Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The domestic citrus industry is continuously threatened by pathogens which cause serious diseases in foreign countries, but that are not present in the United States. The risk of introduction of these pathogens and their insect vectors increases continuously with the increase in international commerce and travel. These 'exotic pathogens' are studied to develop basic knowledge useful to either prevent their introduction into the United States or to facilitate their eradication or management if they are inadvertently introduced into the United States. We are currently working with the bacterial pathogens Xylella fastidiosa (Brazil) , Xanthomonas axonopodis pv citri(worldwide and since 1995 in Florida), Citrus Chlorotic Dwarf Virus (Turkey), Citrus Yellow Mosaic BADNA Virus (India), citrus
greening or huanglongbing (Southern Asia, Brazil and since 2005, Florida). The citrus strains of Xylella fastidiosa and the citrus greening pathogen are select agents under U.S. law, a designation which signifies special concern about the risk posed by these pathogens on the part of the government. Our research is important to the citrus industry, which is a major economic factor in Florida, South Texas, and California. Each of these diseases has potential to seriously damage or destroy the industry. Citrus Variegated Chlorosis Disease, caused by Xylella fastidiosa was first described in Sao Paulo state, Brazil. Since that time, it has established itself as the most serious threat to citriculture in Brazil, which has the largest citrus industry in the world. Current estimates are that 40 percent (88 million) of the orchard trees in Sao Paulo are infected and yield losses can be high. Insects endemic to the United States can transmit this pathogen. Citrus bacterial canker caused by
Xanthomonas axonopodis pv. citri was eradicated from the United States in the first third of this century, because the growers rightly feared its destructive potential. It has since been reintroduced into South Florida. The USDA has abandoned its century long policy of eradicating citrus canker since it has become endemic in Florida. Mild symptoms of this disease prevent the sale of fruit on the fresh market and severe symptoms render the fruit unmarketable. This pathogen is also an object of international phytosanitary quarantine and so complicates international commerce. Citrus Chlorotic Dwarf Virus was discovered in Turkey in commercial lemon groves. It infects and causes disease in all citrus, causing a total yield loss. The insect vector is endemic in the United States. Citrus Yellow Mosaic BADNA Virus has been described in India, and causes a destructive disease there. The insect vector for this virus is endemic in the United States. This virus also has a double stranded DNA
genome, which has implications for the biotechnology industry. Citrus greening or Huanglongbing has also become established in Florida. It kills infected trees, has a long latent period, is spread by insects, and no resistant varieties are available. For these reasons, citrus greening could destroy the citrus industry in Florida. The project has four objectives: 1) Establish the etiology of novel diseases of citrus, 2) Develop novel rapid, sensitive and quantitative diagnostic tests for the presence of exotic pathogens in plant tissues, 3) Identify the virulence mechanisms used by exotic pathogens, and 4) Maintain and facilitate cooperative research on the Exotic Pathogens of Citrus Collection. The research falls under National Program 303 - Plant Diseases 70% and National Program 301- Plant, Microbial & Insect Genetic Res., Genomics, & Genetic Improv. 30%) as described in the National Program Action Plan. Specifically, these are: NP 303, Component 1: Identification and classification
of plant pathogens; Commodity citrus research need 1: Citrus Tristeza Virus (CTV) - Strain Detection, Identification, and Characterization; Commodity citrus research need 4: Maintain and Thoroughly Characterize a Collection of Exotic Pathogens of Citrus. Component 4: Commodity citrus research need #4: Pathogen Characterization and Evaluation of Commercial Scions for Resistance to CVC. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2004) Develop initial rapid PCR tests for Xylella fastidiosa and Huanglongbing. Complete characterization of initial mini-Tn5 mutants. Prioritize accessions; remove duplicates; collaborative research. Year 2 (FY 2005) Complete purification of CCDV. Complete rapid PCR technology; PCR test for CYMV. Confocal microscopy of mutants in planta. Prioritize accessions; remove duplicates; collaborative research; ongoing project. Year 3 (FY2006) Etiology and purification of citrus yellow bark agent. Differentiation of
strains of CYMV. Transfer rapid PCR technology. PCR test for CYMV. Prioritize accessions; remove duplicates; collaborative research. Year 4 (FY 2007) Differentiation of strains of CYMV. Prioritize accessions; remove duplicates; collaborative research. Complete diagnostics for citrus yellow bark agent and CCDV. Identify mutants impaired in pathogenicity. Move collection to new greenhouse. Year 5 (FY 2008) Finish studies and transfer technologies. Prioritize accessions; remove duplicates; collaborative research. 4a List the single most significant research accomplishment during FY 2006. Development and deployment of a rapid and quantitative PCR based assay for citrus greening or Huanglongbing. Our research anticipated the arrival of citrus greening in Florida. As a result, we had developed and validated the first real time and quantitative PCR assay for the greening pathogen. This assay was used by APHIS and the Florida Dept of Agriculture to confirm the presence of citrus
