three or more subspecies of Xf (piercei, multiplex, and pauca) and a wide range of insect vectors, with extensive host ranges for two of the pathogens and the insect vectors. These diseases, especially PD and CVC, are potential threats to the production of major crops in California. A foundation for management of this pest and diseases caused by Xf and its vectors will require a better understanding of the factors that influence disease epidemiology, as well as vector and pathogen biology and ecology.
ARS Emergency Research Response
On January 21, 2000, then Deputy Secretary Richard Rominger and ARS Administrator Floyd P. Horn charged the ARS Pacific West Area and the ARS National Program Staff with assessing the Agency’s research capability and implementation needs for mitigating the impact of the GWSS and PD in California. On February 17-18, 2000, ARS assembled an Emergency Research Response Team (ARS Team) of senior level program managers and technical experts to provide this assessment. Given the magnitude of the invasive species threat to agricultural crop production, and the lack of any effective pre-harvest ARS disease and insect pest management technical capacity in California, response to the complex PD-GWSS epidemic had to be National and complement ongoing research conducted by scientists in the UC system and CDFA. Although some plant pathology research capability and germplasm resources were available in California, key elements necessary for a full response from ARS were lacking. In particular, the Team concluded that the following critical research areas were not being addressed: Xf biology, ecology and pathology; GWSS biology and ecology, including Xf transmission (acquisition and inoculation); biological control of invasive insect species, including vectors of plant pathogens such as GWSS; chemical control of insect vectors, including the effects of insecticides on non-target organisms; molecular biology of exotic plant pathogens, including Xf strains; plant physiological and biochemical responses to Xf infection and disease development; and pathogen and insect vector modeling. The Team also concluded that ARS capacity building should focus on pest (i.e., insect, pathogen) invasion biology.
A Congressional field hearing on the PD problem was held at the St. Supery Winery and Vineyards in Rutherford, Napa Valley, California, on February 22, 2000. The Under Secretary for Marketing and Regulatory Programs represented the USDA at this hearing. Representatives of the ARS Team were present to provide information to the Deputy Under Secretary that they had obtained, primarily, during the February 17-18, 2000, site visit to the Temecula Viticulture Area and through subsequent research planning sessions.
The ARS Team then developed an Action Plan (ARS Emergency PD/GWSS Research Response Plan, 2000) for addressing the PD-GWSS problem in California. The plan included immediate actions and potential, longer-term, research activities based upon available resources. Through increased base-funding and program redirections, ARS now has more than 30 senior level scientists engaged full or part-time on GWSS and PD (and other xylella diseases).
Through a Specific Cooperative Agreement between USDA-ARS and Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) in Brazil, the genome of a grape strain of Xf associated with PD in California was sequence and annotated. Work is continuing to complete sequencing and annotating the genomes of ALSD and OLSD strains of Xf. The genome sequence of the California Xf-PD strain provides information for elucidating the molecular basis of its pathogenicity, as well as for determining the phylogenetic relationships among Xf strains. Genes that have been identified that potentially function in Xf-host interactions include those that code for hemolysins, hemagglutin, adhesions, and cell wall degrading enzymes. All of these, and likely others, are potential targets for disrupting Xf-host interactions. In addition, genome sequences which have been made available through this research have been used to develop new Xf-PD, -ALSD and –OLSD specific primers for the pathogen-based, clinical diagnosis of Xf infection by real-time PCR.
Also, through a contract with Doug Cook (UC-Davis), an inventory of grape genes for transcriptional profiling was initiated. Among more than 60,000 EST’s identified in this work, 6,550 are from Xf-infected, resistant Vitis hybrids. Several genes that appear to be up-regulated in response to Xf infection were identified. Promoters for these genes could be used to drive Xf-induced and/or tissue specific expression of transgenes. In addition, real-time reverse transcriptase PCR for gene expression was developed. Since they detect systemic responses, host transcripts that can serve as markers of Xf infection may be more sensitive than pathogen-based PCR primers for disease diagnosis. Moreover, transcriptional response pathways may be correlated with disease resistance, tolerance or susceptibility. This work has led to testable hypotheses on the importance of drought, pathogen population level, and host vs. pathogen vs. vector contributions to the diseases process.
A transformation system for somatic embryos of grape was developed by Ralph Scorza (ARS-Kearneysville) in collaboration with Dave Ramming (ARS-Parlier) and Dennis Gray (UF-Apopka). Using this methodology, leaf-derived somatic embryos of the grape cultivar Thompson Seedless were transformed through the use of a combined treatment of Agrobacterium tumefaciens infection and microprojectile bombardment. Thompson Seedless plants were produced that expressed the Shiva-1 lytic peptide gene. Plants expressing the lytic peptide gene were evaluated for resistance to PD and showed a potentially useful level of resistance (Scorza, R. and D. J. Gray. United States Patent 6,232,528 B1, issued May 15, 2001).
Vision
Economic, effective protection of agronomic, horticultural and landscape crops from Xf-caused diseases and Xf-vectors, using scientifically-based, environmentally-sound, and cost-effective methods that are worker and consumer safe.
Mission and Goal
Develop effective management strategies to reduce or mitigate losses due to Xf-caused diseases and Xf-vectors during crop production that are safe, environmentally-sound, socially-acceptable, and economical. The overall goal of this research is to find treatments to mitigate or cure diseases caused by Xf, control or reduce the spread of Xf, and control or suppress populations of Xf insect vectors (including, but not necessarily limited to, the GWSS). Included in the goal is the development of PD resistant germplasm with high fruit quality and identification of genes and molecular markers for PD resistance.
Coordination and Communication with Customers, Stakeholders and Partners
ARS program managers and scientists work actively with partners in the scientific community (state and Federal), regulatory agencies/organizations (state and Federal), and industry to develop, review, and coordinate its research on a National level, to effectively meet the needs of its customers, beneficiaries, and stakeholders.
References
Blua, M. J., et al. (1999). A new sharpshooter threatens both crops and ornamentals. California Agriculture 53(2): 22-25.
Blua, M. J., et al. (2001). Seasonal flight activity of two Homalodisca species (Homoptera: Cicadellidae) that spread Xylella fastidiosa in southern California. Journal of Economic Entomology 94(6): 1506-1510.
Chen, J., et al. (1995). Randomly amplified polymorphic DNA analysis of Xylella fastidiosa Pierce's disease and oak leaf scorch pathotypes. Applied and Environmental Microbiology 61(5): 1688-1690.
Chen, J., et al. (2000). Use of 16S rDNA sequences as signature characters to identify Xylella
fastidiosa. Current Microbiology 40(1): 29-33.
da Costa, P. I., et al. (2000). Strains of Xylella fastidiosa rapidly distinguished by arbitrarily
primed-PCR. Current Microbiology 40(4): 279-282.
Hendson, M., et al. (2001). Genetic diversity of Pierce's disease strains and other pathotypes of Xylella fastidiosa. Applied & Environmental Microbiology 67(2): 895-903.
Hill, B. L. and A. H. Purcell (1997). Populations of Xylella fastidiosa in plants required for transmission by an efficient vector. Phytopathology 87: 1197-1201.
Hopkins, D. L. and A. H. Purcell (2002). Xylella fastidiosa: Cause of Pierce's disease of grapevine and other emergent diseases. Plant Disease 86(10): 1056-1066.
Mehta, A. and Y. B. Rosato (2001). Phylogenetic relationships of Xylella fastidiosa strains from different hosts, based on 16S rDNA and 16S-23S intergenic spacer sequences. International Journal of Systematic & Evolutionary Microbiology 51(2): 311-318.
Pooler, M. and J. S. Hartung (1995a). Genetic relationship among strains of Xylella fastidiosa based on RAPD-PCR data. Hortscience 30(2): 192.
Pooler, M. R. and J. S. Hartung (1995b). Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data. Current Microbiology 31(2): 134-137.
Pooler, M. R. and J. S. Hartung (1995c). RAPDs are useful for genetic analysis of Xylella fastidiosa and for development of strain-specific PCR primers. Hortscience 30(4): 783.
Purcell, A. H. (1979). Leafhopper vectors of xylem-borne plant pathogens. Leafhopper Vectors and Plant Disease Agents. K. Maramorosch and K. F. Harris. New York, Academic Press: 603-625.
Purcell, A. H. and D. L. Hopkins (1996). Fastidious xylem-limited bacterial plant pathogens. Annual Review of Phytopathology. R. K. Webster, Annual Reviews Inc., P.O. Box 10139, 4139 El Camino Way, Palo Alto, California 94306, USA: 131-151.
Purcell, A. H., et al. (1979). Pierce's disease bacterium: Mechanism of transmission by leafhopper vectors. Science 206: 839-841.
Purcell, A. H., et al. (1999). Causal role of Xylella fastidiosa in oleander leaf scorch disease. Phytopathology 89(1): 53-58.
Schaad, N.W. et al. (2004). Xylella fastidiosa subspecies: X. fastidiosa subsp. piercei, subsp. nov., X. fastidiosa subsp. multiplex subsp. nov., and X. fastidosa subsp. pauca subsp. nov. Systematic and Applied Microbiology
Sorensen, J. T. and R. J. Gill (1996). A range extension of Homalodisca coagulata (Say) (Hemiptera: Clypeorrhyncha: Cicadellidae) to southern California. Pan-Pacific Entomologist 72(3): 160-161.
Varela, L. G. (1996). Pierce's disease in the north coast, University of California, Cooperative Extension & Statewide IPM Project: 1-11.
RESEARCH COMPONENTS/RESEARCH AREAS
I. Xf Systematics, Genomics, Biology, Ecology, Epidemiology
ARS Research Context:
ARS laboratories in Beltsville, Frederick and Parlier have focused on Xf strain relationships, particularly genetic differentiation of strains and rapid diagnosis, through use of real-time PCR formats and microarray-PCR based systems, and systematics.
Following FAPESP Brazil’s successful sequencing of an Xf-CVC strain from citrus, ARS sponsored the sequencing of an Xf-PD strain from grape, an Xf-ALSD strain from almond, and an Xf-OLSD strain from oleander, thus providing the first opportunity for a comparative genomics approach for understanding the bacterium’s taxonomy and systematics (at ARS-Parlier and ARS-Beltsville), and its interaction with vectors and plant hosts. Rapidly evolving genes in the bacterium are being investigated (UC-Riverside) because they will most likely be the ones useful for identification of strains, and for control. Progress has also been made by ARS scientists at Beltsville and others at UC-Davis in developing transposon mutants for studying Xf genetics. DNA microarrays and mutational analysis are being used to identify Xf virulence genes at UC-Riverside.
The ARS-Parlier laboratory is also investigating the epidemiology of Xf diseases in California, particularly, determining seasonal fluctuations of genotyped strains of Xf in cultivated crops and reservoir hosts as a function of abiotic factors; while UC-Riverside is focusing on strains in landscape plants. The Parlier laboratory also is determining the spatial and temporal patterns of Xf-caused diseases as a function of GWSS and other sharpshooter species. The fundamental mechanism of Xf-PD transmission by GWSS is being studied at UC-Berkeley and ARS-Parlier.
Naturally-occurring Xf-inoculum sources are being determined at UC-Riverside (for southern California), and by ARS scientists at Parlier (for California’s Central Valley), and UC-Davis (for north coastal California). Xf transmission pathways are being identified at ARS-Parlier and UC-Riverside. Epidemiological assessments are being done cooperatively by ARS, CDFA, UC-Cooperative Extension, UC-Riverside and UC-Berkeley (Kearney Agricultural Center) in major grape growing regions in southern California, the Central Valley of California, and north coastal California.
The Almond Board of California is also funding research on the epidemiology and control of almond leaf scorch disease, as well as on transmission of ALSD strains of Xf, at UC-Davis and UC-Berkeley. This research includes collaborations with ARS scientists at ARS-Parlier and UC-Davis.
Goal 1: Clarify the taxonomy/nomenclature of Xf.
Current Situation: The taxonomy of the organism has not changed since it’s original description in 1987 even though several additional diseases and strains have been described, including diseases of shade trees and CVC of citrus. All strains of Xf have been lumped into a single species. The use of a single pathogen name for several different diseases is confusing. Revision of the species is needed in order to develop improved diagnostics and improved host/vector relationships.
Objective 1: Clarify the classification of the species Xf in order to link all information about host, vector, epidemiology, and control to appropriate names.
Approach: ARS will establish the taxonomy of the species/subspecies of Xyllella, determine the appropriate names, and describe new species/subspecies, as appropriate.
Cooperators: This research was and is being done by Norman W. Schaad (ARS-Ft. Detrick) in collaboration with George Lacy (VPI, Blacksburg, VI), M’Barek Fatmi (Hassan II University, Agadir, Morocco), and C.-J. Chang (University Georgia).
Milestones:
2003: Conduct DNA-DNA relatedness assays using high stringency and sequence the 16S-23S intergenic spacer (ITS) region of typical strains of 8-10 different hosts, including grape and almond.
Accomplishments:
The taxonomy of 26 strains from 10 hosts, including grape, almond, alfalfa, peach plum, and citrus has been completed. Results show that the 26 strains can be divided into three genetically and phenotypically unique subspecies, piercei, multiplex, and pauca.
1) Schaad, N.W., Postnikova, E., Lacy, G., Fatmi, M., and Chang, C.J. 2004. Xylella fastidiosa subspecies: X. fastidiosa subsp. piercei, subsp. nov., X. fastidiosa subsp. multiplex subsp. nov., and X fastidiosa subsp. pauca subsp. nov. Systematic and Applied Microbiology (In Press).
Expected Benefits: The taxonomic revision of Xf will be an authoritative source for identification of the causal agents of the different diseases and be useful to researchers, public and private diagnostic laboratories, and quarantine efforts by state and federal officers. Having specific names for each of the causal disease agents will assist in communication.
Goal 2: Improve diagnostic procedures for detection, identification, and differentiation of Xf.
Current Situation: Methods for detecting Xf infections and diagnosing Xf-caused diseases include symptomatology, pathogen isolation, serological assays, and nucleic acid based approaches. Each of these approaches has advantages and disadvantages depending upon the specific need and application. Because of the sensitivity, specificity, adaptability, speed, and relative ease of use, PCR-based assays (including real-time PCR formats) are widely used for Xf detection and genotype identification. However, the application of PCR-based assays for large-scale, rapid, high-throughput uses can be limited by the presence of inhibitors, laborious extraction protocols, and uneven distribution of low levels of the pathogen in infected hosts (especially in the early stages of infection).
Objective 1: Develop real-time PCR formats for clinical detection and identification of Xf.
Approach: Develop and improve rapid, sensitive and quantitative real-time PCR-based assays for Xf for use in the LightCycler instrument from Roche (Hartung, Beltsville) and the Smart Cycler from Cepheid (Schaad, Frederick; vice-Civerolo, Parlier).
Cooperators: This research was and is being done by John S. Hartung (ARS-Beltsville), Norm Schaad (ARS-Frederick), and vice-Civerolo (ARS-Parlier). Evaluation and validation involves grape and wine growers in California, and other ARS and UC scientists.
Milestones:
2001: A standard PCR assay specific for Xf was first described in 1995.
2001: Applied Cleaved Amplification Product Polymorphism (CAPS) analysis to show that the PCR-amplified product can be easily used to distinguish Xf-CVC strain from Xf-PD strains.
2002: Described a real-time, quantitative PCR assay for a one-hour diagnosis of PD using the portable, rapid cycling Smart Cycler platform.
2003: Compared several extraction methods to select the best method to rapidly isolate Xf DNA prior to real-time PCR.
2000-2003: Designed and evaluated additional, new or improved primers for PCR-based differentiation of Xf strains and pathotypes. Designed and made commercially available a dry bead formulation containing all PCR ingredients, including Xf-specific primers and probe for improved, more robust and rapid real-time PCR assay.
Accomplishments:
The real-time PCR method was applied to quantify Xf-CVC in sweet orange seed. Methods have been developed that can rapidly detect, quantify, and distinguish strains of Xf.
1) Li, W.-B., W. D. Pria, Jr., P. M. Lacava, J. S. Hartung. 2003. Presence of Xylella fastidiosa in Sweet Orange Fruit and Seeds and its Transmission to Seedlings. Phytopathology 93 (8):953-958.
2) Qin, X., Miranda, V. S. , Machado, M., Lemos, E. and Hartung, J.S. 2001. An evaluation of the Genetic Diversity of Xylella fastidiosa isolated from Diseased Citrus and Coffee. Phytopathology 91(6):599-605.
3) Schaad, N. W., D. Opgenorth, and P. Gaush. 2002. Real-time polymerase chain reaction for one-hour on-site diagnosis of Pierce’s disease of grape in early season asymptomatic vines. Phytopathology 92:721-728.
Expected Benefits: Any research project that needs to rapidly quantify Xf, for example, in plant samples or insect vectors, will benefit from this procedure.
Objective 2: Develop DNA microarray-PCR based identification and detection systems for Xf.
Approach: Develop a set of gene or DNA sequences, defined as diagnostic sequences, that can be used to identify specific Xf pathotypes. Use complete and annotated Xf genome sequences to select the appropriate DNA sequences. Evaluate these sequences for their potential to differentially identify Xf pathotypes/genotypes. Using these sequences, design and construct DNA microarrays on glass slides, and use these microarrays to analyze genomic variation among Xf variants from broad geographical areas and hosts.
Cooperators: Scientists at ARS-Parlier are collaborating with California grape and almond growers.
Milestones:
2004: Identify diagnostic sequences and evaluate their specificity at Xf species and pathotype levels. Establish and characterize a large collection of Xf strains.
2005: Construct, evaluate, and modify Xf-DNA chips using the available Xf strain collection.
2006: Standardize and continue improvement of Xf-DNA chips and evaluate their potential in clinical application.
Accomplishments: Research has just been initiated.
Expected Benefits: Xf-DNA chips will be used for quick and accurate identification of Xf and its pathotype. The dynamic design ensures newly available research information for Xf identification and characterization.
Goal 3: Elucidate the biotic and abiotic factors that affect Xf biology and ecology.
Current Situation: Xylella diseases are complex pathosystems. Xf has an extensive and diverse host range, including many cultivated horticultural crops and ornamental landscape plants, as well as native plant species, in California. Many of these species are limited propagative hosts for Xf, while only a few are systemic hosts. Also, many non-cultivated native plant species are asymptomatic. The epidemiological significance of these various Xf hosts as inoculum sources for spread of the pathogen to cultivated crops is not fully known. Since its introduction into California around 1990, the invasive, aggressive Xf vector GWSS has become established and spread in several counties in southern California and into other areas of the State (e.g., portions of the San Joaquin Valley). The GWSS is becoming widely distributed, and feeds and oviposits on a wide range of perennial crops (e.g., citrus), ornamental species and non-cultivated native plant species. The presence of this insect vector is expected to affect the occurrence, distribution, and economic importance of xylella diseases. The epidemiology of xylella diseases in California is differentially affected by the presence of native insect vectors and the recently introduced invasive GWSS. Knowledge of the factors that affect the epidemiology of xylella diseases can provide useful information regarding (but not necessarily limited to) primary and secondary inoculum sources, mechanisms of pathogen dispersal, and the dynamics of spatial and temporal disease incidence. This information is critical for developing effective disease management strategies.
Objective 1: Determine the genetic diversity and relatedness of Xf strains in crops and reservoir hosts.
Approach: Symptomatic leaves will be collected from selected vineyards and orchards, and patterns of disease spread will be recorded. Pathogens will be isolated and subjected to genomic variation analysis, such as RAPD analysis. Genomic DNA will be isolated from the various Xf isolates using the CTAB minipreparation. Computer-image analysis of the DNA banding patterns obtained during RAPD and rep (repetitive extragenic palindromic)-PCR analyses will be performed.
Cooperators: This research is conducted by Jianchi Chen with Russ Groves and vice-Civerolo (ARS-Parlier) in cooperation with California grape and almond growers, and Farm Advisors.
Milestones:
2003: Established experimental designs, initiated sample collections, initiated isolation of Xf strains from diverse sources in different geographical areas, and initiated diversity analyses.
2004-2005: Continue and adjust, if necessary, sample collection and diversity analyses.
2006: Complete this phase of characterization of Xf population genetic diversity
Accomplishments: This research has just been initiated.
Expected Benefits: Clarification of whether single, multiple, or mixed populations of Xf strains are associated with PD and ALSD in California. This knowledge is potentially useful for the assessment of the risk of xylella pathogens to horticultural crops other than grapes and almonds (e.g., peaches, plums, citrus, olives). Determination of inoculum sources. This knowledge will be useful in developing improved, effective strategies to manage Xf-caused diseases, not only in California, but elsewhere as well.
Objective 2: Analyze the comparative spatial and temporal patterns of Xf-caused diseases in the presence of indigenous sharpshooter vectors and GWSS.
Approach: Disease incidence is determined by surveys based on visual symptoms. Selected samples are assayed for Xf by isolation, ELISA, and/or PCR to confirm the association of the pathogen with disease symptoms. Two-dimensional maps of the spatial distribution of disease incidence are generated for experimental field units per commodity. Xf vector populations are also monitored (e.g., using yellow sticky traps, sweep nets) and identified. The spatial patterns of disease incidence are interpreted by statistical means (e.g., by ordinary runs analysis, two-dimensional distance class analyses, and geostatistics).
Cooperators: Kayimbi Tubajika (ARS-Parlier) collaborates with Jennifer Hashim and Don Luvisi (UC-Cooperative Extension); Lloyd Wendel, Dave Bartels and Matt Ciomperlik (APHIS), and grape growers in Kern and Santa Barbara Counties in California. Vice-Civerolo and Russ Groves will collaborate with other UC-Riverside and UC-Berkeley scientists to analyze PD and ALSD incidence data collected in the San Joaquin Valley.
Milestones:
2001: Established field plots in 11 vineyards in Kern County (where the GWSS is present); initiated PD incidence surveys; established initial parameters of PD incidence (e.g., % infected plants, % vineyards with disease gradients, % vineyards with apparent ‘edge effects’).
