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United States Department of Agriculture

Agricultural Research Service


Location: Crop Diseases, Pests and Genetics Research

2009 Annual Report

1a. Objectives (from AD-416)
The overall goals of this project are to reduce losses due to Xf-caused diseases during crop production and to develop effective, sustainable disease and insect vector management strategies by characterizing the host-pathogen-vector-environment interactions of these complex pathosystems. Specific objectives for the project are outlined below: Objective 1: Determine the epidemiology of exotic, emerging, re-emerging, and invasive diseases in California, including (but not necessarily limited to) Xf-caused diseases of horticultural, agronomic, and ornamental crops. Objective 2: Determine the nature and mechanism(s) of susceptibility/resistance to Xf infection and subsequent disease development in horticultural and agronomic crops, including (but not necessarily limited to) Vitis species and Prunus species. Objective 3: Develop effective, economical, environmentally sound strategies to manage exotic, emerging, re-emerging, and invasive diseases, including (but not necessarily limited to) xylella diseases.

1b. Approach (from AD-416)
Determine the epidemiology of Xylellae diseases in California. Characterize the complex host-pathogen-insect vector-environment interactions of these pathosystems. Determine the biochemical, physiological, genetic and mechanistic bases of resistance to XF infection and subsequent disease development in Vitis and selected Prunus species. Formerly 5302-22000-007-00D.

3. Progress Report
Seventy polymorphic molecular markers for Pierce’s disease (PD) resistance were run on 154 seedling progeny and parents in the table grape BD5-117 family. Marker-assisted selection resulted in identification of PD-resistant seedlings in the BD5-117 family that will be further characterized for PD resistance and fruit quality. A comparative protein analysis approach was conducted to characterize differentially expressed protein profiles from PD resistant and susceptible grapevines in response to Xf infection. The results indicate that differentially expressed proteins in response to Xf infection are plant genotype, tissue development and Xf infection dependent. Clonal replicates of peach, almond and a hybrid created from them were challenge inoculated with X. fastidiosa. Seedlings from backcrosses of almond hybrid P63-61, almond cultivar Butte, and Prunus webbii have been transplanted to the field to provide future clonal replicates for X. fastidiosa inoculation assays. Plant samples collected in and near almond nurseries were assayed for X. fastidiosa infection to identify potential reservoir hosts that may serve as a source of primary inocula. To date, Xf was detected in Himalayan blackberry in an area highly infested by insect vectors. The role of alfalfa in the epidemiology of xylellae diseases was assessed. Results indicate that alfalfa may serve as a source of insect vectors but is not likely to represent a significant source of the pathogen. Simulation models were used to assess the effectiveness of different management techniques on X. fastidiosa spread. The model indicates that ecological differences among vectors dictate that management practices must be tailored to the specific vector associated with a given crop. Development of a mechanistic model describing vector transmission of X. fastidiosa to plants continued. Confocal microscopy showed the major enzyme in glassy-winged sharpshooter (GWSS) saliva, beta 1,4-glucanase, is highly concentrated in the deepest, thinnest branches of the salivary sheath. Glucanase loosens and penetrates xylem cell walls, forming a tight bond between sheath and xylem. These results suggest that the enzyme is associated with GWSS access to the xylem and may play a key role in inoculation of X. fastidosa to plants. Mobile genetic elements of X. fastidiosa were characterized. A novel phage of the family Podoviridae was identified by electron microscopy. Sequence analysis indicated that phage DNA is integrated into the X. fastidosa genome. Complete sequence of a 25 kilobase pair plasmid resident in mulberry-infecting strains of X. fastidiosa was determined. The plasmid encoded a DNA transfer system, a plasmid maintenance system, and DNA replication functions. Surprisingly, the plasmid shared extensive sequence identity with a plasmid present in a symbiont of earthworms. A new virus species infecting GWSS was purified and characterized. The genome of the new virus was completely sequenced and shown to be most closely related to rice dwarf virus. Surveys indicated that the virus is common in California.

4. Accomplishments
1. Use of Molecular Markers Aides Introgression of Pierce’s disease (PD) Resistance Into Table, Raisin and Wine Grape Germplasm. A quick method to determine PD resistance in plants is needed as evaluation under natural field (5 years) or controlled greenhouse (6 months) conditions are time consuming and costly. Introgression of PD resistance from a homozygous resistant form of Vitis arizonica (b43-17) was observed by ARS scientists in Parlier, CA in a total of 4,321 seedlings from 83 different crosses of resistant V. arizonica selections and high-quality V. vinifera cultivars from the F1 and first and second modified backcross generations. Based on the presence of resistant molecular markers (VVIP26, ctg1026876 and VMC2a5), 1,683 PD resistant seedlings from wine, table, and raisin grape backgrounds were selected without prolonged field or greenhouse studies. This research will aid in development of PD resistant grapes.