greening/Huanglongbing in Florida. It is also in use in both Florida and Brazil for the management of the disease. This research is performed under National Program 303, Plant diseases Research component 1 Identification and Classification of Pathogens. 4b List other significant research accomplishment(s), if any. In collaboration with researchers in Brazil, we have developed a novel PCR based assay for the citrus canker bacterium. This assay is based on a gene that regulates pathogenicity of the bacterium and is the only such assay that is able to detect all strains of canker causing bacteria, including unusual strains found in the Arabian peninsula and eastwards into Iran. This research is performed under National Program 303, Plant diseases Research component 1 Identification and Classification of Pathogens. 4d Progress report. We are making progress in a collaborative project with the University of Florida, to identify lytic peptides effective at controlling citrus
greening/Huanglongbing disease. This work is difficult, long term and expensive, because it must be done in citrus trees in the greenhouse. We have developed a methodology as well as more than 50 unique PCR based assays that can detect the citrus canker bacterium in herbarium specimens deposited as long as a century ago. These data collectively trace the evolution of the pathogen and the course of its distribution around the world. It would be interesting, if funds were available, to apply this methodology to contemporary strains. We have also identified a volatile chemical compound produced in grapefruit leaves infected by Xanthomonas axonopodis citri, the citrus canker pathogen. The compound, phenylacetaldehyde o-methyloxime, may be the basis for continuous monitoring of grapefruit groves for the presence of the disease. A similar approach may be possible to detect the pathogen in commercial shipments of fruit. We have also identified a volatile chemical compound produced in
grapefruit leaves infected by Xanthomonas axonopodis citri, the citrus canker pathogen. The compound, phenylacetaldehyde o-methyloxime, may be the basis for continuous monitoring of grapefruit groves for the presence of the disease. A similar approach may be possible to detect the pathogen in commercial shipments of fruit. 5. Describe the major accomplishments to date and their predicted or actual impact. Established that the sweet orange strain of Xylella fastidiosa completely colonizes the fruit and seed of sweet oranges and can be transmitted through seed to seedling progeny. This is the first demonstration of seed transmission with this emerging pathogen of global significance, and is of regulatory significance to APHIS. This result establishes new lines of research on the mechanisms used by the pathogen to colonize plant tissue and demonstrates a previously unsuspected capability of the pathogen. This was done as part of Project Objective 3 - Identify the virulence mechanisms
used by exotic pathogens and to meet milestone 2004- 2 - to complete rapid PCR tests for Xylella fastidiosa. This research addresses National Program 303, Plant Diseases; Component IV, Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors; Performance measure 3.2.5: The impact of this research has been to provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases. In particular, APHIS and the various state departments of agriculture are aware of these results and may modify procedures used to insure importations of citrus seed do not allow for the introduction of Xylella fastidiosa. Established that the insect Phyllocnistis citrella, the citrus leafminer, is not an efficient vector for citrus bacterial canker disease. This is good news for the citrus industry, since long distance spread of the pathogen by an insect would have complicated the eradication campaign.
This disease is of great regulatory interest at this time, with a continuing eradication campaign in Florida, run by APHIS and the Florida Dept. of Agriculture and closely watched by the California Dept. of Agriculture. This new scientific knowledge contributes towards better management methods for this disease. This was done as part of Project Objective 3 - Identify the virulence mechanisms used by exotic pathogens and to meet milestone 2005-4, collaborative research with the exotic pathogens of citrus collection. This research addresses National Program 303, Plant Diseases; Component IV, Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors; Performance measure 3.2.5: Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases. The impact of this research is high because USDA APHIS and the Florida Department of Agriculture are using this information in the design
of their current eradication campaign against citrus canker disease. We demonstrated that the expression of genes in Xylella fastidiosa is profoundly affected by passage through host plants as compared to laboratory culture. Control of diseases caused by X. fastidiosa is very problematic, in part because virulence mechanisms used by the pathogen are unknown. Our results are consistent with the emerging model that Xylella fastidiosa exists in a biofilm in diseased plants and insects, and that genetic expression in biofilms is different from that in culture. This new scientific knowledge is important because it will lead to a better understanding of the disease process by the scientific community. This was done as part of Project Objective 3 - Identify the virulence mechanisms used by exotic pathogens and to meet milestone 2004-2 Complete characterization of initial mini-Tn5 mutants This research addresses National Program 303, Plant Diseases; Component IV, Pathogen Biology, Genetics,