2002–2003: Continued PD incidence data collection in Kern County. Initiated PD incidence data collection in Santa Maria (Santa Barbara County), vineyards where the GWSS is not present. Began analysis of collected data.
2003: Completed analysis of PD incidence in selected vineyards in Kern County. Continued analysis of PD incidence data collected in Santa Maria.
2004: Complete comparative epidemiological analyses of PD incidence data collected in areas with and without the GWSS (e.g., Kern County, Santa Maria, and Coachella Valley).
Accomplishments:
PD incidence data collected in Kern County from 2001 through 2003 were analyzed by ordinary runs and two-dimensional class analyses, and illustrated using multispectral images. Based on these analyses: 1) PD incidence in Kern County ranged from <1% to 8% in different vineyards; 2) no disease gradient or edge effect was detected in any vineyard; and, 3) spatial disease gradient analyses consistently described the non-randomness of the patterns of PD-affected vines, and an increase in the degree of clustering of PD-affected vines, as disease incidence increased.
Expected Benefits: Improved xylella disease management strategies based on knowledge of disease epidemiology whether or not the GWSS is present.
Objective 3: Determine seasonal fluctuation(s) of Xf in cultivated crops (including, but not necessarily limited to, grapevines and almonds) and reservoir hosts, and relate these fluctuations to abiotic factors.
Approach 1: Evaluate the significance of riparian hosts in the epidemiology of PD in the North Coast grape-growing region of California. Among systemically infected riparian hosts, seasonal differences in Xf population levels likely affect their importance as Xf reservoirs. The efficiency of Xf acquisition and inoculation by Graphocephala atropunctata (blue-green sharpshooter, BGSS) is influenced by the Xf levels in host plants; the higher the concentration, the higher the probability of BGSS acquiring Xf while feeding. Therefore, in riparian hosts, seasonal fluctuations of Xf levels may influence the spread of the pathogen and the incidence of PD by affecting the proportion of BGSS that acquire Xf when feeding on these hosts. Xf acquisition is also influenced by vector host preference; a systemic riparian host that is fed upon more frequently by BGSS will likely serve as a more significant source of Xf. By measuring seasonal concentrations of Xf in riparian plants, it will be determined if, and when, concentrations are high enough for acquisition of the pathogen by BGSS. This research will focus on five systemic riparian hosts: Rubus discolor (Himalayan blackberry), R. ursinus (California blackberry), Sambucus mexicana (blue elderberry), Vinca major (periwinkle), and Vitis californica (California grapevine). Future research will focus on BGSS feeding preference for these riparian hosts.
Cooperators:
Kendra Baumgartner (ARS-Davis) is collaborating with Alexander H. Purcell (UC-Berkeley).
Milestones:
2002: Propagated California grape, California blackberry, Himalayan blackberry, blue elderberry, and periwinkle (100 plants/species) in the greenhouse.
2003: Mechanically-inoculated all plants with the STL strain of Xf (a strain isolated from PD-symptomatic vines in Yountville, CA) and, 4 months later, used PCR to confirm infection. Transferred infected plants from the greenhouse to screenhouses at two sites: Oakville (Napa County, CA) and Hopland (Mendocino County, CA). Began seasonal Xf quantitation using dilution plating and real-time PCR (October, 2003).
2004: Continue seasonal Xf quantitation. Determine the effects of plant species, season, and location on mean Xf concentration using an analysis of variance. Compare Xf quantitation techniques. Begin research on BGSS feeding preference for the five riparian hosts.
Accomplishments:
Determined that populations reached detectable levels in all five riparian host species during October, 2003. Every replicate plant of periwinkle and California grapevine showed leaf scorch symptoms characteristic of PD, and high concentrations of Xf. Since none of the Himalayan blackberry showed symptoms, despite high Xf population levels, Himalayan blackberry may be tolerant of Xf infection.
Determined that Xf concentrations in California grapevine, Himalayan blackberry, and periwinkle are sufficient for acquisition by BGSS in early autumn. Xf isolations coincided with increased flight activity of young adult BGSS, which peaks in mid summer and remains high through early autumn. Assuming BGSS feeds on California grapevine, Himalayan blackberry, and periwinkle in early autumn, Xf may be transmitted from infected riparian plants to adjacent vineyards before the end of the growing season. Late season infections of grapevines are unlikely to result in chronic disease and infected canes are pruned out during the dormant season. However, young adult BGSSs that acquire Xf in mid summer to early autumn and survive the winter are still capable of transmitting Xf the following spring after budbreak.
Expected Benefits: Riparian revegetation management is a method of PD control that focuses on removal of hosts of BGSS and Xf (host plants other than grapevines), followed by revegetation with native, non-hosts. This method has some positive aspects. With lower BGSS populations, fewer insecticide applications are used. Also, some of the plants targeted for removal (Himalayan blackberry and periwinkle) are invasive weeds. However, removal of riparian vegetation is very disruptive to wildlife, and increases the probability of stream bank erosion. Also, some of the riparian hosts are extremely difficult to eradicate. The fewer the riparian plants removed before revegetation, the less disruption there would be to wildlife habitat.
The success of revegetation management depends on a thorough understanding of how riparian hosts contribute to the spread of Xf and the incidence of PD. Although removal of riparian hosts can reduce local populations of BGSS, impact on the riparian area as a reservoir of Xf has not been quantified. Although Xf hosts are known, it is not known if, or when, these hosts attain high enough populations of Xf for acquisition by BGSS in the field. If the results of this research reveal that only a few of the riparian hosts recommended for removal serve as major sources of Xf, grape-growers can concentrate on removing fewer riparian plants, thereby reducing the total amount of riparian habitat disruption.
Approach 2: Small-plot, caged field experiments will be conducted in southern California to investigate the ability of a PD strain of Xf to infect and overwinter within selected annual and perennial non-crop wild plant species and, further, to examine the relationships among time of plant infection and the efficiency of systemic infections. A total of ten plant species have been pre-selected for these experiments based upon information regarding their extensive distribution and abundance in urban and agricultural environments, their ability to serve as ovipositional and feeding hosts for vectors, and their ability to support infections of Xf.
Cooperators: Russ Groves (ARS-Parlier) is collaborating with Jianchi Chen (ARS-Parlier), California grape and almond growers, and Farm Advisors.
Milestones:
2004: Newly-germinated plants of experimental summer annual and perennial species will be needle-inoculated at each of three stages of plant growth – representing vegetative, flowering, and post-flowering developmental stages. Plants will be assayed monthly for infection and GWSS acquisition studies will be conducted.
2005: Continued examination of infection retention in perennial non-crop species and evaluation of winter annual species as potential systemic sources of Xf inoculum.
Accomplishments: This research has just been initiated.
Expected Benefits: Determination of the extent to which different non-crop species support only propagative versus fully systemic infections, and, further, assessment of the retention of Xf infection over the course of the season. This information will supplement our understanding of important reservoir plant hosts by characterizing the extent to which selected non-crop weed species can serve a dual role as overwintering hosts for Xf and as acquisition sources for pathogen spread.
II. Vector Systematics, Genomics, Biology, Ecology, Epidemiology, Mass Rearing
ARS Research Context:
ARS, uniquely, at the Systematics Entomology Laboratory (SEL) in Beltsville, is conducting research on the systematics of leafhoppers, including GWSS. Identification of tiny wasps that parasitize leafhoppers is also being done by scientists at SEL, in cooperation with scientists at ARS-Weslaco and UC-Riverside.
Artificial diet development was the responsibility of Allen Cohen (ARS-Starkville), until his recent retirement; the ARS laboratory at Columbia will now carry on this work. This work complements studies by APHIS-Mission, North Dakota State University at Fargo, and CDFA (Riverside and Bakersfield) which has resulted in identification of plant hosts for mass rearing GWSS.
Because leafhopper biologies vary according to their biotic and abiotic environments, studies of GWSS biology are being done at several laboratories, working with different populations of GWSS, and under different climatic (e.g., temperature, humidity) and geographic (e.g., day length) conditions, cropping systems, and management practices, and, with the intention of using the acquired data for varied research purposes. Major research collaborations in this area are between ARS-Phoenix and UC-Riverside (southern California), ARS-Parlier and the Kearney Agricultural Center in Parlier (California’s Central Valley), ARS-Davis with UC-Berkeley (north coast of California), and with ARS-Weslaco and UC-Riverside. The ARS program at Parlier is comprehensive, as regards consideration of the majority of these biotic and abiotic factors (e.g., addressing all plant hosts – crops, ornamentals, and native plants), while focused on determining host preference and GWSS population density and movement/dispersal [in citrus and grape]. ARS-Phoenix is developing monitoring methods for GWSS, and testing seasonal abundance and comparative dispersal of GWSS and smoke-tree sharpshooter (STSS) in citrus and grape. UC-Riverside is relating GWSS population size to counts obtained from different sampling methods. Traps for GWSS adults and nymphs being developed at UC-Riverside will facilitate design of monitoring schemes.
Such information will be combined with investigations of hosts (tree quality), vectors (crowding, sex ratio, reproductive status), seasonal and environmental conditions, and pathogen interactions, with the goal of predicting disease spread, and finding life cycle targets for interrupting the transmission of the causative bacteria. This work thus complements in-house (ARS-Parlier) and collaborative research between ARS-Fargo and North Dakota State University to determine the physiological, behavioral, anatomical, and ultrastructural mechanism(s) of feeding (e.g., stylet penetration of canes), as well as studies at UC-Davis to determine the insect’s reproductive biology as related to its physiology.
In addition to ARS-Parlier, UC laboratories (Riverside, Berkeley, Davis) are also conducting multifactor studies of GWSS biology and ecology in the San Joaquin Valley. These studies focus on GWSS survival in relation to xylem flux and chemistry in grape, and in relation to the physiology of host selection in citrus. At UF-Quincy, work focuses on GWSS behavior, physiology (especially as related to host selection, nutrients, and malnutrition), and natural enemies as limits to GWSS populations. Researchers there, as well as in California at ARS-Parlier and UC-Riverside, are also looking, comparatively, at California crop phenology as a means of predicting the relative course of disease expression in these different climates. Also, other laboratories, e.g., those at UC-Riverside, focus on the effects of GWSS feeding on grape fruit quality and yield.
ARS-Weslaco is determining what factors account for the natural low population levels of GWSS in its native habitat; molecular markers are being used to characterize the genetic variation among geographic populations of GWSS to determine if there may be a species complex.
Goal 1: Clarify the taxonomy/nomenclature of GWSS.
Current Situation: Three species of the genus Homalodisca are already known to transmit the Xf strain that causes PD. It is expected that all species in the genus have the capacity to be, or become, important Xf vectors. Of the 19 species in the genus, 13 are exotic for the U.S. and 17 are exotic for California. Most species of Homalodisca, were they to be introduced into, and become established in, California, are serious potential economic threats to several important agricultural crops. Current correct names are in flux and the genus has never been revised. Revision of the genus is needed in order to be clear about which species are present and disseminating Xf strains.
Objective 1: Stabilize the classification of the genus Homalodisca in order to link all other information about host plants, ecology, physiology, and genomics to correct names.
Approach: ARS will establish the taxonomic limits of the genus Homalodisca and the limits of all species in the genus, determine their valid names, and describe new species as necessary and/or appropriate. The brochosome structure will be characterized to allow identification of egg masses. Authoritative and accessible identification aids and distribution data will be provided for the genus, as well as specimen support for the study of other sharpshooter leafhoppers as regards their relationship to Homalodisca and their potential to transmit Xf.
Cooperators: Stuart McKamey (ARS-Beltsville) is collaborating with Roman Rakitov and Daniela Takiya (INHS), Andrew Hicks (University of Colorado), Carolina Godoy (Instituto Nacional de Biodiversidad, Costa Rica), Gustavo Moya Raygoza (Universidad de Guadalajara, Mexico), and Marco Gaiani (Facultad de Agricola, Universidad Central de Venezuela, Marcay).
Milestones:
2003: Examine and analyze Homoladisca species and complete preliminary character matrix; request additional material from institutional collections, extract label data, and determine latitude/longitude coordinates. Complete illustrations for three species of Homalodisca. Conduct expedition to Costa Rica for exotic species of Homalodisca.
2004-2005: Conduct expeditions to Mexico and Venezuela (pending restoration of political stability in the latter country), complete character matrix and phylogenetic analysis of Homalodisca species. Postdoctoral fellow (supported by ARS-Beltsville) will complete characterization of brochosomes and submit results for publication. Complete illustrations for remaining species of Homalodisca. Compile identification key for image-driven, web-based access. Complete revision of Homalodisca and submit for publication.
Accomplishments:
The taxonomic revision of Homalodisca is in progress. In addition to the specimens held by the National Museum of Natural History, ARS-Beltsville has borrowed over 1,000 specimens from over a dozen institutions, has extracted locality data, and converted the data to decimal degree geographic coordinates for over 1,500 specimens, has begun characterization of species variation, and has generated a preliminary data matrix. Dr. Rakitov has characterized the egg brochosomes and related behavior of 8 species of Homalodisca. The Costa Rica fieldwork (June-July 2003) by ARS-Beltsville and cooperators Rakitov, Hicks, and Godoy yielded behavioral data, host data, and samples of egg masses, parasites, and fresh material for molecular and morphological analyses of Homalodisca and many close relatives, including several rare genera. The taxonomic revision of Homalodisca will be the authoritative source for identification of all species in the genus and will therefore be useful to many GWSS/PD/xylella researchers, state and Federal agricultural staff, as well as for commodity risk assessments conducted by APHIS and the quarantine efforts of the Department of Homeland Security. The Costa Rica specimens provided more complete geographical data for Homalodisca and are facilitating development of a more stable and predictive classification of all sharpshooters, including leafhopper vectors of Xylella worldwide.
Expected Benefits: Words are the tools of communication and taxonomy is the vocabulary of species. The objective of this aspect of GWSS research is to stabilize the classification of the genus Homalodisca so that all other information gathered (host plants, ecology, physiology, and genomics, which are priorities in solving the Xylella problem) can be linked to correct names for meaningful communication.
Goal 2: Develop sampling procedures to reliably estimate GWSS population densities and movement.
Current Situation: Since its introduction around 1990, the GWSS has become established in several agricultural production areas, including southern California, and Kern County in the San Joaquin Valley. The sharpshooter has also been detected in other important areas, such as Tulare County in the San Joaquin Valley. The sharpshooter has exacerbated the incidence of PD, and may affect the epidemiology of other Xf-caused diseases such as (but not necessarily limited to) almond leaf scorch disease. Sampling procedures to reliably estimate GWSS population densities are needed in order to develop effective pest management strategies based on understanding factors that affect the vector’s behavior and dispersal.
An important behavior of the GWSS that contributes to the rapid epidemic of PD is long distance dispersal flights of the vector into vineyards (Blua et al., 1999; Blua et al., 2000; Sorenson & Gill, 1996). Most current knowledge of these dispersal differences have been obtained by using sampling methodologies in vineyards and citrus orchards [e.g., a UC-Riverside project in Temecula, CA (Blua et al., 2000)], and these studies are well served by the use of qualitative (relative) measurements. Now, however, sampling methods are being used to also determine timing of biorational and convention pesticide treatments and to judge their efficacy [e.g., Temecula, CA, (Hix et al. 2002), and Bakersfield, CA (Wendel et al., 2000)]. This application implies that the sampling method(s) must accurately quantify population numbers. For example, if a given treatment against GWSS results in “zero counts” by beat sampling, does that mean there are no GWSS in the treated area, that they were all killed by the treatment, or could some be left alive – too few to be detected? In the latter case, how many are left alive, enough to vector PD within or out of the treated area? To answer these questions, it is vitally important that the use of sampling methods be validated by total population counts, and to provide information useful for the selection of sampling methods and interpretation of sampling data.
Objective 1: Develop, test, and deliver statistically-sound sampling plans for estimating densities, and inoculum potential, of GWSS for applications to research and management.
Approach 1: Compare four sampling tools for estimating GWSS density in terms of precision and cost. Develop and validate sampling procedures and plans for citrus and grapes for research and decision-making applications. Extend the sampling plans to estimate the proportion of the GWSS population that is inoculative with Xf.
Cooperators: Steve Castle and Steve Naranjo (ARS-Phoenix) are collaborating with Nick Toscano (UC-Riverside) in Riverside, Temecula, and Ventura Counties.
Milestones:
2002-2003: Comparative quantitative analyses were used to identify the bucket and beat net as the most efficient sampling methods for GWSS in citrus. The bucket method has the advantage of being useful for sampling all heights within the tree.
Catches on yellow sticky traps, a common monitoring tool, were correlated to on-plant populations of GWSS in citrus; however, the relationship was variable across years.
Spatial distribution studies revealed that more GWSS are found in the upper half of the citrus tree canopy, and more are found on the south sides of trees. This information was used to refine the sample unit designation.
A preliminary sequential sampling plan was developed for fixed-precision sampling of GWSS on citrus.
A progressive increase in the proportion of adults positive for Xf occurred from the time of adult emergence in late June, 2002, through April, 2003.
2004: Test and refine the sequential sampling plan for GWSS on citrus, develop and test and binomial sampling plan for management application.
Accomplishments:
Four sampling methods were evaluated (bucket, beat net, D-Vac, and A-Vac) to determine which technique is the most reliable and cost efficient. The bucket sampler was the most cost efficient technique and provided good reproducibility for estimating both adult and nymphal populations of GWSS.
Yellow-sticky trap catches were compared with foliage sampler catches to determine the degree of correlation between these techniques. Yellow sticky trap catches of adult GWSS are highly correlated with all foliage sampling methods, but the relationships were variable between years.
The spatial distribution of nymphal and adult GWSS was studied in citrus orchards in Riverside, CA, using a bucket sampling method. On average, about 2.4 times as many GWSS were collected in the upper half of the tree canopy compared with the lower half, and about 1.6 times as many were collected on the south side of trees compared with the north side. The coefficient of variation (CV=SD/mean) was nearly 2 times lower in samples taken from the upper half of the canopy compared with the lower half, but there were no differences in the CVs among different compass directions. These findings were used to refine the sample unit for sampling GWSS in citrus.
Based on the bucket sampling method, density-dependent sample size and sample cost estimates have now been determined, and a preliminary sequential sampling plan for estimating relative population density of GWSS in citrus has been developed. Further work will be needed to independently test the validity of this sampling plan. For pest management application, additional research will be needed to define treatment thresholds.
This sampling program has been applied towards estimating the incidence of Xf in GWSS adults. ELISA, PCR, and culturing techniques for the detection of Xf in GWSS are being carried out to obtain an accurate estimate of the proportion of individuals within the population that are capable of transmitting PD. ELISAs have provided the most consistent results. A progressive increase in the proportion of adults positive for Xf occurred from the time of adult emergence in late June, 2002, through April, 2003. The mean titer of Xf in heads and thoraxes also increased progressively through this period, suggesting that the potential for vectoring Xf may rise as the spring generation of adults age.
Expected Benefits: Sampling is a fundamental component for the study of population dynamics and central to the development of robust strategies for pest management. ARS research has focused on the development of an efficient method for estimating densities of GWSS in citrus. Based on considerations of precision and cost, a bucket sampler has been identified as an efficient sampling method. Further study of the spatial distribution of GWSS within citrus trees has helped to refine the sample unit and further reduce sampling costs. A preliminary sequential sampling plan that will enable researchers and pest managers to precisely estimate the relative density of GWSS at a minimal cost has been completed.
Approach 2: Methods and materials to fumigate GWSS populations in single orange trees were developed and tested, and, using these methods, procedures were developed to compare sampling methods with absolute counts of all GWSS in citrus trees. Obtained data on how many GWSS are actually present in a given block of citrus, obtained total population counts of GWSS from individual trees, and began correlating sample numbers of beat counts to total populations. Correlated sampling methodologies to total population counts. Provided information for the selection of sampling methods and interpretation of sampling data.
Cooperators: David Akey, Tom Henneberry, and James Hagler (ARS-Phoenix) are collaborating with Matthew Blua and Carlos Coviella (UC-Riverside) on sampling research associated with insecticide treatments, primarily at Riverside.
Milestones:
2001: Developed and tested methods and materials to fumigate and count GWSS population in single orange trees; developed procedures to compare sampling techniques with absolute counts of all GWSS in citrus trees.
2002: Obtained data to answer how many GWSS are present in a given block of citrus, obtained total population counts of GWSS from individual trees, and began correlating sample numbers of beat counts to total populations.
2003: Continued with 2002 objectives, obtained data biweekly, analyzed data, and correlated sampling methodologies to total population counts.
2004: Will provide information for the selection of sampling methods and interpretation of sampling data.
Accomplishments:
Methods and materials were developed and tested to elucidate the actual size of a GWSS population in single orange trees, and total population counts of GWSS were obtained from individual trees.
Procedures were developed for comparing sampling techniques with absolute counts of all GWSS in small citrus trees.
Data are beginning to answer how many GWSS are actually present in a given block of citrus.
Efforts are underway to correlate sample numbers from beat counts to total GWSS populations.
Expected Benefits: These studies have already demonstrated that: 1) exceedingly large numbers of GWSS may be present in individual citrus trees (400-600 in 6-7 ft trees and 4000 in conventional sized trees); 2) beat or visual sampling may not detect GWSS at actual population levels of 20 to 80 GWSS or even higher; and, 3)appreciation of these numbers has led to investigations of whether GWSS populations actually do impact citrus harvest. Also, this work will be used to understand how sampling methods in current use relate to actual population numbers, i.e., to interpret qualitative sampling measurement data with respect to quantitative population numbers.
Goal 3: Determine relationships between climatological factors and GWSS overwintering.
Current Situation: The arrival of GWSS in southern California has dramatically changed the epidemiology of PD. The insect is now present in the San Joaquin Valley and was first detected in Kern County in 1998. However, the insect’s rapid population expansion first observed in southern California appears to be constrained to discrete regions within the Central Valley in or adjacent to citrus production areas where overwintering populations are greatest and winter temperatures are relatively mild when compared to expansion at lower elevations in the valley. Presently, there is limited information on the overwintering biology and ecology of GWSS in this large and important agricultural producing region. This lack of knowledge of the basic field ecology of GWSS limits our understanding of the spatial and temporal distribution of GWSS populations and the risk this pest poses for crop (e.g., grape and almond) production.