2. A New Almond Hybrid Cures Itself of Almond Leaf Scorch Bacteria During the Winter Months. Reduced yield and tree mortality make almond leaf scorch disease a critical problem throughout California’s 700,000+ acres. Through conventional plant breeding with a wild almond relative, a hybrid almond was developed by ARS scientists at Parlier, CA that is better at overcoming almond leaf scorch disease through winter-curing as compared with the almond cultivar Butte. The new hybrid has edible almonds that are similar to Butte in color and kernel shape. This hybrid is significant because it demonstrates that through traditional breeding efforts, a resistant almond variety can be achieved. The development of almond varieties resistant to this disease will be an economic benefit for the California almond industry.

3. The Effect of Grapevine Xylem Sap on Xylella fastidiosa (Xf) Culture. Virtually all grape cultivars are susceptible to Pierce’s disease (PD). However, some related grape species from the southern United States are resistant. To assess differences between resistant and susceptible grape species, ARS scientists at Parlier, CA studied the effect of xylem sap collected from PD-resistant and PD-susceptible grapevines on Xf growth, biofilm formation and cell aggregation was investigated. Media containing xylem sap from PD-susceptible plants provided better support for bacterial growth than media supplemented with xylem sap from PD-resistant plants. This study provides new insights on the molecular basis of xylem-limited bacteria pathogenicity and host resistance which facilitates target selection of promising genetic traits in the breeding of PD-resistant grapevines.

4. Theoretical Assessment of Methods to Reduce Spread of Xylella fastidiosa. Patterns of the bacteria that causes Pierce's Disease, Xylella fastidiosa, spread differ with insect vector species in California. A simulation model was used to assess effectiveness of vine removal and insecticide treatments on slowing pathogen spread. This model used different assumptions concerning the extent of within field pathogen spread and vector reproduction in the affected crop. The results provide recommendations for improved control tactics.

5. The Role of Alfalfa in the Epidemiology of Diseases Caused by Xylella fastidiosa (Xf) Was Assessed. Alfalfa has long been considered an important crop in the epidemiology of diseases caused by Xf the bacteria that causes Pierce's disease, but little research has been completed to assess the importance of alfalfa as a source of Xf and/or vectors. Weedy alfalfa fields were found by ARS scientists at Parlier, CA to harbor large numbers of green sharpshooter, a known vector of Xf. Incidence of Xf in alfalfa was low, due to vector preference for weeds over alfalfa. The results provide growers with an assessment on the potential for alfalfa to serve as a source of vectors and Xf inocula.

6. New Hypothesis for Inoculation of Xylella fastidiosa (Xf) by Insect Vectors. Xf inoculation of grape causes Pierce's Disease. The mechanism of vector inoculation of Xf is unknown, slowing research on development of grape varieties resistant to inoculation. ARS scientists at Parlier, CA used electrical penetration graph (EPG) recordings coupled with confocal microscopy to support the hypothesis that sharpshooters secrete saliva into the plant that is then sucked into the foregut region where Xf forms biofilm colonies. A mixture of plant fluid, saliva and loosened bacteria is then forcefully discharged into the plant to cause inoculation of Xf. This research supports the use of EPG recording as a means of screening plants for resistance to inoculation by vector sharpshooters.

7. Discovery and Characterization of Bacterial Phage Infecting Xylella fastidiosa (Xf). Whether viruses (phage) may be used to control Xf(Pierce's disease) populations in infected plants has not been addressed. A novel phage of Xf was identified by ARS scientists at Parlier, CA by genomic analysis and electron microscopy. A survey of phage present in almond-infecting strains of Xf was conducted. Discovery of Xf phage provides a potential new alternative for biological control of Xf.

8. Genome Characterization of a Virus Infecting the Glassy-winged Sharpshooter. Environmentally friendly alternatives to chemical control of the glassy-winged sharpshooter are needed. A new virus species infecting the glassy-winged sharpshooter was isolated by ARS scientists at Parlier, CA from California populations and characterized as a phytoreovirus species most closely related to rice dwarf virus. The complete genome of the new virus was determined and deposited in the Genbank, the NCBI public database. As the new virus does not cause significant mortality of glassy-winged sharpshooter, its potential as a biological control agent appears limited. Nonetheless, population genetic analyses suggest that sequence variation among virus isolates may serve as genetic markers to differentiate populations of the glassy-winged sharpshooter.

Review Publications
Sisterson, M.S. 2008. Egg load dynamics of Homalodisca vitripennis. Environmental Entomology. 37(5):1200-1207(8).