Population Dynamics, Spread, and Relationship with Hosts and Vectors; Performance measure 3.2.5: Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases. The inmpact of this research is high because the new scientific knowledge developed will be used by the scientific community to form better hypotheses to explain the unusual biology of X. fastidiosa and to develop effective methods for disease control. We have developed quantitative and real-time PCR based assays to detect and quantify Xylella fastidiosa and Candidatus Liberibacter spp. Both of these pathogens are on the select agent list and so rapid and sensitive detection methods are of special concern to the USDA. Because these pathogens are either very slow growing in culture, or do not grow at all in culture, culture independent methods of quantification are essential for the research community. This new scientific knowledge contributes to the
continued exclusion of these pathogens from the United States. This was done as part of CRIS Project Objective 2 - Develop novel rapid, sensitive and quantitative diagnostic tests for the presence of exotic citrus pathogens in plant tissues and to meet milestone 2004-1, Develop initial rapid PCR tests for Xylella fastidiosa and Huanglongbing. This research addresses National Program 303, Plant Diseases; Component I, Identification and classification of pathogens; Performance measure 3.2. 6: The impact of this research is high because it provides needed scientific information and technology to producers of agriculturally important plants in support of exclusion, detection and early eradication. The methods developed provide real-time, quantitative and sensitive detection of two pathogens on the USDA APHIS select agents list. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry,
farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Knowledge of Xylella fastidiosa biology (transmission through seed and influence of plant host on expression of genes) and rapid and quantitative PCR-based technology for have been transferred to the scientific community. APHIS, as well as various state departments of agriculture, are aware of our discovery of seed transmission by the select agent pathogen Xylella fastidiosa, and will decide how to modify plant exchange protocols. APHIS, as well as the Florida Department of Agriculture, are aware of our finding that the citrus leafminer does not transmit citrus canker at a measurable level while laying eggs in citrus leaves. APHIS, the Florida Department of Agriculture and Consumer Services, and regulatory officials in Brazil all know of and use our quantitative and real time PCR based assay for Ca. Liberibacter spp., the causal agents of ctrus greening or
huanglongbing disease. This assay is in general use in diagnosis and management of this disease. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Hartung, J.S. 2005. An overview of the Xanthomonas axonopodis pv citri genome. Citrus canker and huanglongbing workshop. USDA-ARS, USDA-APHIS, University of Florida. Orlando, FL. November 8, 2005. Hartung, J.S., Li, Wenbin, Song, Q., Brlansky, R. 2005. Genetic diversity and worldwide proliferation of citrus bacterial canker pathogens. Citrus canker and huanglongbing workshop. USDA-ARS, USDA- APHIS, University of Florida. Orlando, FL. November 9, 2005. Hartung, J.S., Li, W. Levy, L. 2005. Comparison of methods for the detection of Candidatus Liberibacter spp. In plant samples. Citrus canker and huanglongbing workshop. USDA-ARS, USDA-APHIS, University of Florida. Orlando, FL. November 9, 2005.
Hartung, J.S., Folimonov, A.S., Folimonov, S.Y., Dawson, W.O. 2005. Screening of antibacterial peptides in citrus trees for activity against Candidatus Liberibacter asiaticus. against Citrus canker and huanglongbing workshop. USDA-ARS, USDA-APHIS, University of Florida. Orlando, FL. November 9, 2005. Hartung, J.S. 2006. Exotic pathogens, people and the perfect storm: The crisis in the citrus industry. American Phytopathological Society, Potomac Division. March 16, 2006.
Impacts
(N/A)
Publications
- Li, W., Brlansky, R., Hartung, J.S. 2005. Amplification of dna of xanthomonas axonopodis pv citri from historic citrus canker herbarium specimens. Journal of Microbiological Methods. 65:237-246.
- Colletta-Filho, H., Takita, M., De Souza, A., Neto, J., Destefano, S., Hartung, J.S. 2006. Primers based on the rpf gene region provide improved detection of xanthomonas axonopodis pv citri in naturally and artificially infected citrus plants.. Journal of Applied Microbiology. 100(2):279-285.
- Lacava, P., Li, W., Hartung, J.S. 2005. Rapid, specific and quantitative assays for the detection of the endophytic bacterium methylobacterium mesophilicum in plants. Journal of Microbial Methods. 65:535-541.
- Li, W., Teixeira, D., Hartung, J.S., Levy, L. 2006. Detection and identification of candidatus liberibacter species associated with citrus huanglongbing by multiplex real-tim pcr. Journal of Microbiological Methods. 66:104-115.