Objective 1: Determine GWSS population dynamics and overwintering.
Approach: The seasonal population dynamics and overwintering survival of GWSS populations are comparatively examined in agricultural areas of the Central Valley. Adult GWSS feeding and survival in climate-controlled growth chambers are determined in order to establish the threshold for feeding activity under different combinations of host type and temperature regimes. Using this information, describe differential GWSS mortality and fecundity in different regions and among combinations of host species.
Cooperators: ARS-Parlier (J. C. Chen, R. Groves, H. Lin, E. Backus) is collaborating with Marshall Johnson (UC-Riverside).
Milestones:
2003: Experimental GWSS colonies are being established and maintained in a reproductive diapause condition comparable to overwintering populations.
2004: Experimental bioassays will be conducted in environmental chambers to determine the GWSS temperature-dependent feeding activity threshold.
2005: Results will be coupled with high resolution, 1-km scale, climatological data to spatially define overwintering refugia for GWSS.
Accomplishments: This research has just been initiated.
Expected Benefits: The results of these experiments will aid our ability to define the specific environmental constraints that influence the population dynamics and overwintering success of GWSS. Moreover, the results from these experiments will be coupled with high resolution, 1-km scale, climatological data to spatially define locations where GWSS populations may be unable to successfully overwinter, or, conversely, where populations may find overwintering refuge from extended periods of critical temperatures.
Goal 4: Determine the population dynamics of GWSS with respect to Xf transmission and the occurrence of disease. This includes correlating the effects of crowding, sex ratio, reproductive status, host-plant quality, and seasonal and environmental variables with population dynamics and movement of GWSS as an aid to predicting insect and disease spread, and applying control strategies.
Current Situation: GWSS feeds on a wide variety of cultivated crops and landscape ornamental plants, as well as on native plant species, and vectors Xf, which causes PD and many other plant diseases. Effective management of Xf-caused diseases will depend on understanding the biology and ecology of this and other vectors of the causative agent(s). Of particular interest is understanding the behaviors of these vectors as related to the spread of Xf.
Objective 1: Determine and characterize the pattern(s) of utilization/ preferences of plant hosts among cultivated crops and non-cultivated hosts in agricultural production systems.
Approach: The seasonal host utilization patterns of sharpshooter species within and among a variety of cultivated, perennial crop plant species and non-crop, wild plant species will be examined. Crop utilization patterns are being monitored within perennial crop species including grape, citrus (navel and lemon), stonefruit (peach and plum), olive, cherry, pistachio, and avocado at each of three locations for each crop type through weekly sampling for the presence of sharpshooter adults using yellow sticky, beat-net, and sweep-net counts of all lifestages, and visual inspections. At each location, sharpshooter and spittlebug adults associated with orchard ground cover and surrounding non-crop vegetation are sampled using a standard sweep net. The presence of Xf in a subsample of vectors captured on yellow cards and on perennial and non-crop species are determined using PCR and strain specific primers are used to investigate the pathotype profile.
Cooperators: Russ Groves and Jianchi Chen (ARS-Parlier) are collaborating with Kent Daane UC-Berkeley) and Marshall Johnson (UC-Riverside).
Milestones:
2003: Experimental field plots have been established in GWSS-infested areas to monitor seasonal population dynamics and timing of dispersal. Currently, the overwintering population dynamics of vector species on crop and non-crop species is being evaluated.
2004: Continue to evaluate vector population dynamics through a second cropping season and overwintering period. Identify the pathotype profile of Xf from infectious vectors collected in 2003.
2005: Further evaluate the infection status of vectors collected in the 2004 growing season and the 2004-05 winter season.
Accomplishments: This research has just been initiated.
Expected Benefits: The results of these studies will provide further insight into the relative importance of different crop types as predominant overwintering habitats, ovipositional substrates, preferred feeding hosts, and sources for Xf acquisition and transmission to susceptible crops.
Objective 2: Determine how biotic and abiotic factors influence the relative movement of GWSS and the native smoke-tree sharpshooter (STSS) to help understand the dynamics of the spread of Xf.
Approach: Mark-release recapture (MRR) studies with IgG protein markers were used to compare rates of movement of GWSS and STSS in simple and complex host-plant assemblages. The associations between sharpshooter movement and environmental parameters, as well as host-plant characteristics were determined. The spatial scale of movement was analyzed by regression analysis and a diffusion model.
Cooperators: Jackie Blackmer (ARS-Phoenix) is collaborating with James Hagler (ARS-Phoenix), Greg Simmons (APHIS-Phoenix), and Luis Cañas (University of Arizona, Tucson) in conducting the research.
Milestones:
2001: Established the validity of using an IgG protein marker for GWSS dispersal studies. It had no effect on longevity and remained effective for at least 19 days under field conditions.
2002: Established that STSS dispersed further than GWSS in a MRR regime; however, the dispersal of both species was similarly affected by temperature and wind speed.
2003: GWSS dispersed more slowly in a complex host assemblage when compared to a simple assemblage, and the timing of dispersal was correlated most strongly with time of day and fluctuations in xylem pressure.
2004: Further determine how fluctuations in host-plant quality influence sharpshooter movement.
Accomplishments:
Immunoglobulin (IgG) proteins were tested as potential markers for dispersal studies with GWSS and STSS. Both chicken and rabbit IgG proteins were effective in marking sharpshooters. The marker remained effective under field conditions for at least 19 d and had no effect on survivorship of GWSS.
MRR studies with GWSS and STSS were conducted in 2001 in Moreno Valley in a simple landscape (abandoned alfalfa), and in 2002 in a complex landscape (citrus orchard). Both species readily dispersed horizontally to 90 m and vertically to 7 m; the most distant traps contained a larger percentage of the STSS. For these releases, temperatures above 17º C were correlated with increases in take-offs, and wind speeds over 3 m s-1 resulted in a significant reduction in take-off activity. In MRR studies, recapture rates were 12% in the simple landscape and 1.6% in the complex landscape. Linear regressions of recapture data with the diffusion model provided significant fits to the data for all releases except two. Calculations of dispersal distances using the diffusion model showed that 95% of GWSS had moved 90 m in 6 h or less, while 95% of STSS had moved 155 m in the same period. In the complex landscape, GWSS movement was much slower; 95% of GWSS were recaptured within 99 m of the release site, during a 72 h recapture interval.
In 2002, we also investigated whether plant factors (i.e., amino acids, osmolality, xylem pressure) and environmental parameters (i.e., wind speed, temperature, relative humidity, barometric pressure) influenced sharpshooter population dynamics and movement in a citrus grove setting. Number of egg masses and adults were counted on branches that were sampled for xylem sap. Collection date, tree, and cardinal direction were noted, and xylem pressure, and amino acids (total, essential and amides) were measured. In conjunction with xylem sap collections, movement of sharpshooters was monitored with yellow and clear sticky traps at 4-h intervals during the day and throughout the night. During replicated sampling periods, 40 times more sharpshooters were trapped on yellow sticky traps in comparison to clear sticky traps and the majority, regardless of sex, were trapped between 1000 and 1400 h. Higher trap catches were associated with increasing temperatures above 18ºC, but were not significantly associated with changes in wind speed, relative humidity or barometric pressure. Trap catches varied significantly over the trapping season, but did not differ due to trap location, indicating that there was no strong edge effect for GWSS. Relative to xylem sap collections, xylem pressure and amides varied due to collection date and time of day, and xylem pressure was positively correlated with trap catches. Osmolality, total amino acids, essential amino acids, and percent amides had no apparent relationship with trap catch. GWSS egg counts varied significantly due to collection date and cardinal direction, with the majority of eggs were observed on the east and south sides of the trees.
Blackmer, J.L., J.R. Hagler, G.S. Simmons & L.A. Cañas. Comparative dispersal of Homalodisca coagulata and Homalodisca liturata (Homoptera: Cicadellidae). Environ. Entomol. (in press)
Expected Benefits: Environmental variables and host-plant quality influence insect population dynamics and the timing and extent of their dispersal. An understanding of how these factors influence GWSS development and movement will aid us in predicting the spread of PD, as well as aid area-wide management strategies. The new serological method for MRR studies of sharpshooter movement will facilitate efforts to delineate the insect’s dispersal and spread of Xf.
Objective 3: Determine adult GWSS feeding and oviposition preference for, and nymphal development rates and survivorship on, healthy and Xf-infected grape representing different stages of plant infection.
Approach: Plant physiological status and resulting behavioral responses by phytophagous insects can impact vector ecology and patterns of pathogen spread. The proposed study is designed to determine the effects of Xf infection and resulting plant physiology on the distribution, performance, and behavior of GWSS and its associated natural enemies in controlled greenhouse experimental bioassays and in a small-plot field experiments.
Cooperators: ARS-Parlier is collaborating with Matthew Blua (UC-Riverside).
Milestones:
2003: Field plots established in Riverside, CA.
2004: Experiments are planned for 2004-2005.
Accomplishments: This research is just being initiated.
Expected Benefits: Taken together, these analyses will provide a comprehensive evaluation of how plant infection can influence the population dynamics of the vector, and, in return, PD epidemiology. This knowledge will be critical to evaluate the importance of early pathogen detection and development of crop management practices aimed at minimizing the extent of within-field, secondary spread. Elucidation of the preference for, and performance upon, Xf-infected versus healthy Vitis vinifera plants will aid our understanding of the mechanism of spread of PD and the speed with which in-field secondary pathogen spread can occur.
Goal 5: Determine GWSS feeding behavior, and nutritional and storage needs for use in developing mass production systems and in elucidating pathogen transmission.
Current Situation: Control of PD will largely be dependent upon areawide suppression of GWSS until resistant plant varieties are developed. Unfortunately, more than 130 plants can serve as hosts for GWSS and as reservoirs of the disease, often necessitating insecticidal applications in urban and wilderness areas adjacent to commercial fields. Biological control of GWSS in these areas would be more economically and environmentally desirable, and an important IPM strategy for this pest. Mass production of GWSS is needed for use in biological control programs. Production of one of the most effective biological control agents, Gonatocerus spp., parasites of GWSS eggs, would be critical to the biological control approach, but has been hampered by the difficulty and cost associated with producing sufficient numbers of GWSS eggs on which to rear the parasites. Development and formulation of artificial diets are needed for designing improved, reliable GWSS mass rearing protocols. Better knowledge of GWSS nutritional needs and feeding behavior would facilitate mass rearing, and also would help elucidate pathogen transmission mechanisms. (Also, see Research Area III, Goal I, for studies of GWSS feeding in relation to Xf transmission.)
Objective 1: Determine the feeding mechanism of GWSS and study stylet penetration into the host plant by documenting the path of the mouthparts from the epidermal layer to the vascular tissue and determine if feeding includes the parenchymatous or phloem tissue en route to the xylem tissue. Determine the ultrastructural characteristics of the salivary sheath, its chemistry, and interaction with all plant tissues along the stylet path from plant surface to xylem tissue.
Approach: Roger Leopold (ARS-Fargo) rears GWSS at the Bioscience Laboratory, on the North Dakota State University campus. This provides the ARS-Fargo team (including James Buckner) with a continual supply of immature and adult insects feeding on a variety of host plants. For morphology and ultrastructure of sharpshooter mouthparts, immature and adult GWSSs are examined using confocal scanning light microscopy (CLSM) of intact, cleared, and dissected specimens. Cleared specimens allow examination of the stylets within the labium and the location of each stylet in the non-feeding position, and show the mechanism by which the individual (mandibular and maxillary) stylets are anchored and moved. Cleared specimens are extremely valuable in determining the process of stylet movement through the labium and the mechanism of host tissue penetration. For stylet penetration (sites) and stylet function, the processes of stylet penetration into tissues of various host plants are documented using light, confocal scanning light microscopy, and scanning and transmission electron microscopy (SEM, TEM). Penetration sites in a variety of different plants are examined. The path of all cells that are penetrated, from the epidermal layer to the xylem tissue, is observed to determine plant response to penetration, and to determine the degree to which parenchymatous and phloem tissues are penetrated during a probe. Cleared plant tissues are also examined by CLSM, permitting 3-D reconstruction of the entire salivary sheath. This technique shows the position of the mandibular stylets during the feeding process and elucidating their role in anchoring the insect to the host plant when feeding. The ultrastructural details of the salivary sheath in relationship to the tracheary elements are examined using light, and SEM and TEM. Cleared specimens and those prepared for SEM are used to determine the general structural characteristics of the salivary sheath and TEM will be used to describe the ultrastructural relationship between the sheath and the anatomy of the tracheary elements.
Cooperators: James Buckner (ARS-Fargo) is collaborating with Thomas Freeman (Electron Microscope Center, North Dakota State University, Fargo, ND) and Roger Leopold (ARS-Fargo).
Milestones:
2003: A description of GWSS mouthpart morphology and characterization of plant penetration has been completed.
2004: Continue studies on interactions of salivary sheaths with host plant tissues.
2005: Determine ultrastructural characteristics and chemistry of GWSS salivary sheaths.
Accomplishments:
The gross morphology and ultrastructure of the labrum, labium, and stylet fascicle was described. Stylet probing in host tissue was found to be largely intercellular, with salivary sheaths frequently showing multiple branches.
Leopold, R. A., T. P. Freeman, J. S. Buckner, D. R. Nelson. 2003. Mouthpart morphology and stylet penetration of host plants by the glassy-winged sharpshooter, Homalodisca coagulata (Homoptera: Cicadellidae). Arthropod Structure & Development 32(2-3):189-199.
Expected Benefits: Cellular and ultrastructural data on GWSS mouthpart penetration of host tissues is required if we are to fully understand the interactions of the insect, the bacterium, and the host. Benefits will be a better understanding of nutrient uptake, pathogen transmission, and host suitability.
Objective 2: Determine xylem sap components and GWSS feeding biology as clues to developing an artificial diet for GWSS rearing.
Approach: Develop an improved understanding of the specifics of the feeding dynamics of GWSS in order to more adequately satisfy nutritional requirements in an artificial feeding system. The assumptions behind this work are that the profile of components in xylem sap used by GWSS will be an optimal or at least suitable diet for this species, and that details of the feeding biology of the insect will help identify its dietary needs. Studies are conducted to detail the plant’s sap profile before and after feeding.
Cooperators: Allen C. Cohen (ARS-Mississippi State, retired) collaborated with David Morgan (CDFA-Riverside), Isabelle Lauziere (APHIS-Mission), and Stephanie Rill (CDFA-Oswell Street Biocontrol Laboratory).
Milestones:
2002: Gross and fine anatomies, including ultrastructure, of the feeding system of GWSS were studied with light and electron microscopy. Large amounts of peptidase activity in the salivary glands were discovered.
2003: Began research efforts to develop an artificial diet and a flow-through feeding system for GWSS. This work was terminated after the retirement of the project leader, A. Cohen.
Accomplishments:
Examination of 100 salivary sheaths revealed that they are characteristically straight, leading directly from the plant surface to the xylem bundles; this is in contrast with branching seen in aphids and whiteflies. The conspicuous clypeus lies on the anterior and ventral part of the head and marks the region of attachment of the powerful cibarial (sucking) pump muscles, which permit the ingestion of large amounts of xylem sap (which is under negative pressure in the plant’s vascular system). The filter chamber is extremely active in peristaltic movements that evidently increase the efficiency of concentration of the sap and transfer of water to the Malpighian tubules, which remove the water and carry it directly into the hindgut where the water is stored in a bladder-like expansion of the hindgut until it can be discharged. The concentrated sap is processed by the midgut where the final nutrient products are absorbed by microvilli that are on the surface of a highly convoluted series of tubules.
One of the most unexpected and important findings in this work is the discovery of exceptionally great amounts of aminopeptidase activity and general peptidase activity in the salivary glands, filter chamber, anterior midgut, posterior midgut, and Malpighian tubules. GWSS has a much greater amount of amino peptidase activity, indicating that this insect uses nitrogen sources other than free amino acids, most likely peptides or even proteins. As a xylem sap feeder, GWSS was expected to lack ability to digest peptides because xylem sap is not known to contain substantial amounts of peptides or proteins. These findings led to our studies of the interactions between GWSS and host plants which revealed that the profiles of free amino acids in the xylem sap in infested and un-infested sweet potato plants show an increase in the concentrations of most amino acids in the xylem sap of infested plants.
As a result of these studies, further research efforts were made to provide an artificial diet that contained short peptides, including tryptic soy and casein digests, along with sugars, a dilute salt and vitamin mixture, and small amounts of organic acids that characterize the plant xylem sap, the natural food of this species. Also, a flow-through feeding system based on membrane feeding was used successfully to present the diet.
Expected Benefits: Development of a suitable diet for GWSS will lead to techniques for the mass rearing of promising natural enemies of this important pest species.
Objective 3: Develop artificial (diet) eggs for oviposition and rearing of GWSS parasites.
Approach: Develop an artificial diet for the production of artificial “eggs” that could be used to more economically rear Gonatocerus parasites of GWSS. Screen and optimize existing artificial diets for ability to sustain and promote development of Gonatocerus spp. parasites of GWSS. Develop a suitable artificial ovipositional substrate for Gonatocerus spp. Develop bioassays that can be used to screen bactericidal proteins linked to PD, or insecticidal resistance proteins linked to GWSS, resistance for their effects on Gonatocerus spp., and, perhaps, other nontarget insects. This would enable plant breeders to assess the risk of these proteins before incorporating them into commercial varieties.
Cooperators:
This research is planned to be done by Tom Coudron (ARS-Columbia). There are no collaborators at present.
Milestones:
2004: Assess the development of the gregarious egg parasite G. fasciatus and the solitary egg parasites G. ashmeadi and G. triguttatus on GWSS eggs and three artificial diets previously shown (Coudron, unpublished data) to support development of the hymenopteran parasitoids Trichogramma spp., Melittobia spp., and Euplectrus spp.
2005: A bioassay will be developed and used to select the most promising parasitoid/diet combination. That diet will then be optimized for the development of that species.
2006: Develop an artificial substrate for parasite oviposition, and an efficient method to deliver the parasite egg to the diet via development of an “artificial egg” or direct seeding of the parasite egg onto the diet. Also, assess the efficiency of the bioassay to screen proteins linked to GWSS and PD resistance.
Accomplishments: This research is just being initiated.
Expected Benefits: Development of cost-effective and less labor-intensive methods for mass rearing of the parasitoids to be used for the biological control of GWSS.
Objective 4: Develop storage technology for use in the mass rearing of GWSS and its parasitoids.
Approach: Determine the cold tolerance of the egg parasitoids, G. ashmeadi and G. triguttatus within host eggs of GWSS under specific environmental conditions and developmental stages. Assess whether chilling during an immature developmental stage has latent damaging effects on the quality of the adult parasitoid. Determine the efficacy of extending the shelf life of GWSS eggs for use by mass-reared parasitoids, by the method of pre-conditioning females environmentally and/or nutritionally.
Cooperators: Roger Leopold and George Yocum (ARS-Fargo) are collaborating with David Morgan (CDFA-Riverside).
Milestones:
2002: Established laboratory colonies of GWSS, G. ashmeadi and G. triguttatus. Determined optimum host plants for rearing and oviposition. Initiated cold tolerance studies on parasitized eggs of GWSS.
2003: Determined low temperature limits for development of egg parasitoids and GWSS. Identified preferred ovipositional host plants of the GWSS that also have extended cold tolerance as cuttings for storage purposes. Examined acceptability of cold-stored GWSS eggs by egg parasitoids and the subsequent development of egg parasitoids.
2004: Continue studies on extending cold storage for the unparasitized GWSS eggs, and eggs parasitized by Gonatocerus spp. Devise protocol(s) for cold storage.
Accomplishments:
By establishing a laboratory colony of GWSS, an ancillary study on GWSS mouthpart morphology and plant penetration was completed. The study identified previously undescribed accessory structures associated with the labrum, labium, and stylet fascicle that will provide impetus for electrophysiological and TEM studies for understanding the feeding process and host selection by the GWSS.
Expected Benefits: The effectiveness of any biological control agent used for pest control purposes depends on being released at the proper time. Unforeseeable environmental influences, such as those impacting pest migration, population fluctuations, and crop growth, amplifies the need for precise timing, especially when releases of insects are to be integrated into multi-disciplinary control programs. Development of cold storage technology for mass rearing of insects, such as the GWSS and its parasitoids, will allow insectary managers to gain flexibility and enable them to supply a purely biological product on demand.
Goal 6: Determine the geographic origin of the GWSS presently in California and whether the GWSS population may actually be composed of a geographically distinct species complex.
Current situation: To enhance the effectiveness of a biological control program it is critical to ensure that there is a single species and not a species complex involved. The native area of GWSS has been presumed to be the Gulf States and northeastern Mexico. Within this area, there may be genetically distinct GWSS populations that can be identified using modern methods of DNA analysis. Identification of the geographic source(s) of the California infestation could lead to the identification of possible future sources of invasion, and also identify geographic areas with which to look for adapted natural enemies. The possible detection of GWSS as a species complex would have widespread implications.
Objective 1: Develop molecular markers to determine if GWSS is a single species across its range, and, if not, determine if geographically separated populations can be reliably distinguished.
Approach: Collect GWSS from across its geographic range and screen various PCR-based molecular marking techniques to determine if genetically distinct geographic populations can be identified and characterized.
Cooperators: Jesús de León, Walker Jones, and the ARS-Weslaco group are collaborating with David Boyd (ARS-Poplarville), Jesusa Legaspi (ARS-Tallahassee), Rolando Lopez (Clemson University-Charleston), Robert Lynch (ARS-Tifton), David Morgan, (CDFA-Riverside), Greg Simmons (APHIS-Phoenix), and Russ Mizell (UF-Quincy) who have provided samples for testing.
Milestones:
2001-2002: Collected GWSS from its known range for DNA extraction.
2002: Applied various PCR methods (ISSR-PCR, RAMP, SAMPL, RAPD) to compare sensitivity and efficiency.
2003: Performed a geographic genetic population study of GWSS by ISSR-PCR.