Chen, J., Deng,, X., Liu,, S., Pu,, X., Li,, H., Civerolo, E.L. 2009. A phytoplasma related to Candidatus Phytoplasma asteri is associated with citrus showing Huanglongbing (yellow shoot disease) symptoms in Guangdong, P. R. China. Phytopathology. 99:236-242.

Lin, H., Doddapaneni,, H., Munyaneza, J.E., Civerolo, E.L., Venkatesan, S.G., Buchman, J.L., Stenger, D.C. 2009. Molecular characterization and Phylogenetic analysis of 16S rRNA from a new Candidatus Liberibacter strain associated with Zebra chip disease of Potato (Solanum tuberosum L.) and the potato psyllid. Journal of Plant Pathology. 91:215-219.

Yao, J., Lin, H., Vandeyne, A., Doddapaneni, H., Francis, M., Macedo Lemos, E., Civerolo, E.L. 2008. PrimerSNP: a web tool for whole-genome selection of allele-specific and common primers of phylogenetically-related bacterial genomic sequences. BMC Microbiology, 8:185doi:10.1186/1471-2180-8-185.

De Mello Varani, A., Souza, R., Nakaya, H., De Lima, W., De Almeida, L., Kitajima, E., Chen, J., Civerolo, E.L., Vasconcelos, A., Van Sluys, M. 2008. Origins of the Xylella fastidiosa prophage-like regions and their impact in genome differentiation. PLoS One. 3(12):e4059. doi:10.1371/journal.pone.0004059.

Stenger, D.C., Sisterson, M.S., Krugner, R., Backus, E.A., Hunter, W.B. 2009. A New Phytoreovirus Infecting the Glassy-Winged Sharpshooter (Homalodisca vitripennis). Virology. 386:469-477.

Krugner, R., Groves,, R.L., Johnson,, M.W., Flores,, A.P., Hagler, J.R., Morse,, J.G. 2009. Seasonal Population Dynamics of Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) in Sweet Orange Trees Maintained under Continuous Deficit Irrigation. Journal of Economic Entomology. 102(3):960-973.

Sisterson, M.S. 2009. Transmission of Insect-Vectored Pathogens: Effects of Vector Fitness as a Function of Infectivity Status. Environmental Entomology. 38:345-355.

Sandanayaka, W., Backus, E.A. 2008. Quantitative comparison of stylet penetration behaviors of glassy-winged sharpshooter, Homalodisca vitripennis, on four crop plants important in New Zealand and the USA. Journal of Economic Entomology. 98:787-813.

Deng,, X., Chen, J., Li,, H. 2008. Sequestering and characterization of sequence of a ribosomal RNA operon (rrn) from “Candiatus Liberibacter asiaticus”. Molecular and Cellular Probes. 22:338-340.

Ledbetter, C.A., Chen, J., Livingston, S., Groves, R.L. 2009. Wintercuring of Prunus dulcis cv ‘Butte,’ P. webbii and their interspecific hybrid in response to Xylella fastidiosa infections. Euphytica. Available: http://www/

Cheng, D.W., Lin, H., Walker,, M., Stenger, D.C., Civerolo, E.L. 2009. Effects of Grape Xylem Sap and Cell Wall Constituents on In Vitro Growth, Biofilm Formation, and Cellular Aggregation of Xylella fastidiosa. European Journal of Plant Pathology. 125:213-222.

Krugner, R., Johnson, M.W., Daane, K.M., Morse, J.G. 2008. Olfactory responses of the egg parasitoid, Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae), to host plants infested by Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). Biological Control. 47:8-15.

Doddapaneni, H., Liao,, H., Lin, H., Bai,, X., Zhao,, X., Civerolo, E.L., Irey,, M., Coletta-Filho,, H., Pietersen,, G. 2008. Comparative phylogenomics and multi-gene cluster analyses of the CitrusHuanglongbing (HLB)-associated bacterium Candidatus Liberibacter. BMC Research Notes, 1:72.

Sisterson, M.S., Yacoub,, R., Montez,, G., Grafton-Cardwell, E., Groves,, R.L. 2008. Distribution and Management of Citrus in California: Implications for Management of Glassy-winged Sharpshooter. Journal of Economic Entomology. 101:1041-1050.

Riaz, S., Tenscher, A.C., Graziani, R., Krivanek, A.F., Ramming, D.W., Walker, M. 2009. Using Marker-Assisted Selection to Breed Pierce’s Disease-Resistant Grapes. American Journal of Enology and Viticulture. 60(2):199-207.

Cabrera-La Rosa,, J., Johnson,, M., Chen, J., Civerolo, E.L., Groves,, R. 2008. Seasonal Population Dynamics of Draeculacephala minerva (Hemiptera: Cicadellidae) and Transmission Efficiency of Xylella fastidiosa. Journal of Economic Entomology. 101(4):1105-1113.

Last Modified: 2/23/2016
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