- Montero-Astua, M., Hartung, J.S., Li, W., Auilar, E., Chacon, C., Rivera, C. 2006. Variability in colony morphology of xylella fastidiosa isolates from costa rica. American Phytopathological Society. 96(6):S164
- Hartung, J.S., Montera-Astua, M., Aguila, E., Chacon, C., Rivera, C. 2006. Molecular comparison of xylella fastidiosa isolates from costa rica, north and south america. American Phytopathological Society. 96(6):S164.
- Chacon, C., Montero-Astua, M., Hartung, J.S., Li, W., Garita, L., Rivera, C. 2006. Isolation, description, and identification of bacteria associated with diseased coffee (coffee arabica) and avocado (persea americana) plants from costa rica. American Phytopathological Society. 96(6):S162.
- Montero-Astua, M., Aguilar, E., Chacon, C., Garita-Cambronero, J., Garita, L., Villalobos, W., Moreira, L., Li, W., Hartung, J.S., Rivera, C. 2006. Research on xylella fastidiosa and the plant diseases it causes in costa rica. National American Phytopathology Meetings. 96(6):S81.
- Zhang, A., Hartung, J.S. 2005. Phenylacetaldehyde o-methyloxime: a volatile compound from plants infected with citrus canker pathogenic bacterium, xanthomonas axonopodis pv. citri.. Journal of Agricultural and Food Chemistry. 53:5134-5137.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The domestic citrus industry is continuously threatened by pathogens which cause serious diseases in foreign countries, but that are not present in the United States. The risk of introduction of these pathogens and their insect vectors increases continuously with the increase in international commerce and travel. These 'exotic pathogens' are studied to develop basic knowledge useful to either prevent their introduction into the United States or to facilitate their eradication or management if they are inadvertently introduced into the United States. We are currently working with the bacterial pathogens Xylella fastidiosa (Brazil) , Xanthomonas axonopodis pv citri(worldwide and since 1995 in Florida), Citrus Chlorotic Dwarf Virus (Turkey), Citrus Yellow Mosaic BADNA Virus (India), citrus greening or
'huanglongbing' (Southern Asia and Indonesia), and the presumptive virus that causes 'morte subita' or 'sudden death' disease in Brazil. The citrus strains of Xylella fastidiosa and the citrus greening pathogen are 'select agents' under US law. Our research is important to the citrus industry, which is a major economic factor in Florida, South Texas, and California. Each of these diseases has potential to seriously damage or destroy the industry. Citrus Variegated Chlorosis Disease, caused by Xylella fastidiosa was first described in Sao Paulo state, Brazil. Since that time, it has established itself as the most serious threat to citriculture in Brazil, which has the largest citrus industry in the world. Current estimates are that 40 percent (88 million) of the orchard trees in Sao Paulo are infected and yield losses can be high. Insects endemic to the United States can transmit this pathogen. Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri was eradicated from the
United States in the first third of this century, because the growers rightly feared its destructive potential. It has since been reintroduced into South Florida. The USDA is spending 40 million dollars per year in an effort to eradicate this pathogen. Mild symptoms of this disease prevent the sale of fruit on the fresh market and severe symptoms render the fruit unmarketable. This pathogen is also an object of international phytosanitary quarantine and so complicates international commerce. Citrus Chlorotic Dwarf Virus was discovered in Turkey in commercial lemon groves. It infects and causes disease in all citrus, causing a total yield loss. The insect vector is endemic in the United States. Citrus Yellow Mosaic BADNA Virus has been described in India, and causes a destructive disease there. The insect vector for this virus is endemic in the United States. This virus also has a double stranded DNA genome, which has implications for the biotechnology industry. The project has four
objectives: 1) Establish the etiology of novel diseases of citrus, 2) Develop novel rapid, sensitive and quantitative diagnostic tests for the presence of exotic pathogens in plant tissues, 3) Identify the virulence mechanisms used by exotic pathogens, and 4) Maintain and facilitate cooperative research on the Exotic Pathogens of Citrus Collection. The research falls under National Program 303 - Plant Diseases 70% and National Program 301- Plant, Microbial & Insect Genetic Res., Genomics, & Genetic Improv. 30%) as described in the National Program Action Plan. Specifically, these are: NP 303, Component 1: Identification and classification of plant pathogens; Commodity citrus research need 1: Citrus Tristeza Virus (CTV) - Strain Detection, Identification, and Characterization; Commodity citrus research need 4: Maintain and Thoroughly Characterize a Collection of Exotic Pathogens of Citrus. Component 4: Commodity citrus research need #4: Pathogen Characterization and Evaluation of
Commercial Scions for Resistance to CVC. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) Develop initial rapid PCR tests for Xylella fastidiosa and Huanglongbing Complete characterization of initial mini-Tn5 mutants Prioritize accessions; remove duplicates; collaborative research Year 2 (FY 2005) Complete purification of CCDV Complete rapid PCR technology; PCR test for CYMV Confocal microscopy of mutants in planta Prioritize accessions; remove duplicates; collaborative research; ongoing project Year 3 (FY2006) Etiology and purification of citrus yellow bark agent Differentiation of strains of CYMV Transfer rapid PCR technology PCR test for CYMV Prioritize accessions; remove duplicates; collaborative research Year 4 (FY 2007) Differentiation of strains of CYMV Prioritize accessions; remove duplicates; collaborative research Complete diagnostics for citrus yellow bark agent and CCDV Identify mutants impaired in pathogenicity Move collection to