2003: So far amplified and sequenced the mitochondrial COII gene of nine GWSS individuals from different locations and found seven haplotypes.
2004: Confirm results using mitochondrial DNA techniques.
Accomplishments:
de León, J. H. and W. A. Jones. 2003. Detection of DNA polymorphisms in Homalodisca coagulata (Homoptera: Cicadellidae) by PCR-based DNA fingerprinting methods. Accepted in Annuals of the Entomological Society of America.
DNA polymorphisms, for the first time, were detected in GWSS with four PCR-based DNA fingerprinting methods (ISSR-PCR, RAMP, SAMPL, and RAPD). The methods incorporating Simple Sequence Repeats (SSR) were found to be the most sensitive and efficient methods with GWSS template. These methods were not only able to distinguish different sharpshooter species (H. coagulata, H. lacerta, and H. insolita) but most importantly they were able to detect geographic variation in GWSS.
de León, J. H., W. A. Jones, and D. J. W. Morgan. Population genetic study of Homalodisca coagulata (Homoptera: Cicadellidae) performed by ISSR-PCR DNA fingerprinting. Submitted to Annuals of the Entomological Society of America, 2003.
In this study, compound Inter-Simple Sequence Repeat (ISSR) primers containing CA/GT-repeat motifs in their sequences were utilized to estimate the population genetic structure of GWSS from a total of 19 populations (544 total individuals) throughout the U. S. Results showed significant partitioning of gene diversity at three levels, among regions, among populations within regions, and among populations. The results estimated, for the first time, the population genetic structure of GWSS and suggested that a subset of insects in California may have their origins in the southwestern region (Texas) of the U. S.; furthermore, these results were suggestive of more than one founding event in California.
Since the origin of the California GWSS infestation is likely not Florida, the most effective natural enemies for release in California may therefore be found in an area outside Florida, particularly Texas. A separate study of egg parasitism in south Texas showed that an indigenous species parasitizes about 90% of GWSS egg masses throughout the season, perhaps at least partially explaining the uncommon occurrence of GWSS there. It is interesting to note that populations with some degree of genetic differentiation (differences in marker/alleles frequencies) may be considered as races. More work is needed to confirm this possibility.
Expected Benefits: Determination of GWSS genetics provides leads as to the degree to which management techniques need to be defined to a taxonomic unit below the species, and, also, clues as to where to search for parasitoids.
III. Xf-Vector Interactions
ARS Research Context:
The efficiency by which GWSS serves as a vector of Xf depends on its interaction with the pathogen. ARS-Parlier is focusing efforts on understanding vector acquisition and inoculation of the pathogen, using information gleaned through studies of sharpshooter feeding behaviors, as well as through an understanding of GWSS genomics. (Aspects of studies to elucidate feeding behavior in relation to GWSS nutrition are described in Research Area I, Goal 5.)
This work complements studies on the role of attachment factors in pathogenicity (UC-Berkeley). Other factors being studied include: i) the plant, as a substrate, and its affects on vector retention and inoculation of the pathogen (UC-Riverside and UC-Berkeley); and, ii) the impact of multiple strain infections on acquisition and inoculation (UC-Riverside). This work is aided by development of methods for detecting Xf in GWSS, primarily by PCR-based methods, at ARS-Parlier and UC-Riverside.
Goal 1: Determine the mechanisms of transmission (i.e., acquisition and inoculation) of Xf by GWSS (and other insect vectors).
Current Situation: GWSS is xylophagous and has a wide range of monocot and dicot hosts. The fine structure and function of the mouthparts of xylem-feeding insects, including the GWSS, are largely unknown. Information about the external location of feeding sites, stylet penetration (probing) behavior, salivary sheath formation within plant tissues and specific cells ingested, especially as related to acquisition and inoculation of Xf, is inadequate. Also, the role of watery, digestive saliva in inoculation and/or facilitating movement of Xf in plants is unknown.
Objective 1: a) Identify and quantify all probing (stylet penetration)behaviors of GWSS on grape, and b) identify the precise role of probing behavior in Xf acquisition and inoculation.
Approach: Specific probing behaviors are identified and quantified via electropenetration graph (EPG) monitoring. Waveforms are defined by correlation with stylet activities via videotaping of stylet movements and salivary sheath production in transparent diets, and through light and CLSM investigations of salivary sheath placement in plant tissues. Several methods for detecting Xf in the plant (i.e., PCR, culturing, ELISA, and immunocytochemistry) and in the vector (i.e., SEM examination of Xf in the foregut) are combined with EPG to determine the inoculation success of identified probes. In addition, electromyography or laser vibrometry are used with EPG to correlate specific stylet activities with movement of valves and muscles in the insect’s foregut.
Cooperators: Elaine Backus (ARS-Parlier) is collaborating with Greg Walker (UC-Riverside) and Thomas Miller (UC-Riverside).
Milestones:
2001: While at the University of Missouri, E. Backus established sharpshooter colony and plants and acquired equipment.
2002: Performed EPG-histology-diet correlation experiments to identify and define AC EPG waveforms, developed AC-DC monitor and began AC-DC waveform correlations. Performed experiments, via several bacterial detection methods, to determine the phases of probing behavior that are correlated with inoculation by identified probes.
2003: E. Backus assumed a new research position at ARS-Parlier, and moved experiments there. Analyzing results from correlation and probing-inoculation tests.
2004: Complete analysis of experiments by dissecting the heads of test insects to determine appearance of Xf colonies in foregut, to associate that appearance with results of probing-inoculation tests. Correlate electromyography of valve muscle movement with EPG waveforms.
Accomplishments:
Preliminary results show that certain EPG waveforms are correlated with detectable inoculation of Xf, and thus that specific probing behaviors control, in part, the inoculation process. Immunocytochemistry is the most sensitive method, and can successfully detect the presence of Xf cells near 8 of 10 identified probes’ salivary sheaths, only 5 days after the probe. ELISA is the next most sensitive technique, followed by PCR. Culturing is highly insensitive; bacteria are recoverable from only 1 out of 10 identified probes.
Expected Benefits: Definitive knowledge of the mechanisms of transmission (i.e., acquisition and inoculation) may lead to clues to interrupt transmission. Use of EPG to produce standardized, reproducible, identified probes will greatly aid research on transmission and Xf-plant interactions. Also, identifying the EPG waveforms representing inoculation will allow future development of a Stylet Penetration Index, for screening germplasm for resistance to inoculation behavior. Such an index can be used to greatly further research on artificial diets, feeding preferences, and the development of resistant varieties that will reduce crop production and management costs.
Objective 2: a) Determine whether GWSS watery saliva plays a role in the mechanism of Xf inoculation or in subsequent movement of the bacteria in grape. If so, then b), characterize the chemical composition of this saliva to determine which compounds exert the effect(s).
Approach: a) Immunocytochemistry/histology will be used to visualize and localize antibody-labeled Xf cells, saliva, salivary sheaths, and possible cellular abnormalities in the vicinity of EPG-monitored, identified, inoculating GWSS probes. A needle-inoculation bioassay developed by J. Labavitch, a cooperator, will be used to establish an infection /disease time-course. A histological time-course study will determine whether the presence and distribution of saliva is associated with lateral movement of Xf cells from the site of inoculation, and/or possible cellular abnormalities or other signs of cell wall loosening that could result from actions of salivary enzymes and other solubilizing substances. If so, then b) biologically active salivary gland substances will be identified using analytical methodologies, and individual substances and combinations of substances will be bioassayed via the injection device.
Cooperators: Elaine Backus (ARS-Parlier) is collaborating with Thomas Coudron (ARS-Columbia), Wayne Hunter (ARS-Ft. Pierce), and John Labavitch (UC-Davis).
Milestones:
2004: Initiate immunocytochemical/histological time course of saliva.
2005: Analyze data from time course study and begin to characterize saliva composition.
2005: Correlate specific saliva component(s) with increased pathogenic characteristics.
Accomplishments:
Preliminary immunocytochemical/histological results (Objective 1) suggest that Xf cells move more rapidly, laterally, away from the site of insect-inoculation (near salivary sheaths from EPG-identified probes), when compared to movement of the bacteria after needle-inoculation (E. Civerolo, pers. comm.). Studies with other insects have repeatedly shown that oral secretions such as saliva account for the major differences between insect- and needle-inoculation of pathogens. Also, preliminary results from the same experiments suggest that cellular abnormalities occur near GWSS salivary sheaths. Such abnormalities are often initiated by saliva-mediated cell wall loosening. We hypothesize that lateral movement of Xf cells through pit cell membranes in adjoining xylem cells could be facilitated by the interaction of vector saliva with Xf chemistry.
Expected Benefits: Understanding the role of saliva in acquisition and inoculation will better delineate the total role of the insect in Xf etiology. For example, saliva could play an important role in differences in transmission efficiency among different vector species. This knowledge would allow development of a method for rapid, biochemical screening of xylem-feeding insects for vector potential, that could lead to more rapid response to introduction of new, invasive Xf vectors, or existing native vectors, as Xf spreads.
IV. Xf–Host Plant Interactions
ARS Research Context:
In the large research area of Xf-host plant interactions, ARS is focusing on understanding establishment of infection, and determining pathogenicity and virulence factors, which are crucial to the disease process. This research specifically aims at identifying physiological and biochemical responses of grape and almond to Xf infection, determining the basis for, and regulation of, these responses, and identifying host genes involved in the establishment of infection and disease development.
This work is complementary to that at UC-Davis, where researchers are examining Xf-resistant and susceptible Vitis germplasm to better define the process of pathogen establishment and pathogenicity. Another approach taken at UC-Berkeley and UC-Davis is to interfere with Xf cell-to-cell communication in xylem lumina, as a means of inhibiting establishment of colonies in the plant host. This will be greatly facilitated by a better understanding of the Xf cell surface, as being researched at UC-Davis and UC-Berkeley. Exopolysaccharide (xantham) gum genes revealed through the collaborative ARS-FAPESP (Brazil) Xf genome sequencing project are being exploited at UC-Riverside with the goal of degrading or inhibiting the production of gum to prevent clogging of xylem vessels, a purported factor in pathogenicity. Complementing this work, ARS-Ft. Pierce and UF-Quincy are collaborating to study gum gene expression in defined media. The gum gene work is part of a greater effort to determine gene function and regulation as it relates to factors associated with pathogenicity, as researched at ARS-Parlier, UC-Berkeley, UC-Davis, and the University of Florida. Differential inducement of plant genes by putative Xf virulence genes is being studied at UC-Berkeley and UC-Davis. Determining the roles of vessel cavitation, cell wall metabolism, and vessel occlusion and Xf movement in the vessels, as studied at UC-Davis, will also yield clues that can be exploited through interruption of the infection/disease process, or through plant breeding. Xf virulence-related proteins, including outer membrane proteins (e.g., mopB), are being identified through a Specific Cooperative Agreement between ARS-Parlier and UC-Davis. Knowledge of the role(s) of these gene products in Xf virulence or pathogenicity could potentially lead to strategies for mitigating disease development. The effect of Xf strain on host plant specificity is being studied using whole or partial Xf genome DNA microarrays at UC-Berkeley and UC-Riverside, and at ARS-Parlier, based, again, on data obtained through the collaborative ARS-FAPESP (Brazil) Xf genome sequencing project.
Goal 1: Determine the nature of, and basis for, establishment of infection by Xf in grape.
Current Situation: Xylella diseases are complex pathosystems. The nature of, and basis for, colonization of xylem tissue and establishment of infection by Xf is not well understood.
Objective 1: Identify and characterize Xf pathogenicity and virulence factors.
Approach: Xf virulence-related proteins, including outer membrane proteins (e.g., mopB), will be identified, isolated, and characterized, and their role in establishment of Xf infection will be determined through a Specific Cooperative Agreement between ARS-Parlier and UC-Davis. Mutants of Xf will be required in order to elucidate the mechanism underlying the ability of the pathogen to systemically colonize the vascular system and cause disease. The ability of Xf strains to colonize and cause disease in alternate hosts will be explored by experimental inoculation. The extent and speed with which Xf can colonize plant tissues will be studied with a combination of confocal microscopy of green fluorescent protein (GFP)-labeled bacteria, and quantitative PCR.
Cooperators: John S. Hartung (ARS-Beltsville) is conducting the ARS research on Xf mutants. Vice-Civerolo will continue to cooperate with George Bruening (UC-Davis) in research on Xf virulence-related proteins.
Milestones:
2000: In collaboration with George Bruening (UC-Davis), ARS-Parlier demonstrated the chlorosis-inducing-activity of intact Xf cells and a heat-stable, enzyme resistant fraction of Xf cells in Chenopodium quinoa (Cq).
2001: ARS-Beltsville demonstrated transformation of Xf with an Xf/E. coli hybrid plasmid.
Demonstrated a greater range of diversity present in citrus strains of Xf in comparison with strains from grapevine and other host plants.
In collaboration with George Bruening (UC-Davis), ARS-Parlier developed a method to purify, isolate, and characterize a Cq chlorosis-inducing factor from intact Xf cells.
2002: ARS-Beltsville created Tn5 insertion mutants in Xf (citrus strain) using a triparental mating approach for the first time in Xf. These mutants are simultaneously labeled with GFP.
In collaboration with George Bruening (UC-Davis), continued characterization of Cq chlorosis-inducing factor and identified it as mopB, an outer membrane protein.
2003: ARS-Beltsville developed method to rapidly locate the Tn5 insertions in the Xf genome, and identified them by comparison to the published Xf genome.
In collaboration with George Bruening (UC-Davis), ARS-Parlier continued characterization of mopB, and establishment of its role as a virulence or pathogenicity factor.
2004: Publish first GFP-labeled Xf paper as well as paper on localization of these mutants.
Publish paper describing the biological activity, purification, isolation, and biochemical characterization of mopB.
Accomplishments:
The ARS-Beltsville laboratory has demonstrated that the ability of Xf strains to infect plants and incite disease is even greater than previously thought. By doing this, the laboratory has also demonstrated that the symptoms induced by Xf in plant hosts are entirely plant responses to infection, since a single strain (sweet orange) can induce the widely divergent symptoms of CVC, PD, coffee leaf scorch and periwinkle wilt in the appropriate host plant. This is useful, because alternate hosts are needed for the efficient study of these diseases. The laboratory has developed an efficient mutagenesis strategy for Xf, as well as methods to localize the mutants so that gene function can be guessed from genomic sequence data. And the laboratory has developed a novel system to follow such mutants in planta, by using the GFP marker and confocal microscopy.
1) Li, W., Zhou, C., Pria, Jr., W.D., Teixera, D.C., Miranda, V.S., Pereira, A.J., He, C.-X., Costa, P.I. and Hartung, J.S. 2002. Citrus and coffee strains of Xylella fastidiosa induce Pierce’s disease of grapevine. Plant Disease 86:1206-1210.
2) Li, W.B., Pria Jr., W.D., Teixera, D.C., Miranda, V.S., Ayres, A.J., Franco, C.F., Costa, M.G., He, C.X., Costa, P.I. and Hartung, J.S. 2001. Coffee Leaf Scorch caused by a strain of Xylella fastidiosa from citrus. Plant Disease 85 (5):501-505.
3) Qin, X. and Hartung, J.S. 2001. Construction of a shuttle vector and transformation of Xylella fastidiosa with plasmid DNA. Current Microbiology 43:158-162
4) Qin, X., Miranda, V.S. , Machado, M., Lemos, E. and Hartung, J.S. 2001. An evaluation of the Genetic Diversity of Xylella fastidiosa isolated from Diseased Citrus and Coffee. Phytopathology 91(6):599-605.
In collaboration with George Bruening, mopB, an outer membrane protein from Xf, has been associated with chlorosis-inducing activity in C. quinoa, and specific binding to cellulose. These results suggest a role for this protein as a virulence or pathogenicity factor in the establishment of Xf infection.
Expected/Realized Benefits: Exploitation of the genomic sequence information already available will be possible only through the use of defined mutants. Such mutants are being used to study Xf/host interactions. The sweet orange strain of Xf has been demonstrated to cause disease in coffee, grapevine, periwinkle, and tobacco. More than 200 GFP-labeled mutant Xf strains are now available. The GFP label is being used to study colonization of grapevine tissues by confocal microscopy. These ARS-developed methods can be generalized, and will be useful to anyone needing labeled, defined mutants of Xf. Knowledge of the role(s) of gene products in Xf virulence or pathogenicity (e.g., mopB) could potentially lead to strategies for mitigating disease development.
Objective 2: Identification of the role of GUM proteins in disease epidemiology for the development of PD resistant plants.
Approach: Gum gene (GUM) expression in response to specific constituents within a defined medium are monitored with real-time PCR. Medium constituents are correlated with plant xylem components. Expression of GUM proteins in susceptible and resistant plants are evaluated on disease symptom severity and rates of transmission efficiency by GWSS.
Cooperators: Wayne Hunter (ARS-Ft. Pierce) is collaborating with Russell Mizell and Pete Andersen (UF-Quincy).
Milestones:
2000-2001: Developed defined media for growth and study of xylella, particularly for screening gum gene expression (UF-Qunicy).
2003: Completed Phase I trial of real-time PCR analysis of GUM gene expression in two defined media (ARS-Ft. Pierce and UF-Quincy).
2004: Continue analysis of GUM gene expression in media and in planta, and determine role in sharpshooter transmission.
Accomplishments:
Developed defined media for differential growth of xylella (ARS-Ft. Pierce).
Identified role of sulfide bonds in binding of xylella (ARS-Ft. Pierce).
Determined GUM gene expression in xylella in vitro, using real-time PCR (ARS-Ft. Pierce and UF-Qunicy).
Expected Benefits: Understanding the factors that trigger the differential production of GUM proteins by xylella, and the presence or absence of these elements in plant xylem will aid efforts to develop PD resistant varieties of grape and other crop plants.
V. Grape Genomics, Genetics, Physiology, and Resistance to Xf in Grapes, Almonds, and Other Commercially Important Species
ARS Research Context:
With APHIS and CDFA, ARS contracted (2001-2003) with Doug Cook (UC-Davis) to produce grape ESTs, a project now completed; future plans, funding permitting, are to hire a scientist at Parlier to develop and test hypotheses based on this genomic data, e.g., by using microarrays and real-time PCR to test the hypothesis that certain plant varieties have systemic resistance reactions to the pathogen that exacerbate the course of Xf-diseases.
Manipulation of the grape genome was facilitated by development at ARS-Kearneysville (R. Scorza), ARS-Parlier (D. Ramming), and UF-Apopka (D. Gray) of a grape transformation system based on Agrobacterium tumafaciens, primarily for the purpose of determining gene function. UC-Davis is developing use of Agrobacterium rhizogenes-mediated transformation, with the goal of high-throughput screening for genetic resistance to PD in grape, while maintaining desirable grapevine characteristics and grape quality. This method can also be used to rapidly screen for virulence mechanisms in Xf strains.
ARS has wine, table, and raisin grape breeding programs at Geneva (New York), focused on rootstocks and V. vinifera x Muscadina crosses, and at Parlier, focused on resistant V. vinifera scion stock of high fruit quality and seedlessness. Promising rootstocks are then used by colleagues at UC-Davis and ARS-Geneva for grafting V. vinifera scions (e.g., Chardonnay), and V. muscadina and V. vinifera stocks are used at UC-Davis and FAMU to identify resistance genes, and to create genetic maps for marker aided selection (MAS) to accelerate breeding. An expanded genetic map of V. vinifera x V. muscadina for fine scale mapping and characterization of PD resistance is being developed at UC-Davis. Molecular markers, in response to grape infection by Xf, are also being investigated, at ARS-Parlier and UC-Davis.
Goal 1: Identify grape rootstock germplasm and/or varieties that reduce or mitigate PD development in susceptible wine grape scions in PD-prone production areas.
Current Situation: The influence of grape rootstocks on development of PD in grapevines in vineyard settings is unknown. Based on previous research, the longevity and productivity of vines of PD susceptible varieties is enhanced in PD zones by the use of particular rootstock varieties. In addition, rootstock variety reduces the development/incidence of Xylella disease in peach in field trials in Florida. Rootstocks might confer protection against PD development in susceptible scions grafted to them.
Objective 1: Identification of grape rootstock varieties that reduce PD symptom expression or disease development in susceptible scions.
Approach: Overall, grafted vines are planted in a PD prone vineyard and symptoms of PD development on scions are recorded. Susceptible V. vinifera wine grape varieties Cabernet Sauvignon and Chardonnay were grafted to a select group of rootstocks and planted in a vineyard in Tallahassee, Florida, in 2001. Own-rooted rootstocks and scions were also planted. Vines are managed to control foliar fungal diseases. PD pressure is very high in the experimental vineyard, which has vegetated row middles and abundant Xylella vectors. PD symptom expression is scored twice per growing season, for at least three growing seasons.
Cooperators: Peter Cousins (ARS-Geneva) is collaborating with Jiang Lu (FAMU).
Milestones:
2001: Established pilot experimental block at Center for Viticulture (FAMU). Conducted initial PD symptom evaluation and scoring on test vines.
2002: Expanded experimental block at FAMU. Continued PD symptom evaluation and scoring on test vines.
2003: Continued PD symptom evaluation and scoring on test vines.
2004: Complete PD symptom evaluation and scoring on test vines. Identify rootstocks that reduce PD development in susceptible V. vinifera wine grape scion varieties under vineyard conditions.
Accomplishments: Long-term research is in progress.
Expected Benefits: Rootstocks that mitigate PD development in susceptible V. vinifera wine grapes under vineyard conditions. Increased survival and productivity of PD-susceptible scion varieties grafted on rootstock(s) in PD-prone areas. Reduced crop production and management costs.
Goal 2: Identify table and raisin grape germplasm and varieties with enhanced resistance or tolerance to PD that have commercial fruit quality.
Current Situation: PD-resistant/tolerant grape germplasm has been identified in: the collection at the National Clonal Germplasm Repository-Tree Fruit and Nut Crops and Grapes, Davis, CA; selections obtained from breeders in the southeastern U.S.; and selections from the UC grape breeding program at Davis. However, none of this germplasm or these selections has commercial table and raisin fruit quality or other commercially-desirable horticultural traits (e.g., seedlessness, large berry size, firm fruit without undesirable flavors).