new greenhouse Year 5 (FY 2008) Finish studies and transfer technologies Prioritize accessions; remove duplicates; collaborative research 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Complete purification of CCDV. Milestone Not Met Progress slowed by resource limitation (human,fiscal,equipment, etc. 2. Complete rapid PCR technology; PCR test for CYMV. Milestone Substantially Met 3. Confocal microscopy of mutants in planta. Milestone Substantially Met 4. Prioritize accessions; remove duplicates; collaborative research; ongoing project. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 2 (FY 2006) Research on the etiology and purification of citrus yellow bark agent will not be pursued
further because a large research group in Brazil has completed the purification and characterization of this virus. In place of this milestone, research has been initiated to characterize endophytic bacteria from citrus and their interaction with Xylella fastidiosa. Quantitative real-time PCR assays will be developed this year and used to study Methylobacterium spp. In periwinkle plants coinoculated with X. fastidiosa. Research on differentiation of strains of CYMV will not be pursued further because additional strains of CYMV from India have not been available. In place of this milestone, research has been initiated to characterize strains of Xylella fastidiosa that cause diseases of citrus, coffee and grapevine in Costa Rica. During this year, the strains will be characterized at the DNA level and relationships with strains of the same pathogen isolated from the same hosts from the United States and Brazil will be determined. Rapid PCR-based detection methods for X. fastidiosa and
Candidatus Liberibacter asiaticus will be developed and will be offered to APHIS. Research for milestone 4 will be completed using strains of CYMV currently available to us. Prioritize accessions; remove duplicates; collaborative research with and management of the exotic pathogens of citrus collection will continue on an ongoing basis. Year 3 (FY2007) Mutants of bacterial pathogens impaired in pathogenicity or with other interesting properties will continue. The exotic pathogens of citrus collection will be moved into the new greenhouse depending upon the timeline of the current construction project. Prioritize accessions; remove duplicates; collaborative research with and management of the exotic pathogens of citrus collection will continue on an ongoing basis. Year 4 (FY 2008) Finish studies and transfer technologies to APHIS or other interested parties Prioritize accessions; remove duplicates; collaborative research with and management of the exotic pathogens of citrus collection
will continue on an ongoing basis. 4a What was the single most significant accomplishment this past year? Expression of genes in Xylella fastidiosa is profoundly affected by passage through host plants as compared to laboratory culture Control of diseases caused by X. fastidiosa is very problematic, in part because virulence mechanisms used by the pathogen are unknown. Research showed that that the expression of green fluorescent protein was increased in Xylella fastidiosa after passage through both sweet orange and tobacco plants, and that the increased expression was lost upon further subculture in vitro. Our results are consistent with the emerging model that Xylella fastidiosa exists in a 'biofilm' in diseased plants and insects, and that genetic expression in biofilms is different from that in culture. The accomplishment was important because it will lead to a better understanding of the disease process. 4b List other significant accomplishments, if any. The insect Phyllocnistis
citrella, the citrus leafminer, is not an efficient vector for citrus bacterial canker disease. The presence of the leafminer insect is known to increase the severity of citrus canker disease in Florida and elsewhere. However, we have shown that the insect does not transmit the pathogen when laying eggs on susceptible citrus leaves at a detectable level. This is good news for the citrus industry, since long distance spread of the pathogen by an insect would have complicated the eradication campaign. This disease is of great regulatory interest at this time, with a continuing eradication campaign in Florida, and our results contribute towards better management methods for this disease. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Established that the sweet orange strain of Xylella fastidiosa completely colonizes the fruit and seed of sweet oranges and can be transmitted through seed to seedling progeny. This is the
first demonstration of seed transmission with this emerging pathogen of global significance, and is of regulatory significance to APHIS. This result establishes new lines of research on the mechanisms used by the pathogen to colonize plant tissue and demonstrates a previously unsuspected capability of the pathogen. This was done as part of CRIS Project Objective 3 - Identify the virulence mechanisms used by exotic pathogens and to meet milestone 2004-2 - to complete rapid PCR tests for Xylella fastidiosa. This research addresses National Program 303, Plant Diseases; Component IV, Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors; ARS Strategic Plan Performance Measure 3.2.5: The impact of this research has been to provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases. In particular, APHIS and the various state departments of agriculture are aware of these
results and may modify procedures used to insure importations of citrus seed do not allow for the introduction of Xylella fastidiosa. Established that the insect Phyllocnistis citrella, the citrus leafminer, is not an efficient vector for citrus bacterial canker disease. This is good news for the citrus industry, since long distance spread of the pathogen by an insect would have complicated the eradication campaign. This disease is of great regulatory interest at this time, with a continuing eradication campaign in Florida, run by APHIS and the Florida Dept. of Agriculture and closely watched by the California Dept. of Agriculture. This new scientific knowledge contributes towards better management methods for this disease. This was done as part of CRIS Project Objective 3 - Identify the virulence mechanisms used by exotic pathogens and to meet milestone 2005-4, collaborative research with the exotic pathogens of citrus collection. This research addresses National Program 303, Plant
Diseases; Component IV, Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors; Performance measure 3.2.5: Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases. The impact of this research is high because USDA APHIS and the Florida Department of Agriculture are using this information in the design of their current eradication campaign against citrus canker disease. We demonstrated that the expression of genes in Xylella fastidiosa is profoundly affected by passage through host plants as compared to laboratory culture. Control of diseases caused by X. fastidiosa is very problematic, in part because virulence mechanisms used by the pathogen are unknown. Our results are consistent with the emerging model that Xylella fastidiosa exists in a 'biofilm' in diseased plants and insects, and that genetic expression in biofilms is different from that in culture. This new
scientific knowledge is important because it will lead to a better understanding of the disease process by the scientific community. This was done as part of Project Objective 3 - Identify the virulence mechanisms used by exotic pathogens and to meet milestone 2004-2 'Complete characterization of initial mini-Tn5 mutants' This research addresses National Program 303, Plant Diseases; Component IV, Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors; ARS Strategic Plan Performance Measure 3.2.5: Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases. The inmpact of this research is high because the new scientific knowledge developed will be used by the scientific community to form better hypotheses to explain the unusual biology of X. fastidiosa and to develop effective methods for disease control. We have developed quantitative and real-time PCR based assays to
detect and quantify Xylella fastidiosa and Candidatus Liberibacter spp. Both of these pathogens are on the 'select agent list' and so rapid and sensitive detection methods are of special concern to the USDA. Because these pathogens are either very slow growing in culture, or do not grow at all in culture, culture independent methods of quantification are essential for the research community. This new scientific knowledge contributes to the continued exclusion of these pathogens from the United States. This was done as part of CRIS Project Objective 2 - Develop novel rapid, sensitive and quantitative diagnostic tests for the presence of exotic citrus pathogens in plant tissues and to meet milestone 2004-1, Develop initial rapid PCR tests for Xylella fastidiosa and Huanglongbing. This research addresses National Program 303, Plant Diseases; Component I, Identification and classification of pathogens; ARS Strategic Plan Performance Measure 3.2.6: The impact of this research is high
because it provides needed scientific information and technology to producers of agriculturally important plants in support of exclusion, detection and early eradication. The methods developed provide real-time, quantitative and sensitive detection of two pathogens on the USDA APHIS select agents list. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Knowledge of Xylella fastidiosa biology (transmission through seed and influence of plant host on expression of genes) and rapid and quantitative PCR-based technology for have been transferred to the scientific community. APHIS, as well as various state departments of agriculture, are aware of our discovery of seed transmission by the select agent pathogen Xylella fastidiosa, and will decide how to
modify plant exchange protocols. APHIS, as well as the Florida Department of Agriculture, are aware of our finding that the citrus leafminer does not transmit citrus canker while laying eggs in citrus leaves.
Impacts
(N/A)
Publications
- Qin, X., Hartung, J.S. 2004. Use of triparental mating system to introduce green fluorescent protein marked transposon insertions in the plant pathogen xylella fastidiosa.. Applied and Environmental Microbiology. 49 (3):211-216.
- Huang, Q., Brlansky, R.H., Barnes, L., Li, W.N., Hartung, J.S. 2004. First report of Oleander leaf scorch caused by Xylella fastidiosa in Texas. Plant Disease. 88:1049.
- Hartung, J.S., Civerolo, E.L., Dawson, W.O., Garnsey, S.M. 2004. A proven model for working with exotic plant pathogens with biological security and low cost. Phytopathology. 38(12):162-163.