Objective 1: Develop table and raisin grape germplasm selections with enhanced resistance/tolerance to Xf infection and PD development and that have commercial fruit quality.
Approach: Hybridize high quality seedless table and raisin breeding lines (genotypes) as female parents with previously identified sources having PD resistance/tolerance to combine fruit quality and seedlessness with PD resistance. Use embryo rescue methods to allow seedless genotypes to be used as either male or female parents. Screen each breeding cycle for fruit quality and PD resistance. Susceptibility/resistance to Xf infection under greenhouse conditions following artificial inoculation is evaluated by symptomatology and in planta movement of the pathogen is assessed by ELISA. Back cross PD-resistant selections with good fruit quality to table and raisin grape selections for the continued improvement of fruit quality. Evaluate advanced selections in cultural/field trials. Develop genetic markers for PD-resistance and fruit characteristics.
Cooperators: David Ramming (ARS-Parlier) is collaborating with M. Andrew Walker (Department of Viticulture and Enology, UC-Davis).
Milestones:
2001: Identified PD-resistant seedlings in table grape genotypes. Hybridized seedless table and raisin grape selections with southeastern U.S. (SEUS) PD-resistant selections and cultivars.
2002: Backcrossed PD-resistant selections identified in 2001 with seedless table and raisin grape selections. Hybridized seedless table and raisin grape selections with PD-resistant selections from multiple sources of PD-resistance. Evaluated seedlings for fruit quality in the field and PD resistance in the greenhouse.
2003: Backcrossed PD resistant selections from second family with seedless table and raisin grape selections. Hybridized seedless table and raisin grape selections with SEUS PD-resistant selections. Evaluated seedlings for fruit quality in the field and propagated the best selections for determining PD resistance in the greenhouse.
2004: Continue hybridizing seedless table and raisin grape selections from multiple sources of PD-resistant selections, and back crossing to new advanced PD-resistant selections. Evaluate seedlings for fruit quality in the field and PD resistance in the greenhouse. Use information from greenhouse PD evaluation to choose parents giving the highest proportion of resistant seedlings.
Accomplishments:
2000: Hybridized a PD-resistant selection from the UC-Davis grape breeding program with four seedless table grape selections. Additional families were created and grown at UC-Davis from crosses between UC PD-resistant selections and ARS-Parlier seedless selections.
2001: Identified PD-resistant table grape seedlings in populations derived from hybridization of four seedless table grape seedlings with a PD-resistant selection from the UC-Davis grape breeding program. Hybridized seedless raisin and table grape selections with southeastern U.S. grape selections and varieties, a PD-resistant French hybrid, and a seeded V. vinifera x V. rotundifolia hybrid.
2002: Backcrossed PD-resistant selections from the crosses made in 2000 between seedless table grape selections and the UC-Davis PD-resistant selection back to seedless table and raisin grape selections, and produced plants by embryo rescue. Evaluated fruit quality of the first family of a seeded PD-resistant selection from the UC-Davis grape breeding program hybridized with a large seedless table grape from the ARS Parlier breeding program. Several selections within this first generation family 0023 [8909-15 = (V. rupestris x V. arizonica) x B90-116)] that had low in planta populations of Xf and no cane PD symptoms following artificial inoculation were identified, and have been used as parents.
2003: Identified PD-resistant seedlings in populations derived from hybridization of SEUS selections and cultivars. Selections from family 0023 were backcrossed with seedless table and raisin selections. Additional grape selections from the southeastern U.S. were hybridized with seedless table and raisin selections. Numerous seedlings have fruited and the best selected based on fruit quality and low powdery mildew incidence. Twenty-five wine grape selections were made from families whose parents include SEUS breeders from PD-resistant selections crossed with wine varieties. Cuttings have been taken of 80 advanced selections for greenhouse PD-resistance testing.
Expected Benefits: Availability of PD-resistant scion germplasm and varieties with commercially-desirable fruit quality will allow increased survival and productivity of table and raisin grapes in PD-prone areas. Reduced crop production and management costs.
Goal 3: Identify and characterize physiological PD resistance mechanisms in Vitis species.
Current Situation: Host plant resistance is a critical component of integrated crop management.
Traditional breeding is underway to develop PD resistant plants. Many of the native Vitis
species show good PD resistance, but the mechanisms controlling this resistance have not been
well studied. Given the fact that PD resistance exists in a wide range of genetic backgrounds
with different origins, it is expected that the resistance mechanisms [will] may be different
among Vitis species.
Objective 1: Identify the anatomical and biochemical mechanisms involved in grape plant
resistance to Xf.
Approach: To examine the antimicrobial activity of the xylem sap, the sap is extracted
from PD-resistant and PD-susceptible cultivars and used in a variety of microbial bioassays
designed to test Xf sensitivity. This includes both in vitro and in vivo bioassays. Based on
these antimicrobial bioassays, the cultivar/species exhibiting the most Xf antagonistic activity in
the xylem sap is used in reciprocal and interstock grafting experiments with the very
susceptible wine grape V. vinifera cv. Chardonnay. In these experiments, the transmissibility of antimicrobial compounds from resistant rootstock plants to susceptible scions are examined. Appropriate Xf inoculation procedures are carried out in order to challenge the susceptible
scions. Characterization of the observed phenotype in the scion includes quantitative PCR-
based assays to determine Xf populations and vascular movement. In addition, graft unions are examined using both light and electron microscopy to determine if pit membrane connections
present a physical barrier to Xf movement.
Cooperators: Daniel Kluepfel (ARS-Davis) is collaborating with M. Andrew Walker (UC-Davis) and Hong Lin (ARS-Parlier).
Milestones:
2003: Propagate PD resistant and susceptible Vitis species/cultivars. Confirm that the plant material is Xf free. Express xylem sap from all cultivars. Design and execute antimicrobial bioassays using the collected sap. Statistically analyze the Xf inhibition data to select the most resistant cultivars.
2004: Graft resistant plants identified in 2003 to the susceptible wine grape V. vinifera cv. Chardonnay. Inoculate the susceptible scion. Determine Xf population and movement using PCR based bioassay. Determine vascular movement of antimicrobial components in the xylem sap.
Accomplishments: This work is just being initiated.
Expected Benefits: This project will lead to a clearer understanding of PD resistance mechanisms and the extent to which they vary among grape species. More importantly, if we are able to use these resistant plants as rootstocks and express PD resistance in scion plants, a rapid and very effective means of controlling PD will be available to grape growers.
Goal 4: Identify genes responsible for resistance to Xf and GWSS in grapes, and use these genes in traditional or molecularly-based breeding programs.
Current Situation: Host plant resistance is a critical component of disease and insect pest management to ensure overall plant health. Traditional plant breeding has been the primary strategy for developing PD-resistant table, raisin and wine grapes. However, development of commercially-acceptable, pest (e.g., Xf, GWSS)-resistant varieties by conventional plant breeding procedures is time-consuming. This process can be accelerated by marker-assisted selection (MAS) of pest resistant genotypes. However, availability of known genes linked to resistance to Xf infection and/or GWSS-grape interactions for MAS is limited.
There are no resistance gene markers available that are linked to disease and insect resistance. Grapes native to the southeastern U.S. have resistance to PD and other economic diseases of cultivated grapes. Development of a marker-assisted selection program will accelerate the production of new grape varieties with disease and insect resistance, while reducing grower costs.
Objective 1: Identifiy genetic markers linked to disease and sharpshooter resistance for rapid selection of new varieties with PD resistance.
Approach: Overall, total RNA isolation and large scale EST gene sequencing of grape mRNA are done using disease resistant varieties. Identified genes are compared to known genes from Vitis and putative markers are selected. Progeny from crosses of “susceptible x resistant” grape varieties are evaluated using the selected markers. Genetic markers linked to susceptibility or resistance are catalogued. Further sequencing will identify additional markers. In an experimental vineyard that has abundant xylella vectors and high PD pressure, evaluate subsequent progeny from crosses to develop an adequate marker population linked to disease and sharpshooter resistance. Development of marker-assisted selection program. Focus will be on Florida grape varieties.
Cooperators:
Wayne Hunter (ARS-Ft. Pierce) is collaborating with Jiang Lu (FAMU).
Milestones:
2000-2001: Produced F1 progeny from Vitis susceptible x Vitis resistant grape (representing 20-40 different crosses). This represents independent populations and ~5,000-7,000 seedlings planted to the field, at the Center for Viticulture (FAMU). Conducted initial PD symptom evaluation and scoring on F1 population.
2002: First cDNA library extracted, sequenced 2000 ESTs (ARS-Ft. Pierce). Identified 117 putative markers selected for screening progeny populations (FAMU). Collected and processed grape material for further sequencing. Produced progeny (~5,000-7,000 seedlings) from (49 different cross attempts), to expand experimental block at FAMU. Continued PD symptom evaluation and scoring on primary populations from previous crosses.
2003: Completed 15,000 ESTs from a PD resistant grape V. shuttleworthii (ARS-Ft. Pierce). Identified ~220 putative markers to screen for disease resistance. Genes were made public through publication through NCBI. Continued PD symptom evaluation and scoring on primary populations. Produced another 5,000-7,000 seedlings from new crosses, planted second crossed population seedlings, collected and prepared tissues from each individual vine for marker analysis (FAMU).
2004: Complete another 15,000 ESTs from PD resistant V. shuttleworthii. Validate marker set, and continue to screen new markers identified. Make crosses and produce progeny (5,000-7,000 seedlings) from resistant stock for desired fruit quality traits. Evaluate marker-assisted selection capabilities and determine future industry needs.
Accomplishments:
2002: Completed the first cDNA library, representing 2000 ESTs, from PD resistant grape, V. shuttleworthii (ARS-Ft. Pierce).
Identified 117 potential makers, currently being evaluated (ARS-Ft. Pierce).
2003: Completed 15,000 ESTs from PD resistant grape (ARS-Ft. Pierce, UF-Quincy).
Identified 220 potential markers, published in public database (ARS-Ft. Pierce, UF-Quincy, FAMU).
Training sessions provided by ARS-Ft. Pierce to FAMU scientist and staff, “Bioinformatics-Data processing”.
2004: Continue evaluation of markers.
Expected Benefits: A set of genetic markers linked to disease and insect resistance will accelerate the selection of new, disease-resistant grape varieties. A marker-assisted selection program will also save time and money, while providing a valuable new tool to viticulture industries.
Objective 2: Develop DNA molecular markers for grape genes conferring resistance to Xf infection and PD development, and transform grape rootstock with resistance genes.
Approach: Propagate, and establish groups of, grape genotype selections from siblings derived from segregated crosses (e.g., V. vinifera x V. arizonica ) that are highly resistant and highly susceptible to Xf infection and PD symptom development. Plants are inoculated with pathogenic Xf-PD strains. Total RNA is extracted from pooled samples of stems, leaves, and petioles which reflect different tissues at various stages of disease development. The purified mRNA from different genotypes (including uninfected and infected, as appropriate) is used for cDNA library construction by standard protocols (including suppression subtractive hybridization to remove common housekeeping genes, which will enrich differentially expressed genes of interest). Expressed genes of interest are cloned, sequenced, and annotated, and PD-specific transcriptional profiles are developed. cDNA microarrays are used to identify genes linked to possible metabolic pathways and to elucidate the mechanism(s) of PD resistance and pathogenicity.
Cooperators: H. Lin (ARS-Parlier) is collaborating with Jiang Lu (FAMU) and M. Andrew Walker (UC-Davis).
Milestones:
2002-2003: Selected, propagated, and maintained appropriate resistant and susceptible grape genotypes. Inoculated plants with Xf, as needed. Extracted total grape RNA and purified mRNA. Constructed cDNA libraries; prepare and sequence cloned cDNA fragments.
2003-2004: Analyze sequence data, construct expression profile databases, and identify potential markers for resistance to Xf infection, PD development, and GWSS-grape interactions.
2004-2005: Clone and sequence full-length resistance genes. Construct vectors for transforming plants with resistance genes, conduct in vitro gene expression analyses, transform resistance genes into somatic grape tissue embryos, isolate and bioassay transgenic plants, and initiate plant performance evaluation.
Accomplishments: Long-term research is in progress.
Expected Benefits: Identification of DNA markers linked to resistance genes in grapes infected with Xf, PD development, and GWSS-feeding. This will yield molecular markers of resistance genes for marker-assisted selection programs to facilitate breeding for, and rapid selection of, PD- and GWSS-resistant genotypes. PD- and GWSS-resistant grape scions [or transgenic PD- resistant rootstocks (Note, however, that genetically modifying the scion would not currently be accepted by the wine industry)] will enable breeders to develop PD-resistant grapes while maintaining the integrity of wine varieties (e.g., high grape quality) through classical breeding approaches.
Goal 5. Determine level of resistance to xylella diseases in commercial almond varieties, and in selected Prunus species.
Current Situation: Almonds and other Prunus species of commercial importance are high value crops susceptible to Xf-caused leaf scorch diseases. The potential impact of Xf-GWSS on these crops is not known but could be high. Little is known of the relative susceptibility of different commercial varieties to these diseases, nor is it known if there is a source of resistance that could be introduced to mitigate the effects of more widespread disease. The interaction between scion and rootstock is also of interest in influencing the course of infection and disease. For instance, almond trees on peach rootstock infected with Xf decline rapidly. However, a 12-year infected almond/Marianna 2624 tree that annually develops severe scorch symptoms shows an absence of limb dieback and produces edible almonds. Based on PCR assays, the Peerless scion was positive, and the plum rootstock sucker negative, for Xf. Also, Xf was cultured from the scion tissues. It appears that Marianna 2624 is immune to Xf and the tree does not decline because photosynthates flow unimpeded down the phloem into the rootstock, which in turn, develops structural roots and absorptive root hairs to supply water and minerals to the scion.
Objective 1: Determine resistance to Xf infection among inoculated almond varieties and other Prunus species.
Approach: Small almond trees (commercially available bare root) are inoculated with known strains of Xf (some associated with PD and some with ALSD). The ability of the bacteria to infect these plants is followed by real-time PCR and by immunoassays at several times after inoculation. Bacterial populations are localized from several trees of susceptible varieties. A similar approach is used to monitor susceptibility of selected material from the Prunus collection at the USDA ARS Germplasm Repository at Davis using rooted cuttings when feasible. The susceptibility of the California native wild species P. andersonii and P. fremontii to infection by Xf strains will be examined. Native Prunus species from the southern U.S. (e.g. P. mexicana, P. munsonia, P. serotina, and P. umbellata) may be tested for resistance to Xylella as well.
Cooperators: Fred Ryan (ARS-Parlier) will be collaborating with Craig Ledbetter, Hong Lin, and vice-Civerolo (ARS-Parlier), and Bruce Kirkpatrick (UC-Davis).[FR1]
Milestones:
2004: Methods to inoculate trees and assay the presence of Xf will be standardized, and the time period for bacterial growth determined. Six commercial varieties of almonds and six other Prunus species from the USDA Germplasm Repository (Davis) will be tested for their ability to support growth of Xf.
2005: Six more almond accessions, including P. webbii, and six other Prunus species will be analyzed. The relative susceptibility of the plants to Xf will be determined.
Accomplishments: Work was initiated in October 2003.
Expected benefits: Knowledge of varietal differences in susceptibility to ALSD among commercial varieties will allow growers to reduce the impact of this disease if it becomes established and widespread. If resistance to ALSD exists in Prunus species, plant breeders can test and devise methods to incorporate it into developing lines.
Objective 2: Confirm almond varietal susceptibility to Xf caused disease through field observations.
Approach: In collaboration with the work outlined in Research Area I, Goal 2, Objectives 1&2, information will be collected on the variety of almonds affected by xylella diseases. Incidence (number of trees infected and intensity of symptoms) of ALSD will be determined by variety in the growing areas with the highest incidence of ALSD. Leaf samples from selected affected orchards will be collected and dried to corroborate varietal assignments using microsatellite markers or other DNA-based techniques.
Cooperators: Fred Ryan (ARS-Parlier) will be collaborating with Russell Groves and Jianchi Chen (ARS-Parlier), Farm Advisors, individual growers, and the California Almond Board.
Milestones:
2004: Establish working relationships with cooperators and set up databas[FR2] e for reporting.
2005: Complete analysis of reported data on varietal susceptibility to ALSD. Conduct field verification of disease symptoms; verify almond varieties with microsatellite or by other analyses if data is anomalous.
Accomplishments: Work was initiated in October, 2003.
Expected benefits: These results will verify greenhouse studies on resistance to ALSD, providing reliable information to growers on desirable varieties.
Objective 3: Determine mechanism(s) of resistance to Xf among almond varieties and Prunus species.
Approach: If resistance to Xf is found to exist in almonds or Prunus species, it will be studied to determine the mechanism of resistance and its transferability to commercially important Prunus. Assays of xylem fluid will be conducted to determine the presence of a substance inhibitory to the growth of Xf. Physiological responses will be compared in resistant and susceptible varieties and physiological responses will be correlated with bacterial load. Gene expression in resistant Prunus after Xf inoculation will be determined by analysis of mRNA and these results will be compared to those in grape.
Milestones: (This work will not be initiated until resistance to Xf is shown to exist.)
Year 1: The inhibitory effect of xylem fluid is tested on Xf growing on plates. Concentrations of abscisic acid and selected antioxidants is determined in leaves of almond trees after inoculation with Xf.[FR3]
Year 2: mRNA will be isolated from tissue of susceptible and resistant varieties at several times (to be determined) during infection. mRNA will be sequenced for the most abundant messages and compared to sequences from grape tissue.
Accomplishments: This work has not yet been initiated.
Expected Benefits: Identified resistance genes from almond and other Prunus species that can facilitate choice of breeding stock.
Objective 4: Evaluate almond leaf scorch disease (ALSD) on almond trees growing on the plum rootstock Marianna 2624.
Approach: Establish an orchard trial comprised of two almond cultivars (Butte and Peerless) on two rootstocks (peach and Marianna 2624 plum) with two treatments (Xf inoculated and healthy) and 5 replications. Transplant trees in spring 2004 and needle inoculate with an Xf culture (109 CFUs/ml) in mid-summer. Observe for symptoms prior to dormancy and following spring; reinoculate as necessary.
Cooperators: Jerry K. Uyemoto (UC-Davis) is collaborating with Douglas Gubler and Bruce Kirkpatrick, Department of Plant Pathology, and Tom Gradziel, Department of Pomology (UC-Davis). Future collaborations with Fred Ryan (ARS-Parlier) will be developed.
Milestones:
2004: Transplant almond trees in random block design with 5 replications. Needle inoculate trees with Xf or water (as a control). Observe for symptoms 90 days later.
2005: Evaluate trees for leaf symptoms in late July. Reinoculate trees as necessary.
2006: Continue ALSD symptom evaluation, bag nutlets against crow damage, and evaluate nut quality.
2007: Continue evaluation of ALSD effect and nut quality.
Accomplishments: This project has just been initiated.
Expected Benefits: Use of Marianna 2624 rootstock to mitigate effects of ALSD on almond nut quality and as a control strategy in endemic ALS production areas.
VI. Disease and Vector Management
ARS Research Context:
A large number of approaches will be needed to provide long-term, cost-effective management of PD. In the short-term, ARS has focused on GWSS suppression through use of repellents at Kearneysville [this method has proven successful and is mostly complete] and application of foliar and systemic chemicals to grape and citrus (ARS-Phoenix). On citrus and grape, UC-Riverside is also testing neonicotinoids, carbaryl, and other insecticides and insect growth regulators for control of GWSS and other pests. Research at UC-Riverside is focused on determining baseline toxicity and developing monitoring techniques to detect early resistance to insecticides being employed. Attempts are also being made at UC-Riverside (including the UC-Kearney Agricultural Center in Parlier) to determine the effects of these insecticides on natural biological control agents. In addition, scientists at ARS-Phoenix and UC-Riverside are testing sub-lethal doses of neonicotinoids on GWSS feeding and transmission of PD.
A second approach, at ARS-Parlier, is to prevent Xf infection through use of chemicals that induce heightened plant resistance. And UC-Davis is testing a wide variety of prophylactic and therapeutic chemicals for use against Xf in grape.
ARS is also developing bio-based approaches for GWSS management, including, at Weslaco, classical biological control through importation of natural enemies from southern U.S. and southern South America; while UC-Riverside is focusing on agents from the central and eastern U.S. Egg parasitoids of GWSS released in southern California are being surveyed. The biological control effort includes several studies at UC-Riverside on the biology and application of agents (e.g., exotic mymarid parasitoids), and their identification (e.g., of the Gonatocerus ashmeadi complex). ARS-Weslaco is developing molecular markers that can be used to distinguish between the various geographic sources of released native parasitoids, and also methods to distinguish between native and exotic parasitoid species. Cold storage methods for parasitized (and non-parasitized) GWSS eggs are being developed at ARS-Fargo to facilitate mass production of parasitoids for biological control. At ARS-Phoenix and UC-Riverside, ELISA methods are being developed to detect GWSS eggs in the guts of predators.
ARS-Shafter, following earlier work at ARS-Weslaco, is also developing fungi for control of GWSS, and the University of Florida is studying mycopathogens and the effect of their exotoxins on GWSS.
A large pilot demonstration project in Kern Co. is being led by APHIS and UC-Cooperative Extension, with participation by CDFA and Agricultural Commissioners in Kern and Tulare Counties, using techniques developed, in part (e.g., repellents, insecticides, contemporaneous with classically released parasitoids), by ARS, and monitoring schemes by UC-Cooperative Extension. This project has resulted in significant suppression of the GWSS. This areawide GWSS management program is being implemented in Tulare and Ventura Counties. An IPM program in the Temecula Viticulture Area was also developed and implemented by UC-Riverside scientists for areawide implementation. This has been funded, in part, by APHIS (operations) and ARS (monitoring). And ARS-Davis is collaborating with UC scientists at Davis and Berkeley to develop an IPM program for PD in California north coast winegrape-growing areas using biological, chemical, and cultural approaches.