- Belasque, J., Parra-Pedrazzoli, A., Rodrigues, J., Yammamoto, P., Chagas, M., Parra, J., Hartung, J.S. 2005. Adult citrus leafminer (phyllocnistis citrella) are not vectors for citrus canker in experimental microcosms.. Plant Disease. 89(6):590-594.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The domestic citrus industry is continuously threatened by pathogens which cause serious diseases in foreign countries, but that are not present in the United States. The risk of introduction of these pathogens and their insect vectors increases continuously with the increase in international commerce and travel. These 'exotic pathogens' are studied to develop basic knowledge useful to either prevent their introduction into the United States or to facilitate their eradication or management if they are inadvertently introduced into the United States. We are currently working with the bacterial pathogens Xylella fastidiosa (Brazil) , Xanthomonas axonopodis pv citri(worldwide and since 1995 in Florida), Citrus Chlorotic Dwarf Virus (Turkey), Citrus Yellow Mosaic BADNA Virus (India), citrus greening or
'huanglongbing' (Southern Asia and Indonesia), and the presumptive virus that causes 'morte subita' or 'sudden death' disease in Brazil. The citrus strains of Xylella fastidiosa and the citrus greening pathogen are 'select agents' under US law. Our research is important to the citrus industry, which is a major economic factor in Florida, South Texas, and California. Each of these diseases has potential to seriously damage or destroy the industry. Citrus Variegated Chlorosis Disease, caused by Xylella fastidiosa was first described in Sao Paulo state, Brazil. Since that time, it has established itself as the most serious threat to citriculture in Brazil, which has the largest citrus industry in the world. Current estimates are that 40 percent (88 million) of the orchard trees in Sao Paulo are infected and yield losses can be high. Insects endemic to the United States can transmit this pathogen. Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri was eradicated from the
United States in the first third of this century, because the growers rightly feared its destructive potential. It has since been reintroduced into South Florida. The USDA is spending 40 million dollars per year in an effort to eradicate this pathogen. Mild symptoms of this disease prevent the sale of fruit on the fresh market and severe symptoms render the fruit unmarketable. This pathogen is also an object of international phytosanitary quarantine and so complicates international commerce. Citrus Chlorotic Dwarf Virus was discovered in Turkey in commercial lemon groves. It infects and causes disease in all citrus, causing a total yield loss. The insect vector is endemic in the United States. Citrus Yellow Mosaic BADNA Virus has been described in India, and causes a destructive disease there. The insect vector for this virus is endemic in the United States. This virus also has a double stranded DNA genome, which has implications for the biotechnology industry. The project has four
objectives: 1) Establish the etiology of novel diseases of citrus, 2) Develop novel rapid, sensitive and quantitative diagnostic tests for the presence of exotic pathogens in plant tissues, 3) Identify the virulence mechanisms used by exotic pathogens, and 4) Maintain and facilitate cooperative research on the Exotic Pathogens of Citrus Collection. The research falls under National Program 303 - Plant Diseases 70% and National Program 301- Plant, Microbial & Insect Genetic Res., Genomics, & Genetic Improv. 30%) as described in the National Program Action Plan. Specifically, these are: NP 303 Component 1: Identification and classification of plant pathogens; Commodity citrus research need 1: Citrus Tristeza Virus (CTV) - Strain Detection, Identification, and Characterization; Commodity citrus research need 4: Maintain and Thoroughly Characterize a Collection of Exotic Pathogens of Citrus. Component 4: Commodity citrus research need #4: Pathogen Characterization and Evaluation of
Commercial Scions for Resistance to CVC. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) Develop initial rapid PCR tests for Xylella fastidiosa and Huanglongbing Complete characterization of initial mini-Tn5 mutants Prioritize accessions; remove duplicates; collaborative research Year 2 (FY 2005) Complete purification of CCDV Complete rapid PCR technology; PCR test for CYMV Confocal microscopy of mutants in planta Prioritize accessions; remove duplicates; collaborative research; ongoing project Year 3 (FY2006) Etiology and purification of citrus yellow bark agent Differentiation of strains of CYMV Transfer rapid PCR technology PCR test for CYMV Prioritize accessions; remove duplicates; collaborative research Year 4 (FY 2007) Differentiation of strains of CYMV Prioritize accessions; remove duplicates; collaborative research Complete diagnostics for citrus yellow bark agent and CCDV Identify mutants impaired in pathogenicity Move collection to
new greenhouse Year 5 (FY 2008) Finish studies and transfer technologies Prioritize accessions; remove duplicates; collaborative research 3. Milestones: Milestone 2 has been completed; Milestone 1 is partially completed and research towards this milestone continues. Rapid PCR has been developed for Xylella fastidiosa but not yet finalized for huangongbing - preliminary results are promising. Milestone 3 is ongoing work with the exotic pathogens of citrus collection. Year 1 (FY2004) Develop initial rapid PCR tests for Xylella fastidiosa and Huanglongbing Complete characterization of initial mini-Tn5 mutants Prioritize accessions; remove duplicates; collaborative research B. List the milestones (cited in # 2) that you expect to accomplish over the next 3 years (FY 2005, 2006 and 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 2 (FY 2005) Complete purification of CCDV - will continue attempts at purification from different citrus