A unique approach to controlling Xf transmission is to find endosymbionts that might be used as competitors or antagonists of the pathogen, e.g., the use of non-pathogenic strains of Xf, as is being investigated at UC-Riverside. Efforts are also underway at UC-Riverside, Yale University, UC-Berkeley, and California State University (CSU)-Hayward to isolate other symbiotic bacteria from sharpshooters. To prepare for eventual use of this strategy, the environmental fate of a genetically-marked endophyte is being studied at UC-Riverside and CSU-Hayward. At UC-Davis, scientists are also looking for grapevine endophytes and evaluating these for potential biological control of Xf. And chimeric antimicrobial proteins are being investigated at UC-Davis, the Los Alamos National Laboratories, and at ARS-Parlier for possible use against Xf via transformed grapevines.
An alternative to the use of endophytic bacteria is to use viruses that attack the vector or the pathogen. GWSS pathogenic viruses are being isolated and characterized at UC-Davis, and additional searches are underway at CSU-Hayward for GWSS viruses and Xf bacteriophages.
Goal 1: Prevent Xf infection.
Current Situation: Xf strains infect a number of cultivated crops, as well as uncultivated plant hosts. Xf-diseases have been present in California since the mid-1880’s. Despite occasional costly local epidemics, however, growers generally learned to cope with these diseases until the introduction, establishment, and spread of the invasive GWSS. The GWSS is a more aggressive vector of Xf than other sharpshooter insect vectors of Xf that are indigenous to California. Unlike indigenous sharpshooter insect vectors of Xf, the GWSS can build up to very high population levels; has a very wide range of hosts on which it feeds, reproduces and oviposits; can feed on woody tissues (and potentially transmit Xf) throughout the year; and, is a strong aggressive flier. Once the GWSS acquires Xf, it can transmit the bacterium for life, unless or until it molts. Other diseases of important cultivated horticultural crops caused by Xf pose a threat to crop production in California. These include CVC, PPD, and PLSD. In 2002-2003, scorch disease-like symptoms on olive trees in urban landscape settings were associated with Xf infections. Economic, sustainable, and effective strategies to manage Xf diseases (e,g., PD, ALSD) are currently not available, especially in areas where the GWSS occurs.
Objective 1: Develop effective management of Xf diseases based on induced host resistance.
Approach: Establish greenhouse and field trials in commercial vineyards to evaluate the effects of available chemical and biologically-based systemic resistance inducers on infection and disease development. Materials (e.g., Messenger, Actigard) are applied to potted plants (for greenhouse tests) or in-ground plants (e.g., in vineyards, orchards) following manufacturers’ guidelines. Plants are generally inoculated via Xf-inoculative GWSS (or other insect vectors) for 24- and/or 48-hours, after which time the insects are killed by spraying with an insecticide. As necessary or appropriate, plants are inoculated with Xf following a standard pin-pricking method. Plants are monitored and rated or scored for disease symptoms. Visual observations are confirmed by pathogen re-isolation, ELISA, and/or PCR.
Cooperators: vice-Civerolo and Kayimbi Tubajika (ARS-Parlier) are cooperating with Bruce Kirkpatrick (UC-Davis), grape and almond growers, and industry (e.g., Eden Bioscience) representatives.
Milestones:
2000: Established field trial to evaluate the effects of Messenger and Actigard in commercial vineyards in the Temecula Viticulture Area in Southern California.
2001: Established field trials to evaluate the effect of Messenger on PD development in a commercial vineyard in the San Joaquin Valley (Tulare County).
2002: Continued evaluation of Messenger in a commercial vineyard in Tulare County in California. Established field trial to evaluate effect of Messenger on PD development in Santa Maria in the Central Coast of California winegrape-growing region. Established greenhouse test to a evaluate effect of Messenger on Xf infection of grape plants and PD development.
2003: Completed evaluation of Messenger on Xf infection and PD development in commercial vineyards in Temecula, Tulare County, and Santa Maria. Completed first greenhouse experiment to evaluate effect of Messenger on Xf infections and PD development.
2004: Expand greenhouse experiments to evaluate effect of Messenger on Xf infections and disease development in grape and almond plants. Expand field evaluation of Messenger effect on PD development in commercial vineyard setting in an area in which the GWSS is not present (e.g., in a Napa Valley, CA, north coastal winegrape-growing region).
2005: Continue greenhouse experiments to evaluate effect of Messenger and other potential systemic resistance inducers (chemical and biologically-based) on Xf infections and disease development in grape and almond plants.
Accomplishments:
Two seasons after treatment, the mean incidence of PD in grapevines treated with the harpin-containing Messenger (2.2.5-6.5 oz/acre) was significantly lower in the first small field trial than in untreated grapevines.
In the first greenhouse experiment, fewer plants treated with Messenger were infected with Xf and had reduced PD symptoms than untreated control plants following exposure to Xf-inoculative GWSS.
Expected Benefits: An effective treatment for PD based on increased or induced resistance to Xf infection that is safe and environmentally-sound will reduce losses due to the diseases. Potential broad application of induced resistance to other Xf-caused diseases (e.g., ALSD).
Objective 2: Develop increased host resistance to Xf infection based on antimicrobial proteins that target moieties on the Xf cell surface.
Approach: Identify protein(s), carbohydrates, and/or lipids that function in the establishment, growth, colonization, and/or movement of Xf in planta. Construct novel antimicrobial chimeric proteins composed of two moieties, an initial Xf-specific cell recognition domain (e.g., outer membrane protein-recognizing) and a defensin Xf-specific carbohydrate killer domain (e.g., lipid-recognizing). Express chimeric proteins in plant cells and test their efficacy in lysing Xf in vitro. Deliver anti-Xf chimeric protein(s) to grape plants by injection of purified protein(s) or by engineering plants to deliver the protein(s) into the xylem. Test the susceptibility of anti-Xf chimeric treated plants to Xf infection and PD development by standard methods.
Cooperators: vice-Civerolo will cooperate with George Bruening and Abhaya Dandekar (UC-Davis) and Goutam Gupta (Los Alamos National Laboratories, Los Alamos, NM)
Milestones:
2003-2004: Select appropriate Xf-specific domains for chimeric protein construction. Express most promising domains in plants cells and assess the activity of the individual domains.
2004-2005: Construct appropriate chimeric proteins and assess anti-Xf effects in vitro.
2005-2006: Deliver chimeric proteins to Xf-susceptible grape plants. Evaluate susceptibility of chimeric-treated plants to Xf infection and PD development.
Accomplishments:
An Xf outer membrane protein, mopB, has been isolated, purified and characterized. mopB is a candidate for the Xf-recognition domain of an anti-Xf chimeric protein.
Expected Benefits: A protein inhibitor that interrupts Xf-host interactions (or otherwise inactivated the pathogen) is a novel alternative approach to the use of chemical therapeutics (e.g., antibiotics, metal-containing sprays, insecticides) to manage PD and, possibly, other Xf-caused diseases.
Goal 2: Determine GWSS suppression factors, particularly natural enemies.
Objective 1: Determine what parasitoids maintain GWSS at low numbers in their original geographic range.
Approach: Survey egg masses and nymphs on the most important host plants and determine parasitism and predation rates. Colonize the most important natural enemy species identified and determine key biological characteristics, e.g., development, longevity, fecundity and searching efficiency, at different temperatures and habitats (host plants) throughout the native GWSS geographic range.
Cooperators: Walker Jones (ARS-Weslaco) is collaborating with David Morgan (CDFA), who is making releases of Texas parasitoids and will lead post-release evaluations. Lloyd Wendel’s (APHIS) group is rearing Gonatocerus triguttatus for mass release (augmentation trials).
Milestones:
2001: Surveyed egg parasitism and predation throughout the reproductive season on two primary host plants. Released Texas parasitoids in California.
2002: Continued second year of sampling. Determined basic biological data for native egg parasitoids. Released Texas egg parasitoids in California.
2003: Completed third year of sampling. Began recording behavioral traits of egg parasitoids. Released Texas parasitoids in California.
2004: Complete all biological and behavioral studies, identifying the most effective parasitoid under California conditions. Determine if the Texas egg parasitoid, G. triguttatus, has been successfully established in California on GWSS eggs.
2005-2007: Evaluate impact of released egg parasitoids in California.
Accomplishments:
Although nymphs were collected from a large number of host plants, they were seldom abundant. Spiders were the most often observed predator of both nymphs and adults. The preferred oviposition hosts were Texas mountain laurel and crape myrtle. GWSS were rarely seen in citrus orchards. Sampling revealed that nearly 90% of GWSS egg masses were parasitized throughout each season, with most of the eggs in each mass parasitized. The parasitic wasp, G. triguttatus, was the dominant species, but G. morrilli and G. ashmeadi were occasionally recovered. G. triguttatus were provided to CDFA and APHIS for release in California, where it did not previously occur.
Demonstrated that a parasitic wasp attacking the egg stage contributes significantly in keeping GWSS populations under control in the sharpshooter’s native range.
Expected benefits: If G. trigutattus can become well established and as effective in California as it is in Texas, GWSS populations should be significantly and permanently reduced.
Objective 2: Develop molecular genetic markers that can distinguish between geographic populations of native parasitoids.
Approach: Collect native egg parasitoids from across the geographic range of GWSS and screen various PCR-based molecular marking techniques to determine if the parasitoids can be reliably identified using these methods.
Cooperators: Jesús de León and Walker Jones (ARS-Weslaco) are collaborating with Isabelle Lauziere (APHIS-Mission), Russ Mizell (UF-Quincy), and David Morgan (CDFA-Riverside).
Milestones:
2002-2003: Obtained different species of parasitoid specimens preserved in alcohol. Tested different molecular methods (ISSR-PCR, RAPD-PCR, ITS amplification and sequencing) to compare sensitivity.
2004: Determine the genetic structure and identify population/species-specific markers of pre-released native and south American Gonatocerus parasitoids.
2004-2007: Use markers to determine the genetic structure and geographic origin of parasitoids recovered in California following several years of releases.
Accomplishments:
2003: By ISSR-PCR DNA fingerprinting we determined, for the first time, that there is geographic variation within G. ashmeadi (the most widespread egg parasitoid of GWSS). Results indicated that geographic populations were highly differentiated (GST) and, importantly, population-specific markers were identified. It is interesting to note that populations with some degree of genetic differentiation (differences in marker/allele frequencies) may be considered as races. More work is needed to confirm this possibility.
2003: Molecular methods (ISSR-PCR and ITS region amplification) were able to distinguish eight Gonatocerus species.
2003: Molecular methods suggested that Texas and California G. morrilli may be separate species rather than geographic strains.
de León, J. H., and W. A. Jones. The utility of ISSR-PCR in distinguishing and estimating genetic structure in Gonatocerus species (Hymenoptera: Mymaridae): Egg parasitoids of Homalodisca coagulata (Homoptera: Cicadellidae). Biol. Control. (In preparation 2003).
Expected Benefits: Two egg parasitoids (G. ashmeadi and G. morrilli) already occurred in California when GWSS was discovered there. Large numbers of these species from other states were cultured and released. The ISSR-PCR marker technique may allow workers to be able to distinguish released stock from the native population, providing a way to determine the success of the release program. It can also detect whether outcrossing is occurring among native and released parasitoids of the same species. The genetic population of these species can be followed over time to record changes in the genetic structure of these parasitoid species. The technique also makes it possible to identify not only species, but also the geographic origin of species within GWSS eggs prior to emergence.
Objective 3: Explore for exotic egg parasitoids from related sharpshooters that are pre-adapted to California sub-climate and habitat types.
Approach: Using climate-matching resources, an array of sites throughout the grape-growing regions of California are matched to identical sub-climate types in South America, where other species of related sharpshooters are known to occur. Sharpshooters are collected and caged on potted citrus trees for egg deposition. Egg-infested trees are then placed at pre-planned sites. Citrus orchards are also searched for naturally-occurring eggs. Egg masses are shipped to U.S. quarantine for evaluation on GWSS as well as on non-target leafhoppers prior to petition for possible release for establishment in California.
Cooperators: Foreign explorations are being conducted primarily by Guillermo Logarzo (ARS-Buenos Aires) and Eduardo Virla (PROIMI, Tucuman, Argentina), and taxonomic identifications are provided by Serguei Triapitsyn (UC-Riverside), all in collaboration with Walker Jones (ARS-Weslaco). Quarantine handling in Texas was initially done by Isabelle Lausière (APHIS- Mission) and presently by Walker Jones, and in California by S. Triapitsyn and Charlie Pickett (CDFA-Bakersfield). Any release and evaluation will likely be conducted by David Morgan (CDFA-Riverside) and APHIS personnel.
Milestones:
2000-2001: Using CLIMEX software and Klammdiagrams, identified sites in South America that closely match appropriate sub-climate types in California. Surveyed for egg parasitoids from related sharpshooters and sent for identification.
2002-2003: Conducted extensive collecting in target areas in South America to ship live parasitoids to quarantine facilities in Mission, TX and Riverside, CA. Conducted biological studies on GWSS eggs, and host range studies on non-target leafhoppers.
2003-2004: Import parasitoids and evaluate host suitability of several non-target leafhoppers native to California. Provide data to action agencies to determine if release is desirable. Continue explorations in South America for additional parasitoid species.
2004-2007: Release and evaluate effectiveness of selected imported parasitoids. Explore for more germplasm of parasitoid species approved for release.
Accomplishments:
Using CLIMEX software and Klammdiagrams, identified sites in Peru, Chile, and Argentina as closely matching appropriate sub-climate types in California. ARS researchers at the South American Biological Control Lab, Buenos Aires, Argentina, initially found and preserved 9 species of Gonatocerus and several trichogrammatoid species from sharpshooter eggs, most new to science (identified by S. Triapitzyn). Subsequent collecting in Argentina, Peru and Chile (by G. Logarzo and E. Virla) resulted in successful colonization of 7 Gonatocerus spp. in culture on GWSS eggs in quarantine in Mission, TX and Riverside, CA. Biological studies on GWSS eggs were completed and host range studies were initiated. Studies in TX quarantine showed that 2 species of imported parasitoids did not attack 2 species of non-target California leafhoppers (conducted by W. Jones).
Expected Benefits: Although certain native egg parasitoids are highly efficient in finding GWSS eggs in their native locations, they may not be as efficient when released in California; e.g., G. triguttatus occurs only in south Texas and south Florida, both humid, subtropical areas. The imported parasitoids, by contrast, are already pre-adapted to both the climate and habitat (citrus), so the potential for significantly enhanced suppression of GWSS is high. Such natural control would be self-sustaining and benefits would accrue annually, with no additional input.
Objective 4: Identify possible bioactive compounds (infochemicals) from GWSS, their natural enemies, and plants that could be used to enhance biological control and monitoring of GWSS.
Approach: Using wind tunnels and olfactometers, determine if parasitoids are attracted to i) odors from egg masses (kairomones), or ii) plants being fed upon by GWSS (synomones), and if iii) GWSS are attracted to GWSS (pheromones). Identify and field test bioactive compounds.
Cooperators: This research is being conducted by Gary Elzen, Walker Jones and vice-He (ARS-Weslaco). Future collaboration for trap testing is expected from Raymond Hix (UC-Riverside) and Russ Mizell (UF-Quincy).
Milestones:
2001-2004: Acquire suitable equipment for collecting, bioassaying, and identifying bioactive compounds. Develop bioassays for testing orientation behavior of i) parasitoids to GWSS eggs or other life stages, ii) GWSS to GWSS, and iii) parasitoids and predators to plants fed upon by GWSS. Identify bioactive compounds.
2005-2007: Apply egg kairomones to attract parasitoids to targeted habitats being colonized by GWSS. Identify GWSS sex or aggregation pheromones, and suspected plant synomones. Develop and apply an attractant-based trapping system.
Accomplishments:
Acquired equipment, including wind tunnel, olfactometers, GC-MS, HPLC, odor collection systems, electro-antennagraph/GC system. Developed a bioassay in which parasitoids were recorded as being attracted to odors from GWSS eggs. Found 5 compounds from GWSS-fed plants that are not emitted in the absence of GWSS.
Expected Benefits: A volatile egg kairomone could be applied to crops or other host plants just as GWSS begins to colonize an area, attracting parasitoids before GWSS populations increase. Development of an attractant-based GWSS trap has the potential of being used for monitoring when sharpshooters begin moving into a crop or non-crop, and would likely be used as a decision tool for triggering control measures.
Goal 3: Develop kaolin-based particle film technology to suppress GWSS vector populations to levels that reduce or minimize Xf transmission.
Current Situation: GWSS has a broad host range yet citrus harbors this pest throughout the year and is its primary reproductive host. Vineyards that border GWSS infested citrus are especially vulnerable to PD when adult sharpshooters that overwinter in citrus begin migrating into vineyards in the spring. Contact and systemic insecticides are currently used to reduce adult GWSS numbers in grape and citrus; however, they have not been effective in preventing the establishment and spread of PD in grape. GWSS re-infest grape soon after contact insecticides are applied and systemic insecticides risk the spread of PD because GWSS must feed on the plant in order to ingest the insecticide. Investigating novel approaches that repel GWSS adults and prevent their feeding is needed to effectively prevent the spread of PD in grape and other crops.
Objective 1: Investigate the use of kaolin-based protective barriers as an alternative to conventional contact insecticides for the management of GWSS in vineyards.
Approach: A kaolin-based particle film crop protectant (Surround™ WP, Engelhard Corp., Iselin, NJ) that has general insect repelling properties was compared to standard insecticide programs for grape in large replicated field trials. Initial studies were conducted to determine the efficacy of Surround WP (Temecula, CA) and those results were used to develop a particle-film based GWSS management strategy for vineyards (Kern Co., CA).
Cooperators: Gary Puterka and Michael Glenn collaborated with Matt Ciomperlik, David Bartels, and Lloyd Wendell (APHIS-Mission), Don Luvisi (UC-Cooperative Extension Service, Bakersfield), Ed Civerolo (ARS-Parlier), and Kayimbi Tubajika (ARS-Davis).
Milestones:
2000: In Temecula, demonstrated that Surround WP was very effective in reducing sharpshooter and leafhopper levels in grape. Transferred this information to makers of Surround WP (Engelhard Corp., Iselin, NJ) to expand the labeled uses of Surround.
2001: In Kern Co., determined the benefits of using particle film, Surround WP, over conventional insecticides in large plot field trials. Transferred this information to APHIS and CDFA, and assisted these agencies in developing an area-wide pilot study that utilized Surround WP.
2002: In Kern Co., documented the effects of the 2001 area-wide pilot study and concluded that this management program reduced GWSS to non-detectable levels in the study area by 2002. This programs success resulted in its adoption and expansion into new GWSS control districts in Kern Co.
Research on this Objective was terminated at the end of 2002.
Accomplishments:
Conducted large-acreage replicated field trials at 3 grower locations near Temecula and determined that particle film, Surround WP, was very affective in controlling leafhoppers and sharpshooters in grape. Also, determined that season-long applications of Surround did not have negative effects on grape quality and yield and that this material was compatible with other pesticides and spray tank additives typically used in grape. This information was transferred to the makers of Surround WP (Engelhard Corp., Iselin, NJ) which resulted in the material’s registration for use against GWSS in grape and citrus.
Conducted a large-acreage field trial in Kern Co. and determined that three biweekly applications of Surround WP were more effective in controlling GWSS than six weekly applications of conventional insecticides. An 800 ft. barrier treatment of Surround WP in grape bordering citrus prevented GWSS migration from citrus into grape and prevented GWSS movement beyond the barrier. Furthermore, Surround WP prevented GWSS egg deposition in grape so that GWSS cannot establish itself in grape (weekly use of insecticides, by contrast, did not prevent egg deposition). This research established that Surround was effective and that only barrier treatments are necessary to prevent GWSS migration into grape. This information was transferred to CDFA and APHIS where 800-ft. barrier treatments of Surround WP became part of an area-wide GWSS management program.
Continued monitoring of GWSS populations in the area-wide study determined that this program was highly successful and reduced GWSS numbers to undetectable levels by 2002. This program continues to utilize particle film technology as one of its components, and was expanded into new GWSS control districts in Kern Co.
Puterka, G.J., D.M. Glenn, and D. Luvisi. 2001. Particle Film Technology: A new tool for glassy-winged sharpshooter, sunburn, and other ag problems. California Grower Magazine, p. 8-9. 2001.
Puterka, G. J., Reinke, M., Luvisi, D., Ciomperlik, M. A., Bartels, D., Wendel, L., and Glenn, D. M. 2003. Particle film, Surround WP, effects on glassy-winged sharpshooter behavior and its utility as a barrier to sharpshooter infestations in grape. Online. Plant Health Progress doi:10.1094/PHP-2003-0321-01-RS.
Expected Benefits: This project demonstrated that kaolin-based particle films (Surround WP) are very effective alternatives to conventional chemical insecticides in reducing GWSS infestations in grape. In addition, this research arrived at an economical approach to controlling GWSS in grape by establishing that only an 800-ft. barrier treatment of Surround WP in grape was needed to effectively prevent GWSS migration deep into vineyards, thus resulting in a safe and effective approach to GWSS control in grape.
Goal 4: Suppress GWSS vector populations to levels that reduce or minimize Xf transmission in vineyards/citrus and almond groves, to reduce xylella disease incidence.
Current Situation: Chemical control of GWSS is essential as a short-term solution, and as a component of long-term sound management systems that are economically, ecologically, and socially acceptable. Integrated pest management (IPM) strategies are urgently needed that meld cultural and biological components with efficacious GWSS chemical control, insecticide resistance management (IRM), and integrated crop management (ICM) inputs. Most of the promising agents have been evaluated (as of April 2002, Akey et al., 2002) and are available for use (or to develop data for registration/use) against GWSS. In 2002, one application of the “2nd” generation IGR, Novaluronâ, (benzoylphenylurea group), a chitin blocker, had 86% efficacy against large GWSS nymphs. These preliminary results for Novaluronâ appear promising. ARS-Phoenix is attempting to demonstrate insect vector management of GWSS using repellents, insecticides, biological control, and cultural practices at two sites.
Few pathogens for GWSS have been reported. Fungi and insect viruses can be effective in insect suppression. Sharpshooter viral pathogens are only now being discovered. The role of viruses in the biology and ecology of the GWSS is unknown.
Objective 1: Evaluate agents for GWSS control, generate data suitable for registration support as needed, particularly for biorational insecticides.
Approach: Field trials in small plots are set up to evaluate candidate control agents, using complete block randomized designed field trials in vineyards and citrus. Use conventional, commercial spray equipment at application rates and pressures suitable for production. Use beat and visual count sampling methods. Use proper transformations and statistical analysis to determine efficacy evaluations. Larger blocks (e.g., about 20 & 30 acre total) will be used to demonstrate the use of the IGR, Applaud, in citrus in two locations, to provide data to state workers for use in recommendations that include compounds suitable for IPM programs.
Cooperators: David Akey and Tom Henneberry (ARS-Phoenix) are collaborating with Matthew Blua (UC-Riverside), Russell Groves (ARS-Parlier), and David Morgan (CDFA-Riverside). Past collaborators have included Nick Toscano (UC-Riverside), Gary Puterka (ARS-Kearneysville), Lloyd Wendel (APHIS-Mission), B. Grafton-Cardwell (Parlier), and P. Phillips (UC-Cooperative Extension, Ventura). Cooperators include A. Laird (Deputy Agricultural Commissioner, Ventura County), Ben Drake (Drake Enterprises, Temecula, CA), J. Lee (grower Piru, CA), and producers.
Milestones:
2000: Conducted immediate chemical trials (conventional and biorational insecticides) against GWSS adults in grapes for use in control programs. Foliar tests were conducted by air-blast-sprayer on grape vines with 14 agents (Akey et al., 2001a).
2001-2002: Evaluated insecticides against immatures and adults of GWSS in citrus (this is important because almost all GWSS development occurs on citrus in southern and central California). Systemic tests were conducted (2001 only) with 3 agents at 2 doses by injecting agents in the irrigation system of mini-sprinkler irrigated trees [data unpublished], and foliar tests (2001 and 2002) were conducted by air-blast-sprayer on same-sized orange trees with 16 agents, including 3 rate trials (Akey et al., 2001b, 2002).
2003: Demonstrated insect vector management of GWSS using repellents, insecticides, biological control, and cultural practices in an IPM/ICM approach.
Future: Coordinate this suppression goal with GWSS epidemiology and Xf disease incidence goals to determine what GWSS population level reductions must be achieved to effect PD reduction and what are the defining parameters and circumstances that determine those reductions?
Find biorational insecticides that act on adult GWSS in addition to nymphs and/or eggs. To date, true ovicides have not been discovered or evaluated apart from the IGR, pyriproxyfen, Esteem. Efforts need to be made to1) further evaluate Esteem for efficacy and determine if it can be used without adversely affecting beneficial beetle populations, and 2) discover and evaluate new agents with ovicidal activity.
Confirm positive results for Novaluronâ from 2002 work and determine rate.
Accomplishments:
In four field seasons (2000-2003), more than 25 insecticides were evaluated in 12 different chemical action classes.
Efficacy data on conventional chemistries and on environmentally-friendly biorational insecticides have been obtained.
Eighteen trials have been conducted with 12 insecticides against GWSS in the spring/summer of 2002.
Four agents, the pyrethroid, Baythroidâ, and three biorationals, Applaudâ, Agroneemâ, and Neemixâ, had excellent to good efficacies in trials in 2001 and were found efficacious again in 2002 second evaluations.
Peak titers of imidacloprid were observed in citrus trees 6-8 week post application with near-peak titers sustained for an additional 6-10 weeks. Within-tree distributions of imidacloprid were relatively uniform with no significant differences between the top and bottom canopies of trees or among any of 4 quadrants sampled within the trees. The sustained and relatively uniform distribution of imidacloprid within the citrus trees significantly reduced GWSS nymphs and adults.
Imidacloprid work was done by S. Castle and N. Toscano.
2000 work was done by D. Akey, N. Toscano, and T. Henneberry.
2001-2003 work was done by D. Akey, M. Blua, and T. Henneberry
Expected Benefits: Development of tactics for GWSS control on grapes and citrus as IPM components in grape-citrus-GWSS-PD systems. Provide information needed to develop strategies for insecticide resistance management (IRM), and integrated crop management (ICM) for GWSS.
Objective 2: Determine prevalence of naturally occurring entomopathogens of GWSS and develop microbial control.
Approach 1: Surveys of GWSS populations are made to assess the prevalence of naturally occurring fungal entomopathogens of GWSS in California. These pathogens are cultured, tested for infectivity, host range, and heat tolerance under laboratory conditions and then field tested in citrus plots. Ideally, the fungal entomopathogens would be used to help reduce the overwintering population when populations are most vulnerable to microbial pesticides. In addition, isolates from other areas of the US are tested as available.
Cooperators: Mickey McGuire (ARS-Shafter) will be collaborating with Harry Kaya (UC- Davis), Walker Jones (ARS-Weslaco), and Russell Mizell and Drion Boucias (UF-Gainesville).
Milestones:
2003-2004: Conduct surveys for fungal entomopathogens in areas with high GWSS populations. Isolate and identify the fungi and perform bioassays against adult and nymph GWSS.
2003: Identify commercial entomopathogens that may infect GWSS and conduct bioassays.
2004-2005: Determine host range of new isolates of entomopathogenic fungi and determine optimal production and formulation methods.
2005: Conduct field tests with infectious isolates of fungi on citrus trees.
Accomplishments:
GWSS were collected from Kern and Riverside Counties in 2003. Dead insects were placed on water agar and held for fungal emergence. One individual showed signs of infection by an entomopathogenic fungus but the fungus has not been identified nor tested for efficacy. In Weslaco, the fungus Pseudogibellula formicarum, isolated from GWSS in Mississippi, was shown to infect adults and nymphs under laboratory and greenhouse conditions. This fungus has been deposited in the ARSEF (ARS Entomopathogenic Fungus) collection in Ithaca, NY (information provided by Walker Jones).
Assays were done against GWSS with the Beauveria bassiana product Mycotrol. Infections occurred with high doses (109 spores/ml) in a dip test. However, spray assays with up to 108 spores/ml failed to yield any infections. 107 spores/ml (in an ARS-Shafter test) is a very high dose and probably not economical under field conditions. Paecilomyces fumosoroseus was also tested against GWSS but no mortality or infections occurred.
Expected Benefits: If entomopathogenic fungi can be found and developed as microbial insecticides, another tool in the arsenal for managing GWSS populations in an environmentally sound manner will be available. The fungi would be used in an application to reduce overwintering GWSS populations before their movement into PD sensitive crops. Objective 1: To identify and characterize viral pathogens of the GWSS.
Approach 2: Genetic sequencing is being used to identify viral pathogens in sharpshooters. Molecular primers are designed for further detection and monitoring by PCR. Insect cell cultures are used for mass propagation of virus for further biological evaluations and genetic characterizations.
Cooperators: Wayne Hunter (ARS-Ft. Pierce) is collaborating with Alejandro Chaparro, Nacional Universidad Colombia, South America.
Milestones:
2002: Identified GWSS Iridovirus.
2003: Characterized GWSS Iridovirus.
2004: Evaluate Iridiovirus for GWSS, and search for additional leafhopper viruses.
Accomplishments:
The first GWSS virus has now been characterized, and movement and infection of the virus through GWSS has been studied, with manuscripts in preparation.
Expected Benefits: Viral pathogens of insects have been used in other insect systems for long-term management strategies. Viral pathogens of sharpshooters provide a unique opportunity for the development of a new management methodology for growers to use against sharpshooters to aid in the reduction of PD spread.
ARS PD-GWSS Team Contact Information
Dave Akey, Entomologist
Western Cotton Research Laboratory
4135 E. Broadway Rd.
Phoenix, AZ 85040-8803
Tel: (602) 437-0121 x245
Fax: (602) 437-1274
dakey@wcrl.ars.usda.gov
Elaine Backus, Entomologist
San Joaquin Valley Agricultural Sciences Center
Exotic and Invasive Diseases and Pests Research
9611 So. Riverbend Ave.
Parlier, CA 93648
Tel: (596) 559-2925
Fax: (596) 559-2921
ebackus@fresno.ars.usda.gov
Kendra Baumgartner, Plant Pathologist
Crops Pathology and Genetics Research
Department of Plant Pathology
University of California-Davis
One Shields Ave.
Davis, CA 95616
Tel: (530) 754-7461
Fax: (530) 754-7195
kbaumgartner@ucdavis.edu
Allen R. Bennett, NPL-Plant Pathology
USDA/ARS George Washington Carver Center
5601 Sunnyside Ave.
Bldg. 4, Room 2230
Beltsville, MD
Tel: (301) 504-6915
Fax: (301) 504-6191
arb@ars.usda.gov
Jackie Blackmer, Entomologist
Western Cotton Research Laboratory
4135 E. Broadway Rd.
Phoenix, AZ 85040
Tel: (602) 437-0121 x246
Fax: (602) 437-1274
jblackmer@wcrl.ars.usda.gov
David Boyd
Small Fruits Research Laboratory
306 S. High Street
Poplarville, Mississippi
Tel: (601) 795-8751
Fax: (602) 795-4965
dboyd@ars.usda.gov
James Buckner, Chemist
Insect Genetics and Biochemistry Research
1605 Albrecht Blvd.
Fargo, ND 58105
Tel: (701) 239-1280
Fax: (701) 239-1348
bucknerj@fargo.ars.usda.gov
Steve Castle, Entomologist
Western Cotton Research Laboratory
4135 E. Broadway Rd.
Phoenix, AZ 85040
Tel: (602) 437-0121 x238
Fax: (602) 437-1274
scastle@wcrl.ars.usda.gov
Jianchi (JC) Chen, Molecular Biologist/Geneticist
San Joaquin Valley Agricultural Sciences Center
Exotic and Invasive Diseases and Pests Research
9611 So. Riverbend Ave.
Parlier, CA 93648
Tel: (596) 559-2924
Fax: (596) 559-2921
jichen@fresno.ars.usda.gov
Edwin Civerolo, Plant Pathologist
San Joaquin Valley Agricultural Sciences Center
Exotic and Invasive Diseases and Pests Research
9611 So. Riverbend Ave.
Parlier, CA 93648
Tel: (596) 559-2922
Fax: (596) 559-2921
eciverolo@fresno.ars.usda.gov
Allen C. Cohen (recently retired)
Starkville, MS (previously)
Tom Coudron, Chemist
Biological Control of Insects Research
1503 So. Providence, Research Park
Columbia, MO 65203-3535
Tel: (573) 875-5361
Fax: (573) 875-4261
coudront@missouri.edu
Peter Cousins, Plant Geneticist
Plant Genetic Resources
Cornell University
Collier Dr.
Geneva, NY 14456
Tel: (315) 787-2340
Fax: (315) 787-2339
psc9@cornell.edu
Jesus de Leon, Insect Geneticist
Beneficial Insects Research
2413 E. Highway 83
Weslaco, TX 78596
Tel: (956) 969-4856
Fax: (956) 969-4888
jleon@weslaco.ars.usda.gov
Gary Elzen
Beneficial Insects Research
2413 E. Highway 83
Weslaco, TX 78596
Tel: (956) 969-
Fax: (956) 969-4888
gelzen@weslaco.ars.usda.gov
D. Michael Glenn, Soil Scientist
Applachian Fruit Research Station
Innovative Fruit Production, Improvement and Protection
2217 Wiltshire Road
Kearneysville, WV 25430
Tel: (304) 725-3451 x321
Fax: (304) 728-2340
mglenn@afrs.ars.usda.gov
Russell Groves, Entomologist
San Joaquin Valley Agricultural Sciences Center
Exotic and Invasive Diseases and Pests Research
9611 So. Riverbend Ave.
Parlier, CA 93648
Tel: (596) 559-2923
Fax: (596) 559-2921
rgroves@fresno.ars.usda.gov
Kevin J. Hackett, NPL-Biological Control
USDA/ARS George Washington Carver Center
5601 Sunnyside Ave.
Bldg. 4, Room 2228
Beltsville, MD
Tel: (301) 504-4680
Fax: (301) 504-6191
kjh@ars.usda.gov
Jim Hagler, Entomologist
Western Cotton Research Laboratory
4135 E. Broadway Rd.
Phoenix, AZ 85040
Tel: (602) 437-0121 x243
Fax: (602) 437-1274
jhagler@wcrl.ars.usda.gov
John Hartung, Plant Pathologist
Agricultural Research Center
Fruit Laboratory – Bldg 010A, BARC-West
10300 Baltimore Ave.
Beltsville, MD 20705
Tel: (301) 504-6374 x453
Fax: (301) 504-5062
hartungj@ba.ars.usda.gov
Tom Henneberry, Entomologist; Laboratory Director & Research Leader
Western Cotton Research Laboratory
4135 E. Broadway Rd.
Phoenix, AZ 85040-8803
Tel: (602) 437-0121 x236
Fax: (602) 437-1274
thenneberry@wcrl.ars.usda.gov
Wayne Hunter, Entomologist
US Horticultural Laboratory
Subtropical Insects Research
2001 South Rock Road
Ft. Pierce, FL 34945
Tel: (772) 462-5898
Fax: (772) 462-5986
whunter@ushrl.ars.usda.gov
Walker Jones, Entomologist
Beneficial Insects Research Unit
2413 E. Highway 83
Weslaco, TX 78596
Tel: (956) 969-4851
Fax: (956) 969-4888
wjones@weslaco.ars.usda.gov
Daniel Kluepfel
Crops Pathology and Genetics Research
Department of Plant Pathology
354 Hutchinson Hall
University of California-Davis
Davis, CA 95616
Tel: (530) 752-1137
Fax: (530) 752-7195
dakluepfel@ucdavis.edu
Jesusa Legaspi
Insect Behavrior and Biocontrol Research Laboratory
310 S. Perry Paige
Florida A&M; University
Tallahassee, FL
Tel: (850) 219-5752
Fax: (850) 219-5750
jlegaspi@gainesville.usda.ufl.edu
Craig Ledbetter, Plant Geneticist
San Joaquin Valley Agricultural Sciences Center
Postharvest Quality and Genetics Research
9611 So. Riverbend Ave.
Parlier, CA 93648
Tel: (596) 559-2817
Fax: (596) 559-2921
cledbetter@fresno.ars.usda.gov
Roger Leopold, Entomologist
Insect Genetics and Biochemistry Research
1605 Albrecht Blvd.
Fargo, ND 58105
Tel: (701) 239-1284
Fax: (701) 239-1348
leopoldr@fargo.ars.usda.gov
Hong Lin, Plant Physiologist
San Joaquin Valley Agricultural Sciences Center
Exotic and Invasive Diseases and Pests Research
9611 So. Riverbend Ave.
Parlier, CA 93648
Tel: (596) 559-2933
Fax: (596) 559-2921
hlin@fresno.ars.usda.gov
Guillermo Logarzo
South American Biological Control Laboratory
Buenos Aires, Argentina
Tel: (5411) 4662-0999
glogarzo@mail.retina.ar
Robert Lynch (recently retired)
Tifton, GA (formerly)
Stuart McKamey, Entomologist
Systematic Entomology Laboratory
10th St at Constitution Ave., NW
Washington, D.C. 20560
Tel: (202) 382-1779
Fax: (202) 786-9422
smckamey@sel.barc.usda.gov
Mickey McGuire, Entomologist
Western Integrated Cropping Systems Research
17053 N. Shafter Ave.
Shafter, CA 93263
Tel: (661) 746-8001
Fax: (661) 746-119
mrmcguire@ucdavis.edu
Steve Naranjo, Entomologist
Western Cotton Research Laboratory
4135 E. Broadway Rd.
Phoenix, AZ 85040
Tel: (602) 437-0121 x241
Fax: (602) 437-1274
snaranjo@wcrl.ars.usda.gov
Gary Puterka, Entomologist
Appalachian Fruit Research Station
Innovative Fruit Production, Improvement and Protection
2217 Wiltshire Road
Kearneysville, WV 25430
Tel: (304) 725-3451 x361
Fax: (304) 728-2340
gputerka@afrs.ars.usda.gov
David Ramming, Horticulturist
San Joaquin Valley Agricultural Sciences Center
Postharvest Quality and Genetics Research
9611 So. Riverbend Ave.
Parlier, CA 93648
Tel: (596) 559-2823
Fax: (596) 559-2921
dramming@fresno.ars.usda.gov
Fred Ryan
San Joaquin Valley Agricultural Sciences Center
Exotic and Invasive Diseases and Pests Research
9611 So. Riverbend Ave.
Parlier, CA 93648
Tel: (596) 559-2805
Fax: (596) 559-2921
fryan@fresno.ars.usda.gov
Norm Schaad, Plant Pathologist
Foreign Disease-Weed Science Research
1301 Ditto Ave., Room 215
Fort Detrick, MD 21702
Tel: ((301) 619-2847
Fax: 301) 619-2880
nschaad@fdwsr.ars.usda.gov
Ralph Scorza, Horticulturist
Applachian Fruit Research Station
Innovative Fruit Production, Improvement and Protection
2217 Wiltshire Road
Kearneysville, WV 25430
Tel: (304) 725-3451 x322
Fax: (304) 728-2340
rscorza@afrs.ars.usda.gov
Jerry Uyemoto
Crops Pathology and Genetics Research
Department of Plant Pathology
University of California-Davis
One Shields Ave.
Davis, CA 95616
Tel: (530) 754-7461
Fax: (530) 752-0309
jkuyemota@ucdavis.edu
George Yocum, Insect Physiologist
Insect Genetics and Biochemistry Research
1605 Albrecht Blvd.
Fargo, ND 58105
Tel: (701) 239-1301
Fax: (701) 239-1348
yocumg@fargo.ars.usda.gov
ARS Research Team Biographies
David Akey has 40 years of research experience working with world-recognized authorities on insects of medical and veterinary importance, and agricultural insect pests. In 1974, Dr. Akey joined USDA, ARS, Arthropod-borne Animal Diseases Research Laboratory at Denver, CO. and joined the Western Cotton Research Laboratory, Phoenix, AZ, in 1985. He joined the ARS Emergency Response Team on GWSS in 2000. He is the author or co-author of more than 90 papers, 68 abstracts and a team report, and has given over 90 presentations.
Elaine Backus is a Research Entomologist at the San Joaquin Valley Agricultural Sciences Center, Parlier, CA, having recently joined the new Exotic and Invasive Diseases and Pest unit. Formerly a Professor of Entomology at the University of Missouri-Columbia, Dr. Backus has nearly 25 years’ research experience in feeding mechanisms of hemipterans in relation to plant response, the causation of plant disease, and transmission of plant pathogens. She has studied the relationships among insect anatomy, stylet penetration, salivation, plant physiology, and pathogen transmission. She also has designed and marketed instruments, as well as provided instruction in, electrical penetration graph (EPG) monitoring of insect feeding. Dr. Backus is recognized as a world authority on EPG and hemipteran feeding mechanisms. While at the University of Missouri, she garnered nearly $2 million in extramural research grants. She has edited 2 books, as well as published 5 book chapters, 2 review articles, and nearly 50 scientific articles and abstracts.
Kendra Baumgartner is a Research Plant Pathologist with the Crops Pathology and Genetics Research Unit in Davis, CA. Her research program is focused on developing sustainable methods of disease and weed control for winegrapes. Current research projects include examining the role of riparian hosts in the epidemiology of PD, developing effective control practices for Armillaria root disease, evaluating the effects of vineyard floor management practices (weed control, cover crops) on soil chemical, physical, and biological properties, and studying the beneficial effects of mycorrhizal fungi on grapevine growth.
Jackie Blackmer is a Research Entomologist with the Western Cotton Research Laboratory in Phoenix, AZ, where her main focus is on insect behavior, specifically insect-plant interactions and factors that influence dispersal in insects. She currently is investigating plant- or insect-derived stimuli and/or feedback mechanisms that influence feeding, host finding, oviposition, longevity, reproductive development and emigration of cotton insect pests, as well as invasive species (i.e., Homalodisca coagulata). She is also interested in how host-plant morphology, physiology, and nutritional components influence biological processes in insects. She has published more than 50 journal articles/ or book chapters, presented numerous invited and scientific talks, won several awards, and been part of several research teams that have brought in more than $1.2 mil.
David Boyd is a Research Entomologist at the Small Fruit Research Station, Poplarville, MS. His research areas include integrated pest management of arthropod pests of ornamentals in large production nurseries and biological control of insects. Current research projects include development of pest management tools for the strawberry rootworm in azalea production and the metallic flea beetle in crape myrtle production. Other projects include development of host plant resistance in ornamentals to key pests.
James S. Buckner is a Supervisory Research Chemist/Biochemist with the Insect Genetics and Biochemistry Unit, Fargo, ND, and has been with ARS for 27 years. His research contributes to the development of biological and biochemical approaches to improve beneficial insects for the biocontrol of pest insect populations. His expertise is in the identification, function, and biosynthesis of insect cuticular and internal lipids. He has characterized surface and internal lipids associated with pest and beneficial insects, and their natural enemies. His current and future research includes characterizing interactions of pest/beneficial insects and their natural enemies, determining the role of lipids on interactions of natural enemies with their hosts, and characterizing feeding mechanisms and vector transmission for homopteran insect (whiteflies, leafhoppers) to determine resistant characteristics in plants. He has published more than 60 refereed journal articles, three book chapters on the chemistry and biochemistry of lipids and has given numerous invited and scientific presentations.
Steve Castle is a Research Entomologist at the Western Cotton Research Laboratory, Phoenix, AZ. His areas of interest include pest management, population biology, and insect vector/plant pathogen relations. Current research projects include development of pest management tools for combating two invasive insect species in California, improving chemical control methods for silverleaf whiteflies, and establishing baseline susceptibility levels for pink bollworm to transgenic insecticidal cotton cultivars.