hosts. CCDV is a very difficult virus and we will continue to try novel purification and characterization approaches. Complete rapid PCR technology; Will validate Huanglongbing methods with plant samples PCR test for CYMV will be developed based on sequence information obtained earlier. Confocal microscopy of mutants in planta. This is substantially complete. Prioritize accessions; remove duplicates; collaborative research. This is ongoing work with the exotic pathogens of citrus collection. Year 3 (FY2006) Etiology and purification of citrus yellow bark agent. We will attempt to identify the causal agent of citrus yellow bark using greenhouse plants in Beltsville. Test PCR assays for the yellow bark agent. Differentiation of strains of CYMV. We will use multiple strains from India if available. Transfer rapid PCR technology - to interested parties PCR test for CYMV - this will be developed based on sequence of full length genomic clone already available Prioritize accessions;
remove duplicates; collaborative research. This is ongoing work with the exotic pathogens of citrus collection. Year 4 (FY 2007) Complete diagnostics for citrus yellow bark agent and CCDV - These will be validated with plant samples. Identify mutants impaired in pathogenicity - Continue to screen mutants in planta. Move collection to new greenhouse. This depends upon the successful completion of the current greenhouse construction project. This is ongoing work with the exotic pathogens of citrus collection. 4. What were the most significant accomplishments this past year? A. Single most important accomplishment during FY 2004: Established that the sweet orange strain of Xylella fastidiosa can be transmitted through seed to seedling progeny. This accomplishment is important because this is the first demonstration of seed transmission with this emerging pathogen of global significance. Research showed that the pathogen completely colonizes the fruit and seed of sweet oranges. The sweet
orange strain of Xylella fastidiosa is a select agent and is not present in the United States. Our results may be used to improve the biosecurity of US agriculture. Other significant information B. Demonstrated that the expression of genes in Xylella fastidiosa is profoundly affected by passage through host plants as compared to laboratory culture. The accomplishment was important because it will lead to a better understanding of the disease process. Research showed that that the expression of green fluorescent protein was increased in Xylella fastidiosa after passage through both sweet orange and tobacco plants, and that the increased expression was lost upon further subculture in vitro. Our results are consistent with the emerging model that Xylella fastidiosa exists in a 'biofilm' in diseased plants and insects, and that genetic expression in biofilms is different from that in culture. C. Significant activities that support target populations None D. Progress report opportunity to
submit additional programmatic information to your Area Office and NPS (optional for all in-house ('D') projects and the projects listed in Appendix A; Mandatory for all other subordinate projects). None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Established that the sweet orange strain of Xylella fastidiosa completely colonizes the fruit and seed of sweet oranges and can be transmitted through seed to seedling progeny. This is the first demonstration of seed transmission with this emerging pathogen of global significance. These results establish new lines of research on the mechanisms used by the pathogen to colonize plant tissue and demonstrates a previously unsuspected capability of the pathogen. This was done as part of Objective 3 - Identify the virulence mechanisms used by exotic pathogens and to meet milestone 2 of FY 2004 - to complete rapid PCR tests for Xylella fastidiosa. Action Plan components 1 and 4.
6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Knowledge of Xylella fastidiosa biology (transmission through seed and influence of plant host on expression of genes) and rapid and quantitative PCR-based technology for have been transferred to the scientific community.
Impacts
(N/A)
Publications
- Li, W., Pria, W., Lacava, P., Hartung, J.S. 2003. Presence of xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings. Phytopathology. 93(8):953-953.
- Li, W.N., Hartung, J.S. 2004. Green fluorescent protein-labeled strains of xylella fastidiosa colonize citrus, grapevines and periwinkle.. American Phytopathology Society. 94(6):S60.
- Lacava, P.T., Li, W.N., Hartung, J.S. 2004. Pcr assay for methylobacterium mesophilicum in sweet orange trees infected with xylella fastidiosa.. American Phytopathology Society. 94(6):S56.
- Huang, Q., Li, W.N., Hartung, J.S. 2003. First report of xylella fertidiosa in japanese beech bonsai. Canadian Journal of Plant Pathology. 25:401-405.
- Hartung, J.S., Gouin, C., Lewers, K.S., Maas, J., Hokanson, S. 2003. Identification of sources of resitance to bacterial angular leafspot disease of strawberry. Acta Horticulture Proceedings. 626:155-159.
- Li, W.N., Hartung, J.S. 2003. Use of a triparental mating system to introduce marked mutations in xyella fastidiosa. American Phytopathological Society Annual Meeting. 93(6):S51.
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