Jianchi Chen is a Research Molecular Biologist at the San Joaquin Valley Agricultural Sciences Center, Parlier, CA, and recently joined the Unit of Exotic and Invasive Disease and Pest Research. Dr. Chen has been working in the area of population characterization of Xf for over ten years. He was among the first to investigate the genetic diversity and discovered the presence of plasmids in Xf. He has a strong interest in applying molecular biology technology to study and resolve disease problems directly related to crop production.
Edwin Civerolo is Director and Research Plant Pathologist, San Joaquin Valley Agricultural Sciences Center, Parlier, CA. Past research focused primarily on various aspects of bacterial plant diseases, specifically those affecting stone fruits, citrus and strawberry caused by xanthomonads. Current research is directed on diseases (e.g., PD of grape and ALSD) caused by Xf. This has included pathogen characterization, pathogen detection and identification, disease diagnosis, disease epidemiology and disease management with an emphasis on induced host resistance.
Thomas A. Coudron is a Research Chemist and lead scientist located at the Biological Control of Insects Research Laboratory in Columbia, Missouri, and has 25 years of experience in insect biochemistry and developmental regulation. His primary research focus is to elucidate the effects of nutrition and/or the regulatory role of biological substances on the developmental processes of beneficial insects, with the long-term goal of developing cost-effective mechanisms to in vitro rear beneficial organisms for insect and weed control. Currently he is investigating physiological and biochemical links between nutrition and key developmental processes that challenge the production of quality insects via artificial rearing methods. The goal is to optimize artificial diets used to mass rear beneficial insects and to establish diagnostic techniques to enable the rapid detection and identification of the biochemical and physiological parameters correlated with quality insects reared under in vitro methods.
Peter Cousins is a Plant Geneticist in the Plant Genetic Resources Unit, Ithaca, NY. He is charged with breeding, evaluating, selecting, and introducing grape rootstocks, with national responsibility. He trained in grape rootstock breeding and genetics at the University of California, Davis, focusing on describing the genetic control of resistance to the root-knot nematode Meloidogyne incognita in grape rootstocks. Current research projects include screening diverse grape rootstock germplasm for resistance to virulent nematode populations, evaluation of rootstock impact on Pierce’s disease expression in the scion, evaluation of rootstocks for wine, table, and raisin grapes, and breeding improved rootstocks with nematode and phylloxera resistance and desirable horticultural attributes.
Jesús (Jesse) H. de León is a Research Molecular Biologist located at the Beneficial Insects Research Unit, Weslaco, TX. Jesse was recruited from the biomedical field of pharmacogenetics (over 12 years experience) to implement his expertise in molecular genetics and molecular biology toward biological control (Molecular BioControl) of insect pests. Dr. de Leon’s main goals are to incorporate modern molecular genetic/molecular biological methods to support classical biological control programs that reduce the use of pesticides, develop molecular markers and DNA fingerprinting methods to genetically identify both pests and natural enemy populations, and examine the nature of genetic variation within and among pest and natural enemy populations that could impact the success of biocontrol programs.
Current research projects include determining the population genetic structure of both the glassy-winged sharpshooter and its natural enemies (e.g, Gonatocerus spp.) by PCR-based DNA fingerprinting methods and determining the origin of the glassy-winged sharpshooter.
Gary Elzen is a Research Entomologist within the Beneficial Insects Research Unit of the Kika de la Garza Subtropical Agricultural Research Center, Weslaco, Texas. Dr. Elzen conducts toxicological and behavioral studies to evaluate the suitability of insect natural enemies for use in biologically based pest management. Research emphasis is placed particularly on Anthonomus grandis grandis, Heliothis/Helicoverpa spp., Spodoptera spp., Bemisia argentifolii and its parasitoids, spiders, and the glassy-winged sharpshooter, Homolodisca coagulata, and its parasitoids. In addition to conventional toxicological methods, Dr. Elzen utilizes a variety of organic analytical techniques (e.g. HPLC, GC), and novel behavioral bioassays.
Michael Glenn is a Research Soil Scientist at the Appalachian Fruit Research Station, Kearneysville, WV. His research focuses on the response of fruit and shoot growth to chemical and physical changes in the plant environment, particularly in developing management systems that more efficiently utilize the environmental resources, and production systems that minimize pesticide usage. He patented a pest control system based on particle film technology that controls a wide range of disease and insect pests, including the GWSS. The particle film technology also reduces heat and water stress in plants and improves production efficiency. Dr. Glenn has 94 peer-reviewed, scientific publications in the area of soil and water management, plant nutrition, root physiology and particle film technology, 1 patent related to irrigation scheduling technology, and 8 patents establishing particle film technology.
Russell Groves is a Research Entomologist located at the San Joaquin Valley Agricultural Sciences Center, Parlier, CA, and recently joined a new team of scientists in the Exotic and Invasive Disease and Pest Unit. Dr. Groves has over 10 years experience in applied entomology and a strong background in insects as vectors of plant disease. Previously, his research responsibilities focused on seasonal population dynamics and epidemiology of the thrips/Tomato spotted wilt Tospovirus pathosystem emerging as a serious threat in North Carolina plus more recent research responsibilities administered through Cornell University examining aphid-borne, Potato Y Potyvirus epidemiology. Presently, the focus of his research aims at understanding fundamental biology and ecology of the glassy-winged sharpshooter in the Central San Joaquin Valley as it relates to acquisition and transmission of Xylella fastidiosa scorch diseases.
James Hagler is a Research Entomologist located at the Western Cotton Research Laboratory, Phoenix, AZ. His primary research focuses are in the areas of biological control, insect dispersal, and insect behavior. He has senior authored over 50 publications. Dr. Hagler has made significant contributions toward bridging the gap between molecular biology and applied entomology. His molecular probes for detecting prey remains in predator guts are considered state-of-the-art research among his biological control peers. Dr. Hagler is considered a world authority among biological control researchers in the area of evaluating the efficacy of predaceous natural enemies. Over the past 4 years alone, Dr. Hagler has individually or as a team member, secured external funding exceeding $500,000 through highly competitive grant programs.
John Hartung is a Research Plant Pathologist located at the Fruit Laboratory at Beltsville, Maryland. Dr. Hartung conducts research on a broad range of exotic citrus pathogens under quarantine arrangements in Beltsville, and in cooperation with researchers in Japan, France, Brazil and Florida. A major focus of his research has been on strains of Xylella fastidiosa that cause citrus variegated chlorosis and coffee leaf scorch diseases in Brazil. Among the accomplishments of Dr. Hartung’s group were the demonstration that strains of Xylella fastidiosa cause citrus variegated chlorosis and coffee leaf scorch diseases, the characterization of the population of X. fastidiosa associated with sweet orange in Brazil as compared with North American strains, the development of widely used PCR-based detection methods for the pathogen, and the demonstration that strains of X. fastidiosa from sweet orange can induce symptoms similar to those of Pierce’s disease in artificially inoculated ‘Chardonnay’ grapevine. His laboratory has also transformed X. fastidiosa and created green fluorescent protein tagged, defined mutants of X. fastidiosa and observed them using confocal microscopy in inoculated plants. His group has also provided evidence that the sweet orange strain of X. fastidiosa can be transmitted through seed.
Thomas Henneberry is Director and Research Entomologist, Western Cotton Research Laboratory. The laboratory is the focal point in the Western United States for basic and applied research on cotton pest management, as well as the physiological relationships of the cotton plant affecting cotton production. He has authored more than 500 publications in scientific journals and other media, including more than 15 book chapters. He serves on numerous state, federal and industry committees to establish multidiscipline research, priorities and approaches. He has been a member of the Entomological Society of America for 45 years, during which time he has served on Program Committees, the Auditing Committee, Secretary-Elect of Section F, and the Executive Committee of the Pacific Branch. Elected as "Fellow" of the Entomological Society of America in 1991, and recipient of Agricultural Research Service "Outstanding Scientist of the Year" award in 1990. He received the Miles Cotton Research Recognition Award, and the USDA Department Award for Superior Service in 1997, was installed in the ARS Hall of Fame in 1998 and received the President's Senior Executive Service Meritorious Award in 1999.
Wayne Hunter is the Lead Scientist on PD and the GWSS at USHRL, Ft. Pierce, FL. He has focused on using state-of-the-art technologies to rapidly generate information on the biology, development, and pathology of the GWSS to advance the development of environmentally sound methods of GWSS population management aimed to reduce and/or stop the spread of Pierce’s Disease. The focus is on the potential use of insect viral pathogens to develop an Area-wide suppression program against GWSS and other vectors of PD. Dr. Wayne Hunter has accomplished meeting several critical objectives within the first year of this CRIS, focused on Pierce’s Disease and the GWSS. Dr. Hunter has aggressively pursued the rapid production and release of information to meet CRIS objectives concerning the devastating problems associated with Pierce’s Disease and the GWSS. Important biological information produced through the creation and annotation of expression cDNA libraries from two life stages of the GWSS have been completed. Through these focused efforts genes critical to GWSS development and survival have been identified along with the discovery of new viral pathogens.
Walker A. Jones is Supervisory Research Entomologist and Research Leader of the Beneficial Insects Research Unit, Weslaco, TX. He has served with ARS for 22 years, conducting research associated with classical and augmentation biological control using predators, parasitoids, and pathogens. He currently leads a multi-disciplinary team of experts in field ecology, insect pathology, molecular genetics, population biology, biochemistry, insect toxicology, and weed science. He has completed a 3-year survey of the impact of parasitism on the GWSS in one of its areas of origin, finding that egg parasitism is largely responsible for low sharpshooter populations in south Texas. He has led a team that has identified 10 species of sharpshooter egg parasitoids from South America, collected from sub-climate and habitat types identical to that across the grape-growing areas of California, and is currently studying the biology and risk assessment associated with 7 species of parasitoids from Argentina that successfully attack GWSS eggs in quarantine.
Daniel A. Kluepfel is a Research Plant Pathologist in the Crops Pathology and Genetics Research Unit on the campus of the University of California, Davis. Dr. Kluepfel obtained his Ph.D. from the University of Florida, Gainesville, were he worked on Agrobacterium attachment to plant cell surfaces. After post-doctoral positions in Wagenningen, The Netherlands, and the University of Hawaii, he served on the faculty in the Department of Plant Pathology at Clemson University for 16 years. In August of 2003 he accepted the position of Research Leader of the CPGRU and Location Coordinator at U. C. Davis. His research interests involve an examination of the molecular microbial ecology of the rhizosphere as it pertains to the development of biological control agents. Current research projects include efforts to examine global gene expression patterns in Pseudomonas sp. during root colonization. Additional projects involved the study of microbial diversity and its impact on disease incidence.
Craig A. Ledbetter is a Research Geneticist at the San Joaquin Valley Agricultural Sciences Center in Parlier, CA. He has been with ARS for 16 years and has worked on both Prunus and Vitis germplasm breeding and evaluation. Present research activities include the development of new apricot and plum X apricot varieties for the fresh and processing markets. He has introduced five new apricots during the last decade. He is also involved in almond breeding with a major emphasis on developing new self‑compatible varieties. Prunus rootstock breeding is a third emphasis in his research program. With the enormous market for almond nursery stock in the California growing regions, emphasis is placed on well‑anchored root systems and nematode resistance. Enhanced vigor in these almond rootstocks is accomplished through the use of a male‑sterile facilitated system to produce diverse hybrid peach‑almond seed.
Jesusa C. Legaspi is a Research Entomologist with the Insect Behavior and Biocontrol Unit at Gainesville, FL. Her duty station is at Florida A&M; University (FAMU) at Tallahassee, FL. She also is an Adjunct Associate Professor at the joint USDA‑FAMU Center for Biological Control, which was established in 1999. She has over 20 years experience in biological control and integrated pest management of major insect pests in various field crops. For over seven years, she evaluated transgenic sugarcane against an important stalkborer pest and its impact on their natural enemies. Her current research involves ecological and physiological studies of parasites and predators of major pests in vegetables and small fruits such as silverleaf whiteflies and GWSS. She has published over 112 journal articles, book chapters or extension publications, and has presented numerous papers and lectures.
Roger A. Leopold is the Lead Scientist for the Insect Cryobiology Project at the Red River Valley Agricultural Research Center, Fargo, ND. His career in ARS spans > 36 years, most of which were spent in the study of insect reproduction and development. For the past 10 years his specific research interest has been insect cryopreservation and cryobiology. He has given over 70 research presentations at various scientific meetings, seminars, workshops and conferences, 18 of which were invitational at international venues. He has authored or co-authored 60 journal articles, 6 book chapters, 11 proceedings articles, 5 technical reports and misc. articles and 33 published abstracts. He was awarded a CSIRO Senior Scientist Fellowship in 1994 for study in Australia and the CSIRO Sir Frederick McMaster Fellowship for outstanding foreign scientists in 1996. Leopold currently serves on the editorial board of Cell Cryopreservation Technology. In the past 4 years he has obtained about $550,000 in extramural grants. Current research projects include long-term cold storage of tephritid embryos and short-term storage of GWSS eggs and egg parasitoids.
Hong Lin is a Research Plant Physiologist located at the San Joaquin Valley Agricultural Sciences Center, Parlier, CA. He recently joined a new team of scientists in the Exotic and Invasive Disease and Pest Unit. Dr. Lin has 20 years research experience in working with biotic/abiotic stress physiology. His research has encompassed plant physiology, plant biochemistry, population genetics, and molecular biology. Currently the focus of his research aims at understanding mechanisms of PD resistance using functional genomic approaches and evaluating the effect of resistant grape rootstock on PD expression in the scion.
Guillermo Logarzo has been a Research Biologist at the South American Biological Control Laboratory since 1987, searching for, evaluating, and shipping promising agents for biological control of insect pests and weeds. He has conducted projects on biological control of the weeds Xanthium spp., Larrea spp., and Sesbania spp. At present he is searching for natural enemies of the GWSS, tarnished plant bug, and members of the Heliothis/Helicoverpa complex. He has traveled extensively throughout Argentina, from Patagonia to the northern tropical rain forest, including semi‑arid rangelands, and neighboring countries, and is familiar with pests of tobacco, cotton, and corn.
Michael R. McGuire is Supervisory Research Entomologist and Research Leader of the Western Integrated Cropping Systems Research Unit located in Shafter, CA, the heart of the new GWSS/PD infestation area. After receiving his Ph.D. from the University of Illinois in 1985, Dr. McGuire accepted a post‑doc position in Bozeman, MT to study early detection of entomopxvirus, a potential microbial control agent for rangeland grasshoppers. In 1988, he accepted a position in Peoria, IL, as Research Entomologist and Lead Scientist of a CRIS to invent and develop novel formulations for entomopathogens. The formulations he and his staff developed extended the activity of bacteria and viruses and were licensed by private industry. In 1995, Dr. McGuire became Research Leader of the Bioactive Agents Research Unit in Peoria with a staff of 8 SYs and 17‑20 support staff. In 2000, Dr. McGuire became Research Leader and Location Director for the Shafter, CA location. He currently conducts research to develop new, selective biopesticides for the major pests of cotton. Due to his proximity to the newly infested areas, Dr. McGuire was asked to develop a program aimed at finding entomopathogens for control of GWSS. Dr. McGuire is the author or co‑author of approximately 85 peer reviewed publications and eight patents.
Stuart H. McKamey received his B.S. at the University of California, Berkeley in 1985, his M.Sc. at North Carolina State University in 1989, his Ph.D. at the University of Connecticut in 1994, and was a Post-doctoral fellow at the National Museum of Denmark, Copenhagen in 1995-1996. Since 1997 he has been a Research Scientist in the ARS Plant Sciences Institute, Systematic Entomology Laboratory, Beltsville, and is responsible for the leafhoppers, treehoppers, planthoppers, froghoppers, and cicadas (suborder Auchenorrhyncha, order Hemiptera), which include about 40,000 species. His primary responsibilities include: curation of the U.S. National Insect Collection, Smithsonian Institution; service identifications for domestic and foreign researchers, state extension agents, commodity groups, and the Animal and Plant Health Inspection Service; and research on the taxonomy and systematics of Auchenorrhyncha with emphasis on leafhoppers. In addition to over a dozen research papers on the taxonomy and identification of treehoppers and leafhoppers, he has produced a world catalog of treehoppers (3,166 species) and a world checklist of leafhoppers (1758-1955, 11,007 species), which is still the starting point for all taxonomic research on leafhoppers. He is nearing completion of an update of the leafhopper checklist through the year 2000 (approx. 21,000 species).
Steven E. Naranjo is a Research Entomologist at the Western Cotton Research Laboratory, Phoenix, AZ. His areas of emphasis include, population ecology, biological control, sampling, integrated pest management, and systems analysis. The major focus of this research is to understand the contribution of natural enemies to pest population regulation and to integrate biological control with current and developing pest management strategies. Research activities include sampling and description of the seasonal population dynamics of key pests and predators in the cotton system, characterization of the predator complex attacking key cotton pests, evaluation of the impact of insecticides and transgenic cotton on abundance and activity of cotton pest predators and parasitoids, development of decision-aids for whitefly and sticky lint in cotton and development of sampling methods and plans for GWSS in citrus and grapes. Dr. Naranjo has authored 118 publications and has presented numerous papers and lectures. In support of efforts at the Western Cotton Research Laboratory he has individually, or as a team member, secured external funding exceeding $1.6 million through competitive and other grant programs.
Gary J. Puterka is a Research Entomologist in the Appalachian Fruit Research, Kearneysville, WV, with 23 years of experience in field crop and orchard research with 13 years of service with the USDA-ARS that focused primarily on insect-plant interactions, insect genetics, and the development of alternative technologies for arthropod pest management. Senior or co-authored 78 technical publications (42 senior authored) and two book chapters (one senior authored) and authored ten patents on particle film and sugar ester technologies. Over 90 formal presentations with 40 of these being invitations to present his research, or organize and moderate symposiums for professional society meetings. Expertise in developing alternative technologies for arthropod pest management has led to special ARS assignments (GWSS in California) and CRADA’s that transferred new technologies to industry.
David Ramming is a Research Horticulturist in the Postharvest Quality and Genetics Unit, San Joaquin Valley Agricultural Sciences Center, Parlier, CA. Dr. Ramming has been responsible for the grape, peach, nectarine, and plum breeding program for over 28 years. He has released 30 varieties from this program. Current research projects include developing table and raisin grapes with resistance to PD and powdery mildew, improving table grape fruit quality, developing grapes with the natural ability to dry on the vine for raisins, and improved fruit quality for peaches, nectarines and plums.
Fred Ryan is a Research Plant Physiologist with the Exotic and Invasive Disease and Pest Research Unit, Parlier, CA. He recently joined this unit after working with the Postharvest Quality and Genetics Research Unit, Parlier, on the effects of methyl bromide alternative treatments on fresh commodities and developing and applying molecular markers for quality traits for the grape and Prunus breeding programs. He was previously located with the ARS Aquatic Weed Control Research Unit, Davis, CA, and worked on the physiology and control of problem aquatic plants. Using DNA based markers, he also has conducted research on genetic variation, origin, and biological control of Russian thistle in cooperation with other researchers in the U.S. and abroad. In the present assignment, he will be investigating the interaction between Xylella and Prunus species, with particular regard to mechanisms of resistance in the plant.
Norman W. Schaad is a Research Plant Pathologist (bacteriologist) with the Foreign Disease-Weed Science Research Unit, Ft. Detrick, MD. Research interests include molecular characterization, real-time PCR and microarray detection, identification, and systematics of foreign and domestic plant pathogenic bacteria. He has over 20 years experience in working with Xylella fastidiosa. While at University of Georgia he co-developed PW medium and the first serological assay for X. fastidiosa. Current research projects on X. fastidiosa include development of rapid, sensitive real-time PCR assays and the taxonomy of the bacterium. Dr. Schaad received his BS (1964), MS (1966), and Ph.D. (1969) in Plant Pathology from the University of California, Davis.
Ralph Scorza is a Research Horticulturist and lead scientist at the ARS Appalachian Fruit Research Station, Kearneysville, WV. He has worked for over 20 years on the genetic improvement of temperate fruit crops including stone fruits (peach, nectarine, plum), grape, and pear, with most of his effort focused on stone fruits. He has introduced two peach cultivars that are currently the standards for their season in the northeastern and mid‑Atlantic states ('Sentry' and 'Bounty') and has additionally released a plum and nectarine cultivar. He has developed novel new peach tree growth habits for high-density production systems that will be introduced in 2004. Dr. Scorza has developed a transgenic plum cultivar with high-level resistance to plum pox virus. This plum has been tested in European field tests for the last 7 years where it has shown excellent resistance, productivity and high fruit quality. It is the first transgenic virus resistant temperate tree fruit to be developed and provides a blueprint for the deployment of this technology.
Jerry K. Uyemoto is a Research Plant Pathologist in the Crops Pathology and Genetics Research Unit on the campus of the University of California, Davis. After obtaining a Ph.D. from the UC-Davis in 1968, work experience includes: Associate Professor, New York State Agricultural Experimental Station, Cornell University, Geneva NY 14456 (1968-1977) - responsible for virus diseases of apple, grapevine and stone fruit; Professor, Kansas State University, Manhattan DA 66502 (1977-1982) - virus diseases of corn, sorghum, and wheat; Senior Scientist with Advanced Genetic Sciences, Manhattan KS (1982-1984) - protoplast and tissue culture of legumes (guar); Visiting Scientist, UC-Davis (1984-1986) pistachio epicarp lesion and cherry x-disease projects; Research Plant Pathologist, USDA-ARS, UC-Davis (1986-present) virus and virus like diseases of stone fruits and grapevines.
George D. Yocum is a Research Physiologist at the Red River Valley Agricultural Research Center, Fargo, ND. Dr. Yocum has 10 years of experience as an insect physiologist including 4 years with ARS, and has a strong background in diapause and stress physiology. He has authored/coauthored 14 peer-reviewed articles and 6 book chapters, and given 25 presentations at scientific meetings. Past research focused on the molecular mechanisms of both pupal diapause and stress response to high and low temperature exposure. Current research involves characterization of the molecular regulation of adult diapause initiation, and investigating molecular responses to suboptimal diet feeding.
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